Application of horse kidney fruit extract in anti-dengue fever virus drugs

Abstract

The present invention discloses the use of a horse kidney fruit extract in an anti-dengue virus drug, 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 the DENV-2 virus, and reduce the expression of the DENV-2E protein, showing significant anti-dengue virus effects.

Description

Technical Field

[0001] The invention relates to the technical field of antiviral agents, and in particular to application of a horse kidney fruit extract in anti-dengue fever virus drugs. Background Art

[0002] Dengue virus (DENV) is the causative agent of dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS). Transmitted by Aedes aegypti and Aedes albopictus mosquitoes, DENV 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 DENV 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. The dengue virus is divided 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. No antibodies have been reported against DF-2, DF-3, or DF-4. 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 proposes an application of a horse kidney fruit extract in an anti-dengue virus drug.

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

[0005] An application of a horse kidney fruit extract in an anti-dengue virus drug, wherein the structural formula of the horse kidney fruit extract 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 formula horse kidney fruit extract of the present invention is:

[0008] (1) adding the branches and leaves of the schizonepeta to 95% ethanol for extraction 3 to 5 times, each time for 3 to 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) Based on thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that fraction Fr.G contained the compound of the present invention. Fraction Fr.G was separated using an RP-C18 column, and gradient elution was performed with 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. Based on thin layer chromatography and liquid chromatography-mass spectrometry, it was determined that fraction Fr.G3 contained the compound of the present invention.

[0010] (3) Fraction Fr.G3 was separated by silica gel column chromatography using an eluent having a volume ratio of dichloromethane to acetone of 60:40 to obtain the compound of the present invention.

[0011] Furthermore, the horse kidney fruit extract is used in anti-dengue virus drugs, and the drugs at least include the compound of the structural formula described in the present invention.

[0012] The present invention also claims protection for an anti-dengue virus drug, which at least includes the compound of the structural formula described in the present invention.

[0013] Furthermore, the dengue virus is dengue type 2 virus.

[0014] 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.

[0015] 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.

[0016] Furthermore, the medicine includes any dosage form of injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, granule, spray, and aerosol.

[0017] The invention discloses an application of a horse kidney fruit extract in an anti-dengue virus drug, and its beneficial effects are: the invention can significantly inhibit the cytopathic effect induced by DENV-2 on cells, reduce the generation of viral ions, inhibit the replication of DENV-2 virus, reduce the expression of DENV-2E protein, and has a significant anti-dengue virus effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The effect of the horse kidney fruit extract of the structural formula of the present invention on the CPE effect caused by DENV-2 infection in BHK-21 cells;

[0020] Figure 2 The effect of the horse kidney fruit extract of the structural formula of the present invention on the production of progeny viruses after DENV-2 infection of BHK-21 cells;

[0021] Figure 3 The effect of the horse kidney fruit extract of the present invention on DENV mRNA synthesis in BHK-21 cells after DENV-2 infection;

[0022] Figure 4 The invention relates to the effect of the horse kidney fruit extract of the structural formula on the expression of DENV E protein in BHK-21 cells after DENV-2 infects the cells. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] An anti-dengue virus compound, wherein the compound is extracted by:

[0026] (1) adding the branches and leaves of the schizonepeta to 95% ethanol for extraction 3 to 5 times, each time for 3 to 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;

[0027] (2) According to 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 (Aglaiastatin). The 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, it was determined that the fraction Fr.G3 contained the compound of the structural formula of the present invention (Aglaiastatin).

[0028] (3) Fraction Fr.G3 was separated by silica gel column chromatography using a gradient elution method with a volume ratio of dichloromethane to methanol of 60:40 to obtain the compound of the present invention.

[0029] After nuclear and hydrogen spectra confirmation, the structural formula of the compound of the present invention is obtained as follows:

[0030]

[0031] Example 2

[0032] The compound of the structural formula obtained in Example 1 was tested against dengue 2 virus.

[0033] 1. Experimental Materials

[0034] The compound of the structural formula (Aglaiastatin) 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.

[0035] 2. Experimental Methods and Results

[0036] 1. Observe the effect of the compound on the CPE effect caused by the virus.

[0037] 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. A maintenance medium containing Aglaiastatin 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 aglaiastatin treatment significantly inhibited the cytopathic effect induced by DENV-2 in BHK-21 cells.

[0038] 2. Virus plaque assay verifies the effect of compounds on the virus plaque assay.

[0039] 2.1 Sample preparation

[0040] (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.

[0041] (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;

[0042] (3) Discard the supernatant and add PBS for washing. Add 1 mL of maintenance medium containing Aglaiastatin (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.

[0043] 2.2 Plaque assay

[0044] (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.

[0045] (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.

[0046] (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.

[0047] (4) Fix with 4% paraformaldehyde for 30 min, then invert and discard the agarose cover;

[0048] (5) 1% crystal violet staining solution, staining for 20 min, and washing with running water;

[0049] (6) Allow the plate to dry at room temperature.

[0050] 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 Aglaiastatin. Virus plaque assay analysis showed that Aglaiastatin reduced the production of viral particles in the supernatant in a concentration-dependent manner.

[0051] 3. qPCR experiments were used to verify the effect of the compounds on the level of viral nucleic acid replication.

[0052] 3.1 RNA extraction

[0053] (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.

[0054] (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.

[0055] (3) PBS washing. The drug group was treated with maintenance medium containing different concentrations of Aglaiastatin, and the virus group was treated with maintenance medium containing DMSO. The cells were cultured at 37°C for 48 h.

[0056] (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;

[0057] (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.

[0058] (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.

[0059] (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;

[0060] (8) Add 1 mL of anhydrous ethanol to wash the precipitate and centrifuge at 7500 rpm at 4°C for 15 min;

[0061] (9) Remove the supernatant and dry at room temperature for 10 min;

[0062] (10) Add 10 μL RNase-free H2O to each tube to dissolve RNA and mix well;

[0063] (11) RNA concentration was determined using Nanodrop one.

[0064] 3.2 Reverse transcription

[0065] Using Yisheng First-strand cDNA synthesis was performed using 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigesterplus). Experimental procedures were performed according to the kit instructions. Specific experimental steps are provided in the kit instructions.

[0066] 3.3 qRT-PCR detection of DENV-2 RNA copy number ( Figure 4 )

[0067] (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:

[0068] Table 1 Reaction system

[0069]

[0070] The primer sequences are shown in Table 2:

[0071] Table 2 Primer sequences

[0072]

[0073] (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:

[0074] Table 3

[0075]

[0076] (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 Aglaiastatin. The mRNA level of DENV-2E gene was detected by qPT-PCR, and it was found that Aglaiastatin could inhibit the replication of DENV-2 virus in both cell lines.

[0077] 4. WB experiments were used to verify the effects of compounds on viral protein expression levels.

[0078] (1) Cell collection: BHK-21 cells were collected at a rate of 1×10 6 Cells were seeded per well in a 6-cm diameter culture dish and cultured overnight. The next day, the supernatant was discarded and DENV (MOI = 0.05) was added for infection for 2 hours. The supernatant was then discarded and washed with PBS. The drug group was treated with 3 mL of maintenance medium containing various concentrations of aglaiastatin (0.25, 0.5, and 1 nM), while the virus group was treated with an equal amount of maintenance medium containing DMSO. After 24 hours of treatment, the cells were harvested with a scraper and transferred to a 15-mL centrifuge tube. The tubes were centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded.

[0079] (2) Cell lysis: Wash once with PBS, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Lyse the cells on ice for 30 min using RIPA lysis buffer (1 mL) containing protease inhibitors (1:100). Vortex every 10 min, centrifuge at 12000 rpm for 15 min at 4°C, and collect the supernatant.

[0080] (3) BCA protein quantification: Protein quantification was performed using the Biyuntian BCA protein concentration assay kit according to the instructions;

[0081] (4) Preparation of SDS-PAGE: Prepare 8-12% separation gel according to the formula in Table 4-1 based on the protein molecular weight. Prepare stacking gel according to the formula in Table 4-2: First, pour the separation gel into the electrophoresis gel rack and flatten it with water. After the separation gel solidifies, pour the stacking gel and insert the comb. Use a microsyringe to add 50 μg of protein sample to each well and add 5 μL of protein marker.

[0082] Table 4-1 Volume of each component required for preparing 8-10% SDS-PAGE separation gel (mL)

[0083]

[0084]

[0085] Table 4-2 Volumes of various components required to prepare different volumes of SDS-PAGE stacking gel (mL)

[0086]

[0087] (5) Electrophoresis: The electrophoresis condition was set at 100 V (constant voltage) and electrophoresis was performed until bromophenol blue reached the bottom of the gel plate.

[0088] (6) Transfer: Activate the PVDF membrane in methanol for 1 minute, then place it in transfer solution and wait for use. Arrange the membrane in the order of "negative electrode - filter paper - gel - PVDF membrane - filter paper - positive electrode". Since the protein is negatively charged, ensure that the protein (negative electrode) flows to the PVDF membrane (positive electrode). Set the program to a constant current of 220mA and transfer for 120 minutes.

[0089] (7) Blocking: After transfer, wash the membrane with TBS for 1-2 minutes to remove any residual transfer solution. Block with 5% skim milk at room temperature for 1 hour (or overnight at 4°C).

[0090] (8) Primary antibody incubation: Replace the blocking solution with the diluted primary antibody, shake slowly on a shaker, and incubate at 4°C overnight;

[0091] (9) Secondary antibody incubation: Recover the primary antibody and wash with TBS solution on a shaker for 5 minutes. Repeat the wash three times. Then replace the wash solution with the secondary antibody and incubate for 2 hours on a shaker at room temperature in the dark.

[0092] (10) Scanning: The protein membrane was washed with TBS for 5 min in the dark, and the washing was repeated three times. The membrane was scanned and analyzed using a dual-color infrared fluorescence scanning imager. The results were as follows: Figure 4 shown.

[0093] from Figure 4 It can be seen that after BHK-21 cells were infected with DENV-2 virus, they were treated with Aglaiastatin, and the expression level of DENV-2E protein was detected by Western blotting, indicating that Aglaia statin reduced the expression of DENV-2E protein in a gradient-dependent manner.

[0094] 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.

[0095] 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 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 anti-dengue virus drug also includes an excipient.

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

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