hydroxyl-substituted sphingolipids

Abstract

The present invention provides hydroxyl-substituted sphingolipids and their preparation and use, as well as cosmetic formulations comprising them.

Description

Technical Field

[0001] This invention provides hydroxylated sphingolipids, their preparation and uses, and cosmetic formulations in which they are components. Existing technology

[0002] The purpose of skincare cosmetics is to maintain a youthful appearance, such as youthful skin and hair. In principle, there are several ways to achieve this. For example, existing skin damage, such as irregular pigmentation or wrinkles, can be repaired with concealer powder or cream.

[0003] Another approach is to protect the skin from environmental influences that cause permanent damage and thus contribute to skin aging. Therefore, the idea is to take preventative measures to slow down the aging process.

[0004] The most important functions of skin are, on the one hand, to protect the body from uncontrolled water loss, and on the other hand, to prevent the invasion of harmful chemicals, bacteria, and sun exposure. Prolonged exposure of human skin to sunlight can lead to photoinduced skin aging and / or the development of pigmentation disorders.

[0005] This harmful effect of sunlight is in particular attributed to UVB radiation (280-320nm) present in the sunlight spectrum.

[0006] People are still seeking skin care products that can reduce or even prevent the development of signs of skin aging, especially those caused by sun exposure.

[0007] FR2855049 discloses a 6-hydroxy-sphingosine-based ceramide for strengthening the skin barrier.

[0008] FR2874610 discloses N-dihydroxyalkylhydroxyalkanoamide derivatives and their use in cosmetics.

[0009] The purpose of this invention is to provide a substance that reduces signs of aging caused by sun exposure.

[0010] Invention Description

[0011] Surprisingly, the ceramide derivatives described below were found to achieve the objectives of this invention and inhibit the formation of reactive oxygen species (ROS) induced by UV light.

[0012] Accordingly, the present invention provides certain hydroxyl-substituted sphingolipids, their preparation methods, and uses.

[0013] The present invention also provides cosmetic formulations comprising the hydroxyl-substituted sphingolipids.

[0014] The advantage of the sphingolipid of the present invention is its protective effect against DNA damage, especially UV-induced damage, particularly in the skin.

[0015] Another advantage of the sphingolipid of the present invention is its thickening effect, especially in cosmetic formulations.

[0016] Therefore, the present invention provides a sphingolipid of general formula I.

[0017]

[0018] in

[0019] R 1 It is a hydrocarbon group having 2 to 54, preferably 2 to 30, more preferably 2 to 18 carbon atoms, which is substituted by at least one group selected from -OH and -COOH, and optionally may be interrupted by at least one -O-.

[0020] R 2 It is H, phosphoric acid choline, serine, ethanolamine, or sugar, preferably sugar or H, more preferably H.

[0021] X is CH=CH, CH2-CH2, or CH2-HCOH, preferably CH2-HCOH, and

[0022] Y is selected from -OH and H.

[0023] The prerequisite is that when R 1 When X is a linear alkyl group whose ω-position is substituted only by a -OH group, X is CH2-HCOH.

[0024] Sphingolipids of general formula I have multiple stereoisomer centers, all of which are included in general formula I.

[0025] Unless otherwise stated, all percentages (%) are mass percentages.

[0026] The preferred sphingolipid of the present invention is characterized by:

[0027] R 1 Preferably selected from linear or branched alkyl groups having 2 to 54, more preferably 2 to 30, and more preferably 2 to 18 carbon atoms, wherein the alkyl group is substituted with at least one group selected from -OH and -COOH, and preferably...

[0028] R 2 Y is H, particularly preferred and

[0029] X is CH2-HCOH.

[0030] In a preferred embodiment of the present invention, R 1 The alkyl group is preferably straight-chain.

[0031] A further preferred embodiment of the present invention is characterized by:

[0032] R 1 Preferably, it is selected from hydrocarbon groups having 2 to 54, more preferably 2 to 30, and more preferably 2 to 18 carbon atoms, wherein the hydrocarbon group is substituted at the ω-position by at least one group selected from -OH and -COOH, and preferably...

[0033] R 2 Y is H, particularly preferred and

[0034] X is CH2-HCOH.

[0035] In a preferred embodiment of the present invention, R 1 The hydrocarbon group is preferably straight-chain.

[0036] The sphingolipids particularly preferred in this invention are selected from...

[0037] Hydroxybutyryl phytosphingosine

[0038]

[0039] Succinyl Phytosphingosine

[0040]

[0041] gluconoyl phytosphingosine

[0042]

[0043] Lactobionoyl phytosphingosine

[0044] and

[0045] 2-Hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine

[0046]

[0047] Since the sphingolipid of the present invention exhibits outstanding therapeutic effects, the present invention also provides the sphingolipid of the present invention for the treatment of cell damage, particularly for the prevention of cell damage, preferably cell damage caused by UV irradiation, especially skin cell damage, and the cell damage according to the present invention is preferably DNA damage.

[0048] The sphingolipids of the present invention can also be used in pure cosmetic applications. Therefore, the present invention also provides at least one sphingolipid of the present invention for cosmetic non-therapeutic use in preventing skin aging caused by UV irradiation.

[0049] The sphingolipids of the present invention can be readily incorporated into cosmetic formulations. Therefore, the present invention also provides cosmetic formulations comprising at least one sphingolipid of the present invention, wherein the amount of the sphingolipid is preferably from 0.02% to 1.50% by weight, more preferably from 0.03% to 1.00% by weight, and more preferably from 0.05% to 0.50% by weight, the percentages by weight being based on the total formulation.

[0050] The cosmetic formulations of the present invention are particularly formulations for sun protection and therefore preferably contain UV light-protective filters.

[0051] Therefore, the preferred formulations of the present invention comprise the following:

[0052] At least one sphingolipid of the present invention, and

[0053] At least one UV-protective filter substance.

[0054] The UV protection filter used can be, for example, an organic material that can absorb ultraviolet radiation and then re-emit the absorbed energy in the form of longer-wave radiation, such as heat.

[0055] UVB filters can be oil-soluble or water-soluble. Examples of oil-soluble UVB photoprotective filters include:

[0056] 3-Benzyl camphor and its derivatives, such as 3-(4-methylbenzyl)camphor,

[0057] 4-Aminobenzoic acid derivatives, such as 2-ethylhexyl 4-(dimethylamino)benzoate and pentyl 4-(dimethylamino)benzoate,

[0058] Esters of cinnamic acid, such as 2-ethylhexyl 4-methoxycinnamic acid, isopentyl 4-methoxycinnamic acid, and 2-ethylhexyl 2-cyano-3-phenylcinnamic acid (Octocrylene).

[0059] Esters of salicylic acid, such as 2-ethylhexyl salicylic acid, 4-isopropylbenzyl salicylic acid, and menthol salicylic acid.

[0060] Derivatives of benzophenone, such as 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, and 2,2′-dihydroxy-4-methoxybenzophenone.

[0061] Esters of benzyl malonic acid, such as di-2-ethylhexyl 4-methoxybenzyl malonic acid.

[0062] Triazine derivatives, such as 2,4,6-triphenylamino-(p-carbon-2′-ethyl-1′-hexyloxy)-1,3,5-triazine, octyltriazinone, and those described in EP 1180359 and DE 2004 / 027475,

[0063] Propane-1,3-dione, such as 1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione.

[0064] Suitable water-soluble UVB photoprotective filters include:

[0065] 2-Phenylenimazole-5-sulfonic acid and its alkali metal salts, alkaline earth metal salts, ammonium salts, alkyl ammonium salts, alkyl alcohol ammonium salts, and glucose ammonium salts,

[0066] Sulfonic acid derivatives of benzophenone, such as 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts.

[0067] Sulfonic acid derivatives of 3-benzyl camphor, such as 4-(2-oxo-3-iminoborneol)benzenesulfonic acid and 2-methyl-5-(2-oxo-3-iminoborneol)sulfonic acid and their salts.

[0068] Suitable typical UVA photoprotective filters include, in particular, derivatives of benzoylmethane, such as 1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione or 1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione. Mixtures of UV-A and UV-B filters can also be used.

[0069] In addition to the listed soluble substances, insoluble pigments are also suitable for this purpose, namely finely dispersed metal oxides or salts, such as titanium dioxide, zinc oxide, iron oxides, aluminum oxide, cerium oxide, zirconium oxide, silicates (talc), barium sulfate, and zinc stearate. The particles here should have an average diameter of less than 100 nm, for example, between 5 and 50 nm, particularly between 15 and 30 nm. They can be spherical, although elliptical or particles with a shape deviating from spherical shapes in some other way can also be used. A relatively new class of photoprotective filters is micronized organic pigments, such as 2,2′-methylenebis{6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol} with a particle size of <200 nm, which can be obtained, for example, as a 50% aqueous dispersion.

[0070] More suitable UV protection filters can be found in P. Finkel. It was found in the review of 122,543 (1996).

[0071] In relation to the formulations of the present invention, the formulations preferably contain lipophilic, hydrophobic UV light protective filter substances, particularly triazine derivatives.

[0072] The following UV-B filter substances are particularly preferred for use here: 2-ethylhexyl 2-cyano-3-phenylcinnamate, 2,4-bis{[4-(2-ethylhexyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine, dioctylbutylaminotriazinone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxybenzophenone, di-2-ethylhexyl-4-methoxyphenylmalonate (di-2-ethylhexyl-4-methoxyphenylmalonate) 4-methoxybenzmalonate), 2,4,6-tris-[anilino-(p-carbon-2′-ethyl-1′-hexyloxy)]-1,3,5-triazine, 2,4-bis[5,1(dimethylpropyl)benzoxazol-2-yl-(4-phenyl)imino]-6-(2-ethylhexyl)imino-1,3,5-triazine, 2,4-bis-{[4-(2-ethylhexyloxy)-2-hydroxy]phenyl}-6-(4-methoxyphenyl)-1,3,5-triazine, and 2-[4,6-bis(2,4-dimethylphenyl)-1,3,5-triazine-2-yl]-5-(octyloxy)phenol.

[0073] The UV-A filter used is preferably 1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione or 1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione.

[0074] The particularly preferred UV-A filter is 4-(tert-butyl)-4′-methoxydibenzoylmethane (CAS No. 70356-09-1) (it is produced by DSM). The 1789 brand was sold and sold by Merck under the trade name. 9020 (for sale) and hydroxybenzophenone according to DE 102004027475, particularly preferred is 2-(4′-diethylamino-2′-hydroxybenzoyl)hexyl benzoate (also known as: aminobenzophenone), which is available from BASF under the name Uvinul A Plus.

[0075] Other preferred UV filter materials are other so-called broadband filters, i.e., filter materials that absorb both UV-A and UV-B irradiation. In this group, 2,2′-methylenebis(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol is preferred, which is available from Ciba Chemikalien GmbH under the trade name... M obtained, and 2-(2H-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1,3,3,3-tetramethyl-1-[(trimethylsilyl)oxy]disiloxyl]propyl]phenol (CAS No.: 155633-54-8), which has the INCI name Drometrizole Trisiloxane.

[0076] In addition to the two main groups of UV light protection filters mentioned above, secondary light stabilizers of the antioxidant type can also be used, which prevent the photochemical reaction chain that is triggered when UV radiation penetrates the skin.

[0077] The cosmetic formulation of the present invention comprises a UV photoprotective filter in an amount preferably from 0.01% to 15% by weight, more preferably from 0.05% to 10% by weight, and more preferably from 0.1% to 5% by weight, based on the total formulation.

[0078] Preferably, the cosmetic formulation of the present invention comprises a combination of two or more different UV light protective filters.

[0079] When UVA and UVB photoprotective filters are used in the formulations of the present invention, the weight ratio of these filters is preferably 1:2 to 1:4.

[0080] The formulations of the present invention may further comprise at least one additional component selected from the group consisting of:

[0081] Moisturizer

[0082] Emulsifier,

[0083] Thickener / viscosity modifier / stabilizer

[0084] Antioxidants

[0085] Water-soluble substances (or polyols),

[0086] Solids and fillers

[0087] Film-forming agent,

[0088] Pearlescent additives

[0089] Deodorizing and antiperspirant active ingredients,

[0090] Insect repellent,

[0091] Self-tanning agent;

[0092] preservative,

[0093] Conditioner,

[0094] spices,

[0095] dye,

[0096] Odor absorbent

[0097] Cosmetic active ingredients

[0098] Nursing additives

[0099] Fat enrichment agent,

[0100] Solvent.

[0101] Substances that can be used as exemplary representatives of the various groups are known to those skilled in the art and can be found, for example, in German patent application DE 102008001788.4. This patent application is hereby incorporated by reference and thus constitutes a part of this disclosure.

[0102] Regarding other optional components and the amounts of these components used, please refer to relevant manuals known to those skilled in the art, such as K. Schrader, "Grundlagen und Rezepturen der Kosmetika" [Fundamentals and formulations of cosmetics], 2nd edition, pp. 329-341, Hüthig Buch Verlag Heidelberg.

[0103] The dosage of each additive is guided by its intended use.

[0104] Typical framework formulations for specific applications are known from existing technologies and are contained in the manuals of manufacturers of, for example, specific base materials and active ingredients. These existing formulations can usually be used without modification. However, when adjustments and optimizations are required, the desired modifications can be performed directly through simple testing.

[0105] The present invention also provides a method for preparing sphingolipids, and particularly a method for preparing the sphingolipids of the present invention, the method comprising the following steps:

[0106] I) Provide a first component, at least a soluble sphingolipid of general formula II.

[0107]

[0108] in

[0109] R 2b It is H, phosphoric acid choline, serine, ethanolamine, or sugar, preferably sugar or H, more preferably H, and

[0110] X b It is CH=CH, CH2-CH2, or CH2-HCOH, preferably CH2-HCOH, and

[0111] II) Provide a second component, at least of formula R 1b CY b Intramolecular cyclic esters of hydroxycarboxylic acids containing HCOOH.

[0112] in

[0113] R 1b It is a hydrocarbon group having 2 to 54, preferably 2 to 30, more preferably 2 to 18 carbon atoms, which is substituted by at least one group selected from -OH and -COOH, and optionally may be interrupted by at least one -O-.

[0114] Y b Selected from -OH and H, and

[0115] III) React the first component with the second component to obtain a sphingolipid, and optionally...

[0116] IV) Purify the sphingolipid.

[0117] The preferred feature of the method of the present invention is that step III) is carried out in a temperature range of 40°C to 95°C, preferably 50°C to 80°C, and more preferably 60°C to 70°C.

[0118] The present invention also provides an alternative method for preparing sphingolipids, particularly a method for preparing the sphingolipids of the present invention, the method comprising the following steps:

[0119] A) Provide a first component, at least a soluble sphingolipid of general formula II.

[0120]

[0121] in

[0122] R 2b It is H, phosphoric acid choline, serine, ethanolamine, or sugar, preferably sugar or H, more preferably H, and

[0123] X b It is CH=CH, CH2-CH2, or CH2-HCOH, preferably CH2-HCOH, and

[0124] B) Provide a second component, at least of formula R 1b CY b Hydroxycarboxylic acid of HCOOH,

[0125] in

[0126] R 1b It is a hydrocarbon group having 2 to 54, preferably 2 to 30, more preferably 2 to 18 carbon atoms, which is substituted by at least one group selected from -OH and -COOH, and optionally may be interrupted by at least one -O-.

[0127] Y b Selected from -OH and H, and

[0128] C) Using at least one coupling agent for activating the hydroxycarboxylic acid, react the first component with the second component to obtain the sphingolipid, and optionally...

[0129] D) Purify the sphingolipid.

[0130] The preferred feature of the alternative method of the present invention is that, in step C), the coupling agent used is at least one selected from the group consisting of, preferably, the following substances: dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-cyclohexyl-N′-(2′-morpholinoethyl)carbodiimide methyl p-toluenesulfonate, N-benzyl-N′-3′-dimethylaminopropylcarbodiimide hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-ethylcarbodiimide hydrochloride, and carbonyl diimidazole, with dicyclohexylcarbodiimide and diisopropylcarbodiimide being particularly preferred.

[0131] The preferred feature of the alternative method of the present invention is that, in step C), at least one catalyst selected from the group consisting of, and preferably composed of: N-ethyldiisopropylamine, trialkylamine, pyridine, 4-dimethylaminopyridine, and hydroxybenzotriazole, especially hydroxybenzotriazole.

[0132] The preferred feature of the method of the present invention is that step C) is carried out in a temperature range of 40°C to 95°C, preferably 50°C to 80°C, and more preferably 55°C to 65°C.

[0133] The preferred method according to the present invention preferably produces the sphingolipid described above according to the present invention.

[0134] The following embodiments illustrate the invention by way of example, and the scope of the invention is apparent from the entire specification and claims, and is not intended to limit the invention to the embodiments specified in the embodiments.

[0135] The following figures form part of the embodiments:

[0136] Figure 1 ROS is generated after UV irradiation.

[0137] Figure 2 DNA damage after UV irradiation

[0138] Figure 3 The ITA° increased after two, four, and eight weeks of application testing of the formulation.

[0139] Figure 4 L* increased after two, four, and eight weeks of application testing of the formulation.

[0140] Figure 5 Skin roughness decreased after two, four, and eight weeks of application of the tested formulation.

[0141] Figure 6 Skin density increased after two, four, and eight weeks of application of the tested formulation. Example:

[0142] Example 1: 4-Hydroxybutyryl phytosphingosine

[0143] Phytosphingosine and γ-butyrolactone were dissolved in methanol in a 1:1 molar ratio. The mixture was reacted at 65°C until phytosphingosine was no longer detectable. Cooling to room temperature yielded 4-hydroxybutyryl phytosphingosine crystals, which were filtered and washed with a 1:1 THF / water mixture. Yield >80%, purity >90%.

[0144] Example 1b: 6-Hydroxyhexanoyl phytosphingosine

[0145] Phytosphingosine and ε-caprolactone were dissolved in methanol in a 1:1 molar ratio. The mixture was reacted at 65°C until phytosphingosine was no longer detectable. Cooling to room temperature yielded 6-hydroxyhexanoyl phytosphingosine crystals, which were filtered and washed with a 1:1 THF / water mixture. Yield >80%, purity >90%.

[0146] Example 1c (not according to the invention): 3-hydroxypropionyl phytosphingosine

[0147] Phytosphingosine and β-propiolactone were dissolved in methanol in a 1:1 molar ratio. The mixture was reacted at 65°C until phytosphingosine was no longer detectable. Cooling to room temperature yielded 3-hydroxypropionyl phytosphingosine crystals, which were filtered and washed with a 1:1 THF / water mixture. Yield >80%, purity >90%.

[0148] Example 2: Succinyl Phytosphingosine

[0149] Phytosphingosine and succinic anhydride were dissolved in methanol in a 1:1 molar ratio. The mixture was reacted at 65°C until phytosphingosine was no longer detectable. Cooling to room temperature yielded succinyl phytosphingosine crystals, which were filtered and washed with a 1:1 THF / water mixture. Yield >80%, purity >90%.

[0150] Example 3: Glucoyl Phytosphingosine

[0151] Phytosphingosine and d-gluconolactone were dissolved in methanol in a 1:1 molar ratio. The mixture was reacted at 65°C until phytosphingosine was no longer detectable.

[0152] Post-treatment is performed by gradually lowering the temperature to 10°C for crystallization.

[0153] The product was filtered out under vacuum and washed with ethanol. After drying to a constant mass, the yield was >80% and the purity was >90%.

[0154] Example 4: Lactosyl Phytosphingosine

[0155] 71.66 g of lactobionic acid and 75.44 g of phytosphingosine were dissolved in dimethylformamide (DMF). 2 g of N-hydroxysuccinimide (HSU) and 15 ml of N,N-diisopropylcarbodiimide (DIC) were added, and the mixture was reacted at 60 °C for 2 h. 15 ml of water was added, and the mixture was allowed to react further for 1 / 2 h to quench any residual DIC. For post-treatment, the dimethylformamide was distilled off, and the product residue was dissolved in methanol. The product was then crystallized by slow cooling to 10 °C. The resulting product was filtered off, washed with methanol, and dried. Yield >80%, purity >90%.

[0156] Example 5: 2-Hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine

[0157] Phytosphingosine and D-pantothenic acid lactone were dissolved in ethanol at a molar ratio of 1:1. The mixture was reacted at 60°C for 8 h. The product was then crystallized by slow cooling to 10°C. The product was then filtered and dried. Yield >80%, purity >90%.

[0158] Example 5b: 2-Hydroxy-3,3-dimethyl-hydroxybutyryl dihydrosphingosine

[0159] Dihydrosphingosine and D-pantothenic acid lactone were dissolved in ethanol at a molar ratio of 1:1. The mixture was reacted at 60°C for 8 h. The product was then crystallized by slow cooling to 10°C. The product was then filtered and dried. Yield >80%, purity >90%.

[0160] Example 6: Prevention of UV damage

[0161] The extent to which the substances of the present invention can protect normal human epidermal keratinocytes (NHEK) exposed to UV radiation was tested.

[0162] Therefore, biomarkers of reactive oxygen species (ROS) were studied, quantified using 2,7-dichlorodihydrofluorescein diacetate, and DNA damage and repair were quantified using the Comet assay.

[0163] Reactive oxygen species (ROS)

[0164] Intracellular formation of reactive organic substances (ROS) is a major factor contributing to DNA damage caused by UV radiation.

[0165] Keratinocytes were seeded in 96-well plates and cultured in medium for 24 hours, followed by another 24 hours in assay medium. The medium was then replaced with test medium containing either the test compound or a reference (100 μM vitamin E and 10 μM EGCG) or without them (irradiation control), and the cells were pre-incubated for 24 hours. After pre-incubation, the medium was removed and replaced with test medium, and the fluorescent probe (2,7-dichlorodihydrofluorescein diacetate (test medium containing 10 μM 2,7-DCDHF-DA) was added. The cells were then incubated at 37°C for 30 minutes. The cells were then washed with PBS and, in the absence of the test compound or reference, with 100 mJ / cm² solution. 2 UVB + UVA (+0.7 J / cm) 2 Irradiation. The lamp used is from a SOL500 solar simulator equipped with an H2 filter. (AG). After irradiation, the cells were incubated for 30 minutes. Parallel trials were performed on an unirradiated control and a sample-free (background noise) condition.

[0166] All experimental conditions were performed in triplicate.

[0167] use The fluorescence emission intensity was measured using a PerkinElmer microplate reader (ex = 485 nm, em = 538 nm).

[0168] The fluorescence intensity of the metabolically generated sample (DCF) is proportional to the formation of ROS. Therefore, ROS production is expressed as the relative intensity of fluorescence.

[0169] Figure 1 The corresponding results are displayed.

[0170] It can be seen that the production of reactive oxygen species (ROS) in human epidermal keratinocytes is caused by UV light irradiation (corresponding to 0% protection). The vitamin E and epigallocatechin gallate (EGCG) positive controls used in the test are known to have protective effects against UV irradiation, thus showing a 30%-40% protective effect against ROS formation. The substances used in this invention also showed a significant protective effect exceeding 40% with increasing concentration. This effect has not been previously described for sphingolipids / ceramides.

[0171] As a baseline, sphingoid base phytosphingosine (PS) was also tested. No obvious protective effect was observed.

[0172] The following experiments were not conducted in Figure 1 Description in Chinese:

[0173] In the above cases, 2-hydroxy-3,3-dimethyl-hydroxybutyryl dihydrosphingosine showed a 22% protective effect at a concentration of 5 μM, while 2-hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine showed a 38% protective effect at a concentration of 5 μM.

[0174] In the above cases, ceramide 3 (also known as ceramide NP) showed a 4% protective effect at a concentration of 10 μM, and 3-hydroxypropionyl phytosphingosine showed a 5% protective effect at a concentration of 10 μM.

[0175] DNA damage and repair

[0176] Keratinocytes were seeded into 6-well plates and incubated in medium for 48 hours, with medium replenished after 24 hours. The medium was then replaced with medium containing the test compound or a reference (0.3 mM control) or neither (control), and the cells were incubated for 24 hours. After pre-incubation, the medium was removed and the test medium was added again, and the cells were incubated at 250 mJ / cm² in the absence of the compound. 2 UVB + UVA (+1.6 J / cm) 2 The cells were irradiated with a SOL500 solar simulator equipped with an H2 filter. (AG). Parallel tests were conducted under unirradiated control conditions. All experimental conditions were performed in duplicate.

[0177] At the end of irradiation, the culture supernatant was discarded, and the cells were washed with phosphate-buffered saline (PBS) before analysis.

[0178] Cells were digested with trypsin and counted, and the supernatant was removed after centrifugation. Cells were then washed with PBS and resuspended in the same PBS solution to achieve a cell density of 1 × 10⁻⁶ cells / mL. 5Cell concentrations were determined using cells / ml. The cell suspension was then mixed with melted (37°C) 1% low-melting-point agarose gel and pipetted onto Comet microscope slides (two-component analysis was performed for each condition). For cell lysis, the microscope slides were immersed in freshly prepared alkaline solution (200 mM NaOH containing 1 mM EDTA, pH > 13) on electrophoresis support. Gel electrophoresis was performed at 21 volts for 30 minutes. Comet microscope slides were washed twice with water for 5 minutes each time, then washed with 70% ethanol for 5 minutes, and air-dried at 37°C for 15 minutes.

[0179] After electrophoresis, each dried sample was stained with a DNA intercalation fluorescent dye (SYBR Green solution). Then, an automated IN Cell Analyzer was used. TM Cells were observed in epifluorescence using a 2200 microscopy image analyzer (GE Healthcare) (×10 objectives). DNA-binding cells were observed upon excitation (ex 494nm, em 524nm). Green emits green light. In healthy cells, fluorescence is confined to the nucleus: undamaged DNA is supercoiled and therefore does not migrate extensively from the nucleus under the influence of an electric current. If DNA damage occurs, alkaline treatment causes the DNA to coil up, releasing fragments that migrated out of the cell when exposed to an electric field. Negatively charged DNA migrates to the anode, and the extruded length is proportional to the relaxation of the supercoiled structure, an indicator of damage. When alkaline electrophoresis conditions are used, the distribution of DNA between the tail and the comet head can be used to determine the degree of DNA damage.

[0180] Cell images were analyzed using OpenComet in conjunction with ImageJ software. The minimum values ​​of 500 events were analyzed for each replicate sample.

[0181] Figure 2 The corresponding results are displayed.

[0182] DNA damage caused by UV light irradiation of human epidermal keratinocytes is identifiable. Due to its known antioxidant properties, the Tiron positive control was able to protect against DNA damage, thus showing approximately 60% protection against DNA damage after UV irradiation. The substance of this invention also showed over 60% protection. This effect is unknown for sphingolipids / ceramides, and it could not be demonstrated for pure phytosphingosine (PS) in this test.

[0183] The following experiments were not conducted in Figure 2 Description in Chinese:

[0184] In the above cases, 2-hydroxy-3,3-dimethyl-hydroxybutyryl dihydrosphingosine showed a 24% protective effect at a concentration of 5 μM, and 2-hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine showed a 39% protective effect at a concentration of 2 μM.

[0185] In the above cases, ceramide 3 (also known as ceramide NP) showed a 12% protective effect at a concentration of 10 μM, and 3-hydroxypropionyl phytosphingosine showed a 13% protective effect at a concentration of 10 μM.

[0186] Example 7: In vivo data

[0187] For the in vivo study, 24 test subjects (male and female) with skin subjected to sun stress were recruited. To ensure skin was subjected to sun stress, the study was conducted between September and November. The assumption was that skin exhibited the greatest degree of sun stress at the end of summer.

[0188] Test subjects received either two different test formulations, applied to one forearm individually, or one test formulation applied to one forearm while the other forearm remained untreated (control). The test formulations consisted of a mediator and a formulation containing 0.1% hydroxybutyryl phytosphingosine (hydroxybutyryl PS). The various test combinations were randomly assigned to test subjects.

[0189] The composition of the test formulation is shown in Table 1. Test subjects applied the test formulation twice daily to the medial and lateral sides of one forearm in each condition for eight weeks. The following measurements were taken on the forearm before application and at two, four, and eight weeks later:

[0190] 1. Color Measurement: The parameters L* and ITA° on the outer forearm were measured using a colorimeter probe (Skin-Colorimeter CL 400, Courage & Khazaka, Cologne). L* describes the skin's black / white value, and ITA° describes the skin tone. Both values ​​increase as the skin becomes lighter.

[0191] 2. Skin roughness: This parameter is determined using a dedicated camera (Visioscan VC 98, Courage & Khazaka) on the inner forearm. This camera records digital black-and-white images of the skin. The grayscale distribution of the images can be used to determine skin roughness.

[0192] 3. Dermal Density: Dermal density is determined using ultrasound (SkinLab Combo, Cortex Technologies, Denmark) on the lateral forearm. A specialized probe transmits an ultrasound signal to the skin and records the reflection. This reflection is used to determine the skin density value. Decreased skin density is particularly evident in areas of skin that have been heavily exposed to sunlight.

[0193]

[0194] Table 1: Composition of the test preparation

[0195] Figure 3 The results show an increase in ITA° after two, four, and eight weeks of application of the test formulation.

[0196] Figure 4 The increase in L* was shown after two, four, and eight weeks of application of the test formulation.

[0197] The increase in color parameters L* and ITA° in Figures xy1 and xy2 indicates that the skin became brighter throughout the measurement period, with the most significant increase in brightness observed in the skin areas treated with hydroxybutyryl phytosphingosine compared to the mordant formulation or untreated control. In this case, this cannot be attributed to the destruction of melanin in the skin. Skin tanning is most pronounced at the end of summer and then fades in the fall due to the normal skin regeneration cycle. Hydroxybutyryl phytosphingosine enhances this cycle, leading to faster skin brightening in this case.

[0198] Figure 5 The study showed a reduction in skin roughness after two, four, and eight weeks of application of the test formulation.

[0199] from Figure 5 It can be seen that, compared with the untreated control, and especially with the mordant, the reduction in skin roughness was most significant in the test formulation containing hydroxybutyryl phytosphingosine. This supports the skin color measurement results: skin under sun stress is characterized by increased skin roughness. This roughness also decreases in the fall due to normal skin regeneration. Because hydroxybutyryl phytosphingosine contributes to the skin renewal cycle, the reduction in skin roughness is more pronounced than in the mordant formulation or the untreated control.

[0200] Figure 6 The study showed an increase in skin density after two, four, and eight weeks of application of the test formulation.

[0201] Skin areas exposed to more sun exposure showed decreased skin density. Results from skin studies indicated that, after two weeks of use, skin density was significantly increased in the hydroxybutyryl phytosphingosine (HPS) condition compared to untreated controls or mordants.

[0202] The experiment was repeated in the same manner using 6-hydroxyhexanoyl phytosphingosine (Example 1b), 2-hydroxy-3,3-dimethyl-hydroxybutyryl sphingosine (Example 5b), ceramide 3 (also known as ceramide NP), and 3-hydroxypropionyl phytosphingosine (Example 1c).

[0203]

Claims

1. Sphingolipids, which are selected from Hydroxybutyryl phytosphingosine ， Succinyl Phytosphingosine ， Glucoyl phytosphingosine ， Lactosyl phytosphingosine ,and 2-Hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine 。 2. Use of the sphingolipid as described in claim 1 in the preparation of a medicament for treating or preventing cell damage.

3. The use as described in claim 2, characterized in that... The cell damage mentioned is cell damage caused by UV irradiation.

4. The use as described in claim 2, characterized in that The cell damage mentioned refers to skin cell damage.

5. The use as described in claim 2, characterized in that... The cell damage mentioned is DNA damage.

6. At least one sphingolipid as described in claim 1 for cosmetic, non-therapeutic use in preventing skin aging caused by UV irradiation.

7. A cosmetic formulation comprising at least one sphingolipid as described in claim 1.

8. The cosmetic formulation as described in claim 7, characterized in that... The amount of the sphingolipid is from 0.02% to 1.50% by weight, and the weight percentage is based on the total formulation.

9. The cosmetic formulation as described in claim 7, characterized in that... The amount of the sphingolipid is from 0.03% to 1.00% by weight, and the weight percentage is based on the total formulation.

10. The cosmetic formulation as described in claim 7, characterized in that... The amount of the sphingolipid is from 0.05% to 0.50% by weight, and the weight percentage is based on the total formulation.

11. The cosmetic formulation of claim 7, further comprising... At least one UV-protective filter substance.

12. A method for preparing sphingolipids, the method comprising the following steps: I) Provide a first component, at least a soluble sphingolipid of general formula II. Formula II, in R 2b It is H, and X b It is CH2-HCOH, and II) Provide a second component, the second component being selected from γ-butyrolactone, succinic anhydride, d-gluconolactone, and D-pantothenic lactone. III) React the first component with the second component to obtain the sphingolipid, and optionally... IV) Purify the sphingolipid.

13. The method as described in claim 12, characterized in that... The sphingolipids are selected from hydroxybutyryl phytosphingosine, succinoyl phytosphingosine, glucosyl phytosphingosine and 2-hydroxy-3,3-dimethyl-hydroxybutyryl phytosphingosine.

14. The method as described in claim 12, characterized in that Method step III) is carried out in a temperature range of 40℃ to 95℃.

15. The method as described in claim 12, characterized in that... Method step III) is carried out in a temperature range of 50℃ to 80℃.

16. The method as described in claim 12, characterized in that... Method step III) is carried out in a temperature range of 60℃ to 70℃.

17. A method for preparing sphingolipids, the method comprising the following steps: A) Provide a first component, at least a soluble sphingolipid of general formula II. Formula II, in R 2b It is H, and X b CH2-HCOH, and B) provide lactobionic acid as a second component, and C) Using at least one coupling agent for activating the lactobionic acid, react the first component with the second component to obtain the sphingolipid, and optionally... D) Purify the sphingolipid.

18. The method as described in claim 17, characterized in that... The sphingolipid is lactosyl phytosphingosine.

19. The method as described in claim 17, characterized in that... In method step C), the coupling agent used is at least one selected from the group consisting of: dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-cyclohexyl-N'-(2'-morpholinoethyl)carbodiimide methyl p-toluenesulfonate, N-benzyl-N'-3'-dimethylaminopropylcarbodiimide hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-ethylcarbodiimide hydrochloride, and carbonyl diimidazole.

20. The method as described in claim 17, characterized in that... In method step C), the coupling agent used is selected from at least one of the following substances: dicyclohexylcarbodiimide and diisopropylcarbodiimide.

21. The method according to any one of claims 17 to 20, characterized in that In step C), at least one catalyst selected from the group consisting of N-ethyldiisopropylamine, trialkylamine, pyridine, 4-dimethylaminopyridine, and hydroxybenzotriazole is used.

22. The method according to any one of claims 17 to 20, characterized in that... In step C), hydroxybenzotriazole is used as a catalyst.

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