Anti-dengue virus compound and its application

Abstract

The present invention discloses an anti-dengue virus compound and its application, belonging to the technical field of antiviral agents. The present invention can significantly inhibit the cytopathic effect induced by DENV-2 on cells, reduce the production of viral ions, inhibit the replication of DENV-2 virus, and reduce the expression of DENV-2E protein, showing significant anti-dengue virus effects.

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 causative agent of dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS). Transmitted by Aedes aegypti and Aedes albopictus mosquitoes, it is widespread in over 60 tropical and subtropical countries and regions worldwide, infecting over 100 million people annually and threatening over 2.5 billion people. The spread of DEN has become a serious public health problem in these 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 viruses are classified into genotypes DF-1, DF-2, DF-3, and DF-4. Dengue viruses are highly susceptible to dengue fever. Current anti-dengue virus research focuses primarily on developing antibodies against dengue virus, and most of these antibodies are directed against DF-1. Antibodies against DF-2, DF-3, and DF-4 have yet to be reported. Currently, there are no effective clinical treatments for dengue fever, nor are there any specific anti-dengue virus drugs. 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 through 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 kiwi fruit.

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

[0008] (1) adding the branches and leaves of the schizonepeta 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 a gradient elution solvent of petroleum ether and acetone in a volume ratio of 9:1 to 1:1, and combining the fractions 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 the eluent was gradient eluted with a methanol to water volume ratio of 35:65 to 95:5. The fractions were merged 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 protection for the use of an anti-dengue virus compound as an anti-dengue drug, wherein the drug at least comprises 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 solely the 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 present invention discloses an anti-dengue virus compound and its application, which has the beneficial effects of significantly inhibiting the cytopathic effect induced by DENV-2 on cells, reducing the generation of viral ions, inhibiting the replication of DENV-2 virus, and reducing the expression of DENV-2E protein, thereby 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[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 in 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 present invention relates to the effect of the compound (Aglaodoratin I) of the structural formula of the present invention on DENV mRNA synthesis in BHK-21 cells after DENV-2 infection. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall 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 schizonepeta 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 a gradient elution solvent of petroleum ether and acetone in a volume ratio of 9:1 to 1:1, and combining the fractions 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 thin layer chromatography and liquid chromatography-mass spectrometry detection, it was determined that fraction Fr.G contained the compound of the structural formula of the present invention (Aglaodoratin I). Fraction Fr.G was separated using an RP-C18 column, and gradient elution was performed with an eluent having a methanol to water volume ratio 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 thin layer chromatography and liquid chromatography-mass spectrometry detection, it was determined that 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 using a gradient elution method with a volume ratio of dichloromethane to methanol of 100:3 to obtain the 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) was obtained as follows:

[0028]

[0029] Example 2

[0030] The compound of the structural formula obtained in Example 1 was tested against dengue 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 retained 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 the compound on the CPE effect caused by the virus.

[0035] BHK-21 cells were cultured at 2 × 10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate and cultured overnight. DENV-2 (MOI = 0.01) was added to infect the cells for 2 hours. The control group without virus solution was treated with DMEM medium. After 2 hours, the virus solution was removed and the cells were washed twice with PBS. Maintenance medium containing Aglaodoratin I was added and cultured at 37°C, 5% CO2 for 5 days. The cytopathic effect was observed under a microscope. The results are as follows: Figure 1 DENV-2 infection affects the normal morphology of cells, causing them to round, necrotize, and fall off the flask wall. Microscopic observation revealed that aglaodoratin I treatment significantly inhibited the cytopathic effect induced by DENV-2 in 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 1 mL of maintenance medium containing DMSO to the virus group. After 48 h of culture, the supernatant was collected and frozen 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, and add 300 μL of sample to each well. Place in an incubator at 37°C for 2 h.

[0044] (3) Discard the supernatant and wash with PBS. Add 1 mL of a mixture of 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 showed that Aglaodoratin I reduced the production of viral particles in the supernatant in a concentration-dependent manner.

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

[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 treated with maintenance medium containing different concentrations of Aglaodoratin I, and the virus group was treated with maintenance medium containing DMSO. The cells were cultured at 37°C for 48 h.

[0054] (4) Discard the supernatant, wash with pre-cooled PBS, add 0.5 mL of Trizol to each well, let it 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 at a ratio of (1:5) chloroform:Trizol, shake vigorously for 30 seconds, let it stand for 10 minutes, and centrifuge at 4°C, 12,000 rpm, for 15 minutes.

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

[0057] (7) Remove the supernatant and 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 First-strand cDNA synthesis was performed using 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus). Experimental procedures were performed according to the kit instructions. Specific experimental steps are provided in the kit instructions.

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

[0065] (1) Take 2 μL cDNA sample, according to Prepare the qRT-PCR reaction system according to the qPCR Green Master Mix (No Rox) kit instructions. 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) qRT-PCR was performed on the cDNA samples using a Bio-Rad CFX96 PCR instrument, using a two-step amplification method. The setup procedure 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 As can be seen, after BHK-21 and Huh7 cells were infected with DENV-2 virus, they were treated with Aglaodoratin I. The mRNA level of the 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] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, 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 can still be modified, or some of the technical features therein can 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 application of a horse kidney fruit extract in the preparation of an anti-dengue virus drug, characterized in that: The structural formula of the horse kidney fruit extract is: ； The kiwifruit extract is a methanol and / or ethanol extract of kiwifruit branches and leaves; The dengue virus is dengue type 2 virus.

2. The use of the horse kidney fruit extract according to claim 1 in the preparation of anti-dengue virus drugs, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

3. Use of the horse kidney fruit extract according to claim 1 or 2 in the preparation of anti-dengue virus drugs, characterized in that: The medicine includes any dosage form of injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, granule, spray and aerosol.

Images