Lysozyme-based quercetin colon-targeting nanoparticle and preparation method and application thereof

Abstract

The application provides a lysozyme-based quercetin colon-targeting nanoparticle and a preparation method and application thereof. 2+ A metal polyphenol network (MPN) nanocore is sequentially self-assembled with positively charged egg white lysozyme and negatively charged pectin through electrostatic self-assembly to form a nanoparticle with a triple-coating structure. The quercetin encapsulation rate of the nanoparticle ranges from 77% to 98%, the drug loading rate ranges from 13% to 32.2%, and the nanoparticle has excellent gastrointestinal stability and colon targeting. The results of high-fat model animal experiments show that the nanoparticle has good lipid-lowering effect and antioxidant capacity, and protects the structure and function of the intestinal tract. The application effectively solves the problems of poor stability and insufficient targeting in oral delivery of quercetin, and provides a green and efficient solution for colon-targeted delivery of polyphenol functional factors.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nano products, and particularly relates to a lysozyme-based quercetin colon-targeted nanoparticle as well as a preparation method and application thereof. BACKGROUND

[0002] Quercetin is a natural flavonoid compound widely existing in lotus leaves and other plants, and has multiple biological activities such as antioxidant, anti-inflammatory, blood lipid regulation, and blood glucose regulation. It has been proved to have important application prospects in the prevention and control of chronic metabolic diseases. Studies have shown that the activity of quercetin in vivo not only depends on its original structure, but also depends on its metabolic transformation in the intestinal microecosystem, and especially the small molecule metabolites formed by microbial metabolism in the colon are more active. However, due to the poor stability of quercetin itself in the gastrointestinal environment and its easy oxidation and degradation, the bioavailability of quercetin in vivo is extremely low, which significantly limits its practical application in functional products or drugs.

[0003] In recent years, nano delivery technology has provided a new idea for solving this problem. Some studies have attempted to use microcapsules, nanoemulsions, liposomes and other delivery methods to embed and protect quercetin, aiming to improve its gastrointestinal stability and bioavailability, and some achievements have achieved preliminary results in the laboratory stage. However, the existing technology generally lacks the ability to respond to multiple factors such as pH, enzymes, and microbial metabolism in the gastrointestinal tract, and the targeting and controlled release are still not ideal. SUMMARY

[0004] The purpose of the present application is to provide a quercetin-lysozyme-pectin self-assembled nanoparticle, which is prepared by Fe 2+ The metal polyphenol network (MPN) forms a nano core with quercetin, and is then coated with a layer of egg white lysozyme and a layer of pectin in sequence. This structure not only avoids the degradation of quercetin in the gastrointestinal environment, but also realizes colon-targeted release, thereby improving the efficacy of quercetin.

[0005] The present application provides a quercetin-lysozyme-pectin self-assembled nanoparticle, which comprises a nano core and a layer of egg white lysozyme and a layer of pectin coated in sequence from the inside out.

[0006] The nano core is a quercetin-Fe 2+ metal polyphenol network;

[0007] The mass ratio of the nano core, the layer of egg white lysozyme, and the layer of pectin is 3-6:7-10:7-10.

[0008] Preferably, the quercetin-Fe 2+ metal polyphenol network has a Fe 2+The mass ratio of quercetin to ferrous chloride tetrahydrate is 1:1-6.5.

[0009] Preferably, the mass ratio of quercetin to ferrous chloride tetrahydrate is 1:3-4.

[0010] Preferably, the mass ratio of the nanocore, egg white lysozyme layer and pectin layer is 0.4-0.85:1:1.

[0011] The application provides a preparation method of the quercetin-lysozyme-pectin self-assembled nanoparticle, comprising the following steps:

[0012] Mixing and reacting a quercetin solution and a ferrous ion-containing solution to form a quercetin-Fe 2+ a metal polyphenol network, denoted as a first nanoparticle;

[0013] Automatically assembling egg white lysozyme on the surface of the first nanoparticle to form a second nanoparticle;

[0014] Automatically assembling pectin on the surface of the second nanoparticle to obtain a quercetin-lysozyme-pectin self-assembled nanoparticle.

[0015] Preferably, the volume ratio of the quercetin solution to the ferrous ion-containing solution is 2-4:7-9.

[0016] The concentration of the quercetin is 5-10 mg / mL.

[0017] The ferrous ion-containing solution comprises a ferrous chloride tetrahydrate aqueous solution.

[0018] The concentration of the ferrous chloride tetrahydrate aqueous solution is 2-12 mg / mL.

[0019] Preferably, the automatic assembly of egg white lysozyme or the automatic assembly of pectin is accompanied by oscillation.

[0020] The rotation speed of the oscillation is 300-600 rpm, and the oscillation time is 30-90 min.

[0021] Preferably, the coating concentration of egg white lysozyme in the egg white lysozyme layer is 0.5-2 mg / mL.

[0022] The application provides a lipid-lowering product, and the active ingredient comprises the quercetin-lysozyme-pectin self-assembled nanoparticle or the quercetin-lysozyme-pectin self-assembled nanoparticle prepared by the preparation method.

[0023] The application provides application of the quercetin-lysozyme-pectin self-assembled nanoparticle or the quercetin-lysozyme-pectin self-assembled nanoparticle prepared by the preparation method in preparation of a medicine for preventing and / or treating chronic metabolic diseases.

[0024] The application provides a quercetin-lysozyme-pectin self-assembled nanoparticle, which comprises a nanometer core and a lysozyme layer and a pectin layer successively coated from inside to outside; the nanometer core is a quercetin-Fe 2+ a metal polyphenol network; the mass ratio of the nanometer core, the lysozyme layer and the pectin layer is 3-6:7-10:7-10. The nanoparticle has the characteristics of high stability and high delivery efficiency, effectively avoids early degradation of quercetin in a gastrointestinal environment through the protection of the three-layer structure, and improves the structural stability, encapsulation rate and drug loading rate; meanwhile, the lysozyme and the pectin have response ability to pH, enzymes and intestinal bacteria metabolites, have good colon-targeted release behavior, improve the absorption and metabolism of quercetin in the intestinal tract, and are beneficial to improving the availability of quercetin. In addition, the lysozyme and the pectin are of natural origin, are non-toxic and edible, are suitable for oral products, and meet the green edible safety standards.

[0025] The application provides a lipid-lowering product, and active ingredients include the quercetin-lysozyme-pectin self-assembled nanoparticle or the quercetin-lysozyme-pectin self-assembled nanoparticle prepared by the preparation method. In-vivo experimental results of the application show that the nanoparticle can significantly improve the bioavailability of quercetin, improve the body weight and blood lipid level (triglyceride content and cholesterol content) in a high-fat model, stabilize lipid metabolism, and play a lipid-lowering, antioxidant and anti-inflammatory effect better than free quercetin; and improve the movement activity of model animals, reduce the intestinal leakage rate and protect the intestinal barrier. The product has good market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure A is a preparation process flow diagram of the quercetin-lysozyme-pectin self-assembled nanoparticle in the application; Figure 1 Figure B is a SEM diagram of the quercetin-lysozyme-pectin self-assembled nanoparticle, and the scale is 1 μm; Figure 1 Figure C is a TEM diagram of the quercetin-lysozyme-pectin self-assembled nanoparticle; Figure 1 Figure D is a particle size distribution diagram of the quercetin-lysozyme-pectin self-assembled nanoparticle; Figure 1 Figure E is the average particle size of the quercetin-lysozyme-pectin self-assembled nanoparticle; Figure 1 Figure F is the polydispersity index of the quercetin-lysozyme-pectin self-assembled nanoparticle; Figure 1 Figure G is a potential diagram of the quercetin-lysozyme-pectin self-assembled nanoparticle; wherein p <0.05; p <0.005; p <0.05001; ns indicates p >0.05;

[0027] Figure 2 In Figure A, the full-spectrum UV-Vis spectrum of the quercetin-lysozyme-pectin self-assembled nanoparticles in this embodiment of the invention is shown. Figure 2 B in the image represents the FTIR spectrum of quercetin-lysozyme-pectin self-assembled nanoparticles.

[0028] Figure 3 The in vitro simulated digestion release curves of quercetin-lysozyme-pectin self-assembled nanoparticles at different time points in the stomach and small intestine environments in this embodiment of the invention;

[0029] Figure 4 The results of the in vivo lipid-lowering effect of quercetin-lysozyme-pectin self-assembled nanoparticles on high-fat fruit flies in the embodiments of the present invention are as follows; Figure 4 In the middle, A represents the weight measurement result; Figure 4 B represents the result of exercise vitality; Figure 4 C represents the serum triglyceride test result; Figure 4 D represents total cholesterol; Figure 4 E represents the SOD measurement result; Figure 4 F represents the CAT measurement result; Figure 4 G represents the intestinal leakage rate; Figure 4 In the diagram, H represents intestinal leakage in fruit flies. CS indicates the standard culture medium group prepared with corn flour-yeast powder-agar, HFD indicates the high-fat diet control group, MPN indicates the high-fat diet culture medium group containing 0.25% (w / v) quercetin powder, MPN-LYS-PEC indicates the high-fat diet culture medium group containing 0.83% (w / v) quercetin-lysozyme-pectin nanoparticles (MPN-LYS-PEC) powder, and LYS-PEC indicates the high-fat diet culture medium group containing 0.58% lysozyme-pectin nanoparticles powder. p <0.05; p <0.01; p <0.005; p <0.001; ns indicates p >0.05. Detailed Implementation

[0030] The application provides a quercetin-lysozyme-pectin self-assembled nanoparticle (MPN-LYS-PEC), which comprises a nanoparticle core and a lysozyme layer and a pectin layer successively coated from inside to outside.

[0031] The nanoparticle core is quercetin-Fe 2+ metal polyphenol network.

[0032] The mass ratio of the nanoparticle core, the lysozyme layer and the pectin layer is 3-6:7-10:7-10.

[0033] In the application, quercetin is used as an active ingredient in the nanoparticle and plays a biological function. The quercetin is complexed with Fe 2+ to form a metal polyphenol network (MPN), the quercetin contains multiple ortho-phenolic hydroxyl groups and can be coordinated with Fe 2+ ions at multiple points to construct a three-dimensional crosslinked structure. The quercetin-Fe 2+ metal polyphenol network contains Fe 2+ , and the mass ratio of quercetin to ferrous chloride tetrahydrate is preferably 1:1-6.5, and can be 1:3-4. The mass ratio of the quercetin and Fe 2+ complexation can affect the volume of the nanoparticle core. When the proportion of quercetin increases, the number of ligands participating in coordination in the system increases, resulting in enhanced complexation degree, extended network chain, increased particle volume, and more likely aggregation to form larger nanocomposites, which affects the uniformity of the system. Meanwhile, when the proportion of quercetin increases, the Zeta potential changes first increases and then decreases, and the nanoparticle surface has a higher negative charge, stronger electric repulsion and optimal colloidal stability when the medium proportion (1:3.3) is used, which provides an ideal interface state for subsequent self-assembly.

[0034] In the application, the lysozyme layer is electrostatically assembled on the surface of the nanoparticle core. The lysozyme layer is positively charged, and the nanoparticle core is negatively charged, which indicates that as the concentration of lysozyme increases, the density of positive charges in the system increases, which promotes the adsorption and coating of lysozyme on the surface of the nanoparticle core and forms a thicker protein shell layer, thereby causing the particle size to swell. The lysozyme is sensitive to pH, protease and intestinal bacteria metabolites in the intestinal tract and can be rapidly degraded in the intestinal tract, thereby achieving rapid release of the nanoparticle core.

[0035] In the application, the pectin layer is electrostatically assembled on the surface of the lysozyme layer. The lysozyme nanoparticle coated on the nanoparticle core is positively charged, and the pectin is negatively charged, which are combined through electrostatic interaction to achieve encapsulation and drug loading of the nanoparticle core.

[0036] In the present application, the mass ratio of the nanocore, egg white lysozyme layer and pectin layer is preferably 0.4-0.85:1:1. Too high coating concentration of the egg white lysozyme layer will cause the surface of quercetin-lysozyme nanoparticle (MPN-LYS) to be loose, or a large amount of free egg white lysozyme in the system, reducing the surface charge density of the nanoparticle, and further affecting the assembly of the pectin layer. The ratio of the egg white lysozyme layer and the pectin layer affects the structure and encapsulation rate of the nanoparticle and the drug loading property. Experiments show that when the mass ratio of the egg white lysozyme layer and the pectin layer is 1:1, the particle size, potential, quercetin encapsulation rate and drug loading rate are optimal; when the pectin layer is increased by 2 times the mass of the egg white lysozyme, the particle size of the nanoparticle increases, and the quercetin encapsulation rate and drug loading rate decrease; and when the egg white lysozyme layer is increased by 2 times the mass of the pectin, the surface charge of the nanoparticle is not neutralized, the potential increases, the stability of the nanoparticle is poor, and the quercetin encapsulation rate and drug loading rate decrease.

[0037] In the present application, the particle size of the quercetin-lysozyme-pectin self-assembled nanoparticle is 608.2±12.9-697.0±9.7 nm, and the Zeta potential is-17.3±2.9--29.3±3.7 mV. The quercetin encapsulation rate of the nanoparticle is 80.2±3.2-95.3±2.7%, and the drug loading rate ranges from 15.3±2.3 to 30.3±1.9%.

[0038] The present application provides a preparation method of the quercetin-lysozyme-pectin self-assembled nanoparticle, comprising the following steps:

[0039] Mixing and reacting a quercetin solution and a solution containing ferrous ions to form a quercetin-Fe 2+ metallic polyphenol network, denoted as a first nanoparticle;

[0040] Automatically assembling egg white lysozyme on the surface of the first nanoparticle to form a second nanoparticle;

[0041] Automatically assembling pectin on the surface of the second nanoparticle to obtain a quercetin-lysozyme-pectin self-assembled nanoparticle.

[0042] The present application mixes and reacts a quercetin solution and a solution containing ferrous ions to form a quercetin-Fe 2+ metallic polyphenol network, denoted as a first nanoparticle.

[0043] In the present application, the volume ratio of the quercetin solution and the solution containing ferrous ions is preferably 2-4:7-9, and can be 3-8. The concentration of the quercetin is preferably 5-10 mg / mL, and can be 5-8 mg / mL. The solution containing ferrous ions preferably comprises an aqueous solution of ferrous chloride tetrahydrate; the concentration of the aqueous solution of ferrous chloride tetrahydrate is preferably 2-12 mg / mL, and can be 4-10 mg / mL, or 6 mg / mL. The solvent for the mixing reaction is a PB buffer. The concentration of the PB buffer is preferably 10 mM, and the pH value is 7.4. The volume ratio of the quercetin solution, the solution containing ferrous ions, and the PB buffer is 0.3:0.8:10. The temperature of the mixing reaction is 18-27℃, and can be 20-25℃, or 22-24℃. The time of the mixing reaction is preferably 10-30 min, and can be 15-25 min, or 20 min. After the mixing reaction, the first nanoparticles are separated. The separation method of the first nanoparticles is preferably centrifugation. The rotation speed of the centrifugation is preferably 5000-10000 rpm, and can be 6000-9000 rpm, or 7000-8000 rpm. The time of the centrifugation is preferably 5-30 min, and can be 10-25 min, or 15-20 min.

[0044] In the present application, the egg white lysozyme automatic assembly or pectin automatic assembly is preferably accompanied by oscillation. The rotation speed of the oscillation is preferably 300-600 rpm, and can be 400-500 rpm. The time of the oscillation is preferably 30-90 min, and can be 40-80 min, or 50-70 min, or 60 min.

[0045] In the present application, the coating concentration of the egg white lysozyme in the egg white lysozyme layer is preferably 0.5-2 mg / mL, and can be 1-1.5 mg / mL, or 1.2 mg / mL. The coating concentration of the pectin is preferably 0.5-2 mg / mL, and can be 1-1.5 mg / mL, or 1.2 mg / mL.

[0046] In the present application, the prepared quercetin-lysozyme-pectin self-assembled nanoparticles are subjected to morphology observation, and the SEM results show that the nanoparticles are in a spherical structure as a whole, are uniformly distributed, have good particle size consistency, and no obvious aggregation phenomenon is observed. The TEM image further reveals the core-shell layered structure of the particles, and the boundary between the central core layer and the outer coating layer is clear, indicating that the quercetin-Fe 2+ The metal polyphenol network successfully serves as a nanometer core, the outer layer of lysozyme and pectin is completely coated, and the assembly structure is stable and orderly. At the same time, the particle size distribution is in a single peak state, reflecting that the system has good uniformity.

[0047] In the present application, the resistance of the quercetin-lysozyme-pectin self-assembled nanoparticles to gastric juice digestion mainly depends on the stability of the egg white lysozyme layer and the pectin layer, and the binding strength of the egg white lysozyme and the pectin mainly depends on the positive charge of the egg white lysozyme, which is stronger under acidic conditions and weaker under neutral conditions, thereby causing the release speed of the egg white lysozyme under neutral conditions to be faster than that under acidic conditions. In an embodiment of the present application, in vitro gastrointestinal fluid simulation digestion experiments are carried out, and the results show that after gastric juice digestion, the quercetin release rate of the quercetin-lysozyme-pectin self-assembled nanoparticles is only 7.9±1.9%, and the quercetin-lysozyme nanoparticles without pectin coating release 20.6±4.9% of quercetin. When the intestinal juice is digested, the quercetin-lysozyme nanoparticles release 60.4±4.6% of quercetin within 6 hours; the quercetin release rate of the quercetin-lysozyme-pectin self-assembled nanoparticles is only 26.2±5.9% within the same time. This shows that the pectin and the egg white lysozyme can effectively protect the MPN from degradation by pepsin in the SGF.

[0048] The present application provides a lipid-lowering product, and the active ingredient includes the quercetin-lysozyme-pectin self-assembled nanoparticles or the quercetin-lysozyme-pectin self-assembled nanoparticles prepared by the preparation method.

[0049] In the present application, the lipid-lowering product preferably includes a lipid-lowering drug, a lipid-lowering health product or a functional food. The functional food preferably includes a functional drink and a gel candy. The dosage form of the lipid-lowering drug includes a capsule, a tablet, a granule, a powder, an oral liquid and the like. The lipid-lowering drug has the functions of reducing blood lipid levels (triglyceride content and cholesterol content), improving antioxidant capacity, improving body movement activity, and protecting the intestinal barrier. The mass percentage content of the active ingredient in the lipid-lowering product is preferably 1% to 99%, can be 5% to 90%, can also be 10% to 70%, and further can be 20% to 50%.

[0050] The present application provides the application of the quercetin-lysozyme-pectin self-assembled nanoparticles or the quercetin-lysozyme-pectin self-assembled nanoparticles prepared by the preparation method in the preparation of a drug for preventing and / or treating chronic metabolic diseases.

[0051] In the present application, the chronic metabolic disease preferably includes at least one of the following: diabetes, hyperlipidemia and non-alcoholic fatty liver disease (Oxid Med Cell Longev. 2021 Jun 23;2021:6678662. doi:10.1155 / 2021 / 6678662. eCollection 2021.).

[0052] The lysozyme-based quercetin colon-targeting nanoparticle, the preparation method and application thereof provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0053] Example 1

[0054] Preparation method of quercetin-lysozyme-pectin self-assembled nanoparticles

[0055] Under the condition of stirring at 800 rpm, 10 mL of 10 mM phosphate buffer solution (PB buffer solution) at pH 7.4 was taken, and 0.3 mL of a quercetin solution (with methanol as a solvent, at a concentration of 5 mg / mL) and 0.8 mL of a FeCl2·4H2O solution (with deionized water as a solvent, at a concentration of 12 mg / mL) were mixed at a mass ratio of 1:6.4; 0.3 mL of a quercetin solution (with methanol as a solvent, at a concentration of 5 mg / mL) and 0.8 mL of a FeCl2·4H2O solution (with deionized water as a solvent, at a concentration of 6 mg / mL) were mixed at a mass ratio of 1:3.3 in the phosphate buffer solution; and 0.3 mL of a quercetin solution (with methanol as a solvent, at a concentration of 5 mg / mL) and 0.8 mL of a FeCl2·4H2O solution (with deionized water as a solvent, at a concentration of 2 mg / mL) were mixed at a mass ratio of 1:1.1 in the phosphate buffer solution; after reaction for 20 min, centrifugation was performed at 8000 rpm for 15 min to obtain quercetin-Fe 2+ Metal polyphenol network nanoparticles (MPN). The MPN nanoparticles prepared at a mass ratio of 1:3 of quercetin and FeCl2·4H2O and at a concentration of 5 mg / mL of quercetin solution were resuspended in 20 mL of an aqueous egg white lysozyme solution at a concentration of 0.5 mg / mL, 1 mg / mL and 2 mg / mL, and magnetic stirring was performed at 500 rpm for 60 min to complete the first layer of electrostatic assembly, and then centrifugation was performed at 8000 rpm for 15 min to collect the composite particles. The composite particles prepared from the 1 mg / mL aqueous egg white lysozyme solution were dispersed in 20 mL of an aqueous pectin solution at a concentration of 0.5 mg / mL, 1 mg / mL and 2 mg / mL, and magnetic stirring was continued at 400 rpm for 60 min to complete the second layer of self-assembly. Finally, centrifugation was performed at 8000 rpm for 15 min to obtain self-assembled nanoparticles with a three-layer structure of quercetin-lysozyme-pectin, and the preparation process is shown in detail in FIG. 1B. Figure 1

[0056] ​The samples of nanoparticles after freeze-drying were observed by field emission scanning electron microscopy (FE-SEM). In order to better observe the sample morphology, the conductivity of the nanoparticle powder was enhanced by gold spraying treatment. Transmission electron microscopy (TEM) was used to observe the structure of the nanoparticles. 10 μL of nanoparticle dispersion was dropped on a 400 mesh carbon support membrane net, and the excess dispersion was absorbed with filter paper. Then it was observed under an acceleration voltage of 80 kV. The particle size distribution, average particle size, polydispersity index (PDI) and zeta potential were measured by dynamic light scattering (DLS) method on a laser nanoparticle size analyzer Zetasizer Nano-ZS90. In addition, the encapsulation efficiency (EE) and loading efficiency (LE) of quercetin in the obtained nanoparticles were calculated according to the following formulas:

[0057] EE (%) = encapsulated quercetin mass / total quercetin mass added × 100% Formula I;

[0058] LE (%) = encapsulated quercetin mass / total nanoparticle mass × 100% Formula II.

[0059] The test results include:

[0060] Table 1 Quercetin and Fe 2+ Comparison of average particle size, polydispersity index (PDI) and zeta potential of MPN nanoparticles prepared under different mass ratios

[0061]

[0062] Note: The same column represents a significant difference in data P <0.05.

[0063] As shown in Table 1, quercetin and Fe 2+ complexes to form metal polyphenol network (MPN) nanoparticles as the core structure. As the mass ratio of quercetin to Fe 2+ increased from 1:6.4 to 1:1.1, the average particle size of the prepared MPN particles gradually increased, which was 367.0±8.4, 429.9±8.6 and 504.1±9.8 nm, respectively. This reflects that a higher proportion of quercetin promotes the further extension of the network structure. Quercetin molecules contain multiple ortho-phenolic hydroxyl groups, which can coordinate with Fe 2+The ions were coordinated by multiple points to construct a three-dimensional cross-linked structure. When the proportion of quercetin increased, the number of ligands that could participate in coordination in the system increased, leading to enhanced complexation degree, extended network chain, increased particle volume, and more easily occurred aggregation to form larger nano-complexes. This trend was also reflected in the change of polydispersity (PDI): the PDI of 1:5 group and 1:3 group was 0.374 ± 0.030 and 0.359 ± 0.027, respectively, indicating that the particle distribution was relatively uniform; while the PDI of 1:1 group significantly increased to 0.532 ± 0.031, suggesting that the particle aggregation intensified and the system uniformity decreased. In addition, the Zeta potential first increased and then decreased with the change of the proportion, and the 1:3 group reached the most negative potential (-31.1 ± 2.1 mV), which was higher than that of 1:5 (-23.3 ± 1.9 mV) and 1:1 (-20.2 ± 1.3 mV), indicating that the nanoparticles formed under the medium proportion (1:3) had higher surface negative charge, stronger electric repulsion, and optimal colloidal stability, which provided an ideal interface state for subsequent self-assembly.

[0064] Table 2 Preparation of MPN under different concentrations of egg white lysozyme 1:3 -Comparison of average particle size, polydispersity index (PDI) and potential of LYS nanoparticles

[0065]

[0066] Note: The same column, different letters represent significant differences P <0.05.

[0067] As shown in Table 2, when the proportion of quercetin to Fe 2+Under the condition of 1:3 ratio, MPN nanoparticles were electrostatically assembled with different concentrations of lysozyme (0.5, 1, 2 mg / mL), and the average particle size of the obtained MPN-LYS particles increased in turn (467.0 ± 5.4, 550.8 ± 17.2, 609.3 ± 9.2 nm), indicating that with the increase of lysozyme concentration, the positive charge density in the system increased, which promoted the adsorption and coating of the protein shell on the particle surface, resulting in the "expansion" of the particle size. This layer-by-layer self-assembly behavior verified the effective binding between lysozyme and MPN core. At the same time, PDI showed an upward trend (from 0.356 ± 0.030 to 0.462 ± 0.031), indicating that excessive lysozyme may cause non-specific adsorption or particle aggregation, resulting in a wider particle size distribution and a decrease in system stability. In terms of zeta potential, the particle potential of the 1 mg / mL group was the highest (30.4 ± 1.7 mV), showing the best charge neutralization effect and the most complete coating of lysozyme; while at 2 mg / mL, the potential decreased to 26.2 ± 3.3 mV, which may be due to the excessive lysozyme forming a loose adsorption layer on the surface or being dispersed freely in the system, reducing the effective surface charge density. In summary, 1 mg / mL is the optimal concentration for lysozyme assembly, with the advantages of controllable particle size, good dispersion, and balanced charge.

[0068] Table 3 Comparison of average particle size, PDI, potential, quercetin encapsulation efficiency (EE%), and drug loading efficiency (LE%) of MPN-LYS-PEC nanoparticles prepared under different mass ratios of lysozyme and pectin

[0069]

[0070] Note: The same column, different letters represent significant differences P <0.05.

[0071] As shown in Table 3, pectin was further introduced into MPN-LYS nanoparticles for electrostatic layer-by-layer self-assembly. Different mass ratios of egg white lysozyme to pectin (1:2, 1:1, 2:1) significantly affected the structural characteristics and drug loading capacity of the final MPN-LYS-PEC nanoparticles. Among them, the particles with the mass ratio of 1:1 showed the best overall performance, exhibiting the smallest particle size (608.2±12.9 nm), moderate zeta potential (-28.3±2.7 mV), and achieving the highest quercetin encapsulation efficiency (95.3±2.7%) and drug loading rate (30.3±1.9%). This indicates that the shell assembly is balanced and the structure is compact and stable under this ratio, which is conducive to the efficient encapsulation and fixation of quercetin. In contrast, the 1:2 group showed excessive pectin, resulting in an overly thick surface coating and a significantly increased particle size (697.0±9.7 nm). Simultaneously, both the encapsulation efficiency (80.2±3.2%) and drug loading rate (15.3±2.3%) decreased significantly. In the 2:1 group, the relative lack of pectin led to insufficient surface charge neutralization, increasing the zeta potential to -17.3±2.9 mV and decreasing particle stability. Furthermore, the encapsulation efficiency and drug loading rate of quercetin did not reach optimal levels. Comprehensive analysis indicates that a 1:1 mass ratio of egg white lysozyme to pectin achieves a good balance between particle size, surface charge, and loading capacity, representing the optimal outer layer construction condition for this delivery system.

[0072] Based on the above optimization conditions, quercetin and Fe were selected. 2+ MPN-LYS-PEC nanoparticles were constructed with a mass ratio of 1:3.3, a lysozyme concentration of 1 mg / mL, and an egg white lysozyme to pectin mass ratio of 1:1 for subsequent performance evaluation. The nanoparticles prepared under these conditions were analyzed by scanning electron microscopy (SEM). Figure 1 (B) and transmission electron microscopy (TEM) Figure 1 (C) Microscopic morphology characterization. SEM results showed that the nanoparticles exhibited a spherical structure, uniform distribution, and good particle size consistency, with no obvious agglomeration. TEM images further revealed the core-shell layered structure of the particles, with a clear boundary between the central core layer and the outer coating layer, indicating that quercetin-Fe 2+ The metal polyphenol network successfully served as a nanocore, with complete outer coating of lysozyme and pectin, resulting in a stable and orderly assembled structure. Simultaneously, the particle size distribution exhibited a single peak. Figure 1 The mean diameter (D) indicates that the system exhibits good homogeneity. Specifically, the average particle sizes of MPN, MPN-LYS, and MPN-LYS-PEC are 429.9±8.6, 550.8±17.2, and 608.2±12.9 nm, respectively. Figure 1 The PDI values ​​for the three types of nanoparticles were 0.359±0.027, 0.375±0.012, and 0.354±0.035, respectively, all less than 0.5, indicating that the three types of nanoparticles were uniformly distributed.Figure 1 Zeta potentials were -31.1 ± 2.1, 30.4 ± 1.7 and -28.3 ± 2.7 mV (Fig. 4C), respectively. Figure 1 Zeta potentials were -31.1 ± 2.1, 30.4 ± 1.7 and -28.3 ± 2.7 mV (Fig. 4C), respectively.

[0073] Example 2

[0074] Structural characterization of MPN-LYS-PEC nanoparticles prepared in Example 1

[0075] The optical properties of the nanoparticles were analyzed using ultraviolet-visible spectroscopy (UV-Vis). The samples were dispersed in deionized water and uniformly dispersed by ultrasonic treatment. An appropriate amount was added to a quartz cuvette with deionized water as a blank reference. The ultraviolet-visible spectrophotometer was scanned at a wavelength of 200-800 nm, and the characteristic absorption peaks and changes were recorded to evaluate the optical behavior and intermolecular interactions of the nanoparticles. Meanwhile, to further verify the structural characteristics of MPN-LYS-PEC, Fourier transform infrared spectroscopy (FTIR) was used for characterization. About 1 mg of dried sample was mixed with 200 mg of potassium bromide and ground to form a tablet. The Nicolet iN10 FTIR spectrometer was used to scan the sample in the range of 4000-400 cm -1 -400 cm -1 The vibration changes of various functional groups and the formation of chemical bonds were analyzed.

[0076] The test results include: to explore the possible intermolecular interactions of the components in the MPN-LYS-PEC nanoparticles, first, ultraviolet-visible spectroscopy (UV-Vis) was used for analysis (Fig. 4A). Figure 2 The results showed that after complexing with Fe 2+ , the characteristic absorption peak of quercetin at about 370 nm was red-shifted, and an enhanced absorption band was formed at 430 nm, indicating that the phenolic hydroxyl group of quercetin was coordinated with Fe 2+ , successfully constructing a stable metal polyphenol complex structure (MPN). At the same time, the characteristic absorption peak of aromatic amino acid residues (such as tryptophan and tyrosine) in lysozyme was observed near 280 nm. This peak was significantly shifted after assembly and accompanied by changes in intensity, indicating that there was electrostatic adsorption or hydrogen bonding between lysozyme and the MPN core. In addition, the carboxyl absorption peak of pectin at 230 nm also showed a blue shift and intensity change, further indicating that there was electrostatic binding behavior between pectin and lysozyme, forming an outer coating structure.

[0077] The above mechanism was further verified by FTIR spectra. The spectrum of pure quercetin showed typical polyphenol characteristics: 3232 cm -1 was assigned to -OH stretching vibration, 1672 cm -1 was assigned to C=O carbonyl stretching, 1614 cm -1 was assigned to aromatic ring C=C / C-C skeleton vibration, 1018 cm -1 and 1165 cm -1 were assigned to C-O stretching, 1200 cm -1 -1400 cm -1 was assigned to phenolic hydroxyl bending vibration region, and another at 1456 cm -1 , 1372 cm -1 and 833 cm -1 were C-H bending vibration peaks. After complexing with Fe 2+ to form MPN, the -OH stretching peak red shifted to 3360 cm -1 and the intensity decreased, indicating that the phenolic hydroxyl group was deprotonated and involved in coordination; the C=O peak also slightly red shifted, indicating that its electronic environment was disturbed by Fe 2+ . The newly appearing 630 cm -1 absorption peak was assigned to Fe=O bond vibration, further confirming the formation of metal polyphenol complex structure. The FTIR spectrum of egg white lysozyme showed typical protein characteristic peaks: 3302 cm -1 was assigned to N-H / O-H stretching, 1640 cm -1 was assigned to amide I band (C=O), 1526 cm -1 was assigned to amide II band (N-H bending and C-N stretching). After adsorbing on the surface of MPN, the amide I and II bands in the MPN-LYS complex were still clearly visible, and there was a slight shift and enhancement, indicating that lysozyme was combined with MPN through electrostatic adsorption or hydrogen bonding, forming a stable coating. In the FTIR spectrum of pure pectin, 954 cm -1 , 1013 cm -1 , 1043 cm -1 , 1106 cm -1 were assigned to pyran ring C-C stretching, 1149 cm -1 was assigned to C-O glycosidic bond stretching, 1411 cm -1 was assigned to C-O-H plane bending, 1630 cm -1 and 1736 cm -1 corresponded to non-esterified and esterified carboxyl C=O stretching vibration, 2800 cm -1 -3000 cm -1 was assigned to C-H stretching, 3100 cm -1 -3600 cm -1were assigned to O-H region. After pectin coated on MPN-LYS, these characteristic peaks still existed, 1630 and 1736 cm -1 occurred with slight red shift and intensity change, indicating that the carboxyl groups of pectin interacted with the cationic groups such as lysine residues on the surface of lysozyme. The enhancement of C-O-C, C-O and C-C vibrations further supported the formation of stable coating layer of pectin on the surface of MPN-LYS.

[0078] In summary, the results of UV-Vis and FTIR analysis clearly revealed the mechanism of layer-by-layer self-assembly of quercetin, Fe 2+ , egg white lysozyme and pectin in MPN-LYS-PEC nanostructure, and confirmed that the stable nano-complex system was formed through coordination bond, hydrogen bond and electrostatic interaction among the components.

[0079] Example 3

[0080] In vitro simulated gastric and intestinal digestion of MPN-LYS-PEC nanoparticles prepared in Example 1

[0081] In the process of in vitro simulated gastric digestion, first, 2000 U / mL of pepsin was added into 10 mL of SGF solution until it was completely dissolved, then 65 mg of quercetin-lysozyme nanoparticles (MPN-LYS) and 100 mg of quercetin-lysozyme-pectin nanoparticles powder (MPN-LYS-PEC) were added respectively, and the pH of the solution was adjusted to 3. The digestion was carried out in a 37°C water bath shaker, and 2 mL of digestion solution was taken out at different time points of 0 min, 30 min, 60 min, 90 min, 120 min, and the enzyme was inactivated before the quercetin content was determined by ultraviolet visible spectrophotometer at 370 nm. In the process of in vitro simulated intestinal digestion, first, 10 mM of bile was added into 9 mL of SIF solution, and 100 U / mL of trypsin was added until it was completely dissolved, then the remaining 9 mL of SGF solution was mixed with 9 mL of SIF solution, and the pH of the solution was adjusted to 7. The digestion was carried out in a 37°C water bath shaker, and 2 mL of digestion solution was taken out at different time points of 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, and the enzyme was inactivated before the quercetin content was determined by ultraviolet visible spectrophotometer at 370 nm.

[0082] The results of the test include: the system based on the colon-targeted release needs to avoid the upper gastrointestinal absorption of the active substance as much as possible, in general, the gastric transit time is 1-2 hours, the small intestinal transit time is 1-6 hours, the binding strength of egg white lysozyme and pectin mainly depends on the positive charge of lysozyme, and the binding strength is stronger under acidic conditions and weaker under neutral conditions, thereby causing the release speed of lysozyme under neutral conditions to be faster than that under acidic conditions. In this study, the in-vitro simulated SGF digestion time is set to 2 hours, and the SIF digestion time is set to 6 hours, Figure 3 The release curves of MPN-LYS and MPN-LYS-PEC nanoparticles under in-vitro simulated gastrointestinal digestion conditions are shown. The results show that due to the action of pepsin, MPN-LYS releases 20.6±4.9% of quercetin in the SGF stage, while MPN-LYS-PEC only releases 7.9±1.9% of quercetin, indicating that pectin and egg white lysozyme can effectively protect MPN from pepsin degradation in SGF. Then the samples are transferred to SIF. Compared with MPN-LYS which releases 60.4±4.6% of quercetin within 6 hours, MPN-LYS-PEC only releases 26.2±5.9% under the same conditions, indicating that the double-layer carrier of egg white lysozyme and pectin significantly improves the ability of quercetin nanoparticles to resist digestion in the stomach and small intestine, effectively avoids the release of quercetin in the gastrointestinal tract and realizes colon targeting.

[0083] Example 4

[0084] Establishment of high-fat fruit fly model

[0085] To determine the application effect of quercetin-lysozyme-pectin nanoparticles, a high-fat fruit fly model is established for experiments and verification: corn flour-yeast powder-agar is used to configure a standard medium (CS), and 4.4 g of lard is added to the standard medium as a high-fat diet control group (HFD). On the basis of the high-fat diet control medium, 0.25% (w / v) quercetin (MPN) powder, 0.83% (w / v) quercetin-lysozyme-pectin nanoparticle (MPN-LYS-PEC) powder (equivalent to 0.25% MPN), and 0.58% lysozyme-pectin nanoparticle (LYS-PEC) powder are added. After CO2 anesthesia, female fruit flies are selected on a fly picking plate, 20 flies per group, and placed in the above five different culture media for culture and divided into CS group, HFD group, MPN group, LYS-PEC group and MPN-LYS-PEC group. The culture is carried out in a constant temperature and humidity incubator at 25°C with 12 hours of light per day, and the food is replaced every three days.

[0086] Fruit fly weight determination

[0087] Weight experiment was also carried out according to the above grouping, 5 tubes of female fruit flies per group, fruit flies cultured to the 30th day were collected, then transferred to empty culture tubes for 2 h of starvation treatment, then transferred to weighing paper for weighing on an analytical balance, and the average weight of each fruit fly was calculated.

[0088] The test results include: adult weight refers to the body weight of an insect individual that has developed mature after the larval and pupal stages in the life cycle of the insect. The adult weight of fruit flies can be used to evaluate aspects such as the development, metabolism, and health of individuals. In female fruit flies, at 30 days of age, as shown in FIG. 2A, the adult weight of the CS group was reduced by 26.43% compared to the HFD group, with a highly significant difference (P<0.0001); the HFD group also had a highly significant difference (P<0.0001) compared to the MPN-LYS-PEC group, with a reduction of 23.93% in adult weight compared to the HFD group, and the test effect was better than that of the MPN (17.02%) group and the LYS-PEC group (>0.05). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly reduce the adult weight of female fruit flies induced by high-fat diet. Figure 4 p <0.0001). The HFD group also had a highly significant difference (P<0.0001) compared to the MPN-LYS-PEC group, with a reduction of 23.93% in adult weight compared to the HFD group, and the test effect was better than that of the MPN (17.02%) group and the LYS-PEC group (>0.05). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly reduce the adult weight of female fruit flies induced by high-fat diet. p p Example 5

[0089] Drosophila movement activity determination

[0090] Drosophila movement activity determination

[0091] According to the above grouping, 5 tubes of female fruit flies per group, fruit flies cultured to the 30th day were collected, and the number of fruit flies reaching the top of the tube was recorded after 10 s, and the percentage of the total number of fruit flies was calculated. The experiment was repeated three times for each culture tube of fruit flies.

[0092] The test results include: climbing ability refers to the activity and behavior exhibited by an organism within a certain time period, reflecting the ability and enthusiasm of the organism in terms of movement and activity. In the ordinary food group and the high-fat food control group, it was observed that the fruit flies in the high-fat food control group showed a significant decrease in movement activity, as shown in FIG. 2B, in 30-day-old female fruit flies, the HFD group had a decrease of 48.58% in movement activity compared to the CS group (P<0.0001). The MPN-LYS-PEC experimental group had an increase of 94.84% in movement activity compared to the HFD control group (P<0.0001), and this increase was greater than that of the MPN group (51.86%) and the LYS-PEC group (30.66%). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly improve the movement activity of female fruit flies induced by high-fat diet. Figure 4 p <0.0001). The HFD group also had a highly significant difference (P<0.0001) compared to the MPN-LYS-PEC group, with a reduction of 23.93% in adult weight compared to the HFD group, and the test effect was better than that of the MPN (17.02%) group and the LYS-PEC group (>0.05). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly reduce the adult weight of female fruit flies induced by high-fat diet. p The test results include: climbing ability refers to the activity and behavior exhibited by an organism within a certain time period, reflecting the ability and enthusiasm of the organism in terms of movement and activity. In the ordinary food group and the high-fat food control group, it was observed that the fruit flies in the high-fat food control group showed a significant decrease in movement activity, as shown in FIG. 2B, in 30-day-old female fruit flies, the HFD group had a decrease of 48.58% in movement activity compared to the CS group (P<0.0001). The MPN-LYS-PEC experimental group had an increase of 94.84% in movement activity compared to the HFD control group (P<0.0001), and this increase was greater than that of the MPN group (51.86%) and the LYS-PEC group (30.66%). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly improve the movement activity of female fruit flies induced by high-fat diet.​​​

[0093] Example 6

[0094] Determination of triglyceride content and cholesterol content in fruit flies

[0095] Each group of female fruit flies was collected on the 30th day of culture, weighed, and the average weight was calculated. Each group of fruit flies was transferred to a 1.5 mL centrifuge tube, anhydrous ethanol was added at a mass-volume ratio (g:mL) of m1 (fruit fly weight):m2 (anhydrous ethanol) = 1:9, and homogenized and ground using a low-temperature tissue homogenizer. The centrifuge temperature was set to 4°C, and the sample was centrifuged at a speed of 2500 r / min. After 10 min, the supernatant was aspirated, and the content of triglyceride (TG) in Drosophila melanogaster was determined according to the method described in the instructions. The pretreatment for cholesterol (T-CHO) content determination was the same as that for triglyceride, and the total cholesterol content in fruit flies was determined according to the method described in the instructions (TG, T-CHO detection kit provided by Nanjing Jiancheng Biological Engineering Institute).

[0096] The test results include: the triglyceride content refers to the content or concentration of triglyceride in the body of an organism (fruit fly) under specific conditions (e.g., high-fat diet). Its increase can reflect the synthesis and accumulation of lipids, indicating that the fruit fly may have fat deposition or obesity under high-fat diet conditions. By measuring the content of triglyceride in fruit flies, changes in fruit fly lipid metabolism, obesity, and related physiological phenomena can be studied. The results show that in female fruit flies, as shown in Table C, the triglyceride content of the CS group was reduced by 29.28% compared to the HFD group at 30 days of age. The MPN-LYS-PEC group had a triglyceride content reduction of 26.55% compared to the HFD group, also with a significant difference (p<0.01). This effect was better than that of the MPN group (17.22%) and the LYS-PEC group (p>0.05). This indicates that quercetin-pectin-lysozyme self-assembled nanoparticles can significantly reduce the triglyceride content in fruit flies induced by high-fat diet and reduce fat deposition. Figure 4 p <0.01). And this effect is better than that of the MPN group (17.22%) and the LYS-PEC group (p>0.05). This indicates that quercetin-pectin-lysozyme self-assembled nanoparticles can significantly reduce the triglyceride content in fruit flies induced by high-fat diet and reduce fat deposition. p Cholesterol is a lipid substance that plays an important physiological function in the body, including the structural composition of cell membranes, hormone synthesis, and the precursor of bile acids. By measuring the total cholesterol content in fruit flies, the effect of high-fat diet on fruit fly lipid metabolism can be evaluated. As shown in Table D, the total cholesterol content of the CS group was reduced by 50.51% compared to the HFD group at 30 days of age, with a significant difference (p<0.001). At the same time, the HFD group also had a significant difference (p<0.001) compared to the MPN-LYS-PEC group.

[0097] Figure 4 <0.001), and the HFD group also had a significant difference (p<0.001) compared to the MPN-LYS-PEC group. p <0.001). And this effect is better than that of the MPN group (17.22%) and the LYS-PEC group (p>0.05). This indicates that quercetin-pectin-lysozyme self-assembled nanoparticles can significantly reduce the triglyceride content in fruit flies induced by high-fat diet and reduce fat deposition. p ​​<0.01), the total cholesterol level in the MPN-LYS-PEC group was 35.79% lower than that in the HFD group, while there was no significant change in either the MPN or LYS-PEC groups. p >0.05). This indicates that quercetin-lysozyme-pectin self-assembled nanoparticles can significantly reduce total cholesterol levels in female fruit flies induced by a high-fat diet and stabilize lipid metabolism.

[0098] Example 7

[0099] Drosophila enzyme activity assay

[0100] Following the above grouping, five tubes containing 20 female fruit flies were collected from each group. Fruit flies cultured for 30 days were collected, fasted for 2 hours, and then weighed to calculate the average body weight. The fruit flies from each group were transferred to 1.5 mL centrifuge tubes, and anhydrous ethanol was added at a ratio of m1 (fly body weight): m2 (anhydrous ethanol) = 1:9. The mixture was homogenized using a low-temperature tissue homogenizer at 4°C and centrifuged at 2500 r / min for 10 min. The supernatant was collected, and the levels of superoxide dismutase (SOD) and catalase (CAT) in the fruit flies were determined according to the manufacturer's instructions.

[0101] Superoxide dismutase (SOD) is an important antioxidant enzyme that plays a crucial protective role within cells. It scavenge superoxide anion free radicals, reducing oxidative stress and cell damage. Figure 4 As shown in Figure E, in female fruit flies, at 30 days of age, the SOD index value in the CS group was 60.71% higher than that in the HFD group. p <0.001); The SOD index value of the MPN-LYS-PEC group increased by 35.64% compared with the HFD group ( p <0.05, and the effect was better than that of the MPN group ( p >0.05) and LYS-PEC group ( p >0.05). CAT is an important antioxidant enzyme, primarily responsible for degrading intracellular hydrogen peroxide. In female fruit flies, such as... Figure 4 As shown in Figure F, at 30 days of age, the CAT index value in the CS group was 52.07% higher than that in the HFD group. p <0.01); The CAT index value of the MPN-LYS-PEC group increased by 31.99% compared with the HFD group ( p <0.05, and the effect was better than that of the MPN group ( p >0.05) and LYS-PEC group ( p >0.05). This indicates that quercetin-pectin-lysozyme self-assembled nanoparticles can significantly enhance the antioxidant capacity of fruit flies.

[0102] Example 8

[0103] Detection of fruit fly intestinal leakage rate

[0104] According to the above grouping, 5 tubes of female fruit flies, each containing 20 flies, were collected at the 30th day of culture, fasted for 2 hours, and then moved into culture tubes containing 2.5% smurf in sucrose medium (5% sucrose solution) for staining culture. After 10 hours, the fruit fly mouthparts were observed under a microscope to see if there was obvious leakage in the digestive tract and photographed. The spread of the dye was recorded and the leakage rate was calculated.

[0105] The test results include: intestinal leakage of fruit flies refers to the phenomenon that under adverse environments such as high-fat and high-sugar diet, the barrier function of fruit fly intestine is damaged, leading to overflow or leakage of intestinal internal substances and components to the surrounding tissues or hemolymph. Therefore, the intestinal leakage of female fruit flies at the 30th day was studied. It is shown that the quercetin-pectin-lysozyme self-assembled nanoparticles can effectively reduce the intestinal leakage rate of female fruit flies induced by high-fat diet. For example Figure 4 In groups G and H, the intestinal leakage of the HFD group increased by 78.57% compared with the CS group at the 30th day of age p <0.0001); while the intestinal leakage of the MPN-LYS-PEC group decreased by 63.78% compared with the HFD group p <0.0001), which is much lower than that of the MPN group (35.71%) and the LYS-PEC group p >0.05). It is shown that the quercetin-lysozyme-pectin self-assembled nanoparticles can reduce the intestinal leakage rate of female fruit flies induced by high-fat diet and protect the intestinal barrier.

[0106] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. A quercetin-lysozyme-pectin self-assembled nanoparticle, characterized in that, The nano core and the lysozyme layer and the pectin layer are sequentially coated from inside to outside; The nanocore is quercetin-Fe 2+ Metal polyphenol network; The mass ratio of the nano core, the lysozyme layer and the pectin layer is 0.4-0.85:1:1; said quercetin-Fe 2+ Fe in the metal polyphenol network 2+ The mass of said quercetin is 1 / 3~4 of the mass of ferrous chloride tetrahydrate.

2. The method for preparing quercetin-lysozyme-pectin self-assembled nanoparticles according to claim 1, characterized in that, The method comprises the following steps: The quercetin-Fe complex was formed by mixing the quercetin solution and the solution containing ferrous ions 2+ metal polyphenol network, denoted as first nanoparticles; The lysozyme is automatically assembled on the surface of the first nano particle to form a second nano particle; The pectin is automatically assembled on the surface of the second nano particle to obtain the quercetin-lysozyme-pectin self-assembled nano particle.

3. The method of claim 2, wherein, The volume ratio of the quercetin solution and the solution containing ferrous ions is 2-4:7-9; The concentration of the quercetin is 5-10 mg / mL; The solution containing ferrous ions comprises an aqueous solution of ferrous chloride tetrahydrate; The concentration of the aqueous solution of ferrous chloride tetrahydrate is 2-12 mg / mL.

4. The production method according to claim 2, characterized by, The automatic assembly of the lysozyme or the automatic assembly of the pectin is accompanied by oscillation; The rotation speed of the oscillation is 300-600 rpm, and the oscillation time is 30-90 min.

5. The method according to any one of claims 2 to 4, wherein the compound of formula (I) is prepared by the process of claim 1. The coating concentration of the lysozyme in the lysozyme layer is 0.5-2 mg / mL.

6. A lipid lowering product, characterized in that, The active ingredient comprises the quercetin-lysozyme-pectin self-assembled nano particle of claim 1 or the quercetin-lysozyme-pectin self-assembled nano particle prepared by the preparation method of any one of claims 2-5.

7. The use of the quercetin-lysozyme-pectin self-assembled nano particle of claim 1 or the quercetin-lysozyme-pectin self-assembled nano particle prepared by the preparation method of any one of claims 2-5 in the preparation of a drug for preventing and / or treating a chronic metabolic disease, wherein the chronic metabolic disease is hyperlipidemia.

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