Oleanolic acid / cyclodextrin metal organic framework compound as well as preparation method and application thereof

Abstract

The invention discloses an oleanolic acid / cyclodextrin metal organic framework compound and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The preparation method comprises the following steps: dissolving gamma-cyclodextrin and alkaline potassium salt in water to obtain crystallization mother liquor of a cyclodextrin metal organic framework; and then adding oleanolic acid into the crystallization mother liquor of the cyclodextrin metal organic framework for co-crystallization growth to obtain the oleanolic acid / cyclodextrin metal organic framework compound. According to the co-crystallization method provided by the invention, the oleanolic acid can be efficiently encapsulated in the cyclodextrin metal organic framework, and the solubility of the oleanolic acid is improved by 109.81 times by the prepared oleanolic acid / cyclodextrin metal organic framework compound, so that the lipid-lowering activity of the oleanolic acid is further enhanced; the compound provided by the invention has wide application potential in the aspects of preparing medicines for preventing / treating the non-alcoholic fatty liver disease and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an oleanolic acid / cyclodextrin metal organic framework complex and a preparation method and application thereof. Background Art

[0002] Nonalcoholic fatty liver disease is a liver disease caused by metabolic syndrome, which is often associated with obesity, hypertension, diabetes, hyperlipidemia and other metabolic disorders.

[0003] Oleanolic acid (OA) is a natural triterpene acid compound and a common natural active ingredient. It is often distributed in various plants in free and glycoside forms. Studies have shown that oleanolic acid has multiple pharmacological effects such as promoting metabolism, anti-inflammation, regulating blood lipids, relieving fatigue, anti-diabetes and liver protection. In particular, oleanolic acid can improve metabolic-related diseases such as obesity, lipid deposition, and diabetes through various pathways. In recent years, it has been reported that oleanolic acid can alleviate metabolic disorders and hepatic steatosis, thereby improving non-alcoholic fatty liver disease. However, due to the poor solubility of oleanolic acid in water and its low bioavailability in vivo, the application of oleanolic acid is limited.

[0004] Many methods have been reported to improve the solubility of oleanolic acid, including adding cosolvents, molecular modification, and preparing solid dispersions, polymer micelles, self-assembled nanoparticles, and liposomes. In addition, there are also studies on loading oleanolic acid in β-cyclodextrin or cyclodextrin derivatives through host-guest interactions, but the solubilization effect is often limited. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an oleanolic acid / cyclodextrin metal organic framework complex and its preparation method and application. The method provided by the present invention can effectively and significantly improve the hydrophilicity of oleanolic acid, increase the solubility of oleanolic acid by 109.81 times, and further make it have better lipid-lowering activity. The complex provided by the present invention has wide application potential in the preparation of drugs for preventing / treating non-alcoholic fatty liver disease, etc.

[0006] The first aspect of the present invention provides a method for preparing an oleanolic acid / cyclodextrin metal organic framework complex, the preparation method comprising the following steps:

[0007] dissolving γ-cyclodextrin and alkaline potassium salt in water to obtain a crystallization mother solution of a cyclodextrin metal organic framework;

[0008] Oleanolic acid is added to the crystallization mother liquor of cyclodextrin metal organic framework to obtain a mixed solution, the mixed solution is filtered and then an organic solvent is added, and the mixture is cultured at 25-50° C. for 5-14 days to allow oleanolic acid and cyclodextrin metal organic framework to co-crystallize and grow, thereby obtaining an oleanolic acid / cyclodextrin metal organic framework complex.

[0009] Furthermore, the drug loading amount of the oleanolic acid / cyclodextrin metal organic framework composite to oleanolic acid is 2.76wt% to 19.4wt%.

[0010] Furthermore, the molar ratio of the γ-cyclodextrin to the alkaline potassium salt is 1:7.9-8.1.

[0011] Furthermore, the alkaline potassium salt is potassium hydroxide.

[0012] Furthermore, the mixing method is ultrasonic mixing or mechanical stirring mixing.

[0013] Furthermore, the power of the ultrasound is 300-600W, and the treatment time is 40-60min.

[0014] Furthermore, the organic solvent is methanol or ethanol, and the organic solvent is in liquid or vapor form.

[0015] Furthermore, the organic solvent is preferably methanol.

[0016] Furthermore, the culture growth temperature is preferably 45° C., and the culture growth time is preferably 7 days.

[0017] Furthermore, the co-crystal growth further includes centrifugation, crystal collection, washing and drying; the drying temperature is 35 to 50° C. and the drying time is 6 to 12 hours.

[0018] Preferably, the drying temperature is 45° C. and the drying time is 6 hours.

[0019] The second aspect of the present invention provides an oleanolic acid / cyclodextrin metal organic framework complex, wherein the oleanolic acid / cyclodextrin metal organic framework complex is prepared according to the preparation method provided by the first aspect of the present invention.

[0020] The third party of the present invention provides an application of an oleanolic acid / cyclodextrin metal organic framework complex in the preparation of medicines or health products, wherein the medicine is a medicine for preventing / treating non-alcoholic fatty liver disease.

[0021] Furthermore, the medicine or health product has oleanolic acid / cyclodextrin metal organic framework complex as the only effective active ingredient.

[0022] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0023] (1) The present invention provides a method for preparing an oleanolic acid / cyclodextrin metal organic framework complex, which is to directly add oleanolic acid to the crystallization mother liquor of the cyclodextrin metal organic framework, so that the oleanolic acid is captured in the cavity of the cyclodextrin metal organic framework during the crystallization process of the cyclodextrin metal organic framework, and culture for 5 to 14 days at 25 to 50°C to allow the oleanolic acid and the cyclodextrin metal organic framework to co-crystallize and grow, thereby obtaining an oleanolic acid / cyclodextrin metal organic framework complex. The porous structure of the cyclodextrin metal organic framework uses the hydrophilic segment as the shell to protect the oleanolic acid inside its cavity, thereby improving the solubility of the oleanolic acid and the bioavailability in the body. Moreover, it is verified by Experimental Example 1 that, compared with the conventional cyclodextrin drug loading or cyclodextrin metal organic framework drug loading method, the co-crystallization method provided by the present invention achieves a more efficient delivery of oleanolic acid.

[0024] (2) In the method of the present invention, the crystallization mother liquor of the cyclodextrin metal organic framework is prepared by dissolving γ-cyclodextrin and potassium hydroxide in water. The inventors found in their previous studies that when the co-crystallization method provided by the present invention is used to prepare the oleanolic acid / cyclodextrin metal organic framework complex, if γ-cyclodextrin is replaced with β-cyclodextrin, and other preparation steps remain unchanged, the composite crystal of oleanolic acid and cyclodextrin metal organic framework cannot be obtained. This may be because the diameter of the hydrophobic cavity of β-cyclodextrin is smaller than that of γ-cyclodextrin, and its structural mode of forming a metal organic framework is also different from that of γ-cyclodextrin. Its framework formation requires β-cyclodextrin to be arranged in a "T" shape and an "8" double channel arrangement. The addition of oleanolic acid molecules may cause it to form a "head-to-head" inclusion complex with β-cyclodextrin, which may be unfavorable for further construction of a metal organic framework with potassium ions.

[0025] (3) The preparation method of the oleanolic acid / cyclodextrin metal organic framework complex provided by the present invention is simple and easy to operate, and has low cost. The obtained oleanolic acid / cyclodextrin metal organic framework complex not only increases the solubility of oleanolic acid by 109.81 times, but also has good lipid-lowering activity, effectively reduces the dosage of the drug, and the prepared product is safer. Oleanolic acid has multiple pharmacological effects such as promoting metabolism, anti-inflammation, regulating blood lipids, relieving fatigue, anti-diabetes and liver protection. The oleanolic acid / cyclodextrin metal organic framework complex provided by the present invention improves its solubility without destroying the effect of oleanolic acid, and can be used to prepare medicines for preventing / treating non-alcoholic fatty liver disease, anti-inflammation, protecting the liver, etc., and can also be used to prepare health products with functions such as relieving physical fatigue and assisting in lowering blood lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an optical microscope image of the oleanolic acid / cyclodextrin metal organic framework complex in Example 1.

[0027] Figure 2 It is a Fourier transform infrared spectrum of oleanolic acid / cyclodextrin metal organic framework complex; the samples from bottom to top are oleanolic acid / cyclodextrin metal organic framework complexes prepared in Examples 1 to 6.

[0028] Figure 3 The graph is a relationship between the yield and drug loading of oleanolic acid / cyclodextrin metal organic framework complex; wherein OA represents oleanolic acid, and a, b, c, and d represent significant differences between groups, p < 0.05.

[0029] Figure 4 It is the apparent solubility of oleanolic acid / cyclodextrin metal organic framework complex; wherein OA is oleanolic acid, OA@γ-CD is the oleanolic acid / γ-CD complex prepared in Comparative Example 1, OA@CD-MOF-D2 is the oleanolic acid / cyclodextrin metal organic framework complex prepared in Comparative Example 2, OA@CD-MOF-1 is the oleanolic acid / cyclodextrin metal organic framework complex prepared in Example 1, OA@CD-MOF-6 is the oleanolic acid / cyclodextrin metal organic framework complex prepared in Example 6, and a, b, and c indicate that there are significant differences between groups, p < 0.05.

[0030] Figure 5 The lipid-lowering activity result diagram of the oleanolic acid / cyclodextrin metal organic framework complex in the zebrafish animal model in Example 1; wherein the upper and lower rows of Figure A are fluorescence microscope images of zebrafish liver stained with BODIPY and ORO, respectively, Figure B is the fluorescence intensity of BODIPY staining, Figure C is the color intensity of ORO staining, Figure D is the change in total triglyceride (TG) content, and Figure E is the change in total cholesterol (TC) content; CON represents the negative control group fed with Paramecium, HFD represents the high-fat feeding group fed with egg yolk powder, OA represents the treatment group fed with high-fat feed and fed with oleanolic acid, OA@CD-MOF represents the treatment group fed with high-fat feed and fed with the oleanolic acid / cyclodextrin metal organic framework complex prepared in Example 1, AC represents the positive control group fed with high-fat feed and fed with simvastatin, * represents the significant difference between groups, the more * the more significant the difference, and ns represents no difference.

[0031] Figure 6The lipid-lowering activity results of the oleanolic acid / cyclodextrin metal organic framework complex in Example 1 in a mouse animal model; wherein, Figure A is a graph showing the weight of mice in each treatment group, and Figure B is a graph showing the blood glucose level of mice in each treatment group; CON represents a negative control group fed with a normal diet, HFD represents a group fed with a high-fat diet, OA represents a group fed with a high-fat diet and oleanolic acid, and OA@CD-MOF represents a group fed with a high-fat diet and oleanolic acid prepared in Example 1. * represents a significant difference between groups, and the more *s there are, the more significant the difference is, and ns represents no difference. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0033] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0034] Due to the poor solubility of oleanolic acid in water and its low bioavailability in vivo, the application of oleanolic acid is limited. In order to improve the solubility of oleanolic acid, it has been reported that oleanolic acid can be included in β-cyclodextrin or cyclodextrin derivatives, but the simple cyclodextrin inclusion has limited solubilization effect on oleanolic acid. It has been reported that cyclodextrin metal organic framework (CD-MOF) can be used to load drugs to improve drug solubility, but the drugs are often directly loaded on the prepared CD-MOF, and the drug solubilization effect is limited.

[0035] The embodiment of the present invention provides an oleanolic acid / cyclodextrin metal organic framework complex and its preparation method and application. The present invention dissolves γ-cyclodextrin and potassium hydroxide in water to obtain a crystallization mother liquor of a cyclodextrin metal organic framework, then adds oleanolic acid to the crystallization mother liquor of the cyclodextrin metal organic framework, and cultures for 5 to 14 days at 25 to 50°C to allow oleanolic acid and the cyclodextrin metal organic framework to co-crystallize and grow, thereby obtaining an oleanolic acid / cyclodextrin metal organic framework complex. The prepared complex was characterized by optical microscopy and Fourier transform infrared analysis, and experimentally determined that the oleanolic acid / cyclodextrin metal organic framework complex provided by the present invention can increase the solubility of oleanolic acid by 109.81 times, which is more effective than the conventional method of using cyclodextrin to load drugs or cyclodextrin metal organic framework to load drugs to increase drug solubility, and the lipid-lowering activity of the complex was detected using zebrafish model and mouse model.

[0036] Example 1

[0037] A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex comprises the following steps:

[0038] S1. Dissolve 1 mmol of γ-cyclodextrin and 8 mmol of potassium hydroxide in 30 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0039] S2. Add 100 mg of oleanolic acid to the crystallization mother solution obtained in S1, and then ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0040] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0041] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0042] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0043] Example 2

[0044] A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex comprises the following steps:

[0045] S1. Dissolve 1 mmol of γ-cyclodextrin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 8 mmol of potassium hydroxide in 30 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0046] S2. 80 mg of oleanolic acid (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added to the crystallization mother liquor obtained in S1, and then ultrasonicated for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0047] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0048] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0049] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0050] Example 3

[0051] A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex comprises the following steps:

[0052] S1. Dissolve 1 mmol of γ-cyclodextrin and 8 mmol of potassium hydroxide in 30 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0053] S2. Add 120 mg of oleanolic acid to the crystallization mother liquor obtained in S1, and then ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0054] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0055] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0056] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0057] Example 4

[0058] S1. Dissolve 1 mmol of γ-cyclodextrin and 8 mmol of potassium hydroxide in 30 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0059] S2. Add 140 mg of oleanolic acid to the crystallization mother liquor obtained in S1, and then ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0060] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0061] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0062] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0063] Example 5

[0064] S1. Dissolve 1 mmol of γ-cyclodextrin and 8 mmol of potassium hydroxide in 30 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0065] S2. Add 160 mg of oleanolic acid to the crystallization mother liquor obtained in S1, and then ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0066] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0067] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0068] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0069] Example 6

[0070] A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex comprises the following steps:

[0071] S1. Dissolve 0.5 mmol of γ-cyclodextrin and 4 mmol of potassium hydroxide in 15 mL of aqueous solution to obtain a crystallization mother solution of a cyclodextrin metal organic framework.

[0072] S2. Add 228.4 mg of oleanolic acid to the crystallization mother liquor obtained in S1, and then ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain an oleanolic acid / cyclodextrin / potassium hydroxide composite solution.

[0073] S3. Filter the composite solution prepared in S2 using a 0.45 μm water-based microporous filter membrane.

[0074] S4. Place the filtered composite solution obtained in S3 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0075] S5. The solution obtained in S4 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0076] Comparative Example 1

[0077] A method for preparing an oleanolic acid / γ-CD complex comprises the following steps:

[0078] S1. Dissolve 50 mg of oleanolic acid in 20 mL of methanol solution.

[0079] S2. Add 100 mg of γ-cyclodextrin to the solution obtained in S1, and then heat and stir in a water bath at 50°C with a stirring speed of 400 r / min for 4 hours.

[0080] S3. The suspension obtained after stirring S3 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum-dried at 45° C. for 6 h and then collected to obtain an oleanolic acid / γ-cyclodextrin complex (OA@γ-CD).

[0081] Comparative Example 2

[0082] A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex comprises the following steps:

[0083] S1. Dissolve 1 mmol of γ-cyclodextrin and 8 mmol of potassium hydroxide in 30 mL of aqueous solution, and ultrasonicate for 1 h at an ultrasonic power of 600 W to obtain a crystallization mother liquor of a cyclodextrin metal organic framework.

[0084] S2. Filter the crystallization mother liquor prepared in S1 through a 0.45 μm water-based microporous filter membrane.

[0085] S3. Place the filtered composite solution obtained in S2 into a sealed container with a volume of 2200 mL and containing 150 mL of methanol, and then place it in a constant temperature and humidity chamber and culture it at 45°C for 7 days.

[0086] S4. The solution obtained in S3 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and then vacuum dried at 45° C. for 6 h and then collected to obtain a cyclodextrin metal organic framework.

[0087] S5. Dissolve 50 mg of oleanolic acid in 20 mL of methanol solution.

[0088] S6. Add 100 mg of the cyclodextrin metal organic framework obtained in S4 to the solution obtained in S5, and then heat and stir in a water bath at 50°C with a stirring speed of 400 r / min for 4 hours.

[0089] S7. The suspension obtained after stirring S3 was centrifuged to obtain a precipitate, which was washed three times with an ethanol solution, and vacuum-dried at 45° C. for 6 h, and then collected to obtain an oleanolic acid / cyclodextrin metal organic framework complex.

[0090] Experimental Example 1

[0091] Determination of related properties of oleanolic acid / cyclodextrin metal organic framework composite (OA@CD-MOF)

[0092] (1) The OA@CD-MOF prepared in Example 1 was characterized using an optical microscope.

[0093] The results are as follows Figure 1 As shown, the oleanolic acid / cyclodextrin metal organic framework complex presents a micrometer-sized cubic structure, indicating that the encapsulation of oleanolic acid does not destroy the morphology and structure of the cyclodextrin metal organic framework.

[0094] (2) The OA@CD-MOF prepared in Examples 1 to 6 was characterized by Fourier transform infrared analysis (FTIR).

[0095] Test results such as Figure 2 As shown, it can be seen that although the amount of oleanolic acid added in Examples 1 to 6 is different, the infrared spectra of the prepared OA@CD-MOF are consistent as a whole. -1 The broad and strong absorption peak at 3000~2800cm is caused by the OH stretching vibration of the hydroxyl group on cyclodextrin. -1 The absorption peak at 1158 cm is the stretching vibration of carbon and hydrogen on the carbon skeleton. -1 、1080cm -1 and 1030cm -1 The absorption peak at is the stretching vibration of carbon and oxygen.

[0096] (3) The inventors detected the effects of different amounts of oleanolic acid added in the method of the present invention on the drug loading and yield of OA@CD-MOF.

[0097] The experimental results are as follows Figure 3 As shown, the amounts of oleanolic acid (OA) added to OA@CD-MOF prepared in Examples 1 to 5 were 80 mg, 100 mg, 120 mg, 140 mg and 160 mg, respectively. It was found that the drug loading of the oleanolic acid / cyclodextrin metal organic framework complex increased with the increase of the amount of oleanolic acid added, while the yield did not change significantly.

[0098] (4) In order to detect the solubility performance of OA@CD-MOF prepared by the method of the present invention, the apparent solubility of OA@CD-MOF prepared in Example 1, Example 6 and Comparative Example 2 was measured respectively. The OA@CD-MOF prepared in Example 1 was recorded as OA@CD-MOF-1, the OA@CD-MOF prepared in Example 6 was recorded as OA@CD-MOF-6, and the OA@CD-MOF prepared in Comparative Example 2 was recorded as OA@CD-MOF-D2. At the same time, the apparent solubility of OA@γ-CD prepared in Comparative Example 1 was detected, and the solubility performance of OA was used as the control group.

[0099] The results are as follows Figure 4As shown, after OA and CD-MOF were co-crystallized according to the preparation method provided by the present invention, the solubility of Example 1 was increased by 61.62 times (269.27±19.40μg / mL) compared with the solubility of OA in the control group (4.37μg / mL), and the solubility of Example 6 was increased by 109.81 times (479.89±32.52μg / mL); compared with the solubility of OA@γ-CD obtained by coating oleanolic acid with γ-cyclodextrin in Comparative Example 1 (25.26±3.51μg / mL), the solubility of OA@CD-MOF-1 prepared in Example 1 was increased by 109.81 times (479.89±32.52μg / mL). The solubility of OA@CD-MOF-6 prepared in Example 6 is 19 times that of OA@γ-CD; compared with the solubility of OA@CD-MOF-D2 obtained by loading OA on CD-MOF in Comparative Example 2 (141.28±4.52 μg / mL), the solubility of OA@CD-MOF-1 prepared in Example 1 is 1.91 times that of OA@CD-MOF-D2, and the solubility of OA@CD-MOF-6 prepared in Example 6 is 3.4 times that of OA@CD-MOF-D2.

[0100] (5) In order to detect the lipid-lowering activity of OA@CD-MOF prepared by the method of the present invention, since Examples 1 to 6 have similar effects, for the convenience of subsequent discussion and reference, the inventors selected OA@CD-MOF prepared in Example 1 to conduct animal experiments on anti-nonalcoholic fatty liver disease.

[0101] The zebrafish larvae were induced with a high-fat diet to establish a non-alcoholic fatty liver model. Five days after fertilization, the zebrafish larvae were randomly divided into five groups (n=100). Then, they were placed in a 6-well plate containing fish water and 0.1% dimethyl sulfoxide and fed with different feeds until 15 days after fertilization. Group 1 was fed with paramecium, group 2 was fed with egg yolk powder (1 mg / mL), group 3 was fed with egg yolk powder (1 mg / mL) with OA (4 μg / mL), group 4 was fed with egg yolk powder (1 mg / mL) with OA@CD-MOF (containing 4 μg / mL OA) prepared in Example 2, and group 5 was fed with egg yolk powder (1 mg / mL) with simvastatin (0.1 μg / mL) as a positive control. After the experiment, the zebrafish larvae were euthanized with chloroform sulfonic acid. ORO and BODIPY dyes were used to stain and quantify the lipids of the whole fish. Five zebrafish larvae were randomly selected from each group and stained with ORO or 5-fluorescein isothiocyanate, respectively, and the liver staining was observed under a fluorescence microscope (MShot, MF52-N). The color and fluorescence intensity were quantified using ImageJ 1.42 software (Bethesda, MD, USA).

[0102] The results are as follows Figure 5 As shown, Figure 5 The results of fluorescence staining and oil red staining in Figure A reveal the presence of fatty degeneration in the liver of zebrafish larvae. Compared with the CON group, the fluorescence staining intensity of zebrafish larvae in the HFD group ( Figure 5 Figure B) and ORO staining intensity ( Figure 5 C), which indicates that the zebrafish NAFLD model was successfully constructed. The fluorescence staining intensity of the three drug treatment groups was significantly reduced, indicating that OA, OA@CD-MOF and simvastatin can successfully reduce the high-lipid phenotype of zebrafish. Among them, the fluorescence staining intensity of the OA group (1.55) and the AC group (1.57) was similar, which was significantly stronger than the OA@CD-MOF group (0.65). The results suggest that OA@CD-MOF has a good effect in preventing fatty degeneration. Similar trends were also found in the analysis of ORO staining. In addition, the levels of total triglycerides (TG) and total cholesterol (TC) in the liver are important indicators for quantifying the degree of fat accumulation in the liver. Figure 5 The D graph and Figure 5 As shown in Figure E, the TG and TC levels in zebrafish larvae fed with HFD increased significantly, which were 11.57 times and 3.43 times that of the CON group, respectively. This indicates that HFD causes lipid metabolism disorders in zebrafish larvae, which in turn causes fat accumulation. Compared with the HFD group, the TG levels of zebrafish larvae with added drugs (OA, OA@CD-MOF and simvastatin) were reduced to 8.14 times, 6.71 times and 4.86 times that of the CON group, respectively. Similarly, the TC levels were reduced to 2.5 times, 1.86 times and 1.21 times that of the CON group, respectively. Although the liver fat level could not be restored to the level of the CON group, the three drug treatments could effectively alleviate the excessive accumulation of liver fat in NAFLD zebrafish larvae. OA@CD-MOF is significantly better than OA in reducing fat accumulation. This is consistent with the results of BODIPY fluorescence and ORO staining. In summary, the anti-fatty degeneration and anti-lipid accumulation effects of the oleanolic acid / cyclodextrin metal organic framework complex are stronger than those of oleanolic acid, indicating that the OA@CD-MOF provided by the present invention has a more superior lipid-lowering activity than oleanolic acid.

[0103] (6) In order to further detect the lipid-lowering activity of OA@CD-MOF provided by the present invention, the inventors selected OA@CD-MOF prepared in Example 1 to conduct an animal experiment on anti-nonalcoholic fatty liver disease.

[0104] C57BL / 6J mice were induced with a high-fat diet to establish a non-alcoholic fatty liver model. After 1 week of adaptive feeding, the mice were randomly divided into four groups (n=8). The control group was fed with normal feed; the high-fat model group, oleanolic acid group and OA@CD-MOF group prepared in Example 2 were fed with 60% Kca high-fat feed for 8 weeks, and after 8 weeks, they were continuously intervened with feed supplemented with OA and OA@CD-MOF for 4 weeks. During the dietary administration period, the weight of the mice was measured weekly, and blood glucose was tested in the last week.

[0105] The results are as follows Figure 6 As shown, CON is the normal feed group, HFD is the high-fat feed group, OA is the high-fat feed group and treated with oleanolic acid, and OA@CD-MOF is the high-fat feed group and treated with oleanolic acid / cyclodextrin metal organic framework complex. It can be seen that the body weight of mice in the oleanolic acid / cyclodextrin metal organic framework complex group was not significantly different from that in the oleanolic acid group, but the blood sugar was significantly lower than that in the oleanolic acid group.

[0106] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an oleanolic acid / cyclodextrin metal organic framework complex, characterized in that: The preparation method comprises the following steps: dissolving γ-cyclodextrin and alkaline potassium salt in water to obtain a crystallization mother solution of a cyclodextrin metal organic framework; Oleanolic acid is added to the crystallization mother liquor of cyclodextrin metal organic framework to obtain a mixed solution, the mixed solution is filtered and then an organic solvent is added, and the mixture is cultured at 25-50° C. for 5-14 days to allow oleanolic acid and cyclodextrin metal organic framework to co-crystallize and grow, thereby obtaining an oleanolic acid / cyclodextrin metal organic framework complex.

2. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The drug loading amount of the oleanolic acid / cyclodextrin metal organic framework composite to oleanolic acid is 2.76 wt % to 19.4 wt %.

3. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The molar ratio of the gamma-cyclodextrin to the alkaline potassium salt is 1:7.9-8.

1.

4. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The alkaline potassium salt is potassium hydroxide.

5. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The mixing method is ultrasonic mixing or mechanical stirring mixing.

6. The mixing method according to claim 5, characterized in that: The power of the ultrasound is 300-600W, and the treatment time is 40-60min.

7. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The organic solvent is methanol or ethanol, and the organic solvent is in liquid or vapor form.

8. The method for preparing the oleanolic acid / cyclodextrin metal organic framework complex according to claim 1, characterized in that: The co-crystal growth further includes centrifugation, crystal collection, washing and drying; The drying temperature is 35-50° C. and the drying time is 6-12 hours.

9. An oleanolic acid / cyclodextrin metal organic framework complex prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the oleanolic acid / cyclodextrin metal organic framework complex according to claim 9 in the preparation of medicines or health products, wherein the medicine is a medicine for preventing / treating non-alcoholic fatty liver disease.

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