Antiviral Chinese medicine monomer dauricine, and its pharmaceutical composition and application

Abstract

The present invention discloses an antiviral Chinese medicine monomer, which is dauricine or its derivatives. The present invention also discloses an antiviral Chinese medicine composition, which includes active ingredients dauricine and / or its derivatives, and one or more pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers or diluents thereof. The present invention also discloses the use of the antiviral Chinese medicine monomer dauricine and its pharmaceutical composition as a viral infection inhibitor and / or in the preparation of drugs for preventing and treating viral infectious diseases. The present invention provides a potential antiviral drug for the prevention and treatment of various viral infectious diseases. The present invention is an expansion of the clinical application of dauricine.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and relates to an antiviral traditional Chinese medicine monomer dauricine, and a pharmaceutical composition and application thereof. Background Art

[0002] Viral infection refers to the process in which viruses invade the body through various pathways and proliferate in susceptible host cells. Viral infectious diseases are a global public health problem that poses a huge threat to human health. Their pathogens are diverse, including vesicular stomatitis virus, polio virus, HIV, influenza virus, etc. The infection and spread of these viruses have caused huge losses to human health and economic and social development. Although a variety of vaccines and drugs for the prevention and treatment of viral diseases are currently on the market, large-scale viral infections still break out. Therefore, the development of new and effective broad-spectrum antiviral drugs is still of great significance.

[0003] Batryticatine was originally extracted and isolated from the rhizome of the plant Batryticatine of the Menispermaceae family. It is a dibenzyltetrahydroisoquinoline alkaloid. The alkaloid components in the root of northern bean are its characteristic components and bioactive components, with a total content of about 1.7%-2.5%. Among the active ingredients, batryticatine has the highest content and has strong pharmacological activity. Previous studies have found that the pharmacological effects of batryticatine are very wide, and it is often used clinically to treat related diseases such as cardiac ischemia, angina pectoris, arrhythmias and inflammation. Recent reports also show that batryticatine has a protective effect on brain damage, can inhibit the infection of the new coronavirus, and can also act as an autophagy activator to stimulate autophagic cell death.

[0004] There are currently no reports that batryticatine inhibits viruses other than SARS-CoV-2. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide antiviral Chinese medicine monomers.

[0007] Another object of the present invention is to provide an antiviral pharmaceutical composition.

[0008] Another object of the present invention is to provide an antiviral Chinese medicine monomer or composition for use as a viral infection inhibitor and / or in the preparation of drugs for preventing and / or treating viral infectious diseases.

[0009] To this end, the technical solution provided by the present invention is:

[0010] An antiviral Chinese medicine monomer, which is Dauricine (DAC) or its derivatives.

[0011] An antiviral pharmaceutical composition comprising dauricine and / or its derivatives as active ingredients.

[0012] Preferably, the antiviral pharmaceutical composition further comprises one or more pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or diluent thereof.

[0013] Preferably, the antiviral pharmaceutical composition is in the form of a solid preparation (tablet, capsule, pill, granule), injection, inhalation preparation, spray, liquid preparation (oral solution), emulsion or compound preparation.

[0014] In the antiviral Chinese medicine monomer or the antiviral pharmaceutical composition, the dauricine derivative is a pharmaceutically acceptable salt of dauricine, a solvate of dauricine, or a hydrate of dauricine.

[0015] The antiviral Chinese medicine monomer or the antiviral medicine composition is used as a virus infection inhibitor and / or in the preparation of drugs for preventing and / or treating viral infectious diseases.

[0016] Preferably, in the application, the virus includes one or more of vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), and adenovirus (ADV).

[0017] Preferably, the use, inhibitor or drug is a mammalian drug.

[0018] Preferably, in the application, the mammal is bovine, equine, ovine, porcine, canine, feline, rodent or primate.

[0019] The present invention has at least the following beneficial effects:

[0020] The present invention proves that dauricine has the function of inhibiting the proliferation of multiple viruses at both the cellular level and the animal level.

[0021] The present invention provides a potential antiviral drug for the prevention and treatment of various viral infectious diseases. The present invention is an expansion of the clinical application of dauricine.

[0022] definition

[0023] To facilitate understanding of the present invention, the meanings of the terms and phrases used in the present invention are defined as follows:

[0024] Biopharmaceuticals: Biopharmaceuticals refer to products used for prevention, treatment and diagnosis that are manufactured from organisms, biological tissues, cells, organs, body fluids, etc., using the research results of microbiology, biology, medicine, biochemistry, etc., and comprehensively utilizing the scientific principles and methods of microbiology, chemistry, biochemistry, biotechnology, pharmacy, etc. The raw materials of biopharmaceuticals are mainly natural biological materials, including microorganisms, human bodies, animals, plants, marine organisms, etc.

[0025] Pharmacology: Pharmacology is a science that studies the interaction between drugs and the body and the mechanism of drug action. It mainly includes two aspects: pharmacodynamics and pharmacokinetics. The former explains the effects and action principles of drugs on the body, while the latter explains the processes of drug absorption, distribution, biotransformation and excretion in the body, as well as the laws of drug effects and blood drug concentrations over time.

[0026] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a proliferation-toxicity detection diagram of DAC in Example 1 of the present invention at the cellular level (24h).

[0028] Figure 2 The figure is a detection result of DAC inhibiting VSV virus replication at the cellular level in Example 2 of the present invention. Among them, a is the result of detecting the inhibitory effect of DAC on the virus by flow cytometry after co-incubation of VSV-eGFP virus and DAC, i.e., adding drugs throughout the virus infection; b is the result of qRT-PCR detection of the inhibitory effect of DAC on the virus after co-incubation of VSV virus and DAC; c is the result of detecting the inhibitory effect of DAC on the virus after co-incubation of VSV-eGFP virus and DAC by Western blotting.

[0029] Figure 3 This is a flow cytometry experimental result diagram showing that DAC in Example 3 of the present invention inhibits ADV virus replication at the cellular level.

[0030] Figure 4 This is a graph showing the experimental results of qRT-PCR detection of DAC in Example 4 of the present invention inhibiting EMCV virus replication at the cellular level.

[0031] Figure 5The experimental results of DAC in Example 5 of the present invention inhibiting H1N1 virus replication at the cellular level are shown in Figure a. a is the result of qRT-PCR detection of the inhibitory effect of DAC on the virus by co-incubation of H1N1 virus and DAC; b is the result of Western blotting detection of the inhibitory effect of DAC on the virus by co-incubation of H1N1 virus and DAC; c is the result of plaque assay of DAC inhibiting H1N1 virus replication at the cellular level.

[0032] Figure 6 This is a qRT-PCR test result diagram of the inhibition of H1N1 virus replication at the cellular level by various dosing methods of DAC in Example 6 of the present invention, which includes five dosing methods: dosing by DAC pretreatment, dosing during virus adsorption, dosing during virus entry, dosing after virus entry, and dosing throughout virus infection.

[0033] Figure 7 The figure is a test result diagram of DAC in Example 7 of the present invention inhibiting H1N1 virus infection at the animal level. Wherein, a is the weight change of mice after H1N1 infection; b is the lung CT image of mice after virus infection. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0035] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0036] The present invention provides an antiviral Chinese medicine monomer, which is dauricine (DAC) or its derivatives. Dauricine is a dibenzyltetrahydroisoquinoline alkaloid extracted and separated from the rhizome of the plant dauricum of the Menispermaceae family.

[0037] The structural formula of dauricine is shown below:

[0038]

[0039] The present invention also provides an antiviral pharmaceutical composition, which comprises dauricine or its derivatives as an active ingredient.

[0040] In the above scheme, preferably, the antiviral pharmaceutical composition further comprises one or more pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or diluent thereof.

[0041] In the above scheme, preferably, the dosage form of the composition is a solid preparation (such as tablets, capsules, pills or granules), injection, inhalation preparation, spray, liquid preparation (such as oral solution), emulsion or compound preparation.

[0042] The present invention also provides the antiviral Chinese medicine monomer or the antiviral pharmaceutical composition, wherein the derivative of dauricine is a pharmaceutically acceptable salt of dauricine or a solvate of dauricine or a hydrate of dauricine. The derivative of dauricine also includes but is not limited to stereoisomers, tautomers, homologues, prodrugs or polymorphs of dauricine.

[0043] The present invention also provides the use of the antiviral Chinese medicine monomer or the antiviral medicine composition as a virus infection inhibitor and / or in the preparation of drugs for preventing and / or treating viral infectious diseases.

[0044] In the above scheme, preferably, the virus includes one or more of vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), and adenovirus (ADV).

[0045] In the above embodiment, the inhibitor or drug is preferably a drug for mammals.

[0046] In the above scheme, preferably, the mammal is bovine, equine, ovine, porcine, canine, feline, rodent or primate (primates include primitive monkeys, capuchins, giant lemurs, aye-aye, loris, cynomolgus, mouse lemurs, advanced monkeys, apes, humans, white-cheeked gibbons, tarsiers, capuchins, green monkeys, saki-faced monkeys, spider monkeys, monkeys, gibbons, orangutans, and hominids.).

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for illustration:

[0048] Example 1

[0049] DAC proliferation-toxicity assay at the cellular level

[0050] Different concentrations (0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 μmol·L-1) of DAC were added to lung cancer human alveolar basal epithelial cells A549 cultured in 96-well plates, with 3 replicates for each concentration. After incubation for 24 h, CCK8 reagent was added, and the cells were incubated at 37°C for 30 min. The absorbance at 450 nm was detected by a microplate reader.

[0051] like Figure 1The results showed that the IC of DAC in A549 cells after 24 h of incubation 50 99.67 μmol·L -1 .

[0052] Example 2

[0053] Flow cytometry detection of DAC inhibiting VSV viral proliferation at the cellular level

[0054] Vesicular stomatitis virus (VSV) with GFP tag (MOI = 0.05) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after 12h of co-incubation, cells were collected for flow cytometry to detect the percentage of GFP-positive cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0055] qRT-PCR detection of DAC inhibiting VSV viral amplification at the cellular level

[0056] Vesicular stomatitis virus (VSV) with GFP tag (MOI = 0.05) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after incubation for 12 hours, cells were collected to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0057] Western blotting assay to detect the inhibition of VSV viral amplification by DAC

[0058] Vesicular stomatitis virus (VSV) with GFP tag (MOI = 0.05) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 2.5×10 5 / well), and after incubation for 12 h, the cells were harvested to extract proteins, and Western blotting was used to detect changes in viral proteins in the cells.

[0059] like Figure 2As shown, a is the percentage of GFP-positive cells detected by flow cytometry after VSV-eGFP virus and DAC were co-incubated, that is, the drug was added during the whole process of virus infection, and the bar graph is the statistics of flow cytometry results; b is the expression of VSV virus mRNA after qRT-PCR detection of VSV virus and DAC co-incubation; c is the expression of GFP protein detected by Western blotting after VSV-eGFP virus and DAC co-incubation. The results showed that the virus amplified significantly in the control group, while the experimental group adding batricine significantly inhibited VSV virus replication in a dose-dependent manner, among which 20μmol·L -1 The inhibition was most significant when DAC was used as the control group, indicating that DAC can effectively resist VSV virus infection.

[0060] Example 3

[0061] Flow cytometry detected that DAC inhibited ADV virus proliferation at the cellular level

[0062] Adenovirus ADV virus with GFP tag (MOI = 5) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after 12h of co-incubation, cells were collected for flow cytometry to detect the percentage of GFP-positive cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0063] like Figure 3 As shown, dauricine treatment of A549 cells can dose-dependently (5, 10, 20 μmol·L -1 ) reduced the percentage of GFP-positive cells. The bar graph counted the percentage of GFP-positive cells, ***, P<0.001 (extremely significant difference). The higher the GFP-positive ratio, the stronger the ADV virus replication. After adding DAC, the GFP-positive rate decreased, proving that DAC can inhibit ADV virus replication during full administration.

[0064] Example 4

[0065] qRT-PCR detection of DAC inhibiting EMCV virus proliferation at the cellular level

[0066] EMCV virus (MOI = 3) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), incubated for 12 hours, harvested cells to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0067] Figure 4 The results of qRT-PCR test of DAC inhibiting EMCV virus at the cellular level. The results showed that the virus amplified significantly in the control group, while the experimental group with dauricine significantly inhibited the gene expression of EMCV virus in a dose-dependent manner. -1 The inhibition was most significant when DAC was used as the control group, indicating that DAC can effectively resist EMCV virus infection.

[0068] Example 5

[0069] qRT-PCR detection of DAC inhibiting H1N1 virus proliferation at the cellular level

[0070] The H1N1 virus (MOI = 0.05) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after incubation for 12 hours, cells were collected to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0071] Western blotting assay to detect the inhibition of H1N1 virus proliferation by DAC

[0072] The H1N1 virus (MOI = 0.05) and different concentrations (5, 10, 20 μmol·L -1 ) was added to A549 cells (cell volume 2.5×10 5 / well), and after incubation for 12 h, the cells were harvested to extract proteins, and the changes of viral proteins in the cells were detected by Western blotting.

[0073] Plaque assay to detect the inhibition of H1N1 virus proliferation by DAC

[0074] DAC and H1N1 were added to A549 cells (cell volume 2.5×10 5 / well) for 12 hours, and the virus supernatant dilution was added to MDCK cells cultured in 24-well plates (cell volume 1.7×10 5 / well), place the cell culture plate in a cell culture incubator at 37°C and 5% CO2 for 2h, rinse twice with PBS to wash away the unadsorbed virus. Heat and melt 5% agarose, cool to below 40°C, mix evenly with DMEM culture medium at a volume ratio of 1:4, add TPCK-trypsin and mix until the concentration is 1μg / ml. Gently add the culture medium containing agarose to the 24-well plate, 500μl per well. After standing and solidifying at room temperature, invert it in a cell culture incubator and culture it. After culturing for 2-3 days, take it out, add 500μl of 4% paraformaldehyde to each well for 30min, discard the agarose, add 200μl / well of 0.1% crystal violet for staining, wash off the crystal violet after 15min, and observe the number of plaques.

[0075] like Figure 5 The results of a and b showed that dauricine and H1N1 virus co-incubated A549 cells in a dose-dependent manner (5, 10, 20 μmol·L -1 ) Reduce the expression of viral mRNA and protein. The higher the mRNA value and the darker the protein band color, the more the H1N1 virus proliferates. After adding DAC, the mRNA expression decreases and the band color becomes lighter. Figure 5 c The results showed that the supernatant of dauricine co-incubated with H1N1 virus in A549 cells treated MDCK cells in a dose-dependent manner (5, 10, 20 μmol·L -1 ) Reduce the number of plaques. The more plaques there are, the higher the titer of the H1N1 virus is. After adding DAC, the number of plaques decreases. The above results indicate that DAC can inhibit the amplification of the H1N1 virus.

[0076] Example 6

[0077] Detection of DAC's anti-H1N1 virus function by different administration methods

[0078] In order to determine the effects of different DAC administration methods on viral replication, the inventors used five administration methods, including administration during pretreatment, administration during virus adsorption, administration during virus entry, administration after virus entry, and administration throughout virus infection. After incubation for a corresponding period of time, cells were collected to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The specific dosing methods are as follows:

[0079] Drug pretreatment experiment: Different concentrations (10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after incubation for 12 hours, fresh medium was replaced and HIN1 virus (MOI = 0.1) was added for another 12 hours, cells were harvested to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0080] Drug administration during virus adsorption: H1N1 virus (MOI = 0.1) and different concentrations (10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), adsorbed at 4℃ for 2h, then replaced with fresh medium and continued incubation for 12h, cells were harvested to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0081] Drug administration during virus entry: A549 cells were infected with H1N1 virus (MOI = 0.1) and adsorbed at 4°C for 2 h. The medium was replaced with different concentrations of (10, 20 μmol·L -1 ) complete medium containing batryticatine, incubated at 37°C for 1.5h, then replaced with fresh medium and continued incubation for 12h, cells were harvested to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0082] Drug administration after virus entry: A549 cells were infected with H1N1 virus (MOI = 0.1), adsorbed at 4°C for 2 h, and then replaced with fresh medium. The medium was incubated at 37°C for 1.5 h, and different concentrations (10, 20 μmol·L -1 ) complete medium containing batryticatine was added, and the cells were incubated for 12 hours. The cells were harvested to extract RNA, and the changes in viral load in the cells were detected by qRT-PCR. The experiment was repeated 3 times independently. There was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0083] The whole drug treatment: H1N1 virus (MOI = 0.1) and different concentrations (10, 20 μmol·L -1 ) was added to A549 cells (cell volume 1.5×10 5 / well), after incubation for 12 hours, cells were collected to extract RNA, and qRT-PCR was used to detect changes in viral load in cells. The experiment was repeated 3 times independently, and there was a statistical difference between the experimental group and the control group. The results are expressed as mean ± standard error, ***, P < 0.001.

[0084] Figure 6 The results show that DAC inhibits H1N1 virus replication at the cellular level.

[0085] like Figure 6 The results showed that among the three administration methods, namely virus pretreatment, entry process and whole virus infection, DAC had the most significant effect in inhibiting H1N1 virus replication and was dose-dependent.

[0086] Example 7

[0087] Effects of DAC on body weight changes and lung infection severity in H1N1 infected mice

[0088] C57BL / 6J mice (healthy female mice aged 6-8 weeks, weighing about 20-22g) were randomly divided into 6 groups, namely CON group (blank control group), H1N1 group (model group), oseltamivir group (positive drug group), and DAC group (30mg / kg). The mice were infected with H1N1 virus through intranasal drops. The drug-treated group was gavaged with 200μl / mouse according to the corresponding dose, and the CON group and H1N1 group were given the same dose of normal saline. After that, gavage was continued every day until the end of the experiment. The diet, coat color, mental state, respiration, weight changes and death of the mice were observed and recorded daily. Under ethical circumstances, mice were considered dead when their weight dropped by more than 20% compared with before infection. 72 hours after the virus infection, the mice were anesthetized and fixed in CT, and anesthesia was continued with anesthesia masks. Micro-CT was used to image the lung tissue and analyze the progression of lung infection. Figure 7 The results showed that compared with the model group, oral administration of batricine could slow down the weight loss of infected mice and reduce the degree of lung infection in mice, indicating that batricine has a significant protective effect on mice infected with influenza A virus.

[0089] In the present invention, the inventors detected the inhibitory effect of dauricine on viral replication at the cell and animal levels respectively.

[0090] 1. Detection of the antiviral function of dauricine at the cellular level: The inventors selected A549 lung cancer human alveolar basal epithelial cells, a common cell line for antiviral immune response, to conduct pharmacological and efficacy studies. The experiment used H1N1 (influenza A virus), VSV (vesicular stomatitis virus), EMCV (encephalomyocarditis virus) and ADV (adenovirus) to explore the antiviral function of DAC, and further studied the effects of different DAC administration methods on H1N1 virus replication. The experimental results show that the addition of dauricine can significantly inhibit the proliferation of multiple viruses in cells.

[0091] 2. Animal level detection of the antiviral function of dauricine: mice were given corresponding doses of drugs or saline according to groups, and infected with H1N1 virus by nasal drops. The status, weight change and death of mice were observed and recorded daily. The inventors found that the weight change and survival rate of dauricine were significantly better than those of the virus group, indicating that dauricine can effectively resist viral infection at the body level.

[0092] In summary, the research of the present invention proves that dauricine has the function of inhibiting the proliferation of multiple viruses, confirms that dauricine has a strong antiviral effect at the cellular level and animal level, and can be used to prepare new broad-spectrum antiviral infectious disease drugs, thereby providing a new way and means for the prevention and treatment of multiple viral infectious diseases, which has important research and development value and development significance. The present invention provides a potential new antiviral drug for the prevention and treatment of viral infectious diseases.

[0093] The number of modules and processing scales described here are used to simplify the description of the present invention. Modifications and variations of the broad-spectrum antiviral drug, its pharmaceutical composition and application of the present invention are obvious to those skilled in the art.

[0094] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Use of dauricine or its salt in the preparation of a drug for preventing and / or treating viral infectious diseases, characterized in that: The virus is one or more of encephalomyocarditis virus (EMCV) and adenovirus (ADV).

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