A fish swim bladder chondroitin-sulfate dermatan sulfate hybrid chain and a preparation method and application thereof

Abstract

The application discloses a fish swim bladder chondroitin sulfate-Dermatan sulfate hybrid chain and a preparation method and application thereof, and relates to the technical field of medicines. The fish swim bladder chondroitin sulfate (CS) / dermatan sulfate (DS) hybrid chain with anticoagulant activity is composed of iduronic acid, N-acetylgalactosamine, glucuronic acid and N-acetylglucosamine, and the CS / DS hybrid chain is derived from a fish swim bladder, is easily soluble in water, has strong anticoagulant activity, and can be used for preparing medicines for preventing and / or treating thrombotic cardiovascular diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain and a preparation method and application thereof. BACKGROUND

[0002] Thrombotic cardiovascular diseases seriously endanger human health and are the leading cause of death and disability. Anticoagulant and antithrombotic drugs play an important role in the prevention and treatment of venous / arterial thrombosis and other related diseases. Heparin drugs are the earliest and most widely used anticoagulant and antithrombotic drugs in clinical application, occupying a large market share, with a global sales of over 10 billion US dollars. Among them, one of the heparin varieties, enoxaparin sodium Clexane, ranked 66th in the 2022 global generic drug sales Top100. However, existing clinical anticoagulant drugs all have serious side effects such as bleeding tendency. In addition to the serious side effect of bleeding tendency, heparin drugs also have side effects such as platelet aggregation and allergic reactions, and are easily contaminated by viruses and other pathogens during preparation from organs such as pigs, cows, and sheep. Therefore, exploring efficient and low-toxicity anticoagulant drugs from marine organisms such as seaweed, sea cucumbers, and fish swim bladders is still a hot spot for the research and development of new anticoagulant drugs.

[0003] Swim bladder is an important organ of fish and has been used as food for a long time in China, with the earliest record dating back to the Northern Wei Dynasty (386-534 AD) in the book of “Qimin Yaoshu”. In addition, swim bladder has various medicinal values. According to the book of “Bencao Gangmu”, swim bladder can stop bleeding, and swim bladder glue can treat difficult labor, tetanus, vomiting blood, blood stasis and swelling. The medicinal values of swim bladder are also recorded in the books of “Haiyao Bencao” and “Bencao Xinbian”. The medicinal values of swim bladder have been recognized by modern Chinese medicine, and swim bladder has the effects of tonifying essence and blood, stopping bleeding and reducing swelling. In 2004, swim bladder was recommended by the State Administration of Traditional Chinese Medicine as one of the key development Chinese medicinal materials. Generally, most animals contain glycosaminoglycans, which have a wide range of physiological effects and pharmacological activities. However, there are few studies on glycosaminoglycans from swim bladder, and only the research group of Guangdong Ocean University has reported that glycosaminoglycans extracted from swim bladder have anti-angiogenic, anti-coagulation, anti-oxidation, anti-inflammatory, moisture absorption and retention, and other activities and properties (Qu Y et al. Food Industry Science and Technology, 2017, 16: 118-125; Zhou S et al. Food and Machinery, 2018, 34(7): 151-219; Qu Y et al. Journal of Guangdong Ocean University, 2018, 38(1): 47-53). The materials selected in the series of studies are Lateolabrax japonicus or Aristichthys nobilis, and the structure of the obtained glycosaminoglycans is determined as chondroitin sulfate A (CSA) (Zhou S et al. Food Science, 2019, 40(15): 84-91; Chen et al. Food Research International, 2022, 157: 111444).

[0004] The inventors found in the previous studies that the glycosaminoglycans in the swim bladder of certain species are CS / DS hybrid chains, and the DS disaccharide structure unit is dominant, which is different from the previous reports. The CS / DS hybrid chains have strong anti-coagulation activity, can inhibit endogenous factor X enzyme (FXase) and act on heparin cofactor II (HC-II) to inhibit thrombin. The CS / DS hybrid chains described in the present application are expected to become new FXase and HC-II inhibitors for the treatment of thrombotic cardiovascular diseases. SUMMARY

[0005] The purpose of the present application is to provide a novel swim bladder CS / DS hybrid chain, a preparation method and application thereof. The prepared swim bladder CS / DS hybrid chain compound can be used as a potential FXase and HC-II inhibitor for the preparation of a drug for treating thrombotic cardiovascular diseases.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The application provides a fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain with anticoagulant activity, which is composed of iduronic acid, N-acetylgalactosamine, glucuronic acid and N-acetylglucosamine four monosaccharides; and the structure (I) of the fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain is as follows:

[0008]

[0009] [→4)-C-α-(1→3)-D-β-(1→] m The disaccharide units account for 50% to 95% of the total disaccharide content, that is, the disaccharide structural units of dermata sulfate (DS) mainly account for 50% to 95% of the total disaccharide content. In addition, the disaccharide structural units with a higher content [→4)-A-α-(1→3)-E-β-(1→] n Or [→4)-I-β-(1→3)-G-β-(1→] r Account for 5% to 20% and 1% to 30% of the total disaccharide content, respectively.

[0010] Further, the fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain is obtained by extraction, separation and purification from fish swim bladder, and the weight average molecular weight calibrated by dextran is 80 to 150 kDa, the uronic acid content is 20% to 40%, the sulfate ester group content is 15% to 40%, the protein content is less than 3%, and the nucleic acid content is less than 1%.

[0011] The application also provides a preparation method of the fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain, which comprises the following steps:

[0012] S1, the fish swim bladder is extracted by conventional enzymolysis and alkaline hydrolysis: the homogenate liquid or dry powder of the fish swim bladder is added into protease for enzymolysis, and the suspension of the enzymolysis is added into inorganic alkali with a final concentration of 0.2 to 1.0 M for alkaline hydrolysis;

[0013] S2, protein is removed at isoelectric point, and salt precipitation and alcohol precipitation are performed to obtain a fish swim bladder crude polysaccharide extract: the extract is purified by anion exchange column chromatography, and the elution components in 0.5 to 2.0 M NaCl solution are collected, or the dissociation is precipitated by quaternary ammonium salt precipitation method, and repeated alcohol precipitation, concentration and freeze-drying are performed to obtain the fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain.

[0014] Further, the source of the fish swim bladder includes Atlantic cod (Gadus morhua) and barracuda (Sphyraena jello).

[0015] At present, only in the swim bladder of Gadus morhua and Sphyraena jello, the fish swim bladder CS / DS hybrid chain defined in the present application is found, but there are about tens of thousands of fish species in the world. Although the present inventors have carried out the separation and purification of the fish swim bladder CS / DS hybrid chain from a few swim bladder species, the fish swim bladder species containing the fish swim bladder CS / DS hybrid chain defined in the present application are not limited to Gadus morhua and Sphyraena jello. Those skilled in the art can understand that even if the fish derived from other species meet the definition of the fish swim bladder CS / DS hybrid chain defined in the present application, they can also be used to prepare the fish swim bladder CS / DS hybrid chain and pharmaceutically acceptable salts thereof with the structure composition described in the present application.

[0016] Further, the protease includes papain, trypsin, and alkaline protease; and the inorganic base includes NaOH and KOH.

[0017] The present application also provides a drug for treating and / or preventing thrombotic cardiovascular diseases, wherein the effective component of the drug contains the fish swim bladder chondroitin sulfate-dermatan sulfate hybrid chain described above.

[0018] Further, the drug dosage form is a freeze-dried powder injection or an aqueous injection.

[0019] Thrombotic cardiovascular diseases are the leading cause of death and disability in humans, causing a huge social and economic burden. At present, the anti-thrombotic drugs used in clinical practice and under development include three categories such as thrombolytic drugs, anticoagulants, and anti-platelet drugs, but the existing anticoagulants in clinical practice all have serious side effects such as bleeding tendency. There is still an urgent need for safer and more effective anti-thrombotic drugs in clinical practice.

[0020] Therefore, the fish swim bladder CS / DS hybrid chain described in the present application has good anticoagulant activity and can effectively inhibit endogenous FXase and HCII. In general, acting on these two targets has the advantage of low bleeding tendency. Therefore, the fish swim bladder CS / DS hybrid chain described in the present application may have lower toxic side effects, and it has application value for the prevention and treatment of thrombotic cardiovascular diseases in clinical practice.

[0021] The present application provides a drug for treating thrombotic cardiovascular diseases, which contains an effective dose of the fish swim bladder CS / DS hybrid chain and pharmaceutically acceptable salts thereof, and the thrombotic cardiovascular diseases include but are not limited to thrombotic cardiovascular diseases, deep vein thrombosis, pulmonary vein thrombosis, peripheral vein thrombosis, and peripheral arterial thrombosis.

[0022] The present application uses the fish swim bladder CS / DS hybrid chain with anticoagulant activity described above and its preparation method and application, which has the following beneficial technical effects:

[0023] 1. The fish swim bladder CS / DS hybrid chain prepared in this invention is a novel glycosaminoglycan with a weight-average molecular weight of 80–150 kDa as determined by dextran, a uronic acid content of 20%–40%, and a sulfate ester group content of 15%–30%. It is composed of iduronic acid, N-acetylgalactosamine, glucuronic acid, and N-acetylglucosamine, and further, mainly composed of disaccharide structural units of dermatin sulfate (DS). Its structural composition is significantly different from previously disclosed and literature-reported fish swim bladder polysaccharides (chondroitin sulfate A).

[0024] 2. A fish swim bladder CS / DS hybrid chain in this invention can significantly prolong APTT, inhibit FXase and HC-II-dependent thrombin inhibition, providing more options for the development of novel anticoagulant drugs and functional foods.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1 The high-performance liquid chromatograms of the CS / DS hybrid chains of the swim bladders of Atlantic cod and spotted barracuda in this embodiment of the invention are shown.

[0027] Figure 2 The chromatograms for monosaccharide composition analysis of the CS / DS hybrid chains of the swim bladders of Atlantic cod and spotted barramundi in this embodiment of the invention are shown.

[0028] Figure 3 The infrared spectra of the CS / DS hybrid chains of the swim bladders of Atlantic cod and spotted barracuda in this embodiment of the invention;

[0029] Figure 4 This is a diagram showing the disaccharide composition analysis of the CS / DS hybrid chain of the swim bladder of Atlantic cod and spotted barramundi in an embodiment of the present invention;

[0030] Figure 5 The CS / DS hybrid chain of Atlantic cod swim bladder in this embodiment of the invention. 1 H NMR spectrum and its assignment;

[0031] Figure 6 The CS / DS hybrid chain of Atlantic cod swim bladder in this embodiment of the invention. 13 C NMR spectra and their assignments;

[0032] Figure 7 The CS / DS hybrid chain of Atlantic cod swim bladder in this embodiment of the invention. 1 H- 13 C HSQC spectrum and its attribution;

[0033] Figure 8 The CS / DS hybrid chain of the swim bladder of the spotted barracuda in this embodiment of the invention. 1H NMR spectra and assignment;

[0034] Figure 9 For the CS / DS hybrid chain of the swim bladder of Scomberomorus guttatus in the embodiments of the present application 13 C NMR and DEPT135 spectra and assignment;

[0035] Figure 10 For the CS / DS hybrid chain of the swim bladder of Scomberomorus guttatus in the embodiments of the present application 1 H- 13 CHS QC-TOCSY spectra and assignment;

[0036] Figure 11 For the CS / DS hybrid chain of the swim bladder of Scomberomorus guttatus in the embodiments of the present application 1 H- 1 H ROESY spectra and assignment. DETAILED DESCRIPTION

[0037] The technical solutions of the present application are further described below by means of the accompanying drawings and embodiments. The specific embodiments and drawings described herein are only used for in-depth analysis of the present application and are not intended to limit the present application.

[0038] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0039] Example 1: Extraction and purification of swim bladder CS / DS hybrid chain

[0040] Extraction: Dry Gadus macrocephalus, Scomberomorus guttatus swim bladder was crushed, 500g of fish swim bladder dry powder was weighed and placed in a double-layer glass reactor, 5L of pure water was added at 10 times (g / mL) the amount, 55g of papain was added at a final concentration of 1%, and the mixture was stirred and reacted at 55°C for 16h. After enzyme hydrolysis, the temperature was increased to 60°C, and 6M NaOH was slowly added to a final concentration of 0.5M, and the stirring was continued for 2h. After the reaction was completed, the temperature was cooled to room temperature, 6M HCl was slowly added to adjust the pH to 2-3, and the mixture was placed at 4°C for 4h, then centrifuged at 4500rpm for 15min, the supernatant was adjusted to neutral pH with 6M NaOH, and salt precipitation and alcohol precipitation were performed. The precipitate was centrifuged at 4500rpm for 15min, then a certain amount of pure water was added to dissolve the precipitate, and alcohol precipitation was performed again. After repeating several times, the crude polysaccharide was obtained by concentration and freeze-drying.

[0041] Separation and purification: 5 g of crude polysaccharide from swim bladder was dissolved in 60 mL of pure water, and then loaded onto an Amberliter FPA98Cl strong anion exchange column (45 mm x 500 mm) for separation. Gradient elution was performed using pure water, 0.5, 1.0, 2.0, and 3.0 M NaCl, respectively. The 1.0 M NaCl fraction was collected, concentrated, repeatedly precipitated with alcohol, and freeze-dried to obtain the purified CS / DS hybrid chain from swim bladder.

[0042] Results: The crude polysaccharide was obtained from the swim bladder of Atlantic cod and striped bonito through enzymatic hydrolysis, alkaline hydrolysis, deproteinization, and alcohol precipitation. The yield was 0.35% and 0.24%, respectively, based on the dry weight of the raw material. The CS / DS hybrid chain obtained by ion exchange column separation and purification was easily soluble in water and difficult to dissolve in organic solvents such as ethanol, propanol, and ethyl acetate.

[0043] Example 2: Determination of the physicochemical properties of the CS / DS hybrid chain from swim bladder

[0044] The glycuronic acid content of the CS / DS hybrid chain from swim bladder was determined by the m-hydroxybenzene method using GalA as the standard.

[0045] The protein content of the CS / DS hybrid chain sample was determined by the Coomassie brilliant blue method using BSA as the standard.

[0046] The sulfate ester group content of the CS / DS hybrid chain sample was determined by the barium chloride gelatin method using K2SO4 as the standard.

[0047] The molar ratio of sulfate carboxyl groups was determined by the conductivity method.

[0048] The molecular weight of the CS / DS hybrid chain sample was determined by high-performance liquid chromatography. The instrument was LC-2030C3D HPLC, the chromatographic column was Shodex OHpak SB-804HQ (7 μm, 8 x 300 mm), the mobile phase was 0.1 M NaCl solution, the flow rate was 0.5 mL / min, the column temperature was 35°C, and the injection volume was 20 μL.

[0049] The monosaccharide composition of the CS / DS hybrid chain from swim bladder was determined by pre-column derivatization combined with high-performance liquid chromatography. The instrument was LC-2030C 3D HPLC, the chromatographic column was Agilent ZORBAX Eclipse Plus C18 chromatographic column (4.6 x 250 mm, 5 μm), the mobile phase was a mixture of acetonitrile and 0.1 M phosphate buffer at pH 6.7 in a volume ratio of 17:83, the flow rate was 1.0 mL / min, the detector was DAD, the detection wavelength was 245 nm, and the column temperature was 30°C.

[0050] The functional groups of the polysaccharide samples were preliminarily analyzed using infrared spectroscopy. 1-2 mg of fish swim bladder polysaccharide and KBr powder were thoroughly dried, mixed, ground under an infrared lamp, and pressed into transparent thin sheets using a tablet press. The sheets were then placed in a Nicolet iS50 FT-IR spectrometer and analyzed at 4000–4000 cm⁻¹. -1 Scan within the range.

[0051] Results: The purified Atlantic cod swim bladder CS / DS hybrid chain (GMG-1.0) and spotted barracuda swim bladder CS / DS hybrid chain (SJG-1.0) showed single chromatographic peaks by HPLC analysis, with calculated peak molecular weights of 123.13 kDa and 109.32 kDa, respectively. Figure 1 The uronic acid content of GMG-1.0 and SJG-1.0 was 29.49% and 35.04% respectively, as determined by the m-hydroxybiphenyl method; the sulfate ester content of GMG-1.0 and SJG-1.0 was 23.04% and 35.04% respectively, as determined by the barium chloride gelatin method; the molar ratio of sulfate and carboxylic acid in GMG-1.0 and SJG-1.0 was 1.56 and 1.16 respectively, and the sulfate ester content was calculated to be 22.4% and 28.8% respectively, as determined by the conductivity method; no protein was detected.

[0052] The results of the monosaccharide composition determination are as follows: Figure 2 As shown. Comparison with standard monosaccharide elution times revealed that the CS / DS hybrid chains of Atlantic cod and spotted gudgeon swim bladders both contain iduronic acid (IdoA), glucuronic acid (GlcA), N-acetylglucosamine (GalNAc), and N-acetylglucosamine (GlcNAc). Based on the standard curve, the molar ratios of their monosaccharide composition, IdoA:GalNAc:GlcA:GlcNAc, were calculated to be 36.43:26.28:3.95:1.00 and 19.69:22.76:12.48:1.00, respectively.

[0053] Infrared spectra of the CS / DS hybrid chains in the swim bladders of Atlantic cod and spotted wrasse, as shown in... Figure 3 As shown, at 3435cm -1 A strong absorption peak appears at 2943 cm⁻¹, which is due to the stretching vibration of OH groups; the absorption band is at 2943 cm⁻¹. -1 The absorption peak is caused by the stretching vibration of CH; 1643 cm⁻¹ -1 and 1418cm -1 The absorption peak is generated by the C=O asymmetric stretching vibration and the CH bending vibration; at 1260 cm⁻¹ -1 and 843cm -1 There are obvious absorption peaks nearby, which are the tensile vibrations of sulfate S=O and the bending vibrations of COS.

[0054] Example 3: Structural analysis of the CS / DS hybrid chain in fish swim bladder

[0055] The disaccharide composition of the CS / DS hybrid chain of swim bladder was determined by enzymolysis and disaccharide composition analysis. 2.5 mg of swim bladder CS / DS hybrid chain sample was weighed, 0.25 mL of pure water was added to prepare a sample solution of 10 mg / mL, 100 μL was taken into a 2 mL centrifuge tube, 800 μL of Tris buffer and 100 μL (1 mU / μL) of chondroitinase ABC were added in sequence, and 37°C water bath enzymolysis was carried out for 1 h. After the reaction was completed, the enzyme activity was inactivated by boiling water bath for 5 min, the supernatant was obtained by centrifugation to obtain the enzymolysis disaccharide sample, which was filtered through a 0.22 μm filter membrane and analyzed by HPLC. Chromatographic column: Welch XB-SAX (4.6 x 250 mm, 3 μm) strong anion exchange column; mobile phase: A: 2 mM sodium dihydrogen phosphate (pH 3.0), D: 2 mM sodium dihydrogen phosphate containing 1.2 M sodium perchlorate (pH 3.0); column temperature: 40°C; flow rate: 0.6 mL / min; detection wavelength: 232 nm.

[0056] The exact structure of the CS / DS hybrid chain of swim bladder was determined by nuclear magnetic resonance instrument. The swim bladder CS / DS hybrid chain was dissolved in D2O, repeated freeze-drying for 3 times, the fully dried swim bladder polysaccharide sample was dissolved in 0.5 mL D2O, and the exact structure of the swim bladder CS / DS hybrid chain such as the connection mode of various sugar residues and the substitution site of sulfate group was analyzed by one-dimensional NMR spectrum 1 H and 13 C) and two-dimensional NMR spectrum (COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY, HMBC).

[0057] Results: The swim bladder GMG-1.0 was analyzed after chondroitinase ABC enzymolysis as shown in Figure 4 The comparison with the disaccharide standard showed that the GMG-1.0 mainly contained ΔDi4S unit, also contained a small amount of ΔDi4,6diS, ΔDi2,4diS, ΔDi0S unit, in addition, there were very small amounts of ΔUA-2S, ΔDi2,6diS and ΔDi2,4,6triS units. Among them, the main disaccharide composition was ΔDi4S, accounting for about 87.25% (Table 1).

[0058] The swim bladder SJG-1.0 was analyzed after chondroitinase ABC enzymolysis as shown in Figure 4 and Table 1. The SJG-1.0 mainly contained ΔDi4S, accounting for 78.61%, followed by ΔDi6S (8.96%), ΔDi0S (7.56%), ΔDi2,4diS (3.21%), also contained very small amounts of ΔDi4,6diS and ΔDi2,6diS.

[0059] Table 1 Disaccharide composition of GMG-1.0 and SJG-1.0

[0060]

[0061] The NMR spectra of the CS / DS hybrid chains GMG-1.0 and SJG-1.0 from Atlantic cod and striped bonito swim bladder are shown in Figures 5-10 Table 2 and Table 3. Based on the results of monosaccharide composition analysis, disaccharide composition analysis and NMR spectra analysis, the chemical structures of GMG-1.0 and SJG-1.0 are shown as follows.

[0062]

[0063] Table 2. NMR chemical shift assignment results of GMG-1.0

[0064]

[0065] Note: bold represents the connection site; underlined represents the sulfate group connection site.

[0066] Table 3. NMR chemical shift assignment results of SJG-1.0

[0067]

[0068] Note: bold represents the connection site; underlined represents the sulfate group connection site.

[0069] Example 4: Method for determining the anticoagulant activity of swim bladder polysaccharide:

[0070] APTT determination: The colorimetric cup was preheated at 37.0℃, and 5μL of the sample to be tested or negative control Tris-HCl buffer, 45μL of normal coagulation quality control plasma were added, and incubated at 37.0℃ for 2min; then 50μL of APTT reagent preheated at 37.0℃ was added, and incubated at 37.0℃ for 3min, 50μL of 0.02M CaCl2 preheated at 37.0℃ was added to the reaction solution, and the timing was started, and the blood clotting time was recorded.

[0071] PT determination: After the detection tube was preheated at 37.0℃, 5μL of the sample to be tested or Tris-HCl buffer, 45μL of normal coagulation quality control plasma were added, and incubated at 37.0℃ for 2min. The PT reagent was preheated at 37.0℃, and 100μL was added to the reaction solution, and the clotting time was recorded.

[0072] TT assay: The colorimetric cup was preheated at 37.0℃, and the sample solution or Tris-HCl buffer solution 10 μL was added into the preheated colorimetric cup, followed by normal coagulation quality control plasma 90 μL. After incubation at 37.0℃ for 2 min, TT reagent 50 μL preheated at 37.0℃ was added, and the coagulation time was recorded.

[0073] Sample inhibition of endogenous FXase activity detection:

[0074] The sample inhibition of FXase activity was determined by using FVIII:C kit. A 96-well plate was taken, and sample solution 30 μL of gradient concentration, FVIII solution (2 IU / mL) 30 μL, and R2 solution 30 μL were sequentially added. After incubation at 37.0℃ for 2 min, R1 solution 30 μL was added to the reaction solution, and incubation was carried out at 37.0℃ for 1 min. R3 solution 30 μL preheated at 37.0℃ was added to the reaction solution, and the absorbance at 405 nm was continuously read after shaking. Tris-HCl buffer solution was used as a blank control well.

[0075] Sample HC-II-dependent antithrombin activity detection:

[0076] A 96-well plate was taken, and polysaccharide sample solution or negative control Tris-HCl buffer solution 30 μL of gradient concentration was added, followed by HC-II solution 30 μL. The enzyme reader was shaken and mixed, and incubation was carried out at 37.0℃ for 1 min. Then, thrombin solution 30 μL of concentration 20 NIH / mL was added, and the mixture was shaken and mixed and incubated at 37.0℃ for 1 min. Finally, thrombin substrate solution 30 μL preheated at 37.0℃ of concentration 2.5 mg / mL was added, and the mixture was shaken and mixed. The absorbance at 405 nm was continuously read every 30 s. The change rate of absorbance (ΔOD405 / min) was used to represent the activity of thrombin.

[0077] Anti-coagulation activity determination results:

[0078] Both of the CS / DS hybrid chains from the swim bladders of Atlantic cod and striped bonito could significantly prolong APTT and TT (Table 4), suggesting that both of the CS / DS hybrid chains had strong anticoagulant activity. The drug concentrations required for doubling APTT by SJG-1.0 and GMG-1.0 were 0.586 and 0.226 μM, respectively. The activity of SJG-1.0 in prolonging APTT was similar to that of low molecular weight heparin (0.571 μM), while the activity of GMG-1.0 in prolonging APTT was stronger than that of low molecular weight heparin. SJG-1.0 had no TT prolonging activity, while the TT prolonging activity of GMG-1.0 was also significantly lower than that of low molecular weight heparin, suggesting that the CS / DS hybrid chains had weak effects on the common coagulation pathway. According to previous reports, the currently used anticoagulants all affect the common coagulation pathway and thus affect hemostasis, and therefore all have a strong bleeding tendency. The CS / DS hybrid chains have weak effects on the common coagulation pathway, and thus are likely to have a lower bleeding tendency. The EC50 value of GMG-1.0 for inhibiting FXase was 0.058 μM, which was weaker than the activity of low molecular weight heparin, but GMG-1.0 still showed strong FXase inhibitory activity. Both SJG-1.0 and GMG-1.0 showed strong HC-II-dependent anti-FIIa activity, with EC50 values of 20.410 and 6.225 nM, respectively. According to previous studies, anticoagulants that depend on HC-II to inhibit FIIa have the advantage of a low bleeding tendency. Therefore, SJG-1.0 and GMG-1.0 are promising as new anticoagulants. 50 50

[0079] Table 4. Effects of the CS / DS hybrid chains from the swim bladders of Atlantic cod and striped bonito on APTT, TT, endogenous FXase, and factor IIa

[0080] Sample APTT, μM TT, μM anti-tenase, μM anti-IIa (by HC-II), nM LMWH 0.571±0.003 0.658±0.043 0.010±0.001 4.547±0.245 SJG-1.0 0.586±0.001 / / 20.410±5.357 GMG-1.0 0.226±0.016 12.035±0.404 0.058±0.015 6.225±0.657

[0081] Note: " / " represents no activity.

[0082] Example 5: Preparation of fish swim bladder CS / DS hybrid chain sodium salt, potassium salt, calcium salt, and freeze-dried products thereof

[0083] ① Preparation of fish swim bladder CS / DS hybrid chain sodium salt, potassium salt, and calcium salt: 5 g of each of SJG-1.0 and GMG-1.0 was dissolved in 50 mL of deionized water, and the sample solution was exchanged to the hydrogen form by passing through a 732 hydrogen form strong acid cation exchange resin (5.0 cm x 40 cm) at a flow rate of 1 mL / min. The eluate was collected, and 1 M NaOH, KOH, or Ca(OH)2 was added dropwise to the sample solution until the pH of the sample solution was 7.0.

[0084] ​​The fish swim bladder CS / DS hybrid chain SJG-1.0 and GMG-1.0 sodium salt samples each 10 g are weighed, 200 mL of water for injection is added to dissolve, the heat source is removed by filtering through a 22 μm microporous filter, then 2 mL of a Westlin bottle is used for sub-packaging, 0.5 mL per bottle, semi-tight plug, placed in a vacuum freeze dryer, set to -40°C, keep for 3 h, then vacuum to 25 Pa, keep for 6 h, then increase the temperature to 10°C, continue to vacuum, reduce the vacuum degree to the lowest value of the instrument, keep for 24 h, freeze dry, plug, cap.

[0085] Therefore, the fish swim bladder CS / DS hybrid chain and its preparation method and application can be used as potential FXase and HC-II inhibitors to prepare drugs for treating thrombotic cardiovascular diseases.

[0086] 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 the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing a fish swim bladder chondroitin-sulfate dermatan sulfate hybrid chain extract having anticoagulant activity, characterized by, The method comprises the following steps: S1, enzymatic hydrolysis and alkaline hydrolysis of swim bladder: homogenate liquid or dry powder of swim bladder is added into protease for enzymatic hydrolysis, and the suspension of enzymatic hydrolysis is added into inorganic alkali with a final concentration of 0.2-1.0 M for alkaline hydrolysis; S2, isoelectric point protein removal, salt precipitation and alcohol precipitation to obtain swim bladder crude polysaccharide extract, taking swim bladder crude polysaccharide extract, using anion exchange column chromatography purification, eluted with pure water, 0.5 M, 1.0 M, 2.0 M, 3.0 M NaCl gradient, collecting 1.0 M NaCl solution elution component, repeated alcohol precipitation, concentration and freeze-drying to obtain swim bladder chondroitin sulfate-dermatan sulfate hybrid chain extract, the source of the swim bladder is Atlantic cod; the swim bladder chondroitin sulfate-dermatan sulfate hybrid chain extract is composed of iduronic acid, N-acetylgalactosamine, glucuronic acid and N-acetylglucosamine four monosaccharides; the swim bladder chondroitin sulfate-dermatan sulfate hybrid chain extract has the following structure: wherein, [→4)-C-α-(1→3)-D-β-(1→] m The disaccharide units account for 87.25% of the total disaccharide content, [→4)-A-α-(1→3)-E-β-(1→] n The disaccharide units account for 5.05% of the total disaccharide content, [→4)-I-β-(1→3)-G-β-(1→] r The disaccharide units account for 1.54% of the total disaccharide content, and the total disaccharide content is the sum of the contents of ΔDi0S, ΔDi4S, ΔUA-2S, ΔDi2,6diS, ΔDi4,6diS, ΔDi2,4diS and ΔDi2,4,6triS.

2. The production method according to claim 1, characterized by, The swim bladder chondroitin sulfate-dermatan sulfate hybrid chain extract has a right dextran standard weight average molecular weight of 80-150 kDa, wherein the protein content is <3%, and the nucleic acid content is <1%.

3. The preparation method according to claim 1, characterized in that, The protease is selected from papain, trypsin or alkaline protease; and the inorganic alkali is selected from NaOH or KOH.

4. A medicine for treating and / or preventing thrombotic cardiovascular diseases, characterized by, The effective component of the drug is the swim bladder chondroitin sulfate-dermatan sulfate hybrid chain extract prepared by the preparation method of any one of claims 1-3.

5. The medicament according to claim 4, characterized in that, The drug dosage form is a freeze-dried powder injection or an aqueous solution for injection.

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