OA21069A - Protein hydrolyzate from Moringa peregrina seed cake for its application as a medicine, its process for obtaining it and pharmaceutical and dermatological compositions - Google Patents

Abstract

The invention relates to a process for obtaining a particular protein hydrolyzate from Moringa peregrina seed cake. It also relates to a protein hydrolyzate from Moringa peregrina seed cake and its use as a medicine. Finally, the invention relates to a pharmaceutical or dermatological composition, comprising as active agent, an effective quantity of a protein hydrolyzate from the seed cake of Moringa peregrina for its use as a medicine for the treatment of fibrotic diseases and the treatment of inflammation, the treatment of cancer, the treatment of infectious diseases of bacterial or viral type and the treatment of genetic drift and pathologies associated with skin pigmentation.

Description

Description

Title of the invention: Protein hydrolyzate from Moringa peregrina seed cake for its application as a medicine, its process for obtaining it and pharmaceutical and dermatological compositions.

|Technical area

[1] The invention relates to the field of pharmacy and dermatology and more particularly that of active ingredients used in the formulation of medicines. The invention relates to a process for obtaining a protein hydrolyzate from Moringa peregrina seed cake. The invention also relates to the protein hydrolyzate of Moringa peregrina seed cake, the pharmaceutical and dermatological compositions comprising such a hydrolyzate and intended to be used for the treatment of fibrotic diseases, the treatment of inflammation, cancer, bacterial or viral infectious diseases as well as the treatment of genetic drift and pathologies associated with skin pigmentation.

Technology background

[2] The Moringaceae constitute a mono-generic family (a single genus, Moringa Adans), element of the Saharo-Sindian flora, made up of between twelve and fourteen species according to the authors, distributed from eastern Africa to Asia . The genus is classically divided into 3 sections which are, however, not confirmed as monophyletic by phylogenetic analyses. The latter rather highlighted clades focused on certain morphological characters: pachycaules (“bottle trees”); tuberous trees and neither pachycaules nor tuberous trees (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 reality of the species compared to other species in the genus, particularly with regard to the Indian Moringa, Moringa oleifera Lam. (see in particular the articles: Olson, Μ. E., 2002, Combining Data from DNA Sequences and Morphology for a Phylogeny of Moringaceae (Brassicales), Systematic Botany 27(1): p. 55-73; Hassanein, A. M. A. et al. , 2018, Morphological and genetic diversity of Moringa oleifera and Moringa peregrina genotypes, Horticulture,

Environment and Biotechnology 59(2): p. 251-261). A recent article on Moringa peregrina sampled from different localities in Saudi Arabia concluded, using ITS markers, that the species is genetically stable (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.

[3] The species Moringa peregrina is found in rocky environments of Yemen, Oman, and Saudi Arabia, in East Africa, Sudan, Ethiopia, Eritrea, Somalia and Djibouti. Its presence in Iran seems limited to the south-eastern 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 10/23/2019). In the Levant and Egypt, the species today only represents rare, scattered, relict stations (with the exception of a few altitudinal populations), mainly in the areas of the Sudanian area. Moringa peregrina is also considered rare and endangered today in Sudan and Yemen. Moringa peregrina occupies the driest and most inhospitable habitats compared to the other species of its clade. It is apparently more drought tolerant than Moringa oleifera which is planted commercially on a large scale in tropical and subtropical areas. Recent work has shown that the size and size of seeds has a positive impact on germination time and the rate and speed of growth of young individuals (Gomaa, N.H. state, 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 on seed quality rather than number, this which allows Moringa peregrina to reproduce effectively in extreme abiotic (hyperarid) environments. Moringa peregrina seeds have a thicker central mesotesta, in terms of cell layer, than those of Moringa oleifera.

[4] There are some historical mentions which tend to indicate that the oil of

Moringa peregrina was actively traded in the early days of Islam in the AlUla region (Naseef, A. A. S., 1995, Al-Vlâ, A study of Cultural and

Social Heritage). The oil produced from Moringa peregrina is today mainly intended for self-consumption or local markets. In Saudi Arabia, the leaves were traditionally used as a decoction for internal use to treat diabetes, colon diseases, eye diseases and anemia (Abdel-Kader et al., A survey on the traditions! plants used in Al 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 Center for Science and Technology, Riyadh, Saudi Arabia). In Oman, the oil, extracted by women at the end of summer, is used against migraines, fever, burns, lacerations and fractures, constipation and stomach pain, against muscle pain and against hair dryness (Ghazanfar, SA, 1994, Handbook of Arabian Medicinal Plants, ^1st ed., CRC Press, Boca Raton, Ann Harbor, su; Ghazanfar, SA, 1998, Plants of Economic Importance, cap. 15, in Ghazanfar, SA et al. (eds.) Vegetation of the Arabian Peninsula. Geobotany 25, pp. 241-264, Kluwer Academie Publishers, table 11.1, p. 247 and 11.7 p. 251). It was also used in perfume 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 form 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).

[5] A certain number of extracts from the seed of Moringa oleifera are known, particularly useful in the cosmetic field. In the dermatological field, we know from document FR2776519 that protein extracts of Moringa oleifera seeds, known for their clarifying effects on turbid water, present on the skin and mucous membranes a softening, physiological conditioning, hydrating, restructuring, repairing and an anti-pollution effect. In this document the active ingredients are proteins with molecular weights between 6,500 and 8,800 Da which are obtained by aqueous extraction on Moringa oleifera cake.

[6] In the pharmaceutical field, we know from document KR20140143655 a composition containing a water-soluble extract of Moringa oleifera leaves as an active ingredient for treating or preventing cancer. The enzymatic hydrolysis process on Moringa oleifera leaf extracts makes it possible to isolate proteins with molecular weights between 6,000 and 8,000 Da.

[7] Document CN107012190 concerns the use of Moringa oleifera seeds to obtain, after extracting the oil and carrying out enzymatic hydrolysis using microwave radiation, polypeptides from the proteins of the seed. The resulting product is used orally and is useful for anticancer activity. This product in powder form is described as actually having an action to reduce the side effects of chemotherapy and to increase the appetite of patients with liver cancer, or to control the leukocyte index and body temperature.

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

[9] All the aforementioned documents concern the use of the species Moringa oleifera; none describes the use in the pharmaceutical or dermatological field of extracts from the Moringa peregrina species.

[10] We also know the document by Abu Tarbosh et al. XP055753092, 2005 which describes the extraction of a hydrolyzate from shelled Moringa peregrina seeds produced by enzymatic hydrolysis for a period of 10 hours. The aim of this extraction is to obtain a product with a high oil and water absorption capacity.

[11] An extract of Moringa peregrina oil made from Egyptian seeds, with a dichloromethane/methanol mixture (1/1) showed activity on three human cancer cell lines MCF-7 (breast adenocarcinoma). ), HepG2 (hepatocellular carcinoma) and HCT-116 (colon carcinoma) with IC50 values of 2.92, 9.40, and 9.48 pg/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 by DPPH (2,2-diphenyl 1 -picrylhydrazyl), ABTS (anionscavenging capability and reducing power) and anti-proliferative activity. Due to processes for obtaining the extract different from those of the invention, the molecules obtained have very different polarities.

[12] Abou-Hashem and colleagues (Abou-Hashem, Μ. Μ. M. et al., 2019, Induction of sub-GO arrest and apoptosis by seed extract of Moringa peregrina (Forssk.) Fiori in cervical and prostate cancer cell lines, Journal of Integrative Medicine 17: p. 410-422) also demonstrated an activity of inducing subGo arrest and apostosis on cervical (HELA) and prostate (PC-3) cancer cell lines. ). The protocol involved Egyptian Moringa peregrina seeds, powdered and extracted with 95% ethanol, then dissolved in an aqueous solution from which three fractionated extracts were studied (fractions by petroleum ether (PE), CHCl3 and EtOAc) as well as that a hydrolic extract remains from the extraction. The activity was demonstrated for the total chloroform fraction and was attributed to saturated and unsaturated fatty acids and polyphenols without specific study of the mechanistics. Due to processes for obtaining the extract different from those of the present invention, the active molecules obtained have very different polarities.

[13] Considering the above, a problem that the invention proposes to solve is to develop new products based on an extract of the species Moringa peregrina of the Moringa genus and the Moringaceae family which can be used in pharmacy or dermatology and easy to implement.

[14] Thus, the Applicant has surprisingly demonstrated a particular protein hydrolyzate obtained from Moringa peregrina seed cake for its application as a medicine, and in particular for the treatment of fibrotic diseases (as an inhibitor of furin convertase), the treatment of inflammation, cancer, bacterial or viral infectious diseases as well as the treatment of genetic drift and pathologies associated with skin pigmentation,

[15] The species Moringa peregrina grows in very arid climates. Thus, its ability to resist drought and to reproduce in extremophilic conditions allowed it to acquire particular unique characteristics, which the Applicant was able to identify by implementing a specific extraction process on the cake. Moringa peregrina seeds.

The protein hydrolyzate according to the invention turns out to have properties as a drug and it is demonstrated that it has in particular a very high inhibitory activity of furin convertase.

[16] Furin convertase (hereafter named furin) is a type 1 transmembrane protein with 794 amino acids expressed in different 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.orq/ 10.1002/cti2.1073). It is involved in particular in wound healing as well as in the regulation of fibrosis in the treatment of conditions in which fibrosis is a major mechanism of tissue repair or when excessive fibrosis leads to pathological disturbance and tissue dysfunction (see In this regard, document WO200409113 relates to 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 an internal tissue or organ such as the lungs, kidneys, heart, intestines, tendons, or liver. The wound healing process on tissue usually begins with a hemostatic response triggered by damage to blood vessels in the skin. During this process, the connective tissue that forms during the healing process is often fibrous in nature and usually forms into connective tissue scarring (a process called fibrosis). Thus, fibrosis may include pulmonary fibrosis, renal fibrosis, hepatic fibrosis, skin fibrosis, ocular fibrosis, cardiac fibrosis, and other various fibrotic conditions. The protein hydrolysates of the invention may also be useful for treating other furin-mediated disease states, including, but not limited to, hypertension, cancer, infectious diseases (bacterial and viral), and genetic disorders (for example, cystic fibrosis (CF)), and neurodegenerative disorders (see in this regard document WO2019215341 which relates to new 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).

[17] By intergovernmental agreement of April 10, 2018 between the government of the French Republic and the Kingdom of Saudi Arabia, the plaintiff, Agence Française Pour Le Développement d'AIUla (AFALULA) as well as the Royal Commission for AIUIA (RCU) have for a joint project in particular to develop responsible agriculture and the local economy, in particular through the local production of natural products derived from indigenous plants and to protect the biodiversity and rights of the AIUla region of the Kingdom of Saudi Arabia. The Kingdom of Saudi Arabia has been a member of the Nagoya Protocol since October 8, 2020. At the time of writing this patent, the implementing regulations under which the Nagoya Protocol will be incorporated into relevant aspects of local law are in force. exam course. 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 day the patent application is filed, there is no requirement for a certificate of conformity regarding access to genetic resources.

[18] Summary

The first subject of the invention is a process for obtaining a protein hydrolyzate from Moringa peregrina seed cake, comprising the following steps according to which:

a) the unshelled seeds are collected when ripe from the fruit of Moringa peregrina which are dried to obtain an internal humidity level of less than 8%,

b) the dried seeds are pressed so as to separate the oil from the rest of the seed, so as to obtain the cake comprising less than 6% by weight of residual oil,

c) we grind the cake obtained in step b),

d) the ground cake obtained in step c) is dispersed in an aqueous phase.

e) chemical proteolysis is carried out on the aqueous dispersion obtained in step d) for a period of approximately 2 hours, at a pH greater than 13 and at a temperature between 16 and 25°C,

f) proteolysis is neutralized to stabilize the protein hydrolyzate obtained,

g) the protein hydrolyzate is recovered by solid/liquid separation,

h) the protein hydrolyzate is purified by ultrafiltration and/or nanofiltration, then optionally,

i) the protein hydrolyzate obtained in step h) is lyophilized.

The second object of the invention is a hydrolyzate of proteins from the cake of unshelled seeds harvested at maturity from the fruit of Moringa peregrina comprising a majority fraction of amino acid derivatives, amino acids, peptides and glycopeptides whose molecular weight P1 is between 1,500 Da and 5,000 Da, a fraction of approximately 20% whose molecular weight P2 is between 10,000 to 17,000 Da and a fraction of approximately 20% whose molecular weight P3 is approximately 23,000 Da, in that it is obtained by chemical proteolysis at a pH greater than 13 for a period of approximately 2 hours at a temperature between 16 and 25°C and in that it is liquid and has a greater density at 1 and preferably around 1.1.

[19] Thanks to its characteristics, the protein hydrolyzate of Moringa peregrina according to the invention has never been demonstrated in the Moringa genus and the Moringaceae family. It will be demonstrated that the extract of the species Moringa peregrina presents a particular peptide profile different from other species of the genus, in particular from the species Moringa oleifera, which the applicant was able to demonstrate.

[20] The third object of the invention is a protein hydrolyzate from Moringa peregrina seed cake for its application as a medicine.

[21] The fourth subject of the invention is a pharmaceutical or dermatological composition comprising, as active agent, an effective quantity of a protein hydrolyzate from Moringa peregrina seed cake and a physiologically acceptable excipient.

[22] Finally, the fifth object of the invention is a pharmaceutical or dermatological composition intended to be used for the treatment of fibrotic diseases, the treatment of inflammation, cancer, infectious diseases of bacterial or viral type as well as the treatment of genetic drift and pathologies associated with skin pigmentation.

Brief description of the figures

[23] The invention will be better understood, and other aims, details, characteristics and advantages thereof will appear more clearly during the following description of several particular embodiments of the invention, given solely by way of illustration. and non-limiting, with reference to the appended drawings.

[24] [fig.1] represents a diagram of the infection of a crown virus SRASCOV2 (Severe Acute Respiratory Syndrome-Coronavirus2) which demonstrates the anti-infectious and virostatic effect of peregrina protein hydrolyzate according to the invention against SARS COV2 crown pseudovirions.

[25] [fig-2] represents the diagram of furin inhibition by an extract of peregrina oil. In this figure, ** means significantly different from the “control” group (P<0.001).

[26] [fig.3] represents the diagram of furin inhibition by an extract of peregrina cake (96° ethanol). In this figure, * means significantly different from the “control” group (P<0.001).

[27] [fig.4] represents the diagram of furin inhibition by a peregrina meal protein hydrolyzate according to the invention. In this figure, means significantly different from the “control” group (P<0.001).

Description of embodiments

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

[29] 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 enumerating live cells)

- 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 ίο

[30] In the present invention, we mean:

- “mainly amino acid derivatives, amino acids, peptides and glycopeptides” a quantity greater than 20%, preferably even greater than 30% and which can reach approximately 40% (Mass/Mass) of dry matter in derivatives of amino acids, amino acids, peptides and glycopeptides, preferably another 50% of the weight of the dry matter.

- “effective quantity” the necessary quantity of active molecules to obtain the desired result, namely, to ensure the desired therapeutic activity.

- “proteolysis” the segmentation of proteins into peptides, oligopeptides and its basic fragments (amino acids) and its residues via chemical hydrolysis.

- “local route”, the fact that the medication, directly applied to its site of action, exerts its pharmacological effect on the precise site of the condition. The aim of the local route is to limit the diffusion of the active ingredient from its place of administration, allowing a minimum of undesirable effects. The main local routes used are the skin route, the nasal and respiratory route, the ocular route, the ear route, the vaginal route and the oral route.

- “topical application”, the act of applying or spreading the active principle according to the invention, or a composition containing it, to the surface of the skin, a mucous membrane or the integuments.

- “physiologically acceptable” which is 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, allergic response.

- “cake”, the deoiled part of the seed after pressing. This is the solid residue from the extraction of oil from the seeds. It is a co-product of crushing, the oil manufacturing process. It generally represents 50 to 75% of the mass of the seeds.

- “unshelled seeds”, the fact that the shell (pericarp) and the integument of the harvested seeds are maintained around the almonds.

- “at maturity of the fruit”, the fact that the fruit is ripe, preferably when the pod is at the beginning of dehiscence and it takes on a dark beige color at

brown and when a 180° twist on the lower quarter of the pod causes the valves to begin to open.

- “approximately” a margin more or less of 10% to 20% in relation to the information given.

- “pool of active molecules”, “active agent” and also “active principle”, the protein hydrolyzate extracted according to the process of the invention from Moringa peregrina seed cake. This hydrolyzate is responsible for the biological activities described in the present invention.

- “active agent” a sufficient quantity of an extract according to the invention to obtain the biological activities described. Depending on whether the extract is liquid or dried, concentrated or not, the quantities of the active agent can vary in proportions from 0.0001 to 40% by weight relative to the total weight of the composition.

[31] The invention firstly concerns a process for obtaining a protein hydrolyzate from Moringa peregrina seed cake comprising the following steps according to which:

a) the unshelled seeds are collected when ripe from the fruit of Moringa peregrina which are dried to obtain an internal humidity level of less than 8%,

b) the dried seeds are pressed so as to separate the oil from the rest of the seed so as to obtain the cake comprising less than 6% by weight of residual oil,

c) we grind the cake obtained in step b),

d) the ground cake obtained in step c is dispersed in the aqueous phase),

e) chemical proteolysis is carried out on the aqueous dispersion obtained in step d) for a period of approximately 2 hours, at a pH greater than 13 and at a temperature between 16 and 25°C,

f) proteolysis is neutralized to stabilize the protein hydrolyzate obtained,

g) the protein hydrolyzate is recovered by solid/liquid separation,

h) the protein hydrolyzate is purified by ultrafiltration and/or nanofiltration carried out with a cut-off threshold of between 100 and 25,000 Da, then optionally,

i) the protein hydrolyzate obtained in step h) is lyophilized.

[32] We collect the unshelled seeds, that is to say whose shell we keep when the fruit is ripe and preferably when the pod is at the beginning of dehiscence.

[33] The seeds are dried to obtain an internal humidity level of less than 8% and preferably around 6%; drying will preferably be done on a ventilated shelf sheltered from direct sunlight, preferably under a shade in the open air.

[34] The dried seeds are then crushed extemporaneously by cold pressing which makes it possible to mechanically separate the oil from the rest of the compressed seed, namely the cake.

[35] The cake is then crushed mechanically with any type of mechanical crusher such as hammer, flail, knife, crushing/shredding, ball or ball or pestle systems, but also with any type of cryomill.

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

[37] A liquid protein hydrolyzate 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 compounds. nitrogen, in particular volatile nitrite derivatives in a proportion of 0.5 to 1.5%, preferably around 0.8%, as well as 20 mg/liter of polyphenols (0.002%).

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

[39] 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, spinning, filtration.

[40] In one embodiment of the process for obtaining the liquid Moringa peregrina protein hydrolyzate obtained, the protein hydrolyzate is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration to concentrate it into compounds of interest compared to organic materials also extracted, in particular to other derivatives also extracted. These purification steps make it possible to concentrate the pool of compounds of interest at the expense of other extracted compounds as cited.

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

[42] It is also advantageous to separate the three bands by nanofiltration in order to obtain by the process of obtaining a protein hydrolyzate:

- a P1 strip with nanofiltration carried out with a cut-off threshold of between 1,500 Da and 5,000 Da, preferably with a cut-off threshold of between 3,000 Da and 4,500 Da.

- a P2 strip with nanofiltration carried out with a cut-off threshold of between 10,000 Da and 17,000 Da.

- a P3 strip with nanofiltration carried out with a cut-off threshold of between 17,000 Da and 25,000 Da.

[43] In another embodiment of the extraction process according to the invention, the liquid protein hydrolyzate obtained is dried so as to obtain a dry hydrolyzate of the Moringa peregrina seed cake containing a quantity greater than 10%, preferably greater than 20%, preferably even greater than 30% and capable of reaching approximately 40% (Mass/Mass) of dry matter in peptides, oligopeptides, glycopeptides and amino acids or their volatile nitrilated derivatives, preferably still 50% of the weight of the dry matter.

[44] According to one embodiment of the invention, the liquid Moringa peregrina seed meal protein hydrolyzate obtained is dried for example by atomization, lyophilization or zeodration so as to obtain a solid Moringa peregrina seed hydrolyzate, the water being evaporated. Drying can be carried out in the presence of an organic support such as maltodextrin, cyclodextrin or inuiîn, or in the presence of an inorganic support such as phyllosilicate, magnesium silicate or carbonate and its salts.

The invention also relates to the protein hydrolyzate of Moringa peregrina seed cake obtained by the extraction process according to the invention.

The second subject of the invention is a hydrolyzate of proteins from the cake of unshelled seeds harvested at maturity from the fruit of Moringa peregnna, comprising a majority P1 fraction of amino acid derivatives, amino acids, peptides and glycopeptides whose weight molecular weight is between 1,500 Da and 5,000 Da, a P2 fraction of approximately 20% whose molecular weight is between 10,000 to 17,000 Da and a P3 fraction of approximately 20% whose molecular weight is approximately 23,000 Da, in that it is obtained by chemical proteolysis at a pH greater than 13 for a period of approximately 2 hours at a temperature between 16 and 25°C and in that it is liquid and has a greater density at 1 and preferably around 1.1.

[45] According to one embodiment, the liquid protein hydrolyzate obtained is dried so as to obtain a dry hydrolyzate of the Moringa peregrina seed cake containing a quantity greater than 20%, preferably even greater than 30% and which can reach approximately 40% (Mass/Mass) of dry matter in peptides, oligopeptides, glycopeptides and amino acids or their volatile nitrilated derivatives, preferably another 50% of the weight of the dry matter.

[46] According to a preferred embodiment, the protein hydrolyzate also comprises between 0.3% and 3% of volatile compounds, including 50% of these compounds, or between 0.15% and 1.5% of the extract according to the invention, is made up of light nitrilated compounds, mainly isobutyrOnitrile and methylbutanenitrile; of which 5 to 10% of these compounds, or 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, or between 0.003 and 0.15%, is made up of essential oil, mainly Eucalyptol, Menthol and Benzaldehyde.

[47] According to yet another embodiment, the protein hydrolyzate comprises a dry matter content of between 10 and 15%, preferably around 12.5% comprising between 1 and 6% of nitrogen compounds, in particular volatile nitrile derivatives in a proportion of 0.5 to 1.5%, preferably around 5 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 are used for the extraction of their oil, useful for self-consumption or in various traditional medicinal treatments. The cake obtained after the seed has been de-oiled is a waste currently used for animal feed in particular.

[48] According to yet another embodiment, the protein hydrolyzate comprises the majority P1 fraction whose molecular weight is between 1,500 Da and 5,000 Da.

[49] According to yet another embodiment, the protein hydrolyzate comprises the P2 fraction of approximately 20% whose molecular weight is between 10,000 Da and 17,000 Da.

[50] According to yet another embodiment, the protein hydrolyzate comprises the P3 fraction of approximately 20% whose molecular weight is approximately 20 23,000 Da.

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

[52] The third object of the invention is a protein hydrolyzate from the cake of 25 Moringa peregrina seeds for its application as a medicine.

[53] The fourth subject of the invention is a pharmaceutical or dermatological composition comprising, as active agent, an effective quantity of a protein hydrolyzate from Moringa peregrina seed cake according to the invention and a physiologically acceptable excipient.

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

[55] According to a first variant, the different compositions are suitable for ingestion; the composition may be in the form of capsules, syrups, granules or tablets. It may not include an excipient and may consist entirely of the plant extract including the protein hydrolyzate in dried form.

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

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

[58] The main local routes used for the administration of the active agent according to the invention are the cutaneous and percutaneous route, the nasal and respiratory route, the ocular route, the ear route and the vaginal route. Other routes can be considered, in particular the oral route, to administer the compositions via the mucous membranes as well as the subcutaneous route by microinjections.

[59] The compositions according to the invention for pharmaceutical purposes can be presented in all the galenic forms normally used for local application. They can be administered to the mucous membranes, particularly to the nasal or respiratory, ocular, oral, vaginal and ear areas.

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

[61] The compositions incorporating a protein hydrolyzate according to the invention may include ingredients commonly used in this type of formulations locally or cutaneously.

[62] According to a preferred embodiment, the different compositions are suitable 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 propellant under pressure.

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

[64] The compositions according to the invention intended more particularly for local administration contain an acceptable pharmaceutical or dermatological medium, that is to say compatible with the skin and mucous membranes, and cover all appropriate dosage forms. These compositions may in particular be in the form of creams, oil-in-water emulsions, or water-in-oil or multiple emulsions, serums, solutions, suspensions, gels, milks, lotions, sticks, aerosols, sprays or any other product for external application such as that, for example, medical devices or aerosol products also containing a propellant under pressure, or in the form of powders, and 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, dyes, preservatives, perfumes.

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

[66] In all cases, those skilled in the art will ensure that these adjuvants as well as their proportions are chosen in such a way as not to harm the advantageous properties sought from the compositions according to the invention.

[67] In the compositions of the invention, the protein hydrolyzate 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.

[68] In a preferred embodiment, the protein hydrolyzate 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, more preferably in an amount ranging from 0 .01% to 5% by weight relative to the total weight of the composition.

[69] Finally, the fifth object of the invention is a pharmaceutical or dermatological composition for its use as a medicine for the treatment:

[70] - fibrotic diseases and the treatment of inflammation, comprising as active agent an effective quantity of the PI or P2 fraction of the protein hydrolyzate, preferably the P1 fraction.

[71] - cancer, comprising as active agent an effective quantity of the fraction

P3 of protein hydrolyzate.

[72] - infectious diseases of bacterial or viral type, and in particular to inhibit Spike-COV2 proteins, comprising as active agent an effective quantity of the protein hydrolyzate as a whole.

[73] - genetic drift and pathologies associated with skin pigmentation, comprising as active agent an effective quantity of the P1 and P2 fractions of the protein hydrolyzate. By “genetic drift” we mean in particular environmental stress. By “pathologies associated with pigmentation” we mean, for example, carcinomas and lentigo.

[74] For the treatment of fibrotic diseases, fibrosis means pulmonary fibrosis, renal fibrosis, hepatic fibrosis, skin fibrosis, ocular fibrosis, cardiac fibrosis, and other various fibrotic conditions and for the treatment of fibrotic diseases. other furin-mediated disease states, including, but not limited to, hypertension, cancer, infectious diseases including viral and bacterial, genetic disorders (e.g., cystic fibrosis (CF)), and neurodegenerative disorders.

[75] Although the invention has been described in connection with several particular embodiments, it is obvious that it is in no way limited there and that it includes all the technical equivalents of the means described as well as their combinations if these fall within the scope of the invention.

[76] The use of the verb “include”, “understand” or “include” and its conjugated forms does not exclude the presence of other elements or other steps than those set out in a claim.

Examples

[77] Example 1: Preparation of a plant protein hydrolyzate according to the invention from Moringa peregrina cake

[78] Moringa peregrina (Forssk.) Fiori seeds were dried to obtain an internal humidity level of less than 8% and preferably around 6%, then pressed with a mechanical screw press, so as to separate the oil from the rest of the seed to obtain on the one hand the virgin oil and on the other hand a cake. The cake is then isolated in the form of sausages pre-cut into pieces of 1 to 2 cm on which the extraction is carried out.

[79] The raw materials used are as follows.

[80] [Table 1]

Materials % Actual weighed quantity Peregrina cake pre-cut into pieces of 1 to 2 cm 8.4% 33.6 Network 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

[81] Protocol:

[82] a) A concentrated solution of 1 molar of a strong alkaline agent such as sodium hydroxide, potassium hydroxide or calcium hydroxide is prepared, or of an aqueous mixture concentrating 1 molar of strong alkaline agents, but preferably sodium hydroxide; this strong alkaline solution has a pH between 13 and 14,

[83] b) We weigh 9.1% (mass/mass) of peregrina cake precut into pieces of approximately 1cm in 90.9% (mass/mass) of the alkaline solution,

[84] c) we grind the cake obtained in step b),

[85] d) The ground cake obtained in step c) is dispersed in the aqueous phase.

[86] e) chemical proteolysis is carried out on the aqueous dispersion obtained in step d) for a period of 2 hours and at a temperature of 22°C,

[87] f) proteolysis is neutralized to stabilize the protein hydrolyzate obtained,

[88] g) the protein hydrolyzate is recovered by solid/liquid separation by passing it through a 1 pm filter,

[89] h) the protein hydrolyzate is purified by ultrafiltration,

[90] A translucent yellow filtrate is obtained containing approximately 12.48% dry matter. The liquid extract obtained is subsequently named “peregrina protein hydrolyzate according to the invention” or “peregrina protein hydrolyzate” or “extract according to the invention”.

[91] This peregrina protein hydrolyzate 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% of nitrogen 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 dry peregrina protein hydrolyzate according to the invention is given below.

[92] [Table 2]

Tr CAS No. Compounds % DM 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-Methyl-butanenitrile 2.194 14.56 625-28-5 3-Methyl-butanenitrile 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-anal. 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

The peregrina protein hydrolyzate 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. 14851487; Afsharypuor, S. et al., 2010, Volatile Constituents of the Seed Kernel and Leaf of Moringa peregrina (Forssk.) Fîori, 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, notably in the families Brassicaceae, Capparaceae, Caricaceae and Moringaceae as a defense system against attack by pathogens. In the genus Moringa, they have been identified notably in M. oleifera Lam. and M. stenopetala (Baker f.) Cufold.

(Abd Rani, N.Z. et al., 2018, Moringa genus: A review of Phytochemistry and Pharmacology, Frontiers in Pharmacology, vol. 9, art. 108, p. 3-8). Isothiocyanates come from the hydrolysis of glucosinolates by the enzyme myrosinase when 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, Μ. E., Gonzalez-Leon, A. (ed) Recent advances in alternative postharvest technologies to control fungal diseases in fruits &

vegetables. Transworld Research Network, Kerala, India, p. 127-156; TroncosqRojas, 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), anti-microbial, anti-cancer and anti-inflammatory effects (Park, E. J. et al., 2011, Inhibition of lipopolysaccharide induced cyclooxygenase-2 expression and inducible nitric oxide synthase by 4-[(2'-O -acetyla-L-rhamnosyloxy)benzyl]isothiocyanate from M. oleifera, Nutrition and Cancer 63(6), pp. 971-982; Rajan T. S. et al., 2016, Anticancer activity of glucomoringin isothiocyanate in human malignant astrocytoma cells, Fitoterapa 110 , pp. 1-7; Padla, E. P. et al. 2012, Antimicrobial isothiocyanates from the seeds of Moringa oleifera Lam., Zeitschrift fur 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 seeds and in many dried fruits. Carbon disulfide, isobutyronitrile, methyl isobutyrate, methyl butanenitrile and hexanenitrile are volatile compounds derived from amino acids. These are markers of protein degradation. These degradation compounds represent approximately 50% of the volatile compounds in peregrina protein hydrolyzate. This result indicates that the hydrolyzate consists mainly of proteins. Only very small traces of free fats or sugars were detected in the peregrina protein hydrolyzate; the hydrolyzate is mainly made up of proteins and glycoproteins. In the dry matter, the citrate buffer (which is an saccharide compound) represents approximately 50% of the remainder and includes the glycosylated (saccharide) compounds resulting from the degradation by proteolysis of proteins, oligopeptides and amino acids. Finally, it should be noted the presence of approximately 21 mg/liter of polyphenols (0.002%) from Moringa peregrina seeds. Benzoic acid should not be considered in the characterization because it is a stabilizing agent added to the extract.

[93] The dry extract described above is obtained by a gravimetric method based on the mass before and after evaporation present in the liquid extract.

[941 Example 2: Effect of the peregrina protein hydrolyzate according to the invention as an inhibitor of furin convertase (called furin)

[95] The objective of this study is to evaluate the inhibitory activity of the protein hydrolyzate of Moringa peregrina seed cake, obtained according to Example 1.

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

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

[98] 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 test compound:

[99] “Peregrina protein hydrolyzate”; 0.02; 0.2 and 2% (v/v).

[100] At the end of the pre-incubation step, furin substrate was added and the experimental conditions were incubated again at room temperature away from light for 5 minutes. All experiments were performed in triplicate.

[101] Preparation of compounds:

[102] The peregrina protein hydrolyzate tested was solubilized directly in the assay buffer then diluted to reach the test concentration described above.

[103] Evaluation protocol

[104] Cleavage of the furin fluorescent substrate was monitored for 5 min after substrate addition by reading fluorescence at 485 nm/535 nm.

Statistics

Results are expressed in RFU (Relative Fluorescent Units) +/- S.D. (Standard Deviation).

The statistical significance of the difference observed between the “Control” and “Reference product” groups was evaluated by a Student’s t test (p <0.001).

The statistical significance of the difference observed between the “Control” or “Witness” and “Test Compound” groups was assessed by one-way ANOVA analysis, followed by a HolmSidak test (p < 0.05).

The reference furin inhibitor named DecanoyLArg-Val-Lys-ArgCMK _: tested at 100 nm, significantly inhibited furin activity by 97.9% (p <0.001).

This result was expected and validates the study.

[105] The results of furin enzyme activity in relation to basal activity obtained are given below.

[106] [Table 3]

“Peregrina protein hydrolyzate” at 0.02% (v/v) 124.9% (p<0.001) “Peregrina protein hydrolyzate” at 0.2% (v/v) 73.2% (p<0.001) “Peregrina protein hydrolyzate” at 2.0% (v/v) 1.2% (p<0.001)

[107] Conclusion: At a concentration of 2%, the peregrina protein hydrolyzate according to the invention is capable of inhibiting the activity of furin convertase by 98.8%. From a concentration of 0.2%, the peregrina protein hydrolyzate according to the invention is capable of inhibiting the activity of furin by 26.8%.

[108] Example 3: Effect of the peregrina protein hydrolyzate according to the invention to inhibit the enzymes Histone Desacetylases (HDACs) and Sirtuin I

[109] The objective of this study is to demonstrate the inhibitory activity of the peregrina protein hydrolyzate according to the invention on the enzymes HDACs and Sirtuins I, enzymes involved in the control of genetic drift by regulating condensation. / decondensation of chromatin which gives access or not to the genes carried by the DNA. A buffered solution of HDACs & Sirtuin I reacts with a substrate for 20 minutes at 37°C and transforms it to form a compound which colors in the presence of a developer after incubation at 37°C for 10 minutes. The maximum deacetylation activity of Sirtuins can thus be evaluated by measuring the absorbance at 405 nM. The peregrina protein hydrolyzate according to the invention or the reference product “Trichostatin A (STA) inhibitor 1μΜ” are brought into contact with the Sirtuins solution at the same time as the enzyme substrate for 20 minutes at 37°C. ; the substrate transformed by the enzyme is colored by the addition of a developer. The deacetylating 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 &

Sirtuin l in the absence of active ingredient, that is to say only in the presence of the substrate of the enzymes HDACs & Sirtuin I.

[110] Protocol: A solution of Sirtuin enzymes is incubated in its substrate for 20 min in the absence (control) or presence of the reference product, or increasing concentrations of the products under test. The peregrina protein hydrolyzate according to the The invention is tested at the following concentrations: 2%; 1%; 0.1% (V/V). At the end of the incubation period, the activity of the Sirtuin enzymes with and without test or reference product was revealed by coloring using 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 enzymes Histone Deacetysases and Sirtuins I by the product under test is calculated according to the following formula.

[111] [Math. 1 ] Percentage of modulation of the activity of Sirtuin enzymes = 100 x [(OD405 test or reference product) - (OD405 HDACs & Sirtuin I alone)]/DO405 Sirtuins alone.

[112] 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 Desacetylase enzymes (HDACs) are given below.

[113] [Table 4]

Peregrina protein hydrolyzate according to the invention Percentage Inhibition versus Control (%) 2% 15 1% ns 0.10% -18

[114] Conclusion: At 2%, the peregrina protein hydro ysate according to the invention shows significant inhibition of HDACs; this inhibition reveals the capacity to promote self-protection of skin cells against genetic drift.

Thus the extract appears to be useful against one of the most common genetic deviations on the surface of the skin, namely fibrosis which manifests itself by the appearance of flesh “pimples” (fibrotic protuberance). The extract can advantageously interfere against the phenomena of fibrosis on the surface of the skin.

[115] Example 4: Effect of the peregrina protein hydrolyzate according to the invention to inhibit the action of endothelin 1 (ET-1)

[116] 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 (OHBAT. et at., WO2012/081370).

[117] It has been reported in recent years that endothelin type 1 (ET-1) is a bioactive factor that continuously contracts vascular and nonvascular smooth muscles through direct and indirect actions. An increase in endothelin fraction is thought to provide continuous vasoconstriction to blood vessels in peripheral sites, kidneys and brain, and is thought to cause 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. .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, pp. 571-577; Anonymous, 2006, American Society for Biochemistry and Molecular Biology, New Cosmetics Handbook, pp. 527-529). All these peptides have a vasoconstrictor effect and vasopressor actions.

[118] 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 increase in keratinocytes when the skin is exposed to UV irradiation, and have suggested that ET-1 is associated with melanogenesis in melanocytes. exposed to UV irradiation. Accordingly, 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, pp. 32-37; Imokawa, G. et al. al., 1992, Endothelins Secreted from ïO Keratinocytes are Intrinsic Mitogens for Human Melanocytes, Journal of Biological Chemistry, 267, pp. 24675-24680; Gilchrest, B. et al., 1996, Mechanisms of Ultraviolet Light-induced Pigmentation, Photochemistry and Photobioliogy63, p. ΙΙΟ).

[119] The objective is to measure type 1 endothelin in human microvascular endothelial cells after exposure for 24 hours to the peregrina protein hydrolyzate according to the invention.

[120] Protocol: The human microvascular endothelial cells were supplied by the company PELOBiotech and cultured in 96-well plates according to the supplier's production procedures. The 20 extracts were left to act at different concentrations on the endothelial cells at 80% confluence for 24 hours, then quantify endothelin 1 using the PicoKine ELISA kit (EDN1) in cell supernatants. A viability test is carried out beforehand to define the non-toxic doses to be used when measuring endothelin 1. The negative control is carried out using the cells in culture medium without treatment. The positive control in the viability test is S DS at 0.5%. All conditions are prepared in culture media, and the cells are then incubated at 36.5°C / 5% CO2 for 24 hours.

a) Application of the solutions to be tested on endothelial cells:

The products to be tested are placed in contact with the endothelial cells at sub30 confluence in 96-well plates. For each concentration the test is carried out on 3 wells. The plates are incubated for 24 hours ± 1 hour at 36.5°C / 5% CO2.

b) Viability test:

Cell viability is evaluated by the MTT method on the 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. In each well 50 μL of MTT solution at 0.5 mg/ml are added: incubation for 3 hours at 36.5°C l 5% CO2. In each well, 100 μL of isopropanol are added. 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.

c) Dosage of endothelin 1:

The assay is carried out using the ELISA kit. The results of inhibition of endothelin action are given below.

[121] [Table 5]

Extract concentration Cell growth versus control (%) Endothelin 1 versus control (%) Endothelin 1 versus control (pg/ml) Protein hydrolyzate 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

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

Endothelin 1 is assayed in cell supernatants at non-toxic concentrations. The quantity of endothelin 1 for each condition is measured using the ELISA kit.

For the basal rate control cells, the values are around 134.94 pg/ml. For cells treated with different concentrations of hydrolyzate, the basal rate is reduced by 47.09 pg/ml (with 2% of the extract according to the invention) and is reduced by 17.58 pg/m! (with 1% of the extract according to the invention). This demonstrates very significant inhibitions from 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. 'invention.

The results on endothelin and its specific dose-dependent inhibition demonstrate that the protein hydrolyzate according to the invention has an antiangiogenesis and anti-fibrotic effect, due to its capacity to significantly reduce endothelin.

[123] A complementary study for the evaluation of PI, P2, P3 and the ίο P1+P2 complex described in Example 12 is described below on the production of endothelins: Cellular test model on normal human endothelial cells .

[124] As described in Example 12, 3 bands or protein fractions characterized by their mass P1 (has a molecular weight less than 10,000 Da), P2 (has a molecular weight less than 10,000 Da), P2 (has a molecular weight less than 10,000 Da) were isolated from the hydrolyzate according to the invention. molecular between

10,000 and 17,000 Da) and finally P3 (has a molecular weight of approximately 23,000 Da).

These fractions are hereinafter also referred to as “extracts”.

[125] [Table 6]

Witness Extract P1 (%, v/v) 0.1 0.3 1 33.8 29.0 44.0 38.3 Endothelin-I 36.7 34.2 41.5 31.7 (pg/pg of protein) 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 control 100.0 84.6 112.1 94.2

[126] *Statistical significance (p>0.05)

[127] At all concentrations, the standard deviations are high, and the P1 fraction of the protein hydrolyzate does not show significant modulation on type l endothelin.

[128] [Table 7]

Witness Extract P2 (% v/v) 0.1 0.3 1 33.8 34.8 24.7 34.6

Endothelin-I (pg/pg of protein) 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 Witness 100.0 94.9 75.2 104.1

[129] ‘Statistical significance (p>0.05)

[130] “Statistical significance (p<0.05)

[131] At concentrations 0.1% and 1%, the standard deviations are high, and the P2 fraction of the protein hydrolyzate does not show significant modulation on 5 type I endothelin at these concentrations. by 0.3%, P2 inhibits the production of L-type endothelin by 24.8%

[132] [Table 8]

Witness Extracts P1+P2 (50/50) (%, v/v) 0.1 0.3 1 33.8 39.4 42.0 34.5 Endothelin-I 36.7 29.7 34.2 34.7 (pg/pg of protein) 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 Witness 100.0 100.9 103.8 97.2

[133] ‘Statistical significance (p>0.05)

[134] At all concentrations, the standard deviations are high, and the combination of P1 + P2 fractions does not present significant modulation on L-type endothelin

[135] [Table 9]

Witness Extract P3 (%, v/v) 0.1 0.3 1 Endothelin-I (pg/pg 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 Witness 100.0 96.7 47.7 51.5

‘Statistical significance (p>0.05) “*: Statistical significance (p<0.001)

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

[137] Conclusion: The compounds called “Extract P1” and “Extract P2” have no significant action on the modulation of type 1 endothelin and only “Extract P3” significantly reduces the endothein 1 released in the culture medium by normal human endothelial cells in monolayer culture with a score of 52.3% inhibition with 0.3% of the extract according to the invention. No synergy is observed when P1 and P2 are combined.

The P3 extract 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 the peregrina protein hydrolyzate according to the invention to protect stem cells

[138] 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 in renewing organs. ESCs (embryonal stem cells) are essential for the maintenance and regeneration of skin tissues.

[139] 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 ESCs (Epidemnal Stem Cells). Indeed, cells in this layer give rise to all epidermal structures, including the stratified epidermis (also called 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. Mesoderm is the primary source of mesenchymal stem cells that give rise to collagen-producing fibroblasts, subcutaneous adipocytes, and skin immune cells.

[140] Stem cells are undifferentiated cells called pluripotent 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 preserve 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 Yejîn Ge et al. (March 10, 2020, The aging skin microenvironment dictates stem cell behavior, PNAS, Vol. 117, p. 5339-5350). This damage impairs their capacity for self-renewal and differentiation, leading to a decrease in the stem cell pool and ultimately to skin aging.

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

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

[143] 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).

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

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

[146] “Peregrina protein hydrolyzate”: 0.01; 0.05 and 0.15% (v/v).

[147] Preparation of test compound:

[148] The test compound “Peregrina Protein Hydrolyzate” was diluted directly in the incubation medium to achieve the different concentrations described above.

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

[150] The results are presented below as a percentage of viability compared to the experimental condition “Control without UVB” (mean +/- S.D). The level of significance between “Control without UVB” and “Control with UVB” was evaluated using a Student t test (p < 0.05).

[151] [Table 10]

Hydrolyzate concentration Cell growth versus control (%) I Stem cell protection versus control (%) peregrina protein hydrolyzate 0.15% That. -9.2 NS 0.05% -3.6 +30.50 0.01% That. -6 +15.50

[152] Conclusion: Peregrina protein hydro ysate can significantly protect human skin stem cells subjected to cellular stress (UV). Stem cells are cells with preserved, young DNA material. They are at the origin of tissue regeneration, the return to a young and healthy state. Stem cell protection correlates with the ability to preserve DNA material. The peregrina protein hydrolyzate according to the invention maintains the integrity of the stem cells; it is thus involved in the conservation of DNA.

[153] Example 6: Effect of the peregrina protein hydrolyzate according to the invention to preserve DNA

[154] Telomere length dynamics are very important for the regulation of replicative lifespan in cells, especially in long-lived species. Telomere shortening and telomerase activity are important factors in aging and tumorigenesis (Shay, J. W. & Wright, W. E., 2005, Senescence an Immortalization: Role of Telomeres and Telomerase, Carcinogenesis 26(5), p. 867-874). Telomeres are complex nucleotide sequences that cover the ends of chromosomes from degradation, unwanted fusionrecombination, inappropriate activation of the DNA damage response. They also play an essential role in cell division and chromosome stability. There is increasing evidence that the stability of telomeres and their average length can be affected by stresses, particularly environmental stresses, or diseases under environmental influence (Valdes, A.L. et al., July 2005, Obesity, cigarette smoking and telomere length in women , Research Letters, 366(9486), pp. 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. 288296; Shin, D. et. 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. , October2010, Telomer length and cardiovascular disease, Archives of Cardiovascular Diseases, 103(8-9), pp. 454-459). Thus, extremely short telomeres have been associated with neurodegenerative diseases, cardiovascular diseases (CVD) and cancer risk.

[155] 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 in length and include several repeats of the sequence TTAGGG

[156] Telomerase is not expressed in most adult cells and telomere length decreases with successive replication cycles. After a certain number of replication cycles, progressive shortening of telomeres causes cells to enter a phase of telomeric crisis, which in turn leads to cellular senescence. Certain diseases are associated with rapid telomeric loss, leading to premature cellular senescence. It has been demonstrated that the expression of the gene encoding the human telomerase protein in human cells (Blasco M., 2007, Telomere Length, Stem Cells and Aging, Nature Chemical Biology, 3(10), p. 640-649 ) confers a consistent quality phenotype, probably by bypassing the cells' natural senescence pathway. Furthermore, it was shown in the aforementioned study that 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.

[157] The objective of this study is to evaluate the effect of the compound called “peregrina protein hydrolyzate” 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, ultimately resulting in a cell incapable of replication. Measurement of telomere length was performed using quantitative PCR and in comparison with telomere length between cells at passages 2 and 5.

[158] Protocol: Human fibroblast cells were obtained from a 44-year-old donor. To perform the experiments, cells were used at passage 2 and 5. Fibroblasts were cultured for 3 consecutive passages in the absence (control) or presence of an increasing concentration to test of peregrina protein hydrolyzate: 0.01; 0.1 and 0.5% (v/v).

[159] Preparation of the test compound: The test compound “peregrina protein hydrolyzate” was diluted directly in the incubation medium to achieve the different concentrations described above.

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

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

[162] Results are expressed in relative units corresponding to the length of telomeres relative to cells at passage 2 (mean + S.D.). The level of significance versus “Control” at passages 2 and 5 was assessed using a Student t test (*: p <0.05). The level of significance between “control” and “test compound” was assessed independently for each product by one-way analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (*: p < 0.05 ).

[163] [Table 11]

Concentration Cell growth versus control (%) Telomere length versus control (%) Protein hydrolyzate 0.5% +1.9 +16.60 0.1% That. 0 +15.10 0.05% -0.6 +8.90

[164] Results: Under our experimental conditions, the peregrina protein hydrolyzate tested at 0.05%, 0.1% and 0.5% (v/v), significantly reduced the shortening of telomeres in normal human fibroblasts.

[165] Telomere shortening shows an inhibition (compared to control) at 0.05% (v/v) of +8.9% (p <0.05); at 0.1% (v/v) by +15.1% (p <0.01) and at 0.5% (v/v) by +16.6% (p <0.01). Conclusion: In a context of normal multiplication or division of human cells, the peregrina protein hydrolyzate according to the invention demonstrates an ability to significantly increase the length of telomeres. Telomeres are caps involved in the protection of DNA material; Increased telomere length correlates with the ability to preserve DNA material. Peregrina protein hydrolyzate may increase telomere length. This hydrolyzate is therefore involved in the preservation of human genetic material (DNA).

[166] Additional study of the evaluation of extracts P1, P2, P3 and the complex

P1+P2 described in Example 12 on their ability to protect DNA by increasing the length of telomeres after several cell divisions.

[167] [Table 12]

Passage 2 Passage 5 Witness Witness Extract P1 (%; v/v) 0.01% 0.3% 1% Variation in telomere length (change in size from first pass) 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 (in % of Control) 100.0 55.8 59.8 66.6 73.3 % telomere lengthening compared to control 0 9.1 24.3 39.6

*Statistical significance (p>0.05) **: Statistical significance (p<0.01) ***: Statistical significance (p<0.001)

[168] The P1 extract according to the invention after 3 cell divisions from a concentration of 0.3% increases the size of telomeres by 24.3% on a culture model of normal human cells (fibroblasts) At a concentration of 1 %, the P1 extract increases the size of telomeres by 39.6% under the same conditions.

[169] [Table 13]

Passage 2 Passage 6 Witness Witness Extract P2 (%; v/v) 0.01% 0.3% 1% Variation in telomere length (change in size from first pass) 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 (in % of Control) 100.0 55.8 65.3 66.3 75.0

% telomere elongation compared to control 0 21.4 23.7 43.5

‘Statistical significance (p>0.05)

[170] The P2 extract according to the invention after 3 cell divisions on a culture model of normal human cells (fibroblasts) presents a statistically unconfirmed tendency towards telomere elongation for each 5 concentration tested.

[171] [Table 14]

Passage 2 Passage 5 Witness Witness Extract P3 (%; v/v) 0.01% 0.3% 1% Variation in telomere length (change in size from first pass) 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 (in % of Control) 100.0 55.8 55.4 53.6 55.2 % telomere elongation compared to control 0 -0.8 -5.0 -1.3

‘Statistical significance (p>0.05)

The P3 extract according to the invention after 3 cell divisions in a culture model of normal human cells (fibroblasts) has no capacity to promote telomere elongation for each concentration tested.

[172] [Table 15]

Passage 2 Passage 5 Witness Witness Extract P1+P2 (%; v/v) 0.01% 0.3% 1% Variation in telomere length (change in size from first pass) 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 (in % of Control) 100.0 55.8 69.3 70.5 77.5 telomere lengthening compared to control 0 30.5 33.3 49.1

‘Statistical significance (p>0.05)

Statistical significance (p<0.01)

[173] The mixture of extracts P1 and P2 (50/50 by volume) according to the invention after 3 cell divisions at a concentration of 1% (i.e. 0.5% of each extract) increases the size of the telomeres on a culture model of normal human cells (fibroblasts). This score not having been achieved by the individual extracts, a synergistic effect is thus demonstrated by mixing extract P1 and P2.

[174] Conclusion: The compounds named “Extract P1” and “Extract P1 + P2” significantly reduce the shortening of telomeres occurring after 3 consecutive cell passages. A synergy is confirmed by combining P1 and P2.

[175] Example 7: Effect of the peregrina protein hydrolyzate according to the invention to stimulate the ZAG protein

[176] Zinc alpha-2-glycoprotein (ZAG) is a plasma glycoprotein that takes its name from its electrophoretic mobility and its ability to be precipitated by Zn salts. ZAG is a member of the immunoglobulin gene superfamily and has a three-dimensional structure highly homologous to MHC class I and II molecules. ZAG was 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, J. P. et al., 1991, Human Zn-a2-Glycoprotein cDNA Cloning and Expression Analysis in Benign and Malignant Breast Tissues, FEBS Letters 290 (1-2), pp. 247-249). Due to its production by the secretory epithelium, ZAG is present in most body secretions and constitutes respectively 2.5% of saliva proteins and 30% of seminal fluids. It has been described that plasma or serum levels of ZAG vary with age, with reported values ranging from 0.9 to 3.5 mg! dl (fetus) at 7.8 to 12.1 mg/dl (young adults) (Jirka, M. et al., 1974, The Zn-alpha 2Glycoprotein Level in Human Serum During Ontogenesis. Clinica Chimica Acta 56, p. 31 -33; Jirka, M. et al., 1978, Human Serum Zn-a2-Glycoprotein in Amniotic Fluid, Clinica Chimica Acta 85, p. 107-110). ZAG accumulates in breast cyst fluids to a plasma concentration of 30-50 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-a2-Glycoprotein Levels in Breast Cancer Cytosols and Correlation with Clinical, Histological, and Biochemical Parameters, European Journal of Cancer 29A, pp. 1256-1260) and is overexpressed in 40 to 50% of breast carcinomas. It has recently been demonstrated (Susan, M. el al., Zinc-al pha2-glycop rote in 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, leading to elevated serum ZAG levels in prostate cancer patients in basal cell carcinomas. The objective of this study is to evaluate the ability of peregrina protein hydrolyzate to stimulate ZAG.

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

[178] This involves letting the samples act at defined concentrations on the keratinocytes at 80% confluence for 48 hours, then quantifying the ZAG using the ELISA kit in the cell supernatants.

[179] A preliminary viability test is carried out to define the non-toxic doses to be used when measuring ZAG. The negative control is carried out using the cells in culture medium without treatment. The positive control for the viability test is 0.5% SDS.

[180] 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 solutions to be tested on keratinocytes:

- The products to be tested are placed in contact with the subconfluent keratinocytes in the 24 and 96 well plates.

- For each concentration, the test is carried out on 3 wells.

- The plates are incubated for 24 hours and 48 hours at 36.5°C / 5% CO2.

• Viability test:

- Cell viability is evaluated by the MTT method on the cells after incubation with the products.

- After 24 and 48 hours of incubation, the planned wells are rinsed once with 200pL of PBS.

- In each well 50 μI of MTT solution at 0.5 mg/ml are added: incubation for 3 hours at 36.5°C / 5% CO2.

- In each well 100 μΙ of isopropanol are added.

- 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 dosage:

- After 48 hours of incubation, all supernatants are collected and stored at -20°C for assays.

- The assay is carried out using an ELISA kit. The results are given below.

[181] [Table 16]

Concentration Cell growth versus control (%) ZAG versus control (%) ZAG versus control (ng/ml) Protein hydrolyzate 2% -17.88 +337.80 0.390 1% -12.54 +195.73 0.157 0.10% -3.88 +151.83 0.085

[182] Conclusion: Peregrina protein hydrolyzate can significantly increase ZAG production, with good dose dependence and low toxicity to human cells, thus it has anti-fibrotic effect and anti-inflammatory based on its ability to significantly increase the

ZAG.

[183] A complementary study of the stimulation of the ZAG from the protein bands identified in Example 12 is described below on normal human keratinocytes.

[184] [Table 17]

Witness Extract P1 (%, v/v) 0.2 1 6 ZAG (pg/pg 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 Witness 100.0 207.1 147.3 73.5 ‘Significance statistics (p>0.05)

**: Statistical significance (p<0.01)

The P1 extract from 0.2% significantly increases the production of ZAG by more than 107%.

[185] [Table 18]

Witness Extract P2 {%, v/v) 0.2 1 5 ZAG (pg/pg 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 Witness 100.0 180.1 167.5 95.6

‘Statistical significance (p>0.05)

Statistical significance (p<0.05)

The P2 extract from 0.2% significantly increases the production of ZAG by more than 80%.

[186] [Table 19]

Witness Extract P3 {%, v/v) 0.2 1 5 ZAG (pg/pg 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 Witness 100.0 | 78·5 | 90·0 95.2 *Statistical significance (p>0.05)

The P3 extract does not have the capacity to significantly influence ZAG production in this study model.

[187] [Table 20]

Witness Extract P1+P2 (50/50) (%, v/v) 0.2 1 5 ZAG (pg/pg 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 Witness 100.0 140.6 132.9 87.8

*Statistical significance (p>0.05)

The combination of extracts P1 and P2 shows a trend towards activation of ZAG production, but this trend is not statistically significant. There is therefore no synergistic effect in the combination of extracts P1 and P2.

[188] The compounds called “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 by combining P1 and P2.

[189] Conclusion: In the hydrolyzed extract according to the invention, the P1 extract is the most effective extract for increasing the ZAG. It therefore has an anti-fibrotic effect and an anti-inflammatory effect from a dose of 0.2%.

[190] Example 8: Effect of the peregrina protein hydrolyzate according to the invention on the modulation of the dosage of DKK1 and DKK3

[191] The involvement of interactions between melanocytes and fibroblasts in the regulation of melanogenesis is well known and has been studied intensively. Although these interactions are not yet fully understood, they are the cause of the whiteness of the palmo-plantar areas and are now used in dermatology for the development of depigmenting products. Yamaguchi and colleagues (Yamaguchi Y. et al., 2004, MesenchymalEpithelial 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 the fibroblasts of the palmar-foot regions was capable of modifying the differentiation program of the melanocytes of these regions, leading to a reduction in the production of melanin. This messenger was identified by the team as a protein called Dikkopf-1 (DKK-1).

[192] The signaling pathways used by DKK-1 to produce these results are well identified today. Thanks to its antagonistic action on the Wnt receptor, DKK-1 is in fact capable of bypassing the intracellular signaling pathways activated by β-catenin, generally responsible for the regulation of genes involved in melanogenesis. Yamaguchi and colleagues (see above) 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 DKK1. In fact, the more the quantity of DKK-3 in the vicinity of this Wnt receptor increases, the weaker the interactions between DKK-1 and this receptor. Increased DKK3 reduces the inhibitory effects of DKK-1 on melanogenesis. The work of Yamaguchi and colleagues (see above) further suggests that the identification of agents having an influence on the DKK11 DKK3 ratio in cultures of normal human dermal fibroblasts of non-palmo-plantar origin would make it possible to control melanin production. from normal human non-palmoplantar melanocytes.

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

[194] Protocol: Human fibroblast cells are obtained from a 68-year-old donor. To carry out the experiments, the fibroblasts are cultured in monolayer until they reach confluence. Dexamethasone at 100 nm was used as a reference inducer of DKK-1 synthesis and release. The skin discs were incubated for 48 hours in the absence (control) or presence of reference product or product to be tested: “Peregrina protein hydrolyzate”: 0.01; 0.1 and 0.5% (v/v).

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

The test compound “peregrina protein hydrolyzate” was diluted directly into the incubation medium to achieve the different concentrations described above.

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

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

Results are expressed as ng of DKK-1 per mg of protein (mean ± S.D.).

The level of significance between the “control” and the “reference product” was assessed using a Student t test (p <0.05).

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

Under our experimental conditions, the reference product named “Dexamethasone”, tested at 100 nm, significantly increased the DKK-1 released by 181.8% (p < 0.01) compared to “Control”. The results on modulation of DKK1 dosage are given below.

[196] [Table 21]

Concentration Cell growth versus control (%) DKK1 versus control (%) peregrina protein hydrolyzate 0.5% +1.9 +131.50 0.1% That. 0 +32.30 0.05% -0.6 +26.10

The study shows that the hydrolyzate according to the invention significantly increases the level of DKK1 at a dose of 0.05% with 26.1% increase compared to the basal rate and the concentration of 0.5% of the extract according to the invention, an increase of 131.5% in the basal rate is observed

[197] The aim of this study is to evaluate the effect of peregrina protein hydrolyzate on the synthesis and release of DKK-3 in a model composed of normal human fibroblasts in monolayer culture. Human fibroblast cells were obtained from a 68-year-old donor. To carry out the experiments, the fibroblasts were cultured in monolayer until reaching confluence. Human fibroblast cells were obtained from a 68-year-old donor. To carry out the experiments, the fibroblasts were cultured in monolayer until reaching confluence.

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

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

[200] Results are expressed as ng of DKK-3 per mg of protein (mean ± S.D.). The level of significance between the “Control” and the “reference product” was evaluated using a Student t test (*: p <0.05).

[201] The level of significance between the “Control” and the peregrina protein hydrolyzate was evaluated by a one-way analysis of variance (one-way ANCV A) followed by a Holm-Sidak test (*: p < 0.05 ). The results on the modulation of DKK3 dosage are given below.

[202] [Table 22]

Concentration Cell growth versus control (%) DKK3 versus control (%) peregrina protein hydrolyzate 0.5% 101.9 -20.7 0.1% That. 100.0 -8 0.05% 99.4 -3.9

[203] Conclusion: The peregrina protein hydrolyzate according to the invention at 0.5% presents a notable inhibition of around 21% of DKK3 compared to the basal level. The peregrina protein hydrolyzate according to the invention has a strong capacity to manage the genes involved in cell differentiation thanks to the principle of palmar-plantar inhibition (beta-catenin signaling pathway), through its ability to considerably increase DKK1 and decrease significantly DKK3, which increases the DKK1/DKK3 ratio.

[204] Complementary study of the evaluation of P1, P2, P3 and the P1+P2 complex described in Example 10 on the production of DKK1: Cellular test model on

Normal human fibroblasts.

[205] [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 Witness 100.0 110.6 126.6 127.8

*Statistical significance (p>0.05) **: Statistical significance (p<0.01)

The P1 extract significantly increases the production of DKK1 by 26.6% from dose 10 of 0.3%, and by 27.8% at the dose of 1%.

[206] [Table 24]

Witness Extract P2 (%; v/v) 0.01 0.3 1 DKK-1 (ng/mg 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 300.1 306.2* 307.6* 374.1* Standard Deviation 6.1 36.5 41.8 33.8 % of Witness 100.0 101.7 102.5 124.7

*Statistical significance (p>0.05)

The P2 extract does not statistically significantly increase the production of DKK1 at each of the doses tested

[207] [Table 25]

Witness Extract P3 (%; v/v) 0.01 0.3 1 306.7 359.2 387.0 224.7

DKK-1 294.9 371.8 263.7 283.0 (ng/mg protein) 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 Witness 100.0 113.8 104.2 88.5

‘Statistical significance (p>0.05)

The P3 extract does not statistically significantly increase the production of DKK1 at each of the doses tested.

[208] [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 Witness 100.0 107.1 114.6 139.2

‘Statistical significance (p>0.05)

Statistical significance (p<0.01)

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

Conclusion: The compounds called “Extract P1” and “Extract P1 + P2” significantly increased the DKK-1 released into the culture medium by normal human fibroblasts in monolayer culture. A synergy is confirmed by mixing P1 and P2.

[209] Conclusion of the previous activities of the protein hydrolyzate according to the invention:

[210] The most remarkable activity of peregrina protein hydrolyzate demonstrated in in-cell tests is its epigenetic action with its ability to very significantly slow down the process of telomere shortening after cell divisions. The hydrolyzate is a cell protector, more particularly of stem cells, which makes it a very good cell and tissue regenerator.

These properties provide very strong protection of DNA and its genetic material. Peregrina protein hydrolyzate is also a potent modulator of ZAG and type I endothelin production, with antifibrotic and anti-inflammatory effect.

[211] Example 9: Evaluation of the effect of delipidated peregrina meal protein hydrolyzate on Angiotensin convertase 2 (ACE2) - eye study of the inhibitory effect.

[212] Angiotensin convertase 2 is notably involved in the intracellular infection of COVID19 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).

[213] [Table 27]

Samples Average Moy-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% Peregrina hydrolyzed extract 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

Conclusion: The hydrolyzed extract of Peregrina cake is not significantly involved in the direct inhibition of Angiotensin convertase 2 (ACE2). Considering table [32] of example 11 below, the amplitude 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 hydrolyzate of proteins according to the invention as a specific inhibitor of furin convertase.

[214] Example 10: Evaluation of the anti-infectious effect of the hydrolyzed extract to inhibit furin in cellulo by preventing the penetration of “Crown spike pseudo-virus SARS-CoV-2”: In ceilulo model on human cells H EK in the presence of “SARS-CoV-2 pseudotyped lentiviruses”.

[215] This assessment uses two main components. On the one hand, recombinant clonal stable HEK293 cells constitutively express full-length human ACE2 (Genbank# NM_021804.3) with surface expression of ACE2 confirmed by flow cytometry. On the other hand, SARS-CoV-2 Spike pseudotyped lentiviruses were produced with SARS-CoV-2 Spike (Genbank accession number QHD43416.1) as envelope glycoproteins 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 may be used for screening applications in a Biosafety Level 2 facility.

[216] The material used is as follows:

[Table 28]

Catalog Components 79951 ACE2-HEK recomb cell line 60187-1 Thawing medium 1 79801 IN growth medium 79942 Spike (SARS-CoV-2) Pseudotyped Lentivirus (Luciferase Reporter) 79943 Baîd Lentiviral Pseudovirîon (Lucîferase Reporter) 60690-1 ONE-Step luciferase assay system AF933 Human/Mouse/Rat/Hamster ACE-2 Antibody (R&D Systems)

[217] Step 1: Plating of ACE2-HEK cells

[218] ACE2-HEK cells are thawed in thawing medium 1, amplified in 1N growth medium, then harvested and put into white, transparent flat bottom 96-well culture plates at 10,000 cells/well in 50 µl of thawing medium 1. Cells are incubated overnight at 37 °C.

[219] Step 2: Pseudotyped lenti viral infection test

[220] The following day, 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:

[221] [Table 29]

Name of ingredient/co-incubation method Solubility Concentrations in “wells” Ingredient 1 Peregrina /ACE2HEK H20 / culture medium 3% (C1), 0.5% (C2), 0.1% (C3)

222] In a first step, the peregrina protein hydrolyzate is diluted to an intermediate concentration 11x in thawing medium 1, for subsequent transfer of 5 µl into the assay plate and co-incubation with ACE2HEK 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 analysis wells.

[223] As a positive control, the ACE2 blocking mAb is used at a final concentration of 0.5 µM in the assay wells.

[224] Step 3:

[225] 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.

[226] The results are given in Figure 1 for the anti-infectious effect of the peregrina protein hydrolyzate according to the invention against SARS COV2 pseudovirions.

Conclusion: Peregrina protein hydrolyzate shows a convincing anti-infectious effect on SARS COV2 pseudovirions from a dose of 3% (C1) in this study model. A marked inhibition of 60% is demonstrated at the concentration

C1 (3%).

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

[228] The studies carried out on the identified bands did not show any inhibitory action on furin convertase at all the concentrations tested with P1, P2, P3 and P1+P2.

[229] Conclusion: The Furin Convertase inhibition activity is therefore not carried by one of these protein bands. This demonstrates that the inhibitory activity of Furin Convertase is due to the protein hydrolyzate in its totum obtained by the method described.

[230] Protein hydrolyzate as a whole inhibits SARS-CoV2-like SPIKE proteins, notably through the inactivation of furin convertase. These inhibitions provide an anti-infectious and virostatic character.

[231] Example 11: Comparative tests of the inhibition of furin convertase by different preparations obtained from Moringa peregrina seeds

[232] The aim of this study is to evaluate the inhibitory effect of peregrina oil obtained by first cold pressing of seeds with hulls, then of peregrina extract with ethanol (96%) concentrating approximately 1.1% dry matter, this dry matter itself being made up 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 peregrina protein hydrolyzate according to the invention on the activity of furin.

[233] Protocol; Peregrina ethanol extract (96%) and peregrina protein hydrolyzate were stored at +4°C, protected from light until use. Peregrina oil was stored at room temperature in a dark area.

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

[235] 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 tested compounds:

- Peregrina oil at 0.3, 1% and 3% (v! v).

- Peregrina extract with ethanol (96%) at 0.02%, 0.2% and 2% (v/v).

- Peregrina protein hydrolyzate according to the invention at 0.02%, 0.2% and 2% (v! v). At the end of the pre-incubation step, furin substrate was added and the experimental conditions were incubated again at room temperature away from light for 5 min. All experiments were performed in triplicate.

[236] Preparation of the compounds: The peregrina extract with 96% ethanol and the peregrina protein hydrolyzate were solubilized directly in the assay buffer then diluted to reach the concentration to be tested as described below. above. Peregrina oil was solubilized at 3% in a solution of 0.05% Tween20 in assay buffer. The solution was then diluted to reach the concentration described above.

[237] Evaluation protocol: Cleavage of the furin fluorescent substrate was monitored for 5 minutes after substrate addition by reading fluorescence at 485 nm! 535nm. g. Statistics: Results are expressed in RFU (Relative Fluorescent Units) +/- S.D. (Standard Deviation). The statistical significance of the difference observed between the “Control” and “Reference Product” groups was evaluated by a Student’s t test (p <0.001). The statistical significance of the difference observed between the “Control” and “Test Compound” groups was evaluated by a one-way ANOVA analysis, followed by a HolmSidak test (p < 0.05). The results for the peregrina oil are given in Figure 2, those of the 96° peregrina ethanol extract in Figure 3 and finally those of the peregrina protein hydrolyzate according to the invention in Figure 4.

[238] Results:

[239] [Table 30]

Peregrina oil concentration Furin convertase activity 0.3% (v;v) 91.2%** 1% (v:v) 90.6%** 3% (v:v) 92.8%**

240] [Table 31]

Ethanol extract concentration 96° peregrina Furin convertase activity 0.02% (v:v) 97.3%* 0.2% (v:v) 102.0%*

| 2.0% (v:v)__112.8%*

[241] [Table 32]

Peregrina protein hydrolyzate concentration Furin convertase activity 0.02% (v:v) 124.9%*** 0.2% (v:v) 73.2%*** 2.0% (v:v) ¹ 1.2%***

242] ‘Statistical significance (p>0.05)

[243] **: Statistical significance (p<0.01)

[244] ***: Statistical significance (p<0.001)

[245] Conclusion: The present study allowed us to show that only the peregrina protein hydrolyzate according to the invention can significantly inhibit the activity of furin by 98.8% at a concentration of 2%.

[246] Example 12: Separative gel electrophoresis chromatography of peregrina protein hydrolyzate - Protein bands.

[247] Polyacrylamide gel electrophoresis containing sodium dodecysulfate is called SDS-PAGE electrophoresis. This is a technique consisting of migrating proteins denatured by negative charge saturation via SDS in a polyacrylamide gel, under the influence of an electric field, thus allowing their separation. It is a denaturing technique which dissociates non-covalent protein complexes using a negatively charged ionic detergent (SDS). This detergent binds indifferently by hydrophobic bonds to two amino acids. This technique makes it possible to analyze proteins and separate them according to their molecular mass.

Separative chromatography by electrophoresis method:

Deposits:

- MW = size marker

- well 1: Hydrolyzed extract of Peregrina cake

- well 2: supernatant of the hydrolyzed extract of Peregrina cake

- well 3; pellet of the hydrolyzed extract of Peregrina cake

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

Well 2 (supernatant) shows attenuated bands, particularly at intermediate and highest molecular weights; well 2 seems to have concentrated the lowest molecular weights, particularly related to volatiles.

Well 3 (pellet) clearly presents the bands of the highest molecular weights (> 75,000 Da), then a band called P3 of approximately 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:

- The volatiles are logically in the supernatant with the “lightest” compounds; 10 ml of supernatant are collected to be added 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 condensate concentrated in volatiles will be passed into GC/FiD after SPME microextraction

Gas Chromatography study after a SPME extraction method in FID detection

No volatile compounds were detected.

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

[249] 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 into liquid chromatographic analysis coupled with mass spectrometry (LC MS/MS).

[250] The results are obtained from 2 bioinformatics software programs specialized in protein recognition in plants (Mascot and Peaks):

[251] The identification results do not make it possible to precisely identify these proteins; we are in the presence of proteins currently not described in the aforementioned databases.

[252] Example 13: Formulation of an anti-fibrotic dermatological product (liquid cleanser)

[253] [Table 31]

Ingredients % Water 80.7000 Sodium coco sulfate 5.0000 Sodium cocoyl isethionate 4.0000 Bentonite 3.7800 Caprylic / Capric triglyceride 2.0000 Peregrina protein hydrolyzate seion the invention 2.0000 Gfuconotactone 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

[254] Example 14: Formulation of a dermatological product treating topical fibrosis (non-rinsed care product)

[255] [Table 32]

Ingredient % Water 71.5000 Caprylic/ Capric triglyceride 18.0000 Bentonite 4.2000 Cetearyl alcohol 1.5000 Peregrina protein hydrolyzate according to invention 2.0000 Gluconoactone 0.7500 Sodium benzoate 03450

Xanthan gum 0.5000 Scent 0.5000 Sodium stearoyl glutamate 0.2500 Citric acid 0.2500 Calcium gluconate 0.0050

[256] Example 15: Formulation of a composition for subcutaneous injection

[257] Formulation of a product for subcutaneous administration: dry extract according to the invention (containing 60% protein hydrolyzate on an inulin support) packaged in a single-dose bottle under inert gas ready to be solubilized by a physiological environment.

[258] Example 16: Formulation of a composition for subcutaneous injection

[259] Formulation of a liquid injectable product: liquid extract according to the invention dosed at 5% in a physiological medium conditioned in sterilizing conditions, in particular by vacuum filtration with a cut-off threshold of 0 45 pm.

[260] Example 17: Formulation in the form of a patch (bandage 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).

[261] Example 18: Formulation of a medicine in capsule form

[262] Antifibrotic medication in 750 mg capsule containing 100 mg of piperine, 300 mg of dry extract according to the invention (containing 60% protein hydrolyzate on an inulin support), 100 mg of boswellic acid, 250 mg of calcium carbonate.)

[263] Example 19: Formulation of a medication in tablet form

[264] Antifibrotic medication in 1 g tablet: 300 mg of dry extract according to the invention (containing 60% protein hydrolyzate 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.

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

[266] Example 21: Toxicological tests of the protein hydrolyzate according to the invention Preparation of the protein hydrolyzate in accordance with Example 1: Unshelled seeds of Moringa peregrina (Forssk.) Fiori harvested when the fruit was ripe been dried to obtain an internal humidity level of less than 8% and preferably around 6%, then pressed with a mechanical worm press, so as to separate the oil from the rest of the seed to obtain on the one hand virgin oil and on the other hand a cake. The cake is then isolated in the form of sausages pre-cut into pieces of 1 to 2 cm. By following the protocol described in Example 1, we obtain the liquid extract which is used undiluted in the tests which follow.

[267] 1. Determination of mutagenic activity on the bacterial strain Salmonella typhimurium (TA 100) - Bacterial reverse mutation test

The test took place in 3 main phases:

- A preliminary experiment is carried out in order to evaluate the cytotoxicity of the element to be tested and to select the dose range for subsequent experiments,

- A first genotoxicity experiment (Test 1), with and without metabolic activation, with direct incorporation of the test system and the test (or controls) on minimal agar, over the dose range defined by the preliminary study, - A second experiment (Test 2), with pre-incubation of the test system and test item (or controls), with and without metabolic activation, with dose levels defined by the study manager after analysis of the results of the first experiment. This second experiment was carried out in order to confirm or complete the results of the first, in particular when equivocal or negative results were obtained.

The dilutions of the extract according to Example 1 were prepared in water to carry out the cytotoxicity test.

[268] The latter was carried out on the Salmonella typhimurium TA100 strain at concentrations of 5000, 1600, 500, 160 and 50 pg/plate, with and without S9-Mix.

The reagents used for the preparation of S9-Mix were prepared according to the following instructions:

[269] ]Table 33]

Final concentration MgCl2 (0.4 M) + KCl (1.65 M) 8mM + 33mM Glucose 6 Phosphate (0.2 M) 5mM NADP (0.1M) 4mM Phosphate buffer for S9-Mix (pH 7.4 - 0.2 M) 0.1M S9 fraction 10% Water Adjust for final concentration

270] 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, D.M. et a!., 1983, Revised Methods for the Salmonella Mutagenicity Test, Mutation Research/Environmental Mutagenesis and Related Subjects, 113, p.173-215), and was provided by MOLTOX TM. It is stored at a temperature below -70°C. The S9 microsomal fraction was used at a concentration of 10% in S94Vlix. The protocol applied was as follows:

• In 3 hemolysis tubes, was introduced:

o dosage 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).

o dosage 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 background agar previously distributed in Petri dishes.

• Incubate at 37°C±2°C for 48 to 72 hours.

These assays were carried out for each test: preliminary cytotoxicity test, test 1 and test 2. The untreated control, the negative controls and the positive controls carried out 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;

o 0.1 ml of 0.2 M phosphate buffer, pH 7.4, o 0.1 ml of solvent, o 0.1 ml of S9-MÎX. o 0.1 ml of the preparation of the element to be tested at the highest concentration, • A fraction of 2 ml of top agar for S. typhimurium is used to control 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 carried out 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.

[271] Expression of results and interpretation

Many criteria make it possible to determine whether a result is positive, in particular an increase in the number of revertants correlated with the dose of 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, at the end of the verification steps, a dose-effect relationship has been obtained, in a reproducible manner, on one or some 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 equal to 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, after test 1 and test 2, the revertant rate has always remained less than twice the spontaneous reversion rate for all concentrations of the test item tested, for strains TA98, TA100 and TA102 (R <2) and less than three times the spontaneous reversion rate 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 linked to the toxicity of the concentrations tested.

The preliminary study showed no cytotoxicity of the test element; therefore, this concentration range was used for genotoxicity test 1.

[272] Depending on the result obtained for test 1, it was decided to use the same dilution range for test 2. The 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 double the spontaneous reversion rate for TA98, TA100 and TA102 and triple the spontaneous reversion rate for TA1535 and TA1537, with and without metabolic activation,

- No dose response was observed, regardless of test system or test conditions.

[273] In view of the results obtained during this study, the protein hydrolyzate according to Example 1 can be considered as having no mutagenic or pro-mutagenic activity.

[274] 2 In vitro phototoxicity test 3T3 NRU

The principle of the test is based on the comparison of the cytotoxicity of the protein hydrolyzate according to Example 1 in the presence and absence of a non-cytotoxic dose of UVA, on cells in culture. Cytotoxicity is evaluated by determining cell viability using a vital dye: neutral red, 24 hours after treatment with the reference elements and the peregrina protein hydrolyzate according to the invention with or without irradiation with UVA. The cells used are Balb/c 3T3 lineage mouse embryo fibroblasts clone 31 (ATCC - CCL163). The positive control is a solution of chlorpromazine (CAS number: 69-09-0), The negative control is a diluent of the tested and reference extract (buffered saline solution +1- 1% solvent). Peregrina protein hydrolyzate was tested at 8 concentrations on at least four culture wells per concentration studied, in the presence or absence of UVA. The fibroblasts were trypsinized and two 96-well culture plates were seeded with 100 μΙ of a cell suspension at 2.10 ⁵ cells/ml (i.e. 2.10 ^e cells per well) in complete culture medium.

The seeded plates were incubated in the oven for 24 hours at 37°C, 5% CO2. At the end of the incubation, the semi-confluence of the cell layer was checked. The dilutions were prepared just before their deposition on the cells. The PH of the highest concentration was measured; it is between 6.5 and 7.8. The culture medium was removed; each well was carefully rinsed beforehand 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 carried out 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 tracks the UV light emission. Irradiation stops automatically when the energy delivered is equal to the programmed energy. The spectral irradiation of the device under test was measured in the wavelength range 250 to 700 nanometers with a calibrated spectroradiometer.

One of the 2 plates was irradiated with its lid at room temperature, and the other plate was protected from UV rays and kept at room temperature during the irradiation. After irradiation, the treatment medium was aspirated and the cells were rinsed. Then 100 μl of complete culture medium were added gently and the plates were incubated for 18 to 22 hours at 37°C and 5% CO2. The next day, cell viability (growth, morphology, integrity of the monolayer) was evaluated by observations on a phase contrast microscope. The culture medium was removed, each well was previously rinsed and maintained at room temperature before being treated with 100 μΙ of the staining solution. The plates were returned to the incubator for 3 h under the same conditions. The dye solution was removed and the cells were rinsed, then the rinsing solution was removed and 150 μl of desorption solution was added to each well. The plates were shaken until the crystals were completely solubilized. Absorbances were measured at 450 nm.

[275] Test validation:

The sensitivity of the cells to UVA is monitored approximately every 10 passages, by evaluating their viability after exposure to increasing doses of irradiation. The cells are cultured at the density used in the assay. They are irradiated the following day at doses of 2.5 and 9 J/cm ² and cell viability is determined one day later using the NRU test. The cells meet the quality criteria if their viability after irradiation at 5 J/cm ² UVA is greater than or equal to 80% of the viability of the controls kept in the dark; at the highest dose of 9 J/cm ² UVA, the viability must be at least equal to 50% of that of the controls kept in the dark.

[276] Results:

The negative control has an absorbance greater than or equal to 0.4. Chlorpromazine, a positive control, has an Clso of between 0.1 and 2 pg/ml in the presence of UVA and between 7 and 90 pg/ml in the absence of UVA. These results allow the test to be validated. The concentration of the peregrina protein hydrolyzate according to example 1 giving 50% cell death in the presence or absence of UVA cannot be estimated. Mortality never reached 50%. The concentration of peregrina protein hydrolyzate giving 50% cell viability in the presence or absence of UVA cannot be estimated. Viability is always above 50%.

Conclusion: Under the experimental conditions retained, the peregrina protein hydrolyzate according to the invention can be considered non-phototoxic.

[277] 3. Evaluation of the ocular irritant potential by studying in vitro cytotoxicity using the neutral red release method on S1RC cell line

This in vitro study is based on the evaluation of the cytotoxicity of peregrina protein hydrolyzate by determining the concentration leading to 50% cell mortality (Clso) on a cell monolayer using the neutral red release technique. . The cells used are SI RC rabbit corneal fibroblasts (ATCC - CCL60) free of mycoplasmas.

Peregrina protein hydrolyzate was diluted in 25% and 50% saline. The fibroblasts were trypsinized and two 24-well culture plates were seeded with 1 ml of a cell suspension at 2.10 ⁵ cells/ml in complete culture medium. The seeded plates were incubated in the oven overnight at 37°C and 5% CO2. At the end of the incubation, the confluence of the cell layer was checked. The staining solution was prepared at 0.5 mg/ml in complete culture medium. The culture medium was removed; 1 ml of the coloring solution was placed in 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 dye solution was removed 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 μΙ of each dilution of peregrina or reference protein hydrolyzate were deposited in contact with the cell layer. The contact time was 60 seconds (30 seconds for the positive control). The treatment was carried out well by well with the stopwatch triggered at the time of deposition of the peregrina protein hydrolyzate or the reference. The plate was shaken manually throughout the treatment. After 55 seconds (or 25 seconds for the positive control), the dilution was aspirated. At precisely 60 or 30 seconds, 5 successive rinses were carried out (5 x 2 ml of PBS maintained at room temperature). The supernatant was aspirated after each rinse and after the last rinse the wells remained devoid of medium while awaiting the revelation phase. After complete treatment of the culture plate, 1 ml of the desorption solution was placed in each well. The plate is shaken for approximately 15 minutes until a uniform color is obtained. The solutions obtained for each culture well were taken and distributed into 2 wells of a 96-well plate, i.e. 150 μΙ/well.

[278] Results:

The concentration of peregrina protein hydrolyzate giving 50% cell death was evaluated at >50%. The percentage of cell death at 50% peregrina protein hydrolyzate was estimated at 17%.

Conclusion: Under the experimental conditions retained, the cytotoxicity of the peregrina protein hydrolyzate according to the invention can be classified as negligible cytotoxicity.

[279] 4. Evaluation of the skin compatibility of a peregrina protein hydrolyzate according to the invention after single application under an occlusive dressing for 48 hours under dermatological control.

The aim of this study is to evaluate the degree of skin compatibility of peregrina protein hydrolyzate by patch test, carried out on the antero-external side of the arm for 48 hours; and generally evaluate the ability of protein hydrolyzate to maintain the skin in good condition. 10 healthy volunteers, female or male, aged 18 to 65 years, having neither dry nor sensitive skin and free from any dermatological lesion on the treatment area were to be included in the study. The skin compatibility of the peregrina protein hydrolyzate, prepared in the form of a lotion with 5% of the peregrina protein hydrolyzate according to Example 1 and 95% of a Propanediol/Sorbitol mixture, was evaluated 48 hours after the Initial application between 30 to 40 minutes after removal of the dressing. Skin reactions (erythema and edema) were counted from 0 to 3 according to the following scales:

[280] [Table 34]

Quoting Erythema (E) Edema (Oe) 0 No erythema No edema 0.5 Barely noticeable erythema, very slightly pink coloring in part of the patching area Barely noticeable palpable edema 1 Light erythema, pink discoloration over the entire patching surface Palpable and visible edema 2 Moderate erythema, clear discoloration over the entire patching surface Marked edema with or without papules or vesicles 3 Significant erythema, intense coloring over the entire patching area Significant edema spreading outside the patching area with or without papules or vesicles

Any other skin reaction (bubbles, papules, vesicles, dryness, scaling, roughness, soap effect, etc.) was evaluated according to the following scale and reported descriptively:

- 0: no reaction, 15 - 0.5: very light

- 1: light

- 2: moderate

- 3: important.

At the end of the study, an average irritation index (LLM.) was calculated according to the following formula;

[281] [Math. 4]

I.I.M. = Sum of skin reactions (E + Oe + bubbles + papules + vesicles) / Number of volunteers analyzed

The I.I.M. obtained made it possible to classify the peregrina protein hydrolyzate tested according to the scale presented below:

I.I.M. < 0.20 Non-irritating

0.20 < I.I.M. < 0.50 Slightly irritating

0.50 < I.I.M. < 2 Moderately irritating < I.I.M. <3 Very irritating

Results: The Average Irritation Index (I.I.M.) of peregrina protein hydrolyzate is equal to: 0.

Conclusion: Peregrina protein hydrolyzate can be considered non-irritating after 48 consecutive hours of application in 12 volunteers.

[282] General conclusion of the tests:

The results of the tests carried out above are conclusive and demonstrate for the peregrina protein hydrolyzate according to example 1:

) Eye and skin irritation tests are negative

2) Phototoxicity tests are negative

3) Mutagenicity tests are negative.

The safety of the peregrina protein hydrolyzate according to the invention is demonstrated and ideal for large-scale dermatological use without restriction on target populations.]

Claims (20)

1. Claim 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 internai 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 séparation, 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, and then optionally,

   i) the protein hydrolysate obtained in step h) is freeze-dried.
2. Claim 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. Claim 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. Claim 4 · The method as claimed in daims 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
5. Claim 5 The method as claimed in daims 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. Claim 6 A protein hydrolysate from seed cake that has not been shelled and has 10 been harvested from ripe Moringa peregrina fruit, characterised in that it comprises a major fraction P1 (mass/mass) of amino acid dérivatives, 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 15 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 température comprised between 16 and 25°C and in that it is liquid and has a density of more than 1.

   20
7. Claim 7 The protein hydrolysate as claimed in claim 6, characterised in that it comprises a dry matter content of about 12.5% (mass/mass) comprisîng between 1 and 6% of nitrogen-containing compounds, in particular volatile nitrile dérivatives in a proportion of 0.5% to 1.5%, preferably about 0.8%, and 20 mg/liter of polyphenols.

   25
8. Claim 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 dérivatives, 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 30 period of approximately 2 hours at a température 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.

   69 « _f
9. Claim 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 dérivatives, amino acids, peptides and glycopeptides for which the molecular weight is comprised between 5 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 température 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. Claim 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 dérivatives, amino acids, peptides and glycopeptides for which the molecular weight is approximately 23,000 15 Da, in that it is obtained by Chemical proteolysis at pH of more than 13 for a period of about 2 hours at a température 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.

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11. Claim 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. Claim 12 The protein hydrolysate of Moringa peregrina seed cake according to 25 any of claims 6 to 11 for use as a médicament.
13. Claim 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 any of claims 6 to 11, and a physiologically 30 acceptable excipient.
14. Claim 14 The pharmaceutical composition as claimed in claim 13, characterised in that it is formulated for ingestion.
15. Claim 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. Claim 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. Claim 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 ofthe protein hydrolysate as claimed in claim 8 or 9, preferably as claimed in claim 9.
18. Claim 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 ofthe protein hydrolysate as claimed in claim 10.
19. Claim 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 ofthe protein hydrolysate as claimed in claim 6 or 7.
20. Claim 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 ofthe protein hydrolysate as claimed in claim 11.