CN116672329A - Application of aurantiol in preparation of anti-influenza virus drugs - Google Patents

Application of aurantiol in preparation of anti-influenza virus drugs Download PDF

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CN116672329A
CN116672329A CN202210167830.8A CN202210167830A CN116672329A CN 116672329 A CN116672329 A CN 116672329A CN 202210167830 A CN202210167830 A CN 202210167830A CN 116672329 A CN116672329 A CN 116672329A
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influenza
virus
influenza virus
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郭颖
陈姝冰
唐克
莘一婧
王淳正
吴悠
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Institute of Materia Medica of CAMS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Abstract

The invention belongs to the technical field of medicines, and discloses application of aurantium cassia essence in preparation of anti-influenza virus medicines. In particular to the application of aurantiol or pharmaceutically acceptable salt thereof shown in a structural formula (I) in preparing medicines for preventing or treating influenza virus infection. And the application of the pharmaceutical composition containing the aurantiol shown in the structural formula (I) or the pharmaceutically acceptable salt thereof in preparing medicines for preventing or treating influenza virus infection, and further, the pharmaceutical composition also contains other antiviral medicines.

Description

Application of aurantiol in preparation of anti-influenza virus drugs
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of aurantium cassia element (CAS: 67979-25-3) in preparation of a medicine for preventing or treating influenza virus infection. The invention comprises the use of aurantio-obtusin (CAS: 67979-25-3) alone or in combination in the prevention or treatment of influenza virus infection.
Background
Influenza virus (Influenza virus) is an important threat to global public health safety, and about 5-10% of adults and 20-30% of children are infected with Influenza virus every year according to World Health Organization (WHO) statistics, wherein 300-500 thousands of severe patients and about 25-50 thousands of deaths are observed [ Ziegler T, mamahit A, cox NJ:65 years of Influenza surveillance by a world health organization-coordinat ed global network.Influza and other respiratory viruses (2018) 12 (5): 558-565 ]. Influenza viruses belong to the Orthomyxoviridae (Orthomyxoviridae) and are classified into four types a, b, c and t, wherein influenza a, b and c viruses can infect humans and cause respiratory diseases. Influenza a virus has the greatest epidemic range and the greatest hazard. Wherein 5000 tens of thousands of people die in the spanish influenza epidemic situation in 1918; in 2009 swine influenza epidemic, about 57 thousands of deaths were also caused worldwide [ First global estimates of 2009 h1n1 pandemic mortality released by cdc-led collaboration (2012) [ https:// www.cdc.gov/flu/spotlights/handtic-global-estimates, htm ].
Influenza viruses are spherical, about 80-120nm in diameter, enveloped viruses, and their genomes are single stranded segmented RNAs. Influenza A virus has a total of 8 RNAs encoding at least 10 viral proteins [ David M Knope P, peter M Howley, MD: fields virology,6th edition.2.Lippincott Williams&Wilkins (LWW), (2013) ]. Influenza virus envelopes are derived from the host cell membrane, on which three viral proteins are embedded: hemagglutinin protein (HA), neuraminidase (NA), and M2 ion channels. Influenza a viruses are serologically classified according to HA and NA, and 18 HA and 10 NA are known, so that there are 180 subtypes theoretically. Epidemiological data showed that H1N1 subtype (spanish influenza 1918 and swine influenza 2009), H2N2 subtype (asian influenza 1950), H3N2 subtype (hong gang influenza 1960) had caused massive electrocution.
In addition to influenza pandemics, seasonal influenza occurs annually, mainly in winter, most often caused by influenza a or b viruses, with H1N1 and H3N2 being more common. Symptoms after seasonal influenza include sudden fever, cough (usually dry), headache, muscle and joint pain, sore throat, and runny nose. Cough can be severe and can last for 2 weeks or more. Most people recover from fever and other symptoms within a week, and do not need to be treated. However, influenza can lead to severe illness or death in high-risk populations. [ How can I avoid getting the fluWorld Health Organization website https:// www.who.int/news-rotor/q-a-details/how-can-i-avoid-getting-the-flu ]
The existing Anti-influenza virus drugs include three classes of 7 [ Amarelle L, leculona E, sznajder JI: anti-influenza treatment: drugs currently used and under development. Archvos de bronconeumologia (2017) 53 (1): 19-26.]: the M2 ion channel inhibitors amantadine and rimantadine; the neuraminidase inhibitors oseltamivir, zanamivir, peramivir and lanamivir; CAP dependent endonuclease inhibitor Ballo Sha Weima Boc ester (baloxavir marboxil). These 7 anti-influenza drugs share a commonality: all are medicines taking influenza virus proteins as targets, so that when the targets are mutated, the medicine affinity is reduced, and the influenza viruses escape to become drug-resistant viruses. For example, the influenza virus M2 ion channel inhibitors amantadine and rimantadine have been subject to stable drug resistance mutations over long term use, and WHO has not recommended amantadine and rimantadine for the treatment of influenza a virus [ Summary of influenza antiviral susceptibility surveillance findings, september 2010-march 2011 (2011): https:// www.who.int/influhenza/gisrs_major/updates/anti-viral_persistence/en/].
Although there are 7 anti-influenza drugs that have been or are in use, there are still 5-15 hundred million influenza virus infections and diseases per year worldwide. Statistics of the American disease control center, 2010-2018, the number of patients infected with influenza A virus is 930-4900 thousands, death is 1.2-7.9 thousands [ Influenza (flu) (2020): https:// www.cdc.gov/flu/about/burden/index. Html ]; that is, after supply of anti-influenza drugs and injection of influenza vaccines, 5% -20% of the population still suffers from infection, mainly due to the nature and variation of influenza virus itself and recombination (Reassortment) of its RNA genome. For example, neuraminidase inhibitors are the most commonly used anti-influenza A virus drugs in clinic, wherein oseltamivir (darfein) is most widely used, and clinical data show that patients can obtain better curative effects only when the patients take the drug for 48 hours after infection of viruses [ Summary of influenza antiviral susceptibility surveillance findings, september 2010-March 2011 (2011): https:// www.who.int/influzza/gisrs_laboratory/update/anti-persistence/en/].
The artificial intelligence is a rapidly developed front-edge technology in recent years, can carry out high-efficiency analysis and effective extraction on high-dimension and large-magnitude data, provides possibility [ Y.Peng, M.Yuan, J.Xin, et al, screening novel drug candidates for Alzheimer's disease by an integrated network and transcriptome analysis.Bio information 36 (2020) 4626-4632 ] for extracting and condensing characteristic information of disease big data and compound big data, and provides a path for researching and developing medicaments for intervention of influenza virus infection in multiple links. Compound cell libraries are transcriptomic big data of compound-perturbed cells, comprising whole gene expression profiles of compounds after different concentrations, different times of treatment of cells. The invention applies a transcriptome characteristic reverse matching (Transcriptome signature reversion, TSR) strategy [ Koudijs KKM, et al, transcriptome Signature Reversion as a Method to Reposition Drugs Against Cancer for Precision oncology J.2019,25 (2): 116-120], and the transcriptome characteristic gene set of the influenza virus infection cytopathic transcriptome characteristic gene set is reversely matched with the transcriptome characteristic gene set of the 20,000 compound disturbance cells, so that an active compound of the multi-link reverse intervention influenza virus infection characteristic gene is discovered.
Orange-obtusin, also known as 1,3, 7-trihydroxy-2, 8-dimethoxy-6-methylanthracene-9, 10-dione (Aurantia-obtusin). Studies have shown that aurantiin has a hypotensive effect and that the mechanism of action is associated with inhibition of expression of iNOS. [ Li S, li Q, lv X, et al Auransition-obtusin relaxes systemic arteries through endothelial PI K/AKT/ENOS-dependent signaling pathway in rates.J Pharmacol Sci,2015,128 (3): 108-115]. In addition, it has been reported that aurantiol also has hypolipidemic action [ Li Mingyuan, etc.. The hypolipidemic action of aurantiol is studied, pharmacology and clinic of traditional Chinese medicine, 2008,24 (06): 36-37 ]. No report on the anti-influenza virus activity of aurantium is found by the literature search.
The invention firstly extracts the characteristic gene set of the cytopathic effect caused by the infection of the influenza virus, and reversely matches the characteristic gene set of the cytopathic effect caused by the infection of the influenza virus with the compound disturbance cell characteristic gene set, and discovers that the compound aurantiol can reversely regulate the expression of the characteristic gene of the cytopathic effect caused by the infection of the influenza virus. Then, the antiviral activity of the compound is evaluated by using an influenza virus infection model, and the aurantiol is found to have broad-spectrum anti-influenza virus activity and stronger inhibitory activity on influenza A and B virus infection. The data show that the anti-influenza virus activity of the aurantium cassia element is equivalent to that of the first-line antiviral drug ribavirin, and the aurantium cassia element has good safety. The new application value of the aurantium cassia element against influenza virus is considered to be higher, and the application prospect is achieved. The invention relates to an invention patent related to new application of a known compound.
Disclosure of Invention
The invention solves the technical problem of providing application of aurantio-obtusin and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating influenza virus infection.
Specifically, in order to solve the technical problems of the invention, the following technical scheme is adopted:
the first aspect of the technical proposal of the invention provides the application of the aurantium obtusin and the pharmaceutically acceptable salts thereof in preparing the medicines for preventing or treating influenza virus,
the pharmaceutically acceptable salt of the aurantio-obtusin comprises pharmaceutically acceptable organic salts or inorganic salts, wherein the organic salts comprise sulfonate, carboxylate, amino acid salt and fatty acid salt, and the inorganic salts comprise hydrochloride, bromate, iodate, sulfate, bisulfate, phosphate, hydrogen phosphate, dihydrogen phosphate and nitrate. Preferably bisulphates, sulphates, hydrochlorides and iodates.
The sulfonate comprises alkyl sulfonate containing 1-15 carbon atoms, benzene sulfonate, p-toluene sulfonate, o-toluene sulfonate and m-toluene sulfonate; the carboxylate comprises tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate and lactate; amino acid salts include glutamate, aspartate; fatty acid salts include long chain fatty acid salts containing 2 to 18 carbon atoms.
Wherein the influenza virus comprises influenza A virus, influenza B virus, influenza C virus and influenza D virus.
The influenza A virus comprises an H1N1 subtype, an H1N2 subtype, an H2N3 subtype, an H3N1 subtype, an H3N2 subtype, an H3N8 subtype, an H5N1 subtype, an H5N2 subtype, an H5N3 subtype, an H5N6 subtype, an H5N8 subtype, an H5N9 subtype, an H6N1 subtype, an H6N2 subtype, an H7N1 subtype, an H7N2 subtype, an H7N3 subtype, an H7N4 subtype, an H7N7 subtype, an H7N9 subtype, an H9N2 subtype, an H10N7 subtype, an H10N8 subtype, an H11N2 subtype, an H11N9 subtype, an H17N10 subtype and an H18N11 subtype. Wherein the influenza A H1N1 virus comprises A/PurtoRico/8/1934, A/WSN/33, A/Hubei Hongshan/52/2005, A/Beijing Fangzheng/262/1995, A/Guangdong Lou/219/2006 and A/FM/1/47 strains; influenza A H3N2 viruses include strains A/Jiangxi Dong lake/312/2006, A/Ji Fang/15/90, A/Yue Fang/243/1972, A/Han Fang/359/1995, A/New York/238/2015, A/Brisbane/10/07, A/Perth/16/09 and A/Udorn/307/72. Influenza B viruses include the B/Jiangxi New/BV/39/2008, B/Ji Fang/13/1997, B/Shenzhen/155/2005, B/Sichuan/63/2001, B/Zhejiang/2/2001, B/Shandong/7/97, B/Durban/39/98, B/Shandong Taian Taishan/1219/2009, B/Sichuan/34/2001B/Yamagata/16/88, B/Victoria/2/87, B/Johannesburg/1/99 and B/Maputo/1/99 strains.
The second aspect of the technical scheme of the invention provides application of a pharmaceutical composition in preparing anti-influenza virus drugs, which is characterized in that the pharmaceutical composition comprises aurantiol and pharmaceutically acceptable salt thereof shown in a structural formula (I) and a pharmaceutically acceptable carrier or excipient; the pharmaceutical composition may also contain other antiviral agents
Wherein the influenza virus comprises influenza A virus, influenza B virus, influenza C virus and influenza D virus.
The influenza A virus comprises an H1N1 subtype, an H1N2 subtype, an H2N3 subtype, an H3N1 subtype, an H3N2 subtype, an H3N8 subtype, an H5N1 subtype, an H5N2 subtype, an H5N3 subtype, an H5N6 subtype, an H5N8 subtype, an H5N9 subtype, an H6N1 subtype, an H6N2 subtype, an H7N1 subtype, an H7N2 subtype, an H7N3 subtype, an H7N4 subtype, an H7N7 subtype, an H7N9 subtype, an H9N2 subtype, an H10N7 subtype, an H10N8 subtype, an H11N2 subtype, an H11N9 subtype, an H17N10 subtype and an H18N11 subtype. Wherein the influenza A H1N1 virus comprises A/PurtoRico/8/1934, A/WSN/33, A/Hubei Hongshan/52/2005, A/Beijing Fangzheng/262/1995, A/Guangdong Lou/219/2006 and A/FM/1/47 strains; influenza A H3N2 viruses include strains A/Jiangxi Dong lake/312/2006, A/Ji Fang/15/90, A/Yue Fang/243/1972, A/Han Fang/359/1995, A/New York/238/2015, A/Brisbane/10/07, A/Perth/16/09 and A/Udorn/307/72. Influenza B viruses include the B/Jiangxi New/BV/39/2008, B/Ji Fang/13/1997, B/Shenzhen/155/2005, B/Sichuan/63/2001, B/Zhejiang/2/2001, B/Shandong/7/97, B/Durban/39/98, B/Shandong Taian Taishan/1219/2009, B/Sichuan/34/2001B/Yamagata/16/88, B/Victoria/2/87, B/Johannesburg/1/99 and B/Maputo/1/99 strains.
The pharmaceutical compositions may be prepared according to methods well known in the art. Any dosage form suitable for human or animal use may be made by combining the compounds of the invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.
The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by the enteral or parenteral route, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, ocular, pulmonary and respiratory, cutaneous, vaginal, rectal, etc.
The dosage form may be a liquid, solid or semi-solid dosage form. The liquid preparation can be solution (including true solution and colloid solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including injection solution, powder injection and transfusion), eye drop, nasal drop, lotion, liniment, etc.; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like.
The compound of the invention can be prepared into common preparations, slow release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.
For the preparation of the compounds of the present invention into tablets, various excipients known in the art may be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrating agent can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.
The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.
In order to make the administration unit into a capsule, the compound of the present invention as an active ingredient may be mixed with a diluent, a glidant, and the mixture may be directly placed in a hard capsule or a soft capsule. The active ingredient of the compound can be prepared into particles or pellets by mixing with a diluent, an adhesive and a disintegrating agent, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants and glidants used to prepare the tablets of the compounds of the invention may also be used to prepare capsules of the compounds of the invention.
For the preparation of the compound of the present invention into injection, water, ethanol, isopropanol, propylene glycol or their mixture may be used as solvent, and appropriate amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator may be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.
In addition, colorants, preservatives, fragrances, flavoring agents, or other additives may also be added to the pharmaceutical formulation, if desired.
The inventors of the present invention have found that aurantiol can block infection of host cells by influenza virus. Can also be combined with other antiviral drugs.
For the purpose of administration, the drug or the pharmaceutical composition of the present invention can be administered by any known administration method to enhance the therapeutic effect.
The dosage of the pharmaceutical composition of the present invention may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form, etc.
The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents. When the compound of the present invention has a synergistic effect with other therapeutic agents, its dosage should be adjusted according to the actual circumstances.
Beneficial technical effects
The inventor of the invention obtains the compound aurantiamarin which can interfere with the characteristic gene of the cytopathic effect of the influenza virus infection by extracting the characteristic gene set of the cytopathic effect of the influenza virus infection and reversely matching the characteristic gene set of the cytopathic effect of the influenza virus infection with the compound disturbance cell characteristic gene set. Then, an anti-influenza virus activity evaluation model is applied, the aurantiol has stronger inhibitory activity on influenza A virus infection and influenza B virus infection, and the data show that the anti-influenza virus activity of the aurantiol is equivalent to that of the first-line antiviral drug ribavirin, and the safety of the aurantiol is good. The new application value of the aurantium cassia element against influenza virus is considered to be higher, and the application prospect is achieved.
Drawings
FIG. 1. Evaluation results of the activity of aurantium obtusin blocking type A/Puerto Rico/8/1934 (H1N 1) infected MDCK cells.
FIG. 2 shows the results of evaluation of the activity of aurantium obtusin to block MDCK cells infected with A/Jiangxi Dongfu lake/312/2006 (H3N 2).
FIG. 3 shows the results of evaluation of the activity of aurantium obtusion against B/Ji Fang/13/1997 infected MDCK cells.
FIG. 4. Effect of aurantium obtusin on MDCK cell viability
Detailed Description
Example 1 extraction of a characteristic Gene set of cytopathic influenza Virus infection
High throughput sequencing datasets (GSE 61517, GSE 104168) and DNA microarray datasets (GSE 106279, GSE32139, GSE71766, GSE 37571) of Influenza infected cells were retrieved from the GEO database (Gene Expression Omnibus, https:// www.ncbi.nlm.nih.gov/GEO /) using "Influenza", "lung" and "homo sapiens" as keywords. Firstly, respectively carrying out differential expression analysis on transcriptome data of 9 different influenza virus strains in 3 data sets after 6 groups of different cells are infected for 24 hours, and calculating to obtain gene expression change fold (Log) 2 Foldchange value); then apply rank aggregation method based analysis tool Robust Rank Aggregation software package [ Kolde R, laur S, adler P, vilo J. Robust rank aggregation for gene list integration and meta-analysis. Bioinformatics.2012,28 (4): 573-580]Differential gene summary analysis, 111 protein-encoding genes were determined as characteristic gene sets of influenza virus infection cytopathic effect (Table 1) for transcriptome characteristic genesAnd (5) performing reverse matching calculation.
TABLE 1 characteristic Gene sets of influenza Virus-infected lung cells
Example 2 use of transcriptome signature Gene reverse matching to obtain Compounds that intervene in influenza Virus infection cytopathic signature genes
Applying a genome enrichment analysis method (Gene Set Enrichment Analysis, GSEA), applying fgsea [ Subramann A, tamayo P, mootha VK, et al Gene set enrichment analysis: a knowledges-based approach for interpreting genome-width expression profiles.Proc Natl Acad Sci U S A.2005 to the characteristic genome of influenza virus infection cytopathic and compound cell library data; 102 (43) carrying out feature reverse matching on 15545-15550R package, and showing that the enrichment fraction of the compound aurantiol is-0.0315, and suggesting that the aurantiol can reversely regulate the feature gene set of influenza virus infection cytopathic effect, wherein the compound is used for evaluating anti-influenza virus activity.
Example 3 principle of detecting influenza Virus infection model
A/Puerto Rico/8/1934 (H1N 1), A/Jiangxi Dong lake/312/2006 (H3N 2) and B/Ji Fang/13/1997 are classical seasonal influenza strains. The detection model mainly detects the inhibition effect of the compound on MDCK cells infected by influenza A virus (A/Puerto Rico/8/1934 and A/Jiangxi Dong lake/312/2006) and the inhibition effect of the compound on MDCK cells infected by influenza B virus B/Ji Fang/13/1997.
The test model preincubates the compound with cells for 20 hours prior to infection, subsequently infects the cells with virus, and detects MDCK cell viability at 48 hours after infection, and calculates the inhibition rate of the compound on virus infection by comparing the cell viability with that of the solvent control group cells and normal cells not infected with virus.
EXAMPLE 4 Experimental methods and results of A/Puerto Rico/8/1934 (H1N 1) infected MDCK cell model
MDCK cells were used at 4X 10 per well 4 Individual cells were seeded in 96-well plates and after 4 hours, aurantiol was added at final concentrations of 100 μm, 50 μm, 25 μm and 12.5 μm, respectively, the normal cell control group was not added with any compound, the solvent control group was added with an equal volume of DMSO, and the culture was continued for 20 hours. Media from the plates was aspirated, cells were rinsed once with PBS, and A/Puerto Rico/8/1934 virus infection (100X TCID) was added 50 ) Incubation was carried out at 37℃for 1 hour. The medium was aspirated, rinsed once with PBS, medium containing test compound was added, normal cell control was added to medium, and solvent control was added to medium containing equivalent amount of DMSO. After 48 hours CellTiter-Cell viability was detected by luminescence (Promega Corp.) using the cell viability detection kit, i.e.the relative luciferase activity in the cell lysates (relative luminescence units, RLUs). Cytopathic and viral inhibition rates were calculated for each experimental group according to formulas (1) and (2). Analyzing experimental data by using GraphPad Prism software, taking a concentration-inhibition ratio as a scatter diagram, obtaining a dose-response curve by nonlinear fitting, and calculating half-effective concentration EC of a compound to be tested 50 。
(1) Cytopathic rate% = (100-RLUs) Administration group (or RLUs) Solvent control group )/RLUs Normal cell control group )×100％
(2) Percent viral inhibition = (solvent control group cytopathic rate-dosing group cytopathic rate)/solvent control group cytopathic rate x 100%
The results show that the aurantiol can block A/Puerto Rico/8/1934 (H1N 1) from infecting MD CK cells, and the antiviral activity is equivalent to that of ribavirin which is a first-line antiviral drug (the results are shown in Table 2, and the dose-response curves are shown in figure 1).
Evaluation results of Activity of the Compounds of Table 2 on influenza A Virus A/Puerto Rico/8/1934 (H1N 1) infected MDCK cells
Example 5 Experimental methods and results of A/Jiangxi Dong lake/312/2006 (H3N 2) infected MDCK cell model
MDCK cells were used at 4X 10 per well 4 The individual cells were seeded in 96-well plates and after 4 hours, aurantiol was added at final concentrations of 100. Mu.M, 30. Mu.M and 10. Mu.M, respectively, the normal cell control group was not added with any compound, the solvent control group was added with an equal volume of DMSO, and the culture was continued for 20 hours. Media in the plates were aspirated, cells were rinsed once with PBS, and A/Jiangxi Dong lake/312/2006 virus infection (100X TCID) 50 ) Incubation was carried out at 37℃for 1 hour. The medium was aspirated, rinsed once with PBS, medium containing test compound was added, normal cell control was added to medium, and solvent control was added to medium containing equivalent amount of DMSO. After 48 hours CellTiter-Cell viability was detected by luminescence (Promega Corp.) using the cell viability detection kit, i.e.the relative luciferase activity in the cell lysates (relative luminescence units, RLUs). Cytopathic and viral inhibition rates were calculated for each experimental group according to formulas (1) and (2). Analyzing experimental data by using GraphPad Prism software, taking a concentration-inhibition ratio as a scatter diagram, obtaining a dose-response curve by nonlinear fitting, and calculating half-effective concentration EC of a compound to be tested 50 。
(1) Cytopathic rate% = (100-RLUs) Administration group (or RLUs) Solvent control group )/RLUs Normal cell control group )×100％
(2) Percent viral inhibition = (solvent control group cytopathic rate-dosing group cytopathic rate)/solvent control group cytopathic rate x 100%
The results show that the aurantium obtusin can block the MDCK cells infected by A/Jiangxi Dong lake/312/2006 (H3N 2), and the inhibition activity is equivalent to that of the first-line antiviral drug ribavirin (the results are shown in Table 3, and the dose-response curves are shown in figure 3).
Evaluation results of Activity of the Compounds of Table 3 on influenza A Virus A/Jiangxi Dong lake/312/2006 (H3N 2) infected MDCK cells
Example 6 Experimental methods and results for B/Ji Fang/13/1997 infection of MDCK cell model
MDCK cells were used at 4X 10 per well 4 Individual cells were seeded in 96-well plates and after 4 hours, aurantiol was added at final concentrations of 200 μm, 100 μm, 50 μm and 10 μm, respectively, the normal cell control group was not added with any compound, the solvent control group was added with an equal volume of DMSO, and the culture was continued for 20 hours. Media in the plates was aspirated and cells were rinsed once with PBS and B/Ji Fang/13/1997 virus infection (100X TCID) 50 ) Incubation was carried out at 37℃for 1 hour. The medium was aspirated, rinsed once with PBS, medium containing test compound was added, normal cell control was added to medium, and solvent control was added to medium containing equivalent amount of DMSO. After 48 hours CellTiter-Cell viability was detected by luminescence (Promega Corp.) using the cell viability detection kit, i.e.the relative luciferase activity in the cell lysates (relative luminescence units, RLUs). Cytopathic and viral inhibition rates were calculated for each experimental group according to formulas (1) and (2). Analyzing experimental data by Grap hPad Prism software, taking a concentration-inhibition ratio as a scatter diagram, obtaining a dose-response curve by nonlinear fitting, and calculating half-effective concentration EC of a compound to be detected 50 。
(1) Cytopathic rate%＝(100-RLUs Administration group (or RLUs) Solvent control group )/RLUs Normal cell control group )×100％
(2) Percent viral inhibition = (solvent control group cytopathic rate-dosing group cytopathic rate)/solvent control group cytopathic rate x 100%
The results show that the aurantium obtusin can block B/Ji Fang/13/1997 from infecting MDCK cells, and the inhibition activity is equivalent to that of ribavirin which is a first-line antiviral drug (the results are shown in Table 4, and the dose response curves are shown in figure 3).
Evaluation results of Compounds of Table 4 on MDCK Activity against influenza B Virus B/Ji Fang/13/1997 infection
Example 7 detection of the Effect of Compounds on cell viability
Principle of: ATP plays an important role in various physiological processes of cells, provides energy for organisms directly, and is an important index reflecting cell viability and is positively related to the number of living cells. Thus, the number of living cells in the test sample is reflected by the quantitative detection of ATP in the cell lysate.
The model adopts CellTiter-Luminescent Cell Viability Assay luminescence method cell viability assay kit (Promega Corp.) the effect of compounds on MDCK cell viability was assessed by quantifying MDCK cell viability by detecting ATP.
MDCK cells were seeded at 8000 wells/well into 96-well plates with 100. Mu.L of cell fluid per well, 37℃and 5% CO 2 Culturing for 24h. The next day test compounds were added to the cells at different concentrations with equivalent amounts of DMSO (0.1% v/v) as solvent control. After further culturing for 48h, 100. Mu.L of CellTiter-Glo reagent was added to each well, mixed by shaking for 2min, incubated at room temperature for 10min, and RLUs [ Tang K, he S, zhang X, et al, tangeretin, an extract from Citrus peels, blocks cellular entry of arenaviruses that cause viral hemorrhagic fever. Anti-viral Res.201 were assayed in each well8,160:87-93.]. Cell viability of the dosing wells was calculated using DMSO solvent well RLUs values of 100%.
Cell viability% = fluorescence intensity Administration group Fluorescence intensity Solvent control group ×100％。
The experimental result shows that the compound aurantio-obtusin has no influence on the activity of MDCK cells at half of the effective concentration (the result is shown in table 5, and the dose-response curve is shown in figure 4).
TABLE 5 Effect of aurantium obtusin on MDCK cell viability
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Claims (6)

1. The application of the aurantio-obtusin or the pharmaceutically acceptable salt thereof shown in the structural formula (I) in preparing the medicine for preventing or treating influenza virus infection;
2. the use according to claim 1, wherein the pharmaceutically acceptable salts comprise pharmaceutically acceptable organic salts or inorganic salts, wherein the organic salts comprise sulfonates, carboxylates, amino acid salts and fatty acid salts, and the inorganic salts comprise hydrochlorides, bromates, iodates, sulfates, bisulfate, phosphates, hydrogen phosphates, dihydrogen phosphates and nitrates.
3. Use according to claim 2, characterized in that said sulfonates comprise alkyl sulfonates containing 1 to 15 carbon atoms, benzene sulfonates, p-toluene sulfonates, o-toluene sulfonates, m-toluene sulfonates; the carboxylate comprises tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate and lactate; amino acid salts include glutamate, aspartate; fatty acid salts include long chain fatty acid salts containing 2 to 18 carbon atoms.
4. The application of a pharmaceutical composition in preparing a medicament for preventing or treating influenza virus infection is characterized in that the pharmaceutical composition comprises aurantiamarin shown in a structural formula (I) or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient,
5. the use according to claim 4, wherein said pharmaceutical composition further comprises an additional antiviral agent.
6. The use according to any one of claims 1 to 4, wherein the influenza virus comprises influenza a virus, influenza b virus, influenza c virus or influenza d virus.
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