FR3113584A1 - Revitalizing and chemoprotective saline composition derived from the PEDRA DE LUME saline on the island of SAL in CABO VERDE - Google Patents

Abstract

The invention relates to a new dermatological or cosmetic composition which comprises a proportion ranging from 0.001 to 0.5%, preferably from 0.001 to 0.25% and particularly preferably from 0.025 to 0.25% of a saline composition which comes from the PEDRA DE LUME saltworks on the island of SAL in CABO VERDE. Such a composition has surprising and interesting properties, in particular an anti-inflammatory and anti-redness effect, as well as revitalizing, chemoprotective and protective properties against UV radiation, or even healing properties.

Description

Revitalizing and chemoprotective saline composition derived from the PEDRA DE LUME saline on the island of SAL in CABO VERDE

The present invention belongs to the field of dermatology and cosmetics.

The applicant has developed a new dermatological or cosmetic composition surprisingly having a revitalizing and chemoprotective effect and comprising as an active ingredient a saline composition which may come from the PEDRA DE LUME saline on the island of SAL in CABO VERDE which is enriched with certain salts and mineral elements.

Thus, the first subject of the invention is a dermatological or cosmetic composition comprising a proportion (by weight relative to the total weight of the composition) ranging from 0.001 to 10%, preferably from 0.01 to 1% of a saline composition including:
- magnesium at a concentration ranging from 10 to 20 g/L,
- potassium at a concentration ranging from 2 to 4 g/L,
- bromides at a concentration ranging from 0.5 to 1 g/L,
- boron at a concentration ranging from 40 to 60 mg/L,
- manganese at a concentration ranging from 0.1 to 1 mg/L.

According to a preferred embodiment, the saline composition comes from the PEDRA DE LUME saline on the island of SAL in CABO VERDE and the dermatological or cosmetic composition comprises a proportion ranging from 0.001 to 0.5%, preferably from 0.001 to 0.25% and, in a particularly preferred manner, from 0.025 to 0.25% of this saline composition.

According to a second preferred embodiment, the saline composition comprises an NaCl concentration of less than 0.1M, preferably a zero NaCl concentration, and the dermatological or cosmetic composition according to the invention comprises a proportion ranging from 0.05 to 10 %, preferably from 0.15 to 5% and, particularly preferably, from 0.25 to 2.5% of this saline composition.

A second object of the invention relates to a dermatological composition according to the invention for its use as a medicament.

A third object of the invention relates to the cosmetic use of a saline composition as described above as a revitalizing agent.

A fourth object of the invention relates to the cosmetic use of a saline composition as described above as a protective agent against pollution.

A fifth object of the invention relates to the cosmetic use of a saline composition as described above as a photoprotective agent for the skin against UV radiation.
The subject of the present invention is a dermatological or cosmetic composition comprising a proportion ranging from 0.001 to 10%, preferably from 0.01 to 1% (by weight relative to the total weight of the composition) of a saline composition comprising:
- magnesium at a concentration ranging from 10 to 20 g/L,
- potassium at a concentration ranging from 2 to 4 g/L,
- bromides at a concentration ranging from 0.5 to 1 g/L,
- boron at a concentration ranging from 40 to 60 mg/L,
- manganese at a concentration ranging from 0.1 to 1 mg/L.

Advantageously again, the saline composition comprises:

- magnesium at a concentration ranging from 10 to 15 g/L,

- potassium at a concentration ranging from 2 to 3 g/L,

- bromides at a concentration ranging from 0.6 to 0.8 g/L,

- boron at a concentration ranging from 40 to 50 mg/L,

- manganese at a concentration ranging from 0.2 to 0.5 mg/L.

Still advantageously, the saline composition further comprises:

- lithium at a concentration ranging from 0.4 to 1 mg/L, preferably from 0.5 to 0.9 mg/L; and or

- strontium at a concentration ranging from 10 to 25 mg/L.

According to a first preferred embodiment, the saline composition used corresponds to sea water enriched with the elements and mineral salts mentioned above and comes from the PEDRA DE LUME saline on the island of SAL in CABO VERDE. Consequently, the saline composition may include, and in their usual concentration, all the other elements and mineral salts naturally present in seawater such as barium, calcium, chlorine, copper, iron, sodium, or sulfur again. Typically, this saline composition has an NaCl concentration greater than or equal to 0.5M.

In this context, the dermatological or cosmetic composition according to the invention comprises a proportion ranging from 0.001 to 0.5%, preferably from 0.001 to 0.25% and, in a particularly preferred manner, from 0.025 to 0.25% of this saline composition.

According to a second preferred embodiment, the saline composition comprises an NaCl concentration of less than 0.1M, preferably a zero NaCl concentration, and the dermatological or cosmetic composition according to the invention then comprises a proportion ranging from 0.05 to 10%, preferably from 0.15 to 5% and, particularly preferably, from 0.25 to 2.5% of this saline composition.

The composition according to the invention may be in any form known to those skilled in the art in the fields of cosmetics and dermatology, without any particular galenic restriction.

The cosmetic or dermatological composition may be in any galenic form known to those skilled in the art, for example in the form of an aqueous formulation, a serum, a cream, an oil, a milk, an ointment, a powder, a stick, an impregnated tampon, a transdermal patch, a solution, a gel, a spray, an aerosol, a lotion or suspension. It may be for example a cream, a milk, a gel, a lotion, an ointment, a transdermal patch, a balm, a mask.

According to a preferred embodiment, the cosmetic or dermatological composition according to the invention can take the form of an aqueous formulation. As such, the composition according to the invention can be packaged in the form of a spray.

Generally, a composition according to the invention may comprise numerous types of adjuvants or active principles used in cosmetic formulations, whether fatty substances, waxes, organic solvents, thickeners, gelling agents, softeners, antioxidants, opacifiers, stabilizers, foaming and/or detergent surfactants, emollients, superfatting agents, perfumes, ionic or non-ionic emulsifiers, thickening and/or emulsifying polymers, fillers, sequestrants, chelators, preservatives, essential oils, coloring matter, pigments, hydrophilic or lipophilic active agents, humectants such as glycerine or glycols, preservatives, colorants, d cosmetic active ingredients, mineral and/or organic sunscreens, mineral fillers such as iron oxides, titanium oxides and talc, synthetic fillers such as crosslinked or non-crosslinked nylons and poly(methyl methacrylate), elastic silicone omers, plant extracts, lipid vesicles, or any other ingredient usually used in cosmetics.

As examples of fatty substances that can be used in a composition according to the invention, mention may be made of paraffins, isoparaffins, white mineral oils, vegetable oils (from flowers, fruits, vegetables, trees, cereals, oilseeds, etc.), animal oils, synthetic oils, silicone oils and fluorinated oils; and more particularly: oils of plant origin, such as sweet almond oil, coconut oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soybean oil, cottonseed oil, alfalfa oil, l poppyseed oil, pumpkin oil, evening primrose oil, millet oil, barley oil, rye oil, safflower oil, bankoulier oil, passionflower oil, hazelnut oil, palm oil, shea butter, apricot kernel oil, camellia seed oil, calophyllum oil, sysymbrium oil , avocado oil, calendula oil, oils from flowers or vegetables; ethoxylated vegetable oils; oils of animal origin, such as squalene, squalane; mineral oils, such as paraffin oil, petroleum jelly and isoparaffins; synthetic oils, in particular fatty acid esters such as butyl myristate, propyl myristate, cetyl myristate, isopropyl palmitate, butyl stearate, hexadecyl stearate, isopropyl stearate , octyl stearate, isocetyl stearate, dodecyl oleate, hexyl laurate, propylene glycol dicaprylate; esters derived from lanolic acid, such as isopropyl lanolate, isocetyl lanolate, monoglycerides, diglycerides and triglycerides of fatty acids such as glycerol triheptanoate, alkylbenzoates, polyalphaolefins, polyolefins such as polyisobutene, synthetic isoalkanes such as isohexadecane, isododecane, perfluorinated oils and silicone oils. Among the latter, mention may more particularly be made of dimethylpolysiloxanes, methylphenylpolysiloxanes, silicones modified with amines, silicones modified with fatty acids, silicones modified with alcohols, silicones modified with alcohols and fatty acids, silicones modified with with polyether groups, modified epoxy silicones, silicones modified with fluorinated groups, cyclic silicones and silicones modified with alkyl groups.

As fatty substances which can be used in a composition according to the invention, mention may also be made of fatty alcohols, linear or branched, saturated or unsaturated, mixtures of fatty alcohols, linear and/or branched, saturated and/or unsaturated, or acids fatty acids, linear or branched, saturated or unsaturated, mixtures of linear or branched, saturated or unsaturated fatty acids.

Among the thickening and/or emulsifying polymers that can be used, there are, for example, homopolymers or copolymers of acrylic acid or derivatives of acrylic acid, homopolymers or copolymers of methacrylic acid or derivatives of methacrylic, homopolymers or copolymers of acrylamide, homopolymers or copolymers of acrylamide derivatives, homopolymers or copolymers of acrylamidomethyl propanesulfonic acid, homopolymers or copolymers of vinyl monomers, homopolymers or copolymers of trimethylaminoethylacrylate chloride, hydrocolloids of vegetable or biosynthetic origin such as for example xanthan gum, karaya gum, carrageenans, alginates; silicates; cellulose and its derivatives; starch and its hydrophilic derivatives; polyurethanes.

Among the polymers of polyelectrolyte type which can be used in the production of a gelled aqueous phase suitable for use in the preparation of W/O, O/W, W/O/W or O/W/O emulsions, there are, for example, copolymers of acrylic acid and 2-methyl-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid (AMPS), copolymers of acrylamide and 2-methyl-[(1-oxo-2-propenyl)amino] 1-propanesulfonic acid, 2-methyl-[(1-oxo-2-propenyl)amino] acid copolymers 1 -propanesulfonic and (2-hydroxyethyl)acrylate, the homopolymer of 2-methyl-[(l-oxo-2-propenyl)amino]1-propanesulfonic acid, the homopolymer of acrylic acid, copolymers of acryloyl ethyl trimethyl ammonium chloride and acrylamide, copolymers of AMPS and vinylpyrolidone, copolymers of AMPS and N5N Dimethyl acrylamide, terpolymers of AMPS, acrylic acid and N5N Dimethyl acrylamide, copolymers of acrylic acid and acrylates alkyl whose carbon chain comprises between ten and thirty carbon atoms, copolymers of AMPS and alkyl acrylates whose carbon chain comprises between ten and thirty carbon atoms.

Among hydrophilic gelling agents/thickeners, mention may be made in particular of carboxyvinyl polymers (CARBOMER), acrylic copolymers such as acrylate/alkylacrylate copolymers, polysaccharides, natural gums and clays, and, as lipophilic gelling agents/thickeners, mention may be made of modified clays such as bentones, metal salts of fatty acids, and hydrophobic silica.

Among the waxes that can be used in the compositions according to the invention, mention may be made, for example, of natural or modified beeswax, carnauba wax, candelilla wax, ouricoury wax, Japanese wax, jojoba, cork or sugar cane fiber wax, paraffin waxes, lignite waxes, microcrystalline waxes, lanolin wax, ozokerite, polyethylene wax, hydrogenated oils, silicone waxes , vegetable waxes, fatty alcohols and fatty acids solid at room temperature, glycerides solid at room temperature.

Among the emulsifiers which can be used in the compositions according to the invention, mention may be made of:

- fatty esters of optionally alkoxylated alkylpolyglycosides;

- alkoxylated fatty esters;

- polyalkylene glycol carbamates with fatty chains;

- fatty acids, ethoxylated fatty acids, fatty acid and sorbitol esters, ethoxylated fatty acid esters, polysorbates, polyglycerol esters, ethoxylated fatty alcohols, sucrose esters, alkylpolyglycosides, sulfated and phosphated fatty alcohols or mixtures of alkylpolyglycosides and fatty alcohols;

- combinations of emulsifying surfactants chosen from alkylpolyglycosides;

- associations of alkylpolyglycosides and fatty alcohols, esters of polyglycerols or of polyglycols or of polyols.

Among the surfactants that can be used in the compositions according to the invention, mention may be made of: topically acceptable anionic, cationic, amphoteric or nonionic surfactants usually used in cosmetics.

Among the anionic surfactants which can be used in the compositions according to the invention, particular mention will be made of alkali metal salts, alkaline-earth metal salts, ammonium salts, amine salts, amino alcohol salts of the following compounds : alkylether sulphates, alkylsulphates, alkylamidoethersulphates, alkylarylpolyethersulphates, monoglyceride sulphates, alpha-olefin sulphonates, paraffin sulphonates, alkylphosphates, alkyletherphosphates, alkylsulphonates, alkylamidesulphonates, alkylarylsulphonates, alkylcarboxylates, alkylsulfosuccinates, alkylethersulfosuccinates , alkylamidesulfosuccinates, alkylsulfoacetates, alkylsarcosinates, acylisethionates, N-acyltaurates, acyllactylates.

Among the amphoteric surfactants that can be used in the compositions according to the invention, mention may be made of alkylbetaines, alkylamidobetaines, sultaines, alkylamidoalkylsulfobetaines, imidazoline derivatives, phosphobetaines, amphopolyacetates and amphopropionates.

Among the cosmetic active agents which can be used in the composition according to the invention, mention may in particular be made of desquamating agents, moisturizing agents, photoprotective agents, depigmenting or propigmenting agents, antimicrobial agents, anti-glycation agents, NO- synthase, agents stimulating the synthesis of dermal or epidermal macromolecules and/or preventing their degradation, agents stimulating the proliferation of fibroblasts and/or keratinocytes or stimulating the differentiation of keratinocytes, other myorelaxing agents and/or dermo-decontracting agents , tensioning agents, chelating agents, anti-pollution and/or anti-radical agents, agents acting on the microcirculation, agents acting on the energy metabolism of cells, and mixtures thereof.

According to a particular embodiment, the composition according to the invention also comprises an antimicrobial agent, preferably sodium anisate, potassium sorbate, sodium levulinate, phenylpropanol, 1,2 hexanediol, benzoate sodium, phenethyl alcohol, ethylhexylglycerin, caprylyl glycol and glyceryl caprylate.

Examples of such additional compounds are: retinol and its derivatives such as retinyl palmitate, ascorbic acid and its derivatives such as magnesium ascorbyl phosphate and ascorbyl glucoside, tocopherol (Vitamin E) and its derivatives such as tocopheryl acetate, nicotinic acid and its precursors such as nicotinamide, ubiquinone, glutathione and its precursors such as L-2-oxothiazolidine-4-carboxylic acid, plant extracts and in particular vegetable proteins and their hydrolysates, as well as phytohormones, marine extracts such as seaweed extracts, bacterial extracts, sapogenins such as siosgenin and Wild Yam extracts containing them, ceramides, hydroxy acids such as salicylic acid and n-octanoyl-5-salicylic acid, resveratrol, oligopeptides and pseudodipeptides and their acylated derivatives, manganese and magnesium salts, in particular gluconates and mixtures thereof.

Among the photoprotective agents, mention may be made of organic or inorganic compounds, the latter optionally being coated to make them hydrophobic.

The organic photoprotective agents can in particular be chosen from: anthranilates, in particular menthyl anthranilate, benzophenones, in particular benzophenone-1, benzophenone-3, benzophenone-5, benzophenone-6, benzophenone-8 , benzophenone-9, benzophenone-12, and preferably benzophenone-3 (oxybenzone), or benzophenone-4 (UVINUL MS40 available from B.A.S.F.), benzylidene-camphors, in particular 3-benzylidene-camphor, benzylidene camprosulfonic acid, benzalkonium camphor methosulfate (EUZOLEX 6300 available from MERCK), benzimidazoles, in particular benzimidazilate (NEO HELIOPAN AP available from HAARMANN AND REIMER) or phenylbenzimidazole sulfonic acid (EUSOLEX 232 available from MERCK); benzotriazoles, in particular drometrizole trisiloxane, or methylene bis-benzotriazolyltetramethylbutylphenol (TINOSORB M available from CIBA); cinnamates, in particular cinoxate, DEA methoxycinnamate, diisopropyl methylcinnamate, dimethoxycinnamate glyceryl ethylhexanoate, isopropyl methoxycinnamate, isoamyl cinnamate, and preferably ethocrylene (UVINUL N35 available from B.A.S.F.), octylmethoxycinnamate (PARSOL MOX available from HOFFMANN LA ROCHE), or octocrylene (UVINUL 539 available from B.A.S.F.); dibenzoylmethanes, in particular butyl methoxydibenzoylmethane (PARSOL 1789); imidazolines, in particular ethylhexyl dimethoxybenzylidene dioxoimidazoline; PABAs, in particular ethyl Dihydroxypropyl PABA, ethylhexyldimethyl PABA, glyceryl PABA, PABA, PEG-25 PABA, and preferably diethylhexylbutamido-triazone (UVASORB HEB available from 3V SIGMA), ethylhexyltriazone (UVINUL T150 available from B.A.S.F), or ethyl PABA (benzocaine); salicylates, in particular dipropylene glycol salicylate, ethylhexyl salicylate, homosalate, or TEA salicylate; triazines, in particular anisotriazine (TINOSORB S available from CIBA); drometrizole trisiloxane.

The present invention also relates to a dermatological composition according to the invention for its use as a medicament.

Advantageously, the term drug is understood to mean a dermatological drug, intended for topical application.

Now, such a drug can also target the nasal mucosa and be administered intranasally, for example, by aerosol.

Another object of the invention relates to the cosmetic use of a saline composition as described above as a revitalizing agent.

According to another aspect, the invention relates to the cosmetic use of a saline composition as described above as a protective agent against pollution.

The effects of pollution on the skin are varied: there is an acidification of the skin pH, a drop in hydration with an increase in trans-epidermal water loss, an increase in desquamation, a decrease in skin suppleness, a modification of surface lipids by the action of free radicals, a decrease in cellular energy metabolism. The repetition of these aggressions initiates an inflammatory process and predisposes to intolerance reactions. Thus, a cosmetic composition capable of combating and preventing the effects of atmospheric pollution on the skin must be able to combat these various phenomena, and is advantageously capable of reducing the visible damage caused by the pollution on the epidermis.

Another aspect of the invention relates to the cosmetic use of a saline composition as described above as a protective agent for the skin against UV radiation.

For these uses, the saline composition as described above is used in a proportion ranging from 0.001 to 0.5% (by weight relative to the total weight of the composition), preferably from 0.001 to 0.25% and in a particularly preferably 0.025 to 0.25%.

The invention also relates to a dermatological composition according to the invention for preventing or treating inflammation of the skin.

Another object of the invention relates to a dermatological composition according to the invention for treating the inflammation of skin chosen from the group consisting of sensitive skin, fragile skin, reactive skin, intolerant skin, skin prone to rosacea, erythrosis-prone skin and erythro-rosacea-prone skin.

The invention also relates to a dermatological composition according to the invention for its use in the treatment of wounds, preferably cuts, abrasions, superficial scars.

Indeed, a final subject of the invention relates to a dermatological composition according to the invention for promoting healing, in particular for promoting regeneration of the epidermis, reconstruction of the epidermis and/or promoting re-epithelialization skin or mucous membranes affected by a wound. More generally, a composition according to the invention can be used to prevent or treat a skin or mucous membrane wound.

Such a wound may correspond to trauma to the skin or mucous membrane, for example, a cut, a scratch.

The wound can also result from surgery or aesthetic medicine.

Example 1: Preparation of a composition according to the invention:

Different water samples from the PEDRA DE LUME saltworks were analyzed, as well as the composition of seawater in the ocean near the site. The main differences identified are presented in Table 1 below:

Elements Measure 1
Saltworks PEDRA DE LUME Measure 2
Saltworks PEDRA DE LUME
Sea water Li (in mg/L) 0.5 0.9 Approx 0.2 B (mg/L) N.d. 44 6 Mg (eng/L) 13 12.65 Env 1 K (in g/L) 3.1 2.7 0.25 Mn (in mg/L) 0.5 0.4 0 Sr (in mg/L) 13.5 21.5 8.9 Bromides (g/L) N.d. 0.7 0.07 NaCl

It should be noted that the analyzes also show an NaCl concentration greater than 0.5 M.

Based on these analyses, a synthetic composition was also prepared comprising the same components as the water from the PEDRA DE LUME saltworks, based on the results obtained below, but not comprising of NaCl.

Example 2: Anti-inflammatory effect of a composition according to the invention

Principle:

The inflammatory activity of PEDRA DE LUNA saline water is evaluated in vitro on cell lines of immortalized murine macrophages on which an inflammatory reaction is induced using lipopolysaccharides (LPS) from E. coli .

Protocol:

RAW 267.4 murine macrophages (SIGMA-ALDRICH) are cultured in 48-well cell culture plates in DMEM with stable L-glutamine supplemented with penicillin (100 IU/mL) and streptomycin (100 μg/mL), comprising 10% of inactivated calf serum, pH 7.2, at a rate of 2.10 ⁴ cells per well. After 24 h of incubation at 37° C. (5% CO ₂ ), the supernatant is replaced with medium comprising the composition to be tested at an appropriate concentration.

Two compositions are tested, one being a composition according to the invention prepared according to Example 1, and the other being a reference thermal water. The concentration in the medium of compositions to be tested does not exceed 5%.
A negative control is used, this consisting of PBS (SIGMA-ALDRICH) replacing the composition to be tested in the medium.
A positive control is used, this consisting of dexamethasone at concentrations of 1, 5, 10, 50, 100 μM in the medium.

The cells are incubated for 1 hour at 37° C. (5% CO ₂ ). Lipopolysaccharides from E. coli having a pro-inflammatory effect are added to the cells (1 μg/mL). The cells are then incubated at 37° C. (5% CO ₂ ) for 24 hours.

The release of nitrates (NO) is measured in the supernatant by the Griess reaction. 100 µL of supernatants from each well are transferred to a new 96-well cell culture plate, to which 100 µL of Griess reagent (modified) is added. After 15 minutes at room temperature, the absorbance of each well at 540 nm is measured using a fluorescence/luminescence plate reader (INFINITE M200Pro, TECAN). The measured absorbance is converted to a percentage based on the absorbance of the negative control (PBS) corresponding to 100% NO production.
The concentration of the product tested inducing a 50% reduction in the production of NO, IC _{50-production NO} is then calculated.

The cell viability is evaluated using the WST-1 dye reagent (100 μl per well of WST-1 (1/10 dilution) after decantation). After 30 minutes of incubation at 37° C. (5% CO ₂ ), the absorbance of each well at 450 nm is measured using a fluorescence/luminescence plate reader (INFINITE M200Pro, TECAN). The measured absorbance is converted into a percentage based on the absorbance of DMSO control wells corresponding to 100% cell viability.
The concentration of the product tested inducing a 50% decrease in cell viability, IC _{50-cell viability} is then calculated.

The anti-inflammatory ratio corresponding to the ratio between anti-inflammatory activity and toxicity is then calculated by calculating the ratio IC _{50-cell viability} /IC _{50-NO production} .

Results :

The following [table 2] presents the IC _{50-NO production} , the IC _{50-cell viability} as well as the anti-inflammatory ratio calculated for the composition according to the invention, the reference thermal water, dexamethasone (positive control ).

Samples IC _{50-production NO} IC _{50-cell viability} Anti-inflammatory ratio Dexamethasone 5.60±0.22µM 196.85 ± 3.18µM 35.15 Composition according to the invention 0.074 ± 0.07% 0.81 ± 0.06% 10.94 Thermal water 0.28 ± 0.09% > 2.5% > 8.92

Conclusion :

The results show that the saline water has anti-inflammatory activity but with a dilution 5 times less than that of the reference thermal water (0.074 vs 0.28% for the production of NO and 0.81 vs > 2.5%.

Example 3: Healing effect of a composition according to the invention.

Principle:

The evaluation of the healing effect is carried out by means of the evaluation of the capacity of the composition to test, to initiate the growth and the migration of human keratinocytes.
To do this, human keratinocytes are cultured in vitro in a monolayer until confluence, then the monolayer of cells is altered by a mechanical scratch, so as to simulate an injury. A visual follow-up under the microscope is carried out in order to observe the cellular displacements and the filling of the “injured” zone, which allows the evaluation of the healing effect.

Protocol

Human keratinocytes are cultured in 24-well cell culture plates, at a rate of 1.10 ⁶ cells per well, in "Keratinocytes-SFM" medium supplemented with bovine pituitary extract (30 μg/mL) and recombinant epidermal growth factor ( rEGF, 0.2 ng/ml). Calibrated inserts with a thickness of 0.9 mm are present in the wells during the incubation in order to allow the creation of the “wounded” zone. After 24 hours of incubation, when the cells are at confluence, the inserts are removed, as well as the culture medium, which is replaced by 1 mL of non-supplemented "Keratinocytes-SFM" medium (INVITROGEN), to which is added the composition to test at different concentrations (0.5, 1, 5, 10, 15% (v/v) for the reference thermal water and 0.01%, 0.025%, 0.05%, 0.1%, 0, 25% for the composition according to the invention prepared according to example 1).
Two compositions are tested, one being a composition according to the invention prepared according to Example 1, and the other being a reference thermal water.
A negative control is used, this consisting of “keratinocyte-SFM” culture medium (INVITROGEN).
A positive control is used, this consisting of bovine pituitary extract (30 μg/ml) and recombinant epidermal growth factor (rEGF, 0.2 ng/ml).

The cells are then incubated at 37° C. (5% CO ₂ ) for 48 h. After incubation, the culture medium is removed, the cells are washed, then fixed with ethanol and stained with Giemsa. The cells are then observed by microscopy. Measurements of the spacing between the two cell edges of the injured area are carried out using image processing software. Ten measurements are taken per sample in order to calculate an average spacing.

The percentage of healing is finally calculated by taking the ratio between the average spacing measured for the composition to be tested and the average spacing measured for the positive control.

Results :

Table 3 presents the average difference measured (in pixels) and the percentage of healing calculated according to the different compositions tested.

Samples % (v:v) Average deviation measured (in pixels) Healing percentage Negative control 139.91 ± 14.47 0 Positive control 37.39 ± 18.00 100 Dilution of the salt water of PEDRA DE LUNA 0.01% 133.44±6.76 0.47 0.025% 131.31 ± 8.34 2.38 0.05% 131.45 ± 9.93 2.53 0.1% 130.18 ± 14.98 3.84 0.25% 132.48 ± 12.29 1.46 Reference thermal water 0.5% 132.02±11.97 1.94 1% 132.88 ± 12.93 1.05 5% 131.91±12.58 2.05 10% 132.10 ± 13.30 1.86 15% 137.68 ± 12.30 0

Conclusion :

The results show that PEDRA DE LUNA saline water exhibits healing activity at a dilution of 0.01 to 0.25%, with a maximum activity at 0.10%.

Example 4: Revitalizing effect of two compositions according to the invention

Principle:

The revitalizing effect is evaluated by observing the ability of the test composition to keep cells exposed to environmental stress alive.

In detail, the cell viability of human fibroblasts is evaluated after incubation for 4 hours in the presence of the composition to be tested at different concentrations, with a 0.9% NaCl solution as a control. Cellular viability is expressed by the reduction, depending on the concentration, of the integration of the neutral red vital dye, measured 24 hours after incubation with the product to be tested. Neutral red being a weak cationic dye that does not enter cells by passive diffusion, but accumulates intracellularly in lysosomes. Alteration of the cell surface of the sensitive lysosomal membrane leads to lysosomal fragility and other changes that gradually become irreversible. These modifications induced by the action of xenobiotics, lead to a decrease in the fixation of neutral red, on the basis of which one can distinguish living, dead, or damaged cells.

Protocol:

Human fibroblasts are placed in 96-well cell culture plates, at a rate of 2.10 ⁴ cells per well (0.2 mL per well), and incubated for 24 h at 37° C. (5% CO ₂ ) until at confluence, in DMEM culture medium (PAN BIOTECH) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL) and inactivated calf serum.
After incubation, the culture medium is removed and replaced with 200 μL of 0.9% NaCL solution containing a certain concentration of the composition to be tested (from 0.01% to 2.5% (v:v)).
Two compositions are tested, one being a composition according to the invention prepared according to Example 1, and the other being a reference thermal water.
A negative control is used, this one consisting of a 0.9% NaCl solution.
A positive control is used, the latter consisting of complete medium.
The cells are then incubated at 37° C. (5% CO ₂ ) for 4 hours. At the end of the incubation period, the NaCl solution is removed and the cells are washed. A medium containing neutral red (50 μg/mL of neutral red in complete medium) is then added to the wells. The cells are incubated for 3 h at 37° C. (5% CO ₂ ). The medium is then removed and the cells are washed three times with 0.2 mL of PBS per well. A bleaching solution is added to the cells (50% ethanol, 1% acetic acid, 49% distilled water), at a rate of 50 µL per well, and the cell culture plates are shaken for 15-20 minutes in the dark at room temperature. ambient.

The evaluation of membrane damage is evaluated through the absorbance of each well at 540 nm. This is measured using a fluorescence/luminescence plate reader (INFINITE M200Pro, TECAN). The results for the different concentrations of composition to be tested are converted into a percentage relative to the positive control (complete medium), corresponding to a cell viability of 100%.

The revitalizing potential, expressed as a percentage, is then calculated.

Results

Table 4 shows the revitalizing potential (in %) measured according to the different compositions tested. Samples Concentrations (v:v) Revitalizing potential (in %) Positive control (complete medium) 100 Negative control (0.9% NaCl) 0 Dilution of the salt water of PEDRA DE LUNA 0.01 21.44 ± 2.67 0.025 30.81 ± 5.96 0.05 68.29 ± 2.95 0.1 49.58 ± 2.91 0.25 33.76 ± 4.80 0.5 10.32 ± 2.00 1 1.86 ± 0.64 2.5 0.18 ± 0.32 Synthetic composition without NaCl 0.05 6.75 ± 0.30 0.1 22.46 ± 5.07 0.25 68.10 ± 5.19 0.5 80.30 ± 2.20 1 76.79 ± 4.57 2.5 66.62 ± 2.79 5 42.83 ± 2.26 10 38.85 ± 2.29 Reference thermal water 0.01 15.31± 3.51 0.025 22.94 ± 4.91 0.05 50.95 ± 5.80 0.1 55.24±8.98 0.25 54.07±3.90 0.5 43.36 ± 3.79 1 43.17 ± 3.15 2.5 40.96 ± 7.73

Findings

The water from the PEDRA DE LUNA saline exhibits a strong revitalizing activity, with a revitalizing potential of 68.29% ± 2.95 at a concentration of 0.05% and which then rapidly decreases above 0.5% . Now, when you use a synthetic water that does not include NaCl, you then obtain a higher revitalizing potential that can go up to more than 80% at 0.5% (but usable even up to more than 10 It should be noted that the revitalizing potential of the compositions according to the invention is higher than that of a composition comprising the reference thermal water.

Example 5: Protective effect against pollution of two compositions according to the invention

Principle:

The evaluation of the protective activity against pollution of a composition is evaluated by treating in vitro human fibroblasts with such a composition, then by exposing these cells to a toxic extract of urban dust. After 24 hours of incubation, the cell viability is evaluated.
The cytotoxicity generated by increasing concentrations of urban dust is measured using the neutral red vital dye. . Indeed, the internalization of this dye in the lysosomes of a cell is all the higher when it is in good health (this internalization is nil for a dead cell) Thus, the modifications induced by the action of xenobiotics lead to a decrease in the fixation of neutral red, on the basis of which one can distinguish living, dead, or damaged cells.

Protocol:

Human fibroblasts are placed in the wells of a 96-well cell culture plate, at a rate of 2.10 ⁴ cells per well (0.2 mL per well), and incubated for 24 h at 37° C. (5% CO ₂ ) until confluence, in DMEM (PAN BIOTECH) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL) and 10% inactivated calf serum.
After incubation, the culture medium is removed and replaced with 200 μl of complete medium containing a certain concentration of the composition to be tested (0.025%, 0.05%, 0.1%).
Two compositions are tested, one being a composition according to the invention prepared according to Example 1, and the other being a reference thermal water.
A negative control is used, this one consisting of PBS (SIGMA).

The cells are then incubated at 37°C (5% CO2) for 1 hour. At the end of the incubation period, the medium is removed and the cells are washed. Medium containing different concentrations of urban dust extract is added. Such an urban dust extract is prepared from urban dust powder (URBAN DUST POWDER, SIGMA-ALDRICH). 500 mg of this powder is sonicated in 20 mL of isopropanol for 30 minutes, then centrifuged for 10 minutes at 1,200 rpm. The supernatant is recovered, then the isopropanol is completely evaporated. The dry extract is then dissolved in 1 mL of DMSO, then diluted 1/100 in DMSO.

The cells are incubated in this medium at 37° C. for 24 hours. At the end of the incubation period, the culture medium is removed, and a medium containing neutral red (50 µg/mL of neutral red in complete medium) is then added to the wells. The cells are incubated for 3 h at 37° C. (5% CO2). The medium is then removed and the cells are washed three times with 0.2 mL of PBS per well. A bleaching solution is added to the cells (50% ethanol, 1% acetic acid, 49% distilled water), at a rate of 50 µL per well, and the cell culture plates are shaken for 15-20 minutes in the dark at room temperature. ambient.

The evaluation of membrane damage is evaluated through the absorbance of each well at 540 nm. This is measured using a fluorescence/luminescence plate reader (INFINITE M200Pro, TECAN). The results for the different concentrations of composition to be tested are converted into a percentage relative to the positive control (complete medium), corresponding to a cell viability of 100%.

The percentage of cell viability is then calculated for each condition tested, and the median inhibitory concentration IC ₅₀ of urban dust extract is calculated.

Results :

Table 5 below shows the effect of a composition according to the invention and of a reference thermal water on cell viability when the cells are exposed to urban pollution. In detail, the table shows the median inhibitory concentration of IC ₅₀ extract of urban dust and the increase in cell viability generated by a composition according to the invention and the reference thermal water.

Samples Concentrations (%) IC ₅₀ in µg/L of urban dust extract Increase in cell viability (in %) Without treatment 27.09 ± 1.41 - Dilution of the salt water of PEDRA DE LUNA 0.025 34.57±2.75 27.6% 0.05 39.05±2.56 44.12% 0.1 30.07 ± 2.10 11.01% Synthetic composition without NaCl 0.5 36.64 ± 3.06 13.9% 1 48.51±4.06 50.8% 5 36.59 ± 3.21 13.08% Reference thermal water 0.025 29.15 ± 1.45 7.61% 0.05 33.28 ± 1.86 22.8% 0.1 38.42 ± 1.54 41.82% 0.5 45.04 ± 2.04 40.1% 1 47.45 ± 2.14 47.6% 5 41.26 ± 1.98 28.33

Findings:

PEDRA DE LUNA salt water exhibits chemoprotective activity against pollution with an increase in cell viability of 44.12% in cells exposed to urban pollution (0.05% dilution). Now, when we use synthetic water that does not include NaCl, we obtain a chemoprotective activity of more than 50% at 1%. It should be noted that the chemoprotective activity of the composition according to the invention is greater than that of the reference thermal water.

Example 6: Photoprotective effect of a composition according to the invention

Principle:

The photoprotective effect of PEDRA DE LUNA saline water is evaluated through the comet test (COMET ASSAY), which makes it possible to evaluate the ability to protect the DNA of a composition on human keratinocytes exposed to UVA rays having induced DNA damage. A modified version of this test can identify the amount of oxidative DNA damage. For this purpose, the cells are treated with the composition to be tested for an incubation period of two hours, and irradiated with UVA and visible light. After lysis of the cells, the damage caused to the DNA is revealed using an enzymatic treatment with FPG (Formamidopyrimidine [fapy]-DNA glycosylase), which recognizes 7,8-dihydro-8-oxoguanine (8- oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil, and cleaves oxidative bases to generate apurinic sites (AP sites) generating the separation of DNA strands.

Protocol:

Human keratinocytes are incubated for 24 h in 24-well cell culture plates. After incubation, the compositions to be tested are added to the wells at different concentrations (0.01%, 0.1%, 0.5%). The cells are incubated for two hours at 37° C. in a humid incubator (5% CO ₂ ).

The experience plan includes:
- cells without treatment or irradiation;
- cells treated with FPG, without irradiation;
- cells treated with different concentrations of compositions to be tested (composition according to the invention and reference thermal water) and FPG, without irradiation;
- cells without treatment, with irradiation;
- cells treated with FPG, with irradiation;
- cells treated with different concentrations of compositions to be tested (composition according to the invention and reference thermal water) and FPG, with irradiation.

After exposure, cells are treated with trypsin (0.05% trypsin, 0.02% EDTA in PBS) for 3-5 minutes at 37°C. After this treatment, the cells are suspended in PBS and centrifuged for 5 minutes at 1200g. After centrifugation, the cell pellets are resuspended in 75 µL of 0.5% LMP agarose solution and placed on microscope slides, previously covered with a solidified layer of 2% NMP agarose solution. After hardening of the cell layer, a third layer consisting of 85 µL of 0.8% agarose solution is added. After solidification, the slides are irradiated using a SUNTEST CPS+ SOLAR SIMULATOR device (ATLAS MATERIAL TESTING TECHNOLOGY BV) emitting UVA and visible light in the 290-800 nm spectrum so that the slides receive a dose of 150 KJ/m².

The comet test is then performed. To do this, slides are immersed in a lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris (pH10), 1% L-lauroyl sarcosinate, with 10% dimethylsulfoxide and 1% triton X-100 ) for 90 minutes at 4°C. After lysis, the slides are washed three times for 5 minutes in PBS, then three times for 5 minutes in a BSA enzymatic buffer (0.1 M KCl, 0.5 mM Na2 EDTA, 40 mM HEPES, 0.2 mg/mL BSA , pH8 KOH 4M). After these washing steps, the slides are treated with 50 μL of FPG (NEB LABORATORIES) at the rate of 8 units per slide for 30 minutes at 37° C. in a humid chamber.
The slides are then placed horizontally in an electrophoresis tank filled with electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA, pH>13). The slides are left in this buffer for 20 minutes at 25° C. in such a way as to allow the unwinding of the DNA. The electrophoresis is then carried out in the same buffer for 20 minutes at 25 V, 300 mA. The slides are then washed three times for 5 minutes with a 0.4 M Tris buffer.
Finally, the slides are rinsed with pure water, dehydrated in methanol and dried at room temperature for 12 h. Each slide is then stained with 50 μL of 2 μg/μL of ethidium bromide.

The cells are then examined under a fluorescence microscope (OLYMPUS OPTICAL CO). After acquisition, the images are analyzed using dedicated software (KOMET). DNA damage is expressed in OLIVE TAIL MOMENT (OTM).

A protective ratio induced by the compositions to be tested can then be calculated.

Results

Table 6 shows the ratio of radiation protection against UVA in percentage induced by different concentrations of a composition according to the invention.

Samples Concentrations (%) Radiation protection value (in %) Dilution of the salt water of PEDRA DE LUNA 0.01% 9.59 0.05% 14.38 0.1% 28.24 0.5% 7.99

Conclusion :

PEDRA DE LUNA Saline Water exhibits a photoprotective effect, with a protection value of up to 28.24% for a 0.1% dilution. Such a value advantageously provides a protective activity against the mutagenic activity of ultraviolet radiation, thus protecting the genetic material.

Claims (10)

A dermatological or cosmetic composition comprising a proportion ranging from 0.001 to 10% of a saline composition (by weight relative to the total weight of the composition), preferably preferably from 0.01 to 1% comprising:
- magnesium at a concentration ranging from 10 to 20 g/L,
- potassium at a concentration ranging from 2 to 4 g/L,
- bromides at a concentration ranging from 0.5 to 1 g/L,
- boron at a concentration ranging from 40 to 60 mg/L,
- manganese at a concentration ranging from 0.1 to 1 mg/L. The dermatological or cosmetic composition according to any one of the preceding claims, characterized in that the saline composition comprises:
- magnesium at a concentration ranging from 10 to 15 g/L,
- potassium at a concentration ranging from 2 to 3 g/L,
- bromides at a concentration ranging from 0.6 to 0.8 g/L,
- boron at a concentration ranging from 40 to 50 mg/L,
- manganese at a concentration ranging from 0.2 to 0.5 mg/L. The dermatological or cosmetic composition according to any one of the preceding claims, characterized in that the saline composition additionally comprises lithium at a concentration ranging from 0.4 to 1 mg/L and strontium at a concentration ranging from 10 to 25 mg/L. The dermatological or cosmetic composition according to any one of Claims 1 to 3, characterized in that it comprises a proportion ranging from 0.001 to 0.5% of a saline composition which comes from the PEDRA DE LUME saline on the SAL island in CABO VERDE. The dermatological or cosmetic composition according to any one of Claims 1 to 3, characterized in that it comprises a proportion ranging from 0.05 to 10% of a saline composition comprising an NaCl concentration of less than 0.1M, of preferably a zero NaCl concentration. The dermatological composition as defined in any one of claims 1 to 5, for its use as a medicament. A cosmetic use, and as a revitalizing agent for the skin, of a saline composition which comprises:
- magnesium at a concentration ranging from 10 to 15 g/L,
- potassium at a concentration ranging from 2 to 3 g/L,
- bromides at a concentration ranging from 0.6 to 0.8 g/L,
- boron at a concentration ranging from 40 to 50 mg/L, and
- manganese at a concentration ranging from 0.2 to 0.5 mg/L. A cosmetic use, and as a protective agent for the skin against pollution of a saline composition which comprises:
- magnesium at a concentration ranging from 10 to 15 g/L,
- potassium at a concentration ranging from 2 to 3 g/L,
- bromides at a concentration ranging from 0.6 to 0.8 g/L,
- boron at a concentration ranging from 40 to 50 mg/L, and
- manganese at a concentration ranging from 0.2 to 0.5 mg/L. A cosmetic use, and as a photoprotective agent, of a saline composition which comprises:
- magnesium at a concentration ranging from 10 to 15 g/L,
- potassium at a concentration ranging from 2 to 3 g/L,
- bromides at a concentration ranging from 0.6 to 0.8 g/L,
- boron at a concentration ranging from 40 to 50 mg/L, and
- manganese at a concentration ranging from 0.2 to 0.5 mg/L. Use according to any one of Claims 7 to 9, characterized in that the saline composition is used in a proportion ranging from 0.001 to 10%, preferably from 0.01 to 1%.