Total alkaloids from the antiviral Chinese herbal extract *Sophora tonkinensis* and their applications

Abstract

This invention discloses an antiviral traditional Chinese medicine extract, namely total alkaloids from Sophora flavescens root, which can be used to prepare antiviral drugs. This invention also discloses the application of the antiviral traditional Chinese medicine extract, total alkaloids from Sophora flavescens root, as a viral infection inhibitor and / or in the preparation of drugs for the prevention and / or treatment of viral infectious diseases. It can significantly inhibit the amplification of various viruses, including VSV (vesicular stomatitis virus), EMCV (encephalomyocarditis virus), HSV-1 (herpes simplex virus type I), and H1N1 (influenza A virus), at both the cellular and organismal levels.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology. More specifically, the present invention relates to an antiviral traditional Chinese medicine extract, total alkaloids of Menispermi Rhizoma, and its application. Background Art

[0002] Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused heavy losses to the health of all mankind and the development of the world economy. In the early stage when there were no specific drugs or vaccines, traditional Chinese medicine played a crucial role in the prevention and treatment of this disease.

[0003] Therefore, exploring more effective antiviral drugs from the vast treasure house of traditional Chinese medicine, especially antiviral drugs that can treat diseases caused by different virus infections, has great scientific significance and application prospects for combating novel and mutant virus infections that may break out in the future, and has practical feasibility.

[0004] Menispermi Rhizoma is the dried rhizome of Menispermum dauricum DC., which is recorded in the Chinese Pharmacopoeia (2015 edition) as having a faint smell, a bitter taste, being cold in nature, and belonging to the lung, stomach, and large intestine meridians, with the effects of clearing heat and detoxifying, dispelling wind and relieving pain, and can be used to treat sore throat, enteritis dysentery, rheumatic arthralgia and other symptoms. The alkaloid components in Menispermi Rhizoma are its characteristic components and bioactive components, with a total content of about 1.7% - 2.5%. In recent years, studies have found that the pharmacological effects of total alkaloids of Menispermi Rhizoma are very extensive, not only having antibacterial, anti-inflammatory and other pharmacological activities based on traditional effects, but also having bioactivities such as protecting the cardiovascular and cerebrovascular systems, anti-tumor, anti-depression, and anti-Alzheimer's disease. However, whether it has antiviral effects remains unknown. Deeply clarifying the antiviral effects and broad-spectrum nature of total alkaloids of Menispermi Rhizoma is the key to expanding the medicinal value of total alkaloids of Menispermi Rhizoma and also the difficulty in developing antiviral drugs. Summary of the Invention

[0005] One object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0006] To achieve these objects and other advantages in accordance with the present invention, there is provided an antiviral traditional Chinese medicine extract, which is total alkaloids of Menispermi Rhizoma (MRTA).

[0007] An antiviral pharmaceutical composition is provided, comprising total alkaloids of Sophora flavescens, one or more salts of total alkaloids of Sophora flavescens, and / or a carrier or diluent of total alkaloids of Sophora flavescens, and / or a solvate of total alkaloids of Sophora flavescens.

[0008] This invention provides the use of the aforementioned antiviral traditional Chinese medicine extract as a viral infection inhibitor and / or in the preparation of drugs for the prevention and / or treatment of viral infectious diseases.

[0009] The antiviral pharmaceutical composition is provided for use as a viral infection inhibitor and / or in the preparation of a medicament for the prevention and / or treatment of viral infectious diseases.

[0010] Preferably, the virus is one or more of vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), and herpes simplex virus type I (HSV-1).

[0011] This invention provides the use of the aforementioned antiviral traditional Chinese medicine extract in the preparation of products for the prevention and / or treatment of vesicular stomatitis virus, or in the preparation of products as vesicular stomatitis virus inhibitors, or in the preparation of products for inhibiting the replication or reproduction of vesicular stomatitis virus in cells, or in the preparation of products for the prevention and / or treatment of influenza A virus, or in the preparation of products as influenza A virus inhibitors, or in the preparation of products for inhibiting the replication or reproduction of influenza A virus in cells, or in the preparation of products for the prevention and / or treatment of encephalomyocarditis virus, or in the preparation of products as encephalomyocarditis virus inhibitors, or in the preparation of products for inhibiting the replication or reproduction of encephalomyocarditis virus in cells, or in the preparation of products for the prevention and / or treatment of herpes simplex virus type I, or in the preparation of products as herpes simplex virus type I inhibitors, or in the preparation of products for inhibiting the replication or reproduction of herpes simplex virus type I in cells.

[0012] Preferably, the antiviral drug is a human or animal medicine.

[0013] Preferably, the antiviral drug is in the form of an oral dosage form, a solid dosage form, a spray, an inhalation dosage form, an injection dosage form, a topical dosage form, or a compound dosage form.

[0014] The present invention has at least the following beneficial effects:

[0015] First, this invention uses the A549 cell line, a commonly used cell line for antiviral immune response in lung cancer human alveolar basal epithelial cells, to conduct pharmacological and efficacy studies. The addition of total alkaloids from Sophora flavescens can significantly inhibit the amplification of various viruses in cells, including VSV (vesicular stomatitis virus), EMCV (encephalomyocarditis virus), HSV-1 (herpes simplex virus type I), and H1N1 (influenza A virus).

[0016] Secondly, this invention uses animal experiments on mice to detect the antiviral function of total alkaloids from Sophora flavescens at the animal level, indicating that total alkaloids from Sophora flavescens can effectively resist viral infection at the body level.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is a graph showing the proliferation-toxicity detection of MRTA in one embodiment of the present invention after 24 hours of cell treatment;

[0019] Figure 2 This is a graph showing the proliferation-toxicity detection of MRTA after 48 hours of cell treatment in one embodiment of the present invention.

[0020] Figure 3 This is a flow cytometry result of MRTA inhibiting VSV virus replication at the cellular level in one embodiment of the present invention.

[0021] Figure 4 for Figure 3 A bar chart;

[0022] Figure 5 This is a graph showing the qPCR detection results of MRTA inhibiting EMCV virus replication at the cellular level in one embodiment of the present invention.

[0023] Figure 6 This is a graph showing the qPCR detection results of MRTA inhibiting HSV-1 viral replication at the cellular level in one embodiment of the present invention.

[0024] Figure 7 This is a graph showing the qPCR detection results of MRTA inhibiting H1N1 virus replication at the cellular level in one embodiment of the present invention.

[0025] Figure 8 This is a Western blot result of MRTA inhibiting H1N1 virus replication at the cellular level in one embodiment of the present invention.

[0026] Figure 9This is a plaque assay result of MRTA inhibiting H1N1 virus replication at the cellular level in one embodiment of the present invention.

[0027] Figure 10 This is a graph showing the qPCR detection results of MRTA inhibiting H1N1 virus replication at the cellular level using various dosing methods in one embodiment of the present invention.

[0028] Figure 11 This is a survival curve of mice in which MRTA inhibits H1N1 virus infection at the animal level, according to one embodiment of the present invention.

[0029] Figure 12 This is a graph showing the change in body weight of mice infected with H1N1 virus at the animal level using MRTA in one embodiment of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0031] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0032] <Example 1>

[0033] MRTA for proliferation-toxicity assays at the cellular level:

[0034] Different concentrations (0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 μg / ml) of total alkaloids (MRTA) from *Sophora tonkinensis* were added to A549 lung cancer alveolar basal epithelial cells cultured in 96-well plates, with three replicates for each concentration. After incubation for 24 or 48 h, CCK8 reagent (Cell Counting Kit-8) was added, and the cells were incubated at 37 °C for 30 min. The absorbance at 450 nm was then measured using a microplate reader.

[0035] like Figure 1 The results showed that the total alkaloids of *Sophora tonkinensis* var. *chin ... incubated in A549 cells for 24 h had an IC50 value of 100%. 50 It is 147.9 μg / ml; such as Figure 2 The results showed that the total alkaloids from *Sophora benjamina* var. *chinensis*, after incubation in A549 cells for 48 hours, had an IC50 value of 100%. 50 It was 146.1 μg / ml.

[0036] <Example 2>

[0037] MRTA inhibits VSV viral amplification at the cellular level:

[0038] GF-tagged vesicular stomatitis virus (VSV) (MOI = 0.05) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell size 1.5 × 10⁶). 5 Cells were incubated in wells ( / well) for 12 hours, and then flow cytometry was used to detect the percentage of GFP-positive cells. The experiment was independently repeated three times. There was no statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0039] like Figure 3 and Figure 4 The results showed that MRTA treatment of A549 cells reduced the percentage of GFP-positive cells in a dose-dependent manner (5, 10, 20 μg / ml), as indicated by the bar graph ( Figure 4 The percentage of GFP-positive cells was statistically analyzed, and the results showed that the difference was highly significant (p < 0.001). A higher GFP-positive percentage indicates stronger VSV virus replication. The addition of MRTA decreased the GFP-positive rate, demonstrating that MRTA can inhibit VSV virus replication.

[0040] <Example 3>

[0041] MRTA inhibits EMCV viral amplification at the cellular level:

[0042] EMCV virus (MOI=3) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell mass 1.5 × 10⁶). 5 The experimental group consisted of A549 cells cultured in 24-well plates (1.5 × 10⁶ cells per well) incubated for 12 hours. EMCV virus (MOI = 3) was added alone to the cells. 5 Cells incubated for 12 hours (using one well) served as the control group, while A549 cells without EMCV virus and MRTA served as the blank group. RNA was extracted from the cells, and changes in viral load in the cells were detected by qPCR (real-time quantitative polymerase chain reaction). The experiment was independently repeated three times. There was a statistically significant difference between the experimental group and the control group. Results are expressed as mean ± standard error, ***, p < 0.001.

[0043] Figure 5The results showed that EMCV virus amplification was significant in the control group, while the experimental group treated with MRTA significantly inhibited viral gene expression in a dose-dependent manner, with the most significant inhibition observed at a MRTA concentration of 20 μg / ml. After infecting the host, the virus releases its genome and then utilizes host cell resources for progeny viral amplification. The intensity of viral gene expression reflects the viral amplification activity in the host. The inhibition of viral gene expression after MRTA treatment indicates that MRTA is effective in resisting EMCV virus infection.

[0044] <Example 4>

[0045] MRTA inhibits HSV-1 viral amplification at the cellular level:

[0046] HSV virus (MOI=1) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell size 1.5 × 10⁶). 5 The experimental group consisted of cells co-incubated in 24-well plates for 12 hours, with HSV-1 virus (MOI=1) added alone to A549 cells cultured in 24-well plates (cell quantity 1.5×10⁶). 5 Cells incubated for 12 hours (using one well) served as the control group, while A549 cells without HSV-1 virus and MRTA served as the blank group. RNA was extracted from the cells, and changes in viral load were detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0047] Figure 6 The results showed that HSV-1 virus amplification was significant in the control group, while viral gene expression was significantly inhibited in the experimental group with MRTA at a concentration of 20 μg / ml, indicating that MRTA can resist HSV-1 virus infection.

[0048] <Example 5>

[0049] MRTA inhibits H1N1 virus amplification at the cellular level:

[0050] H1N1 virus (MOI = 0.05) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell size 1.5 × 10⁶). 5 The experimental group consisted of A549 cells cultured in 24-well plates and incubated for 12 hours with H1N1 virus (MOI = 0.05) alone (cell mass 1.5 × 10⁶ cells / well). 5( / well) served as the control group, while A549 cells without H1N1 virus and MRTA served as the blank group. RNA was extracted from the cells, and changes in viral load in the cells were detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental group and the control group. Results are expressed as mean ± standard error, ***, p < 0.001.

[0051] Figure 7 The results showed that the H1N1 virus amplified significantly in the control group, while the experimental group with added MRTA significantly inhibited viral gene expression in a dose-dependent manner, with the most significant inhibition at 20 μg / ml, indicating that MRTA can effectively resist H1N1 virus infection.

[0052] To further demonstrate the inhibitory effect of MRTA on H1N1 virus replication, the inventors infected A549 cells (1.5 × 10⁻⁵) cultured in 24-well plates with H1N1 virus (MOI = 0.05). 5 Cells were incubated in MRTA complete medium (DMEM medium + 10% FBS, with the drug added to the medium at a certain concentration) at different concentrations (5, 10, 20 μg / ml) in each well. After incubation for 24 h, the cells were harvested for Western blotting to detect the expression of NP protein of H1N1 virus.

[0053] like Figure 8 The results showed that MRTA significantly inhibited the expression of H1N1 virus NP protein in A549 cells and inhibited H1N1 virus replication in a concentration gradient (5, 10, 20 μg / ml).

[0054] <Example 6>

[0055] Plaque assay shows that MRTA inhibits H1N1 virus amplification:

[0056] A549 cells were treated with 20 μg / ml MRTA and simultaneously infected with H1N1 (MOI = 0.05). After 12 hours, the cell culture supernatant was collected, and the virus-containing supernatant was diluted 100-fold and added to MDCK fibroblast cells (1.7 × 10⁶ cells per well) cultured in 24-well plates. 5The cells were then incubated in a 37°C, 5% CO2 cell culture incubator. After 2 hours of incubation, the cells were washed twice with PBS to remove unadsorbed viruses. 5% agarose was then melted by heating and cooled to below 40°C. It was then mixed with DMEM medium at a 1:4 volume ratio, and TPCK-trypsin was added and mixed to a concentration of 1 μg / ml. The agarose-containing medium was gently added to each well of a 24-well plate (500 μl per well). After solidification at room temperature, the plate was inverted and incubated in a 37°C, 5% CO2 cell culture incubator for 2–3 days. After incubation, 500 μl of 4% paraformaldehyde was added to each well for fixation for 30 minutes. The agarose was then poured out, and 200 μl of 0.1% crystal violet was added per well for staining for 15 minutes. The crystal violet was washed away with distilled water, and the number of empty spots was observed.

[0057] like Figure 9 The results showed that treatment of MDCK cells with the supernatant after co-incubation with H1N1 virus with MRTA reduced the number of plaques in a dose-dependent manner (5, 10, 20 μg / ml). A higher number of plaques indicated a higher H1N1 virus titer; the reduction in plaque number after MRTA addition indicated that MRTA could inhibit the amplification of H1N1 virus.

[0058] <Example 7>

[0059] Detection of MRTA's inhibition of HIN1 virus replication at the cellular level using different administration methods:

[0060] To determine the impact of different MRTA administration methods on viral replication, the inventors employed five administration methods: pretreatment administration, administration during viral adsorption, administration during viral entry, administration after viral entry, and administration throughout the entire viral infection process. After incubation for the corresponding time using each administration method, RNA was extracted from cells, and changes in viral load in the cells were detected by qPCR. Specific drug administration methods are as follows:

[0061] Pretreatment method for drug administration: Different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell mass 1.5 × 10⁻⁶). 5 Cells were incubated in fresh medium for 12 hours, followed by incubation with HIN1 virus (MOI = 0.1) for another 12 hours. RNA was extracted from the cells, and viral load was detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0062] Drug administration during virus adsorption: H1N1 virus (MOI = 0.1) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell mass 1.5 × 10⁻⁶). 5Cells were incubated in fresh medium for 2 hours at 4°C, then incubated for another 12 hours. RNA was extracted from the cells, and changes in viral load were detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0063] Virus administration during entry: A549 cells were infected with H1N1 virus (MOI = 0.1) and incubated at 4°C for 2 h. The medium was then changed to complete medium containing different concentrations (5, 10, 20 μg / ml) of MRTA, and incubated at 37°C for 1.5 h. The medium was then replaced with fresh medium and incubated for another 12 h. Cells were harvested, RNA was extracted, and qPCR was used to detect changes in viral load in the cells. The experiment was independently repeated three times. There was a statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0064] Virus administration after entry: A549 cells were infected with H1N1 virus (MOI = 0.1). After adsorption at 4°C for 2 h, the medium was replaced with fresh medium, and the cells were incubated at 37°C for 1.5 h. The medium was then changed again, and complete medium containing different concentrations (5, 10, 20 μg / ml) of MRTA was added. Incubation was continued for 12 h. Cells were harvested, RNA was extracted, and changes in viral load in cells were detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental group and the control group. Results are expressed as mean ± standard error, ***, p < 0.001.

[0065] Drug administration throughout the viral infection process: H1N1 virus (MOI = 0.1) and different concentrations (5, 10, 20 μg / ml) of MRTA were added to A549 cells cultured in 24-well plates (cell mass 1.5 × 10⁻⁶). 5 Cells were incubated for 12 hours (in wells), and RNA was extracted from the cells. Changes in viral load in the cells were detected by qPCR. The experiment was independently repeated three times. There was a statistically significant difference between the experimental and control groups. Results are expressed as mean ± standard error, ***, p < 0.001.

[0066] like Figure 10 The results showed that, among the two administration methods of administering the drug during viral entry and throughout viral infection, MRTA had the most significant inhibitory effect on H1N1 virus replication, and the effect was dose-dependent.

[0067] <Example 8>

[0068] Effects of MRTA on mortality and weight change rate in H1N1-infected mice:

[0069] C57BL / 6J mice (6-8 week old healthy female mice, weighing 20-22g) were randomly divided into 6 groups: CON group (blank control group), H1N1 group (model group), oseltamivir group (OST, positive control group), low-dose MRTA group (10mg / kg), medium-dose MRTA group (20mg / kg), and high-dose MRTA group (30mg / kg).

[0070] Except for the CON group, mice in other groups were infected with H1N1 virus via nasal drops. Different treatment groups were administered 200 μl / mouse via gavage at the corresponding dose. The CON and H1N1 groups received the same dose of physiological saline. Gavage was continued daily until the end of the experiment. Daily observations and records were kept of the mice's diet, coat color, mental state, respiration, weight changes, and mortality. Under ethical conditions, a mouse was considered dead when its weight decreased by more than 20% compared to its pre-infection weight.

[0071] like Figure 11 The results showed that, compared with the model group, oral MRTA significantly reduced mouse mortality and prolonged survival time. Furthermore, the high-dose MRTA group, positive control group, and blank control group all showed consistent results, with a 100% survival rate. Figure 12 The results showed that oral administration of MRTA could also slow down the weight loss of infected mice, indicating that the total alkaloids of Sophora flavescens have a significant protective effect against influenza virus infection in mice.

[0072] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

[0073] like Figure 1 and Figure 2 As shown, the English explanation is as follows: Cell viability (% of control) represents the cell survival rate under different concentrations of MRTA treatment;

[0074] like Figure 3 As shown, the English explanations are as follows: SSC-A represents lateral scattering, which represents the granularity of the cells, where Con represents the blank group and Con+ represents the control group;

[0075] like Figure 4 As shown, the English explanation is as follows: GFP ratio (100% of control) represents the percentage of GFP-positive cells;

[0076] like Figure 5 As shown, the English explanation is as follows: Rel.mRNA of EMCV indicates the relative abundance of EMCV RNA in cells;

[0077] like Figure 6 As shown, the English explanation is as follows: Rel.mRNA of HSV-1 indicates the relative abundance of HSV-1 RNA in cells;

[0078] like Figure 7 As shown, the English explanation is as follows: Rel.mRNA of H1N1 indicates the relative abundance of H1N1 RNA in cells;

[0079] like Figure 9 As shown, the English meanings are as follows: CON represents the blank group, and VIRUS represents the control group;

[0080] like Figure 10 As shown, the English explanations are as follows: Pretreatment indicates the group receiving medication under pretreatment conditions; Attachment indicates the group receiving medication during virus adsorption; Entry indicates the group receiving medication during virus entry; Post-entry indicates the group receiving medication after virus entry; and The whole process indicates the group receiving medication throughout the entire virus infection process.

[0081] like Figure 11 As shown, the English translation is as follows: Probability of Survival indicates the survival rate of mice at different number of days.

[0082] like Figure 12 As shown, the English explanation is as follows: Body Weight Change (%) indicates the change in mouse body weight over time after administration of the drug via gavage.

Claims

1. Use of total alkaloids of Sophora subprostrata or salts thereof in the preparation of a drug for preventing and / or treating viral infectious diseases, characterized in that, The virus is one or more of H1N1 influenza A virus, encephalomyocarditis virus, and herpes simplex virus type I.

2. Use according to claim 1, wherein The drug is an inhibitor of H1N1 influenza A virus, encephalomyocarditis virus, or herpes simplex virus type I.

3. The use according to claim 1, wherein The drug is used to inhibit replication or propagation of H1N1 influenza A virus, encephalomyocarditis virus, or herpes simplex virus type I in cells.

4. Use according to any one of claims 1 to 3, wherein The drug is a human or animal drug.

5. The use according to claim 4, wherein the compound is ###0002### The drug is in the form of an oral dosage form, an inhalation preparation, an injection dosage form, or a topical dosage form.

6. The use according to claim 4, wherein the compound is ###0002### The drug is in the form of a solid preparation or a spray.

Images