Natural self-assembled hydrogel based on antioxidant, anti-inflammatory and intestinal barrier repair functions and application thereof in treatment of inflammatory bowel disease

Abstract

The invention provides a carrier-free GMR hydrogel drug formed by self-assembly of natural active components glycyrrhizic acid (GA) and rosmarinic acid (RA) mediated by Mg < 2 + >, and the carrier-free GMR hydrogel drug is used for treating inflammatory bowel disease (IBD). The hydrogel medicine is simple in preparation method and good in gelation property. In animal models of ulcerative colitis and Crohn's disease, the GMR hydrogel shows remarkable prevention and treatment effects. The action mechanism of the compound is mainly derived from a cell protection effect, mitochondrial damage caused by oxidative stress can be relieved, and an NLRP3 / caspase-1 / GSDMD mediated pyroptosis pathway in intestinal epithelial cells is inhibited. Meanwhile, the hydrogel can restore the integrity of the intestinal barrier by up-regulating tight junction protein, and can play a comprehensive therapeutic effect on IBD by remodeling intestinal flora, enhancing the diversity of the flora and promoting the generation of short-chain fatty acid. In addition, the GMR hydrogel shows good biological safety and a simple and convenient preparation process, so that the hydrogel based on a natural product is expected to become an IBD treatment strategy with a wide application prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing hydrogels of Mg2+-mediated natural products glycyrrhizic acid (GA) and rosmarinic acid (RA), and their applications as antioxidants, mitochondrial protectants, inhibitors of pyroptosis, and upregulatory tight junction proteins. These hydrogels exhibit significant protective and therapeutic effects against ulcerative colitis (UC) and Crohn's disease (CD). Furthermore, they possess good biocompatibility, further ensuring the safety of clinical use, and belong to the field of pharmaceutical formulations. Background Technology

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disease driven by oxidative stress, intestinal barrier dysfunction, dysbiosis, and immune disorders. Current mainstream therapies, such as 5-aminosalicylic acid, biologics, and IL-23 / JAK inhibitors, all have significant limitations: including insufficient durability of efficacy, a high risk of loss of response, and the predominantly parenteral route of administration. These limitations highlight that existing therapies often cannot fully address the complex pathophysiological mechanisms of IBD, leading to incomplete disease control and frequent relapses. Therefore, developing new drugs that can achieve sustained disease control with good safety profiles has become an urgent clinical need and is currently a major focus of drug development.

[0003] Hydrogels are semi-solid or viscous liquid formulations with gel properties, made from active pharmaceutical ingredients and suitable matrices, and are the mainstream dosage form for intracavitary mucosal medications. Oral gels, as novel oral formulations, can adhere to the gastric surface to form a protective film, promoting rapid drug release and absorption (acetaminophen oral gel has been approved in my country for antipyresis). However, the matrices of existing oral gels (such as polymeric hydrogels, aluminum hydroxide / aluminum phosphate inorganic gels, and common hydrogel materials such as carbomer, cellulose, and poloxamer) are mostly inert excipients, lacking pharmacological activity themselves, and requiring a swelling process during preparation. These matrices generally suffer from easy dehydration and mold growth, and their system stability is easily affected by metal ions (for example, cellulose readily forms insoluble precipitates with cations), thus significantly limiting drug loading and drug performance. Therefore, there is an urgent clinical need for novel oral gel formulations that combine safety and efficacy.

[0004] Compared to traditional hydrogels, supramolecular self-assembled hydrogels spontaneously form three-dimensional network structures through non-covalent interactions (hydrogen bonds, π-π stacking, hydrophobic interactions, etc.), exhibiting high biocompatibility and metabolic safety, dynamic reversibility and stimulus responsiveness, and simple molecular structure with programmable functionality. They hold immense potential for biomedical applications such as drug delivery, wound healing, and tissue engineering. Traditional Chinese medicine, as a vast treasure trove of medicinal resources, provides an ideal source for developing novel functional materials with its abundant pharmacological components. Of particular note is the self-assembly characteristic of many active molecules in traditional Chinese medicine, making them a primary research target for constructing intelligent drug delivery systems and biofunctional materials using a bottom-up approach.

[0005] Given the complex and multifactorial pathogenesis of inflammatory bowel disease (IBD), this invention presents an orally administered magnesium-mediated carrier-free GA-Mg²⁺-RA (GMR) hydrogel. This system integrates natural small-molecule glycyrrhizic acid and rosmarinic acid to synergistically address the aforementioned challenges. This self-assembled hydrogel has demonstrated broad-spectrum and significant therapeutic effects in two established IBD models: ulcerative colitis and Crohn's disease. Its core therapeutic mechanism lies in its powerful cytoprotective effect—simultaneously alleviating oxidative stress-induced mitochondrial damage and inhibiting the NLRP3 / Caspase-1 / GSDMD-mediated pyroptosis pathway in intestinal epithelial cells. Therefore, the GMR hydrogel induces a comprehensive repair effect in vivo: remodeling intestinal barrier integrity by upregulating tight junction proteins (ZO-1, Occludin, and claudin-1); and simultaneously remodeling the gut microbiota by enhancing gut microbiota diversity and promoting the production of key short-chain fatty acids, thereby constructing an anti-inflammatory microenvironment. This hydrogel system combines excellent biocompatibility with a simple preparation process, making it a highly promising drug for the treatment of inflammatory bowel disease. Summary of the Invention

[0006] Based on supramolecular chemistry, this invention prepares a carrier-free supramolecular hydrogel (GMR) using glycyrrhizic acid, rosmarinic acid, and magnesium ions as raw materials. This hydrogel exhibits excellent gelation properties; it can alleviate oxidative stress-induced mitochondrial damage and inhibit pyroptosis; it can remodel the intestinal barrier integrity by upregulating tight junction proteins; and it can remodel the intestinal microbiota by enhancing microbial diversity and promoting the production of key short-chain fatty acids, thereby constructing an anti-inflammatory microenvironment. Furthermore, it has no toxic side effects and holds promise for development into an innovative drug for the treatment of inflammatory bowel disease.

[0007] Therefore, one of the objectives of this invention is to provide a GMR hydrogel.

[0008] The second objective of this invention is to provide a method for preparing GMR hydrogels.

[0009] The third objective of this invention is to provide excellent gel material properties of GMR hydrogels.

[0010] The fourth objective of this invention is to provide GMR hydrogels with good antioxidant activity and to alleviate oxidative stress-induced mitochondrial damage and inhibit pyroptosis.

[0011] The fifth objective of this invention is to provide the effect of GMR hydrogels in remodeling the integrity of the intestinal barrier by upregulating tight junction proteins.

[0012] The sixth objective of this invention is to provide the function of GMR hydrogel in reshaping the gut microbiota by enhancing microbial diversity and promoting the production of key short-chain fatty acids, thereby creating an anti-inflammatory microenvironment.

[0013] The seventh objective of this invention is to provide the application of GMR hydrogel in the prevention and treatment of IBD.

[0014] The eighth objective of this invention is to provide GMR hydrogels with good biocompatibility.

[0015] To achieve this objective, the present invention adopts the following technical solution: 1. A supramolecular hydrogel formed by the self-assembly of glycyrrhizic acid, rosmarinic acid and magnesium salt.

[0016] 2. The magnesium salt is a soluble aqueous or anhydrous inorganic magnesium salt commonly found in the art that can dissociate magnesium ions, including but not limited to magnesium chloride and magnesium sulfate.

[0017] 3. The aforementioned method for self-assembly preparation of supramolecular hydrogels includes the following steps: (1) Weigh a certain amount of glycyrrhizic acid and heat it to dissolve it in water.

[0018] (2) Weigh a certain amount of rosmarinic acid and heat it to dissolve it in water.

[0019] (3) Weigh a certain amount of inorganic magnesium salt and heat it to dissolve it in water.

[0020] (4) Mix the glycyrrhizic acid, rosmarinic acid and magnesium ion aqueous solution prepared in steps (1), (2) and (3) and heat them, then let them stand and cool.

[0021] Preferably, the heating temperature in step (1) is 60-100℃, for example 60℃, 65℃, 70℃, 80℃, 100℃.

[0022] Preferably, the heating temperature in steps (2) and (3) is 25-100℃, for example, 25℃, 30℃, 50℃, 80℃, or 100℃.

[0023] Preferably, the molar ratio of glycyrrhizic acid, rosmarinic acid, and magnesium salt is 2:1:1, 2:2:1, 2:1:2, 4:2:1, or 3:2:1. Experiments have shown that stable hydrogel drugs can only be prepared within these specific ranges; beyond these ranges, the hydrogel will disintegrate. For example, when the ratio of glycyrrhizic acid to magnesium ions exceeds 1:1, flocculent precipitates form, making it impossible to form a stable hydrogel drug system.

[0024] Preferably, the molar concentration of glycyrrhizic acid is 10-100 mM, for example, 10 mM, 20 mM, 40 mM, 70 mM, and 100 mM. Experiments have shown that if the molar concentration of glycyrrhizic acid is below 10 mM, no matter how the ratio is adjusted, it cannot assemble into a hydrogel and remains in a solution state.

[0025] 4. Rheological studies of GMR supramolecular hydrogels. The main research steps are as follows: The gel material properties of GMR supramolecular hydrogels were characterized by frequency scanning, amplitude scanning, and time scanning.

[0026] 5. This invention provides the application of the hydrogel in the preparation of antioxidant drugs. Its antioxidant effect is evaluated by measuring the scavenging rate of different free radicals.

[0027] The evaluation of the antioxidant effect of GMR hydrogel includes the following steps: (1) Prepare a 200 μM DPPH stock solution, then add GMR hydrogel, incubate in the dark for 30 minutes, and measure the absorbance at 517 nm.

[0028] (2) ABTS (7 mM) solution was reacted with potassium persulfate (K2S2O8, 2.45 mM) at room temperature in the dark for 16 h to generate ABTS•+. Then, an appropriate concentration of ABTS•+ solution was reacted with GMR hydrogel in the dark for 10 min. Finally, the absorbance at 734 nm was measured.

[0029] (3) NCM460 cells (5×10⁻⁶) 4 Cells (cells / mL) were seeded in 96-well plates and cultured overnight. After pretreatment with different concentrations of GMR hydrogel (0–40 μM) for 2 hours, oxidative stress was induced by adding 100 μg / mL Rosup for 4 hours. Cell viability was then assessed using the CCK-8 assay to quantify the cell-protective effect of the hydrogel under oxidative stress.

[0030] (4) NCM460 cells were seeded in 24-well plates (5×10⁵ cells / well), treated with GMR hydrogel for 2 hours, and then exposed to 100 μg / mL Rosup for 4 hours. Cells were stained with calcein-AM for 20 minutes and propidium iodide for 5 minutes, washed with PBS, and then observed for live and dead cells using a fluorescence microscope. Furthermore, the Annexin V-FITC / PI apoptosis detection kit was used, combined with flow cytometry to quantitatively analyze the proportion of apoptotic cells, to assess its protective effect against apoptosis.

[0031] 6. The anti-inflammatory effects of different pro-inflammatory factors in an inflammation model were evaluated by measuring their inhibition rates in different cell lines. The main research steps are as follows: Inflammation models were established by inducing mouse macrophages RAW264.7 and human normal colonic epithelial cells NCM460 with lipopolysaccharide (LPS). After incubation with GMR hydrogel for 24 hours, the effect of GMR hydrogel on inflammatory factors produced by LPS-induced macrophages was measured to evaluate its anti-inflammatory activity.

[0032] 7. Evaluate the role of DSS in the prevention and treatment of ulcerative colitis using a DSS-induced ulcerative colitis model. The main steps are as follows: (1) Preventive study: To evaluate the preventive effect, the following experiment was conducted. The experimental animals were divided into five groups (n=6 in each group): (1) control group (normal), (2) PBS group (DSS model), (3) RA group (DSS+RA solution), (4) GM group (DSS+GM hydrogel) and (5) GMR group (DSS+GMR hydrogel). After the animals adapted to the environment for 1 week, they were orally administered the corresponding intervention or 2.5% DSS aqueous solution (changed every 48 hours) for 7 days.

[0033] (2) Therapeutic intervention: To evaluate the therapeutic effect, the following experiment was conducted. Animals were divided into five groups (n=6 in each group): (1) control group (normal), (2) PBS group (DSS model), (3) RA group (DSS+RA solution), (4) GM group (DSS+GM hydrogel) and (5) GMR group (DSS+GMR hydrogel). After the animals adapted to the environment for 1 week, except for the normal control group, the other groups were allowed to drink 2.5% DSS solution freely from day 1 to day 6 to induce colitis. Subsequently, from day 7 to day 12, the corresponding drug interventions were given to each treatment group.

[0034] 8. Evaluate its role in the prevention and treatment of ulcerative colitis using a TNBS-induced Crohn's disease model. The main steps are as follows: Five groups of experimental animals were set up in this study (n=6 in each group): (1) control group (normal), (2) PBS group (TNBS model), (3) RA group (TNBS+RA solution), (4) GM group (TNBS+GM hydrogel) and (5) GMR group (TNBS+GMR hydrogel). After one week of acclimatization, the animals were fasted overnight, and then groups 2–5 were given 2.5% TNBS / ethanol enema to induce Crohn's disease. From the first day after modeling, each group was given gavage intervention for 5 consecutive days: group 2 was given PBS solution, group 3 was given RA solution, group 4 was given GM hydrogel, group 5 was given GMR hydrogel, and the control group was not treated. The animals' weight changes, fecal characteristics (consistency and occult blood), disease activity index score and general physiological status were monitored daily to assess disease progression.

[0035] 9. The regulatory effect of fecal samples from mice in the DSS-induced ulcerative colitis model on gut microbiota was evaluated by 16S rRNA sequencing analysis. The main steps are as follows: Fecal samples were collected from mice in each group, flash-frozen in liquid nitrogen, and used for microbiome analysis. Total genomic DNA was extracted from the samples using the FastDNA SPINKit for Soil, and DNA quality and concentration were validated by agarose gel electrophoresis, Nanodrop 2000, and Qubit 3.0. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 341F / 805R and sequenced on the Illumina NovaSeq 6000 platform. The raw sequences were quality-controlled filtered using DADA2 to generate amplicon sequence variants, which were then annotated using a Naive Bayes classifier based on the SILVA 138.1 database.

[0036] 10. Safety was evaluated through cell safety experiments and pathological sections of mouse liver, heart, spleen, lungs and kidneys.

[0037] (1) The MTT method was used to evaluate the survival rate of NCM460, HT-29 and HK-2 cells after 24 hours of culture in GMR hydrogel and to evaluate the cytotoxicity of the drug.

[0038] (2) Take the heart, liver, spleen, lung and kidney tissues of mice that have been drugged for 12 days, compare the pathological condition of the tissues of normal mice and the drug-treated mice, and evaluate the in vivo safety of the drug. Attached Figure Description

[0039] Figure 1 The GMR hydrogel prepared in Example 1 of this invention.

[0040] Figure 2The data are the gel rheological data of the GMR hydrogel obtained in Example 3 of this invention.

[0041] Figure 3 The fluorescence imaging and flow cytometry data of the antioxidant effect of the GMR hydrogel obtained in Example 4 of this invention on NCM460 cells are shown.

[0042] Figure 4 The relevant data for the prevention of ulcerative colitis by the GMR hydrogel obtained in Example 6 of this invention include modeling method, body weight, colon length, DAI disease index, spleen index, colon immunohistochemical sections, and antioxidant data.

[0043] Figure 5 The relevant data on the therapeutic effect of the GMR hydrogel obtained in Example 7 of this invention on ulcerative colitis include modeling method, body weight, colon length, DAI disease index, spleen index, colon immunohistochemical sections, and antioxidant data.

[0044] Figure 6 The relevant data for treating Crohn's disease with GMR hydrogel obtained in Example 8 of this invention include modeling method, body weight, colon length, DAI disease index, spleen index, and colon immunohistochemical sections. Detailed Implementation

[0045] The following embodiments are intended to further illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of the present invention.

[0046] Example 1 Hydrogels are prepared by a method including the following steps.

[0047] Preparation: Certain masses of glycyrrhizic acid, rosmarinic acid, and inorganic magnesium salt were separately dissolved in water, mixed in a specific ratio, heated, and then allowed to cool. Experiments revealed that if the ratio of glycyrrhizic acid to magnesium ions exceeded 1:1, a flocculent precipitate formed, preventing the formation of a stable hydrogel system. Furthermore, if the final molar concentration of glycyrrhizic acid was below 10 mM, it was difficult to assemble into a hydrogel regardless of the ratio, remaining in a solution state.

[0048] Preparation: Glycyrrhizic acid, rosmarinic acid, and inorganic magnesium salt were weighed and dissolved separately in water. A colorless and transparent hydrogel was prepared by dissolving the glycyrrhizic acid, rosmarinic acid, and inorganic magnesium salt in a ratio of 2:1:1, with a final concentration of glycyrrhizic acid of 10 mM (mmol / L). The macroscopic morphology is as follows: Figure 1As shown. Subsequent examples were conducted using a 10mM sample as the stock solution; the sample concentrations for pharmacological activity-related tests were calculated proportionally based on glycyrrhizic acid content.

[0049] Example 2 The hydrogel obtained in Example 1 was further characterized in terms of its gel material properties, as follows: The prepared GMR hydrogel was placed at room temperature and observed at intervals to determine its stability. The prepared hydrogel was heated to dissolve it and then allowed to cool at room temperature. It was observed whether it could self-assemble into a hydrogel again to determine its thermosensitivity. The prepared hydrogel was shaken to destroy its structure and then allowed to stand at room temperature to determine whether it could recover into a hydrogel to determine its self-healing property. The prepared hydrogel was added to a syringe and then gently ejected to determine its injectability.

[0050] The results showed that the GMR hydrogels remained intact for at least 120 days at room temperature, demonstrating good stability. After heating and then cooling to room temperature, they self-assembled to form hydrogels again, indicating their thermosensitivity. After shaking and then allowing to stand, they recovered to form hydrogels, demonstrating their self-healing properties. The hydrogels could be easily ejected with a syringe, indicating their injectability. These results indicate that GMR hydrogels possess excellent gel properties such as good stability, thermosensitivity, self-healing, and injectability, making them ideal biomedical materials for clinical application.

[0051] Example 3 In this embodiment, the rheological tests of the GMR hydrogel prepared in Example 1 were performed using the following method: The GMR hydrogel was placed on the rheometer measuring plate with a gap of 5 mm and a temperature of 25℃. Time scan: the strain was kept constant at 0.1%, and the measurement frequency range was 1 Hz. Frequency scan: the strain was kept constant at 0.1%, and the measurement frequency range was 0.1 Hz–10 Hz. Amplitude scan: the frequency was set to a constant 1 Hz, and the strain variation range was 0.001%–10%, obtaining the storage modulus (G′) and loss modulus (G′′) as a function of strain and frequency. Under changes in frequency and shear stress, the storage modulus was greater than the loss modulus, indicating that it possesses excellent rheological properties.

[0052] Example 4 In this embodiment, the antioxidant activity of the GMR hydrogel prepared in Example 1 was determined, and the method is as follows: (1) Prepare a 0.2 mmol / L DPPH solution with anhydrous ethanol and store it in the dark. Then prepare test samples of different concentrations with anhydrous ethanol. All samples have the same molar concentration (600 μM). Add 2 mL of the test sample solution and 2 mL of DPPH solution to the same test tube, shake well, and let it stand at room temperature in the dark for 30 minutes before measuring its absorbance A at 517 nm. sample Simultaneously, the absorbance A0 of a mixture of 2 mL DPPH solution and 2 mL anhydrous ethanol was measured. Finally, the absorbance was calculated using the formula: DPPH clearance percentage = (A0 - A) sample ) / A0*100% The specific results are shown in Table 1.

[0053] Table 1: Scavenging rate of DPPH by GMR hydrogel GMR hydrogel (%) GM hydrogel (%) RA (%) GMR solution (%) 97.92±0.26 8.38±1.22 63.25±0.28 64.25±1.48 (2) ABTS (7 mM) solution was reacted with potassium persulfate (K2S2O8, 2.45 mM) at room temperature in the dark for 16 h to generate ABTS•+. Then, an appropriate concentration of ABTS•+ solution was reacted with the test sample (all samples had a molar concentration of 600 μM) under dark conditions for 30 min. Finally, the absorbance at 734 nm was measured. The results were then calculated according to the formula: ABTS•+ clearance percentage = A0 - (A sample -A x0 ) / A0x100% Where A0 represents the absorbance of the blank control group, A sample A represents the absorbance of the sample group. x0 The absorbance is without the sample. See Table 2 for specific results.

[0054] Table 2: Scavenging rate of ABTS•+ by GMR hydrogel GMR hydrogel (%) GM hydrogel (%) RA (%) GMR solution (%) 90.227±1.26 3.25±1.28 59.25±1.28 62.25±1.52 The results showed that the antioxidant activity of GMR hydrogel was significantly enhanced compared to the monomer and the mixture of the two, and it is worthy of further research and clinical development.

[0055] (3) NCM460 cells (5×10⁻⁶) 4 Cells (cells / mL) were seeded in 96-well plates and cultured overnight. After pretreatment with different concentrations of GMR hydrogel (0–40 μM) for 2 hours, oxidative stress was induced for 4 hours with 100 μg / mL Rosup. Cell viability was then assessed using the CCK-8 assay to quantify the cell-protective effect of the hydrogel under oxidative stress. The results showed that the GMR hydrogel could alleviate oxidative stress-induced cell death and increase cell viability. See attached figure for detailed results. Figure 3 .

[0056] (4) NCM460 cells were seeded into 24-well plates (5 × 10⁻⁶ cells per well). 5 Cells / wells were treated with GMR hydrogel (40 μM) for 2 hours and then exposed to 100 μg / mL Rosup for 4 hours. After calcein-AM staining for 20 minutes and propidium iodide staining for 5 minutes, and washing with PBS, live and dead cells were observed under a fluorescence microscope. Furthermore, the proportion of apoptotic cells was quantitatively analyzed using the Annexin V-FITC / PI apoptosis detection kit combined with flow cytometry to assess its protective effect against apoptosis. The results showed that GMR hydrogel could alleviate oxidative stress-induced cell death, increase cell viability, and also exhibit mitochondrial protective effects. Detailed results are attached. Figure 3 .

[0057] Example 5 In this embodiment, the anti-inflammatory activity of the GMR hydrogel prepared in Example 1 was evaluated using different cell lines, and the methods are as follows: 1. Using LPS-induced mouse macrophage RAW 264.7 as a model, the effects of the prepared GMR hydrogel (40 μM) on inflammatory factors (TNF-α, IL-1β, IL-6, and NO) produced by LPS-induced macrophages were measured using an ELISA kit to evaluate its anti-inflammatory activity. Specific results are shown in Table 3.

[0058] Table 3: Effects of GMR hydrogel on inflammatory factors in mouse macrophage RAW 264.7 cells GMR hydrogel GM hydrogel RA GMR solution Model TNF-α (pg / mL) 43.03±1.06 156.05±1.27 236.98±1.25 152.25±1.83 458.18±1.77 IL-1β (pg / mL) 39.04±1.67 105.65±2.56 387.68±0.68 106.73±1.84 428.18±1.77 IL-6 (pg / mL) 8.29±2.67 29.53±0.08 34.55±1.56 31.29±1.48 157.26±2.33 NO (pg / mL) 13.04±1.07 145.09±1.12 168.56±0.47 145.09±0.25 269.94±0.25 2. Using LPS-induced normal human colonic epithelial cells NCM460 as a model, the effects of GMR hydrogel (40 μM) on inflammatory factors (TNF-α, IL-1β, IL-6, and NO) produced by LPS-induced macrophages were measured using an ELISA kit to evaluate its anti-arthritis activity. Specific results are shown in Table 4.

[0059] Table 4: Effects of GMR hydrogel on inflammatory factors in NCM460 cells of normal human colonic epithelial cells GMR hydrogel GM hydrogel RA GMR solution Model TNF-α (pg / mL) 35.34±1.24 94.27±1.56 105.34±1.24 92.36±1.28 156.22±0.79 IL-1β (pg / mL) 24.65±2.23 72.21±2.23 74.32±2.23 74.28±1.23 136.12±0.43 IL-6 (pg / mL) 22.22±2.33 98.37±2.74 88.87±1.08 89.26±1.53 145.29±1.29 NO (pg / mL) 18.23±1.66 89.36±2.64 98.27±1.36 92.25±2.19 247.26±1.57 The above results indicate that GMR hydrogel has a significant inhibitory effect on inflammatory factors produced by LPS-induced mouse macrophages RAW 264.7 and human normal colonic epithelial cells NCM460, exhibiting strong anti-inflammatory activity. Furthermore, since it does not assemble into a hydrogel assembly, it has value for in-depth research and further clinical development.

[0060] Example 6 In this embodiment, the efficacy of the GMR hydrogel prepared in Example 1 in preventing ulcerative colitis was evaluated in vivo, and the method is as follows: Male C57bl / 6J mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. Mice were randomly divided into five groups (n=6 per group): (1) control group (normal), (2) PBS group (DSS model), (3) RA group (DSS+RA solution), (4) GM group (DSS+GM hydrogel) and (5) GMR group (DSS+GMR hydrogel). After the animals acclimatized to the environment for 1 week, group (1) was not treated, while groups (2)-(5) were given free access to 2.5% DSS aqueous solution and were orally administered 300μL of the corresponding intervention by gavage for 7 days. After the 7-day treatment period, the mice were sacrificed by cervical dislocation. All tissues were collected, and colon tissue samples were fixed in 4% paraformaldehyde, dehydrated, cleared in xylene, and embedded in paraffin. Sections with a thickness of 5-10μm were prepared using a microtome and attached to glass slides. Paraffin sections were dewaxed and hydrated, while frozen sections were washed with PBS before staining. All sections were mounted after various staining processes for pathological evaluation of tissue structure and cell morphology.

[0061] The results showed that mice treated with 2.5% DSS experienced significant weight loss compared to the control group and other treatment groups. Compared to the 2.5% DSS group, the GMR treatment group showed significantly reduced weight loss, suggesting that GMR hydrogel may have a therapeutic effect on UC. Colon length is another key indicator for assessing disease severity in IBD models, and all experimental groups showed significant shortening: 2.5% DSS group (5.1±0.37 cm), RA group (5.1±0.40 cm), and GM group (5.8±0.26 cm). Notably, GMR hydrogel intervention effectively maintained colonic structure, restoring its length to 6.4±0.12 cm, indicating its superior efficacy in reducing inflammation-induced colonic contractions. DAI score is a recognized indicator for assessing the severity of colitis. DSS-induced mice exhibited significant clinical deterioration, with a marked increase in diarrhea and hematochezia. Although RA and GM treatments provided limited improvement, the GMR hydrogel group significantly delayed the progression of DAI, indicating its effective regulation of disease progression. Observations of the mouse anus and bedding further confirmed the above findings: DSS-treated mice exhibited severe symptoms such as loose stools, diarrhea, and bloody stools, while GMR hydrogel intervention maintained fecal characteristics and perianal appearance at levels close to those of the healthy control group. Regarding UC-induced inflammation, the spleen index (the ratio of spleen mass to body weight) increases due to splenomegaly. GMR hydrogel demonstrated a significant therapeutic effect during UC treatment. Unlike the 2.5% DSS group, mice treated with RA or GM had higher spleen indices, but there was no significant difference compared to the 2.5% DSS group. In contrast, GMR hydrogel not only significantly inhibited the increase in spleen index but also restored spleen size to a level comparable to the control group. These findings indicate that GMR possesses potent anti-inflammatory properties and can effectively improve the pathological state of UC. Further H&E staining of colonic tissue showed that 2.5% DSS treatment induced significant histopathological changes, including mucosal erosion, dense inflammatory cell infiltration, and crypt structure destruction. Notably, GMR hydrogel treatment not only maintained normal epithelial morphology but also significantly reduced the recruitment of inflammatory cells in the mucosal layer, suggesting its dual role in tissue protection and anti-inflammatory regulation. Furthermore, 2.5% DSS treatment significantly reduced the expression levels of three tight junction proteins, while GMR hydrogel treatment effectively restored the expression of ZO-1, Occludin, and Claudin-1 to near-normal levels. These results collectively demonstrate the superior ability of GMR hydrogel in alleviating the clinical and macroscopic manifestations of IBD. See the appendix for detailed results. Figure 4 .

[0062] Example 7 In this embodiment, the efficacy of the GMR hydrogel prepared in Example 1 for the in vivo treatment of ulcerative colitis was evaluated, and the method is as follows: Male C57bl / 6J mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. Mice were randomly divided into five groups (n=6 per group): (1) control group (normal), (2) PBS group (DSS model), (3) RA group (DSS+RA solution), (4) GM group (DSS+GM hydrogel), and (5) GMR group (DSS+GMR hydrogel). After one week of acclimatization, except for the normal control group, the other groups were given free access to 2.5% DSS solution for days 1-6 to induce colitis. Subsequently, from days 7 to 12, each treatment group was administered 300 μL of the corresponding drug orally via gavage. At the end of the experiment, mice were euthanized by cervical dislocation. Tissues were collected, with colon tissue samples fixed in 4% paraformaldehyde, dehydrated, cleared with xylene, and embedded in paraffin. Sections with a thickness of 5–10 μm were prepared using a microtome and attached to glass slides. Paraffin sections were dewaxed and hydrated, while frozen sections were washed with PBS before staining. All sections were mounted after various staining processes for pathological evaluation of tissue structure and cell morphology.

[0063] Longitudinal monitoring results showed that the mice in each group exhibited different physiological responses. The control group mice showed steady weight gain and a stable disease activity index, confirming their healthy growth. In stark contrast, mice in the 2.5% DSS model group and the RA treatment group showed progressive deterioration, manifested as significant weight loss and a sustained increase in DAI scores. Notably, both GM and GMR hydrogel interventions showed protective effects against these pathological changes, with GMR hydrogel showing more significant effects in reducing weight loss and inhibiting disease progression. Morphological assessment revealed a clear efficacy rank: the colon length in the GMR hydrogel treatment group was closest to that of the healthy control group, significantly superior to other groups. The colonic structure of GMR hydrogel-treated mice was similar to that of normal physiological conditions, and their clinical manifestations were significantly improved, with minimal fecal blood, a stark contrast to the severe bloody stools and diarrhea in other groups, fully establishing the superior efficacy of GMR hydrogel in the management of colitis. Furthermore, quantitative assessment of the spleen index showed that the value in the GMR hydrogel treatment group was highly similar to that of the healthy control group and significantly lower than that in the model group and other treatment groups, indicating that GMR hydrogel has an excellent ability to reduce systemic inflammatory responses. H&E staining of colonic tissue sections revealed a clear treatment gradient among the groups. The mucosa morphology of control mice remained intact, while 2.5% DSS treatment induced typical UC pathological changes. Although RA and GM treatments provided partial protection against these changes, GMR hydrogel achieved the most significant tissue repair. This superior histopathological result makes GMR hydrogel a potential therapeutic candidate that can simultaneously promote anti-inflammatory and regenerative processes in the colonic epithelium. Quantitative immunohistochemical evaluation of intestinal barrier markers showed that GMR hydrogel treatment significantly enhanced the expression of tight junction proteins in the colonic epithelium. This morphological evidence was strongly supported and quantitatively validated by Western blot analysis. The significant restoration of these structural proteins, which are crucial for maintaining epithelial barrier integrity, provides direct molecular evidence that GMR hydrogel can effectively repair the damaged mucosal barrier in experimental colitis, achieving a dual therapeutic benefit of barrier strengthening and inflammation control. ELISA quantitative analysis of colonic tissue cytokines showed that GMR hydrogel treatment significantly downregulated pro-inflammatory mediator levels while restoring the anti-inflammatory factor IL-10 to near-normal levels. This synergistic cytokine regulation provides strong molecular evidence that GMR hydrogels improve UC through a dual mechanism of inhibiting inflammation and alleviating oxidative stress. See attached results for details. Figure 5 .

[0064] Example 8 In this embodiment, the efficacy of the GMR hydrogel prepared in Example 1 for in vivo treatment of Crohn's disease, a clinically relevant IBD subtype characterized by transmural inflammation, is evaluated as follows: Male C57bl / 6J mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. Mice were randomly divided into 5 groups (n=6 per group): (1) control group (normal), (2) PBS group (TNBS model), (3) RA group (TNBS+RA solution), (4) GM group (TNBS+GM hydrogel), and (5) GMR group (TNBS+GMR hydrogel). After one week of acclimatization, the animals were fasted overnight. Groups 2–5 were then given 2.5% TNBS / ethanol enema to induce Crohn's disease. From day 1 after modeling, each group was administered gavage daily for 5 consecutive days: Group 2 received PBS solution, Group 3 received 300μL RA solution, Group 4 received 300μL GM hydrogel, Group 5 received 300μL GMR hydrogel, and the control group received no treatment. Daily monitoring of animal weight changes, fecal characteristics (consistency and occult blood), disease activity index score, and general physiological status is used to assess disease progression.

[0065] A comprehensive assessment of body weight changes, DAI scores, and colon length in TNBS-induced colitis mice demonstrated the therapeutic efficacy of GMR hydrogel. Results showed that mice treated with GMR hydrogel exhibited significantly improved outcomes and reduced body weight loss compared to the TNBS model group, indicating its effective mitigation of colitis progression. Morphometry analysis of colon length revealed outstanding therapeutic effects: GMR hydrogel treatment maintained intestinal size at near-physiological levels, achieving 94.4% of the recovery rate of the healthy control group, a 53.6% improvement compared to untreated TNBS colitis mice. This demonstrates the superior ability of GMR hydrogel to maintain intestinal structure under inflammatory challenges. DAI scores showed a significant increase in TNBS-treated mice by day 5, reflecting the progression of colitis symptoms. However, after GMR hydrogel treatment began on day 2, DAI scores significantly decreased, demonstrating the hydrogel's therapeutic potential in alleviating disease severity. As a primary site of immune cell residence and activation, the spleen exhibited significant pathological changes in the untreated group. Notably, GMR hydrogel treatment showed significant efficacy, with substantial recovery in spleen index and spleen size compared to the untreated control group. These findings indicate that GMR hydrogel not only alleviates local inflammatory damage but also effectively modulates the systemic immune response by regulating spleen function. Histological examination revealed that TNBS administration induced severe mucosal damage, characterized by extensive inflammatory cell infiltration and significant epithelial destruction. Conversely, GMR hydrogel treatment promoted significant tissue repair, with H&E staining revealing epithelial regeneration and restoration of crypt structures. These results collectively suggest that GMR hydrogel exerts a protective effect against TNBS-mediated colonic pathology through structural protection and anti-inflammatory mechanisms. Immunohistochemical staining analysis of intestinal barrier function revealed that GMR hydrogel treatment significantly enhanced the expression of key tight junction proteins in colonic tissue compared to untreated colitis mice. This significant upregulation of key junctional components suggests that GMR hydrogel protects intestinal mucosal integrity by enhancing the structural and functional components of the epithelial barrier. These molecular-level findings, consistent with observed treatment outcomes, confirm that GMR hydrogel-mediated restoration of the tight junction complex plays a crucial role in repairing the damaged intestinal barrier in experimental colitis. Furthermore, dysregulation of the colonic immune response is a key characteristic of ulcerative colitis. The TNBS-induced colitis model is a well-established experimental system capable of reproducing this pathological process: the haptenizing agent TNBS disrupts the epithelial barrier, triggering a strong T-cell-mediated inflammatory response primarily driven by Th1 and Th17 responses. This is reflected in the extensive infiltration of CD3⁺ T cells into the colonic mucosa and submucosa. Against this pro-inflammatory backdrop, our investigation of the efficacy of GMR treatment revealed its significant immunomodulatory effects. Histological evaluation showed that GMR treatment significantly reduced the severity of colitis and substantially decreased CD3⁺ T-cell infiltration.The key is that this suppression of effector T cell responses is accompanied by a significant expansion of FoxP3⁺ regulatory T cells within the lamina propria. This crucial shift in the T cell landscape from a pro-inflammatory to a regulatory phenotype strongly suggests that GMR improves TNBS-induced colitis not merely through widespread immunosuppression, but by actively promoting immune tolerance and restoring mucosal homeostasis, with the potential mechanism possibly related to enhanced Treg-mediated inhibitory function. These findings confirm that GMR hydrogels represent a potential therapeutic strategy for targeted intervention in the pathogenesis of Crohn's disease by modulating inflammatory pathways. Specific results are attached. Figure 6 .

[0066] Example 9 In this embodiment, 16S rRNA sequencing analysis was performed on fecal samples from each group of mice in Example 6 to evaluate its regulatory effect on the gut microbiota. The main steps are as follows: Fecal samples were collected from mice in each group, flash-frozen in liquid nitrogen, and used for microbiome analysis. Total genomic DNA was extracted from the samples using the FastDNA SPINKit for Soil, and DNA quality and concentration were validated by agarose gel electrophoresis, Nanodrop 2000, and Qubit 3.0. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 341F / 805R and sequenced on the Illumina NovaSeq 6000 platform. The raw sequences were quality-controlled filtered using DADA2 to generate amplicon sequence variants, which were then annotated using a Naive Bayes classifier based on the SILVA 138.1 database.

[0067] Microbiome analysis showed that GMR hydrogel treatment significantly promoted gut microbial restoration, a finding consistently corroborated by multidimensional diversity assessments. Alpha diversity analysis revealed that GMR hydrogel treatment significantly increased microbial richness and community diversity compared to the model group. Although the effect of GMR intervention on the Shannon index was not statistically significant, it still showed a trend towards increasing alpha diversity. Beta diversity analysis further indicated that the microbial community composition of the GMR hydrogel-treated group differed significantly from other intervention groups, exhibiting a unique community reorganization pattern. Notably, Venn diagram analysis showed that GMR hydrogel treatment restored bacterial OTU abundance to levels close to those of the healthy control group, a stark contrast to the severe species depletion observed in the DSS model group.

[0068] Further taxonomic analysis revealed the beneficial remodeling capabilities of GMR hydrogels at both the phylum and family levels in the gut microbiota. At the phylum level, DSS-induced dysbiosis manifested as a decrease in Firmicutes and an expansion of Proteobacteria, while GMR hydrogel treatment uniquely enriched Bacteroidetes, a shift conducive to the formation of anti-inflammatory flora. More importantly, family-level analysis showed that GMR hydrogels have a dual regulatory effect: significantly inhibiting pro-inflammatory Enterobacteriaceae while selectively enriching beneficial anti-cancer Mansoniaceae.

[0069] Through LEfSe-LDA biomarker analysis, we identified key microbial taxa at multiple taxonomic levels that define treatment-specific ecological patterns. LDA scores quantitatively measured the contribution of each biomarker to community diversity, revealing how GMR hydrogels induce unique microbial signatures, thus differentiating them from other treatment groups. These findings not only validate the aforementioned β-diversity results but also characterize specific bacterial lineages at the molecular level, including enriched beneficial bacteria and reduced pathogens, thereby mechanistically explaining the therapeutic remodeling of the intestinal ecosystem in colitis by GMR hydrogels.

[0070] Furthermore, heatmaps showed the relative abundance of gut microbiota at the genus and phylum levels for each group. Bar chart analysis at the genus level indicated that GMR hydrogel increased the abundance of beneficial bacteria, including *Akkermansia*, *Verrucous*, and *Bacteroidetes*. Simultaneously, GMR hydrogel treatment significantly suppressed pro-inflammatory bacteria, demonstrating its ability to reverse colitis-associated dysbiosis. This precise microbial landscape remodeling—promoting protective symbiotic bacteria while suppressing disease-associated pathogens—provides a mechanistic basis for the therapeutic efficacy of GMR hydrogel in IBD.

[0071] Short-chain fatty acids (SCFAs) play a crucial role in the pathogenesis of IBD, maintaining intestinal barrier integrity, regulating immune responses, and preserving microbial homeostasis through multiple mechanisms. GC-MS quantitative analysis showed that, compared to the DSS model group, GMR hydrogel treatment significantly increased the concentration of key SCFAs in feces. Butyrate, in particular, became a critical therapeutic mediator through its dual-pathway mechanism: firstly, it directly strengthens intestinal barrier integrity by enhancing tight junction protein expression; secondly, it simultaneously activates the Nrf2 signaling pathway to upregulate key antioxidant enzymes. Through this synergistic effect, GMR hydrogel simultaneously targets two interrelated pathological features of IBD—oxidative stress and inflammation—which constitutes the core mechanism behind its observed therapeutic effect.

[0072] Further Spearman correlation analysis revealed significant microbe-metabolite-immune associations. The analysis showed that *Akkermania* was positively correlated with IL-10 but weakly correlated with SCFAs; *Escherichia coli*-*Shigella* was negatively correlated with butyrate and positively correlated with pro-inflammatory cytokines; *norank_o__Clostridia_UCG-014* was strongly negatively correlated with IL-10 and SCFAs and positively correlated with pro-inflammatory cytokines; *Turicibacter* was strongly negatively correlated with propionic acid and positively correlated with IL-1β. These findings present a pattern of interactions within the gut ecosystem, and while they do not imply causation, they provide valuable information for understanding their intrinsic connections. The positive correlation between *Akkermania* and IL-10 is consistent with experimental observations and warrants further mechanistic exploration.

[0073] Example 10 In this embodiment, the supramolecular hydrogel prepared in Example 1 was evaluated for in vitro safety using the following method: NCM460, HT-29, and HK-2 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) and all cells were placed in a humidified incubator at 37°C and 5% CO2. After the cells reached a suitable density, they were treated with different concentrations (0–40 μM) of GMR hydrogel for 24 hours. The drug-containing medium was discarded, the cells were washed with PBS, and then serum-free medium containing 10% CCK-8 reagent was added, followed by incubation for another hour. Finally, the absorbance was measured using a microplate reader to calculate cell viability.

[0074] The results showed that the hydrogel exhibited excellent cell tolerance in NCM460, HT-29 and HK-2 cell lines, and cell viability was maintained at all tested concentrations.

[0075] Example 11 In this embodiment, the supramolecular hydrogel prepared in Example 1 was evaluated for in vivo safety using the following method: Male C57bl / 6J mice aged 6-8 weeks and weighing 16-18g were used as experimental animals. Mice were randomly divided into four groups: control group (drinking water), RA group, GM group, and GMR group (all administered 300μL of the drug via gavage). After 7 consecutive days of drug administration, the mice were observed for another 7 days, after which they were sacrificed, and samples of major organs and blood were collected for analysis. Histopathological evaluation was performed by H&E staining, and hematological and biochemical parameters of whole blood were detected.

[0076] In vivo safety analysis showed that, compared with the untreated control group, GMR hydrogel did not cause clinically relevant changes in liver and kidney function markers, and the tissue structure of major organs remained normal.

Claims

1. A carrier-free self-assembling hydrogel, characterized in that, The supramolecular hydrogel is composed of glycyrrhizic acid, rosmarinic acid and magnesium salt.

2. The supramolecular hydrogel of claim 1, wherein the molar ratio of glycyrrhizic acid, rosmarinic acid and magnesium salt is 1-5: 1-5: 1-5, preferably the molar ratio of glycyrrhizic acid, rosmarinic acid and magnesium salt is 2: 1: 1, 2: 2: 1, 2: 1: 2, 4: 2: 1 or 3: 2:

1.

3. The supramolecular hydrogel of any one of claims 1-2, wherein the magnesium salt is magnesium sulfate, magnesium chloride or magnesium acetate with or without water.

4. A method for preparing a supramolecular hydrogel, characterized by, The method comprises the following steps: (1) weigh a certain amount of glycyrrhizic acid and heat to dissolve in water; (2) weigh a certain amount of rosmarinic acid and heat to dissolve in water; (3) weigh a certain amount of inorganic magnesium salt and heat to dissolve in water; (4) mix and heat the glycyrrhizic acid, rosmarinic acid and magnesium salt aqueous solutions prepared in steps (1), (2) and (3), and cool.

5. The method of any one of claims 4, wherein the heating temperature in step (1) is 60-100℃, or wherein the heating temperature in steps (2) and (3) is 25-100℃.

6. The supramolecular hydrogel prepared by the self-assembly method of any one of claims 4-5.

7. Use of the supramolecular hydrogel of any one of claims 1-3 or 6 in the preparation of an antioxidant drug.

8. Use of the supramolecular hydrogel of any one of claims 1-3 or 6 in the preparation of an inflammatory bowel disease (including ulcerative colitis and Crohn's disease) drug.

9. The supramolecular hydrogel of any one of claims 1-3 or 6, or the medicament of any one of claims 7, 8, further characterized by Prepared into a pharmaceutically acceptable dosage form, preferably prepared into an oral drug, an external or transdermal drug, further preferably into an oral gel, a tablet, a capsule, a soft capsule, an external gel, an injectable hydrogel. Prepared into a pharmaceutically acceptable dosage form, preferably prepared into an oral drug, an external or transdermal drug, further preferably into an oral gel, a tablet, a capsule, a soft capsule, an external gel, an injectable hydrogel.

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