Porphyra haitanensis polysaccharide PPHP3 with blood fat reducing activity as well as preparation method and application thereof

Abstract

The invention discloses porphyra haitanensis polysaccharide PPHP3 with blood fat reducing activity as well as a preparation method and application of the porphyra haitanensis polysaccharide PPHP3. The weight-average molecular weight of the polysaccharide PPHP3 is 25.208 KDa, monosaccharide of the polysaccharide PPHP3 is composed of galactose, glucose and glucuronic acid, and the molar ratio of the galactose to the glucose to the glucuronic acid is 98.3: 0.46: 1.24. The preparation method comprises the following steps: decoloring and degreasing porphyra haitanensis, carrying out ultrasonic-assisted compound enzyme synergistic extraction, and carrying out alcohol precipitation, deproteinization, dialysis and freeze-drying to obtain crude polysaccharide; and then carrying out two-step fine purification through an anion exchange column and a sephadex filter column in sequence to obtain the purified component PPHP3. In-vitro cell experiments show that PPHP3 has definite blood fat reducing activity. The polysaccharide component is high in purity, clear in structure and remarkable in activity, and a material basis is provided for application of the polysaccharide component in development of blood fat reducing functional products.

Description

Technical Field

[0001] This invention belongs to the field of marine bioactive substance extraction and purification technology, specifically relating to a method for extracting and purifying polysaccharides from *Porphyra yezoensis* and its application in the intervention of hyperlipidemia. This invention provides a highly efficient extraction process combining ultrasound-assisted extraction and compound enzymatic hydrolysis, and utilizes column chromatography to obtain homogeneous polysaccharide components. This method aims to improve the yield and purity of polysaccharides. The obtained product, verified by cell models, exhibits significant lipid metabolism-regulating activity. Background Technology

[0002] Since the beginning of the 21st century, changes in global dietary structures and lifestyles have exacerbated lipid metabolism disorders. Hyperlipidemia, as its core manifestation, is a major risk factor for cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, and stroke, seriously endangering human health. Currently, mainstream lipid-lowering chemical drugs may cause adverse reactions such as liver damage and muscle toxicity with long-term use. Therefore, finding safe and effective lipid-lowering functional components from natural resources has become an important topic in the fields of food science and preventive medicine.

[0003] Laver ( Pyropia haitanensisRich in nutrients, traditional Chinese medicine records its effects of resolving phlegm, softening hard masses, clearing heat, and promoting diuresis. Modern research shows that the polysaccharides in laver are one of its key bioactive substances, possessing various physiological functions such as regulating immunity, anti-tumor activity, anti-oxidation, and regulating lipid metabolism, showing great potential in the development of functional foods and dietary supplements. However, the industrial application of *Porphyra yezoensis* polysaccharides still faces bottlenecks: First, although the traditional hot water extraction method (such as CN109232762A) is simple to operate, it suffers from problems such as high extraction temperature, low efficiency, long time, and high energy consumption. High-temperature and long-term treatment can easily lead to structural damage such as polysaccharide molecular chain breakage and sulfate group removal, affecting its biological activity. At the same time, hot water has limited ability to break down the dense algal cell walls composed of cellulose, hemicellulose, and pectin, making it difficult to fully release intracellular active polysaccharides, resulting in generally low extraction rates and restricting the efficient utilization of raw materials. Second, conventional single enzymatic hydrolysis methods have limited ability to break down the dense cell walls of seaweed (containing cellulose, pectin, etc.), making it difficult to achieve efficient and complete dissolution of active polysaccharide components, resulting in insufficient product yield and efficacy stability. Third, in terms of the correlation between product components and activity, existing technologies are mostly focused on the preparation of crude polysaccharides or the application of simple mixed components. For example, CN109232762A mainly focuses on the beneficial properties of polysaccharides, and the obtained product is still a crude extract with complex components; while CN115505049A discloses a mixture of Porphyra polysaccharides composed of sulfated galactan (PHP1) and sulfated glucose galactan (PHP2) in a specific ratio, but its extraction process is still the conventional hot water method. The extraction efficiency has not been systematically optimized, nor has the key active components been separated and purified. The disclosed product is still a mixture of Porphyra polysaccharides with lipid-lowering activity.

[0004] Therefore, existing technologies suffer from problems such as low cell wall breakdown efficiency, unsatisfactory yield of active polysaccharides, loss of activity, complex product composition, and unclear activity indicators. They fail to provide a method for efficiently and controllably preparing high-purity, structurally defined, and activity-concentrated polysaccharide components from Porphyra yezoensis, which severely limits in-depth research on their structure-activity relationship and the development of high-end products based on clearly defined active ingredients. Summary of the Invention

[0005] To address the shortcomings of the prior art, the technical problem to be solved by the present invention is reflected in the following aspects: 1. Low extraction efficiency and loss of activity: To address the problems of incomplete cell wall breakdown, low polysaccharide extraction rate, and damage to the natural active structure of polysaccharides caused by high temperature or insufficient enzymatic hydrolysis in traditional hot water extraction and single enzymatic hydrolysis methods, a novel extraction process is provided that can efficiently and gently release active polysaccharides from Porphyra yezoensis.

[0006] 2. Problems of complex product composition and unclear activity indicators: To overcome the limitations of existing technologies that mostly stop at obtaining crude polysaccharides or mixtures in a fixed proportion, and cannot provide single polysaccharide components with uniform structure and high purity, we will develop an integrated process that can separate, purify and obtain polysaccharide components with clear and uniform structure from Porphyra yezoensis.

[0007] This invention offers a novel solution to the problems of low cell wall disruption efficiency and unsatisfactory yield of active polysaccharides in existing extraction technologies. For the first time, we synergistically combine ultrasonic cavitation disruption with a composite enzymatic hydrolysis technique (pectinase and cellulase), and systematically optimize key parameters such as ultrasonic temperature, power, and the ratio of the two enzymes using response surface methodology. This method can efficiently and gently disrupt the cell walls of *Porphyra yezoensis*, significantly improving the extraction rate of active polysaccharides and the uniformity of subsequent products. Furthermore, the method utilizes an anion exchange column and dextran... sugar Two-step gel column purification yielded purified polysaccharide PPHP3 with a concentrated molecular weight distribution. Cell model validation demonstrated that this component possesses clear in vitro lipid-lowering activity, providing a material basis and technical support for the development of marine-derived functional products or drugs with lipid metabolism regulation functions.

[0008] To achieve the above objectives, the present invention provides the following technical solution: PPHP3, a polysaccharide from Porphyra yezoensis with lipid-lowering activity, has a weight-average molecular weight of 25.208 kDa. Its monosaccharide composition consists of galactose, glucose, and glucuronic acid, with a molar ratio of 98.3:0.46:1.24.

[0009] A method for preparing the polysaccharide PPHP3 from *Porphyra yezoensis* includes the following steps: (1) Raw material pretreatment: After washing, drying and crushing the laver, the pigments and lipids were removed by reflux with anhydrous ethanol, and the laver powder was obtained after drying. (2) Ultrasonic-compound enzyme extraction and alcohol precipitation: Porphyra powder was mixed with water, a compound enzyme was added, and extraction was carried out under ultrasonic conditions. After enzyme inactivation and centrifugation, the supernatant was collected. Ethanol was added to the supernatant for alcohol precipitation. The precipitate was collected and dried to obtain crude polysaccharide from Porphyra. (3) Deproteinization and preliminary purification: The crude polysaccharide of Porphyra yezoensis was dissolved, the protein was removed, and after neutralization, dialysis and freeze-drying, the deproteinized crude polysaccharide of Porphyra yezoensis was obtained; (4) Ion exchange column chromatography purification: The crude polysaccharide of Porphyra yezoensis after deproteinization was dissolved in Tris-HCl buffer and subjected to anion exchange column chromatography. Gradient elution was performed sequentially using Tris-HCl buffer containing different concentrations of NaCl. The eluted fractions were collected, dialyzed, and lyophilized to obtain the initial pure polysaccharide PHP3. (5) Fine purification by gel filtration: The primary purified polysaccharide PHP3 was dissolved and purified by dextran. sugarGel filtration column chromatography was performed, followed by elution with water. The sugar fraction corresponding to the main peak was collected, dialyzed, and lyophilized to obtain a purified fraction containing the pure polysaccharide PPHP3 from Porphyra yezoensis. The content of polysaccharide PPHP3 in the purified fraction was ≥80% (w / w).

[0010] Preferably, in step (1), the drying temperature is 60-80℃, the pulverization is passed through a 20-60 mesh sieve, and the conditions for decolorization and degreasing by reflux of anhydrous ethanol are a material-to-liquid ratio of 1:15-1:25 (g / mL), a reflux temperature of 85-95℃, and repeated 2-4 times; the drying is performed at 40-50℃.

[0011] According to a specific embodiment of the present invention, in step (2), the composite enzyme is composed of cellulase and pectinase. Preferably, in step (2), the conditions for the ultrasonic-composite enzyme extraction are: material-liquid ratio 1:20-1:40 (g / mL), cellulase addition amount is 0.5-3.0% of the algal powder mass, pectinase addition amount is 0.5-3.0% of the algal powder mass, ultrasonic power 150-250W, ultrasonic temperature 55-75℃, extraction time 60-120 minutes; the alcohol precipitation is performed by adding 2-4 times the volume of anhydrous ethanol and standing at 0-10℃ for 12-36 hours; the centrifugation conditions are 0-10℃, centrifugation at 8000-12000g for 20-40 minutes; the drying temperature is 40-50℃, and the drying time is 20-40 minutes.

[0012] According to a specific embodiment of the present invention, in step (3), protein is removed using the trichloroacetic acid method. Preferably, in step (3), the trichloroacetic acid deproteinization involves adding 15-25% (w / v) trichloroacetic acid solution to a final concentration of 3-5% (v / v); the neutralization involves adjusting the pH to 6.5-7.5 using 1-3M NaOH solution; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-5000 Da and dialyzes in deionized water for 48-96 hours; and the lyophilization time is 48-96 hours.

[0013] According to a specific embodiment of the present invention, in step (4), the anion exchange column is a DEAE anion exchange column, and gradient elution is performed sequentially using Tris-HCl buffer solutions containing different concentrations of NaCl, namely 0M, 0.3M, and 0.5M. Preferably, in step (4), the DEAE anion exchange column is a DEAE... Sepharose FastFlow column; the Tris-HCl buffer concentration is 10-30 mM, pH 7.5-8.5; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-5000 Da.

[0014] According to a specific embodiment of the present invention, in step (5), the dextran gel filter column is a Sephadex G-75 gel column. Preferably, in step (5), the elution conditions of the Sephadex G-75 gel column are: deionized water as the mobile phase, flow rate 0.1 mL / min; the fraction collected is within the main peak range; and the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-5000 Da.

[0015] More preferably, the preparation method of the above-described PPHP3 polysaccharide from Porphyra yezoensis includes: Step 1: Wash and dry the laver, crush it, sieve it, add anhydrous ethanol and reflux to remove pigments and lipids, and then dry it to obtain laver powder. Step 2: Extract the dried Porphyra yezoensis powder using an ultrasonic-assisted compound enzyme method to obtain Porphyra yezoensis extract; precipitate the extract with alcohol overnight, centrifuge, and dry the precipitate to obtain crude Porphyra yezoensis polysaccharide; Step 3: Dissolve the crude polysaccharide of Porphyra yezoensis and remove the protein using the TCA method to obtain a deproteinized solution. Neutralize the deproteinized solution to obtain a neutralized solution. Dialyze the neutralized solution to obtain a dialysate. Freeze-dry the dialysate to obtain deproteinized crude polysaccharide of Porphyra yezoensis. Step 4: Use DEAE to process the crude polysaccharide from the seaweed. Sepharose FastFlow column chromatography purification was performed by gradient elution with buffer, 0.3M NaCl elution buffer, and 0.5M NaCl elution buffer. The eluents were collected separately. The eluent obtained by elution with 0.5M NaCl buffer was dialyzed and then freeze-dried to obtain the primary pure product of Porphyra yezoensis, named PHP3. Step 5: Dissolve the primary purified Porphyra yezoensis product in PHP3, purify it using a Sephadex-G75 column, elute with water, detect the sugar peak and collect the sugar-containing fraction to obtain the purified component.

[0016] Preferably, step 1 includes the following steps: drying the laver at 70°C, pulverizing it, passing it through a 40-mesh sieve, adding anhydrous ethanol at a ratio of 1:20 g / ml and refluxing it three times to remove pigments and lipids, and then drying it at 45°C to obtain degreased and decolorized laver powder.

[0017] Preferably, step 2 includes the following steps: In step 2.1, the ultrasound-assisted compound enzyme method specifically refers to the volume-to-mass ratio of dried laver powder to enzyme solution being 1g:30mL, the amount of cellulase added being 1.480% of the laver powder mass, the amount of pectinase added being 1.47% of the laver powder mass, the ultrasound power being 180W, and the ultrasound temperature being 65.900℃. The overnight alcohol precipitation mentioned in step 2.2 refers to adding 3 times the volume of anhydrous ethanol to the extract and placing it at 4°C for 24 hours; The centrifugation mentioned in step 2.3 refers to centrifugation at 4°C with a centrifugal force of 10000g for 35 minutes. The drying process described in step 2.4 refers to drying at 45°C for 30 minutes.

[0018] Preferably, step 3 includes the following steps: Step 3.1, specifically the TCA deproteinization method, refers to adding 20% ​​TCA solution until the final TCA concentration in the solution is 4%. The neutralization mentioned in step 3.2 refers to adjusting the pH of the deproteinizing solution to 7 using 2M NaOH; The dialysis described in step 3.3 refers to dialysis with the neutralizing solution for 72 hours using a 3500Da dialysis bag; The freeze-drying mentioned in step 3.4 refers to freeze-drying the dialysate for 72 hours; Preferably, step 4 includes the following steps: Step 4.1 describes the use of DEAE Sepharose FastFlow column chromatography purification refers to the purification of DEAE... The Sepharose FastFlow column was connected to the Taidu ACHROM Pure system, with a pressure of 0.3 MPa and a flow rate of 1 mL / min. One tube was collected for every 10 mL. The buffer solution mentioned in step 4.2 is a 20 mM Tris-HCl solution; The 0.3M NaCl elution buffer solution and 0.5M NaCl elution buffer solution mentioned in step 4.3 refer to 20mM Tris-HCl containing 0.3M NaCl / 0.5M NaCl. Step 4.4 refers to the process of collecting the eluent, dialyzing with a 3500 Da dialysis bag for 72 hours, and then lyophilizing for 72 hours. Preferably, step 5 includes the following steps: Step 5.1, which describes dissolving the initial purified PHP3 of laver, refers to dissolving PHP3 in deionized water and filtering it through a 0.45μm filter membrane. Step 5.2, the Sephadex-G75 column purification, refers to connecting the Sephadex-G75 column to the Taidu ACHROM Pure system, setting the pressure to 0.3 MPa and the flow rate to 0.1 mL / min; Step 5.3, which refers to detecting the sugar peak, means using the phenol-sulfuric acid method to detect the sugar content of each collected fraction. The purified component contained 84.34 ± 2.25% polysaccharide PPHP3, 0.89 ± 0.05% protein, and other components mainly consisted of bound water, ash, and trace amounts of inorganic salts.

[0019] The weight-average molecular weight of the purified polysaccharide PPHP3 is 25.208 kDa.

[0020] The purified polysaccharide PPHP3 is mainly composed of galactose (Gal), glucose (Glc), and glucuronic acid (Glc-UA), with a molar ratio of Gal : Glc : Glc-UA = 98.3 : 0.46 : 1.24.

[0021] Another objective of this invention is to evaluate the effects of the aforementioned *Porphyra yezoensis* polysaccharide PPHP3 on regulating lipid metabolism and / or lowering blood lipids. The *Porphyra yezoensis* polysaccharide PPHP3 prepared using this integrated technology, as verified by cell models, exhibits significant activity in regulating lipid accumulation in hepatocytes, providing a superior technical pathway for developing high-value-added marine health products with a clear lipid-lowering function.

[0022] PPHP3, a polysaccharide from Porphyra yezoensis, has the following effects: PPHP3 can significantly inhibit oleic acid-induced lipid accumulation in hepatocytes; PPHP3 can effectively reduce intracellular levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). PPHP3 can increase intracellular high-density lipoprotein cholesterol (HDL-C) levels, thereby exerting a comprehensive effect in regulating lipid metabolism.

[0023] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1) This invention employs a synergistic treatment of ultrasound and compound enzymes (cellulase and pectinase) during the extraction process. The cavitation effect of ultrasound can instantly disrupt the algal cell wall structure, greatly increasing the contact area and efficiency between the enzyme and the substrate, while the compound enzymes can precisely degrade the cellulose and pectin networks in the cell wall. The synergistic effect of these two methods significantly accelerates the release rate of polysaccharides, improves extraction efficiency, and avoids the structural damage to polysaccharides that may occur during traditional high-temperature, long-term extraction.

[0024] 2) In this invention, the raw material is pretreated with ethanol by reflux decolorization and defatting before extraction. This step effectively removes fat-soluble pigments and impurities, reduces the burden on subsequent purification processes, and avoids co-precipitation of impurities during polysaccharide alcohol precipitation, laying a good foundation for obtaining polysaccharide products with light color and high purity.

[0025] 3) This invention employs a two-step purification strategy combining DEAE anion exchange chromatography and Sephadex gel filtration chromatography. First, ion exchange is used for coarse separation based on charge differences, followed by gel chromatography for finer separation based on molecular size. This combined method efficiently separates and removes heteropolysaccharides and residual impurities, ultimately yielding a high-purity polysaccharide component, PPHP3, with a concentrated molecular weight distribution and uniform structure, thus overcoming the selectivity limitations of conventional single purification methods.

[0026] 4) The purified polysaccharide component PPHP3 obtained in this invention has been confirmed by cell experiments to have clear lipid-lowering biological activity. This component can effectively reduce lipid accumulation in hepatocytes and regulate triglyceride and cholesterol metabolism, providing direct and reliable experimental evidence for its application as a natural and safe lipid-lowering functional factor in pharmaceuticals, health foods, or special dietary foods. Attached Figure Description

[0027] Figure 1 This is a diagram showing the monosaccharide composition of PPHP3, a polysaccharide from Porphyra yezoensis prepared in Example 4; Figure 2 This is a graph showing the cytotoxicity of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 against HepG2 cells; Figure 3 The figure shows the effect of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 on lipid droplet distribution in OA-induced HepG2 cells. Figure 4 The effect of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 on intracellular TG induced by OA in HepG2 cells is shown in the figure. Figure 5 The effect of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 on intracellular TC induced by OA in HepG2 cells is shown in the figure. Figure 6 The effect of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 on OA-induced HDL-C in HepG2 cells is shown in the figure. Figure 7 The figure shows the effect of PPHP3 polysaccharide from Porphyra yezoensis prepared in Example 4 on OA-induced intracellular LDL-C in HepG2 cells. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] Example 1: Extraction and crude purification of polysaccharides from heat-resistant Porphyra yezoensis SW-81 (1) Heat-resistant laver ( Pyropia haitanensisAfter washing, the SW-81 strain was dried in a 70℃ forced-air drying oven until constant weight. It was then pulverized using a pulverizer and passed through a 40-mesh sieve. The heat-resistant Pyropia haitanensis SW-81 is described in the literature (Ding H, Fei Q, Zhang P, et al. Isolation and characterization of a heat-resistant strain with high yield of Pyropia haitanensis induced by ultraviolet ray[J]. Aquaculture, 2020, 521: 735050), which is a high-yielding heat-resistant strain (SW-81) isolated from a wild species (WT-10) after ultraviolet irradiation and heat stress treatment.

[0030] (2) Weigh 100g of the above-screened laver and add 2000 mL of anhydrous ethanol at a material-to-liquid ratio of 1:20 (g / mL). Reflux and extract in a 90℃ water bath. Repeat this process three times until the ethanol extract is nearly colorless to fully remove pigments and lipids. Then dry at 45℃ to obtain defatted and decolorized laver powder.

[0031] (3) Weigh 20g of defatted and decolorized laver powder, add 600 mL of deionized water at a material-to-liquid ratio of 1:30 (g / mL), and add cellulase (1.480% of the laver powder mass) and pectinase (1.47% of the laver powder mass). Place the mixture in an ultrasonic extraction device and extract for 90 minutes at 65.9℃ and 180W power.

[0032] (4) After extraction, the mixture was boiled in a water bath for 10 minutes to inactivate the enzyme, cooled and then centrifuged at 4°C and 10,000 g for 35 minutes to collect the supernatant.

[0033] (5) Add 3 times the volume of anhydrous ethanol to the supernatant, mix well, and place in a refrigerator at 4°C for 24 hours to precipitate alcohol. Then centrifuge under the same conditions and collect the precipitate. Dry the precipitate in an oven at 45°C for 30 minutes to obtain crude polysaccharide from Porphyra yezoensis.

[0034] Example 2: Deproteinization of crude polysaccharide from Porphyra yezoensis (1) Dissolve the crude polysaccharide of Porphyra yezoensis obtained in Example 1 in an appropriate amount of deionized water to prepare a solution with a concentration of 20 mg / mL.

[0035] (2) While stirring, slowly add 20% (w / v) trichloroacetic acid (TCA) solution to bring the final concentration of TCA in the mixture to 4% (v / v). Continue stirring for 20 minutes to allow the protein to denature and precipitate fully.

[0036] (3) Centrifuge the above mixture at 4°C and 10,000 g for 20 minutes and carefully collect the supernatant.

[0037] (4) Use 2 M NaOH solution to precisely adjust the pH of the supernatant to 7.0.

[0038] (5) The neutralized solution was placed into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in flowing deionized water for 72 hours, during which the external dialysis solution was changed several times.

[0039] (6) Transfer the solution in the dialysis bag to a freeze-drying bottle, pre-freeze it at -80℃, and freeze-dry it in a freeze dryer for 72 hours to obtain deproteinized crude polysaccharide from Porphyra yezoensis.

[0040] Example 3: Ion exchange column chromatography purification of Porphyra polysaccharides (1) Weigh 200 mg of the deproteinized crude polysaccharide obtained in Example 2, dissolve it in 20 mM Tris-HCl buffer (pH 8.0) and bring the volume to 40 mL to make its concentration 20 mg / mL. Filter the solution through a 0.45 μm filter membrane for later use.

[0041] (2) Pack the prepared DEAE Sepharose Fast Flow packing material into a column (approximately 20 mL in volume) and connect it to the Taidu ACHROM Pure protein purification system. Equilibrate the column with 20 mM Tris-HCl buffer until the baseline is stable.

[0042] (3) Load the sample solution, set the system pressure to 0.3 MPa, and the flow rate to 1 mL / min. Perform stepwise elution with 20 mM Tris-HCl buffer containing 0 M NaCl, 20 mM Tris-HCl buffer containing 0.3 M NaCl, and 20 mM Tris-HCl buffer containing 0.5 M NaCl. Collect the elution fraction in 10 mL increments using an automated collector.

[0043] (4) The sugar content of each collection tube was determined by the phenol-sulfuric acid method, and the elution curve was plotted. The fractions corresponding to the main sugar peaks eluted with 0.5 M NaCl buffer were combined.

[0044] (5) The combined components were placed in a 3500 Da dialysis bag and dialyzed against deionized water for 72 hours to remove salt. The dialysis solution was freeze-dried to obtain a preliminarily purified polysaccharide component, named PHP3.

[0045] Example 4: Fine purification of Porphyra polysaccharide PHP3 by gel filtration column (1) Weigh 20 mg of the PHP3 component obtained in Example 3, dissolve it in deionized water and make up to 10 mL to make its concentration 2 mg / mL. Filter the solution through a 0.45 μm filter membrane.

[0046] (2) Pack Sephadex G-75 packing material into a column (size 2.6 cm × 50 cm), connect it to the purification system, and equilibrate with deionized water.

[0047] (3) Load the sample solution, set the system pressure to 0.3 MPa and the flow rate to 0.1 mL / min, and elute with deionized water. Collect the eluent in 5 mL tubes.

[0048] (4) The sugar content of each tube was determined by the phenol-sulfuric acid method, and the elution peaks were plotted. The fractions within the main peak range were combined.

[0049] (5) The combined distillate solutions were dialyzed in a 3500 Da dialysis bag and then freeze-dried to obtain a refined polysaccharide fraction. In this refined polysaccharide fraction, the content of the polysaccharide PPHP3 was 84.34 ± 2.25%, the protein content was 0.89 ± 0.05%, and the other components were mainly bound water, ash, and trace inorganic salts.

[0050] Example 5: Structural Characterization of PPHP3, a Pure Polysaccharide from Laveria bassiana (1) Molecular weight determination: High performance gel permeation chromatography-multi-angle laser light scattering (HPGPC-MALLS) was used for determination. The purified polysaccharide sample obtained in Example 4 was dissolved in a 0.1 M NaNO3 solution containing 0.02% NaN3 (1 mg / mL), filtered through a 0.45 μm filter, and then injected. Chromatographic conditions: Ohpak SB-805 HQ and SB-803 HQ columns in series, column temperature 45℃, mobile phase was the above NaNO3 solution, flow rate 0.6 mL / min. The weight-average molecular weight (Mw) of PPHP3 was determined to be 25.208 kDa by ASTRA software analysis.

[0051] (2) Monosaccharide composition analysis: Ion chromatography was used for determination. PPHP3 samples were hydrolyzed with 2 M trifluoroacetic acid at 121℃ for 2 hours, derivatized, and then injected. Chromatographic conditions: Dionex CarboPac PA20 column, electrochemical detector, gradient elution. The results showed that PPHP3 was mainly composed of galactose (Gal), glucose (Glc), and glucuronic acid (Glc-UA), with a molar ratio of 98.3 : 0.46 : 1.24.

[0052] Example 6: Verification of the in vitro lipid-lowering activity of pure polysaccharide PPHP3 from Porphyra yezoensis (1) Cell culture and model establishment: Human hepatocellular carcinoma cells (HepG2) were cultured in MEM medium containing 10% fetal bovine serum. Logarithmic growth phase cells were seeded into 96-well or 6-well plates. A blank control group, a model group, a PHP3 intervention group, and a PPHP3 intervention group were set up. The model group and the intervention group were treated with 500 μM oleic acid to establish a lipid accumulation model. The intervention group was treated with different concentrations of PHP3 or PPHP3 solution for 24 hours.

[0053] (2) Cell viability assay: The CCK-8 assay was used. After treatment and incubation with CCK-8 reagent, the absorbance of each group of cells was measured at a wavelength of 450 nm. Figure 2 The results showed that, compared with the control group, the cell survival rate of the PPHP3 intervention group with a concentration of ≤800 μg / mL was higher than 90%, indicating that PPHP3 had no significant cytotoxicity within the experimental concentration range.

[0054] (3) Visualization of lipid accumulation (Oil Red O staining): After treatment, cells were fixed with 4% paraformaldehyde and stained with Oil Red O working solution. Figure 3 Microscopic observation revealed a large number of red lipid droplets in the model group cells; while the number and area of ​​lipid droplets in cells treated with PPHP3 (especially 800 μg / mL) were significantly reduced.

[0055] (4) Quantitative determination of lipid content: Collect cells and use commercial kits to determine the content of total triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in cells. Figures 4-7 The results showed that, compared with the model group, both the PHP3 and PPHP3 intervention groups reduced the levels of TG, TC and LDL-C in HepG2 cells, while increasing the level of HDL-C. Compared with the PHP3 intervention group, the PPHP3 intervention group showed a stronger regulatory effect, demonstrating its clear in vitro lipid metabolism regulation activity.

Claims

1. A polysaccharide PPHP3 from *Porphyra yezoensis* with lipid-lowering activity, characterized in that... Its weight-average molecular weight is 25.208 kDa, and its monosaccharide composition is galactose, glucose and glucuronic acid, with a molar ratio of 98.3 : 0.46 : 1.

24.

2. A method for preparing the PPHP3 polysaccharide from *Porphyra yezoensis* as described in claim 1, characterized in that, Includes the following steps: (1) Raw material pretreatment: After washing, drying and crushing the laver, the pigments and lipids were removed by reflux with anhydrous ethanol, and the laver powder was obtained after drying. (2) Ultrasonic-compound enzyme extraction and alcohol precipitation: Porphyra powder was mixed with water, a compound enzyme was added, and extraction was carried out under ultrasonic conditions. After enzyme inactivation and centrifugation, the supernatant was collected. Ethanol was added to the supernatant for alcohol precipitation. The precipitate was collected and dried to obtain crude polysaccharide from Porphyra. (3) Deproteinization and preliminary purification: The crude polysaccharide of Porphyra yezoensis was dissolved, the protein was removed, and after neutralization, dialysis and freeze-drying, the deproteinized crude polysaccharide of Porphyra yezoensis was obtained; (4) Ion exchange column chromatography purification: The crude polysaccharide of Porphyra yezoensis after deproteinization was dissolved in Tris-HCl buffer and subjected to anion exchange column chromatography. Gradient elution was performed sequentially using Tris-HCl buffer containing different concentrations of NaCl. The eluted fractions were collected, dialyzed, and lyophilized to obtain the initial pure polysaccharide PHP3. (5) Fine purification by gel filtration column: The pure polysaccharide PHP3 is dissolved, filtered by dextran gel filtration column, eluted with water, and the sugar fraction corresponding to the main peak is collected. After dialyzing and freeze drying, the purified fraction containing the pure polysaccharide PPHP3 of Porphyra yezoensis is obtained. The content of polysaccharide PPHP3 in the purified fraction is ≥80% (w / w).

3. The method according to claim 2, characterized in that, In step (1), the drying temperature is 60-80℃, the pulverization is passed through a 20-60 mesh sieve, and the conditions for decolorization and degreasing by reflux of anhydrous ethanol are a material-to-liquid ratio of 1:15-1:25 (g / mL), a reflux temperature of 85-95℃, and repeated 2-4 times; the drying is carried out at 40-50℃.

4. The method according to claim 2, characterized in that, In step (2), the complex enzyme is composed of cellulase and pectinase; in step (3), the protein is removed by trichloroacetic acid method; in step (4), the anion exchange column is a DEAE anion exchange column, and gradient elution is performed sequentially using Tris-HCl buffer containing different concentrations of NaCl of 0M, 0.3M and 0.5M.

5. The method according to claim 4, characterized in that, In step (2), the conditions for ultrasonic-composite enzyme extraction are as follows: material-liquid ratio 1:20-1:40 (g / mL), cellulase addition amount is 0.5-3.0% of algal powder mass, pectinase addition amount is 0.5-3.0% of algal powder mass, ultrasonic power 150-250W, ultrasonic temperature 55-75℃, extraction time 60-120 minutes; the alcohol precipitation is to add 2-4 times the volume of anhydrous ethanol and let stand at 0-10℃ for 12-36 hours; the centrifugation conditions are 0-10℃, centrifugation at 8000-12000g for 20-40 minutes; the drying temperature is 40-50℃, time is 20-40 minutes.

6. The method according to claim 4, characterized in that, In step (3), the deproteinization by trichloroacetic acid method involves adding 15-25% (w / v) trichloroacetic acid solution to a final concentration of 3-5% (v / v); the neutralization involves adjusting the pH to 6.5-7.5 using 1-3M NaOH solution; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000-5000 Da and dialysis in deionized water for 48-96 hours; and the lyophilization time is 48-96 hours.

7. The method according to claim 4, characterized in that, In step (4), the DEAE anion exchange column is a DEAE... Sepharose FastFlow column; the Tris-HCl buffer concentration is 10-30 mM, pH 7.5-8.5; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-5000 Da.

8. The method according to claim 2, characterized in that, In step (5), the dextran gel filter column is a Sephadex G-75 gel column.

9. The method according to claim 8, characterized in that, In step (5), the elution conditions of the Sephadex G-75 gel column are: deionized water as the mobile phase and a flow rate of 0.1 mL / min; the fraction collected is within the main peak range; and the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-5000 Da.

10. The use of the PPHP3 polysaccharide from Porphyra yezoensis according to claim 1 in the preparation of products for regulating lipid metabolism and / or lowering blood lipids.

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