A pH-responsive composite temperature-sensitive hydrogel containing Xiaoyou decoction and a preparation method and application thereof

Abstract

This application discloses a pH-responsive composite thermosensitive hydrogel containing Xiaoyoutang (a traditional Chinese medicine formula), its preparation method, and its application. It belongs to the technical field of traditional Chinese medicine preparations. The pH-responsive composite thermosensitive hydrogel is prepared from the following raw materials: Xiaoyoutang compound liposomes, Xiaoyoutang lyophilized powder, chitosan, poloxamer P407, poloxamer P188, and a 0.5% lactic acid aqueous solution. The Xiaoyoutang is composed of the following raw materials in parts by weight: 20 parts Cnidium monnieri, 15 parts Smilax glabra, 15 parts Sophora flavescens, 12.5 parts Sophora tonkinensis, 12.5 parts Phellodendron chinense, 12.5 parts Lithospermum erythrorhizon, 12.5 parts Stemona japonica, 10 parts Artemisia annua, and 2.5 parts Brucea javanica. This invention solves the technical problems of existing Xiaoyoutang preparations, such as outdated form, easy loss after administration, short duration of action, and uncontrollable release behavior.

Description

Technical Field

[0001] This invention relates to the technical field of traditional Chinese medicine preparations, and in particular to a pH-responsive composite thermosensitive hydrogel containing Xiaoyou Decoction, its preparation method, and its application. Background Technology

[0002] "Xiaoyou Decoction" is an empirical formula composed of multiple traditional Chinese medicines, including Cnidium monnieri, Smilax glabra, Sophora flavescens, Sophora tonkinensis, Phellodendron chinense, Lithospermum erythrorhizon, Stemona japonica, Artemisia annua, and Brucea javanica. Clinically, it is used to treat HPV-related diseases such as condyloma acuminata, and has definite efficacy in clearing heat and detoxifying, removing dampness and relieving itching, and antiviral activity. However, traditional Chinese medicine compound formulas are mostly in the form of decoctions, lotions, or ordinary ointments, which have problems such as inconvenience in use, inaccurate dosage, short residence time of the drug at the lesion site, and low bioavailability, thus limiting their full efficacy.

[0003] In-situ gels, especially thermosensitive hydrogels, are environmentally sensitive smart materials. They are liquid at low or normal temperatures, facilitating drug delivery, and rapidly undergo a phase transition to form a semi-solid gel upon stimulation at body temperature (37°C), thus achieving long-term retention and sustained drug release at the administration site (such as the vagina or skin). However, existing in-situ gel formulations often lack responsive drug release characteristics to the lesion microenvironment (such as the local slightly acidic environment caused by HPV infection), making it difficult to achieve precise targeted drug release.

[0004] Therefore, developing a novel compound formulation that combines thermosensitive gelation, pH-responsive drug release, and the ability to effectively encapsulate the complex active ingredients of "Xiaoyoutang" (including volatile oils and water-soluble alkaloids) is of great significance for improving the efficacy and patient compliance of this compound.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction, its preparation method and application, which can solve the technical problems of existing wart-reducing decoction preparations being outdated in form, easily lost after administration, having a short duration of action and uncontrollable release behavior.

[0007] To achieve the above objectives, the present invention provides a pH-responsive composite thermosensitive hydrogel containing a wart-removing decoction, prepared from the following raw materials:

[0008] The ingredients include liposomes of the traditional Chinese medicine formula Xiaoyoutang, freeze-dried powder of Xiaoyoutang, chitosan, poloxamer P407, poloxamer P188 and 0.5% lactic acid aqueous solution;

[0009] The wart-removing decoction is composed of the following raw materials in parts by weight: 20 parts of Cnidium monnieri, 15 parts of Smilax glabra, 15 parts of Sophora flavescens, 12.5 parts of Sophora tonkinensis, 12.5 parts of Phellodendron chinense, 12.5 parts of Lithospermum erythrorhizon, 12.5 parts of Stemona japonica, 10 parts of Artemisia annua, and 2.5 parts of Brucea javanica.

[0010] In one embodiment of the present invention, each 10 mL of pH-responsive composite thermosensitive hydrogel containing Xiaoyoutang is composed of the following raw materials: 0.5 mL of Xiaoyoutang herbal compound liposomes, 4.58 g of Xiaoyoutang lyophilized powder, 0.06 g of chitosan, 2 g of poloxamer P407, 0.6 g of poloxamer P188, and the remainder is made up to 10 mL by 0.5% lactic acid aqueous solution.

[0011] In one embodiment of the present invention, the mass ratio of egg yolk lecithin to cholesterol in the wart-removing decoction compound liposome is 6:1.

[0012] In one embodiment of the present invention, the average alkaloid content in the freeze-dried powder of the wart-removing decoction is 21.87% to 22.41%.

[0013] This invention also provides a method for preparing a pH-responsive composite thermosensitive hydrogel containing the above-mentioned wart-removing decoction, comprising the following steps:

[0014] S1: Extraction of volatile oil from wart-removing decoction: Remove the raw material of wart-removing decoction, add pure water at a material-to-liquid ratio of 5:1, soak for 0.5 h, extract by gentle boiling for 6 h, collect the volatile oil, and keep the remaining residue for later use;

[0015] S2: Preparation of compound liposomes of traditional Chinese medicine: Egg yolk lecithin and cholesterol were mixed at a mass ratio of 6:1 using the thin film dispersion method, dissolved in anhydrous ethanol, and formed a uniform thin film by rotary evaporation at 60°C. The organic solvent was removed by vacuum drying. The volatile oil of Xiaoyoutang obtained in step S1 and phosphate buffer were added, and after ultrasonic dispersion, the mixture was filtered through a microporous membrane to obtain compound liposomes of traditional Chinese medicine.

[0016] S3: Preparation of freeze-dried powder of wart-removing decoction: Take the residue of wart-removing decoction after volatile oil in step S1, add pure water at a material-to-liquid ratio of 1:8, decoct and extract twice, extract for 1 hour each time, combine the extracts and concentrate, pre-freeze at -80℃ and freeze-dry under vacuum to obtain freeze-dried powder.

[0017] S4: Preparation of pH-responsive composite thermosensitive hydrogel: Chitosan, poloxamer P407 and poloxamer P188 were dissolved in 0.5% lactic acid aqueous solution. Using the traditional Chinese medicine compound liposomes obtained in step S2 as the medium, the mixture was swollen at 4℃ for 24 h to obtain a blank thermosensitive hydrogel. The lyophilized powder of Xiaoyoutang obtained in step S3 was added to the blank thermosensitive hydrogel and stirred evenly at 100 r / min to obtain the pH-responsive composite thermosensitive hydrogel.

[0018] The dosage of chitosan, poloxamer P407, poloxamer P188, and the lyophilized powder of Xiaoyoutang was 0.06 g : 2 g : 0.6 g : 4.58 g / 10 mL of 0.5% lactic acid aqueous solution.

[0019] In one embodiment of the present invention, microporous membrane filtration uses membranes of 0.45 µm and 0.22 µm in sequence.

[0020] In one embodiment of the present invention, the vacuum degree of freeze drying is 0.3 Pa and the time is 24 h.

[0021] This invention provides the application of a pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction prepared according to the above-mentioned method, or a pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction prepared according to the above-mentioned method, in the preparation of a vaginal topical antimicrobial drug.

[0022] Compared with the prior art, the present invention has the following outstanding advantages and significant progress:

[0023] 1. Innovative Dosage Form for Long-Lasting In-Situ Drug Delivery: For the first time, the effective traditional Chinese medicine compound "Xiaoyou Tang" has been formulated into a thermosensitive hydrogel. This formulation is a free-flowing liquid at 4°C, facilitating precise application to the vagina, cervix, or skin lesions via syringe or applicator; at body temperature of 37°C, it rapidly transforms into a semi-solid gel (approximately 65 seconds), perfectly conforming to the irregular lesion surface and significantly prolonging the drug's residence time at the administration site, avoiding the rapid loss problems associated with traditional lotions and ointments.

[0024] 2. pH-responsive drug release for targeted therapy: Utilizing the pH sensitivity of chitosan, the hydrogel releases drugs slowly in a normal physiological environment (pH 7.4) (approximately 49% cumulative release over 16 hours), reducing systemic drug leakage. However, in a slightly acidic local environment (pH 4.5) caused by HPV infection, the chitosan amino groups protonate, the gel network swells, and drug release accelerates (up to 90% cumulative release over 16 hours). This "rapid release in acidic conditions, sustained release in neutral conditions" characteristic enables precise, rapid, and high-concentration drug release at the lesion site, while reducing side effects on normal tissues.

[0025] 3. Highly Efficient Multi-Component Encapsulation and Stable Process: This invention employs a step-by-step strategy of "volatile oil liposome encapsulation + direct loading of water extract lyophilized powder," effectively solving the common challenge of containing both volatile and water-soluble components in traditional Chinese medicine compound formulas. Liposome technology protects the stability of the volatile oil, while the lyophilized powder form ensures uniform content of active ingredients such as water-soluble alkaloids. The preparation process has been systematically optimized (lecithin / cholesterol ratio, rotary evaporation temperature, chitosan and poloxamer dosages, etc.), exhibiting good reproducibility and suitability for industrial production.

[0026] 4. Excellent sustained-release properties enhance efficacy: In vitro release studies show that this hydrogel exhibits an ideal "fast at first, slow later" drug release curve in an acidic lesion simulation environment: the initial moderate burst release (approximately 42.85% released in 2 hours) facilitates rapid onset of action, while the subsequent stable and sustained release for up to 16 hours helps maintain an effective therapeutic concentration. This is expected to reduce the frequency of patient administration, improve treatment compliance, and thus significantly enhance the clinical efficacy of "Wart-Eliminating Decoction". Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the results of the investigation on the extraction time of volatile oils in this invention.

[0028] Figure 2 This is a schematic diagram of the screening results of volatile oil soaking time in this invention.

[0029] Figure 3 This is a schematic diagram of the volatile oil-liquid ratio screening results in this invention.

[0030] Figure 4 This is a schematic diagram of the particle size when the ratio of egg yolk lecithin to cholesterol in this invention is 6:1.

[0031] Figure 5 This is a schematic diagram of the particle size at a rotary evaporation temperature of 60°C in this invention.

[0032] Figure 6 This is a schematic diagram of the cumulative release rate of alkaloids under different pH conditions in this invention. Detailed Implementation

[0033] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0034] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0035] According to a preferred embodiment of the present invention, the pH-responsive composite thermosensitive hydrogel containing Xiaoyoutang (a traditional Chinese medicine formula) is composed of the following raw materials per 10 mL: 0.5 mL of Xiaoyoutang compound liposomes, 4.58 g of Xiaoyoutang lyophilized powder, 0.06 g of chitosan, 2 g of poloxamer P407, 0.6 g of poloxamer P188, with the remainder made up to 10 mL by 0.5% lactic acid aqueous solution;

[0036] The wart-removing decoction is composed of the following raw materials in the indicated weight proportions: 20 parts of Cnidium monnieri, 15 parts of Smilax glabra, 15 parts of Sophora flavescens, 12.5 parts of Sophora tonkinensis, 12.5 parts of Phellodendron chinense, 12.5 parts of Lithospermum erythrorhizon, 12.5 parts of Stemona japonica, 10 parts of Artemisia annua, and 2.5 parts of Brucea javanica.

[0037] Specifically, the preparation method of the traditional Chinese medicine compound liposome is as follows: egg yolk lecithin and cholesterol are mixed at a mass ratio of 6:1, dissolved in anhydrous ethanol, and evaporated at 60°C to form a uniform film. The organic solvent is removed by vacuum drying. The volatile oil of the wart-removing decoction obtained in step S1 and phosphate buffer are added, and the mixture is ultrasonically dispersed and filtered through a microporous membrane to obtain the traditional Chinese medicine compound liposome.

[0038] The extraction method of the volatile oil of Xiaoyou Decoction is as follows: add the Xiaoyou Decoction medicinal materials to pure water at a material-to-liquid ratio of 5:1, soak for 0.5 hours, boil, and then adjust the heat to maintain a gentle boil for 6 hours to extract the volatile oil.

[0039] The preparation method of the freeze-dried powder of the wart-removing decoction is as follows: the residue after removing the volatile oil of the wart-removing decoction is added to pure water at a material-to-liquid ratio of 1:8, and the decoction is boiled and extracted twice, each time for 1 hour. The extracts are combined and concentrated, and then pre-frozen at -80℃ and freeze-dried under vacuum to obtain freeze-dried powder. The average alkaloid content in the freeze-dried powder is 21.87% to 22.41%.

[0040] The preparation method of the pH-responsive composite thermosensitive hydrogel containing wart-removing decoction according to a preferred embodiment of the present invention includes the following steps:

[0041] S1: Extraction of volatile oil from the wart-removing decoction: Remove the raw materials of the wart-removing decoction, add pure water at a material-to-liquid ratio of 5:1, soak for 0.5 hours, extract by gentle boiling for 6 hours, collect the volatile oil, and reserve the remaining residue; wherein, the raw materials of the wart-removing decoction are composed of the following parts by weight: Cnidium monnieri 20 parts, Smilax glabra 15 parts, Sophora flavescens 15 parts, Sophora tonkinensis 12.5 parts, Phellodendron chinense 12.5 parts, Lithospermum erythrorhizon 12.5 parts, Stemona japonica 12.5 parts, Artemisia annua 10 parts, and Brucea javanica 2.5 parts.

[0042] S2: Preparation of compound liposomes of traditional Chinese medicine: Egg yolk lecithin and cholesterol were mixed at a mass ratio of 6:1 using the thin film dispersion method, dissolved in anhydrous ethanol, and formed a uniform thin film by rotary evaporation at 60°C. The organic solvent was removed by vacuum drying. The volatile oil of Xiaoyoutang obtained in step S1 and phosphate buffer were added, and after ultrasonic dispersion, the mixture was filtered through a microporous membrane to obtain compound liposomes of traditional Chinese medicine.

[0043] S3: Preparation of freeze-dried powder of wart-removing decoction: Take the residue after volatile oil removal from the wart-removing decoction in step S1, add pure water at a material-to-liquid ratio of 1:8, decoct and extract twice, each time for 1 hour, combine the extracts and concentrate, pre-freeze at -80℃ and freeze-dry under vacuum to obtain freeze-dried powder;

[0044] S4: Preparation of pH-responsive composite thermosensitive hydrogel: Chitosan, poloxamer P407, and poloxamer P188 were dissolved in 0.5% lactic acid aqueous solution. Using the traditional Chinese medicine compound liposomes obtained in step S2 as the medium, the mixture swelled at 4℃ for 24h to obtain a blank thermosensitive hydrogel. The lyophilized powder of Xiaoyoutang obtained in step S3 was added to the blank thermosensitive hydrogel and stirred evenly at 100 r / min to obtain the pH-responsive composite thermosensitive hydrogel. The amounts of chitosan, poloxamer P407, poloxamer P188, and lyophilized powder of Xiaoyoutang were 0.06g: 2g: 0.6g: 4.58g / 10mL 0.5% lactic acid aqueous solution.

[0045] The pH-responsive composite thermosensitive hydrogel containing anti-wart decoction prepared in this invention can be used to prepare vaginal topical antimicrobial drugs, specifically for vaginal local administration. This hydrogel is adapted to the vaginal physiological environment, rapidly releasing the drug in the acidic vaginal environment (pH 3.8~4.5) to quickly reach an effective therapeutic concentration; and slowly releasing the drug in the neutral human body fluid environment (pH 7.4), reducing off-target drug leakage, while achieving a long-lasting sustained release of 16 hours, effectively reducing the frequency of drug administration.

[0046] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0047] Example 1: Preparation of pH-responsive composite thermosensitive hydrogel containing wart-removing decoction

[0048] 1.1 Raw material ratio: The total mass of raw materials for Xiaoyou Decoction is 112.5 g, and the components are as follows: Cnidium monnieri 20g, Smilax glabra 15g, Sophora flavescens 15g, Sophora tonkinensis 12.5g, Phellodendron chinense 12.5g, Lithospermum erythrorhizon 12.5g, Stemona japonica 12.5g, Artemisia annua 10g, and Brucea javanica 2.5g.

[0049] 1.2 Extraction of volatile oil from wart-removing decoction: Add 562.5g of pure water (material-to-liquid ratio 5:1) to the mixed herbs of wart-removing decoction and soak at room temperature for 0.5 h; connect the volatile oil extraction device, heat to a gentle boil, and continue extraction for 6 h, collect the volatile oil, and keep the residue for later use.

[0050] 1.3 Preparation of Traditional Chinese Medicine Compound Liposomes: Weigh 6g of egg yolk lecithin and 1g of cholesterol (mass ratio 6:1), add anhydrous ethanol and stir until completely dissolved; transfer to a round-bottom flask, place in a rotary evaporator, and evaporate at 60℃ until a uniform film forms on the flask wall; vacuum dry to completely remove organic solvent; add 0.5 mL of the above volatile oil and 4.5 mL of phosphate buffer, and sonicate for 25 min; filter successively through 0.45μm and 0.22μm microporous membranes to obtain clear Traditional Chinese Medicine Compound Liposomes.

[0051] 1.4 Preparation of freeze-dried powder of wart-removing decoction: Take the residue after extracting volatile oil, add 900g of pure water (material-to-liquid ratio 1:8), heat and decoct for 1 hour, filter and collect the filtrate; add an equal amount of pure water to the residue again, decoct for 1 hour, filter, and combine the two filtrates; concentrate the combined filtrate under reduced pressure; dispense the concentrate into freeze-drying trays, pre-freeze at -80℃ for 12 hours; place in a freeze dryer, vacuum degree 0.3Pa, freeze-dry for 24 hours to obtain freeze-dried powder of wart-removing decoction, the average alkaloid content of which was found to be 22.14%.

[0052] 1.5 Preparation of pH-responsive composite thermosensitive hydrogel: Take 0.06g of chitosan, add an appropriate amount of 0.5% lactic acid aqueous solution and stir to dissolve; add 2g of poloxamer P407 and 0.6g of poloxamer P188, and stir to mix well; add the prepared traditional Chinese medicine compound liposome, and add 0.5% lactic acid aqueous solution to make up to 10mL; place the system in a 4℃ refrigerator to swell for 24h to obtain a transparent blank thermosensitive hydrogel; add 4.58g of Xiaoyoutang lyophilized powder to the blank gel, and stir at 100 r / min until the system is uniform to obtain the finished hydrogel.

[0053] Example 2: Optimization of Volatile Oil Extraction Process

[0054] Using volatile oil content as an indicator, a single-factor experiment was conducted to investigate the effects of material-to-liquid ratio, soaking time, and extraction time on volatile oil extraction. 20 g of Cnidium monnieri, 15 g of Smilax glabra, 15 g of Sophora flavescens, 12.5 g of Sophora tonkinensis, 12.5 g of Phellodendron chinense, 12.5 g of Lithospermum erythrorhizon, 12.5 g of Stemona japonica, 10 g of Artemisia annua, and 2.5 g of Brucea javanica were weighed, each portion containing 112.5 g of medicinal materials. Based on preliminary experiments showing that the specific gravity of the volatile oil obtained was less than 1.0, extraction was performed according to Method A of Volatile Oil Determination under Section 2204 of the 2025 edition of the Chinese Pharmacopoeia, Part IV. The extract was placed in a 1000 ml round-bottom flask, connected to a volatile oil extractor and condenser, and soaked in 4 times the amount of water for 30 min. After boiling, the heat was adjusted to a gentle boil for 4 h before heating was stopped, yielding the extract. The volatile oil was collected, allowed to stand for a period of time, and the volume of the volatile oil was accurately recorded.

[0055] 2.1. Extraction time investigation: With other conditions fixed, 112.5 g of medicinal material was weighed for each sample and extracted according to the method in Example 2. The effect of extraction time of 4, 5, 6, 7 and 8 h on the volatile oil extraction amount was investigated, with the volatile oil extraction amount as the indicator.

[0056] The table below shows the results of the screening of volatile oil extraction time (n=3, )

[0057] Extraction time (h) Volatile oil content (mL) 4 0.36±0.04 5 0.45±0.02 6 0.49±0.01 7 0.49±0.01 8 0.49±0.01

[0058] Figure 1 The results of the investigation on the extraction time of volatile oils are shown in the table above and Figure 1 It can be seen that the extraction yield of volatile oil gradually increases with the extension of extraction time, reaching its maximum at 6 hours. Further extending the extraction time to 7 hours and 8 hours did not significantly increase the extraction yield. Considering energy saving and efficiency, 6 hours was chosen as the optimal extraction time.

[0059] 2.2 Soaking time investigation: With other conditions unchanged, 112.5 g of medicinal material was weighed for each sample and extracted according to the method in Example 2. The effect of soaking time of 0, 0.5, 1 and 1.5 h on the volatile oil extraction amount was investigated, using the volatile oil extraction amount as the indicator.

[0060] The table below shows the screening results for volatile oil soaking time (n=3). )

[0061] Soaking time (h) Volatile oil content (mL) 0 0.40±0.02 0.5 0.49±0.01 1 0.49±0.01 1.5 0.48±0.01

[0062] Figure 2 The results of the screening of volatile oil soaking time are shown in the table above. Figure 2 It can be seen that the extraction yield of volatile oil is low when the soaking time is 0 h. As the soaking time increases to 0.5 h, the extraction yield increases significantly. Further extending the soaking time does not result in a significant increase in the extraction yield of volatile oil, indicating that 0.5 h is sufficient for the medicinal material to fully swell. Therefore, 0.5 h is selected as the optimal soaking time.

[0063] 2.3. Liquid-to-solid ratio study: With other conditions fixed, 112.5 g of medicinal material was weighed for each sample and extracted according to the method in Example 2. The effect of liquid-to-solid ratios of 3:1, 4:1, 5:1, 6:1, and 7:1 on the volatile oil extraction yield was investigated, using the volatile oil extraction yield as the indicator.

[0064] The table below shows the screening results for volatile oil-to-liquid ratio (n=3). )

[0065] Material-liquid ratio Volatile oil content (mL) 3：1 0.32±0.02 4：1 0.43±0.02 5：1 0.49±0.01 6：1 0.49±0.01 7：1 0.48±0.01

[0066] Figure 3 The results of the screening of volatile oil-to-liquid ratio are shown in the table above. Figure 3 It can be seen that the extraction yield of volatile oil first increases and then stabilizes with the increase of the liquid-to-solid ratio. Extraction is incomplete at liquid-to-solid ratios of 3:1 and 4:1. The extraction yield is highest at a liquid-to-solid ratio of 5:1. Further increasing the solvent ratio to 6:1 and 7:1 results in a slight decrease in extraction yield. Considering both extraction cost and efficiency, 5:1 is selected as the optimal liquid-to-solid ratio.

[0067] Example 3: Single-factor investigation of liposomes

[0068] 3.1 Matrix Ratio Investigation: Following step 1.3 above, liposomes were prepared by weighing egg yolk lecithin and cholesterol at different mass ratios of 5:1, 6:1, 7:1, and 8:1 to prepare compound liposomes of traditional Chinese medicine. The appearance characteristics were observed. The ratio of egg yolk lecithin to cholesterol was screened using the following evaluation indicators: whether the liposomes formed a film, film thickness and uniformity, residue on the flask wall after hydration, gloss, clarity, zeta potential, PDI, and particle size.

[0069] The table below shows the results of an investigation into different ratios of lecithin and cholesterol in egg yolks.

[0070] Proportion Whether it forms a film thickness Uniformity Residue on the wall gloss Clarity Particle size (nm) PDI (%) Potential (mV) 5：1 yes Thicker Poor less Difference turbid 240.16±4.28 18.70±5.40 -9.42±1.15 6：1 yes thinner better less good clarify 169.16±1.33 20.90±5.12 -12.27±2.36 7：1 yes thinner better More good Clarified 203.21±4.42 20.51±1.50 -10.14±1.82 8：1 yes thinner better More good Clarified 257.93±16.46 21.33±1.77 -9.64±1.43

[0071] Figure 4 The table above shows the particle size when the ratio of egg yolk lecithin to cholesterol is 6:1. Figure 4 It is known that the optimal ratio of egg yolk lecithin to cholesterol is 6:1 for the preparation of liposomes.

[0072] 3.2 Investigation of rotary evaporation temperature: Keeping other conditions constant, liposomes were prepared according to step 1.3 above, and the effects of rotary evaporation temperatures of 45℃, 50℃, 55℃, 60℃ and 65℃ on liposome formation were investigated.

[0073] The table below shows the results of the investigation at different rotary evaporation temperatures.

[0074] Temperature (°C) Whether it forms a film thickness Uniformity Residue on the wall gloss Clarity Particle size (nm) PDI (%) Potential (mV) 45 no - - - - - - - - 50 yes Thicker Poor less Difference turbid 343.94±63.82 41.87±8.05 -7.80±5.03 55 yes thinner better less good Clarified 160.96±2.72 22.90±0.92 -10.12±6.26 60 yes thinner better less good clarify 156.50±0.12 19.87±0.91 -14.71±3.26 65 yes thinner Poor More good Clarified 178.20±8.37 13.37±7.74 -8.78±5.46

[0075] Figure 5 The table above shows the particle size at a rotary evaporation temperature of 60°C. Figure 5 It can be seen that the optimal temperature for preparing liposomes is 60℃.

[0076] Example 4: Process Validation

[0077] Verification experiments were conducted according to the optimal preparation process flow of Example 1. Three batches of volatile oil and traditional Chinese medicine compound liposomes were prepared according to the optimal process flow. The extraction amount of volatile oil and the Zeta potential, PDI and particle size of traditional Chinese medicine compound liposomes were measured.

[0078] The table below shows the results of the process validation test for volatile oils and traditional Chinese medicine compound liposomes (n=3). )

[0079] batch Volatile oil content (mL) Particle size (nm) PDI (%) Potential (mV) 20251201 0.49±0.01 150.53±0.53 13.70±5.91 -8.34±0.80 20251202 0.49±0.01 150.67±0.95 13.67±1.42 -8.98±0.86 20251203 0.49±0.01 153.61±1.09 17.37±4.80 -9.37±0.89

[0080] In the above embodiments, the extraction process of volatile oils from traditional Chinese medicine (TCM) compound preparation and the preparation process of TCM compound liposomes were optimized using a single-factor method. First, using the volatile oil extraction yield as an indicator, the effects of extraction time, soaking time, and liquid-to-solid ratio were investigated to determine the optimal extraction process: soaking time 0.5 h, extraction time 6 h, and liquid-to-solid ratio 5:1. Second, TCM compound liposomes were prepared using a thin-film dispersion method. By evaluating appearance, particle size, PDI, and Zeta potential, the optimal formulation and preparation process for the liposomes were screened: an egg yolk lecithin to cholesterol mass ratio of 6:1 and a rotary evaporation temperature of 60℃. The optimized liposomes exhibited good dispersibility, providing a theoretical basis for subsequent research on the preparation of pH-responsive composite thermosensitive hydrogels.

[0081] Example 5: Optimization of Extraction Process for Compound Traditional Chinese Medicine Solutions

[0082] Using alkaloid content and extract yield as indicators, a single-factor experiment was conducted to investigate the effects of material-to-liquid ratio, extraction time, and extraction times on the alkaloid content and extract yield in a traditional Chinese medicine compound solution. The extract and residue after volatile oil extraction were filtered. The extract was collected, and the residue was decocted with 1000 mL of the original prescription water. The decoction was filtered again, and the residue was added again with 1000 mL of the original prescription water for decoction. The decoctions were then combined and concentrated to 150 mL. The extract yield was measured, and the absorbance was measured. The alkaloid concentration and relative standard deviation (RSD) were calculated using the following equation: Extract yield (%) = (Total weight of the dried extract concentrated to the specified specific gravity / Total weight of the added medicinal materials) × 100%

[0083] 5.1 Study on material-liquid ratio: With other conditions fixed, the extraction was performed according to the method in Example 6 of this paper. The extraction time was fixed at 1 hour and the number of extractions was 2. The effects of material-liquid ratios of 1:7, 1:8, 1:9 and 1:10 on the traditional Chinese medicine compound solution were investigated using alkaloid content and clear extract rate as indicators.

[0084] The table below shows the results of the study on the feed-to-liquid ratio (n=3). )

[0085] Material-liquid ratio Clearance rate (%) Alkaloid content (%) 1：7 18.52±0.45 20.89±0.32 1：8 22.15±0.38 22.12±0.25 1：9 22.31±0.41 22.05±0.28 1：10 22.40±0.35 21.98±0.30

[0086] As shown in the table above, the alkaloid content and clearing rate increase with increasing solvent volume. When the solid-liquid ratio increases from 1:7 to 1:8, the alkaloid transfer rate significantly improves. Further increasing the solvent to 1:9 and 1:10, although the clearing rate increases slightly, the increase in alkaloid content is not significant, and excessive solvent increases the energy and time costs of subsequent concentration. Considering both extraction efficiency and cost, 1:8 is selected as the optimal solid-liquid ratio.

[0087] 5.2 Extraction time investigation: With other conditions unchanged, the extraction was carried out according to the method of Example 6, with the material-to-liquid ratio fixed at 1:8 and the number of extractions at 2. The effects of extraction time of 0.5, 1, 1.5 and 2 h on the traditional Chinese medicine compound solution were investigated, with alkaloid content and clear extract rate as indicators.

[0088] The table below shows the results of the extraction time evaluation (n=3). )

[0089] Extraction time (h) Clearance rate (%) Alkaloid content (%) 0.5 19.85±0.52 20.55±0.41 1.0 22.20±0.35 22.15±0.22 1.5 22.35±0.40 22.18±0.28 2.0 22.42±0.38 21.95±0.33

[0090] As shown in the table above, extraction was incomplete at 0.5 h; when the extraction time was extended to 1.0 h, all indicators reached a high level. Further extending the extraction time to 1.5 h and 2.0 h did not significantly increase the alkaloid content, and prolonged high-temperature boiling may lead to the decomposition of some heat-sensitive components. Therefore, the optimal extraction time was determined to be 1 h.

[0091] 5.3 Investigation of the number of extractions: With other conditions unchanged, extraction was performed according to the method in Example 6 of this paper, with a fixed material-to-liquid ratio of 1:8 and an extraction time of 1 h. The effects of 0, 1, 2 and 3 extractions on the traditional Chinese medicine compound solution were investigated, using alkaloid content and clear extract rate as indicators.

[0092] The table below shows the results of the number of extractions (n=3). )

[0093] Number of extractions (times) Clearance rate (%) Alkaloid content (%) 1 16.80±0.62 17.55±0.55 2 22.18±0.41 22.16±0.26 3 22.95±0.33 22.25±0.20

[0094] As shown in the table above, the drug components are not completely extracted after one extraction; after two extractions, the alkaloid content and the clear extract rate are significantly improved. While the total amount increases slightly after three extractions, considering the production cycle and energy consumption, two extractions are sufficient for complete extraction. Therefore, the optimal number of extractions is determined to be two.

[0095] In summary, the optimal extraction process for the traditional Chinese medicine compound solution is: a material-to-liquid ratio of 1:8, extraction time of 1 hour, and extraction twice. The traditional Chinese medicine compound solution prepared according to this optimal process, when concentrated to 150 mL, had a density of 1.200 g / mL and an alkaloid content of approximately 22.16 ± 0.26%, slightly higher than the results obtained from the pre-prescription study of the raw materials from the Central Plains, indicating that the extraction process has a good enrichment effect.

[0096] Example 6 Preparation of freeze-dried powder of traditional Chinese medicine compound

[0097] The traditional Chinese medicine compound solution was prepared according to the optimization results of Example 5 above. The solution was dispensed into freeze-drying trays, pre-frozen at -80 ℃ overnight, and then freeze-dried under a vacuum of 0.3 Pa for 24 h. The solution was immediately removed to obtain the freeze-dried powder of the traditional Chinese medicine compound. The alkaloid content was used as an indicator for determination.

[0098] The traditional Chinese medicine compound solution was prepared according to the optimized process described in Example 5 above, and after freeze-drying, a brownish-red, loose, powdery traditional Chinese medicine compound freeze-dried powder was obtained. The alkaloid content in the freeze-dried powder was determined to be stable, with an average content of 22.14 ± 0.27% (RSD = 1.2%, n = 3), meeting the requirements for formulation preparation.

[0099] Example 7: Preparation and Process Optimization of pH-Responsive Composite Thermosensitive Hydrogel

[0100] Thermosensitive hydrogels were prepared using a cold-dissolution method. 0.05 g of chitosan was dissolved in a 0.5% (w / v) lactic acid solution (the medium used was a traditional Chinese medicine compound liposome). 1.5 g of poloxamer P407 and 0.5 g of poloxamer P188 were added, and the lactic acid solution was brought to 20 mL. The solution was then expanded at 4 °C for 24 h to obtain a blank pH-responsive thermosensitive hydrogel in a transparent solution state. Subsequently, 4.58 g of lyophilized traditional Chinese medicine compound powder was added to 10 mL of the pH-responsive thermosensitive hydrogel, and the mixture was stirred at 100 r / min to form a pH-responsive composite thermosensitive hydrogel.

[0101] 7.1 Investigation of chitosan dosage: With the final volume of the system fixed at 10 mL and other conditions unchanged, hydrogels were prepared according to the method in Example 7. Poloxamer P407 was fixed at 2 g and poloxamer P188 at 0.6 g. The effects of chitosan dosages of 0.05, 0.06, 0.07, 0.08, and 0.09 g on pH-responsive thermosensitive hydrogels were investigated, using gelation time and gel viscosity at different temperatures (4 ℃ and 37 ℃) as indicators.

[0102] The table below shows the results of the chitosan dosage study (n=3). )

[0103] Chitosan dosage (g / 10mL) Quality score (%) Viscosity at 4 °C (mPa·s) Viscosity at 37℃ (mPa·s) gelation time (s) 0.05 0.5 420±15 2800±120 110±5 0.06 0.6 550±20 4650±150 65±3 0.07 0.7 980±35 5100±180 50±4 0.08 0.8 1550±50 5800±210 45±3 0.09 0.9 2100±80 >6000 40±2

[0104] As shown in the table above, the viscosity of the system increases significantly with the increase of chitosan dosage. When the chitosan dosage is 0.06 g, the viscosity of the hydrogel is less than 600 mPa·s at 4 ℃, exhibiting good fluidity and facilitating injection administration; while at 37 ℃, the viscosity rapidly rises to over 4500 mPa·s, forming a stable semi-solid gel, which can effectively prolong the drug's retention time in the body. Therefore, 0.06 g is selected as the optimal dosage of chitosan.

[0105] 7.2 Investigation of the proportion of hydrogel excipients: With the final volume of the system fixed at 10 mL and other conditions unchanged, the hydrogel was prepared according to the method in Example 7. The amount of chitosan was fixed at 0.06 g. The effects of the proportions of poloxamer P407 and P188 (2 g: 0.7 g, 2 g: 0.6 g, 2 g: 0.5 g, 3 g: 0.6 g, 2.5 g: 0.6 g, 1.5 g: 0.6 g) on ​​the pH-responsive thermosensitive hydrogel were investigated, using gel time and gel viscosity at different temperatures (4 ℃ and 37 ℃) as indicators.

[0106] The table below shows the results of the investigation of the ratio of poloxamer P407 to P188 (n=3). )

[0107] P407:P188 (g:g) Viscosity at 4℃ (mPa·s) Viscosity at 37℃ (mPa·s) gelation time (s) 2 :0.5 480±12 3200±110 115±5 2 :0.6 560±18 4700±130 65±3 2 :0.7 650±22 4900±150 42±4 1.5 :0.6 350±10 1500±80 >300 2.5 :0.6 1800±60 >6000 ＜10

[0108] As shown in the table above, poloxamer P407 is the main framework material for forming the thermosensitive gel, while poloxamer P188 plays a role in regulating the gelation time. When the P407:P188 ratio is 2 g:0.6 g, the gelation time is approximately 65 s. This time is optimal, allowing sufficient time for injection to prevent the drug from clogging the needle due to excessively rapid gelation, while also ensuring rapid phase transition and gel formation after injection, preventing drug diffusion and loss to surrounding tissues before gelation. In contrast, when the P407 concentration is too high (2.5 g) or the P188 ratio increases (0.7 g), the gelation time is significantly shortened (<10 s or 42 s), easily leading to needle clogging or excessive injection resistance; while when the P407 concentration is too low (1.5 g) or the P188 ratio is insufficient (0.5 g), the gelation time is too long or even fails to gel (>300 s or 115 s), making it difficult to form an effective drug reservoir. Therefore, taking into account both injectability and gelling properties, the optimal excipient ratio was determined to be 2 g of P407 and 0.6 g of P188.

[0109] Based on the optimized formulation process described above: 0.06 g of chitosan, 2.00 g of P407, 0.60 g of P188, and 4.58 g of lyophilized compound traditional Chinese medicine powder were weighed, and three batches of pH-responsive composite thermosensitive hydrogels were prepared according to the method in Example 7. The viscosity and gelation time at 4 ℃ and 37 ℃ were measured.

[0110] The table below shows the results of the optimal process validation test (n=3).

[0111] batch number Viscosity at 4℃ (mPa·s) Viscosity at 37℃ (mPa·s) gelation time (s) 20251201 555 4680 63 20251202 562 4710 66 20251203 548 4650 64 average value 555.0 4680.0 64.3 RSD (%) 1.26 0.64 2.38

[0112] The verification results show that the pH-responsive composite thermosensitive hydrogel prepared by the optimized process has good process reproducibility, good fluidity at low temperature (viscosity <600 mPa·s), and can be rapidly transformed into a high-viscosity gel (viscosity >4500 mPa·s) at body temperature, which meets the design requirements.

[0113] Through Examples 5-7, the water extraction process of the traditional Chinese medicine compound was optimized, and the optimal process parameters were determined to be: a material-to-liquid ratio of 1:8, an extraction time of 1 h, and two extractions. Under this process, the relative density of the concentrated liquid reached 1.2, and the alkaloid content remained stable at 22.16 ± 0.26%. Subsequently, using the lyophilized powder of the traditional Chinese medicine compound as a model drug, a pH-responsive composite thermosensitive hydrogel was developed. Through single-factor experiments, the optimal matrix formulation for 10 mL of hydrogel was determined to be: 0.06 g of chitosan, 2 g of poloxamer P407, and 0.6 g of poloxamer P188. Verification experiments showed that the hydrogel prepared by this optimized formulation has obvious thermosensitive properties and suitable viscosity, is easy to inject at low temperatures, and can rapidly gel in situ at body temperature.

[0114] Example 8 In vitro release experiment

[0115] The in vitro drug release behavior of pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction was investigated using the dialysis bag method.

[0116] The lyophilized powder was prepared according to Example 6, and the hydrogel was prepared according to Example 7. 2.0 mL of the pH-responsive composite thermosensitive hydrogel was accurately measured and placed in a pretreated dialysis bag (molecular weight cutoff MWCO: 3.5 kDa), with both ends clamped. The hydrogel was then immersed in beakers containing 30 mL of release medium. The release mediums were phosphate-buffered saline (PBS) at pH 4.5 and pH 7.4, respectively, both containing 1.0% (v / v) Tween-80 to maintain the leak conditions.

[0117] Place the beaker in a constant temperature water bath shaker, set the temperature to (37±0.5)℃ and the shaking rate to 100 r / min. At time points of 0.5, 1, 2, 4, 6, 8, 10, 12 and 16 h, accurately aspirate 2.0 mL of the release medium and immediately replenish it with the same temperature and volume of medium.

[0118] The extracted sample solution was filtered through a 0.45 µm microporous membrane, and the absorbance was measured. The drug concentration at each time point was calculated by substituting the absorbance into the standard curve equation, and the cumulative release rate (Q%) was calculated using the following formula. Each experiment was repeated in triplicate.

[0119]

[0120] In the formula: Cn is the concentration measured at the nth sampling point (mg / mL); V is the total volume of the release medium (30 mL); Vi is the volume of each sampling (2 mL); Wtotal is the total mass of the drug added to the gel (mg).

[0121] Following the method described in Example 8 above, the in vitro release of the pH-responsive composite thermosensitive hydrogel was investigated over 16 hours under different pH environments (pH 4.5 and pH 7.4).

[0122] The table below shows the cumulative release rate of alkaloids under different pH conditions (n=3). )

[0123] Time (h) pH 4.5 Cumulative release rate (%) Cumulative release rate at pH 7.4 (%) 0.5 14.52±1.25 5.21±0.85 1 26.18±2.10 11.85±1.12 2 42.85±2.45 20.52±1.65 4 63.50±3.12 31.20±2.08 6 75.12±2.88 38.65±2.45 8 82.45±2.30 43.50±1.95 10 86.80±1.85 46.25±2.10 12 89.55±1.55 48.10±1.88 16 90.01±1.92 49.06±1.77

[0124] Figure 6 The cumulative release rate of alkaloids under different pH conditions is shown in the figure. The experimental results indicate that the prepared pH-responsive composite thermosensitive hydrogel exhibits obvious pH-dependent release characteristics and good sustained-release performance.

[0125] pH responsiveness analysis: In acidic medium (pH 4.5), the drug release rate was significantly faster than in neutral medium (pH 7.4). At 16 h, the cumulative release rate reached 90.01 ± 1.92% under pH 4.5 conditions, while it was only 49.06 ± 1.77% under pH 7.4 conditions. This is mainly due to the pH sensitivity of chitosan in the gel matrix. Chitosan molecules contain a large number of amino groups (-NH2). In an acidic environment (pH 4.5), these amino groups are protonated to -NH3⁺, enhancing the electrostatic repulsion between molecular chains with the same charge. This leads to swelling and increased porosity in the gel network structure, thereby accelerating drug diffusion and release. However, at pH 7.4, the amino groups are deprotonated, resulting in a denser gel network structure that hinders drug release. This characteristic is particularly advantageous for vaginal delivery systems because the normal vaginal environment is acidic (pH 3.8~4.5), allowing the formulation to achieve rapid and full release of the drug at the lesion site, while reducing leakage in non-target areas (neutral environment).

[0126] Sustained-release characteristics analysis: Under pH 4.5 conditions, drug release exhibits a "rapid initial release followed by slower release" trend. Approximately 42.85% of the drug is released within the first 2 hours; this moderate burst release effect facilitates the rapid achievement of effective therapeutic concentrations. From the subsequent 2 to 16 hours, the drug is released steadily and continuously, with near-complete release by 16 hours. This indicates that the hydrogel system can provide continuous drug delivery for up to 16 hours, helping to maintain drug concentrations at the lesion site, reduce the frequency of administration, and improve patient compliance.

[0127] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A pH-responsive composite thermosensitive hydrogel containing a wart-removing decoction, characterized in that, It is prepared from the following raw materials: The ingredients include liposomes of the traditional Chinese medicine formula Xiaoyoutang, freeze-dried powder of Xiaoyoutang, chitosan, poloxamer P407, poloxamer P188 and 0.5% lactic acid aqueous solution; The wart-removing decoction is composed of the following raw materials in parts by weight: 20 parts of Cnidium monnieri, 15 parts of Smilax glabra, 15 parts of Sophora flavescens, 12.5 parts of Sophora tonkinensis, 12.5 parts of Phellodendron chinense, 12.5 parts of Lithospermum erythrorhizon, 12.5 parts of Stemona japonica, 10 parts of Artemisia annua, and 2.5 parts of Brucea javanica.

2. The pH-responsive composite thermosensitive hydrogel containing wart-removing decoction according to claim 1, characterized in that, Each 10 mL of pH-responsive composite thermosensitive hydrogel containing Xiaoyoutang (a traditional Chinese medicine formula) is composed of the following ingredients: 0.5 mL of Xiaoyoutang compound liposomes, 4.58 g of Xiaoyoutang lyophilized powder, 0.06 g of chitosan, 2 g of poloxamer P407, 0.6 g of poloxamer P188, with the remainder made up to 10 mL by 0.5% lactic acid aqueous solution.

3. The pH-responsive composite thermosensitive hydrogel containing wart-removing decoction according to claim 1, characterized in that, In the compound liposome of the wart-removing decoction, the mass ratio of egg yolk lecithin to cholesterol is 6:

1.

4. The pH-responsive composite thermosensitive hydrogel containing wart-removing decoction according to claim 1, characterized in that, The average alkaloid content in the freeze-dried powder of the wart-removing decoction is 21.87% to 22.41%.

5. A method for preparing a pH-responsive composite thermosensitive hydrogel containing a wart-removing decoction according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1: Extraction of volatile oil from wart-removing decoction: Remove the raw material of wart-removing decoction, add pure water at a material-to-liquid ratio of 5:1, soak for 0.5 h, extract by gentle boiling for 6 h, collect the volatile oil, and keep the remaining residue for later use; S2: Preparation of compound liposomes of traditional Chinese medicine: Egg yolk lecithin and cholesterol were mixed at a mass ratio of 6:1 using the thin film dispersion method, dissolved in anhydrous ethanol, and formed a uniform thin film by rotary evaporation at 60°C. The organic solvent was removed by vacuum drying. The volatile oil of Xiaoyoutang obtained in step S1 and phosphate buffer were added, and after ultrasonic dispersion, the mixture was filtered through a microporous membrane to obtain compound liposomes of traditional Chinese medicine. S3: Preparation of freeze-dried powder of wart-removing decoction: Take the residue of wart-removing decoction after volatile oil in step S1, add pure water at a material-to-liquid ratio of 1:8, decoct and extract twice, extract for 1 hour each time, combine the extracts and concentrate, pre-freeze at -80℃ and freeze-dry under vacuum to obtain freeze-dried powder. S4: Preparation of pH-responsive composite thermosensitive hydrogel: Chitosan, poloxamer P407 and poloxamer P188 were dissolved in 0.5% lactic acid aqueous solution. Using the traditional Chinese medicine compound liposomes obtained in step S2 as the medium, the mixture was swollen at 4℃ for 24 h to obtain a blank thermosensitive hydrogel. The lyophilized powder of Xiaoyoutang obtained in step S3 was added to the blank thermosensitive hydrogel and stirred evenly at 100 r / min to obtain the pH-responsive composite thermosensitive hydrogel. The dosage of chitosan, poloxamer P407, poloxamer P188, and the lyophilized powder of Xiaoyoutang was 0.06 g : 2 g : 0.6 g : 4.58 g / 10 mL of 0.5% lactic acid aqueous solution.

6. The preparation method according to claim 5, characterized in that, In step S2, the microporous membrane filtration uses 0.45 µm and 0.22 µm membranes respectively.

7. The preparation method according to claim 5, characterized in that, In step S3, the vacuum degree of freeze drying is 0.3 Pa, and the time is 24 h.

8. The application of the pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction according to any one of claims 1 to 4, or the pH-responsive composite thermosensitive hydrogel containing wart-reducing decoction prepared by the preparation method according to any one of claims 5 to 7, in the preparation of vaginal topical antimicrobial drugs.

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