Plant polyphenol supramolecular self-assembly system as well as construction method and application thereof

Abstract

The invention relates to the technical field of supramolecular chemistry, and particularly discloses a plant polyphenol supramolecular self-assembly system and a construction method and application thereof, and the plant polyphenol supramolecular self-assembly system is obtained by mixing echinacoside and ferrous gluconate in an environment with pH of 7-11 according to a molar ratio of (5-6): 4, heating and reacting at 40-45 DEG C for 20-24 hours, and dialyzing and drying. The plant polyphenol supramolecular self-assembly system provided by the invention shows an excellent drug release behavior, shows remarkable pH responsiveness in an acid environment, effectively enhances the antioxidant and neuroprotective effects of ECH, and remarkably improves the blood-brain barrier transmittance of ECH.

Description

Technical Field

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

[0002] Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are one of the major challenges facing the global health field. These diseases involve pathological mechanisms such as oxidative stress and inflammatory response and are very complex, and effective treatments are urgently needed. Echinoside (ECH), as a natural extract, has multiple pharmacological activities, especially neuroprotective and antioxidant effects, and has therefore received widespread attention. Many studies have shown that ECH can delay the progression of neurodegenerative diseases by inhibiting oxidative stress and inflammatory response. However, the low bioavailability and limited blood-brain barrier (BBB) ​​permeability of ECH, and the polar groups in ECH limit the absorption and distribution of ECH in the body, resulting in a low effective concentration in the target tissue. In addition, the BBB, as a natural barrier of the central nervous system, further limits the brain delivery of ECH, making it difficult to fully exert its neuroprotective effect in clinical practice. Therefore, it is particularly necessary to explore drug delivery systems that enhance the blood-brain barrier permeability of ECH. Summary of the invention

[0003] In order to develop a drug delivery system that enhances the blood-brain barrier permeability of ECH, the present invention provides a plant polyphenol supramolecular self-assembly system and its construction method and application. 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.

[0004] The invention provides a plant polyphenol supramolecular self-assembly system. The plant polyphenol supramolecular self-assembly system is prepared by mixing echinacea glycoside and ferrous gluconate in a molar ratio of 5 to 6:4 in a liquid environment with a pH value of 7 to 11, heating the mixture at 40 to 45° C. for a reaction for 20 to 24 hours, and then dialyzing and drying the mixture to obtain the system.

[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 blood-brain barrier permeability.

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

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

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

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

[0010] Echinacea glycoside was prepared into ECH stock solution with methanol, and ferrous gluconate was prepared into FG stock solution with purified water. They were diluted to obtain ECH dilution solution and FG dilution solution respectively, and the pH was adjusted. Then, the ECH dilution solution and the FG dilution solution were mixed with the molar ratio of echinacea glycoside to ferrous gluconate being 5 to 6:4. The mixture was heated at 40°C to 45°C for reaction for 20h to 24h, and then dialyzed and dried to obtain the plant polyphenol supramolecular self-assembly system.

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

[0012] Further, the heating reaction was carried out at 100 rpm.

[0013] The present invention also provides the use of the plant polyphenol supramolecular self-assembly system in the preparation of drugs for treating neurodegenerative diseases. The plant polyphenol supramolecular self-assembly system is used as a drug carrier to improve the blood-brain barrier permeability and enhance the anti-oxidation effect of ECH.

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

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

[0016] Compared with the prior art, the present invention has the following beneficial effects:

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

[0018] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The color changes of 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 This is a transmission electron microscope (TEM) image of the ECH@FG system prepared in Example 1;

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

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

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

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

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

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

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

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

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

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

[0032] Figure 6 This is the activity curve of the ECH@FG system prepared in Example 1 in the ABTS free radical scavenging experiment.

[0033] Figure 7 This is the activity curve of the ECH@FG system prepared in Example 1 in the DPPH free radical scavenging experiment.

[0034] Figure 8Effects of the ECH@FG system prepared in Example 1 and ECH alone on HT22 cell viability, Control l: control group; Mode l: 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] Fig. 9 Effects of the ECH@FG system and ECH prepared in Example 1 on the levels of SOD and MDA in HT22 cells, Control l: control group; Mode l: 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 effect of ECH@FG system and ECH on SOD content in HT22 cells;

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

[0038] Fig.10 is the blood-brain barrier permeability of ECH and ECH@FG system;

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

[0040] B is the statistical diagram of blood-brain barrier permeability of ECH and ECH@FG systems. DETAILED DESCRIPTION

[0041] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and 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 10mM echinacoside (ECH) stock solution with 30% volume fraction of methanol, and prepare 10mM ferrous gluconate (FG) stock solution with purified water. Dilute the ECH stock solution to 1mM with purified water to obtain ECH dilution solution, and dilute the FG stock solution to 1mM with purified water to obtain FG dilution solution. Adjust the pH of the ECH dilution solution to 9.5 with 0.05M HCl and 0.05M NaOH solution to ensure stability. Slowly add 4mL of FG dilution solution to 5mL of ECH dilution solution and quickly shake to mix. Heat the entire reaction system at 40℃ and 100rpm for 24 hours to obtain ECH@FG solution. 2 mL of ECH@FG solution was placed in a dialysis bag and stirred at 145 rpm in 200 mL of 10% PBS buffer at 37°C. After 60 min, the dialysis medium (10% PBS buffer) was replaced and the dialysis was repeated 3 times to remove the unassembled drug. The sample in the dialysis bag was collected as the ECH@FG supramolecular self-assembly solution, and the ECH@FG supramolecular lyophilized powder was obtained after lyophilization.

[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 usage ratio of the ECH diluent and the FG diluent 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 the pH of the ECH dilution solution is adjusted 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 plant polyphenol supramolecular self-assembly system.

[0055] 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 1 hour.

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

[0057] 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 30 min.

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

[0059] The preparation steps are basically the same as those in Example 1, except that the usage ratio of the ECH diluent and the FG diluent is 1 mL:1 mL.

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

[0061] The preparation steps are basically the same as those in Example 1, except that the usage ratio of the ECH diluent to the FG diluent is 2 mL:1 mL.

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

[0063] The preparation steps are basically the same as those in Example 1, except that the usage ratio of the ECH diluent and the FG diluent is 5 mL:3 mL.

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

[0065] The preparation steps are basically the same as those in Example 1, except that the pH of the ECH dilution solution is adjusted to 3.

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

[0067] The preparation steps are basically the same as those in Example 1, except that the pH of the ECH dilution solution is adjusted to 5.

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

[0069] The preparation steps are basically the same as those in Example 1, except that the pH of the ECH dilution solution is adjusted to 6.

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

[0071] The preparation steps are basically the same as those in Example 1, except that the pH of the ECH dilution solution is adjusted to 7.

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

[0073] The preparation steps are basically the same as those in Example 1, except that the pH of the ECH dilution solution is adjusted to 8.

[0074] 1. Detecting the effect of heating time on self-assembly, the particle size and PDI value of the plant polyphenol supramolecular self-assembly system prepared in Example 1 and Comparative Examples 1-4 were detected. The detection method and results are as follows.

[0075] 1. Detection method:

[0076] The average particle size and PDI value of the self-assembled system were determined using dynamic light scattering (DLS) technology. The specific operating conditions were: after the sample was diluted to 1 mg / mL, the measurement was performed at 25°C, and each sample was measured three times to obtain the average value. The results are recorded in Table 1.

[0077] Table 1 Effect of heating time on particle size and PDI of 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: PDI 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 the particles is. The closer the value is to 1, the more uneven the particle size distribution is and the greater the size difference between the particles is.

[0080] It can be seen from Table 1 that with the extension of heating time, 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 at this time has good stability and uniformity.

[0081] 2. Detection of the effect of different ratios of ECH and FG on the absorbance of the prepared ECH@FG system

[0082] 1. Detection method:

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

[0084] Table 2 Effect of different ratios of ECH and 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] It can be seen from Table 2 that when the ratio of ECH to FG is 5:4, the absorbance of the system reaches the maximum, and the absorbance peak shifts to 383 nm, indicating that the supramolecular system structure is the most stable and the complexation is the strongest at this time. The pH value of the solution is measured to be 11.

[0087] 3. Effect of different pH on the appearance of the prepared ECH@FG system

[0088] Test results such as Figure 1 As shown, when pH=3, the solution is colorless and transparent. When the pH rises to about 5, the solution is gray-green. When the pH is between 7-9, the solution is dark gray. When the pH>9, the solution turns reddish brown, and no precipitation is produced during the process. From the color change, it can be seen that the ECH@FG self-assembly process is regulated by pH. Under the condition of pH>5, ECH begins to complex with the iron ions in FG and form supramolecular self-assemblies of different forms. Combined with the absorbance value, the best pH is 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: Zeta potential was measured by electrophoresis light scattering at 25°C to analyze the surface charge characteristics of the system. The sample concentration was 1 mg / mL and the electrophoresis solution was 10 mM NaCl solution.

[0092] Morphological observation: The microstructure of the supramolecular system was observed using a transmission electron microscope (TEM) and a scanning electron microscope (SEM) to confirm its morphology after self-assembly. The TEM sample was dropped on a copper grid at a concentration of 1 mg / mL and observed at 200 kV after negative staining; the SEM sample was observed at 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, and the sample was in dry powder form and prepared using the KBr tablet method. The detection wavelength of Raman spectroscopy was 1064 nm, the scanning range was 4000-200 cm-1, and the sample was prepared in a solution at a concentration of 1 mg / mL.

[0094] 2. Experimental results

[0095] (1) Morphological observation

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

[0097] Scanning electron microscopy: In order to further confirm the morphology of the ECH@FG system prepared in Example 1, its microstructure was observed using a scanning electron microscope (SEM). Figure 3 As shown, the ECH@FG system prepared in Example 1 has a smooth surface and shows good morphological consistency, which further confirms its uniform nanoparticle structure.

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

[0099] Table 3 Particle size and PDI value of ECH@FG system prepared at different molar ratios of ECH and FG

[0100]

[0101]

[0102] As shown in Table 3 , when the ECH:FG ratio is 1.25:1, the particle size is 107 nm, indicating that most of the particle sizes are concentrated in this range, indicating that the self-assembly system has 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 is 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, stirred at 145 rpm in 200 mL of buffer at 37°C, and samples were taken at regular intervals to determine the released ECH content. At the same time, the antioxidant capacity of the ECH@FG system was evaluated by ABTS and DPPH free radical scavenging experiments, and compared with ECH alone.

[0116] 2. Detection of neuroprotective effects

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

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

[0119] 3. Blood-brain barrier permeability test

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

[0121] 2. Experimental Results

[0122] 1. pH-responsive drug release behavior

[0123] In the treatment of neurodegenerative diseases such as Alzheimer's disease, the lesion site in the central nervous system (CNS) often presents 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 section systematically studies and analyzes this.

[0124] (1) Drug release experiment design

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

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

[0127] pH Drug release rate (% / h) Cumulative release in 16 hours (%) Cumulative release in 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] like Figure 5 As shown in the figure, the ECH@FG system exhibits significant differences in drug release rates under different pH conditions: the system releases drugs faster in an environment with lower pH values ​​(pH 5.5-6.5), while the release rate slows down significantly in a neutral or weakly alkaline environment (pH 7.0-7.5).

[0129] Under pH 5.5, the drug release rate was the highest, with a cumulative release of 70.52% within 16 hours and 85.16% within 48 hours. This indicates that in an acidic environment, the ECH@FG supramolecular structure is easy to disintegrate or break chemical bonds, thereby promoting the rapid release of drugs.

[0130] Under pH 6.0-6.5, the drug release rate was fast, with cumulative release of 78% and 72% within 48 hours, 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 needs.

[0131] Under pH 7.0, the drug release rate was reduced, and the cumulative release within 48 hours was 60.63%, which showed that under a near-neutral environment, the structure of the ECH@FG system was more stable and the drug release was inhibited to a certain extent.

[0132] At pH 7.5, the drug release rate was the lowest, with the cumulative release within 48 hours being only 50%, which indicates that under more alkaline conditions, the system has higher structural integrity and the drug release is slower.

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

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

[0135] Structural stability: Under low 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, changes in the surface charge of the system may lead to particle surface dissociation, thereby accelerating drug release. Under neutral or alkaline conditions, changes in Zeta potential slow down the diffusion of drugs.

[0137] Coordination complexation: The coordination complexation between iron ions and ECH is more easily disintegrated in an acidic environment, resulting in 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, which can achieve rapid release in acidic environments and slow release in neutral or weakly alkaline environments. 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 effect 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. The present invention evaluates the antioxidant capacity of the ECH@FG system through a variety of free radical scavenging experimental systems to better understand the protective role of the system in oxidative stress-related diseases.

[0141] (1) ABTS free radical scavenging experiment

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

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

[0144] sample IC50(mg / mL) Antioxidant Capacity Vc 0.014 high ECH 0.025 middle ECH@FG 0.03 middle

[0145] As shown in Table 6, the antioxidant capacity of the ECH@FG system still has stronger free radical scavenging ability than ECH.

[0146] like Figure 6 As shown in the figure, with the increase of sample concentration, both ECH and ECH@FG systems showed strong free radical scavenging ability, especially at a concentration of 0.5 mg / mL, the scavenging rate of ECH was close to that of the Vc group, while 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 free radical scavenging experiment

[0148] The results are as follows Figure 7 As shown in the figure, with the increase of sample concentration, each sample showed a higher DPPH free 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 shows that the ECH@FG system not only protects the antioxidant activity of ECH, but also effectively plays a role in free radical scavenging.

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

[0150] sample IC50(mg / mL) Antioxidant Capacity Vc 0.012 high ECH 0.02 middle ECH@FG 0.028 middle

[0151] As shown in Table 7, the antioxidant activity of the ECH@FG system indicated that it had 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 polyphenol compound, the phenolic hydroxyl structure of ECH has a strong free radical scavenging ability. The introduction of FG did not weaken this ability, but may have improved the stability of the system through metal chelation and enhanced 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 also may generate more hydroxyl radicals by participating in the Fenton reaction, thereby enhancing the free radical scavenging ability.

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

[0157] The antioxidant activity of the ECH@FG supramolecular system was fully verified by ABTS and DPPH free radical scavenging experiments. Although the introduction of FG slightly reduced the single scavenging ability of ECH, its overall antioxidant performance still showed excellent results. This shows that the application of the ECH@FG system in oxidative stress-related diseases has broad prospects, especially when long-term 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 HT22 cell model

[0160] The present invention uses CCK-8 method to detect the cell viability of different treatment groups and evaluates the protective effect of ECH and ECH@FG system on oxidative damage. Figure 8 As shown. The cell viability of the control group (Control l) remained close to 100%, while the cell viability of the model group (Mode l) 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 shows that both ECH and ECH@FG have good neuroprotective effects and can significantly reduce glutamate-induced cell oxidative 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 Fig. 9 shown.

[0163] Table 8 Effects of ECH@FG and ECH treatment 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] like Fig. 9 A, the SOD activity of the control group was the highest, and the SOD activity of the model group was significantly reduced to 0.8U / mg. The SOD activity of the ECH and ECH@FG treatment groups was restored, especially the SOD activity of the ECH@FG treatment group reached 1.2U / mg, showing a stronger antioxidant capacity.

[0168] like Fig. 9 B. The MDA content in the model group increased significantly to nearly 2.0 nmol / mg protein, while the MDA content in the ECH and ECH@FG treatment groups was significantly reduced to 1.2 and 1.0 nmol / mg protein, respectively, further demonstrating its protective effect on cells.

[0169] (3) Analysis of neuroprotective mechanisms

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

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

[0172] Reducing lipid peroxidation: MDA is a signature product of intracellular lipid peroxidation. The reduction 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] Protective effect of FG: FG not only makes the supramolecular system more stable, but also enhances the neuroprotective effect of ECH by improving its bioavailability and stability. This synergistic effect makes ECH@FG show a stronger protective effect in response to oxidative stress.

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

[0175] 4. Blood-brain barrier permeability

[0176] The application prospects of the ECH@FG supramolecular system in drug delivery, especially in crossing the blood-brain barrier (BBB), are the key to its potential treatment of central nervous system diseases such as Alzheimer's disease. In order to evaluate the BBB permeability of the ECH@FG system, the present invention constructed an in vitro BBB model and revealed the cross-BBB delivery potential of the system through experiments.

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

[0178] The present invention uses the classic Transwell chamber model to construct an in vitro blood-brain barrier model. HT22 mouse hippocampal neuronal cells are inoculated in the lower layer of the Transwell chamber, and bEnd.3 brain microvascular endothelial cells are inoculated in the upper layer. After 48 hours of co-culture, the integrity of the BBB is confirmed by resistance measurement. When the resistance value exceeds 50Ω, the model is considered to be successfully constructed and can be used for subsequent experiments.

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

[0180] ECH and ECH@FG systems were added to the successfully constructed BBB model for permeation experiments. After 4 hours of incubation, the ECH content in the lower layer liquid was detected to evaluate its BBB permeability. The experimental results are shown in Figure 2. Fig.10 The permeability of ECH in the free state is 4.02%, and the permeability of ECH in the ECH@FG supramolecular system is significantly increased to 12.18%, indicating that the supramolecular system has obvious advantages in enhancing the ability of ECH to deliver across the BBB.

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

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

[0183] Nano-size effect: The nano-scale supramolecular structure formed by the self-assembly of the ECH@FG system is easier to enter the central nervous system through the intercellular space. Nanoparticles can cross the BBB through various pathways such as adsorption, transport, and transmembrane transport.

[0184] Surface charge effect: Zeta potential analysis showed that the ECH@FG system has a moderate positive charge (about 6.73 mV), which may help it interact with the anionic phospholipid bilayer on the BBB, thereby 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 TfR-1 (transferrin receptor 1)-mediated pathway.

[0186] The results of in vitro BBB model studies show that the ECH@FG supramolecular system can significantly improve the blood-brain barrier permeability of ECH, showing excellent drug delivery potential. This discovery 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 the foundation for the further development of drug carriers with efficient BBB-crossing capabilities.

[0187] In summary, the present invention successfully constructed a plant polyphenol supramolecular self-assembly system (ECH@FG system), which exhibited excellent drug release behavior and significant pH responsiveness in an acidic environment. 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 ECH@FG system, which is also called ECH@FG supramolecular self-assembly system or ECH@FG supramolecular system in the present invention.

[0189] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0190] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A plant polyphenol supramolecular self-assembly system, characterized in that: The plant polyphenol supramolecular self-assembly system is obtained by mixing echinacoside and ferrous gluconate in a molar ratio of 5 to 6:4 in a liquid environment with a pH of 7 to 11, heating and reacting at 40 to 45° C. for 20 to 24 hours, and then dialyzing and drying.

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 molar ratio of echinacoside to ferrous gluconate is 5:

4.

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

5. A method for constructing a plant polyphenol supramolecular self-assembly system according to any one of claims 1 to 4, characterized in that: The steps include: Echinacea glycoside was prepared into ECH stock solution with methanol, and ferrous gluconate was prepared into FG stock solution with purified water. They were diluted to obtain ECH dilution solution and FG dilution solution respectively, and the pH was adjusted. Then, the ECH dilution solution and the FG dilution solution were mixed with the molar ratio of echinacea glycoside to ferrous gluconate being 5 to 6:

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

6. The method for constructing the plant polyphenol supramolecular self-assembly system according to claim 5, characterized in that: The concentrations of the ECH dilution solution and the FG dilution solution were both 1 mM.

7. The method for constructing a plant polyphenol supramolecular self-assembly system according to claim 5, characterized in that: The heating reaction was carried out at 100 rpm.

8. Use of the plant polyphenol supramolecular self-assembly system according to any one of claims 1 to 4 in the preparation of drugs for treating neurodegenerative diseases, characterized in that: The plant polyphenol supramolecular self-assembly system is used as a drug carrier to improve the blood-brain barrier permeability and enhance the anti-oxidation of ECH.

9. Use of the plant polyphenol supramolecular self-assembly system according to claim 8 in the preparation of drugs for treating neurodegenerative diseases, characterized in that: The plant polyphenol supramolecular self-assembly system is used for preparing medicine for protecting nerves.

10. Use of the plant polyphenol supramolecular self-assembly system according to claim 8 in preparing drugs for treating neurodegenerative diseases, characterized in that: The neurodegenerative disease is Alzheimer's disease.

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