Nereis glycopeptide SC2-3 as well as preparation method and application thereof

Abstract

The invention discloses a nereis glycopeptide SC2-3 as well as a preparation method and application thereof, and the preparation method comprises the following steps: firstly extracting crude polysaccharide from nereis, and then identifying by means of column chromatography purification, spectroscopy and the like to obtain the nereis glycopeptide SC2-3 with the relative molecular mass of 5061Da. The glycopeptide SC2-3 is obtained by taking nereis as a raw material, and pharmacological experiments show that the nereis glycopeptide SC2-3 has immunomodulatory activity, can improve low mouse immunologic function induced by cyclophosphamide chemotherapeutic drugs, and is expected to be developed into an immunologic adjuvant for assisting in resisting tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide extraction, and relates to polysaccharide substances, their extraction methods and their uses in the preparation of drugs and functional products. Specifically, it relates to a method for extracting glycopeptide SC2-3 from nereis, the glycopeptide extracted by this method, and the use of the glycopeptide as an immune adjuvant. Background Art

[0002] With the continuous improvement of people's attention to health, the importance of immune regulation has become increasingly prominent. Immunity is the first line of defense in the human body's defense mechanism, which can recognize, attack and eliminate viruses, bacteria, fungi and other pathogens, protecting the body from infection. Immune regulation is of great significance in preventing infection, promoting recovery, and preventing chronic diseases. In recent years, significant progress has been made in the research of immune metabolism, neuro-immune regulation, immunosenescence, tumor immunotherapy, traditional Chinese medicine immune regulation, and vaccine adjuvant research and development, providing a theoretical basis and practical guidance for the development of new immune regulators and treatment methods.

[0003] Polysaccharides are high-molecular compounds formed by the linkage of multiple monosaccharide molecules through glycosidic bonds. They belong to a type of carbohydrate and are widely present in organisms such as higher plants, animals, microorganisms, lichens, and seaweeds. For example, in the seed, stem and leaf tissues of plants, animal mucus, the shells of insects and crustaceans, fungi, and the intracellular and extracellular parts of bacteria. Polysaccharides have a variety of important physiological functions, including energy supply, cell structure support, immune regulation, antioxidant, anti-inflammatory, anti-tumor and antiviral effects. In terms of applications, polysaccharides are used as thickeners and stabilizers in the food industry to improve the texture and stability of foods; in the pharmaceutical field, polysaccharides can be used to develop immune regulators, anti-tumor drugs and antiviral drugs; in addition, polysaccharides are also widely used in the development of health products and other industrial fields. Due to their rich sources and diverse functions, they show broad application prospects.

[0004] In recent years, significant progress has been made in the research of polysaccharides in the field of immunomodulation. For example, the combined use of lentinan, pachyman, and tremella polysaccharides can regulate the functions of T cells and B cells, enhance the activity of NK cells, and improve the phagocytic ability of macrophages. Lycium barbarum polysaccharide can enhance blood circulation and cardiac function, thereby improving immunity. Polysaccharides in sea urchin eggs (such as MGSA) can activate the NF-κB and MAPKs (ERK1 / 2 and JNK) signaling pathways, up-regulate the expression of inflammation-related enzymes and pro-inflammatory mediators, and polarize tumor-associated macrophages (TAMs) into the M1 phenotype. Astragalus polysaccharide (APS) has shown significant immunomodulatory activity in in vitro and in vivo experiments, can enhance the M1 polarization of macrophages, and down-regulate the M2 polarization induced by IL-4 and IL-13. These studies reveal that polysaccharides have significant potential in immune enhancement and regulation, and have broad application prospects in the fields of anti-tumor, anti-virus, and vaccine development.

[0005] Nereis, also known as sea centipede, sea leech, sandworm, etc., is a marine annelid living on the muddy beach in the intertidal zone. Nereis is rich in protein, amino acids, unsaturated fatty acids, trace elements, and vitamins, and has high nutritional and medicinal values. Nereis was recorded in ancient herbal literature and was used to treat various diseases, including invigorating the spleen and stomach, enriching blood, promoting diuresis to alleviate edema, expelling wind and removing dampness, etc., and its edible and medicinal values are widely recognized. Research shows that Nereis has anticoagulant, thrombolytic, anti-tumor, and anemia-improving effects, and it has a long application history in the field of traditional Chinese medicine. In recent years, significant progress has also been made in the research of the activities of Nereis polysaccharides, which have antioxidant, immunomodulatory, and liver-protecting activities. At present, there are few reports on the research of the fine component structure and activities of polysaccharides derived from Nereis. The Nereis glycoprotein SC2-3 prepared in this invention has significant immunomodulatory activity, can improve the immune function deficiency induced by cyclophosphamide chemotherapy drugs in mice, and is expected to be developed into an immune adjuvant for assisting anti-tumor, which is beneficial to improving the deep processing and development and utilization value of Nereis. Summary of the Invention

[0006] This invention uses a simple and effective glycoprotein extraction process and method to obtain a glycoprotein SC2-3 from Nereis. Pharmacological experiments show that this Nereis glycoprotein SC2-3 has immunomodulatory activity, can reverse the immune function deficiency induced by cyclophosphamide chemotherapy drugs in mice, and is expected to be developed into an immune adjuvant for assisting anti-tumor.

[0007] One object of this invention is to provide a Nereis glycoprotein SC2-3.

[0008] Another object of this invention is to provide a preparation method of the above-mentioned Nereis glycoprotein SC2-3.

[0009] Another object of the present invention is to provide the use of nereis glycopeptide SC2-3 in the preparation of an immunoadjuvant for promoting inflammation and assisting in enhancing immunity.

[0010] To achieve the above object of the invention, the following technical solutions are adopted:

[0011] The present invention provides a method for preparing nereis glycopeptide SC2-3, comprising the following steps:

[0012] (a) Glycopeptide extraction: Take fresh nereis tissue, homogenize it, treat it with acetone, centrifuge and dry to obtain a defatted sample powder; add water and extract it successively with papain and trypsin to obtain crude nereis polysaccharide; take the crude nereis polysaccharide, dissolve it in water, then add trichloroacetic acid to remove precipitated proteins; dialyze, concentrate, and freeze-dry to obtain protein-free crude nereis polysaccharide SC.

[0013] (b) Glycopeptide purification: Purify the protein-free crude nereis polysaccharide SC through a DEAE cellulose anion column to obtain nereis polysaccharide SC2; purify the nereis polysaccharide SC2 using a Sephacryl S-100 gel chromatography column to obtain nereis glycopeptide SC2-3.

[0014] More specifically, the method for preparing nereis glycopeptide SC2-3 comprises the following steps:

[0015] (a) Glycopeptide extraction: Wash fresh nereis tissue, homogenize it, stir it with acetone at room temperature for 12 h, discard the supernatant, repeat the operation, centrifuge, air-dry the precipitate at room temperature first and then dry it in an oven at 45 °C, pulverize to obtain a defatted sample powder, add 20-40 times the volume of distilled water, add 2.5% (mass fraction) papain, extract at pH = 5-7 and 60 °C for 12 h, inactivate at 100 °C for 15 min. After cooling, adjust the pH to 7-9, add 2.5% (mass fraction) trypsin, stir and extract at 37 °C for 12 h, inactivate at 100 °C for 15 min. After cooling, centrifuge, concentrate the supernatant, dialyze (molecular weight cut-off of the dialysis bag is 3500 Da) for 3 days, and freeze-dry to obtain a crude nereis polysaccharide fraction. Take the crude nereis polysaccharide, dissolve it in deionized water, add an equal volume of 30% trichloroacetic acid, stir at 4 °C for 3 h, centrifuge to remove the precipitate, and adjust the pH to 7. After protein removal, concentrate the sample, dialyze with a 3500 Da dialysis bag for 3 days, concentrate, and freeze-dry to obtain protein-free crude nereis polysaccharide (SC).

[0016] (b) Glycopeptide purification: Take the crude polysaccharide from nereis after protein removal, dissolve it in deionized water, centrifuge it, and separate the supernatant through a DEAE cellulose anion column. Elute it successively with water, 0.2M NaCl, 0.5M NaCl, 1M NaCl, and 2M NaCl. Detect it by the sulfuric acid-phenol method, collect and combine the 0.2M NaCl eluate, concentrate it, dialyze it, and freeze-dry it to obtain nereis polysaccharide SC2. Take nereis polysaccharide SC2, dissolve it in deionized water, centrifuge it, separate and purify the supernatant using a Sephacryl S-100 gel chromatography column, and elute it with a 0.2M NaCl solution. Combine the eluates to obtain nereis glycopeptide SC2-3.

[0017] Preferably, in step (a), add 30 times the volume of deionized water, adjust the pH to 6 and 8 respectively, and then use papain and trypsin for enzymatic extraction.

[0018] Preferably, the nereis glycopeptide SC2-3 is determined by high performance gel permeation chromatography (HPGPC). Its relative molecular mass is 5.061KDa, and the monosaccharide composition by mass ratio is fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid: glucuronic acid = 6.32: 20.6: 23.93: 9.79: 1.79: 7.49: 2.62: 0.24: 4.85. The determination of physical and chemical properties shows that its total sugar content is 24%, the uronic acid content is 16%, and the protein content is 24%. The amino acid analysis results show that it is mainly composed of glycine (14.2%), aspartic acid (11.8%), glutamic acid (9.75%), and threonine (6.1%). Among them, "%" represents mass fraction.

[0019] The present invention also provides a nereis glycopeptide SC2-3, which is prepared by the preparation method of the nereis glycopeptide SC2-3. The relative molecular mass of the nereis polysaccharide SC2-3 is 1.0 - 100KDa, preferably 5.061KDa.

[0020] The present invention also provides the use of the nereis glycopeptide SC2-3 in the preparation of an immunoadjuvant for promoting inflammation and assisting in enhancing immunity, which can reverse the immune function deficiency induced by cyclophosphamide in mice.

[0021] The present invention also provides a pharmaceutical composition, which contains the nereis glycopeptide SC2-3 and uses it as an active ingredient, and also includes pharmaceutically acceptable excipients.

[0022] The present invention also provides a functional product, which contains the nereis glycopeptide SC2-3 and uses it as an active ingredient.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses nereis as a raw material, and proposes a method for extracting glycopeptide SC2-3 from nereis, the glycopeptide extracted by this method, and the use of the obtained glycopeptide in the preparation of an immunoadjuvant for promoting inflammation and assisting in enhancing immunity. Pharmacological experiments show that the nereis glycopeptide SC2-3 has immunomodulatory activity, can reverse the immune function deficiency induced by cyclophosphamide chemotherapy drugs, and is expected to be developed into an immunoadjuvant for assisting in enhancing immunity. Description of the Drawings

[0025] Figure 1 It is the molecular weight diagram of nereis glycopeptide SC2-3 in Example 1.

[0026] Figure 2 It is the IR diagram of nereis glycopeptide SC2-3 in Example 1.

[0027] Figure 3 It is the pro-inflammatory activity results of nereis glycopeptide SC2-3 in Example 2; a) The effect of nereis glycopeptide SC2-3 on the viability of RAW264.7 cells; b) The effect of nereis glycopeptide SC2-3 on the phagocytic viability of RAW264.7 cells; c) The effect of nereis glycopeptide SC2-3 on the NO secretion of RAW264.7 cells; d) The effect of nereis glycopeptide SC2-3 on the TNF-α secretion of RAW264.7 cells; e) The effect of nereis glycopeptide SC2-3 on the IL-1β secretion of RAW264.7 cells; f) The effect of nereis glycopeptide SC2-3 on the IL-6 secretion of RAW264.7 cells; g) The effect of nereis glycopeptide SC2-3 on the ROS secretion of RAW264.7 cells.

[0028] Figure 4 It is the schematic diagram of the body weight change of mice in Example 3; (where CT is the normal control group, CTX is the model control group, LH is the positive control group, SCL is the low-dose glycopeptide group, SCM is the medium-dose glycopeptide group, and SCH is the high-dose glycopeptide group).

[0029] Figure 5 It is the schematic diagram of the immune organ index and blood routine analysis of mice in each group in Example 3; a) The schematic diagram of the change of thymus index of mice in each group; b) The schematic diagram of the change of spleen index of mice in each group; c) The schematic diagram of the change of white blood cell count in the body of mice in each group; d) The schematic diagram of the change of red blood cell count in the body of mice in each group; e) The schematic diagram of the change of hemoglobin concentration in the body of mice in each group; f) The schematic diagram of the change of lymphocyte count in the body of mice in each group; g) The schematic diagram of the change of eosinophil count in the body of mice in each group; h) The schematic diagram of the change of platelet count in the body of mice in each group; i) The schematic diagram of the change of neutrophil count in the body of mice in each group; (where CT is the normal control group, CTX is the model control group, LH is the positive control group, SCL is the low-dose glycopeptide group, SCM is the medium-dose glycopeptide group, and SCH is the high-dose glycopeptide group).

[0030] Figure 6 Schematic diagram of the changes in inflammatory factors (TNF-α, IL-6, IL-1β) in the sera of mice in each group in Example 3; a) Schematic diagram of the change in TNF-α in the sera of mice in each group; b) Schematic diagram of the change in IL-6 in the sera of mice in each group; c) Schematic diagram of the change in IL-1β in the sera of mice in each group; (where CT is the normal control group, CTX is the model control group, LH is the positive control group, SCL is the low-dose glycopeptide group, SCM is the medium-dose glycopeptide group, and SCH is the high-dose glycopeptide group).

[0031] Figure 7 HE staining map of the spleens of mice in each group in Example 3 (where R is red pulp; W is white pulp, n = 3, CT is the normal control group, CTX is the model control group, LH is the positive control group, 50SC is the low-dose glycopeptide group, 100SC is the medium-dose glycopeptide group, and 200SC is the high-dose glycopeptide group).

[0032] Figure 8 HE staining of the thymuses of mice in each group in Example 3; (where n = 3, CT is the normal control group, CTX is the model control group, LH is the positive control group, CT is the normal control group, CTX is the model control group, LH is the positive control group, 50SC is the low-dose glycopeptide group, 100SC is the medium-dose glycopeptide group, and 200SC is the high-dose glycopeptide group).

[0033] Figure 9 HE staining of the intestines of mice in each group in Example 3; (where the representative photograph shows the histomorphology of the ileum by H&E staining, the scale bar represents 100 μm, and the green and red line segments represent the villus length and crypt depth of the ileum, respectively. CT is the normal control group, CTX is the model control group, LH is the positive control group, CT is the normal control group, CTX is the model control group, LH is the positive control group, 50SC is the low-dose glycopeptide group, 100SC is the medium-dose glycopeptide group, and 200SC is the high-dose glycopeptide group). Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1: Preparation of nereis glycopeptide SC2-3

[0036] a. Glycopeptide extraction: 2000 g of fresh nereis tissue was cleaned and homogenized. It was stirred with acetone at room temperature for 12 h, and the supernatant was discarded. The operation was repeated, followed by centrifugation. After precipitation, it was first air-dried at room temperature and then dried in an oven at 45 °C, and then pulverized to obtain 105.613 g of defatted sample powder, with a yield of 5.3%. 30 times the volume of distilled water was added, along with 2.5% papain (10 U / mg). It was extracted at pH = 6 and 60 °C for 12 h and inactivated at 100 °C for 15 min. After cooling, the pH was adjusted to 8, and 2.5% trypsin (≥250 NFU / g) was added. It was stirred and extracted at 37 °C for 12 h and inactivated at 100 °C for 15 min. After cooling, it was centrifuged. The supernatant was concentrated, dialyzed (the molecular weight cut-off of the dialysis bag was 3500 Da) for 3 days, and freeze-dried to obtain 50 g of crude polysaccharide fraction from nereis. 2 g of the crude polysaccharide from nereis was taken, dissolved in 100 ml of water, 100 ml of 30% trichloroacetic acid was added, and it was stirred at 4 °C for 3 h. The precipitate was removed by centrifugation, and the pH was adjusted to 7. It was dialyzed with a 3500 Da dialysis bag for 3 days, concentrated, and freeze-dried to obtain 442 mg of protein-free crude polysaccharide from nereis (SC).

[0037] b. Glycopeptide purification: 800 mg of the protein-free crude polysaccharide from nereis (SC) was taken, dissolved in deionized water, and centrifuged. The supernatant was separated by a DEAE cellulose anion column, and eluted successively with water, 0.2 M NaCl, 0.5 M NaCl, 1 M NaCl, and 2 M NaCl. Detection was carried out by the sulfuric acid-phenol method, and the 0.2 M NaCl eluate was collected and combined, concentrated, dialyzed, and freeze-dried to obtain 283 mg of polysaccharide SC2 fraction from nereis. 200 mg of polysaccharide SC2 from nereis was taken, dissolved in deionized water, and centrifuged. The supernatant was purified by a Sephacryl S-100 gel chromatography column, eluted with a 0.2 M NaCl solution at a flow rate of 0.3 mL / min. The elution curve was plotted by the sulfuric acid-phenol method, and the eluate was collected and combined according to the elution curve to obtain 65 mg of glycopeptide fraction SC2-3 from nereis.

[0038] c. Determination of physicochemical properties of glycopeptide: The total sugar content was determined to be 24% by the sulfuric acid-phenol colorimetric method, the protein content was determined to be 24% by the BCA method, and the uronic acid content was determined to be 16% by the carbazole-sulfuric acid method. The amino acid composition was analyzed using an amino acid automatic analyzer, indicating that it was mainly composed of glycine (14.2%), aspartic acid (11.8%), glutamic acid (9.75%), and threonine (6.1%). (Table 1)

[0039] Table 1 Amino acid composition of SC2-3

[0040]

[0041] d. Glycopeptide structure analysis: HPGPC analysis showed that the molecular weight of SC2-3 was 5.061 KDa ( Figure 1)。Analysis of monosaccharide composition showed that SC2-3 contained fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, with the ratio of fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid: glucuronic acid = 6.32: 20.6: 23.93: 9.79: 1.79: 7.49: 2.62: 0.24: 4.85. The infrared spectrum showed that 3275 cm -1 was the stretching vibration peak of N-H in the proteome and the stretching vibration peak of O-H in the polysaccharide component, and 2934 cm -1 was the bending and stretching vibrations of C-H, and 1637 cm -1 was the stretching vibration of the amide C=O bond, 1540 cm -1 was the bending vibration peak of the N-H bond, 1401 cm -1 was the stretching vibration peak of C-N, 1247 cm -1 was the stretching vibration peak of C-H, 1020 cm -1 was the C-O-C stretching vibration( Figure 2 ).

[0042] Example 2: Immunostimulatory effect of nereidoglycopeptide SC2-3 on RAW264.7 cells (cellular level)

[0043] I. Test materials

[0044] 1. Instruments and reagents

[0045] Instruments: laminar flow hood, centrifuge, cell incubator, carbon dioxide incubator, full wavelength multifunctional microplate reader, flow cytometer, fluorescence microscope

[0046] Reagents: high-glucose DMEM medium (Gibco), fetal bovine serum (Gibco), SC2-3 was synthesized and provided by Shanghai Ocean University (synthesized by the preparation method of Example 1), purity > 98%, neutral red staining solution, cell lysis solution, NO kit, mouse interleukin (IL-6) kit, mouse tumor necrosis factor (TNF-α) kit, mouse interleukin (IL-1β) kit

[0047] 2. Test cells

[0048] Mouse mononuclear macrophages (RAW264.7)

[0049] II. Test methods

[0050] 1. Cell proliferation experiment (CCK-8)

[0051] Growth conditions: DMEM high-glucose medium with 10% FBS, and the medium was changed once a day. Incubator at 37°C with 5% carbon dioxide. The cells should be cultured until the logarithmic phase, then gently pipetted down and evenly distributed onto a 96-well plate.

[0052] CCK-8 assay procedure: The cells were divided into 9 groups, including 1 blank control group (containing only medium), 1 control group (1 μg / mL LPS - lipopolysaccharide), and 7 experimental groups (containing 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1000 μg / mL SC2-3 polysaccharide solution), with 3 replicates in each group. 100 μL of complete medium was added to each well of the 96-well plate to culture 1×10 4 cells. After culturing for 24 h, the old medium was removed, gently washed with PBS, and 100 μL of medium containing polysaccharide solution at different concentrations was added, without adding in the blank group. After continuing to culture for 48 h, the old medium was discarded, and in the dark, 100 μL of medium containing 10% CCK8 was added to each well, and the absorbance was measured at 450 nm to determine cell viability.

[0053] 2. Neutrophil phagocytosis experiment

[0054] The experiment was divided into 5 groups: 1 blank group (containing only medium), 1 control group (containing 1 μg / mL LPS), and 6 experimental groups (containing 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1000 μg / mL SC2-3 polysaccharide solution), with 3 replicates in each group. 100 μL of complete medium was added to each well of the 96-well plate to culture 1×10 4 cells. After culturing for 24 h, the old medium was discarded, gently washed once with PBS, and then incubated with 0.01% neutral red solution at 37°C for 1 hour. Then, the neutral red stain was gently aspirated, washed three times with PBS, 200 μL of lysis solution was added to each well, and after incubating for 2 h, the absorbance was measured at 540 nm using a multifunctional microplate reader.

[0055] 3. NO content detection

[0056] The experiment was divided into 5 groups: 1 blank group (containing only medium), 1 control group (containing 1 μg / mL LPS), and 5 experimental groups (containing 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL SC2-3 polysaccharide solution), with 3 replicates in each group. 100 μL of complete medium was added to each well of the 96-well plate to culture 2×10 4Cells. After culturing for 48 h, the medium was taken out, centrifuged at 10,000 rpm for 3 min at 4 °C, and the supernatant was taken for standby. Then, 50 μL of the sample in each well of the 96-well plate was taken, 50 μL of Griess reagent I was added first, and then 50 μL of Griess reagent II was added. The mixture was homogenized at room temperature and waited for 5 min for the reaction. Finally, the absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0057] 4. Determination of IL-6, TNF-α, and IL-1β levels (ELISA kit)

[0058] Mouse interleukin (IL-6) kit, mouse tumor necrosis factor (TNF-α) kit, and mouse interleukin (IL-1β) kit were used to detect the levels of cell-secreted factors IL-6, IL-1β, and TNF-α. The cell supernatants prepared above were used for standby. According to the kit instructions, the absorbance was measured at 450 nm using an ELISA reader.

[0059] 5. Determination of ROS level

[0060] The experiment was divided into 5 groups: 1 blank group (containing only medium), 1 control group (containing 1 μg / mL LPS), and 4 test groups (containing 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL SC2-3 polysaccharide solution), with 3 replicates in each group. 100 μL of complete medium was added to each well of the 96-well plate to culture 2×10 4 cells. After culturing for 48 h, under light-proof conditions, 100 μL of the culture solution containing 10 μmol / mL DCFHDA was added and incubated at 37 °C for 20 min. Then, the cells were washed 3 times with serum-free medium, 50 μL of PBS was added, and photos were taken with a fluorescence microscope.

[0061] III. Test results

[0062] Figure 3 a The results showed that compared with the blank control group, the cell viability of the SC2-3 treatment group was higher than that of the blank control group, indicating that SC2-3 was non-toxic to RAW264.7 cells at concentrations between 25 and 1000 μg / mL and could improve cell viability. The above suggested that SC2-3 had an immune-promoting effect on RAW264.7 cells.

[0063] Figure 3b The results showed that compared with the blank control group, the phagocytosis rates of cells in the SC2-3 treatment groups were all higher than those in the blank control group and showed a concentration dependence. It indicated that SC2-3 could improve the phagocytosis ability of RAW264.7 cells between the concentrations of 50 and 1000 μg / mL. Macrophages exert immune functions through endocytosis and enhance the body's immune ability by phagocytosing harmful pathogens and aging cells. The above suggested that SC2-3 enhanced the body's immune ability by enhancing the phagocytosis of RAW264.7 cells.

[0064] Figure 3 c The results showed that compared with the blank control group, 1 μg / mL of LPS could significantly promote the NO release of RAW264.7 cells. At the same time, when the concentration of SC2-3 was in the range of 50 - 800 μg / mL, it could also significantly promote the release of NO by RAW264.7 cells and showed a concentration dependence. NO is a key mediator for macrophages to exert bactericidal and antitumor cell functions and enhances cell immune functions. The above suggested that SC2-3 enhanced the immune activity of macrophages to a certain extent by promoting the release of NO by RAW264.7 cells.

[0065] Figure 3 d, e, f The results showed that compared with the blank control group, SC2-3 could promote the secretion of TNF-α, IL-6, and IL-1β by RAW264.7 cells between the concentrations of 50 and 400 μg / mL and showed a concentration dependence. When stimulated, cells secrete small molecular proteins, known as cytokines, which regulate the intensity and duration of the body's inflammatory response. The above suggested that SC2-3 effectively promoted the expression of inflammatory mediators, could cause an inflammatory response of macrophages to a certain extent, and had an immune-enhancing effect.

[0066] Figure 3 g The results showed that compared with the blank, SC2-3 could promote the ROS release of RAW264.7 cells between the concentrations of 50 and 400 μg / mL and showed a concentration dependence. During the activation process of macrophages, ROS is usually produced along with the secretion of NO. The above suggested that SC2-3 enhanced the immune activity of macrophages by promoting the release of ROS by macrophages.

[0067] Example 3: Immunomodulatory effect of nereidoglycopeptide SC2-3 on mice (at the animal level)

[0068] I. Test materials

[0069] 1. Instruments and reagents

[0070] Instruments: Full-wavelength multifunctional microplate reader, centrifuge, analytical balance.

[0071] Reagents: Cyclophosphamide for injection, SC2-3 was synthesized and provided by Shanghai Ocean University (synthesized by the preparation method of Example 1), with a purity > 98%, Levamisole Hydrochloride, Sodium Chloride, ELISA kits (TNF-α, IL-6, IL-1β)

[0072] 2. Experimental animals

[0073] Male BALB / c mice (18 - 22 g, 6 - 8 weeks old)

[0074] II. Experimental methods

[0075] Male BALB / c mice were randomly divided into six groups, with 8 mice in each group: normal control group (CT), model control group (CTX), positive control group (LH), low-dose glycopeptide group (SCL), medium-dose glycopeptide group (SCM), and high-dose glycopeptide group (SCH). After one week of adaptive feeding, the environmental conditions were maintained at about 25°C, relative humidity 50 - 55%, and a 12 h light / dark cycle. During the adaptation period, the mice could freely drink sterile water and a standard laboratory diet.

[0076] Specifically, after one week of adaptive feeding, mice in the CTX group, LH group, SCH group, SCM group, and SCL group were intraperitoneally injected with 80 mg / kg BW / day of cyclophosphamide. Starting from the 8th day, for 3 consecutive days, mice in the CT group were given normal saline in the same manner. Subsequently, mice in the CT group and CTX group were gavaged with sterile water. Mice in the SCH group, SCM group, SCL group, and LH group were gavaged with 200 mg / kg, 100 mg / kg, 50 mg / kg of SC2-3 polysaccharide solution and 40 mg / kg BW of Levamisole Hydrochloride every day for 14 days. The specific operations are shown in Table 2.

[0077] Table 2 Specific arrangements for animal experiments

[0078]

[0079] The body weights of mice in each group were recorded starting from the last day of adaptive feeding and recorded every three days for a total of 6 times. For the last administration, the animals were fasted for 12 h, not water-deprived, and euthanized 24 h later. After the experiment, all mice were sacrificed, and blood, spleen, thymus, and intestinal tissues were collected for further analysis. All dissected tissues were cryopreserved at -80°C for subsequent analysis.

[0080] III. Detection indicators

[0081] 1) Mouse body weight: The body weights of mice in each group were recorded starting from the last day of adaptive feeding and recorded every three days for a total of 6 times.

[0082] 2) Whole blood of mice was collected for routine blood analysis. Blood was collected from the medial canthus vein of the mouse eye into an anticoagulation tube, and a fully automatic blood cell analyzer was used to measure white blood cells, red blood cells, platelets, lymphocytes, mean corpuscular hemoglobin concentration, eosinophils, and neutrophils.

[0083] 3) The thymus and spleen were weighed, and the immune organ index was calculated.

[0084] 4) The contents of TNF-α, IL-1β, and IL-6 in the serum were measured.

[0085] 5) HE staining was used to detect the spleen, thymus, and intestine.

[0086] IV. Test Results

[0087] Figure 4 The results showed that compared with the CT group, the body weights of mice in the CTX, LH, SCL, SCM, and SCH groups gradually decreased in the first 6 days. After the 6th day, the body weights of mice in the CTX, LH, SCL, SCM, and SCH groups all increased, with little difference.

[0088] Figure 5 The results showed that compared with the CT group, the thymus and spleen indices of the CTX group were significantly decreased, indicating that an immunosuppressive model was successfully established in mice of the CTX group. The thymus and spleen indices are key indicators for evaluating the immune status of mice and can reflect the activation or inhibition state of the immune system. An increase in the thymus index usually indicates enhanced immune function, while a decrease in the thymus index may suggest weakened immune function. However, after treatment with LH, SCL, SCM, and SCH, compared with the CTX group, the decreases in the thymus and spleen indices of mice in the LH, SCL, SCM, and SCH groups were all improved. The blood routine indices of mice are important tools for evaluating the immune status and can reflect the activation or inhibition state of the immune system. Compared with the CTX group, the LH, SCL, SCM, and SCH groups significantly increased the numbers of white blood cells, red blood cells, platelets, lymphocytes, eosinophils, and neutrophils. The above suggests that both the polysaccharide treatment group and the positive control group can improve the organ indices and blood cell counts of mice in the CTX group and activate their immune systems.

[0089] Figure 6 The results showed that compared with the CT group, the expression levels of TNF-α, IL-6, and IL-1β in the CTX group were decreased, while the LH, SCL, SCM, and SCH groups all increased the expression of TNF-α, IL-6, and IL-1β in the serum. The above suggests that SC2 can effectively promote the expression of inflammatory mediators, can cause an inflammatory response of macrophages to a certain extent, and has an immune-enhancing effect.

[0090] Figure 7The results showed that the structure of the spleen alveoli in the control group (CT) was intact, and the boundary between the red and white bone marrow was clear. The spleen cords in the red bone marrow were connected as a whole, and the blood cells around the outside of the spleen alveoli were neatly arranged. No obvious spleen alveoli were observed in the spleen of the model group (CTX) mice, and the white and red medulla parts were mixed. After treatment with 50mg / kg SC2-3, the gross structure of the spleen alveoli could be observed, but the boundary between the red and white pulp was still unclear. As the drug concentration increased, the red and white medulla oblongata could be seen, and the edge area of ​​the white pulp gradually widened, indicating that SC2-3 had a certain repair effect on the spleen damage caused by CTX. The spleen is the largest peripheral immune organ in mice and has a regulatory effect on the body's immune response. The above suggests that SC2-3 helps the body maintain normal immune regulation by reducing the spleen damage caused by CTX.

[0091] Figure 8 The results showed that the cortical and medullary structures of the control group were clear and obvious, and obvious thymic vesicles were visible in the medulla. After the injection of cyclophosphamide, the cortical and medullary structures of the model group were diffuse, and a small number or inconspicuous thymic vesicles were visible in the field of vision. After administration, the cortical and medullary structures returned to normal. This shows that SC2-3 has a certain repair effect on thymic damage caused by CTX. The thymus is the main place for T cell development, differentiation and maturation, and plays a core role in specific immunity. The above suggests that SC2-3 helps the body maintain normal immune regulation by reducing thymic damage caused by CTX.

[0092] Figure 9 The results showed that the villi in the CTX group became blunt, the number of intestinal glands increased, and cell shedding, vacuoles, and defective integrity appeared at the tips of the villi. As shown in Table 3, compared with the CT group, the villus length and the ratio of villus length to crypt depth in the CTX group were significantly reduced. Compared with the CTX group, both of the above indicators in the SC group increased. This suggests that SC2-3 has a protective effect on intestinal barrier damage in CTX-treated mice. The intestinal mucosal epithelial cells of mice form a continuous physical barrier through tight junctions, which can prevent pathogens, antigens and other harmful substances in the intestine from entering the body, reduce unnecessary stimulation of the immune system, and enable the immune system to play a role in a relatively stable internal environment. The above suggests that SC2-3 helps the body maintain normal immune regulation by reducing the intestinal barrier damage of mice caused by CTX.

[0093] Table 3 Effects of SC2-3 on ileal villus length, crypt depth and villus length / crypt depth ratio in CTX-treated mice

[0094]

[0095] The above is a preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, all equivalent or modified implementations completed without departing from the spirit disclosed by the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing Nereis glycopeptide SC2-3, characterized in that: The following steps are involved: (a) Glycopeptide extraction: fresh nereid tissue was homogenized and treated with acetone, and centrifuged to obtain defatted sample powder; after adding water, papain and trypsin were used to extract the sample to obtain nereid crude polysaccharide; nereid crude polysaccharide was dissolved in water and then trichloroacetic acid was added to remove precipitated protein; dialyzed, concentrated, and freeze-dried to obtain nereid crude polysaccharide SC without protein; (b) Glycopeptide purification: The crude polysaccharide SC obtained by removing protein from nereidia was purified by DEAE cellulose anion column to obtain nereidia polysaccharide SC2; nereidia polysaccharide SC2 was purified by Sephacryl S-100 gel chromatography column to obtain nereidia glycopeptide SC2-3.

2. The method for preparing the neris glycopeptide SC2-3 according to claim 1, characterized in that: The following steps are involved: (a) Glycopeptide extraction: Fresh lugworm tissue was cleaned and homogenized, acetone was added, and the mixture was stirred at room temperature for 12 h. The supernatant was discarded and the operation was repeated. The mixture was centrifuged and the precipitation was air-dried at room temperature and then dried at 45°C. The sample defatted powder was obtained by crushing. 20-40 times the volume of distilled water and 2.5% papain were added. The mixture was extracted at pH = 5-7 and 60°C for 12 h, and inactivated at 100°C for 15 min. After cooling, the pH was adjusted to 7-9, 2.5% trypsin was added, and the mixture was stirred at 37°C. Extract for 12 hours, inactivate at 100℃ for 15 minutes; centrifuge after cooling, concentrate the supernatant, dialyze for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da, and freeze-dry to obtain a crude polysaccharide component of nereid; take the crude polysaccharide of nereid, dissolve it in deionized water, add an equal volume of 30% trichloroacetic acid, stir at 4℃ for 3 hours, centrifuge to remove the precipitate, and adjust the pH to 7; concentrate the sample after protein removal, dialyze for 3 days using a dialysis bag of 3500Da, concentrate, and freeze-dry to obtain the crude polysaccharide SC without protein of nereid; (b) Glycopeptide purification: Take the crude polysaccharide obtained by removing protein from the nereid, dissolve it in deionized water, centrifuge it, separate the supernatant by passing it through a DEAE cellulose anion column, elute it with water, 0.2M NaCl, 0.5M NaCl, 1M NaCl and 2M NaCl in sequence, detect it with sulfuric acid-phenol, collect and combine the 0.2M NaCl eluate, concentrate it, dialyze it, and freeze-dry it to obtain nereid polysaccharide SC2; take nereid polysaccharide SC2, dissolve it in deionized water, centrifuge it, separate the supernatant by using a Sephacryl S-100 gel chromatography column, purify it, elute it with 0.2M NaCl solution, combine the eluates, and obtain nereid glycopeptide SC2-3.

3. The method for preparing the neriworm glycopeptide SC2-3 according to claim 2, characterized in that: In step (a), 30 volumes of deionized water were added to adjust the pH to 6 and pH to 8, and then papain and trypsin were used for enzyme extraction respectively.

4. The method for preparing the neris glycopeptide SC2-3 according to claim 3, characterized in that: The relative molecular mass of the neriworm glycopeptide SC2-3 determined by HPGPC is 5.061KDa; the monosaccharide composition is calculated by mass ratio as fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid: glucuronic acid = 6.32: 20.6: 23.93: 9.79: 1.79: 7.49: 2.62: 0.24: 4.85; the results of physicochemical property determination show that its total sugar content is 24%, the uronic acid content is 16%, and the protein content is 24%; the amino acid analysis results show that it is mainly composed of 14.2% glycine, 11.8% aspartic acid, 9.75% glutamic acid and 6.1% threonine.

5. A nereid glycopeptide SC2-3, characterized in that It is prepared by the preparation method of lugworm polysaccharide SC2-3 described in any one of claims 1-4.

6. The neriworm glycopeptide SC2-3 according to claim 5, characterized in that The relative molecular mass of the nereid polysaccharide SC2-3 is 1.0-100KDa.

7. The neriworm glycopeptide SC2-3 according to claim 6, characterized in that The relative molecular mass of the nereid polysaccharide SC2-3 is 5.061 KDa.

8. Use of the neris glycopeptide SC2-3 according to any one of claims 5 to 7 in the preparation of an immune adjuvant for assisting anti-tumor function, which can reverse the low immune function of mice induced by cyclophosphamide chemotherapy drugs by improving immunity.

9. A pharmaceutical composition comprising the neris glycopeptide SC2-3 according to any one of claims 5 to 7 as an active ingredient, and further comprising pharmaceutically acceptable excipients.

10. A functional product comprising the neris glycopeptide SC2-3 according to any one of claims 5 to 7 as an active ingredient.

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