Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/34—Alcohols
  • A61K8/345—Alcohols containing more than one hydroxy group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/18—Antioxidants, e.g. antiradicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Definitions

• TITLE Glycosylated bacterioruberins and their industrial applications
• the present invention relates to glycosylated bacterioruberins.
• the present invention relates in particular to the purification or synthesis of glycosylated forms of the carotenoid bacterioruberine, as well as its applications, in particular in the fields of pharmaceuticals, dermocosmetics and nutraceuticals and biotechnology.
• Carotenoids are highly conjugated linear isoprenoid compounds responsible for the majority of yellow, orange and red pigmentation seen in organisms on earth (Armstrong, 1997). Although about a thousand different carotenoids have been identified in nature and they have widely varying structural characteristics, all known carotenoids share a lipophilic linear conjugate backbone, obtained by passing through highly conserved biosynthetic pathways (Britton, 2004 ). Carotenoids are synthesized from the linear condensation of isoprene units, derived from primary metabolism (Armstrong, 1994). Covalent modifications at each end of the chain give rise to the observed structural diversity of known carotenoids (Armstrong, 1997).
• the desaturation of the chains generates the chromophore, characteristic of carotenoids, which results in a region of easily excitable delocalized electrons; these properties are the basis of two fundamental characteristics common to all carotenoids, namely their photochemical properties and their antioxidant action (Britton, 1995).
• carotenoid brings together molecules from the carotene and xanthophyll families.
• Bacterioruberins and their derivatives are found in extremophilic bacteria, in particular the halophilic archaea and certain psychrophilic actinobacteria; in these microorganisms, they play an important role in the protection of DNA and membranes against solar irradiation as well as stresses thermal and osmotic environments that these organisms constantly face (Mandelli et al., 2012).
• glycosylated derivatives that is to say in which the ends of the hydrophobic chains are substituted with sugar residues.
• these glycosylated forms represent a minor fraction of carotenoids and, therefore, have been the subject of little scientific study.
• the very function of these glycosylated forms of carotenoids has not been definitively elucidated. Studies have shown that in this group of photosynthetic bacteria, glycosylated carotenoids are localized to the thylakoid membrane and that sugar residues present at the ends of the isoprenoid chain are required to ensure proper stacking of the chloroplast membrane (Mohamed et al. ., 2005). In non-photosynthetic organisms, the presence of sugars at the ends can provide anchor or attachment points for other molecules on lipid membranes.
• glycosylated carotenoids Despite the gaps in the understanding of the physiological functions of glycosylated carotenoids, it is obvious that given their physicochemical properties and in particular their amphiphilic character, as well as their antioxidant potential, these molecules can find major applications in several fields, particularly in the cosmetics, food, nutraceutical and pharmaceutical industries.
• the present invention aims to solve the technical problem of providing glycosylated bacterioruberins, in particular in isolated or purified form.
• the aim of the present invention is to solve the technical problem consisting in providing compositions comprising one or more glycosylated bacterioruberins, in particular in isolated or purified form.
• the aim of the present invention is to solve the technical problem consisting in providing a method for the separation and purification of at least one glycosylated bacterioruberine.
• the present invention aims to solve the technical problem of providing an in vitro method for stabilizing proteins.
• the Applicant has developed a method for efficiently isolating the glycosylated forms of bacterioruberine.
• the present invention relates to a glycosylated bacterioruberin characterized in that it is isolated and chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated, decaglycosylated bacterioruberins, undecaglycosylated bacterioruberins, and dodecaglycosylated bacterioruberins.
• the present invention relates to a glycosylated bacterioruberin chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins and tetraglycosylated bacterioruberins.
• the glycosylated bacterioruberine is separated and purified from an extract of an extremophilic bacterium, preferably a bacterium from the Micrococcaceae family, advantageously from Arthrobacter agilis.
• the glycosylated bacterioruberine is separated and purified from an extract of carotenoids of ⁇ agilis According to one embodiment, the glycosylated bacterioruberine has a degree of purity of at least 70%, even 80%, preferably 90%, even more advantageously 95%, even 99% by mass.
• the extract is a total extract.
• a total extract is typically an extract from the whole of the bacterium considered.
• one or more glycosylated bacterioruberins isolated according to the invention are synthesized by biotechnology synthesis or by chemical synthesis.
• Bacterioruberine means in particular (all-E)-(2S,2'S)-Bacterioruberine (CAS No. 32719-43-0), also known under the name “a-Bacterioruberine”.
• a-Bacterioruberine has the following structure:
• ⁇ -Bacterioruberine comprises 4 terminal hydroxyl groups, each of which is capable of being substituted by ether bond with a sugar-type group, or even one or more covalently bonded sugars.
• glycosylated form of bacterioruberine or “glycosylated bacterioruberin” is meant a bacterioruberin of which at least one hydroxyl group is substituted with one or more, for example two or three, sugar residues by means of an ether bond between the skeleton bacterioruberine and sugar.
• An “isolated glycosylated bacterioruberin” according to the invention is obtained by synthesis by biotechnology, by chemical synthesis, or by purification of glycosylated bacterioruberin naturally contained in a natural bacterium.
• a “glycosylated bacterioruberine” corresponds to the following structure: [Chem 2] wherein R is independently selected from a hydrogen atom, one or more, for example two or even three, sugar residues and wherein R at least one occurrence represents one or more, for example two or even three sugar residues.
• the sugar is a hexose or a deoxyhexose selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose, fucose, fructose, the fucose.
• the present invention also relates to a composition comprising at least one glycosylated bacterioruberin, said composition essentially comprising no non-glycosylated form of bacterioruberin, and said composition comprising one or more excipients acceptable for a human being.
• said composition comprises at least one isolated glycosylated bacterioruberine as defined according to the present invention.
• said composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins and tetraglycosylated bacterioruberins.
• said composition comprises a mixture of glycosylated bacterioruberins essentially consisting of monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and said mixture preferably comprising 20 to 80% by mass of monoglycosylated bacterioruberins and 20 to 80% by mass of diglycosylated bacterioruberins with respect to the total mass of the mixture of glycosylated bacterioruberins.
• the present invention also relates to any one of the mixtures of two or more isolated glycosylated bacterioruberins chosen from bacterioruberins monoglycosylées, bactériorubérines diglycosylées, bactériorubérines triglycosylées, bactériorubérines tétraglycosylées, bactériorubérines pentaglycosylées, bactériorubérines hexaglycosylées, bactériorubérines heptaglycosylées, bactériorubérines octaglycosylées, bactériorubérines nonaglycosylées, bactériorubérines decaglycosylées, bactériorubérines undecaglycosylées, et bactériorubérines dodecaglycosylées ne commack piping pas de forme non-glycosylée de bactériorubérine.
• said mixture essentially consists of a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, tetraglycosylated bacterioruberins; and preferably a mixture of monoglycosylated bacterioruberins and diglycosylated bacterioruberins.
• the invention also relates to a method for separating and purifying at least one glycosylated bacterioruberine defined according to the invention from an extract of carotenoids containing them, comprising the following steps: i) dissolving said extract in a solvent, and preferably in tetrahydrofuran; ii) Separation of the fractions containing one or more glycosylated bacterioruberins by adsorption chromatography on a stationary phase column of the silica type using an eluent, and preferably using a mixture of dichloromethane/methanol.
• the weight ratio between dichloromethane and methanol is between 20/1 and 1/20, advantageously between 10/1 and 3/7.
• fractions obtained by this purification process can be further analyzed and characterized using qualitative and quantitative methods known to those skilled in the art, such as, for example, thin layer chromatography, HPLC/UV, HPLC/DAD and mass spectrometry.
• glycosylated bacterioruberin(s) are separated and purified from a total carotenoid extract containing the glycosylated bacterioruberins.
• the total extract of carotenoids containing the glycosylated bacterioruberins is a bacterial extract, preferably of Actinobacteria, even more advantageously from the Micrococcaceae family,
• these are the Micrococcus roseus and Arthrobacter agilis species.
• the strains of Arthrobacter agilis used as sources of glycosylated bacterioruberins within the meaning of the invention are strain MB813 (described in Fong et al. 2001) and/or SB5 (described in patent application WO2014167247).
• This species is also known as Micrococcus agilis.
• the methods for obtaining extracts of total carotenoids from these bacterial species are known to those skilled in the art and are for example described in Strand et al. 1997, Fong et al. 2001 as well as patent application WO2014167247.
• the total extract of carotenoids containing the glycosylated bacterioruberins according to the invention corresponds to the extract contained in the starting material MIRORUBERINE marketed by the company GREENTECH and corresponding to the INCI designation Micrococcus lysate.
• the glycosylated bacterioruberins according to the invention can be obtained by biotechnology, or by chemical synthesis, for example by means of controlled glycosylation of the native forms of bacterioruberins, typically ⁇ -bacterioruberins, for example starting from a-bacterioruberine. This glycosylation can be obtained chemically or by biotechnology, preferably by biotechnology using one or more suitable glycosyltransferases.
• the raw material HALORUBINE marketed by the company HALOTEK GMBH can be used as a source of a-bacteriorubérine in the synthesis of glycosylated bacteriorubérines within the meaning of the invention.
• glycosylated bacterioruberins according to the invention have shown significant antioxidant activity; these molecules are able to protect proteins from carbonylation and can contribute to their stabilization. Therefore, glycosylated bacterioruberins lend themselves to different applications in the fields of pharmaceuticals, dermocosmetics, nutraceuticals and biotechnology.
• the present invention relates to an isolated glycosylated bacterioruberin as defined according to the invention, and in particular chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, decaglycosylated bacterioruberins, undecaglycosylated bacterioruberins, and dodecaglycosylated bacterioruberins for use as a medicament.
• the present invention also relates to an isolated glycosylated bacterioruberin selected from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, and tetraglycosylated bacterioruberins for its use as a medicament.
• the present invention also relates to a composition according to the invention for its use as a medicament.
• said composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, and tetraglycosylated bacterioruberins, preferably a mixture of glycosylated bacterioruberins essentially consisting of monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and said mixture preferably comprising 20 to 80% by mass of monoglycosylated bacterioruberins and 20 to 80% by mass diglycosylated bacterioruberins relative to the total mass of the mixture of glycosylated bacterioruberins.
• the invention relates to glycosylated bacterioruberins, their mixture and compositions defined according to the invention, for their use in a method of therapeutic treatment of a human or animal being.
• it is the treatment of neurodegenerative diseases, such as for example Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy or even amyotrophic lateral sclerosis (ALS) as well as ocular neurodegenerative diseases selected from macular degeneration, retinitis pigmentosa and retinopathy.
• neurodegenerative diseases such as for example Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy or even amyotrophic lateral sclerosis (ALS)
• ocular neurodegenerative diseases selected from macular degeneration, retinitis pigmentosa and retinopathy.
• glycosylated bacterioruberins can also be useful and therapeutically effective in the treatment of other pathologies presenting a deregulation in the aggregation of proteins, such as, for example, fibrosis, advantageously pulmonary fibrosis, and diabetes.
• compositions according to the invention are generally presented in dosed form.
• a composition according to the invention can be in the form of a tablet, dragee, capsule, suppository, injectable or drinkable solution, or drop and it is suitable for oral, oromucosal, rectal, vaginal, intramuscular parenteral or ophthalmic administration.
• compositions according to the invention mention will be made more particularly of those which are suitable for oral, oromucosal, parenteral (intravenous, intramuscular or subcutaneous), per or transcutaneous, intravaginal, rectal, nasal, perlingual, buccal, ocular or respiratory.
• compositions according to the invention for parenteral injections include in particular sterile aqueous and non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstituting solutions or dispersions for injection.
• compositions according to the invention for solid oral administration, include in particular simple or coated tablets, sublingual tablets, sachets, capsules, granules, and for oral, nasal, buccal or ocular liquid administration, include in particular emulsions, solutions, suspensions, drops, syrups and aerosols.
• compositions for rectal or vaginal administration are preferably suppositories or ovules, and those for per or transcutaneous administration include in particular powders, aerosols, creams, ointments, gels and patches.
• compositions cited above illustrate the invention but do not limit it in any way.
• inert, non-toxic excipients or vehicles acceptable for a human being or pharmaceutically acceptable, mention may be made, by way of indication and not of limitation, of diluents, solvents, preservatives, wetting agents, emulsifiers, dispersing agents, binders , blowing agents, disintegrating agents, retardants, lubricants, absorbents, suspending agents, colorants, flavorings, etc.
• the useful dosage varies according to the age and weight of the patient, the route of administration, the pharmaceutical composition used, the nature and the severity of the condition.
• the composition according to the invention can be administered once a month, week or day and it may contain from 1 mg to 1 g of glycosylated bacterioruberins or any one of their mixtures.
• glycosylated bacterioruberins according to the invention are suitable for their use in food and nutraceutical supplements.
• a composition according to the invention is a food supplement.
• each dose may contain, by way of example, from 1 mg to 1 g of glycosylated bacterioruberins and/or any of their mixtures.
• microcrystalline cellulose is used as bulking agent. It is used between 10 and 30% by weight relative to the total weight of the food supplement, more advantageously around 20% by weight.
• Dicalcium phosphate and tricalcium phosphate are used as compression agents to prepare tablets.
• the dicalcium phosphate is used from 10 to 30% by weight relative to the total weight of the food supplement, more advantageously around 15% by weight.
• the tricalcium phosphate is used in an amount varying from 2.5 to 7.5% by weight relative to the total weight of the food supplement, and more advantageously around 5% by weight.
• Hydrated silica, magnesium stearate and colloidal silica can advantageously be used as thinners in the food supplement in the form of tablets or capsules. They are introduced in an amount located around 2% by weight, 1% by weight and 0.6% by weight relative to the total weight of the food supplement, respectively.
• adjuvants such as flavorings (natural or chemical, fruit or other flavorings) or pigments are advantageously incorporated into the preparation of the food supplement.
• the envelope of these soft capsules or hard gelatin capsules may contain in particular animal gelatin such as fish gelatin, glycerin, or a material of vegetable such as a cellulose or starch derivative, or a vegetable protein.
• animal gelatin such as fish gelatin, glycerin, or a material of vegetable such as a cellulose or starch derivative, or a vegetable protein.
• one or more glycosylated bacterioruberins according to the invention incorporated into the capsules can be dissolved in a fatty substance, advantageously of caprylic and/or capric triglyceride and preferably stabilized with tocopherol.
• glycosylated bacterioruberins as well as their compositions, are suitable for use in biotechnological applications.
• these molecules can be used to stabilize proteins, for example enzymes, and prevent or slow down their degradation, inactivation, aggregation, etc.
• the present invention also relates to an in vitro method for stabilizing proteins consisting in incubating or storing the proteins to be stabilized in contact with one or more glycosylated bacterioruberins as defined by the invention or with a composition as defined by the invention.
• a composition according to the invention is a cosmetic composition, advantageously suitable for topical application.
• said composition comprises comprising at least one glycosylated bacterioruberin chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, , undecaglycosylated bacterioruberins, and dodecaglycosylated bacterioruberins.
• glycosylated bacterioruberin chosen from monoglycosylated bacterioruberins, diglycosylated bacter
• said composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, tetraglycosylated bacterioruberins; preferably a mixture of glycosylated bacterioruberins essentially consisting of monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and said mixture preferably comprising 20 to 80% by mass of monoglycosylated bacterioruberins and 20 to 80% by mass of diglycosylated bacterioruberins relative to the total mass of the mixture of bacterioruberins glycosylated.
• the glycosylated bacterioruberins according to the invention represent from 0.0001 to 0.5%, advantageously from 0.001 to 0.01% by weight relative to the total weight of the cosmetic composition.
• composition according to the invention further comprises at least one UV screening agent.
• UV filter encompasses organic or mineral compounds capable of filtering UVA, UVB and/or UVC.
• they may just as well be mineral filters as chemical or organic filters.
• compositions according to the invention may contain one or more broad-spectrum UV filters, that is to say compounds or mixtures which absorb UV-A, UV-B, UV-C and optionally visible light. .
• the filters corresponding to the following INCI designations can be used in the context of the invention: tris biphenyl triazine, bis ethylhexyloxyphenol methoxyphenyl triazine, methylene bis-benzotriazolyl tetramethylbutylphenol.
• these are marketed by the company BASF under the names of TINOSORB S®/TINOSORB AQUA®, TINOSORB A2B®, TINOSORB M®, respectively.
• a broad-spectrum filter suitable for the composition according to the invention corresponds to the INCI designation diethylhexyl butamido triazone, for example marketed by the company SIGMA 3V under the name UVASORB HEB®.
• the composition according to the invention comprises at least one filter chosen from the following group of compounds identified by their INCI designation: tris biphenyl triazine, bis ethylhexyloxyphenol methoxyphenyl triazine, and methylene bis-benzotriazolyl tetramethylbutylphenol, diethylhexyl triazone butamido, or mixtures thereof.
• the composition comprises the bis-ethylhexyloxyphenol methoxyphenyl triazine screening agent.
• the composition instead of or in addition to the broad-spectrum filter(s), contains at least one UVA and/or UVB, organic and/or mineral filter, which may be in the aqueous phase (lipophilic) and/or oily (fat-soluble).
• composition according to the invention may contain fat-soluble UV-B filters, capable of contributing to the stabilization or solubilization of broad-spectrum filters or even of stabilizing each other and thereby increasing the sun protection factor (FPS or "SPF" in English).
• FPS sun protection factor
• such filters correspond to the following INCI designations: homosalate, octocrylene, ethylhexyl salicylate, ethylhexyl triazone.
• the composition according to the invention comprises ethylhexyl triazone, marketed by BASF under the name UVINUL T 150®.
• the fat-soluble UV-B filter is a-(trimethylsilyl)- ⁇ -(trimethylsilyloxy)poly[oxy(dimethyl)silylene]-co-[oxy(methyl)(2- ⁇ 4-[ 2,2-bis(ethoxycarbonyl)vinyl]phenoxy ⁇ -1-methyleneethyl)silylene]-co-[oxy(methyl)(2-(4-[2,2-bis(ethoxycarbonyl)vinyl]phenoxy)prop-1- enyl)silylene], a silicone polymer able to filter in the UV-B.
• This filter corresponds for example to the cosmetic raw material Parsol SLX®, marketed by the company DSM under the INCI designation polysilicone-15.
• the composition according to the invention comprises at least one UV-B screening agent chosen from the following group of compounds identified by their INCI designation: homosalate, ethylhexyl salicylate, ethylhexyl triazone, polysilicone-15, or mixtures thereof.
• the composition is free of the following screening agents: 4-methylbenzylidene camphor, benzophenone-2, benzophenone-3, ethylhexyl methoxycinnamate, octocrylene.
• the composition according to the invention comprises at least one UV-A filter, in order to ensure complete filtration of the harmful part of the solar spectrum.
• Advantageous UVA filters within the meaning of the present invention are butyl methoxydibenzoylmethane (I NC I) and diethylamino hydroxybenzoyl hexyl benzoate (INCI), corresponding respectively to the raw materials Parsol 1789® marketed by the company DSM and UVINUL® A+ marketed by the company BASF.
• the UV-A screening agent is bis-(diethylaminohydroxybenzoylbenzoyl)piperazine (INCI) (CAS number 919803-06-8), corresponding for example to the raw material C1332® marketed by the company BASF.
• the composition according to the invention comprises at least one screening agent chosen from the following group of compounds identified by their inci designation: butyl methoxydibenzoylmethane, diethylamino hydroxybenzoyl hexyl benzoate, bis-(diethylaminohydroxybenzoyl benzoyl) piperazine, or their mixtures.
• UV filters within the meaning of the present invention are water-soluble filters, such as for example:
• the basic amino acid represents between 0.5 and 2% by weight of the composition, preferably between 1 and 1.5% by weight of the composition.
• the composition according to the invention comprises at least one water-soluble screening agent chosen from the following group of compounds identified by their INCI designation: disodium phenyl dibenzimidazole tetrasulfonate, phenylbenzimidazole sulfonic acid, or mixtures thereof.
• the inorganic mineral filters, or mineral screens are metal oxides and/or other compounds which are not easily soluble or insoluble in water, in particular titanium oxides (TiOs), zinc oxides (ZnO), advantageously doped with iron, iron (FesOs), zirconium (ZrOs), silicon (SiOs), manganese (for example MnO), aluminum (Al2O3), or cerium (CesOs), bismuth (Bi 2 Os) ⁇
• the inorganic mineral screening agents can be used in the form of an oily or aqueous predispersion available on the market. These predispersions can advantageously be supplemented with dispersion auxiliaries and/or solubilization mediators.
• Inorganic mineral filters can also be surface-treated or encapsulated, in order to give them a hydrophilic, amphiphilic or hydrophobic character.
• This surface treatment may consist in the mineral filters being provided with a thin hydrophilic and/or hydrophobic inorganic and/or organic film.
• the composition according to the invention comprises at least one mineral screen chosen from the following group of compounds identified by their INCI designation: Zinc oxide, Titanium dioxide, or their mixtures.
• the UV screening agents as described above, present in the composition according to the invention represent from 0.1% to 30% by weight of the composition, advantageously between 0.5% and 20%, even more advantageously between 1% and 15%.
• the composition according to the invention has a sun protection factor ("SPF" or SPF) greater than or equal to 10, preferably greater than or equal to 20, advantageously greater than or equal to 30, even more advantageously greater than or equal to 50.
• SPPF sun protection factor
• the composition according to the invention comprises a UV-A/UV-B protection ratio equal to or greater than 1/3.
• the sunscreen composition according to the invention comprises at least one sunscreen solubilizer chosen from the following group of compounds identified by their INCI designation: caprylyl caprylate/caprate, dibutyl adipate, dicaprylyl carbonate, diisopropyl sebacate, dicaprylyl ether, coco-caprylate, C12 -15 alkyl benzoate, propylheptyl caprylate and butylene glycol dicaprylate/dicaprate.
• sunscreen solubilizers are commercially available from several suppliers.
• the following raw materials can be used in the composition according to the invention:
• MIGLYOL® 8810 marketed by the company IOI Oleo GmbH corresponding to the INCI designation butylene glycol dicaprylate/dicaprate.
• the composition according to the invention comprises at least four solubilizers chosen from the following group of compounds identified by their INCI designation: caprylyl caprylate/caprate, dibutyl adipate, dicaprylyl carbonate, diisopropyl sebacate, dicaprylyl ether, coco- caprylate, C12-15 alkyl benzoate, propylheptyl caprylate and butylene glycol dicaprylate/dicaprate.
• the composition according to the invention comprises the solubilizers corresponding to the INCI designations dibutyl adipate, dicaprylyl carbonate, and diisopropyl sebacate.
• the composition according to the invention also comprises at least one other solubilizer chosen from the following group of compounds identified by their INCI designation: propylheptyl caprylate, dicaprylyl ether, coco-caprylate, C12-15 alkyl benzoate, caprylyl caprylate/caprate.
• it is propylheptyl caprylate.
• the solubilizers as defined above represent between 5% and 80% by weight of the total of the composition, advantageously between 10% and 70%, even more advantageously between 15 and 60%.
• composition according to the invention may also comprise an SPF "booster”, that is to say an agent for enhancing the sun protection factor, and/or a photostabilizer, that is to say an ingredient which makes it possible to increase the SPF or light stabilize the filters, such an ingredient not being considered itself a sunscreen.
• SPF sunscreen
• a photostabilizer that is to say an ingredient which makes it possible to increase the SPF or light stabilize the filters, such an ingredient not being considered itself a sunscreen.
• photostabilizer advantageously representing between 0.01% and 10% by weight of the composition, even more advantageously between 0.1% and 2%.
• This raw material is for example marketed by the company HALLSTAR under the name Hallbrite® BHB;
• photostabilizer advantageously representing between 0.01% and 10% by weight of the composition, even more advantageously between 0.1% and 2%.
• This raw material is for example marketed by the company BASF under the name of TINOGARD® TL;
• sold by the company HALLSTAR can be used in the context of the present invention.
• styrene acrylate copolymer preferably representing between 1% and 10% by weight of the composition according to the invention.
• the raw materials SunSpheres® H53 and SunSpheres® PGL Polymer, marketed by the company DOW CHEMICALS, can be used in the context of the present invention;
• the composition according to the invention also comprises one or more substances capable of filtering visible light, in particular blue light.
• the compounds described in the document EP 1 484 051 can be used to provide filtering of blue light.
• the composition according to the invention also comprises other components which can contribute to internal protection by an action which can consist of DNA protection, a reduction in the immunosuppression induced by radiation UV, an antiradical action or a combined effect of these actions.
• the protective action of a preparation according to the invention against oxidative stress or against the effect of free radicals can be further improved if it additionally comprises one or more antioxidants, easily selected by man.
• tocopherol and its derivatives for example from the following list: tocopherol and its derivatives, totarol, magnolol, honokiol, amino acids and their derivatives, peptides (D and/or L-carnosine) and their derivatives (for example l anserine, hypotaurine, taurine), carotenoids, carotenes (a-carotene, p-carotene, lycopene) and their derivatives, chlorogenic acid and its derivatives, lipoic acid and its derivatives (dihydrolipoic acid) , aurothioglucose, propylthiouracil and other thiols (thioredoxin, glutathione, cysteine, cystine, cystamine and their esters glycosyl, N-acety
• the composition according to the invention also contains glycyrrhetinic acid, a derivative or a salt of this acid, used as a soothing agent (anti-inflammatory agent) and representing between 0.01% and 2% by weight of the composition, preferably between 0.1% and 1%.
• the cosmetic and/or dermatological composition contains at least one, or even all of the following constituents exerting a biological activity in vivo on the cells of the skin, lips, hair and/or mucous membranes subjected to UV-A and/or UV-B radiation, respectively:
• an anti-radical agent preserving cell structures such as for example vitamin E and/or its fat-soluble or water-soluble derivatives, in particular tocotrienol and/or tocopherol, advantageously representing between 0.001 and 10% by total weight of the composition, again more advantageously between 0.02 and 2%, preferably of the order of 0.04%;
• an agent limiting immunosuppression such as for example vitamin PP, advantageously representing between 0.001 and 1% by total weight of the composition, even more advantageously from 0.01% to 0.3%;
• a protective agent for the p53 protein such as for example epigallocatechin gallate (EGCG), advantageously representing between 0.001 and 0.1% by total weight of the composition, even more advantageously from 0.005% to 0.05%.
• EGCG epigallocatechin gallate
• composition according to the invention may also also comprise peptide extracts of soya and/or wheat, such as those described in EP 2 059 230.
• the peptide extracts come from soybean and wheat seeds resulting from an enzymatic hydrolysis of said seeds via peptidases which makes it possible to recover peptides with an average size of 700 Daltons.
• the soy peptide extract is the extract identified under CAS number 68607-88-5, just as the wheat peptide extract is the extract identified under CAS number 70084-87-6.
• Wheat and soy extracts may correspond to the INCI designations Hydrolyzed wheat protein and Hydrolyzed soy protein, respectively.
• the soybean and wheat peptide extracts are used together, for example in a weight ratio respectively between 80/20 and 20/80, advantageously between 70/30 and 30/70, preferably equal at 60/40.
• the soybean and/or wheat peptide extracts are free of synthetic GHK tripeptides (glycyl-histidyl-lysine; INCI: Tripeptide-1).
• GHK tripeptides GHK tripeptides
• the peptide extracts of soya and/or wheat representing from 0.01 to 20% by total weight of the composition, advantageously from 0.1% to 10%, even more advantageously from 0.2% to 0.7% .
• composition according to the invention comprises, in accordance with the teachings of document FR 2 865 398, the combination of at least one amino acid chosen from the group consisting of ectoine, creatine, ergothioneine and/or carnosine, or their physiologically acceptable salts, and mannitol or a mannitol derivative.
• the composition according to the invention comprises, in a physiologically acceptable medium, the amino acid or one of its salts, alone or as a mixture in proportions of between 0.001% and 10% by total weight of the composition, and preferably between 0.01% and 5%.
• composition according to the present invention preferably comprises, in a physiologically acceptable medium, mannitol or one of its derivatives, in proportions of between 0.01% and 30% by total weight of the composition, advantageously between 0, 1% and 10%.
• composition according to the invention comprises ectoin and mannitol.
• the composition according to the invention contains one or more other tanning or self-tanning agents. It can be a self-tanner which reacts with the amino acids of the skin according to a Maillard reaction or via a Michael addition, or else a promoter of melanogenesis or a propigmenting compound which promotes tanning natural to the skin.
• Self-tanning substances can be 1,3-dihydroxyacetone (DHA), glycerolaldehyde, hydroxymethylglyoxal, y-dialdehyde, erythrulose, 6-aldo-D-fructose, ninhydrin, 5-hydroxy-1 ,4-naphthoquinone (juglone), 2-hydroxy-1,4-naphthoquinone (lawsone), or their combination.
• DHA 1,3-dihydroxyacetone
• glycerolaldehyde hydroxymethylglyoxal
• y-dialdehyde erythrulose
• 6-aldo-D-fructose ninhydrin
• 5-hydroxy-1 ,4-naphthoquinone juglone
• 2-hydroxy-1,4-naphthoquinone lawsone
• Propigmenting substances can be melanocyte-stimulating hormone (a-MSH), peptide analogs of a-MSH, endothelin-1 receptor agonists, p opiate receptor agonists, cAMP, tyrosinase stimulating agents.
• a-MSH melanocyte-stimulating hormone
• peptide analogs of a-MSH endothelin-1 receptor agonists
• p opiate receptor agonists p opiate receptor agonists
• cAMP tyrosinase stimulating agents.
• the composition according to the invention further comprises, as a self-tanner, a combination of dihydroxy methylchromonyl palmitate and/or dimethyl methoxy chromanol as well as a lipophilic form of tyrosine.
• Dihydroxy methylchromonyl palmitate (CAS number: 1387636-35-2) corresponds, for example, to the cosmetic ingredient marketed by Merck under the name RonaCare® Bronzyl.
• Dimethylmethoxy chromanol (CAS number: 83923-51 - 7) corresponds to the cosmetic ingredient marketed by LIPOTEC SA under the name lipochromone-6.
• the dihydroxy methylchromonyl palmitate or the dimethylmethoxy chromanol is present in the composition according to the invention in the amount of 0.01% to 10% by total weight of the composition, advantageously from 0.05% to 10% , even more advantageously from 0.1% to 5%, more particularly from 0.1% to 0.5%.
• the lipophilic form of tyrosine is an ingredient based on tyrosine and having a more pronounced lipophilic nature than tyrosine.
• the lipophilic form of tyrosine may in particular correspond to oleoyl tyrosine (CAS number: 147732-57-8), which is found, for example, in the liquid cosmetic ingredient TYR-OL, marketed by the company SEDERMA, and which comprises approximately 50% by weight of oleoyl tyrosine in butylene glycol (approximately 30%+approximately 20% oleic acid), or alternatively in the liquid cosmetic ingredient TYR-EXCEL, marketed by the company SEDERMA, which comprises approximately 50% by weight of oleoyl tyrosine, approximately 20% oleic acid (CAS No: 112-80-1) and approximately 30% Luffa cylindrica oil (sponge gourd seed oil; CAS No: 1242417-48 -6). According
• the vegetable oil is oleic sunflower oil, advantageously deodorized.
• the raw material OLEOACTIVE TYROSINE BASE HELIANTHUS ANNUS marketed by the company OLEOS, and corresponding to the INCI designations Helianthus annuus seed oil (and) tyrosine (and) glyceryl stearate, can be used in the context of the present invention.
• the lipophilic form of tyrosine such as in cosmetic ingredients based on oleoyl-tyrosine (advantageously at 50% by weight) or tyrosine formulated in vegetable oil, represents between 0.1% and 10% by total weight of the composition, advantageously between 1 and 3%, even more advantageously between 1% and 1.5%.
• composition according to the invention may also contain active agents having depigmenting properties, such as for example: lysine azeilate, or other derivatives or salts of the azelaic acid of andrographolide, in particular the extract of Andrographis paniculata corresponding to the INCI designation Andrographis paniculata leaf extract; native ascorbic acid (vitamin C) or its derivatives, in particular the derivatives corresponding to the INCI Ascorbyl Glucoside Ethyl ascorbic acid, Ascorbyl methylsilanol pectinate, Sodium ascorbyl phosphate and Ascorbyl tetraisopalmitate; arbutin or a plant extract containing it, in particular bearberry extract corresponding to the INCI designation Arctostaphylos uva-ursi ⁇ eat extract;
• active agents having depigmenting properties such as for example: lysine azeilate, or other derivatives or salts of the azelaic acid of andrographoli
• biomimetic peptides corresponding to the INCI designations hexapeptide 2 and/or nonapeptide-1; an aqueous extract of an alga called Palmaria palmata, in particular the extract corresponding to the INCI designation Palmaria palmata extract; 4-n-butylresorcinol;
• niacinamide also called nicotinamide, and its derivatives
• composition according to the invention may also contain active agents having healing properties such as, for example, an antimicrobial agent chosen from the active agents corresponding to the following INCI designations: copper sulphate, zinc sulphate, sodium hyaluronate, Vitis vinifera (grape) vine extract, or their mixtures.
• active agents having healing properties such as, for example, an antimicrobial agent chosen from the active agents corresponding to the following INCI designations: copper sulphate, zinc sulphate, sodium hyaluronate, Vitis vinifera (grape) vine extract, or their mixtures.
• the cosmetic composition according to the invention may also contain active agents having sebocorrective, keratolytic, sebum-regulating properties and/or an anti-acne activity, in order to allow the formulation of sun products treating acne.
• the cosmetic composition according to the invention may comprise an antimicrobial agent chosen from active agents corresponding to the INCI designations propyl gallate, dodecyl gallate, Ginkgo biloba leaf extract, bakuchiol, dihydromyricetin, zinc gluconate, salicylic acid, or mixtures thereof.
• an antimicrobial agent chosen from active agents corresponding to the INCI designations propyl gallate, dodecyl gallate, Ginkgo biloba leaf extract, bakuchiol, dihydromyricetin, zinc gluconate, salicylic acid, or mixtures thereof.
• composition according to the invention can therefore also comprise at least one ingredient chosen from the following list: at least one polyol chosen from xylitol, rhamnose, sorbitol and mannitol;
• vitamin E or one of its hydrophilic or lipophilic derivatives, or one of their salts, advantageously tocotrienol or tocopherol; salicylic acid or one of its derivatives;
• peptide extract of soya and/or wheat an amino acid selected from the group consisting of ectoine, creatine, ergothioneine, carnosine, tyrosine, decarboxycarnosine, glutamine and their salts;
• composition according to the invention may also contain adjuvants such as those usually used in the field of cosmetics, such as active agents, preservatives, antioxidants, complexing agents, solvents, perfumes, fillers, bactericides, electrolytes, odor absorbers, coloring matter or even lipid vesicles.
• adjuvants such as those usually used in the field of cosmetics, such as active agents, preservatives, antioxidants, complexing agents, solvents, perfumes, fillers, bactericides, electrolytes, odor absorbers, coloring matter or even lipid vesicles.
• composition of the invention is intended for topical application and more particularly for application to the skin, lips, hair and/or mucous membranes.
• composition of the invention can be in all the pharmaceutical forms normally used in the cosmetic and dermatological fields, such as, for example, but in a non-limiting way, in the form of an optionally gelled aqueous solution, of a dispersion of the type lotion, an emulsion (O/W) or vice versa (W/O), more or less fluid, or a multiple emulsion such as for example a triple emulsion (W/O/W or O/W/O), or else in the form of a vesicular dispersion of the ionic (liposomes) and/or non-ionic type, of a two-phase composition devoid of emulsifiers and gelling agents whose immiscible phases separate during storage, of mousse, of stick, of anhydrous oil, spray or mist.
• the composition according to the invention is a W/O emulsion.
• the W/O emulsions according to the invention comprise PEG-30 dipolyhydroxystearate (I NCI) as emulsifier.
• the composition according to the invention is an O/W emulsion.
• the O/W emulsions according to the invention comprise an emulsifier chosen from the following group of compounds identified by their INCI designation: potassium cetyl phosphate, glyceryl stearate, PEG-100 stearate and C20-22 alkyl phosphate/C20-C22 alkyl alcohol , glyceryl stearate citrate, tribehenin PEG-20 esters and mixtures thereof.
• an emulsifier chosen from the following group of compounds identified by their INCI designation: potassium cetyl phosphate, glyceryl stearate, PEG-100 stearate and C20-22 alkyl phosphate/C20-C22 alkyl alcohol , glyceryl stearate citrate, tribehenin PEG-20 esters and mixtures thereof.
• composition according to the invention may also comprise preservatives. Any preservative suitable for use in cosmetic or dermatological compositions can be used in the formulation of a composition according to the invention.
• the preservatives used are alkanediols, even more advantageously 1,2-alkanediols or 1,3-alkanediols, and mixtures thereof.
• the preservative is a 1,2-diol chosen from 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, 1,2-octanediol and 1,2 - decanediol, or mixtures thereof.
• the preservative is a 1,3-diol chosen from 1,3-propanediol and 1,3-butanediol, or mixtures thereof.
• these alkanediols are marketed by several companies, in particular the company SYMRISE or MINACARE.
• the composition according to the invention comprises between 0.01% and 2% by weight of the composition of diols, advantageously between 0.1% and 1%, for example 0.5%.
• the present invention also relates to a cosmetic, non-therapeutic treatment process, advantageously for combating oxidative stress, and in particular cell aging of the skin, consisting in applying a composition according to the invention to the skin of a subject in need thereof. .
• said composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, tetraglycosylated bacterioruberins; and preferably a mixture of glycosylated bacterioruberins essentially consisting of monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and said mixture preferably comprising 20 to 80% by mass of monoglycosylated bacterioruberins and 20 to 80% by mass diglycosylated bacterioruberins relative to the total mass of the mixture of glycosylated bacterioruberins.
• composition advantageously applied topically, is also suitable for the following uses:
• Figure 1 shows the composition of a carotenoid extract from isolate SB5 of A. agilis species.
• BR a-Bacterioruberine
• BR-MonoG monoglycosylated form
• BR-DiG di-glycosylated form
• BR-DiG2 Another form of BR-DiG
• BR-TetraG tetraglycosylated form.
• FIG 2 Figure 2 shows the protection conferred by several carotenoids against UVB-induced protein carbonylation in Normal Human Keratinocytes (KHN).
• DMSO dimethylsulfoxide (solvent control);
• BR a-bacterioruberine;
• BR-DiG diglycosylated bacterioruberins;
• BR-MonoG + BR-DiG mixture of mono- and di-glycosylated bacterioruberins. Examples of realization
• Example I Purification of glycosylated bacterioruberins from a carotenoid extract of the bacterium A. agilis
• the aim of this study is to isolate and quantify the molecules contained in an extract of total carotenoids from the bacterium A. agilis.
• the SBE extract was taken up in tetrahydrofuran (THF) until complete solubilization.
• THF tetrahydrofuran
• a stage of separation of the glycosylated Bactériorubérines by chromatography on silica gel was carried out in a glass column after dilution of the SBE in THF.
• the first step of separation by column chromatography made it possible to collect the various fractions whose purity was confirmed by TLC and by HPLC-DAD, by comparing it to the absorbance spectrum of the native extract; the quantification of the different forms was carried out by UV absorption at 500nm
• the monoglycosylated form BR-MonoG represents >26% of the extract.
• the tetraglycosylated form (BR-TetraG) represents only 0.01% of the extract (Fig.1)
• Example II protection conferred by several carotenoids against UVB-induced carbonylation in human keratinocytes
• the aim of this study is to compare the protective effect of keratinocyte proteins from UV stress of different molecules derived from an extract of carotenoids from the SB5 strain of the actinobacteria A. agilis according to example I.
• the following molecules were studied:
• Total protein carbonylation was measured in the presence or absence of UVB stress.
• UVB 80; 40; 20; 15; 10mJ/cm2
• UVB 80; 40; 20; 15; 10mJ/cm2
• UVB stress 313 nm lamp, 80 dose; 40; 20; 10mJ/cm2 for dose selection and 15mJ/cm2 for molecule testing
• Immortalized NHEK/SVTERT 3-5 keratinocytes were seeded with approximately one million cells/plate in 108 plates (diam: 100 mm, 56 cm 2 ) corresponding to condition 36 in biological triplicates -> cells were cultured until at a confluence of 90 to 95% in keratinocyte medium (Keratinocyte-SFM from Gibco 17005075) at 37° C. and 5% CO 2 .
• the cells were incubated with the molecules to be tested for 2 hours.
• SFM keratinocyte medium was added and the cells were incubated for 24 hours at 37° C., 5% CO 2 .
• the cells were lysed by adding to each tube containing the cell pellet, 100 ⁇ l of UTC lysis buffer (UTC: Urea, thiourea & CHAPS).
• UTC lysis buffer Urea, thiourea & CHAPS.
• the lysis of the cells was carried out on a shaker for microtubes at a temperature of 4°C for 45min; 600rpm.
• the supernatant was transferred to an Amicon 3 kDa filter and rinsed with PBS (approximately 100 ⁇ l of protein for 400 ⁇ l of PBS).
• PBS approximately 100 ⁇ l of protein for 400 ⁇ l of PBS.
• the samples were centrifuged for 40 min at +4°C at 14,000 rpm. The procedure was repeated 3 times for each sample. After the third centrifugation, the Amicon filters were placed in a new tube and centrifuged for 3 minutes at 2000 rpm; the eluates were used in the rest of the analysis.
• Protein concentration was measured with a Bradford protein quantitation assay.
• Each 15 ⁇ g sample of protein was stained with 0.15 ⁇ L of 5 mM CF647 dye. Staining was performed overnight at +4°C, 600 rpm.
• Protein samples were loaded onto 12.5% acrylamide gels. Electrophoresis was performed at 80V until the samples exited the stacking gel, after which the voltage was increased to 120V until the samples reached the end of the gel.
• the gels were then collected and rinsed repeatedly in ultrapure water. Then, the gels were stained with 1x purple dye (SERVA Purple - 43386.01) from a 250x concentrated form to examine protein expression. This step was performed at 50 rpm on a shaker for 10 min at room temperature.
• 1x purple dye SEQUENT - 43386.01
• Example III - O/W SPF50+ emulsion The percentages indicated are given by mass of product relative to the total mass of the composition in the tables below.

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