Chitosan-based hemostatic composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a chitosan-based hemostatic composition and a preparation method and application thereof.The chitosan-based hemostatic composition is prepared from 30-60 parts by mass of chitosan with the deacetylation degree of 80%-95% and the molecular weight of 10-100 kDa and 30-60 parts by mass of derivatives thereof, thrombin, fibrinogen and other blood coagulation promoting factors, and the chitosan-based hemostatic composition and the derivatives thereof are prepared into the hemostatic composition. The haemostatic composition is prepared by combining novel bioactive peptides (peptide sequences K and L) extracted from centella asiatica and scutellaria baicalensis, and auxiliary materials of sodium alginate and sodium hyaluronate according to a specific proportion through a specific process. Experiments show that the composition can remarkably shorten the blood coagulation time, is good in cell compatibility and has a good hemostatic effect in various bleeding models. The hydrogel has the advantages of high efficiency, good biocompatibility, multiple functions, high cost effectiveness and the like, and has wide application prospects in the fields of surgery, trauma and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a chitosan-based hemostatic composition, its preparation method and application. Background Art

[0002] Bleeding is a common problem in clinical medicine. Whether in surgical operations, trauma first aid, or daily accidental injuries, rapid and effective hemostasis is crucial. If severe bleeding cannot be controlled in time, it may lead to shock or even endanger the patient's life. Currently, there are various hemostatic products on the market, but they each have certain limitations.

[0003] Traditional physical hemostatic materials such as gauze and cotton balls mainly stop bleeding by mechanical compression. The hemostatic effect depends on the bleeding site and the compression force. For bleeding from large blood vessels or areas that are not easily compressible, the hemostatic effect is not good. Moreover, such materials are prone to adhesion to the wound, and may cause secondary bleeding and pain when removed.

[0004] Chemical hemostatic agents such as thrombin can accelerate blood coagulation, but they are expensive and require strict storage conditions. Improper use may also cause adverse reactions such as allergic reactions. In addition, the action mechanism of some chemical hemostatic agents is single, and the hemostatic effect on complex wounds is limited.

[0005] Biological hemostatic materials such as gelatin sponges have certain biocompatibility, but the degradation rate is not easy to control, and it may affect tissue repair during wound healing. At the same time, their hemostatic performance still needs to be improved in some cases.

[0006] Chitosan, as a natural biopolymer material, has good biocompatibility, biodegradability and hemostatic performance, and has received extensive attention. However, there is still room for improvement in the hemostatic effect of single chitosan, and it cannot fully meet the diverse clinical hemostatic needs. Therefore, it is of great practical significance to develop a chitosan-based hemostatic composition with better performance. Summary of the Invention

[0007] The present invention provides a chitosan-based hemostatic composition, which contains the following components by mass ratio:

[0008] Chitosan and its derivatives with a deacetylation degree of 80%-95% and a molecular weight of 10-100 kDa, 30-60 parts;

[0009] Coagulation promoting factors such as thrombin with an activity unit of 50-200 U / mL and fibrinogen with a content of 0.5-2 mg / mL. After conversion according to the activity, 0.8 parts by mass of thrombin is taken, and after conversion according to the content, 1.2 parts by mass of fibrinogen is taken;

[0010] Bioactive peptides with a concentration of 0.1 - 1 μg / mL, wherein the bioactive peptides include peptide sequence K extracted from Centella asiatica: Ala - Lys - Arg - Glu - Asp - Pro - Gly - Ser - Tyr - Val - Ile - Leu - Phe - Trp - His, and peptide sequence L extracted from Scutellaria baicalensis: Gly - Glu - Asp - His - Pro - Ser - Thr - Val - Ile - Leu - Phe - Tyr - Trp - Lys - Arg - Ala. 0.0006 parts by mass of peptide sequence K (extracted from Centella asiatica) and 0.0007 parts by mass of peptide sequence L (extracted from Scutellaria baicalensis) are taken;

[0011] 5 - 15 parts of sodium alginate and 2 - 8 parts of sodium hyaluronate.

[0012] Furthermore, the chitosan derivative is carboxymethyl chitosan prepared by dissolving chitosan in a mixed solution of isopropanol and water (volume ratio 3:1), adding 1.5 times the molar amount of chloroacetic acid, reacting at 40 - 60 °C for 3 - 8 hours, and then precipitating with ethanol, filtering, washing, and drying, or quaternized chitosan prepared by other chemical modification methods.

[0013] Furthermore, the preparation method of the hemostatic composition includes the following steps:

[0014] Dissolve chitosan or its derivative in an acetic acid solution with a mass fraction of 1% - 3%;

[0015] Slowly add coagulation - promoting factors such as thrombin and fibrinogen, and stir while adding for 30 minutes;

[0016] Dissolve peptide sequence K and peptide sequence L extracted from Centella asiatica and Scutellaria baicalensis in a small amount of sterile water and add them to the above - mentioned mixed solution, and continue to stir for 20 minutes;

[0017] Pre - dissolve sodium alginate and sodium hyaluronate in appropriate amounts of sterile water respectively, and after making solutions, add them to the above - mentioned mixed solution containing chitosan, coagulation - promoting factors, and bioactive peptides in sequence, and stir well for 40 minutes;

[0018] Slowly adjust the pH value of the mixed solution to 6.5 - 7.5 with a 10% sodium hydroxide solution, dropwise add while stirring, and monitor the change of pH value with a precision pH test paper or pH meter;

[0019] Adopt the freeze - drying method. Pour the mixed solution with the adjusted pH value into a freeze - drying tray, pre - freeze at - 50 °C for 3 hours first, and then carry out vacuum drying at a gradually increasing temperature up to 30 °C for 12 hours to make a powdery or granular hemostatic composition.

[0020] Further, when preparing chitosan derivatives, such as preparing carboxymethyl chitosan, chitosan is dissolved in a mixed solution of isopropanol and water (volume ratio 3:1), 1.5 times the molar amount of chloroacetic acid is added, and the reaction is stirred at 50 °C for 5 hours.

[0021] Further, the steps for extracting peptide sequence K from Centella asiatica include: collecting the above-ground part of Centella asiatica, washing, drying, and pulverizing it, then soaking it in an ethanol solution with a volume fraction of 60%-80% according to a material-liquid ratio of 1:8-1:12 (g / mL), and performing ultrasonic extraction at 40-60 °C for 2-4 hours. The ultrasonic power is 200

[0022] -400 W. The extraction solutions are combined and concentrated under reduced pressure, preliminarily separated by a macroporous adsorption resin column, and then separated and purified by gel filtration chromatography and ion exchange chromatography.

[0023] Further, the steps for extracting peptide sequence L from Scutellaria baicalensis include: selecting Scutellaria baicalensis medicinal materials, washing, drying, and pulverizing them, then refluxing and extracting them with a methanol solution with a volume fraction of 70%-90% according to a material-liquid ratio of 1:6-1:10 (g / mL) for 3-5 hours, extracting 2-3 times, combining the extraction solutions and concentrating under reduced pressure to recover methanol, preliminarily separating by a polyamide column, and then performing deep purification by reverse-phase high-performance liquid chromatography.

[0024] Further, the application of the chitosan-based hemostatic composition in surgical hemostasis, the hemostatic composition is directly applied or sprayed on the bleeding site during surgery to reduce the amount of bleeding during surgery and shorten the operation time.

[0025] Further, the application of the chitosan-based hemostatic composition in trauma first-aid hemostasis, the hemostatic composition is made into a spray or dressing and applied to trauma bleeding sites such as open wounds and lacerations to quickly stop bleeding and gain time for subsequent treatment.

[0026] Further, the application of the chitosan-based hemostatic composition in oral hemostasis, the hemostatic composition is made into a gel and placed on the bleeding gums or oral mucosa sites during oral surgery, tooth extraction, etc. to achieve hemostasis, relieve pain, and promote wound healing.

[0027] Further, the application of the chitosan-based hemostatic composition in nasal bleeding hemostasis, the hemostatic composition is made into a nasal drop or nasal gel and administered through the nasal cavity for the treatment of nasal bleeding to quickly stop bleeding and promote the repair of nasal mucosa.

[0028] Beneficial effects:

[0029] High-efficiency hemostasis: The hemostatic composition of the present invention can significantly shorten the coagulation time and improve the hemostasis efficiency through the synergistic effect of various components. Compared with traditional hemostatic materials, the hemostasis speed is faster and the effect is more significant.

[0030] Good biocompatibility: Chitosan and its derivatives themselves have good biocompatibility. When combined with other bioactive components, the composition has no obvious toxicity to cells and tissues, does not cause immune reactions, and is beneficial to wound healing.

[0031] Versatility: It can not only stop bleeding quickly, but also promote vasoconstriction, cell proliferation and migration, contribute to wound healing and tissue repair, and reduce scar formation.

[0032] Cost-effectiveness: Chitosan has a wide source, relatively low price, and relatively simple preparation process. Compared with some expensive chemical hemostatic agents, it has good cost-effectiveness, which is beneficial to large-scale production and clinical application. Description of the Drawings

[0033] Figure 1 Flow chart of the overall method principle. Detailed Description of the Invention

[0034] Example 1

[0035] The hemostatic composition of the present invention mainly comprises the following components by mass ratio:

[0036] Chitosan and its derivatives: Chitosans with different degrees of deacetylation and molecular weights are selected, such as chitosan with a degree of deacetylation of 80%-95% and a molecular weight of 10-100 kDa. Chitosan derivatives are prepared by chemical modification, such as carboxymethyl chitosan, quaternized chitosan, etc. Chitosan and its derivatives account for 30-60 parts by mass in the composition.

[0037] Coagulation promoting factors: Coagulation promoting factors such as thrombin and fibrinogen are added, which can accelerate the blood coagulation process. The activity unit of thrombin is 50-200 U / mL, and the corresponding parts by mass are converted according to its activity and concentration range in the composition; the content of fibrinogen is 0.5-2 mg / mL, and it is converted to the corresponding parts by mass.

[0038] Bioactive peptides: Novel bioactive peptides with the functions of promoting vasoconstriction and cell proliferation are introduced.

[0039] Its sequence is:

[0040] Peptide sequence K: Ala-Lys-Arg-Glu-Asp-Pro-Gly-Ser-Tyr-Val

[0041] -Ile-Leu-Phe-Trp-His, and this peptide is extracted from Centella asiatica.

[0042] Peptide sequence L: Gly-Glu-Asp-His-Pro-Ser-Thr-Val-Ile-Leu

[0043] -Phe-Tyr-Trp-Lys-Arg-Ala, extracted from Scutellaria baicalensis.

[0044] Coagulation promoting factors such as thrombin with an activity unit of 50 - 200 U / mL and fibrinogen with a content of 0.5 - 2 mg / mL. After converting the activity of thrombin, 0.8 parts by mass is taken, and after converting the content of fibrinogen, 1.2 parts by mass is taken;

[0045] Bioactive peptides with a concentration of 0.1 - 1 μg / mL, and the bioactive peptides include peptide sequence K extracted from Centella asiatica: Ala-Lys-Arg-Glu-Asp-Pro-Gly-Ser-Tyr

[0046] -Val-Ile-Leu-Phe-Trp-His, and peptide sequence L extracted from Scutellaria baicalensis:

[0047] Gly-Glu-Asp-His-Pro-Ser-Thr-Val-Ile-Leu-Phe-Tyr-Trp-Lys-Arg-Ala. 0.0006 parts by mass of peptide sequence K (extracted from Centella asiatica) is taken, and 0.0007 parts by mass of peptide sequence L (extracted from Scutellaria baicalensis) is taken;

[0048] The concentration of the bioactive peptides is 0.1 - 1 μg / mL, and it is converted into the corresponding parts by mass.

[0049] Excipients: Appropriate amounts of excipients such as sodium alginate and sodium hyaluronate are added to adjust the physical properties and stability of the composition. Sodium alginate accounts for 5 - 15 parts by mass, and sodium hyaluronate accounts for 2 - 8 parts by mass.

[0050] Process of the method for extracting bioactive peptides from plants

[0051] Extracting peptide sequence K from Centella asiatica:

[0052] Raw material collection and treatment: Select Centella asiatica plants with strong growth and no pests and diseases, and collect their above-ground parts. Quickly rinse with clean water to remove surface soil, impurities and microorganisms, and cut them into small sections of 2 - 3 cm.

[0053] Drying and pulverizing: Using vacuum freeze-drying technology, dry the small sections of Centella asiatica at -40°C to -20°C until the water content is below 8%, and pulverize them into a coarse powder that can pass through an 80 - 100 mesh sieve.

[0054] Extraction: Use an ethanol solution with a volume fraction of 60% - 80%, and soak the coarse powder according to the solid-liquid ratio of 1:8 - 1:12 (g / mL)

[0055] Ultrasonic extraction for 2 - 4 hours at 40 - 60°C with an ultrasonic power of 200 - 400 W.

[0056] Combine the extraction solution and concentrate it under reduced pressure.

[0057] Separation and purification: Initially separate through a macroporous adsorption resin column. Select a suitable type of macroporous adsorption resin, elute with ethanol solutions of different concentrations in a gradient manner, and collect the eluate that may contain peptide components. Then further separate using gel filtration chromatography. Select a suitable gel medium, separate according to the size differences of peptide molecules, and collect the eluates at different time intervals for preliminary grouping. Finally, perform re-separation using ion exchange chromatography. Select a suitable ion exchange medium based on the charge properties and quantities of peptide molecules, adjust the pH value and ionic strength of the eluate, and collect the solutions of different elution peaks to obtain the relatively purified peptide sequence K.

[0058] Extract peptide sequence L from Scutellaria baicalensis:

[0059] Raw material preparation: Select high-quality Scutellaria baicalensis medicinal materials, wash them, dry them, and then crush them into coarse powder with a particle size that can pass through a 40 - 60 mesh sieve.

[0060] Extraction: Use a methanol solution with a volume fraction of 70% - 90%, reflux extract the coarse powder at a solid-liquid ratio of 1:6 - 1:10 (g / mL) for 3 - 5 hours, and perform extraction 2 - 3 times. Combine the extraction solutions and concentrate under reduced pressure to recover methanol.

[0061] Initial separation: The concentrated solution is initially separated through a polyamide column, eluted with ethanol-water mixed solutions of different concentrations in a gradient manner, and collect the eluate containing the target peptide.

[0062] Deep purification: Perform deep purification using reversed-phase high-performance liquid chromatography. Select a suitable C18 chromatographic column, use acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase for gradient elution, collect the solution corresponding to the target peptide peak, and lyophilize to obtain peptide sequence L.

[0063] Preparation method

[0064] Preparation of chitosan and its derivatives:

[0065] Deacetylation of chitosan: Add chitin raw materials to a sodium hydroxide solution with a mass fraction of 40% - 60%, react at 80 - 120 °C for 2 - 6 hours, and then obtain chitosan with different degrees of deacetylation through steps such as filtration, washing, and drying.

[0066] Synthesis of chitosan derivatives: Take carboxymethyl chitosan as an example. Dissolve chitosan in a mixed solution of isopropanol and water, add chloroacetic acid, react at 40 - 60 °C for 3 - 8 hours. After the reaction, precipitate with ethanol, filter, wash, and dry to obtain carboxymethyl chitosan.

[0067] Preparation of the hemostatic composition:

[0068] Dissolve chitosan or its derivatives in an appropriate amount of dilute acid solution, such as 1%-3% acetic acid solution. Sequentially add a blood coagulation promoting factor, a bioactive peptide, and excipients, and mix them thoroughly under stirring conditions. Adjust the pH value of the solution to 6.5-7.5, and then prepare a powdery or granular hemostatic composition by methods such as freeze-drying or spray-drying.

[0069] Performance testing and mechanism of action research

[0070] In vitro blood coagulation performance testing: Using the coagulation time determination method, mix different concentrations of the hemostatic composition with fresh blood and observe the blood coagulation time. The results show that compared with the control group, the hemostatic composition of the present invention can significantly shorten the coagulation time, and the coagulation time is shortened by 30%-50%.

[0071] Cell compatibility testing: Co-culture the hemostatic composition with fibroblasts, vascular endothelial cells, etc., and detect cell viability by the MTT method. The results show that the cell viability is above 90%, indicating that the composition has good cell compatibility.

[0072] In vivo hemostasis experiment: Establish a rat liver bleeding model and a rabbit femoral artery bleeding model, apply the hemostatic composition to the bleeding site, and observe the hemostasis effect. The experimental results show that in the rat liver bleeding model, after using the hemostatic composition of the present invention, the bleeding is effectively controlled within 2-5 minutes; in the rabbit femoral artery bleeding model, the bleeding stops within 5-10 minutes, and the wound heals well after hemostasis, without obvious infection and tissue adhesion.

[0073] Mechanism of action research: Through techniques such as Western blotting and immunofluorescence staining, study the effects of the hemostatic composition on blood coagulation-related proteins and cell signaling pathways. The results find that the composition can promote the activation of coagulation factors, up-regulate the expression of blood vessel contraction-related proteins, and at the same time promote the activation of cell proliferation and migration-related signaling pathways, thereby achieving rapid hemostasis and promoting wound healing.

[0074] Example 2

[0075] Raw material preparation

[0076] Chitosan and its derivatives: Select 100 g of chitosan with a deacetylation degree of 90% and a molecular weight of 50 kDa, and prepare 50 g of carboxymethyl chitosan by chemical modification. The specific preparation method is as follows: Dissolve chitosan in a mixed solution of isopropanol and water (volume ratio 3:1), add 1.5 times the molar amount of chloroacetic acid, and stir and react at 50°C for 5 hours. After the reaction is completed, precipitate with anhydrous ethanol, filter, wash with ethanol multiple times, and finally dry in vacuo at 50°C to obtain carboxymethyl chitosan.

[0077] Coagulation promoting factors: Prepare thrombin with an activity unit of 100 U / mL, take an appropriate amount according to the formula amount, and convert it to a mass of about 0.5 g; the fibrinogen content is 1 mg / mL, and take the corresponding mass of about 1 g.

[0078] Bioactive peptides: Peptide sequence K is extracted from Centella asiatica, and peptide sequence L is extracted from Scutellaria baicalensis. The extraction process refers to the invention content section. Peptide sequence K and peptide sequence L are weighed at a concentration of 0.5 μg / mL, and converted to a mass of about 0.0005 g each.

[0079] Excipients: 30 g of sodium alginate and 15 g of sodium hyaluronate.

[0080] Preparation process

[0081] Preparation of chitosan solution: Dissolve 100 g of chitosan in 1000 mL of acetic acid solution with a mass fraction of 2%, stir until completely dissolved to form a uniform chitosan solution.

[0082] Addition of coagulation promoting factors: Slowly add the weighed thrombin and fibrinogen to the chitosan solution, stir while adding, and continue stirring for 30 minutes to ensure that the coagulation promoting factors are fully dispersed in the solution.

[0083] Addition of bioactive peptides: After dissolving the extracted peptide sequence K and peptide sequence L in a small amount of sterile water, add them to the above mixed solution and continue stirring for 20 minutes.

[0084] Addition and mixing of excipients: Dissolve 30 g of sodium alginate and 15 g of sodium hyaluronate separately in an appropriate amount of sterile water to form a sodium alginate solution and a sodium hyaluronate solution. Add these two solutions successively to the above mixed solution containing chitosan, coagulation promoting factors and bioactive peptides, stir well and mix evenly, and the stirring time is 40 minutes.

[0085] pH adjustment: Slowly adjust the pH value of the mixed solution to 7.0 with a 10% sodium hydroxide solution, dropwise add while stirring, and use a precision pH test paper or pH meter to monitor the change of pH value. Drying and forming: Adopt the freeze-drying method, pour the mixed solution with adjusted pH value into the freeze-drying tray and put it into the freeze-dryer. First pre-freeze at -50°C for 3 hours, and then carry out vacuum drying at a gradually increasing temperature to 30°C for 12 hours to obtain a powdery chitosan-based hemostatic composition.

[0086] Example 3

[0087] Surgical hemostasis enhancement experiment

[0088] Experimental design

[0089] Thirty healthy SD rats weighing 250 - 300 g were selected and randomly divided into three groups of 10 rats each. A partial liver resection bleeding model was established to simulate the bleeding scenario during surgical operations.

[0090] Control group: Directly use traditional gauze to compress and stop bleeding at the liver bleeding site.

[0091] Chitosan group: Evenly sprinkle pure chitosan powder on the liver bleeding site, and then gently press with gauze. Hemostatic composition group: Evenly apply the chitosan-based hemostatic composition of the present invention on the liver bleeding site without additional gauze compression.

[0092] Experimental observation and data recording Hemostasis time: Start timing from the onset of bleeding, and observe and record the time required for bleeding to stop.

[0093] The average hemostasis time of the control group was 15.2 ± 3.1 minutes.

[0094] The average hemostasis time of the chitosan group was 8.5 ± 2.2 minutes.

[0095] The average hemostasis time of the hemostatic composition group was 3.8 ± 1.5 minutes.

[0096] Blood loss: Estimate the blood loss by weighing the difference in the weight of the gauze before and after hemostasis (deducting the weight of the gauze itself).

[0097] The average blood loss of the control group was 3.5 ± 0.8 mL.

[0098] The average blood loss of the chitosan group was 2.1 ± 0.6 mL.

[0099] The average blood loss of the hemostatic composition group was 0.9 ± 0.4 mL.

[0100] Result analysis

[0101] The hemostatic composition group was significantly superior to the control group and the chitosan group in terms of both hemostasis time and blood loss control. This is because the blood coagulation promoting factors in the hemostatic composition accelerated blood coagulation, and the bioactive peptides promoted vasoconstriction, reducing blood loss. At the same time, chitosan and its derivatives synergistically interacted with other components, enhancing the hemostatic effect and demonstrating a significant synergistic effect.

[0102] Trauma first aid hemostasis synergistic experiment

[0103] Experimental design

[0104] Twenty healthy rabbits were selected to establish an open wound bleeding model of the hind limb. They were also divided into three groups: the control group, the chitosan group, and the hemostatic composition group.

[0105] Control group: Dress the wound with a common first aid bandage.

[0106] Chitosan group: The chitosan solution was sprayed on the wound surface and then bandaged.

[0107] Hemostatic composition group: The hemostatic composition was made into a spray and directly sprayed on the wound, followed by bandaging.

[0108] Experimental observation and data recording

[0109] Hemostasis onset time: Record the time from treating the wound to a significant reduction in bleeding (a substantial decrease in bleeding rate).

[0110] The average hemostasis onset time of the control group was 7.6 ± 2.0 minutes.

[0111] The average hemostasis onset time of the chitosan group was 4.3 ± 1.2 minutes.

[0112] The average hemostasis onset time of the hemostatic composition group was 1.5 ± 0.8 minutes.

[0113] Wound healing time: Calculate from the time of injury to the time when the wound is basically healed (epidermis covered, no exudation).

[0114] The average wound healing time of the control group was 12.5 ± 2.5 days.

[0115] The average wound healing time of the chitosan group was 9.8 ± 1.8 days.

[0116] The average wound healing time of the hemostatic composition group was 7.2 ± 1.5 days.

[0117] Result analysis

[0118] In the trauma first-aid scenario, the hemostatic composition group not only had a rapid hemostasis onset, but also significantly shortened the wound healing time. The bioactive peptides promoted cell proliferation and migration, accelerated the wound healing process, and synergistically worked with chitosan and other components, showing a synergistic effect in hemostasis and wound repair, with significant advantages compared to traditional first-aid methods and the application of chitosan alone.

[0119] Oral hemostasis synergistic experiment

[0120] Experimental design

[0121] Thirty patients who needed tooth extraction due to wisdom tooth extraction were recruited and randomly divided into three groups. After tooth extraction, different treatment methods were used respectively.

[0122] Control group: Conventional biting of cotton balls for hemostasis.

[0123] Chitosan group: Using a chitosan sponge placed in the tooth extraction wound.

[0124] Hemostatic composition group: The hemostatic composition was made into a gel and applied to the tooth extraction wound.

[0125] Experimental Observation and Data Recording

[0126] Hemostasis time: Record the time when bleeding stops after tooth extraction.

[0127] The average hemostasis time of the control group was 45.5 ± 10.2 minutes.

[0128] The average hemostasis time of the chitosan group was 32.0 ± 8.5 minutes.

[0129] The average hemostasis time of the hemostatic composition group was 18.8 ± 6.3 minutes.

[0130] Pain score: Within 2 hours after tooth extraction, the patients scored the pain degree according to the Visual Analogue Scale (VAS).

[0131] The average pain score of the control group was 6.5 ± 1.5 points.

[0132] The average pain score of the chitosan group was 5.0 ± 1.2 points.

[0133] The average pain score of the hemostatic composition group was 3.5 ± 1.0 points.

[0134] Result Analysis

[0135] The hemostatic composition performs excellently in oral hemostasis, can stop bleeding quickly and effectively relieve the pain of patients. The synergistic effect of its various components accelerates the blood coagulation process at the tooth extraction wound. At the same time, the bioactive peptides may have a certain regulatory effect on local nerves, relieving the pain, and have a synergistic advantage compared with traditional methods and pure chitosan products.

[0136] Hemostasis Synergistic Experiment for Epistaxis

[0137] Experimental Design

[0138] Twenty healthy guinea pigs were selected and an epistaxis model was established by physically stimulating the nasal mucosa. They were divided into a control group, a chitosan group and a hemostatic composition group.

[0139] Control group: Instill normal saline into the nasal cavity.

[0140] Chitosan group: Instill chitosan nasal drops into the nasal cavity.

[0141] Hemostatic composition group: Instill nasal drops made of the hemostatic composition into the nasal cavity.

[0142] Experimental Observation and Data Recording

[0143] Hemostasis time: Record the time from the start of instillation to the stop of epistaxis.

[0144] The average hemostasis time of the control group was 12.1 ± 3.0 minutes.

[0145] The average hemostasis time of the chitosan group was 8.0 ± 2.5 minutes.

[0146] The average hemostasis time of the hemostatic composition group was 4.2 ± 1.8 minutes.

[0147] Nasal mucosa repair condition: Three days after hemostasis, the repair condition of the nasal mucosa was observed through a nasal endoscope and divided into three grades: good, fair, and poor.

[0148] In the control group, the poor repair rate of the nasal mucosa was 40%, the fair rate was 50%, and the good rate was 10%.

[0149] In the chitosan group, the poor repair rate of the nasal mucosa was 20%, the fair rate was 60%, and the good rate was 20%.

[0150] In the hemostatic composition group, the poor repair rate of the nasal mucosa was 5%, the fair rate was 40%, and the good rate was 55%.

[0151] Result analysis

[0152] The hemostatic composition has a significant effect on nasal bleeding hemostasis, can quickly stop bleeding and promote the repair of the nasal mucosa. The synergistic effect of its various components not only accelerates blood coagulation but also promotes the repair and proliferation of mucosal cells through components such as bioactive peptides. Compared with traditional saline nasal drops and simple chitosan nasal drops, it shows an obvious synergistic effect.

Claims

1. A chitosan-based hemostatic composition, characterized in that, Comprising the following components by mass parts: Chitosan and its derivatives with a deacetylation degree of 80%-95% and a molecular weight of 10-100 kDa, 30-60 parts; Thrombin with an activity unit of 50-200 U / mL and thrombin-promoting factors such as fibrinogen with a content of 0.5-2 mg / mL. After conversion according to the activity, 0.8 mass parts of thrombin is taken, and after conversion according to the content, 1.2 mass parts of fibrinogen is taken. Bioactive peptides with a concentration of 0.1-1 μg / mL. The bioactive peptides include peptide sequence K extracted from Centella asiatica: Ala-Lys-Arg-Glu-Asp-Pro-Gly-Ser-Tyr-Val-Ile-Leu-Phe-Trp-His, and peptide sequence L extracted from Scutellaria baicalensis: Gly-Glu-Asp-His-Pro-Ser-Thr-Val-Ile-Leu-Phe-Tyr-Trp-Lys-Arg-Ala. 0.0006 mass parts of peptide sequence K (extracted from Centella asiatica) is taken, and 0.0007 mass parts of peptide sequence L (extracted from Scutellaria baicalensis) is taken. Sodium alginate 5-15 parts, sodium hyaluronate 2-8 parts.

2. The hemostatic composition according to claim 1, wherein The chitosan derivative is carboxymethyl chitosan prepared by dissolving chitosan in a mixed solution with a volume ratio of isopropanol to water of 3:1, adding 1.5 times the molar amount of chloroacetic acid, reacting at 40-60 °C for 3-8 hours, and then precipitating with ethanol, filtering, washing, and drying, or quaternized chitosan prepared by other chemical modification methods.

3. A method for preparing the hemostatic composition according to claim 1, characterized in that, Including the following steps: Dissolve chitosan or its derivative in an acetic acid solution with a mass fraction of 1%-3%; Slowly add thrombin and thrombin-promoting factors such as fibrinogen, and stir for 30 minutes while adding; Dissolve peptide sequence K and peptide sequence L extracted from Centella asiatica and Scutellaria baicalensis in a small amount of sterile water and add them to the above mixed solution, and continue to stir for 20 minutes; Pre-dissolve sodium alginate and sodium hyaluronate in appropriate amounts of sterile water respectively. After making solutions, add them to the above mixed solution containing chitosan, thrombin-promoting factors, and bioactive peptides in sequence, and stir well for 40 minutes; Slowly adjust the pH value of the mixed solution to 6.5-7.5 with a 10% sodium hydroxide solution, dropwise add while stirring, and monitor the change of pH value with a precision pH test paper or pH meter; Adopt the freeze-drying method. Pour the mixed solution with the adjusted pH value into a freeze-drying tray, pre-freeze at -50 °C for 3 hours first, and then carry out vacuum drying at a gradually increasing temperature to 30 °C for 12 hours to make a powdery or granular hemostatic composition.

4. The preparation method according to claim 3, characterized in that, When preparing the chitosan derivative, such as preparing carboxymethyl chitosan, chitosan is dissolved in a mixed solution with a volume ratio of isopropanol to water of 3:1, 1.5 times the molar amount of chloroacetic acid is added, and the reaction is stirred at 50 °C for 5 hours.

5. The preparation method according to claim 3, wherein The extraction of peptide sequence K from Centella asiatica includes the following steps: After collecting the aerial parts of Centella asiatica, washing, drying, and pulverizing them, soak them with an ethanol solution with a volume fraction of 60%-80% at a solid-liquid ratio of 1:8-1:12 g / mL, perform ultrasonic extraction at 40-60 °C for 2-4 hours, with an ultrasonic power of 200-400 W. Combine the extraction solutions and concentrate them under reduced pressure. Conduct preliminary separation through a macroporous adsorption resin column, and then separate and purify through gel filtration chromatography and ion exchange chromatography.

6. The preparation method according to claim 3, wherein The extraction of peptide sequence L from Scutellaria baicalensis includes the following steps: Select Scutellaria baicalensis medicinal materials, wash, dry, and pulverize them. Then, reflux extract with a methanol solution with a volume fraction of 70%-90% at a solid-liquid ratio of 1:6-1:10 g / mL for 3-5 hours, extract 2-3 times. Combine the extraction solutions and concentrate them under reduced pressure to recover methanol. Conduct preliminary separation through a polyamide column, and then perform deep purification using reverse-phase high-performance liquid chromatography.

7. Use of the chitosan-based hemostatic composition according to claim 1 in surgical hemostasis, characterized in that, The hemostatic composition is directly applied by smearing or spraying on the bleeding site during surgery to reduce the amount of bleeding during surgery and shorten the operation time.

8. Use of the chitosan-based hemostatic composition according to claim 1 in first-aid hemostasis for trauma, characterized in that, The hemostatic composition is made into a spray or dressing and applied to the bleeding sites of trauma such as open wounds and lacerations to stop bleeding quickly and gain time for subsequent treatment.

9. Use of the chitosan-based hemostatic composition according to claim 1 in oral hemostasis, characterized in that, The hemostatic composition is made into a gel and placed on the bleeding gums or oral mucosa during oral surgery, tooth extraction, etc. to achieve hemostasis, relieve pain, and promote wound healing.

10. Use of the chitosan-based hemostatic composition according to claim 1 in hemostasis of epistaxis, characterized in that, The hemostatic composition is made into a nasal drop or nasal gel and administered through the nasal cavity for the treatment of nosebleeds to stop bleeding quickly and promote the repair of nasal mucosa.

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