Anti-HPV (human papillomavirus) temperature-sensitive gel as well as preparation method and application thereof

Abstract

The invention provides an anti-HPV (human papillomavirus) temperature-sensitive gel as well as a preparation method and application thereof. The invention provides the anti-HPV temperature-sensitive gel as well as the preparation method and application thereof. The gel consists of a natural active extract, a temperature-sensitive polymer, a cross-linking agent and a stabilizer, wherein the natural active extract comprises a mixture of a sophora flavescens extract, an artemisia annua extract and a mushroom extract in a specific mass ratio. These extracts are obtained by specific extraction processes to ensure their antiviral activity. The thermo-sensitive polymer is a mixture of poly (N-isopropylacrylamide) and hydroxypropyl methyl cellulose, and the cross-linking agent and the stabilizer are used for enhancing the performance of the gel. The preparation method of the gel comprises the following steps: preparing a natural active extract emulsion, preparing a wall material solution, emulsifying and microencapsulating, so as to finally form the gel with temperature sensitivity. The gel has specific rheological properties at body temperature, can be used for treating HPV infection, and provides a new local treatment method.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-HPV virus temperature-sensitive gel and a preparation method and application thereof. Background Art

[0002] Human papillomavirus (HPV) infection is a common sexually transmitted disease, which is closely related to the occurrence and development of many diseases such as cervical cancer. In the field of medicine, the treatment of human papillomavirus (HPV) infection has always been a hot topic and difficulty in research. HPV infection is closely related to many diseases, including cervical cancer, genital warts, etc., posing a serious threat to public health. At present, the treatment methods for HPV infection mainly include surgery, physical therapy and drug therapy. Among them, thermosensitive gels in drug therapy have attracted much attention due to their advantages such as convenience of use and obvious local effects. However, they still have many shortcomings in terms of efficacy, safety and ease of use. Among them, gel preparations have shown broad application prospects in the treatment of HPV infection due to their good biocompatibility and local drug delivery characteristics.

[0003] However, existing anti-HPV gel preparations generally have problems such as long gel time, poor rheological properties, and weak anti-HPV ability. Long gel time means that the drug has partially or completely lost its activity before reaching the target site, affecting the efficacy of the drug; poor rheological properties lead to insufficient stability of the gel in the body, making it difficult to form a long-lasting drug release system at the target site, and the uniformity and continuity of drug release are difficult to accurately control; and the existing thermosensitive gel has weak anti-HPV ability, making it difficult to effectively inhibit the replication and spread of the virus, thus limiting its application in clinical treatment. Summary of the invention

[0004] In view of this, the present invention proposes an anti-HPV virus thermosensitive gel and a preparation method and application thereof to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: an anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 40-60 parts of natural active extracts, 15-25 parts of thermosensitive polymers, 3-5 parts of crosslinking agents, and 8-10 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of (3-8): (5-12): (15-20). A crosslinking agent with good compatibility with the thermosensitive polymer and high crosslinking efficiency is added to ensure the stability and uniformity of the gel network structure; the stabilizer can protect the gel from the influence of the external environment, can improve the physical stability of the gel, and thus extend its service life.

[0006] Furthermore, the Sophora flavescens extract is prepared by crushing the Sophora flavescens medicinal material into 80-100 meshes, using ethanol with a mass concentration of 60%-80% as a solvent, mixing the Sophora flavescens powder with ethanol at a solid-liquid ratio of 1:8-12, heating and refluxing extraction at 60-80°C for 2-4 times, each time for 1-3 hours, combining the extracts, and concentrating under reduced pressure to obtain the Sophora flavescens extract.

[0007] Furthermore, the Artemisia annua extract is obtained by drying and crushing Artemisia annua to 100-120 mesh, extracting with supercritical CO2 fluid through an extractor filled with Artemisia annua powder at 10-30MPa and 40-60°C for 2-4h, separating and purifying to obtain the Artemisia annua extract.

[0008] Furthermore, the mushroom extract is prepared by inoculating mushroom strains into a liquid culture medium, culturing at a temperature of 25-30°C and a relative humidity of 60-80% for 7-14 days, collecting mushroom mycelium, crushing and grinding, adding ethanol with a mass concentration of 60-80% to mix, the mass volume ratio of mycelium to ethanol is 1:5-10, stirring and extracting at 40-60°C for 1-3h, filtering, concentrating, and purifying to obtain the mushroom extract.

[0009] Furthermore, the temperature-sensitive polymer is poly (N-isopropylacrylamide) and hydroxypropyl methylcellulose in a mass ratio of 3-5:2.

[0010] Furthermore, the cross-linking agent includes but is not limited to N,N'-methylenebisacrylamide, glutaraldehyde, and divinylbenzene.

[0011] Furthermore, the stabilizer is disodium ethylenediaminetetraacetate.

[0012] Furthermore, the method for preparing the anti-HPV virus thermosensitive gel comprises the following steps:

[0013] (1) Preparing a natural active extract emulsion: dispersing the natural active extract in an anhydrous ethanol-chloroform solution, wherein the volume ratio of anhydrous ethanol to chloroform is 15-25:50, and the mass volume ratio of the natural active extract to the anhydrous ethanol-chloroform solution is 8-12:45, and then stirring at a speed of 400-600 rpm for 15-30 minutes using a stirring device to form a uniform emulsion;

[0014] (2) preparing a wall material solution: dissolving a thermosensitive polymer in hot water at 40-60° C. to form a transparent solution, then cooling the solution to room temperature to keep the solution uniform, dispersing a crosslinking agent and a stabilizer in the polymer solution to form a wall material solution;

[0015] (3) Emulsification: under high-speed stirring, the natural active extract emulsion is slowly added dropwise to the wall material solution, and an emulsifier is added for stirring at a stirring speed of 500-700 rpm to form a coarse emulsion, and then the coarse emulsion is homogenized using a high-pressure homogenizer with a pressure set to 800-1500 bar, and the homogenization cycle is repeated 3-5 times to obtain a uniform fine emulsion;

[0016] (4) Microencapsulation: The pH of the miniemulsion is adjusted to 4.0-4.5, and the temperature is maintained at 30-40°C. Ultrasonic stirring is used for 30-60 min, with a stirring rate of 100-300 rpm, an ultrasonic power of 200-400 W, and an ultrasonic frequency of 60-80 kHz. The temperature is maintained at 30-40°C to cause a phase change and form a gel state to obtain a thermosensitive gel.

[0017] Furthermore, the emulsifier is poloxamer, sodium lauryl sulfate or cetyltrimethylammonium bromide, and the amount used is 1-3% of the mass of the natural active extract.

[0018] Furthermore, the anti-HPV virus thermosensitive gel is used in the preparation of drugs for treating HPV infection.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Shorten gelation time and improve drug utilization: Through the carefully designed formula and preparation process, the thermosensitive gel of the present invention can quickly form a stable gel structure at body temperature, significantly shortening the gelation time. This allows the drug to reach and act on the target site more quickly, reduces the loss of the drug during the transmission process, and improves the bioavailability and efficacy of the drug.

[0021] (2) Optimizing rheological properties and enhancing stability: The thermosensitive gel of the present invention exhibits excellent rheological properties after formation, and can maintain a stable gel state in the body without flowing or deforming easily. This stability helps the drug to form a long-lasting drug release system at the target site, ensuring that the drug can be released continuously and stably, thereby extending the drug's action time and improving the therapeutic effect.

[0022] (3) Enhanced anti-HPV ability: The thermosensitive gel of the present invention introduces a variety of natural ingredients with strong antiviral activity, such as Sophora flavescens extract, Artemisia annua extract and mushroom extract. These ingredients can significantly enhance the anti-HPV ability of the gel through scientific proportion and synergistic effect. Experiments have shown that the gel has a good inhibitory effect on HPV virus, providing strong support for the treatment of HPV infection.

[0023] In summary, the gel of the present invention aims to solve the problems of long gel time, poor rheological properties and weak anti-HPV virus ability in existing gel preparations by scientifically matching natural active extracts, thermosensitive polymers, crosslinking agents and stabilizers, combined with advanced preparation processes. By optimizing the formula and preparation conditions of the gel, the thermosensitive gel of the present invention can quickly form a stable gel structure at body temperature, effectively prolong the residence time of the drug in the target area, improve the bioavailability and efficacy of the drug, and at the same time show excellent rheological properties and strong antiviral ability, providing a new solution for the treatment of HPV infection. DETAILED DESCRIPTION

[0024] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0027] Example 1

[0028] An anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 40 parts of natural active extracts, 15 parts of thermosensitive polymers, 3 parts of crosslinking agents, and 8 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of 3:5:15;

[0029] The sophora flavescens extract is obtained by grinding the sophora flavescens medicinal material into 80 meshes, using ethanol with a mass concentration of 60% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:8, heating and refluxing extraction at 60° C. for 2 times, each time for 1 hour, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract;

[0030] The Artemisia annua extract is prepared by drying and crushing Artemisia annua to 100 meshes, extracting for 2 hours with supercritical CO2 fluid through an extractor filled with Artemisia annua powder under the conditions of 10 MPa and 40° C., separating and purifying to obtain the Artemisia annua extract;

[0031] The mushroom extract is prepared by inoculating mushroom spawn into a liquid culture medium, culturing at a temperature of 25° C. and a relative humidity of 60% for 7 days, collecting mushroom mycelium, crushing and grinding, adding 60% ethanol to mix, the mass volume ratio of mycelium to ethanol being 1:5, stirring and extracting at 40° C. for 1 hour, filtering, concentrating, and purifying to obtain the mushroom extract;

[0032] The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 3:2;

[0033] The cross-linking agent is divinylbenzene;

[0034] The stabilizer is disodium ethylenediaminetetraacetate.

[0035] Example 2

[0036] An anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 60 parts of natural active extracts, 25 parts of thermosensitive polymers, 5 parts of crosslinking agents, and 10 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of 8:12:20;

[0037] The sophora flavescens extract is obtained by grinding the sophora flavescens medicinal material into 100 meshes, using ethanol with a mass concentration of 80% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:12, heating and refluxing at 80° C. for 4 times, each time for 3 hours, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract;

[0038] The Artemisia annua extract is obtained by drying and crushing Artemisia annua to 100-120 meshes, extracting for 4 hours through an extractor filled with Artemisia annua powder under the conditions of 30 MPa and 60° C., separating and purifying to obtain the Artemisia annua extract;

[0039] The mushroom extract is prepared by inoculating mushroom spawn into a liquid culture medium, culturing at a temperature of 30° C. and a relative humidity of 80% for 14 days, collecting mushroom mycelium, crushing and grinding, adding ethanol with a mass concentration of 80% and mixing, wherein the mass volume ratio of mycelium to ethanol is 1:10, stirring and extracting at 60° C. for 3 hours, filtering, concentrating and purifying to obtain the mushroom extract;

[0040] The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 5:2;

[0041] The cross-linking agent is glutaraldehyde;

[0042] The stabilizer is disodium ethylenediaminetetraacetate.

[0043] Example 3

[0044] An anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 50 parts of natural active extracts, 20 parts of thermosensitive polymers, 4 parts of crosslinking agents, and 9 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of 5:10:18;

[0045] The sophora flavescens extract is obtained by grinding the sophora flavescens medicinal material into 90 meshes, using ethanol with a mass concentration of 70% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:10, heating and refluxing extraction at 70°C for 3 times, each time for 2 hours, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract;

[0046] The Artemisia annua extract is prepared by drying and crushing Artemisia annua to 110 meshes, extracting for 3 hours with supercritical CO2 fluid through an extractor filled with Artemisia annua powder under the conditions of 20 MPa and 50° C., separating and purifying to obtain the Artemisia annua extract;

[0047] The mushroom extract is prepared by inoculating mushroom spawn into a liquid culture medium, culturing at a temperature of 28° C. and a relative humidity of 70% for 10 days, collecting mushroom mycelium, crushing and grinding, adding 70% ethanol to mix, the mass volume ratio of mycelium to ethanol being 1:8, stirring and extracting at 50° C. for 2 hours, filtering, concentrating, and purifying to obtain the mushroom extract;

[0048] The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 4:2;

[0049] The cross-linking agent is N,N'-methylenebisacrylamide;

[0050] The stabilizer is disodium ethylenediaminetetraacetate;

[0051] The above examples 1-3 adopt the following preparation method:

[0052] (1) preparing a natural active extract emulsion: dispersing the natural active extract in an anhydrous ethanol-chloroform solution, wherein the volume ratio of anhydrous ethanol to chloroform is 20:50, and the mass volume ratio of the natural active extract to the anhydrous ethanol-chloroform solution is 10:45, and then stirring the mixture at a speed of 500 rpm for 25 minutes to form a uniform emulsion;

[0053] (2) preparing a wall material solution: dissolving a thermosensitive polymer in hot water at 50° C. to form a transparent solution, then cooling the solution to room temperature to keep the solution uniform, dispersing a crosslinking agent and a stabilizer in the polymer solution to form a wall material solution;

[0054] (3) Emulsification: under high-speed stirring, the natural active extract emulsion is slowly added dropwise to the wall material solution, and an emulsifier is added for stirring. The emulsifier is poloxamer, and the amount used is 2% of the mass of the natural active extract. The stirring speed is 600 rpm to form a coarse emulsion, and then the coarse emulsion is homogenized using a high-pressure homogenizer. The pressure is set to 1200 bar, and the homogenization is circulated for 4 times to obtain a uniform fine emulsion;

[0055] (4) Microencapsulation: The pH of the miniemulsion was adjusted to 4.0, and the temperature was maintained at 35°C. Ultrasonic stirring was used for 50 min. The stirring rate was 200 rpm, the ultrasonic power was 300 W, and the ultrasonic frequency was 70 kHz. The temperature was maintained at 35°C to allow a phase change to occur and form a gel state to obtain a thermosensitive gel.

[0056] Example 4

[0057] An anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 50 parts of natural active extracts, 20 parts of thermosensitive polymers, 4 parts of crosslinking agents, and 9 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of 5:10:18;

[0058] The sophora flavescens extract is obtained by grinding the sophora flavescens medicinal material into 90 meshes, using ethanol with a mass concentration of 70% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:10, heating and refluxing extraction at 70°C for 3 times, each time for 2 hours, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract;

[0059] The Artemisia annua extract is prepared by drying and crushing Artemisia annua to 110 meshes, extracting for 3 hours with supercritical CO2 fluid through an extractor filled with Artemisia annua powder under the conditions of 20 MPa and 50° C., separating and purifying to obtain the Artemisia annua extract;

[0060] The mushroom extract is prepared by inoculating mushroom spawn into a liquid culture medium, culturing at a temperature of 28° C. and a relative humidity of 70% for 10 days, collecting mushroom mycelium, crushing and grinding, adding 70% ethanol to mix, the mass volume ratio of mycelium to ethanol being 1:8, stirring and extracting at 50° C. for 2 hours, filtering, concentrating, and purifying to obtain the mushroom extract;

[0061] The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 4:2;

[0062] The cross-linking agent is N,N'-methylenebisacrylamide;

[0063] The stabilizer is disodium ethylenediaminetetraacetate;

[0064] The above-mentioned Example 4 adopts the following preparation method:

[0065] (1) preparing a natural active extract emulsion: dispersing the natural active extract in an anhydrous ethanol-chloroform solution, wherein the volume ratio of anhydrous ethanol to chloroform is 15:50, and the mass volume ratio of the natural active extract to the anhydrous ethanol-chloroform solution is 8:45, and then stirring the mixture at a speed of 400 rpm for 15 minutes to form a uniform emulsion;

[0066] (2) preparing a wall material solution: dissolving a thermosensitive polymer in hot water at 40° C. to form a transparent solution, then cooling the solution to room temperature to keep the solution uniform, dispersing a crosslinking agent and a stabilizer in the polymer solution to form a wall material solution;

[0067] (3) Emulsification: under high-speed stirring, the natural active extract emulsion is slowly added dropwise to the wall material solution, and an emulsifier is added and stirred at the same time. The emulsifier is sodium dodecyl sulfate, and the amount used is 1% of the mass of the natural active extract. The stirring speed is 500 rpm to form a coarse emulsion, and then the coarse emulsion is homogenized using a high-pressure homogenizer. The pressure is set to 800 bar, and the homogenization is circulated for 3 times to obtain a uniform fine emulsion;

[0068] (4) Microencapsulation: The pH of the miniemulsion was adjusted to 4.0, and the temperature was maintained at 30°C. Ultrasonic stirring was used for 30 min, with a stirring rate of 100 rpm, an ultrasonic power of 200 W, and an ultrasonic frequency of 60 kHz. The temperature was continued to be maintained at 30°C to allow a phase change to occur and form a gel state to obtain a thermosensitive gel.

[0069] Example 5

[0070] An anti-HPV virus thermosensitive gel: comprising the following raw materials in parts by weight: 50 parts of natural active extracts, 20 parts of thermosensitive polymers, 4 parts of crosslinking agents, and 9 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of 5:10:18;

[0071] The sophora flavescens extract is obtained by grinding the sophora flavescens medicinal material into 90 meshes, using ethanol with a mass concentration of 70% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:10, heating and refluxing extraction at 70°C for 3 times, each time for 2 hours, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract;

[0072] The Artemisia annua extract is prepared by drying and crushing Artemisia annua to 110 meshes, extracting for 3 hours with supercritical CO2 fluid through an extractor filled with Artemisia annua powder under the conditions of 20 MPa and 50° C., separating and purifying to obtain the Artemisia annua extract;

[0073] The mushroom extract is prepared by inoculating mushroom spawn into a liquid culture medium, culturing at a temperature of 28° C. and a relative humidity of 70% for 10 days, collecting mushroom mycelium, crushing and grinding, adding 70% ethanol to mix, the mass volume ratio of mycelium to ethanol being 1:8, stirring and extracting at 50° C. for 2 hours, filtering, concentrating, and purifying to obtain the mushroom extract;

[0074] The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 4:2;

[0075] The cross-linking agent is N,N'-methylenebisacrylamide;

[0076] The stabilizer is disodium ethylenediaminetetraacetate;

[0077] The above-mentioned Example 5 adopts the following preparation method:

[0078] (1) preparing a natural active extract emulsion: dispersing the natural active extract in an anhydrous ethanol-chloroform solution, wherein the volume ratio of anhydrous ethanol to chloroform is 25:50, and the mass volume ratio of the natural active extract to the anhydrous ethanol-chloroform solution is 12:45, and then stirring at a speed of 600 rpm for 15-30 minutes using a stirring device to form a uniform emulsion;

[0079] (2) preparing a wall material solution: dissolving a thermosensitive polymer in hot water at 60° C. to form a transparent solution, then cooling the solution to room temperature to keep the solution uniform, dispersing a crosslinking agent and a stabilizer in the polymer solution to form a wall material solution;

[0080] (3) Emulsification: under high-speed stirring, the natural active extract emulsion is slowly added dropwise to the wall material solution, and an emulsifier is added for stirring. The emulsifier is hexadecyltrimethylammonium bromide, and the amount is 3% of the mass of the natural active extract. The stirring speed is 700 rpm to form a crude emulsion, and then the crude emulsion is homogenized using a high-pressure homogenizer. The pressure is set to 1500 bar, and the homogenization is circulated for 5 times to obtain a uniform fine emulsion;

[0081] (4) Microencapsulation: The pH of the miniemulsion was adjusted to 4.5, and the temperature was maintained at 40°C. Ultrasonic stirring was used for 60 min, with a stirring rate of 300 rpm, an ultrasonic power of 400 W, and an ultrasonic frequency of 80 kHz. The temperature was maintained at 40°C to allow a phase change to occur and form a gel state, thereby obtaining a thermosensitive gel.

[0082] Comparative Example 1

[0083] An anti-HPV virus thermosensitive gel comprises the following raw materials in parts by weight: 80 parts of natural active extracts, 12 parts of thermosensitive polymers, 8 parts of cross-linking agents, and 12 parts of stabilizers.

[0084] Comparative Example 2

[0085] The difference between this comparative example and Example 3 is that the temperature-sensitive polymer in the raw material of the anti-HPV virus temperature-sensitive gel is chitosan.

[0086] Comparative Example 3

[0087] The difference between this comparative example and Example 3 is that in the preparation method of the anti-HPV virus thermosensitive gel, no microencapsulation preparation is performed, and the details are as follows:

[0088] The natural active extract, thermosensitive polymer, crosslinker and stabilizer were mixed, and ultrasonic stirring was performed at 35°C for 50 min, with a stirring rate of 200 rpm, an ultrasonic power of 300 W, and an ultrasonic frequency of 70 kHz. The temperature was maintained at 35°C to cause a phase change and form a gel state to obtain a thermosensitive gel.

[0089] Test Example 2-Rheological Properties Test

[0090] 1. Sample preparation: Take the anti-HPV virus thermosensitive gel samples of Examples 1-5 and Comparative Examples 1-3 and put them into the sample container of the rheometer to avoid bubbles and uneven loading.

[0091] 2. Gel time: quickly place the container containing the gel sample into a 34°C constant temperature water bath and start the timer;

[0092] When the gel begins to lose fluidity and shows an obvious gel state, stop the timer. The time recorded at this time is the gel time;

[0093] In order to improve the accuracy of the test, multiple repeated tests can be performed and the average value can be taken as the final gel time.

[0094] 3. Steady-state shear test:

[0095] Shear rate range: 0.01s-1 to 100s-1,

[0096] Test temperature: 37℃,

[0097] Data collection points: Select at least 20 data points evenly within the shear rate range to obtain a more accurate curve.

[0098] 4. Dynamic Oscillation Test

[0099] Frequency range: 0.1rad / s to 100rad / s, to investigate the viscoelastic response of the gel at different frequencies.

[0100] Strain amplitude: between 0.1% and 10% to ensure that the test is in the linear viscoelastic region.

[0101] Test temperature: same as the steady-state shear test temperature.

[0102] 5. Temperature scanning test

[0103] Temperature range: 10℃ to 50℃.

[0104] Heating rate: 1-5℃ / min, in order to observe the response of the gel during temperature change.

[0105] Oscillation frequency: frequency sweep from 0.1rad / s to 100rad / s.

[0106] The stress response at different frequencies, including the storage modulus (G') and loss modulus (G"), was recorded.

[0107] 6. Analyze the test data and calculate the zero shear viscosity, consistency coefficient, flow index and storage modulus.

[0108] 7. Test results:

[0109]

[0110]

[0111] Result analysis: Examples 1-5 showed good rheological properties as a whole. Among them, Example 3 performed outstandingly in various indicators and had the best effect.

[0112] Comparative analysis of embodiments:

[0113] Gel time: The gel time of Examples 1-5 is between 29 and 35 seconds, which is relatively stable and within a shorter time range.

[0114] Zero shear viscosity: The zero shear viscosity of Examples 1-5 is between 140-155 Pa·s, among which Example 3 has the highest zero shear viscosity of 155 Pa·s. A higher zero shear viscosity means that the gel has better stability and is less likely to flow at low shear rates. This indicates that Example 3 may have better morphology retention ability during storage and initial use.

[0115] Consistency coefficient: The consistency coefficient of Example 3 is 82, which is higher than that of other examples. The consistency coefficient reflects the viscosity and cohesion of the gel. The higher consistency coefficient indicates that the gel of Example 3 is more viscous, has stronger structural stability, and can better maintain its shape and integrity.

[0116] Flow index: The flow index of Example 3 is 0.28, which is relatively low. The smaller the flow index, the more obvious the shear thinning property of the gel, that is, the viscosity decreases at high shear rates, and it is easy to apply and use. This is very important for the practical application of the anti-HPV virus thermosensitive gel, because it needs to be able to evenly cover the lesion site when used.

[0117] Storage modulus and loss modulus: At low frequency (0.1rad / s) and high frequency (100rad / s), the storage modulus of Example 3 is higher than that of other examples, while the loss modulus is relatively moderate. The storage modulus represents the elastic part of the gel, and the loss modulus represents the viscous part of the gel. A higher storage modulus means that the gel of Example 3 has better elasticity and can quickly restore its original shape after being subjected to external force, which is very important for maintaining the structural stability of the gel and its effectiveness against HPV viruses. At the same time, the moderate loss modulus indicates that when the gel is subjected to external force, it has a certain ability to dissipate energy through viscosity and maintain good elasticity.

[0118] Comparative analysis of embodiments and comparative examples:

[0119] Gel time: By comparing the example group with the comparative example, it can be seen that different formulations and preparation methods have a significant effect on the gel time. The ratio of the natural active extract, the thermosensitive polymer, the crosslinking agent and the stabilizer used in the example and the microencapsulation preparation steps help to shorten the gel time.

[0120] Zero shear viscosity, consistency coefficient and flow index: The zero shear viscosity and consistency coefficient of the comparative group are significantly lower than those of the examples, while the flow index is relatively high. This indicates that the gel of the comparative example is not as good as that of the examples in terms of stability, viscosity and cohesion, and is closer to Newtonian fluid, and the shear thinning property is not obvious. This may make it difficult to apply evenly during use, affecting the release and absorption of the drug.

[0121] Storage modulus and loss modulus: The storage modulus of the comparative group is significantly lower than that of the embodiment at both low and high frequencies, while the loss modulus is relatively high. This indicates that the gel of the comparative group has poor elasticity and relatively high viscosity. When subjected to external forces, it is more easily deformed and difficult to maintain its original shape and structure, thus affecting its therapeutic effect on HPV virus.

[0122] In summary, the embodiment group shows the best performance in terms of zero shear viscosity, consistency coefficient, flow index, storage modulus and loss modulus. Compared with Comparative Examples 1 and 2, it is shown that the thermosensitive polymers of each raw material under reasonable formula design and selected by specific ratios can obtain anti-HPV virus thermosensitive gel with good stability, spreadability and elasticity. Compared with Comparative Example 3, in the preparation process of the present invention, the thermosensitive polymer is made into a wall material solution, and a stable complex is formed with the anti-HPV virus natural active extract to enhance the structural stability of the gel; it has better elasticity and ability to resist deformation.

[0123] The following comparative example was set up to test the anti-HPV virus ability of the thermosensitive gel

[0124] Comparative Example 4

[0125] The difference between this comparative example and Example 3 is that the natural active extract is Sophora flavescens extract.

[0126] Comparative Example 5

[0127] The difference between this comparative example and Example 3 is that the natural active extracts are Sophora flavescens extract, Artemisia annua extract and Mushroom extract in a mass ratio of 1:1:1.

[0128] Test Example 2 - Anti-HPV Virus Ability of Thermosensitive Gel

[0129] 1. Material preparation: Select HeLa cells, HPV16 virus strain, and the thermosensitive gel of Example 3 and Comparative Examples 1-5.

[0130] 2. Cell culture: Cultivate cell suspension and adjust the cell density to 1×10 per well. 4 Add the cell suspension to the culture plate, shake it gently, and place it in an incubator to allow the cells to adhere to the wall and grow.

[0131] 3. Virus infection: The HPV virus strain was cultured and amplified in a suitable cell line, the virus fluid was collected, and the virus titer was determined; the virus fluid was added to the cells in the culture plate, the multiplicity of infection (MOI) was 10, and incubated in a 37°C, 5% CO2 incubator for 8 hours.

[0132] 4. Thermosensitive gel treatment: 24 hours after virus infection, add thermosensitive gel to the cells in the culture plate. For a 96-well plate, add 100 μL of gel. Set up a control group consisting of untreated infected cells and infected cells treated only with culture medium.

[0133] 5. Calculate the inhibition rate of temperature-sensitive gel on HPV virus:

[0134] (1) The formula for calculating the inhibition rate of HPV virus gene expression is: inhibition rate = (virus gene expression level in the control group - virus gene expression level in the treatment group) / virus gene expression level in the control group × 100%.

[0135] (2) The calculation formula for the cytopathic effect inhibition rate is: inhibition rate = (cytopathic effect level of control group - cytopathic effect level of treatment group) / cytopathic effect level of control group × 100%.

[0136] (3) The formula for calculating the cell survival rate improvement rate is: improvement rate = (cell survival rate of the treatment group - cell survival rate of the control group) / cell survival rate of the control group × 100%.

[0137] 6. Results and data

[0138]

[0139] Through the analysis of Example 3 and Comparative Examples 1-5, it can be seen that the anti-HPV virus thermosensitive gel in Example 3 shows significant advantages in terms of HPV virus gene expression inhibition rate, cytopathic inhibition rate and cell survival rate improvement rate. It shows that the thermosensitive gel in Example 3 has an outstanding effect in inhibiting HPV virus gene expression, and can effectively reduce viral replication and transcription, thereby reducing viral activity. It can effectively prevent HPV virus from destroying cells and maintain the normal morphology and function of cells. It shows that the gel of the present invention can promote cell survival and growth, enhance cell resistance, and provide strong support for fighting HPV virus.

[0140] From the comparison of comparative examples 4 and 5, it can be seen that the natural active extracts of the present invention have good antiviral effects after being designed with specific ratios, wherein the Sophora flavescens extract plays an anti-HPV role by interfering with the replication cycle of HPV virus, inhibiting the synthesis of viral proteins or enhancing the immune function of the body; the Artemisia annua extract plays an anti-HPV role by inhibiting the nucleic acid synthesis of HPV virus, destroying the structure of the virus or regulating the immune response of the body; the mushroom extract plays an anti-HPV role by activating the body's immune system, enhancing the function of immune cells or directly inhibiting the growth of HPV virus; the three can synergistically promote the activation and proliferation of immune cells, and enhance the body's immune surveillance and clearance ability of HPV virus. The antiviral effect is significantly enhanced, which helps to enhance the self-cleaning ability and immunity of the vagina, thereby resisting the infection of HPV virus, and can reduce the inflammatory response caused by HPV virus infection.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-HPV virus thermosensitive gel, characterized in that: The invention comprises the following raw materials in parts by weight: 40-60 parts of natural active extracts, 15-25 parts of thermosensitive polymers, 3-5 parts of cross-linking agents, and 8-10 parts of stabilizers; the natural active extracts are Sophora flavescens extracts, Artemisia annua extracts, and mushroom extracts in a mass ratio of (3-8):(5-12):(15-20).

2. The anti-HPV thermosensitive gel according to claim 1, characterized in that: The sophora flavescens extract is prepared by crushing the sophora flavescens medicinal material into 80-100 meshes, using ethanol with a mass concentration of 60%-80% as a solvent, mixing the sophora flavescens powder with ethanol at a solid-liquid ratio of 1:8-12, heating and refluxing extraction at 60-80°C for 2-4 times, each time for 1-3 hours, combining the extracts, and concentrating under reduced pressure to obtain the sophora flavescens extract.

3. The anti-HPV virus thermosensitive gel according to claim 1, characterized in that: The artemisia annua extract is prepared by drying and crushing the artemisia annua to 100-120 meshes, extracting the artemisia annua powder through an extractor containing supercritical CO2 fluid at 10-30 MPa and 40-60° C. for 2-4 hours, separating and purifying the artemisia annua extract.

4. The anti-HPV virus thermosensitive gel according to claim 1, characterized in that: The mushroom extract is prepared by inoculating mushroom strains into a liquid culture medium, culturing for 7-14 days at a temperature of 25-30° C. and a relative humidity of 60-80%, collecting mushroom mycelia, crushing and grinding them, adding ethanol with a mass concentration of 60-80% to mix them, wherein the mass volume ratio of the mycelia to the ethanol is 1:5-10, stirring and extracting at 40-60° C. for 1-3 hours, filtering, concentrating and purifying to obtain the mushroom extract.

5. The anti-HPV thermosensitive gel according to claim 1, characterized in that: The temperature-sensitive polymer is poly-N-isopropylacrylamide and hydroxypropyl methylcellulose in a mass ratio of 3-5:

2.

6. The anti-HPV virus thermosensitive gel according to claim 1, characterized in that: The cross-linking agent includes but is not limited to N,N'-methylenebisacrylamide, glutaraldehyde, and divinylbenzene.

7. The anti-HPV thermosensitive gel according to claim 1, characterized in that: The stabilizer is disodium ethylenediaminetetraacetate.

8. The method for preparing an anti-HPV virus thermosensitive gel according to claim 1, characterized in that: The following steps are involved: (1) Preparing a natural active extract emulsion: dispersing the natural active extract in an anhydrous ethanol-chloroform solution, wherein the volume ratio of anhydrous ethanol to chloroform is 15-25:50, and the mass volume ratio of the natural active extract to the anhydrous ethanol-chloroform solution is 8-12:45, and then stirring at a speed of 400-600 rpm for 15-30 minutes using a stirring device to form a uniform emulsion; (2) preparing a wall material solution: dissolving a thermosensitive polymer in hot water at 40-60° C. to form a transparent solution, then cooling the solution to room temperature to keep the solution uniform, dispersing a crosslinking agent and a stabilizer in the polymer solution to form a wall material solution; (3) Emulsification: under high-speed stirring, the natural active extract emulsion is slowly added dropwise to the wall material solution, and an emulsifier is added for stirring at a stirring speed of 500-700 rpm to form a coarse emulsion, and then the coarse emulsion is homogenized using a high-pressure homogenizer with a pressure set to 800-1500 bar, and the homogenization cycle is repeated 3-5 times to obtain a uniform fine emulsion; (4) Microencapsulation: The pH of the miniemulsion is adjusted to 4.0-4.5, and the temperature is maintained at 30-40°C. Ultrasonic stirring is used for 30-60 min, with a stirring rate of 100-300 rpm, an ultrasonic power of 200-400 W, and an ultrasonic frequency of 60-80 kHz. The temperature is maintained at 30-40°C to cause a phase change and form a gel state to obtain a thermosensitive gel.

9. The method for preparing an anti-HPV virus thermosensitive gel according to claim 8, characterized in that: The emulsifier is poloxamer, sodium lauryl sulfate or hexadecyl trimethyl ammonium bromide, and the amount used is 1-3% of the mass of the natural active extract.

10. Use of the anti-HPV virus thermosensitive gel according to any one of claims 1 to 7 in the preparation of drugs for treating HPV infection.

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