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A group of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity and their application

A technology of adenosine deaminase and extract, which is applied in the field of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity, to achieve the effects of high sensitivity, enhanced activity and simple operation

Active Publication Date: 2021-03-19
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there is no report on the inhibitory activity of Chinese herbal extracts on adenosine deaminase, and it is necessary to further develop new medical uses of Chinese herbal medicines.

Method used

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  • A group of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity and their application
  • A group of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity and their application
  • A group of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Screening of ADA inhibitors

[0049] 1. Experimental method

[0050] 1 Instruments and reagents

[0051] 1.1 Instruments

[0052] Vortex instrument (Votex-Genie 2, USA), BT-25S electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.). KQ-250DB CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). Micropipette (Eppendorf, USA). Milli-Q water purifier (Milli-Q, Millipore Co., Billerica, MA, USA). Microplate constant temperature oscillator (Hangzhou Aosheng Instrument Co., Ltd.).

[0053] 1.2 Reagents

[0054] Adenosine deaminase (ADA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Adenosine (AD), Hypoxanthine ribonucleoside (Hypoxanthine ribonucleoside), chlorhexidine (internal standard, IS), purchased from Sigma-Aldrich (MO, USA). EHNA and 2'-deoxycometamycin (2'-dCF) were purchased from Dalian Meilun Biotechnology Co., Ltd. Chromatographic grade methanol was purchased from Fisher Co. (NJ, USA). Chromat...

Embodiment 2

[0099] (1) Take blood from the orbital venous plexus of SD rats to obtain whole blood by anticoagulation with heparin, and obtain serum and blood cells by centrifugation without anticoagulation. Dilute with 11 mM PBS buffer with pH 7.5 and measure the protein concentration to obtain a protein concentration of 18 mg / mL. Add the substrate adenosine to the test sample, the final concentration of adenosine is 3.740μM, react at 30°C for 22min, immediately stop the reaction with ice methanol at 0°C, take the reaction stop solution, add 3.5 Dilute with twice the volume of water, mix well, and centrifuge to obtain the reaction stop solution supernatant.

[0100] (2) according to the method for embodiment 1, measure the inosine content in the reaction termination liquid supernatant by liquid chromatography tandem mass spectrometry:

[0101] The liquid phase conditions are as follows: Column: 1.8 μm ACQUITY UPLC HSS T3 chromatographic column; Mobile phase: 0.1% formic acid water-methano...

Embodiment 3

[0105] (1) Whole blood was obtained from SD rat orbital venous plexus by heparin anticoagulation, and serum and blood cells were obtained by non-anticoagulant centrifugation, diluted with 9mM PBS buffer pH 7.7, and the protein concentration was determined to obtain a protein concentration of 22 mg / mL. Add the substrate adenosine to the test sample, the final concentration of adenosine is 3.750μM, react at 40°C for 18min, immediately stop the reaction with ice methanol at 0°C, take the reaction stop solution, add 4.5 Dilute with twice the volume of water, mix well, and centrifuge to obtain the reaction stop solution supernatant.

[0106] (2) according to the method for embodiment 1, measure the inosine content in the reaction termination liquid supernatant by liquid chromatography tandem mass spectrometry:

[0107] The liquid phase conditions are as follows: Column: 1.8 μm ACQUITY UPLC HSS T3 chromatographic column; Mobile phase: 0.1% formic acid water-methanol; Elution procedu...

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Abstract

The invention relates to a traditional Chinese medicine extract with adenosine deaminase inhibition activity and an application of the traditional Chinese medicine extract. A preparation method of thetraditional Chinese medicine extract comprises the steps of taking traditional Chinese medicinal material powder, adding methanol, performing ultrasound extraction under a sealed condition, after theultrasound extraction, complementing the lost weight with methanol, performing centrifugation, taking a supernate, performing nitrogen blow-drying to form an extract, namely the traditional Chinese medicine extract, wherein traditional Chinese medicines are radix bupleuri, ligusticum wallichii, Chinese angelica, radix sophorae flavescentis, artemisia capillaris, bighead atractylodes rhizome, folium isatidis, a glossy privet fruit, schisandra chinensis, gardenia, rheum palmatum, sinensis, rhizoma cimicifugae, pollen pini, polygonum cuspidatum or sedum sarmentosum. After determination by established liquid chromatography-tandem mass spectrometry, the traditional Chinese medicine extract has a stronger inhibiting effect on adenosine deaminase, and cell experiments and animal experiments further confirm that the traditional Chinese medicine extract has a significant treatment effect on leukemia and the like.

Description

technical field [0001] The invention relates to the field of traditional Chinese medicines, in particular to a group of traditional Chinese medicine extracts with adenosine deaminase inhibitory activity and applications thereof. Background technique [0002] Adenosine deaminase (Adenosine deaminase; Adenosine aminohydrolase; ADA; EC3.5.4.4) is a sulfhydryl enzyme that has an important relationship with the body's cellular immune activity, and each molecule contains at least 2 active sulfhydryl groups. It is also a key enzyme in the metabolism of purine nucleotides, which can specifically catalyze the deamination of adenosine or deoxyadenosine to inosine or inosine deoxynucleoside, which is then catalyzed by nucleoside phosphorylase to generate Hypoxanthine is finally oxidized to uric acid and excreted. [0003] ADA is widely distributed in many tissues of the human body, especially in the mesentery, liver, spleen, kidney and lymphocytes. , kidney, lung, skeletal muscle are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K36/855A61K36/79A61P35/02A61P31/04A61P37/02A61P3/10A61P1/16A61P35/00A61P25/00A61P25/28A61P9/10A61P9/12G01N30/02
CPCA61K36/068A61K36/15A61K36/195A61K36/232A61K36/233A61K36/236A61K36/282A61K36/284A61K36/315A61K36/41A61K36/489A61K36/57A61K36/638A61K36/704A61K36/708A61K36/71A61K36/744A61K36/79A61K36/855A61K2236/33A61K2236/53G01N30/02A61K2300/00Y02A50/30
Inventor 王长虹刘伟程雪梅戚胜兰管辉达
Owner SHANGHAI UNIV OF T C M
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