A method for isolating and preparing a compound with neuraminidase inhibitory activity from Comfrey

A neuraminidase and inhibitory activity technology is applied in the field of separation and preparation of compounds with neuraminidase inhibitory activity, can solve problems such as discovering neuraminidase inhibitors, and achieves simple and feasible separation and preparation methods, abundant resources, The effect of solving poverty

Active Publication Date: 2018-04-17
SHANDONG ANALYSIS & TEST CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the discovery of neuraminidase inhibitors from comfrey and the quality control of comfrey using it as an indicator

Method used

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  • A method for isolating and preparing a compound with neuraminidase inhibitory activity from Comfrey
  • A method for isolating and preparing a compound with neuraminidase inhibitory activity from Comfrey
  • A method for isolating and preparing a compound with neuraminidase inhibitory activity from Comfrey

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the screening of extraction solvent

[0030] (1) Preparation of comfrey extract

[0031] Grind comfrey, pass through a 40-mesh sieve, accurately weigh 0.5g of the powder, put it in a stoppered conical flask, add 50mL of extraction solvent, ultrasonically extract for 30min, filter the filtrate, and spin-evaporate to dryness at 50°C, weigh . Add 50% ethanol and MES buffer (containing 5% DMSO) to reconstitute for use.

[0032] In this embodiment, the extraction solvents are: pure water, 80% ethanol, ethyl acetate and petroleum ether.

[0033] (2) In vitro enzyme activity test:

[0034] Add comfrey extract, neuraminidase solution and MES buffer obtained from four different extraction solvents into a 96-well plate at a ratio of 3:3:4, mix well and incubate at 37°C for 30 min, add 20 μL of MUNANA substrate and continue to incubate for 1 h , adding 100 μL of terminator, after terminating the reaction, measure in a microplate reader at an excitation wavelength...

Embodiment 2

[0038] Example 2: Isolation and Preparation of Compounds with Neuraminidase Inhibitory Activity from Comfrey

[0039] (1) Preparation of comfrey extract

[0040] Grind comfrey, pass through a 40-mesh sieve, accurately weigh 0.5g of the powder, put it in a stoppered conical flask, add 50ml of petroleum ether, extract it ultrasonically for 30min, filter the filtrate, spin evaporate to dryness at 50°C, and weigh . Add 1.0 mL each of ethanol and MES buffer (containing 5% DMSO) to reconstitute and pass through the membrane for later use;

[0041] (2) Screening of compounds with neuraminidase inhibitory activity

[0042]Add 10 μL of comfrey petroleum ether extract, 50 μL of neuraminidase and 240 μL of buffer solution into a YM-100 ultrafiltration tube with a molecular weight cut-off of 100KD, incubate in a constant temperature incubator at 37°C for 30min, and centrifuge at 8000r / min for 5min. The buffer was centrifuged three times to remove unbound small molecules. Then add 150 ...

Embodiment 3

[0049] Example 3: Verification of the inhibitory activity of the compound on neuraminidase

[0050] Arkanin, acetylarkanin, isobutyryl arcanin, β, β-dimethylacryloyl arcanin, and isovaleryl arcanin prepared in Example 2 were prepared in solutions of different concentrations. According to the method of "in vitro enzyme activity test" in Example 1, the inhibitory activities of these five compounds on neuraminidase were respectively identified.

[0051] The results show that: the above-mentioned 5 compounds have better inhibitory activity on neuraminidase, and its IC 50 They are: 62.85μg / mL, 54.39μg / mL, 200.23μg / mL, 59.44μg / mL, 60.87μg / mL.

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Abstract

The invention discloses a method for separating and preparing a compound with neuraminidase inhibitory activity from radix lithospermi. The method comprises the steps of 1, preparing radix lithospermi extracting solution with petroleum ether as the extraction solvent; 2, evenly mixing the radix lithospermi extracting solution with neuraminidase, conducting ultrafiltration centrifugation after reaction, then adding methyl alcohol to achieve enzyme denaturation, releasing complex compounds, conducting further centrifugal ultrafiltration, conducting HPLC detection on filtrate, and screening out the compound with neuraminidase inhibitory activity through HPLC spectrogram comparison with denatured neuraminidase as a control; 3, preparing the screened compound with neuraminidase inhibitory activity by means of a semipreparative chromatography. By the adoption of the method, the purity of the prepared compound is high and is over 98%, and the prepared compound can be directly used as a standard substance. The neuraminidase inhibitory activity of the separated compound is high, and the compound can be used for preparing neuraminidase inhibitor drugs.

Description

technical field [0001] The invention relates to a method for separating and preparing a compound with neuraminidase inhibitory activity from Zicao. Background technique [0002] Influenza (influenza) is an acute respiratory infection caused by influenza virus. It is highly contagious, has a high incidence rate, and is easy to cause outbreaks or pandemics. Especially in recent years, the outbreak of new influenza has brought serious harm to human health. Neuraminidase is an important viral surface glycoprotein on the surface of influenza virus. It has exoglycosidase activity and can hydrolyze the α-glycosidic bond between sialic acid residues and adjacent oligosaccharides. Assisting mature influenza virus to escape from host cells and infect new cells plays an important role in the life cycle of influenza virus, so neuraminidase is one of the main targets of influenza therapeutic drugs. Neuraminidase inhibitors (neuraminidase, NA) are currently widely used in various countr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C46/10C07C50/32C07C67/48C07C69/18C07C69/30C07C69/533A61P31/16
CPCC07C46/10C07C67/48C07C50/32C07C69/18C07C69/30C07C69/533
Inventor 赵恒强张敏敏王晓闫慧娇王岱杰耿岩玲于金倩
Owner SHANDONG ANALYSIS & TEST CENT
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