Use of a composition comprising clavulanate

Abstract

The present disclosure relates to the fields of skin care products, beauty and medicine, and specifically provides a use of a composition containing corallate. The corallate of the present disclosure has good efficacy in improving skin conditions or appearance.

Description

Technical Field

[0001] The present invention belongs to the fields of skin care products, beauty and medicine, and particularly relates to the use of a composition containing colanate. Background Art

[0002] Colanic acid (CA) is a bacterial exopolysaccharide produced by most Escherichia coli strains and other species of the Enterobacteriaceae family. It is a polysaccharide synthesized by the bacterial cells during their life activities to adapt to environmental changes and improve their survival probability. CA has a relatively large molecular weight and is loosely wrapped on the surface of the bacterial cells, making the cells show a mucous state, preventing cell water loss, protecting the cells and at the same time resisting harmful substances. Under environmental conditions unfavorable for growth, such as dryness, low pressure and low pH, the mucilaginous strains have stronger viability than the wild strains. In 2017, Han et al. reported that feeding purified CA or Escherichia coli that can secrete CA can significantly extend the lifespan of Caenorhabditis elegans. In addition, as a unique active biopolymer, CA has special biological characteristics and physiological parameters and has broad application prospects. Summary of the Invention

[0003] On the one hand, the present disclosure provides the use of colanic acid or its physiologically acceptable salt, or a composition containing colanic acid or its physiologically acceptable salt, in increasing skin water content, wherein the molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 6000 kDa.

[0004] In some embodiments, the content of the colanic acid or its physiologically acceptable salt is 0.001 - 2% (w / v), 0.01 - 1% (w / v), or 0.05 - 0.5% (w / v).

[0005] In some embodiments, the molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 50 kDa or 1000 - 5000 kDa.

[0006] On the other hand, the present disclosure provides the use of colanic acid or its physiologically acceptable salt, or a composition containing colanic acid or its physiologically acceptable salt, in enhancing the protective function of skin cells such as increasing cell viability, wherein the molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 6000 kDa.

[0007] On the other hand, the present disclosure provides the use of colanic acid or its physiologically acceptable salt, or a composition containing colanic acid or its physiologically acceptable salt, in skin anti-aging, wherein the molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 6000 kDa.

[0008] In some embodiments, the skin anti-aging includes skin anti-wrinkle, skin anti-aging, increasing the collagen content in the skin, preferably the content of type I and type III collagen.

[0009] On the other hand, the present disclosure provides the use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, in improving telangiectasia caused by skin allergy or skin inflammation, wherein the molecular weight of the colanic acid or a physiologically acceptable salt thereof is in the range of 10 - 6000 kDa.

[0010] In some embodiments, the molecular weight of the colanic acid or a physiologically acceptable salt thereof is in the range of 10 - 50 kDa, 300 - 500 kDa, 600 - 800 kDa, 1000 - 2000 kDa or 3000 - 5000 kDa.

[0011] In some embodiments, the content of the colanic acid or a physiologically acceptable salt thereof is 0.001 - 10% (w / v), 0.05 - 5% (w / v), or 0.1 - 3% (w / v).

[0012] In some embodiments, the composition contains only colanic acid or a physiologically acceptable salt thereof as the active ingredient.

[0013] In some embodiments, the physiologically acceptable salt of colanic acid is sodium colanate.

[0014] In some embodiments, the composition is formulated for topical, oral, intramuscular, subcutaneous or intravenous administration.

[0015] In some embodiments, the composition is formulated for subcutaneous administration.

[0016] In some embodiments, the composition further contains a cosmetic, beauty or pharmaceutically acceptable additive.

[0017] On the other hand, the present disclosure provides the use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, as described above, in the preparation of a drug or cosmetic for increasing skin water content, enhancing the protective function of skin cells, skin anti-aging, and / or improving telangiectasia caused by skin allergy or skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] With reference to the following drawings, the present invention can be more fully understood.

[0019] Figure 1 Shows the mechanism of the protective effect of macromolecular sodium colanate on epidermal cells.

[0020] Figure 2Shows the effect of sodium caffeate on inhibiting reactive oxygen / nitrogen species in HaCaT cells irradiated with ultraviolet light.

[0021] Figure 3 Shows the effect of sodium caffeate on inhibiting inflammation in human keratinocytes.

[0022] Figure 4 Shows the effect of sodium caffeate on inhibiting the increase of DNA damage markers caused by ultraviolet irradiation and reducing cellular senescence and apoptosis.

[0023] Figure 5 Shows the protective effect of sodium caffeate on cell mitochondria.

[0024] Figure 6 Shows the effect of sodium caffeate on promoting cell proliferation and migration by upregulating cell CD44.

[0025] Figure 7 Shows the results of cell experiments on the procollagen activity of sodium caffeate.

[0026] Figure 8 Shows the effect of sodium caffeate on increasing the content of 3D skin filaggrin (FLG).

[0027] Figure 9 Shows the effect of sodium caffeate on increasing the content of 3D skin aquaporin 3 (APQ3).

[0028] Figure 10 Shows the results of the cytotoxicity test of sodium caffeate.

[0029] Figure 11 Shows the results of the anti-wrinkle efficacy of sodium caffeate.

[0030] Figure 12 Shows the test results of the anti-redness and anti-allergy efficacy of sodium caffeate.

[0031] Figure 13 Shows the test results of the water-locking and moisturizing efficacy of sodium caffeate.

[0032] Figure 14 Shows the skin protection effect of sodium caffeate with different molecular weights.

[0033] Figure 15 Shows the effect of sodium caffeate with different molecular weights on stimulating human fibroblasts to express type I collagen.

[0034] Figure 16 Shows the migration effect of sodium caffeate with different molecular weights on HaCaT cells.

[0035] Figure 17 Shows the results of the skin penetration test of sodium caffeate.

[0036] Figure 18 The experimental results of the mitochondrial localization of sodium corolate are shown.

[0037] In the figure, * indicates P < 0.05 relative to the negative control, ** indicates P < 0.01 relative to the negative control, *** indicates P < 0.001 relative to the negative control, ## indicates P < 0.01 relative to the blank control, and indicates P < 0.001 relative to the blank control. Detailed Description of the Invention

[0038] The various different features and aspects of the present invention will be discussed in more detail below.

[0039] Figure 1 The protective effect of macromolecular corolate on epidermal cells and the anti-aging mechanism on the dermis are shown. For epidermal cells, corolic acid can reduce cell damage of keratinocytes under oxidative stress and also promote keratinocyte migration and proliferation. At the same time, sodium corolate can also penetrate into the dermis and enter the mitochondria of dermal cells, playing the role of promoting collagen synthesis of dermal cells and maintaining the mitochondrial homeostasis of cells. Macromolecular corolate (10 - 6000 kDa) can exert its effects from the following 7 aspects, including inhibiting reactive oxygen / nitrogen species, inflammatory factors, DNA damage, mitochondrial damage, matrix metalloproteinases (MMP3 / 9), malondialdehyde (MDA), etc., and promoting epidermal cells to up-regulate the expression of CD44. These mechanisms can respectively play the roles of inhibiting inflammatory reactions, reducing zombie cells, reducing skin wrinkles, and promoting epidermal integrity.

[0040] In the present disclosure, colanic acid (CA) or a physiologically acceptable salt thereof can be prepared by any method as long as the desired molecular weight can be obtained. Specifically, the molecular weight of colanic acid or a physiologically acceptable salt thereof can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1500 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa, 6000 kDa or within any range constituted by the above molecular weights. For example, the molecular weight of colanic acid or a physiologically acceptable salt thereof can be within any of the following ranges: 10 - 6000 kDa, such as 50 - 5000 kDa, 100 - 5000 kDa, 1000 - 5000 kDa, 1000 - 4000 kDa, 1000 - 3000 kDa, 1000 - 2000 kDa, 2000 - 5000 kDa, 2000 - 4000 kDa, 2000 - 3000 kDa, 3000 - 5000 kDa, 3000 - 4000 kDa, 300 - 500 kDa, 600 - 800 kDa, 10 - 1000 kDa, 10 - 100 kDa, 10 - 50 kDa, 10 - 30 kDa, 10 - 20 kDa, 10 - 18 kDa, 10 - 15 kDa or 10 - 12 kDa.

[0041] The inventors of the present invention unexpectedly found that by selecting colanic acid or a salt thereof with a specified molecular weight, its efficacy in increasing skin moisture, enhancing the protective function of skin cells, and skin anti - aging, especially in increasing the collagen content in the skin, can be further improved.

[0042] In some embodiments, the molecular weight of colanic acid or a physiologically acceptable salt thereof is in the range of 10 - 100 kDa, 10 - 50 kDa, or 10 - 15 kDa.

[0043] In some embodiments, the molecular weight of colanic acid or a physiologically acceptable salt thereof is in the range of 100 - 1000 kDa, 300 - 500 kDa, or 600 - 800 kDa.

[0044] In some embodiments, the molecular weight of colanic acid or a physiologically acceptable salt thereof is in the range of 1000 - 5000 kDa, 1000 - 2000 kDa, or 3000 - 5000 kDa.

[0045] For example, the method of CN115287314B can be used to prepare clavulanic acid or its physiologically acceptable salts. In addition, the clavulanic acid or its physiologically acceptable salts can also be purified by the method of CN114957509A. Those skilled in the art should understand that clavulanic acid or its salts with different molecular weight ranges can be obtained through conventional preparation methods.

[0046] The term "physiologically acceptable salts" can be any salts derived from clavulanic acid. In the present invention, these salts are not limited to specific types as long as they can be used in cosmetic and pharmaceutical compositions. Specific examples thereof include alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium, magnesium, barium, and zinc salts; alkylamine salts, where the alkylamines are, for example, ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, butylamine, tetrabutylamine, pentylamine, and hexylamine; alkanolamine salts, where the alkanolamines are, for example, ethanolamine, diethanolamine, triethanolamine, propanolamine, dipropanolamine, isopropanolamine, and diisopropanolamine; salts of other organic amines such as piperazine and piperidine; salts of basic amino acids such as lysine, arginine, histidine, and tryptophan, etc. In the present disclosure, the physiologically acceptable salt of clavulanic acid is, for example, sodium clavulanate.

[0047] The composition of the present invention can be used as a pharmaceutical composition or a cosmetic composition. The dosage form of the composition can be specifically selected according to needs. For example, the dosage forms of the cosmetic composition can include emulsions, creams, lotions, serums, masks, gels, powders, lipsticks, cosmetic bases, foundations, lotions, ointments, patches, beauty liquids, cleansing foams, cleansing pastes, cleansing waters, soaps, or sprays; the dosage forms of the pharmaceutical composition can include lozenges, capsules, emulsions, dispersants, suspensions, solutions, syrups, granules, transdermal patches, gels, powders, creams, ointments, suppositories, or sprays. The pharmaceutical composition can be formulated for topical, oral, intramuscular, subcutaneous, or intravenous administration.

[0048] In the composition of the present invention, the content of clavulanic acid or its physiologically acceptable salts can be selected according to needs, for example, it can be 0.001 - 10% (w / v), 0.05 - 5% (w / v), or 0.1 - 3% (w / v).

[0049] In addition, according to needs, the pharmaceutical composition or cosmetic composition of the present invention, in addition to containing the necessary active ingredients, can also contain additives commonly added to pharmaceutical compositions or cosmetic compositions. Examples of such additives include oily components, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, antibacterial agents, antioxidants, plant extracts, pH regulators, alcohols, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, and water, etc.

[0050] In some specific embodiments, the composition of the present invention contains only clavulanic acid or its physiologically acceptable salt as the active ingredient. In some specific embodiments, the composition of the present invention further contains other active ingredients, such as boswellic acid, retinol and its derivatives (such as retinol propionate), retinal, retinoic acid, hyaluronic acid, etc.

[0051] In the present disclosure, the molecular weight of clavulanic acid or its physiologically acceptable salt is determined by a liquid chromatography method. The specific method is as follows: accurately weigh the sample and the standard product. The sample is prepared into a 2 mg / mL solution, and after being fully dissolved, it is filtered through a 0.22 μm needle filter into a 1.8 mL injection vial. Chromatographic column: PolySep-GFC-P(35*7.8mm); PolySep-GFC-P 4000(300*7.8mm); PolySep-GFC-P 6000(300*7.8mm); Mobile phase: 0.02M NaCl solution; Flow rate: 0.6 mL / min, Column temperature: 40 °C; Injection volume: 20 μL; Detector: Differential detector 1260-RID.

[0052] Preparation method

[0053] In the following examples, sodium clavulanate (with a molecular weight in the range of 10 - 6000 kDa) is prepared by the following method.

[0054] 1. Solid-liquid separation

[0055] 1) Dilution: Pump the fermentation broth into the storage tank, dilute it 3 - 20 times with 10 - 60% calcium chloride solution (the dosage of calcium chloride is 2% (w / v) of the fermentation broth) and purified water, and stir for 2 - 3 h until fully dissolved.

[0056] 2) Flocculation: After adding solid sodium carbonate to adjust the pH to 9.0 - 12.0, flocculate for 1 - 2 h and then premix diatomaceous earth (the dosage of diatomaceous earth is 1% (w / v) of the fermentation broth).

[0057] 3) Plate and frame filtration: According to the sop of the plate and frame filter press, install the filter cloth and tighten the filter plate. Circulate and rinse the plate and frame filter press system with 0.5M sodium hydroxide solution for 15 min, and then rinse with purified water until the pH test paper shows neutral. Start the feed pump to pump in the diatomaceous earth suspension (the precoating amount is 0.5 kg / m 2 ) and precoat diatomaceous earth on the plate and frame. After the precoating is completed, connect the liquid inlet to the storage tank and pump in the flocculated liquid for circulating filtration through the plate and frame filter press, and control the working pressure < 0.2 MPa. Place the outlet of the liquid collection pipe into the feed tank for circulating filtration. After the liquid at the outlet is clarified (turbidity ≤ 50 NTU), start collecting the filtrate. After completion, rinse with 1 - 2 times the dead volume of purified water.

[0058] 2. Activated carbon adsorption

[0059] 1) Adsorption: Add injection-grade activated carbon at 0.5% of the weight of the fermentation broth, adjust the pH to 4 - 6 with 0.1M HCl, and stir for adsorption for 1 - 2 h. After the adsorption is completed, premix diatomaceous earth (the dosage of diatomaceous earth is 0.1 - 5% (w / v) of the fermentation broth).

[0060] 2) Plate and frame filtration: According to the sop of the plate and frame filter press operation, install the filter cloth and tighten the filter plate. Start the feed pump to pump in the diatomaceous earth suspension (the precoating amount is 0.5 kg / m 2 ), and precoat diatomaceous earth in the plate and frame. After the precoating is completed, connect the inlet to the storage tank and pump in the flocculant for circulating filtration by the plate and frame filter press, controlling the working pressure < 0.2 MPa. Connect the inlet to the filtrate collection tank for circulating filtration. When the liquid at the outlet is clear (turbidity ≤ 30 NTU), start collecting the clarified liquid.

[0061] After completion, rinse with 1 - 2 times the dead volume of purified water.

[0062] 3. Fine filtration

[0063] Adjust the pH of the clarified liquid to 7.0 ± 0.2 with solid sodium carbonate, and then fine-filter the clarified liquid with a 20-inch ultra-high-precision PP filter element in series with a PES filter element. Slowly increase the feed flow rate so that the filtration pressure ≤ 0.1 MPa. If the pressure is too high, backflush and empty the feed liquid, replace the filter and filter again. When continuous liquid flows out of the exhaust port, close the exhaust valve and collect the filtered liquid.

[0064] 4. Ceramic membrane concentration

[0065] 1) Cleaning: Connect the ceramic membrane ultrafiltration system, and circulate and rinse the ceramic membrane system with 2% citric acid, purified water, and 0.5M NaOH solution in sequence for 15 - 30 min, and then rinse with purified water until the pH test paper at the permeate end is neutral.

[0066] 2) Concentration: Use a pump to pump the liquid after fine filtration into the ceramic membrane circulating storage tank, connect the outlet of the reflux end to the circulating storage tank, and then open the bottom valve of the tank. Completely open the reflux valve and one end permeate valve. Start the system, control the frequency at 10 - 80 Hz, so as to control the feed flow rate at 100 - 1500 L / h. Control the inlet pressure < 0.1 MPa by setting the alarm pressure at 0.1 MPa (if overpressure, reduce the feed flow rate). Stop the concentration when the volume of the fermentation broth is concentrated to 1 / 2 or the inlet pressure ≥ 0.1 MPa. Close the permeate end valve, circulate and rinse the ceramic membrane system with 1 - 5 times the dead volume of purified water for 5 - 30 min, empty and collect the liquid at the reflux end again, and repeat the above rinsing and collection operations 2 times. Combine the collected concentrated liquid and rinsing liquid.

[0067] 5. Alcohol precipitation

[0068] 1) Sodium salt treatment: Add a 10 - 40% sodium chloride solution (final concentration of sodium chloride is 1 - 5%) to the concentrated solution and the rinsing solution, and stir evenly. Then, perform fine filtration using a 20 - inch ultra - high - precision PP filter element in series with a PES filter element. Slowly increase the feed flow rate so that the filtration pressure ≤ 0.1 MPa. If the pressure is too high, back - flush and empty the feed liquid, replace the filter and filter again, and collect the filtrate.

[0069] 2) Ethanol precipitation: Circulatingly wash the ethanol precipitation tank with citric acid, purified water, and NaOH solution for 15 - 30 min in sequence, and then rinse with purified water until the pH test paper shows neutral. Pump the filtrate from the previous step into the ethanol precipitation tank, start stirring, and slowly add 95% ethanol with a volume twice that of the feed liquid. Stop stirring after a large amount of white flocculent precipitate is generated. Let the precipitate stand for 2 - 4 h, pump out the supernatant, and collect the precipitate.

[0070] 6. Washing and dehydration

[0071] 1) Washing: Add 1 - 10 times the volume of 70% ethanol (containing 1% sodium chloride) to the CA sugar precipitate, stir and wash for 20 min, then let it stand for 1 - 8 h, pump out the supernatant, and collect the precipitate. Then repeat the washing 2 more times and collect the precipitate.

[0072] 2) Dehydration: Add 1 - 10 times the volume of 95% ethanol to the precipitate, stir for dehydration for 2 h, let it stand for 1 - 2 h, and then pump out the supernatant. Repeat the above dehydration operation 1 more time and collect the precipitate. After the precipitate is vacuum - filtered to reduce the residual liquid, add 1 - 10 times the volume of 95% ethanol for dehydration again. Collect the precipitate, filter again to remove the liquid, and weigh the wet weight of the sample.

[0073] 7. Drying

[0074] Put the sample with wet weight into a vacuum drying oven (the thickness of the sample does not exceed 2 cm), set the drying temperature to 45 °C, and the drying time to 15 h. Turn the material once every 4 h.

[0075] 8. Crushing and sieving

[0076] After the secondary drying is completed, crush the sample again using a crusher, sieve it through a 100 - mesh sieve, and pack it into aluminum foil bags.

[0077] 9. Enzymatic method to obtain sodium caffeate with different molecular weights

[0078] The obtained clavulanic acid product was mixed at 37 °C for 0 - 12 h by adding clavulanic acid degrading enzymes (produced by Baiyin Biology, see SEQ ID No.2 in CN116334039B) at different concentrations (1000 U / L, 2000 U / L, 3000 U / L). Samples were collected and heated to inactivate the enzymes, and then separated and purified using a DEAE ion column to remove a small amount of parts with non-uniform molecular weights and impurities, obtaining clavulanic acid sodium products with different molecular weights of 10 - 6000 kDa. The molecular weights of clavulanic acid sodium were in the ranges of 10 - 50 kDa, 300 - 500 kDa, 600 - 800 kDa, 1000 - 2000 kDa, and 3000 - 5000 kDa respectively. Subsequently, the final clavulanic acid product could be obtained by freeze-drying or spray-drying.

[0079] Experimental method

[0080] (1) Cytotoxicity

[0081] Cell culture: HaCaT cells were cultured in complete DMEM medium (DMEM + 10% FBS + 1% P / S) and placed statically in an incubator at 37 °C and 5% CO2 for culture.

[0082] Sample treatment: CA powder was dissolved in the medium, and after complete dissolution, it was filtered and sterilized with a 0.2 μm sterile filter membrane.

[0083] Experimental treatment: HaCaT cells were seeded into 96-well plates, and sample groups, blank controls, and positive control groups were set. There were at least 3 replicates for each sample for each detection index. After culturing for 24 h, the culture medium was discarded. The corresponding CA solution was added to the sample groups, 10% DMSO solution was added to the positive control group, and complete medium was added to the blank control group. Culturing was continued for 24 h.

[0084] Cell viability detection: The supernatant was discarded, and complete medium containing 10% AlamarBlue was added and incubated in the incubator for 4 h. Then, the absorbance at ex / em = 560 / 590 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0085] (2) Anti-wrinkle (promote collagen)

[0086] Cell culture: HDF cells were cultured in complete DMEM medium (DMEM + 10% FBS + 1% P / S) and placed statically in an incubator at 37 °C and 5% CO2 for culture.

[0087] Sample treatment: CA powder was dissolved in the medium, and after complete dissolution, it was filtered and sterilized with a 0.2 μm sterile filter membrane.

[0088] Experimental treatment: HDF cells were seeded into 96-well plates, and sample groups, blank controls, and positive control groups were set up. For each detection index, each sample had at least 3 replicates. After culturing for 24 h, the culture medium was discarded. In the sample groups, the corresponding CA solution was added; in the positive control group, TGF-β solution was added; and in the negative control group and the blank group, complete medium was added. Culturing was continued for 24 h.

[0089] Detection of type I and III collagens: The supernatants of each well were collected, and the test was performed according to the operation instructions of the ELISA collagen detection kit (Huamei Bio, CSB-E04799h).

[0090] (3) Inflammation (macrophages)

[0091] Cell culture: RAW264.7 cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S), and were placed statically in an incubator at 37 °C and 5% CO2 for culture.

[0092] Sample treatment: CA powder was dissolved in the medium, and after complete dissolution, it was filtered and sterilized with a 0.2 μm sterile filter membrane.

[0093] Experimental treatment: HDF cells were seeded into 96-well plates, and sample groups, blank controls, positive control groups, and negative control groups were set up. For each detection index, each sample had at least 3 replicates. After culturing for 24 h, the culture medium was discarded. In the sample groups, the corresponding CA solution was added; in the positive control group, complete medium containing 100 μg / mL dexamethasone was added; and in the negative control group and the blank group, complete medium was added. After incubation in the incubator for 2 h, 1 μg / mL LPS (lipopolysaccharide) was added to the sample groups, positive control groups, and negative control groups. Culturing was continued for 24 h.

[0094] Detection of inflammatory factors: The supernatants of each well were collected, and the test was performed according to the operation instructions of the ELISA kits (Huamei Bio, CSB-E04740h, CSB-E08053h, CSB-E04638h).

[0095] Detection of reactive oxygen species (ROS): After collecting the supernatants, the cells in each well were washed twice with PBS, and the test was performed according to the operation instructions of the DCFH-DA kit.

[0096] (4) Inflammation (keratinocytes)

[0097] Cell culture: Keratinocytes HaCaT were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S), and were placed statically in an incubator at 37 °C and 5% CO2 for culture.

[0098] Sample treatment: CA powder was dissolved in the medium, and after complete dissolution, it was filtered and sterilized with a 0.2 μm sterile filter membrane.

[0099] Experimental treatment: Cells were seeded into 96-well plates, and sample groups, blank control groups, positive control groups, and negative control groups were set up. For each detection index, there were at least 3 replicates for each sample. After culturing for 24 h, the culture medium was discarded. In the sample groups, the corresponding CA sample solutions were added; in the positive control groups, the corresponding VC solutions (dissolved in the culture medium) were added; and in the negative control groups and blank control groups, complete culture medium was added. After incubation in an incubator for 2 h, hydrogen peroxide solution (0.8 mM HX0640 Sigma-Aldrich) was added to the sample groups, positive control groups, and negative control groups for inflammatory stimulation. Culturing was continued for 24 h.

[0100] Gene detection: The old liquid was aspirated and discarded, and the cells were washed twice with PBS. Cell lysate was added to each well, and after the cells were lysed by pipetting, the samples were collected. RNA was extracted, reverse-transcribed into cDNA, and then fluorescence quantitative PCR was performed for detection. The 2-ΔΔCT method was used for result calculation. Through the calculated 2-ΔΔCt values, the expression differences of the target genes in the two groups of samples were obtained.

[0101] Inflammatory factor detection: The supernatant of each well was collected, and the experiment was carried out according to the operation instructions of the ELISA kit (Huamei Bio, CSB-E04740h, CSB-E04638h, CSB-E10103h).

[0102] (5) Photoinjury

[0103] Cell culture and sample treatment were the same as in (4).

[0104] Experimental treatment: HaCaT cells were seeded into 96-well plates, and sample groups, blank control groups, positive control groups, and negative control groups were set up. For each detection index, there were at least 3 replicates for each sample. After culturing for 24 h, the culture medium was discarded. In the sample groups, the corresponding CA solutions were added; in the positive control groups, VC solutions (dissolved in the culture medium) were added; and in the negative control groups and blank control groups, complete culture medium was added. After incubation in an incubator for 2 h, the sample groups, positive control groups, and negative control groups were irradiated with ultraviolet light at 320 - 400 nm with a power of 40 mW for 4 min. After irradiation, culturing was continued for 24 h.

[0105] Cell viability detection: The supernatant was discarded, and complete culture medium containing 10% AlamarBlue was added. After incubation in an incubator for 4 h, the absorbance at ex / em = 560 / 590 nm was detected with an enzyme-labeled instrument.

[0106] Type I collagen detection: The supernatant of each well was collected, and the experiment was carried out according to the operation instructions of the ELISA collagen detection kit (Huamei Bio, CSB-E08082h).

[0107] Detection of DNA damage markers: Huamei Biotech, CSB-PA833019. NO detection kit: Beyotime, S0021S. ROS detection kit: Solarbio, CA1410. iNOS detection was quantified by ELISA (Huamei Biotech, CSB-E08148h).

[0108] (6) 3D skin model

[0109] The model used in this test was a 3D epidermal skin model Lot number: ES230408, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0110] Transfer the 3D skin model to a 6-well plate (add 0.9 mL of EpiGrowth culture medium in advance), and label the test group numbers on the 6-well plate.

[0111] The BC group was not treated with anything. The PC group was added with the corresponding concentration of working solution under the liquid. The sample group evenly distributed the sample working solution on the surface of the model and incubated it in a CO2 incubator (37 °C, 5% CO2) for 24 h. After incubation, the remaining test substances on the surface of the model were washed with sterile PBS solution, and the remaining liquid inside and outside the model was wiped off with a sterile cotton swab.

[0112] HPLC test

[0113] Cut the model into a 1.5 mL centrifuge tube, add 500 μL of 0.2 mg / mL proteinase K to each tube, and place it in a 50 °C water bath for 2 h. After the stratum corneum falls off, add 500 μL of methanol to each tube, sonicate for 30 min; then centrifuge at 14000 rpm for 10 min. Dry the methanol with nitrogen at 60 °C, re-dissolve it with 500 μL of deionized water, filter it through a 0.22 μm filter membrane, and perform HPLC determination.

[0114] Immunofluorescence test

[0115] Cut and remove the model for detection, fix it with 4% paraformaldehyde. After 24 h of fixation, perform immunofluorescence detection (FLG and AQP3 antibodies were purchased from Abcam), take pictures and observe under a microscope, and collect and analyze the pictures.

[0116] (7) Mitochondrial morphology

[0117] Preparation of JC-10 staining working solution:

[0118] The amount of JC-10 staining working solution required for each well of the six-well plate is 1 mL, and the dosage of JC-10 staining working solution for other culture vessels can be deduced by analogy; for cell suspension, 0.5 mL of JC-10 staining working solution is required for every 500,000 - 1,000,000 cells. Take an appropriate amount of JC-10 (200×), and dilute JC-10 according to the ratio of adding 8 mL of ultrapure water to every 50 μL of JC-10 (200×). Vigorously shake to fully dissolve and mix JC-10. Then add 2 mL of JC-10 staining buffer (5×), and after mixing, it is the JC-10 staining working solution.

[0119] Observation of cell mitochondrial staining:

[0120] (1) For each well of the six-well plate, aspirate the culture medium. If necessary according to the specific experiment, wash the cells once with PBS, and add 1 mL of cell culture medium. The cell culture medium can contain serum and phenol red.

[0121] (2) Add 1 mL of JC-10 staining working solution and mix well. Incubate at 37 °C in a cell culture incubator for 20 min.

[0122] (3) During the incubation period, prepare an appropriate amount of JC-10 staining buffer (1×) according to the ratio of adding 4 mL of distilled water to every 1 mL of JC-10 staining buffer (5×), and place it in an ice bath.

[0123] (4) After the incubation at 37 °C is completed, aspirate the supernatant and wash twice with JC-10 staining buffer (1×).

[0124] (5) Add 2 mL of cell culture medium, and the culture medium can contain serum and phenol red.

[0125] (6) Observe under a fluorescence microscope or a laser confocal microscope.

[0126] (7) Detection with a fluorescence spectrophotometer or a fluorescence microplate reader: After mixing, directly perform a time scan with a fluorescence spectrophotometer, with an excitation wavelength of 485 nm and an emission wavelength of 590 nm.

[0127] (8) CD44 immunoblot

[0128] Cell culture: HaCaT cells are cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S), and are placed statically in an incubator at 37 °C and 5% CO2.

[0129] Sample treatment: CA powder is dissolved in the culture medium, and after complete dissolution, it is filtered and sterilized with a 0.2 μm sterile filter membrane.

[0130] Experimental treatment: HaCaT cells were seeded into 96-well plates, and sample groups, blank control, positive control group, and negative control group were set. For each detection index, there were at least 3 replicate wells for each sample. After culturing for 24 h, the culture medium was discarded, and the corresponding CA solution was added to the sample groups. Then, the cells were incubated in an incubator for 2 h. The cells were taken out and lysed, followed by SDS-PAGE experiment. They were transferred onto PVDF membranes by a transfer apparatus (Thermo), reacted with primary antibody (CD44 antibody, Huamei Biological, CSB-RA292372A0HU) and secondary antibody, and then chromogenic solution was added for observation.

[0131] (9) Cell viability - CCK8 assay

[0132] Seeding plates: After resuscitating and culturing human keratinocytes HaCaT for 1 - 2 days until the cell confluence reached 70%, after trypsin digestion, the keratinocytes were seeded at 5×10 4 / well, and the groups were DMEM, H2O2 - DMEM, and H2O2 - CA.

[0133] H2O2 stimulation: The concentration of H2O2 (Shanghai Lingfeng Chemical Reagent) used was 0.8 mM. First, it was diluted to 10 mM and then added to the culture medium at 100× (the original storage concentration was 8820 mM, 10 μL of H2O2 was added to 8.82 mL of PBS, and then 40 μL of the diluted solution was added to 4 mL of the culture medium). The prepared culture medium containing H2O2 was added to the H2O2 stimulation group in the form of medium replacement, and the cells were cultured for 2 h.

[0134] CA treatment: 50 mg of CA powder sterilized by ultraviolet was pre - added to 5 mL of PBS, shaken and dissolved to prepare a 10 mg / mL solution, and then diluted with DMEM medium to a working concentration of 5 mg / mL. The culture supernatant after H2O2 stimulation was taken out (including the DMEM blank control group). After adding 100 μL of PBS to rinse each sample, 100 μL of the medium containing CA was added to the H2O2 - CA group, and 100 μL of the medium was added to the DMEM and H2O2 - DMEM groups, and the cells were cultured for 24 h.

[0135] CCK8 assay: Calculate the volume of the culture medium required for the assay, and then add CCK8 (Beyotime, C0038) at 10× (i.e., 400 μL of CCK8 was added to 4 mL of the culture medium). After aspirating the culture medium supernatant of each sample, 100 μL / well was added to a 96 - well plate and incubated for 1 - 2 h. Detection was performed at OD450 using an enzyme - linked immunosorbent assay reader in the dark.

[0136] (10) Cell migration

[0137] Cell culture: HaCaT (keratinocytes) were cultured in complete DMEM medium (DMEM + 10% FBS + 1% P / S) and placed in an incubator at 37°C with 5% CO2 for incubation.

[0138] Sample preparation: The samples were prepared with PBS and filtered through a 0.22 μm sterile filter membrane to remove bacteria.

[0139] Experimental treatment: After the cells were plated and cultured for 24 h, a scratch was made, and the samples prepared with serum-free medium were added.

[0140] Photographing: Photographs were taken under a 4-fold microscope at 0 h after the scratch. After 16 h of the scratch, the cells were washed once with PBS and photographed under a 4-fold microscope.

[0141] Data analysis: The ImageJ software was used to calculate the scratch area.

[0142] (11) Transdermal experiment

[0143] Ex vivo skin preparation: The ex vivo skin stored at -20°C was thawed at room temperature with deionized water and repeatedly rinsed with PBS buffer.

[0144] Determination of transdermal absorption ability:

[0145] 1) The ex vivo skin was fixed between the supply chamber and the receiving chamber of the Franz cell diffusion cell, with the stratum corneum side of the skin facing the supply chamber and the dermis side facing the receiving chamber;

[0146] 2) The receiving solution was added to the receiving chamber. After the skin was tightened and fixed, the receiving solution (PBS) was added to the receiving chamber through a sampler, and the air was exhausted to make the dermis of the skin in close contact with the receiving solution;

[0147] 3) Sample addition: The sample was added to the skin surface in the supply chamber (sodium corallate with a molecular weight of 10 - 50 kDa labeled with green fluorescence, at a concentration of 1 mg / mL). The sample was evenly spread from the central part of the skin to the edge;

[0148] 4) Penetration: The electromagnetic stirrer was turned on and stirred at a speed of 300 rpm, and a constant temperature water bath at (32 ± 1)°C was maintained;

[0149] 5) Skin samples were collected at 1 h, 6 h, and 18 h time points. The skin surface was rinsed with PBS, the residual liquid on the surface was wiped dry, and the skin was cut with a blade. Frozen sections were prepared for use.

[0150] Observation and analysis: The skin sections were observed with a fluorescence microscope, photographed and recorded, and the fluorescence intensity was analyzed with Image J.

[0151] (12) Photoaging

[0152] Cultured cells: HDF cells were cultured in complete DMEM medium (DMEM + 10% FBS + 1% P / S) and placed statically in an incubator at 37°C and 5% CO2 for culture.

[0153] Drug administration: According to the experimental grouping, when the cell seeding rate in the 6-well plate reached 40% - 60%, grouping and drug administration were carried out. 2 mL of sample was added to each well, and 3 replicate wells were set in each group. After drug administration, the 6-well plate was placed in the incubator and cultured for another 24 h.

[0154] UVA irradiation: The groups except the blank control (BC) were irradiated with UVA at 30 J / cm 2 and then placed in the incubator for continued culture for 24 h.

[0155] Sample collection: After 24 h of culture, the cell culture supernatant was collected into EP tubes and stored frozen at -80°C.

[0156] ELISA detection: Detection was carried out according to the operation instructions of the ELISA kit. Example

[0157] The following examples are merely illustrative and are not intended to limit the scope or content of the present invention in any way.

[0158] In the following examples, if not otherwise specified, the clavulanic acid used was sodium clavulanate with a molecular weight of 3000 - 5000 kDa.

[0159] Example 1: Photoinjury inhibition

[0160] According to the method of photoinjury in the experimental method, the effect of clavulanic acid on inhibiting reactive oxygen / nitrogen species in ultraviolet-irradiated HaCaT cells was tested, and the results are as Figure 2 shown.

[0161] As can be seen from Figure 2 , clavulanic acid can significantly inhibit the increase in reactive oxygen species in human keratinocytes (HaCaT) caused by ultraviolet irradiation. At the same time, it can also be observed that the expression level of iNOS in HaCaT cells increased significantly after ultraviolet irradiation, while the expression level decreased significantly after adding clavulanic acid. Similarly, the content of NO (reactive nitrogen species) in the cells also decreased significantly.

[0162] Example 2: Inflammation inhibition

[0163] According to the method of inflammation (keratinocytes) in the experimental method, the effect of clavulanic acid on inhibiting inflammation in human keratinocytes was tested, and the results are as Figure 3 shown.

[0164] As can be seen from Figure 3As can be seen, colacic acid significantly inhibits TNF-α, IL-6 and COX-2 in HaCaT cells. At the same time, in the Figure 3 immunoblotting experiment of B, it can also be observed that both IL-1 and p65 in HaCaT cells irradiated with ultraviolet light are significantly up-regulated, while after adding colacic acid, both IL-1 and p65 are significantly down-regulated. It shows that the addition of colacic acid can significantly inhibit the inflammatory response of cells.

[0165] Example 3: Inhibition of photo-damage

[0166] Referring to the method of treating photo-damage in the experimental method, the effect of colacic acid on inhibiting the increase of DNA damage markers caused by ultraviolet irradiation and reducing cell senescence and apoptosis was tested. The results are as Figure 4 shown.

[0167] From Figure 4 it can be seen that colacic acid can effectively inhibit the DNA damage of HaCaT cells caused by ultraviolet irradiation ( Figure 4 A and Figure 4 B). It can be seen that adding colacic acid first and then irradiating with ultraviolet light (UV+CA+) can significantly inhibit the DNA damage marker H2AX. Relatively speaking, irradiating with ultraviolet light first and then adding colacic acid (UV+CA-+) has no obvious effect on this. At the same time, by testing the cell cycle, it can be seen that the cells in the colacic acid-added group are less in the G0 / 1 cycle than the ultraviolet irradiation group, and there is a significant difference, while the cells irradiated with ultraviolet light alone are more in the cell arrest phase ( Figure 4 C), indicating that colacic acid can significantly inhibit cell senescence caused by external environmental stimuli. Similarly, the apoptotic cells also significantly decrease after adding colacic acid ( Figure 4 D).

[0168] Example 4: Mitochondrial protection

[0169] By the method of mitochondrial morphology in the experimental method, the protective effect of colacic acid on cell mitochondria was tested. The results are as Figure 5 shown.

[0170] Through Figure 5 the experimental results of HaCaT cells in A, it is observed that colacic acid has a good protective effect on cell mitochondria. After adding colacic acid, more JC-10 on the mitochondrial membrane presents in the normal aggregated state. Relatively speaking, the mitochondrial JC-10 of cells only irradiated with ultraviolet light changes from the aggregated state to the monomeric form. Similarly, from Figure 5 B and Figure 5 C, it can be seen that colacic acid can significantly reduce mitochondrial fragmentation under oxidative stress conditions. Thus, it can be seen that colacic acid has a good protective effect on mitochondria.

[0171] Example 5: Promoting cell proliferation and migration

[0172] According to the method of CD44 immunoblotting in the experimental method, it was tested that corilagin promotes cell proliferation and migration by upregulating cell CD44, and the results are as Figure 6 shown.

[0173] As can be seen from Figure 6 A, corilagin can significantly upregulate the expression of CD44 in HaCaT cells. As a cell adhesion molecule with broad-spectrum expression, CD44 is involved in biological processes such as cell proliferation, differentiation, migration, and angiogenesis, and plays a key role in mediating cell signal transduction and regulating tissue homeostasis. At the cellular level, Figure 6 B and Figure 6 C's experimental results prove that after adding corilagin, both cell proliferation and migration are significantly upregulated.

[0174] Example 6: Promoting collagen

[0175] According to the method of photoaging in the experimental method, the collagen-promoting activity of corilagin was tested by cell experiments, and the results are as Figure 7 shown. Corilagin also has a significant effect on enhancing the activity of human fibroblasts to synthesize collagen at the cellular level. Under ultraviolet irradiation conditions, the collagen-promoting activity of corilagin is better than that of ectoin. Under normal culture conditions, the collagen-promoting activity of corilagin on human fibroblasts is also very significant.

[0176] Example 7: 3D skin test

[0177] According to the method of 3D skin model in the experimental method, the effect of corilagin on increasing the content of filaggrin (FLG) in 3D skin was tested, and the results are as Figure 8 shown. Figure 8 A is the test result of 3D skin experiment. Compared with the BC blank control group, the content of filaggrin (FLG) significantly increased at a concentration of 0.5% (w / v) of oligomeric corilagin, and the promotion rate was 58.00%. Figure 8 The fluorescence quantitative result of B shows that corilagin has an effect on increasing filaggrin FLG in 3D skin.

[0178] According to the method of 3D skin model in the experimental method, the effect of corilagin on increasing the content of aquaporin 3 (APQ3) in 3D skin was tested, and the results are as Figure 9 shown. Figure 9 A and Figure 9 In B, compared with the BC control group, the content of aquaporin 3 (APQ3) significantly increased at a concentration of 0.5% (w / v) of corilagin, and the promotion rate was 38.00%.

[0179] Example 8: Cytotoxicity

[0180] The cytotoxicity of colanic acid was tested, and the results are as Figure 10 shown. From Figure 10 the test results, it can be seen that when the concentration of colanic acid in the cell culture medium reaches 5 mg / mL or more, it has no obvious effect on cell viability and morphology.

[0181] Example 9: Human experiment

[0182] The human experiment of macromolecular sodium colanate (molecular weight 3000 - 5000 kDa) was carried out according to the following method, and the results are as Figures 11 - 13 shown.

[0183] Subjects: A total of 31 people, 5 males and 26 females, aged 28 to 59 years old, with an average age of 47 ± 8 years old, meeting the volunteer inclusion criteria for subjects.

[0184] Instruments: Cutin hydration measuring instrument (MSC1201, Delfin), transepidermal water loss measuring instrument (SWL5201, Delfin), Antera 3D imager (HC1023Q, Delfin), balance, accuracy 0.1 mg.

[0185] Detection environment: Test environment temperature: 20.0 - 22.0 °C; humidity: 40 - 60%, and real-time dynamic monitoring is carried out.

[0186] Test method: At the first visit, the subjects were given an explanation of the test and signed an informed consent form. Among the subjects participating in the test, those with dry skin or skin flushing were selected, and finally 31 people entered the experimental session. The subjects wiped the test area on the day of the 0-week visit. After balancing for 20 minutes in a constant temperature and humidity environment, the subjects were measured with instruments. After the test was completed, the sample usage instructions were given and the samples were distributed, and the sample weights were confirmed at the start and end of the test. On-site follow-up visits were conducted after the 2nd and 4th weeks of sample use, and instrument measurements were carried out.

[0187] Sample usage instructions: Use once in the morning and once in the evening every day, the usage amount is 0.5 mL, and the concentration is 0.15% (w / v).

[0188] Figure 11 Shows the anti-wrinkle efficacy test results of sodium colanate. From Figure 11 it can be seen that after the subjects continuously used 0.15% macromolecular colanic acid essence for 4 weeks, the length of the eye corner wrinkles decreased by 38.03%, and the number of wrinkles decreased by 35.68%.

[0189] Figure 12 Shows the anti-redness and anti-allergy efficacy test results of sodium colanate. From Figure 12 it can be seen that after the subjects continuously used 0.15% macromolecular colanic acid essence for 4 weeks, the skin heme index decreased by 33.03%.

[0190] Figure 13 shows the water-locking and moisturizing efficacy test of sodium corallinate. From Figure 13 it can be seen that after the subjects continuously used 0.15% macromolecular sodium corallinate essence for 4 weeks, the skin water content increased significantly by 33.35%, and the trans epidermal water loss (TEWL) decreased significantly by 19.84%

[0191] Example 10

[0192] Prepare sodium corallinate with molecular weights of 50 - 5000 kDa, 100 - 5000 kDa, 1000 - 5000 kDa, 1000 - 4000 kDa, 1000 - 3000 kDa, 1000 - 2000 kDa, 2000 - 5000 kDa, 2000 - 4000 kDa, 2000 - 3000 kDa, 3000 - 4000 kDa, 300 - 500 kDa, 600 - 800 kDa, 10 - 1000 kDa, 10 - 100 kDa, 10 - 50 kDa, 10 - 20 kDa, 10 - 15 kDa in the same way as the reference preparation method, and test its properties and efficacy according to the same methods as in Examples 1 - 9.

[0193] Example 11: Comparison of the effects of sodium corallinate with different molecular weights

[0194] Use CCK8 to detect and test the skin protection effect of sodium corallinate (CA) with different molecular weights, and the results are as Figure 14 shown. From Figure 14 it can be seen that sodium corallinate in the molecular weight ranges of 10 - 50 kDa, 1000 - 2000 kDa, and 3000 - 5000 kDa has better skin protection effects.

[0195] According to the anti-wrinkle (promote collagen) method in the experimental method, test the effect of sodium corallinate (CA) with different molecular weights on stimulating the expression of type I collagen in human fibroblasts at a concentration of 3 mg / mL, and the results are as Figure 15 shown. From Figure 15 it can be seen that sodium corallinate in the molecular weight ranges of 10 - 50 kDa, 300 - 500 kDa, and 1000 - 2000 kDa has better collagen-promoting effects.

[0196] According to the cell migration method in the test method, test the effect of sodium corallinate (CA) with different molecular weights on the migration of human fibroblasts, and the results are as Figure 16 shown. From Figure 16 it can be seen that sodium corallinate in the molecular weight ranges of 10 - 50 kDa, 300 - 500 kDa, 600 - 800 kDa, and 1000 - 2000 kDa has better effects on promoting cell migration.

[0197] Example 12: Transdermal Test of Sodium Colate

[0198] Entrust Chutai Biology to carry out green fluorescence labeling on sodium colate (molecular weight 10 - 50 kDa), and then purify to remove free fluorescent molecules. Dissolve it in water to 1 mg / mL and conduct a transdermal experiment test. Figure 17 The results show that sodium colate can effectively penetrate through the skin surface layer and enter the dermis layer. Within 1 - 18 h, the data of skin fluorescence sections show that the fluorescence intensity has increased by nearly 9 times.

[0199] Example 13: Mitochondrial Localization Experiment of Sodium Colate

[0200] Cell culture: HDF cells are cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S), and placed statically in an incubator at 37°C and 5% CO2 for culture.

[0201] Sample treatment: Fluorescently labeled CA powder (molecular weight 10 - 50 kDa) is dissolved in the medium at a concentration of 1 mg / mL, and after complete dissolution, it is filtered and sterilized with a 0.2 μm sterile filter membrane.

[0202] Experimental treatment: Inoculate HDF cells into a 96 - well plate, and set up a sample group and a blank control group (BC). There are at least 3 replicates for each sample for each detection index. After culturing for 24 h, discard the culture medium. Add CA solution to the sample group and culture for 24 h, 48 h, and 72 h respectively. Discard the supernatant, stain the mitochondria with mitotracker, then fix the cells with paraformaldehyde, and then observe the fluorescence with a confocal microscope. Figure 18 The results show that sodium colate represented by green fluorescence and mitochondria represented by red fluorescence overlap spatially. Therefore, it can be considered that sodium colate can enter cells and even play a role inside mitochondria.

[0203] Incorporation by reference

[0204] The entire content of each patent and scientific literature mentioned in this article is incorporated herein by reference for all purposes.

[0205] Equivalence

[0206] The present invention can be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above - mentioned embodiments should be regarded as illustrative in all cases rather than a limitation of the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than the above description, and is intended to cover all changes within the meaning and scope of equivalence of the claims.

Claims

1. Use of clavulanic acid or a physiologically acceptable salt thereof, or a composition comprising clavulanic acid or a physiologically acceptable salt thereof, in the preparation of a medicament or cosmetic for increasing skin water content, characterized in that, The molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 50 kDa or 1000 - 2000 kDa.

2. According to the use described in claim 1, wherein The content of the colanic acid or its physiologically acceptable salt is 0.001 - 2% w / v.

3. According to the use described in claim 1, wherein The content of the colanic acid or its physiologically acceptable salt is 0.01 - 1% w / v.

4. According to the use described in claim 1, wherein The content of the colanic acid or its physiologically acceptable salt is 0.05 - 0.5% w / v.

5. Use of clavulanic acid or a physiologically acceptable salt thereof, or a composition comprising clavulanic acid or a physiologically acceptable salt thereof, in the preparation of a medicament or cosmetic for skin anti-aging, characterized in that, The molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 50 kDa or 1000 - 2000 kDa.

6. The use according to claim 5, wherein The skin anti - aging includes skin anti - wrinkle, skin anti - aging, and increasing the collagen content in the skin.

7. The use according to claim 6, wherein, The skin anti - aging includes increasing the content of type I and / or type III collagen in the skin.

8. The use according to claim 5, wherein, The content of the colanic acid or its physiologically acceptable salt is 0.001 - 10% w / v.

9. The use according to claim 8, wherein The content of the colanic acid or its physiologically acceptable salt is 0.05 - 5% w / v.

10. The use according to claim 9, wherein, The content of the colanic acid or its physiologically acceptable salt is 0.1 - 3% w / v.

11. Use of clavulanic acid or a physiologically acceptable salt thereof, or a composition comprising clavulanic acid or a physiologically acceptable salt thereof, in the preparation of a medicament or cosmetic for improving telangiectasia or skin inflammation caused by skin allergy, characterized in that, The molecular weight of the colanic acid or its physiologically acceptable salt is in the range of 10 - 50 kDa or 1000 - 2000 kDa.

12. The use according to claim 11, wherein, The content of the colanic acid or its physiologically acceptable salt is 0.001 - 10% w / v.

13. According to the use according to claim 12, wherein, The content of the colanic acid or its physiologically acceptable salt is 0.05 - 5% w / v.

14. The use according to claim 13, wherein, The content of the colanic acid or its physiologically acceptable salt is 0.1 - 3% w / v.

15. The use according to any one of claims 1-14, wherein, The composition contains only the colanic acid or its physiologically acceptable salt as the active ingredient.

16. The use according to any one of claims 1 to 14, wherein, When the composition is a drug, it is formulated for topical, oral, intramuscular, subcutaneous or intravenous administration.

17. The use according to claim 16, wherein, When the composition is a drug, it is formulated for subcutaneous administration.

18. Use according to any one of claims 1-14, wherein, When the composition is a cosmetic, it is formulated as an emulsion, paste, facial mask, gel, powder, foundation, lotion, patch, beauty liquid, cleansing foam, cleansing water, soap, spray; or When the composition is a drug, it is formulated as lozenges, capsules, emulsions, suspensions, solutions, syrups, granules, transdermal patches, gels, powders, pastes, suppositories or sprays.

19. Use according to any one of claims 1 - 14, wherein, The composition also contains cosmetic or pharmaceutically acceptable additives.

20. The use according to any one of claims 1-14, wherein, The physiologically acceptable salt of the colanic acid is sodium colanate.

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