Application of chlorogenic acid or pharmaceutically acceptable salt thereof in preparation of products with RNA virus broad-spectrum resistance

Abstract

The invention provides application of chlorogenic acid or pharmaceutically acceptable salts thereof in preparation of products with RNA virus broad-spectrum resistance, and belongs to the technical field of research and development of antiviral preparations. The RNA viruses disclosed by the invention comprise RNA viruses of the coronaviridae and the small RNA viridae, such as SADS-CoV and PEDV of alpha coronavirus of the coronaviridae and EV-D68 of the small RNA viridae. The chlorogenic acid or the pharmaceutically acceptable salt thereof can effectively inhibit the infection of RNA viruses on cells, inhibit the apoptosis induced by the RNA viruses and inhibit the replication of RNA virus nucleocapsid genes, so that the cross-viridae antiviral activity of the chlorogenic acid is proved, and the chlorogenic acid or the pharmaceutically acceptable salt thereof can be used for preparing products with RNA virus broad-spectrum resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-virus preparation, and particularly relates to application of chlorogenic acid or a pharmaceutically acceptable salt thereof in preparation of a product with broad-spectrum resistance to RNA viruses. BACKGROUND

[0002] Chlorogenic acid, also known as coffee tannic acid, is a natural phenolic acid ester medicine condensed by caffeic acid and quinic acid, and widely exists in honeysuckle, eucommia, yinchen and many other Chinese herbal medicines and numerous fruits and vegetables. Studies have shown that chlorogenic acid has various biological activities, such as antioxidant, antibacterial, anti-allergic, liver protection and choleretic, and also shows certain potential in anti-virus. At present, the research on chlorogenic acid is focused on a single virus type, such as hepatitis B virus (HBV, hepadnavirus of hepadnavirus family), dengue virus (DENV, flavivirus of flavivirus family) and influenza virus (H1N1, orthomyxovirus of orthomyxovirus family), and the anti-virus activity has been preliminarily explored, but there are significant limitations, and no research has reported the effect of chlorogenic acid on SADS-CoV, PEDV and viruses of picornavirus family, the activity spectrum is narrow, and there is lack of broad spectrum. SUMMARY

[0003] In order to solve the problems in the prior art, the first purpose of the application is to provide application of chlorogenic acid or a pharmaceutically acceptable salt thereof in preparation of a product with broad-spectrum resistance to RNA viruses.

[0004] In order to achieve the above-mentioned purpose of the application, the application provides the following technical scheme: The application provides application of chlorogenic acid or a pharmaceutically acceptable salt thereof in preparation of a product with broad-spectrum resistance to RNA viruses, wherein the RNA viruses include RNA viruses of coronavirus family and picornavirus family.

[0005] Preferably, the RNA viruses of coronavirus family include RNA viruses of coronavirus alpha genus of coronavirus family.

[0006] Preferably, the RNA viruses of coronavirus alpha genus of coronavirus family include SADS-CoV and PEDV.

[0007] Preferably, the RNA viruses of picornavirus family include EV-D68.

[0008] Preferably, the chlorogenic acid includes the following natural isomers: 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid and cryptochlorogenic acid.

[0009] Preferably, the chlorogenic acid further comprises the following chlorogenic acid modified products: esterification, glycosylation and metal chelation.

[0010] Preferably, the pharmaceutically acceptable salt comprises sodium salt, potassium salt.

[0011] Preferably, the chlorogenic acid or its pharmaceutically acceptable salt inhibits the infection of cells by RNA viruses, inhibits RNA virus-induced apoptosis.

[0012] Preferably, the chlorogenic acid or its pharmaceutically acceptable salt inhibits the replication of RNA virus nucleocapsid genes.

[0013] Preferably, the product comprises a pharmaceutical product, a feed additive.

[0014] Compared with the prior art, the beneficial effects of the technical scheme of the present application are as follows: The present application provides RNA virus broad-spectrum resistance of chlorogenic acid or its pharmaceutically acceptable salt, and it is found that chlorogenic acid or its pharmaceutically acceptable salt has obvious resistance to SADS-CoV and PEDV of Coronaviridae alpha coronavirus genus, EV-D68 of Picornaviridae, can effectively inhibit the infection of cells by RNA viruses, inhibit RNA virus-induced apoptosis, inhibit the replication of RNA virus nucleocapsid genes, and confirm the antiviral activity of chlorogenic acid across virus families, which can be used for preparing products with RNA virus broad-spectrum resistance. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : Cell viability and proliferation detection primary mouse neuron cell survival rate results; Figure 2 : CellTiter-Glo® luminescence method cell viability detection primary mouse neuron cell viability results; Figure 3 : Inhibition rate of chlorogenic acid on EV-D68 virus nucleocapsid VP1 gene in primary mouse neuron cells; Figure 4 : Cell viability and proliferation detection PK15 cell survival rate results; Figure 5 : CellTiter-Glo® luminescence method cell viability detection PK15 cell viability results; Figure 6 : Death rate of PEDV infected PK15 cells treated with chlorogenic acid; Figure 7 : Inhibition rate of chlorogenic acid on PEDV virus nucleocapsid NP gene in PK15 cells; Figure 8 : Cell viability and proliferation detection Vero cell survival rate results; Figure 9 Vero cell viability results from CellTiter-Glo® Luminescent Cell Viability Assay Figure 10 Mortality results of Vero cells infected with SADS-CoV virus treated with chlorogenic acid Figure 11 Inhibition rate results of SADS-CoV virus nucleocapsid NP gene on Vero cells treated with chlorogenic acid DETAILED DESCRIPTION

[0016] The present application provides the use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of a product with broad-spectrum resistance to RNA viruses, including RNA viruses of the Coronaviridae and Picornaviridae.

[0017] The RNA viruses of the Coronaviridae in the present application are preferably RNA viruses including the Coronaviridae alpha coronavirus genus, and more preferably RNA viruses including SADS-CoV and PEDV.

[0018] The RNA viruses of the Picornaviridae in the present application are preferably EV-D68.

[0019] The chlorogenic acid in the present application preferably includes the following natural isomers: 1-caffeoylquinic acid (1-CQA), 3-caffeoylquinic acid (3-CQA, the main active form), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), and cryptochlorogenic acid. It also preferably includes structural modification products: esters (such as ethyl esters), glycosides (such as glucosides), and metal chelates (such as zinc complexes).

[0020] The pharmaceutically acceptable salt of chlorogenic acid in the present application preferably includes metal ion salts, and more preferably includes sodium salt and potassium salt.

[0021] The present application provides the broad-spectrum antiviral activity of chlorogenic acid, which is verified by cross-virus families of Coronaviridae (SADS-CoV, PEDV) and Picornaviridae (EV-D68), indicating that chlorogenic acid or a pharmaceutically acceptable salt thereof can simultaneously inhibit multiple RNA viruses, and has broad-spectrum antiviral activity; the chlorogenic acid or a pharmaceutically acceptable salt thereof can effectively inhibit the infection of RNA viruses on cells, inhibit the apoptosis of cells induced by RNA viruses, and inhibit the replication of RNA virus nucleocapsid genes. Moreover, chlorogenic acid is derived from natural dietary ingredients, and has the characteristics of high safety and low toxicity.

[0022] The product in the present application preferably includes a drug and a feed additive.

[0023] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Apparently, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the protection scope of the present application.

[0024] In specific embodiments of the present application, Graphpad prism 8.0 software is used to statistically analyze the test data. The test results are represented by Mean ± SEM, and single-factor variance analysis is used to test the toxicity of chlorogenic acid in different cells, cell viability, cell mortality and inhibition rate on viruses. "*" represents P <0.05, "**" represents P <0.01, and "***" represents P <0.001.

[0025] In the following examples, unless otherwise specified, all are conventional methods.

[0026] In the following examples, unless otherwise specified, all the materials, reagents, etc. can be obtained from commercial channels.

[0027] Example 1 1. Cell plating (1) Extraction of primary mouse neuron cells: 17-18-day pregnant C57BL / 6 pregnant mice were sacrificed after CO2 anesthesia by cervical dislocation. The abdomen was disinfected with 75% ethanol, and the uterus was taken out under sterile conditions and placed in pre-cooled Hanks' balanced salt solution (HBSS, without calcium and magnesium ions). The fetal skull was separated, and the whole brain tissue was taken out. Under a dissecting microscope, the meninges and vascular tissue were stripped, and the collected cortical tissue blocks were transferred to a 15 mL centrifuge tube. Preheated papain digestion solution was added at 37°C. It was placed in a 37°C water bath for gentle shaking and digestion for 15 min. The tissue blocks were gently washed three times with pre-cooled dissection solution to terminate digestion and remove residual enzyme solution. An appropriate amount of plating liquid containing 10% FBS (2-3 mL) was added to the tissue blocks, and a flame-polished Pasteur pipette was used for gentle and repeated blowing for 15 times until a uniform single-cell suspension was obtained. The viable cell density was calculated. The polyllysine / laminin coated cell culture plate was inoculated at a density of 2.5×10 3 cells / well in a 96-well plate (100 μl of culture medium / well) and 7.5×10 4 cells / well in a 6-well plate (2 ml of culture medium / well) at 37°C, 5% CO2. After 4-6 hours of inoculation, the plating liquid was completely replaced with serum-free Neurobasal™ medium supplemented with 2% B-27™, 0.5 mM glutamine and 1% P / S.

[0028] (2) Porcine kidney epithelial cells (PK15) and monkey epithelial cells (Vero) were seeded in 96-well plates at a density of 2.5 x 105cells / well (100 μl of medium / well) and 6-well plates at a density of 7.5 x 105cells / well (2 ml of medium / well), respectively, and pre-cultured at 37 °C, 5% CO2for 24 h. 3 4

[0029] 2. Virus inoculation EV-D68 virus was obtained from the research group of Professor Huang Yaowei, College of Animal Sciences, Zhejiang University, and PEDV and SADS-CoV viruses were provided by Dr. Zhao Pengwei, School of Medicine, Zhejiang University.

[0030] The cells were washed once with PBS and inoculated with virus suspension at an MOI of 0.1. Primary mouse neurons were inoculated with EV-D68, PK15 with PEDV, and Vero with SADS-CoV. After 2 h of inoculation, the virus-containing medium was removed and replaced with fresh maintenance medium containing 0, 12.5, 25, 50, 100 μM chlorogenic acid (Macklin #C805070). The primary mouse neuron cells were cultured in neural stem cell medium, and PK15 and Vero were cultured in high-glucose DMEM medium containing 2% FBS. Three replicate wells were set up for each concentration, and the control group (0 μM) contained only medium. The cells were incubated at 37 °C, 5% CO2in an incubator for 48 h.

[0031] 3. Cell viability and proliferation detection (CCK-8 method) The cells were cultured in fresh maintenance medium containing 0, 12.5, 25, 50, 100, and 200 μM chlorogenic acid for 48 h. Cell Counting Kit-8 reagent (Yeasen Biotechnology) was used to detect cell viability and proliferation. For toxicity detection, 10 μL of CCK-8 solution was directly added to each well of the 96-well plate to avoid the generation of bubbles, and incubation was continued for 4 h. The absorbance (OD) was measured at 450 nm using a microplate reader. Blank control groups (medium + CCK-8 without cells) and untreated control groups (cells + CCK-8 without compounds) were set up, and all operations were performed in the dark. The cell viability calculation formula was: Cell viability (%) = [(treatment group - blank group) / (untreated group - blank group)] x 100%.

[0032] 4. CellTiter-Glo® luminescence method for cell viability detection ​​Replace the 96-well plate culture medium with 100 μL of fresh DMEM high-sugar medium without phenol red, add an equal volume of 100 μL CellTiter-Glo® reagent (Promega, G7570) to each well, and induce cell lysis by orbital shaking for 2 minutes. Incubate at room temperature for 10 minutes in the dark to stabilize the luminescent signal, and use an enzyme marker (Centro LB960, Berthold Technologies) to detect and read the cold fluorescence values. Set up blank control groups (culture medium + CellTiter-Glo® reagent without cells) and untreated control groups (cells + CellTiter-Glo® reagent without compounds), and perform all operations in the dark. Cell viability is expressed in relative luminescence units (RLU), which are proportional to the ATP content of living cells. Cell viability ATP calculation formula: Cell viability (%) = [(treatment group - blank group) / (untreated group - blank group)] x 100%.

[0033] 5. SYTOX GREEN staining Add SYTOX GREEN (final concentration: 1 μM) at a ratio of 1:5000 to the culture medium in a 96-well black plate (perk inElmer), add 50 μL of culture medium to each cell well, and incubate in the dark for 10 min. Use an enzyme marker to read the fluorescence intensity (excitation / emission wavelength: 504 / 523 nm). Dead cells take up the dye and bind to DNA due to membrane damage, producing green fluorescence, while live cells have no fluorescence signal.

[0034] 6. Fluorescent quantitative PCR detection of nucleocapsid RNA load (1) Wash the cells with PBS twice, add 500 μL Trizol, and mix gently with a pipette to clarify the liquid. Incubate at room temperature for 5 min. Add 150 μL chloroform and vortex for 15 s on a vortex shaker. Incubate at room temperature for 3 min. Centrifuge at 12000 rpm and 4°C for 10 min. Transfer 200 μL of supernatant to a new 1.5 mL EP tube. Add 200 μL of isopropanol, mix thoroughly by gently inverting several times, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm and 4°C for 10 min, and discard the supernatant. Add 1 mL of 70% ethanol, resuspend the RNA precipitate by blowing, centrifuge at 12000 rpm and 4°C for 3 min, and discard the supernatant. Blow dry in a fume hood, add 30 μL of DEPC water, mix well, and incubate at 4°C to dissolve. Use a nucleic acid protein analyzer (Allsheng nano-300) to detect the RNA concentration, and store at -80°C for future use.

[0035] (2) Remove a small amount of residual genomic DNA from the RNA sample: Reaction system: 5x gDNA Eraser Buffer 2 μL, gDNA Eraser 1 μL, 1 μg of total RNA, RNAase Free ddH2O added to 10 μL; Reaction condition: 42℃ for 2 min, 4℃ for preservation.

[0036] (3) Reverse transcription Reaction system: 5x PrimeScript® RT Buffer 2 4 μL, PrimeScript® RT Enzyme Mix I 1 μL, RT Primer Mix 1 μL, Reaction Solution 10 μL, RNAase Free ddH2O added to 20 μL; Reaction condition: 37℃ for 15 min, 85℃ for 5 s, 4℃ for preservation, -20℃ for long-term storage.

[0037] (4) Fluorescence quantitative PCR Reaction system: 2x SYBR® Premix Ex TaqTM 5 μL, PCR Primer Mix (10 μM) 0.4 μL, cDNA template 500 ng, RNAase Free ddH2O added to 10 μL; Reaction condition: pre-denaturation 95℃ for 10 s, denaturation 95℃ for 5 s, annealing 55-65℃ for 10 s, 40 cycles, extension 72℃ for 15 s, final extension 72℃ for 5 min, 4℃.

[0038] The primer sequence (5'→3') is: PEDV-NP-F: 5'-CGCAAAGACTGAACCCACTA-3' (SEQ ID No. 1); PEDV-NP-R: 5'-TTGCCATTACCACGACTCAT-3' (SEQ ID No. 2); Porcine-β-actin-F: 5'-GGACTTCGAGCAGGAGATGG-3' (SEQ ID No. 3); Porcine-β-actin-R: 5'-GCACCGTGTTGGCGTAGAGG-3' (SEQ ID No. 4); EVD68-VP1-F: 5'-GAGGCCTACTTCACCAACGA-3' (SEQ ID No. 5); EVD68-VP1-R: 5'-CGGTGTCTAGTTCCACGTTG-3' (SEQ ID No. 6); Mouse-β-actin-F: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID No. 7); Mouse-β-actin-R: 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID No. 8); SADS-NP-F: 5'-TGCAAGACCCTGACCTACCT-3' (SEQ ID No. 9); SADS-NP-R: 5'-AGGTGGTCCGAATGTCAATC-3' (SEQ ID No. 10); Human-β-actin-F: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID No. 11); Human-β-actin-R: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID No. 12).

[0039] (5) Analysis of fluorescence quantitative PCR data The relative quantitative analysis was performed by using the ΔΔCT method. First, a stable β-actin was selected as an internal reference gene. ΔΔCT is the difference between the ΔCT value of the experimental group and the ΔCT value of the control group (ΔCT: the difference between the CT value of the target gene of the experimental group or the corresponding target gene of the control group and the internal reference gene), and finally 2 -ΔΔCT value is the expression difference of the target gene of the experimental group relative to the target gene of the control group.

[0040] 7、Experimental results (1) Antiviral (EV-D68 virus) detection of chlorogenic acid in primary mouse neurons Cell viability and proliferation detection The survival rate of primary mouse neurons was as shown in Table 1. Figure 1 The results showed that the half toxicity IC 50 of chlorogenic acid on primary mouse neurons was 100 μM.

[0041] CellTiter-Glo® luminescence method for cell viability detection The viability of primary mouse neurons was as shown in Table 2. Figure 2 The results showed that low concentration of chlorogenic acid (12.5 μM) significantly inhibited EV-D68 virus infection of primary mouse neurons, and showed a dose-dependent relationship.

[0042] Inhibition rate of different concentrations of chlorogenic acid on EV-D68 virus nucleocapsid VP1 gene in primary mouse neuron cells was shown in Figure 3 The results showed that low concentration of chlorogenic acid (12.5 μΜ) significantly inhibited EV-D68 virus replication in a dose-dependent manner.

[0043] (2) Anti-virus (PEDV virus) detection of chlorogenic acid in PK15 cells Cell viability and proliferation detection PK15 cell survival rate was shown in Figure 4 The results showed that the half toxicity IC 50 of chlorogenic acid on PK15 cells was >200 μΜ.

[0044] CellTiter-Glo® luminescent cell viability detection PK15 cell viability was shown in Figure 5 The results showed that low concentration of chlorogenic acid (25 μΜ) significantly inhibited PEDV virus infection of PK15 cells in a dose-dependent manner.

[0045] Death rate detection of PEDV infected PK15 cells treated with different concentrations of chlorogenic acid was shown in Figure 6 Using dead cell nucleic acid dye Sytox Green staining, green fluorescence intensity was read by microplate reader, and the results showed that low concentration of chlorogenic acid (25 μΜ) could significantly inhibit virus-induced apoptosis.

[0046] Inhibition rate of different concentrations of chlorogenic acid on PEDV virus nucleocapsid NP gene in PK15 cells was shown in Figure 7 The results showed that low concentration of chlorogenic acid (12.5 μΜ) significantly inhibited PEDV virus replication in a dose-dependent manner.

[0047] (3) Anti-virus (SADS-CoV virus) detection of chlorogenic acid in Vero cells Cell viability and proliferation detection Vero cell survival rate was shown in Figure 8 The results showed that the half toxicity IC 50 of chlorogenic acid on Vero cells was >200 μΜ.

[0048] CellTiter-Glo® luminescent cell viability detection Vero cell viability was shown in Figure 9 The results showed that low concentration of chlorogenic acid (50 μΜ) significantly inhibited SADS-CoV virus infection of Vero cells in a dose-dependent manner.

[0049] Death rate detection of SADS-CoV virus infected Vero cells treated with different concentrations of chlorogenic acid was shown in Figure 10The results showed that low concentration of chlorogenic acid (12.5 μM) could significantly inhibit virus-induced apoptosis.

[0050] The inhibition rates of chlorogenic acid at different concentrations on the SADS-CoV virus nucleocapsid NP gene in Vero cells were as shown in the following table. Figure 11 The results showed that low concentration of chlorogenic acid (12.5 μM) could significantly inhibit SADS-CoV virus replication, and showed a dose-dependent relationship.

[0051] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of products with broad-spectrum resistance to RNA viruses, characterized in that, The RNA viruses mentioned include RNA viruses from the Coronaviridae and Picornaviridae families.

2. The application according to claim 1, characterized in that, The RNA viruses of the Coronaviridae family include RNA viruses of the genus Alphacoronavirus.

3. The application according to claim 2, characterized in that, RNA viruses belonging to the genus Alphacoronavirus of the family Coronaviridae include SADS-CoV and PEDV.

4. The application according to claim 1, characterized in that, The RNA viruses of the Picornaviridae family include EV-D68.

5. The application according to claim 1, characterized in that, The chlorogenic acid includes the following natural isomers: 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid and cryptochlorogenic acid.

6. The application according to claim 1, characterized in that, The chlorogenic acid also includes the following chlorogenic acid modified products: esters, glycosides and metal chelates.

7. The application according to claim 1, characterized in that, Pharmaceutically acceptable salts include sodium salts and potassium salts.

8. The application according to claim 1, characterized in that, The chlorogenic acid or its pharmaceutically acceptable salt inhibits RNA virus infection of cells and inhibits RNA virus-induced apoptosis.

9. The application according to claim 1, characterized in that, The chlorogenic acid or a pharmaceutically acceptable salt thereof inhibits the replication of the nucleocapsid gene of RNA viruses.

10. The application according to claim 1, characterized in that, The products include pharmaceuticals and feed additives.

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