阅读说明：本技术 硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用 (Application of dextran sulfate galacto-oligosaccharide in preparation of anticoagulant and/or antithrombotic drugs ) 是由 张建法 林茜 王磊 于 2021-07-09 设计创作，主要内容包括：本发明公开了一种硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用。所述的硫酸葡半乳寡聚糖的结构式为R-(1)～R-(24)独立的为SO-(3)～(—)或H,但不全为H。本发明的硫酸葡半乳寡聚糖在体内和体外具有明显的抗凝血活性,可用于制备抗凝血和/或抗血栓药物。(The invention discloses an application of dextran sulfate galacto-oligosaccharide in preparation of anticoagulant and/or antithrombotic drugs. The structural formula of the dextran sulfate galacto-oligosaccharide is shown in the specification R 1 ～R 24 Independently is SO 3 — Or H, but not all H. The dextran sulfate galacto-oligosaccharide has obvious anticoagulant activity in vivo and in vitro, and can be used for preparing anticoagulant and/or antithrombotic medicaments.)

1. The application of the dextran sulfate galacto-oligosaccharide in preparing anticoagulant and/or antithrombotic drugs is characterized in that the dextran sulfate galacto-oligosaccharide has the structural formula shown as follows:

wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、R₁₆、R₁₇、R₁₈、R₁₉、R₂₀、R₂₁、R₂₂、R₂₃And R₂₄Independently is SO₃ ^—Or H, but not all H.

2. The use according to claim 1, wherein the antithrombotic agent is an agent for treating acute thromboembolic disorders, or an agent for treating cerebral infarction, formation of atherosclerotic plaques in carotid arteries, pulmonary infarction, myocardial infarction, spleen infarction, formation of mesenteric venous thrombosis, mesenteric arterial embolism, venous thromboembolic disorders, or lower extremity arterial arteriosclerosis obliterans; the anticoagulant is used for treating disseminated intravascular coagulation or is used as an anticoagulant for blood sample samples of blood transfusion, extracorporeal circulation hemodialysis or peritoneal dialysis.

3. The use according to claim 1, wherein the dextran sulfate galacto-oligosaccharide has an average number of sulfate groups per saccharide residue of 0.5 to 3, preferably an average number of sulfate groups per saccharide residue of 0.5 to 2.5, more preferably 2.1 to 2.5.

4. The use of claim 1, wherein the dextran sulfate galacto-oligosaccharide is prepared by sulfating dextran galacto-oligosaccharide with a sulfating agent to produce dextran sulfate galacto-oligosaccharide.

5. Use according to claim 4, characterized in that the sulfating agent is a mixture of pyridine and chlorosulfonic acid.

6. The use of claim 4, wherein the preparation method of the dextran sulfate galacto-oligosaccharide comprises the following specific steps: and (2) taking a mixture of pyridine and chlorosulfonic acid as a sulfating reagent, adding a dimethyl sulfoxide solution of the dextran-galacto-oligosaccharide into the sulfating reagent under stirring, stirring in an oil bath at 50-120 ℃ for reaction, and after the reaction is finished, carrying out post-treatment and purification on the reaction solution to obtain the dextran-galacto-oligosaccharide sulfate.

7. The use of claim 6, wherein the volume ratio of pyridine to chlorosulfonic acid in the mixture of pyridine and chlorosulfonic acid is 3: 1-8: 1, and the ratio of galacto-oligosaccharide to dimethylsulfoxide in a solution of galacto-oligosaccharide is 1: 10-20, g: mL.

8. The use of claim 6, wherein the ratio of galacto-glucan to sulfating agent is 1:20 to 1:60 g/mL.

9. The use according to claim 6, wherein the reaction temperature is 60 ℃ and the reaction time is 2 to 5 hours.

10. Use according to claim 6, characterized in that the work-up and purification process is as follows: adjusting the pH value of the reaction solution to be neutral by using 15% NaOH solution, centrifuging at 8000rpm and 4 ℃ to remove insoluble impurities, collecting supernate, dialyzing by using a 500-1000 Da dialysis bag for 5 days, concentrating and desalting by using an organic ultrafiltration membrane, and performing freeze-drying or ethanol precipitation to obtain the dextran sulfate galacto-oligosaccharide.

Technical Field

The invention belongs to the field of anticoagulant and antithrombotic drugs, and relates to application of dextran sulfate galacto-oligosaccharide in preparation of anticoagulant and/or antithrombotic drugs.

Background

Coagulation refers to the process of changing blood from a fluid state to a gel state which is not fluid, and is essentially the process of converting water-soluble fibrinogen into water-insoluble solid fibrin, which is the process of producing prothrombin activator by the intrinsic and/or extrinsic coagulation pathways, producing thrombin by the action of coagulation factors, and finally converting fibrinogen into fibrin by the action of thrombin.

Thrombi are small patches of blood flow that form on the surface of a denuded or repaired site within a blood vessel of the cardiovascular system. Thrombosis involves local blood clotting in the vascular system, often leading to serious health-related diseases such as heart attack and stroke. Risk factors for thrombosis include abnormal hyperlipidemia, hyperglycemia, and elevated plasma fibrinogen.

Heparin is the most widely used anticoagulant drug and has been used in clinical applications for over 90 years. Heparin is a mucopolysaccharide extracted from intestinal mucosa or liver of animals such as pigs, sheep, cattle and the like, which causes waste of animal resources to a certain extent. Although heparin has a function of preventing and treating thrombus, heparin also has side effects such as easiness in causing spontaneous hemorrhage, various mucosal hemorrhage, joint hemorrhage, wound hemorrhage and the like, and after heparin is injected, the content of blood platelets in blood is greatly reduced, so that the bone mineral density is reduced after the heparin is used for a long time, osteoporosis is caused, and the fracture risk is increased. Low molecular weight heparins are prepared from heparin by controlled depolymerization. Compared with common heparin, the low molecular weight heparin has higher availability and longer half-life period, and becomes the first choice anticoagulant for treating deep venous thrombosis. However, low molecular weight heparin is derived from animal tissue by nature and its anticoagulation is not easily reversible, resulting in the risk of overdose of blood. The ultra-low molecular weight heparin is mainly a chemically synthesized heparinoid, has good pharmacokinetic control and is free from virus impurity pollution. However, these drugs are complicated in chemical synthesis, very expensive, and their anticoagulation effect is not easily reversible. There is therefore a great clinical need to find new antithrombotic and anticoagulant agents with fewer side effects than heparin.

Previous studies by the inventors reported that a galactoglucan and a method for preparing the same have certain properties of molecular weight, low viscosity and good water solubility, and indicate that the galactoglucan has a significant improvement in the biological activity of the Gut microflora as a prebiotic (Wang, L., Cheng, R., Sun, X., et al., Preparation and guide microbial Property of the Oligosaccharide Ricini 31; 69(12): 3667-3676.). International patent application WO2019090203a1 discloses a sulfated heparan sulfate oligosaccharide compound having anti-inflammatory activity, but which does not have anticoagulant activity. The above results indicate that the oligosaccharide after sulfation modification does not necessarily have anticoagulant and antithrombotic activity.

Disclosure of Invention

The invention aims to provide application of dextran sulfate galacto-oligosaccharide in preparation of anticoagulant and/or antithrombotic drugs.

The dextran sulfate galacto-oligosaccharide has good activity of inhibiting thrombin generated by blood coagulation factors, and can be used as an anticoagulant or a medicament for treating or preventing antithrombotic.

The anticoagulant and/or antithrombotic drug provided by the invention is a conventional anticoagulant and/or antithrombotic drug, and specifically can be a drug for treating acute thromboembolic diseases, or a drug for treating cerebral infarction, formation of carotid atherosclerotic plaque, pulmonary infarction, myocardial infarction, spleen infarction, formation of mesenteric venous thrombosis, mesenteric arterial embolism, venous thromboembolic diseases and lower limb arterial sclerosis, or a drug for treating disseminated intravascular coagulation, or an in vitro anticoagulant, such as an anticoagulant for in vitro experiments such as blood transfusion, extracorporeal circulation hemodialysis, blood sample of peritoneal dialysis and the like.

In the invention, the structural formula of the dextran sulfate galacto-oligosaccharide is shown as follows:

wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、R₁₆、R₁₇、R₁₈、R₁₉、R₂₀、R₂₁、R₂₂、R₂₃And R₂₄Independently is SO₃ ^—Or H, but not all H.

In the dextran sulfate galacto-oligosaccharide, the average number of sulfate groups on each sugar residue is 0.5-3. The larger the number of sulfate groups per saccharide residue on average, the higher the inhibition rate of FXIa. In a specific embodiment of the present invention, the number of sulfate groups per saccharide residue is 0.5 to 2.5, more preferably 2.1 to 2.5, on average.

The dextran sulfate galacto-oligosaccharide contains 8 sugar units, including 7 glucose residues and 1 galactose residue. The 1 st glucose residue is linked to a pyruvate group, the 8 th saccharide unit is a galactose residue, and the specific linkage of the 7 glucose residues (Glcp) and the 1 galactose residue (Galp) is as follows:

D-Glcp(4,6-pyr)-β-(1→3)-D-Glcp-β-(1→3)-D-Glcp-β-(1→6)-D-Glcp-β-(1→6)-D-Glcp-β-(1→4)-D-Glcp-β-(1→4)-D-Glcp-β-(1→3)-D-Galp。

the invention also provides a preparation method of the dextran sulfate galacto-oligosaccharide, which is characterized in that the dextran sulfate galacto-oligosaccharide is sulfated by utilizing a sulfating reagent to prepare the dextran sulfate galacto-oligosaccharide.

In the invention, the sulfation degree of the galacto-oligosaccharide can be controlled by controlling the proportion of the galacto-oligosaccharide and the sulfating reagent, the reaction temperature and the reaction time, namely the number of sulfate groups on each sugar residue is controlled on average, and the galacto-oligosaccharide substituted by sulfate groups with different degrees is obtained.

In the present invention, the sulfating agent is a conventionally used sulfating agent in the art, and any agent may be used as long as it can sulfate the galactoglucan. In a particular embodiment of the invention, the sulfating agent used is a mixture of pyridine and chlorosulfonic acid.

In the specific embodiment of the invention, the adopted preparation method of the dextran sulfate galacto-oligosaccharide comprises the following specific steps: taking a mixture of pyridine and chlorosulfonic acid as a sulfating reagent, adding a dimethyl sulfoxide (DMSO) solution of the dextran-galacto-oligosaccharide into the sulfating reagent under stirring, stirring and reacting in an oil bath at 50-120 ℃, and after the reaction is finished, performing post-treatment and purification on a reaction solution to obtain the dextran-galacto-oligosaccharide sulfate.

Preferably, in the mixture of pyridine and chlorosulfonic acid, the volume ratio of pyridine to chlorosulfonic acid is 3: 1-8: 1.

Preferably, the ratio of the galacto-oligosaccharide to the dimethyl sulfoxide in the solution of the galacto-oligosaccharide is 1: 10-20, and g: mL.

Preferably, the ratio of the galacto-glucan to the sulfating agent is 1: 20-1: 60, g: mL.

Preferably, the reaction temperature is 60 ℃.

Preferably, the reaction time is 2 to 5 hours.

Preferably, the work-up and purification process is as follows: adjusting the pH value of the reaction solution to be neutral by using 15% NaOH solution, centrifuging at 8000rpm and 4 ℃ to remove insoluble impurities, collecting supernate, dialyzing by using a 500-1000 Da dialysis bag for 5 days, concentrating and desalting by using an organic ultrafiltration membrane, and performing freeze-drying or ethanol precipitation to obtain the dextran sulfate galacto-oligosaccharide.

Compared with the prior art, the invention has the following advantages:

the invention discovers that the dextran sulfate galacto-oligosaccharide has obvious in-vitro anticoagulation activity for the first time, can selectively inhibit coagulation factor XIa (FXIa), has small bleeding risk, and is suitable for preparing in-vitro anticoagulation medicines; meanwhile, in a mouse model, the dextran sulfate galacto-oligosaccharide shows the function of inhibiting thrombosis and can be used for treating or preventing thrombosis. The dextran sulfate galacto-oligosaccharide is safe and non-toxic, has strong drug effect, and has wide application prospect in anticoagulant and/or antithrombotic drugs.

Drawings

FIG. 1 is a graph showing the results of FXIa inhibitory activity of dextran galacto-oligosaccharide sulfate, which includes the FXIa inhibitory activity of dextran galacto-oligosaccharide and dextran galacto-oligosaccharide sulfate.

FIG. 2 is a graph showing the results of the effect of dextran sulfate galacto-oligosaccharide on APTT, including the effect of three different concentrations of dextran sulfate galacto-oligosaccharide on plasma clotting time.

FIG. 3 shows inhibition of FeCl by dextran sulfate galacto-oligosaccharide₃Graph showing the results of induced arterial thrombosis, wherein (a) group was the thrombosis of mice injected with physiological saline, (b) group was the thrombosis of mice injected with 0.5mg/mL of dextran sulfate galacto-oligosaccharide, and (c) group was the thrombosis of mice injected with 1.0mg/mL of dextran sulfate galacto-oligosaccharide.

Fig. 4 is a graph showing the effect of dextran sulfate galacto-oligosaccharide on bleeding time of mouse tail, which includes the effect of two different concentrations of dextran sulfate galacto-oligosaccharide on bleeding of mouse tail tip.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, the reagents or materials used in the following examples may be synthesized by commercially available methods or by reference to existing methods.

The galacto-glucan of the present invention is prepared by conventional methods known in the art. Specifically, it may be prepared according to the method taught in Wang, l., Cheng, r., Sun, x. et al, j.agric.food chem.,69, pp.3667-3676 (2021).

In the present invention, the term "and/or" means individually or in combination.

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

Example 1

Preparation of dextran sulfate galacto-oligosaccharide:

weighing 1.0g of a galacto-glucan raw material, transferring the raw material into a conical flask, adding 10-20 mL of DMSO, and stirring to dissolve the DMSO to obtain a galacto-glucan oligosaccharide solution. Mixing pyridine and chlorosulfonic acid in a volume ratio of 3:1 in a three-necked flask, and magnetically stirring in an ice-water bath for 30min to obtain a sulfating agent. The above solution of galacto-oligosaccharides was gradually added to 30mL of sulfating agent under stirring, the condenser tube was closed and two additional caps were stoppered, and the three necked flask was placed in a 60 ℃ oil bath and stirred for reaction for 3 hours. Naturally cooling to room temperature, and stopping the reaction. Adjusting the pH value of the solution to be neutral by using 15% NaOH solution, centrifuging at 8000rpm and 4 ℃ to remove insoluble impurities, collecting supernatant, dialyzing by using a 500-1000 Da dialysis bag, concentrating and desalting by using an organic ultrafiltration membrane after dialyzing for 5 days, and performing freeze-drying or ethanol precipitation to obtain the dextran sulfate galactooligosaccharide with the average number of sulfate groups on each sugar residue of 1.65.

According to the preparation method, the galacto-dextran sulfate oligosaccharide and the sulfating reagent are respectively subjected to sulfation reactions according to mass-volume ratios of 1:20, 1:30, 1:45 and 1:60, so that the galacto-dextran sulfate oligosaccharide with substitution degrees (namely the average number of sulfate groups on each sugar residue) of 1.23, 1.65, 2.10 and 2.52 is respectively obtained.

Example 2

Measurement of sulfated substitution degree (Ds) of dextran sulfate galactooligosaccharide.

10mg/mL of dextran sulfate galacto-oligosaccharide solution is prepared, 200 mu L of the dextran sulfate galacto-oligosaccharide solution is put into a glass bottle, equal volume of 4M HCl is added, the complete acid hydrolysis is carried out for 8h at 100 ℃, and after the complete acid hydrolysis is carried out by nitrogen drying, 100 mu L of pure water is used for redissolving. Preparing 0.5% gelatin solution, and adding 0.5% BaCl₂. 100 μ L of the dextran sulfate galacto-oligosaccharide solution and an equal volume of BaCl were taken₂Gelatin solution, OD measured at 360 nm. The degree of substitution is calculated by the following formula:

the dosage of the sulfating agent, the reaction time, the reaction temperature and other conditions can affect the degree of substitution of the sulfation. The sulfating agent is used in a reduced dose and the degree of sulfation substitution is also reduced.

In examples 3 to 6, the employed dextran sulfate galactooligosaccharides were all dextran sulfate galactooligosaccharides having an average number of sulfate groups per saccharide residue of 1.65, unless otherwise specified.

Example 3

And (3) measuring the inhibitory activity of the dextran sulfate galacto-oligosaccharide on the FXIa coagulation factor.

1. Grouping experiments:

the experiment was divided into three groups: the first group was a negative control group (TBS-BSA buffer), a galacto-dextran group and a galacto-dextran sulfate group.

2. Determination of inhibitory Activity

mu.L of HFXIa (human coagulation factor XIa,1nM) and 50. mu.L of a dextran sulfate galactooligosaccharide sample (0-500 nM) were mixed well in a 96-well plate, incubated at 37 ℃ for 1h, added with 50. mu.L of a chromogenic substrate (1mM), and detected at 405nM for 1h, once per minute. The galacto-glucan panel was 100 μ LHFXIa +50 μ L galacto-glucan sample +50 μ L substrate; the negative control group was 100. mu.L of LHFXIa + 50. mu.L of TBS-BSA buffer + 50. mu.L of substrate.

3. Data analysis

Data processing was performed using the software Graphpad Prism 6.0. Regression analysis was performed using the reaction time as abscissa and the absorbance value at 405nm as ordinate.

And (3) detecting the FXIa inhibition activity of the dextran sulfate galactooligosaccharide sample and the dextran galactooligosaccharide by adopting a chromogenic substrate method. The inhibition ratio was calculated as (V0-Vi)/V0 × 100% by taking the slope of the curve as the speed V of the enzymatic reaction, the speed V0 of the negative control reaction, and the speed Vi of the sample reaction. As can be seen from FIG. 1, the inhibition ratio of FXIa by the galacto-dextran sulfate having an average number of sulfate groups of 1.65 per saccharide residue was 57.6% and the inhibition ratio of FXIa by the galacto-dextran sulfate was 9.1%, indicating that the galacto-dextran sulfate has an inhibitory activity against FXIa and that the galacto-dextran sulfate has no inhibitory activity against FXIa. By controlling the ratio of the galacto-glucan to the sulfating agent to control the degree of sulfation of the galacto-glucan sulfate, and table 2 shows the inhibition activity of the galacto-glucan sulfate to FXIa at different degrees of sulfation, it can be seen that the higher the number of sulfate groups per saccharide residue on average, the higher the inhibition rate of FXIa by the galacto-glucan sulfate.

Similarly, the inhibitory activity of the dextran sulfate galacto-oligosaccharide on other enzymes was determined by a chromogenic substrate method to determine whether the dextran sulfate galacto-oligosaccharide has selective specificity for FXIa. The combination of table 1 shows that the dextran sulfate galacto-oligosaccharide has selective specificity to FXIa and no inhibitory activity to other coagulation factors.

TABLE 1 inhibition of different coagulation factors by dextran sulfate galacto-oligosaccharide

Table 2 shows the inhibition rate of FXIa by dextran sulfate galacto-oligosaccharide with different degrees of sulfation

Example 4

The effect of dextran sulfate galacto-oligosaccharide on clotting time.

1. Establishing a mouse model:

the experimental animal is a male C57/BL-6J mouse, and 18-20 g. Feeding under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, and the tail vein administration was confirmed to be complete anesthesia and no eyelid reflex. Randomly dividing mice into three groups, wherein the first group is a negative control group, and injecting normal saline into tail veins; the second group is a low dose group, and 0.25mg/mL of dextran sulfate galacto-oligosaccharide is injected into tail vein; the third group is a medium dose group, and 0.5mg/mL of dextran sulfate galacto-oligosaccharide is injected into tail vein; the fourth group was a high dose group, which was administered 1.0mg/mL dextran sulfate galacto-oligosaccharide by tail vein injection. Blood was collected from the abdominal aorta of mice in silanized tubes moistened with anticoagulant and incubated at 37 ℃.

2. Determination of ATPP

Activated partial thromboplastin time (ATPP) is the time required for observing the coagulation of plasma after adding an activated partial thromboplastin time reagent and calcium ions into the plasma to be detected, and mainly reflects the condition of an endogenous coagulation system. The whole blood was collected, diluted appropriately, centrifuged at 1080rpm for 10min, and the upper platelet-rich plasma was removed. Centrifuging at 3000rpm for 5min, and collecting upper layer anemia platelet plasma (PPP) for blood coagulation time determination. The ATPP assay was performed according to the protocol in the kit instructions, reading the fibrin formation time on the hemagglutination instrument.

3. Data analysis

Data processing was performed using software Graphpad Prism 6.0 software. And drawing an APTT line graph by taking the sample concentration as an abscissa and the plasma coagulation time multiple as an ordinate. A significant difference is that P is less than 0.05.

The clotting time of the saline group was recorded as 1-fold, and the APTT of the dextran sulfate galactooligosaccharide at different concentrations was calculated as a fold of the saline group. Referring to FIG. 2, it can be seen that different concentrations of the galacto-dextran sulfate had a significant effect on APTT, and thus the galacto-dextran sulfate acts on the intrinsic coagulation pathway.

Example 5

The in vivo antithrombotic effect of dextran sulfate galacto-oligosaccharide.

1. Establishing an animal model:

the experimental animal is a male C57/BL-6J mouse, and 18-20 g. Feeding under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, and the tail vein administration was confirmed to be complete anesthesia and no eyelid reflex. Randomly dividing mice into three groups, wherein the first group is a negative control group, and injecting normal saline into tail veins; the second group is a low dose group, and 0.5mg/mL of dextran sulfate galacto-oligosaccharide is injected into tail vein; the third group was a high dose group, which was injected tail vein with 1.0mg/mL dextran sulfate galacto-oligosaccharide. Dissecting mouse carotid artery after tail vein administration, applying filter paper (1 × 2mm) dipped with 6.0% ferric trichloride solution on mouse carotid artery outer wall for induction for 3min, and constructing ferric trichloride induced carotid artery thrombosis model.

2. Determination of vascular occlusion time

The blood flow and thrombosis of the carotid artery of the mouse are observed by using a laser speckle blood flow imager, and the carotid artery of the mouse is observed for 15min under a moorFLPI-2 lens. The carotid artery of the mice that ended the observation was carefully dissected and HE stained.

3. Data analysis

Graphs were prepared using GraphPad Prism 6.0 software, and statistical differences were calculated using one-way anova and Dunnett's multiple comparisons. A significant difference is that P is less than 0.05.

The vessel occlusion time is the time from removal of a piece of filter paper soaked with ferric trichloride to occlusion of the vessel. If no thrombosis was observed 15min after removal of the iron trichloride filter paper sheet, 15min was considered as the vessel occlusion time. Referring to FIG. 3, in the negative control group (saline group), the mean vascular occlusion time was 4.5. + -. 0.23 min. The mean vessel occlusion time of the sample dextran sulfate galacto-oligosaccharide is 5.4 plus or minus 1.37min and 15min respectively under the administration dosage of 0.5 and 1.0 mg/mL. The blood flow is kept unchanged under the administration dose of 1.0mg/mL, which indicates that the dextran sulfate galacto-oligosaccharide has obvious antithrombotic effect.

Example 6

Determination of the bleeding risk of dextran sulfate galacto-oligosaccharide.

1. Establishing an animal model:

the experimental animal is a male C57/BL-6J mouse, and 18-20 g. Feeding under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, and the tail vein administration was confirmed to be complete anesthesia and no eyelid reflex. Randomly dividing mice into three groups, wherein the first group is a negative control group, and injecting normal saline into tail veins; the second group is a dose metering group, and 0.5mg/mL of dextran sulfate galacto-oligosaccharide is injected into tail vein; the third group was a high dose group, which was injected tail vein with 1.0mg/mL dextran sulfate galacto-oligosaccharide.

2. Measuring bleeding time of the tail tip:

after administration for 10min, the mice were placed steadily with their tails left in a natural straightened state, and the tails were cut off at a distance of 3mm from the tip, and immersed in silanized tubes containing physiological saline at 37 ℃. Counting time for 20min from the cutting tail tip, taking a stopwatch to record the accumulated bleeding time t from the cutting tail tip of the mouse, and recording the accumulated bleeding time t as 20min if the bleeding time t is more than 20 min.

3. Data analysis

Data processing was performed using software Graphpad Prism 6.0 software. Samples concentrations were plotted as bleeding time. A significant difference is that P is less than 0.05.

Bleeding time refers to the time required from bleeding to natural hemostasis after capillary damage. In the negative control group (physiological saline group), the cumulative bleeding time was 10.35. + -. 5.48 min. The cumulative bleeding time of the sample dextran sulfate galacto-oligosaccharide is 11.88 plus or minus 4.44min and 11.60 plus or minus 4.13min respectively under the administration dosage of 0.5 and 1.0mg/mL, and the difference is not significant in statistics (P is more than 0.05). Referring to fig. 4, it is shown that the dextran sulfate galacto-oligosaccharide has significant antithrombotic activity without significant risk of bleeding.