Glycyrrhizic acid astragaloside composite hydrogel and application thereof

Abstract

The invention relates to glycyrrhizic acid astragaloside composite hydrogel, and belongs to the technical field of medical materials. The invention aims to solve the problems that astragaloside is poor in water solubility, and hydrogel prepared by a traditional method is uneven in drug entrapment and insufficient in healing promoting performance. According to the technical scheme, the preparation method is characterized in that glycyrrhizic acid is subjected to ultrasonic pretreatment to form nano-micelles, astragaloside is entrapped forcibly through high-pressure homogenization, and finally the composite hydrogel with a compact structure is obtained through programmed cooling. The glycyrrhizic acid astragaloside composite hydrogel prepared by the invention is mainly used for preparing dressings or medicines for promoting skin wound healing, and is particularly suitable for treating diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology. More specifically, this invention relates to a glycyrrhizic acid-astragaloside A composite hydrogel and its applications. Background Technology

[0002] Diabetic ulcerative wounds are a common complication of diabetes, and their healing process is often affected by factors such as persistent inflammation, oxidative stress, and impaired cell migration. Traditional Chinese medicine active ingredients, such as astragaloside A, have effects such as promoting cell migration and regulating immune responses, showing good application potential in the treatment of chronic wounds. However, astragaloside A has poor water solubility, with a solubility of approximately 0.05 mg / mL in water. This physical property severely limits its bioavailability and clinical application. Direct application of astragaloside A powder or simple suspension is difficult to achieve an effective drug concentration at the wound site, and it is easily cleared by body fluids, failing to exert a sustained effect.

[0003] To address its solubility issue, conventional methods attempt to physically mix astragaloside A with various carrier materials to prepare gels or dressings. However, in practice, due to the strong hydrophobicity of astragaloside A, its molecules readily aggregate, making uniform dispersion within the carrier difficult. This inhomogeneity leads to uneven drug distribution in the prepared product, with local concentrations being either too high or too low, affecting the stability and reproducibility of therapeutic efficacy. Furthermore, simple physical mixing typically relies on the spontaneous swelling or encapsulation of the carrier, which generally has low encapsulation efficiency for hydrophobic drugs. A large amount of drug may remain outside the gel network without being effectively encapsulated, leading to rapid release during use and failing to achieve long-term sustained release.

[0004] Therefore, developing a hydrogel formulation that can effectively overcome the hydrophobicity of astragaloside A, ensure its uniform dispersion at high concentrations and stable binding with carrier materials, thereby preparing a uniform structure and controllable drug release performance, is a practical and urgent technical problem in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a glycyrrhizic acid-astragaloside A composite hydrogel and its application, so as to at least solve the above-mentioned problems.

[0006] To achieve the objectives and other advantages of this invention, a glycyrrhizic acid-astragaloside composite hydrogel is provided, prepared from raw materials comprising the following components: glycyrrhizic acid and astragaloside, wherein the concentration of glycyrrhizic acid in the composite hydrogel is 10-30 mg / mL and the concentration of astragaloside is 0.1-2 mg / mL; the preparation method is as follows: S1, dissolving glycyrrhizic acid in preheated PBS buffer at 35-45℃, immediately placing it in an ultrasonic device with a frequency of 40-80 kHz, and treating it at 50-70℃ for 5-15 minutes to obtain a pre-assembled glycyrrhizic acid nanomicelle dispersion; S2, mixing astragaloside powder with the pre-assembled glycyrrhizic acid nanomicelle dispersion obtained in step S1, and then immediately transferring it to a high-pressure homogenizer, and homogenizing it at 100-500... The mixture was cyclically treated 1-5 times under bar pressure to obtain a highly homogeneous mixed solution; S3, the mixed solution was heated in a constant temperature water bath at 65±2℃ for 10 minutes, and then transferred to a programmed cooling device to be slowly cooled to 25℃ at a rate of 0.5-1.5℃ / min to obtain glycyrrhizic acid astragaloside A composite hydrogel.

[0007] Preferably, the concentration of glycyrrhizic acid in the composite hydrogel is 20 mg / mL and the concentration of astragaloside A is 1 mg / mL.

[0008] Preferably, the frequency of the ultrasonic treatment in step S1 is 50 kHz and the treatment temperature is 60°C.

[0009] Preferably, the pressure of the high-pressure homogenization in step S2 is 200 bar, and the process is repeated 3 times.

[0010] Preferably, the cooling rate in step S3 is 1.0°C / minute.

[0011] The present invention also provides the application of the above-mentioned glycyrrhizic acid-astragaloside A composite hydrogel in the preparation of dressings or drugs for promoting skin wound healing.

[0012] Preferably, the skin wound is a diabetic ulcer wound.

[0013] The present invention has at least the following beneficial effects: First, the extreme shear force generated by high-pressure homogenization forces and uniformly encapsulates the hydrophobic astragaloside A molecules within the hydrophobic core of glycyrrhizic acid pre-assembled nanomicelles, forming a stable complex. This fundamentally solves the problem that astragaloside A is difficult to apply to water-based formulations due to its extremely low solubility (0.05 mg / mL), and effectively prevents the aggregation and precipitation of drug molecules, significantly improving the solubility and stability of astragaloside A.

[0014] Secondly, the synergistic effect of ultrasonic pretreatment and high-pressure homogenization ensures that astragaloside A is highly uniformly distributed in the glycyrrhizic acid matrix, avoiding drug agglomeration or uneven distribution that may occur with traditional stirring and mixing, thus ensuring consistency between product batches and reliability of efficacy.

[0015] Third, by using a specific cooling process, the molecular chains are arranged and cross-linked in an orderly manner, ultimately forming a three-dimensional network structure of hydrogel with a dense structure and mechanical strength (such as storage modulus) that is significantly higher than that of hydrogels prepared by conventional rapid cooling methods. It also has good thixotropy (shear thinning) and self-healing ability, and can closely adhere to the wound.

[0016] Fourth, the composite hydrogel prepared by the method of the present invention not only retains the inherent anti-inflammatory and antioxidant activities of glycyrrhizic acid, but also, through efficient encapsulation and uniform dispersion, allows the functions of astragaloside A in promoting cell migration, anti-inflammation and regulating macrophage polarization to be fully exerted. The two produce a synergistic effect in the wound microenvironment, jointly accelerating the healing process of chronic wounds.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is a scanning electron microscope image of the lyophilized products of glycyrrhizic acid astragaloside A composite hydrogel GAS and glycyrrhizic acid hydrogel GA prepared in this invention. Figure 2 The infrared spectra of the glycyrrhizic acid-astragaloside A composite hydrogel GAS, glycyrrhizic acid hydrogel GA, and astragaloside A aqueous solution AS prepared in this invention are shown below. Figure 3 The rheological strain diagrams of the glycyrrhizic acid-astragaloside A composite hydrogel GAS and glycyrrhizic acid hydrogel GA prepared in this invention are shown. Figure 4 This is a rheological frequency scan of the glycyrrhizic acid-astragaloside A composite hydrogel GAS and glycyrrhizic acid hydrogel GA prepared in this invention. Figure 5 The figure shows the biocompatibility test results of the glycyrrhizic acid-astragaloside A composite hydrogel GAS and glycyrrhizic acid hydrogel GA prepared in this invention. Figure 6 The figure shows the cytotoxicity test results of the glycyrrhizic acid-astragaloside A composite hydrogel GAS and glycyrrhizic acid hydrogel GA prepared in this invention. Figure 7 These are wound images on day 0 and day 15 of the glycyrrhizic acid-astragaloside A composite hydrogel GAS, glycyrrhizic acid hydrogel GA, and astragaloside A aqueous solution AS prepared in this invention in an experiment promoting the healing of diabetic ulcer wounds. Figure 8 This is a statistical result of the wound healing area during an experiment on promoting the healing of diabetic ulcers using the glycyrrhizic acid-astragaloside A composite hydrogel GAS, glycyrrhizic acid hydrogel GA, and astragaloside A aqueous solution AS prepared in this invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0022] Example 1: 20 mg of glycyrrhizic acid was dissolved in 1 mL of PBS buffer (10 mM, pH 7.4) preheated to 40°C. The solution was immediately placed in an ultrasonic device at 50 kHz and treated in a 60°C water bath for 10 minutes to obtain a pre-assembled glycyrrhizic acid nanomicelle dispersion. Subsequently, 1 mg of astragaloside A powder was added, and the mixture was gently vortexed. The mixture was then immediately transferred to a high-pressure homogenizer and cyclically treated three times at 200 bar. The homogenized solution was heated in a 65°C constant-temperature water bath for 10 minutes, and then transferred to a temperature-programmed cooling device to slowly cool to 25°C at a rate of 1.0°C / min, forming a dense glycyrrhizic acid-astragaloside A composite hydrogel, labeled GAS.

[0023] Example 2: Based on Example 1, the homogenization pressure was adjusted to 50 bar, while the other treatments remained unchanged. The resulting glycyrrhizic acid-astragaloside A composite hydrogel was labeled GAS1.

[0024] Example 3: Based on Example 1, after adding 1 mg of astragaloside A powder and gently vortexing, the mixture was placed directly in a 65°C constant temperature water bath without high-pressure homogenization. All other treatments remained unchanged, and the resulting sample was labeled GAS2. Observation revealed that the sample contained microscopic astragaloside A particles suspended in the air. After standing for 24 hours, these particles gradually precipitated and failed to form a homogeneous gel.

[0025] Example 4: Weigh 20 mg of glycyrrhizic acid and 1 mg of astragaloside A powder, add them to 1 mL of PBS buffer (10 mM, pH 7.4), mix with magnetic stirring (500 rpm, 60 minutes), heat the mixture in a 65°C constant temperature water bath for 10 minutes, and then let it stand at room temperature (about 25°C) for 30 minutes to allow it to cool naturally into a gel. The resulting glycyrrhizic acid-astragaloside A composite hydrogel is labeled GAS3.

[0026] Example 5: Weigh 20 mg of glycyrrhizic acid and add it to 1 mL of PBS buffer (10 mM, pH 7.4). After stirring evenly, heat the mixture in a 65°C constant temperature water bath for 10 minutes, and then allow it to cool naturally at 25°C room temperature. The resulting glycyrrhizic acid hydrogel is labeled as GA.

[0027] Experimental Example 1: Particle Size Analysis.

[0028] The particle size distribution of astragaloside A in glycyrrhizic acid-astragaloside A composite hydrogel was determined using a laser particle size analyzer. Before the test, 1g of samples of GAS, GAS1 and GAS3 were weighed, diluted 10 times with ultrapure water, and then placed in an ultrasonic cleaner and ultrasonically treated at 300W for 10 minutes. The results showed that the Z-average particle size of GAS was 152.3 ± 3.5 nm, and the polydispersity index (PDI) was 0.121 ± 0.015, indicating a highly uniform drug distribution. The Z-average particle size of GAS1 was 285.7 ± 25.6 nm, and the PDI was 0.283 ± 0.041, indicating that the lower homogenization pressure could not provide sufficient shear force to fully break down the astragaloside A aggregates, resulting in larger particle size and wider distribution. The Z-average particle size of GAS3 was 306.4 ± 31.7 nm, and the PDI was 0.312 ± 0.089, indicating that the traditional heating and cooling methods could not fully break down the astragaloside A aggregates, and compared with GAS1, GAS3 had a larger Z-average particle size and wider distribution.

[0029] Experimental Example 2: Encapsulation efficiency determination.

[0030] The encapsulation efficiency of astragaloside A in glycyrrhizic acid-astragaloside composite hydrogels was determined using ultracentrifugation combined with high-performance liquid chromatography (HPLC). Five g samples of GAS, GAS1, and GAS3 were weighed and placed in pre-weighed centrifuge tubes. The tubes were then centrifuged at 4°C and a relative centrifugal force of 15,000 × g for 60 minutes. This high-speed centrifugation process aimed to completely precipitate free astragaloside A that was not successfully encapsulated within the hydrophobic core of the glycyrrhizic acid nanomicelles, while retaining the intact micelle structure with encapsulated drug in the supernatant. After centrifugation, 500 μL of the supernatant was carefully transferred to a new centrifuge tube. This supernatant contained successfully encapsulated glycyrrhizic acid micelles, free glycyrrhizic acid molecules, and buffer solution. Add twice the volume of organic solvent (such as acetonitrile or methanol) to the supernatant sample and vortex for 10 minutes to ensure complete micelle dissociation and full release of astragaloside A into the solution. Then, filter the mixture through a 0.22 μm microporous membrane and collect the filtrate. Quantitative analysis of the astragaloside A content in the obtained filtrate was performed using a pre-validated HPLC method to calculate the mass of encapsulated astragaloside A in the supernatant sample. Typical chromatographic conditions included: a C18 reversed-phase column with gradient elution of acetonitrile and water as the mobile phase, a column temperature of 30°C, a flow rate of 1.0 mL / min, and a detection wavelength of 200 nm.

[0031] To calculate the encapsulation efficiency, the total dosage of the drug is also required. Accurately weigh an equal amount of astragaloside A reference standard used in the preparation of the 5g GAS hydrogel sample, dissolve it directly in an appropriate amount of acetonitrile-water (e.g., a volume ratio of 70:30) mixed solvent, vortex to ensure complete dissolution, and filter through a 0.22 μm filter membrane. Detect the solution using the same HPLC conditions, calculate the concentration based on the standard curve, and then extrapolate the total dosage.

[0032] The encapsulation rate is calculated as follows: Encapsulation rate (%) = (mass of encapsulated astragaloside A / total dosage) × 100%.

[0033] The results showed that the encapsulation efficiency in GAS was 96.8 ± 1.2%, in GAS1 it was 78.5 ± 3.8%, and in GAS3 it was 68.3 ± 4.5%, indicating that the combination of "ultrasonic pretreatment" and "homogenization under specific high pressure" successfully increased the encapsulation efficiency of astragaloside A from 68.3% in the traditional method (Example 4) to 96.8%. This is likely because ultrasonic pretreatment provides a uniform pre-assembly template for glycyrrhizic acid molecules, while the 200 bar high-pressure homogenization step provides sufficient shear forces, impact forces, and cavitation effects to overcome the van der Waals forces between drug molecules. This powerful physical energy forces the hydrophobic astragaloside A molecules to be effectively "embedded" into the hydrophobic core of the glycyrrhizic acid micelles, achieving near-complete encapsulation. The high encapsulation efficiency means minimal drug waste, with the vast majority of the active ingredient being effectively utilized, indicating that subsequent drug release will be more controllable and prolonged, laying a solid foundation for excellent in vitro and in vivo efficacy.

[0034] Experimental Example 3: Scanning Electron Microscopy Detection.

[0035] Scanning electron microscopy was used to examine the lyophilized products of the glycyrrhizic acid-astragaloside A composite hydrogel GAS prepared in Example 1 and the glycyrrhizic acid hydrogel GA prepared in Example 5. Figure 1 As shown, glycyrrhizic acid hydrogel GA is a uniform nanocube, while glycyrrhizic acid astragaloside A hydrogel GAS exhibits a uniform nanosphere morphology, is smoother and denser with smaller interparticle gaps, indicating that astragaloside A has entered the hydrophobic inner cavity aggregate of glycyrrhizic acid, and the hydrophobic inner cavity of glycyrrhizic acid can encapsulate astragaloside A.

[0036] Experimental Example 4: Fourier Transform Infrared Spectroscopy Detection.

[0037] Fourier transform infrared spectroscopy was used to analyze the chemical structures of glycyrrhizic acid hydrogel GA prepared in Example 5, astragaloside A aqueous solution AS with a concentration of 1 mg / mL, and glycyrrhizic acid-astragaloside A composite hydrogel GAS prepared in Example 1, in the range of 400-4000 cm⁻¹. -1 Scanning is performed within the wavelength range. For example... Figure 2 As shown, glycyrrhizic acid (GA) at 3240 cm⁻¹ -1 The position represents a typical stretching vibration of the hydroxyl (-OH) group, at 1657 cm⁻¹. -1 The stretching vibration of the glucuronic acid carbonyl (-C=O) group occurs at 1422 cm⁻¹. -1 The peak at [location] corresponds to the carboxyl (-COO) group in glycyrrhizic acid (GA) glucuronic acid. Astragaloside A (AS) at 1050 cm⁻¹... -1 A strong absorption peak is observed at 1366 cm⁻¹, representing the CO stretching vibration of the glycosidic bond in astragaloside IV. The glycyrrhizic acid-astragaloside IV composite hydrogel GAS, formed after the reaction of astragaloside IV with glycyrrhizic acid, exhibits this peak. -1The peak at that point disappeared, suggesting that it may have undergone intermolecular interaction with glycyrrhizic acid through hydrogen bonds.

[0038] Experimental Example 5: Rheological Performance Test.

[0039] The rheological properties of the glycyrrhizic acid-astragaloside A composite hydrogel GAS prepared in Example 1 and the glycyrrhizic acid hydrogel GA prepared in Example 5 were tested using a rheometer. Figure 3 The rheological diagram of the hydrogel is shown, where G′ is the storage modulus and G′′ is the loss modulus. Within the linear viscoelastic region, G′ is consistently higher than G′′, indicating that the hydrogel is a solid-like elastic material. The intersection of G′ and G′′ indicates that at strain greater than 5%, the hydrogel's cross-linked network breaks down, resulting in a gel-sol transition. This demonstrates that the GAS hydrogel exhibits shear-thinning properties, facilitating injection administration when applied to wounds. The strain-frequency scan plot shows that the hydrogel possesses good stability, maintaining its gel morphology within a certain frequency range. Figure 4 ).

[0040] Experiment Example 6: Biocompatibility Test.

[0041] Blood compatibility assay: Red blood cells were obtained by centrifugation after blood collection, and the red blood cell suspension was diluted to 5% with physiological saline. Hydrogel extracts were prepared using the glycyrrhizic acid-astragaloside A composite hydrogel GAS prepared in Example 1 and the glycyrrhizic acid hydrogel GA prepared in Example 5, respectively, at a ratio of 1 g gel to 10 mL of physiological saline. 500 μL of the extract and 500 μL of 5% red blood cell suspension were gently mixed and incubated at 37°C for 4 hours, with the sample inverted twice every hour. After 4 hours, the sample was centrifuged to obtain the supernatant, and its absorbance at 540 nm was measured using an ELISA reader. The positive control group was 0.1% Triton X-100, and the negative control was physiological saline. The hemolysis rate was calculated using the following formula: Wherein, ODs, ODn, and ODp represent the hydrogel sample group, negative control group, and positive control group, respectively. The hemolysis rate of the GAS hydrogel was measured to be 0.143±0.046, less than 5%, which meets the international standards for biomaterials. Figure 5 ).

[0042] Experiment Example 7: Cytotoxicity Test.

[0043] The hydrogel extract prepared in Example 4 was sterilized by ultraviolet light and then filtered through a filter membrane for sterilization before use. L929 cells were passaged into 96-well plates at a density of 5,000 cells / well, and live / dead staining was performed and photographed after 24h, 48h, and 72h of culture. Figure 6This is a staining image of live and dead cells. Green fluorescence indicates live cells, and red fluorescence indicates dead cells. The glycyrrhizic acid / astragaloside A (GAS) hydrogel extract showed that the cell survival rate was higher than that of the control group after 72 hours of culture.

[0044] Experiment Example 8: Assessment of promoting wound healing.

[0045] Establishment and treatment of a diabetic rat skin wound model: Male SD rats weighing 190-220g were selected. The rats were housed in a constant temperature environment of 23 ± 2℃ with a normal 12-hour diurnal cycle. All animal experiments were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine. Type I diabetes was induced by intraperitoneal injection of streptozotocin. A blood glucose level ≥ 16.8 mmol / L was considered a successful model of diabetes. Two 15 mm diameter wounds were created on the back of the rats. 50% glacial acetic acid was applied to the wounds twice daily for 3 days, successfully creating diabetic ulcer wounds. Dressings were changed and photographs were taken daily, and the wound healing area was quantitatively analyzed. The control group received PBS solution, the GA group received glycyrrhizic acid hydrogel prepared in Example 5, the GAS group received glycyrrhizic acid-astragaloside composite hydrogel prepared in Example 1, and the AS group received an aqueous solution of 1 mg / mL astragaloside. The wound healing status of each group over time is shown in the figure. Figure 7 The results showed that on day 15 of treatment, the wound healing areas in the control group, GA, AS, and GAS groups were 89.78 ± 1.61%, 90.68 ± 1.02%, 89.83 ± 1.61%, and 95.68 ± 2.15%, respectively. Figure 8 The results indicate that GAS has a good ability to promote rapid wound healing. Compared with the GA group, the healing rate of the AS aqueous solution group was lower throughout the treatment cycle. This may be because the AS aqueous solution has low solubility, resulting in low bioavailability and difficulty in exerting its pharmacological activity. GAS hydrogel can increase the solubility of AS, improve its bioavailability, and better exert its anti-inflammatory and migration-promoting effects.

[0046] The number of devices and processing scale described herein are for simplification of the invention. Applications, modifications, and variations of the glycyrrhizic acid-astragaloside A composite hydrogel and its applications will be readily apparent to those skilled in the art.

[0047] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A glycyrrhizic acid-astragaloside A composite hydrogel, characterized in that, It is prepared from raw materials including glycyrrhizic acid and astragaloside A, wherein the concentration of glycyrrhizic acid in the composite hydrogel is 10-30 mg / mL and the concentration of astragaloside A is 0.1-2 mg / mL; The preparation method is as follows: S1. Dissolve glycyrrhizic acid in preheated PBS buffer at 35-45℃, and immediately place it in an ultrasonic device with a frequency of 40-80 kHz. Treat it at 50-70℃ for 5-15 minutes to obtain a pre-assembled glycyrrhizic acid nanomicelle dispersion. S2. Mix the astragaloside A powder with the pre-assembled glycyrrhizic acid nanomicelle dispersion obtained in step S1, and then immediately transfer it to a high-pressure homogenizer and cycle it 1-5 times under a pressure of 100-500 bar to obtain a highly homogeneous mixed solution. S3. The mixed solution is heated in a constant temperature water bath at 65±2℃ for 10 minutes, and then transferred to a programmed cooling device to slowly cool to 25℃ at a rate of 0.5-1.5℃ / min to obtain glycyrrhizic acid astragaloside A composite hydrogel.

2. The glycyrrhizic acid-astragaloside A composite hydrogel as described in claim 1, characterized in that, The concentration of glycyrrhizic acid in the composite hydrogel was 20 mg / mL, and the concentration of astragaloside A was 1 mg / mL.

3. The glycyrrhizic acid-astragaloside A composite hydrogel as described in claim 1, characterized in that, The ultrasonic treatment in step S1 has a frequency of 50 kHz and a treatment temperature of 60°C.

4. The glycyrrhizic acid-astragaloside A composite hydrogel as described in claim 1, characterized in that, The high-pressure homogenization in step S2 is performed at a pressure of 200 bar, and the process is repeated 3 times.

5. The glycyrrhizic acid-astragaloside A composite hydrogel as described in claim 1, characterized in that, The cooling rate described in step S3 is 1.0℃ / minute.

6. The use of the glycyrrhizic acid-astragaloside A composite hydrogel as described in any one of claims 1-5 in the preparation of dressings or medicines for promoting skin wound healing.

7. The application as described in claim 6, characterized in that, The skin wound is a diabetic ulcer.

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