Aspartic homopolymer ACP-W1 with skin anti-photoaging activity, and preparation method and application thereof

Abstract

This invention discloses a homogeneous asparagus polysaccharide ACP-W1 with anti-photoaging activity, its preparation method, and its applications. The monosaccharide components of this polysaccharide are fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid; its weight-average molecular weight is 1440.0–1640.0 kDa, its number-average molecular weight is 1150.0–1350.0 kDa, and its polydispersity index is 1.1–1.3; it exhibits a stable triple helix conformation and an ordered secondary structure resembling parallel β-sheets in aqueous solution. This polysaccharide is prepared from asparagus root and inhibits UV-induced skin ROS generation and inflammatory responses, improves the skin's photoaging phenotype, and exerts anti-photoaging effects. When used synergistically with sunscreens, it can enhance sun protection and anti-friction properties. It has high safety and clear efficacy, and can be used as a core functional ingredient in anti-photoaging personal care products, demonstrating good application and transformation potential.

Description

Technical Field

[0001] This invention relates to the field of natural product application technology, specifically to an asparagus polysaccharide with anti-photoaging activity in the skin and its application, and more particularly to a homogeneous asparagus polysaccharide ACP-W1 with anti-photoaging activity in the skin and its preparation method and application. Background Technology

[0002] Photoaging is a chronic skin damage process caused by ultraviolet (UVA, UVB) radiation. The core mechanisms involve excessive generation of reactive oxygen species (ROS) induced by ultraviolet radiation, collagen fiber degradation, abnormal activation of matrix metalloproteinases (MMPs) and imbalance of skin cell apoptosis. It ultimately manifests as pathological features such as fine lines, sagging, pigmentation and impaired barrier function. Photoaging protection is the core research direction in the field of personal care products.

[0003] In the field of personal care products, the anti-photoaging protection system consists of sunscreen agents and bioactive ingredients. Each plays a specific role while working synergistically to achieve comprehensive photoaging protection. Sunscreen agents directly block or shield ultraviolet (UV) radiation, reducing photoaging damage at its source and fulfilling the core function of UV protection. Bioactive ingredients (which do not directly absorb / shield UV rays) compensate for the shortcomings of sunscreen agents in damage repair by regulating photoaging-related molecular pathways and repairing UV-induced skin damage, further enhancing the anti-photoaging effect. Currently, commonly used sunscreen agents in this field are mainly divided into two categories: chemical sunscreens and physical sunscreens. Commonly used bioactive ingredients are primarily chemically synthesized. Both types of components face significant technological bottlenecks, which are analyzed in detail below:

[0004] Chemical sunscreens, with organic UV absorbers at their core, absorb UVA / UVB photons through a molecular conjugated system and convert them into heat energy. They are the most widely used type of sunscreen due to their lightweight texture and lack of noticeable white cast. However, they suffer from dual drawbacks in safety and performance stability: on the one hand, most ingredients (such as benzophenone-3, ethylhexyl methoxycinnamate, and octocrylene) have a molecular weight <500. Da, which easily penetrates the stratum corneum of the skin and enters the dermis and even the systemic circulation, contains ingredients such as benzophenone-3, which has potential endocrine-disrupting activity; ethylhexyl methoxycinnamate, which may cause contact dermatitis; and butyl methoxydibenzoylmethane (avobenzone), which is photodegraded under ultraviolet radiation. The free radicals generated exacerbate oxidative stress damage to the skin and induce photosensitive contact dermatitis. On the other hand, the absorption spectrum of a single ingredient is limited, and multi-component compounding is required to achieve broad-spectrum protection. However, when compounding, interactions between ingredients are prone to occur, leading to decreased stability of the sunscreen system, excessively rapid decay of SPF and PA, and affecting the durability of protection. In addition, ingredients such as benzophenone are toxic to marine plankton, which limits their use in outdoor sunscreen products.

[0005] Physical sunscreens use inorganic nanoparticles (zinc oxide, titanium dioxide) as their core, achieving protection through the physical absorption, reflection, and scattering of ultraviolet rays. Because they do not penetrate the skin and have no significant sensitizing properties, they offer significant safety advantages, making them particularly suitable for products targeting sensitive skin, infants, and pregnant women. However, a significant contradiction exists between performance and cost: Firstly, zinc oxide and titanium dioxide have high refractive indices, resulting in strong scattering of visible light. High concentrations can form a noticeable white film. Furthermore, the strong hydrophobicity of inorganic particles and poor compatibility with skincare bases lead to a thick, sticky texture that is prone to pilling, significantly reducing the user's willingness to apply. Firstly, the actual amount applied is only 20% to 50% of the recommended amount, resulting in a much lower actual protective effect than the labeled value. Secondly, inorganic particles are prone to agglomeration and are difficult to disperse evenly. They have poor hydrophilicity and weak waterproof and sweat-resistant properties, are easily washed off by sweat, and tend to settle and separate in the product system, requiring repeated shaking before use. Thirdly, surface modification technologies such as silanization and polyethylene glycol modification are required to improve skin feel and dispersibility, combined with high concentrations of active ingredients, resulting in production costs that are significantly higher than ordinary chemical sunscreens. Furthermore, the protective performance of pure mineral sunscreens is mostly concentrated in the low to medium SPF / PA levels, making it difficult to meet the needs of high-end products.

[0006] As another key component of the anti-photoaging system, the bioactive ingredients commonly used in the personal care product field are mainly chemically synthesized, including ascorbic acid (vitamin C) and its derivatives (such as ascorbate glucoside and ascorbate palmitate), tocopherol (vitamin E) and its derivatives (such as tocopheryl acetate), retinol (vitamin A), retinoic acid (vitamin A acid) and its derivatives (such as retinaldehyde and retinyl palmitate). While these ingredients can exert anti-photoaging effects through different pathways and form synergistic protection with sunscreens—ascorbic acid and its derivatives can inhibit tyrosinase activity and scavenge ROS to achieve whitening and anti-oxidation; tocopherol and its derivatives can block lipid peroxidation to protect skin cell membranes; and retinol, retinoic acid, and their derivatives can regulate the proliferation and differentiation of keratinocytes and promote collagen synthesis in fibroblasts to improve skin laxity and fine lines—they have fundamental technical defects. Firstly, they have extremely poor photostability. Their molecular structure contains unsaturated bonds or phenolic hydroxyl groups, making them prone to photooxidation and photoisomerization under ultraviolet radiation, leading to the destruction of active sites and a significant reduction in bioavailability. Furthermore, retinoic acid components, after photodegradation, can also generate… Firstly, these ingredients can form quinone derivatives that are irritating to the skin. Secondly, they require special encapsulation and drug delivery technologies such as microcapsules and liposomes to maintain their activity, which not only significantly increases R&D and production costs, but also makes the encapsulation system prone to damage under complex environments such as high outdoor temperatures and sweat rinsing, further limiting their practical application effects. More importantly, these ingredients have a single target and can only act on a specific step of photoaging (such as ROS clearance and collagen synthesis), and cannot achieve synergistic regulation of the entire pathway of "ROS generation-collagen degradation-MMPs activation-cell apoptosis". They are difficult to block the photoaging cascade reaction at the molecular level and cannot form a highly efficient synergy with sunscreen agents to meet users' needs for long-lasting, comprehensive, and deep anti-photoaging.

[0007] In summary, in existing personal care product anti-photoaging systems, sunscreens (chemical and physical sunscreens) and bioactive ingredients are equally important, but both have significant drawbacks: sunscreens face bottlenecks such as safety concerns, performance instability, and poor user experience, while bioactive ingredients suffer from poor photostability, limited target sites, and high application costs. These drawbacks overlap, preventing the two from achieving efficient synergy. Current anti-photoaging products fail to achieve the comprehensive technical goals of "highly effective broad-spectrum protection, long-lasting and stable activity, safety, gentleness, and excellent user experience," thus hindering the development of anti-photoaging technology in this field. Therefore, developing novel sunscreens and bioactive ingredients that overcome these shortcomings, and constructing an anti-photoaging technology that is synergistic, multi-target, highly stable, highly safe, and offers a superior user experience, has become a pressing technical challenge in this field. Summary of the Invention

[0008] To solve the above-mentioned technical problems, the primary objective of this invention is to provide an asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity in the skin.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity of the skin.

[0010] Another object of the present invention is to provide the application of the above-mentioned asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity of the skin.

[0011] The technical solution adopted in this invention is: an asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity of the skin, the monosaccharide composition of which is as follows: the molar ratio of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid is 0.34%~0.74%: 15.63%~17.63%: 7.62%~9.62%: 55.12%~57.12%: 4.45%~6.45%: 0.89%~2.89%: 0.97%~2.97%: 7.77%~9.77%; the weight average molecular weight (Mw) is 1440.0~1640.0 kDa, the number average molecular weight (Mn) is 1150.0~1350.0 kDa, and the polydispersity index (PDI) is 1.1~1.3.

[0012] Preferably, the monosaccharide composition of the asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity is as follows: the molar ratio of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid is 0.54%: 16.63%: 8.62%: 56.12%: 5.45%: 1.89%: 1.97%: 8.77%; the weight-average molecular weight (Mw) is 1543.4 kDa, the number-average molecular weight (Mn) is 1254.4 kDa, and the polydispersity index (PDI) is 1.23.

[0013] Preferably, the chemical structural formula of the asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity is shown below:

[0014] ; Wherein, m and n represent the number of repetitions (degree of polymerization) of different sugar chain segments; m is an integer from 4000 to 5800, preferably an integer from 4100 to 5500; n is an integer from 570 to 850, preferably an integer from 580 to 720.

[0015] Preferably, the asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity in the skin can exhibit a stable triple helix conformation in aqueous solution and has a parallel β-sheet-like ordered secondary structure.

[0016] The preparation method of the above-mentioned homogeneous polysaccharide ACP-W1 of Asparagus with anti-photoaging activity includes the following steps: drying, pulverizing, defatting, enzymatically hydrolyzing and water-assisted extraction of Asparagus root, followed by filtration, concentration and alcohol precipitation, centrifugation and resolution, protein removal, decolorization, dialysis to remove small molecules, and then concentration and freeze-drying to obtain crude Asparagus polysaccharide ACP; the aqueous solution of crude Asparagus polysaccharide ACP obtained by dissolving crude Asparagus polysaccharide ACP in water is successively subjected to weak anion exchange column chromatography and gel filtration column chromatography to obtain homogeneous polysaccharide ACP-W1 of Asparagus.

[0017] The preferred drying temperature is 60–65 °C.

[0018] The preferred degree of drying is drying to a constant weight.

[0019] The drying time is preferably 8 to 12 hours.

[0020] The preferred degree of pulverization is that it can pass through a standard sieve of 60-80 mesh.

[0021] The defatting is preferably performed using ethanol; the specific steps are preferably as follows: mix asparagus root powder and anhydrous ethanol at a material-to-liquid mass ratio of 1g:10-15mL, stir to extract fat-soluble pigments and some impurities, separate the solid and liquid, and take the solid.

[0022] The preferred method for solid-liquid separation is centrifugation.

[0023] The preferred centrifugation conditions are 4000–6000 g for 10–20 min.

[0024] The enzymatic hydrolysis-assisted water extraction described herein employs a combination of papain and cellulase. The preferred specific procedure is as follows: a buffer solution with a pH of 4.0–6.0 and a concentration of 0.2 mol / L is prepared using sodium acetate and acetic acid; the buffer solution and defatted asparagus root powder are mixed thoroughly at a material-to-liquid ratio of 1 g: 10–50 mL; then, a compound enzyme equivalent to 0.5–2.5% w / w of the asparagus root powder mass is added, consisting of papain and cellulase in a 1:1 mass ratio; after thorough mixing, the mixture is heated to 45–55 °C for 2–6 h for enzymatic hydrolysis, with continuous stirring during the extraction process. After the reaction is complete, any evaporated water is replenished; the enzyme is then inactivated after hydrolysis.

[0025] The preferred material-to-liquid ratio is 1g:25-30mL.

[0026] The preferred amount of the compound enzyme is 1.6-1.7% w / w of the mass of the asparagus root powder.

[0027] The preferred enzyme activity of the papain is 800 U / mg.

[0028] The cellulase activity is preferably 50 U / mg.

[0029] The preferred conditions for enzymatic extraction are 48–52°C for 5–6 hours.

[0030] The preferred conditions for enzyme inactivation are heating in a boiling water bath for 10–15 minutes.

[0031] The preferred method for filtration is to use a 200-mesh filter cloth.

[0032] The preferred operation for concentration and alcohol precipitation is as follows: after concentrating the filtrate, add ethanol and mix evenly, wherein the final concentration of ethanol is 70-80% by volume, and let stand.

[0033] The preferred method of concentration is vacuum concentration.

[0034] The degree of concentration is preferably 1 / 10 to 1 / 15 of the original volume.

[0035] The preferred mixing conditions are stirring for 20 to 30 minutes.

[0036] The preferred standing conditions are standing at 0-8°C for at least 8 hours; more preferably standing at 4°C for 8-12 hours.

[0037] The centrifugation conditions for centrifugation and reconstitution are preferably centrifugation at a speed of 6000-10000g for 5-25 minutes; more preferably centrifugation at a speed of 8000g for 10 minutes.

[0038] The resolution involves dissolving the precipitate obtained by centrifugation in deionized water to obtain an aqueous solution of asparagine polysaccharide.

[0039] The preferred method for protein removal is the Sevag method. The specific steps are as follows: Add 1 / 4 volume of Sevag reagent to the asparagus polysaccharide aqueous solution, shake thoroughly for 20-30 min, allow to stand for layering, centrifuge at 4000 g for 10-20 min, discard the intermediate protein layer and the lower organic phase each time, collect the upper aqueous phase, and repeat 3-5 times until the intermediate protein layer disappears; the Sevag reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1.

[0040] The decolorization is achieved using a macroporous adsorption resin column.

[0041] The macroporous adsorption resin is preferably AB-8 macroporous adsorption resin.

[0042] The preferred dialysis procedure is as follows: Dialyze in deionized water using a 3000-5000 Da dialysis bag for 24-48 hours, changing the water every 4-6 hours during this period.

[0043] The degree of concentration in the concentrated freeze-drying process is to concentrate to near-dry / without alcohol odor.

[0044] The preferred weak anion exchange column is the DEAE Seplife FF weak anion exchange column.

[0045] The preferred procedure for weak anion exchange column chromatography is as follows: Centrifuge the aqueous solution of crude asparagus polysaccharide ACP, take the supernatant and load it into a weak anion exchange column at a flow rate of 4-5 mL / min, then elute with pure water to obtain the eluent, concentrate and dialyze to obtain the ion-exchange purified asparagus polysaccharide ACP-W.

[0046] The preferred centrifugation conditions are 8000–10000 g for 10–15 min.

[0047] The preferred method of concentration is rotary evaporation concentration.

[0048] The preferred degree of concentration is to concentrate to 1 / 5 of the original volume.

[0049] The preferred dialysis procedure is to use a 3000–5000 Da dialysis bag for dialysis for 24–48 hours to remove small molecule components.

[0050] The preferred gel filter column is the Sephacryl S-400 HR gel filter column.

[0051] The preferred procedure for gel filtration column chromatography is as follows: centrifuge the eluent obtained from weak anion exchange column chromatography, and load the supernatant into a gel filtration column at a flow rate of 1–1.5 mL / min; elute with pure water, collecting one tube for every 10 mL, and collect all eluent; select and confirm the main elution peak by measuring the total sugar content of the eluent in each collection tube, combine the eluents from each collection tube corresponding to the elution peak, and then concentrate, dialyze, and freeze-dry to obtain asparagine homogeneous polysaccharide ACP-W1.

[0052] The preferred centrifugation conditions are 8000–10000 g for 10–15 min.

[0053] The amount of pure water used is preferably 1 to 2 times the column volume; more preferably 1.5 times the column volume.

[0054] The preferred method of concentration is rotary evaporation concentration.

[0055] The preferred degree of concentration is to concentrate to 1 / 5 of the original volume.

[0056] The preferred dialysis procedure is to use a 3000–5000 Da dialysis bag for dialysis for 24–48 hours to remove small molecule components.

[0057] The application of the aforementioned asparagine homogeneous polysaccharide ACP-W1 in the preparation of skin anti-photoaging care products.

[0058] A skin care product for anti-photoaging contains the above-mentioned asparagine homogeneous polysaccharide ACP-W1.

[0059] The types of skin anti-photoaging care products mentioned include toners, sprays, serums, facial masks, lotions, creams, eye creams, and sunscreens.

[0060] The amount of the asparagus homogeneous polysaccharide ACP-W1 added to the skin anti-photoaging care product is 0.1% to 2.0% of the total product mass.

[0061] A sunscreen lotion contains the following components in percentage by weight: disodium EDTA 0.04–0.06%, glycerin 2–4%, butylene glycol 3–5%, allantoin 0.1–0.3%, acrylate / C10-30 alkanol acrylate crosspolymer 0.05–0.15%, and aspartic polysaccharide ACP-W1. 0.2-2%, p-hydroxyacetophenone 0.4-0.6%, 1,2-hexanediol 0.8-1.2%, potassium cetyl phosphate 0.7-0.9%, glyceryl stearate and PEG-100 stearate 2-3%, cetearyl alcohol 1-2%, bis-ethylhexyloxyphenol methoxyphenyl triazine 1-3%, diethylamino hydroxybenzoyl hexyl benzoate 2-4%, ethylhexyl methoxycinnamate 7-9%, C12-15 alcohol benzoate 4-6%, isononyl isononanoate 3-5%, polydimethylsiloxane 3-5%, aminomethylpropanol 0.05-0.07%, fragrance as needed, water balance; preferably containing the following components by mass percentage: disodium EDTA 0.05%, glycerol 3%, butylene glycol 4%, allantoin 0.2%, acrylate / C10-30 alkyl acrylate crosspolymer 0.1%, aspartic homogeneous polysaccharide ACP-W1 0.2-0.5%, p-hydroxyacetophenone 0.5%, 1,2-hexanediol 1%, potassium cetyl phosphate 0.8%, glyceryl stearate and PEG-100 stearate 2.5%, cetearyl alcohol 1.5%, bis-ethylhexyloxyphenol methoxyphenyl triazine 2%, diethylamino hydroxybenzoyl hexyl benzoate 3%, ethylhexyl methoxycinnamate 8%, C12-15 alcohol benzoate 5%, isononyl isononanoate 4%, polydimethylsiloxane 4%, aminomethylpropanol 0.06%, fragrance as needed, water balance.

[0062] The "appropriate amount" mentioned refers to adding based on actual needs, which complies with industry regulations.

[0063] The balance is 100% for water and other components.

[0064] The preparation method of the above sunscreen lotion includes the following steps:

[0065] (1) Mix disodium EDTA, glycerol, butanediol, allantoin, acrylate / C10-30 alkyl acrylate crosspolymer, asparagine homogeneous polysaccharide ACP-W1, p-hydroxyacetophenone, 1,2-hexanediol, potassium cetyl phosphate and water to obtain an aqueous phase;

[0066] (2) Bis-ethylhexyloxyphenol methoxyphenyl triazine, diethylamino hydroxybenzoyl hexyl benzoate and ethylhexyl methoxycinnamate are stirred and dissolved at 75 ℃~85 ℃ until a clear yellow solution is obtained. Then, glyceryl stearate, PEG-100 stearate, cetearyl alcohol, C12-15 alcohol benzoate 4~6%, isononyl isononanoate 3~5% and polydimethylsiloxane 3~5% are added and stirred and dissolved to obtain the oil phase.

[0067] (3) Add the oil phase to the water phase and emulsify it homogenously; after emulsification, cool it down to 50 °C, add aminomethylpropanol and fragrance, stir and disperse evenly to obtain sunscreen emulsion.

[0068] The homogenization conditions described in step (3) are preferably 8000-10000 rpm for 2-5 min; more preferably 9000 rpm for 3 min.

[0069] The advantages and positive effects of this invention are as follows: It provides a novel asparagus homogeneous polysaccharide ACP-W1 with a defined fine structure, which can be used for anti-photoaging of the skin. It can reduce the release of inflammatory factors such as IL-1β, IL-6 and TNF-α by reducing ROS levels and regulating the TNF-α / NF-κB signaling pathway, downregulating the expression of proteins such as MMP-1, MMP-3, TNFR1, TRADD and NF-κB p65 and upregulating IκBα expression, significantly inhibiting the skin ROS level and inflammatory response induced by ultraviolet rays, and improving redness, roughness, sagging and wrinkles of photoaged skin. At the same time, the asparagus homogeneous polysaccharide ACP-W1 also has excellent film-forming properties, which can form a uniform and rigid protective film on the skin surface, help improve the stability of the sunscreen system, prolong the sun protection time and synergistically enhance the sunscreen efficacy. Attached Figure Description

[0070] Figure 1 This is a graph showing the Congo Red test results of different asparagine polysaccharide components ACP-W, ACP-N1, and ACP-N2 obtained by ion chromatography.

[0071] Figure 2 Asparagine polysaccharide ACP-W is effective against superoxide anion (O2). - ·) and ABTS + • Graph showing the free radical scavenging ability.

[0072] Figure 3 This is the HPAEC-PAD map of the homogeneous polysaccharide ACP-W1 of asparagus.

[0073] Figure 4 This is the HPSEC-MALLS-RI diagram of the asparagine homogeneous polysaccharide ACP-W1.

[0074] Figure 5 This is the FT-IR image of ACP-W1, a homogeneous polysaccharide from asparagus.

[0075] Figure 6 This is the NMR spectrum of asparagine homogeneous polysaccharide ACP-W1; where A represents... 1 H NMR spectrum, B is 13 C10 NMR spectra, C is the two-dimensional COSY spectrum, D is the HSQC spectrum, E is the HMBC spectrum, and F is the NOESY spectrum.

[0076] Figure 7 This is the TG / DTG graph of asparagine homogeneous polysaccharide ACP-W1.

[0077] Figure 8 This is the AFM diagram of the asparagine homogeneous polysaccharide ACP-W1.

[0078] Figure 9 This is a SEM image of the asparagine homogeneous polysaccharide ACP-W1.

[0079] Figure 10 This is a diagram showing the Congo Red test results of the asparagus homogeneous polysaccharide ACP-W1.

[0080] Figure 11 This is a circular dichroism chromatogram of asparagus homogeneous polysaccharide ACP-W1.

[0081] Figure 12 These are macroscopic phenotypic photographs of the skin of mice in each group after the modeling period (8 weeks).

[0082] Figure 13 This is a graph showing the effects of inflammatory factors and oxidative stress markers on the skin of mice in each group after the modeling period (8 weeks).

[0083] Figure 14 This is a graph showing the effects of different groups of mice on proteins related to the TNF-α / NF-κB signaling pathway after the modeling period (8 weeks). Detailed Implementation

[0084] To make the objectives, technical solutions, and beneficial effects of this invention clearer and easier for those skilled in the art to understand and implement the technical solutions in this application, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments.

[0085] Example 1

[0086] (1) Complex enzymatic extraction of asparagus polysaccharides

[0087] 1) Raw material drying pretreatment: The tuberous roots of Asparagus medicinal material were placed in a 60 ℃ electric constant temperature oven and dried for 8 hours until constant weight. They were then crushed using a pulverizer, passed through an 80 mesh standard sieve, and the Asparagus powder was collected and stored in a desiccator in a light-proof and sealed container for later use.

[0088] 2) Ethanol defatting: Add anhydrous ethanol to asparagus powder at a mass ratio of 1:10 (g / mL), stir at room temperature to extract fat-soluble pigments and some impurities, centrifuge at 6000 g for 10 min, and collect the precipitate.

[0089] 3) Enzyme-assisted water extraction: Prepare a sufficient amount of buffer solution with pH 5.0 and concentration of 0.2 mol / L using sodium acetate and acetic acid. Add defatted asparagus root powder to the buffer solution, controlling the material-to-liquid ratio at 1:28.8 (g / mL), and stir until homogeneous. Then add a complex enzyme with an enzyme-to-powder ratio of 1.65% (w / w, enzyme mass / asparagus powder mass). The complex enzyme consists of papain (BR, 800 U / mg) and cellulase (BR, 50 U / mg) in a 1:1 mass ratio. After stirring until homogeneous, heat to 50 ℃ and extract for 5 h, stirring continuously during the extraction process. After the reaction is complete, replenish the evaporated water. After enzymatic hydrolysis, heat in a boiling water bath for 10 min to inactivate the enzyme.

[0090] 4) Filtration: The extracted mixture is filtered using a 200-mesh filter cloth, and the filtrate is collected to obtain the crude extract of asparagus polysaccharide.

[0091] 5) Concentration and alcohol precipitation: Concentrate the crude extract of asparagus polysaccharide under reduced pressure to 1 / 10 of the original volume, add 4 times the volume of 95% ethanol (v / v) to the concentrate, mix well to make the final volume fraction of ethanol 70% to 80%, stir for 20 min, and let stand at 4 ℃ for 12 h.

[0092] 6) Centrifugation and reconstitution: Discard the supernatant, centrifuge the lower precipitate at 8000 g for 10 min, discard the supernatant, dissolve the precipitate in deionized water and make up to volume to obtain asparagus polysaccharide extract.

[0093] (2) Hot water extraction of asparagus polysaccharides

[0094] 1) Raw material drying pretreatment: Same as step (1) 1).

[0095] 2) Ethanol defatting: Same as step (1) 2).

[0096] 3) Conventional hot water extraction: Take deionized water, add defatted asparagus root powder to it, control the material-to-liquid ratio to be 1:28.8 g / mL, stir and mix evenly; heat to 90 ℃, extract for 5 h, stirring continuously during the extraction process, and replenish the evaporated water after extraction.

[0097] 4) Filtering: Same as step (1) 4).

[0098] 5) Concentration and alcohol precipitation: Same as step (1) 5).

[0099] 6) Centrifugation and reconstitution: Same as step (1) 6).

[0100] (3) Effect verification

[0101] Compare the polysaccharide extraction rates of steps (1) and (2). The total sugar content of polysaccharides was determined using the phenol-sulfuric acid method. Referring to the standard method for polysaccharide quantification, the absorbance was measured at a wavelength of 490 nm and converted to mass. The polysaccharide yield of the asparagus polysaccharide extract was calculated using the following formula: Wherein, 0.9 is the conversion factor for correcting the total sugar content (glucose equivalent) to polysaccharide mass, calculated from the theoretical molar mass ratio (162 / 180) of glucose monomer dehydration condensation.

[0102] .

[0103] The results showed that the polysaccharide yield was 2.54% when extracted by the compound enzyme method and 1.98% when extracted by hot water, representing an increase of 28.28%. Therefore, the asparagus polysaccharide extract obtained by the compound enzyme method was selected for further study.

[0104] Example 2

[0105] Purification of asparagus polysaccharide extract:

[0106] (1) Protein removal: Add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1, v / v) to the asparagus polysaccharide aqueous solution, shake well for 20 min, let stand to separate the layers, centrifuge at 4000 g for 10 min, discard the middle protein layer and the lower organic phase each time, collect the upper aqueous phase, repeat 3 to 5 times until the middle protein layer disappears.

[0107] (2) Decolorization: The asparagus polysaccharide aqueous solution after protein removal is decolorized by passing it through an AB-8 macroporous adsorption resin column.

[0108] (3) Small molecules: Collect the liquid and dialyze it in deionized water for 48 h using a 3000 Da dialysis bag, changing the water every 4 to 6 h during the process.

[0109] (4) Secondary concentration and freeze drying: The asparagus polysaccharide aqueous solution after impurity removal is concentrated by rotary evaporation under reduced pressure until it is nearly dry / without alcohol odor, to obtain asparagus polysaccharide aqueous concentrate; the concentrate is freeze-dried to obtain asparagus crude polysaccharide ACP after impurity removal.

[0110] Example 3

[0111] (1) DEAE Seplife FF weak anion exchange chromatography:

[0112] The purified asparagus polysaccharide (ACP) was purified by DEAE Seplife FF weak anion exchange chromatography. The purified crude asparagus polysaccharide sample was dissolved in pure water; centrifuged at 10000 g for 10 min, and the supernatant was purified by passing it through an ion exchange column (26 mm × 400 mm) at a flow rate of 4.0 mL / min; the eluent was eluted sequentially with pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions, and collected in 15 mL tubes. The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. Ion purification elution curves were plotted, yielding three characteristic polysaccharide fractions. The eluents from each collection tube corresponding to the elution peak were combined and concentrated to 1 / 5 of their original volume by rotary evaporation. After desalting via dialysis using a 3000 Da dialysis bag for 24–48 h, the fractions were freeze-dried to obtain the ion-exchange purified water-eluted fraction ACP-W, the acidic fraction ACP-N1 eluted with 0.1 mol / L NaCl solution, and the acidic fraction ACP-N2 eluted with 0.2 mol / L NaCl solution. ACP-W was the dominant fraction, accounting for 73.8% by mass, significantly higher than ACP-N1 (15.5%) and ACP-N2 (10.7%).

[0113] An 80 μmol / L Congo red solution was mixed with equal volumes of 1 mg / mL samples (ACP-W, ACPN1, and ACP N2 solutions, respectively), followed by the addition of an appropriate amount of 1 mol / L sodium hydroxide solution. The changes in the maximum absorption wavelength of the mixture were measured using a UV-Vis spectrophotometer (Shanghai Youke Instrument Co., Ltd.) in the wavelength range of 400–600 nm at different sodium hydroxide concentrations (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L). The results are as follows: Figure 1 As shown, ACP-W possesses a triple helix conformation, while this structural feature was not detected in ACP-N1 and ACP-N2. Previous studies have demonstrated that after polysaccharides form a regular and rigid triple helix conformation, their molecular chains can unfold in an orderly manner, fully exposing key active groups such as hydroxyl groups. This enhances their specific recognition and interaction with cell surface receptors and signaling pathway proteins, while simultaneously improving the stability of the molecular skeleton and resisting enzymatic degradation. Therefore, from the perspectives of molecular recognition and functional stability, this synergistically amplifies their immunomodulatory, antioxidant, and anti-photoaging biological activities.

[0114] Given that ACP-W has a high mass content in crude polysaccharides, is the main form of the polysaccharide, and possesses an activity-related triple helix higher conformation, this invention prioritizes ACP-W for in vitro antioxidant activity testing to preliminarily explore its potential application value for skin anti-photoaging.

[0115] Asparagine polysaccharide ACP-W was prepared into polysaccharide solutions of different concentrations and subjected to superoxide anion (O2) ion exchange. - ·) and ABTS + • Free radical scavenging capacity test to evaluate the in vitro antioxidant capacity of asparagine polysaccharide ACP-W.

[0116] 1) Superoxide anion (O2) - • Free radical scavenging ability test

[0117] Referencing superoxide anion (O2) - • Follow the instructions for the free radical scavenging ability test kit. Measure the absorbance of the blank tube and the test tube at 530 nm, and record it as A. 空白 With A 测定 Three parallel tests were conducted for each tube.

[0118] The superoxide anion scavenging rate is calculated using the formula: Scavenging rate (%) = (A... 空白 -A 测定 )÷A 空白 ×100%; plot the curve with sample concentration on the x-axis and superoxide anion scavenging rate on the y-axis, and fit the regression equation (R0). 2 (≥0.9), calculate the sample concentration corresponding to a clearance rate of 50%, which is the IC50. 50 value.

[0119] 2) ABTS + Free radical scavenging ability test

[0120] Asparagine polysaccharide ACP-W was diluted with water to prepare multi-concentration gradient samples. Sample reaction wells (T), sample background wells (T0), solvent reaction wells (C), and solvent background wells (C0) were set up in a 96-well microplate. 100 μL of sample solution was added to each of the sample reaction wells and sample background wells. 100 μL of water was added to each solvent reaction well, and 200 μL of water was added to each solvent background well. Finally, 100 μL of ABTS was added to each of the sample reaction wells and solvent reaction wells. + • For the free radical working solution, add 100 μL of water to each sample well. Set up 3 replicates per group, with 1 replicate for each sample well. Incubate the ELISA plate in the dark for 10 min, and then measure the absorbance of each well at 734 nm using an ELISA reader.

[0121] Calculate ABTS according to the formula +• Free radical scavenging rate, scavenging rate (%) = [1 - (T - T0) / (C - C0)] × 100%. Plot a curve with sample concentration on the x-axis and the corresponding scavenging rate on the y-axis, and fit a regression equation (R²). 2 ≥0.9), calculate the sample concentration at which the clearance rate is 50%, which is the IC50. 50 value.

[0122] Experimental results showed that asparagine polysaccharide ACP-W had an effect on superoxide anion (O2). - ·) and ABTS + • Free radicals all exhibit significant scavenging ability, and this scavenging ability is dose-dependent, with a half-maximal scavenging concentration (IC50) of [a certain value]. 50 The concentrations were 0.13 mg / mL and 6.42 mg / mL, respectively. Figure 2 As shown, the asparagine polysaccharide ACP-W has good in vitro antioxidant activity and can be further purified for in vivo anti-photoaging mouse experiments.

[0123] (2) Sephacryl S-400HR gel chromatography:

[0124] The polysaccharide fraction ACP-W, eluted with pure water after ion exchange purification, was further purified by Sephacryl S-400HR gel chromatography. The purified polysaccharide sample was added to pure water; centrifuged at 10000 g for 10 min, and the supernatant was passed through a gel chromatography column (26 mm × 1000 mm) for separation and purification at a flow rate of 1.0 mL / min. Elution was performed with 1.5 column volumes of pure water, collecting 10 mL tubes at a time, and all eluents were collected. The total sugar content of the eluents in each collection tube was determined using the sulfuric acid-phenol method, and the main elution peak was selected and confirmed. The eluents corresponding to each peak were combined and concentrated to 1 / 5 of the original volume by rotary evaporation. After dialyzing with a 3000 Da dialysis bag for 24–48 h to remove small molecule components, the sample was freeze-dried to obtain aspartic polysaccharide ACP-W1.

[0125] Asparagine polysaccharide ACP-W and asparagine homogeneous polysaccharide ACP-W1 were dissolved in 0.1M NaNO3 aqueous solution containing 0.02% w / w NaN3 to prepare solutions with a final concentration of 1 mg / mL. The solutions were then filtered through a 0.45 μm filter before analysis. A gel permeation chromatography-differential chromatography-multi-angle laser light scattering (LPLC-MS) system was used. The HPLC system was a U3000, the differential detector was an Optilab T-rEX, and the laser light scattering detector was a DAWN HELEOS II. The chromatographic columns were an Ohpak SB-805 HQ (300 × 8 mm) and an Ohpak SB-803 HQ (300 × 8 mm) in series. The column temperature was 45 ℃, the injection volume was 100 μL, the mobile phase was 0.1M NaNO3 aqueous solution containing 0.02% w / w NaN3, the flow rate was 0.6 mL / min, and isocratic elution was performed for 75 min.

[0126] Samples were separated sequentially by molecular weight or size using gel size exclusion chromatography. The sample concentration was detected by a differential detector, and the light scattering information was detected by a multi-angle laser light scattering instrument. The corresponding molecular weight was calculated according to the Mark-Hovink equation. The data were processed using ASTRA 6.1 software, and an absolute molecular weight analysis graph was plotted with retention time as the x-axis and molar mass as the y-axis. The number-average molecular weight Mn, weight-average molecular weight Mw, polydispersity index Mw / Mn, and root mean square radius R were determined and recorded.

[0127] The results showed that the weight-average molecular weight (Mw) of asparagus polysaccharide ACP-W1 was 1543.4 kDa, the number-average molecular weight (Mn) was 1254.4 kDa, and the polydispersity index (PDI) was 1.23; while the weight-average molecular weight (Mw) of asparagus polysaccharide ACP-W was 1520.2 kDa, the number-average molecular weight (Mn) was 1020.1 kDa, and the polydispersity index (PDI) was 1.49. It is evident that asparagus polysaccharide ACP-W1 has a narrower molecular weight distribution and higher uniformity, significantly superior to asparagus polysaccharide ACP-W.

[0128] Based on the above results, the asparagine polysaccharide ACP-W1 was named Asparagine Homogeneous Polysaccharide and further analyzed.

[0129] Example 4

[0130] The structure of the asparagus homogeneous polysaccharide ACP-W1 was analyzed, as follows:

[0131] (1) Monosaccharide composition analysis:

[0132] Accurately weigh an appropriate amount of asparagus homogeneous polysaccharide ACP-W1, place it in a chromatographic bottle, add 1 mL of 2 mol / L trifluoroacetic acid solution, heat at 121 ℃ for 2 h for hydrolysis, and dry under nitrogen. Wash with 99.99% methanol, dry again, and repeat the methanol washing 2-3 times. Redissolve in sterile water, transfer to a chromatographic bottle, and prepare for analysis. A Thermo ICS 5000+ ion chromatography system equipped with an electrochemical detector was used; the chromatographic column was a Dionex. TM CarboPac TM PA20 (150 mm × 3.0 mm, 10 μm); injection volume 5 μL, flow rate 0.5 mL / min, column temperature 30 ℃; mobile phase A is water, mobile phase B is 0.1 mol / L sodium hydroxide solution, and mobile phase C is a solution containing 0.1 mol / L sodium hydroxide and 0.2 mol / L sodium acetate; elution gradient: 0 min, volume ratio of phases A, B, and C is 95:5:0; 26 min, volume ratio is 85:5:10; 42 min, volume ratio is 85:5:10; 42.1 min, volume ratio is 60:0:40; 52 min, volume ratio is 60:40:0; 52.1 min, volume ratio returns to 95:5:0; 60 min, maintain volume ratio is 95:5:0.

[0133] Fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid were used as standards to prepare single-standard stock solutions with a concentration of 10 mg / mL. These solutions were then mixed to prepare a series of mixed standard solutions. The retention time of the standards was used for qualitative analysis, and a standard curve was plotted with the concentration of the standards on the x-axis and the peak area on the y-axis. The external standard method was used for quantification.

[0134] Test results are as follows Figure 3 As shown, the monosaccharide composition (molar ratio) of asparagus polysaccharide ACP-W1 is: fucose: arabinose: rhamnose: galactose: glucose: xylose: mannose: galacturonic acid = 0.54%: 16.63%: 8.62%: 56.12%: 5.45%: 1.89%: 1.97%: 8.77%.

[0135] (2) Molecular weight distribution analysis:

[0136] The molecular weight distribution of asparagus homogeneous polysaccharide ACP-W1 was determined using gel permeation chromatography-differential refractive index-multi-angle laser light scattering (MLLS), as described above. Data were processed using ASTRA 6.1 software. An absolute molecular weight analysis plot was constructed with retention time on the x-axis and molar mass on the y-axis. The red line represents the MLLS signal (unit: V), and the intensity of the scattered light is proportional to the molecular size and molecular weight of the substance. The blue line represents the differential signal (unit: RIU), and the response value depends on the change in the refractive index of the effluent after the column, and is related to the type, concentration, and molecular weight of the substance. The black line represents the molecular weight obtained by fitting the two signals, such as... Figure 4 As shown. The number-average molecular weight Mn, weight-average molecular weight Mw, polydispersity index Mw / Mn, and root-mean-square radius R were measured and recorded. The weight-average molecular weight (Mw) of asparagine polysaccharide ACP-W1 was 1543.4 kDa, the number-average molecular weight (Mn) was 1254.4 kDa, and the polydispersity index PDI was 1.23.

[0137] (3) FTIR analysis: The infrared spectrum of ACP-W1 was recorded using a Fourier transform infrared spectrometer (Bruker, Germany) to characterize its functional groups. Approximately 3 mg of asparagine polysaccharide ACP-W1 was mixed with an appropriate amount of potassium bromide (KBr), ground evenly, and pressed into thin slices. The slices were then analyzed at 4000–400 cm⁻¹. -1 Scanning is performed within the wavenumber range.

[0138] like Figure 5 As shown, the infrared spectrum of the homogeneous asparagus polysaccharide ACP-W1 exhibits the characteristic absorption peaks typical of polysaccharides. (3439 cm⁻¹) -1 The strong, broad peak at 2932 cm⁻¹ is the stretching vibration peak of the hydroxyl group (-OH); -1 Peak for stretching vibration of saturated CH bonds; 1728 cm⁻¹ -1 The absorption peak corresponds to the stretching vibration of the C=O group in uronic acid; 1632 cm⁻¹ -1 The peak represents the OH bending vibration of bound water; 1044 cm⁻¹ -1 The characteristic absorption peak of COC (co-occurrence of pyranose rings and glycosidic bonds) is 890 cm⁻¹. -1 The characteristic peaks indicate that this polysaccharide mainly contains β-glycosidic bonds; 606 cm⁻¹ -1 The peaks represent the vibrational peaks of the sugar ring skeleton. In summary, ACP-W1 is a β-configuration pyranose polysaccharide containing uronic acid.

[0139] (4) Methylation analysis: Weigh about 5 mg of aspartic homopolymer polysaccharide ACP-W1, dissolve it in 1 mL of pure water, add 1 mL of 1-cyclohexyl-2-morpholinoethylcarbodiimide methyl p-toluenesulfonate (CAS: 2491-17-0) with a concentration of 100 mg / mL, and react for 2 h; add 1 mL of 2 M imidazole solution, divide the sample into two equal parts, add 1 mL of 30 mg / mL NaBH4 solution and 1 mL of 30 mg / mL NaBD4 solution respectively, and react for 3 h; add 100 μL of glacial acetic acid to terminate the reaction, dialyze for 48 h and freeze dry; add 500 μL of DMSO to the sample to dissolve it, add 1 mg of NaOH, and incubate for 30 min; add 50 μL of iodomethane and react for 1 h; add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water 3 times; take the lower dichloromethane phase and blow dry with nitrogen; add 100 In a reaction mixture of 2 M trifluoroacetic acid (TFA) and 1 M ammonia solution, the mixture was stirred at 121 °C for 90 min and then evaporated to dryness at 30 °C. 50 μL of 2 M ammonia solution and 50 μL of 1 M NaBD4 solution were added, mixed, and the mixture was allowed to react at room temperature for 2.5 h. The reaction was terminated by adding 20 μL of acetic acid, and the mixture was dried under nitrogen. The mixture was washed twice with 250 μL of methanol and dried under nitrogen. 250 μL of acetic anhydride was added, vortexed, and the mixture was allowed to react at 100 °C for 2.5 h. 1 mL of water was added, and the mixture was allowed to stand for 10 min. 500 μL of dichloromethane was added, vortexed, centrifuged, and the aqueous phase was discarded. The washing was repeated three times. The lower dichloromethane phase was then used for GC-MS analysis.

[0140] An Agilent 6890A-5977B GC-MS system was used, with a BPX70 column (30 m × 0.25 mm × 0.25 µm); the injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium; the column temperature was initially 140 ℃ and held for 2.0 min, then increased to 230 ℃ at a rate of 3 ℃ / min and held for 3 min; the mass spectrometry used an electron impact ionization (EI) source in full scan mode, with an ion source temperature of 200 ℃, an MS quadrupole temperature of 110 ℃, an ionization energy of 50 EV, a transfer line temperature of 210 ℃, and a mass scan range of m / z 50–350.

[0141] Qualitative analysis was performed based on retention time and characteristic fragment ions compared with the database; relative molar amount was calculated by the ratio of peak area to the molecular weight of the corresponding derivative, and relative molar ratio was calculated by the percentage of relative molar amount to the sum of relative molar amounts of each component, thus determining the linkage mode and relative content of sugar residues.

[0142] (5) NMR analysis: An appropriate amount of asparagine polysaccharide ACP-W1 was fully dissolved in heavy water (D2O) to prepare a polysaccharide solution with a concentration of not less than 40 mg / mL. 0.5 mL of this solution was transferred to an NMR tube and detected using a Bruker AVANCE NEO 600M NMR spectrometer at a scanning temperature of 25℃. One-dimensional NMR was then performed. 1 H NMR, 13 C10 NMR spectra and two-dimensional COSY, HSQC, HMBC, and NOESY spectra, based on the NMR signals generated by the absorption of electromagnetic radiation by atomic nuclei in a magnetic field, record the chemical shifts of carbon and hydrogen atoms, which are used to resolve the anomeric configuration, glycosidic bond linkage mode, and linkage sequence of polysaccharides, such as... Figure 6 As shown. Based on the above monosaccharide component analysis, molecular weight distribution, FTIR analysis, methylation analysis, and NMR analysis, the chemical structural formula of the aspartic homogeneous polysaccharide ACP-W1 can be deduced:

[0143] ; m and n represent the number of repetitions (degree of polymerization) of different sugar chain segments, respectively; according to the molecular weight distribution, m should be an integer between 4000 and 5800, concentrated in the range of 4100 to 5500; n should be an integer between 570 and 850, concentrated in the range of 580 to 720.

[0144] (6) TG / DTG analysis: Thermogravimetric analysis (TGA) was used to evaluate thermal stability. Approximately 5.0 mg of asparagine ACP-W1 sample was accurately weighed and placed in a crucible, with nitrogen as the purge gas; during the test, the temperature was increased from 30 ℃ to 600 ℃ at a rate of 10 ℃ / min.

[0145] like Figure 7 As shown, the thermal behavior of the asparagus homogeneous polysaccharide ACP-W1 can be divided into two main stages. Within the 30–120 °C range, a weight loss peak appears at 67.8 °C, with a mass loss of 9.41%, mainly due to the volatilization of free and bound water in the sample. 200 °C is the initial temperature for polysaccharide thermal decomposition, and the main chain thermal pyrolysis stage occurs between 200 and 600 °C, reaching the maximum thermal decomposition rate at 264.8 °C. When the temperature is raised to 600 °C, the residual mass of the sample is 25.85%. The results indicate that ACP-W1 is structurally stable below 200 °C, exhibiting excellent thermal stability. The processing and storage temperatures (generally ≤80 °C) of conventional topical skincare preparations are lower than its thermal decomposition initiation temperature, ensuring high safety during processing and use, making it suitable for applications in skin anti-photoaging personal care products.

[0146] (7) AFM analysis: The asparagine polysaccharide ACP-W1 sample was dissolved in an ethanol aqueous solution to prepare a solution with a concentration of 20 μg / mL, and placed in a 60 ℃ water bath and shaken for 120 min; 10 μL of the treated solution was dropped onto a mica sheet and dried at 25 ℃ for 12 h; AFM scanning was performed using a Bruker ICON atomic force microscope in knock mode at room temperature with a scanning range of 5×5 μm to observe the ultrastructure and molecular aggregation morphology of the sample.

[0147] The results are as follows Figure 8 As shown in the two-dimensional height map and three-dimensional morphology map, ACP-W1 exhibits a flocculent morphology on a mica substrate, with dispersed fine granular aggregates. The longitudinal height ranges from -2.6 to 2.7 nm, significantly higher than the theoretical height of a single linear polysaccharide chain, demonstrating clear entanglement and ordered aggregation between polysaccharide molecules, rather than a single-chain dispersion. This aggregation behavior originates from hydrogen bonds and van der Waals forces generated by its triple-helix, parallel β-sheet ordered structure, confirming that ACP-W1 possesses a stable ordered conformation.

[0148] (8) SEM analysis: Take about 5 mg of asparagine ACP-W1 sample, adhere it to a conductive carbon film with double-sided adhesive, and place it in the sample chamber of the ion sputtering instrument for about 40 s to sputter gold; place the sample in the observation chamber of the ultra-high resolution scanning electron microscope, set the accelerating voltage to 5 kV, and observe the micromorphology of the sample surface at magnifications of 100x, 500x, 2000x, 5000x and 10000x respectively.

[0149] like Figure 9 As shown, the hierarchical aggregation structure of the polysaccharide was verified from the micrometer to the nanometer scale. Under low magnification, the homogeneous aspartic polysaccharide ACP-W1 mainly exhibits an irregularly stacked layered structure, which originates from the directional stacking of molecular chains induced by the ordered parallel β-sheet secondary structure of the polysaccharide. Under medium magnification, spherical and short rod-shaped aggregates are distributed on the surface of the sheets, consistent with the AFM observation results. Under high magnification, fine cracks are visible on the surface of the sheet structure, which is due to the high rigidity of the molecular chains caused by the triple helix and ordered secondary structure of the polysaccharide, resulting in brittleness of the layered aggregates during the drying and film formation process. The above results confirm the multi-level aggregation characteristics of ACP-W1, ranging from nanoscale aggregates to micrometer-scale layered structures.

[0150] (9) Congo Red Analysis: 80 μmol / L Congo red solution was mixed with an equal volume of 1 mg / mL ACP-W1 solution, followed by the addition of an appropriate amount of 1 mol / L sodium hydroxide solution. The changes in the maximum absorption wavelength of the mixture were measured using a UV-Vis spectrophotometer from Shanghai Youke Instrument Co., Ltd. within the wavelength range of 400–600 nm at different sodium hydroxide concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L).

[0151] like Figure 10 As shown, the results indicate that the maximum absorption wavelength (λ) of the ACP-W1-Congo red complex varies under different NaOH concentrations. max The complex λ showed a significant redshift compared to free Congo red. max The concentration of free Congo red increased from approximately 497 nm under 0 mol / L NaOH conditions to approximately 511 nm under 0.4 mol / L NaOH conditions, while the concentration of free Congo red increased with increasing alkalinity. max The spectral shift continues to decrease. This characteristic spectral shift is a hallmark feature of polysaccharides exhibiting a complete triple helix conformation, and the redshift phenomenon remains stable under moderately alkaline conditions, indicating that ACP-W1 possesses a triple helix structure with excellent conformational stability, attributed to a strong intramolecular and intermolecular hydrogen bond network.

[0152] (10) Circular dichroism chromatogram: Take an appropriate amount of asparagine polysaccharide ACP-W1 sample, dissolve it in ultrapure water to prepare a 5 mg / mL solution, centrifuge at 12000 rpm for 5 min, and take the supernatant; use a J-1500 circular dichroism spectrometer to scan, with a detection wavelength range of 180-260 nm, a test temperature of 20 ℃, a step size of 0.1 nm, and a scanning speed of 20 nm / min, and record the circular dichroism chromatogram to analyze the secondary conformation of the polysaccharide.

[0153] like Figure 11 As shown, within the scanning range of 180–260 nm, ACP-W1 exhibits a significant strong positive absorption peak at 204 nm, while the signal is weak at other wavelengths. This positive peak in the 200–210 nm range is a typical characteristic of the ordered asymmetric spatial arrangement of glycosidic bonds and sugar units, corresponding to the ordered secondary structure of polysaccharides resembling parallel β-sheets. This provides the structural basis for the formation of its triple helix higher-order structure, which is consistent with the results of the Congo red experiment, confirming that ACP-W1 possesses a highly ordered non-random coil conformation in aqueous solution.

[0154] Example 6

[0155] The skin anti-photoaging activity of asparagine polysaccharide ACP-W1 was verified through mouse experiments, as follows:

[0156] 1. Experimental Materials

[0157] (1) Laboratory animals

[0158] Thirty-six 6-week-old SPF-grade female BALB / c mice (purchased from Shouzheng Hongyao (Wuhan) Biotechnology Co., Ltd.) were selected. The mice were housed in individually ventilated cages (IVCs) under the following conditions: temperature 20–26 °C, relative humidity 40%–70%, and a 12-hour light / 12-hour dark cycle. The mice were given free access to 60Co irradiated maintenance feed and sterile drinking water. Formal experiments were conducted after one week of acclimatization. All animal ethics guidelines were followed throughout the experiment.

[0159] (2) Reagents and Samples

[0160] This invention includes: asparagine homogeneous polysaccharide ACP-W1; matrix solution (blank matrix); positive control agent ascorbate tetraisopalmitate (VCIP); depilatory cream and razor; UVA / UVB composite light source; ELISA detection kits for IL-1β, IL-6, TNF-α, ROS, etc.; HE, Masson, EVG, Sirius red, SA-β-gal tissue staining kits; RIPA protein lysis buffer, protease / phosphatase inhibitors, primary antibody, HRP-labeled secondary antibody, ECL chemiluminescence solution, and other Western blot-related reagents; and BCA protein quantification kit.

[0161] (3) Main instruments

[0162] UV light irradiation device, multifunctional microplate reader, inverted optical microscope, protein electrophoresis and transfer system, gel imaging system, Image-Pro Plus image analysis software, and ImageJ grayscale analysis software.

[0163] 2. Experimental Grouping

[0164] Thirty-six mice were randomly divided into six groups of six each. The grouping and intervention methods are as follows:

[0165] (1) Group 1 - Blank control group: No sample was applied to the back of the mice, and no UV light was applied;

[0166] (2) Group 2 - Photoaging model group: No sample was applied to the back of the mice, and UV light was used to create the model;

[0167] (3) Group 3 - Matrix control group: The back of the mice was coated with blank matrix solution (polysorbate-60 1%, phenoxyethanol 0.3%, hexanediol 0.8%, acrylate / C10-30 alkanol acrylate crosspolymer 0.1%, aminomethylpropanol 0.06%, the rest is water, % is mass percentage) and UV light was used to create the model;

[0168] (4) Group 4 - Positive drug group: 1.0% (w / w) VCIP matrix solution (solvent is blank matrix solution) was applied to the back of mice and UV light was used to create the model;

[0169] (5) Group 5 - Low-dose polysaccharide group: 0.5% (w / w) ACP-W1 polysaccharide matrix solution (solvent is blank matrix solution) was applied to the back of mice and UV light was used to create the model;

[0170] (6) Group 6 - High-dose polysaccharide group: 1% (w / w) ACP-W1 polysaccharide matrix solution was applied to the back of mice and UV light was used to create the model.

[0171] 3. Construction of a UV-induced mouse skin photoaging model

[0172] Hair removal treatment on the back: The back skin of mice was completely shaved using a razor and hair removal cream, leaving the back skin completely bare; in subsequent experiments, hair removal was performed once every 3 days to maintain the hairless state of the back.

[0173] Minimum Erythema Dose (MED) determination: Eight healthy mice were randomly selected and their back skin was photographed the day after hair removal. A 20 W UVA + 20 W UVB composite light source was used, with a vertical irradiation distance of 20 cm and gradient irradiation duration. Skin erythema was observed 24 h after irradiation. The shortest irradiation time that caused visible erythema on the back of the mice was determined as 1 minimum erythema dose (1 MED).

[0174] Formal photoaging model: Except for the blank control group, mice in other groups were exposed to UV light once every other day for 8 weeks. The initial irradiation dose was 1 MED, and the irradiation intensity was increased by 0.5 times after every 3 irradiations. From the 7th irradiation onwards, the irradiation was fixed at 2 MED, and a total of 14 UV irradiations were performed to establish a stable mouse skin photoaging damage model.

[0175] During the rearing period, closely observe the condition of the mice. Mice with hairless backs are prone to agitation and fighting, and there is a risk of skin damage. Mice with aggressive tendencies should be housed in individual cages in a timely manner to eliminate human interference that could cause skin damage.

[0176] 4. Drug intervention regimen

[0177] 30–60 minutes before each UV light exposure, the mice were given a preventative topical application of the medication to the hairless area on their backs according to their experimental groups. The single dose was 100 mg per mouse. The blank control group and the photoaging model group did not receive any application intervention.

[0178] 5. Detection Indicators and Experimental Methods

[0179] (1) Macroscopic phenotype and double-blind scoring of mouse skin

[0180] After the modeling period, standardized photographs were taken of the mouse back skin to observe photoaging characteristics such as roughness, dryness, wrinkles, erythema, peeling, and ulceration, and to macroscopically evaluate the degree of photoaging damage. Results are as follows: Figure 12As shown.

[0181] Group 1 (blank control group): The mice had intact and healthy skin with smooth texture, uniform color, no obvious erythema, dryness and wrinkles, and presented a normal and healthy skin condition.

[0182] Group 2 (Photoaging Model Group): After continuous UV irradiation, the mouse skin showed typical photoaging phenotypes, including severe dryness, erythema, desquamation, thickening and deep wrinkles, confirming the successful construction of the UV-induced mouse skin photoaging model.

[0183] Group 3 (matrix control group): The macroscopic skin damage of mice was basically the same as that of Group 2, with no significant improvement, indicating that the matrix used in the experiment did not have inherent anti-photoaging activity, thus eliminating the interference of the matrix on the experimental results.

[0184] Group 4 (1% VCIP positive drug group): After VCIP intervention, UV-induced skin damage in mice was significantly alleviated, with reduced erythema, less desquamation, and shallower wrinkles, demonstrating a good anti-photoaging protective effect.

[0185] Group 5 (0.5% low-dose ACP-W1 group) and Group 6 (1% high-dose ACP-W1 group): Both groups showed different degrees of protection against UV-induced skin photoaging; among them, Group 6 (high-dose) showed the most significant macroscopic improvement, and its skin appearance was closest to that of Group 1 (blank control group), followed by Group 4 (positive control group) and Group 5 (low-dose group); although there were slight differences in macroscopic phenotype among the three groups, there was no significant difference.

[0186] (2) Skin tissue pathological examination

[0187] After the experiment, mice were euthanized, and skin tissue from the UV-irradiated area on their backs was collected. 4 μm paraffin sections were prepared and stained with HE, Masson's trichrome, EVG, and Sirius red to observe skin tissue morphology, dermal collagen content and distribution, elastic fiber degeneration, and the ratio of type I / III collagen. 8 μm frozen sections were prepared and stained with SA-β-gal to evaluate the level of skin tissue cell senescence. All sections were photographed with standardized parameters, and double-blind quantitative analysis of histopathological indicators was performed using Image-Pro Plus software.

[0188] EVG staining results: Group 1 collagen fibers were coarse and dense, with a clear bundle structure, while elastic fibers were fine, continuous, and neatly arranged, exhibiting a wavy or loose network morphology; Groups 2 and 3 showed severely disordered collagen fiber bundle structures, accompanied by obvious breakage, curling, and entanglement, and elastic fibers completely lost their normal network structure, exhibiting disordered aggregation, breakage, and fragmentation; Groups 4, 5, and 6 showed well-preserved elastic and collagen fiber structures, with only slight and comparable collagen fiber disorder, almost no fiber breakage or entanglement, and the elastic fiber network structure was basically intact and orderly, with no significant histological differences among the three groups.

[0189] HE staining results: Group 1 showed intact epidermal and dermal structures with regular fibroblast arrangement and almost no inflammatory infiltration; Groups 2 and 3 showed significant epidermal thickening, disordered keratinocyte proliferation, and a large number of inflammatory cells infiltrating the dermis, resulting in severe skin tissue structure disorder; After intervention, the overall morphology of the skin tissue in Groups 4, 5, and 6 was effectively restored, epidermal hyperplasia was reduced, tissue arrangement was regular, and inflammatory infiltration was significantly reduced, with no significant histological differences among the three groups.

[0190] Masson staining and Sirius red staining results: Group 1 showed dense, intact dermal collagen fibers with a tight and orderly arrangement; Groups 2 and 3 showed severe collagen loss, fiber breakage, and disordered arrangement after UV irradiation; Groups 4, 5, and 6 showed effective inhibition of collagen degradation and reconstruction of the dermal collagen network after intervention with VCIP and different doses of ACP-W1, with collagen density, fiber integrity, and arrangement basically restored. The collagen morphology characteristics of the three groups were highly similar with no significant differences.

[0191] SA-β-gal staining results: Senescent cells showed blue positive expression. No obvious blue positive signal was observed in the skin tissue of group 1, indicating a low level of cell senescence; a large number of blue positive signals were observed in the skin tissue of groups 2 and 3, with deep staining and strong positive signals, indicating a significantly increased degree of cell senescence; only a small amount of blue positive signal was observed in the skin tissue of groups 4, 5, and 6, with lighter staining, indicating a significantly reduced degree of cell senescence compared to groups 2 and 3.

[0192] In summary, ACP-W1 can effectively alleviate UV-induced photoaging damage in mouse skin. Among them, 1% high-dose ACP-W1 (group 6) showed the best macroscopic protective effect, while 0.5% low-dose ACP-W1 (group 5) and 1% VCIP (group 4) both showed good anti-photoaging effects. Moreover, there were no significant differences in histopathology among groups 4, 5, and 6. ACP-W1 can exert its anti-photoaging effect by improving the macroscopic phenotype of the skin, reversing epidermal hyperplasia and inflammatory infiltration, and protecting the structural integrity of dermal collagen and elastic fibers.

[0193] (3) ELISA detection of inflammatory factors and oxidative stress indicators

[0194] Skin tissue from mice in each group was collected, homogenized with pre-cooled PBS (0.01 M, pH 7.4) under ice bath conditions, centrifuged at 12000×g for 15 min at 4 ℃, and the supernatant was collected. Total protein concentration was determined using the BCA method for result normalization. The contents of IL-1β, IL-6, TNF-α, and ROS in skin tissue were detected strictly according to the kit instructions using the corresponding commercially available ELISA kits and ROS test kits. Absorbance was measured using a microplate reader, and the absolute concentrations of each indicator were calculated using a standard curve. Results are as follows: Figure 13 As shown.

[0195] Compared with group 1, after UV irradiation, the secretion of three pro-inflammatory factors, IL-1β, IL-6 and TNF-α, in the skin tissue of group 2 was significantly upregulated, and the ROS level was significantly increased. The differences were statistically significant (all p<0.05), indicating that UV irradiation can successfully induce obvious inflammatory response and oxidative stress damage in mouse skin.

[0196] The levels of the above-mentioned inflammatory factors and ROS in the skin tissue of group 3 (matrix control group) were not significantly different from those in group 2, and all showed a significant increasing trend. This confirms that the blank matrix used in the experiment does not affect the UV-induced skin inflammatory response and oxidative stress, and can eliminate the interference of the matrix on the experimental results.

[0197] After intervention, the levels of IL-1β, IL-6, TNF-α and ROS in the skin tissue of mice in groups 4 (1% VCIP), 5 (0.5% ACP-W1) and 6 (1% ACP-W1) were significantly reduced, and the differences were statistically significant compared with those in groups 2 and 3 (all p<0.05), indicating that VCIP and ACP-W1 can effectively inhibit UV-induced skin inflammation and oxidative stress.

[0198] Among them, group 6 (1% dose ACP-W1) showed the most significant inhibitory effect on inflammatory factors and ROS, and its IL-1β, IL-6, TNF-α and ROS levels were closest to those of group 1 (blank control group). Groups 4 and 5 also showed good inhibitory effects.

[0199] (4) Western blot detection of TNF-α / NF-κB signaling pathway protein expression

[0200] Total protein was extracted from skin tissue using RIPA total protein lysis buffer, and protein concentration was determined by the BCA method. 30 μg of total protein from each group was added to 5× loading buffer and denatured in a boiling water bath at 95–100 °C for 5 min. Separation was performed by 10% SDS-PAGE electrophoresis, followed by stacking gel electrophoresis at 80 V and separating gel electrophoresis at 120 V. The protein was then transferred to PVDF membranes at a constant current of 300 mA under ice bath conditions. Blocking with 5% skim milk at room temperature for 1 h; incubation overnight at 4 °C with primary antibodies against TNFR1, TRADD, NF-κB p65, IκBα, MMP-1, MMP-3, and GAPDH; washing three times with TBST for 5 min each time; incubation with HRP-labeled secondary antibody at room temperature for 30 min; washing four times with TBST for 5 min each time; ECL chemiluminescence imaging; exposure in a dark room; development and fixing; and scanning to acquire bands. ImageJ was used to quantify grayscale values, and the relative protein expression level was calculated using GAPDH as an internal reference. Results are as follows: Figure 14 As shown.

[0201] Compared with group 1, the TNF-α / NF-κB signaling pathway was significantly activated in groups 2 and 3 after UV irradiation. Specifically, the expression of IκBα protein was significantly downregulated, while the expression of NF-κB p65, TNFR1, TRADD, MMP-1, and MMP-3 proteins was significantly upregulated. All differences were statistically significant (p<0.05), indicating that UV irradiation can induce abnormal activation of this signaling pathway, thereby causing photoaging damage to the skin.

[0202] After intervention, groups 4 (1% VCIP), 5 (0.5% ACP-W1), and 6 (1% ACP-W1) effectively reversed UV-induced abnormal expression of pathway proteins: IκBα protein expression was significantly restored, and the expression of NF-κB p65, TNFR1, TRADD, MMP-1, and MMP-3 proteins was significantly downregulated, with statistically significant differences compared to groups 2 and 3 (all p < 0.05), indicating that both VCIP and ACP-W1 can effectively inhibit the abnormal activation of the TNF-α / NF-κB signaling pathway. Among them, group 6 (1% ACP-W1) showed the best effect.

[0203] There was no significant difference in the relative expression levels of the core proteins of the above pathway among groups 4, 5, and 6, which is similar to the previous histopathological examination results. This further confirms the regulatory effect of ACP-W1 on the TNF-α / NF-κB signaling pathway, and that it can exert an anti-photoaging effect by inhibiting the activation of this pathway.

[0204] Example 7

[0205] Asparagus homogeneous polysaccharide ACP-W1 was prepared sequentially according to Examples 1 and 2, and was added to the sunscreen cosmetic formulation as the core active ingredient to prepare asparagus polysaccharide sunscreen cosmetics. The formulation is shown in Table 1 below.

[0206] Sunscreen Lotion 1 does not contain ACP-W1, and Sunscreen Lotion 2 does not contain any sunscreen agents. Sunscreen Lotions 3 and 4 are based on Sunscreen Lotion 1 and contain different amounts of ACP-W1. All other ingredients in the formula (except water) are exactly the same, and the formula is 100% hydrating.

[0207] Table 1 Comparative Examples and Examples of Sunscreen Cosmetic Formulations

[0208]

[0209] The preparation methods for sunscreen lotions 1-4 include the following specific steps:

[0210] (1) Dissolve all components of the aqueous phase thoroughly by stirring at 75 ℃~85 ℃;

[0211] (2) The oil phase sunscreen agents “bis-ethylhexyloxyphenol methoxyphenyl triazine, diethylamino hydroxybenzoyl hexyl benzoate, and ethylhexyl methoxycinnamate” were thoroughly stirred and dissolved at 75 ℃~85 ℃ until a clear, transparent yellow solution was obtained. The remaining oil phase components were then added and thoroughly stirred and dissolved to obtain the oil phase. Since sunscreen lotion 2 does not contain oil phase sunscreen agents, the oil phase components were thoroughly stirred and dissolved at 75 ℃~85 ℃ to obtain the oil phase. The oil phase was then slowly and uniformly added to the aqueous phase, while homogenizing and emulsifying at 9000 rpm for 3 min.

[0212] (3) After emulsification, cool down to 50°C, add phase C, stir and disperse evenly, then cool down to below 40°C, and add sunscreen lotion 1 to 4.

[0213] ① Sunscreen efficacy test:

[0214] The procedure was performed according to COLIPA standards. The sunscreen sample was applied evenly to a PMMA plate using a syringe, and then spread evenly on the PMMA plate surface using a latex finger cot. The final coating amount was 1.3 mg / cm². 2 The entire plate was placed in a relatively dark environment at room temperature for 15 minutes. The ultraviolet transmittance of the sample at wavelengths of 290 nm to 400 nm was measured using a UV-2000S ultraviolet transmittance analyzer, and the SPF was calculated through systematic analysis. in vitro and PFA in vitro .

[0215] The results are shown in Table 2.

[0216] Table 2. SPF and PFA values ​​of sunscreens 1-4

[0217]

[0218] The results showed that sunscreen lotion 2, without any added sunscreen agents, had no external sun protection effect. Compared to sunscreen lotion 1, sunscreen lotion 3, with 0.2% ACP-W1, and sunscreen lotion 4, with 0.5% ACP-W1, significantly improved the SPF and PFA of the products, showing a dose-dependent increase. When the asparagus homogeneous polysaccharide ACP-W1 of this invention is used synergistically with sunscreen agents, it can significantly enhance the sun protection effect of the sunscreen agents and increase the SPF and PFA values ​​of the sunscreen products. The asparagus homogeneous polysaccharide ACP-W1 obtained in this invention has high safety and clear efficacy, and can be used as a core functional ingredient in anti-photoaging personal care products, possessing good application transformation potential.

[0219] ② Friction resistance test:

[0220] The abrasion resistance of sunscreen cosmetics was tested using a friction tester and a Labsphere UV-2000S instrument.

[0221] Using HD6 type PMMA board as the substrate, the sample to be tested was subjected to a concentration of 1.3 mg / cm³. 2 The coating was uniformly applied to the surface of an HD6 PMMA board. After equilibration for 15 minutes, the initial SPF value of the sample was measured. Subsequently, the HD6 board with the coated sample was placed on a friction tester. Soft polyurethane material was selected as the friction surface, and the vertical pressure was set to 50 g and the friction speed to 200 mm / min. After one cycle of rubbing, the SPF value of the friction area was measured. The friction resistance of the sample was evaluated by the equivalent film thickness residual rate, and the calculation formula is shown below:

[0222]

[0223] This index represents the ratio of the film thickness after friction to the film thickness before friction; the higher the residual rate, the better the sample's friction resistance.

[0224] The results are shown in Table 3.

[0225] Table 3. Anti-friction properties of sunscreens 1, 3, and 4

[0226]

[0227] The results showed that, compared to sunscreen lotion 1, sunscreen lotion 3 with 0.2% ACP-W1 and sunscreen lotion 4 with 0.5% ACP-W1 significantly improved the anti-friction performance of the original formula. The regular and rigid triple helix structure ensures the orderly arrangement and spatial stability of the ACP-W1 molecular chains. When added to the sunscreen formula, it forms a continuous, dense, mechanically tough, and highly shear-resistant high-molecular-weight breathable protective film on the skin surface. This film itself possesses excellent anti-friction and abrasion resistance, buffering external mechanical friction stress from skin, clothing, and hand rubbing. Simultaneously, it tightly anchors sunscreen particles through intermolecular forces, preventing particle displacement, detachment, and film damage caused by friction. This significantly improves the anti-friction stability, abrasion resistance, and long-lasting makeup effect of sunscreen products, while simultaneously enhancing waterproof and sweat-resistant properties, addressing the pain points of traditional sunscreens such as pilling, friction-induced film removal, and rapid attenuation of protective power.

[0228] It should be particularly noted that the embodiments described below are only some embodiments of the present invention, and not all embodiments. They are only used to explain and exemplify the present invention, and are not intended to limit the scope of protection of the present invention. All other embodiments obtained by those skilled in the art based on the core technical concepts disclosed above should fall within the scope of protection claimed by the present invention.

Claims

1. An asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity in the skin, characterized in that: The monosaccharide composition of the asparagine homogeneous polysaccharide ACP-W1 is as follows: the molar ratio of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid is 0.34%–0.74%: 15.63%–17.63%: 7.62%–9.62%: 55.12%–57.12%: 4.45%–6.45%: 0.89%–2.89%: 0.97%–2.97%: 7.77%–9.77%; the weight-average molecular weight is 1440.0–1640.0 kDa, the number-average molecular weight is 1150.0–1350.0 kDa, and the polydispersity index is 1.1–1.

3.

2. The asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity for skin according to claim 1, characterized in that: The chemical structural formula of the asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity is shown below: ； Where m is an integer between 4000 and 5800, and n is an integer between 570 and 850.

3. The asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity according to claim 1 or 2, characterized in that: The asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity exhibits a stable triple helix conformation in aqueous solution and has a parallel β-sheet-like ordered secondary structure.

4. The method for preparing asparagine homogeneous polysaccharide ACP-W1 with anti-photoaging activity of the skin according to any one of claims 1 to 3, characterized in that... The process includes the following steps: drying, pulverizing, defatting, enzymatically hydrolyzing and water-assisted extraction of asparagus roots, followed by filtration, concentration and alcohol precipitation, centrifugation and resolution, protein removal, decolorization, dialysis to remove small molecules, and then lyophilizing to obtain crude asparagus polysaccharide ACP; the aqueous solution of crude asparagus polysaccharide ACP obtained by dissolving crude asparagus polysaccharide ACP in water is successively subjected to weak anion exchange column chromatography and gel filtration column chromatography to obtain homogeneous asparagus polysaccharide ACP-W1.

5. The method for preparing asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity according to claim 4, characterized in that: The drying temperature is 60–65 °C; The degree of drying refers to drying to a constant weight; The degree of pulverization is such that it can pass through a 60-80 mesh standard sieve; The defatting mentioned above is defatting using ethanol; The enzymatic hydrolysis-assisted water extraction described herein uses a combination of papain and cellulase for extraction. The filtration process involves using a 200-mesh filter cloth. The concentration and alcohol precipitation process is as follows: after concentrating the filtrate, add ethanol and mix well, wherein the final concentration of ethanol is 70-80% by volume, and let stand. The centrifugation conditions for centrifugation and reconstitution are centrifugation at a speed of 6000-10000g for 5-25 minutes; The resolution method involves dissolving the precipitate obtained by centrifugation in deionized water to obtain an aqueous solution of asparagine polysaccharide. The protein removal method described is the Sevag method. The decolorization is achieved through a macroporous adsorption resin column; The dialysis procedure is as follows: Dialyze in deionized water using a 3000-5000 Da dialysis bag for 24-48 hours, changing the water every 4-6 hours during the process; The degree of concentration in the concentrated freeze-drying process is to concentrate to near-dry / without alcohol odor.

6. The method for preparing asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity according to claim 5, characterized in that: The specific steps of defatting are as follows: mix asparagus root powder and anhydrous ethanol at a material-to-liquid mass ratio of 1g:10-15mL, stir to extract fat-soluble pigments and some impurities, separate solids and liquids, and take the solid. The specific operation of the enzymatic hydrolysis-assisted water extraction is as follows: Prepare a buffer solution with a pH of 4.0–6.0 and a concentration of 0.2 mol / L using sodium acetate and acetic acid; mix the buffer solution and defatted asparagus root powder at a material-to-liquid ratio of 1 g: 10–50 mL; then add a compound enzyme equivalent to 0.5–2.5% w / w of the asparagus root powder mass, consisting of papain and cellulase in a 1:1 mass ratio; after mixing thoroughly, heat to 45–55 °C for enzymatic hydrolysis extraction for 2–6 h, continuously stirring during the extraction process; after the reaction is complete, replenish the evaporated water; inactivate the enzyme after hydrolysis. The specific steps for removing proteins are as follows: Add 1 / 4 volume of Sevag reagent to the asparagus polysaccharide aqueous solution, shake thoroughly for 20-30 min, allow to stand for layering, centrifuge at 4000 g for 10-20 min, discarding the intermediate protein layer and the lower organic phase each time, collecting the upper aqueous phase, repeating 3-5 times until the intermediate protein layer disappears; Sevag reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; The macroporous adsorption resin is AB-8 macroporous adsorption resin.

7. The method for preparing asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity according to claim 4, characterized in that: The weak anion exchange column mentioned is a DEAE Seplife FF weak anion exchange column; The operation of the weak anion exchange column chromatography is as follows: centrifuge the aqueous solution of crude asparagus polysaccharide ACP, take the supernatant and load it into the weak anion exchange column at a flow rate of 4-5 mL / min, then elute with pure water to obtain the eluent, concentrate and dialyze to obtain the ion-exchange purified asparagus polysaccharide ACP-W. The gel filter column is a Sephacryl S-400 HR gel filter column; The gel filtration column chromatography process is as follows: the ion-exchange purified asparagus polysaccharide ACP-W is centrifuged, and the supernatant is loaded into a gel filtration column at a flow rate of 1–1.5 mL / min; pure water is used for elution, and each 10 mL tube is collected to collect all the eluent; by measuring the total sugar content of the eluent in each collection tube, the main elution peak is selected and confirmed, and the eluents corresponding to the elution peak are combined. Then, the mixture is concentrated, dialyzed, and freeze-dried to obtain homogeneous asparagus polysaccharide ACP-W1.

8. The method for preparing asparagus homogeneous polysaccharide ACP-W1 with anti-photoaging activity according to claim 7, characterized in that: The centrifugation conditions are 8000-10000 g for 10-15 min; The concentration method is rotary evaporation concentration; The degree of concentration is to concentrate to 1 / 5 of the original volume; The dialysis procedure involves using a 3000–5000 Da dialysis bag for 24–48 hours to remove small molecule components.

9. The use of the asparagine homogeneous polysaccharide ACP-W1 according to any one of claims 1 to 3 in the preparation of skin anti-photoaging care products.

10. A skin anti-photoaging care product, characterized in that: Contains the asparagine homogeneous polysaccharide ACP-W1 as described in any one of claims 1 to 3.

11. A sunscreen lotion, characterized in that... Contains the following components by mass percentage: 0.04-0.06% disodium EDTA, 2-4% glycerol, 3-5% butylene glycol, 0.1-0.3% allantoin, 0.05-0.15% acrylate / C10-30 alkanol acrylate crosspolymer, and the asparagine homogeneous polysaccharide ACP-W1 as described in any one of claims 1-3. 0.2-2%, p-hydroxyacetophenone 0.4-0.6%, 1,2-hexanediol 0.8-1.2%, potassium cetyl phosphate 0.7-0.9%, glyceryl stearate and PEG-100 stearate 2-3%, cetearyl alcohol 1-2%, bis-ethylhexyloxyphenol methoxyphenyl triazine 1-3%, diethylamino hydroxybenzoyl hexyl benzoate 2-4%, ethylhexyl methoxycinnamate 7-9%, C12-15 alcohol benzoate 4-6%, isononyl isononanoate 3-5%, polydimethylsiloxane 3-5%, aminomethylpropanol 0.05-0.07%, fragrance as needed, water balance.

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