*2.2.* β*-Caryophyllene and Cancer*

Both BCP and BCPO have shown cytotoxic activity against various cancer cell lines.

In particular, BCP has significantly decreased the proliferation of two colon cancer cell lines, HT-29 and HCT-116, and a pancreas cancer cell line, PANC-1. Moreover, it has been quite successful on other types of cancer cells [45].

In the intestinal cancer cell line CaCo-2 [46], for example, BCP has not been able to exert a significant e ffect on cell growth, unlike the isomer α-humulene [13].

In human breast cancer cells MCF-7, BCP amplifies the cytotoxicity of the isomers isocaryophyllene and α-humulene [47]. A study realized on obese mice C57BL/6N, injected with melanoma cells, has shown that the phytocannabinoid is able to decrease the precancerous e ffect caused by a high-fat diet [48].

BCP, which is more than 25% of the essential oil from *Pamburus missionis*, has synergistic e ffect with two other important components of the essential oil, phytol (**8**) and aromadendrene oxide (**9**) (Figure 3), resulting in antitumor activity against A431 and HaCaT cell lines, by blocking the cell in phase G0/G1 or sub-G1.

**Figure 3.** Structures of the phytol (**8**) and aromadendrene oxide (**9**).

The mechanism of action is associated to ROS production [69] and the mitochondrial membrane potential loss, by increasing Bax expression and decreasing Bcl-2 expression. Bax and Bak oligomers form pores, which increase the permeability of the external mitochondrial membrane, releasing cytochrome c in the cytoplasm, which is an apoptosis characterizing event [70–72]. The release of cytochrome c from mitochondria to cytosol leads to the formation of apoptosomes, and consequently, the activation of caspase-9, which activates the cascade of the e ffector caspases [49,73,74].

It has been shown that BCP causes the activation of caspase-3 and determines nucleolus fragmentation and the consequent apoptosis in two di fferent cell lines, BS-24-1 (murine cell line of lymphoma) and MoFir (human T cell transformed through Epstein-Barr virus) [49].

BCP, one of the compounds in the essential oil, of *Commiphora gileadensis*, is responsible for antiproliferative exhibited by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay [75–80] and proapoptotic e ffects (exhibited via DNA "ladder" and caspase-3 activation) in tumor cell lines, while there was no apoptosis induction in normal cell lines (FB) [13,50].

Anyhow, numerous studies have revealed that BCP enhances the effectiveness of antitumor drugs. In particular, a research has shown that it increases the activity of paclitaxel in lots of cell lines: MCF-7 (breast cancer), L-929 (mouse fibroblasts), DLD-1 (colon cancer) [47]. In detail, in the latter cell line, the BCP determines a rise of the intracellular concentrations in the drug, probably by increasing the permeability of the cell membrane [13].

BCP has been reported to exert anticancer and hypoglycemic effects in BALB/c mice transplanted with cells of the line CT26 exposed to high levels of glucose, to mimic a colorectal cancer. The sesquiterpene blocks ART1 effects, by inhibiting NF-κB. ART1 (arginine-specific mono-ADP-ribosyl transferase 1) is an enzyme, whose concentrations are higher in patients with type 2 diabetes, involved in the pathogenesis of colorectal cancer. The overexpression of ART1 probably increases glycolysis and energy metabolism, thus regulating the protein kinase B/mammalian target of rapamycin/c-Myc signaling pathway and the expression of glycolytic enzymes. This suggests that BCPO may be a potential treatment for this kind of carcinoma [51].

Further, BCPO is cytotoxic against various cell lines, including: HeLa (human cervical adenocarcinoma cells), HepG2 (human leukaemia cells), AGS (human lung cancer cells), SNU-1 and SNU-16 (human stomach cancer cells) and A-2780 (human ovarian cancer cells). It modulates many fundamental pathways in tumour pathogenesis, such as those involving MAPK, Phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) and Signal Transducer and Activator of Transcription (STAT3) [13].

BCPO, like BCP, shows synergy with antitumor drugs, like paclitaxel and doxorubicin. In particular, it is able to increase the concentration of the two drugs in many cancer cell lines, including CaCo-2.

Anyhow, between the two sesquiterpenes, the oxidised derivative has the greatest antitumor activity, variable according to cell types and dosages. In fact, due to the epoxide function, BCPO binds covalently to amino groups and thiol moieties of proteins and nitrogen bases, which constitute the nucleic acids. In any case, both the molecules achieve their antitumor action by causing apoptosis and blocking the cell cycle [13].

### *2.3.* β*-Caryophyllene and Inflammatory Diseases*

Acute inflammation is a body defence mechanism against various factors, which may evolve in chronic inflammation, resulting in pathological disease [81]. After the acute reaction, monocytes remain in the inflammation site, where they secrete cytokines and chemokines and stimulate macrophages, amplifying the inflammatory response. As for neuroinflammation, the main phlogistic mediators are interleukins IL-1β, IL-6, and TNF-<sup>α</sup>, which increase NF-κB (nuclear factor kappa B) expression, prostaglandins and leukotrienes, synthesized by cyclooxygenases (COX) and 5-lipoxigenase (LOX) [82].

An in vitro study on macrophages of RAW267.4 [83] mice has revealed that BCP, administered in association to other two natural molecules, baicalin (**10**) and (+)-catechin (**11**) (Figure 4), at relatively low doses, suppresses the proliferation of these cells involved in inflammation [52].

**Figure 4.** Structures of the baicalin (**10**) and (+)-catechin (**11**).

The three substances act synergically, since they separately do not exert any significant activity. Their e ffect is achieved through the cell cycle arrest phase G2/M and the modulation of various intracellular pathways, such as PI3K/Akt, extracellular signal-regulated kinases(ERK)/MAPK, and calcium homeostasis. In particular, the decrease of the expression of Akt, MAPK p38 and p44/42, and caspase-3 activation, an important step of apoptosis, have been observed. In addition to these effects, the expression of COX-1 and COX-2 diminishes, and so the activity of the protein p65 of NF-κB family does [52].

According to a recent study, BCP exerted powerful results against the negative e ffects of dyslipidemia and vascular inflammation in mice [53]. In fact, it has been revealed that the treatment with the sesquiterpene, at the dose of 30 mg/kg, prevents the increase of adiposity index, glycemia and insulinemia due to a high fat-diet. It also helps dyslipidemia and reduces all atherogenic risk indexes, even if it does not modify body weight. BCP reduces oxidative stress, by decreasing the concentration of NO and malondialdehyde (MDA), a by-product of lipid peroxidation, and by increasing the level of the endogenous antioxidant glutathione [84]. The phytocannabinoid is able to suppress mediators involved both in inflammation and atherosclerosis, such as TNFα and NF-κB. In this way, it leads to the inhibition of VCAM1 [53], a vascular cell adhesion protein, which promotes the adhesion of white cells of the vascular endothelium and favours atherosclerosis, confirming what was reported in other in vitro researches [54].

BCP normalizes the ratio between endothelial (eNOS) and inducible (iNOS) nitric oxide synthase within the aorta. The latter is activated in the phlogistic process and in the atherosclerosis following the oxidative stress-induced NF-κB activation. Further, iNOS produces a high amount of nitric oxide, which interacts with ROS, generating peroxynitrites, which amplify the oxidative stress.

Furthermore, BCP attenuates the formation of foam cells and the deposition of collagen, which plays a crucial role in the formation and progression of vulnerable atherosclerotic plaques, and protects the integrity of elastic lamina.

All these e ffects are attributable to direct action of BCP, as agonist of cannabinoid receptors CB2, other than of PPAR-γ receptors (receptors activated by peroxisome proliferator-activated receptors), involved in the reduction of blood levels of total cholesterol, low density lipoprotein (LDL) and very low density lipoprotein (VLDL), in the inhibition of vascular inflammation and synthesis of adhesion molecules and in the rebalancing of nitric oxide concentration and nitric oxide synthase isoforms. Moreover, it would seem that BCP is also an agonist of PPARα receptors, reduces fat mass and triglycerides and increases high density lipoprotein (HDL). This happens through the binding of BCP to cannabinoid receptors CB2-R, which activates PGC1- α (coactivator 1 of the peroxisome gamma proliferator) and allows the interaction among PPAR-γ and various transcriptional factors, such as PPAR-<sup>α</sup>, increasing the expression of enzymes with the function of oxidizing fat acids, above all in the liver.

The comparison with the thiazolidinedione pioglitazone (**12**) (Figure 5), a PPAR-γ agonist used for the treatment of type 2 diabetes and atherosclerosis, has shown that BCP is more e ffective than the drug for all the parameters, except for the glutathione levels. In addition, the sesquiterpene does not induce body weight gain, the main side e ffect of pioglitazone [53].

**Figure 5.** Structure of the pioglitazone.

BCP is beneficial in a model of bilateral carotid artery occlusion and reperfusion (BCCAO/R) in Wistar rats. This study has demonstrated that a single dose of BCP prevents plasmatic and

tissue modifications induced by carotid obstruction and reperfusion [55]. It increases tissue levels of endocannabinoids (anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, oleylethanolamide) and cannabinoid receptors CB1 and CB2, reducing anandamide blood concentrations. Furthermore, it preserves the tissue levels of the essential fatty acid docosahexaenoic acid (DHA), increases PPAR-α expression, and decreases lipoperoxidation damage [55].

BCP e ffects have been studied in a rat model of rheumatoid arthritis, characterized by inflammatory response.

BCP and copaiba oil (the sesquiterpene accounts for about 37% of the oil) have the same ability to reduce paw edema, popliteal lymph nodes weight and myeloperoxidase plasmatic activity. BCP, unlike copaiba oil, decreases also the activity of hepatic myeloperoxidase and leukocytes, both the blood ones and those present in the joints. The anti-inflammatory activity is slightly greater in copaiba oil than in BCP, probably because of the presence of other molecules with synergist e ffect. Neither the oil nor the single molecule are able to modify secondary injuries and body weight in rats. Both are capable, at high doses, to:


Copaiba oil, because of the presence of diterpenes, like kaurenoic acid and hardwickiic acid, is hepatotoxic, reducing the liver functionality due to hepatic cholestasis. In this context, the use of isolated BCP is preferable, since it is hepatoprotective [57].

BCP exerts in vitro [45,85,86] and in vivo antioxidant capacities.

As regards the mechanism, BCP, at a dose of 430 mg / kg, exhibits antioxidant activity and acts by exerting various functions: radical scavenging ability in particular with respect to hydroxyl radicals, lipid peroxides and superoxide anions; stimulation of the endogenous antioxidant system, highlighted by the increased content of glutathione in the liver, induced by Nrf. A decrease in inflammation, characterized by reduced activity of myeloperoxidase, is a marker of infiltration of polymorphonuclear cells, and diminished expression of COX-2 and cytokines, such as TNFα, IL-1β, IL-6, and consequently, NF-κB.

Also, BCP e ffects on C57BL/6J mice have been investigated. The mice, fed with an essential amino acid-deficient diet, are a model of non-alcoholic steatohepatitis (NASH), which is a hepatic inflammatory pathology associated to metabolic syndrome predisposing cardio-vascular diseases. In the case of steatohepatitis, the organ undergoes histological changes caused by oxidative stress, inflammation and fibrosis. The treatment with BCP reduced inflammation and fibrosis. Moreover, a decrease in alanine-transaminase (ALT) and cytokine expression has been observed, suggesting that the liver has been less damaged. BCP exerts antioxidant e ffect, by increasing the levels of the enzymes SOD2 (superoxide dismutase 2) and GPx1 (glutathione peroxidase 1), both involved in free radical detoxification. In addition, the enzyme Nox2 (reduced form of nicotinamide adenine dinucleotide phosphate (NADP) oxidase 2), TGF-β*,* and collagen, all elements which contribute to hepatic fibrosis, have been inhibited by the sesquiterpene [58].

### *2.4.* β*-Caryophyllene and Micro-Organisms*

BCP has antimicrobial activity both against Gram-positive bacteria, such as *Staphilococcus aureus* and Gram-negative bacteria, including *Escherichia coli* [59]. It is more e ffective against Gram-positive and the activity of *Artemisia fed dei* extracts, containing BCP, against bacteria responsible for tooth decay and periodontitis has been reported [60]. The antimicrobial activity of the essential oil and some of its compounds was tested against 15 di fferent genera of oral bacteria, including *Streptococcus mutans*, which produces a biofilm, constituted by a variety of extracellular polymeric substances (EPS), which protect micro-organisms of oral cavity. The antibacterial activities of the essential oil and of BCP was determined based on the minimum inhibitory concentrations (MICs) which were determined as the lowest concentration of the test samples that resulted in a complete inhibition of visible growth in the broth. BCP kills *Streptococcus mutans* with a minimum inhibiting concentration (MIC) of 0.32% by penetrating the biofilm, which constitutes a protective barrier against external substances, acting as an ion exchange resin. *Streptococcus mutans* uptakes sucrose, which is necessary for glucan synthesis, through the enzymes GtfB, GtfC and GtfD. The first two are required for the synthesis of insoluble glucans, which facilitate bacterial aggregation. GtfD, on the other hand, promotes the synthesis of water-soluble glucans, which are necessary for biofilm development. The mechanism of action of BCP is to reduce the expression of Gtf genes, thus preventing biofilm synthesis.

### *2.5.* β*-Caryophyllene and Osteoporosis*

BCP exerts e ffects on various tissues, like bone tissue. In fact, it stimulates the di fferentiation of multipotent stem cells (MSC) of bone marrow (which can di fferentiate in many cell types, including adipocytes, osteoblasts, myoblasts, chondrocytes, etc.) in osteoblasts, limiting the formation of adipose cells. In order to modulate this process, regulated by various pathways, BCP activates PPAR-γ. It suppresses osteoclastogenesis, which is the synthesis of osteoclasts, cells derived from haematopoietic progenitors which promote bone resorption. This mechanism, TNF-α-mediated, activates the transcription factor NF-κB in prosteoclasts and it is inhibited in vitro by BCP (in cell cultures of murine bone marrow) in the di fferentiation stage in prosteoclasts.

Therefore, BCP enhances osteoblast mineralization, favouring osteoblastogenesis and suppressing osteoclastogenesis and adipogenesis, which is an interesting property for the potential application in the treatment of osteoporosis, in particular the one associated to obesity and diabetes [61].
