A compound having neuroprotective activity, methods of making and use

Abstract

The application discloses a compound with neuroprotective activity, a preparation method and application. A new compound of diketopiperazine alkaloids is separated from a fermentation product of Aspergillus, the compound is stable in performance, has no cytotoxicity, and has significant neuroprotective activity. The compound can improve the cell survival rate of a nerve injury in vitro model at a lower concentration, inhibit the generation of active oxygen and malondialdehyde, and obviously increase the expression amount of superoxide dismutase, and has the effects of antioxidation and anti-nerve injury, and has a wide application prospect in the preparation of medicines for preventing and treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a compound with neuroprotective activity, its preparation method, and its application. Background Technology

[0002] Neurodegenerative disorders (NDs) are a group of diseases that cause damage to nerve cells or lead to motor and cognitive dysfunction, seriously threatening human health. Parkinson's disease and Alzheimer's disease (AD) are the most common neurodegenerative diseases, prevalent in the elderly, and severely impacting normal life. These diseases not only cause immense suffering for patients but also place a heavy burden on their families and society.

[0003] Currently, there are no effective drugs for the treatment of non-disorders of disease (ND). Drugs used to treat atopic dermatitis (AD), such as donepezil, galantamine, or memantine, can only alleviate early-stage symptoms. Their effects are limited and they cannot prevent, stop, or reverse the progression of AD. Therefore, finding more effective new drugs for prevention and treatment is a hot research topic.

[0004] Therefore, a compound with neuroprotective activity, its preparation method, and its application are urgently needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a compound with neuroprotective activity, a preparation method, and applications.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a compound with neuroprotective activity, the structural formula of which is as follows:

[0008] As shown in Equation 1:

[0009]

[0010] R1 and R2 are each independently selected from hydrogen or hydroxyl groups.

[0011] A second objective of this invention is to provide a method for preparing a compound with neuroprotective activity, comprising the following steps:

[0012] S1. Fermentation culture: Aspergillus sp. fungus is fermented in large quantities;

[0013] S2. Extraction and separation: The fermentation product obtained in S1 was repeatedly soaked in anhydrous ethanol for extraction until the ethanol extract was almost colorless. Then, the extract was concentrated under reduced pressure at a temperature below 50°C using a rotary evaporator to recover the ethanol. The dry extract was extracted with ethyl acetate multiple times and then evaporated under reduced pressure to obtain a crude extract. The crude extract was separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate to obtain the compound of formula 1.

[0014] Preferably, in step S2, a gradient elution of 20:1-0:1 v / v petroleum ether-ethyl acetate is used.

[0015] Preferably, fraction C was obtained by elution with 1:1 v / v petroleum ether-ethyl acetate. Fraction C was then separated by reversed-phase silica gel column chromatography using a methanol-water gradient elution of 20:80, 40:60, 60:40, 80:20, and 100:0 (v / v), with 5 column volumes eluted for each ratio. The fraction was concentrated under reduced pressure, the solvent was recovered, and the fraction was identified by thin-layer chromatography (TLC) to obtain three subfractions, C1-C3. Fraction C2 was purified by Sephadex LH-20 column chromatography (CH2Cl2-MeOH, 1:1, v / v) to obtain 10 major subfractions, C2-1-C2-10, based on TLC analysis. Among these, fraction C2-5 was purified by semi-preparative high-performance liquid chromatography (MeOH-H2O, 62:38, v / v) to obtain compound 1 (16.7 mg, t) as shown in Formula 1-1. R =39min):

[0016]

[0017] A third objective of this invention is to provide the use of a compound with neuroprotective activity in the preparation of a medicament for improving neurodegenerative diseases.

[0018] Preferably, the effective concentration range of the drug is 0.625 μM to 40 μM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention yields a diketopiperazine alkaloid compound from Aspergillus fungi. This compound is stable, non-cytotoxic, and exhibits significant neuroprotective activity. The effective concentration range of this compound is 0.625 μM–40 μM. It shows significant protective effects against H2O2-damaged SH-SY5Y cells, significantly improving cell viability, inhibiting the production of reactive oxygen species (ROS) and malondialdehyde, and significantly increasing superoxide dismutase expression. It possesses antioxidant and anti-neural damage effects and shows broad application prospects in the preparation of drugs for the prevention and treatment of neurodegenerative diseases. Attached Figure Description

[0021] Figure 1 It is compound 1 in this invention.1 H NMR spectral data;

[0022] Figure 2 It is compound 1 in this invention. 13 C NMR and DEPT spectral data;

[0023] Figure 3 This is the HRESIMS spectrum of compound 1 in this invention;

[0024] Figure 4 This is a schematic diagram illustrating the cellular neurotoxicity induced by different concentrations of H2O2 in this invention;

[0025] Figure 5 This is a schematic diagram illustrating the protective and neuroprotective effects of compound 1 of the present invention against H2O2-induced neurotoxicity.

[0026] Figure 6 This is a schematic diagram of the half-maximal effective concentration (EC50) of compound 1 in this invention to resist H2O2-induced neurotoxicity and provide neuroprotection.

[0027] Figure 7 This is a schematic diagram illustrating the effect of compound 1 of the present invention on the activity of superoxide dismutase (SOD) in cells;

[0028] Figure 8 This is a schematic diagram illustrating the effect of compound 1 of the present invention on the malondialdehyde (MDA) content in cells;

[0029] Figure 9 This is a schematic diagram illustrating the effect of compound 1 of the present invention on H2O2-induced reactive oxygen species (ROS-) in cells;

[0030] Figure 10 This is a schematic diagram illustrating the effect of compound 1 of the present invention on H2O2-induced apoptosis. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0032] Example 1: Source of bacterial strain.

[0033] Aspergillus sp. was isolated from the rice grasshopper (Oxya chinensis Thunb) and deposited at the China Center for Type Culture Collection on November 8, 2023, with accession number CCTCCNO: M 20232161.

[0034] Example 2: Fermentation culture and extraction separation.

[0035] Fermentation culture: Aspergillus sp., a fungus derived from Oxya chinensis Thunb, was inoculated onto several potato dextrose agar (PDA) plates and cultured at 28°C for 5 days as the inoculum. The agar was then cut into small pieces, and the inoculum was inoculated into sterilized rice culture medium (250g rice and 250mL distilled water in 1L Erlenmeyer flasks) in a clean bench and cultured at 28°C for 28 days, for a total of 400 flasks.

[0036] Extraction and Separation: Inject 300 mL of anhydrous ethanol into each vial and soak for 24 hours to stop fungal growth. Discard the solvent and concentrate under reduced pressure using a rotary evaporator at below 50°C. Repeatedly soak the culture in the recovered ethanol until the solvent extract is almost colorless. Suspend the dry extract in 2 L of water and extract 10 times with ethyl acetate (1:1, v / v). Evaporate the organic solvent under reduced pressure to obtain a crude extract (500 g). Separate the crude extract by silica gel column chromatography, eluting sequentially with petroleum ether (PE)-ethyl acetate (EtOAc) (20:1, 5:1, 1:1, 0:1, v / v), eluting 5 column volumes for each ratio. Concentrate under reduced pressure to obtain four fractions, numbered A through D. C(PE-EtOAc, 1:1, v / v) was separated by reversed-phase silica gel column chromatography, followed by gradient elution with methanol-water (20:80, 40:60, 60:40, 80:20, 100:0, v / v) systems, with 5 column volumes eluted for each ratio. The solution was concentrated under reduced pressure, the solvent was recovered, and three subfractions (C1-C3) were identified by thin-layer chromatography. Fraction C2 was purified by Sephadex LH-20 column chromatography (CH2Cl2-MeOH, 1:1, v / v), yielding 10 major fractions (C2-1-C2-10). Fraction C2-5 was purified by semi-preparative high-performance liquid chromatography (MeOH-H2O, 62:38, v / v) to obtain compound 1 (16.7 mg, t). R =39min)

[0037]

[0038] Example 3: Structural identification of compound 1.

[0039] 1. Nuclear Magnetic Resonance Imaging (NMR):

[0040] Compound shown in Formula I-1: white powder; high-resolution electrospray ionization mass spectrometry (+)-HRESIMS [M+Na] + m / z 561.0783 (calculated value C) 26 H 22 N₂O₇S₂Na + ,561.0761); [α]2D 5 -96(MeOH, c 0.3); UV(MeOH)λ max (logε):225(3.83),255(3.57),295(3.20)nm; IR(KBr)ν max :3430,2926,2853,1709,1692,1606,1520,1358,1291,1220,1144,1028,899,763,714cm –1 ; 1 H and 13 The C NMR data are shown in Table 1.

[0041] Table 1. Compound 1 1 H NMR and 13 C10 NMR spectral data (δin ppm, J in Hz)

[0042]

[0043]

[0044]

[0045] 1 H NMR, 13 C NMR was measured at 400 MHz and 100 MHz, respectively, using CDCl3 as the solvent.

[0046] Example 4: Preparation of a drug for improving neurodegenerative diseases using compound 1.

[0047] The prepared drug dosage forms may be suitable for use in the following ways: oral administration (e.g., as tablets, capsules, pouches, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and capsules), topical administration (e.g., as creams, ointments, emulsions, solutions, pastes, sprays, foams, and gels), transdermal administration (e.g., via transdermal patches), inhalation administration (e.g., as dry powders, aerosols, suspensions, and solutions), inhalation administration (e.g., as fine powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal, or intramuscular administration), or as suppositories for rectal administration.

[0048] Example 5: Neuroprotective activity.

[0049] H2O2-damaged SH-SY5Y cells were used as a cell model of neurodegenerative diseases. Cell viability was detected by the CCK-8 assay, and the anti-neurodegenerative activity of the compounds was analyzed and evaluated. The steps included:

[0050] (1) Establishing a cell damage model

[0051] SH-SY5Y cells were seeded in 96-well plates and cultured for 24 hours. The model group was treated with different concentrations of H2O2 for 4 hours to induce cell growth, followed by incubation at 37°C for 1 hour with 10 μL of CCK-8 solution. The absorbance (OD) of each group at 450 nm was measured using a microplate reader. Cell viability % = (OD of experimental group or model group – OD of blank group) / (OD of control group – OD of blank group) × 100%; cell viability was obtained from three replicate experiments.

[0052] Results: Compared with the control group, the cell survival rate of the 40μM~1.2mM H2O2 group was significantly reduced, indicating that H2O2 caused damage to SH-SY5Y cells, and the cell damage model was successfully established.

[0053] (2) Activity experiment of compound 1 on hydrogen peroxide-damaged SH-SY5Y cells (a neurodegenerative disease model)

[0054] SH-SY5Y cells were seeded into 96-well plates at a density of 10,000 per well and cultured in a cell culture incubator for 12 hours. Different concentrations of compound 1 were added for prophylactic administration. After standing for 4 hours, 800 μM hydrogen peroxide was added to induce a cell model for 4 hours. CCK-8 solution was added and incubated for 1 hour. The absorbance at 450 nm was then measured using a microplate reader.

[0055] Results: As shown in the figure, compound 1 exhibited a significant protective effect against H2O2-damaged SH-SY5Y cells within the concentration range of 0.625 μM to 40 μM, showing a dose-response relationship. Cell viability increased with increasing compound concentration. Its EC50 was 2.148 μM.

[0056] (3) Detection of superoxide dismutase (SOD) and malondialdehyde (MDA)

[0057] SH-SY5Y cells were seeded at a density of 10,000 cells / well in 96-well plates and cultured for 12 h. Compound 1 (0.625 μM, 1.25 μM, 2.5 μM, and 5 μM) was added, and the cells were cultured for 4 h. Then, H2O2 (800 μM) was added and incubated for another 4 h. Cells from each group were collected into centrifuge tubes. After centrifugation, the supernatant was discarded. Extraction buffer was added according to the corresponding cell count (1 mL of extraction buffer is recommended per 5 million cells). Cells were sonicated (ice bath, 200 W, 3 s sonication, 10 s interval, repeated 30 times), and centrifuged at 8000g at 4℃ for 10 min. The supernatant was collected and placed on ice for analysis. SOD and MDA were detected according to the kit instructions.

[0058] Experimental results showed that, compared with the H2O2 model group, the expression level of MDA in compound 1 was significantly reduced and the expression level of SOD was significantly increased, and the higher the concentration, the more significant the activity.

[0059] (4) ROS detection

[0060] Intracellular ROS were detected using a fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime Biotechnology Co., Ltd.). Cells were seeded at a density of 10,000 per well in 96-well plates and cultured for 12 h. Compound 1 (0.625 μM, 1.25 μM, 2.5 μM, and 5 μM) was added, and the cells were cultured for 4 h, followed by incubation with 800 μM H2O2 for 4 h. The cell supernatant was then discarded, and the cells were washed once with PBS and stained with 10 μM DCFH-DA at 37°C in the dark for 30 min. Cells were collected, washed twice with PBS, and then analyzed by flow cytometry. The results showed that compared with the H2O2 model group, the reactive oxygen species (ROS) level in the compound 1 group was reduced, and the higher the concentration, the more significant the inhibitory activity against ROS.

[0061] (5) DAPI staining

[0062] DAPI (4,6-diamidinyl-2-phenylindole) is a DNA-specific dye that emits blue fluorescence upon binding to DNA. This dye is semi-permeable to cell membranes, allowing it to pass through normal living cells and produce weak blue fluorescence. Apoptotic cells, however, have increased membrane permeability, leading to enhanced uptake and strong blue staining. Normal cell nuclei are round with clear edges and uniform staining, while apoptotic cell nuclei have irregular edges, condensed chromosomes, and heavier staining, accompanied by nuclear pyknosis and increased nucleosome fragmentation. Therefore, both fluorescence intensity and nuclear morphology can be used to identify the typical characteristics of apoptosis.

[0063] To investigate the effect of compound 1 on H2O2-induced apoptosis, DAPI staining was performed. Cells were seeded at a density of 10,000 per well in 96-well plates and cultured for 12 h. Compound 1 was added, and the cells were cultured for 4 h, followed by incubation with H2O2 (800 μM) for 4 h. The cell supernatant was then discarded, and the cells were washed with PBS. Cells in each group were fixed in 4% paraformaldehyde (Solarbio, China) for 20 min. Then, cells were stained with DAPI (Solarbio, China) at 37°C in the dark for 30 min (the recommended working concentration of DAPI is 0.5-10 μg / mL). The cells were then washed with PBS. The apoptotic morphology of the cells after DAPI staining was observed under a fluorescence microscope. The results showed that H2O2 stimulation resulted in significant nuclear shrinkage and increased brightness in the cell nucleus, exhibiting typical apoptotic characteristics. Different concentrations of compound 1 significantly attenuated H2O2-induced apoptosis, with higher concentrations showing a stronger inhibitory effect on apoptosis.

[0064] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a compound with neuroprotective activity in the preparation of a medicament for improving neurodegenerative diseases, characterized in that, The structural formula of the compound is shown in Formula 1: Formula 1.

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