CN111773240A - Application of marine biological source natural sulfated polysaccharide as anti-coronavirus and disease-causing drugs - Google Patents

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of natural sulfated polysaccharide from marine organisms as a medicine for resisting coronavirus and diseases caused by coronavirus. Application of natural sulfated polysaccharide and pharmaceutically acceptable salt derived from marine organisms in preparation of medicines combined with Spike protein (Spike), inhibitor of main protease (Mpro), or papain-like (PLpro) inhibitor. The marine organism-derived natural sulfated polysaccharide provided by the invention has an obvious effect of inhibiting the multiplication of coronavirus, can inhibit the combination of Spike protein (Spike) on the surface of the coronavirus and host cells, and can inhibit the activities of main protease (Mpro) and papain-like (PLpro), thereby effectively blocking the coronavirus from infecting the host cells.

Description

Natural sulfated polysaccharides derived from marine organisms are used as anti-coronavirus and disease-causing drugs applications in

technical field

The invention belongs to the field of biomedicine, and in particular relates to the application of a marine biological source natural sulfated polysaccharide as an anti-coronavirus and a disease-causing drug.

Background technique

The World Health Organization named the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the new coronavirus pneumonia (COVID-19), which was declared a public health emergency of international importance by the World Health Organization on January 30, 2020 . The rapid spread of COVID-19 around the world has posed a great threat to human health and social public health security. However, there is currently no specific drug against the virus. Clinically, supportive treatment and symptomatic treatment to improve symptoms are the mainstays. The research and development of anti-SARS-CoV-2 drugs is urgent. A large number of drug screening experiments have been carried out for the new coronavirus worldwide, but no drugs with good therapeutic effects have been found.

SARS-CoV-2 is an RNA virus with an enveloped structure. Spike glycoproteins on the lipid membrane of virus particles responsible for adhesion to host receptors. Viral genomic RNAs within lipid membranes encode replicase polyproteins that can be cleaved by 3C-like proteases (3CLpro, also known as major protease Mpro) and papain-like proteases (PLpro) to produce functional polypeptides such as RNA Dependent RNA polymerase (RdRp) and helicase (Hel), involved in viral RNA transcription and replication. Therefore, the spike glycoproteins associated with SARS-CoV-2 infection of host cells, the major proteases and papain-like proteases associated with SARS-CoV-2 transcriptional replication are important targets for anti-SARS-CoV-2 drug development.

Mar.Drugs，2015,13,697–712)，但也未发现有抗新冠病毒活性。Marine organisms are rich in resources. Natural carbohydrates from marine organisms, especially acidic carbohydrates, can bind to positively charged proteins of viruses to prevent viruses from replicating or infecting cells. In addition, it has the advantages of low toxicity and is not easy to cause virus resistance, and has broad prospects in the development of antiviral drugs. Literature search found that natural sulfated polysaccharides derived from marine organisms, such as the complex structure fucoidan sulfate derived from brown algae, have anti-influenza virus effects (Publication No. CN 103880975A; Patent No.: ZL201410132608.x), and its main chain structure is composed of Composed of α-1,2-mannose (Man) and β-1,4-glucuronic acid (GlcA), containing a branched α-1,3-linked-sulfated fucose residue at the C3 position of mannose (Jiandong Wu, J Carbohy Chem, 34:303-317; Wei Wang, Sci.Rep.2017, 7:40760), and recently reported a similar structure of fucoidan (RPI) with different molecular weights (100kD and 12kD) -27 and RPI-128) have the activity of inhibiting COVID-COV-2 coronavirus Spike protein, the polysaccharide contains α-1,2-mannose (Man) and β-1,4-glucuronic acid (GlcA) chain, containing a branched α-1,2/1,3-linked-sulfated fucose residue at the C3 position of mannose (Paul S. Kwon, Cell Discovery, 2020, 6:50); found that wakame-derived Fucoidan (Hayashi, K., Int. Immunopharmacol. 2008, 8, 109) has anti-HSV activity. There is a lack of reports on the anti-SARS-CoV-2 research of other types of fucose sulfates. Carrageenan derived from red algae has broad anti-H1N1, HSV, HPV activity (Cui Hao, Rev Med Virol. 2019, 29, e2043), and iota-carrageenan also has anti-porcine blue ear virus (PRRSV) activity (Chunhe Guo , Antivir Ther, 2019, 24(4):261-270), but no anti-new coronavirus activity has been found. Chlorella-derived rhamnose sulfate has anti-children hand, foot and mouth virus (EV71) activity (Shuyao Wang, Carbohydr Polym, 2018, 200: 43-53) and Newcastle disease virus (NDV) activity (Aguilar-

Mar. Drugs, 2015, 13, 697–712), but no anti-new coronavirus activity was found.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of application of marine biological source natural sulfated polysaccharide as anti-coronavirus and disease-causing medicine, using specific marine plants and marine animals as raw materials, through extraction and separation, obtain a series of marine source natural sulfated polysaccharide, Screen its activity in inhibiting coronavirus, especially novel coronavirus (SARS-COV-2), and provide effective drugs and compositions for treating coronavirus, especially novel coronavirus pneumonia (COVID-19).

The technical scheme of the present invention is:

The application of a marine organism-derived natural sulfated polysaccharide as an anti-coronavirus and a disease-causing drug, the marine organism-derived natural sulfated polysaccharide and a pharmaceutically acceptable salt are used in the preparation of a drug combined with a spike protein (Spike), preparation of The main protease (Mpro) inhibitor or the application in the preparation of papain-like (PLpro) inhibitor, the marine organism-derived natural sulfated polysaccharide has any of the following structural characteristics, and its molecular weight ranges from 3 to 600kDa, including:

(1) Fucoidan sulfate (also known as fucoidan), having one of the following structural characteristics: (a) Type I structure fucoidan, the main chain is sulfated fucose linked by α-1,3- (b) Type II structure fucoidan, the main chain is composed of sulfated fucose linked by α-1,3 and α-1,4 alternately; (c) Type III structure fucoidan, the main chain is composed of β -1,4-glucuronic acid and α-1,2-mannose, the branched structure is composed of α-1,3-sulfated fucoid oligosaccharide; (d) other types of fucoidan, the main chain is composed of α-1,3-sulfated fucoidan -1,2 and α-1,3 and/or α-1,4-linked sulfated fucose composition;

(2) Fucosylated chondroitin sulfate, its structural features are as follows: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds Alternately linked chondroitin sulfate disaccharide repeating units, the C4, C6 or C4 and C6 hydroxyl groups of GalNAc contain a sulfate group, and the sulfated fucoid oligosaccharide is linked at the C3 position of GlcA through an α-1,3 glycosidic bond Branched;

(3) Galactan sulfate, the structural characteristics of which are as follows: galactose (Gal) linked by β-1,3- and α-1,4-, or β-1,3-linked with α-1 ,4-linked-3,6-lactone galactose (AnG), and the C2, C4 or C6 positions of galactose contain different sulfate groups;

(4) Rhamnose sulfate: its structural characteristics are as follows: it is formed by alternately connecting rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by Rha and Aidur Uronic acids are alternately linked by α-1,4 glycosidic bonds; or rhamnose (Rha) and xylose (Xyl) are alternately linked by α-1,4 and β-1,4 glycosidic bonds; or It is formed by alternately connecting α-1,2 and α-1,3-rhamnose; and the C2 and/or C3 positions of the above structure Rha contain a sulfate group.

The type I structure fucoidan also has branched chain structures of other sugar residues; the type II structure fucoidan also has branched chain structures of other sugar residues.

The coronavirus is selected from one or more of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

The natural sulfated polysaccharide is derived from marine plants or marine animals.

Described natural sulfated polysaccharide is combined with the spike protein (Spike) of coronavirus, as the inhibitor that inhibits its infection of cells, by inhibiting the activity of coronavirus main protease (Mpro) and/or papain-like protease (PLpro), Inhibits virus replication and amplification.

The application of described natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing coronavirus inhibitor.

The application of described natural sulfated polysaccharide and pharmaceutically acceptable salt in the preparation of a medicine for preventing and/or treating coronavirus infection, or preventing and/or treating a disease or symptom related to coronavirus infection.

The kit or reagent for combining the natural sulfated polysaccharide and pharmaceutically acceptable salt with Spike protein (Spike), the kit or reagent for preparing major protease (Mpro) inhibitor, or the preparation of papain-like protease (PLpro) Use of inhibitors in kits or reagents.

Described natural sulfated polysaccharide, pharmaceutically acceptable salt and another one or more active agents for preventing and/or treating coronavirus infection are mixed to make the pharmaceutical composition for preventing and/or treating coronavirus infection .

The design idea of the present invention is:

The marine organism-derived natural sulfated polysaccharide provided by the present invention can not only inhibit the combination of the spike protein (Spike) on the surface of the coronavirus with the host cell, thereby effectively blocking the infection of the host cell by the coronavirus, but also inhibit the main protease (Mpro) of the coronavirus. and papain-like protease (PLpro) activity, thereby inhibiting the replication and amplification process of the virus. Therefore, the marine natural sulfated polysaccharide provided by the present invention has great potential to be developed into anti-coronavirus and disease-causing drugs.

The advantages and beneficial effects of the present invention are:

(1) The marine organism-derived natural sulfated polysaccharide provided by the present invention can inhibit the combination of the spike protein (Spike) on the surface of the coronavirus with the host cell, and its inhibitory activity level is better than that of the clinical drug heparin and low molecular weight heparin.

(2) The marine organism-derived natural sulfated polysaccharide provided by the present invention has the activity of inhibiting major protease (Mpro) and papain-like protease (PLpro), and its inhibitory activity level is better than that of heparin.

(3) as a whole, the anti-coronavirus activity of the natural sulfated polysaccharide derived from marine organisms provided by the present invention is superior to clinical medicine heparin and low molecular weight heparin, and can be used as the preparation and application of anti-coronavirus and related medicines caused by diseases field.

Description of drawings

Figure 1. Inhibitory activities of fucosan sulfate, galactan sulfate, fucosylated chondroitin sulfate, rhamnose sulfate and heparin against SARS-CoV-2 spike protein. In the figure, the abscissa Log Dose represents the logarithm (μM) of the compound concentration, and the ordinate Relative Luminescence represents the relative luminescence intensity (%).

Figure 2. Inhibitory effect curve of galactan sulfate, fucoidan sulfate, and fucosylated chondroitin sulfate on SARS-CoV-2 main protease. In the figure, the abscissa Time represents time (s), the ordinate Intensity represents fluorescence intensity, Blank represents blank control, Iota-carrageenan represents iota-carrageenan, FCS represents fucosylated chondroitin sulfate, and Fucoidan represents fucoidan Sulfate.

Fig. 3 Determination curve of _IC50 of fucoidan sulfate inhibiting SARS-CoV-2 main protease. In the figure, the abscissa Log concentration represents the logarithm (nM) of the concentration, and IC ₅₀ represents the half inhibitory concentration.

Detailed ways

In the specific implementation process, the present invention relates to the application of marine source natural sulfated polysaccharide and pharmaceutically acceptable salt in the preparation of coronavirus inhibitor, and the natural sulfated polysaccharide has any of the following structures:

(1) Fucoidan sulfate (also known as fucoidan), including: (a) Type I fucoidan, the main chain is composed of α-1,3-linked sulfated fucose, and may also have other Sugar residue branched chain structure; (b) Type II fucoidan, the main chain is composed of sulfated fucose alternately linked by α-1,3 and α-1,4, and can also have other sugar residue branched chain structures (c) Type III fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branch structure is composed of α-1,3-sulfated fucoid oligosaccharide ; (d) other types of fucoidan, the main chain is composed of α-1,2 and α-1,3 and/or α-1,4-linked sulfated fucose.

(2) Fucosylated chondroitin sulfate, its structural features are as follows: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds Alternately linked chondroitin sulfate disaccharide repeat units, and the sulfated fucoid oligosaccharide branch chain is connected by α-1,3 glycosidic bond at the C3 position of GlcA.

(3) Galactan sulfate, the structural characteristics of which are as follows: galactose (Gal) linked by β-1,3- and α-1,4-, or β-1,3-linked with α-1 ,4-linked-3,6-lactone galactose (AnG), and the C2, C4 or C6 positions of galactose contain different sulfate groups.

(4) Rhamnose sulfate, its structural characteristics are as follows: it is formed by alternately connecting rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; Uronic acids are alternately linked by α-1,4 glycosidic bonds; or rhamnose (Rha) and xylose (Xyl) are alternately linked by α-1,4 and β-1,4 glycosidic bonds; or It is formed by alternately connecting α-1,2 and α-1,3-rhamnose; and the C2 and/or C3 positions of the above structure Rha contain a sulfate group.

The present invention also provides marine organism-derived natural sulfated polysaccharides and pharmaceutically acceptable salts in the preparation of drugs combined with spike protein (Spike), the preparation of major protease (Mpro) inhibitors, or the preparation of papain-like protease (PLpro) inhibitors The application in the agent, the marine biological source sulfated polysaccharide has any of the following structures:

(1) Fucoidan sulfate (also known as fucoidan), including: (a) I-structure fucoidan, the main chain is composed of α-1,3-linked sulfated fucose, and may also have Branched structure of other sugar residues; (b) Type II structure of fucoidan, the main chain is composed of sulfated fucose alternately linked by α-1,3 and α-1,4, and can also have branches of other sugar residues Chain structure; (c) Type III structure Fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucoidan Oligosaccharide composition; (d) other types of fucoidan, the main chain is composed of α-1,2 and α-1,3 and/or α-1,4-linked sulfated fucose.

(2) Fucosylated chondroitin sulfate, its structural features are as follows: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds Alternately linked chondroitin sulfate disaccharide repeating units, and the sulfated fucose branch chain is connected by α-1,3 glycosidic bond at the C3 position of GlcA.

(3) Galactan sulfate, the structural characteristics of which are as follows: galactose (Gal) linked by β-1,3- and α-1,4-, or β-1,3-linked with α-1 ,4-linked-3,6-lactone galactose (AnG), and the C2, C4 or C6 positions of galactose contain different sulfate groups.

(4) Rhamnose sulfate: its structural characteristics are as follows: it is formed by alternately connecting rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by Rha and Aidur Uronic acid (IdoA) is alternately linked by α-1,4 glycosidic bonds; or rhamnose (Rha) and xylose (Xyl) are alternately linked by α-1,4 and β-1,4 glycosidic bonds. or by alternately connecting α-1,2 and α-1,3-rhamnose; and the C2 and/or C3 position of the above structure Rha contains a sulfate group.

Application as above, preferably one or more of the spike protein (Spike), main protease (Mpro), papain-like protease (PLpro) in the coronavirus; more preferably, the coronavirus selects One or more of COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

The present invention also provides the application of the above-mentioned marine organism-derived natural sulfated polysaccharide and pharmaceutically acceptable salts in the preparation of coronavirus inhibitors.

The present invention also provides the above-mentioned marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt in the preparation of preventing and/or treating coronavirus infection, or preventing and/or treating diseases or symptoms related to coronavirus infection application in medicines.

For the use as described above, preferably, the coronavirus is selected from COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

The present invention also provides the above-mentioned marine organism-derived natural sulfated polysaccharide and pharmaceutically acceptable salt in the preparation of a kit or reagent combined with a spike protein (Spike), a kit for preparation of a major protease (Mpro) inhibitor or Reagents, or kits or reagents for preparing papain-like protease (PLpro) inhibitors.

The present invention also provides a pharmaceutical composition for preventing and/or treating coronavirus infection, comprising a marine organism-derived natural sulfated polysaccharide, a pharmaceutically acceptable salt, and one or more of the other Active agents for the prevention and/or treatment of coronavirus infections.

Preferably, the molecular weight of the marine organism-derived natural sulfated polysaccharide ranges from 3 to 600 kDa.

The present invention has shown through experimental screening studies that a series of marine organism-derived natural sulfated polysaccharides are effective against coronaviruses, especially the spike protein (Spike), main protease (Mpro or 3CL pro) and PLpro of the novel coronavirus (SARS-CoV-2). have better inhibitory effect. The invention provides the application potential of marine biological source natural sulfated polysaccharide in anti-coronavirus, especially SARS-CoV-2, and confirms that it has a good clinical development and application prospect.

The marine biological source natural sulfated polysaccharide provided by the invention, preferably, its structural characteristics are as follows:

(1) Fucoidan sulfate (also known as fucoidan), including: (a) I-structure fucoidan, the main chain is composed of α-1,3-linked sulfated fucose, and may also have Branched structure of other sugar residues; (b) Type II structure of fucoidan, the main chain is composed of sulfated fucose alternately linked by α-1,3 and α-1,4, and can also have branches of other sugar residues Chain structure; (c) Type III structure Fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucoidan Oligosaccharide composition; (d) other types of fucoidan, the main chain is composed of α-1,2 and α-1,3 and/or α-1,4-linked sulfated fucose.

(2) Fucosylated chondroitin sulfate, its structural features are as follows: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds Alternately linked chondroitin sulfate disaccharide repeating units, and the sulfated fucose branch chain is connected by α-1,3 glycosidic bond at the C3 position of GlcA.

(3) Sulfated galactan, the structural characteristics of which are as follows: β-1,3-linked and α-1,4-linked galactose (Gal), or β-1,3-linked galactose with It is composed of α-1,4-linked-3,6-lactone galactose (AnG), and the C2, C4 or C6 position of galactose contains different sulfate groups.

(4) Rhamnose sulfate: its structural characteristics are as follows: it is formed by alternately connecting rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by Rha and Aidur Uronic acids are alternately linked by α-1,4 glycosidic bonds; or rhamnose (Rha) and xylose (Xyl) are alternately linked by α-1,4 and β-1,4 glycosidic bonds; or It is formed by alternately connecting α-1,2 and α-1,3-rhamnose; and the C2 and/or C3 positions of the above structure Rha contain a sulfate group.

The marine organism-derived sulfated polysaccharides of the present invention all have the effect of inhibiting coronaviruses, especially novel coronaviruses (SARS-CoV-2). The experimental results show that the various marine organism-derived sulfated polysaccharides provided by the present invention have significant anti-new coronavirus effects, the IC ₅₀ value of inhibiting Spike is between 0.01 and 30 μM, and the IC ₅₀ value of inhibiting Mpro is between 1 nM and 100 nM. , the IC ₅₀ value of inhibiting papain-like protease (PLpro) is 0.001～30μM. For example, the IC ₅₀ of fucoidan sulfate for inhibiting Mpro was 5.68 nM, the IC ₅₀ for inhibiting Spike protein was 0.06 μM, and the IC ₅₀ for inhibiting papain-like protease (PLpro) was 7.65 nM, all of which were significantly better than heparin.

As a preferred solution, for the above application, the coronaviruses include but are not limited to novel coronavirus (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (which causes severe acute respiratory syndrome) and MERS-CoV (which causes Middle East respiratory syndrome).

The application of the marine biological source natural sulfated polysaccharide in anti-new coronavirus provided by the present invention can prepare the marine biological source natural sulfated polysaccharide into sprays, injections, capsules, ointments, creams, gels, liniments, paint.

Preferably, a therapeutically effective amount of the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt or stereoisomer of the present invention is administered in a conventional pharmaceutical composition (the pharmaceutical composition) according to the usual route of administration and according to methods known in the art. A dosage form containing an effective amount of the active ingredient and a suitable pharmaceutically acceptable carrier) is formulated for administration to a patient in need of such treatment.

The "therapeutically effective amount" means that when administered, it is sufficient to prevent the development of, or alleviate to some extent, one or more symptoms of the targeted disease. The particular dosage of a compound administered in accordance with the present invention will be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations. In particular, a "therapeutically effective amount of a compound" refers to an amount of the compound sufficient to prevent or to some extent alleviate infection by one or more coronaviruses.

Individual doses, as well as daily doses, vary further depending on the type and severity of the coronavirus infection to be treated and the specific patient's response to drug therapy. Therefore, the exact single dose will be determined according to standard medical principles under the direction of a physician.

The effective daily dose for using the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt of the present invention in the treatment of diseases caused by coronavirus is, 1 mg to about 500 mg, about 5 mg to about 1000 mg, or about 10 mg to about Oral dosage form of 2000 mg for injection or other active agent.

The marine organism-derived natural sulfated polysaccharide and pharmaceutically acceptable salts of the present invention can be used alone or in combination or in combination therapy with other therapeutic agents.

In one embodiment of the present invention, marine organism-derived natural sulfated polysaccharide, a pharmaceutically acceptable salt, is for use in the prevention and/or treatment of coronavirus infection, wherein the prevention or treatment includes administration as the sole active ingredient.

In another embodiment of the present invention, the above-mentioned marine organism-derived natural sulfated polysaccharides, pharmaceutically acceptable salts are used in the prevention and/or treatment of coronavirus infection, wherein the prevention or treatment includes a combination selected from other Combination therapy use of therapeutic agents.

As will be apparent to those skilled in the art, the combination of the present invention comprising the marine organism-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt, and an additional therapeutic agent is not only effective when these active ingredients are used in a single composition and is also effective when used in two different compositions (administered simultaneously, sequentially or separately after a period of time). In addition, those skilled in the art will understand that the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt of the present invention can be prescribed for use with other active ingredients in combination therapy to prevent and/or treat coronavirus infection.

In a particular embodiment, the combination therapy comprises administering to the subject simultaneously, sequentially or separately the marine organism-derived natural sulfated polysaccharide, the pharmaceutically acceptable salt, and the additional therapeutic agent of the present invention. Alternatively, the combination therapy comprises administering to the subject the marine organism-derived natural sulfated polysaccharide of the invention, a pharmaceutically acceptable salt or stereoisomer, and an additional therapeutic agent in a single composition.

In one embodiment of the present invention, the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt or stereoisomer of the present invention can be conveniently administered to a patient. Thus, the compounds for use in the present invention may be in the form of a pharmaceutical composition comprising an effective amount of the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt, of the present invention in combination with a pharmaceutically acceptable excipient or carrier. This aspect can also be expressed as a composition comprising an effective amount of the marine organism-derived natural sulfated polysaccharide, a pharmaceutically acceptable salt of the present invention in combination with a pharmaceutically acceptable excipient or carrier for use in the prevention and/or treatment of coronary Viral infection.

In one embodiment of the invention, the compounds used may be administered orally, injected, subcutaneously, respiratory, transdermal, parenteral, rectal, topical, intravenous, intramuscular or by other way to give. The pharmaceutical composition can be formulated into any pharmaceutical form, such as: tablets, granules, injections, gels, pills, capsules, suppositories, implants, nano-formulations, powder injections. Some dosage forms such as tablets and capsules can be subdivided into appropriate dosage unit forms containing appropriate quantities of the active component, such as an effective amount to achieve the desired purpose.

In another embodiment, the marine organism-derived natural sulfated polysaccharide, the pharmaceutically acceptable salt for the use of the present invention is an injection preparation to be administered to the patient to be treated, and the injection suitable for the present invention refers to a drug with a suitable A solvent or dispersion medium for sterile or sterile solutions, emulsions or suspensions for injection into the human body, and sterile powder preparations for solution or suspension before use. The injections include injection solutions (large-volume injection solutions for intravenous drip are also called intravenous infusion solutions), sterile powders for injection and concentrated solutions for injection.

Carriers include excipients and diluents, and must be of sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the patient to be treated. The carrier may be inert or it may itself possess pharmaceutical benefits.

Types of carriers include, but are not limited to, diluents such as fillers and bulking agents, binders, lubricants, anti-caking agents, disintegrants, sweeteners, buffers, preservatives, solubilizers, isotonic agents, suspending agents and dispersing agents, wetting or emulsifying agents, flavoring and perfuming agents, thickening agents and vehicles.

Optional active agents can be included in the pharmaceutical compositions that do not substantially affect the activity of the compounds of the present invention.

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention 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. The structure of the marine biological source natural sulfated polysaccharide is as follows:

(1) Fucoidan sulfate: the main chain is composed of α-1,3-linked sulfated fucose with a molecular weight of 600 kDa.

(2) ι-Carrageenan: It is composed of β-1,3-linked galactose and α-1,4-linked-3,6-endethergalactose repeating disaccharide units. The galactose residue C4 and internal The ether galactose residue is substituted by sulfate at the C2 position and has a molecular weight of 200 kDa.

(3) Fucosylated chondroitin sulfate: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) alternately connected by β-1,3 and β-1,4 glycosidic bonds Chondroitin sulfate disaccharide repeating unit and fucose branched chain substituted by sulfate group at C2 and C3 positions through α-1,3 glycosidic linkage at C3 position of GlcA, with molecular weights of 3kDa and 50kDa.

(4) Rhamnose sulfate: It is composed of rhamnose (Rha) and glucuronic acid alternately connected by α-1,4 and β-1,4 glycosidic bonds, and the C2 and C3 positions of the above structure Rha contain sulfate esters base; molecular weight is 200kDa.

Example 1 Inhibitory effect of marine biological source natural sulfated polysaccharide on SARS-CoV-2 spike protein

Based on the SARS-CoV-2-spike protein viroid detection system, evaluation of fucoidan sulfate (derived from brown algae), iota-carrageenan (derived from red algae), fucosylated chondroitin sulfate (derived from Sea cucumber), rhamnose sulfate (derived from green algae) blocking the effect of SARS-CoV-2 infected cells.

The sulfated polysaccharide samples used in the present invention can be extracted by conventional reagents, methods and equipment in the technical field.

1) Cell recovery culture: 293T/17 human embryonic kidney cell line, calculated by weight percentage, mixed with 10% fetal bovine serum, 1% double antibody and the balance of DMEM medium for culture, and the cells were cultured for two generations after recovery. .

2) Virus-like preparation: Spread 293T/17 human embryonic kidney cells into a 6-well cell culture plate, when the cell confluence reaches about 60%, use transfection reagent (Lipofiter 3.0) to package and transfect 3 μg of plasmid, then The 6-well cell culture plate was cultured in a carbon dioxide incubator (37°C, 5% CO ₂ ), and the viroid supernatant solution was collected 48 hours after transfection, and stored at -80°C.

3) Transient cell transfection: 293T/17 human embryonic kidney cells were plated into a 6-well cell culture plate, and when the cell confluence reached about 70%, 3 μg of plasmid (ACE2-pcDNA3. 1) Transfected into 293T17 cells, and cultured the 6-well cell culture plate in a carbon dioxide incubator for 48 hours.

4) Cell digestion: The transiently transfected 293T/17 human embryonic kidney cells were digested with trypsin, resuspended in complete medium, counted with a cell counter, and plated into a 96-well cell culture plate with 12,000 cells per well. Cells were cultured for 6-8 h.

5) Preparation of natural sulfated polysaccharides derived from marine organisms with different concentrations: the compounds to be tested are formulated into a mother solution with a molar concentration of 1 mM, and 9 different molar concentrations are prepared in the range of 0.00015-1.00 mM according to the different concentrations detected on the cell line. The dilutions of the concentration gradient were stored in 1.5mL transparent EP tubes and stored at -20°C. The prepared 9 concentration gradient test compounds were diluted 10 times with complete medium respectively, and the same volume of dimethyl sulfoxide (DMSO) solvent was used as a control.

6) Plate plating: Take out the 96-well white cell culture plate that adheres to the wall for 6 to 8 hours in the incubator, take out 60 μL from each well, and then add 10 μL of the test substance to the above-mentioned culture plate containing 40 μL of cell volume, 2 replicates for each concentration gradient. After culturing in an incubator (37°C, 5% CO ₂ ) for 1 hour, 50 μL of the collected viroid supernatant solution was added to each well, wherein the final detection concentration of the compound to be tested was a molar concentration ranging from 0.00152 to 10.00 μM. 9 concentration gradients; after placing the culture plate in an incubator for 24 hours, replace 100 μL of medium and continue to culture for 48 hours before testing.

7) Plate reading: Place the Renilla luciferase detection reagent to room temperature, take out the cell culture plate and place it for 10 minutes, let it equilibrate to room temperature, add 15 μL of detection reagent to each well, and shake the culture plate on an orbital shaker for 2 minutes to induce cells After lysis, the plates were left at room temperature for 10 minutes, and the luminescent signal was measured on a MD (Molecular Devices, Meigu Molecular) SpectraMax Paradigm plate reader.

8) Data analysis: Using SpectraMax Paradigm reading, the corresponding fluorescence value RLU of each well was obtained. Data were processed using the following formula: RLU(%)=(RLU _Drug )/(RLU _DMSO )*100%. The cell viability corresponding to different concentrations of compounds was calculated in EXCEL, and then GraphPad 7.0 Prism software was used to make a curve graph to calculate the IC ₅₀ value of each compound. The results are shown in the table below,

compound IC₅₀(μM) heparin 10.0 Fucoidan Sulfate 0.06 iota-carrageenan 0.27 Fucosylated Chondroitin Sulfate 0.25 rhamnose sulfate 0.44

As shown in Figure 1, a series of marine organism-derived natural sulfated polysaccharides have obvious inhibitory effects on blocking the new coronavirus (SARS-CoV-2) infection of cells, and the compounds fucoidan sulfate and carrageenan, fucosyl Natural sulfated polysaccharides derived from marine organisms such as chondroitin sulfate and rhamnose sulfate are more effective than heparin in blocking virus-infected cells.

Example 2 Inhibitory effect of marine source natural sulfated polysaccharide on SARS-CoV-2 main protease

The SARS-CoV-2 main protease can hydrolyze fluorescent polypeptide substrates to generate fluorescence. The main protease activity was inhibited by marine-derived natural sulfated polysaccharide, and the fluorescence intensity was lower than that of the blank control. Among them, the main protease and fluorescent polypeptide substrate can be obtained by the method reported in the literature W.Dai et al., Science 10.1126/science.abb4489 (2020).

(1) Preparation of TE buffer solution

Add 100 μL of 0.5M disodium ethylenediaminetetraacetate (EDTA-2Na) aqueous solution to 2.5 mL of 1M Tris-HCl buffer, dilute to 50 mL, adjust pH to 7.3, filter with 0.22 μm membrane After that, store at -4°C.

(2) Screening of marine organism-derived sulfated polysaccharides for the inhibitory activity of major proteases

Add 87 μL of TE buffer solution to the 96-well cell culture plate, and then add ₁ μL of main protease with a molar concentration of 19.7 μM respectively. Molar concentration 15μM; molecular weight 260.1kDa), iota-carrageenan (2μL, molar concentration 15μM; molecular weight 200kDa), keratan sulfate from shark bone (2μL, molar concentration 15μM, molecular weight 45.98kDa), fucose from sea cucumber Chondroitin sulfate (2 μL, molar concentration 15 μM, molecular weight 42 kDa) was added to each well in turn, shaken well, and after 30 min of reaction at room temperature, 10 μL of substrate with a molar concentration of 20 μM was added to each well, and the fluorescence intensity was quickly detected by a microplate reader. . The sulfated polysaccharide samples used in the present invention can be extracted by conventional reagents, methods and equipment in the technical field.

(3) Detection conditions of microplate reader

Under the conditions of excitation wavelength of 320 nm, emission wavelength of 405 nm, and detection temperature of 27 °C, the fluorescence intensity of each well was continuously detected for 10 min, and the dynamic curve of time-fluorescence intensity was obtained.

(4) Data processing

Linear fitting was performed based on the obtained time-fluorescence intensity dynamic curve, and the slope k of the corresponding curve of each marine source natural sulfated polysaccharide and the corresponding curve slope _k of the blank control were obtained.

Inhibition rate of natural sulfated polysaccharide (molar concentration 300nM) to main protease=(1-k/k ₀ )×100%

compound Inhibition rate/% heparin 72.2 Fucoidan Sulfate 92.3 iota-carrageenan 62.8 Fucosylated Chondroitin Sulfate 65.9 rhamnose sulfate 75.0

As shown in Figure 2, brown algae-derived fucoidan sulfate, red algae-derived carrageenan, and sea cucumber-derived fucosylated chondroitin sulfate all had a 50% higher inhibition rate on the main protease activity of SARS-CoV-2.

Example 3 Inhibitory effect of different concentrations of fucoidan sulfate on SARS-CoV-2 main protease

(1) Inhibition experiment of fucoidan sulfate on main protease activity

Add 87 μL of TE buffer solution to the 96-well cell culture plate, and then add 1 μL of main protease with a molar concentration of 19.7 μM, respectively. After mixing, add 2 μL of H ₂ O and 9 concentration gradients with a molar concentration ranging from 0.5 nM to 45 μM. Seaweed-derived fucoidan sulfate aqueous solution was added to each well in turn, shaken well, and after 30 min of reaction at room temperature, 10 μL of substrate with a molar concentration of 20 μM was added to each well, and the fluorescence intensity was quickly detected by a microplate reader. The sulfated polysaccharide samples used in the present invention can be extracted by conventional reagents, methods and equipment in the technical field.

(2) Detection conditions of microplate reader

Under the conditions of excitation wavelength of 320 nm, emission wavelength of 405 nm, and detection temperature of 27 °C, the fluorescence intensity of each well was continuously detected for 10 min, and the dynamic curve of time-fluorescence intensity was obtained.

(3) Data processing

Linear fitting was performed based on the obtained time-fluorescence intensity dynamic curve, and the slope k of the corresponding curve of fucoidan sulfate and the corresponding curve slope _k of the blank control at different concentrations were obtained.

Inhibition rate of main protease by different concentrations of fucoidan sulfate=(1-k/k ₀ )×100%

(4) IC ₅₀ calculation

Using the software origin 8.1, the logarithm of the concentration was plotted against the inhibition rate, and a nonlinear fitting was used to obtain the _IC50 value.

(5) Results

As shown in Figure 3, marine organism-derived fucoidan sulfate has a good inhibitory effect on the main protease activity, and its corresponding _IC50 value=5.68±0.43nM (nmol/L)

The experimental results show that the natural sulfated polysaccharide derived from marine organisms provided by the present invention has an obvious inhibitory effect on the proliferation of coronaviruses, which can not only inhibit the combination of the spike protein (Spike) on the surface of the coronaviruses with host cells, but also inhibit the main protease (Mpro). and papain-like protease (PLpro) activity, thereby effectively blocking the infection of host cells by the coronavirus.

Claims (9)

1. the application of a marine organism source natural sulfated polysaccharide as anti-coronavirus and the medicine caused by the disease, it is characterized in that, the marine organism source natural sulfated polysaccharide, pharmaceutically acceptable salt are prepared with spike protein (Spike ) combined medicine, preparation of main protease (Mpro) inhibitor, or preparation of papain-like (PLpro) inhibitor, the marine organism-derived natural sulfated polysaccharide has any of the following structural characteristics, and its molecular weight range is 3～ 600kDa, including: (1) Fucoidan sulfate (also known as fucoidan), having one of the following structural characteristics: (a) Type I structure fucoidan, the main chain is sulfated fucose linked by α-1,3- (b) Type II structure fucoidan, the main chain is composed of sulfated fucose linked by α-1,3 and α-1,4 alternately; (c) Type III structure fucoidan, the main chain is composed of β -1,4-glucuronic acid and α-1,2-mannose, the branched structure is composed of α-1,3-sulfated fucoid oligosaccharide; (d) other types of fucoidan, the main chain is composed of α-1,3-sulfated fucoidan -1,2 and α-1,3 and/or α-1,4-linked sulfated fucose composition; (2) Fucosylated chondroitin sulfate, its structural features are as follows: the main chain is composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds The repeating units of chondroitin sulfate disaccharide are alternately connected, and the sulfated fucoid oligosaccharide branch is connected by α-1,3 glycosidic bond at the C3 position of GlcA; (3) Galactan sulfate, the structural characteristics of which are as follows: galactose (Gal) linked by β-1,3- and α-1,4-, or β-1,3-linked with α-1 ,4-linked-3,6-lactone galactose (AnG), and the C2, C4 or C6 positions of galactose contain different sulfate groups; (4) Rhamnose sulfate: its structural characteristics are as follows: it is formed by alternately connecting rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by Rha and Aidur Uronic acids are alternately linked by α-1,4 glycosidic bonds; or rhamnose (Rha) and xylose (Xyl) are alternately linked by α-1,4 and β-1,4 glycosidic bonds; or It is formed by alternately connecting α-1,2 and α-1,3-rhamnose; and the C2 and/or C3 positions of the above structure Rha contain a sulfate group. 2 . The application according to claim 1 , wherein the type I structure fucoidan also has branched chain structures of other sugar residues; the type II structure fucoidan also has branched chain structures of other sugar residues. 3 . . 3. The application of claim 1, wherein the coronavirus is selected from one of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV species or two or more. 4. application as claimed in claim 1 is characterized in that, natural sulfated polysaccharide is derived from marine plants or marine animals. 5. application as claimed in claim 1 is characterized in that, natural sulfated polysaccharide is combined with the spike protein (Spike) of coronavirus, as the inhibitor that suppresses its infection cell, by suppressing coronavirus main protease (Mpro) and/or papain-like protease (PLpro) activity to inhibit virus replication and amplification. 6. application as claimed in claim 1 is characterized in that, the application of natural sulfated polysaccharide, pharmaceutically acceptable salt in preparing coronavirus inhibitor. 7. application as claimed in claim 1 is characterized in that, natural sulfated polysaccharide, pharmaceutically acceptable salt are in preparation prevention and/or treatment coronavirus infection, or prevention and/or treatment and the disease relevant to coronavirus infection or Symptomatic drug use. 8. application as claimed in claim 1 is characterized in that, the test kit or reagent that natural sulfated polysaccharide, pharmaceutically acceptable salt are combined with spike protein (Spike), the test kit of preparing main protease (Mpro) inhibitor Or reagents, or the use of kits or reagents for preparing papain-like protease (PLpro) inhibitors. 9. application as claimed in claim 1 is characterized in that, natural sulfated polysaccharide, pharmaceutically acceptable salt and another one or more activating agents for preventing and/or treating coronavirus infection are mixed to make Pharmaceutical composition for preventing and/or treating coronavirus infection.

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