Chiral polyhydroxylated carbon-based nanometric clusters and their use in treating musculoskeletal and / or osteoarticular diseases

Abstract

The present invention relates to a polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of diseases and / or disorders characterized by inflammatory processes on an oxidative basis, such as those observed in musculoskeletal and / or osteoarticular diseases and / or disorders, in a mammal in need thereof. The present invention also relates to a feed ingredient comprising said polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials called Micronized I Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of diseases and / or disorders of the musculoskeletal and / or osteoarticular system, in a mammal in need thereof.

Description

[0001] CHIRAL POLYHYDROXYLATED CARBON-BASED NANOMETRIC CLUSTERS AND THEIR USE IN TREATING MUSCULOSKELETAL AND / OR OSTEOARTICULAR DISEASES

[0002] The present invention relates to a chiral poly hydroxylated carbon-based nanometric cluster derived from plantbased materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of diseases and / or disorders characterized by oxidative-based inflammatory processes in a mammal in need thereof.

[0003] Furthermore, the present invention relates to a feed ingredient intended for pets comprising said chiral polyhydroxylated carbon-based nanometric cluster derived from plant-based materials called Micronized Vegetal Carbon NCN (NITE Carbon Nanocluster - NITE NCN Pet)) for use in a method of treatment, preventive and / or adjuvant to the curative process, of musculoskeletal and / or osteoarticular diseases and / or disorders, in a mammal in need thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Musculoskeletal and / or osteoarticular diseases and / or disorders are common in humans and pet animals, arthritis, osteoarthritis, acute, recurrent and / or chronic local osteoarticular inflammation being among the most recurrent diseases / disorders. Related symptoms include cartilage and / or bone degeneration, limb muscle atrophy, reduced motor skills, persistent and / or chronic pain.

[0006] Arthritis, particularly osteoarthritis, is a degenerative joint disease commonly found in humans and pet animals. Osteoarthritis involves the progressive deterioration of articular cartilage, with loss of proteoglycan and collagen and proliferation of new bone, accompanied by a variable inflammatory response within the synovial membrane. It is the most common form of joint and musculoskeletal disease affecting dogs, and, albeit less frequently, cats.

[0007] Chronic joint inflammation causes weakness in the bone structure and poor function of joint tissues, leading to reduced motor skills in the animal; chronic / persistent pain; apathy, fatigue, tendency to inactivity; atrophy of muscles due to inactivity; insistent licking of the painful part (with risk of injury / infection); irritability, restlessness and other psychological repercussions.

[0008] Osteoarthritis, unlike osteoarthritis, can affect both young and old animals, and there are currently no cures that can eliminate this condition but only treatments that can alleviate its symptoms and slow its progress.

[0009] Management of osteoarthritis may include pharmacological treatments, nutraceutical administration, and dietary management. These current management approaches, however, have focused on symptomatic relief and, as a result, have not been fully successful in managing the disease or treating the underlying conditions. Thus, there remains a need for new approaches to the management of osteoarthritis in pet animals, particularly in dogs.

[0010] There are numerous nutraceutical-based feed supplements on the market indicated to support joint metabolism in pet animals, particularly dogs, often with anti-inflammatory and antioxidant actions.

[0011] In addition to inflammation, oxidative stress and oxidative damage are also important underlying mechanisms of osteoarticular problems, including osteoarthritis. WO 2010 / 123179 A1 describes the use of a Vitis vinifera seed extract in the treatment of osteoarthritis or cartilage degeneration.

[0012] Mevel Elsa et al, “Olive and grape seed extract prevents post-traumatic osteoarthritis damage and exhibits in vitro anti IL-1 [3 activities before and after oral consumption." Sci Rep. 2016 Sep 19;6:33527 describes the use of an olive or grape extract in the treatment of osteoarthritis damage, cartilage degeneration, and pain.

[0013] Kato Rina et al, “Effect of olive leaf extract on skeletal muscle in disuse-induced muscle atrophy model rats " FUNCTIONAL FOOD RESEARCH, vol. 18, 2022 describes the effects of an olive leaf extract on skeletal muscle in situations of muscle atrophy.

[0014] None of these documents describe a chiral carbon-based polyhydroxylated nanometric cluster derived from source materials for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute musculoskeletal and / or osteoarticular diseases and / or disorders.

[0015] The Italian patent application 102018000009526 describes a method for the preparation of Micronized Vegetal Carbon NON (NITE Carbon Nano Cluster) but does not describe its use in a method of treatment, preventive and / or adjuvant to the curative process, of acute musculoskeletal and / or osteoarticular diseases and / or disorders.

[0016] A disadvantage associated with the use of some anti-inflammatory substances and many antioxidant substances is related to their pronounced hydrophobic nature, resulting in poor solubility in water and thus low bioavailability in the physiological environment.

[0017] The technical problem that the present invention addresses and solves is to provide a new ingredient, preferably for use in pet feed or food, that is effective and free of side effects, for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms, in a mammal in need thereof.

[0018] The Applicant, following intensive research efforts, has identified the compound called Micronized Vegetal Carbon NCN which is a chiral polyhydroxylated carbon-based nanometric cluster derived from plant- based materials.

[0019] In the context of the present invention, "Micronized Vegetal Carbon NCN” means "NITE Carbon Nano Cluster - NITE NCN Pet.”

[0020] According to the invention, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is an effective ingredient for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal in need thereof.

[0021] Said diseases and / or disorders and / or related symptoms are characterized by inflammatory processes on an oxidative basis. Preferably, said diseases and / or disorders and / or related symptoms are selected from the group comprising or, alternatively, consisting of preferably at least one selected from osteoarthritis, acute, recurrent and / or chronic local osteoarticular inflammation, cartilage and / or bone degeneration, limb muscle atrophy, reduced motor skills, persistent and / or chronic pain.

[0022] Said mammal is a non-human animal, preferably a dog or cat, even more preferably a dog. The ingredient Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster- NITE NCN Pet), in addition to having strong antioxidant activity, also has anti-inflammatory activity, is water soluble, and is characterized by high bioavailability and stability, as well as having a high safety profile.

[0023] The Applicant has verified the efficacy of a feed or pet food ingredient comprising a Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) in a clinical study conducted on dogs with osteoarthritis, demonstrating its ability to reduce many of the most disabling symptoms of osteoarticular disease, improving the animal's quality of life and the impact of osteoarthritis in everyday life, allowing the animal to be more active, feel less pain, and live better both physically and mentally.

[0024] In addition, the feed or animal food ingredients of the invention, being free of side effects and having high tolerability, are particularly suitable for supporting joint metabolism both in cases of osteoarthritis in the elderly / adult dog and in the case of healthy animals but subjected to heavy physical exertion, such as working or rescue dogs.

[0025] These objects and others, which will be clear from the detailed description that follows, are achieved by the novel feed or pet ingredients of the present invention by the technical features claimed in the attached claims.

[0026] DESCRIPTION OF THE FIGURES

[0027] Figure 1 shows a SEM (Scanning Electron Microscope) image of the structure of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet).

[0028] Figure 2 shows the results of an experimental study to evaluate the ROS scavenger capacity of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) compared with two typical ROS scavengers: superoxide dismutase (SOD), scavenger of superoxide anions, and mannitol, scavenger of hydroxyl radicals.

[0029] Figure 3 shows the statistically significant reduction in the pain clinical score after treatment with the preparation (T2) containing Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet), obtained during the clinical study performed. The Pain Clinical Score, as well described by Pollmeier et al., Clinical evaluation of firocoxib and carprofen for the treatment of dogs with osteoarthritis, Veterinary Record, Volume 159, Issue 17 p. 547-551 , Oct. 21, 2006, https: / / doi.Org / 10.1136 / yr.159.17.547, includes the evaluation of several objective parameters fundamental to the global assessment of the joint pain symptom in the pet, such as: degree of lameness, pain on manipulation, pain on palpation, joint swelling, and degree of mobility.

[0030] Figure 4 shows the effects of the combination of sodium hyaluronate and NITE on the radiograph of the knee affected by osteoarthritis (OA) according to the experiment described in Example 9. OA was evaluated with clinic radiographs to demonstrate joint space width (JSW) and osteophyte formation. Radiographs also allow visualization of subchondral sclerosis and subchondral cysts. Knee sections of sham rats showed normal joint tissue architecture (see radiographic score). The MIA+ solvent and MIA+ NITE groups showed decreased JSW and increased osteophyte formation.

[0031] Treatment with 1 % sodium hyaluronate and the combination of HA and NITE at a dose of 150 pig / 25pil significantly reduced the changes induced by MIA administration. Data are mean ± SEM of 10 rats in each group. *P < 0.05 compared with sham, #P < 0.05 compared with MIA + vehicle. Figure 5 shows the effects of the combination of hyaluronic acid and NITE on the histological features of knee tissue affected by osteoarthritis (OA). Knee sections of Sham rats showed normal joint tissue architecture (see Mankin score). The MIA+ solvent and MIA+ NITE groups showed fibrillation of the surface layer, decreased blood cells, multilayer in the transition and radial zones, no pannus formation, and modified Mankin scores. Treatment with 1 % sodium hyaluronate and the combination of HA and NITE at a dose of 150 pig / 25pil significantly reduced the histological changes induced by MIA administration. Histologic scoring was performed by an independent observer. Data are expressed as mean ± SEM of 10 rats in each group. *P < 0.05 compared with sham, #P < 0.05 compared with MIA + vehicle.

[0032] Figure 6 shows the effects of the combination of hyaluronic acid and NITE on cartilage degeneration in knee tissue affected by osteoarthritis (OA). No cartilage degeneration was observed in the sham group. Significant histopathologic changes were evidenced in the MIA+ solvent and MIA+ NITE groups, as indicated by surface irregularity, disorganization of articular cartilage with apparent cloning of chondrocytes in the transition and radial zones, and an intact tidemic ark. Treatment with 1 % sodium hyaluronate and the combination of HA and NITE at a dosage of 150 pig / 25pil significantly prevented damage to cartilage structure, reduced cellular abnormalities, and prevented the tidemark change induced by MIA administration. Cartilage degeneration scoring was performed by an independent observer. Data are expressed as mean ± SEM of 10 rats in each group. *P < 0.05 compared with sham, #P < 0.05 compared with vehicle MIA+.

[0033] Figure 7 shows the effects of the combination of hyaluronic acid and NITE on mast cell staining in osteoarthritis (OA)-affected knee tissue. Compared with the Sham group, the MIA + vehicle and MIA + NITE groups showed a significant increase in the number density of toluidine blue-positive cells. In contrast, treatment with 1 % sodium hyaluronate and the combination of HA and NITE at a dose of 150 pig / 25pil produced a significant reduction in mast cell infiltration in the knees of MIA-treated rats.

[0034] Number of mast cells per unit area of muscle parenchyma (mast cell density). Data are expressed as mean ± SEM of 10 rats in each group. *P < 0.05 compared with sham, #P < 0.05 compared with vehicle MIA+.

[0035] Figure 8 shows the effects of the combination of hyaluronic acid and NITE on plasma cytokines and nerve growth factor (NGF) in rats with osteoarthritis (OA). Increased plasma levels of tumor necrosis factor-alpha (TNF- a), interleukin-1 beta (IL-1 |3) and NGF were detected in the MIA+ solvent and MIA+ NITE groups after MIA administration. OA rats treated with HA 1 % and particularly with the combination NITE and HA 1 % showed a reduction in plasma levels of all measured parameters. Values are shown as mean ± SEM of 10 animals in each group. *P < 0.05 compared with sham, #P < 0.05 compared with MIA+ vehicle.

[0036] Figure 9 shows the effects of combining HA and NITE on plasma levels of matrix metalloproteinases (MMPs). MMPs play a primary role in downstream signaling pathways in osteoarthritis (OA) and cartilage degradation. The MIA+ vehicle and NITE group showed significantly higher expression of MMP-1 , MMP-3 and MMP-9. Compared with the MIA group, the group treated with 1.0% sodium hyaluronate and, in particular, NITE + 1.0% sodium hyaluronate showed decreased expression of MMP-1, MMP-3 and MMP-9. Data are expressed as mean ± SEM of 10 rats in each group. *P < 0.05 compared with Sham, #P < 0.05 compared with Ml A+ vehicle.

[0037] Figure 10 shows the effect of NITE on the time course of carrageenan-induced paw edema.

[0038] NITE was administered orally 30 min before CAR injection. Paw edema was assessed at the time points shown in the figure. NITE produced significant improvements in paw edema (measured as paw volume) compared with CAR at the same time point. # p < 0.05 compared with CAR.

[0039] Figure 11 shows the results of the histological examination performed as described in Example 10. Specifically, the histological examination was performed by hematoxylin and eosin staining: SHAM is the control group; CAR: the intraplantar injection of CAR; CAR with NITE 10mg / kg treatment, respectively; histological score for the different treatment groups. *** p < 0.001 vs. Sham; # p < 0.05 vs. CAR.

[0040] Figure 12 shows the results of the in vitro study and in vivo study described in Example 11 .

[0041] Specifically, Figure 12A shows the % reduction in the level of the biomarkers shown in the figure after treatment with the preparation clustered according to the invention or with HT / PCy of IL-1 p-stimulated primary articular chondrocytes; Figure 12B shows the % improvement / reduction of specific parameters of osteoarthritis and cartilage degradation assessed in mice and rabbits after treatment with the preparation clustered according to the invention or with HT / PCy.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] It is an object of the present invention a polyhydroxylated chiral carbon-based nanometric cluster derived from plant- based materials in the form of an ingredient called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms, in a mammal (preferably a dog or cat, even more preferably a dog) in need thereof.

[0044] In the context of the present invention, "Micronized Vegetal Carbon NCN” means "NITE Carbon Nano Cluster - NITE NCN Pet.”

[0045] It is a further object of the present invention a polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials in the form of an ingredient referred to as Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treating, preventing and / or adjuvant the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal (preferably a dog or cat, even more preferably a dog) in need thereof, wherein said diseases and / or disorders and / or related symptoms are characterized by oxidative-based inflammatory processes. Said diseases and / or disorders and / or related symptoms are preferably selected from the group that includes or, alternately, consists of at least one of osteoarthritis, acute, recurrent and / or chronic local osteoarticular inflammation, cartilage and / or bone degeneration, limb muscle atrophy, reduced motor skills, persistent and / or chronic pain. Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is configured as a plant source of carbon atoms derived from plant waste materials, e.g., grape seeds (V / fe Vinifera) and olive kernels {Olea Europea).

[0046] The Italian patent application 102018000009526, filed on 17-10-2018 and the Italian patent application 102022000014722, filed on 13-07-2022, both fully incorporated in this application as references, describe methods of preparation of said Micronized Vegetal Carbon NCN (NITE Carbon Nano Pet) and the characteristics of the product obtained by such methods of preparation.

[0047] According to the invention, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) has a particle size in the range of from 1 nm to 200 nm, preferably from 1 nm to 120 nm, more preferably from 1 to 80 nm, as measured by Scanning Electron Microscopy (SEM). Preferably, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises an amount of carbon (C) from 90% to 99.9%, more preferably from 95% to 99.9%, more preferably from 98.8% to 99.8%.

[0048] Preferably, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises an amount of hydrogen (H) between 0.1 to 10%, more preferably 0.1% to 5%, more preferably 0.1 % to 0.7%. Preferably, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises: an amount of carbon (C) comprised from 90% to 99.9%, preferably from 95% to 99.9%; and an amount of hydrogen (H) comprised from 0.1 % to 5%.

[0049] More preferably, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is characterized by: a particle size comprised from 1 nm to 200 nm, preferably from 1 nm to 120 nm; and an amount of carbon (C) comprised from 90% to 99.9%, preferably from 95% to 99.9%; and an amount of hydrogen (H) comprised from 0.1 % to 5%.

[0050] From the point of view of molecular structure, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is characterized by a number of hydroxyl (OH) groups preferably comprised from 20 to 700, more preferably from 30 to 600, even more preferably from 50 to 500 and a number of carbonic clusters comprised from 100 to 26,000, preferably from 150 to 25,500, more preferably from 200 to 25,000.

[0051] The number of OH groups in a Carbon Nanocluster does not depend on the amount of material used because it is an intrinsic property of the chemical structure and surface of the individual cluster. Each nanocluster has a specific amount of OH groups, determined by its size, morphology and surface functionalization. Increasing the amount of material increases the total number of nanoclusters, but the ratio of OH groups to nanoclusters remains constant. From the point of view of physical properties, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) preferably appears as a grey-colored ultrafine powder or as or liquid suspension (hydrophilic or lipophilic). Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to the invention exhibits high stability in hydrophilic solutions such as, for example, demineralized water, PVP, glycols and high stability in lipophilic solutions such as, for example, vegetable oils: olive, coconut, avocado.

[0052] Without binding to any theory or mechanism, it appears that the electronic properties and reactivity of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet), endowed with a pronounced free radical scavenger effect, depend on the number of hydroxyl (-OH) groups and their positioning in the carbon cluster structure. The specific behaviour of the NCN is a consequence of the structural flexibility of OH groups, which rotate around axes through C-0 bonds with a distribution in different carbon sites of the NCN surface. NCNs simultaneously have both attractive (C-OH) and repulsive (C-O-) sites. Without wishing to be bound by any theory, it is speculated that acidic protons could be involved in hydrogen bonding interactions with other NCN molecules, leading to the formation of nano-clusters. Indeed, it has been observed that NCN forms nano-clusters in water, despite the increase in the number of hydroxyl groups on the molecular structure, which would decrease the hydrophobic portion of the molecular surface. However, aggregate formation has been observed at micromolar concentrations, suggesting that NCN is more likely to form supramolecular nano-clusters with an average size of 100 nm or more and, often, of different shapes, which depend on the characteristics of the hydroxyl groups (length). The effect of length is related to packing and molecular equilibrium between attractive and repulsive interactions. As disclosed by the above Italian patent applications, NITE technology uses a characteristic production of "NITE Carbon Nano Cluster (NCN).” The products undergo infrared spectroscopy testing, UV-Vis testing, fluorescence testing and elemental analysis to ascertain the number of hydroxyls on the surface of the Carbon Nano Cluster, which is then guaranteed. The higher the number of hydroxyls on the NCN, the better the water solubility of the NCN, and, at the same time, the hydroxyl on the carbon structure is allyl, which gives the NCN moderate electronic affinity. More preferably, the number of hydroxyl groups (OH) present in said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is from 50 to 500 and the number of carbon clusters is from 200 to 25,000.

[0053] These characteristics give Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) good potential in application for free radical removal, antioxidant effect in biological systems.

[0054] A SEM (Scanning Electron Microscope) image of the structure of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is shown in Figure 1.

[0055] More preferably, the Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use according to the present invention, in solid powder form, is characterized by:

[0056] - size: comprised from 1 to 120 nanometers, preferably from 1 to 80 nanometers, more preferably from 1 to 20 nanometers, even more preferably the average size is about 6 nanometers;

[0057] - composition: carbon comprised from 95% to 99.9%, preferably from 98.8% to 99.8%, preferably about 99%; hydrogen comprised from preferably 0.1 % to 5%, preferably from 0.1 % to 0.7% preferably about 0.5%; oxygen comprised from 0.1 % to 0.5%, preferably about 0.5%; - morphology: number of OH hydroxyl groups comprised from 50,000 to 250,000 per gram of material, preferably from 175,000 to 190,000 per gram of material; carbonic nano clusters in the range of from 200,000 to 500,000 clusters per gram of material, preferably from 350,000 to 400,000 clusters per gram of material;

[0058] - relative humidity: 1-2%;

[0059] - predominantly sp2 hybridized carbon atoms.

[0060] Preferably, the Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use according to the present invention is an NCN derived from Olea Europea, preferably from Olea Europea kernels, an NCN derived from Vitis Vinifera, preferably from Vitis Vinifera seeds, or a mixture thereof.

[0061] For example, Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use according to the present invention is NCN DRY (NITE CARBON NANO CLUSTERS DRY), by NITE Technology, Product ID 161293b. Said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster NITE NCN Pet), derived from Vitis Vinifera grape seeds and Olea Europea kernels (CAS No. 7440-44-0), is in the form of a grey powder, with size (bulk dimension, medium size) greater than about 0.15 microns, bulk density of about 2 g / cm3at 20°C and solubility of about 25 mg / ml in DMSO and 1 mg / mg the deionized water. As per the manufacturer's certification, NCN DRY (NITE CARBON NANO CLUSTERS DRY) is non-toxic and therefore has a high safety profile.

[0062] Plant-based carbon atom-based compounds named NCN (NITE Carbon Nano Cluster NCN Pet) developed by NITE TECHNOLOGY OU have demonstrated high antioxidant and anti-inflammatory properties.

[0063] Antioxidant activity

[0064] The antioxidant effect of NCN was evaluated in comparison with known compounds with antioxidant activity by the test described below.

[0065] Antioxidant activity test

[0066] To study the inhibitory activity of NCN on the oxidative degradation of p-carotene, differential absorbance (A Abs) was measured at 460 nm, before and after 30 seconds of irradiation with a 300 W high-pressure Hg lamp in an oxygen atmosphere.

[0067] The p-carotene bleaching assay was performed according to an optimally modified procedure. To a 22 -pl solution of commercial surfactant "Tween 40” (0.2 g / ml in chloroform) in a quartz cell equipped with a screw cap, 11 pl of p- carotene (1.0 mg / ml in chloroform) and 4.4 pl of linoleic acid (0.1 g / ml in chloroform) were added, then the chloroform was removed by vacuum evaporation (p-carotene in linoleic acid matrix).

[0068] A sample of p-carotene in hydrogen peroxide matrix was also prepared.

[0069] An aliquot of the emulsion was immediately diluted with 2.4 ml of phosphate buffer solution (about 0.02M, pH 7.0), 0.1 ml of antioxidant (0.03 pmol, equivalent to NCN) was added to the mixture; the solutions were well mixed and heated to 50°C and then subjected to monitoring.

[0070] The other antioxidants were treated at an equivalent amount of NCN as an active component.

[0071] In this case, the decrease in Abs during photoirradiation could not be monitored. Instead, antioxidant activity was evaluated as the percentage of inhibition compared with a control (% Inh) using the A Abs value.

[0072] Table 1 a). %AOA — 100 (kcontrol " ksample) / ^control b). %lnh — 100 (AAbScontrol—AabSsample) / AAbScontrol

[0073] NCN showed superior antioxidant capacity compared with other tested antioxidants currently used in food compositions.

[0074] Anti-inflammatory Activity

[0075] The anti-inflammatory effect of NCN has been estimated compared with amifostine (AMI) and indomethacin (IND). Amifostine has indirect effects on reducing inflammation through its congruent antioxidant action and is sometimes used as a primary anti-inflammatory agent. NCN significantly increased in a dose-dependent manner the antiinflammatory activity, both compared with that of IND and that of AMI.

[0076] Test of anti-inflammatory activity

[0077] Adult male Wistar rats weighing between 200 and 250 g were used. The animals were housed in plastic cages under standard laboratory conditions before use. Each experimental group consisted of 6 animals.

[0078] Materials

[0079] NCN was dissolved in dimethyl sulfoxide (DMSO) 24 h before administration (NCN in DMSO forms stable nanoaggregates of polyanions 30, 60 and 100 nm in diameter, determined by DLS and AFM).

[0080] The amifostine (AMI) sample was prepared for administration by dissolving the substance in a sterilized, apyrogenic 0.9 percent NaCI solution, ex tempore.

[0081] Indomethacin (IND) was dissolved in DMSO immediately before use.

[0082] Carrageenan was dissolved in saline and prepared as a 0.5% solution.

[0083] NCN, AMI and IND were administered i.p., in doses of 5, 7.5 and 10 mg / kg; 50, 100 and 200 mg / kg; and 2, 3 and 4 mg / kg, respectively. Control rats were administered the corresponding vehicles (DMSO for NCN and IND, and saline for AMI) in a dose of 1 ml / kg, i.p.

[0084] The tested compounds and vehicles were administered 30 min before injecting carrageenan saline (0.5% in a volume of 0.1 ml) into the plantar surface of the rat's right hind paw to induce inflammation.

[0085] Three hours later, the paw volume was measured with a mercury plethysmograph and compared with the preinjection volume of the same paw.

[0086] Swelling was then calculated in the compound-treated animals.

[0087] The percentage of inhibition was derived by comparison with that of the control groups.

[0088] The results obtained were used to calculate the average effective anti-inflammatory doses (ED50) of the tested compounds.

[0089] Table 2

[0090] The results obtained support that, in a model of acute inflammation, NCNs have potent anti-inflammatory activity, higher than the other anti-inflammatory agents tested.

[0091] The results of the above studies, make NCNs good candidates for further investigation as agents for the treatment of various disorders / pathological conditions associated with inflammation and / or oxidative stress.

[0092] Anti-degeneration, repair and regeneration activities of bone and cartilage tissues

[0093] There is much evidence indicating that ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species) are involved in progressive degenerative diseases of various skeletal tissues. In some bone tissues, oxidative stress can initiate or participate in matrix destruction and cell apoptosis, and increased exogenous NO can promote cell apoptosis and suppress cell proteoglycan synthesis. For example, NO has been shown to participate in disc degeneration induced by mechanical stress or interleukin (IL)-1 .

[0094] Therefore, further studies have also been conducted on the ability of NCNs to counteract oxidative stress by acting as potent scavengers of ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species).

[0095] The characteristic of NCNs to form stable polyanionic nano clusters, both in water and other biological media, indicates a basic pathway of antioxidant characteristics in biological models.

[0096] Oxidative stress and oxidative damage are mediators of cellular damage in many pathological conditions. The damaging effects of oxidative stress are associated with mitochondrial depolarization and the subsequent release of pro-apoptotic small molecules, such as cytochrome c, leading to activation of caspase cascades and apoptosis. NCNs are able to associate with mitochondria and induce a cytoprotective antioxidant effect in various oxidative stress-associated matrices.

[0097] The hypothesized mechanism is based on the fact that NCNs have numerous oxygen free electron pairs distributed around NCNs and have a great ability to form coordinative bonds with pro-oxidant metal ions.

[0098] Experimental studies by the Applicant showed that NCNs have a high capacity to remove ROS compared with two typical ROS scavengers: superoxide dismutase (SOD), a scavenger of superoxide anions, and mannitol, a scavenger of hydroxyl radicals (Figure 2).

[0099] The ingredient Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet), by virtue of its pronounced antioxidant, free radical scavenger and anti-inflammatory activities, combined with the characteristics of high water solubility, high bioavailability and stability, as well as a high safety profile (it is a non-toxic and highly tolerable compound of plant origin), has therefore proven to be an optimal candidate for use in a method of treatment, preventive and / or adjuvant to the curative process, of musculoskeletal and / or osteoarticular diseases and / or disorders, in a mammal (preferably a dog or cat, even more preferably a dog) in need thereof.

[0100] Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to the invention is capable of:

[0101] • reducing the effect of ROS, preventing cellular, lipid, protein and DNA damage caused by oxidative stress;

[0102] • acting in parallel as a chelator of metal ions such as Fe and Cu, important players in ROS formation processes themselves; and

[0103] • activating and protecting against the degradation of superoxide dismutase, catalase, and glutathione peroxidase, key enzymes in endogenous antioxidant systems.

[0104] Such effects on antioxidant enzymes determines a significant role in protecting against lipid peroxidation, a critical moment of damaging oxidative processes on cell membranes at all levels.

[0105] In addition, the Applicant has advantageously observed that the antioxidant activities Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to the above-described invention (i.e., free radical scavenging, chelation of metal ions, inhibition of lipid peroxidation, and enhancement of antioxidant enzyme activity) have a synergistic effect in the 'intestinal environment to support a restoration of endogenous microbial balance, promoting the development of eubiotic microbes to the detriment of harmful species. Such antioxidant activity at the gut level can prevent the development of various inflammatory, gastrointestinal or systemic, or metabolic and degenerative diseases, including on the neurological side due to known gut-brain interactions.

[0106] The efficacy of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) has been confirmed in a clinical study conducted on dogs with osteoarthritis (described in detail below), demonstrating its ability to reduce many of the most disabling symptoms of osteoarticular pathology, improving the animal's quality of life and the impact of osteoarthritis in everyday life, allowing the pet to be more active, feel less pain, and live better physically and mentally.

[0107] Advantageously, the ingredient called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) can be fed to a mammal, preferably a dog or cat, even more preferably a dog, which is in need thereof by including it in a feed composition, preferably a highly palatable feed composition.

[0108] It is therefore a further object of the present invention to provide an ingredient for feed or pet food comprising a chiral polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal (preferably a dog or cat, even more preferably a dog) in need thereof.

[0109] It is further an object of the present invention to provide a feed or pet food comprising a chiral polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal (preferably a dog or cat, even more preferably a dog) in need thereof.

[0110] Advantageously, the Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) may be combined, in the composition of a feed or pet food for use according to the present invention, with additional active ingredients such as, preferably, at least one selected from the group comprising or, alternatively, consisting of a vitamin E an omega-3 or a mixture thereof, a fish oil titrated in EPA-DHA, an L-methionine, a manganese salt, a vitamin, preferably a vitamin E, a prebiotic, preferably an inulin from chicory, an ingredient of plant origin, preferably selected from Boswellia, willow {Salix), devil's claw {Harpagophytum procumbens) and mixtures thereof.

[0111] It is therefore a further object of the present invention to have a feed ingredient and / or a feed for use according to the present invention comprising a Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) and at least one further active ingredient selected from the group consisting of or, alternatively, consisting of a vitamin E an omega-3 or a mixture thereof, a fish oil titrated in EPA-DHA, an L-methionine, a manganese salt, a vitamin, preferably a vitamin E, a prebiotic, preferably an inulin from chicory, an ingredient of plant origin, preferably selected from Boswellia, willow {Salix), devil's claw {Harpagophytum procumbens) and mixtures thereof.

[0112] Representative embodiments of feeds comprising the ingredient Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use according to the invention are set out below, for illustrative purposes only and in no way limiting.

[0113] The Applicant has also verified the efficacy of a feed containing a Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) in a clinical study conducted on dogs suffering from osteoarthritis, demonstrating its ability to reduce many of the most disabling symptoms of osteoarticular pathology, improving the animal's quality of life and the impact of osteoarthritis in everyday life, allowing the animal to be more active, feel less pain and live better physically and mentally.

[0114] In addition, the feed ingredients of the invention, being free of side effects and with high tolerability, are particularly suitable for supporting joint metabolism both in the case of osteoarthritis in the elderly / adult dog and in the case of healthy animals under heavy physical strain, such as working rescue dogs.

[0115] A further object of the present invention is a mixture comprising or alternatively consisting of:

[0116] I) a polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials in the form of an ingredient called Micronized Vegetal Carbon NCN NITE (Carbon Nano Cluster - NITE NCN Pet);

[0117] II) hyaluronic acid, or a salt thereof.

[0118] Preferably, said salt is sodium hyaluronate.

[0119] Said Micronized Vegetal Carbon NCN NITE (NITE Carbon Nano Cluster - NITE NCN Pet) in said mixture has one or more of the characteristics described in the present invention.

[0120] Preferably, said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) in the mixture according to the invention:

[0121] - has a particle size from 1 nm to 200 nm, preferably from 1 nm to 120 nm, more preferably from 1 to 80 nm; and / or

[0122] - comprises an amount of carbon (C) from 90% to 99.9%, more preferably from 95% to 99.9%, more preferably from 98.8% to 99.8%; and / or

[0123] - comprises an amount of hydrogen (H) from 0.1 to 10%, more preferably from 0.1 % to 5%, more preferably from 0.1 % to 0.7%; and / or

[0124] - comprises a number of hydroxyl (OH) groups preferably from 20 to 700, more preferably from 30 to 600, even more preferably from 50 to 500 and a number of carbon clusters from 100 to 26,000, preferably from 150 to 25,500, more preferably from 200 to 25,000.

[0125] The number of OH groups in a Carbon Nanocluster does not depend on the amount of material used because it is an intrinsic property of the chemical structure and surface of the individual cluster. Each nanocluster has a specific amount of OH groups, determined by its size, morphology and surface functionalisation. Increasing the amount of material, increases the total number of nanoclusters, but the ratio of OH groups to nanoclusters remains constant. Said mixture can advantageously be used as a medicine. Indeed, as shown in Example 9 and in Figure 4, treatment with 1 % sodium hyaluronate and the combination with NITE at a dose of 150 pig / 25pil significantly reduced the changes induced by the administration of monosodium iodoacetate in an osteoarthritis model.

[0126] Therefore, said mixture comprising or, alternatively, consisting of I) and II) may advantageously be used in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal in need thereof.

[0127] Furthermore, another aspect of the present invention relates to composition comprising said mixture and pharmaceutically or food grade acceptable additives and / or excipients.

[0128] Such a composition may be advantageously used as a medicament, preferably in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular pathologies and / or disorders and / or related symptoms, in a mammal in need thereof.

[0129] Example 1

[0130] 1000 mg divisible chewable tablet (used in the clinical study described below).

[0131] Each tablet contains:

[0132] Main ingredient Dose per tablet

[0133] NCN 2.50 mg

[0134] Vit. E 35.00 mg

[0135] Fish oil (titrated at 65% EPA-DHA) 36.40 mg

[0136] Of which:

[0137] EPA 14.50 mg

[0138] DHA 7.30 mg

[0139] D L-Methionine 20.00 mg

[0140] Manganese 0.56 mg

[0141] Table 3

[0142] Composition:

[0143] Maltodextrin, inactivated yeast, hydrolysed proteins, dicalcium phosphate, fish oil 3.64%, magnesium salt of stearic acid, mono- and triglycerides of fatty acids (glyceryl bennate), Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet).

[0144] Additives:

[0145] Nutritional additives: Vitamins: 3a700 Vit. E (35,000 mg / kg) Amino acids: 3c301 Technically pure D.L-Methionine (20,000 mg / kg) Trace elements: 3b503 Manganese sulphate monohydrate 1.750 mg / kg (Manganese 568.75 mg / kg)

[0146] Technological additives: E460 microcrystalline cellulose, E551 b colloidal silica

[0147] Method of use and daily ration:

[0148] Table 4

[0149] It is recommended to administer the tablet directly into the animal's mouth or in addition to the desired food, according to the daily ration indicated in the table. The high palatability of the chewable, divisible tablets facilitates direct administration to the animal. Recommended period of use: initially up to 3 months, to be repeated as required. It is recommended to seek veterinary advice before use or before extending the period of use. Supplement the daily ration with the usual food. A balanced daily ration is recommended. Always leave fresh water available.

[0150] Example 2

[0151] 1000 mg palatable tablet for use as joint metabolism support in dog / cat osteoarthritis according to the present invention.

[0152] Each tablet contains:

[0153] NCN 2.50 mg

[0154] Vitamin E 50% powder 70.00 mg (pari a 35,00 mg di vitamina E / cpr)

[0155] Fish oil powder

[0156] 26% EPA 13% DHA 80.00 mg

[0157] Manganese sulphate monohydrate 1.75 mg

[0158] D,L Methionine 20.00 mg

[0159] Excipients: hydrolysed animal protein (poultry origin) 60.00 mg brewer's yeast 101.75 mg dibasic calcium phosphate (dicalcium phosp.) 35.00 mg microcrystalline cellulose E460 330.00 mg glyceryl behenate 10.00 mg magnesium stearate 18.00 mg maltodextrin 235.00 mg colloidal silica E551 b 20.00 mg

[0160] TOTAL: 1000.00 mg Example 3

[0161] 1000mg palatable tablet for use as joint metabolism support in dog / cat osteoarthritis according to the present invention.

[0162] Each tablet contains:

[0163] NCN 2.50 mg

[0164] Boswellia serrata powder 25.00 mg

[0165] Vitamina E 50% powder 70.00 mg (pari a 35,00 mg di vitamina E / cpr)

[0166] Fish oil powder

[0167] 26% EPA 13% DHA 80,00 mg

[0168] Manganese sulphate monohydrate 1.75 mg

[0169] D,L Methionine 20.00 mg

[0170] Excipients: hydrolysed animal protein (poultry origin) 60.00 mg brewer's yeast 101.75 mg dibasic calcium phosphate (dicalcium phosphate) 35.00 mg microcrystalline celluloseE460 325.00 mg glyceryl behenate 12.00 mg magnesium stearate 16.00 mg maltodextrin 210.00 mg colloidal silica E551b 25.00 mg

[0171] TOTAL: 1000.00 mg

[0172] Example 4

[0173] 1000mg palatable tablet for use as joint metabolism support in osteoarthritis in dogs according to the present invention.

[0174] Each tablet contains:

[0175] NCN 2.50 mg

[0176] Boswellia serrata d.e. titrated 65% boswellic acids 25.00 mg

[0177] Vitamin E 50% powder 70.00 mg (pari a 35,00 mg di vitamina E / cpr)

[0178] Fish oil powder

[0179] 26% EPA 13% DHA 80.00 mg

[0180] Manganese sulphate monohydrate 1.75 mg

[0181] D,L Methionine 20.00 mg

[0182] Excipients: hydrolysed animal protein (poultry origin) 60.00 mg brewer's yeast 101.75 mg dibasic calcium phosphate (dicalcium phosphate) 35.00 mg microcrystalline celluloseE460 325.00 mg glyceryl behenate 12.00 mg magnesium stearate 16.00 mg maltodextrin 210.00 mg colloidal silica E551b 25.00 mg

[0183] TOTAL: 1000.00 mg

[0184] Example 5

[0185] Feed for use according to the present invention: kibble for puppies and adult dogs up to 10 kg weight. The feed contains (weight / weight percentages):

[0186] Dehydrated protein*: 45% w / w

[0187] Monocalcium phosphate: 23% w / w

[0188] Calcium carbonate: 23% w / w

[0189] Dehydrated hemp: 2% w / w

[0190] Dried chicory: 2% w / w

[0191] NCN: 1% w / w

[0192] Camelina Oil: 1% w / w

[0193] Dehydrated Broccoli: 1 % w / w

[0194] Yucca Schidigera: 1 % w / w

[0195] Olive Oil: 0.5% w / w

[0196] Dehydrated pomegranate: 0.5% w / w

[0197] * Protein from beef, lamb, fish, tuna. Excluding protein from chicken, chicken fat or other poultry.

[0198] Example 6

[0199] Feed ingredient for use according to the present invention. The ingredient contains:

[0200] NCN 3.0 mg

[0201] Inulin from chicory 0.3 mg

[0202] Vitamin E powder 50% 0.6 mg (pari a 0,3 mg vit. E) Methionine hydroxylate 0.1 mg TOTAL 4.0 mg Example 7

[0203] Feed ingredient for use according to the present invention. The ingredient contains:

[0204] NCN 3.0 mg

[0205] Inulin from chicory 0.2 mg

[0206] Vitamin A 0.2 mg

[0207] Vitamin D3 0.2 mg

[0208] Vitamin E 0.3 mg

[0209] Vitamin B1 0.1 mg

[0210] Vitamin B2 0.1 mg

[0211] Vitamin B6 0.1 mg

[0212] Methionine hydroxylate 0.1 mg

[0213] L-Lysine 0.1 mg

[0214] Citric acid 0.1 mg

[0215] TOTAL: 1.5 mg

[0216] Example 8

[0217] Clinical study

[0218] The following clinical study was carried out to evaluate the activity of the ingredient for the support of joint metabolism in osteoarthritis in the dog subject of the invention.

[0219] Study objectives

[0220] This study evaluated the effect of supplementation with Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet), vitamin E, fish oil, methionine, manganese (tablet from Example 1) in dogs suffering from osteoarthritis with the aim of achieving a reduction in symptoms in the absence of oxidative stress generation due to dietary change. The secondary objective was to improve the quality of life of the animals involved, and to assess tolerability and compliance with supplementation.

[0221] Study design

[0222] Enrolment of 10 dogs clinically and radiographically affected by coxofemoral osteoarthritis.

[0223] 10 subjects of the same size were included, treated with the same nutritional supplement containing Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet).

[0224] At the start of the study, at 30 days (T1) and at 60 days (T2) a complete blood profile, evaluation of oxidative stress with specific test, orthopaedic examination (with morphometric examination of muscle circumferences), nutritional examination, pain clinical score were carried out. The Pain Clical Score, as well described by Pollmeier et al., Clinical evaluation of firocoxib and carprofen for the treatment of dogs with osteoarthritis, Veterinary Record, Volume 159, Issue 17, p. 547-551 , 21 October 2006, https: / / doi.org / 10.1136 / yr.159.17.547, includes the evaluation of several objective parameters fundamental for the global assessment of the joint pain symptom in the pet, such as degree of lameness, pain on manipulation, pain on palpation, joint swelling, degree of mobility. The great significance of the Pain Clinical Score, as highlighted by Pollmeier et al., Clinical evaluation of firocoxib and carprofen for the treatment of dogs with osteoarthritis, Veterinary Record, Volume 159, Issue 17, p. 547-551 , 21 October 2006, https: / / dos.Org / 10, 136 / yr , 59.17.547, lies in the significance of the evaluation of a sum of observations and evaluations related to joint pain for each of the parameters described above on a scale from 0 to 3 (0=absent, 1 =slight, 2=moderate, 3=severe) and where the processing of the observed data for a given period compose the global Pain Clinical Score that assigns the degree of incidence of the symptom osteoarticular pain, or its reduction, in the subject suffering from osteoarticular pathology.

[0225] The Pain Clinical Score, moreover, being an overall evaluative index of the degree of lameness, pain on manipulation and / or palpation, joint swelling and ability to move for the subject with osteoarticular disorders, allows for an overall evaluation of the quality of life of the subject with osteoarticular disorders in the absence of treatment or in the presence of specific treatment, reinforcing its overall statistical value.

[0226] Exclusion criteria

[0227] Subjects taking drugs or supplements that interfere with the assessment of oxidative stress were excluded. Subjects suffering from other chronic diseases were also excluded.

[0228] Results

[0229] As shown in Figure 3, the treatment of subjects with osteoarthritis showed a statistically significant reduction in the Pain Clinical Score after administration of the product containing Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet). The great statistical value of the Pain Clinical Score, as argued by Pollmeier et al., Clinical evaluation of firocoxib and carprofen for the treatment of dogs with osteoarthritis, Veterinary Record, Volume 159, Issue 17, p. 547-551 , 21 October 2006, https: / / doi.org / 10.1136 / yr.159.17.547, and as observed in the study for the group treated with Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet), represents the efficacy of the treatment in reducing:

[0230] 1) the degree of lameness;

[0231] 2) pain on manipulation;

[0232] 3) pain on palpation;

[0233] 4) joint swelling;

[0234] 5) the subject's difficulty in movement; thereby globally improving the quality of life of the treated subject, favouring the prevention of recurrent osteoarticular symptoms, with consequent improvement of muscular performance and nutritional status. Ultimately, the Pain Clinical Score appears to be one of the evaluation elements most appreciated by the veterinary world, as it allows for an overall assessment of both the invalidating degree of the osteoarticular pathology and the repercussions in terms of effective benefits following treatment with Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet).

[0235] An increase in muscle mass was also observed in a subgroup of animals (G1). This result is particularly significant as it was observed for a subgroup of animals (G1) consisting of dogs of lower somatic size / weight.

[0236] Furthermore, at the final time of the clinical study (T2) no statistically significant differences were observed on the oxidative stress index (OSI), contrary to what was observed at an early stage, while a trend towards an increase in endogenous antioxidant defences and a reduction in Derivatives of Reactive Oxygen Metabolites (DROMs) remained.

[0237] Adverse effects

[0238] No significant side effects were noted in the study conducted.

[0239] Conclusions:

[0240] The data collected showed that the intake of nutritional support with Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) can be considered a valid support of joint metabolism in the case of osteoarthritis in the animal with reduction of pain that improves the quality of life of the animal and the impact of osteoarthritis in everyday life, allowing the animal to be more active both physically and mentally.

[0241] Example 9 - Effect of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) on a mouse model of osteoarthritis

[0242] The Applicant evaluated the effect of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) on a mouse model of osteoarthritis.

[0243] In the present example, the treatment with Micronized Vegetal Carbon NCN according to the invention is referred to as ‘NITE’.

[0244] MATERIAL AND METHODS

[0245] Animals

[0246] Forty male Lewis rats (Sprague-Dawley, 200-230 g; Envigo, Milan, Italy) were maintained in a monitored environment and received water and standard rodent food. Mice were housed in steel cages in a room maintained at 22 ± 1 °C with a 12-hour light-dark cycle. The mice were acclimatised to their habitat for 1 week and had ad libitum access to water from the standard rodent diet. All animal experiments complied with Italian regulations (D.M. 116192) and those of the European Union (O.J. E.C. L 358 / 1 12 / 18 / 1986).

[0247] Experimental protocol

[0248] Osteoarthritis (OA) was induced by intra-articular injection of monosodium iodoacetate (MIA) into the knee joint. At day 0, rats were anaesthetised with 5.0% isoflurane (Baxter International). A volume of 25 l sterile saline + 3 mg MIA was injected into the knee joint of the rats via the right infrapatellar ligament. An equal volume of sterile 0.9% saline was injected into the left knee of the rats. The MIA was prepared under sterile conditions and injected using a 50 pl Hamilton syringe with a 27-gauge needle that was inserted approximately 2-3 mm into the joint. On day 21 after MIA administration, the knee joints were inspected in detail to determine histopathological changes. The cartilage was stained to evaluate the presence or absence of OA.

[0249] Experimental groups

[0250] Rats were randomly divided into the following groups:

[0251] - MIA + vehicle (solvent solution): rats underwent OA induction as described above and received 25 l of solvent solution in the infrapatellar area of the right knee on days 3, 7, 14 and 21 (n = 10) by intra-articular injection.

[0252] - MIA+ NITE (Nite Carbon Nanocluster 4ppm): Rats underwent OA induction as described above and were treated with intra-articular injection of NITE at a dose of 150 pg / 25 pl on days 3, 7, 14 and 21 after MIA induction (n = 10). MIA NITE + sodium hyaluronate 1 .0%: Rats underwent OA induction as described above and were treated with an intra-articular injection of NITE + sodium hyaluronate 1.0% (high molecular weight sodium hyaluronate, between 1.5 and 2.0 million daltons) at a dose of 150 pg / 25 pl on days 3, 7, 14 and 21 after MIA induction (n = 10).

[0253] MIA+ sodium hyaluronate 1.0 %: rats underwent OA induction as described above and were treated with intraarticular injection of sodium hyaluronate 1.0 % (high molecular weight sodium hyaluronate, between 1.5 and 2.0 million daltons) at a dose of 150 pg / 25 pl on days 3, 7, 14 and 21 after MIA induction (n = 10).

[0254] - MIA+ sodium hyaluronate 1.0 %: Rats underwent OA induction as described above and were treated with intraarticular injection of sodium hyaluronate 1.0 % (high molecular weight sodium hyaluronate, between 1.5 and 2.0 million daltons) at a dose of 150 pg / 25 pl on days 3, 7, 14 and 21 after MIA induction (n = 10).

[0255] - Sham group: Rats were given an intra-articular injection of 0.9% saline (25 pl) instead of MIA and were treated with either vehicle on days 3, 7, 14 and 21 (n = 10).

[0256] Pain measurement

[0257] Mechanical sensitivity was assessed using a dynamic plantar extensometer (Ugo Basile, Comerio, Italy). Rats were placed on a wire mesh surface in a temperature-controlled chamber (22 °C) and were allowed to adapt for 15 minutes before the start of the test. The tactile stimulator part was oriented under the animal. When the extensometer was activated, a plastic monofilament touched the paw in the proximal metatarsal region, exerting a gradually increasing force on the plantar, increasing until the stimulus became painful causing the rat to remove the paw. The force required to produce the paw displacement reflex was automatically recorded and measured in grams. A maximum force of 50 g and a ramp speed of 20 seconds was used for all aesthetic tests.

[0258] A Motor function analysis (Walking Track Analysis)

[0259] The rat was placed on a walking track with a dark end. White office paper was placed on the bottom of the track. The rat's hind limbs were dipped in ink and the rat was made to walk along the track, leaving paw prints on the paper. The test was performed before induction of osteoarthritis (day 0) and at days 3, 7, 14 and 21 after induction. The sciatic nerve function index (SFI), calculated using the Walking Track Analysis, was assessed 60 minutes after the injection on days 3, 7, 14 and 21 : values close to 0 indicate normal function, while values tending to -100 indicate impaired sciatic nerve function.

[0260] Micro-computed tomography

[0261] To assess bone mass and microarchitecture parameters, including bone volume fraction, the proximal and distal parts of the right tibia of rats were scanned with micro-computed tomography (Micro-CT; SkyScan, Belgium). The scanning conditions were as follows: an aluminium filter of 0.5 mm, an X-ray voltage of 50 Kv, an X-ray current of 200 mA and an exposure time of 360 ms. After scanning, transverse slices were reconstructed and three- dimensional analyses were performed with the CTAn SkyScan software.

[0262] Histologic analysis

[0263] On day 21 after MIA administration, rats were sacrificed by overdose of the anesthetic and perfused with a 4% paraformaldehyde solution. The tibiofemoral joints were harvested and postfixed in neutral buffered formalin (containing 4% formaldehyde), decalcified in EDTA and processed as described below. After decalcification, the samples were embedded in kerosene. Mid-coronal tissue sections (5 pm) were stained for evaluation; all histomorphometric analyses will be performed by an observer blinded to the treatment groups. The sections were stained with hematoxylin and eosin and observed under a light microscope (Dialux 22 Leitz; Leica Microsystems SpA, Milan, Italy). Histopathologic analysis of cartilage was evaluated by means of the modified Mankin score (score range 0 to 12, normal to complete disorganization and hypocellularity). Degeneration of the cartilage was assessed by toluidine blue staining and analyzed according to the following criteria described by Janusz et al: 1 = mild in the superficial region; 2 = slightly extensive in the upper center; 3 = moderate in the middle area; 4 = extensive in depth; 5 = severe degeneration.

[0264] Mast cell staining

[0265] For mast cell identification, knee sections will be cut at 5 pm thickness and stained with 0.25% toluidine blue, pH 2.5, for 45 minutes at room temperature. Sections were then dehydrated and mounted for visualization. Three non- consecutive sections were randomly selected for examination from each paw. All sections were evaluated at 200x, while some were photographed at 400x with a Nikon inverted microscope. Mast cell density was expressed as the number of mast cells per unit area of bone tissue.

[0266] Measurement of cytokines, metalloproteinases and nerve growth factor

[0267] Levels of tumor necrosis factor (TNF)-a, interleukin 1 (IL-1), nerve growth factor (NGF) and matrix metalloproteinase (MMP)-1 , MMP-3 and MMP-9 were measured in serum. The tests were performed using commercial colorimetric enzyme-linked immunosorbent assay (ELISA) kits (TNF-a, IL-1 and NGF: Thermo Fisher Scientific; MMP-1 , MMP- 3 and MMP-9: Cusabio). RISULTS

[0268] The results of the present experiment are summarized in Figures 4 - 9.

[0269] All values in the figures were expressed as the mean standard error (s.e.m.) of the mean of n observations. For in vivo studies, n represents the number of animals studied. In experiments involving histology, the figures shown are representative of at least three experiments (histological staining) performed on different experimental days on tissue sections collected from all animals in each group. Data sets were examined with an analysis of variance. A p-value less than 0.05 was considered significant.

[0270] Example 10 - Effect of Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) on a Mouse Model of Acute Inflammation

[0271] Administration of NITE (Nite Carbon Nanocluster) in a mouse model of paw edema was performed to demonstrate the anti-inflammatory and antioxidant effect of NITE in a model of acute inflammation. Paw edema was induced by subplantar injection of CAR (Carrageenan-0.1 mL of 1% suspension in 0.85% saline) into the right hind paw of male Sprague Dawley rats. At the end of the experiment, the animals were killed under anesthesia and the hind paws were fixed in 10% neutral buffered formalin and embedded in paraffin for histological and biochemical examinations.

[0272] The results of the in vivo study demonstrated a significant reduction in CAR-induced histological damage to the paw and neutrophil infiltration in rats treated with NITE at a concentration of 10 mg / Kg.

[0273] These data demonstrated that, at 10 mg / kg, NITE possesses antioxidant and anti-inflammatory properties.

[0274] MATERIALS AND METHODS

[0275] Animals

[0276] Pathogen-free specific Sprague-Dawley rats (Envigo, Milan, Italy) were housed in a controlled environment (22 ± 2 °C, 55 ± 15% relative humidity, 12-h light / dark cycle). After a 1-week acclimatization, rats were fed a standard diet and water. Animal experiments complied with the Italian legislation on the protection of animals used for experimental and other scientific purposes (DM 116192) and with the European legislation (OJ EC L 358 / 1 of 18.12.1986).

[0277] Each rat was placed in individual cages. The cages and the housing room were cleaned periodically. The animals were fed a standard complete diet in pellets provided by the authorized breeder. Tap water filtered from the local network was provided ad libitum.

[0278] Rodent models provide a reliable model to study the inflammatory target after the application of NITE.

[0279] Experimental protocol

[0280] The protocol consisted of administering NITE 4mg / kg to the rats 30 minutes before the induction of paw edema. The animals will be sacrificed 6 hours after the administration of CAR. At time 0, 1 , 2, 3, 4, 5 and 6 hours after the induction of CAR, the paw edema was measured with the plethysmometer.

[0281] At the end of the experiment, several analyses were performed, in particular:

[0282] MPO analysis; and histological examination.

[0283] Edema volume

[0284] Paw edema volume was measured with a plethysmometer (Ugo Basile, Comerio, Varese, Italy) before carrageenan (CAR) injection and every hour for 6 hours. Edema was expressed as the increase in paw volume (mL) after carrageenan injection compared to the pre-injection value for all animals. Scores are expressed as the difference in paw volume (mL) (Figure 10).

[0285] MPO analysis

[0286] Myeloperoxidase (MPO) activity, an index of polymorphonuclear cell accumulation, was determined in the hind paw palm. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was measured as the amount of enzyme that degrades 1 mM peroxide in 1 min at 37 °C and was expressed in units per gram of wet tissue weight.

[0287] From the present analysis, it was observed that increased MPO levels after CAR injection and NITE treatment significantly reduced MPO levels.

[0288] Histological analysis

[0289] Seven-micrometer thick sections stained with hematoxylin and eosin (H&E) were examined under a light microscope associated with an imaging system (AxioVision, Zeiss, Milan, Italy) and evaluated by two researchers in blind. The sections were stained with H&E to allow a complete histological analysis that identified the morphological characteristics of the muscle fibers, from 0 to 5, defined as follows: 0 = no inflammation; 1 = mild inflammation; 2 = mild / moderate inflammation; 3 = moderate inflammation; 4 = moderate / severe inflammation; 5 = severe inflammation (Figure 11).

[0290] RESULTS

[0291] The results of this experiment are reported in figures 10 and 11. In conclusion, this study demonstrated that high dose NITE (1 Omg / kg) exhibits antioxidant and anti-inflammatory properties.

[0292] Example 11 COMPARATIVE STUDY

[0293] The Applicant performed an in vitro study to evaluate the efficacy of the polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials according to the invention (hereinafter referred to as "NCN Preparation" or "Clustered Preparation") compared to a grape seed and olive extract containing hydroxy tyrosol (HT) and procyanidins (PCy) (hereinafter referred to as "HT / PCy").

[0294] Furthermore, the Applicant then performed an in vivo study on a murine model of medial meniscus destabilization (DMM) and on rabbits characterized by anterior cruciate ligament section (ACLT) to evaluate the post-oral administration bioactivity of the clustered preparation vs HT / PCy in the DMM and ACLT animal models.

[0295] Materials and methods

[0296] The NCN preparation was obtained by subcritical hydrothermal enzymatic carbonization of plant species with antioxidant properties (e.g. Vitis Vinifera, Olea Europea, Piper Nigrum) at controlled pressures and temperatures. Agglomeration and clustering were then performed by ultrasonic sonication and high-frequency pulsed electromagnetic fields, obtaining nanoparticles with carbon clusters and active hydroxyl groups.

[0297] For in vitro studies, a dose equivalent to 30 mg / g of NCN preparation was used. Standardized samples with a content of 30 mg of HT / g and 30 mg of PCy / g were used as comparative samples.

[0298] Laboratory Equipment Used

[0299] ELISA System

[0300] Model: Bio-Rad ELISA Reader

[0301] Application: Quantitative analysis of IL-1 p, PGE2 and MMP-13 biomarkers.

[0302] Griess Reaction Spectrophotometer

[0303] Model: Thermo Scientific

[0304] Application: Measurement of NO concentrations in cell cultures.

[0305] RT-qPCR System

[0306] Model: Applied Biosystems QuantStudio 5

[0307] Application: Evaluation of COX-2 and I NOS gene expression levels.

[0308] Optical Microscope with FIJI Software

[0309] Model: Olympus BX53

[0310] Application: Quantification of NITEGE stains by immunohistochemistry.

[0311] Ultrasonicator for Deagglomeration and Clustering

[0312] Model: Hielscher UP200St

[0313] Application: Production of the clustered NCN preparation with parameter control. CO? Cell Incubator

[0314] Model: Thermo Scientific Forma Series II

[0315] Application: Maintenance of cell cultures during in vitro experiments.

[0316] Imaging System for Animal Models

[0317] Model: MS Spectrum CT

[0318] Application: Monitoring of joint lesions in DMM and ACLT models.

[0319] 11.1 In vitro study - evaluation of the level of pro-inflammatory and degradative biomarkers

[0320] Cell Culture Preparation

[0321] The cells used for the present experiment are primary articular chondrocytes (RAC) isolated from cartilage tissue samples.

[0322] • Experimental conditions: o Negative control: Untreated cultures. o Positive control: Cultures treated with standardized HT / PCy extracts (30 mg / g). o Clustered treatment: Cultures treated with the NCN preparation (30 mg / g).

[0323] • Protocol: o Cultures are pretreated for 24 hours with the assigned samples. o Subsequently, stimulation with interleukin-1 beta (IL-1|3) at a concentration of 1 ng / mL is administered to induce an inflammatory response. o The effect of the NCN preparation and HT / PCy extracts on the reduction of the following inflammatory and degradative biomarkers was monitored.

[0324] Biomarkers monitored and related analytical methods:

[0325] IL-113 (lnterleukin-1 beta):

[0326] Method: Enzyme immunoassay (ELISA).

[0327] Equipment: Bio-Rad ELISA Reader.

[0328] NO (Nitric Oxide):

[0329] Method: Griess reaction.

[0330] Equipment: Thermo Scientific spectrophotometer.

[0331] PGE2 (Prostaglandin E2):

[0332] Method: ELISA assay.

[0333] COX (Cyclooxygenase-2) and iNOS (Inducible nitric oxide synthase): Method: Quantification of mRNA levels by RT-qPCR.

[0334] Equipment: Applied Biosystems QuantStudio 5.

[0335] MMP-13 (Metalloproteinase-13):

[0336] Method: ELISA.

[0337] NITEGE (NITE Neo-Generated Epitope):

[0338] Method: Immunohistochemistry and quantification with FIJI software..

[0339] Equipment: Olympus BX53 optical microscope.

[0340] The preliminary results of the in vitro study are shown in Table 5 and depicted in Figure 12A.

[0341] Preliminary In Vitro Results

[0342] Table 5

[0343] 11.2 In vivo study: Assessment of the effect on the reduction of osteoarthritis severity (OARSI score) and cartilage degradation (NITEGE)

[0344] • DMM (Mouse) and ACLT (Rabbit) model

[0345] • Treatments administered: o Negative control: Saline solution (placebo). o HT / PCy treatment: Standardized extracts (100 mg / kg) administered every 2 days orally. o Clustered treatment: NCN preparation (100 mg / kg) administered every 2 days orally.

[0346] • Duration of treatment: 12 weeks for each model

[0347] Experimental Protocol

[0348] Animals were divided into groups based on treatment (control, HT / PCy, clustered NCN).

[0349] Treatments were administered orally according to the dosage and frequency specified.

[0350] During the 12-week period, the parameters of inflammation and cartilage degradation were monitored using histological analysis and imaging techniques and the following parameters were assessed. OARSI score: Assessment of the severity of osteoarthritis based on standardized criteria.

[0351] Method: Analysis of histological sections of the joints.

[0352] NITEGE stains:

[0353] Method: Immunohistochemistry for the quantification of newly generated NITE epitopes, associated with cartilage degradation.

[0354] Cartilage erosion:

[0355] Method: Measurement of cartilage thickness loss and visual analysis of lesions.

[0356] Equipment:

[0357] Imaging system: IVIS Spectrum CT to monitor the progression of joint lesions.

[0358] Preliminary results of the in vivo study are shown in Table 6 and represented in Figure 4B.

[0359] Preliminary In Vivo Results

[0360] Table 6

[0361] Discussion of Results

[0362] 1 . In Vitro: The clustered preparation shows a higher efficacy (up to 100%) compared to HT / PCy in reducing the main inflammatory and degradative biomarkers (IL-1 |3, NO, PGE2, COX-2, INOS, MMP-13 and NITEGE).

[0363] 2. In Vivo: In murine and rabbit models, the clustered preparation significantly reduces the severity of osteoarthritis (OARSI score) and cartilage degradation (NITEGE spots) with a higher efficacy up to 103%.

[0364] 3. Mechanism of Action: The NON clustered nanotechnology increases the bioavailability, stability and cellular uptake capacity, enhancing the inhibitory effect compared to conventional extracts.

[0365] Final Conclusions

[0366] • The NCN clustered preparation demonstrates superiority in efficacy over the HT / PCy extract with an increase of 96%-103% on key biomarkers.

[0367] • The NCN clustered preparation optimizes anti-inflammatory and anti-catabolic properties, making it a promising preventive therapeutic solution.

Claims

CLAIMS1 . A polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials in the form of an ingredient called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms, in a mammal in need thereof.

2. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to claim 1 , wherein said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) has a particle size comprised from 1 nm to 200 nm, preferably from 1 nm to 120 nm, more preferably from 1 to 80 nm.

3. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to claim 1 or 2, wherein said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises a quantity of carbon (C) comprised from 90% to 99.9%, more preferably from 95% to 99.9%, more preferably from 98.8% to 99.8%.

4. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to any one of the preceding claims, wherein said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises an amount of hydrogen (H) comprised from 0.1 to 10%, more preferably from 0.1 % to 5%, more preferably from 0.1 % to 0.7%.

5. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to any one of the preceding claims, wherein said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) comprises a number of hydroxyl groups (OH) preferably comprised from 20 to 700, more preferably from 30 to 600, even more preferably from 50 to 500 and a number of carbon clusters comprised from 100 to 26,000, preferably from 150 to 25,500, more preferably from 200 to 25,000.

6. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) according to any one of the preceding claims, wherein said diseases and / or disorders and / or related symptoms are diseases and / or disorders and / or symptoms characterized by inflammatory processes on an oxidative basis, preferably selected from the group comprising or, alternatively, consisting of osteoarthritis, acute, recurrent and / or chronic local osteoarticular inflammation, cartilage and / or bone degeneration, muscle atrophy in the limbs, reduction of motor skills, persistent and / or chronic pain.

7. The Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) for use according to any one of the preceding claims, wherein said NCN is an Olea Europea derived NCN, preferably from Olea Europea kernels, a Vitis Vinifera derived NCN, preferably from Vitis Vinifera grape seeds, or a mixture thereof.

8. A feed ingredient comprising a polyhydroxylated chiral carbon-based nanometric cluster derived from plantbased materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) as defined in any one of claims 1 to 7, for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal in need thereof.

9. A feed comprising a poly hydroxylated chiral carbon-based nanometric cluster derived from plant-based materials called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) as defined in any one of claims 1 to 7, for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms in a mammal in need thereof.

10. The feed ingredient according to claim 8 and / or the feed according to claim 9, wherein said related diseases and / or disorders and / or symptoms are diseases and / or disorders and / or symptoms characterized by inflammatory processes on an oxidative basis, preferably selected from the group comprising or, alternatively, consisting of osteoarthritis, acute, recurrent and / or chronic local osteoarticular inflammation, cartilage and / or bone degeneration, muscle atrophy in the limbs, reduction of motor skills, persistent and / or chronic pain.

11. The feed ingredient and / or the feed according to any one of claims 8-10 wherein said mammal Is a non-human animal, preferably a dog or a cat, even more preferably a dog.

12. The feed ingredient and / or feed according to any of one claims 8-11 wherein said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) is an Olea Europea derived NCN, preferably from Olea Europea kernels, a Vitis Vinifera derived NCN, preferably from Vitis Vinifera grape seeds, or a mixture thereof.

13. The feed ingredient and / or feed according to any of one claims 8-12 further comprising, in combination with said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster - NITE NCN Pet) at least one further active ingredient selected from the group comprising or, alternatively, consisting of a vitamin E, an omega-3 or a mixture thereof, a fish oil titrated in EPA-DHA, an L-methionine, a manganese salt, a vitamin, preferably a vitamin E, a prebiotic, preferably a chicory inulin, an ingredient of plant origin, preferably selected from Boswellia, willow {Salix), devil's claw (Harpagophytum procumbens) and mixtures thereof.

14. A mixture comprising or, alternatively, consisting of:I) a polyhydroxylated chiral carbon-based nanometric cluster derived from plant-based materials in the form of an ingredient called Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster- NITE NCN Pet);II) hyaluronic acid, or a salt thereof.

15. The mixture according to claim 14, wherein said salt is sodium hyaluronate.

16. The mixture according to claim 14 or 15 wherein:- said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster-NITE NCN Pet) has a particle size comprised from 1 nm to 200 nm, preferably from 1 nm to 120 nm, more preferably from 1 to 80 nm; and / or- said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster-NITE NCN Pet) comprises a quantity of carbon (C) comprised from 90% to 99.9%, more preferably from 95% to 99.9%, more preferably from 98.8% to 99.8%; and / or- said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster-NITE NCN Pet) comprises a quantity of hydrogen (H) comprised from 0.1 to 10%, more preferably from 0.1% to 5%, more preferably from 0.1 % to 0.7%; and / or,- said Micronized Vegetal Carbon NCN (NITE Carbon Nano Cluster-NITE NCN Pet) comprises a number of hydroxyl groups (OH) preferably comprised from 20 to 700, more preferably from 30 to 600, even more preferably from 50 to 500 and a number of carbon clusters comprised from 100 to 26,000, preferably from 150 to 25,500, more preferably from 200 to 25,000.

17. A composition comprising the mixture according to any one of claims 14-16, and additives and / or excipients of acceptable pharmaceutical or food grade.

18. The mixture according to any one of claims 14-16, for use as a medicament.

19. The composition according to claim 17, for use as a medicament.

20. The mixture according to claim 18, for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms, in a mammal in need thereof.

21. The composition according to claim 19, for use in a method of treatment, preventive and / or adjuvant to the curative process, of acute, recurrent and / or chronic musculoskeletal and / or osteoarticular diseases and / or disorders and / or related symptoms, in a mammal in need thereof.