Moisturizing and anti-aging compositions containing neo-agar oligosaccharides, their preparation methods, applications, and cosmetics

Abstract

This invention belongs to the field of cosmetic technology, specifically relating to a moisturizing and anti-aging composition containing neo-agar oligosaccharides, its preparation method, and its application in cosmetics. This invention first studies the efficacy and mechanism of action of neo-agar oligosaccharides in anti-aging, anti-oxidation, and whitening. Then, it applies neo-agar oligosaccharides to a basic formulation and evaluates its efficacy in the cosmetic field through human efficacy testing. Based on the above mechanism, this invention designs and optimizes a proprietary formulation, scientifically combining neo-agar oligosaccharides with hydrolyzed sodium hyaluronate, specific plant fermentation extracts, etc. This not only solves the industry challenges of active ingredient synergy, transdermal absorption, and stability, but also transforms the anti-aging and whitening mechanism discovered in the laboratory into a stable production formulation. Therefore, it provides a complete solution from theoretical basis to productization and efficacy verification for the application of neo-agar oligosaccharides as a high-end cosmetic raw material with a clear mechanism, stable formulation, and proven efficacy.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a moisturizing and anti-aging composition containing neo-agar oligosaccharides, its preparation method, and its application in cosmetics. Background Technology

[0002] Aging is a complex biological process mediated by both endogenous (e.g., genetics, metabolism) and exogenous (e.g., ultraviolet radiation, pollution) factors. At the molecular level, the accumulation of free radicals (reactive oxygen species, ROS) and the oxidative stress they induce are among the core mechanisms driving aging. ROS produced by normal metabolism can attack and damage biomolecules such as DNA, proteins, and lipids. As we age, the body's antioxidant defense systems (such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)) decline in function, leading to the gradual accumulation of oxidative damage and accelerating cellular and tissue aging.

[0003] During skin aging, exogenous stimuli (such as ultraviolet radiation) can induce the excessive production of reactive oxygen species (ROS) within cells, triggering significant oxidative stress. Excessive ROS accumulation not only promotes lipid peroxidation and lipofuscin deposition, forming "age spots," but also activates the expression of matrix metalloproteinases (MMPs), thereby degrading key extracellular matrix components of the dermis—collagen and elastin—leading to loss of skin elasticity, decreased firmness, and wrinkles. Collagen, as the main protein maintaining the integrity of skin structure, experiences decreased synthesis and accelerated degradation with age, making it a significant marker of skin aging. Therefore, supplementing collagen externally or promoting its synthesis internally has become a crucial strategy for delaying skin aging.

[0004] Neo-agar oligosaccharides are low-molecular-weight oligosaccharides derived from seaweed. Existing research confirms that they possess excellent moisturizing properties and can exert a whitening effect by inhibiting tyrosinase activity, thus reducing melanin production. However, their bioactivity is closely related to their degree of polymerization, with oligosaccharides of different degrees of polymerization exhibiting significant differences in efficacy. Currently, there is a lack of clear and in-depth research on whether neo-agar oligosaccharides can directly combat skin aging, particularly their anti-wrinkle mechanism. Products on the market containing this ingredient largely rely on their excellent moisturizing properties and preliminary in vitro antioxidant data, lacking strong evidence of direct action on the dermis, stimulation of collagen regeneration, or repair of elastic fibers—the core anti-wrinkle mechanisms. Furthermore, these products face challenges in practical applications, including unclear transdermal efficiency of the active ingredients, untested long-term stability in formulations, and a lack of rigorously designed efficacy validation. Summary of the Invention

[0005] The purpose of this invention is to provide a moisturizing and anti-aging composition containing neo-agar oligosaccharides, its preparation method, and its application in cosmetics, thereby overcoming the shortcomings of existing technologies. This invention studies the efficacy and mechanism of action of neo-agar oligosaccharides in anti-aging, anti-oxidation, and whitening. Then, it applies neo-agar oligosaccharides to a basic formulation and evaluates its efficacy in the cosmetic field through human efficacy testing. This points the way for the application of neo-agar oligosaccharides in the cosmetic field.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a moisturizing and anti-aging composition containing neo-agar oligosaccharides, comprising the following components in weight percentages: Glycerin 3-6%, 1,3-Butanediol 3-6%, Disodium EDTA 0.01-0.05%, Gel 0.4-1.0%, Hydrolyzed Sodium Hyaluronate 0.2-0.5%, Inositol 0.5-1.0%, PEG / PPG-14 / 7 Dimethyl Ether 0.5-1.0%, Preservative 0.6%-1.2%, Tromethamine 0.2-0.5%, Neo-Agar Oligosaccharide 0.2-1.0%, Oat Fermentation Broth 0.1-0.5%, Gastrodia elata Extract 0.1-0.5%, Yeast / Barley Seed Fermentation Product Filtrate 0.2-0.6%, and Water as Balance.

[0008] This invention optimizes moisturizing and anti-aging effects through a three-layer synergistic system of matrix construction, activity synergy, and stability assurance, as detailed below: Glycerin and 1,3-butanediol form a small-molecule moisturizer that maintains stratum corneum moisture through hygroscopic properties; hydrolyzed sodium hyaluronate, as a large-molecule water-locking agent, forms a moisturizing film on the skin surface; and neo-glucan oligosaccharides enhance moisturizing longevity by forming strong hydrogen bonds with water molecules through hydroxyl groups. These three moisturizing ingredients achieve a complete moisturizing process from hydration to long-lasting water retention. Simultaneously, neo-glucan oligosaccharides, as the core active ingredient, form a multi-target anti-aging network with various plant extracts. Oat ferment broth, rich in β-glucan, can enhance the skin barrier function and work with neo-glucan oligosaccharides to reduce transepidermal water loss. Gastrodia elata root extract has antioxidant activity and can assist neo-glucan oligosaccharides in mitigating oxidative stress damage. Yeast / barley seed ferment filtrate provides amino acids, vitamins, and other nutrients to support cell metabolism.

[0009] 1,3-Butanediol acts as a penetration enhancer to improve the bioavailability of active ingredients; PEG / PPG-14 / 7 dimethyl ether promotes the penetration of macromolecular active ingredients by temporarily altering the stratum corneum structure; inositol, as a skin conditioning agent, optimizes cell membrane fluidity, together forming a graded penetration enhancement system. The preservative combination of p-hydroxyacetophenone and 1,2-hexanediol has broad-spectrum antibacterial properties and low irritation; disodium EDTA prevents oxidative degradation by chelating metal ions; the three-dimensional gel network formed by carbomer and acrylate polymers protects the stability of active ingredients, ensuring efficacy throughout the product's shelf life. The gel matrix formed by the gelling agent delays the sedimentation of active ingredients, ensuring consistent dosage during use. Tromethamine precisely modulates the skin-friendly range, maintaining the stability of active ingredients while avoiding skin irritation.

[0010] In some other embodiments, the preparation method of the new agar oligosaccharide is as follows: engineered strains expressing AgaA enzyme and AgaB enzyme, respectively, are inoculated into a fermentation medium and cultured to OD. 600 The concentration was 0.6-0.8. Isopropyl-β-D-thiogalactoside IPTG was added as an inducer to induce expression and obtain fermentation broth. The fermentation broth was centrifuged to collect the bacterial cells. After homogenization and disruption, the cells were centrifuged and filtered to obtain crude enzyme solutions of AgaA enzyme and AgaB enzyme, respectively. The crude enzyme solution of AgaA enzyme was added to agar substrate and reacted at 55-65℃ for 20-30 h. Then, the temperature was lowered to 40-50℃, and AgaB enzyme was added to continue the reaction for 10-20 h. After the reaction was completed, the cells were sterilized and cooled. After centrifugation and filtration, a new agar oligosaccharide solution was obtained. The solution was concentrated and freeze-dried to obtain the new agar oligosaccharide.

[0011] In some other embodiments, the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, with a mass ratio of p-hydroxyacetophenone to 1,2-hexanediol of 1:(0.8-1.2). Specifically, p-hydroxyacetophenone is 0.3-0.6 wt% and 1,2-hexanediol is 0.3-0.6 wt%.

[0012] In some other embodiments, the gelling agent comprises an acrylate / C10-30 alkyl acrylate cross-linked copolymer and carbomer, wherein the mass ratio of the acrylate / C10-30 alkyl acrylate cross-linked copolymer to carbomer is 1:(0.8-1.2). Specifically, the acrylate / C10-30 alkyl acrylate cross-linked copolymer comprises 0.2-0.5 wt%, and the carbomer comprises 0.2-0.5 wt%.

[0013] In some other embodiments, the oat fermentation broth is prepared as follows: oat seeds and bran are mixed and pulverized into fine powder; the fine powder, carbon source, vitamins, inulin, and water are mixed and sterilized to obtain a sterile culture medium; a compound lactic acid bacteria solution is inoculated into the sterile culture medium, stirred, and cultured at a constant temperature to obtain a first fermentation broth; the fermentation broth is heated to 65-75℃ and ultrasonically treated, then activated carbon aqueous solution is added and stirred and filtered to obtain a second fermentation broth; the second fermentation broth is heat-treated at 80-90℃, and after cooling, the oat fermentation broth is obtained. Alternatively, the preparation method of yeast / barley seed fermentation product filtrate is as follows: Barley seed powder was inoculated into a fermentation medium and inoculated with brewer's yeast. The mixture was then cultured with shaking at a pH of 6-7 and a temperature of 30-35℃ for 36-48 hours to obtain a crude fermentation broth. The crude fermentation broth was centrifuged, and the supernatant was collected. After sterilization and filtration, the yeast / barley seed fermentation product filtrate was obtained.

[0014] In a second aspect, the present invention provides a method for preparing the moisturizing and anti-aging composition containing neo-agar oligosaccharides of the first aspect, comprising the following steps: Glycerin, 1,3-butanediol, gelling agent, hydrolyzed sodium hyaluronate, inositol, and PEG / PPG-14 / 7 dimethyl ether were dispersed in water to form phase A. Mix the preservatives thoroughly to form phase B; The new oligosaccharide, oat fermentation broth, gastrodia extract, yeast / barley seed fermentation product filtrate and tromethamine were dissolved in a portion of water to form phase C; Add phase B to phase A, stir until homogeneous, then add phase C and homogenize to obtain a moisturizing and anti-aging composition containing neo-augment oligosaccharides.

[0015] This invention first fully hydrates gelling agents and basic moisturizers in phase A to construct a stable gel matrix; preservatives are premixed in phase B to ensure uniform distribution and effective antibacterial properties; while sensitive or easily inactivated core active ingredients (such as neo-agar oligosaccharides, plant extracts, and hyaluronic acid) are placed in phase C to avoid premature contact with extreme pH or high-concentration preservative systems. Finally, after phases A and B are mixed to form a stable base, phase C is added and adjusted to the skin-compatible range in one step. This precisely controls the pH of the system to maintain the optimal activity of each component, and the final homogenization step ensures uniform dispersion of active ingredients in the viscous matrix, thus achieving a synergistic unity of high activity retention, good stability, and excellent texture performance in large-scale production.

[0016] Thirdly, the present invention provides the application of the moisturizing and anti-aging composition containing neo-agar oligosaccharides of the first aspect in the preparation of cosmetics.

[0017] In some other embodiments, the moisturizing and anti-aging composition containing neoazo oligosaccharides has the effects of improving dry skin, whitening, reducing fine lines and wrinkles.

[0018] Fourthly, the present invention provides a cosmetic comprising the moisturizing and anti-aging composition containing neo-agar oligosaccharides of the first aspect.

[0019] In some other embodiments, the cosmetic also contains other inactive ingredient components that are permitted to be added in the cosmetic field.

[0020] The beneficial effects of this invention are: (1) Through systematic biochemical and cell experiments, this invention has for the first time clarified the direct mechanism of action of new agar oligosaccharides in anti-oxidation, anti-aging, anti-wrinkle and whitening, which surpasses the limitations of existing technologies that rely only on moisturizing inferences and in vitro data, and lays a solid scientific foundation for its application.

[0021] (2) Based on the mechanism of action of the new oligosaccharide, the present invention designed and optimized a special formula, which scientifically compounded the new oligosaccharide with hydrolyzed sodium hyaluronate, specific plant fermentation extracts, etc. Through the moisturizing and penetration-promoting network constructed by "glycerol / butanediol-gelling agent" and the special process of "phase separation preparation and final pH adjustment", it not only solved the industry problems of active ingredient synergy, transdermal and stability, but also transformed the anti-aging and whitening mechanism discovered in the laboratory into a stable production formulation.

[0022] (3) This invention further evaluated the basic anti-aging and whitening effects of the formula at the human level, realizing the empirical leap from "cellular mechanism" to "human efficacy", thus providing a complete solution from theoretical basis to productization and efficacy confirmation for the application of new oligosaccharide as a high-end cosmetic raw material with clear mechanism, stable formula and empirical efficacy. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 The new agar oligosaccharide in Example 1 of this invention affects DPPH and O 2- The graph shows the scavenging capacity of ·OH radicals, where A represents the scavenging capacity for DPPH and B represents the scavenging capacity for O2. 2- The scavenging capacity diagram is shown for α, and the scavenging capacity diagram for ·OH free radicals is shown for C. Figure 2The diagram shows the anti-aging efficacy of the new agar oligosaccharide in Example 1 of this invention. In the diagram, A is the effect of the CCK-8 assay on cell viability, B is the optimal concentration of H2O2 on HSF determined by the CCK-8 assay, C is the effect of the Edu assay on cell proliferation, D is the H2O2-induced oxidative senescence of HSF cells, with a scale bar of 100 μm, and E is the effect of the new agar oligosaccharide on the activity of intracellular β-galactosidase, with a scale bar of 50 μm. Figure 3 This is a graph showing the antioxidant analysis of the new agar oligosaccharide at the cellular level in Example 1 of the present invention. In the graph, A is the effect of the new agar oligosaccharide on SOD enzyme activity, B is the effect of the new agar oligosaccharide on CPx enzyme activity, and C is the effect of the new agar oligosaccharide on CAT enzyme activity. Figure 4 This is a graph showing the anti-wrinkle and anti-inflammatory effects of neo-Agar oligosaccharides at the cellular level in Example 1 of the present invention. A represents the effect of neo-Agar oligosaccharides on type I collagen, B represents the effect of neo-Agar oligosaccharides on type III collagen, C represents the synthesis of TIMP-1, D represents the synthesis of TIMP-3, E represents the synthesis of MMP-1, F represents the synthesis of MMP-8, G represents the synthesis of MMP-13, H represents the release of TNF-α, and I represents the release of IL-6. Figure 5 This is a diagram illustrating the anti-aging mechanism of the novel agar oligosaccharide in Example 1 of the present invention. In this diagram, A represents the inhibition of ROS production (scale bar: 100 μm), B represents the activation of NRF2 expression (scale bar: 100 μm), C represents the visual representation of the original experimental results, and D represents the relative protein expression level. Figure 6 The diagram shows the whitening mechanism of the new agar oligosaccharide in Example 1 of this invention. A is the inhibition rate of tyrosinase, B is the effect on melanin production, C is the inhibition rate of melanin production, and D is the inhibition of NO production. The scale bar is 100 μm. Figure 7 The following is a diagram showing the effect of the new oligosaccharide moisturizing and anti-aging essence in Example 2 of the present invention. In the diagram, A is the safety verification diagram, B is the skin stratum corneum moisture content diagram, C is the water loss diagram, D is the skin total elasticity diagram, E is the net elasticity diagram, F is the skin color diagram, and G is the ITA change diagram. Figure 8 This is a graph showing the change in the number of wrinkles in the Neo-Agar Oligosaccharide Moisturizing and Anti-aging Essence in Example 2 of the present invention; in, Figures 2-7 In the significance analysis, ns indicates no significant change. P<0.05, P<0.01, P<0.001. Detailed Implementation

[0025] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Specific conditions not specified in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products. The Gastrodia elata extract was purchased from Jinan Ruibeike Biotechnology Co., Ltd.

[0026] The main components of neo-AgaA oligosaccharides are neo-AgaA disaccharide, neo-AgaA tetrasaccharide, neo-AgaA hexasaccharide, neo-AgaA octasaccharide, and neo-AgaA decasaccharide, etc. The specific preparation method includes the following steps: Single colonies containing the target gene are picked to obtain the recombinant expression strain BL21(DE3). The engineered strains expressing AgaA enzyme and AgaB enzyme are inoculated into fermentation medium and cultured at 37 ℃ until OD... 600 The concentration reached 0.6-0.8. IPTG inducer was added, and expression was induced for another 24 h at the same temperature. The bacterial cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation again. The supernatant was filtered through a 209 filter paper to obtain a clear crude enzyme solution of AgaA and AgaB enzymes. A 0.2% (w / v) agar substrate solution was prepared, sterilized at high temperature, and then cooled and maintained at 60 °C. An excess of crude AgaA enzyme solution was added, and the reaction was carried out at this temperature for 24 h. Subsequently, the temperature was lowered to 45 °C, and crude AgaB enzyme solution was added, and the reaction was continued for 12 h. After the reaction was completed, the system was sterilized at high temperature to terminate the reaction. After cooling to room temperature, the system was centrifuged, and the supernatant was collected. The supernatant was filtered through 205 and 209 filter paper sequentially to obtain a clear and transparent oligosaccharide solution. The filtrate was concentrated to 1 / 10 of its original volume at high temperature, and then freeze-dried to finally obtain a new agar oligosaccharide powder.

[0027] The preparation method of oat fermentation broth is as follows: Oat seeds and wheat bran were mixed in a 1:2 mass ratio and pulverized through a 50-mesh sieve to obtain fine powder. Five parts of the fine powder, one part of carbon source, 0.2 parts of vitamin, two parts of inulin, and 90-95 parts of water were mixed evenly and sterilized at 121℃ (0.1 MPa) for 20 minutes to obtain a sterilized culture medium. The compound lactic acid bacteria solution (10...) 9 (CFU / mL, a mixture of *Lactobacillus plantarum* and *Lactobacillus paracasei* in a 1:1 ratio) was inoculated into sterile culture medium and fermented at 35°C under constant temperature conditions. The mixture was then cultured in a shaker at 200 r / min for 48 h to obtain the fermentation broth. The fermentation broth was heated to 70°C, sonicated for 10 min, and then 0.5% (w / w) activated carbon aqueous solution was added. The mixture was stirred at 10000 r / min for 1 h and then filtered through a 5 μm pore size filter to remove residue, resulting in a clear liquid. The clear liquid was then treated at 85°C and cooled to obtain the oat fermentation broth.

[0028] The preparation method of yeast / barley seed fermentation product filtrate is as follows: The culture medium for *Saccharomyces cerevisiae* included 15 g / L glucose, 5 g / L glycerol, 25 g / L soybean peptone, 2.5 g / L yeast extract, and 0.8 g / L dipotassium hydrogen phosphate. Barley seeds were broken down and pulverized to obtain barley seed powder. The barley seed powder was inoculated into the *Saccharomyces cerevisiae* culture medium at a material-to-liquid mass ratio of 1:20 for fermentation. The fermentation conditions were: shaking incubator speed of 10000 r / min, pH of 6.8, temperature of 35 ℃, and fermentation time of 48 h, yielding a crude fermentation broth. The crude fermentation broth was centrifuged (10000 rpm for 10 min) to obtain a clear fermentation product, which was then sterilized in an autoclave at 121 ℃ for 30 min. The filtrate was collected after filtration.

[0029] Example 1 This embodiment first studies the antioxidant, anti-aging, anti-wrinkle, and whitening effects and mechanisms of action of neoagar oligosaccharides at the biochemical and cellular levels. The specific process is as follows: I. Provide the biochemical antioxidant analysis of the new agar oligosaccharide, as detailed below: 1. Scavenging rate of DPPH by neo-Qiong oligosaccharides: Exogenous skin aging is primarily caused by photoaging, which leads to excessive accumulation of free radicals within cells, inducing oxidative stress and ultimately resulting in skin cell aging. Therefore, to investigate whether neo-agar oligosaccharides can inhibit free radical production, the effects of neo-agar oligosaccharides on DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) and O2 were first tested at the biochemical level. 2- The ability to scavenge ·OH free radicals.

[0030] (1) Prepare 0.1 mM DPPH solution and 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2 mg / mL neoagar oligosaccharide solutions respectively; (2) Sample group, control group and blank group were set up respectively; the sample group was 180 µL of different concentrations of new agar oligosaccharide + 90 µL of DPPH, the control group was 180 µL of DMSO (dimethyl sulfoxide) + 90 µL of DPPH, and the blank group was 180 µL of different concentrations of new agar oligosaccharide + 90 µL of anhydrous ethanol. (3) Shake in the dark for 10 minutes, and zero the container with anhydrous ethanol.

[0031] (4) Test absorbance at 520 nm.

[0032] (5) Record and analyze the data. DPPH clearance rate % = A 对照组 -(A 样品组 -A 空白组 ) / A 对照组 ×100%.

[0033] 2. Scavenging rate of neo-Agar oligosaccharides against ·OH: (1) Prepare solutions of 9 mmol / L ethanol-salicylic acid, 9 mmol / L ferrous sulfate, 8.8 mmol / L H2O2 and different concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2 mg / mL of neoagar oligosaccharide respectively; (2) The reaction system was carried out in a 1.5 mL colorimetric tube, and the order of sample addition is shown in Table 1 below; (3) Shake well, incubate in a 37 ℃ water bath for 15 min, and measure its absorbance; the absorbance of the sample is measured as A. X The blank control is A0, and the sample background is A. X0 ; (4) Record and analyze the data. ·OH removal rate % = [A0 - (A X -A X0 )] / A0×100%.

[0034] Table 1 shows the sample addition order for the ethanol-salicylic acid method for determining hydroxyl radical scavenging ability.

[0035] 3. Neo-Qiong oligosaccharides against O 2- Sweep rate: (1) Prepare solutions of 5 mmol / L pyrogallol, 0.1 mol / L Tris-HCl and 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 2 mg / mL neoagar oligosaccharide, respectively; (2) This reaction can be carried out in a cuvette, and the order of sample addition is shown in Table 2; (3) After rapid and uniform mixing, the absorbance of the solution is measured at 325 nm as the first absorbance value. The measurement is repeated every 1 min for 4 min. The increase in absorbance per minute within the linear range is calculated. △A0 is the auto-oxidation rate of pyrogallol; △A is the auto-oxidation rate of pyrogallol after the sample solution is added.

[0036] (4) Record and analyze the data, O 2- Clearance rate (%) = (△A0 - △A) / △A0 × 100%.

[0037] Table 2 shows the sample addition sequence for the determination of superoxide anion scavenging using the pyrogallol method.

[0038] Experimental results are as follows Figure 1 As shown, neo-agar oligosaccharides inhibited DPPH in a dose-dependent manner. Figure 1 A) O 2- ( Figure 1 B) and ·OH free radicals ( Figure 1 The production of C) in which neo-agar oligosaccharides at 0.6 mg / mL affect DPPH and O 2- The scavenging rates of ·OH free radicals reached 80%, 45%, and 25%, respectively; the new agar oligosaccharide at 2 mg / mL showed scavenging effects on DPPH and O2 free radicals. 2- The scavenging rates of ·OH free radicals reached 90%, 75%, and 35%, respectively.

[0039] II. Anti-aging analysis of Neo-Qiong oligosaccharides, as detailed below: Fibroblasts are one of the most abundant cell types in the dermis of the skin. They maintain normal skin structure by promoting the synthesis of collagen and elastin fibers, and maintain skin homeostasis by secreting various extracellular matrix components and related regulatory factors. This embodiment studies the anti-aging effects of neo-agar oligosaccharides by constructing an oxidative aging model.

[0040] 1. CCK-8 assay to detect the effect of neo-agar oligosaccharides on cell viability: (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) Dilute the cell suspension to a concentration of 4×10⁻⁶. 4 After inoculating at a rate of 1 / mL into a 96-well plate, the edge wells were filled with sterile phosphate-buffered saline (PBS), and three replicates were set up. (3) After the cells have completely adhered to the wall and entered the logarithmic growth phase, the control group was replaced with fresh culture medium, and 200 µL of culture medium containing different concentrations of neoagar oligosaccharides was added to the experimental group, and the cells were cultured for another 24 h. (4) After the culture is completed, add 10% CCK-8 solution to each well and incubate again for 2-3 h; (5) Select 490 nm as the detection wavelength and measure the absorbance (OD value) of each well. (6) Cell viability (%) = (OD 实验组 - OD 调零组） / (OD 空白对照组 - OD 调零组） × 100%.

[0041] Figure 2 The results in A showed that, compared with the control group (CTR), neo-agar oligosaccharides had no significant effect on cell viability between 100-600 μM, decreased cell viability between 800-1000 μM, and reached a half-inhibitory concentration for the cells at 1000 μM. However, neo-agar oligosaccharides at 600 µM significantly promoted cell proliferation.

[0042] 2. Edu assay to detect the effect of neo-agar oligosaccharides on cell proliferation: Cell proliferation was detected using the Edu (5-Ethynyl-2'-deoxyuridine) kit (Beyotime, Shanghai, China).

[0043] (1) The cells were seeded at an appropriate density in 12-well plates and treated with neoagar oligosaccharides for 24 h; (2) Add 10 μM Edu to each well, incubate at 37 °C for 2 h, then fix with 4% paraformaldehyde for 30 min, permeate with 0.3% Triton X-100, and wash twice with PBS. (3) Incubate with Click reaction solution in the dark for 30 min, and then stain the DNA with Hoechst 33342; (4) Four fields of view were randomly selected from each well and photographed using a fluorescence microscope (Olympus, Japan); (5) The formula for calculating the Edu-positive cell rate is: (Number of cells incorporating Edu / Number of cells stained with Hoechst) × 100%; Figure 2 The results of C showed that neo-Agar oligosaccharide significantly promoted the fluorescence count of HSF cells, indicating that 600 µM neo-Agar oligosaccharide significantly promoted the proliferation of HSF cells. Therefore, 600 μM neo-Agar oligosaccharide was selected for subsequent experiments.

[0044] 3. The optimal concentration of H2O2 for HSF was determined by the CCK-8 experiment, and the procedure was the same as in step 1 above. Figure 2 The results of B showed that H2O2 at 50-200 μM had no significant effect on cell viability, but at 300 μM it significantly inhibited cell viability. Therefore, 200 μM H2O2 was used to induce oxidative senescence in HSF cells.

[0045] 4. H2O2-induced oxidative senescence model of HSF cells: (1) Seed cells at an appropriate density in a six-well plate and process them after they adhere to the plate. (2) After the experimental group was treated with 200 µM H2O2 for 2-3 h, it was washed with PBS 3 times. The control group was replaced with fresh culture medium. The experimental group was treated with 600 µM agar oligosaccharide solution for 24 h, and then the anti-aging indicators were detected.

[0046] (3) Construction of aging model by β-gal staining experiment: Discard the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining fixative, fix at room temperature for 15 min, wash three times with PBS, prepare staining solution according to the reagent instructions and stain overnight; discard the staining solution, add PBS and take pictures under an optical microscope.

[0047] Figure 2 The results from D showed that, compared with the control group, cells treated with H2O2 had smaller cell volume, looser chromatin structure, and larger nuclei due to the reduction in water content; while the addition of neo-agar oligosaccharides reversed the H2O2-induced senescence of cells.

[0048] β-galactosidase activity increases significantly during cell senescence, and this elevated enzyme activity can serve as a marker of senescence. β-gal staining allows for a more direct observation of the inhibitory effect of neogalactosin oligosaccharides on cell senescence. Figure 2 The results from the study showed that, compared with the control group, the activity of β-galactosidase in cells was significantly increased after H2O2 treatment, indicating that the cell senescence model was successfully constructed. The addition of neo-agar oligosaccharides significantly reduced the activity of β-galactosidase in cells. These results indicate that neo-agar oligosaccharides can not only inhibit fibroblast senescence by promoting cell proliferation, but also delay fibroblast senescence by inhibiting oxidative damage.

[0049] III. Antioxidant analysis of neo-agar oligosaccharides at the cellular level, as detailed below: Excessive accumulation of free radicals promotes cellular aging by inducing oxidative stress. Intracellular antioxidant enzymes can scavenge free radicals generated in the body. For example, catalase (CAT) participates in the oxidation of fatty acids and the decomposition of hydrogen peroxide, clearing peroxides and other toxic substances produced during metabolism; superoxide dismutase (SOD) catalyzes the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen, maintaining metabolic homeostasis; and glutathione peroxidase (GPx) protects cells from oxidative damage by maintaining intracellular glutathione levels. Therefore, antioxidant enzymes play a crucial role in maintaining anti-aging processes. The effects of neoagar oligosaccharides on antioxidant enzyme activity were investigated at the cellular level.

[0050] 1. Effect of neo-agar oligosaccharides on SOD enzyme activity: (1) Seed cells at an appropriate density in a six-well plate and process them after they adhere to the plate. (2) Changes in intracellular SOD activity were detected using a total SOD activity assay kit (Beyotime, Shanghai, China); (3) Prepare the WST-8 enzyme working solution and reaction start-up working solution in advance according to the instructions; (4) After processing according to the steps of constructing the aging model, discard the culture medium and wash several times with PBS. Use the SOD sample preparation solution provided in this kit to properly lyse the cells by pipetting. (5) After centrifugation at 4℃ for 5-10 min, the supernatant was collected for SOD enzyme assay; (6) The order of sample addition is shown in Table 3. After the sample addition is completed, incubate at 37°C for 30 min. (7) Measure the absorbance at 450 nm, record the data, and perform calculations. SOD enzyme activity inhibition rate = [(A 空白对照1 -A 空白对照2 )-(A 样品 -A 空白对照3 )] / (A 空白对照1 -A 空白对照2 ) × 100%, SOD Vitality (U / 10) 4 Cell) = 0.04 Inhibition rate / (1 - Inhibition rate).

[0051] Table 3. Sample addition sequence for the total SOD activity assay kit (WST-8 method)

[0052] In Table 3, "-" indicates "not added".

[0053] 2. Effect of neo-agar oligosaccharides on CAT enzyme activity: (1) Seed cells at an appropriate density in a six-well plate and process them after they adhere to the plate. (2) Changes in intracellular CAT enzyme activity were detected using an H2O2 enzyme detection kit (Beyotime, Shanghai, China); (3) After processing according to the steps of constructing the aging model, the culture medium was discarded and the cells were washed several times with PBS. Cell lysis buffer (P0013) was used to lyse the cells properly by pipetting. (4) After centrifuging at 4 ℃ for 5-10 min, take the supernatant to determine CAT enzyme activity. Take 10 µL of sample into a 1.5 mL plastic centrifuge tube, add H2O2 enzyme detection buffer to a volume of 40 µL, and then add 10 µL of 250 mM H2O2 solution. (5) React at 25 °C for 1-5 min, then add 450 µL of H2O2 reaction termination solution, and mix by inverting or Vortexing to terminate the reaction. (6) Add 40 µL of H2O2 enzyme detection buffer to a clean EP centrifuge tube, then add 10 µL of the above reaction system that has been terminated and mixed, and mix well. (7) Take 10 µL from the above reaction system and add it to one well of a 96-well plate, then add 200 µL of the chromogenic working solution. Incubate at 25 °C for at least 15 min, and then measure its OD value using a microplate reader at a wavelength of 520 nm. (8) Record and analyze the data, CAT(U / 10) 4 cell) = ΔA×V 反总 ÷(ε×d)×10 6 ÷(V 样 ÷V 样总 (×500)÷T = 1.529×ΔA.

[0054] 3. Effect of neo-agar oligosaccharides on GPx enzyme activity: (1) Seed cells at an appropriate density in a six-well plate and process them after they adhere to the plate. (2) Changes in intracellular GPx enzyme activity were detected using a GPx enzyme detection kit (Beyotime, Shanghai, China); (3) Prepare 62.5 mM NADPH solution, 75 mM GSH solution, GPx detection working solution and 30 mM peroxide reagent solution in advance; (4) After processing according to the steps of constructing the aging model, discard the culture medium and wash several times with PBS. Use cell lysis buffer (P0013) and gently pipette to fully lyse the cells. (5) After centrifuging at 4 ℃ for 5-10 min, take the supernatant to determine GPx enzyme activity; (6) The order of sample addition is shown in Table 4. Add 10 µL of 30 mM peroxide reagent solution to each well and mix well. (7) Heat the microplate reader to 25°C and set the wavelength to 340 nm to measure its OD value; (8) GPx (U / 10) 4 (cell) = ΔA determination ÷ (ΔA standard ÷ C standard) × 1000 × V enzyme ÷ (cell number × V sample ÷ V total sample) ÷ T = 200 × ΔA determination ÷ ΔA standard ÷ cell number.

[0055] Table 4 Sample addition sequence for the GPx activity assay kit

[0056] In Table 4, "-" indicates that it does not contain.

[0057] Figure 3 The results showed that, compared with the control group, H2O2-induced fibroblasts significantly inhibited CAT ( Figure 3 C in SOD Figure 3 A) and GPx ( Figure 3The addition of neo-agar oligosaccharides reduced the activity of CAT, SOD, and GPx, while the addition of neo-agar oligosaccharides reversed the activity of CAT, SOD, and GPx. These results indicate that neo-agar oligosaccharides delay the aging process of fibroblasts by resisting oxidative stress, promoting the activity of antioxidant enzymes, and inhibiting the generation of free radicals.

[0058] IV. Analysis of the anti-wrinkle and anti-inflammatory effects of neo-agar oligosaccharides at the cellular level, as detailed below: The reduction of collagen is one of the important mechanisms of skin aging. Supplementing collagen can improve skin elasticity and texture and delay the aging process.

[0059] Collagen is the main structural protein of the skin. With age, the synthesis of collagen slows down while the degradation rate increases, leading to a decrease in skin elasticity and firmness, resulting in wrinkles and sagging. To investigate whether neo-Agar oligosaccharides can reverse collagen loss caused by oxidative stress, Matrixyl-3000 (1%) and recombinant human collagen (0.1%) were used as positive controls. The effects of neo-Agar oligosaccharides on the content of type I and type III collagen were detected by ELISA.

[0060] 1. Neo-Qiong oligosaccharides promote the production of type I and III collagen: (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) After thoroughly mixing the cell suspension, take 10 µL and count the cells using a hemocytometer. Dilute the cell suspension to a concentration of 4 × 10⁻⁶. 4 cells / mL; (3) Mix the cell suspension thoroughly and then seed it into a six-well plate. Set up three replicates for each concentration gradient. Note that after adding a few wells, you need to resuspend the cells by pipetting. (4) The next day, discard the old culture medium, add H2O2 to the aging model group, sample group and positive control group for 1-3 h, replace the blank control group and aging model group with fresh culture medium, add Matrixyl-3000 (1%) and human recombinant collagen (0.1%) to the positive control group, add 600 µM of new agar oligosaccharide to the sample group, and continue to culture in the cell culture incubator for 24 h; (5) After 24 h of treatment, discard the original culture medium, wash the cells twice with PBS, add cell lysis buffer and lyse for 30 min, sonicate for 5-10 min, centrifuge to obtain supernatant for subsequent experiments. (6) After incubation, the corresponding type I and type III collagen ELISA kits were used for experimental determination; (7) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 min, and seal the remaining strips in a self-sealing bag and return them to 4℃; (8) Set up standard wells and sample wells, and add 50 µL of standard at different concentrations to each standard well; (9) Add 50 µL of the sample to be tested to the sample well first; do not add any to the blank well; (10) Except for the blank wells, add 100 µL of horseradish peroxidase (HRP) labeled antibody to each well of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate in a water bath or incubator at 37 ℃ for 60 min. (11) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times. (12) Add 50 µL of substrate A and B to each well and incubate at 37°C in the dark for 15 min; (13) Add 50 µL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min. (14) Standard curve plotting and sample concentration calculation: Establish a standard curve based on the concentration (x, pg / mL) of the standard tube and the absorbance ΔA standard (y, ΔA standard). Based on the standard curve, substitute the ΔA measurement (y, ΔA measurement) into the formula to calculate the sample concentration (x, pg / mL).

[0061] Compared with the control group, H2O2 treatment of HSF cells significantly inhibited type I ( Figure 4 Type A and Type III (in the middle) Figure 4 In the B) collagen synthesis, compared with the H2O2-induced aging model group, the addition of Matrixyl-3000, recombinant human collagen and neo-Qiong oligosaccharides promoted the synthesis of type I and type III collagen. Among them, the effect of neo-Qiong oligosaccharides on the synthesis of type I and type III collagen was comparable to that of the positive control group.

[0062] 2. Neo-Agar oligosaccharides promote collagen synthesis by inhibiting the expression of TIMPs / MMPs: Matrix metalloproteinases (MMPs) accelerate skin aging by breaking down collagen and other ECM components, while matrix metalloproteinase inhibitors (TIMPs) specifically inhibit their activity by covalently binding to MMPs, thus jointly regulating matrix metabolism and maintaining the structure of the dermis. To investigate whether neo-agar oligosaccharides promote collagen regeneration through the TIMPs / MMPs pathway, Matrixyl-3000 and recombinant human collagen were used as positive controls. The expression of matrix metalloproteinases MMP-1, MMP-8, and MMP-13, which mainly degrade type I and type III collagen, and changes in the synthesis of their inhibitors TIMP-1 and TIMP-3 were examined.

[0063] The experimental procedure followed the guidelines for promoting the production of type I and III collagen with neo-Qiong oligosaccharides, and the corresponding ELISA kits for TIMP1 / 3 and MMP-1 / 8 / 13 were used for detection.

[0064] Compared with the control group, H2O2 significantly inhibited intracellular TIMP-1 in HSF cells ( Figure 4 C) and TIMP-3 Figure 4 The synthesis of D in MMP-1 significantly promoted the synthesis of MMP-1 (D in MMP-1). Figure 4 The synthesis of E), MMP-8 and MMP-13; Figure 4 The results showed that, compared with the H2O2 group, the addition of Matrixyl-3000, recombinant human collagen, and neo-Agar oligosaccharides significantly promoted the synthesis of TIMP-1 and TIMP-3, while significantly inhibiting the synthesis of MMP-1 and MMP-8. Figure 4 (F) and MMP-13 ( Figure 4 The synthesis of G in the above results indicates that neo-agar oligosaccharides resist collagen loss caused by H2O2-induced oxidative stress through the TIMPs / MMPs signaling pathway, thus delaying cell aging.

[0065] 3. Neo-agar oligosaccharides inhibit the release of TNF-α and IL-6 inflammatory factors: Senescent skin cells exhibit SASP (Skin Acid-Prone Skin Cells), releasing large amounts of pro-inflammatory cytokines (such as IL-6 and IL-8). These factors activate signaling pathways such as NF-κB, leading to the degradation of collagen and elastin, thereby triggering inflammatory responses and skin aging. To investigate whether neo-agar oligosaccharides have anti-inflammatory effects, an inflammatory cell model was constructed. Mouse RAW264.5 macrophages were treated with neo-agar oligosaccharides for 24 h, followed by stimulation with the endotoxin LPS (1 µg / mL) for 2 h. The release levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were then measured.

[0066] (1) Select RAW264.7 cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) After thoroughly mixing the cell suspension, take 10 µL and count the cells using a hemocytometer. Dilute the cell suspension to a concentration of 4 × 10⁻⁶. 4 cells / mL; (3) Mix the cell suspension thoroughly and then seed it into a six-well plate. Set up three replicates for each concentration gradient. Note that after adding a few wells, you need to resuspend the cells by pipetting. (4) The next day, the old culture medium was removed, and the blank group and negative control group were replaced with fresh culture medium. The sample groups were treated with 600 µM, 1200 µM and 1800 µM of new agar oligosaccharide for 24 h respectively. (5) After 24 hours of treatment, 10-20 µL of 1-5 µg / mL LPS was added to the negative control group and the sample group to stimulate the cells for 2-4 hours. Then, the TNF-α and IL-6 inflammatory factor ELISA kits were used for subsequent detection. (6) The experimental procedure is the same as the ELISA detection procedure for promoting the production of type I and III collagen by neo-Qiong oligosaccharides.

[0067] Compared with the control group, LPS stimulation significantly stimulated intracellular TNF-α ( Figure 4 H) and IL-6 ( Figure 4 In addition to inhibiting the release of inflammatory factors, neo-Qon oligosaccharides significantly reduced the levels of TNF-α and IL-6 at 1800 μM (0.6 mg / mL) in a concentration-dependent manner. These results indicate that neo-Qon oligosaccharides can not only promote the synthesis of type I and type III collagen by inhibiting the TIMPs / MMPs pathway, but also inhibit the expression of pro-inflammatory factors, thereby suppressing the cellular aging process.

[0068] V. The anti-aging mechanism of neo-Qiong oligosaccharides: Studies have shown that external stimuli such as ultraviolet radiation can lead to excessive production of reactive oxygen species (ROS) in skin cells, triggering oxidative stress. Excessive ROS accumulation can not only oxidize the cell membrane system, forming lipofuscin and the common "age spots," but also activate the expression of matrix metalloproteinases (MMPs), degrading the extracellular matrix and promoting the degradation of collagen and elastin in dermal cells, thereby leading to decreased skin elasticity and wrinkle formation. This study investigated the ROS scavenging ability of neoagar oligosaccharides in HSF cells under H2O2-induced oxidative stress.

[0069] 1. The effect of agarbiose on ROS scavenging ability: (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase and seed them at an appropriate density in a six-well plate. After the cells adhere to the plate, they are then processed. (2) After 24 h of treatment following the steps for constructing the aging model, the culture medium was discarded and the sample was washed several times with PBS. (3) Dilute the DCFH-DA probe with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 µM; (4) Discard the original culture medium, add an appropriate volume of diluted DCFH-DA probe, and incubate at 37°C for 20 min.

[0070] (5) Wash the cells three times with serum-free cell culture medium to thoroughly remove the DCFH-DA probes that have not entered the cells; (6) Observe under a fluorescence microscope.

[0071] Figure 5 The results in A showed that, compared with the control group, H2O2 treatment of HSF cells significantly promoted the accumulation of ROS, while the addition of neo-augment oligosaccharides significantly inhibited the production of ROS.

[0072] 2. Neo-Qiong oligosaccharides activate Nrf2 for nuclear translocation: When Nrf2 dissociates from Keap1 and translocates into the cell nucleus, it can activate the expression of various antioxidant enzymes, scavenge excess ROS, and alleviate oxidative stress damage to skin cells. The effect of neo-agar oligosaccharides on Nrf2 was then detected using immunofluorescence assays.

[0073] (1) The cells were loaded with 1×10 4 The density is laid in a six-hole plate; (2) After the cells adhered to the wall, the control group was replaced with fresh culture medium, while the experimental group was treated with 600 µM of new agar oligosaccharide for 24 h. (3) After the culture is completed, remove the supernatant and wash with PBS. Then add 4% paraformaldehyde for fixation. (4) Permeation was performed with 0.3% Triton 100 for 20-30 min. (5) After washing with PBS, add 2% BSA and block for 30 min; (6) Add the NRF2 primary antibody dilution solution and incubate overnight at 4 °C.

[0074] (7) The next day, after discarding the primary antibody, wash with TBST and then add Goat Anti-Rabbit lgGTRITC and incubate in the dark for 1 hour; (8) Then TBST was added for washing, followed by DIPA for nuclear restaining; (9) Take pictures under a fluorescence microscope.

[0075] Figure 5 Results B showed that the new oligosaccharide not only significantly activated NRF2 expression, but also promoted the translocation of Nrf2 from the cytoplasm to the nucleus.

[0076] 3. Neo-Agar oligosaccharides delay fibroblast senescence through the Nrf2 / Keap1 signaling pathway: Nrf2, a key transcription factor, regulates the expression of various antioxidant enzymes, thereby enhancing the antioxidant capacity of cells. Next, Western blot experiments were used to examine the effects of neo-agar oligosaccharides on the expression of Nrf2, Keap1, and the downstream transcription factor HO-1.

[0077] (1) The cells were loaded with 1×10 4 The density is laid in a six-hole plate; (2) After the cells adhered to the wall, the control group was replaced with fresh culture medium, and the experimental group was treated with mL 385 (2µM), new agar oligosaccharide (600 µM), and the combination of the two for 24 h. (3) After the treatment, the cells were collected and RIPA lysis buffer containing PMSF was added. The cells were then placed in a refrigerator at 4°C for lysis for 30 min. (4) After collecting the cell suspension, centrifuge at 4 ℃ and 12000 rpm to obtain the protein supernatant; (5) First, use the BCA protein detection kit to determine the protein concentration in the supernatant; (6) Take the remaining protein supernatant and mix it with 6× SDS-PAGE loading buffer (mix well in proportion, boil in a water bath for 10 min and then place on ice for later use). (7) Take 50-70 μg of total cell protein and perform SDS / PAGE with 10-12% gel, transfer it to PVDF membrane, and block the membrane with 5% skim milk for 60-90 min. (8) The imprinted membrane was incubated overnight at 4 °C using a primary antibody solution of Nf2, Keap1 and GAPDH; (9) After thorough washing with TBS-T, the blot membrane was incubated with a suitable horseradish peroxidase-conjugated secondary antibody for 2 hours.

[0078] (10) Protein bands were visualized by chemiluminescence using an ECL detection kit (Millipore, Billerica, MA, USA).

[0079] Figure 5 The C in the image is a visual representation of the original experimental results for NRF2, Keap1, HO-1, and the internal control protein GAPDH. Figure 5The results showed that neo-Augmentosin oligosaccharides inhibited Keap1 expression and promoted the expression of Nrf2 and the downstream transcription factor HO-1. When the Nrf2 inhibitor mL385 was added, mL385 significantly inhibited the expression of Nrf2 and HO-1 and promoted the release of Keap1, while the addition of neo-Augmentosin oligosaccharides reversed this phenomenon. These experimental results indicate that neo-Augmentosin oligosaccharides combat oxidative stress-induced aging by activating Nrf2 expression, promoting its nuclear shift, and enhancing the expression of downstream antioxidant transcription factors.

[0080] VI. The whitening mechanism of neo-glucan oligosaccharides is as follows: To investigate the skin-whitening effects of neo-Qiong oligosaccharides, the effects of neo-Qiong oligosaccharides on tyrosinase activity and melanin production were examined at both the biochemical and cellular levels.

[0081] 1. Effects of neo-agar oligosaccharides on tyrosinase: (1) Add 800 µL of L-tyrosine solution and 1500 µL of PBS (pH=6.8) buffer to the control group and blank group respectively; add 800 µL of L-tyrosine solution, 1500 µL of PBS (pH=6.8) buffer and 800 µL of test sample to the sample group and blank group respectively, and mix well. (2) Heat each experimental group in a 37°C water bath for 10 min. At the same time, preheat the tyrosinase in a 37°C water bath for 10 min. (3) After the water bath heating is completed, 1000 µL of tyrosinase solution was added to the control group and the sample group respectively, and after mixing, the mixture was heated in a water bath at 37 °C for 40 min. (4) After the reaction is complete, each experimental group is quickly transferred to a 96-well plate and the absorbance is immediately measured at 475 nm using an enzyme-linked immunosorbent assay (ELISA) reader. (5) Formula for tyrosinase activity inhibition rate: Inhibition rate = ((OD 对照组 -OD 空白组） -(OD 样品组 -OD 样品空白组） ) / (OD 对照组 -OD 空白组 ).

[0082] Figure 6 Results A showed that, compared with the positive control group (0.5% arbutin), Neo-Agar oligosaccharide inhibited tyrosinase activity in a concentration-dependent manner. The inhibition rate of tyrosinase by 1 mg / mL Neo-Agar oligosaccharide reached approximately ±50%, achieving an effect comparable to arbutin.

[0083] 2. Effects of CCK-8 assay on the viability of new agar oligosaccharides on melanoma cells: (1) Select B16 cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) Dilute the cell suspension to a concentration of 4×10⁻⁶. 4 After inoculating the cells / mL into a 96-well plate, sterile PBS was used in the edge wells, and three replicates were set up. (3) After the cells have completely adhered to the wall and entered the logarithmic growth phase, the control group was replaced with fresh culture medium, and 200 µL of culture medium containing different concentrations of neoagar oligosaccharides was added to the experimental group, and the cells were cultured for another 24 h. (4) After the culture is completed, add 10% CCK-8 solution to each well and incubate again for 2-3 h; (5) Select 490 nm as the detection wavelength and measure the absorbance (OD value) of each well. (6) Cell viability (%) = (OD 实验组 - OD 调零组） / (OD 空白对照组 - OD 调零组） × 100%.

[0084] Figure 6 The results in section B showed that 100-1000 µM of neo-agar oligosaccharide had no significant effect on B16 cell viability. To ensure consistency with the previous results, 600 µM of neo-agar oligosaccharide was selected to detect its effect on melanin production.

[0085] 3. Neo-agar oligosaccharides inhibit melanin production. (1) Select B16 cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) Dilute the cell suspension to a concentration of 4×10⁻⁶. 4 After inoculating the cells / mL into a 96-well plate, sterile PBS was used in the edge wells, and three replicates were set up. (3) After the cells adhered to the wall, the control group was replaced with fresh culture medium, the experimental group was given culture medium containing 600 µM neoagar oligosaccharide, and the positive control group was given culture medium containing 100 µM arbutin. The cells were placed for 20-24 h. (4) After the treatment, wash the cells twice with PBS, digest the cells with trypsin, and centrifuge them at 4000 rpm for 5 min to obtain cell pellet; (5) Add 200 µL of melanin extract to each tube, shake well, and heat in an 80 ℃ water bath for 1 h; (6) After cooling, centrifuge the droplets on the tube wall under moderate pressure, blow them evenly, and take 150 µL of solution from each centrifuge tube and transfer them into a 96-well plate. Use an ELISA reader to detect the absorbance at 405 nm in each well.

[0086] (7) Calculate the melanin synthesis inhibition rate: Inhibition rate (%) = (CT) / (C-C0) × 100%; Wherein, C is the melanin content of the negative control group (without inhibitor), T is the melanin content of the experimental group (with inhibitor), and C0 is the melanin content of the blank control group.

[0087] Figure 6 The results showed that arbutin and neo-glucan oligosaccharides inhibited melanin production by approximately 25% and 40%, respectively. Neo-glucan oligosaccharides significantly inhibited melanin production in B16 cells without inhibiting melanocyte activity.

[0088] 4. Neo-agar oligosaccharides inhibit NO production. Studies have shown that NO increases intracellular cGMP levels by activating guanylate cyclase, thereby activating tyrosinase and increasing melanin production. Furthermore, NO can enhance melanin production by increasing MC1R expression and stimulating α-MSH secretion. The ability of neo-agar oligosaccharides to scavenge NO can, to some extent, reflect their skin-whitening effect. Therefore, the ability of neo-agar oligosaccharides to scavenge NO in Hacat cells was detected using a fluorescent probe. However, whether neo-agar oligosaccharides inhibit NO production has not yet been reported.

[0089] (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase and seed them at an appropriate density in a six-well plate; (2) After the cells adhered, they were treated for 24 h according to the established cell senescence model group; (3) Dilute the DAF-FM DA probe with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 µM; (4) Discard the original culture medium, add an appropriate volume of diluted DAF-FM DA probe, and incubate at 37°C for 20 min; Wash the cells three times with serum-free cell culture medium to thoroughly remove any DAF-FM DA probes that have not entered the cells; (6) Observe under a fluorescence microscope.

[0090] Figure 6 The results from the study showed that, compared with the control group, H2O2-induced NO production in Hacat cells was significantly increased, while the addition of neo-agar oligosaccharides significantly inhibited NO production.

[0091] VII. Safety Verification of Neo-Qiong Oligosaccharides To further verify the anti-aging effects of Neo-Qiong oligosaccharides at the human level, the safety of low (5 mg / mL), medium (10 mg / mL), and high (50 mg / mL) concentrations of Neo-Qiong oligosaccharides was first verified by taking advantage of the intact, clear, and transparent vascular system of the chorioallantoic membrane of hatched chicken embryos.

[0092] (1) CAM preparation: Select 7-day-old chicken embryos, check by candling, place the air cell end of the chicken embryo upwards, and draw the outline of the air cell of the chicken embryo with a pencil. (2) Drill a hole directly above the air cell of the chicken embryo with pointed tweezers, carefully peel off the eggshell above the air cell along the outline of the air cell with tweezers (be careful not to get too close to the outline of the air cell), blow off the eggshell that has fallen on the air cell membrane with a bulb blower, add 1 mL of physiological saline to moisten the eggshell membrane and discard the residual physiological saline, disinfect the pointed tweezers again with an alcohol lamp and carefully tear off the eggshell membrane (do not puncture the allantoic membrane and blood vessels during this process), exposing the allantoic membrane with a diameter of 2-3 cm; (3) At this point, observe the structure of the vascular system again and make a judgment on its integrity and suitability for the experiment; (4) Endpoint assessment method: Since the sample was a transparent liquid, the time assessment method was selected for detection. That is, 0.3 mL of the above transparent liquid was directly dropped onto the CAM surface, the CAM reaction was observed, and the time of occurrence of each toxic effect within 5 min was recorded; (5) The irritation score (IS) is shown in Table 5: (6) The reaction time method is used to conduct the test. The stimulus score (IS) is calculated using the following formula and the result is kept to two decimal places. According to the IS value, the eye irritation of the test substance is classified according to the following table.

[0093] IS=(301-sec H) 5 / 300+(301-sec L) ×7 / 300+(301-sec C) ×9 / 300; Note: sec H (bleeding time) - the average time observed on the CAM membrane for the onset of bleeding, in seconds (s); sec L (vascular dissolution time) - the average time observed on the CAM membrane for the onset of vascular dissolution, in seconds (s); sec C (clotting time) - the average time observed on the CAM membrane for the onset of clotting, in seconds (s).

[0094] Table 5 Evaluation of Reaction Time Method Results Stimulus rating Stimulus Classification IS<1 Non-irritating IS<5 Mild irritation 5≤IS<10 moderate irritation IS>10 Strongly irritating / corrosive Figure 7Results A showed that the IS scores of the negative control group, positive control group (0.1 mol NaOH), low concentration (5 mg / mL), medium concentration group (10 mg / mL), and high concentration group (50 mg / mL) were relatively consistent. Since the IS score of the positive control group was between 10 and 19, the experiment was considered reliable. The IS values ​​of the sample groups at different concentrations were all less than 1. Therefore, the new agar oligosaccharides at low, medium, and high concentrations were all safe and non-irritating.

[0095] Example 2 This embodiment provides a moisturizing and anti-aging essence containing neo-agar oligosaccharides, the specific composition of which is shown in Table 6. The preparation method of the moisturizing and anti-aging composition containing neo-agar oligosaccharides includes the following steps: Glycerin, 1,3-butanediol, gelling agent, hydrolyzed sodium hyaluronate, inositol, and PEG / PPG-14 / 7 dimethyl ether were dispersed in water to form phase A. Mix the preservatives thoroughly to form phase B; The new oligosaccharide, oat fermentation broth, gastrodia extract, yeast / barley seed fermentation product filtrate and tromethamine were dissolved in a portion of water to form phase C; Add phase B to phase A, stir until homogeneous, then add phase C and homogenize to obtain a moisturizing and anti-aging composition containing neo-augment oligosaccharides.

[0096] Example 3 This embodiment provides a new oligosaccharide moisturizing and anti-aging essence, the specific composition of which is shown in Table 6, and the preparation method is the same as that in Example 2.

[0097] Example 4 This embodiment provides a new oligosaccharide moisturizing and anti-aging essence, the specific composition of which is shown in Table 6, and the preparation method is the same as that in Example 2.

[0098] Table 6. Neo-Gynosyl Oligosaccharide Moisturizing and Anti-aging Essence

[0099] Comparative Example 1 Unlike Example 2, the neo-agar oligosaccharide was replaced with an equal amount of water, while the other components remained unchanged.

[0100] Comparative Example 2 Unlike Example 2, neo-agar oligosaccharide was replaced with neo-agar disaccharide in equal amounts.

[0101] Comparative Example 3 Unlike Example 2, the neo-agar oligosaccharides were replaced with neo-agar disaccharides and neo-agar tetrasaccharides in equal amounts (the mass ratio of neo-agar disaccharides and neo-agar tetrasaccharides was 1:1), while the other components remained unchanged.

[0102] Comparative Example 4 Unlike Example 2, the oat fermentation liquid was replaced with an equal amount of water, while other components remained unchanged.

[0103] Comparative Example 5 Unlike Example 2, the Gastrodia elata extract was replaced with an equal amount of water, while the other components remained unchanged.

[0104] Comparative Example 6 Unlike Example 2, the yeast / barley seed fermentation product filtrate was replaced with an equal amount of water, while other components remained unchanged.

[0105] Experimental Example 1 The human efficacy evaluation of the new agar oligosaccharide anti-aging and whitening essence prepared in Example 2 was conducted using the following specific steps: An essence containing 0.5% neo-glucan oligosaccharides was distributed to 10 eligible volunteers and used for 28 days. Their skin anti-aging and whitening indicators were then tested.

[0106] The moisture content and water loss of the stratum corneum are important indicators for measuring skin hydration. Adequate hydration helps delay the skin aging process and reduce the appearance of fine lines and wrinkles. The changes in stratum corneum moisture content and epidermal water loss rate in subjects after using a serum were detected using a Corneometer CM825 and a Tewameter™ 300.

[0107] Figure 7 Results B showed that during the test, the stratum corneum moisture content of the subjects showed an increasing trend. After 28 days of using the serum, the stratum corneum moisture content of the skin increased significantly, while the epidermal water loss showed a decreasing trend. Figure 7 (C in the text), which is consistent with the results of its detection of increased stratum corneum moisture content.

[0108] Total elasticity of the skin refers to its ability to return to its original state after being subjected to external force. Net elasticity is usually obtained by subtracting certain inelastic factors (such as skin plastic deformation) from total elasticity, and it more directly reflects the functional state of the skin's elastic fibers and collagen. Total and net elasticity are important indicators for assessing skin condition, reflecting the skin's health and degree of aging. Then, the changes in total and net elasticity of the subjects' skin after using a serum were detected using a Cutometer MPA580. After using the serum, the subjects' total skin elasticity ( Figure 7 D) and net elasticity ( Figure 7The levels of E in the samples showed an increasing trend over time, and after 28 days of use, the total elasticity and net elasticity of the subjects' skin significantly increased. These results indicate that after using the Neo-Qiong Oligosaccharide Essence, the subjects' skin showed increased collagen and elastin content, resulting in a tighter skin structure and improved skin moisture retention.

[0109] Skin color and ITA (Intense Powder Tariff) are important indicators for assessing skin pigmentation. A higher skin color value indicates a color closer to white. The ITA value, on the other hand, represents skin whiteness; a higher ITA value indicates a lighter skin tone. Next, the effect of neo-augmented oligosaccharides on pigmentation in the subjects was investigated using the Skin-Colorimeter CL400.

[0110] After 14 days of using the serum, the subjects' skin color ( Figure 7 F) and ITA value ( Figure 7 The levels of G in the samples were elevated, with significant changes in skin color observed after day 14. The ITA values ​​increased in a time-dependent manner, indicating that neo-agar oligosaccharides improve skin color by inhibiting melanin production and delaying pigmentation.

[0111] Finally, to further characterize the anti-aging efficacy of the new oligosaccharide, changes in the number of wrinkles in the subjects could visually demonstrate the anti-aging effect of the ingredient. The VISIA facial image analyzer was used to detect changes in the number of wrinkles in the subjects after using the new oligosaccharide essence. The results are as follows: Figure 8 As shown, among which, Figure 8 The eyebrows and eyes of the people appearing in the video have been obscured and blurred.

[0112] Depend on Figure 8 It can be seen that after using the new oligosaccharide essence for 14 days, the number of wrinkles in the subjects was significantly reduced. On the 28th day, the number of wrinkles in the subjects was reduced by about 40%.

[0113] In summary, the experimental results indicate that neo-Gynostemma oligosaccharides can not only resist oxidative stress through the Nrf2 / Keap1 signaling pathway, but also delay skin aging by promoting collagen production. Furthermore, neo-Gynostemma oligosaccharides can reduce skin pigmentation by decreasing NO production in the stratum corneum and inhibiting melanin production.

[0114] Experiment Example 2 This experiment provides a skin irritation test for moisturizing and anti-aging essences, using a standard skin patch test. The test results are shown in Table 7. The grading criteria for adverse skin reactions in the patch test are as follows: 0 for no reaction, 1 for light erythema, 2 for erythema, infiltration, and papules, 3 for erythema, edema, papules, and vesicles, and 4 for erythema, edema, and bullae.

[0115] Table 7 shows the observation results. Serial Number High concentration group (60 mg / mL) Example 2 0.02 Example 3 0.03 Example 4 0.05 Comparative Example 1 3.02 Comparative Example 2 3.10 Comparative Example 3 2.13 Comparative Example 4 2.27 Comparative Example 5 2.39 Comparative Example 6 2.54 As can be seen from the test results in Table 7, the test levels of Examples 2-4 are all less than 1, indicating no adverse reactions.

[0116] Experimental Example 3 This experiment provides a stability test for moisturizing and anti-aging serums. The test method is as follows: Samples from Examples 1-3 and Comparative Examples 1-8 were placed at room temperature and... 18℃, 4℃, 45℃, hot and cold cycles ( The product was subjected to stability observation at 18℃, 4℃, and 45℃ (cycled every 3 days) for the corresponding time. The stability of the product was judged by whether precipitation occurred. If no precipitation or instability occurred, the product passed the stability test. The specific results are shown in Table 8 below.

[0117] Table 8 Stability

[0118] Note: √ indicates that the stability has passed and there is no precipitation; × indicates slight precipitation; ×× indicates severe precipitation; ○ indicates that the solution color has changed and the color has darkened slightly; ○○ indicates that the solution color has changed and the color has darkened significantly.

[0119] Experiment Example 4 This experiment provides an assessment of the antioxidant and anti-aging properties of a moisturizing and anti-aging serum. The anti-aging assay utilizes elastase inhibition, and the specific experimental method is as follows: Elastase (porcine pancreas) was used as the research subject. Sample groups (Examples 2-4), comparative groups (Examples 1-6), and a negative control group were established, with three replicates for each group. 10 μL of the test sample was added to each well of a 96-well plate. 10 μL of diluent was added to the negative control group. Then, 20 μL of 0.1 U / mL elastase solution was added to each well. The 96-well plates were incubated at 25°C for 15 min. Then, 50 μL of substrate (98% N-succinyl-L-alanyl-L-alanine) at a concentration of 1 mg / mL was added. The absorbance of the samples at 410 nm was measured using a microplate reader, and the inhibition rate of elastase activity by the test samples was calculated.

[0120] The elastase activity inhibition rate is calculated using the following formula: Elastase activity inhibition rate (%) = [(△A-△B) / △A] × 100%; Where △A represents the absorbance value of the negative control group without the test sample, and △B represents the absorbance values ​​of different concentrations of the test solution. The higher the elastase inhibition rate, the better the anti-aging effect of the sample.

[0121] Antioxidant assay was performed using a DPPH free radical scavenging test. According to the "Cosmetic Free Radical (DPPH) Scavenging Test Method (T / SHRH006-2018)," the DPPH free radical scavenging rate (%) of the samples prepared in Examples 2-4, Comparative Examples 1-6, and the blank control group was tested.

[0122] The specific experimental method is as follows: A 96-well plate was used, with three replicates per group, and the total system volume was 200 μL. For the sample group: an appropriate amount of sample was dissolved in 100 μL of distilled water to achieve a final concentration of 1% in the system, and 100 μL of 0.1 mM DPPH solution was added to the reaction system. For the control group: 100 μL of distilled water was used, followed by the addition of 100 μL of 0.1 mM DPPH solution. After the reaction system was constructed, it was shaken in the dark for 10 min, and the absorbance at 520 nm was measured using a microplate reader.

[0123] The clearance rate is calculated as follows: Clearance rate (%) = [(A0-Ax) / A0] × 100%; Where A0 represents the absorbance of the control group and Ax represents the absorbance of the sample group. A higher scavenging rate indicates a better antioxidant effect of the sample.

[0124] The results of the elastase activity inhibition rate combined with the DPPH free radical scavenging test are shown in Table 9.

[0125] Table 9 Moisturizing and Anti-aging Experiments Serial Number Antioxidant Anti-aging Example 2 77.20 80.21 Example 3 75.06 78.22 Example 4 73.24 76.90 Comparative Example 1 50.12 60.93 Comparative Example 2 52.01 60.22 Comparative Example 3 49.98 54.29 Comparative Example 4 47.73 53.20 Comparative Example 5 47.01 42.98 Comparative Example 6 45.55 40.63 This invention investigated the antioxidant, anti-aging, anti-wrinkle, and skin-whitening effects and mechanisms of action of neo-agar oligosaccharides at the biochemical and cellular levels. By applying them to basic formulations, their fundamental anti-aging and skin-whitening effects were evaluated at the human level, pointing the way for the application of neo-agar oligosaccharides in the cosmetics field.

[0126] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A moisturizing anti-aging composition containing neoagarooligosaccharides, characterized in that, It consists of the following components by mass percentage: Glycerin 3-6%, 1,3-Butanediol 3-6%, Disodium EDTA 0.01-0.05%, Gel 0.4-1.0%, Hydrolyzed Sodium Hyaluronate 0.2-0.5%, Inositol 0.5-1.0%, PEG / PPG-14 / 7 Dimethyl Ether 0.5-1.0%, Preservative 0.6%-1.2%, Tromethamine 0.2-0.5%, Neo-Agar Oligosaccharide 0.2-1.0%, Oat Fermentation Broth 0.1-0.5%, Gastrodia elata Extract 0.1-0.5%, Yeast / Barley Seed Fermentation Product Filtrate 0.2-0.6%, and Water Balance; The method for preparing the moisturizing and anti-aging composition containing neo-agar oligosaccharides includes the following steps: Glycerin, 1,3-butanediol, gelling agent, hydrolyzed sodium hyaluronate, inositol, and PEG / PPG-14 / 7 dimethyl ether were dispersed in water to form phase A. Mix the preservatives thoroughly to form phase B; The new oligosaccharide, oat fermentation broth, gastrodia extract, yeast / barley seed fermentation product filtrate and tromethamine were dissolved in a portion of water to form phase C; Add phase B to phase A, stir evenly, then add phase C and homogenize to obtain a moisturizing and anti-aging composition containing neo-augment oligosaccharides. The preparation method of the new agar oligosaccharide is as follows: Engineered strains expressing AgaA and AgaB enzymes, respectively, were inoculated into fermentation medium and cultured to OD0.

05. 600 The concentration was 0.6-0.8, and IPTG inducer was added to induce expression and obtain fermentation broth; The fermentation broth was centrifuged to collect the cells, which were then homogenized and broken down. After centrifugation and filtration, crude enzyme solutions of AgaA enzyme and AgaB enzyme were obtained, respectively. The crude enzyme solution of AgaA enzyme was added to agar substrate and reacted at 55-65℃ for 20-30 h. Then, the temperature was lowered to 40-50℃, and the crude enzyme solution of AgaB enzyme was added to continue the reaction for 10-20 h. After the reaction was completed, the mixture was sterilized and cooled. After centrifugation and filtration, a new agar oligosaccharide solution was obtained. After concentration and freeze-drying, the new agar oligosaccharide was obtained. The new oligosaccharides include new oligosaccharide disaccharide, new oligosaccharide tetrasaccharide, new oligosaccharide hexasaccharide, new oligosaccharide octasaccharide and new oligosaccharide decasaccharide; The oat fermentation broth is prepared as follows: Oat seeds and bran are mixed and ground into a fine powder. The fine powder, carbon source, vitamins, inulin, and water are mixed and sterilized to prepare a sterile culture medium. A compound lactic acid bacteria solution is inoculated into the sterile culture medium, stirred, and cultured at a constant temperature to obtain the first fermentation broth. The fermentation broth is heated to 65-75℃ and ultrasonically treated. An activated carbon aqueous solution is added, and stirring and filtration are continued to obtain the second fermentation broth. The second fermentation broth is heat-treated at 80-90℃ and cooled to obtain the oat fermentation broth. The compound lactic acid bacteria solution is composed of Lactobacillus plantarum and Lactobacillus paracasei in a 1:1 ratio. The method for preparing the yeast / barley seed fermentation product filtrate is as follows: Barley seed powder was inoculated into a fermentation medium and inoculated with brewer's yeast. The mixture was then cultured with shaking at a pH of 6-7 and a temperature of 30-35℃ for 36-48 hours to obtain a crude fermentation broth. The crude fermentation broth was centrifuged, and the supernatant was collected. After sterilization and filtration, the yeast / barley seed fermentation product filtrate was obtained.

2. The moisturizing and anti-aging composition containing neoagar oligosaccharides according to claim 1, characterized in that, The preservatives include p-hydroxyacetophenone and 1,2-hexanediol, with a mass ratio of p-hydroxyacetophenone to 1,2-hexanediol of 1:(0.8-1.2).

3. The NJO-containing moisturizing anti-aging composition of claim 1, wherein The gelling agent comprises acrylate / C10-30 alkyl acrylate cross-linked copolymer and carbomer, wherein the mass ratio of acrylate / C10-30 alkyl acrylate cross-linked copolymer to carbomer is 1:(0.8-1.2).

4. A method of preparing a neoagarooligosaccharide-containing moisturizing anti-aging composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: Glycerin, 1,3-butanediol, gelling agent, hydrolyzed sodium hyaluronate, inositol, and PEG / PPG-14 / 7 dimethyl ether were dispersed in water to form phase A. Mix the preservatives thoroughly to form phase B; The new oligosaccharide, oat fermentation broth, gastrodia extract, yeast / barley seed fermentation product filtrate and tromethamine were dissolved in a portion of water to form phase C; Add phase B to phase A, stir until homogeneous, then add phase C and homogenize to obtain a moisturizing and anti-aging composition containing neo-augment oligosaccharides.

5. The use of the moisturizing and anti-aging composition containing neoagar oligosaccharides according to any one of claims 1-3 in the preparation of cosmetics.

6. Use according to claim 5, characterized in that, The moisturizing and anti-aging composition containing neo-augment oligosaccharides has the effects of improving dry skin, whitening, and reducing fine lines and wrinkles.

7. A cosmetic product, characterized by A moisturizing and anti-aging composition comprising neo-augment oligosaccharides as described in any one of claims 1-3.

8. The cosmetic product according to claim 7, characterized in that, Cosmetics also contain other inactive ingredients that are permitted to be added in the cosmetics industry.

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