A meglumine- luteolin complex, a preparation method and application thereof

Abstract

This invention discloses a meglumine-luteolin complex, its preparation method, and its applications, belonging to the interdisciplinary field of microbiome and drug delivery. Using luteolin as the main material, this invention employs a dual inclusion technique with hydroxypropyl-β-cyclodextrin and meglumine to synergistically prepare a meglumine-luteolin complex with colon-targeted delivery capabilities. This invention significantly improves the delivery efficiency and drug loading of luteolin in the colon through the dual inclusion technique. The preparation process is simple, and it exhibits excellent hypoglycemic effects and colon-targeted release characteristics in the treatment of diabetes and its complications.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microbiome and drug delivery, specifically relating to a meglumine-luteolin complex, its preparation method, and its application. Background Technology

[0002] Meglumine is an amino sugar derived from sorbitol, possessing excellent water solubility and biocompatibility. In the pharmaceutical field, meglumine is primarily used as a solubilizer and cosolvent, capable of forming soluble salts with various poorly soluble acidic drugs, significantly improving drug solubility and facilitating the preparation of carriers. Luteolin is a natural flavonoid compound widely found in vegetables, fruits, and various traditional Chinese medicines. Its molecular structure contains multiple phenolic hydroxyl groups, which give its aqueous solution a slightly acidic pH, facilitating the formation of a complex with meglumine. Studies have shown that luteolin can significantly improve insulin resistance, promote glucose uptake and utilization, regulate glucose and lipid metabolism disorders, and reduce oxidative stress and inflammatory responses in diabetic model animals.

[0003] Existing luteolin preparations face multiple technical bottlenecks in the treatment of diabetes, which severely restrict their clinical efficacy. In terms of drug delivery, conventional oral preparations have significant bioavailability barriers: (1) Luteolin is prone to C-ring opening degradation in the gastric acid environment, and in vitro simulation experiments show that the degradation rate is as high as 68±5% within 2 hours; (2) The colonic delivery efficiency of existing preparations is generally less than 5%. Clinical data show that after oral administration of 200 mg of luteolin, the local drug concentration in the colon is only 0.3-0.5 μg / g, which is far below the threshold concentration required to exert the effect of gut microbiota regulation. Although there are reports that luteolin can improve the α diversity of gut microbiota, metagenomic sequencing analysis shows that its selective regulatory ability on specific functional strains is limited, mainly due to the poor stability of luteolin in gastric juice. Therefore, developing a meglumine-luteolin complex with good solubility, high bioavailability, and excellent anti-diabetic activity is of great significance for overcoming the bottlenecks in the clinical application of luteolin and expanding its application in the field of diabetes treatment. Summary of the Invention

[0004] This invention provides a meglumine-luteolin complex, its preparation method, and its application. This method uses a combination of hydroxypropyl-β-cyclodextrin inclusion and meglumine inclusion technologies to solve the technical problems of low colonic delivery efficiency and insufficient drug loading of luteolin in the prior art.

[0005] (I) This invention provides a method for preparing a meglumine-luteolin complex, comprising the following steps:

[0006] Step a: Add luteolin and hydroxypropyl-β-cyclodextrin to distilled water and stir magnetically for a period of time at room temperature and in the dark to allow luteolin to be fully encapsulated by hydroxypropyl-β-cyclodextrin and form a preliminary inclusion complex dispersion system.

[0007] Step b: Add meglumine aqueous solution dropwise to the inclusion complex dispersion system obtained in step a, and continue stirring for a period of time after the addition is complete;

[0008] Step c: Transfer the mixture obtained in step b to a centrifuge tube, centrifuge in a refrigerated centrifuge, and collect the precipitate;

[0009] Step d, washing: Add deionized water to the precipitate after centrifugation in step c, resuspend and disperse, centrifuge again, and discard the washing liquid; repeat the washing operation multiple times to remove free meglumine, free luteolin and hydroxypropyl-β-cyclodextrin.

[0010] Step e: Place the precipitate obtained after washing in a freeze dryer for freeze drying to obtain meglumine-luteolin complex.

[0011] Furthermore, in step a, the molar ratio of luteolin to hydroxypropyl-β-cyclodextrin is 1:(1-2), and the amount of distilled water used is: 50-100 mL of distilled water for every 1 mmol of luteolin.

[0012] Furthermore, in step a, the magnetic stirring time is 24 hours.

[0013] Furthermore, in step b, the mass ratio of meglumine to luteolin is 1:(5-10); the concentration of the meglumine aqueous solution is 5-20 mg / mL; and the volume ratio of the inclusion complex dispersion system to the meglumine aqueous solution is 1:1 to 1:2.

[0014] Furthermore, in step b, stirring is continued for 30 minutes to promote dissolution and stabilize the complex.

[0015] Furthermore, in steps c and d, the centrifugation conditions of the refrigerated centrifuge are set as follows: 6000-8000 rpm / min, centrifugation for 10-15 minutes, and temperature set to 4℃.

[0016] Furthermore, in step e, the freeze-drying conditions are: freeze-drying at -50 ℃ for 48 hours.

[0017] (ii) The present invention provides a meglumine-luteolin complex prepared by the above preparation method.

[0018] (III) Application of the meglumine-luteolin complex of the present invention, wherein the meglumine-luteolin complex can be used to prepare drugs for treating type 2 diabetes, drugs for pancreatic islet dysfunction, and drugs for liver damage.

[0019] Beneficial effects:

[0020] 1. This invention significantly improves the water solubility of luteolin by introducing meglumine as an auxiliary ligand and employing hydroxypropyl-β-cyclodextrin inclusion technology.

[0021] 2. In vitro simulated release experiments showed that, in a simulated colonic environment (pH 7.4), the meglumine-luteolin complex prepared in this invention exhibited good colon-targeted release characteristics, with a cumulative release rate of up to 88.61±3.99% after 6 hours, which was significantly higher than that of the hydroxypropyl-β-cyclodextrin-luteolin complex, thus facilitating localized and efficient treatment of the drug in the colon.

[0022] 3. Animal pharmacodynamic experiments confirmed that the complex of this invention has a significant hypoglycemic effect on type 2 diabetic mice. After administration by gavage at a dose of 10 mg / kg, the fasting blood glucose of mice decreased significantly from 28.7 mmol / L to 10.1 mmol / L, a reduction of 64.8%, and the effect was significantly better than that of luteolin alone, indicating that the complex can effectively improve the in vivo absorption of drugs and enhance the hypoglycemic effect. Attached Figure Description

[0023] Figure 1 The images shown are scanning electron microscope (SEM) images of luteolin, meglumine, and meglumine-luteolin microspheres from Example 2; where A is luteolin, B is meglumine, and C is meglumine-luteolin microspheres.

[0024] Figure 2 This is a targeted release curve of the meglumine-luteolin complex and the hydroxypropyl-β-cyclodextrin-luteolin complex in the colon in Example 2.

[0025] Figure 3 The effects of meglumine-luteolin complex, hydroxypropyl-β-cyclodextrin-luteolin complex, and luteolin on fasting blood glucose levels, insulin resistance index, and pancreatic β-cell function index in type 2 diabetic mice are shown in Example 2. Wherein, A represents fasting blood glucose level; B represents insulin resistance index; and C represents pancreatic β-cell function index.

[0026] Figure 4 The effects of meglumine-luteolin complex, hydroxypropyl-β-cyclodextrin-luteolin complex, and luteolin on total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the liver of type 2 diabetic mice are shown in Example 2. In this example, A represents total cholesterol, B represents triglycerides, and C represents low-density lipoprotein cholesterol. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0028] In the examples: meglumine, purchased from Sigma-Aldrich, CAS No.: 6284-40-8, molecular formula: C7H 17 NO5, molecular weight: 195.21, structural formula is: ;

[0029] Luteolin, purchased from Nanjing Zelang Biotechnology Co., Ltd., CAS No.: 491-70-3, molecular formula: C 15 H 10 O6, molecular weight: 286.24, structural formula is: ;

[0030] Hydroxypropyl-β-cyclodextrin, purchased from Shandong Binzhou Zhiyuan Biotechnology Co., Ltd., CAS No.: 128446-35-5, average molecular weight approximately 1540, degree of substitution approximately 4-6. The structural formula is as follows: .

[0031] Example 1

[0032] 1. Preparation of meglumine-luteolin complex:

[0033] Step a: Accurately weigh 286.24 mg (1 mmol) of luteolin and 1.54 g (1 mmol) of hydroxypropyl-β-cyclodextrin according to a molar ratio of 1:1. At room temperature (25 ± 2 ℃), place the weighed luteolin and hydroxypropyl-β-cyclodextrin together in 50 mL of distilled water, start a magnetic stirrer, and stir continuously for 24 hours (away from light) to allow luteolin to fully enter the hollow structure of hydroxypropyl-β-cyclodextrin, forming a preliminary inclusion complex dispersion system.

[0034] Step b: Prepare a 5 mg / mL aqueous solution of meglumine. Take 10 mL of this solution (containing 50 mg of meglumine) and slowly add it dropwise to the 50 mL inclusion complex dispersion system obtained in step a. After the addition is complete, continue stirring at room temperature for 30 minutes to allow meglumine to fully exert its solubilizing effect. The amino group of meglumine can interact with the phenolic hydroxyl group of luteolin, further disrupting the crystal structure of the luteolin molecule, and utilizing its glucosyl structure to increase the overall hydration capacity of the complex, promoting the formation of the ternary complex. In step b: the mass ratio of meglumine to luteolin is 50:286.24 ≈ 1:5.72.

[0035] Step c: Transfer the final mixture obtained in step b to a centrifuge tube, place it in a refrigerated centrifuge, and centrifuge at 6000 rpm for 12 minutes at 4°C to ensure complete precipitation of the complex. Discard the supernatant and collect the precipitate.

[0036] Step d: Add 20 mL of deionized water to the centrifuged precipitate, and use a vortex mixer or glass rod to resuspend and disperse the precipitate. Centrifuge again at 4°C and 6000 rpm for 12 minutes, and discard the washing liquid. Repeat the above washing operation 3 times to fully remove unreacted free meglumine, free luteolin, and hydroxypropyl-β-cyclodextrin.

[0037] Step e: Spread the washed complex evenly in a petri dish and freeze-dry in a freeze dryer until the product is completely dry, obtaining the finished product of meglumine-luteolin complex (meglumine-luteolin microspheres). Freeze-drying conditions: -50 ℃ freeze-drying for 48 hours.

[0038] 2. Preparation method of hydroxypropyl-β-cyclodextrin-luteolin complex: Luteolin (286.24 mg) and hydroxypropyl-β-cyclodextrin (1.54 g) were weighed according to a molar ratio of luteolin to hydroxypropyl-β-cyclodextrin of 1:1, and added to 50 mL of distilled water. The mixture was stirred at room temperature for 24 hours (protected from light). The resulting suspension was centrifuged at 4℃ and 6000 rpm for 12 minutes. The precipitate was washed three times with 20 mL of deionized water (centrifuged under the same conditions), and then freeze-dried at -50℃ for 48 hours to obtain the final product.

[0039] 3. Water solubility test: Excess amounts of luteolin raw material, hydroxypropyl-β-cyclodextrin-luteolin complex, and meglumine-luteolin complex were added to 5 mL of distilled water, and the solutions were incubated at 25°C with shaking for 24 hours until saturation. The solutions were then filtered through a 0.45 μm filter membrane. The filtrate was appropriately diluted and the luteolin concentration was determined by high-performance liquid chromatography (HPLC). The results showed that the saturated solubility of luteolin raw material was 0.08 ± 0.01 mg / mL, that of hydroxypropyl-β-cyclodextrin-luteolin complex was 0.95 ± 0.08 mg / mL, and that of meglumine-luteolin complex was 2.35 ± 0.12 mg / mL (n=3).

[0040] 4. In vitro colon-targeted release experiment: The dialysis bag method was used. The meglumine-luteolin complex and the hydroxypropyl-β-cyclodextrin-luteolin complex (containing 10 mg of luteolin equivalent) of this invention were respectively placed in pretreated dialysis bags (molecular weight cutoff 3500 Da), sealed, and placed in 50 mL of simulated colon fluid (pH 7.4 phosphate buffer, containing 0.5% Tween 80 to maintain the leak conditions), and oscillated at 37℃ and 100 rpm. At predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24 h), 1 mL samples were taken, and isothermal fresh medium (simulated colon fluid) was added simultaneously. The luteolin concentration was determined by HPLC, and the cumulative release rate was calculated. The results showed that the cumulative release rate of the meglumine-luteolin complex reached 83.45±4.37% (n=3) after 6 hours, which was higher than the 52.46±5.84% of the control complex.

[0041] 5. Application of meglumine-luteolin complex in hyperglycemia, pancreatic function, and liver damage in type 2 diabetes:

[0042] Pharmacodynamic experiments were conducted using T2DM mice (db / db mice) to evaluate drug efficacy, and the mice were randomly divided into 4 groups (n=10 / group):

[0043] Control group: administered physiological saline by gavage;

[0044] Free luteolin group: luteolin 10 mg / kg (suspended in 0.5% CMC-Na);

[0045] Hydroxypropyl-β-cyclodextrin-luteolin complex group: equivalent to luteolin 10 mg / kg;

[0046] Meglumine-luteolin complex group: equivalent to luteolin 10 mg / kg.

[0047] Administer the medication once daily by gavage for four consecutive weeks. Experimental results showed:

[0048] (1) In terms of glucose metabolism:

[0049] The fasting blood glucose level in the meglumine-luteolin complex group decreased to 11.4±2.2 mmol / L, a 60.6% decrease compared to the control group (28.9±1.3 mmol / L), which was superior to the free luteolin group (22.1±1.3 mmol / L, a decrease of 23.5%) and compared to the hydroxypropyl-β-cyclodextrin-luteolin complex group (16.8±1.9 mmol / L, a decrease of 32.1%).

[0050] The insulin resistance index in the meglumine-luteolin complex group decreased to 15.37±2.4, a decrease of 49.4% compared with the control group (30.38±2.5); compared with the free luteolin group (24.15±2.11, a decrease of 36.4%); and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (19.83±2.32, a decrease of 22.5%).

[0051] The pancreatic β-cell function index in the meglumine-luteolin complex group increased to 30.84±2.72, an increase of 171.2% compared with the control group (11.37±1.16). Compared with the free luteolin group (17.56±1.89, an increase of 75.6%), and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (23.41±2.14, an increase of 31.7%).

[0052] (2) Regarding liver protection:

[0053] The total cholesterol content in the liver of the meglumine-luteolin complex group was 0.038±0.03 mg / g tissue, which was 80.9% lower than that of the control group (0.199±0.052 mg / g); lower than that of the free luteolin group (0.112±0.041 mg / g, a decrease of 66.1%); and lower than that of the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.075±0.035 mg / g, a decrease of 49.3%).

[0054] The liver triglyceride content in the meglumine-luteolin complex group was 0.159±0.028 mg / g tissue, which was 52.4% lower than that in the control group (0.334±0.043 mg / g); lower than that in the free luteolin group (0.261±0.037 mg / g, a decrease of 39.1%); and lower than that in the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.208±0.031 mg / g, a decrease of 23.6%).

[0055] The total cholesterol content of low-density lipoprotein in the liver of the meglumine-luteolin complex group was 0.0134±0.0032 mg / g tissue, which was 62.1% lower than that of the control group (0.0354±0.005 mg / g). Compared with the free luteolin group (0.0245±0.0041 mg / g, a decrease of 45.3%), and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.0189±0.0035 mg / g, a decrease of 29.1%), it was lower.

[0056] Example 2

[0057] 1. Preparation of meglumine-luteolin complex:

[0058] Step a: Accurately weigh 286.24 mg (1 mmol) of luteolin and 2.31 g (1.5 mmol) of hydroxypropyl-β-cyclodextrin according to a molar ratio of 1:1.5. At room temperature (25 ± 2 ℃), place the weighed luteolin and hydroxypropyl-β-cyclodextrin together in 100 mL of distilled water, start a magnetic stirrer, and stir continuously for 24 hours (away from light) to allow luteolin to fully enter the hollow structure of hydroxypropyl-β-cyclodextrin, forming a preliminary inclusion complex dispersion system.

[0059] Step b: Prepare a 10 mg / mL aqueous solution of meglumine. Take 4 mL of this solution (containing 40 mg of meglumine) and slowly add it dropwise to the 100 mL inclusion complex dispersion system obtained in step a. After the addition is complete, continue stirring at room temperature for 30 minutes to allow meglumine to fully exert its solubilizing effect. The amino group of meglumine can interact with the phenolic hydroxyl group of luteolin, further disrupting the crystal structure of the luteolin molecule, and utilizing its glucosyl structure to increase the overall hydration capacity of the complex, promoting the formation of the ternary complex. In step b: the mass ratio of meglumine to luteolin is 40:286.24 ≈ 1:7.16.

[0060] Step c: Transfer the final mixture obtained in step b to a centrifuge tube, place it in a refrigerated centrifuge, and centrifuge at 8000 rpm for 15 minutes at 4°C to ensure complete precipitation of the complex. Discard the supernatant and collect the precipitate.

[0061] Step d: Add 20 ml of deionized water to the centrifuged precipitate, and use a vortex mixer or glass rod to resuspend and disperse the precipitate. Centrifuge again at 4°C and 8000 rpm for 15 minutes, and discard the washing liquid. Repeat the above washing operation 3 times to fully remove unreacted free meglumine, free luteolin, and hydroxypropyl-β-cyclodextrin.

[0062] Step e: Spread the washed complex evenly in a petri dish and freeze-dry in a freeze dryer until the product is completely dry, obtaining the meglumine-luteolin complex product. Freeze-drying conditions: -50 ℃ for 48 hours.

[0063] Figure 1 The images shown are scanning electron microscope (SEM) images of luteolin, meglumine, and meglumine-luteolin microspheres from Example 2; where A is luteolin, B is meglumine, and C is meglumine-luteolin microspheres.

[0064] 2. Preparation method of hydroxypropyl-β-cyclodextrin-luteolin complex: Luteolin (286.24 mg, 1 mmol) and hydroxypropyl-β-cyclodextrin (2.31 g, 1.5 mmol) were accurately weighed according to a molar ratio of luteolin to hydroxypropyl-β-cyclodextrin of 1:1.5. The mixture was added to 100 mL of distilled water and stirred at room temperature for 24 hours (protected from light). The resulting suspension was centrifuged at 8000 rpm for 15 minutes at 4 °C. The precipitate was washed three times with 20 mL of deionized water (centrifuged under the same conditions) and then freeze-dried at -50 °C for 48 hours to obtain the final product.

[0065] 3. Water solubility test: Excess amounts of luteolin raw material, hydroxypropyl-β-cyclodextrin-luteolin complex, and the meglumine-luteolin complex of this invention were added to 5 mL of distilled water and shaken at 25°C for 24 hours until saturation. The mixture was then filtered through a 0.45 μm filter membrane. The filtrate was appropriately diluted and the luteolin concentration was determined by high-performance liquid chromatography (HPLC). The results showed that the saturated solubility of luteolin raw material was 0.08 ± 0.01 mg / mL, that of hydroxypropyl-β-cyclodextrin-luteolin complex was 0.97 ± 0.1 mg / mL, and that of meglumine-luteolin complex was 2.48 ± 0.15 mg / mL (n=3).

[0066] 4. In vitro colon-targeted release experiment: The dialysis bag method was used. The meglumine-luteolin complex and the hydroxypropyl-β-cyclodextrin-luteolin complex (containing 10 mg of luteolin equivalent) of the present invention were respectively placed in pretreated dialysis bags (molecular weight cutoff 3500 Da), sealed, and placed in 50 mL of simulated colon fluid (pH 7.4 phosphate buffer, containing 0.5% Tween 80 to maintain the leak conditions), and oscillated at 37℃ and 100 rpm. 1 mL samples were taken at predetermined time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), and isothermal fresh medium was added simultaneously. The luteolin concentration was determined by HPLC, and the cumulative release rate was calculated. The results showed that the cumulative release rate of the complex of the present invention reached 88.61±3.99% (n=3) after 6 hours, which was higher than the 61.23±4.12% of the control complex.

[0067] Figure 2 This is a targeted release curve of the meglumine-luteolin complex and the hydroxypropyl-β-cyclodextrin-luteolin complex in the colon in Example 2.

[0068] 5. Application of meglumine-luteolin complex in hyperglycemia, pancreatic function, and liver damage in type 2 diabetes:

[0069] Pharmacodynamic experiments were conducted using T2DM mice (db / db mice) to evaluate drug efficacy, and the mice were randomly divided into 4 groups (n=10 / group):

[0070] Control group: administered physiological saline by gavage;

[0071] Free luteolin group: luteolin 10 mg / kg (suspended in 0.5% CMC-Na);

[0072] Hydroxypropyl-β-cyclodextrin-luteolin complex group: equivalent to luteolin 10 mg / kg;

[0073] Meglumine-luteolin complex group: equivalent to luteolin 10 mg / kg.

[0074] Administer the medication once daily by gavage for four consecutive weeks. Experimental results showed:

[0075] (1) In terms of glucose metabolism:

[0076] The fasting blood glucose level in the meglumine-luteolin complex group decreased to 10.1±2.5 mmol / L, which was 64.8% lower than that in the control group (28.7±1.4 mmol / L), and was superior to that in the free luteolin group (20.4±1.6 mmol / L, a decrease of 50.5%); compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (15.3±1.6 mmol / L, a decrease of 34%).

[0077] The insulin resistance index in the meglumine-luteolin complex group decreased to 14.26±2.73, a decrease of 49.6% compared with the control group (28.32±3.36); compared with the free luteolin group (24.86±2.65, a decrease of 42.6%); and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (21.37±3.54, a decrease of 33.3%).

[0078] The pancreatic β-cell function index in the meglumine-luteolin complex group increased to 34.79±2.75, an increase of 203.3% compared with the control group (11.47±1.18). Compared with the free luteolin group (16.85±1.73, an increase of 106.5%), and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (24.58±2.75, an increase of 41.5%).

[0079] Figure 3 The effects of meglumine-luteolin complex, hydroxypropyl-β-cyclodextrin-luteolin complex, and luteolin on fasting blood glucose levels, insulin resistance index, and pancreatic β-cell function index in type 2 diabetic mice are shown in Example 2. Wherein, A represents fasting blood glucose level; B represents insulin resistance index; and C represents pancreatic β-cell function index.

[0080] (2) Regarding liver protection:

[0081] The total cholesterol content in the liver of the meglumine-luteolin complex group was 0.032±0.03 mg / g tissue, which was 82.6% lower than that of the control group (0.184±0.043 mg / g); lower than that of the free luteolin group (0.116±0.038 mg / g, a decrease of 72.4%); and lower than that of the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.071±0.028 mg / g, a decrease of 54.9%).

[0082] The liver triglyceride content in the meglumine-luteolin complex group was 0.147±0.028 mg / g tissue, which was 53% lower than that in the control group (0.313±0.034 mg / g); lower than that in the free luteolin group (0.268±0.041 mg / g, a decrease of 45.1%); and lower than that in the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.195±0.046 mg / g, a decrease of 24.6%).

[0083] The total cholesterol content of low-density lipoprotein in the liver of the meglumine-luteolin complex group was 0.0125±0.0024 mg / g tissue, which was 64% lower than that of the control group (0.0347±0.009 mg / g). It was also 55.5% lower than that of the free luteolin group (0.0281±0.0056 mg / g) and 22.8% lower than that of the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.0162±0.0043 mg / g).

[0084] Figure 4 The effects of meglumine-luteolin complex, hydroxypropyl-β-cyclodextrin-luteolin complex, and luteolin on total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the liver of type 2 diabetic mice are shown in Example 2. In this example, A represents total cholesterol, B represents triglycerides, and C represents low-density lipoprotein cholesterol.

[0085] Example 3

[0086] 1. Preparation of meglumine-luteolin complex:

[0087] Step a: Accurately weigh 286.24 mg (1 mmol) of luteolin and 3.08 g (2 mmol) of hydroxypropyl-β-cyclodextrin according to a molar ratio of 1:2. At room temperature (25 ± 2 ℃), place the weighed luteolin and hydroxypropyl-β-cyclodextrin together in 100 mL of distilled water, start a magnetic stirrer, and stir continuously for 24 hours (away from light) to allow luteolin to fully enter the hollow structure of hydroxypropyl-β-cyclodextrin, forming a preliminary inclusion complex dispersion system.

[0088] Step b: Prepare a 20 mg / mL aqueous solution of meglumine. Take 2 mL of this solution (containing 40 mg of meglumine) and slowly add it dropwise to the 100 mL inclusion complex dispersion system obtained in step a. After the addition is complete, continue stirring at room temperature for 30 minutes to allow meglumine to fully exert its solubilizing effect. The amino group of meglumine can interact with the phenolic hydroxyl group of luteolin, further disrupting the crystal structure of the luteolin molecule, and utilizing its glucosyl structure to increase the overall hydration capacity of the complex, promoting the formation of the ternary complex. In step b: the mass ratio of meglumine to luteolin is 40:286.24 ≈ 1:7.16.

[0089] Step c: Transfer the final mixture obtained in step b to a centrifuge tube, place it in a refrigerated centrifuge, and centrifuge at 7000 rpm for 10 minutes at 4°C to ensure complete precipitation of the complex. Discard the supernatant and collect the precipitate.

[0090] Step d: Add 20 mL of deionized water to the centrifuged precipitate, and use a vortex mixer or glass rod to resuspend and disperse the precipitate. Centrifuge again at 4°C and 7000 rpm for 10 minutes, and discard the washing liquid. Repeat the above washing operation 3 times to fully remove unreacted free meglumine, free luteolin, and hydroxypropyl-β-cyclodextrin.

[0091] Step e: Spread the washed complex evenly in a petri dish and freeze-dry in a freeze dryer until the product is completely dry, obtaining the meglumine-luteolin complex product. Freeze-drying conditions: -50 ℃ for 48 hours.

[0092] 2. Preparation method of hydroxypropyl-β-cyclodextrin-luteolin complex: Luteolin (286.24 mg, 1 mmol) and hydroxypropyl-β-cyclodextrin (3.08 g, 2 mmol) were accurately weighed according to a molar ratio of luteolin to hydroxypropyl-β-cyclodextrin of 1:2. They were added to 100 mL of distilled water and stirred at room temperature for 24 hours (protected from light). The resulting suspension was centrifuged at 7000 rpm for 10 minutes at 4 °C. The precipitate was washed three times with 20 mL of deionized water (centrifuged under the same conditions) and then freeze-dried at -50 °C for 48 hours to obtain the final product.

[0093] 3. Water solubility test: Excess amounts of luteolin raw material, hydroxypropyl-β-cyclodextrin-luteolin complex, and the meglumine-luteolin complex of this invention were added to 5 mL of distilled water and shaken at 25°C for 24 hours until saturation. The mixture was then filtered through a 0.45 μm filter membrane. The filtrate was appropriately diluted and the luteolin concentration was determined by high-performance liquid chromatography (HPLC). The results showed that the saturated solubility of luteolin raw material was 0.08 ± 0.01 mg / mL, that of hydroxypropyl-β-cyclodextrin-luteolin complex was 0.91 ± 0.13 mg / mL, and that of the meglumine-luteolin complex of this invention was 2.38 ± 0.17 mg / mL (n=3).

[0094] 4. In vitro colon-targeted release experiment: The dialysis bag method was used. The meglumine-luteolin complex and the hydroxypropyl-β-cyclodextrin-luteolin complex (containing 10 mg of luteolin equivalent) of this embodiment were respectively placed in pretreated dialysis bags (molecular weight cutoff 3500 Da), sealed, and placed in 50 mL of simulated colon fluid (pH 7.4 phosphate buffer, containing 0.5% Tween 80 to maintain the leak conditions), and oscillated at 37℃ and 100 rpm. 1 mL samples were taken at predetermined time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), and isothermal fresh medium was added simultaneously. The luteolin concentration was determined by HPLC, and the cumulative release rate was calculated. The results showed that the cumulative release rate of the complex of this invention reached 85.74±4.18% (n=3) after 6 hours, which was higher than the 55.47±5.34% of the control complex.

[0095] 2. Application of meglumine-luteolin complex in hyperglycemia, pancreatic function, and liver damage in type 2 diabetes:

[0096] Pharmacodynamic experiments: The efficacy was evaluated using T2DM mice (db / db mice), which were randomly divided into 4 groups (n=10 / group):

[0097] T2DM mice were constructed and their efficacy evaluated. They were randomly divided into 4 groups (n=10 / group):

[0098] Control group: administered physiological saline by gavage;

[0099] Free luteolin group: luteolin 10 mg / kg;

[0100] Hydroxypropyl-β-cyclodextrin-luteolin complex group: equivalent to luteolin 10 mg / kg;

[0101] The meglumine-luteolin complex of the present invention is equivalent to 10 mg / kg of luteolin.

[0102] Administer the medication once daily by gavage for four consecutive weeks. Experimental results showed:

[0103] (1) In terms of glucose metabolism:

[0104] The fasting blood glucose level in the meglumine-luteolin complex group decreased to 11.7±2.9 mmol / L, a 58.9% decrease compared to the control group (28.5±1.6 mmol / L), which was superior to the free luteolin group (21.7±1.8 mmol / L, a 46.1% decrease); and compared to the hydroxypropyl-β-cyclodextrin-luteolin complex group (16.4±2.1 mmol / L, a 28.7% decrease).

[0105] The insulin resistance index in the meglumine-luteolin complex group decreased to 15.58±2.51, a decrease of 47.7% compared with the control group (29.79±3.91); compared with the free luteolin group (24.58±2.85, a decrease of 36.6%); and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (20.86±3.22, a decrease of 25.3%).

[0106] The pancreatic β-cell function index in the meglumine-luteolin complex group increased to 32.47±3.06, an increase of 186.8% compared with the control group (11.32±1.52). Compared with the free luteolin group (17.29±1.96, an increase of 87.8%), and compared with the hydroxypropyl-β-cyclodextrin-luteolin complex group (24.08±2.53, an increase of 34.8%).

[0107] (2) Regarding liver protection:

[0108] The total cholesterol content in the liver of the meglumine-luteolin complex group was 0.036±0.027 mg / g tissue, which was 81.3% lower than that of the control group (0.193±0.048 mg / g); lower than that of the free luteolin group (0.115±0.043 mg / g, a decrease of 68.7%); and lower than that of the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.073±0.031 mg / g, a decrease of 50.7%).

[0109] The liver triglyceride content in the meglumine-luteolin complex group was 0.153±0.026 mg / g tissue, which was 53.2% lower than that in the control group (0.327±0.038 mg / g); lower than that in the free luteolin group (0.265±0.039 mg / g, a decrease of 42.3%); and lower than that in the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.201±0.039 mg / g, a decrease of 23.9%).

[0110] The total low-density lipoprotein cholesterol (LDL-C) content in the liver of the meglumine-luteolin complex group was 0.0131±0.0029 mg / g tissue, a decrease of 63.2% compared to the control group (0.0356±0.008 mg / g). This was also a decrease of 50.2% compared to the free luteolin group (0.0263±0.0049 mg / g) and a decrease of 24.3% compared to the hydroxypropyl-β-cyclodextrin-luteolin complex group (0.0173±0.0039 mg / g).

[0111] Examples 1-3 fully demonstrate that the complex of the present invention significantly improves the effect of luteolin on hyperglycemia, pancreatic function and liver damage in type 2 diabetes by enhancing the stability of luteolin in the gastrointestinal tract.

[0112] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A method for preparing a meglumine-luteolin complex, characterized in that, Includes the following steps: Step a: Add luteolin and hydroxypropyl-β-cyclodextrin to distilled water and stir magnetically for a period of time at room temperature and in the dark to allow luteolin to be fully encapsulated by hydroxypropyl-β-cyclodextrin and form a preliminary inclusion complex dispersion system. Step b: Add meglumine aqueous solution dropwise to the inclusion complex dispersion system obtained in step a, and continue stirring for a period of time after the addition is complete; Step c: Transfer the mixture obtained in step b to a centrifuge tube, centrifuge in a refrigerated centrifuge, and collect the precipitate; Step d, washing: Add deionized water to the precipitate after centrifugation in step c, resuspend and disperse, centrifuge again, and discard the washing liquid; repeat the washing operation multiple times to remove free meglumine, free luteolin and hydroxypropyl-β-cyclodextrin. Step e: Place the precipitate obtained after washing in a freeze dryer for freeze drying to obtain meglumine-luteolin complex.

2. The preparation method according to claim 1, characterized in that, In step a, the molar ratio of luteolin to hydroxypropyl-β-cyclodextrin is 1:(1-2), and the amount of distilled water used is: 50-100 mL of distilled water for every 1 mmol of luteolin.

3. The preparation method according to claim 1, characterized in that, In step a, the magnetic stirring time is 24 hours; in step b, the stirring time is 30 minutes.

4. The preparation method according to claim 1, characterized in that, In step b, the mass ratio of meglumine to luteolin is 1:(5-10); the concentration of the meglumine aqueous solution is 5-20 mg / mL; and the volume ratio of the inclusion complex dispersion system to the meglumine aqueous solution is 1:1 to 1:

2.

5. The preparation method according to claim 1, characterized in that, In steps c and d, the centrifugation conditions of the refrigerated centrifuge are set as follows: 4℃, 6000-8000 rpm / min, centrifugation for 10-15 minutes.

6. The preparation method according to claim 1, characterized in that, In step e, the freeze-drying conditions are: freeze-drying at -50℃ for 48 hours.

7. The meglumine-luteolin complex prepared by the preparation method according to any one of claims 1-6.

8. The use of the meglumine-luteolin complex of claim 7 in the preparation of a medicament for treating type 2 diabetes.

9. The use of the meglumine-luteolin complex of claim 7 in the preparation of a drug for treating pancreatic islet dysfunction.

10. The use of the meglumine-luteolin complex of claim 7 in the preparation of a drug for treating liver injury.

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