CN115778952B - Application of papaya alkaloids in the preparation of anti-coronavirus drugs - Google Patents

Abstract

The invention provides the application of papaya alkaloids in preparing anti-coronavirus drugs, and belongs to the field of medical technology. Experiments of the present invention show that papaya alkaloids (papain, pseudopapain, dehydropapain I, dehydropapain II) can effectively inhibit the binding of the new coronavirus S protein and ACE2, and inhibit the binding of the new coronavirus S protein and ACE2. The pseudovirus of protein infects cells and has low cytotoxicity. Therefore, this application provides the application of papaya alkaloids in the preparation of antiviral drugs, providing new ideas for preventing and treating the new coronavirus.

Description

Application of papaya alkaloids in the preparation of anti-coronavirus drugs

Technical field

The invention belongs to the field of medical technology, and specifically relates to the application of papaya alkaloids in the preparation of anti-coronavirus drugs.

Background technique

Coronaviruses are enveloped viruses with positive single-stranded RNA genomes. Coronaviruses are divided into four genera: α-CoVs, β-CoVs, γ-CoVs and δ-CoVs ^[1] . Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a respiratory disease known as coronavirus disease 2019 (COVID-19), and its spread has resulted in a pandemic. The most common clinical features include cough, Fever and infectious pneumonia, acute respiratory distress syndrome, and even death in severe cases ^[2] . There are currently no specific anti-COVID-19 drugs. Research on drug targets for the novel coronavirus and small drug molecules that specifically block the invasion of the novel coronavirus have yet to be developed.

At present, there are many new coronavirus drugs on the market, such as the viral RNA polymerase inhibitor remdesivir, monegravir, etc., which can inhibit the replication of viral RNA as nucleoside analogs ^[3] ; mifenovir inhibits the Lattice protein-mediated endocytosis inhibits the membrane fusion of the viral envelope and the host cell plasma membrane, preventing the virus from entering the host cell; nematvir and ritonavir inhibit the 3CL protease, the main protease of the new coronavirus, on the translation of viral RNA. Processing of polyproteins ^[4] . It has been confirmed that the antimalarial drugs chloroquine and hydroxychloroquine can inhibit the terminal phosphorylation of ACE2 and increase the pH value in endosomes to inhibit membrane fusion, thus preventing viral infection. Clinical trials are currently underway ^[5] .

The spike glycoprotein of SARS-CoV-2 plays an important role in viral infectivity. SARS-CoV-2 uses the receptor binding domain (RBD) of the spike protein (S protein) to bind to the receptor angiotensin-converting enzyme 2 (ACE2) on the host cell to infect the host cell. The spike glycoprotein may prevent SARS-CoV-2 is a good target for entering host cells ^[6] . Drug screening is carried out with S protein and ACE2 as targets. It inhibits new coronavirus infection by blocking the combination of S protein and ACE2 ^[7] , and cell experiments are used to detect inhibition. The virus invasion effect is an important way to study new coronavirus invasion inhibitors.

Natural products are an important source of antiviral drugs. There are currently reports in the literature that stephanatine, as a naturally occurring plant alkaloid, can target the invasion stage of the new coronavirus and significantly inhibit the combination of the virus and host cells ^[8] . It has low toxicity in animals and has no obvious side effects on the human body. .

It is reported that papaya leaves contain four kinds of alkaloids, namely papayaine, pseudopapainine, dehydropapainine I, and dehydropapainine II, among which the content of papayaine is relatively high ^[9] . At present, studies have shown that papaya has anti-cancer activity, anti-thrombocytopenia and anthelmintic activity. Some studies have confirmed that papaya also has anti-malarial properties ^[10] . However, there are no studies reporting that papaya can inhibit COVID-19.

references

[1] Seyed Hosseini, E.; Riahi Kashani, N.; Nikzad, H.; Azadbakht, J.; Hassani Bafrani, H.; Haddad Kashani, H. The novel coronavirus Disease-2019 (COVID-19): Mechanism of action , detection and recent therapeutic strategies. Virology2020,551,1-9.DOI:10.1016/j.virol.2020.08.011.

[2]Samudrala, P.K.; Kumar, P.; Choudhary, K.; Thakur, N.; Wadekar, G.S.; Dayaramani, R.; Agrawal, M.; Alexander, A. Virology, pathogenesis, diagnosis and in-line treatment of COVID-19.Eur J Pharmacol 2020,883,173375.DOI:10.1016/j.ejphar.2020.173375From NLM Medline.

[3]Ohashi, H.; Watashi, K.; Saso, W.; Shionoya, K.; Iwanami, S.; Hirokawa, T.; Shirai, T.; Kanaya, S.; Ito, Y.; Kim, K.S.; et al. Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment. iScience 2021, 24(4), 102367. DOI: 10.1016/j.isci.2021.102367From NLMPubMed-not-MEDLINE.

[4]McKee, D.L.; Sternberg, A.; Stange, U.; Laufer, S.; Naujokat, C. Candidated drugs against SARS-CoV-2and COVID-19.Pharmacological Research 2020,157.DOI:10.1016/j.phrs .2020.104859.

[5]Wang, N.; Han, S.; Liu, R.; Meng, L.; He, H.; Zhang, Y.; Wang, C.; Lv, Y.; Wang, J.; Li, X. et al. Chloroquine and hydroxychloroquine as ACE2blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus. Phytomedicine 2020, 79, 153333. DOI: 10.1016/j.phymed.2020.153333From NLM Medline.

[6]Ou, X.; Liu, Y.; Lei, X.; Li, P.; Mi, D.; Ren, L.; Guo, L.; Guo, R.; Chen, T.; Hu, J.; et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 2020,11(1),1620.DOI:10.1038/s41467-020-15562-9From NLM Medline.

[7] Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the receptor-binding domain( RBD) of 2019novelcoronavirus:implication for development of RBD protein as a viral attachmentinhibitor and vaccine. Cell Mol Immunol 2020,17(6),613-620.DOI:10.1038/s41423-020-0400-4From NLM Medline.

[8] Fan, H.H.; Wang, L.Q.; Liu, W.L.; An, X.P.; Liu, Z.D.; He, X.Q.; Song, L.H.; Tong, Y.G. related coronavirusmodel.Chin Med J(Engl)2020,133(9),1051-1056.DOI:10.1097/CM9.0000000000000797From NLM Medline.

[9]Munir, S.; Liu, Z.W.; Tariq, T.; Rabail, R.; Kowalczewski, P.L.; Lewandowicz, J.; Blecharczyk, A.; Abid, M.; Inam-Ur-Raheem, M.; Aadil, R.M. Delving into the Therapeutic Potential of Carica papaya Leaf against Thrombocytopenia. Molecules 2022, 27(9). DOI: 10.3390/molecules27092760From NLMMedline.

[10] Zunjar, V.; Dash, R.P.; Jivrajani, M.; Trivedi, B.; Nivsarkar, M. Antithrombocytopenic activity of carpaine and alkaloidal extract of Caricapapaya Linn. leaves in busulfan induced thrombocytopenic Wistar rats. JEthnopharmacol 2016,181, 20-25.DOI:10.1016/j.jep.2016.01.035From NLM Medline.

Contents of the invention

In view of this, the object of the present invention is to provide the application of papaya alkaloids in the preparation of anti-coronavirus drugs.

The invention provides the application of papaya alkaloids in the preparation of anti-coronavirus drugs.

Preferably, the papaya alkaloids include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain with a structural formula shown in Formula II, and dehydrophenine alkaloids with a structural formula shown in Formula III. Papaine I, dehydropapain II with a structural formula shown as Formula IV;

Preferably, the papaya alkaloid includes a hydrate form of papaya alkaloid or a salt form of papaya alkaloid.

Preferably, the salt form of the papaya alkaloids includes one or more of the following: acetate of papaya alkaloids, formate of papaya alkaloids, hydrochloride of papaya alkaloids, papaya alkaloids Phosphates of alkalis and sulfates of papaya alkaloids.

The invention provides the use of an inhibitor that blocks the combination of coronavirus S protein and ACE2 in the preparation of anti-coronavirus drugs. The inhibitor is papaya alkaloid.

Preferably, the papaya alkaloids include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain with a structural formula shown in Formula II, and dehydrophenine alkaloids with a structural formula shown in Formula III. Papaine I, dehydropapain II with a structural formula shown as formula IV;

Preferably, the coronavirus includes the new coronavirus SARS-COV-2.

Preferably, the anti-coronavirus includes preventing coronavirus infection.

The invention provides an antiviral drug, which uses papaya alkaloids as active ingredients and medically acceptable auxiliary materials.

Preferably, the papaya alkaloids include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain with a structural formula shown in Formula II, and dehydrophenine alkaloids with a structural formula shown in Formula III. Papaine I, dehydropapain II with a structural formula shown as formula IV;

The invention provides the application of papaya alkaloids in the preparation of anti-coronavirus drugs. The present invention constructs a new coronavirus pseudovirus screening model to obtain a new coronavirus pseudovirus that uses S protein to wrap the reporter gene Luciferase or GFP, and at the same time constructs an A549 cell strain that stably expresses the ACE2 protein as a host cell. The new coronavirus pseudovirus is used to infect the 549-ACE2 cell line to simulate the virus infection process. The present invention can effectively block S protein and The binding of ACE2 blocks the infection of host cells by pseudovirus. Experimental results show that the above-mentioned papayaine, pseudopapain, dehydropapain I or dehydropapain II have the ability to inhibit the infection of cells by the new coronavirus pseudovirus, with IC ₅₀ of 5.48 μmol/L and 8.21 μmol respectively. /L, 24.3μmol/L, 30.65μmol/L. At the same time, cytotoxicity experiments show that papayaine has good cell administration safety when acting on A549-ACE2 cells.

Description of the drawings

Figure 1 is a statistical graph showing the infection rate of the compound papayaline in Example 1 at different concentrations against novel coronavirus-infected A549-ACE2 cells;

Figure 2 is a statistical diagram of the inhibitory rate of the compound papayaline in Example 1 against novel coronavirus infection of A549-ACE2 cells at different concentrations;

Figure 3 shows the results of the toxic effects of different concentrations of veratrine on A549-ACE2 cells in Example 2.

Detailed ways

The invention provides the application of papaya alkaloids in the preparation of anti-coronavirus drugs.

In the present invention, the papaya alkaloids preferably include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain alkaloids with a structural formula shown in Formula II, and papaya alkaloids with a structural formula shown in Formula III. Dehydropapain I and dehydropapain II having a structural formula as shown in formula IV;

In the embodiment of the present invention, the papayaine, pseudopapainine, dehydropapainine I or dehydropapainine II were purchased from Shaanxi Chenguang Biotechnology Co., Ltd.

In the present invention, the papaya alkaloid preferably further includes a hydrate form of papaya alkaloid or a salt form of papaya alkaloid. The salt form of the papaya alkaloid preferably includes one or more of the following: acetate of papaya alkaloid, formate of papaya alkaloid, hydrochloride of papaya alkaloid, and Phosphates and sulfates of papaya alkaloids. The present invention has no special restrictions on the preparation method of the salt form of the papaya alkaloid, and it is sufficient to adopt the preparation method of the alkaloid salt form well known in the art. The present invention has no special restrictions on the preparation method of the hydrate of the papaya alkaloid, and it is sufficient to adopt the preparation method of the hydrate of the compound well known in the art.

In the present invention, the anti-coronavirus preferably includes preventing coronavirus infection. The coronavirus preferably includes the novel coronavirus SARS-COV-2. In the embodiments of the present invention, at the cellular level, the IC ₅₀ of papayaine, pseudopapainine, dehydropapainine I or dehydropapainine II against the new coronavirus pseudovirus is 5.48 μmol/L, respectively. 8.21μmol/L, 24.3μmol/L, 30.65μmol/L. Cytotoxicity experiments showed that the IC ₅₀ value of papaya on A549-ACE2 cells was 35.08 μmol/L.

In view of the fact that veratrine achieves antiviral effects by blocking the combination of coronavirus S protein and ACE2, the present invention provides an application of an inhibitor that blocks the combination of coronavirus S protein and ACE2 in the preparation of anti-coronavirus drugs. The inhibitor is papaya alkaloid.

In the present invention, the papaya alkaloids preferably include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain alkaloids with a structural formula shown in Formula II, and papaya alkaloids with a structural formula shown in Formula III. Dehydropapain I and dehydropapain II having a structural formula as shown in formula IV;

In the present invention, the coronavirus preferably includes the new coronavirus SARS-COV-2. Said anti-coronavirus preferably includes preventing coronavirus infection.

The invention provides an antiviral drug, which uses papaya alkaloids as active ingredients and medically acceptable auxiliary materials.

In the present invention, the papaya alkaloids preferably include one or more of the following: papaya alkaloids with a structural formula shown in Formula I, pseudopapain alkaloids with a structural formula shown in Formula II, and papaya alkaloids with a structural formula shown in Formula III. Dehydropapain I and dehydropapain II having a structural formula as shown in formula IV;

In the present invention, the dosage form of the drug includes one or more of the following: injections, tablets, pills, capsules, suspensions and emulsions. The medicine also includes medically acceptable excipients. The auxiliary materials include one or more of the following: diluents, disintegrants, fillers, binders, absorption accelerators, surfactants, adsorption carriers, lubricants and synergists. The drugs are administered orally, transdermally, intravenously or intramuscularly.

The application of the papaya alkaloids provided by the present invention in the preparation of anti-coronavirus drugs will be described in detail below with reference to the examples, but they should not be understood as limiting the scope of the present invention.

Establish a new coronavirus pseudovirus screening model, and use the lentiviral packaging system (pCMVΔ8.2+pCMV3-SARS-Cov-2-S+pBOB-Luciferase/GFP) in 293T cells to obtain the new coronavirus pseudovirus that uses S protein to wrap the reporter gene Luciferase or GFP. Virus. The coding sequence of the S protein is shown in SEQ ID NO: 1 (); the specific construction method can be found in the reference literature (Hu J, Gao Q, He C, Huang A, Tang N, Wang K. Development of cell-based pseudovirus entry assay to identify potential viral entryinhibitors and neutralizing antibodies against SARS-CoV-2.Genes Dis.2020Dec;7(4):551-557.doi:10.1016/j.gendis.2020.07.006.Epub 2020Jul 17.PMID:32837985;PMCID: PMC7366953.Ma H,Zhu Z,Lin H,Wang S,Zhang P,Li Y,Li L,Wang J,Zhao Y,HanJ.Pyroptosis of syncytia formed by fusion of SARS-CoV-2spike and ACE2-expressing cells.Cell Discov.2021Aug 24;7(1):73.doi:10.1038/s41421-021-00310-0.PMID:34429403; PMCID:PMC8384103.).

In 293T cells, a lentiviral packaging system (VSVG+REV+pMDLg/pRRE+pBOB-ACE2) was used to obtain the VSVG-wrapped virus fluid containing the ACE2 gene (SEQ ID NO: 2,). After the virus fluid infected A549 cells, Through cell monoclonal screening and identification, an A549 cell line (549-ACE2 cell) that stably expresses ACE2 protein was established. The specific construction method is as follows: Plate 293T cells into a six-well plate on the first day to ensure that the cells are encapsulated with the virus on the second day. The density is about 90%. The encapsulated virus system is as follows. The amount of DNA per well is: pCMVΔ8.2μg, pCMV3-SARS-Cov-2-S 0.5μg, pBOB-Luciferase/GFP 2μg, mixed with 50μl DMEM culture medium without serum and antibiotics. , mix 3 times the amount of PEI with 50 μl of DMEM culture medium without serum and antibiotics, mix the DNA tube and PEI tube evenly, let it stand for 25 minutes, add 293T cells replaced with DMEM with 5% serum, and put it in the incubator After 8 hours of culture, the medium was changed to 30% serum-containing antibiotic medium to continue culturing. After 36 hours, the virus liquid was filtered with a 0.45um filter and stored in a -80°C refrigerator.

Example 1

The inhibitory effect of the compounds papayaine, pseudopapain, dehydropapain I or dehydropapain II on A549-ACE2 cells infected by the new coronavirus at different concentrations.

1Experimental method

After the A549-ACE2 cells in the logarithmic growth phase are digested from a 10 cm diameter culture dish, dilute it to a cell suspension of 10 ⁵ cells/mL with DMEM complete medium containing 10% fetal calf serum according to the cell density, and mix the cells After evenly seeding into a 96-well plate, add 100 μL of cell suspension to each well, add the same volume of PBS buffer to the surrounding wells, gently tap around the culture plate to make the cells evenly distributed, wait until the cells settle to the bottom of the culture plate Place in a 37°C, 5% _CO2 cell culture incubator.

A lentiviral packaging system was used in 293T cells to obtain a safer new coronavirus pseudovirus liquid composed of S protein packaging reporter gene Luciferase.

A549-ACE2 cells were inoculated into a 96-well plate, and the drug was added 24 hours later. Virus infection was carried out at the same time. The virus liquid and culture medium were mixed at a ratio of 2:3 to obtain a pseudovirus solution. Add papayaine and pseudopapapain . Dehydropapain I or dehydropapain II was diluted with a mixture of culture medium and virus liquid to a concentration of 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 100 μL per well, and a blank group was set up ( culture medium), negative control group (DMSO), and positive control group (Stephanatine Ceph and Chloroquine CQ). After infection for 72 hours, a fluorescent microplate reader was used to detect the fluorescence emission value at 492 nm.

The fluorescence intensity values of each group were counted, using GraphPad Prism8 software, with the inhibitor concentration as the abscissa, and the corresponding infection rate as the ordinate. The results are shown in Figure 1; using the logarithm of the inhibitor concentration as the abscissa, the corresponding infection rate is the ordinate, and the results are shown in Figure 2. The inhibitory abilities of each compound against the new coronavirus pseudovirus are shown in Table 1.

Table 1. Papain and its analogues inhibit the activity of new coronavirus

serial number compound Inhibits the activity of new coronavirus (IC ₅₀ ) 1 papayaline 5.48μmol/L 2 pseudopapain 8.21μmol/L 3 Dehydropapain I 24.3μmol/L 4 Dehydropapain II 30.65μmol/L

Example 2

Toxic effect of papaya on A549-ACE2 cells

1Experimental method

1.1 After the A549-ACE2 cells in the logarithmic growth phase are digested from a 10cm diameter culture dish, mix the cell suspension diluted to 10 ⁵ cells/mL and inoculate it into a 96-well plate. Each well has a volume of 100 μL. Culture in a 37 °C, 5% _CO2 cell incubator.

1.2 Cell drug delivery

Dissolve papayaline with DMSO, the concentration of the mother solution is 10mM, and then further dilute it with DMEM culture medium to a concentration of 1μM, 2μM, 5μM, 10μM, 20μM, 50μM, 100μM. Add 100μL to each well of a 96-well plate, and set each dose group Three parallel wells were set up with a blank group and a control group at the same time. They were placed in an incubator for culture. Cytotoxicity testing was performed 48 hours after adding the drug.

1.3MTT detection of cytotoxicity of papayaline

1. After the drugs are treated separately for 48 hours, discard the culture medium, add 10 μL MTT solution to each well, incubate in the incubator for 4 hours, then discard the culture medium in the well, add 100 μL DMSO to each well, and shake on a shaker for 10 minutes to fully dissolve the formazan crystals. , measure the OD value at 490nm with a microplate reader.

2. The microplate reader reads the absorbance value at 490nm wavelength (OD ₄₉₀ ). Calculate the cell survival ratio of the experimental group (i.e., the drug-added group) relative to the control group.

Use GraphPad Prism8 to calculate the cell survival rate and IC ₅₀ value. Cell survival rate = (OD of the experimental group - OD of the blank group) / (OD of the control group - OD of the blank group) × 100%.

The results are shown in Figure 3. The IC ₅₀ value of papaya on A549-ACE2 cells was 35.08 μmol/L.

In summary, veratrine can effectively inhibit the binding of the new coronavirus S protein and ACE2, inhibit the infection of cells by pseudoviruses containing the new coronavirus S protein, and has low cytotoxicity, which shows that veratrine can be used for prevention and/or treatment COVID-19 infection, and then used to prepare drugs for preventing and/or treating COVID-19 infection. At the same time, the compounds papayaine, pseudopapainine, dehydropapainine I or dehydropapainine II have the ability to inhibit the binding of the new coronavirus S protein and ACE2 to varying degrees, so they can be combined with veratrine as an active ingredient to exert anti- The role of the new coronavirus and the preparation of anti-coronavirus drugs.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (4)

1. The application of papaya alkaloids in the preparation of anti-coronavirus drugs. The papaya alkaloids are one or more of the following: papaya alkaloids with a structure as shown in formula I, and pseudopaenine with a structural formula as shown in formula II. Papaine, dehydropapain I with a structural formula shown in formula III and dehydropapain II with a structural formula shown in formula IV; the coronavirus is the new coronavirus SARS-COV-2; Formula I /> Formula II Formula III/> Formula IV. 2. Application according to claim 1, characterized in that the papaya alkaloid is a hydrate form of papaya alkaloid or a salt form of papaya alkaloid. 3. The application according to claim 2, characterized in that the salt form of the papaya alkaloids includes one or more of the following: acetate of papaya alkaloids, formate of papaya alkaloids, saponinate of papaya alkaloids. Papaya alkaloid hydrochloride, papaya alkaloid phosphate and papaya alkaloid sulfate. 4. The application according to claim 1, characterized in that the anti-coronavirus includes preventing coronavirus infection.