CN114306337A

CN114306337A - Application of anisodamine in preparing medicine for treating novel coronavirus

Info

Publication number: CN114306337A
Application number: CN202011044836.3A
Authority: CN
Inventors: 刘冲; 魏巍
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-04-12

Abstract

The invention discloses an application of anisodamine in preparing a medicine for treating novel coronavirus, belonging to the technical field of medicines. Experiments show that the anisodamine has very remarkable inhibitory effect on new coronavirus for the first time, provides a treatment medicament with large safety range and small toxic and side effects for the clinical treatment of the novel coronavirus pneumonia, and also provides experimental support for expanding the clinical application of the anisodamine.

Description

Application of anisodamine in preparing medicine for treating novel coronavirus

Technical Field

The invention relates to the technical field of medicines, in particular to application of anisodamine in preparation of a novel coronavirus treatment medicine.

Background

Anisodamine is alkaloid extracted from Scopolia tangutica of Solanaceae in 1965 by Chinese scientist, and its synthetic product is 654-2. Anisodamine has a structure similar to atropine, and is an ester of tropine acid and organic base, and has asymmetric hydroxyl group at the 6-position carbon atom of tropine group.

Anisodamine belongs to a non-selective M receptor blocker and can block M1-M5 receptor subtype. Therefore, anisodamine has a series of pharmacological actions similar to atropine, such as inhibition of salivary secretion, inhibition of gastric acid and sweat secretion, inhibition of gastrointestinal motility and bladder contraction, and other actions including mydriasis, bronchiectasis, and rescue of organophosphate poisoning. Since 1965The anisodamine is used for treating infectious diseases such as septic shock, disseminated intravascular coagulation and the like, and has good treatment effect. In pediatrics of Beijing friendship hospital, anisodamine is used to treat epidemic cerebrospinal meningitis with mortality rate of 66.9% to 12.4%, and anisodamine is used to treat toxic bacillary dysentery with mortality rate of 20-30% to 0.5%. Researchers attribute the mechanism of action to the inhibition of noradrenaline-induced microvascular spasm and the dilation of small blood vessels, and the action against free radical damage. In recent years, it has been reported that the antishock effect of anisodamine is closely related to reduction of thromboxane synthesis in platelets and inhibition of granulocyte and platelet aggregation, and simultaneously can antagonize the interference of endotoxin on the flow state and function of leukocytes, reduce the rolling and adhesion of leukocyte adhesion, obviously reduce the adhesion coefficient of endothelial cells and leukocytes, and accelerate blood flow rate. Anisodamine can also reduce the content of malondialdehyde in isolated tissues, protect injury of endotoxin-activated polymorphonuclear cells to vascular endothelial cells, and stabilize endothelial cell membrane. These effects can be attributed to the action of relieving small vasospasm, dilating blood vessels, increasing blood flow to improve microcirculation, and blocking Ca²⁺The membrane stabilization by internal flow is the major mechanism known at present.

In recent years, some studies show that anisodamine can play an antishock role through an anti-inflammatory mechanism besides a vascular mechanism, and Kushiya et al found that anisodamine has the capability of remarkably inhibiting the generation of inflammatory factors (TNF alpha, IL-1 beta, IFN-gamma) induced by human peripheral mononuclear cells by type 2 Shiga toxin (Stx 2). Nakagawa et al have proved that anisodamine can significantly inhibit the level of proinflammatory cytokines (TNF alpha, IL-6, IL-1 beta, IFN gamma) in serum of septic shock mice, and can effectively reduce the mortality of septic shock mice. In cultured human umbilical vein endothelial cells, anisodamine can remarkably inhibit the expression of plasminogen activation inhibitor 1(PAI-1) and Tissue Factor (TF) by endotoxin-induced endothelial cells, and the action of anisodamine is presumed to be related to NF-kB. These studies suggest that anisodamine's anti-infective action plays an important role in its anti-shock action.

The international committee for viral classification will name the novel coronavirus causing the novel coronavirus pneumonia as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The WHO announces on the same day that the Disease caused by the virus is formally named as Coronavir Disease 2019 (COVID-19). The shizheng team of the institute for wuhan virus, academy of sciences, china, obtained the whole genome sequence of the virus from 5 early patient samples, and the similarity of the virus sequence from 5 patients reached 99.9%. Although the sequence consistency of the whole genome of the virus and a severe acute respiratory syndrome coronavirus (SARS-CoV)/BJ01 strain is 79.5 percent, the consistency of 7 conserved replicase structural domains in an Open Reading Frame (ORF)1ab used as a coronavirus classification standard and the SARS-CoV amino acid sequence is as high as 94.6 percent, which indicates that the novel coronavirus SARS-CoV belongs to a SARS-related coronavirus (SARSr-CoV) family. The receptor on which SARS-CoV-2 invades the host cell is the same as SARS-CoV, and is also angiotensin-converting enzyme II. Due to the expression profile of ACE2, these 2 coronaviruses predominantly infect ciliated bronchial epithelial cells and human alveolar type II epithelial cells. The COVID-19 epidemic seriously threatens the human health, the society and the economic development; in the face of the severe situation of the global outbreak of COVID-19, the research and development of related drugs and vaccines are very urgent needs for epidemic situation control.

Therefore, the problem to be solved by those skilled in the art is to provide the application of anisodamine in preparing the medicine for treating the novel coronavirus.

Disclosure of Invention

In view of the above, the present invention provides an application of anisodamine in preparing a medicament for treating a novel coronavirus.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of anisodamine in preparing medicine for treating coronavirus is provided.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the anisodamine as a medicament which is clinically applied for many years has clear pharmacological characteristics, large safety range and small toxic and side effects, and the experimental data of the invention show that the anisodamine has very obvious effect of inhibiting the neocoronaviruses for the first time. The invention provides a treatment medicament with large safety range and small toxic and side effects for the clinical treatment of the novel coronary virus pneumonia, and also provides experimental support for expanding the clinical application of the anisodamine.

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, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the effect of anisodamine of various concentrations on cell viability;

FIG. 2 is a diagram showing the result of fluorescent quantitation of the inhibitory effect of anisodamine on novel coronavirus;

wherein, A: a normal group; b: new coronavirus infection group; c: neocoronavirus infection + anisodamine (50 μ g/ml) pretreatment group; d: neocoronavirus infection + anisodamine (100 μ g/ml) pretreatment group; e: neocoronavirus infection + anisodamine (200. mu.g/ml) pretreatment group;

FIG. 3 is a diagram showing the fluorescent quantitative statistics of the inhibition of novel coronavirus by anisodamine according to the present invention; wherein the content of the first and second substances,^***p < 0.001 vs. New coronaviruses.

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

(1) Cell and reagent

The Vero E6 cells were provided by the department of biomedical protection, university of naval medical science, university of naval military medical, university, and research laboratory; the nose/throat swab sample used in the experiment is derived from a COVID-19 suspected pneumonia patient collected by the Changhai hospital of the naval medical university from 1 month to 2 months in 2020, and a SARS-CoV-2 nucleic acid positive sample is detected from the COVID-19 suspected patient nose/throat swab sample to be detected by using a qRT-PCR method by the biomedical protection and research laboratory of the naval medical department of the naval medical university. The samples for SARS-CoV-2 isolation were aliquoted at 200. mu.L per tube and frozen in a-80 ℃ freezer. COVID-19 patient convalescent serum samples were obtained from confirmed COVID-19 patients admitted to the third national Hospital, Zhenjiang, university of Jiangsu (collected 3 weeks after patient treatment recovery discharge). The whole process of virus isolation is carried out in the P3 laboratory of the university of naval military medical science. The SARS-CoV-2(ORF1ab/N gene) fluorescent probe quantitative kit is a product of Shanghai Berger biological medicine, Inc.; the penicillin streptomycin double-resistance is a product of Mei Thermo Fisher company.

(2) CCK8 detection of cell viability

Anisodamine was diluted with PBS and seeded in 96-well plates with Vero E6 cell suspension (100. mu.l/well) at a cell density of about 4X 10⁴Each of the cells was incubated at 24 hours, the cells were changed to each of the final concentrations of anisodamine of 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL and 400. mu.g/mL after 24 hours, 10. mu.l of CCK8 was added to each well according to the instructions of the CCK8 kit (Shanghai Dong Kenren chemical Co., Ltd.), the plate was incubated in an incubator for 0.5 hours, and then absorbance values of cells of each group at 450nm were measured using a microplate reader, and the results are shown in FIG. 1.

The results in FIG. 1 show that there was no significant decrease in cell viability after 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml of anisodamine, suggesting that neither of these concentrations of anisodamine has a toxic effect on cells.

(3) Cell administration and grouping

According to the therapeutic dose obtained by the research of the preliminary experimental study of the subject group and the detection result of CCK8, the administration doses of anisodamine are determined to be divided into the following groups respectively: 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml; the inhibitory effect of anisodamine pretreatment on novel coronaviruses was observed.

(4) Immunofluorescence detection and cytopathic observation

Preparation of the virus: vero E6 cells were cultured in DMEM medium containing 10% FBS (1% streptomycin diabody) in 5% CO₂Culturing in incubator at 37 deg.C and saturated humidity for use. Vero E6 cells were seeded in 96-well cell culture plates at a cell density of about 4X 10 at 100. mu.l/well⁴And (4) adding a nasal/pharyngeal swab sample when the cell confluency is about 90% for virus separation. Taking out a nasal/pharyngeal swab sample frozen in a refrigerator with the temperature of-80 ℃, putting the nasal/pharyngeal swab sample in the refrigerator with the temperature of-80 ℃ for melting, taking out the nasal/pharyngeal swab sample and putting the nasal/pharyngeal swab sample on ice, adding DMEM culture solution (containing 2 mu g/mL of TPCK pancreatin, 1% of streptomycin double antibody and 0.2% of bovine serum albumin) with the same volume, putting the nasal/pharyngeal swab sample in an incubator with the temperature of 37 ℃, sucking the supernatant of the nasal/pharyngeal swab after 1h, and adding a 96-well plate inoculated with Vero E6 cells, wherein each well is 50 mu L. The 96-well plate is placed in an incubator, after 2h, the supernatant is aspirated, the well is washed gently with PBS 3 times, then 100 μ L of serum-free DMEM culture solution containing TPCK pancreatin is added, and the incubation is continued in the incubator. Observing whether cells have cytopathic effect (CPE) every day after 24h, sucking supernatant to avoid touching cell debris when more than 50% of cells have CPE and fall off from the bottom surface of the well, then centrifuging at high speed, carefully sucking 10 mu L of the supernatant to subculture Vero E6 cells inoculated in a 24-well plate, taking 20 mu L of the supernatant, detecting SARS-CoV-2 nucleic acid by using a qRT-PCR method, determining the sample as a SARS-CoV-2 nucleic acid positive sample, and determining the virus titer to be 1x10⁸PFU/ml。

Administration and observation: vero E6 cells were cultured in DMEM medium containing 10% FBS (1% streptomycin diabody) in 5% CO₂Culturing in incubator at 37 deg.C and saturated humidity for use. Anisodamine was added to cultured Vero E6 cells (100. mu.l/well, cell density about 4X 10⁴one/mL) (96-well plate), making the final concentration of anisodamine 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 6 wells per drug concentration, incubating for 1 hour, adding the virus isolated from the nasal/pharyngeal swabs of COVID-19 pneumonia patients into Vero E6 cells (2 μ L/well), and incubating for 24 hours. The 96-well plate was then removed and soaked in methanol and placed in a-20 ℃ freezer for 30 minutes to inactivate the virus and fix the cells. Then using cell immunofluorescence method to detect SARS-CoV-2 protein, using serum of convalescent patient as primary antibody，Alexa

Labeled anti-rabbit and anti-human fluorescent IgG were used as secondary antibodies, and after immunofluorescent staining, observation and photographing were performed using a full-automatic cell Imaging and analysis system (bioteck staining 5 Imaging Reader, BioTek corporation, usa), and the results were shown in fig. 2 to fig. 3.

The results in FIGS. 2-3 show that the expression of SARS-CoV-2 protein is significantly increased after infection with the novel coronavirus, while the expression of SARS-CoV-2 protein was found to be effectively inhibited after pretreatment with anisodamine at different concentrations, especially after pretreatment with anisodamine at 100. mu.g/ml and 200. mu.g/ml, the expression of SARS-CoV-2 protein was significantly inhibited, and the effect thereof was dose-dependent.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of anisodamine in preparing medicine for treating coronavirus is provided.