Porphyra yezoensis polysaccharide PSP-I with anti-ultraviolet damage activity as well as preparation method and application of Porphyra yezoensis polysaccharide PSP-I

Abstract

The invention discloses porphyra yezoensis polysaccharide PSP-I with anti-ultraviolet damage activity as well as a preparation method and application of the porphyra yezoensis polysaccharide PSP-I. The Mw of the PSP-I is 26.149 kDa, the Mn of the PSP-I is 11.267 kDa, and the molar ratio of galactose to glucose to glucuronic acid in monosaccharide composition of the PSP-I is 96.76: 2.31: 0.93. The preparation method comprises the following steps: pretreating a nori raw material, extracting crude polysaccharide by adopting an enzyme method, deproteinizing, dialyzing, collecting a main polysaccharide elution peak by utilizing anion exchange chromatography, further purifying by virtue of sephadex chromatography, collecting the main elution peak, removing impurities and drying, thereby obtaining the nori polysaccharide. The polysaccharide PSP-I can effectively remove active oxygen induced by UVB, inhibit apoptosis and down-regulate expression of matrix metalloproteinase in cells under a safe concentration, shows excellent anti-light aging activity, and can be used for preparing cosmetics or skin care products for resisting ultraviolet damage.

Description

A polysaccharide PSP-Ⅰ from Porphyra yezoensis with anti-UV damage activity, its preparation method and application Technical Field

[0001] This invention relates to the field of plant active ingredient separation and purification technology, specifically to a polysaccharide PSP-Ⅰ from Porphyra yezoensis with anti-ultraviolet damage activity, its preparation method, and its application. Background Technology

[0002] In recent years, with the aggravation of environmental pollution and ozone layer depletion, skin damage caused by ultraviolet radiation has increasingly become a global public health problem. Approximately 2%-5% of UVB rays from sunlight reach the Earth's surface and penetrate the epidermis, becoming a major factor causing photodamage. UVB induces skin cells to produce excessive reactive oxygen species (ROS), disrupting the body's oxidative balance and triggering oxidative stress—this is the core mechanism of UV damage. As consumers increasingly demand higher safety standards for cosmetics, natural active ingredients, due to their safety, low irritation, and good biocompatibility, are gradually becoming a new trend in skin care product development.

[0003] Among numerous natural resources, marine organisms, due to their unique living environment and metabolic pathways, are becoming a treasure trove of natural active ingredients. Marine algae are rich in various bioactive substances, such as polysaccharides, polyphenols, and phycobiliproteins, showing broad application prospects in the food, pharmaceutical, and cosmetic fields. Algal polysaccharides, as one of the most abundant bioactive substances in marine algae, are a class of polyhydroxy polymers and their derivatives composed of more than 20 monosaccharides linked by glycosidic bonds, accounting for more than 76% of their dry weight. Studies have found that algal polysaccharides have various physiological regulatory functions, including antioxidant, lipid-lowering, anti-tumor, and anti-inflammatory effects. Although algae contain a large number of bioactive substances, they are mainly consumed as food and processed as salt or seasonings. However, current research and development of algal active ingredients still faces significant limitations: on the one hand, research is mostly focused on a few traditional cultivated varieties such as *Porphyra yezoensis* and *Porphyra tenera*, or their specific components (such as *Porphyra tenera* polysaccharides and *Porphyra tenera* phycobiliproteins), with insufficient exploration of other algal resources with special and superior traits; on the other hand, existing algal polysaccharide preparation technologies often struggle to simultaneously ensure high product purity, clear structural definition, and in-depth elucidation of the active mechanism. For example, Chinese patent application CN202510453592.0 discloses an alginate with anti-photoaging activity, its preparation method, and its application. This technology uses common alginate as a raw material and employs dielectric barrier discharge plasma for physical degradation to obtain a mixture of degradation products with a wide molecular weight range (10-350 kDa). Although this method is environmentally friendly, its degradation process is random, and the product is a complex and heterogeneous polysaccharide mixture, making precise quality control difficult. Furthermore, its activity verification mainly focuses on preliminary phenotypes such as elastase inhibition and cell viability, lacking systematic research on deeper molecular pathways such as intracellular antioxidant, anti-apoptotic, and extracellular matrix protection. This leads to unclear structure-activity relationships and limits its application in the development of high-value-added products. Therefore, developing research on the bioactivity of algae and achieving high-value utilization of algae is of great significance. In particular, for some specific algae species, such as Porphyra yezoensis, research on the polysaccharide composition, structure, and bioactivity is still insufficient, leaving ample room for the in-depth development of marine polysaccharide resources.

[0004] Pyropia suborbiculata, a species of laver in the genus Pyropia, is a eurythermal seaweed and one of the rarer species of laver. Many algae researchers have found that it has stronger heat resistance compared to traditionally cultivated Pyropia yezoensis and Pyropia haitanensis, and has higher commercial value in the context of global warming and rising sea temperatures. Against this backdrop, in order to address the issue that the leaf shape (often round or kidney-shaped, with relatively small leaves) of wild-type Pyropia suborbiculata strains is unsuitable for commercial cultivation, the laboratory of Professor Ding Hongchang at the College of Fisheries, Shanghai Ocean University, used artificial mutagenesis to isolate several long mutant strains (PS-5) from wild-type strains. They then used 60Co-γ to induce PS-5 growth, and based on growth rate and leaf shape, screened out a new strain, PS-M4, which exhibits rapid growth (Yan N, Ding HC, Yan X H. Selection and characterization a new strain (PS-M4) of pyropia suborbiculata with fast growth based on 60Co-γ ray irradiation[J].Journal of Applied Phycology, 2024, 36(5): 2769-2780.). This strain has already undergone marine testing, and the results show that PS-M4 exhibits significant improvements in heat tolerance, leaf growth, quality, and conchospore release. It meets production requirements in both morphology and yield, making it a promising candidate for large-scale cultivation and a potential candidate for commercial breeding. However, current research on *Porphyra yezoensis* is limited to strain development and breeding; its active ingredients have not yet been systematically studied.

[0005] Therefore, this invention focuses for the first time on the new Porphyra yezoensis strain PS-M4, aiming to provide an efficient and green method for extracting and purifying Porphyra yezoensis polysaccharides, obtaining polysaccharide components with well-defined structures and high purity, and systematically evaluating their activity and mechanism in resisting ultraviolet damage, so as to fill the technological gap in this field and realize the high-value utilization of Porphyra yezoensis resources. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies and realize the high-value utilization of Porphyra yezoensis, the purpose of this invention is to provide an efficient and green extraction and purification method for Porphyra yezoensis polysaccharides and to characterize their structure. The Porphyra yezoensis polysaccharide PSP-Ⅰ provided by this invention has anti-UV photodamage effects and is expected to be applied in anti-photodamage skincare products.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a polysaccharide PSP-Ⅰ from Porphyra yezoensis with anti-ultraviolet damage activity, having a weight-average molecular weight (Mw) of 26.149 kDa and a number-average molecular weight (Mn) of 11.267 kDa, and its monosaccharide composition comprising galactose, glucose and glucuronic acid, with a molar ratio of 96.76:2.31:0.93.

[0008] A method for preparing PSP-I, a polysaccharide from *Porphyra yezoensis* as described above, includes the following steps: S1, raw material pretreatment: drying and pulverizing *Porphyra yezoensis* raw material, extracting with anhydrous ethanol to remove pigment, and drying to obtain depigmented *Porphyra yezoensis* powder; S2, enzymatic extraction: mixing the depigmented *Porphyra yezoensis* powder with a buffer solution of pH 4.5-6.0, adding a complex enzyme for enzymatic hydrolysis; after enzymatic hydrolysis, inactivating the enzyme and centrifuging; collecting the supernatant for alcohol precipitation, drying the precipitate to obtain crude *Porphyra yezoensis* polysaccharide; S3, protein removal and preliminary purification: removing protein from the crude *Porphyra yezoensis* polysaccharide, dialysis, and freeze-drying to obtain pre-purified polysaccharide PSP; S4, anion exchange chromatography purification: subjecting the pre-purified polysaccharide PSP to anion exchange chromatography, collecting the eluent corresponding to the main polysaccharide elution peak obtained by elution with Tris-HCl buffer, i.e., the PSP-0M polysaccharide fraction.

[0009] S5. Gel chromatography purification: The PSP-0M polysaccharide fraction obtained in step S4 is subjected to dextran gel chromatography. The eluent corresponding to the main elution peak is collected. After impurity removal and drying, a purified fraction containing Porphyra polysaccharide PSP-Ⅰ is obtained. The content of polysaccharide PSP-Ⅰ in the purified fraction is ≥80% (w / w).

[0010] Preferably, in step S2, the enzymatic hydrolysis is performed under ultrasound assistance; and / or, the pH of the buffer solution is 5.0; and / or, the complex enzyme comprises cellulase and pectinase.

[0011] Preferably, in step S2, the composite enzyme is composed of cellulase and pectinase, and the ultrasonic conditions are a temperature of 55-65℃ and a power of 170-190W.

[0012] Preferably, in step S2, the composite enzyme is composed of cellulase and pectinase; based on the mass of the depigmented laver powder, the amount of cellulase added is 1.5-2.5%, and the amount of pectinase added is 0.5-1.5%.

[0013] Preferably, in step S3, the protein is removed using the trichloroacetic acid (TCA) method; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-3500 Da.

[0014] Preferably, in step S4, anion exchange chromatography is performed using a DEAE anion exchange column.

[0015] Preferably, in step S5, the dextran gel chromatography uses a Sephadex G-75 gel column and deionized water as the mobile phase for elution.

[0016] More preferably, the preparation method of the above-described round laver polysaccharide PSP-Ⅰ includes: Step 1: Drying and pulverizing round laver, sieving, repeatedly stirring and extracting with anhydrous ethanol, and drying to obtain depigmented round laver powder; Step 2: Taking a certain amount of dried laver powder, adding a certain proportion of citrate-disodium hydrogen phosphate buffer and adjusting the pH to 5.0, adding a complex enzyme (cellulase, pectinase) and enzymatically hydrolyzing under suitable temperature and ultrasonic conditions. Then, inactivating the enzyme by boiling water bath and centrifuging, taking the supernatant and adding anhydrous ethanol for alcohol precipitation, drying the precipitate to obtain crude round laver polysaccharide; Step 3: Removing protein by trichloroacetic acid (TCA) method, then dialyzing, taking the dialysate and freeze-drying to obtain preliminarily purified crude polysaccharide PSP; Step 4: Taking the obtained crude polysaccharide and performing DEAE Sepharose FF anion exchange chromatography using a protein purifier, eluting with Tris-HCl (0M NaCl) solution, dialyzing and freeze-drying the eluent to obtain polysaccharide PSP-0M.

[0017] Step 5: The obtained PSP-0M polysaccharide fraction was subjected to dextran gel chromatography (G-75) using a purification instrument, eluted with deionized water, dialyzed using a dialysis bag, and freeze-dried to obtain a purified fraction containing Poriata polysaccharide PSP-Ⅰ, with a polysaccharide PSP-Ⅰ content ≥80% (w / w) in the purified fraction.

[0018] More preferably, the drying temperature in step 1 is 50-60℃; the drying time is 4-5h; the pulverization and sieving is done through a 40-60 mesh sieve; the repeated stirring and extraction is done 3-5 times, each time for 1-2h; and the drying temperature of the powder is 30-40℃, and the time is 1-2h.

[0019] More preferably, in step 2, the ratio of *Porphyra yezoensis* powder to buffer solution is 1:25-35 (g:mL); the proportion of cellulase added is 1.5-2.5%; the proportion of pectinase added is 0.5-1.5%; the suitable temperature is 55-65℃, and the ultrasonic conditions are 170-190W; the centrifugation is 9000-11000 r / min, and the centrifugation time is 30-40 min; the amount of anhydrous ethanol added is 3-4 times the volume of the supernatant; and the drying of the precipitate is performed by heating at a temperature of 35-55℃ for 1-2 h.

[0020] More preferably, the dialysis in step 3 uses a 3000-3500 Da dialysis bag, and the dialysis time is 48-72 hours; the freeze-drying time is 3-4 days.

[0021] More preferably, when performing DEAE Sepharose FF anion exchange chromatography on the crude polysaccharide described in step 4, the sample concentration is 3-4 mg / mL, the sample volume is 30-40 mL, and Tris-HCl solution is used for elution at a flow rate of 3-5 mL / min.

[0022] More preferably, when performing dextran gel chromatography (G-75) on the polysaccharide described in step 5, the sample concentration is 2-2.5 mg / mL, the sample volume is 8-10 mL, the eluent is deionized water, and the elution rate is 0.5-1 mL / min; the dialysis bag specification is 3000-3500 Da, the dialysis time is 48-72 h, and the freeze-drying time is 3-4 days.

[0023] In terms of preparation technology, this invention constructs a synergistic technology chain of "directional extraction-fine purification," achieving efficient purification, activity preservation, and structural analysis of *Porphyra yezoensis* polysaccharides. This invention employs an ultrasound-assisted enzymatic extraction method, which is mild, highly specific, and effectively disrupts cell walls while maximally preserving the natural active conformation of the polysaccharides. Crucially, this invention does not stop at the crude extract but systematically purifies the obtained crude polysaccharides through fractional purification, sequentially using anion exchange chromatography and dextran gel chromatography. This purification process allows for precise separation based on the charge characteristics and molecular size of the polysaccharides, ultimately yielding purified polysaccharide components with highly homogeneous components, relatively well-defined structures, and concentrated molecular weight distribution. This not only significantly improves the purity and quality of the target product and avoids interference from impurities but also lays a solid foundation for in-depth research on its structure-activity relationship and the development of standardized products, demonstrating significantly superior product controllability and quality compared to degradation mixtures in existing technologies.

[0024] The purified component prepared by the above method contains 84.64 ± 0.52% polysaccharide, 3.72 ± 0.32% protein, and other components are mainly bound water, ash, and trace amounts of inorganic salts.

[0025] The polysaccharide PSP-Ⅰ has a weight-average molecular weight (Mw) of 26.149 kDa, a number-average molecular weight (Mn) of 11.267 kDa, and a polydispersity index (Mw / Mn) of 2.321.

[0026] The polysaccharide PSP-I is mainly composed of galactose, glucose and glucuronic acid, with a molar ratio of galactose: glucose: glucuronic acid = 96.76: 2.31: 0.93.

[0027] The Fourier transform infrared spectrum of the polysaccharide PSP-I shows that it has typical characteristic peaks of polysaccharide structure, and it is mainly a polysaccharide with β-glycosidic bonds.

[0028] Scanning electron microscopy results of the polysaccharide PSP-I show that its microstructure is a porous lamellar structure.

[0029] Another objective of this invention is to evaluate the effect of the aforementioned Porphyra polysaccharide PSP-Ⅰ on anti-ultraviolet damage. Cell model verification showed that Porphyra polysaccharide PSP-Ⅰ exhibited significant anti-ultraviolet damage activity and has potential application in the preparation of anti-photodamage skincare products. Its effects include: (1) The polysaccharide PSP-Ⅰ of this invention can inhibit apoptosis of human immortalized keratinocytes (HaCaT) cells caused by UVB.

[0030] (2) The polysaccharide PSP-I of the present invention can reduce the production of ROS in HaCaT cells caused by UVB and can inhibit cellular oxidative stress caused by photodamage.

[0031] (3) The polysaccharide PSP-I of the present invention can reduce the production of matrix metalloproteinases (MMPs) in HaCaT cells caused by UVB and can inhibit the degradation of cell collagen caused by photodamage.

[0032] This invention provides a product for protecting against UV damage to the skin, comprising the above-mentioned Porphyra polysaccharide PSP-Ⅰ as an active ingredient.

[0033] The PSP-I can be added to products such as cosmetics or skincare products.

[0034] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses an ultrasound-assisted compound enzyme method to extract polysaccharides from laver, which significantly improves the extraction efficiency compared with the traditional hot water extraction method; the compound enzymes (such as cellulase, pectinase, etc.) specifically hydrolyze cell wall components under mild conditions, and combined with ultrasound low-temperature extraction, it is beneficial to maintain the natural molecular structure and biological activity of polysaccharides, and avoid degradation or destruction caused by high temperature or strong chemical treatment; this method reduces the use of acids, alkalis or organic solvents in traditional chemical extraction methods, reduces environmental pollution and subsequent processing costs, and conforms to the concept of green extraction; ultrasound assistance can enhance the mass transfer process, reduce the temperature and time required for enzymatic hydrolysis, the process parameters are easy to control, the reproducibility is good, and it is suitable for large-scale production.

[0035] (2) In the existing technology, research on the extraction of algal polysaccharides mainly focuses on varieties such as *Porphyra yezoensis* and *Porphyra tenera*, while research on the extraction of polysaccharides from the specific species *Pyropia suborbiculata* is still lacking. This invention is the first to use *Pyropia suborbiculata* as the target raw material and systematically develop its polysaccharide extraction process, filling the technological gap in the research and development of active polysaccharides from this specific resource. It opens up a completely new resource library of active substances and provides a reference path for the high-value utilization of *Pyropia suborbiculata*.

[0036] (3) At the activity verification level, this invention, based on high-purity products, has conducted systematic and in-depth research on the anti-photoaging mechanism, providing sufficient biological evidence. Existing technologies focus on elastase inhibition and cell survival rate after ultraviolet irradiation, confirming their basic protective mechanism. This invention, however, utilizes Porphyra polysaccharide in a cell model for a comprehensive evaluation across multiple dimensions and pathways: in addition to cell survival rate, the level of intracellular reactive oxygen species (ROS) was further measured to clarify its antioxidant capacity; the cell apoptosis rate was detected to reveal its role in combating ultraviolet-induced programmed cell death; and the content of key matrix metalloproteinases (MMPs) was measured, directly confirming its ability to inhibit extracellular matrix degradation and prevent the formation of photoaging wrinkles. This series of indicators are interconnected and progressively deepened, not only confirming that the Porphyra polysaccharide of this invention has significant anti-ultraviolet damage activity, but also preliminarily elucidating its molecular mechanism of synergistic action through multiple pathways of "ROS scavenging - apoptosis reduction - matrix protection," laying a solid scientific basis for the application of Porphyra polysaccharide in anti-ultraviolet damage.

[0037] In summary, this invention forms a complete and innovative technical solution, encompassing raw materials (porphyria), extraction methods (ultrasound-assisted compound enzymatic method), purification processes (two-step chromatography), and activity evaluation (multi-index mechanism verification). Compared with existing technologies, this invention not only achieves gentler and more precise control in the preparation method, yielding high-purity active substances with clearer structures, but also achieves a leap from phenomenological observation to mechanism elucidation in efficacy evaluation. This provides ample and reliable data support and material basis for developing high-end, well-defined functional skincare or oral beauty products, demonstrating significant technological progress and application value. Attached Figure Description

[0038] Figure 1. UV scan results of PSP (porphyria var. rubrum) crude polysaccharide.

[0039] Figure 2. Anion exchange chromatography elution curve of crude polysaccharide PSP from Porphyra yezoensis.

[0040] Figure 3. Elution curves of dextran gel chromatography for crude polysaccharides PSP-0M and PSP-0.5M from Porphyra yezoensis.

[0041] Figure 4. Chromatographic analysis results of monosaccharide composition of PSP-Ⅰ from Porphyra yezoensis: (a) Monosaccharide standard solution; (b) PSP-Ⅰ polysaccharide sample.

[0042] Figure 5. FT-IR infrared spectrum of PSP-Ⅰ, a polysaccharide from Porphyra yezoensis.

[0043] Figure 6. Scanning electron microscopy results of PSP-Ⅰ, a polysaccharide from Porphyra yezoensis.

[0044] Figure 7. Cytotoxicity results of PSP-Ⅰ and PSP-Ⅱ polysaccharides from Porphyra yezoensis.

[0045] Figure 8. Results of the repair effects of PSP-Ⅰ and PSP-Ⅱ polysaccharides from Porphyra yezoensis on UVB-damaged cells.

[0046] Figure 9. Effect of PSP-Ⅰ, a polysaccharide from Porphyra yezoensis, on intracellular ROS content in UVB-damaged cells.

[0047] Figure 10. Effect of PSP-Ⅰ, a polysaccharide from Porphyra yezoensis, on the formation of apoptotic bodies in UVB-damaged cells.

[0048] Figure 11. Effect of PSP-Ⅰ on the content of MMPs in UVB-damaged cells. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0050] Example 1: Extraction and Purification of Polysaccharides from Pyropia suborbiculata 1. Pyropia suborbiculata PS-M4 raw material was placed in an oven and dried thoroughly at 55°C until constant weight. It was then pulverized using a pulverizer and passed through a 60-mesh sieve to obtain uniform dried powder. This powder was mixed with anhydrous ethanol at a ratio of 1:3 (g:mL), and extracted with continuous stirring at room temperature for 1 hour each time. This process was repeated 3 times to thoroughly remove fat-soluble pigments and some small molecule impurities. After extraction, the solid material was filtered out and dried again to obtain a light-colored, depigmented dried powder of Pyropia suborbiculata.

[0051] 2. Accurately weigh 5g of the treated dried laver powder and place it in a beaker. Add 150mL of pre-prepared citrate-disodium hydrogen phosphate buffer solution to adjust the pH to 5.0. This pH environment is the optimal condition for subsequent enzymatic hydrolysis. Then, add accurately weighed compound enzymes (containing cellulase and pectinase, with 1.5% cellulase and 1% pectinase) to the system, place it in an ultrasonicator, and carry out the enzymatic hydrolysis reaction at 60℃. Set the ultrasonic power to 180W and the treatment time to 90min.

[0052] 3. After enzymatic hydrolysis, heat the beaker in a boiling water bath for 10 minutes to ensure complete enzyme inactivation. After cooling, centrifuge the solution at 10000 rpm for 35 minutes at 4°C and carefully collect the supernatant. Slowly add 3 volumes of pre-cooled anhydrous ethanol to the supernatant while stirring slowly, and let it stand overnight at 4°C to allow the polysaccharides to fully precipitate. The next day, centrifuge again and collect the precipitate. Dry the precipitate at 37°C for 1 hour to obtain the crude polysaccharide product from *Porphyra yezoensis*. The polysaccharide extraction rate was determined using the phenol-sulfuric acid method, and the extraction rate was 12.342% ± 0.09%.

[0053] Polysaccharide extraction rate = polysaccharide concentration × dilution factor × polysaccharide volume / laver powder mass × 100%.

[0054] 4. Weigh 5g of crude polysaccharide and dissolve it in an appropriate amount of water. Add 20% TCA solution to the solution until the final concentration is 4%, adding it dropwise and mixing thoroughly. Incubate overnight at 4℃. The next day, centrifuge at 1000 rpm for 10 minutes, collect the supernatant, and adjust the pH to 7 with NaOH solution. Dialyze using a 3500 Da dialysis bag for 72 hours. Collect the dialysate and freeze-dry for 3 days to obtain the crude polysaccharide PSP after protein removal. Perform UV scanning at a wavelength of 200-600 nm to detect protein removal. The results are shown in Figure 1. No absorption at 280 nm indicates complete protein removal.

[0055] 5. Take 150 mg of crude polysaccharide PSP and dissolve it in 40 ml of Tris-HCl solution. Perform DEAESepharose FF anion exchange chromatography using a purification instrument. Elute with Tris-HCl solutions containing 0 M, 0.1 M, 0.3 M, 0.5 M, 0.7 M, and 0.9 M NaCl sequentially at a flow rate of 3 ml / min to obtain different polysaccharide fractions. Monitor the elution at 490 nm using the phenol-sulfuric acid method. Based on the yield, dialyze and freeze-dry the 0 M and 0.5 M elution fractions to obtain polysaccharides for further research. Figure 2 shows the elution curves of anion exchange chromatography. It can be observed that when the eluent is 0 or 0.5 mol / L NaCl... When the eluent was NaCl of a certain concentration, a large amount of polysaccharide components from the elution were obtained. These two eluent components were collected and named PSP-0M and PSP-0.5M for subsequent purification experiments. However, when the eluent was NaCl of other concentrations, very few polysaccharide components were separated from the chromatography column. Considering that the amount of polysaccharide used in subsequent experiments was large, these four polysaccharide components were not collected.

[0056] 6. The obtained polysaccharide fractions were subjected to dextran gel chromatography (G-75) using a purification instrument. The loading concentration was 2.5 mg / mL, and the loading volume was 8 mL. Deionized water was used for elution at a flow rate of 1 mL / min. Polysaccharide fractions of different molecular weights were obtained. Figure 3 shows the elution curves of dextran gel chromatography. The figure shows that the elution peak of PSP-0M is very uniform, while the elution peak of PSP-0.5M has some impurity peaks, indicating that the molecular weight of polysaccharides in PSP-0M is more uniform, while the molecular weight in PSP-0.5M is not uniform. Five samples from tubes with the highest peak numbers of the elution peaks of PSP-0M and PSP-0.5M were collected and named PSP-Ⅰ and PSP-Ⅱ, respectively.

[0057] 7. Dialysis was performed using a 3500Da dialysis bag for 72 hours, followed by freeze-drying for 3 days to obtain freeze-dried polysaccharides PSP-Ⅰ and PSP-Ⅱ for subsequent experiments.

[0058] Example 2: Structural Analysis of PSP-I, a Polysaccharide from Porphyra yezoensis 1. Chemical Composition Analysis of the Polysaccharide The polysaccharide content was determined using the phenol-sulfuric acid method, with glucose as the standard. Protein content was determined using the BCA method. Chemical analysis results showed that the polysaccharide content of PSP-I was 84.64 ± 0.52%, the protein content was 3.72 ± 0.32%, and other components mainly consisted of bound water, ash, and trace amounts of inorganic salts.

[0059] 2. Molecular weight determination and monosaccharide composition determination: PSP-I samples were dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filtered through a 0.45 μm filter before analysis. The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an OptilabT-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt technology, CA, USA). Specific column and elution conditions were as follows: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns were used in series. The column temperature was 45℃. After filtration through a 0.45 μm membrane filter, the injection volume was 100 μL. The mobile phase was (0.02% NaN3, 0.1M NaNO3), and the flow rate was 0.6 mL / min. Statistical analysis of the data using ASTRA 6.1 showed that the weight-average molecular weight (Mw) of PSP-Ⅰ was 26.149 kDa, the number-average molecular weight (Mn) was 11.267 kDa, and the polydispersity index (Mw / Mn) was 2.321, indicating that PSP-Ⅰ is a typical polydisperse system of natural polysaccharides with relatively uniform molecular weight.

[0060] Take a clean chromatographic vial, weigh an appropriate amount of polysaccharide sample, add 1 ml of 2M trifluoroacetic acid (TFA) solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with 99.99% methanol, and dry again. Repeat the methanol washing 2-3 times to completely remove TFA. Dissolve in sterile water, filter through a 0.22 μm membrane filter, and transfer to a chromatographic vial for analysis. Analyze by high performance anion exchange chromatography (HPAEC) on a Thermo ICS 5000+ chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Dionex™ CarboPac™ PA-20 anion exchange column (3 × 150 mm, 10 μm) and a pulsed amperometric detector (PAD). 5 μl of the processed sample was injected into the column. The mobile phase consisted of: mobile phase A (H2O), mobile phase B (0.1M NaOH), and mobile phase C (0.1M NaOH, 0.2M NaAc). The flow rate was 0.5 mL / min, and the column temperature was 30 °C. The gradient elution program was as follows: 0 min A / B / C (95:5:0, v / v), 26 min A / B / C (85:5:10, v / v), 42 min A / B / C (85:5:10, v / v), 42.1 min A / B / C (60:0:40, v / v), 52 min A / B / C (60:40:0, v / v), 52.1 min A / B / C (95:5:0, v / v), 60 min A / B / C (95:5:0, v / v). Phase / C phase (95:5:0, v / v). Referring to Figure 4a and Figure 4b, PSP-I is composed of galactose, glucose and glucuronic acid, with a molar ratio of galactose:glucose:glucuronic acid = 96.76:2.31:0.93.

[0061] 3. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis: FT-IR spectra of PSP-I were recorded using a Thermo Nicolet iS10 (Thermo Inc., Waltham, 297 MA, USA) equipped with a deuterated triglycine sulfate detector. All samples were analyzed within the range of 600–4000 cm⁻¹. -1 Tests were conducted within the wavenumber range, with a resolution of 4 cm. -1 A total of 32 scans were performed. The infrared spectrum of PSP-I is shown in Figure 5, and bands similar to those of macroalgal polysaccharides can be observed. PSP-I shows a band at 3368 cm⁻¹. -1 The broad and strong absorption band at 2926 cm⁻¹ can be attributed to the O–H stretching vibrations of numerous hydroxyl groups within the polysaccharide molecule;-1 This is a C–H stretching vibration. 1644 cm -1 The absorption peak at this point is mainly related to the H–O–H bending vibration of bound water, and may also be related to the COO2 of carboxylate. - Asymmetric stretching vibrations overlap; 1411 cm -1 Can be attributed to COO - Symmetric stretching vibrations, consistent with high uronic acid content. 1217, 1150, and 1027 cm⁻¹ -1 These are characteristic peaks in the polysaccharide fingerprint region, mainly corresponding to C–O–C (glycosidic bonds) and C–O stretching vibrations, with the 1217 cm⁻¹ peak being the most prominent. -1 The presence of S=O stretching vibrations in sulfate esters may also indicate that PSP-I possesses a typical polysaccharide backbone structure. Furthermore, 894 cm⁻¹ -1 The characteristic absorption peaks are related to the configuration of β-glycosidic bonds, indicating that PSP-I may be mainly composed of β-glycosidic bonds.

[0062] 4. Scanning Electron Microscopy (SEM) Analysis: PSP-I was fixed, coated with gold, and observed using a Hitachi SU5000 thermal field emission scanning electron microscope (SEM) at an accelerating potential of 5.0 kV. Images were then recorded at different magnifications. SEM analysis further clarified the microstructure of PSP-I. Figure 6 mainly shows the morphology of PSP-I at different magnifications. It can be observed that it exhibits a typical biopolymer aggregation morphology. Its microstructure at different magnifications shows a relatively typical porous lamellar structure with an uneven surface, possibly due to fiber breakage and reorganization caused by ultrasonic treatment. At lower magnifications (×200, ×400), the polysaccharide particles were observed to be irregularly aggregated, with a relatively loose overall structure, showing a large number of broken thin lamellar sheets. This structure is highly consistent with the physical exfoliation caused by ultrasonic treatment. At higher magnifications (×600, ×1500), obvious wrinkles, broken edges, and rough flakes are visible on the surface of the polysaccharide, which also exhibits a porous structure. This may be related to the degradation and loosening of the polysaccharide chains by ultrasonic cavitation and enzymatic hydrolysis, allowing the polysaccharide to be released more completely from the cell without forming highly dense aggregates, which is beneficial to improving its hydration and solubility.

[0063] Example 3: Anti-UV damage activity of polysaccharides from Porphyra yezoensis 1. HaCaT cell culture Human immortalized keratinocytes HaCaT cells were cultured in DMEM medium containing 10% (v / v) serum (FBS) and 1% (v / v) penicillin and streptomycin (antibiotics) at 37°C in a 5% CO2 incubator.

[0064] 2. Determination of polysaccharide cytotoxicity: When HaCaT cells reached a confluence of 80%-90%, the cells were injected with 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [insert density here] in 96-well plates, with 100 μL of PBS added to each well around the perimeter to prevent evaporation. Cell-free wells were reserved in the plates (to account for the influence of DMEM itself on absorbance). Cells were cultured in a cell culture incubator for 24 hours until adherence. Cytotoxicity evaluation of polysaccharides and establishment of a UV damage model were then performed. PSP-I, PSP-II, and HA30 (sodium hyaluronate Mw300,000) were each prepared into 5 mg / mL solutions using complete culture medium, diluted appropriately with culture medium, and added to 96-well plates. The final concentrations of each sample after addition were 1000, 800, 400, 200, 100, and 0 μg / mL, with three replicates for each concentration. A cell control group was also included. Cells were cultured for another 24 hours, and cell viability was assessed using the CCK-8 assay. The viability of positive control cells was calculated as 100%. Figure 7 shows the cytotoxicity results of different concentrations of polysaccharides. The results show that PSP-II has certain cytotoxicity; PSP-I, a polysaccharide from Porphyra yezoensis, is non-toxic in the concentration range of 0 μg / mL to 800 μg / mL, indicating that it has a high safety factor as a raw material for skin care products. Therefore, 200, 400, and 800 μg / mL were set as the low, medium, and high drug concentrations for subsequent cell experiments.

[0065] 3. Study on the repair effect of polysaccharides on UVB-damaged cells: When HaCaT cells grew to a confluence of 80%-90%, the cells were injected with 1×10⁻⁶ polysaccharides. 4Cells were seeded at a density of [insert density here] in 96-well plates. 100 μL of PBS was added to each well around the perimeter to prevent evaporation. The plates were incubated at 37 °C with 5% CO2 for 12 h until cell adhesion. Appropriate concentrations of polysaccharide were added to the 96-well plates to final concentrations of 800, 400, and 200 μg / mL. The positive control group received HA30 at a final concentration of 400 μg / mL. After 6 h of polysaccharide incubation, UV damage was applied. Positive control, control, model, and experimental groups were established. Cells in the model group, positive control, and experimental groups were subjected to 60 s of UVB damage, while the control group received no damage treatment. After damage treatment, different concentrations of polysaccharide were added again for 24 h of incubation. Each group was in triplicate. Cell viability was measured using cck-8 assay, with the viability of positive control cells calculated as 100%. The results are shown in Figure 8. UVB irradiation induced photoaging and cell death in HaCaT cells. The cck-8 assay results showed that UVB irradiation significantly reduced HaCaT cell viability (70.94 ± 1.28%) compared to the control group. However, intervention with PSP-I polysaccharide significantly reversed these conditions, and the cell viability increased with increasing concentration, reaching 93.81±1.23% at 800 μg / mL, close to HA30's 94.59±0.65%. As shown in the figure, the repair effect of PSP-II polysaccharide was significantly weaker than that of PSP-I, with a maximum cell viability of 83.51±4.45%, and a slight decrease in cell viability with increasing concentration. Considering the repair effect and the low purification yield of PSP-II, future research will focus on PSP-I.

[0066] 4. Effects of polysaccharides on intracellular ROS production caused by UVB damage: When HaCaT cells reached a confluence of 80%-90%, the cells were injected with polysaccharides at a rate of 3 × 10⁻⁶ cells / year. 6 Cells were seeded at a density of 1 cell / well in 6-well plates and incubated at 37°C with 5% CO2 for 12 h until cell adhesion. Subsequent treatment was the same as the incubation and injury steps in section 4. After injury, the cells were incubated for 24 h, the cell supernatant was discarded, and the cells were washed once with phosphate-buffered saline (PBS). A 1000-fold diluted DCFH-DA fluorescent probe was added to each well, and the cells were incubated for 20 min. The probe solution was discarded, and the cells were washed three times with PBS to remove unbound probes. The fluorescence intensity was assessed using a fluorescence microscope. Intracellular ROS levels were determined by hydrolyzing the fluorescent probe DCFH-DA into DCFH, which then binds to intracellular ROS. Cells produced more ROS after UVB irradiation, and the addition of PSP-I significantly reduced intracellular ROS (Figure 9) in a concentration-dependent manner. Under high concentrations of polysaccharide (800 μg / mL), the effect of reducing ROS levels was close to that of the positive control. The results showed that PSP-I could effectively reduce the production of ROS in HaCaT cells, and it could alleviate UVB damage by regulating oxidative stress.

[0067] 5. Effects of Polysaccharides on Apoptosis in Cells Damaged by UVB Infection. Cell plating, polysaccharide incubation, and UV damage treatment were performed as described in section 5. After 24 hours of culture following injury, the cell supernatant was discarded, and the cells were washed once with PBS. 10 μg / mL Hoechst 33342 fluorescent dye was added to each well, and the cells were incubated in the dark for 15 minutes. The cells were then washed three times with PBS to remove unbound dye, and the formation of apoptotic bodies was observed using a fluorescence microscope. As shown in Figure 10, compared to the control group, the model group showed a significant increase in induced apoptotic bodies (manifested as enhanced blue fluorescence). However, PSP-I significantly reduced the formation of intracellular apoptotic bodies, thereby inhibiting apoptosis. Therefore, PSP-I alleviates UVB damage by reducing apoptosis.

[0068] 6. Effects of Polysaccharides on Intracellular MMP Production in UVB-Induced Cells. Cell plating, polysaccharide incubation, and UV damage treatment were performed as in step 4. After 24 hours of culture following damage, the cell culture supernatant was centrifuged at 3000 rpm for 20 minutes at 4°C to remove impurities and dead cells. The levels of MMP-1, MMP-3, and MMP-9 were measured using a commercially available ELISA kit. As shown in Figure 11, UVB irradiation significantly increased the MMP levels in HaCaT cells. However, PSP-I significantly reduced the expression of MMP-1, MMP-3, and MMP-9 in UVB-irradiated HaCaT cells in a dose-dependent manner. These results indicate that PSP-I protects HaCaT cells from collagen degradation stimulated by UVB radiation by inhibiting MMP expression.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polysaccharide PSP-Ⅰ from Porphyra yezoensis with anti-UV damage activity, characterized in that, Its weight-average molecular weight is 26.149 kDa, and its number-average molecular weight is 11.267 kDa. Its monosaccharide composition includes galactose, glucose and glucuronic acid, and the molar ratio of the three is 96.76:2.31:0.

93.

2. A method for preparing the Porphyra polysaccharide PSP-I as described in claim 1, characterized in that, Includes the following steps: S1. Raw material pretreatment: Dry and pulverize the raw material of *Porphyra yezoensis*, extract with anhydrous ethanol to remove pigment, and dry again to obtain depigmented *Porphyra yezoensis* powder; S2. Enzymatic extraction: Mix the depigmented *Porphyra yezoensis* powder with a buffer solution of pH 4.5-6.0, add a compound enzyme for enzymatic hydrolysis; after enzymatic hydrolysis, inactivate the enzyme and centrifuge; collect the supernatant for alcohol precipitation, dry the precipitate to obtain crude *Porphyra yezoensis* polysaccharide; S3. Protein removal and preliminary purification: Remove the protein from the crude *Porphyra yezoensis* polysaccharide, dialyze and freeze-dry to obtain preliminarily purified polysaccharide PSP; S4. Anion exchange... Ion exchange chromatography purification: The preliminarily purified polysaccharide PSP is subjected to anion exchange chromatography, and the eluent corresponding to the main polysaccharide elution peak obtained by elution with Tris-HCl buffer is collected, which is the PSP-OM polysaccharide fraction; S5, gel chromatography purification: The PSP-OM polysaccharide fraction obtained in step S4 is subjected to dextran gel chromatography, and the eluent corresponding to the main elution peak is collected. After impurity removal and drying, a purified fraction containing Poriata polysaccharide PSP-Ⅰ is obtained. The content of polysaccharide PSP-Ⅰ in the purified fraction is ≥80% (w / w).

3. The method according to claim 2, characterized in that, In step S2, the enzymatic hydrolysis is performed under ultrasound assistance; and / or, the pH of the buffer solution is 5.0; and / or, the complex enzyme comprises cellulase and pectinase.

4. The method according to claim 3, characterized in that, In step S2, the composite enzyme is composed of cellulase and pectinase, and the ultrasonic conditions are a temperature of 55-65℃ and a power of 170-190W.

5. The method according to claim 3, characterized in that, In step S2, the composite enzyme is composed of cellulase and pectinase; based on the mass of the depigmented dried laver powder, the amount of cellulase added is 1.5-2.5%, and the amount of pectinase added is 0.5-1.5%.

6. The method according to claim 2, characterized in that, In step S3, proteins are removed using the trichloroacetic acid method; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-3500 Da.

7. The method according to claim 2, characterized in that, In step S4, anion exchange chromatography is performed using a DEAE anion exchange column.

8. The method according to claim 2, characterized in that, In step S5, the dextran gel chromatography is performed using a Sephadex G-75 gel column with deionized water as the mobile phase for elution.

9. A product for protecting against UV damage to the skin, characterized in that, It contains PSP-Ⅰ, the polysaccharide from Porphyra yezoensis as described in claim 1, as an active ingredient.

10. The product according to claim 9, characterized in that, The product in question is a cosmetic or skincare product.

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