Quercetin nano-vesicle complex as well as preparation method and application thereof

Abstract

The invention discloses a quercetin nano-vesicle complex as well as a preparation method and application thereof. The preparation method of the nano-vesicles comprises the following steps: separating and purifying extracellular vesicle-like nano-particles from cow milk by combining differential centrifugation with an EDTA precipitation method; quercetin is efficiently loaded in a vesicle nanostructure by adopting an ultrasonic-assisted co-incubation technology, and a drug loading system is characterized through particle size analysis, Zeta potential determination and a transmission electron microscope. The natural lipid bilayer structure of the milk-derived extracellular vesicles is creatively utilized, the constructed quercetin nano-vesicle complex shows excellent stability in simulated gastrointestinal fluid, and the average encapsulation efficiency can reach 60%. In-vitro and in-vivo experiments prove that the nano vesicles can efficiently remove senescent cells. The invention has a wide clinical application prospect in the field of anti-aging and inflammation-related disease treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomedicine, in particular to a quercetin nanovesicle complex, a preparation method and application thereof. BACKGROUND

[0002] Flavonoids, as secondary metabolites widely distributed in the plant kingdom, are an important component of human dietary polyphenols. Their multiple pharmacological properties, such as anti-inflammatory, antioxidant, wound healing, and gut microbiota regulation, make them unique in the treatment of intestinal diseases. Among them, quercetin has become one of the most promising natural drug molecules due to its excellent antioxidant, anti-inflammatory, and anti-aging activities. However, the clinical application of quercetin has been limited by its inherent physicochemical defects: ①extreme hydrophobicity leading to low bioavailability (oral bioavailability in rats <17%, and only about 1% in humans); ②rapid first-pass effect and systemic metabolism leading to insufficient effective concentration at the target site. Although nanodelivery systems (such as liposomes, protein particles, etc.) can partially improve its solubility, existing technologies still have significant limitations: for example, synthetic carriers often require the use of chemical cross-linking agents (such as tripolyphosphate), which can cause potential biological toxicity.

[0003] In view of the above technical bottlenecks, research on extracellular vesicles (EVs) as natural nanocarriers in recent years has provided a new way out of this dilemma. EVs are biologically active lipid bilayer particles naturally released by cells, which can be secreted by almost all living cells and divided into exosomes, microvesicles, and apoptotic bodies according to size, and contain proteins, lipids, DNA, mRNA, miRNA, etc. They are important mediators of intercellular information exchange and play an important role in disease diagnosis and treatment. In particular, food-derived extracellular vesicles have opened up new dimensions in the treatment of intestinal diseases due to their natural oral applicability, especially milk-derived vesicle-like nanoparticles, as an important subclass of FEVs, which have the following industrialization advantages: ①abundant raw materials (up to (1.2-5.6)×10^12 particles per liter of milk); ②low immunogenicity; ③multiple regulatory functions.

[0004] Although the delivery potential of milk-derived extracellular vesicles has been initially verified, their unique advantages in flavonoid drug delivery have not been fully explored. Existing researches focus on carrier engineering (such as ligand modification, molecular chimerization), but ignore the synergistic mechanism of milk-derived extracellular vesicles and specific drug molecules. In particular, in the field of colitis treatment, how to use the natural intestinal targeting of milk-derived extracellular vesicles to achieve efficient delivery of quercetin and synergistically exert the anti-aging-antioxidant dual effect is still a technical problem to be solved. SUMMARY

[0005] Therefore, the present application aims to provide a quercetin nanovesicle complex, a preparation method and application thereof, break through the technical bottleneck of low oral bioavailability of traditional flavonoids, provide a safe and efficient new nano preparation solution for senescent cell clearance, and have a wide clinical application prospect in the field of anti-aging and inflammation-related disease treatment.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] In the first aspect of the present application, a preparation method of a quercetin nanovesicle complex is provided, comprising:

[0008] S1, taking cow milk to perform differential centrifugation, taking the supernatant after centrifugation to incubate with a metal ion chelating agent at a certain volume ratio, collecting the supernatant after ultracentrifugation, filtering, performing density gradient ultracentrifugation, and collecting the precipitate, and resuspending and filtering the precipitate to obtain milk-derived outer vesicles;

[0009] S2, mixing quercetin with the outer vesicles prepared in step S1, co-incubating after ultrasonication, and collecting the precipitate by ultracentrifugation to obtain a quercetin nanovesicle complex.

[0010] Further, in step S1,

[0011] The differential centrifugation is differential centrifugation at a relative centrifugal force of 10000-15000xg at 2-8℃ for 20-40min;

[0012] The ultracentrifugation is centrifugation at 90000-110000xg at 2-8℃ for 60-80min;

[0013] The filtration is all filtration with a 0.22μm filter;

[0014] The density gradient ultracentrifugation is centrifugation at 130000-140000xg at 2-8℃ for 90-100min.

[0015] Further, in step S1, the cow milk is commercially available pasteurized low-fat milk with a fat content of ≤1.5%, the metal ion chelating agent is an EDTA solution with a concentration of 0.3-0.6mol / L, the volume ratio of the centrifuged supernatant to the metal ion chelating agent is 2-4:1, and the incubation with the metal ion chelating agent is incubation of the centrifuged supernatant with the EDTA solution on ice for 10-20min.

[0016] Further, in step S2,

[0017] The mass ratio of quercetin to the outer vesicles prepared in step S1 for mixing is 1-8:1;

[0018] The ultrasonic is: 400-500W ultrasonic, 10-30s on, 30-50s off, 4-8 cycles of drug loading;

[0019] The post-ultrasonic co-incubation is: 37 DEG C, 0-100rpm, incubation for 1-3h;

[0020] The ultracentrifugation is: 130000-170000xg centrifugation for 60-90min.

[0021] In the second aspect of the application, the quercetin nanovesicle complex prepared by any of the above preparation methods is provided.

[0022] In the third aspect of the application, the quercetin nanovesicle complex is applied to the preparation of a drug for treating aging-related diseases.

[0023] Further, the aging-related diseases include inflammatory bowel disease.

[0024] Further, the dosage form of the drug is oral preparation.

[0025] The application further provides an anti-aging composition comprising the quercetin nanovesicle complex.

[0026] In summary, in the application, the preparation method of the quercetin nanovesicle complex comprises: separating and purifying extracellular vesicle-like nanoparticles from bovine milk by differential centrifugation combined with EDTA precipitation method; ultrasonic-assisted co-incubation technology is used to load quercetin into the vesicle nanostructure, and the drug loading system is characterized by particle size analysis, Zeta potential determination and transmission electron microscopy.

[0027] The application has at least the following beneficial effects:

[0028] The application innovatively utilizes the natural lipid bilayer structure of milk-derived extracellular vesicles, and the constructed quercetin nanovesicle complex can be used as a quercetin nanodelivery system, which exhibits excellent stability in simulated gastrointestinal fluid, and the average encapsulation rate can reach 60%. In vitro and in vivo experiments prove that the nanovesicle can efficiently remove senescent cells, and the mechanism involves regulating the p53-p21 signaling pathway to relieve persistent DNA damage, while maintaining the normal expression level of replication-related factors (such as PCNA) to ensure the genomic integrity during DNA synthesis. Animal experiments further show that after oral administration, the nanovesicle can specifically target the inflammation site of the colon, significantly reduce the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and the lipid peroxidation marker malondialdehyde, and at the same time, improve the activity of superoxide dismutase, which is significantly better than free quercetin. The application breaks through the technical bottleneck of low oral bioavailability of traditional flavonoid drugs, provides a safe and efficient new type of nanofomulation solution for senescent cell removal, and has a broad clinical application prospect in the field of anti-aging and inflammation-related disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Extraction schematic diagram of milk-derived extracellular vesicles.

[0030] Figure 2 Preparation schematic diagram of milk-derived extracellular vesicles loaded with flavonoid drugs.

[0031] Figure 3 NTA image of milk-derived extracellular vesicles.

[0032] Figure 4 NTA image of milk-derived extracellular vesicles loaded with flavonoid drugs.

[0033] Figure 5 Drug loading capacity and encapsulation efficiency of milk-derived extracellular vesicles loaded with flavonoid drugs.

[0034] Figure 6 TEM image of milk-derived extracellular vesicles.

[0035] Figure 7 TEM image of milk-derived extracellular vesicles loaded with flavonoid drugs.

[0036] Figure 8 Particle size change of milk-derived extracellular vesicles loaded with flavonoid drugs after incubation in gastrointestinal fluid for 3h.

[0037] Figure 9 PDI change of milk-derived extracellular vesicles loaded with flavonoid drugs after incubation in gastrointestinal fluid for 3h.

[0038] Figure 10TEM image of milk-derived extracellular vesicles loaded with flavonoid drugs after 2h incubation in gastric juice.

[0039] Figure 11 TEM image of milk-derived extracellular vesicles loaded with flavonoid drugs after 2h incubation in intestinal juice.

[0040] Figure 12 Image of intestinal epithelial HCT-116 cell senescence β-galactosidase.

[0041] Figure 13 Image of intestinal epithelial HCT-116 cell p53 immunofluorescence.

[0042] Figure 14 Image of intestinal epithelial HCT-116 cell p21 immunofluorescence.

[0043] Figure 15 Image of intestinal epithelial HCT-116 cell PCNA immunofluorescence.

[0044] Figure 16 Mouse body weight change.

[0045] Figure 17 Disease activity index score, mouse daily body weight, diarrhea, and bloody stool.

[0046] Figure 18 Image of colon and measurement of length of each group of mice after dissection on day 8.

[0047] Figure 19 Inflammatory factor detection of mouse colon tissue.

[0048] Figure 20 Malondialdehyde and superoxide dismutase activity detection of mouse colon tissue.

[0049] Figure 21 Image of p21 immunofluorescence of mouse colon tissue.

[0050] Figure 22 Image of p53 immunofluorescence of mouse colon tissue. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work, fall within the protection scope of the present application.

[0052] In addition, the technical solutions among various embodiments of the present application can be combined with each other, but it must be based on that a person skilled in the art can realize, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the protection scope required by the present application.

[0053] The scheme proposed by the present application is specifically described below through specific embodiments:

[0054] Embodiment 1

[0055] The preparation method of milk-derived extracellular vesicles (mEVs) loaded with quercetin includes the following standardized operation steps:

[0056] Step one, commercially available pasteurized low-fat milk (fat content ≤1.5%) is purchased, and differential centrifugation is performed at 13,000×g relative centrifugal force (RCF) at 4°C for 30 minutes to effectively remove somatic cells and cellular debris. The centrifugal supernatant is incubated with ethylenediaminetetraacetic acid (EDTA, 0.5 mol / L, pH 7.4) at a ratio of 3:1 (v / v) on ice for 15 minutes to induce the disaggregation and precipitation of casein micelles by metal ion chelation. A Beckman Coulter ultracentrifuge (SW32Ti rotor, k-factor=137) is used to centrifuge at 100,000×g at 4°C for 60 minutes to simultaneously remove milk fat globules (MFGs), precipitated proteins, and large vesicles with a particle size >200 nm. The supernatant is filtered through a 0.22μm filter, and the same rotor is used to perform ultracentrifugation at 135,000×g (4°C) for 90 minutes (the solvent is 0.9% NaCl) to effectively enrich the population of milk-derived extracellular vesicles (exosomes) with a diameter of 50-150 nm. The precipitate is resuspended with pre-cooled PBS (0.01M, pH 7.4), and the supernatant is filtered through a 0.22μm filter. As shown in FIG. 1. Figure 1

[0057] ​Step two, preparation of Que-loaded milk-derived extracellular vesicles (EV-Que), Que and milk-derived extracellular vesicles (EV) were mixed at a mass ratio of 1:5, and drug loading was performed at 400 W ultrasonic waves for 20 s on and 40 s off for 6 cycles. Membrane fusion was promoted by incubation at 37°C and 100 rpm for 2 h. The solution was then placed in an ultracentrifuge and centrifuged at 150,000 x g for 75 min to remove free Que. The final preparation (protein concentration < 6 mg / mL) was aliquoted and stored at -80°C. Before use, the membrane permeability was enhanced by liquid nitrogen freeze-thaw cycling 3 times. As shown in Figure 2 .

[0058] Take 500 μL EV-Que and add it to an ultrafiltration centrifuge tube (MWCO: 100 kDa), centrifuge at 14000 x g for 3 min, and use a multifunctional microplate detector (Tecan Spark, Austria) to measure the ultraviolet absorbance of the filtrate at λ 370 nm. Dissolve Que with an ethanol: water = 1:1 solution, take 500 μL of the dissolved solution and ultrafiltrate it under the same conditions, and measure the ultraviolet absorbance of the filtrate at λ 370nm . Prepare a standard curve. Calculate the BD concentration according to the standard curve. Calculate EE% and LC% according to formulas (1) and (2), respectively. Remove free Que by ultrafiltration.

[0059]

[0060] Observe its structure by transmission electron microscopy. Drop the mixed sample on a carbon-supported membrane copper mesh, and let it stand for 5 minutes. Use filter paper to absorb the remaining liquid from the edge of the copper mesh. Drop 2% phosphotungstic acid on the copper mesh, and stain it at room temperature for 30 seconds. Use filter paper to absorb the excess stain, and then place it under a transmission electron microscope for observation. Use Nanosight LM10 (Malvern Instruments Ltd., UK) for nanoparticle tracking analysis (NTA), and the results are shown in Figure 3 and 4 The particle concentration of milk-derived extracellular vesicles is 5.1 x 10 9 particles / mL, and after Que loading, the particle concentration increases to 5.6 x 10 9 particles / mL. The particle size distribution of both samples is uniform, mainly in the range of 100-130 nm, which is consistent with the typical size characteristics of extracellular vesicles.

[0061] The Que-loaded milk-derived extracellular vesicles prepared by the above preparation method are used for oral treatment of inflammatory bowel disease.

[0062] When the feeding ratio of milk-derived extracellular vesicles and Que is 5:1, the encapsulation efficiency and drug loading capacity are as followsFigure 5 As shown, the encapsulation efficiency was 60%, and the drug loading was 10%, achieving effective embedding of Que in milk-derived extracellular vesicles.

[0063] Example 2

[0064] (1) Detection of protein concentration in milk-derived extracellular vesicles.

[0065] The sample concentration was determined by the BCA kit. First, the protein standard was prepared and the BCA working solution was prepared. Then, the protein concentration standard and the milk-derived extracellular vesicle stock solution were added to the 96-well enzyme-labeled plate at different concentrations, the BCA working solution was added in proportion, followed by 37°C constant temperature incubation for 30 min, and the optical density (OD) value was determined at 562 nm using an enzyme-labeled instrument. According to the measured OD value, combined with the standard curve, the dilution multiple of each experimental group was obtained, and the corresponding protein concentration of each group was obtained. Each group was set with three parallel samples to reduce experimental error.

[0066] (2) Characterization of milk-derived extracellular vesicles.

[0067] For the determination of size and polydispersity index, an appropriate amount of milk-derived extracellular vesicles was dissolved in 1.5-2 mL deionized water (the final concentration of milk-derived extracellular vesicles was 0.5-2 mg / mL), and the dynamic light scattering particle size instrument was used to determine the hydrodynamic particle size and polydispersity index of milk-derived extracellular vesicles. The average value was obtained by measuring three times. The hydrodynamic particle size of milk-derived extracellular vesicles was 78.29±0.79 nm, and the particle size of extracellular vesicles loaded with quercetin increased to 87.28±0.49 nm. The polydispersity index before and after drug loading was maintained at

[0068] ~0.2, indicating that the particle system has good monodispersity.

[0069] The structure was observed by transmission electron microscopy (TEM). The mixed sample was dropped on the carbon support film copper mesh, and stood for 5 minutes. The residual liquid was absorbed from the edge of the copper mesh with filter paper. 2% phosphotungstic acid was dropped on the copper mesh, and stained at room temperature for 30 seconds. The excess staining solution was absorbed with filter paper, and then observed under a transmission electron microscope after natural air drying. As shown in Figure 6 , it is shown that milk-derived extracellular vesicles have a cavity structure and a lipid bilayer structure, which are suitable as drug delivery carriers. The TEM image of milk-derived extracellular vesicles loaded with flavonoid drugs is shown in Figure 7 , which shows that the milk-derived extracellular vesicles exhibit an unbroken cup-shaped morphology after drug loading, indicating that the ultrasonic drug loading process does not destroy the cavity structure and lipid bilayer structure of the vesicles.

[0070] (3) Stability of milk-derived extracellular vesicles loaded with quercetin nanoparticles in simulated gastrointestinal fluid

[0071] Preparation of simulated gastrointestinal fluid: 80 mM hydrochloric acid, 35 mM sodium chloride, 0.3% pepsin, and deionized water to 100 mL, pH 1.2; preparation of simulated small intestinal fluid: 15 mM sodium hydroxide, 50 mM potassium dihydrogen phosphate, 1% trypsin, and deionized water to 100 mL, pH 6.8; preparation of simulated colon fluid: 559 mg of potassium phosphate dibasic and 41 mg of potassium dihydrogen phosphate, and deionized water to 100 mL, pH 7.8.

[0072] The milk-derived extracellular vesicles loaded with quercetin nanoparticles were diluted with phosphate buffered saline (PBS), simulated gastrointestinal fluid, simulated small intestinal fluid, and simulated colon fluid to a concentration of 0.5-2 mg / mL, incubated at 37°C, 200 rpm for 1-3 h, and the hydrodynamic particle size and polydispersity index of the milk-derived extracellular vesicles loaded with quercetin nanoparticles were determined using a dynamic light scattering particle size analyzer. The determination was performed three times to obtain an average value; and the structure was observed by transmission electron microscopy. As shown in Figure 8 , it is indicated that the particle size of the milk-derived extracellular vesicles loaded with quercetin nanoparticles in different solutions has no obvious difference, indicating that the structure of the milk-derived extracellular vesicles in different solutions remains stable and has the ability to resist gastrointestinal fluid digestion. As shown in Figure 9 , it is indicated that the polydispersity index of the milk-derived extracellular vesicles in different solutions is less than 0.3, indicating that the milk-derived extracellular vesicles in different solutions all have colloidal stability.

[0073] The structure was observed by transmission electron microscopy, and the results are shown in Figure 10 and Figure 11 , indicating that the extracellular vesicles loaded with quercetin maintain the complete double-membrane structure during the digestion process, indicating that the milk-derived extracellular vesicles have the intrinsic ability to resist the harsh environment of the upper digestive tract.

[0074] Example 3

[0075] Milk-derived extracellular vesicles loaded with flavonoid drugs for anti-aging effect

[0076] Paracrine effects mediated by reactive oxygen species (ROS) can accelerate the aging process of adjacent normal cells. In order to elucidate the anti-aging mechanism of milk-derived extracellular vesicles loaded with flavonoid drugs, a doxorubicin (DOX)-induced intestinal epithelial cell aging model was established, and the regulatory effect of milk-derived extracellular vesicles loaded with flavonoid drugs on cell aging markers was systematically evaluated. In addition, compared with other groups, the milk-derived extracellular vesicles loaded with flavonoid drugs treatment group showed improved SA-β-Gal, indicating the cell aging phenotype Figure 12 . Similarly, the results of immunofluorescence staining are shown in Figure 13 and Figure 14, DOX treatment intuitively up-regulated the expression level of p53 and p21, while quercetin or milk-derived extracellular vesicles loaded with flavonoids treatment effectively reversed the adverse effects of cell cycle arrest. In addition, as shown in Figure 15 Figure 6, cell proliferation can be roughly assessed by detecting the expression of proteins involved in the cell cycle process, such as PCNA. After treatment with milk-derived extracellular vesicles loaded with flavonoids, a significant improvement in PCNA expression was observed, which was superior to other groups. These findings suggest that milk-derived extracellular vesicles loaded with flavonoids can exert a protective effect on persistent DNA damage by regulating the p53-p21 signaling cascade, while maintaining normal expression levels of replication-related factors such as PCNA. This ultimately ensures the genomic integrity during DNA synthesis, thereby playing an anti-aging role.

[0077] Example 4

[0078] Oral administration of milk-derived extracellular vesicles loaded with flavonoids for the removal of senescent cells in the treatment of inflammatory bowel disease

[0079] 25 eight-week-old male C57BL / 6J mice were housed in the Experimental Animal Center of Northwestern Polytechnical University Medical Research Institute. After one week of free feeding adaptation, five mice were randomly selected as a cage for the normal group, and the other experimental mice were randomly divided into five groups: DSS modeling group, Que group, EV group, EV-Que group, and positive drug 5-ASA group. The four groups of mice were established by free drinking of 2% to 3% DSS to establish the mouse model of ulcerative colitis, and each mouse was labeled and administered on the 0th day. The state of the mice was checked every day, and the water leakage of the water bottle was checked.

[0080] The body weight of each mouse was recorded throughout the experiment to calculate the percentage of body weight loss, and the mouse feces were collected to reflect the diarrhea and blood in the stool. The sum of the three scores of body weight loss percentage, diarrhea score, and blood in stool score was used to calculate the disease activity index (Disease activity index, DAI), which is the DAI value. That is, DAI = body weight index + stool shape + bleeding condition. DAI scoring criteria: 1 symptom score per day: 0 points for 0-1% body weight loss, 1 point for 1%-5%, 2 points for 5%-10%, 3 points for 10%-20%, and 4 points for ≥20%; normal stool without abnormalities counts 0 points, loose stool counts 1 point, stool without shape counts 2 points, and water-like stool counts 3 points; normal stool without blood counts 0 points, stool with blood counts 1 point, visible counts 2 points, and massive bleeding counts 3 points. On the eighth day, the mice were sacrificed by cervical dislocation, and the entire colon segment was dissected and rinsed with normal saline. The colon length was measured and photographed, and the colon length was used to represent the inflammatory condition of the mice.

[0081] As shown in Figure 16 ,Figure 17 As shown in the figure, the body weight of the normal control group did not change, the body weight of the negative control DSS group decreased significantly to about 70%, and the body weight decreased significantly, while the body weight of the quercetin-loaded milk-derived vesicle-like nanoparticle group recovered to about 90%, which indicated that the quercetin-loaded milk-derived vesicle-like nanoparticle could inhibit the weight loss of the colitis mice and relieve the symptoms of the colitis mice.

[0082] As shown in the figure, the colon length of the normal control group was about 8 cm, the colon length of the negative control DSS group was about 6.2 cm, and the colon length of the quercetin-loaded milk-derived vesicle-like nanoparticle group was about 7.8 cm, which indicated that the quercetin-loaded milk-derived vesicle-like nanoparticle could inhibit the colon shortening of the colitis mice and relieve the symptoms of the colitis mice. Figure 18 As shown in the figure, the pro-inflammatory factors of the negative control DSS group increased significantly, while the pro-inflammatory factors of the quercetin-loaded milk-derived vesicle-like nanoparticle group decreased significantly. The malondialdehyde activity detection and superoxide dismutase activity detection of the colon tissue of the mice also proved that the quercetin-loaded milk-derived vesicle-like nanoparticle could inhibit the oxidative inflammation index and relieve the symptoms of the colitis mice.

[0083] Figure 19 As shown in the figure, the use of the senolytic positive drug Aby263 for control found that the effect of the quercetin-loaded milk-derived vesicle-like nanoparticle on the senescence-related p53 and p21 was better than that of the positive drug group, which indicated that the quercetin-loaded milk-derived vesicle-like nanoparticle had an anti-aging effect. Figure 20 Therefore, the quercetin-loaded milk-derived vesicle-like nanoparticle and the preparation method and application thereof have a stable lipid bilayer structure in the gastrointestinal simulation solution, and can be used for oral treatment of diseases. The application provides a new strategy for oral application of Que, and has good application prospect.

[0084] Figure 21 It should be noted that in this paper, the term "including", "containing" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device. Without more limitation, the element defined by the statement "including a" does not exclude the presence of another identical element in the process, method, article or device including the element. Figure 22

[0085] Therefore, the quercetin-loaded milk-derived vesicle-like nanoparticle and the preparation method and application thereof have a stable lipid bilayer structure in the gastrointestinal simulation solution, and can be used for oral treatment of diseases. The application provides a new strategy for oral application of Que, and has good application prospect.

[0086] It should be noted that in this paper, the term "including", "containing" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device. Without more limitation, the element defined by the statement "including a" does not exclude the presence of another identical element in the process, method, article or device including the element.

[0087] ​​​The above-mentioned embodiment numbers of the application are only for description, and do not represent the advantages and disadvantages of the embodiments.

[0088] The above embodiments are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalent replaced without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing a quercetin nanovesicle complex, characterized in that, include: S1. Take milk and centrifuge it at a differential speed. After centrifugation, add metal ion chelating agent to the supernatant at a certain volume ratio and incubate. After ultracentrifugation, collect the supernatant, filter it, perform density gradient ultracentrifugation, and collect the precipitate. Resuspend and filter the precipitate to obtain milk-derived extravesicles. S2. Mix quercetin with the outer vesicles prepared in step S1, sonicate and co-incubate, and collect the precipitate by ultracentrifugation to obtain the quercetin nanovesicle complex.

2. The preparation method according to claim 1, characterized in that, In step S1, The differential centrifugation is performed at 2-8℃ with a relative centrifugal force of 10000-15000×g for 20-40 minutes. The ultracentrifugation is performed at 2–8℃ at 90,000–110,000 × g for 60–80 min. All filtration processes employed 0.22μm filters. The density gradient ultracentrifugation is performed at 130,000–140,000 × g for 90–100 min at 2–8 °C.

3. The preparation method according to claim 1, characterized in that, In step S1, the milk is commercially available pasteurized low-fat milk with a fat content ≤1.5%, the metal ion chelating agent is an EDTA solution with a concentration of 0.3-0.6 mol / L, the volume ratio of the centrifuged supernatant to the metal ion chelating agent is 2-4:1, and the incubation with the metal ion chelating agent is incubating the centrifuged supernatant and the EDTA solution on ice for 10-20 minutes.

4. The preparation method according to claim 1, characterized in that, In step S2, The mass ratio of quercetin to the exovesicles prepared in step S1 is 1–8:1; The ultrasound is 400-500W, turned on for 10-30 seconds, turned off for 30-50 seconds, and drug delivery is carried out in 4-8 cycles. The ultrasound-guided co-incubation was performed at 37°C for 1–3 hours at 0–100 rpm. The ultracentrifugation is performed at 130,000–170,000 × g for 60–90 min.

5. The quercetin nanovesicle complex prepared by any one of the methods described in claims 1 to 4.

6. The use of the quercetin nanovesicle complex according to claim 5 in the preparation of a medicament for treating aging-related diseases.

7. The application according to claim 6, characterized in that, The age-related diseases include inflammatory bowel disease, and the drug can specifically target the site of colonic inflammation.

8. The application according to claim 6, characterized in that, The drug is an oral formulation.

9. An anti-aging composition, characterized in that, Including the quercetin nanovesicle complex as described in claim 5.

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