Protein hydrolysate from Moringa peregrina seed cake for use as a medicinal product, process for its preparation, and pharmaceutical and dermatological compositions

Description

Domaine technique

[0001] The invention relates to the fields of pharmacy and dermatology, and more particularly to active ingredients used in drug formulations. The invention relates to a process for obtaining a protein hydrolysate from the seed cake of... Moringa peregrina. The invention also relates to the protein hydrolysate of seed cake Moringa peregrina, pharmaceutical and dermatological compositions comprising such a hydrolysate and intended for use in the treatment of fibrotic diseases, the treatment of inflammation, cancer, and viral infectious diseases. Technological rear plan

[0002] The Moringaceae constitute a monogeneric family (a single genus, MoringaAdans), an element of the Saharan-Sindian flora, comprises between twelve and fourteen species according to different authors, distributed from East Africa to Asia. The genus is classically divided into three sections, which, however, are not confirmed as monophyletic by phylogenetic analyses. These analyses have instead highlighted clades based on certain morphological characteristics: pachycaul ("bottle trees"); tuberous ("tuberous trees"); and neither pachycaul nor tuberous (in English, "slender trees"). The species Moringa peregrina (Forssk.) Fiori belongs to the third group. The few genetic studies on the genus or family confirm the existence of the species in relation to other species in the genus, particularly with regard to the Indian Moringa, Moringa oleiferaLam. (see in particular the articles: OLSON, ME, 2002, Combining Data from DNA Sequences and Morphology for a Phylogeny of Moringaceae (Brassicales), Systematic Botany 27(1): p. 55-73; HASSANEIN, AMA et al., 2018, Morphological and genetic diversity of Moringa oleifera and Moringa peregrina genotypes, Horticulture, Environment and Biotechnology 59(2): pp. 251-261). A recent article on the Moringa peregrina Sampled from different locations in Saudi Arabia, using ITS markers, the species has a genetic stability (ALAKLABI, A., 2015, Genetic diversity of Moringa peregrina species in Saudi Arabia with ITS sequences, Saudi Journal of Biological Sciences 22: p. 186-190) with, however, a high level of intra-population genetic variation.

[0003] The species Moringa peregrinaIt is found in rocky environments in Yemen, Oman, and Saudi Arabia, in East Africa, Sudan, Ethiopia, Eritrea, Somalia, and Djibouti. Its presence in Iran appears limited to the southeastern provinces but requires confirmation (PROTA14 = MUNYANZIZA, E. et al. Vegetable oils / Oléagineux, Moringa peregrina (Forssk.) Fiori, http: / / database.prota.org / protahtml / moringa peregrina_fr.htm, accessed 23 / 10 / 2019). In the Levant and Egypt, the species now represents only a few scattered, relict populations (with the exception of some high-altitude stands), mainly in areas of the Sudanian zone. Moringa peregrina is also considered rare and endangered today in Sudan and Yemen. Moringa peregrina Compared to other species in its clade, it occupies the most arid and inhospitable habitats. It is apparently more drought-resistant than Moringa oleiferawhich is commercially cultivated on a large scale in tropical and subtropical areas. Recent work has shown that seed size and thickness favorably impact germination time and the rate and speed of growth of young trees (GOMAA, NH et al., 2011, Seed germination, seedling traits, and seed bank of the tree Moringa peregrina (Moringaceae) in a hyper-arid environment, American Journal of Botany 98(6): p. 1024-1030), indicating an adjustment in resource allocation based on seed quality rather than quantity, which allows Moringa peregrina to reproduce effectively in extreme abiotic (hyperarid) environments. The seeds of Moringa peregrina have a thicker central mesotesta, in terms of cell layer, than those of Moringa oleifera.

[0004] There are some historical references that tend to indicate that the oil of Moringa peregrinawas the subject of active trade in the early days of Islam in the AlUla region (NASEEF, AAS, 1995, Al-'Ul , A study of Cultural and Social Heritage ) . The oil produced from Moringa peregrinais now intended mainly for self-consumption or local markets. In Saudi Arabia, the leaves were traditionally used in decoction for internal use to treat diabetes, colon diseases, eye diseases and anemias (ABDEL-KADER et al., A survey on the traditional plants used in AI Kobah village, Saudi Pharmaceutical Journal 26(6): p. 817-821) and as a diuretic, rubefacient and astringent (AQEEL, AAM et al., 1984, Plants used in Arabia Folk medicine, Report submitted to Saudi Arabian National Centre for Science and Technology, Riyadh, Saudi Arabia). In Oman, the oil, extracted by women at the end of summer, is used to treat migraines, fever, burns, lacerations and fractures, constipation and stomach aches, muscle pain, and dry hair (GHAZANFAR, SA, 1994, Handbook of Arabian Medicinal Plants, 1st ed., CRC Press, Boca Raton, Ann Harbor, USA).; GHAZANFAR, SA, 1998, Plants of Economic Importance, cap. 15, in GHAZANFAR, SA et al. (ed.) Vegetation of the Arabian Peninsula. Geobotany 25, p. 241-264, Kluwer Academic Publishers, table 11.1, p. 247 and 11.7 p. 251). It was also used in perfumed compositions (GHAZANFAR, SA, 1998, p. 259) and in Oman and Yemen as a facial lotion (GHAZANFAR, SA et al., 1996, Two multi-purpose seed oils from Oman. Plants for Food and Medicine. Paper presented at the joint meeting of the Society for Economic Botany and International Society for Ethnopharmacology, 1-7 July 1996, London).

[0005] We know of a number of extracts derived from the seed of Moringa oleifera particularly useful in the cosmetics field. In the dermatological field, document FR2776519 indicates that protein extracts from seeds of Moringa oleifera,Known for their clarifying effects on turbid waters, they offer a softening, physiological conditioning, moisturizing, restructuring, repairing, and anti-pollution effect on the skin and mucous membranes. In this document, the active ingredients are proteins with molecular weights between 6,500 and 8,800 Da, obtained by aqueous extraction from the press cake of Moringa oleifera.

[0006] In the pharmaceutical field, document KR20140143655 describes a composition containing a water-soluble extract of leaves of Moringa oleifera as an active ingredient to treat or prevent cancer. The enzymatic hydrolysis process on leaf extracts of Moringa oleifera allows the isolation of proteins with molecular weights between 6,000 and 8,000 Da.

[0007] Document CN107012190 concerns the use of seeds of Moringa oleiferaTo obtain, after extracting the oil and performing enzymatic hydrolysis using microwave radiation, polypeptides of the seed proteins are produced. The resulting product is taken orally and is believed to have anticancer activity. This powdered product is described as having an effect on reducing the side effects of chemotherapy and increasing the appetite of patients with liver cancer, as well as controlling white blood cell count and body temperature.

[0008] Finally, we know from document IN2009CH02906 that an aqueous extract of the oilseed cake Moringa oleifera containing glucosinolate combined with cationic proteins can be used as an anti-diabetic product.

[0009] All the aforementioned documents concern the use of the species Moringa oleifera; None of them describe the use in the pharmaceutical or dermatological field of extracts from the species Moringa peregrina.

[0010] We are also familiar with the document by Abu Tarbosh et al. XP055753092, 2005 which describes the extraction of a hydrolysate from seeds of Moringa peregrina The hulled product is produced by enzymatic hydrolysis for a period of 10 hours. The purpose of this extraction is to obtain a product with a high capacity for absorbing oil and water.

[0011] An extract of the oil of Moringa peregrinaPrepared from Egyptian seeds, with a dichloromethane / methanol mixture (1:1), showed activity against three human cancer cell lines: MCF-7 (breast adenocarcinoma), Hep-G2 (hepatocellular carcinoma), and HCT-116 (colon carcinoma), with IC50 values ​​of 2.92, 9.40, and 9.48 µg / min (ABD EL BAKY et al., 2013, Characterization of Egyptian Moringa peregrina seed oil and its bioactivities, International Journal of Management Sciences and Business Research, 2(7): p. 98-108). The authors also demonstrated significant antioxidant activity through assays DPPH (2,2-diphenyl 1-picrylhydrazyl), ABTS (anion-scavenging capability and reducing power) and anti-proliferative. Due to extraction processes different from those of the invention, the molecules obtained have very different polarities.

[0012] ABOU-HASHEM and colleagues (ABOU-HASHEM, MMM et al., 2019, Induction of sub-G0 arrest and apoptosis by seed extract of Moringa peregrina (Forssk.) Fiori in cervical and prostate cancer cell lines, Journal of Integrative Medicine 17: pp. 410-422) also demonstrated sub-G0 arrest and apoptosis induction activity in cervical (HELA) and prostate (PC-3) cancer cell lines. The protocol involved seeds of Moringa peregrinaEgyptian leaves, reduced to powder and extracted to 95% ethanol, were then dissolved in an aqueous solution from which three fractionated extracts were studied (fractions by petroleum ether (PE), CHCl3, and EtOAc) as well as a residual hydrolic extract. Activity was demonstrated for the total chloroform fraction and was attributed to saturated and unsaturated fatty acids and polyphenols without a specific mechanistic study. Due to different extraction methods than those of the present invention, the active molecules obtained have very different polarities. Carrie Waterman et al. ("Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro," Phytochemistry, vol. 103, July 1, 2014, pages 114-122) disclose the preparation of an extract from the leaves of Moringa oleiferarich in isothiocyanate compounds obtained by water or ethanol extraction. The resulting extract demonstrates an anti-inflammatory effect. in vitro.

[0013] Considering the above, one problem that the invention aims to solve is to develop new products based on an extract of the species Moringa peregrina of the kind Moringa and from the Moringaceae family that are usable in pharmacy or dermatology and easy to implement.

[0014] Thus, the Applicant has surprisingly highlighted a particular protein hydrolysate obtained from the seed cake of Moringa peregrina for its application as a medicinal product, and in particular for the treatment of fibrotic diseases (as a furin convertase inhibitor), the treatment of inflammation, cancer, and viral infectious diseases.

[0015] The species Moringa peregrinagrows in very arid climates. Thus, its ability to resist drought and reproduce in extremophile conditions has allowed it to acquire unique characteristics, which the Applicant was able to identify by implementing a specific extraction process on the seed cake of Moringa peregrina. The protein hydrolysate according to the invention is shown to possess properties as a drug and it is demonstrated that it exhibits in particular a very high inhibitory activity of furin convertase.

[0016] Furin convertase (hereafter referred to as furin) is a type 1 transmembrane protein with 794 amino acids expressed in various cell types. Furin is a protein that has been shown to be involved in a large number of biological processes (BRAUN E. et al., 2019, Furin-mediated protein processing in infectious diseases and cancer, Clinical & Translational Immunology https: / / doi.org / 10.1002 / cti2.1073).).It plays a role in wound healing and in regulating fibrosis in the treatment of conditions where fibrosis is a major tissue repair mechanism, or where excessive fibrosis leads to pathological tissue disturbance and dysfunction (see document WO200409113, which concerns the use of convertase to reduce scarring during wound healing and to reduce fibrosis in the treatment of fibrotic conditions). Wound healing in adults is a complex repair process. Wounds can cause damage, injury, or trauma to tissue or internal organs such as the lungs, kidneys, heart, intestines, tendons, or liver. The wound healing process in tissue typically begins with a hemostatic response triggered by damage to the skin's blood vessels.During this process, the connective tissue that forms during the healing process is often fibrous in nature and typically forms a connective tissue scar (a process called fibrosis). Thus, fibrosis can include pulmonary fibrosis, renal fibrosis, hepatic fibrosis, cutaneous fibrosis, ocular fibrosis, cardiac fibrosis, and other diverse fibrotic conditions.Protein hydrolysates according to the invention may also be useful for treating other furin-mediated pathological conditions, including, but not limited to, hypertension, cancer, infectious diseases (bacterial and viral) and genetic disorders (e.g., cystic fibrosis (CF)), and neurodegenerative disorders (see in this regard document WO2019215341 which relates to novel furin convertase inhibitor compounds and pharmaceutical compositions containing them, and which cites numerous publications demonstrating the pharmaceutical activity of inhibitors, which citations are incorporated by reference in the present description).

[0017] By intergovernmental agreement of April 10, 2018, between the Government of the French Republic and the Kingdom of Saudi Arabia, the applicant, the French Agency for the Development of AlUla (AFALULA), and the Royal Commission for AIUIA (RCU) have a joint project aimed at developing responsible agriculture and the local economy, particularly through the local production of natural products derived from indigenous plants, and at protecting the biodiversity and rights of the AlUla region of the Kingdom of Saudi Arabia. The Kingdom of Saudi Arabia has been a party to the Nagoya Protocol since October 8, 2020. At the time of drafting this patent application, the implementing regulations under which the Nagoya Protocol will be incorporated into the relevant aspects of local law are under review. Therefore, at this stage, the Kingdom of Saudi Arabia has no specific requirements with regard to this patent application and the Nagoya Protocol.Thus, on the date of filing the patent application, there is no requirement for a certificate of conformity regarding access to genetic resources. Summary

[0018] The invention's primary object is a process for obtaining a protein hydrolysate from the seed cake of Moringa peregrina, including the following steps according to which: a) The unhulled seeds are collected when ripe from the fruit of Moringa peregrinaa) the seeds are dried to obtain an internal moisture content of less than 8%, b) the dried seeds are pressed to separate the oil from the rest of the seed, so as to obtain a press cake containing less than 6% residual oil by weight, c) the press cake obtained in step b) is ground, d) the ground press cake obtained in step c) is dispersed in an aqueous phase, e) the aqueous dispersion obtained in step d) is chemically proteolyzed for approximately 2 hours at a pH above 13 and a temperature between 16 and 25°C, f) the proteolysis is neutralized to stabilize the resulting protein hydrolysate, g) the protein hydrolysate is recovered by solid / liquid separation, h) the protein hydrolysate is purified by ultrafiltration and / or nanofiltration, and then optionally, i) freeze-dried the protein hydrolysate obtained in step h).

[0019] The invention also relates to a protein hydrolysate of the press cake of unhulled seeds harvested at maturity from the fruit of Moringa peregrina comprising a major fraction of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight P1 between 1,500 Da and 5,000 Da, a fraction of about 20% (mass / mass) with a molecular weight P2 between 10,000 and 17,000 Da and a fraction of about 20% (mass / mass) with a molecular weight P3 of about 23,000 Da, in that it is obtained by chemical proteolysis at pH above 13 for a period of about 2 hours at a temperature between 16 and 25°C and in that it is liquid and has a density greater than 1 and preferably around 1.1.

[0020] Thanks to its characteristics, the protein hydrolysate of Moringa peregrina according to the invention has never been highlighted in the genre Moringaand the Moringaceae family. It will be demonstrated that the extract of the species Moringa peregrina exhibits a particular peptide profile, different from other species of the genus, notably the species Moringa oleifera, which the plaintiff was able to highlight.

[0021] The invention has as its third object a protein hydrolysate of seed cake Moringa peregrina for its application as a medicine.

[0022] The invention has as its fourth object a pharmaceutical or dermatological composition comprising, as an active agent, an effective amount of a protein hydrolysate of the seed cake of Moringa peregrina and a physiologically acceptable excipient.

[0023] The invention also has as its fifth object a pharmaceutical or dermatological composition intended to be used for the treatment of fibrotic diseases, skin fibrosis, the treatment of inflammation, cancer, viral infectious diseases and lentigo. Brief description of the figures

[0024] The invention will be better understood, and other objects, details, features and advantages thereof will become more apparent from the following description of several particular embodiments of the invention, given solely by way of illustration and not limitation, with reference to the accompanying drawings. [ fig.1 [ ] represents a diagram of SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) infection, demonstrating the anti-infective and virostatic effect of protein hydrolysate pilgrim according to the invention against SARS-CoV-2 crown pseudovirions. fig.2] represents the diagram of furin inhibition by a peregrina oil extract. In this figure, ** means significantly different from the "control" group (P<0.001). fig.3 ] represents the diagram of furin inhibition by a peregrina cake extract (96° ethanol). In this figure, * means significantly different from the "control" group (P<0.001). fig.4 Figure ] represents the furin inhibition diagram by a peregrina meal protein hydrolysate according to the invention. In this figure, *** means significantly different from the "control" group (P<0.001). Description of realization modes

[0025] In this description, unless otherwise specified, it is understood that, when an interval is given, it includes the upper and lower bounds of said interval.

[0026] In the present invention, the following abbreviations mean: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (the MTT test is a rapid method for live cell counting) SDS: Sodium Dodecyl Sulfate PBS: Phosphate Buffer Saline ELISA: Enzyme-Linked Immunosorbent Assay PCR: Polymerase Chain Reaction ANOVA: Analysis of Variance MSH: Melanocyte Stimulating Hormone

[0027] In the present invention, the following definitions apply: "Predominantly amino acid derivatives, amino acids, peptides, and glycopeptides" in a quantity greater than 20%, preferably greater than 30%, and potentially reaching approximately 40% (by weight) of dry matter in amino acid derivatives, amino acids, peptides, and glycopeptides, preferably 50% by weight of dry matter. "Effective quantity" the quantity of active molecules necessary to achieve the desired result, namely, to ensure the desired therapeutic activity. "Proteolysis" the segmentation of proteins into peptides, oligopeptides, their basic fragments (amino acids), and their residues via chemical hydrolysis. "Local route" the fact that the drug, applied directly to its site of action, exerts its pharmacological effect on the precise site of the condition. The purpose of the local route is to limit the diffusion of the active ingredient from its site of administration, thus minimizing adverse effects.The main local routes of administration are the cutaneous, nasal and respiratory, ocular, auricular, vaginal, and oral routes. "Topical application" means applying or spreading the active ingredient according to the invention, or a composition containing it, to the surface of the skin, mucous membranes, or hair. "Physiologically acceptable" means suitable for topical use, in contact with human skin, or for use by other routes of administration, for example, the oral route or injection into the skin, without risk of toxicity, incompatibility, instability, or allergic reaction. "Oatcake" means the de-oiled portion of the seed after pressing. It is the solid residue from oil extraction. It is a co-product of crushing, the oil production process. It generally represents 50 to 75% of the seed mass."Unhulled seeds" means that the shell (pericarp) and seed coat of the harvested seeds remain attached to the kernels. "At fruit maturity" means that the fruit is ripe, preferably when the pod is beginning to dehisce and turns a dark beige to brown color, and when a 180° twist on the lower quarter of the pod causes the valves to begin opening. "Approximately" means a margin of plus or minus 10% to 20% compared to the information given. "Pool of active molecules," "active agent," and also "active ingredient" means the protein hydrolysate extracted according to the process of the invention from the seed cake of [unspecified ingredient]. Moringa peregrina.This hydrolysate is responsible for the biological activities described in the present invention. "Active agent" means a sufficient quantity of an extract according to the invention to obtain the described biological activities. Depending on whether the extract is liquid or dried, concentrated or not, the quantities of the active agent can vary from 0.0001 to 40% by weight relative to the total weight of the composition.

[0028] The invention relates primarily to a process for obtaining a protein hydrolysate from the seed cake of Moringa peregrina including the following steps according to which: a) The unhulled seeds are collected when ripe from the fruit of Moringa peregrinaa) the seeds are dried to obtain an internal moisture content of less than 8%, b) the dried seeds are pressed to separate the oil from the rest of the seed to obtain a press cake containing less than 6% residual oil by weight, c) the press cake obtained in step b) is ground, d) the ground press cake obtained in step c) is dispersed in an aqueous phase, e) the aqueous dispersion obtained in step d) undergoes chemical proteolysis for approximately 2 hours at a pH above 13 and a temperature between 16 and 25°C, f) the proteolysis is neutralized to stabilize the resulting protein hydrolysate, g) the protein hydrolysate is recovered by solid / liquid separation, h) the protein hydrolysate is purified by ultrafiltration and / or nanofiltration with a cutoff between 100 and 25,000 Da. Then, according to a particular embodiment, i) the protein hydrolysate obtained in step h is lyophilized.

[0029] The seeds are harvested unshelled, that is to say, those whose shell is kept at the maturity of the fruit and preferably when the pod is at the beginning of dehiscence.

[0030] The seeds are dried to obtain an internal moisture content of less than 8% and preferably around 6%; drying is preferably done on a ventilated rack away from the sun's rays, preferably under a shade structure in the open air.

[0031] The dried seeds are then ground extemporaneously by cold pressing, which allows the oil to be mechanically separated from the rest of the compressed seed, namely the press cake.

[0032] The oilcake is then mechanically ground with any type of mechanical crusher such as hammer mill, flail mill, knife mill, crusher / shredder, ball mill or pellet mill, but also with any type of cryo-mill.

[0033] The aqueous phase dispersion according to step d) and the proteolysis according to step e) are advantageously always carried out under agitation, thus allowing dispersion and homogenization of the solid in the liquid, thereby improving the overall exchange surface and consequently the proteolysis.

[0034] A liquid protein hydrolysate is obtained with a density greater than 1 and preferably around 1.1, comprising a dry matter content of between 10 and 15%, preferably around 12.5%, comprising between 1 and 6% of nitrogen compounds, including volatile nitrile derivatives in a proportion of 0.5 to 1.5%, preferably around 0.8%, as well as 20 mg / litre of polyphenols (0.002%).

[0035] According to a preferred embodiment of the process according to the invention, the temperature of proteolysis in step f) is approximately 22°C.

[0036] According to a preferred embodiment of the process according to the invention, the solid / liquid separation of step g) is carried out by different processes such as centrifugation, dewatering, filtration.

[0037] In one embodiment of the process for obtaining protein hydrolysate Moringa peregrina The resulting liquid, the protein hydrolysate, is purified by distillation, microfiltration, ultrafiltration, and / or nanofiltration to concentrate it in compounds of interest relative to the extracted organic matter, particularly other extracted derivatives. These purification steps allow for the concentration of the pool of compounds of interest at the expense of other extracted compounds, as mentioned.

[0038] According to another preferred embodiment of the process according to the invention, nanofiltration is carried out so as to separate 3 bands or fractions of the protein hydrolysate comprising a band P1 whose molecular weight is less than 10,000 Da, a band P2 whose molecular weight is between 10,000 and 17,000 Da and a band P3 whose molecular weight is about 23,000 Da.

[0039] It is also advantageous to separate the three bands by nanofiltration in order to obtain, via the process of obtaining a protein hydrolysate: a P1 band with nanofiltration carried out with a cut-off threshold between 1,500 Da and 5,000 Da, preferably with a cut-off threshold between 3,000 Da and 4,500 Da. a P2 band with nanofiltration carried out with a cut-off threshold between 10,000 Da and 17,000 Da. a P3 band with nanofiltration carried out with a cut-off threshold between 17,000 Da and 25,000 Da.

[0040] In another embodiment of the extraction process according to the invention, the liquid protein hydrolysate obtained is dried to obtain a dry hydrolysate of the seed cake. Moringa peregrina containing an amount greater than 10%, preferably greater than 20%, preferably even greater than 30% and up to about 40% (W / W) of dry matter in peptides, oligopeptides, glycopeptides and in amino acids or their volatile nitrilated derivatives, preferably even 50% of the dry matter weight.

[0041] According to one embodiment of the invention, the protein hydrolysate of seed cake Moringa peregrina The resulting liquid is dried, for example by spray drying, freeze-drying or zeodration, in order to obtain a seed hydrolysate of Moringa peregrinasolid, the water having evaporated. Drying can be carried out in the presence of an organic support such as maltodextrin, cyclodextrin or inulin, or in the presence of an inorganic support such as phyllosilicate, magnesium silicate or carbonate and its salts.

[0042] The invention also relates to the protein hydrolysate of seed cake Moringa peregrina obtained by the extraction process according to the invention.

[0043] The invention also relates to a protein hydrolysate of the press cake of unhulled seeds harvested at maturity from the fruit of Moringa peregrina,comprising a major fraction P1 of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight between 1,500 Da and 5,000 Da, a fraction P2 of about 20% (mass / mass) with a molecular weight between 10,000 and 17,000 Da and a fraction P3 of about 20% (mass / mass) with a molecular weight of about 23,000 Da, in that it is obtained by chemical proteolysis at pH above 13 for a period of about 2 hours at a temperature between 16 and 25°C and in that it is liquid and has a density greater than 1 and preferably around 1.1.

[0044] According to one embodiment, the liquid protein hydrolysate obtained is dried to obtain a dry hydrolysate of the seed cake. Moringa peregrinacontaining more than 20%, preferably more than 30% and up to about 40% (W / W) of dry matter in peptides, oligopeptides, glycopeptides and in amino acids or their volatile nitrilated derivatives, preferably still 50% of the dry matter weight.

[0045] According to a preferred embodiment, the protein hydrolysate also comprises between 0.3% and 3% of volatile compounds, of which 50% of these compounds, i.e. between 0.15% and 1.5% of the extract according to the invention, consists of light nitrilated compounds, mainly isobutyronitrile and methyl-butanenitrile; of which 5 to 10% of these compounds, i.e. between 0.015% and 0.3% of the extract according to the invention, consists of isothiocyanate derivatives, mainly isopropyl isothiocyanate and isobutyl isothiocyanate; of which 1 to 5% of these compounds, i.e. between 0.003% and 0.15%, consists of essential oil, mainly Eucalyptol, Menthol and Benzaldehyde.

[0046] According to yet another embodiment, the protein hydrolysate comprises a dry matter content of between 10 and 15%, preferably around 12.5%, including between 1 and 6% nitrogenous compounds, in particular volatile nitrile derivatives in a proportion of 0.5 to 1.5%, preferably around 0.8%, as well as 20 mg / liter of polyphenols. In the context of the present invention, the plant part chosen is the seed of Moringa peregrina. It is known that the seeds of Moringa peregrina They are used for the extraction of their oil, useful for personal consumption or in various traditional medicinal treatments. The press cake obtained after the seed has been de-oiled is a waste product currently used, notably for animal feed.

[0047] According to yet another embodiment, the protein hydrolysate comprises the major fraction P1, the molecular weight of which is between 1,500 Da and 5,000 Da.

[0048] According to yet another embodiment, the protein hydrolysate comprises the P2 fraction of about 20% (w / w) whose molecular weight is between 10,000 Da and 17,000 Da.

[0049] According to yet another embodiment, the protein hydrolysate comprises the P3 fraction of about 20% (w / w) whose molecular weight is about 23,000 Da.

[0050] According to yet another embodiment, the protein hydrolysate comprises fraction P1, whose molecular weight is between 1,500 Da and 5,000 Da, and fraction P2, whose molecular weight is between 10,000 and 17,000 Da.

[0051] The invention has as its third object a protein hydrolysate of seed cake Moringa peregrina for its application as a medicine.

[0052] The invention has as its fourth object a pharmaceutical or dermatological composition comprising, as an active agent, an effective amount of a protein hydrolysate of the seed cake of Moringa peregrina according to the invention and a physiologically acceptable excipient.

[0053] The compositions according to the invention can be presented in all the pharmaceutical forms normally used depending on whether the composition is to be ingested, injected or applied to the skin or mucous membranes.

[0054] According to one variant, the various compositions are suitable for ingestion; the composition may be in the form of capsules, syrups, granules, or tablets. It may not contain any excipients and may consist entirely of the plant extract, including the protein hydrolysate in dried form.

[0055] According to a second variant, the different compositions are adapted for injection; the composition can be in the form of an aqueous lotion, an oily lotion or a serum.

[0056] According to a third variant, the different compositions are intended more specifically for local administration, allowing a pharmacological effect on the precise site of the condition via the skin or mucous membranes.

[0057] The principal local routes used for administering the active agent according to the invention are the cutaneous and percutaneous routes, the nasal and respiratory routes, the ocular route, the auricular route, and the vaginal route. Other routes may be considered, including the oral route for administering the compositions via mucous membranes, as well as the subcutaneous route by microinjections.

[0058] The compositions according to the invention for pharmaceutical purposes can be presented in all the pharmaceutical forms normally used for topical application. They can be administered to mucous membranes, in particular nasal or respiratory, ocular, oral, vaginal and auricular.

[0059] The compositions according to the invention for dermatological purposes can be presented in all the pharmaceutical forms normally used in the field of dermatology for cutaneous application. They can be administered transdermally or applied topically to the skin.

[0060] Compositions incorporating a protein hydrolysate according to the invention may include ingredients commonly used in this type of formulation by local or cutaneous route.

[0061] According to a preferred embodiment, the various compositions are adapted for topical administration and include creams, oil-in-water and water-in-oil emulsions, milks, ointments, lotions, oils, balms, aqueous or hydro-alcoholic or glycolic solutions, serums, powders, patches, sprays or any other product for external application such as, for example, medical devices or aerosol products also containing a pressurized propellant.

[0062] According to another preferred embodiment, the various compositions are suitable for subcutaneous injection and transdermal administration; the composition may be in the form of an aqueous lotion, emulsion, or serum. In transdermal systems or patches, the release of the active ingredient(s) is controlled by a permeable and generally adhesive membrane in direct contact with the skin.

[0063] The compositions according to the invention, intended more particularly for topical administration, contain an acceptable pharmaceutical or dermatological medium, i.e., one compatible with the skin and mucous membranes, and cover all suitable dosage forms. These compositions may, in particular, be in the form of creams, oil-in-water or water-in-oil emulsions or multiple emulsions, serums, solutions, suspensions, gels, milks, lotions, sticks, aerosols, sprays, or any other product for external application, such as, for example, medical devices or aerosol products also containing a pressurized propellant, or in the form of powders, and are suitable for application to the skin and mucous membranes. These compositions include the excipients necessary for their formulation, such as solvents, emollients, thickeners, diluents, surfactants, antioxidants, bioactive agents, colorants, preservatives, and perfumes.

[0064] The compositions according to the invention therefore include any additive commonly used in the field of application envisaged as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, sun filters, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, dermatological or pharmaceutical actives, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[0065] In all cases, a person skilled in the art will ensure that these adjuvants and their proportions are chosen in such a way as not to impair the advantageous properties sought in the compositions according to the invention.

[0066] In the compositions of the invention, the protein hydrolysate according to the invention is used in an amount ranging from 0.0001 to 40% by weight relative to the total weight of the composition.

[0067] In a preferred embodiment, the protein hydrolysate according to the invention is used in an amount ranging from 0.001 to 10% by weight relative to the total weight of the composition, preferably again in an amount ranging from 0.01% to 5% by weight relative to the total weight of the composition.

[0068] Finally, the invention has as a fifth object a pharmaceutical or dermatological composition for use as a medicinal product for the treatment of: For fibrotic diseases and the treatment of inflammation, comprising as an active agent an effective amount of the P1 or P2 fraction of the protein hydrolysate, preferably the P1 fraction. For cancer, comprising as an active agent an effective amount of the P3 fraction of the protein hydrolysate. For viral infectious diseases, and in particular for inhibiting the Spike-COV2 proteins of SARS-CoV, comprising as an active agent an effective amount of the entire protein hydrolysate. For lentigo, comprising as active agents an effective amount of the P1 and P2 fractions of the protein hydrolysate.

[0069] "Genetic drift" refers in particular to environmental stress.

[0070] For the treatment of fibrotic diseases, fibrosis is understood to mean pulmonary fibrosis, renal fibrosis, hepatic fibrosis, cutaneous fibrosis, ocular fibrosis, cardiac fibrosis, and other miscellaneous fibrotic conditions, and for the treatment of other furin-mediated pathological conditions, including, but not limited to, hypertension, cancer, infectious diseases including viral and bacterial, genetic disorders (e.g., cystic fibrosis (CF)), and neurodegenerative disorders.

[0071] Although the invention has been described in connection with several particular embodiments, it is clearly evident that it is by no means limited to them and that it includes all technical equivalents of the means described as well as their combinations if these fall within the scope of the invention.

[0072] The use of the verb "comporter", "comprendre" or "include" and its conjugated forms does not exclude the presence of other elements or steps than those stated in a claim. Examples Example 1: Preparation of a plant protein hydrolysate according to the invention from the oilseed cake of Moringa peregrina

[0073] Seeds of Moringa peregrina (Forssk.) Fiori seeds were dried to obtain an internal moisture content of less than 8%, and preferably around 6%, then pressed with a mechanical screw press to separate the oil from the rest of the seed, yielding virgin oil on the one hand and press cake on the other. The press cake is then separated into pre-cut rolls of 1 to 2 cm pieces, from which the extraction is carried out.

[0074] The raw materials used are as follows. [Table 1] Materials % Actual quantity weighed Crab of peregrina pre-cut into 1-2 cm pieces 8.4% 33.6 Mains water 80.68% 322.7 Sodium hydroxide 3,36 % 13.5 Citric acid monohydrate F6000 7.14% 28.7 Sodium benzoate 0.42% 1.7 100 400.2 Protocol:

[0075] a) A concentrated 1 molar solution of a strong alkali, such as sodium hydroxide, potassium hydroxide, or calcium hydroxide, or an aqueous mixture containing 1 molar concentrations of strong alkali, but preferably sodium hydroxide, is prepared; this strong alkali solution has a pH between 13 and 14. b) 9.1% (w / w) of oilseed cake is weighed out. peregrinapre-cut into pieces of approximately 1 cm in 90.9% (w / w) of the alkaline solution, c) the cake obtained in step b is ground, d) the ground cake obtained in step c is dispersed in aqueous phase, e) chemical proteolysis of the aqueous dispersion obtained in step d) is carried out for a period of 2 hours and at a temperature of 22°C, f) the proteolysis is neutralized to stabilize the protein hydrolysate obtained, g) the protein hydrolysate is recovered by solid / liquid separation by passing through a 1 µm filter, h) the protein hydrolysate is purified by ultrafiltration,

[0076] A translucent yellow filtrate containing approximately 12.48% dry matter is obtained. The resulting liquid extract is subsequently referred to as "protein hydrolysate". peregrina according to the invention" or "protein hydrolysate" peregrina » or "extract according to the invention".

[0077] This protein hydrolysate peregrinaThe product according to the invention has a density greater than 1 and preferably around 1.1, comprising a dry matter content of between 10 and 15%, preferably around 12.5%, comprising between 1 and 6% nitrogenous compounds, in particular volatile nitrile derivatives in a proportion of 0.5 to 1.5%, preferably around 0.8%, and 20 mg / liter of polyphenols. The composition of the protein hydrolysate peregrina dry according to the invention is given below. [Table 2] Tr CAS NO. Compounds % MS 4.42. 64-17-5 Ethanol 0.809 5.58 67-64-1 Acetone 0.444 6.10 75-15-0 Carbon disulfide 0.327 7.83 78-93-3 2-Butanone 0.165 8.57 141-78-6 Ethyl acetate 0.047 8.99 78-82-0 Isobutyronitrile 20.720 10.39 78-82-0 Isobutyronitrile 0.127 12.06 547-63-7 Methyl isobutyrate 0.201 14.10 18936-17-9 2-Methylbutanenitrile 2.194 14.56 625-28-5 3-Methylbutanenitrile 27.495 18.77 66-25-1 Hexanal 0.186 21.09 2253-73-8 Isopropyl isothiocyanate 4.590 23.65 628-73-9 Hexanenitrile 0.198 24.43 110-43-0 2-Heptanone 0.093 27.18 4426-79-3 2-Isothiocyanato-Butane 1.058 27.59 80-56-8 Alpha-Pinene 0.048 28.45 591-82-2 isobutyl isothiocyanate 2.299 28.99 100-52-7 Benzaldehyde 1.753 29.77 108-95-2 Phenol 0.413 30.80 13475-82-6 2,2,4,6,6-pentamethyl-Heptane 0.318 32.80 99-87-6 Para-Cymene 0.232 33.10 138-86-3 Limonene 0.133 33.34 470-82-6 Eucalyptol 0.918 36.45 1195-32-0 Para-Cymenene 0.035 36.88 124-19-6 Non-analytical. 0.080 40.26 65-85-0 Benzoic acid 19.150 41.07 1490-04-6 Menthol 0.918 56.29 96-76-4 2,4-Di-telt-butylphenol 0.086 Total 85.035 Protein hydrolysate peregrina contains isopropyl isothiocyanate and isobutyl isothiocyanate at relatively high levels, confirming previous publications on the species ( KJAER, A. et al. 1979, Isothiocyanates in Myrosinase-treated seed extracts of Moringa peregrina, Phytochemistry, 18, p. 1485-1487 ; AFSHARYPUOR, S. et al., 2010, Volatile Constituents of the Seed Kernel and Leaf of Moringa peregrina (Forssk.) Fiori, Agricolt. Cultivated in Chabahar (Iran), Iranian Journal of Pharmaceutical Sciences 6(2): p. 141-144 ; DEHSHAHRI, S. et al., 2012, Determination of volatile glucosinate degradation products in seed coat, stem and in vitro cultures of Moringa peregrina (Forssk.) Fiori, ScienceOpen, Research in Pharmaceutical Sciences 7(1): p. 51-56 Isothiocyanates are compounds produced by various plants belonging to the order Brassicales, particularly in the families Brassicaceae, Capparaceae, Caricaceae, and Moringaceae, as a defense system against pathogen attacks. In the genus Moringa, They were identified in particular in M. oleifera Lam. and Mr. stenopetala (Baker f.) Cufold. ( ABD RANI, NZ et al., 2018, Moringa genus: A review of Phytochemistry and Pharmacology, Frontiers in Pharmacology, vol. 9, art. 108, p. 3-8 Isothiocyanates are produced by the hydrolysis of glucosinolates by the enzyme myrosinase when the plant tissues are damaged. Isothiocyanates have been reported to have various biological effects, such as antifungal activity ( TRONCOSO-ROJAS, R. et al., 2007, Natural compounds to control fungal diseases in fruits & vegetables, in TRONCOSO-ROJAS, R., TIZNADO-HERNANDEZ, M. E., GONZALEZ-LEON, A. (éd) Recent advances in alternative postharvest technologies to control fungal diseases in fruits & vegetables. Transworld Research Network, Kerala, India, p. 127-156 ; TRONCOSO-ROJAS, R. et al., 2005, Analysis of the isothiocyanates present in cabbage leaves extract and their potential application to control Alternaria rot in bell peppers, Food Research International 38, p. 701-708 ), des effets anti-microbiens, anti-cancéreux et anti-inflammatoires ( PARK, E. J. et al., 2011, Inhibition of lipopolysaccharide induced cyclooxygenase-2 expression and inducible nitric oxyde synthase by 4-[(2'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate from M. oleifera, Nutrition and Cancer 63(6), p. 971-982 ; RAJAN TS et al., 2016, Anticancer activity of glucomoringin isothiocyanate in human malignant astrocytoma cells, Fitoterapa 110, p. 1-7 ; Padla, EP et al. 2012, Antimicrobial isothiocyanates from the seeds of Moringa oleifera Lam., Zeitschrift für Naturforschung C, 67, p. 557-564 ; WATERMAN, C. et al., 2014, Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro. Phytochemistry 103, p. 114-122 Furfural is present at a very standard concentration, found in this type of seed and in many nuts. Carbon disulfide, isobutyronitrile, methyl isobutyrate, methyl butanenitrile, and hexanenitrile are volatile compounds derived from amino acids. They are markers of protein degradation. These degradation compounds represent approximately 50% of the volatile compounds in the protein hydrolysate of peregrina. This result indicates that the hydrolysate is primarily composed of protein. Only very small traces of fats or free sugars were detected in the protein hydrolysate. peregrina The hydrolysate is primarily composed of proteins and glycoproteins. In the dry matter, the citrate buffer (which is a glycosidic compound) represents approximately 50% of the remainder and includes glycosylated (glycosidic) compounds resulting from the proteolytic degradation of proteins, oligopeptides, and amino acids. Finally, it should be noted that approximately 21 mg / liter of polyphenols (0.002%) from the seeds are present. Moringa peregrina. Benzoic acid is not to be considered in the characterization as it is a stabilizing agent added to the extract.

[0078] The dry extract described above is obtained by a gravimetric method based on the mass before and after evaporation present in the liquid extract. Example 2: Effect of protein hydrolysate peregrina according to the invention as an inhibitor of furin convertase (also known as furin)

[0079] The objective of this study is to evaluate the inhibitory activity of protein hydrolysate from seed cake of Moringa peregrina, obtained according to example 1.

[0080] Protocol: The study is performed using recombinant human furin, which catalyzes the cleavage of a specific fluorescent reference substrate.

[0081] Decanoyl-Arg-Val-Lys-Arg-CMK at 100 nm was used as the reference inhibitor of furin activity.

[0082] Furin was pre-incubated for 10 minutes at room temperature in the absence (control) or presence of the reference product or increasing concentrations of the tested compound: "Protein Hydrolysate" peregrina » ; 0.02; 0.2 and 2% (v / v).

[0083] At the end of the pre-incubation step, a furin substrate was added and the experimental conditions were incubated again at room temperature in the dark for 5 minutes. All experiments were performed in triplicate. Preparation of compounds:

[0084] Protein hydrolysate peregrina The tested sample was solubilized directly into the titration buffer and then diluted to achieve the concentration to be tested described above. Evaluation Protocol

[0085] The cleavage of the fluorescent furin substrate was monitored for 5 minutes after the addition of the substrate by reading the fluorescence at 485 nm / 535 nm. Statistics

[0086] The results are expressed in RFU (relative fluorescent units) + / - SD (standard deviation).

[0087] The statistical significance of the difference observed between the "Control" and "Reference Product" groups was assessed by a Student's t-test (p <0.001).

[0088] The statistical significance of the observed difference between the "Control" or "Control" and "Trial Compound" groups was assessed by a one-way ANOVA analysis, followed by a HolmSidak test (p <0.05).

[0089] The reference furin inhibitor named Decanoyl-Arg-Val-Lys-Arg-CMK, tested at 100 nm, significantly inhibited furin activity by 97.9% (p <0.001).

[0090] This result was expected and validates the study.

[0091] The results of the furin enzyme activity compared to the basal activity obtained are given below. [Table 3] "Protein hydrolysate" peregrina » at 0.02% (v / v) 124.9% (p<0.001) "Protein hydrolysate" peregrina » at 0.2% (v / v) 73.2% (p<0.001) "Protein hydrolysate" peregrina » at 2.0% (v / v) 1.2% (p<0.001)

[0092] Conclusion: At a concentration of 2%, the protein hydrolysate peregrinaaccording to the invention is capable of inhibiting furin convertase activity by 98.8%. Even at a concentration of 0.2%, the protein hydrolysate peregrina according to the invention is capable of inhibiting furin activity by 26.8%. Example 3: Effect of protein hydrolysate peregrina according to the invention for inhibiting Histone Deacetylase (HDAC) and Sirtuin I enzymes

[0093] The objective of this study is to demonstrate the inhibitory activity of protein hydrolysate peregrina According to the invention, HDACs and Sirtuin I enzymes are involved in controlling genetic drift by regulating chromatin condensation / decondensation, which grants or denies access to the genes carried by the DNA. A buffered solution of HDACs and Sirtuin I reacts with a substrate for 20 minutes at 37°C and transforms it to form a compound that stains in the presence of a developer after incubation at 37°C for 10 minutes. The maximum deacetylation activity of the Sirtuins can thus be evaluated by measuring the absorbance at 405 nM. The protein hydrolysate peregrinaAccording to the invention, or the reference product "Trichostatin A (STA) inhibitor 1µM," the samples are contacted with the Sirtuin solution along with the enzyme substrate for 20 minutes at 37°C. The substrate transformed by the enzyme is stained by adding a developer. The deacetylation activity of HDACs and Sirtuin I in the presence of the active ingredient is then evaluated by measuring the absorbance at 405 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the maximum activity of HDACs and Sirtuin I in the absence of the active ingredient, i.e., solely in the presence of the substrate of the HDACs and Sirtuin I enzymes.

[0094] Protocol: A solution of Sirtuins enzymes is incubated in its substrate for 20 minutes in the absence (control) or presence of the reference product, or at increasing concentrations of the test products. The protein hydrolysate peregrinaThe product according to the invention is tested at the following concentrations: 2%, 1%, and 0.1% (v / v). At the end of the incubation period, the activity of the Sirtuin enzymes with and without the test or reference product was revealed by staining with a developer solution (10 min at 37°C) and evaluated by measuring the absorbance of the reaction media at 405 nm. For each concentration tested, the modulation of the deacetylating activity of the Histone Deacetysases and Sirtuin I enzymes by the test product is calculated according to the following formula.

[0095] If the result is negative, the percentage is expressed as inhibition of the enzymatic reaction; if the result is positive, the percentage is expressed as activation of the enzymatic reaction. The results of the inhibition of Histone Deacetylase (HDAC) enzymes are given below. [Table 4] Protein hydrolysate peregrina according to the invention Percentage Inhibition versus Control (%) 2% 15 1% ns 0.10% -18

[0096] Conclusion: At 2%, the protein hydrolysate peregrina The invention demonstrates a significant inhibition of HDACs; this inhibition reveals the ability to promote the self-protection of skin cells against genetic drift. Thus, the extract appears to be useful against one of the most common genetic drifts on the skin's surface, namely fibrosis, which manifests as the appearance of fleshy "bumps" (fibrotic protuberances). The extract may advantageously interfere with fibrotic phenomena on the skin's surface. Example 4: Effect of protein hydrolysate peregrina according to the invention to inhibit the action of endothelin 1 (ET-1)

[0097] Endothelin is a peptide hormone derived from endothelial cells capable of acting on various cells and tissues via its receptors. For example, endothelin is known to cause an increase in intracellular calcium concentration in vascular smooth muscle cells and other cells (OHBA T. et al., WO2012 / 081370).

[0098] It has been reported in recent years that endothelin type 1 (ET-1) is a bioactive factor that continuously contracts vascular and non-vascular smooth muscles through direct and indirect actions. Increased endothelin activity is thought to provide continuous vasoconstriction to blood vessels in peripheral sites, the kidneys, and the brain, and may underlie various diseases such as hypertension, myocardial infarction, stroke, acute renal failure, Raynaud's syndrome, atherosclerosis, asthma, and prostate cancer (MIYAGAWA K. & EMOTO N. et al., 2014, Current state of endothelin receptor antagonism in hypertension and pulmonary hypertension, Therapeutic Advances in Cardiovascular Diseases, vol. 8(5) 202-216; SCHINZARI F. et al., 2018, Increased Endothelin-1-Mediated Vasoconstrictor Tone in Human Obesity: Effects of Gut Hormones, Physiological Research 67 suppl.1). S69-S81).Three types of endothelin family peptides with similar structures are present in animals, including humans (KADONO, S. et al., 2001, The Role of the Epidermal Endothelin Cascade in the Hyperpigmentation Mechanism of Lentigo Senilis, Journal of Investigative Dermatology 116 4, p. 571-577; ANONYMOUS, 2006, American Society for Biochemistry and Molecular Biology, New Cosmetics Handbook, p. 527-529). All these peptides have vasoconstrictive and vasopressor effects.

[0099] In recent years, the role of endothelin in various cells other than vascular smooth muscle cells has been elucidated. Scientific publications, for example, have reported that the generation of ET-1 and other factors increases in keratinocytes when the skin is exposed to UV radiation, and have suggested that ET-1 is associated with melanogenesis in melanocytes exposed to UV radiation. Consequently, suppression of endothelin expression is considered useful not only for the prevention and / or treatment of the aforementioned diseases, but also for the prevention or improvement of skin pigmentation (IMOKAWA, G. et al., 1995, Endothelin-1 as a New Melanogen: Coordinated Expression of its Gene and the Tyrosinase Gene in UVB-Exposed Human Epidermis, Journal of Investigative Dermatology 1051, p. 32-37; IMOKAWA, G. et al.)., 1992, Endothelins Secreted from Keratinocytes are Intrinsic Mitogens for Human Melanocytes, Journal of Biological Chemistry, 267, p. 24675-24680; GILCHREST, B. et al., 1996, Mechanisms of Ultraviolet Light-Induced Pigmentation, Photochemistry and Photobioliogy63, p. 1-10).

[0100] The objective is to measure type 1 endothelin in human microvascular endothelial cells after 24h exposure to protein hydrolysate peregrina according to the invention.

[0101] Protocol: Human microvascular endothelial cells were supplied by PELOBiotech and cultured in 96-well plates according to the supplier's production procedures. Extracts at various concentrations were infused onto the endothelial cells at 80% confluence for 24 hours, followed by quantification of endothelin-1 using the PicoKine ELISA kit (EDN1) in the cell supernatants. A viability test was performed beforehand to determine the non-toxic doses to be used for endothelin-1 measurement. The negative control was performed using untreated cells in culture medium. The positive control in the viability test was 0.5% SDS. All conditions were prepared in culture media, and the cells were then incubated at 36.5°C / 5% CO2 for 24 hours. a) Application of the solutions to be tested on endothelial cells:

[0102] The products to be tested are brought into contact with subconfluenced endothelial cells in 96-well plates. For each concentration, the test is performed on 3 wells. The plates are incubated for 24 hours ± 1 hour at 36.5°C / 5% CO2. b) Viability test:

[0103] Cell viability is assessed using the MTT method on cells after incubation with the products. After 24 hours of incubation, the supernatants are collected and stored at -20°C for assays. The wells are then rinsed once with 200 µL of PBS. Fifty percent of an MTT solution at 0.5 mg / ml is added to each well and incubated for 3 hours at 36.5°C / 5% CO2. One hundred and ten percent isopropanol is then added to each well. After homogenization, an absorbance reading is taken at 550 nm. For each condition, the ratio of the mean optical density of the cells to the mean optical density of the negative controls determines the viability rate. c) Endothelin 1 assay:

[0104] The assay is performed using an ELISA kit. The results of the endothelin inhibition assay are given below. [Table 5] Concentrated extract Cell growth versus control (%) Endothelin 1 versus control (%) Endothelin 1 versus control ( pg / ml ) Protein hydrolysate according to the invention 2% +8,46 -34,90 -47,09 1% 1,54 -13,03 -17,58 0,10% -1,15 -2,44 -16,79

[0105] Conclusion: The viability test carried out at the end of the treatment did not show any toxic effect for the concentrations tested.

[0106] Endothelin-1 levels are measured in cell supernatants at non-toxic concentrations. The amount of endothelin-1 for each condition is determined using an ELISA kit.

[0107] For cells controlling basal levels, the values ​​are approximately 134.94 pg / ml. For cells treated with different concentrations of hydrolysate, the basal level is reduced by 47.09 pg / ml (with 2% of the extract according to the invention) and by 17.58 pg / ml (with 1% of the extract according to the invention). This demonstrates highly significant inhibitions even at 1% of the extract according to the invention, with approximately 13% inhibition of type 1 endothelin production, and up to 34.90% inhibition with 2% of the extract according to the invention.

[0108] The results on endothelin and its specific dose-dependent inhibition demonstrate that the protein hydrolysate according to the invention has an anti-angiogenic and anti-fibrotic effect, due to its ability to significantly decrease endothelin.

[0109] A further study for the evaluation of P1, P2, P3 and the P1+P2 complex described in Example 12 is described below on endothelin production: Cell assay model on normal human endothelial cells.

[0110] As described in Example 12, three protein bands or fractions characterized by their mass were isolated from the hydrolysate according to the invention: P1 (having a molecular weight less than 10,000 Da), P2 (having a molecular weight between 10,000 and 17,000 Da), and P3 (having a molecular weight of approximately 23,000 Da). These fractions are hereinafter also referred to as "extracts." [Table 6] Witness Extract P1 (%, v / v) 0.1 0.3 1 Endothelin-I (pg / µg of protein) 33.8 29.0 44.0 38.3 36.7 34.2 41.5 31.7 39.7 30.1 38.1 33.9 Average 36.7 31.1* 41.2* 34.6* Standard deviation 3.0 2.7 2.9 3.3 % of the control 100.0 84.6 112.1 94.2 *Statistical significance (p>0.05)

[0111] At all concentrations, the standard deviations are high, and the P1 fraction of the protein hydrolysate does not show significant modulation on type I endothelin. [Table 7] Witness Extract P2 (% v / v) 0.1 0.3 1 Endothelin-I (pg / µg of protein) 33.8 34.8 24.7 34.6 36.7 35.7 28.9 39.3 39.7 34.0 29.2 40.8 Average 36.7 34.9* 27.6** 38.3* Standard deviation 3.0 0.9 2.5 3.2 % of the Witness 100.0 94.9 75.2 104.1 *Statistical significance (p>0.05) **Statistical significance (p<0.05)

[0112] At concentrations of 0.1% and 1%, the standard deviations are high, and the P2 fraction of the protein hydrolysate does not show significant modulation of type I endothelin at these concentrations. At a concentration of 0.3%, P2 inhibits type I endothelin production by 24.8%. [Table 8] Witness Extracts P1+P2 (50 / 50) (%, v / v) 0.1 0.3 1 Endothelin-I (pg / µg of protein) 33.8 39.4 42.0 34.5 36.7 29.7 34.2 34.7 39.7 42.2 38.3 37.9 Average 36.7 37.1* 38.1* 35.7* Standard deviation 3.0 6.6 3.9 1.9 % of the Witness 100.0 100.9 103.8 97.2 *Statistical significance (p>0.05)

[0113] At all concentrations, the standard deviations are high, and the combination of fractions P1 + P2 does not show any significant modulation on type I endothelin. [Table 9] Witness Extract P3 (%, v / v) 0.1 0.3 1 Endothelin-I (pg / µg of proteins) 33.8 36.2 19.5 18.8 36.7 38.9 14.8 20.4 39.7 31.5 18.2 17.6 Average 36.7 35.5* 17.5*** 18.9*** Standard deviation 3.0 3.8 2.4 1.4 % of the Witness 100.0 96.7 47.7 51.5 *Statistical significance (p>0.05) ***: Statistical significance (p<0.001)

[0114] At concentrations of 0.3% and 1%, the P3 fraction of the protein hydrolysate very significantly inhibits type I endothelin production by approximately 50%.

[0115] Conclusion: The compounds designated "Extract P1" and "Extract P2" have no significant effect on the modulation of endothelin-1, and only "Extract P3" significantly reduces endothelin-1 released into the culture medium by normal human endothelial cells in monolayer culture, with an inhibition score of 52.3% with 0.3% of the extract according to the invention. No synergy is observed when P1 and P2 are combined.

[0116] Extract P3 specifically demonstrates anti-cancer activity (BAGNATO A. et al., 2011, Role of the endothelin axis and its antagonists in the treatment of cancer, British Journal of Pharmacology, 163: 220-233). Example 5: Effect of protein hydrolysate peregrina according to the invention to protect stem cells

[0117] Adult tissues, including the skin epidermis, gastrointestinal epithelium, and hematopoietic system, have a high rate of cell turnover. The physiological process of maintaining tissue homeostasis is attributed to maintaining a constant number of cells during organ renewal. Embryonic stem cells (ESCs) are essential for the maintenance and regeneration of skin tissues.

[0118] The epidermis develops from the embryonic surface ectoderm. It begins as a single layer of unspecified progenitor cells covering the embryo after neurulation and develops into the epidermal basal layer. The epidermal basal layer is enriched in epidermal stem cells (ESCs). Indeed, the cells of this layer give rise to all epidermal structures, including the stratified epidermis (also called the interfollicular epidermis) and epidermal appendages, such as hair follicles, sebaceous glands, and sweat glands. The underlying dermis derives primarily from the mesoderm beneath the ectoderm. The mesoderm is the main source of mesenchymal stem cells that give rise to collagen-producing fibroblasts, subcutaneous adipocytes, and skin immune cells.

[0119] Stem cells are undifferentiated, pluripotent cells with a young genotype capable of self-renewal and differentiation to produce an organ or tissue, such as skin. At this stage, they are identified as "multipotent cells." Since 50% of the progeny of the stem cell population remains undifferentiated, stem cells help maintain homeostasis and ensure the renewal of damaged or senescent differentiated cells. However, these epidermal stem cells are frequently affected by the environment. Oxidative stress, such as pollution or ultraviolet radiation, damages their DNA, according to Yejin Ge et al. (March 10, 2020, "The aging skin microenvironment dictates stem cell behavior," PNAS, Vol. 117, pp. 5339-5350). This damage impairs their ability to self-renew and differentiate, leading to a decrease in the stem cell pool and ultimately to skin aging.

[0120] The objective of the study is to evaluate the effect of protein hydrolysate peregrina according to the invention on the protection of epidermal stem cells against UVB irradiation.

[0121] Protocol: Human keratinocyte cells were obtained from a 62-year-old donor. To perform the experiments, the keratinocytes were cultured in a monolayer until 80% confluence was reached.

[0122] The cell culture was then enriched with epidermal stem cells following the method described by GOODELL, M. et al. (1996, Hoescht 33342 HSC staining and stem cell purification protocol Journal of Experimental Medicine 183, p. 1797-806).

[0123] Reference product: Quercetin 1 µM was used as the reference product in this study. Quercetin was purchased from Sigma Aldrich.

[0124] The cells were pre-incubated for 24 hours in the absence ("Control") or presence of the reference product or increasing concentrations of the compound to be tested. At the end of the pre-incubation period, the cells were irradiated with UVB (30 mJ / cm²) and then incubated for 8 days at 37°C in the absence ("Control") or presence of the reference product or increasing concentrations of the compound to be tested.

[0125] "Protein hydrolysate" peregrina » : 0.01; 0.05 and 0.15% (v / v). Preparation of the test compound:

[0126] The test compound "Protein Hydrolysate" peregrina » was diluted directly into the incubation medium to achieve the different concentrations described above.

[0127] At the end of the incubation period, cell viability was measured using Alamar blue, a non-cytotoxic viability indicator based on mitochondrial reduction of rezazurin. Each experimental condition was performed in triplicate (n = 3).

[0128] The results are presented below as a percentage of viability compared to the experimental condition "Control without UVB" (mean + / - SD). The significance level between "Control without UVB" and "Control with UVB" was assessed using a Student's t-test (p < 0.05). [Table 10] Hydrolysate concentration Cell growth versus control (%) I. Stem cell protection versus control (%) Protein hydrolysate peregrina 0.15% ca. -9.2 NS 0.05% -3.6 +30.50 0.01% ca. -6 +15.50

[0129] Conclusion: Protein hydrolysate peregrinacan significantly protect human skin stem cells subjected to cellular stress (UV). Stem cells are cells with preserved, youthful DNA material. They are responsible for tissue regeneration, restoring a youthful and healthy state. Stem cell protection is correlated with the ability to preserve DNA material. Protein hydrolysate peregrina according to the invention maintains the integrity of stem cells; it is thus involved in the preservation of DNA. Example 6: Effect of protein hydrolysate peregrina according to the invention for preserving DNA

[0130] Telomere length dynamics are crucial for regulating replicative lifespan in cells, particularly in long-lived species. Telomere shortening and telomerase activity are important factors in aging and tumorigenesis (SHAY, JW & WRIGHT, WE, 2005, Senescence and Immortalization: Role of Telomeres and Telomerase, Carcinogenesis 26(5), pp. 867-874). Telomeres are complex nucleotide sequences that protect the ends of chromosomes from degradation, unwanted fusion-recombination, and inappropriate activation of the DNA damage response. They also play a vital role in cell division and chromosome stability. There is growing evidence that telomere stability and average length can be affected by stressors, including environmental stressors and environmentally influenced diseases (VALDES, ALet al., July 2005, Obesity, cigarette smoking and telomere length in women, Research Letters,366(9486), p. 662-664 ; PHILLIPS A.C. et al., 2013, Do symptoms of depression predict telomere length? Evidence from the West of Scotland Twenty-07 Study, Psychosomatic Medicine, 75(3), p. 288-296; SHIN, D. et al. May 2019, Effects of inflammation and depression on telomere length in young adults in the United States, Journal of Clinical Med 2019, 8(5), p. 711; SALIQUES, S. et al., October 2010, Telomer length and cardiovascular disease, Archives of Cardiovascular Diseases, 103(8-9), p. 454-459). Ainsi, les télomères extrêmement courts ont été associés à des maladies neurodégénératives, cardiovasculaires (MCV) et au risque de cancer.

[0131] Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repeats to the ends of telomeres. Telomeres are long stretches of repeated sequences that cap the ends of chromosomes and are thought to stabilize the chromosome. In humans, telomeres are typically 7 to 10 kb long and consist of several repeats of the sequence TTAGGG.

[0132] Telomerase is not expressed in most adult cells, and telomere length decreases with successive replication cycles. After a certain number of replication cycles, the progressive shortening of telomeres induces a telomeric crisis, which in turn leads to cellular senescence. Some diseases are associated with rapid telomeric loss, resulting in premature cellular senescence. Expression of the gene encoding the human telomerase protein in human cells has been shown (BLASCO M., 2007, Telomere Length, Stem Cells and Aging, Nature Chemical Biology, 3(10), p. 640-649) to confer a consistent quality phenotype, likely by bypassing the natural cellular senescence pathway.Furthermore, it was demonstrated in the aforementioned study that the expression of the telomerase gene in aging cells with short telomeres produces an increase in telomere length and restores a phenotype generally associated with younger cells.

[0133] The objective of this study is to evaluate the effect of the compound called "protein hydrolysate". peregrina » on telomere shortening in a model composed of normal human fibroblasts in monolayer culture. It is well known that the telomere corresponds to a biological clock. Telomere length gradually decreases with cell divisions, eventually resulting in a cell incapable of replication. Telomere length was measured using quantitative PCR and compared with telomere lengths between cells at passages 2 and 5.

[0134] Protocol: Human fibroblast cells were obtained from a 44-year-old donor. Cells were used in passages 2 and 5 for the experiments. The fibroblasts were cultured for three consecutive passages in the absence (control) or in the presence of an increasing concentration of the protein hydrolysate to be tested. peregrina: 0.01; 0.1 and 0.5% (v / v).

[0135] Preparation of the test compound: The test compound "protein hydrolysate" peregrina » was diluted directly into the incubation medium to achieve the different concentrations described above.

[0136] At the end of incubation, the cells were trypsinized. DNA was extracted from the cells using a dedicated DNA extraction kit. The DNA was quantified by nanodrop.

[0137] Telomere length was measured by quantitative PCR (qPCR). For each sample, telomere length variation was measured by relative quantification using the SCR (single-copy reference) gene as the reference gene. For each sample, one qPCR was performed using a telomere primer set that recognizes and amplifies telomere sequences, and a second qPCR was performed using the SCR primer set that recognizes and amplifies a 100 bp region on human chromosome 17 and serves as a reference for data normalization.

[0138] Results are expressed in relative units corresponding to telomere length relative to cells at passage 2 (mean ± SD). The significance level versus "Control" at passages 2 and 5 was assessed using a Student's t-test (*: p < 0.05). The significance level between "Control" and "Trial compound" was assessed independently for each product by a one-way analysis of variance (ANOVA) followed by a Holm-Sidak test (*: p < 0.05). [Table 11] Concentration Cell growth versus control (%) Telomere length versus control (%) Protein hydrolysate 0.5% +1.9 +16.60 0.1% approx. 0 +15.10 0.05% -0.6 +8.90

[0139] Results: Under our experimental conditions, the protein hydrolysate peregrina tested at 0.05%, 0.1% and 0.5% (v / v), significantly decreased telomere shortening in normal human fibroblasts.

[0140] Telomere shortening exhibited inhibition (compared to the control) at 0.05% (v / v) of +8.9% (p <0.05); at 0.1% (v / v) of +15.1% (p <0.01); and at 0.5% (v / v) of +16.6% (p <0.01). Conclusion: In the context of normal human cell multiplication or division, protein hydrolysate peregrina The invention demonstrates an ability to significantly increase telomere length. Telomeres are caps involved in protecting DNA material; increasing telomere length is correlated with the ability to preserve DNA material. Protein hydrolysate peregrina It can increase telomere length. This hydrolysate is therefore involved in the preservation of human genetic material (DNA).

[0141] Further study of the evaluation of extracts P1, P2, P3 and the P1+P2 complex described in example 12 on their ability to protect DNA by increasing telomere length after several cell divisions. [Table 12] Passage 2 Passage 5 Witness Witness Extract P1 (%; v / v) 0.01% 0.3% 1% Change in telomere length (change in size compared to the first passage) 1.08 0.56 0.57 0.65 0.74 0.86 0.58 0.61 0.64 0.77 1.07 0.54 0.62 0.71 0.70 Average 1.00 0.56 0.60* 0.67** 0.74*** Standard deviation 0.12 0.02 0.03 0.04 0.04 Telomere length (as a percentage of the control) 100.0 55.8 59.8 66.6 73.3 % telomere elongation compared to the control 0 9.1 24.3 39.6 *Statistical significance (p>0.05) **: Statistical significance (p<0.01) ***: Statistical significance (p<0.001)

[0142] Extract P1 according to the invention, after 3 cell divisions, at a concentration of 0.3%, increases telomere length by 24.3% in a normal human cell culture model (fibroblasts). At a concentration of 1%, extract P1 increases telomere length by 39.6% under the same conditions. [Table 13] Passage 2 Passage 5 Witness Witness Extract P2 (%; v / v) 0.01% 0.3% 1% Change in telomere length (change in size compared to the first passage) 1.08 0.56 0.53 0.61 0.66 0.86 0.58 0.70 0.71 0.68 1.07 0.54 0.74 0.68 0.92 Average 1.00 0.56 0.66* 0.67* 0.75* Standard deviation 0.12 0.02 0.11 0.05 0.14 Telomere length (as a percentage of the control) 100.0 55.8 65.3 66.3 75.0 % telomere elongation compared to the control 0 21.4 23.7 43.5 *Statistical significance (p>0.05)

[0143] Extract P2 according to the invention, after 3 cell divisions on a normal human cell culture model (fibroblasts), shows a statistically unconfirmed tendency towards telomere lengthening for each concentration tested. [Table 14] Passage 2 Passage 5 Witness Witness Extract P3 (%; v / v) 0.01% 0.3% 1% Change in telomere length (change in size compared to the first passage) 1.08 0.56 0.51 0.46 0.50 0.86 0.58 0.52 0.54 0.55 1.07 0.54 0.64 0.61 0.61 Average 1.00 0.56 0.56* 0.54* 0.55* Standard deviation 0.12 0.02 0.07 0.07 0.06 Telomere length (as a percentage of the control) 100.0 55.8 55.4 53.6 55.2 % telomere elongation compared to the control 0 -0.8 -5.0 -1.3 *Statistical significance (p>0.05)

[0144] Extract P3 according to the invention, after 3 cell divisions on a normal human cell culture model (fibroblasts), shows no ability to promote telomere lengthening for each concentration tested. [Table 15] Passage 2 Passage 5 Witness Witness Extract P1+P2 (%; v / v) 0.01% 0.3% 1% Change in telomere length (change in size compared to the first passage) 1.08 0.56 0.61 0.72 0.76 0.86 0.58 0.84 0.67 0.71 1.07 0.54 0.63 0.74 0.86 Average 1.00 0.56** 0.70* 0.71* 0.78** Standard deviation 0.12 0.02 0.13 0.04 0.08 Telomere length (as a percentage of the control) 100.0 55.8 69.3 70.5 77.5 telomere elongation compared to the control 0 30.5 33.3 49.1 *Statistical significance (p>0.05) **: Statistical significance (p<0.01)

[0145] The mixture of extracts P1 and P2 (50 / 50 by volume) according to the invention, after three cell divisions at a concentration of 1% (i.e., 0.5% of each extract), increases telomere length by 49.1% in a normal human cell culture model (fibroblasts). Since this result was not achieved by the individual extracts, a synergistic effect is thus demonstrated by mixing extracts P1 and P2.

[0146] Conclusion: The compounds named "Extract P1" and "Extract P1 + P2" significantly reduce telomere shortening occurring after three consecutive cell passages. Synergy is confirmed when combining P1 and P2. Example 7: Effect of protein hydrolysate peregrina according to the invention to stimulate the ZAG protein

[0147] Zinc alpha-2-glycoprotein (ZAG) is a plasma glycoprotein named for its electrophoretic mobility and its ability to be precipitated by zinc salts. ZAG belongs to the immunoglobulin gene superfamily and has a three-dimensional structure highly homologous to MHC class I and II molecules. ZAG has been detected immunohistochemically in normal secretory epithelial cells of the breast, prostate, and liver, in salivary, bronchial, gastrointestinal, and sweat glands, and in normal stratified epithelia, including the epidermis. ZAG mRNA remains uniformly distributed in different cell types (FREIJE, JP et al., 1991, Human Zn-α2-Glycoprotein cDNA Cloning and Expression Analysis in Benign and Malignant Breast Tissues, FEBS Letters 290 (1-2), p. 247-249).Due to its production by the secretory epithelium, ZAG is present in most bodily secretions and constitutes 2.5% of salivary proteins and 30% of seminal fluids. Plasma or serum ZAG levels have been described to vary with age, with reported values ​​ranging from 0.9 to 3.5 mg / dl (fetus) to 7.8 to 12.1 mg / dl (young adults) (JIRKA, M. et al., 1974, The Zn-alpha 2-Glycoprotein Level in Human Serum During Ontogenesis. Clinica Chimica Acta 56, p. 31-33; JIRKA, M. et al., 1978, Human Serum Zn-α2-Glycoprotein in Amniotic Fluid, Clinica Chimica Acta 85, p. 107-110). ZAG accumulates in breast cystic fluids up to a plasma concentration of 30 to 50 times (BUNDRED et al., 1987, An Immunohistochemical Study of the Tissue Distribution of the Breast Cyst Fluid Protein, Zinc Alpha2-Glycoprotein, Histopathology 11, p. 603-610; DIEZ-ITZA, I. et al., 1993, Zn-α2-Glycoprotein Levels in Breast Cancer Cytosols and Correlation with Clinical, Histological, and Biochemical Parameters, European Journal of Cancer 29A, p. 1256-1260) and is overexpressed in 40 to 50% of breast carcinomas. It has recently been shown (SUSAN, M. et al., Zinc-alpha2-glycoprotein Expression as a Predictor of Metastatic Prostate Cancer Following Radical Prostatectomy, 2006, Journal of the National Cancer Institute, Volume 98(19), p. 1420-1424) that ZAG is produced in quantity by most prostate carcinomas, resulting in elevated serum ZAG levels in prostate cancer patients with basal cell carcinomas. The objective of this study is to evaluate the capacity of the protein hydrolysate. peregrina to stimulate the ZAG.

[0148] Protocol: Normal human keratinocytes are isolated from foreskins and cultured in 24 and 96 well plates according to internal procedures.

[0149] This involves allowing the samples to act at defined concentrations on keratinocytes at 80% confluence for 48 hours, then quantifying the ZAGs using the ELISA kit in the cellular supernatants.

[0150] A preliminary viability test is performed to determine the non-toxic doses to be used when assaying ZAGs. The negative control is performed using untreated cells in culture medium. The positive control for the viability test is 0.5% SDS.

[0151] All conditions are prepared in culture media, and the cells are then incubated at 36.5°C / 5% CO2 for 24 hours for the viability test and 48 hours for the ZAG assay. Application of the test solutions to keratinocytes: The test products are brought into contact with keratinocytes in subconfluence in 24- and 96-well plates. For each concentration, the test is performed on 3 wells. The plates are incubated for 24 and 48 hours at 36.5°C / 5% CO2. Viability test: Cell viability is assessed by the MTT method on cells after incubation with the products. After 24 and 48 hours of incubation, the prepared wells are rinsed once with 200 µL of PBS. 50 µL of 0.5 mg / ml MTT solution are added to each well and incubated for 3 hours at 36.5°C / 5% CO2. 100 µL of isopropanol is then added to each well. After homogenization, an absorbance reading is taken at 550 nm. For each condition, the ratio of the average optical densities of the cells to the average optical densities of the negative controls will determine the viability rate.ZAG protein assay: After 48 hours of incubation, all supernatants are collected and stored at -20°C for analysis. The assay is performed using an ELISA kit. The results are given below. [Table 16] Concentration Cell growth versus control (%) ZAG versus control (%) ZAG versus control (ng / ml) Protein hydrolysate 2% -17.88 +337.80 0.390 1% -12.54 +195.73 0.157 0.10% -3.88 +151.83 0.085

[0152] Conclusion: Protein hydrolysate peregrina It can significantly increase ZAG production, with good dose dependence and low toxicity to human cells. It therefore has an anti-fibrotic and anti-inflammatory effect based on its ability to significantly increase ZAG.

[0153] A further study of ZAG stimulation from the protein bands identified in Example 12 is described below on normal human keratinocytes. [Table 17] Witness Extract P1 (%, v / v) 0.2 1 5 ZAG (pg / µg of protein) 2.8 4.1 4.3 2.3 3.0 6.0 3.9 1.1 2.0 6.1 3.4 2.4 Average 2.6 5.4** 3.9* 1.9* Standard deviation 0.5 1.1 0.5 0.7 % of the Witness 100.0 207.1 147.3 73.5 *Statistical significance (p>0.05) **: Statistical significance (p<0.01) Extract P1 at 0.2% significantly increases ZAG production by more than 107%. [Table 18] Witness Extract P2 (%, v / v) 0.2 1 5 ZAG (pg / µg of protein) 2.8 6.2 4.3 2.6 3.0 4.2 4.4 2.7 2.0 3.7 4.5 2.3 Average 2.6 4.7** 4.4** 2.5* Standard deviation 0.5 1.4 0.1 0.2 % of the Witness 100.0 180.1 167.5 95.6 *Statistical significance (p>0.05) **: Statistical significance (p<0.05) Extract P2 at concentrations as low as 0.2% significantly increases ZAG production by more than 80%. [Table 19] Witness Extract P3 (%, v / v) 0.2 1 5 ZAG (pg / µg of protein) 2.8 2.1 2.3 2.3 3.0 1.9 2.7 1.8 2.0 2.2 2.1 3.3 Average 2.6 2.1* 2.4* 2.5* Standard deviation 0.5 0.2 0.3 0.8 % of the Witness 100.0 78.5 90.0 95.2 *Statistical significance (p>0.05) Extract P3 does not have the ability to significantly influence ZAG production in this study model. [Table 20] Witness Extract P1+P2 (50 / 50) (%, v / v) 0.2 1 5 ZAG (pg / µg of protein) 2.8 3.9 4.2 1.7 3.0 3.7 3.5 2.6 2.0 3.5 2.8 2.6 Average 2.6 3.7* 3.5* 2.3* Standard deviation 0.5 0.2 0.7 0.5 % of the Witness 100.0 140.6 132.9 87.8 *Statistical significance (p>0.05) The combination of extracts P1 and P2 shows a tendency towards activation of ZAG production, but this tendency is not statistically significant. Therefore, there is no synergistic effect from the combination of extracts P1 and P2.

[0154] The compounds named "Extract P1" and "Extract P2" significantly increase the ZAG released in culture medium by normal human keratinocytes in monolayer culture, but no synergy is observed when combining P1 and P2.

[0155] Conclusion: In the hydrolyzed extract according to the invention, extract P1 is the most effective extract for increasing ZAG. It therefore has an anti-fibrotic and anti-inflammatory effect even at a dose of 0.2%. Example 8: Effect of protein hydrolysate peregrina according to the invention on the modulation of the dosage of DKK1 and DKK3

[0156] The involvement of interactions between melanocytes and fibroblasts in the regulation of melanogenesis is well known and has been extensively studied. Although these interactions are not yet fully understood, they are responsible for the "whiteness" of the palms and soles and are now being used in dermatology for the development of depigmenting products. Yamaguchi and colleagues (YAMAGUCHI Y. et al., 2004, Mesenchymal-Epithelial Interactions in the Skin: Increased Expression of Dickkopf by Palmoplantar Fibroblasts Inhibits Melanocyte Growth and Differentiation, Journal of Cell Biology 165(2), p. 275-285) demonstrated that a soluble messenger produced by fibroblasts in the palms and soles was able to modify the differentiation program of melanocytes in these regions, leading to a decrease in melanin production. This messenger was identified by the team as a protein named Dikkopf-1 (DKK-1).

[0157] The signaling pathways used by DKK-1 to produce these results are now well identified. Thanks to its antagonistic action on the Wnt receptor, DKK-1 is indeed able to short-circuit the intracellular signaling pathways activated by β-catenin, which are generally responsible for regulating genes involved in melanogenesis. Yamaguchi et al. (cf. above ) have also demonstrated that DKK-3, a molecule like DKK-1 but without effect on the Wnt receptor, could play a regulatory role on the effect of DKK-1. In fact, the greater the amount of DKK-3 in the vicinity of this Wnt receptor, the weaker the interactions between DKK-1 and this receptor. The increase in DKK-3 reduces the inhibitory effects of DKK-1 on melanogenesis. The work of Yamaguchi and colleagues (see above) further suggest that identifying agents influencing the DKK1 / DKK3 ratio in normal human dermal fibroblast cultures of non-palmo-plantar origin would allow control of melanin production from normal non-palmo-plantar human melanocytes.

[0158] The objective of this study is to evaluate the effect of protein hydrolysate peregrina on the synthesis and release of DKK-1 in a model composed of normal human fibroblasts in monolayer culture.

[0159] Protocol: Human fibroblast cells were obtained from a 68-year-old donor. For the experiments, the fibroblasts were cultured in a monolayer until confluence was reached. Dexamethasone at 100 nm was used as a reference inducer for the synthesis and release of DKK-1. Skin discs were incubated for 48 hours in the absence (control) or presence of a reference product or the product to be tested: protein hydrolysate. peregrina » : 0.01; 0.1 and 0.5% (v / v).

[0160] At the end of the incubation, the incubation media were removed to perform the DKK-1 release measurement.

[0161] The test compound "protein hydrolysate" peregrina » was diluted directly into the incubation medium to achieve the different concentrations described above.

[0162] At the end of the 48-hour incubation period, the DKK-1 released into the incubation media was quantified using a sensitive and specific ELISA kit.

[0163] At the end of the incubation period, the proteins contained in the cell lysates were quantified using a spectro-colorimetric method (Bradford method).

[0164] The results are expressed in ng of DKK-1 per mg of protein (mean ± SD).

[0165] The level of significance between the "control" and the "reference product" was assessed using a Student's t-test (p <0.05).

[0166] The significance level between the "Control" and the "test product" was assessed by a one-way analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (p <0.05).

[0167] Under our experimental conditions, the reference product, "Dexamethasone," tested at 100 nm, significantly increased DKK-1 release by 181.8% (p < 0.01) compared to the "Control." The results on the modulation of DKK-1 dosage are given below. [Table 21] Concentration Cell growth versus control (%) DKK1 versus control (%) Protein hydrolysate 0,5% +1,9 +131,50 0,1% approx. 0 +32,30 0,05% -0,6 +26,10 The study shows that the hydrolysate according to the invention significantly increases the DKK1 level at a dose of 0.05%, with a 26.1% increase compared to the baseline level, and at a concentration of 0.5% of the extract according to the invention, a 131.5% increase in the baseline level is observed.

[0168] The aim of this study is to evaluate the effect of protein hydrolysate peregrinaon the synthesis and release of DKK-3 in a model composed of normal human fibroblasts in monolayer culture. The human fibroblast cells were obtained from a 68-year-old donor. For the experiments, the fibroblasts were cultured in monolayer until confluence was reached.

[0169] At the end of the 48-hour incubation period, the DKK-3 released into the incubation media was quantified using a sensitive and specific ELISA kit.

[0170] At the end of the incubation period, the proteins contained in the cell lysates were quantified using a spectro-colorimetric method (Bradford method).

[0171] Results are expressed as ng of DKK-3 per mg of protein (mean ± SD). The significance level between the "Control" and the "reference product" was assessed using a Student's t-test (*: p < 0.05).

[0172] The significance level between the "Control" and the protein hydrolysate peregrina was evaluated by a one-way analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (*: p < 0.05). The results on the modulation of the DKK3 dosage are given below. [Table 22] Concentration Cell growth versus control (%) DKK3 versus control (%) Protein hydrolysate 0.5% 101.9 -20.7 0.1% approx. 100.0 -8 0.05% 99.4 -3.9

[0173] Conclusion: Protein hydrolysate peregrina According to the invention, a 0.5% solution exhibits a significant inhibition of DKK3, on the order of 21%, compared to the baseline level. The protein hydrolysate peregrinaaccording to the invention has a strong ability to manage genes involved in cell differentiation through the principle of palmoplantar inhibition (beta-catenin signaling pathway), by its ability to significantly increase DKK1 and significantly decrease DKK3, thereby increasing the DKK1 / DKK3 ratio.

[0174] Further study of the evaluation of P1, P2, P3 and the P1+P2 complex described in example 10 on the production of DKK1: Cell assay model on normal human fibroblasts. [Table 23] Witness Extract P1 (%; v / v) 0.01 0.3 1 DKK-1 (ng / mg of proteins) 306.7 334.8 399.5 372.9 294.9 293.1 347.9 392.0 298.6 367.6 392.4 385.1 Average 300.1 331.9* 379.9** 383.3** Standard deviation 6.1 37.3 28.0 9.7 % of the Witness 100.0 110.6 126.6 127.8 *Statistical significance (p>0.05) **: Statistical significance (p<0.01) Extract P1 significantly increases DKK1 production by 26.6% at a dose of 0.3%, and by 27.8% at a dose of 1%. [Table 24] Witness Extract P2 (%; v / v) 0.01 0.3 1 DKK-1 (ng / mg of protein) 306.7 291.5 292.6 410.8 294.9 346.5 275.4 367.0 298.6 277.4 354.9 344.4 Average 300.1 305.2* 307.6* 374.1* Standard deviation 6.1 36.5 41.8 33.8 % of the Witness 100.0 101.7 102.5 124.7 *Statistical significance (p>0.05) Extract P2 did not statistically significantly increase DKK1 production at each of the doses tested. [Table 25] Witness Extract P3 (%; v / v) 0.01 0.3 1 DKK-1 (ng / mg of protein) 306.7 359.2 387.0 224.7 294.9 371.8 263.7 283.0 298.6 293.4 287.3 288.6 Average 300.1 341.5* 312.7* 265.5* Standard deviation 6.1 42.1 65.5 35.4 % of the Witness 100.0 113.8 104.2 88.5 *Statistical significance (p>0.05) Extract P3 did not statistically significantly increase DKK1 production at any of the doses tested. [Table 26] Witness Extract P1+P2 50 / 50(%; v / v) 0.01 0.3 1 DKK-1 (ng / mg of proteins) 306.7 305.9 366.6 389.8 294.9 311.1 357.1 479.5 298.6 346.8 308.1 384.2 Average 300.1 321.3* 343.9* 417.8** Standard deviation 6.1 22.2 31.4 53.5 % of the Witness 100.0 107.1 114.6 139.2 *Statistical significance (p>0.05) **: Statistical significance (p<0.01)

[0175] The combination of extracts P1 and P2 at a dose of 1% (i.e., 0.5% for each extract) significantly increases DKK1 production by more than 39%. This result is higher than that achieved with either extract P1 or P2 alone; this therefore reflects a synergistic effect of the combination of the two extracts P1 and P2.

[0176] Conclusion: The compounds designated "Extract P1" and "Extract P1 + P2" significantly increased DKK-1 released into the culture medium by normal human fibroblasts in monolayer culture. Synergy was confirmed by mixing P1 and P2. Conclusion of the previous activities of the protein hydrolysate according to the invention :

[0177] The most remarkable activity of the protein hydrolysate peregrina demonstrated in tests in celluloIts epigenetic action lies in its ability to significantly slow down telomere shortening after cell division. The hydrolysate is a cell protector, particularly of stem cells, making it an excellent cell and tissue regenerator. These properties offer very strong protection for DNA and its genetic material. The protein hydrolysate peregrina is also a potent modulator of ZAG and type I endothelin production, with an anti-fibrotic and anti-inflammatory effect. Example 9: Evaluation of the effect of protein hydrolysate of delipidated oilseed cake peregrina on Angiotensin convertase 2 (ACE2) - cell-free study of the inhibitory effect.

[0178] Angiotensin convertase 2 is notably involved in the intracellular infection of COVID-19 after the activation of spike proteins by other convertases and more particularly furin, see PETER BRADDING et al. (ACE, TMPRSS2, and furin gene expression in the airways of people with asthma - implications of COVID-19, JOURNAL ALLERGY CLINICAL IMMUNOLOGY, July 2020, n°146(1), p. 206-211). [Table 27] Samples Average Medium-white % enzymatic activity % inhibition White 89576,000 0,000 0,00 100% T+ 101450,333 11874,333 100,00 0% T- 93017,667 3441,667 28,98 71% Hydrolyzed extract peregrina 2,0% 98839,000 10687,333 90,00 10% 1,0% 101400,667 17466,333 147,09 NS 0,10% 104515,333 24256,333 204,28 NS

[0179] Conclusion: The hydrolyzed extract of Oilseed Cake peregrinais not significantly involved in the direct inhibition of Angiotensin convertase 2 (ACE2). Considering Table

[32] of Example 11 below, the extent of action of this extract on another convertase, namely furin, was evaluated. The inhibitory action of the extract according to the invention on furin is clearly demonstrated and established in Table

[32] of Example 11. This specific inhibition demonstrated on a single convertase enzyme reinforces the interest of the protein hydrolysate according to the invention as a specific inhibitor of furin convertase. Example 10: Evaluation of the anti-infective effect of the hydrolyzed extract to be inhibited in cellulo Furin prevents the penetration of "SARS-CoV-2 spike crown-like virus": Model in cellulo on HEK human cells in the presence of "SARS-CoV-2 pseudotyped lentiviruses".

[0180] This evaluation utilizes two main components. First, recombinant clonal stable HEK293 cells constitutively express full-length human ACE2 (Genbank # NM_021804.3) with surface ACE2 expression confirmed by flow cytometry. Second, SARS-CoV-2 Spike pseudotyped lentiviruses were produced with SARS-CoV-2 Spike (Genbank accession number QHD43416.1) as the envelope glycoprotein instead of the commonly used VSV-G. These pseudovirions also contain the firefly luciferase gene driven by a CMV promoter; therefore, spike-mediated cell entry can be conveniently measured via luciferase reporter activity. The SARS-CoV-2 Spike pseudotyped lentivirus can be used for screening applications in a biosafety level 2 facility.

[0181] The equipment used is as follows: [Table 28] Catalog Components 79951 ACE2-HEK recomb cell line 60187-1 Defrosting stage 1 79801 1N Growth Medium 79942 Spike (SARS-CoV-2) Pseudotyped Lentivirus (Luciferase Reporter) 79943 Bald Lentiviral Pseudovirion (Luciferase Reporter) 60690-1 ONE-Step luciferase assay system AF933 Human / Mouse / Rat / Hamster ACE-2 Antibody (R&D Systems) Step 1: Plate ACE2-HEK cells

[0182] ACE2-HEK cells are thawed in thawing medium 1, expanded in 1N growth medium, then harvested and placed in 96-well, flat-bottom, white, transparent culture plates at 10,000 cells / well in 50 µl of thawing medium 1. The cells are incubated overnight at 37°C. Step 2: Pseudotyped Lentiviral Infection Test

[0183] The following day, the visual inspection of the homogeneity and integrity of the cell layer is validated using an inverted microscope, and the following test ingredients are prepared according to the following instructions: [Table 29] Ingredient name / co-incubation method Solubility Concentrations in "wells" Ingredient 1 Peregrina / ACE2-HEK H2O / culture medium 3% (C1), 0.5% (C2), 0.1% (C3)

[0184] In the first step, the protein hydrolysate of peregrinais diluted to an intermediate concentration of 11x in thawing medium 1, for subsequent transfer of 5 µl into the test plate and co-incubation with ACE2-HEK cells. After an incubation period of 30 min at 37 °C, 5 µl of undiluted pseudotyped lentivirus (Bald or S1-Spike) are added to the corresponding assay wells.

[0185] As a positive control, the ACE2 blocking mAb is used at a final concentration of 0.5 µM in the analysis wells. Step 3:

[0186] After an incubation period of 48 hours, a volume of 50 µl of luciferase reagent is added to the analysis wells and the luminescent signal is measured using the PolarStar Omega luminofluorimeter.

[0187] The results are given to the figure 1 for the anti-infective effect of protein hydrolysate peregrina according to the invention against SARS-CoV-2 pseudovirions. Conclusion: Protein hydrolysate of peregrina This study model demonstrates a convincing anti-infective effect on SARS-CoV-2 pseudovirions at a dose of 3% (C1). A marked inhibition of 60% is demonstrated at the C1 (3%) concentration.

[0188] Evaluation of the P1, P2, P3 bands identified in example 12 and of the P1+P2 complex on Furin convertase: Cell-free assay model.

[0189] Studies conducted on the identified bands did not show any inhibitory action on furin convertase at any of the concentrations tested with P1, P2, P3 and P1+P2.

[0190] Conclusion: The inhibitory activity of Furin Convertase is therefore not mediated by one of these protein bands. This demonstrates that the inhibitory activity of Furin Convertase is due to the total protein hydrolysate obtained by the described process.

[0191] The protein hydrolysate as a whole inhibits SARS-CoV-2 type SPIKE proteins, notably by inactivating furin convertase. These inhibitions provide anti-infective and virostatic properties. Example 11: Comparative tests of furin convertase inhibition by different preparations obtained from the seeds of Moringa peregrina

[0192] The aim of this study is to evaluate the inhibitory effect of oil peregrina obtained by first cold pressing of the seeds with shells, then of the extract of peregrina by ethanol (96%) concentrating approximately 1.1% of dry matter, this dry matter itself being composed of approximately 55% by weight of 2,5-diformylfuran, 2.5% furfural, 1.2% isopropyl myristate, 4.7% palmitic acid, 11.1% oleic acid and 25.8% triglycerides, and finally protein hydrolysate peregrina according to the invention on the activity of furin.

[0193] Protocol: The extract from peregrina with ethanol (96%) and protein hydrolysate peregrina were stored at +4°C, protected from light until use. The oil of peregrina was kept at room temperature in a dark area.

[0194] The reference product Decanoyl-Arg-Val-Lys-Arg-CMK at 100 nm was used as the reference inhibitor of furin activity.

[0195] The incubation protocol: Furin was pre-incubated for 10 minutes at room temperature in the absence (Control) or presence of the reference product or increasing concentrations of the compounds tested: Oil Peregrina at 0.3%, 1% and 3% (v / v). Excerpt from peregrina in ethanol (96%) at 0.02%, 0.2% and 2% (v / v). Protein hydrolysate peregrinaaccording to the invention at 0.02%, 0.2%, and 2% (v / v). At the end of the pre-incubation step, a furin substrate was added, and the experimental conditions were incubated again at room temperature, protected from light, for 5 minutes. All experiments were performed in triplicate.

[0196] Preparation of the compounds: The extract of peregrina with 96% ethanol and protein hydrolysate peregrina were solubilized directly in the titration buffer and then diluted to reach the concentration to be tested as described above. The oil of peregrina was solubilized to 3% in a 0.05% Tween20 solution in titration buffer. The solution was then diluted to achieve the concentration described above.

[0197] Evaluation Protocol: The cleavage of the furin fluorescent substrate was monitored for 5 minutes after substrate addition by reading the fluorescence at 485 nm / 535 nm. g. Statistics: Results are expressed in RFUs (Relative Fluorescent Units) + / - SD (Standard Deviation). The statistical significance of the observed difference between the "Control" and "Reference Product" groups was assessed using a Student's t-test (p < 0.001). The statistical significance of the observed difference between the "Control" and "Test Compound" groups was assessed using a one-way ANOVA followed by a Holm-Sidak test (p < 0.05). Results for oil peregrina are given to the figure 2 those of the 96° ethanol extract peregrina to the figure 3 and finally those of the protein hydrolysate peregrina according to the invention to the figure 4 . Results :

[0198] [Table 30] Oil concentration peregrina Furin convertase activity 0.3% (v :v) 91.2%** 1% (v :v) 90.6%** 3% (v :v) 92.8%** [Table 31] Concentration of ethanol extract 96° peregrina Furin convertase activity 0.02% (v :v) 97.3%* 0.2% (v :v) 102.0%* 2.0% (v :v) 112.8%* [Table 32] protein hydrolysate concentration peregrina Furin convertase activity 0.02% (v :v) 124.9%*** 0.2% (v :v) 73.2%*** 2.0% (v :v) 1.2%*** *Statistical significance (p>0.05) **: Statistical significance (p<0.01) ***: Statistical significance (p<0.001)

[0199] Conclusion: This study has allowed us to show that only protein hydrolysate peregrina according to the invention can significantly inhibit furin activity by 98.8% at a concentration of 2%. Example 12: Gel chromatography-based electrophoresis of protein hydrolysate peregrina - Protein bands.

[0200] SDS-PAGE is a technique that involves the migration of proteins denatured by negative charge saturation with SDS through a polyacrylamide gel under the influence of an electric field, thus allowing their separation. This denaturing technique dissociates non-covalent protein complexes using a negatively charged ionic detergent (SDS). This detergent binds indiscriminately via hydrophobic bonds to two amino acids. This technique therefore allows for the analysis and separation of proteins based on their molecular weight. Separation chromatography by electrophoresis method : Deposits:

[0201] MW = well size marker 1: Hydrolyzed extract of oilcake Peregrina well 2: supernatant of the hydrolyzed extract of oilseed cake Peregrina well 3: pellet of the hydrolyzed extract of oilseed cake Peregrina

[0202] The hydrolyzed extract in well 1 clearly shows all the expected protein bands.

[0203] Well 2 (supernatant) shows attenuated bands, particularly on the intermediate and highest molecular weights; well 2 appears to have concentrated the lowest molecular weights, particularly those related to volatiles.

[0204] Well 3 (pellet) clearly shows the bands of the highest molecular weights (> 75,000 Da), then a band called P3 of about 23,000 Da, then a band called P2 between 10,000 and 17,000 Da and finally a band below 10,000 Da called P1, estimated between 4,000 and 6,000 Da. Rotavapor preparation:

[0205] The volatiles are logically in the supernatant along with the "lighter" compounds; 10 ml of supernatant are collected and mixed with 10 ml of 96% ethanol; the mixture is extracted under vacuum at 45°C; the volatile compounds are condensed in a vacuum flask. The concentrated condensate will be analyzed by GC / FID after SPME microextraction.

[0206] Gas Chromatography study after SPME extraction method in FID detection: No volatile compounds were detected.

[0207] Conclusion: Isolation of protein bands reveals that volatile compounds are not bound to proteins; volatile compounds do not participate in the molecular structure of the smallest protein bands.

[0208] Determination of three protein bands: Bands P1 (less than 10,000 Da) and P2 (between 10,000 and 17,000 Da) and P3 (approximately 23,000 Da) are prepared and passed through liquid chromatography coupled to mass spectrometry analysis (LC MS / MS).

[0209] The results are obtained from 2 bioinformatics software specialized in protein recognition in plants (Mascot and Peaks): The identification results do not allow for the precise identification of these proteins; we are in the presence of proteins not currently described in the aforementioned databases. Example 13: Formulation of an anti-fibrotic dermatological product (liquid cleanser)

[0210] [Table 31] Ingredients % Water 80,7000 Sodium coco-sulfate 5,0000 Sodium cocoyl isethionate 4,0000 Bentonite 3,7800 Caprylic / Capric triglyceride 2,0000 Protein hydrolysate peregrina according to the invention 2,0000 Gluconolactone 0,7500 Sodium benzoate 0,5450 Scent 0,5000 Xanthan gum 0,2700 Sodium stearoyl glutamate 0,2250 Citric acid 0,2250 Calcium gluconate 0,0050 Example 14: Formulation of a dermatological product treating topical fibrosis (leave-on skincare product)

[0211] [Table 32] Ingredient % Water 71,5000 Caprylic / Capric triglyceride 18,0000 Bentonite 4,2000 Cetearyl alcohol 1,5000 Protein hydrolysate peregrina according to invention 2,0000 Gluconolactone 0,7500 Sodium benzoate 0,5450 Xanthan gum 0,5000 Scent 0,5000 Sodium stearoyl glutamate 0,2500 Citric acid 0,2500 Calcium gluconate 0,0050 Example 15: Formulation of a composition for subcutaneous injection

[0212] Formulation of a product for subcutaneous administration: dry extract according to the invention (containing 60% protein hydrolysate on an inulin support) packaged in a single-dose vial under inert gas ready to be solubilized by a physiological medium. Example 16: Formulation of a composition for subcutaneous injection

[0213] Formulation of a liquid injectable product: liquid extract according to the invention dosed at 5% in a physiological medium conditioned under sterile conditions in particular by vacuum filtration with a cut-off threshold of 0.45µm.

[0214] Example 17: Patch Formulation (A dressing or external medical device stuck to the skin, soaked in the active ingredient which it releases slowly, allowing a slow diffusion effect of the active ingredient). Example 18: Formulation of a drug in capsule form

[0215] Antifibrotic drug in 750 mg capsules containing 100 mg of piperine, 300 mg of dry extract according to the invention (containing 60% protein hydrolysate on an inulin support), 100 mg of boswellic acid, 250 mg of calcium carbonate. Example 19: Formulation of a drug in tablet form

[0216] Antifibrotic drug in 1 g tablet form: 300 mg of dry extract according to the invention (containing 60% protein hydrolysate on an inulin support), 400 mg of calcium carbonate containing 200 IU of vitamin D, 150 mg of magnesium gluconate, 80 mg of inulin and 70 mg of magnesium stearate.

[0217] Example 20: Formulation of a nasal spray medication (solution containing the active ingredient in a medical device that propels the active solution into the nasal cavity by misting).

[0218] Example 21: Toxicological tests of the protein hydrolysate according to the invention Preparation of the protein hydrolysate according to example 1: Unhulled seeds of Moringa peregrina(Forssk.) Fiori, harvested at fruit maturity, were dried to obtain an internal moisture content of less than 8%, preferably around 6%, and then pressed using a mechanical screw press to separate the oil from the rest of the seed, yielding virgin oil on the one hand and press cake on the other. The press cake was then separated into pre-cut rolls of 1 to 2 cm. Following the protocol described in Example 1, the liquid extract was obtained, which was used undiluted in the subsequent tests. 1. Determination of mutagenic activity on the bacterial strain Salmonella typhimurium (TA 100) - Reverse bacterial mutation test

[0219] The test took place in 3 main phases: A preliminary experiment is conducted to evaluate the cytotoxicity of the test element and to select the dose range for subsequent experiments. This involves a first genotoxicity experiment (Test 1), with and without metabolic activation, with direct incorporation of the test system and the test element (or controls) onto minimal agar, at the dose range defined by the preliminary study. A second experiment (Test 2) involves pre-incubation of the test system and the test element (or controls), with and without metabolic activation, at dose levels defined by the study principal investigator after analysis of the results of the first experiment. This second experiment is conducted to confirm or supplement the results of the first, particularly when equivocal or negative results were obtained. Dilutions of the extract according to example 1 were prepared in water to perform the cytotoxicity test.

[0220] The latter was carried out on the stump Salmonella typhimurium TA100 at concentrations of 5000, 1600, 500, 160, and 50 µg / plate, with and without S9-Mix. The reagents used for the preparation of S9-Mix were prepared according to the following instructions: [Table 33] Final concentration MgCl2 (0.4 M) + KCl (1.65 M) 8 mm + 33 mm Glucose 6 Phosphate (0.2 M) 5 mm NADP (0.1 M) 4 mm Phosphate buffer for S9-Mix (pH 7.4 - 0.2 M) 0.1 M S9 fraction 10% Water Adjust for final concentration

[0221] The bacteria were exposed to the test extract according to the invention with and without a metabolic activation system. The metabolic system used is a cofactor-enhanced post-mitochondrial fraction (S9). This S9 fraction, a microsomal fraction of Sprague Dawley rat liver homogenate treated with an enzyme inducer, is prepared according to MARON, DM et al., 1983, Revised Methods for the Salmonella Mutagenicity Test, Mutation Research / Environmental Mutagenesis and Related Subjects, 113, pp. 173–215, and was supplied by MOLTOX TM. It is stored at a temperature below -70°C. The S9 microsomal fraction was used at a concentration of 10% in S9-Mix. The protocol applied was as follows: In 3 hemolysis tubes, the following was introduced: ∘ assay without metabolic activation: 0.1 ml of the different concentrations of the test elements, 0.5 ml of sterile 0.2 M phosphate buffer, pH 7.4, 2 ml of top agar for S. typhimurium,0.1 ml of bacterial inoculum (TA100). ∘ Assay with metabolic activation: 0.1 ml of the different concentrations of the test elements, 2 ml of top agar for S. typhimurium, 0.1 ml of bacterial inoculum (TA100), 0.5 ml of S9-Mix. Mix and pour onto the surface of the base agar previously distributed in Petri dishes. Incubate at 37°C ± 2°C for 48 to 72 hours. These assays were performed for each test: preliminary cytotoxicity test, test 1, and test 2. The untreated control, negative controls, and positive controls performed during the pre-incubation method were incubated for 20 to 30 minutes at 37°C ± 2°C before pouring the top agar. The protocol applied was as follows: In 4 fractions of 2 ml of top agar for S. typhimurium,Introduce: ∘ 0.1 ml of 0.2 M phosphate buffer, pH 7.4, ∘ 0.1 ml of solvent, ∘ 0.1 ml of S9-Mix, ∘ 0.1 ml of the preparation of the element to be tested at the highest concentration, A 2 ml fraction of agar top for S . typhimurium is used to check its sterility. Mix and pour onto the surface of the background agar previously distributed in Petri dishes. Incubate at 37°C ± 2°C for 48 to 72 hours. The test is performed in triplicate. No bacterial growth should be observed. For at least 5 concentrations of the extract to be tested, a test without metabolic activation and a test with metabolic activation were carried out. Expression of results and interpretation

[0222] Numerous criteria can be used to determine if a result is positive, including an increase in the number of revertants correlated with the dose of the item to be tested, or a reproducible increase in the number of revertants at one or more concentrations, with or without metabolic activation. The element to be tested is considered mutagenic if, following the verification steps, a reproducible dose-response relationship has been obtained on one or more of the 5 strains with and / or without metabolic activation. Mutagenicity is only taken into account for a given concentration when the number of revertants is at least twice the spontaneous reversion rate for strains TA98, TA100, and TA102 (R ≥ 2) and three times the spontaneous reversion rate for strains TA1535 and TA1537 (R ≥ 3).The test item is considered non-mutagenic if, at the end of test 1 and test 2, the rate of revertants has always remained less than twice the rate of spontaneous reversion for all concentrations of the tested test item, for strains TA98, TA100 and TA102 (R <2) and less than three times the rate of spontaneous reversion for strains TA1535 and TA1537 (R <3), with and without metabolic activation, and provided that it has been verified that the absence of the mutagenic effect is not related to the toxicity of the concentrations tested. The preliminary study showed no cytotoxicity of the test element; therefore, this concentration range was used for the genotoxicity test 1.

[0223] Based on the results obtained for test 1, it was decided to use the same dilution range for test 2. Analysis of the revertants shows that: No cytotoxic effect was observed. No concentration of the extract to be tested showed an R ratio greater than or equal to at least twice the spontaneous reversion rate for TA98, TA100 and TA102 and three times the spontaneous reversion rate for TA1535 and TA1537, with and without metabolic activation. No dose response was observed, regardless of the test system or test conditions.

[0224] In view of the results obtained during this study, the protein hydrolysate according to example 1 can be considered as having no mutagenic or pro-mutagenic activity. 2. Phototoxicity Test in vitro 3T3 NRU

[0225] The principle of the test is based on comparing the cytotoxicity of the protein hydrolysate (Example 1) in the presence and absence of a non-cytotoxic dose of UVA radiation on cultured cells. Cytotoxicity is assessed by determining cell viability using a vital dye: neutral red, 24 hours after treatment with the reference elements and the protein hydrolysate. peregrina according to the invention, with or without UVA irradiation. The cells used are Balb / c 3T3 clone 31 mouse embryo fibroblasts (ATCC - CCL163). The positive control is a chlorpromazine solution (CAS No. 69-09-0). The negative control is a diluent of the tested and reference extracts (buffered saline + / - 1% solvent). Protein hydrolysate peregrinawas tested at 8 concentrations on at least four culture wells per concentration studied, in the presence or absence of UVA. Fibroblasts were trypsinized and two 96-well culture plates were seeded with 100 µl of a cell suspension at 2.10 5< cells / ml (i.e. 2.10 6< cells per well) in complete culture medium.

[0226] The inoculated plates were incubated in an incubator for 24 hours at 37°C, 5% CO2. At the end of incubation, the semi-confluence of the cell mat was verified. The dilutions were prepared just before being applied to the cells. The pH of the highest concentration was measured; it ranged from 6.5 to 7.8. The culture medium was discarded; each well was first gently rinsed with 150 µl of PBS maintained at room temperature before being treated with 100 µl of each dilution of the extract or reference. The culture plates were incubated in the dark for 1 hour ± 5 minutes at 37°C and 5% CO2. Irradiation was performed using a BIO SUN solar irradiator (Vilber Lourmat RMX3W). The BIO SUN is a system that controls UV irradiation using a programmable microprocessor. The system continuously monitors UV light emission. Irradiation stops automatically when the delivered energy equals the programmed energy.The spectral irradiation of the device under test was measured in the wavelength range of 250 to 700 nanometers with a calibrated spectroradiometer.

[0227] One of the two plates was irradiated with its lid on at room temperature, while the other plate was protected from UVA and kept at room temperature during irradiation. After irradiation, the treatment medium was aspirated and the cells were rinsed. Then, 100 µl of complete culture medium was slowly added, and the plates were incubated for 18 to 22 hours at 37°C and 5% CO2. The following day, cell viability (growth, morphology, monolayer integrity) was assessed by phase-contrast microscopy. The culture medium was removed, each well was rinsed and kept at room temperature before being treated with 100 µl of the staining solution. The plates were returned to the incubator for 3 hours under the same conditions.The dye solution was removed and the cells were rinsed. The rinsing solution was then removed, and 150 µl of desorption solution was added to each well. The plates were shaken until the crystals were completely dissolved. Absorbances were measured at 450 nm. Test validation:

[0228] Cell sensitivity to UVA is checked approximately every 10 passes by assessing viability after exposure to increasing doses of radiation. Cells are cultured at the density used in the assay. They are irradiated the following day at doses of 2.5 and 9 J / cm², and cell viability is determined one day later using the NRU assay. Cells meet quality criteria if their viability after irradiation at 5 J / cm² UVA is greater than or equal to 80% of the viability of controls maintained in the dark; at the highest dose of 9 J / cm² UVA, viability must be at least equal to 50% of that of controls maintained in the dark. Results :

[0229] The negative control has an absorbance greater than or equal to 0.4. Chlorpromazine, the positive control, has a Cl50 between 0.1 and 2 µg / ml in the presence of UVA and between 7 and 90 µg / ml in the absence of UVA. These results validate the test. The concentration of the protein hydrolysate peregrina According to example 1, a 50% cell death rate in the presence or absence of UVA cannot be estimated. Mortality has never reached 50%. The concentration of the protein hydrolysate peregrina The 50% cell viability rate in the presence or absence of UVA cannot be estimated. Viability is always greater than 50%.

[0230] Conclusion: Under the experimental conditions considered, the protein hydrolysate peregrina according to the invention, it can be considered non-phototoxic. 3. Evaluation of ocular irritant potential by cytotoxicity study in vitro according to the neutral red release method on the SIRC cell line

[0231] This in vitro study is based on the evaluation of the cytotoxicity of the protein hydrolysate peregrinaby determining the concentration causing 50% cell death (Cl 50) on a cell monolayer using the neutral red release technique. The cells used are mycoplasma-free SIRC rabbit corneal fibroblasts (ATCC - CCL60).

[0232] Protein hydrolysate peregrinaThe solution was diluted in 25% and 50% physiological saline. Fibroblasts were trypsinized, and two 24-well culture plates were inoculated with 1 ml of a cell suspension at 2 x 10⁵ cells / ml in complete culture medium. The inoculated plates were incubated overnight at 37°C and 5% CO₂. At the end of incubation, the confluence of the cell mat was checked. The staining solution was prepared at 0.5 mg / ml in complete culture medium. The culture medium was discarded, and 1 ml of the staining solution was added to each well. The plates were returned to the incubator at 37°C and 5% CO₂ for 3 hours ± 15 minutes. After this contact time, the staining solution was discarded and replaced with 1 ml of complete culture medium per well. The plates were kept at room temperature for at least 30 minutes to stabilize the system before contact with the extract or reference.Each well was rinsed with 2 ml of PBS, maintained at room temperature, then 500 µl of each protein hydrolysate dilution. peregrina or reference samples were deposited in contact with the cell mat. The contact time was 60 seconds (30 seconds for the positive control). The treatment was performed well by well, with the timer starting upon deposition of the protein hydrolysate. peregrinaor the reference. The plate was agitated manually throughout the entire treatment. After 55 seconds (or 25 seconds for the positive control), the dilution was aspirated. At precisely 60 or 30 seconds, five successive rinses were performed (5 x 2 ml of PBS maintained at room temperature). The supernatant was aspirated after each rinse, and after the last rinse, the wells were left empty of medium pending the development phase. After complete treatment of the culture plate, 1 ml of the desorption solution was added to each well. The plate was agitated for approximately 15 minutes until a homogeneous color was obtained. The solutions obtained for each culture well were taken and distributed into two wells of a 96-well plate, i.e., 150 µl / well. Results :

[0233] The concentration of the protein hydrolysate peregrinaThe percentage of cell death resulting in 50% protein hydrolysate was estimated to be >50%. peregrina was assessed at 17%.

[0234] Conclusion: Under the experimental conditions considered, the cytotoxicity of the protein hydrolysate peregrina according to the invention, it can be classified as having negligible cytotoxicity. 4. Evaluation of the skin compatibility of a protein hydrolysate peregrina according to the invention after single application under occlusive dressing for 48 hours under dermatological control.

[0235] The aim of this study is to evaluate the degree of skin compatibility of the protein hydrolysate peregrina by patch test, performed on the anterolateral aspect of the arm for 48 hours; and generally to evaluate the ability of the protein hydrolysate to maintain healthy skin. Ten healthy volunteers, male or female, aged 18 to 65 years, with neither dry nor sensitive skin and free from any dermatological lesions in the treatment area, were to be included in the study. The skin compatibility of the protein hydrolysate peregrina, prepared as a lotion with 5% protein hydrolysate peregrina According to example 1 and 95% of a Propanediol / Sorbitol mixture, the reaction was assessed 48 hours after initial application and between 30 and 40 minutes after dressing removal. Skin reactions (erythema and edema) were counted from 0 to 3 according to the following scales: [Table 34] Quoting Erythema (E) Edema (Oe) 0 No erythema No edema 0,5 Barely perceptible erythema, very slightly pinkish discoloration on part of the depatching area Palpable edema, barely perceptible 1 Mild erythema, pinkish discoloration over the entire patch area Palpable and visible edema 2 Moderate erythema, clear discoloration over the entire patching area Clear edema with or without papules or vesicles 3 Significant erythema, intense discoloration over the entire patching area Significant edema spreading beyond the patching area, with or without papules or vesicles

[0236] Any other skin reaction (blisters, papules, vesicles, dryness, scaling, roughness, soap effect, etc.) was assessed according to the following scale and reported descriptively: 0: no reaction, 0.5: very slight, 1: slight, 2: moderate, 3: significant.

[0237] At the end of the study, an average irritation index (IIM) was calculated using the following formula:

[0238] The IIM obtained allowed the protein hydrolysate to be classified peregrina tested according to the scale presented below: IIM ≤ 0.20 Non-irritating 0.20 < IIM ≤ 0.50 Slightly irritating 0.50 < IIM ≤ 2 Moderately irritating 2 < IIM ≤ 3 Very irritating

[0239] Results: The Mean Irritation Index (MII) of the protein hydrolysate peregrina is equal to: 0.

[0240] Conclusion: Protein hydrolysate peregrina can be considered non-irritating after 48 consecutive hours of application in 12 volunteers. General conclusion of the tests:

[0241] The results of the tests carried out above are conclusive and demonstrate the properties of the protein hydrolysate. peregrina according to example 1: 1) Eye and skin irritation tests are negative. 2) Phototoxicity tests are negative. 3) Mutagenicity tests are negative.

[0242] The safety of protein hydrolysate peregrinaaccording to the invention is demonstrated and ideal for large-scale dermatological use without restriction on target populations.

Claims

1. A method for obtaining a protein hydrolysate from Moringa peregrina seed cake, characterized in that it comprises the following steps, in which: a) unshelled mature seeds are collected from the ripe Moringa peregrina fruit and dried to an internal moisture content of less than 8%, b) the dried seeds are pressed in a manner such as to separate the oil from the remainder of the seed in a manner such as to obtain the cake comprising less than 6% by weight of residual oil, c) the cake obtained in step b) is ground, d) the ground cake obtained in step c) is dispersed in the aqueous phase in a proportion of 90.9 / 9.1 (mass / mass) respectively, e) the aqueous dispersion obtained in step d) undergoes chemical proteolysis for a period of approximately 2 hours, at a pH greater than 13 and at a temperature comprised between 16 and 25°C, f) the proteolysis is neutralised in order to stabilise the protein hydrolysate obtained, g) the protein hydrolysate is recovered by solid / liquid separation, h) the protein hydrolysate is purified by ultrafiltration and / or nanofiltration carried out with a cut-off threshold comprised between 100 and 25,000 Da.

2. The method according to claim 1, characterized in that the nanofiltration is carried out in a manner such as to separate 3 bands from the protein hydrolysate comprising a P1 band with a molecular weight of less than 10,000 Da, a P2 band with a molecular weight of between 10,000 and 17,000 Da and a P3 band with a molecular weight of about 23,000 Da.

3. The method as claimed in one of claims 1 and 2, characterised in that the nanofiltration step h) is carried out with a cut-off threshold comprised between 1,500 Da and 5,000 Da, preferably with a cut-off between 3,000 Da and 4,500 Da.

4. The method as claimed in any one of claims 1 and 2, characterised in that the nanofiltration step h) is carried out with a cut-off threshold comprised between 10,000 Da and 17,000 Da.

5. The method as claimed in any one of claims 1 and 2, characterised in that the nanofiltration step h) is carried out with a cut-off threshold comprises between 17,000 Da and 25,000 Da.

6. The method as claimed in any one of claims 1 to 5, characterised in that i) the protein hydrolysate obtained in step h) is freeze-dried.

7. A protein hydrolysate from seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, characterised in that it comprises a major fraction P1 (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight is comprised between 1,500 Da and 5,000 Da, a fraction P2 of approximately 20% (mass / mass) for which the molecular weight is comprised between 10,000 and 17,000 Da, and a fraction P3 of approximately 20% (mass / mass) for which the molecular weight is approximately 23,000 Da, in that it is obtained by chemical proteolysis at a pH of more than 13 for a period of approximately 2 hours at a temperature comprised between 16 and 25°C and in that it is liquid and has a density of more than 1.

8. A protein hydrolysate from seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, characterised in that it comprises the major fraction P1 (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight is comprised between 1,500 Da and 5,000 Da, in that it is obtained by chemical proteolysis at pH of more than 13 for a period of approximately 2 hours at a temperature comprised between 16 and 25°C followed by nanofiltration carried out with a cut-off threshold comprised between 1,500 Da and 5,000 Da and in that it is liquid and has a density of more than 1.

9. A protein hydrolysate seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, characterised in that it comprises the P2 fraction of approximately 20% (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight is comprised between 10,000 Da and 17,000 Da, in that it is obtained by chemical proteolysis at pH of more than 13 for a period of approximately 2 hours at a temperature comprised between 16 and 25°C followed by nanofiltration carried out with a cut-off threshold comprised between 10,000 Da and 17,000 Da and in that it is liquid and has a density of more than 1.

10. A protein hydrolysate seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, characterised in that it comprises the P3 fraction of approximately 20% (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight is approximately 23,000 Da, in that it is obtained by chemical proteolysis at pH of more than 13 for a period of about 2 hours at a temperature comprised between 16 and 25°C followed by nanofiltration carried out with a cut-off threshold comprised between 17,000 Da and 25,000 Da and in that it is liquid and has a density of more than 1.

11. A protein hydrolysate seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, characterized in that it comprises in combination the protein hydrolysate of claim 8 and the protein hydrolysate of claim 9.

12. The protein hydrolysate of Moringa peregrina seed cake according to any of claims 7 to 11 for use as a medicament.

13. A pharmaceutical or dermatological composition, characterized in that it comprises, as an active agent, an effective quantity of a protein hydrolysate of the Moringa peregrina seed cake as claimed in one of claims 7 to 11, and a physiologically acceptable excipient.

14. The pharmaceutical composition as claimed in claim 13, characterised in that it is formulated for ingestion.

15. The dermatological composition as claimed in claim 13, characterised in that it is formulated for local application to the skin and the mucous membranes.

16. The composition as claimed in claims 13 to 15, characterised in that the protein hydrolysate of Moringa peregrina seed cake is present in the composition in a concentration of 0.0001 to 40% by weight with respect to the total weight of the composition.

17. A composition as claimed in one of claims 13 to 16, for its use as a drug for the treatment of fibrotic diseases and the treatment of inflammation, characterized in that it comprises as the active agent, an effective quantity of the protein hydrolysate as claimed in claim 8 or 9, preferably as claimed in claim 9.

18. A composition as claimed in one of claims 13 to 16, for its use as a drug for the treatment of cancer, characterised in that it comprises, as the active agent, an effective quantity of the protein hydrolysate as claimed in claim 10.

19. A composition as claimed in one of claims 13 to 16, for its use as a drug for the treatment of infectious diseases of the viral type to inhibit the Spike-COV2 proteins of SARS-COV2, characterized in that it comprises, as the active agent, an effective quantity of the protein hydrolysate as claimed in claim 7.

20. A composition as claimed in one of claims 13 to 16, for its use as a drug for the treatment of skin fibrosis and lentigo, characterized in that it comprises, as the active agent, an effective quantity of the protein hydrolysate as claimed in claim 11.