阅读说明：本技术 一种新型抗病毒产品对抗包膜病毒的模型构建及其应用 (Model construction and application of novel antiviral product to resisting enveloped virus ) 是由 王婧宁 于 2020-07-16 设计创作，主要内容包括：本发明公开了一种新型抗病毒产品对抗包膜病毒的模型构建,包括抗病毒作用机制的构建；所述抗病毒作用机制模型构建包括对细胞毒性的测定、不同作用模式下抗病毒效果的测定、分子对接法测N蛋白相互作用、检测细胞膜中磷脂和胆固醇含量。(The invention discloses a model construction of a novel antiviral product against enveloped viruses, which comprises the construction of an antiviral action mechanism; the antiviral mechanism model construction comprises the steps of measuring cytotoxicity, measuring antiviral effect under different action modes, measuring the interaction of N protein by a molecular docking method and detecting the content of phospholipid and cholesterol in cell membranes.)

1. The model construction of a novel antiviral product against enveloped viruses is characterized by comprising the construction of an antiviral action mechanism; the antiviral mechanism model construction comprises the steps of measuring cytotoxicity, measuring antiviral effect under different action modes, measuring the interaction of N protein by a molecular docking method and detecting the content of phospholipid and cholesterol in cell membranes.

2. The novel antiviral product against enveloped viruses as claimed in claim 1, wherein said novel antiviral product is an antiviral product containing a pentacyclic triterpene compound.

3. The novel antiviral product against enveloped viruses as claimed in claim 1, characterized in that said determination of cytotoxicity comprises the following steps: adding active substances with different concentrations into cells, culturing the cells, and testing the inhibition rate of the active substances on the cells to obtain the maximum concentration which is nontoxic to the cells.

4. The novel antiviral product of claim 1, wherein said mode of action comprises pretreatment of the virus, virus replication, pretreatment of the cells, virus adsorption.

5. The modeling of the novel antiviral product against enveloped viruses as claimed in claim 4, characterized in that said determination of the antiviral effect of the mode of action of the pretreatment virus comprises the following steps: after mixing and incubating the active substance and the enveloped virus, adding the mixture into cells for co-culture, and testing the inhibition rate of the active substance on the enveloped virus to obtain the antiviral effect under the action mode of using the active substance to pretreat the virus.

6. The modeling of a novel antiviral product against enveloped viruses as claimed in claim 4, characterized in that said determination of the antiviral effect of the mode of action of viral replication comprises the following steps: infecting cells with the virus liquid, incubating, removing the virus liquid, adding the active substance to continue culturing the cells, and testing the survival rate of the cells to obtain the effect of the active substance on virus replication.

7. The modeling of novel antiviral products against enveloped viruses as claimed in claim 4, characterized in that said determination of the antiviral effect of the mode of action of the pretreated cells comprises the following steps: adding the active substance into the cells for cell culture, adding virus liquid for continuous culture, and testing the survival rate of the cells after the culture is finished to obtain the antiviral effect under the action mode of using the active substance to pretreat the cells.

8. Modeling of the novel antiviral product against enveloped viruses as claimed in claim 4, characterized in that said determination of the antiviral effect of the mode of action of viral adsorption comprises the following steps: mixing the active substance with virus solution, immediately adding into cells for adsorption, removing the liquid after adsorption, continuously culturing, and testing the survival rate of the cells after the culture is finished to obtain the effect of the active substance on virus adsorption.

9. The modeling of a novel antiviral product against enveloped viruses according to any one of claims 1 to 8, wherein said modeling of a novel antiviral product against enveloped viruses further comprises product development modeling.

10. Use of a novel antiviral product according to any of claims 1 to 9 for modeling against enveloped viruses for predicting the mechanism of action of the antiviral product.

Technical Field

The invention relates to the field of antiviral products, in particular to a model construction and application of a novel antiviral product to resisting enveloped viruses.

Background

The development history of human beings is accompanied by long-term fight with viruses, a novel coronavirus (abbreviated as 2019-nCoV) discovered at the end of 2019 causes the world to be involved in severe epidemic crisis, after the human beings are infected by the virus, the human body symptoms are generally fever, fatigue, dry cough, gradual dyspnea, and severe patients show acute respiratory distress syndrome, septic shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct. In the face of the novel virus, mature preparations and medicaments for inhibiting or even inactivating the virus do not exist at home and abroad.

Researches show that the saponin compound containing the pentacyclic triterpene parent nucleus has wide pharmacological action and important biological activity, particularly shows interesting pharmacological properties in the aspects of anti-inflammation, liver protection and tumor resistance, researches the existing active compound and prepares the active compound into a mature product, can realize quick response to epidemic situations, has huge market prospect under the background of normalization of epidemic prevention, however, the pentacyclic triterpene compound has less research on the aspect of antivirus and is difficult to provide perfect theoretical guidance for the application of the pentacyclic triterpene compound in an antivirus product.

Disclosure of Invention

In order to solve the above problems, the first aspect of the present invention provides a model construction of a novel antiviral product against enveloped viruses, including construction of an antiviral mechanism of action;

the antiviral mechanism model construction comprises the steps of measuring cytotoxicity, measuring antiviral effect under different action modes, measuring the interaction of N protein by a molecular docking method and detecting the content of phospholipid and cholesterol in cell membranes.

As a preferred technical scheme, the novel antiviral product is an antiviral product containing a pentacyclic triterpene compound.

As a preferred technical scheme, the determination of cytotoxicity comprises the following steps: adding active substances with different concentrations into cells, culturing the cells, and testing the inhibition rate of the active substances on the cells to obtain the maximum concentration which is nontoxic to the cells.

As a preferred embodiment, the mode of action includes pretreatment of the virus, virus replication, pretreatment of the cells, and virus adsorption.

As a preferred technical scheme, the determination of the antiviral effect under the action mode of the pretreated virus comprises the following steps: after mixing and incubating the active substance and the enveloped virus, adding the mixture into cells for co-culture, and testing the inhibition rate of the active substance on the enveloped virus to obtain the antiviral effect under the action mode of using the active substance to pretreat the virus.

As a preferred technical scheme, the determination of the antiviral effect under the action mode of virus replication comprises the following steps: infecting cells with the virus liquid, incubating, removing the virus liquid, adding the active substance to continue culturing the cells, and testing the survival rate of the cells to obtain the effect of the active substance on virus replication.

As a preferred technical scheme, the determination of the antiviral effect under the action mode of the pretreated cells comprises the following steps: adding the active substance into the cells for cell culture, adding virus liquid for continuous culture, and testing the survival rate of the cells after the culture is finished to obtain the antiviral effect under the action mode of using the active substance to pretreat the cells.

As a preferred technical scheme, the method for measuring the antiviral effect under the action mode of virus adsorption comprises the following steps: mixing the active substance with virus solution, immediately adding into cells for adsorption, removing the liquid after adsorption, continuously culturing, and testing the survival rate of the cells after the culture is finished to obtain the effect of the active substance on virus adsorption.

As a preferable technical scheme, the model construction of the novel antiviral product against the enveloped virus also comprises the product development model construction.

A second aspect of the invention provides the use of a novel antiviral product as described above for model construction of anti-enveloped viruses for predicting the mechanism of action of the antiviral product.

Has the advantages that: according to the model construction of the novel antiviral product against the enveloped virus, the cytotoxicity of the active matter is firstly determined, so that basic guidance can be provided for the subsequent whole research process, the research time is greatly shortened, and the research efficiency is improved; by testing the antiviral effect of the active substance in different modes and detecting the content of phospholipid and cholesterol in a cell membrane, the effect of the active substance on viruses and cells can be understood from a cell level, and the interaction between the active substance and N protein is analyzed by using a molecular docking method, so that the action mechanism of the active substance is perfected from a molecular level, the antiviral action mechanism of the active substance can be comprehensively predicted by integrating four kinds of detection, and theoretical guidance is provided for the application of the active substance and the research and development of products.

Drawings

To further illustrate the beneficial effects of modeling and using the novel antiviral product against enveloped viruses provided in the present invention, the accompanying drawings are provided, and it should be noted that the drawings provided in the present invention are only selected as individual examples of all drawings, and are not intended to be limiting of the claims, and all other corresponding maps obtained by the drawings provided in the present application should be considered within the scope of protection of the present application.

FIG. 1 is the model construction of the mechanism of action of the antiviral agent of the present invention.

FIG. 2 is a product development model construction in the present invention.

Detailed Description

The invention will be further illustrated below with reference to the detailed description of preferred embodiments of the invention. It should be understood that the detailed description of the embodiments is intended to illustrate the invention and not to limit the scope of the invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Unless defined otherwise, all terms (including technical and scientific terms) used in disclosing the invention have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, definitions of terms are included to better understand the teachings of the present invention.

The term "prepared from …" is used herein synonymously with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In order to solve the problems, the invention provides a novel antiviral product for model construction of anti-enveloped virus, including antiviral action mechanism model construction.

The novel antiviral product in the application refers to an antiviral product containing a pentacyclic triterpene compound, in particular to an antiviral product containing ursolic acid and/or oleanolic acid.

The term "pentacyclic triterpene compound" herein is a triterpene compound having five closed rings formed by six isoprene units connected as a parent.

The term "enveloped virus" as used herein refers to a virus having a layer of envelope outside the viral protein coat, which is primarily derived from the host cell membrane (phospholipid layer and membrane proteins), but also contains some of the virus' own glycoproteins, which can help the virus enter the host cell and maintain the integrity of the virion structure.

The enveloped virus in the present application may be influenza virus, coronavirus, rabies virus, HIV, etc.

Model construction of antiviral mechanism of action

The antiviral action mechanism model in the application is used for determining the inhibition and killing effect of active matters in the antiviral product on viruses, and further guides the product development.

In some preferred embodiments, the active is a pentacyclic triterpene compound; further preferably, the active is ursolic acid and/or oleanolic acid.

As shown in FIG. 1, in some preferred embodiments, the model construction of the antiviral mechanism of action includes measurement of cytotoxicity, measurement of antiviral effect under different action modes, measurement of N protein interaction by molecular docking method, and measurement of phospholipid and cholesterol content in cell membrane.

Determination of cytotoxicity

The inventor finds that the concentration of the active substance in the antiviral product is in direct proportion to the virus inhibition rate within a certain concentration range, but when the concentration of the active substance is too high, the antiviral effect is reduced, because the active substance generates toxicity to cells at high concentration, so that the survival rate of the cells is reduced, and the measurement of the cytotoxicity can guide the dosage range of the active substance in subsequent experiments and product development.

In some preferred embodiments, the determination of cytotoxicity comprises the steps of: adding active substances with different concentrations into cells, culturing the cells, and testing the inhibition rate of the active substances on the cells to obtain the maximum concentration which is nontoxic to the cells.

The formula for the term "inhibition" herein is calculated as: inhibition (%) -.

In some preferred embodiments, the cell is selected from one of Huh7 cell (human hepatoma cell), Vero cell (Vero monkey kidney cell), Hela cell (Hela cell), MDCK cell (canine kidney cell).

Determination of antiviral Effect under different modes of action

The virus inhibition effect of the active substances is tested under different action modes to deduce the action mechanism of the antiviral product, thereby providing theoretical support for product development.

In some preferred embodiments, the mode of action comprises pretreatment of the virus, virus replication, pretreatment of the cells, virus adsorption.

The term "viral replication" as used herein refers to the process of synthesizing viral genome and protein respectively in living cells by using the genome as a template and using enzyme to assemble into complete virus particles. The virus replication cycle mainly comprises six continuous steps of adsorption, penetration, uncoating, biosynthesis, assembly and release, wherein the adsorption step corresponds to the term of virus adsorption, and refers to a process that a virosome is in contact with a cell to carry out electrostatic binding, and a surface site of the virosome after the binding is combined with a corresponding receptor on a host cell membrane.

In some preferred embodiments, the determination of the antiviral effect of the mode of action of the pre-treated virus comprises the steps of: after mixing and incubating the active substance and the enveloped virus, adding the mixture into cells for co-culture, and testing the inhibition rate of the active substance on the enveloped virus to obtain the antiviral effect under the action mode of using the active substance to pretreat the virus.

In some preferred embodiments, the determination of the antiviral effect of the mode of action of viral replication comprises the steps of: infecting cells with the virus liquid, incubating, removing the virus liquid, adding the active substance to continue culturing the cells, and testing the survival rate of the cells to obtain the effect of the active substance on virus replication.

In some preferred embodiments, the determination of the antiviral effect of the pretreated cells in their mode of action comprises the steps of: adding the active substance into the cells for cell culture, adding virus liquid for continuous culture, and testing the survival rate of the cells after the culture is finished to obtain the antiviral effect under the action mode of using the active substance to pretreat the cells.

In some preferred embodiments, the determination of the antiviral effect of the mode of action of viral adsorption comprises the steps of: mixing the active substance with virus solution, immediately adding into cells for adsorption, removing the liquid after adsorption, continuously culturing, and testing the survival rate of the cells after the culture is finished to obtain the effect of the active substance on virus adsorption.

The inventor finds that the research on the action mechanism of the active substance can provide guidance for the type of the product, for example, when the pretreatment of the virus by using the active substance can play a remarkable inhibiting effect, the product can be developed into a disinfectant product for environmental disinfection; when the active matter can obviously inhibit virus replication or virus adsorption, the active matter can be considered to be used for research and development of therapeutic drugs, so that the interaction of the virus and normal cells in vivo is reduced; when the active matter is used for pretreating cells to improve the survival rate of the cells, the active matter can be used for research and development of preventive medicaments, the immunity of a human body to viruses is improved, and the risk of virus infection is reduced.

Molecular docking method for detecting N protein interaction

The term "molecular docking method" herein refers to a method for drug design by the characteristics of a receptor and the interaction pattern between the receptor and a drug molecule, for predicting the binding pattern and binding stability between molecules (such as ligand and receptor), and can be performed using software such as DOCK, AutoDock, FlexX, and the like.

The term "N protein" refers to the nucleocapsid protein, the N protein in coronavirus is the only protein existing in nucleocapsid, and intertwines with viral genome RNA to form viral nucleocapsid, which plays an important role in the synthesis process of viral RNA, and the N protein accounts for the largest proportion in the structural protein of virus, and can be used as the target of virus prevention and treatment drugs.

The inventor finds that the interaction between the active substance and the N protein is researched by adopting a molecular docking method, the action site of the active substance and the N protein can be predicted, and theoretical guidance is provided for research and development work of the active substance and analogues thereof as medicines from a molecular level.

Detecting the content of phospholipid and cholesterol in cell membrane

The inventor finds that enveloped viruses rely to a large extent on lipid envelopes to enter susceptible cells, and when the enveloped viruses infect host cells, the invasion process is mainly the fusion process of the viral envelopes and host cell membranes, so that virus particles invade the host cells, namely, the genetic materials of the viruses enter the cells in the form of nucleocapsids. The surface glycoprotein of the virus mediates the fusion process by identifying and combining with a corresponding specific receptor, and the phospholipid and the cholesterol play important roles in the invasion process of the virus, the effect of the active substance on the content of the phospholipid and the cholesterol in a cell membrane is detected, and the research can be carried out from the perspective of the effect of the active substance on cells so as to perfect the antiviral mechanism of the active substance.

In some preferred embodiments, the detecting the phospholipid and cholesterol levels in the cell membrane comprises the steps of: treating the cells with an active agent, and measuring the phospholipid and cholesterol content before and after treatment; treating the cells with M beta CD, and measuring the contents of phospholipid and cholesterol before and after treatment; the processing ability of the actives was compared to that of M β CD.

The term "M β CD" as used herein refers to methyl- β -cyclodextrin, a compound that destroys cholesterol and phospholipids, which destroys lipid rafts in cells, but has no effect on the integrity of cell membranes.

In some preferred embodiments, the antiviral mechanism of action model construction further comprises summarizing the mechanism of action of the active against viruses.

The inventor finds that the determination of the toxicity of the active substance to the cells can provide basic guidance for the subsequent whole research process, greatly shortens the research time and improves the research efficiency, the antiviral effect of the active substance is tested in different modes, the contents of phospholipid and cholesterol in cell membranes are detected, the effect of the active substance on viruses and cells can be understood from the cell level, the interaction between the active substance and N protein is analyzed by using a molecular docking method, the action mechanism of the active substance is perfected from the molecular level, the antiviral action mechanism of the active substance can be comprehensively predicted by integrating four detections, and theoretical guidance is provided for the application of the next active substance and the research and development of products.

In some preferred embodiments, the model construction of the novel antiviral product against enveloped viruses further comprises a product development model construction.

Product development model construction

In some preferred embodiments, the product development model construction comprises HPLC screening of natural plant material, product development and optimization, and product export.

As shown in fig. 2, in some preferred embodiments, the product development model construction includes HPLC screening of natural plants containing ursolic acid and oleanolic acid, formulation of natural plant raw materials, optimization of pentacyclic triterpene antiviral products, coronavirus killing test, and product output.

The term "HPLC" herein refers to high performance liquid chromatography, which uses liquid as mobile phase, and adopts high pressure infusion system to pump mobile phase such as single solvent with different polarity or mixed solvent, buffer solution, etc. into chromatographic column filled with stationary phase, after each component in the column is separated, it enters into detector for detection, thus realizing analysis of sample.

In some preferred embodiments, the natural plant is selected from the group consisting of loquat leaves, hawthorn, oldenlandia, ligustrum lucidum, persimmon, plantain, pomegranate, smoked plum, and mixtures of one or more of papaya, and any combination thereof.

In some preferred embodiments, the natural plant material is a plant extract of a corresponding natural plant, and the extraction method can be any one of those well known to those skilled in the art, such as water decoction, alcohol extraction, supercritical CO₂Extraction methods, and the like.

The inventor screens natural plants containing ursolic acid and oleanolic acid by an HPLC method, develops products by taking the natural plants with higher active matter content as raw materials, gradually improves the primary formula according to a test for killing coronavirus, and finally obtains the products with excellent virus inactivation performance for production.

A second aspect of the invention provides the use of a novel antiviral product as described above for model construction of anti-enveloped viruses for predicting the mechanism of action of the antiviral product.