Patent Application
20240325477
The invention relates to the field of pharmacy and dermatology; and more particularly to that of active ingredients in the formulation of drugs. The invention pertains to a method for obtaining a protein hydrolysate from Moringa peregrina seed cake. The invention also pertains to the protein hydrolysate from Moringa peregrina seed cake, to pharmaceutical and dermatological compositions comprising a hydrolysate of this type and intended for use in the treatment of fibrotic diseases, in the treatment of inflammation, of cancer, of infectious bacterial or viral types of diseases, as well as for the treatment of genetic drift and pathologies associated with skin pigmentation.
Moringaceae constitute a monogeneric family (a single genus, Moringa Adans), part of the saharo-sindian flora, constituted by between twelve and fourteen species according to the authors, distributed across east Africa and Asia. The genus is conventionally divided into 3 sections which, however, have not been confirmed to be monophyletic by phylogenetic analyses. These latter have rather highlighted clades oriented towards certain morphological features: pachycauls (bottle trees), tuberous shrubs and trees that are neither pachycauls nor tuberous shrubs (slender trees). The species Moringa peregrina (Forssk.) Fiori belongs to the third group. The few genetic studies on the genus or the family confirm the reality of the species compared with other species in this genus, in particular compared with the Indian Moringa, Moringa oleifera Lam. (see in particular the articles: O
The species Moringa peregrina is found in rocky environments in Yemen, Oman and Saudi Arabia, in East Africa, in Sudan, in Ethiopia, Eritrea, Somalia and Djibouti. Its presence in Iran appears to be limited to south-eastern provinces, but needs to be confirmed (PROTA14=M
Historically, there have been a few mentions that tend to indicate that Moringa peregrina oil was actively traded at the inception of Islam in the Al-Ula region (N
A certain number of extracts deriving from the Moringa oleifera seeds are known that more particularly are used in the cosmetics field. In the dermatological field, it is known from the document FR 2 776 519 that protein extracts from Moringa oleifera seeds, known for their clarifying effects on turbid water, have an emollient, physiologically conditioning, hydrating, restructuring, repair and anti-polluting effect on the skin and mucous membranes. In that document, the active principles are proteins with molecular weights comprised between 6500 and 8800 Da, which are obtained by aqueous extraction from Moringa oleifera cake.
In the pharmaceutical field, a composition is known from KR 2014 0143655 that contains a hydrosoluble extract from Moringa oleifera leaves as the active ingredient in order to treat or prevent cancer. The method for enzymatic hydrolysis on the Moringa oleifera leaf extracts can be used to isolate proteins with molecular weights comprised between 6000 and 8000 Da.
The document CN 1 0701 21 90 concerns the use of Moringa oleifera seeds in order to obtain polypeptides from the seed proteins after having extracted the oil and after carrying out an enzymatic hydrolysis using microwave radiation. The product obtained is used orally and is useful for its anti-cancer activity. That product in powder form is in fact described as having an action to reduce the side effects of chemotherapy and to increase appetite in patients presenting with liver cancer or to control the leukocyte index and body temperature.
Finally, from the document IN2009CH02906, an aqueous extract of Moringa oleifera cake comprising glucosinolate combined with cationic proteins is known that can be used as an anti-diabetic product.
All of the documents cited above concern the use of the species Moringa oleifera; none of them describes the use of extracts from the species Moringa peregrina in the pharmaceutical or dermatological fields.
In addition, the document by A
An extract of Moringa peregrina oil produced from seed from Egypt using a (1/1) dichloromethane/methanol mixture exhibited an activity on three human cancer cell lines, MCF-7 (adenocarcinoma of the breast), Hep-G2 (hepatocellular carcinoma) and HCT-116 (colon carcinoma) with 1050 values of 2.92, 9.40 and 9.48 μg/mn (A
A
In view of the foregoing, a problem which the invention proposes to overcome is the development of novel products based on an extract of the species Moringa peregrina from the genus Moringa and the Moringaceae family that can be used in pharmacy or in dermatology and that are easy to use.
Thus, the Applicant has surprisingly developed a particular protein hydrolysate obtained from Moringa peregrina seed cake for its application as a drug, and in particular, for the treatment of fibrotic diseases (as an inhibitor of furin convertase), the treatment of inflammation, of cancer, of infectious diseases of the bacterial or viral type as well as the treatment of genetic drift and of pathologies associated with skin pigmentation.
The species Moringa peregrina grows in very arid climates. Thus, its ability to resist drought and to reproduce under extremophile conditions has enabled it to acquire particular unique characteristics that the Applicant has been able to identify by carrying out a specific extraction method on Moringa peregrina seed cake. The protein hydrolysate in accordance with the invention has been shown to have properties as a drug and it has been demonstrated that in particular, it has a very high furin convertase inhibiting activity.
Furin convertase (hereinafter termed furin) is a type 1 transmembrane protein with 794 amino acids expressed in different cell types. Furin is a protein which has been shown to be involved in a large number of biological processes (B
By means of an intergovernmental agreement between the government of the French Republic and the Kingdom of Saudi Arabia of Apr, 10 2018, the Applicant, Agence Française Pour Le Développement d'AlUla (AFALULA) [French Agency for AlUla] as well as the Commission Royale pour AlUIA (RCU) [Royal Commission for AlUla] instigated a joint project particularly for the development of responsible agriculture and local economy, in particular by the local production of natural products derived from indigenous plants and for the protection of the biodiversity and laws of the AlUla region of the Kingdom of Saudi Arabia. The Kingdom of Saudi Arabia has been a member of the Nagoya Protocol since Oct. 8, 2020. At the time of drafting the present patent, the rules by virtue of which the Nagoya Protocol will be integrated into the pertinent aspects of local law are under consideration. As a consequence, at this stage, the Kingdom of Saudi Arabia has no specific demands as regards this patent application and the Nagoya Protocol. Thus, as at the day of filing of the patent application, there is no requirement for a certificate of conformity regarding access to genetic resources.
In a first aspect, the invention provides a method for obtaining a protein hydrolysate from Moringa peregrina seed cake, comprising the following steps, in which:
In a second aspect, the invention concerns a protein hydrolysate from seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, comprising a major fraction of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight P1 is comprised between 1500 Da and 5000 Da, a fraction of approximately 20% for which the molecular weight P2 is comprised between 10000 and 17000 Da and a fraction of approximately 20% for which the molecular weight P3 is approximately 23000 Da, in that it is obtained by chemical proteolysis at a pH of more than 13 for a period of approximately 2 hours at a temperature comprised between 16° C. and 25° C. and in that it is liquid and has a density of more than 1 and preferably about 1.1.
Because of its characteristics, the Moringa peregrina protein hydrolysate in accordance with the invention has never been disclosed in the genus Moringa and the Moringaceae family. It will be demonstrated that the extract from the species Moringa peregrina has a particular peptide profile that differs from other species of the genus, in particular the species Moringa oleifera, which the Applicant has been able to demonstrate.
In a third aspect, the invention concerns a protein hydrolysate from Moringa peregrina seed cake for its application as a drug.
In a fourth aspect, the invention concerns a pharmaceutical or dermatological composition comprising, as the active agent, an effective quantity of a protein hydrolysate from Moringa peregrina seed cake and a physiologically acceptable excipient.
Finally, in a fifth aspect, the invention concerns a pharmaceutical or dermatological composition intended for use in the treatment of fibrotic diseases, the treatment of inflammation, of cancer, of infectious diseases of the bacterial or viral type as well as in the treatment of genetic drift and of pathologies associated with skin pigmentation.
The invention will be better understood and other aims, details, features and advantages thereof will become more apparent from the following description of several particular embodiments of the invention, given solely by way of non-limiting illustration and with reference to the accompanying drawings.
In this description, unless specified othemise, it should be understood that when a range is given, it includes the upper and lower limits of said range.
In the present invention, the abbreviations below mean the following:
In the present invention:
In a first aspect, the invention concerns a method for obtaining a protein hydrolysate from Moringa peregrina seed cake, comprising the following steps in which:
The unshelled seeds are collected, i.e. wherein the shell is retained when the fruit is ripe and preferably when the hull is starting to split.
The seeds are dried in order to obtain an internal moisture content of less than 8% and preferably about 6%; drying will preferably be carried out on a ventilated rack shielded from the sun's rays, preferably in the shade in the open air.
Next, the dried seeds are ground extemporaneously with cold pressing, which can be used to mechanically separate the oil from the remainder of the compressed seed, namely the cake.
The cake is then ground mechanically with any type of mechanical grinder, such as a hammer mill, flail mill, knife mill, crushing/shredding mill, a ball mill or beater mill, but also with any type of cryomill.
The dispersion to form the aqueous phase in accordance with step d) and the proteolysis in accordance with step e) are advantageously carried out with continuous stirring, thereby enabling dispersion and homogenization of the solid in the liquid, therefore improving the overall surface area for exchange and, as a result, improving proteolysis.
A liquid protein hydrolysate with a density of more than 1 and preferably about 1.1 is obtained, comprising a dry matter content comprised between 10% and 15%, preferably about 12.5%, comprising between 1% and 6% of nitrogen-containing compounds, in particular volatile nitrile derivatives, in a proportion of 0.5% to 1.5%, preferably about 0.8%, as well as 20 mg/liter of polyphenols (0.002%).
In accordance with a preferred embodiment of the method in accordance with the invention, the temperature of the proteolysis of step f) is approximately 22° C.
In accordance with a preferred embodiment of the method in accordance with the invention, the solid/liquid separation of step g) is carried out using different methods such as centrifuging, draining, filtration.
In one embodiment of the method for obtaining the liquid Moringa peregrina protein hydrolysate, the protein hydrolysate is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration in order to concentrate it into compounds of interest over the organic material that is also extracted, in particular over the other derivatives that are also extracted. These purification steps can be used to concentrate the pool of compounds of interest over the other extracted compounds that have been cited.
In accordance with another preferred embodiment of the method in accordance with the invention, nanofiltration is carried out in a manner such as to separate 3 bands or fractions of the protein hydrolysate, comprising a band P1 for which the molecular weight is less than 10000 Da, a band P2 for which the molecular weight is comprised between 10000 and 17 000 Da and a band P3 for which the molecular weight is approximately 23 000 Da.
It is also advantageous to separate the three bands by nanofiltration in order to obtain, by the method for obtaining a protein hydrolysate:
In another embodiment of the extraction method in accordance with the invention, the liquid protein hydrolysate obtained is dried in a manner such as to obtain a dry hydrolysate of Moringa peregrina seed cake containing a quantity of more than 10%, preferably more than 20%, more preferably more than 30% and possibly up to approximately 40% (Weight/Weight) dry matter of peptides, oligopeptides, glycopeptides and amino acids or their volatile nitrile derivatives, more preferably 50% by weight of dry matter.
In accordance with one embodiment of the invention, the liquid protein hydrolysate from Moringa peregrina seed cake obtained is dried for example by atomization, lyophilization or zeodration in a manner such as to obtain a solid hydrolysate of Moringa peregrina seeds, the water being evaporated off. Drying may be carded out in the presence of an organic support such as maltodextrin, cyclodextrin or inulin, or in the presence of an inorganic support such as phyllosilicate, magnesium silicate or carbonate and its salts.
The invention also concerns the protein hydrolysate from Moringa peregrina seed cake obtained by the extraction method in accordance with the invention.
In a second aspect, the invention concerns a protein hydrolysate from seed cake that has not been shelled and has been harvested from ripe Moringa peregrina fruit, comprising a major fraction P1 of amino acid derivatives, amino acids, peptides and glycopeptides for which the molecular weight is comprised between 1500 Da and 5000 Da, a fraction P2 of approximately 20% for which the molecular weight is comprised between 10000 and 17000 Da and a fraction P3 of approximately 20% for which the molecular weight is approximately 23000 Da, in that it is obtained by chemical proteolysis at a pH of more than 13 for a period of approximately 2 hours at a temperature comprised between 16° C. and 25° C. and in that it is liquid and has a density of more than 1 and preferably about 1.1.
In accordance with one embodiment, the liquid protein hydrolysate obtained is dried in a manner such as to obtain a dry hydrolysate from Moringa peregrina seed cake containing a quantity of more than 20%, more preferably more than 30% and possibly up to approximately 40% (Weight/Weight) dry matter of peptides, oligopeptides, glycopeptides and amino acids or their volatile nitrile derivatives, more preferably 50% by weight of dry matter.
In accordance with a preferred embodiment, the protein hydrolysate also comprises between 0.3% and 3% of volatile compounds, wherein 50% of these compounds, i.e. between 0.15% and 1.5% of the extract in accordance with the invention, is constituted by light nitrile compounds, principally with isobutyronitrile and methylbutanenitrile; wherein 5% to 10% of these compounds, i.e. between 0.015% and 0.3% of the extract in accordance with the invention, is constituted by isothiocyanate derivatives, principally with isopropyl isothiocyanate and isobutyl isothiocyanate; wherein 1% to 5% of these compounds, i.e. between 0.003% and 0.15%, is constituted by essential oil, principally with eucalyptol, menthol and benzaldehyde.
In accordance with yet another embodiment, the protein hydrolysate comprises a dry matter content comprised between 10% and 15%, preferably about 12.5%, comprising between 1% and 6% of nitrogen-containing compounds, in particular volatile nitrile derivatives in a proportion of 0.5% to 1.5%, preferably about 0.8%, as well as 20 mg/liter of polyphenols.
In the context of the present invention, the portion of the plant that is selected is the Moringa peregrina seed. It is known that Moringa peregrina seeds are used in order to extract theft oil, which is useful for domestic consumption or for various traditional medicinal treatments. The cake obtained after the seed has been defatted is a waste product that is currently used to feed animals in particular.
In accordance with yet another embodiment, the protein hydrolysate comprises the major fraction P1 for which the molecular weight is comprised between 1500 Da and 5000 Da.
In accordance with yet another embodiment, the protein hydrolysate comprises the fraction P2 of approximately 20% for which the molecular weight is comprised between 10000 Da and 17000 Da.
In accordance with yet another embodiment, the protein hydrolysate comprises the fraction P3 of approximately 20% for which the molecular weight is approximately 23000 Da.
In accordance with yet another embodiment, the protein hydrolysate comprises the fraction P1 for which the molecular weight is comprised between 1500 Da and 5000 Da and the fraction P2 for which the molecular weight is comprised between 10000 and 17000 Da.
In a third aspect, the invention concerns a protein hydrolysate from Moringa peregrina seed cake for its application as a drug.
In a fourth aspect, the invention concerns a pharmaceutical or dermatological composition comprising, as the active agent, an effective quantity of a protein hydrolysate from Moringa peregrina seed cake in accordance with the invention and a physiologically acceptable excipient.
The compositions in accordance with the invention may be in any of the galenical forms that are in normal use, depending upon whether the composition is to be ingested, injected or applied to the skin or to the mucous membranes.
In accordance with a first variation, the various compositions are suitable for ingestion; the composition may be in the form of capsules, syrups, granules or tablets. It does not have to comprise any excipient and could be constituted in its entirety by the plant extract comprising the protein hydrolysate in the dry form.
In accordance with a second variation, the various compositions are suitable for injection; the composition may be in the form of an aqueous or oily lotion, or in the form of a serum.
In accordance with a third variation, the various compositions are more particularly suitable for local administration, enabling a pharmacological effect to be obtained at the exact site of the disorder via the skin or the mucous membranes.
The principal local modes used for administration of the active agent in accordance with the invention are the cutaneous and percutaneous modes, the nasal and respiratory modes, the ocular mode, the auricular mode and the vaginal mode. Other modes may be envisaged, in particular the buccal mode, for the administration of the compositions via the mucous membranes as well as the subcutaneous mode by micro-injections.
The compositions in accordance with the invention for pharmaceutical use may be in any of the galenical forms that are normally used for application via the local route. They may be administered to the mucous membranes, in particular by nasal or respiratory, ocular, buccal, vaginal and auricular administration.
The compositions in accordance with the invention for dermatological use may be in any of the galenical forms that are normally used in the dermatological field for cutaneous application. They may be administered transdermally or applied topically to the skin.
The compositions incorporating a protein hydrolysate in accordance with the invention may comprise ingredients that are routinely used in this type of formulation for local or cutaneous administration.
In accordance with 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 glycolic solutions, serums, powders, patches, sprays or any other product for external application such as, for example, medical devices or aerosol products that also contain a propellant under pressure.
In accordance with another preferred embodiment, the various compositions are suitable for subcutaneous injection and transdermal administration; the composition may be in the form of an aqueous lotion, emulsion or in the form of a serum. In transdermal or patch systems, the release of the active principle or principles is controlled by a permeable membrane that is generally adhesive and in direct contact with the skin.
The compositions in accordance with the invention that are more particularly intended for local administration contain an acceptable pharmaceutical or dermatological medium, i.e. compatible with the skin and the mucous membranes, and encompass all of the appropriate galenical forms. These compositions may in particular be in the form of creams, oil-in-water emulsions or water-in-oil or multiple emulsions, serums, solutions, suspensions, gels, milks, lotions, sticks, aerosols, sprays or any other product for external application such as, for example, medical devices or aerosol products also containing a pressurized propellant, or in fact any form of powder that is suitable for application to the skin and the mucous membranes. These compositions comprise the excipients that are necessary to their formulations, such as solvents, emollients, thickeners, diluents, surfactants, antioxidants, bioactive agents, colorants, preservatives, fragrances.
The compositions in accordance with the invention therefore comprise any additive that is in routine use in the envisaged field of application, as well as the adjuvants that are necessary to their formulations, such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, dermatological or pharmaceutical active agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.
In all cases, the person skilled in the art will take care to ensure that these adjuvants as well as their proportions are selected so that they do not have a deleterious effect on the desired advantageous properties of the compositions in accordance with the invention.
In the compositions of the invention, the protein hydrolysate in accordance with the invention is used in a quantity ranging from 0.0001% to 40% by weight with respect to the total weight of the composition.
In a preferred embodiment, the protein hydrolysate in accordance with the invention is used in a quantity ranging from 0.001% to 10% by weight with respect to the total weight of the composition, more preferably in a quantity ranging from 0.01% to 5% by weight with respect to the total weight of the composition.
Finally, in a fifth aspect, the invention concerns a pharmaceutical or dermatological composition for its use as a drug for the treatment of:
For the treatment of fibrotic diseases, the term fibrosis means pulmonary fibrosis, renal fibrosis, hepatic fibrosis, cutaneous fibrosis, ocular fibrosis, cardiac fibrosis and other various fibrotic states and 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 disorders (for example, cystic fibrosis (CF)), and neurodegenerative disorders.
While the invention has been described with respect to several particular embodiments, it is clear that it is not in any way limited to these and it includes any technical equivalents of the means described as well as their combinations if they fall within the scope of the invention.
The use of the verb “compose of”, “comprise” or “include” and its conjugated forms does not exclude the presence of elements or steps other than those that have been defined in a claim.
Seeds of Moringa peregrina (Forssk.) Fiori were dried in order to obtain an internal moisture content of less than 8% and preferably about 6%, then pressed with an endless screw mechanical press in a manner such as to separate the oil from the remainder of the seed in order to obtain virgin oil on the one hand and cake on the other hand. The cake was then isolated in the form of extrudates precut into 1 to 2 cm pieces, on which the extraction was carried out.
The starting materials used were as follows.
A translucent yellow filtrate containing approximately 12.48% of dry matter was obtained. The liquid extract obtained is known below as the “peregrina protein hydrolysate in accordance with the invention” or the “peregrina protein hydrolysate” or the “extract in accordance with the invention”.
This peregrina protein hydrolysate in accordance with the invention had a density of more than 1 and preferably about 1.1, comprising a dry matter content comprised between 10% and 15%, preferably about 12.5%, comprising between 1% and 6% of nitrogen-containing compounds, in particular volatile nitrile derivatives, in a proportion of 0.5% to 1.5%, preferably about 0.8%, and 20 mg/liter of polyphenols. The composition of the dry peregrina protein hydrolysate in accordance with the invention is given below.
The peregrina protein hydrolysate contains relatively high levels of isopropyl isothiocyanate and isobutyl isothiocyanate, confirming previous publications on the species (K
The dry extract described above was obtained by a gravimetric method based on the mass present in the liquid extract before and after evaporation.
The aim of this study was to evaluate the inhibiting activity of the protein hydrolysate from Maringa peregrina seed cake obtained in accordance with Example 1.
Protocol: The study was carried out using a recombinant human furin, which catalyzes cleavage of a specific fluorescent reference substrate.
100 nM decanoyl-Arg-Val-Lys-Arg-CMK was used as a reference inhibitor for the activity of furin.
The furin was pre-incubated for 10 minutes at ambient temperature in the absence (control) or in the presence of a reference product, or of increasing concentrations of the test compound:
“Peregrina protein hydrolysate”; 0.02; 0.2 and 2% (v/v).
At the end of the pre-incubation step, a furin substrate was added and the experimental conditions were incubated once again at ambient temperature away from light for 5 minutes. All of the experiments were carried out in triplicate.
The test peregrina protein hydrolysate was dissolved directly in the assay buffer then diluted in order to obtain the test concentration described above.
Cleavage of the fluorescent furin substrate was monitored for 5 minutes after addition of the substrate by reading the fluorescence at 485 nm/535 nm.
The results are expressed in RFU (relative fluorescent units)+/−S.D. (standard deviation).
The statistical significance of the difference observed between the groups “Control” and “Reference product” was evaluated by a Student t test (p<0.001).
The statistical significance of the difference observed between the groups “Control” or “Reference” and “Test compound” was evaluated by a one-way ANOVA, followed by a Holm-Sidak test (p<0.05).
The reference inhibitor for furin, termed decanoyl-Arg-Val-Lys-Arg-CMK, tested at 100 nM, significantly inhibited the activity of the furin by 97.9% (p<0.001).
This result was expected and validated the study.
The results for the activity of the furin enzyme compared with the base activity obtained are given below.
“Peregrina protein
Conclusion: At a concentration of 2%, the peregrina protein hydrolysate in accordance with the invention is capable of inhibiting 98.8% of the activity of furin convertase. Beyond a concentration of 0.2%, the peregrina protein hydrolysate in accordance with the invention is capable of inhibiting the activity of furin by 26.8%.
The aim of this study is to demonstrate the inhibiting activity of the peregrina protein hydrolysate in accordance with the invention on the HDAC and sirtuin I enzymes, enzymes that are involved in the control of genetic drift by the regulation of the condensation/decondensation of chromatin, which provides or prohibits access to genes carried by DNA. A buffered solution of HDACs and sirtuin I reacts with a substrate over 20 minutes at 37° C. and transforms it in order to form a compound that colors in the presence of a developer after incubation at 37° C. for 10 minutes. The maximum activity for deacetylation of sirtuins can then be evaluated by measuring the absorbance at 405 nm. The peregrina protein hydrolysate in accordance with the invention or the reference product “Trichostatin A (STA) inhibitor 1 μM” were brought into contact with the solution of sirtuins at the same time as the substrate for the enzyme for 20 minutes at 37° C.; the substrate transformed by the enzyme was stained by the addition of a developer. The deacetylating activity of the HDACs and sirtuin I in the presence of the active ingredient was then evaluated by measuring the absorbance at 405 nm. The modulation of this activity was expressed as a percentage inhibition or activation of the maximum activity of the HDACs and sirtuin I in the absence of active ingredient, i.e. solely in the presence of the substrate for the HDACs & sirtuin I enzymes.
Protocol: A solution of sirtuin enzymes was incubated in its substrate for 20 min in the absence (control) or presence of the reference product, or in increasing concentrations of the products to be tested. The peregrina protein hydrolysate in accordance with the invention was tested at the following concentrations: 2%; 1%; 0.1% (V/V). At the end of the incubation period, the activity of the sirtuin enzymes with and without the test product or reference product was revealed by staining using a developer solution (10 min at 37° C.) and evaluated by measuring the absorbance of the reaction media at 405 nm. For each test concentration, the modulation of the deacetylating activity of the histone deacetylase and sirtuin I enzymes by the test product was calculated using the following formula.
Percentage modulation of the activity of sirtuin enzymes=100×[(OD405 produced in test or reference)−(0D405 HDACs & sirtuin I alone)]/OD405 sirtuins alone. [Math. 1]
If the result is negative, the percentage is expressed as the inhibition of the enzymatic reaction; if the result is positive, the percentage is expressed as the activation of the enzymatic reaction. The results for the inhibition of the histone deacetylase enzymes (HDACs) are given below.
Peregrina protein
Conclusion: At 2%, the peregrina protein hydrolysate in accordance with the invention exhibits significant inhibition of HDACs; this inhibition reveals the capacity for promoting self-protection of cells of the skin against genetic drift. Thus, the extract appears to be useful against one of the most routine genetic drifts on the surface of the skin, namely fibrosis, which is manifested by the appearance of a “lump” of flesh (fibrotic protuberance). The extract could advantageously interfere in fibrosis phenomena on the surface of the skin.
Endothelin is a hormone peptide derived from endothelial cells that is capable of acting on various cells and tissues via its receptors. As an example, endothelin is known to cause an increase in the intraceliular concentration of calcium in smooth muscle vascular cells and other cells (OHBA T. et al., WO 2012/081370).
It has been reported in recent years that endothelin type 1 (ET-1) is a bioactive factor that contracts the smooth vascular and non-vascular muscle cells by direct and indirect actions. It is considered that an increase in the action of endothelin provides continuous vasoconstriction to blood vessels in peripheral sites, in the kidneys and in the brain, and could be at the origin of a variety of diseases such as hypertension, myocardial infarction, cerebrovascular accidents, acute renal insufficiency, Raynaud syndrome, atherosclerosis, asthma and prostate cancer (M
Recently, the role of endothelin in a variety of cells other than smooth vascular muscle cells has been elucidated. As an example, scientific publications have reported that the generation of ET-1 and other factors increases in keratinocytes when the skin is exposed to UV radiation and have suggested that ET-1 might be associated with melanogenesis in melanocytes exposed to UV radiation. As a consequence, the suppression of the expression of endothelin is considered to be useful, not only for the prevention and/or treatment of the aforementioned diseases, but also in order to prevent or improve pigmentation of the (I
The aim is to assay endothelin type 1 in human micro-vascular endothelial cells after exposure to the peregrina protein hydrolysate in accordance with the invention for 24 h.
Protocol: The human micro-vascular endothelial cells were provided by PELOBiotech and cultured in 96-well plates in accordance with the supplier's production procedures. This meant allowing the extracts to act at different concentrations on endothelial cells at 80% confluence for 24 hours, then quantifying endothelin I in the cell supernatants with the aid of the ELISA PicoKine (EDN1) kit. A prior viability test was carried out in order to define the non-toxic doses to be used when assaying the endothelin 1. The negative control was formed by cells in culture medium without treatment. The positive control in the viability test was 0.5% SDS. All of the conditions were prepared in culture media and the cells were then incubated at 36.5° C./5% CO2 for 24 hours.
Conclusion: The viability test carried out at the end of treatment did not exhibit any toxic effects for the concentrations that were tested.
A complementary study for the evaluation of P1, P2, P3 and of the complex P1+P2 described in Example 12 is described below for the production of endothelins: Cell test model on normal human endothelial cells.
As described in Example 12, 3 bands or protein fractions were isolated from the hydrolysate in accordance with the invention, characterized by theft mass P1 (at a molecular weight of less than 10000 Da), P2 (at a molecular weight comprised between 10000 and 17000 Da) and finally P3 (at a molecular weight of approximately 23000 Da). These fractions are hereinafter also termed “extracts”.
At all concentrations, the standard deviations were high and the fraction P1 of the protein hydrolysate did not exhibit any significant modulation on endothelin type 1.
At concentrations of 0.1% and 1%, the standard deviations were high, and the fraction P2 of the protein hydrolysate did not exhibit any significant modulation on endothelin type 1 at these concentrations. At the concentration of 0.3%, P2 inhibits the production of endothelin type 1 by 24.8%.
At all concentrations, the standard deviations were high, and the combination of fractions P1+P2 did not exhibit any significant modulation on endothelin type 1.
At concentrations of 0.3% and 1%, the fraction P3 of the protein hydrolysate very significantly inhibits approximately 50% of the production of endothelin type 1.
Conclusion: The compounds termed “Extract P1” and “Extract P2” did not have a significant action on the modulation of the endothelin type 1, and only the “Extract P3” significantly reduced the endothelin-1 liberated into the culture medium by the normal human endothelial cells in a monolayer culture with a score of 52.3% inhibition with 0.3% of the extract in accordance with the invention. No synergistic effects were observed when P1 and P2 were combined.
The extract P3 specifically exhibited an anti-cancer effect (B
Adult tissues, including the cutaneous epidermis, the gastro-intestinal epithelium and the hematopoietic system, have a very high level of cell renewal. The physiological process of maintaining tissue homeostasis is attributed to maintaining a constant number of cells in organ renewal. Embryonic stem cells (ESC) are essential to maintaining and regenerating cutaneous tissues.
The epidermis develops from the ectodermis of the embryo surface. It starts as a single layer of non-specific progenitor cells covering the embryo after neurulation and develops into the basal epidermal layer. The basal epidermal layer is enriched in ESCs (Epidermal Stem Cells). In fact, the cells of this layer give rise to all of the epidermal structures, including the stratified epidermis (also known as the interfollicular epidermis) and the epidermal appendices, such as the hair follicles, the sebaceous glands and the sweat glands. The subjacent dermis principally derives from the mesodermis under the ectodermis. The mesodermis is the principal source of mesenchymal stem cells, which give rise to the collagen-producing fibroblasts, the subjacent adipocytes and the immune cells of the skin.
Stem cells are undifferentiated cells that are known as pluripotent cells with a young genotype that is capable of self-renewal and of differentiating to produce an organ or a tissue such as the skin. At this stage, they are identified as “multipotent cells”. Given that 50% of the descendants of the stem cell population remains undifferentiated, the stem cells contribute to preserving homeostasis and ensuring renewal of damaged or senescent differentiated cells. However, these epidermal stem cells are frequently affected by the environment. Oxidative stress such as pollution or ultraviolet radiation damages their DNA, according to Y
The aim of the study is to evaluate the effect of the peregrina protein hydrolysate in accordance with the invention on the protection of the epidermal stem cells against UVB irradiation.
Protocol: Human keratinocyte cells were obtained from a 62 year old donor. In order to carry out the experiments, the keratinocytes were cultured in a monolayer until they reached 80% confluence.
The cell culture was then enriched in epidermal stem cells by following the method described by G
Reference product: 1 μM quercetin was used as a reference product in this study. The quercetin was purchased from Sigma Aldrich.
The cells were pre-incubated for 24 hours in the absence (“Control”) or in the presence of the reference product or an increasing concentration of the compound to be tested. At the end of the pre-incubation period, the cells were irradiated with UVB (30 mJ/cm2) then incubated for 8 days at 37° C. in the absence (reference) or in the presence of the reference product or by increasing the concentration of the compound to be tested.
“Peregrina protein hydrolysate”: 0.01; 0.05 and 0.15% (v/v).
The test compound “peregrina protein hydrolysate” was diluted directly in the incubation medium in order to obtain the various concentrations described above.
At the end of the incubation period, the cell viability was measured using Alamar blue, a non-cytotoxic viability indicator based on the reduction of rezazurin by mitochondria. Each experimental condition was carried out in triplicate (n=3).
The results are presented below as a percentage viability with respect to the “Control without UVB” experimental condition (mean+/−S.D). The level of significance between “Control without UVB” and “Control with UVB” was evaluated with the aid of a Student test (p<0.05).
Peregrina
Conclusion: The peregrina protein hydrolysate can significantly protect the stem cells of the human skin subjected to cellular stress (UV). Stem cells are cells with preserved and young DNA material. They are at the origin of tissue regeneration, and a return to a young and healthy state. The protection of stem cells is correlated with the capacity to preserve DNA material. The peregrina protein hydrolysate in accordance with the invention maintains the integrity of the stem cells; it is therefore involved in DNA conversion.
The dynamics of the length of telomeres is very important for the regulation of the replicative lifetime of cells, in particular in species that are long-lived. Shortening of telomeres and the activity of telomerase are important factors in aging and in tumorigenesis (S
Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repetitions to the ends of the telomeres. Telomeres are long sections of repeated sequences that cap the ends of the chromosomes and are known to stabilize the chromosome. In human beings, the telomeres are generally 7 to 10 kb in length and include several repetitions of the sequence -TTAGGG-.
Telomerase is not expressed in the majority of adult cells and the length of the telomeres reduces with successive replication cycles. After a certain number of replication cycles, the progressive shortening of the telomeres causes the cells to enter a phase of telomeric crises, which in turn leads to cell senescence. Certain diseases are associated with a rapid loss of telomeres, causing premature cell senescence. It has been shown that the expression of the gene coding for the human telomerase protein in human cells (B
The aim of this study is to evaluate the effect of the compound termed “peregrina protein hydrolysate” on telomere shortening in a model composed of normal human fibroblasts in a monolayer culture. It is well known that the telomere corresponds to a biological clock. The length of the telomeres reduces progressively with cell divisions, in the end resulting in a cell that is incapable of replication. The measurement of the length of the telomeres was carded out using quantitative PCR and comparison with the length of the telomeres among cells at passes 2 and 5.
Protocol: Human fibroblast cells were obtained from a 44 year old donor. In order to carry out the experiments, the cells were used in passes 2 and 5. The fibroblasts were cultured for 3 consecutive passes in the absence (control) or in the presence of an increasing test concentration of the peregrina protein hydrolysate: 0.01%; 0.1% and 0.5% (v/v).
Preparation of test compound: The “peregrina protein hydrolysate” test compound was diluted directly in the incubation medium in order to obtain the various concentrations described above.
At the end of the incubation, the cells were trypsinized. The DNA was extracted from the cells with the aid of a dedicated DNA extraction kit. The DNA was quantified by nanodrop.
The telomere length was measured by quantitative PCR (q-PCR). For each sample, the variation in the telomere length was measured by relative quantification using the SCR (single copy reference) gene as a reference gene. For each sample, a q-PCR was carried out using a set of telomere primers which recognize and amplify the telomere sequences and a second q-PCR was carried out using the set of SCR primers which recognize and amplify a 100 bp region on the human chromosome 17 and act as a reference for standardization of the data.
The results are expressed in relative units corresponding to the length of the telomeres with respect to the cells in pass 2 (mean±S.D.). The level of significance versus “Control” in passes 2 and 5 was evaluated with the aid of a Student test (*: p<0.05). The level of significance between “control” and “test compound” was evaluated independently for each product by a single factor analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (*: p<0.05).
Peregrina
Results: Under our experimental conditions, the peregrina protein hydrolysate tested at 0.05%, 0.1% and 0.5% (v/v), significantly reduced shortening of normal human fibroblast telomeres.
The shortening of the telomeres exhibited an inhibition (compared with the control) at 0.05% (v/v) of +8.9% (p<0.05); at 0.1% (v/v) of +15.1% (p<0.01) and at 0.5% (v/v) of +16.6% (p<0.01). Conclusion: in a context of normal multiplication or division of human cells, the peregrina protein hydrolysate in accordance with the invention demonstrates a capacity to significantly increase the length of telomeres. Telomeres are plugs involved in the protection of DNA material; increasing the length of the telomeres is correlated with the capacity to preserve DNA material. The peregrina protein hydrolysate can increase the length of the telomeres. This hydrolysate is therefore involved in the preservation of human genetic material (DNA).
Complementary study of the evaluation of extracts P1, P2, P3 and of the complex P1+P2 described in Example 12 on their capacity to protect DNA by increasing the length of telomeres after several cell divisions.
After 3 cell divisions from the concentration of 0.3%, the extract P1 in accordance with the invention increased the size of the telomeres on a normal human cell culture model (fibroblasts) by 24.3%. At the concentration of 1%, under the same conditions, the extract P1 increased the telomere size by 39.6%.
After 3 cell divisions on a normal human cell culture model (fibroblasts), the extract P2 in accordance with the invention had a statistically unconfirmed tendency for elongation of the telomeres for each test concentration.
After 3 cell divisions on a normal human cell culture model (fibroblasts), the extract P3 in accordance with the invention did not have any capacity to promote elongation of the telomeres for each test concentration.
ongation
indicates data missing or illegible when filed
After 3 cell divisions at the concentration of 1% (i.e. 0.5% for each extract), the mixture of extracts P1 and P2 (50/50 by volume) in accordance with the invention increased the size of the telomeres on a normal human cell culture model (fibroblasts) by 49.1%. This score was not reached by the individual extracts, and so a synergistic effect has been demonstrated by mixing the extract P1 and P2.
Conclusion: The compounds termed “Extract P1” and “Extract P1+P2” significantly reduce the shortening of telomeres, occurring after 3 consecutive cell passes. A synergistic effect is confirmed with a combination of P1 and P2.
Zinc alpha-2-glycoprotein (ZAG) is a plasma glycoprotein that takes its name from its electrophoretic mobility and its capacity to be precipitated by Zn salts. ZAG forms part of the superfamily of immunoglobulin genes and has a three-dimensional structure which is highly homologous to CMH class I and II molecules. ZAG has been detected immunohistochemically in normal secretory epithelial cells of the breast, prostate and liver, in the saliva glands, bronchial, gastro-intestinal and sweat glands and in normal stratified epitheliums including the epidermis. The mRNA of ZAG remains uniformly distributed in the various types of cells (F
Protocol: Normal human keratinocytes were isolated from foreskin and cultured in 24 and 96 well plates in accordance with internal procedures.
This involved leaving the samples to act on the keratinocytes at 80% confluence for 48 hours at defined concentrations, then quantifying the ZAGs in the cell supernatants with the aid of the ELISA kit.
A prior viability test was carried out in order to define non-toxic doses to be used when assaying the ZAGs. The negative control was produced with the aid of cultured cells without treatment. The positive control for the viability test was 0.5% SDS.
All of the conditions were prepared in the culture media, and the cells were then incubated at 36.5° C./5% CO2 for 24 hours for the viability test and 48 hours for the assay of the ZAGs.
Peregrina
Conclusion: The peregrina protein hydrolysate can significantly increase the production of ZAG, with good dose dependency and a low toxicity on human cells. For this reason, it has an anti-fibrotic effect and an anti-inflammatory effect based on its capacity to significantly increase ZAG.
A complementary study of the stimulation of ZAG starting from the protein bands identified in Example 12 is described below for normal human keratinocytes.
From 0.2%, the extract P1 significantly increased the production of ZAG by more than 107%.
From 0.2%, the extract P2 significantly increased the production of ZAG by more than 80%.
The extract P3 did not have the capacity to significantly influence the production of ZAG in this study model.
The combination of extracts P1 and P2 exhibited a tendency to activation of the production of ZAG, but this tendency was not significant from a statistical viewpoint. Thus, there was no synergistic effect in the combination of the extracts P1 and P2.
The compounds termed “Extract P1” and “Extract P2” significantly increased the ZAG liberated into the culture medium by normal human keratinocytes in a monolayer culture, but no synergistic effects were observed when combining P1 and P2.
Conclusion: In the hydrolyzed extract in accordance with the invention, the extract P1 is the extract that performs best for increasing ZAG. For this reason, it has an anti-fibrotic effect and an anti-inflammatory effect from a dose of 0.2%.
The implications of the interactions between melanocytes and fibroblasts in the regulation of melanogenesis is well known and has been studied in depth. Although these interactions are not yet fully understood, they are at the origin of the “bleaching” of palmoplantar zones and are now used in dermatology for the development of depigmentation products. Yamaguchi et al (Y
The signaling pathways used by DKK-1 to produce these results have now been identified. Because of its antagonist action on the Wnt receptor, DKK-1 is in fact capable of short-circuiting the intracellular signaling pathways activated by β-catenin, generally responsible for the regulation of the genes involved in melanogenesis. Yamaguchi et al (cf. supra) have also demonstrated that DKK-3, a molecule like DKK-1 but with no effect on the Wnt receptor, could play a regulatory role on the DKK-1 effect. In fact, the larger the quantity of DKK-3 in the vicinity of this Wnt receptor, the weaker are the interactions between DKK-1 and this receptor on melanogenesis. The work by Yamaguchi et al (cf. supra) also suggest that the identification of agents having an influence on the DKK1/DKK3 ratio in cultures of normal human dermal fibroblasts of non-palmoplantar origin could control the production of melanin starting from normal non palmoplantar human melanocytes.
The aim of this study is to evaluate the effect of the peregrina protein hydrolysate on the synthesis and release of DKK-1 in a model compound of normal human fibroblasts in a monolayer culture.
Protocol: Human fibroblast cells were obtained from a 68 year old donor. In order to carry out the experiments, the fibroblasts were cultured in a monolayer to confluence. 100 nM dexamethasone was used as a reference inducer for the synthesis and release of DKK-1. The disks of skin were incubated for 48 hours in the absence (control) or in the presence of the reference product or of the test product: “peregrina protein hydrolysate”: 0.01%; 0.1% and 0.5% (v/v).
At the end of the incubation, the incubation media were removed in order to carry out the DKK-1 liberation method.
The “peregrina protein hydrolysate” test compound was diluted directly in the incubation medium in order to obtain the various concentrations described above.
At the end of the incubation period of 48 hours, the DKK-1 liberated into the incubation medium was quantified with the aid of a sensitive and specific ELISA kit.
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).
The results are expressed in ng of DKK-1 per mg of proteins (mean±S.D.).
The level of significance between the “control” and the “reference product” was evaluated with the aid of a Student test (p<0.05).
The level of significance between the “Control” and the “test product” was evaluated by a single factor analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (p<0.05).
Under our experimental conditions, the reference product denoted “Dexamethasone”, tested at 100 nM, significantly increased the DKK-1 liberated by 181.8% (p<0.01) compared with the “Control”. The results on the modulation of the DKK1 assay are given below.
Peregrina
The study shows that the hydrolysate in accordance with the invention significantly increased the levels of DKK1 at a dose of 0.05% with a 26.1% increase with respect to the base level and at a concentration of 0.5% of the extract in accordance with the invention, an increase of 131.5% of the base level was observed.
The aim of this study was to evaluate the effect of the peregrina protein hydrolysate on the synthesis and the liberation of DKK-3 in a model compound of normal human fibroblasts in a rnonolayer culture. The human fibroblast cells were obtained from a 68 year old donor. In order to carry out the experiments, the fibroblasts were cultured in a monolayer to confluence. The human fibroblast cells were obtained from a 68 year old donor. In order to carry out the experiments, the fibroblasts were cultured in a monolayer to confluence.
At the end of the incubation period of 48 hours, the DKK-3 liberated into the incubation media was quantified with the aid of a sensitive and specific ELISA kit.
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).
The results are expressed in ng of DKK-3 per mg of proteins (mean±S.D.). The level of significance between the “Control” and the “reference product” was evaluated with the aid of a Student test (*: p<0.05).
The level of significance between the “Control” and the peregrina protein hydrolysate was evaluated by a single factor analysis of variance (one-way ANOVA) followed by a Holm-Sidak test (*: p<0.05). The results on the modulation of the DKK3 assay are given below.
Peregrina
Conclusion: The peregrina protein hydrolysate in accordance with the invention at 0.5% exhibited a substantial inhibition of the order of 21% for DKK3 compared with the base level. The peregrina protein hydrolysate in accordance with the invention has a large capacity to manage the genes involved in cell differentiation because of the palmoplantar inhibition principle (beta-catenin signaling pathway), by its capacity to significantly increase DKK1 and significantly reduce DKK3, which increases the ratio DKK1/DKK3.
Complementary study of the evaluation of P1, P2, P3 and of the complex P1+P2 described in Example 10 on the production of DKK1: Cell test model on normal human fibroblasts.
The extract P1 significantly increased the production of DKK1 by 26.6% from the dose of 0.3%, and by 27.8% at a dose of 1%.
The extract P2 did not significantly statistically increase the production of DKK1 in any of the experimental doses.
The extract P3 did not significantly statistically increase the production of DKK1 in any of the experimental doses.
The combination of the extracts P1 and P2 at the dose of 1% (i.e. 0.5% for each of the extracts) increased the production of DKK1 significantly and by more than 39%. This score is more than that obtained with the extract P1 or P2 alone; this therefore means that there is a synergistic effect in the combination of the two extracts P1 and P2.
Conclusion: The compounds termed “Extract P1” and “Extract P1+P2” significantly increased the DKK-1 liberated into the culture medium by normal human fibroblasts in a monolayer culture. A synergistic effect was confirmed when P1 and P2 were mixed.
The most remarkable activity of the peregrina protein hydrolysate demonstrated in the in celluia tests is its epigenetic action with its capacity to very significantly slow down the process of telomere shortening following cell division, The hydrolysate is a cellular protector, more particularly of stem cells; which makes them a very good cell and tissue regenerator. These properties offer very high protection of DNA and its genetic material. The peregrina protein hydrolysate is also a powerful modulator of the production of ZAG and of endothelin type 1, with an anti-fibrotic and anti-inflammatory effect.
Angiotensin convertase 2 is in particular involved in intra-cellular infection by COVID19 after the activation of spike proteins by other convertases and more particularly by furin, see P
peregrina
Conclusion: The hydrolyzed extract of peregrina cake is not significantly involved in the direct inhibition of angiotensin convertase 2 (ACE2). By considering table of Example 11 below, the amplitude of the action of this extract on another convertase, namely furin, was evaluated. The inhibiting action of the extract in accordance with the invention on furin has been clearly demonstrated and is established in Table 32 of Example 11. This specific inhibition demonstrated on a single convertase enzyme reinforces the importance of the protein hydrolysate in accordance with the invention as a specific inhibitor for furin convertase.
The present evaluation used two principal components. On the one hand, stable recombinant clonal HEK293 cells constitutively expressing full length human ACE2 (Genbank #NM_021804.3) with a surface expression of ACE2 confirmed by flow cytometry. On the other hand, the spike SARS-CoV-2 pseudotyped lentivirus was produced with spike SARS-CoV-2 (Genbank access number QHD43416.1) as envelope glycoproteins instead of the VSV-G that is conventionally used. These pseudovirions also contain the gene for luciole luciferase directed by a CMV promoter; as a consequence, cell entry mediated by the peak can be measured in a practical manner via the activity of the luciferase reporter. The spike SARS-CoV-2 pseudotyped lentivirus may be used to screen the applications in a biosafety level 2 facility.
The materials used were as follows:
The ACE2-HEK cells were thawed in the thawing medium 1, amplified in the 1N growth medium then harvested and placed in white, transparent flat bottom 96-well culture plates, in an amount of 10000 cells/well in 50 μL of thawing medium 1. The cells were incubated overnight at 37° C.
The following day, visual monitoring of the homogeneity and integrity of the cell layer was validated with the aid of an inverted microscope and the following test ingredients were prepared in accordance with the following instructions:
In a first step, the peregrina protein hydrolysate was diluted 11× to an intermediate concentration in the thawing medium 1 and subsequently, 5 pt was transferred to the test plate and co-incubation with the ACE2-HEK cells was carried out. After an incubation period of 30 min at 37° C., 5 μL of undiluted pseudotyped lentivirus (Bald or S1-spike) was added to the corresponding wells for analysis.
The mAb blocking ACE2 was used as a positive control in a final concentration of 0.5 μM in the wells for analysis.
After an incubation period of 48 hours, a volume of 50 μL of the reagent luciferase was added to the wells for analysis and the luminescent signal was measured using the PolarStar Omega luminometer.
The results are given in
Conclusion: The peregrina protein hydrolysate exhibits a convincing anti-infectious effect on the pseudovirions of SARS COV2 starting from a dose of 3% (Ci) on the study model A marked inhibition of 60% at the concentration C1 (3%) has been demonstrated.
Evaluation of bands P1, P2, P3 identified in Example 12 and of the complex P1+P2 on furin convertase: acellular test model.
The studies carried out on the identified bands did not demonstrate any inhibiting action on furin convertase in all of the concentrations tested with P1, P2, P3 and P1+P2.
Conclusion: The inhibiting activity of furin convertase is therefore not carded by one of these protein bands. This demonstrates that the inhibiting activity of furin convertase is due to the protein hydrolysate in its entirety obtained by the method described.
The protein hydrolysate in its entirety inhibits the SARS-CoV2 spike type proteins, in particular by inactivation of furin convertase. These inhibitions provide an anti-infectious and virostatic nature.
The aim of this study was to evaluate the inhibiting effect of peregrina oil obtained by first cold pressing of seeds with shells, then of the peregrina extract with ethanol (96%) constituted by approximately 1.1% of dry matter, this dry matter itself being constituted by approximately 55% by weight 2,5-diformylfuran, 2.5% furfural, 1.2% isopropyl myristate, 4.7% palmitic acid, 11.1% oleic acid and 25.8% triglycerides, and finally the peregrina protein hydrolysate in accordance with the invention, on the activity of furin.
Protocol: The peregrina extract in ethanol (96%) and the peregrina protein hydrolysate were stored at +4° C., away from light until use. The peregrina oil was stored at ambient temperature in a dark area.
The reference product: 100 nM decanoyl-Arg-Val-Lys-Arg-CMK was used as the reference inhibitor for the activity of furin.
The incubation protocol: The furin was pre-incubated for 10 minutes at ambient temperature in the absence (Control) or in the presence of the reference product or increasing concentrations of the test compounds:
Preparation of compounds: The peregrina extract in 96% ethanol and the peregrina protein hydrolysate were dissolved directly in the assay buffer then diluted in order to reach the test concentration, as described above. The peregrina oil was dissolved to 3% in a 0.05% Tween20 solution in assay buffer. The solution was then diluted in order to obtain the concentration described above.
Evaluation protocol: Cleavage of the fluorescent furin substrate was monitored for 5 minutes after addition of the substrate by recording the fluorescence at 485 nm/535 nm. g. Statistics: The results are expressed in RFU (Relative Fluorescent Units)+/−S.D. (standard deviation). The statistical significance of the difference observed between the “Control” and “Reference product” groups was evaluated by a Student test (p<0.001). The statistical significance of the difference observed between the “Control” and “Test compound” groups was evaluated by a one-way ANOVA, followed by a Holm-Sidak test (p<0.05). The results for the peregrina oil are given in
Results:
Conclusion: The present study enabled us to demonstrate that only the peregrina protein hydrolysate in accordance with the invention can significantly inhibit the activity of furin by 98.8% at a concentration of 2%.
Gel electrophoresis on polyacrylamide containing sodium dodecyl sulfate is known as SDS-PAGE electrophoresis. It is a technique consisting of causing denatured proteins to migrate in a polyacrylamide gel by saturation of the negative charge via SOS under the influence of an electric field, enabling them to be separated thereby. It is a denaturing technique that dissociates non-covalent protein complexes by using a negatively charged ionic detergent (SOS). This detergent binds indiscriminately via hydrophobic bonds to two amino adds. This technique can therefore be used to analyze proteins and to separate them as a function of their molecular mass.
The hydrolyzed extract in well 1 clearly exhibited all of the expected protein bands.
Well 2 (supernatant) exhibited the expected bands, in particular for the intermediate and highest molecular weights; well 2 appears to have concentrated the lowest molecular weights, in particular with the volatiles.
Well 3 (pellet) clearly exhibited the highest molecular weight bands (>75000 Da), then a band known as P3 of approximately 23000 Da, then a band known as P2 comprised between 10000 and 17000 Da and finally a band at less than 10000 Da known as P1, estimated to be between 4000 and 6000 Da.
No volatile compounds were detected.
Conclusion: The isolation of the protein bands reveals that the volatile compounds are not bound to the proteins; the volatile compounds do not participate in the molecular cavalcade of the smallest protein bands.
Determination of three protein bands: The bands P1 (less than 10000 Da) and P2 (between 10000 and 17000 Da) and P3 (approximately 23000 Da) were prepared and passed on for liquid chromatographic analysis coupled with mass spectrometric analysis (LC MS/MS).
The results were obtained using two specialized software programs for protein recognition in plants (Mascot and Peaks):
The results of the identification did not enable these proteins to be identified precisely; we were in the presence of proteins which have not yet been described in the aforementioned databases.
Peregrina protein hydrolysate
Peregrina protein hydrolysate
Formulation of a product for subcutaneous administration: dry extract in accordance with the invention (containing 60% of protein hydrolysate on an inulin support) packaged in a single dose flask under inert gas ready to be dissolved by a physiological medium.
Formulation of an injectable liquid product: liquid extract in accordance with the invention in a 5% dose in a physiological medium packaged under sterilizing conditions in particular by vacuum filtration with a cutoff threshold of 0.45 μm.
Antifibrotic drug in a 750 mg capsule containing 100 mg of piperine, 300 mg dry extract in accordance with the invention (containing 60% of protein hydrolysate on an inulin support), 100 mg boswellic acid, 250 mg of calcium carbonate).
Antifibrotic drug in 1 g tablet: 300 mg of dry extract in accordance with the invention (containing 60% of protein hydrolysate on an inulin support), 400 mg of calcium carbonate containing 200 IU of vitamin D, 150 mg of magnesium gluconate, 80 mg of inulin and 70 mg of magnesium stearate.
Preparation of the protein hydrolysate in accordance with Example 1: Unshelled seeds of Moringa peregrina (Forssk.) Fiori harvested when the fruit was ripe were dried in order to obtain an internal moisture content of less than 8% and preferably about 6%, then pressed with an endless screw mechanical press in a manner such as to separate the oil from the remainder of the seed in order to obtain the virgin oil on the one hand and a cake on the other hand. The cake was then isolated in the form of extrudates that had been cut into 1 to 2 cm pieces. By following the protocol described in Example 1, the liquid extract that was obtained was used undiluted in the following tests.
The test was carried out in 3 principal phases:
The cytotoxicity test was carried out on the Salmonella typhimurium TA100 strain at concentrations of 5000, 1600, 500, 160 and 50 μg/plate, with and without S9-Mix.
The reagents used for the preparation of the S9-Mix were prepared in accordance with the following instructions:
The bacteria were exposed to the test extract of the invention with and without the metabolic activation system. The metabolic system used was a post-mitochondrial fraction improved with cofactor (S9). This fraction 59, a microsomal fraction of Sprague Dawley rat liver homogenate treated with an enzymatic inducer, was prepared in accordance with M
0.1 mL of the test element preparation at the highest concentration,
A number of criteria can be used to determine whether a result is positive, in particular an increase in the number of revertants correlated with the dose of the item to be tested, or a reproducible increase in the number of revertants at one or more concentrations, with or without metabolic activation.
Because of the result obtained for test 1, it was decided to use the same dilution range for test 2. The analysis of the revertants shows that:
In view of the results obtained during this study, the protein hydrolysate in accordance with Example 1 may be considered to have neither mutagenic nor pro-mutagenic activity.
The principle of the test is the comparison of the cytotoxicity of the protein hydrolysate in accordance with Example 1 in the presence and in the absence of a non-cytotoxic dose of UVA, on cells under culture. The cytotoxicity was evaluated by determining the cell viability with the aid of a vital stain: neutral red, 24 h after treatment with the reference elements and the peregrina protein hydrolysate in accordance with the invention with or without irradiation with UVA. The cells used were mouse embryo fibroblasts from the Balb/c 3T3 clone 31 (ATCC-CCL163) line. The positive control was a solution of chlorpromazine (CAS number: 69-09-0). The negative control was a diluent for the test and reference extract (buffered saline solution+/−1% solvent). The peregrina protein hydrolysate was tested at 8 concentrations on at least four culture wells per concentration being studied, in the presence or absence of UVA. The fibroblasts were trypsinized and two 96-well culture plates were inoculated in an amount of 100 μL of a 2×105 cells/mL (i.e. 2×106 cells per well) cell suspension in complete culture medium. The inoculated plates were incubated in an oven for 24 hours at 37° C., 5% CO2. At the end of the incubation, semi-confluence of the cell mat was verified. The dilutions were prepared just before depositing them onto the cells. The pH of the highest concentration was measured; it was comprised between 6.5 and 7.8. the culture medium was eliminated; each well had already been carefully rinsed with 150 μL of PBS maintained at ambient temperature before being treated with 100 μL of each extract dilution or reference dilution. The culture plates were incubated in the dark for 1 h±5 minutes at 37° C. and 5% CO2. Irradiation was carried out with the aid of a BIO SUN solar irradiation system (Vilber Lourmat RMX3W). The BIO SUN is a system that controls UV irradiation with the aid of a programmable microprocessor. The system continuously monitors the emission of UV light. The irradiation stops automatically when the energy delivered is equal to the programmed energy. The spectral irradiation of the test device was measured in the wavelength range 250 to 700 nanometers with a calibrated spectroradiometer.
One of the 2 plates was irradiated with its cover at ambient temperature, and the other plate was protected from UVA and stored at ambient temperature for the irradiation period. After irradiation, the treatment medium was sucked off and the cells were rinsed. Next, 100 μL of complete culture medium was carefully added and the plates were incubated for 18 to 22 h at 37° C. and 5% CO2. The next day, the cell viability (growth, morphology, integrity of the monolayer) was evaluated by observations using a phase contrast microscope. The culture medium was eliminated, each well had already been rinsed and maintained at ambient temperature before being treated with 100 μL of the staining solution. The plates were replaced in the incubator for 3 h under the same conditions. The staining solution was eliminated and the cells were rinsed; then the rinsing solution was removed and 150 μL of desorption solution was added to each well. The plates were agitated until the crystals had completely dissolved. The absorbances were measured at 450 nm.
The sensitivity of the cells to UVA was monitored over all of the approximately 10 passes, by evaluation of their viability after exposure to increasing doses of irradiation. The cells were cultured to the density used in the test. They were irradiated the next day at a dose of 2.5 to 9 J/cm2 and the cell viability was determined a day later with the aid of a NRU test. The cells satisfied the quality criteria if their viability after irradiation at 5 J/cm2 UVA was 80% or more of the viability of the references kept in the dark; at the higher dose of 9 J/cm2 UVA, the viability had to be at least 50% of that of the references kept in the dark.
The negative control had an absorbance of 0.4 or higher. Chlorpromazine, the positive control, had a Cl50 comprised between 0.1 and 2 μg/mL in the presence of UVA and comprised between 7 and 90 μg/mL in the absence of UVA. These results enabled the test to be validated, The concentration of peregrina protein hydrolysate in accordance with Example 1 providing 50% cell death in the presence or absence of UVA could not be estimated. The mortality never reached 50%. The concentration of peregrina protein hydrolysate giving 50% cell viability in the presence or absence of UVA could not be estimated. The viability was always more than 50%.
Conclusion: Under the experimental conditions employed, the peregrina protein hydrolysate in accordance with the invention may be considered to be non-phototoxic,
This in vitro study was based on an evaluation of the cytotoxicity of the peregrina protein hydrolysate by determining the concentration causing 50% cell death (IC50) on a cellular monolayer with the aid of the neutral red leaching technique. The cells used were SRC, rabbit cornea fibroblasts (ATCC-CCL60) which were free from mycoplasma.
The peregrina protein hydrolysate was diluted with physiological serum to 25% and 50%. The fibroblasts were trypsinized and two 24-well culture plates were inoculated In an amount of 1 mL of a 2×105 cells/mL cell suspension in complete culture medium, The inoculated plates were incubated in an oven overnight at 37° C. and 5% CO2. At the end of incubation, confluence of the cell mat was verified. The staining solution was prepared in a concentration of 0.5 mg/mL in complete culture medium. The culture medium was eliminated; 1 mL of the staining solution was deposited into each well. The plates were replaced in the incubator at 37° C. and 5% CO2 for 3 h+/−15 minutes. After this contact period, the staining solution was eliminated and replaced by 1 mL of complete culture medium per well. The plates were maintained at ambient temperature for at least 30 minutes in order to stabilize the system before contact with the extract or the reference, Each well was rinsed with 2 mL of PBS, maintained at ambient temperature, then 500 μL of each dilution of peregrina protein hydrolysate or reference was deposited in contact with the cell mat, The contact time was 60 seconds (30 seconds for the positive control). The treatment was carried out well by well, starting the clock at the moment of depositing the peregrina protein hydrolysate or the reference. The plate was agitated manually throughout the treatment period. After 55 seconds (or 25 seconds for the positive control), the dilution was sucked off. At exactly 60 or 30 seconds, 5 successive rinses were carried out (5×2 mL of PBS maintained at ambient temperature). The supernatant was sucked off after each rinse and after the last rinse, the wells were kept free from medium while awaiting the revealing phase. After the treatment of the culture plate was complete, 1 mL of the desorption solution was deposited into each well. The plate was agitated for approximately 15 minutes until homogeneous staining was obtained. The solutions obtained for each culture well were removed and divided into 2 wells of a 96-well plate, i.e. 150 μL/well.
The concentration of the peregrina protein hydrolysate providing 50% cell death was evaluated at >50%. The percentage cell death at 50% peregrina protein hydrolysate was evaluated to be 17%.
Conclusion: Under the experimental conditions employed, the cytotoxicity of the peregrina protein hydrolysate in accordance with the invention could be classified as being: negligible cytotoxicity.
The aim of this study was to evaluate the degree of skin compatibility of the peregrina protein hydrolysate by epicutaneous testing, carried out on the outer anterior surface of the arm for 48 hours; and in general to evaluate the capacity of the protein hydrolysate to maintain the skin in a good condition. 10 healthy male or female volunteers aged from 18 to 65 years who had neither dry skin nor sensitive skin and who were free from any dermatological lesions in the treatment zone could be included in the study. The skin compatibility of the peregrina protein hydrolysate, prepared in the form of a lotion with 5% peregrina protein hydrolysate in accordance with Example 1 and 95% of a propane diol/sorbitol mixture were evaluated 48 hours after initial application, 30 to 40 minutes after removing the dressing. The reactions of the skin (erythema and edema) were given a score of 0 to 3 in accordance with the following scales:
All of the skin reactions (blisters, papules, vesicles, dryness, desquamation, roughness, soap effect, etc.) were evaluated in accordance with the following scale and reported descriptively:
At the end of the study, a mean irritation index (M.I.I.) was calculated using the following formula:
M.I.I.=sum of skin reactions (E+Oe+blisters+papules+vesicles)/number of volunteers analyzed [Math. 4]
The M.I.I. obtained enabled the tested peregrina protein hydrolysate to be classified using the following scale:
Results: The Mean Irritation Index (M.I.I.) of the peregrina protein hydrolysate is equal to: 0.
Conclusion: The peregrina protein hydrolysate may be considered to be non-irritating after 48 consecutive hours of application to 12 volunteers.
The results of the tests carried out above are conclusive, and demonstrate for the peregrina protein hydrolysate in accordance with Example 1:
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2005426 | May 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/063704 | 5/21/2021 | WO |
