CN115463125B - Application of 5-methoxy flavone in preparing medicine for preventing or treating influenza virus infection - Google Patents
Application of 5-methoxy flavone in preparing medicine for preventing or treating influenza virus infection Download PDFInfo
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- CN115463125B CN115463125B CN202211034992.0A CN202211034992A CN115463125B CN 115463125 B CN115463125 B CN 115463125B CN 202211034992 A CN202211034992 A CN 202211034992A CN 115463125 B CN115463125 B CN 115463125B
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- XRQSPUXANRGDAV-UHFFFAOYSA-N 5-Methoxyflavone Chemical compound C=1C(=O)C=2C(OC)=CC=CC=2OC=1C1=CC=CC=C1 XRQSPUXANRGDAV-UHFFFAOYSA-N 0.000 title claims abstract description 57
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Abstract
The invention discloses an application of 5-methoxy flavone in preparing a medicine for preventing or treating influenza virus infection, belonging to the field of influenza virus prevention and treatment; the invention uses plaque technique to confirm the anti-influenza virus effect of 5-methoxy flavone; the Luminex technology is adopted to prove that 5-methoxy flavone inhibits the A549 cells infected by the influenza A virus H1N1 from expressing inflammatory factors in vitro; immunoblotting technology shows that 5-methoxy flavone inhibits influenza A virus H1N1 infection A549 cells from activating NF-kappa B, P MAPK signal pathway in vitro; meanwhile, in vivo results show that 5-methoxy flavone can improve influenza virus H1N1 induced lung pathological damage; therefore, the 5-methoxy flavone disclosed by the invention can be developed as a novel anti-influenza drug, and provides a novel drug choice for preventing and treating influenza.
Description
Technical Field
The invention belongs to the field of influenza virus prevention and treatment, and in particular relates to application of 5-methoxy flavone in preparation of a medicament for preventing or treating influenza virus infection.
Background
Influenza viruses are enveloped RNA viruses, belonging to the orthomyxoviridae family and are classified as A, B and C. The acute upper respiratory infectious diseases caused by the A-type virus have strong infectivity and rapid transmission speed, and bring serious threat to public health and serious economic and medical burden to society. Because the gene of influenza virus is easy to mutate, the antigen mutation capability is extremely strong, so that the vaccination of vaccine is delayed and the effect is not obvious. Currently, drugs approved for clinical treatment of influenza infection are M2 ion channel inhibitors: polyadamantane, rimantadine and neuraminidase inhibitors: oseltamivir and zanamivir. But resistance has developed in influenza strains that have been monitored for multiple epidemics. Thus, newly pandemic influenza is likely to outbreak at any time. In view of this situation, it is important to find anti-influenza drugs.
Research at home and abroad shows that the excessive immune response of a host organism is one of main pathogenic mechanisms for causing lung tissue injury. Currently, clinically applied glucocorticoids show good effects in the treatment of acute and chronic inflammation. However, it was reported that in H1N1 influenza viruses that were epidemic in 2009, glucocorticoid treatment was applied to increase the mortality of patients. Thus, the use of glucocorticoids has limited therapeutic efficacy in the treatment of immune inflammation caused by influenza. Influenza viruses act as foreign pathogens and their related molecular structures act as pathogen-associated molecular patterns that stimulate body cell signaling to produce a number of inflammatory responses. Therefore, compounds which can resist viruses and block host cell signal transduction are developed, and novel medicaments can be provided for clinically treating influenza virus infection.
Flavonoid compounds are widely existed in various types of plants, and are receiving attention because of various biological activities such as anti-inflammatory, anti-tumor, bacteriostasis and the like. Notably, the literature reports that the bioactivity and oral bioavailability of methoxylated flavonoids are superior to unmethylated flavonoids. 5-methoxyflavone (5-methoxyflavone, 5-MF) is a flavone compound methylated at the C5 atom, which has been reported to have anticancer properties. However, no respiratory diseases caused by 5-methoxyflavone parainfluenza virus have been reported yet.
Disclosure of Invention
Aiming at the defects of the prior art, the application of the 5-methoxy flavone in preparing medicaments for preventing or treating influenza virus infection is provided.
The aim of the invention can be achieved by the following technical scheme:
use of 5-methoxyflavone in preparing medicine for preventing or treating influenza virus infection is provided.
Further, the structural formula of the 5-methoxy flavone is as follows:
further, the medicine for preventing or treating influenza virus infection is a medicine prepared by taking 5-methoxy flavone as a medicine active ingredient.
Further, the influenza virus is an influenza a virus.
Further, the influenza virus includes H1N1, H3N2, and H9N2.
Further, the 5-methoxy flavone is obtained by separating cogongrass rhizome.
Use of 5-methoxyflavone for the preparation of a medicament for inhibiting an influenza a virus-mediated inflammatory response.
Use of 5-methoxyflavone in inhibiting H1N1 from inducing activation of A549 cell signaling pathway.
Use of 5-methoxy flavone for improving lung lesions caused by H1N1 infection.
A pharmaceutical preparation for preventing or treating influenza virus infection comprises 5-methoxyflavone as pharmaceutically active ingredient.
The invention has the beneficial effects that: the invention uses plaque technique to confirm the anti-influenza virus effect of 5-methoxy flavone; the Luminex technology is adopted to prove that 5-methoxy flavone inhibits the A549 cells infected by the influenza A virus H1N1 from expressing inflammatory factors in vitro; immunoblotting technology shows that 5-methoxy flavone inhibits influenza A virus H1N1 infection A549 cells from activating NF-kappa B, P MAPK signal pathway in vitro; meanwhile, in vivo results show that 5-methoxy flavone can improve influenza virus H1N1 induced lung pathological damage; therefore, the 5-methoxy flavone disclosed by the invention can be developed as a novel anti-influenza drug, and provides a novel drug choice for preventing and treating influenza.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to those skilled in the art that other drawings can be obtained according to these drawings without inventive effort.
FIG. 1 shows the inhibition of different strains of influenza virus by 5-methoxyflavone detected by the cell plaque assay;
FIG. 2 is a graph showing the effect of Luminex technology on detecting 5-methoxyflavone on induction of human A549 cells to express a series of inflammatory genes by influenza A/PR8/3/4 (H1N 1);
FIG. 3 is a graph showing the effect of detection of 5-methoxy flavone on induction of activation of cell signaling pathway by influenza A/PR8/3/4 (H1N 1) by immunoblotting technique;
FIG. 4 is a graph showing the effect of 5-methoxyflavone on lung index of influenza A/PR8/3/4 (H1N 1) infected mice;
FIG. 5 is a graph showing the effect of 5-methoxyflavone on lung pathology in influenza A/PR8/3/4 (H1N 1) infected mice using H & E staining technique.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Separating 5-methoxy flavone from lalang grass rhizome;
the method comprises the following specific steps:
step one: 10kg of cogongrass rhizome is taken, chopped and then extracted by heating and reflux with 95% ethanol for 2 times, the first time is extracted with 10 times of solvent for 2 hours, the second time is extracted with 8 times of solvent for 1.5 hours, the extracting solutions are combined and decompressed and recovered to obtain extract, the extract is suspended in water and sequentially extracted with petroleum ether (60-90 ℃), ethyl acetate and n-butanol to respectively obtain a petroleum ether part (56 g), an ethyl acetate part (49 g) and an n-butanol part (120 g);
step two: subjecting ethyl acetate part to silica gel column chromatography, gradient eluting with chloroform-methanol (60:1-1:1) to obtain A-I parts, subjecting D part to repeated silica gel column chromatography, ODS column chromatography, and eluting with methanolEluting with water (1:1), and purifying with SephadexLH-20 to obtain pale yellow compound 7.3mg, wherein the compound is identified as 5-methoxyflavone (C) 16 H 12 O 3 ) The purity was 98.5%.
And the structural formula of the 5-methoxy flavone is as follows:
detecting the inhibition effect of 5-methoxy flavone on different influenza virus strains by using a cell plaque method;
the method comprises the following specific steps:
step one: preparing MDCK cells; will be longer than 100cm 2 MDCK cells in the culture dish are added with 1mL of pancreatin for digestion for 20min, and the digestion is stopped by 2mL of complete culture medium; transferring into a 15mL centrifuge tube, and centrifuging for 1000rmp multiplied by 5min; adding 10mL of complete culture medium, uniformly mixing, and then, re-suspending cells, and planting the cells into a 6-well plate; culturing in incubator for 24 hr, observing whether the cells are 100% confluent at the bottom of 6-well plate, and if so, starting the following plaque experiment;
step two: diluting the virus; taking an EP tube, marking, and adding 1080 mu L of DMEM culture medium; adding 120 mu L of virus liquid into a first EP pipe, uniformly mixing the mixture on a vortex mixer, adding 120 mu L of the mixture into a next EP pipe, and similarly diluting the virus to 100PFU according to the storage concentration of the virus;
step three: virus infection of MDCK cells; the 6-well plate was removed, the complete medium was discarded and the cells were washed 2 times with PBS; adding 100PFU diluted toxin, placing in an incubator, incubating for 2h, taking out every 20min, and shaking;
step four: paving agar; discarding virus solution, mixing DMEM culture medium (containing 1 μg/mL TPCK-trypsin) with equal volume of 70 deg.C 2% agarose solution, adding into 6-well plate, and standing for 5min; after agar is solidified, placing the 6-hole plate in a 37 ℃ incubator for 48 hours in a reverse way;
step five: fixing and dyeing; taking out the 6-hole plate, adding 2mL of 4% formaldehyde, and fixing for 2h; sucking 4% paraformaldehyde, adding crystal violet, and dyeing for 5min; discarding crystal violet, adding PBS to wash the 6-hole plate, and photographing and preserving the result;
as shown in FIG. 1, 5-methoxyflavone can inhibit the occurrence of plaques caused by different influenza virus strains [ A/PR8/3/4 (H1N 1), A/HK/8/68 (H3N 2), A/HK/Y280/97 (H9N 2) ].
Detecting the effect of 5-methoxy flavone on influenza virus A/PR8/3/4 (H1N 1) to induce human A549 cells to express serial inflammatory genes by using Luminex technology;
the method comprises the following specific steps:
step one: a549 cells were cultured at 5×10 6 Is planted into a 6-hole plate, and is placed in an incubator for culture; after the cells are attached, the original culture medium is discarded, PBS is added for washing twice, and then DMEM/DF12 (1:1) culture medium containing influenza A/PR8/3/4 (H1N 1) without serum is added for adsorbing the cells for 2 hours; washing the cells with PBS 2 times to remove unadsorbed virus;
step two: setting a normal group, a virus group (model group), a 5-methoxy flavone intervention group (10. Mu.M, 20. Mu.M, 30. Mu.M) and a single 5-methoxy flavone treatment group (20. Mu.M); the virosomes were not added with pharmaceutical intervention; in the 5-methoxyflavone alone treated group, A549 cells were not virus adsorbed.
Step three: after 24 hours of drug intervention, the supernatants of each group were collected and transferred to 1.5mLEP tubes; centrifuging at 13000rpm in a centrifuge with pre-cooling at 4deg.C for 15min; after centrifugation, taking the supernatant to split the small parts, and then freezing the supernatant in a refrigerator at the temperature of minus 80 ℃ for use when detection is carried out;
step four: detecting the inflammatory factor content in the cell culture supernatant by using a Bio-plex suspension chip technology;
1) Balancing kit: the Bio-Plex suspension chip assay kit was removed to prevent equilibration at room temperature. Meanwhile, taking out the cells for culturing, and slowly thawing on ice;
2) Setting a detection plate: setting positions of a standard substance hole, a blank hole and a sample hole on a detection plate;
3) Adding 50 μl of detection antibody per well of the assay plate and adding the corresponding sample to the assay plate according to the kit instructions; and placing in a 96-well plate shaker for incubation at 300rpm for 30min;
4) After incubation, the assay plates were washed and reporter antibody was added and incubated for 10min at 300rpm on a 96-well plate shaker.
The experimental results are shown in FIG. 2, and the level of inflammatory factors (IL-6, IL-8, TNF-alpha, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, GM-CSF) induced by influenza virus is obviously inhibited after 5-methoxyflavone intervention; it was suggested that 5-methoxyflavone intervention may exert a therapeutic effect on influenza infection by inhibiting influenza infection to induce an excessive inflammatory response (compare # p <0.01, # p <0.001; compare # p <0.05, # p <0.01, # p <0.001; compare H1N 1).
Detecting the effect of 5-methoxy flavone on inducing activation of cell signaling pathway by influenza A/PR8/3/4 (H1N 1) by immunoblotting technology;
the method comprises the following specific steps:
step one: a549 cells were cultured at 5×10 6 Is planted into a 6-hole plate, and is placed in an incubator for culture; after the cells are attached, the original culture medium is discarded, PBS is added for washing twice, and then DMEM/DF12 (1:1) culture medium containing influenza A/PR8/3/4 (H1N 1) without serum is added for adsorbing the cells for 2 hours; washing the cells with PBS 2 times to remove unadsorbed virus;
step two: setting a normal group, a virus group (model group), a 5-methoxy flavone intervention group (10. Mu.M, 20. Mu.M, 30. Mu.M) and a single 5-methoxy flavone treatment group (20. Mu.M); the virosomes were not added with pharmaceutical intervention; the individual 5-methoxy flavone treated groups, whose a549 cells were not virus adsorbed;
step three: after 24 hours of drug intervention, the cell culture supernatant was discarded and washed twice with cold PBS; after the washing, 200ul of cell lysate RIPA (containing protease inhibitor cocktail, which was added to the cell lysate RIPA at a volume ratio (V/V) of 1:100 and a final concentration of 10. Mu.M of PMSF) was added to extract the total cell proteins;
step four: the cell lysate was transferred to a 1.5EP tube and placed in a pre-chilled centrifuge at 4℃for 15min at 13000 rpm;
step five: after centrifugation, the protein concentration of the total cell extract was determined according to BCA protein assay kit instructions: taking a 96-well plate, adding 10 mu L/well of standard substance and sample (2 multiple wells), adding 200 mu L/well of BCA detection working solution (configured working solution A: B=1:50) hole by hole, uniformly mixing, incubating at 37 ℃ for 30min, and measuring an OD value at the absorbance 572nm by using an enzyme-labeled instrument; drawing a standard curve, and converting the OD value of the sample into concentration according to the standard substance;
step six: preparation of SDS polyacrylamide gel:
preparing 10% SDS polyacrylamide gel electrophoresis separating gel:
after fully and uniformly mixing, pouring the mixture between the separation glass plates, adding ddH20 into the upper layer after pouring to seal the liquid level of the separation gel, and waiting for 30min for the separation gel to coagulate;
step seven: preparing 5% SDS polyacrylamide gel electrophoresis concentrated gel:
pouring concentrated glue between the separation glass plates after fully and uniformly mixing, pouring Bi Charu comb, and waiting for 30min for the separation glue to coagulate;
step eight: denatured protein: mixing 5 Xprotein loading buffer with each group of samples (20 mug) in a ratio of volume to mass (V/V) (μL/μL) (1:4), and placing in a metal heater at 95 ℃ to denature protein for 10min;
step nine: loading and electrophoresis: pulling the comb off and sampling the comb hole by hole; setting a 90V constant-pressure electrophoresis of a Bio-Rad vertical electrophoresis apparatus, and stopping electrophoresis when the bromophenol blue indicator reaches the bottom of the separation gel;
step ten: transferring: pre-activating PVDF film with methanol for 15min; taking off gel after electrophoresis; stacking Whatman3M filter paper-gel-PVDF film-Whatman 3M filter paper in sequence, and placing on a wet-rotating negative plate; in a refrigeration house, a film is transferred for 2 hours by a constant current of 380 mA;
step eleven: after the transfer, adding 5% skimmed milk powder (5% milk/TBST) to seal PVDF membrane for 2h;
step twelve: antibody incubation: 5% BSA/TBST was prepared at 1: the rabbit anti-human p-IKB alpha antibody, the rabbit anti-human p-p65 antibody, the rabbit anti-human p-38 antibody, the rabbit anti-human p-ERK1/2 antibody and the rabbit anti-human ERK1/2 antibody are diluted in 5% BSA according to the proportion of 1000; and placing in a refrigerator at 4 ℃ for overnight incubation;
step thirteen: incubating the secondary antibody; after overnight, taking out the PVDF film and recovering to room temperature; washing the membrane 3 times by using a washing solution TBST, adding horseradish peroxidase (HRP) -coupled goat anti-rabbit secondary antibody after 5 min/time, and incubating for 1h at room temperature;
step fourteen: developing; removing the secondary antibody and washing the membrane 3 times with washing solution TBST for 5 min/time; adding ECL immunofluorescence chemiluminescence color developing agent for developing;
fifteen steps: analyzing and sorting results;
as shown in FIG. 3, 5-methoxyflavone obviously inhibits the activation of the NF- κB and P38MAPK signal channels of the human A549 cell signal channels induced by influenza A/PR8/3/4 (H1N 1).
Detecting the influence of 5-methoxy flavone on the lung pathology of mice infected with influenza A/PR8/3/4 (H1N 1);
the method comprises the following steps:
step one: C57/B6 mice purchased from 4-6 week old SPF class center of medical laboratory animals in guangdong province were randomly divided into five groups, each: normal group, H1N1 group (model group), H1N1+5-methoxyflavone (20 mg/kg/d), H1N1+5-methoxyflavone (40 mg/kg/d);
step two: two days in advance, 5-methoxyflavone (20 mg/kg/d,40 mg/kg/d) was administered by intragastric administration;
step three: influenza virus A/PR8/3/4 (H1N 1) is frozen and thawed and diluted to 5LD50 by serum-free DMEM/F12, and after the mice are anesthetized by 10% chlorohydrol (mass/volume ratio) (M/V), an influenza infection model is prepared by nasal drip of 50 mu L; dripping the same amount of DMEM/F12 into the mixture by a normal group in the same way;
step four: on day 7 of preparation of influenza infection model, mice were anesthetized with 10% hydrochloroaldehyde, and the lung was dissected for lung measurement of lung index (lung index=lung weight (g)/body weight (g) ×100), and changes in body weight and lung index of the mice were evaluated; dissecting lung to fix paraformaldehyde, embedding paraffin, slicing and H & E staining, observing lung lesions of mice under a microscope, and photographing.
The results are shown in fig. 4, where the H1N1 group lung index is significantly elevated compared to the normal group with significant differences (#p < 0.01); the lung index of the 5-methoxyflavone-interfered group was statistically different from that of the viroid (< 0.05).
The lung pathology results are shown in fig. 5, in which the H1N1 group lung is infiltrated with a large number of inflammatory cells and the alveolar structure is disrupted; the 5-methoxy flavone intervention group obviously improves lung lesions caused by H1N1 infection.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.
Claims (6)
- Use of 5-methoxyflavone as sole active ingredient in the manufacture of a medicament for the prevention or treatment of influenza a virus infection.
- 2. The use according to claim 1, wherein the 5-methoxy flavone has the structural formula:。
- 3. the use according to claim 1, wherein the medicament for preventing or treating influenza a virus infection is a medicament prepared by using 5-methoxy flavone as a pharmaceutically active ingredient.
- 4. The use according to claim 1, wherein the influenza a virus comprises H1N1, H3N2 and H9N2.
- 5. The use according to claim 1, wherein the 5-methoxyflavone is isolated from lalang grass rhizome.
- The use of 5-methoxyflavone as sole active ingredient in the manufacture of a medicament for inhibiting influenza a virus mediated inflammatory response.
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