Application of peucedanin in preparation of anti-influenza virus drugs

Abstract

The invention discloses an application of peucedanin in preparation of a medicine for resisting influenza virus and viral pneumonia thereof. The influenza virus is an influenza A virus H1N1. The viral pneumonia is pneumonia induced by influenza A virus. The Nodaken in can be used for reducing the expression of influenza virus mRNA (messenger ribonucleic acid) and influenza virus NP (natriuretic peptide) protein in vitro and inhibiting the pyroptosis process caused by the influenza virus. The lung tissue inflammation pathology caused by influenza virus can be inhibited in vivo, and the lung index is reduced. Due to the fact that the peucedanum decursum has an obvious inhibiting effect on replication of influenza viruses, the peucedanum decursum provided by the invention provides a novel treatment medicine for the anti-influenza virus medicine.

Description

Technical Field

[0001] The invention relates to application of purpurogenol in anti-influenza A virus drugs, belonging to the technical field of medicines. Background Art Influenza (hereinafter referred to as flu) is an acute respiratory infectious disease caused by influenza virus. It is highly contagious, has a high morbidity, is widespread and has a high mortality rate. It is listed as one of the global infectious diseases that has not yet been effectively controlled. Influenza virus belongs to the family of Orthomyxoviridae, a single-stranded negative-strand RNA virus. It is divided into three types: A, B, and C. Among them, type A virus often undergoes antigenic mutation, is highly contagious, spreads rapidly, and is very likely to become a large-scale epidemic. It is the main type of virus that causes influenza. Influenza often causes viral pneumonia, which is caused by the spread of viral infection downward. The prevention and treatment measures for influenza mainly include vaccines and antiviral drugs. However, due to the antigenic drift and antigenic variation of influenza viruses, their high mutation rate and variable transmission patterns lead to the possibility that vaccines with long production cycles may not be immune-matched with the current major circulating virus strains, so the protective effect of influenza vaccines is still limited. There are four main types of antiviral drugs, including M2 ion channel inhibitors, neuraminidase inhibitors, hemagglutinin inhibitors and influenza virus RNA polymerase inhibitors. The first type is M2 ion channel inhibitors, which inhibit viral replication by blocking the influenza virus M2 ion channel. Representative drugs are adamantane and rimantadine. However, studies have shown that long-term use of such drugs may cause serious adverse reactions in the central nervous system and digestive system. In addition, this type of drug has already shown widespread drug resistance. The second type is neuraminidase inhibitors, which inhibit the enzyme activity required for the budding of new virus particles in host cells to prevent viral transmission. Representative drugs include oseltamivir and others. Neuraminidase inhibitors are currently the most commonly used and used drugs for the treatment of human influenza. This type of drug is effective against most influenza A and B strains and has low toxicity. However, influenza viruses can develop resistance to neuraminidase inhibitors by acquiring mutations at the NA catalytic site or the HA receptor binding site, and the resistance rate is gradually increasing globally, resulting in seasonal outbreaks of neuraminidase-resistant virus strains. The third category is hemagglutinin inhibitors, which prevent virus adsorption and invasion of cells by inhibiting the fusion of influenza virus lipid membrane and host cells. The representative drug is Abidor. The side effects of Abidor mainly include diarrhea, abdominal distension, nausea, vomiting and other digestive tract symptoms and abnormal liver function indicators. The fourth category is influenza virus RNA polymerase inhibitors, and the representative drugs are ribavirin, baloxavir, favipiravir, etc. This type of drug still has side effects on the nervous system and digestive system. Therefore, researching new anti-influenza virus drugs and finding new effective drug targets to control influenza A have become urgent problems for mankind. my country is a country with extremely rich natural resources, and has unique advantages in developing antiviral drugs from natural products. Purpuroside belongs to coumarin glycosides and is considered to be the effective active ingredient of many traditional Chinese medicines. It is mainly found in the rhizomes of traditional Chinese medicines such as Purpuroside, Notopterygium wilfordii, and Angelica sinensis. Purpuroside has a wide range of medicinal properties, including anti-inflammatory, anti-tumor, anti-immune, anti-fibrosis, anti-platelet aggregation, regulation of intestinal flora, liver protection, and enhancement of cognitive function. It has certain therapeutic effects on tumors such as liver cancer, cervical cancer, and breast cancer, inflammatory diseases such as rheumatoid arthritis, knee osteoarthritis, asthma, ulcerative colitis, and osteoporosis, obstructive nephropathy, alcoholic cirrhosis, neuropathic pain and other diseases. At present, patent applications for purpurogenol include the use of purpurogenol in the preparation of products for preventing or treating osteoporosis (patent number: CN116098915A) and the use of purpurogenol in the preparation of drugs for reducing the myotoxicity of platinum drugs (patent number: CN118526513A). However, no research has proposed the use of purpurogenol in anti-influenza. The present invention discovers for the first time that purpurogenol has the use of anti-influenza A virus. Experimental results show that purpurogenol can affect the replication of the virus by inhibiting the expression of influenza virus mRNA and NP protein, revealing that it has the prospect of being developed into an anti-influenza virus drug. Summary of the invention The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new use of purpurogenol. The purpurogenol has significant anti-influenza A virus activity and can be developed as a new anti-influenza virus drug with a wide range of application prospects. In order to achieve the above object, the present invention provides, on one hand, the use of purpurogenol in the preparation of anti-influenza virus drugs. Wherein, the influenza virus is influenza A, and the viral pneumonia is pneumonia induced by influenza A virus. Another aspect of the present invention provides the use of purpurogenol in the preparation of a drug for inhibiting the replication of influenza A virus in a host cell. The purpurogenol glycoside can inhibit the replication of influenza A virus H1N1 in host cells. Among them, purslane glycosides inhibit the replication of influenza virus in host cells by inhibiting the transcription of influenza virus M gene and the translation of influenza virus NP protein, thereby playing an antiviral role. Another aspect of the present invention provides the use of purpurogenol in the preparation of influenza virus drugs for inhibiting cell pyroptosis and tissue inflammatory response. Among them, purpurogenol inhibits cell death by reducing the expression of cell pyroptosis-related factors GSDMD and Caspase-1; it plays an anti-inflammatory role in the body by alleviating the inflammatory infiltration of lung tissue after influenza virus infection. The purpurogenol can be prepared into a variety of pharmaceutically acceptable dosage forms, such as tablets, granules, powders, capsules, oral liquids, injections, etc., for the treatment of influenza. The drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field. In summary, the present invention has the following advantages and effects compared with the prior art: 1. Purpurogenol can inhibit the infection of influenza A virus to the host in vitro and in vivo, and has excellent antiviral activity. 2. Purpurogenol has low toxicity to cells. When administered within the concentration range of 200 μg / ml, it has low toxicity to A549 cells. The median lethal concentration (CC) of Purpurogenol to influenza virus H1N1 is 50 The half effective concentration EC is 0.2551 mg / mL. 50 It is 5.521μg / mL, and the therapeutic index TI is 46.21. It can be developed as a safe and effective anti-influenza virus drug. 3. In addition to its direct killing effect on viruses, purpurogenol can also inhibit cell death and tissue inflammatory infiltration caused by viral infection, and alleviate the adverse effects of viral infection on the body in many aspects. It is a potentially effective antiviral drug and viral pneumonia treatment drug. BRIEF DESCRIPTION OF THE DRAWINGS In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application. Figure 1 Cytotoxicity test of purpurogenol. Survival rate of A549 cells under different concentrations of purpurogenol; Figure 2 The activity of purpurogenol against influenza A virus. A. The inhibitory activity of purpurogenol against H1N1; B. The inhibitory effect of purpurogenol on CPE caused by influenza A virus Figure 3 Purpurogenol inhibits influenza virus replication in cells. A. Purpurogenol reduces the expression of viral M mRNA in A549 cells; B. Purpurogenol inhibits the expression of viral NP protein in A549 cells after virus infection (Western Blot method); C. Purpurogenol inhibits the expression of viral NP protein in A549 cells after virus infection (immunofluorescence method) Figure 4 Purpurogenol inhibits the cell pyroptosis process caused by influenza virus. A. Purpurogenol reduces the expression of pyroptosis-related factor Caspase-1 in A549 cells after virus infection. B. Purpurogenol reduces the expression of pyroptosis-related factor GSDMD in A549 cells after virus infection. Figure 5 Curve of the effect of purpurogenol on body weight changes in mice infected with influenza A virus Figure 6 Effect of purpurogenol on lung index of mice infected with influenza A virus Figure 7 Effect of purslane on pathological sections of mice infected with influenza A virus DETAILED DESCRIPTION In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application is further described through the following examples. In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature. Materials used in the present invention 1 Cells and viruses Lung cancer epithelial cells (A549) were grown in F-12K medium containing 10% fetal bovine serum, 100U / L penicillin and streptomycin. Influenza A virus A / FM / 1 / 47 (H1N1) was amplified by passage in SPF chicken embryos in our laboratory and stored at -80°C. All experiments related to live viruses were conducted in a biosafety level 2 facility. Embodiment 1 In this example, the cytotoxicity of purpurogenol was detected. The MTT method was used to detect the cytotoxicity of purpurogenol. The specific method is as follows: A549 cells were collected at 2×10 5 / well inoculated in a 96-well plate, cultured to a monolayer in a constant temperature cell culture incubator at 37°C and 5% CO2, dissolved the purpurogenol powder with 100μl DMSO solution, and then added 9.9mL F-12K culture medium to make a 2mg / mL mother solution. When used, the purpurogenol was diluted 5 times to obtain 4 gradient concentrations of 0.2mg / ml, 0.04mg / ml, 0.008mg / ml, and 0.0016mg / ml. Add different concentrations of purpurogenol solution to a 96-well plate, 100μl per well, and continue to culture for 24h. Observe the cytopathic effect (CPE) under a light microscope, discard the culture supernatant, add 100μL of F-12K culture medium containing 0.5mg / ml MTT to each well, and incubate at 37°C for 4h. The absorbance (OD) at 490 nm was measured using a multifunctional microplate reader (Genios Pro, Tecan, US), and the cell destruction rate (%) was calculated. The results were used to calculate the half-maximal inhibitory concentration (CC) of purslane glycosides on A549 using the nonlinear regression method of GraphPad. 50 ). Cell survival rate % = 1-[(OD value of normal control group - OD value of drug group) / OD value of normal control group] × 100%. The cell survival rate was used as an indicator of the toxicity of purpurogenol on A549 cells. The results are as follows Figure 1 As shown in the figure, purpurogenol has less toxicity to cells. 50 =255.1μg / mL, the safe range of cell medication is within 200μg / mL. The test results showed that the cytotoxicity of purpurogenol was low and the biological safety was high. The concentration range of 200 μg / ml was within the safe range for A549 cells. Embodiment 2 This example tests the activity of purpurogenol against influenza A virus in vitro. First, the cytotoxicity of the drugs to A549 cells (CC 50 ) and virus titer (TCID 50 ). CC calculated based on the results of cytotoxicity experiments 50 200 μg / mL was taken as the maximum non-toxic dose of the efficacy experiment, which was set as the high-dose group, and 100 μg / mL was taken as the low-dose group. A549 cells were inoculated in a 96-well plate, 100 μl per well, and cultured at 37°C and 5% CO2. When they grew to a tight monolayer, the supernatant was discarded. First, 100 TCID 50The virus solution was added to a 96-well plate, 100 μl per well, adsorbed at 35°C for 2 hours, and 100 μl of the drug solution was added after the supernatant was discarded, with 4 replicates for each concentration. The normal group and the virus group discarded the supernatant and added an equal amount of F12-K maintenance medium for 48 hours. The cell morphology changes were observed under an inverted microscope every day, and the cytopathic effect (CPE) was recorded. Ribavirin was used as a positive control. The experiment was repeated 3 times, and the cell destruction rate was calculated using the thiazolyl blue colorimetric method (MTT method) to test the inhibitory effect of the drug on viral replication. The data were regressed using the statistical software GraphPad Prsim9 to obtain the EC value of purslane glycosides. 50 . Therapeutic index TI = CC 50 / EC 50 , thus concluding the effect of purpurogenol on influenza A virus. Experimental results: Purpurogenol can inhibit cell death caused by influenza virus infection The experimental results are as follows Figure 2 shown. Figure 2 A shows that purpurogenol has a significant inhibitory effect on influenza A virus H1N1, and there is a significant dose-effect relationship. The inhibition rate of purpurogenol on the virus increases with the increase of concentration, and the half effective concentration EC 50 =5.521μg / mL, selectivity safety index TI =46.21, can be developed as a safe and effective anti-influenza virus drug. At the same time, the inhibitory effect of purpurogenol on virus-induced cytoviral phenomenon (CPE) was observed. The results are as follows Figure 2 As shown in B, 100TCID 50 The virus solution can cause obvious cytopathic effect 24 hours after infecting cells, while 200μg / ml of purslane can inhibit the pathological effect caused by the virus, and the results are consistent with the positive compound ribavirin. Embodiment 3 This example evaluates the inhibitory effect of purslane on influenza A virus replication. In order to evaluate the inhibitory effect of purpurogenol on influenza virus replication, the present invention uses three methods, Q-PCR, Western blot and immunofluorescence, to detect the effect of purpurogenol on virus replication at the gene and protein expression levels, respectively. The specific methods are as follows: The PCR method is as follows: A549 cells were inoculated in 6-well plates, 2 ml per well, and the cell concentration was 2×10 5The cells were divided into 5 groups: normal group, virus group, ribavirin group, low-dose purpurogenol group, and high-dose purpurogenol group. Each group had 3 replicates. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h. When the cell growth was 80-90%, the plates were washed with PBS and 1 ml of virus dilution was added to the groups other than the normal group. The concentration was 100 TCID 50 ,After 2 hours of virus adsorption, each group of drug solutions were added, with 200μg / ml of high dose and 100μg / ml of low dose of purpurogenol, 1ml per well. After 48 hours of incubation in the incubator, RNA was extracted to detect the expression level of related genes. The immunofluorescence experimental method is as follows: the modeling method is as described in Example 2. After 48 hours of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and washed 3 times with PBS, each time for 5 minutes. Then, they were permeabilized with Triton100 for 20 minutes and washed 3 times with PBS. Blocked with protein-free rapid blocking solution for 1 hour, added with virus NP protein primary antibody (anti-influenza A virus nucleoprotein (NP) antibody), and incubated at 4°C overnight; then added fluorescent secondary antibody, incubated at room temperature for 1 hour, and finally stained with DAPI at room temperature for 10 minutes, and the protein expression of the virus was observed under a fluorescence microscope. The Western Blot experimental method is as follows: the modeling method is as described in Example 2. After 48 hours of culture, the cell protein is extracted, and after electrophoresis, transfer, blocking and other operations, the virus NP protein primary antibody (anti-influenza A virus nucleoprotein (NP) antibody) is added and kept at 4°C overnight; then the secondary antibody is added and the protein bands are photographed under an imaging device. Experimental results: Purpurogenol can inhibit the replication of influenza virus and the expression of influenza virus protein. The results are as follows Figure 3 shown. Figure 3 A is the expression of viral M gene mRNA in A549 cells. Compared with the virus group, the expression of viral M gene mRNA in the high-dose and low-dose purpurogenol groups was reduced, and the inhibitory effect was concentration-dependent. This proves that purpurogenol can reduce the expression of influenza virus mRNA. Figure 3 B and 3C are the results of Western blotting and immunofluorescence. Compared with the virus control group, the fluorescence intensity of influenza virus NP protein expression in the purpurogenous glycoside group was reduced, proving that purpurogenous glycoside can reduce the expression of influenza virus NP protein. This result is consistent with the Q-PCR result. Embodiment 4 In this example, the inhibitory effect of purpurogenol on cell pyroptosis was evaluated. The experimental operation is as described in the PCR method in Example 3. Experimental results: Purpurogenol can induce cell pyroptosis The results are as follows Figure 4 A and 4B. Figure 4 A and 4B are the expressions of Caspase-1 and GSDMD mRNA in A549 cells, respectively. It is proved that purpurogenol can reduce the expression of Caspase-1 and GSDMD mRNA in cells infected with influenza virus and inhibit cell pyroptosis caused by virus infection. Embodiment 5 This example evaluates the anti-influenza virus efficacy of purpurogenol in vivo. Sixty 4-week-old bal B / C female mice weighing 12-15 g were randomly divided into a normal group, a virus group, a ribavirin group, a low-dose purpurogenol group, and a high-dose purpurogenol group, with 10 mice in each group. Adaptive feeding was performed for one day, and 15 LD 50 The mice were anesthetized with FM1 virus dilution of a certain concentration and infected by intranasal drops. The low-dose group of purpurogenol was given 10 mg / Kg, and the high-dose group was orally given 20 mg / Kg twice a day for 4 days. After the mice became ill, their body weight and lung wet weight were weighed, and the lung index and lung tissue pathology section experiments were calculated. Experimental results: Purpurogenol can treat viral pneumonia caused by influenza virus like Figure 5 As shown in the figure, compared with the virus group, the weight loss of mice in the high-dose group of purpurogenol was slower, indicating that purpurogenol can slow down the weight loss trend of mice. Compared with the virus group, the lung index of mice in the high-dose group of purpurogenol was lower, indicating that purpurogenol can reduce the lung index of mice. Figure 6 As shown in the figure, compared with the mice in the virus group, the lung index of the mice in the high-dose and low-dose groups of purslane glycosides was reduced. Figure 7 As shown, compared with the mice in the virus group, the thickening of the alveolar septum and bronchiolar wall in the high-dose and low-dose groups of purpurogenol glycosides was reduced, the infiltration of inflammatory cells was reduced, and the dilation of alveolar wall capillaries was reduced. In summary, Purpurogenol can be developed as a potential anti-influenza virus drug. In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory. In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined. The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. Application of purslane glycosides in the preparation of drugs against influenza virus 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. Application of purpurogenol in the preparation of drugs for inhibiting the replication of influenza A virus in host cells.

4. Application of purslane glycosides in the preparation of influenza virus drugs for inhibiting cell pyroptosis and tissue inflammatory response.

5. The use of purpurogenol in anti-influenza virus and viral pneumonia drugs according to claim 1, characterized in that: The drug can inhibit the inflammatory pathology of lung tissue caused by influenza virus and reduce the lung index.

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