Composition comprising hydrolyzed colanic acid salt and use thereof in skin Anti-aging and skin protection

Abstract

A composition comprising a colanic acid salt and use thereof in skin anti-aging and skin protection. The colanic acid salt has good skin anti-aging and skin protection effects.

Description

Compositions containing hydrolyzed colanate and their use in skin anti-aging and skin protection Technical Field

[0001] The invention belongs to the fields of skin care products and medicine, and particularly relates to a composition containing hydrolyzed colanate and use thereof in skin anti-aging and skin protection. 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 synthesized during bacterial life to adapt to environmental changes and improve survival. CA has a large molecular weight and is loosely coated on the bacterial surface, causing the bacteria to exhibit a mucus-like state, preventing cell dehydration, protecting cells, and defending against harmful substances. Under environmental conditions unfavorable for growth, such as dryness, low pressure, and low pH, mucoid strains exhibit greater survival than wild-type strains. In 2017, Han et al. reported that feeding purified CA or CA-secreting Escherichia coli significantly extended the lifespan of Caenorhabditis elegans. Furthermore, as a unique active biopolymer, CA possesses unique biological properties and physiological parameters, offering broad application prospects.

[0003] Summary of the Invention

[0004] In one aspect, the present disclosure provides use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, for skin anti-aging and / or skin protection, wherein the colanic acid or a physiologically acceptable salt thereof has a molecular weight in the range of 1-10 kDa.

[0005] In some embodiments, the skin anti-aging comprises increasing the collagen content in the skin, preferably the content of type I and type III collagen.

[0006] In some embodiments, the skin protection comprises enhancing the protective function of skin cells such as increasing the viability of skin cells.

[0007] In some embodiments, the skin anti-aging and the skin protection include improving skin condition or appearance.

[0008] In some embodiments, the content of colanic acid or a physiologically acceptable salt thereof is 0.01-10% (w / v), 0.1-5% (w / v), or 0.5-3% (w / v).

[0009] In some embodiments, the physiologically acceptable salt of colanic acid is sodium colanate.

[0010] In some embodiments, the colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, is formulated for topical, oral, intramuscular, subcutaneous, or intravenous administration.

[0011] In some embodiments, the composition comprising colanic acid or a physiologically acceptable salt thereof further comprises a cosmetically or pharmaceutically acceptable additive.

[0012] In another aspect, the present disclosure provides 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 medicament or cosmetic for skin anti-aging and / or skin protection.

[0013] In another aspect, the present disclosure provides a composition comprising colanic acid or a physiologically acceptable salt thereof as an active ingredient, wherein the molecular weight of the colanic acid or the physiologically acceptable salt thereof is in the range of 1-10 kDa.

[0014] In some embodiments, the molecular weight of the colanic acid or a physiologically acceptable salt thereof is in the range of 1-3 kDa or 3-10 kDa.

[0015] In some embodiments, the content of colanic acid or a physiologically acceptable salt thereof in the composition is 0.01-10% (w / v), 0.1-5% (w / v), or 0.5-3% (w / v).

[0016] In some embodiments, the composition comprises only colanic acid or a physiologically acceptable salt thereof as an active ingredient.

[0017] In some embodiments, the composition is formulated for topical, oral, intramuscular, subcutaneous, or intravenous administration.

[0018] In some embodiments, the composition is formulated for topical administration.

[0019] In some embodiments, the composition further contains cosmetically or pharmaceutically acceptable additives.

[0020] In some embodiments, the physiologically acceptable salt of colanic acid is sodium colanate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention can be more fully understood with reference to the following drawings.

[0022] FIG1 shows the results of transcriptome analysis of fibroblasts by large and small molecular weight sodium colanate.

[0023] FIG2 shows the multi-dimensional anti-aging efficacy mechanism of colanic acid.

[0024] FIG3 shows the results of transcriptome analysis of human fibroblasts by oligocorticosteroid sodium.

[0025] FIG4 shows the results of transcriptome analysis of human fibroblasts by oligocorticosteroid sodium (A) and the results of cell viability test after UVA irradiation (B).

[0026] FIG5 shows the inhibitory effect of sodium oligocorticate on macrophage TNF-α and IL-6.

[0027] FIG6 shows the results of transcriptional regulation of sodium oligocorticate on genes related to mitochondrial homeostasis and repair in human fibroblasts.

[0028] FIG7 shows the transcription analysis of oligocorticosteroid sodium on SOD2 and GPX1 / 8 in HDF cells (A) and the scavenging effect on reactive oxygen species (B).

[0029] FIG8 shows data showing that sodium oligocorticate promotes the synthesis of type I collagen in HDF cells and upregulates the transcription of different types of collagen in HDF cells.

[0030] FIG9 shows the phototoxicity test results of oligomeric sodium colanate.

[0031] FIG10 shows the test results of the activity of oligomeric colanic acid in promoting the synthesis of type I collagen and type III collagen by HDF cells under light conditions.

[0032] FIG11 shows the inhibitory effect of sodium oligocorticate on MMP9 in a UV-irradiated skin model.

[0033] FIG12 shows the anti-inflammatory effect of sodium oligocorticate on human keratinocytes.

[0034] FIG13 shows the results of the skin permeability test of oligomeric sodium colanate.

[0035] FIG. 14 shows the results of a localization test of oligomeric sodium colanate in cells.

[0036] FIG15 shows the results of a cytotoxicity test of oligomeric sodium colanate.

[0037] FIG16 shows the effect of oligomeric sodium colanate on increasing the density of the human dermis.

[0038] FIG17 shows the effect of oligomeric sodium colanate on increasing the expression of collagen in the human dermis.

[0039] FIG18 shows the effect of oligomeric sodium colanate on increasing the expression of elastin in the human dermis.

[0040] FIG19 shows the effect of oligomeric sodium colanate on improving the elasticity and fineness of human skin.

[0041] FIG20 shows the effect of sodium oligocorticate on the enhancement of the normalized area of ​​DEJ in human skin.

[0042] FIG21 shows the effect of oligomeric sodium colanate on increasing the thickness of the epidermis of human skin.

[0043] FIG22 shows the effect of sodium oligocorticate on improving redness of human skin.

[0044] FIG. 23 shows the effect of oligomeric sodium colanate on increasing the content of filaggrin (FLG) in 3D skin.

[0045] FIG. 24 shows the effect of oligomeric sodium colanate on increasing the content of aquaporin 3 (AQP3) in 3D skin.

[0046] FIG25 shows the effect of oligomeric sodium colanate on locking moisture and moisturizing human skin.

[0047] FIG. 26 shows the effect of sodium oligocorticate on stimulating the expression of type I collagen in human fibroblasts.

[0048] FIG27 shows the skin protective effect of sodium oligocorticate.

[0049] 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.

[0050] Detailed Description of the Invention

[0051] Various features and aspects of the present invention are discussed in greater detail below.

[0052] Figure 1 shows transcriptome analysis results for small-molecule colanate (molecular weight 1-3 kDa, hereinafter referred to as hydrolyzed or oligomeric colanate, abbreviated as oligomeric CA or Oli-CA) and large-molecule colanate (molecular weight 10-6000 kDa). Specifically, each was added to human fibroblasts (HDF) cultured for 24 hours, then irradiated with UV light and cultured for another 24 hours. The cells were then removed for transcriptome analysis. Based on the test results, statistical analysis of up- or down-regulation of different genes was performed. The results showed that colanate affected 2,500 genes in the cells. In contrast, the control group, cucurbitacin, only affected 426 genes. These results demonstrate that colanate of varying molecular weights has a significant impact on the fibroblast transcriptome.

[0053] Based on transcriptome analysis results and combined with KEGG longevity-related pathways (https: / / www.kegg.jp / pathway / map04211), different anti-aging pathways of colanate were identified, as shown in Figure 2. In Figure 2, gray areas indicate transcriptome mRNA suppression, green areas indicate transcriptome mRNA upregulation, pointed arrows indicate enhanced expression, and blunt arrows indicate suppressed expression. Figure 2 shows that in pathway 1, colanate can promote the transcriptome of NAMPT, a key enzyme in intracellular NAD+ synthesis. Colanate can also upregulate genes such as AMPK and SIRT1, and further inhibit BAX and NF-κ, thereby inhibiting apoptosis and cellular inflammation. It can also upregulate PGC-1α, promoting mitochondrial activity.

[0054] In pathway 2, colanate can inhibit the Rheb and mTOR genes, thereby upregulating the transcription of ATG13 and RB1CC1 genes and promoting cellular autophagy.

[0055] In pathway 3, colanate can upregulate the expression of genes such as AMBRA1, USP8, and ATF4 to promote mitophagy, promote mitochondrial fission by upregulating HIF1 and promoting the transcription of BNIP3 and BCL2L13, and promote the mitochondrial unfolded protein response by promoting the transcription of ATF4 / ATF5 and CHOP. These three aspects synergistically promote mitochondrial homeostasis remodeling.

[0056] In pathway 4, colanate increases the cell's ability to scavenge ROS by upregulating the transcription of SOD2, GPX1, and GPX8. Furthermore, upregulated GPX8 expression not only scavenges reactive oxygen species but also regulates calcium transfer from the endoplasmic reticulum to mitochondria, alleviating mitochondrial damage caused by regulated intracellular calcium loss.

[0057] In pathway 5, colanate promotes the synthesis of extracellular matrix by upregulating the transcriptome expressing collagen and various extracellular matrices, thereby enhancing intercellular communication and providing better support and protection for cell morphology.

[0058] In the present disclosure, colanic acid (CA) or a colanic acid salt can be prepared by any method as long as the desired molecular weight is obtained. Specifically, the molecular weight of colanic acid or a physiologically acceptable salt thereof can be 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa, or any range thereof. A molecular weight of 1-10 kDa, 1-8 kDa, 1-6 kDa, 1-5 kDa, 1-4 kDa, 1-3 kDa, 1-2 kDa, 2-10 kDa, 2-8 kDa, 2-6 kDa, 2-4 kDa, 2-3 kDa, 3-6 kDa, 4-10 kDa, 4-8 kDa, 4-6 kDa, 5-10 kDa, 5-8 kDa, 6-10 kDa, or 6-8 kDa can further enhance the efficacy of colanic acid or a physiologically acceptable salt thereof.

[0059] The inventors of the present invention unexpectedly discovered that by selecting colanic acid or a physiologically acceptable salt thereof having the above-specified molecular weight, its efficacy in skin anti-aging and skin protection (e.g., improving skin condition or appearance, such as skin anti-aging, especially increasing collagen content in the skin) can be further enhanced.

[0060] For example, colanic acid or a physiologically acceptable salt thereof can be prepared using the method of CN115287314B. Furthermore, colanic acid or a physiologically acceptable salt thereof can also be purified using the method of CN114957509A. Those skilled in the art will appreciate that colanic acid or colanate salts of varying molecular weights can be obtained using conventional preparation methods.

[0061] The term "physiologically acceptable salt" refers to any salt derived from colanic 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 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, such as ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, butylamine, tetrabutylamine, pentylamine, and hexylamine; alkanolamine salts, such as ethanolamine, diethanolamine, triethanolamine, propanolamine, dipropanolamine, isopropanolamine, and diisopropanolamine; salts of other organic amines, such as piperazine and piperidine; and salts of basic amino acids, such as lysine, arginine, histidine, and tryptophan. In the present disclosure, the physiologically acceptable salt of colanic acid may be sodium colanate.

[0062] 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 as needed. For example, the dosage form of the cosmetic composition can include an emulsion, cream, lotion, essence, facial mask, gel, powder, lipstick, cosmetic base, foundation, lotion, ointment, patch, beauty serum, decontamination foam, decontamination cream, clean water, soap or spray. The dosage form 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.

[0063] In the composition of the present invention, the content of colanic acid or a physiologically acceptable salt thereof can be selected as needed, for example, 0.01-10% (w / v), 0.1-5% (w / v), or 0.5-3% (w / v).

[0064] In addition, as needed, the pharmaceutical or cosmetic composition of the present invention may contain, in addition to the necessary active ingredients, additives commonly added to pharmaceutical or cosmetic compositions. Examples of such additives include oily ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, antibacterial agents, antioxidants, plant extracts, pH regulators, alcohols, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, and water.

[0065] In some embodiments, the composition of the present invention comprises only colanic acid or a physiologically acceptable salt thereof as an active ingredient. In some embodiments, the composition of the present invention further comprises other active ingredients, such as bosaicin, retinol and its derivatives (such as retinyl propionate), retinal, retinoic acid, hyaluronic acid, ergothioneine, ectoine, etc.

[0066] In the present disclosure, the molecular weight of colanic acid or its physiologically acceptable salt is determined by liquid chromatography. The specific method is as follows: accurately weigh the sample and standard, prepare the sample into a 2 mg / mL solution, fully dissolve it, filter it through a 0.22 μm syringe filter, and place it in a 1.8 mL injection vial. Chromatographic columns: PolySep-GFC-P (35 x 7.8 mm); PolySep-GFC-P 4000 (300 x 7.8 mm); PolySep-GFC-P 6000 (300 x 7.8 mm); mobile phase: 0.02 M NaCl solution; flow rate: 0.6 mL / min, column temperature: 40°C; injection volume: 20 μL; detector: 1260-RID differential detector.

[0067] Preparation method

[0068] In the following examples, oligomeric sodium colanate (1-10 kDa) was prepared by the following method.

[0069] The obtained colanic acid product was added with colanic acid-degrading enzyme (produced by Baiyin Biotechnology, see SEQ ID No. 2 in CN116334039B) at different concentrations (2000 U / L, 3000 U / L, and 5000 U / L) and mixed at 37°C for 10-12 hours. The sample was collected and heated to inactivate the enzyme. The product was then separated and purified using a DEAE ion column to remove a small amount of molecular weight heterogeneity and impurities, yielding sodium colanate products of varying molecular weights. The final product was then freeze-dried or spray-dried. The molecular weight of the obtained sodium colanate is 1-10 kDa, 1-8 kDa, 1-6 kDa, 1-5 kDa, 1-4 kDa, 1-3 kDa, 1-2 kDa, 2-10 kDa, 2-8 kDa, 2-6 kDa, 2-4 kDa, 2-3 kDa, 3-6 kDa, 4-10 kDa, 4-8 kDa, 4-6 kDa, 5-10 kDa, 5-8 kDa, 6-10 kDa, or 6-8 kDa.

[0070] The colanic acid product, macromolecular sodium colanate (3000-6000 kDa), was prepared by the following method.

[0071] 1. Solid-liquid separation

[0072] 1) Dilution: Pump the fermentation broth into a storage tank, dilute it 3-20 times with 10-60% calcium chloride solution (the amount of calcium chloride is 2% (w / v) of the fermentation broth) and purified water, and stir for 2-3 hours until it is fully dissolved.

[0073] 2) Flocculation: Sodium carbonate solid was added to adjust the pH to 9.0-12.0, and flocculation was carried out for 1-2 hours before premixing with diatomaceous earth (the amount of diatomaceous earth was 1% (w / v) of the fermentation broth).

[0074] 3) Plate and frame filtration: Follow the plate and frame filter press operating instructions, install the filter cloth, and press the filter plate. Use 0.5M sodium hydroxide solution to circulate the plate and frame filter press system for 15 minutes, and then rinse with purified water until the pH test paper is neutral. Start the feed pump to pump in the diatomaceous earth suspension (pre-coating amount is 0.5kg / m 2 ) to pre-coat the plates and frames with diatomaceous earth. After pre-coating, connect the liquid inlet to the liquid storage tank and pump in the flocculation liquid for circulation filtration in the plate and frame filter, maintaining the operating pressure at <0.2 MPa. Place the outlet of the liquid collection pipe into the feed tank for circulation filtration. Once the liquid at the outlet is clear (turbidity ≤50 NTU), begin collecting the filtrate. After completion, top-wash with 1-2 times the dead volume of purified water.

[0075] 2. Activated carbon adsorption

[0076] 1) Adsorption: Add 0.5% injection-grade activated carbon to the fermentation broth by weight, adjust the pH to 4-6 with 0.1M HCl, and stir for 1-2 hours. After adsorption, pre-mix with diatomaceous earth (0.1-5% (w / v) of the fermentation broth).

[0077] 2) Plate and frame filtration: According to the plate and frame filter press operation SOP, install the filter cloth and press the filter plate. Start the feed pump to pump in the diatomaceous earth suspension (pre-coating amount is 0.5kg / m 2 ) to pre-coat the plates and frames with diatomaceous earth. After pre-coating, connect the liquid inlet to the liquid storage tank and pump in the flocculation liquid for circulation filtration in the plate and frame filter, controlling the operating pressure to <0.2MPa. Connect the liquid inlet to the filtrate collection tank for circulation filtration. Once the liquid at the discharge port is clear (turbidity ≤30NTU), begin collecting the filtrate.

[0078] After the end, top wash with 1-2 times the dead volume of purified water.

[0079] 3. Fine filtration

[0080] Adjust the pH of the filtrate to 7.0 ± 0.2 with solid sodium carbonate. Then, fine filter the filtrate using a 20-inch ultra-high-precision PP filter element connected in series with a PES filter element. Slowly increase the feed flow rate to maintain a filtration pressure of ≤ 0.1 MPa. If the pressure is too high, backflush the feed, replace the filter, and filter again. When liquid continues to flow out of the exhaust port, close the exhaust valve and collect the filtrate.

[0081] 4. Ceramic membrane concentration

[0082] 1) Cleaning: Connect the ceramic membrane ultrafiltration system and flush the ceramic membrane system with 2% citric acid, purified water, and 0.5M NaOH solution in a cycle for 15-30 minutes, then flush with purified water until the pH test paper at the permeate end is neutral.

[0083] 2) Concentration: Use a pump to pump the finely filtered liquid into the ceramic membrane circulation tank, connect the reflux outlet to the circulation tank, and then open the tank bottom valve. Fully open the reflux valve and the permeate valve at one end. Start the system and control the frequency to 10-80Hz to control the feed flow rate at 100-1500L / h. By setting the alarm pressure to 0.1MPa, control the inlet pressure to <0.1MPa (if overpressure, reduce the feed flow rate). Stop the concentration when the circulation concentration reaches 1 / 2 of the fermentation liquid volume or the inlet pressure is ≥0.1MPa. Close the permeate valve and circulate the ceramic membrane system with purified water 1-5 times the dead volume of the system for 5-30 minutes. Empty and collect the reflux liquid again, and repeat the above flushing and collection operations twice. Combine the collected concentrate and flushing liquid.

[0084] 5. Alcohol precipitation

[0085] 1) Sodium Salt Treatment: Add 10-40% sodium chloride solution (final sodium chloride concentration 1-5%) to the concentrate and rinse solution and stir thoroughly. 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 to maintain a filtration pressure ≤ 0.1 MPa. If the pressure is too high, backflush the feed, replace the filter, filter again, and collect the filtrate.

[0086] 2) Ethanol Precipitation: Clean the alcohol precipitation tank with citric acid, purified water, and NaOH solution in a cycle for 15-30 minutes. Then rinse with purified water until the pH paper reads neutral. Pump the filtrate from the previous step into the alcohol precipitation tank. Start stirring and slowly add 95% ethanol (2 times the volume of the feed solution). Stir until a large amount of white flocculent precipitate forms, then stop stirring. Allow to settle for 2-4 hours. Drain the supernatant and collect the precipitate.

[0087] 6. Washing and dehydration

[0088] 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 minutes, then let it stand for 1-8 hours, remove the supernatant, and collect the precipitate. Repeat the washing process twice and collect the precipitate.

[0089] 2) Dehydration: Add 1-10 volumes of 95% ethanol to the precipitate and dehydrate with stirring for 2 hours. Allow to stand for 1-2 hours, then remove the supernatant. Repeat the dehydration process once more and collect the precipitate. Vacuum filter the precipitate to reduce residual liquid, then add 1-10 volumes of 95% ethanol again and dehydrate. Collect the precipitate, filter again to remove liquid, and weigh the wet weight of the sample.

[0090] 7. Drying

[0091] Place the wet weight sample in a vacuum drying oven (sample thickness not exceeding 2 cm), set the drying temperature to 45°C, and the drying time to 15 hours. Turn the sample over every 4 hours.

[0092] 8. Crushing and screening

[0093] After the secondary drying, the sample was crushed again with a pulverizer, passed through a 100-mesh sieve, and packaged into aluminum foil bags.

[0094] Experimental methods

[0095] (1) Cytotoxicity

[0096] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) in a 37°C, 5% CO2 incubator.

[0097] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0098] Experimental treatment: HDF cells were seeded into 96-well plates and divided into sample groups, blank controls (BC), and positive controls (PC). At least three replicates were performed for each sample and each indicator. After 24 hours of incubation, the culture medium was discarded, and the corresponding CA solution was added to the sample group, 10% DMSO solution was added to the positive control group, and complete culture medium was added to the blank control group. Incubation was continued for another 24 hours.

[0099] Cell viability detection: discard the supernatant, add complete medium containing 10% AlamarBlue, incubate in an incubator for 4 hours, and then detect the absorbance at ex / em=560 / 590 nm using a microplate reader.

[0100] (2) Photodamage

[0101] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) in a 37°C, 5% CO2 incubator.

[0102] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0103] Experimental treatment: HDF cells were seeded into 96-well plates, and sample groups, blank control groups (BC), positive control groups (PC), and negative control groups (NC) were set up. At least 3 replicates were performed for each sample for each test indicator. After culturing for 24 hours, the culture medium was discarded, the corresponding CA solution was added to the sample group, the VC solution (dissolved in culture medium) was added to the positive control group, and complete culture medium was added to the negative control group and the blank control group. Place in an incubator and incubate for 2 hours. The sample group, positive control group, and negative control group were irradiated under a UVA lamp with an irradiation dose of 5J / cm 2 After irradiation, the cells were cultured for 24 h.

[0104] Cell viability detection: discard the supernatant, add complete medium containing 10% AlamarBlue, incubate in an incubator for 4 hours, and then detect the absorbance value at ex / em=560 / 590 using a microplate reader.

[0105] Type I collagen detection: The supernatant of each well was collected and the test was performed according to the operating instructions of the ELISA collagen detection kit (Huamei Biotechnology, CSB-E08082h).

[0106] (3) Anti-wrinkle (promoting collagen)

[0107] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) in a 37°C, 5% CO2 incubator.

[0108] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0109] Experimental treatment: HDF cells were seeded into 96-well plates and divided into sample groups, blank controls (BC), and positive controls (PC). At least three replicates were performed for each sample and each indicator. After 24 hours of incubation, the culture medium was discarded, and the corresponding CA solution was added to the sample group, the TGF-β solution was added to the positive control group, and complete culture medium was added to the negative control group and the blank control group. Incubation was continued for another 24 hours.

[0110] Type I and III collagen detection: The supernatant of each well was collected and the test was performed according to the operating instructions of the ELISA collagen detection kit (Huamei Biotechnology, CSB-E04799h).

[0111] (4) Inflammation (macrophages)

[0112] Cell culture: RAW264.7 cells were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) in a 37°C, 5% CO2 incubator.

[0113] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0114] Experimental treatment: RAW264.7 cells were seeded into 96-well plates and set up sample groups, blank controls, positive controls, and negative controls. At least three replicates were performed for each sample for each test indicator. After 24 hours of incubation, the culture medium was discarded, and the corresponding CA solution was added to the sample group, complete culture medium containing 100 μg / mL dexamethasone was added to the positive control group, and complete culture medium was added to the negative control group and blank group. After incubation in an incubator for 2 hours, 1 μg / mL LPS (lipopolysaccharide) was added to the sample group, positive control group, and negative control group. Culture was continued for 24 hours.

[0115] Inflammatory factor detection: The supernatant of each well was collected and the test was performed according to the operating instructions of the ELISA kit (Huamei Biotechnology, CSB-E04740h, CSB-E08053h, CSB-E04638h).

[0116] Reactive oxygen species (ROS) detection: After collecting the supernatant, the cells in each well were washed twice with PBS, and the test was performed according to the operating instructions of the DCFH-DA kit.

[0117] (5) Inflammation (keratinocytes)

[0118] Cell culture: Keratinocytes HaCaT were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) and placed in a 37° C., 5% CO 2 incubator.

[0119] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0120] Experimental treatment: Cells were seeded into 96-well plates and set up sample groups, blank controls, positive controls, and negative controls. At least three replicates were performed for each sample for each test indicator. After 24 hours of incubation, the culture medium was discarded, and the corresponding CA sample solution was added to the sample group, the corresponding VC solution was added to the positive control group, and complete culture medium was added to the negative control and blank controls. After incubation in an incubator for 2 hours, hydrogen peroxide solution (0.8mM HX0640 Sigma-Aldrich) was added to the sample group, positive control group, and negative control group for inflammatory stimulation. Culture was continued for 24 hours.

[0121] Genetic testing: Discard the remaining solution, wash twice with PBS, add cell lysis buffer to each well, lyse the cells by pipetting, and collect samples. RNA is extracted and reverse-transcribed to cDNA. Quantitative PCR is performed, and the results are calculated using the 2-ΔΔCt method. The calculated 2-ΔΔCt value indicates the difference in expression of the target gene between the two groups of samples.

[0122] (6) Transdermal test

[0123] Ex vivo skin preparation

[0124] The excised skin stored at -20°C was thawed with deionized water at room temperature and repeatedly rinsed with PBS buffer.

[0125] Transdermal absorption capacity determination

[0126] 1) Fix the ex vivo skin between the donor chamber and the receiving chamber of a Franz cell diffusion cell, with the stratum corneum side of the skin facing the donor chamber and the dermis side facing the receiving chamber;

[0127] 2) Add receiving solution to the receiving chamber. After tightening the skin and fixing it, add receiving solution (PBS) to the receiving chamber through the sampler, expel all air, and ensure close contact between the dermis and the receiving solution.

[0128] 3) Sample application: Add the sample to the skin surface in the supply chamber. Spread the sample evenly from the center of the skin to the edge.

[0129] 4) Infiltration: Turn on the electromagnetic stirrer and stir at 300 rpm, and maintain a constant temperature of (32±1)°C in a water bath;

[0130] 5) Collect skin samples at 1 hour, 6 hours, and 18 hours. Wash the skin surface with PBS, wipe off any residual liquid, and circularly cut the skin with a blade. Cryosection and set aside.

[0131] Observation and analysis

[0132] Skin sections were observed under a fluorescence microscope, photographed, and the fluorescence intensity was analyzed using Image J.

[0133] (7) In vitro anti-wrinkle test

[0134] 1) Cell seeding: 2.2×10 5 Fibroblasts were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0135] 2) Dosing: Assemble the test groups according to Table 2. When the cell plating rate in the 6-well plate reaches 40-60%, administer the drug to each group. Add 2 mL of sample to each well, and set up three replicates per group. After dosing, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 24 hours.

[0136] 3) UVA irradiation: According to the experimental grouping, the group requiring UVA irradiation was irradiated with 30 J / c of ​​UVA and placed in an incubator (37°C, 5% CO2) for a further 24 hours.

[0137] 4) Sample collection: After 24 h of culture, the cell culture supernatant was collected into an EP tube and stored in a -80°C freezer.

[0138] 5) ELISA test: The test was performed according to the operating instructions of the ELISA kit (Huamei Biotechnology CSB-E08082h).

[0139] (8) Anti-wrinkle and collagen-promoting under light conditions

[0140] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM+10% FBS+1% P / S) in a 37°C, 5% CO2 incubator.

[0141] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0142] Experimental treatment: HDF cells were seeded into 96-well plates and assigned to sample groups, blank controls, and positive controls. At least three replicates were performed for each sample for each assay. After 24 hours of incubation, the culture medium was discarded, and the corresponding CA solution was added to the sample group, the TGF-β solution was added to the positive control group, and complete culture medium was added to the negative control and blank groups. The sample, positive, and negative controls were irradiated under UVA light at 40 mW for 4 minutes. Following irradiation, the cells were cultured for an additional 24 hours.

[0143] Type I and III collagen detection: The supernatant of each well was collected and the test was performed according to the operating instructions of the ELISA collagen detection kit (Huamei Biotechnology, CSB-E04799h).

[0144] (9) Cell viability-CCK8 assay

[0145] Seed plate: After thawing, culture HaCaT keratinocytes for 1-2 days until the cell confluence reaches 70%. After trypsin digestion, keratinocytes are plated at 5×10 4 / well seed plates, grouped as DMEM, H2O2-DMEM, and H2O2-CA.

[0146] H2O2 stimulation: H2O2 (Shanghai Lingfeng Chemical Reagent) was used at a concentration of 0.8 mM. It was diluted to 10 mM and then added to the culture medium at a 100x dilution (the original stock concentration was 8820 mM; 10 μL of H2O2 was added to 8.82 mL of PBS, and 40 μL of the dilution was added to 4 mL of culture medium). The prepared H2O2-containing culture medium was added to the H2O2 stimulation group as a replacement medium and cultured for 2 h.

[0147] CA treatment: Pre-treatment: 50 mg of UV-sterilized CA powder was added to 5 mL of PBS and vortexed to dissolve to a 10 mg / mL solution. The solution was then diluted in DMEM to a working concentration of 5 mg / mL. The supernatant from the H2O2-stimulated culture (including the DMEM blank control) was removed and rinsed with 100 μL of PBS. Then, 100 μL of CA-containing culture medium was added to the H2O2-CA group, and 100 μL of culture medium was added to the DMEM and H2O2-DMEM groups. The cells were incubated for 24 hours.

[0148] CCK8 Assay: Calculate the required culture medium volume and add CCK8 (Biyuntian, C0038) at a 10x concentration (i.e., add 400 μL of CCK8 to 4 mL of culture medium). Aspirate the supernatant from each sample and add 100 μL / well to a 96-well plate. Incubate for 1-2 hours. Measure at OD450 using a microplate reader in the dark. Example

[0149] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0150] In the following examples, unless otherwise specified, the colanic acid used was sodium colanate having a molecular weight of 1-3 kDa.

[0151] Example 1

[0152] Cell culture: Human dermal fibroblast (HDF) cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) in a 37°C, 5% CO2 incubator.

[0153] Sample treatment: CA powder was dissolved in culture medium and sterilized by filtration using a 0.2 μm sterile filter membrane after complete dissolution.

[0154] Experimental treatment: HDF cells were seeded into 96-well plates, and sample groups, blank controls, positive controls, and negative controls were set up. At least 3 replicates were performed for each sample for each test indicator. After 24 hours of culture, the culture medium was discarded, and the corresponding CA solution was added to the sample group, VC solution (dissolved in culture medium) was added to the positive group, and complete culture medium was added to the negative control group and blank group. The cells were placed in an incubator and incubated for 24 hours. The cells were then removed and frozen, and transcriptome analysis was commissioned to Jin Weizhi.

[0155] Figure 3 shows that oligomeric CA (Oli-CA) promotes the transcription of AMPK, PGC1α, and Cab39 (Figure 3A), as well as NAMPT, sirtuin1, sirtuin6, and sirtuin7 (Figure 3B) genes in fibroblasts under ultraviolet conditions. The results in Figure 3 indicate that oligomeric CA promotes the expression of sirtuin1 in human fibroblasts.

[0156] Example 2

[0157] Fibroblasts were treated with oligocorticosteroids (2.5 mg / mL), HA (hyaluronic acid, 2.5 mg / mL), ectoin (2.5 mg / mL), NC (UV irradiation only), PC (cucurbitacin 1 mg / mL), and BC (cells only). After 24 hours of culture, cell viability was observed under UV irradiation. The effects of oligocorticosteroids on BAX and NF-κB inhibitory protein transcription in human fibroblasts were tested, as shown in Figure 4. The results in Figure 4 demonstrate that oligocorticosteroids inhibit BAX transcription and upregulate NF-κB inhibitory protein transcription (Figure 4A). Cell viability was then assessed after irradiation with oligocorticosteroids, HA, ectoin, and vitamin C (positive control). These experimental results demonstrate that oligocorticosteroids significantly enhance cell survival under UV conditions, demonstrating their efficacy in inhibiting apoptosis.

[0158] Example 3

[0159] According to the inflammation (macrophage) method in the experimental method, the inhibitory effect of oligomeric CA on mouse macrophage inflammation was tested, and the results are shown in Figure 5. As can be seen from Figure 5, oligomeric CA can effectively inhibit the inflammatory response of macrophages induced by LPS (lipopolysaccharide), including TNF-α (Figure 5A) and IL-6 (Figure 5B).

[0160] Example 4

[0161] According to the experimental method of Example 1, the transcriptional regulatory effect of oligomeric CA on genes related to mitochondrial homeostasis and repair in human fibroblasts was tested, and the results are shown in Figure 6. Figure 6 shows that oligomeric CA can upregulate the transcription of a series of genes, including AMBRA1, USP8, ATF4, BNIP3 and BCL2L13, ATF5 and CHOP.

[0162] Example 5

[0163] Oligomeric CA was tested for its effects on the transcriptional analysis of SOD2 and GPX1 / 8 in human fibroblasts, as well as its ROS scavenging effects (Solarbol, ROS Detection Kit). The results are shown in Figure 7. As can be seen from Figure 7, oligomeric CA significantly upregulated the transcriptional levels of SOD2, GPX1, and GPX8 in human fibroblasts (Figure 7A). Simultaneously, the ROS production in human fibroblasts irradiated with UV light was significantly suppressed (Figure 7B). This indicates that oligomeric CA has a strong ability to regulate cellular ROS scavenging activity.

[0164] Example 6

[0165] Following the anti-wrinkle (collagen-promoting) assay described in the experimental methods, oligomeric CA was tested for its ability to promote type I collagen synthesis in HDF cells and upregulate the transcription of various collagen types in HDF cells. The results are shown in Figure 8 . Figure 8 demonstrates that oligomeric CA significantly increases type I collagen synthesis in HDF cells (data obtained by diluting the supernatant 10-fold with culture medium). Figure 8A shows that oligomeric CA exhibits superior collagen-promoting activity compared to pro-xylane and retinyl propionate (RP) at the same concentration. At the same concentration, its collagen-promoting activity is four times that of pro-xylane (2 mg / mL) and three-to-four times that of retinyl propionate (3 mg / mL and 10 mg / mL). Furthermore, transcriptome analysis of HDF cells (Figure 8B) also reveals that oligomeric CA upregulates the transcription of many other collagen types, including types 4, 6, 7, 9, 17, 22, and 25. This result indicates that oligomeric colanic acid has good application prospects in promoting collagen activity.

[0166] Example 7

[0167] The phototoxicity of oligocoranoic acid was tested according to the light damage test described in the experimental methods. The results are shown in Figure 9. Figure 9 shows that when HDF cells were exposed to light after the addition of oligocoranoic acid (CA), no significant cytotoxicity was observed (cell viability >90% is considered non-toxic). In contrast, the cell protective effect increased with increasing oligocoranoic acid dosage, from 90% at 2 mg / mL to 97% at 10 mg / mL. Retinyl propionate, on the other hand, exhibited significant cytotoxicity at both 3 mg / mL and 10 mg / mL.

[0168] Example 8

[0169] According to the method of anti-wrinkle and collagen-promoting under light conditions in the experimental method, the activity of oligomeric colanic acid in promoting the synthesis of type I collagen and type III collagen by HDF cells under light conditions was tested, and the results are shown in FIG10 .

[0170] Figure 10 shows that oligocorticosteroids exhibited stronger activity in promoting type I and type III collagen in an illuminated HDF cell model. Compared to phosphatase, at a concentration of 2 mg / mL, oligocorticosteroids promoted type I collagen production 2.3-fold and type III collagen production 2.8-fold. Compared to retinyl propionate, at the same concentration, oligocorticosteroids promoted type I collagen production 18-fold (at 3 mg / mL) and 2-fold (at 10 mg / mL), and type III collagen production 10-fold (at 3 mg / mL) and 28-fold (at 10 mg / mL). This demonstrates that oligocorticosteroids are not only non-phototoxic but also exhibit activity in promoting both type I and type III proteins, with significantly greater efficacy than phosphatase and retinyl propionate.

[0171] Example 9

[0172] The inhibitory effect of oligomeric colanic acid on MMP9 in a UV-irradiated skin model (Huamei Bio, CSB-PA002676) was tested, and proteomic data were obtained under UV-irradiated skin ex vivo. Figure 11 shows a significant decrease in MMP9 levels in the colanic acid-treated group. MMP9 is a key target for inhibiting DEJ aging. Increased MMP9 skin levels lead to a rapid loss of type VI collagen in the DEJ layer, which in turn causes the DEJ to become smooth. The DEJ layer is the connecting layer between the dermis and epidermis. The smoother it is, the less connection there is between the dermis and epidermis, and the more severe skin aging will be.

[0173] Example 10

[0174] Following the cell culture and modeling methods described in the Inflammation (Keratinocyte) Experimental Methods section, TNF-α, IL-6, and COX-2 were assayed (ELSIA kits: Huamei Biotechnology, CSB-E04740h, CSB-E04638h, CSB-E10103h) to test the anti-inflammatory effect of oligocoronic acid on human keratinocytes. Figure 12 shows that oligocoronic acid exhibits a strong anti-inflammatory effect on human keratinocytes, significantly inhibiting the expression of TNF-α, IL-6, and COX-2 at 1 mg / mL.

[0175] Example 11

[0176] The transdermal permeability of oligocoranic acid was tested using the transdermal assay described in the experimental methods. Figure 13 shows that after fluorescent labeling, the transdermal permeability of oligocoranic acid in vitro was significantly increased within 18 hours, with a nine-fold increase in fluorescence intensity. Furthermore, the molecule was found to effectively penetrate the dermis.

[0177] The intracellular localization of oligomeric colanic acid was tested by the following method.

[0178] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) in a 37°C, 5% CO2 incubator.

[0179] Sample treatment: 1 mg / mL fluorescently labeled CA powder was dissolved in the culture medium. After complete dissolution, the solution was sterilized by filtration using a 0.2 μm sterile filter membrane.

[0180] Experimental treatment: HDF cells were seeded into 96-well plates and set up as sample groups, blank controls, and positive controls. At least three replicates were performed for each sample for each assay. After 24-48 hours of culture, the cells were removed and DAPI and Mitotracker staining solutions were added to stain the nucleus and mitochondria, respectively. After each staining, the cells were washed two to three times with PBS and fluorescence was observed using a light microscope.

[0181] Figure 14 shows that oligocoronic acid can effectively enter cells. Mitotracker staining of mitochondria reveals overlap between oligocoronic acid (green) and mitochondria (red), demonstrating that oligocoronic acid can permeate the cell membrane and enter mitochondria. In contrast, no overlap was observed between oligocoronic acid and the nucleus (blue), suggesting that oligocoronic acid has difficulty entering the nucleus.

[0182] In addition, the cytotoxicity test results of oligocoronic acid ( FIG. 15 ) showed that the molecule had no cytotoxicity at a concentration below 10 mg / mL.

[0183] Example 12: Human Experimental Test

[0184] Human experiments on oligomeric sodium colanate were conducted as follows, and the results are shown in Figures 16-25.

[0185] Sixty-six healthy Chinese male and female subjects with dry, sagging, and lacking facial elasticity (R2 values ​​between 0.35 and 0.55), fine lines or wrinkles, and wrinkles at the canthiform angles of the eyes (grades 2 to 5), as well as a weak skin barrier and sensitive skin, were randomly divided into two groups, 33 participants in each group, ranging in age from 30 to 60 years. Using a before-and-after control and between-group comparison method, the subjects' stratum corneum moisture content, transepidermal water loss, skin elasticity, skin firmness, dermal density, dermal thickness, and total dermal thickness were subjectively assessed, and partial and full-face photographs were taken. The results were compared using statistical tests to determine if there were statistically significant differences.

[0186] Detection site

[0187] (1) Skin stratum corneum moisture content - cheeks

[0188] (2) Transepidermal water loss value - cheek

[0189] (3) Skin elasticity R2 - cheek

[0190] (4) Skin firmness F4 - cheeks

[0191] (5)UC22 dermal density - cheek

[0192] (6)UC22 Dermis Thickness - Cheek

[0193] (7) UC22 full skin thickness - cheek

[0194] (8) PRIMOS CR Under-eye wrinkle area - Under-eye

[0195] (9) PRIMOS CR Under-eye wrinkle volume - Under-eye

[0196] (10) PRIMOS CR cheek roughness Ra - cheek

[0197] (11) VISIA CR skin color L value - full face photo analysis of cheeks

[0198] (12) VISIA CR skin color b value - full face photo analysis of cheeks

[0199] (13) VISIA CR Skin Color ITA° Value - Full Face Photo Analysis of Cheeks

[0200] (14)VISIA CR skin transparency parameters - full face

[0201] (15)VISIA CR skin glossiness parameter - full face photo analysis of cheeks

[0202] (16)VISIA CR skin red area ratio - full face photo analysis of cheeks

[0203] (17)VISIA CR Wrinkle Area Ratio at the Corner of the Eyes – Full Face Photo Analysis of the Corner of the Eyes

[0204] (18) VISIA CR Pore Area Ratio - Full Face Photo Analysis of Cheeks

[0205] (19)VISIA CR jaw line angle - full face photo analysis of the jaw

[0206] (20) Standardized area of ​​the epidermal dermal junction (DEJ) layer DEJI - canthus

[0207] (21) Aging Index ELCOR - Canthus

[0208] (22) Aging Index SAAID - Canthus

[0209] (23) Epidermal thickness TED - canthus

[0210] (24) Elastic fiber fluorescence intensity - canthus

[0211] (25) Collagen fiber harmonic intensity - canthus

[0212] (26) Assessment of eye wrinkle levels – eye corners

[0213] (27) Assessment of wrinkle levels under the eyes – under the eyes

[0214] (28) Glossiness score - cheeks

[0215] (29) Elasticity score - cheek

[0216] (30) Smoothness score - cheeks

[0217] (31) Smoothness score - cheeks

[0218] (32) Self-assessment - Full face

[0219] Day 0:

[0220] When the subjects come for a visit, technicians will assess the condition of their facial skin and select those with dry skin or wrinkles and redness from the participants of the trial;

[0221] Cleansing the face and waiting: The subjects cleansed their face with a cleansing product and dried their skin with dry tissue paper. They then sat quietly in a laboratory at a temperature of 21°C ± 1°C and a humidity of 50% RH ± 10% RH for 20 minutes.

[0222] Collection of baseline skin values ​​of test subjects: Laboratory technicians used SUPERVISION 780, VISIA CR, Cutometer, PRIMOSCR, Corneometer, Vapometer, and UC22 instruments to measure the baseline values ​​of relevant indicators on the test areas of the subjects;

[0223] Trained dermatologists professionally rated and recorded the subjects' eye wrinkles;

[0224] Professional assessors will score and record the subjects' skin gloss, fineness, smoothness, and elasticity;

[0225] Laboratory technicians instructed participants on how to use the product according to the product's instructions and provided written test precautions and instructions. Participants were randomly divided into two groups according to a randomization table. One participant in each group was randomly assigned to apply the product to half of their face. Moisture testing was performed on multiple facial areas 2 and 4 hours after application.

[0226] The subjects received the products and left the laboratory.

[0227] Day 14:

[0228] Subjects returned for a visit, cleansed their face, and waited: cleansed their face with a cleansing product and dried their skin with dry tissues. They sat quietly in a laboratory at a temperature of 21°C ± 1°C and a humidity of 50% RH ± 10% RH for 20 minutes, completing the D14 self-assessment questionnaire during the 20-minute sitting period.

[0229] D14 data collection of the test subjects' skin: Laboratory technicians used SUPERVISION 780, VISIA CR, Cutometer, PRIMOSCR, Corneometer, Vapometer, and UC22 instruments to measure relevant indicators on the test areas of the subjects;

[0230] Trained dermatologists professionally rated and recorded the subjects' eye wrinkles;

[0231] Professional assessors will score and record the subjects' skin gloss, fineness, smoothness, and elasticity;

[0232] Staff checked subjects' usage records and products;

[0233] The subject left the laboratory.

[0234] Day 28:

[0235] Subjects returned for a visit, cleansed their face, and waited: cleansed their face with a facial cleanser and dried their skin with dry facial tissues. They sat in a laboratory at a temperature of 21°C ± 1°C and a humidity of 50% RH ± 10% RH for 20 minutes and completed the D28 self-assessment questionnaire during this 20-minute period.

[0236] D28 data collection of the test subjects' skin: Laboratory technicians used SUPERVISION 780, VISIA CR, Cutometer, PRIMOSCR, Corneometer, Vapometer, and UC22 instruments to measure relevant indicators on the test areas of the subjects;

[0237] Trained dermatologists professionally rated and recorded the subjects' eye wrinkles;

[0238] Professional assessors will score and record the subjects' skin gloss, fineness, smoothness, and elasticity;

[0239] Staff checked and collected the subjects' usage records and products;

[0240] The subject left the laboratory;

[0241] The testing process is complete.

[0242] Figure 16 shows the effect of sodium oligocorticate on increasing the density of the human dermis. As shown in Figure 16, after continuous use of the serum containing 0.5% (w / v) sodium oligocorticate for 14 days, the UC22 dermal density increased by 22.87%, and after 28 days of continuous use, the increase was 24.00%.

[0243] Figure 17 shows the effect of sodium oligocorticate on enhancing collagen expression in the human dermis. As shown in Figure 17, after subjects used a serum containing 0.5% (w / v) sodium oligocorticate for 14 consecutive days, the collagen fiber harmonic intensity increased by 12.31%, and after 28 consecutive days, it increased by 21.61%.

[0244] Figure 18 shows the effect of sodium oligocorticate on enhancing elastin expression in the human dermis. As shown in Figure 18, after subjects used a serum containing 0.5% (w / v) sodium oligocorticate for 14 days, the fluorescence intensity of elastic fibers increased by 17.02%. After 28 days of continuous use, the increase was 22.13%.

[0245] Figure 19 shows the effect of sodium oligocorticate on improving skin elasticity and refinement. As shown in Figure 19, after subjects used a serum containing 0.5% (w / v) sodium oligocorticate for 14 days, skin elasticity (R2) increased by 24.44% and skin firmness (F4) decreased by 6.62%. After 28 days of continuous use, R2 increased by 40.00% and F4 decreased by 12.00%.

[0246] Figure 20 shows the effect of sodium oligocorticate on increasing the normalized area of ​​the epidermal dermal junction (DEJ) in human skin. As shown in Figure 20, after subjects used a serum containing 0.5% (w / v) sodium oligocorticate for 14 consecutive days, the normalized area of ​​the DEJ layer at the epidermal dermal junction (DEJI) increased by 28.70%. After 28 consecutive days, the increase was 36.11%.

[0247] Figure 21 shows the effect of sodium oligocorticate on increasing epidermal thickness in human skin. As shown in Figure 21, after subjects used a serum containing 0.5% (w / v) sodium oligocorticate for 14 days, the epidermal thickness increased by 27.78%, and after 28 days, the increase was 27.29%.

[0248] Figure 22 shows the effect of sodium oligocorticate on improving skin redness in humans. As shown in Figure 22, after subjects used the serum containing 0.5% (w / v) oligocorticate for 28 consecutive days, the red area of ​​the VISIA-CR skin decreased by 10.45%.

[0249] Figure 23 shows the effect of sodium oligocorticate on increasing the content of filaggrin (FLG) in 3D skin. The 3D skin experimental test results in Figure 23 show that compared with the BC control group, sodium oligocorticate at a concentration of 0.5% (w / v) significantly increased the content of filaggrin (FLG), with an increase rate of 49.00%.

[0250] Figure 24 shows the effect of sodium oligocorticate on increasing the content of aquaporin 3 (AQP3) in 3D skin. The 3D skin test results in Figure 24 show that compared with the BC control group, sodium oligocorticate at a concentration of 0.5% (w / v) significantly increased the content of aquaporin 3 (AQP3), with an increase rate of 37.00%.

[0251] Figure 25 shows the moisturizing effect of sodium oligocorticate on human skin. As shown in Figure 25, after 14 days of continuous use of a serum containing 0.5% (w / v) sodium oligocorticate, the water content of the stratum corneum significantly increased by 31.76%. After 28 days of use, the increase was 63.84%. Transepidermal water loss (TEWL) decreased significantly by 12.57% compared to baseline, and after 28 days, the decrease was 20.08%.

[0252] Example 13

[0253] Sodium colanate with a molecular weight of 1-10 kDa, 1-8 kDa, 1-6 kDa, 1-5 kDa, 1-4 kDa, 1-2 kDa, 2-10 kDa, 2-8 kDa, 2-6 kDa, 2-4 kDa, 2-3 kDa, 3-6 kDa, 4-10 kDa, 4-8 kDa, 4-6 kDa, 5-10 kDa, 5-8 kDa, 6-10 kDa, or 6-8 kDa was prepared in the same manner as described in Examples 1-12, and its properties and efficacy were tested.

[0254] Example 14

[0255] According to the anti-wrinkle (collagen-promoting) method described in the experimental method, the effects of sodium colanate (CA) with different molecular weights at a concentration of 3 mg / mL on stimulating the expression of type I collagen in human fibroblasts were tested. The results are shown in Figure 26. As can be seen from Figure 26, oligomeric sodium colanate with molecular weights in the ranges of 1-3 kDa and 3-10 kDa exhibited superior anti-aging and collagen-promoting activity.

[0256] The skin protective effects of sodium colanate (CA) with different molecular weights were tested according to the cell viability-CCK8 method in the experimental method, and the results are shown in Figure 27. As can be seen from Figure 27, oligomeric sodium colanate with molecular weights in the ranges of 1-3 kDa and 3-10 kDa has better skin protective effects.

[0257] Incorporated by Reference

[0258] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0259] Equivalence

[0260] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. Use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, for skin anti-aging and / or skin protection, characterized in that: The molecular weight of the colanic acid or a physiologically acceptable salt thereof is in the range of 1-10 kDa, preferably in the range of 1-3 kDa or 3-10 kDa.

2. The use according to claim 1, wherein The skin anti-aging method includes increasing the collagen content in the skin, preferably the content of type I and type III collagen.

3. The use according to claim 1, wherein The skin protection includes enhancing the protective function of skin cells such as increasing the vitality of skin cells.

4. The use according to claim 1, wherein The skin anti-aging and the skin protection include improving the condition or appearance of the skin.

5. The use according to any one of claims 1 to 4, wherein The content of colanic acid or a physiologically acceptable salt thereof is 0.01-10% (w / v), preferably 0.1-5% (w / v), more preferably 0.5-3% (w / v).

6. The use according to any one of claims 1 to 5, wherein The physiologically acceptable salt of colanic acid is sodium colanate.

7. The composition according to any one of claims 1 to 6, wherein The colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, is formulated for topical, oral, intramuscular, subcutaneous or intravenous administration.

8. The composition according to any one of claims 1 to 7, wherein The composition comprising colanic acid or a physiologically acceptable salt thereof further contains cosmetically or pharmaceutically acceptable additives.

9. A composition, characterized in that The composition comprises colanic acid or a physiologically acceptable salt thereof as an effective ingredient. The molecular weight of the colanic acid or the physiologically acceptable salt thereof is in the range of 1-10 kDa, preferably in the range of 1-3 kDa or 3-10 kDa.

10. The composition according to claim 9, wherein The content of the colanic acid or its physiologically acceptable salt in the composition is 0.01-10% (w / v), preferably 0.1-5% (w / v), more preferably 0.5-3% (w / v).

11. The composition according to claim 9 or 10, wherein The composition contains only colanic acid or a physiologically acceptable salt thereof as an active ingredient.

12. The composition according to any one of claims 9 to 11, wherein The compositions are formulated for topical, oral, intramuscular, subcutaneous or intravenous administration.

13. The composition according to any one of claims 9 to 12, wherein The composition may also contain cosmetically or pharmaceutically acceptable additives.

14. The composition according to any one of claims 9 to 13, wherein The physiologically acceptable salt of colanic acid is sodium colanate.

Images