Applications of dihydromyricetin in preparation of anti-influenza virus drugs

A technology against influenza virus, influenza virus, applied in the field of medicine

Inactive Publication Date: 2018-08-03
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, clinically used anti-influenza virus drugs are mainly: M2 ion channel inhibitors (amantadine, rimantadine); NA inhibitors (oseltamivir, zanamivir, peramivir); Broad-spectrum RNA polymerase inhibitors (ribavirin, favipiravir), but drug-resistant strains are gradually produced during the use of antiviral drugs, and considering the neurotoxicity of these drugs, people are urgently looking for Novel Anti-Influenza A Drugs and Strategies

Method used

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  • Applications of dihydromyricetin in preparation of anti-influenza virus drugs
  • Applications of dihydromyricetin in preparation of anti-influenza virus drugs
  • Applications of dihydromyricetin in preparation of anti-influenza virus drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

[0030] Cytotoxicity detection of embodiment 1 dihydromyricetin

[0031] The cytotoxicity of dihydromyricetin was detected by MTT method, so as to determine the concentration of the drug. The specific method is as follows:

[0032] 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 to a monolayer, dihydromyricetin was diluted with serum-free DMEM or 1640, and then 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 dihydromyricetin to MDCK or A549 cells.

[0033] Cell viability (%)=E / N×100

[0034] E is the absorbance of the drug group, and N is the absorbanc...

Embodiment 2 2

[0037] Example 2 In vitro anti-influenza virus activity detection of dihydromyricetin

[0038] 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:

[0039] 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 50 100 μl per well of PR8-infected cells, incubated at 37°C for 1 h, discarded the virus solution, added dihydromyricetin 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 experiment, it reflects that the antiviral activity of dihydromyricetin is determined by the protective effect of dihydromyricetin on cells, including the observation of dihydromyricetin inhibition of virus-induced cell virus phenomenon (CPE) and the detection of cell Survival rate, and further ca...

Embodiment 3

[0044] Example 3 Dihydromyricetin inhibits the replication of influenza A virus

[0045] In order to evaluate the inhibitory effect of dihydromyricetin on influenza virus replication, the present invention uses three methods of indirect immunofluorescence, Q-PCR and Western blotting to detect the effect of dihydromyricetin on virus replication from the expression levels of genes and proteins respectively. influences. The specific method is as follows:

[0046] 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 dihydromyricetin 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...

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Abstract

The invention discloses applications of dihydromyricetin in preparation of anti-influenza virus drugs, wherein influenza virus is influenza A virus, preferably H1N1, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 or H10N8. According to the present invention, the anti-influenza A virus action mechanism of the dihydromyricetin is that the activity of the ribonucleoprotein complex vRNP is inhibited by binding the dihydromyricetin to the PB2cap protein so as to prepare the drugs for prevention and treatment of influenza, wherein the binding loci of the dihydromyricetin and the PB2cap protein are site-432 histidine (His432), site-357 histidine (His357), site-404 phenylalanine (Phe404), site-323 Phenylalanine (Phe323), site-361 glutamic acid (Glu361), site-376 lysine (Lys376), site-363 phenylalanine (Phe363), and site-429 asparagine (Asn429).

Description

technical field [0001] The invention relates to the application of dihydromyricetin in anti-influenza virus drugs, and belongs to the technical field of medicine. Background technique [0002] As a viral infectious disease, influenza seriously threatens human health and hinders social and economic development. Influenza A virus causes hundreds of millions of cases of infection around the world every year, and has the characteristics of high morbidity and high mortality. There have been four pandemics caused by influenza viruses in history, all of which were caused by animal-derived influenza viruses crossing the host barrier. In 1997, human infection with avian influenza virus H5N1 occurred in Hong Kong, leading to human respiratory diseases and death, and spread to many countries, with a fatality rate as high as 50%, which aggravated people's panic about highly pathogenic avian influenza viruses. In March 2013, the first case of human infection with H7N9 occurred in China...

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 杨洁刘叔文田元新刘淼淼陈飞敏陈芳昭
Owner SOUTHERN MEDICAL UNIVERSITY
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