Ph-driven co-assembly of pharmafood flavonoid components into hydrogel and application thereof

Abstract

This invention relates to hydrogels, and more particularly to a pH-driven co-assembly hydrogel of medicinal and edible flavonoid components and its applications. This invention is the first to discover that the natural medicinal and edible flavonoid components rutin and quercetin can effectively co-assemble into RQ nanoparticles within a pH range of 8.0-8.9, without any carrier or chemical cross-linking agent. The RQ nanoparticles cross-link glycyrrhizic acid through intermolecular hydrogen bonds to form a GRQ hydrogel network, preserving the complete structure of the nanoparticles and exhibiting excellent injectability, adhesion, and viscoelasticity. The GRQ-gel components of this invention are all derived from natural flavonoid components, exhibiting high biocompatibility. This invention relates to a hydrogel of medicinal and edible flavonoid components that significantly inhibits myocardial fibroblast antigen presentation and suppresses the progression of myocardial fibrosis, possessing outstanding substantial characteristics, optimal practical effects, and high economic value.

Description

Technical Field

[0001] This invention relates to hydrogels, and more particularly to a pH-driven co-assembled hydrogel of medicinal and edible flavonoid components and its applications. Background Technology

[0002] Myocardial fibrosis (MF) is a major determinant of adverse clinical outcomes and a common pathological feature in many cardiac diseases, such as hypertension, myocardial infarction, and heart failure. It is characterized by excessive collagen deposition in myocardial tissue, leading to cardiac sclerosis and decreased systolic and diastolic function. Even with standard treatment, fibrosis may persist in patients with heart failure. However, a safe and effective strategy for directly treating MF remains lacking.

[0003] In recent years, the advantages of Traditional Chinese Medicine (TCM) in treating myocardial infarction (MF) have gradually become apparent, playing an important role in inhibiting fibroblast activation and reducing myocardial collagen deposition. The TCM concept of "medicine and food sharing the same origin" first appeared in the *Huangdi Neijing* (Yellow Emperor's Inner Classic), providing a theoretical basis for disease treatment and health preservation in later generations. "Medicine and food sharing the same origin" refers to substances that can be used as both food and medicine, possessing clear therapeutic effects, low side effects, and flexible dosage forms, attracting much attention due to their dual nutritional and medicinal value. Flavonoids, as a natural and safe bioactive substance, have shown significant potential in antioxidation, anti-inflammation, neuroprotection, and cardioprotection. Because flavonoids are natural and difficult to synthesize, such flavonoid dietary supplements have been commercialized in many countries worldwide. Rutin and quercetin are two representative flavonoids derived from various medicinal and food plants, such as Sophora japonica flowers, mulberry leaves, honeysuckle, hawthorn, and kudzu root. Rut can significantly inhibit fibrosis, promote angiogenesis, inhibit myocardial remodeling, and improve cardiac function in mice with diabetic cardiomyopathy. Rut can significantly reduce the expression of matrix metalloproteinase 2 and matrix metalloproteinase 9, thereby preventing lipopolysaccharide-induced interstitial fibrosis. Que exerts its anti-fibrotic effect by regulating the SIRT3 / TGF-β / Smad3 signaling pathway. Glycyrrhizic acid can improve cardiac function after myocardial infarction, reduce myocardial injury markers (CK-MB / LDH), improve the degree of myocardial fibrosis, and protect microvessels. Its mechanism of action is related to the activation of the Nrf-2 / HO-1 antioxidant pathway and the inhibition of the NF-κB inflammatory signaling pathway, thereby reducing myocardial oxidative stress and inflammatory damage.

[0004] However, the clinical application of Rut, Que, and glycyrrhizic acid still cannot avoid common limitations, such as near-insolubility in water and extremely low in vivo absorption. Although various drug delivery systems have been developed to improve the physicochemical properties and in vivo behavior of Rut and Que, including nanoparticles, liposomes, solid lipid nanoparticles, and hydrogels, these systems have low drug loading capacity, the organic solvents are toxic, and high-dose carriers still pose potential safety issues, limiting their practical application in clinical practice.

[0005] Therefore, it is necessary to break through the bottleneck of traditional drug delivery strategies and develop a new drug delivery system for flavonoids with high safety and high drug loading capacity, which has become a technical challenge in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing a hydrogel (GRQ-gel) based on medicinal and edible flavonoids, loaded with glycyrrhizic acid, rutin, and quercetin, effectively solving the problems mentioned in the background section.

[0007] The technical solution adopted by the present invention to solve the above problems is as follows: A pH-driven co-assembled hydrogel of medicinal and edible flavonoid components is characterized by the following steps: Rutin and quercetin in different molar ratios are added to 10 mL of water and stirred at 1000 rpm for 10 minutes at 80°C using a magnetic stirrer. The pH is adjusted with 0.2 mol / L NaOH to ensure complete drug dissolution, followed by sonication at 200 W for 5 minutes to prepare RQ nanoparticles. Subsequently, the obtained RQ nanoparticle solution is freeze-dried to obtain a powder. The RQ powder and glycyrrhizic acid (GA) are weighed and added to water, heated to 80°C to completely dissolve, and cooled to room temperature to obtain GRQ-gel.

[0008] The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components is characterized in that when the molar ratio of Rut to Que is 1:1, the RQ nanoparticles assembled under alkaline conditions of pH 8-9 have the most uniform particle size distribution and the largest absolute value of zeta potential.

[0009] The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components is characterized in that Rut and Que molecules are co-assembled into RQ nanoparticles through π-π stacking and intermolecular hydrogen bonding interactions.

[0010] The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components is characterized in that 5 mg / mL LRQ nanoparticles and 30 mg / mL GA are co-assembled to form GRQ-gel.

[0011] The method is characterized in that the GRQ-gel has a network gel structure composed of a large number of intermolecular hydrogen bonds and π-π stacking interactions.

[0012] Application of the pH-driven co-assembled hydrogels of medicinal and edible flavonoid components in the preparation of drugs that inhibit myocardial infarction, myocardial fibrosis or vascular damage.

[0013] The application is characterized in that it improves fibrosis symptoms by inhibiting antigen presentation by fibroblasts, thereby blocking the inflammatory response mediated by CD4+ T cells.

[0014] The assembly process of the GRQ-gel only requires a simple heating-cooling method, without the need for any organic reagents or chemical cross-linking agents, and is characterized by being green and environmentally friendly, simple to prepare, and highly biosafe.

[0015] Beneficial effects Cardiovascular disease is the leading cause of death worldwide. Myofibrosis (MF), characterized by excessive accumulation of extracellular matrix proteins in the myocardium, is a common pathological process in almost all cardiovascular diseases and has been shown to predict the severity of cardiovascular disease. In acute injury events (such as myocardial infarction or certain types of myocarditis), the death of a large number of cardiomyocytes triggers an inflammatory response, thereby activating fibroblasts to migrate to the damaged area, proliferate, and form fibrous tissue. Under the influence of inflammatory cytokines, cardiac fibroblasts differentiate into myofibroblasts, promoting increased synthesis of extracellular matrix (such as type I and type III collagen). Fibroblasts are non-cardiomyocytes whose classic function is to synthesize and degrade extracellular matrix to maintain tissue homeostasis. Under pathological conditions such as pressure load, infection, and ischemia, the proliferation and activation capacity of fibroblasts are significantly enhanced, which is closely related to the occurrence of myocardial fibrosis. Activated fibroblasts secrete excessive extracellular matrix and deposit it in the myocardial matrix, thereby reducing myocardial compliance and leading to deterioration of cardiac function. CD4+ T cell-mediated immune responses can directly affect the repair process of myocardial tissue damage. CD4+ T cells can be activated by specialized antigen-presenting cells (APCs). In a mouse model of heart failure induced by stress overload, the development of myocardial fibrosis (MF) is consistent with the infiltration of CD4+ T cells into the myocardium, while CD4+ T cell deficiency can prevent MF from occurring in mice. Conditional knockout of MHC-II in cardiac fibroblasts can improve remodeling and dysfunction caused by cardiac stress overload. Therefore, therapeutic strategies targeting fibroblast antigen presentation can effectively inhibit the process of myocardial fibrosis and prevent further deterioration of cardiac function.

[0016] Unlike traditional nanoprecipitation methods, this study proposes for the first time a pH-driven co-assembly method of rutin and quercetin. In this method, rutin and quercetin serve as the framework materials for RQ nanoparticles. Furthermore, the inventors dissolved glycyrrhizic acid and the aforementioned RQ nanoparticles in water and heated the solution to form a hydrogel.

[0017] This invention relates to a special hydrogel formed by rutin, quercetin, and glycyrrhizic acid. All three components possess pharmaceutical activity and also form the skeletal structure of the hydrogel. This hydrogel, as a drug, eliminates the need for organic solvents and additional additives, solving the problems of rutin, quercetin, and glycyrrhizic acid's poor water solubility and low drug loading capacity. Furthermore, the three components exhibit a synergistic effect, resulting in a therapeutic efficacy superior to GA-gel alone. GRQ-gel can inhibit antigen presentation by fibroblasts, thereby blocking the inflammatory response mediated by CD4+ T cells and improving fibrosis symptoms. Additionally, the toxicity of long-term use of GRQ-gel is negligible.

[0018] Specifically: Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. 1 ¹H-NMR spectroscopy analysis results show that Rut and Que in a molar ratio of 1:1 co-assemble into nanoparticles with a particle size range of 100-120 nm and a polydispersity index of 0.182 under alkaline conditions through intermolecular hydrogen bonding and π-π stacking.

[0019] Subsequently, RQ nanoparticles were loaded into the GA hydrogel framework. SEM imaging revealed that the GRQ-gel formed a porous network structure composed of surface-loaded RQ nanoparticles. Within the frequency range of 0.1–100 rad / s, the storage modulus (G') of the GRQ-gel consistently exceeded the loss modulus (G''), confirming its viscoelastic characteristics. In the long term, the GRQ-gel offers advantages such as being environmentally friendly, easy to prepare, and highly biosafe. As a proof-of-concept, the GRQ-gel can be applied to sites of myocardial fibrosis, significantly reducing collagen deposition and improving cardiac function. Furthermore, the GRQ-gel significantly reduced the expression of MHC-II antigen in myocardial fibroblasts, inhibited the response of CD4+ T cells, and downregulated the expression of inflammatory factors IL-17 and IL-22. Overall, the co-assembly strategy proposed in this study provides a new approach for constructing carrier-free flavonoid drug delivery systems based on the concept of "medicine and food homology," and offers insights for developing drugs targeting cardiac fibroblast antigen presentation. Currently, there are no reports on the development of pH-driven hydrogels containing flavonoids that are both medicinal and edible.

[0020] This invention produces a hydrogel of flavonoid components that are both medicinal and edible, possessing outstanding substantial characteristics and optimal efficacy. It shows great promise for the treatment of myocardial infarction, myocardial fibrosis, and vascular injury. This invention utilizes the self-assembly of small-molecule natural flavonoids to form the hydrogel, making it a green, simple, and commercially viable method. Attached Figure Description

[0021] Figure 1 For the preparation and characterization of RQ nanoparticles formed by Rut and Que in different molar ratios in this invention, (A) Tyndall effect, (B) particle size distribution, (C) zeta potential, (D) circular dichroism spectroscopy, (E) ultraviolet-visible spectroscopy, (F) Fourier transform infrared spectroscopy, (G) X-ray diffraction spectroscopy. Figure 2 This is a TEM image of the RQ nanoparticles (Rut:Que=1:1) in this invention; Figure 3 For the stability analysis of RQ nanoparticles (Rut:Que=1:1) in this invention, A is the particle size and B is the PDI; Figure 4 For the molecular dynamics simulation of RQ nanoparticles (Rut:Que=1:1) in this invention, (A) the change of RQ nanoparticle structure over time during the molecular dynamics simulation (purple molecule: PAE, green molecule: GA), (B) the structural changes of RQ nanoparticles at 0 and 100 ns, (C) the intermolecular interaction analysis of RQ nanoparticles, (D) the RMSD of RQ nanoparticles during the simulation time, (E) SASA, (F) hydrogen bonds, (G) RG; Figure 5 The following are the (A) gel diagram, (B) injectability, and (C) adhesion of the GRQ-gel in this invention; Figure 6 The assembly mechanism of GRQ-gel in this invention is shown. (A) UV-Vis spectra of GA, RQ and GRQ-gel, (B) UV-Vis spectra of GRQ-gel at different concentrations, (C) Fourier transform infrared spectra of GA, RQ and GRQ-gel, (D) X-ray diffraction spectra of GA, RQ and GRQ-gel; Figure 7 This is a TEM image of the GRQ-gel in this invention; Figure 8 Rheological analysis of GRQ-gel in this invention; Figure 9 This is a biosafety evaluation of GRQ-gel in this invention.

[0022] Figure 10 This is a pharmacodynamic evaluation of GRQ-gel in treating isoproterenol-induced myocardial fibrosis according to the present invention.

[0023] Figure 11 This study evaluates the mechanism by which GRQ-gel inhibits myocardial fibroblast antigen presentation and improves myocardial fibrosis in this invention. Detailed Implementation

[0024] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0025] Example 1: Construction of a pH-driven hydrogel co-assembled with food-grade flavonoids and its application in treating myocardial fibrosis after myocardial infarction. 1. Preparation of RQ nanoparticles co-assembled with rutin and quercetin Rutin and quercetin in different molar ratios were added to 10 mL of water and stirred with a magnetic stirrer at 1000 rpm for 10 minutes at 80 °C. Simultaneously, 0.2 mol / L NaOH solution was added dropwise using a pipette to adjust the pH of the system to 8.5. The mixture was then sonicated at 200 W for 5 minutes to prepare RQ nanoparticles. The Tyndall effect of the samples was observed using a laser pointer. Finally, the obtained RQ nanoparticle solution was freeze-dried into powder and stored at 4 °C.

[0026] Result: See Figure 1 A. Although Rut and Que can spontaneously assemble into nanoparticles under alkaline conditions and exhibit a significant Tyndall effect, the Tyndall effect optical path of RQ nanoparticles prepared at a 1:1 ratio is clearer than that of Rut and Que at 2:1 and 1:2 molar ratios.

[0027] 2. Particle size distribution and zeta potential analysis of rutin-quercetin co-assembled RQ nanoparticles Take appropriate amounts of Rut, Que, and RQ nanoparticles (2:1, 1:1, 1:2), dilute them 3 times, and test them using a high-sensitivity Zeta potential and particle size analyzer.

[0028] Results: According to dynamic light scattering analysis, the nanoparticles formed by Rut and Que exhibited uneven particle size distribution, with significant large particles. When the molar ratio of Rut to Que was 2:1, 1:1, and 1:2, the resulting RQ nanoparticles had particle sizes of approximately 106 nm (PDI: 0.243), 112 nm (PDI: 0.182), and 150 nm (PDI: 0.278), respectively. Figure 1 B). Furthermore, the zeta potentials of the Rut and Que nanoparticles are approximately -6.78 mV and -11.22 mV, respectively. Figure 1C). Co-assembled RQ nanoparticles prepared using different Rut to Que molar ratios exhibit significantly higher zeta potentials than those prepared using Rut or Que alone, especially at a 1:1 molar ratio. This phenomenon indicates that RQ nanoparticles prepared under this molar ratio condition possess the highest zeta potential and the best stability.

[0029] 3. Circular dichroism chromatographic analysis of rutin-quercetin co-assembled RQ nanoparticles Appropriate amounts of Rut, Que, and RQ nanoparticles (2:1, 1:1, 1:2) were placed in quartz cuvettes, ensuring the samples were homogeneous and free of air bubbles. A circular dichroism spectroscopy (CDS) instrument was used for scanning, with typical parameter settings including a wavelength range of 200-400 nm and a scan rate of 100 nm / min.

[0030] Result: See Figure 1 D. Individual Rut nanoparticles exhibit a distinct positive peak at 207 nm and a broad peak in the 250–275 nm range. In contrast, the original spectrum of individual Que nanoparticles shows a relatively flat baseline peak. However, after co-assembly of Rut and Que, the peak intensities of RQ nanoparticles in different molar ratios significantly increased in the 207 nm and 250–275 nm ranges, and shifted to varying degrees, especially at a 1:1 molar ratio. Compared to individual Rut and Que nanoparticles, the co-assembled RQ nanoparticles showed two strong negative peaks at 233 nm and 278 nm, indicating the presence of π-π stacking and hydrogen bonding interactions between the two compounds.

[0031] 4. UV-Vis absorption spectroscopy analysis of rutin-quercetin co-assembled RQ nanoparticles Appropriate amounts of Rut, Que, and RQ nanoparticles (2:1, 1:1, 1:2) were taken, diluted 200 times, and placed in a UV spectrophotometer to measure the UV-Vis absorption spectra in the wavelength range of 200-800 nm.

[0032] Result: See Figure 1 In the UV-Vis spectrum of Rut, three distinct absorption peaks were observed at approximately 268 nm, 325 nm, and 397 nm. Similarly, the characteristic peaks of Que were located at 252 nm, 310 nm, and 327 nm. After co-assembly, a significant blue shift was observed in the characteristic peak at 397 nm, indicating a strong π-π stacking interaction between Rut and Que.

[0033] 5. Fourier Infrared Spectroscopy Analysis of RQ Nanoparticles Co-assembled with Rutin and Quercetin Take appropriate amounts of Rut, Que, and RQ nanoparticles (2:1, 1:1, 1:2) and compress them together with potassium bromide into tablets. Scan the samples using a Fourier Infrared Spectrometer in the wavelength range of 4000-400 cm⁻¹. -1 The map was obtained by internal scanning.

[0034] Result: See Figure 1 F, at 3000-3700 cm -1 Within the range, both Rut and Que exhibit broad and strong stretching vibration peaks of the phenolic hydroxyl group (-OH), with Que showing a peak at 1650 cm⁻¹. -1 A carbonyl (C=O) stretching vibration peak appears at [location missing]. Compared to Rut or Que alone, the hydroxyl absorption peak in the assembled complex is significantly broadened and shifted towards lower wavenumbers (from 3400 cm⁻¹). -1 Dropped to 3380 cm -1 In addition, a slight red shift was observed in the carbonyl absorption peak. (Fingerprint region (1200-1500 cm⁻¹)) -1 The peak shape and relative intensity of the aromatic ring skeletal vibrations also changed. This phenomenon indicates that Rut and Que formed intermolecular hydrogen bonds after co-assembly.

[0035] 6. X-ray diffraction analysis of rutin-quercetin co-assembled RQ nanoparticles Appropriate amounts of Rut, Que, and RQ nanoparticles (2:1, 1:1, 1:2) were tested at 40 kV and 40 mA on an X-ray diffractometer at room temperature using Cu-Kα radiation, with a 2θ diffraction angle range of 5°-80°.

[0036] Result: See Figure 1 G, Rut, and Que exhibit multiple sharp diffraction peaks in the 10–50° range, suggesting their crystalline structure. However, the co-assembled RQ nanoparticles show significant broad peaks in the 20–30° range, indicating that these nanoparticles have an amorphous structure. In particular, compared with molar ratios of 1:1 and 1:2, the nanoparticles prepared at a molar ratio of 2:1 still show a distinct crystalline diffraction peak at approximately 31.48°, indicating incomplete co-assembly. Based on the results of the zeta potential of RQ nanoparticles prepared with different molar ratios (R:Q = 2:1, 1:1, 1:2), subsequent studies selected a molar ratio of R:Q = 1:1.

[0037] 7. TEM analysis of rutin-quercetin co-assembled RQ nanoparticles RQ nanoparticles were placed on a copper mesh support film, allowed to dry at room temperature, and then negatively stained with sodium tungstate phosphate (or uranium oxyacetate). Their microstructure was observed by TEM.

[0038] Result: See Figure 2RQ samples exhibit spherical nanoparticles with a diameter of approximately 40-50 nm. Compared to DLS analysis, TEM measurements revealed smaller particle sizes in the dry state.

[0039] 8. Stability analysis of RQ nanoparticles co-assembled with rutin and quercetin An appropriate amount of RQ nanoparticles were placed in 10% serum for 48 hours, and the particle size, polydispersity index (PDI), and zeta potential were measured.

[0040] Result: See Figure 3 For A and 3B, after 48 hours of storage under in vitro serum conditions, the particle size, polydispersity index (PDI), and zeta potential of RQ nanoparticles did not show significant changes, indicating that they have good stability and meet the requirements for in vitro and in vivo administration.

[0041] 9. Molecular dynamics simulation of the co-assembly mechanism of Rut and Que Molecular dynamics simulations were performed using the GPU-accelerated program pmemd.cuda in AMBER 24. Force field parameters for Rut and Que molecules were generated by Anttecamber, and atomic charges were calculated using the AM1-BCC method. The force field type was set to the general AMBER force field GAFF, with missing parameters supplemented using parmchk2. The solvation model of the system adopted the TIP3P water model. The molar ratio of Rut to Que was set to 1:1, meaning the system contained 45 Rut molecules and 45 Que molecules. First, 90 solute molecules were randomly placed in an 80×80×80 Å cubic region using Packmol, and then a 10 Å thick TIP3P water layer was added outside the system using tleap. Energy minimization was performed on the system before the formal simulation. The minimization process was conducted under periodic boundary conditions, with a maximum of 20,000 iterations. The steepest descent method was used for the first 10,000 steps, after which the conjugate gradient method was used for further optimization. The RMS gradient convergence criterion was set to 0.1 kcal·mol⁻¹. -1 Å -1 Subsequently, the system underwent 1 ns NPT equilibrium simulations at 300 K and 1 bar, followed by 1 ns NVT equilibrium simulations at 300 K. Temperature was controlled using the Langevin dynamics method, and the collision frequency was 2.0 ps. -1The pressure in the NPT simulation was controlled using the Monte Carlo pressure constant method, with a pressure relaxation time of 2.0 ps. Chemical bonds involving hydrogen atoms were constrained using the SHAKE algorithm, thus the integration step size was set to 2 fs. After equilibrium was reached, a 100 ns NPT generation simulation was performed at 300 K and 1 bar. The cutoff radius for nonbonded interactions was set to 12.0 Å, and long-range electrostatic interactions were handled using the particle-grid Ewald method. Trajectory coordinates were saved every 25,000 steps (i.e., every 50 ps), therefore the 100 ns generation component trajectory contained 2000 frames. After the simulation, the molecular structures at times 0, 20, 40, 60, 80, and 100 ns were extracted using CPPTRAJ, and parameters such as RMSD, solvent-accessible surface area, radius of rotation, and hydrogen bonds between Rut and Que molecules were further calculated.

[0042] Result: See Figure 4 As shown in A and 4B, Rut and Que were fully aggregated after 60 ns of simulation. The mean root mean square deviation (RMSD) was 4.53 ± 0.2 nm. After 60 ns, the RMSD value tended to stabilize, indicating that the RQ nanoparticle system maintained structural stability throughout the simulation. Figure 4 D). Furthermore, from the start of the simulation to 20 ns, the solvent accessible surface area (SASA) increased from 110 mm². 2 Reduced to 220 mm 2 The SASA value stabilized after 40 ns, indicating the formation of a stable and dense aggregated structure within the system. Figure 4 C). In the co-assembly system of RQ nanoparticles, the benzene ring structures of Rut and Que favor π-π stacking, while the hydroxyl functional groups in their structures provide binding sites for intermolecular hydrogen bonds. For example... Figure 4 As shown in Figure E, the number of hydrogen bonds in the RQ nanoparticles reaches a maximum of 45 within 100 ns after 40 ns. Subsequently, the time-varying radius of rotation (RG) was used to assess the transition from molecular diffusion to a more compact aggregated state. From the start of the simulation up to 20 ns, the RG value rapidly decreased, indicating a strong interaction between Rut and Que that promotes their bonding, likely due to non-covalent interactions. Thereafter, the RG value stabilized at approximately 4, indicating that a stable aggregate had formed between Rut and Que. Figure 4 F). Overall, this study successfully constructed nanospheres assembled from two natural flavonoids (Rut and Que) without the need for a carrier or organic solvent. The preparation process is simple and controllable, providing a research foundation for the construction of flavonoid nanocarriers.

[0043] 10. Preparation, injectability, and adhesiveness of GRQ-gel GRQ-gel was prepared by loading RQ nanoparticles with glycyrrhizic acid (GA) as the gel matrix.

[0044] Specifically, 5 mg of lyophilized RQ nanoparticles and 30 mg of glycyrrhizic acid (GA) were added to 1 mL of water, heated to 80°C until completely dissolved, and then cooled to room temperature to obtain GRQ-gel. The success of hydrogel preparation can be preliminarily determined by observing its appearance and injection properties.

[0045] Result: See Figure 5 Specifically, it employs the classic heating and cooling method. For example... Figure 5 As shown in A and 5B, GRQ-gel presents as a uniform, light yellow hydrogel with good injectability, a crucial prerequisite for local administration. Simultaneously, GRQ-gel effectively adheres to the major organs of mice (…). Figure 5 C).

[0046] 11. UV-Vis absorption spectroscopy analysis of GRQ-gel An appropriate amount of GRQ-gel sample was taken, diluted 200 times, and placed in a UV spectrophotometer to measure the UV-Vis absorption spectrum in the wavelength range of 200-800 nm.

[0047] Result: See Figure 6 The UV-Vis spectra of A and GA show a distinct absorption peak at 259 nm, attributed to the π-π* transition in the enone conjugated structure. The UV-Vis spectrum of GRQ-gel contains two representative absorption peaks at 259 nm and 358 nm. This indicates that GRQ-gel retains the original hydrogel framework structure of GA. Furthermore, the absorption band I of flavonoids in RQ nanoparticles shifts from 364 nm to 358 nm, suggesting the presence of π-π stacking interactions in the hydrogel system. Further, the hydroxyl groups of GA can form hydrogen bonds with the phenolic hydroxyl groups of RQ nanoparticles, thereby promoting a blue shift in the absorption band I of flavonoids in RQ nanoparticles. Moreover, with increasing GRQ-gel concentration (overall hydrogel concentration), the characteristic peak of the GA framework in GRQ-gel shows a red shift (from 259 nm to 272 nm), while the absorption band I of flavonoids in RQ nanoparticles shows a blue shift (from 364 nm to 356 nm), indicating enhanced encapsulation performance of RQ nanoparticles. Figure 6 B).

[0048] 12. Fourier transform infrared spectroscopy analysis of GRQ-gel Take appropriate amounts of RQ nanoparticles, GA and GRQ-gel samples and compress them together with potassium bromide into tablets. Scan the wavelength range of 4000-400 cm⁻¹ using a Fourier transform infrared spectroscopy. -1 The map was obtained by internal scanning.

[0049] Result: See Figure 6 The FTIR spectra of C and GA are in the range of 3683–3033 cm⁻¹. -1 (O–H), 2934 / 2866 cm -1 (C–H), 1647cm -1 (C=O) and 1035 cm -1 Characteristic peaks are observed at (C–O). RQ nanoparticles exhibit peaks at 3674–3070 cm⁻¹. -1 The O–H peak is shown at 1718 cm⁻¹. -1 A C=O peak for flavonoids appeared at 1718 cm⁻¹, along with an aromatic C=C stretching vibration peak at 1604 cm⁻¹. Multiple peak shifts were observed after GA and RQ nanoparticles were co-assembled. First, the C=O peak for flavonoids shifted from 1718 cm⁻¹ to 1604 cm⁻¹. -1 Moved to 1721 cm -1 Secondly, the C=O peak of GA is from 1647 cm⁻¹. -1 Moved to 1650 cm -1 Meanwhile, the O–H peaks broaden and shift towards the lower wavenumber region (3628–3000 cm⁻¹). -1 Meanwhile, the C–O peak decreased from 1035 cm⁻¹. -1 Slightly shifted to 1031 cm -1 The blue shift of these two C=O stretching vibrations indicates that the carbonyl functional groups of GA and RQ act as hydrogen bond acceptors, interacting with the hydroxyl groups of their respective compounds. The broadening and red shift of the O–H peaks further confirm the formation of an enhanced hydrogen bond network within the GRQ hydrogel. These results provide direct spectroscopic evidence that hydrogen bonding is the primary driver of RQ nanoparticle embedding within the GA hydrogel framework.

[0050] 13. X-ray diffraction analysis of GRQ-gel RQ nanoparticles, GA and GRQ-gel were tested at 40kV and 40mA on an X-ray diffractometer, respectively, with Cu-Kα radiation at room temperature and a 2θ diffraction angle range of 5°-80°.

[0051] Result: See Figure 6 The XRD pattern of D and GA showed a distinct diffraction peak at 14.65°. GRQ-gel retained the diffraction peak characteristic of GA, indicating that the addition of RQ nanoparticles did not affect the basic structural framework of the GA hydrogel.

[0052] 14. SEM analysis of GRQ-gel After freeze-drying the GRQ-gel, a fresh cross-section was obtained by liquid nitrogen embrittlement. After gold sputtering, the cross-section was observed under an accelerating voltage of 5 kV.

[0053] Result: See Figure 7 GRQ-gel forms a porous network structure composed of surface-loaded RQ nanoparticles.

[0054] 15. Rheological evaluation of GRQ-gel The GRQ-gel was placed in a rheometer for testing. Frequency scanning: shear strain was constant at 0.1%, frequency was 0.1-10 Hz, amplitude scanning: frequency was 1 Hz.

[0055] Result: See Figure 8 In the frequency range of 0.1–100 rad / s, the storage modulus (G') of GRQ-gel is always higher than the loss modulus (G''), confirming its viscoelastic properties.

[0056] 16. Biosafety Analysis of GRQ-gel Healthy C57BL / 6J mice were administered GA, RQ, and GRQ-gel for 30 days, respectively. A control group received no treatment. After 30 days of treatment, hearts, livers, spleens, lungs, and kidneys were collected from each group for HE staining.

[0057] Result: See Figure 9 No abnormal or damaged internal organs were observed in any of the groups, indicating that the toxicity of long-term use of GRQ-gel is negligible. This may be attributed to its composition of bioactive ingredients extracted from natural medicines and food-derived herbs.

[0058] 17. Pharmacodynamic evaluation of GRQ-gel in the treatment of myocardial fibrosis A myocardial infarction (MF) mouse model induced by ISO was established by subcutaneous injection of 50 mg / kg / day of isoproterenol (ISO) for 14 days. Healthy mice served as the control group (CON). The MF mice were further divided into ISO, GA gel, and GRQ-gel groups. Mice in the drug groups received myocardial injections of GA-gel and GRQ-gel, respectively, while the ISO group received an equal volume of saline. After 14 days of treatment, echocardiography was performed on all mice. Subsequently, the hearts of all mice were collected, fixed, embedded, and sectioned. Myocardial sections were stained with hematoxylin and eosin (HE), Masson's stain, and Sirius red stain to assess myocardial damage and collagen deposition.

[0059] The explanation for why only GA-gel was used as a control, while there was no control for rutin-quercetin nanoparticles, is that the administration method is direct myocardial injection. Therefore, two hydrogels, GA-gel and GRQ-gel, were selected. GRQ nanoparticles are not in a gel state, so they are not suitable for myocardial injection.

[0060] Result: See Figure 10Compared with the control group, the ISO group showed significant cardiac dysfunction and left ventricular remodeling, manifested as a significant decrease in ejection fraction (EF) (45.31%), a significant decrease in ventricular fractional shortening (FS) (22.07%), and a significant increase in left ventricular end-diastolic diameter (LVESD) (2.74 mm) and left ventricular end-diastolic diameter (LVEDD) (3.52 mm). MF mice treated with GRQ-gel showed a significant increase in ejection fraction (EF%) (71.27%), a significant increase in ventricular fractional shortening (FS%) (39.57%), and a significant decrease in left ventricular end-diastolic diameter (LVESD) (1.95 mm), indicating that its therapeutic effect was superior to GA-gel alone. However, we noted that although LVEDD values ​​decreased after GA or GRQ-gel treatment compared to the ISO group, the difference was not statistically significant. Subsequently, HE staining, Masson staining, and Sirius Red staining were used to examine the effects of GRQ-gel treatment on myocardial tissue damage and collagen deposition in MF mice. In the CON group, cardiomyocytes were uniformly red, tightly packed, clearly structured, and of consistent size. However, as cardiac function deteriorated, the ISO group showed fibrosis in its myocardial tissue, with loosely arranged and unevenly sized cardiomyocytes, indicating significant damage. After GRQ-gel treatment, myocardial damage in MF mice was significantly reduced. Masson and Sirius Red staining results showed that both GA and GRQ-gel reduced collagen deposition compared to the ISO group.

[0061] 18. GRQ-gel inhibits myocardial fibroblast antigen presentation and improves myocardial fibrosis. Fourteen days after treatment, myocardial tissue was removed from each group of mice, and immunofluorescence staining was performed to detect CD4, IL-17, IL-22, MHC-II, and Vimentin.

[0062] Result: See Figure 11During myocardial fibroblast (MF), myocardial fibroblasts highly express MHC-II, thereby promoting the presentation of CD4+ T cells. Co-localization staining of the fibroblast markers Vimentin and MHC-II showed that, compared with the control group (CON), the co-localization expression of MHC-II and Vimentin in myocardial tissue was significantly increased in the ISO group. Compared with the control group, the ISO group also showed a significant increase in CD4+ T cells, along with significantly elevated levels of IL-17 and IL-22. However, after treatment with GRQ-gel, the proportion of positive CD4+ T cells significantly decreased, and the expression of inflammatory factors IL-17 and IL-22 was also significantly reduced. Furthermore, the co-localization expression of MHC-II and Vimentin in myocardial tissue was significantly decreased in the GRQ-gel group. These results indicate that GRQ-gel can inhibit antigen presentation by fibroblasts, thereby blocking the CD4+ T cell-mediated inflammatory response and improving fibrosis symptoms.

Claims

1. A pH-driven hydrogel co-assembled with medicinal and edible flavonoid components, characterized in that, It includes RQ nanoparticles and glycyrrhizic acid.

2. The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components according to claim 1, characterized in that, RQ nanoparticles are formed by the co-assembly of rutin and quercetin and are prepared by the following steps: rutin and quercetin are added to 10 mL of water at a molar ratio of 2:1-1:2, stirred at 1000 rpm for 5-10 minutes at 60-80℃ using a magnetic stirrer, the pH is adjusted to 8.0-8.9 with 0.2 mol / L NaOH, and then sonicated at 200 W for 5-10 minutes to obtain RQ nanoparticles.

3. The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components according to claim 1, characterized in that, The molar ratio of rutin to quercetin is 1:

1.

4. The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components according to claim 1, characterized in that, It is prepared by the following steps: RQ nanoparticles and glycyrrhizic acid are mixed in a mass ratio of 1:2-1:8, added to 1 mL of water, heated to 60-80℃ to completely dissolve, and cooled to room temperature to obtain the hydrogel GRQ-gel.

5. The pH-driven co-assembled hydrogel of medicinal and edible flavonoid components according to claim 4, characterized in that, 5 mg / mL LRQ nanoparticles were co-assembled with 30 mg / mL GA to form GRQ-gel.

6. The application of the pH-driven co-assembled hydrogel of medicinal and edible flavonoid components according to any one of claims 1-5 in the preparation of drugs for inhibiting myocardial infarction, myocardial fibrosis or vascular damage.

7. The application according to claim 6, characterized in that, It improves fibrosis symptoms by inhibiting antigen presentation by fibroblasts, thereby blocking the inflammatory response mediated by CD4+ T cells.

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