2.1. β-Caryophyllene and Nervous System
Many studies report the beneficial effects of BCP on central nervous system [22
], 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
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
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
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
]. 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-α, 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-α, 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 management (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
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 suggest 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 effect is due to the activation of CB2-R [41
Various studies have shown muscle relaxant effect 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 effect [9
]. 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
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 effect on cell growth, unlike the isomer α
In human breast cancer cells MCF-7, BCP amplifies the cytotoxicity of the isomers isocaryophyllene and α
]. A study realized on obese mice C57BL/6N, injected with melanoma cells, has shown that the phytocannabinoid is able to decrease the precancerous effect caused by a high-fat diet [48
BCP, which is more than 25% of the essential oil from Pamburus missionis
, has synergistic effect 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.
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
]. 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 effector caspases [49
It has been shown that BCP causes the activation of caspase-3 and determines nucleolus fragmentation and the consequent apoptosis in two different 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
] and proapoptotic effects (exhibited via DNA “ladder” and caspase-3 activation) in tumor cell lines, while there was no apoptosis induction in normal cell lines (FB) [13
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-α, 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
The three substances act synergically, since they separately do not exert any significant activity. Their effect 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 effects 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 TNF-α 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 effects 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 PPAR-α 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-α, 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 effective 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 effect of pioglitazone [53
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 effects 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 effect. 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
] 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 TNF-α, IL-1β, IL-6, and consequently, NF-κB.
Also, BCP effects 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 effect, 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