WO2009095600A2

WO2009095600A2 - Novel cellular growth factor of non-animal origin and applications

Info

Publication number: WO2009095600A2
Application number: PCT/FR2009/050105
Authority: WO
Inventors: Jean-Noël THOREL; Hugues Gatto; Jean-Claude Hubaud
Original assignee: Developpement Industrialisation Et Promotion De Technologies Avancees
Priority date: 2008-01-23
Filing date: 2009-01-23
Publication date: 2009-08-06
Also published as: FR2926461A1; WO2009095600A3; FR2926461B1

Abstract

The invention relates to a cellular growth factor of non-animal origin, characterized in that it contains L-Alanyl-L-Glutamine, associated with at least one β-glucane.

Description

NOVEL NON-ANIMAL CELL GROWTH FACTOR AND APPLICATIONS

The subject of the invention is a novel "cell growth factor" of non-animal origin and its applications. In particular, the growth factor is intended to be used in anti-aging cosmetic compositions, especially as a substitute for retinoids, but also in normal nutrient media of fibroblasts and / or normal human keratinocytes.

In the field of cosmetics, numerous compositions have been developed to fight against aging of the skin, against the appearance of wrinkles as well as to promote the regeneration of the skin, in particular in the elderly. These different applications are generally grouped under the "anti-aging" indication. Several mechanisms can be behind this indication. One of them consists in stimulating, directly or indirectly, fibroblast growth, or the synthesis of structural proteins.

Among the molecules capable of stimulating the growth of fibroblasts, the retinoids have in particular been described. Although the effect on the growth of fibroblasts is effective, the major disadvantage of retinoids remains their phototoxic and potentially mutagenic action, which is documented in the article

"Photomutagenicity of retinyl palmitate by ultraviolet irradiation in mouse lymphoma cells" Nan Mei et al Toxicological Sciences 88 (1), 142-149 (2005).

Furthermore, the Applicant has described in WO96 / 21421, an innovative concept consisting in developing aqueous nutrient media comprising essentially vitamins, amino acids and minerals for incorporation into cosmetic compositions, said nutrient media being suitable for create an extracellular environment perfectly adapted to the epidermis. These nutrient media have the essential feature of being completely devoid of animal growth factors, such as fetal calf serum and beef pituitary stalk extract. Also excluded from the medium are medicated growth factors not usable in cosmetics, such as EGF, insulin, cholera toxin, hydrocortisone, ethanolamine, phosphoethanolamine, etc. The stimulating activity of the cell growth is demonstrated with respect to normal human keratinocytes. These media can therefore be incorporated in cosmetic compositions but can also be used as such, as culture medium of normal human keratinocytes in vitro.

In FR-A-2 864 445, the Applicant has described an improved medium of the same type as the previous one, in which growth is observed not only of keratinocytes but also of normal human fibroblasts. The medium is more particularly adapted to the general indication "anti-aging".

The two nutrient media illustrated in these documents contain an essential amino acid, in this case L-glutamine, used as a source of cellular energy. This component is the component in greater proportion after water and sodium chloride and represents respectively 0.17% (WO96 / 21421) and 0.2% (FR-A-2,864,445) by weight of the medium. The major disadvantage of this amino acid remains its instability over time, requiring adjustment before use

The Applicant has now developed a new cell growth factor of non-animal origin based on stabilized glutamine and β glucan favoring the stimulation of the growth of fibroblasts and keratinocytes.

Such a product, which has been shown to be non-phototoxic, is therefore, in cosmetics, a potential substitute for retinoids and all products having an activity on preventing the effects of age on the skin. . This new growth factor also has performances at least comparable to those of animal-derived cell growth factors, thus making it a candidate for the substitution of fetal calf serum and pituitary beef stem extracts in standard culture of fibroblasts or keratinocytes. From this property, it follows from the possibility of using the growth factor of the invention in bases or nutrient media of the same type as those described in WO96 / 21421 and FR-A-2 864 445, said growth factor additionally being substituted in whole or in part for L glutamine present in these media.

In other words, the invention relates to a cell growth factor of non-animal origin, which is characterized in that it contains, as glutamine, stabilized glutamine, in this case L alanyl Glutamine, advantageously combined, in a solvent, with at least one β glucan.

The β glucan is advantageously β 1.3 glucan, preferably β 1.3, β 1.6 glucan, that is to say a branched β 1-3 glucan with β 1.6 glucans.

In a particular embodiment, the β 1.3, β 1.6 glucan is laminarine. As a laminarin source, the invention advantageously uses a laminar extract.

The Applicant has found that all stabilized glutamines are unsuitable. In particular, N acetyl L glutamine associated with a laminar extract has no effect on cell growth. L alanyl L glutamine is in practice in solution, although it can also be used in the form of a powder. In practice, the concentration of pure L alanyl L glutamine in the growth factor is between 0.01 and 99.9% by weight.

The laminar is advantageously chosen as Laminaria digitata. It is a large brown alga rarely emerged, growing on the rocks of the subtidal floor to a depth of 10 m, in areas of moderate or high current. The laminar contains in practice between 10 and 30% by weight of laminarin.

According to the invention, the laminar extract is obtained from thalli or parts of thalli in fresh, frozen, dry, whole, fragmented or milled form. The extract is an extract obtained by any technique known to those skilled in the art, such as by example maceration, decoction, leaching in a solvent, in particular water, followed in particular by a concentration. The extract can be in dry or liquid form. In an advantageous embodiment, the extract is an aqueous extract preferably containing between 20 and 80% by weight of dry matter, advantageously between 40 and 60% by weight. Such an aqueous extract in practice represents between 0.01% and 99.9% by weight of the growth factor.

The subject of the invention is also a cosmetic composition containing the growth factor described above, possibly in the presence of at least one cosmetic vehicle.

In an advantageous embodiment, the cosmetic composition does not contain retinoids.

In practice, the growth factor, which consists of pure L alanyl L glutamine representing between 0.01 and 99.99% of its weight, the 100% complement being constituted by a β glucan, advantageously β 1.3 glucan, of preferably β 1.3, β 1.6, in practice pure laminarine or in the form of an aqueous laminar extract, the latter representing between 0.05 and 20%, advantageously from 0.5 to 5% by weight of the cosmetic composition.

The cosmetic composition of the invention is generally applied topically.

It may be in any galenical form normally used for topical application to the skin, especially in the form of an oil-in-water or water-in-oil or multiple emulsion, a silicone emulsion, a microemulsion or nanoemulsion. This composition may be more or less fluid and have the appearance among others of a white or colored cream, an ointment, a milk, a lotion, a serum or a gel.

The composition of the invention may contain the usual vehicles in the cosmetic and dermatological fields, such as fats, emulsifiers and co-emulsifiers, hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers, hydrophilic and lipophilic filters, dyestuffs, neutralizers, protective agents penetrants, and polymers.

The cosmetic composition may also contain active ingredients. In this case, the presence of the growth factor makes it possible to enhance or potentiate the action of the other active ingredients. As active agents, depigmentants, anti-radicals, emollients, moisturizers, anti-seborrhoeic agents, anti-inflammatories, anti-acne agents, keratolytic and / or desquamating agents, anti-wrinkle agents and tensors, draining agents, anti-irritant agents, soothing agents, slimming agents, vitamins and their mixtures, mattifying agents, cicatrizants, antiseptics and essential oils.

The composition of the invention can be advantageously used to combat aging of the skin and / or the appearance of wrinkles and / or to promote the regeneration of the skin and more generally as an anti-aging.

The subject of the invention is also the use of the previously described growth factor for the manufacture of a healing medicament.

In a particular embodiment, the cosmetic composition contains a nutrient base of the same type as that described in document FR-A-2 864 445, in which the growth factor of the invention is incorporated.

More specifically, the invention also relates to a cosmetic composition which is characterized in that it comprises an aqueous nutrient base excluding the presence of growth factor of animal origin, as well as the following growth factors: EGF, insulin, toxin cholera, hydrocortisone, ethanolamine, phosphoethanolamine, said base consisting of amino acids, vitamins, inorganic components, and additionally containing the growth factor based on L alanyl L glutamine and laminar extract, as described above . In this embodiment, the growth factor contains 10 to 100 times less pure L alanyl L glutamine than aqueous laminar extract. In an advantageous embodiment, the proportions of L alanyl L glutamine and extract in the growth factor and the proportion of growth factor as such in the base, calculated to obtain a concentration of 0.005 to 10%, advantageously equal to 0.01% by weight of L alanyl L glutamine and a concentration of 0.1 to 5%, advantageously equal to 1% by weight of an aqueous laminar extract. For this purpose, the growth factor contains, for example, from 0.1 to 10% by weight of L alanyl L glutamine, the 100% complement consisting of the aqueous laminar extract, the growth factor representing in this case between 0.001 and 2% by weight of the nutritional base.

In this case, the cosmetic composition may be used as it is, in particular in the form of sprayable or formulated lotions in the presence of cosmetic vehicle (s). It can also be incorporated into a cosmetic base, to formulate a cream or other.

As already stated, the growth factor of the invention also finds application as a substitute for calf serum or beef pituitary stalk extract in in vitro cell culture or survival medium.

The invention therefore also relates to an in vitro culture medium of normal human fibroblasts containing, as a growth factor, the previously described growth factor, that is to say a medium excluding calf serum extracted from beef pituitary stalk, EGF, insulin, cholera toxin, hydrocortisone, ethanolamine, phosphoethanolamine.

The subject of the invention is also an in vitro culture medium of normal human keratinocytes containing, as a growth factor, the previously described growth factor. Finally, the subject of the invention is also a medium for the survival of normal human keratinocytes intended for the survival maintenance of cutaneous substitutes (epithelial sheets) before grafting, in particular for burn victims.

The invention also relates to a normal human fibroblast survival medium intended for the survival or culturing of cutaneous pre-transplantation substitutes, in particular for burn victims (in particular fibroblast cultures in collagen matrices).

When the growth factor is used in culture or survival media, it represents between 0.001 and 20% by weight of the medium, the growth factor itself containing from 0.1 to 10% by weight of L alanyl L glutamine, the complement 100% consisting of β glucans, in practice present in the form of an aqueous laminar extract. In practice, the growth factor represents between 0.05 and 2%, advantageously 1% by weight of the fibroblast culture medium. Similarly and always in practice, the growth factor represents between 0.005 and 1%, advantageously 0.1% by weight of the keratinocyte culture medium.

The invention and the advantages resulting therefrom will emerge more clearly from the following exemplary embodiments, in support of the appended figures.

Figure 1 is a graphic representation of the effect of the aqueous Laminar / L Glutamine extract association on the growth of normal human fibroblasts in DMEM media without FCS.

Figure 2 is a graphical representation of the effect of Laminar / L Alanyl L Glutamine aqueous extract association on the growth of normal human fibroblasts in DMEM without SVF.

Figure 3 is a graphical representation of the effect of the aqueous Laminar / N Acetyl L Glutamine combination on the growth of normal human fibroblasts in DMEM without FCS. Figure 4 is a graphical representation of the effect of Laminar / L Glutamine aqueous extract association on the growth of normal human fibroblasts in PBS medium.

Figure 5 is a graphical representation of the effect of the aqueous Laminar / L Alanyl L Glutamine extract association on the growth of normal human fibroblasts in the nutrient base designated EVE Pharma.

Figure 6 is a graphical representation of the effect of the aqueous Laminar / L Alanyl L Glutamine extract association on the growth of normal human keratinocytes in the EVE Pharma nutrient base. Figure 7 is a graphical representation of the effect of the aqueous extract of Laminaria combination / L Alanyl L Glutamine on the growth of normal human fibroblasts in DMEM medium without FCS versus RETINOL to 10 ^"6 M and ^{10" 7} M added to DMEM medium without FCS.

FIG. 8 is a graphical representation of the effect of the aqueous Laminar / L Alanyl L Glutamine extract association on the growth of normal human fibroblasts in DMEM without SVF versus TRANS RETINAL (Retinaldehyde) at 10 ^-6 M and 10 ^{" 7} M added to DMEM medium without FCS.

Figure 9 is a graphical representation of the effect of Laminar / L Alanyl L Glutamine aqueous extract association on the growth of normal human fibroblasts in DMEM media without SVF versus RETINOL PALMITATE at 10 ^"6 M and 10 ^"

⁷ M added to DMEM medium without FCS.

Figure 10 is a graphical representation of the effect of the aqueous extract of Laminaria combination / L Alanyl L Glutamine on the growth of normal human fibroblasts in DMEM medium without FCS versus RETINYL ACETATE 10 ^"6 M and ^{10" 7} M added in the middle DMEM without SVF.

Material:

The products used in the examples are as follows: The total concentrations are expressed relative to the medium. Aqueous extract of laminaria 40% dry matter obtained by cold maceration then concentration from fresh seaweed. L Glutamine (G7513 Sigma 100X) Alanyl L 200mM Glutamine (PAN) N Acetyl L Glutamine (Kyowa Hakko) DMEM (Sigma) PBS

Nutritional base "EVE Pharma":

Example 1: Influence of Glutamine Type Associated with Laminar Extract on the Growth of Normal Human Fibroblasts

1 / Objective

The objective of this study is to evaluate the effect of the aqueous Laminar extract, associated or not with three types of Glutamine, L. Glutamine, L Alanyl L Glutamine, N Acetyl L-Glutamine on growth. normal human fibroblasts cultured for 6 days. Each of the Glutamines is added to the medium at the final concentration of 2 mM.

The study is carried out on a culture carried out in the standard fibroblast culture medium, DMEM, without addition of fetal calf serum, as well as in a PBS buffer medium (with CaCl ₂ and MgCl ₂ ) and in which are added the different assets studied. The study is carried out in comparison with a culture performed under standard culture conditions (DMEM medium supplemented with 10% fetal calf serum).

2 / Technique The fibroblasts are inoculated at a low density (5000 cells / well) in a 96-well plate in standard medium (DMEM + 10% FCS) and grow for 24 hours after seeding in this medium. In the 2 ^nd day, the cells are placed in different conditions studied.

• Control: DMEM (containing L Glutamine at 2 mM) + SVF 10%

• DMEM, without L Glutamine, without FBS

• DMEM, without L Glutamine, without FBS + 1% Laminar Extract

• DMEM, without L Glutamine, without FBS + L Glutamine (2mM) + Laminar Extract 1% • DMEM, without L Glutamine, without SVF + L Alanyl L Glutamine (2 mM)

• DMEM, without L Glutamine, without SVF + L Alanyl L Glutamine (2 mM) + Laminar Extract 1%

• DMEM, without L Glutamine, without SVF + N Acetyl L Glutamine (2 mM) • DMEM, without L Glutamine, without FBS + N Acetyl L Glutamine (2 mM) + Laminar Extract 1%

• PBS buffer (with CaCl ₂ and MgCl ₂ , NaHCO ₃ , Glucose (4.5 g / l) = concentrations identical to those of DMEM) • PBS + L Glutamine buffer (2 mM)

• PBS + L Glutamine Buffer (2mM) + 1% Laminar Extract

Each condition is done in quadruplicate. The media are not renewed during the experiment.

Cell density is measured 24 h after cell seeding, prior to contact different study conditions (= TO) and then fibroblast growth is assessed the ^era '4 ^th' 5 ^th and 6 ^th days of culture by the conversion method of WST-I (reading at 450 nm).

3 / Results

FIG. 1 presents the results obtained for the culture of normal human fibroblasts in DMEM medium in the presence of aqueous extract of Laminar +/- addition of L Glutamine.

> In the absence of L Glutamine, Laminar extract at the concentration of 1% in the DMEM medium, does not allow cell growth.

> The addition of L. Glutamine (2 mM) to DMEM stimulates the growth of normal human fibroblasts.

> L Glutamine (2 mM) and 1% Laminar aqueous extract in DMEM strongly stimulate the growth of normal human fibroblasts.

Figure 2 shows the results obtained for the culture of normal human fibroblasts in DMEM medium in the presence of L Alanyl L Glutamine +/- addition of aqueous Laminar extract. > The results obtained with L Alanyl L Glutamine, alone or in combination with the aqueous Laminar extract, are in all respects similar to those obtained with L. Glutamine.

Figure 3 shows the results obtained for the culture of normal human fibroblasts in DMEM medium in the presence of N-acetyl L Glutamine +/- addition of aqueous Laminar extract.

> The addition of N Acetyl L Glutamine (2 mM) to DMEM medium does not stimulate the growth of normal human fibroblasts.

The addition of 1% Laminar aqueous extract combined with N-acetyl L Glutamine (2 mM) in DMEM does not stimulate the growth of normal human fibroblasts.

Figure 4 shows the results obtained for the culture of normal human fibroblasts in PBS buffer in the presence of Glutamine +/- addition of aqueous Laminar extract.

In PBS medium (with CaCl ₂ and MgCl ₂ , NaHCO ₃ , Glucose (4.5 g / l) = concentrations identical to those of DMEM), there is an absence of growth of normal human fibroblasts.

Addition of L. glutamine (2 mM) to PBS buffer allows mild growth of normal human fibroblasts.

The combination of L Glutamine (2 mM) and 1% Laminar aqueous extract in PBS buffer increases the growth stimulating effect of normal human fibroblasts observed with L Glutamine.

Since Figures 1 and 2 show that substitution of L Glutamine by L Alanyl L Glutamine gives identical results, it can also be expected that the use of L Alanyl L Glutamine in PBS medium gives identical results to those obtained with L Glutamine. EXAMPLE 2 Effect of L Alanyl L Glutamine Associated with a Laminar Extract on the Growth of Normal Human Fibroblasts Cultured in the Nutrient Base (EVE Pharma)

The objective of this study is to evaluate the effect of Alanyl L Glutamine Laminar aqueous extract on the growth of normal human fibroblasts cultured for 7 days in EVE PHARMA.

The nutrient base contains, as Glutamine, L Alanyl L Glutamine (stabilized Glutamine).

The study is conducted under the same conditions as in Example 1

Figure 5 shows the results obtained for culturing normal human fibroblasts in the nutrient base in the presence of L Alanyl L Glutamine and aqueous Laminar extract.

> The addition of aqueous laminar extract at a concentration of 1% to the nutrient base (containing Alanyl L Glutamine), with or without MPC (MiIk Peptide Complex) allows the growth of normal human fibroblasts over 7 days of cultures. .

EXAMPLE 3 Effect of Alanyl L Glutamine Associated with a Laminar Extract on the Growth of Normal Human Keratinocytes Cultured in the Nutrient Base EVE Pharma.

The objective of this study is to evaluate the effect of Alanyl L Glutamine Laminar aqueous extract on the growth of normal human keratinocytes cultured for 5 days in the EVE Pharma nutrient base.

The nutrient base contains as Glutamine, L Alanyl L Glutamine (stabilized glutamine). The study is conducted under the same conditions as in Example 1 but on keratinocytes.

Figure 6 shows the results obtained for culturing normal human keratinocytes in the nutrient base in the presence of L Alanyl L Glutamine and aqueous Laminar extract.

> The addition of aqueous Laminar extract at a concentration of 0.1% to the EVE nutrient base (containing Alanyl L Glutamine) without MPC (MiIk Peptide Complex) allows the growth of normal human keratinocytes comparable to that obtained in the EVE nutrient base (with MPC).

Example 4 Effect of L Alanyl L Glutamine Associated with a Laminar Extract on the Growth of Normal Human Fibroblasts in DMEM Medium versus 4 Retinoids

1 / Objective of the study

The objective of this study is to evaluate the effect of 4 retinoids on the growth of normal human fibroblasts, comparative study versus effect of the aqueous extract complex of Laminar and Alanyl L Glutamine.

The effect of retinoids on cell growth is evaluated on a culture performed in the standard fibroblast culture medium, DMEM, without addition of fetal calf serum, and in which retinoids are added at concentrations of 10 ^-6 M and ^10-7 M (non-cytotoxic tested concentrations). The effect of the complex of the invention is evaluated on a culture performed in DMEM medium without FCS, in the presence of L Alanyl L Glutamine and in which the 1% Laminar extract is added. 2 / Technical

The fibroblasts are inoculated at a low density (3000 cells / well) in a 96-well plate in standard medium (DMEM + 10% FCS) and grow for 24 hours after seeding in this medium.

In the 2 ^nd day, the cells are placed in different conditions studied.

• Control: DMEM + SVF 10% (DMEM complete) • DMEM + DMSO at 0.0001% (solvent control retinoids)

• DMEM without SVF + L Alanyl L 2mM Glutamine + 1% Laminar Extract

• DMEM without SVF + RETINOL at 10 ^"6 M (DMSO solvent)

• DMEM without SVF + RETINOL at 10 ^"7 M (DMSO solvent) • DMEM without SVF + TRANS RETINAL at 10 ^{" 6} M (DMSO solvent)

• DMEM without SVF + TRANS RETINAL at 10 ^"7 M (DMSO solvent)

• DMEM without SVF + RETINOL PALMITATE at 10 ^" 6M (DMSO solvent)

• DMEM without SVF + RETINOL PALMITATE at 10 ^"7 M (DMSO solvent)

• DMEM without SVF + RETINYL ACETATE at 10 ^"6 M • DMEM without SVF + RETINYL ACETATE at 10 ^{" 7} M

Each condition is made in triplicate.

Cell density was assessed 24h after seeding of the cells, prior to contacting the different conditions of study (= TO) and the growth of fibroblasts is assessed ^1, 2 ^nd 3 ^rd and 5 ^th days of culture the conversion method of WST-I (reading at 450 nm). 3 / Results

Cell growth is objectified by the measurement of cell viability at different times of the experiment.

As shown in FIGS. 8 to 11, the 1% Laminar aqueous extract, combined with Alanyl L Glutamine (2 mM) in DMEM medium without FCS, allows a high growth of normal human fibroblasts up to 30%. the 5 ^th day of culturing and so superior to 4 retinoids tested at concentrations of 10 ^"6 M and ^{10" 7} M.

EXAMPLES 5 Evaluation of the UVA Internal Photoprotection (1.0 J / cm ² ) of the Aqueous Extracted Laminate (Identical Extract to that of the Previous Examples) and Retinol Palmitate by the Comet Test on Normal Human Fibroblasts

Culture of normal human fibroblasts

Normal Human Fibroblast (HNF) cultures are made from foreskins of infants and newborns with phimosis. Fibroblasts obtained from skin fragments are placed in DMEM medium (Sigma St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (Dominique Dutscher, Brumath, France). The cultures are maintained in an incubator at 37 ° C. and under an atmosphere containing 5% CO ₂ .

Preparation of slides and irradiation of cells

After treatment with a trypsin / EDTA mixture (0.05% / 0.02%) for 2 to 3 minutes, the cells are recovered by centrifugation and placed in PBS buffer without Ca ²⁺ or Mg ²⁺ (Sigma). Following a second centrifugation, the cells (ranging in number from 4.5 x 10 ^{4 to} 5.0 x 10 ⁴ ) are suspended in 0.5% Low Melting Point agarose, LMP (Sigma). The mixture is directly deposited on microscope slides which are covered with a pre-layer of agarose (1.6%) dried overnight at room temperature and freshly precoated with a second layer of agarose (0.8%). A third layer of LMP agarose is finally deposited to trap the keratinocytes.

UVA irradiations are generated by a Bio-Sun UV irradiator (Vilbert Lourmat, Marne la Vallée). This unit is equipped with monochromatic lamps that emit at 365 nm wavelengths. The lamps deliver a calculated energy, using a radiometer type RMW-365/312 of 4.0 mW / cm ² for UVA: the energies delivered are then 0.8 J / cm ² . The cells are irradiated by UVA on an ice bath and the comet test is performed immediately after exposure.

Comet test (dry blade technique)

The comet test protocol is that of De Meo and CoIl (Meo M, Laget M, Castegnaro M, Dumenil G., Genotoxic activity of potassium permanganate in acidic solutions, Mutation Res 1991; 260; 295-306) by incorporating the technical

"Dry blades" (Klaude M, Ericksson S, Nygren J, Annstrom G. The cornet assay: mechanism and technical consideration, Mutation Res 1996; 363; 89-96). The slides are placed, after the irradiations, in a lysis bath containing 2.5 M NaCl, 100 mM Na ₂ EDTA, 10 mM Tris-HCl pH 10, 1% sodium sarcosinate, 1% triton X- 100 and 10% DMSO. Cell lysis was performed at 4 ° C for 60 min followed by denaturation of DNA at room temperature for 20 min in a strongly alkaline solution comprising 1 mM Na ₂ EDTA and 300 mM NaOH at pH greater than 13.0. . After electrophoresis (25V, 300 mA) for 20 min, the slides are neutralized with Tris-HCl buffer (0.4 M, pH 7.4) and dehydrated in absolute ethanol or methanol.

Microscopic observation and image analysis

The slides are stained with ethidium bromide solution and observed with a BH2-RFL fluorescence microscope (Olympus, Japan) equipped with a 20BG-W dichroic filter (excitation: 515-560 nm; : 590 nm) and an Apo D-Plan 2Ox lens. Image analysis is performed with a high-sensitivity monochrome CCD camera (Cohu 4912-5000) coupled with a Matrox IP-8 acquisition card. The set is driven by Fenestra Komet software (Kinetic Imaging, Liverpool, UK, version 3.1). A total of 100 cells per sample (50 cells per slide) is analyzed. The parameter used is "Olive Tail Moment" (OTM which is defined as the product of the percentage of DNA in the comet's tail by the length thereof expressed in μm). For each series of experiments, a negative control (non-irradiated cells) and a positive control (irradiated cells without a screen) are included.

Internal photoprotection was measured after a 120 min treatment at 37 ° C. For UVA irradiation (320 nm - 400 nm), the total irradiation dose was 1.0 J / cm ² for a period of 4 min. Negative controls included untreated fibroblasts and non-irradiated and fibroblasts treated with laminar extract (0.1%), and retinol palmitate (10 ^-6 M) and non-irradiated. The genomic protection coefficients ( _UVA CPG) are calculated with the formula:

(χ ² OTM _e - χ ² OTM _Ce )

GIC = 1 - x100 (χ ² OTM _{c +} - χ ² OTM _c _)

With χ ² 0TM _e : χ ² OTM of the sample; χ ² 0TM _ce : χ ² OTM of the non-irradiated control of the sample; χ ² 0TM _C -: χ ² OTM of non-irradiated control; χ ² OTM _{C +} : χ ² OTM of the irradiated control.

χ ² OTM: parameter calculated by regression of OTM distributions according to a χ ² function (see: Bauer and CoIl (Bauer E, Recknagel RD, Fiedler U, Wollweber L, Bock C, Greulich KO; cell electrophoresis (cornet assay) obeys a chi-square (χ ² ) not a gaussian distribution, mutation Res 1998; 398: 101-110)).

Under these experimental conditions, only DNA strand breaks caused by radical species were examined. Direct DNA damage induced mainly by UVB radiation was not detected. The results are summarized in Table 1.

Control / NI cells grown in DMEM medium and not irradiated.

DMSO (DMEM) ZNI cells treated directly in DMEM → - DMSO medium (solubilizing solvent PR) for two hours at 37 ° C and not irradiated.

LaminarZNI cells treated with 0.1% laminar aqueous extract for two hours at 37 ° C and unirradiated.

Witness ZUVA cells grown in DMEM medium and irradiated by UVA radiation (1.0 JZcm ² ).

DMSOZUVA cells in DMEM + DMSO medium irradiated by UVA radiation (1.0 JZcm ² ).

LaminarZUVA cells treated with 0.1% laminar aqueous extract for two hours at 37 ° C and then irradiated with UVA radiation (1.0 JZcm ² ). io-6 M retinol palmitateZ NI: cells treated with 10-6 M retinol palmitate for two hours at 37 ° C and unirradiated. M-6 M retino 1 palmitate ZuvA: cells treated with 10-6 M retinol palmitate for two hours at 37 ° C and then irradiated with UVA radiation (1.0 JZcm ² ).

*** significantly different from WitnessZUVA or DMSOZUVA (P <0.001); NS not significant

Examples 6: Cosmetic Compositions

Composition 1

Growth factor 0.75% - 67% aqueous laminar extract (50Z50)

- 33% Alanyl glutamine

Isostearyl Isostearate 5.00%

Titanium oxide (MTlOOZ) 5.00%

Hyaluronic acid 2.00% Dimethicone trimethylsiloxysilicate 3.00%

Tocopheryl Acetate 0.20%

Purified water qs 100% Sucroester 5.00%

Glycerin 5.00%

Methylparaben 0.10%

Phenoxyethanol 0.40% Perfume 0.20%

Composition 2

Growth factor 1.65% - 90% aqueous laminar extract (50/50) +

- 10% Alanyl glutamine

Glyceryl Stearate 4.00%

Cetyl alcohol 0.50%

Dimethicone 0.50% Coco Caprylate / Caprate 8.00%

PVP / Eicosene copolymer 2.00%

Potassium Cetyl Phosphate 2.00%

Methylparaben 0.25%

Disodium EDTA 0, 10% Purified water 27.05%

Acrylic polymer 10.00%

Propylene Glycol 5.00%

Potassium Hydroxide 0.45%

Tocopheryl Acetate 2.50% Panthenol 1.00% Perfume qs 100%

Composition 3

Caprylic / Capric Triglyceride 4.00%

Octyl stearate 3.00%

Growth factor 0.85%

- 94% aqueous laminar extract (50/50) +

- 6% Alanyl Glutamine Perfume 0.30% Polyglyceryl-3 Diisostearate 4.00%

PEG-20 Glyceryl Laurate 1.00%

Water 66.95%

0.4% Acrylic Polymer

Hexanediol 2.00%

Phenoxyethanol 0.50%

Methylparaben 0.25%

Xanthan gum 0.30%

Triethanolamine 0.85%

Purified water 3.85%

Acetyl tyrosine 2%

Composition 4:

Glycerol stearate 1.50%

Octyldodecanol 5.00%

0.01% BHA

Potassium cetyl phosphate 0.93%

Magnesium stearate 0.70% Demineralized water qs 100%

Growth factor 0.02%

- 50% aqueous extract of laminaria (50/50)

- 50% Alanyl Glutamine Cyclomethicone 3.00% Matricium® 3.00% Simulgel NS 0.50% Phenonip 0.50%

Composition 5:

Cetearyl alcohol and cetearyl glucoside 4.00%

Jojoba oil 5.00%

Sweet almond oil refined 5.00%

Polyacrylamide and Isoparaffin C13-C14 and laureh-7 (Sepigel 2.00%

0.01% BHA Demineralized water qs 100%

Growth factor 1.05%

- 95% aqueous laminar extract (50/50) +

- 5% Alanyl Glutamine Cyclomethicone 3.00%

Bacterial polysaccharide 7.00%

EDTA 0.10%

Citric acid 0.045%

Methyl paraben 0.30% Phenoxyethanol 0.40%

Composition 6:

Demineralized water qs 100% Green tea extract 0.90%

Growth factor 0.55%

- 90% aqueous extract of laminaria (50/50) +

- 10% Alanyl glutamine

Stabilized Vitamin C 1.00% Methyl Paraoxybenzoate Sodium 0.20%

Cyclomethicone 5.00%

Simulgel NS 4.00%

Phenonip 0.65%

Lactic acid qsp pH = 6.5

Claims

1 / cell growth factor of non-animal origin, characterized in that it contains L alanyl L Glutamine, associated with at least one β glucan.

2 / Growth factor according to claim 1, characterized in that the β glucan is a β 1.3 glucan.

3 / growth factor according to claim 2, characterized in that the β 1.3 glucan is β 1.3, β 1.6 glucan

4 / Growth factor according to claim 3, characterized in that the β 1.3, β 1.6 glucan is laminarine.

5 / growth factor according to claim 4, characterized in that it contains as a source of laminarine, a Laminar extract.

6 / growth factor according to claim 5, characterized in that the Laminaria is Laminaria digitata.

Growth factor according to claim 5, characterized in that the laminar extract is an aqueous extract.

8 / growth factor according to claim 5, characterized in that the L alanyl L glutamine is pure and is between 0.01 and 99.99% by weight of the factor, the complement to 100% being constituted by the aqueous extract of laminar.

9 / Cosmetic composition containing the growth factor of one of claims 1 to 8, optionally in the presence of at least one cosmetic vehicle. 10 / cosmetic composition according to claim 9 characterized in that the growth factor is between 0.05 and 20%, preferably 0.5 to 5% by weight of the cosmetic composition.

11 / Cosmetic composition according to claim 9, characterized in that it comprises an aqueous nutrient base consisting of amino acids, vitamins, inorganic components, and a growth factor according to one of claims 1 to 4.

12 / Cosmetic composition according to claim 9, characterized in that the concentration of L alanyl L glutamine in the nutritive base is between 0.005 and 10%, advantageously equal to 0.01% by weight and in that it contains an aqueous extract of wherein the laminar is from 0.1 to 5%, preferably 1% by weight of the nutrient base.

13 / Use of the composition object of one of claims 9 to 12, to combat aging of the skin and / or the appearance of wrinkles and / or to promote the regeneration of the skin.

14 / In vitro culture medium of normal human fibroblasts and / or keratinocytes containing, as a growth factor, the growth factor according to one of claims 1 to 8

15 / A survival medium of normal human keratinocytes intended for the maintenance of survival of skin substitutes before grafting, in particular to a large burnt containing, as a growth factor, the growth factor according to one of claims 1 to 8.

16 / Media according to one of claims 14 or 15, characterized in that the growth factor contains from 0.1 to 10% by weight of L alanyl L glutamine, the complement to 100% consisting of an aqueous laminar extract, said factor representing between 0.001% and 20% by weight of the medium.