CN119632999A

CN119632999A - Application of sisal sapogenin in the preparation of drugs for treating influenza and viral pneumonia

Info

Publication number: CN119632999A
Application number: CN202411842290.4A
Authority: CN
Inventors: 徐培平; 冯文文; 张悦瑶
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2024-12-13
Filing date: 2024-12-13
Publication date: 2025-03-18
Anticipated expiration: 2044-12-13
Also published as: CN119632999B

Abstract

The invention discloses an application of sisal sapogenin in preparing medicaments for treating influenza and viral pneumonia thereof, wherein the influenza virus is influenza A, and the viral pneumonia is influenza A viral pneumonia. The sisal sapogenin (Tigogenin) can reduce the expression of influenza virus mRNA and influenza virus NP protein in vitro. Can inhibit lung tissue inflammation pathology caused by influenza virus in vivo, and reduce lung index. Because the sisal sapogenin has obvious inhibiting effect on replication of influenza viruses, the sisal sapogenin provided by the invention provides a novel medicament for treating influenza and viral pneumonia thereof.

Description

Application of sisal sapogenin in preparation of medicine for treating influenza and viral pneumonia thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and in particular relates to an antiviral drug molecule taking influenza A virus NP protein as a target spot and a preparation method thereof.

Background

Influenza A Virus (IAV) infection can cause respiratory disease, its host is widespread, contagious, and over the past hundred years, there are several IAV pandemics worldwide, and it causes death in at least tens of millions of people. Influenza causes seasonal outbreaks and unpredictable pandemics each year, and has high morbidity and mortality, which seriously threatens public health safety of humans.

The neuraminidase inhibitor of the current global anti-influenza virus medicament is mainly an M2 enzyme inhibitor, and comprises oseltamivir, zanamivir, peramivir and 4 kinds of ranimivir which are sialic acid derivatives, and has the advantages of better activity, more chiral centers in the structure and more complex synthesis. And various mutants of 4 kinds of on the market ceramidase inhibitor drugs having drug resistance, such as H274Y subtype of H1N1 or H5N1 and W119V subtype of H3N2 influenza viruses are most conventional M mutant strains, and are resistant to zanami Wei Jiao stave, so that the cooperative application of oral drug oseltamivir is severely limited, and therefore, development of new targets and traditional Chinese medicine inhibitors suitable for influenza viruses is urgently needed.

Sisal sapogenin Tigogenin is an effective component extracted from rhizoma anemarrhenae in Yinqiao antiphlogistic powder patent prescription through early network pharmacology in a laboratory, the main component of the sisal sapogenin is tikenine (Tigogenin), the chemical name of the sisal sapogenin is 5 alpha, 25D-spirostan-3 b hydroxyl, the sisal sapogenin is a medical intermediate and an important raw material for synthesizing steroid hormone medicines, and pharmacological research shows that the sisal sapogenin has obvious biological activities of resisting inflammation, resisting bacteria, stopping bleeding, resisting aging and reducing blood sugar. The patent application with publication number CN118852316A discloses a sisal sapogenin derivative, a preparation method and application thereof in hypoglycemic drugs or preparations, and in addition, another published patent application states that Tigogenin-cellobioside, a preparation method of heptaacetate thereof and patent retrieval information of pharmaceutical compositions containing Tigogenin-cellobioside. However, no research has been reported on the mechanism of action and prevention and control of sisal sapogenin in anti-influenza viruses.

The invention discovers that the sisal sapogenin has the purpose of resisting influenza A virus for the first time, and experimental results show that the sisal sapogenin influences the replication of the virus by inhibiting the expression content of mRNA and NP proteins of the influenza virus, has the function of resisting the influenza virus in vitro and in vivo, and has the prospect of being developed into medicaments for treating influenza and viral pneumonia induced by the influenza virus.

Disclosure of Invention

The invention aims to overcome the defects existing in the prior art and provide a new application of sisal sapogenin. The sisal sapogenin has remarkable activity of resisting influenza A virus, can be developed as a new generation of anti-influenza virus drug, and has wide application prospect.

To achieve the above object, in one aspect, the present invention provides an anti-influenza virus drug, wherein the influenza virus is influenza a virus.

In another aspect, the invention provides the use of sisal sapogenin in the manufacture of a medicament for inhibiting infection and replication of influenza virus pneumonia.

The invention also provides the use of sisal sapogenin for inhibiting the M gene expression of influenza virus H1N1 so as to achieve antiviral effect.

The invention also provides the inhibition effect of the sisal sapogenin on the NP protein of the influenza virus.

The sisal sapogenin also inhibits the expression of influenza virus mouse pneumonia in vivo.

In summary, compared with the prior art, the invention has the following advantages and effects:

1. Sisal sapogenin can inhibit infection of influenza A virus H1N1 to cells, and has spectral antiviral activity

2. Sisal sapogenin has small cytotoxicity, has obvious advantage on the inhibition rate of H1N1 infected cells under the condition of almost nontoxic micro dose (12.5 ng/ml concentration), and can be used for developing safe, effective and high-cost-performance antiviral drugs.

3. Sisal sapogenin can act on NP protein targets of influenza viruses, and has obvious inhibition effect on expression of the sisal sapogenin.

4. Sisal sapogenin can inhibit lung tissue inflammation pathology caused by influenza virus, and reduce lung index.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides an application of sisal sapogenin in preparing anti-influenza virus drugs, wherein the structural molecular formula of the sisal sapogenin is as follows:

Sisal hemp sapogenin molecular structure formula

The invention discloses the following technical effects:

the invention discloses an application of sisal sapogenin in preparing medicaments for treating influenza and viral pneumonia thereof, wherein the influenza virus is influenza A, and the viral pneumonia is influenza A viral pneumonia. The sisal sapogenin has obvious inhibition effect on influenza virus, reduces the expression of influenza virus NP protein, and can inhibit lung tissue inflammation pathology of mice infected with influenza virus.

Drawings

In the drawings, the same reference numerals refer to the same or similar parts or elements throughout the several views unless otherwise specified. The figures are not necessarily drawn to scale. It is appreciated that these drawings depict only some embodiments according to the disclosure and are not therefore to be considered limiting of its scope.

FIG. 1Tigogenin A549 cytotoxicity assay

FIG. 2Tigogenin anti-influenza Activity assay

FIG. 3PCR assay Tigogenin for inhibition of H1N 1M Gene

FIG. 4WB assay Tigogenin inhibition of H1N1 NP protein

FIG. 5 immunofluorescence assay Tigogenin inhibition of H1N1 NP protein

FIG. 6Tigogenin graph of body weight change in H1N1 influenza mice

FIG. 7Tigogenin lung index graph of H1N1 influenza mice

FIG. 8Tigogenin pathological section of H1N1 influenza mice

Detailed Description

For a better understanding of the present invention, the use of sisal sapogenin in anti-influenza A virus is further described below in conjunction with the experiments and experimental results.

The material cells and viruses used in the invention:

Cell strain and virus strain are provided by the Ghancinolone acetonide life technology Co., ltd, and influenza A/FM 1/47/(H1N 1) is provided by Guangzhou Chinese medicine university technical innovation center. The reagent comprises F-12K culture medium (GIBCO), penicillin-streptomycin double antibody (GIBCO), fetal bovine serum (GIBCO), PBS solution (GIBCO), 0.25% EDTA pancreatin (GIBCO), dimethyl sulfoxide (sigma), TPCK treated pancreatin (sigma), thiazole blue (sigma) and the like. All virus-related experiments were performed in biosafety class 2 laboratories.

Example 1

The cytotoxicity of sisal sapogenin was tested in this example.

The detection of the cytotoxicity of the sisal sapogenin adopts an MTT method, and the specific method is as follows:

A549 cells are respectively inoculated in 96-well plates, 100ul of each well has the cell concentration of 2 multiplied by 10 ⁵ per ml, the cells are cultured for 24 hours in a 37 ℃ and 5 percent CO2 incubator, the next experiment can be carried out on the cell with the cell length of 80 to 90 percent, the active ingredient of the sisalagenin is dissolved by using a DMSO cosolvent, the F-12K cell culture solution is added for diluting the medicine to the concentration of 1mg per ml, and the medicine is diluted by using a 0.22um filter for filtering and sterilizing and then is used as mother liquor, and the medicine is diluted by 5 times to obtain medicine liquid with 8 gradient concentrations. After the cells A549 grow to 80-90%, respectively adding the prepared liquid medicine, 100ul of each hole, 5 compound holes with each concentration, simultaneously setting a 6-hole blank control group and a 10-hole normal control group, and detecting absorbance at 490nm by adopting a multifunctional enzyme-labeled instrument after culturing in an incubator for 48 hours. Cell viability was used as an indicator of toxicity of agamogenin to A549 cells

Cell viability was (%) = drug group absorbance-blank absorbance/normal group absorbance-blank absorbance =100%

As shown in FIG. 1, the sisal sapogenin has almost no toxicity to A549 cells in the concentration range of 200ng/ml, and the concentration of the selected medicine in the experimental study of the invention is within 12.5ng/ml, which is the development experiment in the safe and nontoxic concentration range

Example 2

The present examples examined the activity of sisal sapogenin against influenza A virus in vitro.

The specific method comprises the following steps:

A549 cells were inoculated at a concentration of 2×10 ⁵/ml into 96 wells, 100ul per well, incubated in a 37 ℃ 5% co ₂ incubator for 24h, 80-90% of the cell length allowed for the next experiment, infected with 100TCID ₅₀ in FM1 strain virus dilutions (containing 1ug/ml TPCK) of influenza a virus, after 2h virus adsorption, virus solution was discarded, washed with PBS, gradient diluted sisal sapogenin dilutions were added per well, and incubation was continued for 48h. The antiviral activity of sisal sapogenin is determined by the protective effect of sisal sapogenin on cells, including observing cytopathic effect (CPE) caused by cytoviruses caused by sisal sapogenin inhibitory viruses and detecting the survival rate of cells, and further calculating half-effective concentration IC ₅₀, oseltamivir as a positive control.

As shown in figure 2, the sisal sapogenin has obvious inhibition effect on influenza virus H1N1 at the concentration of 6.25ng/ml, and the inhibition rate is more than 90%.

Example 3

The present example evaluates the inhibition of replication of influenza a virus by sisalagenin.

In order to evaluate the inhibition effect of sisal sapogenin on replication of influenza virus, the invention adopts three experiments of q-PCR, westernblotting and immunofluorescence to detect the influence of sisal sapogenin on replication of M gene and NP protein of influenza virus from the expression level of the genes and the proteins, and the specific method is as follows:

Inoculating A549 cells into a 6-well plate, wherein each well has 2ml of cell concentration of 3X 10 ⁵ cells/ml, and the cells are divided into a normal group, a virus group, an oseltamivir group, a Tigogenin low-dose group, a Tigogenin medium-dose group and a Tigogenin high-dose group, wherein each group has 3 compound wells, culturing the cells in a 37 ℃ and 5% CO ₂ incubator for 24 hours, adding 1ml of virus diluent into each other outside the normal group after plate PBS washing, and extracting the expression level of the RNA detection related genes after the plates are taken, wherein the concentration is 100TCID ₅₀, the virus is absorbed for 2 hours, and the Tigogenin is cultured in a high-medium-low molding concentration of 0.0125 mug/ml, 0.00625 mug/ml and 0.003125 mug/ml in each 1ml incubator for 48 hours.

The primers were designed as follows:

H1N 1M gene:

Forward primer CTAAGGCTATGGAGCAAAT

Reverse primer CACTGGAGCTAGGATGAGT

IL-6 gene:

Forward primer GAACTCCTTCTCCACAAGCG

Reverse primer CCGTCGAGGATGTACCGAAT

as shown in fig. 3, compared with oseltamivir, tigogenin has obvious therapeutic effect on different genes, although the therapeutic trend of low, medium and high concentrations has fluctuation, and Tigogenin has obvious therapeutic effect on related genes of virus infection in the whole view of experimental error reasons.

And (3) detecting the expression level of related signal pathway proteins in the cells by using Western-blot, namely collecting total proteins of cell-extracted cells/lung tissues and measuring the protein concentration. Samples were loaded, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred using a semi-dry electrotransfer membrane apparatus. After blocking, the relevant protein of interest [ viral-related protein (NP); 4℃overnight was added. Washing the membrane for 3 times, adding horseradish peroxidase-labeled secondary antibody diluted by the rinse solution, and oscillating. PVDF membranes were incubated with shaking at room temperature. ECL color development, X-ray film exposure, development, fixation, scanning and then observation of the results. And carrying out absorbance analysis on target bands of the scanned Image by using Image-J software, wherein the absorbance ratio of each target band to B-action is the relative expression quantity of the target protein.

As a result, FIG. 4 shows that Tigogenin administration groups showed various degrees of protein expression reduction in detecting protein expression of NP protein, indicating that Tigogenin has an effect of treating influenza virus.

Immunofluorescence detection moulding method As described above, after culturing cells in incubator for 48h, fixing and permeabilizing, soaking cover glass with climbed cells in culture plate 3 times with 1 XPBS, fixing climbed piece with 4% paraformaldehyde for 15min, soaking slide with 1 XPBS 3 times for 3min each time. Cells were permeabilized at room temperature for 15min at 0.5% Triton X-100 (1 XPBS) and slides were rinsed 3 times for 3min each. Blocking, the 1 XPBS was blotted with a blotter paper, 5% normal serum (identical or similar to the secondary antibody species source) was added drop wise to the slide, and blocked for 1h at room temperature. Antibody incubation, in which the blocking solution is sucked off by the absorbent paper and not washed, a sufficient amount of diluted primary antibody is added dropwise to each slide and placed in a wet box, and incubated at 4 ℃ overnight. Adding a fluorescent secondary antibody, namely soaking and washing the climbing sheet for 3 times for 3min each time, and dripping the diluted fluorescent secondary antibody after the water absorption paper absorbs excessive liquid on the climbing sheet, incubating for 1h at 37 ℃ in a wet box, and soaking and washing the climbing sheet for 3 times for 3min each time by using the PBST. Fixing photographing, namely counterstaining, namely dripping DAPI, incubating for 5min in dark, and staining the sample, and washing off redundant DAPI by PBST for 5min multiplied by 4 times. The liquid on the climbing sheet is sucked by water absorbing paper, and the sealing sheet liquid sealing sheet containing anti-fluorescence quenching agent is used for observing and collecting images under a fluorescence microscope.

As shown in FIG. 5, tigogenin has a good effect of inhibiting the expression of the virus in the detection of the expression of the Influenza gene compared with the virus group, and the result is statistically significant.

Example 4

The present example investigated the anti-influenza effect of sisal sapogenin Tigogenin in vivo.

60 SPF-class BALB/C mice of 4 weeks of age were prepared, weighing 12-15g, and females were randomly divided into a normal group, a viral group, an oseltamivir group, an E7820 group, a Tigogenin low dose group, and a Tigogenin high dose group, each group of 10. Adaptively feeding for one day, anesthetizing and nasal-infecting mice with FM1 virus diluent with concentration of 15LD ₅₀ in the next day, carrying out intraperitoneal injection administration of Tigogenin with concentration of 10mg/Kg at low dose and 20mg/Kg at high dose twice a day for 4 days, weighing the weight and the wet lung weight of the mice after the mice are ill, and calculating lung index and lung tissue pathological section experiment.

The results are shown in fig. 8, where the mice infected with influenza virus had significantly increased interstitial congestion, edema, inflammatory infiltrates, and various degrees of relief from the oseltamivir control group, tigogenin, and asterisks indicate significant levels, P <0.05, P <0.01, P <0.001, as compared to the normal group.

The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily recognize that various changes and substitutions are possible within the scope of the present application. Therefore, the protection scope of the application is subject to the protection scope of the claims.

Claims

1. Application of sisal saponin (Tigogenin) in the preparation of drugs for treating influenza and viral pneumonia.

2. The use according to claim 1, characterized in that the influenza is influenza A, and the viral pneumonia is pneumonia induced by influenza A virus.

3. Use of sisal sapogenin according to claim 1 in the preparation of a drug for inhibiting the replication of influenza A virus in host cells, wherein the drug can inhibit the expression of the M gene and NP protein of N1N1.

4. The use of sisal sapogenin according to claim 1 in the preparation of a drug for treating influenza and viral pneumonia thereof, characterized in that the drug can inhibit the inflammatory pathology of lung tissue caused by influenza virus and reduce the lung index.