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

Composition comprising hydrolyzed colanic acid salts and uses thereof in skin anti-aging and skin protection TECHNICAL FIELD

[0001] The present invention belongs to the field of skin care and medicine, in particular to a composition comprising hydrolyzed colanic acid salts and uses thereof in skin anti-aging and skin protection. BACKGROUND

[0002] Colanic acid (CA) is a bacterial exopolysaccharide produced by most strains of Escherichia coli and other species of Enterobacteriaceae. It is a polysaccharide synthesized by the bacteria during their life activities to adapt to changes in the environment and improve their survival rate. CA has a large molecular weight and loosely wraps around the surface of the bacteria, making the bacteria show a mucous state, preventing cell dehydration, protecting cells, and resisting harmful substances. In adverse environmental conditions, such as dryness, low pressure, and low pH, mucoid strains have stronger survivability than wild-type strains. In 2017, Han et al. reported that feeding purified CA or E. coli that can secrete CA can significantly prolong the lifespan of Caenorhabditis elegans. In addition, CA, as a unique active biopolymer, has special biological characteristics and physiological parameters, and has a wide application prospect.

[0003] SUMMARY

[0004] In one aspect, the present disclosure provides uses of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, in skin anti-aging and / or skin protection, wherein the colanic acid or the 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, preferably type I and type III collagen content, in the skin.

[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 comprise improving the skin condition or appearance.

[0008] In some embodiments, the colanic acid or the physiologically acceptable salt thereof is present in an amount of 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 the colanic acid is sodium colanic acid.

[0010] In some embodiments, the colanic acid or a physiologically acceptable salt thereof, or a composition comprising the 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 the colanic acid or a physiologically acceptable salt thereof further contains a cosmetically or pharmaceutically acceptable additive.

[0012] In another aspect, the present disclosure provides use of the colanic acid or a physiologically acceptable salt thereof, or a composition comprising the colanic acid or a physiologically acceptable salt thereof as described above in the manufacture of a medicament or a cosmetic for skin anti-aging and / or skin protection.

[0013] In another aspect, the present disclosure provides a composition comprising the colanic acid or a physiologically acceptable salt thereof as an effective ingredient, the molecular weight of the colanic acid or a physiologically acceptable salt thereof being 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 the 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 contains only the 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 a cosmetically or pharmaceutically acceptable additive.

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

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

[0022] FIG. 1 shows the results of transcriptome analysis of fibroblasts by size molecules of sodium colanic acid.

[0023] FIG. 2 shows the multi-dimensional efficacy mechanism of colanic acid anti-aging.

[0024] Figure 3 shows the results of oligo sodium roloxate on human fibroblast transcriptome analysis.

[0025] Figure 4 shows the results of oligo sodium roloxate on human fibroblast transcriptome analysis (A) and cell viability test results after UVA irradiation (B).

[0026] Figure 5 shows the inhibitory effect of oligo sodium roloxate on macrophage TNF-a and IL-6.

[0027] Figure 6 shows the transcriptional regulation results of oligo sodium roloxate on human fibroblast mitochondrial homeostasis repair related genes.

[0028] Figure 7 shows the transcriptional analysis of SOD2 and GPX1 / 8 of HDF cells by oligo sodium roloxate (A) and the scavenging effect on reactive oxygen species (B).

[0029] Figure 8 shows the transcriptional data of oligo sodium roloxate promoting HDF cells to synthesize type I collagen and upregulating different types of collagen in HDF cells.

[0030] Figure 9 shows the phototoxicity test results of oligo sodium roloxate.

[0031] Figure 10 shows the activity test results of oligo sodium roloxate promoting HDF cells to synthesize type I and type III collagen under light conditions.

[0032] Figure 11 shows the inhibitory effect of oligo sodium roloxate on MMP9 of ultraviolet irradiated skin model.

[0033] Figure 12 shows the anti-inflammatory effect of oligo sodium roloxate on human keratinocytes.

[0034] Figure 13 shows the transdermal test results of oligo sodium roloxate.

[0035] Figure 14 shows the intracellular localization test results of oligo sodium roloxate.

[0036] Figure 15 shows the cytotoxicity test results of oligo sodium roloxate.

[0037] Figure 16 shows the effect of oligo sodium roloxate on improving the density of human dermal layer.

[0038] Figure 17 shows the effect of oligo sodium roloxate on improving the expression of collagen in human dermal layer.

[0039] Figure 18 shows the effect of oligo sodium roloxate on improving the expression of elastin in human dermal layer.

[0040] Figure 19 shows the effect of oligo sodium roloxate on improving skin elasticity and skin delicacy of human skin.

[0041] Figure 20 shows the effect of oligomeric sodium colaurate on the normalized area of the DEJ of human skin.

[0042] Figure 21 shows the effect of oligomeric sodium colaurate on the epidermis layer thickness of human skin.

[0043] Figure 22 shows the effect of oligomeric sodium colaurate on the skin redness of human skin.

[0044] Figure 23 shows the effect of oligomeric sodium colaurate on the 3D skin fibulin (FLG) content.

[0045] Figure 24 shows the effect of oligomeric sodium colaurate on the 3D skin aquaporin 3 (AQP3) content.

[0046] Figure 25 shows the effect of oligomeric sodium colaurate on the water retention of human skin.

[0047] Figure 26 shows the effect of oligomeric sodium colaurate on stimulating human fibroblast to express collagen type I.

[0048] Figure 27 shows the skin protection effect of oligomeric sodium colaurate.

[0049] In the figures, * means P<0.05 relative to the negative control, ** means P<0.01 relative to the negative control, *** means P<0.001 relative to the negative control, ## means P<0.01 relative to the blank control, and ### means P<0.001 relative to the blank control.

[0050] DETAILED DESCRIPTION

[0051] The various features and aspects of the present application are discussed in more detail below.

[0052] Figure 1 shows the results of the transcriptome analysis of small molecule colaurate (molecular weight of 1-3 kDa, also referred to as hydrolyzed or oligomeric colaurate, simply oligomeric CA or Oli-CA) and large molecule colaurate (molecular weight of 10-6000 kDa). Specifically, each was added to human fibroblast cells (HDF) that had been cultured for 24 h, then subjected to UV irradiation, and then cultured for another 24 h, after which the cells were taken for transcriptome analysis. Statistical analysis of the up- or down-regulation of different genes according to the test results showed that colaurate affected 2500 genes of the cells. In contrast, the control group, which was treated with white propolis, affected only 426 genes. These results show that colaurate of different molecular weights has a very large impact on the transcriptome of fibroblast cells.

[0053] According to the results of transcriptome analysis, and combined with the KEGG longevity-related pathways (https: / / www.kegg.jp / pathway / map04211), the different anti-aging pathways of colanic acid salt were sorted out, as shown in Figure 2. The gray part in Figure 2 represents the inhibition of transcriptome mRNA, the green part represents the up-regulation of transcriptome mRNA, the sharp arrow represents the promotion of expression, and the blunt arrow represents the inhibition of expression. Figure 2 shows that in pathway 1, colanic acid salt can promote the transcriptome of NAMPT, a key enzyme for intracellular synthesis of NAD+, while colanic acid salt can also up-regulate AMPK, sirt1 and other genes, and further inhibit BAX and NF-κ, thereby inhibiting the effects of cell apoptosis and cell inflammation, and also up-regulating PGC-1α to promote mitochondrial activity.

[0054] In pathway 2, colanic acid salt can inhibit Rheb and mTOR genes, thereby up-regulating the expression of ATG13 and RB1CC1 gene transcription to promote autophagy.

[0055] In pathway 3, colanic acid salt can up-regulate the expression of AMBRA1, USP8, ATF4 and other gene transcription to promote mitochondrial autophagy, promote mitochondrial fission by up-regulating HIF1 and promoting BNIP3 and BCL2L13 transcription, and promote mitochondrial unfolded protein response by promoting ATF4 / ATF5 and CHOP transcription. Mitochondrial homeostasis remodeling is promoted by the above three aspects.

[0056] In pathway 4, colanic acid salt can increase the activity of cells to clear ROS by up-regulating the transcription of SOD2, GPX1 and GPX8. In addition to being able to clear reactive oxygen species, the up-regulation of GPX8 expression can also regulate the transfer of calcium ions from the endoplasmic reticulum to the mitochondria, and alleviate the damage to mitochondria caused by the loss of calcium ions in cells.

[0057] In pathway 5, colanic acid salt can promote the synthesis of extracellular matrix by up-regulating the expression of collagen and various extracellular matrix transcripts, enhance cell-cell communication and have a better supporting and protective effect on cell morphology.

[0058] In the present disclosure, colanic acid (CA) or a colanic acid salt can be prepared by any method as long as a desired molecular weight is obtained. Specifically, the molecular weight of the 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 constituted by the above-mentioned molecular weights. Among them, the 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 improve the effect of the colanic acid or the physiologically acceptable salt thereof.

[0059] The inventors of the present application have unexpectedly found that by selecting the colanic acid or the physiologically acceptable salt thereof with the above-mentioned specified molecular weight, the effect thereof in skin anti-aging and skin protection (e.g., improving the skin condition or appearance, such as skin anti-aging, especially increasing the collagen content in the skin) can be further improved.

[0060] For example, the colanic acid or the physiologically acceptable salt thereof can be prepared by the method of CN115287314B. In addition, the colanic acid or the physiologically acceptable salt thereof can also be purified by the method of CN114957509A. It should be understood by those skilled in the art that different molecular weight ranges of colanic acid or colanic acid salt can be obtained by conventional preparation methods.

[0061] The term "physiologically acceptable salt" can be any salt derived from colanic acid. In the present application, these salts are not limited to a specific kind 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 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; salts of basic amino acids such as lysine, arginine, histidine, tryptophan, and the like. In the present disclosure, the physiologically acceptable salt of the colanic acid can be sodium colanic acid.

[0062] The composition of the present application can be a pharmaceutical composition or a cosmetic composition. The dosage form of the composition can be selected as necessary. For example, the dosage form of the cosmetic composition can include emulsion, cream, lotion, serum, pack, gel, powder, lip balm, cosmetic base, foundation, wash, ointment, patch, toner, detergent foam, detergent cream, cleansing water, soap, or spray. The dosage form of the pharmaceutical composition can include tablet, capsule, emulsion, dispersion, suspension, solution, syrup, granule, transdermal patch, gel, powder, cream, ointment, suppository, or spray. The pharmaceutical composition can be formulated for topical, oral, intramuscular, subcutaneous, or intravenous administration.

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

[0064] In addition, the pharmaceutical composition or the cosmetic composition of the present application can contain, as necessary, additives generally added to pharmaceutical compositions or cosmetic compositions, in addition to the essential active ingredient. Examples of the additives include oily ingredients, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, antibacterial agents, antioxidants, plant extracts, pH adjustors, alcohols, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, and water, etc.

[0065] In some embodiments, the composition of the present application contains only cariporide or a physiologically acceptable salt thereof as an active ingredient. In some embodiments, the composition of the present application further contains other active ingredients, for example, bovine somatotropin, retinol and its derivatives (e.g., retinol propionate), retinal, retinoic acid, hyaluronic acid, ergothioneine, ectoine, etc.

[0066] In the present disclosure, the molecular weight of cariporide or a physiologically acceptable salt thereof is determined by a liquid chromatography method. The specific method is as follows: accurately weigh the sample and the standard, prepare a 2 mg / mL solution of the sample, filter it into a 1.8 mL injection vial with a 0.22 μm needle filter after complete dissolution. Chromatographic column: PolySep-GFC-P (35*7.8 mm); PolySep-GFC-P 4000 (300*7.8 mm); PolySep-GFC-P 6000 (300*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: differential refractometer 1260-RID.

[0067] Preparation method

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

[0069] The obtained colforsate product is mixed with different concentrations (2000 U / L, 3000 U / L, 5000 U / L) of colforsate degrading enzyme (Baiyin Bioproducts, see SEQ ID No. 2 in CN116334039B) at 37°C for 10-12 h, the sample is collected and heated to inactivate the enzyme, then purified by DEAE ion column, and the small amount of non-uniform molecular weight part and impurities are removed to obtain sodium colforsate products with different molecular weights. The final product can be obtained by lyophilization or spray drying. The obtained sodium colforsate has 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.

[0070] wherein the large molecular weight sodium colforsate (3000-6000 kDa) is prepared by the following method.

[0071] 1. Solid-liquid separation

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

[0073] 2) Flocculation: After adjusting the pH to 9.0-12.0 by adding sodium carbonate solid, flocculate for 1-2 h, and then pre-mix diatomite (diatomite is 1% (w / v) of the fermentation broth).

[0074] 3) Plate and frame filtration: According to the plate and frame filter operation sop, install the filter cloth and compress the filter plate. Circulate the plate and frame filter system with 0.5 M sodium hydroxide solution for 15 min, and then wash with purified water until the pH test paper is neutral. Start the feed pump to pump in the diatomite suspension (pre-coating amount is 0.5 kg / m 2 ), and pre-coat diatomite in the plate and frame. After pre-coating is completed, connect the inlet to the storage tank to pump in the flocculation liquid for plate and frame filter circulation filtration, and control the working pressure <0.2 MPa. Place the outlet of the liquid collection pipe into the feed tank for circulation filtration, and after the liquid at the outlet is clear (turbidity ≤50 NTU), start collecting the filtrate. After completion, wash with 1-2 times the dead volume of purified water.

[0075] 2. Activated carbon adsorption

[0076] 1) Adsorption: 0.5% (w / v) of injection grade activated carbon was added to the fermentation broth, pH was adjusted to 4-6 with 0.1 M HC1, and the mixture was stirred for 1-2 h. After adsorption, diatomite was added (0.1-5% (w / v) of diatomite was added to the fermentation broth).

[0077] 2) Plate and frame filtration: according to the operation SOP of the plate and frame filter, the filter cloth was installed, and the filter plate was compressed. The feed pump was started to pump the diatomite suspension (0.5 kg / m2of pre-coating amount) into the plate and frame to pre-coat diatomite. After pre-coating, the inlet was connected to the liquid storage tank to pump the flocculation liquid for plate and frame filter circulation filtration, and the working pressure was controlled to be <0.2 MPa. The inlet was connected to the filtrate collection tank for circulation filtration. When the liquid at the outlet was clear (turbidity ≤30 NTU), the filtrate was collected. 2

[0078] After filtration, 1-2 times the dead volume of purified water was used for backwashing.

[0079] 3. Precision filtration

[0080] The filtrate was adjusted to pH 7.0±0.2 with sodium carbonate, and then the filtrate was subjected to precision filtration using a 20-inch ultra-high-precision PP filter core in series with a PES filter core. The feed flow rate was slowly increased so that the filtration pressure was ≤0.1 MPa. If the pressure was too high, the filter was back-flushed to empty the liquid, and the filter was replaced for filtration again. When liquid continuously flowed out of the exhaust port, the exhaust valve was closed to collect the filtrate.

[0081] 4. Ceramic membrane concentration

[0082] 1) Cleaning: the ceramic membrane ultrafiltration system was connected, and the system was sequentially washed with 2% citric acid, purified water, and 0.5 M NaOH solution for 15-30 min. Then, the system was washed with purified water until the pH of the filtrate at the permeation end was neutral.

[0083] 2) Concentration: the filtrate after precision filtration was pumped into the circulation liquid tank of the ceramic membrane system, the outlet at the backflow end was connected to the circulation liquid tank, and then the tank low valve was opened. The backflow valve and the valve at one end of the permeation end were fully opened. The system was started, and the frequency was controlled to be 10-80 Hz to control the feed flow rate to be 100-1500 L / h. The inlet pressure was controlled to be <0.1 MPa by setting the alarm pressure to 0.1 MPa (if the pressure was too high, the feed flow rate was reduced). When the volume of the fermentation broth was reduced to 1 / 2 or the inlet pressure was ≥0.1 MPa, the concentration was stopped. The valve at the permeation end was closed, and the ceramic membrane system was washed with 1-5 times the dead volume of the system with purified water for 5-30 min. The liquid at the backflow end was collected again, and the above washing and collection operations were repeated twice. The concentrated liquid and the washing liquid were combined and collected.

[0084] 5. Alcohol precipitation

[0085] ​1) Sodium salt treatment: Add 10-40% sodium chloride solution (final concentration of sodium chloride 1-5%) to the concentrated solution and the washing solution, and stir well. Then, perform fine filtration using 20-inch ultra-high-precision PP filter cartridges in series with PES filter cartridges. Slowly increase the feed flow rate so that the filtration pressure is ≤0.1 MPa. If the pressure is too high, backflush the filtrate and replace the filter cartridges to perform filtration again, and collect the filtrate.

[0086] 2) Ethanol precipitation: Wash the alcohol precipitation tank with citric acid, purified water, and NaOH solution in sequence for 15-30 min, and then wash with purified water until the pH test paper is neutral. Pump the filtrate in the previous step into the alcohol precipitation tank, start stirring, and slowly add 2 times the volume of 95% ethanol. After stirring until a large amount of white flocculent precipitate is generated, stop stirring. Let the precipitate stand for 2-4 h, remove 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 for 20 min, let stand for 1-8 h, remove the supernatant, and collect the precipitate. Then repeat the washing 2 more times and collect the precipitate.

[0089] 2) Dehydration: Add 1-10 times the volume of 95% ethanol to the precipitate, stir for 2 h, let stand for 1-2 h, and remove the supernatant. Repeat the above dehydration operation once more, and collect the precipitate. After vacuum filtration of the precipitate to reduce the residual liquid, add 1-10 times the volume of 95% ethanol again for dehydration. Collect the precipitate, remove the liquid by filtration again, and weigh the wet sample.

[0090] 7. Drying

[0091] Place the wet sample in a vacuum drying oven (the sample thickness should not exceed 2 cm), set the drying temperature to 45°C, and dry for 15 h. Turn the sample over every 4 h.

[0092] 8. Crushing and sieving

[0093] After the second drying is complete, crush the sample again using a crusher, sieve it through a 100-mesh sieve, and pack it into aluminum foil bags.

[0094] Experimental method

[0095] (1) Cytotoxicity

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

[0097] Sample treatment: CA powder was dissolved in culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0098] Experimental treatment: HDF cells were inoculated into a 96-well plate, and sample groups, blank controls (BC), and positive control groups (PC) were set up. Each sample had at least 3 replicate wells for each detection index. After 24 h of culture, the culture solution was discarded, and the sample groups were added with corresponding CA solutions, the positive control groups were added with 10% DMSO solution, and the blank control groups were added with complete culture medium. The culture was continued for 24 h.

[0099] Cell viability detection: The supernatant was discarded, and complete culture medium containing 10% AlamarBlue was placed in an incubator for 4 h of incubation, after which the absorbance value at ex / em = 560 / 590 nm was detected by a microplate reader.

[0100] (2) Light damage

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

[0102] Sample treatment: CA powder was dissolved in culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0103] Experimental treatment: HDF cells were inoculated into a 96-well plate, and sample groups, blank control groups (BC), positive control groups (PC), and negative control groups (NC) were set up. Each sample had at least 3 replicate wells for each detection index. After 24 h of culture, the culture solution was discarded, and the sample groups were added with corresponding CA solutions, the positive control groups were added with VC solution (dissolved in culture medium), the negative control groups and the blank control groups were added with complete culture medium. The culture was incubated in an incubator for 2 h. The sample groups, the positive control groups, and the negative control groups were placed under a UVA lamp for irradiation, and the irradiation amount was 5 J / cm 2 . After irradiation, the culture was continued for 24 h.

[0104] Cell viability detection: The supernatant was discarded, and complete culture medium containing 10% AlamarBlue was placed in an incubator for 4 h of incubation, after which the absorbance value at ex / em = 560 / 590 nm was detected by a microplate reader.

[0105] Collagen type I detection: The supernatant of each well was collected, and the test operation was performed according to the operation instruction of the ELISA collagen detection kit (Huami Biotechnology, CSB-E08082h).

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

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

[0108] Sample processing: CA powder was dissolved in the culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0109] Experimental treatment: HDF cells were inoculated into a 96-well plate, and sample groups, blank controls (BC), and positive control groups (PC) were set up. Each sample had at least 3 replicate wells for each detection index. After 24 h of culture, the culture solution was discarded, the sample group was added with the corresponding CA solution, the positive control group was added with TGF-β solution, and the negative control group and the blank group were added with complete culture medium. Continue to culture for 24 h.

[0110] I, III type collagen detection: The supernatant of each well was collected, and the test operation was performed according to the operation instruction of the ELISA collagen detection kit (Huawmei Biological, 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℃, 5% CO2 incubator.

[0113] Sample processing: CA powder was dissolved in the culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0114] Experimental treatment: RAW264.7 cells were inoculated into a 96-well plate, and sample groups, blank controls, positive control groups, and negative control groups were set up. Each sample had at least 3 replicate wells for each detection index. After 24 h of culture, the culture solution was discarded, the sample group was added with the corresponding CA solution, the positive control group was added with complete culture medium containing 100 g / mL dexamethasone, and the negative control group and the blank group were added with complete culture medium. After 2 h of incubation in the incubator, 1 μg / mL LPS (lipopolysaccharide) was added to the sample group, the positive control group, and the negative control. Continue to culture for 24 h.

[0115] Inflammatory factor detection: The supernatant of each well was collected, and the test operation was performed according to the operation instruction of the ELISA kit (Huawmei Biological, CSB-E04740h, CSB-E08053h, CSB-E04638h).

[0116] Active oxygen ROS detection: After collecting the supernatant, the cells in each well were washed twice with PBS, and the test operation was performed according to the operation instruction of the DCFH-DA kit.

[0117] (5) Inflammation (keratinocytes)

[0118] Cell culture: HaCaT cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) and incubated at 37℃ in a 5% CO2 incubator.

[0119] Sample processing: CA powder was dissolved in the culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0120] Experimental treatment: Cells were seeded into a 96-well plate, and sample groups, blank control groups, positive control groups, and negative control groups were set up. Each sample had at least 3 replicate wells for each detection index. After 24 h of culture, the culture medium was discarded, and the sample group was added with the corresponding CA sample solution, the positive control group was added with the corresponding VC solution, and the negative control group and the blank control group were added with complete culture medium. After 2 h of incubation in the incubator, hydrogen peroxide solution (0.8 mM HX0640 Sigma-Aldrich) was added to the sample group, the positive control group, and the negative control group for inflammation stimulation. Continue to culture for 24 h.

[0121] Gene detection: The old liquid was aspirated, washed twice with PBS, and 10 μL of cell lysis buffer was added to each well. After lysing the cells by blowing, the sample was collected. After extracting RNA and reverse transcribing to cDNA, fluorescence quantitative PCR detection was performed, and the results were calculated using the 2-△△CT method. The calculated 2-△△Ct value was used to obtain the expression difference of the target gene in the two groups of samples.

[0122] (6) Transdermal experiment

[0123] Preparation of ex vivo skin

[0124] The ex vivo skin stored at -20℃ was thawed at room temperature with deionized water and repeatedly washed with PBS buffer.

[0125] Determination of transdermal absorption capacity

[0126] 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 facing the supply chamber and the dermis layer side facing the receiving chamber;

[0127] 2) Add receiving liquid to the receiving chamber. After the skin was tightly fixed, add receiving liquid (PBS) to the receiving chamber through the sampler, and exhaust the air to make the dermis layer of the skin in close contact with the receiving liquid;

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

[0129] 4) Penetration: Start the electromagnetic stirrer at a speed of 300 rpm and maintain a constant temperature water bath at (32±1)℃;

[0130] 5) At 1h, 6h, 18h time points, collect skin samples, wipe the skin surface with PBS, wipe off the residual liquid on the surface, and cut the skin with a blade. Freeze section and wait for use.

[0131] Observation analysis

[0132] Observe the skin sections with a fluorescence microscope, take pictures, and analyze the fluorescence intensity with Image J.

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

[0134] 1) Cell inoculation: Inoculate fibroblasts into a 6-well plate at a seeding density of 2.2 x 10 5 cells / well, and incubate in an incubator (37°C, 5% CO2) overnight.

[0135] 2) Dosing: According to the test grouping in Table 2, when the plating rate of cells in the 6-well plate reaches 40-60%, group dosing is performed, 2mL per well, and 3 replicates per group. After dosing, place the 6-well plate in an incubator (37°C, 5% CO2) for 24h.

[0136] 3) UVA irradiation: According to the test grouping, UVA irradiation of 30 J / cm2 is performed on groups that require UVA irradiation, and the plate is placed in an incubator (37°C, 5% CO2) for 24h.

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

[0138] 5) ELISA detection: Detection is performed according to the operating instructions of the ELISA kit (Huamaibio CSB-E08082h).

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

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

[0141] Sample treatment: Dissolve CA powder in the culture medium, filter sterilize with a 0.2μm sterile filter after complete dissolution.

[0142] Experimental treatment: HDF cells were inoculated into 96-well plates, and sample groups, blank controls, and positive control groups were set up. Each detection index had at least 3 replicate wells for each sample. After 24 h of culture, the culture solution was discarded, and the corresponding CA solution was added to the sample groups, the TGF-β solution was added to the positive control group, and the complete culture medium was added to the negative control group and the blank group. The sample groups, the positive control group, and the negative control group were placed under a UVA lamp for irradiation at a power of 40 mW for 4 min. After irradiation, the culture was continued for 24 h.

[0143] I, III type collagen detection: The supernatant of each well was collected, and the experimental operation was performed according to the operation instruction of the ELISA collagen detection kit (Huawmei Biological, CSB-E04799h).

[0144] (9) Cell viability-CCK8 detection

[0145] Plate seeding: After the keratinocyte HaCaT was recovered and cultured for 1-2 days to a cell confluence of 70%, the keratinocytes were trypsinized and seeded at 5×10 4 / well, and were grouped into DMEM, H2O2-DMEM, and H2O2-CA.

[0146] H2O2 stimulation: H2O2 (Shanghai Lingfeng Chemical Reagent) was used at a concentration of 0.8 mM, which was diluted to 10 mM and then added to the culture medium at 100× (the original storage concentration was 8820 mM, 8.82 mL of PBS was added to 10 μL of H2O2, and then 40 μL of the diluted solution was added to 4 mL of culture medium). The prepared culture medium containing H2O2 was added to the H2O2 stimulation group in the form of medium replacement, and the culture was continued for 2 h.

[0147] CA treatment: 50 mg of CA powder sterilized by ultraviolet was added to 5 mL of PBS, shaken to dissolve to form a liquid at a concentration of 10 mg / mL, and then diluted with DMEM culture medium to a working concentration of 5 mg / mL. After the culture supernatant after H2O2 stimulation was removed (containing the DMEM blank control group), each sample was rinsed with 100 μL of PBS, 100 μL of culture medium containing CA was added to the H2O2-CA group, and 100 μL of culture medium was added to the DMEM and H2O2-DMEM groups, and the culture was continued for 24 h.

[0148] CCK8 detection: The volume of the culture medium required for detection was calculated, and CCK8 (Biyun Tian, C0038) was added at 10×, i.e., 400 μL of CCK8 was added to 4 mL of culture medium. After the culture medium supernatant of each sample was removed, it was added to a 96-well plate at 100 μL / well, and incubated for 1-2 h. The detection was performed by an enzyme marker at OD450 under light shielding. Example

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

[0150] In the following examples, unless otherwise specified, the orotic acid used is sodium orotate with 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) and incubated at 37 degrees in a 5% CO2 incubator.

[0153] Sample processing: CA powder was dissolved in the culture medium and sterilized by filtering through a 0.2 pm sterile filter after complete dissolution.

[0154] Experimental treatment: HDF cells were seeded into a 96-well plate, and sample groups, blank controls, positive control groups, and negative control groups were set up. Each sample had at least 3 replicate wells for each detection index. After 24 hours of culture, the culture medium was discarded, and the sample groups were added with the corresponding CA solution, the positive group was added with VC solution (dissolved in culture medium), and the negative control group and blank group were added with complete culture medium. Incubate in the incubator for 24 hours. Then the cells were taken out and frozen, and the transcriptome analysis was entrusted to Kintech.

[0155] Figure 3 shows that oligomeric CA (Oli-CA) promotes the transcription of AMPK, PGC1a, and Cab39 (Figure 3A), and NAMPT, sirtuin1, sirtuin6, and sirtuin7 (Figure 3B) genes in fibroblasts under ultraviolet conditions. The results of Figure 3 show that oligomeric orotic acid promotes the expression of sirtuin1 in human fibroblasts.

[0156] Example 2

[0157] After adding oligomeric orotic acid (2.5 mg / mL), HA (hyaluronic acid, 2.5 mg / mL), Ectoin (2.5 mg / mL), NC (only ultraviolet light), PC (ubiquinol 1 mg / mL), and BC (only cells) in fibroblasts for 24 hours, then irradiated by ultraviolet light, the cell survival rate was observed, and the transcriptional effect of oligomeric CA on human fibroblast BAX and NF-kB inhibitor-related proteins was tested, and the results are shown in Figure 4. The results of Figure 4 show that oligomeric CA inhibits the transcription of BAX and upregulates the transcription of NF-kB inhibitor protein (Figure 4A); cell viability test after cells are irradiated after adding oligomeric CA, HA, Ectoin, and vitamin C (positive control). From the above experimental results, it can be seen that oligomeric orotic acid can significantly improve the survival rate of cells under ultraviolet conditions and has good efficacy in inhibiting cell apoptosis.

[0158] Example 3

[0159] According to the method of inflammation (macrophage) in the experimental method, the oligo CA was tested for its effect on inhibiting inflammation of mouse macrophages, and the results are shown in Figure 5. As can be seen from Figure 5, oligo colanic acid can effectively inhibit the inflammatory response of macrophages caused by LPS (lipopolysaccharide), including TNF-a (Figure 5A) and IL-6 (Figure 5B) and the like.

[0160] Example 4

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

[0162] Example 5

[0163] The transcriptional analysis of SOD2 and GPX1 / 8 of human fibroblasts and the effect of oligo CA on the removal of reactive oxygen species (Solebo, ROS detection kit) were tested, and the results are shown in Figure 7. As can be seen from Figure 7, oligo CA can greatly up-regulate the transcriptional level of SOD2, GPX1 and GPX8 of human fibroblasts (Figure 7A), and at the same time, the reactive oxygen species produced by the human fibroblasts under ultraviolet irradiation is significantly inhibited (Figure 7B). This shows that oligo colanic acid has good activity in regulating the removal of reactive oxygen species by cells.

[0164] Example 6

[0165] According to the experimental method of anti-wrinkle (collagen promotion) in the experimental method, the transcriptional data of oligo CA for promoting HDF cells to synthesize type I collagen and up-regulating different types of collagen of HDF cells were tested, and the results are shown in Figure 8. Figure 8 shows that oligo CA can significantly increase the amount of type I collagen synthesized by HDF cells (the data in the figure is measured by diluting the supernatant with culture medium by 10 times). As can be seen from Figure 8A, the collagen regeneration activity of oligo CA is higher than that of Pro-Xylane and retinyl propionate (RP) under the same concentration conditions. Its collagen promotion activity is 4 times that of Pro-Xylane (2 mg / mL) and 3-4 times that of retinyl propionate (3 mg / mL, 10 mg / mL) under the same concentration. In addition, the transcriptional up-regulation of oligo CA on many other types of collagen, including types 4, 6, 7, 9, 17, 22, 25 and the like can also be observed from the transcriptional analysis results of HDF cells (Figure 8B). This result shows that oligo colanic acid has good application prospect in collagen promotion activity.

[0166] Example 7

[0167] The phototoxicity of oligo-collalic acid was tested according to the experimental method of photodamage in the experimental method, and the results are shown in Figure 9. Figure 9 shows that HDF cells were irradiated after adding oligo-collalic acid CA, and no obvious cytotoxicity was observed (cell activity > 90% can be considered non-toxic), on the contrary, with the increase of the amount of oligo-collalic acid added, the protective effect on cells increased from 90% at 2 mg / mL to 97% at 10 mg / mL. Retinyl propionate has significant cytotoxicity at 3 mg / mL and 10 mg / mL.

[0168] Example 8

[0169] According to the method of anti-wrinkle collagen promotion under light irradiation in the experimental method, the activity of oligo-collalic acid in promoting the synthesis of type I and type III collagen of HDF cells under light irradiation was tested, and the results are shown in Figure 10.

[0170] Figure 10 shows that oligo-collalic acid shows stronger activity in promoting type I and type III collagen in the light irradiation HDF cell model. Compared with bovine, at a concentration of 2 mg / mL, oligo-collalic acid promotes type I collagen by 2.3 times and type III collagen by 2.8 times. Compared with retinyl propionate, at the same concentration, it promotes type I collagen by 18 times (3 mg / mL) and 2 times (10 mg / mL), and type III collagen by 10 times (3 mg / mL) and 28 times (10 mg / mL). Therefore, oligo-collalic acid not only has no phototoxicity, but also has the activity of promoting type I and type III collagen at the same time, and its efficacy is significantly higher than that of bovine and retinyl propionate.

[0171] Example 9

[0172] The inhibitory effect of oligo-collalic acid on MMP9 of the ultraviolet irradiated skin model (Huawmei Biological, CSB-PA002676) was tested, and the proteomic data under the condition of ultraviolet irradiation of the ex vivo skin was obtained. From Figure 11, it can be observed that the content of MMP9 in the collalic acid added group is significantly reduced. MMP9 is a key target for inhibiting DEJ aging. The increase of MMP9 content in the skin will cause the rapid loss of type VI collagen in the DEJ layer, and then cause the DEJ to become smooth. The DEJ layer is the connecting layer between the dermis layer and the epidermis layer, and the smoother it is, the less contact there will be between the true epidermis, and the skin aging will be more severe.

[0173] Example 10

[0174] According to the experimental method of inflammation (keratinocytes) in the experimental method of cell culture and modeling, TNF-a, IL-6 and COX-2 (ELSIA kit: Huamei Biology, CSB-E04740h, CSB-E04638h, CSB-E10103h) were detected to test the anti-inflammatory effect of oligo colanic acid on human keratinocytes. Figure 12 shows that oligo colanic acid has good anti-inflammatory effect on human keratinocytes, and can significantly inhibit the expression of TNF-a, IL-6 and COX-2 at 1 mg / mL.

[0175] Example 11

[0176] According to the method of transdermal experiment in the experimental method, the transdermal property of oligo colanic acid was tested. Figure 13 shows that after the fluorescence labeling of oligo colanic acid, the transdermal amount of the molecule can be observed to increase significantly within 18h by observing the transdermal test of ex vivo skin, and the fluorescence intensity is enhanced by 9 times. At the same time, it can be observed that the molecule can effectively penetrate into the dermis.

[0177] The localization of oligo colanic acid in cells was tested by the following method.

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

[0179] Sample treatment: 1 mg / mL of fluorescently labeled CA powder was dissolved in the culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter to remove bacteria.

[0180] Experimental treatment: HDF cells were inoculated into a 96-well plate, and sample groups, blank controls and positive control groups were set up. Each sample had at least 3 replicate wells for each detection index. After 24-48h of culture, the cells were removed, and DAPI and Mitotracker staining solution was added to stain the cell nucleus and mitochondria, respectively. After each staining, the cells were washed with pbs for two to three times, and finally the fluorescence was observed under an optical microscope.

[0181] Figure 14 shows that oligo colanic acid can effectively enter the cell interior, and through the mitochondrial staining of mitotracker, it can be observed that oligo colanic acid (green) and mitochondria (red) have overlap, from which it can be seen that oligo colanic acid can penetrate the cell membrane and enter the mitochondria. On the contrary, colanic acid and the nucleus (blue) do not have overlap phenomenon, and thus we believe that oligo colanic acid is difficult to enter the nucleus.

[0182] In addition, the results of the cytotoxicity test of oligo colanic acid (Figure 15) show that the molecule has no cytotoxicity below the concentration of 10 mg / mL.

[0183] Example 12: Human experimental test

[0184] The human experiment of oligo sodium coloate was carried out according to the following method, and the results are shown in Figures 16-25.

[0185] 66 Chinese healthy male / female subjects with dry, loose, lack of elasticity, skin elasticity R2 between 0.35 and 0.55 on the face; fine lines or wrinkles on the face, such as wrinkles around the eyes, grade 2 to 5; weak skin barrier, sensitive skin, were randomly divided into two groups, 33 people in each group, aged 30 to 60 years old. Using the method of before and after control, inter-group control, the water content of the stratum corneum of the subjects, the skin transepidermal water loss value, the skin elasticity, the skin tightness, the dermal density, the dermal thickness, the whole skin thickness were measured, and the local and whole face photos were taken. The test results were compared by statistical test method to determine whether there was a statistical difference.

[0186] Detection site

[0187] (1) Stratum corneum water content - cheek

[0188] (2) Skin transepidermal water loss value - cheek

[0189] (3) Skin elasticity R2 - cheek

[0190] (4) Skin tightness F4 - cheek

[0191] (5) UC22 dermal density - cheek

[0192] (6) UC22 dermal thickness - cheek

[0193] (7) UC22 whole 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 - whole face photograph analysis cheek

[0198] (12) VISIA CR skin color b value - whole face photograph analysis cheek

[0199] (13) VISIA CR skin color ITA ° value - whole face photograph analysis cheek

[0200] (14) VISIA CR skin transparency parameter - whole face

[0201] (15) VISIA CR Skin Gloss Parameter - Full Face Photograph Analysis Cheek

[0202] (16) VISIA CR Skin Red Area Percentage - Full Face Photograph Analysis Cheek

[0203] (17) VISIA CR Periorbital Wrinkle Area Percentage - Full Face Photograph Analysis Periorbital

[0204] (18) VISIA CR Pore Area Percentage - Full Face Photograph Analysis Cheek

[0205] (19) VISIA CR Jawline Angle - Full Face Photograph Analysis Mandible

[0206] (20) Epidermal-Dermal Junction (DEJ) Layer Standardized Area DEJI - Periorbital

[0207] (21) Aging Index ELCOR - Periorbital

[0208] (22) Aging Index SAAID - Periorbital

[0209] (23) Epidermal Layer Thickness TED - Periorbital

[0210] (24) Elastin Fiber Fluorescence Intensity - Periorbital

[0211] (25) Collagen Fiber Harmonic Intensity - Periorbital

[0212] (26) Periorbital Wrinkle Grade Assessment - Periorbital

[0213] (27) Suborbital Wrinkle Grade Assessment - Suborbital

[0214] (28) Glossiness Score - Cheek

[0215] (29) Elasticity Score - Cheek

[0216] (30) Smoothness Score - Cheek

[0217] (31) Radiance Score - Cheek

[0218] (32) Self-Assessment - Full Face

[0219] Day 0:

[0220] The subject visits, and the skin condition of the face of the subject is evaluated by the technician, and among the subjects participating in the experiment, those with dry skin or redness and wrinkles are selected;

[0221] Clean face and wait: subjects clean their face with cleansing product and dry the skin with dry facial tissue, sit in the laboratory for 20 min at temperature 21℃±1℃, humidity 50%RH±10%RH;

[0222] Test subject skin baseline data collection: the laboratory technician operates the SUPERVISION 780, VISIA CR, Cutometer, PRIMOS CR, Corneometer, Vapometer, UC22 instruments to measure the relevant indicators of the test site of the subjects;

[0223] Professional rating of eye wrinkles of the subjects by trained dermatologists and record;

[0224] Score and record the skin glossiness, delicacy, smoothness, elasticity of the subjects by professional assessors;

[0225] The laboratory technician guides the subjects to use the product according to the product use requirements, and provides written test precautions and product use instructions. According to the random table, the subjects are randomly divided into two groups, and one person is randomly selected from each group to use the product on half of the face. 2h and 4h after using the product, the subjects are tested for facial multi-point moisture.

[0226] The subjects take the product and leave the laboratory.

[0227] Day 14:

[0228] The subjects return for a visit, clean their face and wait: clean their face with cleansing product and dry the skin with dry facial tissue, sit in the laboratory for 20 min at temperature 21℃±1℃, humidity 50%RH±10%RH, and complete the D14 self-evaluation questionnaire during the 20 min sitting period;

[0229] Test subject skin D14 data collection: the laboratory technician operates the SUPERVISION 780, VISIA CR, Cutometer, PRIMOS CR, Corneometer, Vapometer, UC22 instruments to measure the relevant indicators of the test site of the subjects;

[0230] Professional rating of eye wrinkles of the subjects by trained dermatologists and record;

[0231] Score and record the skin glossiness, delicacy, smoothness, elasticity of the subjects by professional assessors;

[0232] The staff checks the use records and products of the subjects;

[0233] The subjects leave the laboratory.

[0234] Day 28:

[0235] Subject returns for visit, cleans face and waits: Cleans face with facial cleanser and dries skin with a dry paper towel, sits for 20 min in a laboratory at temperature 21 °C ± 1 °C, humidity 50% RH ± 10% RH, and completes the D28 self-assessment questionnaire during the 20 min sit;

[0236] D28 data collection on test subject skin: Laboratory technician operates SUPERVISION 780, VISIA CR, Cutometer, PRIMOS CR, Corneometer, Vapometer, UC22 instruments to measure relevant parameters on test subject’s test site;

[0237] Professional grading of eye wrinkles on test subject by trained dermatologist and recorded;

[0238] Professional grader scores and records skin shine, smoothness, elasticity, and radiance on test subject;

[0239] Staff checks and recovers test subject’s usage records and product;

[0240] Test subject leaves the laboratory;

[0241] End of test procedure.

[0242] Figure 16 shows the effect of oligo sodium chondroitin sulfate on the density of human dermis. As can be seen from Figure 16, the UC22 dermal density of the test subject increased by 22.87% after 14 days of continuous use of the serum containing 0.5% (w / v) oligo sodium chondroitin sulfate, and increased by 24.00% after 28 days of continuous use.

[0243] Figure 17 shows the effect of oligo sodium chondroitin sulfate on the expression of collagen in human dermis. As can be seen from Figure 17, the harmonic intensity of collagen fibers of the test subject increased by 12.31% after 14 days of continuous use of the serum containing 0.5% (w / v) oligo sodium chondroitin sulfate, and increased by 21.61% after 28 days of continuous use.

[0244] Figure 18 shows the effect of oligo sodium chondroitin sulfate on the expression of elastin in human dermis. As can be seen from Figure 18, the fluorescence intensity of elastin fibers of the test subject increased by 17.02% after 14 days of continuous use of the serum containing 0.5% (w / v) oligo sodium chondroitin sulfate, and increased by 22.13% after 28 days of continuous use.

[0245] Figure 19 shows the effect of oligo sodium colate on the elasticity and skin fineness of human skin. As can be seen from Figure 19, after the subjects continuously used the serum containing 0.5% (w / v) oligo sodium colate for 14 days, the skin elasticity R2 increased by 24.44%, and the skin tightness F4 decreased by 6.62%; after 28 days, R2 increased by 40.00%, and F4 decreased by 12.00%.

[0246] Figure 20 shows the effect of oligo sodium colate on the standardized area of DEJ of human skin. As can be seen from Figure 20, after the subjects continuously used the serum containing 0.5% (w / v) oligo sodium colate for 14 days, the standardized area DEJI of the epidermal dermal junction DEJ layer increased by 28.70%; after 28 days, it increased by 36.11%.

[0247] Figure 21 shows the effect of oligo sodium colate on the thickness of the epidermis layer of human skin. As can be seen from Figure 21, after the subjects continuously used the serum containing 0.5% (w / v) oligo sodium colate for 14 days, the thickness of the epidermis layer increased by 27.78%, and after 28 days, it increased by 27.29%.

[0248] Figure 22 shows the effect of oligo sodium colate on the improvement of skin redness of human skin. As can be seen from Figure 22, after the subjects continuously used the serum containing 0.5% (w / v) oligo sodium colate for 28 days, the area ratio of VISIA-CR skin red region decreased by 10.45%.

[0249] Figure 23 shows the effect of oligo sodium colate on the 3D skin fibulin (FLG) content. As can be seen from the 3D skin test results of Figure 23, compared with the BC control group, the fibulin (FLG) content significantly increased by 49.00% at a concentration of 0.5% (w / v) of oligo sodium colate.

[0250] Figure 24 shows the effect of oligo sodium colate on the 3D skin aquaporin 3 (AQP3) content. As can be seen from the 3D skin test results of Figure 24, compared with the BC control group, the aquaporin 3 (AQP3) content significantly increased by 37.00% at a concentration of 0.5% (w / v) of oligo sodium colate.

[0251] Figure 25 shows the effect of oligo sodium colate on the water retention and moisturizing of human skin. As can be seen from Figure 25, after the subjects continuously used the serum containing 0.5% (w / v) oligo sodium colate for 14 days, the water content of the stratum corneum skin significantly increased by 31.76%; after 28 days, it increased by 63.84%. The transepidermal water loss value decreased by 12.57% compared with the base value; after 28 days, it decreased by 20.08%.

[0252] Example 13

[0253] Sodium colaurum of molecular weight 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 were prepared similarly with reference to the preparation method and tested for their properties and efficacy similarly as in Examples 1-12.

[0254] Example 14

[0255] The effect of sodium colaurum (CA) of different molecular weights in stimulating the expression of collagen type I by human fibroblasts was tested at a concentration of 3 mg / mL according to the method for anti-wrinkle (pro-collagen) in the experimental method, and the results are shown in Figure 26. As can be seen from Figure 26, oligomeric sodium colaurum with molecular weight in the range of 1-3 kDa and 3-10 kDa has better anti-aging pro-collagen activity.

[0256] The skin protection effect of sodium colaurum (CA) of different molecular weights was tested according to the method for cell viability-CCK8 in the experimental method, and the results are shown in Figure 27. As can be seen from Figure 27, oligomeric sodium colaurum with molecular weight in the range of 1-3 kDa and 3-10 kDa has better skin protection effect.

[0257] INCORPORATED BY REFERENCE

[0258] The entire contents of each patent and scientific document referred to herein is incorporated by reference herein for all purposes.

[0259] EQUIVALENCIES

[0260] The present application can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the foregoing embodiments are to be considered in all respects only as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency 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, in skin anti-aging and / or skin protection, characterized in that, 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.

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

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

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

5. The use according to any one of claims 1 to 4, wherein, The content of the colanic acid or the 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 the colanic acid is sodium colanic acid.

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

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

9. A composition characterized in that, The composition comprises the colanic acid or the 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 of claim 9, wherein, The content of the colanic acid or the physiologically acceptable salt thereof 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 comprises only the colanic acid or the physiologically acceptable salt thereof as an active ingredient.

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

13. The composition according to any one of claims 9-12, wherein, The composition further contains a cosmetically or pharmaceutically acceptable additive.

14. The composition according to any one of claims 9-13, wherein, The physiologically acceptable salt of the colanic acid is sodium colanic acid. The composition comprises the colanic acid or the 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. The content of the colanic acid or the physiologically acceptable salt thereof in the composition is 0.01-10% (w / v), preferably 0.1-5% (w / v), more preferably 0.5-3% (w / v). The composition comprises only the colanic acid or the physiologically acceptable salt thereof as an active ingredient. The composition is formulated for topical, oral, intramuscular, subcutaneous or intravenous administration. The composition further contains a cosmetically or pharmaceutically acceptable additive. The physiologically acceptable salt of the colanic acid is sodium colanic acid.

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