Inulin-carboxymethyl chitosan modified urolithin A liposome as well as preparation method and application thereof

Abstract

The invention provides an inulin-carboxymethyl chitosan modified urolithin A liposome and a preparation method and application thereof.The preparation method includes the steps that S1, urolithin A, egg yolk lecithin and beta-sitosterol are mixed and dissolved in absolute ethyl alcohol, and a mixed solution is obtained; performing water bath rotary evaporation on the mixed solution until the ethanol is removed, observing that a layer of oily film is formed in a container, adding a tween-80 aqueous solution, and stirring and heating until the mixed solution is completely hydrated to form crude liposome; performing ultrasonic treatment and centrifugal treatment on the crude lipidosome to obtain supernatant liquid, namely urolithin A lipidosome; s2, adding a carboxymethyl chitosan solution into the urolithin A liposome, and uniformly stirring, so as to obtain carboxymethyl chitosan modified urolithin A liposome; and S3, adding an inulin solution into the carboxymethyl chitosan modified urolithin A liposome, and uniformly stirring to obtain the inulin-carboxymethyl chitosan modified urolithin A liposome. The entrapment efficiency and the bioavailability of the urolithin A liposome can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of urolithin A delivery technology, and in particular to an inulin-carboxymethyl chitosan modified urolithin A liposome, its preparation method and application. Background Technology

[0002] Urolithin A is one of the metabolites produced by the degradation of ellagic tannins by intestinal microorganisms in vivo. Current research shows that urolithin A has various biological activities, such as anti-inflammatory, anti-aging, anti-tumor, and immunomodulatory effects. However, due to its poor water solubility and low bioavailability, it is not easily absorbed by the human body and cannot fully exert its functional properties, thus limiting its application in the food, pharmaceutical, and other fields.

[0003] Liposomes are closed vesicles with a bilayer structure formed by the self-assembly of amphiphilic phospholipids dispersed in an aqueous phase. Compared to other delivery systems, the closed vesicle structure of liposomes can simultaneously encapsulate both lipid-soluble and water-soluble substances, improving the bioavailability and stability of active substances.

[0004] Traditional unmodified liposomes are sensitive to the external environment and prone to aggregation, leading to leakage of the encapsulated material. Modifying liposomes with polysaccharides can effectively improve their encapsulation efficiency, stability, and bioavailability. How to prepare layer-by-layer modified liposomes to improve the encapsulation efficiency and bioavailability of urolithin A is of great significance for the clinical application of urolithin A. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides inulin-carboxymethyl chitosan-modified urolithin A liposomes, their preparation method, and their applications. This invention can significantly improve the encapsulation efficiency and bioavailability of urolithin A liposomes.

[0006] The technical solution of the present invention is as follows: The first objective of this invention is to provide a method for preparing inulin-carboxymethyl chitosan modified urolithin A liposomes, comprising the following steps: S1, urolithiasis A, egg yolk lecithin and β-sitosterol are mixed and dissolved in anhydrous ethanol to obtain a mixed solution; The mixed solution was subjected to rotary evaporation in a water bath until the ethanol was removed, and an oily film was observed to form in the container. Tween-80 aqueous solution was added, and the mixture was stirred and heated until it was completely hydrated to form crude liposomes. The crude liposomes were subjected to ultrasonic and centrifugation to obtain the supernatant as urolithin A liposomes; S2, add carboxymethyl chitosan solution to urolithin A liposomes and stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; S3, add inulin solution to carboxymethyl chitosan-modified urolithin A liposomes and stir evenly to obtain inulin-carboxymethyl chitosan-modified urolithin A liposomes.

[0007] In one embodiment of the present invention, in step S1, the mass ratio of urolithin A, egg yolk lecithin and β-sitosterol is 1:10:2.

[0008] In one embodiment of the present invention, in step S1, after urolithiasis A, egg yolk lecithin and β-sitosterol are mixed and dissolved in anhydrous ethanol, magnetic stirring is performed at a speed of 300 r / min.

[0009] In one embodiment of the present invention, in step S1, the conditions for water bath rotary evaporation are: temperature 30-37°C and rotation speed 50-80 r / min.

[0010] In one embodiment of the present invention, in step S1, the concentration of the Tween-80 aqueous solution is 0.5-1.5%, and the amount of Tween-80 aqueous solution used is 500-1000 times the mass of urolithin A.

[0011] In one embodiment of the present invention, in step S1, the conditions for stirring and heating are: heating temperature of 55°C and stirring speed of 200 r / min.

[0012] In one embodiment of the present invention, in step S1, the ultrasonic treatment of crude liposomes is carried out under ice bath conditions, with an ultrasonic power of 180W and an ultrasonic treatment time of 10min; the centrifugation temperature is 4℃, the centrifugation speed is 4000r / min, and the centrifugation time is 10min.

[0013] In one embodiment of the present invention, in step S2, the concentration of the carboxymethyl chitosan solution is 3-7 mg / mL.

[0014] In one embodiment of the present invention, in step S2, the method for preparing the carboxymethyl chitosan solution is as follows: carboxymethyl chitosan is dissolved in deionized water and stirred at 300 r / min for 1 h to prepare a carboxymethyl chitosan solution of 3-7 mg / mL.

[0015] In one embodiment of the present invention, in step S2, the volume ratio of carboxymethyl chitosan solution to urolithin A liposomes is 1:1.

[0016] In one embodiment of the present invention, in step S3, the concentration of the inulin solution is 3-7 mg / mL.

[0017] In one embodiment of the present invention, in step S3, the inulin solution is prepared by dissolving inulin in deionized water and stirring at 300 r / min for 1 h to obtain an inulin solution of 3-7 mg / mL.

[0018] In one embodiment of the present invention, in step S3, the volume ratio of inulin solution to carboxymethyl chitosan-modified urolithin A liposomes is 1:1.

[0019] The second objective of this invention is to provide an inulin-carboxymethyl chitosan-modified urolithin A liposome prepared by the above method.

[0020] A third objective of this invention is to provide an application of the above-mentioned inulin-carboxymethyl chitosan modified urolithin A liposomes for the preparation of a drug to prevent immune decline.

[0021] A fourth objective of this invention is to provide a combination pharmaceutical composition containing urolithin A liposomes modified with inulin-carboxymethyl chitosan as described above for the prevention of immunodeficiency.

[0022] The beneficial technical effects of this invention are as follows: This invention uses egg yolk lecithin as the wall material and urolithin A as the core material to prepare urolithin A liposomes; the urolithin A liposomes are modified with inulin and carboxymethyl chitosan. The modified inulin-carboxymethyl chitosan urolithin A liposomes have good stability, high encapsulation efficiency, and high bioavailability.

[0023] The present invention has the following advantages: (1) Improve the encapsulation efficiency of urolithin A: Urolithin A liposomes were modified with inulin and carboxymethyl chitosan. Experimental data showed that the encapsulation efficiency could be significantly improved.

[0024] (2) Enhanced bioavailability: The closed vesicle structure of liposomes inherently improves the bioavailability and stability of active substances, and this is further optimized after modification with inulin and carboxymethyl chitosan. This modification improves the performance of liposomes, enabling urolithin A to be better absorbed and utilized after entering the human body, thus maximizing its efficacy.

[0025] (3) Overcoming the limitations of urolithin A application: Due to its poor water solubility and stability, urolithin A is limited in its application in the food, pharmaceutical and other fields. The modified liposome technology of this invention improves the stability of urolithin A, improves the problem of poor water solubility, and expands the application range of urolithin A in multiple fields.

[0026] (4) Optimizing liposome performance: Traditional liposomes have poor resistance to changes in the external environment and are prone to aggregation, leading to leakage of the encapsulated material. This invention utilizes polysaccharide modification to enhance the resistance of liposomes to changes in the external environment, reduce aggregation and leakage of the encapsulated material, and improve the overall performance of liposomes. Attached Figure Description

[0027] Figure 1 The following are the characterization and stability results of the inulin-carboxymethyl chitosan modified urolithin A liposomes of the present invention: A is a transmission electron micrograph of the liposomes; B is a graph showing the change in particle size during storage stability; C is a graph showing the change in zeta potential during storage stability; and D is a graph showing the change in encapsulation rate during storage stability.

[0028] Figure 2 Coca-2 cells take up urolithin A liposomes, carboxymethyl chitosan-liposomes, and inulin-carboxymethyl chitosan-modified urolithin A liposomes.

[0029] Figure 3 Figures showing the results of inulin-carboxymethyl chitosan-modified urolithin A liposomes in preventing symptoms of immunodeficiency: A is the animal experimental protocol; B is the mouse body weight change; C is the physical image of the spleen in each group; D is the thymus index of each group; E is the spleen index of each group.

[0030] Figure 4 HE section and flow cytometry results of spleen tissue in mice with immunodeficiency, modified with inulin-carboxymethyl chitosan liposomes for the prevention of urolithin A liposomes: A is HE section of spleen tissue in each group of mice; B is flow cytometry results of CD4+ T cells in spleen tissue in each group of mice.

[0031] Figure 5 The following are flow cytometry results of the spleens of mice in each group: A shows the flow cytometry results of Th1 cells in the spleens of mice in each group; B shows the flow cytometry results of Th2 cells in the spleens of mice in each group. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0033] The experimental animals used in the following examples were male Balb / c mice, obtained from Liaoning Changsheng Biotechnology Co., Ltd.

[0034] Example 1 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0035] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0036] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0037] (4) Add an equal volume of 5 mg / mL carboxymethyl chitosan solution to the liposomes, stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; then add an equal volume of 3 mg / mL inulin solution to the carboxymethyl chitosan modified urolithin A liposomes, stir evenly, and filter through a 0.45 μm filter membrane to obtain inulin-carboxymethyl chitosan modified urolithin A liposomes.

[0038] Example 2 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0039] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0040] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0041] (4) Add an equal volume of 5 mg / mL carboxymethyl chitosan solution to the liposomes, stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; then add an equal volume of 5 mg / mL inulin solution to the carboxymethyl chitosan modified urolithin A liposomes, stir evenly, and filter through a 0.45 μm filter membrane to obtain inulin-carboxymethyl chitosan modified urolithin A liposomes (IC-NL).

[0042] Example 3 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0043] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0044] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0045] (4) Add an equal volume of 5 mg / mL carboxymethyl chitosan solution to the liposomes, stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; then add an equal volume of 7 mg / mL inulin solution to the carboxymethyl chitosan modified urolithin A liposomes, stir evenly, and filter through a 0.45 μm filter membrane to obtain inulin-carboxymethyl chitosan modified urolithin A liposomes.

[0046] Example 4 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0047] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0048] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0049] (4) Add an equal volume of 3 mg / mL carboxymethyl chitosan solution to the liposomes, stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; then add an equal volume of 5 mg / mL inulin solution to the carboxymethyl chitosan modified urolithin A liposomes, stir evenly, and filter through a 0.45 μm filter membrane to obtain inulin-carboxymethyl chitosan modified urolithin A liposomes.

[0050] Example 5 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0051] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0052] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0053] (4) Add an equal volume of 7 mg / mL carboxymethyl chitosan solution to the liposomes, stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; then add an equal volume of 5 mg / mL inulin solution to the carboxymethyl chitosan modified urolithin A liposomes, stir evenly, and filter through a 0.45 μm filter membrane to obtain inulin-carboxymethyl chitosan modified urolithin A liposomes.

[0054] Comparative Example 1 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0055] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0056] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant, filter through a 0.45 μm filter membrane to obtain urolithin A liposome solution (NL).

[0057] Comparative Example 2 (1) Place 20mg of urolithiasis A, 200mg of egg yolk lecithin and 40mg of β-sitosterol into a flask, add 100ml of anhydrous ethanol until completely dissolved to form a mixed solution.

[0058] (2) Evaporate at 60 r / min in a 37°C water bath until the ethanol is removed. An oily film is observed to form in the container. Add 20 ml of deionized water containing 1% Tween-80 and heat at 200 r / min at 55°C for 1 h until complete hydration to obtain crude liposomes.

[0059] (3) Stir and heat at 200 r / min at 55℃ for 1 h until completely hydrated to obtain crude liposomes. Sonicate at 180 W for 10 min, then centrifuge at 4000 rpm for 10 min at 4℃. Discard the precipitate and keep the supernatant to obtain urolithin A liposome solution.

[0060] (4) Add an equal volume of 5 mg / mL carboxymethyl chitosan solution to the liposomes, stir well, and filter through a 0.45 μm filter membrane to obtain carboxymethyl chitosan modified urolithin A liposomes (C-NL).

[0061] Test Example 1 The particle size, zeta potential, and PDI of the liposomes prepared in Comparative Examples 1-2 and Examples 1-5 were determined using a multi-angle particle size and potential analyzer. The liposomes were disrupted and the drug dissolved using DMSO-C2H5OH (1:9) solution, and then centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was collected, and the absorbance at 256 nm was measured using a UV-Vis spectrophotometer to calculate the drug encapsulation efficiency. The results are shown in Table 1.

[0062] Table 1

[0063] As shown in Table 1, the drug encapsulation efficiency of Examples 1-4 was improved compared to Comparative Examples 1-2. Furthermore, the PDI of all examples was less than 0.3, indicating that the system was homogeneous. The absolute potential values ​​of the examples were also greater than 30 mV, indicating that the liposome systems of the examples were stable.

[0064] Test Example 2 The liposomes prepared in Example 1 and Comparative Examples 1-2 were diluted 10-fold. 10 μL of the solution was dropped onto a copper grid and allowed to air dry. Then, 10 μL of 2% potassium phosphate solution was dropped onto the copper grid, and staining was performed for 3 minutes. The morphology of the product was observed under an electron microscope. Figure 1 As shown in Figure A, the liposome nanoparticles are nearly spherical and uniform in size.

[0065] Test Example 3 The liposomes prepared in Example 2 and Comparisons 1-2 were stored at 4 °C for 21 days, and samples were taken at days 0, 7, 14, and 21. The particle size and zeta potential changes of the inulin-carboxymethyl chitosan-modified urolithin A liposomes were measured using a multi-angle particle size and potential analyzer. The liposomes were disrupted and the drug dissolved using DMSO-C2H5OH (1:9) solution, and then centrifuged at 12000 rpm for 15 minutes at 4 °C. The supernatant was collected, and the absorbance at 256 nm was measured using a UV-Vis spectrophotometer.

[0066] The results show that... Figure 1As shown in Figure BD, the encapsulation efficiency of liposomes decreased with increasing storage time. However, liposomes modified with both inulin and carboxymethyl chitosan exhibited higher encapsulation efficiency than unmodified liposomes and those modified with only carboxymethyl chitosan. The particle size of liposomes increased with increasing storage time. The zeta potential of liposomes also increased with increasing storage time, indicating that the stability of liposomes deteriorates with prolonged storage. However, the absolute value of the zeta potential of liposomes modified with both inulin and carboxymethyl chitosan still exceeded 30 mV after 21 days of storage, indicating that the liposomes remained stable.

[0067] Test Example 4 Caco-2 cells were fed at a rate of 2 × 10 5 Caco-2 cells were seeded at a density of 1 mL in 35 mm cell culture dishes. After incubation for 24 hours, the culture medium was carefully removed, and the cells were washed twice with 1 mL PBS. The cells were then treated with Rh 123 (5 μM) loaded with -NL (Rh123-NL), Rh 123 (5 μM) loaded with C-NL (Rh 123-C-NL), and Rh 123 (5 μM) loaded with -IC-RNL (Rh 123-IC-RNL), and further incubated at 37 °C for 2 hours. The sample was then removed, and the cells were washed three times with 1 mL PBS. The cells were then fixed with 4% paraformaldehyde (250 μL) for 15 minutes, stained with DAPI for 10 minutes, and washed twice more with PBS. Finally, the Caco-2 cells were examined under a fluorescence microscope. The results of loading Rh-123 into NL, C-NL, and IC-NL (Example 1) are as follows: Figure 2 As shown, compared with C-NL and NL, IC-NL exhibits more green fluorescence signal, indicating that IC-NL has higher uptake efficiency.

[0068] Test Example 5 1. The effect of inulin-carboxymethyl chitosan-modified urolithin A liposomes on symptoms of immunodeficiency. Experimental Methods: To evaluate the effect of inulin-carboxymethyl chitosan-modified urolithin A liposomes on immunodeficiency, animals were randomly divided into the following groups: Control group (Ctrl): Mice were fed normally; Model group (Mod): injected with cyclophosphamide (50 mg / kg); Intervention group (UA): Cyclophosphamide (50 mg / kg) by injection + urolithiasis A (20 mg / kg) by gavage; Intervention group (UA-NL): Cyclophosphamide (50 mg / kg) was injected and urolithin A liposomes modified with inulin-carboxymethyl chitosan prepared in Example 1 were administered by gavage (20 mg / kg).

[0069] From day 1 to 14, administer inulin-carboxymethyl chitosan-modified urolithin A liposomes or urolithin A via gavage. Starting day 15, administer cyclophosphamide via gavage and injection for 3 consecutive days. Figure 3 A. The mice were observed and their weight was recorded daily during the period. On day 17, they were euthanized after a 12-hour fast, and their spleen and thymus were collected and weighed.

[0070] Experimental results are as follows Figure 3 Compared with the blank group, the liver index, spleen index and thymus index of mice in the model group were significantly decreased, while the liver index, spleen index and thymus index of the intervention group were significantly increased compared with the model group, indicating that inulin-carboxymethyl chitosan modified urolithin A liposomes can effectively prevent immune decline.

[0071] 2. HE staining was used to further observe changes in the mouse spleen. The group experiment followed the same steps as above. At 18 days, the spleen of the mouse was taken, embedded in paraffin, and stained with hematoxylin and eosin (HE).

[0072] The experimental results, as shown in tissue sections 4A, revealed a reduction in the red pulp of the spleen in the model group, with blurred boundaries between the red and white pulp, and an increase in megakaryocytes. Compared to the model group, the red pulp was more abundant, and the boundary between the red and white pulp was clearer. This indicates that inulin-carboxymethyl chitosan-modified urolithin A liposomes can effectively prevent spleen damage.

[0073] 3. Changes in spleen cell subtypes after prevention The group experiment followed the same steps as above. On day 21, mouse spleens were harvested and immersed in pre-chilled PBS, gently washed to remove fat and connective tissue. After adding 3 mL of PBS, the spleens were gently ground, and the cell suspension was filtered through a 70 μm sieve. The filtrate was then transferred to 15 mL centrifuge tubes, gently washed with 2 mL of PBS, and centrifuged at 1000 rpm for 5 minutes to pellet the cells. Red blood cells were lysed with 3 mL of Tris-NH4Cl for approximately 2 minutes, followed by the addition of 6 mL of RPMI 1640 complete medium to neutralize the lysis. The cells were then centrifuged at 800 rpm for 5 minutes, resuspended in RPMI 1640 complete medium, and their density was adjusted to 1 × 10⁻⁶ cells using a cell counter. 6 Cells / mL. Antibody was added to the suspension for labeling, the mixture was gently mixed, and then incubated on ice in the dark for 1 h. Lymphocytes were finally detected by flow cytometry.

[0074] Experimental results are as follows Figure 4 B and Figure 5 As shown, compared with the blank group, CD4 in the spleen of mice in the model group was higher. +T and Th1 cell expression was significantly decreased, while Th2 cell expression was significantly increased. Furthermore, the CD4 expression of cells in the inulin-carboxymethyl chitosan-modified urolithin A liposome group was significantly lower than that in the model group. + T and Th1 expression were significantly increased, while Th2 cell expression was significantly decreased, indicating that inulin-carboxymethyl chitosan-modified urolithin A liposomes can restore CD4 expression. + The number of T cells and the Th1 / Th2 balance are restored, thereby enhancing immunity.

[0075] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing inulin-carboxymethyl chitosan modified urolithin A liposomes, characterized in that, Includes the following steps: S1, urolithiasis A, egg yolk lecithin and β-sitosterol are mixed and dissolved in anhydrous ethanol to obtain a mixed solution; The mixed solution was subjected to rotary evaporation in a water bath until the ethanol was removed, and an oily film was observed to form in the container. Tween-80 aqueous solution was added, and the mixture was stirred and heated until it was completely hydrated to form crude liposomes. The crude liposomes were subjected to ultrasonic and centrifugation to obtain the supernatant as urolithin A liposomes; S2, add carboxymethyl chitosan solution to urolithin A liposomes and stir evenly to obtain carboxymethyl chitosan modified urolithin A liposomes; S3, add inulin solution to carboxymethyl chitosan-modified urolithin A liposomes and stir evenly to obtain inulin-carboxymethyl chitosan-modified urolithin A liposomes.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of urolithin A, egg yolk lecithin, and β-sitosterol is 1:10:

2.

3. The preparation method according to claim 1, characterized in that, In step S1, the conditions for water bath rotary evaporation are: temperature 30-37℃, rotation speed 50-80r / min.

4. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the Tween-80 aqueous solution is 0.5-1.5%, and the amount of Tween-80 aqueous solution used is 500-1000 times the mass of urolithin A.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of the carboxymethyl chitosan solution is 3-7 mg / mL.

6. The preparation method according to claim 1, characterized in that, In step S2, the volume ratio of carboxymethyl chitosan solution to urolithin A liposomes is 1:

1.

7. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the inulin solution is 3-7 mg / mL.

8. The preparation method according to claim 1, characterized in that, In step S3, the volume ratio of inulin solution to carboxymethyl chitosan-modified urolithin A liposomes is 1:

1.

9. A urolithin A liposome modified with inulin-carboxymethyl chitosan prepared by any one of claims 1-8.

10. The application of the inulin-carboxymethyl chitosan-modified urolithin A liposome according to claim 9, characterized in that, Used to prepare drugs for preventing immune decline.

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