Thermosensitive propolis gel solution and preparation method thereof

Abstract

The preparation method comprises the following steps: firstly, respectively preparing a propolis extracting solution, a polyvinyl alcohol solution and a chitosan solution, then uniformly mixing the polyvinyl alcohol solution and the chitosan solution, then adding the propolis extracting solution to obtain a mixed solution, and finally, filtering the mixed solution, standing at low temperature, compounding at ultrahigh pressure, and cooling to obtain the temperature-sensitive propolis gel solution. And carrying out gelation on the propolis gel solution, so as to obtain the temperature-sensitive propolis gel solution. The temperature-sensitive propolis gel solution prepared by the method can be subjected to phase change at the body temperature of a human body, a sol state is converted into a gel state, controlled release of active ingredients of propolis is completed according to the change of a pH value, and a higher drug concentration is directionally provided in a bacterial infection area for a long time. And the chitosan / polyvinyl alcohol composite carrier has excellent properties such as strong antibacterial ability and good stability, and can generate a synergistic effect after being combined with the propolis, thereby effectively enhancing the antibacterial effect of the whole thermo-sensitive propolis gel solution and improving the physical properties of the propolis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a temperature-sensitive propolis gel solution and a preparation method thereof. Background Art

[0002] Propolis, due to its excellent chemical composition, wide range of pharmacological activities and biological activities, is widely used in the fields of wound treatment, antibacterial, antiviral, antioxidant, anti-inflammatory, hemostatic, antispasmodic, anti-tumor and immunomodulatory. In particular, in the field of wound treatment, due to the multi-component and multi-target characteristics of propolis, it can comprehensively improve the wound repair environment, inhibit bacteria and inflammation, promote skin repair, thereby quickly stopping bleeding, and enhance the proliferation, activation and growth ability of skin cells, accelerate wound healing, and due to its natural source, non-toxicity and good human tolerance, the potential of propolis in the field of medical treatment has received increasing attention. However, the therapeutic use of propolis is subject to various restrictions, such as the drug concentration of propolis may not be sufficient to be effective in all cases of use; and due to the strong viscosity of propolis, it may cause pain when changing dressings, or damage new tissues due to excessive adhesion; in addition, the low water solubility of propolis may lead to its poor dissolution and absorption in the body, thereby affecting its bioavailability, etc. To this end, a temperature-sensitive propolis gel solution and a preparation method are provided to solve the above problems. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a temperature-sensitive propolis gel solution and a preparation method. Propolis is loaded on a chitosan / polyvinyl alcohol composite carrier through ultrahigh pressure technology to form a temperature-sensitive supramolecular hydrogel, which solves the problem in the prior art that the use of propolis is limited and cannot maximize its own therapeutic effect. The release rate of propolis can be adjusted according to the pH changes of the local environment, thereby providing a higher drug concentration in the bacterial infection area, providing an efficient and multifunctional solution for wound treatment and pathogen control.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for preparing a temperature-sensitive propolis gel solution comprises the following steps:

[0006] Step S 1 , respectively preparing propolis extract, polyvinyl alcohol solution and chitosan solution;

[0007] Step S 2 , firstly mixing the polyvinyl alcohol solution and the chitosan solution evenly, and then adding the propolis extract and mixing evenly to obtain a mixed solution;

[0008] Step S 3, filtering the mixed solution, standing it at low temperature, compounding it under ultra-high pressure, and cooling it to obtain a temperature-sensitive propolis gel solution.

[0009] Furthermore, the homogeneous working voltage of the ultra-high voltage composite is 150-200MPa.

[0010] Furthermore, the weight ratio of the polyvinyl alcohol solution to the chitosan solution is 1-3:1.

[0011] Furthermore, the propolis extract accounts for 0.5-4.0% in the mixed solution.

[0012] Furthermore, the mixed solution is mixed uniformly by stirring, the stirring temperature is 80-90° C., and the stirring speed is 400-1000 rpm.

[0013] Furthermore, the preparation of the propolis extract comprises the following specific steps:

[0014] Step S 11 , mixing propolis and a solvent in a ratio of 1:2-8, soaking and extracting at room temperature for 2-3 days, and then filtering to obtain a first extract, wherein the propolis is measured in grams and the solvent is measured in milliliters;

[0015] Step S 12 , mixing the first extract with aqueous ethanol at a ratio of 1:4-8, and extracting by ultrasonic for 0.25-1h to obtain a filtrate, wherein the aqueous ethanol is a 70%-95% ethanol solution in milliliters, and the first extract is in grams; after repeating at least three times, combining the filtrates obtained from each ultrasonic extraction, and evaporating the combined filtrates at 50-65°C to obtain a propolis extract;

[0016] Step S 13 , adding the propolis extract into polyethylene glycol for mixing and fully dissolving, and then filtering to obtain the propolis extract.

[0017] Furthermore, the preparation of the polyvinyl alcohol solution includes the following specific steps: adding polyvinyl alcohol to water to swell for 12-24 hours and then fully dissolving, then adding disodium hydrogen phosphate solution to adjust the pH to 6-7, and stirring at 80-90°C at a speed of 600-1000 rpm for at least 12 hours until the polyvinyl alcohol solution is obtained.

[0018] Furthermore, the preparation of the chitosan solution comprises the following specific steps: adding chitosan into acetic acid solution for mixing, and stirring at 50-80° C. at a speed of 400-1000 rpm for at least 12 hours until a transparent and clear chitosan solution is obtained.

[0019] In order to achieve the above object, the present invention also adopts the following technical solutions:

[0020] A temperature-sensitive propolis gel solution comprises, by weight percentage, 0.5-4.0% of propolis extract, 1.5-5.0% of a polymer of polyvinyl alcohol and chitosan, and the balance being water.

[0021] Further, the pH value is 4.5-7.0;

[0022] The temperature-sensitive propolis gel solution is placed in the human body temperature range or in the human body environment to form a gel-like temperature-sensitive propolis gel;

[0023] The microstructure of the thermosensitive propolis gel is a three-dimensional network structure.

[0024] In summary, compared with the prior art, the present invention has at least the following beneficial effects:

[0025] The present invention relates to a thermosensitive propolis gel solution and a preparation method thereof, wherein a propolis extract, a polyvinyl alcohol solution and a chitosan solution are prepared respectively, and then the polyvinyl alcohol solution and the chitosan solution are mixed evenly and then added to the propolis extract, and mixed evenly to obtain a mixed solution, and finally the mixed solution is filtered, left to stand at low temperature, subjected to ultrahigh pressure compounding, and cooled to obtain a propolis gel solution, and the propolis gel solution is gelled to obtain a thermosensitive propolis gel solution. The thermosensitive propolis gel solution manufactured by the method has a unique network structure and stimulus responsiveness, can undergo a phase change at human body temperature, and can be transformed from a sol state to a gel state, and can complete the controlled release of the active ingredients of propolis according to the pH value change, and can achieve a higher drug concentration in a bacterial infection area in a long-term manner, so that the therapeutic effect of propolis can be maintained for a long time, and frequent replacement can be reduced, and the use effect of propolis can be maximized. In addition, the chitosan / polyvinyl alcohol composite carrier itself has excellent properties such as strong antibacterial ability and good stability. After combining with propolis, it produces a synergistic effect, effectively enhancing the antibacterial effect of the overall thermosensitive propolis gel solution, accelerating the wound healing process, and effectively improving the physical properties of propolis. For example, the viscosity of the thermosensitive propolis gel solution is significantly lower than that of propolis, so that it can be firmly attached to the wound when applied to the wound, and is easy to remove painlessly when changing the dressing, or the stability is enhanced, effectively extending the service life. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1This is a sustained-release diagram of Test Example 2 provided in one embodiment of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention provides a method for preparing a temperature-sensitive propolis gel solution, comprising the following steps:

[0030] Step S 1 , respectively preparing propolis extract, polyvinyl alcohol solution and chitosan solution;

[0031] Step S 2 , firstly, uniformly mix the polyvinyl alcohol solution and the chitosan solution, and then add the propolis extract and mix uniformly to obtain a mixed solution;

[0032] Step S 3 , filter the mixed solution, let it stand at low temperature, compound it under ultra-high pressure, and cool it to obtain a temperature-sensitive propolis gel solution.

[0033] Specifically, the method for preparing the thermosensitive propolis gel solution disclosed in the present invention is particularly suitable for wound treatment and pathogen treatment. The core of the technology is to combine the active ingredients in propolis with chitosan-polyvinyl alcohol polymers using ultra-high pressure homogenization technology. Ultra-high pressure homogenization technology uses extremely high pressure to pass a mixture of propolis, chitosan and polyvinyl alcohol through a small hole after filtration and low-temperature standing to generate high-speed shear force and impact force. These forces can break the particles and cell structures in the material to achieve the purpose of refinement and uniform dispersion. In the preparation process of the gel, ultra-high pressure homogenization technology can be used to improve and optimize the network structure of the gel, so that it has more uniform and finer pores, thereby improving the mechanical strength and stability of the gel. In addition, it can also be used to improve the dispersibility and uniformity of the active ingredients in the gel, thereby improving the efficacy and performance of the gel. In the present invention, ultra-high pressure technology is used to cause chitosan and polyvinyl alcohol to form a supramolecular hydrogel with a unique network structure and stimulus responsiveness. This hydrogel can slowly release the propolis loaded on chitosan / polyvinyl alcohol, and undergo a phase change at human body temperature, from a sol state to a gel state, so that the therapeutic effect of propolis can be maintained for a long time, reducing frequent replacement, and providing a higher drug concentration in the bacterial infection area according to the pH value change, and accurately controlling the drug release to maximize the use effect of propolis. In addition, the thermosensitive propolis gel solution prepared by the method exhibits multiple innovative functions such as antibacterial, tissue adhesion, material transport, and anti-inflammatory, has no damage to human skin, no pollution to the environment, and no stimulation, and does not contain chemical antibacterial agents, is non-toxic to the human body, has no side effects, and does not produce drug resistance, and is safe and reliable.

[0034] It should be noted that chitosan / polyvinyl alcohol polymer is selected as a carrier because its polymer has a synergistic effect in many aspects to improve the efficacy of propolis. Specifically: (1) The bacterial infection area of ​​the patient is generally an acidic environment, and chitosan, as a temperature-sensitive and pH-sensitive biomaterial, can be transformed into a gel state due to the effect of body temperature when it contacts the wound, which helps the gel to better fit the wound surface, provide a physical barrier, and thus prevent bacterial invasion. In an acidic environment, it can be used as a sustained-release system to control the release of active ingredients in propolis, so that the gel prepared by the method of the present invention can maintain its therapeutic effect for a long time and reduce the number of frequent changes; (2) It can effectively promote wound healing. Since chitosan itself has antibacterial activity, it can effectively enhance the overall antibacterial effect after combining with propolis, and can stimulate cell proliferation and tissue regeneration. In addition, chitosan can increase the water solubility of propolis, thereby improving its dispersibility and permeability in the wound site, further playing a role in promoting wound healing. (3) Improve patient comfort: Since propolis has strong viscosity, tearing it off when changing it will cause pain, or damage the new tissue due to excessive adhesion. The prepared thermosensitive gel containing chitosan is in a liquid state for easy even application when applied, and then turns into a gel at body temperature. The viscosity of the composite gel is significantly lower than that of propolis, so that it can be firmly attached to the wound when applied, and can be easily removed painlessly when replaced. Furthermore, through the composite use of chitosan and polyvinyl alcohol, the cell affinity and adhesion ability of polyvinyl alcohol and the above-mentioned advantages of chitosan itself are combined, and the mechanical properties of chitosan can be improved. In addition, chitosan and polyvinyl alcohol are relatively environmentally friendly materials that can be degraded in the body to reduce the impact on the environment.

[0035] In certain embodiments of the present invention, the homogenous working voltage of the ultra-high voltage composite is 150-200 MPa.

[0036] In certain embodiments of the present invention, the filter screen used for filtering the mixed solution is 80-100 mesh.

[0037] In certain embodiments of the present invention, the low temperature standing and cooling method is an ice bath.

[0038] In certain embodiments of the present invention, the gelling temperature of the propolis gel solution is human body temperature, more preferably 37°C.

[0039] In some embodiments of the present invention, step S 2 The mixed solution is mixed uniformly by stirring at a temperature of 80-90° C. and a stirring speed of 400-1000 rpm.

[0040] In certain embodiments of the present invention, the weight ratio of the polyvinyl alcohol solution to the chitosan solution is 1-3:1, including but not limited to 1:1, 2:1, 3:1, etc.

[0041] In certain embodiments of the present invention, the propolis extract accounts for 0.5-4.0% in the mixed solution.

[0042] In certain embodiments of the present invention, stirring is performed using a magnetic stirrer.

[0043] In addition, in certain embodiments of the present invention, the preparation of propolis extract comprises the following steps:

[0044] Step S 11 , mixing propolis and a solvent in a ratio of 1:2-8, soaking and extracting at room temperature for 2-3 days, and then filtering to obtain a first extract, wherein the propolis is measured in grams and the solvent is measured in milliliters;

[0045] Step S 12 , mixing the first extract with aqueous ethanol at a ratio of 1:4-8, and extracting by ultrasonic wave for 0.25-1h to obtain a filtrate;

[0046] Step S 13 , add the propolis extract into polyethylene glycol, mix and fully dissolve it, and then filter to obtain the propolis extract.

[0047] It is worth mentioning that through strict standardized extraction of the chemical components of propolis and combining it with the stabilizing properties of chitosan / polyvinyl alcohol, the concentration of the product's active ingredients can be ensured to be consistent and batch-to-batch differences can be reduced.

[0048] The solvent is used to remove the n-hexane phase, wax and other impurities in propolis. In some embodiments of the present invention, the solvent can be n-hexane, petroleum ether, ether, etc., which are not limited here. Optimally, n-hexane is selected.

[0049] In certain embodiments of the present invention, the aqueous ethanol is a 70%-95% ethanol solution, which can more fully extract the effective ingredients in propolis.

[0050] In certain embodiments of the present invention, the aqueous ethanol is measured in milliliters and the first extract is measured in grams.

[0051] In certain embodiments of the present invention, the ratio of propolis extract to polyethylene glycol is 1:4 by weight.

[0052] In certain embodiments of the present invention, the ultrasonic extraction is repeated at least three times, the filtrates obtained from each ultrasonic extraction are combined, and the combined filtrates are evaporated at 50-65° C. to obtain the propolis extract.

[0053] In certain embodiments of the present invention, the preparation of the polyvinyl alcohol solution comprises the following steps:

[0054] Step S 141. Add polyvinyl alcohol to water and swell for 12-24 hours, then heat at 80-90°C to fully dissolve it, then add disodium hydrogen phosphate solution, and stir at 80-90°C at 600-1000 rpm for at least 12 hours until a transparent and clear polyvinyl alcohol solution is obtained.

[0055] In certain embodiments of the present invention, water is only used to swell the polyvinyl alcohol while the proportion of the total formula remains unchanged. The proportion is not strictly limited and is approximately 1:5 or more.

[0056] In certain embodiments of the present invention, the heating method is water bath heating.

[0057] In certain embodiments of the present invention, a magnetic stirrer is used for stirring and the device is sealed during stirring.

[0058] In certain embodiments of the present invention, disodium hydrogen phosphate is used as a pH regulator to adjust the pH of the dissolved polyvinyl alcohol solution to 6-7. Preferably, 3-10 mL of 0.2 mol / L sodium dihydrogen phosphate solution is added.

[0059] In certain embodiments of the present invention, the preparation of chitosan solution comprises the following specific steps:

[0060] Step S 15 , add chitosan to the acetic acid solution and mix, and stir at 50-80°C at a speed of 400-1000 rpm for at least 12 hours until a transparent and clear chitosan solution is obtained.

[0061] In certain embodiments of the present invention, acetic acid is preferably used as a pH adjuster to adjust the pH to 4-6 due to its weak acidity and low toxicity.

[0062] In certain embodiments of the present invention, the acetic acid concentration is preferably 90%.

[0063] In certain embodiments of the present invention, stirring is performed using a magnetic stirrer.

[0064] The present invention also provides a thermosensitive propolis gel solution, which is prepared by the preparation method of any one of the above technical solutions. Specifically, by weight percentage, the thermosensitive propolis gel solution comprises: 0.5-4.0% of propolis extract, 1.5-5.0% of a polymer of polyvinyl alcohol and chitosan, and the balance is water. Furthermore, the mass ratio of polyvinyl alcohol to chitosan in the polymer is 1-3:1.

[0065] It should be noted that in the preparation of propolis thermosensitive gel, the mass ratio of polyvinyl alcohol to chitosan and the total concentration of the polymer are key factors, which together determine the formation time and properties of the gel. Specifically, as the total concentration of the polymer decreases, the gel time will be prolonged. When the polymer concentration exceeds 5%, the gel will form immediately; when the concentration is lower than 1.5%, gelation will not occur even within 30 minutes. Moreover, when the total polymer concentration is constant, increasing the content of polyvinyl alcohol or reducing the content of chitosan will also lead to a longer gel time. In particular, when the mass ratio of polyvinyl alcohol to chitosan in the polymer is lower than 1:1, the gel will form immediately; and when the mass ratio exceeds 3:1, gelation will not occur within 30 minutes. Therefore, choosing to control the appropriate total polymer concentration or the mass ratio of polyvinyl alcohol to chitosan in the polymer can achieve effective regulation of the gel time.

[0066] In certain embodiments of the present invention, the pH values ​​of the polymer and the thermosensitive propolis gel solution are consistent, both of which are 4.5-7.0.

[0067] In certain embodiments of the present invention, the thermosensitive propolis gel solution can form a gel-like thermosensitive propolis gel when placed within the human body temperature range during the experiment. Similarly, when used by a patient, the thermosensitive propolis gel can also form a gel-like thermosensitive propolis gel when placed within the human body environment.

[0068] In certain embodiments of the present invention, the microstructure of the thermosensitive propolis gel is a three-dimensional network structure.

[0069] In certain embodiments of the present invention, the thermosensitive propolis gel is used as a dressing in wound care, which can provide a moist healing environment for the bacterial infection area, and gradually control the release of the antibacterial and healing components in the propolis according to the changes in body temperature, so as to promote the cleaning and healing of local wounds. Or it can be used in the form of a facial mask, eye cream or oral capsule in beauty skin care and health products, which can effectively provide anti-inflammatory, antioxidant and anti-aging effects. Or it can be used in other similar fields, which are not limited here.

[0070] In order to further illustrate the present invention, a temperature-sensitive propolis gel solution and a preparation method provided by the present invention are described in detail below in conjunction with examples, but it should not be understood as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] Step S 11 , taking 100 g of propolis raw material, adding 400 ml of n-hexane to mix, soaking and extracting at room temperature for 2 days and then filtering to obtain a first extract;

[0073] Step S 12, mixing the first extract with 400 ml of 95% aqueous ethanol, and extracting the propolis by ultrasonic extraction for 0.75 h, repeating the process three times, combining the filtrates obtained by each ultrasonic extraction, and evaporating the combined filtrates at 60° C. to obtain a propolis extract;

[0074] Step S 13 1. Take 1g of propolis extract and add it to 15ml of polyethylene glycol, mix and fully dissolve it in a water bath at 80℃, and then filter it through a 100-mesh filter to obtain the propolis extract;

[0075] Step S 14 1. Add 0.5 g of polyvinyl alcohol to 40 ml of water and swell for 12 h. Heat in a water bath at 85°C to dissolve. After complete dissolution, add 5 mL of 0.2 mol / L sodium dihydrogen phosphate solution. Seal the mixture and place in a magnetic stirrer. Stir at 800 rpm for 12 h at 85°C until a transparent and clear polyvinyl alcohol solution is obtained.

[0076] Step S 15 , add 0.5 g chitosan into 40 ml 90% acetic acid solution, mix, seal and place in a magnetic stirrer at 60°C and 600 rpm for 12 h, until a transparent and clear chitosan solution is obtained;

[0077] Step S 2 , slowly pour the polyvinyl alcohol solution into the chitosan solution and stir evenly at a stirring temperature of 85°C and a stirring speed of 800 rpm, then slowly pour the propolis extract into the chitosan solution and continue stirring evenly to obtain a mixed solution;

[0078] Step S 3 , filter the mixed solution through a 100-mesh filter, place it in an ice water bath and let it stand for 15 minutes, then perform ultra-high pressure compounding through an ultra-high pressure homogenizer with a homogenization working voltage of 150MPa, place the treated solution in an ice bath to cool down, and obtain a temperature-sensitive propolis gel solution.

[0079] Example 2

[0080] Step S 11 , taking 100 g of propolis raw material, adding 600 ml of n-hexane to mix, soaking and extracting at room temperature for 2 days and then filtering to obtain a first extract;

[0081] Step S 12 , mixing the first extract with 600 ml of 80% aqueous ethanol, and extracting the propolis by ultrasonic extraction for 0.5 h, repeating the process three times, combining the filtrates obtained by each ultrasonic extraction, and evaporating the combined filtrates at 65° C. to obtain a propolis extract;

[0082] Step S 132. Take 2g of propolis extract and add it to 15ml of polyethylene glycol, mix and fully dissolve it in a water bath at 85℃, and then filter it through a 60-mesh filter to obtain the propolis extract;

[0083] Step S 14 1. Add 2g of polyvinyl alcohol to 40ml of water and swell for 12h. Heat in a water bath at 85℃ to dissolve. After complete dissolution, add 6mL of 0.2mol / L disodium hydrogen phosphate solution. Seal and place in a magnetic stirrer and stir at 600rpm for 12h at 90℃ until a transparent and clear polyvinyl alcohol solution is obtained.

[0084] Step S 15 1.0 g of chitosan was added into 40 ml of 90% acetic acid solution, and the mixture was sealed and placed in a magnetic stirrer at 70° C. and 600 rpm for 12 h until a transparent and clear chitosan solution was obtained;

[0085] Step S 2 , slowly pour the polyvinyl alcohol solution into the chitosan solution and stir evenly at a stirring temperature of 80° C. and a stirring speed of 600 rpm, then slowly pour the propolis extract into the chitosan solution and continue stirring evenly to obtain a mixed solution;

[0086] Step S 3 , filter the mixed solution through an 80-mesh filter, place it in an ice water bath and let it stand for 15 minutes, then perform ultra-high pressure compounding through an ultra-high pressure homogenizer with a homogenization working voltage of 180 MPa, place the treated solution in an ice bath to cool down, and obtain a temperature-sensitive propolis gel solution.

[0087] Example 3

[0088] Step S 11 , taking 100 g of propolis raw material, adding 200 ml of n-hexane to mix, soaking and extracting at room temperature for 3 days and then filtering to obtain a first extract;

[0089] Step S 12 , mixing the first extract with 800 ml of 85% aqueous ethanol, and extracting the propolis by ultrasonic extraction for 0.25 h, repeating the process three times, combining the filtrates obtained by each ultrasonic extraction, and evaporating the combined filtrates at 60° C. to obtain a propolis extract;

[0090] Step S 13 1. Take 4g of propolis extract and add it to 15ml of polyethylene glycol, mix and fully dissolve it in a water bath at 80℃, and then filter it through a 100-mesh filter to obtain the propolis extract;

[0091] Step S 141. Add 3g of polyvinyl alcohol to 40ml of water and swell for 12h. Heat in a 90℃ water bath to dissolve. After complete dissolution, add 7mL of 0.2mol / L disodium hydrogen phosphate solution. Seal and place in a magnetic stirrer and stir at 1000rpm for 12h at 90℃ until a transparent and clear polyvinyl alcohol solution is obtained.

[0092] Step S 15 1.5 g chitosan was added into 40 ml 90% acetic acid solution, and the mixture was sealed and placed in a magnetic stirrer, and stirred at 80°C and 1000 rpm for 12 h until a transparent and clear chitosan solution was obtained;

[0093] Step S 2 , slowly pour the polyvinyl alcohol solution into the chitosan solution and stir evenly at a stirring temperature of 90° C. and a stirring speed of 1000 rpm, then slowly pour the propolis extract into the solution and continue stirring evenly to obtain a mixed solution;

[0094] Step S 3 , filter the mixed solution through a 100-mesh filter, place it in an ice water bath and let it stand for 15 minutes, then perform ultra-high pressure compounding through an ultra-high pressure homogenizer with a homogenization working voltage of 200MPa, place the treated solution in an ice bath to cool down, and obtain a temperature-sensitive propolis gel solution.

[0095] Example 4

[0096] Step S 11 , taking 100 g of propolis raw material, adding 800 ml of n-hexane to mix, soaking and extracting at room temperature for 2 days and then filtering to obtain a first extract;

[0097] Step S 12 , mixing the first extract with 600 ml of 90% aqueous ethanol, and extracting the propolis by ultrasonic extraction for 0.75 h, repeating the process three times, combining the filtrates obtained by each ultrasonic extraction, and evaporating the combined filtrates at 50° C. to obtain a propolis extract;

[0098] Step S 13 1. Take 3g of propolis extract and add it to 15ml of polyethylene glycol, mix and fully dissolve it in a water bath at 80℃, and then filter it through a 100-mesh filter to obtain the propolis extract;

[0099] Step S 14 1. Add 3g of polyvinyl alcohol to 15ml of water and swell for 12h. Heat in a water bath at 80℃ to dissolve. After complete dissolution, add 7mL of 0.2mol / L disodium hydrogen phosphate solution. Seal and place in a magnetic stirrer and stir at 900rpm for 12h at 80℃ until a transparent and clear polyvinyl alcohol solution is obtained.

[0100] Step S 15 1. Take 1 g of chitosan and add it to 40 ml of 90% acetic acid solution, mix them, seal them and place them in a magnetic stirrer, stir them at 900 rpm at 80°C for 12 h, until a transparent and clear chitosan solution is obtained;

[0101] Step S 2 , slowly pour the polyvinyl alcohol solution into the chitosan solution and stir evenly at a stirring temperature of 80° C. and a stirring speed of 900 rpm, then slowly pour the propolis extract into the chitosan solution and continue stirring evenly to obtain a mixed solution;

[0102] Step S 3 , filter the mixed solution through a 100-mesh filter, place it in an ice water bath and let it stand for 15 minutes, then perform ultra-high pressure compounding through an ultra-high pressure homogenizer with a homogenization working voltage of 150MPa, place the treated solution in an ice bath to cool down, and obtain a temperature-sensitive propolis gel solution.

[0103] Example 5

[0104] Step S 11 , taking 100 g of propolis raw material, adding 600 ml of n-hexane to mix, soaking and extracting at room temperature for 3 days and then filtering to obtain a first extract;

[0105] Step S 12 , mixing the first extract with 500 ml of 75% aqueous ethanol, and extracting the propolis by ultrasonic extraction for 0.75 h, repeating the process three times, combining the filtrates obtained by each ultrasonic extraction, and evaporating the combined filtrates at 55° C. to obtain a propolis extract;

[0106] Step S 13 1. Take 0.7g of propolis extract and add it to 15ml of polyethylene glycol, mix and fully dissolve it in a 75℃ water bath, and then filter it through an 80-mesh filter to obtain the propolis extract;

[0107] Step S 14 1. Add 1.5 g of polyvinyl alcohol to 40 ml of water and swell for 12 h. Heat in a water bath at 85°C to dissolve. After complete dissolution, add 5 mL of 0.2 mol / L sodium dihydrogen phosphate solution. Seal and place in a magnetic stirrer and stir at 700 rpm for 12 h at 85°C until a transparent and clear polyvinyl alcohol solution is obtained.

[0108] Step S 15 1.5 g chitosan was added into 40 ml 90% acetic acid solution, and the mixture was sealed and placed in a magnetic stirrer, and stirred at 700 rpm for 12 h at 85°C until a transparent and clear chitosan solution was obtained;

[0109] Step S 2 , slowly pour the polyvinyl alcohol solution into the chitosan solution and stir evenly at a stirring temperature of 85° C. and a stirring speed of 700 rpm, then slowly pour the propolis extract into the chitosan solution and continue stirring evenly to obtain a mixed solution;

[0110] Step S 3 , filter the mixed solution through an 80-mesh filter, place it in an ice water bath and let it stand for 15 minutes, then perform ultra-high pressure compounding through an ultra-high pressure homogenizer with a homogenization working voltage of 200MPa, place the treated solution in an ice bath to cool down, and obtain a temperature-sensitive propolis gel solution.

[0111] Comparative Example 1

[0112] The difference between this comparative example and Example 1 is that only step S is performed. 11 -S 13 , that is, only the propolis extract is obtained without compounding with chitosan / polyvinyl alcohol, step S 11 -S 13 The parameters in the operation were the same, and the remaining ingredients were replaced by water according to the total formula to make a comparison sample.

[0113] Comparative Example 2

[0114] The difference between this comparative example and Example 1 is that step S is not performed. 3 The ordinary propolis and chitosan / polyvinyl alcohol solutions were obtained by ultrahigh pressure compounding related operations, and the other parameters were the same.

[0115] The propolis solutions prepared in Examples 1-5 and Comparative Examples 1-2 are marked as Example 1, Example 2, Example 3, Example 4, Example 5, Comparative Example 1 and Comparative Example 2, respectively. Then, the performance of Examples 1-5 and Comparative Examples 1-2 is tested. The specific testing methods and test items are as follows:

[0116] 1. Stability test

[0117] The embodiments of the present invention and the comparative examples were subjected to accelerated stability studies under the conditions of 40°C ± 2°C / 75 ± 5% RH for 6 months. The stability samples (n = 3) were analyzed at 0 days, 30 days, 60 days, 90 days and 180 days to evaluate their physical appearance, including uniformity, color, consistency, separation, etc., and their pH value and viscosity changes were also evaluated. The results are shown in Table 1. The experimental results show that the pH value and viscosity of the obtained product, the thermosensitive propolis gel solution (Examples 1-5), did not change significantly, and there was no phase separation in the appearance. In addition, the samples were intact within the prescribed temperature range of six weeks, and no precipitation, fault, water precipitation, coarse particles, fading, etc. occurred, and the product stability was good. Comparative Example 1 is a propolis polyethylene glycol solution, which has a certain antibacterial effect, but has a strong fluidity and cannot be used as a wound dressing; Comparative Example 2 showed water precipitation and stratification after 30 days, and the pH value and viscosity could not be accurately determined, and the product stability was poor.

[0118] In addition, by comparison, it was found that the thermosensitive propolis gel solution made by loading propolis on chitosan / polyvinyl alcohol polymer was compared with the propolis solution alone. The gel encapsulated the propolis, which greatly reduced its viscosity when it came into contact with the wound. The viscosity range of the composite gel enables the thermosensitive propolis gel to be firmly attached when applied to the wound, and to be easy to remove painlessly when changing the dressing.

[0119] Table 1

[0120]

[0121] 2. Release experiment

[0122] The release of Examples 1-5 and Comparative Example 1 was evaluated by dialysis bag diffusion technique, using phosphate buffer with pH = 7.4 as the release medium, with continuous stirring at 37 ± 0.5 ° C and a rotation speed of 100 rpm. 80 mg of each sample was loaded into a dialysis bag membrane (molecular weight cutoff value was 12,000 Da), and the bag was immersed in the release medium. Then, 2 mL of sample was taken at 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours and 6 hours. The sample was filtered through qualitative filter paper, the resulting solution was diluted, and the concentration of propolis was estimated. The results of the propolis release pattern are shown in the attached Figure 1As shown, the release pattern of propolis in the hydrogel formula is closely related to the drug interaction and matrix structure. In Comparative Example 1, the propolis extract was observed to be completely released within the first 60 minutes, while in Examples 1-5, the initial burst release pattern of the drug was observed within 120 minutes, and the release was basically completed after about 4 hours. It shows that the thermosensitive propolis gel solution prepared by the present invention can be used as a sustained release system to control the release of active ingredients in propolis loaded on chitosan / polyvinyl alcohol in an acidic environment, and has an obvious sustained release effect. The gel prepared by the method of the present invention can maintain its therapeutic effect for a long time, reducing the number of frequent changes.

[0123] 3. Antibacterial experiment

[0124] (1) Bacterial species: Staphylococcus aureus (ATCC 6538) and Escherichia coli (8099) were used as test bacteria;

[0125] (2) Test method: Take sterile and dry filter paper. Add 20 μl of antibacterial solution of actual concentration to each piece, then place the filter paper flat in a clean sterile plate, open the cover and place in an incubator (37°C) to dry, or place it at room temperature to dry naturally for later use. Preparation of negative control sample: Take sterile and dry filter paper, add 20 μl of sterile distilled water to each piece, dry and set aside. For each test, place one infected plate, 4 test samples and 1 negative control sample on each plate, for a total of 5 samples. Use sterile tweezers to take the sample and place it on the surface of the plate. The distance between the centers of each sample is more than 25 mm, and the distance from the edge of the plate is more than 15 mm. After placing it, use sterile tweezers to gently press the sample to make it close to the surface of the plate. Cover the plate, place it in an incubator at 37°C, incubate for 16-18 hours and observe the results. Use a vernier caliper to measure the diameter of the antibacterial ring (including the patch) and record it. When measuring the antibacterial ring, you should choose a uniform and completely sterile antibacterial ring. The diameter should be measured with the outer edge of the antibacterial ring as the boundary.

[0126] (3) Determination of antibacterial effect: When the diameter of the antibacterial ring is greater than 7 mm, it is judged to have antibacterial effect; when the diameter of the antibacterial ring is less than or equal to 7 mm, it is judged to have no antibacterial effect.

[0127] The results are shown in Table 2. The experimental results show that Examples 1-5 and Comparative Examples 1-2 have significant inhibitory effects on Staphylococcus aureus and Escherichia coli. Examples 1-5 show that the antibacterial ability is mainly related to the amount of propolis added; and the comparison between Example 1 and Comparative Example 1 shows that the chitosan / polyvinyl alcohol composite carrier itself has excellent properties such as strong antibacterial ability and good stability. After combining with propolis, a synergistic effect is produced, which effectively enhances the antibacterial effect of the overall temperature-sensitive propolis gel solution and accelerates the wound healing process. Comparative Document 2 was not subjected to ultra-high pressure treatment, and the newly prepared solution also had a good antibacterial effect, but the state was unstable.

[0128] Table 2

[0129]

[0130] In summary, it can be seen from each test example of comparative example and each embodiment that the present invention combines the active ingredient in propolis with chitosan-polyvinyl alcohol polymer by ultrahigh pressure homogenization technology to compare propolis monomers, and the thermosensitive propolis gel solution can complete the controlled release of propolis active ingredients according to human body temperature and pH value changes, and lasting realization provides higher drug concentration in bacterial infection area, so that the therapeutic effect of propolis can be maintained for a long time, and frequent replacement is reduced, and the use effect of propolis is maximized. And the chitosan / polyvinyl alcohol composite carrier itself has antibacterial activity, and after being combined with propolis, a synergistic effect may be produced, so that the thermosensitive propolis gel solution has a stronger antibacterial effect, and the wound healing process is accelerated. In addition, the stability of the thermosensitive propolis gel solution is also significantly improved, and its service life can be effectively extended. Compared with the prior art, the present invention has significant progress.

[0131] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a temperature-sensitive propolis gel solution, characterized in that: The following steps are involved: Step S1, preparing propolis extract, polyvinyl alcohol solution and chitosan solution respectively; Step S2, firstly mixing the polyvinyl alcohol solution and the chitosan solution evenly, and then adding the propolis extract and mixing evenly to obtain a mixed solution; Step S3, filtering the mixed solution, standing it at low temperature, compounding it under ultra-high pressure, and cooling it to obtain a temperature-sensitive propolis gel solution.

2. The preparation method according to claim 1, characterized in that The homogeneous working voltage of ultra-high voltage composite is 150-200MPa.

3. The preparation method according to claim 1, characterized in that: The weight ratio of the polyvinyl alcohol solution to the chitosan solution is 1-3:

1.

4. The preparation method according to claim 1, characterized in that: The propolis extract accounts for 0.5-4.0% of the mixed solution.

5. The preparation method according to claim 1, characterized in that: The mixed solution is mixed uniformly by stirring, the stirring temperature is 80-90° C., and the stirring speed is 400-1000 rpm.

6. The preparation method according to claim 1, characterized in that: The preparation of the propolis extract comprises the following specific steps: Step S 11 , mixing propolis and a solvent in a ratio of 1:2-8, soaking and extracting at room temperature for 2-3 days, and then filtering to obtain a first extract, wherein the propolis is measured in grams and the solvent is measured in milliliters; Step S 12 , mixing the first extract with aqueous ethanol at a ratio of 1:4-8, and extracting by ultrasonic for 0.25-1h to obtain a filtrate, wherein the aqueous ethanol is a 70%-95% ethanol solution in milliliters, and the first extract is in grams; after repeating at least three times, combining the filtrates obtained from each ultrasonic extraction, and evaporating the combined filtrates at 50-65°C to obtain a propolis extract; Step S 13 , adding the propolis extract into polyethylene glycol for mixing and fully dissolving, and then filtering to obtain the propolis extract.

7. The preparation method according to claim 1, characterized in that: The preparation of the polyvinyl alcohol solution comprises the following specific steps: adding polyvinyl alcohol to water to swell for 12-24 hours and then fully dissolving, then adding disodium hydrogen phosphate solution to adjust the pH to 6-7, and stirring at 80-90° C. at a speed of 600-1000 rpm for at least 12 hours until the polyvinyl alcohol solution is obtained.

8. The preparation method according to claim 1, characterized in that: The preparation of the chitosan solution comprises the following specific steps: adding chitosan into acetic acid solution for mixing, and stirring at 50-80° C. at a rotation speed of 400-1000 rpm for at least 12 hours until a transparent and clear chitosan solution is obtained.

9. A temperature-sensitive propolis gel solution, characterized in that: Calculated by weight percentage, the invention comprises: 0.5-4.0% of propolis extract, 1.5-5.0% of a polymer of polyvinyl alcohol and chitosan, and the balance of water.

10. The temperature-sensitive propolis gel solution according to claim 9, characterized in that pH value is 4.5-7.0; The temperature-sensitive propolis gel solution is placed in the human body temperature range or in the human body environment to form a gel-like temperature-sensitive propolis gel; The microstructure of the thermosensitive propolis gel is a three-dimensional network structure.

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