Composite quick-acting styptic powder as well as preparation method and application thereof

Abstract

The invention discloses composite quick-acting styptic powder. The composite quick-acting styptic powder comprises oxidized konjac glucomannan and hydrazide hyaluronic acid. The invention further provides a preparation method and application of the composite quick-acting styptic powder. The composite quick-acting styptic powder disclosed by the invention has excellent hemostasis and blood coagulation effects.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and particularly relates to a composite fast-acting hemostatic powder and a preparation method and application thereof. Background Art

[0002] Currently, massive bleeding caused by accidental injuries remains one of the key factors contributing to the rapidly increasing mortality rate. For minor, everyday wounds, the body's natural coagulation mechanism effectively stops bleeding naturally and subsequently repairs itself. However, in the case of severe trauma, if uncontrolled bleeding occurs, the patient faces a higher risk of death due to the difficulty of providing first aid on the spot.

[0003] In the research, development, and application of hemostatic materials, conventional hemostatic materials primarily include cotton gauze and tourniquets. Meanwhile, biohemostatic materials, as a new class of hemostatic materials, are attracting widespread attention and in-depth research. Ideal biohemostatic materials should be able to rapidly aggregate blood cells and platelets, accelerating the conversion of fibrinogen to fibrin in the blood, thereby forming a physical barrier and effectively preventing further bleeding.

[0004] Hyaluronic acid is a substance naturally present in human connective tissue. It has the characteristics of high water retention, non-immunogenicity, biodegradability with non-toxic degradation products, promotion of tissue repair, and good adhesion. Konjac gum is a natural ingredient extracted from konjac tubers and has advantages such as high water absorption, biocompatibility, and adhesion. Both have good biocompatibility and biodegradability, making them ideal raw materials for the preparation of hemostatic biomaterials. Currently, the hemostatic effect of single-component konjac gum or hyaluronic acid is relatively limited, and the material strength is not sufficient to withstand the pressure caused by arterial blood impact. Summary of the Invention

[0005] One object of the present invention is to provide a hemostatic material with good hemostatic and coagulation effects in response to the above technical problems.

[0006] Another object of the present invention is to provide a method for preparing the hemostatic material.

[0007] Another object of the present invention is to provide applications of the hemostatic material.

[0008] In order to achieve the above objectives, the present invention provides a composite fast-acting hemostatic powder, which comprises oxidized konjac gum and hydrazide-modified hyaluronic acid.

[0009] According to the composite fast-acting hemostatic powder of the present invention, preferably, the mass ratio of oxidized konjac gum to hydrazide hyaluronic acid is (1-3):(1-3). More preferably, the mass ratio of oxidized konjac gum to hydrazide hyaluronic acid is 1:1 to 3:1, and most preferably 2:1.

[0010] According to the composite fast-acting hemostatic powder of the present invention, preferably, the oxidation degree of the oxidized konjac gum is 20%-30%.

[0011] According to the composite fast-acting hemostatic powder of the present invention, preferably, the degree of hydrazide of the hydrazide hyaluronic acid is 15%-30%.

[0012] According to the composite fast-acting hemostatic powder of the present invention, preferably, the molecular weight of the hyaluronic acid in the fast-acting hemostatic powder is 150,000-200,000.

[0013] According to the composite fast-acting hemostatic powder of the present invention, preferably, the oxidized konjac gum is prepared by the following steps: The konjac gum is oxidized by using sodium periodate, and then dialyzed and freeze-dried to obtain the oxidized konjac gum.

[0014] According to the composite fast-acting hemostatic powder of the present invention, preferably, the hydrazide hyaluronic acid is prepared by the following steps: Hyaluronic acid is modified with adipic acid dihydrazide, and then dialyzed and freeze-dried to obtain the hydrazide hyaluronic acid.

[0015] On the other hand, the present invention also provides a method for preparing the composite fast-acting hemostatic powder, which comprises the following steps: Oxidized konjac gum and hydrazide hyaluronic acid are frozen in liquid nitrogen respectively and then ground into powder. The two powders are then mixed evenly according to a certain proportion and fully ground to obtain the composite fast-acting hemostatic powder.

[0016] On the other hand, the present invention also provides use of the composite fast-acting hemostatic powder for hemostasis.

[0017] Compared with the existing technology, the advantages of the present invention are as follows: the raw materials are natural polymer materials, the biocompatibility is good, the preparation steps are simple, and it has a more significant hemostatic effect than the same type of hemostatic products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The graph shows the comparison of the clotting time of the composite fast-acting hemostatic powder of the present invention and each individual powder.

[0019] Figure 2 The figure shows the aggregation of blood cells by the composite fast-acting hemostatic powder of the present invention.

[0020] Figure 3 The results show the survival of live and dead stained cells of the composite fast-acting hemostatic powder of the present invention.

[0021] Figure 4 The results show that the composite fast-acting hemostatic powder of the present invention can stop bleeding in the liver of BALB / C mice.

[0022] Figure 5 The results show that the composite fast-acting hemostatic powder of the present invention can stop bleeding in the abdominal aorta of BALB / C mice.

[0023] Figure 6 The results show that the composite quick-acting hemostatic powder of the present invention can stop bleeding in the liver of SD rats.

[0024] Figure 7 The results show that the composite fast-acting hemostatic powder of the present invention can stop bleeding in the abdominal aorta of SD rats.

[0025] Figure 8 The results show that the composite fast-acting hemostatic powder of the present invention can stop bleeding in the iliac artery of SD rats. DETAILED DESCRIPTION

[0026] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of patent protection of the present invention.

[0027] Unless otherwise specified, the reagents, methods and equipment used in the practice of the present invention are conventional reagents, methods and equipment in the technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0029] The invention provides a novel biological hemostatic material, which comprises oxidized konjac gum and hydrazide hyaluronic acid.

[0030] In the present invention, oxidized konjac glucomannan is oxidatively modified konjac glucomannan, is obtained by oxidizing the ortho-hydroxyl group of konjac glucomannan to aldehyde group.The oxidation degree of oxidized konjac glucomannan is preferably 20~60%.For example, the oxidation degree of oxidized konjac glucomannan can be 20%, 30%, 40%, 50%, 60% etc.

[0031] In the present invention, the preparation method of oxidized konjac glucomannan preferably comprises the following steps: konjac glucomannan is dissolved in the solution configured to 1~5wt% in water, oxidant is added under lucifuge, the concentration of oxidant in the reaction system is 0.1~1wt%, and stirring is continued for 12~24 hours, then terminating reaction agent is added, the concentration of terminating reaction agent in the reaction system is 1~5wt%, and stirring is continued for 0.5~2 hour termination reaction, dialysis bag is used for dialysis, freeze drying, obtains oxidized konjac glucomannan. Oxidant is preferably sodium periodate. Terminating reaction agent is preferably ethylene glycol.

[0032] In the present invention, hydrazide hyaluronic acid is hydrazide-modified hyaluronic acid obtained by activating the carboxyl groups of a polysaccharide and causing an amide reaction. The degree of hydrazide hyaluronic acid is preferably 10% to 50%, for example, 10%, 20%, 30%, 40%, 50%, etc.

[0033] In the present invention, the preparation method of hydrazide hyaluronic acid preferably comprises the following steps: dissolving hyaluronic acid in water to prepare a 1-5wt% solution, then adding an activator and a hydrazide reagent, wherein the concentration of the activator and the hydrazide reagent in the reaction system is 0.1-1wt%, adjusting the pH to 4.75, continuously stirring for 12-24 hours, dialysis using a dialysis bag, and freeze-drying to obtain hydrazide hyaluronic acid. The hydrazide reagent is preferably adipic acid dihydrazide (ADH), and the activator is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT), preferably at a concentration of 1-5wt%. The pH is preferably adjusted with sodium hydroxide and hydrochloric acid.

[0034] The preparation method of the composite quick-acting hemostatic powder of the present invention comprises the following steps: (1) Cut the oxidized konjac gum into pieces and put it into a grinding bottle and freeze it with liquid nitrogen for 5-10 min. Use a tissue grinder to grind it 5 times at an oscillation frequency of 60 Hz. Wait for it to cool and then take it out to obtain OKGM powder.

[0035] (2) Cut the acylhydrazide hyaluronic acid into pieces and put it into a grinding bottle and freeze it with liquid nitrogen for 5-10 minutes. Use a tissue grinder to grind it 5 times at an oscillation frequency of 60 Hz. Wait for it to cool and then take it out to obtain ADHHA powder.

[0036] (3) The above two powders are stirred and mixed evenly in proportion and fully ground to obtain the composite fast-acting hemostatic powder of the present invention (also known as OKGM@ADHHA fast-acting hemostatic powder).

[0037] Example 1 A composite fast-acting hemostatic powder is prepared by the following steps: (1) 5 g of konjac gum (KGM) was thoroughly stirred with 500 mL of deionized water until completely dissolved to prepare a 1% mass concentration konjac gum solution. Then, 1.32 g of NaIO4 was added to the konjac gum solution, and the mixture was stirred at 30 °C in the dark for 12 hours. Next, 10 mL of ethylene glycol was added to terminate the reaction, and the mixture was stirred for another 2 hours. The resulting solution was then dialyzed in deionized water for 3 days, and the resulting solution was freeze-dried to obtain oxidized konjac gum (OKGM). The resulting solution was chopped and placed in a grinding bottle and frozen in liquid nitrogen for 5-10 minutes. The mixture was ground 5 times using a tissue grinder at an oscillation frequency of 60 Hz, and then cooled and removed to obtain OKGM powder.

[0038] (2) Dissolve 2 g of hyaluronic acid (HA) in 200 mL of sterile water. Add 2.3 g of ADH to the solution. While stirring continuously, slowly add 1.84 g of ADH and 1.43 g of HOBT (dissolved in 10 mL of deionized water and then added dropwise to the HA solution) to the HA solution to adjust the pH to 4.75. Continue to slowly add 0.91 g of EDC (dissolved in 10 mL of deionized water and then added dropwise to the mixed solution) to the solution. Adjust the pH with 1 M HCl and 1 M NaOH to maintain pH 4.75 for 4 hours. Continue stirring at room temperature for 24 hours. After the reaction is completed, dialyze with deionized water for 3 days, freeze at -80 ° C, and freeze-dry using a freeze dryer to obtain hydrazide hyaluronic acid (ADHHA). Cut it into pieces and put it into a grinding bottle and freeze it with liquid nitrogen for 5-10 minutes. Use a tissue grinder to grind it 5 times at an oscillation frequency of 60 Hz. Wait for it to cool and take it out to obtain ADHHA powder.

[0039] (3) Take ADHHA and OKGM powders in a weight ratio of 1:1 and grind them thoroughly and mix them evenly. Then add deionized water and stir evenly. Let it stand and observe the gel formation.

[0040] Example 2 A composite fast-acting hemostatic powder, the preparation method of which is different from the preparation method in Example 1 only in that the ratio of oxidized konjac gum powder is changed; in step (3), the ratio of oxidized konjac gum to hydrazide hyaluronic acid is 2:1.

[0041] Example 3 A composite fast-acting hemostatic powder, the preparation method of which is different from the preparation method in Example 1 only in that the concentration of oxidized konjac gum is changed; in step (3), the ratio of oxidized konjac gum to hydrazide hyaluronic acid is 3:1.

[0042] Example 4 A composite fast-acting hemostatic powder is prepared by the following steps: (1) Add 5 g of konjac gum (KGM) to 500 mL of deionized water and stir until completely dissolved. Then, add 1.32 g of NaIO4 and stir the mixture in the dark at 30 °C for 12 h. Next, add 10 mL of ethylene glycol to terminate the reaction and stir the mixture for another 2 h. The resulting solution is then dialyzed in deionized water for 3 days and freeze-dried to obtain OKGM. Cut the solution into pieces and place it in a grinding bottle and freeze it in liquid nitrogen for 5-10 min. Grind it five times using a tissue grinder at an oscillation frequency of 60 Hz. Wait for it to cool and then remove it to obtain OKGM powder.

[0043] (2) Dissolve 2 g of hyaluronic acid in 200 mL of sterile water. Add 2.3 g of ADH to the solution. While stirring continuously, slowly add 1.84 g of ADH and 1.43 g of HOBT (dissolved in 10 mL of deionized water and then added dropwise to the HA solution) to the HA solution to adjust the pH to 4.75. Continue to slowly add 0.91 g of EDC (dissolved in 10 mL of deionized water and then added dropwise to the mixed solution) to the solution. Adjust the pH with 1 M HCl and 1 M NaOH to maintain pH 4.75 for 4 hours. Continue stirring at room temperature for 24 hours. After the reaction is completed, dialyze with deionized water for 3 days, freeze at -80 ° C, and freeze-dry using a freeze dryer to obtain ADHHA. Cut it into pieces and put it into a grinding bottle and freeze it with liquid nitrogen for 5-10 minutes. Use a tissue grinder to grind it 5 times at an oscillation frequency of 60 Hz. Wait for it to cool and take it out to obtain ADHHA powder.

[0044] (3) Take ADHHA and OKGM powder in a ratio of 1:2 and grind them thoroughly and mix them evenly. Then add deionized water and stir evenly. Let it stand and observe the gel formation.

[0045] Example 5 A composite fast-acting hemostatic powder, the preparation method of which is different from the preparation method of Example 4 only in that the ratio of oxidized konjac gum to hydrazide hyaluronic acid powder is changed; in step (3), the weight ratio of oxidized konjac gum to hydrazide hyaluronic acid is 1:3.

[0046] Performance Testing The gelling effects of the samples obtained in the above-mentioned embodiments 1-5 are shown in Table 1. Table 1: Comparison of gelation time of hemostatic powder in various examples

[0047] As shown in Table 1, the gelation of the composite fast-acting hemostatic powders of Examples 1-5 changes with the concentration of ADHHA and OKGM. When the weight ratio of OKGM to ADHHA is 2:1, the gelation time of the fast-acting hemostatic powder is significantly shortened, the gelation is more stable, and the gelation is optimal. This is of great significance for the preparation of fast-acting hemostatic powders for use in severe bleeding.

[0048] The various properties of the composite fast-acting hemostatic powders of Examples 1 to 5 were measured. The results showed that the composite fast-acting hemostatic powders of Examples 1 to 5 all had good coagulation and hemostasis effects.

[0049] The performance test results of the composite fast-acting hemostatic powder of Example 2 (hereinafter referred to as OKGM@ADHHA fast-acting hemostatic powder) are used as an example for description.

[0050] The coagulation of the prepared OKGM and ADHHA powders was compared with the OKGM@ADHHA fast-acting hemostatic powder of Example 2. The anticoagulant blood and the powder were added to a centrifuge tube, and the blood coagulation was recorded by inverting the centrifuge tube and recording the time. Figure 1 It can be seen that the performance of OKGM and ADHHA powder alone in coagulation is not obvious, and the coagulation time is more than 5 minutes, which has a coagulation effect compared with the blank group; when the two powders are mixed in a specific proportion, the coagulation time is greatly shortened, and the coagulation effect can be achieved in only 28 seconds. This is mainly because when the fast-acting hemostatic powder comes into contact with blood, the two powders are quickly cross-linked into gel through Schiff base, and the positive charge carried by Schiff base aggregates platelets and red blood cells. It can also quickly absorb water in the blood, concentrate thrombin and fibrinase in the blood, and increase the coagulation speed to achieve rapid hemostasis.

[0051] Mouse whole blood was reacted with quick-acting hemostatic powder to form gel, which was fixed with a fixative and dehydrated, and then the aggregation of blood cells was observed using a scanning electron microscope. Figure 2 It can be seen that when the OKGM@ADHHA fast-acting hemostatic powder of Example 2 absorbs blood to form a gel, a layer of fibrin and other substances can be clearly seen on the surface, and blood cells are obviously aggregated into clusters. The blood cell morphology is not damaged and the morphology is intact, which proves that the fast-acting hemostatic powder not only has good blood compatibility, but also has a significant procoagulant effect ( Figure 2 In the figure, A: magnification 500 times; B: magnification 1000 times).

[0052] The cytotoxicity was investigated. Calcein-AM / PI live-dead staining was used to directly sterilize OKGM, ADHHA, and the OKGM@ADHHA fast-acting hemostatic powder of Example 2 with UV light and then added to a 5×10 5 After 24 hours of treatment, 3T3 cells were placed in a 6-well plate and stained with Calcein-AM and PI, respectively, and cell survival was observed using a fluorescence microscope. Figure 3 It can be seen that the OKGM@ADHHA fast-acting hemostatic powder of Example 2 has no effect on cells, the cell survival rate is high, and direct contact does not cause cell death, which indicates that the OKGM@ADHHA fast-acting hemostatic powder of Example 2 has no cytotoxicity.

[0053] To investigate hemostasis, BALB / C mice were used for the experiment. A 6 mm long wound was created at the apex of the liver (left lobe) using a scalpel. The OKGM@ADHHA fast-acting hemostatic powder (20 mg) from Example 2 was immediately placed on the bleeding point, and the wound surface was carefully observed for bleeding. No other treatment was given to the blank group, while the control group received the US-listed product BleedStop. TM Hemostatic powder. Figure 4From Table 2, it can be concluded that the mouse liver will bleed profusely without any treatment. When treated with OKGM@ADHHA fast-acting hemostatic powder, it can be observed that the mouse liver stops bleeding in 15 seconds, and no obvious bleeding is found later. TM When the hemostatic powder was sprinkled on the bleeding site, the bleeding did not stop immediately. The blood continued to ooze out, and the bleeding marks were obvious. The hemostatic time was 35s, which was significantly longer. Therefore, the quick-acting hemostatic powder prepared by the present invention had a good hemostatic effect.

[0054] Table 2: Statistical data on the hemostatic effect of OKGM@ADHHA fast-acting hemostatic powder on mouse and rat bleeding models

[0055] The hemostasis of the abdominal aorta of BALB / C mice was further investigated. A massive bleeding model was created in the abdominal aorta of mice using a needle. The OKGM@ADHHA fast-acting hemostatic powder (20 mg) from Example 2 was sprinkled on the bleeding area and the hemostasis was observed. The control group was treated with BleedStop. TM Hemostatic powder. Figure 5 As shown in Table 2, the abdominal aorta of mice bleeds a lot without any treatment. When treated with OKGM@ADHHA fast-acting hemostatic powder, it can be observed that the bleeding of the abdominal aorta of mice slows down and stops, and the surrounding blood quickly coagulates into clots. The hemostatic effect is achieved in 50 seconds, and no blood exudation is found later. The hemostatic effect is significant. TM During the treatment with hemostatic powder, the bleeding stops after 70 seconds, no blood clots appear around the wound, and the blood clots adhere to the abdominal cavity and are difficult to clean. Therefore, the quick-acting hemostatic powder prepared by the present invention has a better hemostatic effect.

[0056] To further investigate the hemostasis of the liver in SD rats, a 6 mm long wound was created at the tip of the liver (left lobe) using a scalpel. The OKGM@ADHHA fast-acting hemostatic powder (30 mg) from Example 2 was then immediately placed on the bleeding point. The bleeding on the wound was carefully observed. The blank group received no other treatment, while the control group received BleedStop®. TM Hemostatic powder.

[0057] pass Figure 6 From Table 2, it can be concluded that when the control hemostatic powder BleedStop TM When the hemostatic powder was used, the rat liver wound continued to bleed until the end of the experiment without achieving a hemostatic effect, and the hemostatic powder did not form a gel. When the OKGM@ADHHA fast-acting hemostatic powder of Example 2 was used, after the wound was made and bleeding was confirmed, the rat liver was sprinkled with the fast-acting hemostatic powder. It was observed that the bleeding in the rat liver stopped 40 seconds later, and no subsequent bleeding occurred. In addition, debridement revealed that the fast-acting hemostatic powder formed a gel on the wound surface. Therefore, the fast-acting hemostatic powder of the present invention has significant hemostatic and coagulation effects.

[0058] The hemostasis of the abdominal aorta of SD rats was further investigated. A massive bleeding model was created by puncturing the abdominal aorta with a needle. Then, the OKGM@ADHHA fast-acting hemostatic powder (100 mg) of Example 2 was immediately placed on the bleeding point, and the bleeding on the wound was carefully observed. The blank group received no other treatment, while the control group received BleedStop. TM Hemostatic powder.

[0059] pass Figure 7 As shown in Table 2, the OKGM@ADHHA fast-acting hemostatic powder of Example 2 and the control BleedStop TM The hemostatic powder can stop bleeding in the abdominal aorta. The fast-acting hemostatic powder can stop bleeding in 60 seconds. TM The hemostatic powder stops bleeding in 90 seconds. The hemostatic powder of the present invention stops bleeding quickly and in a short time. TM Hemostatic powder is easily left in the body or wound if it does not form a gel during wound debridement; however, quick-acting hemostatic powder is easier to remove after absorbing blood and forming a gel, greatly reducing the probability of residue.

[0060] The hemostasis of the iliac artery in SD rats was further investigated. A massive bleeding model was created by puncturing the iliac artery with a needle in the abdominal aorta. Then, the OKGM@ADHHA fast-acting hemostatic powder (100 mg) of Example 2 was immediately placed on the bleeding point, and the bleeding on the wound was carefully observed. The blank group received no other treatment, while the control group received BleedStop. TM Hemostatic powder.

[0061] pass Figure 8 As shown in Table 2, using BleedStop TM When hemostatic powder was applied to the rat's iliac artery, bleeding did not stop immediately after application. Instead, blood continued to ooze outward, ultimately achieving cessation within 60 seconds. During debridement, a large amount of hemostatic powder remained, and no gelling occurred. When OKGM@ADHHA fast-acting hemostatic powder was applied to the iliac artery, the hemostasis time was ≤30 seconds, and no significant blood oozing was observed subsequently. During debridement, the OKGM@ADHHA fast-acting hemostatic powder formed a gel, allowing for direct removal, reducing the probability of residual blood. Therefore, the fast-acting hemostatic powder of the present invention not only has excellent hemostatic effects but also facilitates secondary treatment.

[0062] The various technical features of the above-described embodiments can be combined in any combination. For the sake of brevity, all technical features of the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be deemed to be within the scope of this specification. It should be pointed out that for ordinary technicians in this field, many variations and improvements can be made without departing from the concept of the present invention, which all fall within the scope of protection of this patent. Therefore, the scope of protection of the patent of this invention shall be based on the attached claims.

Claims

1. A composite fast-acting hemostatic powder, characterized in that Includes oxidized konjac gum and hydrazide hyaluronic acid.

2. The composite fast-acting hemostatic powder according to claim 1, characterized in that The mass ratio of oxidized konjac gum to hydrazide hyaluronic acid is (1-3): (1-3).

3. The composite fast-acting hemostatic powder according to claim 1, characterized in that The oxidation degree of the oxidized konjac gum is 20%-30%.

4. The composite fast-acting hemostatic powder according to claim 1, characterized in that The hydrazide degree of the hydrazide hyaluronic acid is 15%-30%.

5. The composite fast-acting hemostatic powder according to claim 1, characterized in that: The molecular weight of the hyaluronic acid in the quick-acting hemostatic powder is 150,000-200,000.

6. The composite fast-acting hemostatic powder according to claim 1, characterized in that: Described oxidized konjac gum is prepared by following steps: The konjac gum is oxidized by using sodium periodate, and then dialyzed and freeze-dried to obtain the oxidized konjac gum.

7. The composite fast-acting hemostatic powder according to claim 1, characterized in that: The hydrazide hyaluronic acid is prepared by the following steps: Hyaluronic acid is modified with adipic acid dihydrazide, and then dialyzed and freeze-dried to obtain the hydrazide hyaluronic acid.

8. The method for preparing the composite fast-acting hemostatic powder according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: Oxidized konjac gum and hydrazide hyaluronic acid are frozen in liquid nitrogen respectively and then ground into powder. The two powders are then mixed evenly according to a certain proportion and fully ground to obtain the composite fast-acting hemostatic powder.

9. Use of the composite fast-acting hemostatic powder according to any one of claims 1 to 7 for hemostasis.

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