A fully natural aggregation-induced emission licorice acid active hydrogel and a preparation method and application thereof

Abstract

The application discloses a kind of all natural aggregation-induced emission glycyrrhizic acid active hydrogel and its preparation method and application. Contain the following steps: 1) by stirring and dissolving glycyrrhizic acid or its derivative and berberine derivative at a certain temperature, obtain transparent aqueous solution, and adjust pH to neutral;2) glycyrrhizic acid or its derivative solution and berberine derivative solution are mixed according to certain proportion;3) the pH of composite solution is adjusted to acid, and it is stationary at room temperature, and all natural aggregation-induced emission glycyrrhizic acid active hydrogel is obtained by using the interaction between the two co-assemblies.It has excellent AIE characteristics, high mechanical strength, excellent fluorescent environmental responsiveness, excellent injectable and structure recovery characteristics, significant photodynamic synergistic antibacterial ability, excellent immunomodulatory and anti-inflammatory ability, can promote skin wound healing.The raw material of the hydrogel is natural, biocompatibility is high, preparation process is simple, condition is mild, easy to scale production, has potential application prospect in medical gel field.

Description

Technical Field

[0001] This invention belongs to the field of biomedical antibacterial and anti-inflammatory materials and gel materials, specifically relating to an all-natural aggregation-induced luminescence glycyrrhizic acid active hydrogel and its preparation method and application. Background Technology

[0002] Fluorescent hydrogels are a novel type of hydrogel that possesses not only traditional hydrogel properties (such as an internal gel network structure and a certain level of mechanical strength) but also photoluminescent properties. These hydrogels are playing an increasingly important role in drug delivery, wound dressings, and tissue engineering. However, due to the aggregation quenching (ACQ) effect that occurs when fluorophores are aggregated within the gel network, most fluorescent hydrogels currently exhibit weaker fluorescence than the fluorescent material alone, limiting their practical applications. In recent years, the discovery of aggregation-induced emission (AIE) has provided new insights into the preparation of fluorescent hydrogels with enhanced photoluminescence capabilities (i.e., AIE hydrogels). AIE hydrogels can be prepared by adding AIE fluorescent molecules to the gel matrix or by utilizing their covalent or non-covalent interactions with the gel building blocks. In AIE hydrogels, the AIE material is compatible with the semi-solid or condensed-state properties of the gel. The dense gel network structure restricts the intramolecular movement of AIE molecules, inhibiting energy dissipation through non-radiative transitions, ultimately leading to enhanced overall fluorescence properties in the hydrogel. Novel AIE hydrogels, especially those that generate reactive oxygen species (ROS) after short-term light exposure or exhibit high photothermal conversion efficiency, demonstrate stable and broad-spectrum photodynamic or photothermal antibacterial capabilities, attracting significant attention for treating bacterial wound infections. It is noteworthy that, to date, most AIE hydrogels are based on semi-synthetic or synthetic AIE-like substances combined with a hydrogel matrix, which presents a series of problems including high cost, complex synthesis processes, poor biocompatibility, and potential toxicity. Therefore, developing an all-natural, non-toxic AIE hydrogel is crucial for expanding its applications in food and biomedicine.

[0003] In recent years, the construction of co-assembled supramolecular hydrogels using small molecules, especially natural medicinal and edible small molecules with high bioactivity, biocompatibility, and biodegradability, has attracted widespread attention. To achieve precise control over the properties of small molecule hydrogels and synergistic effects, scientists have begun to utilize the co-assembly of small molecules to prepare small molecule-small molecule co-assembled hydrogels with various biological applications. Most notably, the assembly behavior between natural fluorescent small molecules can effectively prepare all-natural AIE hydrogels, which have broad potential applications in antibacterial, anti-inflammatory, and wound dressing fields.

[0004] Glycyrrhizic acid (GA) is a natural triterpenoid saponin with various biological activities. Its monomeric molecule is composed of the hydrophobic glycosidic aglycone 18β-glycyrrhetinic acid and the hydrophilic diglucuronic acid. Saha et al. reported the self-assembly behavior of glycyrrhizic acid in aqueous solution and found that under non-covalent bonding, chiral GA molecules exhibit unique self-assembly characteristics in aqueous solution. First, they form long nanofibers with a thickness of 2.5 nm, and then assemble into supramolecular hydrogels with a three-dimensional network structure (Saha A, Adamcik J, Bolisettty S, et al. Fibrillar networks of glycyrrhizic acid for hydrid nanomaterials with catalytic features[J]. Angewandte Chemie International Edition, 2015, 54(18): 5408-5412). However, GA hydrogels alone have disadvantages such as weak mechanical properties and limited functionality (e.g., no fluorescence), which seriously limits their application in antibacterial and wound dressing fields. To expand the application range of glycyrrhizic acid hydrogels, CN118340934A discloses a method for preparing self-adhesive glycyrrhizic acid hydrogels and their applications. This method involves preparing self-adhesive glycyrrhizic acid hydrogels by combining hydrophilic or water-soluble glycyrrhizic acid salts and their modifications, metal ions, aldehyde-modified polysaccharides, and acylhydrazides, hydroxylamines, or primary amine derivatives. The resulting hydrogels exhibit good biocompatibility, biofunctional activity, and biodegradability, promoting wound healing in biological tissues. However, the hydrogels prepared by this patent have complex compositions and lack sufficient antibacterial efficacy and unique functionalities (such as aggregation-induced emission properties). Therefore, it is necessary to explore new preparation strategies (such as utilizing the assembly behavior between glycyrrhizic acid or its derivatives and other natural small molecules) to construct AIE hydrogels for developing novel glycyrrhizic acid-based functional hydrogels with practical clinical application value. Summary of the Invention

[0005] The purpose of this invention is to address the problems of weak mechanical properties, poor functionality (such as fluorescence luminescence properties), and limited applications of existing glycyrrhizic acid hydrogels, while also solving the problems of poor biocompatibility and complex preparation processes of current AIE hydrogels. This invention provides a novel all-natural aggregation-induced emission (AIE) glycyrrhizic acid active hydrogel. Specifically, this AIE glycyrrhizic acid active hydrogel is a novel glycyrrhizic acid hydrogel possessing AIE properties, tunable environmentally responsive fluorescence, high mechanical strength, excellent injectability and structural recovery characteristics, significant traditional and photodynamic antibacterial abilities, excellent immunomodulatory and anti-inflammatory abilities, and wound-healing properties.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing an all-natural aggregation-induced luminescent glycyrrhizic acid active hydrogel includes the following steps:

[0008] (1) Dissolution: Glycyrrhizic acid or its derivatives are uniformly dispersed in water and stirred at a certain temperature to obtain a transparent solution of glycyrrhizic acid or its derivatives; berberine derivatives are uniformly dispersed in water and stirred at a certain temperature until the berberine derivatives are completely dissolved to obtain a berberine derivative solution.

[0009] (2) Combination: The pH of the glycyrrhizic acid or its derivative solution and the berberine derivative solution obtained in step (1) is adjusted to neutral by using an alkaline pH adjuster, and the two are mixed at room temperature in a certain proportion to obtain a composite solution;

[0010] (3) Preparation of hydrogel: The composite solution obtained in step (2) is stirred continuously, and the pH is adjusted back to acidic using an acidic pH adjuster until natural AIE glycyrrhizic acid hydrogel is obtained.

[0011] Preferably, in step (1), the glycyrrhizic acid or its derivatives include one of glycyrrhizic acid, ammonium glycyrrhizate, ammonium glycyrrhizate salt, monopotassium glycyrrhizate, dipotassium glycyrrhizate, and trisodium glycyrrhizate.

[0012] Preferably, in step (1), the berberine derivative includes one of berberine hydrochloride hydrate, berberine chloride hydrate, Coptis chinensis extract, berberine sulfate hydrate, berberine bisulfate, and berberine tannate.

[0013] Preferably, in step (1), the stirring temperature is 60-90℃, the stirring speed is 100-500rpm, and the stirring time is 5-15min.

[0014] Preferably, in step (1), the concentration of the glycyrrhizic acid or its derivative solution is 2%-6%; and the concentration of the berberine derivative solution is 0.5%-2%.

[0015] Preferably, in step (2), the alkaline pH adjuster is one of sodium hydroxide solution, potassium hydroxide solution, disodium hydrogen phosphate solution, and sodium bicarbonate; the concentration of the alkaline pH adjuster is 0.5-4 mM, and the pH of the glycyrrhizic acid or its derivative solution and the berberine derivative solution is adjusted to 6.0-8.0.

[0016] Preferably, in step (2), the combined concentration ratio of the glycyrrhizic acid or its derivative solution and the berberine derivative solution is 1000:1-4:1.

[0017] Preferably, in step (2), the concentration of glycyrrhizic acid or its derivative solution in the composite solution is 0.1%-1%.

[0018] Preferably, in step (2), the concentration of the berberine derivative solution in the composite solution is 0.001%-0.25%.

[0019] Preferably, in step (3), the stirring speed is 400-1000 rpm and the stirring time is 2-10 min.

[0020] Preferably, in step (3), the acidic pH adjuster includes one of hydrochloric acid, sulfuric acid, citric acid, tartaric acid, acetic acid, and gluconolactone, and the concentration of the acidic pH adjuster is 0.5-2mM to adjust the pH of the composite solution to 3.0-5.0.

[0021] The present invention provides a method for preparing an all-natural aggregation-induced luminescence glycyrrhizic acid active hydrogel.

[0022] Preferably, the all-natural aggregation-induced emission glycyrrhizic acid active hydrogel is a glycyrrhizic acid hydrogel with aggregation-induced emission properties, excellent mechanical and structural recovery properties, environmentally responsive fluorescence properties, photodynamic antibacterial and anti-inflammatory functions.

[0023] The present invention provides an all-natural aggregation-induced luminescence glycyrrhizic acid active hydrogel dressing that can be applied to drug carriers, antibacterial and anti-inflammatory materials, smart fluorescent materials or medical gel dressings.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial technical effects:

[0025] 1. This invention ingeniously utilizes the interaction between glycyrrhizic acid or its derivatives, a natural food and medicinal compound, and berberine derivatives to co-assemble a novel aggregation-induced emission (AIE) functional glycyrrhizic acid hydrogel. The inventors discovered that berberine molecules, which originally exhibit AIE effects only in organic solvents but not in aqueous solutions, can co-assemble into assemblies and hydrogels by combining glycyrrhizic acid or its derivatives with berberine derivatives, leveraging the interaction between the two. The formation of these assemblies effectively inhibits the movement of berberine molecules, prompting them to release energy via radiative transitions (light energy), ultimately endowing the hydrogel with excellent AIE properties.

[0026] 2. The raw materials used in this invention are all naturally derived small molecules that are both food and medicine, which are non-toxic and harmless, and have good biocompatibility and broad pharmacological activity.

[0027] 3. The preparation method of this invention is simple, rapid, and requires no additional gelling agents. It utilizes the co-assembly behavior of small molecules to form hydrogels. Specifically, glycyrrhizic acid or its derivatives are mixed with berberine derivatives under alkaline conditions, and an AIE glycyrrhizic acid hydrogel is prepared using pH adjustment technology. During pH adjustment, glycyrrhizic acid molecules assemble to form glycyrrhizic acid fibers, while simultaneously forming assemblies with berberine molecules through electrostatic interactions. The fiber network and assemblies in the system effectively prevent the movement of berberine molecules, promoting energy dissipation through radiative transitions (light energy), ultimately forming the AIE hydrogel.

[0028] 4. The hydrogel of the present invention exhibits unique aggregation-induced emission properties. Compared with glycyrrhizic acid or its derivative hydrogels alone or berberine aqueous solution, the hydrogel prepared by the present invention has stronger photoluminescence properties.

[0029] 5. The AIE hydrogel of the present invention has excellent injectability and structural recovery characteristics, as well as temperature and pH responsive fluorescence properties, and has great application prospects in biomedical smart fluorescent materials.

[0030] 6. The hydrogel of this invention exhibits sufficient mechanical properties and strong intrinsic biological activity. Compared with glycyrrhizic acid or its derivative hydrogels alone, or aqueous solutions of berberine derivatives, the hydrogel prepared by this invention exhibits superior antibacterial and anti-inflammatory effects, especially its ability to achieve photodynamic synergistic antibacterial and bactericidal effects, further enhancing the antibacterial and anti-inflammatory capabilities of the hydrogel.

[0031] 7. The hydrogel of the present invention has good biocompatibility, is easy to degrade, and has no cytotoxicity, and can be widely used in the field of biomedical materials.

[0032] 8. The AIE hydrogel in this invention can cover the wound surface, reduce the risk of infection during the wound healing process, improve the healing rate of infected wounds, and play a practical application and commercial value in the field of medical gel dressings. Attached Figure Description

[0033] Figure 1 The images show the appearance of the hydrogels in Comparative Examples 1 and 2 and Examples 2 and 3 of this invention.

[0034] Figure 2 This is the fluorescence emission spectrum of the AIE glycyrrhizic acid hydrogel in Example 1 of this invention.

[0035] Figure 3 The fluorescence emission spectrum of the berberine derivative solution in Comparative Example 3 of this invention is shown.

[0036] Figure 4 The images show the fluorescence emission spectra of the glycyrrhizic acid hydrogel in Comparative Example 1 and the AIE glycyrrhizic acid hydrogel in Example 2 of this invention.

[0037] Figure 5 The frequency scan diagrams are of the glycyrrhizic acid hydrogel in Comparative Example 2 and the AIE glycyrrhizic acid hydrogel in Example 3 of this invention.

[0038] Figure 6 Viscosity curves of the glycyrrhizic acid hydrogel in Comparative Example 2 and the AIE glycyrrhizic acid hydrogel in Example 3 of this invention.

[0039] Figure 7 The image shows the thixotropic recovery curve of the AIE glycyrrhizic acid hydrogel in Example 3 of this invention.

[0040] Figure 8 This is the fluorescence emission spectrum of the AIE glycyrrhizic acid hydrogel in Example 4 of this invention during the pH decrease process.

[0041] Figure 9 This is a graph showing the fluorescence intensity change of the AIE glycyrrhizic acid hydrogel in Example 5 of this invention during the cyclic heating-cooling process.

[0042] Figure 10 The bacterial survival rate was obtained by testing the antibacterial properties of glycyrrhizic acid hydrogel alone in Comparative Example 2 and AIE glycyrrhizic acid hydrogel in Example 3 against Staphylococcus aureus.

[0043] Figure 11 This invention relates to the effects of glycyrrhizic acid hydrogel alone (Comparative Example 2) and AIE glycyrrhizic acid hydrogel (Example 3) on TNF-α inflammatory factor levels.

[0044] Figure 12 This is a graph showing the changes in wound area of ​​MRSA-infected wounds treated with different hydrogels according to the present invention. Detailed Implementation

[0045] To better understand the present invention, the following description, in conjunction with the accompanying drawings and embodiments, will further illustrate the present invention, but the implementation of the present invention is not limited thereto.

[0046] Example 1

[0047] A fully natural aggregation-induced luminescence active glycyrrhizic acid hydrogel, the preparation method of which includes the following steps:

[0048] (1) Add ammonium glycyrrhizate powder to deionized water and stir for 5 minutes at a stirring speed of 100 rpm and a temperature of 90°C until a clear and transparent ammonium glycyrrhizate solution (6%) is obtained. At the same time, berberine hydrochloride hydrate is uniformly dispersed in water and stirred for 5 minutes at a stirring speed of 200 rpm and a temperature of 90°C to obtain a completely dissolved berberine hydrochloride hydrate solution (2%).

[0049] (2) Adjust the pH of the ammonium glycyrrhizate solution and the berberine hydrochloride hydrate solution obtained in step (1) to about 7.0 with potassium hydroxide (1mM). Then mix the ammonium glycyrrhizate solution and the berberine hydrochloride hydrate solution at 25°C at concentration ratios of 100:1, 50:1, 25:1 and 10:1, so that the concentrations of the ammonium glycyrrhizate solution in the system are 0.1%, 0.25%, 0.5% and 1%, and the concentration of the berberine solution is 0.01%.

[0050] (3) The composite solutions with different concentrations of ammonium glycyrrhizate solution obtained in step (2) were stirred at 1000 rpm, and the pH of all samples was adjusted to 4.0 with 0.5 mM hydrochloric acid. After stirring for 2 min, the samples were allowed to stand to obtain natural AIE glycyrrhizic acid hydrogels, which were denoted as hydrogel 1, hydrogel 2, hydrogel 3 and hydrogel 4 respectively.

[0051] Example 2

[0052] A fully natural aggregation-induced luminescent glycyrrhizic acid active hydrogel, the preparation method of which includes the following steps:

[0053] (1) Add dipotassium glycyrrhizate powder to deionized water and stir for 15 minutes at a stirring speed of 500 rpm and a temperature of 60°C until a clear and transparent dipotassium glycyrrhizate solution (2%) is obtained. At the same time, uniformly disperse berberine sulfate hydrate in deionized water and stir for 15 minutes at a stirring speed of 500 rpm and a temperature of 60°C to obtain a completely dissolved berberine sulfate hydrate solution (0.5%).

[0054] (2) Adjust the pH of the dipotassium glycyrrhizate solution and the berberine sulfate hydrate solution obtained in step (1) to about 6.0 using disodium hydrogen phosphate (1mM). Then mix the dipotassium glycyrrhizate solution and the berberine sulfate hydrate solution at 25°C with concentration ratios of 1000:1, 500:1, 200:1, and 100:1, so that the concentration of the dipotassium glycyrrhizate solution in the system is 1%, and the concentration of the berberine sulfate hydrate solution is 0.001%, 0.0025%, 0.05%, and 0.01%, respectively.

[0055] (3) The composite solutions with different concentrations of sulfated berberine hydrate obtained in step (2) were stirred at 400 rpm, and the pH of all samples was adjusted to 3.5 with 0.5 mM hydrochloric acid. After stirring for 10 min, the samples were allowed to stand to obtain natural AIE glycyrrhizic acid hydrogels, which were denoted as hydrogel 5, hydrogel 6, hydrogel 7 and hydrogel 8 respectively.

[0056] Example 3

[0057] A fully natural aggregation-induced luminescent glycyrrhizic acid active hydrogel, the preparation method of which includes the following steps:

[0058] (1) Add ammonium glycyrrhizate powder to deionized water and stir for 10 min at a stirring speed of 200 rpm and a temperature of 80°C until a clear and transparent ammonium glycyrrhizate solution (2%) is obtained. At the same time, uniformly disperse berberine sulfate hydrate in deionized water and stir for 10 min at a stirring speed of 200 rpm and a temperature of 80°C to obtain a completely dissolved berberine sulfate hydrate solution (2%).

[0059] (2) Adjust the pH of the glycyrrhizic acid ammonium solution obtained in step (1) to about 8.0 with sodium hydroxide (4mM). Then mix the glycyrrhizic acid ammonium solution and the sulfated berberine hydrate solution at 25°C with concentration ratios of 100:1, 40:1, 20:1, 10:1, and 4:1, so that the concentration of the glycyrrhizic acid ammonium solution in the system is 1%, and the concentration of the sulfated berberine hydrate solution is 0.01%, 0.025%, 0.05%, 0.1%, and 0.25%, respectively.

[0060] (3) The composite solutions with different concentrations of sulfated berberine hydrate obtained in step (2) were stirred at 1000 rpm, and the pH of all samples was adjusted to 4.5 with 0.5 mM hydrochloric acid. After stirring for 3 min, the samples were allowed to stand to obtain natural AIE glycyrrhizic acid hydrogels, which were named hydrogel 9, hydrogel 10, hydrogel 11, hydrogel 12 and hydrogel 13, respectively.

[0061] Example 4

[0062] A fully natural aggregation-induced luminescent glycyrrhizic acid active hydrogel, the preparation method of which includes the following steps:

[0063] (1) Add ammonium glycyrrhizate powder to deionized water and stir for 10 min at a stirring speed of 400 rpm and a temperature of 70°C until a clear and transparent ammonium glycyrrhizate solution (2%) is obtained. At the same time, uniformly disperse berberine sulfate hydrate in deionized water and stir for 10 min at a stirring speed of 200 rpm and a temperature of 70°C to obtain a completely dissolved berberine sulfate hydrate solution (2%).

[0064] (2) Adjust the pH of the ammonium glycyrrhizate solution and the berberine sulfate hydrate solution obtained in step (1) to about 7.0 with sodium hydroxide (0.5mM). Then mix the ammonium glycyrrhizate solution and the berberine sulfate hydrate solution at 25°C at a concentration ratio of 100:1, so that the concentration of the ammonium glycyrrhizate solution in the system is 1% and the concentration of the berberine sulfate hydrate solution is 0.01%.

[0065] (3) The composite solution obtained in step (2) was stirred at 1000 rpm, and gluconolactone was added to slowly adjust the pH of the sample to 4.5. After stirring for 5 min, it was allowed to stand to obtain AIE glycyrrhizic acid hydrogel, wherein the concentration of gluconolactone in the system was 0.5 mM.

[0066] Example 5

[0067] A fully natural aggregation-induced luminescent glycyrrhizic acid active hydrogel, the preparation method of which includes the following steps:

[0068] (1) Add glycyrrhizic acid powder to deionized water and stir for 10 min at a stirring speed of 300 rpm and a temperature of 80°C until a clear and transparent glycyrrhizic acid solution (2%) is obtained. At the same time, berberine chloride hydrate is uniformly dispersed in deionized water and stirred for 10 min at a stirring speed of 300 rpm and 80°C to obtain a completely dissolved berberine chloride hydrate solution (2%).

[0069] (2) Adjust the pH of the glycyrrhizic acid solution and berberine chloride hydrate solution obtained in step (1) to about 7.0 with sodium hydroxide (0.5mM), and then mix them at 25°C at a concentration ratio of 20:1, so that the concentration of glycyrrhizic acid solution in the system is 1% and the concentration of berberine chloride hydrate solution is 0.05%.

[0070] (3) Add citric acid (0.5mM) to the composite solution obtained in step (2) and slowly adjust the pH of the sample to 3.5 while stirring at 1000 rpm. After stirring for 3 min, let it stand to obtain AIE glycyrrhizic acid hydrogel.

[0071] Comparative Example 1

[0072] A dipotassium glycyrrhizate supramolecular hydrogel is prepared by the following steps:

[0073] Dipotassium glycyrrhizate powder was added to deionized water and stirred for 15 minutes at 60°C and 200 rpm until a clear and transparent dipotassium glycyrrhizate solution (1%) was obtained. After standing and cooling at room temperature, dipotassium glycyrrhizate supramolecular hydrogel was obtained, which was named control hydrogel 1.

[0074] Comparative Example 2

[0075] An ammonium glycyrrhizate supramolecular hydrogel is prepared by the following steps:

[0076] Ammonium glycyrrhizate powder was added to deionized water and stirred for 10 minutes at 80°C and 200 rpm until a clear and transparent ammonium glycyrrhizate solution (1%) was obtained. After cooling at room temperature, ammonium glycyrrhizate supramolecular hydrogel was obtained and named control hydrogel 2.

[0077] Comparative Example 3

[0078] A berberine derivative solution, the preparation method of which includes the following steps:

[0079] Berberine hydrochloride hydrate was uniformly dispersed in water and stirred at 90°C and 200 rpm for 5 minutes to obtain a completely dissolved berberine hydrochloride hydrate solution (2%). This solution was then diluted with deionized water to a concentration of 0.01%. The pH of the berberine hydrochloride hydrate solution was then adjusted to approximately 7.0 using sodium hydroxide (1 mM). This final solution was designated as control solution 1.

[0080] Performance testing:

[0081] (1) AIE glycyrrhizic acid hydrogel fluorescence intensity test

[0082] The fluorescence emission spectrum of the hydrogel was measured using a fluorescence spectrophotometer (F-7100, Hitachi, Japan). The excitation wavelength was selected as 405 nm, the emission wavelength range was set to 450-700 nm, and the slit width of the emission spectrum was set to 2.5 nm. Pure glycyrrhizic acid hydrogel and berberine solution were used as controls in the experiment.

[0083] (2) AIE glycyrrhizic acid hydrogel rheological frequency scanning test

[0084] Frequency sweep tests (frequency range 0.1-10 Hz) were performed on the hydrogel using a HAKKE MARS 60 rheometer. The hydrogel was placed on the rheometer sample stage, and a parallel plate with a diameter of 35 mm was used for testing. The gap between the sample stage and the parallel plate was 1 mm. Frequency sweep test conditions: the test stress value was fixed (0.1%, within the linear range of the modulus), the frequency range was set to 0.1-10 Hz, and the trends of elastic modulus (G') and viscous modulus (G”) as a function of frequency were recorded.

[0085] Examples 1-3 illustrate a series of AIE glycyrrhizic acid hydrogels prepared using different preparation processes (e.g., different derivative types and different component concentrations). Examples 1-3 obtained AIE hydrogels with semi-solid properties by combining glycyrrhizic acid or its derivatives with berberine derivatives. Figure 1 As shown, compared with the appearance of the hydrogels in Comparative Examples 1 and 2, the hydrogels formed in Examples 2 and 3 under the same conditions exhibit stronger stereoforming properties and emit bright green fluorescence under ultraviolet light irradiation (Comparative Examples 1 and 2 do not emit bright green fluorescence). This indicates that the hydrogels prepared in Examples 2 and 3 of this invention can effectively improve the weak stereoforming properties of glycyrrhizic acid supramolecular hydrogels alone and endow the hydrogels with new fluorescence emission properties. The fluorescence emission spectra of the hydrogel prepared in Example 1 and the berberine solution alone in Comparative Example 3 are shown below. Figure 2 and Figure 3 As shown, it can be seen that compared to Comparative Example 3 alone, the 0.01% berberine solution ( Figure 3 In Example 1, all hydrogel systems exhibited significantly higher fluorescence intensity, and the fluorescence intensity of these hydrogels increased significantly as the concentration of glycyrrhizic acid derivative in the system increased from 0.1% to 1%. Furthermore, Figure 4 The fluorescence spectra of the AIE glycyrrhizic acid hydrogel of Example 2 and the pure glycyrrhizic acid hydrogel of Comparative Example 1 are shown. It can be seen that increasing the concentration of the berberine derivative effectively enhances the fluorescence intensity of the hydrogel. These data collectively confirm that the fluorescent hydrogel co-assembled from glycyrrhizic acid or its derivatives and berberine derivatives possesses significant aggregation-induced emission properties, and a stronger AIE effect can be achieved by increasing the concentration of glycyrrhizic acid or its derivatives or berberine derivatives in the system.

[0086] The rheological frequency scanning curve of the AIE glycyrrhizic acid hydrogel prepared in Example 3 is shown below. Figure 5 As shown in the figure (solid figures represent elastic modulus G', hollow figures represent viscous modulus G"), it can be seen from the figure that G' of the hydrogel is greater than G”, further proving the formation of hydrogels and their semi-solid viscoelastic properties. Compared with Comparative Example 2, the hydrogel prepared in Example 3 has larger elastic modulus and viscous modulus values, indicating that increasing the content of berberine derivatives in the hydrogel can significantly enhance the mechanical strength of the hydrogel.

[0087] (3) AIE glycyrrhizic acid hydrogel viscosity test

[0088] The hydrogel was placed on the sample stage of the rheometer, with a 1mm gap between the sample stage and the parallel plate (35mm in diameter). The hydrogel was measured at shear rates of 0.01-100 s⁻¹ using a HAKKE MARS 60 rheometer. -1 The viscosity curve below.

[0089] (4) AIE glycyrrhizic acid hydrogel thixotropic recovery test

[0090] The thixotropic recovery properties of the hydrogel were tested using a HAKKE MARS 60 rheometer. The procedure was as follows: the hydrogel was placed on the rheometer's sample stage, with a 1mm gap between the stage and the parallel plate (35mm in diameter). The thixotropic recovery test conditions were: the changes in G′ and G″ over time were measured under alternating low strain (0.1%, within LVR) and high strain (10%, beyond LVR).

[0091] Taking the hydrogel obtained in Example 3 as an example, its viscosity curve was tested. Figure 6 The viscosity curve of the hydrogel obtained in Example 3 is shown. Figure 6The data shows that the viscosity of the hydrogel decreases with increasing shear rate, exhibiting typical shear-thinning behavior. Shear-thinning behavior is a classic characteristic of injectable materials, which also indirectly confirms that the AIE hydrogel prepared in this invention has excellent injectability, providing the hydrogel's ability to cover various irregular skin wounds. Alternating strains (0.1%, 10%, and 0.1%) were further applied to the AIE hydrogel prepared in Example 3, and the changes in its elastic modulus (G') and viscous modulus (G'') were observed to explore the structural recovery ability of these hydrogels. Figure 7 The thixotropic recovery test results shown (solid graphic represents G', hollow graphic represents G”) demonstrate that the elastic modulus and viscous modulus of the AIE hydrogel obtained in Example 3 can still be significantly restored to their initial state after cyclic action of small strain (0.1%) - large strain (10%) - small strain (0.1%). This indicates that the AIE hydrogel co-assembled from these two drug-food homologous molecules has excellent structural recovery ability. The ideal mechanical strength and excellent structural recovery performance of this AIE hydrogel make it possible for it to be used as an ideal dressing to adhere to wounds and achieve rapid self-healing.

[0092] (5) pH-responsive fluorescence test of hydrogel

[0093] In the study of pH-responsive fluorescence performance, as shown in Example 4, gluconate-δ-lactone needs to be added to the composite solution to slowly reduce the pH value. During the pH reduction process, the fluorescence intensity change of the hydrogel system at the emission wavelength of 545.5 nm is measured using a fluorescence spectrophotometer.

[0094] Figure 8 The figure shows the fluorescence emission spectrum of the AIE hydrogel during pH reduction. As can be seen, the fluorescence intensity of the hydrogel gradually increases with a slow decrease in pH. During the pH reduction process, the glycyrrhizic acid network becomes more tightly packed and entangled, which further increases the aggregation degree of the hydrogel, hinders the movement of berberine molecules, and prompts them to release more energy through non-radiative transitions, resulting in brighter fluorescence and a stronger fluorescence intensity.

[0095] (6) Temperature response fluorescence test of hydrogel

[0096] In the temperature-response fluorescence test, the temperature of the hydrogel in Example 5 was adjusted to the corresponding temperatures (25°C and 80°C). The hydrogel was first gradually heated from 25°C to 80°C, and then cooled to 25°C. This heating-cooling cycle was repeated five times, and the change in fluorescence intensity (emission wavelength 545.5 nm) of the hydrogel at 25°C and 80°C during this process was measured.

[0097] Taking the hydrogel prepared in Example 5 as an example, the temperature-responsive fluorescence properties of the hydrogel were investigated. Figure 9 The graph shows the fluorescence intensity of the hydrogel at 25℃ and 80℃ during the cyclic heating-cooling process. As can be seen from the graph, during the five cycles of heating (80℃) and cooling (25℃), the hydrogel consistently showed a relatively higher fluorescence intensity at 25℃. Once the temperature was increased to 80℃, the fluorescence intensity of the hydrogel decreased again, indicating that the hydrogel has temperature-controllable AIE behavior.

[0098] (7) Antibacterial test of AIE glycyrrhizic acid active hydrogel

[0099] The bactericidal ability of the hydrogel of this invention was evaluated by observing bacterial colonies on plates, with Staphylococcus aureus as a representative example. Deionized water was passed through a membrane (0.22 μm aqueous membrane) to obtain sterile water. The prepared sterile water was used instead of the deionized water in Example 3, and the hydrogel was prepared according to the method of Example 3. The hydrogel was irradiated under a UV lamp for 30 min to obtain a sterile hydrogel. The bacterial suspension was first diluted multiple times with sterile PBS solution to obtain the final OD of the bacterial suspension. 600 The value was close to 0.65. Subsequently, 100 μL of the obtained bacterial suspension was added to the surface of an AIE glycyrrhizic acid hydrogel (0.2 g). Then, these samples with added bacteria were divided into a dark group, a 30 min light exposure group, and a 60 min light exposure group. The dark group was incubated at 37°C in the dark for 4 h, while the 30 min and 60 min light exposure groups were first exposed to white light (20 mW / cm²). 2 Irradiate the bacteria for 30 and 60 minutes respectively, then incubate them in the dark for an additional time (total time 4 hours). Finally, take 100 μL of each bacterial suspension and spread it on an agar plate, incubate at 37°C for 24 hours, record the number of bacteria and calculate the bacterial survival rate.

[0100] Figure 10 This study evaluated the antibacterial activity of the AIE glycyrrhizic acid hydrogel prepared according to the method in Example 3 of this invention against Staphylococcus aureus. The results showed that compared with the control (PBS treatment) and glycyrrhizic acid hydrogel alone (control hydrogel 2), the bacterial survival rate treated with AIE hydrogel was significantly reduced, and this phenomenon became more pronounced with increasing berberine derivative concentration, indicating that the AIE glycyrrhizic acid hydrogel possesses excellent antibacterial and bactericidal capabilities. Notably, compared with the dark hydrogel group and the corresponding light-treated blank samples, the light-treated hydrogel groups, namely the AIE glycyrrhizic acid hydrogel groups exposed to light for 30 min and 60 min, both exhibited lower bacterial survival rates. This indicates that the aggregation-induced emission glycyrrhizic acid hydrogel possesses photodynamic antibacterial capabilities, which can further enhance the antibacterial effect on top of the hydrogel's inherent antibacterial ability.

[0101] (8) In vitro anti-inflammatory experiment of AIE glycyrrhizic acid active hydrogel

[0102] The in vitro anti-inflammatory capacity of the hydrogel was evaluated using a lipopolysaccharide (LPS) cell inflammation model. RAW264.7 macrophages seeded in 96-well plates were cultured at 37°C and 5% CO2 for 24 h. After inducing an inflammatory response with LPS (100 μL, 1 μg / mL), the cells were incubated for another 24 h. Then, fresh DMEM medium containing the hydrogel extract was added to the cells and cultured for 24 h. Finally, the level of pro-inflammatory cytokines (TNF-α) in the supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0103] The anti-inflammatory ability of the hydrogel was evaluated by measuring the level of the TNF-α inflammatory factor. The results are as follows: Figure 11 As shown in the data, the LPS group exhibited increased TNF-α inflammatory factor levels compared to the control group, indicating the successful construction of the inflammation model. After treatment with hydrogel 2 and hydrogel 13 (from Example 3), the TNF-α inflammatory factor levels significantly decreased, demonstrating that both glycyrrhizic acid hydrogel alone and AIE glycyrrhizic acid active hydrogel exhibited excellent anti-inflammatory capabilities. Notably, the TNF-α inflammatory factor levels treated with AIE glycyrrhizic acid active hydrogel were significantly lower than those in the glycyrrhizic acid hydrogel group, indicating that the introduction of berberine derivatives endows the hydrogel with higher anti-inflammatory capabilities.

[0104] (9) Experiment on wound repair in infection with drug-resistant Staphylococcus aureus

[0105] Twenty-four 6-week-old nude mice were subjected to a 10mm diameter circular full-thickness wound on their backs using a punch, and then infected with drug-resistant Staphylococcus aureus (MRSA). 8 Mice were randomly divided into four groups (cfu / mL), with six mice in each group. All mice were housed individually and treated with PBS, comparative hydrogel 2, hydrogel 13 of Example 3, and light (20 mW / cm²), respectively. 2 The hydrogel was treated for 10 min) for 13 days, and the wound closure status was recorded every other day.

[0106] Figure 12The figure shows the statistical results of wound area changes in the above-treated infected skin wound repair experiment. After 11 days of treatment, compared with wounds treated with PBS and control hydrogel 2, wounds treated with hydrogel 13, especially those treated with a combination of hydrogel 13 and light irradiation (i.e., hydrogel 13-light irradiation), showed smaller wound area and faster wound healing effect. This indicates that the hydrogel prepared by the present invention has a better ability to promote wound healing. The photodynamic antibacterial ability of AIE glycyrrhizic acid hydrogel can further enhance the role of hydrogel in promoting the healing of infected wounds, which has great practical application and commercial value in the field of medical materials.

[0107] In summary, the hydrogel dressing prepared by this invention possesses excellent aggregation-induced emission ability, strong mechanical properties, shear-thinning injectability, high structural recovery ability, temperature / pH responsive fluorescence characteristics, significant hydrogel antibacterial properties, photodynamic synergistic antibacterial ability, and excellent anti-inflammatory ability. It can be used as a novel functional AIE hydrogel dressing to promote the healing of infected wounds, demonstrating potential practical application value in medical gel materials.

[0108] The embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a fully natural aggregation-induced emission licorice acid active hydrogel, characterized in that, Comprising the following steps: (1) Dissolution: uniformly disperse glycyrrhizinic acid or its derivative in deionized water, stir to obtain a transparent glycyrrhizinic acid or its derivative solution; uniformly disperse berberine derivative in deionized water, stir until the berberine derivative is completely dissolved to obtain a berberine derivative solution; the glycyrrhizinic acid or its derivative includes one of glycyrrhizinic acid, ammonium glycyrrhizinate, monopotassium glycyrrhizinate, dipotassium glycyrrhizinate; the berberine derivative includes one of berberine hydrochloride hydrate, berberine chloride hydrate, sulfated berberine hydrate; (2) Complexing: use an alkaline pH adjuster to adjust the pH of the glycyrrhizinic acid or its derivative solution and the berberine derivative solution obtained in step (1) to 6.0-8.0, mix the two at room temperature to obtain a complex solution; (3) Preparing a hydrogel: use an acidic pH adjuster to adjust the pH of the complex solution obtained in step (2) to 3.0-5.0 under continuous stirring, stir until a natural AIE glycyrrhizinic acid hydrogel is obtained.

2. The preparation method of the all-natural aggregation-induced emission licorice acid active hydrogel according to claim 1, characterized in that, In step (1), the stirring temperature is 60-90℃, the stirring speed is 100-500 rpm, and the stirring time is 5-15 min.

3. The method according to claim 1, wherein the method is characterized by, In step (1), the concentration of the glycyrrhizinic acid or its derivative solution is 2%-6%; the concentration of the berberine derivative solution is 0.5%-2%.

4. The preparation method of the all-natural aggregation-induced emission licorice acid active hydrogel according to claim 1, characterized in that, In step (2), the alkaline pH adjuster is one of sodium hydroxide solution, potassium hydroxide solution, disodium hydrogen phosphate solution, sodium bicarbonate; the concentration of the alkaline pH adjuster is 0.5-4 mM.

5. The method for preparing an all-natural aggregation-induced luminescence glycyrrhizic acid active hydrogel according to claim 1, characterized in that, In step (2), the complexing concentration ratio of the glycyrrhizinic acid or its derivative solution and the berberine derivative solution is 1000:1-4:1; the concentration of the glycyrrhizinic acid or its derivative solution in the complex solution is 0.1%-1%; the concentration of the berberine derivative solution in the complex solution is 0.001%-0.25%.

6. The method of claim 1, wherein the method is characterized by, In step (3), the stirring speed is 400-1000 rpm, and the stirring time is 2-10 min; the acidic pH adjuster is one of hydrochloric acid, sulfuric acid, citric acid, tartaric acid, acetic acid, gluconic acid lactone, and the concentration of the acidic pH adjuster is 0.5-2 mM.

7. The fully natural aggregation-induced emission glycyrrhizinic acid active hydrogel prepared by the preparation method of any one of claims 1-6.

8. The use of the fully natural aggregation-induced emission glycyrrhizinic acid active hydrogel of claim 7 in the preparation of a drug carrier, an antibacterial and anti-inflammatory material, a smart fluorescent material, or a medical gel dressing.

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