CN115531384A - Application of dibenzyl isoquinoline alkaloid in preparation of anti-African swine fever virus medicine - Google Patents
Application of dibenzyl isoquinoline alkaloid in preparation of anti-African swine fever virus medicine Download PDFInfo
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- CN115531384A CN115531384A CN202211021536.2A CN202211021536A CN115531384A CN 115531384 A CN115531384 A CN 115531384A CN 202211021536 A CN202211021536 A CN 202211021536A CN 115531384 A CN115531384 A CN 115531384A
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- CN
- China
- Prior art keywords
- asfv
- alkaloid
- swine fever
- bisbenzylisoquinoline
- african swine
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Abstract
The invention belongs to the technical field of veterinary medicines, and particularly relates to application of dibenzylisoquinoline alkaloids in preparation of an anti-African Swine Fever Virus (ASFV) medicine. The bisbenzylisoquinoline alkaloid comprises the following components: liensinine, isoliensinine, neferine, dauricine, cepharanthine, tetrandrine, fangchinoline and berbamine can remarkably inhibit ASFV proliferation, and the effective inhibiting concentration for half of ASFV is 0.31-2.34 μ M. In addition, the dibenzyl isoquinoline alkaloid has small toxic effect on ASFV natural target cell porcine alveolar macrophages, can have remarkable antiviral activity on ASFV under the concentration without cytotoxicity, and has high safety; in addition, the bisbenzylisoquinoline alkaloid is derived from common traditional Chinese medicines, has wide source and low cost, and is very suitable for developing drugs for preventing and treating African swine fever.
Description
Technical Field
The invention belongs to the technical field of veterinary drugs. More particularly, relates to an application of dibenzylisoquinoline alkaloids in preparing an anti-African swine fever virus medicament.
Background
African Swine Fever (ASF) is a virus disease with extremely high infectivity caused by African Swine Fever Virus (ASFV), the disease incidence rate is high, diseased pigs often show acute, febrile and hemorrhagic clinical symptoms, organs of the diseased pigs show serious vascular diseases, such as lymph node hemorrhage, kidney hemorrhage, disseminated intravascular coagulation, thrombocytopenia and the like, and the death rate is close to 100%. ASF is a legal report disease of the world animal health Organization (OIE), is a type of animal epidemic disease in our country, and is listed as a priority for preventing and treating epidemic diseases in the plan for preventing and treating long-term animal epidemic disease in the country (2012-2020). African swine fever was first discovered in 1921 in african kenya; in 1957, ASF first erupted outside the african continent at the grapple; in 2007, ASFV was again introduced from the continental Africa into the Caucasian bordered region; in 2018, the first ASF epidemic outbreak occurs in China, and then the first ASF epidemic outbreak occurs in the surrounding countries. ASF has become the most serious infectious disease for the swine industry worldwide, causing serious economic losses.
Vaccines and antiviral drugs are effective means for preventing and controlling viral diseases. For example, chinese patent application CN110093324A discloses an attenuated ASFV with gene deletion and an application thereof as a vaccine, ASF Chinese epidemic strain Pig/CN/HLJ/2018 is adopted to delete virulence gene of the ASFV through a gene engineering technology, thereby obtaining a gene deletion virus with MGF360-505R deletion and CD2V and MGF360-505R combined deletion, and further taking the virus as a vaccine to protect ASF Chinese epidemic virulent strain by 100 percent of immunity; similarly, chinese patent application CN114107228a discloses an attenuated ASFV vaccine strain lacking twelve genes and a vaccine containing the vaccine strain, which is prepared by attenuating an epidemic strain in china, ASFV CN/GS 2018 strain to prepare a corresponding ASFV vaccine. However, because of various reasons such as safety, virulence reversion and the like, the ASF attenuated vaccine is not approved to be marketed in China at present, and along with continuous recombination and evolution of viruses, the attenuated vaccine may lose immune protection effect on new variant subtype ASFV. Except for vaccines, chinese patent application CN113797184A discloses an application of danye emodin in preparing drugs for preventing and treating ASFV, and experiments prove that the danye emodin can effectively inhibit the infection and proliferation of the ASFV in cells under the condition of lower than the maximum nontoxic dose. However, few research reports on the ASF prevention and treatment medicines exist at present, and no medicine with an ASFV (advanced respiratory syndrome Virus) resistance effect exists in clinic. Therefore, the research and development of anti-ASFV drugs is in urgent need.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of a bisbenzylisoquinoline alkaloid in preparing an anti-ASFV medicament aiming at the situation that the existing anti-ASFV medicament is lack of medicaments and no medicament is available.
Therefore, the invention aims to provide the application of the bisbenzylisoquinoline alkaloid in preparing the ASF prevention and treatment medicine.
The above purpose of the invention is realized by the following technical scheme:
bisbenzylisoquinoline alkaloids (Bisbenzylisoquinoline alkaloids) are a large group of naturally occurring alkaloids with wide distribution range in plant kingdom and changeable structural types and various physiological activities, and exist in Ranunculaceae, fangke, berberidaceae, magnoliaceae, annonaceae and other plants. The bisbenzylisoquinoline alkaloid is formed by connecting two benzylisoquinoline units through an oxygen bridge, and is divided into 26 structural types according to the difference of the ether bond number, the substitution condition and the connecting position of the alkaloid, and the common bisbenzylisoquinoline alkaloid comprises the following components: liensinine (Liensinine), isoliensinine (Isoliensinine), neferine (Neferine), dauricine (Dauricine); stephanine (Cepharanthine), tetrandrine (Tetrandrine), fangchinoline (Fangchinoline), berbamine (Berbamine), etc. The existing pharmacological research shows that the bisbenzylisoquinoline alkaloid has the effects of calcium ion antagonism, blood pressure reduction, cancer resistance, neuroprotection, sedation, osteoporosis resistance, allergy resistance, inflammation resistance, oxidation resistance and the like.
The inventor finds that the above bisbenzylisoquinoline alkaloid: liensinine, isoliensinine, neferine, dauricine, cepharanthine, tetrandrine, fangchinoline, berbamine can significantly reduce ASFV-p30 protein synthesis and ASFV-B646L gene duplication, and significantly inhibit ASFV proliferation in its target cell Porcine Alveolar Macrophage (PAMs). In addition, the bisbenzylisoquinoline alkaloid is derived from common traditional Chinese medicines, and has wide source and low cost. Meanwhile, the dibenzylisoquinoline alkaloids have small toxic effect on normal cells, good selection index, obvious antiviral activity on ASFV under the condition of no cytotoxicity concentration and high safety.
Therefore, the invention claims the application of the bisbenzylisoquinoline alkaloid in preparing the ASFV resistant medicament, wherein the bisbenzylisoquinoline alkaloid is liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline and berbamine.
Specifically, the bisbenzylisoquinoline alkaloid has any structure from the following formulas (I) to (VIII):
further, the bisbenzylisoquinoline alkaloids also include pharmaceutically acceptable salts, solvates, isomers or esters of liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline or berbamine.
In the present invention, the term "pharmaceutically acceptable salt" generally refers to any salt (which, in general, means that it is non-toxic) that is physiologically tolerable when used in an appropriate manner for therapy, in particular when applied or used in humans and/or mammals. These physiologically acceptable salts may be formed with cations or bases and in the context of the present invention, especially when administered in humans and/or mammals, they are to be understood as being salts formed from at least one compound provided according to the invention, usually an acid (deprotonated), such as an anion, and at least one physiologically tolerated cation, preferably an inorganic cation. These physiologically acceptable salts may also be formed with anions or acids, and in the context of the present invention, in particular when administered in humans and/or mammals, they are to be understood as being salts formed by at least one compound provided according to the invention, usually protonated (e.g. on nitrogen), such as a cation and at least one physiologically tolerable anion. In the context of the present invention, salts formed from physiologically tolerable acids, i.e. salts of the particular active compounds with physiologically tolerable organic or inorganic acids, may be included in particular, but not exclusively, with hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.
In the context of the present invention, the term "solvate" refers in general to any form of substance obtained by non-covalent bonding of an active compound according to the invention to another molecule, generally a polar solvent, and may in particular include, but is not limited to, hydrates and alcoholates, such as methanolate.
Further, the bisbenzylisoquinoline alkaloids inhibit the replication of ASFV.
Further, the effective half concentration (EC) of the bisbenzylisoquinoline alkaloid on ASFV 50 ) 0.31 to 2.34 mu M.
Furthermore, the Selection Index (SI) of the bisbenzylisoquinoline alkaloid is 15.58-65.90.
Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the excipient comprises an excipient, a disintegrant, a binder, a lubricant, a flavoring agent, a coloring agent, an emulsifier, or a diluent.
Further, the medicine is an injection preparation, an oral preparation, an aerosol inhalation preparation or a transdermal preparation.
In addition, the invention also claims application of the bisbenzylisoquinoline alkaloid in preparation of the medicine for preventing and treating ASF, wherein the bisbenzylisoquinoline alkaloid is liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline or berbamine.
Further, the bisbenzylisoquinoline alkaloids also include pharmaceutically acceptable salts, solvates, isomers or esters of liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline or berbamine.
The invention has the following beneficial effects:
the invention relates to a bisbenzylisoquinoline alkaloid: liensinine, isoliensinine, neferine, dauricine, cepharanthine, tetrandrine, fangchinoline, and berbamine can significantly reduce ASFV-p30 protein synthesis and ASFV-B646L gene replication, inhibit virus proliferation in PAMs, and achieve significant antiviral effect. In addition, the dibenzylisoquinoline alkaloids have small toxic effect on normal cells, have good selection index, have obvious antiviral activity on ASFV under the concentration without cytotoxicity and have high safety; in addition, the bisbenzylisoquinoline alkaloid is derived from common traditional Chinese medicines, has wide source and low cost, and is very suitable for developing the medicine for preventing and treating ASF.
Drawings
FIG. 1 is a data statistical chart showing the results of experiments of different concentrations of dibenzylisoquinoline alkaloids in PAMs on the inhibition of ASFV-p30 protein synthesis in example 2 by IFA analysis.
FIG. 2 is a data statistical chart of the results of the ASFV-B646L gene replication inhibition assay in PAMs using real-time fluorescent quantitative PCR assay for different concentrations of bisbenzylisoquinoline alkaloids in example 3.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: half-Cytotoxic Concentration (CC) of 8 bis-benzylisoquinoline alkaloids on PAMs 50 ) anti-ASFV activity half Effective Concentration (EC) 50 ) And Selection Index (SI)
1. half-Cytotoxic Concentration (CC) of dibenzylisoquinoline alkaloids on PAMs 50 ) Measurement of
Resuscitating Porcine Alveolar Macrophages (PAMs) (obtained by killing pigs, separating lung, and storing in liquid nitrogen tank in advance for use), resuspending cells with RPMI-1640 culture solution (containing 10% fetal calf serum, 100U/mL penicillin, 100U/mL streptomycin), and mixing 2 × 10 5 Each/mL of PAMs was inoculated in a 96-well plate at 100. Mu.L/well, 37 ℃, 5% CO 2 Culturing in a cell culture box. After 6h of incubation, the test compound was diluted by a double ratio with a 1640 maintenance solution containing 2% FBS, and 8 dibenzylisoquinoline alkaloid drug groups were set to 5 concentration gradients of 1.1 to 90. Mu.M, a solvent control group containing 3% DMSO and a blank control group, each at 100. Mu.L, at 37 ℃ and 5% CO 2 After 48 hours of incubation in the incubator, the supernatant was discarded and 0.5mg/mL MTT solution was added to 100. Mu.L per well, and incubated at 37 ℃ in the dark. After 4h, terminating incubation, removing supernatant, adding 150 μ L of DMSO into each well, and oscillating for 10min by a low-speed oscillator to fully dissolve formazan crystals; the OD value of the cells was measured at a wavelength of 570nm by a full-wavelength microplate reader, and the cell viability was calculated. Half-maximal Cytotoxic Concentration (CC) was calculated by nonlinear regression function using GraphPad Prism 9.0 software 50 )。
The calculation formula is as follows:
2. half Effective Concentration (EC) of dibenzylisoquinoline alkaloids on PAMs for resisting ASFV activity 50 ) Measurement of
The PAMs were resuscitated as above, using RPMI-1640 medium to resuspend the cells and add them at 5X 10 5 Seed/well in 48-well plates; setting cell control group and virus group at 100. Mu.L/well, 37 ℃ and 5% CO 2 Culturing in a cell culture box. After 6h of culture, washing with PBS for 2 times, infecting PAMs in 48-well plate with 10MOI ASFV, after 2h of infection, removing supernatant virus liquid, diluting with maintenance liquid (RPMI-1640 culture liquid containing 2% fetal calf serum) to prepare bisbenzylisoquinoline alkaloids with different concentrations, placing at 37 deg.C, and reacting with 5% CO 2 The incubator was incubated for another 48h and the culture was terminated. IFA detection was performed with 4% paraformaldehyde fixation and observed using a fluorescence inverted microscope and recorded by photography. Fluorescence intensity (blue and green) of each well was quantified using Image J software, DMSO-treated virus control group was set as 100%, other groups were compared to DMSO-treated group, and half Effective Concentration (EC) was determined by quantified drug-treated group cytoprotection rate 50 ) Values were calculated by nonlinear regression function using GraphPad Prism 9.0 software. The calculation formula is as follows:
3. selection Index (SI) of bisbenzylisoquinoline alkaloids against ASFV in cells
Based on half the Cytotoxic Concentration (CC) of dibenzylisoquinoline alkaloids to PAMs 50 ) And half Effective Concentration (EC) of dibenzylisoquinoline alkaloids on PAMs against ASFV activity 50 ) Calculating a Selection Index (SI) by the formula: SI = CC 50 /EC 50 . See table 1 for results.
TABLE 1 CC of Bisbenzylisoquinoline alkaloids 50 And EC 50 And SI
| Compound (I) | CC 50 (μM) | EC 50 (μM) | SI |
| Liensinine | 36.39 | 1.5 | 24.26 |
| Isoliensinine | 26.16 | 1.17 | 22.36 |
| Neferine | 36.46 | 2.34 | 15.58 |
| Dauricine | 18.28 | 0.79 | 23.14 |
| Cepharanthine | 33.04 | 1.03 | 32.07 |
| Tetrandrine | 62.83 | 1.38 | 45.52 |
| Fangchinoline | 34.6 | 1.4 | 24.71 |
| Berbamine | 20.43 | 0.31 | 65.9 |
As can be seen from the table, the 8 types of bisbenzylisoquinoline alkaloids have remarkable antiviral activity on ASFV under the concentration without cytotoxicity, and SI is between 15.58 and 65.90; the dibenzyl isoquinoline alkaloid has obvious antiviral activity on ASFV under the concentration without cytotoxicity.
Example 2: immunofluorescence method for evaluating inhibition effect of different concentrations of dibenzylisoquinoline alkaloids on ASFV-p30 protein synthesis in PAMs cells
Reference example 1 procedures for resuscitating PAMs, resuspending the cells in RPMI-1640 medium and culturing at 5X 10 5 One/well inoculated in 48-well plate; setting cell control group (Mock group, no test medicine and no ASFV), virus group (ASFV group, and no test medicine), and bisbenzylisoquinoline alkaloid group with different concentrations, wherein each group has 3 repeats; 37 ℃ and 5% of CO 2 Culturing for 6h in a cell culture box; infecting PAMs in 48-well plate with 10MOI ASFV, absorbing supernatant virus solution for 2h, adding 8 kinds of bisbenzylisoquinoline alkaloids prepared by diluting with maintenance solution (RPMI-1640 culture solution containing 2% fetal calf serum) at different concentrations, and reacting at 37 deg.C and 5% CO 2 The incubator was incubated for another 48h and the culture was terminated. Discarding the supernatant, adding 4% of the mixture into each wellFixing the paraformaldehyde for 30min at 400 mu L; washing with PBS for 3 times, adding 200 μ L of Triton X-100 permeable membrane with mass concentration of 0.25%, and treating at room temperature for 30min; adding BSA at a mass concentration of 5% to block the hybrid proteins, blocking for 1h at room temperature; washing with PBS 3 times, adding murine ASFV-p30 monoclonal antibody (1; washing with PBS 3 times, adding secondary antibody (Alexa) in dark
488-labeled anti-mouse IgG,1, 1000), incubating at 37 ℃ for 1h in the dark, and washing with PBS; and when the protein is observed under a fluorescence microscope, green fluorescence represents ASFV-p30 protein.
The test result is shown in figure 1, and it can be seen from the figure that the 8 types of bisbenzylisoquinoline alkaloids have obvious inhibition effect on ASFV-p30 protein synthesis under the concentration without cytotoxicity, and present obvious dose dependence relationship, which indicates that the bisbenzylisoquinoline alkaloids have obvious inhibition effect on ASFV protein synthesis under the concentration without cytotoxicity.
Example 3: fluorescent quantitative PCR method for evaluating ASFV-B646L gene replication inhibition effect of dibenzylisoquinoline alkaloids with different concentrations in PAMs
The method of reference example 1 recovers cultured PAMs, incubates ASFV, sucks supernatant, washes with PBS 2 times, adds dibenzylisoquinoline alkaloid diluted by maintenance solution with different concentration 2h after toxin attacking, sets Mock group (without adding test drug, without adding ASFV) and DMSO group (with ASFV, without adding test drug), sets three parallels for each test time point; and is left at 37 ℃ with 5% CO 2 The incubator was incubated for 48h and the culture was terminated. And (3) repeatedly freezing and thawing for three times, extracting DNA by using a DAN extraction kit (Nanjing NuoZan Biotechnology GmbH), carrying out fluorescent quantitative PCR (polymerase chain reaction) by using primers B646L-F and B646L-R to detect the CT value of the B646L gene, and evaluating the influence of different concentrations of bisbenzylisoquinoline alkaloids in PAMs on ASFV replication.
Wherein, the upstream and downstream primer sequences of ASFV UPL are as follows:
B646L-F:5’-CCCAGGRGATAAAATGACTG-3’
B646L-R:5’-CACTRGTTCCCTCCACCGATA-3’
the results of the assay are shown in figure 2, where P <0.05 compared to the virus-infected control group; * P <0.01; p <0.001, indicating significance of difference; ns indicates no significance of difference. As can be seen from the figure, compared with a DMSO virus control group, the 8 types of dibenzylisoquinoline alkaloids reduce the copy number of ASFV-B646L gene under the concentration without cytotoxicity, and show obvious dose dependence; the dibenzyl isoquinoline alkaloid is shown to have an inhibition effect on the replication of ASFV DAN under the concentration without cytotoxicity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of the dibenzyl isoquinoline alkaloid in the preparation of the medicine for resisting African swine fever virus is characterized in that the dibenzyl isoquinoline alkaloid is liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline or berbamine.
2. The application of the dibenzyl isoquinoline alkaloid in the preparation of the anti-African swine fever virus medicament is characterized in that the dibenzyl isoquinoline alkaloid has any structure from the following formula (I) to the formula (VIII):
3. the use of claim 1, wherein said bisbenzylisoquinoline alkaloid further comprises a pharmaceutically acceptable salt, solvate, isomer or ester of said liensinine, isoliensinine, neferine, dauricine, cepharanthine, tetrandrine, fangchinoline or berbamine.
4. The use according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the bisbenzylisoquinoline alkaloid with hydrochloric, hydrobromic, sulphuric, methanesulphonic, formic, acetic, oxalic, succinic, malic, tartaric, mandelic, fumaric, lactic or citric acid.
5. The use according to claim 1, wherein the bisbenzylisoquinoline alkaloid inhibits the replication of African swine fever virus.
6. The use according to any one of claims 1 to 5, wherein the dibenzylisoquinoline alkaloid is present at a half-effective concentration of 0.31 to 2.34 μ M against African swine fever virus.
7. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.
8. The use of claim 1, wherein the medicament is an injectable formulation, an oral formulation, an aerosol inhalation formulation, or a transdermal formulation.
9. The application of the bisbenzylisoquinoline alkaloid in preparing the medicine for preventing and treating African swine fever is characterized in that the bisbenzylisoquinoline alkaloid is liensinine, isoliensinine, neferine, dauricine, stephanine, tetrandrine, fangchinoline or berbamine.
10. The use of claim 9, wherein said bisbenzylisoquinoline alkaloid further comprises a pharmaceutically acceptable salt, solvate, isomer or ester of said liensinine, isoliensinine, neferine, dauricine, cepharanthine, tetrandrine, fangchinoline or berbamine.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115770245A (en) * | 2022-12-28 | 2023-03-10 | 华南农业大学 | Application of dibenzyl isoquinoline alkaloid in preparation of drug for preventing and treating African swine fever virus |
| CN115887459A (en) * | 2023-02-09 | 2023-04-04 | 南京农业大学 | Application of cepharanthine and pharmaceutical composition thereof in preparation of anti-African swine fever drugs |
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