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Application of myricetin in preparation of anti-influenza-virus drugs

An anti-influenza virus, influenza virus technology, applied in the field of medicine, can solve the problems of anti-influenza virus application that has not been reported, has not been widely used, drug resistance and side effects are limited in use, etc.

Inactive Publication Date: 2018-04-13
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Clinically used anti-influenza drugs can be divided into three categories, the classic two types of anti-influenza virus drugs: neuraminidase (NA) inhibitors and M2 ion channel inhibitors, due to drug resistance and side effects limit their use. use
The third category is broad-spectrum RNA polymerase inhibitors, including ribavirin and favipiravir, which are not widely used at present
Myricetin, a class of flavonoids widely present in dicotyledons, has multiple biological activities. It has made some research progress in liver protection, anti-oxidation, immune regulation, tumor, antibacterial and antiviral, etc., but in anti-influenza Virus application has not been reported yet

Method used

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  • Application of myricetin in preparation of anti-influenza-virus drugs
  • Application of myricetin in preparation of anti-influenza-virus drugs
  • Application of myricetin in preparation of anti-influenza-virus drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The detection of embodiment 1 myricetin to cytotoxicity

[0029] The cytotoxicity of myricetin was detected by MTT method, so as to determine the concentration of the drug. The specific method is as follows:

[0030] MDCK or A549 cells by 1×10 4 / well seeded in 96-well plate at 37°C, 5% CO 2 Cultured in a constant temperature cell incubator until monolayer, myricetin was diluted with serum-free DMEM or 1640 gradient and added to a 96-well plate, 200 μl per well, and continued to culture for 48 hours. Discard the culture supernatant, add 100 μL of 1640 or DMEM medium containing 0.5 mg / ml MTT to each well, and incubate at 37°C for 4 hours. The absorbance at 570 nm was detected with a multifunctional microplate reader (Genios Pro, Tecan, US). The survival rate of the cells was used as the index of the toxicity of myricetin to MDCK or A549 cells.

[0031] Cell viability (%)=E / N×100

[0032] E is the absorbance of the drug group, and N is the absorbance of the cell con...

Embodiment 2

[0035] Example 2 Detection of Myricetin Anti-Influenza A Virus Activity in Vitro

[0036] In the in vitro antiviral experiment of the present invention, various subtypes of influenza A viruses are involved, including H1N1 and H3N2, and the specific methods are as follows:

[0037] MDCK cells by 2 x 10 4 / well seeded in 96-well plate at 37°C, 5% CO 2 cultured to a monolayer in a constant temperature cell incubator. Use 100TCID 50100 μl per well of PR8-infected cells, incubated at 37°C for 1 h, discarded the virus solution, added myricetin serially diluted in DMEM (containing 1 μg / ml TPCK), 200 μl per well, and continued to incubate for 48 h. Combined with MTT method and plaque test, the antiviral activity of myricetin was detected. By observing myricetin's inhibition of virus-induced cellular virus phenomenon (CPE) and detecting the survival rate of cells, evaluate the protective effect of myricetin on cells, and further calculate the half effective concentration IC 50 . ...

Embodiment 3

[0042] Example 3 Myricetin inhibits the replication of influenza A virus

[0043] In order to evaluate the inhibitory effect of myricetin on influenza virus replication, the present invention uses three methods of indirect immunofluorescence, Q-PCR and Western blotting to detect the influence of myricetin on virus replication from the expression levels of genes and proteins. The specific method is as follows:

[0044] Indirect immunofluorescence method: MDCK cells were divided into 5×10 4 / well was seeded in a 48-well plate at 37°C, 5% CO 2 cultured to a monolayer in a constant temperature cell incubator. Use 100TCID 50 Infect the cells with PR8, 1ml per well, incubate at 37°C for 1h, discard the virus solution, add myricetin serially diluted in DMEM (containing 1μg / ml TPCK), 500μl per well, and continue to incubate for 24h. Afterwards, fix with 4% paraformaldehyde for 20 minutes, stain the NP protein and cell nucleus, and randomly select multiple fields of view to photogr...

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Abstract

The invention discloses application of myricetin in the preparation of anti-influenza-virus drugs. Influenza A virus is referred to herein, preferably, H1N1, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9,H9N2 or H10N8. The acting mechanism of myricetin to resist influenza A virus is to inhibit activity of ribonucleoprotein complex vRNP by combination with PB2cap protein; myricetin is applicable to thepreparation of drugs to prevent and treat influenza viruses, wherein sites of myricetin combined with influenza virus PB2cap protein are histidine at 357 (His357), phenylalanine at 404 (Phe404), phenylalanine at 323 (Phe323), glutamic acid at 361 (Glu361), lysine at 376 (Lys376), phenylalanine at 363 (Phe363) and asparagine at 429 (Asn429).

Description

technical field [0001] The invention relates to the application of myricetin in anti-influenza virus drugs, and belongs to the technical field of medicine. Background technique [0002] Influenza disease has always been the focus of people's attention, especially the outbreak caused by influenza A virus is a serious threat to human health. There have been several outbreaks of influenza caused by influenza A viruses in history. The "Spanish flu" that broke out from 1918 to 1919 swept the world, causing tens of millions of deaths, and then the "Asian flu" that broke out, "Hong Kong Flu", "Mexican Flu". Because the antigen of influenza A virus is easy to change and drift, the situation of influenza prevention and control is still not optimistic. At present, the prevention and control strategies of influenza are mainly the vaccination of influenza vaccine and the use of anti-influenza drugs. Use a total lag of 0.5 to 1 year. Scientists are also currently working on a univers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/352A61P31/16
CPCA61K31/352
Inventor 杨洁刘叔文刘淼淼
Owner SOUTHERN MEDICAL UNIVERSITY
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