Preparation method of hydrophobic natural small molecule self-assembled hydrogel sustained-release delivery system and application of hydrophobic natural small molecule self-assembled hydrogel sustained-release delivery system in skin wound repair

Abstract

The invention relates to a preparation method of a hydrophobic natural-drug-effect micromolecule self-assembled hydrogel sustained-release drug delivery system and wound repair application. Belongs to the technical field of biological materials and pharmaceutical preparations. The hydrogel has antibacterial, anti-inflammatory and wound repairing capabilities, can selectively inhibit drug-resistant bacteria and fungi, and does not influence the activity of probiotics; the entrapment performance on exosomes and various pharmacodynamic molecules is good. Wherein the coptisine and chrysin self-assembled hydrogel is uniform and stable in form, has dual antibacterial activity superior to those of penicillin G, ketoconazole and the like, and has a value of being developed into a carrier-free supramolecular nano-drug. In the preparation process, compared with the traditional technology, the method is simpler and more environmentally friendly, high in repeatability and has industrial production potential. In application, the sustained-release delivery system can remarkably shorten the wound healing period and reduce the infection risk, and shows good applicability and treatment effect on complex wounds such as acute skin wounds, chronic ulcers and diabetic feet.

Description

Technical Field

[0001] This invention relates to a method for preparing a hydrophobic natural pharmaceutical small molecule self-assembled hydrogel sustained-release drug delivery system and its application in wound repair. The hydrophobic natural pharmaceutical small molecule self-assembled hydrogel possesses excellent antimicrobial, anti-inflammatory, and wound-healing capabilities. While selectively inhibiting drug-resistant bacteria and fungi, it does not affect the activity of beneficial intestinal bacteria, such as Bacillus subtilis and Enterococcus faecalis; and it exhibits good encapsulation capacity for exosomes and various pharmaceutical molecules. Specifically, the hydrogels self-assembled from the hydrophobic berberine and styraxin exhibit uniform morphology, good dispersibility and stability, and excellent dual antibacterial activity, superior to existing antibacterial drugs such as penicillin G and ketoconazole, making it valuable for development into carrier-free supramolecular nanomedicines with dual antibacterial properties. In terms of preparation method, this invention employs an innovative process, achieving controllable self-assembly of the hydrogel by precisely controlling key parameters such as small molecule concentration, solvent polarity, and pH value. Compared to traditional hydrogel preparation techniques, this method is simple to operate, environmentally friendly, highly reproducible, and has the potential for industrial production. In wound healing applications, this sustained-release delivery system continuously releases antibacterial components and repair-promoting factors, significantly shortening the wound healing cycle and reducing the risk of infection. Whether for acute skin trauma, chronic ulcers, or complex wounds such as diabetic foot, the system demonstrates good applicability and therapeutic efficacy. It belongs to the field of biomaterials and pharmaceutical formulation technology. Background Technology

[0002] As the body's first natural line of defense, the skin is the tissue that directly contacts the external environment, playing a vital role in providing a barrier to protect the body from external stimuli and damage. Wound management, as a resource-intensive medical activity, places significant pressure on healthcare systems worldwide. With an aging population and increased life expectancy, the prevalence of chronic wounds continues to rise, bringing unprecedented stress to healthcare systems. When wounds are not treated promptly, bacterial and fungal infections and suppuration can easily occur, leading to systemic infection or sepsis in severe cases. Post-traumatic bacterial and fungal infections are a major cause of trauma mortality globally. Data from 2019 shows that bacterial infections caused 7.7 million deaths worldwide, accounting for 13.6% of all deaths globally, making it the second leading cause of death globally. Therefore, finding wound dressings that can quickly stop bleeding and effectively prevent infection has become a hot research topic. Although antibiotics are effective in treating bacterial infections, overuse of antibiotics has led to the emergence of superbugs or superfungi, and their effects are not long-lasting. Therefore, the use of long-acting nano-antibacterial agents or other non-pharmaceutical antibacterial materials is urgently needed. To promote wound healing, hospitals currently commonly use dry non-woven fabric wound dressings. However, these materials not only excessively absorb wound exudate and dry out, adhering to the wound surface and causing secondary damage during dressing changes, but also fail to prevent moisture evaporation, leading to wound dehydration. To overcome the shortcomings of dry dressings, research into moist dressings has gained more attention. For rapid and effective wound repair, an ideal wound dressing should first promote wound healing; secondly, it should keep the wound moist to prevent dehydration; thirdly, it should be soft and easy to change; and fourthly, it should not easily break or break. Finally, an ideal wound dressing should also possess certain biochemical properties or other functions beneficial to wound recovery, such as antibacterial properties, hemostasis, and promotion of cell proliferation and differentiation.

[0003] Hydrogels, with their three-dimensional network structure similar to the extracellular matrix and their hydrophilicity, have attracted considerable interest from researchers due to their potential for modification and the ability to acquire multiple functions, significantly overcoming the limitations of traditional dry dressings. Furthermore, changes in the cross-linking mechanisms and innovative components of hydrogels can meet the diverse requirements of different types of wound healing and repair processes, making the development of novel multifunctional hydrogels a mainstream trend. However, previously reported hydrogels are mostly based on high-molecular-weight polymers, and reports of hydrogels formed by the self-assembly of natural active small molecules have become a new research hotspot. Research on self-assembled hydrogels has almost entirely focused on single-component systems, with research on two-component self-assembled hydrogels still in its early stages. Single-component self-assembled hydrogels often only provide a single function, such as antibacterial properties or cell adhesion promotion, making it difficult to meet the complex and varied physiological needs during wound repair. For example, while chitosan-based single-component hydrogels possess good antibacterial properties, they perform poorly in anti-inflammatory applications; and some single-component hydrogels with pro-angiogenic functions show weak effects in resisting infection. Two-component or multi-component self-assembled hydrogel systems can achieve multifunctional integration through the synergistic effects of different components. The self-assembly of two or more small molecules with different bioactivities can endow hydrogels with multiple functions, including antibacterial, anti-inflammatory, and cell proliferation and tissue regeneration promotion. Furthermore, the proportions and assembly methods of the components in a two-component system can be flexibly controlled, making the hydrogels more suitable for the repair needs of different types of wounds in terms of mechanical properties and degradation rates, greatly enhancing the biomedical application value of hydrogels. Currently, the few reported two-component systems all contain typical hydrophilic structures (carboxylic acids, phosphates, glycosides), and research on hydrophobic two-component self-assembled hydrogel systems has not been reported. This not only limits the application expansion of hydrogels in complex physiological environments but also hinders the development of novel hydrogels with waterproof, long-lasting antibacterial, and tissue repair functions, urgently requiring further exploration and breakthroughs. Summary of the Invention

[0004] Building upon existing technologies, this invention aims to develop a hydrophobic, self-assembled hydrogel sustained-release drug delivery system using natural pharmaceutical small molecules. It combines hydrophobic flavonoid aglycones and their analogues from Scutellaria baicalensis, which possess cell proliferation-promoting, anti-inflammatory, and antioxidant properties, with alkaloids from Coptis chinensis, which exhibit significant antimicrobial activity. By precisely controlling parameters such as small molecule concentration, centrifugation speed, and pH, the hydrogel achieves controllable self-assembly. This hydrogel exhibits uniform morphology, good dispersibility, and stability. It selectively inhibits drug-resistant bacteria and fungi without affecting the activity of beneficial intestinal bacteria, and can effectively encapsulate exosomes and various pharmaceutical molecules. Its antibacterial and antifungal activity surpasses that of traditional antibacterial drugs such as penicillin G and ketoconazole. The preparation method is simple, environmentally friendly, and highly reproducible. In the field of wound repair, it can continuously release effective components for complex wounds such as acute skin trauma, chronic ulcers, and diabetic foot, shortening the healing cycle and reducing the risk of infection. This invention has significant research implications for the discovery and development of structurally well-defined natural nanomedicines from natural Chinese medicines.

[0005] One of the objectives of this invention is to provide supramolecular hydrogels formed by flavonoid aglycones and berberine alkaloids, as well as a method for their preparation, and a green, environmentally friendly, easy-to-operate, and industrially scalable hydrogel preparation process, overcoming the limitations of traditional hydrogel preparation technologies.

[0006] The second objective of this invention is to provide applications for the obtained supramolecular hydrogel compound, specifically to develop a hydrogel with excellent antibacterial properties, especially against drug-resistant bacteria and fungi, while also protecting beneficial intestinal bacteria, promoting cell proliferation, and possessing multiple functions such as anti-inflammation and anti-oxidation, effectively shortening the healing cycle of various wounds and improving the treatment effect of wounds.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] 1. By exploring different pH values, molar ratios of feed materials, centrifugal speeds, and heating temperatures, the preparation process for forming supramolecular hydrogel compounds from flavonoid aglycones and berberine alkaloids was determined. High-performance liquid chromatography (HPLC), ultraviolet spectroscopy, mass spectrometry (MS), nuclear magnetic resonance (NMR), and isothermal calorimetric titration were used to determine its structural information.

[0009] The preparation method includes the following steps:

[0010] (1) Heat and sonicate the Coptis chinensis alkaloids, and add DMSO dropwise to help dissolve it in water.

[0011] (2) Flavonoid aglycones are suspended in water, ultrasonically heated, and the pH is adjusted to obtain a clear and transparent solution.

[0012] (3) The aqueous solution of Coptis chinensis alkaloids prepared in steps (1) and (2) is allowed to stand and then cooled down before being mixed. After ultrasonic stirring, the mixture is centrifuged at a constant speed to obtain a supramolecular hydrogel.

[0013] Preferably, the concentration of DMSO in step (1) is 0.5% of the total solution system.

[0014] Preferably, the heating temperature in steps (1) and (2) is 80-100℃, for example, 80℃, 90℃, 95℃, or 100℃.

[0015] Preferably, the molar ratio of flavonoid aglycones to berberine alkaloids is 1:0.1 to 1:10, further preferably 1:0.5 to 1:3, and specifically 1:0.5, 1:1 and 1:2.

[0016] Preferably, the ultrasonic frequency in steps (1), (2), and (3) is 30-40 kHz.

[0017] Preferably, the pH value in step (2) is 7.0-9.0, such as pH=7.5, pH=8.0, pH=8.5, etc.

[0018] Preferably, the pH adjuster in step (2) is an inorganic base, such as sodium hydroxide, sodium carbonate, sodium bicarbonate, and ammonia.

[0019] Preferably, the specific temperature for cooling in step (3) is 10-12℃.

[0020] Preferably, the centrifugation speed during centrifugation purification in step (3) is 3000-10000 rpm, such as 3000 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm or 9500 rpm.

[0021] 1. Rheological properties and hydrogel performance of the "Coptis chinensis alkaloid-flavonoid aglycone" supramolecular hydrogel were evaluated by rheological testing.

[0022] 2. The antibacterial effect of the obtained "Coptis chinensis alkaloid-flavonoid aglycone" supramolecular hydrogel compound was evaluated by determining the minimum inhibitory concentration (MIC) values ​​of different bacterial species.

[0023] The evaluation of antibacterial efficacy includes the following steps:

[0024] The minimum inhibitory concentrations (MICs) of the compound and control group against Candida albicans, multidrug-resistant Candida auris, Candida krusei, Candida dublin, Candida tropicalis, Candida glabrata, Escherichia coli, Bacillus subtilis, and methicillin-resistant Staphylococcus aureus were determined by dilution method.

[0025] 3. Taking the most difficult-to-inhibit and eliminate multidrug-resistant Candida auris as an example, the in vitro antibacterial effect of the supramolecular hydrogel compound "Coptis chinensis alkaloid-flavonoid aglycone" on multidrug-resistant Candida auris and other pathogenic bacteria was observed by scanning electron microscopy.

[0026] 4. Taking the most difficult-to-inhibit and eliminate multidrug-resistant Candida auris as an example, the inhibitory and elimination effects of supramolecular hydrogel compounds on the biofilm of multidrug-resistant Candida auris were observed by scanning electron microscopy.

[0027] 5. Taking the most difficult-to-inhibit and eradicate multidrug-resistant Candida auris as an example, the combination of the strain with a supramolecular hydrogel compound of "Coptis chinensis alkaloid-flavonoid aglycone" under high salinity, high alkalinity, and high sugar conditions affected the strain's own osmotic stress response and reduced its tolerance to high osmotic stress. The apoptosis status of the strain was observed by flow cytometry and apoptosis staining.

[0028] 6. Taking the berberine-capillary supramolecular hydrogel compound as an example, the in vitro antibacterial effect of the supramolecular hydrogel compound against various pathogenic bacteria such as multidrug-resistant Candida auris and biofilms was observed by scanning electron microscopy. Taking multidrug-resistant Candida auris, which is the most difficult to inhibit and eliminate, as an example, a mouse skin wound infection animal model was established. The wound healing rate, fungal growth, and skin pathological tissue changes in mice after treatment were observed to evaluate its antibacterial effect against multidrug-resistant Candida auris, Candida albicans, methicillin-resistant Staphylococcus aureus, and Escherichia coli, among other fungi and bacteria.

[0029] 7. The safety of supramolecular hydrogel compounds is evaluated through cell safety experiments, in vitro hemolysis experiments, and staining of organ tissue sections.

[0030] Taking the berberine-capsicum supramolecular hydrogel compound with the best antibacterial effect as an example, the in vitro antibacterial effect of the supramolecular hydrogel compound of this application against bacteria and fungi such as multidrug-resistant Candida auris, Candida albicans, methicillin-resistant Staphylococcus aureus, and Escherichia coli, the evaluation of its inhibition and clearance effects on bacterial or fungal biofilms, and the effect of the supramolecular hydrogel compound on promoting the healing of various types of wounds include the following steps:

[0031] (1) The effect of supramolecular hydrogel compounds on the growth rate of various bacteria and fungi at 12h was determined by microplate reader.

[0032] (2) The bactericidal effect of supramolecular hydrogel compounds on various bacteria and fungi was detected by agar plate coating.

[0033] (3) Scanning electron microscopy was used to observe the morphological effects of supramolecular hydrogel compounds on bacteria and fungi.

[0034] (4) The effect of supramolecular hydrogel compounds on the growth rate of bacteria and fungi was determined by enzyme-linked immunosorbent assay (ELISA).

[0035] (5) Combine live and dead cell staining experiments, solid agar plate coating and flow cytometry experiments to evaluate the induction of bacterial and fungal apoptosis by supramolecular hydrogel compounds and their combination with osmotic stress reagents.

[0036] (6) The effects of supramolecular hydrogel compounds on cell cycle progression were evaluated by a combination of flow cytometry and morphological statistics.

[0037] (7) The XTT method was used to evaluate the inhibitory and scavenging effects of supramolecular hydrogel compounds on fungal and bacterial biofilms.

[0038] (8) Scanning electron microscopy was used to observe the inhibitory and scavenging effects of supramolecular hydrogel compounds on bacterial and fungal biofilms.

[0039] (9) Establish rat deep burn wound healing model, diabetic wound healing model, microbial infection wound healing model and diabetic microbial infection wound healing model respectively to comprehensively evaluate the wound healing activity of supramolecular hydrogel compounds. The skin wound recovery process of mice was detected by HE sectioning, Masson staining and immunofluorescence sectioning.

[0040] (10) The survival rate of HUVEC, HaCat and Raw264.7 cells cultured for 24h and 48h after drug administration was evaluated by the MTT method to assess the cytotoxicity of supramolecular hydrogel compounds.

[0041] (11) The supramolecular hydrogel compound was incubated with rat erythrocytes, and the hemolysis rate of each drug administration group at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the hemolytic activity of the supramolecular hydrogel compound.

[0042] (12) To test the biosafety of supramolecular hydrogel compounds on the heart, liver, spleen, lungs and kidneys of mice during the skin wound healing process.

[0043] The specific research results of this invention are described below:

[0044] I. Formation of supramolecular hydrogel compounds

[0045] This invention discovers that berberine alkaloids and flavonoid aglycones can form supramolecular hydrogel compounds. After process optimization and preparation, combinations of raw material compounds that can form hydrogels include, but are not limited to: berberine-salicylic acid, berberine-baicalein, berberine-chiplostigmine A, berberine-robinin, berberine-5,4'-hydroxyflavone, berberine-baicalein, berberine-daidzein, berberine-genistein, etc. The selected berberine alkaloids (such as berberine, berberine, palmatine, and berberine) and flavonoid aglycones (such as salicylic acid, baicalein, and wogonin) are structurally similar, but subtle changes in functional groups lead to the formation of hydrogel network structures with varying fiber thicknesses and lengths, exhibiting different gel and rheological properties.

[0046] II. Antibacterial and antifungal effects of supramolecular hydrogel compounds

[0047] Table 2 in the examples systematically presents the effects of each supramolecular hydrogel compound on pathogenic bacteria and probiotics. Studies have confirmed that the berberine-capsicum supramolecular hydrogel prepared in this invention exhibits highly selective inhibitory capabilities against bacteria and fungi, with minimal impact on beneficial bacteria. Its antibacterial activity is significantly superior to traditional antibacterial drugs and raw material monomers, demonstrating unique clinical application potential. For methicillin-resistant Staphylococcus aureus (MRSA), a clinically challenging pathogen, the berberine-capsicum supramolecular hydrogel has a minimum inhibitory concentration (MIC) of 5-10 μM, significantly superior to many first-line clinical antibiotics. Vancomycin has an MIC of 40-60 μM for MRSA, penicillin-G has 60-80 μM, and doxycycline has 40-60 μM, while the inhibitory concentration of this hydrogel is only 1 / 8-1 / 4 of these drugs, demonstrating a strong inhibitory ability against drug-resistant Gram-positive bacteria. Compared to the raw material monomers, berberine has an MIC of 160-200 μM for MRSA, while capsicum is completely... No inhibitory effect (MIC>320μM), and no activity in physical mixtures (MIC>320μM), confirming that supramolecular self-assembly produced a significant synergistic effect; against common Gram-negative pathogenic bacteria Escherichia coli, the MIC of this hydrogel is 2-5μM, which is much lower than that of traditional drugs: vancomycin, penicillin-G, and doxycycline against Escherichia coli are all 100-120μM, and berberine and berberine monomers are 200-240μM, while the antibacterial activity of the hydrogel is increased by 20-60 times, which can effectively inhibit the excessive proliferation of opportunistic pathogens and avoid disrupting the balance of intestinal flora. In the field of antifungal treatment, berberine-capsicum supramolecular hydrogel exhibits particularly strong inhibitory effects against multidrug-resistant strains. Against multidrug-resistant Candida auris ("superfungus"), its inhibitory concentration (MIC) is 5-10 μM, far exceeding that of first-line clinical antifungal drugs. Amphotericin B has an MIC of 300-320 μM, and ketoconazole has an MIC >320 μM. The hydrogel's inhibitory concentration is only 1 / 30-1 / 60 of both, solving the problem of poor efficacy of traditional drugs against drug-resistant Candida. For Candida albicans, the hydrogel's MIC is 2-5 μM. It is significantly superior to amphotericin B (100-120 μM) and ketoconazole (100-120 μM), with an antibacterial activity that is 20-60 times higher. Moreover, the activity of the raw material monomers berberine (200-240 μM) and apigenin (>320 μM) is much lower than that of the hydrogel. For Trichophyton indicum (a common dermatophyte), the MIC of the hydrogel is 5-10 μM, while the MIC of amphotericin B is 250-280 μM and that of ketoconazole is 300-320 μM. Its activity advantage can effectively address the problem of drug resistance in skin fungal infections.Biofilms are a significant cause of drug resistance and chronic infections in pathogenic bacteria. The berberine-capsicum supramolecular hydrogel demonstrated excellent performance in biofilm experiments. Against MRSA biofilms, at a concentration of 100 μM, it achieved an inhibition rate of 89.41%, significantly higher than vancomycin (63.09%), berberine (52.12%), and capsicum (12.12%). In terms of clearance rate, at the same concentration, the hydrogel's clearance effect on mature MRSA biofilms far exceeded that of traditional drugs, further confirming its therapeutic potential for refractory infections. Against Candida auris biofilms, at a concentration of 100 μM, it achieved an inhibition rate of 85.58%, far higher than ketoconazole (39.20%), berberine (41.28%), and capsicum (10.23%). Its clearance rate against mature biofilms was also significantly better than drugs such as ketoconazole, solving the problem of refractory infections caused by fungal biofilms. This hydrogel exhibits a weak inhibitory effect on beneficial intestinal bacteria, with MICs >320 μM against Bacillus subtilis and Enterococcus faecium, while the MIC against the opportunistic pathogen Escherichia coli is only 2-5 μM. This "precise targeting" characteristic can both inhibit the excessive proliferation of pathogenic bacteria and maintain the balance of intestinal flora, reducing the damage to the intestinal microecology caused by traditional broad-spectrum antibacterial drugs. Compared with traditional antibacterial drugs, the mechanism of action of berberine-capsicum supramolecular hydrogel is fundamentally different: it causes bacterial cell lysis by disrupting the surface structure of bacteria, and induces cell cycle arrest and cell wall synthesis defects in fungi, rather than the single target of traditional drugs (such as cell wall synthesis or nucleic acid metabolism). This may be the key reason why it still maintains high activity against multidrug-resistant strains. In addition, this hydrogel can self-assemble without excipients, reducing the potential toxicity of carrier materials and improving drug safety and formulation convenience. In summary, berberine-salicylic acid supramolecular hydrogels demonstrate comprehensive advantages over traditional antibacterial drugs by effectively inhibiting drug-resistant bacteria and fungi, powerfully clearing biofilms, and selectively protecting the intestinal flora. They provide a novel strategy for the treatment of multidrug-resistant infections and have extremely high clinical translational value. Attached Figure Description

[0048] Figure 1 The macroscopic gel state, 40-day stability and pattern shaping characterization of one of the representative supramolecular hydrogel compounds (berberine-salicylic acid supramolecular hydrogel) prepared in Examples 1 and 2 of this invention are shown to have good uniformity, stability and injectability.

[0049] Figure 2 The microstructure (scanning electron microscope image) of one of the representative supramolecular hydrogel compounds (berberine-salicylic acid supramolecular hydrogel) prepared in Examples 1 and 2 of this invention is shown.

[0050] Figure 3The rheological properties and hydrogel performance of one of the representative supramolecular hydrogel compounds (berberine-salicylic acid supramolecular hydrogel) prepared in Examples 1 and 2 of this invention are described. A, B, C, and D are time-strain test, oscillatory shear rheological test, dynamic frequency scan test, viscosity test, step strain test, and temperature stability test, respectively.

[0051] Figure 4 This describes the antibacterial coating effect of each test group against the drug-resistant fungus Candida auris in Example 4 of the present invention. 1), 2), 3), and 4) are respectively the blank group, the berberine-capsicum physical mixture group, the positive control drug ketoconazole group, and the berberine-capsicum supramolecular hydrogel compound group.

[0052] Figure 5 The images show the inhibitory effects of each experimental group on the formation of drug-resistant fungus *Candida auris* biofilm observed under a scanning electron microscope in Example 5 of this invention. Images 1), 2), and 3) are scanning electron microscope images of *Candida auris* biofilms from the blank group, the berberine-salicylate physical mixture group, and the berberine-salicylate supramolecular hydrogel compound group, respectively.

[0053] Figure 6 This illustrates the anti-Candida auris effect of the berberine-capsicum supramolecular hydrogel compound combined with various osmotic stress agents in Example 5 of this invention. A, B, C, and D represent the solid agar spotting test, turbidimetric inhibition rate test, apoptosis fluorescence staining test, and flow cytometry apoptosis test, respectively.

[0054] Figure 7 The images show the actual healing process of skin wounds and the statistical chart of wound healing rate in mice using the diabetic microbial infection wound healing model in Example 7 of this invention.

[0055] Figure 8 The images show the evaluation results of the hemolysis experiment of the berberine-salicylic acid supramolecular hydrogel compound in Example 8 of this invention. Figures A and B are a macroscopic diagram of hemolysis and a statistical diagram of the hemolysis rate, respectively.

[0056] Figure 9 The images show the biosafety evaluation results of HUVEC, HaCat, and Raw264.7 cells observed under a laser confocal microscope in Example 8 of this invention. A, B, C, and D represent the fluorescence staining images of HUVEC, HaCat, and Raw264.7 cells in each group, as well as the survival rates of HUVEC, HaCat, and Raw264.7 cells after treatment in each group. Detailed Implementation

[0057] The following embodiments are intended to further illustrate the present invention. Those skilled in the art should understand that these embodiments are merely illustrative of the invention and should not be construed as limiting the invention.

[0058] Example 1

[0059] Coptis chinensis alkaloids and scutellaria baicalensis flavonoid aglycones and their analogues were weighed out in a molar ratio of 1:0.1 to 1:10 and dissolved separately in water. Coptis chinensis alkaloids were dissolved in water by heating and sonicating to 100°C and adding DMSO dropwise. Flavonoid aglycones were dissolved by adjusting the pH to 8.5 with an inorganic base such as sodium carbonate and then heating and sonicating. After obtaining the aqueous solutions of coptis chinensis alkaloids and flavonoid aglycones, the solutions were allowed to stand, cooled to 10°C, mixed, and sonicated for 20 minutes before centrifugation to obtain the coptis chinensis alkaloid-flavonoid aglycone supramolecular hydrogel compound.

[0060] Example 2

[0061] Weigh out molar amounts of berberine and flavonoids such as baicalin, scutellarin, wogonin, apigenin, robinin, 5,4'-hydroxyflavone, and chickpea extract A in a ratio of 1:0.1 to 1:10 and dissolve them separately in water. Adjust the pH to 7-9 with flavonoid aglycone to obtain a clear and transparent solution. Mix the berberine solution with the flavonoid aglycone solution separately at 90-100℃, stir ultrasonically, and centrifuge at 8000 r / min to obtain the berberine-flavonoid aglycone hydrogel compound. Specific examples include, but are not limited to, a series of supramolecular hydrogel compounds such as berberine-baicalin, berberine-scutellarin, berberine-chickpea extract A, berberine-robinin, berberine-5,4'-hydroxyflavone, berberine-baicalin, berberine-daidzein, berberine-genistein, and palmatine-apigenin.

[0062] Example 3

[0063] The structural information of the representative supramolecular hydrogel compound in Example 2 was confirmed by the following steps.

[0064] Mass spectrometry analysis of various supramolecular hydrogel compounds was performed using static spray-HRMS from Waters Corporation, USA. Positive ion mode was used for mass spectrometry detection, with the following parameters: capillary voltage 3.5 kV, cone voltage 40 V, ion source temperature 120 °C, collision energy 35 eV, cone gas flow rate 50 L / h, and desolvation gas flow rate 800 L / h. The mass acquisition range was 50–2000. Samples were directly injected for analysis without column separation. The molecular ion peaks and molecular structures of the basic unit cells of the supramolecular hydrogel compounds are shown in Table 1.

[0065] Table 1. Structural information of eight representative supramolecular hydrogel compounds prepared in Example 2.

[0066]

[0067]

[0068] Some supramolecular hydrogel compounds were characterized using proton nuclear magnetic resonance spectroscopy (Avance IIIHD 400MHz spectrometer, Bruker, America). The results are as follows:

[0069] NMR Assignment of Berberine-Salicin Supramolecular Hydrogel Compound 1 H NMR(400MHz,DMSO-d6):12.84(s,1H,5-OH,CHR),10.91(s,1H,7-OH,CHR),8.07(m,2H,2'-H,6'-H,CHR),7.60(m,3H,3'-H ,4'-H,5'-H,CHR),6.97(s,1H,3-H,CHR),6.53(d,J=2.4Hz,1H,8-H,CHR),6.24(d,J=2.4Hz,1H,6-H,CHR),9.97(s,1H,8- H,COP),8.99(s,1H,13-H,COP),8.03(d,J=8.0Hz,1H,12-H,COP),7.84(d,J=8.0Hz,1H,11-H,COP),7.79(s,1H,1-H,COP) ,7.08(s,1H,4-H,COP),6.54(s,2H,14-H,COP),6.17(s,2H,H-15,COP),4.90(brs,2H,H-6,COP),3.20(brs,2H,H-5,COP).

[0070] NMR assignment of berberine-robinin supramolecular hydrogel compound 1H NMR(400MHz,DMSO-d6):3.86(s,3H,-CH3,ACA),6.21(d,1H,6-H,ACA),6.51(d,1H,8-H,ACA),6.89(s,1H,-CH=,A CA),7.12(d,2H,3′,5′-H,ACA),8.05(d,2H,2′,6′-H,ACA)12.97(s,1H,5-OH,ACA),9.97(s,1H,8-H,COP),8.99(s ,1H,13-H,COP),8.03(d,J=8.0Hz,1H,12-H,COP),7.84(d,J=8.0Hz,1H,11-H,COP),7.79(s,1H,1-H,COP),7.08(s ,1H,4-H,COP),6.54(s,2H,14-H,COP),6.17(s,2H,H-15,COP),4.90(brs,2H,H-6,COP),3.20(brs,2H,H-5,COP).

[0071] Berberine-Chickpea Sprout A supramolecular hydrogel compound NMR classification 1 H NMR(400MHz,DMSO-d6):12.96(s,1H,5-OH,BIO),8.34(d,1H,H-2,BIO),7.36(d,2H,H-2′,6′,BIO),7.02(d,2H,H- 3′,5′,BIO),6.37(s,1H,H-8,BIO),6.22(s,1H,H-6,BIO),3.81(s,3H,-OCH3,BIO),9.97(s,1H,8-H,COP),8.99(s ,1H,13-H,COP),8.03(d,J=8.0Hz,1H,12-H,COP),7.84(d,J=8.0Hz,1H,11-H,COP),7.79(s,1H,1-H,COP),7.08(s ,1H,4-H,COP),6.54(s,2H,14-H,COP),6.17(s,2H,H-15,COP),4.90(brs,2H,H-6,COP),3.20(brs,2H,H-5,COP).

[0072] NMR Assignment of Berberine-Baicalein Supramolecular Hydrogel Compound 1H NMR(400MHz,DMSO-d6):6.69(1H,s,H-3,BA),6.77(1H,s,H-8,BA),8.05~8.07(2H,m,6-OH,BA),7.59~7 .61(4H,m,H-3',4',5',BA),12.77(H,s,5-OH,BA).9.97(s,1H,8-H,COP),8.99(s,1H,13-H,COP),8.03 (d,J=8.0Hz,1H,12-H,COP),7.84(d,J=8.0Hz,1H,11-H,COP),7.79(s,1H,1-H,COP),7.08(s,1H,4-H,C OP),6.54(s,2H,14-H,COP),6.17(s,2H,H-15,COP),4.90(brs,2H,H-6,COP),3.20(brs,2H,H-5,COP).

[0073] NMR Assignment of Baicalein-Symplocane Supramolecular Hydrogel Compound 1 H NMR(400MHz,DMSO-d6):6.69(1H,s,H-3,BA),6.77(1H,s,H-8,BA),8.05~8.07(2H,m,6-OH,BA),7.59~7.61(4H,m,H- 3',4',5',BA),12.77(H,s,5-OH,BA).6.51(s,1H,4-H,JAT),7.71(s,1H,1-H,JAT),3.15(t,J=8.0Hz,4Hz,1H,H-5,JA T),4.93(t,J=8.0Hz,4Hz,1H,6-H,JAT),9.86(s,1H,8-H,JAT),8.03(d,J=8.0Hz,1H,11-H,JAT),8.19(d,J=8.0Hz,1- H,H-12,JAT),9.03(s,1H,H-13,JAT),4.1(s,3H,9-OCH3,JAT),4.07(s,3H,10-OCH3,JAT),3.95(s,3H,2-OCH3,JAT).

[0074] NMR Assignment of Berberine-Dyelignin Supramolecular Hydrogel Compound 1H NMR(400MHz,DMSO-d6):12.47(1H,s,5-0H,GEN),10.09(1H,s,4-OH,GEN),8.26 1H,s,2-OH,GEN),7.38(2H,s,H-2,H-6,6.85(1H,d,H-8,GEN),6.81(2H,d,H-3,H-5,GEN),6.18(1H,d,H-6 ,GEN),9.91(s,1H,8-H,BBR),8.97(s,1H,13-H,BBR),8.19(d,J=9.2Hz,1H,11-H,BBR),8.01(d,J=8.0Hz, 1H,12-H,BBR),7.79(s,1H,1-H,BBR),7.08(s,1H,4-H,BBR),6.18(s,2H,15-CH2,BBR),4.95(t,J=6.0Hz, 2H,6-CH2,BBR),4.10(s,3H,9-OCH3,BBR),4.07(s,3H,10-OCH3,BBR),3.21(t,J=6.0Hz,2H,5-CH2,BBR).

[0075] Parmatine-apigenin supramolecular hydrogel compound NMR classification 1 H NMR(400MHz,DMSO-d6):12.47(1H,s,5-0H,API),10.09(1H,s,4-OH,API),8.26 1H,s,2-OH,API),7.38(2H,s,J=8.6Hz,H-2,H-6,API),6.85(1H,d,J=2.3Hz,H-8,API),6.81(2H,d,J=8.6Hz,H-3,H- 5,API),6.18(1H,d,J=2.1Hz,H-6,API).9.92(s,1H,8-H,PA),9.15(s,1H,13-H,PA),8.20(d,J=8.0Hz,1H,11-H,PA) ,8.06(d,J=8.0Hz,1H,12-H,PA),7.74(s,1H,1-H,PA),7.09(s,1H,4-H,PA),4.98(d,2H,6-CH2-,PA),4.11(s,3-H,9 -OCH3,PA),4.08(s,3H,10-OCH3,PA),3.24(brs,2H,5-CH2-,PA),3.95(s,3-H,3-OCH3,PA),3.88(s,3H,2-OCH3,PA).

[0076] NMR Assignment of Berberine-Roseflavin Supramolecular Hydrogel Compound 1H NMR(400MHz,DMSO-d6):9.91(s,1H,8-H,BBR),8.97(s,1H,13-H,BBR),8.19(d,J=9.2Hz,1H,11-H,BBR),8.01(d,J=8.0Hz,1H,12-H ,BBR),7.79(s,1H,1-H,BBR),7.08(s,1H,4-H,BBR),6.18(s,2H,15-CH2,BBR),4.95(t,J=6.0Hz,2H,6-CH2,BBR),4.10(s,3H,9-OCH 3,BBR),4.07(s,3H,10-OCH3,BBR),3.21(t,J=6.0Hz,2H,5-CH2,BBR),10.8(1H,s,3-OH,FI),9.32(2H,s,3′-OH,4′-OH,FI),7.93( 1H,d,J=9.30Hz,H-5,FI),7.69(1H,d,J=2.10Hz,H-2′,FI),7.55(1H,dd,J=8.50,2.10Hz,H-6′,FI),6.91(3H,m,H-5′,H-6,H-8,FI)

[0077] Example 4

[0078] To evaluate the in vitro antimicrobial properties of the supramolecular hydrogel compounds prepared in Examples 1 and 2, their antimicrobial activity was determined using turbidimetry and plate coating experiments, respectively. The microorganisms involved included multidrug-resistant Candida auris, methicillin-resistant Staphylococcus aureus, Escherichia coli, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton indicum, Candida albicans, Candida parapsilosis, Candida dublin, Candida krusei, the probiotic Bacillus subtilis, and Enterococcus faecalis. The specific experimental procedures are as follows:

[0079] Turbidimetric assay and plate coating assay for antibacterial activity: This experiment selected conditionally pathogenic bacteria *Escherichia coli*, methicillin-resistant *Staphylococcus aureus*, *Staphylococcus aureus*, and probiotics *Bacillus subtilis* and *Enterococcus faecalis* as research subjects. All strains were provided by the School of Traditional Chinese Medicine, Beijing University of Chinese Medicine. The supramolecular hydrogel compound powders prepared in Examples 1 and 2 were sealed and stored at 4°C. Before use, they were dissolved in LB medium containing 1% DMSO to prepare a 10 mM stock solution. In the bacterial culture stage, each strain was first dispersed in LB medium and cultured overnight in a constant temperature shaker at 37°C and 200 rpm. Subsequently, the bacterial concentration was adjusted to 2 × 10⁻⁶ using the plate count method and LB medium. 6Bacterial stock solutions were prepared using CFU / mL. The minimum inhibitory concentration (MIC) of the supramolecular hydrogel compound was determined using a dilution method. The specific procedure was as follows: In 48-well plates, 1 mL of compound solutions with concentration gradients of 300, 280, 250, 220, 200, 180, 150, 120, 100, 80, 60, 50, 40, 35, 30, 25, 20, 15, 10, 5, 2, and 1 μM were prepared using LB medium. Then, 30 μL of bacterial stock solution was added to each well, and the plates were incubated at 37°C in a humidified incubator for 12 h. After incubation, the OD value was measured at 600 nm using a microplate reader. The experiment was repeated three times, and the concentration corresponding to a bacterial viability greater than 80% was determined as the minimum inhibitory concentration. The experiment included a blank control group with no drugs and no bacteria, and a blank bacterial group with bacteria but no drugs. The bacterial survival rate was calculated using the following formula: Bacterial survival rate (%) = (Absorbance value of sample group - Absorbance value of blank group) / (Absorbance value of blank bacterial group - Absorbance value of blank group) × 100%.

[0080] Turbidimetric assay and plate coating were used to determine antifungal activity: Human pathogenic fungi *Candida albicans*, multidrug-resistant *Candida auris*, *Trichophyton rubrum*, *Trichophyton mentagrophytes*, and *Trichophyton indicum* were selected as test strains. These strains were obtained from the Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, Beijing Normal University. The strains were incubated overnight and diluted to 1×10⁻⁶. 7 CFU / mL suspensions of *Candida albicans* or multidrug-resistant *Candida auris* were added to 96-well plates and mixed with supramolecular hydrogel compound solutions to create a final concentration gradient of 320, 300, 280, 250, 220, 200, 180, 150, 120, 100, 80, 60, 50, 40, 35, 30, 25, 20, 15, 10, 5, 2, and 1 μM. After incubation at 30°C for 12 hours, the OD value was measured at 600 nm using a microplate reader. The experiment was repeated three times, and the concentration corresponding to a fungal survival rate greater than 80% was recorded as the minimum inhibitory concentration (MIC). The control group was set up in the same manner as the antibacterial experiment. The fungal survival rate was calculated as follows: Fungal survival rate (%) = (Absorbance value of sample group - Absorbance value of blank group) / (Absorbance value of blank group - Absorbance value of blank group) × 100%. The YPD medium used in this experiment was prepared with 1% yeast extract, 2% peptone, and 2% glucose. The Sabouraud dextrose agar (SDA) medium used in this experiment consisted of 4% glucose, 1% peptone, and 2% agar. The potato dextrose agar (PDA) medium consisted of 20% potato extract, 2% glucose, and 2% agar. Detailed experimental results are shown in Table 2.

[0081] Table 2: Antibacterial effects of the six supramolecular hydrogel compounds prepared in Examples 1 and 2 against different microorganisms.

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Note: * indicates that no relevant drug activity was detected.

[0088] The results showed that the supramolecular hydrogel compound, self-assembled from berberine alkaloids, flavonoid aglycones, and their analogues without other excipients, exhibited significantly more significant antimicrobial properties compared to its precursors and physical mixtures. This supramolecular hydrogel compound demonstrated highly selective inhibitory activity against pathogenic bacteria, while also exhibiting excellent antibacterial activity against clinically resistant bacteria and fungi, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Candida auris, and Trichophyton indicum, maintaining good sensitivity. Regarding the regulation of intestinal microbiota, it showed moderate antibacterial activity against the opportunistic pathogen Escherichia coli, effectively inhibiting its excessive proliferation while maintaining the ecological balance of the intestinal flora; while showing only weak inhibitory effects against beneficial intestinal bacteria such as Bacillus subtilis and Enterococcus faecalis, minimizing damage to beneficial bacteria. Notably, the berberine-capsicum supramolecular hydrogel compound exhibited particularly outstanding antibacterial activity. Amphotericin B, a polyene antifungal drug, exerts its antibacterial effect by disrupting cell membrane integrity. Ketoconazole, an imidazole antifungal drug, exhibits broad-spectrum antifungal activity but has no effect on bacteria or other microorganisms. It works by blocking ergosterol synthesis. Both berberine and ketoconazole are widely used in clinical indications, drug safety, and resistance management for fungal infections. Against drug-resistant Candida auris (multidrug-resistant Candida auris), the berberine-capillary supramolecular hydrogel compound has a minimum inhibitory concentration (MIC) of 5-10 μM, significantly superior to the two aforementioned first-line antifungal drugs (Amphotericin B MIC: 300-320 μM, ketoconazole MIC: 300-320 μM). Furthermore, against drug-resistant Trichophyton indicum, the berberine-capillary supramolecular hydrogel compound also shows significantly superior MIC compared to the two aforementioned first-line antifungal drugs (hydrogel MIC: 5-10 μM, Amphotericin B MIC: 250-280 μM, ketoconazole MIC: 300-320 μM). The berberine-capillary supramolecular hydrogel exhibits a minimum inhibitory concentration (MIC) of 5-10 μM against methicillin-resistant Staphylococcus aureus, significantly superior to first-line antibiotics such as penicillin-G, vancomycin, and doxycycline (MIC 40-80 μM). Against the non-drug-resistant pathogenic bacterium Candida albicans, the compound's MIC is 2-5 μM, again far exceeding traditional antifungal drugs such as ketoconazole and amphotericin B (MIC 100-120 μM). Similarly, against the non-drug-resistant pathogenic bacterium Escherichia coli, the berberine-capillary supramolecular hydrogel also shows a MIC of 2-5 μM, significantly exceeding traditional antibacterial drugs such as penicillin-G, vancomycin, and doxycycline (MIC 100-120 μM). Simultaneously, the berberine-capillary supramolecular hydrogel shows a weak inhibitory effect on beneficial intestinal bacteria such as Bacillus subtilis and Enterococcus faecalis (MIC > 320 μM).In summary, the series of supramolecular hydrogel compounds prepared in this study have shown outstanding application potential in the field of antimicrobial therapy. Their unique selective antibacterial properties and excellent activity provide a solid research foundation for further in-depth mechanistic exploration and clinical translation, and have high scientific research value and development prospects.

[0089] Example 5

[0090] Biofilm formation was induced using RPMI-1640 medium under conditions of 37℃, 5% CO2, and saturated humidity. In bacterial or fungal biofilm inhibition experiments, *Candida auris* or methicillin-resistant *Staphylococcus aureus* were used at a concentration of 1×10⁻⁶. 6 CFU / mL concentration was suspended in culture medium. 100 μL of the cell suspension was mixed with an equal volume of culture medium containing different concentrations of samples and inoculated into 96-well plates. Methicillin-resistant Staphylococcus aureus biofilms were cultured for 24 hours, and Candida auris for 48 hours. In bacterial or fungal biofilm inhibition experiments, 100 μL of 1×10⁻⁶ CFU / mL solution was first inoculated. 6 Microorganisms at CFU / mL were cultured for 48 hours to allow the biofilm to mature, followed by the addition of the drug and another 24 hours of culturing. Biofilm activity was quantified using an XTT reduction assay kit: 50 μL of XTT-menaquinone solution was added to the cleaned biofilm pores, and the mixture was incubated at 37°C in the dark for 4 hours. The absorbance at 450 nm was then measured. The biofilm inhibition / scavenging rate was calculated using the formula: Inhibition / Scavenging Rate (%) = 100 - [(OD...] 样品 -OD 溶剂 ) / (OD 空白菌 -OD 溶剂 )]×100%

[0091] The specific results are shown in Table 3-6.

[0092] Table 3: Inhibition rates of different drug groups on methicillin-resistant Staphylococcus aureus biofilms

[0093]

[0094] Note: * indicates that no relevant drug activity was detected.

[0095] Table 4: Inhibition rates of different drug groups on Candida auris biofilm

[0096]

[0097] Note: * indicates that no relevant drug activity was detected.

[0098] Table 5: Clearance rates of methicillin-resistant Staphylococcus aureus biofilms from different drug groups

[0099]

[0100]

[0101] Note: * indicates that no relevant drug activity was detected.

[0102] Table 6: Clearance rate of Candida auris biofilm by different drug groups

[0103]

[0104] Note: * indicates that no relevant drug activity was detected.

[0105] Example 6

[0106] To investigate the regulatory effect of hydrogels on the migration ability of L929 and HUVEC cells, a cell scratch assay was designed. The experiment included a control group and an experimental group, with the experimental group additionally supplemented with a supramolecular hydrogel compound. Cells were sputtered at 3 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1:1 in 6-well culture plates. Once cell confluence exceeded 90%, a 200 μL sterile pipette tip was used to streak lines perpendicularly to the plate surface at a constant speed to create regular scratches. The plates were then gently rinsed three times with PBS buffer to remove suspended cells caused by the scratches and ensure a clear field of view. 1 mL of hydrogel extract prepared in DMEM medium was added to each well. Images of the scratched areas were acquired under an inverted microscope at time points of 0 h, 4 h, 8 h, 12 h, and 24 h. Using ImageJ image processing software, the scratch area at different time points was accurately calculated using threshold segmentation and area measurement functions. Cell migration rate was quantitatively analyzed based on a formula to systematically evaluate the effect of the hydrogel on the migration of two types of cells.

[0107] Example 7

[0108] Using the berberine-salicylic acid supramolecular hydrogel prepared in Example 2 as the research object, an in vivo pharmacodynamic evaluation of the supramolecular hydrogel compound of the present invention related to skin healing was conducted. The skin healing-related experiments involved in this study included: a rat deep burn wound healing model, a diabetic wound healing model, a microbial infection wound healing model, and a diabetic microbial infection wound healing model. Specific operating steps are as follows:

[0109] In a rat model of deep burn wound healing, rats were first anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mL / kg). Hair removal cream was applied to the rat's back and left for 8 minutes before being gently wiped clean with physiological saline. A rat model of deep third-degree burns was constructed using a constant temperature and pressure burn instrument. The burn conditions were set as follows: pressure 500 g, temperature 100℃, and time 10 s. A 2.5 cm deep burn was created 1 cm lateral to the spine on both sides of the back. 2The scalded wounds of the rats were observed to turn white at the hair removal sites, and the next day, the skin in the burned area hardened, scabbed, and turned dark reddish-brown. HE staining showed coagulative necrosis in the dermis and vacuolar degeneration in the subcutaneous tissue, indicating successful modeling. The successfully modeled rats were randomly divided into three groups: a control group, a model group, and a berberine-salicylic acid supramolecular hydrogel treatment group. The control group rats did not undergo any scalding procedure; the model group rats received no treatment after scalding; and the hydrogel group rats had 5 mM berberine-salicylic acid hydrogel applied to the wound. Five rats (n=5) were included in each group as parallel controls, and the entire treatment period was set at 12 days. During the treatment period, the size of the rat wounds was recorded every 3 days, and the wound healing rate was calculated using ImageJ. After the 12-day treatment period, the rats were euthanized, and the skin from the wound on the rat's back was cut using sterile surgical scissors, flattened, and fixed in paraformaldehyde fixative. Wound healing rate = (S t -S0) / S0×100%, where S0 is the initial wound area, S t It represents the wound area at that point in time.

[0110] In a diabetic wound healing model, 5-week-old male C57BL / 6 mice were fed a high-sugar, high-fat diet for one month. After one month, they were intraperitoneally injected with 50 mg / kg streptozotocin for 5 consecutive days to establish a type 2 diabetes model. Mice with blood glucose levels exceeding 16.7 mmol / L were selected. After anesthetizing the mice with sodium pentobarbital, the hair on the backs of the mice was removed with depilatory cream, and the wounds were disinfected with 75% alcohol, creating circular wounds with a diameter of 8 mm on the dorsal skin of the mice. The experimental mice were grouped according to the procedures used in the rat deep burn wound healing model experiment.

[0111] In a microbial wound healing model, Candida albicans was selected as the experimental pathogen due to its difficulty in eradication and high morbidity rate after infection. Female Balb / c mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. Before the experiment, the fur on the backs of the mice was carefully shaved with electric clippers, and then anesthetized with sodium pentobarbital via intraperitoneal injection at a dose of 50 mg / kg. After the mice exhibited typical signs of anesthesia, such as lethargy and deep, slow breathing, the modeling process began. The shaved areas on the backs of the mice were gently wiped with cotton balls soaked in 75% alcohol for skin disinfection. After the alcohol had completely evaporated and the skin was dry, a circular area was marked on the back using an 8mm circular punch, and then a circular piece of skin tissue with a diameter of 8mm was precisely cut along the marked line. 100μL of a 2×10⁻⁶ solution was pipetted into the tissue. 6CFU / mL Candida albicans bacterial suspension was uniformly inoculated onto the wound surface of each mouse. After standing for 1 hour to allow sufficient infection, the Candida albicans infection mouse model was established. The successfully modeled mice were randomly divided into three groups: a control group, a model group, and a berberine-salicylic acid hydrogel treatment group (Gels), with 5 mice in each group (n=5). Mice in the control group underwent no bacterial infection; mice in the model group underwent only Candida albicans infection and received no further intervention; mice in the hydrogel treatment group had 5mM berberine-salicylic acid hydrogel uniformly applied to the wound surface. The entire treatment period was set at 11 days. On days 3, 5, 7, and 11, the major and minor axes of the mouse wounds were precisely measured using calipers, and the wound healing rate was calculated using ImageJ. After the treatment period, mice were euthanized by cervical dislocation, and skin tissue from the back wound was completely excised using sterile surgical scissors, flattened and fixed in paraformaldehyde fixative for subsequent histological analysis. The hydrogels prepared in this invention can effectively inhibit inflammatory responses caused by bacterial or fungal infections, reduce inflammatory factor levels, and promote wound healing. Given that the foregoing embodiments have verified that various hydrogels in this invention have inhibitory effects on multiple bacteria, those skilled in the art can reasonably expect that the hydrogels of this invention will also inhibit infections and inflammation caused by various fungi or bacteria, and promote wound healing.

[0112] Example 8

[0113] This embodiment uses the berberine-capsicumine supramolecular hydrogels prepared in Examples 1 and 2 as the research object to conduct a safety evaluation from the aspects of cytotoxicity and in vitro hemolytic properties. The specific experimental protocol is as follows:

[0114] Cytotoxicity assay: HUVECs (human umbilical vein endothelial cells), HaCat (human immortalized keratinocytes), and Raw264.7 (mouse mononuclear macrophages) were treated with berberine-salicylic acid supramolecular hydrogels at gradient concentrations (5–320 μM). The three cell types were seeded into 96-well plates. After cell attachment, different concentrations of hydrogel samples were added, and incubation was performed for 24 h and 48 h, respectively. After incubation, 100 μL of serum-free medium containing 0.5 mg / mL MTT was added to each well, and incubation continued for 4 h. Subsequently, 150 μL of DMSO was added to dissolve formazan crystals. The absorbance was measured at 490 nm using a microplate reader. Cell viability (%) was calculated using the formula: (OD0.05) = (OD0.05) / (OD0.05) / (OD0.05). 给药组 -OD 空白组 ) / (OD 正常组 -OD 空白组 Cell viability was calculated as (%) × 100%. The results showed that after treatment with different concentrations of hydrogel for 24 h and 48 h, the survival rate of the three cell types was higher than 90%, indicating that the supramolecular hydrogel had no obvious cytotoxicity.

[0115] In vitro hemolysis experiment: Fresh rat blood was used to assess the hemolytic properties of the samples. Red blood cells were separated by centrifugation at 3000 r / min for 15 min. After washing three times with physiological saline, 3 mL of red blood cells were mixed with 11 mL of physiological saline to prepare a 4% red blood cell stock solution. Berberine-salicylic acid supramolecular hydrogel was diluted with physiological saline to four concentration gradients: 40, 160, 240, and 320 μM. 100 μL of each gradient was mixed with the red blood cell stock solution and incubated at 37℃ for 4 h. After incubation, the sample was centrifuged again at 3000 r / min for 15 min, and the absorbance of the supernatant was measured at 570 nm. Deionized water (100% hemolysis positive control) and physiological saline (0% hemolysis negative control) were used in the experiment. The blood compatibility of the hydrogel was systematically evaluated using the hemolysis rate calculation formula. Hemolysis rate (%) = (A... 给药组 -A PBS组 ) / (A 去离子水组 -A PBS组 The 100% hemolysis test results showed that the berberine-capsicum supramolecular hydrogel did not exhibit significant hemolytic properties. Even at concentrations exceeding 10 times the MIC, the hemolysis rate remained below the internationally recognized standard of 5%.

[0116] The applicant declares that this invention illustrates the self-assembled carrier-free supramolecular hydrogel compound, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials used, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A hydrogel compound having the structure shown in general formula 1: in, R1 and R2 are selected from -OH, CH3-, and OCH3. R3, R4, R5, and R6 are each independently selected from -OH or -OCH3, or R3 and R4 or R5 and R6 together with their respective attached oxygen atoms form a methylenedioxy group.

2. The compound of claim 1, wherein its structure may further be as shown in general formulas 2-5: in, R1 and R2 are each preferably selected independently from -OH or -OCH3.

3. The preferred structure of the hydrogel compound as described in claims 1 and 2 is shown in Figures 1-7:

4. The hydrogel compound according to claim 1, 2 or 3, further characterized in that, Without the addition of excipients, drug molecules can self-assemble to form binary supramolecular hydrogels.

5. A composition comprising the hydrogel compound of any one of claims 1, 2, 3 or 4.

6. A method for preparing the hydrogel compound according to any one of claims 5, characterized in that, Includes the following steps: Take flavonoid aglycone and coptis alkaloid in a molar ratio of 1:0.1 to 1:10, and prepare flavonoid aglycone solution and coptis alkaloid solution respectively; Add an appropriate amount of distilled water to the flavonoid aglycone and adjust the pH of the solution to obtain a clear and transparent solution; The alkaloids were dissolved in water to obtain a Coptis chinensis alkaloid solution. The two solutions were mixed, the aggregates were collected, and washed with deionized water.

7. The hydrogel compound according to any one of claims 1, 2, 3 or 4 in the preparation of antibacterial and / or antifungal drugs.

8. The application as described in claim 7, characterized in that, The drug is used to treat infections caused by methicillin-resistant Staphylococcus aureus, Escherichia coli, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton indicum, Candida albicans, Candida parapsilosis, Candida dublinii, Candida krusei, and multidrug-resistant Candida auris.

9. The use of the hydrogel compound according to any one of claims 1, 2, 3 or 4 in the preparation of a skin wound healing medicament.

10. The application as described in claim 7, 8, or 9, characterized in that, The drug is used in the treatment of acute skin trauma, chronic ulcers, diabetic wounds, and bacterial and / or fungal skin infections, as well as in drug carriers.

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