Red algae biomass extract, and its cosmetic use to remove skin imperfections.

Abstract

The present invention relates to a photosensitive polar biomass extract obtained from phycocyanin-producing organisms, particularly from unicellular red algae (URA) or cyanobacteria, and its cosmetic use for preventing and / or eliminating skin defects, i.e., for improving the appearance of the skin.

Description

[Technical Field]

[0001] The present invention relates to a photosensitive polar biomass extract obtained from phycocyanin-producing organisms, particularly from unicellular red algae (URA) or cyanobacteria, and its cosmetic use for preventing and / or eliminating skin defects, i.e., for improving the appearance of the skin. [Background technology]

[0002] Acne is associated with excessive sebum secretion from the sebaceous glands, leading to clogged pores in the skin. The etiology of acne is multifactorial. Lesions include comedones, papules, pustules, and even nodules in the most severe forms.

[0003] Acne lesions can be aggravated by inflammatory responses resulting from abnormal bacterial growth in the sebum, which may be associated with Propionibacterium acnes, Xerosis bacteria, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pyogenes, Group B Streptococcus, or Demodex mites.

[0004] During puberty, acne begins when the sebaceous glands mature due to hormonal stimulation by androgens. In adults, acne results from irritation of the sebaceous glands, combined with decreased sebum secretion due to increased synthesis of adrenocortical hormones as a result of cosmetics or stress.

[0005] Various acne treatments are available, including photodynamic therapy (PDT). This therapy involves exposing a photosensitizer to light, which in turn produces singlet oxygen and other reactive oxygen species, causing the death of nearby microorganisms.

[0006] In such PDT treatments for acne treatment, a photosensitizer is applied topically to the skin surface to be treated. This skin surface is then exposed to an irradiation source, such as a laser or pulsed light, to allow the release of reactive oxygen species. The presence of singlet oxygen leads to the death of acne-causing bacteria and local desquamation of the skin, and frees clogged pores.

[0007] Examples of acne treatment using photodynamic therapy are described in European Patents No. 1755676, No. 2152259, No. 3082788, and No. 3558374.

[0008] It is important that photosensitizers protect skin cells as much as possible and do not increase inflammation caused by acne.

[0009] The main drawback of photodynamic therapy is the occurrence of undesirable side effects, particularly on the treated surface. These include erythema, swelling, edema, burning, itching, severe scaling, hyperpigmentation, irritation, and / or hypersensitivity.

[0010] Therefore, there is a real need to provide new substances that limit the occurrence of undesirable effects.

[0011] Furthermore, while the use of microalgae extracts in phototherapy for the treatment of acne has already been reported, methods for preparing such extracts present technical limitations, such as the need to use an inert atmosphere to avoid the degradation of active molecules and the need for a long maceration phase (for example, International Patent Application Publication No. 2021 / 209441 shows the use of a polar extract of Skeletonema).

[0012] Therefore, there is a real need to develop new materials that are easier and cheaper to manufacture, and whose manufacturing methods reduce the environmental impact.

[0013] To satisfy the needs of the prior art, the inventors have demonstrated that polar extracts of biomass from phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria, can be used to prevent and / or eliminate skin defects in subjects, i.e., to improve the appearance of the skin. [Overview of the project]

[0014] The present invention relates to photosensitive polar biomass extracts from phycocyanin-producing organisms, particularly from unicellular red algae (URA) or cyanobacteria.

[0015] Advantageously, the biomass is URA biomass, and the URA is selected from Gardieria or Ideyukogome, more preferably from Gardieria, the genus Cyanidia or Cyanidium, and even more preferably from Gardieria.

[0016] Advantageously, URA belongs to the Gardieria sulphuraria species.

[0017] Advantageously, the photosensitive polar extract is a solution comprising a polar organic solvent, preferably an alcohol, particularly methanol, ethanol, and isopropanol, a volatile organic acid, particularly formic acid, acetic acid, a primary or secondary amine, and a protic polar organic solvent selected from PEG (polyethylene glycol) and mixtures thereof.

[0018] Advantageously, the photosensitive polar extract contains up to 95% by weight, preferably 90% to 65% by weight, and more preferably 80% to 90% by weight, of lipids relative to the total dry weight of the extract.

[0019] Advantageously, the lipids include monogalactosyl diglycerides, digalactosyl diglycerides, phosphatidylcholine, and ceramides.

[0020] Advantageously, the photosensitive polar extract contains pheophorbide-a and its derivatives in amounts of 350 mg / g to 0.5 mg / g, preferably 175 mg / g to 1 mg / g, and more preferably 85 mg / g to 5 mg / g, relative to the total weight of the extract.

[0021] The present invention also provides a method for preparing a photosensitive polar crude extract, comprising culturing the biomass of phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria, and subsequently, a) A step of collecting biomass by separating the culture medium for obtaining live biomass, b) A step of dissolving cells from the live biomass obtained from step (a) to obtain dissolved biomass, c) Optionally diluting the dissolved biomass from step (b) to obtain solubilized dissolved biomass; d) Recovering insoluble matter suspended in the dissolved biomass from step (b) or the solubilized dissolved biomass from step (c) to obtain extracted biomass; e) Extracting the extracted biomass obtained in step d) by contacting it with a polar solvent, and then recovering an aqueous fraction by separating insoluble matter in the suspension to obtain a photosensitive polar crude extract; relates to a method comprising.

[0022] Advantageously, the polar organic solvent used in step e) of the method is a polar organic solvent selected from alcohols, especially methanol, ethanol, and isopropanol, volatile organic acids, especially formic acid, acetic acid, primary amines or secondary amines, and PEG (polyethylene glycol) and mixtures thereof.

[0023] Advantageously, the method further comprises a step of heating the biomass before or during the extraction step e), i.e., in any of steps a), b), c), d) or e).

[0024] Another object of the present invention is the photosensitive polar extract as defined above, which can be obtained by the method as defined above.

[0025] The present invention also relates to a topical composition comprising the photosensitive polar extract as defined above and a topically acceptable carrier.

[0026] The present invention also relates to a photosensitive polar extract or a composition containing it for use in the prevention and / or treatment of acne.

[0027] The present invention relates to a photosensitive polar extract or a composition containing it for use in the prevention and / or removal of skin defects.

[0028] Advantageously, the photosensitive polar extract or a composition containing it is applied to the treated skin surface of the subject, and then the surface is exposed to a light source. [Modes for carrying out the invention]

[0029] definition In the context of this invention, the term “biomass” refers to a collection of microalgae cells preferentially produced by fermentation in a biological reactor. This biomass can be considered a mass of single-celled organisms. The biomass can undergo various treatments and may be raw biomass, lysed biomass, or extracted biomass. In the context of this application, the characteristics of the biomass correspond to the average characteristics of all the cells that make up the biomass; in other words, lysed biomass is biomass containing at least 50% lysed cells with respect to the total number of cells, and raw biomass may contain lysed cells resulting from the harvesting step, but will not be considered lysed biomass as long as the number of lysed cells remains small compared to the number of non-lysed cells, i.e., less than 50%.

[0030] "Raw biomass" refers to biomass obtained after harvesting, that is, after the recovery of the culture and the separation of cells from at least a portion of the culture medium.

[0031] "Dissolved biomass" refers to microalgae biomass in which at least 50% of the cells are dissolved, preferably at least 70%, and more preferably at least 80%, 85%, 90%, 95%, and up to 100% of the cells are dissolved.

[0032] In the context of the present invention, "solubilized biomass" or "solubilized product" refers to dissolved biomass that has undergone a dilution step with an aqueous solution of a neutral, acidic, or basic pH.

[0033] "Extracted biomass" refers to the insoluble fraction recovered from dissolved biomass after washing it one or more times with an aqueous solution of neutral, acidic, or basic pH, particularly to extract water-soluble components such as phycocyanin.

[0034] According to the present invention, the term "dried biomass" refers to microalgae biomass that has been dried using methods known to those skilled in the art, and in which the water content relative to the total weight of the biomass is less than 10%, preferably less than 7%, and more preferably between 5% and 1%. Known drying methods include natural air drying, spray drying, fluidized air bed drying, roller dryer drying, and freeze-drying. The dried biomass may be dried raw biomass, dried dissolved biomass, or dried extracted biomass.

[0035] The term "thawed biomass" refers to biomass that has been frozen, in some cases for storage and / or transport reasons, and then thawed to a temperature suitable for the preparation of polar extracts.

[0036] "Polar biomass extract" refers to a composition obtained by extracting biomass using a polar organic solvent.

[0037] A "photosensitized extract" refers to an extract (or composition) that enables the generation of reactive oxygen species when exposed to a light source. One way to characterize the photosensitizing properties of an extract is to determine the minimum inhibitory concentration (MIC) of the extract against growing bacteria. In particular, in the context of this invention, the bacteria used can be selected from those known to be involved in acne exacerbation, such as Propionibacterium acnes or Staphylococcus aureus.

[0038] "Unicellular red algae" or "URA" refers to eukaryotic microalgae of the URA taxonomy that can be cultivated industrially to produce biomass and by-products such as proteins or phycobiliproteins like phycocyanin.

[0039] Cyanobacteria, also known as blue-green algae, are prokaryotic microalgae that can be cultivated industrially to produce biomass and by-products such as proteins and phycocyanin.

[0040] A “topical composition” refers to a composition intended to be applied to the skin of a target for use. It contains “topically acceptable” components suitable for use on the skin of a target, including one or more active ingredients and excipients or carriers. In the context of the present invention, a “cosmetic composition” refers to a topical composition consisting of components suitable for cosmetic use, intended exclusively or primarily for contact with the surface parts of the human body (epidermis, hair, nails, etc.) or teeth and oral mucosa, for the purpose of cleansing, fragrance, modifying their appearance, protecting them, maintaining them in good condition, or correcting body odor. Therefore, the selection of components in the composition is very important and distinguishes medical compositions from those suitable for topical cosmetic use. Cosmetic compositions may also be sterilized to prevent the carrying of pathogens that may develop during use.

[0041] According to the present invention, exposure to a light source or illumination refers to exposure to natural light or artificial light. In the case of exposure to artificial light, this is selected from illumination including lasers, pulsed light, or light-emitting diodes (LEDs), and preferably illumination including one or more LEDs.

[0042] "Skin defects" refer to wrinkles, benign skin conditions such as brown spots or moles and purpura, hidradenitis, rosacea, and skin irregularities, especially those associated with acne, such as enlarged pilaris, blackheads (open comedones), whiteheads (closed comedones), papules (red pimples), pustules (white pimples), nodules, and cysts.

[0043] Photosensitized polar extract The present invention relates to photosensitive polar biomass extracts of phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria, preferably unicellular red algae (URA) such as Cyanidioschizomon, Cyanidium, or Gardieria.

[0044] Photosensitive polar biomass extracts from phycocyanin-producing organisms, particularly URAs or cyanobacteria, are obtained by extraction of biomass extracted from phycocyanin-producing organisms with polar organic solvents.

[0045] In particular, the extract can be obtained by the methods according to the present invention as described in the remainder of this specification. According to one embodiment, the biomass is cyanobacteria, in particular biomass of the genus Althrospira, and is preferably selected from the species Althrospira pratensis (also known as Spirulina), Althrospira fusiformis, and Althrospira maxima.

[0046] Preferably, the biomass is URA biomass selected from the families Ideyukogoceae or Gardieriaceae, and more preferably from the genera Cyanidioschizonium, Cyanidium, or Gardieria.

[0047] According to a more preferred embodiment, the URA is of the genus Gardieria, more preferably the species Gardieria sulfuraria.

[0048] According to another embodiment, the URA is of the genus Cyanidioschizono, preferably of the species Cyanidioschizono merolae.

[0049] Polar organic solvents are either aprotic or protic solvents, with protic polar solvents being preferred.

[0050] Polar aprotic solvents that can be used according to the present invention include, preferably, acetone, butanone, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, acetonitrile, ethyl acetate, triethylamine and pyridine, 4-hydroxy-4-methyl-2-pentanone, and mixtures thereof.

[0051] The protic polar organic solvents used in the present invention are preferably alcohols, particularly methanol, ethanol, and isopropanol; volatile organic acids, particularly formic acid, acetic acid, primary or secondary amines; and PEG (polyethylene glycol) and mixtures thereof.

[0052] Microalgae, particularly those of the Idycogomales order, such as those of the genera Gadieria, Cyanidioschizole, and Cyanidium, especially Gadieria sulphuraria, Cyanidioschizole merolae, and Cyanidium caldarium, produce phycocyanin. The biomass of these microalgae contains phycocyanin. However, since phycocyanin is soluble in water, the biomass extracted from these microalgae, especially the biomass obtained by the method of the present invention, contains little to no phycocyanin. This is because the latter has already been removed during the preceding washing step with an aqueous solution.

[0053] The photosensitive polar extract according to the present invention can be concentrated by removing all or part of the polar organic solvent used in the extraction, for example by distillation, in which case it is called a concentrated extract.

[0054] The photosensitive polar extract according to the present invention is photosensitive and can generate reactive oxygen species when irradiated with light of one or more wavelengths, preferably 380-800 nm (white light), and preferably 380-450 nm (blue light) or 620-780 nm (red light).

[0055] Preferably, the photosensitive polar extract according to the present invention comprises pigments and lipids.

[0056] Preferably, the photosensitizing polarity extract according to the present invention comprises one or more chlorophyll-a (chl-a) decomposition derivatives, particularly pheophorbide-a (phb-a) and pheophytin-a (pht-a), as well as their respective derivatives such as methyl ester pheophorbide, hydroxypheophorbide, pyropheophorbide, or pyropheophytin, and mixtures thereof.

[0057] Preferably, the photosensitizing polarity extract according to the present invention contains 350 mg / g to 0.5 mg / g, more preferably 175 mg / g to 1 mg / g, and more preferably 85 mg / g to 5 mg / g of pheophorbide-a and its derivatives (also called total pheophorbide-a) based on the total weight of the extract.

[0058] Preferably, the photosensitizing polarity extract according to the present invention contains 250 mg / g to 0.35 mg / g by weight, more preferably 125 mg / g to 0.7 mg / g, and more preferably 60 mg / g to 3.5 mg / g of pheophorbide-a relative to the total weight of the extract.

[0059] Preferably, the photosensitive polarity extract according to the present invention contains 70% by weight of pheophorbide-a relative to the sum of total pheophorbide-a (corresponding to pheophorbide-a and its derivatives) and total pheophytin-a (corresponding to pheophytin-a and its derivatives), more preferably 40% to 90% by weight, and more preferably 60% to 75% by weight, relative to the total weight of the extract.

[0060] Preferably, the photosensitive polarity extract according to the present invention contains 95% by weight, more preferably 90% to 65% by weight, and more preferably 80% to 90% by weight of lipids (i.e., fat content, FC) based on the total dry mass of the extract.

[0061] Preferably, the photosensitized polarity extract according to the present invention, in particular, in the case of a photosensitized polarity extract of biomass extracted from Gardieria, preferably Gardieria sulfuraria, comprises monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG), phosphatidylcholine (PC), and ceramide (lipid amide).

[0062] Preferably, the photosensitive polar extract according to the present invention contains fatty acids. In particular, in the case of a photosensitive polar biomass extract of biomass extracted from Gardieria, preferably Gardieria sulfuraria, the extract contains, in dominant order, palmitic acid, oleic acid, gamma-linolenic acid, then alpha-linolenic acid, and stearic acid. More preferably, this photosensitive polar extract contains fatty acids, and the combined content of palmitic acid, oleic acid, gamma-linolenic acid, alpha-linolenic acid, and stearic acid represents at least 80% by weight of the total fatty acid content of the extract, and more preferably 85-95% of the total fatty acid content of the extract (GC-FID fatty acid analysis, percentage of fatty acids in the form of methyl esters (%FAME)).

[0063] Preferably, the photosensitive polarity extract according to the present invention contains less than 1% by weight of carotenoids (carot) and their derivatives (also called total carotenoids) based on the total dry weight of the extract.

[0064] Topical composition The present invention also relates to a topical composition comprising a photosensitive polar extract as described above and in the examples, and a topically acceptable carrier, i.e., a carrier suitable for application to the skin.

[0065] The topical composition according to the present invention is, in particular, a cosmetic composition.

[0066] Advantageously, the topical composition according to the present invention contains, by dry weight, 0.0001‰ to 10‰, preferably 0.001‰ to 2‰, and particularly 0.01‰ to 0.5‰ of the photosensitizing polarity extract according to the present invention, relative to the total weight of the composition.

[0067] Advantageously, the topical composition according to the present invention contains pheophorbide-a at a concentration of 1 nM to 50 μM, preferably 10 nM to 10 μM, and particularly 25 nM to 5 μM, relative to the total weight of the composition.

[0068] Advantageously, the topical composition according to the present invention, comprising the photosensitizing polarity extract according to the present invention, has a pH of 4.0 to 7.5, preferably 5.5 to 7.5.

[0069] Advantageously, the topical composition contains water. Depending on the selected form, it preferably contains 60-98% water.

[0070] The topical compositions according to the present invention may optionally further include one or more water-miscible solvents in addition to the polar organic solvent that can be provided by the biomass extract according to the present invention. These solvents (also known as additional solvents) include glycerin, alcohols (especially C1-C4 alcohols), organic solvents, polyols, glycols or mixtures thereof, particularly alcohols and polyols, such as ethyl alcohol, isopropyl alcohol, propyl alcohol, benzyl alcohol, phenylethyl alcohol, or glycols or glycol ethers, such as monomethyl, monoethyl and monobutyl ethers of ethylene glycol, propylene glycol or ethers thereof, such as propylene glycol monomethyl ether, butylene glycol monomethyl ether, hexylene glycol monomethyl ether, dipropylene glycol monomethyl ether and diethylene glycol alkyl ethers, such as diethylene glycol monoethyl ether or diethylene glycol monobutyl ether, or polyhydric alcohols, such as 1,2,6-hexanetriol, trimethylolpropane, and ethylene glycol , propylene glycol, diethylene glycol, butylene glycol, hexylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, dipropylene glycol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 3-methyl-1,3-butanediol, 1,5-pentanediol, tetraethylene glycol, 1,6-hexanediol, 2-methyl-2,4-pentanediol, polyethylene glycol, 1,2,4-butanetriol, 1,2,6-hexanetriol, 2-butene-1,4-diol, 2-ethyl-1,3-hexanediol, 2-methyl-2,4-pentanediol, (caprylyl glycol), 1,2-hexanediol, 1,2-pentanediol, and 4-methyl-1,2-pentanediol, alkyl alcohols having 1 to 4 carbon atoms, such as ethanol, methanol, butanol, propanol and isopropanol;Glycol ethers, such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monomethyl ether acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol mono-n-propyl ether, ethylene glycol mono-isopropyl ether, diethylene glycol mono-isopropyl ether, ethylene glycol mono-n-butyl ether, ethylene glycol mono-t-butyl ether, diethylene glycol mono-t-butyl ether, 1-methyl-1-methoxybutanol, propylene glycol monomethyl ether Examples include propyl glycol monoethyl ether, propylene glycol mono-t-butyl ether, propylene glycol mono-n-propyl ether, propylene glycol mono-isopropyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, dipropylene glycol mono-n-propyl ether, and dipropylene glycol mono-isopropyl ether, 2-pyrrolidone, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, formamide, acetamide, dimethyl sulfoxide, sorbitol, sorbitan, acetin, diacetin, triacetin, sulfone, or mixtures thereof.

[0071] The topical composition according to the present invention may optionally further comprise one or more thickening agents or gelling agents selected from gums such as xanthan gum, cellulose gum, acacia senegal gum, guar gum, sclerotium gum, dehydroxanthan gum, gellan gum, agar, algin, polymer-based synthetic thickeners, polyvinylidene chloride / acrylonitrile, acrylic acid copolymers, polyorganosiloxanes, and combinations thereof.

[0072] If the topical composition is in emulsion form, it generally contains at least one emulsifier selected from amphoteric, anionic, cationic, or nonionic emulsifiers, used alone or in mixtures, and optionally a co-emulsifier. The emulsifier is selected according to the emulsion to be obtained (W / O or O / W). The emulsifier and co-emulsifier are generally present in the topical composition in a proportion ranging from 0.3 to 30% by weight, preferably 0.5 to 20% by weight, based on the total weight of the composition.

[0073] In the case of W / O emulsions, examples of emulsifiers include dimethicone copolyols, such as the mixture of cyclomethicone and dimethicone copolyol sold by Dow Corning under the name "DC 5225 C", and alkyl-dimethicone copolyols, such as the laurylmethicone copolyol sold by Dow Corning under the name "Dow Corning 5200 Formulation Aid", and the cetyldimethicone copolyol sold by Goldschmidt under the name Abil EM 90R. Alternatively, a crosslinked solid elastomeric organopolysiloxane containing at least one oxyalkylene group can be used, for example, one obtained by the methods described in Examples 3, 4, and 8 of U.S. Patent No. 5,412,004 and Examples of U.S. Patent No. 5,811,487, in particular the product of Example 3 (synthesis example) of U.S. Patent No. 5,412,004, and one commercially available from Shin Etsu under the reference name KSG21.

[0074] In the case of O / W emulsions, examples of emulsifiers include nonionic emulsifiers such as oxyalkylene (especially polyoxyethylene) glycerol fatty acid esters, oxyalkylene (oxyethylene and / or oxypropylene) fatty acid esters, oxyalkylene (oxyethylene and / or oxypropylene) fatty alcohol ethers, sugar esters such as sucrose stearate, and mixtures thereof, such as mixtures of glyceryl stearate and PEG-40 stearate.

[0075] The topical compositions according to the present invention optionally include one or more natural or synthetic oils, particularly sunflower oil, malt oil, grape seed oil, sesame oil, corn oil, apricot oil, castor oil, shea oil, cotton oil, hazelnut oil, macadamia oil, jojoba oil, avocado oil, olive oil, soybean oil, sweet pea oil, palm oil, rapeseed oil, alfalfa oil, poppy oil, pumpkin seed oil, pumpkin seed oil, blackcurrant oil, evening primrose oil, millet oil, barley oil, quinoa oil, rye oil, safflower oil, candlenut oil, passionflower oil, and rosehip oil, or microalgae oils, such as those rich in ω-3; essential oils, such as jasmine, juniper, lavender, lemongrass, marjoram, sunflower, sesame, peppermint oil, and macadamia nut, tea tree, evening primrose, sage, rosemary, and coriander oil. It may further contain: corn, thyme, pimento berry, rose, anise, balsam, lime, mandarin, bergamot, rosewood, cedar, chamomile, sage, clary sage, clove, cypress, eucalyptus, fennel, sea fennel, frankincense, geranium, ginger, grapefruit, myrrh, neroli, orange, patchouli, pepper, black pepper, petitgrain, pine, rose otto, rosemary, sandalwood, spearmint, narl, vetiver, wintergreen, and ylang-ylang; silicone oils, such as dimethicone, cyclomethicone, polysilicone-11, phenyl trimethicone, trimethylsilyl amodimethicone, and stearoxytrimethylsilane; mineral oils, such as paraffin and its derivatives; and mixtures of these oils.

[0076] The topical composition according to the present invention may optionally further contain one or more pH adjusters, acids or bases, or pH buffers, such as citrate or phosphate buffers, preservatives, chelating agents, such as EDTA, and other adjuvants commonly used in cosmetic or pharmaceutical compositions.

[0077] The topical composition according to the present invention is preferably in the form of a gel, an oil-in-water or water-in-oil emulsion, a lotion, or a solution. More preferably, the composition according to the present invention is in the form of a gel.

[0078] Those skilled in the art can determine the components used to formulate a topical composition, whether it be a gel, ointment, or lotion.

[0079] The topical composition according to the present invention, in addition to a photosensitive polar biomass extract, contains one or more cosmetically or therapeutically active compounds, such as antiacids, such as asiatic acid, monoethanolamine salt of 1-hydroxy-4-methyl-6-trimethylpentyl-2-pyridone, 10-hydroxy-2-decanoic acid, sodium ursate, zinc oxide, 2,4,4'-trichlor-2'-hydroxydiphenyl ether (or triclosan), 1-(3',4'-dichlorophenyl)-3-(4'-chlorophenyl)urea (or triclocarban), 3,4,4'-trichlorcarban The following may be optionally included: lido, 3',4',5'-trichlorsalicylanilide, metronidazole and its salts, miconazole and its salts, itraconazole, terconazole, econazole, ketoconazole, saperconazole, fluconazole, clotrimazole, butoconazole, oxyconazole, sulfaconazole, sulconazole, terbinafine, cyclopirox, cyclopiroxolamine, undecylenic acid and its salts, resorcinol, octoxyglycerin or octoglycerin, octanoylglycine, and mixtures thereof.

[0080] The topical composition according to the present invention may optionally further contain one or more UV filtering components, so-called anti-UV filters, such as ethylhexyl triazone, ethylhexyl salicylate, butyl methoxydibenzoylmethane, bis-ethylhexyloxyphenol methoxyphenyl triazine, diethylamino hydroxybenzoylhexyl benzoate, phenylene bis-diphenyl triazine, and mixtures thereof.

[0081] biomass According to the present invention, "phycocyanin-producing organisms" include URAs and cyanobacteria.

[0082] URA includes microalgae of the Idycogomales order. The Idycogomales order encompasses the Idycogomalaceae and Gardieriaceae families, which themselves are subdivided into the genera Cyanidia, Cyanidium, and Gardieria, in particular the species Cyanidioschizono melorae, Cyanidia caldarum, Cyanidia didarum, Cyanidia maximum, Cyanidia partitum, Cyanidia lumpen, Gardieria didara, Gardieria maxima, Gardieria partita, and Gardieria sulfuraria (especially UTEX2919 and its varieties).

[0083] The microalgae of the genus Gardieria are unicellular red algae (URA) belonging to the subphylum Cyanidiofytina, class Idycogomei, order Idycogomeales, and family Gardieriaceae.

[0084] Preferably, the microalgae of the genus Gardieria are selected from Gardieria daedara, Gardieria maxima, Gardieria partita, or Gardieria sulfuraria, with a more favorable selection from Gardieria sulfuraria.

[0085] Methods for producing URA biomass, particularly biomass from microalgae of the genus Gardieria, are described in particular in International Patent Application Publications 2017 / 050917, 2017 / 050918, and 2017 / 093345.

[0086] Preferably, the biomass culture medium according to the present invention includes a carbon source and a nitrogen source. In particular, the carbon source is glucose.

[0087] Method for preparing an extract according to the present invention In another embodiment, the present invention provides a method for preparing a photosensitive polar crude extract comprising culturing the biomass of phycocyanin-producing organisms, particularly URAs or cyanobacteria, and subsequently, a) A step of collecting biomass by separating the culture medium for obtaining live biomass, b) A step in which, optionally, cells from the live biomass obtained from step (a) are preferably dissolved by mechanical grinding to obtain dissolved biomass, c) A step of optionally diluting the dissolved biomass from step (b) to obtain solubilized dissolved biomass, d) A step of recovering insoluble material suspended in the dissolved biomass from step (b) or in the solubilized dissolved biomass from step (c) to obtain extracted biomass, e) The extracted biomass obtained in step d) is extracted by contacting it with a polar solvent, and then the aqueous fraction is recovered by separating the insoluble matter from the suspension to obtain a photosensitive polar extract. This includes methods.

[0088] The use of the extracted biomass obtained in step d) of the present invention further enables the adoption of a sustainable development approach by using by-products (extracted biomass) that would normally be discarded from the production cycle as waste. Therefore, the present invention makes it possible to reduce the production cost of photosensitive polar extracts.

[0089] According to one particular embodiment, the method may include a biomass heating step before or during extraction step e), i.e., in any of steps a), b), c), d), or e), preferably during extraction step e).

[0090] According to another specific embodiment, the method may include a step of drying the biomass obtained in one of steps a), b), c), or d) to obtain dried biomass, i.e., dried raw biomass, dried dissolved biomass, or dried extracted biomass.

[0091] The present invention relates to a method for preparing a photosensitive polar biomass extract extracted from phycocyanin-producing organisms, particularly URAs or cyanobacteria, comprising the steps of: contacting the extracted biomass, whether dry or not, with a polar solvent, particularly under the above conditions; then separating the solvent from insoluble substances to recover a crude extract, wherein the crude extract may be concentrated or dried.

[0092] The present invention also relates to a method for preparing a photosensitive polar extract of biomass extracted from phycocyanin-producing organisms, particularly URAs or cyanobacteria, comprising the steps of preparing extracted, but optionally dried, biomass, and extracting a polar solvent.

[0093] a) Collect biomass The live biomass according to the present invention is obtained by culturing and then harvesting the biomass (step a) of the method of the present invention).

[0094] Unicellular red algae can be harvested using any technique known to those skilled in the art, possibly including gravimetric or reduced-pressure filtration, decantation, precipitation, followed by gravimetric filtration or centrifugation.

[0095] Therefore, biomass recovery corresponds to the recovery of the culture, followed by the isolation of biomass cells from at least a portion of the culture medium.

[0096] This step produces raw biomass. The raw biomass thus collected can also be preferably washed with water to remove certain soluble impurities.

[0097] After collection, the raw biomass obtained after one or more optional washes contains at least 70% by weight of water, and up to 90% by weight of water, with a preference for 75-88% by weight of water, relative to the total weight of the raw biomass.

[0098] Preferably, the raw biomass according to the present invention has a dry material content of 5 to 30% by weight, generally, still preferentially 10 to 25% by weight, and more preferably 10 to 20% by weight, relative to the total weight of the raw biomass.

[0099] b) Cell lysis The soluble biomass according to the present invention is obtained after an optional step of cellular lysis of raw biomass (step b) of the method of the present invention). The raw biomass may be washed, frozen, thawed, dried, and / or rehydrated. In other words, in the context of the present invention, the raw biomass may be thawed and / or dried biomass.

[0100] Cell lysis can be carried out by any lysis method known to those skilled in the art, in particular by enzymatic, mechanical, and / or chemical means.

[0101] Mechanical means that can be used in accordance with the present invention include ball mills, high-shear mixers, high-pressure homogenizers, bag mills, pin mills, impact mills, ultrasonics, or pulsed electric fields. For apparatus to carry out these methods, see the ball mill: Discus-100 from Netzsch or ECM-AP60 from WAB; the high-pressure homogenizer: Ariete from GEA; the high-shear mixer: 700-X from Silverson; the pin mill: Contraplex from Hosokawa; and the impact mill: Condux from Netzsch.

[0102] Preferably, for the preparation of a topical composition containing a photosensitive polar extract according to the present invention, cell lysis is carried out by mechanical lysis, more preferably by grinding, particularly using a ball mill.

[0103] Preferably, the resulting dissolved biomass has a dry material content of 5-30% by weight, preferably 10-25% by weight, and more preferably 10-20% by weight, relative to the total weight of the dissolved biomass.

[0104] c) Biomass dilution According to the present invention, optionally dissolved biomass can optionally undergo a dilution step (step c) of the method of the present invention). In the context of the present invention, the dilution step refers to the addition of a solution to the optionally dissolved biomass to reduce its dry matter content. Advantageously, the dilution step is carried out by adding an aqueous solution. Preferably, the aqueous solution is water.

[0105] Preferably, if present, the dilution step c) is performed on dissolved biomass having a dry material content of 5–30% by weight, preferably 12–25% by weight, relative to the total weight of dissolved biomass.

[0106] The aqueous solution may further contain one or more pH-adjusting compounds. The term "pH-adjusting compound" refers to any organic or inorganic compound (acidity corrector, acid, base, neutralizing agent, or buffer) used to correct pH. Examples of such compounds include sulfuric acid, acetic acid, citric acid, phosphoric acid, sodium citrate, potassium lactate, potassium malate, sodium chloride, disodium phosphate, and potassium phosphate. The aqueous solution will have an acidic or basic pH depending on the pH-adjusting compounds present in the solution.

[0107] Preferably, if present, step c) diluting the dissolved biomass is carried out using an aqueous solution having a pH of 8 or less, particularly 0-6, preferably 1-6, and more preferably 2-5. The pH of the aqueous solution added to the dissolved biomass or the dissolved biomass itself may be about 2, about 3, about 4, about 5, about 6, or about 7. Examples of acidic solutions that can be added to the dissolved biomass are acid-containing solutions such as those already described above.

[0108] Depending on the circumstances, the solubilized biomass may have a neutral, acidic, or basic pH, or a pH close to neutral.

[0109] Preferably, solubilized biomass has an acidic pH below 7, especially between 1 and 6, more preferably between 2 and 5, and even more preferably between 3 and 4.

[0110] Preferably, the resulting solubilized biomass has a dry material content of 1-15% by weight, preferably 3-12% by weight, and more preferably 4-8% by weight, relative to the total weight of the solubilized biomass.

[0111] d) Recovery of extracted biomass The biomass extracted according to the present invention is obtained after processing the biomass to remove the aqueous extract containing the target water-soluble molecules such as phycobiliproteins, particularly phycocyanin (step d of the method of the present invention).

[0112] Dissolved biomass includes an aqueous phase and an insoluble fraction. To obtain extracted biomass, the insoluble fraction of the dissolved biomass is recovered by one of the methods known to those skilled in the art. These methods include front filtration, tangential filtration, decantation, and centrifugation, with centrifugation being the preferred method for separation.

[0113] Preferably, the recovery of the insoluble fraction of dissolved biomass to obtain extractable biomass is carried out on solubilized or unsolubilized dissolved biomass having an acidic pH, i.e., less than 7, preferably 1-6, more preferably 2-5, and even more preferably 3-4.

[0114] - Biomass drying The extracted biomass obtained in step d) may be subjected to a drying step before the polar extraction step. In this case, drying is carried out at a temperature of 30-200°C, preferably 100-200°C.

[0115] If the extracted biomass undergoes a drying process, this is preferably carried out for less than 5 minutes, more preferably between 0.1 seconds and 2 minutes, and even more preferably between 0.2 seconds and 1 minute.

[0116] According to one embodiment of the present invention, a photosensitive polar extract is obtained from dried biomass.

[0117] In this case, the photosensitive polar extract is preferentially obtained from the extracted biomass that has undergone a drying step in its preparation method.

[0118] - Biomass heating Advantageously, the biomass is heated before or during the polar extraction step e), i.e., in any of steps a), b), c), d), or e), preferably during the extraction step e). Preferably, the extracted biomass obtained in method step d) may optionally be heated before or during step e), preferably during step e).

[0119] If a heating process is involved, heating should be performed at temperatures above 40°C, preferably between 40 and 100°C, and even more preferably between 45 and 90°C.

[0120] If the biomass, preferably extracted biomass, undergoes a heating process, this is carried out for a period of at least 5 minutes, preferably at least 10 minutes, and up to 24 hours. Preferably, the heating is carried out for less than 12 hours, and more preferably for 2 to 6 hours.

[0121] In very high priority, this heating step is performed during the polar extraction step e).

[0122] According to a particular embodiment, the drying process and the heating process are performed simultaneously.

[0123] e) Extraction with polar solvents Preferably, the extraction step (step e) of the method according to the present invention is carried out using a polar organic solvent for at least 5 minutes, preferably at least 10 minutes. The maceration time is advantageously less than 24 hours, preferably less than 12 hours, and more preferably about 2 to 6 hours. The higher the extraction temperature, the greater the extraction of compounds derived from chlorophyll-a decomposition. Preferably, in order to obtain the best compromise between energy consumption and the extraction efficiency of chlorophyll-a decomposition derivatives, this extraction with a polar organic solvent is carried out at a temperature above 15°C, preferably 15°C to 120°C, and more preferably 40 to 90°C.

[0124] According to the present invention, the extraction is advantageously carried out using at least 1 L of polar solvent per kilogram of biomass dry material, preferably at least 2 L of polar solvent per kilogram of biomass dry material, and particularly preferably at least 4 L of polar solvent per kilogram of biomass dry material.

[0125] According to the present invention, in order to obtain the best compromise between the volume of polar extract recovered and the extraction efficiency of compounds derived from chlorophyll-a decomposition, the extraction is advantageously carried out using 1 L to 200 L of polar solvent per kilogram of biomass dry material, preferably 2 L to 50 L of polar solvent per kilogram of biomass dry material, and particularly preferably 4 L to 20 L of polar solvent per kilogram of biomass dry material. In fact, for the same amount of polar solvent, as the amount of biomass to be extracted increases, the extraction efficiency of compounds derived from chlorophyll-a decomposition increases, but the volume of extract recovered decreases.

[0126] The extract is obtained by conventional solvent extraction methods.

[0127] Preferably, the extraction step of the method according to the present invention is carried out with stirring to improve the extraction yield of chlorophyll-α decomposition derivatives.

[0128] Extraction is preferable to be carried out at room temperature, but can also be performed at low or high temperatures. Heating can promote the extraction of biomass components, but it should not affect the photosensitizing properties of the extract.

[0129] In one preferred embodiment, extraction is performed. At temperatures of -40℃ to 90℃, -For 2 to 4 hours, -While stirring, - Biomass dried material: The mass ratio of polar organic solvent is 1:5 to 1:10, - Polar organic solvents are preferred to be used, in particular, with practical polar organic solvents, especially alcohols.

[0130] Advantageously, the extraction is carried out at room temperature, which makes this method easier to implement and reduces the energy required.

[0131] According to a preferred embodiment of the present invention, the polar organic solvent is selected from alcohols, particularly ethanol, and isopropanol. The alcohol used may contain water in a proportion of less than 60% by mass, preferably less than 50% by mass, and more preferably less than 10% by mass, relative to the total mass of the alcohol / water mixture.

[0132] Insoluble materials are separated by known methods, particularly by filtration and / or centrifugation.

[0133] Advantageously, in order to obtain a polar extract, the method according to the present invention further includes, after extraction, a step for separating insoluble matter in the suspension, particularly by filtration (e.g., 0.3 mm, 0.8 mm, or other filters) and / or centrifugation (with recovery of only the supernatant).

[0134] The extract obtained after biomass separation is a solution called a "crude extract."

[0135] The obtained crude extract can be concentrated by complete or partial removal of the solvent. This is known as a “concentrated extract.” In this case, preferably, after the polar solvent extraction step of the extraction method according to the present invention, the photosensitive polar crude extract is distilled to remove all or part of the polar solvent. Advantageously, this step allows for the preparation of a concentrated extract containing less than 50% by weight, preferably less than 20% by weight, and more preferably 0.5–1% by weight of the polar solvent relative to the total weight of the concentrated extract.

[0136] The resulting crude or concentrated extract may undergo various treatments, including sterilization by conventional techniques, before being used as a cosmetic.

[0137] According to the present invention, the photosensitive polar extract of biomass is obtained from extracted biomass and preferably has undergone a drying and / or heating step in the preparation method described above.

[0138] Advantageously, according to the present invention, the photosensitive polar biomass extract is obtained from extracted biomass and, as described above, undergoes a pH adjustment step to an acidic pH in its preparation method.

[0139] Method for preparing a topical composition containing a photosensitive polar extract from biomass. In another aspect, the present invention relates to a method for preparing a topical composition comprising a photosensitive polar extract of biomass extracted from phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria, - A step of extracting the extracted biomass with a polar organic solvent to obtain the above-mentioned photosensitive polar extract according to the present invention, and -The present invention relates to a method comprising the step of mixing the above extract with at least one locally acceptable compound, such as those described above.

[0140] use The present invention also relates to a photosensitive polar extract or a composition containing the same, for use as a pharmaceutical.

[0141] The present invention also relates to a photosensitive polar extract or composition comprising the present invention for use in the prevention and / or treatment of acne, particularly hormonal and / or inflammatory acne. The present invention further relates to the use of a photosensitive polar extract or composition comprising the present invention for the manufacture of a pharmaceutical for the prevention and / or treatment of acne, particularly hormonal and / or inflammatory acne. The present invention also relates to a method for treating acne, particularly hormonal and / or inflammatory acne in a subject requiring such treatment, comprising the step of administering a therapeutically effective amount of a photosensitive polar extract or composition comprising the present invention to the subject.

[0142] Pheophorbide-α and its derivatives are known for their photosensitizing effect. When exposed to a light source, they produce reactive oxygen species that cause the death of nearby microorganisms, thereby preventing and / or treating acne.

[0143] Surprisingly, in contrast to pheophorbide-a alone, the photosensitive polar extract according to the present invention exhibits antimicrobial activity both in the presence and absence of light. In other words, after application to the skin surface to be treated, the photosensitive polar extract or composition containing the same according to the present invention exhibits antimicrobial activity against skin bacteria, regardless of whether the skin surface to which the extract or composition containing the same has been applied is exposed to a light source.

[0144] The present invention also relates to the cosmetic use of an effective amount of a photosensitive polarity extract or composition containing the same according to the present invention for preventing and / or eliminating skin defects, i.e., for improving the appearance of the skin.

[0145] The use of the photosensitizing polarity extract according to the present invention in phototherapy involves applying it to the skin surface to be treated, or applying a composition containing the extract, and then exposing this surface to a light source. In this case, to limit the associated side effects, the extract may be applied as a topical treatment for the defect.

[0146] The photosensitive polarity extract according to the present invention can also be used, for example, as a night cream in the absence of a light source, in which case use involves applying the extract or a composition containing it to the skin surface to be treated.

[0147] The present invention also relates to the use of the photosensitive polar extract or composition containing the same according to the present invention for its antimicrobial, particularly bacteriostatic and / or bactericidal effects. Accordingly, the present invention relates to the photosensitive polar extract or composition containing the same according to the present invention for use as an antimicrobial agent, particularly for its bacteriostatic and / or bactericidal effects. The present invention further relates to the use of the photosensitive polar extract or composition containing the same according to the present invention for the manufacture of antimicrobial agents, particularly bacteriostatic and / or bactericidal pharmaceuticals. The present invention also relates to a method for antimicrobial treatment, particularly bacteriostatic and / or bactericidal treatment, in a subject requiring such treatment, comprising the step of administering a therapeutically effective amount of the photosensitive polar extract or composition containing the same according to the present invention to the subject.

[0148] The present invention also relates to a photosensitive polarity extract or composition containing the same for killing and / or inhibiting the growth of certain bacteria on a light-exposed surface, particularly Staphylococcus aureus and / or Propionibacterium acnes and / or Xerosis bacteria and / or Staphylococcus epidermidis.

[0149] The present invention also relates to the use of an effective amount of a photosensitive polarity extract or composition containing the same according to the present invention to kill and / or inhibit the growth of certain bacteria, particularly Staphylococcus aureus and / or Propionibacterium acnes and / or Xerosis bacteria and / or Staphylococcus epidermidis, on surfaces not exposed to light.

[0150] Exposure to a light source activates photosensitized extracts to help prevent and / or eliminate skin defects, especially acne.

[0151] According to certain embodiments, the present invention relates to a method for preventing or treating a skin defect, particularly acne, in a subject, comprising applying a photosensitive polarity extract or a composition containing the same according to the present invention to a portion of the skin of the subject to be treated, and then exposing the surface of the skin to which the extract or composition containing the same has been applied to a light source.

[0152] Advantageously, the illumination wavelength is preferably 380-800 nm (white light), and even more preferably 380-450 nm (blue light) or 620-780 nm (red light).

[0153] Advantageously, the illumination wavelength is 380–450 nm, preferably 400–440 nm, and more preferably 405–420 nm. This makes it possible to limit the occurrence of undesirable effects associated with the combination of use of the extract or composition containing it according to the present invention and exposure to the light source. In fact, this or these selection of irradiation wavelengths makes it possible to obtain local antibacterial and / or bacteriostatic effects on the surface of the skin, because wavelengths of 200–400 nm reach only the epidermal layer of the skin, wavelengths of 400–600 nm penetrate the skin to the dermis layer, and wavelengths of 600–700 nm can reach the subcutaneous tissue of the skin (Francisco et al., 2021).

[0154] When the illumination wavelength is 620-780 nm, it is preferably 650-700 nm, and more preferably 670-685 nm.

[0155] According to the present invention, exposure to a light source is preferably performed 10 seconds to 240 minutes, preferably 1 minute to 180 minutes, and more preferably 2 to 60 minutes after application of the extract or composition containing it, in order to obtain a sufficient antibacterial effect.

[0156] Exposure to a light source, especially artificial light, can last for 1 to 90 minutes, preferably 10 to 30 minutes, and more preferably 15 to 25 minutes.

[0157] Exposure to a light source, especially natural light, can continue for 10 to 420 minutes, preferably 60 to 120 minutes, and more preferably 15 to 45 minutes.

[0158] For cosmetic procedures, the illumination dose from light exposure should preferably be between 0.01 and 100 J / cm². 2 , preferentially 50 J / cm 2 Less than 10-30 J / cm², especially between 10 and 30 J / cm². 2 That is the case.

[0159] The intensity of light exposure during cosmetic procedures is preferably 10-900 W / m². 2 Prioritizing 15-400W / m 2 Especially 20-100 W / m 2 That is the case.

[0160] It is understood that the procedure can be repeated, generally every 2-7 days, for several weeks, especially for up to 12 weeks or more, depending on the desired results. This can be repeated 3-6 months after the last treatment.

[0161] After exposure to a light source, the photosensitive polar extract or a composition containing the same can be advantageously removed from the skin surface by any conventional skin cleansing means.

[0162] In certain embodiments, the skin surface of the target to be treated is pre-treated to facilitate the absorption of a photosensitive polar extract into the superficial layer of the skin. This pre-treatment can be carried out by "chemical" means by any suitable means to achieve surface exfoliation, in particular by applying compositions that promote this surface exfoliation, such as compositions containing exfoliants, fruit acids, alpha-hydroxy acids (AHAs), such as glycolic acid and lactic acid, azelaic acid, retinoids, such as tretinoin, adapalene, tazarotene, or vitamin D3 derivatives. Surface exfoliation can also be achieved by mechanical means such as scraping, (micro) abrasion (e.g., using appropriate abrasive paper), micropuncture (e.g., using a skin roller), tape stripping, pan scrubbing, exfoliation scrubbing, or low-energy non-exfoliating lasers.

[0163] Surprisingly, the present invention relates to a photosensitive polar extract according to the present invention for use in skin regeneration, particularly due to its keratinocyte proliferation-stimulating properties. In fact, the epidermis is composed of different, overlapping layers that reflect the progressive differentiation of keratinocytes migrating from the basal layer to the surface layer. Epidermal regeneration depends on a delicate balance between the proliferation and differentiation of epidermal stem cells and the removal of dead cells from the surface by desquamation. This helps maintain and / or restore the structure and function of the epidermis. Various factors such as genetics, environment, lifestyle, acne, inflammation, scarring, and aging are known to influence the quality and rate of epidermal regeneration.

[0164] Accordingly, the present invention also relates to the use of the photosensitive polar extract or composition containing the same according to the present invention for the manufacture of pharmaceuticals that enable skin regeneration, particularly by their keratinocyte proliferation stimulating properties. The present invention also relates to a method for regenerating skin in a subject in need, particularly by stimulating keratinocyte growth, comprising the step of administering to the subject a therapeutically effective amount of the photosensitive polar extract or composition containing the same according to the present invention.

[0165] Furthermore, and surprisingly, the polar extract according to the present invention promotes the production of mitochondrial proteins involved in skin energy production, and thus in the repair, regeneration, and protection of the skin, as well as the production of proteins involved in the proteasome, a proteolytic complex that is important for the degradation and recycling of proteins, and thus ensures their regeneration.

[0166] As a result of these remarkable effects, in relation to skin regeneration, the polar extracts according to the present invention can also be used to prevent and / or eliminate skin defects, in particular to prevent and / or eliminate signs of aging such as wrinkles, or to promote skin healing. The present invention further relates to the photosensitive polar extracts according to the present invention for use in skin healing. The present invention also relates to the use of the photosensitive polar extracts according to the present invention for the manufacture of skin healing pharmaceuticals. The present invention also relates to a method of skin healing in a subject requiring skin healing, comprising the step of administering to the subject a therapeutically effective amount of the photosensitive polar extract according to the present invention.

[0167] Removal of photosensitizer Advantageously, the photosensitive polar extract and / or composition containing the same according to the present invention is removed from the skin surface to which it is applied.

[0168] Removal of surface stains The present invention also relates to the use of polar photosensitized extracts according to the present invention for the removal of inert surface stains by applying them to a surface to be treated and then, optionally, exposing them to a light source to enable the activation of the photosensitized extract.

[0169] For the purposes of this invention, the term "inert surface" refers to all hard surfaces in medical, public, institutional (school, kitchen, etc.) and home environments, preferably in medical environments, such as a doctor's operating room, hospital, and medical institution.

[0170] Preservatives The present invention also relates to the use of photosensitive polar extracts according to the present invention as preservatives in compositions, particularly cosmetic compositions.

[0171] Culture medium The present invention also relates to the use of photosensitive polar extracts according to the present invention as growth boosters for eukaryotic cells or mammalian cells, particularly keratinocytes.

[0172] Biological control The present invention also relates to the use of photosensitive polar extracts according to the present invention as biological control agents for preventing and / or treating diseases affecting the production of agriculturally important plants. The present invention also relates to photosensitive polar extracts according to the present invention for use as biological control agents for preventing and / or treating diseases affecting the production of agriculturally important plants.

[0173] Examples include Pseudomonas syringe, which causes various diseases in many plants including tomatoes, peppers, and cherries, such as bacterial spot, root nodule, and bacterial leaf spot; Xanthomonas bacteria, which cause bacterial spot and root nodule in various crops such as tomatoes, citrus fruits, cabbage, peppers, and beans; Erwinia amylovola, which causes fire blight, a serious disease that mainly affects apple and pear trees; Bacterial wilt, which causes bacterial wilt, a destructive disease that affects many crops including potatoes, tomatoes, eggplants, and bananas; and bacteria that mainly affect grapevines and potato crops. This includes the treatment of Agrobacterium tumefaciens, which causes plant galls and is a disease that causes verrucosa; Clavibacter misiganensis, which causes bacterial verrucosa and other diseases in various plants including grapevines, olives, and citrus fruits; Pierce's disease, which causes Pierce's disease in grapevines and other diseases in various plants including olives and citrus fruits; Dikea and Pectobacterium species, which cause soft rot and black rot in crops such as potatoes, carrots, and melons; and bacterial blight, which causes bacterial grain rot and mainly affects rice crops. [Brief explanation of the drawing]

[0174] [Figure 1] This graph shows the analysis of the fatty acid profiles of photosensitized polar extracts prepared according to the method of the present invention at different temperatures (20°C, 50°C, and 80°C). [Figure 2] This graph shows the absorbance spectrum of the photosensitive polarity extract according to the present invention, extracted with isopropanol or ethanol. [Figure 3]This graph shows the fatty acid profile analysis of photosensitive polar extracts prepared using the method of the present invention with different solvents (ethanol or isopropanol). [Figure 4] This photograph shows the identification of lipid classes present in the photosensitive polar extract of Gardieria sulfuraria according to the present invention by thin-layer chromatography (5 μL (left band) or 10 μL (right band) of the extract is deposited in layers and compared with standards of glucocerebroside, phosphatidylcholine (PC), ceramide, digalactosyl diglyceride (DGDG), monogalactosyl diglyceride (MGDG), and glycosylsterol). [Figure 5] This graph shows the effect of the polar extract of Gardieria sulfuraria according to the present invention on the growth of S. aureus (Staphylococcus aureus) under different conditions, such as with and without light. [Figure 6] This graph shows the effect of the polar extract of Gardieria sulfuraria according to the present invention on the growth of C. acnes (Propionibacterium acnes) under different conditions, such as with and without light. [Figure 7] This graph shows the effect of the polar extract of Gardieria sulfuraria according to the present invention (at different concentrations) on the growth of C. xerosis (xydermatitis fungus) with and without light. [Figure 8] This graph shows the effect of the polar extract of Gardieria sulfuraria according to the present invention (at different concentrations) on the growth of S. epidermidis (Staphylococcus epidermidis) with and without light. [Figure 9] This graph shows the effects of different polar extracts (polar extracts of biomass extracted according to the present invention and comparative polar extracts of dissolved biomass) and pheophorbide-a on a bacterial mixture in the absence of light. [Figure 10] This graph shows the effects of different polar extracts (polar extracts of biomass extracted according to the present invention and comparative polar extracts of dissolved biomass) and pheophorbide-a on a bacterial mixture after exposure to light. [Figure 11]This graph shows the effects (at different concentrations) of the polar extract of Gardieria sulfuraria according to the present invention on NHEK-p keratinocyte cells, compared to culture medium (positive control) or TX-100 (negative control). [Figure 12-1] This graph shows a comparative study of the proteomes of keratinocytes exposed to a polar biomass extract obtained according to the present invention and keratinocytes not exposed to it. [Figure 12-2] This graph shows a comparative study of the proteomes of keratinocytes exposed to a polar biomass extract obtained according to the present invention and keratinocytes not exposed to it. [Examples]

[0175] The present invention will be better understood by reading the following examples, which illustrate the invention in a non-limiting manner.

[0176] Example 1: Preparation of polar extracts under different time, temperature, and concentration conditions The extracted Gardieria sulfuraria biomass powder is treated with ethanol to prepare polar extracts by varying three parameters: extraction temperature, extraction time, and the amount of biomass per unit of ethanol (10 mL).

[0177] Extraction efficiency is evaluated by measuring the absorbance of the centrifuged sample at 410 nm and the volume of the recovered solvent.

[0178] Various experimental conditions and the results obtained are shown in Table 1 below.

[0179] Experiment 15 was repeated three times (Experiments 1.15.1, 1.15.2, and 1.15.3) to determine the experimental variance of the experiment. Analysis of the variance for the response of maximum absorbance and recovered solvent volume shows that the selected factors are helpful in understanding the variability in the experimental results.

[0180] [Table 1]

[0181] The above results demonstrate that the method according to the present invention enables good extraction of chlorophyll-α decomposition derivatives regardless of temperature and time conditions.

[0182] The extraction of chlorophyll-α decomposition derivatives is further improved when the temperature exceeds 50°C and the extraction time exceeds 250 minutes.

[0183] Example 2: Study of the composition of various polar extracts of Gardieria sulfuraria The extracted Gardieria sulfuraria biomass powder was treated with ethanol to prepare photosensitive polar extracts by stirring at 20°C (Test 2.1), 50°C (Test 2.2), and 80°C (Test 2.3) for 2 hours. The volume ratio of powder to anhydrous ethanol was 1:5 for all three conditions.

[0184] The obtained extract was distilled at 35°C and 30 mbar until the ethanol completely evaporated, then redissolved in DSMO, and analyzed by absorption spectroscopy to determine the maximum absorbance at 410 nm. The percentage of total lipids (%DM) relative to the total weight of the dry material and the fatty acid profile were analyzed by the Fortsch method and GC-FID, respectively, after transmethyl esterification and internal calibration. The method used to analyze and quantify the pigment composition of the extract is described by Van Heukelem & Thomas (Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J Chromatogr A. 2001 Feb 23;910(1):31-49).

[0185] The results are shown in Table 2 and Figure 1 below.

[0186] [Table 2]

[0187] The results in the table and Figure 1 above indicate that the obtained extracts, when subjected to homogeneous stirring, have similar pigment and fatty acid compositions regardless of the temperature used (20°C, 50°C, or 80°C).

[0188] Example 3: Comparison of absorbance spectra and fatty acid composition of polar extracts obtained using different polar solvents. The extracted Gardieria sulfuraria biomass powder is treated with ethanol or isopropanol and stirred at 50°C for 2 hours to prepare a polar extract. The volume ratio of powder to solvent is 1:5. The resulting extract is distilled at 35°C and 30 mbar until the solvent (ethanol or isopropanol) is completely evaporated, and then analyzed by absorbance spectroscopy. The fatty acid profiles of the resulting extracts are also analyzed. These analyses are performed by the Folch method and GC-FID, respectively, after transmethyl esterification and internal calibration.

[0189] The results are shown in Figures 2 and 3. No significant differences were found in the appearance of the absorbance spectrum or the studied lipid properties between the extract obtained by extraction with ethanol and the extract obtained by extraction with isopropanol.

[0190] Example 4: Comparison of polar extract according to the present invention with polar extract according to prior art The polar extract prepared in Example 2 (Test 2.3) is analyzed to identify the different lipid families present in this extract according to the present invention, in addition to its pigment composition. Thin-layer chromatography is performed. To do this, the sample is taken at a concentration of 10 mg / mL in a chloroform / methanol mixture (volume ratio 2 / 1), and then deposited twice on a plate in 5 μL and 10 μL portions, respectively. Standards of glucocerebroside, ceramide, phosphatidylcholine (PC), monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG), and glycosylsterol are deposited individually on the plate. The mobile phase used for electrophoresis is a chloroform / methanol / water / acetic acid mixture (volume ratio 65 / 16 / 2 / 1). The plate is then air-dried, transferred to bypass reagent (H2SO4, 50%), drained, and heated at 140°C for 5 minutes. Heating is stopped when spots appear.

[0191] The results are shown in Figure 4.

[0192] Ambrosino et al. have disclosed the preparation and characterization of a polar extract of unextracted dissolved biomass of Galdieria sulphuraria using acetone (Ambrosino et al., Galdieria sulphuraria: An Extremophilic Alga as a Source of Antiviral Bioactive Compounds. Mar. Drugs 2023, 21, 383). This extract was reproduced in the laboratory, and its pigment composition was analyzed as described in Example 2. The results are shown in Table 3 below.

[0193] [Table 3] 1 Results presented by Ambrosino et al.

[0194] By comparing the migration profile of the polar extract produced according to the present invention with that of a standard (Figure 4), it is possible to determine that the lipid fraction mainly consists of MGDG, DGDG, PC, and ceramide (lipid amide).

[0195] Therefore, the lipid composition of the extract according to the present invention differs from the lipid composition of the extract prepared according to Ambrosino et al. In fact, the latter consists only of lipid amides, free fatty acids, and chlorophyll-α derivatives.

[0196] The concentration of chlorophyll-a derivative in the extract prepared according to the present invention (Example 2.3) is also different from the concentration in the extract prepared according to Ambrosino. The latter has a ratio of less than 40% pheophorbide-a (phb-a) to (total pheophorbide-a + total pheophytin-a (pht A)).

[0197] Example 5: Pilot-scale production of photosensitive polar extracts The extracted Gardieria sulfuraria biomass powder is treated with ethanol to prepare a photosensitive polar extract, which is stirred at 75°C for 120 minutes in a powder-to-solvent volume ratio of 1:10. This extract is distilled at 35°C and 30 mbar until the ethanol has completely evaporated, and then analyzed by absorbance spectroscopy (Test 5.1). The obtained extract is also analyzed for the percentage of fat relative to dry mass (%) (Folch method), the fat profile compared to the extract obtained according to the present invention on a laboratory scale (Test 2.3) (GC-FID after transmethyl esterification and internal calibration), and its pigment composition (as described in Example 2).

[0198] The results are shown in Tables 4 and 5.

[0199] [Table 4]

[0200] [Table 5]

[0201] Using a high-performance pilot mixing system, it is possible to observe that extraction is more efficient than on a smaller scale, and it is possible to obtain at least three times higher pheophorbide content from the same initial material (Tables 2 and 5).

[0202] The main fatty acids represented in extract 5.1 are palmitic acid, oleic acid, linoleic acid, alpha-linolenic acid, and stearic acid, which account for approximately 92% of the total fatty acid content (%FAME).

[0203] Example 6: Study of the bactericidal and / or bacteriostatic effects of a polar extract of biomass extracted from Gardieria sulfuraria against various bacteria. The pre-solubilized polar extract 2.3 from Example 2 is dissolved in Mueller-Hinton medium.

[0204] Five different concentrations of polar extracts of the extracted biomass were added to a microbial suspension of Staphylococcus aureus (ATCC6538): 0% (control), 0.0014 wt‰, 0.007 wt‰, 0.036 wt‰, and 0.36 wt‰ (n=3) relative to the total weight of the medium, corresponding to 0 nM, 0.17 nM, 83 nM, 0.41 μM, and 4.1 μM pheophorbide-a relative to the volume of the medium. Each culture was then pre-incubated at 37°C at 280 rpm for 3 hours, followed by exposure to white light (10 or 25 J / cm2) or no exposure. Finally, the cultures were incubated for a further 24 hours at 37°C.

[0205] To monitor microbial growth, optical density at 600 nm is measured over time (microplate spectrometer, EPOCH2, BioTek Instruments), and the area under the curve (AUC) is calculated from t=0 to t=16 hours.

[0206] The same test is independently applied to the following three other bacteria: Propionibacterium acnes (ATCC 6919), Malassezia furfur (ATCC373), and Staphylococcus epidermidis (ATCC14990). In the case of the Malassezia furfur strain and the Propionibacterium acnes strain, 5% sheep blood is added to the medium, and incubation is continued at 37 °C for 72 hours instead of 24 hours.

[0207] The results are shown in Figures 5, 6, 7, and 8, respectively.

[0208] It is noted that in the absence of light, when the pro-acne strain, Staphylococcus aureus, and Propionibacterium acnes are brought into contact with the polar extract according to the invention, the area under the curve is significantly reduced compared to the control condition without the extract. This indicates that the growth of these strains is significantly reduced in the presence of the polar extract according to the invention. In particular, depending on the concentration used and the strain concerned, it was observed that the growth of these bacteria was inhibited by 13% to 74%. Therefore, in the absence of light, it can be concluded that the polar extract has bacteriostatic properties against Staphylococcus aureus and Propionibacterium acnes and limits the growth of these bacteria and the occurrence of acne (Figures 5 and 6). 10 J / cm 2 and 25 J / cm 2 In the presence of light of, this bacteriostatic effect is even more pronounced (Figures 5 and 6).

[0209] As described above, in the presence of light and the polar extract according to the invention, the growth of Malassezia furfur and Staphylococcus epidermidis is reduced or even completely inhibited (Figures 7 and 8).

[0210] Example 7: Study on the bactericidal and / or bacteriostatic effects of a polar extract of biomass extracted from Galdieria sulphuraria, a polar extract of biomass not extracted from Galdieria sulphuraria, and pure pheophorbide-a against a mixture of bacteria.

[0211] Unextracted Gardieria sulfuraria biomass powder was treated with ethanol to prepare a photosensitive polar extract with stirring at 80°C (Test 7.1) for 2 hours. The volume ratio of powder to anhydrous ethanol was 1:5 for all three conditions.

[0212] The resulting extract is distilled at 35°C and 30 mbar until the ethanol has completely evaporated.

[0213] The method used to analyze and quantify the pigment composition of the extract is described by Van Heukelem & Thomas (Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J Chromatogr A. 2001 Feb 23;910(1):31-49).

[0214] The pure pheophorbide-a used was a commercially available product from ChemCruz (batch K0223, purity over 90%). The polar extract sample of extracted Gardieria sulfuraria biomass was the same as that used in Test 2.3 of Example 2. All samples were pre-diluted in DMSO to obtain equivalent pheophorbide-a concentrations for each sample. The polar extract and pheophorbide-a solution were resolubilized and dissolved in Epilife medium. The bacteria used were the same as those used in Example 6 and were added to the medium at equivalent optical density.

[0215] A polar extract of the extracted biomass at a concentration of 0.036‰, corresponding to 0.41 μM pheophorbide-a, was added to the culture medium (n=3). The cultures were then pre-incubated at 37°C and 280 rpm for 3 hours, followed by exposure to white light (25 J / cm²). 2 ) Expose or do not expose. Finally, incubate the culture at 37°C for a further 20 hours.

[0216] To monitor microbial growth, optical density at 600 nm is measured over time (microplate spectrometer, EPOCH2, BioTek Instruments).

[0217] The results are shown in Figures 9 and 10.

[0218] Monitoring the growth of bacterial mixtures in the presence of different polar extracts and pure pheophorbide-a clearly demonstrates the bacteriostatic effect of the polar extracts of extracted biomass compared to control conditions in the absence of light (Figure 9). This confirms the results obtained in independent cultures of the same bacteria (Example 6). It can also be noted that polar extracts of unextracted biomass, as well as pure pheophorbide-a, do not affect bacterial growth in the absence of light. This indicates that the bacteriostatic effect is not solely related to the presence of pheophorbide-a in the culture medium. In fact, the same concentration of this element is present under all tested conditions except the control conditions. Since polar extracts of unextracted biomass have no effect in the presence of light, it is demonstrated that the bacteriostatic activity is also related to the fact that the biomass was pre-treated.

[0219] After exposure to light, bacterial cultures incubated with polar extracts of unextracted biomass and pure pheophorbide-a show a significant growth delay of up to approximately 10 hours during the first growth phase (Figure 10). Since the growth dynamics during this phase are similar, it can be concluded that in both cases the effect is directly related to the photoactivation of pheophorbide-a. After this phase, a recovery of growth to a level similar to that observed under control conditions can be observed. The growth dynamics of cells incubated with polar extracts of extracted biomass differ in that there is no growth delay during the first 4–6 hours, but growth is very rapid, reaching a plateau phase between 10–12 hours and showing either cell division arrest or cell death.

[0220] Example 8: Effects of the polar extract according to the present invention on keratinocytes Normal adult human keratinocytes from a single donor (Promocell) were cultured and maintained as a monolayer at a concentration of less than 75% in the recommended medium. The keratinocytes were cultured at 37°C and 5% CO2. The cells were subcultured with purified trypsin and a trypsin / BSA inhibitor combination and used at less than 6 subgenerations.

[0221] Amounts of the polar extract according to the present invention (Test 2.3), prepared according to Example 2, corresponding to 0.00084‰, 0.0042‰, 0.0084‰, and 0.042‰, were added to the culture medium. The cells were then cultured for 24 hours. Each condition was tested in at least three consecutive sets using a positive reference control and an untreated control. The cultures were not irradiated with light.

[0222] Cell viability was measured using CellTox® Green Cytotoxicity Assay (Promega) according to the manufacturer's instructions. CellTox Green Cytotoxicity Assay measures cell death using a fluorescent dye that permeates dead cells, binds to DNA, and emits fluorescence. This fluorescence correlates with the number of dead cells, enabling quantitative assessment of cytotoxicity. Cells were treated with 0.1% v / v (water) Triton®-X100 (Sigma-Aldrich) as appropriate, which induces membrane damage and cell death. Fluorescence reading and imaging were performed using a Cytation multimode cell imaging reader (Agilent / Biotek).

[0223] The experiments and data processing were conducted by ELYSIA BIOSCIENCE (40 AV. Ferdinand de Lesseps 33610 CANEJAN).

[0224] The results are shown in Figure 11.

[0225] The results show a decrease in cell markers indicating cell death after Triton®-X100 treatment (negative control). Surprisingly, when the extracted biomass is incubated with polar extracts at concentrations of 0.00084‰ to 0.0084‰ according to the present invention, a significant increase in the number of cells can be observed compared to the control. Therefore, it can be concluded that polar extracts of biomass extracted from Gardieria sulfuraria according to the present invention promote keratinocyte growth and thus accelerate the skin regeneration cycle under the above conditions.

[0226] Example 9: Study of keratinocyte proteome after cell exposure to polar extracts of extracted biomass. Reconstructed human epidermis (RHE, Episkin) was treated with a 0.036‰ polar extract according to the present invention (Test 2.3), prepared according to Example 2, in a sterile cell culture environment under specified application conditions. Proteins were extracted under denaturation conditions suitable for SDS-PAGE and downstream proteomics applications. Protein concentrations were determined by the BCA (bicinchoninic acid) method and standardized for all samples. Samples were separated by SDS-PAGE and digested overnight. The resulting peptides were acidified and separated using a Fusion Lumos mass spectrometer (Thermo Fisher) with a gradient of 146 minutes.

[0227] Mass spectra were analyzed using Proteome Discoverer (version 2.5). The obtained MS / MS data were compared to the Homo sapiens UP000005640 proteome (revised entry 20371). Study parameters included monoisotopic mass; trypsin as the cleaving enzyme; up to two cleavage errors; cysteine ​​carbamide methylation as a fixed modification; and N-terminal acetylation and methionine oxidation as variable modifications. Results were filtered according to the single peptide score of more than two and the overall peptide score.

[0228] Using a proprietary bioinformatics processing pipeline (including protein database searches, protein interaction networks, quantitative analysis, and statistics), we analyzed and constructed a comprehensive interpretation of the relevant biological activity of the tested products.

[0229] The experiments and data processing were conducted by ELYSIA BIOSCIENCE (40 AV. Ferdinand de Lesseps 33610 CANEJAN).

[0230] The results are shown in Figure 12.

[0231] The protein abundance in each group is represented by white circles if there is no change from the control conditions, and by gray circles if the abundance has increased. The size of the circles indicates the relative number of proteins associated with each group.

[0232] After exposure of keratinocyte cells to polar extracts of extracted biomass, a 6–10% increase in the abundance of proteins involved in energy production, such as the TCA cycle, respiration, and mitochondrial organization, was observed (Figure 12).

[0233] An increase in the abundance of mitochondrial proteins elevates the skin's energy levels, which can promote overall skin repair, regeneration, and protection. Energy-dependent processes are also essential for maintaining the integrity of the skin barrier, and this is reflected in a 14-15% increase in the abundance of proteins involved in maintaining the skin barrier.

[0234] The abundance of proteins involved in the proteasome also increases by 4-5%. The proteasome is a proteolytic complex crucial for protein degradation and recycling, ensuring the quality control of intracellular proteins. It degrades and recycles abnormal or damaged proteins, preventing their accumulation.

[0235] Overall, these results indicate that polar extracts of Gardieria sulfuraria have beneficial effects on various cellular metabolic processes involved in skin repair, regeneration, and protection.

Claims

1. Photosensitive polar biomass extracts from phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria.

2. The photosensitive polarity extract according to claim 1, characterized in that the biomass is URA biomass, and the URA is selected from the family Gardieriaceae or Ideyukogoceae, more preferably from Gardieria, the genus Cyanidia or Cyanidium, and even more preferably from Gardieria.

3. The photosensitive polar extract according to claim 2, characterized in that the URA is of the species Gardieria sulfuraria.

4. A photosensitive polar extract according to any one of claims 1 to 3, characterized by being a solution comprising a polar organic solvent, preferably an alcohol, particularly methanol, ethanol, and isopropanol, a volatile organic acid, particularly formic acid, acetic acid, primary or secondary amines, and PEG (polyethylene glycol), and a protic polar organic solvent selected from mixtures thereof.

5. A photosensitive polar extract according to any one of claims 1 to 4, characterized in that it contains lipids in an amount of up to 95% by weight, preferably 90% to 65% by weight, and more preferably 80% to 90% by weight, relative to the total dry weight of the extract.

6. The photosensitive polar extract according to claim 5, characterized in that the lipid comprises monogalactosyl diglyceride, digalactosyl diglyceride, phosphatidylcholine, and ceramide.

7. A photosensitive polarity extract according to any one of claims 1 to 6, characterized in that it contains 350 mg / g to 0.5 mg / g, preferably 175 mg / g to 1 mg / g, and more preferably 85 mg / g to 5 mg / g, of pheophorbide-a and its derivatives, based on the total weight of the extract.

8. A method for preparing a photosensitive polar crude extract, comprising culturing the biomass of phycocyanin-producing organisms, particularly unicellular red algae (URA) or cyanobacteria, and subsequently, a) A step of collecting biomass by separating the culture medium for obtaining live biomass, b) A step of dissolving the cells of the live biomass from step (a) to obtain dissolved biomass, c) A step of optionally diluting the dissolved biomass from step (b) to obtain solubilized dissolved biomass, d) A step of recovering insoluble matter suspended in the dissolved biomass from step (b) or in the solubilized dissolved biomass from step (c) to obtain extracted biomass, e) Extracting the biomass obtained in step d) by contacting it with a polar solvent, and then recovering the aqueous fraction by separating the insoluble matter from the suspension to obtain the photosensitive polar crude extract, Methods that include...

9. The method according to claim 8, characterized in that the polar organic solvent is a protic polar organic solvent selected from alcohols, particularly methanol, ethanol, and isopropanol, volatile organic acids, particularly formic acid, acetic acid, primary or secondary amines, and PEG (polyethylene glycol) and mixtures thereof.

10. The method according to claim 8 or 9, further comprising a step of heating the biomass before or during the extraction step e), i.e., in any of steps a), b), c), d), or e).

11. A photosensitive polar extract according to any one of claims 1 to 7, which can be obtained by the method described in any one of claims 8 to 10.

12. A topical composition comprising a photosensitive polar extract according to any one of claims 1 to 7 or 11 and a locally acceptable carrier.

13. A photosensitive polar extract according to any one of claims 1 to 7 or 11, or a topical composition according to claim 12, for use in the prevention and / or treatment of acne.

14. A photosensitive polar extract according to any one of claims 1 to 7 or 11, or a topical composition according to claim 12, for use in the prevention and / or removal of skin defects.

15. A photosensitive extract for use or topical composition for use according to claim 13 or 14, characterized in that the extract or composition is applied to a skin surface to be treated, and the surface is subsequently exposed to a light source.

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