Hydrolyzed extract of hemp seed cake

Abstract

Hydrolyzed extract of hemp seed cake The present invention relates to a hydrolyzed extract of deoiled hemp seed cake obtained by alkaline hydrolysis at a pH greater than or equal to 11, its peptide fractions, the association of this extract or its peptide fractions with an ethanolic extract of hemp seed cake and a pharmaceutical composition comprising it. It further relates to a method for manufacturing the hydrolyzed extract according to the invention. Finally, it relates to the hydrolyzed extract according to the invention or one of its peptide fractions or the association or composition comprising it for its use as a medicament.

Description

Title of the invention: Hydrolyzed extract of hemp seed cake Technical field

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

[0002] 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.

[0003] It is known from the article by Girgih et al. (J. Am. Oil Chem Soc (2011) 88:381-389) that certain 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 weak antioxidant activity. Furthermore, 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.

[0004] The article by Rodriguez-Martin et al. (Food Funct, 2019, 10, 6732-6739) suggests that certain 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 NF-kB pathway mediated by TLR-4. 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. In addition, the tests carried out are not sufficient to prove a real neuroprotective activity because they do not correspond to a 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 supporting neuron degeneration; it is therefore very clearly an indirect effect with respect to the neuroprotective action.

[0005] Calpains are a family of Ca2+-dependent intracellular soluble cysteine proteases. They respond to Ca2+ signals by cleaving many specific proteins, thereby irreversibly modifying their function(s). They are involved in various Ca2+-regulated cellular processes 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, repeated concussion-induced neuropathy, and cancer. Calpains represent potential therapeutic targets for drug discovery.

[0006] 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 in 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.

[0007] Furthermore, deregulation of calpain activity has been implicated in tumorigenesis, suggesting its impact on cancer and metastases (Ivan Shapovalov et al., EXPERT OPINION ON THERAPEUTIC TARGETS, 2022, VOL. 26, NO. 3, 217-231).

[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 via 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] Pathological overactivation of calpains is also known to be involved in certain fibrotic diseases, including cardiac fibrosis and idiopathic pulmonary fibrosis. Calpain upregulates transforming growth factor beta 1 (TGF-beta) 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).

[0010] Consequently, it would be interesting to find new products that can inhibit the activity of calpain.

[0011] The inventors surprisingly discovered that a particular hydrolyzed extract of deoiled hemp seed cake, and more particularly two of its peptide fractions, had a strong power to inhibit the activity of calpain, in particular 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. Statement of the invention

[0012] 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 12.

[0013] In the present application, the expressions "between ... and..." and "from ... to..." must be understood to include limits unless explicitly stated otherwise.

[0014] The term "hemp seed cake" means the co-product obtained after extraction of oil from hemp seeds, in particular from Cannabis saliva L. The extraction of oil from cannabis seeds is carried out by techniques known from a person 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 of this extraction is called "co-product" within the meaning of the present invention and contains all of the compounds not extracted by said technique. By co-product, we therefore mean the residue obtained after extraction of the oil. Thus, the cake therefore has a fat content lower 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.

[0015] 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.

[0016] 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.

[0017] 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 the company Hemp Acres LLC or from the company Ecoprod.

[0018] 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.

[0019] In an advantageous embodiment, before being hydrolyzed in the context of the present invention, the hemp seed cake according to the invention is extracted with ethanol, advantageously 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. This de-oiled residue is also called "de-oiled hemp seed cake" in the context of the present description. The extraction with ethanol according to the invention can be implemented by different extraction methods known to those skilled in the art, such as maceration, with or without stirring, or hot decoction. Preferably, 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, more preferably for a period of 1 hour to 3 hours, and even more advantageously for a period of 2 hours. The extraction can be carried out at a temperature of between 15 and 35°C, advantageously between 20°C and 30°C, more advantageously at 25°C.The extraction can 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. 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° ethanol) is macerated with stirring in 96° ethanol for a period of 2 hours at a temperature of 25°C, in particular under the conditions as described in Example 1.

[0020] 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.

[0021] 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 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 between 1 hour 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.

[0022] 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 par- particular between 4 and 5.

[0023] Advantageously, a preservative such as sodium benzoate or potassium sorbate is then added to the hydrolyzate. This preservative makes it possible to avoid microbial contamination or fermentation of the hydrolyzate obtained and therefore to stabilize it sustainably.

[0024] The hydrolyzate obtained can then be filtered in particular by a 10 μm filter.

[0025] Advantageously, the proteins are not previously extracted from the cake before the hydrolysis step.

[0026] The quantity by mass of deoiled hemp seed cake used for carrying out the 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.

[0027] 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 (10 μm), under the conditions as described in Example 1.

[0028] 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, advantageously 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.

[0029] Steps a) and b) therefore correspond to the extraction with ethanol and to the hydrolysis as described above.

[0030] 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 - 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 separative method by electrophoresis on denaturing SDS-gel 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: - Concentration 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 - Separation gel (Tris-HCl 375mM, pH 8.8, SDS 0.1%, acrylamide-bisacrylamide 15%, ammonium persulfate 0.01%, TEMED 0.001%)

[0031] 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.

[0032] 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.

[0033] Advantageously, the hydrolyzed extract according to the invention also has a total protein content of between 0.8 and 2.4% by weight, more advantageously between 1 and 2%, even more advantageously between 1.2 and 1.6% by weight, relative to the total weight of the hydrolyzed extract.

[0034] 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.

[0035] 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.

[0036] 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 of between 9 and 12 kDa.

[0037] The present invention further relates to the peptide fraction (or peptide or oli gopeptide) 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, of between 16 and 18 kDa, advantageously between 16.5 and 17.5 kDa, in particular 17KDa.

[0038] 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 of between 16 and 18 kDa.

[0039] 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 of hemp seed cake, advantageously obtained in step a) of the process according to the present invention.

[0040] The ethanolic extract of hemp seed cake according to the invention is a lipophilic extract. Advantageously, it contains polar lipids.

[0041] 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).

[0042] Advantageously, the dry matter (without solvent) of the ethanolic 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.

[0043] Advantageously, the dry matter (without solvent) of the ethanolic 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.

[0044] Advantageously the dry matter (without solvent) of the ethanolic 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.

[0045] Advantageously, the dry matter (without solvent) of the ethanolic 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.

[0046] The dry matter (without solvent) of the extract according to the invention can therefore be obtained by extraction with ethanol 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 which is recovered and not the deoiled residue. The ethanolic extract can be used as is in the composition according to the invention or it can be concentrated to increase its 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 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, relative to the total mass of the extract.The concentrated ethanolic 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.

[0047] 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 combination according to the invention and a pharmaceutically acceptable excipient.

[0048] In the present invention, "pharmaceutically acceptable" is intended to mean that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary as well as human pharmaceutical use.

[0049] 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 (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 according to the condition in question.

[0050] Said excipients are chosen according to the pharmaceutical form and the desired method of administration.

[0051] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous administration, topical, intratracheal, intranasal, transdermal, local or rectal, 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 can 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 can be used in creams, ointments or lotions.

[0052] According to usual practice, the dosage appropriate for each patient is determined by the physician according to the method of administration, the mass and the response of said patient.

[0053] When preparing a solid composition in tablet form, the main 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.

[0054] A capsule preparation is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard capsules.

[0055] A preparation in the form of a syrup or elixir may contain the active ingredient together with a sweetener, an antiseptic, as well as a suitable flavoring agent and coloring agent.

[0056] The water-dispersible powders or granules may contain the active ingredient in admixture with dispersing agents or wetting agents, or suspending agents, as well as with flavor correctors or sweeteners.

[0057] For intranasal or intraocular parenteral administration, aqueous suspensions, isotonic saline solutions or sterile, injectable solutions which contain pharmacologically compatible dispersing agents and / or wetting agents, for example propylene glycol or butylene glycol, may be used.

[0058] For rectal administration, suppositories are used which are prepared with binders which melt at rectal temperature, for example cocoa butter or polyethylene glycols.

[0059] The active ingredient may also be formulated in the form of microcapsules, optionally with one or more additive carriers.

[0060] 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, advantageously 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.

[0061] Steps a) and b) are as described previously with regard to obtaining the deoiled hemp seed cake (step a) and the alkaline hydrolysis (step b).

[0062] 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 combination according to the invention or the pharmaceutical composition according to the invention for its use as a medicament.

[0063] 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 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 anti-cancer agent and / or healing agent and / or anti-fibrotic agent, more advantageously for treatment or prevention; - 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, particularly after burns, and cancer, particularly 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 can lead to the development of HELLP syndrome or type II glutaric acidemia, cardiomyopathies and muscle deficiency. - diseases associated with tyrosinase modulation such as hyperpigmentation of the skin (lentigo) and retina.

[0064] 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 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.

[0065] The present invention will be better understood on reading the following examples which are given for non-limiting information purposes.

[0066] Examples

[0067] Example 1: preparation of the hydrolyzed extract of deoiled hemp seed cake according to the invention

[0068] Step a): Defatting of the hemp seed cake by extraction with ethanol

[0069] 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 cellulose and 10.50% by mass of crude fat. This is the residue obtained after cold pressing of hemp seeds.

[0070] Step a) consists of grinding and macerating with stirring at room temperature between 20 and 25 °C in a mixer with a 4-blade propeller for 2 hours 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 makes it possible to remove 2.8 g of fat from the 75 g of cake, or approximately 3.7% of the mass of the cake. The yield of this step is 55.8%.

[0071] 102.87 g of the filtrate obtained is concentrated by total evaporation of the ethanol at a temperature 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.

[0072] Step b): alkaline hydrolysis of the deoiled hemp seed cake

[0073] A solution of IM sodium hydroxide (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, which therefore corresponds to a yield of 71.98% by mass. The dry extract of the filtrate is 12.5% by mass.

[0074] Example 2: characterization of the hydrolyzed extract of deoiled hemp seed cake obtained in example 1

[0075] 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 is observed, which correspond to molecular masses of 17 kDa and 11 kDa.

[0076] The SDS PAGE denaturing gel electrophoresis method used is as follows: - Gel preparation: The gels used are 1.5 mm 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-bis-acrylamide 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 (50mM Tris-HCl pH6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, [3-mercaptoethanol 3%). 40pL of each preparation is deposited in the wells of the gels previously placed in a tank containing the reservoir solution (25mM Tris-HCl, 0.25M glycine, pH 8.3, 0.1% SDS). 5pL of a commercial size marker is deposited 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 R250 0.25%, ethanol / water / acetic acid 45 / 45 / 10 by volume). Excess dye is removed by successive baths of a mixture of ethanol / acetic acid / water (50 / 10 / 40 by volume).

[0077] Example 3: separation of the 11 kDa peptide fraction

[0078] The gel bands obtained with the separation method by denaturing gel electrophoresis in SDS PAGE as described in Example 2 are cut at the level of 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).

[0079] Example 4: separation of the 17 kDa peptide fraction

[0080] The gel bands obtained with the separation method by denaturing gel electrophoresis in SDS PAGE as described in example 2 are cut at the level of 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).

[0081] Example 5: preparation of a comparative hydrolyzate of hemp seed according to the article by Girgih et al. (HPI)

[0082] 10 g of hemp seed cake marketed by the company EcoProd under the denomination "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 whole 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 recovered and filtered on 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 on a pleated filter and has a dry matter content of 0.12%. It is freeze-dried to obtain a powder.

[0083] Example 6: acellular in vitro test for evaluating calpain activity.

[0084] ANASPEC's SensoLyte® 520 Calpain Assay Kit provides a convenient assay for the assessment of calpain activity (mixture of calpains 1 and 2) thus enabling screening of enzyme inhibitors or continuous calpain activity assay 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 longer fluorescence wavelength of calpain is less impacted / interfered with by T autofluorescence of constituents in biological samples and test compounds.

[0085] MATERIALS

[0086] 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).

[0087] 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).

[0088] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK.

[0089] METHODS

[0090] Test system

[0091] 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). The activity of Calpain is thus evaluated.

[0092] 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.

[0093] The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Calpain in the absence of active ingredient, i.e. only in the presence of the enzyme substrate.

[0094] Incubation protocol

[0095] 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.

[0096] Evaluation of the effects

[0097] 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).

[0098] For each concentration tested, the modulation of Calpain activity by the test product is calculated according to the following formula:

[0099] [Math.l] Percentage modulation of Caipame activity = 100 x [(00. test or reference product ~ OO Calpain alone) / OO Calpame alone]

[0100] 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.

[0101] Results

[0102] The results are presented in the following Tables 1 to 3:

[0103] [Tables] Samples Average Fluorescence Intensity (RFU) Average Fluorescence Intensity Sample - Fluorescence Blank Enzymatic activity (%) Inhibition of enzymatic activity (%) Blank 2835.33 0.000 0.00 100.00 T+ (reference product) (enzyme + substrate without inhibitor) 52200.00 49364.67 100.00 0.00 T (control) (reference inhibitor of the detection KIT = cal-pastatin peptide B27-WT) 2797.67 -37.67 -0.08 100.08 Example 1 (hydrolyzed extract according to the invention) at 5.0% by mass in the reaction medium 2029.33 1165.67 2.36 97.64 Example 1 (hydrolyzed extract according to the invention) at 5.0% by mass in the reaction medium the invention) at 2.0% by mass in the reaction medium 13075.33 11555.33 23.41 76.59 Example 1 (hydrolyzed extract according to the invention) at 0.5% by mass in the reaction medium 46925.00 44197.33 89.53 10.47 actionable

[0104] In the presence of the reference inhibitor T (control) the activity of Calpain is inhibited by 100%. This inhibition makes it possible to validate 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.

[0105] [Tables2] Samples Average Fluorescence Intensity (RFU) Average Fluorescence Intensity Sample - Fluorescence Blank Enzymatic activity (%) Inhibition of enzymatic activity (%) Blank 1512.33 0.000 0.00 100.00 T+ (reference product) (enzyme + substrate without inhibitor) 61093.33 59581.00 100.00 0.00 T (control) (reference inhibitor of the detection KIT = cal-pastatin peptide B27-WT) 1522.333 10.00 0.02 99.98 Example 3 (Eluate band 11 kDa according to the invention) at 10.0% by mass in the reaction medium 1845.00 73.33 0.12 99.88 Example 4 (Eluate band 17 kDa according to the invention) 10.0% by mass in the reaction medium 1886.00 172.67 0.29 99.71 Mixture of example 3 and example 4 (1:1, v:v) at 10% by mass in the 1861.33 189.00 0.32 99.68 reaction medium

[0106] 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.

[0107] 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.

[0108] 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.

[0109] At the tested concentration of 10.0% of the 50 / 50 mixture of the 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.

[0110] [Tables3] Samples Average Fluorescence Intensity (RFU) Average Fluorescence Intensity Sample - Fluorescence Blank Enzymatic activity (%) Inhibition of enzymatic activity (%) Blank 1413.67 0.000 0.00 100.00 T+ (reference product) (enzyme + substrate without inhibitor) 42990.00 41756.33 100.00 0.00 T (control) (enzyme + substrate without inhibitor) 1478.33 64.67 0.16 99.84 Example 5 (HPI extract according to Girgig publication) at 3.0% by mass in the reaction medium 45316.67 43935.33 100.00 0.00 Example 5 (HPI extract according to Girgig publication) at 1.0% by mass in the reaction medium 43501.00 42091.33 100.00 0.00 Example 5 (HPI extract according to the Girgig publication) at 0.3% by mass in the reaction medium 39602.00 38171.33 91.81 8.19 Example 5 (extract 40769.33 39388.67 94.74 5.26 HPI according to the Girgig publication) at 0.1% by mass in the reaction medium

[0111] In the presence of the reference inhibitor T (control), the activity of Calpain is inhibited by 99.84%. This inhibition allows the test to be validated.

[0112] At all concentrations tested from 3.0% to 0.1% by mass in the reaction medium, no significant inhibitory activity of Calpain is observed for the comparative hydrolyzate.

[0113] Example 7: acellular in vitro test for evaluating antioxidant activity according to the DPPH method.

[0114] The objective of this study is to evaluate the modulation of antioxidant activity by one or more samples in an acellular In Vitro colorimetric model using the DPPH radical, 2,2-Diphenyl-l-picrylhydrazyl as well as the reference antioxidant, ascorbic acid.

[0115] MATERIALS

[0116] The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0117] 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.

[0118] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH.

[0119] METHODS

[0120] Test system

[0121] 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-l-picrylhydrazine which will be colorless or light yellow.

[0122] 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.

[0123] The modulation of this activity is expressed as a percentage of stimulation of the antioxidant activity by the active ingredient tested, with the maximum antioxidant activity obtained in the presence of ascorbic acid (T+) as a reference.

[0124] Incubation protocol

[0125] 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.

[0126] Evaluation of effects

[0127] 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.

[0128] For each concentration tested, the modulation of the antioxidant activity by the product under test is calculated according to the following formula

[0129] [Math.2] Percentage modulation of F antioxidant activity = 100 x Control - DOs® (Product to be tested) / DOs® (Reference product)

[0130] If the result is negative, the product to be tested will be considered as an oxidant; if the result is positive, the percentage is expressed as stimulation of anti-radical activity.

[0131] Results

[0132] The results are presented in the following Table 4:

[0133] [Tables4] Samples Average OD (nm) OD DPPH -OD Sample (nm) Antioxidant power (%) T+ (Ascorbic acid) 0.429 0.569 100.00 T- (DPPH) 0.998 0.000 0.00 Example 1 at 5.0% by mass in the reaction medium 0.785 0.176 30.93 Example 1 at 2.0% by mass in the reaction medium 0.866 0.096 16.81 Example 1 at 0.5% by mass in the reaction medium 0.851 0.111 19.45

[0134] Under the experimental conditions, the hydrolyzed extract of seed cake of deoiled hemp according to the invention exhibits antioxidant activity at concentrations of 5.0%, 2.0% and 0.5% by mass in the reaction medium.

[0135] Example 8: acellular in vitro test for evaluating anti-inflammatory (soothing) activity on the lipoxygenase enzyme.

[0136] 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 analysis kit "Lipoxygenase Inhibitor Screening Assay Kit" from the company CAYMAN / INTERCHIM. Lipoxygenase is a key enzyme upstream of the inflammatory process triggered by the arachidonic cascade.

[0137] MATERIALS

[0138] The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0139] The reagents used are provided in the CAYMAN / INTERCHIM kit and are as follows: - 0.1M Tris-HCl buffer; - chromogen 1; - chromogen 2; - enzyme: 15-lipoxygenase; - substrate: arachidonic acid - potassium hydroxide - NDGA (Nordihydroguaiaretic) inhibitor.

[0140] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK.

[0141] METHODS

[0142] Test system

[0143] A buffered solution of Lipoxygenase reacts with a specific substrate, arachidonic acid, and transforms it to form a compound that binds to a chromogen, under stirring at room temperature. The activity of Lipoxygenase can thus be evaluated by measuring the absorbance at 500 nm.

[0144] The sample or the inhibitory reference product "Nordihydroguaiaretic" (NDGA) are 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 the Lipoxygenase in the presence / absence of the sample, or the reference product is then evaluated by measuring the absorbance at 500 nm.

[0145] The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Lipoxygenase in the absence of active ingredient, i.e. only in the presence of the enzyme substrate (arachidonic acid).

[0146] Incubation protocol

[0147] 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.

[0148] Evaluation of effects

[0149] 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.

[0150] For each concentration tested, the modulation of the activity of the Lipoxygenase enzyme by the product under test is calculated according to the following formula:

[0151] [Math.3] Percentage modulation of Lipoxygenase enzyme activity = 100 x [(OD of test or reference product — OD of Lipoxygenase alone] / OD of Lipoxygenase alone]

[0152] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0153] Results

[0154] The results are presented in the following Table 5:

[0155] [Tables5] Samples Average OD Average OD Sample - OD Blank % enzyme activity % inhibition of enzyme activity 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% by mass in the reaction medium 0.202 0.037 25.00 75.00 Example at 2.00% by mass in the reaction medium 0.163 0.105 71.82 28.18

[0156] 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.

[0157] Example 9: acellular in vitro test for evaluating anti-inflammatory (soothing) activity on the phospholipase A2 enzyme.

[0158] 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 the company CAYMAN / INTERCHIM. Phospholipase A2 is a key enzyme upstream of the inflammatory process triggered by the arachidonic cascade.

[0159] MATERIALS

[0160] The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0161] 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 Img in lOOpl Ethanol; -substrate: arachidonic acid - sPLA2 assay buffer (10x).

[0162] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH and the INCU-SHAKER MINI from BENCHMARK.

[0163] METHODS

[0164] Test system

[0165] 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. The activity of phospholipase A2 can thus be evaluated by measuring the absorbance at 413 nm.

[0166] 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.

[0167] The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Phospholipase A2 in the absence of active ingredient, i.e. only in the presence of the enzyme substrate (diheptanoyl thio-PC).

[0168] Incubation protocol

[0169] 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 sample tested, then the chromogen DTNB is incorporated before a 15-minute incubation at 25°C.

[0170] Evaluation of effects

[0171] 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.

[0172] 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:

[0173] [Math.4] Percentage modulation of Phospholipase A2 enzyme factor = 100 x [(&O405 test or reference product - DC405 sPLAZ soot) / D0405 sPlA2 alone]

[0174] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0175] Results

[0176] The results are presented in the following Table 6:

[0177] [Tableauxô] Samples Average OD Average OD Sample - OD Blank Enzymatic activity (%) Inhibition of enzymatic activity (%) Blank 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% by mass in the reaction medium 0.890 0.510 37.69 62.31 Example 1 at 2.00% by mass in the reaction medium 0.963 0.281 20.79 79.21 Example 1 at 0.50% by mass in the reaction medium 1.066 0.806 59.55 40.45

[0178] 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.

[0179] Example 10: acellular in vitro test for evaluating tyrosinase activity.

[0180] The objective of this study is to evaluate the modulation of the activity of the enzyme ty rosinase by a sample in a cell-free in vitro model using tyrosinase, L-tyrosine and hydroquinone.

[0181] MATERIALS

[0182] The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0183] The reagents used are as follows: - monobasic potassium phosphate supplied by SIGMA; - hydroquinone supplied by SIGMA ALDRICH; - L-tyrosine supplied by SIGMA ALDRICH; - mushroom tyrosinase provided by SIGMA; - potassium hydroxide; - citric acid; - NaOH.

[0184] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH.

[0185] METHODS

[0186] Test system

[0187] A buffered solution of Tyrosinase is brought into contact with the substrate L-tyrosine to form a compound which will be our positive control.

[0188] 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.

[0189] In parallel, the sample is brought into contact with the Tyrosinase solution at the same time as the enzyme substrate.

[0190] The whole is incubated at 23°C for 60 minutes.

[0191] 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.

[0192] 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.

[0193] Evaluation of effects

[0194] For each concentration tested, the modulation of the activity of the Tyrosinase enzyme by the test product is calculated according to the following formula:

[0195] [Math.5] Percentage modulation of ^activity of the enzyme Tyrosinase = 100 x [(DO product: for testing or reference - DO Tyrosinase sede) / DO Tyrosinase seute]

[0196] If the result is negative, the percentage is expressed as inhibition of the enzyme.

[0197] Results

[0198] The results are presented in the following Table 7:

[0199] [Tables7] Samples Average OD Average OD Sample - OD Blank % enzyme activity % inhibition of enzyme activity 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% by mass in the reaction medium 0.071 0.013 6.90 93.10 Example 1 at 2.0% by mass in the reaction medium 0.099 0.062 33.94 66.06 Example 1 at 0.5% by mass in the reaction medium 0.179 0.151 82.40 17.60

[0200] 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.

[0201] In view of the results, this inhibitory activity is dose-dependent, which tends to demonstrate a specific inhibitory action on the active site of the enzyme.

[0202] Example 11: study of cytotoxicity on HELA cells

[0203] HeLa cells are an international reference human cancer cell line in cell biology.

[0204] 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).

[0205] MATERIALS

[0206] The sample tested is the hydrolyzed extract of deoiled hemp seed cake according to the invention (example 1).

[0207] The reagents used are as follows: - HeLa9903 cells supplied by Ephyla; - DMEM supplied by PanBiotech; - FBS CS provided 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 provided by Carlo Erba; - DMSO supplied by Fisher bioreagents; - PBS 10X supplied by biosolve; - Formaldehyde 37% supplied by Carlo Erba; - Purple crystal provided by Carlo Erba; - Na2HPO4 provided by Carlo Erba; - Na2HPO4.H2O provided by Carlo Erba; - Acetic acid provided by Carlo Erba.

[0208] The spectrophotometer used is the POLARSTAT OMEGA from BMG LABTECH.

[0209] METHODS

[0210] cell culture

[0211] The cells are maintained in 15ml of DMEM supplemented with 10% FBS-CS, 2mM L-Glutamine, 0.8mg / ml Geneticin, 16qg / ml Blasticidin S, in a 75cm2 flask 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. 100ql of this suspension is seeded in 60 of the 96 wells of the plate. The number of cells per well is 10,000. The perimeter of the plate (i.e. lines A and H and columns 1 and 12) is filled with 100qL 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).

[0212] After 24 hours of incubation, the treatment of the cells is carried out 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 carried out in duplicate in order to observe the effect of 24 hours and 48 hours of treatment on cell survival.

[0213] The cells are reincubated for 24 or 48 hours.

[0214] Crystal violet marking

[0215] The culture medium is removed from each well. The living cells, attached to the bottom of the plate, are rinsed with 100 μL of PBS and then fixed by adding 100 μL of a formaldehyde solution (10% in PBS) for 10 min. The cells thus fixed are rinsed 3 times with deionized water and then labeled with 50 μL of a 1% solution of crystal violet dissolved in a 200 mM sodium phosphate buffer, pH 6 for 20 to 30 min. The crystal violet not fixed to the cells is removed by 3 immersions of the plate in water (tap). The plate is dried and then the crystal violet incorporated in the cells is dissolved in 100 μL of a 1% solution of acetic acid for 20 to 30 min with moderate shaking. 80 μL of the crystal violet / acetic acid solution are transferred to a new 96-well plate in order to read the absorbance at 590 nm.

[0216] Evaluation of effects

[0217] For each concentration tested, the viability of HeLa cells is calculated according to the following formula:

[0218] [Math.6] Percentage of HeLa cell viability = 100 x (DCb® Sample / DO593 Control)

[0219] The sample will be considered toxic if the result is less than 50%.

[0220] Results

[0221] Results at 24 hours

[0222] The results are presented in the following Table 8:

[0223] [Tables8] 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

[0224] Results at 48 hours

[0225] The results are presented in the following Table 9:

[0226] [Tables9] Samples Average 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

[0227] In view of the homogeneity of the results obtained at 24h and 48h, from 24h the values are considered usable and representative.

[0228] 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.

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 it is obtained by the process comprising the following steps: a- defatting of the hemp seed cake by extraction with ethanol, advantageously 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.

3. Hydrolyzed extract according to any one of claims 1 or 2, characterized in that it comprises a peptide fraction with a molecular mass of between 9 and 12 KDa, advantageously of HKDa, and a peptide fraction with a molecular mass of between 16 and 18 KDa, advantageously of 17 kDa, measured by the separation method by denaturing gel electrophoresis in SDS-PAGE.

4. Hydrolyzed extract according to any one of claims 1 to 3, 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.

5. Peptide fraction of the hydrolyzed extract according to any one of claims 1 to 4, 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.

6. Peptide fraction of the hydrolyzed extract according to any one of claims 1 to 4, 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 17KDa.

7. Association of the hydrolyzed extract according to any one of the claims- dications 1 to 4 or one or more of its peptide fractions according to any one of claims 5 or 6 and an ethanolic extract of hemp seed cake, advantageously obtained in step a) of the process of claim 2.

8. Pharmaceutical composition comprising the hydrolyzed extract according to any one of claims 1 to 4 or one or more of its peptide fractions according to any one of claims 5 or 6 or the combination according to claim 7 and a pharmaceutically acceptable excipient.

9. Process for manufacturing the hydrolyzed extract according to any one of claims 1 to 4, characterized in that it comprises the following steps: a- defatting the hemp seed cake by extraction with ethanol, advantageously 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.

10. Hydrolyzed extract according to any one of claims 1 to 4 or peptide fraction of the extract according to any one of claims 5 or 6 or combination according to claim 7 or composition according to claim 8 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 anti-cancer agent and / or healing agent and / or anti-fibrotic agent, more advantageously for the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.