Snake tail glycosaminoglycan and derivative, pharmaceutically acceptable salt, preparation method and application thereof

Abstract

The invention discloses a sea snake tail glycosaminoglycan as well as a derivative, a pharmaceutically acceptable salt, a preparation method and an application of the sea snake tail glycosaminoglycan. The sea snake tail glycosaminoglycan has a structure as shown in a formula (I), is prepared by extracting, separating and purifying the sea snake tail, and is further subjected to deacetylation, carboxyl reduction, peroxidation depolymerization, deacylation and deamination depolymerization and gel column chromatography to obtain various derivatives of the sea snake tail glycosaminoglycan. The glycosaminoglycan compound and the derivative thereof show remarkable anticoagulation and antithrombotic activity, are low in bleeding risk, mainly act on an endogenous blood coagulation factor X enzyme complex (FXase) and a heparin cofactor II (HCII), and can provide a new thought for preparing medicines or functional foods for treating and / or preventing thrombotic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a sea snake tail glycosaminoglycan and a pharmaceutically acceptable salt thereof, a preparation method and application in preparing a drug for treating and / or preventing thrombotic diseases. BACKGROUND

[0002] Thrombotic diseases, such as deep vein thrombosis, pulmonary embolism, ischemic stroke, etc., have become a "silent killer" threatening human health. The incidence of these diseases continues to rise, with high disability and mortality rates, placing a heavy and increasing burden on the global public health system. In clinical practice, ordinary heparin and low molecular weight heparin, as anticoagulants, can effectively block thrombus formation, but they are also accompanied by side effects such as bleeding, thrombocytopenia, allergic reactions, and hyperkalemia. The existence of these side effects forces the medical community to constantly seek safer and more effective alternatives.

[0003] Sulfated polysaccharides from marine sources have become a star molecule in the research of new anticoagulant drugs due to their unique advantages of "natural low toxicity and structural diversity". Among them, the glycosaminoglycans (GAG) secreted by echinoderms have attracted much attention due to their novel sulfation patterns and different anticoagulation pathways from heparin. Sea snake tail (Ophiuroidea) is a widely distributed ancient echinoderm that can form a dominant population in various seas. In 1955, Nature first reported that sea snake tail could secrete "heparin-like" sulfated glycosaminoglycans (Fontaine, Nature, 1955, 176: 606-607). However, due to the small size of individuals and difficulties in catching, related research has almost stagnated in the following six decades. In 2014, Ramachandra et al. identified chondroitin sulfate / dermatan sulfate hybrid chains from four species of sea snake tail (Ramachandra et al., Glycobiology, 2014, 24: 195-207), suggesting that there may be unknown and highly active glycosaminoglycans in their bodies.

[0004] Therefore, exploring glycosaminoglycans in sea snake tail is also one of the important research ideas for developing drugs for treating thrombotic diseases. SUMMARY

[0005] The purpose of the present application is to provide a sea snake tail glycosaminoglycan and a pharmaceutically acceptable salt thereof, a preparation method and application. The sea snake tail glycosaminoglycan can strongly inhibit endogenous FXase, significantly prolong APTT and TT, and does not affect the bleeding time, showing excellent characteristics of "strong anticoagulation and low bleeding risk", providing a new choice for developing drugs for preventing and treating clinical thrombotic diseases.

[0006] To achieve the above purpose, the technical scheme of the present application is as follows:

[0007] The present application provides a sea snake tail glycosaminoglycan having a structural formula of formula (I),

[0008] (I)

[0009] In formula (I), IU is α-L-IdoA or its carboxyl-reduced derivative α-L-Ido;

[0010] GU is α-D-GalA or its carboxyl-reduced derivative α-D-Gal;

[0011] G is β-D-GalNAc or its deacetylated derivative β-D-GalN;

[0012] GN is α-D-GlcN;

[0013] R is -H or -SO3 - ;

[0014] R1 is -COO - or -CH2OH;

[0015] R2 is -H or -COCH3;

[0016] m is an integer of 2-50, and n and o are respectively integers of 0-20;

[0017] R3 is -H or one of the three groups of the depolymerized derivative of formula (II):

[0018] (II)

[0019] In formula (II), T is α-D-anTal-ol;

[0020] M is α-D-anMan-ol.

[0021] Preferably, the monosaccharide composition comprises iduronic acid, N-acetylgalactosamine, galacturonic acid, glucosamine and galactose, wherein the molar ratio of iduronic acid to N-acetylgalactosamine is 1.0:(1±0.5).

[0022] Preferably, the sea snake tail glycosaminoglycan has a weight average molecular weight ranging from 3 to 70 kDa, and a sulfate group content of 20-60%.

[0023] The second aspect of the present application provides a sea snake tail glycosaminoglycan derivative, which is:

[0024] The sea snake tail glycosaminoglycan described above is subjected to carboxyl reduction, and the hexuronic acid therein is converted into a corresponding sea snake tail glycosaminoglycan carboxyl-reduced derivative;

[0025] Or

[0026] The sea snake tail glycosaminoglycan described above is subjected to deacetylation treatment to remove part of the acetyl group, to obtain a sea snake tail glycosaminoglycan deacetylated derivative;

[0027] Or

[0028] The sea snake tail glycosaminoglycan described above is subjected to hydrogen peroxide depolymerization, or deacyl and deamination depolymerization, to obtain a sea snake tail glycosaminoglycan low-molecular-weight depolymerized derivative.

[0029] Preferably, in the carboxyl reduction process, the activated carboxyl reagent includes but is not limited to carbodiimides such as 1-ethyl-(3-dimethylaminopropyl) carbodiimide, or a reducing reaction is carried out using but not limited to 1-cyclohexyl-2-morpholinoethyl diimide p-toluenesulfonate to generate a diimide intermediate; the reducing agent is optionally sodium borohydride or potassium borohydride.

[0030] Preferably, in the deacetylation treatment process, hydrazine is used for removal. The new glycosaminoglycan can be dissolved in anhydrous or aqueous hydrazine, and a catalyst such as hydrazine sulfate and / or hydrazine hydrochloride is used for stirring reaction, the reaction temperature is 60-100°C, and the reaction time is 12-48h.

[0031] Preferably, in the hydrogen peroxide depolymerization process, H2O2 with a final concentration of 1-10wt% is used for treatment, and the catalyst is iron ion, copper ion or vitamin C.

[0032] Preferably, in the deacyl and deamination depolymerization process, the new glycosaminoglycan is first treated with hydrazine to cause deacetylation reaction of part of the acetyl galactosamine therein, and then the deacetylated derivative is treated with freshly prepared nitrous acid solution with pH 2-5 for 5-60min, to obtain a deamination depolymerization product. Generally, the obtained deacetyl and deamination depolymerization product is a mixed depolymerization product containing multiple different molecular weights. To obtain a purer or more uniform depolymerization product, separation and purification treatment are further included.

[0033] Preferably, in the process of preparing the derivative described above, the obtained product is further subjected to dialysis and / or ultrafiltration to remove small molecular substances or salts, to obtain a purified target product.

[0034] The third aspect of the present application provides a pharmaceutically acceptable salt of ophiocordyceps sinensis glycosaminoglycan, which is prepared from the ophiocordyceps sinensis glycosaminoglycan or the ophiocordyceps sinensis glycosaminoglycan derivative described above, and the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt or an organic salt.

[0035] The fourth aspect of the present application provides a preparation method of the ophiocordyceps sinensis glycosaminoglycan described above, which comprises the following steps:

[0036] An ophiocordyceps sinensis individual is taken, and then subjected to enzymatic hydrolysis, alkaline extraction, protein removal, salting-out and alcohol precipitation, decolorization and alcohol precipitation to obtain an ophiocordyceps sinensis crude polysaccharide extract; the extract is further purified by column chromatography to obtain the glycosaminoglycan.

[0037] In the present application, the ophiocordyceps sinensis individual is derived from Trichaster palmiferus of the Ophiura family of the Ophiuroidea class of echinoderms. Ophiocordyceps sinensis is distributed in all major oceans around the world, from the intertidal zone to the deep sea, and there are more than 2000 species, which is the largest family of echinoderms. Those skilled in the art should know that the number of ophiocordyceps sinensis species for glycosaminoglycan separation and purification is limited at present, however, the ophiuroidea animal species containing the novel glycosaminoglycan defined in the present application is not limited to Trichaster palmiferus.

[0038] Preferably, in the enzymatic extraction, the enzyme can be a protease, and the protease is one or a combination of papain, trypsin and alkaline protease.

[0039] Preferably, after enzymatic hydrolysis, alkaline extraction is performed, which specifically includes: the enzyme hydrolysis suspension is extracted by adding an inorganic base, and the inorganic base can be NaOH or KOH, or other inorganic bases.

[0040] Preferably, the protein removal method can be selected from isoelectric point method, Sevag method, trichloroacetic acid method and the like.

[0041] Preferably, the decolorization method can be selected from hydrogen peroxide decolorization, macroporous resin decolorization or activated carbon decolorization, and other methods can also be selected according to the situation.

[0042] Preferably, the alcohol precipitation after decolorization can be carried out by using ethanol, methanol, acetone or a combination thereof, or a quaternary ammonium salt precipitation method.

[0043] Preferably, the column chromatography can be selected from anion exchange column chromatography and / or gel column chromatography to obtain purified or partially purified ophiocordyceps sinensis glycosaminoglycan.

[0044] The fifth aspect of the present application provides a pharmaceutical composition comprising the above-mentioned sea snake tail glycosaminoglycan and / or carboxyl-reduced derivative thereof and / or deacetylated derivative thereof and / or low molecular weight depolymerization product thereof and / or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0045] The sixth aspect of the present application provides use of the above-mentioned sea snake tail glycosaminoglycan or pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing thrombotic diseases or a functional food for anticoagulation.

[0046] The above-mentioned thrombotic cardiovascular diseases include, but are not limited to, thrombotic cardio-cerebral vascular diseases, venous thrombosis, pulmonary vein thrombosis, peripheral vein thrombosis, peripheral artery thrombosis, disseminated intravascular coagulation, cardio-cerebral vascular embolism.

[0047] Thrombotic diseases are the leading cause of death and disability in humans, causing a huge social and economic burden. Currently, the clinically used and under-researched anti-thrombotic drugs include three major categories such as thrombolytic drugs, anticoagulants and anti-platelet drugs, but the existing drugs all have serious side effects such as bleeding tendency. There is still an urgent need for safer and more effective therapeutic drugs in clinical practice. Therefore, the present application provides a sea snake tail glycosaminoglycan, including the original sea snake tail glycosaminoglycan, carboxyl-reduced product, deacetylated product, hydrogen peroxide depolymerized sea snake tail glycosaminoglycan mixture and derivative thereof, deacetylated and deaminated depolymerized sea snake tail glycosaminoglycan mixture and derivative thereof, and pharmaceutically acceptable salt, which has good anticoagulant and anti-thrombotic activity and low bleeding tendency, and has application value in the clinical prevention and treatment of thrombotic diseases.

[0048] The present application has the following beneficial technical effects:

[0049] 1. The glycosaminoglycan provided by the present application is a new type of glycosaminoglycan and derivative thereof isolated from a large-sized palm branch snake tail in the Beibu Gulf of Guangxi for the first time. The polysaccharide is different from the reported sea snake tail GAG, and is also different from heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, CS / DS hybrid chain and hyaluronic acid. The sulfation site is unique, which fills the gap in the study of active polysaccharides of sea snake tail.

[0050] 2. Through in vitro and animal model experiments, it is shown that the sea snake tail glycosaminoglycan compound, derivative thereof and pharmaceutically acceptable salt thereof obtained by the present application can strongly inhibit endogenous FXase and the like, significantly prolong APTT and TT, but does not affect the bleeding time, i.e. shows significant anticoagulant and anti-thrombotic activity, and has low bleeding tendency, which can provide a candidate drug molecule with new structure, strong activity and high safety for the development of drugs for preventing and treating clinical thrombotic diseases, and also provides more choices for the research of anticoagulant functional foods. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 HPGPC chromatogram of sea snake tail glycosaminoglycan TPG.

[0052] Figure 2 Monosaccharide composition chromatogram of sea snake tail glycosaminoglycan TPG.

[0053] Figure 3 Infrared spectrum of sea snake tail glycosaminoglycan TPG.

[0054] Figure 4 NMR spectrum of sea snake tail glycosaminoglycan TPG. Wherein Figure A is 1 H spectrum, Figure B is 13 C spectrum.

[0055] Figure 5 Characterization chart of sea snake tail glycosaminoglycan TPG carboxyl reduction derivative. Wherein Figure A is HPGC chromatogram, Figure B is monosaccharide composition chromatogram.

[0056] Figure 6 HPGPC chromatogram of sea snake tail glycosaminoglycan TPG peroxide depolymerization product.

[0057] Figure 7 One-dimensional NMR spectrum of sea snake tail glycosaminoglycan TPG hydrogen peroxide depolymerization product hdTPG-2. Wherein Figure A is 1 H spectrum, Figure B is 13 C spectrum.

[0058] Figure 8 HSQC NMR spectrum of sea snake tail glycosaminoglycan TPG hydrogen peroxide depolymerization product hdTPG-2.

[0059] Figure 9 HMBC NMR spectrum of sea snake tail glycosaminoglycan TPG hydrogen peroxide depolymerization product hdTPG-2.

[0060] Figure 10 Characterization chart of sea snake tail glycosaminoglycan TPG deacetylation derivative. Wherein Figure A is HPGC chromatogram, Figure B is 1 H spectrum.

[0061] Figure 11 HPGPC chromatogram of sea snake tail glycosaminoglycan TPG deacyl deamination depolymerization product.

[0062] Figure 12 One-dimensional NMR spectrum of sea snake tail glycosaminoglycan TPG deacyl deamination depolymerization product ddTPG-1. Wherein Figure A is 1 H spectrum, Figure B is 13 C spectrum.

[0063] Figure 13 HSQC NMR spectrum of the depolymerization product ddTPG-1 of the sea snake tail glycosaminoglycan TPG.

[0064] Figure 14 HMBC NMR spectrum of the depolymerization product ddTPG-1 of the sea snake tail glycosaminoglycan TPG.

[0065] Figure 15 One-dimensional NMR spectrum of the depolymerization product ddTPG-2 of the sea snake tail glycosaminoglycan TPG. Wherein Fig. A is 1 H spectrum, Fig. B is 13 C spectrum.

[0066] Figure 16 HSQC NMR spectrum of the depolymerization product ddTPG-2 of the sea snake tail glycosaminoglycan TPG.

[0067] Figure 17 HMBC NMR spectrum of the depolymerization product ddTPG-2 of the sea snake tail glycosaminoglycan TPG.

[0068] Figure 18 Activity diagram of the sea snake tail glycosaminoglycan TPG in inhibiting thrombosis of the inferior vena cava of rats.

[0069] Figure 19 Bleeding tendency diagram of the sea snake tail glycosaminoglycan TPG. DETAILED DESCRIPTION

[0070] The technical solutions of the present application are further described below by means of the accompanying drawings and examples. The specific examples and drawings described herein are only used for in-depth analysis of the present application, and are not intended to limit the present application.

[0071] Unless otherwise defined, the technical terms or scientific terms used in the present application shall be understood as the usual meanings understood by persons having ordinary skills in the art to which the present application belongs.

[0072] Example 1: Extraction and purification of sea snake tail glycosaminoglycan

[0073] Extraction: 3000 g of dried sea snake tail powder was crushed and placed in a double-layer glass reactor. 15 L of pure water (1:5, g / mL) was added, followed by the addition of 18 g of papain (final concentration 0.1 %). The mixture was stirred at 55°C for 16 h. After enzymatic hydrolysis, the temperature was increased to 60°C, and 6 M NaOH was slowly added to the system to a final concentration of 0.5 M. The stirring was continued for 2 h. The temperature was then cooled to room temperature, and 6 M HCl was added to adjust the pH to 2-3. The mixture was then placed at 4°C for 4 h. The supernatant was obtained by centrifugation at 4500 rpm for 15 min. The pH was adjusted to neutral with 6 M NaOH, and the mixture was salted out and alcohol precipitated. The precipitate was obtained by centrifugation, and the precipitate was dissolved in pure water. The pH was adjusted to 10-11 by the dropwise addition of 6 M NaOH. The temperature was increased to 50°C, and 30 % H2O2 was slowly added to a final concentration of 3 %. The reaction was carried out for 2 h to decolorize. The supernatant was collected by centrifugation, and the pH was adjusted to neutral. The mixture was alcohol precipitated and centrifuged to obtain the precipitate. The precipitate was dissolved in pure water, concentrated by rotary evaporation, and freeze-dried to obtain the crude polysaccharide from sea snake tail.

[0074] Isolation and purification: 5 g of crude polysaccharide was dissolved in 60 mL of pure water and loaded onto an Amberlite FPA98Cl strong anion exchange column. The column was eluted with 0, 0.5, 1.0, 2.0, and 3.0 M NaCl, respectively, and the eluate was monitored by the phenol-sulfuric acid method. The 2.0 M NaCl eluate was combined, concentrated, and repeatedly alcohol precipitated and freeze-dried to obtain the purified glycosaminoglycan, which was designated as TPG.

[0075] Results: According to the extraction and purification steps described in this example, the sea snake tail was extracted by enzymatic hydrolysis-alkaline hydrolysis, the protein was removed by isoelectric point method, the mixture was salted out and alcohol precipitated, decolorized, and alcohol precipitated to obtain the crude polysaccharide. The yield was 0.42 % based on the dry weight of the raw material. The crude polysaccharide was further purified by ion exchange chromatography to obtain a new glycosaminoglycan TPG, and the yield was about 10 %. The glycosaminoglycan was easily soluble in water and difficultly soluble in organic solvents such as ethanol, propanol, and ethyl acetate.

[0076] Example 2: Determination of the physicochemical properties of sea snake tail glycosaminoglycan

[0077] 2.1 Method

[0078] 2.1.1 Determination of the molecular weight of TPG by high-performance gel molecular exclusion chromatography

[0079] Instrument: LC-2030C 3D HPLC; column: Shodex OHpak SB-804 HQ (8 mm x 300 mm); mobile phase: 0.1 mol / L NaCl; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 20 μL.

[0080] 2.1.2 Determination of the sulfate group content

[0081] Barium chloride-gelatin method with K2SO4 as the standard.

[0082] 2.1.3 OSO3 - / COO - Molar ratio determination

[0083] Conductivity titration.

[0084] 2.2 Results

[0085] 2.2.1 As Figure 1 As shown, TPG exhibits a single symmetrical chromatographic peak with a peak molecular weight of 30.3 kDa.

[0086] 2.2.2 Barium chloride-gelatin method: sulfate ester content 31.7%.

[0087] 2.2.3 Conductivity titration: OSO3 - / COO - =1.67, which translates to a sulfate content of 32%.

[0088] Example 3: Structural characterization of galactosidase from sea brittle stars

[0089] 3.1 Monosaccharide Composition Analysis

[0090] The monosaccharide composition of TPG was determined by pre-column derivatization-high performance liquid chromatography (LC-2030C 3D HPLC). Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-0.1 mol / L phosphate buffer (pH 6.7) 17:83 (V / V); flow rate: 1.0 mL / min; column temperature: 30℃; DAD detection wavelength: 245 nm; injection volume: 20 μL.

[0091] Result: As Figure 2 As shown, compared with the standard monosaccharide, TPG is mainly composed of iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc), with a molar ratio of 48.27:51.73; it also contains small amounts of galacturonic acid (GalA), galactose (Gal) and glucosamine (GlcN).

[0092] 3.2 Infrared Spectroscopy (FT-IR)

[0093] Mix 1–2 mg of TPG with dry KBr, compress into tablets, and analyze on a Nicolet iS50 spectrometer at 4000–400 cm⁻¹. -1 scanning.

[0094] Result: As Figure 3 As shown, 3464cm -1 (νO–H), 2924cm -1 (νC–H), 1634cm-1 with 1418 cm -1 (νC=O asymmetric stretch and δC-H), 1260 cm -1 (νS=O) and 825 cm -1 (δC-O-S) confirmed the presence of sulfate groups.

[0095] 3.3 Methylation analysis

[0096] Desulfurized-reduced sample was subjected to four times of Hakomori methylation→ 2 mol / L TFA hydrolysis at 120°C for 2 h→ NaBH4 reduction→ acetic anhydride-pyridine acetylation, and the product was dissolved in 1 mL of dichloromethane for analysis by 7890B-5977B GC-MS system. Chromatographic column: HP-5ms (30 m x 0.25 mm x 0.25 μm); programmed temperature: 50°C (1 min)→ 50°C / min→ 130°C→ 3°C / min→ 230°C (2 min); carrier gas He, 1.0 mL / min; EI source 70 eV, scanning 50-600 m / z.

[0097] Results: Six types of glycosidic bonds, T-IdoA, 1,4-Gal, 1,4-IdoA, 1,3-Gal, T-GalNAc and 1,3-GalNAc were detected (Table 1).

[0098] Table 1. Partial methylation glycol acetate result analysis of desulfurized-reduced TPG product

[0099]

[0100] 3.4 Nuclear magnetic resonance (NMR)

[0101] 30 mg of TPG was dissolved in 0.5 mL of D2O and placed in a nuclear magnetic resonance spectrometer for determination.

[0102] In the 1H NMR spectrum of TPG (Fig. 4A), δ 4.6-5.4 ppm was attributed to the end group proton signal, and the δ 3.4-4.6 ppm region was the sugar ring proton peak. The δ 2.11 ppm sharp single peak corresponded to the -NHCOCH3methyl protons of acetyl amino hexose (GalNAc). Figure 4 The 13C NMR spectrum (Fig. 4B) showed that the acetyl methyl carbon was located at δ 25.4 ppm; the end group carbon signal was distributed at δ 96-107 ppm; the sugar ring carbon was concentrated at δ 53-85 ppm; and the characteristic peaks of the carbonyl carbons of uronic acid (IdoA) and acetyl amino appeared at δ 177.2-178.4 ppm.

[0103] 3.5 Chemical structural formula of sea snake tail glycosaminoglycan TPG

[0104] ​

[0105] The above-mentioned monosaccharide composition, methylation analysis, and nuclear magnetic resonance data of depolymerization products show that the sea snake tail glycosaminoglycan TPG has the following structural formula:

[0106] (I)

[0107] In formula (I), IU is α-L-IdoA or a carboxyl-reduced derivative thereof, α-L-Ido;

[0108] GU is α-D-GalA or a carboxyl-reduced derivative thereof, α-D-Gal;

[0109] G is β-D-GalNAc or a deacetylated derivative thereof, β-D-GalN;

[0110] GN is α-D-GlcN;

[0111] R is -H or -SO3 - ;

[0112] R1 is -COO - or -CH2OH;

[0113] R2 is -H or -COCH3;

[0114] m is an integer of 2-50, and n and o are integers of 0-20, respectively;

[0115] R3 is -H or one of the three groups of the depolymerized derivative of formula (II):

[0116] (II)

[0117] In formula (II), T is α-D-anTal-ol;

[0118] M is α-D-anMan-ol.

[0119] Example 3: Preparation of a new glycosaminoglycan reduced derivative

[0120] 3.1 Preparation method and analysis method

[0121] Accurately weigh TPG 50 mg, place in a 25 mL round-bottom flask, add 5 mL of 0.05 mol / L 2-morpholinoethanesulfonic acid (MES) buffer, and magnetically stir until completely dissolved. Add 2 mL of 500 mg / mL 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide p-toluenesulfonate (CMC) solution, and react at 25-30°C for 2 h; then slowly dropwise add 5 mL of 4 mol / L imidazole solution, and cool in an ice bath for 5 min. Freshly prepare 5 mL of 60 mg / mL NaBH4 solution, and add dropwise, and continue to react at room temperature for 3 h. After the reaction is complete, add glacial acetic acid dropwise to quench the excess NaBH4 and adjust the pH to neutral, transfer into a MWCO 3500 Da dialysis bag, dialyze thoroughly, and lyophilize to obtain the reduced derivative reTPG.

[0122] Determine the molecular weight of reTPG by high-performance gel permeation chromatography according to Example 2; and analyze the monosaccharide composition by PMP pre-column derivatization-HPLC according to Example 3.

[0123] 3.2 Results

[0124] Perform 1, 2, and 4 reduction cycles, respectively, to obtain depolymerized derivatives reTPG-1, reTPG-2, and reTPG-3, as shown in Figure 5 A, and the corresponding molecular weights of reTPG-1, reTPG-2, and reTPG-3 are 26.5 kDa, 25.3 kDa, and 23.3 kDa, respectively. As shown in Figure 5 B, with increasing number of reduction cycles, the IdoA signal gradually weakens and is converted to Ido, confirming that the carboxyl group is selectively reduced.

[0125] Example 4: Preparation of a novel glycosaminoglycan depolymerization product by hydrogen peroxide depolymerization and structural analysis

[0126] 4.1 Preparation method and analysis method

[0127] Accurately weigh TPG 100 mg, place in a 50 mL round-bottom flask, dissolve in 13.34 mL of pure water, and then add 6.67 mg of copper acetate, and stir until completely dissolved. Slowly dropwise add 30% H2O2 0.33 mL, and magnetically stir to react at 35°C. Take samples every 1 h to monitor the change in molecular weight by HPGPC; when the target molecular weight is reached, immediately add 20 mg of EDTA-Na2 to terminate the reaction. Adjust the pH to neutral with 5 mol / L NaOH, and repeatedly precipitate with 4 volumes of anhydrous ethanol until the precipitate is white, and then dissolve in an appropriate amount of pure water to obtain a depolymerized polysaccharide solution.

[0128] The depolymerized polysaccharide solution was loaded onto a pre-equilibrated Amberlite 732 strong acid type cation exchange resin column to remove residual copper ions, the eluate was collected, pH was adjusted to neutral, concentrated, MWCO 3500 Da dialyzed, the bag liquid was concentrated and freeze-dried to obtain the degradation product hdTPG.

[0129] The molecular weight was determined by high performance gel permeation chromatography according to the method of Example 2.

[0130] 4.2 Results

[0131] The depolymerization time was controlled for 720, 240, 90 and 50 min, and hdTPG-1, hdTPG-2, hdTPG-3 and hdTPG-4 were obtained in turn, with yields of 49.75%, 71.43%, 84% and 67.5% respectively. Figure 6 As shown in the HPGPC, each product showed a single, symmetrical peak, and the molecular weight was 5.3, 9.9, 13.5 and 16.7 kDa in turn.

[0132] The 1H NMR comparison showed that the spectra of TPG and each hdTPG were highly similar, only the molecular weight was reduced, and the main structure was not significantly changed. The medium molecular weight hdTPG-2 was selected for one-dimensional and two-dimensional nuclear magnetic resonance analysis, and the results are shown in Figures 7-9 As shown in the table 2, the hdTPG-2 was mainly composed of D-GalNAc 4S -β1,4-D-IdoA 2S3S / 2S -α1,3-{D-GalNAc 4S -β1,4-D-IdoA 2S3S / 2S} m -α1,3-{D-GalNAc 4S -β1,4-GalA} n -α1,3-D-GalNAc 4S , L-IdoA 2S3S -α1,3-{D-GalNAc 4S -β1,4-D-IdoA 2S3S / 2S} m -α1,3-{D-GalNAc 4S -β1,4-D-GlcA} n -α1,3-{D-GalNAc 4S -β1,4-GlcN 0S / 6S} o -α1,3-D-GalNAc 4S sugar fragments.

[0133] Wherein m≈10, n≈2, o≈4.

[0134] Table 2. hdTPG-2 of Example 1 1 H, 13 C signal attribution

[0135]

[0136] Example 5: Preparation of novel depolymerized glycosaminoglycan products by deacylation and deamination and structural analysis

[0137] 5.1 Preparation method and analysis method

[0138] TPG 1.50 g was weighed into a 100 mL rotary evaporation flask, 37.5 mL of hydrazine hydrate and 0.38 g of hydrazine sulfate were added, and the reaction was carried out under nitrogen protection at 90°C with magnetic stirring for 12-48 h. After cooling to room temperature, 4 times the volume of anhydrous ethanol was added for alcohol precipitation, the precipitate was dissolved in 30 mL of pure water, MWCO 3500 Da dialysis was carried out for 48 h, the cut-off liquid was concentrated by rotary evaporation and freeze-dried to obtain deacetylated polysaccharide. The degree of deacetylation was determined by comparing the integrals before and after deacetylation by 1H NMR.

[0139] The deacetylated sample was dissolved in 25 mL of pure water, 50 mL of pH 4 nitrous acid solution was added under ice bath, and the reaction was terminated after stirring for 5 min; anhydrous ethanol was added to a final concentration of 80%, and repeated alcohol precipitation was carried out 4 times. The precipitate was dissolved in pure water, desalted on a Sephadex G-25 column, and the polysaccharide component was concentrated and freeze-dried to obtain the depolymerized product.

[0140] The depolymerized product was dissolved in 25 mL of pure water, 50 mL of 0.25 mol / L NaBH4 was added dropwise, and stirred at 50°C for 2 h. After cooling, the excess NaBH4 was destroyed by adjusting the pH to 3 with 1 mol / L HCl, and then adjusted to neutral with 1 mol / L NaOH, desalted on a Sephadex G-25 column, concentrated and freeze-dried to obtain the reduction end product. Subsequently, further fractionation was carried out on Bio-Gel P10 and P6 gel columns to obtain the purified depolymerized component ddTPG.

[0141] The molecular weight of ddTPG was determined by high-performance gel molecular exclusion chromatography. Instrument: LC-2030C 3D HPLC; chromatographic column: Superdex TM 30 Increase 10 / 300 GL; mobile phase: 0.2 mol / L NaCl; flow rate: 0.4 mL / min; column temperature: 35°C; injection volume: 10-100 μL.

[0142] 5.2.1. Deacetylation stage

[0143] Two products with different degrees of deacetylation were obtained, as shown in Figure 10 A-B:

[0144] dAcTPG-1 (25% deacetylation, Mw 25.9 kDa)

[0145] dAcTPG-2 (47% deacetylation, Mw 20.6 kDa)

[0146] 5.2.2 Deamination-depolymerization stage

[0147] After deamination by nitrous acid and reduction by NaBH4, combined with Bio-Gel P10 / P6 chromatography, five oligosaccharide components were obtained:

[0148] ddTPG-1→ddTPG-5, with yields of 1.69%, 5.86%, 7.03%, 3.90%, and 6.38%, respectively.

[0149] As shown in Figure 11 , HPGPC showed that ddTPG-1-3 were multiple peaks, suggesting that they were mixtures of oligosaccharides with different degrees of polymerization and similar molecular weights, and were difficult to separate further; ddTPG-4 and ddTPG-5 were both single symmetrical peaks.

[0150] 5.3 Structural analysis

[0151] The deacetylation-deamination process converted the reducing end amino sugar into a 2,5-anhydro-talitol (anTal-ol) or 2,5-anhydro-mannitol (Man-ol) end, and the structural characteristics varied with the degree of depolymerization. Low molecular weight ddTPG-1 and ddTPG-2 were selected for one-dimensional and two-dimensional NMR determination, and the results are shown in Figure 12 -17, and the signal assignments are shown in Tables 3-4, and the main sequences are as follows:

[0152] ddTPG-1:

[0153] →3)-L-IdoA 2S3S -α(1→3)-D-anTal-ol 4S / 6S ;

[0154] →3)-L-IdoA 2S -α(1→3)-D-anTal-ol 4S ;

[0155] →3)-D-GalA-α(1→3)-D-anTal-ol 4S

[0156] →4)-D-GalNAc 4S -β(1→4)-D-Man-ol

[0157] ddTPG-2:

[0158] →3)-L-IdoA 2S3S / 2S- a(1→3)-D-GalNAc 4S / 6S - β(1→4)-L-IdoA 2S3S / 2S - a(1→3)-D-Tal-ol 4S / 6S

[0159] → 3)-L-IdoA 2S3S / 2S - a(1→3)-D-GalNAc 4S - β(1→4)-D-GalA-a(1→3)-D-GalNAc 4S-β(1→4)-D-Man-ol

[0160] → 3)-D-GalNAc 4S - β(1→4)-D-GlcN-a(1→3)-D-GalNAc 4S - β(1→4)-D-Man-ol

[0161] → 3)-D-GalNAc 4S - β(1→4)-D-GlcN 6S - a(1→3)-D-Tal-ol 4S

[0162] Table 3. ddTPG-1 1 H, 13 C signal assignment

[0163]

[0164] Table 4. ddTPG-2 1 H, 13 C signal assignment

[0165]

[0166] Example 6: Anti-coagulant activity determination of novel glycosaminoglycans

[0167] 6.1 Determination method

[0168] 6.1.1 Coagulation three-item test

[0169] (1) Activated partial thromboplastin time (APTT)

[0170] The colorimetric cup was preheated at 37°C, and the sample to be tested or Tris-HCl buffer 5 μL, normal coagulation quality control plasma 45 μL were added in turn, and incubated at 37°C for 2 min; 37°C preheated APTT reagent 50 μL was added, and incubated for 3 min; then 0.02 mol / L CaCl2 50 μL was added at 37°C and immediately timed, and the coagulation time was recorded.

[0171] (2) Prothrombin time (PT)

[0172] Detection tube 37℃ preheating, add the sample to be tested or Tris-HCl buffer 5 μL, normal coagulation quality control plasma 45 μL, 37℃ incubation for 2 min; add 37℃ preheated PT reagent 100 μL, record the clotting time.

[0173] (3) Thrombin time (TT)

[0174] Colorimetric cup 37℃ preheating, add the sample to be tested or Tris-HCl buffer 10 μL, normal coagulation quality control plasma 90 μL, 37℃ incubation for 2 min; add 37℃ preheated TT reagent 50 μL, record the clotting time.

[0175] 6.1.2 Target protein related activity assay

[0176] (1) Inhibition of endogenous factor X enzyme (FXase) activity

[0177] 96-well plate sequentially add 30 μL of a series of concentrations of the sample to be tested, 30 μL of FVIII solution (2 IU / mL), 30 μL of R2 solution (60 nM IXa, containing IIa, Ca²⁺, PC / PS), 37℃ oscillation incubation for 2 min; add 30 μL of R1 solution (50 nM FX, containing direct IIa inhibitor), 37℃ oscillation incubation for 1 min; finally add 30 μL of 37℃ preheated R3 solution (8.4 mM SXa-11), immediately read the absorbance at 405 nm continuously. Tris-HCl buffer as blank control.

[0178] (2) Hepatocutaneous factor II (HCII) dependent inhibition of activated coagulation factor II (FIIa) activity

[0179] 96-well plate add 30 μL of a series of concentrations of the sample to be tested, 30 μL of HCII solution (66.7 μg / mL), 37℃ incubation for 1 min; add 30 μL of thrombin solution (20 NIH / mL), 37℃ incubation for 1 min; finally add 30 μL of 37℃ preheated substrate solution (2.5 mg / mL), read the absorbance at 405 nm every 30 s. Tris-HCl buffer as blank control.

[0180] 6.2 Results

[0181] The results of the assay are shown in Table 5, which shows that TPG can significantly prolong APTT and TT, with doubling concentrations of 3.24 μg / mL and 1.27 μg / mL, respectively, comparable to low molecular weight heparin (LMWH, 2.44 μg / mL), but weaker than heparin (HP, 0.49 μg / mL and 0.19 μg / mL); no effect on PT (>128 μg / mL).

[0182] IC of TPG to inhibit FXase 50 was 38.75 ng / mL, which was superior to LMWH (62.96 ng / mL), indicating that it could efficiently inhibit the key rate-limiting enzyme of the endogenous coagulation pathway and was safer.

[0183] Table 5. Effects of TPG on APTT, TT, PT and FXase

[0184]

[0185] With the increase of carboxyl reduction degree, the APTT and TT prolongation effects of TPG carboxyl reduction derivatives gradually weakened, but there was no linear relationship between the two and the carboxyl content; even if the carboxyl content had been greatly reduced, the derivatives still retained considerable activity. Compared with the original TPG, the APTT, TT prolongation and FXase inhibition of deacetylated derivatives were slightly reduced, but still strong. The hydrogen peroxide depolymerization products had the best activity in the 10-30 kDa range, and the anticoagulant effect decreased as the molecular weight continued to decrease, but the overall APTT prolongation ability was still good. The deacyl-deaminated oligosaccharides also showed the same trend: the smaller the molecular weight, the lower the activity, except for ddTPG-1, the APTT of other deacyl-deaminated depolymerization derivatives was significantly prolonged and could still be maintained.

[0186] IC of TPG to inhibit FIIa in the presence of HCII 50 was 40.99 ± 1.87 ng / mL, which was much lower than that of low molecular weight heparin, 200.43 ± 13.96 ng / mL; its significant HCII-dependent anti-FIIa effect was one of the important mechanisms for prolonging thrombin time.

[0187] Example 7: In vivo anti-thrombus activity determination of new glycosaminoglycans

[0188] Healthy SPF grade SD male rats weighing 200 ± 10 g were selected. They were adaptively fed for 7 days before the experiment, with free access to water and food, a humidity of 50-60% and a temperature of 24 ± 2°C in the animal room, and a 12 h light cycle.

[0189] After the adaptation, the body weight was weighed and the rats were randomly divided into groups: blank control group (normal saline), positive control group (enoxaparin sodium), polysaccharide group (TPG).

[0190] After 1 h of administration, the rats were anesthetized, the abdominal hair was shaved, and the abdomen was disinfected with 75% alcohol. The abdominal wall was incised along the midline. The inferior vena cava and abdominal aorta were separated below the renal vein, a silk thread was prepared, and all visible collateral branches were ligated. Thrombus was induced by injecting 2% rabbit brain powder extract 1.5 mL / kg into the femoral vein, and the inferior vena cava was ligated 20 s later. The blood vessel was clamped 2 cm below the ligature line for 20 min, then longitudinally sectioned and the thrombus was removed. The surface blood was absorbed with filter paper and dried at 50°C for 24 h, and the dry weight was measured.

[0191] Results: As shown in Figure 18 Table 2, TPG can significantly inhibit thrombosis in a dose-dependent manner. The thrombosis inhibition rates at 1.8 mg / kg and 3.6 mg / kg were 74.2% and 84.7%, respectively, and there was no statistically significant difference between the high-dose TPG group and the enoxaparin sodium 3.6 mg / kg group.

[0192] Example 8: Bleeding tendency determination of novel glycosaminoglycan

[0193] SPF male KM mice, weighing 18±2 g, were allowed to adapt to free water and food. After the adaptation period, the mice were randomly divided into groups: blank control group (normal saline), positive control group (heparin, enoxaparin sodium), polysaccharide group (TPG).

[0194] After 60 min of administration, the mice were fixed, the tail tip was cut off 5 mm, and the mouse tail was immediately immersed in 37℃ 40 mL purified water for timing, with continuous stirring for 60 min. The centrifuge tube was taken out and left to stand for 60 min, and the OD 540 was measured by ultraviolet spectrophotometry. The bleeding volume was calculated by substituting the standard curve.

[0195] Results: As shown in Figure 19 Table 3, compared with the blank control group, the bleeding volume of the heparin and enoxaparin sodium groups increased significantly, and heparin > enoxaparin, which is consistent with the clinical bleeding side effects. The bleeding volume of TPG was not significantly different from that of the blank group, indicating that TPG has low bleeding risk and high safety.

[0196] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements will not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A glycosaminoglycan from sea brittle stars, characterized in that: The gypsum glycosaminoglycan has the structural formula (I). （I） In formula (I), IU is α-L-iduronic acid or its carboxyl-reduced derivative α-L-iduronic acid; GU is α-D-galacturonic acid group or its carboxyl reduced derivative α-D-galactosyl; G is β-D-acetaminogalactose or its deacetylated derivative β-D-aminogalactose; GN stands for α-D-glucosamine; R is -H or -SO3 - ; R1 is -COO - or -CH2OH; R2 is -H or -COCH3; m is an integer between 2 and 50, and n and o are integers between 0 and 20 respectively; R3 is -H or one of the three groups of the deacylated and deamination depolymerized derivative shown in formula (II): (Ⅱ) In formula (II), T is 2,5-dehydrated tarostitol; M is 2,5-anhydromannitol.

2. The glycosaminoglycan from *Hydrangea macrophylla* according to claim 1, characterized in that: The monosaccharide composition includes iduronic acid, N-acetylgalactosamine, galacturonic acid, glucosamine and galactose, wherein the molar ratio of iduronic acid to N-acetylgalactosamine is 1.0:(1±0.5).

3. The galactosium glycosaminoglycan according to claim 1, characterized in that: The weight-average molecular weight of the galbestrol glycosaminoglycan ranges from 3 to 70 kDa, and the sulfate content is 20% to 60%.

4. A derivative of galactosidase, characterized in that, The derivative is: The carboxyl group of the galbestrol glycosaminoglycan according to any one of claims 1-3 is reduced to convert the hexuronic acid therein into the corresponding galbestrol glycosaminoglycan carboxyl-reduced derivative. or The glycosaminoglycan of any one of claims 1-3 is subjected to deacetylation treatment to remove some acetyl groups, thereby obtaining a deacetylated derivative of glycosaminoglycan; or The glycosaminoglycans of any one of claims 1-3 are subjected to hydrogen peroxide depolymerization, or deacylation and deamination depolymerization, or further purified by column chromatography to obtain low molecular weight depolymerized derivatives of glycosaminoglycans of glycosaminoglycans.

5. A pharmaceutically acceptable salt of galbuckae glycosaminoglycan, characterized in that, The glycosaminoglycan of any one of claims 1-3 or the derivative of the glycosaminoglycan of claim 4 is prepared into a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt or organic salt.

6. The method for preparing galactosidase as described in any one of claims 1-3, characterized in that... Includes the following steps: Individual brittle stars were collected and extracted by enzymatic and alkaline hydrolysis. After protein removal, salt precipitation, alcohol precipitation, decolorization, and alcohol precipitation, crude polysaccharide extract of brittle stars was obtained. The extract was further purified by column chromatography to obtain the glycosaminoglycans.

7. A pharmaceutical composition comprising the squalene of brittle star anise according to any one of claims 1-3 and / or a derivative of the squalene of brittle star anise according to claim 4 and / or a pharmaceutically acceptable salt of the squalene of brittle star anise according to claim 5, and a pharmaceutically acceptable carrier.

8. The use of the saccharin of any one of claims 1-3, or a derivative of the saccharin of claim 4, or a pharmaceutically acceptable salt of the saccharin of claim 5, or the pharmaceutical composition of claim 7, in the preparation of a medicament for the treatment and / or prevention of thrombotic diseases or an anticoagulant functional food.

9. The application according to claim 8, characterized in that, The thrombotic diseases mentioned are thrombotic cardiovascular and cerebrovascular diseases, venous thrombosis, pulmonary venous thrombosis, peripheral venous thrombosis, peripheral arterial thrombosis, disseminated intravascular coagulation, or cardiovascular and cerebrovascular embolism.

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