CN116211966A

CN116211966A - Application of plant extract, novel coronavirus main protease inhibitor and screening method

Info

Publication number: CN116211966A
Application number: CN202111495923.5A
Authority: CN
Inventors: 李冰麟; 戚歆宇; 李海宁; 郭美静; 张小里; 张甜甜; 赵宇科; 蒋沛阳
Original assignee: NORTHWEST UNIVERSITY
Current assignee: NORTHWEST UNIVERSITY
Priority date: 2021-12-04
Filing date: 2021-12-04
Publication date: 2023-06-06

Abstract

The examples of the present application disclose the use of plant extracts, novel coronavirus main protease inhibitors and screening methods. The application obtains the plant extract with obvious inhibition effect on the novel coronavirus main protease through molecular docking screening, and the found plant extract can be further used for preparing medicines for resisting SARS-CoV-2 virus or treating diseases caused by SARS-CoV-2 virus, thereby providing a new choice for treating novel coronavirus pneumonia.

Description

Application of plant extract, novel coronavirus main protease inhibitor and screening method

Technical Field

The present application relates generally to the field of pharmaceutical technology, and in particular to the use of plant extracts, novel coronavirus main protease inhibitors and screening methods.

Background

The novel coronavirus (SARS-CoV-2) is a single stranded RNA plus-stranded enveloped beta coronavirus and members of the same group include SARS-CoV and middle east respiratory syndrome coronavirus, which has about 79.6% homology to the SARS-CoV genomic sequence. The main protease for controlling the replication of coronaviruses has important roles in the life cycle of the viruses, is a key enzyme for the replication of SARS-CoV-2, is encoded by polypeptides, is responsible for processing the polypeptides into functional proteins, and can be used as an important target for treating novel coronavirus pneumonia.

The current medicines for treating the novel coronavirus pneumonia mainly comprise antibodies, vaccines and small-molecule medicines, the production period of the antibodies is long, the cost is high, the vaccine has the function of preventing diseases, but not treating diseases, and the small-molecule medicines including inhibitors are expected to become main therapeutic medicines.

However, few drugs targeting SARS-CoV-2 main protease are currently available, and new effective drugs remain to be developed.

Disclosure of Invention

In view of the above-mentioned drawbacks or shortcomings of the prior art, the present application provides a use of a plant extract in the preparation of a novel coronavirus main protease inhibitor, a screening method of the inhibitor and a use of the inhibitor.

In one aspect of the present application, there is provided a use of a plant extract in preparing a novel coronavirus main protease inhibitor, the plant extract being at least one selected from the group consisting of astragalus extract, silybum marianum extract, jujube extract, yam extract, motherwort extract, aloe extract, anemarrhena extract, rheum officinale extract, magnolia officinalis extract, gastrodia elata extract, fritillaria extract, nux vomica extract, nutmeg extract, evodia rutaecarpa extract, pueraria extract, forsythia extract, yew extract, rhodiola rosea extract, honeysuckle extract, coptis extract, hypericum perforatum extract, licorice extract and ginkgo extract.

The application finds that the plant extracts all have the effect of inhibiting the activity of SARS-CoV-2 main protease, the inhibition of the activity of the main protease is concentration-dependent, and the inhibition of the main protease is gradually enhanced along with the increase of the concentration of the plant extract, so that the plant extract can be used for preparing the SARS-CoV-2 main protease inhibitor.

In another aspect of the present application, the present application provides a novel coronavirus main protease inhibitor containing one or more plant extracts selected from the group consisting of astragalus extract, silybum marianum extract, jujube extract, yam extract, motherwort extract, aloe extract, anemarrhena extract, rheum officinale extract, magnolia officinalis extract, gastrodia tuber extract, fritillaria extract, nux vomica extract, nutmeg extract, evodia rutaecarpa extract, pueraria root extract, forsythia extract, yew extract, rhodiola rosea extract, honeysuckle extract, coptis extract, hypericum perforatum extract, licorice extract and ginkgo extract as an active ingredient.

In another aspect of the present application, there is provided a method of screening for a novel coronavirus main protease inhibitor comprising the steps of: screening small molecule ligands meeting the conditions from a ZINC library according to the drug-like rules, and establishing a first database; acquiring a three-dimensional structure of a novel coronavirus main protease, and determining an active pocket; utilizing a molecular docking program to perform docking scoring on small molecular ligands in the first database, and establishing a second database; determining a framework structure corresponding to each small molecule ligand in the second database, and determining a plant extract to be tested based on the framework structure; and (3) carrying out biological activity screening on the tested plant extract outside the cells to obtain the inhibitor. The method realizes high-throughput screening of the novel coronavirus main protease, remarkably accelerates the research speed of medicines and saves the research and development cost.

In another aspect of the present application, the present application relates to a method of treating a novel coronavirus pneumonia comprising administering an inhibitor of a novel coronavirus main protease to a patient in need thereof, e.g., by oral or injection methods. The application finds that when the inhibitor acts on the main protease in vitro, the activity of the inhibitor is obviously inhibited, so that the infection force of coronaviruses can be reduced or inhibited by preventing the main protease from being over expressed to block SARS-CoV-2 replication. Thus, the application provides the application of the inhibitor in preparing medicines for resisting SARS-CoV-2 virus or treating diseases caused by SARS-CoV-2 virus.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the following drawings, in which:

FIG. 1 is a graph showing the results of the enzyme inhibition activity of 23 plant extracts screened in the present application on a primary protease;

FIG. 2 is a graph showing IC50 results of rhizoma anemarrhenae extract;

FIG. 3 is a graph showing IC50 results of ginkgo extract;

FIG. 4 is a graph showing IC50 results of Astragalus extract;

FIG. 5 is a graph showing IC50 results of radix Puerariae extract;

FIG. 6 is a diagram of the ligand-protein complex of Sarsasapogenin;

FIG. 7 is another ligand-protein complex diagram of Sarasaponin;

FIG. 8 is a schematic diagram of ligand-protein complex of Mangiferi;

FIG. 9 is another ligand-protein complex diagram of Mangiferi;

FIG. 10 is a diagram showing the structure of ligand-protein complex for Quercetin;

FIG. 11 is a diagram of another ligand-protein complex of Quercetin;

FIG. 12 is a diagram showing the structure of ligand-protein complex of Ginkgetin;

FIG. 13 is a diagram showing another ligand-protein complex structure of Ginkgetin.

Detailed Description

The present application is described in further detail below with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. It should be noted that endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and that such range or value should be understood to include values approaching such range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In a first aspect of the present application, there is provided the use of a plant extract selected from at least one of astragalus extract, silybum marianum extract, chinese date extract, chinese yam extract, leonurus extract, aloe extract, anemarrhena extract, rheum officinale extract, magnolia officinalis extract, gastrodia elata extract, fritillaria extract, nux vomica extract, nutmeg extract, evodia rutaecarpa extract, pueraria extract, forsythia extract, yew extract, rhodiola rosea extract, honeysuckle extract, coptis extract, hypericum perforatum extract, licorice extract and ginkgo extract in the preparation of a novel coronavirus main protease inhibitor.

In the embodiments of the present application, astragalus refers to dried roots of astragalus membranaceus and astragalus mongholicus of the genus astragalus of the family leguminosae. Silybum marianum refers to Silybum marianum (L.) Gaertn. Fructus Jujubae refers to fruit of ziphus jujuba Mill of Zizyphus of Rhamnaceae. Yam refers to Dioscorea oppositifolia l of the genus dioscorea in the family dioscoreaceae, also known as yam. Herba Leonuri refers to Leonurus artemisia (laur.) s.y.hu F of Leonurus of Labiatae. Aloe refers to Aloe vera var. Rhizoma anemarrhenae refers to Anemarrhena asphodeloides of Anemarrhena genus of Liliaceae. Radix et rhizoma Rhei refers to dry root of Rheum palmatum L. Magnolia bark refers to Magnolia officinalis red.et wils. Gastrodia elata refers to Gastrodia elata of Gastrodia genus of Orchidaceae family. Fritillaria is the fritillary of Fritillaria genus of the family liliaceae. Nux-vomica L. Nutmeg refers to Myristica fragrans route. The evodia rutaecarpa refers to Evodia rutaecarpa (juss.) benth of evodia genus of rutaceae family. Radix Puerariae refers to dried root of Pueraria lobata Ohwi of Leguminosae. Fructus forsythiae refers to Forsythia suspensa (thunder.) Vahl of the genus Forsythia of the family Oleaceae. Taxus chinensis refers to Taxus chinensis (Pilger) Red. Rhodiola rosea is Rhodiola rosea l. The flos Lonicerae refers to Lonicera japonica of Lonicera of Caprifoliaceae. Coptis chinensis Franch refers to Coptis chinensis Franch of Coptis genus of Ranunculaceae family. Hypericum perforatum refers to Hypericum perforatum L. Glycyrrhrizae radix refers to Glycyrrhiza uralensis Fisch of Glycyrrhiza of Papilionaceae. Ginkgo refers to the leaves of Ginkgo biloba Linn.

In some embodiments of the present application, the astragalus extract, the pueraria extract and the rheum officinale extract are extracts of dried roots of astragalus, pueraria and rheum officinale, respectively. The fructus Jujubae extract is extract of fructus Jujubae fruit. The ginkgo extract is an extract of ginkgo leaf. The extracts of Silybum marianum, rhizoma Dioscoreae, herba Leonuri, aloe, rhizoma anemarrhenae, cortex Magnoliae officinalis, rhizoma Gastrodiae, bulbus Fritillariae Cirrhosae, semen Strychni, semen Myristicae, wu Caiyu, fructus forsythiae, taxus chinensis, radix Rhodiolae, flos Lonicerae, coptidis rhizoma, herba Hyperici perforati and Glycyrrhrizae radix can be prepared by extracting any part (such as root, rhizome, stem, leaf, bark, flower, fruit, seed) of these plants with solvent. In the extraction, multiple parts can be combined for use.

In other embodiments of the present application, the silybum marianum extract is preferably an extract of silybum marianum fruit. The rhizoma Dioscoreae extract is preferably extract of rhizome of rhizoma Dioscoreae. The herba Leonuri extract is preferably extract of herba Leonuri stem. The aloe extract is preferably aloe stem and leaf extract. The rhizoma anemarrhenae extract is preferably rhizome of rhizoma anemarrhenae extract. The Magnolia bark extract is preferably extract of Magnolia bark, root bark and/or branch bark. The rhizoma Gastrodiae extract is preferably rhizoma Gastrodiae rhizome extract. The Bulbus Fritillariae Cirrhosae extract is preferably Bulbus Fritillariae Cirrhosae bulb extract. The semen Strychni extract is preferably semen Strychni seed extract. The semen Myristicae extract is preferably extract of semen Myristicae kernel. The Evodia rutaecarpa extract is preferably Evodia rutaecarpa fruit extract. The fructus forsythiae extract is preferably extract of fructus forsythiae fruit. The Taxus chinensis extract is preferably Taxus chinensis bark extract. The radix Rhodiolae extract is preferably extract of radix Rhodiolae rhizome. The flos Lonicerae extract is preferably extract of flos Lonicerae bud. The Coptidis rhizoma extract is preferably extract of Coptidis rhizoma rhizome. The herba Hyperici perforati extract is preferably herba Hyperici perforati whole plant extract. The Glycyrrhrizae radix extract is preferably Glycyrrhrizae radix extract.

In the embodiment of the present application, the plant extraction method for obtaining the plant extract is not particularly limited, and may be obtained by a usual extraction method used for extracting the plant component. The extraction method may be appropriately set, and the extraction conditions are not particularly limited. For example, the plant extract is preferably obtained by extracting the above plant with a solvent (extraction solvent) at normal temperature or at elevated temperature. In the preparation of the plant extract, the plant as a raw material may be directly added to the extraction step, or may be pulverized, cut or dried and then added to the extraction step. In the present application, the plant extract may be an extract obtained by directly extracting the plant, or an extract obtained by extracting the plant after being crushed, cut or dried. Preferably, the extract is obtained from the plant obtained by pulverizing, cutting or drying the plant.

In the embodiments of the present application, the extraction solvent used in the preparation of the plant extract may be appropriately selected, and materials generally used in the extraction of plant components may be used. Examples of the extraction solvent include water; monohydric alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propanol, butanol, etc.; polyhydric alcohols having 2 to 5 carbon atoms such as ethylene glycol, propylene glycol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, and 2, 3-butanediol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; squalane, and the like. These may be used alone, or a mixed solvent (mixed solution) in which 2 or more kinds are combined may be used. The monohydric alcohol having 1 to 5 carbon atoms is preferably a substance having 1 to 4 carbon atoms, more preferably a substance having 2 to 4 carbon atoms, and still more preferably ethanol. The polyhydric alcohol having 2 to 5 carbon atoms is preferably a dihydric or trihydric alcohol having 2 to 4 carbon atoms, more preferably a dihydric alcohol, and still more preferably 1, 3-butanediol. Among them, water, monohydric alcohol having 1 to 5 carbon atoms, polyhydric alcohol having 2 to 5 carbon atoms, and mixed solvents of 2 or more of these are preferable as the extraction solvent, water, monohydric alcohol having 2 to 4 carbon atoms or aqueous solution thereof, dihydric alcohol having 2 to 4 carbon atoms or aqueous solution thereof are more preferable, and water, ethanol, aqueous ethanol solution, 1, 3-butanediol, aqueous 1, 3-butanediol solution, and particularly water, aqueous ethanol solution, or aqueous 1, 3-butanediol solution are preferable. In one embodiment, the ethanol aqueous solution and the 1, 3-butanediol aqueous solution preferably have an ethanol or 1, 3-butanediol concentration of 10 to 98% by volume, more preferably 30 to 90% by volume, still more preferably 30 to 70% by volume. As the plant extract of the present application, the above-mentioned solvent extract can be preferably used.

Wherein, acid or alkali can be added to adjust pH of the extraction solvent during extraction. After extraction, the plant residue (extracted plant or part thereof) is preferably removed from the extract. The method for removing plant residues from the extract is not particularly limited, and known separation methods such as filtration and centrifugal separation can be used.

As an example of the extraction method of a plant, the following method can be used, for example. The plant or the dried plant is directly pulverized, and an extraction solvent is added in an amount of 0.1 to 30 times by weight, and the extract is preferably extracted at room temperature for 10 minutes to 15 days, more preferably 30 minutes to 10 days, still more preferably 1 hour to 7 days, or about the boiling point of the extraction solvent, preferably 10 minutes to 1 day (more preferably 10 minutes to 2 hours) at normal pressure, and then filtered to obtain a filtrate. The extraction may be carried out by standing or stirring. The obtained filtrate (plant extract) may be directly used as a plant extract, or may be diluted, concentrated, dried, or the like as needed.

In the embodiment of the present application, the plant extract obtained by extraction may be used as a plant extract as it is. In addition, the composition may be diluted, concentrated or dried by a known method to prepare a diluted solution, concentrate or powder, or may be prepared into a paste for use, as long as the effects of the present application are not impaired. Examples of the drying method include freeze drying and spray drying. Further, the plant extract, concentrate, dry powder, or the like may be further purified, if necessary, by deodorization, decolorization, or the like, as long as the effects of the present application are not impaired. Such a purification method can be carried out by any method selected from usual methods.

The plant extract of the present application includes various solvent extracts, dilutions thereof, concentrates thereof or dried powders thereof, refined products thereof, obtained by the above extraction method. The extract may be diluted or dissolved in a solvent different from the extraction solvent.

It should be understood that the plant extracts are commercially available, and commercially available products may be used.

In a second aspect of the present application, there is provided a novel coronavirus main protease inhibitor comprising one or more plant extracts selected from the first aspect as an active ingredient.

It is to be understood that the inhibitor of the present application may be composed of the above plant extracts, or may be further formulated into other components, additives, or the like to be used in the form of a composition.

In a third aspect of the present application, there is provided a method of screening for a novel coronavirus main protease inhibitor comprising the steps of:

step S101: screening small molecule ligands meeting the conditions from a ZINC library according to the drug-like rules, and establishing a first database;

step S102: acquiring a three-dimensional structure of a novel coronavirus main protease, and determining an active pocket;

step S103: utilizing a molecular docking program to perform docking scoring on small molecular ligands in the first database, and establishing a second database;

step S104: determining a framework structure corresponding to each small molecule ligand in the second database, and determining a plant extract to be tested based on the framework structure;

step S105: and (3) carrying out biological activity screening on the tested plant extract outside the cells to obtain the inhibitor.

Specifically, in step S101 of the method of the present application, establishing a first database means establishing a small molecule ligand library for docking; the method comprises the steps of obtaining traditional Chinese medicine components from a ZINC 15 database, performing ADMET analysis, and analyzing drug properties based on Lipinski 5 rule (RO 5), wherein websites used for ADMET analysis are SwissADDME and admetSAR, and screening to obtain 155 traditional Chinese medicine components; using the skeleton structure of 155 traditional Chinese medicine components as a matrix, and topologically obtaining 1,045,468 small molecule ligands to form a first database; among these, autodock Tools (v.1.5.6) can be further used to optimize the ligand, for example, adding a Gasteiger PEOE moiety charge and polar hydrogen atoms to the ligand.

In step S102 of the method, the three-dimensional structure of the master protease is searched and obtained in a protein database (http:// www.rcsb.org) to determine the active pocket. Wherein the three-dimensional structure of the main protease is preferably a three-dimensional structure with PDB code of 6LU7, and the active pocket can be determined by comparing the three-dimensional structure by adopting an Align/Superpose module in MOE 2016.08. Wherein the main protease can be hydrogenated and water molecules deleted using the Protein preparation wizard module of maestro 10.1 (2015-01,version 10.1,http:// www.com) in the schrodinger package, and then optimized using Autodock Tools (v.1.5.6), for example adding a gaseiger PEOE moiety charge and polar hydrogen atoms to the main protease.

In step S103 of the method, an AutoDock Vina software (v.1.1.2) is used to perform molecular docking on the small molecule ligands and the main protease in the first database by using a semi-flexible docking method, wherein the main protease is a rigid body, and rotatable bonds in all the small molecule ligands are sampled. In each docking simulation, the first 20 complexes were selected according to docking affinity.

The docking scoring can be obtained by sequencing the binding free energy of each small molecule ligand and the main protease, and a plurality of small molecule ligands with the binding free energy sequenced at the front are selected to form a second database. Illustratively, small molecule ligands are ranked by self-preference to inferior of binding free energy; and selecting small molecule ligands ranked within a predetermined proportion to form a second database, the remaining small molecule ligands being eliminated, optionally, the predetermined proportion being no more than 50%, optionally no more than 40%, no more than 30%, or no more than 20%, or no more than 10%.

In some preferred embodiments of the present application, the docking scoring of the small molecule ligands in the first database using a molecular docking procedure, and the establishing the second database specifically includes:

molecular docking is carried out on the small molecule ligands in the first database and the main protease by adopting AutoDock Vina software, so that the binding free energy of each small molecule ligand in the first database and the main protease is obtained;

and (3) obtaining the binding free energy of the positive control and the main protease, selecting a small molecule ligand with the binding free energy absolute value larger than that of the positive control and the main protease, and establishing a second database.

The positive control is a reported drug with an inhibition or obvious inhibition effect on the main protease, and the binding free energy of the positive control and the main protease is obtained through molecular docking of the positive control and the main protease, or can be obtained through reference.

Wherein, in some preferred embodiments of the present application, the positive control is Connipotene, and the free energy of binding of the positive control to the main protease is-8.2 Kcal/mol.

That is, in step S103 of the method of the present application, small molecule ligands having binding free energies of more than 8.2Kcal/mol absolute are selected to constitute a second database, and a total of 465 small molecule ligands are selected to constitute the second database.

In step S104 of the method, since the small molecule ligands to be tested are obtained by topology of skeleton structures of 155 traditional Chinese medicine components, the skeleton structure of each small molecule ligand in the second database obtained by screening can be determined, and 61 skeleton structures in total are obtained by screening, and the 61 skeleton structures are considered to have expected better capability of binding to the main protease. Further, searching for an active ingredient having a corresponding skeleton structure, and searching for a plant extract containing or containing the active ingredient as an active ingredient, and determining a test plant extract. Illustratively, screening finds that the [6/8/6] tricyclic framework structure shown in the following formula has a desired better ability to bind to the main protease, searches for paclitaxel having the framework structure, and further finds that paclitaxel is contained in the Taxus extract, thereby determining the Taxus extract as a test plant extract.

In chemical concepts, the backbone (Scaffold) refers to the core structure of a small molecule. The skeleton is considered as a structural core with multiple points of diversity. Their chemical modification produces a number of building blocks, with subsequent functional modification providing the final compound. In this application, the backbone structure refers to the cyclic core structure in the compound.

In step S105 of the method of the present application, according to the screening result of step S104, an in vitro biological activity test, for example, an enzyme inhibition test, is performed on the test plant extract, and finally, a plant extract having a main protease inhibition activity is screened. Illustratively, the plant extract obtained by the final screening should have an inhibitory effect on the inhibitory activity of the main protease of more than 50%.

In some preferred embodiments of the present application, the inhibitory activity of each compound on the primary protease is detected using a fluorescence resonance energy transfer system.

Specifically, MCA (methoxycoumarin acetic acid) is a fluorescence donor, dnp (2, 4-dinitrophenyl) is a fluorescence acceptor, and absorption spectra of the two fluorescent groups are overlapped to a certain extent. When the distance between these two fluorophores is appropriate (typically 7-10 nm), the fluorescence energy will be transferred from the donor group to the acceptor group, resulting in a decay of the fluorescence intensity of the donor. MCA and Dnp are ligated to the ends of the Mpro natural substrate, i.e.the MCA-AVLQSFGFR-Lys (Dnp) -Lys-NH2 complex is formed. When Mpro does not cleave the substrate, the two groups are close enough to cause fluorescence resonance energy transfer, meaning that Dnp can quench the fluorescence of MCA without causing fluorescence detection. If an inhibitor of a main protease is added to the reaction system, the generation of fluorescence is inhibited, and the fluorescence intensity is inversely proportional to the inhibition effect of the inhibitor, so that the inhibition effect of the inhibitor can be detected.

And finally, 23 plant extracts with obvious inhibition effect on the main protease are obtained by screening from the tested plant extracts through detection of a fluorescence resonance energy transfer system.

In a fourth aspect of the present application, the present application provides the use of the above-described inhibitor in the manufacture of a medicament for use against SARS-CoV-2 virus or for use in the treatment of a disease caused by SARS-CoV-2 virus.

In the embodiments of the present application, "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. "treating" as used herein encompasses diseases in mammals, particularly humans, including inhibiting the disease, e.g., arresting the development of the disease; or to alleviate a disease, e.g., to alleviate symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug as described herein to an individual in need thereof.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Example 1

Each plant extract was dissolved in DMSO solution to prepare a solution with a mass concentration of 0.35 mg/mL. mu.L of the extract solution of the Chinese medicine, 92. Mu.L of assay buffer, and 1. Mu.L of 2019-nCoV Mpro were added to a 96-black plate. And 2. Mu.L of fluorogenic substrate was rapidly added to each well and mixed well. After incubation at 37℃for 10 minutes in the absence of light, the signal stabilized, the fluorescence signal was measured using a multifunctional microplate reader at an excitation wavelength of 325nm and an emission wavelength of 393 nm. The inhibitory activity of the extract was calculated with the enzyme catalytic activity without inhibitor as 100% catalytic activity, and the experimental results are shown in fig. 1. It can be seen that the inhibition activity of the rhizoma anemarrhenae extract is up to 90.1%, and at the concentration, the inhibition activity of the 23 plant extracts screened by the application on the main protease is higher than 50%.

Inhibition (%) = (RFU) _{100% enzyme Activity control} -RFU _{Sample of} )/(RFU _{100% enzyme Activity control} -RFU _{Blank control} )×100％。

Example 2

The rhizoma anemarrhenae extract is dissolved in DMSO solution to prepare solutions with mass concentration of 0.15mg/mL,0.25mg/mL,0.35mg/mL,0.45mg/mL and 0.55mg/mL respectively. mu.L of rhizoma anemarrhenae extract solution with different concentrations, 92 mu.L of assay buffer and 1 mu.L of 2019-nCoV Mpro/3CLpro are added into a 96-black plate. And 2. Mu.L of fluorogenic substrate was rapidly added to each well and mixed well. After incubation at 37℃for 10 minutes in the absence of light, the signal stabilized, the fluorescence signal was measured using a multifunctional microplate reader at an excitation wavelength of 325nm and an emission wavelength of 393 nm. The inhibitory activity of the extract was calculated with the enzyme catalytic activity without inhibitor as 100% catalytic activity, and the experimental results are shown in fig. 2. It can be seen that the IC50 of the rhizoma anemarrhenae extract is 0.22mg/mL.

Example 3

The ginkgo extract is dissolved in DMSO solution to prepare solutions with mass concentration of 0.15mg/mL,0.25mg/mL,0.35mg/mL,0.55mg/mL,0.75mg/mL,0.95mg/mL and 1.5mg/mL respectively. mu.L of ginkgo extract solution with different concentrations, 92 mu.L of assay buffer and 1 mu.L of 2019-nCoV Mpro/3CLpro are added to a 96-black plate. And 2. Mu.L of fluorogenic substrate was rapidly added to each well and mixed well. After incubation at 37℃for 10 minutes in the absence of light, the signal stabilized, the fluorescence signal was measured using a multifunctional microplate reader at an excitation wavelength of 325nm and an emission wavelength of 393 nm. The inhibitory activity of the extract was calculated with the enzyme catalytic activity without inhibitor as 100% catalytic activity, and the experimental results are shown in fig. 3. The IC50 of the ginkgo extract can be seen to be 0.31mg/mL.

Example 4

The astragalus extract is dissolved in DMSO solution to prepare solutions with mass concentration of 0.15mg/mL,0.25mg/mL,0.35mg/mL,0.45mg/mL and 0.55mg/mL respectively. mu.L of ginkgo extract solution with different concentrations, 92 mu.L of assay buffer and 1 mu.L of 2019-nCoV Mpro/3CLpro are added to a 96-black plate. And 2. Mu.L of fluorogenic substrate was rapidly added to each well and mixed well. After incubation at 37℃for 10 minutes in the absence of light, the signal stabilized, the fluorescence signal was measured using a multifunctional microplate reader at an excitation wavelength of 325nm and an emission wavelength of 393 nm. The inhibitory activity of the extract was calculated with the enzyme catalytic activity without inhibitor as 100% catalytic activity, and the experimental results are shown in fig. 4. The IC50 of the astragalus extract can be seen to be 0.25mg/mL.

Example 5

The kudzuvine root extract is dissolved in DMSO solution to prepare solutions with mass concentration of 0.15mg/mL,0.25mg/mL,0.35mg/mL,0.45mg/mL and 0.55mg/mL respectively. mu.L of ginkgo extract solution with different concentrations, 92 mu.L of assay buffer and 1 mu.L of 2019-nCoV Mpro/3CLpro are added to a 96-black plate. And 2. Mu.L of fluorogenic substrate was rapidly added to each well and mixed well. After incubation at 37℃for 10 minutes in the absence of light, the signal stabilized, the fluorescence signal was measured using a multifunctional microplate reader at an excitation wavelength of 325nm and an emission wavelength of 393 nm. The inhibitory activity of the extract was calculated with the enzyme catalytic activity without inhibitor as 100% catalytic activity, and the experimental results are shown in fig. 5. The IC50 of the radix Puerariae extract was found to be 0.28mg/mL.

Example 6

Molecular docking of the major active ingredient (sarsasapogenin, mangiferin, quercetin) present in rhizoma anemarrhenae was performed and visualized using pymol software.

The ligand-protein complex structure of Sarsasapogenin is shown in fig. 6 and 7, and it can be seen from fig. 6 and 7 that this ligand forms a hydrogen bond with the protruding part of the active pocket of the main protease to anchor in the active pocket. sarsasapogenin forms a hydrogen bond with the active pocket L141, N142, S144 amino acid residues. Since the sarsasapogenin structure is a non-rotatable rigid loop structure, it cannot enter the interior of the active pocket completely, but because the amino acid residues forming hydrogen bonds are key amino acid residues, the docking affinity is better, and the free energy of binding is-8.8 Kcal/mol.

The ligand-protein complex structure of Mangiferi is shown in FIGS. 8 and 9, and it can be seen from FIGS. 8 and 9 that the molecular skeleton is small and has a single bond which is rotatable, so that the whole structure enters the inside of the active pocket and is in close contact with the active pocket. Mangiferin forms multiple hydrogen bonds with amino acid residues in the protruding portion of the primary protease active pocket, F140, L141, S144, H163, E166, respectively.

The ligand-protein complex structure of quercetin is shown in fig. 10 and 11, the docking affinity of quercetin is-8.9 Kcal/mol, and forms multiple hydrogen bonds with G143, S144, L145, T26 in the active pocket. Quercetin has three ring structures from the framework structure and is closely arranged. Thus, it is possible to easily enter the binding pocket of the main protease and form strong pi-pi stacking and hydrogen bonding. Thus, from a computer simulation perspective, rhizoma anemarrhenae is a potential candidate for a novel coronavirus main protease inhibitor.

Example 7

Among 1,045,468 small molecule ligands virtually screened through molecular docking, ginkgetin is the highest in docking affinity score of-10.3 Kcal/mol, which is far higher than that of a contrast drug Connetant-8.2 Kcal/mol. Ginkgetin is widely existing in ginkgo and has strong physiological activity. As shown in fig. 1, the ginkgo extract has an inhibition rate of 64.2% in the enzyme inhibition test, and the active ingredient thereof has the highest docking fraction although the inhibition rate is not the highest, so that the active ingredient ginkgetin ligand-protein complex thereof is subjected to visual analysis, as shown in fig. 12 and 13, to form a plurality of hydrogen bonds with T26, H41, Y54, H163 in the active pocket of the main protease. Ginkgetin belongs to biflavone compounds, and the compounds are characterized by containing benzopyran ring structures. The carbonyl and meta-hydroxy groups at the benzopyran end can form multiple hydrogen bonds with the carbonyl and amino groups in T26. Ginkgetin has a plurality of rigid ring structures that cannot perfectly fill the area of all active pockets by reasonable bond rotation, but functional groups in the terminal ring structure are prone to hydrogen bonding due to its lower steric hindrance. When the ligand structure is more rigid, although the steric hindrance of the whole molecule increases, the steric hindrance of the middle region of the molecule decreases, increasing the probability of hydrogen bonding of functional groups in these regions.

The foregoing description is only of the preferred embodiments of the present application and is presented as a description of the principles of the technology being utilized. It will be appreciated by persons skilled in the art that the scope of the invention referred to in this application is not limited to the specific combinations of features described above, but it is intended to cover other embodiments in which any combination of features described above or equivalents thereof is possible without departing from the spirit of the invention. Such as the above-described features and technical features having similar functions (but not limited to) disclosed in the present application are replaced with each other.

Claims

1. Use of a plant extract for the preparation of a novel coronavirus main protease inhibitor, characterized in that the plant extract is at least one selected from the group consisting of:

astragalus extract, silybum marianum extract, jujube extract, chinese yam extract, motherwort extract, aloe extract, rhizoma anemarrhenae extract, rheum officinale extract, magnolia officinalis extract, gastrodia elata extract, fritillaria extract, nux vomica extract, nutmeg extract, evodia rutaecarpa extract, kudzu vine root extract, fructus forsythiae extract, taxus chinensis extract, rhodiola rosea extract, honeysuckle extract, coptis chinensis extract, hypericum perforatum extract, liquorice extract and ginkgo extract.

2. A novel coronavirus main protease inhibitor is characterized by comprising more than one plant extract selected from astragalus extract, silybum marianum extract, chinese date extract, chinese yam extract, motherwort extract, aloe extract, anemarrhena extract, rheum officinale extract, magnolia officinalis extract, gastrodia elata extract, fritillaria extract, nux vomica extract, nutmeg extract, evodia rutaecarpa extract, pueraria lobata extract, forsythia suspensa extract, taxus chinensis extract, rhodiola rosea extract, honeysuckle extract, coptis extract, hypericum perforatum extract, licorice extract and ginkgo extract as an active ingredient.

3. A method of screening for a novel coronavirus main protease inhibitor according to claim 2, comprising the steps of:

screening small molecule ligands meeting the conditions from a ZINC library according to the drug-like rules, and establishing a first database;

acquiring a three-dimensional structure of a novel coronavirus main protease, and determining an active pocket;

utilizing a molecular docking program to perform docking scoring on small molecular ligands in the first database, and establishing a second database;

determining a framework structure corresponding to each small molecule ligand in the second database, and determining a plant extract to be tested based on the framework structure;

and (3) carrying out biological activity screening on the tested plant extract outside the cells to obtain the inhibitor.

4. A method according to claim 3, wherein the three-dimensional structure of the main protease is a three-dimensional structure with PDB ID 6LU 7.

5. A method according to claim 3, wherein the scoring of the small molecule ligands in the first database by means of a molecular docking procedure, and wherein creating the second database comprises:

6. The method of claim 5, wherein the positive control is kanettan and the free energy of binding of positive control to the primary protease is-8.2 Kcal/mol.

7. The method according to claim 3, wherein the screening of the test plant extract for biological activity outside the cells to obtain the inhibitor comprises:

and detecting the inhibition activity of each plant extract on the main protease by using a fluorescence resonance energy transfer system.

8. Use of an inhibitor according to claim 2 for the manufacture of a medicament for the treatment of SARS-CoV-2 virus or for the treatment of a disease caused by SARS-CoV-2 virus.