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Method for high-throughput screening of A beta fibrogenesis inhibitor

An inhibitor, high-throughput technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of time-consuming and laborious, achieve the effect of good repeatability, easy popularization and application, and accelerate the process of discovery and development

Active Publication Date: 2021-09-17
SOUTHWEST MEDICAL UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is: the process of finding biologically active components that inhibit the formation of Aβ fibers from Chinese herbal medicines or formulas with complex chemical components in the prior art is time-consuming Another laborious problem, providing a method for high-throughput screening of inhibitors of Aβ fibril formation

Method used

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  • Method for high-throughput screening of A beta fibrogenesis inhibitor
  • Method for high-throughput screening of A beta fibrogenesis inhibitor
  • Method for high-throughput screening of A beta fibrogenesis inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Validation and screening of Aβ fibrosis inhibitors in Scutellaria baicalensis

[0106] Yu L, Wu A G, Wong K W, et al. The New Application of UHPLC-DAD-TOF / MS in Identification of Inhibitors on β-Amyloid Fibrillation From Scutellaria baicalensis [J]. other, 2019, 10. In this journal, potential inhibitors of Aβ fibrosis in Scutellaria baicalensis were screened by pre-incubating Aβ(1-42) with Scutellaria baicalensis extract and then analyzed by UHPLC-DAD-Q / TOF-MS / MS. Compared with Scutellaria baicalensis extract alone, xanthosides and xanthosides were found to be significantly lower in the Aβ-containing culture medium, indicating that xanthosides and xanthosides are potent inhibitors of Aβ fibrosis in Scutellaria baicalensis.

[0107] Embodiment 1 uses the high-throughput screening method provided by the present invention to verify

[0108] Preparation of Scutellaria baicalensis extract

[0109] Scutellaria baicalensis was weighed and pulverized, then distilled and e...

Embodiment 2

[0125] Preparation of Kaixinsan Extract

[0126] Weigh and crush Kaixin San (the Kaixin San contains ginseng, Polygala, Shichangpu, and Poria cocos), then distill and extract with 10 times the volume of water for 1.5 hours, repeat the extraction 4 times, combine the extracts, filter, concentrate, and then The concentrated extract was dissolved into a 400 mg / mL solution with DMSO (dimethyl sulfoxide) to obtain the Chinese herbal medicine extract to be tested.

[0127] Preparation of biotinylated Aβ(1-42) peptide

[0128] 5 mg of Aβ(1-42) peptide was dissolved in hexafluoroisopropanol (HFIP, Sigma) solution, then the HFIP solution was dispensed into new 1.5 mL test tubes and dried under nitrogen flow, then, in the test tubes Add 10 μL of DMSO (dimethyl sulfoxide) and an appropriate volume of PBS (comprising 15% DMSO and 0.02% Tween 20) to obtain a working concentration of 100 mg / mL of Aβ(1-42).

[0129] Biotin (EZ-LinkNHS-LC-LC-Biotin (Thermo Scientific, USA)) with a molar...

Embodiment 3

[0146] Isolation and purification of PPAC, DTA and TA from Poria cocos

[0147] Firstly, take Poria cocos powder, soak it with ethyl acetate reagent, and then use a rotary evaporator to rotate and collect, and repeat 3 times to obtain Poria cocos extract. The ethyl acetate extract of Poria cocos was roughly separated using a silica gel chromatography column, and 100ml fractions were collected and divided into one group, and then the target compound was detected by mass spectrometry, and finally purified by ultra-fast high-performance liquid phase. Finally, three compounds were successfully isolated, including pyripolic acid C, dehydroturmoic acid, and turmolic acid.

[0148] Figure 9 The representative total ion chromatograms in show the UHPLC-DAD-Q / TOF-MS / MS analysis of PPAC, DTA, TA and Poria cocos extract. Among them, S1: Poria cocos extract; S2: PPAC; S3: DTA; S4: TA. from Figure 10 It shows that the measured accurate masses of PPAC, DTA and TA are [MH]-481.3388, [...

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Abstract

The invention relates to a method for high-throughput screening of an A beta fibrogenesis inhibitor, which comprises the following steps of 1, fixing a to-be-detected Chinese herbal medicine extract working solution and biotinylated A beta (1-42) peptide on a biosensor, and carrying out binding-dissociation to obtain binding and dissociation graphs and kinetic constants, obtaining the binding condition of the to-be-detected Chinese herbal medicine extract and the biotinylated A beta (1-42) peptide, 2, liquid chromatography-mass spectrometry analysis: carrying out N times of binding-dissociation operations on the to-be-detected Chinese herbal medicine extract by adopting the same method in the step 1, and collecting dissociation liquid, then carrying out drying treatment on the collected dissociation liquid, and then, redissolving the dried dissociation liquid by using an organic solvent, putting the redissolved liquid 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 the A beta fibrogenesis inhibitor compound. The method has the advantages of simple operation, less occupied time, high accuracy, high precision and good repeatability, and is convenient for wide popularization and application.

Description

technical field [0001] The invention relates to the technical field of high-throughput screening, in particular to a method for high-throughput screening of Aβ fibril formation inhibitors. Background technique [0002] The pathology of Alzheimer's disease is closely related to the occurrence of neuroinflammation and neuronal death caused by accumulating misfolded protein aggregates such as β-amyloid (Aβ) and Tau. Aβ is produced by the sequential cleavage of β- and γ-secretases from the amyloid precursor protein (APP). The resulting fragments including Aβ(1-40) and Aβ(1-42) are thought to be the most toxic form, forming Aβ fibrils and accumulating extracellularly, eventually in the brains of Alzheimer's patients age spots. In the early stages of Alzheimer's disease, Aβ can still be cleared through the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome pathway (UPP) in neurons, or phagocytized by microglia. However, both ALP and UPP become dysregulated with age so t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N30/8675G01N2030/324
Inventor 吴安国秦大莲吴建明周晓刚邱文乔唐勇郭垊松朱凤丹
Owner SOUTHWEST MEDICAL UNIVERISTY
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