A plant polyphenol supramolecular self-assembly system and its construction method and application

Abstract

The present invention relates to the technical field of supramolecular chemistry, and specifically discloses a plant polyphenol supramolecular self-assembly system, a construction method and an application thereof. The plant polyphenol supramolecular self-assembly system is obtained by mixing echinacoside and ferrous gluconate at a molar ratio of 5-6:4 in an environment with a pH of 7-11, heating and reacting at 40°C-45°C for 20 h-24 h, and then through dialysis and drying. The plant polyphenol supramolecular self-assembly system provided by the present invention exhibits excellent drug release behavior, shows significant pH responsiveness in an acidic environment, effectively enhances the antioxidant and neuroprotective effects of ECH, and significantly improves its blood-brain barrier permeability.

Description

Technical Field

[0001] The present invention relates to the technical field of supramolecular chemistry, and particularly relates to a plant polyphenol supramolecular self-assembly system, a construction method thereof and an application thereof. Background Art

[0002] Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are one of the major challenges in the global health field currently. Such diseases involve pathological mechanisms such as oxidative stress and inflammatory responses and are very complex, and there is an urgent need to find effective treatment means. Echinacoside (ECH), as a natural extract, has a variety of pharmacological activities, especially neuroprotective and antioxidant effects, and thus has received extensive attention. A number of studies have shown that ECH can delay the progression of neurodegenerative diseases by inhibiting oxidative stress and inflammatory responses. However, due to the low bioavailability of ECH and the limited permeability through the blood-brain barrier (BBB), the polar groups in ECH limit the absorption and distribution of ECH in the body, resulting in a low effective concentration in the target tissues. In addition, as a natural barrier of the central nervous system, the BBB further restricts the delivery of ECH to the brain, making it difficult to fully exert its neuroprotective effect in clinical practice. Therefore, it is particularly necessary to explore a drug delivery system that enhances the permeability of ECH through the blood-brain barrier. Summary of the Invention

[0003] To develop a drug delivery system that enhances the permeability of ECH through the blood-brain barrier, the present invention provides a plant polyphenol supramolecular self-assembly system, a construction method thereof and an application thereof. The plant polyphenol supramolecular self-assembly system provided by the present invention exhibits excellent drug release behavior, shows significant pH responsiveness in an acidic environment, effectively enhances the antioxidant and neuroprotective effects of ECH, and significantly improves its permeability through the blood-brain barrier.

[0004] The present invention provides a plant polyphenol supramolecular self-assembly system, which is obtained by mixing echinacoside and ferrous gluconate in a liquid environment with a pH of 7-11 at a molar ratio of 5-6:4, heating and reacting at 40°C-45°C for 20h-24h, and then performing dialysis and drying.

[0005] The plant polyphenol supramolecular self-assembly system provided by the present invention exhibits excellent drug release behavior, shows significant pH responsiveness in an acidic environment, effectively enhances the antioxidant and neuroprotective effects of ECH, and significantly improves its permeability through the blood-brain barrier.

[0006] Further, the pH is 9-11.

[0007] Further, the molar ratio of echinacoside to ferrous gluconate is 5:4.

[0008] Furthermore, the heating reaction time is 24 h.

[0009] The present invention also provides a method for constructing the plant polyphenol supramolecular self-assembly system, comprising the following steps:

[0010] Prepare an ECH stock solution by dissolving echinacoside in methanol, and prepare an FG stock solution by dissolving ferrous gluconate in purified water. Dilute them respectively to obtain an ECH dilution and an FG dilution, adjust the pH, then mix the ECH dilution and the FG dilution with the molar ratio of echinacoside to ferrous gluconate being 5-6:4, and heat and react at 40 °C - 45 °C for 20 h - 24 h, and then obtain the plant polyphenol supramolecular self-assembly system through dialysis and drying.

[0011] Furthermore, the concentrations of both the ECH dilution and the FG dilution are 1 mM.

[0012] Furthermore, the heating reaction is carried out at 100 rpm.

[0013] The present invention also provides the application of the plant polyphenol supramolecular self-assembly system in the preparation of drugs for treating neurodegenerative diseases. The plant polyphenol supramolecular self-assembly system improves the blood-brain barrier permeability as a drug carrier and enhances the antioxidant property of ECH.

[0014] Furthermore, the plant polyphenol supramolecular self-assembly system is used for preparing drugs for protecting nerves.

[0015] Furthermore, the neurodegenerative disease is Alzheimer's disease.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The ECH@FG supramolecular system demonstrated significant neuroprotective effects in an in vitro HT22 cell model. This system not only improved the antioxidant capacity of the cells, but also significantly reduced the oxidative damage of the cells and decreased the incidence of cell apoptosis.

[0018] The plant polyphenol supramolecular self-assembly system provided by the present invention demonstrated excellent drug release behavior, showed significant pH responsiveness in an acidic environment, effectively enhanced the antioxidant and neuroprotective effects of ECH, and significantly improved its blood-brain barrier permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 Color changes during the heating reaction of the ECH@FG system prepared at different pH values. The test tubes from left to right represent pH values of 3, 5, 7, 9, and 11 respectively.

[0021] Figure 2 Transmission electron microscope (TEM) image of the ECH@FG system prepared in Example 1;

[0022] In the figure, A is the 50 nm transmission electron microscope (TEM) image of the ECH@FG system;

[0023] B is the 100 nm transmission electron microscope (TEM) image of the ECH@FG system;

[0024] C is the 200 nm transmission electron microscope (TEM) image of the ECH@FG system.

[0025] Figure 3 Scanning electron microscope (SEM) image of the ECH@FG system prepared in Example 1;

[0026] In the figure, A is the 100 μm SEM image of the ECH@FG system prepared in Example 1;

[0027] B is the 50 μm SEM image of the ECH@FG system prepared in Example 1.

[0028] Figure 4 Fourier transform infrared spectroscopy and Raman spectroscopy diagrams of the ECH@FG system prepared in Example 1;

[0029] In the figure, A is the Fourier transform infrared spectroscopy diagram of the ECH@FG system prepared in Example 1;

[0030] B is the Raman spectroscopy diagram of the ECH@FG system prepared in Example 1.

[0031] Figure 5 In vitro drug release curves of the ECH@FG system prepared in Example 1 under different pH conditions.

[0032] Figure 6 Activity curves of the ECH@FG system prepared in Example 1 in the ABTS radical scavenging experiment.

[0033] Figure 7 Activity curves of the ECH@FG system prepared in Example 1 in the DPPH radical scavenging experiment.

[0034] Figure 8Effects of the ECH@FG system prepared in Example 1 and ECH alone on the viability of HT22 cells. Control: control group; Model: model group; ***: extremely significant difference compared with the blank group, P < 0.001; : extremely significant difference compared with the model group, P < 0.001.

[0035] Figure 9 Effects of the ECH@FG system prepared in Example 1 and ECH on the levels of SOD and MDA in HT22 cells. Control: control group; Model: model group; ***: extremely significant difference compared with the blank group, P < 0.001; : extremely significant difference compared with the model group, P < 0.001;

[0036] In the figure, A shows the effects of the ECH@FG system and ECH on the SOD content in HT22 cells;

[0037] B shows the effects of the ECH@FG system and ECH on the MDA content in HT22 cells.

[0038] Figure 10 Blood-brain barrier permeability of ECH and the ECH@FG system;

[0039] In the figure, A is a schematic diagram of the blood-brain barrier permeability experiment;

[0040] B is a statistical chart of the blood-brain barrier permeability of ECH and the ECH@FG system. Detailed implementation manners

[0041] The following describes the detailed implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the detailed implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0042] Example 1: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0043] Prepare a 10 mM echinacoside (ECH) stock solution with methanol at a volume fraction of 30%, and prepare a 10 mM ferrous gluconate (FG) stock solution with purified water. Dilute the ECH stock solution with purified water to 1 mM to obtain an ECH dilution, and dilute the FG stock solution with purified water to 1 mM to obtain an FG dilution. Adjust the pH of the ECH dilution to 9.5 with 0.05 M HCl and 0.05 M NaOH solutions to ensure stability. Slowly add 4 mL of the FG dilution to 5 mL of the ECH dilution and quickly mix by shaking. Heat the entire reaction system at 40 °C and 100 rpm for 24 hours to obtain an ECH@FG solution. Place 2 mL of the ECH@FG solution in a dialysis bag and stir at 145 rpm in 200 mL of 10% PBS buffer at 37 °C. After 60 min, change the dialysis medium (10% PBS buffer) and repeat dialysis 3 times to remove unassembled drugs. Collect the sample inside the dialysis bag, which is the ECH@FG supramolecular self-assembly solution. After lyophilization, obtain the ECH@FG supramolecular lyophilized powder.

[0044] Example 2: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0045] The preparation steps are basically the same as those in Example 1, except that: the entire reaction system is heated at 40 °C and 100 rpm for 20 hours.

[0046] Example 3: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0047] The preparation steps are basically the same as those in Example 1, except that: the dosage ratio of the ECH dilution to the FG dilution is 10 mL:3 mL.

[0048] Example 4: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0049] The preparation steps are basically the same as those in Example 1, except that: adjust the pH of the ECH dilution to 9.

[0050] Comparative Example 1: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0051] The preparation steps are basically the same as those in Example 1, except that: the entire reaction system is heated at 40 °C and 100 rpm for 4 hours.

[0052] Comparative Example 2: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0053] The preparation steps are basically the same as those in Example 1, except that: the entire reaction system is heated at 40 °C and 100 rpm for 2 hours.

[0054] Comparative Example 3: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0055] It is basically the same as the preparation steps of Example 1, except that the entire reaction system is heated at 40 °C and 100 rpm for 1 hour.

[0056] Comparative Example 4: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0057] It is basically the same as the preparation steps of Example 1, except that the entire reaction system is heated at 40 °C and 100 rpm for 30 min.

[0058] Comparative Example 5: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0059] It is basically the same as the preparation steps of Example 1, except that the dosage ratio of the ECH diluent to the FG diluent is 1 mL:1 mL.

[0060] Comparative Example 6: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0061] It is basically the same as the preparation steps of Example 1, except that the dosage ratio of the ECH diluent to the FG diluent is 2 mL:1 mL.

[0062] Comparative Example 7: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0063] It is basically the same as the preparation steps of Example 1, except that the dosage ratio of the ECH diluent to the FG diluent is 5 mL:3 mL.

[0064] Comparative Example 8: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0065] It is basically the same as the preparation steps of Example 1, except that the pH of the ECH diluent is adjusted to 3.

[0066] Comparative Example 9: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0067] It is basically the same as the preparation steps of Example 1, except that the pH of the ECH diluent is adjusted to 5.

[0068] Comparative Example 10: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0069] It is basically the same as the preparation steps of Example 1, except that the pH of the ECH diluent is adjusted to 6.

[0070] Comparative Example 11: A method for constructing a supramolecular self-assembly system of plant polyphenols.

[0071] It is basically the same as the preparation steps of Example 1, except that: the pH of the ECH dilution is adjusted to 7.

[0072] Comparative Example 12: A method for constructing a plant polyphenol supramolecular self-assembly system.

[0073] It is basically the same as the preparation steps of Example 1, except that: the pH of the ECH dilution is adjusted to 8.

[0074] I. Detect the influence of heating time on self-assembly, and detect the particle size and PDI values of the plant polyphenol supramolecular self-assembly systems prepared in Example 1 and Comparative Examples 1-4. The detection methods and results are as follows.

[0075] 1. Detection method:

[0076] Use dynamic light scattering (DLS) technology to measure the average particle size and PDI value of the self-assembly system. The specific operating conditions are: after diluting the sample to 1 mg / mL, measure it at 25 °C, and measure each sample three times and take the average value. The results are recorded in Table 1.

[0077] Table 1 Influence of heating time on the particle size and PDI of the ECH@FG system

[0078] Heating time Particle size (nm) PDI value 30 minutes 120.4 0.23 1 hour 108.9 0.2 2 hours 107.5 0.18 4 hours 107.2 0.16 24 hours 107 0.13

[0079] Note: The PDI value is the polydispersity coefficient. The closer the value is to 0, the more uniform the particle size distribution is, and the smaller the size difference between particles; while the closer the value is to 1, the more uneven the particle size distribution is, and the larger the size difference between particles.

[0080] It can be seen from Table 1 that as the heating time prolongs, the particle size of the ECH@FG system gradually decreases, while the PDI value tends to decrease, indicating that the dispersibility of the system is improved. When the heating time reaches 24 hours, the particle size and PDI value reach the optimal state, indicating that the self-assembly system has good stability and uniformity at this time.

[0081] II. Detect the influence of different ratios of ECH to FG on the absorbance of the prepared ECH@FG system

[0082] 1. Detection method:

[0083] Taking the methods of Example 1, Example 3, Comparative Example 5, Comparative Example 6 and Comparative Example 7 as the research objects, the effects of different molar ratios of ECH:FG (1:1, 2:1, 5:3, 10:3, 5:4) on the absorbance of the supramolecular self-assembly system of plant polyphenols (ECH@FG system) prepared were studied. The pH of the ECH@FG system was adjusted to 11, heated at 40 °C for 30 min, 1 mL of the sample was taken, diluted to 3 mL with methanol with a volume fraction of 50%, and the absorbance values at 190 - 800 nm were scanned. It was observed that the shift of the maximum absorption peak occurred between 300 - 400 nm. When ECH:FG > 2:1, the main body of the absorption peak shifted to 380 nm, and the maximum absorbance value appeared at ECH:FG = 5:4, which was 383 nm.

[0084] Table 2 Effects of different ratios of ECH to FG on the absorbance of the prepared ECH@FG system

[0085] Grouping ECH: FG Maximum absorbance Absorbance peak shift (nm) Comparative Example 5 1:1 0.45 330 Comparative Example 6 2:1 0.53 340 Comparative Example 7 5:3 0.6 360 Example 3 10:3 0.72 380 Example 1 5:4 0.85 383

[0086] As can be seen from Table 2, when the ratio of ECH to FG was 5:4, the absorbance of the system reached the maximum, the absorbance peak shifted to 383 nm, indicating that the structure of the supramolecular system was the most stable at this time and the complexation was the strongest, and the pH value of the solution was measured to be 11.

[0087] III. Effects of different pH values on the appearance of the prepared ECH@FG system

[0088] The detection results are as Figure 1 shown. When pH = 3, the solution was colorless and transparent. When the pH rose to about 5, the solution was gray-green. When the pH was between 7 - 9, the solution was dark gray. When pH > 9, the solution turned reddish-brown, and no precipitation occurred during the process. It can be seen from the color change that the self-assembly process of ECH@FG was regulated by pH. Under the condition of pH > 5, ECH began to complex with iron ions in FG and formed supramolecular self-assemblies with different morphologies. Combining with the absorbance value, the best result was obtained when the pH was 11.

[0089] IV. Characterization of the ECH@FG system prepared in Example 1

[0090] 1. Zeta potential, morphological observation and chemical bond characterization

[0091] Zeta potential: The Zeta potential was measured by electrophoresis light scattering method at 25 °C to analyze the surface charge characteristics of the system. During the measurement, the sample concentration was 1 mg / mL, and the electrophoresis solution was 10 mM NaCl solution.

[0092] Morphological observation: The transmission electron microscope (TEM) and scanning electron microscope (SEM) were used to observe the microstructure of the supramolecular system and confirm its morphology after self-assembly. The TEM samples were dropped onto copper grids at a concentration of 1 mg / mL and observed at 200 kV after negative staining; the SEM samples were observed at a voltage of 10 kV after gold spraying.

[0093] Chemical bond characterization: Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy were used to analyze the metal chelation between ECH and FG. The FT-IR measurement range was 4000 - 400 cm-1, the sample was in dry powder form, and the KBr pellet method was used for preparation. The detection wavelength of Raman spectroscopy was 1064 nm, the scanning range was 4000 - 200 cm-1, and the sample was prepared as a solution at a concentration of 1 mg / mL.

[0094] 2. Experimental results

[0095] (1) Morphological observation

[0096] Transmission electron microscope: The transmission electron microscope (TEM) image of the ECH@FG system prepared in Example 1 is as Figure 2 shown. The plant polyphenol supramolecular self-assembly system prepared in Example 1 exhibited a uniform spherical structure with a particle size concentrated around 100 nm, indicating that the optimized self-assembly system had good morphological and structural consistency. This further verified the effectiveness of the above self-assembly conditions.

[0097] Scanning electron microscope: To further confirm the morphology of the ECH@FG system prepared in Example 1, the scanning electron microscope (SEM) was used to observe its microstructure. As Figure 3 shown, the surface of the ECH@FG system prepared in Example 1 was smooth, showing good morphological consistency, further confirming its uniform nanoparticle structure.

[0098] (2) Particle size and PDI values of the ECH@FG systems prepared at different ratios of ECH to FG.

[0099] Table 3 Particle size and PDI values of the ECH@FG systems prepared at different molar ratios of ECH to FG

[0100]

[0101]

[0102] As shown in Table 3, when ECH:FG was 1.25:1, the particle size was 107 nm, indicating that most of the particle sizes were concentrated in this range, suggesting that the self-assembly system had good dispersion stability.

[0103] (3) Zeta potential determination

[0104] The surface charge characteristics of the ECH@FG system prepared in Example 1 were detected, and it was found that the Zeta potential of the ECH@FG system was 6.73±1.55 mV, indicating that the system had good stability in solution. The positive charge of the Zeta potential further verified the successful complexation of iron ions and ECH in the system, which helped to improve the stability of the system and prevent particle aggregation. As shown in Table 4.

[0105] Table 4 Zeta potential measurement data of ECH@FG system

[0106] Sample Zeta potential (mV) Standard deviation (mV) ECH@FG system 6.73 ±1.55

[0107] (4) Chemical bond characterization

[0108] The ECH@FG system prepared in Example 1 was analyzed by Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy to verify the metal chelation between ECH and FG.

[0109] The results are as follows Figure 4 As shown in the FT-IR spectrum, the double peak of the benzene ring vibration of the ECH@FG system at 1600 cm-1 shifted significantly, indicating that metal complexation led to a change in the molecular structure ( Figure 4 A). FT-IR shows that the vibration peak at 1600 cm-1 changes from the unchelated state to the single peak after complexation, which supports the binding of iron ions to ECH.

[0110] Raman spectroscopy further confirmed the metal-benzene coordination between ECH and FG, with characteristic peaks clearly appearing at 566 cm-1 and 633 cm-1, verifying the successful chelation of iron ions and ECH in the system ( Figure 4 B). The characteristic peaks of Raman spectrum verify the existence of metal-benzene coordination, further illustrating the successful construction of supramolecular system.

[0111] It can be seen that the ECH@FG system prepared by the present invention forms nanoparticles with good particle size distribution, stability and clear chemical bonding. These characterization results provide strong support for the application of the ECH@FG system in drug delivery systems. Further in vitro and in vivo studies will help to verify its practical application effect and explore its potential value in the field of biomedicine.

[0112] Application Example 1: Study on the antioxidant, neuroprotective effect, blood-brain barrier permeability and pH-responsive drug release behavior of the plant polyphenol supramolecular self-assembly system prepared in Example 1.

[0113] 1. Experimental Methods

[0114] 1. In vitro drug release and antioxidant activity detection

[0115] The in vitro drug release behavior of the ECH@FG system prepared under different pH conditions was investigated. The specific process was as follows: 10% PBS buffer was used as the release medium, and the pH was adjusted to 5.5, 6.0, 6.5, 7.0, and 7.5 respectively. 2 mL of the ECH@FG solution obtained in Example 1 was placed in a dialysis bag and stirred at 145 rpm in 200 mL of buffer at 37°C. Samples were taken at regular intervals and the content of ECH released was determined. At the same time, the antioxidant ability of the ECH@FG system was evaluated by ABTS and DPPH free radical scavenging experiments and compared with that of ECH alone.

[0116] 2. Detection of neuroprotective effect

[0117] The neuroprotective effect of the ECH@FG system was evaluated using a glutamate-induced oxidative damage model of HT22 cells. HT22 cells were seeded in 96-well plates. After 24 hours of culture, the sample group, model group, and control group were added respectively. The sample group included the ECH group and the ECH@FG group, and were pretreated with medium containing 100 mM ECH and 100 mM ECH@FG for 4 h respectively. After 4 h, 15 mM glutamate was added and treated for 4 h. The model group was pretreated with medium for 4 h after adding HT22 cells, and 15 mM glutamate was added and treated for 4 h after 4 h. The control group had no other treatment. After the treatments of each group, the cell viability was detected by the CCK-8 method (n = 6).

[0118] The cell viability was detected by the CCK-8 method, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in the cells were measured to verify the mechanism of action of ECH@FG in neuroprotection.

[0119] 3. Blood-brain barrier permeability experiment

[0120] To verify the blood-brain barrier permeability of the ECH@FG system, an in vitro blood-brain barrier (BBB) model was established in the present invention. HT22 cells were seeded in the lower layer of the Transwell chamber, and bEnd.3 cells were seeded in the upper layer. After 48 hours, the integrity of the blood-brain barrier was confirmed by resistance detection. After the successful establishment of the blood-brain barrier model, ECH and ECH@FG were added to the upper layer liquid respectively, and the content of ECH in the lower layer liquid was detected after incubation for 4 hours to evaluate its blood-brain barrier permeability rate.

[0121] II. Experimental results

[0122] 1. pH-responsive drug release behavior

[0123] When treating neurodegenerative diseases such as Alzheimer's disease, the lesion sites in the central nervous system (CNS) often exhibit a slightly acidic environment. Therefore, studying the drug release behavior of the ECH@FG system under different pH conditions is an important aspect of evaluating the effectiveness of its drug delivery system. This part conducts a systematic study and analysis on this.

[0124] (1) Design of drug release experiment

[0125] To evaluate the pH-responsive drug release behavior of the ECH@FG system obtained in Example 1, the present invention selects five representative pH values: 5.5, 6.0, 6.5, 7.0, and 7.5 to simulate different physiological and pathological environments. In the experiment, 10% PBS buffer was used as the release medium. 2 mL of the ECH@FG solution was placed in a dialysis bag and stirred at 145 rpm in 200 mL of the corresponding pH buffer at 37 °C to simulate the in vivo drug release environment. Every 5 minutes, 200 μL of the sample was taken to detect the content of the released ECH, and an equal volume of the buffer was used for supplementation. The results are shown in Table 5 and Figure 5 as follows.

[0126] Table 5 Drug release rate and cumulative release amount of the ECH@FG system under different pH conditions

[0127] pH value Drug release rate (% / h) Cumulative release amount at 16 hours (%) Cumulative release amount at 48 hours (%) 5.5 4.41 70.52 85.16 6 3.25 62.34 78 6.5 2.95 58.24 72 7 2.11 40.41 60.63 7.5 1.65 30.32 50

[0128] As Figure 5 shown, the ECH@FG system shows significant differences in drug release rate under different pH conditions: the system has a faster drug release rate in an environment with a lower pH value (pH 5.5 - 6.5), while the drug release rate is significantly slowed down in a neutral or weakly alkaline environment (pH 7.0 - 7.5).

[0129] At pH 5.5, the drug release rate is the highest, and the cumulative release amount reaches 70.52% within 16 hours and as high as 85.16% within 48 hours. This indicates that in an acidic environment, the supramolecular structure of ECH@FG is prone to disintegration or the breaking of chemical bonds, thus promoting the rapid release of the drug.

[0130] At pH 6.0 - 6.5, the drug release rate is relatively fast, and the cumulative release amounts within 48 hours are 78% and 72% respectively. These results are consistent with the slightly acidic environment in the CNS, indicating that the drug release rate of the ECH@FG system at the lesion site can meet the treatment requirements.

[0131] At pH 7.0, the drug release rate decreases, and the cumulative release amount within 48 hours is 60.63%. This indicates that in an environment close to neutral, the structure of the ECH@FG system is more stable, and the drug release is inhibited to a certain extent.

[0132] At pH 7.5, the drug release rate is the lowest, and the cumulative release amount within 48 hours is only 50%. This indicates that under more alkaline conditions, the system has higher structural integrity and the drug is released more slowly.

[0133] (2) Analysis of the H-responsive drug release mechanism

[0134] The differences in drug release exhibited by the ECH@FG system under different pH conditions are mainly attributed to the following aspects:

[0135] Structural stability: Under lower pH conditions, the metal chelation between ECH and FG is weakened, resulting in an unstable supramolecular structure and easier drug release. Under neutral and alkaline conditions, the system structure is relatively stable and drug release is inhibited.

[0136] Charge effect: Under acidic conditions, the change in the surface charge of the system may lead to particle surface dissociation, thereby accelerating drug release. Under neutral or alkaline conditions, the change in Zeta potential slows down the drug diffusion rate.

[0137] Coordination complexation: The coordination complexation between iron ions and ECH is more likely to dissociate in an acidic environment, leading to accelerated drug release. This mechanism has important application potential in the acidic CNS environment.

[0138] In summary, the ECH@FG supramolecular system exhibits significant pH-responsive drug release behavior, enabling rapid release in an acidic environment and showing sustained release characteristics in a neutral or weakly alkaline environment. This pH responsiveness makes the ECH@FG system particularly suitable for targeted treatment of neurodegenerative diseases, especially in drug delivery systems that require controlled release, and has potential clinical application value. By further optimizing the structure and chemical properties of the system, its effectiveness and stability in practical applications can be further improved.

[0139] 2. Antioxidant activity

[0140] The antioxidant activity of the ECH@FG system is an important aspect in evaluating its potential in biomedical applications. In this invention, the antioxidant ability of the ECH@FG system was evaluated through a variety of free radical scavenging experimental systems to better understand the protective effect of this system in oxidative stress-related diseases.

[0141] (1) ABTS free radical scavenging experiment

[0142] The antioxidant ability of the ECH@FG system was evaluated through the ABTS free radical scavenging experiment. ABTS free radical is a model free radical widely used in the evaluation of antioxidant activity. The green radical cation ABTS+ generated by it under the action of an oxidant can be reduced by an antioxidant, thus fading. The experimental results are shown in Table 6 and Figure 6As shown

[0143] Table 6 IC50 values of ECH, ECH@FG and Vc in ABTS radical scavenging experiment

[0144] Sample IC50 (mg / mL) Antioxidant capacity Vc 0.014 High ECH 0.025 Medium ECH@FG 0.03 Medium

[0145] As shown in Table 6, the antioxidant capacity of the ECH@FG system still has a strong radical scavenging ability compared to ECH

[0146] As Figure 6 shown, with the increase in sample concentration, both the ECH and ECH@FG systems showed strong radical scavenging abilities. Especially at a concentration of 0.5 mg / mL, the scavenging rate of ECH was close to that of the Vc group, and the ECH@FG system also reached 83.7%, indicating that the presence of FG had no significant effect on the antioxidant capacity of ECH

[0147] (2) DPPH radical scavenging experiment

[0148] The results are as Figure 7 shown. With the increase in sample concentration, each sample showed a high DPPH radical scavenging ability. At a concentration of 1 mg / mL, the scavenging rate of ECH was close to 100%, and the scavenging rate of the ECH@FG system also reached about 90%. This indicates that while the ECH@FG system protects the antioxidant activity of ECH, it also effectively plays a role in scavenging free radicals

[0149] Table 7 IC50 values of ECH, ECH@FG and Vc in DPPH radical scavenging experiment

[0150] Sample IC50 (mg / mL) Antioxidant capacity Vc 0.012 High ECH 0.02 Medium ECH@FG 0.028 Medium

[0151] As shown in Table 7, the antioxidant activity of the ECH@FG system indicates its significant antioxidant potential

[0152] (3) Analysis of antioxidant activity mechanism

[0153] The excellent antioxidant activity of the ECH@FG system is mainly attributed to the following aspects

[0154] Free radical scavenging ability of polyphenol structure: As a polyphenolic compound, ECH has a strong free radical scavenging ability due to its phenolic hydroxyl structure. The introduction of FG does not weaken this ability, but may improve the stability of the system through metal chelation and enhance the antioxidant effect

[0155] Synergistic effect of iron ions: The metal chelation between iron ions and ECH not only maintains the structural integrity of the supramolecular system, but may also generate more hydroxyl radicals through the Fenton reaction, thus enhancing the free radical scavenging ability

[0156] Protective effect of the supramolecular structure: The supramolecular structure of the ECH@FG system may provide a protective microenvironment for ECH, preventing it from being prematurely metabolized or inactivated in the body, thereby maintaining its antioxidant capacity.

[0157] Through ABTS and DPPH free radical scavenging experiments, the antioxidant activity of the ECH@FG supramolecular system was fully verified. Although the introduction of FG slightly reduced the single scavenging ability of ECH, its overall antioxidant performance still showed excellent results. This indicates that the ECH@FG system has broad prospects for application in oxidative stress-related diseases, especially in cases where long-term and stable release of antioxidants is required. Further research will focus on exploring the in vivo antioxidant activity of the system and its potential clinical application value.

[0158] 3. Neuroprotective effect

[0159] (1) Neuroprotective effect in the HT22 cell model

[0160] In the present invention, the CCK-8 method was used to detect the cell viability of different treatment groups, and the protective effects of ECH and the ECH@FG system against oxidative damage were evaluated. The experimental results are as Figure 8 shown. The cell viability of the control group (Control) remained close to 100%, while the cell viability of the model group (Model) treated with glutamate decreased significantly, only 73.15 - 79.64%. The ECH treatment group significantly increased the cell viability, with a survival rate of 91.37 - 97.81%. The cell viability of the ECH@FG system treatment group was slightly lower than that of the ECH group, but still significantly higher than that of the model group, with a survival rate of 80.01 - 85.16%. This indicates that both ECH and ECH@FG have good neuroprotective effects and can significantly reduce glutamate-induced oxidative cell damage.

[0161] (2) Determination of SOD and MDA activities

[0162] To further verify the neuroprotective effect of the ECH@FG system, the present invention also measured the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in HT22 cells. The experimental results are shown in Table 8 and Figure 9 shown.

[0163] Table 8 Effects of ECH@FG and ECH treatments on SOD activity and MDA content in HT22 cells

[0164]

[0165]

[0166] Table 8 shows the detection results of SOD and MDA, indicating that ECH@FG plays an important role in neuroprotection by enhancing the antioxidant defense of cells and reducing oxidative damage.

[0167] As Figure 9 shown in A, the SOD activity was the highest in the control group, and significantly decreased to 0.8 U / mg in the model group. The SOD activities in the ECH and ECH@FG treatment groups were both restored, especially in the ECH@FG treatment group where the SOD activity reached 1.2 U / mg, showing stronger antioxidant ability.

[0168] As Figure 9 shown in B, the MDA content in the model group increased significantly to nearly 2.0 nmol / mg protein, while the MDA contents in the ECH and ECH@FG treatment groups decreased significantly to 1.2 and 1.0 nmol / mg protein respectively, further demonstrating their protective effects on cells.

[0169] (3) Analysis of neuroprotective mechanism

[0170] The neuroprotective effects of the ECH@FG supramolecular system are mainly manifested in the following aspects:

[0171] Enhancing antioxidant defense: The ECH@FG system enhances the antioxidant ability within cells by increasing the SOD activity, thereby effectively reducing the damage of oxidative stress to nerve cells.

[0172] Reducing lipid peroxidation: MDA is a marker product of intracellular lipid peroxidation, and the decrease in its content indicates that the ECH@FG system can effectively reduce the damage of free radicals to cell membranes and protect cell integrity.

[0173] The protective effect of FG: FG not only makes the supramolecular system more stable, but may also enhance its neuroprotective effect by improving the bioavailability and stability of ECH. This synergistic effect makes ECH@FG show a stronger protective effect in response to oxidative stress.

[0174] In summary, the ECH@FG supramolecular system demonstrated significant neuroprotective effects in the in vitro HT22 cell model. This system not only improved the antioxidant ability of cells, but also significantly reduced cellular oxidative damage and the incidence of apoptosis. This study provides strong support for the potential application of the ECH@FG system in the treatment of neurodegenerative diseases. Follow-up studies will further explore its neuroprotective effects in vivo and its potential clinical application value.

[0175] 4. Blood-brain barrier permeability

[0176] The application prospect of the ECH@FG supramolecular system in drug delivery, especially in crossing the blood-brain barrier (BBB), is crucial for its potential treatment of central nervous system diseases such as Alzheimer's disease. To evaluate the BBB permeability of the ECH@FG system, the present invention constructs an in vitro BBB model and reveals the trans-BBB delivery potential of this system through experiments.

[0177] (1) Construction of the blood-brain barrier model

[0178] The present invention constructs an in vitro blood-brain barrier model using the classical Transwell chamber model. HT22 mouse hippocampal neuron cells are seeded on the lower layer of the Transwell chamber, and bEnd.3 brain microvascular endothelial cells are seeded on the upper layer. After 48 hours of co-culture, the integrity of the BBB is confirmed by measuring the electrical resistance. When the resistance value exceeds 50 Ω, the model is considered successfully constructed and can be used for subsequent experiments.

[0179] (2) Blood-brain barrier permeability of the ECH@FG system

[0180] The ECH and ECH@FG systems are respectively added to the successfully constructed BBB model for permeability experiments. After 4 hours of incubation, the content of ECH in the lower-layer liquid is detected to evaluate its BBB permeability. The experimental results are as Figure 10 shown. The permeability of free ECH is 4.02%, and the permeability of ECH in the ECH@FG supramolecular system is significantly increased to 12.18%, indicating that this supramolecular system has obvious advantages in enhancing the trans-BBB delivery ability of ECH.

[0181] (3) Analysis of the mechanism of blood-brain barrier permeability ability

[0182] The ECH@FG system can significantly improve the BBB permeability of ECH, which may involve the following mechanisms:

[0183] Nanoscale size effect: The nanoscale supramolecular structure self-assembled by the ECH@FG system is more likely to enter the central nervous system through intercellular spaces. Nanoparticles can cross the BBB through various pathways such as adsorption, transport, and transmembrane transport.

[0184] Surface charge effect: Zeta potential analysis shows that the ECH@FG system has a moderate positive charge (about 6.73 mV), which may contribute to its interaction with the anionic phospholipid bilayer on the BBB, thus promoting its transmembrane transport.

[0185] Metal chelation effect: The chelation between iron ions and ECH may enhance the adsorption of the system on the BBB, further improving the permeability. This metal chelation effect may also promote the trans-BBB transport of the supramolecular system through the pathway mediated by TfR-1 (transferrin receptor 1).

[0186] The results of in vitro BBB model studies showed that the ECH@FG supramolecular system could significantly improve the blood-brain barrier permeability of ECH, demonstrating excellent drug delivery potential. This finding provides important theoretical support for the application of the ECH@FG system in the treatment of central nervous system diseases such as Alzheimer's disease, and also lays a foundation for the further development of drug carriers with high trans-BBB ability.

[0187] In summary, the present invention successfully constructed a plant polyphenol supramolecular self-assembly system (ECH@FG system). The ECH@FG system demonstrated excellent drug release behavior and showed significant pH responsiveness in acidic environments. In addition, the ECH@FG system effectively enhanced the antioxidant and neuroprotective effects of ECH and significantly improved its blood-brain barrier permeability. It showed significant advantages in drug delivery efficiency and targeted therapy, providing new possibilities for the application of natural compounds in the treatment of neurodegenerative diseases.

[0188] It should be noted that the plant polyphenol supramolecular self-assembly system in the present invention is abbreviated as the ECH@FG system, and is also referred to as the ECH@FG supramolecular self-assembly system or the ECH@FG supramolecular system in the present invention.

[0189] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concepts.

[0190] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and deformations.

Claims

1. A plant polyphenol supramolecular self-assembly system, characterized in that: The plant polyphenol supramolecular self-assembly system is obtained by mixing echinacea glycoside and ferrous gluconate in a molar ratio of 5:4 in a liquid environment with a pH of 7 to 11, heating the mixture at 40 to 45° C. for reaction for 20 to 24 hours, and then dialyzing and drying the mixture.

2. The plant polyphenol supramolecular self-assembly system according to claim 1, characterized in that: The pH is 9-11.

3. The plant polyphenol supramolecular self-assembly system according to claim 1, characterized in that: The heating reaction time is 24 h.

4. A method for constructing a plant polyphenol supramolecular self-assembly system according to any one of claims 1 to 2, characterized in that: The steps include: Echinacea glycoside was prepared into an echinacea glycoside stock solution with methanol, and ferrous gluconate was prepared into a ferrous gluconate stock solution with purified water. The echinacea glycoside dilution solution and the ferrous gluconate dilution solution were diluted respectively, and the pH was adjusted. The echinacea glycoside dilution solution and the ferrous gluconate dilution solution were then mixed with the echinacea glycoside and ferrous gluconate dilution solution in a molar ratio of 5:

4. The mixture was heated at 40°C to 45°C for 20 h to 24 h, and then dialyzed and dried to obtain the plant polyphenol supramolecular self-assembly system.

5. The method for constructing a plant polyphenol supramolecular self-assembly system according to claim 4, characterized in that: The concentrations of the echinacoside dilution solution and the ferrous gluconate dilution solution were both 1 mM.

6. The method for constructing a plant polyphenol supramolecular self-assembly system according to claim 4, characterized in that: The heating reaction was carried out at 100 rpm.

7. Use of the plant polyphenol supramolecular self-assembly system according to any one of claims 1 to 3 in the preparation of drugs for treating neurodegenerative diseases, characterized in that: The neurodegenerative disease is Alzheimer's disease.

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