CN114246859A - Application of andrographolide in preparing product for treating coronavirus infection - Google Patents
Application of andrographolide in preparing product for treating coronavirus infection Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Abstract
The invention discloses application of andrographolide in preparing a product for treating coronavirus infection. The invention provides an application of andrographolide or pharmaceutically acceptable salt thereof or a substance taking andrographolide or pharmaceutically acceptable salt thereof as an active ingredient in any one of the following substances: preparing a product for inhibiting coronavirus; preparing a product capable of treating a disease caused by coronavirus infection; a product is prepared which is capable of ameliorating the symptoms caused by coronavirus infection. The invention screens out andrographolide which is an active medicine and has the effect of resisting SARS-CoV-2 from three medicines. The invention has important clinical application value for the treatment of COVID-19.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of andrographolide in preparation of a product for treating coronavirus infection.
Background
Coronaviruses are a class of nonsegmented, positive-sense RNA viruses with envelope that infect a variety of mammalian and avian hosts, including SARS-CoV and MERS-CoV, which are reported to have bat as their natural host. It has been previously known that 6 coronaviruses can infect humans, including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, and MERS-CoV.
The outbreak of the novel viral pneumonia epidemic situation in the end of 2019 is caused by a new coronavirus strain, the world health organization named the strain 2019 novel coronavirus (2019-nCoV for short in English) in 1 month and 12 days in 2020, and the disease caused by the strain is named COVID-19. On the 2 nd month of 2020, on the "Nature" miscellaneous aspiration journal "microbiology", the coronavirus research group of the International Committee on Virus Classification was formally named SARS-CoV-2.
SARS-CoV-2 belongs to Sarbecovirus subtypes of the order of nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), and the genus of beta coronavirus, and has an envelope, wherein virus particles are spherical or elliptical, have polymorphism, and have a diameter of 60-140 nm. SARS-CoV-2 is a single positive-strand RNA virus with a genome of 29,903bp in total length, and has 78.7% and 48.7% sequence similarity with SARS-CoV and MERS-CoV, respectively. At present, no specific medicine aiming at novel coronary pneumonia (COVID-19) caused by SARS-CoV-2 infection exists, and the development of effective vaccine and specific medicine are important for inhibiting the spread of diseases and treating diseases.
A novel coronavirus has been separated and cultured from pangolins by Beijing university of chemical industry Mytilus edulis team and is named as xCov (which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 19295; the preservation date is 2.14.2020; the address is No. 3 Xilu No. 1. on the North Cheng of the Korean-Yang district in Beijing City; the taxonomic nomenclature is coronavirus), and the whole genome sequence analysis result shows that: the virus has 92 percent of homology with the S protein of 2019-nCoV, and is the virus which has the highest homology with the S protein of 2019-nCoV and is successfully separated and cultured so far. Further experiments showed that the receptors of the virus-infected cells were identical to 2019-nCoV, and were angiotensin-converting enzyme 2(ACE 2). The virus does not infect people, is very safe, and can be used for screening and evaluating medicaments, screening and evaluating vaccines and preparing attenuated and inactivated vaccines of 2019-nCoV viruses. xCov, after deposition, has been disclosed in two documents: Hua-Hao Fan, Li-Qin Wang, Wen-Li Liu, Xiao-Ping An, Zhen-Dong Liu, Xiao Qi He, Li-Hua Song, Yi-Gang Tong, reproduction of clinical approved drugs for treatment of coronavirus disease 2019in a 2019-novel coronavirus (2019-nCoV) related coronavirus. Lam, T.T., Jia, N., Zhang, Y.et al.identifying SARS-CoV-2-related coronaviruses in Malayan specifications. Nature 583, 282-285 (2020) https:// doi.org/10.1038/s 41586-020-. The virus GX _ P2V/pangolin/2017/Guangxi in reference 1 and GX/P2V in reference 2 are the same strain as xCov in the deposited certificate. In document 1, it is reported that 2,406 existing drugs are screened by using the model (GX _ P2V/pangolin/2017/Guangxi), a plurality of old drugs which can effectively inhibit coronavirus are found and verified, and the results of two models are confirmed to be consistent by comparing part of the drugs with the screening model of new coronavirus.
In recent years, the research on the antiviral property of traditional Chinese medicines at home and abroad is increasing day by day, and as the traditional Chinese medicines have the advantages of low toxic and side effects, more active ingredients, various action targets, abundant resources and the like, part of the traditional Chinese medicines are applied to the field of antiviral infection. The traditional Chinese medicines exert great potential in the epidemic situation, and three medicines with obvious curative effects, such as Jinhua Qinggan granule, Lianhua Qingwen Capsule, Xuebijing injection, Lung clearing and toxin expelling decoction, dampness eliminating and toxin expelling formula, Lung diffusing and toxin expelling formula and the like, are screened at present. However, the traditional Chinese medicine components are complex and various, and part of the traditional Chinese medicines may also contain toxic and harmful substances, so that identification of effective components capable of playing an antiviral role has important significance for prevention and treatment of the new coronary pneumonia.
Disclosure of Invention
The invention aims to provide application of andrographolide in preparing a product for treating coronavirus infection.
In a first aspect, the present invention claims the use of Andrographolide (Andrographolide; Andrographis) or a pharmaceutically acceptable salt thereof, or a substance comprising Andrographolide or a pharmaceutically acceptable salt thereof as an active ingredient, in any one of the following:
(A1) preparing a product for inhibiting coronavirus, or inhibiting coronavirus;
(A2) preparing a product capable of treating a disease caused by a coronavirus infection, or treating a disease caused by a coronavirus infection;
(A3) preparing a product capable of ameliorating symptoms due to coronavirus infection, or ameliorating symptoms due to coronavirus infection.
Wherein, the inhibiting coronavirus can be inhibiting coronavirus at an organism level or a cell level. The inhibiting coronavirus is inhibiting coronavirus replication.
The symptoms due to coronavirus infection may be fever, cough, shortness of breath, and/or dyspnea, among others.
Further, the inhibition of coronavirus is effected after coronavirus enters the host or host cell (i.e., after entry into the cell). Andrographolide is therefore suitable for the preparation of a product intended for the treatment of diseases caused by coronavirus infections.
In the present invention, the coronavirus may be SARS-CoV-2 virus or isolated pangolin GX _ P2V/pangolin/2017/Guangxi or other similar coronavirus.
When the coronavirus is SARS-CoV-2 virus, the disease caused by its infection is COVID-19.
In the invention, the andrographolide is derived from Xiyanping injection in three medicines.
In such applications, the product may be in particular a medicament.
In a second aspect, the invention claims a product.
The product claimed by the invention has the active ingredient of andrographolide or pharmaceutically acceptable salt thereof; the product has any one of the following uses:
(a1) inhibiting coronavirus;
(a2) treating diseases due to coronavirus infection;
(a3) improving symptoms caused by coronavirus infection.
Further, the inhibition of coronavirus is effected after coronavirus enters the host or host cell (i.e., after entry into the cell). Andrographolide is therefore suitable for the preparation of a product intended for the treatment of diseases caused by coronavirus infections.
In the present invention, the coronavirus may be SARS-CoV-2 virus or isolated pangolin GX _ P2V/pangolin/2017/Guangxi or other similar coronavirus.
When the coronavirus is SARS-CoV-2 virus, the disease caused by its infection is COVID-19.
The product may in particular be a medicament.
The structural formula of the andrographolide is shown as a formula I.
The invention screens out andrographolide with SARS-CoV-2 resisting activity from three medicine parties based on a pangolin coronavirus xCoV (namely a pangolin coronavirus isolate GX _ P2V/pangolin/2017/Guangxi) medicine screening model. The invention has important clinical application value for the treatment of COVID-19.
Drawings
FIG. 1 shows the virus replication inhibition rate of andrographolide as a monomer of traditional Chinese medicine on the pangolin coronavirus xCoV.
FIG. 2 is EC of andrographolide50、CC50And SI.
FIG. 3 shows the results of the experiment of the dosing time of andrographolide.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The coronavirus squama Manis isolate GX _ P2V/pangolin/2017/Guangxi used in the examples below has been disclosed in two documents:
1.Hua-Hao Fan,Li-Qin Wang,Wen-Li Liu,Xiao-Ping An,Zhen-Dong Liu,Xiao Qi He,Li-Hua Song,Yi-Gang Tong.Repurposing of clinically approved drugs for treatment of coronavirus disease 2019in a 2019-novel coronavirus(2019-nCoV)related coronavirus.
lam, T.T., Jia, N., Zhang, Y.et al.identifying SARS-CoV-2-related coronaviruses in Malayan specifications. Nature 583, 282-285 (2020) https:// doi.org/10.1038/s 41586-020-. The GX _ P2V/pangolin/2017/Guangxi used in the invention is firstly subjected to strain preservation and then published in a paper, and xCov in a preservation certificate is virus GX _ P2V/pangolin/2017/Guangxi in document 1 and GX/P2V in document 2.
The xCov is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 19295; the preservation date is as follows: year 2020, month 2, day 14; address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; taxonomic nomenclature: a coronavirus.
Example 1 screening of "three drugs three parties" for drug monomers with anti-SARS-CoV-2 Activity
First, experiment method
1. Cell culture and virus culture
The African green monkey kidney cell line Vero E6 was obtained from American model culture Collection (ATCC, No. 1586) at 37 ℃ with 5% CO2In DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).
Squama Manis isolate xCoV (i.e., GX _ P2V/pangolin/2017/Guangxi) was propagated in Vero E6 cells and virus titers were determined using the plaque assay. All infection experiments were performed in a biosafety class 2 (BLS2) laboratory.
The COVID-19 resistant Chinese medicinal compound library (product number L6720, containing 389 Chinese medicinal monomers separated and purified from three-medicine three-part) is a product of Shanghai ceramic Biotechnology limited company. The initial concentration of all drugs was 10mM (millimoles per liter).
2. Screening potential anti-novel coronavirus drug from COVID-19 traditional Chinese medicine compound library by using pangolin coronavirus xCoV (GX _ P2V/pangolin/2017/Guangxi) with high SARS-CoV-2 homology
Seeded 2.5X 10 in 96-well cell plates4Vero cells were infected 24 hours later with xCoV (GX _ P2V/pangolin/2017/Guangxi) with MOI of 0.01, 389 kinds of Chinese medicinal monomers were added to each well at a final concentration of 50. mu.M, cytopathic effect was observed under a microscope on day 2, RNA was extracted from cells and supernatant from culture wells without significant cytopathic effect, and virus replication and GAPDH expression were measured by qRT-PCR. In the absence of significant cytotoxicity, inhibition of viral replication by more than 90% is considered a potential anti-neocoronavirus drug.
3. Potential antiviral drug EC50And CC50Measurement of
Seeded 2.5X 10 in 96-well cell plates4Vero cells were infected with xCoV (GX _ P2V/pangolin/2017/Guangxi) with MOI 0.01 after 24 hours, while Chinese medicinal monomers were added thereto at final concentrations of 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M and 0.390625. mu.M, respectively, and cytopathic effect was observed under a microscope on day 3 for no apparent fine linesRNA in cells and supernatant was extracted from the culture wells of cytopathic disease, and viral replication and expression of the intracellular reference gene GAPDH in the cells and supernatant were determined by qRT-PCR.
EC50The medicine concentration is effective in inhibiting 50% of cell infection virus, and the smaller the numerical value is, the better the virus inhibition effect is.
CC50Is the concentration of drug that causes 50% of the cells to become diseased, with higher numbers indicating lower toxicity to the cells.
And (3) SI: selectivity index of CC50And EC50The larger the value of (A) indicates the higher the possibility of drug formation.
4. Experiment of dosing time
Seeded 2.5X 10 in 24-well cell plates5Vero cells were infected 24 hours later with xCoV (GX _ P2V/pangolin/2017/Guangxi) with MOI 0.01, and potential effective drugs were added thereto at a concentration of 10. mu.M before the full infection cycle (at the time of virus addition and after 2 hours of virus addition incubation), before the cell entry (at the time of virus addition), after the cell entry (after 2 hours of virus addition incubation), and cytopathic effect was observed on day 3 under a microscope, and RNA was extracted from the cells and supernatant from the culture wells without significant cytopathic effect, and virus replication and expression of intracellular reference gene GAPDH were measured by qRT-PCR.
5. Viral RNA extraction and real-time quantitative RT-PCR (qRT-PCR)
AxyPrep was used according to manufacturer's instructionsTMHumoral virus DNA/RNA miniprep kit (Axygen, product number AP-MN-BF-VNA-250) and AxyPrepTMA multipurpose total RNA micro-preparation kit (Axygene, product number AP-MN-MS-RNA-250G) collects cell culture supernatant and Vero cells for RNA extraction. Reverse transcription was performed using a Hifair II 1 chain cDNA synthesis kit with gDNase (Shanghai assist san Biotech Co., Ltd., product No. 11121ES60), and qPCR was performed using a Hieff-qPCR-SYBR-Green-Master Mix (Shanghai assist san Biotech Co., Ltd., product No. 11202ES08) or a two-step Taqman probe detection qRT-PCR system (Applied-Biosystem), and sequence information of primers used is shown in Table 1. After sequencing confirmation, the sequence is determined by northThe PCR product was inserted into a T-vector by Biotech, Inc., of Kyoto Boxing, Kyoto, to generate a standard plasmid. Standard curve is determined by serial dilution of plasmid (10)3-109) And the Ct value obtained by qRT-PCR can be used for calculating the virus copy number by a standard curve.
The SYBR-Green method amplification program is as follows: 95 ℃ for 5min, 40 cycles, 95 ℃ for 10s, 55 ℃ for 20s, 72 ℃ for 31 s.
The Taqman method: 2min at 50 ℃, 10min at 95 ℃,40 cycles, 10s at 95 ℃ and 1min at 60 ℃. Data were analyzed using GraphPad-Prism 8.3.0 software.
Primer sequences used in the study of Table 1
| Primer name | Sequence (5 '-3') |
| xCoV-F | GGTGATTGCCTTGGTGATATTG |
| xCoV-R | GCAAGTAGTGCAGAAGTGTATTG |
| xCoV-P | TCTGTGAGCAAAGGCGGTAGAACC(5’-FAM，3’-TAMRA) |
| GAPDH-F | AGCCTCAAGATCATCAGCAATG |
| GAPDH-R | ATGGACTGTGGTCATGAGTCCTT |
| GAPDH-P | CCAACTGCTTAGCACCCCTGGCC(5’-FAM，3’-TAMRA) |
Second, experimental results
It was found by real-time quantitative PCR detection that 50. mu.M of andrographolide inhibited viral replication by 99.97% 48 hours after 0.01 of the multiplicity of infection of xCoV (i.e., GX _ P2V/pangolin/2017/Guangxi) infected cells (FIG. 1).
EC of andrographolide50、CC50And SI of 5.285 μ M,>50μM、>9.46 (fig. 2).
The dosing time experiments showed that andrographolide mainly acted at the post-viral entry stage (figure 3).
Third, discuss
In the present invention, the inventors have conducted screening of an anti-coronavirus active drug in a SARS-CoV-2-associated coronavirus, i.e., Pangolin coronavirus xCoV (i.e., GX _ P2V/pangolin/2017/Guangxi) model. Based on the results of preliminary laboratory studies, it was found that xCoV (i.e., GX _ P2V/pangolin/2017/Guangxi) infected mammalian cell Vero (Vero cell) can cause very obvious cytopathic effect. Based on the characteristic, the inventor uses xCoV (namely GX _ P2V/pangolin/2017/Guangxi) to infect Vero cells in a 96-well cell culture plate at the previous stage, and simultaneously adds drug monomers (389 traditional Chinese medicine monomers) separated from three drugs and three parties into each cell culture well to carry out potential active drug screening for inhibiting virus replication. Cytopathic effect is observed under a microscope on the 2 nd day, and the result shows that the andrographolide has obvious inhibition on virus infected cells and can be used as a potential antiviral drug. Further, the real-time fluorescent quantitative PCR technology detects that the inhibition rate of 50 mu M andrographolide on virus replication is 99.97% 48 hours after xCoV with the multiplicity of infection of 0.01 infects cells. Since xCoV is highly homologous to current SARS-CoV-2 and the receptor of xCoV-infected cells is identical to SARS-CoV-2, if a drug has inhibitory effect on xCoV-infected cells, it also has inhibitory effect on SARS-CoV-2 infection (references: Hua-Hao Fan, Li-Qin Wang, Wen-Li Liu, Xiao-Ping An, Zhen-Dong Liu, Xiao Qi He, Li-Hua Song, Yi-gan Tong.
Meanwhile, EC of andrographolide was determined to further verify its effect50And CC50. EC of andrographolide50、CC50And SI of 5.285 μ M,>50μM、>9.46. These results indicate that andrographolide has good antiviral activity and low toxic and side effects, and has potential clinical application value.
Finally, the effect stage of andrographolide is explored. Andrographolide is mainly responsible for the stage after the virus has entered the cell. This result further strongly demonstrates the potential antiviral activity of andrographolide.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The application of andrographolide or pharmaceutically acceptable salt thereof or a substance taking andrographolide or pharmaceutically acceptable salt thereof as an active ingredient in any one of the following substances:
(A1) preparing a product for inhibiting coronavirus, or inhibiting coronavirus;
(A2) preparing a product capable of treating a disease caused by a coronavirus infection, or treating a disease caused by a coronavirus infection;
(A3) preparing a product capable of ameliorating symptoms due to coronavirus infection, or ameliorating symptoms due to coronavirus infection.
2. Use according to claim 1, characterized in that: the inhibition of coronavirus is effected upon entry of the coronavirus into the host or host cell.
3. Use according to claim 1 or 2, characterized in that: the coronavirus is SARS-CoV-2 virus.
4. Use according to claim 1 or 2, characterized in that: the coronavirus is coronavirus pangolin isolate GX _ P2V/pangolin/2017/Guangxi or other similar coronavirus.
5. Use according to claim 3, characterized in that: the disease due to the coronavirus infection is COVID-19.
6. Use according to any one of claims 1 to 5, characterized in that: the product is a medicament.
7. A product contains andrographolide or its pharmaceutically acceptable salt as active ingredient; the product has any one of the following uses:
(a1) inhibiting coronavirus;
(a2) treating diseases due to coronavirus infection;
(a3) improving symptoms caused by coronavirus infection.
8. The product of claim 7, wherein: the inhibition of coronavirus is effected upon entry of the coronavirus into the host or host cell.
9. The product according to claim 7 or 8, characterized in that: the coronavirus is SARS-CoV-2 virus or coronavirus pangolin isolate GX _ P2V/pangolin/2017/Guangxi or other similar coronavirus.
10. The product according to any one of claims 7-9, wherein: the product is a medicament.
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| CN1454592A (en) * | 2003-06-06 | 2003-11-12 | 中国科学院上海药物研究所 | Medical use of andrographolide and its derivatives |
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| CN1454592A (en) * | 2003-06-06 | 2003-11-12 | 中国科学院上海药物研究所 | Medical use of andrographolide and its derivatives |
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| Title |
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| 蔡楠;李云鹃;周桂荣;辛辰;谢静;周洪浩;缪兴龙;周水平;何毅;王成;: "穿心莲内酯类制剂抗新型冠状病毒肺炎的相关理论依据和作用特点" * |
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