Glycosaminoglycans and / or series of derivatives thereof, and methods of preparation and use thereof

Abstract

This invention relates to the fields of natural product chemistry, glycochemistry, and biomedicine, specifically providing a glycosaminoglycan and / or its series of derivatives, its preparation method, and applications. The glycosaminoglycan is extracted from *Aster sclerotium*. The preparation method includes: extraction using papain combined with alkaline hydrolysis, followed by separation and purification by ion exchange column chromatography and size exclusion column chromatography to obtain ALHX-2M. Further deacetylation, deamination, and gel column chromatography separation and purification yield the ALHX-2M series of derivatives. Its application in the prevention or treatment of thrombotic diseases is also disclosed. The glycosaminoglycan and its derivatives of this invention target the endogenous coagulation factor X enzyme complex (FXase), exhibiting superior anticoagulant and antithrombotic activities compared to low molecular weight heparin. Furthermore, at equivalent antithrombotic doses, the risk of bleeding is significantly reduced, making it suitable as a drug or functional food for the prevention and treatment of thrombotic cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry, glycochemistry and biomedicine, specifically to a glycosaminoglycan ALHX-2M and / or its series of derivatives, its preparation process, and its application in the preparation of drugs or functional foods for venous thrombosis, disseminated intravascular coagulation, cardiovascular and cerebrovascular embolism or improving microcirculation. Background Technology

[0002] Heparin (HP) and low molecular weight heparin (LMWH) are currently the first-line drugs for the clinical prevention and treatment of venous thromboembolism (VTE), but their shortcomings are becoming increasingly apparent: (1) High bleeding risk: They bind to antithrombin III (AT-III) and act on thrombin (IIa) and coagulation factor Xa (FXa), which are related to hemostasis; (2) Limited drug source: They are mainly extracted from the small intestinal mucosa of pigs, and potential contamination by viruses such as African swine fever and foot-and-mouth disease makes the supply chain fragile; (3) Drug resistance / allergic reactions: Long-term use can induce heparin-induced thrombocytopenia (HIT). Therefore, the development of new anticoagulants that are "highly effective, low-bleeding, safe in origin, and structurally controllable" has always been an urgent global need.

[0003] In the past decade, marine invertebrates have become a research hotspot due to their absence of terrestrial pathogens and novel polysaccharide structures. *Anthenoides laevigatus*, widely distributed in the South China Sea, is a common predator in economically important shellfish farming areas, and its outbreaks can lead to benthic ecological imbalance and economic losses in fisheries. Currently, apart from a small amount used for feed or compost, over 99% of this starfish is discarded for low-value purposes, resulting in a huge waste of biological resources.

[0004] This invention discovers a novel glycosaminoglycan containing a 2,3-O-disulfated uronic acid motif from the starfish species. No natural sources of this type of compound have been reported to date, and there is no research on its anticoagulant activity through its FXase targeting mechanism. Summary of the Invention

[0005] The purpose of this invention is to provide a glycosaminoglycan ALHX-2M and / or its series derivatives, and its preparation process. This novel glycosaminoglycan and / or its derivatives further prepared from the glycosaminoglycan exhibit potent anticoagulant and antithrombotic activities both in vivo and in vitro, and can effectively inhibit endogenous coagulation factor X enzyme (FXase). It can be used to prepare drugs or functional foods for the treatment of thrombotic cardiovascular diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In one aspect, the present invention provides a glycosaminoglycan and / or a series of derivatives thereof, said glycosaminoglycan being a mixture of homologous polysaccharides having formula (I).

[0008] (I)

[0009] In formula (I), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; and R are independently -H or -SO3. － ;

[0010] and / or

[0011] The series of glycosaminoglycan derivatives are partially deacetylated derivatives of glycosaminoglycans, and are mixtures of homologous polysaccharides of formula (II).

[0012] (II)

[0013] In formula (II), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; R are independently -H or -SO3. － R' is H or -COCH3;

[0014] and / or

[0015] The series of glycosaminoglycan derivatives are deacylated and deamination-depolymerized derivatives of the glycosaminoglycans, and are mixtures of homologous polysaccharides having the structure of formula (III).

[0016] (III)

[0017] In formula (III), m+n are integers with a mean of 2 to 12; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; R are independently -H or -SO3. － R1 is -H or one of the three groups shown in formula (IV):

[0018] (IV)

[0019] In formula (IV), anTal-ol is 2,5-dehydrated tarottitol; anMan-ol is 2,5-dehydrated mannitol.

[0020] Furthermore, the glycosaminoglycan or its series derivatives have a weight-average molecular weight of 1-50 kDa and a polydispersity index between 1.0 and 1.9; the sulfate content is 20-60%.

[0021] Furthermore, the monosaccharide composition includes L-α-iduronic acid, D-β-glucuronic acid, and D-β-N-acetaminogalactosamine; the content ratio of the monosaccharide composition of the glycosaminoglycan is as follows, based on the molar ratio: D-β-glucuronic acid: L-α-iduronic acid: D-β-N-acetaminogalactosamine = 1:(1.97±1.5):(3.36±2.0).

[0022] The second aspect of the present invention provides a method for preparing the above-mentioned glycosaminoglycans and / or their series derivatives. The method for preparing the glycosaminoglycans includes the following steps: taking the dried, smooth asterisk body wall, crushing it, adding solvent and protease for enzymatic extraction, then heating it to 40-95°C, adding an inorganic base for alkaline hydrolysis, cooling it after alkaline hydrolysis, separating the liquid part, removing the protein from the liquid part, separating the liquid part again, adjusting the pH of the liquid part to neutral, and obtaining the glycosaminoglycans by alcohol precipitation, washing, and purification.

[0023] Further, the purification includes the following steps: after washing, crude starfish polysaccharide is obtained; the crude starfish polysaccharide is dissolved in pure water, filtered through a microporous membrane, and loaded onto a strong anion exchange column; eluted sequentially with pure water, 0.5M NaCl solution, 1.0M NaCl solution, and 2.0M NaCl solution at a flow rate of 0.5-3 mL / min; the elution fraction eluted with 2.0M NaCl solution is combined; dialyzed with deionized water for 40-56 h; concentrated; loaded onto a gel permeation column; isocratically eluted with 0.15M NaCl solution at a flow rate of 0.1-0.5 mL / min; a symmetrical single peak is collected; dialyzed and dried to obtain the glycosaminoglycan.

[0024] In short, the preparation method of glycosaminoglycans involves using smooth starfish as raw material, extracting it with papain combined with alkaline hydrolysis, and then separating and purifying it by ion exchange column chromatography and size exclusion column chromatography to obtain high-purity glycosaminoglycans, named ALHX-2M.

[0025] In the above-mentioned method for preparing glycosaminoglycans, the pulverized smooth aster body wall is further extracted using water as a solvent, and the weight of water is preferably 5-20 times the weight of the pulverized and dried smooth aster body wall.

[0026] Furthermore, the amount of papain used is 0.3-5% (by weight) of the total weight of the crushed smooth starfish body wall and the solvent.

[0027] Furthermore, the enzymatic extraction time is 6-48 hours.

[0028] Furthermore, the optimal temperature for enzymatic hydrolysis is 30-60℃.

[0029] Furthermore, alkaline hydrolysis methods include: adding an inorganic base to adjust the OH- concentration in the reaction system. - The final concentration is 0.2-1 mol / L, and the reaction is stirred for 1-5 hours. The preferred inorganic base is sodium hydroxide or potassium hydroxide.

[0030] Furthermore, centrifugation was used to separate the liquid portion after alkaline hydrolysis.

[0031] Furthermore, the method for removing protein includes: taking the liquid portion and adjusting the pH to 2–3 with 3–8 mol / L HCl, and letting it stand at 2–6℃ for 3–9 hours to remove protein.

[0032] Furthermore, the method of alcohol precipitation includes: adding 2-5 times the volume of 80-95% ethanol pre-cooled to 2-6℃, and letting it stand at 2-6℃ for 4-24 hours.

[0033] Furthermore, the washing method after alcohol precipitation includes: collecting the precipitate after alcohol precipitation and washing it 2-5 times with 80-95% ethanol.

[0034] Furthermore, the drying process after washing is preferably carried out by vacuum freeze drying.

[0035] Furthermore, the protease may be selected from papain, trypsin, or alkaline protease; the inorganic base is preferably sodium hydroxide.

[0036] Furthermore, the packing materials used in strong anion exchange columns include, but are not limited to, diethylaminoethyl (DEAE) packing materials and FPA98Cl ion exchange resin; the packing materials used in gel permeation columns can be selected from gel media such as Sepharose CL-6B or Sephadex G-100.

[0037] Furthermore, the method for preparing the partially deacetylated derivative of the glycosaminoglycan includes the following steps:

[0038] Anhydrous hydrazine and / or hydrated hydrazine are treated at high temperature under nitrogen or inert gas protection to partially deacetylate the aminohexose contained therein, thereby obtaining a partially deacetylated derivative of the glycosaminoglycan.

[0039] In the preparation method of the above-mentioned partially deacetylated derivatives of glycosaminoglycans, the high-temperature treatment temperature is 70-95℃ and the reaction time is 6-48h.

[0040] Furthermore, the weight ratio of the glycosaminoglycan to anhydrous hydrazine and / or hydrated hydrazine is 2:0.2-2.

[0041] Further, after the deacetylation reaction is completed, the reaction solution is repeatedly precipitated with 75-90% ethanol until the nitrite test paper no longer shows color. The solution is then desalted using a G-25 gel column and dried to obtain a partially deacetylated derivative of high-purity glycosaminoglycans.

[0042] Furthermore, the preparation method of the deacylated and deamination-depolymerized derivative of the glycosaminoglycan includes the following steps:

[0043] The partial deacetylated derivatives of the glycosaminoglycan are treated with nitrous acid to induce a deamination and depolymerization reaction, thereby obtaining low molecular weight deacylated and deamination-depolymerized derivatives of the glycosaminoglycan or the glycosaminoglycan series of derivatives.

[0044] The low molecular weight deacylated and deamination depolymerized derivatives are further separated using size exclusion gel column and / or ion exchange chromatography column to obtain deacylated and deamination depolymerized derivatives of the glycosaminoglycan with the structure of formula (III), where m+n is an integer from 2 to 12.

[0045] The packing material used in the size exclusion gel column is Bio-gel P6 / P10 or Sephadex G-50.

[0046] In the preparation method of the above-mentioned deacylated and deamination depolymerization derivatives of glycosaminoglycans, the concentration of nitrous acid is further 2-6 mol / L.

[0047] Furthermore, the reaction solution after the deamination and depolymerization reaction is repeatedly precipitated with 75-90% ethanol until the nitrite test paper no longer shows color. The solution is then desalted using a G-25 gel column, dried, and the deacylated and deamination-depolymerized derivative of glycosaminoglycan is obtained.

[0048] Currently, ALHX-2M conforming to the definition of this invention has only been identified in the smooth-flowered starfish (Anthenoides laevigatus). However, starfish are an abundant marine resource, with approximately 1600 known species worldwide. Although the inventors have only isolated and identified ALHX-2M from a limited number of starfish species, it is reasonable to infer that ALHX-2M conforming to the structural characteristics described in this invention may be widely present in other starfish species. Therefore, those skilled in the art should understand that any starfish-derived ALHX-2M conforming to the structural composition described in this invention, regardless of its species origin, can be used to prepare the ALHX-2M and its pharmaceutically acceptable salts described in this invention.

[0049] The third aspect of this invention provides the use of the above-described glycosaminoglycans and / or their series derivatives in the preparation of medicaments or functional foods for the prevention and treatment of thrombotic cardiovascular diseases or for improving microcirculation.

[0050] Furthermore, thrombotic cardiovascular diseases include, but are not limited to: thrombotic cardiovascular and cerebrovascular diseases, venous thrombosis, pulmonary venous thrombosis, peripheral venous thrombosis, peripheral arterial thrombosis, disseminated intravascular coagulation, and cardiovascular and cerebrovascular embolism.

[0051] A fourth aspect of the present invention provides a pharmaceutical composition comprising the glycosaminoglycans of any one of claims 1-3 and / or their derivatives, and a pharmaceutically acceptable carrier.

[0052] Furthermore, the pharmaceutical compositions described above can be prepared into pharmaceutically acceptable dosage forms.

[0053] Preferably, the dosage form is an injection, including lyophilized powder for injection or aqueous solution for injection.

[0054] Thrombotic cardiovascular disease is currently the leading cause of death and disability in humans, imposing a heavy burden on society and the economy. Existing clinical antithrombotic drugs mainly fall into three categories: thrombolytics, anticoagulants, and antiplatelet drugs, but they generally have serious side effects such as high bleeding risk. The glycosaminoglycans and / or their derivatives disclosed in this invention exhibit significant anticoagulant activity, effectively inhibiting intrinsic coagulation factor Xase (FXase). Since this target has a low correlation with bleeding tendency, using the glycosaminoglycans and / or their derivatives as FXase inhibitors is expected to become a novel antithrombotic drug candidate with low toxicity and side effects, showing good clinical application prospects and providing a broader approach for developing new antithrombotic drugs with higher safety and better efficacy.

[0055] In summary, the pharmaceutical composition provided by the present invention comprises an effective dose of the above-described glycosaminoglycans and / or their series derivatives, and can be used to treat thrombotic cardiovascular diseases, including but not limited to: thrombotic cardiovascular and cerebrovascular diseases, venous thrombosis, pulmonary venous thrombosis, peripheral venous thrombosis, peripheral arterial thrombosis, disseminated intravascular coagulation, and cardiovascular and cerebrovascular embolism.

[0056] This invention reveals for the first time the unique value of glycosaminoglycans extracted from starfish, and / or derivatives further prepared from these glycosaminoglycans, in the field of anticoagulation, and brings the following significant technical advantages:

[0057] 1. Novel structure: The obtained glycosaminoglycans and their series of derivatives are composed of iduronic acid, glucuronic acid, N-acetylgalactosamine, glucosamine, etc. The O-2 and O-3 of the uronic acid are both modified by sulfation, which has both a high degree of sulfation and a clear sequence. It is a new glycosaminoglycan entity that has not been reported to date, and lays a material basis for subsequent structure-activity relationship studies.

[0058] 2. Outstanding anticoagulant efficacy and excellent safety: In vitro experiments have confirmed that the glycosaminoglycans and their series derivatives of this invention can prolong APTT in a dose-dependent manner and selectively inhibit endogenous FXase. In animal models, their antithrombotic activity is superior to that of enoxaparin sodium, a first-line clinical drug, while significantly reducing bleeding side effects. They have the dual advantages of "high efficiency and low toxicity", providing an ideal candidate for the development of new-generation anticoagulant drugs and functional food ingredients. Attached Figure Description

[0059] Figure 1 The high-performance liquid chromatogram of glycosaminoglycan ALHX-2M having formula (I) is shown in the embodiment of the present invention.

[0060] Figure 2 The chromatogram for monosaccharide composition analysis of glycosaminoglycan ALHX-2M having formula (I) in an embodiment of the present invention is shown.

[0061] Figure 3 The infrared spectrum of ALHX-2M, a glycosaminoglycan of formula (I), is shown in the embodiment of the present invention.

[0062] Figure 4 The glycosaminoglycan ALHX-2M having formula (I) in the embodiments of the present invention 1 H NMR spectrum and its assignment.

[0063] Figure 5 The glycosaminoglycan ALHX-2M having formula (I) in the embodiments of the present invention 13 C NMR spectrum and its assignment.

[0064] Figure 6 The glycosaminoglycan ALHX-2M having formula (I) in the embodiments of the present invention 1 H- 13 C HSQC spectrum and its classification.

[0065] Figure 7 The glycosaminoglycan ALHX-2M having formula (I) in the embodiments of the present invention 1 H- 1 H ROESY spectrum and its classification.

[0066] Figure 8 The glycosaminoglycan ALHX-2M having formula (I) in the embodiments of the present invention 1H- 13 C HMBC spectrum and its classification.

[0067] Figure 9 HPGPC chromatograms of DE-1, DE-2, and DE-3, which are deacetylated products at different degrees.

[0068] Figure 10 The deacetylated products DE-1, DE-2, and DE-3 at different degrees 1 H NMR spectrum.

[0069] Figure 11 The HPGPC chromatogram of the deacylated and deamination depolymerization product dALHX-2M-1 is shown.

[0070] Figure 12 HPGPC chromatogram of the oligosaccharide fraction isolated from the deacylated and deamination depolymerization product dALHX-2M-1.

[0071] Figure 13 OF1, a low molecular weight oligosaccharide component isolated from the deacylated and deamination depolymerization product dALHX-2M-1. 1 H- 13 CHSQC-TOCSY spectrum and its classification.

[0072] Figure 14 OF1, a low molecular weight oligosaccharide component isolated from the deacylated and deamination depolymerization product dALHX-2M-1. 1 H- 13 CHMBC spectrum and its classification.

[0073] Figure 15 OF2, a low molecular weight oligosaccharide component isolated from the deacylated and deamination depolymerization product dALHX-2M-1. 1 H- 1 HTOCSY spectrum and its attribution.

[0074] Figure 16 OF2, a low molecular weight oligosaccharide component isolated from the deacylated and deamination depolymerization product dALHX-2M-1. 1 H- 13 CHSQC spectrum and its classification.

[0075] Figure 17 OF2, a low molecular weight oligosaccharide component isolated from the deacylated and deamination depolymerization product dALHX-2M-1. 1 H- 13 CHMBC spectrum and its classification.

[0076] Figure 18Figure 1 shows the in vivo antithrombotic activity of ALHX-2M; (A) Comparison of thrombus weight; (B) Comparison of the proportion of black tail length to tail length in each treatment of the intraperitoneal injection group.

[0077] Figure 19 The following are the bleeding tendency diagrams for ALHX-2M: (A) Comparison of blood loss among treatment groups during subcutaneous injection; (B) Comparison of bleeding time among treatment groups during subcutaneous injection; (C) Comparison of blood loss among treatment groups during intraperitoneal injection; (D) Comparison of bleeding time among treatment groups during intraperitoneal injection. Detailed Implementation

[0078] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. The specific embodiments and drawings described herein are merely for in-depth analysis of the present invention and are not intended to limit the present invention.

[0079] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0080] Example 1: Preparation, purification, and physicochemical properties of glycosaminoglycans (ALHX-2M) having formula (I).

[0081] 1.1 Preparation method:

[0082] Extraction: 500g of dried, smooth body wall tissue from *Anthenoides laevigatus* was pulverized and passed through an 80-mesh sieve. The tissue was then placed in a 10L double-walled glass reactor, and 5L of pure water was added (material-to-liquid ratio 1:10, w / v). The mixture was stirred and heated to 55℃. 55g of papain (final concentration 1%, w / v) was added, and the mixture was kept at 55℃ for 24 hours to obtain the enzymatic hydrolysate. The hydrolysate was then heated to 60℃, and 6M NaOH was slowly added dropwise until the final concentration in the hydrolysate reached 0.5M. Stirring continued for 2 hours to complete the alkaline hydrolysis, yielding the reaction solution. The reaction solution was cooled to room temperature in an ice-water bath, centrifuged at 4℃ and 4000rpm for 20 minutes to remove residue, and the supernatant was adjusted to pH 2–3 with 6M HCl. The solution was allowed to stand at 4℃ for 4 hours to remove protein, and then centrifuged again at 4℃ and 8000rpm for 20 minutes. The supernatant was adjusted to pH 7.0 with 6M NaOH, and then 3 times the volume of pre-cooled 95% ethanol was slowly added. The mixture was allowed to stand overnight at 4°C. The precipitate was centrifuged, collected, and washed three times with 80% ethanol. It was then freeze-dried under vacuum to obtain 3.45 g of crude starfish polysaccharide, with a yield of 0.69% (based on the dry weight of the raw material).

[0083] Separation and purification: 5 g of crude polysaccharide was dissolved in 60 mL of pure water, filtered through a 0.45 µm microporous membrane, and then loaded onto an Amberlite™ FPA98Cl strong anion exchange column (Ø 45 mm × 500 mm). Elution was performed sequentially with pure water, 0.5 M, 1.0 M, and 2.0 M NaCl solutions at a flow rate of 2 mL / min, collecting 10 mL per tube. The phenol-sulfuric acid method was monitored in real time. The 2.0 M NaCl eluent was combined, dialyzed against deionized water for 48 h (MWCO 3.5 kDa), concentrated, and then loaded onto a Sepharose column. TM A CL-6B gel permeation column (Ø1.5cm×170cm) was used for isocratic elution with 0.15M NaCl solution at a flow rate of 0.3mL / min. A symmetrical single peak was collected, dialyzed, and freeze-dried to obtain a white flocculent pure product, named ALHX-2M, with a weight of 218mg and a recovery rate of 4.4% (based on crude polysaccharide).

[0084] 1.2 Physicochemical Properties and Structural Analysis:

[0085] 1.2.1 Molecular weight distribution

[0086] HPLC system: Shimadzu LC-2030C 3D; Column: Shodex OHpak SB-804 HQ (8×300mm); Mobile phase: 0.1M NaCl, 0.5mL / min, 35℃; Injection volume: 20µL. Dextran standard curve calibration results are as follows... Figure 1 As shown, ALHX-2M exhibits a single symmetrical peak with a peak molecular weight of 43.9 kDa.

[0087] 1.2.2 Chemical Composition

[0088] Glucuronic acid: The content was determined to be 28.59±0.42% by the m-hydroxybiphenyl method, using galacturonic acid (GalA) as the standard.

[0089] Sulfate ester group: Barium chloride-gelatin method, with K2SO4 as standard, the content was measured to be 38.53±0.37%.

[0090] Protein: Coomassie Brilliant Blue G-250 method, not detected (<0.1%).

[0091] 1.2.3 Monosaccharide Composition

[0092] Pre-column PMP derivatization-HPLC analysis: Agilent ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 µm); mobile phase acetonitrile-0.1 M phosphate buffer (pH 6.7) 17:83 (v / v), 1.0 mL / min, 245 nm, 30 °C. Results are as follows. Figure 2As shown, compared with the standard monosaccharide, ALHX-2M is composed of iduronic acid (IdoA), glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), with a molar ratio of GlcA:IdoA:GalNAc = 1:1.97:3.36.

[0093] 1.2.4 Infrared Spectroscopy

[0094] Nicolet iS50 FT-IR spectrometer, KBr pellet, 4000–4000 cm⁻¹ -1 Scan. Results as follows Figure 3 As shown, typical characteristics of sulfated glycosaminoglycans are displayed:

[0095] -1640cm -1 (C=O stretching / N-acetyl)

[0096] -1430cm -1 (COO⁻ Symmetrical Expansion)

[0097] -1250cm -1 (S=O telescopic)

[0098] -840cm -1 (Axial C–O–S bending)

[0099] 1.2.5 NMR Analysis of ALHX-2M

[0100] NMR data for the ALHX-2M were acquired at 298 K on a Bruker AVANCE NEO 800 MHz spectrometer (QCI-F cryogenic probe). (Routine...) 1 ¹H NMR: Approximately 5 mg of lyophilized sample was dissolved in 0.5 mL of D₂O (99.9% D) and transferred to a 5 mm tube. Before the two-dimensional experiment, 10–20 mg of sample was thoroughly deuterated by three cycles of dissolution-lyophilization in 0.5 mL of D₂O, and finally dissolved in 0.5 mL of D₂O containing 0.05% TSP (w / v). The NMR was obtained using Bruker standard pulsed sequencing. 1 H– 1 H TOCSY, COSY, ROESY 1 H– 13 C HSQC and HMBC spectra; number of scans: 13 C: 1200, DEPT-135: 512, COSY: 4, TOCSY: 8, ROESY: 32, HSQC: 4, HMBC: 64.

[0101] Results and Analysis: The results are as follows Figure 4 –8 is shown. The signal attribution results are shown in Table 1.

[0102] Table 1. Chemical shift assignments of ALHX-2M nuclear magnetic resonance for *Aster scleroderma*.

[0103]

[0104] Note: Bold text indicates linkage sites; underlined text indicates sulfate ester linkage sites.

[0105] The above data confirm that ALHX-2M is a highly sulfated, novel starfish-derived glycosaminoglycan. Its structural composition is {D-GlcA}. 2S3S -β1,3-D-GalNAc 4S -β1,4-} m -{L-IdoA 2S3S -α1,3-D-GalNAc 4S -β1,4-} n -{D-GlcN-α1,3-D-GalNAc 4S -β1,4-} o .

[0106] It is a mixture of homologous polysaccharides with the structure of formula (I):

[0107] (I)

[0108] In formula (I), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; and R are independently -H or -SO3. － .

[0109] Example 2 Preparation of partially deacetylated derivatives of glycosaminoglycans and deacylated / deamination depolymerized oligomers

[0110] 2.1 Preparation method

[0111] 2.1.1 Preparation method of deacetylated derivatives of glycosaminoglycans with different degrees of acetylation

[0112] ALHX-2M (≈20 mg) was placed in a 10 mL round-bottom flask, and 5 mg of hydrazine sulfate and 0.5 mL of hydrazine hydrate were added sequentially. Under nitrogen protection, the mixture was reacted at 250 rpm and 90 °C for 12 h, 24 h, and 48 h, respectively. After the reaction was complete, the mixture was precipitated three times with 80% (v / v) ethanol, desalted using a G-10 gel column, and lyophilized to obtain the deacetylated products after 12 h, 24 h, and 48 h, respectively, and designated as DE-1, DE-2, and DE-3.

[0113] 2.1.2 Preparation of deacylated and deamination-depolymerized oligomers of ALHX-2M

[0114] Deacetylation intermediate: Same as method 2.1.1, but with the reaction time fixed at 24 h, to obtain deacetylation intermediate DE-2.

[0115] Deamination and depolymerization: Dissolve intermediate DE-2 in 1 mL of deionized water, add 2 mL of HNO2 solution (5.5 M, pH 4), and react at room temperature for 15 min to obtain a reaction solution; add 1 M NaOH to neutralize the reaction solution to neutral (test with pH paper), and then precipitate repeatedly with 80% ethanol 5 times (until no color develops on nitrous acid paper), dissolve the precipitate in distilled water, desalt it through a G-25 gel column, and freeze-dry to obtain the depolymerized oligomer dALHX-2M-1.

[0116] 2.1.3 Separation and preparation of oligosaccharide components from deacylated and deamination-depolymerized oligomers of ALHX-2M

[0117] Approximately 400 mg of dALHX-2M-1 was taken, and its oligosaccharide components were separated using a Bio-Gel P-6 or P-10 column with 0.2 M NaCl as the eluent at a flow rate of 8.0 mL / h, collecting 3 mL per tube. Fractions 1-7 (OF1 to OF7) were combined, desalted using a Sephadex G-25 column, lyophilized, and analyzed by HPLC on a Superdex™ Peptide 10 / 300 GL column. The low molecular weight oligosaccharide components OF1 and OF2 were structurally resolved using NMR.

[0118] The above preparation method is only an example of the deacylase and deamination depolymerization derivative of glycosaminoglycans. The deacetylation intermediate used can be intermediates with different degrees of deacetylation, such as DE-1, DE-3, or a mixture thereof.

[0119] 2.2 Results and Analysis:

[0120] 2.2.1 Deacetylated derivatives

[0121] Hydrazolysis selectively removes the acetyl group from N-acetylgalactose, generating a free amino group. The degree of deacetylation gradually increases with reaction time: DE-1 44.9%, DE-2 64.5%, DE-3 90% (…). 1 H NMR, Figure 10 HPGPC analysis showed the following results: Figure 9 As shown, the lower the acetyl content, the smaller the molecular weight of the product, indicating that the deacetylation process is accompanied by sugar chain breakage.

[0122] 2.2.2 Deacylated and deamination depolymerization derivatives

[0123] like Figure 11 As shown, HPGPC analysis revealed that the elution time of dALHX-2M-1 (19.2 min) was significantly later than that of the prototype polysaccharide (15.8 min), confirming successful deacylation and deamination, and yielding low molecular weight oligomers.

[0124] Seven low molecular weight oligosaccharide fractions, OF1–OF7, were isolated and purified from dALHX-2M-1. Their HPGPC chromatograms are shown below. Figure 11 and 12 The HSQC-TOCSY and HMBC spectra of OF1 are shown in Figure 1. Figure 13 , 14 The TOCSY, HSQC, and HMBC spectra of OF2 are shown below. Figure 15 -17, the corresponding signal attributions are summarized in Tables 2 and 3. Analysis of the NMR spectra shows that OF1 is derived from L-IdoA. 2S3S -α1,3-D-anTal-ol 4S / 6S L-GlcA 2S3S -β1,3-D-anTal-ol 4S L-IdoA 2S3S -α1,3-D-GalNAc-ol 4S Composed of sugar fragments. OF2 is composed of L-IdoA. 2S3S -α1,3-D-GalNAc 4S -β1,4-L-IdoA 2S3S -α1,3-D-Tal-ol 4S D-GlcA-β1,3-D-GalNAc 4S -β1,4-L-IdoA 2S3S -α1,3-D-Tal-ol 4S D-GalNAc 4S -β1,4-L-IdoA 2S3S -α1,3-D-Tal-ol 4S L-IdoA 2S3S -α1,3-D-GalNAc 4S -β1,4-D-GlcA-β1,3-D-GalNAc 4S -β1,4-L-IdoA 2S3S -α1,3-D-Tal-ol 4S / 6S It is composed of sugar fragments.

[0125] Table 2. Chemical shift assignments of oligosaccharide component OF1

[0126]

[0127] Note: Italics represent sulfate ester linkage sites, and bold represents glycosidic bond linkage sites.

[0128] Table 3. Chemical shift assignments of the oligosaccharide component OF2

[0129]

[0130] Note: Italics represent sulfate ester linkage sites, and bold represents glycosidic bond linkage sites.

[0131] Based on the analysis of chemical reaction results and image analysis, the partially deacetylated derivatives of glycosaminoglycans are a mixture of homologous polysaccharides with formula (II).

[0132] (II)

[0133] In formula (II), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; R are independently -H or -SO3. － R' is H or -COCH3;

[0134] Deacylated and deamination-depolymerized derivatives of glycosaminoglycans are mixtures of homologous polysaccharides having the structure of formula (III).

[0135] (III)

[0136] In formula (III), m+n are integers with a mean of 2 to 12; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; R are independently -H or -SO3. － R1 is -H or one of the three groups shown in formula (IV):

[0137] (IV)

[0138] In formula (IV), anTal-ol is 2,5-dehydrated tarostitol; anMan-ol is 2,5-dehydrated mannitol.

[0139] Example 3: Evaluation of the anticoagulant activity of ALHX-2M and its derivatives

[0140] 3.1 Method:

[0141] 3.1.1 Activated partial thromboplastin time (APTT)

[0142] After preheating the cuvette to 37°C, add the following ingredients in sequence:

[0143] 5 μL of ALHX-2M and its derivatives (prepared in Tris-HCl buffer with gradient concentrations of 0–160 μg / mL), HP (prepared in Tris-HCl buffer with gradient concentrations of 0–51.2 μg / mL), LMWH (prepared in Tris-HCl buffer with gradient concentrations of 0–320 μg / mL), or Tris-HCl buffer (negative control).

[0144] 45 μL of normal coagulation quality control plasma

[0145] Incubate at 37℃ for 2 min → Add 50 μL of APTT reagent (phospholipid + activator) preheated at 37℃ → Incubate at 37℃ for 3 min → Add 50 μL of 0.02 mol / L CaCl2 preheated at 37℃, and immediately start timing to record the time (in seconds) required for fibrin clots to appear. Perform three replicates per concentration and take the average.

[0146] 3.1.2 Prothrombin Time (PT)

[0147] After preheating the test tube to 37°C, add the following in sequence:

[0148] ALHX-2M and its derivatives (prepared in Tris-HCl buffer at gradient concentrations of 0–1280 μg / mL), HP (prepared in Tris-HCl buffer at gradient concentrations of 0–640 μg / mL), LMWH (prepared in Tris-HCl buffer at gradient concentrations of 0–1280 μg / mL), or 5 μL of Tris-HCl buffer.

[0149] 45 μL of normal plasma

[0150] Incubate at 37℃ for 2 min → Add 100 μL of PT reagent (tissue factor + phospholipid) preheated at 37℃, start timing immediately, and record the coagulation time (seconds).

[0151] 3.1.3 Thrombin Time (TT)

[0152] After preheating the cuvette to 37°C, add the following ingredients in sequence:

[0153] ALHX-2M and its derivatives (prepared in Tris-HCl buffer at gradient concentrations of 0–20 μg / mL), HP (prepared in Tris-HCl buffer at gradient concentrations of 0–1.6 μg / mL), LMWH (prepared in Tris-HCl buffer at gradient concentrations of 0–6.4 μg / mL), or 10 μL of Tris-HCl buffer.

[0154] 90 μL of normal plasma

[0155] Incubate at 37°C for 2 min → Add 50 μL of TT reagent (standardized thrombin 2.0 NIH U / mL) preheated at 37°C, start timing immediately, and record the coagulation time (seconds).

[0156] 3.1.4 Inhibition of endogenous coagulation factor FXase (FVIIIa-FIXa complex) assay

[0157] Commercially available FVIII:C chromogenic substrate kit (Siemens), 96-well plate method:

[0158] Step 1: 30 μL of serial gradient samples + 30 μL of FVIII standard (2 IU / mL) + 30 μL of R2 (containing FIXa, phospholipids, and Ca) 2+ ), and incubate at 37°C with shaking for 2 min to form the FXase complex;

[0159] Step 2: Add 30 μL of R1 (FX substrate) and incubate at 37°C for 1 min;

[0160] Step 3: Add 30 μL of R3 (chromogenic substrate S-2765) preheated at 37 °C, and immediately read the absorbance (ΔA / min) continuously at 405 nm.

[0161] According to OD 405 The change value was used to calculate the inhibition rate of the sample on FXase activity.

[0162] Based on the gradient concentrations and corresponding inhibition rates, an inhibition rate-concentration curve was completed, and the IC50 was calculated. 50 .

[0163] 3.2 Results and Analysis

[0164] 3.2.1 Basic Coagulation Screening

[0165] Table 2 shows that ALHX-2M significantly prolongs human plasma APTT and TT at the ng–μg / mL level, while having almost no effect on PT (PT prolongation <5% at 128 μg / mL), suggesting that it selectively acts on the intrinsic and common pathways without interfering with the extrinsic pathway. The concentration required for 2×APTT (doubling clotting time) is 2.96±0.05 μg / mL, comparable to low molecular weight heparin (LMWH, 2.65±0.02 μg / mL); the concentration required for 2×TT is 1.65±0.08 μg / mL, weaker than unfractionated heparin (HP, 0.34±0.02 μg / mL) and low molecular weight heparin (0.74±0.02 μg / mL).

[0166] The deacetylated derivatives of ALHX-2M, DE-1, DE-2, and DE-3, all exhibited significant anticoagulant effects in in vitro human plasma systems: the concentrations required for a 2-fold extension of APTT and TT were all below 20 μg / mL. With increasing degree of deacetylation, the anticoagulant activity showed a moderate decreasing trend (DE-1>DE-2>DE-3), which is presumably related to the simultaneous β-elimination cleavage of the glycans in a strongly alkaline hydrazine environment, leading to a gradual decrease in the number-average molecular weight (Mn). The decrease in molecular weight reduces the density of binding sites between the glycans and coagulation factors / antithrombin, thereby weakening the anticoagulant efficacy.

[0167] The deacylated and deamination-deamination oligomer derivative of ALHX-2M, dALHX-2M-1 (Mn≈3.2kDa), retains remarkable anticoagulant activity despite its significantly shortened chain length: the required concentrations for 2×APTT and 2×TT are 17.31±0.34μg / mL and 44.26±1.92μg / mL, respectively, suggesting that molecular weight is not the sole determining factor. This may be because, at the oligomer scale, the sulfated / aminated sequences sufficient to recognize intrinsic coagulation factors (FXase complex, thrombin, etc.) are still retained, thus continuing to exert a highly effective and low-bleeding-prone antithrombotic effect.

[0168] Among the low-molecular-weight oligosaccharides isolated from dALHX-2M-1, OF1 showed no anticoagulant activity; OF2 exhibited moderate activity, with a 2×APTT value of 55.03 ± 2.25 μg / mL. OF3–OF7 all showed potent anticoagulant activity, requiring only 4–26 μg / mL for a 2×APTT; among them, only OF5–OF7 also had a significant TT prolongation effect.

[0169] 3.2.2 FXase inhibitory activity

[0170] ALHX-2M exhibits potent inhibitory activity against FXase, IC50 50 The concentration was 57.1 ± 9.3 ng / mL, and the activity was approximately LMWH (IC50). 50The concentration was approximately 177.1 ± 12.3 ng / mL, which is three times higher than that of the previous study. This result is highly consistent with the APTT prolongation effect, further confirming that ALHX-2M mainly exerts its anticoagulant effect by blocking FXase, the rate-limiting enzyme in the intrinsic coagulation cascade.

[0171] Among the series of deacetylated derivatives of ALHX-2M, the inhibitory activity of FXase decreased significantly with increasing degree of deacetylation: DE-1 (44.9% deacetylation) still retained strong inhibitory ability, with an IC50 value of [missing value]. 50 The concentration was 327.3 ± 44.6 ng / mL; DE-2 (degree of deacetylation 64.5%) activity further decreased, IC50... 50 The concentration reached 1227.1±273.5 ng / mL; while DE-3 (90% deacetylation) almost lost its significant inhibitory efficacy.

[0172] The oligomer dALHX-2M-1 obtained by deacetylation-deamination depolymerization, although its molecular weight has been significantly reduced, still retains a certain degree of FXase inhibitory activity, and its IC50 value is [not specified]. 50 The concentration was 1116.5 ± 131.5 ng / mL, similar to that of DE-2. This result suggests that even at lower degrees of polymerization, specific sulfation / amylation sequences are sufficient to maintain partial recognition and inhibition of FXase, providing a structural basis and activity evidence for the subsequent development of novel low-molecular-weight, low-bleeding-risk FXase-targeted anticoagulants.

[0173] Of the low-molecular-weight oligosaccharides isolated from dALHX-2M-1, OF4 can inhibit FXase, IC50. 50 The concentration was 1132.0 ± 134.7 ng / mL; the activity of OF5–OF7 was significantly enhanced, and the IC50 was 1132.0 ± 134.7 ng / mL. 50 As low as 117–447 ng / mL.

[0174] 3.2.3 Security Potential

[0175] Existing clinical anticoagulants (such as UFH and DOACs) inhibit the extrinsic pathway (prolonged PT) to varying degrees, thereby increasing the risk of bleeding. ALHX-2M has almost no effect on PT within the effective anticoagulant concentration range, and its prolongation of TT is weaker than that of UFH and LMWH, suggesting that its bleeding tendency may be lower. In addition, FXase is a key node in thrombus formation, rather than a major component of physiological hemostasis. Targeting this enzyme can effectively inhibit thrombus formation while preserving extrinsic hemostasis. Therefore, ALHX-2M holds promise for development as a next-generation safe FXase inhibitor for the prevention and treatment of venous thromboembolism and contact thrombosis-related diseases.

[0176] Table 2. Effects of ALHX-2M on APTT, TT, and endogenous FXase in *Starfish scleroderma*

[0177]

[0178] Example 4: In vivo anticoagulant activity and bleeding tendency of ALHX-2M

[0179] 4.1 Method:

[0180] 4.1.1 Rat tissue thromboplastin-induced venous thrombosis

[0181] Male Sprague-Dawley rats were randomly assigned to three groups, receiving either saline (as a solvent, Control group), ALHX-2M (0.9 or 1.8 mg / kg, administered subcutaneously), or low molecular weight heparin (3.6 mg / kg, also administered subcutaneously). One hour after administration, anesthesia was induced by intraperitoneal injection of 10% chloral hydrate (0.3 mL / kg), followed by ligation of the inferior vena cava and immediate intravenous injection of tissue thromboplastin. After 20 minutes, the vein was reopened, the formed thrombus was removed, and the thrombus was dried at 50°C for 24 hours before being photographed and weighed. The inhibition rates of thrombus formation by ALHX-2M and low molecular weight heparin were calculated compared to the saline group.

[0182] 4.1.2 Carrageenan-induced tail thrombosis in rats

[0183] Mice were assigned to two different administration regimens. Intraperitoneal injection group: For 7 consecutive days, mice received either saline (Control group) or ALHX-2M (dose of 10, 20, or 40 mg / kg, corresponding to the ALHX-2M-L, ALHX-2M-M, and ALHX-2M-H groups, respectively) or LMWH (dose of 40 mg / kg, positive control group) via intraperitoneal injection. Intravenous injection group: Mice received ALHX-2M (dose of 40 mg / kg) intravenously on days 1, 3, 5, and 7. In both groups, carrageenan (dose of 20 mg / kg) was injected intraperitoneally 30 minutes after the last administration on day 7 to induce thrombosis. Forty-eight hours later, the length of the necrotic tail was measured; a 4 cm tail tip segment was fixed, paraffin-embedded, and stained with hematoxylin and eosin.

[0184] 4.1.3 Bleeding tendency assessment

[0185] Mice were randomly assigned to receive either intraperitoneal injection of ALHX-2M (40 mg / kg, 0.1 mL / 10 g) or subcutaneous injection (18 mg / kg, 0.1 mL / 10 g), or intraperitoneal injection of LWMH (40 mg / kg, 0.1 mL / 10 g) or subcutaneous injection (36 mg / kg, 0.1 mL / 10 g) as a positive control. Sixty minutes after administration, under brief isoflurane anesthesia, the mice's tails were severed 5 mm distally, and the severed end was immediately immersed in a container containing 40 mL of 37°C distilled water with gentle agitation to simulate physiological conditions. The absorbance of the solution was measured spectrophotometrically at 540 nm and compared with a pre-established hemoglobin standard curve to quantitatively calculate blood loss. Bleeding time is defined as the time interval from the start of the tail cut to the complete cessation of bleeding and a duration of ≥30 seconds; if the bleeding time exceeds 60 minutes, it is uniformly recorded as 60 minutes.

[0186] 4.2 Results and Analysis

[0187] 4.2.1 In the venous thrombosis model, ALHX-2M significantly inhibited thrombus formation in a dose-dependent manner: inhibition rates of 79.9% and 89.7% were achieved at doses of 0.9 mg / kg and 1.8 mg / kg, respectively. The positive control, low molecular weight heparin (LMWH), showed an inhibition rate of 89.5% at a dose of 3.6 mg / kg. Figure 18 A). Notably, ALHX-2M achieved antithrombotic efficacy comparable to the clinical gold standard at dose levels far below LMWH. This "dose-saving" effect is attributed to its retained FXase-targeting inhibitory activity (IC50). 50 The combined effects of ALHX-2M (57.1 ng / mL) indicate that it has a stronger potential for inhibiting venous thrombosis in vivo.

[0188] 4.2.2 In a carrageenan-induced mouse tail thrombosis model, ALHX-2M significantly shortened the length of the black tail at 48 h ( Figure 18 B). H&E staining of tail tissue further confirmed that ALHX-2M significantly reduced the thrombus area within the tail vessels. Notably, ALHX-2M, administered intraperitoneally at 10 mg / kg, was equivalent to 40 mg / kg LMWH in inhibiting black-tail thrombus formation, while the former required only one-quarter the dose of the latter, further highlighting its superior unit-dose activity. Furthermore, to investigate the effect of the route of administration on efficacy, we added an intravenous administration experiment: in the same model, ALHX-2M administered intravenously at 40 mg / kg significantly inhibited black-tail thrombus formation compared to the solvent control group.

[0189] 4.2.3 In the safety assessment of hemorrhage in mice, ALHX-2M (18 mg / kg, subcutaneous injection) was compared with the clinical dose of LMWH (36 mg / kg, subcutaneous injection). The results showed that LMWH significantly prolonged bleeding time and increased blood loss compared to the normal control group, while ALHX-2M at a dose of 18 mg / kg had no significant effect on either indicator (e.g., ...). Figure 19 (As shown in A and B). Further increasing the intraperitoneal dose of ALHX-2M to 40 mg / kg did not result in a significant increase in bleeding parameters compared to the control group (e.g., ...). Figure 19 (As shown in C and D). The above data suggests that ALHX-2M has the potential to become a novel, low-bleeding-risk anticoagulant. Considering its potent in vitro FXase inhibitory activity, ALHX-2M is expected to become a naturally derived, highly effective, and low-bleeding-risk FXase inhibitor, providing a unique candidate molecule for the development of new antithrombotic drugs.

[0190] Therefore, the glycosaminoglycan ALHX-2M disclosed in this invention is the first to demonstrate that this compound can be used as a highly active and selective FXase inhibitor, and can be applied to the development of a new generation of low-bleeding-risk anticoagulant drugs for the prevention and treatment of thrombotic cardiovascular diseases.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art can still modify or make equivalent substitutions to the technical solutions; however, any improvements and changes made based on the spirit of the present invention should not depart from the protection scope of the technical solutions of the present invention.

Claims

1. A glycosaminoglycan and / or its series of derivatives, characterized in that: The glycosaminoglycan is a mixture of homologous polysaccharides having formula (I). （I） In formula (I), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; and R are independently -H or -SO3. － ; and / or The series of glycosaminoglycan derivatives are partially deacetylated derivatives of glycosaminoglycans, and are mixtures of homologous polysaccharides of formula (II). (II) In formula (II), m+n+o are integers with a mean of 3 to 24; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; β-D-GlcN is β-D-2-amino-2-deoxyglucosyl; R are independently -H or -SO3. － R' is H or -COCH3; and / or The series of glycosaminoglycan derivatives are deacylated and deamination-depolymerized derivatives of the glycosaminoglycans, and are mixtures of homologous polysaccharides having the structure of formula (III). (III) In formula (III), m+n are integers with a mean of 2 to 12; α-L-IdoA is α-L-iduronic acid; β-D-GlcA is β-D-glucuronic acid; β-D-GalNAc is β-D-2-acetamido-2-deoxygalactosyl; β-D-GalN is β-D-2-amino-2-deoxygalactosyl; R are independently -H or -SO3. － R' is H or -COCH3; R1 is -H or one of the three groups shown in formula (IV): (IV) In formula (IV), anTal-ol is 2,5-dehydrated tarottitol; anMan-ol is 2,5-dehydrated mannitol.

2. The glycosaminoglycan and / or its series derivatives according to claim 1, characterized in that: The glycosaminoglycan or its series derivatives have a weight-average molecular weight of 1-50 kDa and a polydispersity index between 1.0 and 1.9; the sulfate content is 20-60%.

3. The glycosaminoglycan and / or its series derivatives according to claim 1, characterized in that: The monosaccharide composition includes L-α-iduronic acid, D-β-glucuronic acid, and D-β-N-acetaminogalactosamine; the content ratio of the monosaccharide composition of the glycosaminoglycan is as follows, based on the molar ratio: D-β-glucuronic acid: L-α-iduronic acid: D-β-N-acetaminogalactosamine = 1:(1.97±1.5):(3.36±2.0).

4. A method for preparing glycosaminoglycans and / or their series derivatives according to any one of claims 1-3, characterized in that: The preparation method of the glycosaminoglycan includes the following steps: take the dry, smooth body wall of the starfish, crush it, add solvent and protease for enzymatic hydrolysis and extraction, then heat it to 40-95℃, add inorganic base for alkaline hydrolysis, cool it after alkaline hydrolysis, separate the liquid part, take the liquid part to remove protein, separate the liquid part again, adjust the pH of the liquid part to neutral, and obtain the glycosaminoglycan by alcohol precipitation, washing and purification.

5. The method for preparing glycosaminoglycans and / or their series derivatives according to claim 4, characterized in that: The purification process includes the following steps: After washing, crude starfish polysaccharide is obtained. The crude starfish polysaccharide is dissolved in pure water, filtered through a microporous membrane, and then loaded onto a strong anion exchange column. Elution is performed sequentially with pure water, 0.5 M NaCl solution, 1.0 M NaCl solution, and 2.0 M NaCl solution at a flow rate of 0.5-3 mL / min. The elution fraction from the 2.0 M NaCl solution is combined, dialyzed with deionized water for 36-72 h, concentrated, and then loaded onto a gel permeation column. Isocratic elution is performed with 0.15 M NaCl solution at a flow rate of 0.1-0.5 mL / min. A symmetrical single peak is collected, dialyzed, and dried to obtain the glycosaminoglycan.

6. The method for preparing glycosaminoglycans and / or their series derivatives according to claim 4, characterized in that: The method for preparing the partially deacetylated derivative of the glycosaminoglycan includes the following steps: treating the glycosaminoglycan at high temperature under nitrogen or inert gas protection with anhydrous hydrazine and / or hydrated hydrazine to cause partial deacetylation of the aminohexose contained therein, thereby obtaining the partially deacetylated derivative of the glycosaminoglycan.

7. The method for preparing glycosaminoglycans and / or their series derivatives according to claim 6, characterized in that: The method for preparing the deacylated and deamination-depolymerized derivative of the glycosaminoglycan includes the following steps: The glycosaminoglycan is partially deacetylated by nitrous acid to cause a deamination and depolymerization reaction, thereby obtaining the glycosaminoglycan or a series of glycosaminoglycan derivatives of low molecular weight deacylated and deamination depolymerization. The low molecular weight deacylated and deamination depolymerized derivatives are further separated using size exclusion gel column and / or ion exchange chromatography column to obtain deacylated and deamination depolymerized derivatives of the glycosaminoglycan with the structure of formula (III), where m+n is an integer from 2 to 12.

8. The use of the glycosaminoglycans and / or their derivatives as described in any one of claims 1-3 in the preparation of medicaments or functional foods for the prevention and treatment of thrombotic cardiovascular diseases or for improving microcirculation.

9. A pharmaceutical composition comprising the glycosaminoglycans of any one of claims 1-3 and / or their derivatives, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9 is prepared into a pharmaceutically acceptable dosage form.

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