1. A method for high throughput screening of a β fibril formation inhibitors comprising the steps of:

step 1, biofilm interference analysis

Preparing a Chinese herbal medicine extract to be detected, and then diluting the Chinese herbal medicine extract to be detected by using a phosphate buffer solution to obtain a working solution;

preparing biotinylated Abeta (1-42) peptide;

injecting the working solution and biotinylated Abeta (1-42) peptide into a biosensor, and performing association-dissociation to obtain an association and dissociation diagram and a kinetic constant to obtain the association condition of the Chinese herbal medicine extract to be detected and the biotinylated Abeta (1-42) peptide;

step 2, LC-MS analysis

Continuously carrying out N times of combination-dissociation operations on the Chinese herbal medicine extract to be detected and biotinylated Abeta (1-42) peptide in a biosensor by adopting the same method in the step 1, and collecting dissociation liquid, wherein N is a natural number which is more than or equal to 5;

then drying the collected dissociation liquid under nitrogen flow;

and then, redissolving the dried dissociation solution by using an organic solvent, putting the redissolved solution into an ultra-high performance liquid chromatography tandem mass spectrometry detection instrument to obtain a representative total ion chromatogram, analyzing the chromatogram, and screening out a compound having a direct binding effect with the A beta fiber, namely the A beta fiber formation inhibitor.

2. The method for high-throughput screening of the A beta fibrogenesis inhibitor according to claim 1, wherein in the step 1, the Chinese herbal medicine extract to be tested is prepared by the following method: weighing and crushing the Chinese herbal medicine to be detected, distilling and extracting for 1-2h by using water, ethanol or methanol with the volume of 8-12 times, repeatedly extracting for 3-5 times, combining extracting solutions, filtering, concentrating, and dissolving the concentrated extract into a solution of 200-500 mg/mL by using DMSO (dimethyl sulfoxide), so as to obtain the Chinese herbal medicine extract to be detected.

3. The method for high-throughput screening of A beta fibrogenesis inhibitor according to claim 1, wherein in step 1, the biotinylated A beta (1-42) peptide is prepared mainly by the following method:

biotin with the molar ratio of 2-6:1 and A beta (1-42) peptide solution are mixed, and then incubated for 30min to 60min at room temperature to obtain biotinylated A beta (1-42) peptide.

4. The method for high-throughput screening of A beta fibrogenesis inhibitors according to claim 3, wherein the A beta (1-42) peptide solution is prepared by the following method:

placing the Abeta (1-42) peptide in hexafluoroisopropanol, subpackaging the obtained mixed solvent in a test tube, drying under nitrogen flow to obtain a dried film, and adding DMSO and a phosphate buffer in the test tube to obtain an Abeta (1-42) peptide solution.

5. The method for high throughput screening of A β fibrogenesis inhibitors according to claim 1, wherein in step 1, when the response value in the association and dissociation graph is increased, indicating that the herbal extract to be tested is bound to the biotinylated A β (1-42) peptide, the analysis of step 2 is performed.

6. The method for high-throughput screening of A beta fibrogenesis inhibitors according to claim 5, wherein the kinetic constant obtained in step 1 is 1x 10⁵And (3) if the Chinese herbal medicine extract to be detected is combined with the biotinylated Abeta (1-42) peptide and the combination capacity is strong, analyzing in the step 2.

7. The method for high-throughput screening of A beta fibrogenesis inhibitors according to claim 1, wherein in step 2, N is a natural number of 5 to 10.

8. The method for high-throughput screening of A β fibril formation inhibitors according to claim 1, wherein in step 2, the peak area ratio of the detection results of the HPLC is analyzed to obtain the RBA values of all compounds, and the RBA values of different compounds are compared, wherein the compound with the larger RBA value is a compound having a direct binding effect with A β fibrils.

9. The method for screening a β fibril formation inhibitor with high throughput according to claim 8, wherein the method for determining the compound having a larger RBA value and a higher fault height is: arranging RBA values of different compounds from high to low to form a 1 st compound, an N +1 th compound, an N +2 th compound and an N +3 th compound, wherein when the RBA value of the nth compound is more than 100% higher than that of the N +1 th compound, the 1 st to N compounds are screened A beta fiber formation inhibitor compounds, wherein N is a natural number which is more than or equal to 1, and N is 0 or more than or equal to 1.

10. The method for screening high throughput for an a β fibril formation inhibitor according to claim 9, wherein when the RBA value of the nth compound is higher than the RBA value of the n +1 st compound by 200% or more, the 1 st to n compounds are screened for the a β fibril formation inhibitor compound.

Background

The pathology of alzheimer's disease is closely related to the occurrence of neuroinflammation and neuronal death caused by the accumulation of misfolded protein aggregates, such as beta-amyloid (a β) and Tau. A β is produced by the sequential cleavage of β -and γ -secretase from Amyloid Precursor Protein (APP). The resulting fragments, including A β (1-40) and A β (1-42), are considered to be the most toxic forms, forming A β fibrils and accumulating extracellularly, eventually producing senile plaques in the brains of Alzheimer's patients. In the early stages of alzheimer's disease, Α β can still be cleared by the autophagosomal pathway (ALP) and the ubiquitin-proteasome pathway (UPP) in neurons, or phagocytosed by microglia. However, both ALP and UPP are imbalanced with age, so that a β clearance cannot be performed efficiently. Thus, targeted inhibition of a β fibril formation has become a promising therapeutic strategy for alzheimer's disease.

It has been found that a number of Chinese medicinal materials or components thereof exhibit potent neuroprotective effects in vitro and in vivo models of Alzheimer's disease. The process of finding bioactive components from herbal medicines or formulations with complex chemical compositions is time consuming and laborious. To date, many screening methods for traditional Chinese medicines have been reported, including Cell Membrane Chromatography (CMC), network pharmacology and molecular docking, affinity ultrafiltration with target drugs, and high performance liquid chromatography-mass spectrometry (AUF-HPLC-MS/MS), generation of magnetic liposomes in combination with bubbles of LC-MS, and the like. In general, most methods have been developed based on the application of advanced analytical instruments such as HPLC, MS and Nuclear Magnetic Resonance (NMR).

Therefore, it has become very important to develop an efficient, time-saving, reliable and simple method for screening a β fibril formation inhibitor from a traditional Chinese medicine.

Disclosure of Invention

The invention aims to: aiming at the problem that the process of finding the bioactive component for inhibiting the formation of the A beta fiber from the Chinese herbal medicine or the formula with complex chemical components in the prior art is time-consuming and labor-consuming, the method for screening the A beta fiber formation inhibitor in high throughput is provided. The method is simple to operate, time-saving, effective, reliable and convenient to popularize and apply.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for high throughput screening of a β fibril formation inhibitors comprising the steps of:

step 1, biofilm interference analysis

Preparing a Chinese herbal medicine extract to be detected, and then diluting the Chinese herbal medicine extract to be detected by using a phosphate buffer solution to obtain a working solution;

preparing biotinylated Abeta (1-42) peptide;

fixing the working solution and biotinylated Abeta (1-42) peptide on a biosensor, and performing association-dissociation to obtain an association and dissociation graph and a kinetic constant to obtain the association condition of the Chinese herbal medicine extract to be detected and the biotinylated Abeta (1-42) peptide;

step 2, LC-MS analysis

Continuously carrying out N times of combination-dissociation operations on the Chinese herbal medicine extract to be detected and biotinylated Abeta (1-42) peptide in a biosensor by adopting the same method in the step 1, and collecting dissociation liquid, wherein N is a natural number which is more than or equal to 5;

then drying the collected dissociation liquid under nitrogen flow;

and then, redissolving the dried dissociation solution by using an organic solvent, putting the redissolved solution into an ultra-high performance liquid chromatography tandem mass spectrometry detection instrument to obtain a representative total ion chromatogram, analyzing the chromatogram, and screening out a compound having a direct binding effect with the A beta fiber, namely the A beta fiber formation inhibitor.

The high-throughput method for screening the A beta fiber formation inhibitor mainly comprises the steps of analyzing the combined use result of biomembrane interference analysis and LC-MS analysis, wherein what needs to be detected is the combination condition of various unknown compounds in a Chinese herbal medicine extract to be detected and biotinylated A beta (1-42) peptide, designing repeated combination-dissociation operation for many times according to the combination condition, collecting dissociation liquid, carrying out LC-MS analysis on the collected dissociation liquid, and screening out monomers directly combined with the A beta fiber according to the analysis of a representative total ion chromatogram. And screening out the monomer with inhibiting effect by using subsequent molecular biology experiments. The high-throughput screening method provided by the invention is simple to operate, small in occupied time, high in accuracy, high in precision, good in repeatability and convenient for wide popularization and application, and has very important significance in easily screening the A beta fibrogenesis inhibitor from the traditional Chinese medicine.

Further, in the step 1, in the process of preparing the Chinese herbal medicine extract to be detected, the extraction solvent is water, ethanol or methanol.

Further, in the step 1, the Chinese herbal medicine extract to be detected is prepared by the following method: weighing and crushing the Chinese herbal medicine to be detected, distilling and extracting for 1-2h by using water, ethanol or methanol with the volume of 8-12 times, repeatedly extracting for 3-5 times, combining extracting solutions, filtering, concentrating, and dissolving the concentrated extract into a solution of 200-500 mg/mL by using DMSO (dimethyl sulfoxide) to obtain the Chinese herbal medicine extract to be detected.

Further, in the step 1, the concentration of the working solution is 25-4000 mug/mL.

Further, in step 1, the biotinylated a β (1-42) peptide is prepared mainly by the following method:

biotin with the molar ratio of 2-6:1 is mixed with the A beta (1-42) peptide solution, and then the mixture is incubated for 30-60 min at room temperature to obtain biotinylated A beta (1-42) peptide.

Further, the A beta (1-42) peptide solution is mainly prepared by the following method:

placing the Abeta (1-42) peptide in hexafluoroisopropanol, subpackaging the obtained mixed solvent in a test tube, drying under nitrogen flow to obtain a dried film, and adding DMSO (dimethyl sulfoxide) and a phosphate buffer in the test tube to obtain an Abeta (1-42) peptide solution. Further, the concentration of the A beta (1-42) peptide solution is 100-200 mg/mL.

Further, in step 1, when the response value in the association and dissociation graph is increased to indicate that the Chinese herbal medicine extract to be tested is combined with the biotinylated Abeta (1-42) peptide, the analysis of step 2 is performed. Preferably, in step 1, the kinetic constant obtained is1 × 10⁵And (3) if the Chinese herbal medicine extract to be detected is combined with the biotinylated Abeta (1-42) peptide and the combination capacity is strong, analyzing in the step 2.

Further, in the step 1, working solutions with different concentrations are adopted to perform multiple binding-dissociation, so as to obtain multiple binding and dissociation graphs and kinetic constants. Experiments are carried out through working solutions with different concentrations, the combination condition of the Chinese herbal medicine extract to be detected and the biotinylated Abeta (1-42) peptide can be fully and accurately judged, and the stability and the accuracy of the experiments are further improved.

Further, in the step 2, N is a natural number of 5-10. Through a great deal of experimental research and exploration of the inventor, when the times of repeated operation of combination-dissociation are less than 5 times, the detection limit of a mass spectrometer cannot be reached, and the error is large and inaccurate. However, the number of times of binding and dissociation cannot be excessive, and when the number of times exceeds 10 times, interference of non-specific binding monomers in the dissociation solution on mass spectrum detection is increased, so that the detection structure error is large, and the stability is poor.

Further, the ultra performance liquid chromatography tandem mass spectrometry detection instrument is an ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry instrument (ultra performance liquid chromatography-DAD-Q/TOF-MS/MS).

Further, in the step 2, the organic solvent is ethanol or methanol.

Further, in the step 2, the conditions of the ultra high performance liquid chromatography analysis are as follows:

c18 column, the temperature of the column is 30-45 ℃; the fluidity is composed of mobile phase A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile solution);

the flow rate is 0.2-0.5 mL/min.

Preferably, in the step 2, the ultra high performance liquid chromatography analysis conditions are as follows:

zorbax EcLipse Plus C18 column 100mm × 2.1mm × 1.8 um; flow rate: 0.3 mL/min; the column temperature was set at 40 ℃ and the mobile phase consisted of A (0.1% aqueous formic acid) and B (0.1% aqueous formic acid in acetonitrile).

Further, in the step 2, the conditions of the mass spectrometry are as follows:

the Duo Spray source is in negative electrospray ionization mode, and electrospray ionization is performed according to the following parameters: spray voltage, -4,500V; ion source temperature, 550 ℃; air curtain air, 35 psi; atomizing gas (GS 1), 55 psi; heater gas (GS 2), 55 psi; depolymerization Potential (DP), -100V; the mass range of the MS scan was set to m/z 100-1,600 Da.

Further, in the step 2, analyzing the peak area ratio of the detection result of the high performance liquid chromatography to obtain the RBA values of all compounds, and comparing the RBA values of different compounds, wherein the compounds with larger RBA values and increased layer breaking are compounds with direct binding effect with A beta fibers.

Further, a method for determining a compound having a large RBA value and a fault height is: arranging RBA values of different compounds from high to low to form a 1 st compound, an N +1 th compound, an N +2 th compound and an N +3 th compound, wherein when the RBA value of the nth compound is more than 100% higher than that of the N +1 th compound, the 1 st to N compounds are screened A beta fiber formation inhibitor compounds, wherein N is a natural number which is more than or equal to 1, and N is 0 or more than or equal to 1.

Preferably, when the RBA value of the nth compound is more than 200% higher than the RBA value of the n +1 th compound, then the 1 st to n compounds are screened as a β fibril formation inhibitor compounds.

More preferably, when the RBA value of the nth compound is higher than the RBA value of the (n + 1) th compound by 250% or more, the 1 st to n compounds are screened as a β fibril formation inhibitor compounds. For example, 300% or more, 350% or more.

The explanation text presents the abbreviated Chinese meaning:

SB-Scutellariae radix; KXS KAIXIN powder; DTA-dehydrotemmoic acid; TA-Tumoic acid; PPAC-grifolic acid C; Poria-Poria cocos; cur-curcumin.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the high-throughput method for screening the A beta fibrogenesis inhibitor provided by the invention is used by combining biomembrane interference analysis and LC-MS analysis, and finally, the A beta fibrogenesis inhibitor compound can be screened out according to the analysis of a representative total ion chromatogram. The screening method is simple to operate, small in occupied time, high in accuracy, good in repeatability and convenient to popularize and apply widely, has very important significance for easily screening the A beta fibrogenesis inhibitor from the traditional Chinese medicine, and accelerates the discovery and development process of future anti-AD medicines.

Drawings

FIG. 1 is a graph showing the dynamic combination and dissociation patterns of different concentrations of Scutellariae radix extract in example 1.

FIG. 2 is a chromatogram of representative total ions of Scutellaria baicalensis extract in example 1.

FIG. 3 is a bar graph of RBA of the fraction detected in Scutellaria baicalensis extract of example 1.

FIG. 4 is a graph of the dynamic binding and dissociation profiles of kadsura extract at different concentrations in example 2.

FIG. 5 is a representative total ion chromatogram of the extract of KAIXIN POWDER of example 2.

FIG. 6 is an Extracted Ion Chromatogram (EICS) of DTA in S1, S2, and S3 in example 2.

FIG. 7 is EICS of TAs in S1, S2 and S3 in example 2.

FIG. 8 is EICS of PPAC in S1, S2 and S3 in example 2.

FIG. 9 is a TIC map of PPAC, DTA, TA and Poria extract of example 3.

FIG. 10 is a mass spectrum of PPAC, DTA and TA in example 3.

FIG. 11 is a molecular structural diagram of PPAC, DTA and TA in example 3.

FIG. 12 is a graph showing the fluorescence intensity of each of the analytes at the time point of 0h in example 3.

FIG. 13 is a graph showing the fluorescence intensity of each of the analytes at the 24-hour time point in example 3.

FIG. 14 is a graph showing the fluorescence intensity of each of the analytes at the 48h time point in example 3.

FIG. 15 is a graph showing cytotoxicity reactions of each test substance measured by the MTT method in example 3.

FIG. 16 is a bar graph of the percent cell viability of PC-12 cells from example 3.

FIG. 17 is a graph of the percent cell death of PC-12 cells in example 3.

FIG. 18 is the ratio of PI/Hoechst signals of PC-12 cells in example 3.

FIG. 19 is a graph showing the rate of paralysis of the A.beta. -inducing nematode strain CL4176 tested in example 3 with each test agent.

FIG. 20 is a graph of the change in nematode perceived food rate of the A.beta. -induced control line nematode CL2122 under the effect of each test article tested in example 3.

FIG. 21 is a graph of the change in food rate perceived by A β -induced CL2355 C.elegans under the influence of each test substance tested in example 3.

Fig. 22 is a fluorescence image of a β deposits in the nematode CL2331 strain affected by each test substance in example 3.

FIG. 23 is a three-dimensional interaction and docking 2D model of PPCA, DA and TA with A β (1-42) active site residues in example 3.

FIG. 24 is a graph of the kinetic binding sensation of TA concentration increased from 12.5. mu.M to 400. mu.M in example 3.

FIG. 25 is a UHPLC-DAD-Q/TOF-MS/MS analysis spectrum of TA in example 3.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Chemical and reagent sources in the following examples:

3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide (MTT, M2128) and thioflavin T (ThT, 596200) were purchased from Sigma (St. Louis, Mo., USA).

Milli-Q water was prepared by a Milli-Q integrated water purification system (Millipore, Billerica, Mass., USA).

Acetonitrile was purchased from Anaqua Chemicals Supply (Houston, Tex., USA).

A β (1-42) was obtained from China Peptides co., Ltd. (shanghai, China).

EZ-LinkNHSLC-LC-Biotin was purchased from Saimer Feishel technologies, Inc. (Waltham, Mass.).

Streptavidin (SA) biosensors were purchased from Fort BIO from PALL Life Sciences, Washington harbor, N.Y..

Hoechst33342 (B2261) and propidium iodide (PI, P4170) were purchased from Sigma (st louis, missouri, usa).

Annexin V-FITC/PE apoptosis detection kit was purchased from BD Biosciences (san Jose, Calif., USA).

The cell culture in the following examples was mainly obtained by the following method:

PC-12 cells were purchased from American type culture Collection (ATCC; Rokville, Md.).

PC-12 cells were cultured in DMEM medium containing 10% horse serum, 5% fetal bovine serum, 50U/mL penicillin and 50. mu.g/mL streptomycin. All cells were maintained in a 5% carbon dioxide humidified incubator at 37 ℃.

MTT detection procedure

100 μ L of PC-12 cells were seeded into 96-well plates at a density of 30,000 cells/ml. The following day, PC-12 cells were treated with the test drug for 24 hours in the absence or presence of A β (1-42). After treatment, 10. mu.L of MTT solution (5mg/mL) was added to the wells, followed by incubation at 37 ℃ for 4 hours. The medium was removed and 100 μ L DMSO was added to dissolve the blue crystals. The Optical Density (OD) value of the solution was measured at a wavelength of 570nm with a microplate reader (BioTek, VT Lab, USA). Cell viability was calculated according to the following formula. Cell viability (%) × [ OD value (experiment) -OD value (blank) ]/[ OD value (control) -OD value (blank) ].

ThT fluorescence detection

2 μ L of A β (1-42) (1mM) was diluted with PBS with or without test drugs (including PPAC, DTA and TA, curcumin, Poria or Kaixiong extract) and the final volume was set at 100 μ L. At incubation time points of 0, 24 and 48 hours, 50 microliters ThT (20 microliters) were diluted with PBS (pH 7.4) and mixed with 100 microliters of the above solution and added to a black 96-well plate. The OD of the solution was then read with a microplate reader (BioTek, VT Lab, USA) at an excitation wavelength of 450nm and an emission wavelength of 490 nm.

Flow cytometry analysis

PC-12 cells seeded in 6-well plates were treated with A β (1-42) in the absence or presence of the test drug for 24 hours, and the treated cells were trypsinized and centrifuged at 2000 rpm. Cells were resuspended in 250 microliters of 1X buffer and stained with 2 microliters of Propidium Iodide (PI) and 1 microliter of isothiocyanic fluorescing agent (FITC) reagent (BD Biosciences, San Jose, california, usa) for 15 minutes in the dark. The cells were then analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Data acquisition and analysis was performed by Flowjo 7.6.1 software (Tree Star, San Carlos, california, usa).

Hochest 33342/PI staining

Death of PC-12 cells was measured by Hochest 33342/PI staining. Briefly, PC-12 cells seeded on coverslips in 6-well plates were treated with test drugs for 24 hours in the absence or presence of A β (1-42). After treatment, the cells were fixed with fresh 4% Paraformaldehyde (PFA) solution for 15 minutes and washed 3 times with PBS. Then, cells were stained with 5 mg/l Hoechst33342 and 5 mg/l PI solution for 5 minutes. After incubation, representative images showing nuclei with blue fluorescence in the same area and cells showing red fluorescence were captured and merged using an ImageXpress Micro4(Molecular Devices, usa). The death of PC-12 cells was then measured by calculating the percentage of cells with PI signal to cells with blue signal.

Strains and maintenance conditions of nematodes

Nematode strains include N2 (WT); CL4176, dvIs27[ myo-3p: A-Beta (1-42): let-8513' UTR) + rol-6(su1006) ] X; CL2122, dvIs15[ (pPD30.38) unc-54(vector) + (pCL26) mtl-2:: GFP ]; CL2355, dvIs50[ pCL45(snb-1:: Abeta 1-42::3' UTR (long) + mtl-2:: GFP ] I; and CL2331, dvIs37[ myo-3p:: GFP:: A-Beta (3-42) + rol-6(su1006) ] were all obtained from the C.elegans genetics center (CGC.) unless otherwise stated, all worms were maintained on Nematode Growth Medium (NGM) plates and fed with E.coli (E.coli) OP50 in an incubator at 20 ℃.

Paralysis test

Whether the drug improves a β deposition in vivo is tested by measuring the paralysis rate of the AD caenorhabditis elegans CL 4176. Briefly, CL4176 nematodes containing a heat-inducible human Α β (1-42) transgene expressed in muscle cells were transferred to NGM and treated with the test drug. The temperature of the incubator was changed from 15 ℃ to 25 ℃ to stimulate a β expression and aggregation. After exposure at 25 ℃ for 36 hours, the number of paralysis of the nematodes was counted and finally the rate of paralysis was counted. Nematodes fail to move their body when touched or exhibit a "halo" that clears bacteria when fed are manifested as paralysis.

The behavior of nematodes appears to be paralysed, either by failing to move their body on contact or by exhibiting a "halo" that clears bacteria on feeding.

Food perception assay

The improvement effect of the test drug on food-sensing behavior was studied using CL2355 strain expressing human Abeta (1-42) and its vector control CL2122 strain. Briefly, E.coli OP50 was spread on an annular plate having an inner diameter of about 1 cm and an outer diameter of about 8 cm. After 48 hours of treatment with PPAC, DTA, TA or Poria extract, the worms were washed with M9 buffer and placed in the center of NGM agar plates with or without E.coli OP 50. After 5 minutes, the number of body bends of the worms was counted for 20 s. The slowing of body curvature is calculated according to the following formula. Deceleration rate (N without food-N with food)/N without food. N represents the total number of nematode body bends.

Association analysis of Abeta (3-42)

Aggregation of A.beta.3-42 was studied by using CL2331 strain, which expresses GFP bound to human A.beta.3-42 in body wall muscles in temperature sensitive conditions. Briefly, PPAC, DTA, TA or Poria extract treated nematodes were incubated to induce aggregation of A β (3-42) at 23 ℃ until the next day of adult life. After treatment, the nematodes were fixed on slides containing 0.1% NaN 3. Representative images of worms were captured by fluorescence microscopy (Leica DM6B, Leica Microsystems GmbH, germany). The total number of pro-zone Α β deposits was then calculated.

Molecular docking

To further explore the interaction mechanism and binding pattern of PPAC, DTA and TA with Α β, we performed molecular docking studies using the SYBYLX 2.0 molecular modeling program. The crystal structure (ID: 1FIQ) of A.beta.1-42 was retrieved from the Protein Database (PDB) of the research cooperative organization for structural bioinformatics (RCSB). SDF files of ligands, including PPAC (CID: 9805290), DTA (CID: 15225964) and TA (CID: 12314446), are all available from the NCBI Pubchem database. The Surflax-Dock program was used to study potential interaction patterns between A β and PPAC, DTA, TA. Visualization of the docking conformation was done by Chimera molecular graphics software and Ligplot software.

Statistical analysis

All data from three or more independent experiments are presented as means ± Standard Deviation (SD). Using GraphPad Prism 8.0 statistical software (san diego, california, usa), one-way analysis of variance was employed followed by Tukey's test. P <0.5 is considered statistically significant between the comparison groups.

Example 1

Verification and screening of Abeta fibrosis inhibitor in Scutellaria baicalensis

Yu L, Wu A G, Wong K W, et al, the New Application of UHPLC-DAD-TOF/MS in Identification of inhibition on beta-Amyloid fibre disruption From Scutellaria basilica [ J ] other,2019, 10. In this journal, potential inhibitors of A β fibrosis in Scutellaria baicalensis were screened by pre-incubating A β (1-42) with Scutellaria baicalensis extracts and then analyzing UHPLC-DAD-Q/TOF-MS/MS. A significant reduction in the amount of xanthosine and luteolin found in the culture broth containing A β compared to the use of the Scutellariae radix extract alone indicates that xanthosine and luteolin are effective inhibitors of A β fibrosis in Scutellariae radix.

Example 1 validation Using the high throughput screening method provided by the invention

Preparation of Scutellaria baicalensis Georgi extract

Weighing and crushing scutellaria baicalensis, distilling and extracting for 1h by using water with the volume being 10 times of that of the scutellaria baicalensis, repeatedly extracting for 3 times, combining extracting solutions, filtering, concentrating, and dissolving the concentrated extract into a solution with the concentration of 400mg/mL by using DMSO (dimethyl sulfoxide) to obtain the Chinese herbal medicine extract to be detected.

Preparation of biotinylated Abeta (1-42) peptide

5mg of A β (1-42) peptide was dissolved in hexafluoroisopropanol (HFIP, Sigma) solution, the HFIP solution was then dispensed into new 1.5mL tubes and dried under a stream of nitrogen, and then 10. mu.L of DMSO (dimethyl sulfoxide) and an appropriate volume of PBS (including 15% DMSO and 0.02% Tween 20) were added to the tubes to obtain a working concentration of 100mg/mL of A β (1-42).

Biotin (EZ-LinkNHS-LC-LC-Biotin (Thermo Scientific, USA)) in a molar ratio of 5:1 was mixed with the Abeta (1-42) peptide solution, followed by incubation at room temperature for 30min to obtain biotinylated Abeta (1-42) peptide.

High throughput screening

Biofilm interference analysis

The Scutellariae radix extract was diluted with PBS (containing 15% DMSO and 0.02% Tween 20) to obtain working solutions of different concentrations of 25, 50, 100, 200, 400. mu.g/mL.

The working solution and biotinylated Abeta (1-42) peptide are injected into the biosensor and are combined and dissociated, and a combination and dissociation graph is obtained and is shown in figure 1, and the association/dissociation combination curve of the scutellaria baicalensis indicates that the optical thickness (nm) of the biosensor layer is increased in a dose-dependent manner, which indicates that the scutellaria baicalensis extract is directly combined with the Abeta. The kinetic constants calculated by Forte. mu.l BIO data analysis software, including binding affinity (K D), association rate constant (Kon) and dissociation rate constant, of 199. mu.g/mL, 6.18X 10, respectively, are shown in Table 1⁺²1/Ms and 1.23X 10^-11/s, indicating that the scutellaria baicalensis extract and A beta have direct and reversible interaction.

TABLE 1

TCM	KD(μg/mL)	Kon(1/Ms)	Kdis(1/s)
				SB extract	199	6.18×10+2	1.23×10-1

LC-MS analysis

Performing continuous 5 times of binding-dissociation operations on 400 mug/mL of scutellaria baicalensis extract working solution and biotinylated Abeta (1-42) peptide in a biosensor, collecting dissociation solution, preparing 400 mug/mL of scutellaria baicalensis extract working solution for equal volume in parallel as control solution, injecting 10 muL of dissociation solution and control solution into a UHPLC-DAD-Q/TOF-MS/MS instrument for analysis, and obtaining a representative total ion chromatogram map as shown in FIG. 2, S1: dissociation buffer without SB extract, S2: SB extract-containing dissociation buffer, S3: SB extract for BLI analysis. Several compounds were detected in the dissociation buffer compared to the scutellaria baicalensis extract alone, and the retention time, accuracy, molecular weight, chemical name and formulation of the components detected in the scutellaria baicalensis extract are listed in table 2. Among them, fig. 3 shows that the RBA of baicalin and luteolin in Scutellariae radix is highest. Therefore, the conclusion that xanthosine and luteolin are effective a β fibrosis inhibitors in scutellaria was confirmed by example 1 using the high throughput screening method provided by the present invention.

TABLE 2

Example 2

Preparation of extract of Kaixin powder

Weighing and crushing the Kaixin (containing ginseng, polygala tenuifolia, rhizoma acori graminei and poria cocos), distilling and extracting for 1.5h by using water with the volume 10 times that of the Kaixin, repeating the extraction for 4 times, combining extracting solutions, filtering, concentrating, and dissolving the concentrated extract into a 400mg/mL solution by using DMSO (dimethyl sulfoxide) to obtain the Chinese herbal medicine extract to be detected.

Preparation of biotinylated Abeta (1-42) peptide

5mg of A β (1-42) peptide was dissolved in hexafluoroisopropanol (HFIP, Sigma) solution, the HFIP solution was then dispensed into new 1.5mL tubes and dried under a stream of nitrogen, and then 10. mu.L of DMSO (dimethyl sulfoxide) and an appropriate volume of PBS (including 15% DMSO and 0.02% Tween 20) were added to the tubes to obtain a working concentration of 100mg/mL of A β (1-42).

Biotin (EZ-LinkNHS-LC-LC-Biotin (Thermo Scientific, USA)) in a molar ratio of 5:1 was mixed with the Abeta (1-42) peptide solution, followed by incubation at room temperature for 30min to obtain biotinylated Abeta (1-42) peptide.

High throughput screening

Biofilm interference analysis

The extract of Kaoxing san was diluted with PBS (containing 15% DMSO and 0.02% Tween 20) to give working solutions of varying concentrations of 125, 250, 500, 1000, 2000, 4000. mu.g/mL.

The working solution and biotinylated Abeta (1-42) peptide were injected into the biosensor and subjected to association-dissociation, resulting in an association and dissociation pattern as shown in FIG. 4, and the association/dissociation binding curve of the Kaixiong extract showed a dose-dependent increase in the optical thickness (nm) of the biosensor layer, indicating that the Kaixiong extract was directly bound to Abeta. The kinetic constants calculated by Forte. mu.l BIO data analysis software, including binding affinity (K D), association rate constant (Kon) and dissociation rate constant, 170. mu.g/mL, 1.15X 10, respectively, are shown in Table 3²l/Ms and 2.6X 10^-21/s, indicating that the Kaixiong extract has direct and reversible interaction with A beta.

TABLE 3

TCM	KD(μg/mL)	Kon(1/Ms)	Kdis(1/s)
				KXS extract	170	1.15×10+2	2.6×10-2

LC-MS analysis

Continuously performing 5 times of combination-dissociation operations on a 4000 mu g/mL working solution of the extract of the kadsura longipedunculata and biotinylated Abeta (1-42) peptide in a biosensor, collecting dissociation solution, simultaneously preparing the working solution of the extract of the kadsura longipedunculata for the same volume as the 4000 mu g/mL working solution in parallel as control solution, injecting 10 mu L of the dissociation solution and the control solution into a UHPLC-DAD-Q/TOF-MS/MS instrument for analysis, and obtaining a representative total ion chromatogram map which is shown in figure 5, wherein S1: dissociation buffer without kaixuan extract, S2: dissociation buffer containing kaixuan extract, S3: kaikaya extract for BLI analysis, some compounds were detected in the dissociation buffer compared to kaikaya extract alone, and the retention time, accuracy, molecular weight, chemical name and formulation of the components detected in kaikaya extract are listed in table 4.

As can be seen from fig. 6 to 8, among the combined chemical components, three compounds of which RBA is of a fault height, including grifola acid c (ppac), dehydro-temeric acid (DTA) and Temeric Acid (TA), showed the strongest binding force with a β. Using the extract solution of the kaixiong powder.

The triterpenic acid compound is selected from at least one of grifola acid C, dehydro-temmoic acid and temmoic acid as an A beta fibrillation inhibitor by the high-throughput screening method.

TABLE 4

Example 3

Isolation and purification of PPAC, DTA and TA from Poria

Firstly, soaking Poria cocos powder in ethyl acetate reagent, collecting by rotating a rotary evaporator, and repeating for 3 times to obtain Poria cocos extract. The poria cocos ethyl acetate extract is subjected to crude separation by using a silica gel chromatographic column, 100ml fractions are collected into a group, then a target compound is detected by using a mass spectrum, and finally, the purification is carried out by using an ultra-fast high performance liquid phase. Finally, three compounds, including grifol acid C, dehydro-temmoic acid and temmoic acid, were successfully isolated.

Representative total ion chromatograms in FIG. 9 show UHPLC-DAD-Q/TOF-MS/MS analysis of PPAC, DTA, TA and Poria extracts. Wherein, S1: poria cocos wolf extract; s2: PPAC; s3: DTA; s4: and TA. From FIG. 10, it is shown that the exact masses of PPAC, DTA and TA measured are [ MH ] -481.3388, [ MH ] -483.3510 and [ MH ] -485.3368, respectively, and their molecular structures are shown in FIG. 11, which is consistent with the reported compounds.

ThT fluorescence detection

The inhibitory effect of PPAC, DTA and TA on A.beta. (1-42) fibrillation was investigated by the ThT method. Meanwhile, the extracts of Poria cocos, Kaixiong powder and Scutellaria baicalensis were used for comparison. Binding of ThT to a β (1-42) produces an intense fluorescent signal at a wavelength of 482nm, reflecting a β fibril formation.

The fluorescence intensity of the solution was measured with a spectrophotometer at time points of 0h, 24h and 48 h. As shown in FIGS. 12 to 14, A β (1-42) significantly increased ThT fluorescence, while PPAC, DTA, TA, Poria, KANGXIN powder and treatment with curcumin extracts decreased fluorescence intensity. Among these tested drugs, TA and curcumin extracts had the best inhibitory effect.

PC-12 cell death assay

There is increasing evidence that extracellular a β fibrils accumulate and induce cell-dead neurons. In the present experiments, we investigated the protective effect of PPAC, DTA and TA on the viability of A β (1-42) -induced PC-12 cells.

First, the cytotoxicity of PPAC, DTA, TA, Poria, KAIXIN SAN and curcumin extracts in PC-12 cells was measured by MTT, and the results are shown in FIG. 15.

Then, after the PC-12 cells induced by Abeta (1-42) were treated with PPAC (0.5. mu.M), DTA (0.5. mu.M), TA (0.5. mu.M), Poria (20. mu.g/mL), KAIXINSAN (20. mu.g/mL) and curcumin (0.5. mu.M) for 48 hours, the improvement effect on the cell viability was examined by measuring the cell viability using the tetramethylazoazolium salt colorimetry, and the results are shown in FIG. 16, in which PPAC, DTA, TA, Poria, KAIXINSAN and the extract of Scutellaria baicalensis significantly improve the cell viability of PC-12 cells.

In addition, after the PC-12 cells induced by Abeta (1-42) were treated with PPAC (0.5. mu.M), DTA (0.5. mu.M), TA (0.5. mu.M) or Poria cocos (20. mu.g/mL) for 48h, the cells were collected and analyzed by flow cytometry using Annexin V-FITC/PE apoptosis kit, and we found that the Poria cocos extract, PPAC, DTA and TA significantly reduced the cell death of the PC-12 cells treated with Abeta (1-42), and the test results are shown in FIG. 17.

In addition, A β (1-42) -induced PC-12 cells were stained with Hoechst33324/PI for 5min after treating with PPAC (0.5. mu.M), DTA (0.5. mu.M), TA (0.5. mu.M) or Poria (20. mu.g/mL) for 48 h. Representative images of cells with Hoechst or PI signals in the same field were collected and fused (magnification × 10). Scale bar: 100 μm. The histogram shows the ratio of PI/Hoechst signals of PC-12 cells, and the Hoechst/PI staining results indicate that Poria cocos extract, PPAC, DTA and TA can inhibit cell death, which is revealed by the decrease in the number of cells with PI signals, and the test results are shown in FIG. 18.

Taken together, the data show that PPAC, DTA and TA inhibit A β (1-42) -induced death of PC-12 cells.

Behavioral competence test for caenorhabditis elegans

To investigate the neuroprotective effects of PPAC, DTA and TA in an in vivo model of AD, caenorhabditis elegans, a model organism widely used in neurodegenerative diseases, was used.

As shown in FIG. 19, representative nematode images were taken under a microscope (magnification: 10X) after A β (1-42) -induced CL4176 nematodes were treated with Poria cocos extract, PPAC, DTA or TA, respectively, at a certain concentration for 72 h. Scale bar: 100 μm. The histogram shows the percentage of number of non-paralyzed nematodes (n > 60). In addition, a β induced caenorhabditis elegans CL2355 and its control strain CL2122 were used to study the food search behavior, and as a result, as shown in fig. 20, a β induced nematode CL2355 and control strain CL2122 were treated with poria cocos extract, PPAC, DTA and TA at certain concentrations for 72h, respectively, and then their food perception behavior was evaluated by counting the number of nematode bends on the NGM plate per 20s with or without food. The histogram shows the rate of decrease in velocity of nematodes after sensing food. No significance was observed between the control and treated groups of CL2122 strain. As shown in fig. 21, treatment of poria cocos extract, PPAC, DTA and TA can significantly improve the slowing rate of CL2355 strain. Taken together, the above data indicate that PPAC, DTA or TA improves the behavioral function of the C.elegans model.

Assay for abeta deposit change in caenorhabditis elegans

In addition to behavioral capabilities, we also measured the deposition of A.beta.in the nematode CL2331 strain, which expresses human A.beta.conjugated with GFP in somatic muscle cells with temperature sensitivity (1-42), and the results of the test are shown in FIG. 22, in which nematode CL233172h was treated with Poria cocos extract, PPAC, DTA, TA at a certain concentration and representative images of the head of the nematode were taken with a fluorescence microscope (magnification: 40X). Histogram shows total number of Α β deposits in front of nematode CL 2331; bars, s.d.,. p.ltoreq.0.001, n.20. The results showed that the total of Poria cocos extract, PPAC, DTA and TA significantly reduced the deposition of A.beta.in the pro-region, indicating that PPAC, DTA and TA inhibited the formation of A.beta.deposits in vivo.

Experimental test for binding to Abeta (1-42)

To find a more ideal PPAC, DTA and TA bind to the a β protein at the binding site and determine their theoretical binding pattern, calculations based on molecular docking were performed. As shown in FIG. 23, the binding positions of complexes formed by PPCA, DA and TA with A.beta.1-42. Top: 3D interaction shape and polarity of ligand binding pocket surface, bottom: docking 2D model details of the interactive component. PPCA, DA and TA are shown as rods, with key residues highlighted as rods. The dotted line represents a hydrogen bond. These compounds were shown to interact with the hydrophobic pocket of the a β protein. For example, the figures show that PPAC and ASP a: 7 and HIS A: 14 residues form hydrogen bonds and react with hydrogen bonds such as PHE a: 19, GLU A: 22, GLNA A: 15 and GLU A: 11, etc. form van der waals forces. DTA and GLUA: 11, ASNA: 27 and HISA: 14 residues form hydrogen bonds and bind to residues such as ASP a: 7, GLN A: 15, GLU A: 22 form van der waals forces. And GLU A: 11, ASN A: 27, ASP A: residues 23 form hydrogen bonds and bind to residues such as ASP a: 23, GLN A: 15, GLU A: 22 and PHE A: 19 form van der waals forces. In addition, the binding fractions of PPAC, DTA and TA were 4.06, 4.76 and 5.05, respectively.

Thus, PPAC, DTA and TA have strong binding properties to a β. Based on the in vitro and in vivo results above, we further verified TA binding to Α β (1-42) using BLI technology. As shown in fig. 24, the response/binding (Nm) represents the optical thickness of the sensor layer as reflected by the spectral shift (β) when TA interacts with a λ (1-42). Equilibrium binding signal (REQ) represented by a flat curve when the association rate equals the steady state response upon dissociation). TA exhibits direct and reversible interactions with Α β (1-42) as revealed by a dose-dependent increase in the optical thickness (nm) of the sensor layer. In addition, Table 5 shows kinetic constants including binding affinity (K D), association rate constant (Kon) and dissociation rate, 49.9. mu.M, 1.49X 10⁺²1/Ms and 7.42X 10^-31/s. In addition, as shown in fig. 25, S1 represents TA, S2 represents dissociation buffer, and S3 represents blank buffer. UHPLC-DAD-Q/TOF-MS/MS results show that TA can be detected in the dissociated solution.

In general, TA is the compound with the highest binding affinity to A β among the compounds screened from the extract of kadsura.

In example 3, DTA, TA and PPAC were shown to inhibit A β fibril formation, reduce A β cytotoxicity in PC-12 cells, and improve the ability of C.elegans. In the future, this approach will also be used to screen for potential inhibitors of traditional Chinese medicine targeting other pathological proteins such as Tau, alpha-synuclein and Huntingtin (HTT) associated with neurodegenerative diseases.

TABLE 5

Compound	KD(μM)	Kon(1/Ms)	Kdis(1/s)
				TA	49.9	1.49×10+2	7.42×10-3

The high-throughput method for screening the A beta fibrogenesis inhibitor provided by the invention is used by combining biomembrane interference analysis and LC-MS analysis, and finally, the A beta fibrogenesis inhibitor compound can be screened out according to the analysis of a representative total ion chromatogram. The screening method is simple to operate, small in occupied time, high in accuracy, good in repeatability and convenient to popularize and apply widely, has very important significance for easily screening the A beta fibrogenesis inhibitor from the traditional Chinese medicine, and accelerates the discovery and development process of future anti-AD medicines.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.