Application of a Dryopteris latifolia extract in the preparation of a drug with anti-new coronavirus effect

Abstract

The present invention relates to the technical field of natural drug chemistry, and specifically discloses an application of a Dryopteris latifolia extract in the preparation of a drug with an anti-new coronavirus effect. The preparation method of the Dryopteris latifolia extract comprises the following steps: extracting the Dryopteris latifolia with ethanol, and after the extraction, concentrating and drying the extract to obtain the Dryopteris latifolia ethanol extract, that is, the Dryopteris latifolia extract. Studies have shown that the Dryopteris latifolia ethanol extract, the Dryopteris latifolia extract, the Dryopteris latifolia silica gel column chromatography effective part and the Dryopteris latifolia extract prepared according to the method of the present invention have anti-SARS‑CoV‑2 virus effects; therefore, using it as an active ingredient for the preparation of a drug with anti-SARS‑CoV‑2 virus effects has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of natural drug chemistry, and specifically to an application of a Dryopteris latifolia extract in the preparation of a drug with anti-novel coronavirus effects. Background Art

[0002] Dryopteris championii (Benth.) C.Chr. is a plant of the Dryopteris genus of the Dryopteridaceae family. It is widely distributed in Shandong, Jiangsu, Zhejiang, Jiangxi, Fujian, Henan, Hunan and other places in my country, and is also distributed in Japan and North Korea. This plant is included in the "Chinese Traditional Medicine Resources" as a traditional Chinese medicine resource. It has the effects of clearing away heat and detoxifying, relieving cough and relieving asthma. It can be used to treat colds, asthma, blood in the stool, burns and scalds. Dryopteris is a large genus of ferns in my country. It has a long history of medicinal use and was first recorded in the "Shennong Bencao Jing". The original medicinal value was mainly used as an anthelmintic. Because of its good antiviral, antitumor, antibacterial and anti-inflammatory activities, it has gradually attracted the attention of contemporary researchers, especially its antiviral activity.

[0003] However, there are currently no reports on the use of Dryopteris latifolia for anti-SARS-CoV-2 virus; therefore, using the traditional Chinese medicine Dryopteris latifolia as raw material to develop an extract with anti-SARS-CoV-2 virus effect, especially an extract with excellent anti-SARS-CoV-2 virus effect, has important application value. Summary of the invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention first provides a Dryopteris latifolia extract.

[0005] The above technical problems to be solved by the present invention are achieved through the following technical solutions:

[0006] The present invention first provides an application of a Dryopteris latifolia extract in preparing a drug having an anti-novel coronavirus effect, and the preparation method of the Dryopteris latifolia extract comprises the following steps:

[0007] The method comprises extracting Dryopteris latifolia with ethanol, and after the extraction is completed, concentrating and drying the extract to obtain the Dryopteris latifolia ethanol extract, namely the Dryopteris latifolia extract.

[0008] The inventors were surprised to find in the study that the Dryopteris latifolia ethanol extract obtained by extracting the Dryopteris latifolia with ethanol showed a certain anti-SARS-CoV-2 virus effect.

[0009] Preferably, the ethanol refers to ethanol with a volume fraction of 50% to 95%.

[0010] Most preferably, the ethanol refers to ethanol with a volume fraction of 95%.

[0011] Preferably, the usage ratio of ethanol to Dryopteris latifolia is 8-20L:1Kg.

[0012] Most preferably, the ratio of ethanol to Dryopteris latifolia is 10L:1Kg.

[0013] Preferably, the method for preparing the Dryopteris latifolia extract further comprises the following steps:

[0014] The ethanol extract of Dryopteris latifolia is suspended in water, and then extracted with petroleum ether to obtain a petroleum ether extract; the petroleum ether extract is concentrated and dried to obtain a Dryopteris latifolia extract, namely the Dryopteris latifolia extract.

[0015] The inventors further discovered in the study that the anti-SARS-CoV-2 virus effect of the Dryopteris latifolia extract obtained by extracting the ethanol extract of Dryopteris latifolia with petroleum ether was significantly improved.

[0016] Preferably, the dosage ratio of Dryopteris latifolia ethanol extract to water is 0.1-1L:1Kg; and the volume ratio of petroleum ether to the suspension is 3-10:1.

[0017] Most preferably, the dosage ratio of Dryopteris latifolia ethanol extract to water is 0.3L:1Kg; and the volume ratio of petroleum ether to the suspension is 3:1.

[0018] Preferably, the extraction is performed 1 to 6 times.

[0019] More preferably, the extraction is performed 3 to 5 times.

[0020] Preferably, the method for preparing the Dryopteris latifolia extract further comprises the following steps:

[0021] The extract of Dryopteris latifolia is loaded onto a silica gel column, and firstly eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 to 1000:10 for 5 to 15 times the column volume; then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:10 to 1000:30 for 5 to 15 times the column volume, and the eluted fraction is collected, and after concentrating and drying, the effective fraction of the Dryopteris latifolia silica gel column chromatography is obtained, that is, the Dryopteris latifolia extract is obtained.

[0022] Most preferably, the method for preparing the Dryopteris latifolia extract further comprises the following steps:

[0023] The extract of Dryopteris latifolia is loaded onto a silica gel column, and first eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 for 8 times the column volume; then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:20 for 10 times the column volume, and the eluted fraction is collected, concentrated and dried to obtain the effective fraction of the Dryopteris latifolia silica gel column chromatography, that is, the Dryopteris latifolia extract.

[0024] The inventors further discovered in the study that the anti-SARS-CoV-2 virus effect of the effective fraction of Dryopteris latifolia prepared by eluting the extract of Dryopteris latifolia under the silica gel column elution conditions described in this method was further significantly improved.

[0025] The inventors hereby need to explain that the selection of elution conditions in the silica gel column elution method is very critical. Only when the effective fraction of Dryopteris latifolia silica gel column chromatography prepared by elution under the above-mentioned silica gel column elution conditions of the present invention can its anti-SARS-CoV-2 virus effect be further greatly improved. However, the effective fraction of Dryopteris latifolia silica gel column chromatography prepared by elution under other silica gel column elution conditions may not necessarily be further greatly improved in its anti-SARS-CoV-2 virus effect.

[0026] Preferably, the method for preparing the Dryopteris latifolia extract further comprises the following steps:

[0027] The effective part of Dryopteris latifolia silica gel column chromatography is placed on a gel column and eluted with a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1, and the eluate of the 3rd to 5th column volume is collected, concentrated and dried to obtain the Dryopteris latifolia extract.

[0028] The inventors further discovered in the study that the Dryopteris latifolia extract prepared by eluting the effective part of Dryopteris latifolia through silica gel column chromatography under the gel column elution conditions described in this method has a very excellent anti-SARS-CoV-2 virus effect, and its anti-SARS-CoV-2 virus effect has been further significantly improved.

[0029] Preferably, the gel in the gel column is cross-linked dextran gel LH-20.

[0030] Preferably, the novel coronavirus is SARS-CoV-2 virus.

[0031] Beneficial effects: The present invention discovers for the first time that the ethanol extract of Dryopteris scaly has a certain anti-SARS-CoV-2 virus effect. In further research, it is found that the Dryopteris scaly extract and the effective part of Dryopteris scaly silica gel column chromatography prepared by the method of the present invention have gradually and significantly improved anti-SARS-CoV-2 virus effects; in particular, the Dryopteris scaly extract obtained by further elution with a gel column has a very excellent anti-SARS-CoV-2 virus effect. Since the Dryopteris scaly ethanol extract, the Dryopteris scaly extract, the effective part of Dryopteris scaly silica gel column chromatography and the Dryopteris scaly extract prepared according to the method of the present invention have anti-SARS-CoV-2 virus effects; therefore, using them as active ingredients for the preparation of drugs with anti-SARS-CoV-2 virus effects has important application value. DETAILED DESCRIPTION

[0032] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0033] Example 1 Preparation of Dryopteris latifolia extract

[0034] (1) extracting the Dryopteris latifolia with 95% ethanol by volume for 2 days, concentrating and drying the extract after the extraction to obtain the ethanol extract of the Dryopteris latifolia; the ratio of ethanol to the Dryopteris latifolia is 10L:1Kg;

[0035] (2) suspending the ethanol extract of Dryopteris scaly obtained in step (1) in water, with the ratio of water to extract being 0.3 L:1 kg, extracting with petroleum ether 5 times the volume of the suspension, absorbing the petroleum ether layer extract, recovering and concentrating it; repeating the extraction and recovery steps 4 times, combining the extracts to obtain the Dryopteris scaly extract;

[0036] (3) applying the extract of Dryopteris scaly to a silica gel column (the silica gel column is filled with 300 mesh silica gel), firstly eluting and removing impurities with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 for 8 times the column volume; then eluting with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:20 for 10 times the column volume, collecting the eluted fraction, concentrating and drying to obtain the effective fraction of Dryopteris scaly by silica gel column chromatography;

[0037] (4) The effective fraction of Dryopteris latifolia obtained in step (3) is placed on a gel column (cross-linked polysaccharide gel LH-20), eluted with a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1, and the eluate of 3 to 5 times the column volume is collected, concentrated and dried to obtain the Dryopteris latifolia extract.

[0038] Comparative Example 1 Preparation of Dryopteris latifolia extract

[0039] (1) extracting the Dryopteris latifolia with 95% ethanol by volume for 2 days, concentrating and drying the extract after the extraction to obtain the ethanol extract of the Dryopteris latifolia; the ratio of ethanol to the Dryopteris latifolia is 10L:1Kg;

[0040] (2) suspending the ethanol extract of Dryopteris scaly obtained in step (1) in water, with the ratio of water to extract being 0.3 L:1 kg, extracting with petroleum ether 5 times the volume of the suspension, absorbing the petroleum ether layer extract, recovering and concentrating it; repeating the extraction and recovery steps 4 times, combining the extracts to obtain the Dryopteris scaly extract;

[0041] (3) applying the extract of Dryopteris scaly to a silica gel column (the silica gel column is filled with 300 mesh silica gel), firstly eluting and removing impurities with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:10 for 8 times the column volume; then eluting with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:50 for 10 times the column volume, collecting the eluted fraction, concentrating and drying to obtain the effective fraction of Dryopteris scaly by silica gel column chromatography;

[0042] (4) The effective fraction of Dryopteris latifolia obtained in step (3) is placed on a gel column (cross-linked polysaccharide gel LH-20), eluted with a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1, and the eluate of 3 to 5 times the column volume is collected, concentrated and dried to obtain the Dryopteris latifolia extract.

[0043] The difference between Comparative Example 1 and Example 1 is that the elution conditions of the silica gel column chromatography in step (3) are different, so the effective components enriched are also different. Example 1 is firstly eluted and impurities are removed with a mixed solvent system consisting of petroleum ether and ethyl acetate with a volume ratio of 1000:0 for 8 times the column volume; and then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate with a volume ratio of 1000:20 for 10 times the column volume and collected the eluted part. Comparative Example 1 is firstly eluted and impurities are removed with a mixed solvent system consisting of petroleum ether and ethyl acetate with a volume ratio of 1000:10 for 8 times the column volume; and then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate with a volume ratio of 1000:50 for 10 times the column volume and collected the eluted part.

[0044] Comparative Example 2 Preparation of Dryopteris latifolia extract

[0045] (1) extracting the Dryopteris latifolia with 95% ethanol by volume for 2 days, concentrating and drying the extract after the extraction to obtain the ethanol extract of the Dryopteris latifolia; the ratio of ethanol to the Dryopteris latifolia is 10L:1Kg;

[0046] (2) suspending the ethanol extract of Dryopteris scaly obtained in step (1) in water, with the ratio of water to extract being 0.3 L:1 kg, extracting with petroleum ether 5 times the volume of the suspension, absorbing the petroleum ether layer extract, recovering and concentrating it; repeating the extraction and recovery steps 4 times, combining the extracts to obtain the Dryopteris scaly extract;

[0047] (3) applying the extract of Dryopteris scaly to a silica gel column (the silica gel column is filled with 300 mesh silica gel), firstly eluting and removing impurities with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 for 8 times the column volume; then eluting with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:20 for 10 times the column volume, collecting the eluted fraction, concentrating and drying to obtain the effective fraction of Dryopteris scaly by silica gel column chromatography;

[0048] (4) The effective fraction of Dryopteris latifolia obtained in step (3) is subjected to a gel column (cross-linked polysaccharide gel LH-20) by silica gel column chromatography, eluted with methanol, and the eluate of 3 to 5 times the column volume is collected, and the extract is obtained after concentration and drying.

[0049] The difference between Comparative Example 2 and Example 1 is that the elution conditions of the gel column in step (4) are different. Comparative Example 2 uses only methanol for elution, while Example 1 uses a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1 for elution.

[0050] Experimental Example 1 Anti-SARS-CoV-2 Virus Activity Test

[0051] The extract of Dryopteris latifolia prepared according to the method of Example 1 (experimental group 1), the effective fraction of Dryopteris latifolia prepared according to step (3) of Example 1 by silica gel column chromatography (experimental group 2), the extract of Dryopteris latifolia prepared according to step (2) of Example 1 (experimental group 3), the ethanol extract of Dryopteris latifolia prepared according to step (1) of Example 1 (experimental group 4), and the extract of Dryopteris latifolia prepared according to the methods of Comparative Examples 1 and 2 (experimental groups 5 and 6) were tested respectively.

[0052] Experimental methods:

[0053] Vero E6 cells were diluted to 4 × 10 4 / well were inoculated in a 24-well plate and placed in a constant temperature incubator at 37°C and 5% CO2 for 16-24 hours. When the cells reached 90% confluence, the supernatant was discarded, and then a virus (SARS-CoV-2) dilution with MOI = 0.05 was added for infection. After 1 hour of infection, the virus solution was discarded, the cells were washed twice with fresh PBS, and then a series of gradient concentrations of extract solutions were added. After incubation for 48 hours, the cell supernatant was collected for qRT-PCR analysis, and the concentration that inhibited 50% SARS-CoV-2 DNA expression, i.e., IC 50 The specific results are shown in Table 1.

[0054] Table 1. Anti-SARS-CoV-2 activity test results

[0055]

[0056] From the IC in Table 1 50 It can be seen from the experimental data that the ethanol extract of Dryopteris scaly obtained by extracting Dryopteris scaly with ethanol shows a certain anti-SARS-CoV-2 virus effect. The Dryopteris scaly extract obtained by extracting the ethanol extract of Dryopteris scaly with petroleum ether has a significantly improved anti-SARS-CoV-2 virus effect. Furthermore, the Dryopteris scaly silica gel column chromatography effective part prepared by eluting the Dryopteris scaly extract under the silica gel column elution conditions described in this method has a further significantly improved anti-SARS-CoV-2 virus effect. In particular, the Dryopteris scaly extract prepared by eluting the effective part of the Dryopteris scaly silica gel column chromatography under the gel column elution conditions described in the present invention has a very excellent anti-SARS-CoV-2 virus effect, and its anti-SARS-CoV-2 virus effect has been further significantly improved.

[0057] It can also be seen from the experimental data in Table 1 that the Dryopteris latifolia extract prepared according to Example 1 has an IC value of 1.0 for SARS-CoV-2. 50 The value is much smaller than that of Comparative Example 1; and also much smaller than that of Dryopteris scaly. However, compared with the Dryopteris scaly extract, the IC 50Although the value is reduced, the reduction is much smaller than that in Example 1. This shows that the silica gel column elution conditions of the present invention are very critical; only when the extract of Dryopteris scaly is eluted under the silica gel column elution conditions of the present invention and then eluted by the gel column to obtain the Dryopteris scaly extract, it has a very excellent anti-SARS-CoV-2 virus effect, and its anti-SARS-CoV-2 virus effect can be further greatly improved. However, the Dryopteris scaly extract prepared by eluting the extract of Dryopteris scaly under other silica gel column elution conditions and then eluting by the gel column does not have a very excellent anti-SARS-CoV-2 virus effect, and its anti-SARS-CoV-2 virus effect cannot be further greatly improved.

[0058] It can also be seen from the experimental data in Table 1 that the Dryopteris latifolia extract prepared according to Example 1 has an IC value of 1.0 for SARS-CoV-2. 50 The value is significantly smaller than that of Comparative Example 2; its IC 50 The value is significantly lower than that of the Dryopteris scaly-scaled fern extract prepared according to Comparative 2 relative to the effective part of the silica gel column chromatography. This shows that: in the present invention, the elution conditions of the gel column are also very critical; the Dryopteris scaly-scaled fern extract prepared by eluting the effective part of the silica gel column chromatography with a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1 and collecting the 3rd to 5th column volumes of the eluate can further significantly improve the anti-SARS-CoV-2 virus effect and have a very excellent anti-SARS-CoV-2 virus effect; while the Dryopteris scaly-scaled fern extract prepared by eluting the effective part of the silica gel column chromatography with other solvents cannot further significantly improve the anti-SARS-CoV-2 virus effect and does not have a very excellent anti-SARS-CoV-2 virus effect.

[0059] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An application of a Dryopteris latifolia extract in the preparation of a drug having an anti-new coronavirus effect, characterized in that: The preparation method of the Dryopteris latifolia extract comprises the following steps: The method comprises extracting Dryopteris latifolia with ethanol, and after the extraction is completed, concentrating and drying the extract to obtain the Dryopteris latifolia ethanol extract, namely the Dryopteris latifolia extract.

2. The use according to claim 1, characterized in that: The ethanol mentioned refers to ethanol with a volume fraction of 50% to 95%.

3. The use according to claim 1, characterized in that: The usage ratio of ethanol to Dryopteris latifolia is 8-20L:1Kg.

4. The use according to claim 1, characterized in that: The preparation method of the Dryopteris latifolia extract further comprises the following steps: The ethanol extract of Dryopteris latifolia is suspended in water, and then extracted with petroleum ether to obtain a petroleum ether extract; the petroleum ether extract is concentrated and dried to obtain a Dryopteris latifolia extract, namely the Dryopteris latifolia extract.

5. The use according to claim 4, characterized in that: The dosage ratio of Dryopteris latifolia ethanol extract to water is 0.1-1L:1Kg; the volume ratio of petroleum ether to the suspension is 3-10:

1.

6. The use according to claim 4, characterized in that: The preparation method of the Dryopteris latifolia extract further comprises the following steps: The extract of Dryopteris latifolia is loaded onto a silica gel column, and firstly eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 to 1000:10 for 5 to 15 times the column volume; then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:10 to 1000:30 for 5 to 15 times the column volume, and the eluted fraction is collected, and after concentrating and drying, the effective fraction of the Dryopteris latifolia silica gel column chromatography is obtained, that is, the Dryopteris latifolia extract is obtained.

7. The use according to claim 4, characterized in that: The preparation method of the Dryopteris latifolia extract further comprises the following steps: The extract of Dryopteris latifolia is loaded onto a silica gel column, and first eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:0 for 8 times the column volume; then eluted with a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 1000:20 for 10 times the column volume, and the eluted fraction is collected, concentrated and dried to obtain the effective fraction of the Dryopteris latifolia silica gel column chromatography, that is, the Dryopteris latifolia extract.

8. The use according to claim 6, characterized in that: The preparation method of the Dryopteris latifolia extract further comprises the following steps: The effective part of Dryopteris latifolia silica gel column chromatography is placed on a gel column and eluted with a mixed solvent system consisting of chloroform and methanol in a volume ratio of 2:1, and the eluate of the 3rd to 5th column volume is collected, concentrated and dried to obtain the Dryopteris latifolia extract.

9. The use according to claim 8, characterized in that: The gel in the gel column is cross-linked dextran gel LH-20.

10. The use according to claim 8, characterized in that: The novel coronavirus is the SARS-CoV-2 virus.

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