Composition comprising beta-caryophyllene, docosahexaenoic acid and eugenol for analgesia, anti-inflammation and anti-oxidation of central and peripheral nervous system

Abstract

The present invention describes a composition comprising beta-caryophyllene, docosahexaenoic acid and eugenol for use as analgesic and anti-inflammatory antioxidants for the central and peripheral nervous system, administered by oral route, the compounds are useful as analgesics for chronic pain disorders, chronic inflammatory pain, chronic neuralgia and as anti-inflammatory antioxidants for the central and peripheral nervous systems.

Description

TECHNICAL FIELD

[0001] The present invention relates to a composition comprising beta-caryophyllene, docosahexaenoic acid and eugenol as analgesic and anti-inflammatory antioxidant of the central and peripheral nervous system. In particular, said composition comprising beta-caryophyllene, docosahexaenoic acid and eugenol is administered by oral route and it is effective as analgesic of chronic pain conditions, chronic inflammatory pain, chronic neuropathic pain and as anti-inflammatory antioxidant of the central and peripheral nervous system. BACKGROUND

[0002] It is estimated that about 10% of the world population suffers from chronic pain [Wong et al. Exploring the impact of low back pain and mental health symptoms on healthcare resource use and costs: a population-based cohort study protocol. BMJ Open 2019, 9, e031749]. All pain syndromes, including arthritis, back pain, neck pain, fibromyalgia, interstitial cystitis, migraine, neuropathic pain, bursitis and vulvodynia, have specific inflammatory features. The "Pain Theory" states that the root of all pain is inflammation and the response to inflammation. Activation of nociceptors, transmission and modulation of pain signals, neural plasticity and central sensitization are a continuum of inflammation and inflammatory response [Omoigui S. Biochemical origins of pain - proposing a new law of pain: the root of all pain is inflammation and the response to inflammation. Part 2 (of 3) - the unifying law of pain. Med Hypotheses. 2007, 69(1), 70-82].

[0003] Specialized primary afferent neurons with the function of detecting noxious chemicals, thermal stimuli and mechanical stimuli are called nociceptors. Their cell bodies are mainly located in the trigeminal nerve and dorsal root ganglion (DRG) and provide sensory innervation to almost all tissues except the brain parenchyma. Specialized receptors, channels and synthetic pathways contribute to determine the specificity of a particular nociceptor subtype, thus enabling the detection and signaling of acute and persistent noxious stimuli. Two main receptors-channels have been identified. The first is TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1), which is considered an integrator of multiple noxious stimuli, as it is modulated by multiple inflammatory products [Schumacher et al. TRPV1 splice variants: structure and function. Front Biosci (Landmark Ed). 2010, 15(3): 872-882]. The second is the Transient Potential Associated Channel expressed on nociceptors, TRPA1 (Transient Receptor Potential Cation Channel Subfamily A Member 1), which is considered a gatekeeper of inflammation. TRPA1 is considered to play an important role in the development and maintenance of inflammatory pain conditions and can complement TRPV1; in fact, TRPA1 is activated by exogenous and endogenous inflammatory mediators [such as reactive oxygen species (ROS)]. TRPA1 is involved in models of persistent inflammatory pain, mechanical and thermal hyperalgesia, inflammatory muscle pain and pain induced by pancreatitis [Bautista DM, et al. TRPA1: the gatekeeper of inflammation. Annu Rev Physiol. 2013, 75: 181-200; Koivisto A, et al. TRPA1: a transducer and amplifier of pain and inflammation. Basic Clin Pharmacol Toxicol. 2014, 114(1): 50-55].

[0004] In chronic pain, immune system cells not only have an impact on the function of neurons at the site of inflammation, but also on damaged peripheral nerves and the central nervous system.

[0005] Signs and symptoms of inflammation include cell migration, edema, fever, erythema, pain and hyperalgesia. With the development of inflammation, spontaneous pain often occurs and the inflamed tissue can exhibit hyperalgesia, i.e. an increase in pain after stimulation, including an increase in pain after noxious stimuli and a decrease in pain threshold (allodynia).

[0006] The cells responsible for inflammatory pain are mast cells, which release inflammatory mediators, particularly cytokines [Stassen M, et al. Classical and alternative pathways of mast cell activation. Crit. Rev. Immunol. 2002, 22: 115-140] and activated macrophages, which can release a number of inflammatory mediators, such as pro-inflammatory cytokines, particularly tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1 β), nerve growth factor (NGF), nitric oxide (NO), and prostaglandins. Macrophage activation appears to have a significant impact on the subsequent recruitment and activation of other types of cells (neutrophils) at the site of inflammation [Thomazzi SM, et al. Tumor necrosis factor, interleukin-1 and interleukin-8 mediate the injurious activity of LPS-stimulated macrophage supernatants. Mediators Inflamm. 1997, 6: 195-200]. Neutrophils are the first type of inflammatory cell to infiltrate tissues from the blood and control acute and early inflammatory responses. They can produce several inflammatory factors, including lipoxygenase products, NO, cytokines, and chemokines, and it is known that preventing the accumulation of neutrophils in inflammation can reduce the associated inflammatory pain [Chou et al. Anti-inflammatory and analgesic effects of baicalin in carrageenan-induced thermal hyperalgesia. Anesth. Analg. 2003, 97: 1724-1729]. T and B cells can also produce inflammatory cytokines and chemokines and contribute to the innate immune response [Sibilia J. New concepts and therapeutic approaches in autoimmune diseases: ten key points. Joint Bone Spine 2004, 71: 511-517].

[0007] Immune cells release a number of potential pain-producing substances in inflamed tissue, including TNF-α, IL-1 β, NGF, prostaglandin E2 (PGE2), lipoxygenase products (leukotriene B), bradykinin, serotonin, cytokines (IL-6 and leukemia inhibitory factor), chemokines (CCL2, CXCL8, and GCSF), and NO [McMahon et al. Chapter 3 in McMahon SB, Koltzenburg M, eds. Pain. Elsevier, London, 2006; Omote et al. Changes in peripheral nerve nitric oxide in carrageenan-induced inflammation. Brain Res. 2001, 912: 171-175]. Antagonism of each of these pain-producing mediators produces a significant antihyperalgesic effect, often approaching 100%.

[0008] Mast cells, neutrophils, macrophages and T cells also participate in neuropathic pain caused by various forms of nerve injury. Several studies have shown a correlation between the levels of TNF-a, considered a prototypic pro-inflammatory cytokine, and the development of allodynia or hyperalgesia [George et al. Sequence of changes in tumor necrosis factor-a content in rat sciatic nerve after chronic constriction injury. Exp. Neurol. 1999, 160: 124-132]. Other cytokines such as IL-1 β and IL-6 are involved in neuropathic pain [Schafers et al. Additive effects of anti-TNF-α and anti-IL-1 receptor neutralizing antibodies in attenuating nerve pathologic pain in mice. Neurosci. Lett. 2001, 310: 113-116; De Jongh et al. Role of interleukin-6 in nociception and pain. Anesth Analg. 2003, 96: 1096-1103].

[0009] Neuropathic pain conditions resulting from peripheral nerve injury cause pathology not only in the dorsal root ganglion (DRG) and the injured peripheral nerve itself, but also in a series of changes in the processing of sensory information in the central nervous system. These changes are indirect, as the CNS itself is not directly injured. Such central changes include alterations in immune cell function, with hematopoietic-derived leukocytes and microglia being of particular interest. Microglia, oligodendrocytes, and astrocytes together comprise a large class of glial cells in the central nervous system. Microglia express the same surface markers as macrophages / monocytes and can be activated by events such as CNS injury, microbial invasion, and certain pain states, which in turn increase the production of a variety of inflammatory cytokines, chemokines, and other potentially pain-causing substances. Activation of microglia has been implicated in the development of neuropathic pain following peripheral nerve injury. A number of studies have shown that specific inhibitors and / or modulators of microglia can block and / or reverse neuropathic pain states [Meller et al. Possible role for glial cells in nociception and hyperalgesia as revealed by intrathecal tnf-α factor administration. Neuropharmacology 1994, 33: 1471-1478], and that microglia are responsible for the initiation of neuropathic pain, while astrocytes are involved in its maintenance. Released cytokines such as TNF-a and IL-6 can in turn also participate in the activation of microglia [Winkelstein et al. Similar spinal glial activation and selective cytokine upregulation occur after nerve injury in both the L5 spinal nerve and chronic constriction injury models of neuropathic pain. J. Comp. Neurol. 2001, 439: 127-139]. Peripheral nerve injury associated with neuropathic pain can induce activation of microglia in the spinal cord, followed by activation of astrocytes in the affected spinal segments. This process appears to be critical for the full development of neuropathic pain. Under pathophysiological conditions, microglia can release a variety of mediators, such as IL-1β, TNF-a, PGE2, and NO, which can modulate the process of spinal pain in a variety of ways.

[0010] Injuries and diseases directly affecting the CNS can also trigger a strong immune response. In particular, spinal cord injury and multiple sclerosis, Parkinson's disease and Alzheimer's disease can be associated with abnormal sensitivity to pain. As for other tissues, spinal cord injury leads to local inflammatory reactions and activation of immune cells, which are similar to those described above, including microglial cell activation, mitochondrial dysfunction, release of pro-inflammatory cytokines and production of reactive oxygen species [Seo et al. Neuroinflammation in exercise and disease. Int. Neurourol. J. 2019, 23: 82-S92]. Immune cells can then act at many anatomical levels: in the peripheral tissues during the inflammatory phase, in the peripheral nerves and spinal cord in the case of peripheral neuropathies, and in certain forms of bone marrow injury. Under these conditions, a wide range of immune mediators is released, some of which can affect the pain signaling system.

[0011] In summary, chronic pain, such as inflammatory pain, neuropathic pain or pain associated with CNS injury, is a debilitating disease in which the role of immune mediators, in particular pro-inflammatory cytokines, is important.

[0012] First-line pharmacological treatments for chronic pain include opioids and non-steroidal anti-inflammatory drugs (NSAIDs), both of which have serious side effects, such as tolerance / addiction for opioids and gastric pain for NSAIDs [Aloisi et al. Hormone replacement therapy for opioid-induced hypogonadism in male patients with chronic pain. Reproductive Biology and Endocrinology, 2011, 9: 26; de Maddalena et al. Opioid-induced hypogonadism: why and how to treat. Pain Physician, 2012, 15(3): ES111-ES118].

[0013] Therefore, there is an urgent need to develop new pharmacological approaches to limit the progression of the disease and reduce pain, without generating side effects.

[0014] Among these approaches, substances of plant origin and plant extracts, often referred to as natural analgesics, are of particular interest.

[0015] The combination of two natural source substances, beta-caryophyllene (BCP) and docosahexaenoic acid (DHA), has been studied for its analgesic efficacy both in vitro and in vivo [Fiorenzani et al. In vitro and in vivo characterization of a new analgesic combination: beta-caryophyllene and docosahexaenoic acid. Evid Based Complement Alternat Med. 2014, 2014: 596312]. This study showed that BCP, used alone or in combination with DHA, significantly reduced the formalin-induced pain response. Moreover, in vitro acute toxicity tests (performed on fibroblasts and astrocytes) showed that BCP is toxic at high concentrations, but this toxicity is completely eliminated when it is administered in combination with DHA at a weight ratio of about 1 : 1. The authors speculate that the synergistic effect of the BCP+DHA combination could be due to the competitive binding / interaction of the two substances on the same receptor, since this modulatory effect has also been observed in vitro. SUMMARY

[0016] In order to further improve the analgesic efficacy of the BCP+DHA combination, the present inventors found that the addition of a specific amount of eugenol to the BCP+DHA combination significantly enhances the analgesic efficacy and anti-inflammatory antioxidant properties.

[0017] This specific combination composed of BCP, DHA and eugenol is particularly effective in the treatment of chronic pain and as an anti-inflammatory, antioxidant for the central and peripheral nervous system, since it induces the production of anti-inflammatory / resolution-promoting cytokines and reduces pro-inflammatory cytokines.

[0018] Therefore, the object of the present invention is to provide a composition comprising beta-caryophyllene (BCP), docosahexaenoic acid (DHA) and eugenol for the analgesic and anti-inflammatory, antioxidant for the central and peripheral nervous system. In particular, the composition comprises 0.9 - 1.1 parts by weight of BCP, 0.9 - 1.1 parts by weight of DHA and 1.3 - 2.0 parts by weight of eugenol.

[0019] More preferably, the composition of the present invention comprises equal parts of BCP and DHA and 1.5 parts by weight of eugenol. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1A - bar graph showing the IL-6 concentration (pg / mL) measured in the supernatant of U373-MG cells cultured in the presence of samples 1-11.

[0021] Figure 1B - bar graph showing the percentage change of IL-6 of samples 5-11 compared to the positive control (IL-1 beta - sample 4).

[0022] Figure 2A – Bar graph showing IL-10 concentrations (pg / mL) measured in supernatants of U373-MG cells cultured in the presence of samples 1-11.

[0023] Figure 2B – Bar graph showing the percent change in IL-10 for samples 5-11 compared to the positive control (IL-1β - sample 4).

[0024] Figure 3A - Bar graph showing IL-4 concentrations (pg / mL) measured in supernatants of U373-MG cells cultured in the presence of samples 1-11.

[0025] Figure 3B – Bar graph showing the percent change in IL-4 for samples 5-11 compared to the positive control (IL-1β - sample 4).

[0026] Figure 4A - Bar graph showing the TNF-α concentration (pg / mL) measured in the supernatants of U373-MG cells cultured in the presence of samples 1-11.

[0027] Figure 4B – Bar graph showing the percent change in TNF-α for samples 5-11 compared to the positive control (IL-1β - sample 4). DETAILED DESCRIPTION

[0028] The present invention provides a composition comprising β-caryophyllene (BCP), docosahexaenoic acid (DHA), and eugenol for analgesic, anti-inflammatory, and antioxidant effects on the central and peripheral nervous systems. Specifically, the composition comprises 0.9-1.1 parts by weight of BCP, 0.9-1.1 parts by weight of DHA, and 1.3-2.0 parts by weight of eugenol.

[0029] More preferably, the composition object of the present invention comprises equal amounts of BCP and DHA and 1.5 parts by weight of eugenol.

[0030] BCP, DHA and eugenol are naturally derived substances that are widely known and have been used for many years for their various beneficial properties.

[0031] β-Caryophyllene Beta-caryophyllene (herein also referred to as BCP) is a bicyclic sesquiterpene widely distributed in the plant kingdom, the source of the unique aroma of essential oils, and plays a major role in the survival and evolution of higher plants. As a selective agonist of cannabinoid receptor 2 (CB2), it has multiple pharmacological activities, such as antibacterial (e.g. against Helicobacter pylori), antioxidant, anti-inflammatory, analgesic (e.g. against neuropathic pain), anti- neurodegenerative and anti-tumor properties. An increasing number of studies describe the multiple protective effects of BCP in various metabolic and neurological diseases. In particular, these diseases are mostly characterized by chronic inflammation [Scandiffio et al. (E)-beta-caryophyllene (BCP) protects against chronic inflammation. Nutrients. 2020, 12(11): 3273]. Experimental results show that BCP is able to reduce pro-inflammatory mediators such as TNF-alpha, IL-1 beta, IL-6, nuclear factor NF-kappa B, thus improving chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through binding to the CB2 cannabinoid receptor and interaction with members of the family of Peroxisome Proliferator-Activated Receptors (PPAR, in particular PPAR alpha and gamma), BCP shows beneficial effects in obesity, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), diabetes, cardiovascular diseases, chronic pain and other neurological diseases [Gertsch et al. Beta-caryophyllene is a dietary cannabinoid. Proc. Natl. Acad. Sci. USA 2008, 105: 9099-9104; Youssef et al. Beta-caryophyllene is involved in the protection of rats from diet-induced dyslipidemia and vascular inflammation through CB2 and PPAR-gamma receptors. Chem. Biol. Interact. 2019, 297: 16-24].

[0032] Therefore, it is known that BCP can act on multiple molecular pathways involved in the inflammatory response and can effectively reduce various pro-inflammatory mediators, including IL-1 beta, IL-6, TNF-alpha and NF-kappa B. There are research data that show that BCP can exert its strong anti-inflammatory action through various mechanisms, most of which start from the binding of BCP to the CB2 receptor.

[0033] DHA The beneficial properties of omega-3 polyunsaturated fatty acids (PUFAs) in the diet, especially docosahexaenoic acid (DHA), have been known for decades and their metabolic dysfunction has been linked to many diseases, including inflammation and neurodegenerative diseases [Echeverria et al. Docosahexaenoic acid (DHA), an essential fatty acid for the brain: new dietary sources. Prostaglandins Leukot Essent Fatty Acids. 2017, 124: 1-10]. DHA is an important component of the neural membrane, as it modulates the fluidity, permeability and viscosity of the synaptic membrane and plays a key role in regulating neurotransmission and synaptic function. Although it is still not clear why DHA is particularly enriched in brain phospholipids and lacks DPA (docosapentaenoic acid) and EPA (eicosapentaenoic acid), the enrichment phenomenon is preserved in all species, suggesting a high degree of specificity of neuronal membranes for this essential fatty acid [Farkas et al. Molecular species of phospholipids containing docosahexaenoic acid in vertebrate brains. Proc Natl Acad Sci USA. 2000, 97: 6362-6366].

[0034] Once transformed into metabolites with biological activity, EPA and DHA become substances necessary to resolve inflammation through natural endogenous modulators of the immune system; in fact, the lack of these PUFAs is associated with chronic inflammatory diseases [Zhang et al. Resolvins: anti-inflammatory and pro-resolving mediators derived from omega-3 polyunsaturated fatty acids. Annual Review of Nutrition. 2012, 32: 203-227]. Most of the neuroprotective effects of omega-3 oils come from the DHA component rather than the EPA component. DHA has strong antioxidant and anti-inflammatory properties [Cole et al. DHA or can prevent age-related dementia. Journal of Nutrition. 2010, 140(4): 869-874], also increases lipoxins, inhibits NF-kB and produces neuroprotectin-D and resolvin, which are very important for the complete resolution of the inflammatory process, thus minimizing the risk of chronicity and pain exacerbation [Kumar et al. Robbins and Cotran Pathologic Basis of Disease. 7th ed. W.B. Saunders, Philadelphia, PA, USA, 2005].

[0035] DHA is essential for CNS function, including neuronal growth, neuronal survival, synaptic integrity and neurotransmission, as well as protection from neuroinflammatory processes and cognitive impairment [Wu et al. Beneficial effects of DHA dietary supplementation on cognitive function, neural plasticity, and membrane homeostasis after brain trauma. J Neurotrauma. 2011, 28(10): 2113-2122].

[0036] The role of DHA in inflammation is well known. In fact, DHA plays a role as metabolic precursor of resolvins, neuroprotectins and maresins, which are anti-inflammatory molecules involved in the resolution process of inflammation, which is spontaneous but yet active, mainly involving myeloid cells such as macrophages and glial cells [Serhan C.N. Proresolving lipid mediators are leading substances for resolving physiology. Nature. 2014, 510(7503): 92-101]. Defects in the systemic synthesis of DHA in mice induce neuronal plasticity dysfunction and brain inflammation. In a mouse model Elovl2- / - (key enzyme for DHA synthesis), a significant downregulation of several neuroplasticity factors (Arc-1, Egr-1 and BDNF) and a concomitant upregulation of major inflammatory markers (TNF-a, IL-1b, iNOS and Casp1) were found. In this model, the re-introduction of DHA in KO mice significantly increased the expression levels of Arc-1 mRNA and reduced the expression levels of IL-1b and Casp1, which showed levels comparable to wild-type mice, compared to KO mice [Talamonti et al. DHA synthesis impairment alters expression of mouse neuronal plasticity markers and brain inflammatory status. FASEB J. 2020, 34(2): 2024-2040].

[0037] Eugenol As previously mentioned, the inventive feature of the present disclosure lies in the combined use of eugenol with BCP and DHA.

[0038] Eugenol is a phenolic aromatic substance, belonging to allylbenzenes, naturally present in Lamiaceae, Lauraceae, Rutaceae, Myrtaceae, Zingiberaceae, Asteraceae, and others. eugenol is one of the main components of clove essential oil (Syzygium aromaticum L.). Eugenol is classified as GRAS (Generally Recognized as Safe) and is widely used at industrial level as a flavoring agent (in food and cosmetics) and in the pharmaceutical and dental fields (as an antibacterial and local anesthetic agent). Eugenol is known to have remarkable pharmacological properties, as it has proven to be effective in the treatment of various diseases of the reproductive, nervous, digestive and respiratory systems, capable of modulating glucose and cholesterol levels in the blood, and having antihypertensive, antioxidant, anti-inflammatory, antibacterial and antitumor properties [Petrocelli et al. Molecules. 2021, 26: 885. Moleculars from essential oils for prevention and treatment of colorectal cancer (CRC)]. Eugenol can interfere with many intracellular signaling pathways by counteracting oxidative stress and chronic inflammation. At the chemical level, it has extraordinary reducing activity, as it provides a hydroxyl group that reacts with free radicals. Given these properties, eugenol is considered an “enhancer” of the functioning of the immune system.

[0039] After administration of eugenol, a decrease in the expression of several mediators of the inflammatory process (such as TNF-a, NF-KB, COX2, IL-1 b, IL-5, IL-6, iNOS and NO) was observed, accompanied by an increase in the content of antioxidant enzymes (such as superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase).

[0040] Eugenol acts on inflammatory mediators derived from the arachidonic acid (AA) cascade by inhibiting the production of prostaglandins and leukotrienes. In a mouse model of inflammation induced by LPS, eugenol reduced lung infiltration of neutrophils / macrophages and inhibited the release of inflammatory cytokines (TNF-a, IL-1 b and IL-6) by inhibiting the activation of the NF-KB signaling pathway [Nisar et al. Oxidative Medicine and Cellular Longevity. 2021, 2021: 2497354. Pharmacological properties and health benefits of eugenol: a comprehensive review]. In addition, eugenol also showed an antipyretic effect [Feng et al. Eugenol: antipyretic activity in rabbits. Neuropharmacology. 1987, 26(12): 1775-1778].

[0041] Eugenol also has neuroprotective effects. By virtue of its hydrophobicity, orally administered eugenol is able to cross the blood-brain barrier, exerting a local effect in the brain, inhibiting lipid peroxidation, and enhancing endogenous antioxidant mechanisms [Singh et al. Antioxidant and neuroprotective effects of 4-allyl-2-methoxyphenol against clomazone-induced neurotoxicity in rat brain in vivo. Mol Cell Biochem. 2014, 388: 61-74]. For example, in a mouse model of aluminum-induced brain damage, the neuroprotective effect of eugenol was attributed to its antioxidant, anti-apoptotic potential, and neurotrophic properties [Said et al. Neuroprotective effects of eugenol against aluminum-induced neurotoxicity in rats. Arh Hig Rada Toksikol. 2017, 68(1): 27-37].

[0042] Eugenol is able to protect neural cells by inhibiting apoptosis and the secretion of proinflammatory cytokines, related to its ability to counteract oxidative stress and modulate the expression of inflammatory cytokines. In fact, eugenol inhibits the production of proinflammatory mediators in activated macrophages and inhibits the induction of NF-κB activation by tumor necrosis factor (TNF-a) in human acute promyelocytic leukemia cell lines [Li et al. Inhibitory effect of eugenol on nitric oxide production in RAW264.7 macrophages. Biomed Res. 2006, 27: 69-74; Chainy et al. Thymol blocks tumor necrosis factor-mediated early and late cellular responses: effects on NF-κB, AP-1, JNK, MAPKK, and apoptosis. Oncogene. 2000, 19: 2943-2950]. Overall, eugenol exerts multiple direct and indirect effects to restore normal neurotrophic and serotonergic balance. It also normalizes acetylcholinesterase levels and shows beneficial effects on glycogen basal synthesis in mouse primary astrocytes [Sartorius et al. Cinnamon extract improves insulin sensitivity and reduces liver fat content in an obese mouse model. PLoS One. 2014, 9: e92358]. In addition, eugenol can have a direct or indirect role in protecting astrocytes from induced neurotoxicity.

[0043] In a mouse neurodegenerative model of Parkinson's disease-like induced by neurotoxin (6-OHDA) treatment, eugenol was able to improve motor function and counteract body weight loss [Moreira Vasconcelos et al. Eugenol and its association with levodopa on the behavioral and neurochemical alterations of 6-hydroxydopamine-induced hemiparkinsonian rats. Basic Clin Pharmacol Toxicol. 2020, 127(4): 287-302]. It is known that the damage caused by 6-OHDA is closely related to a significant increase in the rate of lipid peroxidation and nitrite / nitrate content [Souza et al. Neuroprotective effects of sulfated agar from the marine red alga Gracilaria cornea in a rat model of Parkinson's disease induced by 6-hydroxydopamine: behavioral, neurochemical, and transcriptional level alterations. Basic Clin Pharmacol Toxicol. 2017, 120: 159-170]. The treatment with eugenol was able to effectively counteract these effects, further confirming its effect in reducing the levels of nitric oxide and its derivatives and the content of malondialdehyde (a byproduct of lipid oxidation) [Prasad et al. Neurorestorative effects of eugenol, a bioactive constituent of a spice: evidence in cellular models and its efficacy as an intervention molecule to alleviate oxidative dysfunction in the brain of streptozotocin diabetic rats. Neurochem Int. 2016, 95: 24-36]. It is known that eugenol can have a direct or indirect antioxidant effect. Its direct action is related to the capture of free hydroxyl radicals, which can interfere with the formation of intermediate fatty acids, a possible mechanism for preventing lipid peroxidation [Ito et al. Antioxidant effects of eugenol derivatives: role of metal ions in the inhibition of lipid peroxidation. Food Chem Toxicol. 2005, 43: 461-466]. In addition, it is hypothesized that eugenol can have an action to prevent the degradation of IκB-a, a protein that inhibits NF-κB, which indirectly ultimately leads to a reduction in NF-κB inhibition and iNOS expression, thus reducing the levels of nitric oxide derivatives [Irie Y. Eugenol's effects on the central nervous system: its potential application in the treatment of Alzheimer's disease, depression, and Parkinson's disease. Curr Bioact Compd. 2006, 2: 57-66].In contrast, the indirect action of eugenol is related to its possible induction of glutathione-S-transferase activity, leading to an increase in the levels of available glutathione and the formation of reduced glutathione (GSH), an important endogenous antioxidant [Vidhya et al. Antioxidant effect of eugenol in the rat gut. Indian J Exp Biol. 1999, 37: 1192-1195]. In mouse models of Parkinson's disease, a decrease in GSH content in the brain has been observed. Treatment of Parkinson's disease with levodopa is usually accompanied by side effects such as dyskinesia [Smith et al. Levodopa peak dose-induced dyskinesia-related striatal mRNA expression patterns in the 6-OHDA hemiparkinsonian rat model. Neuroscience. 2016, 324: 238-251]. The use of eugenol in combination with low doses of levodopa can reduce these side effects, suggesting a possible neuroprotective effect.

[0044] Combination The combination of BCP, DHA and eugenol on which the present invention is based is unique in that eugenol is added in a specific amount, thus producing a synergistic effect compared to the use of BCP and DHA alone.

[0045] In particular, said specific amount is in the range of 1.3 - 2.0 parts by weight.

[0046] It is worth noting that when the amount of eugenol is significantly increased (for example, to triple), no substantial improvement compared to the BCP+DHA combination alone is observed, thus losing the synergistic effect of the addition of eugenol.

[0047] As shown in in vitro experiments on human astrocyte cell lines stimulated with IL-1 beta inflammation (Figures 1-4), this synergistic effect manifests itself in a significant reduction of pro-inflammatory cytokines (IL-6 and TNF-alpha), with a corresponding increase in anti-inflammatory cytokines (IL-10 and IL-4).

[0048] Formulation In the composition of the present invention, the three substances BCP, DHA and eugenol can be used in isolated and / or purified form, optionally also of synthetic origin, or in the form of extracts or essential oils of natural origin containing these ingredients.

[0049] As mentioned previously, the weight ratio between BCP, DHA and eugenol is specific and represents an essential feature of the composition of the present invention.

[0050] In fact, the amount of eugenol is in excess with respect to BCP and DHA, but this excess must be in the range of 1.3 - 2.0 to obtain the best results in terms of synergistic efficacy. Increasing the amount of eugenol reduces the efficacy of the composition, as can be clearly seen in the comparison between the combination BCP:DHA:eugenol = 1 : 1 : 1.5 and BCP:DHA:eugenol = 1 : 1 : 5.0 (Figures 1-4).

[0051] As mentioned above, eugenol is naturally present in the essential oils of several plants, in particular it is one of the main components of clove essential oil (Syzygium aromaticum L.). Clove essential oil is widely used in the perfume, cosmetic, health, medical, spice and food industry. Eugenol represents at least 50% of clove essential oil. The remaining 10-40% is composed of eugenol acetate, BCP and a-humulene. Therefore, from a practical point of view, the use of clove essential oil containing both eugenol and BCP would be particularly advantageous for the preparation of the composition of the present application.

[0052] However, the clove essential oil commonly used does not allow to achieve the required weight ratio between eugenol and BCP.

[0053] The present inventors found that it is feasible to achieve the specific weight ratio between BCP and eugenol by using essential oils having a high concentration (e.g. 80%). These essential oils are obtained by steam distillation.

[0054] Such essential oils are then mixed in the appropriate proportions to reach the desired ratio, preferably microencapsulated according to the conventional techniques before the final formulation.

[0055] A particularly preferred mixture of essential oils rich in BCP and eugenol is exemplified by: consisting of 60% w / w of high purity eugenol (80%) clove oil and 40% w / w of high purity BCP (80%) clove oil.

[0056] Then, 100 grams of this “EUG / BCP” oil contain 48 grams of eugenol and 32 grams of BCP (eugenol:BCP ratio = 1.5: 1).

[0057] DHA is preferably used in the form of a powder with a purity grade > 75%.

[0058] A particularly preferred example is the microencapsulated DHA powder with a titer of 75.8% sold by the company Nutraceuticasrl.

[0059] The final formulation in its preferred form, with a BCP:DHA:eugenol ratio of 1 : 1 : 1.5, can then be obtained by mixing 100 g of microencapsulated “EUG / BCP” oil with 42.7 g of microencapsulated DHA powder.

[0060] Subsequently, one or more carriers and / or excipients can be optionally added to the mixture containing the BCP, DHA and eugenol composition of the application, depending on the final formulation form chosen.

[0061] The composition of the application is administered orally, and the carriers / excipients will therefore be suitably chosen from those commonly used in oral administration forms, such as tablets, capsules, soft capsules and solutions / suspensions.

[0062] Preferably, the composition of the application is administered in microencapsulated form or as an emulsion filled in soft gelatin capsules (soft capsules), each capsule containing a unit dose of the composition of between 350 and 1000 mg, such as 350 mg (100 mg BCP, 100 mg DHA and 150 mg eugenol), 437.5 mg (125 mg BCP, 125 mg DHA and 187.5 mg eugenol), 525 mg (150 mg BCP, 150 mg DHA and 225 mg eugenol), etc.

[0063] The composition of the application is preferably administered 1-3 times a day, more preferably twice a day.

[0064] Experimental Section Study objectives The inflammatory / proalgesic and anti-inflammatory / proresolutive responses of a human astrocyte cell line were tested after an inflammatory stimulus (IL-1 β).

[0065] The production of pro- and anti-inflammatory cytokines was tested in the presence of the inflammatory stimulus alone and in co-culture with eugenol (2.5 μΜ and 0.75 μΜ), β-caryophyllene (BCP - 0.4 μΜ), docosahexaenoic acid (DHA - 0.25 μΜ) and combinations thereof.

[0066] Materials and methods The cell line U373-MG (derived from a human astrocytic glioma) was cultured in DMEM (Dulbecco's Modified Eagle Medium) with the addition of 10% fetal bovine serum, 1% L-glutamine-streptomycin and 1% non-essential amino acids and incubated at 37°C with 5% CO2. When the cells reached the confluence state, they were washed with 0.1 M PBS, detached from the support with a trypsin solution, centrifuged at 1500 rpm for 5 minutes at room temperature and resuspended in complete medium (1 : 15 dilution ratio). In each well of a 24-well plate, 15000 suspended cells were seeded in complete medium. The volume of medium per well was adjusted to 1 mL and the cells were incubated in an incubator at 37°C and 5% CO2for 24 hours.

[0067] At the end of the incubation, to each sample was added: Sample 1 : Negative control Sample 2: 0.1% ethanol (solvent control) Sample 3: 0.1% DMSO (solvent control) Sample 4: IL-1 β 1 ng / mL (positive control, inflammation stimulus only) Sample 5: Eugenol [2.5 μΜ] + IL-1 β [1 ng / mL] Sample 6: Eugenol [0.75 μΜ] + IL-1 β [1 ng / mL] Sample 7: BCP [0.4 μΜ] + IL-1 β [1 ng / mL] Sample 8: DHA [0.25 μΜ] + IL-1 β [1 ng / mL] Sample 9: BCP [0.4 μΜ] + DHA [0.25 μΜ] + IL-1 β [1 ng / mL] Sample 10: BCP [0.4 μΜ] + Eugenol [2.5 μΜ] + DHA [0.25 μΜ] + IL-1 β [1 ng / mL] Sample 11 : BCP [0.4 μΜ] + Eugenol [0.75 μΜ] + DHA [0.25 μΜ] + IL-1 β [1 ng / mL] The correspondence between micromolar concentration and w / V concentration is as follows: Beta-Caryophyllene 0.4 μΜ = 81.744 μg / L DHA 0.25 μΜ = 82.122 μg / L Eugenol 2.5 μΜ = 410 μg / L Eugenol 0.75 μΜ = 123.15 μg / L Therefore, sample 10 contains BCP, DHA and eugenol in a weight ratio of 1 : 1 : 5, while sample 11 contains BCP, DHA and eugenol in a weight ratio of 1 : 1 : 1.5.

[0068] The plates were then further incubated for 24 hours at 37°C in 5% CO2. After collection of the supernatant of each sample, the analysis was performed using the Bio-Plex Multiplex Immunoassay System (Bio-Rad Laboratories srl, Segrate, MI, Italy).

[0069] Four replicates were set up for each sample and the following cytokines were evaluated: IL-6, IL-10, IL-4, IL-8 and TNF-a.

[0070] IL-6 (pg / ml) Figure 1A and 1B ) IL-6 is secreted by macrophages upon stimulation by specific signals, such as during infection. IL-6 can promote the synthesis of acute phase proteins and the production of neutrophils in the bone marrow. It can promote B cell growth and antagonize regulatory T cells. IL-6 promotes inflammatory and autoimmune processes in a variety of diseases, such as multiple sclerosis, diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, rheumatoid arthritis and cerebral hemorrhage. In the CNS, astrocytes are one of the main inducible sources of IL-6. While low concentrations of IL-6 are beneficial to the CNS due to its neurotrophic properties, its overexpression is generally detrimental, exacerbating the pathophysiological processes associated with CNS diseases. Many factors are known to induce overexpression of IL-6 by astrocytes, particularly the proinflammatory cytokines TNF-a and IL-1 b, so their regulation is essential [Van Wagoner et al. Production of interleukin-6 (IL-6) by astrocytes: autocrine regulation by IL-6 and soluble IL-6 receptor. J Neurosci. 1999, 19(13): 5236-44]. In fact, overexpression of IL-6 mediates demyelination, axonal damage and promotes CNS inflammation and damage during progressive autoimmune encephalomyelitis. Studies have shown that blocking IL-6 has therapeutic potential in limiting this pathology and the functional impairment and tissue damage in multiple sclerosis [Constantinescu et al. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 2011, 164(4): 1079-106].

[0071] Figure 1A and 1B IL-6 values (pg / ml) and IL-6 percentage change (pg / ml) are shown for human astrocytoma cell line after incubation with samples 1-11 Figure 1A ) and IL-6 percentage change (pg / ml) are shown for human astrocytoma cell line after incubation with samples 1-11 Figure 1B ).

[0072] Sample 1 (negative control) and samples 2-3 (solvent control) showed no significant differences.

[0073] Sample 4 (IL-1 b only) represents an inflammatory stimulus, inducing a significant increase in IL-6 (P<0.05).

[0074] Sample 5-6 (eugenol), sample 7 (BCP) and sample 8 (DHA) did not cause a significant decrease in IL-6.

[0075] On the contrary, sample 9 (BCP+DHA) and sample 10-11 (BCP+DHA+eugenol) showed a significant decrease in IL-6 compared to sample 4. In particular, sample 11 (combination of the invention) showed the greatest decrease in IL-6, also significant compared to sample 9 (BCP+DHA) and sample 10.

[0076] IL-10 (Fig. 4) Figure 2A and 2B ) IL-10 is a neuroprotective anti-inflammatory cytokine. It is produced by monocytes, mast cells and certain activated T and B cell subsets and plays a fundamental role in regulating inflammatory and immune responses. IL-10 mainly inhibits the induction of pro-inflammatory cytokines TNF-a, IL-1 b, IL-12 and IFN-g secreted by myeloid cells activated by TLRs (Toll-like receptors) mediated by LPS and bacterial products. In the CNS, IL-10 is mainly produced by astrocytes and microglia and is upregulated after various injuries, such as experimental autoimmune encephalomyelitis, middle cerebral artery occlusion, excitotoxicity and traumatic brain injury. Astrocyte production of IL-10 influences microglial reactivity and recruitment of lymphocytes and ultimately has a beneficial effect on neuronal survival [Villacampa et al. IL-10 production targeting astrocytes can change microglial reactivity and reduce motoneuron death after facial axotomy. Glia. 2015, 63(7): 1166-84]. In vitro studies have shown that BCP, as a CB2 receptor agonist, is able to downregulate IL-1 b and TNF-a and reduce iNOS expression and ROS production in primary mouse microglia, protecting them from LPS-induced inflammation. On the contrary, IL-10 and Arg1 (arginase) are upregulated. With the increase in Arg1 and the decrease in iNOS, the production of urea increases and the production of NO decreases, changes that clearly indicate a shift in microglial phenotype towards M2 (pro-resolution of inflammation) [Askari et al. Protective effects of b-caryophyllene on lipopolysaccharide-induced M1 / M2 imbalance in primary microglia: a mechanistic evaluation. Life Sci. 2019, 219: 40-73].

[0077] Figure 2A and 2B shows the IL-10 values (Fig. 5) Figure 2A ) and the percentage change in IL-10 (Fig. 6) Figure 2B ) of a human astrocyte cell line after incubation with samples 1-11.

[0078] Sample 1 (negative control) and samples 2-3 (solvent controls) showed no significant difference.

[0079] Sample 4 (IL-1 β only) represents an inflammatory stimulus and did not produce a significant effect compared to the controls.

[0080] Samples 7 (BCP), 9 (BCP + DHA) and 10-11 (BCP + DHA + Eugenol) all significantly increased IL-10 production.

[0081] In particular, sample 11 (combination of the invention) produced the highest increase in IL-10 and was significantly different compared to samples 7, 9 and 10.

[0082] IL-4 (Fig. 2) Figure 3A and 3B ) IL-4 is a cytokine that induces differentiation of naive helper T cells (Th0) into Th2 cells. Upon activation by IL-4, Th2 cells subsequently produce more IL-4 in a positive feedback loop. IL-4 is produced primarily by mast cells, Th2 cells, eosinophils, and basophils. IL-4 is a key regulator of humoral and adaptive immunity with multiple biological actions, including stimulating the proliferation of activated B and T cells and differentiation of B cells into plasma cells. IL-4 induces B cell class switching, produces IgE, modulates class II MHC expression, and suppresses the production of proinflammatory cytokines such as TNF-a, IFN-g, and IL-17. IL-4 can also promote the expression of growth factors in the CNS by astrocytes, such as nerve growth factor. Astrocytes have a marked response to IL-4, but the expression of its receptor and factors that modulate receptor expression are not well understood. IL-4 can exert proinflammatory and anti-inflammatory effects on astrocytes, depending on the treatment and timing. In primary mouse astrocytes, pre-treatment with IL-4 reduced NO and iNOS production and TNF-a secretion following LPS stimulation. Similarly, simultaneous treatment of human fetal primary astrocytes with IL-4 reduced NO production caused by IL-1b, TNF-a, or IFN-g stimulation. However, in another study, primary mouse astrocytes treated with IL-1b showed higher IL-6 production when subsequently treated with IL-4, while treatment with IL-10 and dexamethasone produced immunosuppressive effects. However, the conditioned media of astrocytes treated with IL-4 had dose-dependent neuroprotective effects. Thus, the literature data show a complex interaction between IL-1b and IL-4 in the context of neuroprotection [Chen et al. Interleukin 4 influences epilepsy through regulation of glial cells: potential roles and possible mechanisms. Front Mol Neurosci. 2020, 13: 554547; Gadani et al. IL-4 in the brain: a cytokine worth remembering. J Immunol. 2012, 189(9): 4213-9].

[0083] Figure 3A and 3B shows IL-4 values (pg / ml) Figure 3A ) and IL-4 percent change (pg / ml) Figure 3B ).

[0084] Sample 1 (negative control) and samples 2-3 (solvent controls) did not show significant differences.

[0085] Sample 4 (IL-1b only) represents an inflammatory stimulus, inducing a significant increase in IL-4 (P < 0.05).

[0086] Sample 5-6 (eugenol), sample 7 (BCP), sample 8 (DHA), sample 9 (BCP+DHA) and sample 10 (BCP:DHA:eugenol = 1 : 1 :5) all caused a non-significant increase in IL-4 levels.

[0087] In contrast, sample 11 (combination of the invention) significantly increased IL-4 production compared to inflammation stimulation and sample 9.

[0088] TNF-a (Fig. 2) Figure 4A and 4B ) TNF-a is produced by macrophages as well as various cells, including lymphoid cells, mast cells, endothelial cells, cardiomyocytes, adipose tissue, fibroblasts and neurons. It is released upon stimulation by lipopolysaccharide, other bacterial products and IL-1 b. It has multiple effects on multiple organs, often synergizing with IL-1 b and IL-6. In the hypothalamus, it activates the hypothalamic-pituitary-adrenal axis, stimulates the release of corticotropin-releasing hormone CRH, suppresses appetite and induces fever. In the liver, it stimulates the acute phase response, but also induces insulin resistance. It is a strong chemoattractant for neutrophils and promotes the expression of adhesion molecules on endothelial cells, facilitating neutrophil migration. In macrophages, it stimulates phagocytosis and the production of IL-1 b and PGE2. Increased local TNF-a concentrations result in the classical signs of inflammation: fever, swelling, redness, pain and loss of function. TNF-a promotes the inflammatory response, which in turn leads to many of the clinical problems associated with autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma.

[0089] In the CNS, TNF-a can have neuroprotective effects, but can also cause neurotoxic effects by activating microglia. Indeed, the occurrence of cognitive impairment after CNS inflammation or infection is associated with increased levels of TNF-a. Local TNF-a increase in the hippocampal dentate gyrus activates astrocytic TNF-a type 1 receptor (TNFR1), which in turn triggers astrocyte-neuron signaling cascades leading to persistent functional alterations of hippocampal excitatory synapses. The hippocampal synaptic alterations and accompanying memory impairment observed in experimental autoimmune encephalomyelitis (EAE, an animal model of multiple sclerosis, MS) are dependent on astrocytic TNFR1 signaling. This process can be involved in the pathogenesis of cognitive impairment in multiple sclerosis and other central nervous system diseases with inflammation and infection [Habbas et al. Neuroinflammatory TNFa impairs memory function through astrocytic signaling pathways. Cell. 2015, 163(7): 1730-41].

[0090] Figure 4A and 4B shows TNF-a values (pg / ml) of human astrocyte cell line after incubation with samples 1-11 Figure 4A ) and TNF-a percentage change (pg / ml) Figure 4B ).

[0091] Sample 1 (negative control) and samples 2-3 (solvent control) did not show significant differences.

[0092] Sample 4 (IL-1 β only) represents an inflammatory stimulus, inducing a significant increase in TNF-a (P<0.05).

[0093] Samples 5-6 (eugenol), sample 7 (BCP) and sample 8 (DHA) did not cause a significant decrease in TNF-a levels.

[0094] On the contrary, samples 9 (BCP+DHA) and 10-11 (BCP+DHA+eugenol) caused a significant decrease in TNF-a levels compared to sample 4.

[0095] The best effect was obtained with sample 11 (composition of the invention), which showed a decrease in TNF-a that was not only significantly lower than sample 4, but also significantly lower than sample 9.

[0096] The results of the in vitro experiments performed on the human astrocyte cell line show that the composition of BCP, DHA and eugenol of the invention has a protective effect on the cell line treated with IL-1 β, being able to promote the production of anti-inflammatory / pro-resolution cytokines and reducing the release of pro-inflammatory cytokines.

Claims

1. A composition for analgesia, anti-inflammation and antioxidant of the central and peripheral nervous systems, characterized in that: Contains beta-caryophyllene (BCP), docosahexaenoic acid (DHA) and eugenol.

2. The composition according to claim 1, characterized in that Contains 0.9-1.1 parts by weight of BCP, 0.9-1.1 parts by weight of DHA, and 1.3-2.0 parts by weight of eugenol.

3. The composition according to claim 1 or 2, characterized in that The weight parts of BCP and DHA are equal, and the weight part of eugenol is 1.5 parts.

4. The composition according to any one of the preceding claims, characterized in that BCP and eugenol were used in the form of clove essential oil.

5. The composition according to claim 4, characterized in that The clove essential oil is a mixture of clove essential oil with high BCP titer and clove essential oil with high eugenol titer.

6. The composition according to claim 5, characterized in that The mixture of high titer clove essential oil is in microencapsulated form.

7. The composition according to any one of the preceding claims, characterized in that DHA is used in the form of a microencapsulated powder.

8. The composition according to any one of the preceding claims, characterized in that It is an oral dosage form, particularly a capsule dosage form.

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