Application of polyketide natural product compounds in the preparation of CVB3 virus drugs

Abstract

This invention discloses the use of polyketide natural product compounds: (3S,4S)-Sclerotinin A and Ligustrone B, in the preparation of anti-Coxsackievirus B3 (CVB3) drugs. Experimental studies of the anti-CVB3 activity of (3S,4S)-Sclerotinin A and Ligustrone B showed that both polyketide natural product compounds inhibited the cytopathic effect (CPE) produced by CVB3 in Hep-2 host cells, enhanced cell survival, and reduced progeny virus production, demonstrating their potential for use in the preparation of anti-CVB3 drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral drugs, and in particular to the application of two polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B in anti-CVB3 virus drugs. Background Art

[0002] Coxsackieviruses are members of the Enterovirus genus of the Picornaviridae family. Infection with coxsackieviruses can cause a variety of diseases, including hand, foot, and mouth disease, aseptic meningitis, encephalitis, myocarditis, myositis herpeticum, and herpangina. There are 29 reported serotypes of coxsackieviruses, which can be divided into two groups, A and B, based on their pathogenicity in suckling mice and their cellular sensitivities: CVA (CVA1-22, 24) and CVB (CVB1-6). Infection with CVBs is the most common, with CVB3 being the most pathogenic of the six CVB serotypes and the leading cause of viral myocarditis. According to statistics from the US Centers for Disease Control and Prevention (CDC), CVB (types 1-6) cause approximately 5 million cases of enteric disease annually, 10%-20% of which are caused by acute myocarditis caused by CVB3. In recent years, the incidence of hand, foot, and mouth disease (HFMD) caused by CVB3 has been increasing, with numerous reports of CVB3-induced epidemics in my country. Currently, there are no specific drugs for coxsackievirus infection, and no targeted clinical treatments are available. Many researchers have identified numerous compounds that inhibit CVB3 activity in vitro and in vivo, but these are still in the early stages of laboratory testing and are far from practical clinical application. Therefore, the development of specific and effective anti-CVB3 drugs is imperative.

[0003] Polyketides are a class of secondary metabolites produced by plants or microorganisms. They are widely found in nature, with tens of thousands of species discovered to date. Polyketides have complex structures and possess a variety of biological activities, including antibacterial, antitumor, and antioxidant properties. They are widely used in agriculture, food industry, and healthcare. (3S,4S)-Sclerotinin A is a natural isocoumarin product that was first isolated from Penicillium citrinum ( Penicillium citrinum ) and Sclerotinia sclerotiorum ( Sclerotinia Sclerotiorum). As a plant growth regulator, (3S,4S)-Sclerotinin A promotes the growth of rice seedlings at a concentration of 5 ppm (Curtis et al. J. Chem. Soc. C. 1968, 85-93; Sassa et al. Agric. Biol. Chem. 1968, 32, 1432-1439). Ngan et al. found that (3S,4S)-Sclerotinin A has little anti-inflammatory activity (Ngan et al. Arch. Pharm. Res. 2017, 40, 328-337). Ligustrone B is a rare fungal metabolite with a chromone and indone structure in its skeleton. As the main product of the plant pathogen Cercospora ligustrina, the biological activity of Ligustrone B has been rarely reported (Arnone et al. Gazz. Chim. Ital. 1975, 105, 1093).

[0004] We evaluated the inhibitory activity of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B against CVB3 virus. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing an anti-Coxsackievirus B3 drug based on a polyketide natural product compound in response to the above situation.

[0006] The polyketide natural product compounds include (3S, 4S)-Sclerotinin A and Ligustrone B.

[0007] The chemical structure of (3S,4S)-Sclerotinin A is shown in formula (I):

[0008] Formula (I).

[0009] The chemical structural formula of Ligustrone B is shown in formula (II):

[0010] Formula (II).

[0011] The purpose of the present invention is achieved by testing the activity of polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B by combining cytopathic effect (CPE) inhibition analysis with MTT assay to determine cell viability.

[0012] Through extensive biological experiments, the applicants discovered that the polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B have anti-CVB3 virus activity. Both compounds can strongly inhibit the cytopathic effect caused by the CVB3 virus and enhance the survival rate of infected cells. They also strongly inhibit the replication and proliferation of the CVB3 virus in cells and reduce the production of progeny viruses. This suggests that the polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B have the potential to be used as specific therapeutic agents against CVB3 infection and have great clinical application prospects.

[0013] The present invention has the following advantages:

[0014] 1. The extraction process of these compounds is simple, economical and rapid, and easy to promote large-scale production.

[0015] 2. The antiviral activity of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B has not been reported, which has a certain guiding role in the development of their anti-CVB3 viral activity.

[0016] 3. Searching for anti-CVB3 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

[0017] Figure 1 The effects of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B on the survival rate of Hep-2 cells treated with CVB3.

[0018] Figure 2 The inhibitory effect of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B on CPE of Hep-2 cells induced by CVB3.

[0019] Figure 3 The inhibitory effect of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B on the production of CVB3 progeny virus. DETAILED DESCRIPTION

[0020] The polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B used in the present invention are separated from the rice solid fermentation product of Talaromyces funiculosus R5SSF2.

[0021] The present invention relates to the application of polyketide-containing natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B in the preparation of anti-CVB3 virus drugs.

[0022] The application refers to a preparation containing (3S, 4S)-Sclerotinin A and Ligustrone B and adding pharmaceutically acceptable excipients and carriers for preparing an anti-CVB3 virus preparation.

[0023] The preparation is granules, tablets, pills, capsules, injections or dispersions.

[0024] The (3S,4S)-Sclerotinin A of the present invention has the chemical structure shown in formula (I):

[0025] Formula (I).

[0026] Ligustrone B of the present invention has the chemical structural formula shown in formula (II):

[0027] Formula (II).

[0028] The present invention discloses compounds containing (3S,4S)-Sclerotinin A and Ligustrone B as antiviral agents against CVB3. These compounds potently inhibit the cytopathic effect (CPE) produced by CVB3 in Hep-2 host cells, enhancing cell survival. They also strongly inhibit CVB3 viral replication and proliferation within cells, reducing progeny viral production. These studies demonstrate the potential for further development of these compounds into effective treatments for CVB3 infection.

[0029] The applicant has conducted an anti-CVB3 activity study experiment on the above compounds, and the experimental results are as follows: In the following, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art.

[0030] 1. Test content:

[0031] Analysis of compound anti-CVB3 activity: The present invention combines cytopathic effect analysis and MTT cell viability assay to evaluate the anti-CVB3 activity of polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B.

[0032] 2. Test method:

[0033] 2.1.1 Toxicity of polyketide natural product compounds to host Hep-2 cells

[0034] Hep-2 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 polyketide natural product compounds was added and continued to culture. After 48 hours, the cytotoxicity of the compounds was observed under a microscope and recorded. The cell survival rate was determined by the MTT assay. The median cytotoxic concentration (CC) of the drugs 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 polyketide natural product compounds against CVB3

[0036] Hep-2 cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator until the cells grew into a monolayer. The culture medium was discarded and 100 TCID 50 Cells were infected with the CVB3 virus solution for 1 hour. Different concentrations of the test polyketide natural product compound (ribavirin was used as a positive control drug) were then added and incubated. After approximately 48 hours of incubation, when approximately 90% CPE was observed in the virus control wells, cytopathic effect (CPE) was observed under a microscope. CPE was recorded as follows: no cytopathic effect was scored as -, less than 25% cytopathic effect was scored as +, 25%-50% cytopathic effect was scored as ++, 50%-75% cytopathic effect was scored as +++, and greater than 75% cytopathic effect was scored as ++++.

[0037] After CPE observation, the MTT method was used to detect the inhibitory rate of drugs on CVB3. The specific steps were as follows: 50 μL of MTT (5 mg·mL-1) was added to each well, incubated for 3-4 hours, 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 drugs on CVB3 was calculated using the following formula. The concentration for 50% of maximal effect (EC) was calculated using SPSS 11.5 software. 50 ).

[0038]

[0039] 2.1.3 Therapeutic Index (SI) of Drugs

[0040] SI = CC 50 / EC 50 The higher the therapeutic index, the greater the antiviral potential.

[0041] 3. Test results

[0042] Table 1 Cytotoxicity and anti-CVB3 activity of (3S,4S)-Sclerotinin A and Ligustrone B on Hep-2 cells

[0043]

[0044] The results of the compound cytotoxicity and anti-CVB3 activity tests are shown in Table 1. The concentration-dependent effects of the compounds on the survival rate of Hep-2 cells induced by CVB3 are shown in Table 1. Figure 1 The present invention detected that both (3S,4S)-Sclerotinin A and Ligustrone B have certain inhibitory activity against CVB3, and their therapeutic index is improved. The experimental results provide guidance for the development of more effective and less toxic anti-CVB3 drugs through structural modification.

[0045] The polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B inhibited the CPE effect of Hep-2 cells induced by CVB3. Figure 2 As shown in the figure, Hep-2 cells infected with CVB3 became rounded and detached from the cell wall. Hep-2 cells treated with 200 μM / mL (3S,4S)-Sclerotinin A and 200 μM / mL Ligustrone B maintained healthy growth, with morphological characteristics nearly identical to those of the non-infected control group. This indicates that both compounds have a significant inhibitory effect on the cytopathic effect induced by CVB3 infection.

[0046] The applicant has conducted in-depth research on the anti-CVB3 activity of polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B, and conducted tests on the inhibitory effects of (3S, 4S)-Sclerotinin A and Ligustrone B on the production of CVB3 progeny viruses. The test results are as follows:

[0047] 1. Test content

[0048] The inhibitory effects of (3S,4S)-Sclerotinin A and Ligustrone B on the production of CVB3 progeny viruses were detected after CVB3 infection of Hep-2 cells.

[0049] 2. Test methods

[0050] Hep-2 cells in the logarithmic growth phase were plated in 24-well plates. After the cells grew into a confluent monolayer, 100 TCID 50The cells were infected with CVB3 and incubated at 37°C for 1.5 h. After that, the virus solution was removed and the cells were washed three times with PBS. Cell maintenance medium containing 200 μM / mL (3S,4S)-Sclerotinin A and 200 μM / mL Ligustrone B was added. The cells and supernatant culture medium were collected at 12 h and 36 h, respectively. After three freeze-thaw cycles at -20°C and 37°C, the TCID 50 Methods The CVB3 virus titer was determined.

[0051] 3. Test results

[0052] like Figure 2 As shown, the cell survival rate of Hep-2 cells infected with CVB3 treated with (3S,4S)-Sclerotinin A and Ligustrone B was significantly increased compared with the virus control group, indicating that the above compounds can significantly inhibit the activity of CVB3.

[0053] like Figure 3 As shown, the viral titers of Hep-2 cells treated with the polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B were significantly reduced compared to the virus control group, with decreases of 3.5 log and 4.4 log, respectively, showing a strong inhibitory effect, indicating that these compounds can strongly inhibit the replication and proliferation of the virus in cells.

[0054] In summary, the polyketide natural product compounds (3S,4S)-Sclerotinin A and Ligustrone B exhibited strong CVB3 inhibitory activity, including suppressing the CVB3-induced Hep-2 cytopathic effect, significantly reducing progeny virus production, and exhibiting a high therapeutic index. This suggests that these compounds have the potential to be used in the development of clinically effective anti-CVB3 infection drugs.

Claims

1. Use of a polyketide natural product compound in the preparation of an anti-CVB3 virus drug, wherein the polyketide natural product compound is selected from (3S,4S)-Sclerotinin A and Ligustrone B; The chemical structure of (3S,4S)-Sclerotinin A is shown in formula (I): Formula (I); The chemical structural formula of Ligustrone B is shown in formula (II): Formula (II).

2. The use of the polyketide natural product compound according to claim 1 in the preparation of an anti-CVB3 virus drug, characterized in that: The polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B have anti-CVB3 virus activity.

3. The use of the polyketide natural product compound according to claim 2 in the preparation of an anti-CVB3 virus drug, characterized in that: The polyketide natural product compound (3S, 4S)-Sclerotinin A has an inhibition rate of 88% on CVB3 at 200 μM / mL; and the inhibition rate of Ligustrone B has an inhibition rate of 92% on CVB3 at 200 μM / mL.

4. The use of the polyketide natural product compound according to claim 3 in the preparation of an anti-CVB3 virus drug, characterized in that: The polyketide natural product compounds (3S, 4S)-Sclerotinin A and Ligustrone B are used as CVB3 virus inhibitors and are added with pharmaceutically acceptable excipients to prepare an anti-CVB3 virus preparation.

5. Use of the polyketide natural product compound according to claim 4 in the preparation of an anti-CVB3 virus drug, characterized in that: The preparation is any one of granules, tablets, pills, capsules, injections or dispersions.

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