Food supplement based on ursolic acid, tomatine and polyphenols from grape pomace, indicated for neurogenic muscle atrophy

Abstract

The present patent application describes a nutraceutical composition comprising ursolic acid, tomatine and polyphenols from grape pomace. Said composition resulted to be useful in counteracting muscle atrophy associated with neuropathic pain.

Description

[0001] PATENT APPLICATION FOR INDUSTRIAL INVENTION WITH TITLE:

[0002] FOOD SUPPLEMENT BASED ON URSOLIC ACID, TOMATINE AND POLYPHENOLS FROM GRAPE POMACE, INDICATED FOR NEUROGENIC MUSCLE ATROPHY

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to the field of food supplements, in particular to food supplements aimed at counteracting muscle atrophy associated with neuropathic pain.

[0005] STATE OF THE ART

[0006] Ursolic acid is a pentacyclic triterpenoid widely present in the peel of many fruits, especially herbs and spices. Ursolic acid has demonstrated antioxidant and antiinflammatory effects; it promotes tumor cell apoptosis and regulates immune function. Recent studies have highlighted that ursolic acid can promote neuronal regeneration in injury-induced animal models (Liu et al., 2013). In particular, mice with sciatic nerve injury were treated with ursolic acid and observed for 8 weeks. The results revealed recovery of the myelin sheath, while assessments of muscle mass index demonstrated a significant reduction in muscle atrophy, as compared to control mice. Biochemical investigations have highlighted that ursolic acid promotes sustained expression of S100 proteins in spinal cord cells, in a dose-dependent manner, following peripheral nerve injury in mice, thus preventing nerve demyelination and providing favorable conditions for myelin sheath repair and regeneration. S100 proteins are a family of proteins capable of binding calcium, undergoing conformational changes and exposing the binding site of the target protein, with various biological effects. S100 protein concentrations in biological fluids can be used as an indicator of functional activation following a nerve injury. Nerve injury can trigger the activation of S100 protein overexpression and promotion of neural regeneration. Murine model studies showed that S100 protein expression levels were significantly increased after treatment with high- and medium-dose ursolic acid, suggesting that ursolic acid can activate S100 protein overexpression, thereby promoting neural regeneration. Experimental results showed that the number, integrity, and thickness of myelin sheaths were increased with ursolic acid administration. The number of myelinated nerve fibers and their average diameter in the high- and medium-dose ursolic acid groups were significantly higher than in the low-dose ursolic acid groups. This provides evidence that ursolic acid can strongly promote the growth of damaged nerve myelin. Nerve injury is accompanied by partial muscle atrophy. As nerve regeneration occurs and neurological function recovers, muscle atrophy also improves. Experimental results showed that muscle fibers in the high- and medium-dose ursolic acid groups were in good physiological condition, and recovery from muscle atrophy was significantly more apparent than in the low-dose ursolic acid groups. These results suggest that nerve regeneration and functional recovery are improved following ursolic acid treatment.

[0007] WO2024 / 142004 describes a process for extracting ursolic acid (UA) from freeze- dried grape pomace, specifically of the Fiano di Avellino DOCG variety, into sunflower seed oil. The resulting oleolyte and the use thereof in cosmetic preparations are also described.

[0008] Tomatidine is a steroidal alkaloid, the aglycone of a-tomatine, a glycoalkaloid abundant in tomato plants, which mediates plant’s defense against fungi, bacteria, viruses, and predatory insects. When consumed by animals, a-tomatine is hydrolyzed by stomach acids and intestinal bacteria into tomatidine, which is absorbed by the intestine.

[0009] Recent animal studies demonstrated that tomatidine can stimulate skeletal muscle anabolism in vivo (Dyle et al., 2014). Young mice (7 weeks old) were treated with tomatidine for 5 weeks by feeding standard chow supplemented with 0.05% (w / w) tomatidine. At the end of treatment, the effect of tomatidine on skeletal muscle was assessed. Tomatidine promoted an increase in mTORCI activity, as evidenced by increased S6K phosphorylation, total cellular proteins, mitochondrial DNA, IGF-1 , and PGC-1 a1 mRNA. As consistent with its effects on mTORCI signaling, tomatidine significantly increased skeletal muscle fiber size and increased muscle weight in the tibialis anterior, gastrocnemius, quadriceps, and triceps muscles. Further examination of skeletal muscle fibers revealed that tomatidine stimulated hypertrophy of glycolytic muscle fibers (types lib and llx) in the extensor digitorum longus muscle, and oxidative muscle fibers (types I and Ila) in the soleus muscle. In addition to increasing skeletal muscle mass, tomatidine significantly increased grip strength in vivo and specific muscle strength ex vivo. Furthermore, tomatidine increased the distance run on an accelerating treadmill. Taken together, these data indicate that tomatidine stimulates skeletal muscle anabolism in vivo, leading to muscle hypertrophy, increased strength, and improved physical exercise capacity. To maintain proper function, such as nerve impulse conduction, peripheral nerves require a local and regular energy supply. In vivo studies demonstrated that this energy supply is ensured by a highly developed, multi-level intraneural blood vessel system, reinforced by fragmentary, local extrinsic vessels (Paulson et al., 2002). The microcirculatory system supplying blood to the peripheral nervous system is made of a two-level circulatory system, the intrinsic and extrinsic. The extrinsic system is made of regional arteries that supply blood to the internal nervous system. The intrinsic system consists of epineurial system, transperineurial arterioles, and endoneurial system. Together, they both supply blood to the entire peripheral nerve tract. This high blood flow makes the nerve fiber resistant to ischemia. However, a decrease or interruption of blood flow can lead to reduced neuronal activity, which can in turn lead to neuropathy. Experiments were carried out in rat models of streptozotocin-induced diabetic neuropathy to study the role of peripheral circulation in diabetic neuropathy. The results showed that diabetic neuropathy is due to blood vessel destruction. Diabetes reduces nitric oxide synthase expression in the blood vessels of peripheral nerves. Reduced nitric oxide synthesis leads to excessive activity of vasoconstrictors, such as norepinephrine, and reduced vessel relaxation. All these studies suggest that there is a severe damage to the microcirculatory blood vessels in peripheral nerves, which is the cause of neuropathic pain in diabetes, and that medical intervention preventing this angiolysis (blood vessel damage) can reverse diabetic neuropathic pain. An important chemical messenger in the control of nerve impulse conduction, vascular function, management of diabetic neuropathy, and pain perception is cyclic guanosine monophosphate (cGMP), which is hydrolyzed by the phosphodiesterase-5 enzyme. Pathophysiological impairment of the NO-sGC-cGMP signaling pathway contributes to dysregulation of vascularization. It is also important to underline that the activity of the sGC enzyme is highly sensitive to conditions of cellular oxidative stress which, by converting the Fe2+atom of the heme group to Fe3+, favors a reduced sGC’s responsiveness to NO, resulting in reduced cGMP production which leads to difficult maintaining of functional homeostasis of the affected organ, including vascularization. On the other hand, increased oxidative stress conditions can either alter the functionality of the eNOS enzyme, resulting in reduced endogenous NO production, or oxidize the produced NO into peroxynitrite (O=NO-O); these factors lead to reduced cGMP production, with all the resulting pathological consequences, including vascularization. Consequently, dietary supplementation with antioxidant molecules may be another useful approach for maintaining physiological homeostatic conditions. In fact, the intake of these compounds could promote protection against the oxidative stress state, in particular by preventing the oxidation of the Fe2+atom of the heme group, thus maintaining the activity of the sGC enzyme and the consequent production of cGMP; this results into the possibility of maintaining the functional homeostasis of the affected organ, and in particular the regulation of blood pressure. Natural polyphenols may be particularly useful for this purpose, as they are hydrophilic molecules capable of diffusing at the cytosolic level and interacting with the catalytic sites of protein subunits, thus preventing the oxidation of physiological metal species, including the iron ion. A study carried out to examine the potential of phosphodiesterase-5 inhibitors in improving diabetic peripheral neuropathy reported that they were able to restore nerve vascularization (Patil et al., 2004). In addition to these, it was also reported that free radical scavengers can increase blood flow in peripheral nerves and therefore alleviate peripheral neuropathy. One category of active ingredients suggested for the treatment of neuropathy are vasodilators. Several studies have recently examined the idea that vasodilator therapy can improve nerve function in experimental diabetes.

[0010] EP3884955 describes a maltodextrin-microencapsulated grape pomace extract (MaGPE), with high bioavailability of polyphenols.

[0011] Yadav et al. (Eur. J. Pharmacol. 2002, 925, 174995) discloses that natural products, such as polyphenols, terpenoids, flavonoids, alkaloids, vitamin D, etc., exhibit strong potential against skeletal muscle atrophy.

[0012] The purpose of the present invention is to provide a nutraceutical composition that may be useful for counteracting muscle atrophy associated with neuropathic pain or as an adjuvant in combination with commercial products already known for the same purpose.

[0013] SUMMARY OF THE INVENTION

[0014] It was surprisingly found that a synergistic modulation of muscle atrophy associated with neuropathic pain can be achieved by a blend of natural components based on ursolic acid, tomatine, and grape pomace polyphenols. Therefore, the object of the present patent application is a nutraceutical composition comprising ursolic acid, tomatine, and grape pomace polyphenols. This composition resulted to be useful in counteracting muscle atrophy associated with neuropathic pain.

[0015] DETAILED DESCRIPTION OF THE INVENTION

[0016] For the purposes of the present invention, the natural sources used to extract ursolic acid are preferably selected from the group consisting of plant sources rich in ursolic acid, especially edible fruits or their processing waste. Preferably, the natural source is grape pomace from any winemaking process of any cultivar. Preferably, the ursolic acid for the purposes of this invention is an Optimized Grape Pomace Oleolyte (OGPO) from freeze-dried grape pomace as described in WO2024 / 142004, using a process comprising an extraction time of 1 -96 hours and a temperature of 20-80 °C, preferably 65-70 hours and 60-65°C, which allows for optimal extraction of UA to obtain an optimized freeze-dried grape pomace oleolyte (OGPO). The process involves contacting a defined amount of freeze-dried grape pomace with a certain volume of sunflower oil (g / m I), preferably in a 1 :4 ratio, i.e. 4 ml of sunflower oil is used for each gram of freeze-dried grape pomace. After maceration of the matrix in the oil, the mixture is subjected to solid-liquid separation, preferably by centrifugation.

[0017] The OGPO oleolyte is preferably included in soft capsules in an amount of 200-600 mg, preferably 400 mg. The excipients are preferably selected from the group consisting of purified water, glycerol. The soft capsule constituents preferably include gelatin, hexitol, sorbitans, sodium ethyl p-hydroxybenzoate, sodium propyl p-hydroxybenzoate, titanium dioxide (E171 ), yellow iron oxide (E172).

[0018] For the purposes of the invention, lecithin-microencapsulated ursolic acid may alternatively be used. Commercial extracts obtained from any plant source containing ursolic acid, preferably rosemary extracts titrated to 50% ursolic acid, may be used.

[0019] Preferably, an aliquot of soy lecithin (3-5 g) and an aliquot of rosemary extract titrated to 50% ursolic acid, preferably in a 4:1 ratio, are suspended in an appropriate amount of hydroalcoholic mixture containing 30% ethyl alcohol. The mixture is stirred at 75-85°C for 4-6 hours. The mixture is subjected to freeze-drying.

[0020] Various plant matrices may be used in freeze-dried form, such as concentrated sources of tomatine, preferably unripe tomatoes, tomato seedling sprouts (harvested up to 21 days after germination), and tomato seedling microgreens (harvested up to 21 days after germination). The plant matrix is introduced into semiindustrial and industrial freeze-dryers, under controlled temperature (between -50 and -60 °C) and pressure (0.05-0.15 mbar).

[0021] The freeze-dried plant matrix described above can be extracted with a suitable solvent, under specific temperature and time conditions. The solvents can be water, ethyl alcohol, or a mixture of both in any proportion, preferably water. Temperatures vary in a range of 25-100 °C, preferably 60 °C. Extraction times vary in a range of 5-60 min, preferably 15 min. The liquid extract is ready for use in soft capsules. The extract is dried using a freeze-dryer or a spray dryer.

[0022] The finely powdered freeze-dried plant matrix or the freeze-dried extract thereof, which contain 2-20 mg / g of tomatine, preferably 10 mg / g, can be used to formulate powders, capsules, and tablets, using well known techniques (encapsulation, compression, controlled release, microgranules, nanocapsules, multilayers) as described in the pharmacopoeia for the preparation of gastro-resistant pharmaceutical formulations for oral use.

[0023] For the purposes of the present invention, polyphenols are those contained in a maltodextrin-microencapsulated grape pomace extract (MaGPE) as described in EP3884955, and microencapsulated in co-presence of maltodextrins at a concentration of 5-20% to obtain an extract preferably having a peculiar polyphenol profile such that 1000 mg of dry extract contains:

[0024] Resveratrol 2.5-200 mcg;

[0025] Gallic acid 2000-6000 mcg;

[0026] Catechin 10000-30000 mcg; Chlorogenic acid 900-1800 mcg;

[0027] Quercetin 900-2000 mcg;

[0028] Procyanidin B2 2000-5000 mcg;

[0029] Procyanidin B1 6000-15000 mcg;

[0030] Procyanidin C1 4000-8000 mcg;

[0031] Epicatechin 10000-20000 mcg

[0032] Quercetin-3-O-glucoside 2000-4000 mcg;

[0033] Para-coumaric acid 2000-4500 mcg;

[0034] Syringic acid 5000-10000 mcg;

[0035] Rutin 50-200 mcg.

[0036] A specific example of pharmaceutical formulation according to the invention consists of a gastro-resistant hard capsule, containing:

[0037] 200-600 mg, preferably 400 mg, of ursolic acid microencapsulated in lecithin;

[0038] 200-600 mg of a freeze-dried extract containing 2-20 mg / g of tomatine, preferably 400 mg;

[0039] 100-350 mg of MaGPE, preferably of Aglianico grape, preferably 300 mg.

[0040] Preferably, excipients are selected from the group consisting of silicon dioxide, com starch, microcrystalline cellulose, calcium phosphate, talc, and aluminum silicate. The composition, according to the invention, is obtained by simply mixing the components in the required amounts using standard mixers and then filling the powdered mixture into hard capsules.

[0041] Another specific example of pharmaceutical formulation according to the invention consists of a gastro-resistant soft capsule, containing:

[0042] 200-600 mg of a freeze-dried extract containing 2-20 mg / g di tomatine, preferably 400 mg;

[0043] 100-350 mg of MaGPE, preferably of Aglianico grape, preferably 300 mg;

[0044] 200-600 mg of an optimized freeze-dried grape pomace oleolyte (OGPO), preferably 400 mg.

[0045] The present invention can be better understood in the light of the following embodiment examples. BRIEF DESCRIPTION OF THE FIGURES

[0046] Fig. 1 . Effects of treatments on the capacity to sustain physical effort, as measured in terms of distance covered on treadmill per unit of time. OGPO: ursolic acid based oleolyte; TL: freeze-dried tomato; MaGPE: maltdextrinated grape polyphenolic extract; Mix: treatment with OGPO + TL + MaGPE. The different numbers above the bars indicate significantly statistical differences. P < 0.05.

[0047] Fig. 2. Effect of treatments on blood levels of the muscle autophagy protein marker Atrogin-1. OGPO: ursolic acid based oleolyte; TL: freeze-dried tomato; MaGPE: maltdextrinated grape polyphenolic extract; Mix: treatment with OGPO + TL + MaGPE. The different numbers above the bars indicate significantly statistical differences. P < 0.05.

[0048] Fig. 3. Effect of treatments on blood levels of the muscle autophagy protein marker MuRF-1. OGPO: ursolic acid based oleolyte; TL: freeze-dried tomato; MaGPE: maltdextrinated grape pomace extract; Mix: treatment with OGPO + TL + MaGPE. The different numbers above the bars indicate significantly statistical differences. P < 0.05.

[0049] Fig. 4. Effect of treatments on the number of simultaneous receptor interactions (colocalizations) of synaptophysin and a-bungarotoxin at the pre-synaptic and post- synaptic levels, respectively. OGPO: ursolic acid based oleolyte; TL: freeze-dried tomato; MaGPE: maltdextrinated grape pomace extract; Mix: treatment with OGPO + TL + MaGPE. The different numbers above the bars indicate significantly statistical differences. P < 0.05.

[0050] Fig. 5. Effect of treatments on the neuromuscular vascularization phenomenon. OGPO: ursolic acid based oleolyte; TL: freeze-dried tomato; MaGPE: maltdextrinated grape polyphenolic extract; Mix: treatment with OGPO + TL + MaGPE. The different numbers above the bars indicate significantly statistical differences. P < 0.05.

[0051] EXPERIMENTAL PART

[0052] EXAMPLE 1 - Ursolic acid from grape pomace

[0053] An optimized oleolyte from freeze-dried pomace (Optimized Grape Pomace Oleolyte - OGPO) was obtained as detailed in WO2024 / 142004, in particular in Examples 2A, 3A e 4A EXAMPLE 2 - Ursolic acid microencapsulated in lecithin

[0054] Ursolic acid can be embedded in food-grade soy lecithin microspheres to obtain ursolic acid microencapsulated in soy lecithin. Commercial extracts obtained from any plant source containing ursolic acid, preferably rosemary extracts titrated to 50% ursolic acid, may be used.

[0055] EXAMPLE 2.1 - Preparation of lecithin-microencapsulated ursolic acid

[0056] An aliquot of soy lecithin (5 g) and an aliquot of rosemary extract (1 g) titrated to 50% ursolic acid, in a 4:1 ratio, are suspended in 50 mL of a hydroalcoholic mixture containing 30% ethyl alcohol. This is left under stirring for 5 hours at 80 °C. The mixture is freeze-dried.

[0057] EXAMPLE 2.2 - Evaluation of particle size and degree of inclusion

[0058] The average diameter of the obtained microspheres was measured by Fourier transform infrared spectroscopy (FTIR). The ursolic acid peak was compared with that of the ursolic acid-lecithin mixture, in a range of 4000-400 cm’1for 100 scans. The average microsphere diameter was 73 pm. The degree of inclusion of ursolic acid in the microspheres was assessed by size exclusion technique. An aliquot of the freeze-dried product was suspended in distilled water and filtered through systems of porous septa filters with 0.45 pm diameter. The filtrate was freeze-dried and solubilized in pure ethyl alcohol. The alcoholic solution was subjected to HPLC analysis under the following conditions. The chromatographic analysis of OGPO was performed using a Jasco Extrema LC-4000 HPLC system (Jasco Inc., Easton, MD, USA), coupled with an autosampler, a binary solvent pump, and a diode array detector (DAD). Elution was performed on a Kinetex® C18 column (250 mm x 4.6 mm, 5 pm; Phenomenex, Torrance, CA, USA). The mobile phases were water with 0.1 % formic acid (A) and acetonitrile (B). The elution gradient was performed under the following conditions: 0-3 min, isocratic with 60% B; 3-20 min, linear gradient from 60 to 90% B; 20-24 min, isocratic with 90% B; 24-29, isocratic with 60% B for column reconditioning. The separation parameters were as follows: the column temperature was set at 30 °C, the injection volume was 20 pL, and the flow rate was set at 1 mL / min. Quantification of ursolic acid was performed at 205 nm.

[0059] The result indicates an inclusion degree of ursolic acid in the microspheres of approximately 98%. EXAMPLE 3 - TOMATINE

[0060] Various plant matrices can be used in freeze-dried form as concentrated sources of tomatine, preferably unripe tomatoes, tomato seedling sprouts (harvested up to 21 days after germination), and tomato seedling microgreens (harvested up to 21 days after germination). The example under study involves unripe tomatoes harvested 30-35 days after flowering. The plant matrix is introduced into semi-industrial and industrial freeze-dryers, under controlled temperature (-55°C) and pressure (0.1 mbar) conditions. The freeze-dried plant matrix is finely pulverized and is ready to be used for the formulation of powders, capsules, and tablets, using well known techniques (encapsulation, compression, controlled release, microgranules, nanocapsules, multilayers) as described in the pharmacopoeia for the preparation of gastro-resistant pharmaceutical formulations for oral use. Excipients (silicon dioxide, corn starch, microcrystalline cellulose, calcium phosphate, talc, aluminum silicate) q.s.. The composition, according to the invention, is obtained simply by mixing the components in the required amounts using standard mixers. The powder contains 2-20 mg / g of tomatine, preferably 10 mg / g. The powder is included in an amount of 200-600 mg, preferably 400 mg.

[0061] EXAMPLE 3.1 - Tomatine extract from freeze-dried plant matrix

[0062] The freeze-dried plant matrix described above can be extracted with a suitable solvent, under specific temperature and time conditions. The solvents may be water, ethyl alcohol, or a mixture of both in any proportion, preferably water. Temperatures vary in a range of 25-100 °C, preferably 60 °C. Extraction times vary in a range of 5-60 min, preferably 15 min. The liquid extract is ready to be used in soft capsules. The extract is dried using a freeze-dryer or a spray-dryer.

[0063] The dry extract is ready to be used in the formulation of powders, capsules, and tablets, using well known techniques (encapsulation, compression, controlled release, microgranules, nanocapsules, multilayers) as described in the pharmacopoeia for the preparation of gastro-resistant pharmaceutical formulations for oral use. Excipients (silicon dioxide, com starch, microcrystalline cellulose, calcium phosphate, talc, aluminum silicate) q.s.. The composition, according to the invention, is obtained simply by mixing the components in the required amounts using standard mixers. The dry extract contains 2-20 mg / g of tomatine, preferably 10 mg / g. The dry extract is included in an amount of 200-600 mg, preferably 400 mg.

[0064] EXAMPLE 4 - POLYPHENOLS FROM GRAPE POMACE

[0065] A maltodextrin-microencapsulated grape pomace extract (MaGPE) was prepared as described in EP3884955 and microencapsulated in co-presence of maltodextrin at a concentration of 5-20% to obtain an extract having a peculiar polyphenol profile so that 1000 mg of dry extract contains:

[0066] Resveratrol 2.5-200 mcg;

[0067] Gallic Acid 2000-6000 mcg;

[0068] Catechin 10000-30000 mcg

[0069] Chlorogenic acid 900-1800 mcg;

[0070] Quercetin 900-2000 mcg;

[0071] Procyanidin B2 2000-5000 mcg;

[0072] Procyanidin B1 6000-15000 mcg;

[0073] Procyanidin C1 4000-8000 mcg;

[0074] Epicatechin 10000-20000 mcg

[0075] Quercetin-3-O-glucoside 2000-4000 mcg;

[0076] Para-coumaric acid 2000-4500 mcg;

[0077] Syringic acid 5000-10000 mcg;

[0078] Rutin 50-200 mcg.

[0079] EXAMPLE 5 - In vivo Experimentation

[0080] Twelve-week-old male C57BL / 6J mice, purchased from Jackson Laboratory (Bar Harbor, ME, USA) were selected. Animals were handled according to National and European communities guidelines and were approved by the Animal Research Committee of the University of Naples “Federico II”. Mice were housed in cages under controlled conditions, with a temperature of 20-24°C and a relative humidity of 50-60%. Food and water were available ad libitum daily to all animals. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol No. 354 / 2019-PR). Eighty mice were selected and divided into four groups of 20 mice each. Group 1 : treatment with ursolic acid-based oleolyte (OGPO) according to Example 1 ; Group 2: treatment with freeze-dried tomato (TL) according to Example 3; Group 3: treatment with MaGPE according to Example 4; Group 4: treatment with a mix of OGPO + TL + MaGPE. The OGPO treatment consisted of kibble (1 g each) containing 175 pL OGPO (ursolic acid, 0.5 mg / mL). The TL treatment consisted of kibble (1 g each) containing 5 mg TL (tomatine, 6 mg / g). The MaGPE treatment consisted of drinking water containing 800 mg / L MaGPE. The mixed treatment consisted of kibble containing OGPO + TL and drinking water containing MaGPE. Each treatment period lasted 30 days. Before starting treatment, mice were subjected to sciatic nerve ligation technique, which causes interruption of electrical conduction to the leg muscle, neurodegeneration, muscle atrophy, and neuropathic pain, simulating the clinicopathological state of sciatica. At the end of the treatment period, the following tests were performed: treadmill test, consisting of measuring the distance (m) covered by mice on a treadmill in a predetermined time; biochemical and physiological analyses to measure specific markers and parameters of: muscle atrophy (Atrogin-1 , MuRF-1 ); neuronal degeneration (number of co-localizations for a-bungarotoxin and synaptophysin receptors); neuromuscular revascularization (measurement of neuromuscular blood flow).

[0081] Results

[0082] The treadmill test measured the mice’s resistance to physical exercise on the treadmill, in terms of meters covered per unit of time, until exhaustion. Fig. 1 shows that the mix of the three components increased the mice’s resistance to physical exercise, in terms of distance covered in a predetermined time, with a clear synergistic effect.

[0083] To evaluate the effects of the treatments on muscle atrophy induced by the nerve ligation technique, blood levels of specific markers of muscle degeneration, such as Atrogin-1 and MuRF-1 , were measured. These two markers represent an index of muscle protein autophagy, which is a direct consequence of the interruption of nerve impulse conduction to the muscle following the ligation. Figs. 2 and 3 clearly indicate that the mix of the three components is capable of synergistically reducing the levels of these markers.

[0084] The potential effects of inducing neuronal regeneration were assessed by measuring the number of ligand interactions with specific receptors at the neuromuscular junction. For presynaptic receptors, the interaction capacity of synaptophysin, an endogenous ligand typical of the animal world, was assessed. For postsynaptic receptors, the interaction capacity of a-bungarotoxin, an exogenous ligand typical of the venom of certain snakes, was assessed. The potential for new neuromuscular junction formation, and therefore neuronal regeneration, was assessed in terms of the number of simultaneous receptor interactions (co-localizations) at the presynaptic and postsynaptic levels. Figure 4 shows that the mix of the three treatments is capable of promoting the greatest number of co-localizations, in a clear synergistic manner.

[0085] The effects of the treatments on neuromuscular vascularization were measured using a polarography technique with hydrogen microelectrodes. Fig. 5 demonstrates that the mix of the three treatments induces an increase in blood flow in the innervation site under exam, with a clear synergistic effect.

[0086] Bibliographic References

[0087] - Liu B, Liu Y, Yang G, Xu ZM, Chen JJ. Ursolic acid induces neural regeneration after sciatic nerve injury. Neural Regen Res. 2013;8(27):251 CI- 2519.

[0088] - Michael C. Dyle, Scott M. Ebert, Daniel P. Cook, Steven D. Kunkel, Daniel K. Fox, Kale S. Bongers, Steven A. Bullard, Jason M. Dierdorff, and Christopher M. Adams. Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy. The Journal of Biological Chemistry, 2014; 289, 14913-14924.

[0089] - P.E. Paulson, K.L. Casey, T.J. Morrow, Long-term changes in behavior and regional cerebral blood flow associated with painful peripheral mononeuropathy in the rat, Pain, 2002; 95 (1-2), 31-40.

[0090] - C.S. Patil, V.P. Singh, S. Singh, S.K. Kulkarni, Modulatory effect of the PDE- 5 inhibitor sildenafil in diabetic neuropathy, Pharmacology, 2004; 72 (3), 190- 195.

Claims

CLAIMS1 . A nutraceutical composition comprising ursolic acid, tomatine and polyphenols.

2. The composition according to claim 1 , wherein ursolic acid is extracted from a plant source rich in ursolic acid.

3. The composition according to claim 2, wherein the ursolic acid is an optimized extract in sunflower seed oil from freeze-dried grape pomace (optimized grape pomace oleolite - OGPO).

4. The composition according to claim 2, wherein the ursolic acid is an extract microencapsulated in soy lecithin.

5. The composition according to any one of claims 1 -4, wherein the tomatine is in the form of freeze-dried unripe tomatoes, tomato seedling sprouts or tomato seedling microgreens.

6. The composition according to any one of claims 1 -4, wherein the tomatine is in the form of a freeze-dried extract of unripe tomatoes, tomato seedling sprouts or tomato seedling microgreens.

7. The composition according to any one of claims 1 -6, wherein the polyphenols are those contained in a maltodextrin-microencapsulated grape pomace extract (MaGPE).

8. The composition according to any one of claims 1 -7 in the form of a gastro- resistant hard capsule, containing:200-600 mg, of ursolic acid microencapsulated in soy lecithin;200-600 mg of a freeze-dried extract containing 2-20 mg / g of tomatine;100-350 mg of MaGPE.

9. The composition according to any one of claims 1 -7 in the form of a gastro- resistant soft capsule containing:200-600 mg of a freeze-dried extract containing 2-20 mg / g of tomatine;100-350 mg of MaGPE;200-600 mg of a optimized freeze-dried grape pomace oleolyte (OGPO).

10. A composition according to any one of claims 1 -9 for use in the treatment of muscle atrophy associated with neuropathic pain.

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