Syngnathus glycoprotein with blood replenishing activity as well as preparation method and application of syngnathus glycoprotein

Abstract

The invention discloses syngnathus glycoprotein with blood replenishing activity as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, syngnathus glycoprotein with different molecular weights is prepared through an enzymolysis method or a water extraction method and alcohol precipitation in combination with optimization of a sephadex column separation process, and verification experiments prove that the syngnathus glycoprotein prepared by the invention is composite glycoprotein modified by N-glycosylation and chondroitin sulfate double sugar chains; the glycoprotein contains rich N-sugar chains and is also combined with rich glycosaminoglycans, the content of sulfated chondroitin sulfate at the site 4 and the site 6 is rich, and the total sulfation degree is 0.4-0.6. The syngnathus glycoprotein prepared by the invention has a remarkable blood replenishing effect, provides a scientific basis for clinical blood replenishing drug research and functional food development, and has great application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a sea dragon glycoprotein with blood supplementing activity as well as a preparation method and application thereof. BACKGROUND

[0002] Anemia refers to a common clinical symptom that the content of hemoglobin per unit volume, red blood cell count and hematocrit in peripheral blood are lower than the normal standard. As is known to all, the oxygen required by human tissues and organs needs to be carried and transported by red blood cells in the blood. Anemia can cause the carrying capacity of blood oxygen in the human body to be poor, and further cause fatigue, dizziness, pale complexion, loss of appetite and the like. Long-term anemia can affect the function of various organs of the body. In addition, anemia can also affect the treatment and prognosis of various diseases. Therefore, the prevention and treatment of anemia is urgent.

[0003] The main causes of anemia are EPO production damage and iron homeostasis disruption. EPO is mainly synthesized by the kidney and a small amount is produced by the liver. It is a hematopoietic cell growth factor that regulates red blood cell proliferation, differentiation and maturation. It acts on bone marrow hematopoietic stem cells to promote red blood cell production. When the body is in a state of hypoxia (such as high altitude environment or anemia), the kidney can sense the low oxygen state and increase the synthesis of EPO through the hypoxia-inducible factor HIF pathway. There are three typical cases of EPO response in anemia patients: patients with renal anemia have absolute EPO deficiency due to kidney damage; patients with anemia due to inflammation have sufficient EPO but have iron metabolism disorders; and patients with aplastic anemia have low bone marrow response to EPO. At present, the main strategies for clinical treatment of anemia are as follows: iron agents are suitable for absolute iron deficiency anemia, but there is a risk of oxidative stress; recombinant human erythropoietin (rhEPO) is suitable for the treatment of renal anemia and chemotherapy-related anemia, but it is expensive; HIF-PH inhibitors (such as Roxadustat) indirectly increase endogenous EPO by stabilizing HIF and improve iron metabolism, but the long-term cardiovascular safety needs to be confirmed and the price is expensive.

[0004] The ocean is the source of life, and the ocean contains rich resources of products. A variety of bioactive substances such as glycoproteins can be obtained from numerous marine organisms. Glycoprotein is a kind of glycoconjugate connected by covalent bond between sugar chain and polypeptide chain. The sugar chain structure of glycoprotein is mainly divided into two types of N-glycosidic bond and O-glycosidic bond. The oligosaccharide chain of glycoprotein contains various structural information and plays an important role in functional activity. Glycoprotein is widely derived from different sources, and the composition and structure of glycoprotein from different sources are different, resulting in significant differences in biological activity function. The special environment of high salinity and low temperature in the ocean endows marine glycoprotein with special structure. In recent years, the research on marine glycoprotein has gradually become a hot spot. The biological activity of marine glycoprotein is usually related to its specific composition and structural characteristics. Therefore, clarifying the structural characteristics and biological activity of marine glycoprotein is an important basis for its high-value utilization.

[0005] H. n. has a sweet and salty taste, and is warm in nature, and belongs to the liver and kidney channels. It has the effects of warming the kidney and strengthening yang, resolving masses and swelling. It is used for kidney yang deficiency, impotence and spermatorrhea, accumulation of masses, scrofula, phlegm nodes, and injury from falls and bruises; and is used for external treatment of carbuncles, boils and furuncles. Modern pharmacological studies have mainly focused on sex hormone-like effects, anti-fatigue, immune enhancement, and anti-tumor effects, but there have been no reports on the activity of H. n. glycoprotein in improving blood supplementing and its material basis. SUMMARY

[0006] The purpose of the present application is to provide a H. n. glycoprotein with blood supplementing activity, a preparation method and application thereof. The prepared H. n. glycoprotein has N-glycosylation modification and chondroitin sulfate modification, can activate the human embryonic kidney HEK 293 cell hypoxia response element HRE, promote the expression of HIF-alpha, and also can promote the secretion of human embryonic kidney HEK 293 cell EPO, which has important significance for the preparation of food or drugs with blood supplementing effect.

[0007] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted:

[0008] The present application provides a H. n. glycoprotein with blood supplementing activity, the molecular structure of the H. n. glycoprotein has N-glycosylation modification and chondroitin sulfate modification, the total glycosaminoglycan content in the H. n. glycoprotein accounts for 0.3%-0.4% of the mass ratio of the H. n. glycoprotein, and the chondroitin sulfate sulfation degree in the H. n. glycoprotein is 0.4-0.6.

[0009] Further, the H. n. glycoprotein contains 38 N-glycan chains, the N-glycan chains include high mannose type N-glycan chains, fucose type N-glycan chains, sialylation N-glycan chains and at least one of fucose and sialylation N-glycan chains, and the proportion of sialylation N-glycan chains is more than 15%.

[0010] Further, there are 13 N-glycan chains with a relative content of more than 1% in the H. n. glycoprotein, and the high mannose type N-glycan chains, fucose type N-glycan chains, sialylation N-glycan chains and fucose and sialylation N-glycan chains account for more than 65% of the N-glycan chains in the H. n. glycoprotein.

[0011] Further, the molecular weight of the H. n. glycoprotein is 0.2-220kDa.

[0012] The present application also provides a preparation method of the H. n. glycoprotein, which specifically comprises the following steps:

[0013] S1: cutting H. n. into pieces, soaking in water, and homogenizing to obtain H. n. homogenate;

[0014] S2: extracting the H. n. homogenate by enzymatic method or water extraction method to obtain supernatant;

[0015] S3: alcohol precipitation of the supernatant in S2, separation of the precipitate after centrifugation;

[0016] S4: drying of the precipitate, re-dissolution in water, centrifugation, and separation of the supernatant;

[0017] S5: Sephadex gel column separation of the supernatant in S4, water elution, and collection of the eluate;

[0018] S6: concentration and drying of the eluate to obtain the sea dragon glycoprotein having blood supplementing activity.

[0019] Further, the sea dragon in step S1 is at least one of Dendrochirus brachyurus, Dendrochirus zebra, Dendrochirus stearnsi, and Dendrochirus biocellatus; and the ratio of sea dragon to water is 1:5-25.

[0020] Further, the biological protease in the enzymatic method includes pepsin and trypsin.

[0021] Further, the specific operation mode of the enzymatic method is: a. adjusting the pH of sea dragon homogenate, adding biological protease for extraction to obtain sea dragon extract; b. heating the sea dragon extract to inactivate the enzyme, cooling to room temperature, removing the precipitate by centrifugation of the extract, and separating the supernatant.

[0022] Further, the biological protease is pepsin and trypsin, and the amount of biological protease is 500-2000 U per gram of sea dragon, the pH value is adjusted to 1-8, the enzymolysis temperature is 30-45°C, the enzymolysis time is 0.5-3 h, and the stirring speed is 50-400 rpm.

[0023] Further, step S5: the supernatant obtained in step S4 is added to a G25 column, and pure water is used for elution at a flow rate of 1 mL / min; the collected eluate is detected for absorbance at 210 nm, 254 nm, and 280 nm; and four different elution components are prepared:

[0024] (1) all eluates with absorbance greater than 0.05 at the above three wavelengths are collected, which are HL-W;

[0025] (2) the eluate with absorbance greater than 0.05 at the above three wavelengths in the first peak of the absorbance spectrum is collected, which is component HL-W1;

[0026] (3) the eluate with absorbance greater than 0.05 at the above three wavelengths in the middle part between the first peak and the second peak of the absorbance spectrum is collected, which is component HL-W2;

[0027] (4) Collect all eluate whose absorbance at the above three wavelengths after the second peak on the absorbance spectrum is greater than 0.05, i.e. component HL-W3;

[0028] S6: Concentrate the eluate of the four different elution components in step S5 under reduced pressure at 50°C and 30 hPa, and freeze-dry at 20-40 Pa and -60°C to -40°C for 2-3 days to obtain 0.7-31 kDa total glycoprotein extract of sea dragon, i.e. HL-W, 17-31 kDa sea dragon glycoprotein component, i.e. HL-W1, 4-19 kDa sea dragon glycoprotein component, i.e. HL-W2, and less than 4.5 kDa sea dragon glycoprotein component, i.e. HL-W3.

[0029] Further, the specific operation steps of the water extraction method are as follows: A homogenate of sea dragon is extracted at 30-100°C and 0-1 Mpa for 0.5-5 hours, and the extraction is performed 1-3 times to obtain sea dragon extract; B remove the precipitate impurities by centrifugation to obtain the supernatant.

[0030] Further, the final concentration of ethanol in step S3 is 40-90%.

[0031] Further, the drying method in steps S4 and S6 is at least one of reduced pressure drying, freeze-drying, and spray drying.

[0032] Further, the reduced pressure drying condition is -0.1-0 Mpa and 30-60°C, and the drying is performed for 2-3 days.

[0033] Further, the freeze-drying condition is 20-40 Pa and -60°C to -40°C, and the freeze-drying is performed for 2-3 days.

[0034] Further, step S5: The supernatant obtained in step S4 is added to a G25 column, and pure water is used for elution at a flow rate of 1 mL / min. The eluate is collected, and the obtained eluate is detected for absorbance at 210 nm, 254 nm, and 280 nm to prepare eluate of four different elution components:

[0035] (1) Collect all eluate whose absorbance at the above three wavelengths is greater than 0.05, i.e. HL-B;

[0036] (2) Collect the eluate whose absorbance at the above three wavelengths is greater than 0.05 at the first peak on the absorbance spectrum, i.e. component HL-B1;

[0037] (3) Collect the eluate whose absorbance at the above three wavelengths is greater than 0.05 at the intermediate part between the first peak and the second peak on the absorbance spectrum, i.e. component HL-B2;

[0038] (4) Collect all the eluate whose absorbance at the above three wavelengths after the second peak on the absorbance spectrum is greater than 0.05, and the component is HL-B3;

[0039] S6: The eluate of the four different elution components in step S5 is concentrated under reduced pressure at 50 DEG C and 30 hPa, and is freeze-dried under the conditions of 20-40 Pa and -60 DEG C to -40 DEG C for 2-3 days to obtain 0.2-215 kDa total sea dragon glycoprotein extract, namely HL-B, 31-190 kDa sea dragon glycoprotein component, namely HL-B1, 20-33 kDa sea dragon glycoprotein component, namely HL-B2, and less than 21 kDa sea dragon glycoprotein component, namely HL-B3.

[0040] The application further provides application of the sea dragon glycoprotein in preparation of food and / or medicine with blood supplementing efficacy.

[0041] Further, the sea dragon glycoprotein can activate the hypoxia response element HRE of human embryonic kidney HEK 293 cells, promote the expression of HIF-alpha, and promote the secretion of EPO of human embryonic kidney HEK 293 cells.

[0042] Compared with the prior art, the application has the following advantages and beneficial effects:

[0043] 1. The application provides a simple, efficient and operable preparation method of sea dragon glycoprotein with blood supplementing efficacy.

[0044] 2. The sea dragon glycoprotein prepared by the water extraction and alcohol precipitation combined with the Sephadex gel column fractionation method has prominent high mannose type, fucose type, sialylation and fucose and sialylation modification in all N-glycosylation modifications, and the proportion of sialylation N-glycosylation modification is more than 15%, and the sea dragon glycoprotein is also combined with rich glycosaminoglycans, the total glycosaminoglycan content is greater than 3.4 mg / g, the 4-position and 6-position sulfated chondroitin sulfate content is rich, and the chondroitin sulfate sulfation degree is 0.4-0.6.

[0045] 3、The application first prepares and identifies the sea dragon glycoprotein, the molecular structure of the sea dragon glycoprotein has N-glycosylation modification and chondroitin sulfate modification at the same time, the modification helps to improve the biological activity and stability of the glycoprotein, prolongs the in-vivo action time, provides data support for the research on the medicinal material basis of the sea dragon, and verifies that the sea dragon glycoprotein can promote the generation of EPO and has significant blood supplementing effect; and the sea dragon glycoprotein with a molecular weight less than 21kDa obtained by the water extraction process combined with column fractionation and / or the sea dragon glycoprotein with a molecular weight less than 4.5kDa obtained by the enzymatic hydrolysis process combined with column fractionation is obviously higher than the total sea dragon glycoprotein extract in blood supplementing activity, can be used for developing blood supplementing drugs and functional foods in succession, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The ultraviolet absorption graph of the sea dragon glycoprotein HL-W and the fractionated components prepared by the enzymatic hydrolysis process.

[0047] Figure 2 The ultraviolet absorption graph of the sea dragon glycoprotein HL-B and the fractionated components prepared by the water extraction process.

[0048] Figure 3 The mass spectrum graph of N-glycan in the sea dragon glycoprotein.

[0049] Figure 4 The sialylated N-glycan with a peak area greater than 1% in the sea dragon glycoprotein and the relative content.

[0050] Figure 5 The content of CS-0S, CS-4S and CS-6S in the sea dragon glycoprotein.

[0051] Figure 6 The chondroitin sulfate (CS) content of different sulfation sites in the sea dragon glycoprotein.

[0052] Figure 7 The evaluation result of the in-vitro EPO regulation effect of the sea dragon glycoprotein.

[0053] Figure 8 The influence of the sea dragon glycoprotein HL-W and the fractionated components prepared in Example 1 on the expression of hypoxia inducible factor HIF-α.

[0054] Figure 9 The influence of the sea dragon glycoprotein HL-B and the fractionated components prepared in Example 2 on the expression of hypoxia inducible factor HIF-α. DETAILED DESCRIPTION

[0055] The present application will be further described in detail below with reference to the accompanying drawings and specific examples, it should be noted that the following examples should not be understood as limiting the present application. The sea dragon and water feed liquid ratio of the present application refers to the mass volume ratio of sea dragon and water, the mass unit is g, and the volume unit is mL.

[0056] Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0057] Example 1: Preparation of sea dragon glycoprotein HL-W and its fraction components

[0058] In this embodiment, sea dragon glycoprotein HL-W is prepared by enzymatic hydrolysis process, and the specific operation is as follows:

[0059] S1: Take sea dragon chunks, soak in water for 2 hours according to the feed liquid ratio of 1:20, homogenize for 10 minutes, and obtain sea dragon homogenate.

[0060] S2: Adjust the pH of the sea dragon homogenate to 2.0, add pepsin, 25 U of pepsin per gram of sea dragon, extract at 37℃ and stirring speed of 150 rpm for 2 hours; adjust the pH to 7.5, add trypsin, 1000 U of trypsin per gram of sea dragon, extract at 37℃ and stirring speed of 150 rpm for 2 hours, and obtain sea dragon extract.

[0061] S3: Heat the sea dragon extract to 100℃, inactivate the enzyme for 10 minutes, cool to room temperature, centrifuge at 3000 rpm for 10 minutes, and separate the supernatant.

[0062] S4: Add ethanol to the supernatant to a final concentration of 90%, stand overnight, centrifuge at 3000 rpm for 20 minutes, and separate the precipitate.

[0063] S5: Dry the precipitate at -0.1 MPa ~0MPa, 30℃~60℃ for 1~2 days to remove ethanol, redissolve in water, centrifuge at 12000 rpm for 20 minutes to remove precipitate impurities, and separate the supernatant.

[0064] S6: Separate the supernatant obtained in step S5 by G25 column with 4 column volumes, elute with pure water, control the flow rate of peristaltic pump at 1 mL / min, automatically collect 1 tube every 4 min, elute for 6 h, collect the eluate, detect the absorbance of the obtained eluate at 210 nm, 254 nm and 280 nm, and draw the absorbance spectrum, and prepare 4 different elution components of the eluate in parallel:

[0065] (1) Collect all eluate (HL-W) with absorbance greater than 0.05 at the above three wavelengths;

[0066] (2) Collect eluate (component HL-W1) with absorbance greater than 0.05 at the above three wavelengths at the first peak on the absorbance spectrum;

[0067] (3) Collect eluate (component HL-W2) with absorbance greater than 0.05 at the above three wavelengths at the middle part between the first peak and the second peak on the absorbance spectrum;

[0068] (4) Collect all eluate (component HL-W3) with absorbance greater than 0.05 at the above three wavelengths after the second peak on the absorbance spectrum;

[0069] S7: Concentrate the eluate of the four different elution components in step S6 under reduced pressure at 50°C and 30 hPa, and freeze-dry at 20-40 Pa and -60°C to -40°C for 2-3 days to obtain sea dragon glycoprotein with blood supplementing activity, specifically including 0.7-31 kDa sea dragon glycoprotein total extract (HL-W), 17-31 kDa sea dragon glycoprotein component (HL-W1), 4-19 kDa sea dragon glycoprotein component (HL-W2), and less than 4.5 kDa sea dragon glycoprotein component (HL-W3); Figure 1 To analyze the ultraviolet absorption of sea dragon glycoprotein and its fractionated components in the enzymatic process.

[0070] Example 2: Preparation of sea dragon glycoprotein HL-B and its fractionated components

[0071] In this example, sea dragon glycoprotein HL-B is prepared by water extraction process, and the specific operation includes the following steps:

[0072] S1: Take the cut pieces of sea dragon, soak in water at a material to liquid ratio of 1:15 for 2 hours, and homogenize for 10 minutes to obtain sea dragon homogenate.

[0073] S2: Extract the sea dragon homogenate at 100°C and 0.4 Mpa for 2 hours, and extract twice to obtain sea dragon extract.

[0074] S3: Cool the extract to room temperature, filter to remove precipitated impurities using a 120-mesh filter screen, and separate the supernatant;

[0075] S4: Add ethanol to the supernatant to a final concentration of 80%, let stand overnight, centrifuge at 3000 rpm for 20 minutes, and separate the precipitate.

[0076] S5: Freeze-dry the precipitate at 20-40 Pa and -60°C to -40°C for 2-3 days to remove ethanol, resuspend in water, centrifuge at 12000 rpm for 20 minutes to remove precipitated impurities, and separate the supernatant.

[0077] S6: The supernatant obtained in step S5 was added to a G25 column for 4 column volumes, and pure water was used for elution, with a flow rate of 1 mL / min controlled by a peristaltic pump, 1 tube was automatically collected every 4 min, elution was performed for 6 h, and the eluate was collected. The absorbance of the obtained eluate was detected at wavelengths of 210 nm, 254 nm, and 280 nm, and an absorbance spectrum was drawn. Four different elution components were prepared in parallel:

[0078] (1) All eluates with an absorbance greater than 0.05 at the above three wavelengths were collected (HL-B);

[0079] (2) The eluate with the first peak in the absorbance spectrum and an absorbance greater than 0.05 at the above three wavelengths was collected (component HL-B1);

[0080] (3) The eluate with an absorbance greater than 0.05 at the above three wavelengths in the middle part between the first peak and the second peak in the absorbance spectrum was collected (component HL-B2);

[0081] (4) All eluates with an absorbance greater than 0.05 at the above three wavelengths after the second peak in the absorbance spectrum were collected (component HL-B3);

[0082] S7: The eluate of the four different elution components in step S6 was concentrated under reduced pressure at 50°C and 30 hPa, and was freeze-dried at 20-40 Pa and -60°C to -40°C for 2-3 days to obtain sea dragon glycoprotein with blood supplementing activity, specifically including 0.2-215 kDa sea dragon glycoprotein total extract (HL-B), 31-190 kDa sea dragon glycoprotein component (HL-B1), 20-33 kDa sea dragon glycoprotein component (HL-B2), and less than 21 kDa sea dragon glycoprotein component (HL-B3); Figure 2 The ultraviolet absorption of the water extraction process sea dragon glycoprotein and the classified components.

[0083] Example 3: Sea dragon glycoprotein molecular weight analysis

[0084] The molecular weights of the different products of sea dragon glycoprotein prepared in Examples 1 and 2 were determined by high performance liquid chromatography. The chromatographic conditions were as follows: Agilent 1260 Infinity high performance liquid chromatograph, TSK-Gel G3000PW (7.5 x 300 mm, 5 μm) chromatographic column, column temperature 30 °C; 0.2% trifluoroacetic acid aqueous solution and acetonitrile (v / v = 7:3) were used as the mobile phase, the flow rate was 0.5 mL / min; the injection volume was 10 μL; the detection wavelength was 220 nm, and the chromatogram was recorded; using uracil, bacitracin, aprotinin, cytochrome C, and bovine serum albumin as the reference substances, the regression equation was y = -0.3275x + 8.5118 (r = 0.9992) with the retention time as the abscissa x and the logarithm of the molecular weight as the ordinate y. The sea dragon glycoprotein sample was determined, the peak time was recorded, and the molecular weight was calculated by entering the regression equation.

[0085] It was determined that the molecular weight distribution of the different products of sea dragon glycoprotein calculated is shown in Table 1.

[0086] Table 1: Yield and molecular weight distribution of sea dragon glycoprotein fractionated components in Examples 1 and 2

[0087]

[0088] Example 4: Monosaccharide composition of sea dragon glycoprotein

[0089] The monosaccharide composition of sea dragon glycoprotein was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization high performance liquid chromatography. Specifically, HL-B prepared in Example 2 was treated by acid hydrolysis to break the sugar chains into free monosaccharides, and then PMP derivatization reagent was added for derivatization reaction to convert the monosaccharides into PMP derivatives with ultraviolet absorption. The derivatized monosaccharides were separated by a reversed-phase C18 chromatographic column (Agilent ZORBAX SB-C18), gradient eluted with acetonitrile-phosphate buffer as the mobile phase, and the chromatographic peaks of each monosaccharide derivative were detected by a diode array detector at a wavelength of 254 nm. Qualitative analysis was performed according to the retention time and standard monosaccharide comparison, and the relative content of each monosaccharide was calculated by peak area external standard method, so as to determine the types and proportions of monosaccharides in sea dragon glycoprotein.

[0090] The experimental results show that the sea dragon glycoprotein is mainly composed of 7 kinds of monosaccharides, and the molar mass ratio of each monosaccharide is mannose (Man) : glucosamine (GlcN) : glucuronic acid (GlcA) : galactosamine (GalN) : glucose (Glc) : galactose (Gal) : fucose (Fuc) = 14.72:18.98:3.97:4.14:25.02:29.23:3.94. The presence of galactosamine (GalN) and glucuronic acid (GlcA) in the monosaccharide composition indicates that the sample contains glycosaminoglycans (GAGs).

[0091] Example 5: Sea dragon glycoprotein N-glycan sugar profile analysis

[0092] PGC-HESI-MS / MS method was used to analyze the N-glycan sugar profile of sea dragon glycoprotein. Specifically, HL-B prepared in Example 2 was used as the sample. PNGase F enzyme was used to release N-glycan from HL-B, and 2-AA was used for labeling. Vanquish Flex ultra-high performance liquid chromatography (UHPLC, Thermo Scientific, USA) equipped with Orbitrap Exploris 240 mass spectrometer was used for detection. Hypercarb PGC column (150 mm x 2.1 mm i.d., 3 μm, Thermo Scientific, USA) was used for N-glycan separation, and the column temperature was 35°C. 10 mM ammonium bicarbonate solution (A) and 80% acetonitrile solution (containing 20% 10 mM ammonium bicarbonate, B) were used as the mobile phase, and the flow rate was 0.3 mL / min. Gradient elution (0-2 min, 12% B; 2-15 min, 12%-28% B; 15-16 min, 28%-90% B; 16-18 min, 90% B; 18-19 min, 90%-12% B; 19-22 min, 12% B). Mass spectrometry data acquisition was performed in negative ion mode (ESI⁻), with a spray voltage of 2500 V. The full scan range was 150-2000 m / z, with a resolution of 120,000 (m / z 200), and the acquisition mode was profile mode. MS / MS analysis was performed using HCD collision mode with a collision energy of 30 V and a resolution of 30,000. Ion source parameters: sheath gas 35 arb (units), auxiliary gas 10 arb (units), purge gas 0 arb (units); ion transmission tube temperature 320°C, gasification temperature 350°C, radio frequency lens 70%.

[0093] The mass spectrum is as follows: Figure 3As shown in the figure, the characteristic peaks in the figure can be used to qualitatively determine the type of sugar chain, and the relative content of the relative amount of sugar chain. The analysis results are as follows Figure 4 As shown, the relative content of each type of N-glycan is obtained by calculating the peak area, and there are 38 N-glycans in the halymen glycoprotein, of which 13 N-glycans have a relative content greater than 1%. High mannose type N-glycan, fucose type N-glycan, sialylation N-glycan and fucose and sialylation N-glycan are the main ones, more than 65%, of which sialylation N-glycan accounts for more than 15%. The results show that the halymen glycoprotein contains a relatively rich N-glycan. The high mannose type N-glycan structure can be specifically recognized by the mannose receptor due to its unique spatial configuration and high mannose residue; and the sialic acid residue at the end of the sialylation N-glycan can prolong the circulation half-life of the glycoprotein in the blood, improve its biological stability and help to regulate erythropoiesis and improve hematopoietic function.

[0094] Example 6: Analysis of halymen glycoprotein glycosaminoglycan disaccharide composition

[0095] The halymen glycoprotein glycosaminoglycan disaccharide composition was analyzed by multiple reflection monitoring mass spectrometry. The specific operation is as follows: the HL-B prepared in Example 2 is subjected to enzymatic hydrolysis to generate glycosaminoglycan disaccharides by using a glycosaminoglycan lyase mixed solution (containing heparin lyase I, II, III and chondroitinase ABC, AC, B, 40 mU of each enzyme). The obtained disaccharides and standard products are labeled by 2-aminoacridone reduction amination method. The UltiMate 3000 ultra-high performance liquid chromatography (UHPLC) separation system equipped with a TSQ-Quantiva mass spectrometer (Thermo Scientific, USA) is used for liquid chromatography-mass spectrometry (LC-MS) analysis, and the multiple reaction monitoring (MRM) mode is used. Poroshell 120 C18 chromatographic column (3.0×150 mm i.d, 2.7 µm, Agilent, USA), column temperature 45°C. The mobile phase A is water / methanol (85:15, v / v), and the mobile phase B is water / methanol (35:65, v / v), both containing 50 mM ammonium acetate (pH adjusted to 6.5 with acetic acid), flow rate 0.2 mL / min; gradient elution (0-2 min, 0%B; 2-8 min, 0%-12% B; 8-15 min, 12%-25% B). Data acquisition is performed in negative ion mode (ESI⁻), with a spray voltage of 3kV; ion source gas parameters: sheath gas flow rate 35 arb (units), auxiliary gas flow rate 10 arb (units); ion transmission tube temperature and gasification temperature are both 350°C.

[0096] The results are shown in Table 2. In the glycosaminoglycan analysis, the total content of glycosaminoglycan in the sea dragon glycoprotein was greater than 3.4 mg / g, and chondroitin sulfate (CS) was the main component. In the glycosaminoglycan subtype distribution, the 4-sulfated chondroitin sulfate accounted for the highest proportion of 91.5%, which was the main type of CS; followed by 6-sulfated chondroitin sulfate, accounting for 6.9% (see Figure 5 ). From the sulfation site, the 4-O sulfation of CS was mainly about 3.1 mg / g, and the 6-O sulfation was about 0.2 mg / g (see Figure 6 ). The CS sulfation degree of the sea dragon glycoprotein was calculated to be 0.4-0.6.

[0097] Table 2 Glycosaminoglycan disaccharide composition analysis of sea dragon glycoprotein

[0098]

[0099] These results show that the sea dragon glycoprotein contains rich glycosaminoglycans, of which chondroitin sulfate (CS) is the main component, accounting for the vast majority of total glycosaminoglycans. From the subtype distribution, the content of 4-sulfated chondroitin sulfate (CS-4S) is significant, and there is also a relatively rich 6-sulfated chondroitin sulfate (CS-6S), and the sulfation degree of chondroitin sulfate is 0.4-0.6. Combined with the fact that the biological activity of glycosaminoglycan is closely related to its sulfation pattern, the sulfation site affects protein interaction, the abundance of specific CS chain units regulates biological function, and CS-4S and CS-6S have multiple biological significance, the high content of CS and the characteristic sulfation pattern in the sea dragon glycoprotein, as well as the high proportion of CS-4S and CS-6S, suggest that it may have significant biological activity, providing an important basis for further exploring its role in related physiological function regulation.

[0100] Example 7: Regulation of sea dragon glycoprotein on TNF-α induced HEK 293 cell inflammation to inhibit EPO expression

[0101] A TNF-α induced HEK 293 cell inflammation to inhibit EPO expression model was used to investigate the effect of the sea dragon glycoprotein prepared in Examples 1 and 2 on EPO expression regulation. The experimental principle is that inflammation can inhibit the production of red blood cells by damaging the differentiation of erythroid hematopoietic stem cells and shortening the life span of mature red blood cells; EPO is a hematopoietic cell growth factor that regulates the proliferation, differentiation and maturation of red blood cells, mainly synthesized by the kidney and a small amount by the liver. It is generally believed that TNF-α is the main inflammatory cytokine that inhibits the production of EPO, and EPO is mainly produced by the kidney, so human embryonic kidney HEK 293 cells were selected for the experiment, and recombinant human TNF-α was used as an EPO inhibitor to establish a cell model.

[0102] Specific experimental method: HEK 293 cells in the logarithmic growth phase were harvested and cultured at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were seeded in 96-well plates and divided into several groups: a control group (cell culture medium), a model group (cell culture medium + TNF-α 100 ng / mL), a positive control group (cell culture medium + TNF-α 100 ng / mL + roxadustat 40 μM), and a hemangioprotein-treated group (cell culture medium + TNF-α 100 ng / mL + HL-W 500 μg / mL; cell culture medium + TNF-α 100 ng / mL + HL-B 500 μg / mL). Real-time PCR was used to detect EPO expression in the cells. Primer sequences are shown in Table 3.

[0103] Table 3. PCR primers for relative gene quantification

[0104]

[0105] according to Figure 7 The results showed that, compared with the blank control group, the expression level of EPO in the model group was significantly reduced ( ### P<0.001, indicating successful model establishment; compared with the model group, both seahorse glycoproteins HL-W and HL-B significantly restored EPO expression at a drug concentration of 500 μg / ml. ** P<0.01) indicates that seahorse glycoprotein can promote the secretion of EPO, suggesting that it has a good blood-tonifying effect.

[0106] The experimental results of Examples 5 and 6 show that sea dragon glycoprotein contains a relatively rich variety of N-glycans, including highly mannose-type N-glycans with unique spatial configurations and sialylated N-glycans. It also contains abundant glycosaminoglycans, primarily chondroitin sulfate at position 4, and relatively abundant chondroitin sulfate at position 6. These glycan structures suggest that sea dragon glycoprotein not only enhances biological stability but also helps regulate erythropoiesis and improve hematopoietic function, thus possessing hematopoietic effects. This invention, through in vitro hematopoietic efficacy verification, confirms that sea dragon glycoprotein has significant hematopoietic activity, and its activity characteristics are consistent with its glycan structure.

[0107] Example 8: Effects of sea dragon glycoprotein and its fractionated components on the expression of hypoxia-inducible factor HIF-α

[0108] The human embryonic kidney cell HEK 293 cell line was used to construct a hypoxia response element (HRE) regulated luciferase reporter gene model to evaluate the effects of the sea dragon glycoprotein and different fractions obtained in Examples 1 and 2 on the expression of HIF-α. The model inserts a firefly luciferase reporter gene in the downstream sequence of HRE. After the tested substance stabilizes HIF-α, HRE is activated, and the firefly luciferase reporter gene is expressed. The intensity of the luciferase can reflect the expression level of HIF-α. The expression level of HIF-α can further promote the secretion of EPO, and thus can be used as an evaluation index of blood supplementing activity. The details are as follows.

[0109] The HEK293 cell strain was inoculated in a 96-well plate at 2x10 4 cells per well. The sea dragon glycoprotein obtained in Examples 1 and 2 was added to the cells using a high-throughput sampling needle (the final concentration of the drug was 62.5, 250, and 1000 μg / mL), and Roxadustat (the final concentration was 40 μM) was used as a positive drug. The cells were incubated at 37°C in a culture condition of 5% CO2 for 24 h. The luciferase assay substrate was added to each well, and the plate was incubated in a dark incubator for 5 min. The SpectraMax L enzyme marker was used to determine the luciferase activity. The relative luciferase activity represented the blood supplementing activity. The relative luciferase activity = sample group luciferase activity / blank control group luciferase activity x 100%.

[0110] The experimental results showed that compared with the blank control group (the luciferase activity was 100%), the sea dragon glycoprotein HL-B prepared by the water extraction process, the HL-B1 with a molecular weight of 31-190 kDa, the HL-B2 with a molecular weight of 20-33 kDa, and the HL-B3 with a molecular weight of less than 21 kDa could significantly improve the relative luciferase activity, and the HL-B3 with a molecular weight of less than 21 kDa had the strongest effect ( Figure 9 ). In addition, compared with the blank control group, the sea dragon glycoprotein HL-W prepared by the enzymatic hydrolysis process and the HL-W3 with a molecular weight of less than 4.5 kDa could significantly improve the relative luciferase activity, and the HL-W3 with a molecular weight of less than 4.5 kDa had the strongest effect ( Figure 8 ).

[0111] The above shows that the HL-B3 with a molecular weight of less than 21 kDa obtained after the fractionation and purification by the dextran gel column and the HL-W3 with a molecular weight of less than 4.5 kDa have stronger promoting effects on the expression of HIF-α and better blood supplementing activity than the HL-B and HL-W.

[0112] From the above, the sea dragon glycoprotein prepared by enzymatic hydrolysis or water extraction and alcohol precipitation process belongs to the complex glycoprotein with N-glycosylation and chondroitin sulfate double sugar chain modification, contains a variety of N-glycan, mainly high mannose type N-glycan, fucose type N-glycan, sialylation N-glycan and fucose and sialylation N-glycan, and the above four types account for more than 65% of all N-glycans, and the proportion of sialylation N-glycan is more than 15%; meanwhile, the glycoprotein is also combined with rich glycosaminoglycans, the total glycosaminoglycan content is greater than 3.4 mg / g, and the 4-sulfated chondroitin sulfate is the main type, and the 6-sulfated chondroitin sulfate is also rich, and the overall sulfation degree is 0.4-0.6. The above characteristic sulfation mode of the sea dragon glycoprotein prepared in the application helps to improve the biological stability of itself, and helps to promote erythropoiesis and improve hematopoietic function.

[0113] The application proves that the sea dragon glycoprotein significantly promotes the secretion of EPO through in vitro efficacy evaluation, which indicates that the sea dragon glycoprotein has good blood supplementing efficacy. The efficacy evaluation results are consistent with the correlation between the structure characteristics and efficacy of the sea dragon glycoprotein.

[0114] Further, the sea dragon glycoprotein HL-B obtained by the water extraction process and the sea dragon glycoprotein HL-W obtained by the enzymatic hydrolysis process are separated by a dextran gel G25 column to obtain sea dragon glycoprotein components with different molecular weights, and through in vitro efficacy evaluation, it is verified that the activity of HL-B3 with a molecular weight less than 21 kDa is significantly better than that of HL-B, and the activity of HL-W3 with a molecular weight less than 4.5 kDa is significantly better than that of HL-W, therefore, the sea dragon glycoprotein with a molecular weight less than 21 kDa is more conducive to blood supplementing efficacy. The above shows that the glycoprotein component with better blood supplementing activity can be obtained after the dextran gel G25 column fractionation and purification.

[0115] Therefore, the experimental results of the application not only provide a reference for the research on the material basis of the blood supplementing efficacy of sea dragon, but also provide a delicate process of the blood supplementing active component of the sea dragon glycoprotein, which can be widely applied in the preparation of drugs and foods with blood supplementing efficacy, and has a broad application prospect.

[0116] The above-described embodiments are only a part of the embodiments of the application, not all the embodiments, and do not limit the application. Based on the embodiments of the application, those skilled in the art can make several improvements and supplements without departing from the principles of the application, and these improvements and supplements should also be regarded as the protection scope of the application.

Claims

1. A sea dragon glycoprotein with hematopoietic activity, characterized in that, The molecular structure of the sea dragon glycoprotein has both N-glycosylation modification and chondroitin sulfate modification. The total glycosaminoglycans in the sea dragon glycoprotein account for 0.3%-0.4% of the mass of the sea dragon glycoprotein, and the degree of chondroitin sulfate sulfate sulfate in the sea dragon glycoprotein is 0.4-0.

6.

2. The sea dragon glycoprotein according to claim 1, characterized in that, The molecular weight of the scorpion glycoprotein is 0.2-220 kDa.

3. The method for preparing the sea dragon glycoprotein according to claim 1, characterized in that, Specifically, the following steps are included: S1: Cut the sea dragon into pieces, soak in water, and homogenize to obtain sea dragon homogenate; S2: Extract the seahorse homogenate using enzymatic hydrolysis or water extraction to obtain the supernatant; S3: Add ethanol to the supernatant from step S2 for alcohol precipitation, and separate the precipitate by centrifugation to obtain the precipitate; S4: Dry the precipitate, reconstitute it with water, centrifuge, and separate the supernatant; S5: Separate the supernatant from step S4 using a dextran gel column, elute with water, and collect the eluent; S6: Concentrate and dry the eluent to obtain scorpion glycoprotein with hematopoietic activity.

4. The preparation method according to claim 3, characterized in that, In step S1, the sea dragon is at least one of the following: sea dragon, pseudo sea dragon, Shu's sea dragon, and Baojia sea dragon; the ratio of the sea dragon to water is 1:5-25.

5. The preparation method according to claim 3, characterized in that, The specific steps of the enzymatic hydrolysis method in step S2 are as follows: a) Adjust the pH of the sea dragon homogenate, add biological protease for extraction, and obtain sea dragon extract; b) Heat the sea dragon extract to inactivate the enzyme, cool it to room temperature, centrifuge to remove precipitate impurities, and obtain supernatant.

6. The preparation method according to claim 5, characterized in that, The biological protease is pepsin and trypsin, calculated per gram of seahorse mass. The amount of biological protease used is 500-2000 U, the pH is adjusted to 1-8, the enzymatic hydrolysis temperature is 30-45℃, the enzymatic hydrolysis time is 0.5-3h, and the stirring speed is 50-400 rpm.

7. The preparation method according to claim 3, characterized in that, Step S5: Add the supernatant obtained in step S4 to a G25 column and elute with pure water at a flow rate of 1 mL / min. Measure the absorbance of the collected eluent at wavelengths of 210 nm, 254 nm, and 280 nm to prepare eluents with four different elution components: (1) Collect all eluents with absorbance greater than 0.05 at the above three wavelengths, i.e., HL-W; (2) Collect the eluent with the first peak on the absorbance spectrum and the absorbance at all three wavelengths above is greater than 0.05, i.e. component HL-W1; (3) Collect the eluent between the first and second peaks on the absorbance spectrum, and the absorbance at all three wavelengths is greater than 0.05, i.e. component HL-W2; (4) Collect all the eluents whose absorbance is greater than 0.05 at the above three wavelengths after the second peak on the absorbance spectrum, i.e., component HL-W3; Step S6: The eluents of the four different elution components in step S5 are concentrated under reduced pressure at 50°C and 30 hPa, and then freeze-dried at 20~40 Pa and -60°C to -40°C for 2~3 days to obtain the total extract of 0.7-31 kDa seahorse glycoprotein (HL-W), the 17-31 kDa seahorse glycoprotein component (HL-W1), the 4-19 kDa seahorse glycoprotein component (HL-W2), and the seahorse glycoprotein component less than 4.5 kDa (HL-W3).

8. The preparation method according to claim 3, characterized in that, The specific operating steps of the water extraction method are as follows: A. Extract the sea dragon homogenate at 30-100℃ and 0-1 MPa for 0.5-5 hours, and extract 1-3 times to obtain sea dragon extract; B. Centrifuge the sea dragon extract to remove precipitated impurities and obtain supernatant.

9. The preparation method according to claim 3, characterized in that, Step S5: Add the supernatant obtained in step S4 to a G25 column and elute with pure water at a flow rate of 1 mL / min. Collect the eluent and measure its absorbance at wavelengths of 210 nm, 254 nm, and 280 nm to prepare eluents with four different elution components: (1) Collect all eluents with absorbance greater than 0.05 at the above three wavelengths, i.e., HL-B; (2) Collect the eluent with the first peak on the absorbance spectrum and the absorbance at all three wavelengths above is greater than 0.05, i.e. component HL-B1; (3) Collect the eluent between the first and second peaks on the absorbance spectrum, and the absorbance at all three wavelengths is greater than 0.05, i.e. component HL-B2; (4) Collect all the eluents whose absorbance is greater than 0.05 at the above three wavelengths after the second peak on the absorbance spectrum, i.e., component HL-B3; S6: The eluents of the four different elution components in step S5 are concentrated under reduced pressure at 50℃ and 30hPa, and then freeze-dried at 20~40Pa and -60℃~-40℃ for 2~3 days to obtain the total extract of 0.2-215kDa seahorse glycoprotein, namely HL-B; the seahorse glycoprotein component of 31-190kDa, namely HL-B1; the seahorse glycoprotein component of 20-33kDa, namely HL-B2; and the seahorse glycoprotein component of less than 21 kDa, namely HL-B3.

10. The use of the sea dragon glycoprotein according to claim 1 in the preparation of food and / or medicine with hematopoietic effects, characterized in that, The scorpion glycoprotein can promote the expression of HIF-α and the secretion of EPO in human embryonic kidney HEK 293 cells, thus exerting a blood-tonifying effect.

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