Application of compound in aspect of reducing neurotoxicity of amyloid protein
Hyperaminoside changes the conformation of β-amyloid 1-42, inhibits its toxicity, solves the neurotoxicity problem caused by abnormal accumulation of β-amyloid β-amyloid, significantly reduces its neurotoxicity, provides protection for nerve cells, and has similar effects. Know the drug curcumin.
Patent Information
- Application Number
- CN202411397981.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-01-03
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Abnormal accumulation of amyloid beta leads to neurotoxicity, and the prior art is difficult to effectively inhibit its toxicity, leading to the occurrence of neurodegenerative diseases such as Alzheimer's disease.
Hypericin is used to change the conformation of β-amyloid 1-42, inhibit its secondary structure and oligomer formation, and reduce neurotoxicity.
Hypericin significantly inhibits the monomer aggregation and secondary structure formation of Aβ protein, reduces its neurotoxicity, and has a protective effect on nerve cells. The effect is similar to the known drug curcumin, and its isomer isoquercetin also shows Similar inhibitory effects.
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Figure CN119235888A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical technology, and in particular, relates to the use of hyperoside in changing the conformation of beta-amyloid protein 1-42 (Aβ42) to prepare a drug for reducing the neurotoxicity of amyloid protein. Background Art
[0002] Amyloid β oligomers are neurotoxic [1] Under normal physiological conditions, the generation and degradation of Aβ, which is hydrolyzed by β- and γ-cleavage enzymes of β-amyloid precursor protein (APP), is in a state of equilibrium. Under pathological conditions, APP metabolism is abnormal, and Aβ generation increases sharply. Since it cannot be cleared in time, it accumulates in the body. The formation of toxic oligomers causes neurofibrillary tangles, astrocytes and microglia to proliferate, and releases inflammatory factors (such as TNF-α, IL-6, IL-2), causing a cascade reaction, which in turn enhances the generation of Aβ. [2] , causing neurodegenerative diseases. Therefore, clearing the neurotoxicity of Aβ in the body becomes an important way to treat neurodegenerative diseases. [3] One of the typical diseases is Alzheimer's disease (AD).
[0003] Epidemiological studies have found that natural active ingredients in some fruits, vegetables and Chinese herbs can reduce the risk of neurodegenerative diseases [4] Curcumin can inhibit the accumulation of Aβ protein in vitro and reduce the toxic effects of Aβ oligomers [5] It was also found that resveratrol, the main polyphenol component of grape seed extract, can also inhibit the accumulation of Tg2576Aβ and improve memory function impairment [6] These evidences indicate that the development of small molecule compounds that inhibit Aβ accumulation and reduce its toxicity can become a promising research direction for the treatment of neurodegenerative diseases.
[0004] Hyperoside (English name Hyperoside, chemical name 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one, molecular formula C21H20O12), also known as quercetin-3-O-β-D-pyranogalactoside, belongs to flavonol glycosides. Hyperoside is widely distributed and has multiple physiological activities such as anti-inflammatory, antispasmodic, diuretic, antitussive, antihypertensive, cholesterol-lowering, protein assimilation, local and central analgesic, and protective effects on heart and brain blood vessels. It is an important natural product. In the present invention, hyperoside is used in the preparation of drugs for reducing amyloid neurotoxicity based on changing the conformation of β-amyloid protein 1-42 (Aβ42).
[0005] References
[0006] 1. PB, SoderoACR, CordeiroY. Natural products targeting amyloid-β oligomer neurotoxicity in Alzheimer's disease. EurJMedChem.2024
[0007] 2.Mehlhorn G, Holborn M, et al. Induction of cytokines in glial cells surrounding corticalbeta-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology, Int J Dev Neurosci. 200018(4-5):423-31
[0008] 3.Wang J, Ho L, et al. (2006a) Moderate consumption of Cabernet Sauvignonattenuate A betaneuropathology in a mouse model of Alzheimer's disease. FASEBJ20:2313-2320
[0009] 4.Assuncao M, Santos Marques MJ, et al.Green tea averts age-dependentdecline of
[0010] hippoca-mpal signaling systens related to antioxidant defenses andsurvival.Free Radic Biol Med.2010.48:831-838
[0011] 5.Venus Singh Mithu,Bidyut Sarkar,et al.Curcumin alters thesaltbridge-contain-ing turn region in amyloidbeta1-42 aggregates.JBC.2014.289(16):11122-11131
[0012] 6. Liu P, kEMPER LJ, et al. Grape seed polyphenolic extract specifically decreases Aβ56 in the brains of Tg2576 mice. JAlzheimer Dis. 2011.26(4):657-66 Summary of the invention
[0013] 1. Application of hyperoside in changing the conformation of Aβ, changing its neurotoxicity, and treating neurodegenerative diseases
[0014] 2. Compared with the prior art, the beneficial effect of the present invention is that: the present invention proves that hyperoside can inhibit the secondary structure of Aβ protein and the formation of oligomers, thereby reducing its neurotoxicity and having a significant protective effect on nerve cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 These are the results of the Thioflavin T experiment (ThT) in which hyperoside inhibits Aβ42 monomer aggregation and the MTT experiment in which it inhibits Aβ42 oligomer-mediated neurotoxic damage.
[0016] Figure 2 The results of circular dichroism spectroscopy detection of hyperoside inhibiting the β-folding formation of Aβ42 monomer (CD).
[0017] Figure 3 Hyperoside disaggregates the formed Aβ polymer structure (ThT on the left, TEM on the right). DETAILED DESCRIPTION
[0018] The following embodiments of the present invention are used in conjunction with the accompanying drawings to further illustrate the essence of the present invention, but the present invention is not limited thereto. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.
[0019] Example 1 Thioflavin T test (ThT) of hyperoside inhibiting Aβ42 monomer aggregation and MTT test of hyperoside inhibiting Aβ42 oligomer-mediated neurotoxic injury
[0020] The protein Aβ protein was synthesized and provided by Nanjing Taopu Biological Co., Ltd. with a purity greater than 95%. The compound hyperoside and its isomer isoquercetin were provided by Shanghai Yuanye Biological Co., Ltd. The Aβ monomer powder was stored at -80°C. When taken out for the first time, it was allowed to stand at room temperature for 30 minutes, then diluted to 1.0 mg / mL with hexafluoroisopropanol (HFIP), allowed to stand for at least 2 hours, and ultrasonicated for 30 minutes to destroy the existing polymer structure. After standing and drying at low temperature, it was immediately stored at -20°C. Thioflavin T was purchased from Sigma (Cat. No. T3516-5g). Weigh 0.032g of ThT powder and dissolve it in 20mL 20mMTris-HCl (pH 7.4) solution to obtain a 5mM ThT mother solution, which was diluted to 5uM when used.
[0021] When used, HFIP-treated Aβ protein and compound hyperoside were dissolved with 100ul respectively, and diluted with PBS buffer (100nM phosphate, 10mMNaCl, pH7.4). The final concentration of Aβ in the mixed solution was 20uM, and the monomer drug concentration was 0uMControl control group and 10uM (Low), 20uM (Middle), 40uM (High) experimental groups. The total system of each sample was 30ul. The prepared mixed solution was incubated at 37°C with slow shaking. After 24 hours, 270ul 5uM ThT was added and detected by a fluorescence analysis tester. The excitation wavelength Ex = 430nm, the emission wavelength Em = 480nm measured its fluorescence value, and each sample was tested 5 times to take the average value.
[0022] The fluorescence intensity results of each group detected by the above method are shown in the attached Figure 1, lower left. It can be seen that compared with the control group without drug addition, the fluorescence intensity of the experimental group with the addition of hyperoside compound decreased significantly and was concentration-dependent, which is similar to the result of curcumin, a positive drug that inhibits Aβ protein aggregation. Several other compounds with similar structures to hyperoside are not as effective as hyperoside. The test results show that the compound hyperoside can significantly inhibit the effective aggregation of Aβ protein monomers. In addition, the isomer of hyperoside, isoquercetin, has a similar inhibitory effect on Aβ aggregation.
[0023] The cells used in the Aβ42 oligomer-mediated neurotoxic injury experiment were human neuroma blast cells SH-SY5Y, which were incubated at 37°C in DMEM medium containing 10% FBS. The mother liquor of the control group without Aβ protein and the experimental group with drug was slowly shaken and cultured at 37°C overnight, and then transferred to a 96-well cell culture plate containing SH-SY5Y. The final concentration of Aβ protein was 1uM, and the final concentration of drug was 0.25uM, 0.5uM, 1uM and 2uM. Three parallel wells were set for each concentration, the action time was 3 hours, and the MTT method was tested 5 times at a wavelength of 490nm to analyze the cell proliferation.
[0024] The absorbance values of each group tested using the above method are shown in the attached Figure 2 , lower right. It can be seen that compared with the control group without drug addition, the absorbance value of the experimental group with the addition of hyperoside compound increased significantly. The drug hyperoside significantly inhibited the neurotoxic reaction mediated by Aβ protein aggregation to form oligomers. At low concentrations, the effect was stronger than the positive control curcumin. Another group of cholinesterase inhibitor drugs Aricept (donepezil hydrochloride, HCl-done) had no effect on the cytotoxicity caused by the aggregation of Aβ protein. The isomer of hyperoside, isoquercetin, had a similar inhibitory effect on the toxicity of Aβ oligomers. The test results showed that the compound hyperoside can significantly inhibit the neurotoxic effect mediated by the aggregation of Aβ protein monomers.
[0025] Example 2 Circular dichroism spectroscopy experiment of hyperoside and Aβ protein (CD)
[0026] Aβ monomers were mixed with drugs, with a final Aβ concentration of 20uM, and the final drug concentrations were 0uM, 10uM, 20uM and 40uM, respectively. After slow shaking and incubation at 37°C for 12 hours, the samples were detected using a J-810 spectrometer (Jasco, Japan). The wavelength range was 190-250nm, the resolution was 0.5nm, the spectral width was 2nm, the scanning speed was 100nm / min, the response time was 1s, and the measurement temperature was room temperature. The solution was added to a quartz cuvette with an optical path of 0.1mm for measurement, and each experiment was repeated three times. The buffer curve was subtracted from the scanning curve obtained each time as the experimental result. Each experiment was repeated three times, and the average value was given.
[0027] The data were analyzed using the software Jascow32 provided by the instrument manufacturer (see Appendix Figure 2 ), Aβ protein monomers mainly form β-folded structures. Compared with the control group without hyperoside, the addition of hyperoside can significantly reduce the formation of β-folded structures, and it is concentration-dependent. Another group of cholinesterase inhibitor drugs Aricept (donepezil hydrochloride) has no effect on the formation of monomeric β-folded structures of Aβ protein. The isomer of hyperoside, isoquercetin, has a similar inhibitory effect on the secondary structure of Aβ. This shows that the compound hyperoside can significantly inhibit the formation of secondary β-folded structures of Aβ protein monomers.
[0028] Example 3: Disaggregation of the formed Aβ fiber structure by hyperoside
[0029] Pretreatment was as in Example 1. Aβ42 protein was first incubated at 37°C for 48 hours before adding drugs. Samples were taken every 6 hours and stored at -20°C. After 48 hours, ThT and transmission electron microscopy (TEM) were performed uniformly. In the TEM experiment, 5ul of Aβ protein sample was dropped on the surface of a 300-mesh carbon film copper grid, air-dried, negatively stained with 2% (w / v) sodium phosphotungstate for 30 seconds, and left to stand at room temperature for about 4 hours. The JEM-100CXII transmission electron microscope system (JEOL inc., Tokyo, Japan) was used for detection and photography, with an accelerating voltage of 80kV.
[0030] The fluorescence intensity results of each group detected by the above method are shown in the attached Figure 3 , above; TEM results are attached Figure 3 , below. It can be seen that compared with the control group without drug addition, the fluorescence intensity of the experimental group (HYP) with the addition of hyperoside compound remained basically unchanged, and the TEM results after 48 hours of action found that hyperoside could not depolymerize the formed Aβ fibers. Similarly, isoquercetin (ISO) had no significant change in the depolymerization of the formed Aβ fibers.
Claims
1. To protect the application of a compound, hyperoside, in the preparation of drugs for reducing the neurotoxicity of amyloid protein.