CN113648305A

CN113648305A - Application of luteolin in preparation of anti-A6 Coxsackie virus medicine

Info

Publication number: CN113648305A
Application number: CN202111092566.8A
Authority: CN
Inventors: 李菲; 田景振
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-11-16
Also published as: CN112472694B; CN113648299A; CN112472694A; CN113786401A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a compound luteoloside in preparation of an anti-A6 Coxsackie virus medicine. The invention discovers the biological activity of the luteolin for resisting the A6 type coxsackie virus medicine for the first time, researches the interaction between the luteolin and a virus target protein, and discloses the mechanism of resisting the A6 type coxsackie virus.

Description

Application of luteolin in preparation of anti-A6 Coxsackie virus medicine

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of compounds of artemisinic acid, artemisinin, chlorogenic acid, geniposide and luteolin in preparation of an anti-A6 Coxsackie virus medicine.

Background

Coxsackie virus A6 (CV-A6) is an important class of Coxsackie viruses. Since 2008, CV-A6 began to become prevalent throughout the world, spreading gradually from Europe to Asia, with an increasing incidence of hand-foot-and-mouth disease caused by CV-A6 infection. In 2008, CV-A6 positive hand-foot-and-mouth disease is firstly exposed in Finland, and in 2009, Taiwan in China, Spain in 2010, Japan in 2011, Guangdong Shenzhen in 2013, France in 2014 and the like, hand-foot-and-mouth disease epidemic caused by CV-A6 infection is successively exposed. CV-A6 became one of the main causes of the outbreak of hand-foot-and-mouth disease since 2013. The incidence rate of hand-foot-and-mouth diseases caused by domestic CV-A6 infection is on the rise, and the incidence rate of the hand-foot-and-mouth diseases caused by CV-A6 infection is 27.4% higher than that of patients suffering from the hand-foot-and-mouth diseases in 2009-2012. Monitoring data of the foot-and-mouth disease epidemic situation in the continental areas of China in 2013-2017 show that CV-A6 becomes a main epidemic strain in most areas, and some areas exceed EV71 and CV-A16 and become the first pathogen causing the foot-and-mouth disease, for example, the CV-A6 positive rate in a Zhejiang province detection sample in 2013 is 51.3%.

The mean age of the onset of CV-A6 infection is (2.91. + -. 2.12) years of age, and may be infected by immunocompromised adults as well as children. The clinical symptoms caused by CV-A6 were widespread and accompanied by a shift in demethylation from the early-aged herpangina to skin rash. Analysis of the VP1 gene by PuenpaJ et al showed that recombination of the CV-A6 genome increased the genetic differences between sequences. Bayesian phylogenetic analysis shows that the current recombination groups (RF-E, -F, -H, -J and-K) can all be traced to a common ancestor (RF-A). The full length genes of the 39 different RFs all recombined between the 2A-2C and 5' UTR. Gaunt et al_[92]The study showed that by 2014 CV-a6 developed 8 forms of recombination, which occurred in the 2A-2C region, inside VP3, non-coding region and VP1 region, and that recombination of non-structural regions could be a potential cause of clinical symptom changes by CVA 6. Bian et al divides CV-A6 into five branches A-E according to different genome sequences, and divides the genotype of E into E1 and E2 subclusters. Currently, the major prevalent CV-A6 in the world is the E2 subclusters. The board of south of the east China establishes phylogenetic tree through ML method, and analysis finds that from 1992 to today the epidemic subtype of Shandong province CV-A6 has obvious alternation, and the alternation pattern is: subtype B in 1992 → subtype C in 1996 → subtype D in 2010 → subtype E2 so far, and the prevalence of CV-a6, have enabled a shift from non-dominant to dominant strains. E2 subclusterCVA6 infection often leads to serious respiratory and nervous system diseases such as herpangina, aseptic meningitis, brainstem encephalitis, and acute flaccid paralysis.

However, the related research of CV-A6 is less, and the research of anti-CV-A6 drugs is urgently needed.

The honeysuckle flower, sweet wormwood herb and cape jasmine fruit formula is derived from famous and old traditional Chinese medicine empirical formula, comprises honeysuckle flower, sweet wormwood herb and cape jasmine fruit and has the effects of clearing heat and removing toxicity, dispelling wind and relieving exterior syndrome. The preparation is successfully developed into traditional Chinese medicine preparations such as Reduning injection (RDN) and the like, is widely applied to the treatment of upper respiratory tract infection (exogenous wind-heat syndrome), and achieves good social and economic benefits. However, no research on anti-CV-A6 has been reported.

Artemisinin (Artemisinin) has tradename of Artemisinin and luteolin. The compound preparation is mainly used for controlling symptoms of vivax malaria and malignant malaria and treating chloroquine-resistant strains, and can also be used for treating dangerous malignant malaria such as encephalopathy and jaundice. Artemisinin molecular formula C₁₅H₂₂O₅Molecular weight of 282, structural formula

The antiviral activity of artemisinin and its derivatives is concerned, and research results show that artemisinin has the effect of inhibiting various viruses such as cytomegalovirus, herpes simplex virus, hepatitis B virus, hepatitis C virus and the like. However, no research report on the anti-CV-A6 exists at present.

Artemisic acid (English name of Artemisic acid) with molecular formula C₁₅H₂₂O₂Molecular weight of 234, structural formula

No research report on the resistance of the antibody to CV-A6 exists at present.

Chlorogenic acid molecular formula C₁₆H₁₈O₉Molecular weight of 354, structural formula

Chlorogenic acid (CGA), also known as caffeotannic acid, is a class of phenylpropanoid compounds found in a variety of plants. Hockey et al (2001) studied the in vitro antiviral effects of CGA and found that CGA significantly inhibited syncytial virus, Coxsackie group B type 3, adenovirus type 7, etc., and also inhibited adenovirus type 3 and Coxsackie type B5 to some extent. Chiang LC et al (2002) also found that CGA has a strong inhibitory effect on adenovirus type 11. Menu et al (2007) report that chlorogenic acid extracted from honeysuckle can effectively inhibit influenza A virus strain FM 1. The Wang scholars et al (2008) reported that chlorogenic acid obtained from honeysuckle by water boiling and alcohol extraction has an in vitro effect on Porcine Parvovirus (PPV). Studies such as contained shaking and discharged shaking (2008) show that chlorogenic acid has obvious antiviral effect on HSV-1 infection in-vitro experiments, can effectively prevent virus infection, but has no direct killing effect on viruses. WangGF (2009) reports that chlorogenic acid has an inhibitory effect on hepatitis B virus-HBsAg and HBeAg, and has a significant inhibitory effect on HBsAg and HBeAg secreted by HepG2.2.15 cells. Juan silk tree et al (2009) reported that chlorogenic acid has a significant effect of resisting human cytomegalovirus in vitro. However, no research report on the anti-CV-A6 exists at present.

Geniposide with English name and molecular formula C₁₇H₂₄O₁₁Molecular weight of 404, structural formula

Geniposide has anti-H1N 1 and anti-EV 71 activities. However, no research on anti-CV-A6 has been reported.

Luteolin (luteolin) named as Luteoloside, molecular formula C₂₁H₂₀O₁₁Molecular weight 448, structural formula

The luteolin can inhibit herpes simplex keratitis, influenza virus pneumonia, influenza A virus, porcine reproductive and respiratory syndrome virus, newcastle disease virus, respiratory syncytial virus, influenza and human cytomegalovirus, etc., thereby achieving the antiviral effect. However, no research report on the anti-CV-A6 exists at present.

Disclosure of Invention

The inventor finds that the inhibition rate of arteannuic acid, artemisinin, chlorogenic acid, geniposide and luteolin on CV-A6 is obviously higher than that of a control drug ribavirin through a large number of in vitro cell test researches and continuous exploration.

The invention aims to provide an application of a compound in preparation of an anti-A6 Coxsackie virus medicament, wherein the compound is one or more of arteannuic acid, artemisinin, chlorogenic acid, geniposide and luteolin, and the arteannuic acid, the artemisinin, the chlorogenic acid, the geniposide and the luteolin are all effective components of a honeysuckle flower artemisia apiacea gardenia prescription.

The invention also aims to provide the application of the artemisinic acid in preparing the anti-A6 coxsackie virus medicament.

The invention also aims to provide the application of artemisinin in preparing a medicament for resisting coxsackie virus A6.

The invention also aims to provide application of chlorogenic acid in preparing a medicament for resisting coxsackie virus A6.

The invention also aims to provide the application of the geniposide in preparing the anti-A6 Coxsackie virus medicament.

The invention also aims to provide the application of the luteoloside in preparing the medicaments for resisting the coxsackie virus A6.

Compared with the prior art, the invention discovers the biological activities of the arteannuic acid, the artemisinin, the chlorogenic acid, the geniposide and the luteolin for resisting the coxsackie virus A6 for the first time, researches the interaction between the arteannuic acid, the artemisinin, the chlorogenic acid, the geniposide and the luteolin and a virus target protein, and discloses the mechanism of resisting the coxsackie virus A6.

Drawings

FIG. 1 is a schematic diagram ofThe inhibition ratio of CV-a6 by the compound, n-3,

(Note: P vs. ribavirin control group;)<0.01*,***P<0.001)；

FIG. 2 is a two-dimensional and three-dimensional schematic view of molecular docking (in the figure, 1-chlorogenic acid; 2-geniposide; 3-artemisinic acid; 4-artemisinin; 5-luteolin).

Detailed Description

The invention will be further described with reference to the accompanying drawings and specific embodiments so that those skilled in the art may better understand the invention, but the invention is not limited thereto.

Example 1 Artemisinic acid, artemisinin, chlorogenic acid, geniposide and luteolin anti-coxsackie virus type A6 experiment

1.1 medicinal materials

The flos Lonicerae is dried bud of Lonicera japonica Thunb of Lonicera of Caprifoliaceae; the herba Artemisiae Annuae is dried aerial part of Artemisia annua L of Artemisia of Compositae; the fructus Gardeniae is dried fruit of Gardenia jasminoides Elis of Gardenia of Rubiaceae. The above herbs were purchased from Tai-Union pharmacia, Tai-city.

1.2 viruses, cells and animals

CV-A6 was introduced from the virus seed room of China center for disease prevention and control; human Rhabdomyoma (RD) cells, introduced by the chinese institute of preventive medicine virus research.

1.3 preparation of the solution

Cell culture solution: MEM medium fetal bovine serum-diabody 89:10: 1.

Cell maintenance solution: MEM medium fetal bovine serum-diabody 97:2: 1.

Cell cryopreservation solution: fetal bovine serum DMSO: MEM: 1: 8.

Weighing arteannuic acid, artemisinin and luteolin respectively at a ratio of 20mg, dissolving in dimethyl sulfoxide to obtain solution with a concentration of 20 mg/mL^-1The stock solution of (1). Taking appropriate amount of stock solution of arteannuic acid and artemisinin, and diluting with cell maintenance solution to obtain 2.5-100 μ g/mL^-1There were 7 different concentration solutions. Diluting with cell maintenance solution to obtain 2.0-20 μ g/mL^-1There were 7 different concentration solutions.

Respectively weighing chlorogenic acid and geniposide each 20mg, adding PBS to dissolve to obtain a solution with a concentration of 10 mg/mL^-1The stock solution of (1). Taking appropriate amount of stock solution, and diluting with cell maintenance solution to obtain 10-320 μ g/mL^-1There were 7 different concentration solutions.

2 method of experiment

2.1 culture of cells

2.1.1 cell recovery

The cell freezing tube is taken out from the liquid nitrogen, immediately placed in a water bath at 37 ℃ and quickly shaken until the cell freezing tube is completely thawed, and centrifuged for 5min at the room temperature of 1000 r/min. Discarding the supernatant, adding cell culture solution, slowly blowing and resuspending, placing in a cell culture box with temperature of 37 ℃ and 5% CO2, and replacing with new culture solution after 12 h.

2.1.2 cell passages

When the cells grew to more than 90% of the bottom area of the cell bottle, the culture solution was discarded, 0.01MPBS was added for washing 2 times, 1ml of 0.25% trypsin was added, and the mixture was placed in a cell incubator at 37 ℃ and 5% CO2 for 1 min. Discarding trypsin, placing in a cell culture box with 37 deg.C and 5% CO2, observing under an inverted microscope until cell gap is obvious, adding culture solution, blowing to make cells uniformly dispersed, adjusting cell density to 2 × 10⁴Perml, placed in a cell incubator at 37 ℃ with 5% CO 2.

2.1.3 cell cryopreservation

The abundance is more than 90%, and after the cells in logarithmic phase are digested by pancreatin, the cells are added with cell growth liquid and blown and dispersed uniformly. Adjusting the cell density to 4 multiplied by 106/mL, centrifuging for 10min at 1000r/min, abandoning the supernatant, adding the precooled frozen stock solution to resuspend the cells, and transferring the cells to a freezing tube. Gradient cooling at 4 deg.C for 20min, -20 deg.C for 30min, -80 deg.C overnight, and transferring into liquid nitrogen for storage.

2.2 determination of cell viability

Cell viability was determined by the CCK8 method. And (3) adding PBS (phosphate buffer solution) into a 96-well plate waste solution, washing for 2 times, adding 100 mu L of maintenance solution and 10 mu L of LCCK8 into each well, culturing for 1h in a cell culture box with the temperature of 37 ℃ and the concentration of 5% CO2, measuring the OD value of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

Cell viability ═ 100% x (OD experimental-OD blank)/(OD normal cell control-OD blank).

2.3 propagation of viruses

The solution was discarded when the RD cell abundance in logarithmic growth phase reached about 80%, and washed 2 times with 0.01 MPBS. Adding 2mL of maintenance solution and 200 μ of LCV-A6 virus solution, mixing, stirring at 37 deg.C with 5% CO₂The cell culture box is used for culturing for 2 hours. 2mL of maintenance solution is added, and the mixture is placed in a cell culture box with 37 ℃ and 5% CO2 for continuous culture. When more than 75% of cells have pathological changes, sucking the supernatant, placing the supernatant in a sterile centrifuge tube, centrifuging for 5min at 8000r/min, filtering the supernatant through a 0.22 mu m microporous filter membrane, subpackaging the filtrate, and freezing and storing at-80 ℃.

2.4 viral virulence assay

Taking RD cells in logarithmic growth phase, adding growth medium to adjust the density to 5 × 104/ml through trypsinization, connecting to a 96-well plate (100 μ L/well), and culturing in a cell culture box at 37 ℃ and 5% CO2 for 24 h. Diluting CV-A6 virus solution with a maintenance solution, wherein the dilution is 10-1-10, each well is 100 μ L, each dilution is 8 multiple wells, and a normal cell control is set at the same time, and incubating for 2h in a cell incubator with 5% CO2 at 37 ℃. Discarding the supernatant, adding 100 μ L of maintenance solution into each well, standing at 37 deg.C and 5% CO₂The culture of the cell culture box is carried out, and the liquid is changed once every 24 hours. Cell morphology changes were observed at 72h after inoculation, the number of wells for characteristic cytopathic effect (CPE) was recorded, and TCID50 of CV-A6 in RD cells was calculated according to the Reed-Muench method.

2.5 cytotoxicity assays

Collecting cells in log phase, adjusting cell density to 5 × 10 with cell growth medium₄Perml, 100. mu.L per well was plated in 96-well plates. Placing at 37 deg.C with 5% CO₂The cells were cultured in a cell culture chamber for 24 hours, discarded, and washed with PBS 2 times. Adding medicinal solution of different formula extracts and control ribavirin solution into cells, adding 100 μ L of each well, adding maintenance solution into control group, and standing at 37 deg.C containing 5% CO₂Culturing for 48h in a cell culture box, measuring the cell survival rate according to the method under the item 2.2, and calculating the maximum non-toxic concentration.

2.6 in vitro antiviral action

2.6.1 viral infection and mode of action of the drug

The logarithmic phase RD cells were collected, adjusted to a cell density of 5X 104/mL using growth medium, inoculated into a 96-well plate at 100. mu.L per well, and cultured in a cell culture chamber containing 5% CO2 at 37 ℃ for 24 hours. Discard solution, wash 2 times with PBS. The 50 μ g/mL drug solution and the virus solution 1:1 were mixed and added to a 96-well plate at 100 μ L per well (MOI ═ 1), and a normal cell control group and a virus control group were set. Placing at 37 deg.C with 5% CO₂The cell culture box of (2) for 1 hour. Discarding the solution, adding maintenance solution 100 μ L per well, standing at 37 deg.C and containing 5% CO₂The cell culture chamber of (3) was incubated to 48 hpi.

2.6.2 evaluation index

Observing cytopathic condition under microscope, measuring cell survival rate according to the method under item 2.2, and calculating virus inhibition rate.

Percent virus inhibition ═ 100% (sample group OD-virus control group OD)/(cell control group OD-virus control group OD)%

3. Results

3.1 Virus virulence assay

The CPE of CV-A6 infected RD cells for 72h is shown in Table 1.

TABLE 1 CV-A6 infection of RD cells for 72h CPE

Distance ratio (more than 50% >, positive percentage-50)/(more than 50% >, positive ratio-less than 50% >)

logTCID₅₀Logarithm of virus dilution greater than 50% + distance ratio x logarithm of dilution factor

TCID of CV-A6 in RD cells₅₀Is 10^-8.41/0.1mL。

3.2 cytotoxicity of Compounds

Maximum nontoxic concentration TC of arteannuic acid, artemisinin, geniposide, chlorogenic acid and luteolin on RD cells₀The results are shown in Table 2.

TABLE 2 cytotoxicity of Compounds on RD cells

3.3 antiviral Activity of Compounds

The results of the virus inhibition rates of artemisinic acid, artemisinin, chlorogenic acid, geniposide and luteolin are shown in figure 1.

As can be seen from figure 1, the inhibition rate of arteannuic acid, artemisinin, chlorogenic acid, geniposide and luteolin on CV-A6 is significantly higher than that of control ribavirin (P <0.01 or 0.001), and the arteannuin-A inhibitor has a good effect of resisting CV-A6.

3.4 interaction between active ingredients and viral target proteins

CV-A6 belongs to EVs group A of Picornaviridae, and the polyprotein encoded by the virion genome is hydrolyzed into 3 protein precursors (P1-P3). P1 is further hydrolyzed into 4 structural proteins (VP 1-VP 4), which together constitute the viral capsid. VP4 is located inside the capsid of the virus, and VP 1-VP 3 are located on the capsid surface, and are the main sites where antigenic determinants are present. Among them, the surface-exposedtools and C-terminal structure of VP1 are the main immunogenic sites as the binding sites for molecular docking. The two-dimensional structure of the compound is converted into a three-dimensional structure, the compound is used as a ligand to be docked with a receptor protein, and the two-dimensional action schematic diagram and the three-dimensional structure schematic diagram of molecular docking are shown in figure 2.

As can be seen from FIG. 2, hydrogen bonds exist between 6 amino acid residues of the chlorogenic acid molecule and the ASN, TYR and the like of the VP1 protein, and hydrogen bonds are formed between 7 amino acids of the geniposide molecule and the MET, ASP and the like, so that the affinity is strong. The artemisinic acid molecule and amino acids ARG, GLU form hydrogen bond, and van der Waals attraction exists between ASP, TYR and other residues. The luteolin molecules have hydrogen bond interaction with 5 amino acids such as ASN and CYS, and simultaneously have carbon-hydrogen bond, van der Waals attraction, Pi-sulfurr interaction with other amino acids. The interaction between the molecules of the compound and the amino acid residues ensures that the molecules and the amino acid residues have good affinity, so that the compound plays a role in inhibiting the VP1 protein.

The molecular docking result shows that multiple hydrogen bonds, van der Waals attraction and other actions exist between arteannuic acid, artemisinin, chlorogenic acid, geniposide and luteolin molecules and CV-A6 capsid protein, so that the arteannuic acid, artemisinin, chlorogenic acid, geniposide and luteolin molecules have good affinity, the generated compound is stable, the virus capsid protein can be inhibited, and the antiviral action is exerted.

Claims

1. Application of luteoloside in preparing medicine for resisting coxsackie A6 virus is provided.