Sargassum muticum immunomodulatory peptide, and preparation method and application thereof

Abstract

The present application belongs to the technical field of biotechnology, and particularly relates to a sargassum thunbergii immunomodulatory peptide as well as a preparation method and application thereof. The present application extracts sargassum thunbergii crude protein by ultrasonic treatment combined with pectinase treatment, and obtains three polypeptides with excellent immunomodulatory activity from sargassum thunbergii by molecular docking screening of complex enzymolysis of alkaline protease and flavor protease. The amino acid sequences of the polypeptides are shown as SEQ ID NO. 1-3. The docking energy of the immunomodulatory peptide with the receptor TLR4 / MD2 is all ≤-9.0 kcal / mol, and the binding energy with the TLR2 receptor is also all ≤-7.4 kcal / mol. After screening and verification, the above immunomodulatory polypeptides all show significant performance in promoting RAW 264.7 cell proliferation, phagocytic ability, secretion of TNF-α and IL-6 and NO release, and show strong immunomodulatory activity. The present application has important significance for the development of novel immunomodulatory peptide products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immunomodulatory peptide from Sargassum fusiforme, its preparation method, and its application. Background Technology

[0002] Sargassum is one of the most widely distributed and diverse genera in the Phaeophyte phylum, found extensively in temperate and tropical seas worldwide, and is an important component of nearshore marine ecosystems. Copper algae ( Sargassum horneri As a dominant species in the shallow seaweed communities of warm temperate waters in my country, Sargassum plays an irreplaceable ecological role in maintaining marine biodiversity and ecosystem stability. However, in recent years, the large-scale explosive growth of Sargassum has created a "golden tide" phenomenon, posing a serious threat to coastal aquaculture, fisheries, coastal tourism, and marine ecological security. Therefore, how to transform this "algal scourge" into a resource and achieve the high-value comprehensive utilization of Sargassum has become an important issue in the field of marine biological resource development.

[0003] Sargassum is rich in polysaccharides, polyphenols, proteins, minerals, and various trace active ingredients, showing broad application prospects in food, feed, medicine, and energy. In the food industry, Sargassum can be processed into functional seaweed foods, seasonings, and dietary fiber supplements. In the feed industry, its protein can be used as a substitute for traditional protein sources such as fishmeal to develop high-value marine feed. In the pharmaceutical and health field, Sargassum extract has been proven to have various pharmacological activities such as antioxidant, anti-inflammatory, immunomodulatory, and anti-osteoporosis effects. In addition, Sargassum can also be used as a bioenergy feedstock to produce bioethanol and bio-oil, or processed into seaweed fertilizer through fermentation technology. With the gradual development of artificial aquaculture technology, the resource utilization of Sargassum is steadily advancing towards intensive processing and high-value utilization.

[0004] Although some progress has been made in the comprehensive utilization of Sargassum, current research mainly focuses on polysaccharides and polyphenols, while the in-depth development of its protein components—especially the targeted preparation and functional evaluation of bioactive peptides—is still in its early stages. Bioactive peptides, due to their small molecular weight, easy absorption, strong targeting, and high safety, have become a hot topic in the research and development of functional foods and drugs. Existing research shows that marine-derived protein peptides have significant immunomodulatory, hypotensive, antibacterial, and antioxidant effects, but specific research on immunomodulatory peptides derived from Sargassum is extremely rare. Existing technologies (such as CN109593810A) disclose a method for extracting active peptides from Sargassum, employing low-temperature freezing, ultrasonic disruption, high-pressure grinding combined with complex hydrolysis by Monascus purpureus and protease, followed by nanofiltration purification to obtain the peptide product. However, the product obtained by this method is a mixed polypeptide with a wide molecular weight distribution of 500-5000 Da, and it is mixed with a large number of colloidal impurities such as seaweed polysaccharides and alginate. It fails to achieve the precise and targeted preparation of a single peptide with a clear sequence and specific immune activity, which limits its practical application in the pharmaceutical field. Summary of the Invention

[0005] To achieve the above-mentioned objectives, this invention provides a technical solution for Sargassum immunomodulatory peptides, their preparation method, and applications, specifically including the following: The first aspect of the present invention provides a Sargassum immunomodulatory peptide, the immunomodulatory peptide comprising at least one of the peptides whose amino acid sequences are shown in SEQ ID NO.1-3.

[0006] A second aspect of the present invention provides a composition comprising the above-described immunomodulatory peptide and pharmaceutically acceptable excipients.

[0007] A third aspect of the present invention provides the use of the above-described immunomodulatory peptides or compositions in the preparation of immunomodulatory drugs.

[0008] A fourth aspect of the present invention provides a method for preparing the above-mentioned immunomodulatory peptide or composition, comprising the following steps: S.1 Extract crude protein from Sargassum, salt out, and freeze dry; S.2 Add alkaline protease and flavor protease to the protein obtained in S.1. After enzymatic hydrolysis, heat in a boiling water bath, centrifuge the hydrolysate and collect the supernatant, adjust the pH, add anhydrous ethanol, concentrate and freeze dry. S.3 Ultrafiltration removes peptides with a molecular weight cutoff of less than 3 kDa, separates and purifies them, and identifies the peptide sequences; S.4 The peptide described in S.3 is molecularly docked with the receptor TLR2 / TLR4, and the most immunomodulatory active peptide is screened and synthesized to obtain the Sargassum immunomodulatory peptide.

[0009] Furthermore, in S.1, crude protein from Sargassum is extracted by ultrasonication combined with pectinase enzymatic hydrolysis. The specific conditions are: enzyme dosage of 600-2400 U, material-to-liquid ratio of 1:10-30 g / mL, temperature of 30-60℃, pH of 3-6, and reaction time of 2-6 h; preferably, enzyme dosage of 1200 U, material-to-liquid ratio of 1:15 g / mL, temperature of 50℃, pH of 5, and reaction time of 4 h.

[0010] Furthermore, the salting out described in S.1 involves adding 10%-80% saturated ammonium sulfate to the crude protein extract of Sargassum; preferably 40%.

[0011] Furthermore, the total amount of alkaline protease and flavor protease added in S.2 is 3000-4000 U / g; specifically, crude protein and deionized water are mixed at a ratio of 1:10-30 w / v, alkaline protease is added first, and the reaction is carried out at pH=8-10 and temperature 45-55℃ for 2-4 h; after the reaction is completed, the protein is heated in a boiling water bath to inactivate the enzyme; after cooling to room temperature, flavor protease is added, and the protein is enzymatically hydrolyzed at pH=6-8 and temperature 45-55℃ for 2-4 h.

[0012] Furthermore, in S.3, peptides with a molecular weight less than 3 kDa were separated and purified by Sephadex G-15 gel filtration chromatography and RP-HPLC; the obtained peptide sequences were identified by LC-MS / MS.

[0013] Furthermore, in S.4, the most immunomodulatory active peptides were screened according to the following conditions: (1) amino acid sequence length of 3-10 amino acid residues; (2) Peptide Ranker score > 0.8; (3) Pre AIP score > 0.35; (4) rich in hydrophobic amino acids and Toxin Pred hydrophobic score > 0.2.

[0014] Furthermore, the amino acid sequence of the Sargassum immunomodulatory peptide is shown in SEQ ID NO.1-3.

[0015] The present invention has the following beneficial effects: This invention extracts crude protein from Sargassum fusiforme through ultrasonic treatment combined with pectinase, followed by enzymatic hydrolysis with alkaline protease and flavor protease, and molecular docking screening. Three polypeptides with excellent immunomodulatory activity were obtained from Sargassum fusiforme. The immunomodulatory peptides shown in SEQ ID NO. 1-3 all exhibited docking energies ≤-9.0 kcal / mol with the TLR4 / MD2 receptor and ≤-7.4 kcal / mol with the TLR2 receptor, demonstrating their stable binding to both TLR4 / MD-2 and the TLR2 receptor. Screening and verification showed that these immunomodulatory peptides significantly promoted RAW 264.7 cell proliferation, phagocytic capacity, TNF-α and IL-6 secretion, and NO release, exhibiting strong immunomodulatory activity. Attached Figure Description

[0016] Figure 1 In the figure, A represents the protein extraction rate of Sargassum fusiforme treated by different methods; Figure 1 In section B, the effect of the solid-liquid ratio on the protein extraction rate from Sargassum fusiforme is represented. Figure 1 In the middle, C represents the effect of temperature on the extraction rate of Sargassum protein; Figure 1 D represents the effect of reaction time on the protein extraction rate from Sargassum fusiforme; Figure 1 E represents the effect of enzyme dosage on Sargassum protein extraction rate; Figure 1 F represents the effect of pH value on the extraction rate of Sargassum protein.

[0017] Figure 2 For the screening and characterization of different enzymatic hydrolysis components; Figure 2 In section A, the DH curves of four proteases are shown. Figure 2 In Figure B, the effects of four hydrolysis products on the proliferation of RAW264.7 cells are shown. Figure 2 In the middle, C represents the HPLC elution chromatogram of the molecular weight standard; Figure 2 D represents the HPLC elution chromatograms of single-enzyme hydrolysis products and complex-enzyme hydrolysis products; Figure 2 E represents the effect of molecular weight distribution on cell proliferation activity; Figure 2 F represents the effect of the DH curve on cell proliferation activity.

[0018] Figure 3 In Figure A, the effects of three ultrafiltration components on RAW264.7 cells are shown. Figure 3 Figure B shows the separation and purification process of Sephadex G-15. Figure 3 In the middle, C represents the effect of the three components of Sephadex G-15 on RAW264.7 cells; Figure 3 Diagram D in the middle is the RP-HPLC separation and purification chromatogram; Figure 3 The effect of three components in RP-HPLC on RAW264.7 cells is shown in Figure E.

[0019] Figure 4The total ion current chromatogram of R-F3 was analyzed by LC-MS / MS.

[0020] Figure 5 The effects of six synthetic peptides on the immune activity of RAW264.7 cells; Figure 5 In the middle, A represents the effect on the proliferation rate; Figure 5 In the middle, B represents the effect on the phagocytosis rate; Figure 5 C represents the effect on NO secretion.

[0021] Figure 6 In section A, the effect of immunomodulatory peptides on cellular ROS production is discussed. Figure 6 In the middle section, B represents the effect of immunomodulatory peptides on cellular NO production.

[0022] Figure 7 The effects of different concentrations (10, 50, 100 and 200 μg / mL) of LNFPLL, GPLFW and LPLLF on RAW264.7 cells; Figure 7 In the middle, A represents the effect on TNF-α; Figure 7 B represents the effect on IL-6. Detailed Implementation

[0023] The technical solution of this invention can be described and verified in detail through the following specific embodiments. Unless otherwise specified, the test samples, basic reagents, and instruments used in the following embodiments can all be routinely purchased through commercial channels. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0024] Example 1: Extraction and purification of Sargassum immunomodulatory peptides 1.1 Materials and Instruments Table 1 Main Materials RAW 264.7 cells Wuhan Pronosei Life Science Technology Co., Ltd. RMPI-1640 culture medium Wuhan Pronosei Life Science Technology Co., Ltd. RAW 264.7 cell culture medium Wuhan Pronosei Life Science Technology Co., Ltd. DMEM medium Wuhan Pronosei Life Science Technology Co., Ltd. LPS Shanghai Biyuntian Biotechnology Co., Ltd. Concanavalin A Shanghai Biyuntian Biotechnology Co., Ltd. CCK-8 reagent kit Shanghai Biyuntian Biotechnology Co., Ltd. Nitric oxide detection kit Shanghai Biyuntian Biotechnology Co., Ltd. Neutral erythrocyte proliferation kit Shanghai Biyuntian Biotechnology Co., Ltd. Reactive oxygen species detection kit Shanghai Biyuntian Biotechnology Co., Ltd. TLR4, MyD88, P-p65 antibodies Shanghai Biyuntian Biotechnology Co., Ltd. Horseradish peroxidase-labeled goat anti-rabbit IgG Shanghai Biyuntian Biotechnology Co., Ltd. Green fluorescent probe for cell membrane Shanghai Biyuntian Biotechnology Co., Ltd. DAPI staining solution Shanghai Biyuntian Biotechnology Co., Ltd. Cyclophosphamide Shanghai Aladdin Biochemical Technology Co., Ltd. Cytokine (IL-6, TNF-α) Detection Kit Wuhan Yilairuit Biotechnology Co., Ltd. IgG enzyme-linked immunosorbent assay kit Wuhan Yilairuit Biotechnology Co., Ltd. sheep red blood cell SRBC Shanghai Yuanye Biotechnology Co., Ltd. Guinea pig serum Shanghai Yuanye Biotechnology Co., Ltd. .

[0025] Table 2 Main Instruments and Equipment ELISA reader SpectraMax Bio-Rad, Inc. (USA) Clean bench ZHJH-C1209 Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd. Inverted microscope CKX4 OLYMPUS Japan Fluorescence microscopy Axio Imager A2 Carl Zeiss AG, Germany slicer RM2135 Leica, Germany <![CDATA[CO₂ incubator]]> Forma3111 Thermo Technologies, Inc. .

[0026] 1.2 Experimental Methods 1.2.1 Preparation of Sargassum protein 1.2.1.1 Treatment of Sargassum algal powder Rinse the dried Sargassum fusiforme repeatedly with water to remove a large amount of mud and sand, dry it in a 50℃ blower dryer, grind it into powder using a multi-functional grinder, pass it through a 60-mesh sieve, and store it in a desiccator.

[0027] 1.2.1.2 Extraction of Sargassum protein This invention employs autolysis, enzymatic hydrolysis, ultrasound, and a combination of ultrasound and enzymatic hydrolysis to process algae powder. The processing details are as follows: Autolysis: Dissolve 5g of algae powder in a phosphate buffer solution with a pH of 5.0 at a material-to-liquid ratio of 1:15 (g / mL), and control the temperature at 45-50 ℃ for 4 hours.

[0028] Cellulase treatment: Dissolve 5g of algal powder in phosphate buffer at a material-to-liquid ratio of 1:15 (g / mL), add cellulase at a dosage of 1500U, and enzymatically hydrolyze at 45-50℃ for 4 hours.

[0029] Pectinase treatment: Dissolve 5g of algae powder in phosphate buffer at a material-to-liquid ratio of 1:15 (g / mL), add pectinase at a dosage of 1200U, and hydrolyze at 45-50℃ for 4 hours.

[0030] Ultrasonic treatment: Dissolve 5g of algae powder in phosphate buffer at a material-to-liquid ratio of 1:15 (g / mL), and treat it three times with an ultrasonic instrument (360W, 40kHz), each time for 20min, with a 30s interval.

[0031] Ultrasonic treatment with cellulase: Dissolve 5g of algae powder in phosphate buffer at a ratio of 1:15 (g / mL) and place it in an ultrasonic instrument (360W power, 40kHz frequency) for treatment as above. After ultrasonic treatment, add cellulase at a dosage of 1500U and enzymatically hydrolyze at 45-50℃ for 4 hours.

[0032] Ultrasonic treatment with pectinase: Dissolve 5g of algae powder in phosphate buffer at a ratio of 1:15 (g / mL) and place it in an ultrasonic instrument (360W, 40kHz) for treatment as above. After ultrasonic treatment, add pectinase at a rate of 1200U and hydrolyze at 45-50℃ for 4 hours.

[0033] Ultrasonic treatment with dual enzymes: Dissolve 5g of algae powder in phosphate buffer at a material-to-liquid ratio of 1:15 (g / mL), place it in an ultrasonic instrument (360W, 40kHz) and treat as above. After ultrasonic treatment, add cellulase and pectinase at a ratio of 1500U and 1200U respectively, and enzymatically hydrolyze at 45-50℃ for 4 hours.

[0034] After all treatment groups were completed, they were centrifuged (8000 r / min, 4℃, 15 min), and the supernatant was collected. The protein content was determined by the BCA method. The effects of different treatment methods on the protein extraction rate of Sargassum were compared, and the treatment method with the highest protein extraction rate was optimized to serve as a method for large-scale extraction of Sargassum protein in the later stage.

[0035] 1.2.1.3 Optimization of protein extraction conditions Using protein extraction rate as an indicator, the reaction conditions were optimized, including the material-to-liquid ratio (1:10, 1:15, 1:20, 1:25, 1:30 g / mL), enzymatic hydrolysis temperature (30, 40, 50, 60 ℃), extraction time (2, 3, 4, 5, 6 h), enzyme dosage (600, 1200, 1800, 2400 U), and pH value (3, 4, 5, 6).

[0036] 1.2.1.4 Protein isolation and purification The ammonium sulfate saturation in the crude Sargassum protein extract was adjusted to 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%, respectively. The separation effect of ammonium sulfate with different saturations on the protein in the crude Sargassum protein extract was compared. The salted-out protein was desalted by dialysis, concentrated, and freeze-dried for the next enzymatic hydrolysis step.

[0037] 1.2.2 Determination of protein content The protein content in the crude Sargassum protein extract was determined by the BCA method, and the total protein content was determined by the Kjeldahl method. Based on the calculated total amount of BCA working solution required, BCA working solutions A and B were prepared in a 50:1 ratio and thoroughly mixed. Bovine serum albumin (BSA) standard solutions with concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 mg / mL were prepared. 25 μL of protein standards at different concentrations and a certain concentration of crude Sargassum protein extract were added to a 96-well plate, followed by 200 μL of BCA working solution. The plate was incubated at 37°C for 30 min, cooled to room temperature, and the absorbance was measured at 562 nm using a multi-mode microplate reader. A standard curve was constructed based on the absorbance values ​​of different BSA standard concentrations, yielding the linear equation y = 1.0231x - 0.138, with a correlation coefficient R0. 2 =0.9985, calculate the protein content in the crude Sargassum extract. The formula for calculating the protein extraction rate is as follows: P: Protein content in the supernatant P tot Total protein content of Sargassum.

[0038] 1.2.3 Preparation of Sargassum protease hydrolysate peptides Crude S. horneri protein was dissolved in deionized water at a material-to-liquid ratio of 1:20 (w / v). Alkaline protease, neutral protease, trypsin, and flavor protease were added for enzymatic hydrolysis, with the enzyme dosage controlled at 3500 U / g (based on substrate protein). Each enzyme was hydrolyzed under its optimal reaction conditions (Table 3) (pH adjusted to the initial value every 30 min), and the hydrolysis time was 4 h. After hydrolysis, the enzymes were inactivated by heating in a boiling water bath for 15 min. The hydrolysate was centrifuged at 4 ℃ and 10000 r / min for 15 min, and the supernatant was collected and the pH adjusted to 7.0. Anhydrous ethanol was added to the supernatant to a final concentration of 65% (v / v), and the mixture was allowed to stand at 4 ℃ to precipitate, removing large molecular weight polysaccharides, polyphenols, and incompletely hydrolyzed large molecular weight proteins. The supernatant was concentrated and freeze-dried to obtain the S. horneri proteolytic peptides. The obtained polypeptide components were screened using hydrolysis degree, molecular weight distribution, and RAW264.7 macrophage proliferation activity as evaluation indicators to determine the polypeptide components with the best immune activity.

[0039] Complex enzymatic hydrolysis: A stepwise enzymatic hydrolysis strategy was adopted while maintaining the material-to-liquid ratio (1:20, w / v) and total enzyme dosage (3500 U / g). First, alkaline protease was added and reacted at its optimal pH and temperature for 3 hours. After the reaction, the enzyme was inactivated by heating in a boiling water bath for 15 minutes. After cooling to room temperature, flavor protease was added, and enzymatic hydrolysis continued for 3 hours under its optimal conditions. Subsequent treatments were performed as described above after enzymatic hydrolysis.

[0040] Table 3. Conditions for enzymatic hydrolysis of Sargassum protein by different proteases .

[0041] 1.2.4 Determination of degree of hydrolysis The degree of hydrolysis of Sargassum protein was determined using the ninhydrin colorimetric method. The ninhydrin colorimetric reagent binds to free α-amino groups to form a stable blue-violet complex, which is absorbed at a wavelength of 570 nm and can be used to determine the content of free amino acids in the enzymatic hydrolysate.

[0042] The preparation method of ninhydrin colorimetric reagent is as follows: Accurately weigh 0.5 g ninhydrin hydrate, 10 g Na2HPO4·10H2O, 6 g K2HPO4 and 0.3 g fructose, dissolve them in deionized water sequentially, and finally adjust the volume to 100 mL. After preparation, it should be stored away from light and used immediately.

[0043] A 20 μg / mL glycine solution was prepared as a standard solution. 0, 200, 400, 600, 800, 1000, 1500, and 2000 μL of glycine standard solution and different time-phase digests of Sargassum protein were added to centrifuge tubes wrapped in aluminum foil. Each tube was diluted to 2 mL with pure water, and 1 mL of colorimetric reagent was added. After mixing, the tubes were placed in boiling water for 15 min, cooled to room temperature in an ice-water bath, and 5 mL of 40% ethanol was added and mixed. After standing for 15 min, the absorbance at 570 nm was measured using a UV spectrophotometer. The blank was marked zero. A standard curve was constructed based on the absorbance values ​​of different concentrations of glycine standards, yielding the linear equation y = 0.0293x + 0.0369, with a correlation coefficient R0. 2 =0.9919, calculate the nitrogen content of amino groups in the Sargassum protein hydrolysate; the total nitrogen content of Sargassum protein was determined by the Kjeldahl method.

[0044] The degree of hydrolysis (DH) of enzymatically hydrolyzed Sargassum protein was determined using the ninhydrin colorimetric method. The ninhydrin colorimetric reagent binds to free α-amino groups to form a stable blue-violet complex, which is absorbed at a wavelength of 570 nm and can be used to determine the content of free amino acids in the enzymatic hydrolysate.

[0045] The preparation method of ninhydrin colorimetric reagent is as follows: Accurately weigh 0.5 g ninhydrin hydrate, 10 g Na2HPO4·10H2O, 6 g K2HPO4 and 0.3 g fructose, dissolve them in deionized water sequentially, and finally adjust the volume to 100 mL. After preparation, it should be stored away from light and used immediately.

[0046] Dilute an appropriate amount of hydrolysate to 2 mL with pure water, add 1 mL of colorimetric reagent, mix well, and react in boiling water for 15 min. Cool to room temperature in an ice-water bath, add 5 mL of 40% ethanol and mix well. After standing for 15 min, measure the absorbance at 570 nm using a UV spectrophotometer. Mark the blank as zero. The number of free amino groups is determined using a glycine standard curve. The DH calculation formula is as follows:

[0047] h1: Number of free peptide bonds in the solution after hydrolysis h0: Number of free peptide bonds in the solution before hydrolysis h tot The total number of peptide bonds in each gram of raw protein.

[0048] 1.2.5 Determination of molecular weight (MW) The molecular weight distribution of three enzymatic hydrolysis products was determined using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system equipped with a TSK-Gel 2000SWXL column (300 mm × 7.8 mm, 5 μm, TOSOH Corporation) and a 218 nm UV detector. Mobile phase A consisted of ultrapure water containing 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B consisted of acetonitrile containing 0.1% (v / v) TFA, with a ratio of A:B = 70:30 (v / v). Isocratic elution was performed at a flow rate of 0.5 mL / min and a column temperature of 35 °C. Samples were dissolved in ultrapure water and diluted to a concentration of 1 mg / mL, filtered through a 0.22 μm microporous membrane, and 10 μL was injected for HPLC analysis. Using cytochrome C (12400 Da), aprotinin (6511.44 Da), bacitracin (1422.69 Da), and reduced L-glutathione (307.3 Da) as molecular weight standards, and after analysis under the same chromatographic conditions, a standard curve was plotted with retention time as the x-axis and the logarithm of molecular weight as the y-axis. The molecular weight distribution of the test samples was calculated from the standard curve based on their respective retention times.

[0049] 1.2.6 Cell Culture RAW264.7 macrophages were cultured in high-glucose DMEM medium (containing 10% heat-inactivated fetal bovine serum and 100 U / mL penicillin-streptomycin) at 37 °C in a 5% CO2 incubator. The medium was replaced with fresh medium every 48 h, and cell morphology and proliferation were observed periodically using an inverted microscope. When cell confluence reached 80% or higher, the cells were passaged at an appropriate ratio to ensure they were in the logarithmic growth phase for subsequent experiments.

[0050] 1.2.7 Determination of cell proliferation The proliferative activity of RAW264.7 macrophages was determined using the CCK-8 assay. RAW264.7 cells in logarithmic growth phase were cultured at a concentration of 5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / well in 96-well plates, with sterile PBS added to the edge wells to reduce evaporation. Five replicates were set up for each group. Cells were incubated at 37 ℃ in a 5% CO2 incubator for 12 h. After cell attachment, the original culture medium was discarded, and different concentrations (50, 200, 500 μg / mL) of peptide sample solution were added. Lipopolysaccharide (LPS) at 1 μg / mL was used as a positive control. A blank group (containing only culture medium) and a control group (containing cells and complete culture medium) were also set up. After 24 h of intervention, the supernatant was discarded, and 10% (v / v) CCK-8 solution was added to each well. Cells were incubated at 37 ℃ in the dark for 1 h, and the absorbance at 450 nm was measured using a microplate reader. The relative proliferation rate of each group was calculated using the following formula: .

[0051] 1.2.8 Separation and purification of hydrolysis products 1.2.8.1 Ultrafiltration The hydrolysate was diluted with distilled water and passed through a 0.22 μm aqueous filter membrane, followed by ultrafiltration using a TFF Cogent μScale ultrafiltration system. Based on the molecular weight distribution of the Sargassum protein hydrolysate components, ultrafiltration membranes with a molecular weight ratio (MW) of 10 kDa and 3 kDa were used sequentially. The hydrolysate was ultimately separated into three components: MW > 10 kDa, 3 kDa < MW < 10 kDa, and MW < 3 kDa. These components were concentrated, lyophilized, and stored at -20°C for later use. The relative proliferation rate of RAW264.7 macrophages was used as an evaluation index to screen for the ultrafiltration component with the highest immunomodulatory activity.

[0052] 1.2.8.2 G-15 Gel Chromatography The ultrafiltration fraction with the highest immunomodulatory activity was further separated and purified using a Sephadex G-15 dextran gel column (1.6 cm inner diameter × 70 cm column height). The column was equilibrated with ultrapure water as the mobile phase at a flow rate of 1.0 mL / min, with an equilibration volume of 5 column volumes. The sample to be separated was prepared into a 25 mg / mL solution with ultrapure water, filtered through a 0.22 μm aqueous microporous membrane, and 2 mL was loaded onto the column. Isocratic elution was performed with ultrapure water as the mobile phase at a flow rate of 1.0 mL / min, collecting one eluent tube every 3 min using an automated fraction collector. The absorbance of each solution was measured at 214 nm, and an elution curve was plotted with the number of tubes on the x-axis and absorbance on the y-axis. The fractions corresponding to each elution peak were combined, freeze-dried, and stored at -20 °C for later verification of immunomodulatory activity. 1.2.8.3 Reversed-phase high-performance liquid chromatography (RP-HPLC) The fraction G-F3, which exhibited the highest immunomodulatory activity after gel filtration chromatography separation, was further purified using reversed-phase high-performance liquid chromatography (RP-HPLC). A galaksil EP-C18H column (10 μm, 20 mm × 250 mm) was used, with the column temperature set at 25 ℃ and the detection wavelength at 214 nm. The lyophilized sample was dissolved in ultrapure water to prepare a 4 mg / mL solution, filtered through a 0.22 μm aqueous microporous membrane, and loaded with 2 mL at an elution flow rate of 1.0 mL / min. Mobile phase A consisted of ultrapure water containing 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B consisted of acetonitrile containing 0.1% (v / v) TFA. The gradient elution program was set as follows: 0–10 min, 10% B (equilibration); 10–12 min, 10% B (injection); 12–42 min, 10%–50% B (linear gradient elution); 42–82 min, 50%–100% B (linear gradient elution); 82–92 min, 100% B (rinse). Each fraction was collected according to the elution peak, and after freeze-drying, its immunomodulatory activity was validated at low, medium, and high concentrations (100 μg / mL, 200 μg / mL, and 500 μg / mL) to identify the most active target peptide fraction.

[0053] 1.3 Results and Discussion 1.3.1 Extraction of Sargassum protein By comparing different extraction methods, such as Figure 1 From A, it can be seen that compared with the protein extraction rate obtained by autolysis at 50 °C, other protein extraction methods all showed significant improvements. Among them, the protein extraction rates of cellulase (5.53±0.3%) and pectinase (5.65±0.4%) treatments were significantly lower than those of ultrasonic treatment (6.53±0.3%), indicating that ultrasonic treatment remains an effective method for extracting algal proteins. Based on this, this invention further explored the extraction of Sargassum protein by combining ultrasound with enzymatic hydrolysis. The protein extraction rates of ultrasound + pectinase and ultrasound + dual enzymes were significantly higher than those of single treatments (P<0.01), at 8.07±0.3% and 7.97±0.4%, respectively. This indicates that ultrasound combined with glycosidase treatment can efficiently break down cell walls, allowing for the full dissolution of intracellular proteins. However, there was no significant difference in protein extraction rate between ultrasound combined with two enzyme treatments and ultrasound combined with pectinase treatment (P>0.05). Therefore, the ultrasonic combined with pectinase enzymatic hydrolysis method was ultimately selected for extracting crude protein from Sargassum.

[0054] The extraction rate of Sargassum protein can be improved by optimizing the reaction conditions of ultrasound combined with pectinase hydrolysis: such as... Figure 1As shown, the protein extraction rate reached its highest when the material-to-liquid ratio was 1:15 (g / mL). However, the extraction rate decreased linearly with increasing material-to-liquid ratio. This may be because an excessively high solvent ratio reduced the contact area between the algae powder and the enzyme, thus decreasing the enzymatic hydrolysis efficiency. When the reaction temperature was controlled at 50°C... The protein extraction rate was highest at ℃, and decreased slightly with further heating, possibly because this temperature exceeded the optimal reaction temperature of pectinase, leading to reduced enzyme activity. The protein extraction rate showed an upward trend in the first 4 hours, then leveled off or slightly decreased after the 4th hour, possibly due to prolonged reaction time causing decomposition of soluble proteins. The protein extraction rate initially increased, then decreased, and finally leveled off with the amount of pectinase added. For cost considerations, subsequent experiments used an enzyme dosage of 1200U. Finally, comparing the effects of different pH values ​​on the protein extraction rate of Sargassum, the protein extraction rate initially increased and then decreased with increasing pH, reaching its maximum at pH 5. Excessively high or low pH values ​​affected pectinase activity, thus affecting the protein extraction rate.

[0055] In summary, the method of ultrasonic treatment combined with pectinase was finally selected for the extraction of crude protein from Sargassum. The reaction conditions were set as follows: enzyme dosage 1200U, material-to-liquid ratio 1:15 (g / mL), temperature 50 ℃, pH 5, and reaction time 4h.

[0056] 1.3.2 Isolation and purification of Sargassum protein The crude extract of Sargassum protein contains high levels of impurities such as polysaccharides and polyphenols. Salting-out precipitation can separate the proteins in the crude extract without altering their properties. As shown in Table 4, when the ammonium sulfate saturation in the solution is between 10% and 40%, the protein content in the crude Sargassum protein increases with increasing ammonium sulfate saturation, reaching a peak at 40% saturation, where the protein content increases to 51.32 ± 2.06%. Further increases in ammonium sulfate saturation gradually decrease the protein content. Therefore, the protein precipitated at 40% ammonium sulfate saturation was collected, desalted, and freeze-dried for further enzymatic hydrolysis.

[0057] Table 4 Results of ammonium sulfate fractionation precipitation of Sargassum protein Protein content (%) 31.64±3.26 33.78±1.26 42.32±1.56 51.32±2.06 47.64±1.26 28.65±1.35 18.34±1.64 11.37±1.87 .

[0058] 1.3.3 Preparation and Screening of Sargassum Immunomodulatory Peptide Components This invention uses four commonly used proteases to hydrolyze Sargassum protein, and screens proteases with higher hydrolysis efficiency by measuring the degree of hydrolysis. For example... Figure 2As shown in Figure A, the degree of hydrolysis of the four proteases all showed an increasing trend within the first 120 min of the reaction; after 120 min, the increase in the degree of hydrolysis of each treatment group slowed down significantly or tended to level off. The final results showed that the flavor protease had the highest degree of hydrolysis (23.48% ± 0.87%), followed by alkaline protease (21.93% ± 0.68%) and neutral protease (20.84% ​​± 0.72%), while trypsin had the lowest degree of hydrolysis (15.28% ± 0.58%).

[0059] This invention performs preliminary screening of the immunomodulatory activity of four enzymatic hydrolysates. For example... Figure 2 As shown in Figure B, compared with the control group, none of the four enzymatic hydrolysates showed a significant proliferation-promoting effect on macrophages at a low concentration of 50 μg / mL (P>0.05); however, at a high concentration of 500 μg / mL, all four components exhibited a significant proliferation-promoting effect (P<0.01). Among them, the alkaline protease component had the highest proliferation rate (132.4% ± 3.55%), followed by the flavor protease component (129.0% ± 4.51%), while the neutral protease (114.8% ± 6.24%) and trypsin (111.8% ± 3.42%) components had relatively low activities. Considering both the degree of hydrolysis and cell proliferation activity results, the alkaline protease and flavor protease not only have high hydrolysis efficiency, but their enzymatic hydrolysates also exhibit excellent immunomodulatory activity, and therefore were selected for subsequent compound enzymatic hydrolysis experiments.

[0060] Figure 2 China E and Figure 2 The results showed that, compared with the single enzymatic hydrolysis component, the composite enzymatic hydrolysis component had a higher proportion of low molecular weight peptides, with the 0-1 kDa component accounting for 49% and the 1-3 kDa component accounting for 24.41%. Simultaneously, the degree of hydrolysis of the composite enzymatic hydrolysis (24.1% ± 0.68%) and the rate of macrophage proliferation promotion by its hydrolysis products (135.2% ± 4.35%) were significantly higher than those of the single enzymatic hydrolysis component (P<0.01). These results indicate that composite enzymatic hydrolysis can more fully hydrolyze Sargassum protein, producing more low molecular weight peptides and exhibiting superior immunomodulatory activity. Therefore, the peptide component prepared by composite enzymatic hydrolysis of alkaline protease and flavor protease was selected for subsequent experiments.

[0061] 1.3.3.1 Ultrafiltration Fractionation and Screening of Active Components To identify key immunomodulatory peptides in Sargassum protein hydrolysate, this invention first fractionated the hydrolysate using ultrafiltration to obtain three peptide fractions with different molecular weight ranges: >10 kDa, 3-10 kDa, and <3 kDa. The effects of each fraction on the proliferation activity of RAW264.7 cells were then evaluated. Figure 3As shown in Figure A, compared with the control group, all three components significantly promoted cell proliferation at a concentration of 500 μg / mL (P<0.01). Among them, the <3 kDa component exhibited the strongest proliferative activity, with a cell proliferation rate of 142.4% ± 3.97%, significantly higher than the other two components (P<0.05 or P<0.01), and showed no significant difference from the positive control group. Low molecular weight peptides generally have better migration and diffusion capabilities, which is beneficial for interaction with target molecules. These results indicate that <3 kDa peptides are the main contributing components to the immunomodulatory activity of Sargassum protein hydrolysate; therefore, this component was selected for subsequent separation and purification.

[0062] 1.3.3.2 Sephadex G-15 gel filtration chromatography separation To further purify the component with strong immunomodulatory activity, G-15 gel filtration chromatography was used to separate the peptide component <3 kDa. The elution curve was monitored at a wavelength of 214 nm, and three main elution peaks were obtained, named G-F1, G-F2, and G-F3, respectively. Figure 3 (B) Figure 3 The effects of each peak component on the proliferation activity of RAW264.7 cells were evaluated. Compared with the control group, all three components significantly promoted cell proliferation within the tested concentration range (P<0.01). Among them, the G-F3 component showed significantly better proliferative effects than G-F1 and G-F2 under high concentration conditions, with a cell proliferation rate of 135.8% ± 4.53%, which was 1.35 times that of the control group. This result indicates that the G-F3 component is rich in bioactive peptides with immune activation potential, and therefore it was selected as the target component for further isolation and purification.

[0063] 1.3.3.3 RP-HPLC purification and acquisition of target peptide components RP-HPLC is widely used in the separation of bioactive peptides. To obtain highly active immunomodulatory peptides, the G-F3 fraction was separated and purified using RP-HPLC. For example... Figure 3 As shown in Figure D, G-F3 was separated by RP-HPLC, yielding three main absorption peaks, named R-F1, R-F2, and R-F3, respectively. To evaluate the immunomodulatory activity of each component, its effect on macrophage proliferation was detected at concentrations of 50, 200, and 500 μg / mL. Figure 3As shown in Figure E, all three components exhibited significant proliferative activity compared to the control group. R-F3 showed the highest proliferation rate (138.8% ± 2.31%) at a concentration of 200 μg / mL, which was 1.38 times that of the blank control group, and was slightly higher than the other two components at all concentrations. The retention time of R-F3 was approximately 60 min, corresponding to a 50%–100% acetonitrile linear gradient elution, indicating its strong hydrophobicity. These results demonstrate that the R-F3 peptide component exhibits stronger hydrophobicity and superior immunomodulatory activity. R-F3 was subsequently collected for further identification of the peptide components.

[0064] Example 2: Identification, virtual screening, and in vivo activity screening of Sargassum immunomodulatory peptides 2.1 Identification of immunomodulatory peptide sequences by HPLC-MS / MS The R-F3 peptide fraction separated from RP-HPLC was redissolved in 0.1% formic acid solution, centrifuged at high speed, and the supernatant was desalted by C18 chromatography and lyophilized before analysis by LC-MS / MS equipped with an online nanospray ionization source. The entire system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC1200 in series. 3 μL of sample was packed into an analytical column (Acclaim PepMap C18, 75 μm × 25 cm), with a column flow rate controlled at 300 nL / min, a column temperature of 40 °C, and an electrospray voltage of 2 kV. The chromatographic gradient is shown in the table below (mobile phase A: 0.1% formic acid aqueous solution; phase B: ACN solution containing 0.1% formic acid): Table 5 Chromatographic gradient 1 0 98.0 2.0 2 75.00 68.0 32.0 3 84.00 50.0 50.0 4 85.00 0.0 100.0 5 90.00 0.0 100.0 .

[0065] Note: Acetonitrile and water used were Fisher mass spectrometry grade reagents, and formic acid was Sigma chromatographic grade reagent.

[0066] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: scan range (m / z) = 150-1600; resolution = 60000; AGC target = 300; maximum injection time = 50ms; scan charge = 1-6; (2) resolution = 120000; isolation window = 1.6m / z; AGC target = 100; maximum injection time = 50ms; collision energy = 35.

[0067] Tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). First, the possible amino acid combinations were calculated based on the molecular weight information of the fragments in each MS / MS spectrum, and then compared with the protein data from the sample source. Error: 0.02 Da, precursor ion mass tolerance: 10 ppm, maximum missed cuts: 2, variable modifications: Oxidation (M) 15.99, Acetylation (Protein N-term) 42.01. Peptide kinematic value: -10lgP ≥ 15.

[0068] 2.2 Computerized screening of immunomodulatory peptide sequences Based on bioactive peptide sequence analysis, low molecular weight peptides were found to be more active than high molecular weight peptides, and most contained 3-10 amino acid residues. Therefore, sequences with <10 amino acid residues were selected for further analysis. The Peptide Ranker software (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict and identify the potential biological activity of peptide sequences; peptide sequences with a prediction score >0.8 were considered to have potential biological activity. Furthermore, the Pre AIP software (http: / / www.thegleelab.org / AIPpred / ) was used to predict the anti-inflammatory activity of peptides; a prediction value >0.35 was considered to indicate good anti-inflammatory activity. Finally, the immunomodulatory activity of bioactive peptides was closely related to the amino acid sequence composition, the proportion of hydrophobic amino acids, and the number of basic amino acid residues. The Toxin Pred software (http: / / crdd.osdd.net / raghava / toxinpred / ) was used to predict the potential toxicity of peptides and analyze their physicochemical properties, such as molecular weight, isoelectric point, hydrophobicity, and charge ratio.

[0069] Therefore, peptide sequences with potential immunomodulatory activity should meet the following conditions: (1) amino acid sequence length of 3-10 amino acid residues; (2) Peptide Ranker score > 0.8; (3) Pre AIP score > 0.35; (4) rich in hydrophobic amino acids and Toxin Pred hydrophobic score > 0.2.

[0070] 2.3 Molecular docking of immunomodulatory peptides and receptors TLR2 / TLR4 Two-dimensional chemical structures of candidate peptides were drawn using ChemDraw 20.0 software, and then imported into Chem3D 20.0 software for conformational optimization and energy minimization to generate three-dimensional structures, which were exported in PDB format as docking ligands. The three-dimensional crystal structures of Toll-like receptor proteins TLR2 (PDB ID: 1FYW) and TLR4 / MD-2 (PDB ID: 5IJD) were downloaded from the RCSB protein database (http: / / www.rcsb.org / pdb). Receptor structures were processed using PyMOL 2.5.2 software to remove water molecules and small organic molecules, and exported in PDB format. Subsequently, hydrogen atoms were added to the receptor structures using AutoDockTools 1.5.7 software, and the structures were exported in pdbqt format as docking receptors. Molecular docking of the receptors and ligands was performed using AutoDock Vina 1.1.2 software, and the visualization analysis of the docking results was completed using PyMOL 2.5.2 and Discovery Studio 2019 software.

[0071] 2.4 Synthesis of Immunomodulatory Peptides The immunomodulatory peptides LLFP, GPLFW, VPVFF, LPLLF, LPVPF, and LNFPLL, identified by LC-MS / MS, were synthesized by Wuxi Maituo Biotechnology Co., Ltd. (Wuxi, Jiangsu). The purity of the synthesized peptides was determined by high-performance liquid chromatography (HPLC), and the purity was >95%. The synthesized peptides were stored at -80°C before use. Their effects on the proliferation and phagocytosis of RAW264.7 macrophages, as well as their ability to regulate NO release, were further screened to identify the immunomodulatory peptides with optimal activity.

[0072] 2.5 Assay of phagocytic activity The phagocytic capacity of RAW264.7 macrophages was assessed using neutral red staining. Logarithmically growing RAW264.7 cells were cultured at a concentration of 5 × 10⁻⁶ cells / cells. 4Cells were seeded at a density of 1:10 / well in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. Subsequently, following the above method, the original culture medium was discarded, and different concentrations (50, 100, 200 μg / mL) of synthetic peptide sample solution or LPS (1 μg / mL) were added for treatment. A blank group (cell-free culture medium) and a control group (complete culture medium) were also set up, and the cells were cultured for another 24 h. After culture, the supernatant was discarded, and the cells were washed twice with PBS. 220 μL of neutral red staining working solution (neutral red: cell culture medium = 1:10, v / v) was added to each well, and the cells were incubated at 37 ℃ in the dark for 2 h. The staining solution was discarded, and the cells were washed three times with PBS. 200 μL of cell lysis buffer (acetic acid: ethanol = 1:1, v / v) was added to each well, and the cells were shaken at room temperature for 15 min until complete lysis. The absorbance of each well was measured at 540 nm. The phagocytic rate of each group was calculated using the following formula: .

[0073] 2.6 Measurement of cellular NO, TNF-α and IL-6 secretion RAW 264.7 cells were divided into groups of 1×10⁻⁶. 5 Cells were seeded at a density of 24 wells. After the cells were fully adhered, they were treated with different concentrations of synthetic peptide sample solution or LPS as described in the previous section, and a blank control (complete culture medium) was set up. After culturing for another 24 hours, the cell culture supernatant of each well was collected.

[0074] Subsequently, 50 μL of cell supernatant from each group was taken, and 50 μL of Griess Reagent I and Griess Reagent II reagents were added sequentially. The mixture was incubated at room temperature in the dark for 10 minutes, and the absorbance of each well was measured at a wavelength of 540 nm. The concentration of NO metabolites in the sample was calculated according to the sodium nitrite standard curve. The results are expressed in μmol / L (µM).

[0075] Take 100 µL of supernatant from each group and strictly follow the ELISA kit instructions to detect the concentrations of TNF-α and IL-6: after serial dilution of pre-coated plates with standards, sample incubation (37℃, 1 h), biotinylated antibody binding (37℃, 1 h), horseradish peroxidase-labeled streptavidin color development for 20 min, and TMB substrate to terminate the reaction, the absorbance was measured at 450 nm.

[0076] 2.7 Discussion of Results 2.7.1 Sequence Identification and Computer Simulation Screening of Immunomodulatory Peptides To further understand the peptide composition and amino acid sequence of R-F3, it was characterized using LC-MS / MS. Figure 4The total ion current (TIC) chromatogram of R-F3 is shown. Denova identified 581 peptide chains (ALC > 99%) with molecular weights ranging from 300 Da to 1000 Da. Toxin Pred software prediction showed that all identified peptides were non-toxic. Subsequently, peptides were used for activity prediction using Peptide Ranker software, typically screened with an activity score > 0.8. After screening, 168 peptides had scores > 0.8. Furthermore, these peptides were used to predict anti-inflammatory potential using the Pre AIP database, and peptides with prediction scores > 0.35 were retained. Finally, the hydrophobicity and isoelectric point of the peptides were predicted using Toxin Pred software, and after comparing the structural characteristics of the peptides, 34 potential immunomodulatory peptides (SEQ ID NO. 1-SEQ ID NO. 34) were screened.

[0077] These 34 peptides were molecularly docked with TLR4 / MD-2 and TLR2. The docking results (Table 6) showed that most peptides strongly bound to the TLR4 / MD-2 and TLR2 receptors. The docking results of six peptides—LLFP, GPLFW, VPVFF, LPLLF, LPVPFPF, and LNFPLL—were particularly significant. Their binding energies to the TLR4 / MD-2 receptor were all ≤-9.0 kcal / mol, and their binding energies to the TLR2 receptor were all -7 kcal / mol or lower. This demonstrates their stable binding to the TLR4 / MD-2 and TLR2 receptors, suggesting they may be the most promising immunomodulatory peptides. Therefore, these six peptides were ultimately selected for solid-phase synthesis and further validated through in vitro immune cell experiments.

[0078] Table 6. Computer simulation analysis and molecular docking binding energy of potential immunomodulatory peptides SEQ ID NO.1 GPLFW 5 618.8 0.98782 0.32 0.4395 -9.0 -7.4 SEQ ID NO.2 LNFPLL 6 715.98 0.903662 0.25 0.4116 -9.2 -7.8 SEQ ID NO.3 LPLLF 5 601.86 0.910462 0.43 0.5233 -9.4 -8.6 SEQ ID NO.4 LLFP 4 488.68 0.898147 0.40 0.5721 -9.0 -7.1 SEQ ID NO.5 LLFF 4 538.74 0.981767 0.57 0.5256 -9.2 -5.6 SEQ ID NO.6 VWPL 4 513.69 0.885102 0.34 0.4488 -7.3 -4.8 SEQ ID NO.7 FPSF 4 496.6 0.985146 0.22 0.5535 -8.2 -6.5 SEQ ID NO.8 WPLL 4 527.72 0.967302 0.34 0.5953 -5.6 -6.6 SEQ ID NO.9 LPYF 4 538.69 0.951316 0.27 0.4744 -7.9 -4.8 SEQ ID NO.10 FPGL 4 432.57 0.972542 0.31 0.4093 -8.1 -5.9 SEQ ID NO.11 LLFY 4 554.74 0.845495 0.42 0.5372 -6.7 -6.3 SEQ ID NO.12 LPLW 4 527.72 0.949268 0.34 0.4395 -7.1 -6.0 SEQ ID NO.13 LGFP 4 432.57 0.945991 0.31 0.5674 -7.9 -4.5 SEQ ID NO.14 LLLF 4 504.73 0.884124 0.55 0.5953 -5.4 -2.1 SEQ ID NO.15 FFGY 4 532.64 0.987309 0.35 0.4605 -7.8 -5.4 SEQ ID NO.16 FPFP 4 506.64 0.994573 0.27 0.4698 -7.7 -6.1 SEQ ID NO.17 FPFF 4 556.7 0.998038 0.44 0.4651 -7.6 -4.6 SEQ ID NO.18 NLFF 4 539.68 0.969944 0.28 0.4372 -6.7 -3.1 SEQ ID NO.19 NLLF 4 505.67 0.820842 0.26 0.5372 -7.3 -5.7 SEQ ID NO.20 TPFF 4 510.63 0.971129 0.24 0.4814 -7.2 -4.5 SEQ ID NO.21 WGPV 4 457.58 0.897943 0.25 0.4209 -7.0 -6.1 SEQ ID NO.22 WPVL 4 513.69 0.880808 0.34 0.5023 -6.9 -5.4 SEQ ID NO.23 SYLF 4 528.65 0.903725 0.23 0.514 -6.8 -5.2 SEQ ID NO.24 VPVFF 5 607.81 0.866925 0.45 0.4488 -9.1 -7.0 SEQ ID NO.25 TPLFF 5 623.81 0.945039 0.30 0.5186 -8.5 -6.3 SEQ ID NO.26 LPGYL 5 561.75 0.821452 0.23 0.4372 -8.6 -5.8 SEQ ID NO.27 FPVLL 5 587.83 0.830173 0.43 0.5419 -8.3 -6.3 SEQ ID NO.28 TPVFF 5 609.78 0.914647 0.30 0.5116 -8.2 -7.3 SEQ ID NO.29 GPVFF 5 565.73 0.963814 0.37 0.4256 -8.2 -7.2 SEQ ID NO.30 VPLLF 5 587.83 0.821033 0.43 0.4884 -8.1 -5.3 SEQ ID NO.31 LPGGF 5 489.64 0.956181 0.28 0.4419 -7.0 -6.8 SEQ ID NO.32 FPGSF 5 553.67 0.977085 0.21 0.4837 -8.1 -4.6 SEQ ID NO.33 LPVPPF 6 668.91 0.914647 0.24 0.3721 -9.1 -7.1 SEQ ID NO.34 DVAFPF 6 694.85 0.904959 0.20 0.4000 -8.3 -6.5 .

[0079] 2.7.2 Synthesis of Immunomodulatory Peptides To verify the immunomodulatory potential of the selected peptides, six candidate peptides were synthesized based on their presence after hydrolysis and the safety and activity predicted by computer analysis. Specifically, LLFP (97.28%), GPLFW (99.94%), VPVFF (98.24%), LPLLF (99.41%), LPVPF (97.53%), and LNFPLL (97.10%), all with purities exceeding 97% and molecular weight ratios (MW) of 488.68, 618.8, 607.81, 601.86, 668.91, and 715.98 Da, respectively.

[0080] 2.7.3 Immunomodulatory activity of immunomodulatory peptides on RAW264.7 cells The effects of six synthetic peptides on the proliferation rate of RAW 264.7 macrophages are shown in [the table below]. Figure 5 Compared with the control group, except for LLFP and LPVPF, the other four synthetic peptides significantly increased cell proliferation (P<0.01). Among them, LNFPLL and LPLLF showed the most prominent promoting effects, with cell proliferation rates reaching 119.2±2.56% and 120.6±4.22% at a concentration of 200 μg / mL, respectively, which were 1.19 times and 1.20 times that of the control group. In addition, LPLLF showed a significant dose-dependent enhancing effect in the concentration range of 50–200 μg / mL. The above results indicate that synthetic peptides VPVFF, GPLFW, LNFPLL, and LPLLF can effectively improve the survival rate and promote the proliferation of RAW 264.7 cells.

[0081] This invention uses neutral red staining to evaluate the effects of six synthetic peptides on the phagocytic function of RAW 264.7 macrophages. The results are as follows: Figure 5 As shown in Figure B, the phagocytic rate of cells in the LPS-treated group was significantly increased compared to the control group (P<0.01), reaching 1.59 times that of the control group. Notably, except for the peptide LPVPF, the phagocytic rates of the other five peptide treatment groups were all significantly increased; among them, the enhancing effects of peptides GPLFW, LNFPLL, and LPLLF were particularly prominent. Specifically, the phagocytic rates of GPLFW and LNFPLL reached 125.6±5.27% and 127.0±2.82%, respectively, under low concentration conditions; while the phagocytic rates of LPLLF reached the highest at concentrations of 100 and 200 μg / mL, at 133.4±5.76% and 130.6±4.46%, respectively, both significantly increased compared to the control group (P<0.01). These results indicate that the synthetic peptides GPLFW, LNFPLL, and LPLLF can significantly enhance the phagocytic capacity of RAW 264.7 cells, suggesting that they may exert immunomodulatory activity by enhancing phagocytic function.

[0082] The effect of synthetic peptides on NO release in RAW 264.7 cells, such as Figure 5As shown in Figure C, compared with the control group, the NO release levels in the LPS group and each peptide treatment group were increased to varying degrees. The LPS group significantly induced NO release (P<0.01); in the synthetic peptide treatment groups, except for LPF PPF, the other five peptides significantly promoted NO secretion. Further analysis showed that GPLFW at a high concentration (200 μg / mL) significantly increased cellular NO release levels, reaching 10.36±0.91 μM; LNFPLL at concentrations of 100 and 200 μg / mL significantly increased NO release, reaching 11.70±0.86 μM and 11.50±0.62 μM, respectively; while LPLLF showed the most significant promoting effect on NO production at high concentrations, reaching a maximum value of 12.96±0.81 μM. These results indicate that the synthetic peptides GPLFW, LNFPLL, and LPLLF can significantly enhance the NO release capacity of RAW 264.7 cells, suggesting that they may be the most immunomodulatory active peptides.

[0083] The above experimental results show that the three peptides LPLLF, GPLFW, and LNFPLL significantly promoted the proliferation, phagocytic capacity, and NO release of RAW 264.7 cells, demonstrating strong immunomodulatory activity. This may be related to the high content of hydrophobic amino acids and immunostimulation-related residues in their sequences. Therefore, LNFPLL, GPLFW, and LPLLF were selected for subsequent experiments to further verify their immunomodulatory effects and explore their mechanisms of action.

[0084] 2.7.4 Effects of Immunomodulatory Peptides on Cellular ROS Production This invention uses the DCFH-DA fluorescent probe to detect the intracellular ROS levels in RAW 264.7 cells after treatment with different concentrations of peptides. The results are as follows: Figure 6 As shown in Figure A, compared with the blank control group, the relative fluorescence intensity of intracellular ROS in each peptide treatment group increased to varying degrees. Among them, the ROS-specific fluorescence signal in the LPLLF and LNFPLL treatment groups showed a significant dose-dependent enhancement. The relative fluorescence intensity of LPLLF at a concentration of 200 μg / mL reached 48.7±0.9%, which was significantly higher than that of the blank control group (P<0.01). The relative fluorescence intensity of GPLFW reached a maximum of 39.64±0.69% at 100 μg / mL, but the fluorescence intensity decreased significantly after further increasing the concentration, suggesting that this peptide may inhibit ROS-related signaling pathways under high concentration conditions. The above results indicate that the three immunomodulatory peptides may enhance the immune response by activating the redox signaling network of RAW 264.7 cells and promoting ROS production. Their mechanism of action may be related to the regulation of the downstream NF-κB signaling pathway.

[0085] 2.7.5 Effects of Immunomodulatory Peptides on Cellular NO Production like Figure 6 As shown in Figure B, after treatment with LNFPLL, GPLFW, and LPLLF, NO secretion in RAW264.7 cells exhibited a concentration-dependent increase, with NO release increasing from a minimum of 6.84±0.68 μM to 13.16±0.48 μM. The promoting effect was particularly significant at high concentrations, with NO release increasing to 1.78, 1.65, and 1.99 times that of the blank control group (6.62±0.24 μM), respectively, after treatment with the three peptides. The results indicate that the three bioactive peptides significantly promoted NO production in RAW264.7 cells.

[0086] 2.7.6 Effects of immunomodulatory peptides on cytokine production like Figure 7 China A and Figure 7 As shown in Figure B, compared with the blank control group, the secretion of TNF-α (75.27 pg / mL) and IL-6 (381.65 pg / mL) in RAW264.7 cells treated with LPS was significantly increased (P < 0.01). Peptide treatment also had the same promoting effect. In terms of TNF-α, the LPLLF group had the highest TNF-α secretion (47.71 pg / mL) (200 μg / mL), which was 2.55 times that of the blank control group. Among them, the TNF-α secretion of cells in the LNFPLL and GPLFW treatment groups showed a trend of first increasing and then decreasing with the increase of peptide concentration. This may be because the high peptide concentration induces cell stress or early apoptosis, thereby damaging cell metabolism and immune response activity. Regarding IL-6 secretion, the GPLFW group, under high concentration (200 μg / mL) conditions, achieved an IL-6 secretion level of 142.48 pg / mL, significantly higher than the blank control group (P < 0.01), which was 2.88 times that of the blank control group. LPLLF and LNFPLL also significantly increased IL-6 secretion levels at concentrations of 200 μg / mL and 100 μg / mL, respectively, to 2.51 times and 2.31 times that of the blank control group. The results indicate that all three bioactive peptides can stimulate RAW264.7 cells to secrete TNF-α and IL-6, and regulate the immune response through related signaling pathways.

[0087] In summary, the three immunomodulatory peptides LNFPLL, GPLFW, and LPLLF screened from Sargassum in this invention all exhibit significant immunoactivating activity. They can dose-dependently promote the proliferation of RAW 264.7 macrophages, enhance phagocytic capacity, and effectively induce the release of immune effector molecules such as NO, TNF-α, and IL-6, while upregulating intracellular ROS levels, suggesting that they may exert their immunomodulatory effects by activating the macrophage redox signaling network and the downstream NF-κB pathway. Molecular docking results further show that these three peptides have strong binding affinity to TLR4 / MD-2 and TLR2 receptors (binding energies ≤-9.0 kcal / mol and ≤-7.4 kcal / mol, respectively), suggesting that TLR-mediated signal transduction may be one of the key molecular mechanisms by which they exert their activity. In conclusion, the peptides represented by SEQ ID NO. 1-3, as novel marine-derived immunomodulators, have broad application prospects in the field of immunotherapy.

Claims

1. A Sargassum immunomodulatory peptide, characterized in that, The immunomodulatory peptides include at least one of the peptides whose amino acid sequences are shown in SEQ ID NO. 1-3.

2. A composition, characterized in that, The composition comprises the immunomodulatory peptide of claim 1 and pharmaceutically acceptable excipients.

3. The use of the immunomodulatory peptide as described in claim 1 or the composition as described in claim 2 in the preparation of immunomodulatory drugs.

4. A method for preparing an immunomodulatory peptide as described in claim 1 or a composition as described in claim 2, characterized in that, Includes the following steps: S.1 Extract crude protein from Sargassum, salt out, and freeze dry; S.2 Add alkaline protease and flavor protease to the protein obtained in S.

1. After enzymatic hydrolysis, heat in a boiling water bath, centrifuge the hydrolysate and collect the supernatant, adjust the pH, add anhydrous ethanol, concentrate and freeze dry. S.3 Ultrafiltration removes peptides with a molecular weight cutoff of less than 3 kDa, separates and purifies them, and identifies the peptide sequences; S.4 The peptide described in S.3 is molecularly docked with the receptor TLR2 / TLR4, and the most immunomodulatory active peptide is screened and synthesized to obtain the Sargassum immunomodulatory peptide.

5. The preparation method according to claim 4, characterized in that, In S.1, crude protein from Sargassum is extracted by ultrasound combined with pectinase hydrolysis. The specific conditions are: enzyme dosage of 600-2400 U, material-to-liquid ratio of 1:10-30 g / mL, temperature of 30-60℃, pH of 3-6, and reaction time of 2-6 h.

6. The preparation method according to claim 4, characterized in that, The salting out described in S.1 involves adding 10%-80% saturated ammonium sulfate to the crude protein extract of Sargassum.

7. The preparation method according to claim 4, characterized in that, The total amount of alkaline protease and flavor protease added in S.2 is 3000-4000 U / g; specifically, crude protein and deionized water are mixed at a ratio of 1:10-30 w / v, alkaline protease is added first, and the reaction is carried out at pH=8-10 and temperature 45-55℃ for 2-4 h; after the reaction is completed, the protein is heated in a boiling water bath to inactivate the enzyme; after cooling to room temperature, flavor protease is added, and the protein is enzymatically hydrolyzed at pH=6-8 and temperature 45-55℃ for 2-4 h.

8. The preparation method according to claim 4, characterized in that, In S.3, peptides with a molecular weight less than 3 kDa were separated and purified by Sephadex G-15 gel filtration chromatography and RP-HPLC. The obtained peptide sequences were identified by LC-MS / MS.

9. The preparation method according to claim 4, characterized in that, In S.4, the most immunomodulatory active peptides were screened according to the following conditions: (1) amino acid sequence length of 3-10 amino acid residues; (2) Peptide Ranker score > 0.8; (3) Pre AIP score > 0.35; (4) rich in hydrophobic amino acids and Toxin Pred hydrophobic score > 0.

2.

10. The preparation method according to claim 4, characterized in that, The amino acid sequence of the Sargassum immunomodulatory peptide is shown in SEQ ID NO.1-3.

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