Preparation method of traditional Chinese medicine external preparation for treating arthralgia based on promoting blood circulation to remove blood stasis

Abstract

The invention relates to the technical field of traditional Chinese medicine pharmacy, and discloses a preparation method of a traditional Chinese medicine external preparation for arthralgia based on blood circulation promoting and blood stasis removing, which comprises the following steps: extracting ferulic acid from angelica sinensis and ligusticum wallichii by adopting a supercritical CO2 fluid extraction process; extracting safflower, frankincense and other medicinal materials to obtain a synergistic active ingredient extract; the ferulic acid is used as a first hydrogen bond donor to react with lauric acid used as a temperature-sensitive phase change hydrogen bond donor and choline chloride used as a hydrogen bond acceptor, and the temperature-sensitive bioactive ternary eutectic solvent is prepared; loading the synergistic active ingredient extract in the solvent at low temperature to form a medicine mother solution; and finally, mixing the medicine mother liquor with auxiliary materials to prepare the external preparation. Ferulic acid in medicinal materials serves as a structural unit of a functional solvent, the solvent prepared under the mild condition has biological activity and temperature-sensitive phase change characteristics, a medicine storage cavern can be formed on the skin surface in situ, long-acting slow release and efficient transdermal absorption of active ingredients are achieved, and the curative effect and safety of a preparation are improved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a method for preparing a topical preparation of traditional Chinese medicine for joint pain based on promoting blood circulation and removing blood stasis. Background Technology

[0002] Joint pain is a common clinical symptom, and topical Chinese herbal preparations that promote blood circulation and remove blood stasis are a common means of relieving this pain. In existing technologies, the preparation of such preparations typically employs traditional processes such as water decoction or alcohol extraction. However, these methods require relatively high temperatures, which can lead to the structural destruction and reduced content of some heat-labile active ingredients in the compound herbs, such as safflower and frankincense. Simultaneously, there is a contradiction between the choice of solvent and the complexity of the herbal components; a single solvent cannot efficiently dissolve all the active ingredients of different polarities simultaneously. This results in the final preparation failing to fully retain the material basis of the herbs, thus affecting the synergistic effect between the components.

[0003] In terms of dosage form, traditional Chinese medicine gels or creams, after being applied to the skin, suffer from short adhesion time and are easily removed by friction from clothing, resulting in insufficient effective contact time between the drug and the skin. Their drug release pattern is typically a rapid initial release, making it difficult to establish a stable drug concentration at the application site to maintain a long-lasting effect. Furthermore, many active ingredients in traditional Chinese medicine compound formulas have large molecular weights or poor lipid solubility, leading to low efficiency in crossing the skin's stratum corneum barrier, thus limiting their ultimate bioavailability and clinical efficacy.

[0004] To address the issue of low transdermal absorption efficiency, current technologies often incorporate chemical penetration enhancers such as azone and propylene glycol into formulations. These chemical penetration enhancers promote drug penetration by altering the structure of the stratum corneum, but they themselves can irritate the skin. Long-term or large-area use can easily trigger adverse reactions such as erythema, itching, and allergies. This non-specific interference with the skin barrier increases the safety risks of medication use and reduces patient compliance, making it particularly unsuitable for patients with sensitive or broken skin. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing topical Chinese medicine preparations for joint pain based on promoting blood circulation and removing blood stasis. This method solves the problems in existing Chinese medicine compound preparation processes, which often employ high-temperature decoction and concentration methods, resulting in the loss of active ingredients in medicinal materials, poor product stability, and the enrichment of irritating components.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, comprising: S1. Angelica sinensis and Ligusticum chuanxiong were taken and extracted and purified using supercritical CO2 fluid extraction technology at an extraction pressure of 20-35 MPa and an extraction temperature of 45-65℃ to obtain ferulic acid as the first hydrogen bond donor HBD1. S2. Take safflower, frankincense, myrrh and Panax notoginseng, extract them by supercritical CO2 fluid extraction or subcritical fluid extraction, and then concentrate them under reduced pressure at a vacuum pressure of -0.08MPa to -0.095MPa to prepare synergistic active ingredient extract A'. S3. The ferulic acid obtained in step S1, along with the pre-provided lauric acid and choline chloride, are mixed in a reaction flask and stirred at 75-95℃ for 1-3 hours to obtain a thermosensitive bioactive ternary eutectic solvent. S4. Add the synergistic active ingredient extract A' obtained in step S2 to the thermosensitive bioactive ternary eutectic solvent prepared in step S3 in portions, and stir to dissolve or disperse at 40-50℃ to form a drug mother liquor. S5. In a homogenizing emulsification vessel, the drug mother liquor prepared in step S4 is mixed with the aqueous dispersion of carbomer 940, and then triethanolamine is added to neutralize and form a gel. Finally, after homogenization, a topical preparation of traditional Chinese medicine for joint pain is prepared.

[0007] By employing the above technical solution, this invention constructs a functionally integrated drug delivery system. Its core mechanism lies in the specific reaction in step S3, where ferulic acid, the active ingredient derived from the principal drug, serves as one of the structural frameworks of the solvent. Through intermolecular hydrogen bonding, it self-assembles with lauric acid and choline chloride to form a homogeneous and stable ternary eutectic solvent. This solvent not only carries the subsequently added synergistic active ingredient extract A', but also possesses biological activity itself due to the presence of ferulic acid, thus achieving a unified function of both the carrier and the drug.

[0008] Furthermore, the introduction of lauric acid endows the solvent system with thermosensitive phase change properties. At room temperature, it is a low-viscosity liquid; upon application to the skin surface, it transforms into a semi-solid structure driven by surface temperature, forming a drug reservoir in situ at the application site. This achieves long-acting sustained release and efficient transdermal absorption of the active ingredient. Simultaneously, the entire preparation method, particularly the supercritical or subcritical extraction and low-temperature concentration processes used in steps S1 and S2, and the low-temperature loading in step S4, are all carried out under mild conditions. This avoids the damage to thermally unstable components in the compound medicinal materials caused by traditional high-temperature processes, ensuring the integrity of the active ingredients in the final formulation.

[0009] Preferably, in step S1, the Angelica sinensis, Ligusticum chuanxiong, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, and Panax notoginseng are all pulverized into powder with a mesh size of 40-60. Furthermore, in the extraction and purification of ferulic acid, the supercritical CO2 fluid extraction process also includes the use of anhydrous ethanol as an entrainer, with the amount of entrainer being 5-15% (v / v) of the CO2 flow rate.

[0010] By adopting the above technical solution, pre-crushing the medicinal materials increases the specific surface area of ​​contact between the extraction solvent and the medicinal materials, thereby improving the extraction efficiency. Adding anhydrous ethanol as an entrainer can change the polarity of the supercritical CO2 fluid, thus significantly improving the solubility and extraction yield of medium polar molecules such as ferulic acid.

[0011] Preferably, in step S3, both the mixing and stirring reactions are carried out under nitrogen protection, and the stirring speed is 200-400 rpm until a clear, homogeneous liquid phase is formed. By adopting the above technical solution, the introduction of inert nitrogen gas can effectively isolate oxygen, prevent the oxidative degradation of phenolic acid active ingredients such as ferulic acid during heating, and ensure the bioactivity of the solvent and the stability of the final formulation.

[0012] Preferably, in step S4, the synergistic active ingredient extract A' is added in equal portions 2-4 times to a temperature-sensitive bioactive ternary eutectic solvent, and the mixture is continuously stirred at 300-500 rpm for 30-60 minutes. By adopting the above technical solution, the portioned addition method avoids the aggregation or precipitation caused by excessively high local concentrations due to the instantaneous addition of a large amount of extract, ensuring that the extract achieves uniform molecular-level dispersion in the solvent.

[0013] Preferably, in step S5, the specific implementation method for forming the topical Chinese medicine preparation for joint pain is as follows: First, carbomer 940 is dispersed in purified water and allowed to stand and swell for 4-6 hours to form an aqueous dispersion of carbomer 940. Then pump in the drug stock solution and mix it evenly with the aqueous dispersion of Carbomer 940; Triethanolamine was then added dropwise to neutralize the solution until the pH reached 5.5-7.0, forming a gel. Finally, homogenize the sample under vacuum conditions at a speed of 1000-2000 rpm for 5-10 minutes.

[0014] By adopting the above technical solution, sufficient swelling and precise pH control are the basis for forming a stable gel skeleton, while the subsequent vacuum homogenization process can eliminate air bubbles trapped in the preparation process and further reduce the droplet size, so that the final formulation has a uniform, delicate and stable texture.

[0015] Preferably, lauric acid is used as the thermosensitive phase transition hydrogen bond donor HBD2, choline chloride is used as the hydrogen bond acceptor HBA, and the molar ratio of ferulic acid, lauric acid and choline chloride is (0.4-0.6):(0.4-0.6):(0.9-1.1).

[0016] By adopting the above technical solution, this specific molar ratio is the key structural basis for forming a stable, homogeneous eutectic solvent with a suitable phase transition temperature, ensuring the fluidity of the formulation at room temperature and its semi-solid gel properties at body surface temperature.

[0017] Preferably, in step S1, the mass ratio of Angelica sinensis to Ligusticum chuanxiong is 1:1; and in step S2, the mass ratio of Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, and Panax notoginseng is 1:1:1:1.

[0018] Preferably, in step S5, the mass ratio of the drug mother liquor, the aqueous dispersion of carbomer 940, and triethanolamine is (10-30):(0.8-1.5):(0.8-1.5).

[0019] Preferably, in step S2, the reduced pressure concentration is carried out under a vacuum pressure of -0.08 MPa to -0.095 MPa.

[0020] This invention provides a method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis. It has the following beneficial effects: 1. This invention utilizes ferulic acid as the first hydrogen bond donor in constructing a thermosensitive bioactive ternary eutectic solvent, achieving dual active functions as both solvent and solute. Ferulic acid not only serves as the solvent framework but also exerts pharmacological effects of promoting blood circulation and removing blood stasis, creating a synergistic effect with the synergistic active ingredient extract A'. Simultaneously, the entire preparation method is carried out under mild conditions, avoiding the destruction of heat-labile components in medicinal materials such as safflower and frankincense by traditional high-temperature processes, ensuring the integrity and high content of the compound active ingredients in the final formulation.

[0021] 2. The present invention utilizes a thermosensitive bioactive ternary eutectic solvent. Due to the introduction of the thermosensitive phase transition hydrogen bond donor HBD2 lauric acid, this solvent exhibits unique thermosensitive phase transition characteristics. At room temperature, it is a low-viscosity liquid that is easy to apply. After being applied to the skin surface, it undergoes a physical phase transition to a semi-solid structure under the influence of the skin surface temperature. This semi-solid structure forms a drug reservoir in situ at the application site, prolonging the contact time between the drug and the skin, and releasing the active ingredient slowly in a controlled manner, thus achieving long-lasting sustained release and improving the transstratum corneum penetration efficiency of the active ingredient.

[0022] 3. This invention eliminates traditional chemical penetration enhancers or organic solvents that can cause skin irritation. The solvent system components ferulic acid, lauric acid, and choline chloride are all biologically derived or possess high biocompatibility, making the thermosensitive, bioactive ternary eutectic solvent itself highly skin-compatible and low-allergenic. Therefore, the prepared topical Chinese medicine preparation for joint pain effectively delivers the active ingredients while reducing the risk of damage and irritation to the skin barrier, thus improving medication safety and patient compliance. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Examples 1-3 Example 1: This invention provides a method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, comprising: S1. Preparation of ferulic acid: Take 500g of Angelica sinensis and 500g of Ligusticum chuanxiong, pulverize them and pass them through a 50-mesh sieve. Mix them evenly and place them in the extraction vessel of a supercritical CO2 fluid extraction device. Set the extraction pressure to 30MPa and the extraction temperature to 55℃. Use anhydrous ethanol as the entrainer, with an entrainer amount of 10% (v / v) of the CO2 flow rate, and extract for 4 hours. Collect the extract, concentrate it under reduced pressure, and then purify it by silica gel column chromatography to finally obtain ferulic acid powder with a purity of 98.8%.

[0025] S2, Preparation of synergistic active ingredient extract A': Take 200g of safflower, 200g of frankincense, 200g of myrrh, and 200g of Panax notoginseng, pulverize them, and pass them through a 50-mesh sieve. Mix them evenly and place them in the extraction vessel of a supercritical CO2 fluid extraction device. Set the extraction pressure to 30MPa and the extraction temperature to 55℃, and extract for 4 hours. Collect the extract and transfer it to a vacuum concentration vessel. Concentrate under vacuum conditions of -0.09MPa and 60℃ to obtain a brownish-yellow viscous extract, which is the synergistic active ingredient extract A'.

[0026] S3. Preparation of temperature-sensitive bioactive ternary eutectic solvent: In a reaction flask equipped with a stirrer and a nitrogen inlet tube, 97.1 g of ferulic acid, 100.2 g of lauric acid, and 139.6 g of choline chloride prepared in step S1 were added according to a molar ratio of ferulic acid:lauric acid:choline chloride of 0.5:0.5:1.0. Nitrogen gas was introduced for protection, and stirring was started at 300 rpm. The reaction flask was placed in a constant temperature water bath at 85°C and heated and stirred for 2 hours until the solid in the system completely disappeared, forming a clear, homogeneous, pale yellow transparent liquid, which is the thermosensitive bioactive ternary eutectic solvent.

[0027] S4. Preparation of drug mother liquor: The temperature-sensitive bioactive ternary eutectic solvent prepared in step S3 was cooled to 45°C. 100g of the synergistic active ingredient extract A' prepared in step S2 was divided into three equal portions. At a stirring speed of 400 rpm, the first portion of extract A' was added to the solvent and stirred until completely dissolved and dispersed. Then, the next portion was added, and so on, until all three portions were added. Stirring was continued at 45°C and 400 rpm for 45 minutes to obtain a homogeneous and stable drug stock solution.

[0028] S5. Formulation of topical Chinese medicine preparations for joint pain: Based on a final formulation of 1000g, 12.0g of Carbomer 940 was weighed and dispersed in 776.0g of purified water. The dispersion was allowed to swell for 5 hours to form a uniform aqueous dispersion of Carbomer 940. This dispersion was transferred to a vacuum homogenizing emulsifier, and 200.0g of the drug stock solution prepared in step S4 was slowly pumped in and stirred until homogeneous. Subsequently, 12.0g of triethanolamine was added dropwise under stirring, monitored with a pH meter, until the pH of the system reached 6.5, forming a uniform gel. The vacuum system of the homogenizing emulsifier was turned on, and homogenization was performed at 1500rpm for 8 minutes. After degassing, the traditional Chinese medicine external preparation for joint pain was obtained.

[0029] Example 2: This invention provides a method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, comprising: S1. Preparation of ferulic acid: The extraction pressure was set to 20 MPa, the extraction temperature to 45°C, and the entrainer dosage to be 5% (v / v) of the CO2 flow rate. The remaining steps were the same as in Example 1.

[0030] S2, Preparation of synergistic active ingredient extract A': The vacuum pressure for vacuum concentration was set to -0.08 MPa. The remaining steps were the same as in Example 1.

[0031] S3. Preparation of temperature-sensitive bioactive ternary eutectic solvent: The reactants were fed in a ferulic acid:lauric acid:choline chloride molar ratio of 0.4:0.4:1.1. The reaction temperature was set at 75°C, the stirring speed at 200 rpm, and the reaction time at 1 hour. The remaining steps were the same as in Example 1.

[0032] S4. Preparation of drug mother liquor: Extract A', the synergistic active ingredient, was added in two batches. The dissolution or dispersion temperature was set at 40°C, the stirring speed at 300 rpm, and the stirring time at 30 minutes. The remaining steps were the same as in Example 1.

[0033] S5. Formulation of topical Chinese medicine preparations for joint pain: Weigh out 100.0g of the drug stock solution, 8.0g of carbomer 940, 8.0g of triethanolamine, and 884.0g of purified water, based on a final formulation volume of 1000g. Neutralize the pH to 5.5. Set the homogenization speed to 1000rpm and the homogenization time to 5 minutes. The remaining steps are the same as in Example 1.

[0034] Example 3: This invention provides a method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, comprising: S1. Preparation of ferulic acid: The extraction pressure was set to 35 MPa, the extraction temperature to 65°C, and the entrainer dosage to be 15% (v / v) of the CO2 flow rate. The remaining steps were the same as in Example 1.

[0035] S2, Preparation of synergistic active ingredient extract A': The vacuum pressure for vacuum concentration was set to -0.095 MPa. The remaining steps were the same as in Example 1.

[0036] S3. Preparation of temperature-sensitive bioactive ternary eutectic solvent: The reactants were fed in a ferulic acid:lauric acid:choline chloride molar ratio of 0.6:0.6:0.9. The reaction temperature was set at 95°C, the stirring speed at 400 rpm, and the reaction time at 3 hours. The remaining steps were the same as in Example 1.

[0037] S4. Preparation of drug mother liquor: The synergistic active ingredient extract A' was added in four batches. The dissolution or dispersion temperature was set at 50°C, the stirring speed at 500 rpm, and the stirring time at 60 minutes. The remaining steps were the same as in Example 1.

[0038] S5. Formulation of topical Chinese medicine preparations for joint pain: Weigh out 300.0g of the drug stock solution, 15.0g of carbomer 940, 15.0g of triethanolamine, and 670.0g of purified water, based on a final formulation yield of 1000g. Neutralize the pH to 7.0. Set the homogenizer speed to 2000rpm and the homogenization time to 10 minutes. The remaining steps are the same as in Example 1.

[0039] Comparative Examples 1-3: Comparative Example 1: Compared with Example 1, the difference is that steps S1 to S4 are not performed in this comparative example. All the medicinal materials used in Example 1 (Angelica sinensis, Ligusticum chuanxiong, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, Panax notoginseng) are mixed and an extract is prepared using the traditional hot reflux extraction method. This extract is then directly used in step S5 for formulation. All other steps are the same.

[0040] Comparative Example 2: The difference between this example and Example 1 lies in the preparation of the eutectic solvent in step S3. This comparative example uses glycerol and choline chloride (molar ratio 2:1), conventional in the art, to prepare the eutectic solvent, which is used in place of the thermosensitive bioactive ternary eutectic solvent described in this invention. All other steps are the same.

[0041] Comparative Example 3: The difference between this example and Example 1 lies in the preparation of the eutectic solvent in step S3. In this comparative example, lauric acid is not added during the preparation of the eutectic solvent; instead, ferulic acid and choline chloride are used to prepare a binary eutectic solvent that does not possess thermosensitive phase transition properties. All other steps are the same.

[0042] Test Examples 1-4: Test Example 1: Evaluation of the basic physicochemical properties of the formulation The samples prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to the following physicochemical property determinations.

[0043] Appearance characteristics: Take an appropriate amount of each group of samples and place them in a clean, dry petri dish. Observe their color, state, uniformity, and whether there are bubbles or insoluble matter under natural light.

[0044] pH measurement: A calibrated PXS-25 precision pH meter was used. Accurately weigh 1.0 g of each sample and place it in a 50 mL beaker. Add 10 mL of purified water and stir thoroughly with a glass rod to disperse the sample evenly. After standing for 5 minutes, insert the pH electrode into the sample dispersion. Record the pH value after the reading stabilizes. Perform three parallel measurements and take the average value.

[0045] Phase transition temperature determination: An MCR302 rotational rheometer was used. An appropriate sample was placed on the test platform of the rheometer, and the test gap was set to 1.0 mm. Temperature sweep mode was adopted, with the oscillation frequency set to 1 Hz and the strain set to 1%. The test temperature range was set to 25℃ to 45℃, and the heating rate was 2℃ / min. The phase transition temperature was defined as the intersection of the storage modulus (G') and the loss modulus (G''), or the temperature at which G' undergoes a sudden change.

[0046] Table 1. Evaluation results of the basic physicochemical properties of the formulations in each example and comparative example.

[0047] As shown in Table 1, the samples of Examples 1-3 prepared by the method of the present invention have a uniform and fine appearance, and their pH values ​​are all within the range of 5.5-7.0. This indicates that the preparation method can stably disperse the synergistic active ingredient extract A' in the matrix, forming a formulation with stable physical properties and compatibility with the physiological environment of the skin surface. In contrast, Comparative Example 1, prepared by the conventional process, has poor appearance, which is directly related to the fact that the active ingredient was not effectively loaded into a specific solvent system.

[0048] The test results further showed that the formulations of Examples 1-3 all exhibited clear physical phase transition behavior within the temperature range of 33-36°C. This characteristic stems from the design of step S3 in their preparation process, which involves the self-assembly of ferulic acid (as the first hydrogen bond donor), lauric acid (as the thermosensitive phase transition hydrogen bond donor), and choline chloride (as the hydrogen bond acceptor) to construct a ternary eutectic solvent with an internal hydrogen bond network that is temperature-responsive. In Comparative Example 3, lauric acid was not added during the preparation process, and no phase transition temperature was detected in its final product, confirming that the introduction of lauric acid is the structural basis for this temperature-responsive characteristic.

[0049] In summary, this invention, through an ordered, low-temperature preparation process, particularly by constructing a specific ternary eutectic solvent as a functional carrier, not only yields a physicochemically stable topical formulation, but more importantly, pre-determines the thermosensitive phase transition physical property at the molecular structure level of the formulation. This property is determined by its specific component composition and molar ratio, and has been verified through rheological testing, providing a prerequisite for the subsequent controllable release and efficient penetration of active ingredients at specific temperatures.

[0050] Test Example 2: In vitro drug release test This test case aims to evaluate the in vitro release characteristics of the active ingredients in each formulation.

[0051] Experimental instruments and reagents: TK-12C Transdermal Diffusion Tester High-performance liquid chromatography (HPLC) equipped with a UV detector Phosphate-buffered saline (PBS, pH 7.4) Dialysis membrane (molecular weight cutoff 8000-14000 Da) Ferulic acid reference standard Experimental steps: Preparation: Soak the dialysis membrane in purified water for 24 hours and cut it to the appropriate size before use. Fill the receiving cell of the Franz diffusion cell with 12.0 mL of preheated PBS solution (pH 7.4) at 37°C and place it in a constant temperature water bath at 32±0.5°C. Turn on the magnetic stirrer at 100 rpm.

[0052] Sample loading: Fix the prepared dialysis membrane between the supply and receiving cells of the diffusion cell. Accurately weigh 1.0 g of each sample from Examples 1-3 and Comparative Examples 1-3, and evenly coat them onto the dialysis membrane.

[0053] Sampling and Analysis: At time points of 2, 4, 8, 12, and 24 hours, 1.0 mL of the receiving solution was aspirated from the sampling port of the receiving cell, and immediately replenished with 1.0 mL of blank PBS solution at the same temperature. The samples were filtered through a 0.45 μm microporous membrane, and the concentration of ferulic acid was determined by HPLC.

[0054] Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol: 0.5% acetic acid aqueous solution (volume ratio 40:60); flow rate: 1.0 mL / min; detection wavelength: 321 nm; column temperature: 30 ℃.

[0055] Calculation: Calculate the cumulative release percentage (Qn%) at each time point based on the measured concentration.

[0056] Table 2. In vitro cumulative release results of each example and comparative formulation (n=3, %)

[0057] As shown in Table 2, the in vitro release data of the samples prepared in Examples 1-3 all exhibited significant sustained-release characteristics. Within 24 hours, the cumulative release of the indicative component, ferulic acid, ranged from 78% to 85%, and the release process was stable. This result is attributed to the drug delivery system constructed using the method of this invention. At an experimental temperature of 32°C, the formulation underwent a physical phase transition, forming a semi-solid gel reservoir structure. The increased matrix viscosity slowed the diffusion rate of the internal active ingredient into the release medium, thereby achieving sustained release over a long period.

[0058] In contrast, Comparative Example 1, prepared using a traditional process, exhibited uneven dispersion and difficult dissolution of the active ingredient in the gel matrix, resulting in a cumulative release of only 26.3% over 24 hours, demonstrating extremely low bioavailability. Comparative Example 2, using a conventional glycerol-based eutectic solvent, improved drug dissolution, but the cumulative release exceeded 86% within 12 hours, indicating rapid release and a lack of effective controlled-release mechanism. This demonstrates that the solvent system constructed in this invention, with ferulic acid as the structural unit, possesses unique advantages in drug release control.

[0059] The results of Comparative Example 3 further confirmed the intrinsic mechanism of the technical solution of the present invention. This comparative example did not add lauric acid during solvent preparation, and its final formulation showed a cumulative release of over 91% within 12 hours, exhibiting a rapid and almost uncontrolled release pattern. This phenomenon confirms the crucial function of the thermosensitive phase-change hydrogen bond donor lauric acid in the system. It is precisely the presence of lauric acid that enables the hydrogen bond network of the entire solvent system to acquire temperature responsiveness, allowing for structural rearrangement at specific temperatures to form a semi-solid matrix with sustained-release function. Otherwise, the system would be unable to form an effective drug reservoir, leading to the rapid release of the active ingredient.

[0060] Test Example 3: In vitro transdermal absorption test This test case aims to evaluate the ability of each formulation to promote the penetration of the active ingredient into the skin's stratum corneum barrier.

[0061] Experimental instruments and materials: TK-12C Transdermal Diffusion Tester High-performance liquid chromatography (HPLC) equipped with a UV detector Ex vivo full-thickness abdominal skin of Kunming mice Phosphate-buffered saline (PBS, pH 7.4) Ferulic acid reference standard Experimental steps: Skin preparation: Healthy Kunming mice were euthanized by cervical dislocation. Full-thickness abdominal skin was harvested, and subcutaneous fat and connective tissue were carefully removed with surgical scissors. The skin was rinsed thoroughly with physiological saline and cut into pieces with an effective area of ​​3.14 cm². 2 Prepare the round pieces for later use.

[0062] Experimental setup: Prepared mouse skin was fixed between the supply and receiving cells of a Franz diffusion cell, with the stratum corneum facing the supply cell and the dermis facing the receiving cell. 12.0 mL of preheated PBS solution to 37°C was added to the receiving cell, and the entire diffusion cell was placed in a constant-temperature circulating water bath at 37±0.5°C. Magnetic stirring was turned on at 100 rpm to maintain the skin surface temperature at 32±1°C.

[0063] Sample application: Accurately weigh 0.5g of each of the samples from Examples 1-3 and Comparative Examples 1-3, and evenly apply them to the surface of the stratum corneum of the skin in the supply pool.

[0064] Sample collection and analysis: At time points of 2, 4, 8, 12, and 24 hours, 0.5 mL of receiving liquid was aspirated from the sampling port of the receiving cell, and immediately replenished with 0.5 mL of blank PBS solution at the same temperature. After filtration through a 0.45 μm microporous membrane, the concentration of ferulic acid in the collected samples was determined using the HPLC method established in Test Example 2.

[0065] Data Calculation: Based on the measured concentration data, calculate the cumulative permeability (Q) of ferulic acid per unit area for each formulation over 24 hours. 24 μg / cm 2） .

[0066] Table 3. Cumulative in vitro permeation results of each example and comparative formulation (n=3, μg / cm³) 2 )

[0067] As shown in Table 3, the in vitro transdermal experimental data of the samples prepared by the method of this invention in Examples 1-3 all showed higher cumulative permeation per unit area over 24 hours than all comparative samples. This result indicates that the formulation obtained by the preparation method described in this invention can efficiently deliver the active ingredient across the skin barrier. The underlying mechanism of this phenomenon lies in the fact that the thermosensitive bioactive ternary eutectic solvent, as a whole, interacts with the stratum corneum of the skin. Its component, lauric acid, can reversibly disturb the orderly arrangement of lipids in the stratum corneum, forming temporary microchannels. Meanwhile, the high drug loading capacity of the solvent system itself maintains a long-term, high-concentration drug concentration gradient on the skin surface, jointly driving the efficient penetration of the active ingredient.

[0068] Comparative Example 1, using a traditional process, showed the lowest cumulative penetration amount, as the active ingredient was difficult to release effectively from the matrix and penetrate the skin. Comparative Example 2, using a conventional glycerol-based eutectic solvent, achieved a slightly increased penetration amount compared to Comparative Example 1, but it was still far lower than that of Examples 1-3. This indicates that the solvent system constructed in this invention, with ferulic acid as the structural unit, possesses a structural functional advantage in promoting transdermal absorption. This structure allows the active ingredient to achieve a high level of local thermodynamic activity, which is not found in conventional solvent systems.

[0069] In Comparative Example 3, lauric acid was not used in its preparation. Although its permeation was better than that of Comparative Example 2, it was still lower than that of Examples 1-3. This result directly confirms the crucial role of the thermosensitive phase transition property in promoting transdermal absorption. Without lauric acid as a hydrogen bond donor for the thermosensitive phase transition, the formulation cannot undergo a physical phase transition from a low-viscosity liquid to a semi-solid structure on the skin surface, failing to form an effective drug reservoir. This results in a shortened effective contact time between the drug and the skin, limiting the total permeation. Therefore, the synergistic effect of thermosensitive phase transition behavior and high bioactive loading achieved by the present invention through the selection of specific components and the design of molar ratios forms the structural and functional basis for obtaining high transdermal absorption efficiency.

[0070] Test Example 4: Skin Irritation Test This test case aims to evaluate the skin compatibility and safety of each formulation.

[0071] Laboratory animals and materials: Healthy New Zealand White rabbits weigh 2.0-2.5kg, with an equal number of males and females.

[0072] Samples prepared in Examples 1-3 and Comparative Examples 1-3.

[0073] Negative control: physiological saline.

[0074] Positive control: 20% sodium dodecyl sulfate (SDS) aqueous solution.

[0075] Experimental steps: Animal preparation: 24 hours before the experiment, approximately 100 cm of the spine was removed from both sides of the rabbit's back using electric clippers. Within the area covered by fur, be careful to avoid damaging the skin.

[0076] Sample application: Divide the hair removal area into several sections. The test area. Accurately weigh 0.5g of each example, comparative example, and control group sample, spread evenly in the corresponding test area, cover with two layers of gauze and one layer of cellophane, and then fix with non-porous tape.

[0077] Observation and Scoring: After 24 hours of application, the residual sample was removed by washing with warm water. At 1, 24, 48, and 72 hours after sample removal, the erythema and edema of the skin in each test area were observed and recorded, and scored according to the following criteria: Erythema scoring criteria: 0 points (no erythema), 1 point (mild erythema), 2 points (obvious erythema), 3 points (moderate to severe erythema), 4 points (severe erythema, deep red with eschar formation).

[0078] Edema scoring criteria: 0 points (no edema), 1 point (mild edema), 2 points (significant edema), 3 points (moderate edema), 4 points (severe edema, with swelling exceeding 1 mm and extending to the surrounding area).

[0079] Data calculation: The average erythema score and average edema score of each sample at all observation time points were calculated, and the two were added together to obtain the single stimulus intensity index of the sample.

[0080] Table 4. Skin irritation test results of each example and comparative formulation.

[0081] According to the skin irritation test data in Table 4, the single-use irritation intensity index of samples 1-3 was all below 0.5, falling into the non-irritating category. The irritation intensity index of all comparative samples was higher than that of the example samples. This result indicates that the topical formulations obtained by the preparation method described in this invention have better skin compatibility than formulations prepared using conventional processes or other solvent systems.

[0082] The low irritation of the formulation of this invention is primarily attributed to its unique material basis and preparation process. Compared to the conventional hot reflux extraction process used in Comparative Example 1, the supercritical CO2 fluid extraction used in steps S1 and S2 of this invention is conducted under conditions free from the risk of organic solvent residue, and the entire process avoids high-temperature treatment, reducing the generation and dissolution of potentially irritating impurities in the medicinal materials. Furthermore, the thermosensitive bioactive ternary eutectic solvent constructed in this invention comprises ferulic acid, lauric acid, and choline chloride, all of which are highly biocompatible materials. Compared to Comparative Example 2, which uses conventional chemical solvents, this fundamentally reduces the irritation risk of the carrier itself.

[0083] The thermosensitive bioactive ternary eutectic solvent, as a homogeneous and stable liquid-phase system, can disperse and encapsulate multiple components in the synergistic active ingredient extract A' at the molecular level in step S4. This encapsulation effect allows the final formulation to slow down the direct and rapid penetration of some potentially irritating active ingredients into the stratum corneum upon skin contact, thereby reducing the intensity of the irritation reaction. Comparative Example 3, which did not use lauric acid, had a solvent system with lower structural integrity and stability than the ternary system, and therefore its irritation index was higher than that of Examples 1-3. This indicates that a structurally complete and stable ternary solvent system provides structural support for mitigating the overall irritation of the formulation.

Claims

1. A method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, characterized in that, Includes the following steps: S1. First, Angelica sinensis and Ligusticum chuanxiong are taken and extracted and purified using supercritical CO2 fluid extraction technology to obtain ferulic acid as the first hydrogen bond donor HBD1. The parameters of the supercritical CO2 fluid extraction process are: extraction pressure of 20-35 MPa and extraction temperature of 45-65℃. S2. Then, take safflower, frankincense, myrrh and Panax notoginseng, extract them in an extraction vessel by supercritical CO2 fluid extraction or subcritical fluid extraction, and then concentrate them in a vacuum concentration vessel to prepare synergistic active ingredient extract A'. S3. The ferulic acid obtained in step S1, along with the pre-provided lauric acid and choline chloride, are mixed in a reaction flask and stirred at 75-95°C for 1-3 hours to obtain a thermosensitive bioactive ternary eutectic solvent. S4. The synergistic active ingredient extract A' obtained in step S2 is added in portions to the thermosensitive bioactive ternary eutectic solvent prepared in step S3, and stirred at 40-50°C to dissolve or disperse it to form a drug mother liquor. S5. In a homogenizing emulsification vessel, the drug stock solution prepared in step S4 is mixed with an aqueous dispersion of carbomer 940, followed by the addition of triethanolamine to neutralize and form a gel. Finally, after homogenization, the topical preparation for joint pain of traditional Chinese medicine is obtained.

2. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... In step S1, the Angelica sinensis, Ligusticum chuanxiong, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, and Panax notoginseng are all pulverized into powder to a fineness of 40-60 mesh. Furthermore, in the extraction and purification of ferulic acid, the supercritical CO2 fluid extraction process further includes using anhydrous ethanol as an entrainer, wherein the amount of the entrainer is 5-15% (v / v) of the CO2 flow rate.

3. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... In step S3, the mixing and stirring reactions are carried out under nitrogen protection, and the stirring speed is 200-400 rpm until a clear and homogeneous liquid phase is formed in the system.

4. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... In step S4, the synergistic active ingredient extract A' is added in portions to the temperature-sensitive bioactive ternary eutectic solvent, and stirred continuously at a speed of 300-500 rpm for 30-60 minutes.

5. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... In step S5, the specific implementation method for forming the topical Chinese medicine preparation for joint pain is as follows: First, carbomer 940 is dispersed in purified water and allowed to stand and swell for 4-6 hours to form an aqueous dispersion of carbomer 940. The drug stock solution is then pumped in and mixed evenly with the aqueous dispersion of Carbomer 940; Triethanolamine was then added dropwise to neutralize the solution until the pH reached 5.5-7.0, forming a gel. Finally, the traditional Chinese medicine external preparation for joint pain is prepared by homogenizing under vacuum conditions at a speed of 1000-2000 rpm for 5-10 minutes.

6. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... The lauric acid is used as the thermosensitive phase transition hydrogen bond donor HBD2, the choline chloride is used as the hydrogen bond acceptor HBA, and the molar ratio of ferulic acid, lauric acid and choline chloride is (0.4-0.6):(0.4-0.6):(0.9-1.1).

7. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that... In step S1, the mass ratio of Angelica sinensis to Ligusticum chuanxiong is 1:1; Furthermore, in step S2, the mass ratio of safflower, frankincense, myrrh, and Panax notoginseng is 1:1:1:

1.

8. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that, In step S4, the synergistic active ingredient extract A' is added in equal amounts 2-4 times.

9. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that, In step S5, the mass ratio of the drug mother liquor, the aqueous dispersion of carbomer 940, and triethanolamine is (10-30):(0.8-1.5):(0.8-1.5).

10. The method for preparing a topical Chinese medicine preparation for joint pain based on promoting blood circulation and removing blood stasis, as described in claim 1, is characterized in that, In step S2, the vacuum concentration is carried out under a vacuum pressure of -0.08 MPa to -0.095 MPa.

Images