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Method for screening lipase inhibitor

A technology of lipase inhibitors and screening methods, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that cannot be adapted to the simultaneous screening of drugs, the range of test samples is small, and the effective ingredients are not clear, so as to achieve easy identification and memory, low experimental cost and low price

Inactive Publication Date: 2013-03-20
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional in vivo and in vitro screening methods have high requirements on the purity and application amount of effective compounds, while the active ingredients of natural medicines are not clear, and most of them are multi-component and multi-target synergistic effects, giving natural The screening of drug-derived lipase inhibitors brings difficulties, and this screening method needs to consume a large number of samples, which is labor-intensive and cannot be adapted to the simultaneous screening of a large number of drugs
When using UV spectrophotometry and MTT colorimetry for lipase screening, it is limited by solvent and sample concentration, so the range of tested samples is small

Method used

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  • Method for screening lipase inhibitor
  • Method for screening lipase inhibitor
  • Method for screening lipase inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Preparation of enzymatic buffer solution: Accurately weigh 1.212g of tris hydroxymethylaminomethane powder and 0.455g of anhydrous calcium chloride powder as a solvent in deionized water, adjust the pH value of the mixture to 6.5 with hydrochloric acid, and dissolve The solution was finally adjusted to 500mL, prepared into an enzymatic reaction buffer (pH=6.5) with a tris concentration of 20mmol / L, and stored in a refrigerator at 2-4°C for future use.

[0029] Preparation of substrate solution: 2-naphthyl myristate powder was dissolved in petroleum ether to prepare a substrate solution with a concentration of 16 mmol / L.

[0030] Preparation of lipase solution: Add porcine pancreatic lipase to the above enzymatic buffer to prepare a solution with a concentration of 10u / mL.

[0031] Preparation of developer solution: Dissolve Fast Blue B salt powder in deionized water to prepare a developer solution with a concentration of 0.5 mg / mL.

[0032] Preparation of the test solu...

Embodiment 2

[0037] Preparation of enzymatic buffer solution: Accurately weigh 3.029g of Tris powder and 0.91g of anhydrous calcium chloride powder as a solvent in deionized water, adjust the pH value of the mixed solution to 7.8 with hydrochloric acid, and dissolve The solution was finally adjusted to 500mL, prepared into 50mmol / L enzymatic reaction buffer (PH=7.8), and stored in a refrigerator at 2-4°C.

[0038] Preparation of substrate solution: 2-naphthyl myristate powder was dissolved in petroleum ether to prepare substrate solutions with concentrations of 0.25mmol / L and 350mmol / L respectively.

[0039] Lipase solution: Add porcine pancreatic lipase to the above-mentioned enzymatic buffer solution to prepare solutions with concentrations of 5u / ml and 200u / ml respectively.

[0040] Preparation of developer solution: Dissolve Fast Blue B salt powder in deionized water to prepare developer solutions with concentrations of 0.1 mg / ml and 100 mg / ml respectively.

[0041]Preparation of the ...

Embodiment 3

[0047] Preparation of enzymatic buffer solution: Accurately weigh 3.029g of Tris powder and 0.91g of anhydrous calcium chloride powder in deionized water, adjust the pH value of the mixture to 7.0 with hydrochloric acid, and dissolve The solution was finally adjusted to 500mL, prepared into 10mmol / L enzymatic reaction buffer (PH=7.0), and stored in a refrigerator at 2-4°C.

[0048] Preparation of substrate solution: 2-naphthyl myristate powder was dissolved in petroleum ether to prepare a substrate solution with a concentration of 25 mmol / L.

[0049] Lipase solution: add porcine pancreatic lipase to the above enzymatic buffer to prepare a solution with a concentration of 35u / ml.

[0050] Preparation of developer solution: Dissolve Fast Blue B salt powder in deionized water to prepare a developer solution with a concentration of 0.2 mg / ml.

[0051] Preparation of the test solution: Dissolve Orlistat (Orlistat, O4139-25MG) powder in 95% ethanol to prepare 4×10 -8 mol / L, 2×10 ...

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Abstract

The invention relates to a method for screening a lipase inhibitor and particularly relates to a method for screening a lipase inhibitor by using a thin-layer chromatography bioautography. The method comprises the following steps of: dissolving a sample to be tested into water and / or an organic solvent, dispensing liquid of the sample to be tested on a thin-layer plate, placing the plate in a developing agent for developing, taking out the plate, and airing the plate for later use; and dipping the obtained thin-layer plate into substrate liquid, dipping the thin-layer plate into lipase liquid after completing solvent evaporation, taking out the thin-layer plate and subjecting the thin-layer plate to an enzymatic reaction at the temperature of 20-40 DEG C, and dipping the thin-layer plate into developing agent liquid for color reaction after completing the enzymatic reaction, thereby obtaining a white and / or brown-yellow spotted result diagram with a purple background. The method for screening the lipase inhibitor has the advantages that complicated instruments and devices are not required, the operation is simple, and the testing cost is low, so that the method is suitable for general laboratories; and the sensitivity and specificity are higher than those of conventional screening methods, and screened results are visual and easily recognized and remembered.

Description

technical field [0001] The invention relates to a method for screening lipase inhibitors, in particular to a method for screening lipase inhibitors using thin-layer chromatography bioautographic technology. Background technique [0002] Lipase, also known as triacylglycerol acyl hydrolase, is a type of water-soluble esterase that promotes the hydrolysis or synthesis of lipids (such as triglycerides, fats, oils, etc.). It is ubiquitous in animals, plants and microorganisms. The pancreas and adipose tissue of the human body contain more lipase, and a certain amount of lipase is also contained in intestinal juice and gastric juice. Various lipases control the processes of digestion, absorption, fat reconstruction and lipoprotein metabolism. In recent years, with the improvement of people's living standards and changes in lifestyle, the prevalence of lipid metabolism disorders has risen rapidly. It is not only an important risk factor for cardiovascular and cerebrovascular dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/90
Inventor 吴弢唐吉鹤陶宏迅
Owner SHANGHAI UNIV OF T C M
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