Sargassum fusiforme polysaccharide with significant anti-photoaging activity, and preparation method and application thereof

Abstract

The present application relates to the technical field of marine bioactive substance extraction and skin care, and particularly relates to a kind of Sargassum fusiforme polysaccharide with significant anti-photoaging activity and its preparation method and application.In preparation, the Sargassum fusiforme raw material is first degreased with ethanol, and then subjected to complex enzymolysis of papain and cellulase combined with ultrasonic-assisted extraction.The extracted solution is subjected to deproteinization and decolorization, and then gradient alcohol precipitation fractionation is performed.The precipitate component with a final ethanol volume fraction of 50% is collected, and the target Sargassum fusiforme polysaccharide is obtained by freeze-drying.The obtained polysaccharide has a weight average molecular weight of 25-55 kDa, a high active group retention rate, can significantly scavenge active oxygen, improve antioxidant enzyme activity, inhibit matrix metalloproteinase expression, and promote collagen synthesis, and has an anti-photoaging effect superior to that of vitamin C.The present application has a mild and controllable process, stable polysaccharide yield and activity, and can be applied to skin care cosmetics, external preparations and health foods.

Description

Technical Field

[0001] This invention relates to the field of marine bioactive substance extraction and skin care technology, specifically to a Sargassum fusiforme polysaccharide with significant anti-photoaging activity, its preparation method, and its application. Background Technology

[0002] Photoaging of the skin is oxidative damage and degenerative changes in skin structure caused by long-term exposure to ultraviolet (UV) radiation. Clinically, it manifests as dry, rough skin, deepened wrinkles, and decreased elasticity. Its molecular mechanism is directly related to the excessive generation of reactive oxygen species (ROS) induced by UV radiation, imbalance of the endogenous antioxidant system, and overexpression of matrix metalloproteinases (MMPs). Specifically, MMPs can degrade collagen and elastin fibers in the dermis, ultimately disrupting the normal skin structure. With the continuous upgrading of public demand for anti-aging products, the development of natural, highly effective, and low-irritation anti-photoaging active ingredients has become one of the core research directions in the skincare and biotechnology fields.

[0003] Sargassum fusiforme is an economically important brown alga widely distributed along the southeastern coast of my country, with abundant resources and low procurement costs. Its core active ingredient is brown algae polysaccharide. Existing research has confirmed that Sargassum fusiforme polysaccharides possess antioxidant, immunomodulatory, and lipid-lowering activities. However, targeted research on its anti-photoaging effects on the skin is severely lacking, and existing extraction processes have significant shortcomings: traditional hot water extraction methods involve high temperatures and long extraction times, resulting in polysaccharide yields generally below 8%. Furthermore, high temperatures can damage active groups such as sulfate ester bonds and uronic acid carboxyl groups on the polysaccharide chains, directly leading to a decrease in the product's biological activity. While single enzymatic hydrolysis or ultrasonic extraction can slightly improve the yield, their effect on cell wall disruption is limited, and they cannot selectively enrich highly active polysaccharide components, resulting in uneven product activity and poor batch-to-batch stability.

[0004] Currently available marine polysaccharide anti-photoaging technologies mostly focus on kelp polysaccharides and fucoidan oligosaccharides. They do not combine the structural characteristics of Sargassum fusiforme polysaccharides for process optimization and targeted activity screening, nor do they clarify the effective components and structure-activity relationship of anti-photoaging, making it difficult to support the development of high-end skincare products and stable industrial production. Summary of the Invention

[0005] To address the aforementioned technical issues, this invention proposes a method for preparing Sargassum fusiforme polysaccharides with significant anti-photoaging activity. This method employs a gentle extraction process combining enzymatic hydrolysis and ultrasound assistance to maximize polysaccharide yield while preserving active functional groups. Furthermore, a gradient alcohol precipitation fractionation method is used to screen for specific molecular weight components with optimal anti-photoaging activity. Cell and animal experiments have verified that its anti-photoaging effect is significantly superior to that of commonly used positive control vitamin C. Moreover, the process is simple, controllable, and suitable for industrial-scale production.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing Sargassum fusiforme polysaccharide with significant anti-photoaging activity includes the following steps: S1, Raw material pretreatment: Take dried Sargassum fusiforme, remove impurities, crush and sieve, then degrease by reflux with ethanol aqueous solution, and dry the filter residue for later use. S2, Compound Enzymatic Hydrolysis: Defatted Sargassum powder and deionized water are mixed according to the material-to-liquid ratio, a compound enzyme is added, and the pH of the system is adjusted before constant-temperature enzymatic hydrolysis. The compound enzyme is a mixture of papain and cellulase in a mass ratio of 2:1, and the amount of enzyme added is 2%~3% of the mass of Sargassum powder. S3, Ultrasonic-assisted extraction: After enzymatic hydrolysis, the system is subjected to intermittent ultrasonic extraction. S4, Separation and purification: After enzyme inactivation and centrifugation, the supernatant of the extract is taken and subjected to deproteinization and decolorization treatments in sequence, and then concentrated under reduced pressure to obtain polysaccharide concentrate. S5, Gradient alcohol precipitation fractionation: Anhydrous ethanol is added to the polysaccharide concentrate in portions for gradient alcohol precipitation. The precipitates at different ethanol concentrations are collected, washed, and freeze-dried to obtain Sargassum fusiforme polysaccharide fractions with different molecular weights. The precipitate fraction corresponding to a final ethanol volume fraction of 50% is the target Sargassum fusiforme polysaccharide.

[0007] Preferably, in step S1, the volume fraction of the ethanol-water solution is 75%~85%, the material-to-liquid ratio is 1:8~1:12, and the degreasing is carried out twice by reflux, each time for 1.5~2.5 hours.

[0008] Preferably, in step S2, the material-to-liquid ratio is 1:20 to 1:30, the pH of the system is adjusted to 5.0 to 6.0, the enzymatic hydrolysis temperature is 45 to 55°C, and the enzymatic hydrolysis time is 80 to 100 min.

[0009] Preferably, in step S3, the ultrasonic extraction power is 250~350 W, the extraction temperature is 50~60℃, the total ultrasonic duration is 15~25 min, and the ratio of working time to intermittent time is 1:1.

[0010] Preferably, in step S4, the deproteinization is repeated 3 to 4 times using the Sevag method; the decolorization is performed using AB-8 macroporous resin for static decolorization; and the reduced pressure concentration temperature is 55 to 65°C, concentrating to 1 / 4 to 1 / 6 of the original volume.

[0011] Preferably, in step S5, the gradient ethanol precipitation is as follows: first, add anhydrous ethanol to the concentrate until the final volume fraction of ethanol is 30%, let it stand at 4°C for 10-14 h, and then centrifuge to collect the first component precipitate; continue to add anhydrous ethanol to the supernatant until it reaches 50%, and then centrifuge under the same conditions to collect the second component precipitate, which is the target Sargassum polysaccharide; continue to add anhydrous ethanol to the supernatant until it reaches 70%, and then centrifuge under the same conditions to collect the third component precipitate.

[0012] To better implement this invention, the following technical solutions are also provided: Sargassum fusiforme polysaccharide with significant anti-photoaging activity is prepared by any of the above preparation methods; the Sargassum fusiforme polysaccharide has a weight-average molecular weight of 25-55 kDa, a sulfate group mass fraction of 12%-18%, and a uronic acid mass fraction of 8%-12%.

[0013] This invention also provides the application of the above-mentioned Sargassum polysaccharide in the preparation of anti-photoaging products; the anti-photoaging products include skin care cosmetics, topical skin preparations or health foods.

[0014] Preferably, the anti-photoaging refers to resisting UVB-induced skin photoaging damage, specifically including at least one of the following: inhibiting intracellular reactive oxygen species generation, enhancing the activity of skin antioxidant enzymes, inhibiting the overexpression of matrix metalloproteinases, promoting collagen fiber synthesis, and improving skin photoaging tissue structure damage.

[0015] The present invention has at least the following beneficial effects: This invention employs a papain and cellulase complex system, which can separately degrade the protein backbone and cellulose network of the cell wall, synergistically breaking down the cell wall barrier. Combined with the cavitation effect of gentle ultrasound to enhance mass transfer, efficient extraction can be achieved at a low temperature of around 55℃, increasing the polysaccharide yield by more than 40% compared to traditional hot water extraction methods. Simultaneously, the low-temperature process avoids the damage to sulfate ester bonds, uronic acid, and other active groups caused by high temperatures, increasing the retention rate of sulfate groups and uronic acid by more than 50%, providing a structural basis for anti-photoaging activity.

[0016] This invention separates crude polysaccharides from Sargassum fusiforme into components with three molecular weight ranges through gradient alcohol precipitation. Targeted screening using an in vitro cell model revealed that the medium molecular weight component (25-55 kDa) exhibited the best anti-photoaging activity. This molecular weight range retains the complete spatial conformation of the active groups in the polysaccharide molecule while possessing excellent cell permeability, thus solving the problems of uneven activity and unstable effects of crude polysaccharides.

[0017] This invention was validated using an in vitro human skin fibroblast photoaging model and an in vivo mouse photoaging model. The Sargassum fusiforme polysaccharide of this invention can reduce oxidative damage by scavenging ROS and increasing the activity of antioxidant enzymes such as SOD. At the same time, it significantly downregulates the expression of MMP-1 and MMP-3 and promotes the synthesis of type I collagen. At the same concentration, its anti-photoaging effect is better than that of vitamin C, and it has no obvious cytotoxicity or skin irritation, making it highly safe for application.

[0018] The reagents used in this invention are all common industrial-grade raw materials, the enzyme preparations are inexpensive, and the ethanol can be recycled; each process is a mature industrial unit operation, requiring no special equipment, the process parameters are controllable, the batch stability is good, and it is easy to scale up production; the raw material Sargassum fusiforme is abundant, and the overall production cost is low. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the preparation method steps of the present invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0021] The raw materials and reagents used in the following examples are as follows: dried Sargassum fusiforme was purchased from Dongtou Aquatic Products Market, Wenzhou, Zhejiang Province; papain (enzyme activity 100,000 U / g) and cellulase (enzyme activity 20,000 U / g) were purchased from Shandong Sukehan Biotechnology Co., Ltd.; AB-8 macroporous resin was purchased from Tianjin Nankai University Chemical Plant; CCK-8 kit, ROS detection kit, and SOD / MDA detection kit were purchased from Beyotime Biotechnology Co., Ltd.; human skin fibroblasts (HSF) were purchased from the Cell Bank of the Chinese Academy of Sciences; MMP-1 and type I collagen ELISA kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.; all other reagents were of analytical grade.

[0022] Main instruments: UV-2550 UV-Vis spectrophotometer (Shimadzu, Japan); KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Labconco freeze dryer (USA); Tecan Infinite M200 microplate reader (Switzerland); UVP UVB ultraviolet irradiator (USA); Eppendorf 5810R high-speed refrigerated centrifuge (Germany); Agilent 1260 high-performance liquid chromatograph (equipped with gel chromatography column).

[0023] Example 1

[0024] The purpose of this embodiment is to verify the effects of different extraction processes on the yield of Sargassum fusiforme polysaccharides and the content of active groups.

[0025] This embodiment compares the polysaccharide yield and active group retention of four extraction processes to verify the synergistic advantages of the process of the present invention.

[0026] In this embodiment, the experimental groups are as follows: The process of this invention is an experimental group, and specifically includes the following steps: (1) Raw material pretreatment: Remove sand and stone impurities from dried Sargassum fusiforme and crush it through a 40-mesh sieve; weigh 100 g Sargassum fusiforme powder, add 1000 mL of 80% ethanol, reflux at 80℃ twice for 2 hours each time, filter and air dry the filter residue to obtain defatted Sargassum fusiforme powder.

[0027] (2) Compound enzymatic hydrolysis: Take 50 g defatted Sargassum powder, add 1250 mL of deionized water at a material-to-liquid ratio of 1:25, stir evenly, and then add 1.25 g of compound enzyme (papain: cellulase = 2:1, mass ratio); adjust the pH to 5.5 with 0.1 mol / L HCl, and place in a 50℃ constant temperature water bath for enzymatic hydrolysis for 90 min.

[0028] (3) Ultrasonic-assisted extraction: After the enzymatic hydrolysis is completed, the system is transferred into an ultrasonic cleaner, the power is set to 300 W and the temperature is 55℃, and the ultrasonic is intermittently used for 20 min (working for 2 s and stopping for 2 s).

[0029] (4) Separation and purification: After extraction, the enzyme was inactivated by boiling water bath for 10 min, and after cooling, the supernatant was collected by centrifugation at 4000 r / min for 15 min; 1 / 5 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added, and the mixture was shaken for 20 min and then centrifuged to remove denatured protein. This process was repeated 3 times; the deproteinized polysaccharide solution was mixed with the pretreated AB-8 macroporous resin at a ratio of 1:5, and statically decolorized for 4 h. The effluent was collected by filtration; the solution was concentrated under reduced pressure at 60℃ to 1 / 5 of the original volume to obtain the polysaccharide concentrate.

[0030] (5) Gradient alcohol precipitation: Anhydrous ethanol was slowly added to the concentrate with stirring until the final volume fraction of ethanol was 30%. The mixture was allowed to stand at 4°C for 12 h, centrifuged at 4000 r / min for 10 min, and the precipitate was collected and recorded as SFP-1. Anhydrous ethanol was added to the supernatant until the volume fraction was 50%, and the mixture was allowed to stand and centrifuged under the same conditions. The precipitate was collected and recorded as SFP-2. Anhydrous ethanol was added to the supernatant until the volume fraction was 70%, and the precipitate was collected and recorded as SFP-3. Each precipitate was washed twice with anhydrous ethanol and acetone, and then freeze-dried for 24 h to obtain pure Sargassum fusiforme polysaccharide. The total yield of crude polysaccharide was calculated by combining the three components.

[0031] Control group 1 used the traditional hot water extraction method, without enzymatic hydrolysis and ultrasonic treatment, and instead used a 90℃ constant temperature water bath for 3 hours. The remaining defatting, deproteinization, alcohol precipitation and drying steps were exactly the same as those of the experimental group.

[0032] Control group 2 used a single cellulose enzymatic hydrolysis method, adding only 1.25 g of cellulase for enzymatic hydrolysis, without ultrasonic assistance, and the rest of the operation was the same as the experimental group.

[0033] Control group 3 used a simple ultrasonic extraction method without adding enzyme preparations, and only performed ultrasonic extraction for 30 minutes. The rest of the operation was the same as the experimental group.

[0034] The detection indicators and methods in this embodiment are set as follows: Polysaccharide yield: determined by the phenol-sulfuric acid method, using glucose as the standard, calculated as "polysaccharide mass / defatted Sargassum powder mass × 100%".

[0035] Sulfate content: determined by gelatin-turbidimetric method, with potassium sulfate as standard.

[0036] Glucuronic acid content: determined by the m-hydroxybiphenyl method, with glucuronic acid as the standard.

[0037] Weight-average molecular weight: determined by high-performance gel permeation chromatography (HPGPC), with a standard curve plotted using a series of dextran as standards.

[0038] The results of the extraction effects of each group are shown in Table 1. According to HPGPC determination, the weight-average molecular weight of SFP-1 is approximately 92 kDa, SFP-2 is approximately 38 kDa, and SFP-3 is approximately 14 kDa.

[0039] Table 1. Polysaccharide yield and active group content of different extraction processes

[0040] As shown in the table above, the polysaccharide yield in the experimental group of this invention reached 11.23%, which is 43.1% higher than that of the traditional hot water extraction method, and also significantly higher than that of single enzymatic hydrolysis and simple ultrasonic methods, proving that the combined enzymatic hydrolysis and ultrasonic assistance have a synergistic effect in breaking down cell walls. Meanwhile, the sulfate and uronic acid contents in the experimental group were significantly higher than those in the traditional hot water group, with increases of 53.0% and 52.5%, respectively, indicating that the mild process of low-temperature enzymatic hydrolysis-ultrasound can effectively reduce the degradation and damage of active groups.

[0041] From a production perspective, this process has a short extraction time and low temperature, resulting in lower overall energy consumption compared to long-term high-temperature extraction. The enzymes used are bulk industrial enzymes with low procurement costs, and the ethanol can be recovered and recycled through distillation, resulting in less waste and making it suitable for industrial-scale mass production.

[0042] Example 2

[0043] The purpose of this embodiment is to screen the in vitro anti-photoaging activity of Sargassum polysaccharide components with different molecular weights.

[0044] This embodiment is based on a UVB-induced human skin fibroblast (HSF) photoaging model to screen for polysaccharide components with the best anti-photoaging activity.

[0045] The cell model was established and grouped in this embodiment as follows: HSF cells in logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / cells. 4 Inoculate 96-well plates at a density of 100 cells / mL, incubate for 24 h, then discard the original culture medium and replace with PBS buffer at 30 mJ / cm². 2 A cell photoaging model was established by UVB irradiation. After irradiation, the cells were cultured in complete culture medium containing different samples for another 24 hours.

[0046] A blank control group was set up, without UVB irradiation, and cultured routinely without adding samples; Set up a model control group, irradiated with UVB, without adding samples; Set up the SFP-1 group, UVB irradiation + 100μg / mL SFP-1 (30% alcohol-precipitated component); Set up the SFP-2 group, UVB irradiation + 100μg / mL SFP-2 (50% alcohol-precipitated component); Set up the SFP-3 group, UVB irradiation + 100μg / mL SFP-3 (70% alcohol-precipitated component); A positive control group was set up, which received UVB irradiation plus 100 μg / mL vitamin C (VC).

[0047] The detection indicators are set as follows in this embodiment: Cell viability: determined by CCK-8 assay, with absorbance measured at 450 nm using an ELISA reader. The relative viability of each group was calculated with the survival rate of the blank group being 100%.

[0048] Intracellular ROS levels: Fluorescence intensity was measured by flow cytometry using the DCFH-DA fluorescent probe method, and the relative level was calculated with the fluorescence intensity of the blank group as 100%.

[0049] MMP-1 secretion and type I collagen content: Cell culture supernatant was collected, and the results were measured using an ELISA kit according to the manufacturer's instructions.

[0050] The results of the detection of photoaging-related indicators of cells in each group are shown in Table 2.

[0051] Table 2. Protective effects of different polysaccharide components on photoaged HSF cells (n=6)

[0052] This indicates that UVB irradiation significantly reduced cell survival rate in the model group, substantially increased ROS and MMP-1 levels, and decreased type I collagen content, demonstrating the successful establishment of the photoaging model. Among the three polysaccharide components, the SFP-2 component exhibited the strongest cell-protective effect, increasing cell survival rate from 62.35% to 89.67%. It also significantly scavenged ROS, inhibited MMP-1 secretion, and promoted collagen synthesis. All indicators were significantly superior to those of the SFP-1 and SFP-3 components, and also superior to the positive control vitamin C at the same concentration.

[0053] Structure-activity relationship analysis showed that high molecular weight SFP-1 had significant steric hindrance, making it difficult to penetrate cell membranes and exert its effect; while low molecular weight SFP-3 had good permeability, it suffered from numerous polysaccharide chain breaks, loss of active groups such as sulfate groups, and conformational disruption leading to decreased activity; medium molecular weight SFP-2 combined good permeability with an intact active structure, thus exhibiting the best anti-photoaging activity. This embodiment clarified the effective components of Sargassum fusiforme polysaccharides for anti-photoaging, providing a precise material basis for subsequent product development.

[0054] Example 3

[0055] The purpose of this embodiment is to verify the dose-dependent protective effect of Sargassum polysaccharide SFP-2 on photoaged HSF cells.

[0056] In this embodiment, SFP-2 with different concentration gradients was set up to explore its dose-effect relationship, and the mechanism of action was verified from the perspectives of oxidation-antioxidant balance and matrix degradation.

[0057] In this embodiment, the experimental groups are as follows: The cell seeding and modeling methods are the same as in Example 2, and the following settings are made: Blank control group and model control group (same as in Example 2); SFP-2 low-dose group: 25 μg / mL; SFP-2 medium dose group: 50 μg / mL; SFP-2 high-dose group: 100 μg / mL; SFP-2 ultra-high dose group: 200 μg / mL; Positive control group: 100 μg / mL Vitamin C.

[0058] The detection indicators are set as follows in this embodiment: LDH leakage rate: Measured using a lactate dehydrogenase kit, reflecting the degree of cell membrane damage.

[0059] SOD activity and MDA content: Cell lysates were collected and measured according to the kit instructions to reflect the cell's antioxidant capacity and lipid peroxidation level.

[0060] MMP-1 and MMP-3 mRNA expression levels were determined by qRT-PCR, with GAPDH as an internal control. The relative expression level was calculated using the (ΔΔCt) method.

[0061] The results of cell damage and oxidative stress indicators in each group are shown in Table 3, and the results of matrix metalloproteinase gene expression are shown in Table 4.

[0062] Table 3. Indicators related to oxidative damage in cells of each group (n=6)

[0063] Table 4. Relative expression levels of MMPs mRNA in each group (n=3)

[0064] It is evident that SFP-2 exhibits a significant dose-dependent protective effect against UVB-induced fibroblast damage: as the concentration increased from 25 μg / mL to 100 μg / mL, the cellular LDH leakage rate gradually decreased, SOD activity continued to recover, MDA content steadily declined, and the gene expression of MMP-1 and MMP-3 was also significantly downregulated with increasing concentration; when the concentration reached 200 μg / mL, the improvement in various indicators tended to level off, indicating that 100 μg / mL was close to the optimal concentration.

[0065] Compared with the positive control at the same concentration, 100 μg / mL SFP-2 was significantly superior to vitamin C in reducing cell membrane damage, enhancing antioxidant capacity, and inhibiting matrix metalloproteinase expression. Furthermore, no cytotoxicity was observed at a concentration of 200 μg / mL, demonstrating its strong activity and wide safety window. From a mechanism of action perspective, SFP-2 alleviates UV oxidative damage by scavenging ROS and enhancing the activity of endogenous antioxidant enzymes, and reduces collagen degradation by inhibiting MMP overexpression, thus exerting a synergistic anti-photoaging effect through multiple pathways.

[0066] Example 4

[0067] The purpose of this embodiment is to verify the in vivo protective effect of Sargassum polysaccharide SFP-2 against photoaging of mouse skin.

[0068] This embodiment constructs a UVB photoaging model of mouse dorsal skin to verify the in vivo anti-photoaging effect and topical safety of SFP-2.

[0069] In this embodiment, the experimental animals and group settings are as follows: Forty SPF-grade female KM mice, 6-8 weeks old and weighing 18-22 g, were acclimatized for 3 days. Their dorsal hair (approximately 2 cm × 3 cm) was removed with depilatory cream. They were then randomly divided into 5 groups of 8 mice each. Blank control group: No UVB irradiation was performed, and 0.2 g of blank cream base was applied to the back daily; Model control group: UVB irradiation + daily application of 0.2 g blank cream base; SFP-2 low-dose group: UVB irradiation + daily application of 0.2 g of cream containing 0.5% SFP-2; SFP-2 high-dose group: UVB irradiation + daily application of 0.2 g of cream containing 2% SFP-2; Positive control group: UVB irradiation + daily application of 0.2 g of cream containing 5% vitamin C.

[0070] In this embodiment, the modeling and drug administration are as follows: Irradiation was performed using a UVB irradiation device at a distance of 15 cm, with an initial dose of 20 mJ / cm². 2 Increased by 10 mJ / cm per week 2 Maintain at 50 mJ / cm from week 4 onwards. 2 Irradiation was performed 5 times per week for 8 consecutive weeks, with a total cumulative dose of approximately 12.6 J / cm². 2 Apply the corresponding cream 30 minutes before daily irradiation; the blank control group applies the base simultaneously.

[0071] The detection indicators are set as follows in this embodiment: Skin appearance rating: After the experiment, the skin on the back was observed with the naked eye and rated from 0 to 4 points according to the roughness, wrinkle depth and peeling. The higher the score, the more serious the photoaging.

[0072] Skin moisture content: Take skin tissue from the same area of ​​the back, weigh it, and dry it to constant weight. Calculate the moisture content according to "(wet weight - dry weight) / wet weight × 100%".

[0073] Hydroxyproline (Hyp) content: determined by alkaline hydrolysis method, reflecting the total collagen content of the skin.

[0074] MMP-1 content in skin: Skin tissue homogenate was prepared and measured using an ELISA kit.

[0075] Histopathological observation: Skin tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under an optical microscope to examine the epidermal thickness and dermal collagen fiber morphology.

[0076] The results of the detection of photoaging-related indicators in the skin of mice in each group are shown in Table 5.

[0077] Table 5 Comparison of photoaging indices in mouse skin among different groups (n=8)

[0078] Pathological observation revealed the following results: In the control group, the epidermis was thin and clearly structured, with abundant and neatly arranged collagen fibers in the dermis; in the model group, the epidermis was significantly thickened, with broken, disordered, and reduced collagen fibers in the dermis, accompanied by inflammatory cell infiltration; in the high-dose SFP-2 group, the epidermal thickness was close to normal, the dermal collagen fibers were abundant and neatly arranged, and the degree of damage improvement was significantly better than that of the positive control group. During the experiment, no mice in any group showed skin redness, swelling, ulceration, allergic reactions, or other irritant reactions.

[0079] Therefore, in vivo experiments have confirmed that continuous UVB irradiation for 8 weeks successfully established a mouse skin photoaging model, and topical application of SFP-2 cream can improve skin photoaging symptoms in a dose-dependent manner; the 2% high-dose group can significantly increase skin moisture content, increase hydroxyproline content, and inhibit MMP-1 overexpression, repairing collagen fiber damage at the tissue structure level, with overall effects superior to the 5% concentration of vitamin C positive control.

[0080] This embodiment verifies the anti-photoaging activity of Sargassum polysaccharide SFP-2 at the in vivo level, and shows that it is non-irritating when applied topically, indicating that it has both good efficacy and safety as a skin care ingredient and has practical product development value.

[0081] Example 5

[0082] The purpose of this embodiment is to verify the stability and application performance of Sargassum polysaccharide SFP-2 in skin care matrix.

[0083] This embodiment examines the stability, transdermal properties, and moisturizing effect of SFP-2 in cream bases from a production application perspective, and verifies its formulation compatibility.

[0084] The sample preparation in this embodiment is as follows: An O / W type base cream matrix was prepared (2% stearic acid, 1% monoglyceride, 5% white oil, 5% glycerin, 0.2% triethanolamine, 0.2% carbomer, and deionized water to make up the difference), and three groups of samples were prepared by adding active ingredients respectively: SFP-2 group: Add 1% SFP-2; Crude polysaccharide group: 1% traditional water-extracted Sargassum fusiforme crude polysaccharide was added (Product of control group 1 in Example 1); HA group: Added 1% sodium hyaluronate (molecular weight 1 million Da, a commonly used skin care moisturizing ingredient).

[0085] The testing method in this embodiment is as follows: (1) Stability test: The three groups of samples were stored in constant temperature incubators at 4℃, 25℃ and 45℃ for 30 days. The appearance was observed on days 0, 10, 20 and 30, the pH value was measured, and the polysaccharide retention rate was determined by phenol-sulfuric acid method (HA group was calculated by uronic acid content).

[0086] (2) In vitro transdermal experiment: The modified Franz diffusion cell was used, with the isolated porcine back skin as the transdermal barrier, the stratum corneum facing the supply cell, and pH 7.4 PBS buffer added to the receiving cell; 1 g of sample was added to the supply cell, and the mixture was stirred at 32℃ and 300 r / min. Samples were taken at 2, 4, 6, 8, 12 and 24 h to determine the polysaccharide content in the receiving solution and to calculate the cumulative transdermal amount over 24 h.

[0087] (3) Human body moisturizing test: 12 healthy volunteers had 4 test areas on the inner side of their forearms. They were coated with equal amounts of three groups of samples and blank matrix. The skin moisture tester measured the stratum corneum moisture content before application and 2 h, 4 h and 8 h after application, and calculated the moisture growth rate.

[0088] The experimental results are as follows: (1) Stability: Under the conditions of 4℃ and 25℃, the three groups of samples were uniform in appearance after 30 days, without layering or precipitation, and the pH was stable at 5.5~6.0; after being placed at 45℃ for 30 days, the polysaccharide retention rate of the SFP-2 group was 92.3±1.2%, that of the crude polysaccharide group was 76.5±1.8%, and that of the HA group was 90.7±1.5%; the crude polysaccharide group showed a slight yellowing phenomenon, while the appearance of the SFP-2 group and the HA group did not change significantly.

[0089] (2) Transdermal performance: The cumulative transdermal dose in the SFP-2 group over 24 hours was 128.6 ± 4.3 μg / cm³. 2 The crude polysaccharide content was 75.2 ± 3.1 μg / cm³. 2 The HA group had a concentration of 98.4 ± 3.7 μg / cm³. 2 SFP-2 showed significantly better transdermal performance than the other two groups, which is closely related to its moderate molecular weight and uniform molecular structure.

[0090] (3) Moisturizing performance: After 8 hours of application, the skin moisture growth rate of the SFP-2 group was 35.6±2.1%, that of the crude polysaccharide group was 18.2±1.7%, that of the HA group was 28.7±1.9%, and that of the blank matrix group was 10.3±1.2%. SFP-2 has a better long-lasting moisturizing effect than sodium hyaluronate, and has the dual effects of moisture absorption and water retention as well as anti-photoaging.

[0091] It is evident that the Sargassum polysaccharide SFP-2 prepared by this invention exhibits good stability in commonly used skincare cream matrices and superior heat resistance compared to crude polysaccharides produced by traditional processes, thus meeting product shelf-life requirements. Furthermore, its transdermal absorption and moisturizing effects are superior to those of commonly used moisturizing ingredient sodium hyaluronate, enabling it to more effectively reach the dermis and exert its anti-photoaging effect.

[0092] From the perspective of formulation development, this raw material has good compatibility and can be directly added to the aqueous phase to prepare various skin care products without the need for a special solubilizing system. It has strong adaptability to production processes and has good prospects for industrial application.

[0093] The terms "upper," "lower," "outer," "inner," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish relative positional relationships and are not necessarily qualitative. It should be understood that such data can be interchanged where appropriate so that embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion.

[0094] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing Sargassum fusiforme polysaccharide with significant anti-photoaging activity, characterized in that, Includes the following steps: S1, Raw material pretreatment: Take dried Sargassum fusiforme, remove impurities, crush and sieve, then degrease by reflux with ethanol aqueous solution, and dry the filter residue for later use. S2, Compound Enzymatic Hydrolysis: Defatted Sargassum powder and deionized water are mixed according to the material-to-liquid ratio, a compound enzyme is added, and the pH of the system is adjusted before constant-temperature enzymatic hydrolysis. The compound enzyme is a mixture of papain and cellulase in a mass ratio of 2:1, and the amount of enzyme added is 2%~3% of the mass of Sargassum powder. S3, Ultrasonic-assisted extraction: After enzymatic hydrolysis, the system is subjected to intermittent ultrasonic extraction. S4, Separation and purification: After enzyme inactivation and centrifugation, the supernatant of the extract is taken and subjected to deproteinization and decolorization treatments in sequence, and then concentrated under reduced pressure to obtain polysaccharide concentrate. S5, gradient alcohol precipitation: anhydrous ethanol was added to the polysaccharide concentrate in portions for gradient alcohol precipitation. The precipitates at different ethanol concentrations were collected, washed, and freeze-dried to obtain Sargassum polysaccharide components with different molecular weights. The precipitate was collected when the final volume fraction of ethanol was 50%, and the target Sargassum polysaccharide was obtained.

2. The preparation method according to claim 1, characterized in that: In step S1, the volume fraction of the ethanol-water solution is 75%~85%, the material-to-liquid ratio is 1:8~1:12, and the degreasing is carried out twice by reflux, each time for 1.5~2.5 hours.

3. The preparation method according to claim 1, characterized in that: In step S2, the material-to-liquid ratio is 1:20 to 1:30, the pH of the system is adjusted to 5.0 to 6.0, the enzymatic hydrolysis temperature is 45 to 55°C, and the enzymatic hydrolysis time is 80 to 100 min.

4. The preparation method according to claim 1, characterized in that: In step S3, the ultrasonic extraction power is 250~350 W, the extraction temperature is 50~60℃, the total ultrasonic duration is 15~25 min, and the ratio of working time to intermittent time is 1:

1.

5. The preparation method according to claim 1, characterized in that: In step S4, deproteinization is performed using the Sevag method repeated 3-4 times; decolorization is performed using AB-8 macroporous resin for static decolorization. The vacuum concentration temperature is 55~65℃, and the volume is concentrated to 1 / 4~1 / 6 of the original volume.

6. The preparation method according to claim 1, characterized in that: In step S5, the gradient ethanol precipitation is as follows: first, add anhydrous ethanol to the concentrate until the final volume fraction of ethanol is 30%, let it stand at 4°C for 10-14 h, and then centrifuge to collect the first component precipitate; continue to add anhydrous ethanol to the supernatant until it reaches 50%, and then centrifuge under the same conditions to collect the second component precipitate, which is the target Sargassum polysaccharide; continue to add anhydrous ethanol to the supernatant until it reaches 70%, and then centrifuge under the same conditions to collect the third component precipitate.

7. A Sargassum fusiforme polysaccharide with significant anti-photoaging activity, characterized in that, The polysaccharide is prepared by any one of the preparation methods described in claims 1 to 6; the weight-average molecular weight of the polysaccharide is 25-55 kDa, the mass fraction of sulfate is 12%-18%, and the mass fraction of uronic acid is 8%-12%.

8. The application of Sargassum polysaccharide according to claim 7 in the preparation of anti-photoaging products; the anti-photoaging products include skin care cosmetics, topical skin preparations, or health foods.

9. The application of Sargassum fusiforme polysaccharide according to claim 7 in the preparation of anti-photoaging products; characterized in that, The term "anti-photoaging" refers to resisting UVB-induced skin photoaging damage, specifically including at least one of the following: inhibiting intracellular reactive oxygen species generation, enhancing skin antioxidant enzyme activity, inhibiting matrix metalloproteinase overexpression, promoting collagen fiber synthesis, and improving skin photoaging tissue structure damage.

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