Iron ion-isoliquiritigenin nanocomposite, preparation method thereof and application thereof in preparation of medicine for treating inflammatory bowel disease

Abstract

The application discloses an iron ion-liquiritigenin nanocomposite, which is prepared from liquiritigenin or a derivative thereof and Fe 3+ salt as raw materials in the presence of polyvinylpyrrolidone, and is formed through coordination of the iron ion and the liquiritigenin or the derivative thereof. The iron ion-liquiritigenin nanocomposite can effectively target inflammatory lesions in the intestinal tract and stay in intestinal tissues, effectively treat intestinal inflammation, reverse the fibrosis process, relieve iron deficiency caused by inflammatory bowel disease, realize multidimensional treatment of the inflammatory bowel disease, and synchronously block the vicious cycle of "inflammation-barrier damage-fibrosis". The application further discloses application of the iron ion-liquiritigenin nanocomposite in preparation of a medicine for treating the inflammatory bowel disease and iron deficiency caused by the inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the fields of nanomaterials for pharmaceuticals and medicine, and relates to an iron ion-isoglycyrrhizin nanocomposite (ISL-Fe), its preparation method and its application in the preparation of drugs for treating inflammatory bowel disease. Specifically, it relates to an iron ion-isoglycyrrhizin nanocomposite, its preparation method and the application of the composite in treating inflammatory bowel disease by targeting the inflammatory site and regulating cannabinoid type 2 receptors. Background Technology

[0002] Inflammatory bowel disease (IBD) mainly includes ulcerative colitis (UC) and Crohn's disease (CD). It is a global disease characterized by chronic, relapsing gastrointestinal inflammation, and its complex pathogenesis poses a great challenge to clinical treatment.

[0003] Currently, available clinical treatments for IBD mainly include surgical and drug therapies, such as aminosalicylic acid, glucocorticoids, immunomodulators, and biologics. Surgical treatment is expensive and carries risks such as postoperative complications and decreased quality of life, resulting in poor patient adherence. Clinical drug therapies generally suffer from low overall efficacy and significant side effects; long-term use may cause serious toxicity to normal tissues. Safe and highly effective treatments remain scarce in clinical practice.

[0004] Studies have shown that cannabinoid receptor 2 (CB2R) is an effective target for the treatment of colitis. However, most CB2R ligands have poor pharmacokinetic properties, exhibiting high lipid solubility, low solubility, high in vivo clearance, low oral bioavailability, and lack of targeting ability, which limits their further application in the treatment of IBD.

[0005] Compared to intraperitoneal, intravenous, and rectal administration, oral administration is considered an ideal route for treating gastrointestinal diseases due to its significant advantages, including safety, convenience, and the absence of the need for specialized medical intervention. However, the unique characteristics of the gastrointestinal tract, including its exposure to gut microbiota, rhythmic peristalsis, acid-base changes, absorption, and excretion, along with its physiological environment, can easily lead to drug degradation, efflux, off-target effects, and low or short exposure levels at pathological sites, severely impacting drug efficacy. Therefore, developing a novel formulation that not only treats colitis but also protects the drug from gastric acid degradation and achieves targeted accumulation against inflammation has become crucial for overcoming the current challenges in IBD treatment.

[0006] Intestinal fibrosis is a serious complication of IBD progression, characterized by thickening and rigidity of the intestinal wall, ultimately leading to intestinal stenosis and bowel dysfunction. Studies have shown that chronic, recurrent inflammation in the intestines of IBD patients can drive fibroblast activation and excessive extracellular matrix deposition, thereby triggering fibrotic changes. However, research on the mechanisms of intestinal fibrosis and the development of intervention strategies remain significant gaps in IBD treatment. Existing research largely focuses on controlling acute inflammation, with insufficient attention paid to fibrotic lesions caused by chronic colitis, and there are very few reports on drug delivery systems with both anti-inflammatory and anti-fibrotic functions.

[0007] Furthermore, IBD patients often experience iron deficiency due to factors such as chronic intestinal blood loss, inflammatory factors inhibiting iron absorption, and decreased appetite. Iron deficiency can worsen fatigue symptoms and affect quality of life, while routine oral iron supplementation may exacerbate intestinal inflammation, creating a treatment dilemma. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing an iron-isoglycyrrhizin nanocomposite (ISL-Fe). This ISL-Fe nanocomposite is a nanocomposite formed by the coordination self-assembly of the flavonoid isoliquiritigenin (ISL) with iron ions. The metal polyphenol coordination nanostructure improves the water solubility, in vivo pharmacokinetic properties, and therapeutic effect on inflammatory bowel disease (IBD) by targeting the inflammatory site and regulating CB2R. Specifically, isoliquiritigenin exerts its therapeutic effect on IBD by regulating CB2R. The coordination of ferric ions improves the water solubility and in vivo pharmacokinetic properties of isoliquiritigenin after oral administration. The introduction of polyvinylpyrrolidone makes the nanocomposite electrically neutral, which facilitates the retention of nanoparticles in the colon, providing a structural basis for further prolonging the residence time of isoliquiritigenin in colonic tissue.

[0009] To address the problems of the prior art, the present invention provides the following technical solution:

[0010] An iron ion-isoglycyrrhizin nanocomposite, wherein the iron ion-isoglycyrrhizin nanocomposite is made from isoliquiritin or its derivatives and iron salts, in the presence of polyvinylpyrrolidone, by isoliquiritin or its derivatives and iron salts. 3+ Coordination-formed nanocomposite materials.

[0011] The mass ratio of isoliquiritigenin or its derivative to iron salt is (1-4):(1-4), preferably (2-2.5):(0.8-1.2), and more preferably 2:1-2.5:1.

[0012] The mass ratio of polyvinylpyrrolidone to isoliquiritigenin is (4-12):(0.5-2), preferably (5-8):(0.8-1.2), and more preferably 6:1.

[0013] In the aforementioned iron ion-isoglycyrrhizin nanocomposite, the Fe atom content is 6.0 wt%, and each 2 molecules of isoglycyrrhizin are coordinated and complexed with 3 Fe atoms. 3+ .

[0014] The molecular formula of isoliquiritigenin is C 15 H 12 O4, the structural formula is shown in equation (Ⅰ):

[0015] ;

[0016] (I).

[0017] The isoliquiritigenin derivatives are pharmaceutically acceptable salts, esters, or prodrugs of isoliquiritigenin, or phenolic hydroxyl alkylated, acylated, or glycosylated derivatives of isoliquiritigenin.

[0018] The iron salt mentioned is ferric chloride.

[0019] The Fe in the iron ion-isoglycyrrhizin nanocomposite 3+ Replace with Zn 2+ Cu 2+ or Mn 2+ .

[0020] The polyvinylpyrrolidone mentioned above can be polyvinylpyrrolidone K30 (Povidone K30, PVP K30).

[0021] The polyvinylpyrrolidone can be replaced with polyethylene glycol, polyvinyl alcohol, or poloxamer.

[0022] Another object of the present invention is to provide a method for preparing the iron ion-isoglycyrrhizin nanocomposite, comprising: using anhydrous lower alcohol as a reaction solvent, stirring isoglycyrrhizin or its derivative with iron salt at room temperature overnight in the presence of polyvinylpyrrolidone; after the reaction is completed, placing the reaction solution into a dialysis bag, dialyzing and purifying it in ultrapure water, and freeze-drying to obtain the iron ion-isoglycyrrhizin nanocomposite.

[0023] The lower alcohols mentioned are C1 to C4 alcohols.

[0024] Specifically, the lower alcohol can be methanol.

[0025] The ratio of isoliquiritigenin or its derivatives to lower alcohols is (2:1 to 20:1) mg / mL, preferably 10:1 mg / mL.

[0026] The molecular weight cutoff of the dialysis bag is 2 kDa.

[0027] Specifically, a method for preparing the iron ion-isoglycyrrhizin nanocomposite includes: dissolving polyvinylpyrrolidone in anhydrous lower alcohol; then, adding iron salt powder to the solution and stirring until completely dissolved, then adding isoglycyrrhizin, mixing evenly, and stirring overnight at room temperature; after the reaction is complete, placing the reaction solution into a dialysis bag, dialyzing it in ultrapure water for 48 h, changing the ultrapure water every 12 h, collecting the liquid in the dialysis bag, and freeze-drying it to obtain the iron ion-isoglycyrrhizin nanocomposite.

[0028] The iron-isoglycyrrhizin nanocomposite of this invention can be used to prepare drugs for treating inflammatory bowel disease. The principle is as follows: based on the high stability, electroneutrality, and ultra-small nanoscale size of the iron-isoglycyrrhizin nanocomposite, it can effectively target inflammatory lesions in the intestine and remain in the intestinal tissue. By regulating CB2R, it effectively treats intestinal inflammation, reverses the fibrosis process, and simultaneously alleviates iron deficiency associated with inflammatory bowel disease, achieving multi-dimensional treatment of inflammatory bowel disease and simultaneously blocking the vicious cycle of "inflammation-barrier damage-fibrosis." Pharmacodynamic validation has been conducted in various models, including a DSS (dextrose sulfate)-induced acute ulcerative colitis model, a DSS-induced recurrent chronic ulcerative colitis model, and a TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced Crohn's disease-like colitis model. This invention is the first to propose a synergistic treatment scheme that simultaneously treats colitis, reverses intestinal barrier damage, and alleviates fibrosis by targeting inflammatory sites and regulating CB2R. It can also effectively restore iron deficiency caused by inflammatory bowel disease, showing promising clinical application prospects and providing an effective approach for the design of multifunctional biomaterials based on natural active products.

[0029] Another object of the present invention is to provide the use of the iron ion-isoglycyrrhizin nanocomposite in the preparation of a medicament for treating inflammatory bowel disease and inflammatory bowel disease complicated with iron deficiency.

[0030] Preferably, the iron ion-isoglycyrrhizin nanocomposite is used in the preparation of drugs that target cannabinoid type 2 receptors and treat inflammatory bowel disease by effectively restoring intestinal barrier damage, remodeling the intestinal barrier microenvironment, and alleviating fibrosis.

[0031] Preferably, the iron ion-isoglycyrrhizin nanocomposite is used in the preparation of a drug for treating inflammatory bowel disease that exerts anti-inflammatory and anti-fibrotic effects.

[0032] The inflammatory bowel diseases mentioned are acute ulcerative colitis, chronic relapsing ulcerative colitis, and Crohn's disease.

[0033] Another object of the present invention is to provide a pharmaceutical composition, wherein the iron ion-isoglycyrrhizin nanocomposite is used as the active ingredient or main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0034] The dosage form of the pharmaceutical composition is an oral preparation; the dosage form of the oral preparation is any one of tablets, capsules, granules, oral solutions, oral suspensions, dry suspensions, powders, pills, drop pills, or orally disintegrating tablets.

[0035] As a further preferred embodiment of the pharmaceutical composition of the present invention, the active ingredient of the pharmaceutical composition further includes a drug with anti-inflammatory activity, wherein the drug with anti-inflammatory activity is selected from small molecules, peptides, nucleic acids (DNA and RNA), proteins (including antibody proteins), and medical isotopes with anti-inflammatory activity.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) By means of the stable coordination of isoliquiritigenin and iron ions, the present invention obtains a stable nanocomposite ISL-Fe, which not only improves the water solubility of isoliquiritigenin, but also resists gastric acid degradation, making it suitable for preparation into oral administration formulations. It achieves targeted accumulation by enhancing the permeability of the inflamed site, and prolongs the retention time of isoliquiritigenin in the colon tissue by means of the electroneutrality of the nanocomposite, thereby improving the in vivo kinetic characteristics of oral administration of isoliquiritigenin and exerting a highly efficient and long-lasting therapeutic effect.

[0038] (2) The nanocomposite ISL-Fe of this invention controls inflammation by targeting the site of inflammation and regulating CB2R, thereby blocking the vicious cycle of "inflammation-barrier damage-fibrosis" and is expected to become a new generation of composite nanomaterials for the treatment of inflammatory bowel disease.

[0039] (3) The nanocomposite ISL-Fe of the present invention effectively restores iron deficiency caused by inflammatory bowel disease.

[0040] (4) The preparation method of the nanocomposite of the present invention is simple and easy to implement, and can synthesize nanocomposite on a large scale and with high efficiency.

[0041] (5) The nanocomposite material of the present invention is combined or mixed with an anti-inflammatory preparation to form a multimodal drug delivery system. The preparations that can be combined include: small molecules, peptides, nucleic acids (DNA and RNA), proteins (including antibody proteins), medical isotopes, etc. Attached Figure Description

[0042] Figure 1 The images show the UV-Vis spectra of ISL-Fe liquid and the reconstituted lyophilized powder.

[0043] Figure 2 This is a transmission electron microscope image of ISL-Fe.

[0044] Figure 3 The results are X-ray photoelectron spectroscopy (XPS) analysis of the nanocomposite ISL-Fe; where A: XPS full spectrum scan, showing the characteristic photoelectron peaks of C, N, O and Fe elements; B: Fe 2p high-resolution XPS spectrum (including peak fitting results), used to analyze the chemical valence state and coordination environment of iron.

[0045] Figure 4 The infrared spectra of ISL, ISL-Fe, and PVP are shown.

[0046] Figure 5 The dissolution of ISL and ISL-Fe in water is shown; where, left: ISL, right: ISL-Fe.

[0047] Figure 6 This is a statistical graph showing the release rate of Fe in ISL-Fe in an acidic buffer medium.

[0048] Figure 7 The effect of ISL-Fe on the expression of tight junction proteins in a TNF-α / IFN-γ-induced UC in vitro model was investigated. A: Western blot protein bands; B: statistical analysis of E-cadherin expression level; C: statistical analysis of Occludin expression level. All analyses were performed using one-way ANOVA. ns indicates no significant difference (p>0.05), *p<0.05, **p<0.01, ***p<0.001.

[0049] Figure 8 The effects of ISL and ISL-Fe on body weight and intestinal length in mice with DSS-induced acute ulcerative colitis were investigated. A: Schematic diagram of the experimental design; B: Statistical graph of mouse body weight changes, analyzed using two-way ANOVA; C: Representative colon photograph; D: Statistical graph of colon length, analyzed using one-way ANOVA. p < 0.0001.

[0050] Figure 9 The effects of ISL and ISL-Fe on the histopathology of colon tissue in mice with DSS-induced acute ulcerative colitis.

[0051] Figure 10The effects of CB2R inhibitors and ISL-Fe on body weight and intestinal length in mice with DSS-induced acute ulcerative colitis were investigated. A: Schematic diagram of the experimental design; B: Statistical graph of mouse body weight changes, analyzed using two-way ANOVA; C: Representative colon photographs; D: Statistical graph of colon length, analyzed using one-way ANOVA. ns indicates no significant difference (p>0.05), ****p<0.0001.

[0052] Figure 11 The effects of CB2R inhibitors and ISL-Fe on colonic histopathology in mice with DSS-induced acute ulcerative colitis.

[0053] Figure 12 The effects of ISL and ISL-Fe on body weight and intestinal length in mice with DSS-induced chronic ulcerative colitis were investigated. A: Schematic diagram of the experimental design; B: Statistical graph of mouse body weight changes, analyzed using two-way ANOVA; C: Representative colon photograph; D: Statistical graph of colon length, analyzed using one-way ANOVA. ns indicates no significant difference (p>0.05), ****p<0.0001.

[0054] Figure 13 The effects of ISL and ISL-Fe on colonic tissue pathology in mice with DSS-induced chronic ulcerative colitis.

[0055] Figure 14 To investigate the restorative effect of ISL-Fe on iron deficiency caused by chronic ulcerative colitis, a one-way ANOVA was used for statistical analysis. ns indicates no significant difference (p>0.05), **p<0.01.

[0056] Figure 15 The effects of ISL-Fe on the histopathology of colon tissue in mice with TNBS-induced Crohn's disease are shown in the following figures: A: Schematic diagram of experimental design; B: Statistical graph of mouse body weight change, analyzed using two-way ANOVA; C: Representative colon photograph; D: Representative H&E staining of mouse colon sections.

[0057] Figure 16 Fluorescence imaging (A) and quantitative fluorescence map (B) of ISL-Fe-RhB in healthy and colitis mice at different time points.

[0058] Figure 17 The mean drug concentration-time curves of ISL in plasma after oral administration of ISL and ISL-Fe to mice are shown.

[0059] Figure 18The concentrations of ISL in various parts of the body after oral administration of ISL and ISL-Fe to mice. Detailed Implementation

[0060] The following embodiments can enable those skilled in the art to more fully understand the technical solutions of the present invention, but do not limit the scope of protection of the present invention to the embodiments.

[0061] Example 1

[0062] Synthesis and characterization of iron ion-isoglycyrrhizin nanocomposite (ISL-Fe)

[0063] Weigh out 240 mg of PVP K30, 40 mg of ISL, and 40 mg of anhydrous FeCl3 in a mass ratio of 6:1:1. Dissolve 240 mg of PVP K30 in 20 mL of anhydrous methanol to obtain a PVP solution. Then, add 40 mg of anhydrous FeCl3 powder to the PVP solution and stir until completely dissolved. Add 40 mg of ISL and stir overnight at room temperature. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 2 kDa and dialyze it in 2 L of ultrapure water for 48 h, changing the ultrapure water every 12 h. Collect the liquid in the dialysis bag, which is the iron ion-isoglycyrrhizin nanocomplex solution. Further freeze-dry the solution to obtain the freeze-dried iron ion-isoglycyrrhizin nanocomplex product.

[0064] UV spectra of the iron ion-isoglycyrrhizin nanocomposite solution and the aqueous complex solution of the lyophilized product Figure 1 To (see) Figure 1 This indicates that the iron ion-isoglycyrrhizin nanocomposite has good freeze-drying stability.

[0065] ICP-MS analysis showed that the iron content in the freeze-dried product of the iron ion-isoglycyrrhizin nanocomposite was 6 wt%. Based on the isoglycyrrhizin content in the composite, the molar ratio of iron to isoglycyrrhizin was calculated to be 2:1, meaning that every 2 molecules of ISL were mixed with 3 molecules of Fe. 3+ Coordination complexation. The zeta potential measured after ISL-Fe dissolved in purified water was -0.11±0.21 mV, and its absolute value was less than 1 mV, indicating that the net charge on the surface of the nanocomposite was close to zero and the whole was electrically neutral.

[0066] Morphology and size were observed and measured using transmission electron microscopy (TEM), such as... Figure 2 As shown, ISL-Fe consists of uniformly dispersed ultra-small nanodots with a diameter of approximately 1 nm.

[0067] X-ray photoelectron spectroscopy (XPS) revealed peaks in the nanocomposite associated with elements C (286 eV), N (400 eV), O (532 eV), and Fe (710 eV). Figure 3 A). High-resolution Fe spectra confirmed the presence of three different valence states of Fe in the ISL-Fe nanocomposite ( Figure 3 B).

[0068] Infrared spectrum ( Figure 4 The data shows that ISL is 3389cm. -1 1628 cm -1 1543 cm -1 1510cm -1 1291-1141cm -1 The peaks at 3450 cm⁻¹ represent the main characteristic peaks for the stretching vibrations of hydroxyl, carbonyl, benzene ring, C=C, and CO, respectively. PVP is at 3450 cm⁻¹. -1 1667 cm -1 1373cm -1 1463 and 1423cm -1 The main characteristic peaks are the stretching vibrations of hydroxyl, carbonyl, CN, and methylene groups. For the ISL-Fe nanocomposite, in addition to the characteristic peaks of ISL and PVP, there are also peaks at 1664 cm⁻¹. -1 1557 cm -1 1025 cm -1 843 cm -1 793 cm -1 The stretching vibrations are those of the carbonyl group, C=C, and CH, respectively, with the stretching vibration of OH starting from 3389 cm⁻¹. -1 and 3450cm -1 Moved to 3435 cm -1 The results indicate that covalent bonds, hydrogen bonds, and metal coordination bonds are formed among ISL, PVP, and Fe. The FTIR results prove that a nanocomposite with ISL and Fe as raw materials and PVP as the framework has been successfully prepared.

[0069] ISL (300 μg / mL) forms a yellow suspension in water and precipitates rapidly; while the nanocomposite ISL-Fe (based on an equal volume of ISL) forms a clear brown solution in water without precipitation, indicating that the nanocomposite significantly improves the water solubility of ISL (e.g., Figure 5 (As shown).

[0070] Example 2

[0071] Release rate of Fe from the ISL-Fe nanocomposite in an acidic buffer medium

[0072] Prepare an ISL-Fe aqueous solution with an ISL concentration of 1.5 mg / mL. Take 5 mL of the ISL-Fe aqueous solution and add it to a dialysis bag with a molecular weight cutoff of 2 kDa. Make three copies.

[0073] Prepare an acidic buffer medium with pH 1.2: 8 g sodium chloride and 28 mL hydrochloric acid, diluted with water to 4 L. Measure 400 mL of the pH 1.2 acidic buffer medium into a 500 mL beaker. Place a dialysis bag containing ISL-Fe aqueous solution into the beaker (three portions). At room temperature, stir at 250 rpm. At 10, 30, 60, and 120 min, transfer 1 mL of the acidic buffer medium from outside the dialysis bag, and simultaneously replenish with an equal volume of acidic buffer medium. Measure the iron ion content in the samples at different sampling time points and plot the iron ion release rate graph, as shown below. Figure 6 As shown, the release rate of iron ions in ISL-Fe was <10% within 2 h, indicating that the complex has good stability in acidic media and can resist gastric juice degradation, providing a theoretical basis for its function in the intestine.

[0074] Example 3

[0075] Effects of the nanocomposite ISL-Fe on an in vitro model of TNF-α / IFN-γ-induced ulcerative colitis (UC) by modulating CB2R

[0076] HT29 cells in the logarithmic growth phase were divided into groups of 6 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and cultured for 24 hours, at which point the cells were fully adherent. The cells were then divided into a control group, a model group (Vehicle), an inhibitor group, an ISL-Fe group, and an ISL-Fe combined with an inhibitor group. The old culture medium was discarded, and the control group was replaced with FBS-free DMEM medium. Each well of the model group was given DMEM medium containing TNF-α (30 ng / mL) and IFN-γ cytokines (30 ng / mL), and each well of the inhibitor group was given DMEM medium containing TNF-α (30 ng / mL) and IFN-γ cytokines (30 ng / mL). The cells were incubated in DMEM medium containing TNF-α (30 ng / mL), IFN-γ cytokines (30 ng / mL), and ISL-Fe (5 μM) per well. The ISL-Fe combined with inhibitor group was incubated in DMEM medium containing TNF-α (30 ng / mL), IFN-γ cytokines (30 ng / mL), ISL-Fe (5 μM), and SR144528 (1 μM) per well. All groups were incubated for 24 h, and cell samples were collected. The expression level of intestinal tight junction protein regulated by CB2R was detected by Western blotting.

[0077] Figure 7 The results showed that the expression levels of tight junction proteins (E-cadherin and occludin) in the TNF-α and IFN-γ-induced in vitro UC model were significantly decreased compared with the control group, and the downregulation of occludin protein expression was more pronounced in the inhibitor group, confirming the association between CB2R regulation and intestinal barrier repair. In the TNF-α and IFN-γ-induced model, ISL-Fe could significantly restore the expression of E-cadherin and occludin proteins. Even under CB2R inhibition, ISL-Fe still had a partial restorative effect on tight junction proteins. This indicates that ISL-Fe can effectively repair intestinal barrier damage by regulating CB2R.

[0078] Example 4

[0079] The therapeutic effect of the nanocomposite ISL-Fe on DSS-induced acute ulcerative colitis in mice.

[0080] like Figure 8 As shown in Figure A, 8-week-old male C57BL / 6 mice of similar weight were randomly divided into four groups (n=9): control group, vehicle group, prototype drug group (ISL, dose 50 mg / kg), and nanocomposite group (ISL-Fe, dose 15 mg / kg). The dose of ISL-Fe is expressed in ISL, and the same applies below.

[0081] Mice were acclimated for 7 days, with normal food and water intake. One day before the experiment, mice in the prototype drug group and the nanocomposite group were given prophylactic administration via gavage. After the experiment began, control group mice had normal food and water intake, while mice in the model group, prototype drug group, and nanocomposite group were given free access to 3.5% dextran sulfate sodium salt (DSS) aqueous solution to induce a colitis model. The prototype drug group and the nanocomposite group continued to receive the corresponding drugs daily for 7 consecutive days. Daily changes in mouse body weight were observed and recorded. Specific administration methods are as follows:

[0082] Model group: Starting one day before the experiment, 0.5% CMC-Na solution was administered by gavage at a rate of 1 mL / 100g.

[0083] Prototype drug group: ISL was ground and dispersed with 0.5% CMC-Na aqueous solution to prepare a suspension of 5 mg / mL. Starting one day before the start of the experiment, it was administered by gavage at a dose of 50 mg / kg.

[0084] Nanocomposite group: The lyophilized product of ISL-Fe nanocomposite (Example 1) was dissolved in purified water to prepare an aqueous solution with an ISL content of 1.5 mg / mL. Starting one day before the start of the experiment, it was administered by gavage at a dose of 15 mg / kg.

[0085] During the drug administration period, the body weight of mice in each group was measured daily; after the treatment, the mice were sacrificed, and the severity indicators of colitis in each group were assessed. Compared with the control group, the body weight of mice in the model group and the original drug group showed a continuous decreasing trend, while the body weight of mice treated with ISL-Fe increased steadily during the experimental period. Figure 8 B). The colon lengths of mice in the control group, model group, original drug group, and nanocomposite group were 8.57±0.76, 5.36±0.27, 6.63±0.30, and 7.73±0.44 cm, respectively. The colon in the model group was significantly shortened and accompanied by congestion. The colon in the original drug group showed some relief from congestion, but its effect on improving colon shortening was limited. In contrast, after treatment, the colon shortening and congestion caused by DSS were significantly restored in the nanocomposite group. Figure 8 C Figure 8 D). Colon pathological section staining shows ( Figure 9 In the model group, the colon of mice showed severe inflammatory damage, characterized by typical pathological changes such as inflammatory cell infiltration, widening of the submucosal space, disintegration of the lamina propria, hemorrhage and ulceration, destruction of intestinal villi structure, significant reduction of goblet cells, and thickening of the intestinal wall. In the original drug group, the colonic tissue structure of mice was significantly restored, but the number of goblet cells was still small, and local inflammation remained. In contrast, the colonic structure of mice in the nanocomposite group was intact, the villi were tightly connected to the intestinal wall, and the villi separation phenomenon was significantly reversed. This indicates that the nanocomposite ISL-Fe can effectively alleviate intestinal inflammation and repair the intestinal barrier, and has a significant therapeutic effect on inflammatory bowel disease.

[0086] In summary, compared with isoliquiritigenin, the nanocomposite ISL-Fe can more significantly restore the intestinal microvilli structure and improve the integrity of tight junctions at a lower concentration of the original drug, thereby more effectively repairing the intestinal barrier and demonstrating superior therapeutic effects.

[0087] Example 5

[0088] Validating the target of the ISL-Fe nanocomposite in treating DSS-induced acute ulcerative colitis in mice

[0089] like Figure 10As shown in Figure A, 8-week-old male C57BL / 6 mice of similar weight were randomly divided into four groups (n=8): control group (Water), model group (DSS), inhibitor group (DSS + SR144528, dose 5 mg / kg), nanocomposite group (DSS + ISL-Fe, dose 15 mg / kg), and combination therapy group (DSS + SR144528 + ISL-Fe, SR144528 dose 5 mg / kg, ISL-Fe dose 15 mg / kg). Mice were acclimated for 7 days with normal food and water intake. Mice in the inhibitor group, nanocomposite group, and combination therapy group received prophylactic medication one day before the experiment. After the experiment began, except for the control group, mice in all other groups freely drank 3.5% dextran sulfate sodium aqueous solution to induce an acute colitis model and continued to receive the corresponding drugs daily for 7 consecutive days. Mice in the control group had normal food and water intake without any treatment. Details are as follows:

[0090] Model group: Starting one day before the experiment, 0.5% CMC-Na aqueous solution was administered by gavage at a volume of 1 mL / 100g.

[0091] Inhibitor group: SR144528 was prepared into a stock working solution of 50 mg / mL using DMSO, and then diluted with physiological saline to obtain a drug working solution with a concentration of 0.5 mg / mL. Starting one day before the start of the experiment, the drug was administered intraperitoneally at a dose of 5 mg / kg.

[0092] Nanocomposite group: The lyophilized product of ISL-Fe nanocomposite (Example 1) was dissolved in purified water to prepare an aqueous solution with an ISL content of 1.5 mg / mL. Starting one day before the start of the experiment, it was administered by gavage at a dose of 15 mg / kg.

[0093] Combined drug administration group: Starting one day before the start of the experiment, SR144528 working solution was injected intraperitoneally at a dose of 5 mg / kg. Six hours later, ISL-Fe nanocomposite lyophilized product was administered by gavage at a dose of 15 mg / kg.

[0094] During the treatment period, the body weight changes of mice were observed and recorded daily. After the treatment, the severity of colitis-related indicators in each group of mice were assessed. The statistical results of mouse body weight changes are shown below. Figure 10B shows that compared with the control group, the body weight of mice in the model group, inhibitor group, and combined drug administration group showed a continuous decreasing trend, while the body weight of mice treated with the nanocomposite ISL-Fe steadily increased during the experimental period. Furthermore, the colon lengths of mice in the control group, model group, inhibitor group, combined drug administration group, and nanocomposite group were 8.59±0.49, 6.63±0.42, 7.03±0.36, 6.21±0.61, and 8.60±0.27 cm, respectively. Compared with the control group, the colon lengths of mice in the model group, inhibitor group, and combined drug administration group were significantly shortened, while the colon length of mice in the nanocomposite group returned to normal levels. Figure 10 D). In the model group mice, the colon was significantly shortened and accompanied by congestion. The congestion was more severe in the inhibitor group. However, after treatment, the DSS-induced colon shortening and congestion in the nanocomposite group mice were significantly restored. In addition, the colonic congestion in the combined drug group mice was also alleviated to some extent. Figure 10 C). Colon pathological section staining shows ( Figure 11 In the model group and the inhibitor group, mice exhibited severe inflammatory damage in the colon. In contrast, the nanocomposite effectively alleviated intestinal inflammation and repaired the intestinal barrier, demonstrating a significant therapeutic effect on inflammatory bowel disease. Furthermore, colon tissue sections from the combined administration group showed partial restoration of the intestinal villus structure, indicating that the nanocomposite ISL-Fe reversed the intestinal damage exacerbated by the inhibitor, suggesting that it may exert its therapeutic effect by regulating CB2R, which is acted upon by the inhibitor.

[0095] In summary, the nanocomposite ISL-Fe not only effectively improves the symptoms of DSS-induced acute colitis, but also reverses the intestinal damage exacerbated by the selective CB2R inhibitor (SR144528), suggesting that the therapeutic effect of the nanocomposite ISL-Fe on inflammatory bowel disease may be closely related to the regulation of CB2 receptor expression or function.

[0096] Example 6

[0097] Reversal effect of the nanocomposite ISL-Fe on intestinal fibrosis induced by chronic ulcerative colitis

[0098] like Figure 12As shown in Figure A, 8-week-old male C57BL / 6 mice of similar weight were randomly divided into four groups (n=8): control group, vehicle group, prototype drug group (ISL, 50 mg / kg), and nanocomposite group (ISL-Fe, 15 mg / kg). A chronic relapsing ulcerative colitis model was established: After one week of acclimatization, except for the control group which had normal drinking water, the other groups of mice freely drank 2.0% sodium dextran sulfate (DSS) aqueous solution from day 0 to day 5 for model induction. From day 6 to day 11, the mice were given normal drinking water. This constituted one cycle, and a total of 3 cycles (36 days in total) were performed to induce chronic ulcerative colitis. The drug was administered every 2 days starting from day 5 of the experiment and continued until day 36, at which point the experiment ended.

[0099] The specific administration method is as follows:

[0100] Model group: 0.5% CMC-Na aqueous solution was administered by gavage at a volume of 1 mL / 100g;

[0101] Prototype drug group: ISL was ground and dispersed with 0.5% CMC-Na aqueous solution to prepare a suspension of 5 mg / mL, and administered by gavage at a dose of 50 mg / kg.

[0102] Nanocomposite group: The lyophilized product of ISL-Fe nanocomposite (Example 1) was dissolved in purified water to prepare an aqueous solution with an ISL content of 1.5 mg / mL, and administered by gavage at a dose of 15 mg / kg.

[0103] During the treatment period, the weight changes of mice were observed and recorded daily. After the treatment, the severity of colitis-related indicators in each group of mice were assessed. Mice experienced a significant decrease in weight during the free access to DSS water phase. Although some mice gained weight during the recovery period, due to colitis recurrence, the model group and the original drug group experienced a significant decrease in weight after the second and third rounds of DSS modeling. In contrast, the nanocomposite group experienced a slight decrease in weight after the second round of modeling, but its weight steadily increased after the third round, showing a highly significant improvement over the model group and no difference from the control group. Figure 12 B). Statistical results of colon length are shown below. Figure 12 C shows that the colon length of mice in the prototype drug group was not significantly improved, but the colon length of mice in the nanocomposite group was significantly restored and was no different from that of the control group. Figure 12 D is a representative colon photograph, showing that the colon of the model group mice was shortened and the intestinal wall was thickened. The colon length and intestinal wall thickening of the mice in the prototype drug group were slightly restored, while the colon length of the mice in the nanocomposite group was completely restored and no intestinal wall thickening was observed.

[0104] Figure 13The stained colonic section images show that the colon of mice in the model group exhibited typical pathological changes, including inflammatory cell infiltration, destruction of intestinal villus structure, thickening of the intestinal wall, compensatory abnormal increase in goblet cells, and collagen fiber hyperplasia. In contrast, the intestinal villus structure of mice in the prototype drug group was partially restored without significant collagen fiber hyperplasia, but with compensatory abnormal increase in goblet cells, which may be due to disordered intestinal epithelial repair. In mice in the nanocomposite group, the intestinal villi were tightly connected to the intestinal wall, the intestinal structure was intact, villus separation was effectively reversed, colonic inflammation was significantly alleviated, and there was no abnormal increase in goblet cells or collagen fiber hyperplasia. These results indicate that the nanocomposite ISL-Fe can effectively alleviate intestinal inflammation, repair the intestinal barrier, and reverse intestinal fibrosis caused by DSS-induced colitis, achieving simultaneous blocking of the vicious cycle of "inflammation-barrier damage-fibrosis".

[0105] Example 7

[0106] The restorative effect of the nanocomposite ISL-Fe on iron deficiency in chronic ulcerative colitis.

[0107] A chronic relapsing ulcerative colitis model was constructed according to the method in Example 6. After the experiment, serum samples were collected from mice in the control group, model group, prototype drug group (ISL, dose 50 mg / kg) and nanocomposite group (ISL-Fe, dose 15 mg / kg). Serum iron content was detected using a fully automated biochemical analyzer.

[0108] The results are as follows Figure 14 As shown, serum iron levels in the model group mice were significantly lower than those in the control group, indicating iron deficiency in mice with DSS-induced chronic ulcerative colitis. Serum iron levels in the prototype drug group showed no significant improvement compared to the model group, suggesting that ISL cannot effectively alleviate iron deficiency associated with IBD. However, serum iron levels in the nanocomposite group mice recovered to levels close to the control group, indicating that the nanocomposite ISL-Fe can effectively correct hypoironemia associated with chronic ulcerative colitis. Its specific mechanism may involve the absorption and utilization of iron in the composite, or indirectly regulate iron metabolism through anti-inflammatory effects.

[0109] Example 8

[0110] The therapeutic effect of the nanocomposite ISL-Fe on TNBS-induced Crohn's disease in mice

[0111] like Figure 15 As shown, 8-week-old male C57BL / 6 mice of similar weight were randomly divided into three groups (n=4): control group, model group (TNBS), and nanocomposite group (TNBS + ISL-Fe, dose calculated as ISL 15 mg / kg). The mice were acclimatized for 7 days with normal food and water intake.

[0112] Mice in the model group and nanocomposite group were fasted overnight for two days before the experiment. One day before the experiment, they were given an enema of a 2% TNBS solution (prepared with 50% ethanol and water) to induce the Crohn's disease model. Mice in the nanocomposite group were given prophylactic medication starting one day before the experiment and continued for five days after the experiment began. Mice in the control group had normal food and water intake and received no treatment.

[0113] The body weight of mice was observed and recorded daily during the experiment. After treatment, the severity of colitis-related indicators in each group of mice were assessed. Compared with the control group, the overall body weight of mice in the model group decreased, while the body weight of mice significantly improved after treatment with the nanocomposite. Figure 15 B). Figure 15 C is a representative image of the colon, showing that compared to the control group, the colon length of the mice in the model group was shortened, while the colon length of the mice in the nanocomposite group was restored. Figure 15 D shows a stained section of colonic pathology, revealing severe damage to the colon in the model group mice, specifically manifested as crypt destruction and widening of the submucosal space. In contrast, the colonic intestinal structure of the nanocomposite group mice remained intact, with regular crypt arrangement and no obvious inflammatory infiltration in the mucosa and submucosa. In conclusion, the ISL-Fe nanocomposite can effectively alleviate TNBS-induced Crohn's disease-like colitis in mice.

[0114] Example 9

[0115] In vivo imaging of small animals reveals the inflammatory targeting effect of the nanocomposite ISL-Fe.

[0116] Preparation of the iron ion-isoglycyrrhizin-rhodamine B nanocomposite (ISL-Fe-RhB): 240 mg of PVP K30, 40 mg of anhydrous FeCl3 powder, 40 mg of ISL, and 3 mg of Rhodamine B (RhB) were weighed according to a mass ratio of PVP K30, anhydrous FeCl3, ISL, and Rhodamine B (RhB) of 6:1:1:0.075. 240 mg of PVP K30 was dissolved in 20 mL of anhydrous methanol. Then, 40 mg of anhydrous FeCl3 powder was added to the solution and stirred until completely dissolved. Next, 40 mg of ISL was added and stirred to mix. 3 mg of Rhodamine B (RhB) was dissolved in 2 mL of anhydrous methanol and added dropwise to the above system. The mixture was stirred overnight at room temperature under light-protected conditions. After the reaction was completed, the reaction solution was added to a dialysis bag with a molecular weight cutoff of 2 kDa. Under light-protected conditions, the solution was purified by dialyzing in 2 L of ultrapure water for 48 h. The ultrapure water was replaced every 12 h. The liquid in the dialysis bag was collected, which was ISL-Fe-RhB. The solution was then freeze-dried to obtain the lyophilized product of the iron ion-isoglycyrrhizin-rhodamine B nanocomplex.

[0117] Eight-week-old male C57BL / 6 mice of similar weight were randomly divided into a control group and a model group (Vehicle) after one week of acclimatization. At the start of the experiment, mice in the model group had free access to 3.5% DSS aqueous solution for 4 days to induce an acute colitis model, while mice in the control group had normal food and water intake. Four days after modeling, ISL-Fe-RhB (15 mg / kg) was administered to both the control and model groups via gavage. At different time points, the mice were imaged and fluorescence intensity was quantified using an IVIS Spectrum instrument. The results are as follows: Figure 16 As shown, Figure 16 In Figure A, there are 3 mice in each group, and each row represents the in vivo fluorescence imaging of the same mouse at different time points.

[0118] In vivo fluorescence imaging results showed that after oral administration, the abdominal fluorescence signal initially increased and then decreased over time, reaching a peak at 4 hours post-administration, and then gradually weakened. Compared with normal mice in the control group, the fluorescence intensity in the colonic region of colitis-affected mice was significantly enhanced, suggesting that the ISL-Fe nanocomposite possesses an inflammatory-responsive targeted accumulation capacity. This selective enrichment property may be jointly mediated by inflammation-induced increases in intestinal mucosal permeability and alterations in the inflammatory microenvironment. In conclusion, the ISL-Fe nanocomposite can effectively target inflammatory areas, thereby reducing the risk of off-target drug exposure while maximizing the therapeutic benefit of the lesion area.

[0119] Example 10

[0120] In vivo pharmacokinetics and tissue distribution studies of ISL and ISL-Fe in mice

[0121] C57BL / 6 mice (male, 7 weeks old, 20-22g) were purchased from Zhejiang Vital River Co., Ltd. Before the experiment, the C57BL / 6 mice were acclimatized for one week in an environment with a room temperature of 22±2℃ and a humidity of 55%±5% to adapt to laboratory conditions. Bedding, food, and drinking water were changed regularly according to the animal husbandry requirements of the Ethics Committee of China Pharmaceutical University. The mice were randomly divided into two groups of 20 C57BL / 6 mice each. All mice were fasted for 12 hours before administration but allowed free access to water. Both groups of mice were administered a single oral gavage of 0.5% CMC-Na suspension of ISL and ISL-Fe aqueous solution, respectively, at a dose of 30 mg / kg (based on ISL). Whole blood (150 μL) was collected from the orbital venous plexus at 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration and placed into heparin sodium anticoagulant Eppendorf tubes. The blood was inhaled at 8000 r·min. -1Mouse blank plasma was obtained by centrifugation for 10 min, and collected under the same conditions. The plasma was then stored at -80℃. At 10 min, 1 h, 4 h, and 8 h post-collection, mouse hearts, livers, spleens, lungs, kidneys, stomachs, duodenums, and colons were collected, rinsed with physiological saline, and stored at -80℃. 20 mg of tissue sample was weighed and added to 200 μL of physiological saline. The mixture was homogenized thoroughly. After homogenization, 1 mL of methanol was added at a volume ratio of 1:5, and the mixture was shaken at 1500 rpm for 5 min to precipitate proteins. The mixture was centrifuged twice, each time at 4℃ and 18000 rpm for 10 min. 90 μL of the supernatant was transferred to a sample vial for analysis by LC-MS / MS. Take 50 μL of plasma sample, add 150 μL of methanol at a volume ratio of 1:3, shake at 1500 rpm for 5 min to fully precipitate proteins; centrifuge twice, 4℃, 18000 rpm for 10 min, transfer 90 μL of supernatant to a sample vial, and analyze by LC-MS / MS. After LC-MS / MS analysis, pharmacokinetic parameters were calculated using non-clinical small animal sparse sampling NCA in Phoenix WinNonLin 8.1 (USA): elimination half-life (t 1 / 2 (h) and peak time (T) max (h) Peak concentration (C) max (μg / L), area under the curve (AUC) from 0 to t. 0→t (μg / L·h), area under the curve (AUC) from 0 to ∞. 0→∞ (μg / L·h), apparent volume of distribution (V) d (L / kg), clearance rate (CL, L / h / kg), and mean residence time from 0 to t (MRT) 0→t (h) and the average residence time from 0 to ∞ (MRT) 0→∞ (h). The results are shown in Table 1. Figure 17 and Figure 18 The results showed that the nanocomposite ISL-Fe exhibited significant advantages in gastrointestinal tissue retention, long-lasting effect, and increased systemic exposure.

[0122] Table 1. Pharmacokinetic parameters of ISL and ISL-Fe in mice after oral administration

[0123]

[0124] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An iron ion-isoglycyrrhizin nanocomposite, characterized in that: The iron ion-isoglycyrrhizin nanocomposite is made from isoliquiritin or its derivatives and iron salts in the presence of polyvinylpyrrolidone, consisting of isoliquiritin or its derivatives and Fe. 3+ Coordination-formed nanocomposite materials.

2. The iron ion-isoglycyrrhizin nanocomposite according to claim 1, characterized in that: The mass ratio of isoliquiritin or its derivative to iron salt is (1-4):(1-4); the mass ratio of polyvinylpyrrolidone to isoliquiritin is (4-12):(0.5-2).

3. The iron ion-isoglycyrrhizin nanocomposite according to claim 2, characterized in that: The mass ratio of isoliquiritin or its derivative to iron salt is (2-2.5):(0.8-1.2); the mass ratio of polyvinylpyrrolidone to isoliquiritin is (5-8):(0.8-1.2).

4. The iron ion-isoglycyrrhizin nanocomposite according to claim 3, characterized in that: The mass ratio of isoliquiritin or its derivative to iron salt is 2:1 to 2.5:1; the mass ratio of polyvinylpyrrolidone to isoliquiritin is 6:

1.

5. A method for preparing the iron ion-isoglycyrrhizin nanocomposite according to claim 1, characterized in that: include: Using anhydrous lower alcohols as the reaction solvent, isoliquiritigenin or its derivatives were stirred with iron salts overnight at room temperature in the presence of polyvinylpyrrolidone. After the reaction was completed, the reaction solution was placed in a dialysis bag, purified by dialyzing in ultrapure water, and freeze-dried to obtain an iron ion-isooliquiritigenin nanocomposite.

6. The use of the iron ion-isoglycyrrhizin nanocomposite according to claim 1 in the preparation of a drug for treating inflammatory bowel disease and inflammatory bowel disease complicated with iron deficiency.

7. The application according to claim 6, characterized in that: The application of the iron ion-isoglycyrrhizin nanocomposite in the preparation of drugs that target cannabinoid type 2 receptors and effectively restore intestinal barrier damage, reshape the intestinal barrier microenvironment, and alleviate fibrosis in the treatment of inflammatory bowel disease.

8. The application according to claim 6, characterized in that: The inflammatory bowel diseases mentioned are acute ulcerative colitis, chronic relapsing ulcerative colitis, and Crohn's disease.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition described herein uses the iron ion-isoglycyrrhizin nanocomposite as the active ingredient or main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage form of the pharmaceutical composition is an oral preparation; the dosage form of the oral preparation is any one of tablets, capsules, granules, oral solutions, oral suspensions, dry suspensions, powders, pills, drop pills, or orally disintegrating tablets.

Images