Method for separating SARS coronavirus main proteinase inhibitor from traditional Chinese medicine

A coronavirus and main protease technology, applied in the direction of antiviral agents, pharmaceutical formulations, biochemical equipment and methods, etc., to achieve the effect of increasing the relative concentration

Active Publication Date: 2010-05-05
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no application report on the screening of SARS coronavirus main protease inhibitors from heat-clearing and detoxifying traditional Chinese medicines such as Scutellaria baicalensis based on the in vitro inhibitory activity assay of SARS coronavirus main protease

Method used

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  • Method for separating SARS coronavirus main proteinase inhibitor from traditional Chinese medicine
  • Method for separating SARS coronavirus main proteinase inhibitor from traditional Chinese medicine
  • Method for separating SARS coronavirus main proteinase inhibitor from traditional Chinese medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Expression, purification of embodiment 1.SARS coronavirus main protease

[0046] According to literature (Yang H etc.. Proc Natl Acad Sci.2003 Nov; 100 (23): 13190-5) carry out the expression, purification of SARS coronavirus main protease, the amino acid sequence of this SARS coronavirus main protease is as follows:

[0047] Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly Cys

[0048] 1 5 10 15

[0049] Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp Leu

[0050]20 25 30

[0051] Asp Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu Asp

[0052] 35 40 45

[0053] Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn His

[0054] 50 55 60

[0055] Ser Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile Gly His

[0056] 65 70 75 80

[0057] Ser Met Gln Asn Cys Leu Leu Arg Leu Lys Val Asp Thr Ser Asn Pro

[0058] 85 90 95

[0059] Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr Phe

[0...

Embodiment 2

[0105] Example 2. Preparation of Radix Scutellariae Extract

[0106] Take 500 grams of dry and pulverized Scutellaria baicalensis, extract with 4500 milliliters of 95% ethanol under reflux, extract 3 times, each time for 2 hours, combine the extracts, and concentrate to obtain the extract (i.e. the total extract of Scutellaria baicalensis 95% ethanol);

[0107] Weigh 10 grams of this extract, suspend it with 150 milliliters of double distilled water, pour it into a 1000 milliliter separatory funnel, extract it with ethyl acetate, use 650 milliliters of ethyl acetate for each extraction, shake it well, and let it stand for 6 hours. layer, extracted 3 times in total, separated the ethyl acetate phase and the water phase, combined the water phase, and set aside, combined each ethyl acetate extract, and evaporated the ethyl acetate extract to remove the solvent with an EYELAN1001 rotary evaporator to obtain a dry paste Scutellaria baicalensis ethyl acetate extract sample (i.e. Scu...

Embodiment 3

[0109] Example 3. Screening SARS coronavirus main protease inhibitors from Scutellaria baicalensis extract

[0110] The above-mentioned ethyl acetate extraction sample, aqueous macroporous resin 95% ethanol elution sample and Scutellaria baicalensis 95% ethanol total extract were carried out in vitro inhibitory activity test of SARS coronavirus main protease, and the inhibition curve is shown in figure 1 . Assay method specifically comprises as follows: (1). Add SARS coronavirus main protease in the buffer solution containing 50mM Tris-HCl, pH 7.3, 1mM EDTA, final concentration is 0.5 μ M, then in different buffer solutions containing this protein Add ethyl acetate extraction sample, aqueous macroporous resin 95% ethanol elution sample and Scutellaria baicalensis 95% ethanol total extract respectively, and their concentration is 100 μ g / mL; (2). Add substrate MCA-AVLQSGFRL (DNP) L-NH 2 In the buffer solution, the final concentration is 16 μ M; and (3). measure the in vitro i...

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Abstract

The invention provides a method for separating an SARS coronavirus main proteinase inhibitor from a traditional Chinese medicine. The method comprises the following steps of: A. measuring the exosomatic suppressive activities of SARS coronavirus main proteinase inhibitors of various extractives from a single traditional Chinese medicine; B. selecting an extractive with the best exosomatic suppressive activity; and C. separating and selecting the extractives selected in the step B at least once. The compound separated by the method has exosomatic suppressive activities on SARS coronavirus mainproteinase, thereby being a possibly optimal medicament or a potential prodrug on sale.

Description

technical field [0001] The present invention relates to a method for screening drugs and small molecule inhibitors with the same structural skeleton. More specifically, it relates to the screening of SARS coronavirus main protease inhibition from traditional Chinese medicines such as Scutellaria baicalensis based on the in vitro inhibitory activity assay of SARS coronavirus main protease. agent method, and a series of flavonoid inhibitors derived from it. Background technique [0002] SARS (Severe Acute Respiratory Sydrome) is an abbreviation for causing severe acute respiratory syndrome (atypical pneumonia), and its pathogen is a virus of the genus Coronaviridae (Peiris J. et al. Lancet, 2003, 361: 1319-1325). Coronaviruses are positive-strand RNA viruses. Coronaviruses belong to the family Coronaviridae. Among the currently known positive-strand RNA viruses, their genomes are the largest (Siddell, S.G. et al. Meulen, V.) 823-856 (Hodder Arnold, London, 2005). This genus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37A61K31/352A61P31/14
Inventor 饶子和马明娄智勇孙玉娜崔庆新薛飞
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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