阅读说明：本技术 一种海胆多糖在抗新型冠状病毒或sars病毒感染药物中的应用 (Application of sea urchin polysaccharide in medicine for resisting novel coronavirus or SARS virus infection ) 是由 吴宁 张全斌 王清池 王晶 耿丽华 岳洋 于 2021-05-14 设计创作，主要内容包括：本发明属于生物医药技术领域,涉及一种海胆生殖腺多糖在制备抗新型冠状病毒(SARS-CoV-2)或SARS病毒(SARS-CoV)感染药物或药物组合物中的应用。本发明从黄海胆的生殖腺中提取得到了一种葡聚糖(HJ2A),对其进行了抗SARS-CoV-2病毒或SARS病毒的活性测定。结果表明,HJ2A能竞争性地抑制SARS-CoV-2病毒或SARS病毒的受体结合域与靶细胞表面受体结合,有效阻断病毒进入细胞,具有显著的抗SARS-CoV-2病毒或SARS病毒能力。本发明的海胆多糖可用于制备拮抗新型冠状病毒或SARS病毒的药物,应用于预防和治疗新型冠状病毒或SARS病毒感染及其肺部并发症,具有良好的开发利用价值。(The invention belongs to the technical field of biological medicine, and relates to application of sea urchin gonadal polysaccharide in preparation of a medicine or a medicine composition for resisting novel coronavirus (SARS-CoV-2) or SARS virus (SARS-CoV). The invention extracts a glucan (HJ2A) from gonads of sea urchin and measures the activity of the glucan against SARS-CoV-2 virus or SARS virus. The result shows that HJ2A can competitively inhibit the binding of SARS-CoV-2 virus or the receptor binding domain of SARS virus with the receptor on the surface of target cell, effectively block the virus from entering the cell, and has obvious SARS-CoV-2 virus or SARS virus resisting capacity. The sea urchin polysaccharide can be used for preparing medicaments for antagonizing novel coronavirus or SARS virus, is applied to preventing and treating the novel coronavirus or SARS virus infection and pulmonary complications thereof, and has good development and utilization values.)

1. Application of sea urchin polysaccharide in preparing medicine for resisting novel coronavirus or SARS virus infection is provided.

2. The use of claim 1, wherein said sea urchin polysaccharide is a glucan extracted and purified from the gonad of sea urchin, and has a relative molecular weight of 100-500 kDa, a total sugar content of 80-100% in sea urchin glucan, a protein content of 0-20%, preferably a total sugar content of 85-95%, and a protein content of 5-15%.

3. The use according to claim 2, wherein said echinacon polysaccharide has a highly branched glycogen structure with a backbone glucose residue linked by an α -1,4 glycosidic linkage, branched at the C-6 position; the repeating structural unit comprises 4-10 glucose residues, wherein the repeating structural unit contains 1-2 branch point residues, and the number of side chain glucose residues is 1-3.

4. The use according to claims 2-3, wherein said sea urchin polysaccharides are derived from one or more of the species Hemicentrotus Flaccida (Glyphnodaris crassipes), Hemicentrotus Macrophyllus (Strongylocentrotus nudus) or Strongylocentrotus intermedius (Strongylocentrotus intermedius).

5. The use of claim 1, wherein said sea urchin polysaccharide can be used for preparing a medicament or a pharmaceutical composition for preventing or treating a novel coronavirus or SARS virus.

6. Use according to claim 1 or 3, characterized in that: the concentration of the sea urchin polysaccharide in the medicine or the medicine composition for resisting and preparing the medicine or the medicine composition for resisting the novel coronavirus SARS-CoV-2 or SARS virus is 0.1-10 nmol/L, and the preferable concentration is 5-10 nmol/L.

7. The use of claim 6, wherein the sea urchin polysaccharide of claim 2 to 3 is mixed with pharmaceutically or food acceptable adjuvants or auxiliary additive components to prepare a medicament or pharmaceutical composition having an anti-new coronavirus effect or SARS virus infection.

8. Use according to claim 1 or 5, characterized in that: sea urchin polysaccharide and angiotensin converting enzyme 2 are combined with the receptor binding domain of the novel coronavirus SARS-CoV-2 or SARS virus competitively to inhibit the novel coronavirus SARS-CoV-2 or SARS virus from entering cells.

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to application of sea urchin Glyptocidaris crenularis polysaccharide in medicines or medicine combinations for resisting novel coronavirus SARS-CoV-2 or SARS virus.

Background

The new type of coronavirus (SARS-CoV-2) is a type B coronavirus which is common to both human and livestock and is transmitted through the air and fecal ports. SARS-CoV-2 infection has symptoms of fever, cough, shortness of breath and dyspnea, and can also lead to pneumonia, severe acute respiratory syndrome, organ failure, and even death. The basis for well controlling the epidemic situation is to recognize the virus infection rule and the pathogenic mechanism as soon as possible. The Spike protein (Spike, S-protein) on the surface of SARS-CoV-2 recognizes and binds to the target cell receptor, induces the membrane fusion of virus and cell, is the first step of virus invasion into host cell, and is the key target for preventing and treating virus infection.

Angiotensin converting enzyme 2(ACE2) is a human cell surface receptor that has been identified as the primary entry point for SARS-CoV-2 virions, membrane proteins such as ACE2 mediate viral entry into cells by binding to the SARS-CoV-2 or SARS-CoV spike protein receptor domain (RBD), but blocking ACE2 can lead to serious cardiovascular toxicity. Therefore, a safe, high-efficiency and non-toxic and side-effect medicine for resisting the novel coronavirus is clinically needed. In addition, the research shows that S-protein can also interact with glycosaminoglycan (GAG) and Heparin Sulfate (HS) on the surface of host cells besides being combined with ACE2, and the virus is promoted to enter the host cells. This also demonstrates that some polysaccharide species specifically bind to S-protein, but competitively bind to the RBD of SARS-CoV-2 or S-protein of SARS-CoV, inhibiting the binding of S-protein receptor to ACE2, is an effective means of inhibiting viral entry into body cells.

Coronavirus infection can lead to Acute Respiratory Distress Syndrome (ARDS), and more severe cases can progress to uncontrolled systemic inflammatory reactions with shock, vascular leakage, disseminated intravascular coagulation and multiple organ failure, which are important factors leading to death of new severe patients with coronary pneumonia. 2019-nCoV severely infected patients have pulmonary diseases with diffuse alveolar damage and pulmonary hyaline membrane formation, and the overall pathological manifestations of the lung are similar to SARS. While SARS-CoV-2 infection is manifested as acute diffuse alveolar injury in the early stage and diffuse alveolar injury and acute fibrinous pneumonia in the later stage.

The gonads of sea urchins contain various polysaccharides, mainly including fucoidan sulfate, galactan sulfate, glycosaminoglycan, glucan and heteropolysaccharide, and have wide biological activity. In recent years, researches show that the high-branch glycogen in sea urchin, clam and oyster can remarkably enhance the immune response of the organism and has the potential of being developed into an immunopotentiator. Mammalian glycogen and vegetable starch have no such activity, indicating that glycogen activity is affected by its source. The inventor finds out in recent research that: sea urchin polysaccharide HJ2A competitively binds with angiotensin converting enzyme 2 with novel coronavirus SARS-CoV-2 or the receptor binding domain of SARS virus, thereby blocking the binding of novel coronavirus SARS-CoV-2 or the receptor binding domain of SARS virus with angiotensin converting enzyme 2, and inhibiting the novel coronavirus SARS-CoV-2 or SARS virus from entering cells. Moreover, the inventor finds that the sea urchin polysaccharide HJ2A can be combined with S protein on the surface of SARS-CoV-2 virus to inhibit the combination of the S protein and ACE2 and inhibit the virus activity, and can inhibit pulmonary fibrosis by inhibiting TGF-beta mediated intercellular transformation, thereby protecting the function of lung cells while resisting virus, and the sea urchin polysaccharide HJ2A has obvious basic research and clinical significance.

Disclosure of Invention

The invention aims to provide application of sea urchin polysaccharide in medicine or medicine composition for resisting novel coronavirus SARS-CoV-2 or SARS virus.

In order to achieve the purpose, the invention adopts the technical scheme that:

the sea urchin polysaccharide is glucan extracted and purified from sea urchin gonads, the relative molecular weight of the glucan is 100-500 kDa, the total sugar content of sea urchin glucan is 80-100%, the protein content is 0-20%, preferably the total sugar content is 85-95%, and the protein content is 5-15%.

The sea urchin polysaccharide has a high-branch glycogen structure, main chain glucose residues are connected through alpha-1, 4 glycosidic bonds and are branched at the C-6 position; the repeating structural unit comprises 4-10 glucose residues, wherein the repeating structural unit contains 1-2 branch point residues, and the number of side chain glucose residues is 1-3.

The sea urchin polysaccharide is derived from one or more of yellow sea urchin (Glyphosaris crenularis), Strongylocentrotus nudus (Strongylocentrotus nudus) and Strongylocentrotus intermedius (Strongylocentrotus intermedius).

The sea urchin polysaccharide can be used for preparing a medicine or a medicine composition for preventing or treating novel coronavirus or SARS virus.

The concentration of the sea urchin polysaccharide in the medicine or the medicine composition for resisting and preparing the medicine or the medicine composition for resisting the novel coronavirus SARS-CoV-2 or SARS virus is 0.1-10 nmol/L, and the preferable concentration is 5-10 nmol/L.

The pharmaceutical composition is prepared by mixing sea urchin polysaccharide serving as an active ingredient with pharmaceutically or food acceptable auxiliary materials or auxiliary additive ingredients, and can be used for preparing a medicine or a pharmaceutical composition with an anti-new coronavirus effect or SARS virus infection according to a conventional preparation method.

The sea urchin polysaccharide can be combined with angiotensin converting enzyme 2 competitively with novel coronavirus SARS-CoV-2 or a receptor binding domain of SARS virus, thereby blocking the combination of the novel coronavirus SARS-CoV-2 or the receptor binding domain of SARS virus and the angiotensin converting enzyme 2, and inhibiting the novel coronavirus SARS-CoV-2 or SARS virus from entering cells.

Wherein the preparation process of the polysaccharide comprises the following steps:

(1) extraction of polysaccharides

Dissolving the sea urchin gonad degreasing powder in 10-20 times volume of pure water, extracting in 90-95 deg.C water bath under stirring for 4-6h, recovering to room temperature after extraction, centrifuging (4000-8000rpm, 15-20min), and collecting extraction residue. Adding 10-20 times of NaOH solution into residue obtained after hot water extraction of sea urchin gonad, stirring and extracting at 60-80 ℃ for 2-4 hours, neutralizing the extracting solution after the extraction, centrifuging and removing the residue, collecting supernatant, concentrating to 1/6-1/8 with the appropriate volume, adding three times of anhydrous ethanol into the concentrated solution, precipitating with ethanol, and standing at 4-6 ℃ for 24-48 hours. Centrifuging, collecting precipitate, re-dissolving the precipitate, centrifuging again, collecting supernatant, dialyzing (MWCO:3-6kDa), concentrating, and lyophilizing to obtain crude polysaccharide of sea urchin gonad.

(2) Purification of polysaccharides

The polysaccharide was purified by using a DEAE Fast Flow (5.0 cm. times.50 cm; 1.6 cm. times.50 cm) strong anion exchange chromatography column. Filtering polysaccharide with microporous membrane of 0.22 μm, loading, eluting with deionized water, 0.02-0.10mol/L NaCl, 0.10-0.20 mol/L NaCl and 2.0mol/L NaCl solution for 1-3 Column Volumes (CV), and controlling flow rate at 3 mL/min. The fractions eluted from 0.02 to 0.10mol/L NaCl were collected using an automated fraction collector, and the objective polysaccharide fraction was shown by an arrow in FIG. 1.

And (2) further purifying the target component by Sephadex G75 gel filtration column chromatography, detecting the polysaccharide by a sulfuric acid phenol method by using 0.1mol/L ammonium bicarbonate as a mobile phase at the flow rate of 0.5mL/min, and collecting the sugar-containing component to obtain the purified polysaccharide.

The invention has the advantages that:

the invention provides the application of sea urchin polysaccharide HJ2A in preparing medicine or medicine composition for resisting novel coronavirus SARS-CoV-2 or SARS virus for the first time. HJ2A is abundant in sea urchin, is alpha-1, 4-glucan, highly branched at C-6 position, and can bind to various receptors. Experiments prove that HJ2A can be combined with spike protein of novel coronavirus, competitively inhibit the combination of angiotensin 2 and virus spike protein, and organize virus invasion. At the same concentration, HJ2A has higher inhibitory activity against the new coronavirus than the reported heparin and galactofucan sulfate. At the same time, the cell fibrosis process is prevented by inhibiting TGF-beta/Smad signal path. Therefore, the medicine has certain prevention and treatment effects on the prevention and treatment of new coronavirus infection and the occurrence and development of pulmonary fibrosis induced by SARS virus infection, and has good development and utilization values.

Drawings

FIG. 1 is an elution chromatogram of sea urchin polysaccharide on DEAE Fast Flow weak anion exchange column chromatography provided in the example of the present invention;

FIG. 2 is an elution chromatogram of sea urchin polysaccharide on Sephadex G75 gel filtration column chromatography provided in the example of the present invention;

FIG. 3 is a chromatogram for determining monosaccharide composition of sea urchin polysaccharide provided in the embodiment of the present invention;

FIG. 4 is a chromatogram for measuring the molecular weight of sea urchin polysaccharide provided by the embodiment of the invention;

FIG. 5 is an infrared spectrum of sea urchin polysaccharide provided by an embodiment of the present invention;

FIG. 6 is an infrared spectrum of methylated Hemicentrotus Seu Strongylocentrotus polysaccharide provided in an embodiment of the invention;

FIG. 7 is a total ion flow graph of the gas chromatography of echinacon polysaccharide provided by the embodiment of the present invention;

FIG. 8 is a mass spectrum diagram of a methylation analysis of echinacea polysaccharide (A: Glc- (1 →; B: → 4) -Glc- (1 →; C: → 4,6) -Glc- (1 →) according to an embodiment of the present invention;

fig. 9 is a mass spectrum of nuclear magnetic resonance spectroscopy of echinacea polysaccharide provided in the example of the present invention (a:¹H NMR；B：¹³C NMR；C：DEPT 135；D：¹H–¹H COSY；E：¹H–¹H TOCSY；F：¹H–¹³C HSQC)；

FIG. 10 is a graph showing the tendency of binding between echinoglycosomes and Spike proteins provided in the examples of the present invention;

FIG. 11 is a kinetic affinity curve of the binding of echinoglycosomes to Spike protein provided by embodiments of the present invention;

FIG. 12 is a diagram showing the activity of sea urchin polysaccharide inhibiting SARS-CoV-2 virus according to the embodiment of the present invention.

FIG. 13 shows the inhibitory effect of in vitro administration of echinacon polysaccharides provided by the examples of the present invention on TGF- β induced epithelial to mesenchymal transition of lung fibroblasts HELF.

FIG. 14 is an electron microscope image of the sea urchin polysaccharide for treating TGF-beta induced lung fibroblast HELF fibrosis provided by the embodiments of the present invention.

Detailed Description

The invention is further explained below with reference to the figures and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.

Example 1 (this process is conventional)

The preparation method of sea urchin polysaccharide HJ2A comprises the following specific steps:

(3) extraction of polysaccharides

Dissolving the defatted powder of gonad of sea urchin with 10 times volume of pure water, extracting in 90 deg.C water bath under stirring for 4 hr, recovering to room temperature, centrifuging (4000rpm, 15min), and collecting the extraction residue. Adding 10 times of 0.2mol/L NaOH solution into residue obtained after hot water extraction of sea urchin gonad, stirring and extracting at 70 ℃ for 2 hours, neutralizing the extracting solution after the extraction is finished, centrifuging and removing the residue, collecting supernatant, concentrating 1/6 of the original volume, adding three times of volume of absolute ethyl alcohol for alcohol precipitation, and standing at 4 ℃ for 24 hours. Centrifuging, collecting precipitate, re-dissolving the precipitate, centrifuging again, collecting supernatant, dialyzing (MWCO:3.5kDa), concentrating, and lyophilizing to obtain crude polysaccharide of sea urchin gonad.

(4) Purification of polysaccharides

The polysaccharide was purified by using a DEAE Fast Flow (5.0 cm. times.50 cm; 1.6 cm. times.50 cm) strong anion exchange chromatography column. After being filtered by a microporous filter membrane with the diameter of 0.22 mu m of polysaccharide, the polysaccharide is loaded, and is eluted by 2 Column Volumes (CV) respectively by deionized water, 0.06mol/L NaCl, 0.15mol/L NaCl and 2.0mol/L NaCl solution, and the flow rate is 3 mL/min. The fraction eluted with 0.06mol/L NaCl was collected using an automated fraction collector, and the objective polysaccharide fraction was shown by an arrow in FIG. 1.

And (2) further purifying the target component by Sephadex G75 gel filtration column chromatography, detecting the polysaccharide by a sulfuric acid phenol method by using 0.1mol/L ammonium bicarbonate as a mobile phase at a flow rate of 0.5mL/min, and collecting the sugar-containing component to obtain purified polysaccharide, namely HJ2A (shown as an arrow in figure 2).

Example 2

Analyzing the physicochemical property of sea urchin polysaccharide.

(1) Analysis of Total sugar content

A sulfuric acid-phenol method is adopted, 5.00mg of each of a standard reference substance (glucose) and a polysaccharide sample is accurately weighed, and the volume is determined in a 50mL volumetric flask to obtain a standard solution for total sugar determination. 25, 50, 100, 200, 300, 400 and 500. mu.L of standard solutions and 500. mu.L of sample solutions were precisely measured in a clean glass tube, each with water to 500. mu.L. Subsequently, 500. mu.L of a 5% phenol solution and 2.5mL of concentrated sulfuric acid were precisely measured and added to the test tube in this order. When adding concentrated sulfuric acid, the concentrated sulfuric acid is added along the wall of the test tube at a constant speed to prevent the solution from boiling and splashing. The solutions were mixed well by gentle shaking and heated in a boiling water bath for 15 min. The solution was taken out and cooled to room temperature, and the same volume of the solution was measured for each tube and added to a 96-well plate, and the absorbance (wavelength: 492nm) of the solution was measured for each tube by means of a microplate reader. And 3 parallel groups are arranged in total, the average absorbance value of each group is calculated respectively, a curve is drawn according to the concentration of the standard solution, and the total sugar content of the sample solution is calculated. From the results, the total sugar content of HJ2A ranged from 80% to 100%.

(2) Protein content analysis

The protein content of the sea urchin polysaccharide is determined by using a BCA method. Measuring the BCA reagent and the CuSO4 solution according to the actual number of the samples to be measured, and measuring the volume ratio of 50: 1, and fully mixing to prepare a BCA experimental solution. Bovine Serum Albumin (BSA) stock solution at 5mg/mL was diluted with PBS to a concentration of 0.5mg/mL and was diluted in a gradient to standard solutions at 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5 mg/mL. 20. mu.L of each standard solution was accurately measured and added to a 96-well plate, and then 200. mu.L of the prepared BCA test solution was measured and added to each well. Bathing at 37 deg.C for 30min, measuring absorbance value of each well at 562nm wavelength within 15min, setting up 3 parallel groups, calculating average value of absorbance of each group, drawing standard curve according to concentration of each standard solution, and calculating protein concentration of each sample. From the results, it was found that the protein content of HJ2A ranged from 0% to 20%.

(3) Monosaccharide composition analysis

And (3) adopting PMP pre-column derivation high performance liquid chromatography to measure monosaccharide composition of sea urchin polysaccharide. The method comprises the following specific steps: a plurality of 5mL clean ampoules are prepared, 5.0mg of sample is accurately weighed and added with 500 mu L of water to be completely dissolved, then 500 mu L of 4.0mol/L trifluoroacetic acid (TFA) is added, the ampoules are sealed and degraded at 105 ℃ for 4h, 200 mu L of degradation liquid is taken after neutralization, the degradation liquid is uniformly mixed with 0.5mol/L PMP methanol solution (240 mu L) and 0.3mol/L NaOH solution (200 mu L) and sealed, and then the ampoules are reacted in 70 ℃ water bath for 1h, and the degradation product or the monosaccharide standard is subjected to derivatization. The derivative is extracted with chloroform for 3 times, the chloroform layer is discarded, and the water layer is injected after passing through a 0.22 mu m microporous filter membrane. Chromatographic conditions are as follows: ZORBAX SB-AQ C18 column (4.6 mm. times.250 mm,5 μm); a detector: SPD-20A UV detector (254 nm); mobile phase: 0.1mol/L PBS (pH 6.8): acetonitrile 83:17(v/v,%); flow rate: 0.8 mL/mL; column temperature: 30 ℃; sample loading amount: 20 μ L. The HPLC is shown in FIG. 3, and it can be seen that the monosaccharide composition HJ2A is glucose, indicating that HJ2A is dextran.

(4) Relative molecular weight analysis

The relative molecular mass is determined by high performance gel permeation chromatography-multi-angle laser light scattering method (HPGPC-MALLS) sea urchin polysaccharide. A chromatographic column: shodex Ohpak SB-806HQ chromatography column (8 mm. times.300 mm,13 μm); mobile phase: 0.1mol/L Na₂SO₄A solution; flow rate: 0.5 mL/min; column temperature: 35 ℃; a detector: G1362A shows the difference detector and HelEos-11 eighteen angle laser detector; sample introduction amount: 20 μ L. As a result, the weight average molecular weight of HJ2A was found to be 100 to 500 kDa. (FIG. 4).

Example 3

Structural characterization of HJ 2A.

(1) Infrared spectroscopic analysis (FT-IR)

Weighing about 2mg of polysaccharide sample in a 1mL centrifuge tube, placing the centrifuge tube in a vacuum drying oven with phosphorus pentoxide in the vacuum drying oven with an opening, and drying the polysaccharide sample for 1-2 days at 50 ℃ under reduced pressure. Placing the sample in a sample hole of an infrared spectrometer by using a sample spoon, and performing infrared spectrum scanning, wherein the wave number is 400-4000 cm^-1. FT-IR spectra of HJ2A are shown in FIG. 5, respectively. O-H (3297 cm)^-1)、C-H (2923cm^-1) And C-O-C (1002 cm)^-1) The stretching vibration of (2) is characteristic absorption of polysaccharide, and is at 1002cm^-1、1075cm^-1And 1143cm^-13 narrow strong peaks at (B), indicating that the sugar ring is pyran-type and appears at 840cm^-1The left and right peaks (-C-H oscillations) confirm that HJ2A is composed of alpha-D-glucopyranose.

(2) Methylation analysis

Methylation reaction was performed on each polysaccharide sample using a modified Hakomari method. Weighing polysaccharide sample 2mg in a reaction flask, and placing in a vacuum drying oven (containing P)₂O₅) And (3) drying for 48h, adding 1mL of anhydrous DMSO into the dried sample, protecting with nitrogen, and magnetically stirring for 1h at room temperature to fully dissolve the polysaccharide. 20mg of dried NaH powder was added, and the reaction was stirred at 50 ℃ for 1 hour, whereby the reaction solution was dark green. And (3) placing the reaction bottle on an ice bath, dropwise adding 0.5mL of methyl iodide after the solution in the reaction bottle is completely solidified, and reacting at room temperature in a dark place until the solution becomes bright yellow.

After the reaction, 1mL of deionized water was added to terminate the reaction, and the reaction solution was transferred to a test tube. Using CHCl₃Extracting the reaction solution twice, and combining CHCl₃And (3) a layer. Washing CHCl with deionized water₃Collecting CHCl for 3-6 times₃And drying the layer under reduced pressure. To ensure complete methylation of the sample, the above procedure was repeated. Taking a sample, and performing infrared spectrum detection on the sample if 3300cm^-1The strong and broad O-H vibration absorption peak almost disappeared while 2900cm^-1The sum of the absorption peak of methyl vibration and the sum of the absorption peak of methyl vibration is 1000-1400 cm^-1The C-O shock absorption peak in between was significantly enhanced, indicating that the polysaccharide sample had been completely methylated.

The fully methylated samples were transferred to a 5mL ampoule, dissolved well by adding 0.5mL water and 0.5mL 4mol/L TFA, heat sealed using an alcohol burner, and hydrolyzed in an oven at 110 ℃ for 4 h. The hydrolysis solution was evaporated to dryness using a rotary evaporator and methanol was added repeatedly until TFA was completely evaporated. 0.5mL of NaOH solution (0.05mol/L) was added to the deacidified hydrolysate, and the sample was dissolved by shaking. 5mg of NaBD was added₄And reacting at room temperature for 4 h. After the reaction is finished, glacial acetic acid is added dropwise until the solution becomes weakly acidic and no gas is generated. And adding methanol into the reaction solution, and repeatedly evaporating until the boric acid and the glacial acetic acid are completely volatilized. During the period, the pH test paper can be used for detection, and when the pH value is close to neutral, the acid is removed completely. The product was dried in a vacuum oven for 48 h.

400 μ L of anhydrous pyridine was added to the reaction flask containing the reduced sample, and the reaction was carried out in an oil bath at 100 ℃ for 0.5 hour in a sealed manner. After the reaction is finished, the reaction solution is cooled to room temperature and is coagulatedAfter the reaction solution solidified to white and translucent, 400. mu.L of anhydrous acetic anhydride was added thereto, and the reaction was sealed at 100 ℃ for 1 hour. After the reaction was completed, 1mL of deionized water was added to terminate the reaction. Methanol was added and evaporated to dryness repeatedly to remove pyridine and acetic acid. The reaction was charged with 2mL CH₂Cl₂Dissolving out, washing with deionized water for 3-6 times, and collecting CH₂Cl₂The layer was concentrated to dryness under reduced pressure, dried in a vacuum oven and analyzed by gas chromatography (GC-MS).

FT-IR spectrum of methylated HJ2A, 3230cm^-1The strong shock absorption peaks at the left and right disappeared, and the C-H shock absorption peak at 2920cm-1 was enhanced, indicating that HJ2A had been completely methylated. After acid hydrolysis, reduction and acetylation, the derivatives were analyzed by GC-MS and the total ion current gas chromatogram was shown in FIG. 7. The mass spectra of each peak were compared with the partially methylated sugar alcohol acetyl ester derivative (PMAA) in the CCRC spectral database to find 3 matching linkages, as shown in fig. 8. Analysis results by GC-MS showed that HJ2A has a typical glycogen structure, and that the repeating structural unit containing Glc- (1 →, → 4) -Glc- (1 →, and → 4,6) -Glc- (1 →, whose molar ratio is 2.19:3.18: 1.00. HJ2A contains 6 Glc residues, including 1 branch point residue.

(3) Nuclear magnetic resonance spectroscopy (NMR)

Weighing about 30mg of solid polysaccharide, placing in a 50mL rotary steaming bottle, adding 1mL of heavy water (D)₂O) dissolving, rotary evaporating to dryness, and adding D again₂Dissolving O, repeatedly evaporating to dryness for 3 times, and adding 500 μ L D₂Dissolving O, centrifuging, and transferring to a nuclear magnetic tube. Recording spectra by nuclear magnetic spectroscopy, as shown in FIG. 9, including¹D spectrum (1H NMR,¹³C NMR and DEPT 135 ℃ and 2D correlation spectra ((C-NMR and DEPT 135))¹H-¹H COSY、¹H-¹³C HMQC、¹H-¹³C HMBC and¹H-¹H TOCSY)。

¹in the H NMR spectrum, 2-group anomeric proton signals were clearly seen at chemical shifts 5.25(A) and 4.86(B) ppm, with a peak area integration ratio of 3.70:1.00, and the configuration of MSGA was presumed to be alpha-type from its chemical shifts, which is consistent with the results of methylation analysis. The other proton (H2-H6) signal of the sugar ring is at 3.30 to 3.90 ppm.¹³In the C NMR spectrum, 3 anomeric carbon signals appear at 99.90ppm, 99.77ppm and 98.48ppm, while the chemical shifts of C2-C6 are between 60 and 77 ppm. According to the glycosidation shift effect, the chemical shifts of C4(77.36ppm) and C6(67.45 ppm) shift to low fields due to the formation of glycosidic bonds, and the signal of unsubstituted C6 appears at 60.54 ppm.

From¹H-¹H COSY and¹H-¹the signals of H1-H3 in the spin system can be easily identified in H TOCSY, but the signals of H2-H6 are distributed densely between 3.30 ppm and 3.90ppm and are difficult to identify (FIG. 4 and FIG. 9C).¹H-¹³The C HSQC spectra (FIGS. 4, 9C, D) show a direct correlation with the C, H signal, e.g., the anomeric protons of residues A and B are both directly related to the anomeric carbon at chemical shifts 99.90 and 99.77ppm, respectively, and the anomeric proton of sugar residue C is directly related to the carbon atom at 98.48 ppm. The signals of two protons of C6 are between 3.60 and 3.80ppm, and the signals related to H4/C4 of A and B residues are 3.53/77.36 ppm.

Example 4

SPR detects the interaction of sea urchin polysaccharide and SARS-CoV-2Spike protein.

Sea urchin polysaccharide was assayed for binding affinity to SARS-CoV-2 by Surface Plasmon Resonance (SPR). Placing a CM5 chip in the chip compartment of a Biacore T200 instrument; SARS-CoV-2Spike protein is dissolved in HBS-EP + buffer solution to prepare 400. mu.g/mL mother liquor, diluted to 20. mu.g/mL with sodium acetate solution with pH 5.5, and protein coupling is carried out by amino coupling mode for 420s at flow rate of 10. mu.L/min. After the chip was activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), Spike proteins were coupled to channel 2 and unbound sites were blocked with ethanolamine. Channel 2 was treated identically with a buffer without Spike protein as a blank. The polysaccharide samples were diluted separately and subjected to a binding assay using PBSP buffer and injected into the chip at a flow rate of 30. mu.L/min. At the end of sample injection, the same buffer flows over the chip surface to facilitate dissociation. After 60s of dissociation, glycine-hydrochloric acid buffer (10mmol/L, pH 1.5) was injected to regenerate the sensor surface for 30 s. Data were fit analyzed using software using a 1:1 binding mode.

The real-time dynamic sensorgram of polysaccharide binding to S-protein is shown in FIG. 10. It can be seen that as the concentration of polysaccharide increases, the amount of bound polysaccharide to Spike protein also increases and reaches a saturation state with time. Kinetic affinity curves of each polysaccharide to S-protein were plotted according to the global fit curve and 1:1 binding model (fig. 11). The dissociation constant is an important parameter for determining the interaction between a ligand and a receptor, and a smaller value indicates a stronger binding ability. HJ2A had a KD of 7.133X 10^-7. SPR data showed that echinacon polysaccharide has a strong affinity for S-protein and interacts in a dose-dependent manner.

Example 5

Effect of sea urchin polysaccharide on infection of HEK293 cells by pseudoviruses

Echinacon and positive controls were thawed at 4 ℃, diluted to the concentrations required for the experimental design using Opti-MEM medium (0.06, 0.024, 0.098, 0.391, 1.563, 6.250, 25.000, and 100.00 μ g/mL) and added to 96-well plates while negative and blank controls were set. Pseudoviruses were removed from liquid nitrogen and quickly thawed in a 37 ℃ water bath with gentle shaking, similarly diluted with Opti-MEM medium, 25. mu.L was added to each well of the sample and control groups, and the blank group was added to the same volume of Opti-MEM medium. The solution was mixed well by gentle shaking and incubated at room temperature for 60 min. During this period, the Opti-HEK293/ACE2 cells were thawed and, after centrifugation, diluted to 6X 10 cells by adding DMEM complete medium⁵one/mL. The cells are fully and uniformly mixed, added into a 96-well plate, 50 mu L of each well, placed into a carbon dioxide incubator to be incubated for 48h, and after 24h of incubation, complete culture medium is added once, wherein 50 mu L of each well is added. The 96-well plate was bottomed, and the medium was carefully aspirated, 50. mu.L of luciferase developing solution was rapidly added, allowed to stand at room temperature for 5min, and the fluorescence intensity was measured with the aid of a microplate reader.

The effect of echinacon HJ2A on the binding of Spike protein to ACE2 is shown in FIG. 12, and the polysaccharides reported to date (galacto-fucoidan sulfate and heparin) with inhibitory activity against new coronaviruses were selected as controls. Researchers have simulated the SARS-CoV-2S-protein on third generation lentiviral (pLV) vectors and tested the effect of various sulfated polysaccharides on mammalian cell transduction efficiency. The result shows that the galactofucan sulfate and the heparin have obvious concentration-dependent inhibition effect on pLV-S signal transduction, which indicates that the galactofucan sulfate and the heparin have the capacity of inhibiting the combination of viruses and cells. Fucoidan has no effect on S. As a negative control, the logarithm of the fluorescence signal intensity after the S-protein and ACE2 are combined to infect cells, and the figure shows that HJ2A can obviously reduce the fluorescence signal intensity, and the inhibitory activity to viruses is higher than that of galactofucan sulfate and heparin under the same concentration. The sea urchin polysaccharide can competitively inhibit the combination of S-protein and ACE2 to a certain extent, and inhibit SARS-CoV-2 virus from entering cells.

Example 6

The echinacon polysaccharide has the therapeutic effect on TGF-beta 1-induced human lung cancer cell A549 fibrosis by in vitro administration.

Taking A549 cells in logarithmic phase of growth, blowing and beating to prepare single cell suspension, counting the cells, and then, dividing the cells into 5 multiplied by 10⁵Cells per mL were cultured in 6-well plates at a density of about 50%, and when the cells grew, the old medium was replaced with 100. mu.L of FBS-free medium, and starved for 12 hours. The experiment was divided into a negative control group, a positive control group (TGF-. beta.1), and a drug-treated group (TGF-. beta.1 + HJ 2A). The administration was continued for 24 h. After treatment, cells were observed using an olympis inverted microscope and a scanning electron microscope for morphological analysis. Scanning electron microscopy analysis cells were washed with PBS, alcohol gradient dehydrated, sampled and observed by electron microscopy (Hitachi-S-3400N).

As shown in FIG. 13, the negative control HELF cells were cobblestone-shaped, and stimulated with TGF-. beta.1, the cells became spindle-shaped and fibrosis occurred. The low concentration of HJ2A (1.25nmol/L) has certain inhibition effect on the fibrosis of HELF cells, and compared with cells of a TGF-beta 1 single treatment group, the long spindle-like transformation induced by the cells through TGF-beta 1 is inhibited by the low concentration of HJ2A administration group; increasing the concentration of HJ2A to 5nmol/L epithelial to mesenchymal transition of lung fibroblasts induced by TGF- β 1 was significantly inhibited, with most cells being cobblestone-like, rather than the long spindle type induced by TGF- β 1. The cell morphology observed under an electron microscope is clearer, as shown in fig. 14, the cells of the negative control group are irregular oval spheres, and are obviously fibrillated and long-strip-shaped after being stimulated by TGF-beta 1. The low concentration of HJ2A (1.25nmol/L) shows certain effect on the fibrosis treatment effect of HELF cells, and the high concentration of HJ2A (5nmol/L) can obviously stimulate fibrosis and cell morphology change caused by TGF-beta 1, and the cells recover oval sphere shape.

Experiments show that the high-concentration HJ2A can inhibit the occurrence of pulmonary fibrosis to a certain extent, and has prevention and treatment effects on the pulmonary fibrosis process caused by the infection of new coronary pneumonia.