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Application of Resazurin as Inhibitor of Bovine Insulin Amyloid Fibrils

A technology for insulin and amyloid, applied in the field of biomedicine

Active Publication Date: 2021-07-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are basically no drugs for the treatment of such diseases on the market at present, so the development of amyloid fibril inhibitors that are effective and have little side effects on the human body is of great significance to biomedicine

Method used

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  • Application of Resazurin as Inhibitor of Bovine Insulin Amyloid Fibrils
  • Application of Resazurin as Inhibitor of Bovine Insulin Amyloid Fibrils
  • Application of Resazurin as Inhibitor of Bovine Insulin Amyloid Fibrils

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Experimental program
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Effect test

Embodiment 1

[0010] PBS buffer formulation: 19mL, 0.2M NaH 2 PO 4 aqueous solution and 81 mL, 0.2M Na 2 HPO 4 Mixing of aqueous solutions.

[0011] 25mM NaCl / HCl solution formulation: use HCl to adjust the pH of the NaCl aqueous solution to 1.6, and the NaCl concentration in the NaCl aqueous solution is 25mM.

[0012] Prepare different concentrations of resazurin solutions (solvent is ultrapure water): 0mM, 0.05mM, 0.1mM, 0.2mM.

[0013] Bovine insulin was added to 0.95mL NaCl / HCl (25mM, pH=1.6) solution, the concentration of bovine insulin was 0.5mM, 0.05mL of resazurin solution of different concentrations were added, and incubated at 65°C for 240min. Take 0.05mL of each sample solution, add 0.05mL of ThT (1mM, thioflavin, thiosulfonin T) solution, and finally dilute to 1mL with 0.9mL of PBS buffer solution, and test the fluorescence of the above-mentioned diluted samples in a fluorescence spectrometer Intensity (λ ex =440nm), the fluorescence spectrogram shows that with the increas...

Embodiment 2

[0015] Since the mechanism of amyloid fibril-related diseases is still being explored, and the dynamics of amyloid fibrils may provide relevant information for the mechanism, resazurin was added to monitor the formation of bovine insulin amyloid fibrils with ThT. Dissolve bovine insulin in 0.95mL NaCl / HCl (25mM, pH=1.6) solution, the concentration of bovine insulin is 0.5mM, add different concentrations of resazurin solution 0.05mL respectively, and fibrillate at 65°C. Take 0.05mL samples at different time points, then add 0.05mL ThT (1mM) solution, and finally dilute to 1mL with 0.9mL PBS buffer solution, make 3 parallel samples for each group of samples, and measure all the above samples on a fluorescence spectrometer. The fluorescence intensity. It can be found that with the increase of resazurin concentration, not only the fluorescence intensity gradually decreases, but also the time of fibers appearing gradually delayed, as shown in figure 2 shown. The kinetic process ...

Embodiment 3

[0017] Take bovine insulin solid and dissolve it in 0.95mL pH=1.6, 25mM NaCl / HCl solution, the sample concentration is 0.5mM, add 0.05mL different concentrations of resazurin solution respectively, the final volume is 1mL, at a temperature of 65°C, pH= Fibrosis was carried out under the conditions of 1.6. Take 0.05mL samples at 60min, 150min, 240min, and 420min time points, and dilute to 1mL with PBS buffer solution. The measured concentration of bovine insulin is 0.025mM. Then the circular dichroism spectrum was measured with a circular dichroism spectrometer, and the spectrum was collected in the range of 195-260 nm. The path length of the quartz cuvette used was 1 mm, and the step length was 1, and the background was subtracted with PBS buffer solution. The result is expressed by molar ellipticity, calculated by the following formula:

[0018] [θ] λ =θ(M / 10dcn)

[0019] [θ] λ (deg.cm2.dmol-1) is the molar ellipticity of protein; θ is the directly measured protein ellipt...

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Abstract

The application of resazurin as an inhibitor of bovine insulin amyloid fibrils belongs to the field of biomedical technology. The invention discloses that after adding different concentrations of resazurin in the process of preparing bovine insulin amyloid, and then adding ThT to detect the fluorescence intensity, the fluorescence spectrum shows that the fluorescence intensity decreases successively with the increase of the resazurin concentration. The circular dichroism chromatogram showed that the secondary structure of bovine insulin added with resazurin was delayed in the time of β-sheet formation. The topography images obtained by atomic force irradiation also show that the addition of resazurin makes bovine insulin stay in the nucleation stage, that is, the addition of resazurin obviously inhibits the formation of bovine insulin amyloid fibrils.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of resazurin as an inhibitor of bovine insulin amyloid fibrils. Background technique [0002] Whether in the field of biomedicine or in the field of chemistry, the study of amyloid fibrils is in a crucial position. Because more and more diseases are directly directed at amyloid fibrils, such as Alzheimer's disease, spongiform encephalopathy, Parkinson's Huntington's disease, cardiac arrhythmia, type II diabetes, atherosclerosis, rheumatoid arthritis , prolactinoma and multiple psychosis. However, there are basically no drugs for the treatment of such diseases on the market at present, so the development of amyloid fibril inhibitors that are effective and have little side effects on the human body is of great significance to biomedicine. Contents of the invention [0003] Resazurin, also known as azoresorcinol, is a known structural compound, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/538A61P25/28A61P25/16A61P25/14A61P9/06A61P3/10A61P9/10A61P19/02A61P29/00A61P35/00A61P25/18
CPCA61K31/538A61P3/10A61P9/06A61P9/10A61P19/02A61P25/14A61P25/16A61P25/18A61P25/28A61P29/00A61P35/00
Inventor 陈志俊王浩杰
Owner JILIN UNIV
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