Preparation and application of gamma-cyclodextrin / genistein inclusion compound for relieving oxidative stress
The self-assembly of γ-cyclodextrin and lignin form an inclusion compound, which solves the problem of low bioavailability of lignin, significantly improves its antioxidant activity and cell protection effect, and provides a new solution for the treatment of oxidative stress-related diseases.
Patent Information
- Application Number
- CN202510461573.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-06-27
AI Technical Summary
Due to its hydrophobic structure, lignin has poor water solubility and low bioavailability, making it difficult to effectively enter cells to exert antioxidant effects, limiting its application in the treatment of diseases related to oxidative stress.
The γ-cyclodextrin and lignin are self-assembled in the solvent to form a γ-cyclodextrin/lignin inclusion compound, thereby improving the solubility, stability and biological activity of lignin.
γ-cyclodextrin/lignin inclusions significantly improve the antioxidant activity of lignin and can effectively protect cells from oxidative stress damage, providing new ideas and solutions for the treatment of related diseases caused by oxidative stress.
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Figure CN120204430A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical and health research, and particularly relates to a method for alleviating oxidative stress by preparing a γ-cyclodextrin / glycitein inclusion complex and its application. Background Art
[0002] Oxidative stress refers to a pathological state in which free radicals or reactive oxygen species in the body are produced in excess, exceeding the scavenging ability of the antioxidant defense system, resulting in cell and tissue damage. Oxidative stress is closely related to the occurrence of various chronic diseases, including cardiovascular diseases, diabetes, neurodegenerative diseases, etc. In recent years, the role of antioxidants in alleviating oxidative stress has attracted extensive attention. Polyphenolic compounds in natural products have become a research hotspot due to their excellent antioxidant properties.
[0003] Glycitein is a flavonoid compound derived from plants, which has significant antioxidant activity, can effectively scavenge free radicals, and reduce oxidative stress. However, due to the hydrophobic structure of glycitein, its water solubility is poor, its bioavailability is low, and it is difficult to effectively enter cells to play an antioxidant role, which limits its application in the treatment of oxidative stress-related diseases. In the prior art, although various methods have been proposed to improve the solubility and low antioxidant activity of glycitein, such as using solvents or surfactants, etc., these methods often have problems such as potential toxicity of the solvent, unstable solubility, poor carrier selectivity, etc., and it is difficult to effectively improve the antioxidant activity of glycitein, and may produce unnecessary side effects.
[0004] As a natural cyclic sugar molecule, γ-cyclodextrin can form inclusion complexes with a variety of hydrophobic compounds due to its unique molecular structure, thereby improving the solubility, stability and biological activity of hydrophobic compounds. γ-Cyclodextrin has good biocompatibility and biodegradability and is widely used in the field of drug carriers. However, there are few studies on the inclusion complex of γ-cyclodextrin and glycitein in alleviating oxidative stress in the existing relevant reports, and its potential application value has not been fully explored. Therefore, the present invention aims to provide a preparation method of a γ-cyclodextrin / glycitein inclusion complex and explore its application in alleviating oxidative stress. By preparing this inclusion complex, the antioxidant ability of glycitein can be effectively improved, providing new ideas and solutions for the prevention and treatment of related diseases. Summary of the Invention
[0005] The purpose of the present invention is to provide a preparation method and application of a γ-cyclodextrin / glycitein inclusion complex for alleviating oxidative stress, so as to solve the problems of low solubility and low stability of glycitein, resulting in poor functional performance.
[0006] The present invention provides a method for preparing a γ-cyclodextrin / glycitein inclusion complex for alleviating oxidative stress, which forms an inclusion complex by the self-assembly of γ-cyclodextrin and glycitein in a solvent. It is determined through a cell oxidative damage model and in vitro antioxidant experiments that it can improve the antioxidant activity of glycitein and alleviate oxidative stress in cells.
[0007] The γ-cyclodextrin / glycitein inclusion complex of the present invention can improve the in vitro antioxidant activity of glycitein and effectively protect cells from oxidative stress damage. It includes the following steps:
[0008] Step 1. Preparation of a glycitein stock solution
[0009] Weigh 0.5 - 5 mg of glycitein, add 25 - 75% alcohol, ultrasonically treat this solution with an ultrasonic cell disruptor for 10 - 20 s, dilute it 2 - 10 times with distilled water, and centrifuge it at 10000 g for 5 - 10 minutes. The supernatant is the glycitein stock solution.
[0010] Step 2. Preparation of the γ-cyclodextrin / glycitein inclusion complex
[0011] Weigh 1 - 50 mg of γ-cyclodextrin and add it to distilled water to prepare a γ-cyclodextrin solution. Continuously stir for 30 min, then gradually add the glycitein solution dropwise to the γ-cyclodextrin solution. After centrifugation, take the supernatant to obtain the self-assembled γ-cyclodextrin / glycitein inclusion complex.
[0012] Step 3. The γ-cyclodextrin / glycitein inclusion complex has the effect of alleviating oxidative stress
[0013] An oxidative damage model is constructed using PC12 cells, and the damaged cells are treated with glycitein and the γ-cyclodextrin / glycitein inclusion complex respectively. The experimental results show that, compared with glycitein, the γ-cyclodextrin / glycitein inclusion complex significantly improves the cell survival rate, indicating that this inclusion complex has the effect of alleviating oxidative stress in cells under oxidative damage conditions. The in vitro antioxidant activities of glycitein and the γ-cyclodextrin / glycitein inclusion complex are further measured. The results show that, compared with glycitein, the iron-reducing ability and DPPH free radical scavenging rate of the γ-cyclodextrin / glycitein inclusion complex increase, and with the increase of concentration, both its iron-reducing ability and DPPH free radical scavenging rate show an increasing trend. In summary, the γ-cyclodextrin / glycitein inclusion complex has potential in antioxidant and alleviating oxidative stress in cells, and is used for treating related diseases caused by oxidative stress.
[0014] The present invention has the following beneficial effects:
[0015] 1. Using γ-cyclodextrin and glycitein as raw materials, a γ-cyclodextrin / glycitein inclusion complex is formed by self-assembly in a liquid.
[0016] 2. The γ-cyclodextrin / glycitein inclusion complex can effectively protect cells from oxidative stress damage in a cell oxidative damage model; in an in vitro antioxidant experiment, it significantly increases the iron reduction ability and DPPH free radical scavenging rate.
[0017] 3. As a natural carrier, γ-cyclodextrin, when acting together with glycitein, can not only improve the antioxidant activity of glycitein, but also provide a new treatment strategy for the prevention of oxidative stress-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The survival rate of PC12 cells when glycitein and the γ-cyclodextrin / glycitein inclusion complex are used to treat an oxidative damage cell model;
[0019] Figure 2 The iron reduction ability of glycitein and the γ-cyclodextrin / glycitein inclusion complex;
[0020] Figure 3 The DPPH free radical scavenging rate of glycitein and the γ-cyclodextrin / glycitein inclusion complex. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0021] The present invention will be described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments.
[0022] Example 1: Preparation of a glycitein stock solution
[0023] (1) Weigh 0.5 - 5 mg of glycitein and add it to 25 - 75% alcohol to prepare a glycitein solution.
[0024] (2) Ultrasonicate this solution for 10 - 20 s with an ultrasonic cell disruptor. Dilute it 2 - 10 times with distilled water and centrifuge at 10000 g for 5 - 10 minutes. The supernatant is the glycitein stock solution.
[0025] Example 2: Preparation of the γ-cyclodextrin / glycitein inclusion complex
[0026] (1) Weigh 1 - 50 mg of γ-cyclodextrin and add it to distilled water to prepare a γ-cyclodextrin solution.
[0027] (2) Under the condition of continuous stirring for 30 min, dropwise add the glycitein solution into the γ-cyclodextrin solution, and centrifuge for 5 - 10 minutes. The supernatant is the self-assembled γ-cyclodextrin / glycitein inclusion complex.
[0028] Example 3: Construction of a PC12 cell oxidative damage model
[0029] Use organic hydrogen peroxide to construct a cell oxidative damage model. Seed PC12 cells at a density of 1 - 6×106 Inoculate 1 cell / well into a six-well plate with a bottom streak and culture for 8 - 15 h. Add 200 - 300 μM of organic hydrogen peroxide to the six-well plate to treat the cells for 8 - 15 h, and the final concentration of the organic hydrogen peroxide is 20 - 30 μM, thereby constructing an oxidative damage model.
[0030] Example 4: Application of low-dose γ-cyclodextrin / glycitein inclusion complex in alleviating oxidative stress
[0031] (1) Cell viability detection: Seed PC12 cells in a 96-well plate at a culture density of 1 - 6×10 5 cells / well. Treat the cells with different concentrations of glycitein (0.4, 0.8 mg / mL) and γ-cyclodextrin / glycitein inclusion complex (0.4, 0.8 mg / mL) for 8 - 15 h, then add organic hydrogen peroxide to treat the cells for 8 - 15 h, and subsequently add 20 μL of MTS and incubate for 1 - 2 h. Measure the absorbance at 490 nm using a microplate reader and calculate the cell viability.
[0032] (2) Iron-reducing ability: Prepare FeSO4 solutions with different concentrations of FeSO4·7H2O (0.15 - 1.5 mM) and draw a standard curve at 593 nm (R 2 > 0.99). Add 5 μL of different concentrations of glycitein (0.4, 0.8 mg / mL) and γ-cyclodextrin / glycitein inclusion complex (0.4, 0.8 mg / mL) to a 96-well plate, then add 180 μL of FRAP working solution, incubate at 37 °C for 3 - 5 minutes, measure the absorbance at 593 nm, and substitute it into the standard curve to calculate the iron-reducing ability.
[0033] (3) DPPH radical scavenging ability: Add 20 μL of glycitein (0.4, 0.8 mg / mL) or γ-cyclodextrin / glycitein inclusion complex (0.4, 0.8 mg / mL) to 90 μL of DPPH solution in a 96-well plate and incubate in the dark at room temperature for 30 - 40 minutes. Use absolute ethanol instead of DPPH solution as the control group and use a nitrogen radical extraction solution instead of the sample as the blank group. Finally, measure the absorbance at 517 nm and calculate the DPPH radical scavenging rate.
[0034] (4) Statistical analysis: Data are expressed as the mean ± standard deviation (Mean ± SD) of three independent experiments. Use SPSS 22.0 software to analyze the differences between samples, compare the significance between samples by one-way analysis of variance (ANOVA), and select Duncan's test for significant difference analysis. Statistical significance is set at p < 0.05. Different letters indicate significant differences.
[0035] (5) Experimental results:
[0036] An organic hydroperoxide was used to construct a cellular oxidative damage model. The experimental results showed that after treating cells with 0.4 and 0.8 mg / mL of genistein, the cell viability was lower than that of the model group. However, after treating cells with γ-cyclodextrin / genistein inclusion complex at the same concentration, the cell viability was significantly higher than that of the model group. This indicates that low-dose γ-cyclodextrin / genistein inclusion complex can protect cells from hydrogen peroxide-induced damage and thus alleviate cellular oxidative stress( Figure 1 ).
[0037] The ferric reducing ability of genistein and γ-cyclodextrin / genistein inclusion complex was evaluated using a total antioxidant capacity (FRAP method) kit. The experimental results showed that compared with the absence of genistein (0.4, 0.8 mg / mL), γ-cyclodextrin / genistein inclusion complex could significantly increase the FRAP value at 0.4 and 0.8 mg / mL concentrations, indicating that the inclusion of genistein by γ-cyclodextrin improved its antioxidant activity and thus enhanced its ferric reducing ability( Figure 2 ).
[0038] The antioxidant activity of genistein and γ-cyclodextrin / genistein inclusion complex was evaluated using a DPPH free radical scavenging rate kit. The experimental results showed that compared with genistein (0.4, 0.8 mg / mL), γ-cyclodextrin / genistein inclusion complex significantly increased its DPPH free radical scavenging rate at 0.4 and 0.8 mg / mL concentrations. Moreover, as the concentration of γ-cyclodextrin / genistein inclusion complex increased, the DPPH free radical scavenging rate increased. The above results indicate that γ-cyclodextrin / genistein inclusion complex exhibits stronger DPPH free radical scavenging ability than genistein alone by enhancing the antioxidant activity of genistein, and its antioxidant effect increases with the increase in concentration( Figure 3 ).
[0039] Example 5: Application of high-dose γ-cyclodextrin / genistein inclusion complex in alleviating oxidative stress
[0040] (1) Cell viability detection: PC12 cells were seeded in 96-well plates at a culture density of 1 - 6×10 5 cells / well. Cells were treated with different concentrations of genistein (1.2, 1.6 mg / mL) and γ-cyclodextrin / genistein inclusion complex (1.2, 1.6 mg / mL) for 8 - 15 h, then treated with organic hydroperoxide for 8 - 15 h, and subsequently incubated with 20 μL MTS for 1 - 2 h. The absorbance at 490 nm was measured using a microplate reader, and the cell viability was calculated.
[0041] (2) Ferric reducing ability: Ferrous sulfate solutions were prepared with different concentrations of FeSO4·7H2O (0.15 - 1.5 mM), and a standard curve was plotted at 593 nm (R2 >0.99). Add 5 μL of genistein at different concentrations (1.2, 1.6 mg / mL) and γ-cyclodextrin / genistein inclusion complex (1.2, 1.6 mg / mL) to a 96-well plate, and then add 180 μL of FRAP working solution. Incubate at 37 °C for 3 - 5 minutes, measure the absorbance at 593 nm, and substitute it into the standard curve to calculate the iron reduction ability.
[0042] (3) DPPH radical scavenging ability: Add 20 μL of genistein (1.2, 1.6 mg / mL) or γ-cyclodextrin / genistein inclusion complex (1.2, 1.6 mg / mL) to 90 μL of DPPH solution in a 96-well plate respectively, and incubate in the dark at room temperature for 30 - 40 minutes. Use absolute ethanol instead of DPPH solution as the control group, and use the nitrogen radical extraction solution instead of the sample as the blank group. Finally, measure the absorbance at 517 nm and calculate the DPPH radical scavenging rate.
[0043] (4) Statistical analysis: The data are expressed as the mean ± standard deviation (Mean ± SD) of three independent experiments. Use SPSS 22.0 software to analyze the differences between samples, compare the significance between samples through one-way analysis of variance (ANOVA), and select Duncan's test for significant difference analysis. The statistical significance is set at p < 0.05. Different letters indicate significant differences.
[0044] (5) Experimental results:
[0045] An oxidative damage model of cells was constructed using organic hydrogen peroxide. The experimental results showed that after treating cells with genistein at 1.2 and 1.6 mg / mL, the cell survival rate was lower than that of the model group, while after treating cells with γ-cyclodextrin / genistein inclusion complex at the same concentration, the cell survival rate was significantly higher than that of the model group. This indicates that a high dose of γ-cyclodextrin / genistein inclusion complex can protect cells from hydrogen peroxide-induced damage, thereby alleviating oxidative stress in cells ( Figure 1 ).
[0046] The total antioxidant capacity (FRAP method) kit was used to evaluate the iron reduction ability of genistein and γ-cyclodextrin / genistein inclusion complex. The experimental results showed that compared with the group without genistein added (1.2, 1.6 mg / mL), the γ-cyclodextrin / genistein inclusion complex could significantly increase the FRAP value at concentrations of 1.2 and 1.6 mg / mL, indicating that the inclusion of genistein by γ-cyclodextrin improved its antioxidant activity, thereby enhancing its iron reduction ability ( Figure 2 ).
[0047] The antioxidant activities of genistein and γ-cyclodextrin / genistein inclusion complex were evaluated using a DPPH free radical scavenging rate kit. The experimental results showed that, compared with genistein (1.2, 1.6
[0048] mg / mL), the γ-cyclodextrin / genistein inclusion complex significantly increased its DPPH free radical scavenging rate at concentrations of 1.2 and 1.6 mg / mL. Moreover, as the concentration of the γ-cyclodextrin / genistein inclusion complex increased, the DPPH free radical scavenging rate increased. The above results indicated that the γ-cyclodextrin / genistein inclusion complex exhibited stronger DPPH free radical scavenging ability than genistein alone by enhancing the antioxidant activity of genistein, and its antioxidant effect increased with the increase in concentration( Figure 3 ).
[0049] The above are only the preferred embodiments of the present invention. For those of ordinary skill in the art, various improvements and refinements can be made to the above embodiments without departing from the principles and spirit of the present invention, and these improvements and refinements all fall within the protection scope of the present invention.
Claims
1. A method for preparing a γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress, characterized in that: The following steps are involved: Step 1. Preparation of genistein stock solution Weigh an appropriate amount of genistein, add 25-75% alcohol, sonicate the solution with an ultrasonic cell crusher, dilute it 2-10 times with distilled water, centrifuge and take the supernatant to obtain a genistein stock solution; Step 2: Preparation of γ-cyclodextrin / genistein inclusion complex Weigh 1-50 mg of γ-cyclodextrin and add it to distilled water to prepare a γ-cyclodextrin solution; continue stirring for 30 minutes and then add the genistein solution dropwise into the γ-cyclodextrin solution; after centrifugation, take the supernatant to obtain a self-assembled γ-cyclodextrin / genistein inclusion complex.
2. The method for preparing the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 1, characterized in that: The mass ratio of gamma-cyclodextrin to genistein used in the process of forming the inclusion complex is 1:1-1:
10.
3. A γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress, characterized in that: It is prepared according to the method for preparing the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 1.
4. The use of the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 3, characterized in that: Application of γ-cyclodextrin / genistein inclusion complex in alleviating cellular oxidative stress: An oxidative damage model was constructed using PC12 cells, and the oxidatively damaged cells were treated with genistein and γ-cyclodextrin / genistein inclusion complex, respectively. The in vitro antioxidant activity of genistein and γ-cyclodextrin / genistein inclusion complex was determined using iron reducing ability and DPPH free radical scavenging rate, which proved that the inclusion complex increased the antioxidant activity of genistein and had the effect of alleviating cellular oxidative stress. Compared with genistein, γ-cyclodextrin / genistein inclusion complex could alleviate cellular oxidative stress.
5. The use of the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 4, characterized in that: Compared with genistein, the γ-cyclodextrin / genistein inclusion complex increases the cell survival rate by 30%-44% when the PC12 cells are treated with the inclusion complex for 12 hours.
6. The use of the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 4, characterized in that: The gamma-cyclodextrin / genistein inclusion complex alleviates the cell oxidative stress caused by organic hydrogen peroxide, and increases the cell survival rate by 12%-18%.
7. The use of the γ-cyclodextrin / genistein inclusion complex for alleviating oxidative stress according to claim 4, characterized in that: The γ-cyclodextrin / genistein inclusion complex showed higher iron reducing ability and DPPH free radical scavenging rate than genistein; among them, the iron reducing ability increased by 4%-23%, and the DPPH free radical scavenging rate increased by 3%-6%.