AU2021101092A4 - Antithrombotic heparinoid extracted from short necked clam, preparation method and use thereof - Google Patents

Abstract

The present disclosure discloses an antithrombotic heparinoid extracted from short necked clam, a preparation method and use thereof. The heparin tetrasaccharide has a structural formula as shown in formula (I) ~<OSO~ 00 OR 0 MXY HO O~ OR, 1 Oil HOO . The preparation method thereof includes the following steps: dissolving a short necked clam meat powder sample in distilled water, adjusting the pH to 7.8-8.2, enzymolyzing the sample with alkaline protease and papain to obtain an enzymatic hydrolysate, inactivating enzymes, centrifuging, and collecting a supernatant; transferring the supernatant into a equilibrated chromatography column packed with macroporous ion exchange resin, where the distilled water is a solvent in the column, and combining and collecting elution fractions of a 1.5 M NaC solution; concentrating collected elution fractions, adding ethanol, leaving the elution fractions to stand, centrifuging, and collecting a precipitate, alternately washing the precipitate with acetone and ethanol, dissolving the precipitate with water, centrifuging to remove insolubles, and repeating the operation 3-4 times; and finally, dialyzing, and freeze-drying to obtain the antithrombotic heparinoid product extracted from short necked clam. The present disclosure has the advantages of low bleeding side effect, mild anticoagulant effect and strong fibrinolysis.

Description

ANTITHROMBOTIC HEPARINOID EXTRACTED FROM SHORT NECKED CLAM, PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

The present disclosure relates to an antithrombotic heparinoid, and in particular to an antithrombotic heparinoid extracted from short necked clam, a preparation method and use thereof.

BACKGROUND

Thrombotic diseases seriously threaten human the life and health, and incidence thereof ranks the highest among various diseases. The incidence of thrombotic diseases has been reduced after the introduction of antithrombotic drugs such as heparin, but compared with malignant tumors, thrombotic diseases are still an important cause of death, and in recent years, there is an increasing trend, becoming one of the focuses and hot spots in contemporary medical research.

Heparin is a glycosaminoglycan that exists in animals and has diverse biological activities. Heparin is widely used clinically, especially as an anticoagulant. At present, there is no product that can completely replace heparin. Therefore, heparin for treating thrombotic diseases mainly depends on powerful anticoagulant effect thereof. At present, most of the heparin used clinically comes from pig small intestine and bovine lung, but heparin derived from porcine small intestines and bovine lungs has too strong anticoagulant effect, which inevitably causes side effects such as bleeding effect and thrombocytopenia. In response to the problem that heparin for treating thrombotic diseases causes bleeding side effects, it is urgent to develop a heparin or heparinoid that does not depend on the anticoagulant effect and exerts antithrombotic functions.

SUMMARY

The technical problem to be solved by the present disclosure is to provide an antithrombotic heparinoid extracted from short necked clam with low bleeding side effect, mild anticoagulant effect and strong fibrinolysis, a preparation method and use thereof.

The technical solutions adopted by the present disclosure to solve the above technical problems are as follows:

An antithrombotic heparinoid extracted from short necked clam is provided, where the heparin tetrasaccharide has the following structural formula as shown in (I):

OS~00' -OR,

H0L_ OH- OR1 J OHj OI

where Ri is SO3- or H, R2 is SO3- and R3 is SO3-; alternatively, R2 is H and R3 is HAC.

A method for preparing the antithrombotic heparinoid is provided, including the following steps:

step 1, raw material treatment: washing raw materials of short necked clams, shelling, homogenizing and freeze-drying shellfish meat to obtain a powder; step 2, enzymolysis: taking a dried short necked clam meat powder sample, dissolving the sample in distilled water in a solid-to-liquid ratio of 1:6 to 1:10, adjusting pH to 7.8-8.2 with NaOH, and enzymolyzing the sample with 0.4%-0.7 %% alkaline protease and 0.4%-0.7 %% papain, by mass of short necked clam meat powder, for 20-25 h at 45-55°C to obtain an enzymatic hydrolysate; step 3, enzyme inactivation and centrifuging: placing the enzymatic hydrolysate in boiling water to inactivate enzymes for 5-15 min, and centrifuging to collect a supernatant; step 4, sample loading: transferring the supernatant to an equilibrated chromatography column packed with AMBERLITE FPA98 CIIon Exchange Resin, where the distilled water is a solvent in the column, and sample flow rate is 5-40 mL/min; step 5, elution: conducting gradient elution with 0 M, 1.0 M, 1.5 M, and 3.5 M NaCl solutions successively at an elution flow rate of 5-15 mL/min, combining and collecting elution fractions of the 1.5 M NaCI solution; step 6, concentration and alcohol precipitation: concentrating collected elution fractions, adding 0.4 times the concentrated solution volume of ethanol, leaving the solution to stand for 20-28 h at 0.5-7.5°C, and centrifuging for 10-20 min at 6,000-8,000 rpm to collect a precipitate; step 7, redissolution: washing the precipitate alternately with acetone and ethanol twice or thrice, dissolving the precipitate with water, centrifuging to remove insolubles, and repeating the operation 3 to 4 times; step 8, desalting: dialyzing a redissolved extract in a dialysis bag for 48-96 h; and step 9, drying: freeze-drying a dialysate to obtain the antithrombotic heparinoid extracted from short necked clam. Use of the above antithrombotic heparinoid in the preparation of antithrombotic products with low bleeding side effect is provided.

Compared with the prior art, the present disclosure has the following advantages: the present disclosure provides an antithrombotic heparinoid extracted from short necked clam, a preparation method and use thereof, where heparinoid with higher purity is separated from the short necked clam, and blood anticoagulation experiments show that the heparinoid has a mild anticoagulant effect; in vivo and in vitro fibrinolysis experiments show that the heparinoid has significant fibrinolytic activity, which is about 3.8 times the that of mammalian-derived commercial heparin; mouse tail amputation experiment shows that the heparinoid has a weak bleeding side effect, and possesses market application value in the preparation of heparinoid with low bleeding side effect or other medical supplies and health products with antithrombotic function.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an ultraviolet spectrum of short necked clam heparin;

FIG. 2 is an cellulose acetate membrane electropherogram of short necked clam heparinoid, where HP is a heparin reference standard, CS is a chondroitin sulfate reference standard, and DS is dermatan sulfate reference standard;

FIG. 3 is a high performance gel permeation chromatogram (HPGPC) of short necked clam heparinoid;

FIG. 4 illustrates a monosaccharide composition of short necked clam heparinoid; note: the numbers in the figure represent: 1, PMP; 2, mannose; 3, rhamnose; 4, glucosamine; 5, glucuronic acid; 6, iduronic acid; 7, N-acetylglucosamine; 8, glucose; 9, galactose; 10, arabinose; and 11, fucose;

FIG. 5 is an infrared spectrum of short necked clam heparinoid;

FIG. 6 is an NMR spectrum of short necked clam heparinoid, where a is1 H NMR, b is1 3 C NMR, c is 1H-1H COSY, d is 1 H- 1 3C HSQC, e is 1H-1H TOCSY, and f is 1H- 1H NOESY;

FIG. 7 is a schematic diagram showing the linear relationship between concentration of short necked clam heparinoid and activated partial thromboplastin time (APTT value);

FIG. 8 is a schematic diagram showing the linear relationship between concentration of short necked clam heparinoid and prothrombin time (PT value);

FIG. 9 is a schematic diagram showing the linear relationship between concentration of short necked clam heparinoid and thrombin time (TT value);

FIG. 10 illustrates the bleeding side effect of short necked clam heparinoid;

FIG. 11 illustrates the in vitro fibrinolytic activity of short necked clam heparinoid. NOTE: (A) The dissolved area produced by urokinase at different concentrations, and the standard curve plotted according to the concentration of urokinase and the size of the fibrinolytic zone; (B) the area of the fibrinolytic zone produced by the sample and HP at a concentration of 12 mg/mL.

DETAILED DESCRIPTION

The present disclosure will be described in further detail below with reference to the accompanying drawings and examples.

Example 1

A method for preparing an antithrombotic heparinoid extracted from short necked clam included the following steps:

step 1, raw material treatment: raw materials of short necked clams were washed, and shelled, and shellfish meat was homogenized and freeze-dried to obtain a powder;

step 2, enzymolysis: 100 g of dried short necked clam powder was dissolved in 8 L of distilled water, and pH was adjusted to 8.0 with NaOH to obtain a feed solution; the feed solution was enzymolyzed with 0.5% 2709 alkaline protease and 0.5% papain, by mass of dried short necked clam powder, for 20 h at 50°C to obtain an enzymatic hydrolysate;

step 3, enzyme inactivation and centrifuging: the enzymatic hydrolysate was placed in boiling water to inactivate enzymes for 10 min, and centrifuged for 20 min at 8,000 rpm to collect a supernatant;

step 4, sample loading: the supernatant was transferred to an equilibrated chromatography column packed with AMBERLITE FPA98 CI Ion Exchange Resin, where the distilled water was a solvent in the column, and sample flow rate was 5-40 mL/min;

step 5, elution: gradient elution was conducted with 0 M, 1.0 M, 1.5 M, and 3.5 M NaCl solutions successively at an elution flow rate of 5-15 mL/min, and 1.5 M fractions were combined and collected;

step 6, concentration and alcohol precipitation: the 1.5 M elution fractions were concentrated, mixed with 0.4 times the concentrated solution volume of ethanol, left to stand for 20-28 h at 0.5 7.5°C, and centrifuged to collect a precipitate;

step 7, redissolution: the precipitate was washed alternately with acetone and ethanol twice or thrice, dissolved with water, and centrifuged to remove insolubles, and the operation was repeated 3 to 4 times; step 8, desalting: a redissolved extract was dialyzed in a dialysis bag for 48-96 h; and step 9, drying: a dialysate was freeze-dried to obtain the antithrombotic heparinoid extracted from short necked clam.

Except for the above example, the solid-to-liquid ratio of distilled water added may also be 1:6, 1:10, or any value within a range of 1:6 to 1:10; the pH value may be 7.8, 8.2, or any value within a range of 7.8 to 8.2; the amount of alkaline protease added may be 0.4%, 0.7%, or any value within a range of 0.4% to 0.7%; the amount of papain added may be 0.4%, 0.7%, or any value within a range of 0.4% to 0.7%; the enzymolysis may be conducted at 45°C, 55°C, or any value within a range of to 55°C, and may last for 20 h, 25 h, or any value within a range of 20 to 25 h; the centrifugal speed may be: 6,000 rpm to 8,000rpm or any value within a range of 6,000 to 8,000 rpm; the centrifugation may last for 10 min, 20 min, or any value within a range of 10 to 20min.

Example 2

Structural characterization of short necked clam heparinoid

1. Ultraviolet (UV) spectroscopy of short necked clam heparinoid

The short necked clam heparinoid prepared in Example 1 was formulated into a 1-3 mg/mL solution with distilled water, the distilled water was used as a zeroing vial, and the sample was subjected to UV spectroscopy on an ultraviolet-visible spectrophotometer. As shown in FIG. 1, the UV spectrum shows that the short necked clam heparinoid has higher purity and contains no protein or nucleic acid.

2. Cellulose acetate membrane electrophoresis of short necked clam heparinoid

3 mg of the short necked clam heparinoid prepared in Example 1 was formulated to 3 mg/mL with distilled water. A cellulose acetate membrane (8 x 2 cm) was immersed in 0.1 mol/L pyridine 0.47 mol/L formic acid (pH 3.0) electrophoresis buffer and taken out after 30 min, and the excess buffer was absorbed with clean filter paper; the sample was loaded on the matte side of the cellulose acetate membrane 1.5 cm away from the cathode end; the loaded membrane was placed on the electrophoresis tank holder, with the smooth side being faced upward; electrophoresis was conducted at 7 mA current for 20 min; after electrophoresis, the membrane was stained in 0.5% Alison blue staining solution (2% acetic acid solution as solvent) for 30 min, and finally destained with 2% aqueous acetic acid for 30 min. As shown in FIG. 2, the cellulose acetate membrane electropherogram shows that the short necked clam heparinoid has similar electrophoretic mobility to heparin.

3. Molecular weight determination of short necked clam heparinoid

High performance gel permeation chromatography (HPGPC) was used to determine the purity and molecular weight of short necked clam heparinoid. Chromatographic conditions were as follows: column: Waters Ultrahydrogel Column 500 (7.8 mm x 300 mm); column temperature: 35°C; mobile phase: 0.2 mol/L sodium sulfate; flow rate: 0.6 mL/min; detector: Agilent 1200 Refractive Index Detector; injection volume: 10 pL.

Heparin reference standards of different molecular weight (3,500, 5,000, 8,000, 15,000, and ,000 Da) and samples were formulated into 5 mg/mL solutions with 0.2 mol/L sodium sulfate, filtered through a 0.22 pm syringe filter and injected. The retention time of the reference standards and samples was recorded for data processing. The results are shown in FIG. 3. The high performance gel permeation chromatogram of short necked clam heparinoid shows a single peak, indicating that the compound has high purity, and the molecular weight of the compound is measured to be 30.99 kDa.

4. Analysis of the monosaccharide composition of short necked clam heparinoid

3.0 mg of the sample prepared in the Example 1 was weighed in a 15 mL sample vial, mixed with 9 mL of 1.5 mol/L trifluoroacetic acid, hydrolyzed in an oven at 110°C for 4-8 h; hydrolysate was blown dry with nitrogen, and the evaporated substance was dissolved in ultrapure water and stored at -20°C for later use. The processed sample and a monosaccharide reference standard were determined by PMP derivatization-reversed-phase high-performance liquid chromatography (HPLC).

Chromatographic conditions for HPLC were as follows: column: ZORBAX Eclipse XDB-C18 column (4.6 x 250 nm, 5 m); mobile phase: phosphate buffer saline (PBS) (0.05 mol/L, pH 6.74)/acetonitrile (V: V = 83:17); flow rate: 1 mL/min; column temperature: 30°C; detection wavelength: 245 nm, UV detector; injection volume: 10 L. The results are shown in FIG. 4. The short necked clam heparinoid is mainly composed of glucosamine, glucuronic acid, and iduronic acid, which is consistent with the main characteristics of the monosaccharide composition of heparin.

5. Infrared spectroscopy of short necked clam heparinoid

After mixing the short necked clam heparinoid prepared in Example 1 and the heparin reference standard with potassium bromide in a 1:100 ratio, using potassium bromide as a blank, infrared spectroscopy was performed in the range of 4,000-400 cm- 1. The results are shown in FIG. 5. In the infrared spectrum, the short necked clam heparinoid is mainly composed of hydroxyl, amino, amide, carboxyl, and sulfate groups; a strong absorption peak with O-S stretching vibration is displayed at 1,240 cm-1, characteristic absorption of heparin is shown at 890 and 940 cm-1 , and absorption bands of the stretching vibration of the COS system in sulfate groups on aminohexose are displayed at 800

850 cm-1, being consistent with the infrared absorption characteristics and functional group characteristics of the heparin.

6. NMR spectroscopy of short necked clam heparinoid

50 mg of the short necked clam heparinoid sample prepared in Example 1 was dissolved in 1 mL of D20. After freeze-drying thrice, ID (H and 13C) and 2D NMR spectra (H-H COSY, 'H-1 3C HSQC,'H-'H TOCSY, and'H-'HNOESY) were measured using anAscend 700M Nuclear Magnetic Resonance Spectrometer at 25°C. The results are shown in FIG. 6,

Analysis of the NMR spectra shows that the short necked clam heparinoid is mainly composed of repeating disaccharides of -- 4)-a-IdoA2S-(1-4)-a-GcNS6S-(1--, ->4)-a-IdoA2S-(1-*4)-a GlcNS3S6S-(--, -*4)-p-GlcA-(1-*4)-a-GlcNS6S-(1--*, and -4)-p-GlcA-(1-*4)-a-GlcNAc(l-*, and the ratio thereof is 1.03: 0.48: 0.76: 0.02. The tetrasaccharide structure of thereof is: -4)-a IdoA2S -(1-*4)-a-GlcNS3S6S(or GlcNS6S)-(1-4)-p-GlcA-(1-*4)-a-GlcNS6S(or GlcNAC)-(1-*, which has not been reported so far. The specific structure is shown in the following structural formula (I):

Oso 0 OR,

HOH

(I), where Ri is S03- or H, R2 is S03- and R3 is S03-; alternatively, R2 is H and R3 is HAC.

Example 3

Anticoagulant activity of short necked clam heparinoid

The effect of the short necked clam heparinoid prepared in Example 1 on three indicators of plasma coagulation (APTT, PT, and TT)

The short necked clam heparinoid and the heparin reference standard were dissolved in normal saline, and formulated into 10 pg/mL, 20 pg/mL, 40 pg/mL, 80 pg/mL, and 500 pg/mL, respectively; with the normal saline as a negative control and heparin reference standard solution as a positive control, APTT, PT, and TT were determined according to the kit instructions, respectively.

1. Determination of activated partial thromboplastin time (APTT value): 20 pL of sample, 0.1 mL of sheep plasma, and 0.1 mL of APTT reagent were added to a test tube, mixed well, incubated in a 37°C water bath for 3 min, and shaken gently; 0.1 mL of 0.025 mol/L calcium chloride solution was added and shaken immediately while starting timing, and the test tube was shaken in the water bath continuously; after about 30 s, the test tube was slowly tilted from time to time; the flow state of the test tube was observed, and the timing was stopped and time was recorded once the liquid did not flow. The results are shown in FIG. 7. From FIG. 7, the short necked clam heparinoid has weaker prolonged effect on plasma APTT than mammalian heparin.

2. Determination of prothrombin time (PT value): PT reagent and sheep plasma were preheated in a 37°C water bath for 3 min; 0.1 mL of plasma and 20 L of sample, followed by 0.2 mL of PT reagent, were added into a preheated test tube, and mixed well while starting the stopwatch; after 8 s, the test tube was removed from time to time to observe the flow state of the plasma. Once the flow stopped, time was recorded, namely the PT value. The results are shown in FIG. 8. From FIG. 8, at low concentrations, there is no significant difference in the prolonged effect of plasma PT between short necked clam heparinoid and mammalian heparin.

3. Determination of thrombin time (TT value): TT reagent and sheep plasma were preheated in a 37°C water bath for 3 min; 0.1 mL of plasma and 20 L of sample, followed by 0.2 mL of TT reagent, were added into a preheated test tube, and mixed well while starting the stopwatch; the test tube was removed from time to time to observe the flow state of the plasma. Once the flow stopped, time was recorded, namely the TT value. The results are shown in FIG. 9. From FIG. 9, the short necked clam heparinoid has slightly weaker prolonged effect on plasma TT than the mammalian heparin.

In summary, the short necked clam heparinoid mainly exerts an anticoagulant effect by prolonging plasma APTT and TT. Compared with the mammalian heparin, the short necked clam heparinoid has a weaker effect of prolonging plasma APTT and TT. At a high concentration (500

[g/ml), the prolonged effect of the short necked clam heparinoid on PT is slightly stronger. According to the analysis of three indicators of blood coagulation, the heparinoid extracted from short necked clam provided by the present disclosure has a milder anticoagulant effect than the mammalian heparin.

Example 4

Bleeding side effect of short necked clam heparinoid

The effect of the prepared short necked clam heparinoid on the tail bleeding time of mice: Mice were divided into 7 groups of 5 mice. The blank group was injected with 10 mL/kg normal saline; the control group was injected with 10 mL/kg heparin reference standard (from National Institutes for Food and Drug Control) solution (20 mg/kg); sample groups were injected with 10 mg/kg sample solutions with concentrations of 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, and 80 mg/kg, respectively. The injection method was tail vein injection. At 30 min after administration, each mouse tail was cut at 3 mm away from the tip of the tail, and the bleeding time of each mouse was recorded. The analysis of bleeding side effect of the short necked clam heparinoid is shown in FIG. 10, and after testing, the heparinoid extracted from short necked clam provided by the present disclosure has a far lower bleeding side effect than the mammalian heparin.

Example 5

The fibrinolytic activity of the short necked clam heparinoid prepared in Example 1

1. Determination of in vitro fibrinolytic activity: The in vitro fibrinolytic activity of the short necked clam heparinoid was determined by the fibrin-agarose plate assay. 0.3 g of agarose was mixed with 20 mL of 0.01 M PBS in an Erlenmeyer flask, heated and dissolved under microwave. After cooling to 55-60°C, 10 mL of fibrinogen solution (0.15%) and 1 mL of thrombin solution (10 U/mL) were quickly added to the flask, mixed well, and transferred to a 9 cm Petri dish. After the flask was left to stand for 1 h at room temperature, wells with a diameter of 3 mm were punched on the solidified plate using a puncher. 20 L each of normal saline, urokinase solution (200 U/mL), sample solutions of different concentrations (1 mg/mL, 6 mg/mL, and 12 mg/mL), and heparin reference standard solution were added to the wells. The Petri dish was incubated in a 37°C constant temperature incubator for 18 h, stained with Coomassie brilliant blue staining solution (0.25% Coomassie brilliant blue R-250, 5% acetic acid, and 4.5% methanol), and destained with destaining solution (45% methanol, 45% distilled water, and 10% acetic acid); the diameter of the fibrinolytic zone was measured by a vernier caliper. A standard curve was plotted according to the area of thefibrinolytic zone produced by urokinase of different activity (5, 10, 20, 40, 80, and 160 U/ml), and the fibrinolytic activity of the sample was calculated quantitatively based on the standard curve and the area of the fibrinolytic zone of the sample. The in vitrofibrinolytic activity of the short necked clam heparinoid is shown in FIG. 11. It has been determined that the in vitrofibrinolytic activity of the short necked clam heparinoid is 1.96 0.11 IU/mg, and that of the mammalian heparin is 0.51 0.02 IU/mg. The in vitro fibrinolytic activity of the short necked clam heparinoid is 3.85 times that of the mammalian heparin.

2. Determination of in vivo fibrinolytic activity: SD rats (220-250 g) were randomly divided into 7 groups (6 rats in each group) and fasted for 24 h before the experiment. The rats were injected intraperitoneally with chloral hydrate (0.15 mL/100 g). After the rats were anesthetized, normal saline, heparin reference standards (1 mg/kg, 6 mg/kg, and 12 mg/kg), and short necked clam heparinoid (1 mg/kg, 6 mg/kg, and 12 mg/kg) were injected through the femoral vein at an injection dose of 0.1 mL/100 g. At 2 h after administration, the rats were fixed in the supine position; the rats were dissected, and blood was collected from the abdominal aorta using vacuum blood collection tubes. The content of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor-1 (PAI-1) in rat blood was determined according to the kit instructions, and the t-PA/PAI-i was calculated.

Table 1 The effects of short necked clam heparinoid on fibrinolytic system in rats Indicator Sample Concentration (mg/kg)

0 1 6 12

t-PA (ng/mL) Heparin 23.86+3.80 46.97+5.07 65.24 + 10.75 65.72+4.23

Short necked clam heparin-like compounds 23.86+3.80 35.64+4.66 41.22 +8.16 62.73+2.66

u-PA (ng/mL) Heparin 1.12+0.06 1.01+0.07 1.23+0.09 1.41+0.14

Short necked clam heparin-like compounds 1.12+0.64 1.22+0.11 1.39+0.24 1.58+0.17

PAI- I(ng/mL) Heparin 12.801 1.57 37.50 + 17.56 33.21 + 11.95 24.05+5.93

Short necked clam heparin-like compounds 12.801 1.57 25.63+6.46 9.46+0.52 6.031 1.74

t-PA/PAI-1 Heparin 1.86+0.07 1.25+0.68 1.96+0.39 2.73+0.31

Short necked clam heparin-like compounds 1.86+0.07 1.39+0.44 4.35+0.62 10.401 1.26

The above results show that the short necked clam heparinoid can significantly promote the release of t-PA and u-PA in rats, and can further inhibit the release of PA-1. Moreover, it can be seen from t-PA/PAI-i that the short necked clam heparinoid has far greater promoting effect on the fibrinolytic system in rats compared with the mammalian heparin. Therefore, the present disclosure has excellent potential application value in the preparation of antithrombotic heparin with low side effects or other medical products with strong antithrombotic function. Unless otherwise specified, the reagents, methods and equipment used in the present disclosure are conventional in the art.

The above description does not limit the present disclosure, and the present disclosure is not limited to the above examples. Variations, modifications, additions, or replacements made by those of ordinary skill in the art within the essential scope of the present disclosure should also fall within the protection scope of the present disclosure.

Claims (3)

What is claimed is:

1. An antithrombotic heparinoid extracted from short necked clam, wherein the heparin tetrasaccharide has the following structural formula as shown in (I):

-01,0 0 7,0 0 HO-N. OH ()So,- O o OR mo 1

wherein Ri is S03- or H, R2 is S03- and R3 is S03-; alternatively, R2 is H and R3 is HAC.

2. A method for preparing the antithrombotic heparinoid according to claim 1, comprising the following steps:

step 1, raw material treatment: washing raw materials of short necked clams, shelling, homogenizing and freeze-drying shellfish meat to obtain a powder;

step 2, enzymolysis: taking a dried short necked clam meat powder sample, dissolving the sample in distilled water in a solid-to-liquid ratio of 1:6 to 1:10, adjusting pH to 7.8-8.2 with NaOH, and enzymolyzing the sample with 0.4%-0.7 %% alkaline protease and 0.4%-0.7 %% papain, by mass of short necked clam meat powder, for 20-25 h at 45-55°C to obtain an enzymatic hydrolysate;

step 3, enzyme inactivation and centrifuging: placing the enzymatic hydrolysate in boiling water to inactivate enzymes for 5-15 min, and centrifuging to collect a supernatant;

step 4, sample loading: transferring the supernatant to an equilibrated chromatography column packed with AMBERLITE FPA98 CI Ion Exchange Resin, wherein the distilled water is a solvent in the column, and sample flow rate is 5-40 mL/min;

step 5, elution: conducting gradient elution with 0 M, 1.0 M, 1.5 M, and 3.5 M NaCl solutions successively at an elution flow rate of 5-15 mL/min, combining and collecting elution fractions of the 1.5 M NaCl solution;

step 6, concentration and alcohol precipitation: concentrating collected elution fractions, adding 0.4 times the concentrated solution volume of ethanol, leaving the solution to stand for 20-28 h at 0.5-7.5°C, and centrifuging for 10-20 min at 6,000-8,000 rpm to collect a precipitate; step 7, redissolution: washing the precipitate alternately with acetone and ethanol twice or thrice, dissolving the precipitate with water, centrifuging to remove insolubles, and repeating the operation 3 to 4 times; step 8, desalting: dialyzing a redissolved extract in a dialysis bag for 48-96 h; and step 9, drying: freeze-drying a dialysate to obtain the antithrombotic heparinoid extracted from short necked clam.

3. Use of the antithrombotic heparinoid according to any one of claim 1 or 2 in the preparation of antithrombotic products with low bleeding side effect.

Short necked clam heparinoid 2021101092

Wavelength (nm)

FIG. 1

Sample

FIG. 2

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FIG. 3

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Reference standard

Sample

FIG. 4

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FIG. 5

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FIG. 6

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FIG. 6 cont’d

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Clotting time (s) Clotting time (s)

FIG. 8 FIG. 7

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Normal saline Heparin Sample (mg/kg)

FIG. 10

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