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Micellar electrokinetic chromatography method for screening prolyl hydroxylase 2 inhibitor from traditional Chinese medicine extract

A technology of prolyl hydroxylase and electrokinetic chromatography, applied in the direction of resisting vector-borne diseases, instruments, measuring devices, etc., can solve problems such as capillary electrophoresis, and achieve accurate and reliable screening results, precise screening, and a wide range of applications Effect

Active Publication Date: 2022-06-03
LANZHOU INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Currently, there is no report on the use of capillary electrophoresis for the screening of prolyl hydroxylase 2 inhibitors

Method used

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  • Micellar electrokinetic chromatography method for screening prolyl hydroxylase 2 inhibitor from traditional Chinese medicine extract
  • Micellar electrokinetic chromatography method for screening prolyl hydroxylase 2 inhibitor from traditional Chinese medicine extract
  • Micellar electrokinetic chromatography method for screening prolyl hydroxylase 2 inhibitor from traditional Chinese medicine extract

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Embodiment Construction

[0025] The following uses the hypoxia-inducible factor-1α peptide containing prolyl at position 564 (sequence 556-574) as the substrate of prolyl hydroxylase 2 inhibitor to optimize the enzymatic reaction conditions and measure the enzyme activity. , Enzyme inhibition research and screening of inhibitors from traditional Chinese medicine.

[0026] 1. Establish a screening method

All experiments were performed on an Agilent capillary electrophoresis instrument equipped with a UV detector, and instrument operation and data analysis were performed on an Agilent ChemStation (Rev. B 04.02). CE separation (capillary electrophoresis) was performed in a fused silica capillary with an inner diameter of 50 μm and a total length of 48.5 cm (effective length of 40 cm). New capillaries should be activated by rinsing sequentially with methanol, 1 M sodium hydroxide, and deionized water for 60 minutes at 50 mbar. Before daily experiments, capillaries should be rinsed with 1 M NaOH, deioni...

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Abstract

The invention discloses a method for accurately screening a prolyl hydroxylase 2 inhibitor in a traditional Chinese medicine extract by micellar electrokinetic chromatography, which comprises the following steps: sequentially injecting a prolyl hydroxylase 2 solution, a to-be-screened traditional Chinese medicine extract solution and an enzymatic reaction buffer solution into a centrifugal tube, then putting the centrifugal tube into a water bath at 20-45 DEG C to react for 0.5-5 hours, taking out the centrifugal tube, adding a quencher to terminate the reaction, and filtering to obtain the prolyl hydroxylase 2 inhibitor. Carrying out pressure sample introduction on a sample in a capillary electrophoresis apparatus, selecting a background buffer solution, applying a voltage of 10-30 kilovolts, carrying out separation detection, and representing enzyme activity by using a peak area of a product at 210 nm; whether the traditional Chinese medicine extract to be screened has the prolyl hydroxylase 2 inhibitory activity or not can be judged according to the change of the peak area of the product. The method is verified by a model inhibitor and investigated by methodology, the screening result is accurate and reliable, and the consumption of samples and reagents is small. Screening objects comprise mixtures, so that the application range is wider, and the method has important significance on development of traditional Chinese medicines and treatment of hypoxia-related diseases.

Description

technical field [0001] The invention relates to a screening method and application of prolyl hydroxylase 2 inhibitors, in particular to a micellar electrokinetic chromatography method for screening prolyl hydroxylase 2 inhibitors from traditional Chinese medicine extracts, belonging to the technical field of medicine. Background technique [0002] Hypoxia is a physiological state in which oxygen supply is insufficient relative to the body's needs, and is one of the factors affecting human health. In recent years, it has been reported that hypoxia is closely related to the occurrence and evolution of various diseases, such as renal anemia, cancer, neovascular eye disease, cardiovascular disease, etc. (Document 1: Exp. Cell Res. 2017, 356, 116- 121; Literature 2: Cell. 2019, 176, 1248-1264.). Although hypoxia is a potentially lethal factor, the body also possesses defense mechanisms against hypoxia that can sustain survival in relatively acute or tolerable conditions. In 201...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/60G01N30/64G01N30/86
CPCG01N30/02G01N30/6078G01N30/64G01N30/8675Y02A50/30
Inventor 杨军丽张瑛王伟峰
Owner LANZHOU INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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