Method for screening compound with lipase inhibition activity by magnetic bead separation and application of method
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A technology for inhibiting activity and separation of magnetic beads, used in material separation, analytical materials, measurement devices, etc.
Inactive Publication Date: 2012-12-26
ZHEJIANG UNIV
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Existing lipase inhibitor screening methods mainly use in vitro enzyme activity inhibition experiments, which can only evaluate the activity of pure compounds, and are difficult to find lipase inhibitors in complex mixtures such as traditional Chinese medicine.
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Embodiment 1
[0021] Example 1 Preparation of magnetic beads bound to lipase
[0022] 1. Reagents:
[0023] Lipase (extracted from porcine pancreatin), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2-(N-morpholine)ethanesulfonic acid (MES ), N-hydroxysuccinimide (NHS) were purchased from Simga Company; carboxyl magnetic beads (specification: 10 mg / ml) were purchased from Invitrogen Company; other reagents were of analytical grade.
[0024] Preparation of 25 mmol / L MES solution: Weigh an appropriate amount of MES, add water to dissolve, add 1 mol / L sodium hydroxide solution to adjust the pH to 6, and prepare a MES solution with a concentration of 25 mmol / L.
[0025] Preparation of 50 mg / ml EDC solution: Weigh an appropriate amount of EDC and quickly dissolve it in cold 25 mmol / L MES solution to prepare an EDC solution with a concentration of 50 mg / ml. Prepared fresh just before use.
[0026] Preparation of 50 mg / ml NHS solution: Weigh an appropriate amount of NHS and ...
Embodiment 2
[0036] Compound screening with lipase inhibitory activity in the total flavonoids extract of Fructus Aurantii in embodiment 2
[0037] 1. Drugs and reagents:
[0038] The standard products of naringin and 4-methylumbelliferone oleate were purchased from Sigma, the standard products of hesperidin and neohesperidin were purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., and the standard products of citrusin were purchased from Shanghai Tongtian Biotechnology Co., Ltd., the total flavonoid extract of Aurantii Fructus was purchased from Changsha Kangerjia Pharmaceutical Group Co., Ltd.
[0039] Preparation of 10 mmol / L phosphate (PBS) buffer solution Weigh an appropriate amount of sodium dihydrogen phosphate, add water to dissolve, and make a solution of sodium dihydrogen phosphate with a concentration of 10 mmol / L, and weigh an appropriate amount of disodium hydrogen phosphate, add water to dissolve, Prepare a disodium hydrogen phosphate solution with ...
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Abstract
The invention provides a method for screening a compound with lipase inhibition activity by magnetic bead separation. The method is used for rapidly screening the compound with lipase inhibition activity rapidly from fructus aurantii by adopting a magnetic bead separation and liquid chromatography-mass spectrometry combination method. Compared with the traditional method and the prior art, the method disclosed by the invention has the advantages of being capable of rapidly screening and identifying the compound with potential lipase inhibition activity from a mixture, further separating and preparing the target compound and carrying out activity verification, thereby greatly reducing the blindness of separating effective ingredients and improving the activity screening efficiency. During screening, the compound does not need to be prepared and separated, therefore the screening efficiency is remarkably improved and the screening time is shortened. In addition, the screening method has the characteristics of high speed, accuracy, good reproducibility and the like and can be popularized to be used for rapidly screening the compound with lipase inhibition activity from natural medicines or Chinese medicine mixture. The compound with lipase inhibition activity, obtained by using the method disclosed by the invention, can be applied to preparation of diet pills and lipid-lowering medicaments.
Description
technical field [0001] The invention belongs to the field of drug screening methods, and relates to the application of magnetic bead separation and liquid chromatography-mass spectrometry techniques to analyze target protein conjugates, and in particular to the screening of compounds with lipase inhibitory activity in Fructus aurantii. Background technique [0002] Obesity is manifested by a large accumulation of triglycerides in adipocytes, increased adipocyte enlargement and impaired lipolysis in adipose tissue. The abnormal regulation of lipolysis can lead to an increase in the concentration of free fatty acids in the body, which will aggravate the disturbance of carbohydrate and fat metabolism in the liver and muscle, and lead to the occurrence of chronic diseases such as type 2 diabetes. [0003] Lipase is the key enzyme that catalyzes the first step in the hydrolysis of triacylglycerol in vivo. The fat in food is hydrolyzed into monoacylglycerol and free fatty acid un...
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