Application of labdane diterpenoid compound in resisting enterovirus 71

Abstract

The invention discloses an application of a diterpenoid compound in resisting enterovirus 71 (EV71). Through anti-EV71 virus activity research experiments of labdane type diterpenoid compounds including atralaabdan A, atralaabdan B and atralaabdan C, the compounds atralaabdan A, atralaabdan B and atralaabdan C can inhibit cytopathic effect (CPE) generated by EV71 on host cells RD, enhance the cell survival rate and reduce the progeny virus yield, and the labdane type diterpenoid compounds have the potential to be applied to preparation of anti-EV71 virus drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral drugs, and specifically refers to the use of atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds, in resisting enterovirus 71. Background Art

[0002] Enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family. It is one of the most important pathogens causing hand, foot and mouth disease in infants and young children, sometimes accompanied by serious central nervous system complications, including aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurological cardiopulmonary failure, etc., and even death. Since its first report in 1974, EV71 infectious diseases have broken out and spread many times around the world. In 2008, the Chinese government classified hand, foot and mouth disease as a Class C infectious disease and included it in management, and formulated a series of relevant laws and regulations to strictly control the spread of hand, foot and mouth disease. At present, there is no specific drug for the treatment of diseases infected by EV71. The relevant vaccine was only launched in 2015, and its preventive effect needs further investigation. Therefore, it is imperative to develop specific and effective anti-EV71 drugs.

[0003] Atralabdan A, atralabdan B and atralabdan C belong to the class of diterpenoid compounds. Diterpenoid compounds are widely present in nature and exhibit a variety of biological activities. Among them, paclitaxel, as a commonly used diterpenoid drug in clinical practice, is well-known for its significant antitumor activity. It effectively exerts its antitumor effect by interfering with the mitotic process of tumor cells and has been widely used in the treatment of various malignant tumors. Andrographolide has multiple pharmacological effects such as anti-inflammatory, antiviral, antibacterial and immunomodulatory, and is often used to treat inflammatory diseases such as colds, pharyngitis, and gastroenteritis. Ginkgolide has multiple pharmacological effects such as vasodilation, improvement of microcirculation, and protection of nerve cells. Ginkgolide has good application prospects in the treatment of cardiovascular and cerebrovascular diseases and Alzheimer's disease. Triptolide has multiple pharmacological effects such as anti-inflammatory, immunosuppressive and anti-tumor, and has potential application value in the treatment of rheumatic diseases and kidney diseases [4]. However, although diterpenoids have shown significant pharmacological activities in many fields, there are few reports on their inhibitory activity against enterovirus 71 (EV71). Summary of the invention

[0004] The purpose of the present invention is to provide a use of atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds, in the prevention of enterovirus 71. In the scheme of the present invention, the cymene-type diterpenoid compounds are used as EV71 virus screening compounds.

[0005] In the scheme of the present invention, the inhibitory activity of the hemipentane-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C against EV71 virus was evaluated.

[0006] To achieve the above object, the scheme of the present invention is as follows: In a first aspect, the present invention provides atralabdan A, atralabdan B and atralabdan C, which are cymene-type diterpenoid compounds.

[0007] The chemical structure of atralabdan A is shown in formula (I): Formula (I).

[0008] The chemical structure of atralabdan B is shown in formula (II): Formula (II).

[0009] The chemical structure of atralabdan C is shown in formula (III): Formula (III).

[0010] In a second aspect, the present invention provides an application of the above-mentioned anthraquinone natural products in anti-EV71, characterized in that the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C have anti-EV71 viral activity.

[0011] As a preferred embodiment, when the concentration of the compound atralabdan A is 6.25 μM / mL, the inhibition rate of EV71 reaches 65%; when the concentration of atralabdan B is 12.5 μM / mL, the inhibition rate of EV71 reaches 75%; when the concentration of atralabdan C is 10.3 μM / mL, the inhibition rate of EV71 reaches 70%.

[0012] Furthermore, the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C are used as EV71 virus inhibitors and are used to prepare anti-EV71 virus preparations after adding pharmaceutically acceptable excipients and carriers.

[0013] Furthermore, the preparation is any one of granules, tablets, pills, capsules, injections or dispersions.

[0014] The object of the present invention is achieved in that the activity of the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C is tested by a standard virus activity test method.

[0015] Through a large number of biological experiments, the present invention finds that the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C have anti-EV71 virus activity. Specifically, they can inhibit the cytopathic effect caused by the EV71 virus, enhance the survival rate of infected cells, inhibit the replication and proliferation of the EV71 virus in cells, and reduce the yield of progeny viruses. This shows that this series of compounds has the potential to prepare specific therapeutic drugs against EV71 infection and has great clinical application prospects.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The anti-picornavirus activity of hemipenem-type diterpenoids has not been reported, but it has a certain guiding role in the development of their anti-EV71 virus activity.

[0017] 2. It is easy to find anti-EV71 drugs from compounds with similar structures through structure-activity relationship research to find their targets, which provides certain reference significance for further drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of atralabdan A, atralabdan B and atralabdan C, a semi-floral diterpenoid compounds, on the survival rate of RD cells treated with EV71.

[0019] Figure 2 The inhibitory effect of the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C on EV71-induced CPE of RD cells.

[0020] Figure 3The inhibitory effect of the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C on the production of EV71 progeny viruses. DETAILED DESCRIPTION

[0021] The diterpenoid natural products atralabdan A, atralabdan B and atralabdan C used in the present invention are obtained by genome mining strategy. Streptomyces atratus A terpenoid gene cluster in Gö66 was heterologously expressed in S. albus J1074 and isolated from a liquid fermentation extract of S. albus J1074 containing the terpenoid gene cluster.

[0022] The present invention discloses atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds, as anti-EV71 virus applications. Atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds, inhibit the cytopathic effect (CPE) produced by EV71 in host cell RD and enhance cell survival rate. They strongly inhibit the replication and proliferation of EV71 virus in cells and reduce the yield of progeny viruses. These studies indicate that these compounds have the potential to be developed into effective drugs for treating EV71 infection.

[0023] Example 1 Experimental study on the anti-EV71 activity of atralabdan A, atralabdan B and atralabdan C, a semi-floral diterpenoid compounds.

[0024] 1. Test content: Analysis of the anti-EV71 activity of compounds: The present invention combines cytopathic effect analysis and MTT cell viability assay to evaluate the anti-EV71 activity of the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C.

[0025] 2. Test methods: 2.1.1 Toxicity of compounds to host RD cells RD cells were plated on 96-well plates and cultured in a 37°C, 5% CO2 incubator until the cells grew into a monolayer. The cell culture medium was discarded and cell maintenance medium containing different concentrations of the test compound was added to continue the culture. After 48 h, the cytotoxicity was observed and recorded under a microscope. The cell survival rate was determined by the MTT method. SPSS 11.5 software was used to calculate the median toxic concentration (CC) of the drug for cells.50 ). Cell survival rate = (mean OD of drug group 492 Value / average OD of cell control group 492 value) × 100%.

[0026] 2.1.2 Inhibitory activity of compounds against EV71 RD cells were plated on 96-well plates and cultured in a 37°C, 5% CO2 incubator until the monolayer was full. The culture medium was discarded, and the cells were infected with 100TCID50 EV71 virus solution for 1.5 hours. Different concentrations of test compounds (ribavirin as a positive control drug) were added to incubate the cells. After continuing to culture for about 48 hours, when about 90% CPE lesions appeared in the virus control wells, the cytopathic effect (CPE) was observed under a microscope. The observation and recording method of CPE: no cytopathic effect is recorded as -, cytopathic effect below 25% is recorded as +, cytopathic effect between 25% and 50% is recorded as ++, cytopathic effect between 50% and 75% is recorded as +++, and cytopathic effect above 75% is recorded as +++.

[0027] After the CPE observation was completed, the MTT method was used to detect the inhibition rate of the drug on EV71. The specific steps were as follows: 50 μL (5 mg mL -1 ), incubate for 3-4 h, remove the supernatant, add an equal volume of DMSO to dissolve the precipitate. Read the corresponding absorbance (OD) at 492 nm using an enzyme reader. 492 The inhibition rate of the drug on EV71 was calculated using the following formula. The half effective concentration (EC) of the drug was calculated using SPSS 11.5 software. 50 ).

[0028] 2.1.3 Therapeutic Index (TI) of Drugs TI = CC 50 / EC 50 The higher the therapeutic index, the greater the antiviral potential.

[0029] 3. Experimental results Table 1 Cytotoxicity and anti-EV71 activity of cymene-type diterpenoids

[0030] The results of the compound cytotoxicity and anti-EV71 activity tests are shown in Table 1. The concentration-dependent effects of the compounds on the survival rate of RD cells acted upon by EV71 are shown in Table 1. Figure 1The present invention detected that the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C have certain inhibitory activity against EV71. When the concentration of the compound atralabdan A is 6.25 μM / mL, the inhibition rate against EV71 reaches 65%; when the concentration of atralabdan B is 12.5 μM / mL, the inhibition rate against EV71 reaches 75%; when the concentration of atralabdan C is 10.3 μM / mL, the inhibition rate against EV71 reaches 70%.

[0031] Example 2 Experiment on the inhibitory effect of atralabdan A, atralabdan B and atralabdan C, a semi-floral diterpenoid compounds, on the production of EV71 progeny viruses.

[0032] This example conducts an in-depth study on the anti-EV71 activity of the cymene-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C, and implements a test on the inhibitory effect of atralabdan A, atralabdan B and atralabdan C on the production of EV71 progeny viruses. The test results are as follows: 1. Test content The inhibitory effects of atralabdan A, atralabdan B and atralabdan C on the production of EV71 progeny viruses after EV71 infection of RD cells were tested.

[0033] 2. Test methods RD cells in the logarithmic growth phase were plated in 24-well plates. After the cells grew to a confluent monolayer, 100 TCID 50 EV71 infected cells were incubated at 37°C for 1.5 h, then the virus solution was removed, washed three times with PBS, and cell maintenance medium containing 6.25 μM / mL atralabdan A, 12.5 μM / mL atralabdan B, and 10.3 μM / mL atralabdan C was added. After 48 h, the cells and supernatant culture medium were collected and lysed three times at -20°C and 37°C, and the TCID 50 Methods The EV71 virus titer was determined.

[0034] 3. Test results like Figure 2As shown, the cell survival rate of RD cells infected with EV71 treated with the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C was significantly increased compared with the virus control group, indicating that the above compounds can significantly inhibit the activity of EV71.

[0035] like Figure 3 As shown, the viral titers of RD cells treated with the semi-floral diterpenoid compounds atralabdan A, atralabdan B and atralabdan C were significantly reduced compared with the virus control group, which were reduced by 3log, 2.5log and 2.2 log respectively.

[0036] In summary, the hemiaran-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C have strong inhibitory activity against EV71, can inhibit RD cell death caused by EV71 virus, and significantly reduce the production of progeny viruses, and have the potential to prepare a clinically effective drug against EV71 infection.

Claims

1. A hemi-florin-type diterpenoid compound, comprising atralabdan A, atralabdan B and atralabdan C, Features: The chemical structure of atralabdan A is shown in formula (I): Formula (I); The chemical structure of atralabdan B is shown in formula (II): Formula (II); The chemical structure of atralabdan C is shown in formula (III): Formula (III).

2. A use of the cymene-type diterpenoid compound as claimed in claim 1 in resisting enterovirus 71, Features: The cymene-type diterpenoid compound has anti-enterovirus 71 (EV71) virus activity.

3. The use of the cymene-type diterpenoid compound according to claim 2 in resisting EV71 virus, Features: When the concentration of the compound atralabdan A is 6.25 μM / mL, the inhibition rate of EV71 reaches 65%; when the concentration of atralabdan B is 12.5 μM / mL, the inhibition rate of EV71 reaches 75%; when the concentration of atralabdan C is 10.3 μM / mL, the inhibition rate of EV71 reaches 70%.

4. The use of the hemimeric diterpenoid compound according to claim 3 in resisting EV71 virus, Features: The cymene-type diterpenoid compounds are used as EV71 virus inhibitors and are used to prepare anti-EV71 virus preparations after adding pharmaceutically acceptable excipients and carriers.

5. Use of the cymene-type diterpenoid compound according to claim 4 in resisting enterovirus 71, Features: The preparation is any one of granules, tablets, pills, capsules, injections or dispersions.

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