Cyclovirobuxine hydrochloride D-loaded temperature-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a temperature-sensitive hydrogel loaded with cyclovirobuxine hydrochloride D as well as a preparation method and application of the temperature-sensitive hydrogel, and belongs to the technical field of medicines. The preparation method comprises the following steps: dissolving glycerol, polyvinylpyrrolidone, poloxamer 407 and poloxamer 188 in water, stirring until the poloxamer is completely dissolved, adding hydrochloric acid after the poloxamer is swelled, and adjusting the pH value to prepare blank temperature-sensitive gel; and adding cyclovirobuxine hydrochloride D into the prepared blank temperature-sensitive gel, stirring for dissolving, and standing to obtain the temperature-sensitive hydrogel loaded with cyclovirobuxine hydrochloride D. According to the invention, characterization, quality evaluation and biocompatibility research are carried out on the material, and the material is found to have good structure and thermal stability. Meanwhile, the hydrogel disclosed by the invention has a treatment effect of cyclovirobuxine D hydrochloride on vaginitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a temperature-sensitive hydrogel for loading cyclovirobuxine D hydrochloride, a preparation method thereof, and an application thereof. Background Art

[0002] Cyclovirobuxine D (CVB-D), also known as buxine, is a traditional Chinese medicine monomer derived from Buxus microphylla, and belongs to pregnane alkaloids. Its molecular formula is: C 26 H 46 N2O, relative molecular mass: 402.66. CVB-D is a colorless or off-white needle-like crystal, soluble in methanol, poorly soluble in water, and has poor oral bioavailability. Therefore, its hydrochloride salt can be prepared to improve its water solubility, and cyclovirobuxine D hydrochloride is its hydrochloride salt. Cyclovirobuxine D has various pharmacological activities such as antibacterial, anti-inflammatory, and anti-tumor.

[0003] Constructing an excellent drug delivery system for cyclovirobuxine D hydrochloride and improving its bioavailability are the prerequisites for the application of cyclovirobuxine D. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a temperature-sensitive hydrogel for loading cyclovirobuxine D hydrochloride, a preparation method thereof, and an application thereof.

[0005] By establishing a rat model of candidal vaginitis, experiments have confirmed that cyclovirobuxine D hydrochloride has an improving effect on vaginitis in rats infected with Candida albicans. Therefore, cyclovirobuxine D hydrochloride may become an ideal drug for the treatment of candidal vaginitis.

[0006] The hydrogel prepared by the present invention has quite suitable temperature-responsive characteristics, which is beneficial to combine with the body temperature of the human body. Thus, by utilizing the temperature-sensitive effect and the characteristic that the drug in the hydrogel state has a phase transition, the vaginal temperature-sensitive hydrogel is in a liquid state at room temperature but in a gel state at body temperature. This makes the low-viscosity liquid state when it first enters the vagina beneficial for the drug to spread on the wrinkled vaginal mucosa. The hydrogel of the present invention has the therapeutic effect of cyclovirobuxine D hydrochloride on vaginitis, and vaginal administration has the advantages of increasing the residence time of the drug at the action site and controlling the drug release rate.

[0007] Technical Solution: The object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a preparation method of a temperature-sensitive hydrogel for loading cyclovirobuxine D hydrochloride, comprising the following steps:

[0009] (1) Dissolve glycerol, polyvinylpyrrolidone, poloxamer 407, and poloxamer 188 in water, stir until the poloxamer is completely dissolved, add hydrochloric acid after it swells, and adjust the pH to prepare a blank thermosensitive gel.

[0010] (2) Add cyclovirobuxine D hydrochloride to the blank thermosensitive gel prepared in step (1), stir to dissolve, and let it stand to obtain the thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride.

[0011] Preferably, in step (1), the mass ratio of glycerol to water is 2% - 10%.

[0012] Preferably, in step (1), the mass ratio of polyvinylpyrrolidone to water is 0.2% - 0.5%.

[0013] Preferably, in step (1), the mass ratio of poloxamer 407 to water is 16% - 20%.

[0014] Preferably, in step (1), the mass ratio of poloxamer 188 to water is 3% - 5%.

[0015] Preferably, in step (1), the temperature of stirring is 4°C, the swelling time is 24 h, and the pH is adjusted to 4.0 - 4.5.

[0016] Preferably, in step (2), the mass ratio of cyclovirobuxine D hydrochloride to the water in step (1) is 0.2% - 0.4%.

[0017] Preferably, in step (2), the temperature of stirring is 4°C, the stirring time is 1 h; the standing time is 12 h.

[0018] The present invention also provides a thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride prepared by the above preparation method.

[0019] The present invention also provides the application of the above thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride in the preparation of drugs for treating vaginitis.

[0020] Beneficial effects:

[0021] The hydrogel of the present invention has the therapeutic effect of cyclovirobuxine D hydrochloride on vaginitis. Vaginal administration has the advantages of increasing the residence time of the drug at the action site and controlling the drug release rate. Based on the anatomical and physiological characteristics of the vagina, ordinary preparations are unevenly distributed on the mucosa and are easily diluted by vaginal fluid, resulting in leakage, so that the residence time of the drug is short. The hydrogel of the present invention is in close contact with the vaginal mucosa through the gel dosage form, so that the amount of drug passing through the vaginal mucosa increases and the local drug concentration increases. The thermosensitive gel has the characteristic of phase transition. When it first enters the vagina, the low-viscosity liquid state is beneficial to the spreading of the drug on the folded vaginal mucosa, which can solve the problem of uneven drug administration due to vaginal folds. Then, when it reaches the gelation temperature, it quickly becomes a semi-solid and adheres to the mucosa, increasing the residence time of the drug and achieving the purpose of slow drug release. Description of the Drawings

[0022] Figure 1 Photographs of the blank thermosensitive hydrogel prepared in Example 1 and the drug-loaded hydrogel prepared in Example 2;

[0023] Figure 2 SEM images of the blank hydrogel prepared in Example 1 and the drug-loaded hydrogel prepared in Example 2;

[0024] Figure 3 Infrared spectra of hydrochloric acid CVB-D, the blank thermosensitive hydrogel prepared in Example 1, and the drug-loaded hydrogel prepared in Example 2;

[0025] Figure 4 Effect of different contents of poloxamer 407 on the gelation temperature T;

[0026] Figure 5 Effect of different contents of poloxamer 188 on the gelation temperature T;

[0027] Figure 6 Effect of different contents of glycerol on the gelation temperature T;

[0028] Figure 7 Effect of different contents of polyvinylpyrrolidone on the gelation temperature T;

[0029] Figure 8 Thermogravimetric analysis diagrams of hydrochloric acid CVB-D, the blank thermosensitive hydrogel prepared in Example 1, and the drug-loaded hydrogel prepared in Example 2;

[0030] Figure 9 Erosion curve of the drug-loaded hydrogel prepared in Example 2;

[0031] Figure 10 Drug release curve of the drug-loaded hydrogel prepared in Example 2;

[0032] Figure 11Drug release and erosion linear graph of the drug-loaded hydrogel prepared in Example 2;

[0033] Figure 12 Results of the cytotoxicity test of the drug-loaded hydrogel prepared in Example 2;

[0034] Figure 13 Results of the hemolysis test of the hydrogel prepared in Example 2;

[0035] Figure 14 Results of Gram staining smears of Candida albicans and vaginal irrigation fluids of each group (×100, scale bar 10 μm);

[0036] Figure 15 Results of Gram staining smears of vaginal irrigation fluids of each group (×20, scale bar 50 μm);

[0037] Figure 16 Results of HE staining of vaginal tissue sections of each group of rats. Detailed implementation manners

[0038] The technical solution of the present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited to the described examples.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0040] Reagent specifications and sources used in the examples:

[0041] The purity of cyclovirobuxine D hydrochloride is 98.3%, self-made in the laboratory (referring to CN112521440A, a purification method of cyclovirobuxine D);

[0042] Poloxamer 407, FEIYUBIO, subcontracted by imported BASF;

[0043] Poloxamer 188, FEIYUBIO, subcontracted by imported BASF;

[0044] Term explanation: Room temperature: It has the meaning well known in the art, generally referring to 25 ± 2 °C.

[0045] Preparation of the blank thermosensitive hydrogel in Example 1

[0046] Accurately weigh 0.4310 g of glycerol, 0.0211 g of polyvinylpyrrolidone, 1.7800 g of poloxamer 407, and 0.4440 g of poloxamer 188, dissolve them in 10 mL of distilled water, stir at 4 °C until the poloxamer is completely dissolved, place it in a 4 °C refrigerator to swell for 24 h until the bubbles generated during stirring disappear, add hydrochloric acid, and adjust the pH to 4.0 - 4.5 to obtain 12.7 g of blank thermosensitive gel; store it at 4 °C. The physical picture of the prepared blank thermosensitive hydrogel is shown in Figure 1 .

[0047] The microstructure of the obtained blank thermosensitive hydrogel was observed by scanning electron microscopy, and the results are shown in Figure 2 A, Figure 2 B, Figure 2 C. Among them, Figure 2 A is the blank gel (×10 μm); Figure 2 B is the blank gel (×2 μm); Figure 2 C is the blank gel (×1 μm). It can be seen from the figure that the hydrogel presents interconnected spherical mesh-like structures, which can promote the release of drugs.

[0048] The blank thermosensitive hydrogel was analyzed by Fourier transform infrared spectrometer, and the results are shown in Figure 3 .

[0049] Example 2 Preparation of drug-loaded thermosensitive hydrogel

[0050] (1) Accurately weigh 0.4310 g of glycerol, 0.0211 g of polyvinylpyrrolidone, 1.7800 g of poloxamer 407, and 0.4440 g of poloxamer 188, dissolve them in 10 mL of distilled water, stir at 4 °C until the poloxamer is completely dissolved, place it in a 4 °C refrigerator to swell for 24 h until the bubbles generated during stirring disappear, add hydrochloric acid, and adjust the pH to 4.0 - 4.5 to obtain 12.7 g of blank thermosensitive gel; store it at 4 °C.

[0051] (2) Add 0.03 g of cyclovirobuxine D hydrochloride to the blank thermosensitive gel prepared in step (1), stir at 4 °C for 1 h until it is dissolved, and let it stand at 4 °C for 12 h to obtain a thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride. The physical picture of the prepared drug-loaded thermosensitive hydrogel is shown in Figure 1 .

[0052] The microstructure of the obtained hydrogel was observed by scanning electron microscopy, and the results are shown in Figure 2 D, Figure 2 E, Figure 2 F. Among them, Figure 2 D is the drug-loaded gel (×10 μm); Figure 2 E is the drug-loaded gel (×2 μm); Figure 2F is the drug-loaded hydrogel (×1 μm).

[0053] As can be seen from the figure, the hydrogel presents interconnected spherical mesh-like structures, and the addition of cyclovirobuxine D hydrochloride does not change the microstructure of the hydrogel.

[0054] The drug-loaded hydrogel was analyzed spectroscopically using a Fourier transform infrared spectrometer, and the results are shown in Figure 3 . From Figure 3 , it can be seen that the characteristic absorption peaks of cyclovirobuxine D hydrochloride (i.e., cyclovirobuxine D hydrochloride) are the stretching vibration of N-H at 3400 cm -1 , the bending vibration at 1600 cm -1 , the deformation vibration at 1500 cm -1 , C-N (1168 cm -1 , 1091 cm -1 ), O-H (3340 cm -1 , 1410 cm -1 ), C-O (1032 cm -1 ), and C-H (2950 cm -1 ). After adding cyclovirobuxine D hydrochloride to the drug-loaded hydrogel, a strong broadband bending vibration peak of secondary amine salt appears at 2360 cm -1 , showing a blue shift compared with the blank hydrogel. The characteristic absorption peaks of the drug-loaded hydrogel at 2740 cm -1 , 2360 cm -1 , and 1280 cm -1 prove the successful binding of cyclovirobuxine D hydrochloride to the hydrogel, that is, the hydrogel successfully loaded the drug cyclovirobuxine D hydrochloride. Example 3 Effects of different contents of poloxamer 407, poloxamer 188, glycerol, and polyvinylpyrrolidone on the gelation temperature of the hydrogel

[0055] (1) Investigation of the dosage of poloxamer 407 (P407)

[0056] Weigh poloxamer 407 and dissolve it in distilled water. Stir at 4 °C until poloxamer is completely dissolved, then place it in a 4 °C refrigerator to swell for 24 h until the bubbles generated during stirring disappear. Add hydrochloric acid to adjust the pH to 4.0 - 4.5 to prepare the hydrogel.

[0057] The effects of different contents of poloxamer 407 on the gelation temperature of the hydrogel are shown in Figure 4As can be seen from the figure, when the content of P407 is in the range of 16% to 20%, hydrogels can be formed, and the gelation temperature will decrease with the increase of the P407 content. This phenomenon is related to the gelation mechanism of P407 in water. Poloxamer is polymerized from different segments. When the temperature is higher than a certain value, the hydrophobic PPO segment dehydrates, and the molecules aggregate to form spherical micelles with dehydrated polyoxyethylene nuclei inside and hydrated and swollen polyoxyethylene chains outside. They are orderly stacked to form a gel, completing the sol-gel transition, and this process is reversible. When the temperature is lower than this value, it will change from a semi-solid gel to a liquid state. When the content of poloxamer 407 is lower than 15%, its aqueous solution cannot form a gel state at any temperature.

[0058] Since the gel prepared in this experimental design is to be used for vaginal administration, considering the local vaginal environmental temperature, and according to the results of preliminary experiments, poloxamer 407 (16%, 17%, and 18%) with a relatively high gelation temperature result is selected as the three levels of P407 for the subsequent response surface experiment. In addition, according to the results of preliminary experiments, 16% P407 with the highest current gelation temperature is selected as the research basis for the experiment on the effect of different contents of P188 on the gelation temperature of the hydrogel.

[0059] (2) Investigation of the dosage of poloxamer 188 (P188)

[0060] Fix the content of poloxamer 407 (16%), weigh poloxamer 188, dissolve it in water, stir at 4°C until poloxamer is completely dissolved, place it in a 4°C refrigerator and swell for 24 h until the bubbles generated during stirring disappear, add hydrochloric acid, and adjust the pH to 4.0 - 4.5 to prepare a hydrogel.

[0061] The results of the effect of different contents of poloxamer 188 on the gelation temperature of the hydrogel are shown in Figure 5 As can be seen from the figure, when the content of poloxamer 407 is fixed (16%), the gelation temperature of the hydrogel will increase with the increase of the content of poloxamer 188; and compared with the hydrogel formed by single P407, the gelation temperature of the hydrogel formed by the combination of P407 and P188 changes significantly. This may be related to the ratio of hydrophilic groups and hydrophobic groups in P188. The proportion of the hydrophilic PEO segment in the copolymer molecule of P188 is high, and the proportion of the hydrophobic PPO segment is low, resulting in a change in the ratio of hydrophilic and hydrophobic groups in the overall gel system, thus increasing the gelation temperature.

[0062] Since the gel prepared in this experimental design is to be used for vaginal administration, considering the local vaginal environmental temperature, poloxamer 188 (3%, 4%, 5%) with a relatively high gelation temperature result is selected as the three levels of P188 for the subsequent response surface experiment. Furthermore, based on the results of this preliminary experiment, 5% P188 with the highest current gelation temperature result is selected as the research basis for the experiment on the effect of different contents of glycerol on the gelation temperature of the hydrogel.

[0063] (3) Investigation of the dosage of glycerol

[0064] Fix the content of poloxamer 407 (16%) and the content of poloxamer 188 (5%). Weigh glycerol, dissolve it in water, stir at 4 °C until poloxamer is completely dissolved, place it in a 4 °C refrigerator and swell for 24 h until the bubbles generated during stirring disappear. Then add hydrochloric acid and adjust the pH to 4.0 - 4.5 to prepare the hydrogel.

[0065] The results of the influence of different contents of glycerol on the gelation temperature of the hydrogel are shown in Figure 6 . It can be seen from the figure that when the content of poloxamer 407 (16%) and the content of poloxamer 188 (5%) are fixed, the gelation temperature of the hydrogel will decrease with the increase of the glycerol content. Since the gel prepared in this experimental design is to be used for vaginal administration, considering the local vaginal environmental temperature, glycerol with relatively better gelation temperature results (2%, 4%, 6%) is selected as the three levels of glycerol for the subsequent response surface experiment. And based on the results of this preliminary experiment, 2% glycerol is selected as the research basis for the experiment on the influence of different contents of polyvinylpyrrolidone on the gelation temperature of the hydrogel.

[0066] (4) Investigation of the dosage of polyvinylpyrrolidone (PVP)

[0067] Fix the content of poloxamer 407 (16%), the content of poloxamer 188 (5%) and the content of glycerol (2%). Weigh polyvinylpyrrolidone, dissolve it in water, stir at 4 °C until poloxamer is completely dissolved, place it in a 4 °C refrigerator and swell for 24 h until the bubbles generated during stirring disappear. Then add hydrochloric acid and adjust the pH to 4.0 - 4.5 to prepare the hydrogel.

[0068] The results of the influence of different contents of polyvinylpyrrolidone on the gelation temperature of the hydrogel are shown in Figure 7 . It can be seen from the figure that when poloxamer 407 (16%), poloxamer 188 (5%) and glycerol (2%) are fixed, the gelation temperature of the hydrogel will increase with the increase of the PVP content. Since the gel prepared in this experimental design is to be used for vaginal administration, considering the local vaginal environmental temperature, PVP with gelation temperature results close to 37 °C (0.1%, 0.2%, 0.3%) is selected as the three levels of polyvinylpyrrolidone for the subsequent response surface experiment.

[0069] (5) Results of the response surface experiment

[0070] The factor levels of the response surface experiment are shown in Table 1. 29 groups of prescription experimental groups were obtained through Box-Behnken design, and the prescription process tests were carried out according to the table. The gelation temperatures of each group were measured, and the results are shown in Table 2. Using Design Expert 13 software to perform regression and variance analysis on the experimental results, the regression equation of the hydrogel gelation temperature T(Y) with respect to the content of poloxamer 407 (A), the content of poloxamer 188 (B), the content of glycerol (C), and the content of polyvinylpyrrolidone (D) is as follows:

[0071] Y = 31.66 - 3.56A + 1.42B - 1.12C + 0.18D + 0.22AB - 0.60AC - 0.10AD + 0.52BC - 0.68BD - 0.22CD + 1.00A 2 - 0.02B 2 + 0.56C 2 + 0.46D 2 , R 2 = 0.9872.

[0072] Performing variance analysis and significance test on the regression model, the results are shown in Table 3. It can be seen from Table 3 that the F value of the regression model is 77.08 and the P value < 0.01, indicating that the total regression model is significant. The F value of the lack-of-fit term is 1.05 and the P value is 0.5252 > 0.05, indicating that the lack-of-fit term is not significant. The regression model R 2 = 0.9872, the adjusted model Adj.R 2 = 0.9744, the prediction model Pred.R 2 = 0.9410, R adj 2 - R pred 2 = 0.0334 < 0.2, indicating that the model is significantly reliable and has high precision, and can be used for analysis and prediction. That is, this regression model can significantly fit the effects of the four factors of the content of poloxamer 407, the content of poloxamer 188, the content of glycerol, and the content of polyvinylpyrrolidone on the hydrogel gelation temperature and accurately predict the experimental results. By comparing the magnitudes of the F values between various factors, the primary and secondary nature of the influence of different factors on the gelation temperature can be judged. F A = 800.34, F B = 128.35, F C = 78.82, F D= 2.12, indicating that the order of the influence of these four factors on the gelation temperature of the hydrogel should be Poloxamer 407 > Poloxamer 188 > Glycerol > Polyvinylpyrrolidone. Considering that the gel will be diluted by vaginal fluid after entering the vagina, resulting in an increase in the gelation temperature. According to the results of the preliminary experiment, the target gelation temperature of the hydrogel was set at 30 °C, entered into the software, and predicted through the regression model. The predicted optimal factor level combination for hydrogel preparation was 17.8% P407 / 4.44% P188 / 4.31% GLY / 0.211% PVP. The hydrogel was prepared according to the predicted combination, and the gelation temperature was measured for verification. Repeated three times, the results are shown in Table 4. The measured results are close to the predicted values, and the results are available. Therefore, the predicted combination: 17.8% Poloxamer 407 / 4.44% Poloxamer 188 / 4.31% Glycerol / 0.211% Polyvinylpyrrolidone was determined as the optimal blank gel preparation prescription.

[0073] Table 1 Factor-Level Table of Response Surface Experiment

[0074]

[0075] Table 2 Results of Response Surface Experiment

[0076]

[0077]

[0078] Table 3 Variance Analysis and Significance Test of Regression Model

[0079]

[0080]

[0081] Table 4 Verification Experiment of Gelation Temperature of Blank Hydrogel

[0082] NO. P407(％) P188(％) GLY(%) PVP(%) T(℃) 1 17.77 4.443 4.311 0.211 29.9 2 17.77 4.443 4.311 0.211 30.3 3 17.77 4.443 4.311 0.211 30.2

[0083] Example 4 Thermogravimetric Analysis of Blank Thermosensitive Hydrogel and Drug-Loaded Thermosensitive Hydrogel

[0084] Take a small amount of CVB-D hydrochloride, the blank hydrogel prepared in Example 1, and the drug-loaded hydrogel prepared in Example 2, and place them in a thermogravimetric balance respectively. Under a nitrogen environment, thermogravimetric measurement was carried out in the temperature range of 25 - 800 °C at a heating rate of 10 °C / min.

[0085] The decomposition behavior was monitored by plotting weight (%) against temperature (°C), and the results are shown in Figure 8As can be seen from the results, the initial mass loss of CVB-D hydrochloride occurred at 93 °C, with a weight loss of 3.95%. After 280 °C, CVB-D hydrochloride began to melt and decompose until complete weight loss at 450 °C. The blank gel had mass loss at 140 °C, while the mass loss temperature of the drug-loaded gel increased by 20 °C. This indicates that the addition of CVB-D hydrochloride improved the thermal stability of the hydrogel.

[0086] Investigation on in vitro drug release of drug-loaded thermosensitive hydrogel in Example 5

[0087] a) Preparation of simulated vaginal fluid: Accurately weigh 1.4 g of potassium hydroxide, 0.222 g of calcium hydroxide, 0.018 g of bovine serum albumin, 3.51 g of sodium chloride, 5.0 g of glucose, 0.16 g of glycerol, 2.00 g of lactic acid, 0.4 g of urea, and 1.00 g of acetic acid into a 1 L beaker, add water to 1 L, stir to dissolve, and adjust the pH value to 4.2 with hydrochloric acid.

[0088] b) Reference substance solution: Take an appropriate amount of CVB-D hydrochloride reference substance, accurately weigh it, dissolve it with simulated vaginal fluid, quantitatively dilute it, and shake well to obtain the reference substance solution.

[0089] c) According to the membrane-free dissolution method (refer to the guiding principles for sustained-release, controlled-release, and delayed-release preparations in the Chinese Pharmacopoeia 2020 Edition 9013), determine the erosion situation of the hydrogel in simulated vaginal fluid at each time point, and record the weight change at each time point.

[0090] Take the drug-loaded thermosensitive gel prepared in Example 2, accurately weigh 5 g of the gel, add it to a centrifuge tube with a known mass, and record the mass of the centrifuge tube as W0. Place the centrifuge tube containing the CVB-D hydrochloride gel in a 37 °C water bath for 10 min, wait for it to undergo sol-gel transition, and after completely forming a semi-solid gel, dry the surface of the centrifuge tube and weigh it. At this time, record the mass as W1. Slowly add 5 mL of isothermal simulated vaginal fluid along the tube wall, place it in a shaker at 37 °C. Set the oscillation frequency to 100 r / min. Take out the centrifuge tube every 0.5 h, pour out all the release medium (upper layer solution) in the tube and store it separately, dry the surface of the tube, quickly place it on the balance and weigh it, and record the mass at this time as W t Slowly add 5 mL of simulated vaginal fluid at the same temperature along the tube wall, immediately place it in the shaker and continue the previous operation until all the solution in the tube is poured out. Calculate the mass difference between adjacent sampling time points, calculate the cumulative erosion rate of the gel according to the formula, and draw the erosion curve of the gel over time. The erosion rate calculation formula is:

[0091]

[0092] Plot the time point against the eroded weight to obtain the erosion curve as shown in Figure 9 It can be preliminarily judged from the figure that the hydrogel can be slowly released in simulated vaginal fluid.

[0093] Example 6 Determination of the Drug Release Curve of the Drug-Loaded Thermosensitive Hydrogel

[0094] a) Accurately measure the release solutions at each time point in Example 5 c), prepare the test solutions according to Example 5 b), use high performance liquid chromatography to determine the concentration of CVB-D hydrochloride in the release solutions at each time point, calculate the drug release amount, plot the cumulative released drug amount against each time point, and obtain the drug release curve as shown in Figure 10 . It can be seen from the figure that the release of the drug-loaded gel has a phase-dependent property. The release rate of CVB-D hydrochloride is relatively fast in the first 3 hours, and the rate decreases in the following 2 hours showing an obvious sustained release. The release degree can reach about 95% at around 5 hours.

[0095] To study the correlation between the erosion of the gel and the drug release, taking the erosion rate as the abscissa (X) and the cumulative release rate as the ordinate, a linear correlation graph was plotted, and the results are shown in Figure 11 , and its regression equation is Y = 0.8801X + 2.2501, r = 0.9987.

[0096] Suppose there are three time points, t1, t2, t3. Use m1, m2, m3 to represent the mass of the drug released in the dissolution cup at these three points. c1, c2, c3 are the concentrations of the drug solution in the dissolution cup at the three points. The sampling volume is V, that is, the volume to be replenished. Then the calculation method of the cumulative release rate is as follows:

[0097] The cumulative release amount at the third point is: m3 + c2V + c1V

[0098] The second point: m2 + c1V

[0099] The first point: m1

[0100] The results show that the release of CVB-D hydrochloride is synchronized with the erosion of the gel, and both show zero-order kinetic characteristics. This indicates that the release of CVB-D hydrochloride is mainly controlled by the erosion of the gel in vaginal fluid.

[0101] The chromatographic conditions in step a) are as follows:

[0102] Chromatographic column: Welch Xtimate C18 (4.6 mm × 250 mm, 5 μm);

[0103] Mobile phase: Phosphate buffer [a solution prepared by mixing 0.01 mol / L potassium dihydrogen phosphate solution and 0.01 mol / L sodium heptanesulfonate in equal amounts (containing 0.4% triethylamine, adjusted to pH 3.5 with phosphoric acid)] - acetonitrile (75:25, v / v);

[0104] Detection wavelength: 206 nm;

[0105] Flow rate: 1.0 mL / min;

[0106] Column temperature: 30 °C;

[0107] Sample injection volume: 10 μL;

[0108] Collection time: 20 min.

[0109] Example 7 Cytotoxicity test

[0110] The CCK-8 method was used to detect the effects of the blank gel prepared in Example 1 and the drug-loaded gel prepared in Example 2 on the viability of RAW264.7 cells, and the results are shown in Figure 12 .

[0111] The cell viability of the blank gel group (Gel group) was 75.81% when the mass-volume ratio of the hydrogel to the DMEM medium was 1:10, and 67.60% when the mass-volume ratio was 1:5; the cell viability of the drug-loaded gel group (CVB-D Gel group) was 71.96% when the mass-volume ratio of the hydrogel to the medium was 1:10, and 61.54% when the mass-volume ratio was 1:5.

[0112] The above results indicate that the thermosensitive hydrogel has good cell compatibility.

[0113] Example 8 Hemolysis test

[0114] Fresh orbital blood of SD rats (purchased from Jiangsu Qinglongshan Animal Experiment Center) was taken into an anticoagulant EP tube containing sodium heparin, centrifuged at 1500 r / min for 10 min, the supernatant was removed, and the lower-layer red blood cells were transferred to a 10 mL centrifuge tube, washed and centrifuged with physiological saline repeatedly until the supernatant was colorless and clear. The supernatant was removed, and physiological saline was added to dilute it into a 5% (v / v) red blood cell suspension. The blank gel and the drug-loaded gel were prepared according to Example 1 and Example 2. 150 μL of the hydrogel and 500 μL of the red blood cell solution were added to the EP tube, and the EP tube was placed in an incubator at 37 °C and 100 rpm for 2 h. After taking out and centrifuging, 100 μL of the supernatant was aspirated and added to a 96-well plate. The positive control tube was 150 μL of pure water and 500 μL of the red blood cell solution, and the negative control tube was 150 μL of physiological saline and 500 μL of the red blood cell solution. The absorbance was measured at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the hemolysis rate (HR, %) was calculated according to the formula, and repeated three times. The hemolysis rate calculation formula is:

[0115]

[0116] Among them, A sample is the absorbance of the gel tube to be measured, A negative is the absorbance of the negative control physiological saline tube, A positiveis the absorbance of the positive control pure water tube.

[0117] The results of the hemolysis test are shown in Figure 13 The cells in the pure water group showed hemolysis, while the upper layers of the normal saline group, drug-loaded gel group and blank gel group were all clear, without obvious hemolysis. The hemolysis rate results obtained by using an ELISA instrument are shown in Table 5:

[0118] Table 5 Hemolysis test results

[0119]

[0120] It can be seen from the table that the hemolysis rates of the blank gel and the drug-loaded gel are both lower than 5%, and the gel has good blood compatibility and meets the safety requirements.

[0121] Example 9 Study on the in vivo anti-Candida albicans activity of cyclovirobuxine D hydrochloride

[0122] a) Animal preparation: 25 SPF-grade SD rats, female, weighing (200±20) g, mature and unmated, were purchased from Jiangsu Qinglongshan Animal Experiment Center. The rats were fed adaptively for one week.

[0123] b) Preparation of strains: Resuscitate Candida albicans (purchased from Puxitang, C12000-1EA) and adjust the bacterial solution concentration to 2×10 8 CFU / mL, set aside. Prepare slides according to Gram staining method and use oil immersion lens (×100) to observe the morphology of strains. The microscopic examination results are shown in Figure 14 A, Candida albicans was oval in shape under 100x oil immersion microscope and was Gram-positive.

[0124] c) Animal modeling: rats in each group were subcutaneously injected with 0.5 mg of estradiol benzoate in the morning to enter pseudoestrus. Except for the blank group, rats in other groups were injected with 50 μg of streptomycin through the vagina; 8 hours later, 100 μL of 2×10 8 CFU / mL bacterial suspension. Stimulate continuously for 4 days. 24 hours after the fourth infection, vaginal secretions were smeared on the surface of nutrient agar plates and Sabouraud glucose agar plates with a sterile cotton swab. After 24 hours of routine culture, bacterial colonies of varying sizes were observed on the plates by naked eye, proving that the infection was successful. The rats with successful modeling were randomly divided into 4 groups: model group, CVB-D low-dose group, CVB-D high-dose group, and compound metronidazole vaginal suppository group, with 5 animals in each group. A blank control group was also set up.

[0125] d) Drug administration: Starting from the 6th day, the successfully infected rats were given corresponding drugs once a day for 5 consecutive days.

[0126] Compound metronidazole group: each was given 70 mg compound metronidazole vaginal suppository;

[0127] CVB-D low-dose group: Each rat was given 0.4 mg of CVB-D hydrochloride solution;

[0128] CVB-D high-dose group: Each rat was given 0.7 mg of CVB-D hydrochloride solution;

[0129] Blank group and model group: Each rat was given an equal amount of 0.9% sodium chloride injection.

[0130] e) Microscopic examination: Before and after the administration, vaginal lavage fluid of rats was taken for smearing, Gram staining, and microscopic examination, and the conditions of Candida albicans and epithelial cells in the lavage fluid were observed and recorded. If no Candida albicans and epithelial cells were seen under the microscope, it was recorded as negative conversion; otherwise, it was recorded as not negative conversion. The microscopic examination results were recorded, and the negative conversion rate was calculated according to the formula.

[0131] The calculation formula is as follows:

[0132]

[0133] The microscopic examination results of rat vaginal fluid are shown in Figure 14 and Figure 15 . Among them, Figure 14 B, 15A are the microscopic examinations (×10μm, ×50μm) of the vaginal lavage fluid of 5 rats in the blank group after staining. A large number of epithelial cells were visible in 5 cases, and no bacteria and hyphae were visible, and the infection rate was 0; Figure 14 C, 15B are the microscopic examinations (×10μm, ×50μm) of the vaginal lavage fluid of 5 rats in the model group after staining. Epithelial cells and a large number of dense Candida albicans were visible in 5 cases, and the infection rate was 100%; Figure 14 D, 15C are the microscopic examinations (×10μm, ×50μm) of the vaginal lavage fluid of 5 rats in the compound metronidazole vaginal suppository group after staining. A small amount of epithelial cells and a very small amount of bacteria were visible in 3 cases, and the negative conversion rate was 60%; Figure 14 E, 15D are the microscopic examinations (×10μm, ×50μm) of the vaginal lavage fluid of 5 rats in the CVB-D low-dose group after staining. A small amount of epithelial cells and a very small amount of bacteria were visible in 2 cases, and the negative conversion rate was 40%; Figure 14 F, 15E are the microscopic examinations (×10μm, ×50μm) of the vaginal lavage fluid of 5 rats in the CVB-D high-dose group after staining. A small amount of epithelial cells and a very small amount of bacteria were visible in 4 cases, and the negative conversion rate was 80%.

[0134] f) Determination of rat vaginal pH: Before and after the administration, vaginal secretions of rats were taken, and the pH of the vagina was measured with a 0.1-grade pH test paper. The pH of the vaginal fluid of rats in each group after administration was measured, and the results are shown in Table 6:

[0135] Table 6 Vaginal pH values of rats in each group

[0136]

[0137] *Note: Compared with the blank group, a P < 0.01; compared with the model group, b P < 0.01.

[0138] As can be seen from the table: The vaginal fluid pH values of the blank group, model group, compound metronidazole vaginal suppository group, CVB-D low-dose group, and CVB-D high-dose group were 4.10 ± 0.21, 5.74 ± 0.21, 5.10 ± 0.21, 5.16 ± 0.25, and 5.04 ± 0.25, respectively. After inoculating rats with Candida albicans, the vaginal environment changed, and the vaginal pH value changed significantly, indicating successful infection. After short-term treatment with compound metronidazole suppository and different concentrations of cyclovirobuxine D hydrochloride, the pH values of the three groups of rats were greatly improved. Compared with the model group at the same time, the pH values of both the CVB-D low-dose group and the CVB-D high-dose group were significantly decreased (P < 0.01); compared with the compound metronidazole group, the improvement of the pH in the high-dose group was also better. The experimental results show that cyclovirobuxine D hydrochloride can improve the vaginal pH change caused by Candida albicans infection.

[0139] g) Detection of the content of each factor in rat serum: After the last administration, the rats were fasted for 12 h without water restriction. Anesthetized by intraperitoneal injection of 10% chloral hydrate, dissected, and abdominal aortic blood of the rats was taken. After standing for 2 h, it was centrifuged at 3000 r / min for 20 min. The supernatant was taken to obtain rat serum. The levels of TNF-α, IL-1β, IL-6, IL-8, and EGFR in rat serum were detected according to the ELISA kit (enzyme immunoassay mmbio), the concentration standard curve was drawn, and the content of the corresponding factor to be measured was calculated. The determination results of the inflammatory factor content in the serum of each group of rats are shown in Table 7:

[0140] Table 7 Content of inflammatory factors in the serum of each group of rats (pg / mL, x ± s)

[0141]

[0142] *Note: Compared with the blank group, a P < 0.01; compared with the model group, b P < 0.01, c P < 0.05; compared with the compound metronidazole vaginal suppository group, d P < 0.01, e P < 0.05.

[0143] As shown in the table, compared with the blank group, the levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum of rats in the model group were higher, and the differences were statistically significant (P < 0.01). TNF-α, IL-1β, IL-6, and IL-8 are pro-inflammatory cytokines that can coordinate the body's response to infection. TNF-α is a pleiotropic pro-inflammatory factor that participates in various immune processes. When the body is infected or stimulated, IL-1β can induce various pro-inflammatory mediators and activate secondary pro-inflammatory mediators such as IL-6, causing an acute-phase response. The levels of serum inflammatory factors in the model group rats were significantly increased, indicating successful Candida albicans infection and an inflammatory response. By comparing the levels of various factors in the serum of rats in each group after drug administration, it was found that the levels of TNF-α, IL-1β, and IL-6 in the serum of the compound metronidazole group, the high-dose CVB-D group, and the low-dose CVB-D group were lower than those in the model group rats, and the differences were statistically significant (P < 0.05). The results showed that CVB-D hydrochloride and the positive drug compound metronidazole had an improving effect on the inflammatory response caused by Candida albicans infection. By comparing the CVB-D administration group with the positive drug group, it was found that the levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum of the high-dose CVB-D group were lower than those in the compound metronidazole group, and the differences were statistically significant (P < 0.05); there were statistically significant differences in the levels of IL-6 and IL-8 between the low-dose CVB-D group and the compound metronidazole group (P < 0.05), and there were no statistically significant differences in TNF-α and IL-1β (P > 0.05). The results indicated that CVB-D hydrochloride had a better effect on improving the increase in the levels of serum inflammatory factors in rats after Candida infection than compound metronidazole. The above results showed that CVB-D hydrochloride could reduce the levels of serum inflammatory factors in rats and improve the inflammatory response in rats caused by infection.

[0144] The content of EGFR in the serum of rats in each group was measured, and the results are shown in Table 8:

[0145] Table 8 Content of EGFR in the serum of rats in each group (ng / mL, x±s)

[0146]

[0147]

[0148] *Note: Compared with the blank group, a P < 0.01; compared with the model group, b P < 0.01, c P < 0.05; compared with the compound metronidazole vaginal suppository group, d P < 0.01.

[0149] As can be seen from the table, compared with the blank group, the content of EGFR in the serum of rats in the model group was higher, and the difference was statistically significant (P < 0.01). EGFR plays a regulatory role in processes such as cell proliferation and signal transduction. As can be seen from the table, the concentration level of EGFR in the rats of the model group increased significantly, indicating that Candida infection could lead to high expression of EGFR. By comparing each drug-administered group, it was found that the content of EGFR in the serum of rats in the high-dose CVB-D group, low-dose CVB-D group, and compound metronidazole group was less than that in the model group, and the difference was statistically significant (P < 0.05). The results showed that after drug treatment, the high expression of EGFR could be improved. By comparing the CVB-D drug-administered group with the positive drug group, it was found that the content of EGFR in the high-dose CVB-D group was lower than that in the compound metronidazole group, and the difference was statistically significant (P < 0.01). The results indicated that high-dose CVB-D hydrochloride was superior to the positive drug in improving the increase in the content of EGFR in the serum of rats after Candida infection. The above results showed that CVB-D hydrochloride could reduce the content of EGFR in the serum of rats and improve the high expression of EGFR caused by infection.

[0150] h) Pathological observation of rat vaginal tissue: The rats were sacrificed, and the vagina and uterus were taken. The connective tissue on the outer edge of the vagina was removed, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned transversely through the vagina and stained with HE, and the pathological changes were observed under the microscope and scored and statistically analyzed.

[0151] The HE staining results of the sections of rat vaginal tissue are shown in Figure 16 . Among them Figure 16 A (×200μm), Figure 16 B (×50μm) are the HE staining results of the sections of the vaginal tissue of rats in the blank group. As can be seen from the figure, the structures of each layer of the vaginal tissue of rats in the blank group were clear, the mucosal epithelium was intact, the connective tissue in the lamina propria was arranged tightly, and a very small amount of inflammatory cell infiltration (red arrow) was visible in the lamina propria, and no congestion or edema was seen. Figure 16 C (×200μm), Figure 16 D (×50μm) are the HE staining results of the sections of the vaginal tissue of rats in the model group. As can be seen from the figure, the structures of each layer of the vaginal tissue of rats in the model group were clear, the mucosal epithelium was intact, the cytoplasm of a large number of epithelial cells was loose, necrosis of a very small amount of mucosal epithelial cells was visible, the cell nuclei were fragmented and dissolved, the cytoplasm was eosinophilic enhanced (black arrow), a large amount of inflammatory cell infiltration (red arrow) was visible in the lamina propria, epithelial cell clusters (yellow arrow) were visible in the lumen, and mild bleeding (green arrow) was visible. Figure 16 E (×200μm), Figure 16F (×50μm) shows the HE staining results of the vaginal tissue sections of rats in the metronidazole vaginal suppository group. As can be seen from the figure, the structures of each layer of the vaginal tissue in the compound metronidazole vaginal suppository group are clear, the mucosal epithelium is intact, the cytoplasm of a large number of epithelial cells is loose, and necrosis of more mucosal epithelial cells can be seen. The nuclei are fragmented and dissolved, and the eosinophilia of the cytoplasm is enhanced (black arrow). A large number of inflammatory cell infiltrations can be seen in the lamina propria (red arrow), and epithelial cell clusters can be seen in the lumen (yellow arrow). Figure 16 G (×200μm), Figure 16 H (×50μm) shows the HE staining results of the vaginal tissue sections of rats in the low-dose CVB-D group. As can be seen from the figure, the structures of each layer of the vaginal tissue in the low-dose CVB-D group are clear, the mucosal epithelium is intact, the cytoplasm of a large number of epithelial cells is loose, and necrosis of a very small amount of mucosal epithelial cells can be seen. The nuclei are fragmented and dissolved, and the eosinophilia of the cytoplasm is enhanced (black arrow). More inflammatory cell infiltrations can be seen in the lamina propria (red arrow), and mild bleeding can be seen in the lumen (green arrow). Figure 16 I (×200μm), 16J (×50μm) show the HE staining results of the vaginal tissue sections of rats in the high-dose CVB-D group. As can be seen from the figure, the structures of each layer of the vaginal tissue in the high-dose CVB-D group are clear, the mucosal epithelium is intact and arranged closely, the eosinophilia of the cytoplasm is enhanced (black arrow), the connective tissue in the lamina propria is arranged closely, and a small amount of inflammatory cell infiltrations can be seen in the lamina propria (red arrow).

[0152] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A preparation method of a thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride, characterized in that, It includes the following steps: (1) Dissolve glycerol, polyvinylpyrrolidone, poloxamer 407, and poloxamer 188 in water, stir until poloxamer is completely dissolved, add hydrochloric acid after it swells, and adjust the pH to prepare a blank thermosensitive gel; (2) Add cyclovirobuxine D hydrochloride to the blank thermosensitive gel prepared in step (1), stir to dissolve, and let it stand to obtain the thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride.

2. The preparation method according to claim 1, wherein In step (1), the mass ratio of glycerol to water is 2% - 10%.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of polyvinylpyrrolidone to water is 0.2% - 0.5%.

4. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of poloxamer 407 to water is 16% - 20%.

5. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of poloxamer 188 to water is 3% - 5%.

6. The preparation method according to claim 1, wherein In step (1), the temperature of stirring is 4°C, the swelling time is 24 h, and the pH is adjusted to 4.0 - 4.

5.

7. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of cyclovirobuxine D hydrochloride to the water in step (1) is 0.2% - 0.4%.

8. The preparation method according to claim 1, characterized in that, In step (2), the temperature of stirring is 4°C, the stirring time is 1 h; the standing time is 12 h.

9. The thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the thermosensitive hydrogel loaded with cyclovirobuxine D hydrochloride according to claim 9 in the preparation of a drug for treating vaginitis.

Images