Application of hemicyclane-type diterpenoids in the treatment of enterovirus 71

Abstract

The present invention discloses the use of a diterpenoid compound in the treatment of enterovirus 71. Experimental studies on the anti-EV71 activity of atralabdan-type diterpenoid compounds, including atralabdan A, atralabdan B, and atralabdan C, have shown that the compounds inhibit the cytopathic effect (CPE) produced by EV71 on host cell RD, enhance cell survival, and reduce progeny virus production, demonstrating their potential for use in the preparation of anti-EV71 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), a member of the Enterovirus genus in the Picornaviridae family, is one of the most common pathogens causing hand, foot, and mouth disease (HFMD) in infants and young children. This disease is sometimes accompanied by severe central nervous system complications, including aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurological cardiopulmonary failure, and even death. Since its first report in 1974, EV71 has caused numerous outbreaks and epidemics worldwide. In 2008, the Chinese government designated HFMD a Category C infectious disease and enacted a series of relevant laws and regulations to strictly control its spread. Currently, there are no specific treatments for EV71-related diseases, and a vaccine was only released in 2015, and its effectiveness in preventing them remains to be determined. Therefore, the development of specific and effective anti-EV71 drugs is imperative.

[0003] Atralabdan A, atralabdan B and atralabdan C belong to the class of diterpenoid compounds of the genus Atralabdan. 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 anti-tumor activity. It effectively exerts its anti-tumor effect by interfering with the mitosis 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 dilating blood vessels, improving microcirculation, and protecting nerve cells. Ginkgolide shows good application prospects in the treatment of cardiovascular and cerebrovascular diseases, Alzheimer's disease, etc. Triptolide has multiple pharmacological effects such as anti-inflammatory, immunosuppressive, and anti-tumor, and has potential application value in the treatment of rheumatic diseases, kidney diseases, etc. [4]. However, although diterpenoids have shown significant pharmacological activity in multiple fields, there are few reports on their inhibitory activity against enterovirus 71 (EV71). Summary of the Invention

[0004] In view of the above situation, the present invention aims to provide a use of atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds, in the treatment 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 cyperane-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:

[0007] In a first aspect, the present invention provides atralabdan A, atralabdan B and atralabdan C, a cymene-type diterpenoid compounds.

[0008] The chemical structure of atralabdan A is shown in formula (I):

[0009] Formula (I).

[0010] The chemical structure of atralabdan B is shown in formula (II):

[0011] Formula (II).

[0012] The chemical structure of atralabdan C is shown in formula (III):

[0013] Formula (III).

[0014] In a second aspect, the present invention provides an application of the above-mentioned anthraquinone natural product in anti-EV71, characterized in that the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C have anti-EV71 viral activity.

[0015] 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%.

[0016] 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.

[0017] Furthermore, the preparation is any one of granules, tablets, pills, capsules, injections or dispersions.

[0018] The object of the present invention is achieved by testing the activity of the atralabdan-type diterpenoid compounds atralabdan A, atralabdan B and atralabdan C using a standard viral activity test method.

[0019] Through extensive biological experiments, the present invention discovered that the cyperane-type diterpenoid compounds atralabdan A, atralabdan B, and atralabdan C exhibit anti-EV71 viral activity. Specifically, they inhibited the cytopathic effect caused by the EV71 virus, enhanced the survival rate of infected cells, inhibited the replication and proliferation of the EV71 virus within cells, and reduced the production of progeny viruses. This suggests that this series of compounds has the potential to be used as a specific therapeutic agent against EV71 infection and has great clinical application prospects.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. There is no report on the anti-picornaviral activity of hemicyclane-type diterpenoids, which has a certain guiding role in the development of their anti-EV71 virus activity.

[0022] 2. Searching for anti-EV71 drugs from compounds with similar structures can easily identify their targets through structure-activity relationship studies, providing a certain reference for further drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effects of atralabdan A, atralabdan B and atralabdan C, a type of diterpenoid compounds, on the survival rate of RD cells infected with EV71 were studied.

[0024] Figure 2 The inhibitory effect of the hemi-petroleum diterpenoid compounds atralabdan A, atralabdan B and atralabdan C on EV71-induced CPE of RD cells.

[0025] Figure 3It is the inhibitory effect of the hemi-petroleum diterpenoid compounds atralabdan A, atralabdan B and atralabdan C on the production of EV71 progeny viruses. DETAILED DESCRIPTION

[0026] The diterpenoid natural products atralabdan A, atralabdan B and atralabdan C used in the present invention were 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.

[0027] The present invention discloses the use of the cymene-type diterpenoid compounds atralabdan A, atralabdan B, and atralabdan C as antiviral agents against EV71. These compounds inhibit the cytopathic effect (CPE) produced by EV71 in host cells (RD), enhancing cell survival. They also strongly inhibit EV71 replication and proliferation within cells, reducing progeny virus production. These studies demonstrate the potential of these compounds as effective therapeutics for EV71 infection.

[0028] Example 1

[0029] Experimental study on the anti-EV71 activity of atralabdan A, atralabdan B and atralabdan C, a semi-floral diterpenoid compounds.

[0030] 1. Test content:

[0031] Analysis of compound anti-EV71 activity: The present invention combines cytopathic effect analysis and MTT cell viability assay to evaluate the anti-EV71 activity of the cyperane-type diterpenoid compounds atralabdan A, atralabdan B, and atralabdan C.

[0032] 2. Test method:

[0033] 2.1.1 Toxicity of compounds to host RD cells

[0034] RD cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator until a confluent monolayer was formed. The cell culture medium was discarded and cell maintenance medium containing different concentrations of the test compound was added and cultured continuously. After 48 h, the cytotoxicity was observed and recorded under a microscope. The cell survival rate was determined by the MTT assay. The median toxic concentration (CC) of the drug for the cells was calculated using SPSS 11.5 software. 50 ). Cell viability = (mean OD of drug group 492 Value / average OD of cell control group 492 value) × 100%.

[0035] 2.1.2 Inhibitory activity of compounds against EV71

[0036] RD cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator until confluent monolayers were achieved. The culture medium was then discarded and the cells were infected with 100 TCID50 of EV71 virus for 1.5 hours. The cells were then incubated with varying concentrations of the test compound (ribavirin was used as a positive control). After approximately 48 hours of incubation, when approximately 90% of the virus control wells showed CPE, the cells were observed microscopically. CPE was recorded as follows: no cytopathic effect (CPE) was scored as -, less than 25% cytopathic effect (CPE) as +, 25%-50% cytopathic effect (CPE) as ++, 50%-75% CPE as +++, and greater than 75% CPE as ++++.

[0037] 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 of MTT (5 mg mL -1 After incubation for 3-4 h, the supernatant was removed and an equal volume of DMSO was added to dissolve the precipitate. The corresponding absorbance (OD) was read at 492 nm using a microplate reader. 492 The inhibition rate of the drug on EV71 was calculated using the following formula. The concentration for 50% of maximal effect (EC) was calculated using SPSS 11.5 software. 50 ).

[0038] 2.1.3 Therapeutic Index (TI) of Drugs

[0039] TI = CC 50 / EC 50 The higher the therapeutic index, the greater the antiviral potential.

[0040] 3. Experimental results

[0041] Table 1 Cytotoxicity and anti-EV71 activity of cymanane-type diterpenoids

[0042]

[0043] 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 induced by EV71 are shown in Table 1. Figure 1 The present invention detected that the cyperane-type diterpenoid compounds atralabdan A, atralabdan B, and atralabdan C had certain inhibitory activity against EV71. When the concentration of atralabdan A was 6.25 μM / mL, the inhibition rate against EV71 reached 65%; when the concentration of atralabdan B was 12.5 μM / mL, the inhibition rate against EV71 reached 75%; and when the concentration of atralabdan C was 10.3 μM / mL, the inhibition rate against EV71 reached 70%.

[0044] Example 2

[0045] Experiment on the inhibitory effect of atralabdan A, atralabdan B and atralabdan C, a type of diterpenoid compounds, on the production of EV71 progeny viruses.

[0046] This example conducts an in-depth study on the anti-EV71 activity of the cyperane-type diterpenoid compounds atralabdan A, atralabdan B, and atralabdan C. Tests were conducted on the inhibitory effects of atralabdan A, atralabdan B, and atralabdan C on the production of EV71 progeny viruses. The test results are as follows:

[0047] 1. Test content

[0048] The inhibitory effects of atralabdan A, atralabdan B, and atralabdan C on the production of EV71 progeny viruses were tested after EV71 infection of RD cells.

[0049] 2. Test methods

[0050] RD cells in the logarithmic growth phase were plated in 24-well plates, and 100 TCID 50EV71 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. The TCID 50 Methods The EV71 virus titer was determined.

[0051] 3. Test results

[0052] like Figure 2 As 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.

[0053] like Figure 3 As shown, the viral titers of RD cells treated with the hemi-petroleum diterpenoid compounds atralabdan A, atralabdan B and atralabdan C were significantly reduced compared with the virus control group, with a decrease of 3log, 2.5log and 2.2log respectively compared with the virus control.

[0054] In summary, the hemi-petroleum 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. They have the potential to prepare a clinically effective drug against EV71 infection.

Claims

1. A use of a cymene-type diterpenoid compound in the preparation of an anti-enterovirus 71 drug, characterized in that: The atralabdan-type diterpenoid compound is selected from atralabdan A, atralabdan B and atralabdan C; 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. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The medicine is any one of granules, tablets, pills, capsules, injections or dispersants.

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