STARCH-BASED HEMOSTATIC POWDER AND PREPARATION METHOD THEREOF

Abstract

Disclosed is a preparation method of a starch-based hemostatic powder, comprising the following steps: (1) starch modification; (2) emulsification and crosslinking; (3) separation, purification, drying and sterilization. Provided is a starch-based hemostatic powder, which has a simple preparation process and a controllable cost. After being modified, raw material medical potato starch has a high degree of emulsification and crosslinking and a good and uniform spherical shape, and has a porous structure. Moreover, the product has good biocompatibility, low biological cytotoxicity, and obvious antibacterial effect; the product has a high liquid absorption ratio, and quickly forms a gel to cover the wound surface after liquid absorption, thus achieving a good hemostatic effect. The starch-based hemostatic powder is an excellent biomedical product, and can be used for clinical wound treatment, surgery to prevent organ adhesion, and bleeding from various wounds.

Claims

1. A preparation method of a starch-based hemostatic powder, comprising the following Steps: (1) gelatinizing and modifying potato starch raw material to obtain modified starch; (2) emulsifying and crosslinking the modified starch, obtained in Step (1), in an emulsifier, thus obtaining cross-linked starch; and (3) carrying out separation, purification, drying and sterilization on the cross-linked starch obtained in Step (2), thus obtaining the starch-based hemostatic powder.

2. The preparation method according to claim 1, wherein in Step (1), the mass concentration of the starch is between 1% and 20%, the gelatinization temperature is between 40° C. and 80° C., and the gelatinization time is between 20 minutes and 120 minutes; after the gelatinization treatment, the pH is adjusted to a range from 8 to 12.

3. The preparation method according to claim 1, wherein in Step (1), a starch modifier is 2-chloroethyldiethylamine with a mass concentration between 1% and 10%, and the reaction time is between 5 h and 24 h.

4. The preparation method according to claim 1, wherein in Step (2), the emulsifier is one or more of Tween-60, Tween-80, Span-60, and Span-80.

5. The preparation method according to claim 4, wherein in Step (2), the emulsifier is first dissolved in an oil phase, heated and stirred uniformly for 10-50 minutes, and then the modified starch obtained in Step (1) is added dropwise to the solution.

6. The preparation method according to claim 5, wherein the oil phase is one of liquid paraffin, aviation kerosene, and vegetable oil; the emulsifier accounts for 0.1%-10% of the oil phase in a mass ratio, and the volume ratio of the oil phase to the aqueous solution of the starch is 3-6:1.

7. The preparation method according to claim 1, wherein in Step (2), a crosslinking agent is epichlorohydrin, and the crosslinking agent accounts for 0.2%40% of the oil phase in a mass ratio.

8. The preparation method according to claim 1, wherein in Step (2), the modified starch is emulsified for 0.5-12 h at a temperature between 35° C. and 80° C., the stirring speed of the emulsification and crosslinking process is between 100 rpm/min and 1000 rpm/min, and the crosslinking time is between 1 h and 72 h.

9. A starch-based hemostatic powder, prepared by the method according to claim 1.

10. An application of the starch-based hemostatic powder according to claim 9, wherein the starch-based hemostatic powder is used for providing one effect of hemostatic, anti-adhesion, antibacterial, and wound sealing effects on bloody wounds or their combinations; the blooding wound refers to the body surface, tissues and organs in the body, or tissues in the body cavity or organs in the body cavity of mammals, birds, and reptiles.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0028] FIG. 1 is a scanning electron micrograph of the starch-based hemostatic powder prepared in Example 2;

[0029] FIG. 2 is an in vitro hemostatic test of the starch-based hemostatic powder prepared in Example 2; and

[0030] FIG. 3 is a skin irritation test of the starch-based hemostatic powder prepared in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The above-mentioned contents of the present invention will be further described in detail through the specific implementation solutions provided in the following embodiments. Those skilled in the art should not understand that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments. Any technology implemented on the basis of the above-mentioned contents of the present invention belongs to the scope of the present invention.

[0032] The experimental methods adopted in the following embodiments, unless otherwise specified, are conventional methods; the reagents, materials, instruments and the like used in the following embodiments, if not specifically stated, are commercially available.

Example 1

[0033] (1) Gelatinization of starch: 10 g of potato starch was weighed and dissolved in 600 mL of purified water, and then heated and stirred to be gelatinized at 75° C. for 30 min; the resulting solution was stirred uniformly and adjusted with a sodium hydroxide solution until the pH reached a range from 8 to 12, thus obtaining a gelatinized starch solution.

[0034] (2) Modification of starch: 1 g of 2-chloroethyldiethylamine was weighed and dissolved in 10 mL of water, the resulting solution was added to the gelatinized starch solution to further react at 70° C. for 5 h, thus obtaining a modified starch solution.

[0035] (3) Preparation of oil phase: 500 mL of liquid paraffin was weighed and, added with 2 g of Tween-80, and the resulting material was stirred at 50° C. for 10 min and set aside for later use.

[0036] (4) Emulsification and crosslinking: the modified starch was dropwise added to the oil phase and stirred to react at 70° C. for 40 min, 10 mL of epichlorohydrin was added to carry out crosslinking reaction for 2 h, and the reaction solution was set still after the reaction.

[0037] (5) Separation of precipitation: after the reaction solution was set still, the upper flowing liquid was removed and the lower milky white liquid was centrifuged and separated.

[0038] (6) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0039] (7) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

Example 2

[0040] (1) Gelatinization of starch: 10 g of potato starch was weighed and dissolved in 800 mL of purified water, and then heated and stirred to be gelatinized at 50° C. for 60 min; the resulting solution was stirred uniformly and adjusted with a sodium hydroxide solution until the pH reached a range from 8 to 12, thus obtaining a gelatinized starch solution.

[0041] (2) Modification of starch: 3 g of 2-chloroethyldiethylamine was weighed and dissolved in 50 mL of water, the resulting solution was added to the gelatinized starch solution to further react at 70° C. for 10 h, thus obtaining a modified starch solution.

[0042] (3) Preparation of oil phase: 1200 mL of aviation kerosene was weighed and, added with 10 g of Tween-80 and 10 g of Span-80, and the resulting material was stirred at 60° C. for 30 min and set aside for later use.

[0043] (4) Emulsification and crosslinking: the modified starch was dropwise added to the oil phase and stirred to react at 60° C. for 60 min, 50 mL of epichlorohydrin was added to carry out crosslinking reaction for 5 h, and the reaction solution was set still after the reaction.

[0044] (5) Separation of precipitation: after the reaction solution was set still, the upper flowing liquid was removed and the lower milky white liquid was centrifuged and separated.

[0045] (6) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0046] (7) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

[0047] The obtained starch-based hemostatic powder was photographed by a scanning electron microscope, as shown in FIG. 1. The particle size of the starch-based hemostatic powder microspheres is between 20 μm and 180 μm, and the microspheres with a particle size between 50 μm and 150 μm account for not less than 70% of the total microsphere particles.

[0048] The obtained starch-based hemostatic powder was tested for hemostasis, and the hemostatic effect is shown in FIG. 2.

[0049] 2 mL of rabbit blood was taken and placed in a 10 mL test tube, 0.5 mg of heparin sodium was added, and after being mixed, the rabbit blood and heparin took a long-lasting anticoagulant effect and achieved good blood fluidity. Then, 50 mg of the above hemostatic powder was taken and added to the rabbit blood, and the rabbit blood was then shaken gently for 5-10 seconds. It was observed that a large number of blood clots were formed, indicating that the coagulation mechanism was quickly activated. Under a microscope, as shown in the photomicrograph in the left image of FIG. 2, red blood cells in normal blood are evenly distributed without aggregation; however, the red blood cells of blood added with the hemostatic powder aggregate obviously, the local concentration increases, and the activation of coagulation process is accelerated.

Example 3

[0050] (1) Gelatinization of starch: 15 g of potato starch was weighed and dissolved in 600 mL of purified water, and then heated and stirred to be gelatinized at 80° C. for 20 min; the resulting solution was stirred uniformly and adjusted with a sodium hydroxide solution until the pH reached a range from 8 to 12, thus obtaining a gelatinized starch solution.

[0051] (2) Modification of starch: 4 g of 2-chloroethyldiethylamine was weighed and dissolved in 50 mL of water, the resulting solution was added to the gelatinized starch solution to further react at 70° C. for 5 h, thus obtaining a modified starch solution.

[0052] (3) Preparation of oil phase: 1800 mL of liquid paraffin was weighed and, added with 4 g of Tween-80 and 4 g of Span-80, and the resulting material was stirred at 60° C. for 30 min and set aside for later use.

[0053] (4) Emulsification and crosslinking: the modified starch was dropwise added to the oil phase and stirred to react at 60° C. for 60 min, 50 mL of epichlorohydrin was added to carry out crosslinking reaction for 5 h, and the reaction solution was set still after the reaction.

[0054] (5) Separation of precipitation: after the reaction solution was set still, the upper flowing liquid was removed and the lower milky white liquid was centrifuged and separated.

[0055] (6) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0056] (7) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

Comparative Example 1

[0057] (1) Gelatinization of starch: 10 g of potato starch was weighed and dissolved in 500 mL of purified water, and then stirred to be gelatinized; 5 g of sodium carboxymethyl cellulose was added, and the pH of the resulting solution was adjusted to 11 with a sodium hydroxide solution.

[0058] (2) Preparation of oil phase: 500 mL of vegetable oil was added to a reaction kettle and heated to 70° C., and a mixture containing 10 g of a mixture of Span-80 and 10 g of Tween-80 was added.

[0059] (3) Emulsification and crosslinking: the gelatinized starch was mixed with the above oil phase, and 20 mL of epichlorohydrin was then added to react for 24 h, and the resulting material was taken.

[0060] (4) Separation of precipitation: absolute ethanol and ethyl acetate were added to separate the resulting material, and the upper oil phase was poured out.

[0061] (5) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0062] (6) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

Comparative Example 2

[0063] (1) Gelatinization of starch: 10 g of potato starch was weighed and dissolved in 800 mL of purified water, and then heated and stirred to be gelatinized at 50° C. for 60 min; the resulting solution was stirred uniformly and adjusted with a sodium hydroxide solution until the pH reached a range from 8 to 12, thus obtaining a gelatinized starch solution.

[0064] (2) Modification of starch: 3 g of 2-chloroethyldiethylamine was weighed and dissolved in 50 mL of water, the resulting solution was added to the gelatinized starch solution to further react at 70° C. for 10 h, thus obtaining a modified starch solution.

[0065] (3) Preparation of oil phase: 1200 mL of aviation kerosene was weighed and, added with 10 g of Tween-80 and 10 g of Span-80, and the resulting material was stirred at 60° C. for 30 min and set aside for later use.

[0066] (4) Emulsification and crosslinking: the modified starch was dropwise added to the oil phase and stirred to react at 60° C. for 60 min, 50 mL of epichlorohydrin was added to carry out crosslinking reaction for 5 h, and the reaction solution was set still after the reaction.

[0067] (5) Esterification modification: 5 mL of sodium trimetaphosphate solution was added to the modified starch after crosslinking, and the resulting solution was stirred to react at 35° C. to prepare a hydroxypropyl distarch phosphate solution;

[0068] (6) Ultrasonic treatment: the hydroxypropyl distarch phosphate solution (ice bath) was placed in an ultrasonic cell pulverizer and treated at a power of 450 w for 3 min (running for 2 s with a stop of 2 s).

[0069] (7) Separation of precipitation: after the reaction solution was set still, the upper flowing liquid was removed and the lower milky white liquid was centrifuged and separated.

[0070] (8) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0071] (9) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

Comparative Example 3

[0072] (1) Enzymatic hydrolysis of starch: 100 g of potato starch and 10 g of amylase were added in 250 mL of phosphate buffer (pH=5), the resulting solution was stirred evenly to take a hydrolysis reaction at 45° C. for 8 h, the reaction solution was subjected to centrifugal separation at a rotating speed of 4000 r/min, the precipitation was then dried in vacuum to obtain hydrolyzed starch.

[0073] (2) Preparation of oil phase: 1000 mL of vegetable oil was weighed and added with 10 g of Tween-80 and 10 g of Span-80, and the resulting material was stirred at 60° C. for 30 min and set aside for later use.

[0074] (3) Emulsification and crosslinking: 10 g of hydrolyzed starch was dropwise added to the oil phase and stirred to react at 60° C. for 20 min, 20 mL of sodium trimetaphosphate was added to carry out crosslinking reaction for 5 h, and the reaction solution was set still after the reaction.

[0075] (4) Separation of precipitation: after the reaction solution was set still, the upper flowing liquid was removed and the lower milky white liquid was centrifuged and separated.

[0076] (6) Purification and drying: the milky body after centrifugation was washed with absolute ethanol and then subjected to suction filtration and vacuum drying at 40° C. The dried powder was sieved with a screen to obtain the product before packaging.

[0077] (7) Packaging and sterilization: the sieved product was packaged in polyethylene bottles and then plastic-sealed, and then sterilized.

Comparative Example 4

[0078] (1) Enzymatic hydrolysis of starch: 100 g of potato starch and 10 g of amylase were added in 250 mL of phosphate buffer (pH=5), the resulting solution was stirred evenly to take a hydrolysis reaction at 45° C. for 8 h, the reaction solution was subjected to centrifugal separation at a rotating speed of 4000 r/min, the precipitation was then dried in vacuum to obtain hydrolyzed starch.

[0079] (2) Carboxymethylation of starch: 8 g of monochloroacetic acid was added in ethanol, the resulting solution was adjusted to be neutral with 0.3 mol/L sodium hydroxide solution, and then mixed with 80 g of ethanol-dissolved microporous starch prepared above; the mixture was then stirred thoroughly and transferred to a constant temperature tank; next, the mixture was continuously stirred at 50° C., 45 r/min for reaction; after 4 h, the reaction was completed, acetic acid was added to neutralize the reaction solution until the pH was 6.5, the reaction solution was then filtered, washed 3 times with ethanol, and dried for 24 h, thus obtaining carboxymethyl microporous starch.

[0080] (3) Boiling agglomeration: the above-mentioned carboxymethyl starch was placed in a boiling machine at 45° C. and added with distilled water to be coagulated and pelletized, thus obtaining a modified starch material. 50-500 μm microspheres accounted for no less than 90% of the total microsphere particles.

[0081] 1. Detection of Saline Absorption Rate and Water Absorption Rate of Hemostatic Powder:

[0082] The saline absorption rate of starch was determined by natural filtration method. 0.5 g of the above hemostatic powder was weighed accurately and put into 100 mL of saline (0.9% NaCl solution) respectively. The centrifuge tube was sealed and the hemostatic powder solution was fully swelled at room temperature. The solution was then filtered with a stainless steel sieve until there was almost no water drop; then, the mass of the hemostatic powder was determined, and the water absorption rate was calculated as follows:

Q=(m.sub.2−m.sub.1)/m.sub.1,

[0083] In the formula, Q is the water absorption rate, in the unit of g/g; m.sub.1 is the mass of the hemostatic powder before water absorption, in the unit of g; m.sub.2 is the mass of the hemostatic powder after water absorption, in the unit of g.

[0084] The water absorption rate of starch was measured by the Sessile Drop method, using the OCA40 Micro video contact angle measuring instrument from the German company Dataphysics. The results of the saline absorption rate and water absorption rate of the above-mentioned hemostatic powder are shown in Table 1.

TABLE-US-00001 TABLE 1 Performance comparison of hemostatic powder in saline absorption rate and water absorption rate Saline absorption Water absorption rate (mL/s) Group rate First 20s Second 20s Third 20s Example 1 8.3 0.0062 0.006 0.0055 Example 2 9.1 0.0078 0.0075 0.007 Example 3 8.2 0.0058 0.0056 0.0048 Comparative 8.2 0.0053 0.0052 0.004 Example 1 Comparative 7.4 0.0054 0.005 0.004 Example 2 Comparative 6.7 0.004 0.0035 0.0025 Example 3 Comparative 6.5 0.0056 0.004 0.0032 Example 4

[0085] The modified starch-based hemostatic powder prepared by the method of the present invention has a saline absorption rate and a water absorption rate close to or higher than that of the hemostatic powder prepared in Comparative Examples 1-4, and has a high water absorption rate and is more effective.

[0086] 2. Skin Irritation Test and Sensitization Test of Hemostatic Powder

[0087] The starch-based hemostatic powder obtained in the above Examples 1-3 was tested in accordance with GB/T 14233.2-2005 and GB/T 16886.10-2005 “Biological Evaluation of Medical Devices Part 10: Stimulation and Delayed Hypersensitivity Test”. Specifically, in addition to the absorption capacity, the extraction medium (extraction medium: physiological saline and vegetable oil) was added at a ratio of 0.2 g/mL for extraction, and the test solution was prepared at (37±1°) C. for (72±2) h; the test solution was taken for test in accordance with the test method specified in GB/T 16886.10-2005.

[0088] The test sample was directly contacted with the skin on both sides of the rabbit spine for 24 h, and the gauze piece was contacted with the skin on both sides of the rabbit spine in the same way as a control. (1±0.1) h, (24±2) h, (48±2) h, and (72±2) h after the contact, erythema and edema at the contact site were scored, and the primary formic acid irritation index (PII) was scored. The test results showed that the primary irritation index (PII) of the rabbits in contact with the hemostatic powder sample was 0.0, indicating that the hemostatic powder test solutions prepared in Examples 1-3 had no skin sensitization reaction.

[0089] 3. Cytotoxicity Test of Hemostatic Powder

[0090] The starch-based hemostatic powder obtained in the above Examples 1-3 was tested according to the MTT colorimetry specified in GB/T 16886.5-2016 “Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test”. Specifically, the prepared L929 fibroblast cell suspension was inoculated into a culture plate and cultured for 24 h and the supernatant was then removed. The positive control group was added with starch-based hemostatic powder test solution, the negative control group was added with negative control test solution, and the blank control group was added with fresh cell culture solution, and the culture was continued for 72 h. By observing the cell morphology and calculating the relative cell proliferation rate, the results showed that the cytotoxicity grade of the hemostatic powder prepared in Examples 1-3 was grade 1, which met the requirements for clinical use.

[0091] 4. The Antibacterial Effect of Hemostatic Powder

[0092] The starch-based hemostatic powder prepared in the above Examples 1-3 was subjected to an antibacterial test, and the antibacterial effect was evaluated using the minimum inhibitory concentration MIC (mg/L). By the method of microwell dilution, 100 μL of hemostatic powder prepared in Examples 1-3 with different concentrations were added to 96 microwell plates, 10 μL of bacterial culture solution of the same concentration (10.sup.4 CFU/mL) was added to each microwell, incubated at 37° C. for 24 h and observed. The smallest concentration of the culture solution in the microwell without visible bacterial growth was the minimum inhibitory concentration (MIC).

TABLE-US-00002 TABLE 2 Comparison of the hemostatic powder solutions of Examples 1-3 in antibacterial performance MIC(mg/L) Group E. coli S. aureus P. aeruginosa Example 1 15.773 51.352 62.240 Example 2 15.987 63.175 66.002 Example 3 14.403 51.093 61.526

[0093] The hemostatic powder prepared in the embodiments of the present invention has obvious antibacterial effect and low toxicity to human cells.

[0094] 5. Hemostatic Effect of Hemostatic Powder on Animal Bleeding Model (Rabbit Femoral Artery Injury Model)

[0095] The test group used the starch-based hemostatic powder obtained in the above Examples 1-3. The control group did not use the starch-based hemostatic powder.

[0096] Two New Zealand albino rabbits were used in the test, and the femoral arteries of the two rabbits were punctured with a 7-gauge needle to squirt blood. After using the hemostatic powder, the test group was covered with gauze and pressed, and the control group was directly covered with gauze and pressed. The hemostatic effect was observed three minutes later.

[0097] The hemostatic powder used for the test group absorbed blood immediately after coming in contact with blood, and formed a viscous gel with blood to effectively cover the wound. It could effectively control wound bleeding within 1 minute. Moreover, after coming in contact with blood, the hemostatic powder adhered closely to the wound tissue to promote blood clotting to form a sealing effect on the damaged blood vessels of the bleeding point on the wound. The clot was not adhered to the pressing gloves or gauze dressing. When the gloves or gauze are removed, the clot was not damaged to cause secondary bleeding. The bleeding of the control group was not stopped, and the bleeding point was still bleeding after three minutes.

[0098] The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person familiar with the profession may use the technical content disclosed above to change or modify them into the equivalent embodiments of equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention still belong to the protection scope of the technical solution of the present invention.