Use of composition comprising colanate

Abstract

The present disclosure relates to the fields of skin care products, beauty treatments, and pharmaceuticals and specifically provides use of a composition comprising a colanate. The colanate of the present disclosure has a good effect of improving the skin condition or appearance.

Description

Use of a composition comprising colanic acid salt

[0001] Cross-reference to Related Applications

[0002] The present disclosure claims priority to Chinese application CN2024108514793 filed on June 27, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure belongs to the field of skin care, cosmetics and medicine, and specifically relates to a use of a composition comprising colanic acid salt. BACKGROUND

[0004] 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 life activities to adapt to environmental changes 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 viability than wild-type strains. In 2017, Han et al. reported that feeding purified CA or feeding CA-secreting E. coli could 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. SUMMARY

[0005] In one aspect, the present disclosure provides a use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, in increasing skin water content, wherein the colanic acid or the physiologically acceptable salt thereof has a molecular weight in the range of 10-6000 kDa.

[0006] In some embodiments, the colanic acid or the physiologically acceptable salt thereof is present in an amount of 0.001-2% (w / v), 0.01-1% (w / v), or 0.05-0.5% (w / v).

[0007] In some embodiments, the colanic acid or the physiologically acceptable salt thereof has a molecular weight in the range of 10-50 kDa or 1000-5000 kDa.

[0008] In another aspect, the present disclosure provides a use of colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof, in enhancing the protective function of skin cells, such as increasing cell viability, wherein the colanic acid or the physiologically acceptable salt thereof has a molecular weight in the range of 10-6000 kDa.

[0009] 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, in skin anti-aging, wherein the molecular weight of the colanic acid or the physiologically acceptable salt thereof is in the range of 10-6000 kDa.

[0010] In some embodiments, the skin anti-aging comprises skin anti-wrinkling, skin anti-aging, increasing collagen content, preferably type I and / or type III collagen content, in the skin.

[0011] 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, in improving red blood vessels caused by skin allergy or skin inflammation, wherein the molecular weight of the colanic acid or the physiologically acceptable salt thereof is in the range of 10-6000 kDa.

[0012] In some embodiments, the molecular weight of the colanic acid or the physiologically acceptable salt thereof is in the range of 10-50 kDa, 300-500 kDa, 600-800 kDa, 1000-2000 kDa, or 3000-5000 kDa.

[0013] In some embodiments, the content of the colanic acid or the physiologically acceptable salt thereof is 0.001-10% (w / v), 0.05-5% (w / v), or 0.1-3% (w / v).

[0014] In some embodiments, the composition comprises only the colanic acid or the physiologically acceptable salt thereof as an active ingredient.

[0015] In some embodiments, the physiologically acceptable salt of the colanic acid is sodium colanic acid.

[0016] In some embodiments, the composition is formulated for topical, oral, intramuscular, subcutaneous, or intravenous administration.

[0017] In some embodiments, the composition is formulated for subcutaneous administration.

[0018] In some embodiments, the composition further contains a cosmetically, cosmetically, or pharmaceutically acceptable additive.

[0019] 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 manufacture of a medicament or a cosmetic for increasing skin water content, enhancing the protective function of skin cells, skin anti-aging, and / or improving red blood vessels caused by skin allergy or skin inflammation.

[0020] In another aspect, the present disclosure provides a method of increasing skin moisture content, enhancing the protective function of skin cells, skin anti-aging, and / or improving red blood vessels or skin inflammation caused by skin allergy, which comprises providing colanic acid or a physiologically acceptable salt thereof, or a composition comprising colanic acid or a physiologically acceptable salt thereof as described above. BRIEF DESCRIPTION OF DRAWINGS

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

[0022] FIG. 1 shows the mechanism of protection of epidermal cells by macromolecular sodium colanic acid.

[0023] FIG. 2 shows the effect of sodium colanic acid on inhibiting reactive oxygen species / reactive nitrogen species in ultraviolet irradiated HaCaT cells.

[0024] FIG. 3 shows the effect of sodium colanic acid on inhibiting inflammation in human keratinocytes.

[0025] FIG. 4 shows the effect of sodium colanic acid on inhibiting the rise of DNA damage markers caused by ultraviolet irradiation and reducing cell aging and apoptosis.

[0026] FIG. 5 shows the protective effect of sodium colanic acid on cell mitochondria.

[0027] FIG. 6 shows the effect of sodium colanic acid on promoting cell proliferation and migration by upregulating cell CD44.

[0028] FIG. 7 shows the results of cell experiments on the collagen activity-promoting effect of sodium colanic acid.

[0029] FIG. 8 shows the effect of sodium colanic acid on increasing the content of 3D skin fibulin (FLG).

[0030] FIG. 9 shows the effect of sodium colanic acid on increasing the content of 3D skin aquaporin 3 (AQP3).

[0031] FIG. 10 shows the results of cytotoxicity tests of sodium colanic acid.

[0032] FIG. 11 shows the results of anti-wrinkle efficacy tests of sodium colanic acid.

[0033] FIG. 12 shows the results of redness and sensitivity reduction efficacy tests of sodium colanic acid.

[0034] FIG. 13 shows the results of water retention and moisturizing efficacy tests of sodium colanic acid.

[0035] FIG. 14 shows the skin protection effects of sodium colanic acid with different molecular weights.

[0036] FIG. 15 shows the effect of sodium colanic acid with different molecular weights on stimulating human fibroblasts to express type I collagen.

[0037] Figure 16 shows the effect of sodium colaurate of different molecular weights on the migration of HaCaT cells.

[0038] Figures 17A to 17C show the effect of sodium colaurate of different molecular weights on the concentration of TNF-a, IL-6 and IL-1 b.

[0039] Figure 18 shows the results of skin penetration test of sodium colaurate.

[0040] Figure 19 shows the results of mitochondrial localization experiment of sodium colaurate.

[0041] In the figures, * indicates P<0.05 relative to the negative control, ** indicates P<0.01 relative to the negative control, *** indicates P<0.001 relative to the negative control, ## indicates P<0.01 relative to the blank control, and ### indicates P<0.001 relative to the blank control. DETAILED DESCRIPTION

[0042] Various different features and aspects of the present application are discussed in more detail below.

[0043] Figure 1 shows the protective effect of macromolecular colaurate on epidermal cells and the anti-aging mechanism on the dermal layer. For epidermal cells, colaurate can reduce cell damage of keratinocytes under oxidative stress, and can also promote keratinocyte migration and proliferation. At the same time, sodium colaurate can also penetrate into the dermal layer and into the mitochondria of dermal cells, exerting the effect of promoting collagen synthesis of dermal cells and maintaining cell mitochondrial homeostasis. Macromolecular colaurate (10-6000 kDa) can exert effects in the following 7 aspects, including inhibition of reactive oxygen species / reactive nitrogen species, inflammatory factors, DNA damage, mitochondrial damage, metal matrix protease (MMP3 / 9), malondialdehyde (MDA), etc. and promotion of epidermal cells to up-regulate CD44 expression. These mechanisms can respectively play the effects of inhibiting inflammatory response, reducing zombie cells, reducing skin wrinkles and promoting epidermal integrity.

[0044] In the present disclosure, colanic acid (CA) or a physiologically acceptable salt thereof can be prepared by any method as long as a desired molecular weight is obtained. Specifically, the molecular weight of the colanic acid or the physiologically acceptable salt thereof can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1500 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa, 6000 kDa, or within any range constituted by the aforementioned molecular weights. For example, the molecular weight of the colanic acid or the physiologically acceptable salt thereof can be within any of the following ranges: 10-6000 kDa, such as 50-5000 kDa, 100-5000 kDa, 1000-5000 kDa, 1000-4000 kDa, 1000-3000 kDa, 1000-2000 kDa, 2000-5000 kDa, 2000-4000 kDa, 2000-3000 kDa, 3000-5000 kDa, 3000-4000 kDa, 300-500 kDa, 600-800 kDa, 10-1000 kDa, 10-100 kDa, 10-50 kDa, 10-30 kDa, 10-20 kDa, 10-18 kDa, 10-15 kDa, or 10-12 kDa.

[0045] The inventors of the present application have surprisingly found that by selecting colanic acid or a salt thereof having a specified molecular weight, the efficacy of the same in increasing skin moisture, enhancing the protective function of skin cells, skin anti-aging, and in particular, increasing the collagen content in the skin can be further improved.

[0046] In some embodiments, the molecular weight of the colanic acid or the physiologically acceptable salt thereof is within the range of 10-100 kDa, 10-50 kDa, or 10-15 kDa.

[0047] In some embodiments, the molecular weight of the colanic acid or the physiologically acceptable salt thereof is within the range of 100-1000 kDa, 300-500 kDa, or 600-800 kDa.

[0048] In some embodiments, the molecular weight of the colanic acid or the physiologically acceptable salt thereof is within the range of 1000-5000 kDa, 1000-2000 kDa, or 3000-5000 kDa.

[0049] For example, the colanic acid or a physiologically acceptable salt thereof can be prepared by the method of CN115287314B. In addition, the colanic acid or a physiologically acceptable salt thereof can be purified by the method of CN114957509A. It will be understood by those skilled in the art that the colanic acid or a salt thereof can be obtained in different molecular weight ranges by conventional preparation methods.

[0050] The term "physiologically acceptable salt" can be any salt derived from the colanic acid. In the present disclosure, the 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 is, for example, sodium colanic acid.

[0051] The composition of the present disclosure can be 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 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; and 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.

[0052] In the composition of the present disclosure, the content of the colanic acid or a physiologically acceptable salt thereof can be selected as needed, for example, can be 0.001-10% (w / v), 0.05-5% (w / v), or 0.1-3% (w / v).

[0053] In addition, the pharmaceutical composition or the cosmetic composition of the present disclosure can contain, as needed, additives commonly 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.

[0054] In some embodiments, the composition of the present application only contains colanic acid or its physiologically acceptable salt as an active ingredient. In some embodiments, the composition of the present application also contains other active ingredients, such as bovine, retinol and its derivatives (such as retinol propionate), retinal, retinoic acid, hyaluronic acid, etc.

[0055] In the present disclosure, the molecular weight of colanic acid or its physiologically acceptable salt is determined by liquid chromatography method. The specific method is as follows: accurately weigh the sample and standard, prepare the sample into a 2 mg / mL solution, filter it into a 1.8 mL sample 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℃; sample size: 20 μL; detector: differential detector 1260-RID.

[0056] Preparation method

[0057] In the following examples, sodium colanic acid (molecular weight in the range of 10-6000 kDa) is prepared by the following method.

[0058] 1. Solid-liquid separation

[0059] 1) Dilution: Pump the fermentation broth prepared according to the method of CN115287314B into a storage tank, dilute 3-20 times with 10-60% calcium chloride solution (calcium chloride dosage is 2% (w / v) of the fermentation broth) and purified water, and stir for 2-3 h until complete dissolution.

[0060] 2) Flocculation: After adjusting the pH to 9.0-12.0 by adding sodium carbonate solid, pre-mixing diatomite (diatomite dosage is 1% (w / v) of the fermentation broth) after flocculation for 1-2 h.

[0061] 3) Plate and frame filtration: according to the plate and frame filter press operation sop, install the filter cloth and press the filter plate. Recycle the plate and frame filter press 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 the diatomite suspension (pre-coating amount is 0.5 kg / m 2 ) into the plate and frame filter press. After pre-coating is completed, connect the liquid inlet to the liquid storage tank to pump the flocculation liquid into the plate and frame filter press for circulating filtration, and control the working pressure <0.2 MPa. Put the outlet of the liquid collection pipe into the feed tank for circulating filtration. When the liquid at the outlet is clear (turbidity ≤50 NTU), start collecting the filtrate. After the end, wash with 1-2 times the dead volume of purified water.

[0062]

[0062] 2. Activated carbon adsorption

[0063] 1) Adsorption: 0.5% of injection grade activated carbon was added to the fermentation broth, pH was adjusted to 4-6 with 0.1 M HC1, and the broth was stirred for 1-2 h. After adsorption, diatomite was pre-mixed (0.1-5% (w / v) diatomite was added to the broth).

[0064] 2) Plate and frame filtration: according to the plate and frame filter operation SOP, filter cloth was installed and filter plate was compressed. The feed pump was started to pump the diatomite suspension (0.5 kg / m2was pre-coated) into the plate and frame filter to pre-coat diatomite. After pre-coating, the inlet was connected to the liquid storage tank to pump the flocculation liquid into the plate and frame filter for cycle filtration, and the working pressure was controlled to be <0.2 MPa. The inlet was connected to the filtrate collection tank for cycle filtration, and after the liquid at the outlet was clear (turbidity <30 NTU), the filtrate was collected. 2

[0065] After filtration, 1-2 times the dead volume of purified water was used for backwashing.

[0066] 3. Polishing filtration

[0067] The filtrate was adjusted to pH 7.0 ± 0.2 with sodium carbonate, and then the filtrate was subjected to polishing 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, and if the pressure was too high, the filter was back-flushed and then replaced for filtration again. When liquid continuously flowed out of the exhaust port, the exhaust valve was closed to collect the filtrate.

[0068] 4. Ceramic membrane concentration

[0069] 1) Cleaning: the ceramic membrane ultrafiltration system was connected, and the system was sequentially cleaned with 2% citric acid, purified water, and 0.5 M NaOH solution for 15-30 min, and then the system was cleaned with purified water until the pH of the filtrate at the permeation end was neutral.

[0070] 2) Concentration: the filtrate after polishing filtration was pumped into the cycle liquid storage tank of the ceramic membrane system, the outlet at the backflow end was connected to the cycle liquid storage 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 be 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 cleaned 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 cleaning and collection operations were repeated twice. The concentrated liquid and the cleaning liquid were combined and collected.

[0071] ​5. Alcohol precipitation

[0072] 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 empty liquid, replace the filter, and perform filtration again to collect the filtrate.

[0073] 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 produced, stop stirring. Let the precipitate stand for 2-4 h, remove the supernatant, and collect the precipitate.

[0074] 6. Washing and dehydration

[0075] 1) Washing: Add 1-10 times the volume of 70% ethanol (containing 1% sodium chloride) to the CA sugar precipitate, stir for 20 min, and then let stand for 1-8 h. Remove the supernatant and collect the precipitate. Then repeat the washing operation 2 more times and collect the precipitate.

[0076] 2) Dehydration: Add 1-10 times the volume of 95% ethanol to the precipitate, stir for 2 h, and then let stand for 1-2 h. Remove the supernatant. Repeat the dehydration operation once more and collect the precipitate. Vacuum filter the precipitate to reduce the residual liquid, add 1-10 times the volume of 95% ethanol again for dehydration, collect the precipitate, and then filter again to remove the liquid. Weigh the wet sample.

[0077] 7. Drying

[0078] 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.

[0079] 8. Crushing and sieving

[0080] After the second drying is complete, crush the sample again using a crusher and sieve it through a 100-mesh sieve. Package the sample in aluminum foil bags.

[0081] 9. Enzymatic preparation of different molecular weight carbenoxolide sodium

[0082] The obtained colanic acid product is mixed with colanic acid degrading enzyme (Baiyin Biological Production, see SEQ ID No. 2 in CN116334039B) of different concentrations (1000 U / L, 2000 U / L, 3000 U / L) at 37°C for 0-12 h, the sample is collected and heated to inactivate the enzyme, then DEAE ion column is used for separation and purification, and a small amount of molecular weight heterogeneous part and impurities are removed to obtain colanic acid sodium products of different molecular weights of 10-6000 kDa. The molecular weight of the colanic acid sodium is in the range of 10-50 kDa, 300-500 kDa, 600-800 kDa, 1000-2000 kDa, and 3000-5000 kDa, respectively. Subsequently, the final colanic acid product can be obtained by freeze-drying or spray-drying.

[0083] Experimental method

[0084] (1) Cell toxicity

[0085] Cell culture: HaCaT cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) and incubated in a 37°C, 5% CO2 incubator.

[0086] Sample treatment: 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.

[0087] Experimental treatment: HaCaT 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 h of culture, the culture medium was discarded, the sample group was added with the corresponding CA solution, the positive control group was added with 10% DMSO solution, and the blank control group was added with complete culture medium. Continue to culture for 24 h.

[0088] Cell viability detection: discard the supernatant, add complete culture medium containing 10% AlamarBlue, and incubate in the incubator for 4 h, then detect the absorbance value at ex / em = 560 / 590 nm with a microplate reader.

[0089] (2) Anti-wrinkle (collagen promotion)

[0090] 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.

[0091] Sample treatment: 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.

[0092] Experimental treatment: HDF cells were inoculated into 96-well plates, 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 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 TGF-β solutions, and the negative control groups and blank groups were added with complete culture medium. The incubation was continued for 24 h.

[0093] I, III 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).

[0094] (3) Inflammation (macrophages)

[0095] Cell culture: RAW264.7 cells were cultured in DMEM complete culture medium (DMEM + 10% FBS + 1% P / S) and incubated in a 37°C, 5% CO2 incubator.

[0096] Sample treatment: CA powder was dissolved in culture medium, and after complete dissolution, it was filtered with a 0.2 μm sterile filter membrane to remove bacteria.

[0097] Experimental treatment: HDF cells were inoculated into 96-well plates, 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, and the sample groups were added with corresponding CA solutions, the positive control groups were added with complete culture medium containing 100 g / mL dexamethasone, and the negative control groups and blank groups were added with complete culture medium. After 2 h of incubation in the incubator, 1 μg / mL LPS (lipopolysaccharide) was added to the sample groups, positive control groups, and negative controls. The incubation was continued for 24 h.

[0098] Inflammatory factor detection: The supernatant of each well was collected, and the experimental operation was performed according to the operation instruction of the ELISA kit (Huawmei Biological, CSB-E04740h, CSB-E08053h, CSB-E04638h).

[0099] Active oxygen ROS detection: After the supernatant was collected, the cells in each well were washed twice with PBS, and the experimental operation was performed according to the operation instruction of the DCFH-DA kit.

[0100] (4) Inflammation (keratinocytes)

[0101] Cell culture: Keratinocytes HaCaT 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 membrane to remove bacteria.

[0103] Experimental treatment: Cells were seeded into 96-well plates, and sample groups, blank control groups, positive control groups, and negative control groups were set up. 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 sample solution was added to the sample groups, the corresponding vitamin C solution (dissolved in the culture medium) was added to the positive control groups, and the complete culture medium was added to the negative control groups and the blank control groups. After incubation in the incubator for 2 h, hydrogen peroxide solution (0.8 mM HX0640 Sigma-Aldrich) was added to the sample groups, the positive control groups, and the negative control groups for inflammation stimulation. The culture was continued for 24 h.

[0104] Gene detection: The old liquid was aspirated, washed twice with PBS, and cell lysis solution 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.

[0105] 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-E04638h, CSB-E10103h).

[0106] Western blotting experiment: HaCaT cells were seeded into 96-well plates, and sample groups, blank control groups, positive control groups, and negative 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. After incubation in the incubator for 2 h, the cells were removed and broken, followed by SDS-PAGE experiment, transfer to PVDF membrane by transfer instrument (Thermo), reaction with primary antibody (IL-1 antibody: Huawmei Biological, CSB-PA829263; p65 antibody: Huawmei Biological, BA1055) and secondary antibody (Dr. Deki, BA1055), and then observation after adding color developing solution.

[0107] (5) Light damage

[0108] Cell culture and sample treatment were the same as in (4).

[0109] Experimental treatment: HaCaT cells were inoculated into 96-well plates, and sample groups, blank control groups, positive control groups and negative control groups were set up. Each detection index had at least 3 replicate wells for each sample. After 24h of culture, the culture solution was discarded, the corresponding CA solution was added to the sample group, the VC solution (dissolved in the culture medium) was added to the positive control group, and the complete culture medium was added to the negative control group and the blank control group. Incubate in the incubator for 2h. Place the sample group, positive control group and negative control group under a 320-400nm ultraviolet lamp for irradiation at a power of 40mW for 4min. After irradiation, continue to culture for 24h.

[0110] Cell viability detection: discard the supernatant, add complete culture medium containing 10% AlamarBlue to the incubator for 4h, and then detect the absorbance value at ex / em = 560 / 590nm with a microplate reader.

[0111] DNA damage marker detection: Huawen Bio, CSB-PA833019. NO detection kit: Biyun, S0021S. ROS detection kit: Solabio, CA1410. iNOS detection was quantified by ELISA (Huawen Bio, CSB-E08148h).

[0112] (6) 3D skin model

[0113] The model used in this test is a 3D epidermal skin model Batch number: ES230408, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0114] Transfer the 3D skin model to a 6-well plate (add 0.9mL EpiGrowth culture solution in advance), and label the test group number on the 6-well plate.

[0115] The BC group was not treated, the PC group was added with the corresponding concentration of working solution under the liquid, and the sample group was evenly distributed on the surface of the model. Incubate in a CO2 incubator (37℃, 5% CO2) for 24h. After incubation, wash the residual test substances on the surface of the model with sterile PBS solution, and wipe off the residual liquid inside and outside the model with a sterile cotton swab.

[0116] HPLC test

[0117] Cut the model ring into a 1.5mL centrifuge tube, add 500μL of 0.2mg / mL proteinase K to each tube, and place it in a 50℃ water bath for 2h. After the cuticle falls off, add 500μL of methanol to each tube and ultrasonic for 30min; then centrifuge at 14000rpm for 10min. Dry the methanol with nitrogen at 60℃, reconstitute with 500μL of deionized water, pass through a 0.22μm filter membrane, and determine by HPLC.

[0118] Immunofluorescence test

[0119] The model ring for detection was cut off, fixed with 4% paraformaldehyde for 24 h, and then subjected to immunofluorescence detection (FLG and AQP3 antibodies were purchased from Abeam). Photographs were taken under a microscope, and the pictures were collected and analyzed.

[0120] (7) Mitochondrial morphology

[0121] Preparation of JC-10 staining working solution:

[0122] The amount of JC-10 staining working solution required for each well of a six-well plate is 1 mL, and the amount of JC-10 staining working solution for other culture vessels is calculated accordingly; for cell suspension, 0.5 mL of JC-10 staining working solution is required for 50-100 million cells. Dilute the JC-10 (200x) by adding 8 mL of ultrapure water for every 50 μL of JC-10 (200x). Shake vigorously to dissolve and mix well. Then add 2 mL of JC-10 staining buffer (5x), mix well, and you have the JC-10 staining working solution.

[0123] Cell mitochondrial staining observation:

[0124] (1) For each well of a six-well plate, remove the culture solution, wash the cells with PBS if necessary, and add 1 mL of cell culture solution. The cell culture solution can contain serum and phenol red.

[0125] (2) Add 1 mL of JC-10 staining working solution and mix well. Incubate at 37°C in a cell incubator for 20 min.

[0126] (3) During incubation, prepare an appropriate amount of JC-10 staining buffer (1x) by adding 4 mL of distilled water to every 1 mL of JC-10 staining buffer (5x) and placing it in an ice bath.

[0127] (4) After incubation at 37°C, remove the supernatant and wash twice with JC-10 staining buffer (1x).

[0128] (5) Add 2 mL of cell culture solution, which can contain serum and phenol red.

[0129] (6) Observe under a fluorescence microscope or a laser confocal microscope.

[0130] (7) Detect with a fluorescence spectrophotometer or a fluorescence microplate reader: After mixing, directly perform time scan with a fluorescence spectrophotometer, with an excitation wavelength of 485 nm and an emission wavelength of 590 nm.

[0131] (8) CD44 immunoblotting

[0132] Cell culture: HaCaT cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) in a 37°C, 5% CO2 incubator.

[0133] 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.

[0134] Experimental treatment: HaCaT 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 medium was discarded, and the sample group was added with the corresponding CA solution. Incubate in the incubator for 2 h. The cells were removed and broken, and then SDS-PAGE was performed. Transfer to a PVDF membrane by a transfer instrument (Thermo), react with a primary antibody (CD44 antibody, Huamei Biological, CSB-RA292372A0HU) and a secondary antibody (Dr. De, BA1055), and then add color developing solution for observation.

[0135] (9) Cell viability-CCK8 detection

[0136] Plating: After recovering, human keratinocytes HaCaT were cultured for 1-2 days to a cell confluence of 70%, and after trypsinization, the keratinocytes were plated at 5×10 4 / well, and divided into DMEM, H2O2-DMEM, and H2O2-CA groups.

[0137] H2O2 stimulation: H2O2 (Shanghai Lingfeng Chemical Reagents) was used at a concentration of 0.8 mM, diluted to 10 mM, and then added to the culture medium at 100x (the original storage concentration was 8820 mM, 8.82 mL PBS was added to 10 μL H2O2, and then 40 μL of the diluted solution was added to 4 mL of culture medium). The prepared H2O2-containing culture medium was added to the H2O2 stimulation group as a replacement, and cultured for 2 h.

[0138] CA treatment: 50 mg of UV-sterilized CA powder was added to 5 mL of PBS, shaken to dissolve, and diluted to 10 mg / mL. After H2O2 stimulation, the culture supernatant was removed (containing the DMEM blank control group), each sample was rinsed with 100 μL of PBS, and then 100 μL of CA-containing medium was added to the H2O2-CA group, and 100 μL of medium was added to the DMEM and H2O2-DMEM groups, and cultured for 24 h.

[0139] CCK8 detection: calculate the volume of culture medium needed for detection, then add CCK8 (Bi Yun Tian, C0038) at 10x, that is, 400 μL CCK8 is added to 4 mL of culture medium. After the supernatant of each sample culture medium is removed, 100 μL / well is added to a 96-well plate and incubated for 1-2 h. Detection is performed at OD450 using an enzyme marker under light protection.

[0140] (10) Cell migration detection

[0141] Cell culture: HaCaT (keratinocytes) were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) and incubated at 37°C in a 5% CO2 incubator.

[0142] Sample treatment: The sample was prepared with PBS and filtered with a 0.22 μm sterile filter to remove bacteria.

[0143] Experimental treatment: After the cells were plated and cultured for 24 h, scratches were made and the sample prepared with serum-free medium was added.

[0144] Photography: After scratching, photographs were taken under a 4x lens at 0 h, and after scratching for 16 h, the skin was washed once with PBS and photographs were taken under a 4x lens.

[0145] Data analysis: ImageJ software was used to calculate the scratch area.

[0146] (11) Transdermal experiment

[0147] Preparation of ex vivo skin: The ex vivo skin (Linxi County Jingde Agricultural Products Sales Co., Ltd.) stored at -20°C was thawed at room temperature with deionized water and repeatedly washed with PBS buffer.

[0148] Transdermal absorption capacity determination:

[0149] (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;

[0150] (2) Add receiving liquid to the receiving chamber, and after the skin is 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;

[0151] (3) Sample addition: Add the sample (green fluorescent labeled sodium carboxymethylcellulose with a molecular weight of 10-50 kDa, concentration is 1 mg / mL) to the surface of the skin in the supply chamber. The sample is evenly spread from the center of the skin to the edge;

[0152] (4) Penetration: Start the electromagnetic stirrer at a speed of 300 rpm and keep the constant temperature water bath at (32±1) °C;

[0153] (5) At 1h, 6h, 18h time points, collect skin samples, wash the skin surface with PBS, wipe off the surface residual liquid, and cut the skin with a blade. Freeze section and wait for use.

[0154] Observation and analysis: Observe the skin sections with a fluorescence microscope, take photos, and record the fluorescence intensity with Image J.

[0155] (12) Photoaging

[0156] 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.

[0157] Dosing: According to the test grouping, when the cell plating rate in the 6-well plate reached 40%-60%, the grouping was dosed, 2mL was added to each well, and 3 replicate wells were set for each group. After dosing, the 6-well plate was placed in the incubator for continued culture for 24h.

[0158] UVA irradiation: UVA irradiation of 30J / cm 2 was performed on groups other than the blank control (BC), and they were placed in the incubator for continued culture for 24h.

[0159] Sample collection: After 24h of culture, the cell culture supernatant was collected in an EP tube and stored at -80°C.

[0160] ELISA detection: According to the operating instructions of the ELISA collagen detection kit (Huawmei Biological, CSB-E08082h).

[0161] (13) Anti-inflammatory effect detection

[0162] Cell culture: RAW264.7 cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) and incubated in a 37°C, 5% CO2 incubator.

[0163] Sample treatment: According to the experimental requirements, working solutions of different molecular weight CA (1-2kDa, 10-50kDa, 1000-2000kDa, 3000-5000kDa) were prepared.

[0164] Experimental treatment: 8x10 4The RAW264.7 cells were inoculated into a 24-well plate at a seeding density of 1 cell / well, and incubated in an incubator (37°C, 5% CO2) overnight. When the cell plating rate in the 24-well plate reached 40%-60%, 400 μL of DMEM medium containing the CA working solution to be tested was added to the plate, and the final concentration of CA was 1 mg / mL. Each group had 3 replicate wells. After the administration was completed, the plate was placed in an incubator (37°C, 5% CO2) for continuous culture for 24 h. 400 μL of 2x LPS working solution was added to the plate, and the plate was shaken left and right to mix the drug in the plate. The final concentration of LPS was 1 μg / mL. After the administration was completed, the plate was placed in an incubator (37°C, 5% CO2) for continuous culture for 24 h. After the incubation was completed, the cell culture supernatant was collected in an EP tube, centrifuged at 1000 rpm for 5 min, and the sample was stored in a -80°C freezer after collection.

[0165] TNF-α, IL-6, IL-1β content detection: The detection was performed according to the experimental method described in the instruction manual of the Doctor De kit (Mouse TNF alpha ELISA Kit-EK0527, Mouse IL-6 ELISA Kit-EK0411, Mouse IL-1beta ELISA Kit-EK0394). Dexa 100 μg / mL was used as a positive control, and the concentration was determined according to the reference cosmetic soothing efficacy test-in vitro TNF-α inflammatory factor content determination lipopolysaccharide-induced macrophage RAW264.7 test method (T / SHRH 033-2021).

[0166] (14) Mitochondrial morphology test

[0167] Mito Tracker Red CMXRos staining and mitochondrial morphology analysis

[0168] HaCaT cells were diluted with DMEM complete medium to 1x105cells per well, and plated in a 35-mm-diameter glass-bottom μ-Dish (Ibidi) at a constant density, and incubated in a 37°C, 5% CO2 incubator until the cells adhered. The experiment was divided into a blank group, a UVB radiation group, and a UVB radiation+clammy (CA) group, with at least 3 replicate wells in each group. The blank group did not need UVB irradiation, and was cultured with normal DMEM complete medium; the UVB radiation group was irradiated with a UV light therapy instrument, and the radiation dose was 360 mJ / cm 2; UVB radiation + clavulanic acid (CA) group was pretreated with 1 mg / ml CA for 24 hours before equal dose of UVB radiation. MitoTracker Red CMXRos probe 50 nM (Life technology) was used to label cell mitochondria, and the probe was loaded at room temperature for 30 minutes, then the cells were washed with PBS, and then fresh culture medium was added, and DAPI dye (1:1000) was used for nucleus staining. Cell mitochondria imaging was performed on Zeiss 880 scanning confocal microscope with 63x oil objective. Data processing was performed by MiNA analysis tool set of Fiji software (https: / / github.com / ScienceToolkit / MiNA) for semi-automatic analysis of mitochondrial network in HaCaT cells. MiNA tool can convert images to binary images, and use the "Analyze Particles" function to measure and calculate the size and shape symbol of all mitochondria in each cell, from which the median area, aspect ratio, length, roundness and total area of mitochondria are determined to quantitatively capture the morphological skeleton data of mitochondrial network morphology, and mitochondria are divided into Fragmented, Tubular and Elongated, and statistical analysis is performed by One-way ANOVA, and P<0.05 is considered to be statistically significant difference.

[0169] (15) qPCR detection of inflammatory gene expression

[0170] (1) Plating: select well-grown human immortalized keratinocytes HaCaT, trypsinize and count cells, plate cells (12-well plate, 2x10 5 cells per well, 1 mL per well), and place in a carbon dioxide incubator for 24 h.

[0171] (2) Modeling: discard the supernatant, add complete culture medium containing hydrogen peroxide and stimulate for 2 h, and the blank group is not stimulated.

[0172] (3) Sample addition: set the test sample group to 0.5 mg / mL and 1 mg / mL, the positive control group to 0.05% vitamin E, the negative control group, and the blank control group. Remove the culture medium in the culture plate, add complete culture medium to the negative control group and blank control group, and add DMEM complete culture medium containing the corresponding concentration of test sample to the test sample group and positive control group, with a liquid volume of 1 mL per well. After sample addition, place the 12-well plate back in the carbon dioxide incubator for 24 h.

[0173] (4) Detection: detect the expression of the target gene by RT-qPCR method, calculate the relative expression of each target gene according to the 2–ΔΔCt method and plot.

[0174] (16) Cell cycle detection method

[0175] (1) Preparation of cell sample: For suspension cells, centrifugally separated cells are suspended in 1 mL of ice-bath pre-cooled PBS; for adherent cells, after trypsinization and centrifugal separation, the cells are suspended in 1 mL of ice-bath pre-cooled PBS, and the supernatant is carefully aspirated.

[0176] (2) Cell fixation: 1 mL of ice-bath pre-cooled 70% ethanol is added, and the mixture is mixed gently by blowing and uniformly, and fixed at 4°C for 30 minutes or more. The cells are centrifugally precipitated at 1000 g for 3-5 minutes. The supernatant is carefully aspirated, and about 1 mL of ice-bath pre-cooled PBS is added to resuspend the cells. The cells are again centrifugally precipitated, and the supernatant is carefully aspirated.

[0177] (3) Preparation of propidium iodide staining solution: for 1 sample, 0.5 mL of staining buffer, 25 μL of propidium iodide staining solution (20X), and 10 μL of RNase A (50X) are mixed. It is stored at 4°C and used on the same day.

[0178] (4) Staining: 0.5 mL of propidium iodide staining solution is added to each cell sample, and the cell precipitate is slowly and thoroughly resuspended, and incubated at 37°C in the dark for 30 minutes. It can then be stored at 4°C or in an ice bath in the dark. After staining, flow detection is completed within 24 hours.

[0179] (5) Flow detection and analysis: the red fluorescence is detected at an excitation wavelength of 488 nm using a flow cytometer, and light scattering is simultaneously detected. Appropriate analysis software is used for cell DNA content analysis and light scattering analysis.

[0180] Example

[0181] The following examples are merely illustrative and are not intended to limit the scope or content of the present application in any way.

[0182] In the following examples, if not otherwise specified, the used colanic acid is sodium colanic acid with a molecular weight of 3000-5000 kDa.

[0183] Example 1: Inhibition of photodamage

[0184] According to the method of photodamage in the experimental method, the effect of colanic acid on inhibiting reactive oxygen / active nitrogen in HaCat cells irradiated by ultraviolet light was tested, and the results are shown in Figure 2.

[0185] As can be seen from Figure 2, colanic acid can significantly inhibit the increase of reactive oxygen in human keratinocytes (HaCaT) caused by ultraviolet irradiation. At the same time, it can also be observed that the expression amount of iNOS in HaCaT cells after ultraviolet irradiation increases significantly, and the expression amount decreases significantly after the addition of colanic acid. Similarly, the content of NO (active nitrogen) in the cells is also significantly down-regulated.

[0186] Example 2: Inflammation inhibition

[0187] According to the method of inflammation (keratinocytes) in the experimental method, the effect of colanic acid on inhibiting inflammation of human keratinocytes was tested, and the results are shown in Figure 3.

[0188] As can be seen from Figure 3A, colanic acid significantly inhibited TNF-a, IL-6 and COX-2 of HaCaT cells. At the same time, in the Western blot experiment of Figure 3B, it can also be observed that IL-1 and p65 of the ultraviolet irradiated HaCaT cells are significantly up-regulated, and after adding colanic acid, IL-1 and p65 are significantly down-regulated. It shows that the addition of colanic acid can significantly inhibit the inflammatory response of cells.

[0189] Example 3: Photo-damage inhibition

[0190] Referring to the method of photo-damage treatment in the experimental method, the effect of colanic acid on inhibiting the increase of DNA damage markers caused by ultraviolet irradiation and reducing cell aging and apoptosis was tested, and the results are shown in Figure 4.

[0191] As can be seen from Figure 4, colanic acid can effectively inhibit DNA damage (Figure 4A and Figure 4B) of HaCaT cells caused by ultraviolet irradiation. It can be seen that adding colanic acid before ultraviolet irradiation (UV+CA+) can significantly inhibit the DNA damage marker H2AX, and relatively, adding colanic acid after ultraviolet irradiation (UV+CA-+) is not obvious. At the same time, by testing the cell cycle, it can be seen that the cells in the G0 / 1 cycle of the colanic acid added group are less than those of the ultraviolet irradiation group, and there is a significant difference, while the cells of the ultraviolet irradiation alone group are more in the cell block period (Figure 4C), indicating that colanic acid can significantly inhibit cell aging caused by external environmental stimulation. Similarly, the apoptotic cells after adding colanic acid are also significantly reduced (Figure 4D).

[0192] Example 4: Mitochondrial protection

[0193] By the method of mitochondrial morphology in the experimental method, the protective effect of colanic acid on cell mitochondria was tested, and the results are shown in Figure 5.

[0194] As can be seen from the experimental results of HaCaT cells in Figure 5A, colanic acid has a good protective effect on cell mitochondria, and after adding colanic acid, more JC-10 on the mitochondrial membrane of the cells presents in the normal aggregated state, and relatively, the mitochondrial JC-10 of the cells irradiated only by ultraviolet light changes from the aggregated state to the monoploid form. Similarly, as can be seen from Figure 5B and Figure 5C, colanic acid can significantly reduce the fragmentation of mitochondria under oxidative stress. It can be seen that colanic acid has a good protective effect on mitochondria.

[0195] Example 5: Promoting cell proliferation and migration

[0196] According to the method of CD44 immunoblotting and cell migration detection in the experimental method, the kola acid was tested to promote cell proliferation and migration by up-regulating cell CD44, and the results are shown in Figure 6.

[0197] As can be seen from Figure 6A, kola acid can significantly up-regulate the expression of CD44 in HaCaT cells. CD44, as a widely expressed cell adhesion molecule, is involved in cell proliferation, differentiation, migration, angiogenesis and other biological processes, and plays a key role in mediating cell signal transduction, regulating tissue homeostasis and other functions. At the cellular level, the experimental results of Figure 6B and Figure 6C demonstrate that the proliferation and migration of cells are significantly up-regulated after the addition of kola acid.

[0198] Example 6: Collagen promotion

[0199] According to the method of photoaging in the experimental method, the collagen promotion activity of kola acid was tested by cell experiment, and the results are shown in Figure 7. Kola acid also has a significant activity in enhancing the synthesis of collagen in human fibroblasts at the cellular level. Under ultraviolet irradiation conditions, the collagen promotion activity of kola acid is superior to that of icodextrin, and under normal culture conditions, the collagen promotion activity of kola acid on human fibroblasts is also very significant.

[0200] Example 7: 3D skin test

[0201] According to the method of 3D skin model in the experimental method, the effect of kola acid on the promotion of 3D skin fibulin (FLG) content was tested, and the results are shown in Figure 8. Figure 8A is the test results of 3D skin experiment, compared with the BC blank control group, the content of fibulin (FLG) significantly increased at the concentration of 0.5% (w / v) of oligomeric kola acid, and the promotion rate was 58.00%. The fluorescence quantitative results of Figure 8B show that kola acid has a promotion effect on the fibulin FLG of 3D skin.

[0202] According to the method of 3D skin model in the experimental method, the effect of kola acid on the promotion of 3D skin aquaporin 3 (AQP3) content was tested, and the results are shown in Figure 9. In Figure 9A and Figure 9B, compared with the BC control group, the content of aquaporin 3 (AQP3) significantly increased at the concentration of 0.5% (w / v) of kola acid, and the promotion rate was 38.00%.

[0203] Example 8: Cell toxicity

[0204] The cell toxicity of kola acid was tested, and the results are shown in Figure 10. As can be seen from the test results of Figure 10, when the concentration of kola acid in the cell culture medium reaches 5 mg / mL or more, it has no obvious effect on cell viability and morphology.

[0205] Example 9: Human experiment

[0206] The human experiment of macromolecular sodium carrageenan (molecular weight of 3000-5000 kDa) was carried out according to the following method, and the results are shown in Figures 11-13.

[0207] Subjects: A total of 31 people, 5 men and 26 women, aged 28-59, with an average age of 47±8, who met the voluntary selection criteria of the subjects.

[0208] Instruments: Stratum corneum hydration meter (MSC1201, Delfin), transdermal water loss meter (SWL5201, Delfin), Antera 3D imager (HC1023Q, Delfin), balance with accuracy of 0.1 mg.

[0209] Test environment: Test environment temperature: 20.0-22.0℃; humidity: 40-60%, and real-time dynamic monitoring was carried out.

[0210] Test method: At the first visit, the subjects were given test instructions and signed the informed consent form. Among the subjects participating in the test, those with dry skin or skin redness were selected, and finally 31 people entered the experimental stage. The subjects were tested on the test day at 0 weeks, and after 20 min of constant temperature and humidity environment balance, the subjects were instrumentally measured. After the test was completed, the sample usage instructions were given and the sample was issued, and the sample weight was confirmed at the beginning and end of the test. On-site follow-up was conducted after 2 and 4 weeks of sample use, and instrumentally measured.

[0211] Sample usage instructions: Use once a day in the morning and evening, with a usage amount of 0.5 mL and a concentration of 0.15% (w / v).

[0212] Figure 11 shows the anti-wrinkle efficacy test results of sodium carrageenan. As shown in Figure 11, after the subjects used 0.15% macromolecular sodium carrageenan essence for 4 weeks, the length of the wrinkles at the corners of the eyes was reduced by 38.03%, and the number of wrinkles was reduced by 35.68%.

[0213] Figure 12 shows the anti-red and anti-sensitive efficacy test results of sodium carrageenan. As shown in Figure 12, after the subjects used 0.15% macromolecular sodium carrageenan essence for 4 weeks, the skin hemoglobin index decreased by 33.03%.

[0214] Figure 13 shows the water retention and moisturizing efficacy test of sodium carrageenan. As shown in Figure 13, after the subjects used 0.15% macromolecular sodium carrageenan essence for 4 weeks, the skin water content increased significantly by 33.35%, and the trans epidermal water loss (TEWL) decreased significantly by 19.84%

[0215] Example 10

[0216] Sodium capecitabine with a molecular weight of 50-5000 kDa, 100-5000 kDa, 1000-5000 kDa, 1000-4000 kDa, 1000-3000 kDa, 1000-2000 kDa, 2000-5000 kDa, 2000-4000 kDa, 2000-3000 kDa, 3000-4000 kDa, 300-500 kDa, 600-800 kDa, 10-1000 kDa, 10-100 kDa, 10-50 kDa, 10-20 kDa, 10-15 kDa were prepared according to the preparation method, and their properties and efficacy were tested according to the same method as in Examples 1-9.

[0217] Example 11: Comparison of effects of sodium capecitabine with different molecular weights

[0218] The skin protection effects of sodium capecitabine (CA) with different molecular weights were tested by CCK8 detection, and the results are shown in FIG. 14. As can be seen from FIG. 14, sodium capecitabine with a molecular weight of 10-50 kDa, 1000-2000 kDa and 3000-5000 kDa has better skin protection effect.

[0219] According to the anti-wrinkle (collagen promotion) method in the experimental method, the effects of sodium capecitabine (CA) with different molecular weights on stimulating human fibroblasts to express collagen type I were tested at a concentration of 3 mg / mL, and the results are shown in FIG. 15. As can be seen from FIG. 15, sodium capecitabine with a molecular weight of 10-50 kDa, 300-500 kDa and 1000-2000 kDa has better collagen promotion effect.

[0220] According to the method of cell migration detection in the experimental method, the effects of sodium capecitabine (CA) with different molecular weights on the migration of human fibroblasts were tested, and the results are shown in FIG. 16. As can be seen from FIG. 16, sodium capecitabine with a molecular weight of 10-50 kDa, 300-500 kDa, 600-800 kDa and 1000-2000 kDa has better effect on promoting cell migration.

[0221] According to the anti-inflammatory effect detection method in the experimental method, the effects of sodium capecitabine (CA) with different molecular weights on anti-inflammatory effect were tested, and the results are shown in FIGS. 17A-17C. As can be seen from FIGS. 17A-17C, sodium capecitabine with a molecular weight of 10-50 kDa or 1000-5000 kDa can achieve better anti-inflammatory effect at the same concentration.

[0222] Example 12: Transdermal test of sodium capecitabine

[0223] The green fluorescent labeled sodium colforsate (10-50 kDa) was purified to remove free fluorescent molecules. It was dissolved in water to 1 mg / mL for transdermal test. The results of Figure 18 show that sodium colforsate can effectively penetrate the skin surface and enter the dermis layer. Within 1-18 h, the skin fluorescence section data shows that the fluorescence intensity has increased nearly 9 times.

[0224] Example 13: Mitochondrial localization experiment of sodium colforsate

[0225] Cell culture: HDF cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% P / S) and incubated at 37°C in a 5% CO2 incubator.

[0226] Sample processing: The fluorescent labeled CA powder (10-50 kDa) was dissolved in the culture medium at a concentration of 1 mg / mL, and sterilized by filtering through a 0.2 μm sterile filter after complete dissolution.

[0227] Experimental treatment: HDF cells were seeded in a 96-well plate, and sample groups and blank control groups (BC) were set up. Each test index had at least 3 replicate wells for each sample. After 24 h of culture, the culture solution was discarded, and the sample groups were added with CA solution and cultured for 24 h, 48 h and 72 h, respectively. The supernatant was discarded, and the mitochondria were stained with mitotracker. Then the cells were fixed with paraformaldehyde, and the fluorescence was observed under a confocal microscope. The results of Figure 19 show that the green fluorescence representing sodium colforsate and the red fluorescence representing mitochondria overlap in space, so it can be considered that sodium colforsate can enter the cell and even the mitochondria to exert its function.

[0228] Incorporated by reference

[0229] The entire contents of each patent and scientific document referred to herein is incorporated by reference for all purposes.

[0230] Equivalents

[0231] The present application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments should be considered in all respects 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. The use of colacid or a physiologically acceptable salt thereof, or a composition comprising colacid or a physiologically acceptable salt thereof, in increasing skin hydration, characterized in that, The molecular weight of the colacid or its physiologically acceptable salt is in the range of 10-6000 kDa, preferably 10-50 kDa or 1000-5000 kDa.

2. The use according to claim 1, wherein, The content of the colacid or its physiologically acceptable salt is 0.001-2% (w / v), preferably 0.01-1% (w / v), and more preferably 0.05-0.5% (w / v).

3. The use of colacid or a physiologically acceptable salt thereof, or a composition comprising colacid or a physiologically acceptable salt thereof, in enhancing the protective function of skin cells, such as increasing cell vitality, characterized in that, The molecular weight of the colacid or its physiologically acceptable salt is in the range of 10-6000 kDa, preferably 10-50 kDa or 1000-5000 kDa.

4. The use of colacid or a physiologically acceptable salt thereof, or a composition comprising colacid or a physiologically acceptable salt thereof, in skin anti-aging, characterized in that, The molecular weight of the colacid or its physiologically acceptable salt is in the range of 10-6000 kDa, preferably 10-50 kDa or 1000-5000 kDa.

5. The use according to claim 4, wherein, The skin anti-aging includes skin anti-wrinkle, skin anti-aging, increasing the collagen content in the skin, preferably type I and / or type III collagen.

6. The use of colacid or a physiologically acceptable salt thereof, or a composition comprising colacid or a physiologically acceptable salt thereof, in improving rosacea or skin inflammation caused by skin allergies, characterized in that, The molecular weight of the colacid or its physiologically acceptable salt is in the range of 10-6000 kDa, preferably 10-50 kDa or 1000-5000 kDa.

7. The use according to any one of claims 3-6, wherein, The content of the colacid or its physiologically acceptable salt is 0.001-10% (w / v), preferably 0.05-5% (w / v), and more preferably 0.1-3% (w / v).

8. The use according to any one of claims 1-7, wherein, The composition contains only colacid or its physiologically acceptable salt as the active ingredient.

9. The use according to any one of claims 1-8, wherein, The composition is formulated for topical, oral, intramuscular, subcutaneous or intravenous application, preferably for subcutaneous application.

10. The use according to any one of claims 1-9, wherein, The composition also contains cosmetically or pharmaceutically acceptable additives.

11. The use according to any one of claims 1-10, wherein, The physiologically acceptable salt of the colacid is sodium colacid.

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