FR3100716A1 - Cosmetic use of jellyfish collagen. - Google Patents

Abstract

The present invention relates to the cosmetic use of Rhizostoma pulmo collagen as an anti-aging agent, as well as its use as a restructuring, regenerating, revitalizing, moisturizing, anti-wrinkle and / or strengthening agent, elasticity. and / or the barrier effect of the skin. The invention also relates to a cosmetic composition comprising this collagen, as well as to an anti-aging cosmetic care process consisting in applying the composition according to the invention to an area of the skin.

Description

Cosmetic use of jellyfish collagen.

The present invention relates to the cosmetic use of collagen from Rhizostoma pulmo as an anti-aging agent, as well as its use as a restructuring, regenerating, revitalizing, moisturizing, anti-wrinkle and/or firming, elasticity and/or the barrier effect of the skin. The invention also relates to a cosmetic composition comprising this collagen, as well as an anti-aging cosmetic care process consisting in the application of the composition according to the invention to an area of the skin.

Skin aging is a complex phenomenon that is due to many intrinsic (genetic factors) and extrinsic (such as the sun, diet, exposure to cigarette smoke) factors.

Clinically, we observe the appearance of wrinkles and fine lines, a loss of skin elasticity, a relaxation of the skin and subcutaneous tissues as well as less good healing. Many research avenues are proposed to fight against skin aging, including protection against the environment (sun, pollution, etc.), activation of cell regeneration, strengthening of the extracellular matrix (collagen and elastin) . Recently, studies have shown the importance of the adhesion of cells to each other on the one hand, and of the adhesion between cells and the extracellular matrix on the other hand, in the process of skin aging and therefore of the need strengthen them to prevent or even treat sagging skin.

Interactions between cells and the extracellular matrix (ECM) play an important role in controlling cell behavior. This control is carried out by specific interactions between the matrix molecules and the cells at the level of transmembrane receptors, mainly of the integrin family, whose intracellular domain is linked to the constituents of the cytoskeleton. This allows the matrix to ensure the transmission of intracellular signals modulating, depending on the case, the adhesion, migration, proliferation, differentiation or apoptosis of the cells of the dermis. This control of cellular behavior is crucial during development but also during tissue remodeling.

Collagen is the main protein of the extracellular matrix (ECM) and the most abundant in animals, representing 25% of the total protein content and between 70 to 80% of that of the skin (in dry matter). The central feature of all collagen molecules is their rigid three-stranded helical structure. Types I and III are the main types of collagen found in connective tissue and make up 90% of all collagen in the body.

Collagen is made by several types of specialized cells, including fibroblasts, and is assembled in connective tissues. Fibroblasts secrete collagen, elastin, growth factors, but also enzymes to degrade the extracellular matrix, renew it and reorganize it. The loss of collagen associated with aging is often concomitant with a loss of elastin and hyaluronic acid. Overall, these phenomena lead to a reduction in the hydration, suppleness and elasticity of the skin and cause a thinning of the face.

The cosmetics industry is constantly awaiting new compositions capable of effectively increasing the adhesion of cells to the extracellular matrix and/or the adhesion of cells to each other, and therefore of having an anti-aging, restructuring action. , regenerating, revitalizing, moisturizing, anti-wrinkle and/or reinforcing firmness, elasticity and/or the barrier effect of the skin.

The Applicant has identified, surprisingly and unexpectedly, that the collagen from the R. Pulmo jellyfish has anti-aging, restructuring, regenerating, revitalizing, moisturizing, anti-wrinkle and/or reinforcing firmness, elasticity and/or the barrier effect of the skin.

According to a first aspect, the invention relates to the cosmetic use of collagen from Rhizostoma pulmo as an anti-aging agent.

Collagen is one of the most abundant proteins in the extracellular matrix of animal bodies. The central feature of all collagen molecules is their rigid triple-stranded helical structure. Types I and III are the main types of collagen found in connective tissue and make up 90% of all collagen. Besides its role in mechanical resistance, collagen functions as a signaling molecule, regulating cell migration (Greenberg, et al., 1981), differentiation (Bosnakovski, et al., 2005) and proliferation (Pozzi, et al. , 1998), and functions in hemostasis (Farndale, et al., 2004).

" Rhizostoma pulmo " is a scyphomedusa of the family Rhizostomatidae . It is found in the northeastern and southern Atlantic, in the Adriatic, the Mediterranean, the Black Sea and the Sea of Azov. It is blue and generally measures up to 40 cm in diameter, its umbrella can measure up to one meter. It has four arms that are divided into eight arms welded to the manubrium, and has no tentacles around its umbrella. Small fish, such as bugs, amberjacks and horse mackerel, take shelter under his umbrella or between his arms. The ends of its eight arms contain symbiotic algae: zooxanthellae, which in exchange for housing and light produce food, the unused excess of which is consumed by jellyfish. R. pulmo collagen is made up of two chains 1 and 2 and contains a heparin binding site which would not be located in the triple helix but rather in the linear portion of the protein.

By anti-aging agent is meant according to the invention an agent having an action of prevention and/or delay and/or limitation of the signs of skin aging, in particular accelerated (exogenous) aging caused by environmental stress or by factors environmental stresses, for example ultraviolet rays (photo-induced stress), or chronological aging (endogenous stress). This anti-ageing action can go through an action of regenerating the skin and/or reducing the slackening of the skin or the loss of firmness of the skin or a loss of elasticity of the skin, in particular a prevention and/or a delay and/or a limitation of relaxation, and/or a firming action and/or an improvement in the biomechanical qualities of the skin. Advantageously, it can promote firming of the structures of the skin and/or a reduction in the formation of wrinkles in the skin, its appendages or the mucous membranes. The term "improving the appearance of the skin", within the meaning of the present invention, means any effect making it possible to obtain an advantage on the appearance of the skin compared to the appearance of the skin before the use of the collagen according to the invention. The term “skin regeneration”, within the meaning of the present invention, means the metabolic stimulation of epidermal or dermal skin cells, the renewal of which is directly or indirectly linked or targeted by intrinsic or extrinsic ageing. The term "reduction in relaxation/loss of firmness/loss of elasticity" within the meaning of the present invention, an action of inhibition of matrix metalloproteinases (or metalloproteinases) (MMP, for example MMP3) which degrade the collagen and/or elastin of the skin, its appendages and/or mucous membranes. It may involve a protective action, and/or maintenance action, and/or reinforcement of the existing fibers in the dermis (collagen and/or elastin) produced by the dermal cells. Advantageously, this action can make it possible to protect, to make it last and/or to improve the biomechanical qualities of the dermis, in particular the firmness, the volume, the density and the resistance of the tissue. The cosmetic use of the invention can advantageously make it possible to improve the surface, and therefore aesthetic, qualities of the epidermis, such as smoothing the skin and/or softening the skin. The term “smoothing the surface of the skin”, within the meaning of the present invention, means the qualitative and quantitative reduction of the microreliefs of the skin, in particular of their depth. It may be for example a reduction in the depth of wrinkles and/or fine lines. The term "softening the skin", within the meaning of the present invention, the obtaining of a silkier effect of the skin to the touch after use of the extract, and/or a reduction in the roughness effect of the skin to the touch.

Preferably, the cosmetic use of collagen from Rhizostoma pulmo is as a preventive and/or curative anti-aging agent.

Preferably, said Rhizostoma pulmo collagen is intended to be used for:
To. promote the synthesis of the extracellular matrix, the organization and the structuring of collagen fibers by activating the COL3A1, FBN1, FBN2, SPARC, MMP3 and LUM genes;
b. promote better dermal cohesion and better structuring of the basal layer of cells by activating the COL7A1, LAMA5 and CDC42 genes;
vs. support the renewal and protection of the dermis through the activation of epithelial growth factor (EGF), SIRT4 and SIRT6;
d. support the improvement of the structure of the dermal cell junction by the activation of the EPPK and LOR genes; and or
e. reduce inflammation by reducing the expression of IL-8, IL6, IL10, and IL-18, in sensitive skin.

According to a second aspect, the invention relates to the cosmetic use of collagen from Rhizostoma pulmo as a restructuring, regenerating, revitalizing, moisturizing, anti-wrinkle agent and/or reinforcing the firmness, the elasticity and/or the barrier effect of the skin.

By restructuring agent is understood according to the invention a compound making it possible to structure the different layers of the skin and its elements, for example by improving the connection between collagen and elastin in the dermis, by improving the organization of collagen fibers in the dermis or the basal lamina, and/or by reinforcing the adhesion of the cells to each other and to the proteins of the epidermis and of the dermis. This can be achieved by improving in particular the synthesis of genes such as the genes coding for collagens, or fibrillins.

By regenerating agent is meant according to the invention a compound allowing the metabolic stimulation of epidermal or dermal skin cells, the renewal of which is directly or indirectly linked or targeted by intrinsic or extrinsic ageing.

By revitalizing agent is understood according to the invention a compound making it possible to stimulate cellular metabolism, resulting in an improvement in the appearance of the skin, including radiance and shine.

By moisturizing agent is understood according to the invention an agent which makes it possible to maintain or increase the water content of the various layers of the skin, in particular the superficial layers, and to retain this water in the skin.

By anti-wrinkle agent is meant according to the invention a compound allowing the increase in the synthesis and / or the activity of certain proteins (for example fibrillins or collagen), when it is brought into contact with an area of skin wrinkles, this effect having the consequence of reducing the appearance of wrinkles and/or fine lines. This agent can also have a tightening and/or smoothing effect on the skin.

By agent reinforcing the firmness of the skin is understood according to the invention a compound allowing the increase in the synthesis of the proteins involved in the firmness of the skin, such as elastin.

By agent reinforcing the elasticity of the skin is understood according to the invention a compound allowing the increase in the synthesis of the proteins involved in the elasticity of the skin, such as elastin.

By agent reinforcing the barrier effect of the skin is understood according to the invention a compound making it possible to act in particular on the stratum corneum or stratum corneum, for example by reducing the insensible loss of water, or by activating genes coding for proteins involved in the formation of this layer of the skin. Among the genes involved, we can name the Loricrin gene (LOR).

According to a third aspect, the invention relates to an anti-ageing cosmetic composition comprising collagen from Rhizostoma pulmo .

Preferably, said collagen is in the form of a hydrolyzate. Even more preferably, said hydrolyzate is in an acid medium.

By hydrolyzate is understood according to the invention a product resulting from any chemical reaction in which a molecule of water breaks one or more chemical bonds. This term covers substitution, elimination and fragmentation reactions in which water is the nucleophile.

Preferably, said hydrolyzate is in the form of a solution, hydrogel or freeze-dried.

By hydrogel is understood according to the invention a network of soluble collagen fibers which are prevented from dissociating by entanglement of polymers and/or covalent crosslinking. They can also be formed from colloidal suspensions. The physical properties of hydrogels can be tuned depending on the manufacturing route.

By freeze-dried is understood according to the invention that the water has been removed from the collagen according to the invention, by a freezing step followed by a vacuum step.

Preferably, said collagen is present from about 0.01% to about 5%, preferably from about 0.1% to 1%, even more preferably from about 0.4% to 0.6% , by weight of the composition.

Preferably, said composition comprises at least one other cosmetically acceptable agent, preferably chosen from soothing agents, restructuring agents, regenerating agents, revitalizing agents, sun filters, anti-wrinkle agents, moisturizing agents, anti-aging agents. -ages, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents enhancing the firmness, elasticity and/or barrier effect of the skin, antioxidants , opacifiers, stabilizers, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestrants and chelators, perfumes, filters, essential oils, coloring materials, pigments, hydrophilic active ingredients or lipophilic, lipid vesicles encapsulating one or more active agents and/or preservatives.

Preferably according to the invention, said cosmetic composition is in the form of a cream, an ointment, an ointment, a mask, a milk, a lotion, a serum, an a paste, a mousse, an aerosol, a stick, a shampoo, a conditioner, patches, an aqueous hydroalcoholic or oily solution, an oil- in water or water-in-oil or multiple, of an aqueous or oily gel, of a liquid, pasty or solid anhydrous product, and/or of a dispersion of oil in an aqueous phase using spherules, these spherules possibly being polymeric nanoparticles such as nanospheres and nanocapsules or lipid vesicles of the ionic and/or non-ionic type.

The composition of the invention may also contain the usual adjuvants in the cosmetic and dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers, filters , pigments, odor absorbers and dyestuffs. The amounts of these various adjuvants are those conventionally used in the fields considered, and for example from 0.01 to 20% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the fatty phase, into the aqueous phase, into the lipid vesicles and/or into the nanoparticles.

According to a fourth aspect, the invention relates to an anti-aging cosmetic care process consisting of the application to an area of the skin of a composition according to the invention.

Preferably, an amount of approximately 0.1 to 50 mg/cm2, preferably approximately 0.25 to 2.5 mg/cm2, of cosmetic composition according to the invention is applied to the area of the skin, so as to even more preferably about 0.5 to 1 mg/cm2.

Preferably, the cosmetic composition according to the invention is applied once or twice a day, preferably twice a day, for at least 7 days, preferably at least 15 days, more preferably at least one month, from particularly preferably at least 1 month, and more particularly preferably at least 2 months. Very particularly preferably, the cosmetic composition is applied in the process according to the invention twice a day for at least 1 month.

Preferably according to the invention, said area of the skin is the face.

According to a fifth aspect, the invention relates to a method for manufacturing the hydrolyzed collagen of R. Pulmo according to the invention, comprising the steps of:
To. Heating R. Pulmo Soluble Collagen to between about 40°C to about 100°C for 10 minutes to 60 minutes, preferably between about 60°C to about 80°C for 45 minutes to 60 minutes;
b. Recovery of said hydrolyzate.

According to one embodiment;
- Step a. includes the addition of a protease; And
- The method comprises a step a' of purification of the composition obtained at the end of step a.

Proteases, or peptidases, are enzymes that catalyze the hydrolysis of peptide bonds found in proteins and polypeptides. Proteases are divided into two groups: exopeptidases and endopeptidases, depending on the site of action on the polypeptide chains. Exopeptidases act on the ends of polypeptide chains and endopeptidases act randomly in the inner regions of polypeptide chains (Raveendran et al. 2018). Preferably, the proteases according to the invention are capable of hydrolyzing marine invertebrate collagen, for example pepsin, matrix metallopeptidase, papain and/or procollagen peptidase. More preferably, said protease is pepsin or a pepsin homolog.

Other characteristics, objects and advantages of the invention will emerge from the description which follows, which is purely illustrative and not limiting, and which must be read in conjunction with the appended drawings in which:

Figure 1 shows immunostaining results for General Morphology. A indicates the result of the check at T0; B indicates the control batch on day 8 (D8); C indicates the batch with P1 to J8; D indicates batch P2 on day 8; E indicates batch P3 on day 8 (D8).

Figure 2 shows immunostaining results for total collagen. A indicates the result of the check at T0; B indicates the control batch on day 8 (D8); C indicates the batch with P1 to J8; D indicates batch P2 on day 8; E indicates batch P3 on day 8 (D8).

Figure 3 shows the results of immunostaining for elastin. A indicates the result of the Negative Control without anti-elastin antibodies; B indicates the control batch on day 8 (D8); C indicates the control batch at D8; D indicates batch P1 on day 8; E indicates batch P2 on day 8 (D8), F indicates batch P3 on day 8 (D8).

Figure 4 shows the results of immunostaining for Loricrin. A indicates the result of the negative control without primary antibody; B indicates the control batch on day 0 (D0); C indicates the control batch on day 8 (D8); D indicates batch P1 on day 8 (D8); E indicates batch P2 on day 8 (D8).

Figure 5 shows the results of SIRT-4 immunostaining. A indicates the result of the negative control without primary antibody; B indicates the control batch on day 0 (D0); C indicates the control batch on day 8 (D8); D indicates batch P1 on day 8 (D8); E indicates batch P2 on day 8 (D8).

Figure 6 shows the results of SIRT-6 immunostaining. A indicates the result of the negative control without primary antibody; B indicates the control batch on day 0 (D0); C indicates the control batch on day 8 (D8); D indicates batch P1 on day 8 (D8); E indicates batch P2 on day 8 (D8).

Figure 7 shows the results of collagen VII immunostaining. A indicates the result of the negative control without primary antibody; B indicates the control batch on day 0 (D0); C indicates the control batch on day 8 (D8); D indicates batch P1 on day 8 (D8); E indicates batch P2 on day 8 (D8).

Figure 8 shows the results of collagen III immunostaining. A indicates the result of the negative control without primary antibody; B indicates the control batch on day 0 (D0); C indicates the control batch on day 8 (D8); D indicates batch P1 on day 8 (D8); E indicates batch P2 on day 8 (D8).

Figure 9 shows the results of the immunostaining of collagen V. A shows the result of the negative control without the primary antibody; B shows the control batch on day 0 (D0); C shows the control batch on day 8 (D8); D shows batch P1 on day 8 (D8); E shows batch P2 on day 8 (D8).

FIG. 10 presents the comparative results of the study of the synthesis of collagen by human fibroblasts under the conditions of Example 3 (application of collagen according to the invention), in light gray for 1, 5, 10, 100 and 500 μg/ml, reference ID-16/11341, and example 4 (Peptan® SR Marine application), in dark gray for 10 and 100 μg/ml reference ID-16/09538. A positive control for TGF-β at 10 μg/ml is also indicated.

Figure 11 shows the results of an immunoassay evaluating IL-8 production in dTHP-1 human macrophage cells in the presence and absence of hydrolyzed R. Pulmo collagen according to the invention and activated with LPS (endotoxin) in the presence or not of marine invertebrate collagen according to the invention. CRPH stands for Hydrolyzed R. Pulmo Collagen.

FIG. 12 presents the results of an immunoassay evaluating IL-1 Beta production in dTHP-1 human macrophage cells in the presence and in the absence of hydrolyzed R. Pulmo collagen according to the invention and activated with LPS (endotoxin ) in the presence or absence of marine invertebrate collagen according to the invention. CRPH stands for Hydrolyzed R. Pulmo Collagen.

Figure 13 shows the results of an immunoassay evaluating IL-18 production in dTHP-1 human macrophage cells in the presence and absence of hydrolyzed R. Pulmo collagen according to the invention and activated with LPS (endotoxin) in the presence or not of marine invertebrate collagen according to the invention. CRPH stands for Hydrolyzed R. Pulmo Collagen.

FIG. 14 presents the results of the SDS PAGE analysis of the collagens of R. pulmo according to the invention. 1A: Lane 1 is the molecular weight marker (from top to bottom: 250, 150, 100, 75, 50, 37 kD); lanes 2-4 are 1, 5 and 10 μg of the jellyfish proteins respectively). Please note that molecular weights are approximate. 1B: SDS PAGE of collagen extracted from R. Pulmo showing the bands of collagen present in the solution. Lanes 1 and 2: Reference scale for high molecular weight proteins. Lanes 3-6: non-collagenous proteins removed from solution before testing. Lanes 7-10: Collagen solution containing 1, 2, 1 and γ collagen chains.

Examples:

Example 1: Evaluation of anti-aging activity by immunostaining

Material and method :

The aim is to evaluate the anti-aging activity of a solution based on hydrolyzed Rhizostoma pulmo collagen from ex vivo skin explants.

The activity is evaluated by:
- A control of cell viability after staining with Masson's trichrome
- Observation of total collagen after picro-Sirius staining
- Immunolabelling of elastin

Concentrations and denomination of the products tested:

Product Concentrations P1 0.1mg/mL P2 1mg/mL P3 8.25mg/mL

The products were stored at 4°C during and after the duration of the study.

1. Preparation of explants:

15 explants of about 1cm in diameter were prepared from an abdominoplasty of a 51-year-old woman with dark skin.

The explants were left to survive in BEM medium (BIO-EC's Explants Medium) at 37°C in a humid atmosphere, enriched with 5% CO2.

Distribution of the explants into 5 lots, as follows:

Batches Designation Concentration Number of explants Stop T0 Plasty control / 3 D0 T Unprocessed control / 3 D8 P1 Product 1 0.1mg/mL 3 D8 P2 Product 2 1mg/mL 3 D8 P3 Product 3 8.25mg/mL 3 D8

2. Application of products:

On D0, D1, D5 and D7, the explants were treated by topical application of products P1 to P3, at the rate of 2 mg per explant spread out using a spatula.

The untreated batches did not receive any treatment, except half the medium renewal (1ml per well) on D1, D5 and D7.

The treatment and sampling days have been modified to adapt the study schedule to working days.

3. Samples:

On day D0, 3 explants from the T0 batches were removed and cut in two. One part was fixed in a buffered formalin solution, the other was stored at –80°C.

On D8, 3 explants from each batch were removed and treated in the same way.

4. Histological treatments:

After 24 hours in buffered formalin, the samples were dehydrated and impregnated with paraffin using a Leica PEARL dehydration machine.

They were blended using a Leica EG 1160 embedding station.

5 μm sections were made using a Minot type microtome, Leica RM2125 and mounted on Superfrost® histological glass slides.

The microscopic observations were carried out by optical microscopy, using a Leica type DMLB or Olympus BX43 microscope. The shots were taken with an Olympus DP72 camera and Cell^D software.

To. Examination of general morphology (viability check)

General morphology was examined on paraffin sections stained with Masson's trichrome variant of Goldner, in order to assess the viability of dermal and epidermal structures.

General morphology was assessed by microscopic examination.

b. Picro-Sirius staining

Staining of total collagen was performed on paraffin sections with a solution of picro-Sirius

Staining was assessed by microscopic observation and image analysis.

vs. Immunolabelling of elastin

Elastin was labeled on formalin paraffin sections with a polyclonal anti-elastin antibody (Novotec, ref. 25011), diluted to 1/400th in PBS-BSA 0.3%-Tween 20 at 0.05% for 1 hour at room temperature, and revealed in AlexaFluor 488 (Lifetechnologies, ref. A11008).

Labeling was performed using an immunolabeling machine (Autostainer, Dako).

Labeling was assessed by microscopic examination.

Results :

1. General morphology (cell viability)

The results are shown in Figure 1.

General morphology:

Batches Cell viability Comments Epidermis Papillary dermis T0 B B / TJ8 B B 3 to 4 cell layers P1J8 B B 6 to 7 cell layers - Fairly marked hyperplastic and hypertrophic acanthosis P2J8 B B 4 to 5 cell layers - Slight hyperplastic and hypertrophic acanthosis P3J8 B B 4 to 5 cell layers - Slight hyperplastic and hypertrophic acanthosis Morpho legend: B= good, AB= fairly good, TLA= very slightly altered, LA= slightly altered, MA= moderately altered, ANA= fairly markedly altered, NA= markedly altered, TNA= very markedly altered

On D0, the viability of the cells of the epidermis and the papillary dermis is good.

On D8, on the control batch TD8, the viability of the cells of the epidermis and of the papillary dermis is good.

Effect of the products on cell viability compared to the control batch TJ8:
- The P1 product does not induce alterations in cell viability, but induces a fairly marked increase in the number of cells (hyperplastic acanthosis) and in the size of the cells of the epidermis (hypertrophic acanthosis).
- The P2 product does not induce alterations in cell viability, but induces a slight increase in the number of cells (hyperplastic acanthosis) and in the size of cells in the epidermis (hypertrophic acanthosis).
- The P3 product does not induce alterations in cell viability, but induces a slight increase in the number of cells (hyperplastic acanthosis) and in the size of cells in the epidermis (hypertrophic acanthosis).

2. Total Collagen (Sirius)

The results are shown in Figure 2.

On D0, the staining of total collagen represents 91.78% of the surface of the dermis.

Effect on total collagen

Total collagen (sirius) T0 TJ8 P1J8 P2J8 P3J8 Mean 91.78 94.27 92.43 95.57 94.28 Standard deviation 2.08 2.20 1.23 0.77 1.06

On D8, on control batch TJ8, collagen synthesis is significantly increased by 3% (p<0.1 (90%)) compared to batch T0.

Effect of the products on collagen synthesis compared to the control batch TJ8:
- Product P1 induces a significant reduction of 2%* (p<0.1 (90%)).
- Product P2 induces a non-significant increase of 1%.
- The P3 product does not induce variation.

3. Elastin

The results are shown in Figure 3.

On D0, on the control group T0, the labeling of elastin is quite clear to clear in the papillary dermis.

On D8, on the TJ8 control batch, the expression of elastin is quite markedly reduced compared to the T0 batch.

Effect of the products on the expression of elastin compared to the control batch TJ8:
- Product P1 induces a net increase
- The product P2 induces a very clear increase
- The P3 product induces a strong increase

Findings:

Conclusions under the control operating conditions above and compared to the control batch T on D8 (TJ8):

Variation vs TJ8 Jellyfish Collagen Concentration = 0.1 mg/mL (P1) Jellyfish Collagen Concentration = 1 mg/mL (P2)
Jellyfish Collagen Concentration = 8.25 mg/mL (P3) General morphology (viability check) Fairly clear increase in the thickness of the epidermis Slight increase in the thickness of the epidermis Slight increase in the thickness of the epidermis Total collagen (picrosirius) -2% (significant with p<0.05 (95%)) 1% (not significant) Elastin Marked increase in elastin expression Very clear increase in the expression of elastin Strong increase in the expression of elastin

Scale = Strong Decrease < Very Definite < Definite < Fairly Definite < Moderate < Slight Decrease < No Variation < Slight Increase < Moderate < Fairly Definite < Definite < Very Definite < Strong Increase

Thus, the hydrolyzed jellyfish collagen according to the invention induces the expression of elastin, in a dose-dependent manner, with the strongest activity at 8.25 mg/mL (P3). According to this study, there does not seem to be any significant activity on the synthesis of total collagen. This product also induces an increase in the thickness of the epidermis, with the strongest activity at 0.1 mg/ml.

Example 2: Evaluation by immunolabeling of the synthesis of loricrin, sirtuins-4 and 6, and collagens III, VII and V.

This study is the continuation of Example 1, and aims to achieve the following immunostaining:
- Immunolabelling of loricrin
- Immunolabelling of sirtuin-4
- Immunolabelling of sirtuin-6
- Immunolabelling of collagen VII
- Immunolabelling of collagen III
- Immunolabelling of collagen V.

The products P1 (0.1 mg/mL) and P2 (1 mg/mL) of example 1 are studied in this example, on the same explants as those used in example 1, and also for 8 days, against a plasty control (T0), and an untreated control (T).

1. Histological treatments

5 μm sections were made using a Minot type microtome, Leica RM2125 and mounted on Superfrost® histological glass slides.

7 μm sections were made using a Leica CM 3050 cryostat and mounted on Superfrost® Plus silanized histological glass slides.

The microscopic observations were carried out by optical microscopy, using a Leica type DMLB or Olympus BX43 microscope. The shots were taken with an Olympus DP72 camera and Cell^D software.

To. Immunolabelling of loricrin

Loricrin was labeled on formalin paraffin sections with a polyclonal anti-loricrin antibody (Covance, ref. PRB-14P), diluted to 1/1600th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 hour at room temperature , with a biotin/streptavidin enhancer system, revealed in VIP, a purple peroxidase substrate (Vector, ref. SK 4600).

The labeling was carried out using an immunolabeling machine (Autostainer, Dako) and it was evaluated by microscopic examination.

b. Immunolabelling of sirtuin-4 (SIRT-4)

Sirtuin-4 was labeled on formalin paraffin sections with a polyclonal anti-sirtuin-4 antibody (GeneTex, ref. GTX51798), diluted to 1/100th in PBS-0.3% BSA-0.05% Tween 20 for 1 h at room temperature, with a biotin/streptavidin enhancer system, revealed in VIP, a violet peroxidase substrate (Vector, ref. SK 4600).

The labeling was carried out using an immunolabeling machine (Autostainer, Dako) and it was evaluated by microscopic examination.

Batches concerned: T0, TJ8, P1J8 and P2J8.

vs. Sirtuin-6 (SIRT-6) immunostaining

Sirtuin-6 was labeled on formalin paraffin sections with a polyclonal anti-sirtuin-6 antibody (Novus Biologicals, ref. NB100-2522), diluted to 1/100th in PBS-0.3% BSA-0.05% Tween 20 for 1 h at room temperature, with a biotin/streptavidin enhancer system, revealed in VIP, a violet peroxidase substrate (Vector, ref. SK 4600).

The labeling was carried out using an immunolabeling machine (Autostainer, Dako) and it was evaluated by microscopic examination.

Batches concerned: T0, TJ8, P1J8 and P2J8.

d. Immunolabelling of collagen VII

Collagen VII was labeled on frozen sections with an anti-collagen VII monoclonal antibody (Santa Cruz, ref. sc-53226, clone LH 7.2), diluted to 1/100th in PBS-BSA 0.3%-Tween 20 at 0.05% for 1 h at room temperature, and revealed in AlexaFluor 488 (Lifetechnologies, ref. A11008).

The labeling was carried out using an immunolabeling machine (Autostainer, Dako) and it was evaluated by microscopic examination.

Batches concerned: T0, TJ8, P1J8 and P2J8.

e. Immunolabelling of collagen III

Collagen III was labeled on frozen sections with a polyclonal anti-collagen III antibody (SBA, ref. 1330-01), diluted to 1/100th in PBS-0.3% BSA-0.05% Tween 20 for 1 hour at room temperature. ambient, with a biotin/streptavidin enhancer system, revealed in VIP, a violet substrate of peroxidase (Vector, ref. SK 4600).

Labeling was done manually and was assessed by microscopic examination.

Batches concerned: T0, TJ8, P1J8 and P2J8.

f. Immunolabelling of collagen V

Collagen V was labeled on formalin paraffin sections with a polyclonal anti-collagen V antibody (Novotec, ref. 20511), diluted to 1/100th in PBS-BSA 0.3%-Tween 20 at 0.05% for 1 hour at room temperature. , with a biotin/streptavidin enhancer system, revealed in VIP, a purple peroxidase substrate (Vector, ref. SK 4600).

The labeling was carried out using an immunolabeling machine (Autostainer, Dako) and it was evaluated by microscopic examination.

Batches concerned: T0, TJ8, P1J8 and P2J8.

Results and Conclusions:

• Results for loricrin are shown in Figure 4.
  On D0, on the control batch T0, the loricrin labeling is fairly clear to clear in the granular layer.
  On D8:
  On the control group TJ8, the expression of loricrin is moderate in the granular layer.
  Effect of the products on the expression of loricrin, compared to the control batch TJ8:
  - Product P1 induces a slight increase
  - Product P2 induces a slight decrease.
• The results for SIRT-4 are shown in Figure 5.
  On D0, on the control batch T0, the labeling of sirtuin-4 is quite clear in the epidermis.
  On D8,
  On the control group TJ8, the expression of sirtuin-4 is fairly clear to clear in the epidermis.
  Effect of the products on the expression of sirtuin-4, compared to the control batch TJ8:
  - Product P1 induces a slight decrease
  - The product P2 induces a moderate reduction
• The results for SIRT-6 are shown in Figure 6.
  On D0, on the control batch T0, the labeling of sirtuin-6 is fairly clear to clear in the epidermis.
  On D8,
  On the control group TJ8, the expression of sirtuin-6 is clear in the epidermis.
  Effect of the products on the expression of sirtuin-6, compared to the control group TJ8:
  - Product P1 induces a slight decrease
  - The product P2 does not induce any variation.
• The results for collagen VII are shown in Figure 7.
  On D0, on the control batch T0, the labeling of collagen VII is weak along the dermo-epidermal junction.
  On D8,
  On the control batch TJ8, the expression of collagen VII is low to moderate along the dermo-epidermal junction.
  Effect of the products on the expression of collagen VII, compared to the control group TJ8:
  - Product P1 induces a moderate increase
  - Product P2 induces a moderate increase.
• The results for collagen III are shown in Figure 8.
  On D0, on the control batch T0, the labeling of collagen III is quite clear in the papillary dermis.
  On D8,
  On the control group TJ8, the expression of collagen III is markedly weak in the papillary dermis.
  Effect of the products on the expression of collagen III, compared to the control batch TJ8:
  - Product P1 does not induce any variation
  - The product P2 does not induce any variation.
  
• The results for collagen V are shown in Figure 9.
  On D0, on the control group T0, the labeling of collagen V is clear to very clear in the papillary dermis.
  On D8,
  On the control group TJ8, the expression of collagen V is clear in the papillary dermis.
  Effect of the products on the expression of collagen V, compared to the control batch TJ8:
  - Product P1 induces a moderate reduction
  - The product P2 induces a fairly clear decrease

A summary of the results of these immunolabels is presented in Table 6.

Results of immunostaining for products P1 and P2:

Variation vs TJ8 P1 P2 Loricrin Slight increase
Slight decrease
Sirtuin-4 Slight decrease Moderate decrease Sirtuin-6 Slight decrease No variation Collagen VII Moderate increase Moderate increase Collagen III No change No change Collagen V Moderate decrease Quite a marked decrease

Scale = Strong Decrease < Very Clear < Clear < Quite Clear < Moderate < Slight Decrease < No Variation < Slight Increase < Moderate < Fairly Clear < Clear < Very Clear < Strong Increase

In conclusion, the hydrolyzed collagen according to the invention shows, whatever its concentration, an effect of moderate increase in the synthesis of collagen VII, and a slight increase in the synthesis of loricrin at 0.1 mg/mL.

Example 3: In vitro study of the effect of the collagen according to the invention on a primary culture of human fibroblast.

A fter application of the collagen according to the invention on the cells for 24 hours, the intracellular and extracellular collagen is quantified with the Sirius red dye (Direct red 80) which has a particular affinity for the triple helix structure (Gly-XY) _n of the native collagen (collagen type I to V). The absorbance of the dye-collagen complex is measured at 540 nm. The total protein fraction is evaluated, after sonication, by the Bradford method (Bradford et al., 1976).

• Test system:

Cells: primary human fibroblasts prepared in accordance with current work instruction IL 14.

The cells are cultured in DMEM medium 4.5 g/l glucose, 2 mM L-glutamine or stabilized glutamine, 10% heat-inactivated fetal calf serum (FCS), 50 IU/ml penicillin, 50 μg/ml of streptomycin.

The cells are maintained in a humid atmosphere (37° C., 5% CO2).

The cells are free of mycoplasma. The mycoplasma detection test was carried out according to the current work instruction IL 07.

Cells were used at passage 6.

• Reference Items:

Negative control: 1% FCS culture medium.

Positive control: Transforming growth factor β1 (TGFβ) at 10 ng/ml in culture medium containing 1% FCS

• Definition of series:

5 concentrations of the collagen according to the invention were tested. The initial concentration of hydrolyzed collagen (8.25 mg/ml) was reduced to 500, 100, 10, 5 and 1 μg/ml.

Each condition of the collagen according to the invention and of the reference elements was tested on three culture wells.

• Test protocol:

Seeding of the cells : The cells were seeded at 50,000 cells/cm2 in 24-well culture plates and then were incubated overnight (37° C., 5% CO2).

Contact between the cells and the test element or the reference elements : The dilutions of the test element or the reference elements were carried out in culture medium at 1% FCS. The culture medium was aspirated and replaced with 500 μl of the different concentrations of the test element or the reference elements. The plates were incubated for 24 hours ± 1 hour (37°C, 5% CO2).

Evaluation of collagen synthesis and cell density: The culture medium of each well was removed and the cell layer was rinsed with 500 μl of 2-fold concentrated protease inhibitor cocktail. The whole, culture medium and inhibitor, was pooled in the same tube.

The cell layer of each well was recovered by scraping in 500 μl of concentrated protease inhibitor cocktail once and the wells were rinsed again with 500 μl of cocktail once. The two volumes, which constitute the extracellular matrix, were brought together in the same tube and treated with an ultrasound probe for 40 seconds.

The collagen from each fraction was complexed with Sirius red dye and, after centrifugation and washing with 0.1M HCl, the pellet was solubilized in 0.5M NaOH. The absorbance of the dye-collagen complex was measured at 540 nm. A calibration range was established under the same conditions between 0 and 10 μg per tube of collagen.

The amount of total protein was assessed using Bradford's method (Bradford et al. Anal Biochem 1976; 72:248-54) against a calibration range established from a 400 μg BSA solution /ml in PBS. 30 μl of each sample was mixed with 280 μl of Bradford's reagent in a 96 well plate. The plate was incubated for approximately 15 minutes at room temperature in the dark. Absorbances were measured at 620 nm against a reagent blank consisting of Bradford's reagent.

• Results :

Stimulation of collagen synthesis by collagen hydrolyzate from Rhhisostoma pulmo

Protein Concentration Cell matrix collagen Collagen culture medium Concentration μg protein /well/ Mean
Standard deviation μg collagen /
well/ % stimulation /
Negative control Mean
Standard deviation μg collagen /
well/ % stimulation /
Negative control Collagen according to the invention 500 μg/ml
54.78
3.8 0.256
0.015 2.60 73 0.233
0.023 2.37 0 100 μg/ml
64.64
9, 0.251
0.042 2.55 70 0.250
0.040 2.54 7 10 μg/ml
62.17
3.5 0.272
0.022 2.77 84 0.252
0.050 2.56 0 5 μg/ml
56.81
3.9 0.205
0.020 2.09 39 0.226
0.028 2.30 0 1 μg/ml
50.58
7.0 0.195
0.043 1.99 32 0.225
0.032 2.29 0 Positive control
TGF-β 10ng/ml 65.87
2.8 0.210
0.031 2.14 42 0.217
0.029 2.20 0 Negative control 50.14
4.1 0.148
0.029 1.50

Concerning the effect of the collagen according to the invention, one can notice a significant increase in the synthesis of collagen in the cellular matrix with a maximum at 84% for the concentration at 10 μg/ml of product (Figure 10 in light gray for 1 , 5, 10, 100 and 500μg/ml, reference ID-16/11341).

Example 4: Comparative data against hydrolyzed fish collagen (Peptan SR Marine).

The same experiments as those carried out in Example 3 were carried out for hydrolyzed fish collagen ( ^Peptan® SR Marine, marketed by Rousselot) at 500, 100 and 10 μg/ml.

The results are as follows:

Stimulation of collagen synthesis by hydrolyzed fish collagen.

Protein Concentration Cell matrix collagen Collagen culture medium Concentration μg protein /well/ Mean
Standard deviation μg collagen /
well/ % stimulation /
Negative control Mean
Standard deviation μg collagen /
well/ % stimulation /
Negative control 500 μg/ml
54.78
3.8 0.256
0.015 2.60 73 0.233
0.023 2.37 0 100 μg/ml
64.64
9, 0.251
0.042 2.55 70 0.250
0.040 2.54 7 10 μg/ml
62.17
3.5 0.272
0.022 2.77 84 0.252
0.050 2.56 0 5 μg/ml
56.81
3.9 0.205
0.020 2.09 39 0.226
0.028 2.30 0 1 μg/ml
50.58
7.0 0.195
0.043 1.99 32 0.225
0.032 2.29 0 Hydrolyzed fish collagen 500 μg/ml
95.22
1.2 0.144
0.019 1.47 0 0.254
0.021 2.58 9 100 μg/ml
66.09
0.6 0.214
0.030 2.18 45 0.212
0.015 2.15 0 10 μg/ml
50.87
6.1 0.249
0.012 2.53 68 0.222
0.031 2.26 0 Positive control
TGF-β 10ng/ml 65.87
2.8 0.210
0.031 2.14 42 0.217
0.029 2.20 0 Negative control 50.14
4.1 0.148
0.029 1.50

Thus, hydrolyzed fish collagen exhibits a maximum effect of 68% at a concentration of 10 μg/ml. The response seems inversely proportional to the concentration, and remains lower than the values obtained with the collagen according to the invention (Figure 10).

Example 5: Inflammatory activation of macrophages by the hydrolyzed collagen according to the invention

Human macrophages, dTHP-1 cells, were cultured in the presence and absence of hydrolyzed R. Pulmo collagen according to the invention and activated with LPS (endotoxin) - again in the presence or absence of marine invertebrate collagen according to the invention.

These cells are of human origin and constitute a highly valued and validated cell model used on a large scale in the study of inflammatory diseases.

Interleukin-1Beta, IL6, IL8 and IL18 were all determined by standard immunoassays and IL10 by RT-PCR.

Figure 11 shows that R. Pulmo's collagen hydrolyzate induces a reduction in IL-8 secretion, alone or in the presence of LPS.

No secretion of IL6 or IL10 was demonstrated with marine cells treated with invertebrate collagen hydrolyzate.

These results confirm that this collagen has no pro-inflammatory action on NF-kB mediated inflammatory or regulatory cytokines.

Given these results, two other cytokines activated by the NLRP3 inflammasomal pathway rather than NF-kB, IL1-Beta and IL18, were examined.

This pathway is an alternative to the NF-kB pathway and is attracting growing interest in the pathology of many diseases.

Figure 12 shows that the R. Pulmo collagen hydrolyzate according to the invention does not induce a significant increase in the secretion of IL-1 Beta, in the presence of LPS.

Figure 13 shows that the collagen hydrolyzate of R. Pulmo according to the invention induces a reduction in the secretion of IL-18, in the presence of LPS.

These results confirm that the R. Pulmo collagen hydrolyzate according to the invention does not act in a pro-inflammatory manner in the presence of macrophages.

Moreover, these data are not limited to a single inflammatory pathway but are related to the NF-kB and NLRP3 inflammatory pathways.

Example 6: Evaluation of the collagen according to the invention on the expression of genes within human skin explants, by microfluidic RT-qPCR, at a lower concentration and compared to fish collagen.

Biological model

The NativeSkin® skin explant is used, which is a biopsy of human skin bathed in a solid and nourishing matrix and whose epidermal surface is kept in contact with the air to allow topical application.

The explants come from an abdominoplasty of a 32-year-old woman corresponding to a pigmentation of 3 on the Fitzpatrick scale.

The hydrolyzed collagens used in this example were concentrated at 100 μg/ml.

Product evaluation

The experiment was carried out in triplicate under the following conditions:
- Skin explant (control) untreated (n=3)
- Skin explant + hydrolyzed collagen according to the invention (n=3)
- Skin explant + hydrolyzed fish collagen (n=3)

The explants were treated with the product for 2 days: one application in the morning and one application in the evening.

NB: Hydrolyzed fish collagen seems to weaken the epidermis. Indeed, after treatment, the explants were prepared for RNA extraction by perforating them to remove the skin not exposed to the product. Note that the epidermis was slightly detached from the dermis.

At the end of the culture phase, the explants were removed from their inserts and microfluidic RT-qPCR was used to characterize the gene expression of the skin explants.

Results / Conclusions

Data analysis

The specificity of each pair of primers is checked for all conditions by analysis of the dissociation curve and problematic points with double peaks are eliminated from the analysis. Each CT is then normalized by the reference genes and the control condition to calculate the ΔΔCT and the relative expression is calculated as a function of the ΔΔCT by the formula 2-∆∆CT.

For each condition, the standard error with respect to the mean (SEM) is calculated and the points having an excessive SEM (> 40% difference between the SEM and the mean of the 3 values) are eliminated from the analysis. The results are expressed as percentage of overexpression or underexpression relative to the reference condition.

The results are presented here by dividing them into 4 categories, in view of the gene expression for each of these indications:
- Beneficial activity: AB
- Globally positive activity temporized by negative activities: AGP
- Globally negative activity, temporized by beneficial activities: AGN
- Negative activity: AN

Effects of hydrolyzed collagen according to the invention, and of hydrolyzed fish collagen.

Products tested Concentration Healing Anti-Aging (AAC) Preventive anti-aging (PAA) AAFC + AAP Synthesis and Organization MEC Cohesion and Basal Lamina MEC Protection and Basal Lamina Hydration Survival, Growth Renewal Antioxidant Protection Protection (Anti-infl) Differentiation (Barrier, Physical Protection) Hydrolyzed collagen according to the invention 100 μg/ml AGP AB AGN / AGP AGP AGN AB Hydrolyzed fish collagen 100 μg/ml AGN AGP AGN YEAR AGP AGP YEAR AGN

These results therefore show an effect of the hydrolyzed collagen according to the invention in curative anti-aging on the Synthesis and Organization aspects of the extracellular membrane (ECM) and cohesion and basal lamina, as a preventive measure in survival, growth and cell renewal and antioxidant protection, as well as a joint effect in differentiation.

Among the genes found to be positively influenced by collagen according to the invention are:

Effect of hydrolyzed collagen according to the invention on the gene expression of human skin explants.

Genoa % increase or decrease in expression COL3A1 +60% COL5A1 +110% DCN +140% FBN1 +80% FBN2 +490% LOX +80% LUM +270% SPARC +100% CDC42 +60% COL7A1 +170% LAMA5 +70% EGF +130% SIRT4 +340% SIRT6 +60% MGST1 +230% EPPK1 +100% EVPL +80% FLG +60% LOR +130% MMP3 -80%

Effect of hydrolyzed fish collagen on gene expression of human skin explants.

Genoa % increase or decrease in expression LOXL1 +180% P4HA1 +120% COL1A1 - 90% COL5A1 -60% DCN -70% SPARC -70% CD44 +60% CDC42 +80% COL4A1 +120% CTNNA1 +90% ITGA2 +170% LAMC2 +430% PXN +200% SDC4 +440% COL17A1 -70% COL7A1 -90% FN1 -60% LAMA5 -90% SDC1 -60% MMP9 -80% PI3 +3230% TIMP1 +130% MMP1 +2390% MMP3 +180% AQP1 -60% AQP3 -60% HBEGF +1000% SIRT6 +140% TINF2 +70% IGFBP6 +140% GLRX +70% SIRT3 +80% SOD2 +570% CAT -70% MSRB2 -90% COX2 +240% IL1A +4350% IL6 +380% IL8 +9280% S100A7 +790% TNFA +1150% IVL +540% TGM1 +520% EVPL -60% FLG -90% LOR -60% POMC +90% DKK1 +200%

The collagen according to the invention is capable of increasing the synthesis of LOXL1, P4HA1, CD44, CDC42, COL4A1, CTNNA1, ITGA2, LAMC2, PXN, SDC4, PI3, TIMP1, MMP1, MMP3, HBEGF, SIRT6, TINF2, IGFBP6, GLRX, SIRT3, SOD2, COX2, IL1A, IL6, IL8, S100A7, TNFA, IVL, TGM1, POMC, DKK1, and decrease the synthesis of COL1A1, COL5A1, DCN, SPARC, COL17A1, COL7A1, FN1, LAMA5, SDC1, MMP9 , AQP1, AQP3, CAT, MSRB2, EVPL, FLG, LOR.

The increase in the expression of the EPPK1, EVPL, FLG, LOR genes has an effect on the barrier function. Indeed, these genes code for proteins involved in the formation of the stratum corneum and thus promote the barrier function of the epidermis, the first defense of the skin against external aggressions and ensure the maintenance of skin hydration. The barrier function allows both a preventive effect but also a restructuring and moisturizing effect. The collagen according to the invention promotes the water balance of the skin.

The increase in the expression of the genes COL3A1, COL5A1, DCN, FBN1, FBN2, LOX, LUM and SPARC characterizes an effect on the neosynthesis and the structuring of the dermal matrix concerning all its components: Collagen fibers, elastic fibers and proteoglycans . The dermal structuring activity is also very strong since LOX, involved in fiber assembly, is stimulated. The two structural proteins Fibrillin 1 (FBN1) and 2 (FBN2) are also activated. Activation of the SPARC gene which encodes osteonectin is also observed.

The increase in the expression of the CDC42, COL7A1 and LAMA5 genes characterizes an effect on the restructuring of the basal lamina which constitutes the dermo-epidermal junction and the strengthening of the anchoring of cells to this basal lamina. This stimulates the dermis/epidermis interactions which diminish with age.

The reduction in the expression of the MMP3 gene promotes the protection of the extracellular matrix and the masal lamina, since this gene codes for an enzyme capable of degrading many components of the extracellular matrix: fibronectin, laminin, collagens and proteoglycans.

The increase in the expression of the EGF, SIRT4 and SIRT6 genes has an effect on cell longevity. Sirtuins are proteins that globally protect cells.

Hydrolyzed fish collagen increases and decreases the expression of different genes, and therefore has different properties.

The properties of the collagen according to the invention, and of the hydrolyzed fish collagen can be summarized in the following table 12:

Summary of the effect of collagen according to the invention and of hydrolyzed fish collagen.

Regeneration of the dermis – anti wrinkles Restructuring / firming Regenerating / revitalizing Preventive anti-aging Barrier function Collagen from R. Pulmo hydrolyzed +++ ++ ++ / ++ Hydrolyzed fish collagen / ++ +

Example 7: Jellyfish collagen according to the invention.

The collagen according to the invention is produced from soluble collagen from R. Pulmo, by heating the latter between approximately 40° C. to approximately 100° C. for 10 minutes to 60 minutes, preferably between approximately 60° C. to approximately 80° C. for 45 minutes to 60 minutes, followed by a step for recovering said hydrolyzate.

The physico-chemical characteristics of the collagens of R. pulmo (hydrolyzed or not) are given in table 13.

Physico-chemical characteristics of R. pulmo collagens

Features Collagen (JCOL) Hydrolyzed Collagen (HJCOL) Aspect Liquid Liquid Color Colorless to pale yellow Colorless to pale yellow Solvent 0.1M Acetic Acid 0.5M Acetic Acid Concentration 3-6 mg.mL ^-1 3-10 mg.mL ^-1 (8.25 mg.mL ^-1 ) Turbidity Clear to slightly hazy Clear to slightly hazy Protein rate > 80% > 80% pH 2.8-3.2 2.8-3.5

SDS-PAGE analyzes of these collagens were carried out (FIG. 14). They show very distinct bands of the different collagen chains, which allows us to suggest that the collagen is not degraded during its extraction.

Jellyfish collagen was also tested for cytotoxicity. The biocompatibility of jellyfish collagen (3.7 mg.mL ^-1 ) was evaluated by measuring cytotoxicity on a cell culture (L929 murine fibroblasts), according to the ISO 10993-5 2009 method by Indirect Contact. The results do not indicate that any of the samples tested produced cytotoxicity under the assay conditions used. This indicates that jellyfish collagen is not toxic to this cell culture.

Example 8: In vivo evaluation of the effect of the application of composition C according to the invention.

The composition according to the invention in serum form comprises between about 0.4 and 0.6% by weight of the hydrolyzed collagen composition of R. Pulmo.

A serum placebo is prepared comprising the same composition without the hydrolyzed collagen from R. Pulmo.

28 female volunteers, with an average age of 55 years [40 to 75 years], with a heterogeneous complexion, applied the above composition twice a day for 1 month on the half-face and on a forearm against a placebo. Single-blind study:

Evaluation by expert judges (4):
- Firmness
- Skin elasticity
- Glow and shine of the skin
- Appearance of wrinkles
- Smoothing of the skin
- Hydrated appearance of the skin
- General anti-aging aspect

After 1 month of application, 4 expert judges complete a questionnaire on 5 criteria. For each criterion, a score between 0 and 7 is assigned.

After 1 month of application of this composition, an improvement on all the criteria presented is observed compared to T0 and the placebo. The expert judges observe visible results on all these criteria.

Skin firmness:

After 1 month, according to the expert judges, the composition according to the invention improves the firmness of the skin.

Skin Elasticity:

After 1 month, according to expert judges, the skin seems to have regained better elasticity.

Skin radiance and shine

After 1 month of application of the composition according to the invention, according to the expert judges, the skin has an improved radiance and is shinier.

Appearance of wrinkles:

According to the expert judges, the composition according to the invention improves the appearance of wrinkles, which are less pronounced, after only 1 month of application.

Skin smoothing

After 1 month, according to the expert judges, the composition according to the invention allows the skin to appear smoother, and to reduce its microreliefs.

Hydrated appearance of the skin

After 1 month of application of the composition according to the invention, according to the expert judges, the skin appears more hydrated.

General anti-aging aspect

According to the expert judges, after 1 month of application, the composition according to the invention improves the general anti-ageing appearance of the skin.

Claims (10)

Cosmetic use of Rhizostoma pulmo collagen as an anti-aging agent. Cosmetic use of collagen from Rhizostoma pulmo as a restructuring, regenerating, revitalizing, moisturizing, anti-wrinkle agent and/or reinforcing the firmness, elasticity and/or barrier effect of the skin. Anti-aging cosmetic composition comprising collagen from Rhizostoma pulmo . Cosmetic composition according to the preceding claim, characterized in that the said collagen is in the form of a hydrolyzate. Cosmetic composition according to the preceding claim, characterized in that the said hydrolyzate is in the form of a solution, a hydrogel or freeze-dried. Cosmetic composition according to Claims 3 to 5, characterized in that the said collagen is present from approximately 0.01% to approximately 5%, preferably from approximately 0.1% to 1%, even more preferably from approximately 0.4% to 0.6%, by weight of the composition. Cosmetic composition according to Claims 3 to 6, characterized in that it comprises at least one other cosmetically acceptable agent, preferably chosen from soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents reinforcing firmness, elasticity and/or skin barrier effect, antioxidants, opacifiers, stabilizing agents, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestrants and chelators, perfumes, filters, essential oils, dyestuffs, pigments, hydrophilic or lipophilic active agents, lipid vesicles encapsulating one or more active agents and/or preservatives. Cosmetic composition according to Claims 3 to 7, in the form of a cream, an ointment, an ointment, a mask, a milk, a lotion, a serum, a paste , a mousse, an aerosol, a stick, a shampoo, a conditioner, patches, an aqueous hydroalcoholic or oily solution, an oil-in-water emulsion or water-in-oil or multiple, of an aqueous or oily gel, of a liquid, pasty or solid anhydrous product, and/or of a dispersion of oil in an aqueous phase using spherules, these spherules which may be polymeric nanoparticles such as nanospheres and nanocapsules or lipid vesicles of the ionic and/or non-ionic type. Anti-ageing cosmetic care process consisting in the application to an area of the skin of a composition according to claims 3 to 8. Anti-ageing cosmetic care process according to the preceding claim, characterized in that the said area of the skin is the face.