Hydrolysed extract of hemp seed cake

Abstract

The present invention relates to a hydrolysed extract of de-oiled hemp seed cake obtained by alkaline hydrolysis at a pH of greater than or equal to 11, the peptide fractions thereof, the combination of this extract or the peptide fractions thereof with an ethanolic extract of hemp seed cake, and a pharmaceutical composition comprising same. The invention further relates to a method for producing the hydrolysed extract according to the invention. Finally, the invention relates to the hydrolysed extract according to the invention or a peptide fraction thereof or the combination or composition comprising same, for use as a drug.

Description

Hydrolyzed extract of hemp seed cake Technical field

[0001] The invention relates to a hydrolyzed extract of hemp seed cake and its uses. Prior art

[0002] Calpain, a key enzyme in the regulation of many cellular functions, plays a central role in various pathologies, particularly when it is overactive. To improve therapeutic prospects, and in particular to treat or prevent the pathologies concerned, it is therefore necessary to target the inhibition of this enzyme.

[0003] Calpains are a family of soluble intracellular Ca-dependent cysteine ​​proteases 2+ They respond to Ca signals. 2+ by cleaving many specific proteins, thus irreversibly modifying their function(s). They are involved in various cellular processes regulated by Ca 2+such as cell proliferation, cell differentiation, cell cycle progression, autophagy, apoptosis, and platelet activation. Changes in calpain activation levels are implicated in various pathological phenomena, such as ischemic injury, muscular dystrophy, diabetes, diabetic endotheliopathy, cataracts, atherosclerosis, Alzheimer's disease, repetitive concussion-induced neuropathy, and cancer. Calpains represent potential therapeutic targets for drug discovery.

[0004] Calpains are involved in many cellular mechanisms, including the CDK5 / P35 complex. Indeed, in the absence of pathological conditions, the CDK5 / P35 complex is involved in certain normal neuronal functions. In the presence of pathological conditions involving calpain hyperactivity, the latter cleaves P35, producing the P25 protein. The CDK5 / P25 complex is involved in pathological mechanisms such as the formation of hyperphosphorylated tau protein, increased neuroinflammation, or neuronal death.

[0005] In particular, there are two ubiquitous calpain isoforms in the brain: calpain-1 and calpain-2. Results obtained over the last 30 years have led to the conclusion that these two calpain isoforms have opposing functions in the brain. Activation of calpain-1 is required for some forms of synaptic plasticity and learning and memory, while activation of calpain-2 limits the extent of plasticity and learning. Calpain-1 is neuroprotective during postnatal development and adulthood, while calpain-2 is neurodegenerative. Several key protein targets participating in these opposing functions have been identified and linked to known pathways involved in synaptic plasticity and neuroprotection / neurodegeneration (Wang et al., Cells 2020, 9, 2698, pages 1-16 – Wang et al. Journal of Neurotrauma 34:1-13, 2017; Wang et al., Sci. Adv.2020; 6: eaba5547- July 1, 2020). Thus, the metabolic pathway associated with calpains directly targets neurons and more particularly at the level of synapses. Its action is therefore direct and targeted to generate neuroprotective activity.

[0006] Furthermore, deregulation of calpain activity has been implicated in tumorigenesis, suggesting its impact on cancer and metastasis (Ivan Shapovalov et al., EXPERT OPINION ON THERAPEUTIC TARGETS, 2022, VOL. 26, NO. 3, 217–231).

[0007] Pathological overactivation of calpains is also known to be involved in some fibrotic diseases, including cardiac fibrosis and idiopathic pulmonary fibrosis. Calpain upregulates transforming growth factor beta 1 (TGF-beta1) at both transcriptional and post-translational levels to induce profibrotic activities. Furthermore, calpain inhibitors have been shown to prevent hypertrophic scar formation after burn injuries (Cheong Hoon Seo et al. Int. J. Mol. Sci. 2021, 22, 5771).

[0008] Furthermore, calpain activity has been shown to contribute to the process of many diseases associated with inflammation and in the majority of cases, calpain inhibitor plays a protective role against the disease. It has been concluded that calpain is involved in the inflammation process through various mechanisms, including regulation of macrophages, neutrophils, lymphocyte migration and apoptosis, modulation of inflammatory mediator activation, degradation of certain associated proteins, autophagy, and induction of cell apoptosis. Calpain is thus associated with many inflammatory diseases such as asthma, atherosclerosis, rheumatoid arthritis, and multiple sclerosis (JINGJING JI et al., BIOMEDICAL REPORTS 5: 647-652, 2016).

[0009] In particular, calpains are known for their involvement in the development of neurodegenerative diseases such as Alzheimer's disease (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630) or Parkinson's disease (Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27;8(1):18083).

[0010] Furthermore, it is also known from the prior art that acetylcholinesterase (AChE) inhibition is a well-established strategy in the symptomatic treatment of Alzheimer's disease. In clinical evaluations, this inhibition leads to a temporary improvement in cognitive symptoms in patients with mild to moderate Alzheimer's disease (Rioux et al., Les insecteurs de l'acide cholinesterase, Formation continue, Le médecin du Québec, 2002: 37(4):89-94, Briks et al., Cholinesterease inhibitors for Alzheimer's disease, Cochrane Database Syst Rev. 2006 Jan 25;2006(1)CD005593). However, this solution involves many disadvantages.Among other things, AChE inhibition leads to numerous adverse effects (such as disruption of the cardiovascular system or the gastrointestinal system), only symptomatic benefits, only short-term efficacy, involves only a single target mechanism, and can only (symptomatically) treat a limited number of neurodegenerative diseases including Alzheimer's disease, but not Parkinson's disease (Saini et al., The structural hybrids of acetylcholinesterase inhibitors in the treatment of Alzheimer's disease, Med Res Rev. 2019 Mar;39(2):608-630, Briks et. al., Cholinesterease inhibitors for Alzheimer's disease, Cochrane Database Syst Rev. 2006 Jan 25;2006(1)CD005593, Rioux et al., Acetylcholinesterase inhibitors, Continuing education, Le médecin du Québec, 2002: 37(4):89-94).

[0011] Since the role of calpain is upstream of the reaction cascades of neurodegenerative diseases, its inhibition has an effect on all levels of the downstream cascades. Calpain inhibition therefore involves a comprehensive neuroprotective effect, an action on several target mechanisms, a direct action on pathological mechanisms, a long-term effect, an action on several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, an action of treatment and prevention of the disease, and not only a symptomatic action, as well as limited adverse effects (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630, Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27;8(1):18083, Trinchese et al., Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest. 2008; 118(8):2796-807). .

[0012] As an illustration and not an exhaustive list, calpain inhibition improves spatial memory and synaptic transmission in models of Alzheimer's disease, and restores normal phosphorylation levels of the transcription factor CREB, which is crucial for memory (Trinchese et al., Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer's disease. J Clin Invest. 2008; 118(8):2796-807). Calpain inhibition protects dopaminergic neurons in the substantia nigra in Parkinson's disease (Zaman et al., Inhibition of Calpain Attenuates Degeneration of Substantia Nigra Neurons in the Rotenone Rat Model of Parkinson's Disease. Int J Mol Sci. 2022;23(22):13849). In addition, by preventing the abnormal degradation of several key proteins in Alzheimer's and Parkinson's diseases that are essential for neuronal function, such as Tau protein or alpha-synuclein protein, calpain inhibition has a more comprehensive neuroprotective effect than that provided by AChE inhibitors. Furthermore, calpain inhibition reduces alpha-synuclein aggregation in Parkinson's disease, reduces neuronal inflammation, promotes protective microglial differentiation, reduces neuronal death, and reduces synaptic dysfunction (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630, Trinchese et al., Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer's disease. J Clin Invest. 2008; 118(8):2796-807, Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27;8(1):18083).Thus, all of the prior art shows that calpain inhibition more directly targets the pathological mechanisms underlying neurodegenerative diseases, unlike AChE inhibition which mainly aims to improve cholinergic neurotransmission, but also that calpain inhibition offers multiple protection, and finally that it acts on the fundamental pathological mechanisms rather than just the symptoms.

[0013] Also for illustrative and non-exhaustive purposes, unlike AChE inhibition, calpain inhibition does not cause cardiovascular side effects. On the contrary, calpain inhibition even causes beneficial effects on the latter (Rioux et al., Acetylcholinsterase inhibitors. Continuing education. Le médecin du Québéc. 2002;37(4):89-94, Potz et al., Calpains and coronary vascular diseases, Circ J. 2016;80:4-10).

[0014] Consequently, it would be interesting to find new products that can inhibit calpain activity in order to treat or prevent several neurodegenerative diseases such as Alzheimer's and Parkinson's, by acting upstream of several reaction cascades and avoiding side effects, particularly on the cardiovascular level, and even having a positive effect on the latter.

[0015] The inventors surprisingly discovered that a particular hydrolyzed extract of deoiled hemp seed cake, and more particularly two of its peptide fractions, advantageously of higher molecular masses than those described in the prior art, had a strong power to inhibit the activity of calpain, in particular of the mixture of isoforms 1 and 2. In addition, this extract also has a strong antioxidant, anti-inflammatory and tyrosinase inhibitory activity, without exhibiting any cytotoxicity.

[0016] Hemp belongs to the order Rosales, the family Cannabaceae, the genus Cannabis, and the species Cannabis sativa. Its botanical classification gives it its unique properties, and in particular those described in the context of the present invention. Hemp seed cake is a by-product obtained after oil extraction that does not contain cannabinoid compounds. It is therefore of little interest at present and is mainly used for animal feed.

[0017] It is known from the article by Girgih et al. (J. Am. Oil Chem Soc (2011) 88:381-389) that some peptide fractions of hemp seed meal protein hydrolysate obtained by enzymatic hydrolysis have antioxidant activity. The peptide fractions of the hydrolysate obtained thus have a molecular mass between <1 kDa and 5–10 kDa. However, this document teaches that it is the smallest peptide fractions that have the best activity and that the unfractionated hydrolysate has only a weak antioxidant activity. In addition, the enzymatic hydrolysis is an acid hydrolysis carried out at pH 2 with a first enzyme (pepsin), then at pH 7.5 with a second enzyme (pancreatin), and finally an overall cooking at 95°C at pH 4 for 15 minutes. This method does not allow the dissociation of the enzymatic elements (themselves composed of peptides) from the hemp peptides.Thus the activities described in this article are attributable not to the peptide fractions of the hydrolysate but to a hydrolytic complex combining hemp protein with protein enzymes.

[0018] Similarly, the article by Santos-Sanchez et al. (Trends in Food Science & Technology, Hempseed (Cannabis sativa) protein hydrolysates: A valuable source of bioactive peptides with pleiotropic health-promoting effects, 127 (2022) 303-318) discusses the extraction of hemp with the aim of obtaining low molecular weight peptides, namely peptides with a maximum weight of 1291.58 Da according to table 7 page 315. This document certainly describes that these peptides have a neuroprotective effect but attributes these effects either to their antioxidant activity or to their AChE inhibition activity. Furthermore, this document emphasizes the fact that the length of the peptide is responsible for these effects and that low molecular weight peptides have the highest inhibitory effect.

[0019] Furthermore, no prior art describes or suggests using larger peptide fractions. On the contrary, similar to the article by Girgih et al., it is generally recommended to use smaller fractions for better activity. However, the inventors surprisingly discovered that larger peptide fractions had an impact on calpain inhibition.

[0020] Finally, the article by Rodriguez-Martin et al. (Food Funct, 2019, 10, 6732-6739) suggests that some hemp seed meal protein hydrolysates obtained by enzymatic hydrolysis have neuroprotective activity by improving the neuroinflammatory state of LPS-stimulated BV-2 microglial cells through downregulation of the TLR-4-mediated NF-κB pathway. However, this document also teaches that it is the presence of low molecular weight peptides (between 300–400 Da and 1.45 kDa) in these hydrolysates that allows this activity to be obtained. In addition, the enzymatic hydrolysis is a basic hydrolysis carried out at pH 8 with a first enzyme (alcalase), then at pH 7 with a second enzyme (flavourzyme), and finally an overall cooking at 85°C at pH 7 for 15 minutes. This method does not allow the dissociation of the enzymatic elements (themselves composed of peptides) from the hemp peptides.Thus, the activities described in this article are attributable not to the peptide fractions of the hydrolyzate but to a hydrolytic complex combining hemp protein with protein enzymes. Furthermore, the tests carried out are not sufficient to prove a real neuroprotective activity because they do not correspond to one. direct action pathway on a metabolic pathway involved in the neurodegenerative process itself. Indeed, microglial cells belong to the brain's immune system and are related to macrophages. Modulating the inflammatory state of these cells therefore acts indirectly on brain stress, which itself can lead to the degeneration of neurons; it is therefore very clearly an indirect effect with respect to the neuroprotective action. Description of the invention

[0021] The present invention therefore relates to a hydrolyzed extract of deoiled hemp seed cake obtained by alkaline hydrolysis at a pH greater than or equal to 11, advantageously greater than or equal to 11.5, in particular greater than or equal to 12.

[0022] In this application, the expressions “between ... and ...” and “from ... to ...” and “in the range ... - ...”, must be understood to include limits unless explicitly stated otherwise.

[0023] The term "hemp seed cake" means the co-product obtained after extraction of the oil from hemp seeds, in particular from Cannabis sativa L. The extraction of the oil from cannabis seeds is carried out by techniques known to those skilled in the art (such as cold pressing) which makes it possible to isolate the oily fraction of the compound to be extracted in order to use it for various applications. The residue from this extraction is called "co-product" within the meaning of the present invention and contains all the compounds not extracted by said technique. By co-product, we therefore mean the residue obtained after extraction of the oil. Thus, the cake has a lower fat content than that of the hemp seed.Advantageously, the cake according to the invention comprises less than 20% by mass of fat, advantageously less than 15% by mass of fat, even more advantageously less than 12% by mass of fat, in particular between 9% and 12% by mass of fat, more particularly 10.5% by mass of fat, relative to the total mass of the cake.

[0024] The hemp seed cake according to the invention comprises proteins, advantageously at least 20% by mass of proteins, more advantageously at least 25% by mass of proteins, even more advantageously at least 27% by mass of proteins, in particular between 27 and 32% by mass of proteins, more particularly 30.70% by mass of proteins, relative to the total mass of the cake.

[0025] The hemp seed cake according to the invention may further comprise fibers, in particular cellulose, advantageously at least 15% by mass of fibers, more advantageously at least 20% by mass of fibers, even more advantageously at least 21% by mass of fibers, in particular between 21% and 28% by mass of fibers, more particularly 21.7% by mass of fibers, advantageously cellulose, relative to the total mass of the cake.

[0026] The hemp seed cake according to the invention may also contain sugars and mineral salts such as iron, calcium, and potassium. Advantageously, it is commercially available from Hemp Acres LLC or Ecoprod.

[0027] Particularly advantageously, the hemp seed cake according to the invention does not contain cannabinoids, in particular chosen from the group consisting of tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), cannabicyclol (CBL), cannabivarol (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol (CBGM) and mixtures thereof.

[0028] In an advantageous embodiment, before being hydrolyzed within the framework of the present invention, the hemp seed cake according to the invention is extracted with ethanol or with supercritical carbon dioxide (CO2), preferably with ethanol.

[0029] Extraction with ethanol or supercritical CO2 can effectively defat a substance such as hemp seed cake. Compared to other solvents such as chloroform and methanol, only or in mixture, extraction in the presence of ethanol or with supercritical CO2 allows better delipidation, in particular by elimination of polar lipids.

[0030] Extraction with ethanol is more interesting than with supercritical CO2 because it is easier to implement.

[0031] The extraction with ethanol according to the invention can be carried out by different extraction methods known to those skilled in the art, such as maceration, with or without stirring, or hot decoction.

[0032] Advantageously, the extraction with ethanol is carried out at at least 90 degrees, more advantageously at least 96 degrees, so as to obtain an ethanolic extract of hemp seed cake and a de-oiled residue.

[0033] Advantageously, the extraction with supercritical CO2 is carried out at a pressure between 10 MPa (100 bars) and 25 MPa (250 bars), so as to obtain a supercritical CO2 extract of hemp seed cake and a de-oiled residue.

[0034] The term "extraction with supercritical carbon dioxide (CO2)" means an extraction in the presence of CO2 under supercritical conditions. Extraction with supercritical carbon dioxide is based on the principle of using supercritical carbon dioxide, the properties of which are similar to those of a liquid solvent, as a solvent for the extraction. A person skilled in the art knows how carbon dioxide can be brought to the supercritical state. In particular, the temperature and pressure must be set simultaneously to appropriate values. The temperature is preferably above 40°C at a pressure of more than 100 bar (10 MPa), preferably above 45°C at more than 250 bar (25 MPa), preferably between 45 and 65°C at a pressure of 250 to 350 bar (25 to 35 MPa). The extraction carried out with supercritical CO2 makes it possible to defat the hemp seed cake.

[0035] The deoiled residue is also referred to as "deoiled hemp seed meal" or "defatted hemp seed meal" within the scope of this specification.

[0036] The term "delipidation" or "deoiling" means the elimination of lipids or their derivatives from a substance, at least partially, at most completely, preferably completely. For example, the delipidation of hemp seed cake corresponds to the elimination, at least partially, at most completely, preferably completely, of lipids or their derivatives from the cake. Thus, "better delipidation" means greater elimination of lipids.

[0037] In an advantageous embodiment, the extraction is carried out by maceration, in particular with stirring. The extraction can be carried out for a period of 30 minutes to 24 hours, preferably 30 minutes to 12 hours. Advantageously, the extraction can be carried out for a period of more than 1 hour, preferably greater than or equal to 1 hour 15 minutes, even more preferably greater than or equal to 1 hour 30 minutes, even more preferably greater than or equal to 1 hour 45 minutes, even more preferably greater than or equal to 2 hours. Advantageously, the extraction can be carried out for a period of 1 hour to 3 hours, preferably between 1 hour 15 minutes and 3 hours, more preferably between 1 hour 30 minutes and 3 hours, even more preferably between 1 hour 45 minutes and 3 hours, even more preferably between 2 hours and 3 hours, and even more advantageously for a period of 2 hours.The extraction may be carried out at a temperature between 15 and 35°C, advantageously between 20°C and 30°C, more advantageously at 25°C. The extraction may be carried out from a quantity of 0.1% to 30% by mass of fresh or dry matter, preferably dry, advantageously from 1% to 28%, still advantageously from 10% to 26%, very advantageously from a quantity of 25% by mass of dry matter, of the hemp seed cake, relative to the total mass of the hemp seed cake and the solvent.

[0038] In a particularly advantageous embodiment of the invention, the deoiled hemp seed cake according to the invention is obtained by ethanolic extraction as follows: 25% by mass of hemp seed cake (relative to the total mass of the cake + 96 degree ethanol) is macerated with stirring in 96 degree ethanol for a period 2 hours at a temperature of 25°C, in particular under the conditions as described in Example 1.

[0039] Advantageously, the deoiled hemp seed cake according to the invention comprises less than 11% by mass of fat, advantageously less than 10% by mass of fat, even more advantageously less than 9% by mass of fat, in particular between 6 and 9% by mass of fat, more particularly 6.8% by mass of fat, relative to the total mass of the cake.

[0040] The hydrolyzed extract of deoiled hemp seed cake according to the invention, also called deoiled hemp seed cake hydrolysate in the context of the present description, is therefore obtained by alkaline hydrolysis of the deoiled hemp seed cake according to the invention at a pH greater than or equal to 11, advantageously greater than or equal to 11.5, more advantageously greater than or equal to 12, advantageously using a base chosen from sodium hydroxide, magnesium hydroxide, calcium hydroxide and potassium hydroxide, more particularly sodium hydroxide, in particular 1M. Advantageously, the duration of the hydrolysis is greater than 1 hour, preferably greater than or equal to 1 hour 15 minutes, preferably still greater than or equal to 1 hour 30 minutes, even more preferably greater than or equal to 1 hour 45 minutes, even more preferably still greater than or equal to 2 hours.Advantageously, the duration of the hydrolysis is between 1 hour and 3 hours, preferably between 1 hour 15 minutes and 3 hours, more preferably between 1 hour 30 minutes and 3 hours, even more preferably between 1 hour 45 minutes and 3 hours, even more preferably between 2 hours and 3 hours, more particularly it is 2 hours. The alkaline hydrolysis is advantageously carried out in water as the sole solvent. In an advantageous embodiment, the hydrolysis is carried out at a temperature between 15°C and 35°C, advantageously between 20°C and 30°C, more advantageously at 25°C.

[0041] Alkaline hydrolysis is stopped by neutralization using an acid, in particular a weak acid such as citric acid, so as to obtain a pH lower than 7, in particular between 4 and 5.

[0042] Advantageously, a preservative such as sodium benzoate or potassium sorbate is then added to the hydrolyzate. This preservative prevents microbial contamination or fermentation of the resulting hydrolyzate and therefore stabilizes it over the long term.

[0043] The hydrolyzate obtained can then be filtered, in particular through a 10 µm filter.

[0044] Advantageously, the proteins are not previously extracted from the cake before the hydrolysis step.

[0045] The quantity by mass of deoiled hemp seed cake used for carrying out alkaline hydrolysis is between 1% and 20%, advantageously between 5% and 10%, still advantageously between 8% and 10%, by mass relative to the total mass of the solvent and the cake.

[0046] In a particularly advantageous embodiment of the invention, the hydrolysate is obtained by alkaline hydrolysis as follows: 9% by mass of deoiled hemp seed cake according to the invention relative to the total mass of cake and water are subjected to alkaline hydrolysis for a period of 2 hours with stirring at a temperature of 25°C, at pH 12, in the presence of 1M sodium hydroxide. The solution is then neutralized so as to stop the hydrolysis by adding citric acid monohydrate in an amount necessary to obtain a pH of 4.8. Sodium benzoate is then added, the mixture is left to settle for 12 hours and then filtered (with a cut-off threshold of 10 µm), under the conditions as described in Example 1.

[0047] The alkaline hydrolysis according to the invention makes it possible to obtain peptide fractions without residue. Indeed, unlike other hydrolyses such as enzymatic hydrolysis, alkaline hydrolysis does not result in peptides from the enzymes used for hydrolysis. Thus, the data analyzed in the presence of peptide fractions obtained by alkaline hydrolysis can be directly attributed to the peptide fractions from the extract studied, and not from other elements such as the enzymes used for hydrolysis.

[0048] Thus, in an advantageous embodiment, the hydrolyzed extract according to the invention is obtained by the process comprising the following steps: a- defatting the hemp seed cake by extraction with ethanol or with supercritical carbon dioxide (CO2), advantageously with ethanol, more advantageously with ethanol at least 96 degrees; b- alkaline hydrolysis at a pH greater than or equal to 11, advantageously greater than or equal to 11.5, in particular greater than or equal to 12, of the deoiled cake obtained in step a), advantageously using a base chosen from sodium hydroxide, magnesium hydroxide, calcium hydroxide and potassium hydroxide.

[0049] Steps a) and b) therefore correspond to extraction with ethanol or with supercritical CO2 and to hydrolysis as described above.

[0050] The hydrolyzed extract according to the invention advantageously comprises: - a peptide fraction with a molecular mass of between 9 and 12 kDa, advantageously between 10 and 12 kDa, more particularly between 10.5 and 11.5 kDa, in particular 11 kDa, and / or, advantageously and, - a peptide fraction with a molecular mass of between 16 and 18 kDa, advantageously between 16.5 and 17.5 kDa, in particular 17 kDa, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE, a method well known to those skilled in the art. In particular, the gels used in this method are composed of acrylamide-bisacrylamide. More particularly, the concentration gel contains 4.5% by mass of acrylamide-bisacrylamide relative to the total mass of the concentration gel and the separation gel contains 15% by mass of acrylamide-bisacrylamide relative to the total mass of the separation gel.Even more particularly, the gels have the following composition: - Concentrating gel (Tris-HCl 125mM, pH 6.8, SDS (Sodium Dodecyl Sulfate) 0.1%, acrylamide-bisacrylamide 4.5%, ammonium persulfate 0.1%, TEMED (N,N,N',N'-Tetramethylethylenediamine) 0.025%) and - Separating gel (Tris-HCl 375mM, pH 8.8, SDS 0.1%, acrylamide-bisacrylamide 15%, ammonium persulfate 0.01%, TEMED 0.001%).

[0051] Advantageously, the proteins are diluted in Laemmli buffer (Tris-HCl 50mM pH6.8, SDS 2%, bromophenol blue 0.1%, glycerol 10%, 2-mercapoethanol 3%) and the reservoir solution has the following composition: 25mM Tris-HCl, 0.25M glycine, pH 8.3, SDS 0.1%. In particular, the migration is carried out at room temperature at a voltage of 40V for 1h then 2h at 100V.

[0052] Advantageously, the hydrolyzed extract according to the invention also has a dry matter content of between 10.5 and 15% by mass, more advantageously between 12 and 14% by mass, even more advantageously between 12.5 and 13% by mass, relative to the total mass of the hydrolyzed extract.

[0053] Advantageously, the hydrolyzed extract according to the invention also has a total protein content of between 0.8 and 2.4% by mass, more advantageously between 1 and 2%, even more advantageously between 1.2 and 1.6% by mass, relative to the total mass of the hydrolyzed extract.

[0054] The hydrolyzed extract according to the invention, in particular as prepared in example 1), is thus in liquid form. Optionally, the hydrolyzed extract can then be dried, for example by lyophilization or by atomization, alone or in the presence of an excipient. The hydrolyzed extract is then in powder form.

[0055] The present invention further relates to the peptide fraction (or peptide or oligopeptide) of the hydrolyzed extract according to the invention having a molecular mass, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE, of between 9 and 12 kDa, advantageously between 10 and 12 kDa, more particularly between 10.5 and 11.5 kDa, in particular 11 kDa.

[0056] This fraction can be obtained by purification of the hydrolyzed extract according to the invention by methods well known to those skilled in the art. In particular, it can be centrifugation followed by ultrafiltration with membranes with a cut-off threshold for peptides with a molecular mass between 9 and 12 kDa.

[0057] The present invention further relates to the peptide fraction (or peptide or oligopeptide) of the hydrolyzed extract according to the invention having a molecular mass, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE, in particular as described above, between 16 and 18 kDa, advantageously between 16.5 and 17.5 kDa, in particular 17 KDa.

[0058] This fraction can be obtained by purification of the hydrolyzed extract according to the invention by methods well known to those skilled in the art. In particular, it can be centrifugation followed by ultrafiltration with membranes with a cut-off threshold for peptides with a molecular mass between 16 and 18 kDa.

[0059] The present invention further relates to the combination of the hydrolyzed extract according to the present invention or of one or more of its peptide fractions according to the present invention and of an ethanolic extract or supercritical CO2 extract of hemp seed cake, advantageously obtained in step a) of the process according to the present invention.

[0060] The ethanolic extract or supercritical CO2 extract of hemp seed cake according to the invention is a lipophilic extract. Advantageously, it contains polar lipids.

[0061] In particular, it comprises phospholipids which may be present in a content of between 8 and 10% by mass, relative to the total mass of the dry extract without solvent, advantageously in a content of 9.38% by mass, relative to the total mass of the dry extract without solvent.Among these phospholipids, we can find phosphatidylserine (advantageously in a content of between 0.8 and 1% by mass relative to the total mass of the extract), phosphatidylinositol (advantageously in a content of between 1.9 and 2.2% by mass relative to the total mass of the extract), phosphatidylglycerol (advantageously in a content of between 0.4 and 0.5% by mass relative to the total mass of the extract), phosphatidylethanolamine (advantageously in a content of between 0.8 and 1% by mass relative to the total mass of the extract), phosphatidylcholine (advantageously in a content of between 3.5 and 4% by mass relative to the total mass of the extract) and phosphatidate. (advantageously in a content of between 1% and 1.2% by mass relative to the total mass of the extract).

[0062] Advantageously, the dry matter (without solvent) of the ethanolic extract or supercritical CO2 extract according to the invention comprises ceramides, in particular in a content of between 0.1 and 0.5% by mass, more particularly 0.33% by mass, relative to the total mass of the dry extract without solvent.

[0063] Advantageously, the dry matter (without solvent) of the ethanolic extract or supercritical CO2 extract according to the invention comprises cardiolipins, in particular in a content of between 0.01% and 0.07% by mass, more particularly 0.04% by mass, relative to the total mass of the extract.

[0064] Advantageously, the dry matter (without solvent) of the ethanolic extract or supercritical CO2 extract according to the invention comprises diacylglycerol, in particular in a content of between 7% and 13% by mass, more particularly 10.82% by mass, relative to the total mass of the extract.

[0065] Advantageously, the dry matter (without solvent) of the ethanolic extract or supercritical CO2 extract according to the invention comprises triglycerides, in particular in a content of between 75% and 82% by mass, more particularly 79.41% by mass, relative to the total mass of the extract.

[0066] The dry matter (without solvent) of the extract according to the invention can therefore be obtained by extraction with ethanol or with supercritical CO2 of the hemp seed cake by the process as described above in the context of the recovery of the deoiled hemp seed cake, except that this time, it is the ethanolic extract or supercritical CO2 extract which is recovered, and not the deoiled residue. The ethanolic extract or supercritical CO2 extract can be used as is in the composition according to the invention or it can be concentrated to increase the dry matter by evaporation of the solvent, in particular by evaporation of the solvent at a temperature for example between 45°C and 48°C. The ethanolic extract or supercritical CO2 extract in liquid form according to the invention has a dry matter content of between 0.5% and 3% by mass, advantageously between 1% and 2% by mass. mass, relative to the total mass of the extract. The concentrated ethanolic extract or supercritical CO2 extract according to the invention has a dry matter content of between 65% and 75% by mass, advantageously between 67% and 72% by mass, relative to the total mass of the extract.

[0067] The present invention further relates to the association of the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention, in particular in liquid form, with a compound chosen from: - ginseng, advantageously black ginseng, more advantageously ginseng root, - harpagophytum, advantageously harpagophytum root , - turmeric, advantageously turmeric root, - black pepper, advantageously black pepper berries, more advantageously in the form of dried fruit, - zinc gluconate, - Ginkgo biloba, and - a mixture of two or more of these compounds.

[0068] Advantageously, the association comprises 80 to 98%, particularly 85% to 95%, more particularly 90%, by mass of hydrolyzed extract or one or more of its peptide fractions, and 2 to 20%, particularly 5 to 15%, more particularly 10%, by mass of this or these compound(s), relative to the total mass of the association.

[0069] Ginseng is a perennial plant of the Araliaceae family and the genus Panax. Examples of ginseng include the species Panax ginseng, Panax quinquefolius, Panax japonicus, Panax vietnamensis, and Panax pseudoginseng. Ginseng can be found in raw form or in extract form, particularly in raw form, especially in powder form.

[0070] The term "harpagophytum" ​​refers to a plant of the Pedaliaceae family and of the genus harpagophytum, and of the genus Panax. The term "harpagophytum" ​​includes, for example, the species Harpagophytum procumbens, or Harpagophytum zeyheri. Harpagophytum can be found in raw form or in the form extract, especially in raw form, more particularly in powder form.

[0071] The term "turmeric" refers to a perennial rhizomatous herbaceous plant belonging to the Zingibeaceae family and the genus Curcuma. The term "turmeric" includes, among others, the species Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma caesia, and Curcuma amada. Turmeric acts, among other things, as an anti-inflammatory, antioxidant, anti-cancer, bile secretor, appetite stimulant, and acts on the reduction of cholesterol levels. Turmeric can be found in raw form or in extract form, particularly in raw form, more particularly in powder form.

[0072] Black pepper refers to a plant belonging to the Piperaceae family, genus Piper, and species Piper nigrum. Black pepper can be found in raw or extracted form, particularly in raw form, especially in powder form.

[0073] The term "zinc gluconate" refers to the molecule with formula C12 H 22 O 14 Zn. Zinc gluconate can be of natural origin or produced by a technical process such as glucose fermentation or electrolytic oxidation. Zinc gluconate acts among other things as an anti-inflammatory, in particular as an anti-inflammatory in the treatment and / or prevention of acne, in particular to reduce acne symptoms such as burning, itching and / or redness. Zinc gluconate is advantageously found in powder form.

[0074] Ginkgo biloba is a species of plant in the gymnosperm phylum, order Ginkgoales, and family Ginkgoaceae.

[0075] In an advantageous embodiment, the association according to the invention consists of: - the association of the hydrolyzed extract according to the invention or one or more of its peptide fractions and ginseng as described above, or - the association of the hydrolyzed extract according to the invention or one or more of its peptide fractions and harpagophytum as described above, or - the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and black pepper and / or turmeric as described above, in particular the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions, black pepper and turmeric, said combination advantageously comprising 88 to 92%, particularly 90%, by weight of hydrolyzed extract or one or more of its peptide fractions, 1 to 3%, particularly 2%, by weight of black pepper and 7 to 9%, particularly 8%, by weight of turmeric, relative to the total mass of the combination, or - the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and zinc gluconate, or - the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and harpagophytum and / or black pepper and / or turmeric as described above,in particular harpagophytum, black pepper and turmeric, or - the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and zinc gluconate and / or black pepper and / or turmeric as described above, in particular zinc gluconate, black pepper and turmeric.

[0076] The present invention further relates to a macerate of the association according to the invention, advantageously the hydrolyzed extract or one or more of its peptide fractions is the solvent of the maceration.

[0077] More particularly, the association according to the invention of the hydrolyzed extract according to the invention or of one or more of its peptide fractions with one or more of these compounds has undergone maceration, so as to obtain a macerate. Indeed, the hydrolyzed extract or one or more of its peptide fractions can serve as a maceration solvent for said compound(s). Advantageously, the maceration is carried out at ambient temperature and pressure, with or without stirring, preferably with stirring, even more preferably with light stirring, over a period of 12 hours to 30 hours, preferably from 18 hours to 28 hours, even more preferably from 22 hours to 26 hours, in particular 24 hours. More particularly, the hydrolyzed extract according to the invention or one or more of its peptide fractions is in liquid form and serves as a solvent for this maceration.

[0078] In an advantageous embodiment, the macerate has undergone filtration, so as to obtain a liquid filtrate, the solid phase being eliminated. Advantageously, the filtration involves the use of a filter having a cut-off threshold of between 9 and 11 µm, preferably 10 µm.

[0079] In a further advantageous embodiment, the macerate or macerates according to the invention, in particular filtered, is (are) mixed with the hydrolyzed extract according to the invention or one or more of its peptide fractions, in particular with a content of macerate(s) in the range 3 - 15%, particularly 5 - 15%, in particular 10%, by mass relative to the total mass of the mixture.

[0080] The present invention further relates to a mixture of the macerate according to the invention with a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention.

[0081] In an advantageous embodiment, the mixture according to the invention thus comprises the hydrolyzed extract according to the invention or one or more of its peptide fractions, in particular in liquid form and: - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and ginseng as described above, or - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and harpagophytum as described above, or - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions, black pepper and turmeric as described above, or - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and zinc gluconate,or - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and harpagophytum as described above, and the macerate of the combination of the hydrolyzed extract according to the invention or, one or more of its peptide fractions, black pepper and turmeric as described above, or - the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions and zinc gluconate, and the macerate of the combination of the hydrolyzed extract according to the invention or one or more of its peptide fractions, black pepper and turmeric as described above.

[0082] In an advantageous embodiment, the mixture according to the invention may further comprise: - sorbitol, in particular in a content of between 5 and 20%, in particular between 9 and 15%, by weight relative to the total mass of the mixture, and / or - glycerin, in particular in a content of between 2 and 9%, in particular 6%, by weight relative to the total weight of the mixture, and / or - guar gum, in particular in a content of between 0.1% and 0.5%, in particular 0.25%, by weight relative to the total mass of the mixture, and / or - xanthan gum, in particular in a content of between 0.1% and 0.5%, in particular 0.25%, by weight relative to the total mass of the mixture.

[0083] In an advantageous embodiment of the invention, the mixture may be in the form of a syrup or a gel.

[0084] Advantageously, in the case where the mixture according to the invention has the form of a syrup, it comprises the macerate(s) according to the invention, the hydrolyzed extract according to the invention or one or more of its peptide fractions in liquid form and sorbitol, in particular in the contents indicated above.

[0085] Advantageously, in the case where the mixture according to the invention has the form of a gel, it comprises the macerate(s) according to the invention, the hydrolyzed extract according to the invention or one or more of its peptide fractions in liquid form, sorbitol, glycerin, xanthan gum and guar gum, in particular in the contents indicated above.

[0086] In an advantageous embodiment of the invention, the mixture according to the invention in syrup form can be administered in an amount ranging from 1 mL to 10 mL per day, preferably 1.5 mL to 8 mL per day, still preferably 2 mL to 5 mL per day or 5 mL to 6 mL per day, for example 1 to 2 teaspoons per day or for example 2 teaspoons in the morning.

[0087] In an advantageous embodiment of the invention, the mixture according to the invention in gel form can be administered in an amount ranging from 3 to 7 g per day, preferably 5 g per day.

[0088] In one embodiment of the invention, the association, macerate or mixture according to the invention, in particular in the form of gel or syrup, is administered to a human, preferably once or twice a day.

[0089] Advantageously, the hydrolyzed extract according to the invention or one or more of its peptide fractions, the association, the macerate or the mixture according to the invention, in particular comprising ginseng, makes it possible to treat and / or prevent veisalgia.

[0090] Advantageously, the hydrolyzed extract according to the invention or one or more of its peptide fractions, the association, the macerate or the mixture according to the invention, in particular comprising harpagophytum, black pepper and turmeric, makes it possible to reduce and / or prevent joint pain.

[0091] Advantageously, the hydrolyzed extract according to the invention or one or more of its peptide fractions, the association, the macerate or the mixture according to the invention, in particular comprising zinc gluconate, black pepper and turmeric, makes it possible to treat and / or prevent acne, in particular to reduce the symptoms of acne such as burning, itching and / or redness.

[0092] The present invention further relates to a pharmaceutical composition comprising the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the association or the macerate or the mixture according to the invention and a pharmaceutically acceptable excipient.

[0093] In the present invention, the term "pharmaceutically acceptable" is intended to mean that which is useful in the preparation of a composition pharmaceutical that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and that is acceptable for veterinary as well as human pharmaceutical use.

[0094] Thus, these pharmaceutical compositions contain an effective dose of the hydrolyzed extract according to the invention or of one or more of its peptide fractions according to the invention or of the combination according to the invention or the macerate according to the invention (as active ingredient), and one or more acceptable pharmaceutical excipients. These compositions can be formulated for administration to mammals, including humans. The dosage varies according to the treatment and the condition in question.

[0095] The said excipients are chosen according to the pharmaceutical form and the desired method of administration.

[0096] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, intranasal, transdermal, local or rectal administration, the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention may be administered in unit administration form, in admixture with conventional pharmaceutical excipients, to animals and humans. Suitable unit administration forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual, buccal, intratracheal, intranasal or intraocular administration forms, subcutaneous, intramuscular or intravenous administration forms and rectal administration forms.For topical application, the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention can be used in creams, ointments or lotions.

[0097] According to usual practice, the appropriate dosage for each patient is determined by the physician according to the method of administration, the mass and the response of the said patient.

[0098] When preparing a solid composition in tablet form, the principal active ingredient is mixed with a pharmaceutical excipient, such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets may be coated with sucrose, a cellulose derivative, or other suitable materials or may be treated so that they have prolonged or delayed activity and continuously release a predetermined amount of active ingredient. The tablets may be made by various techniques, direct compression, dry granulation, wet granulation or hot melting.

[0099] A capsule preparation is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard capsules.

[0100] A preparation in the form of a syrup or elixir, in particular a syrup, may contain the active ingredient together with a sweetener, in particular sorbitol, an antiseptic, as well as a flavoring agent and a suitable coloring agent. Advantageously, it is a syrup comprising sorbitol.

[0101] In one embodiment according to the invention, the preparation in the form of syrup or elixir is administered from 1 to 10 mL per day, preferably from 1.5 to 8 mL per day, more preferably from 2 to 5 mL per day or from 5 to 6 mL per day.

[0102] The term “syrup” or “elixir” means a liquid galenic form, in particular intended for oral administration.

[0103] A gel preparation may contain the active ingredient together with a gelling agent, in particular with xanthan gum, guar gum and / or glycerin, more particularly with a mixture of xanthan gum, guar gum and glycerin.

[0104] In one embodiment according to the invention, the preparation in gel form is administered from 1 to 10 grams per day, preferably from 2 to 8 grams per day, more preferably from 4 to 6 grams per day, even more preferably from 5 grams per day.

[0105] A "gel" is a pharmaceutical form gelled using a lipophilic or hydrophilic gelling agent. Gelling agents may, for example, be derived from plant extracts such as pectins, alginates, starch, agar-agar or carrageenans, or from animal bone extracts such as pectin. They may also be xanthan gum, guar gum or glycerin. The gel is particularly intended for oral administration.

[0106] Water-dispersible powders or granules may contain the active ingredient in admixture with dispersing or wetting agents, or suspending agents, as well as with flavor correctors or sweeteners.

[0107] For intranasal or intraocular parenteral administration, aqueous suspensions, isotonic saline solutions or sterile injectable solutions containing pharmacologically compatible dispersing and / or wetting agents, e.g., propylene glycol or butylene glycol, may be used.

[0108] For rectal administration, suppositories are used, which are prepared with binders that melt at rectal temperature, for example cocoa butter or polyethylene glycols.

[0109] The active ingredient can also be formulated in the form of microcapsules, possibly with one or more additive carriers.

[0110] The present invention further relates to the process for manufacturing the hydrolyzed extract according to the invention, characterized in that it comprises the following steps: a- defatting the hemp seed cake by extraction with ethanol or with supercritical carbon dioxide (CO2), advantageously with ethanol, more advantageously with ethanol at least 96 degrees, b- alkaline hydrolysis at a pH greater than or equal to 11, in particular greater than or equal to 11.5, more particularly greater than or equal to 12, of the deoiled cake obtained in step a), advantageously using a base chosen from sodium hydroxide, magnesium hydroxide, calcium hydroxide and potassium hydroxide.

[0111] Steps a) and b) are as described previously with regard to obtaining the deoiled hemp seed cake (step a) and alkaline hydrolysis (step b).

[0112] The present invention also relates to the use of a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention, as a food supplement.

[0113] The present invention finally relates to a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the association according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention for its use as a medicament.

[0114] Advantageously, the pharmaceutical composition according to the invention comprises a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention and a compound chosen from ginseng, harpagophytum, turmeric, black pepper, zinc gluconate, Ginkgo biloba and a mixture of one or more of these compounds and optionally sorbitol and / or glycerin and / or xanthan gum and / or guar gum.

[0115] Advantageously, the present invention relates to a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention for its use as an antioxidant and / or depigmenting agent and / or non-steroidal anti-inflammatory agent and / or anti-neurodegenerative agent and / or neuroprotective agent and / or anticancer agent and / or healing agent and / or antifibrotic agent and / or for the treatment and / or prevention of veisalgia (hangover) and / or for the reduction and / or prevention of joint pain and / or for the treatment and / or prevention of acne, in particular to reduce the symptoms of acne such as burns, itching and / or redness, more advantageously for the treatment or prevention of: - diseases associated with calpain modulation such as ischemic injury, muscular dystrophy, diabetes, diabetic endotheliopathy, cataract, atherosclerosis, autophagy dysregulation, neuropathy induced by repeated concussion, neurodegenerative diseases (e.g. Alzheimer's disease or Parkinson's disease), inflammatory diseases such as asthma, atherosclerosis, rheumatoid arthritis and multiple sclerosis, fibrotic diseases, including cardiac fibrosis, idiopathic pulmonary fibrosis and hypertrophic scarring, especially after burns, and cancer, especially metastatic cancer;- diseases associated with modulation of the phospholipase A2 enzyme and / or modulation of the lipoxygenase enzyme, such as inflammatory diseases, bronchopulmonary and ENT diseases (asthma, laryngitis, sinusitis, COPD, etc.), rheumatological and neurological diseases (rheumatoid arthritis, Horton's disease, multiple sclerosis, etc.), gastrointestinal diseases (Crohn's disease, ulcerative colitis), dermatological diseases (urticaria, eczema) and kidney diseases;- oxidation-related diseases such as beta-oxidation cycle disorders that may lead to the development of HELLP syndrome or glutaric acidemia type II, cardiomyopathies and muscle impairment. - diseases associated with tyrosinase modulation such as hyperpigmentation of the skin (lentigo) and retina - veisalgia (hangover) - joint pain and / or - acne and / or acne symptoms such as burning, itching and / or redness. ;

[0116] More advantageously, the present invention relates to a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the association according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention. for use in the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease and / or for the reduction and / or prevention of joint pain and / or for the treatment and / or prevention of veisalgia and / or for the treatment and / or prevention of acne, in particular for reducing the symptoms of acne such as burning, itching and / or redness.

[0117] Even more advantageously, the present invention relates to a hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention for its use in the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, without inhibition of acetylcholinesterase, and with inhibition of calpain.

[0118] Calpain inhibition results in a long-term therapeutic effect, with action on several target mechanisms, a direct action on pathological mechanisms, and limited side effects (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630, Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27;8(1):18083, Trinchese et al., Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer's disease. J Clin Invest. 2008; 118(8):2796-807). Among other things, calpain inhibition does not cause cardiovascular side effects, particularly vagotonic effects, and even has beneficial effects on the cardiovascular system (Potz et al., Calpains and coronary vascular diseases, Circ J. 2016;80:4-10).

[0119] Advantageously, the product according to the invention, in particular by the size (or molecular mass) characteristic of its peptides, has an inhibitory action on the active site of calpain. The product according to the invention also makes it possible to limit or even eliminate any side effects. Advantageously, the product according to the invention has no action on cathepsins.

[0120] In an advantageous embodiment of the invention, the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention makes it possible to treat or prevent veisalgia, in particular headaches, nausea and / or fatigue.

[0121] A hangover or veisalgia refers to symptoms caused by alcohol poisoning. These symptoms include headaches, nausea, and / or fatigue. Alcohol poisoning refers to the amount of alcohol consumed that is necessary to induce these symptoms.

[0122] In an advantageous embodiment according to the invention, the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention makes it possible to reduce or prevent joint pain.

[0123] In an advantageous embodiment according to the invention, the hydrolyzed extract according to the invention or one or more of its peptide fractions according to the invention or the combination according to the invention or the macerate according to the invention or the mixture according to the invention or the pharmaceutical composition according to the invention reduces or prevents acne, in particular reduces the symptoms of acne such as burning, itching and redness.

[0124] The present invention will be better understood by reading the following examples which are given for non-limiting information purposes. EXAMPLES Example 1: preparation of the hydrolyzed extract of deoiled hemp seed cake according to the invention

[0125] Step a): Defatting of hemp seed cake by extraction with ethanol: The hemp seed cake used for extraction is marketed by the company EcoProd under the name “organic hemp cake”. It contains 30.70% by mass of crude protein, 21.70% by mass of crude fiber and 10.50% by mass of crude fat. It is the residue obtained after cold pressing of hemp seeds.

[0126] Step a) consists of grinding and maceration with stirring at room temperature between 20 and 25°C in a mixer with a 4-blade propeller for 2 hours of 75 g of this hemp seed cake in 225 g of ethanol at 96 degrees then filtration on a pleated filter (cut-off threshold: 10 µm) so as to recover on one side the filtrate (167.5 g with a dry mass of 1.67% by mass) which corresponds to the ethanolic extract and on the other the solid residue (71.3 g) which corresponds to the deoiled hemp seed cake. This step allows 2.8 g of fat to be removed from the 75 g of cake, or approximately 3.7% of the mass of the cake. The yield of this step is 55.8%.

[0127] 102.87 g of the filtrate obtained is concentrated by total evaporation of the ethanol at a temperature of 45 to 48 °C, which makes it possible to obtain 3.01 g of concentrated ethanolic extract having a dry extract of 69.91% by mass.

[0128] Step b): Alkaline hydrolysis of deoiled hemp seed cake: A 1M sodium hydroxide solution (1613 g of water + 67.2 g of sodium hydroxide) is prepared to obtain a pH of 12. 160 g of deoiled hemp seed cake obtained in step a) is added. The mixture is stirred at 25°C for 2 hours with a 4-blade mixer. Then 142.8 g of citric acid monohydrate is added to neutralize the mixture and obtain a pH of 4.81. 8.4 g of sodium benzoate is then added. The pH obtained is then 4.78. The mixture is left to settle for 12 hours before being filtered through a pleated filter with a cut-off threshold of 10 µm. This gives 1440 g of filtrate in liquid form, which corresponds to a yield of 71.98% by mass. The dry extract of the filtrate is 12.5% ​​by mass. Example 2: characterization of the hydrolyzed extract of deoiled hemp seed cake obtained in example 1

[0129] The extract is characterized by its specific protein bands according to the separation method by denaturing gel electrophoresis in SDS PAGE. The presence of characteristic bands of the active ingredient which correspond to molecular masses of 17 kDa and 11 kDa is observed.

[0130] The SDS PAGE denaturing gel electrophoresis method used is as follows: - Gel preparation: The gels used are 1.5mm thick gels with 10 wells (8 cm x 7 cm) and formed of a concentration gel and a separation gel composed of 4.5 and 15% by mass of acrylamide-bisacrylamide, respectively, relative to the total mass of the gel. The composition of the gels is as follows: Concentration gel (Tris-HCl 125mM, pH 6.8, SDS 0.1%, acrylamide-bisacrylamide 4.5%, ammonium persulfate 0.1%, TEMED 0.025%); Separation gel (Tris-HCl 375mM, pH 8.8, SDS 0.1%, acrylamide-bisacrylamide 15%, ammonium persulfate 0.01%, TEMED 0.001%) - Sample preparation: Proteins are diluted in Laemmli buffer (Tris-HCl 50mM pH6.8, SDS 2%, bromophenol blue 0.1%, glycerol 10%, β-mercapoethanol 3%).40µL of each preparation is placed in the wells of the gels previously placed in a tank containing the reservoir solution (25mM Tris-HCl, 0.25M glycine, pH 8.3, SDS 0.1%). 5µL of a commercial size marker is placed in the first well (BlueStar Prestained Protein Marker, Nippon Genetics). - Migration conditions: Migration is carried out at room temperature at a voltage of 40V for 1h then 2h at 100V. At the end of the migration, the gels are stained with Coomassie Blue (Coomassie brilliant blue R2500, 25%, ethanol / water / acetic acid 45 / 45 / 10 by volume). The excess dye is removed by successive baths of a mixture of ethanol / acetic acid / water (50 / 10 / 40 by volume). Example 3: separation of the 11 kDa peptide fraction.

[0131] The gel bands obtained with the denaturing gel electrophoresis separation method in SDS PAGE as described in Example 2 are cut at the line corresponding to 11 kDa to be eluted in an elution medium (50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, pH 7.5). Example 4: separation of the 17 kDa peptide fraction

[0132] The gel bands obtained with the denaturing gel electrophoresis separation method in SDS PAGE as described in Example 2 are cut at the line corresponding to 17 kDa to be eluted in an elution medium (50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, pH 7.5). Example 5: Preparation of a comparative hemp seed hydrolysate according to the article by Girgih et al. (HPI)

[0133] 10 g of hemp seed cake marketed by the company EcoProd under the name "organic hemp cake" is ground and then dispersed in 200 g of reverse osmosis water. The initial pH of 6.36 is adjusted to 10.20 by adding a 2M NaOH solution. The mixture is stirred at 37 °C for 2 hours. Then the mixture is centrifuged for 60 minutes (7000 g at 4 °C) and the supernatant is collected and filtered through a pleated filter. The pH is then adjusted to 4.96 with a 2M HCl solution to precipitate the proteins. The mixture is then centrifuged (7000 g at 4 °C) for 40 minutes and 0.21 g of precipitate is obtained. This precipitate is then redispersed in 45.65 g of reverse osmosis water and the pH is adjusted to 7 by adding 2M NaOH. The extract is filtered through a pleated filter and has a dry matter content of 0.12%. It is freeze-dried to obtain a powder. Example 6: acellular in vitro test for evaluating calpain activity.

[0134] The ANASPEC SensoLyte® 520 Calpain Assay Kit provides a convenient assay for the assessment of calpain activity (mixture of calpain 1 and 2), enabling screening for enzyme inhibitors or continuous calpain activity assays using a fluorogenic substrate. Upon cleavage by calpain, this substrate generates the fluorophore 5-FAM (5-carboxyfluorescein) with fluorescence that can be detected at excitation / emission wavelengths=490 nm / 520 nm. The increase in fluorescence signal is proportional to calpain activity. The fluorescence wavelength Greater calpain is less impacted / disturbed by the autofluorescence of constituents in biological samples and test compounds.

[0135] MATERIALS: The samples tested are the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1), the peptide fractions according to the invention at 11 kDa (example 3) and at 17 kDa (example 4), a mixture of these peptide fractions (1:1, v:v) and the comparative hydrolyzate (example 5).

[0136] The reagents used are provided in the ANASPEC kit and are as follows: - component A = 5-FAM / QXL™ Calpain Substrate (substrate); - component C = Assay Buffer (buffer); - component D = Human Calpain; - component E = Calpain Inhibitor; - component F = DTT (DiThioThreitol).

[0137] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK. METHODS

[0138] Test System: A buffered solution of Calpain reacts with a specific substrate, 5-FAM / QXL™ 520 FRET substrate, to form a fluorogenic compound: 5-FAM. Fluorescence intensities are collected using an excitation filter passing wavelengths of 490 nm (Exc485-12 filter) and an emission filter passing wavelengths of 520 nm (Em535-30 filter). Calpain activity is thus evaluated.

[0139] The sample or the inhibitory reference product is brought into contact with the Calpain solution at the same time as the enzyme substrate. The activity of Calpain in the presence / absence of the sample or the reference product is then evaluated.

[0140] The modulation of this activity is expressed as the percentage of inhibition or activation of Calpain activity in the absence of active ingredient, i.e. only in the presence of the enzyme substrate.

[0141] Incubation protocol: A solution of Calpain is incorporated into its substrate, 5-FAM / QXL™ 520 FRET substrate. Gentle agitation for 30 seconds is carried out and then the whole is incubated at room temperature for 60 minutes.

[0142] Evaluation of effects: At the end of the incubation period, the activity of Calpain with and without test or reference product was evaluated by measuring fluorescence intensities (expressed in RFU: relative fluorescence unit).

[0143] For each concentration tested, the modulation of Calpain activity by the test product is calculated according to the following formula:

[0144] If the result is negative, the percentage is expressed as inhibition of the enzyme; if the result is positive, the percentage is expressed as activation of the enzyme.

[0145] Results: The results are presented in the following Tables 1 to 3:

[0146] [Table 1] Mean Mean Intensity Inhibition of Enzymatic Intensity of Enzymatic Intensity of Fluorescence Inhibition of Enzymatic Activity Samples Sample Fluorescence – Fluorescence (%) (RFU) Blank (%) Blank 2835.33 0.000 0.00 100.00 T + (reference product) (enzyme 52200.00 49364.67 100.00 0.00 + substrate without inhibitor) T- (control) (reference KIT inhibitor of 2797.67 -37.67 -0.08 100.08 detection = calpastatin peptide B27-WT) Example 1 (hydrolyzed extract according to the invention) at 5.0% 2029.33 1165.67 2.36 97.64 by mass in the reaction medium Example 1 (hydrolyzed extract according to the invention) at 2.0% 13075.33 11555.33 23.41 76.59 by mass in the reaction medium Example 1 (hydrolyzed extract according to the invention) at 0.5% 46925.00 44197.33 89.53 10.47 by mass in the reaction medium

[0147] In the presence of the reference inhibitor T- (control) the activity of Calpain is inhibited by 100%. This inhibition validates the test. At the tested concentrations of 5.0%, 2.0% and 0.5% by mass (in the reaction medium) a Calpain inhibitory activity is observed for the hydrolyzed extract of deoiled hemp seed cake according to the invention.

[0148] [Table 2] Mean Mean Intensity Inhibition of Enzymatic Fluorescence Intensity of Enzymatic Fluorescence Intensity of Enzymatic Activity Samples Sample – Fluorescence (%) (RFU) Blank (%) Blank 1512.33 0.000 0.00 100.00 T + (reference product) (enzyme 61093.33 59581.00 100.00 0.00 + substrate without inhibitor) T- (control) (inhibitor of 1522.333 10.00 0.02 99.98 KIT reference of detection = calpastatin peptide B27-WT) Example 3 (Eluate 1845.00 73.33 0.12 99.88 band 11 kDa according to the invention) at 10.0% by mass in the reaction medium Example 4 (Eluate band 17 kDa according to the invention) 10.0% by mass in the reaction medium Mixture of example 3 and example 4 (1:1, v: 1861.33 189.00 0.32 99.68 v) at 10% by mass in the reaction medium

[0149] In the presence of the reference inhibitor T- (control) the activity of Calpain is inhibited by 99.98%. This inhibition allows the test to be validated.

[0150] At the tested concentration of 10.0% of the 11 kDa protein band eluate by mass (in the reaction medium) a Calpain inhibitory activity is observed with 99.98% inhibition of the activity.

[0151] At the tested concentration of 10.0% of the 17 kDa protein band eluate by mass (in the reaction medium) a Calpain inhibitory activity is observed with 99.71% inhibition of the activity.

[0152] At the tested concentration of 10.0% of the 50 / 50 mixture of 11 and 17 kDa protein band eluates by mass (in the reaction medium) a Calpain inhibitory activity is observed with 99.68% inhibition of the activity.

[0153] [Table 3] Samples Moyenne Average IntensityActivity Inhibition of fluorescence Fluorescence intensity of the enzyme activity – Fluorescence (%) Enzyme sample Blank (RFU) (%) Blank 1413.67 0.000 0.00 100.00 T +(reference product) (enzyme 42990.00 41756.33 100.00 0.00 + substrate without inhibitor) T- (control) (enzyme + substrate 1478.33 64.67 0.16 99.84 without inhibitor) Example 5 (HPI extract according to Girgih publication) at 45316.67 43935.33 100.00 0.00 3.0% by mass in the reaction medium Example 5 (HPI extract according to Girgih publication) at 43501.00 42091.33 100.00 0.00 1.0% by mass in the reaction medium Example 5 (HPI extract according to Girgih publication) at 39602.00 38171.33 91.81 8.19 0.3% by mass in the reaction medium Example 5 (HPI extract according to the Girgih publication) at 40769.33 39388.67 94.74 5.26 0.1% by mass in the reaction medium

[0154] In the presence of the reference inhibitor T- (control), the activity of Calpain is inhibited by 99.84%. This inhibition validates the test.

[0155] At all tested concentrations from 3.0% to 0.1% by mass in the reaction medium, no significant inhibitory activity of Calpain is observed for the comparative hydrolysate.

[0156] Calpain is known to be involved in mechanisms inducing a neurodegenerative effect (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630, Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27; 8(1):18083). Thus, the results of Example 6 demonstrate that the hydrolyzed extract, the peptide fraction of the extract, the combination of the latter two or the composition, according to the invention, has an anti-neurodegenerative and neuroprotective effect.

[0157] In particular, calpain inhibition reflects an improvement in the treatment and / or prevention of Alzheimer's disease (Mahaman et al., Involvement of calpain in the neuropathogenesis of Alzheimer's disease, Med Res Rev. 2019; 39:608-630) and Parkinson's disease (Hassen et al., Effects of novel calpain inhibitors in transgenic animal model of Parkinson's disease / dementia with Lewy bodies, Sci Rep. 2018 Dec 27;8(1):18083). Thus, the results of Example 6 demonstrate that the hydrolyzed extract, the peptide fraction of the extract, the combination of the latter two or the composition, according to the invention, has a positive effect on the treatment and / or prevention of neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Example 7: In vitro acellular test for evaluating antioxidant activity according to the DPPH method.

[0158] The objective of this study is to evaluate the modulation of antioxidant activity by one or more samples in a cell-free in vitro colorimetric model using the DPPH radical, 2,2-Diphenyl-1-picrylhydrazyl, and the reference antioxidant, ascorbic acid.

[0159] MATERIALS: The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0160] The reference product used is ascorbic acid supplied by SIGMA ALDRICH. The reagents used are DPPH supplied by SIGMA ALDRICH and DMSO supplied by FISHER BIOREAGENTS.

[0161] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH. METHODS

[0162] Test system: The method used is called inhibition. In fact, it is based on the degradation of the oxidizing radical DPPH, with a violet color absorbing at 540nm, by a reference antioxidant, ascorbic acid. This reaction, which will serve as a positive control, leads to the formation of the compound 2,2-diphenyl-1-picrylhydrazine which will be colorless or light yellow.

[0163] The sample and the reference product “Ascorbic Acid” are brought into contact with the DPPH solution for 30 minutes at 40°C. The antioxidant activity is then evaluated by absorbance measurement at 540nm.

[0164] The modulation of this activity is expressed as a percentage of stimulation of the antioxidant activity by the active ingredient tested, with as a reference the maximum antioxidant activity obtained in the presence of ascorbic acid (T + ).

[0165] Incubation protocol: A DPPH solution is incubated for 30 minutes at 40°C, in the absence (control), in the presence of the reference product (T + ) and at decreasing concentrations of the sample tested.

[0166] Evaluation of effects: At the end of the incubation period, the antioxidant activity in the presence of the reference product and in the presence or absence of the test product was revealed by coloring after 30 minutes at 40°C. It was thus evaluated by measuring the absorbance of the reaction medium at 540 nm.

[0167] For each concentration tested, the modulation of antioxidant activity by the test product is calculated according to the following formula

[0168] If the result is negative, the product to be tested will be considered an oxidant; if the result is positive, the percentage is expressed as stimulation of anti-radical activity.

[0169] Results: The results are presented in the following Table 4:

[0170] [Table 4] Samples Average DO (nm) DO DPPH Power -DO Sample (nm) antioxidant (%) T+ (Ascorbic acid) 0.429 0.569 100.00 T- (DPPH) 0.998 0.000 0.00 Example 1 at 5.0% in 0.785 0.176 30.93 Mass in the reaction medium Example 1 at 2.0% in 0.866 0.096 16.81 Mass in the reaction medium Example 1 at 0.5% in 0.851 0.111 19.45 Mass in the reaction medium

[0171] Under the experimental conditions, the hydrolyzed extract of deoiled hemp seed cake according to the invention exhibits antioxidant activity at concentrations of 5.0%, 2.0% and 0.5% by mass in the reaction medium. Example 8: acellular in vitro test for evaluating anti-inflammatory (soothing) activity on the lipoxygenase enzyme.

[0172] The objective of this study is to evaluate the modulation of the anti-inflammatory activity of the lipoxygenase enzyme by one or more samples in an acellular in vitro model using the CAYMAN / INTERCHIM Lipoxygenase Inhibitor Screening Assay Kit. Lipoxygenase is a key enzyme upstream of the inflammatory process triggered by the arachidonic cascade.

[0173] MATERIALS: The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0174] The reagents used are provided in the CAYMAN / INTERCHIM kit and are as follows: - 0.1M buffer, Tris-HCl; - chromogen 1; - chromogen 2; - enzyme: 15–lipoxygenase; - substrate: arachidonic acid - potassium hydroxide - NDGA inhibitor (Nordihydroguaiaretic).

[0175] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK. METHODS

[0176] Test System: A buffered solution of Lipoxygenase reacts with a specific substrate, arachidonic acid, and converts it to form a compound that binds to a chromogen, while stirring at room temperature. Lipoxygenase activity can thus be assessed by measuring the absorbance at 500 nm.

[0177] The sample or the reference inhibitor product "Nordihydroguaiaretic" (NDGA) is brought into contact with the Lipoxygenase solution at the same time as the enzyme substrate. The substrate transformed by the enzyme is colored using the chromogen by stirring at room temperature. The activity of Lipoxygenase in the presence / absence of the sample, or the reference product is then evaluated by measuring the absorbance at 500 nm.

[0178] The modulation of this activity is expressed as a percentage of inhibition or activation of Lipoxygenase activity in the absence of active ingredient, i.e. only in the presence of the enzyme substrate (arachidonic acid).

[0179] Incubation protocol: A solution of Lipoxygenase enzyme is incubated in its substrate, arachidonic acid, for 10 minutes, in the absence or presence of the reference inhibitor and the sample tested, then the chromogen is incorporated before a 35-minute incubation at room temperature on an orbital shaker.

[0180] Evaluation of effects: At the end of the incubation period, the activity of the Lipoxygenase enzyme with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 500 nm.

[0181] For each concentration tested, the modulation of the activity of the Lipoxygenase enzyme by the test product is calculated according to the following formula:

[0182] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0183] Results: The results are presented in the following Table 5:

[0184] [Table 5] Samples Average OD Average OD % activity % inhibition of Sample enzyme activity – OD Blank enzyme Blank 0.251 0.000 0.00 100.00 T+ 0.398 0.147 100.00 0.00 T- 0.179 -0.073 / 0.000 0.00 149.55 Example 1 at 4.76% in 0.202 0.037 25.00 75.00 Mass in the reaction medium Example at 2.00% in 0.163 0.105 71.82 28.18 Mass in the reaction medium

[0185] At the tested concentrations of 2.00% and 4.76% by mass in the reaction medium, an inhibitory activity of the key enzyme of the arachidonic cascade, lipoxygenase, is observed for the hydrolyzed extract of deoiled hemp seed cake according to the invention.

[0186] The results of this example reflect a non-steroidal anti-inflammatory effect, capable of containing the fibrotic runaway during healing under the impulse of the production of leukotrienes (induced by the healing phenomenon) by lipoxygenase, of the hydrolyzed extract, the peptide fraction of the extract, the combination of these last two or the composition, according to the invention. Example 9: acellular in vitro test for evaluating the anti-inflammatory (soothing) activity on the phospholipase A2 enzyme.

[0187] The objective of this study is to evaluate the modulation of the anti-inflammatory activity of the phospholipase A2 enzyme by one or more samples in an acellular in vitro model using the analysis kit "SPLA2 (type V) Inhibitor Screening Assay Kit" from CAYMAN / INTERCHIM. Phospholipase A2 is a key enzyme upstream of the inflammatory process triggered by the arachidonic cascade.

[0188] MATERIALS: The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0189] The reagents used are provided in the CAYMAN / INTERCHIM kit and are as follows: - sPLA2 DTNB (2-nitrobenzoic acid); - sPLA2 Diheptanoyl thio-PC (substrate); - sPLA2 assay (human type V); - Thioetheramide-PC 1mg in 100µl Ethanol; - substrate: arachidonic acid - sPLA2 assay buffer (10x).

[0190] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK. METHODS

[0191] Test System: A buffered solution of Phospholipase A2 reacts with a specific substrate, diheptanoyl thio-PC, and transforms it to form a compound that binds to a chromogen, DTNB, under stirring at room temperature. ambient. The activity of phospholipase A2 can thus be assessed by measuring the absorbance at 413 nm.

[0192] The sample or the inhibitor reference product "Thioetheramide-PC" is brought into contact with the Phospholipase A2 solution at the same time as the enzyme substrate. The substrate transformed by the enzyme is colored using the chromogen DTNB by stirring at room temperature. The activity of Phospholipase A2 in the presence / absence of the sample, or the reference product is then evaluated by measuring the absorbance at 413 nm.

[0193] The modulation of this activity is expressed as the percentage of inhibition or activation of Phospholipase A2 activity in the absence of active ingredient, i.e. only in the presence of the enzyme substrate (diheptanoyl thio-PC).

[0194] Incubation protocol: A solution of phospholipase A2 enzyme is incubated in its substrate, diheptanoyl thio-PC, in the absence or presence of the reference inhibitor and the test sample, then the chromogen DTNB is incorporated before a 15-minute incubation at 25°C.

[0195] Evaluation of effects: At the end of the incubation period, the activity of the phospholipase A2 enzyme with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 413 nm.

[0196] For each concentration tested, the modulation of the activity of the phospholipase A2 enzyme by the test product is calculated according to the following formula:

[0197] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0198] Results: The results are presented in the following Table 6:

[0199] [Table 6] Samples Mean OD Mean OD Activity Inhibition of Enzyme Sample (%) activity – OD Enzyme Blank (%) White 0.155 0.000 0.00 100.00 T + 1.508 1.353 100.00 0.00 T- 0.538 0.383 28.33 71.67 Example 1 at 4.35% 0.890 0.510 37.69 62.31 by Mass in the reaction medium Example 1 at 2.00% 0.963 0.281 20.79 79.21 by Mass in the reaction medium Example 1 at 0.50% 1.066 0.806 59.55 40.45 by Mass in the reaction medium

[0200] At the tested concentrations of 4.35%, 2.00 and 0.50% by mass in the reaction medium, an inhibitory activity of the key enzyme of the arachidonic cascade, Phospholipase A2 is observed for the hydrolyzed extract of deoiled hemp seed cake according to the invention. A dose-dependent activity in "Gaussian" is observed for the hydrolyzed extract of deoiled hemp seed cake according to the invention which tends to demonstrate a specific inhibitory activity on the active site of the enzyme.

[0201] The results of this example reflect a non-steroidal anti-inflammatory effect, such as to contain the fibrotic runaway of the arachidonic cascade during healing under the impulse of the production of inflammatory mediators (induced by the healing phenomenon) by phospholipase A2, of the hydrolyzed extract, the peptide fraction of the extract, the combination of the latter two or the composition, according to the invention. Example 10: acellular in vitro test for evaluating tyrosinase activity.

[0202] The objective of this study is to evaluate the modulation of tyrosinase enzyme activity by a sample in a cell-free in vitro model using tyrosinase, L-tyrosine and hydroquinone.

[0203] MATERIALS: The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0204] The reagents used are as follows: - potassium phosphate monobasic supplied by SIGMA; - hydroquinone supplied by SIGMA ALDRICH; - L-tyrosine supplied by SIGMA ALDRICH; - mushroom tyrosinase supplied by SIGMA; - potassium hydroxide; - citric acid; - NaOH.

[0205] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH. METHODS

[0206] Test System: A buffered solution of Tyrosinase is brought into contact with the substrate L-tyrosine to form a compound that will be our positive control.

[0207] For the inhibition control, hydroquinone is the reference inhibitor. It is first brought into contact with the tyrosinase solution and then with the substrate L-tyrosine.

[0208] In parallel, the sample is brought into contact with the Tyrosinase solution at the same time as the enzyme substrate.

[0209] The whole thing is incubated at 23°C for 60 minutes.

[0210] Tyrosinase activity leads to the transformation of the substrate L-tyrosine into melanin pigment, which colors the mixture. Tyrosinase activity is then evaluated by measuring the absorbance at 475 nm.

[0211] The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Collagenase in the absence of active ingredient, i.e. only in the presence of the enzyme substrate.

[0212] Evaluation of effects: For each concentration tested, the modulation of the activity of the enzyme Tyrosinase by the test product is calculated according to the following formula:

[0213] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0214] Results: The results are presented in the following Table 7:

[0215] [Table 7] Samples Average OD Average OD % activity % inhibition of Sample enzyme activity – OD Blank enzyme Blank 0.021 0.000 0.00 100.00 T+ 0.205 0.184 100.00 0.00 T- 0.021 0.000 0.18 99.82 Example 1 at 5.0% 0.071 0.013 6.90 93.10 by mass in the reaction medium Example 1 at 2.0% 0.099 0.062 33.94 66.06 by mass in the reaction medium Example 1 at 0.5% 0.179 0.151 82.40 17.60 by mass in the reaction medium

[0216] At the tested concentrations of 5.0%, 2.0% and 0.5% by mass in the reaction medium, a Tyrosinase inhibitory activity is observed for the hydrolyzed extract of deoiled hemp seed cake according to the invention.

[0217] Based on the results, this inhibitory activity is dose-dependent, which tends to demonstrate a specific inhibitory action on the active site of the enzyme.

[0218] Tyrosinase inhibition is known to have an effect on skin diseases that lead to hyperpigmentation. Thus, the results of Example 10 demonstrate that the hydrolyzed extract, the peptide fraction of the extract, the combination of the latter two or the composition, according to the invention, comprises a depigmenting activity, systemic or local, which may be useful in the treatment of these diseases. Example 11: study of cytotoxicity on HELA cells

[0219] HeLa cells are an international reference human cancer cell line in cell biology.

[0220] The objective of this study is to evaluate the cytotoxicity of a sample on a culture of HeLa9903 cells and to define if possible an EC50 (median effective concentration).

[0221] MATERIALS: The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0222] The reagents used are as follows: - HeLa9903 cells supplied by Ephyla; - DMEM supplied by PanBiotech; - FBS CS supplied by Dutscher; - L-glutamine supplied by Dutscher; - Geneticin supplied by Corning; - Blasticidin S supplied by PanReac; - Trypsin 0.25% EDTA 0.02% in PBS supplied by PanBiotech; - Acetone supplied by Carlo Erba; - DMSO supplied by Fisher bioreagents; - PBS 10X supplied by biosolve; - Formaldehyde 37% supplied by Carlo Erba; - Crystal violet supplied by Carlo Erba; - Na2HPO4 supplied by Carlo Erba; - Na2HPO4.H2O supplied by Carlo Erba; - Acetic acid supplied by Carlo Erba.

[0223] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH. METHODS

[0224] Cell culture: Cells are maintained in 15ml of DMEM supplemented with 10% FBS-CS, 2mM L-Glutamine, 0.8mg / ml Geneticin, 16µg / ml Blasticidin S, in a 75cm balloon 2 then incubated at 37°C under an atmosphere containing 5% CO2. At confluence, the cells are trypsinized and counted on Malassey cells. A dilution of the cells is carried out in supplemented DMEM so as to obtain a cell suspension at 105 cells / ml. 100µl of this suspension is seeded in 60 of the 96 wells of the plate. The number of cells per well is 10000. The perimeter of the plate (i.e. lines A and H and columns 1 and 12) is filled with 100µL of 1X PBS. The cells are incubated at 37°C in an atmosphere containing 5% CO2 for 24 hours (time of attachment of the cells to the support).

[0225] After 24 hours of incubation, the cells are treated in triplicate by adding 1 µL of the different dilutions of the samples to be tested. For the control, 1 µL of dilution solvent is added instead of the sample. Each plate is prepared in duplicate to observe the effect of 24 and 48 hours of treatment on cell survival.

[0226] The cells are then incubated for 24 or 48 hours.

[0227] Crystal violet labeling: The culture medium is removed from each well. The living cells, attached to the bottom of the plate, are rinsed with 100µL of PBS and then fixed by adding 100µL of a formaldehyde solution (10% in PBS) for 10 min. The fixed cells are rinsed 3 times with deionized water and then labeled with 50µL of a 1% solution of crystal violet dissolved in 200mM sodium phosphate buffer, pH 6 for 20 to 30 min. The crystal violet not attached to the cells is removed by immersing the plate in tap water 3 times. The plate is dried and then the crystal violet incorporated in the cells is dissolved in 100µL of a 1% acetic acid solution for 20 to 30 min with moderate shaking. 80µL of the crystal violet / acetic acid solution is transferred into a new 96-well plate to read the absorbance at 590nm.

[0228] Evaluation of effects: For each concentration tested, the viability of HeLa cells is calculated according to the following formula:

[0229] The sample will be considered toxic if the result is less than 50%. Results

[0230] 24-hour results: The results are presented in the following table 8:

[0231] [Table 8] Samples Mean OD Viability of HeLa cells (%) Control 0.598 100.00 Example 1 at 1.00% by mass in the culture medium 0.575 96.04 Example 1 at 0.75% by mass in the culture medium 0.648 108.25 Example 1 at 0.50% by mass in the culture medium 0.624 104.35

[0232] Results at 48 hours: The results are presented in the following table 9:

[0233] [Table 9] Samples Mean OD Viability of HeLa cells (%) Control 1.024 100.00 Example 1 at 1.00% by mass in the culture medium 0.784 76.62 Example 1 at 0.75% by mass in the culture medium 0.966 94.37 Example 1 at 0.50% by mass in the culture medium 0.958 93.59

[0234] Given the homogeneity of the results obtained at 24 and 48 hours, the values ​​after 24 hours are considered usable and representative.

[0235] Under the experimental conditions, the hydrolyzed extract of deoiled hemp seed cake according to the invention is not cytotoxic on HeLa cells at concentrations of 0.50%, 0.75% and 1.00% by mass in the culture medium. Example 12: Association according to the invention comprising black ginseng in syrup form

[0236] Step 1: maceration: 10% by mass of dry black ginseng root powder was macerated in 90% by mass of a hydrolyzed hemp seed extract prepared according to example 1 in liquid form (% by mass relative to the total mass of the extract + black ginseng mixture).

[0237] The maceration was carried out at room temperature with gentle stirring for 24 hours.

[0238] Step 2: Filtration: The macerate obtained in step 1 was filtered (this filtrate representing 10% by mass of the finished assembled product). The filtrate obtained is a liquid filtrate, the solid phase having been eliminated during filtration. The filter used has a cut-off threshold of 10 µm.

[0239] Step 3: Assembly: The filtrate, in a mass content of 10%, was assembled with sorbitol in a mass content of 15%, and with a hydrolyzed extract of hemp seed prepared according to Example 1 in liquid form in a mass content of 75%, the latter acting inter alia as a solvent, the percentages being expressed in mass relative to the total mass of the final mixture obtained.

[0240] The resulting combination is in the form of syrup.

[0241] It can be administered at a dosage of 2 teaspoons in the morning, or approximately 5 to 6 ml per day, particularly in the treatment and / or prevention of veisalgia. Example 13: Association according to the invention comprising black ginseng in gel form

[0242] Steps 1 and 2 of maceration and filtration are identical to those of example 12.

[0243] Step 3: Assembly: The filtrate, in a mass content of 10%, was assembled with sorbitol in a mass content of 9%, glycerin in a mass content of 6%, xanthan gum in a mass content of 0.3%, guar gum in a mass content of 0.25%, and with a hydrolyzed extract of hemp seed prepared according to Example 1 in liquid form in a mass content of 74.45%, the latter acting inter alia in as a solvent, the percentages being expressed by mass relative to the total mass of the final mixture obtained.

[0244] The resulting combination is in the form of a gel.

[0245] It can be administered at a dosage of 5 g per day, in particular in the treatment and / or prevention of veisalgia. Example 14: Association according to the invention comprising harpagophytum, turmeric and black pepper in the form of syrup

[0246] Step 1: maceration of 10% by mass of dry powder of harpagophytum root was macerated in 90% by mass of a hydrolyzed extract of hemp seed prepared according to example 1 in liquid form (% by mass relative to the total weight of the extract + harpagophytum mixture).

[0248] Maceration 2: 8% by mass of dry turmeric root powder and 2% by mass of dry black pepper berry powder in the form of dried fruit were macerated in 90% by mass of a hydrolyzed hemp seed extract prepared according to Example 1 in liquid form (% by mass relative to the total mass of the extract + black pepper + turmeric mixture).

[0249] The macerations were carried out at room temperature with gentle stirring for 24 hours.

[0250] Step 2: Filtration: The two macerates obtained in step 1 were filtered, resulting in two macerates (each of the two filtrates will represent 5% by mass of the finished assembled product). The two filtrates obtained are liquid filtrates, their solid phase having been eliminated during filtration. The filter used has a cut-off threshold of 10 µm.

[0251] Step 3: Assembly: The filtrate of macerate 1 in a mass content of 5% and the filtrate of macerate 2 in a mass content of 5% were assembled with sorbitol in a mass content of 15%, and with a hydrolyzed extract of hemp seed prepared according to example 1 in liquid form in a mass content of 75%, the latter acting inter alia as a solvent, the percentages being expressed in mass relative to the total mass of the final mixture obtained.

[0252] The resulting combination is in the form of syrup.

[0253] It can be administered at a dosage of 1 to 2 teaspoons per day, or approximately 2 to 5 ml per day, particularly in the reduction and / or prevention of joint pain. Example 15: Association according to the invention comprising harpagophytum, turmeric and black pepper in gel form

[0254] Steps 1 and 2 of maceration and filtration are identical to those of example 14.

[0255] Step 3: Blending: The filtrate of macerate 1 in a mass content of 5% and the filtrate of macerate 2 in a mass content of 5% were blended with sorbitol in a mass content of 9%, glycerin in a mass content of 6%, xanthan gum in a mass content of 0.3%, guar gum in a mass content of 0.25%, and with a hydrolyzed extract of hemp seed prepared according to Example 1 in liquid form in a mass content of 74.45%, the latter acting inter alia as a solvent, the percentages being expressed in mass relative to the total mass of the final mixture obtained.

[0256] The resulting combination is in the form of a gel.

[0257] It can be administered at a dosage of 5 g per day, particularly in the reduction and / or prevention of joint pain. Example 16: Combination according to the invention comprising zinc gluconate, turmeric and black pepper in the form of syrup

[0258] Step 1: maceration

[0259] Maceration 1: 10% by mass of dry zinc gluconate powder was macerated in 90% by mass of a hydrolyzed hemp seed extract prepared according to example 1 in liquid form (% by mass relative to the total mass of the extract + zinc gluconate mixture).

[0260] Maceration 2: 8% by mass of dry turmeric root powder and 2% by mass of dry black pepper berry powder in the form of dried fruit were macerated in 90% by mass of a hydrolyzed hemp seed extract prepared according to Example 1 in liquid form (% by mass relative to the total mass of the extract + black pepper + turmeric mixture).

[0261] The macerations were carried out at room temperature with gentle stirring for 24 hours.

[0262] Step 2: Filtration: The two macerates obtained in step 1 were filtered, resulting in two macerates (each of the two filtrates will represent 5% by mass of the finished assembled product). The two filtrates obtained are liquid filtrates, their solid phase having been eliminated during filtration. The filter used has a cut-off threshold of 10 µm.

[0263] Step 3: Blending: The filtrate of macerate 1 in a mass content of 5% and the filtrate of macerate 2 in a mass content of 5% were blended with sorbitol in a mass content of 15%, and with a hydrolyzed hemp seed extract prepared according to Example 1 in liquid form in a mass content of 75%, the latter acting inter alia as a solvent, the percentages being expressed in mass relative to the total mass of the final mixture obtained.

[0264] The resulting combination is in the form of syrup.

[0265] It can be administered at a dosage of 1 to 2 teaspoons per day, or approximately 2 to 5 ml per day, particularly in the treatment and / or prevention of acne, more particularly to reduce the symptoms of acne such as burning, itching and redness. Example 17: Combination according to the invention comprising zinc gluconate, turmeric and black pepper in gel form

[0266] Steps 1 and 2 of maceration and filtration are identical to those of example 16.

[0267] Step 3: Blending: The filtrate of macerate 1 with a mass content of 5% and the filtrate of macerate 2 with a mass content of 5% were blended. with sorbitol in a mass content of 9%, glycerin in a mass content of 6%, xanthan gum in a mass content of 0.3%, guar gum in a mass content of 0.25%, and with a hydrolyzed hemp seed extract prepared according to Example 1 in liquid form in a mass content of 74.45%, the latter acting inter alia as a solvent, the percentages being expressed by mass relative to the total mass of the final mixture obtained.

[0268] The resulting combination is in gel form. It can be administered at a dosage of 5 g per day, particularly in the treatment and / or prevention of acne, especially to reduce acne symptoms such as burning, itching and redness.

Claims

Claims

1. Hydrolyzed extract of deoiled hemp seed cake obtained by alkaline hydrolysis at a pH greater than or equal to 11, advantageously greater than or equal to 12.

2. Hydrolyzed extract according to claim 1, characterized in that the duration of the hydrolysis is greater than 1 hour, preferably greater than or equal to 1 hour 15 minutes, preferably still greater than or equal to 1 hour 30 minutes, even more preferably greater than or equal to 1 hour 45 minutes, even more preferably still greater than or equal to 2 hours.

3. Hydrolyzed extract according to claim 1 or 2, characterized in that it is obtained by the process comprising the following steps: a- defatting of the hemp seed cake by extraction with ethanol or with supercritical CO2, advantageously with ethanol at least 96 degrees, b- alkaline hydrolysis at a pH greater than or equal to 11, in particular greater than or equal to 12, of the deoiled cake obtained in step a), advantageously using a base chosen from sodium hydroxide, magnesium hydroxide, calcium hydroxide and potassium hydroxide.

4. Hydrolyzed extract according to any one of claims 1 to 3, characterized in that it comprises a peptide fraction with a molecular mass of between 9 and 12 KDa, advantageously 11 KDa, and a peptide fraction with a molecular mass of between 16 and 18 KDa, advantageously 17 kDa, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE.

5. Hydrolyzed extract according to any one of claims 1 to 4, characterized in that: - its dry matter content is between 10.5 and 15% by mass, relative to the total mass of the hydrolyzed extract; - its total protein content is between 0.8 and 2.4% by mass. advantageously between 1.2 and 1.6% by mass, relative to the total mass of the hydrolyzed extract.

6. Peptide fraction of the hydrolyzed extract according to any one of claims 1 to 5, characterized in that its molecular mass, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE is between 9 and 12 KDa, advantageously it is 11 KDa.

7. Peptide fraction of the hydrolyzed extract according to any one of claims 1 to 5, characterized in that its molecular mass, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE is between 16 and 18 KDa, advantageously it is 17 KDa.

8. Association of the hydrolyzed extract according to any one of claims 1 to 5 or of one or more of its peptide fractions according to any one of claims 6 or 7 and of an ethanolic extract of hemp seed cake, advantageously obtained in step a) of the process of claim 3.

9. Association of the hydrolyzed extract according to any one of claims 1 to 5 or of one or more of its peptide fractions according to any one of claims 6 or 7 with a compound chosen from: - ginseng, - harpagophytum, - turmeric, - black pepper, - zinc gluconate, - Ginkgo biloba, and - a mixture of two or more of these compounds.

10. Macerate of the association according to claim 9.

11. Mixture of the macerate according to claim 10 with a hydrolyzed extract according to any one of claims 1 to 5 or one or. several of its peptide fractions according to any one of claims 6 or 7.

12. Pharmaceutical composition comprising the hydrolyzed extract according to any one of claims 1 to 5 or one or more of its peptide fractions according to any one of claims 6 or 7 or the combination according to any one of claims 8 or 9 or the macerate according to claim 10 or the mixture according to claim 11 and a pharmaceutically acceptable excipient.

13. Process for manufacturing the hydrolyzed extract according to any one of claims 1 to 5, characterized in that it comprises the following steps: a- delipidation of the hemp seed cake by extraction with ethanol or with supercritical CO2, advantageously with ethanol at least 96 degrees; b- alkaline hydrolysis at a pH greater than or equal to 11, in particular greater than or equal to 12, of the de-oiled cake obtained in step a),advantageously using a base chosen from sodium hydroxide, magnesium hydroxide, calcium hydroxide and potassium hydroxide.

14. Hydrolyzed extract according to any one of claims 1 to 5 or peptide fraction of the extract according to any one of claims 6 or 7 or combination according to any one of claims 8 or 9 or macerate according to claim 10 or mixture according to claim 11 or composition according to claim 12 for its use as a medicament, advantageously as an antioxidant and / or depigmenting agent and / or non-steroidal anti-inflammatory agent and / or anti-neurodegenerative agent and / or neuroprotective agent and / or anticancer agent and / or healing agent and / or antifibrotic agent and / or for the treatment and / or prevention of vesicalgia and / or for the reduction and / or prevention of joint pain and / or for the treatment and / or prevention of acne,in particular for reducing the symptoms of acne such as burning, itching and / or redness, more advantageously for the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease or, Parkinson's disease and / or for the reduction and / or prevention of joint pain and / or for the treatment and / or prevention of vesalgia and / or for the treatment and / or prevention of acne, in particular to reduce the symptoms of acne such as burning, itching and / or redness.

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