CN113975312B - Antiviral drug composition, method for inhibiting in vitro virus activity and application - Google Patents

Antiviral drug composition, method for inhibiting in vitro virus activity and application Download PDF

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CN113975312B
CN113975312B CN202111116895.1A CN202111116895A CN113975312B CN 113975312 B CN113975312 B CN 113975312B CN 202111116895 A CN202111116895 A CN 202111116895A CN 113975312 B CN113975312 B CN 113975312B
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高秀梅
姚荣妹
吕彬
王涛
何俊
韩立峰
郭金赫
杜耀东
邱峰
刘志东
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Tianjin University of Traditional Chinese Medicine
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Abstract

The invention discloses an antiviral drug composition, a method for inhibiting in vitro virus activity and application. Wherein the pharmaceutical composition comprises an extract of perilla leaf. The perilla leaf extract disclosed by the invention has low cytotoxicity, has an obvious inhibiting effect on respiratory viruses (including influenza A viruses and human coronavirus) and enteroviruses under a non-cytotoxic concentration, can improve the cytopathic condition, improve the survival rate of virus infected host cells, effectively inhibit virus replication, shows a good dose-effect relationship and has an obvious antiviral effect.

Description

Antiviral drug composition, method for inhibiting in vitro virus activity and application
Technical Field
The invention relates to the field of medicines, in particular to an antiviral medicine composition, a method for inhibiting in-vitro virus activity and application.
Background
Viruses causing viral diseases, such as influenza, respiratory virus infection, enterovirus infection, and the like, not only cause periodic pandemics around the world, but also some viruses cause clinical acute attacks accompanied by symptoms such as hyperpyrexia, disturbance of consciousness, convulsion, spasticity, and meningeal irritation, and serious patients often have sequelae. Because viruses are strictly parasitic and resistant to antibiotics, the treatment of viral infections based on antibiotics is generally ineffective or minimally effective. Traditional Chinese medicine has been developed for thousands of years in China, and has an irreplaceable role in resisting viruses.
Because the traditional Chinese medicine adopts individualized treatment, the traditional Chinese medicine has more pertinence to the state of an illness. Just because of the diversification of the effective components of the traditional Chinese medicine in the treatment process, the virus is difficult to generate drug resistance to the traditional Chinese medicine, so that the traditional Chinese medicine has obvious advantages in the aspect of treating virus infectious diseases and has wide clinical application prospect.
Chinese patent publication CN113181303A discloses a Chinese medicinal composition for treating or preventing influenza virus infection and its application, wherein the Chinese medicinal composition is prepared from ten Chinese medicinal materials including herba Schizonepetae, radix Saposhnikoviae, flos Lonicerae, fructus forsythiae, bupleuri radix, Scutellariae radix, rhizoma Phragmitis, lalang grass rhizome, folium Perillae and herba Menthae by mixing, and can be used for treating or preventing influenza virus infection.
Chinese patent publication CN111450166A discloses a new application of a Chailian preparation in resisting enterovirus. The Chaiyin preparation contains Chinese medicinal materials of bupleurum root, lonicera flower, scutellaria root, pueraria root, schizonepeta, sweet wormwood, forsythia fruit, etc. and has a certain effect of resisting enterovirus. However, there is still a need for an antiviral pharmaceutical composition with strong antiviral action pertinence, wide application range, high safety, comprehensive action and simple preparation process.
Disclosure of Invention
In order to solve at least some technical problems in the prior art, the inventors of the present invention have found through intensive studies that a pharmaceutical composition containing an extract of perilla leaf can effectively prevent and treat respiratory and enteric viruses. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided an antiviral pharmaceutical composition comprising a perilla leaf extract.
According to certain embodiments of the present invention, the extract in the antiviral pharmaceutical composition is an aqueous extract.
According to the antiviral pharmaceutical composition of the present invention, preferably, the aqueous extract is prepared by the following method: mixing folium Perillae with water at a ratio of 6-14mL per gram of folium Perillae, heating under reflux, decocting for 1-3 hr, extracting for 2-3 times, filtering, mixing filtrates, concentrating into extract, and vacuum drying to obtain folium Perillae water extract. It is also preferred that the perilla leaf is mixed with water in a proportion of 8-12mL of water per gram of perilla leaf.
According to certain embodiments of the present invention, the antiviral pharmaceutical composition is an alcoholic extract.
According to the antiviral pharmaceutical composition of the present invention, preferably, the alcohol extract is prepared by the following method: mixing folium Perillae with alcohol solvent at a ratio of 6-10mL per gram of folium Perillae, heating and reflux-extracting for 1.5-2.5 hr for 2-3 times, filtering, mixing filtrates, concentrating into extract, and vacuum drying to obtain folium Perillae extract. Also preferably, the perilla leaf is mixed with the alcohol solvent according to the proportion of 7-9mL of the alcohol solvent per gram of perilla leaf,
According to the antiviral drug composition, preferably, the alcohol solvent is an alcohol-water mixed solvent, wherein the volume concentration of the alcohol is 60-80%. Also preferably, the alcohol solvent is a 60% -80% ethanol aqueous solution.
Preferably, the virus is selected from the group consisting of respiratory virus and enterovirus.
Preferably, the virus includes influenza a virus, human coronavirus HCoV-229E and coxsackie virus.
In a second aspect of the invention, there is provided a method for inhibiting viral activity in vitro comprising the step of administering to a virus an extract of perilla leaf.
The method for inhibiting viral activity in vitro according to the second aspect, preferably, the virus is an intracellular virus. In vitro cells include, but are not limited to: human embryonic lung fibroblasts (MRC-5), human lung cancer cells (A549), canine kidney cells (MDCK), Vero cells (Vero).
In a third aspect of the invention, there is provided use of an extract of perilla leaf for the preparation of an antiviral medicament. It is understood that one skilled in the art can combine the perilla leaf extract or a composition containing the perilla leaf extract of the present application with other extracts/compositions having antiviral active ingredients to achieve further antiviral effects, as desired.
The perilla leaf extract of the invention not only shows low cytotoxicity, but also has significant inhibitory effect on respiratory viruses. In the invention, the tested medicine has obvious inhibition effect on influenza A virus H1N1 and influenza A virus H3N2 and human coronavirus HCoV-229E under the condition of non-cytotoxic concentration, can improve the cytopathic condition, improve the survival rate of virus infected host cells, effectively inhibit virus replication and show better dose-effect relationship.
In addition, the perilla leaf extract of the present invention has an inhibitory effect on enteroviruses. In the invention, the tested medicine has obvious inhibiting effect on the coxsackie virus under the condition of non-cytotoxic concentration, can improve the cytopathic condition and the survival rate of virus infected host cells, and simultaneously shows better dose-effect relationship. Therefore, the perilla leaf extract has obvious antiviral effect.
Drawings
FIG. 1 shows the toxic effect of the perilla leaf alcohol extract on in vitro cultured cells (x 20).
FIG. 2 shows the effect of perilla leaf alcohol extract on MDCK cytopathic effect of influenza A virus H1N1 FM 1.
Fig. 3 shows the effect of perilla leaf alcohol extract on the survival rate of MDCK cells caused by influenza a virus H1N1 FM1 (N-6).
FIG. 4 shows the effect of perilla leaf alcohol extract on influenza A virus H1N1 FM1 viral load.
FIG. 5 shows the effect of perilla leaf alcohol extract on A549 cell pathology caused by influenza A virus H1N1 PR 8.
Fig. 6 shows the effect of perilla leaf alcohol extract on the survival rate of MDCK cells caused by influenza a virus H1N1 PR8 (N-6).
FIG. 7 shows the effect of perilla leaf alcohol extract on the viral load of influenza A virus H1N1 PR 8.
FIG. 8 shows the effect of perilla leaf alcohol extract on A549 cytopathic effect of influenza A virus H3N 2.
Fig. 9 shows the effect of perilla leaf alcohol extract on the survival rate of a549 cells caused by influenza a virus H3N2 (N ═ 6).
FIG. 10 is a graph showing the effect of perilla leaf alcohol extract on the viral load of influenza A virus H3N 2.
FIG. 11 shows the effect of the perilla leaf alcohol extract on MRC-5 cytopathy caused by human coronavirus HCoV-229E.
FIG. 12 shows the effect of perilla leaf alcohol extract on the survival rate of MRC-5 cells caused by human coronavirus HCoV-229E (n-6).
FIG. 13 shows the effect of perilla leaf alcohol extract on the viral load of human coronavirus HCoV-229E.
FIG. 14 shows the effect of perilla leaf alcohol extract on Coxsackie virus B 5 Influence on Vero cytopathic effect.
FIG. 15 shows the effect of perilla leaf alcohol extract on coxsackie virus B 5 The influence on Vero cell survival (n-6).
FIG. 16 shows the effect of perilla leaf alcohol extract on coxsackie virus B 4 Influence on Vero cytopathic effect.
FIG. 17 shows the effect of perilla leaf alcohol extract on Coxsackie virus B 4 The influence on Vero cell survival (n-6).
FIG. 18 shows the effect of perilla leaf alcohol extract on Coxsackie virus B 3 Influence on Vero cytopathic effect.
FIG. 19 shows the effect of perilla leaf alcohol extract on coxsackie virus B 3 The influence on Vero cell survival (n-6).
FIG. 20 shows the effect of perilla leaf alcohol extract on coxsackie virus A 16 Cause Vero cell diseaseThe effect of the variation.
FIG. 21 shows the effect of perilla leaf alcohol extract on coxsackie virus A 16 The influence on Vero cell survival (n-6).
FIG. 22 shows the effect of perilla leaf water extract on MDCK cytopathic effect of influenza A virus H1N1 FM 1.
Fig. 23 is a graph showing the effect of perilla leaf water extract on survival of MDCK cells from influenza a virus H1N1 FM1 (N-6).
FIG. 24 shows the effect of aqueous perilla leaf extract on A549 cytopathic effect of influenza A virus H3N 2.
Fig. 25 is a graph showing the effect of aqueous perilla leaf extract on the survival of a549 cells caused by influenza a virus H3N2 (N-6).
FIG. 26 shows the effect of aqueous extracts of perilla leaves on MRC-5 cytopathy caused by human coronavirus HCoV-229E.
FIG. 27 is a graph showing the effect of aqueous extracts of perilla leaf on the survival of MRC-5 cells caused by human coronavirus HCoV-229E (n-6).
FIG. 28 shows the effect of an aqueous extract of perilla leaves on coxsackie virus B 5 Influence on Vero cytopathic effect.
FIG. 29 shows the effect of an aqueous extract of perilla leaf on Coxsackie virus B 5 The influence on Vero cell survival (n-6).
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
It will be appreciated by those skilled in the art that other steps or operations may be included in the extraction process of the present invention, for example to further optimize and/or improve the process of the present invention, so long as the objectives of the present invention are achieved. For example, the extraction method may further comprise the step of heating and refluxing the perilla leaves and the water/alcohol mixture for a plurality of times.
Example 1
The embodiment is a research on pharmacodynamic tests of perilla leaf alcohol extract for resisting respiratory tract viruses and enteroviruses in vitro, and the corresponding in vitro antiviral pharmacodynamic tests are designed based on an in vitro cell virus infection model according to the pathogenic characteristics of each virus strain and the characteristics of traditional Chinese medicines. Four doses of 100, 10, 1 and 0.1mg/mL of perilla leaf alcohol extract are designed, the cell survival rate and the virus replication inhibition effect on 11 respiratory viruses and 6 enteroviruses are observed, and the antiviral efficacy is evaluated as follows:
firstly, extracting
Weighing a proper amount of perilla leaves, and mixing the perilla leaves according to the weight ratio of 1 g: mixing perilla leaf with 70 vol% ethanol water solution according to the proportion of 8mL, heating and refluxing for 2 hours, extracting for 2 times, filtering, combining filtrates, concentrating into an extract, and vacuum drying to obtain the perilla leaf extract.
Second, the effectiveness research of perilla leaf alcohol extract for preventing and treating respiratory tract and enterovirus
1. Test materials
1.1 cells
Figure BDA0003275661960000041
1.2 Virus strains
Figure BDA0003275661960000042
Figure BDA0003275661960000051
1.3 reagents
Figure BDA0003275661960000052
1.4 instruments
Figure BDA0003275661960000053
Figure BDA0003275661960000061
1.5 test drugs: folium Perillae 70% ethanol extract. Storage conditions were as follows: and (5) drying at normal temperature.
1.6 Positive control: reidesciclovir, available from Targetmol USA, with a production lot number: 144250, respectively; CAS: 1809249-37-3; molecular weight: 602.58, respectively; the molecular formula is as follows: C27H35N6O 8P; specification: 5 mg/tube; the characteristics are as follows: a white powder; storage conditions were as follows: storing at-20 deg.C. The pharmacological action is as follows: nucleoside analogs having antiviral activity on HAE cells, and EC of SARS-CoV and MERS-CoV 50 Is 74. mu.M.
Oseltamivir phosphate capsules (tamiflu), produced by Delpharm Milano s.r.l. italy, packaged by shanghai roche pharmaceutical limited; production batch number: m1066; subpackage batch number: SH 0089; specification: 75 mg/pellet, 10 pellets/box; the characteristics are as follows: grey and light yellow capsules, the content is white to yellowish white powder; storage conditions were as follows: storing at below 25 deg.C; the production date is as follows: 2019.06.05, effective period to: 2024.06.04. indications are as follows: antiviral agents for the prevention and treatment of influenza. The usage and dosage are as follows: it is administered orally at a dose of 1 granule for adult and 2 times daily.
Ribavirin granules, produced by Sichuan Baili pharmaceutical Co., Ltd; production batch number: 191205, respectively; specification: 50 mg/bag, 18 bags/box; the characteristics are as follows: white or off-white soluble particles; storage conditions were as follows: sealing and storing in a dry place; the production date is as follows: 2019.12.05, effective period to: 2021.11. the indications are as follows: viral pneumonia and bronchitis caused by respiratory syncytial virus, and skin herpes infection.
2. Experimental method
2.1 dose design
Perilla leaf alcohol extract: according to the results of the previous experiments, four doses of 100, 10, 1 and 0.1 mu g/mL are externally applied, and the medicinal powder is diluted to the corresponding concentration during the experiment.
Tamiflu: the lowest dilution at which no significant lesions appear was determined to be the maximum non-Toxic Concentration (TC) on the cells involved in the experiment 0 ) At TC 0 And the subsequent 2-3 concentrations are the drug concentration of the positive control wells. In the experiment, the tamiflu is TC on A549 and MDCK cells 0 Both were 78.125. mu.g/mL.
Ribavirin: the lowest dilution at which no significant lesions appear was determined to be the maximum non-Toxic Concentration (TC) on the cells involved in the experiment 0 ) At TC 0 And the subsequent 2-3 concentrations are the drug concentration of the positive control wells. In the experiment, the ribavirin is TC on Hep-2, Hela and Vero cells 0 Both 312.5. mu.g/mL, TC on RD cells 0 The concentration was 156.25. mu.g/mL.
Reidesciclovir: the instructions suggest that it is on HAE cells, EC of SARS-CoV and MERS-CoV 50 Is 74 nM. In the experiment, three doses of 296, 148 and 74nmol/L are arranged outside the RudeSewei, and the medicine powder is diluted to corresponding concentration during the experiment.
2.2 criteria for cytopathic judgment
Cytopathic effect is judged according to 6-grade standard:
-: the cells grow normally and no disease is generated;
+ -: cytopathic effects are less than 10% of the entire monolayer;
+: cytopathic effects account for less than about 25% of the entire monolayer of cells;
++: cytopathic effects comprise less than about 50% of the total monolayer of cells;
+++: cytopathic effects account for less than about 75% of the total monolayer of cells;
++++: cytopathic effects account for more than about 75% of the total monolayer of cells.
2.3 statistical analysis
Data analysis was performed using GraphPad Prism 6.0 software. Measure data to
Figure BDA0003275661960000071
Showing that the difference between groups was measured by t-test, P<A difference of 0.05 is statistically significant.
Figure BDA0003275661960000072
Figure BDA0003275661960000073
3. Results of the experiment
3.1 toxicity test of Perilla leaf alcohol extract on in vitro cultured cells
The tested drug (original concentration 50mg/mL) is diluted by culture solution in a ratio of 1:2-1:512, and then added into a monolayer-grown A549, Vero, MDCK, MRC-5, Hep-2, Hela and RD cell culture plate (96 wells) with 100 muL/well, 6 multiple wells are made for each dilution liquid medicine, and a normal cell control is set. The plates were placed at 37 ℃ in 5% CO 2 Culturing in incubator, observing cytopathic condition under inverted microscope every day, and determining the lowest dilution factor of cell without obvious pathological changes as maximum nontoxic concentration (TC) 0 ) 50% cytotoxic concentration (TC) was calculated by the Reed-Muench method 50 ). FIG. 1 shows the toxic effect of the perilla leaf alcohol extract on in vitro cultured cells (x 20).
The results show that: TC of perilla leaf alcohol extract on A549 cells (72h) 0 195.31 μ g/mL, TC 50 472.03 μ g/mL; TC for Vero cells (48h) 0 195.31 μ g/mL, TC 50 472.03 μ g/mL μ g/mL; TC for MDCK cells (72h) 0 390.63 μ g/mL, TC 50 931.04 μ g/mL; TC for MRC-5 cells (72h) 0 97.66. mu.g/mL, TC 50 262.41 μ g/mL; TC for Hep-2 cells (48h) 0 390.63 μ g/mL, TC 50 561.01 μ g/mL; TC in Hela cells (72h) 0 390.63 μ g/mL, TC 50 931.04 μ g/mL; TC for RD cells (72h) 0 195.31 μ g/mL, TC 50 It was 523.56. mu.g/mL.
3.2 respiratory Virus test results
3.2.1 Effect of perilla leaf alcohol extract on cytopathic effect of influenza A virus H1N1 FM1 Strain (CPE method)
Collecting MDCK cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Influenza A virus H1N1 FM1 virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine, 100 μ L/well, and setting normal cell control, virus control, and Tamiflu control. Placing at 37 ℃ with 5% CO 2 Continuously culturing in the incubator, observing the pathological condition of the cells under an inverted microscope after 72h, recording the pathological condition result, detecting the survival condition of the cells (CCK8), and sucking cell supernatant to detect the virus load, wherein the specific result is shown in Table 1 and figure 2.
TABLE 1 Effect of perilla leaf alcohol extract on MDCK cytopathy caused by influenza A virus H1N1 FM1 (CPE method)
Figure BDA0003275661960000074
Figure BDA0003275661960000081
In fig. 2: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; galaphenanthrene (39.06. mu.g/mL).
The results in table 1 and figure 2 show that: after MDCK cells are infected by influenza A virus H1N1 FM1 for 72H, the cells are in lesions such as shrinkage, necrosis and desquamation. After the perilla leaf alcohol extract is subjected to dry prognosis, cytopathy caused by viruses can be obviously inhibited, the drug effect is optimal with 100 and 10 mu g/mL doses, and the four doses of 100, 10, 1 and 0.1 mu g/mL show good dose-effect relationship.
Fig. 3 shows the effect of perilla leaf alcohol extract on the survival rate of MDCK cells from influenza a virus H1N1 FM1 (N ═ 6), where P is <0.05 and P is <0.01, compared to model controls. The results in the figure show that: after MDCK cells are infected by influenza A virus H1N1 FM1 for 72 hours, the cells are obviously diseased and even necrotized, and the number of living cells is reduced. After the dry prognosis of the perilla leaf alcohol extract, the cell survival rate is increased, and the three dose groups of 100, 10 and 0.1 mu g/mL have statistical difference compared with a virus control group (p is less than 0.01 or p is less than 0.05).
Fig. 4 is a graph of the effect of perilla leaf alcohol extract on influenza a virus H1N1 FM1 viral load, where N is 4, repeated 1 time per sample; p <0.05, P <0.01 compared to model control. The results in the figure show that: after MDCK cells are infected with influenza A virus H1N1 FM1, the virus load in the supernatant is obviously increased, and compared with a normal control group, the virus load is obviously different (p < 0.01). After the perilla leaf alcohol extract is intervened for 72 hours, the virus load in cell supernatants of the 100 and 10 mu g/mL dose groups is obviously reduced, and compared with a model control group, the virus load is statistically different (p is less than 0.05 or p is less than 0.01), the virus inhibition rates are 80.86 percent and 20.58 percent respectively, and the virus inhibition rate of the Tamiflu group is 93.16 percent.
3.2.2 Effect of perilla leaf alcohol extract on cytopathic effect of influenza A virus H1N1 PR8 Strain (CPE method)
Collecting A549 cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Influenza A virus H1N1 PR8 virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, and adding 4 dilutions of folium Perillae ethanol extractThe liquid medicine is 100 mu L/hole, and is provided with a normal cell control, a virus control and a Tamiflu control. Placing at 37 ℃ with 5% CO 2 Continuously culturing in an incubator, detecting the survival condition of the cells (CCK8) after 72h, observing the pathological condition of the cells under an inverted microscope after 96h, recording the pathological condition result, sucking cell supernatant to detect the virus load, and specifically referring to the table 2 and the figure 5.
TABLE 2 Effect of perilla leaf alcohol extract on A549 cytopathic effect of influenza A virus H1N1 PR8 (CPE method)
Figure BDA0003275661960000082
Figure BDA0003275661960000091
In fig. 5: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; galaphenanthrene (39.06. mu.g/mL).
Table 2 and fig. 5 results show: after influenza A virus H1N1 PR8 infects A549 cells for 96H, the cells have the lesions of cell shrinkage, necrosis, desquamation and the like, and the cell density is reduced. After the dry prognosis of the perilla leaf alcohol extract, three dose groups of 100, 10 and 1 mu g/mL can obviously inhibit cytopathic effect caused by viruses and show good dose-effect relationship.
Fig. 6 shows the effect of perilla leaf alcohol extract on the survival rate of MDCK cells from influenza a virus H1N1 PR8 (N ═ 6), wherein P <0.01, compared to model control group. The results in the figure show that: after MDCK cells are infected by influenza A virus H1N1 PR8 for 72H, the cells are necrosed, and the number of living cells is reduced. After the dry prognosis of the perilla leaf alcohol extract, the cell survival rate is increased, and the three dose groups of 100, 10 and 0.1 mu g/mL have statistical difference compared with a virus control group (p is less than 0.01).
FIG. 7 shows the effect of perilla leaf alcohol extract on the viral load of influenza A virus H1N1 PR 8. Where n is 4, each sample is repeated 1 time; p <0.01 compared to model control group. The results in the figure show that: after A549 cells are infected by influenza A virus H1N1 PR8, the virus load in the supernatant is obviously increased, and compared with a normal control group, the virus load is obviously different (p < 0.01). After chlorogenic acid intervenes for 96 hours, the virus load in cell supernatants of three dose groups of 100, 10 and 0.1 mu g/mL is obviously reduced, and compared with a model control group, the virus load is obviously different (p is less than 0.01), the virus inhibition rates are respectively 30.24%, 35.28% and 23.76%, and the virus inhibition rate of the Tamiflu group is 54.15%.
3.2.3 Effect of perilla leaf alcohol extract on cytopathic effect of influenza A virus H3N2 strain (CPE method)
Collecting A549 cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Influenza A virus H3N2 virus solution with 100 μ L/well, making 6 compound wells for each dilution liquid medicine, placing at 37 deg.C 5% CO 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine, 100 μ L/well, and setting normal cell control, virus control, and Tamiflu control. Placing at 37 ℃ with 5% CO 2 Continuously culturing in the incubator, observing the pathological condition of the cells under an inverted microscope after 72h, recording the pathological condition result, detecting the survival condition of the cells (CCK8), and sucking cell supernatant to detect the virus load. See table 3 and fig. 8 for specific results.
TABLE 3 Effect of perilla leaf alcohol extract on A549 cytopathic effect caused by influenza A virus H3N2 (CPE method)
Figure BDA0003275661960000092
In fig. 8: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; galaphenanthrene (39.06. mu.g/mL).
The results in table 3 and fig. 8 show that: after influenza A virus H3N2 infects A549 cells for 72H, the cells have lesions of cell shrinkage, necrosis and desquamation, and the cell density is reduced. After the dry prognosis of the perilla leaf alcohol extract, three dose groups of 100, 10 and 1 mu g/mL have certain inhibition effect on cytopathic effect caused by viruses.
Fig. 9 shows the effect of perilla leaf alcohol extract on the survival of a549 cells caused by influenza a virus H3N2 (N ═ 6), wherein P is <0.05 and P is <0.01, compared to model control group. The results in the figure show that: after influenza A virus H3N2 infects A549 cells for 72H, the cells have lesions such as round shrinkage, necrosis and the like, and the cell density is reduced. After the prognosis of the perilla leaf alcohol extract, the cell survival rate is obviously improved, and the 100 and 1 mu g/mL dose groups have statistical difference compared with a virus control group (p is less than 0.05). 100. The four dose groups of 10, 1 and 0.1 mug/mL do not show good dose-effect relationship.
Fig. 10 is a graph of the effect of perilla leaf alcohol extract on influenza a H3N2 viral load, where N is 4, repeated 1 time per sample; p <0.01 compared to model control group. The results in the figure show that: after A549 cells are infected with influenza A virus H3N2, the virus load in the supernatant is obviously increased, and compared with a normal control group, the virus load is obviously different (p < 0.01). After the perilla leaf alcohol extract is intervened for 72 hours, the virus load in cell supernatants of four dose groups of 100, 10, 1 and 0.1 mu g/mL is obviously reduced, compared with a model control group, the virus load is obviously different (p is less than 0.01), and the virus inhibition rates are 47.96%, 71.80%, 58.86% and 58.22% respectively. The inhibition rate of the duffy virus is 93.92%.
3.2.4 Effect of Perilla leaf alcohol extract on cytopathic effects of human coronavirus (CPE method)
Collecting MRC-5 cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, and inoculating with 100TCID 50 Human coronavirus HCoV-229E virus solution, 100 mu L/well, 6 multiple wells for each dilution liquid medicine, placing at 37 ℃ with 5% CO 2 Adsorbing in incubator for 2 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine, 100 μ L/well, and setting normal cell control, virus control, and Rdesivir control. Placing at 37 ℃ with 5% CO 2 Culturing in the incubator, observing the pathological condition of the cells under an inverted microscope after 72h, recording the pathological condition result, detecting the survival condition of the cells (CCK8), and sucking cell supernatant to detect the virus load, which is shown in Table 4 and FIG. 11.
TABLE 4 Effect of perilla leaf alcohol extract on MRC-5 cytopathy caused by human coronavirus HCoV-229E (CPE method)
Figure BDA0003275661960000101
In fig. 11: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; g Ruidexiwei (148 mu mol/L).
Table 4 and fig. 11 results show: after MRC-5 cells are infected by the human coronavirus HCoV-229E for 72 hours, the cells have the pathological changes of round shrinkage, enhanced refractive index, flaking, necrosis and the like. After the perilla leaf alcohol extract is dried, cytopathy caused by viruses can be inhibited, wherein three doses of 100, 10 and 1 mu g/mL have obvious inhibition effect on HCoV-229E in vitro.
Figure 12 is a graph of the effect of perilla leaf alcohol extract on MRC-5 cell survival by human coronavirus HCoV-229E (n-6), wherein P is <0.05 compared to model group. The results in the figure show that: after HCoV-229E infects MRC-5 cells for 72h, the cells have obvious lesion and necrosis, and the number of living cells is reduced. After the prognosis of the perilla leaf alcohol extract, the cell survival rate of the group with the dose of only 100 mu g/mL is obviously improved compared with the virus control group (p is less than 0.05), the cell survival rate of the two groups with the dose of 10 mu g/mL and 1 mu g/mL is increased compared with the virus control group, and the statistical difference is not generated compared with the virus control group (p is more than 0.05).
Fig. 13 is a graph of the effect of perilla leaf alcohol extract on the viral load of human coronavirus HCoV-229E, wherein n is 4, and each sample was repeated 1 time; p <0.05, P <0.01 compared to model control. The results in the figure show that: after MRC-5 cells are infected with human coronavirus HCoV-229E, the virus load in the supernatant is obviously increased, and compared with a normal control group, the virus load is obviously different (p is less than 0.01). After the perilla leaf alcohol extract is intervened for 72 hours, the virus load in the cell supernatant of the 100 mu g/mL dose group is obviously reduced, and compared with a model control group, the statistical difference is realized (p is less than 0.05), and the virus inhibition rate is respectively 34.91%.
3.3 Enterovirus test results
3.3.1 Perilla leaf alcohol extract against Coxsackie virus B 5 Cell disease caused by strainVariable influence (CPE method)
Collecting Vero cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Coxsackie virus B 5 (Cox B 5 ) Virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and ribavirin control. Placing at 37 ℃ with 5% CO 2 The culture was continued in the incubator, and after 48 hours, the pathological changes of the cells were observed under an inverted microscope, and the pathological changes were recorded, and the survival of the cells was examined (CCK8), and the results are shown in Table 5 and FIG. 14.
TABLE 5 Perilla leaf alcohol extract against Coxsackie virus B 5 Effect on Vero cytopathy (CPE method)
Figure BDA0003275661960000111
In fig. 14: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; g ribavirin (156.25. mu.g/mL).
The results in table 5 and fig. 14 show: coxsackie virus B 5 After the Vero cells are infected for 48 hours, the cells have lesions of shrinking, necrosis, desquamation and the like, and no living cells with normal forms are seen on the bottom layer. After the dry prognosis of the perilla leaf alcohol extract, the cell pathological changes caused by viruses can be obviously inhibited, the 100 and 10 mu g/mL dose group can be seen as normal living cells with the shape of the sheet distribution, and the 1 mu g/mL dose group can be seen as a small amount of normal living cells with the shape of the scattered distribution. 100. The four doses of 10, 1 and 0.1 mug/mL show good dose-effect relationship.
FIG. 15 shows the effect of perilla leaf alcohol extract on coxsackie virus B 5 Effect on survival of Vero cells (n-6), wherein P is compared to model control group<0.01. The results in the figure show that: coxsackie virus B 5 After 48h of Vero cell infection, the cells showed severe lesions with only a few viable cells. After intervention of perilla leaf alcohol extractThe cell survival rate is obviously improved, and the three dose groups of 100, 10 and 0.1 mu g/mL have statistical difference compared with the virus control group (p)<0.01 or p<0.05)。
3.3.2 Perilla leaf alcohol extract against Coxsackie virus B 4 Effect of Strain induced cytopathy (CPE method)
Collecting Vero cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Coxsackie virus B 4 (Cox B 4 ) Virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and ribavirin control. Placing at 37 ℃ with 5% CO 2 The culture was continued in the incubator, after 48h, the pathological changes of the cells were observed under an inverted microscope, the pathological changes were recorded, and the survival of the cells was examined (CCK8), for details, see Table 6 and FIG. 16.
TABLE 6 Perilla leaf alcohol extract against Coxsackie virus B 4 Effect on Vero cytopathy (CPE method)
Figure BDA0003275661960000121
In fig. 16: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; g ribavirin (156.25. mu.g/mL).
The results in table 6 and fig. 16 show: coxsackie virus B 4 After the Vero cells are infected for 48 hours, the cells have lesions of shrinking, necrosis, desquamation and the like, and no living cells with normal forms are seen on the bottom layer. After the perilla leaf alcohol extract is subjected to dry prognosis, cytopathy caused by viruses can be obviously inhibited, and the groups with 100 and 10 mu g/mL doses can show normal living cells with clustered distribution.
FIG. 17 shows the effect of perilla leaf alcohol extract on Coxsackie virus B 4 Effect on survival of Vero cells (n-6), wherein P is compared to model control group<0.05，**P<0.01. The results in the figure show that: korsa (Korsak) K.K.K.KOdd virus B 4 After 48h of Vero cell infection, the cells showed severe lesions with only a few viable cells. The survival rate of cells after the dry prognosis of the perilla leaf alcohol extract is increased, and the three dose groups of 100, 10 and 0.1 mu g/mL are all statistically different from the virus control group (p)<0.01 or p<0.05), wherein the drug effect of the 100 mu g/mL dose group is particularly remarkable.
3.3.3 Perilla leaf alcohol extract to Coxsackie virus B 3 Effect of Strain induced cytopathy (CPE method)
Collecting Vero cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Coxsackie virus B 3 (Cox B 3 ) Virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and ribavirin control. Put at 37 ℃ in 5% CO 2 The culture was continued in the incubator, and after 48 hours, the pathological changes of the cells were observed under an inverted microscope, and the pathological changes were recorded, and the survival of the cells was examined (CCK8), as shown in Table 7 and FIG. 18.
TABLE 7 Perilla leaf alcohol extract against Coxsackie virus B 3 Effect on Vero cytopathy (CPE method)
Figure BDA0003275661960000122
Figure BDA0003275661960000131
In fig. 18: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; g ribavirin (156.25. mu.g/mL).
The results in table 7 and fig. 18 show: coxsackie virus B 3 After the Vero cells are infected for 48 hours, the cells have lesions of shrinking, necrosis, desquamation and the like, and only a small amount of living cells with normal forms are seen on the bottom layer. Perilla frutescens (L.) BrittAfter the leaf alcohol extract is subjected to dry prognosis, cytopathy caused by viruses can be inhibited, a large amount of morphologically normal living cells distributed on the bottom layer of a 100 and 10 mu g/mL dose group can be seen, and a small amount of morphologically normal living cells distributed in a scattered manner can be seen in a 1 and 0.1 mu g/mL dose group. The four dosages of the perilla leaf alcohol extract show good dose-effect relationship.
FIG. 19 shows that the perilla leaf extract has an effect on coxsackie virus B 3 Effect on survival of Vero cells (n-6), wherein P is compared to model control group<0.05，**P<0.01. The results in the figure show that: coxsackie virus B 3 After 48h of Vero cell infection, the cells showed severe lesions with only a few viable cells. After the dry prognosis of the perilla leaf alcohol extract, the cell survival rate is obviously improved, and the four dose groups of 100, 10, 1 and 0.1 mu g/mL have statistical difference (p) compared with a virus control group<0.01 or p<0.05) and shows better dose-effect relationship.
3.3.4 Perilla leaf alcohol extract to Coxsackie virus A 16 Effect of Strain induced cytopathy (CPE method)
Collecting Vero cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Coxsackie virus A 16 (Cox A 16 ) Virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 2 hr, discarding virus solution, adding 4 dilutions of folium Perillae ethanol extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and ribavirin control. Placing at 37 ℃ with 5% CO 2 Culturing in the incubator, observing cytopathic condition under an inverted microscope after 120h, recording pathological change results, and detecting cell survival (CCK8), see Table 8 and FIG. 20.
TABLE 8 Perilla leaf alcohol extract against Coxsackie virus A 16 Effect on Vero cytopathy (CPE method)
Figure BDA0003275661960000132
In fig. 20: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf alcohol extract; d, extracting perilla leaf with alcohol 10 microgrammes/mL; e, 1 mu g/mL of perilla leaf alcohol extract; f, perilla leaf alcohol extract of 0.1 mu g/mL; g ribavirin (156.25. mu.g/mL).
The results in table 8 and fig. 20 show: coxsackie virus A 16 After Vero cells are infected for 120 hours, the cells have lesions such as round shrinkage, necrosis and desquamation. After the perilla leaf alcohol extract is dried, the cell lesion caused by virus can be inhibited, and the drug effect of the two dose groups of 10 and 1 mu g/mL is better.
FIG. 21 shows the effect of perilla leaf alcohol extract on coxsackie virus A 16 Effect on survival of Vero cells (n-6), wherein P is compared to model control group<0.01. The results in the figure show that: coxsackie virus A 16 After Vero cells are infected for 120 hours, the cells are diseased, necrotic and have reduced cell density. After the prognosis of the perilla leaf alcohol extract, the cell survival rate is obviously improved, and the two dose groups of 10 and 1 mu g/mL have statistical difference (p) compared with a virus control group<0.01)。
4. Conclusion
The perilla leaf alcohol extract shows low cytotoxicity on A549 cells, Vero cells, MDCK cells, MRC-5 cells, Hep-2 cells, Hela cells and RD cells, TC0 cells are 97.66-390.63 mu g/mL cells, and TC50 cells are 262.41-931.04 mu g/mL cells.
The folium Perillae alcoholic extract has effect in inhibiting respiratory tract virus. The tested medicine has obvious inhibiting effect on influenza A virus H1N1(FM1 and PR8 strains) under the condition of no cytotoxicity concentration, has obvious inhibiting effect on influenza A virus H3N2 and human coronavirus HCoV-229E, can improve the cytopathic condition, improve the survival rate of virus infected host cells and effectively inhibit virus replication, and shows better dose-effect relationship in four dose groups of 100, 10, 1 and 0.1 mu g/mL.
The tested medicine has obvious inhibiting effect on coxsackie virus B5 and B4 strains, obvious inhibiting effect on coxsackie virus B3 and A16 strains, improvement on cytopathic condition and improvement on the survival rate of virus infected host cells under the condition of no cytotoxicity concentration, and four dosage groups of 100, 10, 1 and 0.1 mu g/mL show better dose-effect relationship.
The results of this experiment were combined to draw conclusions: the perilla leaf alcohol extract has obvious antiviral effect on influenza A virus, human coronavirus and Coxsackie virus.
Example 2
The embodiment is a research on pharmacodynamic tests of perilla leaf water extracts for resisting respiratory tract viruses and enteroviruses in vitro, and the corresponding in vitro antiviral pharmacodynamic tests are designed based on in vitro cell virus infection models according to the pathogenic characteristics of various virus strains and the characteristics of traditional Chinese medicines. Four doses of 100, 10, 1 and 0.1mg/mL of perilla leaf aqueous extract are designed, and the effects of the perilla leaf aqueous extract on cytopathic effect (CPE) caused by 11 respiratory viruses and 6 enteroviruses and the cell survival rate are observed and the antiviral efficacy of the perilla leaf aqueous extract is evaluated. The method comprises the following specific steps:
Firstly, extracting
Weighing a proper amount of perilla leaves, and mixing the raw materials according to the weight ratio of 1g of perilla leaves: mixing folium Perillae with water at a ratio of 10mL, heating under reflux, decocting for 2 hr, extracting for 2 times, filtering, mixing filtrates, concentrating into extract, and vacuum drying to obtain folium Perillae water extract.
Second, the research on the effectiveness of perilla leaf water extract in preventing and treating respiratory tract and intestinal tract viruses
Effect of perilla leaf aqueous extract on cytopathic effect of influenza A virus H1N1 FM1 strain (CPE method)
Collecting MDCK cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 75TCID 50 Influenza A virus H1N1 FM1 virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae water extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and Tamiflu control. Placing at 37 ℃ with 5% CO 2 Culturing in the incubator, observing the pathological condition of the cells under an inverted microscope after 72h, recording the pathological condition result, and detecting the survival condition of the cells (CCK8), wherein the specific results are shown in a table 9 and a figure 22.
TABLE 9 Effect of aqueous Perilla leaf extracts on MDCK cytopathic effects of influenza A virus H1N1 FM1 (CPE method)
Figure BDA0003275661960000141
Figure BDA0003275661960000151
In fig. 22: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf water extract; d, 10 mu g/mL of perilla leaf water extract; e, 1 mu g/mL of perilla leaf water extract; f, 0.1 mu g/mL of perilla leaf water extract; galaphenanthrene (39.06. mu.g/mL).
The results in table 9 and fig. 22 show: after MDCK cells are infected by influenza A virus H1N1 FM1 for 72H, the cells are in lesions such as shrinkage, necrosis and desquamation. The dry prognosis of the perilla leaf water extract can obviously inhibit cytopathy caused by viruses, the drug effect is optimal with 100 and 10 mu g/mL doses, and the four doses of 100, 10, 1 and 0.1 mu g/mL show good dose-effect relationship.
Fig. 23 is a graph of the effect of perilla leaf water extracts on the survival of influenza a virus H1N1 FM1 induced MDCK cells (N ═ 6), wherein P <0.01 compared to model control. The results in the figure show that: after MDCK cells are infected by influenza A virus H1N1 FM1 for 72 hours, the cells are obviously diseased and even necrotized, and the number of living cells is reduced. After the dry prognosis of the perilla leaf water extract, the cell survival rate is increased, and the three dose groups of 100, 10 and 0.1 mu g/mL have statistical difference compared with a virus control group (p is less than 0.01). 1.2 Effect of Perilla leaf Water extract on cytopathic Effect of influenza A virus H3N2 Strain (CPE method)
Collecting A549 cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 75TCID 50 Influenza A virus H3N2 virus solution with 100 μ L/well, making 6 compound wells for each dilution liquid medicine, placing at 37 deg.C 5% CO 2 Adsorbing in the incubator for 1h, discarding virus solution, adding 4 dilutions of folium Perillae water extract, 100 μ L/well, and setting normal cell control, virus control, and Tamiflu control. Put at 37 ℃ in 5% CO 2 The culture was continued in the incubator, and after 72 hours, the pathological changes of the cells were observed under an inverted microscope, and the pathological changes were recorded, and the survival of the cells was examined (CCK8), and the results are shown in Table 10 and FIG. 24.
TABLE 10 Effect of aqueous Perilla leaf extract on influenza A Virus H3N 2-induced A549 cytopathic effect (CPE method)
Figure BDA0003275661960000152
In fig. 24: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf water extract; d, 10 mu g/mL of perilla leaf water extract; e, 1 mu g/mL of perilla leaf water extract; f, 0.1 mu g/mL of perilla leaf water extract; galaphenanthrene (39.06. mu.g/mL).
The results in table 10 and fig. 24 show: after influenza A virus H3N2 infects A549 cells for 72H, the cells have lesions of cell shrinkage, necrosis and desquamation, and the cell density is reduced. After the dry prognosis of the perilla leaf water extract, two dosage groups of 100 and 10 mu g/mL have obvious inhibition effect on cytopathic effect caused by virus.
Fig. 25 is a graph of the effect of aqueous perilla leaf extract on the survival of a549 cells from influenza a virus H3N2 (N ═ 6), where P <0.05 and P <0.01, compared to model controls. The results in the figure show that: after influenza A virus H3N2 infected A549 cells for 72H, the cells showed a lot of necrosis. After the dry prognosis of the perilla leaf water extract, the cell survival rate is obviously improved, and the 100 and 10 mu g/mL dose groups have statistical difference compared with the virus control group (p is less than 0.05 or p is less than 0.01).
1.3 Effect of aqueous extracts of Perilla leaf on cytopathic effects of human Corona Virus (CPE method)
Collecting MRC-5 cell culture plate with monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, and inoculating with 100TCID 50 Human coronavirus HCoV-229E virus solution, 100 mu L/well, 6 multiple wells for each dilution liquid medicine, placing at 37 ℃ with 5% CO 2 Adsorbing in incubator for 2 hr, discarding virus solution, adding 4 dilutions of folium Perillae water extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and Rudexiwei control. Placing at 37 ℃ with 5% CO 2 The culture was continued in the incubator, and after 72 hours, the pathological changes of the cells were observed under an inverted microscope, and the pathological changes were recorded, and the survival of the cells was examined (CCK8), as shown in Table 11 and FIG. 26.
TABLE 11 Effect of aqueous extracts of perilla leaf on MRC-5 cytopathy caused by human coronavirus HCoV-229E (CPE method)
Figure BDA0003275661960000161
In fig. 26: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf water extract; d, 10 mu g/mL of perilla leaf water extract; e, 1 mu g/mL of perilla leaf water extract; f, 0.1 mu g/mL of perilla leaf water extract; g Ruideciclovir (148 mu mol/L).
The results in table 11 and fig. 26 show: after MRC-5 cells are infected by the human coronavirus HCoV-229E for 72 hours, the cells have the pathological changes of round shrinkage, enhanced refractivity, flaking, necrosis and the like. The perilla leaf water extract can inhibit cytopathy caused by viruses after dry prognosis, wherein three doses of 100, 10 and 1 mu g/mL have obvious inhibition effect on HCoV-229E in vitro.
Figure 27 is a graph of the effect of aqueous perilla leaf extract on MRC-5 cell survival by human coronavirus HCoV-229E (n ═ 6), where P is <0.05 and P is <0.01 compared to model groups. The results in the figure show that: after HCoV-229E infects MRC-5 cells for 72h, the cells have obvious lesion and necrosis, and the number of living cells is reduced. After the dry prognosis of the perilla leaf water extract, the cell survival rate of the 100 and 10 mu g/mL dose groups is obviously improved compared with that of the virus control group (p <0.05 or p <0.01), the cell survival rate of the 10 and 1 mu g/mL dose groups is increased compared with that of the virus control group, and the cell survival rate of the 10 and 1 mu g/mL dose groups is not statistically different from that of the virus control group (p > 0.05).
2. Results of enterovirus experiments
Perilla leaf water extract for treating Coxsackie virus B 5 Effect of Strain induced cytopathy (CPE method)
Collecting Vero cell culture plate with grown monolayer, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, inoculating 100TCID 50 Coxsackie virus B 5 (Cox B 5 ) Virus liquid, 100 mu L/hole, 6 compound holes for each dilution liquid medicine, and placing 5% CO at 37 DEG C 2 Adsorbing in incubator for 1 hr, discarding virus solution, adding 4 dilutions of folium Perillae water extract liquid medicine at 100 μ L/well, and setting normal cell control, virus control, and ribavirin control. Placing at 37 ℃ with 5% CO 2 Continuously culturing in the incubator, and observing cytopathic condition under an inverted microscope after 48hThe lesion results were recorded and cell survival was examined (CCK8), as shown in table 12 and fig. 28.
TABLE 12 aqueous extract of perilla leaf for coxsackie virus B 5 Influence on Vero cytopathy (CPE method)
Figure BDA0003275661960000171
In fig. 28: a normal control; comparing the model B; c, 100 mu g/mL of perilla leaf water extract; d, 10 mu g/mL of perilla leaf water extract; e, 1 mu g/mL of perilla leaf water extract; f, 0.1 mu g/mL of perilla leaf water extract; g ribavirin (156.25. mu.g/mL).
Table 12 and fig. 28 results show: coxsackie virus B 5 After the Vero cells are infected for 48 hours, the cells have lesions of shrinking, necrosis, desquamation and the like, and no living cells with normal forms are seen on the bottom layer. After the dry prognosis of the perilla leaf water extract, cytopathy caused by viruses can be obviously inhibited, the 100 mu g/mL dose group can be used for forming morphologically normal living cells which are distributed in a sheet shape, and the 10 mu g/mL dose group and the 1 mu g/mL dose group can be used for forming morphologically normal living cells which are distributed in a scattered shape.
FIG. 29 shows the effect of an aqueous extract of perilla leaf on Coxsackie virus B 5 Effect on survival of Vero cells (n-6), wherein P is compared to model control group<0.01. The results in the figure show that: coxsackie virus B 5 After 48h of Vero cell infection, the cells showed severe lesions with only a few viable cells. After the dry prognosis of the perilla leaf water extract, the cell survival rate is obviously improved, and the two dose groups of 100 and 10 mu g/mL have statistical difference compared with the virus control group (p) <0.01)。
The folium Perillae water extract has effect in inhibiting respiratory tract virus. The tested medicine has obvious inhibiting effect on influenza A virus H1N1(FM1 strain) and influenza A virus H3N2 and human coronavirus HCoV-229E under the condition of no cytotoxicity concentration, can improve the cytopathic condition and improve the survival rate of virus infected host cells.
The folium Perillae water extract has effect in inhibiting enterovirus. The tested medicine has no cytotoxicity to coxsackie virus B 5 The strain has remarkable inhibitory effect and can improve cellsPathological conditions, increasing the survival rate of virus infected host cells.
The result of the experiment is integrated to obtain the conclusion that: the perilla leaf water extract has obvious antiviral effect on influenza A virus, human coronavirus and Coxsackie virus, the effect is particularly obvious in 100 and 10 mu g/mL dose groups, and the four 100, 10, 1 and 0.1 mu g/mL dose groups show better dose-effect relationship.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments without departing from the scope or spirit of the present invention. The scope of the invention should be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims (5)

1. The application of the perilla leaf extract in preparing the medicine for resisting the coxsackie virus is characterized in that the extract is an aqueous extract or an alcohol extract, wherein the solvent of the alcohol extract is an ethanol aqueous solution with the volume concentration of 60-80%.
2. Use according to claim 1, characterized in that said aqueous extract is prepared by the following method: mixing folium Perillae with water at a ratio of 6-14mL per gram of folium Perillae, heating under reflux, decocting for 1-3 hr, extracting for 2-3 times, filtering, mixing filtrates, concentrating into extract, and vacuum drying to obtain folium Perillae water extract.
3. Use according to claim 1, characterized in that the alcoholic extract is prepared by: mixing folium Perillae with ethanol water solution at a ratio of 6-10 mL/g folium Perillae, heating and reflux-extracting for 1.5-2.5 hr for 2-3 times, filtering, mixing filtrates, concentrating into extract, and vacuum drying to obtain folium Perillae extract.
4. The use according to claim 1, wherein the coxsackievirus is coxsackievirus B 3 、B 4 、B 5 Or coxsackievirus A 16 。
5. A method for inhibiting in vitro coxsackie virus activity, characterized in that a perilla leaf extract is administered to a virus in vitro cells selected from at least one of human embryonic lung fibroblasts, human lung cancer cells, canine kidney cells and vero kidney cells;
Wherein the extract is an aqueous extract or an alcoholic extract, and the solvent of the alcoholic extract is an ethanol aqueous solution with the volume concentration of 60-80%.
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