APPLICATION OF TANNIC ACID IN PREPARATION OF MEDICAMENT AGAINST RESPIRATORY VIRUSES

Abstract

The present invention provides an application of tannic acid in preparing a medicament against respiratory viruses. The present invention show through live virus experiments that tannic acid has a significant inhibitory effect on respiratory viruses including SARS-CoV-2 coronavirus, Influenza A H1N1 virus, etc., with an exact curative effect, and thus has a broad application prospect in the field of preparation of medicaments against respiratory viruses. In addition, tannic acid exists in a variety of plants, which is a natural active compound existing in nature, and has also been used as a food additive, with a high safety and good foundation for medicament development.

Claims

1-16. (canceled)

17. An application of tannic acid in preparation of a medicament against a novel coronavirus SARS-CoV-2.

18. An application of tannic acid and an antiviral metal salt in preparation of a medicament against a novel coronavirus SARS-CoV-2, wherein the antiviral metal salt is a zinc salt, iron salt, calcium salt, magnesium salt, tungsten salt salt or rubidium salt; wherein, the zinc salt is zinc sulfate or zinc gluconate; the iron salt is ferrous gluconate; the calcium salt is calcium gluconate; the tungsten salt is sodium tungstate; and the rubidium salt is rubidium iodide.

19. The application according to claim 18, wherein the medicament further comprises a pharmaceutically acceptable adjuvant.

20. The application according to claim 19, wherein the medicament is an oral preparation.

21. The application according to claim 19, wherein the medicament is a spray.

22. The application according to claim 19, wherein the medicament is an aerosol.

23. The application according to claim 19, wherein the medicament is an injection.

Description

DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0045] Active virus experiments of the present invention are all entrusted to Guangdong Provincial Center for Disease Control and Prevention to perform, and microbial materials are provided by Guangdong Provincial Center for Disease Control and Prevention.

[0046] Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0047] Embodiment 1: Determination of toxicity of tannic acid to African green monkey kidney cells (Vero-E6 cells)

[0048] 1. Experimental Materials

[0049] (1) Medicaments: experimental group, tannic acid; control group, Remdesivir (purchased from Shanghai TopScience Technology Co., Ltd.);

[0050] (2) Cell line: African green monkey kidney cells (Vero-E6 cells);

[0051] (3) Others: MEM medium; 96-well culture plate.

[0052] 2. Experimental steps

[0053] (1) Cell culture was carried out by a microculture method.

[0054] (2) Medicament dilution: tannic acid and Remdesivir in 12 centrifuge tubes was respectively times diluted to in total 12 concentrations of tannic acid and Remdesivir solutions with a cell culture medium [component requirements: MEM medium (Gibco Invitrogen), 1% double antibody (Gibco Invitrogen), 2% fetal bovine serum (FBS; Gibco Invitrogen)].

[0055] (3) Three parallel wells were set for each concentration, and cell control wells were set at the same time (without medicament, with culture medium only). (4) After 48 hours, 10 ul of CCK-8 was added to each 100 ul of cell culture medium, incubated at 37° C. for 1 hour, and OD450 was measured with a spectrophotometer, and a maximum non-toxic concentration (TC.sub.0) and a median toxic concentration (TC50) of tannic acid and Remdesivir were calculated.

[0056] 3. Experimental results

TABLE-US-00001 TABLE 1 Determination results of toxicity of tannic acid and Remdesivir to African green monkey kidney cells Group TC.sub.0 (μM) TC.sub.50 (μM) Tannic acid 11.8 23.5  Remdesivir 52.8 105.6 

[0057] Embodiment 2: Efficacy test of tannic acid against SARS-CoV-2 virus

[0058] 1. Experimental materials

[0059] (1) Medicaments: experimental group, tannic acid; control group, Remdesivir (purchased from Shanghai TopScience Technology Co., Ltd.);

[0060] (2) Cell line: African green monkey kidney cells (Vero-E6 cells);

[0061] (3) Virus: SARS-CoV-2 virus;

[0062] (4) Others: MEM medium; 96-well culture plate.

[0063] 2. Experimental steps

[0064] (1) Vero-E6 cells were pre-cultured into monolayer cells in a 96-well culture plate (a number of cells: 2x10.sup.4 cells/well);

[0065] (2) The medicament solution with the maximum non-toxic dose (TD.sub.0) was selected as the medicament to be tested, and was respectively times diluted to 12 concentrations with a cell maintenance solution, and a virus infection titer is 100 TCID50;

[0066] (3) There were 3 wells for each concentration of the medicament. After the virus was adsorbed for 1 hour, the virus solution was discarded. After washing once with PBS, each well was supplemented with 0.2 ml of maintenance solution containing the medicament. At the same time, a cell control (with maintenance solution only), medicament control (without virus) and virus control (without medicament solution) were set, and cultured in a 37° C., 5% CO.sub.2 incubator.

[0067] (4) After 48 hours, a cell culture supernatant was collected for viral nucleic acid extraction, and a relative quantification of the viruses was carried out using a COVID-19 fluorescence quantitative PCR kit (already obtained a clinical medical device registration certificate), and an effect of tannic acid on inhibition of SARS-CoV was calculated; parameters such as median inhibitory concentration (IC.sub.50) and a selection index (SI) were calculated.

[0068] Selection Index (SI)=TC.sub.50/IC.sub.50

[0069] IS.sub.50 (50% toxic concentration): calculated by Reed-Muench formula

[0070] IS.sub.50 (median inhibitory concentration): calculated by Reed-Muench formula

[0071] 3. Experimental results

[0072] As shown in Table 2, tannic acid shows a relatively high antiviral activity against SARS-CoV-2 virus with an IC50 value against SARS-CoV-2 virus being 0.0032 μM and a SI being 7343.75, which is significantly higher than an antiviral activity of the positive control group Remdesivir, and tannic acid also has a relatively high selection index.

TABLE-US-00002 TABLE 2 Activity parameters of tannin acid and Remdesivir against viruses Group IC.sub.50 (μM) SI Tannic acid  0.0032 ± 0.0002** 7343.75  Positive control 0.651 ± 0.013* 162.21  (Remdesivir) Note: Compared with the positive control group, *P < 0.05, **P < 0.01.

[0073] Embodiment 3: Determination of toxicity of tannic acid to canine kidney epithelial cells (MDCK cells)

[0074] 1. Experimental materials

[0075] (1) Medicaments: experimental group, tannic acid; control group, Ribavirin (purchased from ROMIT Pharmaceutical Corporation Jiangsu);

[0076] (2) Cell line: canine kidney epithelial cells (MDCK cells);

[0077] (3) Others: DMEM medium; 96-well culture plate.

[0078] 2. Experimental steps

[0079] (1) Cell culture was carried out by a microculture method.

[0080] (2) Medicament dilution: tannic acid and Ribavirin in 12 centrifuge tubes was respectively times diluted to in total 12 concentrations of tannic acid and Ribavirin solutions with a cell culture medium [component requirements: DMEM medium (Gibco Invitrogen), 1% double antibody (Gibco Invitrogen), 10% fetal bovine serum (FBS; Gibco Invitrogen)].

[0081] (3) Medicaments of each dilution were added into wells of MDCK cells, 3 wells for each dilution, 100 μL per well, and normal cell control wells were set at the same time, and cultured in a 37° C., 5% CO.sub.2 incubator.

[0082] (4) Cytopathic changes were observed and recorded every day: (−), no cytopathic changes; (+), 0 to 1/4 of cells were cytopathic; (++), ¼ to ½ of cells were cytopathic; (+++), ½ to ¾ of cells were cytopathic; (++++), ¾ to 1 of cells were cytopathic. The maximum non-toxic concentration (TC.sub.0) of the medicament was taken as the minimum dilution times of the medicament without cytopathic changes, and the median toxic concentration (TC50) of the medicament was calculated by the Reed-Muench formula.

[0083] 3. Experimental results

TABLE-US-00003 TABLE 3 Determination results of toxicity of tannic acid and Ribavirin to canine renal epithelial cells Group TC.sub.0 (μM) TC.sub.50 (μM) Tannic acid 376.21 752.41  Ribavirin 655.17 1310.34 

[0084] Embodiment 4: Efficacy test of tannic acid against Influenza A H1N1 virus

[0085] 1. Experimental materials:

[0086] (1) Medicaments: experimental group, tannic acid; control group, Ribavirin (purchased from ROMIT Pharmaceutical Corporation Jiangsu);

[0087] (2) Cell line: canine kidney epithelial cells (MDCK cells);

[0088] (3) Virus: Influenza A H1N1 virus;

[0089] (4) Others: DMEM medium; 96-well culture plate. 2. Experimental steps

[0090] (1) Monolayer MDCK cells were digested with 0.25% trypsin, inoculated and cultured at a cell concentration of 1 x10.sup.5 cells/mL, and the culture medium was taken away when the cells grew to a desired density (80% to 90%), and washed twice with PBS;

[0091] (2) They were divided into cell group, virus group, Ribavirin group and tannic acid group, a medicament solution with the maximum non-toxic dose (TD.sub.0) was selected as the medicament to be tested, and prepared into 12 concentrations of medicament-containing serum after times diluted;

[0092] (3) 100 TCID50/100 μL Influenza A H1N1 virus was used to infect cells of each group (the cell group was not infected, but added with 100 μL of a maintenance solution); after incubator adsorption, PBS washing and other operations, Ribavirin serum, tannic acid serum, and cell maintenance solution was added respectively, 3 wells of each medicament concentration, 100 μL/well, cultured in a 37° C., 5% CO.sub.2 incubator;

[0093] (4) After 48 hours, the cell culture supernatant was collected for viral nucleic acid extraction, and a relative quantification of the viruses was carried out using an influenza A H1N1 fluorescence quantitative PCR kit, and an effect of tannic acid on inhibition of A H1N1 virus was calculated; parameters such as median inhibitory concentration (IC50) and selection index (SI) were calculated.

[0094] Selection Index (SI)=TC.sub.50/IC.sub.50

[0095] IC50 (50% toxic concentration): calculated by Reed-Muench formula

[0096] IC50 (median inhibitory concentration): calculated by Reed-Muench formula

[0097] 3. Experimental results:

[0098] As shown in Table 4, tannic acid showed a relatively high antiviral activity against influenza A H1N1 virus with an IC50 value against influenza A H1N1 virus being 0.0032 μM and a SI being 7343.75, which is significantly higher than an antiviral activity of the positive control group Ribavirin, and tannic acid also has a relatively high selection index.

TABLE-US-00004 TABLE 4 Activity parameters of tannic acid and Remdesivir against viruses Group IC.sub.50 (μM) SI Tannic acid  0.17 ± 0.02** 4425.94  Positive control 22.60 ± 0.04* 57.98 (Ribavirin) Note: Compared with the positive control group, *P < 0.05, **P < 0.01.

[0099] Embodiment 5: Determination of toxicity of tannic acid composition to African green monkey kidney cells (Vero-E6 cells)

[0100] 1. Experimental materials

[0101] (1) Medicaments: in the experimental group, tannic acid was mixed with catechin, resveratrol, and zinc gluconate according to a mass ratio of 1:1 to prepare into three pharmaceutical compositions; in the control group, Remdesivir (purchased from Shanghai TopScience Technology Co., Ltd.);

[0102] (2) Other experimental materials were the same as those in Embodiment 1.

[0103] 2. Experimental steps: refer to Embodiment 1.

[0104] 3. Experimental results

TABLE-US-00005 TABLE 5 Determination results of toxicity of tannic acid compositions to African green monkey kidney cells Group TC.sub.0 (μg/ml) TC.sub.50 (μg/ml) Tannic acid + catechin 21.562 43.132 Tannic acid + resveratrol 22.177 44.350 Tannic acid + 15.132 30.265 zinc gluconate Remdesivir 31.816 63.633

[0105] Embodiment 6: Efficacy test of tannic acid compositions against SARS-CoV-2

[0106] 1. Experimental materials

[0107] (1) Medicaments: same as Embodiment 5;

[0108] (2) Other experimental materials were the same as those in Embodiment 2.

[0109] 2. Experimental procedure: refer to Embodiment 2.

[0110] 3. Experimental results:

[0111] As shown in Table 6, the tannic acid compositions show a relatively high antiviral activity against SARS-CoV-2 virus, which is significantly higher than an antiviral activity of the positive control group Remdesivir, and the tannic acid compositions also have a relatively high selection index.

TABLE-US-00006 TABLE 6 Activity parameters of tannic acid compositions against SARS-CoV-2 virus Group IC.sub.50 (μg/ml) SI Tannic acid + catechin  0.00412 ± 0.00003** 10468.93   Tannic acid + resveratrol  0.00335 ± 0.00004* 13238.81   Tannic acid +  0.00124 ± 0.00007** 24407.26   zinc gluconate Remdesivir  0.392 ± 0.003** 162.21  Note: Compared with the positive control group, *P < 0.05, **P < 0.01.

[0112] Embodiment 7: Determination of toxicity of tannic acid compositions to canine kidney epithelial cells (MDCK cells)

[0113] 1. Experimental materials

[0114] (1) Medicaments: same as Embodiment 5;

[0115] (2) Other experimental materials were the same as those in Embodiment 3.

[0116] 2. Experimental procedure: refer to Embodiment 3.

[0117] 3. Experimental results

TABLE-US-00007 TABLE 7 Determination results of toxicity of tannic acid compositions and Ribavirin to canine renal epithelial cells Group TC.sub.0 (μg/ml) TC.sub.50 (μg/ml) Tannic acid + catechin 650.01 1300.56  Tannic acid + resveratrol 665.43 1330.68  Tannic acid + 573.35 1147.21  zinc gluconate Ribavirin 160.00 320.01 

[0118] Embodiment 8: Efficacy test of tannic acid compositions against influenza A H1N1 virus

[0119] 1. Experimental materials

[0120] (1) Medicaments: same as Embodiment 5;

[0121] (2) Other experimental materials were the same as those in Embodiment 4.

[0122] 2. Experimental procedure: refer to Embodiment 4.

[0123] 3. Experimental results

[0124] As shown in Table 8, the tannic acid compositions show a relatively high antiviral activity against influenza A H1N1 virus, which is significantly higher than an antiviral activity of the positive control group Ribavirin, and the tannic acid compositions also have a relatively high selection index.

TABLE-US-00008 TABLE 8 Activity parameters of tannic acid and Remdesivir against viruses Group IC.sub.50 (μg/ml) SI Tannic acid + catechin  0.24 ± 0.02** 5419.00  Tannic acid + resveratrol  0.19 ± 0.01** 7003.58  Tannic acid +  0.11 ± 0.01** 10429.20   zinc gluconate Ribavirin  5.52 ± 0.03* 57.98 Note: Compared with the positive control group, *P < 0.05, **P < 0.01.

[0125] Embodiment 9: An antiviral spray

[0126] An antiviral spray includes the following components in weight percentages: 10% of tannic acid, 5% of catechin, 3% of vitamin C, and deionized water as the rest.

[0127] Embodiment 10: An antiviral spray

[0128] An antiviral spray includes the following components in weight percentages: 5% of tannic acid, 5% of resveratrol, 3% of rosmarinic acid, and deionized water as the rest.

[0129] Embodiment 11: An antiviral aerosol

[0130] An antiviral aerosol includes the following components in weight percentages: 5% of tannic acid, 1% of zinc gluconate, 8% of deionized water, 2% of ethanol, and a propellant tetrafluoroethane as the rest.

[0131] Embodiment 12: An antiviral oral preparation

[0132] An antiviral oral preparation includes the following components in weight percentages: 10% of tannic acid, 10% of zinc gluconate, 2% of an adjuvant, and distilled water as the rest.

[0133] The above-mentioned embodiments are preferred embodiments of the present invention, but the implementations of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications should be equivalent replacement manners, which are all included in the protection scope of the present invention.