Analgesic hemostatic agent and method for preparing the same

Abstract

The application provides an analgesic hemostatic agent and a preparation method thereof, and belongs to the technical field of medicines. A decellularized material is coupled with a snake venom thrombin and ibuprofen, embedded in a pH-responsive polymer to prepare pH-responsive microspheres, and added into a temperature-sensitive hydrogel together with a traditional Chinese medicine fermentation product to prepare an analgesic hemostatic agent water agent. Porous calcium carbonate microspheres with fixed solid acid are mixed with hydroxyapatite to prepare a spray analgesic hemostatic agent powder, which is used in combination with the analgesic hemostatic agent water agent to prepare the analgesic hemostatic agent. The analgesic hemostatic agent prepared by the application adopts a combination of the powder and the water agent, can better maintain the drug activity, has good hemostatic, analgesic, anti-inflammatory and antibacterial effects, and has a slow and controlled release and a long-acting effect, so that the drug application amount is reduced, the patient compliance is improved, and the analgesic hemostatic agent has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a kind of analgesic hemostatic and preparation method thereof. BACKGROUND

[0002] Hemostasis is a complex physiological process, mainly affected by blood coagulation, platelet aggregation and vasoconstriction and other important factors. When blood vessel injury occurs, the body's own coagulation mechanism is fully manifested, the activity of prothrombin activator is enhanced, the formation is increased, and then prothrombin is converted into thrombin, which catalyzes fibrinogen to convert into fibrin, and also makes the body produce factors against fibrinolysis process. Any drug that can promote bleeding to stop is called hemostatic, which mainly promotes coagulation by enhancing or inhibiting coagulation factors, so as to achieve the purpose of hemostasis. The natural hemostatic drugs used in clinic mainly shorten the coagulation time, promote local vasoconstriction, enhance the resistance of capillary and reduce permeability, inhibit fibrinolysis process, increase platelet count, shorten thrombin generation time and enhance platelet third factor activity to achieve hemostasis. Most of the commonly used hemostatic drugs have single efficacy and cannot fully meet the clinical needs. Some natural hemostatic drugs not only have hemostatic effect, but also can promote blood dissolution and inhibit platelet aggregation, showing the unique efficacy of hemostasis and blood circulation in clinic.

[0003] Chinese patent application CN104324409A discloses a new type of rapid hemostatic adhesive plaster, which comprises a hemostatic layer, a microporous polytetrafluoroethylene layer and a separation layer, the microporous polytetrafluoroethylene layer is located between the hemostatic layer and the separation layer, the hemostatic layer, the microporous polytetrafluoroethylene layer and the separation layer are bonded by an adhesive, the hemostatic layer comprises the following raw materials by weight: pullulan 15-35 parts, chitosan quaternary ammonium salt 10-25 parts, succinyl chitosan 15-30 parts, dextran 18-30 parts, sodium hyaluronate 10-15 parts, indigo 3-8 parts, antibacterial agent 3-8 parts, anti-inflammatory agent 3-8 parts, starch 5-15 parts and plasticizer 5-10 parts. The invention is suitable for medical hemostasis, antibiosis and promotion of wound healing.

[0004] Chinese patent application CN104307027A discloses a medical chitosan rapid hemostatic composite dressing, which comprises the following raw materials by weight: chitosan 15-25 parts, dextran 10-20 parts, hydroxypropyl chitosan 15-25 parts, antibacterial agent 1-5 parts, calming anti-inflammatory agent 2-8 parts and mannose 2-10 parts. The dressing provided by the invention is suitable for medical rapid hemostasis, antibiosis and anti-inflammatory and promotion of wound healing.

[0005] The above patent products can play a role in hemostasis, but have problems such as single pharmacological action, and the hemostatic effect still needs to be further improved, and the effect is poor for hemorrhagic wounds accompanied by pain such as hemorrhoids, which limits the application. SUMMARY

[0006] The present application aims to provide a pain-relieving hemostatic agent and a preparation method thereof, which adopts the form of powder and water agent, can better maintain drug activity, has good hemostatic, analgesic, anti-inflammatory and bacteriostatic effects, is a sustained-release drug, has long-acting effect, reduces the amount of drug application, improves patient compliance, and has a wide application prospect.

[0007] The technical solution of the present application is as follows:

[0008] The present application provides a preparation method of a pain-relieving hemostatic agent, which couples decellularized material with snake venom thrombin and ibuprofen, embeds them in a pH-responsive polymer to prepare pH-responsive microspheres, adds traditional Chinese medicine fermentation into a temperature-sensitive hydrogel to prepare a pain-relieving hemostatic agent water agent, mixes porous calcium carbonate microspheres fixed with solid acid with hydroxyapatite to prepare a pain-relieving hemostatic agent powder, and uses the pain-relieving hemostatic agent water agent and the pain-relieving hemostatic agent powder in combination to prepare the pain-relieving hemostatic agent.

[0009] As a further improvement of the present application, the following steps are included:

[0010] S1. Preparation of decellularized material: wash, depilate and wash pigskin, remove the epidermis cuticle layer and subcutaneous fat layer, take the dermis layer, chop, soak and shock treat with sodium hydroxide solution, trypsin enzymolysis, filter, add a surfactant solution to the solid, freeze with liquid nitrogen, room temperature remelt, repeat the freezing-remelting operation 2-3 times, filter, wash, freeze-dry, and prepare the decellularized material;

[0011] S2. Coupling: add the decellularized material and snake venom thrombin to water, add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDC), stir and activate, add ibuprofen, stir and react, and prepare the coupling material;

[0012] S3. Preparation of traditional Chinese medicine fermentation: add notoginseng, madder, Japanese knotweed and mugwort leaves to water, ball mill, sterilize, inoculate Bacillus subtilis seed liquid, ferment and culture, filter, dry the filtrate, and prepare the traditional Chinese medicine fermentation;

[0013] S4. Preparation of porous calcium carbonate microspheres: add a porogen, an emulsifier and a calcium salt to water to prepare a solution; drop the solution into fish oil, emulsify, drop carbonate solution, ultrasonic react, stand at room temperature, centrifuge, wash, dry, and prepare the porous calcium carbonate microspheres;

[0014] S5. Ball milling: mix the porous calcium carbonate microspheres and the solid acid by ball milling to obtain mixed microspheres;

[0015] S6. Preparation of pH-responsive microspheres: dissolve chitosan in an acid solution, add silk fibroin peptides, stir and mix uniformly to obtain a chitosan-silk fibroin peptide solution, add monomers to water to obtain a monomer solution; mix the chitosan-silk fibroin peptide solution and the monomer solution uniformly, add coupling materials and emulsifiers, drop into fish oil, emulsify, add an initiator, stir and react, centrifuge, wash, dry, and obtain pH-responsive microspheres;

[0016] S7. Preparation of analgesic and hemostatic agent: add carboxymethyl chitosan to water, add poloxamer P407 and poloxamer P188, add traditional Chinese medicine fermentation product and pH-responsive microspheres, stir and mix uniformly to obtain an analgesic and hemostatic agent water preparation, mix the mixed microspheres and hydroxyapatite powder uniformly to obtain an analgesic and hemostatic agent powder preparation, and when used, evenly apply the analgesic and hemostatic agent powder preparation to the affected area, and then spray the analgesic and hemostatic agent water preparation.

[0017] As a further improvement of the present application, the concentration of the sodium hydroxide solution in step S1 is 2-4wt%, the final concentration of the trypsin is 0.5-1wt%, and the concentration of the surfactant solution is 1-3wt%, and the surfactant is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, sodium dodecyl sulfonate, sodium tetradecyl benzene sulfonate, sodium hexadecyl sulfate, and sodium octadecyl sulfate.

[0018] As a further improvement of the present application, the mass ratio of the decellularized material, snake venom thrombin, NHS, EDC, and ibuprofen in step S2 is 10:3-5:2-4:2-4:1-3, the stirring activation time is 20-40min, and the stirring reaction time is 10-15h.

[0019] As a further improvement of the present application, the mass ratio of the radix notoginseng, rubia cordifolia, agrimony, and artemisia leaf in step S3 is 3-5:1-2:2-4:5-7, the bacterial content of the bacillus subtilis seed liquid is 10 8 -10 9 cfu / mL, the inoculation amount is 2-4v / v%, and the fermentation culture conditions are 20-25℃, 50-150r / min, and 36-72h of fermentation culture.

[0020] As a further improvement of the present application, the mass ratio of the pore forming agent, emulsifier, calcium salt, carbonate salt in step S4 is 0.5-1:1-2:10-15:18-20, the calcium salt is calcium chloride or calcium nitrate, the carbonate salt is sodium carbonate or potassium carbonate, the pore forming agent is selected from at least one of sodium polystyrene sulfonate or polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, Tween-85.

[0021] As a further improvement of the present application, the mass ratio of the porous calcium carbonate microspheres and solid acid in step S5 is 10:4-7, and the solid acid is selected from at least one of oxalic acid, citric acid, malic acid, and tartaric acid.

[0022] As a further improvement of the present application, the mass ratio of the chitosan, silk fibroin peptide, monomer, coupling material, initiator, EDC, and emulsifier in step S6 is 7-10:2-4:8-10:3-5:0.1-0.2:0.3-0.7:0.5-1, the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate, the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85, the monomer includes methyl methacrylate, butyl acetate, and acrylamide, and the mass ratio is 2-4:1-3:2-3, and the stirring reaction time is 3-5h.

[0023] As a further improvement of the present application, the mass ratio of the carboxymethyl chitosan, poloxamer P407, poloxamer P188, traditional Chinese medicine fermentation, and pH-responsive microspheres in step S7 is 15-20:3-5:4-6:4-6:3-5, and the mass ratio of the mixed microspheres and hydroxyapatite powder is 4-6:8-10.

[0024] The present application further protects a pain-relieving hemostatic agent prepared by the above preparation method.

[0025] The present application has the following beneficial effects:

[0026] The application adopts a method of repeated freeze-thawing, enzymolysis, surfactant treatment and alkali treatment to prepare a kind of decellularized material, and the water in the cell forms ice crystals at low temperature, so that the cell membrane is mechanically damaged, and with the temperature rising, the ice crystals melt, the cell structure is destroyed, and the cell contents are released, so as to achieve the purpose of removing cells. Compared with other powerful decellularization methods, repeated freeze-thawing can remove most of the cell components without using chemical reagents, better protect the three-dimensional structure of the extracellular matrix and the active ingredients therein, and maximize the retention of collagen, elastin, glycosaminoglycans and fibronectin and other protective components. Enzymolysis is usually carried out under relatively mild conditions, and the damage to the extracellular matrix is relatively small. When combined with other methods, the decellularization efficiency can be improved, and the impact on the overall structure of the tissue can be reduced. Surfactants can insert into the lipid bilayer of the cell membrane, destroy the structure and integrity of the cell membrane, and make the cell contents leak out, thereby achieving the purpose of decellularization. Alkaline solution can denature and dissolve non-collagen proteins, thereby removing them from the extracellular matrix, and also destroying the structure of the cell membrane, making the cell contents more easily released and removed. The prepared decellularized material has a rich pore structure, such as decellularized dermal matrix sponge, which can absorb a large amount of blood, so that the protein, platelets, red blood cells and other components in the blood are enriched in the material, thereby increasing the concentration of local coagulation factors and accelerating the formation of thrombus, achieving the purpose of hemostasis. The chemical groups such as amino and carboxyl groups in the material can react with the components such as hemoglobin in the blood to form hydrogen bonds and other interactions, further enhancing the binding ability with the blood components, promoting the activation, aggregation and morphological change of platelets, and accelerating the coagulation process. At the same time, the infiltration of inflammatory cells and the release of inflammatory factors are inhibited, thereby reducing the inflammatory response of the wound, forming a relatively humid environment on the wound, avoiding the drying of the wound, reducing the friction and irritation of the wound with the outside world, and being beneficial to the healing of the wound, thereby reducing pain.

[0027] The application prepares a traditional Chinese medicine ferment by fermenting notoginseng, madder, agrimony and mugwort leaf, wherein the madder has the effects of cooling blood, removing blood stasis and stopping bleeding, the mugwort leaf can warm the channels and stop bleeding, and the agrimony has the effect of astringing and stopping bleeding; the notoginseng can remove blood stasis, activate blood and relieve pain, is called a “hemostatic god drug”, and has the effects of stopping bleeding without leaving blood stasis and removing blood stasis without damaging normal functions, and is widely used for internal and external bleeding (such as hemoptysis, hematemesis and external trauma bleeding) and blood stasis pain (such as contusion and injury and heart and abdominal pain). Active water-soluble components, including notoginseng saponins, notoginseng polysaccharides, alizarin, hydroxyalizarin, agrimol, tannin, eucalyptol and thujaketone, are obtained by fermentation and extraction, have good effects of astringing and stopping bleeding, antibacterial and anti-inflammatory, dispelling cold and relieving pain, and have good water solubility and can be completely dissolved in an aqueous solution and uniformly sprayed on the affected area, thereby playing the medicinal effects.

[0028] The present invention uses porous calcium carbonate microspheres as a CO2 source. Its porous structure is loaded with solid acid, such as citric acid. When the mixed microspheres in the powder come into contact with the aqueous solution, the solid acid dissolves and releases H + , reacting with calcium carbonate to generate micron-sized bubbles. This controllable gas generation mechanism causes the hydrogel to expand to several times its original volume in a short period of time, generating expansion pressure, effectively sealing the bleeding site and quickly stopping bleeding. Simultaneously, the carbon dioxide bubbles can propel the hydrogel to actively penetrate deep tissue spaces and blind spot bleeding points, resolving the problem of traditional materials being unable to reach hidden bleeding sites.

[0029] The analgesic and hemostatic aqueous solution of the present invention is a thermosensitive hydrogel, which is in a solution state at room temperature and solidifies into a gel state at body temperature, thereby being able to slowly release pH-responsive microspheres and Chinese medicinal ferments. At the same time, the gel state can also better stop bleeding. The pH-responsive microspheres are a type of coupling material that rapidly degrades in the acidic environment of the wound site and in the acidic environment after the powder and the aqueous solution are mixed, and locally releases the coupling material. At the same time, the pH-responsive microspheres can also well protect the coupling material from degradation during storage. The coupling material is a decellularized material coupled with the carboxyl portion of ibuprofen and snake venom thrombin, thereby achieving slow and synchronous release of analgesics and hemostatic drugs, reducing the dosage and side effects of a single component, and realizing time-space controlled release of drugs.

[0030] The analgesic and hemostatic agent prepared by the present invention adopts the form of a combination of powder and aqueous solution, which can not only better maintain the activity of the drug, but also has good hemostatic, analgesic, anti-inflammatory and antibacterial effects. It is a slow-release drug with long-lasting effect, reduces the amount of drug applied, improves patient compliance, and has broad application prospects. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0032] Trypsin, 20,000 U / g, Bacillus subtilis, 20 billion cfu / g. Example 1

[0033] This embodiment provides a method for preparing an analgesic and hemostatic agent, comprising the following steps:

[0034] S1. Preparation of decellularized material: pigskin is washed, depilated, washed, and the epidermis cutin layer and subcutaneous fat layer are removed, the dermis layer is taken, chopped, soaked and shaken with 2wt% sodium hydroxide solution, trypsin is added for enzymatic hydrolysis, the final concentration of trypsin is 0.5wt%, filtered, the solid is added with 1wt% sodium dodecyl sulfate solution, frozen with liquid nitrogen, thawed at room temperature, and the freezing-thawing operation is repeated twice, filtered, washed, and freeze-dried to obtain the decellularized material;

[0035] S2. Coupling: 10g of the decellularized material, 3g of the snake venom thrombin are added to 200mL of water, 2g of NHS and 2g of EDC are added, stirred for 20min, 1g of ibuprofen is added, and stirred for 10h to obtain the coupled material;

[0036] S3. Preparation of traditional Chinese medicine fermentation product: 3g of Sanqi, 1g of madder, 2g of Xianhecao, and 5g of wormwood are added to 200mL of water, ball-milled for 2h, sterilized, inoculated with Bacillus subtilis seed liquid, the bacterial content is 10 8 -10 9 cfu / mL, the inoculation amount is 2v / v%, 20℃, 50r / min, and fermented for 36h, filtered, and the filtrate is dried to obtain the traditional Chinese medicine fermentation product;

[0037] S4. Preparation of porous calcium carbonate microspheres: 0.5g of polystyrene sodium sulfonate, 1g of Tween-40, and 10g of calcium nitrate are added to 250mL of water to obtain a solution; the solution is added dropwise to 500mL of fish oil, emulsified at 8000r / min for 15min, 150mL of an aqueous solution containing 18g of potassium carbonate is added dropwise, ultrasonically reacted at 1000W for 10min, and left to stand at room temperature for 12h, centrifuged, washed, and dried to obtain the porous calcium carbonate microspheres;

[0038] S5. Ball milling: 10g of the porous calcium carbonate microspheres and 4g of citric acid are mixed and ball-milled for 2h to obtain the mixed microspheres;

[0039] S6. Preparation of pH-responsive microspheres: 7g of chitosan is dissolved in 200mL of 2wt% acetic acid solution, 2g of silk fibroin peptide is added, and stirred to mix uniformly to obtain a chitosan-silk fibroin peptide solution; 8g of monomers is added to 100mL of water to obtain a monomer solution; the chitosan-silk fibroin peptide solution and the monomer solution are mixed uniformly, 3g of the coupled material and 0.5g of Tween-40 are added, and the mixture is added dropwise to 500mL of fish oil, emulsified at 8000r / min for 15min, 0.1g of potassium persulfate and 0.3g of EDC are added, stirred for 3h, centrifuged, washed, and dried to obtain the pH-responsive microspheres;

[0040] The monomers include methyl methacrylate, butyl acetate, and acrylamide, and the mass ratio is 2:1:2;

[0041] S7. Preparation of an analgesic and hemostatic agent: 15 g of carboxymethyl chitosan was added to water, along with 3 g of poloxamer P407 and 4 g of poloxamer P188, 4 g of a fermented traditional Chinese medicine product, and 3 g of pH-responsive microspheres, and the mixture was stirred to obtain an analgesic and hemostatic aqueous solution. 4 g of the mixed microspheres and 8 g of hydroxyapatite powder were then mixed to obtain an analgesic and hemostatic powder. Example 2

[0042] This embodiment provides a method for preparing an analgesic and hemostatic agent, comprising the following steps:

[0043] S1. Preparation of acellular material: Porcine skin was cleaned, dehaired, and rinsed to remove the epidermal stratum corneum and subcutaneous fat. The dermis was minced and soaked in a 4 wt% sodium hydroxide solution with shaking. Trypsin was added to the solution to a final concentration of 1 wt%. The solution was filtered and the solid was added to a 3 wt% sodium tetradecylbenzenesulfonate solution. The solution was frozen in liquid nitrogen and thawed at room temperature. The freeze-thaw cycle was repeated three times. The solution was filtered, washed, and freeze-dried to produce acellular material.

[0044] S2. Coupling: 10 g of decellularized material and 5 g of snake venom thrombin were added to 200 mL of water, followed by 4 g of NHS and 4 g of EDC. The mixture was stirred and activated for 40 min. 3 g of ibuprofen was added and the reaction was stirred for 15 h to prepare the coupling material.

[0045] S3. Preparation of Chinese herbal fermentation products: 5g of Panax notoginseng, 2g of Rubia cordifolia, 4g of Agrimoniae herba, and 7g of Artemisia argyi were added to 200mL of water, ball-milled for 2h, sterilized, and inoculated with Bacillus subtilis seed solution to a bacterial count of 10 8 -10 9 cfu / mL, the inoculation amount was 4 v / v%, 25 °C, 150 r / min, the fermentation culture was carried out for 72 h, the filtrate was filtered, and the filtrate was dried to obtain the Chinese medicine fermentation product;

[0046] S4. Preparation of porous calcium carbonate microspheres: 1 g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 2 g of Tween-80, and 15 g of calcium chloride were added to 250 mL of water to prepare a solution. This solution was added dropwise to 500 mL of fish oil and emulsified at 8000 rpm for 15 min. Then, 150 mL of an aqueous solution containing 20 g of potassium carbonate was added dropwise. The mixture was sonicated at 1000 W for 20 min, allowed to stand at room temperature for 12 h, centrifuged, washed, and dried to prepare porous calcium carbonate microspheres.

[0047] S5 ball milling: 10g of porous calcium carbonate microspheres and 7g of oxalic acid were mixed and ball milled for 2h to obtain mixed microspheres;

[0048] S6. Preparation of pH-responsive microspheres: 10 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 4 g of silk fibroin peptide was added, and the mixture was stirred until uniform, to obtain a chitosan-silk fibroin peptide solution. 10 g of monomers was added to 100 mL of water to obtain a monomer solution. The chitosan-silk fibroin peptide solution and the monomer solution were mixed uniformly, 5 g of coupling material and 1 g of Tween-80 were added, and 500 mL of fish oil was added dropwise. The mixture was emulsified at 8000 r / min for 15 min, 0.2 g of ammonium persulfate and 0.7 g of EDC were added, and the mixture was stirred for 5 h. After centrifugation, washing and drying, pH-responsive microspheres were obtained.

[0049] The monomers include methyl methacrylate, butyl acetate and acrylamide, and the mass ratio is 4:3:3.

[0050] S7. Preparation of analgesic and hemostatic agent: 20 g of carboxymethyl chitosan was added to water, 5 g of poloxamer P407 and 6 g of poloxamer P188 were added, 6 g of traditional Chinese medicine ferment and 5 g of pH-responsive microspheres were added and stirred until uniform to obtain an analgesic and hemostatic agent water preparation. 6 g of mixed microspheres and 10 g of hydroxyapatite powder were mixed uniformly to obtain an analgesic and hemostatic agent powder. Example 3

[0051] The present embodiment provides a preparation method of an analgesic and hemostatic agent, comprising the following steps:

[0052] S1. Preparation of decellularized material: pigskin was washed, depilated and washed, and the epidermis and subcutaneous fat layers were removed. The dermis layer was taken, chopped, soaked and shaken in a 3 wt% sodium hydroxide solution, and trypsin was added for enzymatic hydrolysis. The final concentration of trypsin was 0.7 wt%. The solid was added to a 2 wt% sodium octadecyl sulfate solution, frozen in liquid nitrogen, and thawed at room temperature. The freezing-thawing operation was repeated three times. The mixture was filtered, washed, and freeze-dried to obtain a decellularized material.

[0053] S2. Coupling: 10 g of decellularized material and 4 g of snake venom thrombin were added to 200 mL of water. 3 g of NHS and 3 g of EDC were added, and the mixture was stirred for 30 min. 2 g of ibuprofen was added, and the mixture was stirred for 12 h to obtain a coupling material.

[0054] S3. Preparation of traditional Chinese medicine ferment: 4 g of Sanqi, 1.5 g of Rubia cordifolia, 3 g of Agrimonia pilosa and 6 g of Artemisia argyi were added to 200 mL of water, and ball milling was performed for 2 h. The mixture was sterilized, inoculated with Bacillus subtilis seed liquid, and the bacterial content was 10 8 -10 9 cfu / mL. The inoculation amount was 3 v / v%. The mixture was fermented at 22℃ and 100 r / min for 56 h. The mixture was filtered, and the filtrate was dried to obtain a traditional Chinese medicine ferment.

[0055] S4. Preparation of porous calcium carbonate microspheres: 0.7 g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 1.5 g of Tween-85, and 13 g of calcium nitrate were added to 250 mL of water to prepare a solution; the solution was added dropwise to 500 mL of fish oil, emulsified at 8000 r / min for 15 min, 150 mL of an aqueous solution containing 19 g of sodium carbonate was added dropwise, ultrasonicated at 1000 W for 15 min, and left to stand at room temperature for 12 h; centrifugation, washing, and drying were performed to prepare the porous calcium carbonate microspheres.

[0056] S5. Ball milling: 10 g of porous calcium carbonate microspheres and 5.5 g of malic acid were mixed and ball milled for 2 h to prepare mixed microspheres.

[0057] S6. Preparation of pH-responsive microspheres: 8.5 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution, 3 g of silk fibroin peptide was added, and stirring was performed until the mixture was uniform to prepare a chitosan-silk fibroin peptide solution; 9 g of monomers was added to 100 mL of water to prepare a monomer solution; the chitosan-silk fibroin peptide solution and the monomer solution were mixed uniformly, 4 g of coupling material and 0.7 g of Tween-85 were added, and the mixture was added dropwise to 500 mL of fish oil, emulsified at 8000 r / min for 15 min, 0.15 g of sodium persulfate and 0.6 g of EDC were added, stirring was performed for 4 h, centrifugation, washing, and drying were performed to prepare the pH-responsive microspheres.

[0058] The monomers include methyl methacrylate, butyl acetate, and acrylamide, and the mass ratio is 3:2:2.5.

[0059] S7. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan was added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 were added, 5 g of traditional Chinese medicine fermentation product and 4 g of pH-responsive microspheres were added, stirring was performed until the mixture was uniform to prepare an analgesic and hemostatic agent water preparation, 5 g of mixed microspheres and 9 g of hydroxyapatite powder were mixed uniformly to prepare an analgesic and hemostatic agent powder preparation.

[0060] Comparative Example 1

[0061] Compared with Example 3, the difference is that no repeated freeze-thaw treatment is used in step S1.

[0062] The details are as follows:

[0063] S1. Preparation of decellularized material: pigskin was washed, depilated, and washed again to remove the epidermis, the stratum corneum, and the subcutaneous fat layer, and the dermis layer was taken, chopped, and treated by soaking and oscillation in a 3 wt% sodium hydroxide solution; trypsin was added for enzymatic hydrolysis, the final concentration of trypsin was 0.7 wt%, and filtration was performed; the solid was added to a 2 wt% sodium octadecyl sulfate solution, stirring was performed for 3 h, and filtration, washing, and freeze-drying were performed to prepare the decellularized material.

[0064] Comparative Example 2

[0065] The difference compared with Example 3 is that no ibuprofen is coupled in step S2.

[0066] The details are as follows:

[0067] S2. Coupling: 10 g of decellularized material, 6 g of snake venom thrombin were added to 200 mL of water, 3 g of NHS and 3 g of EDC were added, stirred for 30 min of activation, and reacted for 12 h with stirring to prepare the coupled material.

[0068] Comparative Example 3

[0069] The difference compared with Example 3 is that no snake venom thrombin is coupled in step S2.

[0070] The details are as follows:

[0071] S2. Coupling: 10 g of decellularized material was added to 200 mL of water, 3 g of NHS and 3 g of EDC were added, stirred for 30 min of activation, 6 g of ibuprofen was added, and reacted for 12 h with stirring to prepare the coupled material.

[0072] Comparative Example 4

[0073] The difference compared with Example 3 is that step S2 is not performed.

[0074] The details are as follows:

[0075] S1. Preparation of decellularized material: pigskin was washed, depilated, and washed to remove the epidermis, cuticle layer and subcutaneous fat layer, and the dermis layer was taken, chopped, and soaked and shaken in a 3wt% sodium hydroxide solution, then trypsin was added for enzymatic hydrolysis, the final concentration of trypsin was 0.7wt%, then filtered, the solid was added to a 2wt% sodium octadecyl sulfate solution, frozen with liquid nitrogen, and thawed at room temperature, the freezing-thawing operation was repeated 3 times, then filtered, washed, and freeze-dried to prepare the decellularized material;

[0076] S2. Preparation of traditional Chinese medicine fermentation product: 4 g of Sanqi, 1.5 g of madder, 3 g of Xianhecao, and 6 g of mugwort were added to 200 mL of water, ball-milled for 2 h, sterilized, inoculated with Bacillus subtilis seed liquid with a bacterial content of 10 8 -10 9 cfu / mL, inoculated at 3v / v%, and fermented at 22℃, 100r / min for 56 h, then filtered, and the filtrate was dried to prepare the traditional Chinese medicine fermentation product;

[0077] S3. Preparation of porous calcium carbonate microspheres: 0.7 g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 1.5 g of Tween-85 and 13 g of calcium nitrate were added to 250 mL of water to prepare a solution; the solution was added dropwise to 500 mL of fish oil, emulsified at 8000 r / min for 15 min, 150 mL of an aqueous solution containing 19 g of sodium carbonate was added dropwise, ultrasonicated at 1000 W for 15 min, and then left to stand at room temperature for 12 h; centrifugation, washing and drying were performed to obtain the porous calcium carbonate microspheres;

[0078] S4. Ball milling: 10 g of porous calcium carbonate microspheres and 5.5 g of malic acid were mixed and ball milled for 2 h to obtain the mixed microspheres;

[0079] S5. Preparation of pH-responsive microspheres: 8.5 g of chitosan was dissolved in 200 mL of 2wt% acetic acid solution, 3 g of silk fibroin peptide was added, and the mixture was stirred and mixed uniformly to obtain a chitosan-silk fibroin peptide solution; 9 g of monomers were added to 100 mL of water to obtain a monomer solution; the chitosan-silk fibroin peptide solution and the monomer solution were mixed uniformly, 4 g of decellularized material and 0.7 g of Tween-85 were added, and the mixture was added dropwise to 500 mL of fish oil, emulsified at 8000 r / min for 15 min, 0.15 g of sodium persulfate and 0.6 g of EDC were added, and the mixture was stirred and reacted for 4 h; centrifugation, washing and drying were performed to obtain the pH-responsive microspheres;

[0080] The monomers include methyl methacrylate, butyl acetate and acrylamide, and the mass ratio is 3:2:2.5;

[0081] S6. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan was added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 were added, 5 g of traditional Chinese medicine fermentation product and 4 g of pH-responsive microspheres were added, and the mixture was stirred and mixed uniformly to obtain an analgesic and hemostatic agent water preparation; 5 g of mixed microspheres and 9 g of hydroxyapatite powder were mixed uniformly to obtain an analgesic and hemostatic agent powder.

[0082] Comparative Example 5

[0083] Compared with Example 3, the difference is that step S6 is not performed.

[0084] The details are as follows:

[0085] S1. Preparation of decellularized material: pigskin was washed, depilated and washed, and the epidermis and subcutaneous fat layers were removed; the dermis layer was taken, chopped, soaked and shaken in a 3wt% sodium hydroxide solution, and trypsin was added for enzymatic hydrolysis; the final concentration of trypsin was 0.7wt%; filtration was performed, and the solid was added to a 2wt% sodium octadecyl sulfate solution; liquid nitrogen was used for freezing, and the mixture was thawed at room temperature; the freezing-thawing operation was repeated 3 times; filtration, washing and freeze-drying were performed to obtain the decellularized material;

[0086] S2. Coupling: 10 g of decellularized material, 4 g of snake venom thrombin were added to 200 mL of water, 3 g of NHS and 3 g of EDC were added, stirred for activation for 30 min, 2 g of ibuprofen was added, and stirred for reaction for 12 h to prepare a coupling material;

[0087] S3. Preparation of traditional Chinese medicine fermentation product: 4 g of Sanqi, 1.5 g of madder, 3 g of Xianhecao, and 6 g of wormwood were added to 200 mL of water, ball-milled for 2 h, sterilized, inoculated with Bacillus subtilis seed liquid, the bacterial content was 10 8 -10 9 cfu / mL, the inoculation amount was 3 v / v%, 22°C, 100 r / min, fermentation culture for 56 h, filtration, drying of the filtrate, to prepare a traditional Chinese medicine fermentation product;

[0088] S4. Preparation of porous calcium carbonate microspheres: 0.7 g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 1.5 g of Tween-85, and 13 g of calcium nitrate were added to 250 mL of water to prepare a solution; the solution was added dropwise to 500 mL of fish oil, emulsified at 8000 r / min for 15 min, 150 mL of an aqueous solution containing 19 g of sodium carbonate was added dropwise, ultrasonic reaction was carried out at 1000 W for 15 min, room temperature standing for 12 h, centrifugation, washing, drying, to prepare porous calcium carbonate microspheres;

[0089] S5. Ball milling: 10 g of porous calcium carbonate microspheres and 5.5 g of malic acid were mixed and ball-milled for 2 h to fix, to prepare mixed microspheres;

[0090] S6. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan was added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 were added, 5 g of traditional Chinese medicine fermentation product and 4 g of coupling material were added and stirred to mix uniformly, to prepare an analgesic and hemostatic agent water agent, 5 g of mixed microspheres and 9 g of hydroxyapatite powder were mixed uniformly, to prepare an analgesic and hemostatic agent powder.

[0091] Comparative Example 6

[0092] Compared with Example 3, the difference is that no traditional Chinese medicine fermentation product is added in step S7.

[0093] Specifically as follows:

[0094] S7. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan was added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 were added, 9 g of pH-responsive microspheres were added and stirred to mix uniformly, to prepare an analgesic and hemostatic agent water agent, 5 g of mixed microspheres and 9 g of hydroxyapatite powder were mixed uniformly, to prepare an analgesic and hemostatic agent powder.

[0095] Comparative Example 7

[0096] The difference compared with Example 3 is that no pH-responsive microspheres are added in step S7.

[0097] The preparation is as follows:

[0098] S7. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan is added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 are added, 5 g of traditional Chinese medicine fermentation is added, 4 g of pH-responsive microspheres are added, and stirring and mixing are performed to obtain an analgesic and hemostatic agent.

[0099] Comparative Example 8

[0100] The difference compared with Example 3 is that no powder is applied.

[0101] The preparation is as follows:

[0102] S7. Preparation of analgesic and hemostatic agent: 17 g of carboxymethyl chitosan is added to water, 4 g of poloxamer P407 and 5 g of poloxamer P188 are added, 5 g of traditional Chinese medicine fermentation is added, 4 g of pH-responsive microspheres are added, and stirring and mixing are performed to obtain an analgesic and hemostatic agent.

[0103] Test Example 1 Analgesic experiment

[0104] Take Kunming mice, half male and half female, and randomly divide them into a model group, an aspirin group, Example 1-3 groups, and Comparative Example 1-8 groups, 10 mice in each group. The mice in each group are shaved on the abdomen, the shaved area is about 1.5 cm x 1.5 cm, and the drugs are applied to the depilated area on the abdomen of the mice. The mice in the model group are applied with 2 mL / kg of normal saline, the mice in the aspirin group are applied with 0.6 g / kg of aspirin, and the mice in the Example 1-3 and Comparative Example 1-8 groups are first applied with 0.5 g / kg of powder and then sprayed with 2 mL / kg of water agent. After 1 h of administration, the mice in each group are given 0.6% acetic acid (0.1 mL / 10 g) intraperitoneally. The number of writhing times of the mice within 15 min is recorded (the writhing reaction is manifested as that the abdomen of the mice is concave, the trunk and hind legs are stretched, and the hips are raised). The writhing inhibition rate is calculated as follows: writhing inhibition rate = (number of writhing times of the model group - number of writhing times of the administration group) / number of writhing times of the model group x 100%. The results are shown in Table 1.

[0105] Table 1 Comparison of writhing inhibition rates of various agents

[0106]

[0107] As shown in the above table, the analgesic and hemostatic agent prepared in Example 1-3 has good analgesic effect under the synergistic action of the water agent and the powder.

[0108] Test Example 2 Hemostatic effect

[0109] Take Kunming mice 50, half male and female, randomly divided into model group, Yunnan white medicine group, example 1-3 group, comparative example 1-8 group, 10 in each group. The bleeding time determination is carried out by the tail cutting method, that is, the mouse is fixed, the mouse tail is exposed to the outside, and the mouse tail tip 3mm is transversely cut by a sharp scissors, and immediately smeared, the model group is smeared with 2mL / kg normal saline, the Yunnan white medicine group is smeared with 0.77g / kg, the example 1-3 and the comparative example 1-8 group are first smeared with 0.5g / kg powder, and then sprayed with 2mL / kg water agent, and the blood is overflowed by itself to start timing, and the blood drops are absorbed by filter paper every 30s, until the blood flow stops naturally (no blood is absorbed by filter paper), that is, the bleeding time. The results are shown in Table 2.

[0110] Table 2 Comparison table of various bleeding times

[0111]

[0112] Note: * compared with the model group, P<0.05.

[0113] From the above table, the analgesic hemostatic agent prepared by the example 1-3 of the application has good hemostatic effect under the synergistic effect of the water agent and the powder.

[0114] Test example 3 Anti-inflammatory effect

[0115] Take NIH male mice (SPF level), randomly divided into negative control group, positive control group, example 1-3 group, comparative example 1-8 group, 10 in each group. Distilled water is used as the negative control group, flucin ointment is used as the positive control group, and the analgesic hemostatic agent prepared by example 1-3 and comparative example 1-8 is used as the test group.

[0116] The right auricle of each mouse is uniformly smeared with dimethylbenzene to cause inflammation, and the dose is 100μL / one, and the left ear is not treated as a blank control group. After 30min of dimethylbenzene inflammation, each group of animals is given corresponding test substances (first smear 0.2g / one powder, and then spray 0.4mL / one water agent) on the right ear, and pay attention to uniform smearing on the inner and outer surfaces of the right auricle. The negative control group of animals is given distilled water on the right auricle, and the dose is 0.1mL / one. After 1h of giving the test substances, the animals are killed by cervical dislocation, and the two auricles are cut off, the test substances on the right auricle are washed with normal saline, and the water is wiped dry. The left and right ear pieces are punched by a puncher with a diameter of 8mm, and the two ear pieces are weighed, and the swelling value is calculated, and the smaller the swelling value, the better the anti-inflammatory effect. The results are shown in Table 3.

[0117] Swelling value = m 右耳耳片 -m 左耳耳片 .

[0118] Table 3 Comparison table of various swelling values

[0119]

[0120] Note: * P<0.05 compared with the model group.

[0121] From the above table, it can be seen that the analgesic hemostatic agent prepared by the examples 1-3 of the present application has good anti-inflammatory effect under the synergistic effect of the water agent and the powder.

[0122] The above merely describes the preferred embodiments of the present application, but not to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of preparing an analgesic styptic medicament, characterized in that, The method comprises the following steps: S1. Preparation of decellularized material: pigskin is washed, depilated, washed, and the epidermis and subcutaneous fat layers are removed, and the dermis layer is taken, chopped, soaked and shaken in a sodium hydroxide solution, trypsin enzymolysis, filtration, solid addition of a surfactant solution, liquid nitrogen freezing, room temperature remelting, repeated freezing-remelting operation 2-3 times, filtration, washing, freeze-drying, and decellularized material is prepared; S2. Coupling: the decellularized material and snake venom thrombin are added to water, NHS and EDC are added, stirring is activated, ibuprofen is added, stirring is reacted, and coupled material is prepared; S3. Preparation of traditional Chinese medicine fermentation product: Sanqi, madder, Japanese knotweed, and mugwort leaves are added to water, ball milling is performed, sterilization is performed, Bacillus subtilis seed liquid is inoculated, fermentation culture is performed, filtration is performed, and the filtrate is dried to prepare a traditional Chinese medicine fermentation product; S4. Preparation of porous calcium carbonate microspheres: a porogen, an emulsifier, and a calcium salt are added to water to prepare a solution; the solution is added dropwise to fish oil, emulsified, a carbonate solution is added dropwise, ultrasonic reaction is performed, room temperature standing is performed, centrifugation is performed, washing is performed, and drying is performed to prepare porous calcium carbonate microspheres; S5. Ball milling: the porous calcium carbonate microspheres and solid acid are mixed and ball milled to fix, and mixed microspheres are prepared; S6. Preparation of pH-responsive microspheres: chitosan is dissolved in an acid solution, silk fibroin peptide is added, stirring is performed, and a chitosan-silk fibroin peptide solution is prepared; monomers are added to water to obtain a monomer solution; the chitosan-silk fibroin peptide solution and the monomer solution are mixed uniformly, coupled material and an emulsifier are added, dropwise addition is performed to fish oil, emulsification is performed, an initiator is added, stirring is reacted, centrifugation is performed, washing is performed, and drying is performed to prepare pH-responsive microspheres; S7. Preparation of analgesic and hemostatic agent: carboxymethyl chitosan is added to water, poloxamer P407 and poloxamer P188 are added, traditional Chinese medicine fermentation product and pH-responsive microspheres are added, stirring is performed, and an analgesic and hemostatic agent aqueous preparation is prepared; mixed microspheres and hydroxyapatite powder are mixed uniformly to prepare an analgesic and hemostatic agent powder preparation.

2. The production method according to claim 1, characterized by, The concentration of the sodium hydroxide solution in step S1 is 2-4 wt%, the final concentration of the trypsin is 0.5-1 wt%, the concentration of the surfactant solution is 1-3 wt%, and the surfactant is at least one selected from Tween-20, Tween-40, Tween-60, Tween-80, sodium dodecyl sulfonate, sodium tetradecyl benzene sulfonate, sodium hexadecyl sulfate, and sodium octadecyl sulfate.

3. The preparation method according to claim 1, characterized in that The mass ratio of the decellularized material, snake venom thrombin, NHS, EDC, and ibuprofen in step S2 is 10:3-5:2-4:2-4:1-3, the stirring activation time is 20-40 min, and the stirring reaction time is 10-15 h.

4. The method of claim 1, wherein, The mass ratio of the three seven, madder, Japanese knotweed, and wormwood leaf in step S3 is 3-5:1-2:2-4:5-7, the bacterial content of the bacillus subtilis seed liquid is 10 8 -10 9 cfu / mL, the inoculation amount is 2-4 v / v%, and the fermentation culture condition is 20-25°C, 50-150 r / min, and fermentation culture for 36-72 h.

5. The preparation method according to claim 1, characterized in that The mass ratio of the pore-forming agent, emulsifier, calcium salt, carbonate salt in step S4 is 0.5-1:1-2:10-15:18-20, the calcium salt is calcium chloride or calcium nitrate, the carbonate salt is sodium carbonate or potassium carbonate, the pore-forming agent is selected from at least one of sodium polystyrene sulfonate or polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, Tween-85.

6. The method of claim 1, wherein, The mass ratio of the porous calcium carbonate microspheres and solid acid in step S5 is 10:4-7, and the solid acid is selected from at least one of oxalic acid, citric acid, malic acid, and tartaric acid.

7. The preparation method according to claim 1, characterized in that The mass ratio of the chitosan, silk fibroin peptide, monomer, coupling material, initiator, EDC, and emulsifier in step S6 is 7-10:2-4:8-10:3-5:0.1-0.2:0.3-0.7:0.5-1, the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate, the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85, the monomer includes methyl methacrylate, butyl acetate, and acrylamide, and the mass ratio is 2-4:1-3:2-3, and the stirring reaction time is 3-5 h.

8. The method of claim 1, wherein, The mass ratio of the carboxymethyl chitosan, poloxamer P407, poloxamer P188, traditional Chinese medicine ferment, and pH-responsive microspheres in step S7 is 15-20:3-5:4-6:4-6:3-5, and the mass ratio of the mixed microspheres and hydroxyapatite powder is 4-6:8-10.

9. An analgesic hemostatic agent prepared by the preparation method of any one of claims 1-8.

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