An enzyme-modified starch-based hemostatic powder and its preparation method

Abstract

The present invention discloses an enzyme-modified starch-based hemostatic powder and a preparation method thereof, belonging to the field of bio-modified starch. The steps are as follows: the starch is subjected to alkali washing, acid hydrolysis, and enzymatic hydrolysis and then loaded with procoagulant factors to obtain the starch-based hemostatic powder. The method only adopts processes such as stirring, heating, and filtration, with simple steps and convenient operation, and can conveniently achieve large-scale production. The starch-based hemostatic powder has a significant hemostatic effect. Compared with the products prepared by other methods, it has no potential toxicity risk to the human body. This method provides new ideas for the field of bio-modified starch and the field of medical hemostasis.

Description

Technical Field

[0001] The present invention belongs to the field of biologically modified starch, and particularly relates to an enzyme-modified starch-based hemostatic powder and a preparation method thereof. Background Art

[0002] In addition to traditional hemostatic means, various forms of hemostatic materials have been developed to cope with traumatic blood loss under different conditions and improve the hemostatic efficiency. At present, the hemostatic materials on the market can be roughly classified into the following categories according to the different active ingredients: polysaccharide materials, protein materials, platelet activator materials, synthetic polymer materials, inorganic materials, etc. As a renewable polysaccharide, starch can be degraded by amylase in the human body, and starch has no cytotoxicity and good biocompatibility. Therefore, starch-based hemostatic materials have received extensive attention in the past 20 years.

[0003] At present, starch-based hemostatic materials are mainly produced by modifying starch through different chemical reactions. The production process is complex and the raw materials used have potential toxicity. Chinese Patent CN102139123 obtains a hemostatic powder by gelatinizing, enzymolyzing, emulsifying and crosslinking, and solvent extraction of starch. The preparation process requires high-temperature heating and closed reaction. Ethyl acetate and petroleum ether used in extraction are harmful to the human body. Chinese Patent CN104606723 obtains a hemostatic powder by emulsifying and crosslinking native starch and carboxymethyl starch and washing with an organic solvent. The preparation process of carboxymethyl starch is complex and consumes a large amount of ethanol, and the production cost is high. The etherifying agent used has a carcinogenic risk. Chinese Patent CN103720705 prepares a starch microsphere hemostatic powder by water-in-water emulsification of cassava starch solution and polyethylene glycol. The cassava starch solution needs to be gelatinized at a higher temperature, and the emulsification reaction is unstable and difficult to control. Chinese Patent CN105816902 obtains an aqueous solution by ball milling cassava starch and etherified starch, and then obtains an absorbable hemostatic powder by emulsifying and crosslinking. The production process is relatively complex and requires a large amount of ethanol to wash vegetable oil and emulsifier. Chinese Patent CN110935055 cures and crosslinks polyvinyl alcohol and porous starch to obtain a hemostatic sponge, and the hemostatic sponge is easy to cause secondary injury when swelling at the wound.

[0004] When preparing a hemostatic powder by modifying starch through a chemical reaction, there are many reaction steps, so the process of washing starch is increased. In addition, due to the particularity of starch, chemical reagents are still relatively easy to remain after washing. At present, most starch-based hemostatic materials adopt an emulsifying and crosslinking process. After emulsification, the starch needs to remove organic solvents through ethyl acetate, alcohol, etc. Emulsifiers, crosslinking agents and ethyl acetate not only have potential toxicity, but also these reagents are relatively expensive, thus increasing the production cost. [[ID=I6]]

[0005] Therefore, the existing technology urgently needs a method to simplify the production process of starch-based hemostatic powder, and it is necessary to avoid using potentially toxic reaction reagents during the production process to improve the production efficiency of starch-based hemostatic powder, reduce production costs, and reduce potential harm to the human body. Summary of the Invention

[0006] Technical Problem

[0007] The present invention aims to obtain a process for efficiently preparing starch-based hemostatic powder, and to prepare a starch-based hemostatic powder capable of quickly stopping bleeding by using this process, and to establish a scientific method for evaluating the in vitro hemostatic effect of samples.

[0008] Technical Solution

[0009] To solve the above problems, the present invention provides a method for preparing an enzyme-modified starch-based hemostatic powder, which comprises the following steps:

[0010] (1) Dispersing starch granules in a buffer solution to obtain a starch milk, raising the temperature of the starch milk to the preheating temperature, and stirring;

[0011] (2) Cooling the starch milk, and then adding amylase to the starch milk for reaction to obtain Product 1;

[0012] (3) Dispersing Product 1 in an aqueous solution containing a blood coagulation factor, stirring, drying, and performing a sterilization treatment to obtain a starch-based hemostatic powder.

[0013] In an embodiment of the present invention, the starch in step (1) is one of ordinary corn starch, mung bean starch, canna starch, waxy corn starch, cassava starch, potato starch, rice starch, and wheat starch. Specifically, cassava starch can be selected.

[0014] In an embodiment of the present invention, in step (1), the buffer solution is one or more of a phosphoric acid-citric acid buffer solution, a citric acid-sodium citrate buffer solution, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a glycine-sodium hydroxide buffer solution, an acetic acid-sodium acetate buffer solution, and a Tris-HCl buffer solution. Specifically, a phosphoric acid-citric acid buffer solution can be selected.

[0015] In an embodiment of the present invention, in step (1), the mass-volume ratio of starch to the buffer solution is 18 g:(30 - 360 mL). Specifically, 18 g:120 mL can be selected.

[0016] In an embodiment of the present invention, in step (1), the preheating temperature is 40 - 70 °C, and the stirring time is 5 - 150 min.

[0017] In an embodiment of the present invention, in step (2), the temperature after cooling is 25 - 60 °C.

[0018] In one embodiment of the present invention, the addition amount of amylase in step (2) is 100 - 800 U / g.

[0019] In one embodiment of the present invention, the amylase in step (2) is one or more of α-amylase, β-amylase, γ-amylase, cyclodextrin glycosyltransferase, isoamylase, glucoamylase, pullulanase.

[0020] In one embodiment of the present invention, the amylase in step (2) can specifically be 100 U / g (dry basis starch) α-amylase and 200 U / g (dry basis starch) glucoamylase.

[0021] In one embodiment of the present invention, the reaction time in step (2) is 10 - 18 h.

[0022] In one embodiment of the present invention, the procoagulant factor in step (3) is one or more of thrombin, tannic acid, calcium chloride, tranexamic acid, hyaluronic acid, chitosan, tea polyphenols, curcumin, polyphosphate.

[0023] In one embodiment of the present invention, the procoagulant factor in step (3) can specifically be calcium chloride and tannic acid.

[0024] In one embodiment of the present invention, the mass concentration of the procoagulant factor in the aqueous solution of the procoagulant factor in step (3) is 1 - 10%.

[0025] In one embodiment of the present invention, the mass concentration of the aqueous solution of the procoagulant factor in step (3) can specifically be 10 wt% calcium chloride aqueous solution and 5 wt% tannic acid aqueous solution.

[0026] In one embodiment of the present invention, the mass-volume ratio of product 1 to the aqueous solution of the procoagulant factor in step (3) is 10 g : (10 - 100) mL.

[0027] In one embodiment of the present invention, the mass-volume ratio of product 1 to the aqueous solution of the procoagulant factor in step (3) can specifically be 10 g : 40 mL.

[0028] In one embodiment of the present invention, the stirring time in step (3) is 2 - 6 h.

[0029] The present invention also provides an enzyme-modified starch-based hemostatic powder prepared by the above method.

[0030] The application of the enzyme-modified starch-based hemostatic powder provided by the present invention in the preparation of medical hemostatic products.

[0031] Beneficial effects

[0032] 1. The present invention prepares a starch-based hemostatic powder through enzymatic modification. The production process is simple to operate, the product yield is relatively high, and the reagents used are green and safe. The amylase is used to modify starch, without introducing other chemical groups. The starch granules show the cleavage of α-1,4-glycosidic bonds inside the molecule and the reassembly of α-1,6-glycosidic bonds.

[0033] 2. The present invention provides a process method for preparing a starch-based hemostatic powder based on enzymatic modification. The enzymatic reaction is carried out at a suitable reaction temperature. The production process is simple, and the product can be obtained only through simple operations such as stirring, washing, and drying. And due to the high specificity of the enzymatic reaction, through the reasonable design of the enzyme variety and the enzyme addition amount, the products of different batches have strong quality stability.

[0034] 3. The present invention makes full use of the hydrolysis characteristics of different amylases to achieve the purpose of synergistic effect, enhance the reaction efficiency, can significantly increase the porosity of starch granules, enhance the water absorption rate of starch granules, and the water absorption rate can reach 166.38%.

[0035] 4. The present invention provides a method for evaluating the in vitro hemostatic effect of a sample.

[0036] 5. The present invention uses amylase to act on granular starch in sequence, and only uses a small amount of enzyme addition to achieve high-concentration starch modification treatment, improving the production efficiency, and providing a new idea and means for the biological modification to prepare a starch-based hemostatic powder, which is green, low-carbon, energy-saving, consumption-reducing, and environmentally friendly.

[0037] 6. The present invention uses amylase to modify starch, increases the specific surface area of granular starch, greatly improves the adsorption property of granular starch, and on this basis, loads the procoagulant substance on the enzyme-modified starch granules, which can significantly shorten the coagulation time. The shortest coagulation time obtained by the in vitro coagulation test provided by the present invention is 3.24 min. Description of the Drawings

[0038] Figure 1 It is the scanning electron microscope image of the cassava starch-based hemostatic powder. 2]

[0039] Figure 2 It is the hemostatic time of different samples (blank sample, cassava starch-based hemostatic powder, Yunnan Baiyao hemostatic powder) for the severed tail.

[0040] Figure 3 It is the hemostatic time of different samples (blank sample, cassava starch-based hemostatic powder, Yunnan Baiyao hemostatic powder) for the back wound of rats.

[0041] Figure 4 It is the hemostatic time of different samples (blank sample, cassava starch-based hemostatic powder, Yunnan Baiyao hemostatic powder) for the liver wound of rats

[0042] Figure 5 Schematic diagram of tail amputation for hemostasis. Specific implementation mode

[0043] To make the objectives, technical solutions and advantages of the present invention clearer, the following will describe the implementation modes of the present invention in detail with reference to examples. However, the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0044] Source of raw materials:

[0045] α-amylase (4000 U / g), glucoamylase (100000 U / mL), β-amylase (9000 U / mL), isoamylase (≥100000000 U / mg), γ-amylase (100000 U / mL), purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; cyclodextrin glycosyltransferase, obtained by fermentation of a strain derived from Bacillus circulans; Yunnan Baiyao hemostatic powder (4 g / bottle), purchased from Yunnan Baiyao Group Co., Ltd.; tannic acid, calcium chloride, tranexamic acid, chitosan, hyaluronic acid, purchased from Sinopharm Chemical Reagent Co., Ltd.; potato starch, purchased from Inner Mongolia Huaou Starch Industry Co., Ltd.; corn starch, purchased from Shandong Shouguang Jueneng Golden Corn Development Co., Ltd.; mung bean starch, wheat starch, purchased from Xinliang Grain and Oil Processing Co., Ltd.; canna starch, purchased from Kunming, Yunnan; cassava starch, purchased from Guangxi Hongfeng Starch Co., Ltd.

[0046] Example 1

[0047] (1) Disperse 18 g of cassava starch in 60 mL of 100 mM phosphate-citrate buffer to obtain a starch milk with a mass / volume concentration of 30%. Stir and heat the starch milk to 60 °C and continuously stir for 30 min.

[0048] (2) Cool the 60 mL of starch milk to 50 °C, and add 100 U / g (dry basis starch) of α-amylase and 200 U / g (dry basis starch) of glucoamylase. After reacting for 16 h, filter and wash to obtain Product 1.

[0049] (3) Disperse 10 g of Product 1 in 40 mL of a 10% aqueous solution of CaCl2, stir for 4 h, and then dry to obtain Product 2. Disperse 10 g of Product 2 in 40 mL of a 5% aqueous solution of tannic acid, stir for 4 h, and after freeze-drying, pulverizing, and Co 60 Sterilization to obtain starch-based hemostatic powder.

[0050] Example 2

[0051] Adjust the type of buffer in the reaction system, replace the buffer in Example 1 with phosphate-citrate buffer, citrate-sodium citrate buffer, disodium hydrogen phosphate-monobasic potassium phosphate buffer, glycine-sodium hydroxide buffer, acetic acid-sodium acetate buffer, Tris-HCl buffer, and keep other steps unchanged.

[0052] Example 3

[0053] Adjust the concentration of starch milk, replace the concentration of 30% (mass / volume) in step 1 of Example 1 with 5%, 10%, 15%, 30%, 45%, and keep other steps unchanged.

[0054] Example 4

[0055] Adjust the preheating temperature, replace heating to 60°C in step 1 of Example 1 with heating to 30°C, 40°C, 50°C, 55°C, 60°C, and keep other steps unchanged.

[0056] Example 5

[0057] Adjust the preheating time, replace continuous stirring for 2 h in step 1 of Example 1 with continuous stirring for 30 min, 60 min, 90 min, 120 min, 150 min, and keep other steps unchanged.

[0058] Example 6

[0059] Adjust the temperature of enzymatic hydrolysis reaction, replace cooling to 50°C in step 1 of Example 1 with adjusting the temperature to 25°C, 35°C, 45°C, 50°C, 60°C, and keep other steps unchanged.

[0060] Example 7

[0061] Adjust the enzyme dosage, replace adding 100 U / g (dry basis starch) of α-amylase and 200 U / g (dry basis starch) of glucoamylase in step 2 of Example 1 with the combinations shown in the following table, and keep other steps unchanged.

[0062]

[0063] Example 8

[0064] Adjust the type of enzyme, replace adding 100 U / g (dry basis starch) of α-amylase and 200 U / g (dry basis starch) of glucoamylase in step 2 of Example 1 with the combinations shown in the following table, and keep other steps unchanged.

[0065]

[0066] Example 9

[0067] Adjust the procoagulant factors, and replace the 10% mass concentration of calcium chloride aqueous solution and 5% mass concentration of tannic acid aqueous solution in Step 3 of Example 1 with 10 wt% calcium chloride aqueous solution, 10 wt% tannic acid aqueous solution, 10 wt% tranexamic acid aqueous solution, 10 wt% chitosan aqueous solution, 10 wt% sodium hyaluronate aqueous solution, 10 wt% calcium chloride aqueous solution + 10 wt% sodium hyaluronate aqueous solution (volume ratio 2:1), and keep other steps unchanged.

[0068] Comparative Example 1

[0069] Adjust the starch type, and replace the cassava starch in Example 1 with potato starch, corn starch, mung bean starch, wheat starch, and canna starch, and keep other steps unchanged.

[0070] Comparative Example 2

[0071] This comparative example is the method for preparing hemostatic powder described in Chinese invention patent CN 113667706.

[0072] The specific steps are as follows:

[0073] (1) Weigh 2 g of starch, dissolve it in 100 mL of disodium hydrogen phosphate-citric acid buffer solution with pH = 4.5, perform water bath at 50 °C, mechanically stir at 500 rpm, add 50 KNU / g of α-amylase and 10000 U / g of glucoamylase, stir for 6 h, add 17 mL of 0.5 M sodium hydroxide solution to make the pH value 12, stir for 30 min, centrifuge at 5500 rpm * 5 min, wash three times with deionized water, and freeze-dry to obtain enzymatically hydrolyzed starch;

[0074] (2) Dissolve 10 g of BOC tranexamic acid in 100 mL of dichloromethane, perform reflux magnetic stirring at 30 °C and 500 rpm for 10 min, add 200 μL of N,N-dimethylformamide, and stir with 5.646 mL of thionyl chloride for 3 h, then perform rotary evaporation to obtain BOC carbamyl chloride powder;

[0075] (3) Weigh 1 g of enzymatically hydrolyzed starch, perform magnetic stirring at 80 °C and 500 rpm, add 3 g of BOC carbamyl chloride, and stir for 8 h, then centrifuge

[0076] at 5500 rpm * 10 min, wash 3 times with deionized water, centrifuge, and freeze-dry at 60 °C for 24 h to obtain tranexamic acid-loaded cross-linked porous starch hemostatic powder.

[0077] Example 10

[0078] Evaluation method for in vitro hemostatic effect of the sample:

[0079] The specific steps are as follows:

[0080] Weigh 10 mg of the sample into a 5 mL centrifuge tube and preheat it in a water bath at 37 °C for 5 min. Then add 1 mL of anticoagulated whole blood, incubate for 3 min, and then add 100 μL of 0.2 M calcium chloride solution and start timing immediately. To determine whether the blood has coagulated, tilt the test tube every 15 s. The coagulation time is the time when the plasma completely loses its fluidity. The anticoagulated whole blood without any treatment is the blank group, and three parallel experiments are conducted for each sample. After testing, the coagulation time of the anticoagulated whole blood without any treatment is 20.17 min.

[0081] Evaluate the in vitro hemostatic effect of the starch-based hemostatic powders prepared in Examples 1-9 and Comparative Example 1 according to the above test steps. Each comparison of test data includes a tapioca starch control sample, which is tapioca starch without any treatment.

[0082] Table 1 Results of treating tapioca starch-based hemostatic powder with different buffer systems

[0083]

[0084]

[0085] Table 1 shows the comparison of test data between Example 1 and Example 2. It can be found that when using phosphoric acid-citric acid buffer, the liquid absorption rate is the highest at 166.38%, which is 90.28% higher than that of tapioca starch, and the coagulation time is 3.24 min, which is 4.46 min shorter than that of tapioca starch. This indicates that when the phosphoric acid-citric acid buffer is used in the system, the enzyme activity stability of amylase is relatively high, and it can act on starch granules more efficiently.

[0086] Table 2 Influence of starch milk concentration on tapioca starch-based hemostatic powder

[0087]

[0088] Table 2 shows the comparison of test data between Example 1 and Example 3. It can be found that when the starch milk concentration is 30%, the liquid absorption rate is the highest at 166.38%, which is 90.28% higher than that of tapioca starch, and the coagulation time is 3.24 min, which is 4.46 min shorter than that of tapioca starch. This indicates that when the starch milk concentration in the reaction system is appropriate, the starch granules can fully contact the reaction solution, and the reaction efficiency is relatively high.

[0089] Table 3 Influence of preheating temperature on tapioca starch-based hemostatic powder

[0090]

[0091] Table 3 shows the comparison of test data between Example 1 and Example 4. It can be found that when the preheating temperature is 60 °C, the highest liquid absorption rate is 166.38%, which is 90.28% higher than that of cassava starch. The coagulation time is 3.24 min, which is 4.46 min shorter than that of cassava starch. This indicates that the preheating temperature has a great influence on the swelling of cassava starch granules. If the preheating temperature is too low, the degree of granule swelling is insufficient, resulting in insufficient enzymatic hydrolysis. If the preheating temperature is too high, the granules expand close to the gelatinized state, with a high degree of enzymatic hydrolysis, leading to serious granule damage and thus a low liquid absorption rate of the granules.

[0092] Table 4 Influence of preheating time on cassava starch-based hemostatic powder

[0093]

[0094] Table 4 shows the comparison of test data between Example 1 and Example 5. It can be found that when the preheating time is 30 min, the liquid absorption rate is relatively high at 166.38%, which is 90.28% higher than that of cassava starch. The coagulation time is 3.24 min, which is 4.46 min shorter than that of cassava starch. When the preheating time is further extended, the water absorption rate of the sample does not increase significantly, and the shortening ratio of the coagulation time is relatively low. Considering the energy consumption generated by heating samples in large quantities in industry, it is preferable to select a preheating time of 30 min.

[0095] Table 5 Influence of enzymatic hydrolysis reaction temperature on cassava starch-based hemostatic powder

[0096]

[0097] Table 5 shows the comparison of test data between Example 1 and Example 6. It can be found that when the reaction temperature is 50 °C, the highest liquid absorption rate is 166.38%, which is 90.28% higher than that of cassava starch. The coagulation time is 3.24 min, which is 4.46 min shorter than that of cassava starch. This indicates that the mixed enzyme of α-amylase and glucoamylase acts stably on cassava starch at 50 °C, resulting in more pores in the cassava starch granules.

[0098] Table 6 Results of different enzyme addition amounts on cassava starch-based hemostatic powder

[0099]

[0100]

[0101] Table 6 shows the comparison of test data between Example 1 and Example 7. It can be found that when the addition amount of α-amylase is 100 U / g and the addition amount of glucoamylase is 200 U / g, the highest liquid absorption rate is 166.38%, which is 90.28% higher than that of cassava starch. The coagulation time is 3.24 min, which is 4.46 min shorter than that of cassava starch.

[0102] Table 7 Results of Different Enzyme Types on Cassava Starch-based Hemostatic Powder

[0103]

[0104] Table 7 shows the comparison of test data between Example 1 and Example 8. It can be found that when using α-amylase and glucoamylase, the highest liquid absorption rate is 166.38%, which is 90.28% higher than that of cassava starch. The coagulation time is 3.24 min, which is 4.46 min shorter than that of cassava starch. Both α-amylase and glucoamylase can act on raw starch granules under suitable conditions, causing pits, gaps, holes, etc. on the starch surface, thereby increasing the liquid absorption rate of cassava starch granules.

[0105] Table 8 Effects of Coagulation Promoting Factors on Potato Starch-based Hemostatic Powder

[0106]

[0107] Table 8 shows the comparison of test data between Example 1 and Example 9. It can be found that when the coagulation promoting factors are calcium chloride and tannic acid, the coagulation time of cassava starch-based hemostatic powder is the shortest. Tannic acid is a natural polyphenol derivative with good anti-inflammatory, antibacterial and other properties. It is reported that tannic acid can stably interact with thrombin through hydrogen bonds in the physiological environment, thus causing platelet activation. Moreover, tannic acid can form a complex with calcium ions. After the complex is mixed with swollen cassava starch granules, they aggregate into a relatively viscous complex, thereby accelerating blood coagulation.

[0108] Table 9 Effects of Different Types of Starch-based Hemostatic Powders

[0109]

[0110] Table 9 shows the comparison of test data between Example 1 and Comparative Example 1. It can be found that the coagulation time of the hemostatic powder prepared from cassava starch is the shortest, which is 3.24 min. After enzymatic hydrolysis of cassava starch, there are more pits and cracks on the surface, and a single large pore appears at one end of many granules. Therefore, the enzymatically hydrolyzed cassava starch granules have a larger specific surface area and adsorption capacity, which can quickly absorb blood and enrich coagulation factors, thus shortening the coagulation time.

[0111] Table 10 In Vitro Coagulation Time of Starch-based Hemostatic Powder

[0112]

[0113] Table 10 shows the comparison of test data of the samples with the best hemostatic effect in Examples 1, 2-9, Comparative Example 1 and Comparative Example 2. According to Example 1, the coagulation time of anticoagulated whole blood without any treatment was 20.17 s after adding calcium chloride to activate the coagulation system, while Examples 1-9 were all starch-based hemostatic powders prepared by the present invention, and their in vitro coagulation time was obviously much shorter than 20.17 s. In addition, in Comparative Example 1, for the starch-based hemostatic powder prepared from mung bean starch, although the hemostatic effect was not much different from that of Examples 2-8 in terms of in vitro coagulation time, it was still inferior to the hemostatic effects of Example 1 and Example 9. In Comparative Example 2, a hemostatic powder was prepared by the method described in Chinese Patent CN 113667706, and its in vitro coagulation time was 8.61 min, which was obviously longer than that of Examples 1-9. To sum up, cassava starch has a significant hemostatic effect after enzymatic hydrolysis and then loading with procoagulant factors.

[0114] Example 11

[0115] Rat tail amputation wound hemostasis experiment

[0116] The rats were maintained under anesthesia with isoflurane at a concentration of 2-2.5% and a flow rate of 500-700 mL / min. After successful anesthesia, the rats were placed flat on the experimental table, and their four limbs were fixed with rubber bands. The rats' tails were disinfected with iodophor and alcohol. Then, at a distance of 2 cm from the end of the rat tail, the rat tail was cut off at one time with scissors. After 3 s of bleeding from the incision, the blood was wiped off, and the tail wound was immediately inserted into a centrifuge tube containing hemostatic powder, and the stopwatch was started. The wound was observed every 30 s to see if it had stopped bleeding. The filter paper was gently touched to the wound. If no blood stain was left on the filter paper, it indicated that hemostasis was successful. The time from starting the stopwatch to successful hemostasis was the hemostasis time.

[0117] Rat back wound hemostasis experiment

[0118] Use an electric hair clipper to remove the hair on the rats' backs for skin preparation (with the spinal column as the midline, and the skin preparation areas on both sides are 4 cm * 5 cm). Then disinfect with iodophor and alcohol. After the skin is slightly dry, use a biopsy punch to remove the skin in a circular area with a diameter of about 1 cm on both sides of the rat spinal column. Use tissue forceps to lift the skin inside the round hole, and use the pointed end of a tissue scissors to pierce a hole deep into the fascia layer, and then cut off the skin in the circular area along the hole to create a wound, and then use the hemostatic powder to stop the bleeding and record the hemostasis time.

[0119] Rat liver wound hemostasis experiment

[0120] The rat's abdomen was disinfected with iodine and ethanol. A longitudinal incision approximately 2 cm long was then made in the middle of the abdomen. A disposable drape made of sterile gauze was placed over the wound. The abdomen was gently squeezed to expose the liver. The left (right) lobe of the liver was squeezed out of the incision. The left (right) lobe of the liver was then held and secured with sterile gauze wrapped in saline. A linear wound 1 cm long and 5 mm deep was created on the surface of the liver lobe with a blade. After the incision allowed to bleed freely for 3 seconds, the blood was immediately wiped away. An appropriate amount of hemostatic powder was applied flatly, and two 2 cm x 2 cm filter papers were placed on the surface. The pressure was applied and a stopwatch was started. Hemostasis was observed by removing one filter paper every 30 seconds until bleeding ceased. Complete hemostasis was defined as the absence of bright red blood oozing within 3 minutes without pressure. The time of complete hemostasis was recorded.

[0121] According to the above three hemostatic experiments, blank samples, cassava starch-based hemostatic powder, and Yunnan Baiyao hemostatic powder were set up for experiments. Figure 2 、 3 As shown in Figure 4, the hemostatic time of cassava starch-based hemostatic powder for tail cutting is 1.87 minutes, the hemostatic time of back trauma is 11.3 seconds, and the hemostatic time of liver injury is 0.45 minutes, while the hemostatic time of Yunnan Baiyao hemostatic powder for tail cutting is 6.73 minutes, the hemostatic time of back trauma is 15.3 seconds, and the hemostatic time of liver injury is 0.60 minutes. This shows that the hemostatic effect of cassava starch-based hemostatic powder in rat tail, back, and liver experiments is better than that of Yunnan Baiyao hemostatic powder.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or adapt the above-disclosed technical content to produce equivalent experimental examples with equivalent variations. However, any simple modifications, equivalent variations, and improvements to the above-disclosed embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention shall remain within the scope of protection of the present invention.

Claims

1. A preparation method of an enzyme-modified starch-based hemostatic powder, the method comprising the following steps: (1) Dispersing starch granules in a buffer solution to obtain a starch milk, heating the starch milk to a preheating temperature, and stirring; the starch is cassava starch; the buffer solution is a phosphoric acid-citric acid buffer; the mass-volume ratio of the starch to the buffer solution is 18 g: 30-360 mL; the preheating temperature is 40-70 °C, and the stirring time is 5-150 min; (2) Cooling the starch milk, and then adding 100 U / g of α-amylase and 200 U / g of glucoamylase to the starch milk for reaction to obtain Product 1; the temperature after cooling is 25-60 °C; (3) Dispersing Product 1 in an aqueous solution containing a blood coagulation factor, stirring, drying, and performing sterilization treatment to obtain a starch-based hemostatic powder; the blood coagulation factor is tannic acid and calcium chloride.

2. The method according to claim 1, characterized in that, In step (3), the mass concentration of the blood coagulation factor in the aqueous solution of the blood coagulation factor is 1-10%.

3. The method according to claim 1, wherein In step (3), the mass-volume ratio of Product 1 to the aqueous solution of the blood coagulation factor is 10 g: 10-100 mL.

4. The method according to claim 1, wherein In step (3), the stirring time is 2-6 h.

5. An enzyme-modified starch-based hemostatic powder prepared by the method according to any one of claims 1-4.

6. Use of the enzyme-modified starch-based hemostatic powder according to claim 5 in the preparation of medical hemostatic products.

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