CN118370749A - Application of tucin in preparation of anti-influenza A virus infection medicines - Google Patents

Abstract

The invention provides application of tucin in preparing a medicine for resisting influenza A virus infection, and belongs to the technical field of medicines. Experiments prove that the tuotoxin can obviously inhibit the activity of influenza A virus, improve the survival rate of mice infected with H1N1PR8, prolong the survival time of mice infected with H1N1PR8, and has wide application in preventing and/or treating influenza A virus infectious diseases.

Description

Application of phytotoxin in the preparation of drugs against influenza A virus infection

Technical Field

The invention belongs to the field of medical technology, and specifically relates to the application of cephalotoxin in the preparation of drugs for resisting influenza A virus infection.

Background technique

Influenza A virus (IAV), the pathogen of influenza A, belongs to the family Orthomyxoviridae and is a negative-sense, single-stranded, segmented RNA virus. Influenza A is a highly contagious acute febrile respiratory disease with typical symptoms including high fever, myalgia, cough, sore throat and rhinitis. Many patients also develop severe pneumonia and even lead to failure of multiple organs such as the heart and kidneys, so the mortality rate is high. IAV is mainly transmitted through droplets such as sneezing and coughing, and can also be directly or indirectly infected through mucous membranes such as the mouth, nose, and eyes.

Currently, there are two main types of anti-IAV drugs in clinical practice, which act on the viral protein M2 ion channel and neuraminidase (NA) respectively. However, drug resistance has emerged in the spread of IAV, and the use of M2 ion channel inhibitors (aminopyrimidine and limatidine) is no longer recommended, and NA inhibitor (oseltamivir and zanamivir) resistant strains have also appeared. Therefore, it is urgent to develop new anti-IAV drugs.

Rottlerin is a natural product extracted from the fruit and hair of Euphorbia euphorbia. It is a phenolic compound with anti-fertility effects and can also kill tapeworms and flukes. Studies have shown that rottlerin can act as a direct mitochondrial uncoupler, stimulating autophagy by targeting the signal cascade upstream of mTORC1. In addition, rottlerin can also inhibit HIV-1 integration and rabies virus (RABV) infection.

However, there are currently no reports on the inhibition of IAV infection by tadalafil toxin.

Summary of the invention

The present invention aims to provide a new application of thiamin toxin.

The invention provides the use of schizotoxin in preparing medicines for resisting influenza A virus.

The present invention also provides an application of schizotoxin in the preparation of medicines for preventing and/or treating influenza A virus infectious diseases.

Furthermore, the subtype of the influenza A virus is one of H1N1, H3N2, H5N1, H7N2, and H7N9.

Furthermore, the subtype of the influenza A virus is H1N1.

Furthermore, the drug is a preparation prepared with tamarind toxin as an active ingredient and pharmaceutically acceptable excipients.

Furthermore, the auxiliary material is one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix.

Furthermore, the dosage form of the preparation is one of tablets, capsules, pills, powders, granules, injections, sprays, and aerosols.

The present invention also provides a medicine for preventing and/or treating influenza A virus infectious diseases, and further, the medicine contains sarcoma toxin.

Furthermore, the drug also includes pharmaceutically acceptable excipients.

Furthermore, the auxiliary material is one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix.

The present invention provides an application of eriocin in the preparation of an anti-influenza A virus infection drug. The present invention verifies through experiments that eriocin can significantly inhibit the activity of influenza A virus, improve the survival rate of mice infected with H1N1 PR8, and prolong the survival time of mice infected with H1N1 PR8, and is widely used in the prevention and/or treatment of influenza A virus infectious diseases.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Toxicity of tadalafil toxin to A549 human lung cancer cells.

Figure 2. Anti-H1N1 PR8 activity of tadalafil toxin in A549 human lung cancer cells.

Figure 3. Anti-H1N1 PR8 activity of tadalafil toxin on other cell lines.

Figure 4. Effects of tadalafil toxin on body weight changes in mice infected with H1N1.

Figure 5. Effect of serotype-2 toxin on the survival rate of mice infected with H1N1.

Detailed ways

The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

The chemical structural formula of the chelidon toxin (CAS 82-08-6) is as follows:

1. Viruses, drugs, reagents and other materials

(1) Virus: Influenza A virus A/PR/8/34, hereinafter referred to as H1N1 PR8, was provided and preserved by the College of Medical Technology of Chengdu University of Traditional Chinese Medicine.

(2) Abridol, Abridol and sodium carboxymethylcellulose were purchased from MCE, USA. (3) A549 human lung cancer cells, MDCK canine kidney cells, Huh7 human hepatoma cells and SH-SY5Y human neuroblastoma cells were purchased from Hunan Fenghui Biotechnology Co., Ltd. and preserved by the School of Medical Technology of Chengdu University of Traditional Chinese Medicine.

(4) DMEM cell culture medium was a product of Wuhan Pronocell Life Science Co., Ltd., supplemented with 10% fetal bovine serum purchased from Thermo Fisher, USA, non-essential amino acids, ampicillin and streptomycin (100 U/mL each), and culture medium additives were all products of Beijing Solaibao Technology Co., Ltd.

(5) Cell digestion solution, containing 0.25% trypsin, was purchased from Cytiva, USA.

(6) CCK8 cell activity and proliferation detection kits are products of Wuhan Sanying Biotechnology Co., Ltd. (7) Anti-Influenza A Virus Nucleoprotein antibody, Goat Anti-Mouse IgG H&L (Alexa 488) is a product of Abcam, UK.

(8) BALB/c mice aged 6 to 8 weeks were purchased from SpE Biotechnology Co., Ltd.

Example 1: Cell experiment

1. Experimental methods (I) Toxicity of phytotoxin to A549 human lung cancer cells

(1) Cultured A549 human lung cancer cells were seeded into a 96-well plate at 10 ^{4 cells} /well.

(2) On the next day, the toxin was diluted into seven concentration gradients of 0.04, 0.2, 1, 5, 25, 125 and 625 μM using 1640 complete medium (the drug was dissolved in DMSO), and the same concentration of DMSO was used as a control.

(3) Discard the supernatant in the well plate, add the diluted drugs of different concentrations to the well plate, and culture in an incubator for 18 h.

(4) Discard the supernatant, mix 1640 complete medium and CCK-8 reagent in a ratio of 10:1, add 100 μL/well of the mixture, and incubate in a cell culture incubator for 1.5 h.

(5) Use a multifunctional microplate reader to detect the optical density (OD value) of each well at 450 nm.

(6) Calculate the relative cell activity of each well: relative cell activity = [(OD _compound - OD _{blank well} ) / (OD _DMSO - OD _{blank well} )] × 100%.

(7) The 50% cytotoxic concentration (CC ₅₀ ) of each drug was calculated using the GraphPad Prism 10 software based on the relative cell activity values.

The results of the toxicity test of octotoxin on A549 human lung cancer cells are shown in FIG1 . The toxicity of octotoxin on A549 human lung cancer cells is low, and the calculated CC ₅₀ of octotoxin is 224.70 μM.

(II) Anti-H1N1 PR8 Activity of tadalafil toxin in A549 Human Lung Cancer Cells

(1) Cultured A549 human lung cancer cells were inoculated into a 96-well plate at 10 ⁴ cells/well.

(2) On the second day, cells were infected with H1N1 PR8 (MOI=1), and five concentration gradients of 0.04, 0.2, 1, 5 and 25 μM of tadalafil were added to the wells, and DMSO of the same concentration was used as a control.

(3) Incubate in a cell culture incubator for 18 h.

(4) Perform immunofluorescence detection.

(5) Count the positive clones.

(6) Calculate the inhibition rate of each compound on H1N1 PR8 at different concentrations based on the number of positive clones:

Inhibition rate = [(b-a)/b] × 100%

a is the number of positive clones in the wells with drug added; b is the number of positive clones in the wells with the same concentration of DMSO added.

(7) Based on the inhibition rate, the 50% effective concentration (EC ₅₀ ) of each drug was calculated using GraphPad Prism 10 software.

(8) Calculate the selectivity index (SI) of each compound based on its CC ₅₀ and EC ₅₀ .

SI = _CC50 / _EC50

The experimental results are shown in FIG2 , and it can be seen that the toxin has significant anti-H1N1 PR8 activity, and the calculated EC ₅₀ of the toxin is 0.75 μM, and the SI is 301.29.

(III) Anti-H1N1 PR8 Activity of tadalafil Toxin in Other Cell Lines

(1) Cultured MDCK canine kidney cells, Huh7 human hepatoma cells, and SH-SY5Y human neuroblastoma cells were seeded into 96-well plates at a density of 10 ⁴ /well.

(2) On the second day, cells were infected with H1N1 PR8 (MOI = 1), and 5 concentrations of 0.04, 0.2, 1, 5 and 25 μM of tadalafil were added, and DMSO of the same concentration was used as a control. The cells were incubated in a cell culture incubator for 18 h, and immunofluorescence detection was performed.

(3) Calculate the number of positive clones in each well and the inhibition rate of toxin on the virus in each cell line.

The experimental results are shown in Figure 3. The toxin still has anti-H1N1 PR8 activity in other cell lines. The calculated EC ₅₀ values of toxin in MDCK canine kidney cells, Huh7 human liver cancer cells, and SH-SY5Y human neuroblastoma cells are 1.36μM, 65.80μM, and 56.15μM, respectively. The experimental results show that toxin can effectively inhibit the activity of H1N1 PR8 virus in different cell lines.

Example 2: Animal Experiment

1. Experimental Methods

(I) Grouping of mice

BALB/c mice aged 6 to 8 weeks were randomly divided into 5 groups, including the PR8+Pre-Rottlerin group given before inoculation with H1N1 PR8, the PR8+Pre-Abridol group given before inoculation with H1N1 PR8, the PR8+Post-Rottlerin group given after inoculation with H1N1 PR8, the experimental control group (PR8+Vehicle group) and the blank control group (Blank group), with 10 mice in each group.

(II) Virus inoculation

The infection was carried out via intranasal route. The infection dose for each mouse was 5 LD ₅₀ in a volume of 50 μL. The day of virus inoculation was recorded as 0 day.

3. Drug administration to mice

(1) Administration method: intragastric administration.

(2) Specific administration time:

PR8+Pre-Rottlerin group: mice were given the drug once 24 hours before H1N1 PR8 infection, and then given the drug 12 hours after H1N1 PR8 infection for 6 times in a row, once a day;

PR8+Pre-Abridol group: mice were given the drug once 24 hours before H1N1 PR8 infection, and then given the drug 12 hours after H1N1 PR8 infection for 6 times in a row, once a day;

PR8+Post-Rottlerin group: mice were given 6 consecutive doses starting 12h after H1N1 PR8 infection, once a day;

PR8+Vehicle group: mice were given 0.5% sodium carboxymethyl cellulose 6 times continuously starting 12h after H1N1 PR8 infection, once a day;

Blank group: mice were not treated with any treatment.

(3) Dosage: The dosage of both arbidol and tadalafil is 30 mg/kg/d.

(4) Drug solvent: 0.5% sodium carboxymethyl cellulose.

2. Experimental Results

After mice were infected with H1N1 PR8, their weight and death time were recorded every day until 10 days. The preventive and therapeutic effects of the toxin in mice were evaluated by the weight change curve and survival curve.

As can be seen from Figures 4 and 5, the weight of mice in the PR8+Vehicle group gradually decreased, and all died on the 7th day of infection, proving that H1N1 PR8 successfully infected mice. All mice in the PR8+Post-Rottlerin group died on the 9th day, which shows that rottlerin toxin can prolong the survival time of infected mice and has a certain therapeutic effect on influenza A virus infection.

The body weight of mice in the PR8+Pre-Abridol group and PR8+Pre-Rottlerin group decreased first and then gradually increased. On the 10th day, only 2 mice in the PR8+Pre-Abridol group survived, while 3 mice in the PR8+Pre-Rottlerin group survived, indicating that tadalafil toxin is more effective than Abridol in preventing and treating H1N1 PR8 infection.

The results showed that as a preventive drug, jatropha toxin significantly increased the survival rate of mice infected with the H1N1 PR8 virus, and as a therapeutic drug, it could prolong the survival time of mice.

The present invention provides an application of eriocin in the preparation of an anti-influenza A virus infection drug. The present invention provides an application of eriocin in the preparation of a drug for preventing and/or treating influenza A virus infectious diseases. The present invention has verified through experiments that eriocin can significantly inhibit the activity of influenza A virus, improve the survival rate of mice infected with H1N1 PR8, and prolong the survival time of mice infected with H1N1 PR8, and is widely used in preventing and/or treating influenza A virus infectious diseases.

Claims (10)

1. The application of schizotoxin in the preparation of drugs against influenza A virus. 2. Use of schizotoxin in the preparation of drugs for preventing and/or treating influenza A virus infectious diseases. 3. The use according to claim 1 or 2, characterized in that the subtype of influenza A virus is one of H1N1, H3N2, H5N1, H7N2, and H7N9. 4. The use according to claim 3, characterized in that the subtype of influenza A virus is H1N1. 5. The use according to claim 4, characterized in that the drug is a preparation prepared with tadalafil as the active ingredient and pharmaceutically acceptable excipients. 6. The use according to claim 5, characterized in that the auxiliary material is one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix. 7. The use according to claim 5, characterized in that the dosage form of the preparation is one of tablets, capsules, pills, powders, granules, injections, sprays, and aerosols. 8. A drug for preventing and/or treating influenza A virus infectious diseases, characterized in that the drug contains sarcoma toxin. 9. The drug for preventing and/or treating influenza A virus infectious diseases according to claim 8, characterized in that the drug further comprises pharmaceutically acceptable excipients. 10. The drug for preventing and/or treating influenza A virus infectious diseases according to claim 8, characterized in that the auxiliary material is one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix.