CN111110669A

CN111110669A - Application of polyiodinated iodocarboxylic acid in resisting EV71 virus

Info

Publication number: CN111110669A
Application number: CN201911380434.8A
Authority: CN
Inventors: 魏艳红; 李妮; 王海杰; 胡康洪; 王龙胜; 朱茂春
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-05-08
Anticipated expiration: 2039-12-27
Also published as: CN111110669B

Abstract

The invention provides application of polyiodiiodocarboxylic acid in resisting EV71 virus. Through research experiments on the inhibition activity of the EV71 viruses of the multi-iodo iodocarboxylic acids L6 and L7, the multi-iodo iodocarboxylic acids L6 and L7 are proved to be capable of inhibiting cytopathic effect caused by the EV71 viruses, enhancing the survival rate of infected cells and inhibiting the replication and proliferation of the EV71 viruses in the cells. The poly-iodo-carboxylic acid L6 and L7 provided by the invention have potential in preparing specific therapeutic drugs for resisting EV71 infection, and have good clinical application prospects.

Description

Application of polyiodinated iodocarboxylic acid in resisting EV71 virus

Technical Field

The invention belongs to the technical field of antiviral drugs, and particularly relates to application of polyiodiated iodocarboxylic acid in resisting EV71 virus.

Background

Enterovirus type 71 (EV71), a member of the Enterovirus genus (Enterovirus) of the Picornaviridae family (Picornaviridae), is one of the most prominent pathogens causing hand-foot-and-mouth disease in infants and young children, sometimes with severe central nervous system complications including aseptic meningitis, encephalitis, polio-like paralysis, neurological heart-lung failure, etc., and even death. Since the first report in 1974, the EV71 virus infectious disease has been frequently outbreaks and epidemics worldwide, and the situation is severe in Asia-Pacific region, especially China. In view of the great harm brought to the life and health of people in China by the spread and epidemic of the hand-foot-and-mouth disease, the government of China has listed the hand-foot-and-mouth disease as a third class infectious disease in 2008 and brings into management, and a series of relevant laws and regulations are formulated to strictly control the spread and epidemic of the hand-foot-and-mouth disease. There is currently no specific drug for the treatment of diseases infected by EV71, and related vaccines are marketed in 2015, and their preventive effects are yet to be further investigated. Therefore, the development of specific and effective anti-EV 71 medicaments is imperative.

Iodine-containing carboxylic acids are a class of organic compounds with biological activity. For example, p-iodobenzoic acid is a potent inhibitor of cinnamate-4-HYDROXYLASE (CINNAMATE 4-HYDROXYLASE), a key enzyme in the phenylpropanoid pathway for the synthesis of lignin building blocks (Dorien Van de Wouwer, et al. plant Physiology,2016,172, 198-220); the metal salt of m-iodobenzoic acid shows better inhibitory activity to Saccharomyces cerevisiae, Hansenula anomala, Escherichia coli and Bacillus subtilis (P.Koczon, et.al.J.Agric.food chem.2001,49, 2982-2986). For example, 2,3, 5-triiodobenzoic acid (TIBA) is an excellent plant growth regulator (asherman, et al. plant physiological communications, 1958,3, 27-30). 3, 5-bis (acetamido) -2,4, 6-triiodobenzoic acid, also known as diatrizoic acid (diatrizoic acid), is an important contrast agent, and is prepared into diatrizoate sodium and diatrizoate meglumine injection, which can be used for imaging urinary system, cardiovascular system, cerebrovascular system and peripheral blood vessels (I Charles, et al, 1986, US 4567034). However, the antiviral activity of iodine-containing carboxylic acids, including the inhibitory activity against the EV71 virus, has not been reported so far.

Disclosure of Invention

The invention aims to provide application of multi-iodo iodic carboxylic acid in resisting EV71 virus, wherein the multi-iodo iodic carboxylic acid comprises 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7), and the multi-iodo iodic carboxylic acid L6 and L7 have an inhibiting effect on EV71 virus, have potential preparation of specific treatment medicines for resisting EV71 infection, and have a great clinical application prospect.

The invention adopts the following technical scheme:

the application of multi-iodo iodic carboxylic acid in resisting EV71 virus comprises 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7), wherein the chemical structural formulas of L6 and L7 are shown as follows,

further, the multi-iodo iodocarboxylic acid is used as an anti-EV 71 virus drug, and when the concentrations of the multi-iodo iodocarboxylic acid L6 and L7 are both 40 mug/mL, the inhibition rates of the multi-iodo iodocarboxylic acid on EV71 virus are 46% and 64.8%, respectively.

Further, any one of the multi-iodo iodocarboxylic acids L6 and L7 is used as an EV71 virus inhibitor, and is added with pharmaceutically acceptable auxiliary materials and carriers to prepare a preparation for resisting EV71 virus.

Further, the pharmaceutical preparation is any one of granules, tablets, pills, capsules, injections or dispersing agents.

The invention evaluates the inhibitory activity of a series of multi-iodo iodocarboxylic acids including 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7) on EV71 virus through a plurality of biological experiments, proves that the multi-iodo iodocarboxylic acids including 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7) have the activity of resisting EV71 virus, particularly show that the multi-iodo iodocarboxylic acids can inhibit the cytopathic effect caused by EV71 virus, enhance the survival rate of infected cells, inhibit the replication and proliferation of 71 virus in cells, and the inhibitory effect of L6 and L7 on EV71 is better than that of ribavirin which is a positive control medicament, wherein the L7 has the best inhibitory effect, the inhibitory rate of L71 is 64.8 percent at the concentration of 40 mug/mL, and the compounds L6 and/or L7 have the potential of preparing specific therapeutic drugs for resisting EV71 infection, has a wide clinical application prospect.

Compared with the prior art, the invention has the following beneficial effects:

(1) the multi-iodo iodic carboxylic acid 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7) have simple synthesis processes and are easy to produce and popularize on a large scale.

(2) The antiviral activity of the multi-iodo iodic carboxylic acid is not reported, and the multi-iodo iodic carboxylic acid has a certain guiding effect on the development of the anti-EV 71 virus activity.

(3) An anti-EV 71 drug is searched from a compound with a similar structure, so that an action target of the anti-EV 71 drug is easy to find through structure-activity relationship research, and a certain reference significance is provided for further drug development.

Drawings

FIG. 1 is a graph showing the effect of polyiodinated iodocarboxylic acids L6, L7 on RD cell survival of EV71 action in example 1 of the present invention;

FIG. 2 shows the inhibitory effect of polyiodinated iodocarboxylic acids L6 and L7 on RD cell CPE caused by EV71 in example 1 of the present invention;

FIG. 3 shows the inhibitory effect of poly-iodo iodocarboxylic acid L7 on the yield of EV71 progeny virus in example 2 of the present invention.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. In the following examples, materials and methods of operation used in the following examples are well known in the art, unless otherwise specified.

The invention researches the inhibition activity of multi-iodo iodic carboxylic acid L6 and L7 against EV71 virus, and L6 and L7 inhibit the cytopathic effect (CPE) of EV71 in host cells RD, enhance the cell survival rate, inhibit the replication and proliferation of EV71 virus in cells, and have the potential to be developed into a medicament for effectively treating EV71 infection.

The invention relates to application of polyiodinated iodocarboxylic acid L6 and L7 in preparation of an anti-EV 71 virus medicament. The application refers to that pharmaceutically acceptable auxiliary materials and carriers are added into multi-iodo iodic carboxylic acid L6 and L7 to prepare the preparation for resisting EV71 viruses. The preparation is granule, tablet, pill, capsule, injection or dispersant.

[ example 1 ] analysis of anti-EV 71 Virus Activity of polyiodiiodocarboxylic acids L6 and L7

1. The test contents are as follows:

the inhibition activity of multi-iodo iodocarboxylic acids L6 and L7 on EV71 virus is evaluated by combining a cytopathic effect analysis method and an MTT (methyl thiazolyl tetrazolium) determination cell survival rate detection method.

2. The test method comprises the following steps:

2.1.1 toxicity of Polyiodoform iodocarboxylic acids L6, L7 to host RD cells

RD cells were plated in 96-well plates at 37 ℃ with 5% CO₂After the culture box is cultured to grow a monolayer, cell culture solution is discarded, cell maintenance solutions containing multi-iodo iodocarboxylic acid L6 and L7 with different concentrations are respectively added for continuous culture, the cytotoxicity is visually observed and respectively recorded by a microscope after 48 hours, and the cell survival rate is determined by an MTT method. The SPSS11.5 software calculates the Median cytoxicity concentration (CC 50) of drug (polyiodinated iodocarboxylic acid L6, L7) cells. Cell survival rate ═ (mean OD of drug groups)₄₉₀Value/cell control mean OD₄₉₀Value) × 100%.

2.1.2 inhibitory Activity of Polyiodoform-containing Carboxylic acids L6, L7 on EV71

RD cells were plated in 96-well plates at 37 ℃ with 5% CO₂After the culture box is cultured to grow a full monolayer, the culture solution is discarded, and cells are infected by EV71 virus solution of 100TCID50 for 1h and incubated by ribavirin serving as a positive control drug. After the culture is continued for about 48 hours, the cytopathic effect (CPE) is observed under a microscope when about 90% of CPE lesions appear in the virus control wells. Observation and recording method of CPE: no cytopathic effect is recorded as-below 25% cytopathic effect, 25% -50% cytopathic effect is recorded as +++, 50% -75% cytopathic effect is recorded as +++, and more than 75% cytopathic effect is recorded as ++++.

After CPE observation is finished, the inhibition rate of the drug on EV71 is detected by using an MTT method. The method comprises the following specific steps: mu.L of MTT (5 mg. multidot.mL-1) was added to each well, and after incubation for 3-4h, the supernatant was removed and an equal volume of DMSO was added to dissolve the precipitate. The absorbance (OD) at 490nm was read with a microplate reader₄₉₀Value). The inhibition rate of the drug on EV71 was calculated using the following formula. Using SPSS11.5 software meterCalculate the half effective Concentration of the drug (Concentration for 50% of maximum effect, EC 50).

2.1.3 Therapeutic Index (TI) of Polyiodocarboxylic acids (L6, L7)

TI CC50/EC 50. A higher therapeutic index indicates greater antiviral potential.

3. Test results

TABLE 1 Polyiodoform iodocarboxylic acid cytotoxicity and anti-EV 71 Activity

And (4) analyzing results: (1) the test results of the cytotoxicity of the multi-iodo iodic carboxylic acids L6 and L7 and the activity of anti-EV 71 are shown in Table 1, and the influence of the concentration-dependent multi-iodo iodic carboxylic acids L6 and L7 on the survival rate of RD cells with EV71 action is shown in figure 1, the invention detects that the multi-iodo iodic carboxylic acids L6 and L7 have certain inhibitory activity on EV71, have higher therapeutic index and are superior to a positive control medicament ribavirin, wherein L7 has the best inhibitory effect, and when the concentration is 40 mug/mL, the inhibitory rate on EV71 is 64.8%; (2) as shown in fig. 2, RD cells infected with EV71 become rounded and detached from the cell plate wall, and 40 μ g/mL L6 and L7 treatments have different degrees of inhibitory effects on the pathological effects of RD cells caused by inhibition of EV71 by L6 and L7. The results suggest that the polyiodinated iodocarboxylic acids L6 and L7 strongly inhibit the cytopathic effect (CPE) of the EV71 in the host cell RD, enhance the cell survival rate, strongly inhibit the replication and proliferation of the EV71 virus in the cells, and have the potential to be developed into a medicine for effectively treating EV71 infection.

Example 2 test of inhibitory Effect of Polyiodocarboxylic acid L7 on yield of EV71 progeny Virus

1. Content of the experiment

The inhibition effect of multi-iodo iodocarboxylic acid L7 on the yield of EV71 progeny virus after EV71 infects RD cells was tested.

2. Test method

RD cells in logarithmic growth phase are plated on a 24-well plate, 100TCID50 EV71 infected cells grow into a monolayer, virus solution is removed after incubation for 1.5h at 37 ℃, PBS is washed for three times, and cell maintenance solution containing 40 mu g/mL multi-iodo iodocarboxylic acid L7 is added. After 36h, cells and supernatant culture were collected, and EV71 virus titer was determined by the TCID50 method after three freeze-thaw lysis at-20 ℃ and 37 ℃.

3. Test results

As shown in fig. 3, the virus titer of the RD cells treated with the multi-iodo iodocarboxylic acid L7 was significantly reduced compared to the virus control group, indicating that the multi-iodo iodocarboxylic acid L7 has a significant inhibitory effect on the yield of EV71 progeny virus.

In conclusion, the polyiodinated iodocarboxylic acids L6 and L7 have certain EV71 inhibiting activity, wherein the polyiodinated iodocarboxylic acid L7 has the best inhibiting effect, can strongly inhibit RD cytopathic effect caused by EV71 virus, enhance the cell survival rate, remarkably reduce the yield of progeny virus, and have the potential to further develop and prepare a medicament for clinically and effectively resisting EV71 virus infection.

Claims

1. The application of polyiodinated iodic carboxylic acid in resisting EV71 virus is characterized in that: the multi-iodo iodic carboxylic acid comprises 3, 5-diiodosalicylic acid (L6) and 2,3, 5-triiodobenzoic acid (L7), wherein the chemical structural formulas of L6 and L7 are shown as follows,

2. the use of claim 1, wherein: the polyiodinated iodocarboxylic acid is used as an anti-EV 71 virus drug, and when the concentrations of polyiodinated iodocarboxylic acid L6 and L7 are both 40 mug/mL, the inhibition rates of the polyiodinated iodocarboxylic acid to EV71 virus are respectively 46% and 64.8%.

3. Use according to claim 2, characterized in that: any one of the polyiodinated iodocarboxylic acid L6 and L7 is used as an EV71 virus inhibitor, and is added with pharmaceutically acceptable auxiliary materials and carriers to prepare a preparation for resisting EV71 virus.

4. Use according to claim 3, characterized in that: the pharmaceutical preparation is any one of granules, tablets, pills, capsules, injections or dispersing agents.