Liver targeting porous starch-luteolin compound as well as preparation method and application thereof

Abstract

The invention discloses a liver targeting porous starch-luteolin compound and a preparation method and application thereof, and belongs to the technical field of drug delivery, corn porous starch is used as a carrier, and the compound with the liver targeting function is prepared through OSA hydrophobic modification, luteolin loading and epsilon-polylysine-galactoside targeting modification. The drug loading capacity and liver targeting property of luteolin are remarkably improved through triple modification, the production process is simple, the cost is low, and excellent blood sugar reducing and liver protecting effects are shown in diabetes liver injury treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery, and particularly relates to a liver-targeted porous starch-luteolin complex, a preparation method and applications thereof. Background Art

[0002] Luteolin, a potential treatment for diabetic liver damage, faces significant delivery barriers: its extremely poor water solubility (only 0.01 mg / mL at 25°C) results in an oral bioavailability of less than 5%. Furthermore, due to a significant first-pass effect in the liver, the liver accumulation rate of standard formulations is less than 2%. Existing delivery technologies, such as cyclodextrin inclusion compounds, can improve solubility but lack organ targeting, making it impossible to achieve the required liver drug concentrations (≥5 μg / g tissue) for treatment. Furthermore, luteolin's poor gastrointestinal stability makes it difficult for existing delivery systems to achieve both sustained intestinal release and liver targeting, severely limiting its clinical application.

[0003] Traditional porous starch carriers exhibit significant limitations in drug delivery: relying solely on physical adsorption results in drug loadings generally below 5%, and unmodified starch surfaces are unable to achieve active liver targeting, with liver uptake efficiency less than 3%. Furthermore, due to the uneven pore size distribution (10-500 nm), these carriers suffer from severe burst drug release (>40% release in simulated intestinal fluid within 2 hours), failing to maintain stable blood drug concentrations or meet the sustained drug exposure required for the treatment of diabetic liver injury. Existing technologies have neither developed high-drug-loading porous starch carriers suitable for luteolin nor addressed the key challenges of liver-targeted delivery, which has become a major bottleneck restricting the improvement of the drug's therapeutic efficacy. Summary of the Invention

[0004] The purpose of the present invention is to provide a liver-targeted porous starch-luteolin complex, a preparation method and its application. The present invention achieves liver-specific delivery through hydrophobic modification and galactoside modification, solving the technical problems of low bioavailability and insufficient liver accumulation of luteolin in the prior art.

[0005] Technical solution: A method for preparing a liver-targeted porous starch-luteolin complex, the preparation method comprising:

[0006] Using corn porous starch as a carrier, a liver-targeted porous starch-luteolin complex was obtained through hydrophobic modification with octenylsuccinic anhydride (OSA), loading of luteolin and targeting modification with ε-polylysine-galactoside (Gal-PLL).

[0007] Furthermore, the preparation method specifically comprises the following steps:

[0008] Step a, dispersing corn porous starch in a pH 8.5 borate buffer, adding octenylsuccinic anhydride (OSA), reacting at 35° C. for 6 hours, and washing with ethanol to obtain hydrophobically modified porous starch;

[0009] Step b, dissolving luteolin in 30% ethanol solution, and co-loading it with the hydrophobically modified porous starch obtained in step a at 50° C. under shaking conditions for 2 hours to obtain a drug-loaded intermediate;

[0010] Step c: dispersing the drug-loaded intermediate in deionized water, adding ε-polylysine-galactoside (Gal-PLL) solution, and spray-drying after electrostatic adsorption at 25° C. for 1 hour to obtain a liver-targeted porous starch-luteolin complex.

[0011] Furthermore, in step a, n(OSA):n(starch glucose unit)=1:20-1:50;

[0012] In step b, m(luteolin):m(hydrophobically modified porous starch)=1:3-1:5;

[0013] In step c, m(Gal-PLL):m(drug-loaded intermediate)=1:10-1:20.

[0014] Furthermore, in the drug-loaded intermediate obtained in step b, the drug loading amount = m(luteolin):m(drug-loaded intermediate) = 15-25%.

[0015] The liver-targeted porous starch-luteolin complex prepared by the present invention can be used to prepare a drug for preventing or treating abnormal liver function caused by type 2 diabetes.

[0016] The beneficial effects of the present invention are:

[0017] 1) The hydrophobic modification of OSA significantly enhances the binding ability to luteolin, with a drug loading capacity of 22.5±0.6%, which is 4.4 times that of traditional porous starch carriers;

[0018] 2) The Gal-PLL targeting molecule specifically recognizes the asialoglycoprotein receptor on the surface of hepatocytes, increasing the liver accumulation rate to 15.7%, an 8-fold increase compared to the unmodified vector;

[0019] 3) Within 6 hours, the release rate of luteolin in simulated gastric fluid was 28.4%, and the release rate in simulated small intestinal fluid was 92.9%, showing an ideal sustained-release curve;

[0020] 4) Animal experiments have confirmed that compared with luteolin, this complex can significantly lower the fasting blood glucose level of diabetic mice, improve pancreatic β-cell function, and reduce total cholesterol, liver triglycerides and liver low-density lipoprotein cholesterol levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are scanning electron micrographs of luteolin, porous starch, and porous starch-luteolin complex in Example 2 at a 5000x field of view; (A) is a scanning electron micrograph of luteolin, (B) is a scanning electron micrograph of porous starch, and (C) is a scanning electron micrograph of the porous starch-luteolin complex.

[0022] Figure 2 The release curves of luteolin from the porous starch-luteolin complex prepared in Example 2 in simulated gastric fluid and simulated small intestinal fluid, respectively; (A) is the release curve of luteolin in simulated gastric fluid, and (B) is the release curve of luteolin in simulated small intestinal fluid;

[0023] Figure 3 The porous starch-luteolin complex prepared in Example 2 affects the fasting blood glucose level, insulin resistance index and pancreatic β-cell function index of type 2 diabetic mice; wherein, A represents the effect on the fasting blood glucose level, B represents the effect on the insulin resistance index, and C represents the effect on the pancreatic β-cell function index.

[0024] Figure 4 The effects of the porous starch-luteolin complex prepared in Example 2 on total cholesterol, liver triglycerides and liver low-density lipoprotein cholesterol in type 2 diabetic mice; wherein A is the effect on total cholesterol, B is the effect on liver triglycerides, and C is the effect on liver low-density lipoprotein cholesterol. DETAILED DESCRIPTION

[0025] The technical solutions of the present invention are described in detail below through examples, but the protection scope of the present invention is not limited to the examples.

[0026] In the following examples, luteolin, corn-derived porous starch, borate buffer, OSA, ethanol, Gal-PLL and other drugs were purchased from Anaiji Chemical Technology (Shanghai) Co., Ltd.

[0027] Example 1

[0028] 1. Preparation of liver-targeted porous starch-luteolin complex:

[0029] Step a: 10 g of corn-derived porous starch (pore size 60 ± 3 nm) was placed in a 250 mL three-necked flask. 100 mL of 0.1 M borate buffer (pH 8.5) was added and ultrasonically dispersed for 15 minutes until uniformly suspended. OSA was added to the suspension at a ratio of n(OSA):n(starch glucose units) of 1:20. The mixture was stirred in a 35°C water bath for 6 hours. After the reaction, the precipitate was collected by centrifugation, washed three times with 70% ethanol until the eluate reached a neutral pH, and dried under vacuum at 60°C for 12 hours to obtain hydrophobically modified porous starch.

[0030] Step b: Dissolve 2 g of luteolin in 66.7 mL of 30% ethanol in water and heat in a 50°C water bath with stirring until completely dissolved. Add the hydrophobically modified porous starch obtained in step a at a ratio of m(luteolin):m(hydrophobically modified porous starch) of 1:3, and shake at 50°C for 2 hours. Remove unbound drug by centrifugation, wash the precipitate with deionized water, and freeze-dry to obtain a drug-loaded intermediate with a drug loading of 16.1±0.4%.

[0031] Step c: 0.5 g of the drug-loaded intermediate was redispersed in 50 mL of deionized water, and a Gal-PLL solution (10 mg / mL, pH 5.0 acetate buffer) was slowly added dropwise at a m(Gal-PLL):m(drug-loaded intermediate) ratio of 1:10. Electrostatic adsorption was performed at 25°C for 1 hour. The final product was dried using a spray dryer with the following parameters: inlet temperature 130°C, outlet temperature 70°C, and feed rate 5 mL / min. The final product was injected intravenously into 4-week-old healthy male mice. The product reached the liver through the bloodstream and bound to the asialoglycoprotein receptor on the surface of hepatocytes. After autopsy, the luteolin content in the liver tissue was determined by liquid chromatography, revealing a liver accumulation rate of 10.4%.

[0032] 2. Application of liver-targeted porous starch-luteolin complex in diabetic liver injury:

[0033] Four-week-old healthy male mice were selected and fed a high-fat diet for four weeks to induce insulin resistance. This diet, combined with a low-dose of streptozotocin, destroyed pancreatic beta cells to establish a type 2 diabetes model. Furthermore, while inducing type 2 diabetes, the high-fat diet and streptozotocin can cause liver dysfunction, manifested as abnormal liver function indicators. The type 2 diabetic mice were randomly divided into a control group (n=10 / group, saline), a free luteolin group (n=10 / group, 10 mg / kg), and a group containing the compound of the present invention (n=10 / group, equivalent to luteolin 10 mg / kg). The mice were administered orally once daily for four consecutive weeks for efficacy evaluation. The experimental results showed:

[0034] like Figure 3As shown, in terms of glucose metabolism, the fasting blood glucose in the complex group dropped to 11.8±4.2 mmol / L, a decrease of 40.1% compared with the control group (19.7±1.9 mmol / L) (p<0.01), which was significantly better than the free luteolin group (14.1±1.4 mmol / L, a decrease of 16.3%); at the same time, the insulin resistance index dropped to 15.7±3.2, a decrease of 30.2% compared with the control group (22.5±2.1); the pancreatic β-cell function index increased to 18.64±3.247, an increase of 60.6% compared with the control group (11.61±1.135).

[0035] like Figure 4 As shown, in terms of liver protection, the total cholesterol content in the liver of the complex group was 0.051±0.04 mg / g tissue, which was 67.9% lower than that in the control group (0.159±0.054 mg / g) (p<0.01); the triglyceride content in the liver was 0.169±0.025 mg / g tissue, which was 41.3% lower than that in the control group (0.288±0.037 mg / g) (p<0.01); and the total low-density lipoprotein cholesterol content in the liver was 0.0134±0.0056 mg / g tissue, which was 55.2% lower than that in the control group (0.0299±0.007 mg / g) (p<0.01).

[0036] Example 2

[0037] 1. Preparation of liver-targeted porous starch-luteolin complex:

[0038] Step a: 10 g of corn-derived porous starch (pore size 60 ± 3 nm) was placed in a 250 mL three-necked flask. 100 mL of 0.1 M borate buffer (pH 8.5) was added and ultrasonically dispersed for 15 minutes until uniformly suspended. OSA was added to the suspension at a ratio of n(OSA):n(starch glucose units) of 1:30. The mixture was stirred in a 35°C water bath for 6 hours. After the reaction, the precipitate was collected by centrifugation, washed three times with 70% ethanol until the eluate reached a neutral pH, and dried in a vacuum oven at 6°C for 12 hours to obtain the hydrophobically modified porous starch.

[0039] Step b: Dissolve 2 g of luteolin in 66.7 mL of 30% ethanol in water, heat in a 50°C water bath, and stir until completely dissolved. The hydrophobically modified porous starch obtained in step a was prepared with a ratio of m(luteolin):m(hydrophobically modified porous starch) of 1:4. Shake the mixture at 50°C for 2 hours. Remove any unbound drug by centrifugation, wash the precipitate with deionized water, and freeze-dry to obtain a drug-loaded intermediate with a drug loading of 22.5 ± 0.6%.

[0040] Step c: 0.8 g of the drug-loaded intermediate was re-dispersed in 50 mL of deionized water, and a Gal-PLL solution (10 mg / mL, pH 5.0 acetic acid buffer) was slowly added dropwise, m(Gal-PLL):m(drug-loaded intermediate) = 1:16, and electrostatic adsorption was performed at 25°C for 1 hour. Drying was performed using a spray dryer, with the following parameters: inlet temperature 130°C, outlet temperature 70°C, and feeding rate 5 mL / min, to obtain the final product. The final product was injected into mice (4-week-old healthy male mice) by intravenous injection, circulated to the liver through the blood, and combined with the asialoglycoprotein receptor on the surface of liver cells. After dissection, the content of luteolin in the liver tissue was determined by liquid chromatography, and the liver accumulation rate was 15.7% at this time.

[0041] 2. Application of the liver-targeting porous starch-luteolin complex in diabetic liver injury:

[0042] 4-week-old healthy male mice were selected, and insulin resistance was induced by a high-fat diet for 4 weeks, and then combined with a small dose of streptozotocin to destroy the islet β cells, to construct a type 2 diabetes model. In addition, during the induction of type 2 diabetes, a high-fat diet and streptozotocin can cause liver function disorder, manifested as abnormal liver function indicators. The type 2 diabetic mice were randomly divided into a control group (n=10 / group, normal saline), a free luteolin group (n=10 / group, 10 mg / kg), and a complex group (n=10 / group, equivalent luteolin 10 mg / kg) of the application, and the drug was administered once a day for 4 consecutive weeks for efficacy evaluation. The experimental results show that:

[0043] As shown in Figure 3 in terms of glucose metabolism, the fasting blood glucose of the complex group decreased to 10.4±2.3 mmol / L, which was 47.2% lower than that of the control group (19.7±1.9 mmol / L) (p<0.01), and was significantly better than that of the free luteolin group (14.1±1.4 mmol / L, a decrease of 26.2%); at the same time, the insulin resistance index decreased to 13.1±2.3, which was 41.8% lower than that of the control group (22.5±2.1); and the islet β cell function index increased to 24.94±2.714, which was 114.8% higher than that of the control group (11.61±1.135).

[0044] As shown in Figure 4As shown, in terms of liver protection, the total cholesterol content in the liver of the complex group was 0.034±0.02 mg / g tissue, which was 78.6% lower than that in the control group (0.159±0.054 mg / g) (p<0.01); the triglyceride content in the liver was 0.139±0.023 mg / g tissue, which was 51.7% lower than that in the control group (0.288±0.037 mg / g) (p<0.01); and the total low-density lipoprotein cholesterol content in the liver was 0.0104±0.0029 mg / g tissue, which was 65.2% lower than that in the control group (0.0299±0.007 mg / g) (p<0.01).

[0045] Figure 1 The scanning electron microscope images of luteolin, porous starch, and porous starch-luteolin complex in Example 2 at 5000 times magnification are shown. A is the scanning electron microscope image of luteolin, B is the scanning electron microscope image of porous starch, and C is the scanning electron microscope image of porous starch-luteolin complex. Figure 1 It can be clearly seen in Figure B that there are a lot of pores around the porous starch. Figure 1 In Figure C, it can be seen that luteolin has been adsorbed inside and on the outer wall of the pores of porous starch, indicating that luteolin has been successfully adsorbed by the porous starch.

[0046] Example 3

[0047] 1. Preparation of liver-targeted porous starch-luteolin complex:

[0048] Step a: 10 g of corn-derived porous starch (pore size 60 ± 3 nm) was placed in a 250 mL three-necked flask. 100 mL of 0.1 M borate buffer (pH 8.5) was added and ultrasonically dispersed for 15 minutes until a homogeneous suspension was achieved. OSA was added to the suspension at a ratio of n(OSA):n(starch glucose units) of 1:50. The mixture was stirred in a 35°C water bath for 6 hours. After the reaction, the precipitate was collected by centrifugation, washed three times with 70% ethanol until the eluate reached a neutral pH, and dried under vacuum at 60°C for 12 hours to obtain the hydrophobically modified porous starch.

[0049] Step b: Dissolve 2 g of luteolin in 66.7 mL of 30% ethanol in water. Heat in a 50°C water bath with stirring until completely dissolved. The hydrophobically modified porous starch obtained in step a was loaded at a ratio of m(luteolin):m(hydrophobically modified porous starch) of 1:5. Shake and incubate at 50°C for 2 hours. Remove unbound drug by centrifugation. Wash the precipitate with deionized water and freeze-dry to obtain the drug-loaded intermediate with a drug loading of 19.6 ± 0.7%.

[0050] Step c: 1 g of the drug-loaded intermediate was redispersed in 50 mL of deionized water, and a Gal-PLL solution (10 mg / mL, pH 5.0 acetate buffer) was slowly added dropwise at a m(Gal-PLL):m(drug-loaded intermediate) ratio of 1:20. Electrostatic adsorption was performed at 25°C for 1 hour. The final product was dried using a spray dryer with the following parameters: an inlet temperature of 130°C, an outlet temperature of 70°C, and a feed rate of 5 mL / min. The final product was injected intravenously into mice, where it reached the liver through the bloodstream and bound to the asialoglycoprotein receptor on the surface of hepatocytes. After autopsy, the luteolin content in the liver tissue was determined by liquid chromatography, revealing a liver accumulation rate of 13.9%.

[0051] 2. Application of liver-targeted porous starch-luteolin complex in diabetic liver injury:

[0052] Four-week-old healthy male mice were selected and fed a high-fat diet for four weeks to induce insulin resistance. This diet, combined with a low-dose of streptozotocin, destroyed pancreatic beta cells to establish a type 2 diabetes model. Furthermore, while inducing type 2 diabetes, the high-fat diet and streptozotocin can cause liver dysfunction, manifested as abnormal liver function indicators. The type 2 diabetic mice were randomly divided into a control group (n=10 / group, saline), a free luteolin group (n=10 / group, 10 mg / kg), and a group containing the compound of the present invention (n=10 / group, equivalent to luteolin 10 mg / kg). The mice were administered orally once daily for four consecutive weeks for efficacy evaluation. The experimental results showed:

[0053] like Figure 3 As shown in the results, in terms of glucose metabolism, the fasting blood glucose level in the complex group dropped to 11.2±3.1 mmol / L, a decrease of 43.1% compared with the control group (19.7±1.9 mmol / L) (p<0.01), which was significantly better than that in the free luteolin group (14.1±1.4 mmol / L, a decrease of 20.6%). At the same time, the insulin resistance index dropped to 14.7±2.8, a decrease of 34.7% compared with the control group (22.5±2.1); the pancreatic β-cell function index increased to 20.51±3.247, an increase of 76.7% compared with the control group (11.61±1.135).

[0054] like Figure 4As shown, in terms of liver protection, the total cholesterol content in the liver of the complex group was 0.037±0.03 mg / g tissue, which was 76.7% lower than that in the control group (0.159±0.054 mg / g) (p<0.01); the triglyceride content in the liver was 0.148±0.029 mg / g tissue, which was 48.6% lower than that in the control group (0.288±0.037 mg / g) (p<0.01); and the total low-density lipoprotein cholesterol content in the liver was 0.0126±0.0043 mg / g tissue, which was 57.9% lower than that in the control group (0.0299±0.007 mg / g) (p<0.01).

[0055] Example 4

[0056] The porous starch-luteolin complex prepared in Example 2 was placed in simulated gastric fluid and simulated small intestinal fluid, respectively, to study the release effect of luteolin. The experimental process is as follows:

[0057] Weigh 0.2 g of sodium chloride and dissolve it in 80 mL of deionized water. Add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 1.2-1.5. Add 3.2 g of pepsin and dilute to 100 mL with deionized water. Filter and sterilize to obtain simulated gastric fluid.

[0058] Dissolve 0.68 g of potassium dihydrogen phosphate in 800 mL of deionized water, adjust the pH to 6.8 with 1 mol / L sodium hydroxide solution, add 10 g of trypsin and 0.5 g of bile salts, dilute to 1000 mL with deionized water, and filter sterilize to obtain simulated small intestinal fluid.

[0059] 0.5 g of porous starch-luteolin complex was added to 100 mL of simulated gastric fluid and simulated small intestinal fluid, respectively. Samples were taken at 1, 2, 3, 4, 5, and 6 hours to detect the quality of luteolin, and the release rate was calculated as m(luteolin) / m(porous starch-luteolin complex).

[0060] The results are as follows Figure 2 As shown, Figure 2 These are the release curves of luteolin from the porous starch-luteolin complex prepared in Example 2 in simulated gastric fluid and simulated small intestinal fluid, respectively, where A is the release curve of luteolin in simulated gastric fluid, and B is the release curve of luteolin in simulated small intestinal fluid.

[0061] The results showed that the porous starch-luteolin complex had a low release rate in gastric fluid, with a release rate of less than 10% in the second hour, but a higher release rate in the small intestine, exceeding 40% in the second hour. Within 6 hours, the release rate of luteolin in simulated gastric fluid was 28.4%, and in simulated small intestinal fluid was 92.9%, demonstrating an ideal sustained-release curve.

[0062] In Example 2, hydrophobic modification of OSA significantly enhanced its binding to luteolin, achieving a drug loading of 22.5±0.6%. The Gal-PLL targeting molecule specifically recognized the asialoglycoprotein receptor on the surface of hepatocytes, increasing the liver accumulation rate to 15.7%. These data demonstrate that the complex of the present invention significantly enhances the therapeutic efficacy of luteolin against diabetic liver damage by improving liver targeting and sustained-release properties.

[0063] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing a liver-targeted porous starch-luteolin complex, characterized in that: Using corn porous starch as a carrier, a liver-targeted porous starch-luteolin complex was obtained through hydrophobic modification with octenylsuccinic anhydride, loading of luteolin and targeted modification with ε-polylysine-galactoside.

2. The method according to claim 1, wherein: The steps include: Step a, dispersing corn porous starch in a pH 8.5 borate buffer, adding octenylsuccinic anhydride, reacting at 35° C. for 6 hours, and washing with ethanol to obtain hydrophobically modified porous starch; Step b, dissolving luteolin in 30% ethanol solution, and co-loading it with the hydrophobically modified porous starch obtained in step a at 50° C. under shaking conditions for 2 hours to obtain a drug-loaded intermediate; Step c: dispersing the drug-loaded intermediate in deionized water, adding ε-polylysine-galactoside solution, and subjecting to electrostatic adsorption at 25° C. for 1 hour and then spray drying to obtain a liver-targeted porous starch-luteolin complex.

3. The method according to claim 2, characterized in that In step a, the molar ratio of octenylsuccinic anhydride to glucose units in the porous corn starch is 1:20-1:50; In step b, the mass ratio of luteolin to hydrophobically modified porous starch is 1:3-1:5; In step c, the mass ratio of ε-polylysine-galactoside to the drug-loaded intermediate is 1:10-1:

20.

4. A liver-targeted porous starch-luteolin complex prepared by the method according to any one of claims 1 to 3.

5. Use of the liver-targeted porous starch-luteolin complex according to claim 4 in the preparation of a drug for preventing or treating liver damage in type 2 diabetes.

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