Anti-dengue virus compound and application thereof

Abstract

The invention discloses an anti-dengue virus compound and application thereof, and belongs to the technical field of antiviral agents. According to the present invention, the cytopathic effect induced by DENV-2 on cells can be significantly inhibited, the generation of virus ions can be reduced, the duplication of DENV-2 virus can be inhibited, the expression of DENV-2E protein can be reduced, and the significant dengue virus resistance effect can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral agents, and in particular to an anti-dengue virus compound and application thereof. Background Art

[0002] Dengue virus (DEN) is the pathogen of dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS). It is transmitted by Aedes aegypti and Aedes albopictus. It is widely prevalent in more than 60 tropical and subtropical countries and regions around the world. More than 100 million people are infected each year, and more than 2.5 billion people are threatened. The spread of dengue virus has now become a serious public health problem in tropical and subtropical regions. Dengue fever is divided into common dengue fever and hemorrhagic dengue fever, the latter of which has a higher mortality rate. Dengue virus is differentiated into DF-1, DF-2, DF-3, and DF-4 virus types. Dengue virus is prone to cause dengue fever. At present, the research on anti-dengue virus is mainly to develop antibodies against dengue virus, and most of them are antibodies against DF-1 type, while DF-2, DF-3, and DF-4 virus types have not been reported. There is currently no specific drug for clinical treatment of dengue fever, and there is no specific anti-dengue virus drug. Summary of the invention

[0003] In order to solve the above problems, the present invention provides an anti-dengue virus compound and application thereof.

[0004] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0005] An anti-dengue virus compound, the structural formula of which is:

[0006] Furthermore, the compound is an ethanol extract of the branches and leaves of the spatholobiaceae.

[0007] Furthermore, the extraction method of the structural compound (Aglaodoratin I) of the present invention is:

[0008] (1) adding the branches and leaves of the spatholobus to 95% ethanol for extraction 3-5 times, each time for 3-4 days, recovering the ethanol and concentrating it to obtain an extract; subjecting the extract to silica gel column chromatography, using an eluent gradient elution with a volume ratio of petroleum ether to acetone of 9:1 to 1:1, and combining them according to the thin layer chromatography performance to obtain 8 fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H;

[0009] (2) According to the detection of thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that the fraction Fr.G contained the compound Aglaodoratin I. The fraction Fr.G was separated using an RP-C18 column, and gradient elution was performed with an eluent having a volume ratio of methanol to water of 35:65 to 95:5. The fractions were combined according to the thin layer chromatography performance to obtain five fractions: Fr.G1, Fr.G2, Fr.G3, Fr.G4, and Fr.G5. According to the detection of thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that the fraction Fr.G4 contained Aglaodoratin I.

[0010] (3) Fraction Fr.G4 was separated by silica gel column chromatography using an eluent having a volume ratio of dichloromethane to methanol of 100:3 to obtain compound Aglaodoratin I.

[0011] The present invention also claims to protect the use of an anti-dengue virus compound as an anti-dengue drug, wherein the drug at least includes the compound Aglaodoratin I of the structural formula described in the present invention.

[0012] Furthermore, the dengue virus is dengue type 2 virus.

[0013] Furthermore, the anti-dengue virus drug of the present invention can be a single horse kidney fruit extract of the structural formula described in the present invention.

[0014] Furthermore, the anti-dengue virus drug of the present invention can be composed of the horse kidney fruit extract of the structural formula described in the present invention and a pharmaceutical carrier and / or excipient.

[0015] Furthermore, the medicine includes any dosage form of injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, granule, spray and aerosol.

[0016] The invention discloses an anti-dengue virus compound and application thereof, and has the beneficial effects of being able to significantly inhibit the cytopathic effect induced by DENV-2 on cells, reducing the generation of viral ions, inhibiting the replication of DENV-2 virus, reducing the expression of DENV-2E protein, and having a significant anti-dengue virus effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1The effect of the compound of the structural formula of the present invention (Aglaodoratin I) on the CPE effect caused by DENV-2 infection of BHK-21 cells;

[0019] Figure 2 The effect of the compound of the structural formula of the present invention (Aglaodoratin I) on the production of progeny viruses after DENV-2 infection of BHK-21 cells;

[0020] Figure 3 The invention discloses an effect of the compound of the structural formula of the invention (Aglaodoratin I) on the synthesis of DENV mRNA in BHK-21 cells after DENV-2 infects the cells. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1

[0023] An anti-dengue virus compound, wherein the compound is extracted by:

[0024] (1) adding the branches and leaves of the spatholobus to 95% ethanol for extraction 3-5 times, each time for 3-4 days, recovering the ethanol and concentrating it to obtain an extract; subjecting the extract to silica gel column chromatography, using an eluent gradient elution with a volume ratio of petroleum ether to acetone of 9:1 to 1:1, and combining them according to the thin layer chromatography performance to obtain 8 fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H;

[0025] (2) According to the detection of thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that the fraction Fr.G contained the compound of the structural formula of the present invention (Aglaodoratin I), and the fraction Fr.G was separated using an RP-C18 column, and the eluent was gradient eluted with a methanol to water volume ratio of 35:65-95:5. The fractions were combined according to the thin layer chromatography performance to obtain 5 fractions: Fr.G1, Fr.G2, Fr.G3, Fr.G4, and Fr.G5; according to the detection of thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that the fraction Fr.G4 contained the compound of the structural formula of the present invention (Aglaodoratin I).

[0026] (3) Fraction Fr.G4 was separated by silica gel column chromatography, and gradient elution was performed with a volume ratio of dichloromethane to methanol of 100:3 to obtain the structural formula compound of the present invention (Aglaodoratin I).

[0027] After nuclear and hydrogen spectra confirmation, the structural formula of the compound of the present invention (Aglaodoratin I) is obtained as follows:

[0028]

[0029] Example 2

[0030] The compound of the structural formula obtained in Example 1 was tested against dengue type 2 virus.

[0031] 1. Experimental Materials

[0032] The compound of the structural formula (Aglaodoratin I) obtained in Example 1, the baby hamster kidney cells BHK-21 cells were kept in the laboratory, and the DENV-2 virus was amplified by C6 / 36 larval mosquito cells and stored at -80°C.

[0033] 2. Experimental Methods and Results

[0034] 1. Observe the effect of compounds on the CPE effect caused by viruses.

[0035] BHK-21 cells were cultured at 2×10 5 The cells were inoculated at a density of 100 / well into a 12-well plate and cultured overnight. DENV-2 (MOI = 0.01) was added to infect the cells for 2 hours, and DMEM medium was added to the control group without virus solution. After 2 hours, the virus solution was removed and the cells were washed twice with PBS. Aglaodoratin I-containing maintenance medium was added and cultured at 37°C and 5% CO2 for 5 days. The cytopathic effect was observed under a microscope. The results are as follows: Figure 1 As shown in the figure, DENV-2 infection of cells affects the normal morphology of cells, causing them to become rounded, necrotic, and fall off the bottle wall. Microscopic observation showed that aglaodoratin I treatment could significantly inhibit the cytopathic effect induced by DENV-2 on BHK-21 cells.

[0036] 2. Virus plaque assay verifies the effect of compounds on the virus plaque assay.

[0037] 2.1 Sample preparation

[0038] (1) BHK-21 cells were collected at 2×10 5 The cells were plated in 12-well plates and cultured overnight in a 37°C, 5% CO2 incubator.

[0039] (2) DMEM medium was added to the virus-free control group, and DENV-2 (MOI = 0.01) was added to the drug group and virus group. After adsorption at 37°C for 2 h, the supernatant was removed and the cells were washed twice with PBS;

[0040] (3) Discard the supernatant and add PBS for washing. Add 1 mL of maintenance medium containing Aglaodoratin I (0.25, 0.5, 1, 2, 4 μM) to the drug group, and add 1 mL of maintenance medium containing DMSO to the virus group. After culturing for 48 h, take the supernatant and freeze it at -80°C;

[0041] 2.2 Plaque assay

[0042] (1) BHK-21 cells were collected at 2×10 5 The cells were plated in 12-well plates and cultured overnight in a 37°C, 5% CO2 incubator.

[0043] (2) Discard the supernatant, wash with PBS, add 300 μL of sample to each well, and place in an incubator at 37°C for 2 h;

[0044] (3) Discard the supernatant and wash with PBS. Add 1 mL of a mixed culture medium containing 4% FBSDMEM medium and 2% low-melting-point agarose to each well. After solidification, place in a 37°C incubator and culture for 6-7 days.

[0045] (4) Fix with 4% paraformaldehyde for 30 min, then invert and discard the agarose cover;

[0046] (5) 1% crystal violet staining solution, staining for 20 min, and washing with running water;

[0047] (6) Allow the plate to dry at room temperature.

[0048] The results of the empty plaque control were as follows Figure 2 As shown, BHK-21 cells were infected with DENV-2 virus and then treated with Aglaodoratin I. Virus plaque assay analysis revealed that Aglaodoratin I reduced the production of viral particles in the supernatant in a concentration gradient-dependent manner.

[0049] 3. qPCR experiments were used to verify the effects of compounds on viral nucleic acid replication levels.

[0050] 3.1 RNA extraction

[0051] (1) BHK-21 cells and Huh7 cells were cultured in DMEM medium at a rate of 2×10 cells per well. 5 The cells were plated in 12-well plates and cultured overnight in a 37°C, 5% CO2 incubator.

[0052] (2) Discard the supernatant and wash with 1× PBS, add 300 μL of DENV-2 (MOI = 0.01) virus dilution to each well, and adsorb at 37°C for 2 h;

[0053] (3) PBS washing. The drug group was added with maintenance medium containing different concentrations of Aglaodoratin I, and the virus group was added with maintenance medium containing DMSO, and cultured at 37°C for 48 h;

[0054] (4) Discard the supernatant, wash with pre-cooled PBS, add 0.5 mL Trizol to each well, let stand for 15 min, and then transfer to a 1.5 mL enzyme-free EP tube;

[0055] (5) Add chloroform (trichloromethane) to the EP tube in a ratio of (1:5) chloroform:Trizol, shake vigorously for 30 seconds, let stand for 10 minutes; centrifuge at 4°C, 12,000 rpm for 15 minutes;

[0056] (6) The liquid after centrifugation is divided into three layers. The transparent layer (upper layer) is pipetted into a clean enzyme-free EP tube. An equal amount of pre-cooled isopropanol is added to each tube. The tube is inverted 10 times and placed on ice for 15 min. Centrifuge at 4°C, 12,000 rpm for 15 min.

[0057] (7) Remove the supernatant, add 1 mL of freshly prepared 75% anhydrous ethanol to wash the precipitate; centrifuge at 4°C, 7500 rpm for 15 min;

[0058] (8) Add 1 mL of anhydrous ethanol to wash the precipitate and centrifuge at 7500 rpm at 4°C for 15 min;

[0059] (9) Remove the supernatant and dry at room temperature for 10 min;

[0060] (10) Add 10 μL RNase-free H2O to each tube to dissolve RNA and mix well;

[0061] (11) RNA concentration was determined using Nanodrop one.

[0062] 3.2 Reverse transcription

[0063] Use Yisheng Ⅲ1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) was used for first-strand cDNA synthesis, and the experimental operation was performed according to the instructions of the kit. For specific experimental steps, please refer to the instructions for use of the kit.

[0064] 3.3 qRT-PCR detection of DENV-2 RNA copy number ( Figure 3 )

[0065] (1) Take 2 μL cDNA sample and qPCR Green Master Mix (No Rox) kit instructions, prepare qRT-PCR reaction system. The reaction system is shown in Table 1:

[0066] Table 1 Reaction system

[0067]

[0068] The primer sequences are shown in Table 2:

[0069] Table 2 Primer sequences

[0070]

[0071] (2) Bio-Rad CFX96 performed qRT-PCR on the cDNA samples and amplified them in a two-step method in the PCR instrument. The setting program is shown in Table 3:

[0072] Table 3

[0073]

[0074] (3) Data processing: 2 -△△Ct The data were processed and plotted using GraphPad 9.5. Figure 3 As shown. Figure 3 It can be seen that after BHK-21 and Huh7 cells were infected with DENV-2 virus, they were treated with Aglaodoratin I. The mRNA level of DENV-2E gene was detected by qPT-PCR, and it was found that Aglaodoratin I could inhibit the replication of DENV-2 virus in both cell lines.

[0075] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0076] Finally, it should be noted that the embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An anti-dengue virus compound, characterized in that: Its structural formula is:

2. The anti-dengue virus compound according to claim 1, characterized in that: The compound is a methanol and / or ethanol extract of the branches and leaves of Psoralea corylifolia.

3. Use of an anti-dengue virus compound as an anti-dengue drug, characterized in that: At least comprising the compound of the structural formula described in claim 1.

4. The use according to claim 3, characterized in that: The dengue virus is dengue type 2 virus.

5. The use according to claim 3, characterized in that: Pharmaceutically acceptable carriers and / or excipients are also included.

6. The use according to claim 3 or 5, characterized in that: The medicine includes any dosage form of injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, granule, spray and aerosol.

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