Donkey-hide gelatin heparan sulfate capable of inhibiting binding activity of new spike protein and preparation method of donkey-hide gelatin heparan sulfate

Abstract

The invention discloses colla corii asini heparan sulfate capable of inhibiting the activity of new spike protein and a preparation method of the colla corii asini heparan sulfate. The colla corii asini heparan sulfate is prepared by the following steps: performing enzymolysis on a colla corii asini solution by using streptomyces protease, and performing separation and purification by using DEAE anion exchange chromatography to obtain colla corii asini glycosaminoglycan; according to the invention, hexuronic acid and glucosamine are used as raw materials, then chondroitin sulfate ABC enzyme is used for enzymolysis, DEAE anion exchange chromatography is used for separation and purification, the average molecular weight of the hexuronic acid is 13.2 Kda, the hexuronic acid is rented by a disaccharide unit formed by connecting hexuronic acid and glucosamine through a 1-4 glucosidic bond, and the total sulfation degree of C-2-oxygen site of hexuronic acid and the total sulfation degree of C-6-oxygen site of glucosamine are 28.2%, 44.3% and 38.9% respectively. The colla corii asini heparinoid sulfate has competitive activity of inhibiting binding of new coronal spike protein and heparin, can effectively prevent initial infection of new coronal virus, and lays an important foundation for development of new clinical application of colla corii asini.

Description

Technical Field

[0001] The present invention belongs to the field of preparation and purification of natural products, and specifically relates to a donkey-hide gelatin sulfate heparan with the activity of inhibiting the binding of new coronavirus spike protein and a preparation method thereof. Background Art

[0002] Donkey-hide gelatin is a traditional health food and Chinese medicine ingredient. Its main pharmacological effects include nourishing blood, improving calcium metabolism balance, resisting shock, and enhancing immunity. It is mostly used in the treatment of gynecological diseases and has been proven to have effects in the treatment of blood diseases, anti-inflammation, anti-coagulation and anti-aging. The main components of donkey-hide gelatin include amino acids, proteins, polysaccharides, trace elements and polypeptides. At present, the research on the active ingredients in donkey-hide gelatin mainly focuses on amino acids, proteins and trace elements, and there is little research on heparin sulfate in donkey-hide gelatin.

[0003] Heparan sulfate is a highly complex sulfated polysaccharide composed of repeated disaccharide units. Its disaccharide unit is composed of hexuronic acid (GlcA / IdoA) and glucosamine (GlcN) connected by 1-4 glycosidic bonds, in which the sulfation sites are at the C-2-oxygen position of hexuronic acid, the C-6-oxygen position of glucosamine, the C-3-oxygen position of glucosamine, and the N-position of glucosamine. Heparan sulfate interacts with more than 700 proteins in the body and has important regulatory functions in immune response, inflammation regulation, viral infection, thrombosis, and angiogenesis. Many viruses, such as herpes simplex virus, dengue virus, HIV, and various coronaviruses, including the new coronavirus that has been prevalent in recent years, use heparan sulfate as a receptor or auxiliary receptor, and bind to various viral proteins through its specific sequence structure and control the invasion of multiple viral infections. Therefore, heparan sulfate / heparin derivatives with specific structures can be used as competitive inhibitors to block or interfere with the binding of the new crown spike protein to the auxiliary receptor, thereby inhibiting the infection of the new crown virus.

[0004] The present invention prepares and purifies donkey-hide gelatin heparan sulfate and finds that it has a new function of inhibiting the binding activity of the new coronavirus spike protein. Since there are no reports at home and abroad on the inhibition of the binding activity of the new coronavirus spike protein by donkey-hide gelatin heparan sulfate, the present invention can lay an important foundation for the development of new clinical uses of donkey-hide gelatin. Summary of the invention

[0005] The purpose of the present invention is to provide a donkey-hide gelatin sulfate heparan with the activity of inhibiting the binding of the new coronavirus spike protein and a preparation method thereof, which realizes the new function of donkey-hide gelatin polysaccharide in the field of new coronavirus and lays the foundation for developing new clinical uses of donkey-hide gelatin.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A donkey-hide gelatin sulfate heparan with inhibitory activity on the binding of the new coronavirus spike protein. It is a heterogeneous mixture with an average molecular weight of 13.2 Kda. It is composed of disaccharide units connected by 1-4 glycosidic bonds between hexuronic acid (GlcA / IdoA) and glucosamine (GlcN). The total sulfation degrees at the C-2-oxygen position of hexuronic acid, the C-6-oxygen position of glucosamine and the N-position are 28.2%, 44.3% and 38.9%, respectively.

[0008] The method for preparing donkey-hide gelatin heparan sulfate comprises the following steps: 1) Preparation and extraction of donkey-hide gelatin glycosaminoglycan: After heating the donkey-hide gelatin in a water bath, it is enzymatically hydrolyzed with Streptomyces protease, then centrifuged to obtain the supernatant, which is separated and purified using DEAE anion exchange chromatography, and then dialyzed and freeze-dried to obtain donkey-hide gelatin glycosaminoglycan; 2) Preparation and extraction of donkey-hide gelatin heparan sulfate: The donkey-hide gelatin glycosaminoglycan obtained in step (1) is hydrolyzed with chondroitin sulfate ABC enzyme, and the supernatant is centrifuged and separated and purified by DEAE anion exchange chromatography, and then dialyzed and freeze-dried to obtain the donkey-hide gelatin heparan sulfate.

[0009] Specifically, step 1) includes the following operations: a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 ml of ultrapure water in a 50-70°C water bath and keep warm for 30-60 minutes; b) dissolving 1-5 mg of pronase in 1-5 ml of pronase hydrolysate, then adding it to the donkey-hide gelatin solution obtained in step a), heating in a water bath at 36-40° C. for 24-48 h, and then inactivating it with boiling water; c) centrifuging the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collecting the supernatant for later use; d) adding the supernatant obtained in step c) to a DEAE anion exchange column, and eluting and separating the supernatant with a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl, detecting the collected fractions with ultraviolet light of 212 nm wavelength, and then dialyzing the collected target fractions with a 1000-5000 Da dialysis membrane at 2-5° C. for 2-4 days (during which the deionized water is replaced every five hours) to remove NaCl in the eluent, and finally freeze-drying to obtain donkey-hide gelatin glycosaminoglycans.

[0010] The operation of step 2) includes: using 300-500 mU chondroitin sulfate ABC enzyme to enzymatically hydrolyze the obtained donkey-hide gelatin glycosaminoglycan for 24-48 hours at 36-40°C, inactivating the obtained donkey-hide gelatin glycosaminoglycan with boiling water after the enzymatic hydrolysis, and centrifuging at 10,000 r for 10-20 minutes to obtain the supernatant; adding the supernatant to a DEAE anion exchange column, eluting and separating the obtained donkey-hide gelatin glycosaminoglycan with a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl, detecting the collected fractions under ultraviolet light at a wavelength of 212 nm, and then dialyzing the collected target fractions with a 5000-8000 Da dialysis membrane at 2-5°C for 2-4 days to remove NaCl in the eluent, and finally freeze-drying the obtained donkey-hide gelatin heparan sulfate.

[0011] Furthermore, the Streptomyces protease hydrolysate contains 0.24 M sodium acetate and 1.9 M sodium chloride, and its pH is 6.5.

[0012] The obtained donkey-hide gelatin heparan sulfate can inhibit the binding activity of the new coronavirus spike protein with the auxiliary receptor, and can therefore be used as an inhibitor of the binding activity of the new coronavirus spike protein.

[0013] The significant advantages of the present invention are: The present invention prepares a new donkey-hide gelatin sulfate heparin through conventional operations such as enzymatic hydrolysis, ion exchange chromatography separation and purification, and dialysis. The new donkey-hide gelatin sulfate heparin has the ability to competitively inhibit the binding activity of the new coronavirus spike protein and heparin, can effectively prevent the early infection of the new coronavirus, and can lay an important foundation for the development of new clinical uses of donkey-hide gelatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the elution curve of the extracted donkey-hide gelatin glycosaminoglycan (A) and donkey-hide gelatin heparan sulfate (B) prepared in Example 1.

[0015] Figure 2 This is the mass spectrometry analysis diagram of the disaccharide structure obtained in Example 2.

[0016] Figure 3 The electrophoresis diagram (A) and standard curve (B) of the donkey-hide gelatin heparan sulfate obtained in Example 3.

[0017] Figure 4 This is a comparison chart of the response curves of the inhibitory activity of donkey-hide gelatin sulfate heparan obtained in Example 4 on spike protein and heparin chip. DETAILED DESCRIPTION

[0018] A donkey-hide gelatin sulfate heparin, the preparation method of which comprises the following steps: 1) Preparation and extraction of donkey-hide gelatin glycosaminoglycans: a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 ml of ultrapure water in a 50-70°C water bath and keep warm for 30-60 minutes; b) dissolving 1-5 mg of pronase in 1-5 ml of pronase hydrolysate (0.24 M sodium acetate and 1.9 M sodium chloride, pH 6.5), then adding the hydrolysate to the donkey-hide gelatin solution obtained in step a), heating in a water bath at 36-40°C for 24-48 h, and then inactivating the solution with boiling water; c) centrifuging the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collecting the supernatant for later use; d) adding the supernatant obtained in step c) to a DEAE anion exchange column (column volume 5-20 mL) equilibrated with 10-50 mM sodium phosphate buffer containing 0.1 M NaCl, and after sufficient elution with the equilibrated solution, elution and separation are performed with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl, collecting the eluted fractions at 3 mL / tube, and detecting the collected fractions with ultraviolet light of 212 nm wavelength, and then dialyzing the collected target fractions with a 1000-5000Da dialysis membrane at 2-5°C for 2-4 days (during which deionized water is replaced every five hours) to remove NaCl in the eluent, and finally freeze-drying to obtain donkey-hide gelatin glycosaminoglycans.

[0019] 2) Preparation and extraction of donkey-hide gelatin heparan sulfate: At 36-40°C, 300-500 mU of chondroitin sulfate ABC enzyme was used to enzymatically hydrolyze the obtained donkey-hide gelatin glycosaminoglycan for 24-48 hours. After the enzymatic hydrolysis, the gelatin was inactivated by boiling water and centrifuged at 10000 r for 10-20 minutes to obtain the supernatant. The supernatant was added to a DEAE anion exchange column (column volume 5-10 mL) equilibrated with a 10-50 mM sodium phosphate buffer containing 0.2 M NaCl, and after sufficient elution with the equilibration solution, elution and separation were performed with a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. The collected fractions were detected under ultraviolet light with a wavelength of 212 nm, and then the collected target fractions were dialyzed with a 5000-8000 Da dialysis membrane at 2-5°C for 2-4 days to remove NaCl in the eluent, and finally freeze-dried to obtain donkey-hide gelatin sulfate heparin.

[0020] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0021] Example 1 Preparation and extraction of donkey-hide gelatin heparin sulfate using donkey-hide gelatin as raw material (1) Preparation and extraction of glycosaminoglycans from donkey-hide gelatin: Soak 1 g of donkey-hide gelatin in 10 ml of ultrapure water and heat in a 70°C water bath for 30 min. Dissolve 1 mg of Streptomyces protease in 1 ml of Streptomyces protease hydrolysate (0.24 M sodium acetate and 1.9 M sodium chloride, pH 6.5) and add it to the prepared donkey-hide gelatin solution. Heat in a 37°C water bath for 48 h. After the hydrolysis is completed, inactivate it with boiling water and centrifuge at 10,000 rpm for 15 min to collect the supernatant for later use. Add the resulting supernatant to DEAE-Sephacel equilibrated with 50 mM phosphate buffer containing 0.1 M NaCl. TM After sufficient elution with the equilibrium solution in an anion exchange column (column volume 20 mL), separate and elute with 50 mM sodium phosphate buffer containing 2.0 M NaCl, collect the eluted fractions in 3 mL / tube, and perform UV detection of the collected fractions at a wavelength of 212 nm to draw an elution curve. Collect the target fractions according to the elution curve (see Figure 1 The collected target fractions were dialyzed with a 3000 Da dialysis membrane at 4°C for 4 days to remove NaCl in the eluent, during which deionized water was replaced every five hours, and finally freeze-dried to obtain donkey-hide gelatin glycosaminoglycans.

[0022] (2) Preparation and extraction of heparan sulfate from donkey-hide gelatin: 300 mU of chondroitin sulfate ABC enzyme was used to specifically enzymatically hydrolyze the donkey-hide gelatin glycosaminoglycan obtained in step (1) for 24 h at 37°C to degrade the chondroitin sulfate, dermatan sulfate and hyaluronic acid contained therein into disaccharides. After the enzymatic hydrolysis, the gelatin was inactivated by boiling water and centrifuged at 10,000 r for 10 min to obtain the supernatant. The supernatant was added to DEAE-Sephacel equilibrated with 50 mM sodium phosphate buffer containing 0.2 M NaCl. TM After sufficient elution with the equilibrium solution, the anion exchange column (column volume 10 mL) was used to separate and elute with 50 mM sodium phosphate buffer containing 2.0 M NaCl. The eluted fractions were detected by UV at 212 nm, and the elution curve was drawn and the target fractions were collected (see Figure 1 (B) The collected target fractions were dialyzed with a 5000 Da dialysis membrane for 4 days at 4°C to remove NaCl, and finally freeze-dried to obtain donkey-hide gelatin sulfate heparan.

[0023] Example 2 Analysis of the structure and composition of heparan sulfate disaccharide in donkey-hide gelatin The heparin sulfated donkey-hide gelatin prepared in Example 1 was hydrolyzed with heparinase to obtain heparin sulfated disaccharides, and the disaccharides were collected and purified by size exclusion chromatography. The composition and structure of the heparin sulfated donkey-hide gelatin were analyzed by LC / MS-ITTOF. The specific operation was to hydrolyze the heparin sulfated donkey-hide gelatin with 10 mU heparinase for 48 h at 37°C to obtain heparin sulfated disaccharides, inactivate the donkey-hide gelatin with boiling water after the hydrolysis, and centrifuge at 10000 r for 10 min to obtain the supernatant. The disaccharides were collected and purified by size exclusion chromatography, and the chromatographic column used was a Superdex™ peptide column, and the mobile phase was 0.1 M NH 4 HCO 3 , flow rate 0.4 mL / min, collect disaccharide peak components, evaporate and remove the chromatographic mobile phase at 65℃ to obtain donkey-hide gelatin sulfate disaccharide. The composition and structure of donkey-hide gelatin sulfate disaccharide were analyzed by LC / MS-ITTOF, and the chromatographic conditions were as follows: chromatographic column ACQUITY UPLC BEH C18 (2.1×150mm 1.7 μm) column; mobile phase A was an aqueous solution (pH 8.86) containing 30 mM N-hydroxyethyl acrylamide (HXA), and mobile phase B was an 80% acetonitrile aqueous solution (pH 8.86) containing 30 mM HXA; flow rate 0.1 mL / min; column temperature 45℃, mass spectrometry conditions: negative ion mode scanning mode; CDL and Heat Block temperature 100-200℃; spray gas flow rate 1.5 mL / min; cone voltage -3.5 kV. According to the standard curve, the structure and component content of the disaccharide were analyzed. The mass spectrometry analysis chart and disaccharide component table are shown in Figure 2 and Table 1.

[0024] Table 1 Disaccharide composition

[0025] The results showed that 8 heparan sulfate disaccharides were detected, among which ΔUA-GlcNAc had the highest content (30.4%), while the contents of the three disaccharides ΔUA-GlcNS, ΔUA-GlcNS(6S) and ΔUA(2S)-GlcNS(6S) were the second, at 16.7%, 16.7% and 15.2%, respectively. The total sulfation degree of the C-2-oxygen position of hexuronic acid, the C-6-oxygen position of glucosamine and the N-position were 28.2%, 44.3% and 38.9%, respectively. These results indicate that donkey-hide gelatin heparan sulfate has a low-sulfation structure and has a similar structure to the auxiliary co-receptor of the new crown spike protein on the cell surface, so it is expected to become a competitive inhibitor of the new crown spike protein.

[0026] Example 3 Determination of Molecular Weight of Donkey-hide Gelatin Heparan Sulfate The electrophoresis analysis was performed using a discontinuous gel system with a gel thickness of 1 mm, including 4% T for the concentrated gel and 15% for the separated gel. The electrophoresis buffer was 0.25 M tris-glycine pH 8.3. The sample of donkey-hide gelatin heparan sulfate was 40 μg (EJ), and dp18 (3 μg), low molecular weight heparin (15 μg), heparin (10 μg), CS (10 μg), and DS (10 μg) with known molecular weights were used as standards (represented as S1, S2, S3, S4, and S5, respectively). It was stained with 0.5% alcian blue (W / V) for 30 min and faded with a large amount of distilled water. The standard curve drawn using glycosaminoglycans of known molecular weight and their electrophoretic mobility was used to calculate the molecular weight of donkey-hide gelatin heparan sulfate based on this standard curve.

[0027] like Figure 3 As shown, the donkey-hide gelatin heparan sulfate is a heterogeneous mixture (molecular weight distribution range is 20.4 Kda-6.0Kda), and its average molecular weight is 13.2Kda. This indicates that the molecular weight of some donkey-hide gelatin heparan sulfate is lower than that of heparin, and is closer to low molecular weight heparin. It has been reported that the competitive inhibitory activity of low molecular weight heparin with spike protein is 200 times higher than that of heparin. Therefore, the donkey-hide gelatin heparan sulfate has the potential to become a competitive inhibitor with higher activity.

[0028] Example 4 Application of donkey-hide gelatin sulfate heparin in inhibiting the binding activity of novel coronavirus spike protein and auxiliary receptor Using the donkey-hide gelatin heparan sulfate prepared in Example 1 as a competitive inhibitor, the SPR heparin-SA-chip system was used to simulate the interaction between the new coronavirus spike protein and its auxiliary co-receptor heparin / heparan sulfate in vivo, and the inhibitory response curves of different concentrations of donkey-hide gelatin heparan sulfate on the spike protein and heparin chip were characterized, and the inhibitory activity was calculated.

[0029] The heparin-SA chip used was prepared by the following method: ① Preparation of biotinylated heparin: 2 mg of heparin and biotin (amine-PEG3-Biotin) were dissolved in 200 μL ultrapure water, 10 mg of sodium cyanoborohydride was added, and the mixture was reacted in a 70 °C water bath for 24 h. Then, 10 mg of sodium cyanoborohydride was added. After cooling to room temperature, the biotinylated heparin was purified using a 3 kDa dialysis bag and freeze-dried for later use. ② Direct cross-linking of biotinylated heparin on the SA chip: At 25 °C, 10 μg / mL of biotinylated heparin in HBS-EP solution was injected in manual mode, with an injection flow rate of 10 μL / min and an injection time of 120 s. The mobile phase was HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4). After the injection, the chip surface was regenerated with 2 mol / L NaCl solution for 3 min. Finally, the chip was equilibrated with HBS-EP buffer. When the baseline response value increased by more than 200 RU and the baseline remained unchanged, it indicated that the heparin-SA chip was successfully fixed with the ligand.

[0030] Different concentrations of donkey-hide gelatin sulfate heparan solution were prepared and mixed with spike protein (final reaction concentration was 85 nM), and then injected into the heparin-SA chip. The sample flow rate was 30 μL / min, the binding time was 120 s, and the dissociation time was 300 s. After the analysis, the chip surface was regenerated with 2 mol / L NaCl solution for 3 min. The inhibition response curve of donkey-hide gelatin sulfate heparan on spike protein and heparin chip is shown in Figure 4 The inhibition rate of the spike protein was calculated based on the response value of the blank group.

[0031] The experimental results showed that compared with the 0nM control sample, the inhibition rates of 29nM and 145nM donkey-hide gelatin sulfate heparan solutions on the binding of the new coronavirus spike protein to the heparin-SA chip were 67.6% and 82.4%, respectively. After simulation calculation, the inhibition rate of donkey-hide gelatin sulfate heparan on the binding of the new coronavirus spike protein to heparin was obtained. 50 Reaching 12nM, its inhibition rate is 3-5 times higher than the binding activity of heparin and other heparan sulfates. The above content shows that donkey-hide gelatin heparan sulfate has a strong activity in inhibiting the binding of spike proteins, which can prevent and inhibit the initial infection of the new coronavirus.

[0032] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A donkey-hide gelatin sulfate heparan with the ability to inhibit the binding activity of the novel coronavirus spike protein, characterized in that: The donkey-hide gelatin heparan sulfate is a heterogeneous mixture with an average molecular weight of 13.2 Kda. It is composed of disaccharide units connected by hexuronic acid and glucosamine with 1-4 glycosidic bonds, wherein the total sulfation degrees of the C-2-oxygen position of hexuronic acid, the C-6-oxygen position of glucosamine and the N-position are 28.2%, 44.3% and 38.9% respectively.

2. A method for preparing donkey-hide gelatin heparan sulfate as claimed in claim 1, characterized in that: The following steps are involved: 1) Preparation and extraction of donkey-hide gelatin glycosaminoglycan: After heating the donkey-hide gelatin in a water bath, it is enzymatically hydrolyzed with Streptomyces protease, then centrifuged to obtain the supernatant, which is separated and purified using DEAE anion exchange chromatography, and then dialyzed and freeze-dried to obtain donkey-hide gelatin glycosaminoglycan; 2) Preparation and extraction of donkey-hide gelatin heparan sulfate: The donkey-hide gelatin glycosaminoglycan obtained in step (1) is hydrolyzed with chondroitin sulfate ABC enzyme, and the supernatant is centrifuged and separated and purified by DEAE anion exchange chromatography, and then dialyzed and freeze-dried to obtain the donkey-hide gelatin heparan sulfate.

3. The method for preparing donkey-hide gelatin heparan sulfate according to claim 2, characterized in that: Step 1) The specific operations include: a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 ml of ultrapure water in a 50-70°C water bath and keep warm for 30-60 minutes; b) dissolving 1-5 mg of pronase in 1-5 ml of pronase hydrolysate, then adding it to the donkey-hide gelatin solution obtained in step a), heating in a water bath at 36-40° C. for 24-48 h, and then inactivating it with boiling water; c) centrifuging the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collecting the supernatant for later use; d) adding the supernatant obtained in step c) to a DEAE anion exchange column, and eluting and separating the supernatant with a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl, detecting the collected fractions with ultraviolet light at a wavelength of 212 nm, and then dialyzing the collected target fractions with a 1000-5000 Da dialysis membrane to remove NaCl in the eluent, and finally freeze-drying the obtained fractions.

4. The method for preparing donkey-hide gelatin heparan sulfate according to claim 3, characterized in that: The Streptomyces protease hydrolysate contains 0.24 M sodium acetate and 1.9 M sodium chloride, and its pH is 6.

5.

5. The method for preparing donkey-hide gelatin heparan sulfate according to claim 2, characterized in that: The specific operation of step 2) includes: using 300-500 mU chondroitin sulfate ABC enzyme to enzymatically hydrolyze the obtained donkey-hide gelatin glycosaminoglycan for 24-48 hours at 36-40°C, inactivating the obtained donkey-hide gelatin glycosaminoglycan with boiling water after the enzymatic hydrolysis, centrifuging at 10000 r for 10-20 minutes to obtain the supernatant; adding the supernatant to a DEAE anion exchange column, eluting and separating the obtained supernatant with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl, detecting the collected fractions under ultraviolet light at a wavelength of 212 nm, and then dialyzing the collected target fractions with a 5000-8000 Da dialysis membrane to remove NaCl in the eluent, and finally freeze-drying the obtained fractions.

6. An application of the donkey-hide gelatin sulfate heparan as claimed in claim 1 as an inhibitor of the binding activity of the new coronavirus spike protein.

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