Application of chaenomeles speciosa extract in anti-dengue virus medicine

Abstract

The invention discloses an application of a fruit of przewalskia cupana extract in anti-dengue virus drugs, 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 invention relates to the technical field of antiviral agents, and in particular to application of a kiwifruit extract in an anti-dengue virus drug. Background Art

[0002] Dengue virus (DENV) 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 proposes the use 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 by 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 spatholobiaceae.

[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 nephrolepis oleifera 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 an eluent gradient elution with a volume ratio of petroleum ether to acetone 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 is determined that the fraction Fr.G contains the compound of the structural formula of the present invention, and the fraction Fr.G is separated using an RP-C18 column, and the eluent is gradient eluted with a volume ratio of methanol to water of 35:65 to 95:5, and the fractions are 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 is determined that the fraction Fr.G3 contains the compound of the structural formula 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 an anti-dengue virus drug, wherein the drug at least includes 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 a single 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 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.

[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 of 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 infects BHK-21 cells;

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

[0022] Figure 4 The invention discloses an 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 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.

[0024] Example 1

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

[0026] (1) adding the branches and leaves of the nephrolepis oleifera 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 an eluent gradient elution with a volume ratio of petroleum ether to acetone 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 the detection of thin layer chromatography and liquid chromatography-mass spectrometry, it is determined that the fraction Fr.G contains the compound of the structural formula of the present invention (Aglaiastatin), and the fraction Fr.G is separated using an RP-C18 column, and the eluent is gradient eluted with a volume ratio of methanol to water of 35:65 to 95:5, and the fractions are 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 is determined that the fraction Fr.G3 contains the compound of the structural formula of the present invention (Aglaiastatin);

[0028] (3) Fraction Fr.G3 was separated by silica gel column chromatography, and gradient elution was performed 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 obtained by the present invention is:

[0030]

[0031] Example 2

[0032] The compound of the structural formula obtained in Example 1 was tested against dengue type 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 mosquito larvae cells and stored at -80°C.

[0035] 2. Experimental Methods and Results

[0036] 1. Observe the effect of compounds on the CPE effect caused by viruses.

[0037] BHK-21 cells were cultured at 2×10 5 The cells were inoculated at a density of 100 cells / 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. Aglaiastatin-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 aglaiastatin treatment could significantly inhibit the cytopathic effect induced by DENV-2 on 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 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;

[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, add 300 μL of sample to each well and place in an incubator at 37°C for 2 h;

[0046] (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.

[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 revealed that Aglaiastatin reduced the production of viral particles in the supernatant in a concentration gradient-dependent manner.

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

[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 added with maintenance medium containing different concentrations of Aglaiastatin, and the virus group was added with maintenance medium containing DMSO, and cultured at 37°C for 48 h;

[0056] (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;

[0057] (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;

[0058] (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.

[0059] (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;

[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] Use Yisheng Ⅲ1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigesterplus) 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.

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

[0067] (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:

[0068] Table 1 Reaction system

[0069]

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

[0071] Table 2 Primer sequences

[0072]

[0073] (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:

[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 It can be seen that 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 verified the effects of compounds on viral protein expression levels.

[0078] (1) Cell collection: BHK-21 cells were plated at 1×10 6 100 cells / well were planted in a 6-cm diameter culture dish and cultured overnight. The next day, the supernatant was discarded, DENV (MOI = 0.05) was added for infection for 2 hours, the supernatant was discarded and PBS was washed, 3 mL of maintenance medium containing different concentrations (0.25, 0.5, 1 nM) of Aglaiastatin was added to the drug group, and an equal amount of maintenance medium containing DMSO was added to the virus group. After 24 hours of action, the cells were collected with a scraper and transferred to a 15 mL centrifuge tube, 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 once every 10 min, centrifuge at 12000 rpm for 15 min at 4°C, and collect the supernatant;

[0080] (3) BCA protein quantification: Use the Biotech BCA protein concentration assay kit to quantify protein 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 the concentrated gel according to the formula in Table 4-2: First, pour the separation gel into the electrophoresis gel rack, press it flat with water, and after the separation gel solidifies, pour the concentrated gel and insert the comb teeth. Use a microsyringe to add 50μg of protein sample to each well, and add 5μL 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 Volume of each component 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 min, and then place it in the transfer solution for use. Arrange 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) moves toward the PVDF membrane (positive electrode). Set the program to a constant current of 220 mA and transfer for 120 min;

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

[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 min. Repeat the washing three times. Then replace the washing solution with the secondary antibody, shake slowly on a shaker at room temperature, and incubate for 2 h 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 two-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] 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.

[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 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. Application of a horse kidney fruit extract in anti-dengue virus drugs.

2. The use of the horse kidney fruit extract in anti-dengue virus drugs according to claim 1, characterized in that: The structural formula of the horse kidney fruit extract is:

3. The use of the horse kidney fruit extract in anti-dengue virus drugs according to claim 1, characterized in that: The dengue virus is dengue type 2 virus.

4. An anti-dengue virus drug, characterized in that: At least comprising the compound of the structural formula described in claim 2.

5. The anti-dengue virus drug according to claim 4, characterized in that: Pharmaceutically acceptable carriers and / or excipients are also included.

6. The anti-dengue virus drug according to claim 4 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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