Preparation method of 18β-glycyrrhetinic acid derivative antibacterial hydrogel

Abstract

The present invention discloses a preparation method of 18β-glycyrrhetinic acid derivative antibacterial hydrogel, comprising the following steps: S1, dissolving poloxamer and 18β-glycyrrhetinic acid derivative in an appropriate amount of anhydrous ethanol, stirring evenly to obtain a mixed solution; S2, pouring the mixed solution into a rotary evaporator, evaporating at 60°C to obtain a co-evaporated product; S3, dissolving the co-evaporated product in 60°C water and self-assembling into micelles; S4, after the micelles filter out the precipitation, collecting the filtrate and freeze-drying to obtain an inclusion compound of poloxamer inclusion 18β-glycyrrhetinic acid derivatives; S5, dissolving modified carboxymethyl chitosan in deionized water to obtain a modified carboxymethyl chitosan solution of 1wt%-10wt%, then taking 10mg of the inclusion compound in step S4 and dissolving it in 1mL of modified carboxymethyl chitosan solution, mixing evenly and irradiating with blue light of 450nm wavelength to solidify into an antibacterial hydrogel. The antibacterial hydrogel preparation process of the present invention is simple, and has good antibacterial activity and antioxidant activity.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing an antibacterial hydrogel of an 18β-glycyrrhetinic acid derivative. Background Art

[0002] The skin, the largest organ in the human body, serves as a vital barrier against external invasion; however, damage resulting from surgery, burns, and other accidents can mediate adverse health events, including bleeding and microbial infection. Uncontrolled infection and other proinflammatory factors can lead to a chronic inflammatory response and significantly delay wound healing. During the inflammatory phase, immune cells secrete proinflammatory cytokines, inducing the production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radicals (OH), and superoxide anion radicals. Excessive accumulation of ROS in infected wounds overwhelms the natural antioxidant defense system, intensifying the inflammatory response, thereby delaying wound healing and causing scarring, leading to skin dysfunction and poor cosmetic appearance. ROS-scavenging biomaterials, including polyphenols and nanozymes, have demonstrated enhanced therapeutic efficacy by boosting malfunctioning or depleted natural antioxidant mechanisms, demonstrating their utility in addressing delayed wound healing and scarless wound healing. Therefore, the development of a multifunctional therapeutic material system combining antimicrobial capabilities, ROS scavenging, and skin regeneration promotion is highly desirable.

[0003] In the past, wound treatment for chronic wounds caused by bacterial infection mostly used conventional dressings such as bandages, gauze, and cotton. Sometimes antibiotics, growth factors, exosomes, anti-inflammatory drugs, etc. were added to the dressings to further improve the treatment effect. However, these treatment strategies are limited by factors such as high cost, side effects, drug resistance, difficulty in obtaining, and instability of bioactive drugs. In recent years, the widespread development of tissue-engineered wound dressings (TEWD), such as nanofibers, gels, foams, microneedle patches, and scaffolds, has provided new treatment strategies for wound treatment of chronic wounds caused by bacterial infection. These emerging treatment strategies aim to reduce oxidative damage, promote angiogenesis, immunomodulate the ratio of M1 / M2 macrophages, and regulate inflammatory-related cytokines and extracellular matrix metalloproteinases.

[0004] Among them, gels have attracted widespread attention worldwide. Gels have considerable mechanical properties, can maintain a stable moist microenvironment for chronic wounds, and have stimulus-responsive functions, responding to external physical or chemical signals such as temperature, pH, light, reactive oxygen species (ROS), redox, enzymes, and glucose.

[0005] 18β-glycyrrhetinic acid (GA) is a major component of the root of the licorice root, a plant with medicinal and edible properties. It is the in vivo metabolite of glycyrrhizic acid in licorice after hydrolysis and removal of two molecules of glycosides. It is also a minor component of licorice. It is a white crystalline powder at room temperature with a molecular formula of C 30 H 46 O4. As an organic small molecule, its poor water solubility limits its further application. Moreover, the molecule does not show any antibacterial activity against Gram-negative bacteria. In order to increase the solubility of 18β-glycyrrhetinic acid, the method currently used is to melt disodium hydrogen phosphate and other substances at 110°C, then add 18β-glycyrrhetinic acid, and spray it out after melting. 18β-glycyrrhetinic acid is unstable at 110°C, which affects its antibacterial activity. In addition, gel is formed by self-assembly in water and organic solvents. This gel requires an organic solvent as a solvent, which increases its biological toxicity. This type of gel has poor mechanical properties and is easily deformed under external pressure, which greatly limits its practical application value. Therefore, there is an urgent need for a method for preparing an antibacterial hydrogel of glycyrrhetinic acid derivatives with simple process, good antibacterial activity and antioxidant activity. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing an antibacterial hydrogel of a glycyrrhetinic acid derivative with simple process, good antibacterial activity and antioxidant properties.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A method for preparing an antibacterial hydrogel of an 18β-glycyrrhetinic acid derivative comprises the following steps:

[0009] S1. dissolving poloxamer and 18β-glycyrrhetinic acid derivative in an appropriate amount of anhydrous ethanol and stirring to obtain a mixed solution;

[0010] S2. Pour the mixed solution into a rotary evaporator and evaporate at 60°C to obtain a co-evaporated product;

[0011] S3, dissolving the co-evaporated product in water at 60°C and self-assembling into micelles;

[0012] S4, after filtering out the precipitate through micelles, collecting the filtrate and freeze-drying it to obtain a poloxamer inclusion complex of the 18β-glycyrrhetinic acid derivative;

[0013] S5. Dissolve the modified carboxymethyl chitosan in deionized water to obtain a 1 wt%-10 wt% modified carboxymethyl chitosan solution. Then, take 10 mg of the inclusion compound in step S4 and dissolve it in 1 mL of the modified carboxymethyl chitosan solution. After mixing evenly, irradiate with 450 nm wavelength blue light to solidify into an antibacterial hydrogel.

[0014] Preferably, the poloxamer is Pluronic F-127.

[0015] Pluronic F-127 is a triblock copolymer consisting of a central poly(propylene oxide) (PPO) block flanked by two poly(ethylene oxide) (PEO) units. Self-assembled spherical Pluronic F-127 micelles exhibit excellent biocompatibility, making them excellent drug carriers. The introduction of Pluronic F-127 increases the water solubility of glycyrrhetinic acid derivatives.

[0016] Preferably, the 18β-glycyrrhetinic acid derivative is GA-O-09, with the structural formula

[0017]

[0018] Preferably, the modified carboxymethyl chitosan solution in step S5 is prepared by dissolving carboxymethyl chitosan CMCS in pure water to obtain a 10% carboxymethyl chitosan aqueous solution, slowly adding methacrylic anhydride to the carboxymethyl chitosan aqueous solution, reacting in an ice-water bath under weak alkalinity for 24 hours, and freeze-drying to obtain methacrylic anhydride-modified carboxymethyl chitosan CSMA.

[0019] Preferably, in step S5, the volume ratio of the carboxymethyl chitosan aqueous solution to methacrylic anhydride is 1:3.

[0020] Preferably, before freeze-drying in step S5, the product after the reaction is transferred to a dialysis bag and soaked in deionized water for 5 days to remove the reactants.

[0021] Preferably, the pH value of the methacrylic anhydride and carboxymethyl chitosan solution during the reaction is 9.

[0022] Preferably, the mass ratio of Pluronic F-127 to GA-O-09 is 1:1.

[0023] Beneficial effects of the present invention:

[0024] 1. By encapsulating the poorly water-soluble 18β-glycyrrhetinic acid derivative with poloxamer, its water solubility is improved, so that the 18β-glycyrrhetinic acid derivative can be directly dissolved in an aqueous solution without the need to add additional organic solvents, thereby improving the hydrophilicity and bioavailability of the drug molecule and avoiding the biological toxicity caused by the use of organic solvents.

[0025] 2. The preparation is simple and fast. It only needs to be mixed in an aqueous solution in a certain proportion and irradiated with a blue light. No high-temperature heating is required to avoid affecting the antibacterial activity of 18β-glycyrrhetinic acid derivatives due to high temperature.

[0026] 3. The raw materials are easily available and low in cost, which is beneficial to industrial mass production. Moreover, each component is environmentally friendly and non-toxic and does not cause any pollution.

[0027] 4. It has good biocompatibility and bioactivity. Experiments have shown that it has good antioxidant and antibacterial activity and has the potential to be developed into a dressing for chronic wounds infected with bacteria.

[0028] 5. Compared with the existing preparation method of 18β-glycyrrhetinic acid derivative antibacterial hydrogel, the antibacterial hydrogel of the present invention has better mechanical properties (self-healing, plasticity, adhesion), and the introduction of Pluronic F-127 and carboxymethyl chitosan enhances the antibacterial and antioxidant properties of GA-O-09, so that it has certain antibacterial activity against Gram-negative bacteria and also has good antioxidant properties, and its biological activity is better and more comprehensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron microscope image of Pluronic F-127 inclusion GA-O-09 solid particles.

[0030] Figure 2 Antibacterial hydrogels prepared from 18β-glycyrrhetinic acid derivatives with modified carboxymethyl chitosan at different mass concentrations.

[0031] Figure 3 This is a diagram showing the shaping of antibacterial hydrogel.

[0032] Figure 4 This is a diagram showing the self-healing properties of antibacterial hydrogel.

[0033] Figure 5 A diagram demonstrating the injectability of antibacterial hydrogel.

[0034] Figure 6 Demonstration of the adhesion of antibacterial hydrogel on cotton fabric.

[0035] Figure 7 Scanning electron micrographs of gels prepared with modified carboxymethyl chitosan at different mass concentrations: (a) 1%, (b) 2.5%, (c) 5%, and (d) 10%.

[0036] Figure 8 Hemolysis test of antibacterial hydrogel.

[0037] Figure 9 Cytotoxicity test of antibacterial hydrogel.

[0038] Figure 10 Antioxidant activity test of antibacterial hydrogel.

[0039] Figure 11 Antibacterial activity test of antibacterial hydrogel. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Equal mass (1 g) of Pluronic F-127 and GA-O-09 were dissolved in 50 mL of anhydrous ethanol and stirred; the solution was then evaporated in a rotary evaporator at 60°C. During this process, a uniform co-evaporate was obtained, and GA-O-09 was distributed in the Pluronic F-127 copolymer as an amorphous substance. Subsequently, the co-evaporate was dissolved in water at 60°C and self-assembled into Pluronic F-127 micelles coated with GA-O-09. The precipitate was filtered through a 0.22 mm syringe filter (Millex-LG, Millipore Co., USA), and the solution was collected, freeze-dried and stored in a refrigerator at 4°C; the Pluronic F-127 / GA-O-09 inclusion complex was prepared, and an appropriate amount of the Pluronic F-127 / GA-O-09 inclusion complex was taken for scanning electron microscopy testing, and the results are shown as follows. Figure 1 shown.

[0042] Modified carboxymethyl chitosan was prepared by dissolving a certain amount of carboxymethyl chitosan in pure water to obtain a 10% solution. Methacrylic anhydride (MA) was slowly added to the carboxymethyl chitosan aqueous solution at a mass / volume ratio of 1:3. The mixed solution was maintained at pH 9 and reacted in an ice-water bath for 24 hours. The mixed solution was then transferred to a dialysis bag (MWCO: 3500Da) and dialyzed with deionized water for 5 days to remove unreacted reactants. Finally, the bag was transferred to a freeze dryer and freeze-dried to obtain modified carboxymethyl chitosan (CSMA).

[0043] Different amounts of modified carboxymethyl chitosan powder were dissolved in deionized water to obtain CSMA solutions with mass concentrations of 1wt%, 2.5wt%, 5wt%, and 10wt%, respectively. Subsequently, 10mg of Pluronic F-127 / GA-O-09 inclusion complex was added to 1mL of the four CSMA solutions with different mass concentrations.

[0044] A certain amount of CSMA solution with inclusion complex was added to a beaker and irradiated with a blue light flashlight with a wavelength of 450nm, causing the solution to solidify into gel instantly. Figure 2 shown.

[0045] The antibacterial hydrogel prepared from 10 wt% CSMA solution was tested for plasticity, self-healing, injectability and adhesion. Figure 3-6 As shown, Figure 3The "WYU" model has clear contours and good plasticity; Figure 4 When the two parts of the antibacterial hydrogel approach each other, they converge with each other and have a high bonding strength; Figure 5 It shows that the antibacterial hydrogel can be continuously injected using a syringe; Figure 6 The antibacterial hydrogel has good adhesion to cotton fabrics commonly used in the medical field.

[0046] Figure 7 These are scanning electron micrographs of antibacterial hydrogels prepared from CSMA solutions with four different mass concentrations. As the mass concentration of the CSMA solution increases, the antibacterial hydrogel mixed with Pluronic F-127 / GA-O-09 inclusion complex has a richer cross-linked structure and better mechanical properties.

[0047] The hemocompatibility of the gels was determined using a hemolysis test. The samples were divided into five groups: PBS as a negative control, Triton X-100 as a positive control, and CSMA, CSMA / PF-127, and CSMA / PF-127 / GA-O-09 groups as experimental groups. The CSMA group gel was prepared using 10% modified carboxymethyl chitosan followed by photocrosslinking. The CSMA / PF-127 and CSMA / PF-127 / GA-O-09 groups, respectively, added 10 mg of pure Pluronic F-127 and 10 mg of the Pluronic F-127 / GA-O-09 inclusion complex to the CSMA group. Briefly, fresh rabbit blood was mixed with 3.8% sodium citrate (1:9, v / v) to prepare anticoagulated whole blood. 1 mL was transferred to a centrifuge tube and centrifuged (1000 rpm, 10 min) to obtain red blood cells, which were then washed three times with PBS (1000 rpm, 10 min). Afterwards, the red blood cells were resuspended in 5% v / v PBS and added to a 48-well plate. 500 μL of gel was placed on the bottom of the plate. After incubation for 60 minutes, the red blood cell suspension was removed and centrifuged at 1000 rpm for 10 minutes. The absorbance of the supernatant was measured at 540 nm using a microplate reader. A negative control was prepared with 500 μL of PBS, and a positive control was prepared with 500 μL of 0.1% Triton X-100. The hemolysis rate was calculated as follows:

[0048]

[0049] Where AS, APC, and ANC represent the absorbance values ​​of Triton X-100 and PBS at 540 nm, respectively. All experiments were performed three times, and the results were averaged.

[0050] The results are as follows Figure 8As shown in the figure, the hemolysis rate of each component of the gel was tested and the results showed that the hemolysis rate of each component of the gel was less than 5%, reflecting that the 18β-glycyrrhetinic acid derivative antibacterial hydrogel has good blood compatibility and will not cause red blood cells to rupture, which is conducive to its further application as a biomaterial. In particular, the hemolysis rate of the CSMA / PF-127 / GA-O-09 group was the lowest among the three groups of antibacterial hydrogels. Due to the low water solubility of GA-O-09, the hemolysis experiment was not performed on the CSMA / GA-O-09 group.

[0051] Cytotoxicity Assay: Samples were divided into four groups (blank control, CSMA group, CSMA / PF-127 group, and CSMA / PF-127 / GA-O-09 group). The CSMA group gel was prepared using 10% modified carboxymethyl chitosan followed by photocrosslinking. The CSMA / PF-127 group and the CSMA / PF-127 / GA-O-09 group, respectively, added 10 mg of pure Pluronic F-127 and 10 mg of the Pluronic F-127 / GA-O-09 inclusion complex to the CSMA group. Equal amounts of gel samples for cell-based experiments were lyophilized, sterilized with UV light, and then diluted in sterile PBS buffer to a series of concentrations (0.05, 0.1, 1, 2, 5, 10, 20, 30, and 50 mg / mL). 3T3 fibroblasts were seeded into 96-well plates at a density of 5×104 cells / well and placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 24 hours. After the cells adhered, 10 μL of sample solutions of different concentrations were added. Three parallel control groups were set up for each sample. After culturing the cells for 12 hours, the culture medium was discarded and washed with PBS. According to the operating method of the MTT kit, 10 μL of MTT solution and 100 μL of DMEM culture medium were added to each well and incubated at 37°C in the dark for 4 hours. Subsequently, the culture medium was removed, 150 μL of dimethyl sulfoxide was added to each well, and the plates were placed in an oscillating incubator (37°C, 1200 rpm) and incubated in the dark for 15 minutes. The absorbance was detected at 570 nm using an enzyme reader. In addition, 10 mg / mL of gel sample solutions of different components were selected to test the effects on cell activity at different times (1 day, 3 days, 5 days). The results are as follows: Figure 9 shown.

[0052] Through cytotoxicity tests, it was found that the sample solutions of each component of the 18β-glycyrrhetinic acid derivative antibacterial hydrogel did not cause obvious cytotoxicity to normal 3T3 fibroblasts within the gradient concentration range, and the cell viability of the fibroblasts could be maintained above 80%, reflecting that the improved antibacterial hydrogel has good biocompatibility.

[0053] The in vitro antioxidant activity of 18β-glycyrrhetinic acid derivative antibacterial hydrogels was evaluated by DPPH and PTIO scavenging assays. For the DPPH scavenging assay, freeze-dried gels of different compositions (CSMA group, CSMA / PF-127 group, and CSMA / PF-127 / GA-O-09 group; the CSMA group gel was prepared by photocrosslinking with 10% modified carboxymethyl chitosan; the CSMA / PF-127 group and the CSMA / PF-127 / GA-O-09 group were prepared by adding 10 mg of pure Pluronic F-127 and 10 mg of Pluronic F-127 / GA-O-09 inclusion complex to the CSMA group, respectively) were ground into powder. Thirty mg of the freeze-dried gel powder of each composition was treated with 3 mL of 100 μM DPPH ethanol solution and incubated in the dark for 30 minutes. After centrifugation at 3000 rpm for 10 minutes, the absorbance of the supernatant was measured at 517 nm using a microplate reader. Similarly, in the PTIO· scavenging experiment, freeze-dried gel (30 mg) was added to a PTIO· solution (3 mL, 100 μM), incubated in the dark for 60 minutes, and the absorbance of the supernatant was recorded at 557 nm. The negative and positive controls for the two experiments were PBS (300 μL) and vitamin C solution (300 μL, 400 μmL-1), respectively. The DPPH· or PTIO· scavenging rate was calculated as follows:

[0054]

[0055] Where ANC and AS are the absorbance values ​​of PBS and sample, respectively.

[0056] The results are as follows Figure 10 As shown in the antioxidant test, the sample solutions of the 18β-glycyrrhetinic acid derivative antibacterial hydrogels showed excellent DPPH· or PTIO· scavenging efficiency, and the resulting hydrogels exhibited excellent antioxidant activity. The CSMA / PF-127 / GA-O-09 group had the highest scavenging rate. Comparison between the CSMA group and the CSMA / PF-127 group showed that the addition of PF-127 enhanced the antioxidant activity of CSMA.

[0057] Antibacterial activity test: The in vitro antibacterial activity of the 18β-glycyrrhetinic acid derivative antibacterial hydrogel was evaluated using Escherichia coli (ATCC 8739) and Salmonella (ATCC 43845) as representative Gram-negative bacteria, and Staphylococcus aureus (ATCC 6538) and methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) as representative Gram-positive bacteria. The test samples were divided into five groups: PBS as a negative control, gatifloxacin as a positive control, and CSMA, CSMA / PF-127, and CSMA / PF-127 / GA-O-09 groups as experimental groups. The CSMA group gels were prepared using 10% modified carboxymethyl chitosan by photocrosslinking. The CSMA / PF-127 and CSMA / PF-127 / GA-O-09 groups added 10 mg of pure Pluronic F-127 and 10 mg of the Pluronic F-127 / GA-O-09 inclusion complex, respectively, to the CSMA group gels. Before the experiment, equal amounts of gels (1 g) in 24-well plates were sterilized under UV irradiation for 4 hours and washed with PBS. A diluted bacterial suspension (10 μL, 10⁶ CFU mL⁻¹) was evenly applied to the gel surface. After incubation at 37°C for 4 hours, the suspension was diluted 100-fold by adding 1 mL of PBS. Spread 10 μL of bacterial suspension on an LB agar plate and observe the colony growth after 12 hours.

[0058] Antibacterial results such as Figure 11 As shown in the figure, the antibacterial activity test shows that the components of the 18β-glycyrrhetinic acid derivative antibacterial hydrogel have different degrees of inhibitory effects on Gram-positive bacteria and Gram-negative bacteria, and with the introduction of the drug molecule GA-O-09, the bacterial inhibition effect is most obvious, which shows that the 18β-glycyrrhetinic acid derivative antibacterial hydrogel has good antibacterial activity; the addition of PF-127 is conducive to the improvement of the antibacterial activity of CSMA.

[0059] The above is a specific implementation of the present invention, and its description is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention, and these obvious alternative forms are all within the scope of protection of the present invention.

Claims

1. A method for preparing an antibacterial hydrogel of an 18β-glycyrrhetinic acid derivative, characterized in that: The following steps are included: S1. Dissolve poloxamer and 18β-glycyrrhetinic acid derivative in an appropriate amount of anhydrous ethanol and stir to obtain a mixed solution; S2. Pour the mixed solution into a rotary evaporator and evaporate at 60°C to obtain a co-evaporated product; S3, dissolving the co-evaporated product in water at 60°C and self-assembling into micelles; S4, after filtering out the precipitate through micelles, collecting the filtrate and freeze-drying it to obtain a poloxamer inclusion complex of the 18β-glycyrrhetinic acid derivative; S5, dissolving the modified carboxymethyl chitosan in deionized water to obtain a 1wt%-10wt% modified carboxymethyl chitosan solution, then dissolving 10 mg of the inclusion compound in step S4 in 1 mL of the modified carboxymethyl chitosan solution, mixing well, and irradiating with 450 nm wavelength blue light to solidify into an antibacterial hydrogel; the poloxamer is Pluronic F-127; the 18β-glycyrrhetinic acid derivative is GA-O-09, with the structural formula The mass ratio of the Pluronic F-127 to GA-O-09 is 1:

1.

2. The method for preparing the antibacterial hydrogel of 18β-glycyrrhetinic acid derivative according to claim 1, characterized in that: The modified carboxymethyl chitosan solution in step S5 is prepared by dissolving carboxymethyl chitosan CMCS in pure water to obtain a carboxymethyl chitosan aqueous solution with a mass fraction of 10%, slowly adding methacrylic anhydride to the carboxymethyl chitosan aqueous solution, reacting in an ice-water bath under weak alkalinity for 24 hours, and freeze-drying to obtain methacrylic anhydride-modified carboxymethyl chitosan CSMA.

3. The method for preparing the antibacterial hydrogel of 18β-glycyrrhetinic acid derivative according to claim 2, characterized in that: In step S5, the volume ratio of the carboxymethyl chitosan aqueous solution to the methacrylic anhydride is 1:

3.

4. The method for preparing the antibacterial hydrogel of 18β-glycyrrhetinic acid derivative according to claim 2, characterized in that: Before freeze-drying in step S5, the reaction product was transferred to a dialysis bag and soaked in deionized water for 5 days to remove unreacted reactants.

5. The method for preparing the antibacterial hydrogel of 18β-glycyrrhetinic acid derivative according to claim 2, characterized in that: During the reaction of the methacrylic anhydride and the carboxymethyl chitosan solution, the pH value is 9.

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