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

Application of marine biological source natural sulfated polysaccharide as anti-coronavirus and disease-causing drugs Download PDF

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CN111773240A
CN111773240A CN202010825078.2A CN202010825078A CN111773240A CN 111773240 A CN111773240 A CN 111773240A CN 202010825078 A CN202010825078 A CN 202010825078A CN 111773240 A CN111773240 A CN 111773240A
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fucoidan
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CN111773240B (en
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于广利
刘婵娟
蔡超
王鑫
李国云
蒋昊
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Ocean University of China
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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

海洋生物来源天然硫酸多糖在作为抗冠状病毒及所致疾病药 物中的应用Natural sulfated polysaccharides derived from marine organisms are used as anti-coronavirus and disease-causing drugs applications in

技术领域technical field

本发明属于生物医药领域,具体涉及一种海洋生物来源天然硫酸多糖在作为抗冠状病毒及所致疾病药物中的应用。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.

背景技术Background technique

世界卫生组织将严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的疾病命名为新冠肺炎(COVID-19),2020年1月30日世界卫生组织宣布其为国际突发公共卫生事件。COVID-19在世界范围内快速蔓延,给人类健康和社会公共卫生安全产生了极大的威胁。但目前尚无针对该病毒的特效药,临床上主要以支持治疗和改善症状的对症治疗为主,抗SARS-CoV-2药物的研发刻不容缓。全球范围内针对新冠病毒已开展了大量的药物筛选实验,但仍未发现具有良好治疗效果的药物。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是一种具有包膜结构的RNA病毒。病毒颗粒脂质膜上的刺突糖蛋白,负责与宿主受体的粘附。脂质膜内的病毒基因组RNA可编码复制酶多聚蛋白,这些多聚蛋白可被类3C蛋白酶(3CLpro,也称主蛋白酶Mpro)和类木瓜蛋白酶(PLpro)切割,产生功能性多肽,如RNA依赖的RNA聚合酶(RdRp)和解旋酶(Hel),参与病毒RNA转录与复制。因此,与SARS-CoV-2侵染宿主细胞相关的刺突糖蛋白、与SARS-CoV-2转录复制相关的主蛋白酶和类木瓜蛋白酶是抗SARS-CoV-2药物研发的重要靶点。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.

海洋生物资源丰富,来源于海洋生物的天然糖类化合物特别是酸性糖类化合物可以与病毒带有正电荷的蛋白结合从而阻止病毒复制或侵染细胞。此外,其具有毒性低、不易引起病毒产生耐药性等优势,在抗病毒药物开发中具有广阔的前景。文献检索发现,海洋生物来源天然硫酸多糖,如褐藻来源的复杂结构岩藻聚糖硫酸酯具有抗多种流感病毒作用(公开号CN 103880975A;专利号:ZL201410132608.x),其主链结构是由α-1,2-甘露糖(Man)和β-1,4-葡糖糖醛酸(GlcA)组成,在甘露糖C3位含有支链α-1,3-连接-硫酸化岩藻糖残基(Jiandong Wu,J Carbohy Chem,34:303-317;Wei Wang,Sci.Rep.2017,7:40760),且最近报道了一种类似结构不同分子量(100kD和12kD)的褐藻糖胶(RPI-27及RPI-128)具有抑制COVID-COV-2冠状病毒Spike蛋白活性,该多糖含有α-1,2-甘露糖(Man)和β-1,4-葡糖糖醛酸(GlcA)主链,在甘露糖C3位含有支链的α-1,2/1,3-连接-硫酸化岩藻糖残基(PaulS.Kwon,Cell Discovery,2020,6:50);发现裙带菜来源的褐藻糖胶(Hayashi,K.,Int.Immunopharmacol.2008,8,109)具有抗HSV活性。关于其他类型结构的岩藻糖硫酸酯在抗SARS-CoV-2方面的研究尚缺乏报道。红藻来源的卡拉胶具有广泛的抗H1N1、HSV,HPV活性(Cui Hao,Rev Med Virol.2019,29,e2043),以及iota-卡拉胶还具有抗猪蓝耳病毒(PRRSV)活性(Chunhe Guo,Antivir Ther,2019,24(4):261-270),但还未发现有抗新冠病毒活性。绿藻来源的硫酸鼠李聚糖具有抗儿童手足口病毒(EV71)活性(Shuyao Wang,Carbohydr Polym,2018,200:43-53)和新城疫病毒(NDV)活性(Aguilar-

Figure BDA0002635906480000021
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-
Figure BDA0002635906480000021
Mar. Drugs, 2015, 13, 697–712), but no anti-new coronavirus activity was found.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种海洋生物来源天然硫酸多糖在作为抗冠状病毒及所致疾病药物中的应用,以特定海洋植物、海洋动物为原料,经提取分离,获得系列海洋来源天然硫酸多糖,筛选其在抑制冠状病毒,特别是新型冠状病毒(SARS-COV-2)的活性,提供用于治疗冠状病毒,尤其是新型冠状病毒肺炎(COVID-19)的有效药物及其组合物。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).

本发明的技术方案是:The technical scheme of the present invention is:

一种海洋生物来源天然硫酸多糖在作为抗冠状病毒及所致疾病药物中的应用,海洋生物来源的天然硫酸多糖、药学上可接受的盐在制备与刺突蛋白(Spike)结合的药物、制备主蛋白酶(Mpro)的抑制剂、或制备类木瓜蛋白酶(PLpro)抑制剂中的应用,所述海洋生物来源天然硫酸多糖具有如下任一结构特点,其分子量范围为3~600kDa,包括: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)岩藻聚糖硫酸酯(又称褐藻糖胶),具备如下结构特征之一:(a)I型结构褐藻糖胶,主链由α-1,3-连接的硫酸化岩藻糖组成;(b)Ⅱ型结构褐藻糖胶,主链由α-1,3和α-1,4交替连接的硫酸化岩藻糖组成;(c)Ⅲ型结构褐藻糖胶,主链由β-1,4-葡萄糖醛酸和α-1,2-甘露糖组成,分支结构是由α-1,3-硫酸化岩藻寡糖组成;(d)其他类型褐藻糖胶,主链由α-1,2以及α-1,3和/或α-1,4-连接方式的硫酸化岩藻糖组成;(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)岩藻糖基化硫酸软骨素,其结构特征如下:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,GalNAc的C4、C6或C4与C6位羟基含有硫酸酯基,且在GlcA的C3位通过α-1,3糖苷键连接硫酸化岩藻寡糖支链;(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)硫酸半乳聚糖,其结构特征如下:由β-1,3-连接和α-1,4-连接的半乳糖(Gal),或由β-1,3-连接与α-1,4-连接-3,6-内醚半乳糖(AnG)组成,且半乳糖的C2、C4或C6位含有不同硫酸酯基;(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)硫酸鼠李聚糖:其结构特征如下:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成;或者由Rha和艾杜糖醛酸通过α-1,4糖苷键交替连接而成;或者由鼠李糖(Rha)和木糖(Xyl)通过α-1,4和β-1,4糖苷键交替连接而成;或者由α-1,2和α-1,3-鼠李糖交替连接而成;且上述结构Rha的C2和/或C3位含有硫酸酯基。(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.

所述的I型结构褐藻糖胶中,还具有其它糖残基支链结构;Ⅱ型结构褐藻糖胶中,还具有其它糖残基支链结构。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.

所述的冠状病毒选自SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV之一种或两种以上。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.

所述的天然硫酸多糖来源于海洋植物或海洋动物。The natural sulfated polysaccharide is derived from marine plants or marine animals.

所述的天然硫酸多糖通过与冠状病毒的刺突蛋白(Spike)结合,作为抑制其侵染细胞的抑制剂,通过抑制冠状病毒主蛋白酶(Mpro)和/或木瓜蛋白酶样蛋白酶(PLpro)活性,抑制病毒的复制和扩增。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.

所述的天然硫酸多糖、药学上可接受的盐在制备冠状病毒抑制剂中的应用。The application of described natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing coronavirus inhibitor.

所述的天然硫酸多糖、药学上可接受的盐在制备预防和/或治疗冠状病毒感染,或预防和/或治疗与冠状病毒感染相关的疾病或症状的药物中的应用。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.

所述的天然硫酸多糖、药学上可接受的盐与刺突蛋白(Spike)结合的试剂盒或试剂、制备主蛋白酶(Mpro)抑制剂的试剂盒或试剂、或制备木瓜蛋白酶样蛋白酶(PLpro)抑制剂的试剂盒或试剂中的应用。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.

所述的天然硫酸多糖、药学上可接受的盐、和另外的一种或两种以上预防和/或治疗冠状病毒感染的活性剂,混合制成预防和/或治疗冠状病毒感染的药物组合物。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 .

本发明的设计思想是:The design idea of the present invention is:

本发明提供的海洋生物来源的天然硫酸多糖不仅可以抑制冠状病毒表面的刺突蛋白(Spike)与宿主细胞结合,从而有效阻断冠状病毒感染宿主细胞,而且还能抑制冠状病毒主蛋白酶(Mpro)和类木瓜蛋白酶(PLpro)活性,进而抑制病毒的复制和扩增过程。因此,本发明提供的海洋天然硫酸多糖具有开发成抗冠状病毒及所致疾病药物的巨大潜力。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.

本发明的优点及有益效果是:The advantages and beneficial effects of the present invention are:

(1)本发明提供的海洋生物来源的天然硫酸多糖可以抑制冠状病毒表面的刺突蛋白(Spike)与宿主细胞结合,其抑制活性水平优于临床药物肝素和低分子肝素。(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)本发明提供的海洋生物来源的天然硫酸多糖具有抑制主蛋白酶(Mpro)和类木瓜蛋白酶(PLpro)的活性,其抑制活性水平优于肝素。(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)整体来讲,本发明提供的海洋生物来源的天然硫酸多糖的抗冠状病毒活性与优于临床药物肝素和低分子肝素,可以用作抗冠状病毒及所致疾病相关药物的制备和应用领域。(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.

附图说明Description of drawings

图1岩藻聚糖硫酸酯、硫酸半乳聚糖、岩藻糖基化硫酸软骨素、硫酸鼠李聚糖和肝素对SARS-CoV-2刺突蛋白抑制活性。图中,横坐标Log Dose代表化合物浓度的对数(μM),纵坐标Relative Luminescence代表相对发光强度(%)。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 (%).

图2硫酸半乳聚糖、岩藻聚糖硫酸酯、岩藻糖基化硫酸软骨素对SARS-CoV-2主蛋白酶的抑制作用曲线。图中,横坐标Time代表时间(s),纵坐标Intensity代表荧光强度,Blank代表空白对照,Iota-carrageenan代表iota-卡拉胶,FCS代表岩藻糖基化硫酸软骨素,Fucoidan代表岩藻聚糖硫酸酯。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.

图3岩藻聚糖硫酸酯抑制SARS-CoV-2主蛋白酶IC50测定曲线。图中,横坐标Log浓度代表浓度的对数(nM),IC50代表半数抑制浓度。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.

具体实施方式Detailed ways

在具体实施过程中,本发明涉及海洋来源天然硫酸多糖、药学上可接受的盐在制备冠状病毒抑制抑制剂中的应用,所述天然硫酸多糖具有如下任一结构: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)岩藻聚糖硫酸酯(又称褐藻糖胶),包括:(a)I型褐藻糖胶,主链由α-1,3-连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(b)Ⅱ型褐藻糖胶,主链由α-1,3和α-1,4交替连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(c)III型褐藻糖胶,主链由β-1,4-葡萄糖醛酸和α-1,2-甘露糖组成,分支结构是由α-1,3-硫酸化岩藻寡糖组成;(d)其他类型褐藻糖胶,主链由α-1,2以及α-1,3和/或α-1,4-连接方式的硫酸化岩藻糖组成。(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.

Figure BDA0002635906480000051
Figure BDA0002635906480000051

(2)岩藻糖基化硫酸软骨素,其结构特征如下:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,且在GlcA的C3位通过α-1,3糖苷键连接硫酸化岩藻寡糖支链。(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.

Figure BDA0002635906480000052
Figure BDA0002635906480000052

(3)硫酸半乳聚糖,其结构特征如下:由β-1,3-连接和α-1,4-连接的半乳糖(Gal),或由β-1,3-连接与α-1,4-连接-3,6-内醚半乳糖(AnG)组成,且半乳糖的C2、C4或C6位含有不同硫酸酯基。(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.

Figure BDA0002635906480000061
Figure BDA0002635906480000061

(4)硫酸鼠李聚糖,其结构特征如下:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成;或者由Rha和艾杜糖醛酸通过α-1,4糖苷键交替连接而成;或者由鼠李糖(Rha)和木糖(Xyl)通过α-1,4和β-1,4糖苷键交替连接而成;或者由α-1,2和α-1,3-鼠李糖交替连接而成;且上述结构Rha的C2和/或C3位含有硫酸酯基。(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.

Figure BDA0002635906480000062
Figure BDA0002635906480000062

本发明还提供了海洋生物来源天然硫酸多糖、药学上可接受的盐在制备与刺突蛋白(Spike)结合的药物、制备主蛋白酶(Mpro)抑制剂、或制备木瓜蛋白酶样蛋白酶(PLpro)抑制剂中的应用,所述海洋生物来源硫酸多糖具有如下任一结构: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)岩藻聚糖硫酸酯(又称褐藻糖胶),包括:(a)I型结构褐藻糖胶,主链由α-1,3-连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(b)Ⅱ型结构褐藻糖胶,主链由α-1,3和α-1,4交替连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(c)III型结构褐藻糖胶,主链由β-1,4-葡萄糖醛酸和α-1,2-甘露糖组成,分支结构是由α-1,3-硫酸化岩藻寡糖组成;(d)其他类型褐藻糖胶,主链由α-1,2以及α-1,3和/或α-1,4-连接方式的硫酸化岩藻糖组成。(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.

Figure BDA0002635906480000071
Figure BDA0002635906480000071

(2)岩藻糖基化硫酸软骨素,其结构特征如下:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,且在GlcA的C3位通过α-1,3糖苷键连接硫酸化岩藻糖支链。(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.

Figure BDA0002635906480000072
Figure BDA0002635906480000072

(3)硫酸半乳聚糖,其结构特征如下:由β-1,3-连接和α-1,4-连接的半乳糖(Gal),或由β-1,3-连接与α-1,4-连接-3,6-内醚半乳糖(AnG)组成,且半乳糖的C2、C4或C6位含有不同硫酸酯基。(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.

Figure BDA0002635906480000073
Figure BDA0002635906480000073

(4)硫酸鼠李聚糖:其结构特征如下:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成;或者由Rha和艾杜糖醛酸(IdoA)通过α-1,4糖苷键交替连接而成;或者由鼠李糖(Rha)和木糖(Xyl)通过α-1,4和β-1,4糖苷键交替连接而成;或者由α-1,2和α-1,3-鼠李糖交替连接而成;且上述结构Rha的C2和/或C3位含有硫酸酯基。(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.

Figure BDA0002635906480000081
Figure BDA0002635906480000081

如上所述的应用,优选的是冠状病毒中的刺突蛋白(Spike)、主蛋白酶(Mpro)、木瓜蛋白酶样蛋白酶(PLpro)之一种或两种以上;更优选的,所述冠状病毒选自COVID-19(SARS-CoV-2)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV之一种或两种以上。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.

本发明还提供了如上所述的海洋生物来源天然硫酸多糖、药学上可接受的盐在制备冠状病毒抑制剂中的应用。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.

本发明还提供了如上所述的一种海洋生物来源天然硫酸多糖、药学上可接受的盐在制备预防和/或治疗冠状病毒感染,或预防和/或治疗与冠状病毒感染相关的疾病或症状的药物中的应用。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.

如上所述的用途,优选的,所述冠状病毒选自COVID-19(SARS-CoV-2)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV。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.

本发明还提供了如上所述的海洋生物来源天然硫酸多糖、药学上可接受的盐在制备与刺突蛋白(Spike)结合的试剂盒或试剂、制备主蛋白酶(Mpro)抑制剂的试剂盒或试剂、或制备木瓜蛋白酶样蛋白酶(PLpro)抑制剂的试剂盒或试剂中的应用。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.

本发明还提供了一种预防和/或治疗冠状病毒感染的药物组合物,其中包含所述的一种海洋生物来源天然硫酸多糖、药学上可接受的盐、和另外的一种或两种以上预防和/或治疗冠状病毒感染的活性剂。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.

优选的,如上所述的海洋生物来源天然硫酸多糖的分子量范围为3~600kDa。Preferably, the molecular weight of the marine organism-derived natural sulfated polysaccharide ranges from 3 to 600 kDa.

本发明通过实验筛选研究表明,系列海洋生物来源天然硫酸多糖对冠状病毒,尤其是新型冠状病毒(SARS-CoV-2)的刺突蛋白(Spike)、主蛋白酶(Mpro或者3CL pro)以及PLpro均有较好的抑制作用。本发明提供了海洋生物来源天然硫酸多糖在抗冠状病毒,尤其是SARS-CoV-2中的应用潜力,证实其具有很好的临床开发应用前景。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.

本发明提供的海洋生物来源天然硫酸多糖,优选的,其结构特征如下:The marine biological source natural sulfated polysaccharide provided by the invention, preferably, its structural characteristics are as follows:

(1)岩藻聚糖硫酸酯(又称褐藻糖胶),包括:(a)I型结构褐藻糖胶,主链由α-1,3-连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(b)Ⅱ型结构褐藻糖胶,主链由α-1,3和α-1,4交替连接的硫酸化岩藻糖组成,还可以具有其它糖残基支链结构;(c)III型结构褐藻糖胶,主链由β-1,4-葡萄糖醛酸和α-1,2-甘露糖组成,分支结构是由α-1,3-硫酸化岩藻寡糖组成;(d)其他类型褐藻糖胶,主链由α-1,2以及α-1,3和/或α-1,4-连接方式的硫酸化岩藻糖组成。(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.

Figure BDA0002635906480000091
Figure BDA0002635906480000091

(2)岩藻糖基化硫酸软骨素,其结构特征如下:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,且在GlcA的C3位通过α-1,3糖苷键连接硫酸化岩藻糖支链。(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.

Figure BDA0002635906480000101
Figure BDA0002635906480000101

(3)硫酸半乳聚糖,其结构特征如下:由β-1,3-连接和α-1,4-连接的半乳糖(Gal),或由β-1,3-连接的半乳糖与α-1,4-连接-3,6-内醚半乳糖(AnG)组成,且半乳糖的C2、C4或C6位含有不同硫酸酯基。(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.

Figure BDA0002635906480000102
Figure BDA0002635906480000102

(4)硫酸鼠李聚糖:其结构特征如下:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成;或者由Rha和艾杜糖醛酸通过α-1,4糖苷键交替连接而成;或者由鼠李糖(Rha)和木糖(Xyl)通过α-1,4和β-1,4糖苷键交替连接而成;或者由α-1,2和α-1,3-鼠李糖交替连接而成;且上述结构Rha的C2和/或C3位含有硫酸酯基。(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.

Figure BDA0002635906480000103
Figure BDA0002635906480000103

本发明的海洋生物来源硫酸多糖均具有抑制冠状病毒,尤其是新型冠状病毒(SARS-CoV-2)的作用。实验结果显示,本发明提供的各种海洋生物来源硫酸多糖均具有显著的抗新型冠状病毒作用,抑制Spike的IC50值在0.01~30μM之间,抑制Mpro的IC50值在1nM~100nM之间,抑制木瓜蛋白酶样蛋白酶(PLpro)的IC50值在0.001~30μM。如岩藻聚糖硫酸酯抑制Mpro的IC50为5.68nM,抑制Spike蛋白的IC50为0.06μM,抑制木瓜蛋白酶样蛋白酶(PLpro)的IC50为7.65nM,且效果均明显好于肝素。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.

作为优选方案,以上所述的应用,所述的冠状病毒包括但不限于新型冠状病毒(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。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).

本发明提供的海洋生物来源天然硫酸多糖在抗新型冠状病毒中的应用,可以将海洋生物来源天然硫酸多糖制备成喷雾剂、注射剂、胶囊剂、软膏剂、乳膏剂、凝胶剂、搽剂、涂剂。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.

优选地将治疗有效量的本发明海洋生物来源天然硫酸多糖、药学上可接受的盐或立体异构体,根据通常给药途径并且根据本领域已知方法以常规药物组合物(该药物组合物包含有效量的活性成分和合适的药用载体)和剂型配制而施用至需要这样治疗的患者。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.

所述“治疗有效量”是指当施用时,其足以防止所针对的疾病的一种或两种以上症状的发展,或在某种程度上缓解所述一种或两种以上症状。根据本发明施用的化合物的具体剂量将由围绕该病例的具体情况确定,所述情况包括所施用的化合物、给药途径、治疗的具体病况以及类似的考虑因素。特别地,“治疗有效量的化合物”是指足以防止或在某种程度上缓解一种或两种以上冠状病毒感染的化合物的量。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.

进一步根据待治疗的冠状病毒感染的类型和严重性以及具体患者对药物治疗的反应,单个剂量以及日剂量不同。因此,将根据在医生的指导下的标准医学原理来确定准确的单个剂量。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.

用于在治疗冠状病毒所致疾病中使用本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐的人体有效日剂量为,1mg至约500mg、约5mg至约1000mg、或约10mg至约2000mg的注射或其他活性剂的口服剂型。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.

本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐可以单独使用或组合使用或与其他治疗剂的联合疗法使用。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.

在本发明的一个实施方案中,海洋生物来源天然硫酸多糖、药学上可接受的盐用于在预防和/或治疗冠状病毒感染中使用,其中所述预防或治疗包括施用作为唯一活性成分使用。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.

在本发明的另一个实施方案中,上述海洋生物来源天然硫酸多糖、药学上可接受的盐用于在预防和/或治疗冠状病毒感染中使用,其中所述预防或治疗包括以与选自其它的治疗剂的联合疗法使用。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.

如对于本领域技术人员将明显的是,包含本发明海洋生物来源天然硫酸多糖、药学上可接受的盐与另外的治疗剂的本发明的组合不仅在这些活性成分以单一组合物使用时是有效的,而且在以两个不同组合物(同时、依次或在一段时间之后分开地施用)使用时也是有效的。此外,本领域技术人员将理解,本发明海洋生物来源天然硫酸多糖、药学上可接受的盐可以开处方为与联合疗法中的其他活性成分一起使用,以预防和/或治疗冠状病毒感染。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.

在一个特别的实施方案中,联合疗法包括向受试者同时、依次或分开地施用本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐和另外的治疗剂。备选地,联合疗法包括向受试者施用在单一组合物中的本发明海洋生物来源天然硫酸多糖、药学上可接受的盐或立体异构体和另外的治疗剂。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.

在本发明的一个实施方案中,本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐或立体异构体可以方便地施用至患者。因此,用于本发明的用途的化合物可以为包含与药用赋形剂或载体组合的有效量的本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐的药物组合物的形式。这一方面也可以表达为,包含与药用赋形剂或载体组合的有效量的本发明的海洋生物来源天然硫酸多糖、药学上可接受的盐的组合物用于在预防和/或治疗冠状病毒感染。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.

在本发明的一个实施方案中,使用的化合物可以以包含常规药用载体的剂量单位制剂通过口腔、注射、皮下、呼吸道、透皮、非肠道、直肠、局部外用、静脉、肌肉或通过其它方式来给予。可以将药物组合物配制成任何药用形式,如:片剂、颗粒剂、注射剂、凝胶剂、丸剂、胶囊剂、栓剂、植入剂、纳米制剂、粉针剂。诸如片剂和胶囊剂的一些剂型可以再分成包含诸如达到期望目的的有效量的适当量活性组分的适当剂量单位剂型。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.

在另一个实施方案中,用于本发明的用途的海洋生物来源天然硫酸多糖、药学上可接受的盐为待施用至要治疗的患者的注射制剂,适用于本发明的注射剂是指药物与适宜的溶剂或分散介质制成供注入人体内的灭菌或无菌溶液、乳状液或悬浮液,以及供使用前配制成溶液或悬浮液的粉末无菌制剂。所述注射剂包括注射液(其中供静脉滴注用的大体积注射液也称静脉输液)、注射用无菌粉末与注射用浓溶液。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.

载体包括赋形剂和稀释剂,并且必须具有足够高的纯度和十分低的毒性以使它们适于被给予待治疗的患者。载体可以是惰性的或其可以本身具有药用益处。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.

载体的种类包括但不限于:稀释剂如填料和疏松剂、粘合剂、润滑剂、抗结块剂、崩解剂、增甜剂、缓冲剂、防腐剂、增溶剂、等张剂、悬浮剂和分散剂、润湿剂或乳化剂、调味剂和芳香剂、增稠剂和媒介物。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.

可选的活性剂可以包括在药物组合物中,其基本上不影响本发明的化合物的活性。Optional active agents can be included in the pharmaceutical compositions that do not substantially affect the activity of the compounds of the present invention.

以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,以下实施例所用试剂和材料均为市购。所用海洋生物来源天然硫酸多糖结构如下: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)岩藻聚糖硫酸酯:主链由α-1,3-连接的硫酸化岩藻糖组成,分子量为600kDa。(1) Fucoidan sulfate: the main chain is composed of α-1,3-linked sulfated fucose with a molecular weight of 600 kDa.

(2)ι-卡拉胶:由β-1,3-连接的半乳糖与α-1,4-连接-3,6-内醚半乳糖重复二糖单元组成,半乳糖残基C4位和内醚半乳糖残基C2位被硫酸根取代,分子量为200kDa。(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)岩藻糖基化硫酸软骨素:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,且在GlcA的C3位通过α-1,3糖苷键连C2和C3位被硫酸根取代的岩藻糖支链,分子量为3kDa和50kDa。(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)硫酸鼠李聚糖:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成且上述结构Rha的C2和C3位含有硫酸酯基;分子量为200kDa。(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.

实施例一海洋生物来源天然硫酸多糖对SARS-CoV-2刺突蛋白抑制作用Example 1 Inhibitory effect of marine biological source natural sulfated polysaccharide on SARS-CoV-2 spike protein

基于SARS-CoV-2-刺突蛋白类病毒检测体系,评价岩藻聚糖硫酸酯(来源于褐藻)、ι-卡拉胶(来源于红藻)、岩藻糖基化硫酸软骨素(来源于海参)、硫酸鼠李聚糖(来源于绿藻)阻断SARS-CoV-2感染细胞的效果。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.

本发明采用的硫酸多糖样品可采用本技术领域常规试剂、方法和设备提取。The sulfated polysaccharide samples used in the present invention can be extracted by conventional reagents, methods and equipment in the technical field.

1)细胞复苏培养:293T/17人胚肾细胞株,按重量百分比计,采用10%胎牛血清、1%双抗和余量的DMEM培养基混合进行培养,细胞复苏后培养两代,备用。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)类病毒制备:将293T/17人胚肾细胞铺到6孔细胞培养板中,当细胞汇合度达到60%左右时,用转染试剂(Lipofiter 3.0)将3μg质粒进行包装转染,然后将6孔细胞培养板在二氧化碳培养箱中(37℃,5%CO2)培养,转染48h后收取类病毒上清溶液,-80℃保存。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)细胞瞬时转染:将293T/17人胚肾细胞铺到6孔细胞培养板中,当细胞汇合度达到70%左右时,使用转染试剂(Lipofiter 3.0)将3μg质粒(ACE2-pcDNA3.1)转染到293T17细胞,将6孔细胞培养板在二氧化碳培养箱培养48h。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)细胞消化:用胰酶将瞬时转染的293T/17人胚肾细胞消化,用完全培养基重悬细胞后,利用细胞计数仪进行计数,铺到96孔细胞培养板,每孔12000个细胞,培养6~8h。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)不同浓度海洋生物来源天然硫酸多糖的制备:待测化合物配成摩尔浓度为1mM的母液,根据在细胞株上检测浓度的不同,在摩尔浓度为0.00015~1.00mM范围内配制成9个不同浓度梯度的稀释液,分别储存于1.5mL的透明EP管中,-20℃保存。将制备好的9个浓度梯度待测化合物,分别用完全培养基稀释10倍,同时采用同等体积的二甲基亚砜(DMSO)溶剂作为对照。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)铺板:取出培养箱中贴壁6~8h的96孔白色细胞培养板,每孔取出60μL,然后将10μL待测物加入上述含有40μL细胞体积的培养板中,每个浓度梯度2个复孔,在培养箱(37℃、5%CO2)中培养1小时后,每孔再加入50μL收取的类病毒上清溶液,其中待测化合物最终检测浓度为摩尔浓度范围为0.00152~10.00μM的9个浓度梯度;将培养板放置于培养箱中孵育24小时后,更换100μL培养基继续培养48h后检测。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)读板:将Renilla luciferase检测试剂放置至室温,取出细胞培养板放置10分钟,使其平衡至室温,每孔加入15μL的检测试剂,将培养板在轨道摇床上振摇2分钟,诱导细胞裂解,培养板室温放置10分钟,在MD(Molecular Devices,美谷分子)SpectraMaxParadigm读板器上测定发光信号。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)数据分析:利用SpectraMax Paradigm读数,得出对应的每孔荧光值RLU。数据采用下列公式来处理:RLU(%)=(RLUDrug)/(RLUDMSO)*100%。在EXCEL中计算不同浓度化合物对应的细胞活率,然后用GraphPad 7.0Prism软件作曲线图,计算各化合物的IC50值。结果见下表,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,

化合物compound IC<sub>50</sub>(μM)IC<sub>50</sub>(μM) 肝素heparin 10.010.0 岩藻聚糖硫酸酯Fucoidan Sulfate 0.060.06 iota-卡拉胶iota-carrageenan 0.270.27 岩藻糖基化硫酸软骨素Fucosylated Chondroitin Sulfate 0.250.25 硫酸鼠李聚糖rhamnose sulfate 0.440.44

如图1所示,系列海洋生物来源天然硫酸多糖均对阻断新型冠状病毒(SARS-CoV-2)感染细胞具有明显抑制效果,且化合物岩藻聚糖硫酸酯和卡拉胶、岩藻糖基化硫酸软骨素、硫酸鼠李聚糖等海洋生物来源天然硫酸多糖阻断病毒感染细胞效果强于肝素(Heparin)。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.

实施例二海洋来源天然硫酸多糖对SARS-CoV-2主蛋白酶的抑制作用Example 2 Inhibitory effect of marine source natural sulfated polysaccharide on SARS-CoV-2 main protease

SARS-CoV-2主蛋白酶可以水解荧光多肽底物从而使底物产生荧光。主蛋白酶活性被海洋来源天然硫酸多糖抑制,荧光强度较空白对照降低。其中,主蛋白酶和荧光多肽底物可以采用文献W.Dai et al.,Science 10.1126/science.abb4489(2020)报道方法获得。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)TE缓冲溶液配制(1) Preparation of TE buffer solution

将100μL摩尔浓度为0.5M乙二胺四乙酸二钠(EDTA-2Na)水溶液加入2.5mL摩尔浓度为1M的Tris-HCl缓冲液中,定容至50mL,调节pH至7.3,0.22μm滤膜过滤后,-4℃保存。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)海洋生物来源硫酸多糖对主蛋白酶抑制活性的筛选(2) Screening of marine organism-derived sulfated polysaccharides for the inhibitory activity of major proteases

在96孔细胞培养板中加入87μL TE缓冲溶液,再分别加入1μL摩尔浓度为19.7μM的主蛋白酶,混匀后,将H2O(2μL)、海藻来源岩藻聚糖硫酸酯水溶液(2μL,摩尔浓度15μM;分子量260.1kDa)、麒麟菜来源iota-卡拉胶(2μL,摩尔浓度15μM;分子量200kDa)、鲨鱼骨来源硫酸角质素(2μL,摩尔浓度15μM,分子量45.98kDa)、海参来源岩藻糖基化硫酸软骨素(2μL,摩尔浓度15μM,分子量42kDa)依次分别加入各孔,摇匀,室温反应30min后,每孔均加入10μL摩尔浓度为20μM的底物,迅速采用酶标仪检测荧光强度。本发明采用的硫酸多糖样品可采用本技术领域常规试剂、方法和设备提取。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)酶标仪检测条件(3) Detection conditions of microplate reader

在激发波长320nm、发射波长405nm、检测温度为27℃条件下连续检测10min内各孔荧光强度,获得时间-荧光强度动态曲线。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)数据处理(4) Data processing

基于获得的时间-荧光强度动态曲线进行线性拟合,得到各海洋来源天然硫酸多糖对应曲线的斜率k和空白对照对应曲线斜率k0 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.

天然硫酸多糖(摩尔浓度300nM)对主蛋白酶的抑制率=(1-k/k0)×100%Inhibition rate of natural sulfated polysaccharide (molar concentration 300nM) to main protease=(1-k/k 0 )×100%

化合物compound 抑制率/%Inhibition rate/% 肝素heparin 72.272.2 岩藻聚糖硫酸酯Fucoidan Sulfate 92.392.3 iota-卡拉胶iota-carrageenan 62.862.8 岩藻糖基化硫酸软骨素Fucosylated Chondroitin Sulfate 65.965.9 硫酸鼠李聚糖rhamnose sulfate 75.075.0

如图2所示,褐藻来源岩藻聚糖硫酸酯、红藻来源的卡拉胶、海参来源岩藻糖基化硫酸软骨素对SARS-CoV-2主蛋白酶活性抑制率均超高50%。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.

实施例三不同浓度的岩藻聚糖硫酸酯对SARS-CoV-2主蛋白酶的抑制作用Example 3 Inhibitory effect of different concentrations of fucoidan sulfate on SARS-CoV-2 main protease

(1)岩藻聚糖硫酸酯对主蛋白酶活性抑制实验(1) Inhibition experiment of fucoidan sulfate on main protease activity

在96孔细胞培养板中加入87μL TE缓冲溶液,再分别加入1μL摩尔浓度为19.7μM的主蛋白酶,混匀后,分别将2μLH2O和9个浓度梯度的摩尔浓度范围为0.5nM~45μM的海藻来源的岩藻聚糖硫酸酯水溶液依次分别加入各孔,摇匀,室温反应30min后,每孔均加入10μL摩尔浓度为20μM的底物,迅速采用酶标仪检测荧光强度。本发明采用的硫酸多糖样品可采用本技术领域常规试剂、方法和设备提取。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)酶标仪检测条件(2) Detection conditions of microplate reader

在激发波长320nm、发射波长405nm、检测温度为27℃条件下连续检测10min内各孔荧光强度,获得时间-荧光强度动态曲线。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)数据处理(3) Data processing

基于获得的时间-荧光强度动态曲线进行线性拟合,得到各不同浓度下岩藻聚糖硫酸酯对应曲线的斜率k和空白对照对应曲线斜率k0 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.

不同浓度岩藻聚糖硫酸酯对主蛋白酶抑制率=(1-k/k0)×100%Inhibition rate of main protease by different concentrations of fucoidan sulfate=(1-k/k 0 )×100%

(4)IC50计算(4) IC 50 calculation

采用软件origin 8.1,以浓度的对数对抑制率作图,并采用非线性拟合,获得IC50值。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)结果(5) Results

如图3所示,海洋生物来源的岩藻聚糖硫酸酯对主蛋白酶活性具有良好的抑制作用,其相应的IC50值=5.68±0.43nM(nmol/L)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)

实验结果表明,本发明提供的海洋生物来源的天然硫酸多糖具有明显的抑制冠状病毒增殖作用,不仅可以抑制冠状病毒表面的刺突蛋白(Spike)与宿主细胞结合,而且能抑制主蛋白酶(Mpro)和类木瓜蛋白酶(PLpro)活性,从而有效阻断冠状病毒感染宿主细胞。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)

1.一种海洋生物来源天然硫酸多糖在作为抗冠状病毒及所致疾病药物中的应用,其特征在于,海洋生物来源的天然硫酸多糖、药学上可接受的盐在制备与刺突蛋白(Spike)结合的药物、制备主蛋白酶(Mpro)的抑制剂、或制备类木瓜蛋白酶(PLpro)抑制剂中的应用,所述海洋生物来源天然硫酸多糖具有如下任一结构特点,其分子量范围为3~600kDa,包括: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)岩藻聚糖硫酸酯(又称褐藻糖胶),具备如下结构特征之一:(a)I型结构褐藻糖胶,主链由α-1,3-连接的硫酸化岩藻糖组成;(b)Ⅱ型结构褐藻糖胶,主链由α-1,3和α-1,4交替连接的硫酸化岩藻糖组成;(c)Ⅲ型结构褐藻糖胶,主链由β-1,4-葡萄糖醛酸和α-1,2-甘露糖组成,分支结构是由α-1,3-硫酸化岩藻寡糖组成;(d)其他类型褐藻糖胶,主链由α-1,2以及α-1,3和/或α-1,4-连接方式的硫酸化岩藻糖组成;(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)岩藻糖基化硫酸软骨素,其结构特征如下:主链由葡萄糖醛酸(GlcA)和N-乙酰半乳糖胺(GalNAc)通过β-1,3和β-1,4糖苷键交替连接而成的硫酸软骨素二糖重复单元,且在GlcA的C3位通过α-1,3糖苷键连接硫酸化岩藻寡糖支链;(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)硫酸半乳聚糖,其结构特征如下:由β-1,3-连接和α-1,4-连接的半乳糖(Gal),或由β-1,3-连接与α-1,4-连接-3,6-内醚半乳糖(AnG)组成,且半乳糖的C2、C4或C6位含有不同硫酸酯基;(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)硫酸鼠李聚糖:其结构特征如下:由鼠李糖(Rha)和葡萄糖醛酸通过α-1,4和β-1,4糖苷键交替连接而成;或者由Rha和艾杜糖醛酸通过α-1,4糖苷键交替连接而成;或者由鼠李糖(Rha)和木糖(Xyl)通过α-1,4和β-1,4糖苷键交替连接而成;或者由α-1,2和α-1,3-鼠李糖交替连接而成;且上述结构Rha的C2和/或C3位含有硫酸酯基。(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.如权利要求1所述的应用,其特征在于,I型结构褐藻糖胶中,还具有其它糖残基支链结构;Ⅱ型结构褐藻糖胶中,还具有其它糖残基支链结构。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.如权利要求1所述的应用,其特征在于,冠状病毒选自SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV之一种或两种以上。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.如权利要求1所述的应用,其特征在于,天然硫酸多糖来源于海洋植物或海洋动物。4. application as claimed in claim 1 is characterized in that, natural sulfated polysaccharide is derived from marine plants or marine animals. 5.如权利要求1所述的应用,其特征在于,天然硫酸多糖通过与冠状病毒的刺突蛋白(Spike)结合,作为抑制其侵染细胞的抑制剂,通过抑制冠状病毒主蛋白酶(Mpro)和/或木瓜蛋白酶样蛋白酶(PLpro)活性,抑制病毒的复制和扩增。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.如权利要求1所述的应用,其特征在于,天然硫酸多糖、药学上可接受的盐在制备冠状病毒抑制剂中的应用。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.如权利要求1所述的应用,其特征在于,天然硫酸多糖、药学上可接受的盐在制备预防和/或治疗冠状病毒感染,或预防和/或治疗与冠状病毒感染相关的疾病或症状的药物中的应用。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.如权利要求1所述的应用,其特征在于,天然硫酸多糖、药学上可接受的盐与刺突蛋白(Spike)结合的试剂盒或试剂、制备主蛋白酶(Mpro)抑制剂的试剂盒或试剂、或制备木瓜蛋白酶样蛋白酶(PLpro)抑制剂的试剂盒或试剂中的应用。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.如权利要求1所述的应用,其特征在于,天然硫酸多糖、药学上可接受的盐、和另外的一种或两种以上预防和/或治疗冠状病毒感染的活性剂,混合制成预防和/或治疗冠状病毒感染的药物组合物。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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CN113181207A (en) * 2021-02-06 2021-07-30 中国海洋大学 Use of oligomannuronic acid phosphate for the preparation of a medicament for the prevention and treatment of novel coronavirus infections
CN114097832A (en) * 2021-11-30 2022-03-01 常德集智生物科技有限公司 Long-acting inactivated virus spray and preparation method thereof
CN114316081A (en) * 2021-12-07 2022-04-12 大连工业大学 A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application
CN114732827A (en) * 2022-06-10 2022-07-12 中国海洋大学 Application of sulfated polysaccharides from different marine organisms and pharmaceutical composition thereof
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CN115835870A (en) * 2020-06-30 2023-03-21 葛兰素史克消费者健康有限责任公司 Antiviral pharmaceutical composition
WO2024078312A1 (en) * 2022-10-14 2024-04-18 华东理工大学 Anti-sars-cov-2 use of sulfonated polysaccharide
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CN115835870A (en) * 2020-06-30 2023-03-21 葛兰素史克消费者健康有限责任公司 Antiviral pharmaceutical composition
CN112220799A (en) * 2020-11-02 2021-01-15 江苏泰德医药有限公司 A product and use for inhibiting virus
CN113181207A (en) * 2021-02-06 2021-07-30 中国海洋大学 Use of oligomannuronic acid phosphate for the preparation of a medicament for the prevention and treatment of novel coronavirus infections
WO2022219071A1 (en) * 2021-04-13 2022-10-20 Philip Morris Products S.A. Composition comprising a eukaryotic microalgae or an extract thereof for use in the treatment of a viral disease
WO2022217763A1 (en) * 2021-04-14 2022-10-20 大连工业大学 Sulfated abalone polysaccharide and application thereof in inhibiting sars-cov-2
CN114097832A (en) * 2021-11-30 2022-03-01 常德集智生物科技有限公司 Long-acting inactivated virus spray and preparation method thereof
CN114316081A (en) * 2021-12-07 2022-04-12 大连工业大学 A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application
CN114732827A (en) * 2022-06-10 2022-07-12 中国海洋大学 Application of sulfated polysaccharides from different marine organisms and pharmaceutical composition thereof
CN114732827B (en) * 2022-06-10 2022-09-27 中国海洋大学 Application of sulfated polysaccharide from different marine organisms and its pharmaceutical composition
WO2024078312A1 (en) * 2022-10-14 2024-04-18 华东理工大学 Anti-sars-cov-2 use of sulfonated polysaccharide
CN118085116A (en) * 2024-01-16 2024-05-28 中欣安德森(广东)生物技术有限公司 Use of kelp polysaccharide in antiviral and sterilization

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