CN113797193A

CN113797193A - Application of trimethoxy quercetin in preparation of anti-coronavirus medicines

Info

Publication number: CN113797193A
Application number: CN202111257144.1A
Authority: CN
Inventors: 顾玮; 李玉环; 郝小江; 熊燕; 陈俊磊; 黄烈军; 苑春茂
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2021-12-17
Anticipated expiration: 2041-10-27
Also published as: CN113797193B

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of 7,3 ', 4' -trimethoxyquercetin in preparation of an anti-coronavirus drug, wherein the structural formula of the 7,3 ', 4' -trimethoxyquercetin is shown as (I), and the application of the 7,3 ', 4' -trimethoxyquercetin in preparation of an anti-coronavirus drug is used for treating IC of H460 cells infected with HCoV-OC43₅₀The concentration was 1.23. + -. 0.16. mu.g/mL. The invention has the advantages that the natural product 7,3 ', 4' -trimethoxyquercetin is selected for the activity research of anti-human coronavirus, and the 7,3 ', 4' -trimethoxyquercetin can effectively inhibit the human coronavirus HCoV-OC43 infected cells in vitro, and has obvious curative effect. In addition, the source of the 7,3 ', 4' -trimethoxy quercetin is wide and low in price, and more choices are provided for preparing anti-coronavirus medicines.

Description

Application of trimethoxy quercetin in preparation of anti-coronavirus medicines

Technical Field

The invention relates to the technical field of natural active compound application, in particular to application of 7,3 ', 4' -trimethoxyquercetin in preparation of an anti-coronavirus drug.

Background

Human coronavirus (Humancoronavirus; HCoV) is one of the important pathogens causing respiratory tract infection in humans. There are 7 kinds of coronavirus which are known to infect humans, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoVHKU1, SARS-CoV-1, MERS-CoV, and 2019 novel coronavirus (SARS-CoV-2). Of these, HCoV-OC43 and HCoV-229E were discovered as early as 1966 and 1967, but the coronavirus was not widely noticed until 2003 after a global outbreak of SARS. Because the disease fatality rate caused by the coronavirus is low, people do not pay enough attention, and no vaccine or specific medicine aiming at the virus exists at present. Due to the high variability of these viruses, it is also of great importance to study drugs that are capable of having a broad spectrum of anti-coronaviruses. The traditional Chinese medicine has unique advantages in the aspect of treating viral diseases, has wider target compared with western medicines, can eliminate the damage of viruses to bodies from various ways, improves the immunity of organisms, has the advantages of small side effect, no drug resistance and the like. Therefore, natural antiviral drugs found from the traditional Chinese medicines have better prospects.

At present, antiviral drugs such as ribavirin and arbidol which are commonly used have certain effect on coronavirus, but are easy to generate side effects such as hemolytic anemia and the like. The active substance extracted from the plant has low toxicity and high efficiency, and the 7,3 ', 4' -trimethoxyquercetin is a flavonoid compound from natural sources, and can be separated and prepared in large quantities in Chinese medicines such as mistletoe, blumea balsamifera, medicinal dandelion, common sow thistle, acacia rattan and the like. At present, no report is made on the application of the coronavirus.

Disclosure of Invention

The invention aims to solve the problems in the background art and provides application of 7,3 ', 4' -trimethoxyquercetin in preparation of an anti-coronavirus medicine.

In order to achieve the purpose, the invention adopts the technical scheme that: the application of 7,3 ', 4' -trimethoxyquercetin in preparing anti-coronavirus medicines is characterized in that the coronavirus is human coronavirus HCoV-OC43, the structural formula of the 7,3 ', 4' -trimethoxyquercetin is shown as (I), and the IC of the 7,3 ', 4' -trimethoxyquercetin on H460 cells infected with HCoV-OC43 is shown as the specification₅₀The concentration is 1.23 +/-0.16 mu g/mL

It is understood by those skilled in the art that the inclusion of a pharmaceutically acceptable salt of 7,3 ', 4' -trimethoxyquercetin and/or a pharmaceutically acceptable carrier is within the scope of the present invention.

Further, the anti-coronavirus drug is an oral preparation, an injection, an inhalation preparation or an external preparation.

The beneficial technical effects of the invention are as follows: the test data provided by the invention show that the 7,3 ', 4' -trimethoxy quercetin anti-coronavirus drug has good effect, and the in vitro anti-human coronavirus HCoV-OC43 effect is obvious (IC)₅₀1.23 ± 0.16 μ g/mL), the therapeutic index is higher. Meanwhile, as the 7,3 ', 4' -trimethoxy quercetin is a chemical component in the Chinese herbal medicine, has higher content in a plurality of plants, simple preparation method, low price, easy obtainment and no obvious toxic or side effect, provides more choices for preparing the medicine for preventing and treating the coronavirus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a NMR spectrum of 7,3 ', 4' -trimethoxyquercetin in accordance with the present invention;

FIG. 2 is a NMR carbon spectrum of 7,3 ', 4' -trimethoxyquercetin of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The present invention provides a method for extracting and preparing a pure product of 7,3 ', 4' -trimethoxyquercetin from a plant, which comprises the following specific steps:

heating and refluxing the dry blumea balsamifera leaves by 80% methanol for 3 times, wherein each time is 4 hours, recovering under reduced pressure to obtain an extract, adding water into the extract to prepare a suspension, extracting for 4-5 times by using petroleum ether and ethyl acetate, and concentrating under reduced pressure to obtain a petroleum ether extract and an ethyl acetate extract respectively. Subjecting the petroleum ether extract to 40-80 mesh normal phase silica gel column chromatography, sequentially subjecting to petroleum ether: ethyl acetate (1: 0-2: 1), dichloromethane: methanol (1:0-0:1) was eluted to give 12 samples, designated Fr-1 to Fr-12 (FIG. 1-2).

Fr-9 was separated on C18 reverse phase silica gel (methanol: water: 40:1-90:1) to give 10-stage samples, and Fr-9-3 was separated multiple times on normal phase silica gel (petroleum ether: ethyl acetate: 15: 1) to give compound 1(815.3 mg). Compound 1 was identified as 7,3 ', 4' -trimethoxyquercetin by 1D-NMR in combination with literature data. The structural formula is shown as the following (I)

Example 2

The purpose of this example was to test the in vitro anti-coronavirus activity of 7,3 ', 4' -trimethoxyquercetin.

1. Virus strain: human coronavirus OC43(HCoV-OC43) was supplied by ATCC.

2. Cell: the passaged human lung cancer cells H460 are all subcultured in virus chamber of institute of medical and biotechnology of Chinese academy of medical sciences, and are cultured in DMEM medium containing 10% fetal bovine serum (inactivated total serum) and 1% double antibody (penicillin and streptomycin) at 37 deg.C and 5% CO₂Culturing in an incubator, and carrying out subculture once every 2-3 days.

3. Sample treatment: the sample 7,3 ', 4' -trimethoxyquercetin is light yellow powder, is prepared by separating from traditional Chinese medicine blumea balsamifera in the early stage of the laboratory, and has the purity of more than 98 percent. The DMSO is prepared into mother solution before use, and the culture solution is diluted to a certain concentration and then diluted by 3 times during detection, and the dilution is 8 (100.0ug/mL, 33.3ug/mL, 11.1ug/mL, 3.7ug/mL, 1.23ug/mL, 0.41ug/mL, 0.14ug/mL, 0.05ug/mL) in total.

4. Positive control drug: ribavirin injection is purchased from Tianjin Jinyao Hubei Tian medicine pharmaceutical industry Co., Ltd, with the batch number of 31712252 and the specification of 100mg/mL, is diluted to the required concentration when used, and is stored in a refrigerator at 4 ℃.

5. Cell culture: adding 3mL of 0.25% Trypsin-EDTA into a culture bottle full of H460 cells, digesting for 1-2 minutes at 37 ℃, removing digestive juice, adding culture solution, beating by blowing, carrying out passage according to the ratio of 1:3, and then carrying out 5% CO treatment at 37 DEG C₂Culturing, subculturing once for 2-3 days, preparing into 15 ten thousand cells per mL during plating, inoculating 96-well cell culture plate with 0.1mL per well, 37 deg.C, and 5% CO₂The cells were cultured overnight and the experiment was performed after the cells had grown into a monolayer.

6. Activity assay against HCoV-OC43 (CPE method)

The experiment was performed in passaged H460 cells at 1.5X 10⁴Inoculating one cell/well in a 96-well plate, infecting cells in the 96-well plate with 100TCID50 HCoV-OC43 virus liquid after overnight culture, diluting a drug to be detected with the culture liquid, simultaneously administering the drug to be detected during infection, carrying out experiments on samples of which the doses are 8 times diluted with the drug to be detected, setting 2 parallel wells for each dose, observing results when the lesions of a virus control group reach 4+, recording and calculating the half inhibition concentration of the drug to the virus by a Reed-Muench method (the formula isIC50 ═ AntiLog (a + D × (B-50)/(50-C))) and selection index (SI ═ IC50/TC 50).

Wherein: a is the drug concentration with the cumulative inhibition rate of less than 50%, B is the inhibition rate with the cumulative inhibition rate of > 50%, C is the inhibition rate with the cumulative inhibition rate of less than 50%, and D is the log dilution factor.

7. Screening results

The results of in vitro screening for the inhibitory effect of 7,3 ', 4' -trimethoxyquercetin on human coronavirus HCoV-OC43 are shown in Table 1. IC of 7,3 ', 4' -trimethoxyquercetin on HCoV-OC43 in H460 cells₅₀1.23 +/-0.16 mu g/ml, and the selection index SI is 20.95; IC of RBV versus HCoV-OC43₅₀2.13. + -. 0.76. mu.g/ml, selection index SI of 27.0, IC₅₀Is obviously higher than the positive drug RBV.

TABLE 17, 3 ', 4' -trimethoxyquercetin anti-coronavirus HCoV-OC43 Activity screening results

In general, the 7,3 ', 4' -trimethoxyquercetin provided by the invention is applied to preparation of anti-coronavirus medicines, a cell experiment of anti-human coronavirus HCoV-OC43 is carried out by exogenously adding 7,3 ', 4' -trimethoxyquercetin, and the experimental result shows that the 7,3 ', 4' -trimethoxyquercetin has remarkable effects on the two coronaviruses, and IC (integrated circuit) of the 7,3 ', 4' -trimethoxyquercetin₅₀1.23 +/-0.16 mu g/mL (HCoV-OC43), and the drug effect is obviously higher than the inhibition effect (IC) of broad-spectrum antiviral drug Ribavirin (RBV) on HCoV-OC43₅₀＝2.13±0.76μg/mL)。

Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.

Claims

The application of 7,3 ', 4' -trimethoxyquercetin in preparing anti-coronavirus medicines, wherein the coronavirus is human coronavirus HCoV-OC43, and is characterized in that: the structural formula of the 7,3 ', 4' -trimethoxyquercetin is shown as (I), and the IC of the 7,3 ', 4' -trimethoxyquercetin on H460 cells infected with HCoV-OC43₅₀The concentration is 1.23 +/-0.16 mu g/mL
2. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable salt of 7,3 ', 4' -trimethoxyquercetin and/or a pharmaceutically acceptable carrier.
3. The use according to any one of claims 1-2, wherein the anti-coronavirus agent is an oral formulation, an injection, an inhalation formulation or an external formulation.