CN109106712B

CN109106712B - Application of brevifolin methyl phenolic acid in preparation of anti-influenza virus medicine

Info

Publication number: CN109106712B
Application number: CN201810858676.2A
Authority: CN
Inventors: 段文军; 杨洁; 陈芳昭; 杨洛萍; 翟玲燕; 陈云; 陈金香; 谢宝平; 孙斌; 谢扬
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2018-07-31
Filing date: 2018-07-31
Publication date: 2021-03-16
Anticipated expiration: 2038-07-31
Also published as: CN109106712A

Abstract

The invention discloses application of brevifolin methyl phenolic acid in preparation of anti-influenza virus drugs. The inventor finds that the brevifolin phenolic acid A is prepared by experimentsThe ester shows stronger anti-influenza A virus H1N1 subtype effect in the range of 0.78-100 mu g/mL and IC thereof₅₀A value of 18.90 + -5.65 μmol/L, and a control drug of ribavirin (IC)₅₀0.56 ± 1.10 μmol/L) was slightly lower. The result of the plaque experiment shows that the brevifolin methyl ester has an inhibitory effect on the plaque formed by MDCK cells (canine kidney epithelial cells) caused by H1N1 virus and has concentration dependence. The brevifolin methyl ester can inhibit the expression of virus NP protein and PB2 polymerase in MDCK cells, and the inhibition effect is enhanced along with the increase of the concentration. At the same time, methyl hematoxylin has inhibitory effect on H3N2, and its IC₅₀＝71.74±1.48μg/ml。

Description

Application of brevifolin methyl phenolic acid in preparation of anti-influenza virus medicine

Technical Field

The invention relates to a new application of a compound, in particular to an application of brevifolin methyl phenolic acid in preparation of an anti-influenza virus medicine.

Background

Methyl brevifolin phenolic acid methyl ester (methyl brevifolin carboxylic acid) with molecular formula of C₁₄H₁₀O₈. CAS number 154702-76-8. The system is named as methyl7,8,9-trihydroxy-3,5-dioxo-1,2,3,5-tetrahydrocyclopenta [ c]isochromene-1-carboxylate. The structural formula is as follows:

the methyl brevifolinate is extracted from fructus Canarii albi. Olives, the fruit of olive (Canarium album (Lour.) Raeusch) belonging to the genus Canarium of the family Burseraceae, are recorded in the 2015 edition of Chinese pharmacopoeia, and are commonly used for treating sore throat, cough, polydipsia, fish and crab poisoning, etc.

The literature has shown that brevifolin methyl ester has the activity of inhibiting HSV-1STAKER and HSV-2SAR strains [ Asahun, Lifeng, Yaoqiang, et al.

The invention provides a preparation method and application of an ailanthus altissima extract and an active component thereof, namely brevifolin, and application thereof, and CN102659746A [ P ].2012. Biological activity research shows that the compounds have remarkable antioxidant, anti-inflammatory and anti-tumor effects. The experimental result shows that the ailanthus extract (including the effective part) and the active compound ailanthus element can be used for preparing the medicines and health products for resisting oxidation, inflammation, tumor and other related diseases.

The influence of different treatments on the germination of Phyllanthus urinaria seeds [ J ] Chinese traditional medicine journal, 2009,34(7):916 one 918, discloses Phyllanthus urinaria which is a plant of Phyllanthus of Euphorbiaceae, and has the effects of clearing liver, improving vision, promoting urination, reducing blood sugar, lowering blood pressure, detoxifying, removing food retention and the like. The active ingredients mainly comprise phenols, terpenoids, flavones, alkaloids and lipid compounds, wherein the brevifolin methyl phenolate and other ingredients have obvious anti-hepatitis B virus activity and liver protection effect. Hepatitis B Virus (HBV) is a pathogen causing hepatitis B (hepatitis B for short), belongs to the hepadnaviridae, and is mainly transmitted through blood, blood products and the like, vertical transmission of mothers and babies, close contact transmission and iatrogenic transmission. HBV adheres to the surface of hepatocytes through low-affinity receptors (such as heparan sulfate, proteoglycan and the like), and mediates endocytosis of the cells to viruses through binding of the preS1 region of large envelope proteins to viral receptors. Fusion between the endocytoviral envelope and the membrane of the endocytotic membrane releases the capsid into the cytoplasm, the capsid is transported to the nuclear pore complex, and the internal viral genomic rcDNA is released into the nucleus. Within the nucleus, rcDNA may be converted to covalently closed circular DNA (cccdna) by the DNA replication machinery of the cell. The virus uses cccDNA to transcribe mRNA of 3.5kb, 2.4kb, 2.1kb and about 0.8kb, wherein 3.5kb is pregenomic RNA (pgRNA), genome DNA can be reversely transcribed and used as a template for coding virus core protein and polymerase protein, HBsAg is polymerized in rough endoplasmic reticulum after being synthesized and transported to Golgi apparatus anterior cavity to package core particles, the assembled HBV particles and subviral particles are transported to Golgi apparatus to carry out glycosylation modification of HBsAg, and finally the complete virion is secreted out of host cells in a budding mode to complete the life cycle.

Influenza viruses are representative species of the Orthomyxoviridae family (Orthomyxoviridae) and include human influenza viruses, which are classified into three types, a (a), B (B), and C (C), and animal influenza viruses. Influenza a viruses often have antigenic variation and can be further divided into subtypes H1N1, H3N2, H5N1, H7N9 and the like (wherein H and N represent two surface glycoproteins of influenza viruses, respectively). The target of influenza virus invasion is respiratory mucosal epithelial cells. The main transmission route is droplets with influenza virus, which enter the body through the respiratory tract. A few may also be infected by indirect contact with common handkerchiefs, towels, etc. After the influenza virus invades the body, the influenza virus is adsorbed on the surface of a host cell by virtue of hemagglutinin and enters cytoplasm through endocytosis; after entering cytoplasm, the virus envelope fuses with the cell membrane to release the contained ss-RNA; eight segments of ss-RNA encode components such as RNA polymerase, nucleoprotein, matrix protein, membrane protein, hemagglutinin, neuraminidase, nonstructural protein and the like in cytoplasm; the matrix protein, membrane protein, hemagglutinin, neuraminidase and other coding proteins assemble M protein and envelope on endoplasmic reticulum or Golgi apparatus; in the cell nucleus, the genetic material of the virus is continuously replicated and forms a virus core together with nucleoprotein, RNA polymerase and the like; finally, the virus core binds to the membrane M protein and envelope, and is released outside the cell via budding, with a replication cycle of approximately 8 hours. The serious influenza virus has the characteristics of quick transmission and high lethality rate.

The anti-influenza virus drugs commonly used in China at present mainly comprise vaccines, ion channel M2 protein suppressors and neuraminidase inhibitors.

Vaccine: at present, three types of influenza vaccines such as virus live vaccines, split vaccines and subunit vaccines are mainly available in China. The whole virus live vaccine has the best clinical treatment effect, but can induce the children under 12 years old to have adverse reactions such as diarrhea, abdominal pain, dizziness, headache and the like.

Ion channel M2 protein repressor: amantadine and rimantadine are used for blocking the combination of influenza virus M2 protein and host cell protein and blocking the release of influenza virus core RNA and related enzymes, thereby interrupting the replication and propagation effects of influenza virus and achieving the antiviral effect. However, after a large amount of the traditional Chinese medicine is taken for a long time, the patients can have the adverse reactions of confusion, dizziness, headache, somnolence, nausea and the like.

Neuraminidase inhibitors: the most commonly used neuraminidase inhibitors in clinical practice are mainly oseltamivir and zanamivir. By inhibiting the activity of neuraminidase, the activity of influenza virus can be effectively reduced, and the infection of the influenza virus to normal cells can be reduced. Clinical studies find that the number of patients suffering from adverse reactions such as nausea and vomiting of oseltamivir for old patients suffering from heart disease and chronic non-obstructive pulmonary disease is obviously higher than that of patients using placebo. Zanamivir can be administered by various routes such as oral administration, intranasal administration and intravenous administration, wherein the oral administration has high bioavailability, short half-life and low excretion rate through the kidney, and can cause bronchospasm of patients suffering from chronic non-obstructive pulmonary diseases, so that patients suffering from asthma or chronic obstructive pulmonary diseases should be administered with a rapid organ expansion drug simultaneously.

Other classes: the barbidol hydrochloride can inhibit fusion of influenza virus lipid membranes and host cell surface proteins so as to play a role in resisting viruses, and IFN-beta interferon can effectively activate intracellular antiviral protein genes so as to inhibit synthesis of viral proteins.

Most of the medicines are chemically synthesized medicines and have certain side effects, so that the extraction of the effective components for resisting the influenza virus from natural substances has great significance.

Disclosure of Invention

The invention aims to provide application of brevifolin methyl phenolic acid in preparation of anti-influenza virus drugs.

The inventor finds that the brevifolin methyl ester shows stronger effect of resisting H1N1 subtype influenza A virus in the range of 0.78-100 mu g/mL and the IC of the brevifolin methyl ester₅₀The value is 18.90 +/-5.65 mu mol/L, and the comparison drug is ribavirin (IC)₅₀0.56 ± 1.10 μmol/L) was slightly lower. The result of the plaque experiment shows that the brevifolin methyl ester has an inhibitory effect on the plaque formed by MDCK cells (canine kidney epithelial cells) caused by H1N1 virus and has concentration dependence. The brevifolin methyl ester can inhibit the expression of virus NP protein and PB2 polymerase in MDCK cells, and the inhibition effect is enhanced along with the increase of the concentration. Meanwhile, the brevifolin methyl ester has inhibitory effect on H3N2 subtype virus, and its IC₅₀＝71.74±1.48μg/ml。

The technical scheme adopted by the invention is as follows:

application of brevifolin phenolic acid ester in preparing medicine for resisting influenza virus is provided.

As a further improvement of the application, the brevifolin phenolic acid ester is a pharmaceutically acceptable ester of brevifolin phenolic acid.

As a further improvement of the application, the pharmaceutically acceptable ester of brevifolin acid ester is C1-C4 ester thereof.

As a further improvement of the application, the brevifolin phenolic ester is brevifolin methyl ester.

A composition for treating influenza comprises brevifolin acid ester and at least one active component for resisting influenza virus or improving influenza symptom.

As a further improvement of the composition, the pharmaceutically acceptable ester of brevifolin acid ester is C1-C4 ester thereof. Further, the brevifolin phenolic ester is brevifolin methyl ester.

The invention has the beneficial effects that:

the brevifolin methyl phenolate shows stronger effect of resisting H1N1 subtype influenza A virus within the range of 0.78-100 mu g/mL, and IC of the brevifolin methyl phenolate₅₀The value is 18.90 +/-5.65 mu mol/L, and the comparison drug is ribavirin (IC)₅₀0.56 ± 1.10 μmol/L) was slightly lower. The result of the plaque experiment shows that the brevifolin methyl ester has an inhibitory effect on the plaque formed by MDCK cells (canine kidney epithelial cells) caused by H1N1 virus and has concentration dependence. The brevifolin methyl ester can inhibit the expression of virus NP protein and PB2 polymerase in MDCK cells, and the inhibition effect is enhanced along with the increase of the concentration. Meanwhile, the brevifolin methyl ester has inhibitory effect on H3N2 subtype virus, and its IC₅₀＝71.74±1.48μg/ml。

Drawings

FIG. 1 is a graph of the effect of methyl brevifolinate on MDCK cell viability;

FIG. 2 shows the H1N1 inhibitory effect of methyl brevifolinate;

FIG. 3 shows that methyl brevifolinate inhibits the formation of virus-induced plaques;

FIG. 4 shows the inhibitory effect of methyl brevifolinate on the expression of the viruses NP and PB2 in MDCK cells;

FIG. 5 shows the inhibitory effect of methyl brevifolinate on H3N 2.

Detailed Description

The technical scheme of the invention is further explained by combining experiments.

Anti-influenza virus activity

Cell and virus:

MDCK cells: canine kidney epithelial cells were stored in liquid nitrogen in this laboratory, and were cultured in DMEM high-glucose medium containing 10% FBS, 1% penicillin, and streptomycin after recovery.

Strain: influenza A virus A/Puerto Rico/8/34(H1N1)

1. Cytotoxicity of Compounds

MTT method for determining toxicity of brevifolin methyl ester on MDCK cells:

1) MDCK cells were cultured at 1X 10⁴One/well inoculated in 96-well plates at 5% CO₂Culturing in a cell culture box at 37 ℃ for 24 hours;

2) adding 2-fold gradient diluted methyl brevifolinate into 96-well plate, culturing for 48 hr, discarding supernatant, adding 100 μ l MTT solution (0.5mg/mL) per well, and adding 5% CO₂Continuously culturing for 4h in a cell culture box at 37 ℃;

3) after carefully removing the culture supernatant and adding 150. mu.l DMSO, the culture plate was placed in a microplate shaker and shaken for 10min to completely dissolve formazan deposited in the cells; the light absorbance of each hole is detected by a multifunctional microplate reader at the wavelength of 570nm, so that the influence of the compound on the cell survival rate can be indirectly reflected.

As shown in FIG. 1, the half-toxic concentration (CC50) of methyl brevifolinate on MDCK cells was more than 200. mu.g/mL.

MTT assay Compounds for H1N1 inhibitory Activity

1) MDCK cells at 2 × 10⁴One/well inoculated in 96-well plates at 5% CO₂Culturing at 37 deg.C for 24 hr, removing culture supernatant, washing with PBS 2 times, adding 100 μ l influenza virus solution (MOI 0.01) per well, and adding 5% CO₂Adsorbing the cells for 1h in a cell culture box at 37 ℃;

2) the virus solution was discarded and a concentration gradient of compound (prepared in DMEM with 1. mu.g/mL TPCK-pancreatin) in 5% CO was added₂Continuously culturing for 48h in a cell culture box at 37 ℃;

3) the supernatant was discarded and 100. mu.l of MTT solution (0.5mg/mL) was added to each well in 5% CO₂Continuously culturing for 4h in a cell culture box at 37 ℃;

4) carefully removing the culture supernatant, adding 150. mu.L DMSO, and placing the culture plate in a microplate shaker for 10min to completely dissolve formazan deposited in the cells; the absorbance of each well was measured at a wavelength of 570nm using a multifunctional microplate reader. Calculating the IC of the compound to the virus according to the magnitude of the light absorption value₅₀。

The results are shown in FIG. 2, and the MTT results show that the brevifolin methyl ester shows stronger anti-H1N 1 effect in the range of 0.78-100 μ g/mL and the IC thereof₅₀A value of 18.90 + -5.65 μmol/L, and a control drug of ribavirin (IC)₅₀0.56 ± 1.10 μmol/L) was slightly lower.

3. Plaque assay for detecting inhibition of viral infection of cells by compounds

After influenza viruses infect cells, the influenza viruses propagate in the cells in large quantities to cause changes in cell metabolism, which can cause the cells to have pathological changes, namely cell swelling and rounding, cell gap enlargement, cell nucleus shrinkage or rupture, and in severe cases, the cells can be partially or completely shed. Therefore, in order to investigate the anti-influenza a virus activity of brevifolin methyl ester, the inventors observed its inhibitory effect on virus-induced cell plaque. The experimental method is as follows:

1)MDCK cells at 2X 10⁵One/well inoculated in 12-well plates at 5% CO₂Culturing at 37 deg.C for 24 hr, removing culture supernatant, washing with PBS 2 times, adding 0.5mL virus solution (MOI 0.01) in 5% CO₂Adsorbing the cells for 1h in a cell culture box at 37 ℃;

2) discard virus solution, add 1.5mL of 2-fold gradiently diluted compound, 2% agar, 0.05% DEAE and 1. mu.g/mL TPCK-pancreatin 2 XMEM per well in 5% CO₂Continuously culturing for 72 hours in a cell culture box at 37 ℃;

3) removing agar covering layer, staining with 0.1% crystal violet prepared from 4% paraformaldehyde at 37 deg.C for 30 min-1 h, sucking off staining solution, comparing the number of plaques formed by drug treatment group and virus group, and detecting antiviral activity of the compound.

As shown in FIG. 3, the results of the experiments show that brevifolin methyl ester has inhibitory effect on plaque formation by virus and has concentration dependence.

Western blotting assay Compounds inhibit viral replication in MDCK cells

The influenza virus ribonucleoprotein complex (vRNP) forms the core of the virus, and the vRNP complex comprises RNA polymerase (PB1, PB2 and PA) and NP protein, so that the detection of the expression of PB2 and NP protein in a sample cell can reflect the inhibition effect of the compound on the virus. The influence of the methyl brevifolinate on the protein expression of the virus in the host cell is detected by a Western Blotting experiment. The experimental method is as follows:

1) culturing and processing cells: MDCK cells were cultured at 4X 10⁵One/well inoculated in 6-well plates at 5% CO₂Culturing in a cell culture box at 37 ℃ for 24 hours; the culture supernatant was discarded, washed 2 times with PBS, and 1mL of virus solution (MOI 0.01) was added thereto in 5% CO₂Adsorbing the cells for 1h in a cell culture box at 37 ℃; abandoning virus liquid, adding the compound diluted by 2 times of gradient into a 6-hole plate, arranging a cell control hole and a virus control hole, and continuing to culture for 24 hours; discarding the supernatant, washing with precooled PBS 3 times;

2) protein extraction and concentration determination: discarding PBS, adding 100 μ l RIPA lysis buffer (containing protease and nuclease inhibitor) into each well, placing on ice for lysis for 2min, scraping off cells from the culture dish with a cell scraper, and collecting into a sterile 1.5mL EP tube; placing the EP tube in a refrigerated centrifuge precooled to 4 ℃, centrifuging for 15min at 12000rpm, and transferring 80 mu l of a supernatant protein sample into a new 1.5mL EP tube; sucking 2.5 mul of protein sample, using BCA protein quantitative reagent, drawing a standard curve according to the kit instruction, determining the protein concentration, and carrying out normalization quantification on the protein sample; adding a quantitative loading buffer solution and 2% beta-mercaptoethanol into a protein sample, carrying out denaturation for 5min in a metal bath at 100 ℃, and using the sample for Western blotting;

3) western blotting: SDS-PAGE electrophoresis: at 10% separation gel and 4% concentration gel, 20 μ l was loaded at 50 μ g total protein per lane. Switching on a power supply, concentrating the gel at 85V, and performing constant-voltage electrophoresis for about 15 min; separating gel at 120V, and continuing electrophoresis until the protein standard substance is fully separated; electrotransformation membrane (wet process): taking out the gel, cutting a PVDF membrane with the size consistent with that of the separation gel, pre-marking the front and back sides of the protein, preparing a membrane rotating sandwich in a mode of cathode plastic splint-sponge cushion layer-filter paper-gel-PVDF membrane-filter paper-sponge cushion layer-anode plastic splint, placing the membrane rotating sandwich in a membrane rotating groove, and rotating the membrane for 75min at a constant pressure of 100V; after the membrane transfer is finished, taking out the PVDF membrane, immediately transferring the PVDF membrane into 5% skim milk, and sealing for 1h at room temperature; incubating primary antibody: washing with TBS-T buffer solution at room temperature for 3 times, adding primary antibody containing detection protein (prepared by 5% bovine serum albumin solution), and shaking overnight at 4 deg.C; hatching a secondary antibody: taking out the detected protein band, washing with TBS-T buffer solution for 3 times (1 time per 10 min); adding 5% skimmed milk containing corresponding secondary antibody, and incubating at room temperature for 1 h; the strip was removed and washed with TBS-T buffer 1 times every 5min for 6 times; and (3) chemiluminescence development: and soaking the PVDF film in ECL luminous liquid for 2min, taking out, sucking, wrapping with a preservative film, and placing in a developing cartridge. In a dark room, an X-ray film is placed above the protein film, a clamp is covered, exposure is carried out for a certain time, the film is taken out, developed in a developing solution, and fixed in a fixing solution.

As shown in fig. 4, the brevifolin methyl ester can inhibit the expression of NP and PB2 in MDCK cells, and the inhibition effect is enhanced with the increase of the concentration, compared with the virus control group.

5. Inhibitory Activity of Compounds on H3N2

As shown in FIG. 5, brevifolin methyl ester also has inhibitory effect on H3N2, and its IC₅₀71.74 ± 1.48 μ g/mL. The experimental procedure was as described for "2. MTT test Compounds for their inhibitory activity against H1N 1", but with the H1N1 strain being replaced by the H3N2 strain.

By combining the medicines, better curative effect can be obtained. It is expected that the combination of brevifolin methyl ester with other anti-influenza virus drugs or drugs for improving cold symptoms can be better used for treating influenza.

Claims

1. The application of brevifolin phenolic acid ester in preparing anti-influenza virus medicine comprises H1N1 as influenza virus, and brevifolin phenolic acid ester as methyl brevifolin phenolic acid ester with an onset concentration of 0.78-100 μ g/mL.