CN108743599A - A kind of application of compound in terms of reducing albumen and building up treatment senile dementia - Google Patents

Abstract

The invention discloses the application of hyperoside in antagonizing the accumulation of beta amyloid protein, improving cognitive memory ability and reducing senile plaques. Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurofibrillary tangles, β-amyloid (Aβ) plaque deposition, and cognitive decline. Toxic accumulation is considered to be one of the main links in the pathogenesis of AD. The present invention finds that hyperoside and its isomer isoquercetin can significantly inhibit the accumulation of β-amyloid 1‑42 monomers to form fibers, inhibit the formation of β secondary structures, and improve the spatial memory of AD mice Learning ability and can significantly clear the amyloid plaques in the brain tissue of APP/PS1 double transgenic AD model mice, which shows that hyperoside can be applied to the treatment of Alzheimer's disease and the development of related drugs.

Description

Application of a compound in reducing protein accumulation to treat senile dementia

technical field

The invention belongs to the technical field of medicines, and specifically relates to the application of hyperoside in the preparation of medicines for treating Alzheimer's disease by antagonizing the accumulation of β-amyloid 1-42 (Aβ42) as a target.

Background technique

Alzheimer's disease (AD), also known as Alzheimer's disease, is clinically manifested as psychomotor abnormalities, language barriers, cognitive and memory impairment, and is a common progressive neurological decline in the elderly. . According to the "2009 World Alzheimer's Report" report, by 2050, the number of people suffering from the disease will surge to 150 million ^[1] .

The most widely accepted theory about the pathogenesis of AD is the Aβ theory marked by the oligomerization of β-amyloid protein and the formation of fibrous precipitates ^[2] . Under normal physiological conditions, the generation and degradation of Aβ from the hydrolysis of β-amyloid precursor protein (APP) by β- and γ-cleaving enzymes are in balance. Under pathological conditions, the metabolism of APP is abnormal, and the generation of Aβ increases sharply, which accumulates in the brain because it cannot be cleared in time. The release of inflammatory factors (such as TNF-α, IL-6, IL-2) causes a cascade reaction, which in turn will enhance the production of Aβ ^[3] and trigger neurodegenerative diseases. Therefore, clearing the accumulation of Aβ in the brain has become an important way to treat the disease ^[4] .

At present, there is no specific drug for the treatment of AD clinically. Existing drugs for the treatment of AD such as: cholinesterase inhibitor donepezil hydrochloride, non-steroidal anti-inflammatory drug aspirin, calcium antagonist nimodipine can only slow down the onset of AD and improve symptoms, but cannot prevent the progression of the disease. Based on the Aβ hypothesis, design β-secretase and γ-secretase inhibitors to reduce the generation of Aβ, but the reduction of secretase itself is accompanied by relatively large side effects; immunotherapy, Aβ antibodies, peptides, etc. are designed to affect how antibodies and peptides pass through the blood brain The effective accumulation and toxic side effects of the barrier in the brain are another challenge for the treatment of Alzheimer's disease.

Epidemiological studies have found that some natural active ingredients of fruits, vegetables and Chinese herbal medicines can reduce the risk of AD ^[5] . Curcumin can inhibit the accumulation of Aβ protein in vitro and can clear the senile plaques in the brain of AD mice and reduce the toxic effect of Aβ oligomers ^[6] . It was also found that resveratrol, the main polyphenol component of grape seed extract, can also inhibit the accumulation of Tg2576Aβ and improve its memory function impairment ^[7] . These evidences all indicate that the development of small molecule compounds that inhibit Aβ accumulation and reduce its toxicity can become a promising research direction for AD treatment.

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) hyperoside, also known as quercetin-3-O-β-D-galactopyranoside. Belongs to flavonol glycosides. The aglycon is quercetin, the sugar group is galactopyranose, and the 3-position O atom of quercetin is connected to the sugar group by a β-glycosidic bond. Hyperoside is widely distributed and has various physiological activities such as anti-inflammatory, antispasmodic, diuretic, cough-relieving, blood pressure-lowering, cholesterol-lowering, protein assimilation, local and central analgesia, and protection of heart and cerebrovascular. It is an important natural product, but there is no report on the application of antagonizing Aβ protein accumulation as a target to treat AD. Based on the AD amyloid hypothesis, the present invention systematically studies the effect of hyperoside on AD treatment through in vivo and in vitro experiments.

references

1. World Alzheimer's Report 2009. Martin Prince. Jim Jackson et al. 2009

2. Hardy J, Selkoe DJ. Theamyloid hypothesis of Alzheimer's disease: progress and problem on the road to therapeutics. Science. 2002: 297: 353-356

3. MehlhornG, HolbornM, et al. Induction of cytokinesinglial cells surrounding cortical beta-amyloid plaques in transgenic Tg2576 mice with Alzheimerpathology, IntJDevNeurosci. 200018(4-5):423-31

4. WangJ, HoL, et al. (2006a) Moderate consumption of Cabernet Sauvignon attenuate Abeta neuropathology in mouse model of Alzheimer's disease. FASEBJ20: 2313-2320

5. Assuncao M, Santos Marques MJ, et al.

6. VenusSinghMithu, BidyutSarkar, et al. Curcuminaltersthesaltbridge-contain-ingturnregion anamyloidbeta1-42aggregates. JBC.2014.289(16):11122-11131

7. LiuP, kEMPERLJ, et al.Grapeseed polyphenolic extract specifically decreases Aβ56 in the brains of Tg2576 mice. JAlzheimerDis.2011.26(4):657-66

Contents of the invention

1. The application of hyperoside in the treatment of Alzheimer's disease by antagonizing the accumulation of Aβ protein 1-42

2. Application of hyperin in the preparation of drugs for the treatment of Aβ amyloid deposition in the brain

3. Compared with the prior art, the beneficial effect of the present invention is that: the present invention is based on the hypothesis of AD amyloid protein, and the effect of hyperin in the treatment of AD has been tested from both in vitro and in vivo experiments. Systematic research. It has been confirmed that hyperoside can affect the secondary structure of Aβ protein, the polymerization of monomers, the neurotoxicity of oligomers, and the formation of fiber structures, and the Aβ of Alzheimer's disease APP/PS1 double transgenic AD mice B6C3-Tg The formation of amyloid deposition has obvious inhibitory effect, and can significantly improve the learning and memory ability of AD mice.

4. The present invention uses APP/PS1 double transgenic mice as an animal model of hyperoside's curative effect on senile dementia, and feeds them for a long time through intragastric administration, and determines the effect of hyperin on Aβ protein senile plaques in the brains of AD mice. Improvement in clearing effect and its learning and memory function.

Description of drawings:

Figure 1 shows the results of hyperin inhibiting Aβ42 monomer aggregation in Thioflavin T assay (ThT) and MTT assay in inhibiting Aβ42 oligomer-mediated neurotoxic injury

Figure 2 shows the results of transmission electron microscopy (TEM) of hyperin inhibiting the formation of fiber structures by Aβ42 monomers

Figure 3 is the result of circular dichroism detection of hyperin inhibiting the formation of Aβ42 monomer β sheet (CD)

Figure 4 shows the depolymerization of hyperin on the formed Aβ fiber structure (left ThT, right TEM)

Figure 5 is the water maze test to detect the improvement of hyperin on the memory ability of APP/PS1 transgenic mice (MorrisWater Maze)

Figure 6 shows the water maze test to detect the improvement of hyperin on the memory ability of APP/PS1 transgenic mice (YMaze)

Figure 7 is the results of thioflavin S staining detection of hyperin on the removal of senile plaques in the brains of APP/PS1 transgenic mice (ThS)

Figure 8 is the results of Congo red staining detection of hyperin on the removal of senile plaques in the brains of APP/PS1 transgenic mice (CongoRed)

Detailed ways:

Below in conjunction with the accompanying drawings, the embodiments of the present invention are used to further illustrate the essence of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction and replacement can also be made, which should be regarded as belonging to the protection scope of the present invention.

Example 1 Thioflavin T assay (ThT) for hyperoside inhibiting the aggregation of Aβ42 monomers and MTT assay for inhibiting neurotoxic damage mediated by Aβ42 oligomers

The protein Aβ protein was synthesized by Nanjing Taopu Biological Co., Ltd., with a purity greater than 95%. The compound hyperin and its isomer isoquercetin (Isoquercetin) were provided by Shanghai Yuanye Biological Company. The Aβ monomer powder is stored at -80°C. When it is taken out for the first time, it should be allowed to stand at room temperature for 30 minutes, then diluted with hexafluoroisopropanol (HFIP) to 1.0 mg/ml, left to stand for at least 2 hours, and ultrasonicated for 30 minutes to destroy the existing excess Polymer structure, after drying at low temperature, store at -20°C immediately. Thioflavin T (ThioflavinT) was purchased from Sigma (product number T3516-5g). Weigh 0.032g of ThT powder and dissolve it in 20ml of 20mM Tris-HCl (pH7.4) solution to obtain 5mM ThT mother solution, which is diluted to 5uM when used.

When in use, the HFIP-treated Aβ protein and the compound hyperoside were dissolved in 100ul respectively, diluted with PBS buffer (100nM phosphate, 10mMNaCl, pH7.4), the final concentration of Aβ in the mixed solution was 20uM, and the concentration of the monomer drug was 0uM Control control group and 10uM (Low), 20uM (Middle), 40uM (High) experimental groups, the total system of each sample is 30ul. Incubate the prepared mixture with slow shaking at 37°C. After 24 hours, add 270ul5uMThT, and detect it through a fluorescence analyzer. average value.

Using the above method, the results of the detected fluorescence intensity of each group are shown in Figure 1, lower left. It can be seen that, compared with the Control group without drug addition, the fluorescence intensity of the experimental group adding hyperoside compounds decreased significantly and was concentration-dependent, which was consistent with the results of curcumin (curcumin), a positive drug that inhibits Aβ protein aggregation. Similar, the effect of several other compounds similar to hyperoside structure is not as good as that of hyperoside. The test results show that the compound hyperoside can significantly inhibit the effective aggregation of Aβ protein monomers, and can be used for the prevention and treatment of Alzheimer's disease. In addition , isoquercetin, the isomer of hyperoside, has similar inhibitory effect on Aβ accumulation.

The cells used in the Aβ42 oligomer-mediated neurotoxic injury experiment were human neuroma blasts SH-SY5Y, incubated in DMEM medium with 10% FBS at 37°C. The Aβ protein control group without drugs and the drug-added experimental group mother solution were cultured overnight at 37°C with slow shaking, and 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 490nm wavelength to analyze the cell proliferation.

Using the above method, the results of the detected absorbance values of each group are shown in Figure 2, lower right. It can be seen that compared with the control group without drugs, the absorbance value of the experimental group added hyperoside compound increased significantly, and the drug hyperoside significantly inhibited the neurocytotoxic reaction mediated by Aβ protein aggregation to form oligomers, At low concentrations, the effect is stronger than that of the positive control curcumin, while another group of cholinesterase inhibitors, Aricept (donepezil hydrochloride, HCl-done), has no effect on the cytotoxicity caused by the aggregation of Aβ protein. Glycoside isomer isoquercetin has similar inhibitory effect on Aβ oligomer toxicity. The detection results show that the compound hyperoside can significantly inhibit the neurocytotoxicity mediated by the aggregation of Aβ protein monomers, and can be used for the prevention and treatment of Alzheimer's disease.

Example 2 Hyperin and Aβ Protein Transmission Electron Microscopy Experiment (Transmissionelectronmicroscopy, TEM)

The pretreatment was as in Example 1, and then, 5ul of the Aβ protein sample incubated at 37°C for 24 hours was dropped on the surface of a 300-mesh carbon-coated copper grid, air-dried, and negatively stained with 2% (w/v) sodium phosphotungstic acid After 30 seconds, let stand at room temperature for about 4 hours, use JEM-100CXII transmission electron microscope system (JEO Linc., Tokyo, Japan) to detect and take pictures, and the accelerating voltage is 80kV.

The observation results are shown in Figure 2. Compared with the control group without drug addition, the experimental group (HPY) Aβ protein fiber density, length and width decreased significantly in a concentration-dependent manner with the addition of hyperoside compounds, while the other group of cholinesterase inhibitors Aricept (donepezil hydrochloride) has no effect on the inhibition of Aβ protein fibril formation, and isoquercetin (ISO), an isomer of hyperoside, has a similar inhibitory effect on Aβ fibril structure. The test results show that the compound hyperin can obviously inhibit the formation of fiber structure by Aβ protein monomers, and can be used for preventing and treating Alzheimer's disease.

Example 3 Hyperin and Aβ protein circular dichroism experiment (CD)

Aβ monomer was mixed with drugs, the final concentration of Aβ was 20uM, and the final concentrations of drugs were 0uM, 10uM, 20uM and 40uM respectively, and after incubation at 37°C for 12 hours with slow shaking, the J-810 spectrometer (Jasco, Japan) was used to detect. The wavelength range is 190-250nm, the resolution is 0.5nm, the spectral width is 2nm, the scanning speed is 100nm/min, the response time is 1s, and the measurement temperature is normal temperature. The solution was added to a quartz cuvette with an optical path of 0.1 mm for measurement, and each experiment was repeated three times. Subtract the buffer curve from the scan curve obtained each time as the experimental result, each experiment was repeated three times, and the average value was given.

Using the software Jascow32 data analysis provided by the instrument manufacturer, it was found (attached figure 3) that the Aβ protein monomer mainly forms a β-sheet structure. Compared with the control group without adding hyperoside, adding hyperin can significantly reduce the β-sheet structure The formation of the structure is concentration-dependent, while another group of cholinesterase inhibitors, Aricept (donepezil hydrochloride), has no effect on the formation of the monomeric β-sheet structure of the Aβ protein, and the isomers of hyperoside Isoquercetin has a similar inhibitory effect on Aβ secondary structure. It shows that the compound hyperoside can obviously inhibit the formation of the secondary β sheet structure of the Aβ protein monomer, and can be used to prevent and treat Alzheimer's disease.

Example 4 Hyperin depolymerizes the formed Aβ fiber structure

The pretreatment was the same as in Examples 1 and 2, except that the Aβ42 protein was first incubated at 37°C for 48 hours before adding drugs, and samples were taken every 6 hours and stored at -20°C. After 48 hours, ThT and TEM tests were carried out uniformly.

Using the above method, the fluorescence intensity results of each group detected are shown in Figure 4, top; the TEM results are shown in Figure 4, bottom. It can be seen that compared with the Control group without drug addition, the fluorescence intensity of the experimental group (HYP) added with hyperoside compounds remained basically unchanged, and the TEM results after 48 hours of action found that hyperoside could not treat the already The formed Aβ fibers were depolymerized, and similarly, isoquercetin (ISO) had no significant change in the depolymerization of the formed Aβ fibers.

Example 5 Hyperoside Behavioral Experiments on APP/PS1 Transgenic Mice

The wild-type (WT) and APP/PS1 double transgenic mice used in the experiment of the present invention were purchased from the Model Animal Research Institute of Nanjing University, and the mouse genes were identified strictly according to the PCR method provided by the supplier. Eight-month-old APP/PS1 transgenic mice were set as hyperin administration group (low concentration group HYPL, high concentration group HYPH), isoquercetin administration group (low concentration group ISO L, high concentration group ISOH) , Aricept administration group, normal saline as the control group 1, WT wild-type mice as the control group 2, the serum brain drug treatment group (YX) found in this laboratory is the positive control group 3, continuous intragastric administration 2 months.

In the Y maze experiment, three black bar-shaped long arms with the same appearance are set up in a Y-shaped combination. The three long arms are marked as A, B, and C. The mice are put in from the A arm, and if they turn back from the other long arm during free movement It is recorded as an error, such as ABA, BCB, etc. When an error occurs, it will be recalculated with this arm as the starting point, such as ABACBABCBA. The experiment time for each mouse was 8 minutes. When the experiment was completed, the Y maze was cleaned and disinfected with alcohol, and the next mouse experiment was performed after drying.

Morris water maze experiment, two days of mouse water maze training (data not recorded), the mice were put into the pool from 4 water entry points (quadrants) facing the pool wall, the platform was hidden at 1cm underwater, and the water temperature was kept at 23 ℃, the training lasted for 2 minutes, if the mouse failed to find the platform within 2 minutes, it was guided to the hidden platform with a tool and stayed for 20 seconds, and then the next training was carried out. The experimental stage began on the third day, and the experiment was carried out continuously for 6 days. Each mouse was tested once a day. During the experiment, the mice were put into the pool from 4 water entry points (quadrants) facing the pool wall, and the mice were recorded from the time of entering the water. The time required to find and stay on the underwater hidden platform is taken as the incubation period. On the eighth day, the platform was removed, and each mouse entered the water from the farthest point where the original platform was located, and swam for 2 minutes freely. Micro cameras track and record and analyze.

The results of the Morris water maze experiment found that after hyperin treatment, the latency period of AD mice to find the platform was significantly reduced. Initially, the latent period after hyperin treatment of AD mice gradually decreased (lower left of Figure 5). After the platform was removed, the mice underwent positioning and navigation experiments. It was found that the AD mice in the hyperin treatment group crossed the virtual platform significantly. Significant improvement (bottom right of Figure 5), the effect is close to that of the positive control drug nourishing blood brain, while in comparison, the Aricept drug group has limited improvement in the symptoms of AD mice. The effect of isoquercetin and hyperoside The effect is similar. Based on the above behavioral results, it can be seen that hyperoside can significantly improve the learning and memory abilities of AD mice, indicating that hyperoside can be applied to the research and development of drugs for the prevention and treatment of Alzheimer's disease.

The statistics of the Y maze test results are shown in Figure 6. The correct rate of the wild mice (WT) is about 82%, and the correct rate of the Y maze test of the AD mice administered with normal saline is only about 58%. , the correct rate of Y fans in AD mice increased significantly, and the effect was slightly better than that of the Aricept treatment group due to the nourishing serum brain drug treatment group, indicating that hyperoside and its isomer isoquercetin can improve AD mice cognitive It has the potential to become a drug for the prevention and treatment of Alzheimer's disease.

Example 6 Pathological Experiment of the Effects of Hyperin on Brain Tissue of APP/PS1 Transgenic Mice

10-month-old AD mice were anesthetized with 0.4% pentobarbital sodium, blood was collected by perfusing the atrium, the whole body was fixed by perfusion of the heart with 4% paraformaldehyde, the brain tissue was taken and weighed, and half of the brain was placed in 4% paraformaldehyde solution It was used for histopathology, and the other half was stored in a cryopreservation tube at -80°C for biochemical analysis. The 4% paraformaldehyde-fixed brain tissue was embedded in wax and sectioned, and the deposition plaques in the cerebral cortex and hippocampus were stained by Thioflavin S and Congo red. Pathological images were acquired by digital microscope and fluorescence microscope and analyzed quantitatively.

The results of Thioflavin S staining and Congo red staining of brain tissue are shown in Figure 7 and Figure 8. The number of stained Aβ deposition plaques in the hyperoside treatment group, whether in the cortex or hippocampus, was significantly higher than that of those only administered normal saline. For the AD mice of the control group, all showed a significant reduction, and the effect was statistically significant, and the effect was slightly better than that of the positive control group, which was treated with blood serum and brain drugs. Significant changes, and the senile plaque removal effect of isoquercetin is similar to that of hyperoside, indicating that hyperoside can inhibit the deposition of Aβ protein in brain tissue, and can be used in the research and development of drugs for the prevention and treatment of Alzheimer's disease .

Claims (8)

1. The application of a compound hyperoside in the treatment of Alzheimer's disease by antagonizing the accumulation of β-amyloid protein 1-42. 2. The senile dementia described in claim 1, especially Alzheimer's disease, which is called Alzheimer's disease in English. 3. The application of the compound hyperin in the research and development of Alzheimer's disease-related drugs. 4. Alzheimer's disease treatment and drug development of the compound hyperin in inhibiting the neurotoxic damage mediated by the accumulation of Aβ1-42 monomers to form oligomers. 5. Compound hyperoside in the treatment of Alzheimer's disease and drug research and development in terms of inhibiting the formation of β-sheets of Aβ1-42 monomers and inhibiting the accumulation of Aβ1-42 monomers to form oligomers and fibers. 6. Therapeutic effect of the compound hyperoside in inhibiting the accumulation of Aβ protein to form senile plaques in Alzheimer's disease and the development of related drugs. 7. The therapeutic effect of the compound hyperoside on improving cognitive and memory impairment in Alzheimer's disease and the development of related drugs. 8. The molecular structure basis of the compound hyperoside and its analogues in the treatment of Alzheimer's disease and drug development, and the role of the molecular structure of hyperin in the treatment of Alzheimer's disease and drug development. The chemical name of hyperoside is Hyperoside, also known as Hyperin; 482-36-0; Quercetin 3-galactoside; Quercetin-3-O-galactoside; Hyperosid, international compound identification InChI=1S/C21H20O12/c22-6-13-15 (27)17(29)18(30)21(32-13)33-20-16(28)14-11(26)4-8(23)5-12(14)31-19(20)7 -1-2-9(24)10(25)3-7/h1-5,13,15,17-18,21-27,29-30H,6H2/t13-,15+,17+,18- ,21+/m1/s1, molecular weight 464.379g/mol, molecular formula C21H20O12. At the same time, the application of compounds with hyperoside-like structures in the treatment of Alzheimer's disease and drug development. For the isomer isoquercetin formed by changing the hydroxyl group on the glycoside of the hyperoside molecule, or simply replacing other hydroxyl groups on the hyperoside or increasing or decreasing the group -CH2- to bring Similar compounds formed by structural changes.