Isoprenyl flavonoid compound, preparation method and application thereof

Abstract

The application discloses a kind of isoprenyl flavonoids compounds, preparation method and application, the compound is by activating Keap-Nrf2 / HO-1 signal path and exerting neuroprotective effect.Observed compound is isolated from Hypericum perforatum L. (Hypericum perforatum L.) And compound 1 is new compound.Neuroprotective experiment of H2O2 induced PC12 cell oxidative stress, cell apoptosis experiment, lactate dehydrogenase inhibition experiment, Western blotting experiment, molecular dynamics simulation and zebra fish in vivo model evaluate the neuroprotective activity of compound 1 and 2.Compound 1 and 2 have obvious neuroprotective effect on H2O2 induced PC12 cell, and resist damage caused by oxidative stress through Keap-Nrf2 / HO-1 signal.In zebra fish model, compound 1 significantly inhibits H2O2 caused zebra fish brain neuron cell apoptosis.The above research results show that compound 1, first discovered, is a potential neuroprotective lead compound.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an isopentenyl flavonoid compound, its preparation method and application. Specifically, it relates to isopentenyl biflavonoids extracted and isolated from the aerial parts of Hypericum perforatum, its preparation method and its neuroprotective effect in neurodegenerative diseases. Background Technology

[0002] Neurodegenerative diseases (NDDs) are a class of diseases caused by the loss of neurons and / or their myelin sheaths. As the disease progresses, functional impairments are manifested, such as Alzheimer's disease (AD) and Parkinson's disease (PD). They are recognized as serious brain diseases and are a global health care challenge. AD is the most common cause of dementia, accounting for 60-80% of dementia cases. PD is the second most common neurodegenerative disease in the world after AD, with an incidence rate of about 1-2% in people over 50 years old. The pathogenesis of this type of disease is complex, and the search for potential drugs for treatment and prevention has become a research hotspot. The inducing factors of NDD include: (1) Oxidative stress: Free radicals cannot be cleared in a timely and effective manner, causing damage to cells and tissues. In recent years, it has been found that oxidative damage to nerve tissues exists in both AD and PD. (2) Mitochondrial dysfunction: Mitochondrial DNA defects, such as breaks, base deletions, and missense mutations; increased number of mitochondria, and the appearance of lamellar bodies and crystalline inclusion bodies in the structure. This is related to oxidative stress damage and neuronal apoptosis caused by ROS release. (3) Excitotoxicity: Excessive glutamate in the intercellular space can be toxic to neurons, causing them to degenerate and die. Glutamate is an excitotoxic amino acid, and its excitotoxicity is also related to nerve damage. (4) Immune inflammation: Inflammation is an important factor in inducing AD, which can activate the innate immune system and cause immune damage to the brain.

[0003] For centuries, St. John's wort has been used in traditional medicine. Modern research has shown that extracts and monomeric compounds from this plant (such as hypericin and flavonoids like rutin, hyperoside, and quercetin) possess neuroprotective activity. [1 4] These effects are achieved through direct action via multiple mechanisms, such as antioxidant or anti-apoptotic effects, playing a crucial role in the prevention and treatment of neurodegenerative diseases. Therefore, further research into the chemical composition of Hypericum perforatum, aiming to discover lead compounds with novel structures and significant anti-neuritis and neuroprotective activities, is an important strategy for developing drugs to treat neurodegenerative diseases. The isopentenyl flavonoids involved in this invention possess excellent neuroprotective effects, and no patents or literature reports have been found to date.

[0004] References:

[0005] [1]Liu, R., Zhang, T. T., Zhou, D., et al. 2013. Quercetin protects against the Abeta(25-35)-induced amnesic injury: involvement of inactivation of rage-mediated pathway and conservation of the NVU. Neuropharmacology, 67, 419–431. [2]Liu, Z., Tao, X., Zhang, C., Lu, Y. 2005. Protective effects of hyperoside (quercetin-3-o-galactoside) on PC12 cells against cytotoxicity induced by hydrogen peroxide and tert-butyl hydroperoxide. Biomedicine & Pharmacotherapy. 59, 481–490. [3]Oliveira, A.I., Pinho, C., Sarmento, B., 2016. Neuroprotective activity of Hypericum perforatum and its major components. Frontiers in Plant Science. 7, 1004. [4]Zeng, K. W., Wang, X. M., Ko, H., et al. 2011. Hyperoside protects primary rat cortical neurons from neurotoxicity induced by amyloid beta-protein via the PI3K / Akt / Bad / Bcl(XL)-regulated mitochondrial apoptotic pathway [J]. European Journal of Pharmacology. 672, 45–55. Summary of the Invention This invention provides an isopentenyl flavonoid compound, its preparation method, and its application. Specifically, it describes the isopentenyl flavonoid compound and its neuroprotective effect in neurodegenerative diseases by activating the Keap-Nrf2 / HO-1 signaling pathway.

[0006] The specific plan includes: Isoprene flavonoids, wherein the compounds include: ; and / or .

[0007] The method for preparing isopentenyl flavonoids according to the present invention involves isolating the isopentenyl compounds from Hypericum perforatum.

[0008] Optionally, the isopentenyl flavonoids are all isolated from the ethyl acetate phase of the ethanol extract of Hypericum perforatum.

[0009] Optional, specifically including: The aerial parts of St. John's wort were extracted with 95% ethanol to obtain a total ethanol extract. The total ethanol extract was then suspended in water and extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was eluted using a dichloromethane-methanol gradient under the following conditions: v / v , 100:0 → 0:100; 4 fractions Fr Ⅰ are obtained. IV; Fr Ⅳ was eluted with a dichloromethane / ethyl acetate gradient under the following conditions: v / v , 50:1→1:1; 8 fractions Fr Ⅳ1~Ⅳ8 were obtained, and then Fr Ⅳ5 was passed through a reverse-phase RP-18 column with MeOH-H2O to v / v 20% → 100% elution, Sephadex LH-20 gel column elution at CHCl3 / MeOH volume ratio 1:1, and semi-preparative HPLC separation and purification at MeOH-H2O volume concentration of 60%-100% yielded compounds 1 and 2.

[0010] The isopentenyl flavonoids described in this invention are used in the preparation of neuroprotective drugs.

[0011] The isopentenyl flavonoids described in this invention are used in the preparation of drugs for treating neurodegenerative diseases.

[0012] The isopentenyl flavonoids described in this invention are used in the preparation of drugs for treating Alzheimer's disease and / or Parkinson's disease.

[0013] The isopentenyl flavonoids described in this invention are used in the preparation of drugs that regulate the Keap-Nrf2 / HO-1 signaling pathway.

[0014] A medicine for treating neurodegenerative diseases, the medicine comprising the isopentenyl flavonoid compound described in this invention.

[0015] A neuroprotective drug, wherein the drug contains the isopentenyl flavonoid compound described in this invention.

[0016] The isopentenyl flavonoids of this invention, as small molecule neuroprotective drugs, have the potential for development and application in the prevention and treatment of neurodegenerative diseases. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings: Figure 1 The effects of compounds 1 and 2 on H2O2-induced cell viability, lactate dehydrogenase (LDH) levels, and apoptosis in PC-12 cells were investigated. Figure 1 A represents the effects of compounds 1 and 2 on H2O2-induced cell survival of PC-12 cells; Figure 1 B represents the effects of compounds 1 and 2 on LDH levels induced by H2O2 in PC-12 cells; Figure 1 C represents the effect of compounds 1 and 2 on the apoptosis rate induced by H2O2 in PC-12 cells; Figure 1 D represents the quantitative fluorescence values ​​of compounds 1 and 2 in inhibiting apoptosis; data are expressed as the mean ± standard deviation of three independent experiments. # p <0.05, ## p <0.01, ### p <0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the H2O2 treatment group; Figure 2 The effects of compounds 1 and 2 on the levels of antioxidant proteins induced by H2O2 in PC-12 cells; among which Figure 2 A and Figure 2 B represents the effects of compounds 1 and 2 on NQO-1 and Cat protein levels; Figure 2 C and Figure 2D represents the effects of compounds 1 and 2 on Nrf2 and HO-1 protein levels; data are expressed as mean ± standard deviation of three independent experiments. # p <0.05, ## p <0.01, ### p <0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the H2O2 treatment group; Figure 3 Molecular docking of compound 1 with Keap1 protein (PDB: 7PNP); Figure 3 A is a three-dimensional diagram of the interaction between compound 1 and the Keap1 protein molecule; Figure 3 B is a two-dimensional diagram of the interaction between compound 1 and the Keap1 protein molecule; Figure 4 Molecular dynamics simulations of compound 1 and Keap1 protein; Figure 4 A is the root mean square deviation (RMSD) plot; Figure 4 B is the root mean square fluctuation (RMSF) plot; Figure 4 C is the radius of gyration (Rg); Figure 4 D is the solvent accessible surface area (SASA); Figure 4 E is a hydrogen bond; Figure 4 F is the combined free energy diagram; Figure 5 The effect of compound 1 on the apoptosis rate of brain cells in a H2O2-induced zebrafish model; Figure 6 The hydrogen spectrum of compound 1; Figure 7 The carbon spectrum of compound 1; Figure 8 The DEPT spectrum of compound 1; Figure 9 For compound 1 1 H- 1 H COSY spectrum; Figure 10 The HSQC spectrum of compound 1; Figure 11 The HMBC spectrum of compound 1; Figure 12 The NOESY spectrum of compound 1; Figure 13 The HRESIMS spectrum of compound 1; Figure 14The hydrogen spectrum of compound 2; Figure 15 This is the carbon spectrum of compound 2. Detailed Implementation

[0018] This invention provides isopentenyl flavonoids and their neuroprotective effects in neurodegenerative diseases by activating the Keap-Nrf2 / HO-1 signaling pathway.

[0019] The structural formula of the isopentenyl flavonoid compound of this invention is shown below: ; Physicochemical properties of compound (1) of the present invention: C 52 H 60 O 12 Yellow amorphous powder; [α] 20 D + 40.7 ( c 0.015MeOH); HR-ESIMS (positive) m / z 877.4158 [M + H] + (Calculated value C) 52 H 61 O 12 , 877.4157); 1 H-NMR spectra and 13 C-NMR spectral data are shown in Table 1, and spectra are attached. Figure 6-13 .

[0020] The compound of the present invention (2″) S )-5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone (2) Physicochemical properties: C 26 H 30 O6, red amorphous powder; HR-ESIMS (positive) m / z 439.2076 [M + H] + ; 1 H-NMR spectra and 13 The C-NMR spectral data are shown in Table 2, and the spectra are attached. Figure 14-15 .

[0021] The present invention also provides a method for preparing the compound, comprising the following steps: (1) Using St. John's wort as raw material, the extract was heated and refluxed three times with 95% ethanol for 2 hours each time. The ethanol extract was concentrated under reduced pressure and then suspended in an equal proportion of water to obtain an aqueous solution.

[0022] (2) Add equal volumes of petroleum ether, ethyl acetate, and n-butanol to each extract three times. Concentrate the ethyl acetate extract to obtain an extract.

[0023] (3) The ethyl acetate extract was subjected to multiple silica gel column chromatography tests, using dichloromethane-methanol solution (100:0) 1:1, v / v Gradient elution was performed. Sephadex LH-20 gel chromatography (CH2Cl2 / MeOH, 1:1) was used to remove pigments and separate them into different fractions.

[0024] (4) The target fraction was purified by ODS high performance liquid chromatography, and compound 1 was obtained by using acetonitrile-water as the mobile phase (83% CH3CN).

[0025] (5) The target fraction was passed through a Sephadex LH-20 gel column (CH2Cl2 / MeOH, 1:1) and a normal-phase silica gel column at CH2Cl2 / MeOH (50:1) 2:1, v / v Gradient elution was performed, followed by purification via semi-preparative liquid chromatography (MeOH:H2O = 82:18). v / v ), to obtain compound 2.

[0026] Structural analysis of compound 1: High-resolution mass spectrometry in m / z [M + H] appears at position 877.4158. + Peak, combined 1 H and 13 C-NMR data show that the molecular formula of the compound is C 52 H 60 O 12 The degree of unsaturation is 23. From 1 ¹H NMR revealed that the compound had 6 methyl signals. δ H 1.69 (3H, s); 1.65 (3H, s); 1.62 (3H, s); 1.60 (3H, s); 1.55 (3H, s); 1.51 (3H, s);] (numbers sorted from largest to smallest), 2 methoxy groups [3.79 (3H, s); 3.75 (3H, s)], 4 olefin protons [5.00 (1H, overlap); 5.00 (1H, overlap); 7.70 (1H, d, J = 15.5 Hz); 8.03 (1H, d, J = 15.5 Hz), 4 terminal double hydrogen bonds [4.58, 4.54 (2H, d, J= 2.1 Hz); 4.52, 4.48 (2H, d, J = 2.1 Hz). In combination 13 C10 NMR and HSQC spectra indicate that the compound contains 52 carbon atoms, including 6 methyl groups, 7 methylene groups (2 terminal double bonds), 14 methine groups (2 alkene carbons), 2 methoxy carbons, and 23 quaternary carbons (2 carbonyl carbons and 10 oxidized quaternary carbons). 1 H NMR showed that, δ H 6.94 (1H, d, J = 6.0 Hz, H-6"'), 6.31 (1H, s, H-3"') and 6.26 (1H, d, J = 6.0 Hz, H-5"'), indicating the presence of a benzene ring in the ABX system. Four monoaromatic protons. δ H 7.24 (1H, s, H-6'), 6.30 (1H, s, H-3'), 5.95 (1H, s, H-6) and 5.95 (1H, s, H-6'') are 1,2,4,5-tetrasubstituted or pentasubstituted benzene rings, and δ H 7.70 (1H, d, J = 15.5Hz), 8.03 (1H, d, J = 15.5 Hz), indicating the presence of chalcones. α , β -Unsaturated ketone structural units. In addition to the carbons mentioned above, in δ C (194.5, 162.3, 163.9, 108.5, 166.0, 106.4, 115.6, 159.9, 125.7, 124.2, 139.8, 205.4, 162.6, 166.7, 108.7, 163.9, 106.2, 48.8, 34.0) indicates that the compound is a flavonoid composed of one molecule of chalcone and one molecule of dihydrochalcone. From H-β ( δ H 5.01) / C-5' ( δ C 125.67) and C-6' (129.71), H-α ( δ H 3.69) / C-5' ( δ CThe HMBC correlation of 125.6) clearly identifies that these two units are connected via C. 5' -C β The bonds are linked. Further analysis of the compound's 2D NMR spectrum ( 1 H- 1 H COSY, HSQC, HMBC) analysis, in 1 H- 1 The correlations between H-2a / H-3a / H-4a and H-9a / H-10a in H COSY, and the long-range coupling relationships between H-7a / C-5a, 4a and H-2a / C-1a, C-10a in the HMBC spectrum, confirm that the compound contains a lavender group. Furthermore, the long-range coupling between H-1a and C-7, C-8, C-9 indicates that this group is attached at the C-8 position. Two methoxy δ- groups... H 3.79 and 3.75 (3H, s) are respectively related to C-5 (δ C 162.34), C -5" (δ C (162.34) Linked. Based on the above 1D and 2D NMR comprehensive analysis, the planar structure of the compound was determined as shown in the figure. It is a dimer formed by two monomers, 7,9,2',4'-tetrahydroxy-8-lavandulyl-5-methoxyflavanone and 7'',9'',2''',4'''-tetrahydroxy-8''-lavandulyl-5''-methoxydihydrochalcone, linked by C- bonds. The ECD spectrum of the compound shows positive absorption in the range of 205-215 nm and negative absorption in the range of 240 nm, which determines C- β It has an S-configuration.

[0027] Table 1: Compound 1 1 H NMR (400 MHz, Methanol- d 4) and 13 C NMR (100 MHz, Methanol-) d 4) Data

[0028] Table 2: Compound 2 1 H NMR (400 MHz, Methanol- d 4) and 13 C NMR (100 MHz, Methanol-) d 4) Data

[0029] The isopentenyl flavonoids described in this invention have good neuroprotective activity, and their mechanism of action was further studied using H2O2-induced PC12 cell oxidative stress model and zebrafish model.

[0030] The drug for treating neurodegenerative diseases contains the isopentenyl flavonoid compound described in this invention.

[0031] Example 1: Preparation of compounds 1 and 2 Using the dried aerial parts of *Hypericum perforatum* as raw material, the extract was heated to reflux with 95% ethanol three times, 2 hours each time. The extracts were combined and concentrated under reduced pressure to obtain an ethanol extract, which was then suspended in an equal proportion of water to obtain an aqueous solution. Equal volumes of petroleum ether, ethyl acetate, and n-butanol were added separately for extraction three times each. The ethyl acetate extracts were combined and concentrated to obtain a paste. The ethyl acetate extract was subjected to silica gel column chromatography (200-300 mesh) with dichloromethane-methanol solution (100:0 → 0:100). v / v Gradient elution was performed, and the fractions were separated into four fractions (FrⅠ). FrⅣ). FrⅣ was further separated by silica gel column chromatography, using dichloromethane-ethyl acetate as eluent (50:0 → 1:1, v / v The fraction was divided into 8 fractions by gradient elution (Fr Ⅳ1) FrⅣ8), FrⅣ3 was further eluted with Sephadex LH-20 gel (CH2Cl3 / MeOH, 1:1) to separate into 5 fractions (FrⅣ1A). FrⅣ8E). FrⅣ8B was further separated by silica gel column chromatography, using dichloromethane-methanol as the eluent (100:0 → 10:1). v / v The fraction was divided into four fractions (FrⅣ8Ba – FrⅣ8Bd) by gradient elution. FrⅣ8Bb was purified by ODS high-performance liquid chromatography with acetonitrile-water as the mobile phase (83% CH3CN) to give compound 1. t R = 16 min, 37.0 mg). Fr Ⅳ8Bc was further purified by gel chromatography with Sephadex LH-20 (CH2Cl2 / MeOH, 1:1) and normal-phase silica gel chromatography with CH2Cl2 / MeOH (50:1). 2:1 (v / v) gradient elution, and finally semi-preparative high performance liquid chromatography with methanol-water as the mobile phase (82% MeOH) at a flow rate of 2 ml / min to obtain compound 2 ( t R = 30 min, 159.9 mg).

[0032] Example 2: Pharmacological activity of the compound Test methods 1. Cell viability assay The protective effect of compounds against neuronal cell damage was studied by inducing oxidative stress in PC12 cells using H2O2-induced oxidation. The effective concentration of the compound against H2O2-induced cell damage was assessed using the MTT assay. (1.5 × 10⁻⁶) 5 Cells were seeded at a density of 100 μL / well in 96-well plates and pretreated with the compound for 6 hours, followed by co-treatment with H2O2 (800 μM) and the compound for 14 hours. The following groups were established: blank control (DMSO), model group (H2O2 group), positive control group (epidermal growth factor: NGF, resveratrol: Res), and treatment groups (compounds 1 and 2). After treatment, MTT was added and incubated for another 4 hours. The absorbance (OD value) at 450 nm was read using a microplate reader to calculate the viability of PC-12 cells.

[0033] 2. Lactate dehydrogenase (LDH) inhibition test Five experimental groups were set up: negative group (no PC-12 cells, only culture medium), blank group (PC-12 cells, DMSO), model group (PC-12 cells, H2O2 induced), positive group (PC-12 cells + 40 μM NGF / Res + H2O2), and sample group (PC-12 cells + 10, 20, 40 μM 1 / 2 + H2O2).

[0034] ; PC12 cells were seeded in 96-well plates (1.5 × 10⁻⁶ cells per well). 5 Cells were incubated in 100 μL per well at 37 °C with 5% CO2 for 24 hours. After cell attachment, the cells were pretreated with different concentrations of compounds (10, 20, 40) and positive control for 6 hours, followed by stimulation with H2O2 (800 μM) for 14 hours. After treatment, 10 μL of LDH release reagent from the kit was added to each well, and the mixture was gently pipetted several times to mix. The cells were then incubated in a constant temperature incubator in the dark for 1 hour. Subsequently, 120 μL of supernatant was transferred from each well to a new 96-well plate, and the detection solution was added sequentially. The plates were then shaken at room temperature in the dark for 30 min. The absorbance (OD value) at 450 nm was read using a microplate reader, and the inhibition rate was calculated.

[0035] 3. Apoptosis assay Hoechst 33258 fluorescent dye binds to intracellular DNA, exhibiting bright blue fluorescence. This fluorescence allows for clear visualization of nucleus morphology changes under a fluorescence microscope and is commonly used for apoptosis detection. The study included a control group (PC12 cells + DMSO treatment), a model group (PC12 cells + H2O2 treatment), a positive control group (PC12 cells + 40 μM NGF / Res + H2O2), and sample groups (PC12 cells + 10, 20, and 40 μM 1 / 2 + H2O2). PC12 cells were seeded in 24-well plates (6 × 10⁻⁶ wells). 5 Cells were incubated in wells at 37 °C and 5% CO2 for 24 hours at a concentration of 10, 20, and 40 μM of compound and positive control for 6 hours, followed by stimulation with 800 μM H2O2 for 14 hours. After treatment, 100 μL of prepared Hoechst fluorescent dye was added to each well, and the cells were incubated in a constant temperature incubator in the dark for 20 minutes. The morphological changes of the cell nuclei were observed under an inverted microscope.

[0036] 4. Western blotting test PC12 cells were seeded in 9 cm culture dishes at a density of 1 × 10⁻⁶ cells / cm². 6Cells / mL were collected and mediated separately into DMSO group, H2O2 stimulation group, positive control group, and groups with different concentrations of compounds. After treatment, the culture medium was discarded, and the cells were washed twice with PBS buffer. Lysis buffer containing 1% PMSF was added to each well, and the cells were incubated on ice for 30 minutes for lysis. The cells were then gently pipetted to completely detach the cells, and the samples were collected. The samples were centrifuged at 12,000 rpm for 10 minutes at 4 °C, and the supernatant, i.e., total protein, was collected. The total protein concentration was determined using a BCA kit, and polyacrylamide (SDS-PAGE) gel electrophoresis was performed at a protein concentration of 20 μg. The separated gel was transferred to a polyvinylidene fluoride (PVDF) membrane, and the electrophoresis tank was placed on ice for transfer at a current of 120 mA. The transfer time was determined according to the protein molecular weight: 2 h for proteins above 100 KD and 1 h for proteins below 60 KD. Subsequently, the membrane was incubated with primary antibody at 4 °C for 12 h, followed by incubation with the corresponding secondary antibody for 2 h. Finally, the developing solution was prepared, and the corresponding bands on the PVDF membrane were detected using a chemiluminescence immunoassay analyzer. The grayscale values ​​of each band were analyzed using ImageJ 2.0, and the data were statistically analyzed using SPSS 20 software. The main primary antibodies used were as follows: rabbit monoclonal antibody Nrf2 (1:1000, CST, US), rabbit monoclonal antibody HO-1 (1:1000, CST, US), rabbit monoclonal antibody NQO1 (1:1000, Abways, China), and rabbit monoclonal antibody CAT (1:500, Abways, China).

[0037] 5. Molecular docking and molecular dynamics simulation experiments The interaction between the compound and Keap1 was simulated using AutoDock 4.2.6 software. The structure of the compound was converted into a three-dimensional model. The crystal structure of Keap1 (PDB ID: 7P5P) was downloaded from the protein database (http: / / www.rcsb.org / ). The optimal free energy for the binding of the two compounds was evaluated using the AutoDock tool (ADT 1.5.6).

[0038] Stability analysis of the complex formed by the compound and Keap1 protein was performed using molecular dynamics simulations with the GAFF2-TIP3P force field in GROMACS software. The binding affinity between the compound and Keap1 protein was evaluated using calculated parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF) plots, radius of gyration (Rg), solvent accessible surface area (SASA), hydrogen bonding, and binding free energy over a simulation period of 200 ns.

[0039] 6. In vivo testing Six-day-old zebrafish were placed in 48-well plates, 10 zebrafish per well. They were treated for 6 hours with compounds (5, 10, 20, 40 μM), a positive control (40 μM), and DMSO, followed by treatment with H2O2 (500 μM) at 28.5 °C for 18 h. After treatment, the zebrafish were rinsed three times with purified water and then stained with AO (20 µg / mL) for 20 minutes in the dark. Finally, the zebrafish were washed three times with PBS and anesthetized with tricaine before photography. Apoptosis in the zebrafish brain cells was observed using a fluorescence microscope (Nikon, Japan).

[0040] Test results 1. The protective effect of the compound against H2O2-stimulated PC-12 The MTT assay was used to detect the neuroprotective effect of compounds on H2O2-damaged PC-12 cells. For example... Figure 1 As shown in Figure A, pretreatment of PC12 cells with the compounds for 6 hours significantly improved cell viability at 10 μM and exhibited the best neuroprotective effect in a dose-dependent manner. More importantly, the neuroprotective effect of compounds 1 / 2 (10 μM) was stronger than that of NGF and resveratrol (40 μM). Furthermore, compounds 1 and 2 did not exhibit cytotoxicity at these concentrations.

[0041] 2. The compound inhibits the release of lactate dehydrogenase and apoptosis. H2O2 stimulation of PC-12 cells can induce apoptosis and cell membrane damage, leading to the release of enzymes from the cytoplasm into the culture medium, including lactate dehydrogenase (LDH). Therefore, LDH release is detected to assess the protective effect of compound treatment. Figure 1 As shown in Figure B, H2O2 treatment of PC12 caused cell membrane damage, leading to an increase in lactate dehydrogenase levels in the cytoplasm. Compared with the H2O2-stimulated group, treatment with compounds 1 and 2 significantly reduced LDH release. These results indicate that compounds 1 and 2 can resist cell membrane damage induced by H2O2 stimulation, and compounds 1 / 2 at a concentration of 10 μM have stronger cell membrane protective capabilities than NGF and resveratrol (40 μM). Apoptosis was also used as an indicator to evaluate the neuroprotective effects of the compounds. Figure 1 C and Figure 1 D. Compared to the control group, H2O2 stimulation clearly showed nuclear shrinkage and the formation of apoptotic bodies. However, treatment with different concentrations of the compounds (10, 20, 40 μM) significantly reduced the number of apoptotic bodies. In conclusion, compounds 1 and 2 have good neuroprotective effects.

[0042] 3. The compound exerts its neuroprotective effect by activating the Keap1-Nrf2 / HO-1 signaling pathway. Nrf2 is a key transcriptional activator of antioxidant genes, playing a crucial role in antioxidant protection. When the body is stimulated by oxidative stress, Nrf2 and Keap1 dissociate and translocate to the cell nucleus, binding with ARE to form a co-activator complex to exert neuroprotective effects. Conversely, antioxidant proteins, including HO-1, NQO-1, and CAT, are activated to scavenge free radicals and protect cells from oxidative damage. Figure 2 A Figure 2 As shown in Figure D, compared with the control group, the relative expression levels of antioxidant proteins HO-1, NQO-1, and CAT increased in PC-12 cells after H2O2 stimulation. Compounds 1 and 2 significantly upregulated the expression of these antioxidant proteins in a dose-dependent manner after drug administration. These results indicate that compounds 1 and 2 exert neuroprotective effects through the Nrf2 / HO-1 pathway.

[0043] The Keap1 protein contains five domains: the N-terminal region (NTR), the intervening region (IVR), the BTB (Broad complex, Tramtrack, and Bric-à-Brac) region, the double glycinate repeat region (DGR), and the C-terminal region (CTR). The DGR region, also known as the Kelch region, is the binding region between Keap1 and the Neh2 site of Nrf2. Molecular docking was used to study the interaction between compound 1 and the Kelch region of the Keap1 protein, such as... Figure 3 As shown in Figure A, compound 1 occupies the Kelch region precisely, preventing the binding of Keap1 protein to Nrf2. Figure 3 As shown in Figure B, compound 1 binds to the active site residues TYR 572, SER602, SER555, and GLN 530 of the Keap1 protein via hydrogen bonds, with a binding affinity of -8.5 kcal / mol. Simultaneously, molecular dynamics simulations were used to assess the conformational changes during ligand-protein interactions. Compared to molecular docking, trajectory analysis can elucidate the structural changes during the formation of the compound-Keap1 complex. RMSD reflects the degree of positional change of the protein or ligand-protein complex over time. Larger RMSD values ​​indicate instability of the examined structure. Figure 4 As shown in Figure A, the calculated average RMSD of the Keap1 protein was observed to be 3.52 Å, and that of the ligand-protein complex was 2.55 Å. Furthermore, the structural changes within the complex were more stable after ~20 ns. The RMSF values ​​elucidated the degree of fluctuation in atomic motion within the molecule, revealing fluctuations of 19.91 Å and 14.31 Å for the unbound and bound structures, respectively. Figure 4 B). The radius of gyration (Rg) explains the overall stability of the structure and is used to characterize the compactness of the protein structure during simulation. Significant fluctuations were observed until ~25 ns, after which a more stable conformation was obtained upon completion of the simulation. According to Rg... Figure 4 C, the calculated value for the protein is 17.88 Å, and the calculated value for the ligand-protein complex is 17.76 Å. The decrease in radius of gyration reveals the compactness of the structural rearrangement up to 200 ns, indicating that the studied complex has enhanced stability. The calculated solvent-accessible surface area (SASA) value decreases in the complex structure, indicating that the results are consistent with Rg ( Figure 4 D). Furthermore, the strength of the interaction between the compound and the protein is estimated using hydrogen bonds. Figure 4 E indicates that the number of hydrogen bonds in the complex is increased compared to the protein. Finally, the binding free energy of the protein-ligand complex was calculated using the MM-PBSA method. The calculated binding energy of the protein-ligand complex is -24.22 ± 6.74 kcal / mol. Figure 4 F).

[0044] 4. Zebrafish model demonstrates that the compound inhibits nerve cell apoptosis. To assess the protective effect of the compound against H2O2-induced brain injury, apoptosis was measured in zebrafish by AO staining with either the compound and H2O2 or H2O2 alone. Figure 5 As shown, apoptotic bodies, i.e., green fluorescent dots, were clearly observed after H2O2 stimulation compared to the control. However, the number of apoptotic bodies was significantly reduced after drug treatment. These results indicate that compound 1 has a neuroprotective effect.

[0045] In conclusion, isopentenyl flavonoids can be developed as promising lead compounds for the prevention and treatment of neurodegenerative diseases.

[0046] The embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0047] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0048] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. An isopentenyl flavonoid compound, characterized in that, The compound is selected from: ； and .

2. The method for preparing the isopentenyl flavonoid compound according to claim 1, characterized in that, The isopentenyl compounds were isolated from Hypericum perforatum, and specifically include: The aerial parts of St. John's wort were extracted with 95% ethanol to obtain a total ethanol extract. The total ethanol extract was then suspended in water and extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was eluted using a dichloromethane-methanol gradient under the following conditions: v / v , 100:0 → 0:100; 4 fractions FrⅠ-Ⅳ were obtained; Fr Ⅳ was eluted with a gradient of dichloromethane / ethyl acetate under the following conditions: v / v , 50:1→1:1; 8 fractions Fr Ⅳ1~Ⅳ8 were obtained, and then Fr Ⅳ5 was passed through a reverse-phase RP-18 column with MeOH-H2O to v / v 20% → 100% elution, Sephadex LH-20 gel column elution at CHCl3 / MeOH volume ratio 1:1, and semi-preparative HPLC separation and purification at MeOH-H2O volume concentration of 60%-100% yielded compounds 1 and 2.

3. The use of the isopentenyl flavonoid compound of claim 1 in the preparation of neuroprotective drugs.

4. The use of the isopentenyl flavonoid compound of claim 1 in the preparation of a drug for treating neurodegenerative diseases.

5. The use of the isopentenyl flavonoid compound of claim 1 in the preparation of a drug for treating Alzheimer's disease and / or Parkinson's disease.

6. The use of the isopentenyl flavonoid compound of claim 1 in the preparation of drugs that regulate the Keap-Nrf2 / HO-1 signaling pathway.

7. A drug for treating neurodegenerative diseases, characterized in that, The drug contains the isopentenyl flavonoid compound as described in claim 1.

8. A neuroprotective drug, characterized in that, The drug contains the isopentenyl flavonoid compound as described in claim 1.

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