Preparation method and application of naringin-loaded hydrotalcite inorganic nanoparticles

Abstract

The invention relates to the field of nano-drug preparation, in particular to a preparation method and application of naringin-loaded hydrotalcite inorganic nanoparticles. The preparation method comprises the following steps: S1, mixing magnesium salt and aluminum salt to obtain a mixed solution; adding the mixed solution into an alkali metal hydroxide solution, and stirring to obtain a mixed precipitate; s2, collecting the mixed precipitate in the step S1, washing and re-suspending to obtain a suspension liquid; s3, carrying out hydrothermal treatment on the turbid liquid in the step S2 to obtain hydrotalcite inorganic nanoparticles; the preparation method comprises the following steps: adding a naringin aqueous solution into a hydrotalcite nanoparticle aqueous solution, then adding a bovine serum albumin aqueous solution, mixing and stirring, and centrifuging to obtain the naringin-loaded hydrotalcite inorganic nanoparticles. The naringin-loaded hydrotalcite inorganic nanoparticles prepared by the preparation method disclosed by the invention are clear in component, uniform in particle size and simple in preparation method, and have a stronger treatment advantage of relieving cell oxidative stress injury.

Description

Technical Field

[0001] The invention relates to the field of nano drug preparation, and in particular to a preparation method of naringin-loaded hydrotalcite inorganic nanoparticles and application thereof. Background Art

[0002] Quzhike is the dried immature fruit of Changshan Huyou, a Rutaceae plant. It is pungent, sweet, bitter and warm in nature and flavor, and enters the spleen, stomach and large intestine meridians. It has the effects of promoting qi and blood circulation, removing phlegm and dispersing knots, regulating qi and relieving fullness, and relieving stagnation and swelling. Naringin is one of the main components of Quzhike flavonoids, which has pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor, and free radical scavenging. However, conventional preparations of naringin are poorly absorbed and have low bioavailability, which limits its clinical application. This limits the therapeutic effect of naringin and the development of naringin preparations. However, loading naringin on other substances is a solution.

[0003] For example, Chinese patent CN114903911A discloses a method for preparing a naringin complex loaded with octenyl succinic acid modified porous starch ester, (S01) dissolving an appropriate amount of naringin in solvent I to obtain solution A; (S02) adding an appropriate amount of octenyl succinic acid porous starch to solvent II, dispersing and mixing to obtain suspension B; (S03) placing solution A in step (S01) and suspension B in step (S02) in an ultrasonic mixing device for ultrasonic mixing, and centrifuging and drying to obtain an octenyl succinic acid modified porous starch ester loaded with naringin complex. However, naringin is currently mostly loaded in organic matter, but loading in certain organic matter may make it difficult for naringin to achieve effective controlled release, which may lead to uneven distribution of the drug in the body and affect the efficacy. Moreover, the use of organic carriers is not only costly but also complicated in preparation.

[0004] Secondly, the current preparation technology of naringin nano-preparations mainly involves synthesizing phospholipid bilayer nanoliposome films, and quickly injecting flavonoid compounds such as naringin into liposome solutions to synthesize nanoliposome drugs encapsulating naringin. For example, in an article titled "Research Progress of Naringin Preparations" published in the China Medical Herald in 2020 by Xu Kunyong et al., it was mentioned that preparing naringin into naringin hydrophilic gel skeleton tablets, naringin liposome gels, naringin-lecithin inclusion complexes, naringin solid lipid nanoparticles, naringin nanoemulsions, etc. can improve bioavailability and enhance sustained release and targeting effects. However, the naringin liposomes synthesized by the existing technology have limited encapsulation rates, complex synthesis steps, and a large number of organic reagents used in the process. If the subsequent steps are not completely removed, it is likely to cause toxic side effects to the body.

[0005] Hydrotalcite is a new type of inorganic layered nanomaterial with a two-dimensional structure. It has a large specific surface area and can accommodate many different types of compounds. Hydrotalcite has many good biological properties, including high biocompatibility, high chemical stability, high loading capacity, and low toxicity. It has certain anti-inflammatory and antioxidant effects. After loading drugs, the dosage of drugs can be greatly reduced. In the field of biomedicine, hydrotalcite is a good drug delivery carrier. Small molecules, genes, peptides and other drugs can be loaded onto hydrotalcite through surface electrostatic adsorption or interlayer anion exchange, and have been used in the treatment and research of various diseases.

[0006] Although the current research mainly focuses on the preparation and performance improvement of hydrotalcite-based materials, these studies provide a theoretical basis and technical support for the development of hydrotalcite inorganic nanoparticles loaded with naringin. Future research can further explore the loading mechanism of naringin and methods to improve loading efficiency on this basis.

[0007] As mentioned in the article "Preparation of hydrotalcite-based supported catalysts and their applications in catalytic reactions" published in Acta Chemica Sinica by Yu Jun et al. in 2019, hydrotalcite is a type of anionic two-dimensional layered inorganic functional material with characteristics such as adjustable layer element ratio, high dispersion of metal cations and structural topological transformation. In heterogeneous catalysis, it has broad application prospects as a precursor or carrier of supported catalysts. However, there are few studies on loading naringin into hydrotalcite to improve the overall bioavailability.

[0008] In summary, based on the problems existing in the prior art, we are committed to providing a method for preparing hydrotalcite inorganic nanoparticles loaded with naringin, which has a simple synthesis method, safe synthetic reagents and a high drug loading amount, and can be used in the preparation of anti-inflammatory, antioxidant and anti-tumor products. Summary of the invention

[0009] The present invention aims to provide a method for preparing naringin-loaded hydrotalcite inorganic nanoparticles and their application in view of the problems existing in the prior art. The naringin-loaded hydrotalcite inorganic nanoparticles prepared by the method have stable structure, uniform particle size distribution, simple preparation, low cost, high safety, and can effectively improve the bioavailability and safety of naringin.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] In one aspect, the present invention provides a method for preparing hydrotalcite inorganic nanoparticles loaded with naringin, the preparation method specifically comprising the following steps:

[0012] S1, mixing a magnesium salt and an aluminum salt to obtain a mixed solution; adding the mixed solution to an alkali metal hydroxide solution, and stirring to obtain a mixed precipitate;

[0013] S2, collecting the mixed precipitate in step S1, washing it and resuspending it to obtain a suspension;

[0014] S3, subjecting the suspension in step S2 to hydrothermal treatment to obtain hydrotalcite inorganic nanoparticles;

[0015] S4, adding the naringin aqueous solution to the hydrotalcite nanoparticle aqueous solution, and then adding the bovine serum albumin aqueous solution, mixing and stirring, and centrifuging to obtain the naringin-loaded hydrotalcite inorganic nanoparticles.

[0016] Preferably, the alkali metal hydroxide in step S1 is sodium hydroxide.

[0017] Preferably, the stirring time in step S1 is 15-30 min.

[0018] More preferably, the stirring time in step S1 is 15 minutes.

[0019] Preferably, in step S1, the magnesium salt is selected from one of magnesium chloride, magnesium nitrate, magnesium carbonate, magnesium phosphate and magnesium sulfate; the aluminum salt is selected from one of aluminum chloride, aluminum nitrate, aluminum carbonate, aluminum phosphate and aluminum sulfate.

[0020] More preferably, the concentration of the magnesium salt is 0.1-0.3M, the concentration of the aluminum salt is 0.03-0.1M, the volume of the mixed solution is 10mL, and the concentration of the sodium hydroxide solution is 0.06-0.2M.

[0021] Most preferably, the concentration of the magnesium salt is 0.3 M, the concentration of the aluminum salt is 0.1 M, the volume of the mixed solution is 10 mL, the concentration of the sodium hydroxide solution is 0.2 M, and the molar ratio of magnesium ions to aluminum ions in the magnesium salt and aluminum salt is 3:1.

[0022] Preferably, in step S2, the centrifugal force of the centrifugation is not less than 4750g.

[0023] More preferably, in step S2, the centrifugal force of the centrifugation is 4750g.

[0024] Preferably, in step S2, the method for resuspending the mixed precipitate is selected from stirring, vortexing or ultrasound.

[0025] Preferably, the volume of the ultrapure water in step S2 is 10-40 mL; more preferably 30 mL.

[0026] Preferably, in step S3, the water temperature of the hydrothermal treatment is 60-200° C., and the reaction time of the hydrothermal treatment is 2 h-16 h.

[0027] More preferably, in step S3, the water temperature of the hydrothermal treatment is 100° C., and the reaction time of the hydrothermal treatment is 16 h.

[0028] Preferably, in step S4, the mass concentration of the bovine serum albumin aqueous solution is 5-10 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; the mass concentration of the naringin aqueous solution is 2-4 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; and the mixing and stirring time is 8-14 hours.

[0029] More preferably, the mass of the bovine serum albumin aqueous solution is 5 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; the concentration of the naringin aqueous solution is 4 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; and the mixing and stirring time is 12 hours.

[0030] On the other hand, the present invention also provides the application of the above preparation method in loading naringin.

[0031] In another aspect, the present invention also provides a nanomedicine of hydrotalcite inorganic nanoparticles loaded with naringin, wherein the nanomedicine comprises the hydrotalcite inorganic nanoparticles loaded with naringin obtained by the above preparation method; and application of the nanomedicine in the preparation of anti-inflammatory, antioxidant and anti-tumor products.

[0032] Wherein, the loading substance of the nano drug can be selected from one of flavonoid compounds, small molecule chemical drugs, DNA or RNA.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The hydrotalcite inorganic nanoparticles loaded with naringin in the present invention have clear components, uniform particle sizes and a particle size distribution of about 100 nm.

[0035] 2. The preparation method of the hydrotalcite inorganic nanoparticles loaded with naringin in the present invention is simple, the process is easy to control, the cost is low, and the whole process is safe and controllable;

[0036] 3. The hydrotalcite inorganic nanoparticles loaded with naringin in the present invention have a stronger therapeutic advantage in alleviating cell oxidative stress damage.

[0037] 4. The hydrotalcite inorganic nanoparticles loaded with naringin in the present invention have high biological safety. After co-culture with cells, the cell survival rate is still greater than 90% at a higher concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The transmission electron microscopy image of hydrotalcite inorganic nanoparticles loaded with naringin;

[0039] Among them, Figure A is Example 1, Figure B is Example 2, Figure C is Example 3, Figure D is Example 4, Figure E is Example 5, Figure F is Comparative Example 1, and Figure G is Comparative Example 2.

[0040] Figure 2 This is the particle size distribution diagram of hydrotalcite inorganic nanoparticles loaded with naringin.

[0041] Figure 3 The stability test results of hydrotalcite inorganic nanoparticles loaded with naringin in cell culture medium after 24 hours; Note: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001, ns, no significant difference.

[0042] Figure 4 This is the infrared spectrum of hydrotalcite inorganic nanoparticles loaded with naringin.

[0043] Figure 5 The figure shows the cellular uptake results of hydrotalcite inorganic nanoparticles loaded with naringin;

[0044] Among them, Figure A is Example 1, Figure B is Example 2, Figure C is Example 3, Figure D is Example 4, Figure E is Example 5, Figure F is Comparative Example 1, and Figure G is Comparative Example 2.

[0045] Figure 6 The figure is a cytotoxicity test result of hydrotalcite inorganic nanoparticles loaded with naringin;

[0046] Among them, Figure A is Example 1, Figure B is Example 2, Figure C is Example 3, Figure D is Example 4, Figure E is Example 5, Figure F is Comparative Example 1, and Figure G is Comparative Example 2.

[0047] Figure 7 This is a test result diagram of the hydrotalcite inorganic nanoparticles loaded with naringin to increase the intracellular glutathione level;

[0048] Among them, Figure A is Example 1, Figure B is Example 2, Figure C is Example 3, Figure D is Example 4, Figure E is Example 5, Figure F is Comparative Example 1, and Figure G is Comparative Example 2.

[0049] Figure 8 This is a test result diagram of the ability of hydrotalcite inorganic nanoparticles loaded with naringin to increase the level of superoxide dismutase in cells;

[0050] Among them, Figure A is Example 1, Figure B is Example 2, Figure C is Example 3, Figure D is Example 4, Figure E is Example 5, Figure F is Comparative Example 1, and Figure G is Comparative Example 2. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below with reference to specific embodiments.

[0052] Example 1 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0053] The preparation method specifically comprises the following steps:

[0054] S1, 0.3M MgCl 2 6 Η 2 O and 0.1 M AlCl 3 6 Η 2 O to obtain 10 mL of a mixed solution, which was quickly added to a 0.2 M sodium hydroxide solution and stirred for 15 min at room temperature to obtain a mixed precipitate of magnesium hydroxide and aluminum hydroxide;

[0055] S2. Collect the mixed precipitate by centrifugation (centrifugal force is 4750 g), wash with 30 mL of ultrapure water and resuspend the precipitate by stirring until there are no more particles visible to the naked eye in the suspension;

[0056] S3, using a hydrothermal kettle for hydrothermal treatment, wherein the water temperature is 100° C. and the reaction time is 16 h to obtain hydrotalcite inorganic nanoparticles;

[0057] S4. Adding a 4 mg / mL naringin aqueous solution to a 1 mg / mL hydrotalcite nanoparticle aqueous solution, and then adding a 5 mg / mL bovine serum albumin aqueous solution to obtain bovine serum albumin-loaded hydrotalcite inorganic nanoparticles, mixing and stirring for 12 hours, and centrifuging to obtain naringin-loaded hydrotalcite inorganic nanoparticles.

[0058] Example 2 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0059] The preparation method specifically comprises the following steps:

[0060] S1. Mix 0.1M magnesium nitrate and 0.03M aluminum nitrate to obtain 10mL of a mixed solution, quickly add the solution to 0.06M sodium hydroxide solution, and continue stirring at room temperature for 30 minutes to obtain a mixed precipitate of magnesium hydroxide and aluminum hydroxide;

[0061] S2. Collect the mixed precipitate by centrifugation (centrifugal force is 5000 g), wash with 10 mL of ultrapure water and resuspend the precipitate by ultrasonic S until there are no more particles visible to the naked eye in the suspension;

[0062] S3, using a hydrothermal kettle to perform hydrothermal treatment, wherein the water temperature is 60° C. and the reaction time is 2 h, to obtain hydrotalcite inorganic nanoparticles;

[0063] S4. Adding a 2 mg / mL naringin aqueous solution to a 1 mg / mL hydrotalcite nanoparticle aqueous solution, and then adding a 10 mg / mL bovine serum albumin aqueous solution, the mixture was stirred for 12 hours, and centrifuged to obtain naringin-loaded hydrotalcite inorganic nanoparticles.

[0064] Example 3 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0065] The preparation method specifically comprises the following steps:

[0066] S1. Mix 0.3M magnesium carbonate and 0.1M aluminum carbonate to obtain 10mL of a mixed solution, quickly add the solution to 0.2M sodium hydroxide solution, and continue stirring at room temperature for 15 minutes to obtain a mixed precipitate of magnesium hydroxide and aluminum hydroxide;

[0067] S2 is consistent with Example 1;

[0068] S3, using a hydrothermal kettle to perform hydrothermal treatment, wherein the water temperature is 200° C. and the reaction time is 2 h, to obtain hydrotalcite inorganic nanoparticles;

[0069] S4. The same as in Example 1.

[0070] Example 4 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0071] S1. Mix 0.1M magnesium phosphate and 0.03M aluminum phosphate to obtain 10mL of a mixed solution, quickly add the solution to 0.06M sodium hydroxide solution, and continue stirring at room temperature for 30 minutes to obtain a mixed precipitate of magnesium hydroxide and aluminum hydroxide;

[0072] The other steps are consistent with those in Example 1.

[0073] Example 5 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0074] S1. Mix 0.1M magnesium sulfate and 0.03M aluminum sulfate to obtain 10mL of a mixed solution, quickly add the solution to 0.06M sodium hydroxide solution, and continue stirring at room temperature for 30 minutes to obtain a mixed precipitate of magnesium hydroxide and aluminum hydroxide;

[0075] The other steps are consistent with those in Example 1.

[0076] Comparative Example 1 Preparation method of hydrotalcite inorganic nanoparticles loaded with naringin

[0077] The only difference from Example 1 is that in step S1, MgCl 26 Η 2 O concentration is 0.1M, AlCl 3 6 Η 2 The concentration of O is 0.3 M. The other steps are consistent with Example 1.

[0078] Comparative Example 2

[0079] The only difference from Example 1 is that in step S2, the centrifugal force is 4500g. The other steps are the same as those in Example 1.

[0080] Effect Experiment

[0081] Experiment 1 Naringin loading

[0082] Experimental methods

[0083] The particle morphology of the samples was determined using a transmission electron microscope, and the particle size of the samples was determined using a dynamic light scattering analyzer, Malvern Panalytical Zetasizer Pro. The equilibrium time was 40 seconds, and each sample was measured three times and the average value was taken. The particle size of the samples that were placed in the cell culture medium for 0-24 hours was determined using a dynamic light scattering analyzer, and the infrared spectrum of the samples was determined using a Fourier transform infrared spectrometer.

[0084] Experimental results analysis

[0085] The results of transmission electron microscopy are shown in Figure 1 ,from Figure 1 It can be seen that the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Examples 2-5, Comparative Examples 1-2 and the present invention are hexagonal particles with uniform size and uniform distribution;

[0086] The particle size of the sample was measured by a dynamic light scattering analyzer. The particle size of the simple hydrotalcite nanoparticles was about 80 nm, the particle size of the hydrotalcite nanoparticles loaded with bovine serum albumin was about 90 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 1 of the present invention was uniformly distributed at about 100 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 2 was uniformly distributed at about 102 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 3 was uniformly distributed at about 111 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 4 was uniformly distributed at about 114 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 5 was uniformly distributed at about 108 nm, the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Comparative Example 1 was uniformly distributed at about 119 nm, and the particle size of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Comparative Example 2 was uniformly distributed at about 120 nm. The results are shown in Table 1. Figure 2 .

[0087] The particle size of the sample placed in the cell culture medium for 0-24h was measured by a dynamic light scattering analyzer. The particle sizes of Example 1 at 0h, 6h, 12h, 18h, and 24h in the cell culture medium were 114nm, 110nm, 112nm, 111nm, and 111nm, respectively, and the stability was good. The particle sizes of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 2 at 0h, 6h, 12h, 18h, and 24h in the cell culture medium were 115nm, 110nm, 112nm, 111nm, and 111nm, respectively. The particle sizes of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Example 3 after 0h, 6h, 12h, 18h, and 24h in the cell culture medium were 105nm, 111nm, 108nm, 110nm, and 125nm, respectively. The particle sizes of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Example 4 after 0h, 6h, 12h, 18h, and 24h in the cell culture medium were 105nm, 111nm, 108nm, 110nm, and 125nm, respectively. The particle sizes of the inorganic hydrotalcite nanoparticles loaded with naringin in Example 5 were 125 nm, 111 nm, 118 nm, 110 nm and 108 nm in the cell culture medium at 0 h, 6 h, 12 h, 18 h and 24 h, respectively. The particle sizes of the inorganic hydrotalcite nanoparticles loaded with naringin in Comparative Example 1 were 125 nm, 111 nm, 118 nm, 110 nm and 108 nm in the cell culture medium at 0 h, 6 h, 12 h, 18 h and 24 h, respectively. The particle sizes of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Comparative Example 2 at 0h, 6h, 12h, 18h, and 24h in the cell culture medium were 125nm, 121nm, 128nm, 110nm, and 118nm, respectively; the particle sizes of the examples and the comparative examples after 24h in the cell culture medium were significantly different, and the results are shown in FIG. Figure 3 The results show that the particle size stability of the embodiment is significantly higher than that of the comparative example.

[0088] The infrared spectrum of the sample was analyzed by Fourier transform infrared spectrometer. Figure 4 It can be seen that the hydrotalcite inorganic nanoparticles loaded with naringin obtained in the present invention have the characteristic peaks of 2933 cm-1 of naringin and hydrotalcite inorganic nanoparticles. -1 , 1650cm -1 , 1060cm -1 and 1518-1322cm -1 Multiple peaks were detected, indicating the successful loading of naringin.

[0089] Experiment 2

[0090] Experimental methods

[0091] The hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention were incubated with normal mouse liver cells at 37°C for 24 hours to evaluate the in vitro uptake of the hydrotalcite inorganic nanoparticles loaded with naringin by the cells, thereby preliminarily determining the bioavailability of the material. The results are shown in Figure 5 .

[0092] Experimental results analysis

[0093] like Figure 5 As shown, 100 μg / mL hydrotalcite inorganic nanoparticles loaded with 170 μg of naringin were co-incubated with cells. As the incubation time increased, the cell uptake of the hydrotalcite inorganic nanoparticles loaded with naringin in Example 1 gradually increased, with an uptake of 30% after 2 hours, 75% after 4 hours, and nearly 90% after 8 hours. The uptake of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 2 was 22% after 2 hours, 65% after 4 hours, and nearly 80% after 8 hours. The uptake of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 3 was 25% after 2 hours, 60% after 4 hours, and nearly 85% after 8 hours. The uptake of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 4 was 25% after 2 hours, 67% after 4 hours, and nearly 82% after 8 hours. The uptake of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Example 5 after 2 hours was 28%, the uptake after 4 hours was 75%, and the uptake after 8 hours was close to 88%. The uptake of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Comparative Example 1 after 2 hours was 15%, the uptake after 4 hours was 55%, and the uptake after 8 hours was close to 70%. The uptake of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in Comparative Example 1 after 2 hours was 19%, the uptake after 4 hours was 58%, and the uptake after 8 hours was close to 65%, indicating that the naringin-loaded hydrotalcite inorganic nanoparticles obtained in the present invention have a high cellular uptake efficiency in a short time, and can greatly improve the bioavailability of naringin. The uptake of the comparative example is obviously not as good as that of the example, so the concentration of the magnesium salt and the aluminum salt and the setting of the centrifugal force need to be within the limited range of the present invention to greatly improve the bioavailability of naringin.

[0094] Experiment 3

[0095] Experimental methods

[0096] The hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention were incubated with normal mouse liver cells at 37°C for 24 hours to evaluate the toxic side effects of the hydrotalcite inorganic nanoparticles loaded with naringin on the cells, so as to preliminarily determine the biosafety of the material. A hydrotalcite inorganic nanoparticle group loaded with only bovine serum albumin (BL) and a free naringin group (Nar) were set as control groups to determine the biosafety of the embodiment and the comparative example (BLN), respectively. The results are shown in Figure 6 .

[0097] Experimental results analysis

[0098] like Figure 6 As shown, the hydrotalcite inorganic nanoparticles loaded with naringin 0.34-850 μg obtained in Example 1 at 2-500 μg / mL have no obvious toxicity to cells, and the cell survival rates are all above 90%, which are 111.2%, 110.8%, 110.5%, 110.1%, 109.5%, 109.1%, 105.2%, 100.3% and 95.2% respectively. When the concentration of the hydrotalcite inorganic nanoparticles loaded with naringin is lower than 125 μg / mL, it even has a certain effect of promoting cell proliferation, and the cell survival rate is between 120%-100%. At a higher dose of 500 μg / mL, the cell survival rate is still greater than 90%. The toxicity of the hydrotalcite inorganic nanoparticles loaded with naringin obtained in Example 2 to cells is 111.0%, 110.8%, 105.5%, 110.1%, 107.5%, 100.1% respectively. , 99.2%, 98.3% and 95.2%, the toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin obtained in Example 3 to cells was 109.0%, 108.8%, 105.5%, 106.1%, 107.5%, 102.1%, 99.2%, 99.3% and 95.2%, respectively, and the toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin obtained in Example 4 to cells was 112.0%, 110.8%, 112.5%, 110.1% , 107.5%, 105.1%, 99.2%, 98.3% and 95.2%, the toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin obtained in Example 5 to cells was 111.0%, 110.8%, 105.5%, 110.1%, 107.5%, 100.1%, 99.2%, 98.3% and 95.2%, respectively, and the toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin obtained in Comparative Example 1 to cells was 98.0%, 96.8%, respectively. The toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin to cells was 92.5%, 90.1%, 89.5%, 90.1%, 88.2%, 88.3% and 85.2%, respectively. The toxicity of the inorganic hydrotalcite nanoparticles loaded with naringin obtained in Comparative Example 2 to cells was 97.0%, 96.8%, 94.5%, 92.1%, 90.5%, 90.1%, 88.2%, 87.3% and 85.2%, respectively, indicating that the inorganic hydrotalcite nanoparticles loaded with naringin obtained in the present invention have high biocompatibility and biosafety.

[0099] Experiment 4

[0100] Experimental methods

[0101] The hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention were incubated with normal mouse liver cells damaged by 1 mM acetaminophen (APAP) at 37°C for 24 hours to evaluate the recovery effect of the hydrotalcite inorganic nanoparticles loaded with naringin on the glutathione level of damaged cells, so as to preliminarily determine the in vitro therapeutic effect of the material. A hydrotalcite inorganic nanoparticle group loaded with bovine serum albumin (BL), a free naringin group (Nar), and a mouse group not damaged by APAP (Ctrl) were set as control groups to determine the in vitro therapeutic effects of the materials of the embodiment and the comparative example (BLN).

[0102] Experimental results analysis

[0103] like Figure 7As shown, the glutathione content of each group in Example 1 is 5.2 μmol / gprot, 1.1 μmol / gprot, 1.9 μmol / gprot, 1.7 μmol / gprot and 4.2 μmol / gprot, respectively. It can be seen that the hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention have a significant effect on the recovery of glutathione levels in damaged cells; and only when serum albumin and free naringin are indispensable and synergistic, they will have a therapeutic effect on damaged cells. Compared with the Nar group, BLN loaded with 170 μg of naringin at 100 μg / mL showed a stronger effect of increasing glutathione, which can increase the intracellular glutathione level to 4.2 μmol / gprot, which is about 2.2 times that of the Nar group. The glutathione contents of each group in Example 2 were 5.3 μmol / gprot, 1.5 μmol / gprot, 1.9 μmol / gprot, 1.8 μmol / gprot and 4.1 μmol / gprot, respectively; the glutathione contents of each group in Example 3 were 5.4 μmol / gprot, 1.8 μmol / gprot, 2.0 μmol / gprot, 1.7 μmol / gprot and 4.4 μmol / gprot, respectively; the glutathione contents of each group in Example 4 were 5.4 μmol / gprot, 1.4 μmol / gprot, 2.2 μmol / gprot, 1.7 μmol / gprot and 4.5 μmol / gprot, respectively. In Example 5, the content of glutathione in each group is 5.4 μmol / gprot, 1.4 μmol / gprot, 2.5 μmol / gprot, 1.9 μmol / gprot and 4.8 μmol / gprot, in Comparative Example 1, the content of glutathione in each group is 5.0 μmol / gprot, 1.4 μmol / gprot, 1.8 μmol / gprot, 2.0 μmol / gprot and 3.5 μmol / gprot, and in Comparative Example 2, the content of glutathione in each group is 5.0 μmol / gprot, 1.5 μmol / gprot, 2.0 μmol / gprot, 2.5 μmol / gprot and 3.0 μmol / gprot. However, the improvement of intracellular glutathione level in the comparative example is not as good as that in the embodiment, so the concentration of magnesium salt and aluminum salt and the centrifugal force need to be within the range set by the present invention, and the hydrotalcite inorganic nanoparticles loaded with naringin will have a stronger therapeutic advantage in alleviating cell oxidative stress damage.

[0104] Experiment 5

[0105] Experimental methods

[0106] The hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention were incubated with normal mouse liver cells damaged by 1 mM APAP at 37°C for 24 hours to evaluate the recovery effect of the hydrotalcite inorganic nanoparticles loaded with naringin on superoxide dismutase in damaged cells, so as to preliminarily determine the in vitro therapeutic effect of the material. Hydrotalcite inorganic nanoparticles loaded with bovine serum albumin (BL), a free naringin group (Nar), and a mouse group not damaged by APAP (Ctrl) were set as control groups to determine the in vitro therapeutic effects of the materials of the embodiment and the comparative example (BLN), respectively. The results are shown in Figure 8 .

[0107] Experimental results analysis

[0108] like Figure 8As shown, the content of superoxide dismutase in each group of Example 1 is 13.8U / mgprot, 4.9U / mgprot, 10.2U / mgprot, 8.5U / mgprot and 12.5U / mgprot, respectively. It can be seen that the hydrotalcite inorganic nanoparticles loaded with naringin prepared by the present invention have a significant effect on the recovery of superoxide dismutase in damaged cells, and only when serum albumin and free naringin work together, they can have a therapeutic effect on damaged cells. Compared with the naringin free drug treatment group, the 100μg / mL hydrotalcite inorganic nanoparticles loaded with 170μg of naringin showed a stronger effect of increasing superoxide dismutase, which can increase the intracellular superoxide dismutase content to about 12.5U / mgprot, and restore it to 13.8U / mgprot close to the normal level, which is about 1.2 times that of the free naringin drug group. The content of superoxide dismutase in each group of Example 2 was 12.8U / mgprot, 4.9U / mgprot, 11.2U / mgprot, 8.5U / mgprot and 12.4U / mgprot, respectively, and the content of superoxide dismutase was higher than that in the Ctrl group; the content of superoxide dismutase in each group of Example 3 was 13.4U / mgprot, 4.5U / mgprot, 11.9U / mgprot, 8.5U / mgprot and 12.6U / mgprot, respectively; the content of superoxide dismutase in each group of Example 4 was 13.4U / mgprot, 5.2U / mgprot, 11.2U / mgprot, 9.5U / mgprot and 13.6U / mgprot, respectively. .0U / mgprot; the content of superoxide dismutase in each group of Example 5 is 13.2U / mgprot, 5.0U / mgprot, 10.2U / mgprot, 8.5U / mgprot and 12.0U / mgprot respectively; the content of superoxide dismutase in each group of Comparative Example 1 is 13.1U / mgprot, 4.2U / mgprot, 8.2U / mgprot, 9.1U / mgprot and 10.1U / mgprot respectively; the content of superoxide dismutase in each group of Comparative Example 2 is 13.0U / mgprot, 3.2U / mgprot, 7.5U / mgprot, 8.6U / mgprot and 9.7U / mgprot respectively. The comparative examples show that the preparation conditions of the hydrotalcite inorganic nanoparticles loaded with naringin (such as the setting of centrifugal force, the setting of the concentration of magnesium salt and aluminum salt) need to be within the range set by the present invention to have a stronger therapeutic advantage in alleviating cell oxidative stress damage.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing hydrotalcite inorganic nanoparticles loaded with naringin, characterized in that: The preparation method specifically comprises the following steps: S1, mixing a magnesium salt and an aluminum salt to obtain a mixed solution; adding the mixed solution to an alkali metal hydroxide solution, and stirring to obtain a mixed precipitate; S2, collecting the mixed precipitate in step S1, washing it and resuspending it to obtain a suspension; S3, subjecting the suspension in step S2 to hydrothermal treatment to obtain hydrotalcite inorganic nanoparticles; S4, adding the naringin aqueous solution to the hydrotalcite nanoparticle aqueous solution, and then adding the bovine serum albumin aqueous solution, mixing and stirring, and centrifuging to obtain the naringin-loaded hydrotalcite inorganic nanoparticles.

2. The preparation method according to claim 1, characterized in that: The alkali metal hydroxide solution described in step S1 is a sodium hydroxide solution, and the stirring time is 15-30 minutes; the magnesium salt is selected from one of magnesium chloride, magnesium nitrate, magnesium carbonate, magnesium phosphate and magnesium sulfate; and the aluminum salt is selected from one of aluminum chloride, aluminum nitrate, aluminum carbonate, aluminum phosphate and aluminum sulfate.

3. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the magnesium salt is 0.1-0.3M, the concentration of the aluminum salt is 0.03-0.1M, the volume of the mixed solution is 10mL, and the concentration of the sodium hydroxide solution is 0.06-0.2M.

4. The preparation method according to claim 1, characterized in that: In step S1, the molar ratio of magnesium ions to aluminum ions in the magnesium salt and aluminum salt is 1-10:

1.

5. The preparation method according to claim 1, characterized in that: In step S2, the collection is carried out by centrifugation, and the centrifugal force of the centrifugation is not less than 4750g.

6. The preparation method according to claim 1, characterized in that: In step S2, the resuspension method is selected from one of stirring, vortexing and ultrasound; the washing is performed using ultrapure water, and the volume of the ultrapure water is 10-40 mL.

7. The preparation method according to claim 1, characterized in that: In step S3, the hydrothermal treatment adopts a hydrothermal kettle, the water temperature of the hydrothermal treatment is 60-200° C., and the time of the hydrothermal treatment is 2h-16h.

8. The preparation method according to claim 1, characterized in that: The mass concentration of the bovine serum albumin aqueous solution in step S4 is 5-10 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; the mass concentration of the naringin aqueous solution is 2-4 times the mass concentration of the hydrotalcite inorganic nanoparticle aqueous solution; and the mixing and stirring time is 8-14 hours.

9. Use of the preparation method according to any one of claims 1 to 8 in loading naringin.

10. A nanomedicine of hydrotalcite inorganic nanoparticles loaded with naringin, characterized in that: The invention comprises hydrotalcite inorganic nanoparticles loaded with naringin obtained by the preparation method according to any one of claims 1 to 8; and application of the nanomedicine in the preparation of anti-inflammatory, antioxidant and anti-tumor products.

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