Protein hydrolysate of Moringa arabica seed cake for its use as a medicament, method for obtaining same, and pharmaceutical and dermatological compositions

Abstract

The present invention relates to a method for obtaining a specific protein hydrolysate from Moringa oleifera seed cake. The present invention also relates to a protein hydrolysate from Moringa oleifera seed cake and its use as a medicament. Finally, the present invention relates to a pharmaceutical or dermatological composition comprising an effective amount of a protein hydrolysate from Moringa oleifera seed cake as an active agent, for its use as a medicament for treating fibrotic diseases and inflammatory conditions, treating cancer, treating bacterial or viral infectious diseases, and treating genetic drift and pathologies associated with skin pigmentation.

Description

Technical Field

[0001] The present invention relates to the fields of pharmacy and dermatology, and more specifically to the field of active ingredients in pharmaceutical preparations. The present invention relates to a method for obtaining a protein hydrolysate from Moringa peregrina seed cake. The present invention also relates to a protein hydrolysate from Moringa peregrina seed cake, to pharmaceutical and dermatological compositions containing such hydrolysates, and to compositions intended for use in the treatment of fibrotic diseases, inflammatory diseases, cancer, infectious bacterial or viral diseases, and for the treatment of genetic drift and pathologies associated with skin pigmentation. Background Art

[0002] The Moringaceae family (Moringa) constitutes a monogeneric family (single genus, Moringa) and is part of the Saharo-Sindian flora. According to the authors, it consists of between twelve and fourteen species distributed across eastern Africa and Asia. The genus is conventionally divided into three sections, however, phylogenetic analysis has not confirmed monophyletic agreement between these three sections. The latter section features prominent branching toward certain morphological characteristics: pachycaul (bottle tree), tuberous shrub, and tree that is neither pachycaul nor tuberous shrub (slender tree). The species Moringa peregrina (Forssk.) Fiori belongs to this third group. The few genetic studies of this genus or family confirm the existence of this species compared to other species in the genus, particularly the Indian Moringa and Moringa oleifera Lam. (See in particular the articles: OLSON, ME, 2002, Combining Data from DNA Sequences and Morphology for a Phylogeny of Moringaceae (Brassicales), Systematic Botany 27(1): p.55-73; Hassanein, AMA et al., 2018, Morphological and genetic diversity of Moringa oleifera and Moringa peregrinagenotypes, Horticulture, Environment and Biotechnology 59(2): p.251-261). Recent articles on Moringa oleifera sampled at different locations in Saudi Arabia and using ITS markers concluded that the species is stable (A LAKLABI ,A.,2015,Genetic diversity of Moringa peregrina species inSaudi Arabia with ITS sequences,Saudi Journal of Biological Sciences 22:p.186-190), however, there is a high level of genetic variation within the population.

[0003] The species Moringa arabica is found in rocky environments in Yemen, Oman, Saudi Arabia, East Africa, Sudan, Ethiopia, Eritrea, Somalia, and Djibouti. Its presence in Iran appears to be limited to the southeastern provinces, but this needs to be confirmed (PROTA14=M UNYANZIZA ,E.et al.Vegetable oils / oilseed plants,Moringaperegrina (Forssk.) Fiori,http: / / database.prota.org / protahtml / moringaperegrina_fr.htm,accessed on 23 / 10 / 2019). In the Levant and Egypt, the species is found only as rare scattered relict plants (except for a few high-altitude populations), mainly in areas of Sudan. Today, Arabian Moringa is also considered rare and endangered in Sudan and Yemen. Compared with other species in its branch, Arabian Moringa occupies the driest and most harsh habitats. It is obviously more drought-resistant than the large-scale commercial cultivation of Moringa in tropical and subtropical regions. Recent work shows that seed size and weight will have a favorable effect on germination time and the growth rate and rate of individual seedlings (G OMAA NH et al., 2011, Seed germination, seedling traits, and seed bank of the tree Moringa peregrina (Moringaceae) in a hyper-ariden environment, American Journal of Botany 98(6): p.1024-1030), indicating that resource allocation is adjusted towards seed quality rather than seed quantity, which enables Moringa arabica to reproduce efficiently in extreme abiotic environments (hyperarid environments). In terms of cell layers, Moringa arabica seeds have a central mesotesta that is thicker than those of Moringa oleifera.

[0004] History, there are some references that tend to indicate that Arabian Moringa oil was actively traded in the Al-Ula region during the early Islamic period. ASEEF , AAS, 1995, Al-'Ulā, A study of Cultural and Social Heritage). The oil produced from Moringa arabica is now mainly intended for domestic consumption or for the local market. In Saudi Arabia, the leaves have traditionally been used as a decoction for internal use to treat diabetes, colon diseases, eye diseases and anemia (A BDEL -K ADERet al.,Asurvey on the traditional plants used in Al Kobah village,Saudi Pharmaceutical Journal 26(6):p.817-821) and as a diuretic, rubefacient and astringent (A QEEL In Oman, women use the oil extracted in late summer to combat migraines, fevers, burns, lacerations and fractures, constipation and stomach aches, as well as muscle pain and dry scalp (G HAZANFAR ,SA,1994,Handbook of ArabianMedicinal Plants,1 st edition, CRC Press, Boca Raton, Ann Harbor, USA; G HAZANFAR ,SA,1998,Plants of Economic Importance,chapter 15,in Ghazanfar,SAet al.(ed.)Vegetation of the Arabian Peninsula.Geobotany 25,p.241-264,Kluwer AcademicPublishers,table 11.1,p.247and 11.7p.251). It is also used in aromatic compositions (G HAZANFAR ,SA,1998,p.259) and as a facial wash in Oman and Yemen (G HAZANFAR , SA et al., 1996, Two multi-purpose seed oils from Oman. Plants for Food and Medicine. Paper presented at the joint meeting of the Society for Economic Botany and International Society for Ethnopharmacology, London, July 1–7, 1996.

[0005] A number of Moringa seed extracts are known, particularly for use in cosmetics. In the field of dermatology, document FR 2776519 describes protein extracts from Moringa seeds as being known for their ability to clarify turbid water and possessing emollient, physiologically regulating, moisturizing, restructuring, repairing, and anti-pollution properties on the skin and mucous membranes. In this document, the active ingredient is a protein with a molecular weight between 6500 and 8800 Da, obtained through aqueous extraction from Moringa cakes.

[0006] In the pharmaceutical field, a composition from KR20140143655 is known to contain a water-soluble extract from Moringa oleifera leaves as an active ingredient for treating or preventing cancer. Enzymatic hydrolysis of Moringa oleifera leaf extract can be used to separate proteins with a molecular weight between 6000 and 8000 Da.

[0007] Document CN 107012190 relates to the use of Moringa oleifera seeds to obtain peptides from seed proteins, followed by enzymatic hydrolysis using microwave radiation and subsequent oil extraction. The resulting product is intended for oral administration and has anticancer activity. In fact, this powdered product is described as having the effects of reducing chemotherapy side effects, increasing appetite in liver cancer patients, and controlling white blood cell counts and body temperature.

[0008] Finally, from the document IN2009CH02906, an aqueous extract of Moringa cake containing glucosinolates in combination with cationic proteins can be used as an antidiabetic product.

[0009] All of the above-mentioned references relate to the use of the species Moringa oleifera; none of them describe the use of extracts from the species Moringa arabicum in the pharmaceutical or dermatological fields.

[0010] In addition, A BU T ARBOUSH The document XP055753092, 2005, by et al., is known, which describes the extraction of a hydrolysate from shelled Moringa oleifera seeds produced by enzymatic hydrolysis over a period of 10 hours. The purpose of this extraction is to obtain a product with a high oil and water absorption capacity.

[0011] The extract of Moringa arabicum oil produced from Egyptian seeds using a (1 / 1) dichloromethane / methanol mixture showed activity on three human cancer cell lines: MCF-7 (breast cancer), Hep-G2 (hepatocellular carcinoma), and HCT-116 (colon cancer), with IC 50 The values ​​were 2.92, 9.40 and 9.48 μg / mn (A BD el B AKYet 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 high antioxidant activity using DPPH (2,2-diphenyl 1-picrylhydrazyl), ABTS (anion scavenging and reducing capacity), and antiproliferative assays. Since the method used to obtain the extract differs from that of the present invention, the molecules obtained have very different polarity.

[0012] A BOU -H ASHEM et al. (A BOU -H ASHEM , MMMet al., 2019, Induction of sub-G0 arrest and apoptosis by seed extract of Moringa peregrina (Forssk.) Fiori in cervical and prostate cancer cell lines, Journal of Integrative Medicine 17: p. 410-422) also demonstrated induction activity of sub-G0 arrest and apoptosis on cervical (HELA) and prostate (PC-3) cancer cell lines. The protocol involved reducing the seeds of Moringa peregrina (Forssk.) Fiori to a powder and extracting them with 95% ethanol, then dissolving them in an aqueous solution, from which three fractionated extracts (fractions using petroleum ether (PE), CHCl3, and EtOAc) as well as the hydroalcoholic extraction residue were studied. The activity was confirmed in the total chloroform fraction and was attributed to saturated and unsaturated fatty acids and polyphenols, without specific investigation of the mechanism. Since the method of obtaining the extract is different from that of the present invention, the molecules obtained have very different polarities.

[0013] In view of the above, the problem that the present invention proposes to overcome is to develop new products based on extracts of Moringa oleifera, a species of the genus Moringa and the family Moringaceae, which products can be used in pharmacy or dermatology and are easy to use.

[0014] Thus, the Applicant has surprisingly developed a specific protein hydrolysate obtained from Moringa oleifera seed cake for its use as a medicament, and in particular for the treatment of fibrotic diseases (as an inhibitor of furin converting enzymes), for the treatment of inflammatory diseases, cancer, infectious diseases of bacterial or viral type, and for the treatment of genetic drift and pathologies associated with skin pigmentation.

[0015] The Moringa oleifera species grows in very arid climates. Consequently, its drought tolerance and ability to thrive under extreme conditions have given it unique characteristics, which the applicants have been able to identify by applying a specific extraction method to Moringa oleifera seed cake. The protein hydrolysate according to the invention has been shown to possess pharmaceutical properties and, in particular, has been shown to possess very high furin-convertase inhibitory activity.

[0016] Furin convertase (hereinafter referred to as 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.org / 10.1002 / cti2.1073)。 It is particularly involved in the healing of wounds and the treatment of diseases in which fibrosis is the main tissue repair mechanism or excessive fibrosis causes pathological destruction and tissue dysfunction (in this regard, referring to WO 2004 / 09113 file, which relates to the use of invertase to reduce fibrosis in the healing and treatment fibrotic state to reduce fibrosis in the injury healing process). The wound healing of adults is a complex repair process. The wound may relate to the injury, damage or trauma of tissue or internal organs such as the lungs, kidneys, heart, intestines, tendons or liver. The wound healing process in tissue is usually initiated by the hemostatic reaction caused by skin vascular damage. In this process, the connective tissue formed in the healing process is usually fibrous in nature, and is usually formed as connective tissue scar (called fibrotic process). Therefore, fibrosis can include pulmonary fibrosis, kidney fibrosis, liver fibrosis, skin fibrosis, eye fibrosis, cardiac fibrosis and other various fibrotic states. The protein hydrolysates of the present invention can also be used to treat other pathological conditions mediated by furin, including but not limited to: hypertension, cancer, infectious diseases (bacterial and viral) and genetic diseases (such as cystic fibrosis (CF)) and neurodegenerative diseases (in this regard, see WO 2019 / 215341, which relates to novel furin converting enzyme inhibitors or compounds and pharmaceutical compositions containing them, and which cites a number of publications demonstrating the pharmaceutical activity of the inhibitors; these citations are hereby incorporated by reference into this specification).

[0017] Through an intergovernmental agreement concluded on April 10, 2018, between the Governments of the French Republic and the Kingdom of Saudi Arabia, the applicants, the French Agency for AlUla Development (AFALULA) and the Royal Commission for AlUla (RCU), have launched a joint project specifically aimed at developing responsible agriculture and the local economy, particularly through the local production of natural products derived from native plants, and protecting the biodiversity and legal status of the AlUla region in the Kingdom of Saudi Arabia. The Kingdom of Saudi Arabia has been a party to the Nagoya Protocol since October 8, 2020. At the time of drafting this patent, implementing regulations regarding the incorporation of the Nagoya Protocol into relevant aspects of local law were taken into consideration. Therefore, at this stage, the Kingdom of Saudi Arabia has no specific requirements with respect to this patent application and the Nagoya Protocol. Consequently, at the date of filing this patent application, there was no requirement to obtain a certificate of compliance with genetic resources. Summary of the Invention

[0018] In a first aspect, the present invention provides a method for obtaining a protein hydrolysate from Moringa oleifera seed cake, comprising the following steps, wherein:

[0019] a) collecting unshelled mature seeds from ripe Moringa oleifera fruits and drying them to obtain an internal moisture content of less than 8%,

[0020] b) pressing the dried seeds in such a way that the oil is separated from the rest of the seeds, in such a way that a cake comprising less than 6% by weight of residual oil is obtained,

[0021] c) grinding the cake obtained in step b),

[0022] d) dispersing the milled cake obtained in step c) in an aqueous phase,

[0023] e) subjecting the aqueous dispersion obtained in step d) to a chemical proteolysis at a pH greater than 13 and at a temperature comprised between 16° C. and 25° C. for a period of about 2 hours,

[0024] f) neutralizing the protein hydrolysis to stabilize the obtained protein hydrolysate,

[0025] g) recovering the protein hydrolysate by solid / liquid separation,

[0026] h) purifying the protein hydrolysate by ultrafiltration and or nanofiltration, and then optionally,

[0027] i) freeze-drying the protein hydrolysate obtained in step h).

[0028] In a second aspect, the present invention relates to a protein hydrolysate from seed cake that has been harvested from ripe Moringa oleifera fruits and not shelled, comprising a major fraction P1 of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight comprised between 1500 Da and 5000 Da, a fraction P2 of about 20% with a molecular weight comprised between 10000 and 17000 Da, a fraction P3 of about 20% with a molecular weight of about 23000 Da, wherein it is obtained by chemical protein hydrolysis under conditions of pH greater than 13 at a temperature comprised between 16° C. and 25° C. for a period of about 2 hours, and wherein it is liquid and has a density greater than 1, preferably of about 1.1.

[0029] Due to its characteristics, the Moringa arabica protein hydrolysate according to the invention has never been disclosed in the genus Moringa and the family Moringaceae. It will be demonstrated that the extract of the species Moringa arabica has a specific peptide profile that is different from that of other species of this genus, in particular the species Moringa oleifera that the applicant has been able to demonstrate.

[0030] In a third aspect, the present invention relates to the use of a protein hydrolysate from Moringa oleifera seed cake as a medicament.

[0031] In a fourth aspect, the present invention relates to a pharmaceutical or dermatological composition comprising as active agent an effective amount of a protein hydrolysate of Moringa oleifera seed cake, and a physiologically acceptable excipient.

[0032] Finally, in a fifth aspect, the present invention relates to a pharmaceutical or dermatological composition for the treatment of fibrotic diseases, the treatment of inflammation, cancer, infectious diseases of bacterial or viral type, and the treatment of genetic drift and pathologies associated with skin pigmentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Other objects, details, features and advantages of the present invention will become more apparent from the following description of several particular embodiments of the invention, given by way of non-limiting illustration only and with reference to the accompanying drawings.

[0034] [ Figure 1 ] is a schematic diagram of SARS-COV2 (Severe Acute Respiratory Syndrome Coronavirus 2) coronavirus infection, which shows the anti-infection and virus inhibition effects of the Moringa arabicum protein hydrolyzate according to the present invention on SARS COV2 coronavirus pseudovirions.

[0035] [ Figure 2 ] represents the inhibition graph of furin by using the extract of Moringa arabicum oil. In this figure, ** represents a significant difference from the "control" group (P<0.001).

[0036] [ Figure 3 ] represents the inhibition of furin by using Arabica oleifera cake extract (96° ethanol). In this figure, * indicates significant difference from the "control" group (P<0.001).

[0037] [ Figure 4 ] represents the inhibition graph of furin using the protein hydrolyzate from Moringa oleifera cake according to the present invention. In this figure, *** indicates significant difference from the "control" group (P<0.001). DETAILED DESCRIPTION

[0038] In this description, unless otherwise stated, it is understood that when a range is given, it includes the upper and lower limits of the range.

[0039] In the present invention, the following abbreviations refer to the following:

[0040] -MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT test is a rapid method for counting viable cells)

[0041] -SDS: Sodium dodecyl sulfate

[0042] -PBS: Phosphate buffered saline

[0043] -ELISA: Enzyme-linked immunosorbent assay

[0044] -PCR: polymerase chain reaction

[0045] -ANOVA: Analysis of variance

[0046] -MSH: melanocyte-stimulating hormone

[0047] In the present invention:

[0048] - "predominantly amino acid derivatives, amino acids, peptides and glycopeptides" means amino acid derivatives, amino acids, peptides and glycopeptides in an amount of more than 20%, more preferably more than 30% and possibly up to about 40% (w / w) of dry matter, more preferably 50% by weight of dry matter.

[0049] - "Effective amount" refers to the amount of active molecule necessary to achieve the desired result, ie to ensure the desired therapeutic activity.

[0050] - "Proteolysis" refers to the fragmentation of proteins into peptides, oligopeptides and their base fragments (amino acids) and residues thereof by chemical hydrolysis.

[0051] Topical administration refers to the direct application of a drug to its site of action, where it exerts its pharmacological effect at the precise location of the ailment. The goal of topical administration is to limit the diffusion of the active ingredient from the site of administration, thereby minimizing adverse effects. The main routes of topical administration are through the skin, nasal and respiratory routes, ocular routes, auricular routes, vaginal routes, and oral routes.

[0052] "Topical application" means applying or spreading the active ingredient according to the invention or a composition comprising the same onto the surface of the skin, mucous membranes, nails or hair.

[0053] - "Physiologically acceptable" in the context of topical use means contact with human skin, or in the context of other routes of administration, such as oral administration or by injection into the skin, means zero risk of toxicity, incompatibility, instability or allergic reaction.

[0054] - "Cake" refers to the defatted seeds after pressing. It is the solid residue left after the oil is extracted from the seeds. It is a by-product of milling (the oil production process). It usually accounts for 50% to 75% of the seed mass.

[0055] - "Unhulled seeds" refers to seeds that are harvested with the shell (pericarp) and skin remaining around the kernel.

[0056] - "When the fruit is ripe" means that the fruit is ripe, preferably when the shell is ready to split open and is dark beige to brown, and when the lower quarter of the shell is twisted 180°, triggering the valve to open.

[0057] - “Approximately” means plus or minus 10% to 20% of the given information.

[0058] - "Active molecule pool", "active agent" and also "active ingredient" refer to the protein hydrolysate extracted starting from Moringa oleifera seed cake according to the method of the invention. This hydrolysate is responsible for the biological activities described in the present invention.

[0059] - "Active agent" means a sufficient amount of an extract according to the invention to obtain the biological activity described. Depending on whether the extract is liquid or dry, concentrated or otherwise, the amount of active agent may vary from 0.0001% to 40% by weight relative to the total weight of the composition.

[0060] In a first aspect, the present invention relates to a method for obtaining a protein hydrolysate from Moringa oleifera seed cake, comprising the following steps, wherein:

[0061] a) collecting unshelled mature seeds from ripe Moringa oleifera fruits and drying them to obtain an internal moisture content of less than 8%,

[0062] b) pressing the dried seeds in such a way that the oil is separated from the rest of the seeds, in such a way that a cake comprising less than 6% by weight of residual oil is obtained,

[0063] c) grinding the cake obtained in step b),

[0064] d) dispersing the milled cake obtained in step c) in an aqueous phase,

[0065] e) subjecting the aqueous dispersion obtained in step d) to a chemical proteolysis at a pH greater than 13 and at a temperature comprised between 16° C. and 25° C. for a period of about 2 hours,

[0066] f) neutralizing the protein hydrolysis to stabilize the obtained protein hydrolysate,

[0067] g) recovering the protein hydrolysate by solid / liquid separation,

[0068] h) purifying the protein hydrolysate by ultrafiltration and / or nanofiltration with a cut-off threshold comprised between 100 and 25,000 Da, and then optionally,

[0069] i) freeze-drying the protein hydrolysate obtained in step h).

[0070] The seeds are collected unhulled, i.e. with the shell remaining when the fruit is ripe and preferably when the shell begins to split.

[0071] The seeds are dried to obtain an internal moisture content of less than 8% and preferably about 6%; drying is preferably carried out on a ventilated rack protected from light, preferably in the shade of the open air.

[0072] Next, the dried seeds are immediately ground using a cold press, which serves to mechanically separate the oil from the rest of the pressed seeds (ie, the cake).

[0073] The cake is then mechanically milled using any type of mechanical mill such as a hammer mill, flail mill, knife mill, crusher / chopper mill, ball mill or beater mill, and also using any type of cryomill.

[0074] The dispersion to form the aqueous phase according to step d) and the protein hydrolysis according to step e) are advantageously carried out under continuous stirring, so as to disperse and homogenize the solid in the liquid, thus improving the total surface area for exchange and, therefore, the protein hydrolysis.

[0075] A liquid protein hydrolysate is obtained having a density greater than 1 and preferably of about 1.1, comprising a dry matter content of between 10% and 15%, preferably of about 12.5%, said dry matter content comprising between 1% and 6% of nitrogenous compounds, in particular volatile nitrile derivatives in a proportion of 0.5% to 1.5%, preferably of about 0.8%, and 20 mg / l of polyphenols (0.002%).

[0076] According to a preferred embodiment of the method of the present invention, the protein hydrolysis temperature in step f) is about 22°C.

[0077] According to a preferred embodiment of the method of the present invention, the solid / liquid separation in step g) is performed by various methods such as centrifugation, dehydration or filtration.

[0078] In one embodiment of the method for obtaining a liquid Moringa oleifera protein hydrolysate, the protein hydrolysate is purified by distillation, microfiltration, ultrafiltration, and / or nanofiltration to concentrate it into the desired compound over the extracted organic matter, particularly over other extracted derivatives. These purification steps can be used to concentrate the desired compound library over the other extracted compounds cited.

[0079] According to another preferred embodiment of the method according to the present invention, the nanofiltration is carried out in a manner such that three bands or fractions of the protein hydrolysate are separated, including a band P1 with a molecular weight of less than 10,000 Da, a band P2 with a molecular weight comprised between 10,000 and 17,000 Da and a band P3 with a molecular weight of approximately 23,000 Da.

[0080] It is also advantageous to separate three bands by nanofiltration in order to obtain by the process for obtaining the protein hydrolysate:

[0081] - Nanofiltration with band P1 with a cut-off value comprised between 1500 Da and 5000 Da, preferably with a cut-off value comprised between 3000 Da and 4500 Da.

[0082] - Strip P2, nanofiltration with a cut-off threshold comprised between 10000 Da and 17000 Da.

[0083] - Band P3, nanofiltration with a cut-off threshold comprised between 17000 Da and 25000 Da.

[0084] In another embodiment of the extraction process according to the invention, the liquid protein hydrolysate obtained is dried in such a way that a dry hydrolysate of Moringa oleifera seed cake is obtained, which dry hydrolysate comprises oligopeptides, glycopeptides and amino acids or volatile nitrile derivatives thereof in an amount of more than 10%, preferably more than 20%, more preferably more than 30% and possibly up to about 40% (weight / weight) of dry matter, more preferably 50% by weight of dry matter.

[0085] According to one embodiment of the present invention, the liquid protein hydrolysate obtained from the Moringa oleifera seed cake is dried by atomization, freeze drying or boiling, for example, in order to obtain a solid hydrolysate of the Moringa oleifera seeds from which the water has been evaporated. The drying can be carried out in the presence of an organic carrier such as maltodextrin, cyclodextrin or inulin, or in the presence of an inorganic carrier such as phyllosilicates, magnesium silicates or carbonates and their salts.

[0086] The present invention also relates to a protein hydrolysate from Moringa oleifera seed cake obtained by the extraction method according to the invention.

[0087] In a second aspect, the present invention relates to a protein hydrolysate from seed cake that has not been shelled and has been harvested from ripe Moringa oleifera fruits, comprising a major fraction P1 of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight comprised between 1500 Da and 5000 Da, a fraction P2 of about 20% with a molecular weight comprised between 10000 and 17000 Da, and a fraction P3 of about 20% with a molecular weight of about 23000 Da, wherein it is obtained by chemical protein hydrolysis under conditions of pH greater than 13 at a temperature comprised between 16°C and 25°C for a time of about 2 hours, and wherein it is liquid and has a density greater than 1, preferably of about 1.1.

[0088] According to one embodiment, the liquid protein hydrolysate obtained is dried in such a way that a dry hydrolysate is obtained from Moringa oleifera seed cake containing peptides, oligopeptides, glycopeptides and amino acids or volatile nitrile derivatives thereof in an amount of more than 20%, more preferably more than 30% and possibly up to about 40% (weight / weight) of dry matter, more preferably 50% by weight of dry matter.

[0089] According to another embodiment, the protein hydrolysate also comprises between 0.3% and 3% of volatile compounds, of which 50% of these compounds, i.e. between 0.15% and 1.5% of the extract according to the invention, are composed of light nitrile compounds, mainly isobutyronitrile and methylbutyronitrile; of which 5% to 10% of these compounds, i.e. between 0.015% and 0.3% of the extract according to the invention, are composed of isothiocyanate derivatives, mainly isopropyl isothiocyanate and isobutyl isothiocyanate; and of which 1% to 5% of these compounds, i.e. between 0.003% and 0.15%, are composed of essential oils, mainly eucalyptol, menthol and benzaldehyde.

[0090] According to another embodiment, the protein hydrolysate comprises a dry matter content of between 10% and 15%, preferably about 12.5%, comprises between 1% and 6% nitrogenous compounds, in particular volatile nitrile derivatives in a proportion of 0.5% to 1.5%, preferably about 0.8%, and 20 mg / l of polyphenols.

[0091] In the context of the present invention, the plant part of choice is the Moringa oleifera seed. It is known that the Moringa oleifera seed is used to extract its oil, which is used for domestic consumption or various traditional medicine treatments. The cake obtained after de-oiling the seeds is a waste product, which is currently used in particular for animal feed.

[0092] According to yet another embodiment, the protein hydrolysate comprises a major fraction P1 having a molecular weight comprised between 1500 Da and 5000 Da.

[0093] According to yet another embodiment, the protein hydrolysate comprises about a 20% fraction of P2 having a molecular weight comprised between 10,000 Da and 17,000 Da.

[0094] According to yet another embodiment, the protein hydrolysate comprises about 20% of a fraction P3 having a molecular weight of about 23,000 Da.

[0095] According to yet another embodiment, the protein hydrolysate comprises a main fraction P1 having a molecular weight comprised between 1500 and 5000 Da and a fraction P2 having a molecular weight comprised between 10000 and 17000 Da.

[0096] In a third aspect, the present invention relates to the use of a protein hydrolysate from Moringa oleifera seed cake as a medicament.

[0097] In a fourth aspect, the present invention relates to a pharmaceutical or dermatological composition comprising as active agent an effective amount of a protein hydrolysate from Moringa oleifera seed cake according to the invention, and a physiologically acceptable excipient.

[0098] The composition according to the invention may be in any galenical form normally used, depending on whether the composition is to be ingested, injected or applied to the skin or mucous membranes.

[0099] According to a first variant, the various compositions are suitable for ingestion; they may be in the form of capsules, syrups, granules or tablets. They do not necessarily contain any excipients and may be entirely composed of a plant extract comprising a protein hydrolysate in dry form.

[0100] According to a second variant, the various compositions are suitable for injection; they may be in the form of an aqueous or oily lotion, or in the form of a serum.

[0101] According to a third variant, the various compositions are more particularly suitable for topical administration, making it possible to obtain the pharmacological action at the precise site of the disorder through the skin or mucous membranes.

[0102] The main topical modes for administering the active agent according to the invention are dermal and transdermal modes, nasal and respiratory modes, ocular modes, auricular modes and vaginal modes. Other modes can be envisaged, in particular the oral mode, for administering the composition through the mucosa and the subcutaneous mode by microinjection.

[0103] The compositions for pharmaceutical use according to the invention may be in any galenic form usually used for administration by topical route. They may be administered to the mucous membranes, in particular through the nose or respiratory tract, the eyes, the mouth, the vagina and the ears.

[0104] The compositions for dermatological use according to the invention may be in any galenic form commonly used in the dermatological field for cutaneous application. They can be administered transdermally or applied topically to the skin.

[0105] The compositions incorporating the protein hydrolysate according to the present invention may include ingredients conventionally used in such formulations for topical or dermal administration.

[0106] According to a preferred embodiment, the various compositions are suitable for topical administration and include creams, oil-in-water and water-in-oil emulsions, milks, ointments, lotions, oils, balms, aqueous or hydroalcoholic or ethanolic 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.

[0107] According to another preferred embodiment, the various compositions are suitable for subcutaneous injection and transdermal administration; the compositions may be in the form of aqueous lotions, emulsions, or serums. In transdermal or patch systems, the release of the active ingredient or ingredients is controlled by a permeable membrane that is usually adhesive and in direct contact with the skin.

[0108] More specifically be intended to for topical administration according to the composition of the present invention contain acceptable medicine or dermatological medium, promptly compatible with skin and mucous membrane, and comprise all suitable galenic forms.These compositions can be especially the form of emulsifiable paste, oil-in-water emulsion or water-in-oil emulsion or composite emulsion, serum, solution, suspension, gel, emulsion, lotion, stick, aerosol, spray or any other product for external application, such as for example, medical device or also contain the aerosol product of pressurized propellant, or in fact be suitable for any form of powder being applied to skin and mucous membrane.These compositions comprise the necessary excipient of its preparation, such as solvent, emollient, thickening agent, diluent, surfactant, antioxidant, bioactivator, coloring agent, preservative and essence.

[0109] The compositions according to the invention therefore comprise any additives customary in the envisaged field of application and the adjuvants required for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, dermatologically or pharmaceutically active agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[0110] In all cases, the skilled person will take care to ensure that these adjuvants and their proportions are chosen so that they do not deleteriously affect the desired beneficial properties of the compositions according to the invention.

[0111] In the composition of the invention, the protein hydrolysate according to the invention is used in an amount ranging from 0.0001% to 40% by weight relative to the total weight of the composition.

[0112] In a preferred embodiment, the protein hydrolysate according to the invention is used in an amount ranging from 0.001% to 10% by weight relative to the total weight of the composition, more preferably from 0.01% to 5% by weight relative to the total weight of the composition.

[0113] Finally, in a fifth aspect, the present invention relates to a pharmaceutical or dermatological composition for use as a medicament for the treatment of:

[0114] - Treatment of fibrotic diseases and inflammatory diseases comprising as active agent an effective amount of fraction P1 or P2 of a protein hydrolysate, preferably fraction P1.

[0115] - cancer, comprising as an active agent an effective amount of a fraction P3 of a protein hydrolysate.

[0116] - Infectious diseases of bacterial or viral type, in particular for the inhibition of the Spike-COV2 protein, comprising as active agent an effective amount of the protein hydrolysate as a whole.

[0117] - Genetic drift and pathologies associated with skin pigmentation, comprising as active agent an effective amount of fraction P1 or P2 of a protein hydrolysate. The term "genetic drift" refers in particular to environmental stress. The term "pathologies associated with pigmentation" refers, for example, to cancer and lentigines.

[0118] For the treatment of fibrotic diseases, the term fibrosis refers to pulmonary fibrosis, renal fibrosis, liver fibrosis, skin fibrosis, eye fibrosis, cardiac fibrosis and other various fibrotic states, as well as for the treatment of other furin-mediated pathological states, in particular but not limited to hypertension, cancer, infectious diseases including viral and bacterial diseases, genetic diseases (such as cystic fibrosis (CF)) and neurodegenerative diseases.

[0119] Although the present invention has been described with respect to several specific embodiments, it is obvious that the invention is not limited in any way to these embodiments and comprises any technical equivalents of the described means and combinations thereof if they fall within the scope of the invention.

[0120] Use of the verb "compose of," "comprise" or "include" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim.

[0121] Example

[0122] Implementation Example 1: Preparation of a plant protein hydrolysate according to the invention starting from Moringa oleifera cakes

[0123] The seeds of Moringa oleifera (Forssk.) are dried to an internal moisture content of less than 8% and preferably about 6%, and then pressed using a headless screw mechanical press in such a way as to separate the oil from the rest of the seed, in order to obtain, on the one hand, virgin oil and, on the other hand, a cake. The cake is then separated into pieces of 1 to 2 cm in the form of pre-cut extrudates, on which the extraction is carried out.

[0124] The starting materials used were as follows.

[0125] [Table 1]

[0126]

[0127] plan:

[0128] a) preparing a concentrated 1 molar solution of a strong base such as sodium hydroxide, potassium hydroxide or calcium hydroxide, in particular or an aqueous mixture with a 1 molar concentration of a strong base, but preferably sodium hydroxide; this strong alkaline solution has a pH between 13 and 14,

[0129] b) weighing 9.1% (w / w) Moringa cakes pre-cut into 1 cm pieces into approximately 90.9% (w / w) alkaline solution,

[0130] c) milling the cake obtained in step b),

[0131] d) dispersing the milled cake obtained in step c) in an aqueous phase,

[0132] e) subjecting the aqueous dispersion obtained in step d) to chemical proteolysis at a temperature of 22° C. for a period of 2 hours,

[0133] f) neutralizing the protein hydrolysis to stabilize the obtained protein hydrolysate,

[0134] g) recovering the protein hydrolysate by solid / liquid separation by passing through a 1 μm filter,

[0135] h) purifying the protein hydrolysate by ultrafiltration,

[0136] A translucent yellow filtrate containing approximately 12.48% of dry matter was obtained. The liquid extract obtained is hereinafter referred to as "Moringa oleifera protein hydrolysate according to the present invention" or "Moringa oleifera protein hydrolysate" or "extract according to the present invention".

[0137] The Moringa oleifera protein hydrolysate according to the invention has a density greater than 1 and preferably about 1.1, comprises a dry matter content of between 10% and 15%, preferably about 12.5%, comprises between 1% and 6% nitrogenous compounds, in particular volatile nitrile derivatives in a proportion of 0.5% to 1.5%, preferably about 0.8%, and 20 mg / L of polyphenols. The composition of the dried Moringa oleifera protein hydrolysate according to the invention is given below.

[0138] [Table 2]

[0139] Rt CAS number Compound %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-Methylbutyronitrile 2.194 14.56 625-28-5 3-Methylbutyronitrile 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-Butyl isothiocyanate 1.058 27.59 80-56-8 α-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 22466-pentamethylheptane 0.318 32.80 99-87-6 p-Cymene 0.232 33.10 138-86-3 limonene 0.133 33.34 470-82-6 Eucalyptol 0.918 36.45 1195-32-0 Dehydro-p-cymene 0.035 36.88 124-19-6 Nonanal 0.080 40.26 65-85-0 benzoic acid 19.150 41.07 1490-04-6 Menthol 0.918 56.29 96-76-4 24-Di-tert-butylphenol 0.086 total 85.035

[0140] Moringa oleifera protein hydrolysates contained relatively high levels of isopropyl and isobutyl isothiocyanates, confirming previous publications on this species (KJAER, A. et al. 1979, Isothiocyanates in Myrosinase-treated seed extracts of Moringa peregrina, Phytochemistry, 18, p. 1485-1487; AFSHARYPUOR, S. et al., 2010, Volatile Constituents of the Seed Kernel and Leaf of Moringa peregrina (Forssk.) Fiori, Agricolt. Cultivated in Chabahar (Iran), Iranian Journal of Pharmaceutical Sciences 6(2): p. 141-144; DEHSHAHRI, S. et al., 2012, Determination of volatile glucosinate degradation products in seed coat, stem and in vitro cultures of Moringa peregrina (Forssk.) Fiori, Science Open, Research in Pharmaceutical Sciences 7(1): p.51-56). Isothiocyanates are compounds produced by various plants belonging to the order Brassicales, especially in the families Cruciferae, Capparaceae, Caricaceae and Moringaceae, as a defense system against pathogen attacks. In the genus Moringa, especially in Moringa oleifera Lam. and Moringa stenopetala (Baker f.) Cufold (A BD They were identified in RANI, NZ et al., 2018, Moringa genus: A review of Phytochemistry and Pharmacology, Frontiers in Pharmacology, vol. 9, art. 108, p. 3-8). When plant tissues are damaged, isothiocyanates are derived from the hydrolysis of glucosinolates by myrosinase. Isothiocyanates are reported to have various biological effects, such as antifungal activity (T RONCOSO -ROJAS , R. et al., 2007, Natural compounds to control fungal diseases in fruits & vegetables, in TRONCOSO - ROJAS, R., TIZNADO - HERNANDEZ, M.E., GONZALEZ - LEON, A. (ed) Recent advances in alternative postharvest technologies to control fungal diseases in fruits & vegetables. Transworld Research Network, Kerala, India, p. 127–156; TRONCOSO - ROJAS, R. et al., 2005, Analysis of the isothiocyanates present in cabbage leaves extract and their potential application to control Alternaria rot in bell peppers, Food Research International 38, p. 701–708), antibacterial, anticancer and anti - inflammatory effects (P ARK , E.J. et al., 2011, Inhibition of lipopolysaccharide induced cyclooxygenase - 2 expression and inducible nitric oxide synthase by 4 - [(2′ - Ο - acetyl - α - L - rhamnosyloxy)benzyl]isothiocyanate from M. oleifera, Nutrition and Cancer 63(6), p. 971 - 982; R AJAN(Ts et al., 2016, Anticancer activity of glucomoringin isothiocyanate in human malignant astrocytoma cells, Fitoterapa 110, pp. 1-7; PADLA, EP et al., 2012, Antimicrobial isothiocyanates from the seeds of Moringa oleifera Lam., Zeitschrift für Naturforschung C, 67, pp. 557–564; WATERMAN, C. et al., 2014, Stable, water-extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro. Phytochemistry 103, pp. 114–122). Furfural is present in highly standard concentrations found in this type of seed and many dried fruits. Carbon disulfide, isobutyronitrile, methyl isobutyrate, methylbutyronitrile, and hexanenitrile are volatile compounds derived from amino acids. They are markers of protein degradation. These degradation compounds account for approximately 50% of the volatile compounds of the Moringa oleifera protein hydrolysate. This result indicates that the hydrolysate is primarily composed of protein. Only very trace amounts of fat or free sugars were detected in the Moringa oleifera protein hydrolysate; the hydrolysate is primarily composed of protein and glycoproteins. In the dry matter, citrate buffer (which is a glycoside compound) accounts for approximately 50% of the remainder and includes glycosylated (glycoside) compounds resulting from proteolytic degradation of proteins, oligopeptides, and amino acids. Finally, the presence of polyphenols obtained from Moringa oleifera seeds should be noted at approximately 21 mg / liter (0.002%). Benzoic acid was not considered in the characterization because it is a stabilizer added to the extract.

[0141] The above dry extract is obtained by a gravimetric method based on the mass present in the liquid extract before and after evaporation.

[0142] Implementation Example 2: Moringa oleifera protein hydrolysate according to the present invention as furin converting enzyme (referred to as furin) The role of inhibitors

[0143] The aim of this study was to evaluate the inhibitory activity of protein hydrolysate from Moringa oleifera seed cake obtained according to Example 1.

[0144] Protocol: The study was performed using recombinant human furin, which catalyzes the cleavage of a specific fluorescent reference substrate.

[0145] 100 nM Decanoyl-Arg-Val-Lys-Arg-CMK was used as a reference inhibitor for furin activity.

[0146] Furlin was preincubated for 10 minutes at ambient temperature in the absence (control) or presence of reference product or increasing concentrations of test compound:

[0147] "Moringa oleifera protein hydrolysate"; 0.02; 0.2 and 2% (v / v).

[0148] At the end of the pre-incubation step, furin substrate was added and the experimental conditions were incubated again for 5 minutes at ambient temperature away from light. All experiments were performed in triplicate.

[0149] Preparation of compounds:

[0150] The tested Moringa oleifera protein hydrolysates were directly dissolved in assay buffer and then diluted to obtain the above-mentioned test concentrations.

[0151] Evaluation plan

[0152] Cleavage of the fluorogenic furin substrate was monitored by reading fluorescence at 485 nm / 535 nm for 5 minutes after addition of the substrate.

[0153] statistics

[0154] The results are expressed as RFU (relative fluorescence units) + / - SD (standard deviation).

[0155] The statistical significance of the differences observed between the "Control" and "Reference Product" groups was assessed by t-test (p<0.001).

[0156] The statistical significance (p<0.05) of the differences observed between the "Control" or "Reference Product" and "Test Compound" groups was assessed by one-way ANOVA followed by the Holm-Sidak test.

[0157] A reference inhibitor of furin, called decanoyl-Arg-Val-Lys-Arg-CMK, tested at 100 nM significantly inhibited furin activity by 97.9% (p<0.001).

[0158] This result was expected and validated the study.

[0159] The results of the furin activity compared to the alkaline activity obtained are given below.

[0160] [Table 3]

[0161]

[0162] Conclusion: At a concentration of 2%, the Moringa oleifera protein hydrolysate according to the present invention can inhibit the activity of furin convertase by 98.8%. At a concentration of more than 0.2%, the Moringa oleifera protein hydrolysate according to the present invention can inhibit the activity of furin convertase by up to 26.8%.

[0163] Implementation Example 3: Moringa oleifera protein hydrolysate according to the present invention inhibits histone deacetylase Role of HDAC and sirtuin I enzymes

[0164] The purpose of this study was to demonstrate the inhibitory activity of a Moringa oleifera protein hydrolysate according to the present invention against HDAC and sirtuin I enzymes, which are involved in controlling genetic drift by regulating chromatin condensation / decondensation, thereby providing or blocking access to DNA-borne genes. A buffered solution of HDAC and sirtuin I was reacted with a substrate at 37°C for over 20 minutes to form a compound. After incubation at 37°C for 10 minutes, the compound developed color in the presence of a chromogenic agent. Thus, the maximum deacetylation activity of sirtuins can be assessed by measuring absorbance at 405 nm. A Moringa oleifera protein hydrolysate according to the present invention or a reference product, "Trichostatin A (STA) inhibitor 1 μM," was simultaneously exposed to a sirtuin solution as an enzyme substrate at 37°C for 20 minutes. The enzymatically converted substrate was stained by the addition of a chromogenic agent. The deacetylation activity of HDAC and sirtuin I in the presence of the active ingredients was then assessed by measuring absorbance at 405 nm. The modulation of this activity is expressed as a percentage inhibition or a percentage activation of the maximum activity of HDAC and sirtuin I in the absence of active ingredient, ie only in the presence of substrates for the HDAC and sirtuin I enzymes.

[0165] Protocol: A solution of sirtuin enzymes is incubated in its substrate for 20 min in the absence (control) or presence of a reference product or increasing concentrations of the product to be tested. The Moringa oleifera protein hydrolysate of the present 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 enzyme with and without the test product or reference product is visualized by staining with a developer solution (10 min at 37°C) and evaluated by measuring the absorbance of the reaction medium at 405 nm. For each test concentration, the modulation of the deacetylase activity of the test product on the histone deacetylase and sirtuin I enzymes is calculated by using the following formula.

[0166] [Mathematical formula 1] Percentage adjustment of sirtuin enzyme activity = 100 x [(OD produced in test or reference) 405 )–(OD 405 HDAC and sirtuin I only)] / OD only 405sirtuin.

[0167] 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 for inhibition of histone deacetylase (HDAC) are given below.

[0168] [Table 4]

[0169]

[0170] Conclusion: At 2%, the Moringa oleifera protein hydrolysate according to the invention exhibited significant HDAC inhibition; this inhibition suggests a potential for promoting skin cells' ability to protect themselves against genetic drift. Therefore, the extract appears to be useful in combating one of the most common genetic drifts on the skin surface, namely fibrosis, which manifests itself through the appearance of fleshy "lumps" (fibrotic protuberances). The extract can advantageously interfere with fibrosis on the skin surface.

[0171] Implementation Example 4: Effect of Moringa oleifera protein hydrolysate on the inhibition of endothelin 1 (ET-1) according to the present invention Effect

[0172] Endothelin is a hormone peptide derived from endothelial cells that can act on various cells and tissues through its receptors. For example, endothelin is known to increase the intracellular concentration of calcium in smooth muscle vascular cells and other cells (OHBA T. et al., WO 2012 / 081370).

[0173] In recent years, it has been reported that endothelin type 1 (ET-1) is a bioactive factor that constricts smooth blood vessels and non-vascular muscle cells through direct and indirect actions. It is believed that the increase in endothelin action provides sustained vasoconstriction for blood vessels in peripheral sites, in the kidneys, and in the brain, and may be the origin of various diseases such as hypertension, myocardial infarction, cerebrovascular accident, acute renal insufficiency, Raynaud's syndrome, atherosclerosis, asthma, and prostate cancer (M IYAGAWAK.&Emoto N.et al., 2014, Current state of endothelin receptor antagonism in hypertension and pulmonary hypertension, Therapeutic Advances in Cardiovascular Diseases, vol. 8(5) 202-216; SCHINZARI F.et al., 2018, Increased Endothelin-1-Mediated Vasoconstrictor Tone in Human Obesity: Effects of Gut Hormones, Physiological Research 67 suppl. 1: S69-S81). Three types of peptides from the endothelin family with similar structures exist in animals, including humans (K ADONO ,S.et al.,2001,The Role of the Epidermal Endothelin Cascade in the Hyperpigmentation Mechanism of LentigoSenilis,Journal of Investigative Dermatology 116 4,p.571-577;A NONYMOUS , 2006, American Society for Biochemistry and Molecular Biology, New Cosmetics Handbook, p. 527-529). All of these peptides have vasoconstrictor and angiostatic effects.

[0174] Recently, the role of endothelin in a variety of cells other than smooth vascular muscle cells has been elucidated. For example, scientific publications report that when the skin is exposed to UV radiation, the production of ET-1 and other factors increases in keratinocytes, and suggest that ET-1 may be involved in melanin production in melanocytes exposed to UV radiation. Therefore, inhibiting the expression of endothelin is considered to be useful not only for preventing and / or treating the above-mentioned diseases, but also for preventing or improving (I MOKAWA,G.etal.,1995,Endothelin-1as a New Melanogen:Coordinated Expression of its Geneand the Tyrosinase Gene in UVB-Exposed Human Epidermis,Journal of Investigative Dermatology 1051,p.32-37;IMOKAWA,G.etal.,1992,EndothelinsSecreted from Keratinocytes are Intrinsic Mitogens for Human Melanocytes,Journal of Biological Chemistry,267,p.24675-24680;G ILCHREST ,B.et al.,1996,Mechanisms of Ultraviolet Light-Induced Pigmentation,Photochemistry andPhotobioliogy 63,p.1-10).

[0175] The aim was to determine type 1 endothelin in human microvascular endothelial cells after 24 h of exposure to the Moringa oleifera protein hydrolysate according to the invention.

[0176] Protocol: Human microvascular endothelial cells were provided by PELOBiotech and cultured in 96-well plates according to the supplier's manufacturing procedures. This means that the extracts were allowed to act on the endothelial cells at 80% confluence for 24 hours, after which endothelin-1 was quantified in the cell supernatant using the ELISA PicoKine (EDN1) kit. A previous viability test was performed to define the non-toxic dose to be used in the endothelin-1 assay. Untreated cells in culture medium formed a negative control. The positive control in the viability test was 0.5% SDS. All conditions were prepared in culture medium and the cells were subsequently incubated at 36.5°C / 5% CO2 for 24 hours.

[0177] a) Applying the test solution to the endothelial cells:

[0178] The test product was placed in contact with subconfluent endothelial cells in a 96-well plate. Three wells were tested for each concentration. The plates were incubated at 36.5°C / 5% CO2 for 24 hours ± 1 hour.

[0179] b) Viability test:

[0180] After incubation with the product, the cells were assessed for cell viability using the MTT assay. After a 24-hour incubation, the supernatant was recovered and stored at -20°C for use in the assay. The wells were then rinsed once with 200 μL of PBS. 50 μL of a 0.5 mg / mL MTT solution was added to each well and incubated at 36.5°C / 5% CO₂ for 3 hours. 100 μL of isopropanol was added to each well. After homogenization, the absorbance was recorded at 550 nm. For each condition, the ratio of the mean optical density of the cells to the mean optical density of the negative control was used to determine the viability ratio.

[0181] c) Endothelin-1 assay:

[0182] The assay was performed with the aid of an ELISA kit. The results of the inhibition of endothelin action are given below.

[0183] [Table 5]

[0184]

[0185] Conclusion: Viability tests performed at the end of the treatment did not show any toxic effects at the concentrations tested.

[0186] Endothelin-1 assay was performed in cell supernatants at non-toxic concentrations. The amount of endothelin-1 under each condition was determined using an ELISA kit.

[0187] For control cells, the basal level was approximately 134.94 pg / mL. For cells treated with different concentrations of the hydrolyzate, the basal level was reduced by 47.09 pg / mL (with 2% of the extract according to the invention) and by 17.58 pg / mL (with 1% of the extract according to the invention). This demonstrates a very significant inhibitory effect of the 1% extract according to the invention, with an inhibitory effect of approximately 13% on type 1 endothelin production, and a 2% extract according to the invention with an inhibitory effect of up to 34.90%.

[0188] The results of endothelin and its specific dose-dependent inhibition indicate that the protein hydrolysate according to the present invention has anti-angiogenic and anti-fibrotic effects due to its ability to significantly reduce endothelin.

[0189] The following describes additional studies for evaluating P1, P2, P3 and the complex P1+P2 described in Example 12 for the production of endothelin: a cellular test model of normal human endothelial cells.

[0190] As described in Example 12, three bands or protein fractions were isolated from the hydrolysate according to the invention, characterized by their masses P1 (molecular weight less than 10,000 Da), P2 (molecular weight comprised between 10,000 and 17,000 Da) and finally P3 (molecular weight of approximately 23,000 Da). These fractions are also referred to hereinafter as "extracts".

[0191] [Table 6]

[0192]

[0193]

[0194] Statistically significant (p>0.05)

[0195] At all concentrations, the standard deviations were high and fraction P1 of the protein hydrolysate did not show any significant modulation of endothelin type 1.

[0196] [Table 7]

[0197]

[0198] * Statistically significant (p>0.05)

[0199] ** Statistically significant (p<0.05)

[0200] At concentrations of 0.1% and 1%, the standard deviations were high and at these concentrations the protein hydrolysate fraction P2 did not show any significant modulation of endothelin type 1. At a concentration of 0.3%, P2 inhibited endothelin type 1 production by 24.8%.

[0201] [Table 8]

[0202]

[0203] * Statistically significant (p>0.05)

[0204] At all concentrations, the standard deviation was high and the combination of fraction P1 + P2 did not show any significant modulation of type 1 endothelin.

[0205] [Table 9]

[0206]

[0207] * Statistically significant (p>0.05)

[0208] ***: Statistically significant (p<0.001)

[0209] At concentrations of 0.3% and 1%, fraction P3 of the protein hydrolysate very significantly inhibited the production of type 1 endothelin by approximately 50%.

[0210] Conclusion: The compounds designated "Extract P1" and "Extract P2" had no significant effect on the regulation of type 1 endothelin. Only "Extract P3," containing 0.3% of the extract according to the present invention, significantly reduced the release of endothelin-1 from normal human endothelial cells into the culture medium in monolayer culture, with an inhibition score of 52.3%. No synergistic effect was observed when P1 and P2 were combined.

[0211] Extract P3 showed anticancer effect in particular (B AGNATO A. et al., 2011, Role of theendothelin axis and its antagonists in the treatment of cancer, British Journal of Pharmacology, 163: 220-233).

[0212] Example 5: Effect of Moringa oleifera protein hydrolysate on stem cell protection according to the present invention

[0213] Adult tissues, including the skin epidermis, gastrointestinal epithelium, and hematopoietic system, have very high levels of cell turnover. The physiological process of maintaining tissue homeostasis is attributed to maintaining a constant number of cells during organ renewal. Embryonic stem cells (ESCs) are crucial for maintaining and regenerating skin tissue.

[0214] The epidermis develops from the ectoderm layer of the embryonic surface. It begins as a single layer of nonspecific progenitor cells that covers the embryo after neurulation and develops into the basal epidermis. The basal epidermis is rich in ESCs (epidermal stem cells). In fact, the cells of this layer give rise to all epidermal structures, including the stratified epidermis (also known as the interfollicular epidermis) and epidermal appendages such as hair follicles, sebaceous glands, and sweat glands. The lower dermis is primarily derived from the mesoderm layer beneath the ectoderm. The mesoderm is the main source of mesenchymal stem cells, which give rise to fibroblasts that produce collagen, lower-layer fat cells, and immune cells of the skin.

[0215] Stem cells are undifferentiated cells, known as pluripotent cells, that have a youthful genotype and are able to both self-renew and differentiate to generate organs or tissues such as skin. At this stage, they are identified as "pluripotent cells." Given that 50% of the stem cell population's descendants remain undifferentiated, stem cells help maintain homeostasis and ensure the renewal of damaged or aging differentiated cells. However, these epidermal stem cells are often affected by the environment. According to Y EJINGe et al. (10 March 2020, The aging skin microenvironment dictates stem cell behavior, PNAS, Vol. 117, p. 5339-5350), oxidative stress such as pollution or ultraviolet radiation damages their DNA. This damage changes their self-renewal and differentiation abilities, leading to a decrease in the stem cell pool and ultimately causing skin aging.

[0216] The aim of this study was to evaluate the effect of Moringa oleifera protein hydrolysate according to the present invention on the protection of epidermal stem cells against UVB irradiation.

[0217] Protocol: Human keratinocytes were obtained from a 62-year-old donor. For experiments, keratinocytes were cultured in a monolayer until they reached 80% confluence.

[0218] The cell culture was then enriched for epidermal stem cells according to 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).

[0219] Reference product: This study used 1 μM quercetin as a reference product. Quercetin was purchased from Sigma Aldrich.

[0220] Cells were pre-incubated for 24 hours in the absence ("control") or presence of reference product or increasing concentrations of test compound. At the end of the pre-incubation period, cells were irradiated with UVB (30 mJ / cm 2 ) irradiated cells and then incubated at 37°C for 8 days in the absence (reference) or presence of a reference product or with increasing concentrations of the test compound.

[0221] "Moringa oleifera protein hydrolysate": 0.01; 0.05 and 0.15% (v / v).

[0222] Preparation of test compounds:

[0223] The test compound "Moringa oleifera protein hydrolysate" was diluted directly in the incubation medium to obtain the various concentrations mentioned above.

[0224] 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 performed in triplicate (n=3).

[0225] The results are presented below as percentage of viability relative to the "control without UVB" experimental condition (mean + / - SD). The significance level between "control without UVB" and "control with UVB" was assessed by means of a t-test (p<0.05).

[0226] [Table 10]

[0227]

[0228] Conclusion: Moringa oleifera protein hydrolysate significantly protects human skin stem cells exposed to cellular stress (UV). Stem cells are cells with preserved and youthful DNA material. They are the source of tissue regeneration and restoration to a youthful and healthy state. Stem cell protection is associated with the ability to preserve DNA material. The Moringa oleifera protein hydrolysate according to the present invention maintains the integrity of stem cells, thus contributing to DNA turnover.

[0229] Implementation Example 6: Effect of Moringa oleifera protein hydrolysate according to the present invention on DNA preservation

[0230] The dynamics of telomere length are very important for regulating the replicative lifespan in cells, especially in the case of long-lived species. Telomere shortening and telomerase activity are important factors in aging and tumorigenesis (SHAY, JW & Wright, WE, 2005, Senescence and Immortalization: Role of Telomeres and Telomerase, Carcinogenesis 26 (5), p. 867-874). Telomeres are complex nucleotide sequences that cover the ends of chromosomes to protect them from degradation, unwanted fusion recombination, and inappropriate activation of the DNA damage response. They also play an important role in cell division and chromosome stability. There is increasing evidence that the stability of telomeres and their average length are affected by stress, especially diseases under environmental stress or environmental influences. (V ALDES,ALet al.,July 2005,Obesity,cigarette smoking and telomere length in women,Research Letters.366(9486),p.662-664;PHILLIPS ACet al.,2013,Do symptoms of depression predict telomerelength? Evidence from the West of Scotland Twenty-07 Study,PsychosomaticMedicine,75(3),p.288-296;SHIN,D.et al.May 2019,Effects of inflammation and depression on telomere length in young adults in the United States,Journal ofClinical Med 2019,8(5),p.711;SALIQUES,S.et al.,October 2010, Telomer length and cardiovascular disease, Archives of Cardiovascular Diseases, 103(8-9), p.454-459). Consequently, critically short telomeres have been linked to neurodegenerative diseases, cardiovascular disease (CVD), and cancer risk.

[0231] Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repeats to the ends of telomeres. Telomeres are long stretches of repetitive sequences that cap the ends of chromosomes and are known to stabilize chromosomes. In humans, telomeres are typically 7 to 10 kb long and consist of multiple repeats of the sequence -TTAGGG-.

[0232] Telomerase is not expressed in most adult cells, and the length of telomeres decreases with successive replication cycles. After a certain number of replication cycles, the progressive shortening of telomeres causes cells to enter a telomere crisis phase, leading to cellular senescence. Certain diseases are associated with the rapid loss of telomeres, leading to premature aging of precocious cells. It has been shown that the expression of the gene encoding the human telomerase protein in human cells (B LASCOM., 2007, Telomere Length, Stem Cells and Aging, Nature Chemical Biology, 3(10), p. 640-649) produces a phenotype with constant mass, possibly by reversing the natural aging process of cells. In addition, it has been demonstrated in the studies cited above that expression of the telomerase gene in senescent cells with short telomeres results in an increase in telomere length and restoration of a phenotype normally associated with young cells.

[0233] The purpose of this study was to evaluate the effects of a compound called "Moringa oleifera protein hydrolysate" on telomere shortening in a model composed of normal human fibroblasts grown in monolayer culture. Telomeres are known to act as a biological clock. Telomere length gradually decreases with cell division, ultimately leading to cell replication failure. Telomere length was measured using quantitative PCR and compared to telomere length between passage 2 and passage 5 cells.

[0234] Protocol: Human fibroblasts were obtained from a 44-year-old donor. For the experiments, cells from passages 2 and 5 were used. Fibroblasts were cultured for three consecutive passages in the absence (control) or presence of increasing concentrations of Moringa oleifera protein hydrolysate: 0.01%, 0.1%, and 0.5% (v / v).

[0235] Preparation of test compounds: The "Moringa oleifera protein hydrolysate" test compound was directly dissolved in the incubation medium to obtain the various concentrations mentioned above.

[0236] At the end of the incubation period, the cells were trypsinized and DNA was extracted from the cells using a dedicated DNA extraction kit. DNA was quantified using nanodrop.

[0237] Telomere length was measured by quantitative PCR (q-PCR). For each sample, the SCR (single copy reference) gene was used as a reference gene to measure the change in telomere length by relative quantification. For each sample, q-PCR was performed using a set of telomere primers that recognize and amplify telomere sequences, and a second q-PCR was performed using a set of SCR primers that recognize and amplify a 100bp region on human chromosome 17 and serve as a reference for data normalization.

[0238] The results are expressed as relative units corresponding to the length of telomeres at passage 2 (mean ± SD). The significance level of passages 2 and 5 compared to the "control" was assessed by Student's tactile test (*: p < 0.05). The significance level between the "control" and the "test compound" of each product was independently evaluated by one-way analysis of variance (one-way ANOVA) followed by the Holm-Sidak test (*: p < 0.05).

[0239] [Table 11]

[0240]

[0241] Results: Under our experimental conditions, Moringa oleifera protein hydrolysate tested at 0.05%, 0.1% and 0.5% (v / v) significantly reduced telomere shortening in normal human fibroblasts.

[0242] Telomere shortening (compared to the control) was inhibited by +8.9% at 0.05% (v / v) (p<0.05); by +15.1% at 0.1% (v / v) (p<0.01); and by +16.6% at 0.5% (v / v) (p<0.01). Conclusion: The Moringa oleifera protein hydrolysate according to the present invention demonstrated the ability to significantly increase telomere length in the context of normal proliferation or division of human cells. Telomeres are involved in protecting DNA material; increasing telomere length is associated with the ability to preserve DNA material. Moringa oleifera protein hydrolysate is able to increase telomere length. Therefore, this hydrolysate participates in the preservation of human genetic material (DNA).

[0243] Complementary studies were conducted on the ability of extracts P1, P2, P3 and the complex P1+P2 described in Example 12 to protect DNA after several generations of cell division by increasing telomere length.

[0244] [Table 12]

[0245]

[0246] * Statistically significant (p>0.05)

[0247] **: Statistically significant (p<0.01)

[0248] ***: Statistically significant (p<0.001)

[0249] Extract P1 according to the invention increased telomere size by 24.3% after three cell divisions at a concentration of 0.3%. At a concentration of 1%, extract P1 increased telomere size by 39.6% under the same conditions.

[0250] [Table 13]

[0251]

[0252]

[0253] * Statistically significant (p>0.05)

[0254] After 3 cell divisions in a normal human cell culture model (fibroblasts), the extract P2 according to the invention showed a statistically unconfirmed tendency for telomere elongation at each concentration tested.

[0255] [Table 14]

[0256]

[0257] * Statistically significant (p>0.05)

[0258] After 3 cell divisions in a normal human cell culture model (fibroblasts), the extract P3 according to the invention had no ability to promote telomere elongation at each tested concentration.

[0259] [Table 15]

[0260]

[0261]

[0262] * Statistically significant (p>0.05)

[0263] **: Statistically significant (p<0.01)

[0264] A mixture of extracts P1 and P2 according to the invention (50 / 50 by volume) increased telomere size by 49.1% in a normal human cell culture model (fibroblasts) after three cell divisions at a concentration of 1% (i.e. 0.5% of each extract). This score was not achieved by the individual extracts, and a synergistic effect was therefore demonstrated by mixing extracts P1 and P2.

[0265] Conclusion: The compounds designated "Extract P1" and "Extract P1+P2" significantly reduced telomere shortening after three consecutive cell generations. The combination of P1 and P2 demonstrated a synergistic effect.

[0266] Implementation Example 7: Effect of Moringa oleifera protein hydrolysate according to the present invention on stimulating ZAG protein

[0267] Zinc alpha-2-glycoprotein (ZAG) is a plasma glycoprotein that derives its name from its electrophoretic mobility and its ability to be precipitated by zinc salts. ZAG is a member of the immunoglobulin gene superfamily and has a three-dimensional structure that is highly homologous to class I and class II CMH molecules. ZAG has been immunohistochemically detected in normal secretory epithelial cells of the breast, prostate, and liver, in salivary glands, bronchi, gastrointestinal tract, and sweat glands, and in normal stratified epithelial cells including the epidermis. ZAG mRNA remains uniformly distributed in various cell types (FREIJE , JP et al., 1991, Human Zn-α2-Glycoprotein cDNA Cloning and Expression Analysis in Benign and Malignant Breast Tissues, FEBS Letters 290 (1-2), p. 247-249). Since it is produced by secretory epithelia, ZAG is present in most body secretions and accounts for 2.5% of the protein in saliva and 30% of the protein in semen, respectively. It is reported that the level of ZAG in plasma and serum varies with age, with reported values ​​ranging from 0.9 to 3.5 mg / dl (fetus) to 7.8 to 12.1 mg / dl (young people) (J IRKA , M. et al., 1974, The Zn-alpha 2-Glycoprotein Level in Human Serum During Ontogenesis. Clinica Chimica Acta 56, p. 31-33; Jirka, M. et al., 1978, Human Serum Zn-α2-Glycoprotein in Amniotic Fluid, Clinica Chimica Acta 85, p. 107-110). ZAG accumulates in breast cyst fluid at levels 30 to 50 times higher than plasma concentrations (B UNDRED et al.,1987,AnImmunohistochemical Study of the Tissue Distribution of the Breast Cyst FluidProtein,Zinc Alpha2-Glycoprotein,Histopathology 11,p.603-610; D IEZ -I TZA, I.etal.,1993,Zn-α2-Glycoprotein Levels in Breast Cancer Cytosols and Correlationwith Clinical, Histological, and Biochemical Parameters,European Journal of Cancer 29A,p.1256-1260), and is overexpressed in 40% to 50% of breast cancers. It has recently been shown (SUSAN, M.el al.,Zinc-alpha2-glycoprotein Expression as a Predictor of Metastatic Prostate Cancer Following Radical Prostatectomy,2006,Journal of the National Cancer Institute,Volume 98(19),p.1420–1424) that ZAG is produced in large quantities by most prostate cancers, which leads to increased serum ZAG levels in basal cell carcinomas in prostate cancer patients. The purpose of this study was to evaluate the ability of Moringa oleifera protein hydrolysate to stimulate ZAG.

[0268] Protocol: Normal human keratinocytes were isolated from foreskin and cultured in 24-well and 96-well plates according to in-house procedures.

[0269] This involves subjecting keratinocytes to samples at the indicated concentrations for 48 hours at 80% confluence, followed by quantification of ZAG in the cell supernatant using an ELISA kit.

[0270] A previous viability test was performed to define the non-toxic dose to be used in the ZAG assay. Negative controls were generated with the help of untreated cultured cells. The positive control for viability testing was 0.5% SDS.

[0271] All conditions were prepared in culture medium, and cells were subsequently incubated at 36.5° C. / 5% CO 2 for 24 hours for viability testing and 48 hours for ZAG assays.

[0272] Apply the test solution to the keratinocytes:

[0273] - The test product was brought into contact with subconfluent keratinocytes in 24- and 96-well plates.

[0274] - For each concentration, the test was performed in 3 wells.

[0275] - Plates were incubated at 36.5°C / 5% CO2 for 24 hours and 48 hours.

[0276] Survivability test:

[0277] - After incubation with the product, the cell viability of the cells was assessed using the MTT method.

[0278] - After 24 and 48 hours of incubation, the provided wells were rinsed once with 200 μL PBS.

[0279] - Add 50 μL of 0.5 mg / mL MTT solution to each well: incubate for 3 hours at 36.5° C. / 5% CO 2 .

[0280] - Add 100 μL of isopropanol to each well.

[0281] -After homogenization, the absorbance was recorded at 550 nm.

[0282] - For each condition, the ratio of the mean optical density value of the cells to the mean optical density value of the negative control determines the viability ratio.

[0283] Determination of ZAG protein:

[0284] - After 48 hours of incubation, all supernatants were recovered and stored at -20°C for assay.

[0285] - The assay was performed with the help of an ELISA kit. The results are given below.

[0286] [Table 16]

[0287]

[0288] Conclusion: Moringa oleifera protein hydrolysate significantly increased ZAG production in a dose-dependent manner and exhibited low toxicity to human cells. Consequently, it possessed anti-fibrotic and anti-inflammatory effects based on its ability to significantly increase ZAG.

[0289] Complementary studies for the stimulation of ZAG in normal human keratinocytes starting from the protein band identified in Example 12 are described below.

[0290] [Table 17]

[0291]

[0292] * Statistically significant (p>0.05)

[0293] **: Statistically significant (p<0.01)

[0294] Starting from 0.2%, extract P1 significantly increased the production of ZAG by more than 107%.

[0295] [Table 18]

[0296]

[0297] * Statistically significant (p>0.05)

[0298] **: Statistically significant (p<0.05)

[0299] Starting from 0.2%, extract P2 significantly increased the yield of ZAG by more than 80%.

[0300] [Table 19]

[0301]

[0302] * Statistically significant (p>0.05)

[0303] Extract P3 had no ability to significantly affect the production of ZAG in this study model.

[0304] [Table 20]

[0305]

[0306] * Statistically significant (p>0.05)

[0307] The combination of extracts P1 and P2 showed a trend towards activating ZAG production, but this trend was not statistically significant. Therefore, the combination of extracts P1 and P2 did not have a synergistic effect.

[0308] The compounds designated "Extract P1" and "Extract P2" significantly increased ZAG release into the culture medium by normal human keratinocytes in monolayer culture, but no synergistic effect was observed with the combination of P1 and P2.

[0309] Conclusion: Among the hydrolyzed extracts according to the present invention, extract P1 is the best extract in terms of increasing ZAG. For this reason, it has anti-fibrotic and anti-inflammatory effects starting from a dose of 0.2%.

[0310] Example 8: Effect of Moringa oleifera protein hydrolysate on the regulation of DKK1 and DKK3 assays according to the present invention use

[0311] The involvement of interactions between melanocytes and fibroblasts in the regulation of melanogenesis is well known and has been intensively studied. Although these interactions are not yet fully understood, they are responsible for the “whitening” of the palmoplantar area and are now used in dermatology for the development of depigmentation products. Yamaguchi et al (Y AMAGUCHI Y. et al., 2004, Mesenchymal-Epithelial Interactions in the Skin: Increased Expression of Dickkopf by Palmoplantar Fibroblasts Inhibits Melanocyte Growth and Differentiation, Journal of Cell Biology 165(2), p.275-285) have demonstrated that a soluble messenger produced by fibroblasts in the palmoplantar region can modify the melanocyte differentiation program in these areas, thereby reducing melanin production. The messenger was identified by the team as a protein called Dickkopf-1 (DKK-1).

[0312] The signaling pathways used by DKK-1 to produce these results have now been clearly defined. By virtue of its antagonistic effect on Wnt receptors, DKK-1 is able to effectively short-circuit the intracellular signaling pathways activated by β-catenin, which are normally responsible for regulating genes involved in melanogenesis. Yamaguchi et al. (cf. supra) also demonstrated that DKK-3, a molecule similar to DKK-1 but without any effect on Wnt receptors, may mediate the effects of DKK-1. In fact, the greater the amount of DKK-3 in the vicinity of this Wnt receptor, the weaker the interaction between DKK-1 and this receptor on melanogenesis. The work of Yamaguchi et al. (cf. supra) also suggests that identifying agents that affect the DKK1 / DKK3 ratio in cultures of normal human dermal fibroblasts of non-palmar origin would make it possible to control melanin production starting from normal human non-palmar melanocytes.

[0313] The aim of this study was to evaluate the effect of Moringa oleifera protein hydrolysates on the synthesis and release of DKK-1 in a model consisting of normal human fibroblasts in monolayer culture.

[0314] Protocol: Human fibroblasts were obtained from a 68-year-old donor. For the experiments, fibroblasts were cultured as monolayers until confluent. 100 nM dexamethasone was used as a reference inducer of DKK-1 synthesis and release. Skin discs were incubated for 48 hours in the absence (control) or presence of a reference product or test product: "Moringa oleifera protein hydrolysate": 0.01%, 0.1%, and 0.5% (v / v).

[0315] At the end of the incubation period, the incubation medium was removed to proceed with the DKK-1 release method.

[0316] The "Moringa oleifera protein hydrolysate" test compound was directly dissolved in the incubation medium to obtain the various concentrations mentioned above.

[0317] At the end of the 48 h incubation period, DKK-1 released into the incubation medium was quantified with the aid of a sensitive and specific ELISA kit.

[0318] At the end of the incubation period, the proteins contained in the cell lysates were quantified with the aid of a spectrocolorimetric method (Bradford method).

[0319] Results are expressed as ng DKK-1 per mg protein (mean ± SD).

[0320] The significance level between the “control” and “reference product” was assessed with the help of Student's test (p<0.05).

[0321] The significance level (p<0.05) between "Control" and "Test Product" was evaluated by one-way analysis of variance (One-way ANOVA) followed by the Holm-Sidak test.

[0322] Under our experimental conditions, the reference product annotated "Dexamethasone" tested at 100 nm significantly increased released DKK-1 by 181.8% relative to "Control" (p<0.01).The results of the modulation of the DKK1 assay are given below.

[0323] [Table 21]

[0324]

[0325]

[0326] The studies showed that the hydrolysate according to the invention significantly increased the levels of DKK1 at a dose of 0.05%, by 26.1% relative to the basal level, and at a concentration of 0.5% of the extract according to the invention, an increase of 131.5% of the basal level was observed.

[0327] The purpose of this study was to evaluate the effects of Moringa oleifera protein hydrolysate on the synthesis and release of DKK-3 in a model composed of normal human fibroblasts in monolayer culture. Human fibroblasts were obtained from a 68-year-old donor. For the experiments, the fibroblasts were cultured in monolayers until confluence. Human fibroblasts were obtained from a 68-year-old donor. For the experiments, the fibroblasts were cultured in monolayers until confluence.

[0328] At the end of the 48-hour incubation period, DKK-3 released into the incubation medium was quantified by a sensitive and specific ELISA kit.

[0329] At the end of the incubation period, the proteins contained in the cell lysates were quantified by means of a spectrocolorimetric method (Bradford method).

[0330] The results are expressed as ng DKK-3 per mg protein (mean ± SD).The significance level between "Control" and "Reference Product" was assessed with the aid of Student's t-test (*: p<0.05).

[0331] The significance level between the "control" and the Moringa oleifera protein hydrolysate was evaluated by one-way analysis of variance (One-way ANOVA) followed by the Holm-Sidak test (*: p<0.05). The results of the DKK3 regulation assay are given below.

[0332] [Table 22]

[0333]

[0334] Conclusion: The Moringa oleifera protein hydrolysate according to the present invention showed a significant inhibition of DKK3 by approximately 21% at 0.5% compared to the basal level. The Moringa oleifera protein hydrolysate according to the present invention has a great ability to regulate genes involved in cell differentiation due to the palmoplantar inhibition principle (β-catenin signaling pathway), and through its ability to significantly increase DKK1 and significantly decrease DKK3, it increased the DKK1 / DKK3 ratio.

[0335] Complementary studies evaluating the production of DKK1 by P1, P2, P3 and the complex P1+P2 described in Example 10: Cellular test model of normal human fibroblasts.

[0336] [Table 23]

[0337]

[0338] * Statistically significant (p>0.05)

[0339] **: Statistically significant (p<0.01)

[0340] The production of DKK1 was significantly increased by 26.6% at a dose of 0.3% by extract P1 and by 27.8% at a dose of 1%.

[0341] [Table 24]

[0342]

[0343] * Statistically significant (p>0.05)

[0344] Extract P2 did not significantly increase DKK1 production at any dose tested.

[0345] [Table 25]

[0346]

[0347] * Statistically significant (p>0.05)

[0348] Extract P3 did not significantly increase DKK1 production at any dose tested.

[0349] [Table 26]

[0350]

[0351] * Statistically significant (p>0.05)

[0352] **: Statistically significant (p<0.01)

[0353] The combination of extracts P1 and P2 significantly increased DKK1 production by more than 39% at a dose of 1% (i.e., 0.5% of each extract). This score was higher than that obtained with either extract P1 or P2 alone; therefore, this means that the combination of the two extracts P1 and P2 has a synergistic effect.

[0354] Conclusion: The compounds designated "Extract P1" and "Extract P1+P2" significantly increased the release of DKK-1 into the culture medium from normal human fibroblasts in monolayer culture. No synergistic effect was observed when P1 and P2 were mixed.

[0355] The above-mentioned activities of the protein hydrolysate of the present invention lead to the following conclusions:

[0356] The most notable activity of Moringa oleifera protein hydrolysate demonstrated in cellulose testing is its epigenetic effect, which significantly slows the process of telomere shortening after cell division. The hydrolysate is a cytoprotective agent, particularly for stem cells, making it an excellent regenerator of cells and tissues. These properties provide exceptional protection for DNA and its genetic material. Moringa oleifera protein hydrolysate is also a powerful regulator of ZAG and endothelin type 1 production, exhibiting anti-fibrotic and anti-inflammatory effects.

[0357] Example 9: Evaluation of the effect of protein hydrolysate from defatted Moringa oleifera cake on angiotensin-converting enzyme 2 (ACE2) - Cell-free study of the effects of inhibitors.

[0358] Angiotensin-converting enzyme 2 is specifically involved in the intracellular infection of COVID-19 following activation of the spike protein by other converting enzymes, more specifically 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).

[0359] [Table 27]

[0360]

[0361] Conclusion: The hydrolyzed extract of Moringa oleifera cake is not significantly involved in the direct inhibition of angiotensin-converting enzyme 2 (ACE2). By considering Table

[32] of Example 11 below, the amplitude of the action of the extract on another converting enzyme, namely furin, was evaluated. The inhibitory effect of the extract according to the invention on furin has been clearly demonstrated and established in Table

[32] of Example 11. This specific inhibition demonstrated on a single converting enzyme reinforces the importance of the protein hydrolyzate according to the invention as a specific inhibitor of furin convertase.

[0362] Example 10: Evaluation of the hydrolyzed extract by preventing the penetration of "Spike SARS-CoV-2 pseudo-coronavirus" Inhibition of the anti-infective effect of furin in cellulose: Inhibition of the anti-infective effect of furin in cellulose in human HEK cells in the presence of SARS-CoV-2 pseudotyped lentivirus cellulose model.

[0363] Two main components were used in this review. On the one hand, stable recombinant cloned HEK293 cells (Genbank #NM_021804.3) constitutively expressing full-length human ACE2, with surface expression of ACE2 confirmed by flow cytometry. On the other hand, spike SARS-CoV-2 pseudotyped lentiviruses were generated using spike SARS-CoV-2 (Genbank accession number QHD43416.1) as the envelope glycoprotein instead of the traditionally used VSV-G. These pseudovirions also contain the firefly luciferase gene driven by the CMV promoter; therefore, spike-mediated cell entry can be measured in a practical manner through the activity of the luciferase reporter. Spike SARS-CoV-2 pseudotyped lentiviruses can be used for screening applications in biosafety level 2 facilities.

[0364] The starting materials used were as follows:

[0365] [Table 28]

[0366]

[0367] Step 1: Plate ACE2-HEK cells

[0368] ACE2-HEK cells were thawed in Thawing Medium 1, expanded in 1N Growth Medium, and then harvested and plated into a white, clear, flat-bottomed 96-well culture plate at 10,000 cells / well in 50 μL of Thawing Medium 1. The cells were incubated overnight at 37°C.

[0369] Step 2: Pseudotyped lentiviral infection assay

[0370] The next day, the homogeneity and integrity of the cell layer were verified by visual monitoring using an inverted microscope, and the following test components were prepared as described below:

[0371] [Table 29]

[0372]

[0373]

[0374] In the first step, the Moringa oleifera protein hydrolysate was diluted 11X to a medium concentration in thawing medium 1, and then 5 μL was transferred to the test plate and co-incubated with ACE2-HEK cells. After incubation at 37°C for 30 minutes, 5 μL of undiluted pseudotyped lentivirus (naked or S1-spike) was added to the corresponding wells for analysis.

[0375] A mAb that blocks ACE2 was used as a positive control at a final concentration of 0.5 uM in the wells for analysis.

[0376] Step 3:

[0377] After 48 hours of incubation, a volume of 50 μL of luciferase reagent was added to the wells for analysis, and the luminescence signal was measured using a PolarStar Omega luminometer.

[0378] Figure 1 The results given in Figure 3 are the anti-infective effects of the Moringa oleifera protein hydrolysate according to the present invention against the pseudovirus of SARS COV2.

[0379] Conclusion: Moringa oleifera protein hydrolysate showed convincing anti-infective effect against SARS-CoV-2 pseudovirus starting from 3% (C1) dose in the research model. A significant inhibition of 60% was demonstrated at concentration C1 (3%).

[0380] Evaluation of bands P1, P2, P3 and the complex P1+P2 identified in Example 12 on furin convertase: cell-free test model.

[0381] Investigations of the identified bands did not demonstrate any inhibitory effect on furin convertase at all concentrations tested with P1, P2, P3 and P1+P2.

[0382] Conclusion: Therefore, none of these protein bands showed inhibitory activity against furin convertase. This suggests that the inhibitory activity against furin convertase is due to the entirety of the protein hydrolysate obtained by the described method.

[0383] The protein hydrolysate inhibits the SARS-CoV-2 spike protein in its entirety, particularly by inactivating the furin convertase. These inhibitions provide anti-infective and antiviral properties.

[0384] Example 11: Comparative study of the inhibition of furin convertase by different preparations obtained from Moringa oleifera seeds

[0385] The aim of the present study was to evaluate the inhibitory effect on furin activity of Moringa oleifera oil obtained first by cold pressing of the shelled seeds, then of an extract of Moringa oleifera with ethanol (96%) consisting of approximately 1.1% of dry matter, which itself consists of approximately 55% by weight of 2,5-diformylfuran, 2.5% of furfural, 1.2% of isopropyl myristate, 4.7% of palmitic acid, 11.1% of oleic acid and 25.8% of triglycerides, and finally of a hydrolyzate of Moringa oleifera proteins according to the invention.

[0386] Protocol: Moringa oleifera extract and Moringa oleifera protein hydrolysate in ethanol (96%) were stored protected from light at +4°C until use. Moringa oleifera oil was stored in a dark area at ambient temperature.

[0387] Reference product: 100 nM Decanoyl-Arg-Val-Lys-Arg-CMK was used as a reference inhibitor for furin activity.

[0388] Incubation protocol: Furlin was pre-incubated for 10 minutes at ambient temperature in the absence (control) or presence of reference product or increasing concentrations of test compound:

[0389] - 0.3%, 1% and 3% (V / V) Moringa oleifera oil.

[0390] - 0.02%, 0.2% and 2% (V / V) of Moringa oleifera extract in ethanol (96%).

[0391] - 0.02%, 0.2% and 2% (v / v) of Moringa oleifera protein hydrolysate according to the present invention. At the end of the pre-incubation step, the furin substrate was added and the experimental conditions were incubated again at ambient temperature in the dark for 5 minutes. All experiments were performed in triplicate.

[0392] Compound Preparation: Moringa oleifera extract and Moringa oleifera protein hydrolysate in 96% ethanol were dissolved directly in assay buffer and then diluted to achieve the test concentrations as described above. Moringa oleifera oil was dissolved in 0.05% Tween 20 solution in assay buffer to 3%. The solution was then diluted to achieve the above concentrations.

[0393] Evaluation protocol: After addition of substrate, the cleavage of the fluorescent furin substrate was monitored for 5 minutes by reading the fluorescence at 485 nm / 535 nm. g. Statistics: The results are expressed as RFU (Relative Fluorescence Units) + / - SD (Standard Deviation). The statistical significance of the differences observed between the "Control" and "Reference Product" groups was evaluated by Student's test (p < 0.001). The statistical significance of the differences observed between the "Control" or "Reference Product" and "Test Compound" groups was evaluated by one-way ANOVA followed by the Holm-Sidak test (p < 0.05). The results for Moringa oleifera oil are shown in Table 1. Figure 2 shown; in Figure 3 The results of 96° ethanol Moringa oleifera extract and the final Figure 4 Results are given for the Moringa oleifera protein hydrolysate according to the present invention.

[0394] result:

[0395] [Table 30]

[0396] Concentration of Moringa arabicum oil Furin convertase activity 0.3% (V:V) 91.2％** 1% (V:V) 90.6％** 3% (V:V) 92.8％**

[0397] [Table 31]

[0398]

[0399] [Table 32]

[0400]

[0401] * Statistically significant (p>0.05)

[0402] **: Statistically significant (p<0.01)

[0403] ***: Statistically significant (p<0.001)

[0404] Conclusion: This study allowed us to demonstrate that only the Moringa oleifera protein hydrolysate according to the invention was able to significantly inhibit the activity of furin by 98.8% at a concentration of 2%.

[0405] Example 12: Separation of Moringa oleifera protein hydrolysate-protein bands by electrophoresis gel chromatography

[0406] Gel electrophoresis performed on polyacrylamide containing sodium dodecyl sulfate is called SDS-PAGE. It is a technique that involves saturating the polyacrylamide gel with negative charges under the influence of an electric field with SDS, causing denatured proteins to migrate, thus enabling their separation. This is a denaturing technique that uses a negatively charged ionic detergent (SDS) to dissociate non-covalent protein complexes. This detergent binds non-selectively to two amino acids via hydrophobic bonds. Therefore, this technique can be used to analyze proteins and separate them according to their molecular weight.

[0407] Separation chromatography by electrophoresis:

[0408] Deposition:

[0409] -MW = size mark

[0410] - Well 1: Hydrolyzed Moringa oleifera cake extract

[0411] - Well 2: Supernatant of hydrolyzed Moringa oleifera cake extract

[0412] - Well 3: Precipitation of hydrolyzed Moringa oleifera cake extract

[0413] The hydrolyzed extract from well 1 clearly showed all the expected protein bands.

[0414] Well 2 (supernatant) showed the expected bands, particularly for the middle and highest molecular weights; well 2 appeared to be concentrated in the lowest molecular weights, particularly with volatiles.

[0415] Well 3 (precipitate) clearly shows the highest molecular weight band (>75,000 Da), followed by a band of approximately 23,000 Da called P3, then a band called P2 comprised between 10,000 and 17,000 Da, and finally a band called P1 of less than 10,000 Da, estimated to be between 4,000 and 6,000 Da.

[0416] Rotary evaporator preparation:

[0417] - Volatiles are logically in the supernatant along with the "lightest" compounds; recover 10 mL of supernatant and add 10 mL of 96° ethanol;

[0418] The mixture was extracted under vacuum at 45°C; the volatile compounds were condensed in a vacuum flask.

[0419] - The condensate concentrated in volatiles was subjected to GC / FID after SPME microextraction.

[0420] Gas chromatography studies following SPME extraction and FID detection

[0421] No volatile compounds were detected.

[0422] Conclusion: The separation of the protein bands indicates that the volatile compounds are not bound to proteins; the volatile compounds do not participate in the molecular cavalcade of the smallest protein band.

[0423] Three protein bands were determined: bands P1 (less than 10,000 Da), P2 (between 10,000 and 17,000 Da), and P3 (approximately 23,000 Da) were prepared and analyzed by liquid chromatography coupled to mass spectrometry (LC MS / MS).

[0424] The results were obtained using two specialized software programs for protein identification in plants (Mascot and Peaks):

[0425] The results of the identification did not allow the precise identification of these proteins; we were faced with proteins that had not yet been described in the above-mentioned databases.

[0426] Example 13: Formulation of Dermatological Anti-Fibrotic Product (Liquid Cleanser)

[0427] [Table 31]

[0428]

[0429] Example 14: Formulation of a dermatological product for treating localized fibrosis (leave-on care product)

[0430] [Table 32]

[0431]

[0432]

[0433] Example 15: Formulation of a subcutaneously injectable composition

[0434] Formulation of the product for subcutaneous administration: The dry extract according to the invention (containing 60% of protein hydrolysate on an inulin carrier) is packaged in single-dose flasks and prepared for dissolution with a physiological medium under inert gas.

[0435] Example 16: Formulation of a subcutaneously injectable composition

[0436] Preparation of the injectable liquid product: The liquid extract according to the invention is packaged at a dose of 5% in a physiological medium under sterile conditions, in particular by vacuum filtration with a cut-off threshold of 0.45 μm.

[0437] Example 17: Preparation in the form of a patch (A dressing or external medical device attached to the skin, impregnated with an active ingredient, which is slowly released to provide a slow diffusion effect of the active ingredient).

[0438] Example 18: Formulation for drug in capsule

[0439] Antifibrotic drug in 750 mg capsules containing 100 g of piperine, 300 mg of a dry extract according to the invention (containing 60% protein hydrolysate on an inulin carrier), 100 mg of boswellic acid, and 250 mg of calcium carbonate).

[0440] Example 19: Formulation of a medicament for use as a tablet

[0441] Antifibrotic drug in a 1 g tablet: 300 mg of a dry extract according to the invention (containing 60% protein hydrolysate on an inulin carrier), 400 mg of calcium carbonate containing 200 IU vitamin D, 150 mg of magnesium gluconate, 80 mg of inulin and 70 mg of magnesium stearate.

[0442] Example 20: Formulation of a drug for use as a nasal spray (a solution containing an active ingredient in a medical device that propels the active ingredient by spraying the active solution into the nasal cavity).

[0443] Example 21: Toxicity test of the protein hydrolysate according to the present invention

[0444] Preparation of a protein hydrolysate according to Example 1: Unshelled seeds of Moringa oleifera (Forssk.) are dried at maturity to an internal moisture content of less than 8%, preferably approximately 6%, and then pressed using a headless screw mechanical press to separate the oil from the rest of the seed, yielding, on the one hand, virgin oil and, on the other, a cake. The cake is then separated into extrudates in the form of 1 to 2 cm pieces. Following the protocol described in Example 1, a liquid extract is obtained, which is used undiluted in the following tests.

[0445] 1 . Determine the mutagenicity against the bacterial strain Salmonella typhimurium (TA 100) Activity – Reverse bacterial mutation test

[0446] Testing is conducted in 3 main phases:

[0447] - Conduct preliminary experiments to evaluate the cytotoxicity of the test element and select the dose range for subsequent experiments,

[0448] - a first genotoxicity study (Test 1) with and without metabolic activation, with direct incorporation of the test system and the test (or control) on a minimal gel within the dose range defined in the preliminary study,

[0449] - A second experiment (Test 2) in which the test system and the test element (or control) are pre-incubated with and without metabolic activation at dosage levels defined by the study leader after analyzing the results of the first experiment. This second experiment is performed to confirm or supplement the results of the first experiment, especially when equivalent or negative results are obtained.

[0450] Dilutions of the extract according to Example 1 were prepared in water for cytotoxicity testing

[0451] Cytotoxicity tests were performed on Salmonella typhimurium TA100 strain at concentrations of 5000, 1600, 500, 160 and 50 μg / plate, with and without S9-Mix.

[0452] Prepare the reagents for making S9-Mix according to the following instructions:

[0453] [Table 33]

[0454]

[0455]

[0456] Bacteria were exposed to the test extracts of the present invention with and without a metabolic activation system. The metabolic system used was a post-mitochondrial fraction modified with a cofactor (S9). The S9 fraction was a microsomal fraction of Sprague Dawley rat liver homogenate treated with an enzyme inducer and was prepared according to MARON The test was prepared by DM et al., 1983, Revised Methods for the Salmonella Mutagenicity Test, Mutation Research / Environmental Mutagenesis and Related Subjects, 113, pp. 173-215, and supplied by MOLTOX™. It was stored at a temperature below -70°C. The microsomal fraction S9 was used at a concentration of 10% in S9-Mix. The administration protocol was as follows:

[0457] Introduce the following into the 3 vasolytic tubes:

[0458] o Assays without metabolic activation:

[0459] -0.1mL of test elements at various concentrations,

[0460] -0.5 mL of sterile phosphate buffer, 0.2 M, pH 7.4,

[0461] -2 mL of top agar for S. typhimurium,

[0462] - 0.1 mL of bacterial inoculum (TA100).

[0463] o Assays with metabolic activation:

[0464] -0.1mL of test elements at various concentrations,

[0465] -2 mL of top agar for S. typhimurium,

[0466] - 0.1 mL of bacterial inoculum (TA100),

[0467] -0.5 mL of S9-Mix.

[0468] Mix and pour onto the bottom agar surface already placed in the petri dish.

[0469] Incubate at 37°C ± 2°C for 48 to 72 hours.

[0470] These determinations are performed for each test: preliminary cytotoxicity test, test 1 and test 2. Untreated controls, negative controls generated during the pre-incubation process and positive controls are incubated at 37°C ± 2°C for 20 to 30 minutes before pouring the top agar.

[0471] The regimen for administration is as follows:

[0472] For S. typhimurium, introduce the following into four 2 mL portions of top agar:

[0473] ο0.1 mL of phosphate buffer, 0.2 M, pH 7.4,

[0474] ο0.1 mL of solvent,

[0475] o 0.1 mL of S9-Mix.

[0476] o 0.1 mL of the test element preparation at the highest concentration,

[0477] • Use 2 mL of the top agar fraction to control the sterility of Salmonella typhimurium.

[0478] Mix and pour onto the bottom agar surface already placed in the petri dish.

[0479] Incubate at 37°C ± 2°C for 48 to 72 hours.

[0480] Tests were performed in triplicate:

[0481] No bacterial growth was observed.

[0482] At least five concentrations of the test extracts were tested without and with metabolic activation.

[0483] Presentation and interpretation of results

[0484] A number of criteria can be used to determine whether a result is positive, particularly an increase in the number of revertants that is related to the dose of the test item, or a reproducible increase in the number of revertants at one or more concentrations, with or without metabolic activation.

[0485] - The test element is considered mutagenic if, at the end of the validation step, a dose-effect relationship is obtained in a reproducible manner on one or more of the five strains with and / or without metabolic activation. A given concentration is considered mutagenic only if the number of revertants is at least equal to twice the spontaneous reversion rate (R≥2) for strains TA98, TA100 and TA102 and at least equal to three times the spontaneous reversion rate (R≥3) for strains TA1535 and TA1537.

[0486] - The test element is considered non-mutagenic if, at the end of Tests 1 and 2, the number of revertants is still less than twice the spontaneous reversion rate for all concentrations tested (R<2) for strains TA98, TA100 and TA102 and less than three times the spontaneous reversion rate for strains TA1535 and TA1537 (R<3) with and without metabolic activation and provided that the mutagenicity has been demonstrated to be related to the toxicity of the tested concentrations.

[0487] Preliminary studies did not demonstrate any cytotoxicity of the test elements; therefore, this concentration range was used for genotoxicity testing1.

[0488] Due to the results obtained in Test 1, it was decided to use the same dilution range for Test 2. Analysis of the revertants showed:

[0489] - No cytotoxic effects were observed,

[0490] - no concentration of the tested extract showed a ratio R greater than or equal to at least twice the spontaneous reversion rate for TA98, TA100 and TA102, or greater than or equal to three times the spontaneous reversion rate for TA1535 and TA1537, both with and without metabolic activation,

[0491] - No dose response was observed regardless of the test system or test conditions.

[0492] In view of the results obtained during the present study, the protein hydrolysate according to Example 1 can be considered to have neither mutagenic nor pro-mutagenic activity.

[0493] 2. In vitro 3T3 NRU phototoxicity test

[0494] The test principle is to compare the cytotoxicity of the protein hydrolysate according to Example 1 on cells in culture in the presence and absence of non-cytotoxic doses of UVA. Cytotoxicity was assessed 24 hours after treatment with a reference element and the Moringa oleifera protein hydrolysate according to the present invention, with or without UVA irradiation, using the vital dye, neutral red, to assist in determining cell viability. The cells used were Balb / c 3T3 clone 31 mouse embryonic fibroblasts (ATCC CCL163). A positive control was a chlorpromazine solution (CAS No. 69-09-0). Negative controls were dilutions of the test and reference extracts (buffered saline + / - 1% solvent). The Moringa oleifera protein hydrolysate was tested in the presence or absence of UVA at eight concentrations in at least four wells per concentration studied. The fibroblasts were trypsinized and seeded in two 96-well plates with 2×10 cells in 100 μL of complete culture medium. 5 cells / mL (i.e. 2x10 6 cells / well) of the cell suspension.

[0495] The incubated plates were incubated in an oven at 37°C, 5% CO2 for 24 hours. At the end of the incubation period, the cell mats were checked for semi-confluence. Dilutions were prepared before depositing them on the cells. The pH at the highest concentration was measured; it was between 6.5 and 7.8. The culture medium was removed; each well was carefully rinsed with 150 μL of PBS maintained at ambient temperature and then treated with 100 μL of each extract dilution or reference dilution. The culture plates were incubated in the dark at 37°C and 5% CO2 for 1 hour ± 5 minutes. Irradiation was carried out with the help of a BIO SUN solar irradiation system (Vilber Lourmat RMX3W). BIO SUN is a system that controls UV irradiation with the help of a programmable microprocessor. The system continuously monitors the emission of UV light. Irradiation automatically stops when the energy delivered is equal to the programmed energy. The spectral irradiance of the test device was measured using a calibrated spectroradiometer in the wavelength range of 250 to 700 nanometers.

[0496] One of the two plates was irradiated at ambient temperature with its cover, and the other plate was protected from UVA and maintained at ambient temperature during irradiation. After irradiation, the treatment medium was extracted and the cells were rinsed. Next, 100 μL of complete culture medium was carefully added and the plate was incubated at 37°C and 5% CO2 for 18 to 22 hours. The next day, cell viability (growth, morphology, monolayer integrity) was evaluated by observing with a phase contrast microscope. The culture medium was removed, and each well was rinsed and maintained at ambient temperature before being treated with 100 μL of staining solution. Under the same conditions, the plate was returned to the incubator for 3 hours. The staining 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 plate was shaken until the crystals were completely dissolved. The absorbance value was measured at 450 nm.

[0497] Test verification:

[0498] The sensitivity of the cells to UVA was monitored over approximately 10 generations by assessing the viability of the cells after exposure to increasing doses of irradiation. The cells were cultured at the density used in the test. The next day, the cells were exposed to increasing doses of irradiation at 2.5 to 9 J / cm 2 They were irradiated at a dose of 5 J / cm and cell viability was determined one day later by means of the NRU test. 2 The cells met the quality criteria if their viability after UVA irradiation was greater than 80% of that of the reference kept in the dark; at the highest dose of 9 J / cm 2 Under UVA, the viability must be at least 50% of the viability of the reference kept in the dark.

[0499] result:

[0500] The negative control has an absorbance of 0.4 or higher. The positive control, chlorpromazine, has a CI of 50 The values ​​were between 0.1 and 2 μg / ml, and between 7 and 90 μg / ml in the absence of UVA. These results validate the test. It was not possible to estimate the concentration of the Moringa oleifera protein hydrolysate according to Example 1 that would provide 50% cell death in the presence or absence of UVA. The mortality rate never reached 50%. It was not possible to estimate the concentration of the Moringa oleifera protein hydrolysate that would produce 50% cell viability in the presence or absence of UVA. Viability was always greater than 50%.

[0501] Conclusion: Under the experimental conditions used, the Moringa oleifera protein hydrolysate according to the present invention can be considered to be non-phototoxic.

[0502] 3 . Ocular irritation was evaluated by in vitro cytotoxicity studies based on the neutral red leaching method on the SIRC cell line. Irritability

[0503] This in vitro study was based on the determination of 50% cell death (IC 50 The cytotoxicity of Moringa oleifera protein hydrolysate was evaluated at different concentrations. The cells used were SIRC, rabbit corneal fibroblasts (ATCC-CCL60), which are mycoplasma-free.

[0504] Moringa oleifera protein hydrolysate was diluted to 25% and 50% with physiological serum. Fibroblasts were trypsinized and seeded in two 24-well culture plates at a volume of 2×10 cells / mL in complete culture medium. 5The cells were suspended in a 0.5 mg / mL cell suspension. The incubated plates were incubated in an oven at 37°C and 5% CO2. At the end of the incubation period, the cell mat was checked for confluence. A staining solution was prepared at a concentration of 0.5 mg / mL in complete culture medium. The culture medium was removed; 1 mL of the staining solution was deposited in each well. The plates were returned to an incubator at 37°C and 5% CO2 for 3 h ± 15 min. After this contact time, the staining solution was removed and replaced with 1 mL of complete culture medium per well. The plates were kept at ambient 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, kept at ambient temperature, and then 500 μL of each dilution of the Moringa oleifera protein hydrolysate or reference was deposited in contact with the cell mat. The contact time was 60 seconds (30 seconds for the positive control). Treatment was performed well by well, with the timer starting at the moment the Moringa oleifera protein hydrolysate or reference was deposited. The plates were manually shaken throughout the treatment period. After 55 seconds (or 25 seconds for the positive control), extract the diluent. At exactly 60 or 30 seconds, rinse 5 times in sequence (5x2mL PBS, maintained at ambient temperature). Extract the supernatant after each rinse, and after the last rinse, when waiting for the display stage, keep that there is no medium in the hole. After completing the processing of the culture plate, deposit 1mL of staining solution in each hole. The plate was shaken for about 15 minutes, until uniform staining was obtained. The solution obtained in each culture well was taken out and divided into 2 holes of a 96-well plate, i.e. 150 μL / hole.

[0505] result:

[0506] The concentration of Moringa oleifera protein hydrolysate that provided 50% cell death was estimated as >50%.The percentage of cell death at 50% for the Moringa oleifera protein hydrolysate product was estimated to be 17%.

[0507] Conclusion: Under the experimental conditions used, the Moringa oleifera protein hydrolysate according to the present invention can be classified as having negligible cytotoxicity.

[0508] 4 . The Arabidopsis thaliana according to the invention was evaluated 48 hours after a single application under bandage therapy under dermatological control. Skin compatibility of Moringa protein hydrolysate

[0509] The purpose of this study was to evaluate the degree of skin compatibility of the protein hydrolysate of Moringa oleifera by means of an epicutaneous test over 48 hours on the anterior outer surface of the arm; and to generally evaluate the ability of the protein hydrolysate of Moringa oleifera to maintain the skin in good condition. Ten healthy female or male volunteers aged 18 to 65 years, without dry skin or sensitive skin and without any dermatological lesions in the treatment area, were included in the study. The skin compatibility of the protein hydrolysate of Moringa oleifera was evaluated 48 hours after initial application for 30 to 40 minutes after removal of the dressing. The protein hydrolysate of Moringa oleifera was prepared in the form of a lotion having 5% of the protein hydrolysate of Moringa oleifera according to Example 1 and 95% of a propylene glycol / sorbitol mixture. Skin reactions (erythema and edema) were scored from 0 to 3 according to the following scale:

[0510] [Table 34]

[0511]

[0512] All skin reactions (blisters, papules, vesicles, dryness, desquamation, roughness, soap effect, etc.) were evaluated and reported descriptively according to the following scale:

[0513] -0: No reaction,

[0514] -0.5: Very mild

[0515] -1: Mild

[0516] -2: Moderate

[0517] -3: Severe.

[0518] At the end of the study, the mean irritation index (M.I.I.) was calculated using the following formula:

[0519] [Mathematical formula 4]

[0520] M.I.I. = sum of skin reactions (E + Oe + blisters + papules + vesicles) / number of volunteers analyzed

[0521] The M.I.I. obtained enabled the protein hydrolysate of Moringa oleifera tested to be classified using the following scale:

[0522] M.I.I. ≤ 0.20 Non-irritant

[0523] 0.20 < M.I.I. ≤ 0.50 Mildly irritant

[0524] 0.50 < M.I.I. ≤ 2 Moderately irritant

[0525] 2 < M.I.I. ≤ 3 Highly irritant

[0526] Results: The mean stimulation index (MII) of Moringa oleifera protein hydrolysate was equal to: 0.

[0527] Conclusion: Moringa oleifera protein hydrolysate could be considered non-irritating after 48 hours of continuous administration to 12 volunteers.

[0528] General conclusions of the test:

[0529] The results of the tests performed above are conclusive for the Moringa oleifera protein hydrolysate according to Example 1 and demonstrate that:

[0530] 1) Eye and skin irritation tests were negative

[0531] 2) Phototoxicity test is negative

[0532] 3) Mutagenicity test was negative.

[0533] The safety of the Moringa oleifera protein hydrolysate according to the present invention has been demonstrated and it is ideal for large-scale dermatological use without restriction of the target group.

Claims

1. A method for obtaining a protein hydrolysate from Moringa oleifera seed cake, wherein the protein hydrolysate comprises amino acid derivatives, amino acids, peptides and glycopeptides having a molecular weight between 100 Da and 25,000 Da, characterized in that: It comprises the following steps, wherein: a) collecting unshelled mature seeds from ripe Moringa oleifera fruits and drying them to an internal moisture content of less than 8%, b) pressing the dried seeds in such a way that the oil is separated from the rest of the seeds, in such a way that a cake comprising less than 6% by weight of residual oil is obtained, c) grinding the cake obtained in step b), d) dispersing the milled cake obtained in step c) in an alkaline solution in a ratio of 9.1 / 90.9 (mass / mass) between the milled cake and the alkaline solution, e) subjecting the aqueous dispersion obtained in step d) to a chemical proteolysis at a pH greater than 13 and at a temperature comprised between 16° C. and 25° C. for a period of 2 hours, f) neutralizing the protein hydrolysis to stabilize the obtained protein hydrolysate, g) recovering the protein hydrolysate by solid / liquid separation, h) purifying the protein hydrolysate by ultrafiltration and / or nanofiltration with a cut-off threshold comprised between 100 and 25,000 Da.

2. The method for obtaining protein hydrolysate from Moringa arabica seed cake according to claim 1, wherein It comprises the further steps of: i) freeze-drying the protein hydrolysate obtained in step h).

3. The method according to claim 1, characterized in that The nanofiltration was performed in such a way that three bands were separated from the protein hydrolysate, including a band P1 having a molecular weight of less than 10,000 Da, a band P2 having a molecular weight between 10,000 and 17,000 Da, and a band P3 having a molecular weight of 23,000 Da.

4. The method according to claim 1, wherein Said nanofiltration step h) is performed with a cut-off threshold comprised between 1,500 Da and 5,000 Da.

5. The method according to claim 1, wherein Said nanofiltration step h) is performed with a cut-off threshold comprised between 10,000 Da and 17,000 Da.

6. The method according to claim 1, wherein Said nanofiltration step h) is performed with a cut-off threshold comprised between 17,000 Da and 25,000 Da.

7. A protein hydrolysate from unshelled seed cake harvested from ripe Moringa oleifera fruits, characterized in that It comprises a main fraction P1 of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight comprised between 1,500 Da and 5,000 Da, a 20% (mass / mass) fraction P2 with a molecular weight comprised between 10,000 and 17,000 Da, a 20% (mass / mass) fraction P3 with a molecular weight of 23,000 Da, wherein it is obtained by a nanofiltration step carried out according to the method of claim 1 with a retention threshold comprised between 1,500 Da and 5,000 Da, between 10,000 Da and 17,000 Da and between 17,000 Da and 25,000 Da, and wherein it is liquid and has a density greater than 1.

8. The protein hydrolysate according to claim 7, characterized in that It comprises a dry matter content of 12.5% ​​(mass / mass), including between 1 and 6% nitrogen-containing compounds, and 20 mg / litre of polyphenols.

9. A protein hydrolysate from unshelled seed cake harvested from ripe Moringa oleifera fruits, characterized in that It comprises a main portion P1 of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight comprised between 1,500 Da and 5,000 Da, wherein it is obtained by nanofiltration performed by the method according to claim 1 with a cut-off threshold comprised between 1,500 Da and 5,000 Da, and wherein it is liquid and has a density greater than 1.

10. A protein hydrolysate seed cake harvested from ripe Moringa oleifera fruits and not shelled, characterized in that It comprises a P2 fraction of 20% (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides with a molecular weight comprised between 10,000 Da and 17,000 Da, wherein it is obtained by nanofiltration according to the method of claim 1 with a cut-off threshold comprised between 10,000 Da and 17,000 Da, and wherein it is liquid and has a density greater than 1.

11. A protein hydrolysate seed cake harvested from ripe Moringa oleifera fruits and not shelled, characterized in that It comprises a P3 fraction of 20% (mass / mass) of amino acid derivatives, amino acids, peptides and glycopeptides having a molecular weight of 23,000 Da, wherein it is obtained by nanofiltration using a method according to claim 1 with a cut-off threshold comprised between 17,000 Da and 25,000 Da, and wherein it is liquid and has a density greater than 1.

12. A protein hydrolysate seed cake harvested from ripe Moringa oleifera fruits and not shelled, characterized in that The invention comprises a combination of the protein hydrolysate according to claim 9 and the protein hydrolysate according to claim 10.

13. A pharmaceutical composition or dermatological composition, characterized in that It comprises an effective amount of a protein hydrolyzate of Moringa oleifera seed cake according to any one of claims 7 to 12 as an active agent, and a physiologically acceptable excipient, wherein the protein hydrolyzate of Moringa oleifera seed cake is present in the composition at a concentration of 0.0001 to 40% by weight relative to the total weight of the composition.

14. The pharmaceutical composition according to claim 13, characterized in that It is formulated for ingestion.

15. The dermatological composition according to claim 13, characterized in that It is formulated for topical administration to the skin or mucous membranes.

16. Use of the composition according to any one of claims 13 to 15 in the preparation of a medicament for treating skin fibrosis, characterized in that: The composition comprises an effective amount of the protein hydrolysate according to claim 9 or 10 as an active agent.

17. Use of the composition according to any one of claims 13 to 15 in the preparation of a medicament for treating viral infectious diseases to inhibit the spike COV2 protein of SARS-COV2, characterized in that The composition comprises an effective amount of the protein hydrolysate according to claim 7 or 8 as an active agent.

18. Use of the composition according to any one of claims 13 to 15 in the preparation of a medicament for treating skin fibrosis, characterized in that: The composition comprises an effective amount of the protein hydrolysate according to claim 12 as an active agent.

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