CN111494456B - Application of herba Moslae aqueous extract in preparing medicine for resisting novel coronary virus - Google Patents
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Abstract
The invention discloses application of a herba elsholtziae aqueous extract in preparing a novel anti-coronary virus medicament, and also discloses application of the herba elsholtziae aqueous extract in preparing a novel coronary virus prevention medicament. The research of the invention finds that the herba elsholtziae aqueous extract can directly kill the novel coronavirus outside the cells, can inhibit the novel coronavirus from replicating in the cells, and has better capability of inhibiting the replication of the virus than ribavirin. Meanwhile, the research of the invention finds that after the elsholtzia water extract is adopted to pre-treat cells, the replication capacity of the virus in the cells is inhibited after the cells are infected with the novel coronavirus; the invention provides the application of the herba elsholtziae aqueous extract in preparing the medicine for preventing the novel coronavirus for the first time so as to relieve the symptoms of the body infected with the novel coronavirus.
Description
Technical Field
The invention belongs to the technical field of virus infection treatment, and particularly relates to application of an elsholtzia water extract in preparation of a novel coronavirus resistant medicine.
Background
A2019 Novel Coronavirus (NCP), namely "COVID-19", was discovered due to 2019 viral pneumonia cases and named by the world health organization on 1 month and 12 days 2020. People infected with 2019 new coronavirus may have symptoms of different degrees, mild people only have fever or slight cough, severe people may develop pneumonia, and more severe people may die.
2019 the novel coronavirus is a coronavirus newly discovered in the end of 2019, although the epidemic situation is well controlled, no specific medicine exists at present. Since the discovery of the 2019 novel coronavirus, intensive research has been actively and rapidly conducted in various research units and organizations in order to expect to find a drug having a therapeutic effect on the 2019 novel coronavirus infection.
To treat such a virus having a strong infectivity, an important research direction for a therapeutic drug is to newly use an old drug in order to enable rapid application. Herba Moslae (Elsholtzia citia (Thunb.) Hyland.) is a common Chinese medicinal material, and has effects of inducing sweat, relieving exterior syndrome, eliminating dampness, regulating stomach, inducing diuresis, and relieving swelling; the main treatment is as follows: summer cold, cold fluid, headache, fever, aversion to cold, no sweating, fullness in the chest, abdominal pain, vomiting, diarrhea, edema and beriberi.
In addition, the literature (Xuleqing, Jianweier, the research on the anti-influenza virus effect of the water extract of herba elsholtziae. Zhejiang J.J.2013, 48 (4): 273-.
At present, no report on the resistance of an aqueous extract of Elsholtzia splendens (Thunb.) Hyland) to the novel coronavirus is available.
Disclosure of Invention
The invention aims to provide a new application of the elsholtzia water extract and also provides a new active ingredient source for developing a novel coronavirus control medicament.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the application of herba Moslae chinensis aqueous extract (Mosla chinensis Maxim.) in preparing novel anti-coronary virus drugs is provided.
And secondly, the application of the elsholtzia water extract in preparing a novel medicine for preventing the coronary virus.
The invention researches and discovers that the herba elsholtziae aqueous extract can directly kill the novel coronavirus outside the cells, and after the herba elsholtziae aqueous extract acts on the cells infected with the novel coronavirus, the herba elsholtziae aqueous extract can also inhibit the novel coronavirus from replicating in the cells, and the capability of inhibiting the replication of the virus is superior to that of ribavirin. Meanwhile, the invention also researches and discovers that after the elsholtzia water extract is adopted to pre-treat cells, the replication capacity of the virus in the cells is inhibited after the cells are infected with the novel coronavirus; the invention provides the application of the herba elsholtziae aqueous extract in preparing the medicine for preventing the novel coronavirus for the first time so as to relieve the symptoms of the body infected with the novel coronavirus.
The preparation method of the elsholtzia chinensis aqueous extract is well known to those skilled in the art, and in order to implement the present invention, in the above application, the elsholtzia chinensis aqueous extract is prepared by the following method:
reflux-extracting herba Moslae with water as solvent by hot reflux extraction method, and filtering to obtain extractive solution; adding new solvent to reflux and extract the dregs at least once again, combining all the extracting solutions, and concentrating to obtain the elsholtzia chinensis aqueous extract.
In the application, when reflux extraction is carried out for the first time, the mass ratio of water to the elsholtzia splendens medicinal material is 30:1, and reflux extraction is carried out for 1-2 h; when the herb residue is subjected to reflux extraction, the amount of water added can be appropriately reduced.
In addition, the invention also provides a novel anti-coronary virus drug and a novel coronary virus prevention drug, and the effective components of the novel anti-coronary virus drug and the novel coronary virus prevention drug at least comprise an elsholtzia aqueous extract.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention researches and discovers that the herba elsholtziae aqueous extract can directly kill the novel coronavirus outside the cells, and after the herba elsholtziae aqueous extract acts on the cells infected with the novel coronavirus, the herba elsholtziae aqueous extract can also inhibit the novel coronavirus from replicating in the cells, and the capability of inhibiting the replication of the virus is superior to that of ribavirin.
(2) The research of the invention finds that after the elsholtzia water extract is adopted to pre-treat cells, the replication capacity of the virus in the cells is inhibited after the cells are infected with the novel coronavirus; the invention provides the application of the herba elsholtziae aqueous extract in preparing the medicine for preventing the novel coronavirus for the first time so as to relieve the symptoms of the body infected with the novel coronavirus.
Drawings
FIG. 1 is a graph showing the effect of an aqueous extract of Elsholtzia splendens on the resistance to novel coronavirus;
wherein, the killing group is the evaluation result of the direct killing capability of the herba elsholtziae aqueous extract to the novel coronavirus, the inhibition group is the evaluation result of the inhibition capability of the herba elsholtziae aqueous extract to the replication of the novel coronavirus in cells, and the prevention group is the evaluation result of the prevention effect of the herba elsholtziae aqueous extract to the novel coronavirus.
Detailed Description
The technical scheme of the invention is further explained in detail by combining the drawings and the detailed implementation mode.
Example 1
The novel coronavirus strain ZJ001 used in this example was isolated from a specimen of a new coronary pneumonia patient in 2020 by Zhejiang disease prevention and control center, and the virus strain was subjected to full-sequence gene sequencing and confirmed by the Chinese disease prevention and control center. The virus titer was 105.5TCID500.1mL, and stored at-80 ℃ for later use.
Vero cells (ATCC source, P128-P135 as the passage number used in the experiment) were used in this example and were able to support the propagation of the novel coronavirus strain ZJ 001.
1. Preparing herba Moslae aqueous extract
Weighing 100g of herba Moslae (dried aerial part of herba Moslae), extracting with water as solvent under reflux by hot reflux extraction method.
During the first extraction, 30 times of water is added for reflux extraction for 1 hour, and the extracting solution is filtered under reduced pressure; then adding 20 times of water to repeatedly extract the residues for 2 times, and filtering the extract under reduced pressure; the extracts obtained in 3 times were combined and concentrated to a crude drug content of 1g/ml to obtain the aqueous extract of elsholtzia chinensis of this example, which was then refrigerated at-20 ℃ for further use.
2. Maximum nontoxic concentration (TC) of drug on Vero cell0)
Before the test, the Vero cell maintenance solution (containing 2% fetal calf serum) is adopted to dilute the elsholtzia aqueous extract (with the concentration of 1g/mL) to 20mg/mL, 10mg/mL, 5mg/mL, 2.5mg/mL and 1.25mg/mL respectively for later use.
Inoculating Vero cells into a 24-hole plate, after the Vero cells grow into a single layer, removing the growth solution, respectively adding the elsholtzia chinensis aqueous extract with each concentration gradient into the cells, wherein the addition amount is 1 ml/hole, and meanwhile, setting a blank cell control; then, the cells were cultured at 37 ℃ for 48 hours, and the morphology of the cells was observed. TC of drug at the highest concentration that does not cause morphological changes in cells0. TC of aqueous extract of Elsholtzia splendens in Vero cells in this experiment0It was 10 mg/mL.
3. Evaluation of anti-novel coronavirus effect of herba Moslae chinensis aqueous extract
(1) Evaluation of direct killing ability of herba Moslae aqueous extract on novel coronavirus
Mixing 10mg/mL herba Moslae chinensis water extract with 100TCID500.1mL of the novel coronavirus is mixed in equal volume to be used as a test drug group; simultaneously, Vero cell maintenance solution and 100TCID are mixed500.1mL of the novel coronavirus was mixed in equal volume and used as a negative control group (i.e., virus control group).
Respectively placing the mixed liquor of the test drug group and the negative control group at 37 ℃ for incubation for 1h, respectively sucking 0.2mL of drug virus mixed liquor (namely the test drug group) and maintenance solution virus mixed liquor (namely the negative control group) and adding the mixed liquor into the grown single-layer Vero cells, adsorbing the Vero cells at 37 ℃ for 1h, then discarding the supernatant (namely the virus solution), washing the cells twice (1 mL/hole) by PBS, adding 1mL of Vero cell maintenance solution, and culturing at 37 ℃ for 48 h; harvesting the culture, freezing for 1 time at-80 deg.C, extracting virus RNA from 200uL of the harvested culture solution, detecting by fluorescent quantitative RT-PCR method, and comparing the virus nucleic acid levels of the negative control group and the test drug group.
Experiment is carried out by setting bottom hole of virus, culturing 2-hole Vero cells on a cell culture plate to form a monolayer, discarding growth medium, adding 100TCID500.1mL of novel coronavirus, the addition amount is 0.1 mL/hole, the novel coronavirus is cultured for 1h at 37 ℃, culture solution is collected, the supernatant is discarded by centrifugation, cells are washed twice by 1 mL/hole PBS, free viruses outside the cells are removed, and the plate is frozen at minus 80 ℃ after 1 mL/hole Vero cell maintenance solution is added and is used as the background for adsorbing the viruses into the cells. The nucleic acid level of the background is used as a reference value in relative quantification; the same applies below.
The results of the comparison are shown in table 1 and fig. 1.
TABLE 1
| Background of the invention | Test drug groups | Negative control group | |
| Ct value | 32.8 | 32.10 | 9.01 |
| Ct-Vero | 15.00 | 15.40 | 15.51 |
| ΔCt | 17.80 | 16.70 | -6.50 |
| ΔΔCt | 0 | -1.10 | -24.30 |
| 2-ΔΔCt | 1 | 2.14 | 20655175.75 |
| Log 2-ΔΔCt | 0 | 0.33 | 7.32 |
As can be seen from Table 1 and FIG. 1, the test drug group showed a 10-mg/mL reduction in viral nucleic acid levels after 48h treatment with 10mg/mL of the aqueous extract of Elsholtzia stauntoni compared to the negative control group6.99Multiple, substantially near background; the herba Moslae aqueous extract has the capability of directly killing novel coronavirus outside cells.
(2) Evaluation of inhibition ability of herba Moslae chinensis aqueous extract on novel coronavirus intracellular replication
Growing Vero cells in a single layer, discarding the growth liquid, adding 100TCID500.1mL of the novel coronavirus, wherein the addition amount is 0.1 mL/hole; after incubation at 37 ℃ for 1h, the supernatant was discarded and the cells were washed twice with 1 ml/well PBS to remove the virus free from the cells. Then adding 1mL of 10mg/mL herba Moslae aqueous extract (test drug group), 1mL of ribavirin (positive control group, concentration 1mg/mL) or VCulturing an ero cell maintenance solution (a negative control group) at 37 ℃ for 48h, harvesting a culture, freezing at-80 ℃ for 1 time, extracting viral RNA from 200uL of culture solution, detecting by a fluorescent quantitative RT-PCR method, and comparing the levels of viral nucleic acid in a test drug group, a positive control group and a negative control group, wherein the comparison result is shown in table 2 and figure 1.
TABLE 2
| Background of the invention | Test drug groups | Negative control group | Positive control group | |
| Ct value | 32.8 | 21.18 | 9.28 | 17.84 |
| Ct-Vero | 15.00 | 16.79 | 15.51 | 15.64 |
| ΔCt | 17.80 | 4.39 | -6.23 | 2.20 |
| ΔΔCt | 0 | -13.41 | -24.30 | 15.60 |
| 2-ΔΔCt | 1 | 10884.59 | 17129740.97 | 49667.00 |
| Log 2-ΔΔCt | 0 | 4.04 | 7.23 | 4.70 |
As can be seen from Table 2 and FIG. 1, the test drug group showed a 10-mg/mL reduction in viral nucleic acid levels after 48h treatment with 10mg/mL of the aqueous extract of Elsholtzia stauntoni compared to the negative control group3.19Doubling; while the level of viral nucleic acid in the positive control group was reduced by 102.53Doubling; the herba Moslae aqueous extract has intracellular ability of inhibiting virus replication, and the inhibition ability is superior to ribavirin.
(3) Evaluation of preventive effect of herba Moslae aqueous extract on novel coronavirus
Test drug groups: growing into a single layer of Vero cells, removing the growth liquid, adding 10mg/mL of herba Moslae aqueous extract with the addition amount of 1 mL/hole, and culturing at 37 ℃ for 24h to allow the medicine to enter the cells; after 24h, the herba Moslae liquid medicine on the cells is discarded, the cells are washed twice by 1 mL/hole PBS, and then 100TCID is added500.1mL of new coronavirus-infected cells, the amount of the cells added is 0.1 mL/well, and the cells are placedAfter culturing for 1h at 37 ℃, abandoning the virus solution, washing the cells twice by 1 mL/hole PBS, and then adding 1 mL/hole cell maintenance solution; the negative control group does not carry out drug treatment on the Vero cells in advance, and only adds the cell maintenance liquid all the time; culturing at 37 deg.C for 24h, harvesting culture, freezing at-80 deg.C for 1 time, extracting viral RNA from 200uL culture medium, detecting by fluorescence quantitative RT-PCR method, and comparing viral nucleic acid levels of virus control group and drug group, with the comparison results shown in Table 3 and FIG. 1.
TABLE 3
| Background of the invention | Test drug groups | Negative control group | |
| Ct value | 32.8 | 32.50 | 21.30 |
| Ct-Vero | 15.00 | 15.52 | 15.51 |
| ΔCt | 17.80 | 16.98 | 5.79 |
| ΔΔCt | 0 | -0.82 | -12.01 |
| 2-ΔΔCt | 1 | 1.77 | 4124.49 |
| Log 2-ΔΔCt | 0 | 0.25 | 3.62 |
As can be seen from Table 3 and FIG. 1, the virus nucleic acid level of the test drug group was reduced by 10 hours after the cells were pretreated with 10mg/mL of Elsholtzia splendens aqueous extract for 24 hours, compared with the negative control group3.37Doubling; the herba Moslae aqueous extract has effect in preventing novel coronavirus infection in vitro.
Claims (6)
1. Application of herba Moslae chinensis (Mosla chinensis Maxim.) water extract in preparing medicine for resisting novel coronary virus is provided.
2. The use of an aqueous extract of elsholtzia as claimed in claim 1 in the preparation of a medicament against novel coronary viruses, wherein said aqueous extract of elsholtzia is prepared by the following method:
reflux-extracting herba Moslae with water as solvent by hot reflux extraction method, and filtering to obtain extractive solution; adding water to reflux and extract the dregs of a decoction at least once again, combining all extracting solutions, and concentrating to obtain the elsholtzia chinensis aqueous extract.
3. The use of an aqueous extract of herba Moslae in the preparation of a medicament for treating a novel coronary virus disease as claimed in claim 2, wherein the mass ratio of water to herba Moslae is 30:1, and the reflux extraction is carried out for 1-2 h.
4. Use of herba Moslae chinensis aqueous extract in preparing medicine for preventing novel coronary virus is provided.
5. The use of an aqueous extract of herba Moslae in the preparation of a medicament for preventing a novel coronary virus according to claim 4, wherein the aqueous extract of herba Moslae is prepared by the following method:
reflux-extracting herba Moslae with water as solvent by hot reflux extraction method, and filtering to obtain extractive solution; adding water to reflux and extract the dregs of a decoction at least once again, combining all extracting solutions, and concentrating to obtain the elsholtzia chinensis aqueous extract.
6. The use of the aqueous extract of herba Moslae in the preparation of a medicament for preventing and treating coronary heart disease as claimed in claim 4, wherein the mass ratio of water to herba Moslae is 30:1, and reflux extraction is carried out for 1-2 h.
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| 石香薷水提物抗流感病毒作用研究;徐军烈等;《浙江中医杂志》;20130430;第48卷(第4期);第273-274页 * |
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