Application of cepharanthine in preparation of anti-pseudorabies virus medicine

Abstract

The invention discloses an application of cepharanthine in preparation of an anti-pseudorabies virus drug, cepharanthine has no significant toxicity to cells in a concentration range of 0-8 [mu] M, and the cepharanthine can significantly reduce pseudorabies virus infection in a concentration range of 2-4 [mu] M by inhibiting viral genome replication, protein synthesis and progeny virus titer, so that the application of cepharanthine in preparation of the anti-pseudorabies virus drug is realized. The cepharanthine can be used as a safe and effective anti-pseudorabies virus drug, and has the advantages of safety, few toxic and side effects, low drug residue, no pollution and the like.

Description

Technical Field

[0001] The present invention generally relates to the technical field of biological medicines, and in particular to a use of stephania cerebroside in the preparation of an anti-pseudorabies virus drug. Background Art

[0002] Pseudorabies (PR) is an acute infectious disease caused by the pseudorabies virus (PRV) infecting a variety of wild animals and domestic animals. The primary clinical symptoms are fever, severe itching, and encephalitis / myelitis. Pigs are the only natural reservoir and primary source of PRV infection. After infection (without severe itching), piglets primarily experience high fever and neurological disorders, pregnant sows primarily experience reproductive problems, and adult pigs primarily experience respiratory problems. However, these pigs are highly resistant and survive the infection, becoming lifelong carriers of the virus.

[0003] "Bencao Shiyi" (Compendium of Materia Medica) is the earliest ancient text to record Stephania japonica, a perennial vine of the genus Stephania japonica in the Menispermaceae family. Traditional Chinese medicine (TCM) theory posits that Stephania japonica has the effects of clearing heat and detoxifying, dispelling wind and relieving pain, promoting blood circulation and reducing swelling. It is commonly used to treat sore throats, rheumatic pain, and snake bites. Modern pharmacological studies have shown that cepharanthine (CEP), its core alkaloid active ingredient, exhibits significant immunomodulatory, anti-inflammatory, anti-tumor, antioxidant, and broad-spectrum antiviral effects. However, there are currently no studies or reports on the application of CEP in the treatment of PRV. Summary of the Invention

[0004] In view of the above-mentioned defects or deficiencies in the prior art, it is desired to provide a use of stephania quinata in the preparation of an anti-pseudorabies virus drug.

[0005] The present invention provides a use of stephaniarine in preparing an anti-pseudorabies virus drug.

[0006] Preferably, in the anti-pseudorabies virus drug, the concentration of cepharanthine that is non-toxic to cells is 0-8 μM.

[0007] Preferably, in the anti-pseudorabies virus drug, the concentration of cepharanthine used to inhibit the proliferation of pseudorabies virus is 2-4 μM.

[0008] Preferably, the stephanothine is used to prepare a drug for inhibiting the proliferation of the pseudorabies virus.

[0009] Preferably, the anti-pseudorabies virus drug comprises a pharmaceutical composition or compound preparation containing stephanothine as an active ingredient.

[0010] Preferably, the concentration of stephania pine in the pharmaceutical composition or compound preparation is 2-4 μM.

[0011] Preferably, the pharmaceutical composition or compound preparation further includes pharmaceutically acceptable excipients or auxiliary ingredients.

[0012] Preferably, the dosage form of the anti-pseudorabies virus drug includes tablets, powders, granules, suspensions, emulsions, capsules, oral solutions, injections or sustained-release preparations.

[0013] Beneficial effects This study reveals for the first time that the traditional Chinese medicine stephaniarine has anti-pseudorabies virus activity and provides its use in the preparation of anti-pseudorabies virus drugs. Stephaniarine exhibits no significant toxicity to PK-15 cells at concentrations ranging from 0 to 8 μM. At concentrations of 2 to 4 μM, it significantly reduces PRV infection by inhibiting viral genome replication, protein synthesis, and progeny virus titers, with the effects being dose- and time-dependent.

[0014] Stephaniocarbazone is safe for use against pseudorabies virus and has few toxic side effects. Stephaniocarbazone is an ingredient extracted from traditional Chinese medicine. Unlike hormones, antibiotics, and chemically synthesized drugs, it has no obvious toxic side effects on the body.

[0015] Stephaniocarbazone is used to treat pseudorabies virus with low drug residue and no pollution; stephaniocarbazone is an organic molecular compound that is easily absorbed by the animal body, has a high biological metabolic rate, and is excreted without pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Other features, objects and advantages of the present application will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 The toxicity test results of stephanotine on PK-15 cells provided in the examples of this application; Figure 2 Fluorescence microscopy provided in the examples of the present application was used to detect the effect of stephanotisine on the infection of PK-15 cells against PRV-GFP (×100 μm); wherein, Figure 2 (a) is the DMSO group, Figure 2 The concentration of cepharanthine in (b) is 2 μM. Figure 2 The concentration of cepharanthine in (c) was 4 μM; Figure 3 The absolute fluorescence quantitative detection results of the inhibition of PRV genome copy number by stephania cerebroside provided in the examples of this application; Figure 4 The results of Western blotting detection of the inhibition of PRV gE protein expression by stephania cerebroside provided in the examples of this application are as follows; Figure 5 The TCID titer of PRV by cepharanthine provided in the examples of this application is 50In vitro proliferation inhibition results. DETAILED DESCRIPTION

[0017] The present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the invention are shown in the accompanying drawings.

[0018] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0019] Pseudorabies virus (PRV) belongs to the order Herpesvirales ( Herpesvirales ), α-herpesvirus subfamily ( Alphaherpesvirinae PRV is an enveloped, double-stranded DNA virus with a genome size of approximately 143 kb, containing over 70 open reading frames (ORFs) encoding both structural proteins (such as envelope glycoproteins) and nonstructural proteins. The envelope glycoproteins are key targets for viral attachment to host cells and immune evasion. Given its large genome and its ability to evade host immune responses through multiple mechanisms, PRV prevention and control strategies rely heavily on vaccination, while targeted therapeutics remain scarce. Consequently, since the emergence of PRV strains in my country in 2011, significant genetic variation has resulted in a significant reduction in the cross-protective efficacy of commercial vaccines developed based on early genotype I strains (such as Bartha-K61). Traditional live attenuated vaccines have also significantly reduced the neutralizing antibody titers induced by these vaccines, and are unable to effectively block latent infection, resulting in significant economic losses for my country's swine industry. In recent years, a growing number of studies have demonstrated that PRV also carries a high zoonotic risk. Although rare, clear reports in my country have essentially confirmed that, under certain conditions, PRV can cross species and cause human infection. Symptoms of infection primarily include visual impairment and viral encephalitis, and most cases have a poor prognosis. Therefore, PRV strains with significant genetic variation pose a potential threat to both humans and animals, making the development of antiviral drugs for the prevention and treatment of PRV of great significance.

[0020] "Compendium of Materia Medica" is the earliest ancient text to record Stephania japonica, which has a long history of use in traditional Chinese medicine. It is a perennial vine of the genus Stephania japonica, belonging to the Menispermaceae family. Traditional Chinese medicine theory posits that Stephania japonica possesses the effects of clearing heat and detoxifying, dispelling wind and relieving pain, promoting blood circulation and reducing swelling. It is commonly used to treat sore throats, rheumatic pain, and snake bites. Modern pharmacological studies have shown that cepharanthine (CEP), its core alkaloid active ingredient, exhibits significant immunomodulatory, anti-inflammatory, anti-tumor, antioxidant, and broad-spectrum antiviral activities. Studies have shown that cepharanthine can inhibit the release of inflammatory factors by regulating signaling pathways such as NF-κB and exert antiviral activity by interfering with the viral replication cycle. It has demonstrated potential therapeutic value against HIV, influenza virus, and coronavirus infections. However, there are currently no reports on the application of CEP in the treatment of PRV.

[0021] The innovation of this invention lies in revealing for the first time the specific inhibitory effect of CEP on PRV. Relevant experiments have confirmed that it can significantly reduce the viral load and block host cell infection, providing a new strategy for the development of anti-PRV drugs and has important clinical application and industrialization prospects.

[0022] The present invention provides a use of cepharanthine in preparing an anti-pseudorabies virus drug, wherein the chemical structure of cepharanthine is shown in formula (1): Formula (1) In the present invention, porcine kidney-15 (PK-15) cells were used as a cell model for research. The results of cytotoxicity experiments showed that cepharanthine had no obvious cytotoxicity in the concentration range of 0-8 μM. In the concentration range of 2-4 μM, fluorescence microscopy (Fluorescence Microscopy), Western Blot (WB), TCID 50 Both RT-PCR and qPCR results showed that stephanotine significantly inhibited PRV proliferation, and the antiviral effect was positively correlated with its concentration. Stephanotine could be developed as a safe and effective anti-PRV drug, and the development of new anti-PRV drugs based on the existing application conditions of stephanotine has good application prospects.

[0023] The following examples further illustrate the specific embodiments of the present invention. Unless otherwise specified, the instruments and equipment used in the examples of this application are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods.

[0024] The main experimental materials and instruments used in the examples of the present invention are: (1) Main experimental materials The green fluorescent protein-expressing strain (PRV-GFP), the wild-type strain (PRV-HeNLH / 2017), porcine kidney cells (Porcinekidney-15, PK-15), the pEASY-Blunt-UL54 standard plasmid, and the PRV gE mouse monoclonal antibody were all stored in our laboratory. Stephania cypermethrin (HY-N6972, MCE); mouse β-tubulin monoclonal antibody (M20005, Abmart); CCK-8 kit (CA1210-500T), dimethyl sulfoxide (DMSO) (D8371), and high-glucose DMEM medium (12100) were purchased from Solebro. Electrophoresis buffer (WB52001), 20× fast transfer buffer (WB4600), ultrasensitive ECL chemiluminescence kit (P10300), and serum-free cell freezing medium (C40100) were purchased from New Cyme. Fluorescence quantitative reagent SYBR qPCR Master Mix (Q312-02 / 03), cell / tissue nucleic acid extraction kit (DC102-01), and viral nucleic acid extraction kit (RC311) were purchased from Novozymes.

[0025] (2) Main instruments and equipment A CO2 constant-temperature cell culture incubator was purchased from SHELLAB; a real-time fluorescence quantitative PCR instrument and a biological safety cabinet were purchased from ThermoFisher Scientific; a microplate reader was purchased from BioTek Instruments; a high-speed refrigerated centrifuge was purchased from Eppendorf; an inverted fluorescence microscope was purchased from OLYMPUS; an electrophoresis apparatus was purchased from BIO RAD; a clean bench was purchased from Suzhou Antai Air Technology Co., Ltd.; a horizontal refrigerator-freezer conversion cabinet, a medical low-temperature storage box, a medical refrigerator, and a double-opening refrigerator were purchased from Haier; a digital constant-temperature water bath was purchased from Jiangsu Yitong; an integrated chemiluminescence imaging analysis system was purchased from Baijing Biological Co., Ltd.; and a centrifuge for a blood bank was purchased from Changsha Yingtai Co., Ltd.

[0026] Example 1 Results of cytotoxicity test of stephanine on PK-15 cells PK-15 cells in good growth condition were seeded into 96-well plates at an appropriate density the night before, and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 until the cell abundance reached about 80% the next day. Different concentrations of cepharanthine diluted with DMSO were added (at least 3-4 replicates were set for each concentration): 1 μM, 2 μM, 4 μM, 8 μM and 16 μM. A negative control group (Mock group) without any treatment was also included. No cepharanthine was added to the negative control group. The culture plate was returned to the incubator and incubated for 48 hours. After that, 10 μL of CCK-8 reagent was added to each well, and the culture plate was returned to the incubator for another 2 hours. According to the instructions of the CCK-8 kit, the absorbance value at 450 nm (OD 450 GraphPad software was used for analysis and drawing, and Student t test was used for statistical analysis.

[0027] The results are as follows Figure 1 As shown in the results, compared with the Mock group, cepharanthine had no significant effect on cell activity when used in the concentration range of 0-8 μM, that is, cepharanthine had low cytotoxicity and high biosafety within this concentration range.

[0028] Example 2 Fluorescence microscopy detection of the effect of stephanotine on the infection of PK-15 cells with PRV-GFP Well-growing PK-15 cells were seeded at an appropriate density in a 96-well plate the night before and cultured in a 37°C, 5% CO2 incubator until the next day, when cell confluence reached approximately 95%. The cells were then added with cepharanthine at the indicated concentrations (0, 2 μM, and 4 μM) diluted in DMSO (three to four replicates for each concentration). The cells were then infected with the PRV-GFP strain at a multiplicity of infection (MOI) of 0.1. After 1 hour at 37°C, the virus solution was discarded. The cells were then rinsed three times with phosphate-buffered saline (PBS) and cultured for an additional 24 hours in a cell maintenance medium containing the indicated concentration of cepharanthine. The culture medium was discarded, the cells were rinsed three times with PBS, and 50 μL of 4% paraformaldehyde was added to each well. The cells were fixed at room temperature for 20 minutes and rinsed three times with PBS. Finally, the 96-well plate was placed under an inverted fluorescence microscope and observed for green fluorescence intensity to determine viral proliferation.

[0029] The results are as follows Figure 2 As shown, compared with the DMSO group (without cepharanthine), cepharanthine inhibited the proliferation of PRV-GFP strain under the used concentration conditions (2 μM and 4 μM), that is, cepharanthine has good antiviral activity; among them, the fluorescence signal at the 4 μM concentration condition was weakened compared with the 2 μM concentration, and the inhibitory effect at the 4 μM concentration condition was more significant than that at 2 μM, indicating that the antiviral effect of cepharanthine increased with increasing concentration.

[0030] It can be seen from the above Examples 1 and 2 that in the anti-pseudorabies virus drugs provided in the examples of the present application, the concentration of cepharanthine that is non-toxic to cells is 0-8 μM, and the concentration of cepharanthine that inhibits the proliferation of pseudorabies virus is 2-4 μM, indicating that the anti-pseudorabies virus drugs provided in the present application can be effective at a safe dose, and cepharanthine can be used to prepare drugs that inhibit the proliferation of pseudorabies virus.

[0031] Example 3 Absolute fluorescence quantitative detection of the inhibition of PRV genome copy number by stephania pine Well-growing PK-15 cells were seeded at an appropriate density in 24-well plates the night before and cultured in a 37°C, 5% CO2 incubator until the next day, when cell confluence reached approximately 95%. The designated concentrations (0, 2 μM, and 4 μM) of cepharanthin diluted in DMSO were added (three replicates for each concentration). The cells were then infected with wild-type PRV at a multiplicity of infection (MOI) of 0.01. After 1 hour at 37°C, the virus solution was discarded, the cells were rinsed three times with PBS, and cell maintenance medium containing the corresponding concentration of cepharanthin was added for continued culture. The culture plates were removed at 6 and 12 hours, the culture medium was discarded, and cell samples from each well were collected for total DNA extraction according to the cell / tissue nucleic acid extraction kit. Viral proliferation was assessed by qPCR.

[0032] The results are as follows Figure 3 As shown, the viral load showed that compared with the DMSO group (without cepharanthine), cepharanthine inhibited the proliferation of PRV under the used concentration conditions (2 μM and 4 μM), and the viral copy number at the 4 μM concentration of cepharanthine was significantly lower than that at the 2 μM concentration, and the inhibitory effect at 12 hours was more significant than that at 6 hours, that is, the anti-PRV effect of cepharanthine was dose- and time-dependent.

[0033] Example 4 Western blotting detection of the inhibitory effect of stephanotine on PRV gE protein expression PK-15 cells in good growth condition were seeded into 24-well plates at an appropriate density the night before and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 until the cell abundance reached about 95% the next day. Then, cepharanthine at the specified concentrations (0, 2 μM, 4 μM) diluted with DMSO was added. At the same time, the cells were infected with PRV wild-type strains at a multiplicity of infection of 0.01 (MOI=0.01). After 1 hour at 37°C, the virus solution was discarded. After rinsing with PBS three times, cell maintenance medium containing the corresponding concentration of cepharanthine was added and continued to be cultured. According to the requirements of the protein immunoblotting operation, the culture medium was carefully aspirated and discarded at 24 hours and 36 hours respectively. The cell samples in each well were collected to prepare whole cell lysates, and anti-gE (PRV gE protein antibody, Figure 4 upper) and anti-β-tubulin (microtubule protein antibody, Figure 4 The expression of gE and β-tubulin proteins was detected by mouse monoclonal antibodies (bottom).

[0034] The results are as follows Figure 4 As shown in the data, compared with the DMSO group (without cepharanthine), cepharanthine at concentrations of 2 μM and 4 μM significantly inhibited the expression of PRV gE protein in a dose- and time-dependent manner.

[0035] Example 5 Effect of stephanotine on PRV titer TCID 50 In vitro proliferation inhibition results PK-15 cells in good growth condition were seeded into 24-well plates at an appropriate density the night before and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 until the cell abundance reached about 95% the next day. Then, cepharanthine at the specified concentration (0, 2 μM, 4 μM) diluted with DMSO was added (three replicates were set for each concentration). At the same time, the cells were infected with the wild-type PRV at a multiplicity of infection of 0.01 (MOI=0.01). After 1 hour at 37°C, the virus solution was discarded, and the cells were rinsed three times with PBS and then added with cell maintenance medium containing the corresponding concentration of cepharanthine and continued to culture. According to the 50% tissue culture infectious dose (TCID 50 ) Operation requirements: the culture plates were collected at 24 hours and 36 hours respectively, and the samples were frozen and thawed three times, then centrifuged to obtain the supernatant to obtain the whole toxic sample, and finally the TCID 50 The test determines the titer of progeny virus.

[0036] The results are as follows Figure 5As shown, the progeny virus titers showed that compared with the DMSO group (without stepholamine), stepholamine inhibited the proliferation of progeny viruses at both the concentrations used (2 μM and 4 μM). The virus titer at 4 μM was significantly lower than that at 2 μM, and the inhibitory effect at 36 hours was stronger than that at 24 hours. This indicates that stepholamine's anti-PRV effect is dose- and time-dependent. Therefore, stepholamine can be used as a drug to inhibit pseudorabies virus proliferation in vitro.

[0037] In summary, the stephanine provided in the examples of the present application has no significant toxicity to PK-15 cells in the concentration range of 0~8 μM, among which, stephanine significantly reduces PRV infection by inhibiting viral genome replication, protein synthesis and progeny virus titer in the concentration range of 2~4 μM (such as 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM), and the effect is dose- and time-dependent.

[0038] The present invention provides a use of a traditional Chinese medicine, cepharanthine, in a drug for inhibiting the proliferation of pseudorabies virus in vitro. Cepharanthine is found for the first time to be useful in the development of related antiviral drugs for inhibiting the proliferation of pseudorabies virus in vitro. Anti-pseudorabies virus drugs are pharmaceutical compositions with cepharanthine as the sole active ingredient, or containing cepharanthine. Anti-pseudorabies virus drugs refer to drugs for preventing and treating infection by the virus. The present invention uses pseudorabies virus-infected PK-15 cells to detect the antiviral activity of cepharanthine, demonstrating that at a safe concentration of not less than 2 μM, cepharanthine significantly reduces the viral DNA content, viral titer, and structural protein expression level after pseudorabies virus infection compared with the control group, indicating that cepharanthine can be used to inhibit the proliferation of pseudorabies virus in vitro and has good application prospects.

[0039] In some embodiments, the anti-pseudorabies virus drug provided in the embodiments of the present application includes a pharmaceutical composition or compound preparation containing cepharanthine as an active ingredient.

[0040] In some embodiments, the concentration of cepharanthine in the pharmaceutical composition or compound preparation is 2-4 μM.

[0041] In some embodiments, the pharmaceutical composition or compound preparation further includes pharmaceutically acceptable excipients or auxiliary ingredients. For example, excipients include diluents (such as lactose, microcrystalline cellulose), disintegrants (sodium carboxymethyl starch), lubricants (magnesium stearate), pH adjusters (sodium citrate), etc.

[0042] In some embodiments, the dosage form of the anti-pseudorabies virus drug includes tablets, powders, granules, suspensions, emulsions, capsules, oral solutions, injections, or sustained-release preparations.

[0043] In some embodiments, the anti-pseudorabies virus drug can be administered by a drug injection method known in the art, including but not limited to subcutaneous, intramuscular, and intravenous administration, such as enteral, oral (e.g., pills, tablets, buccal, sublingual, disintegrating powders, capsules, films, liquid solutions or suspensions, powders, solid crystals or liquids), rectal (e.g., suppositories, enemas), via injection (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal), via inhalation (e.g., intrabronchial), topical, vaginal, on-dermal or intranasal administration of the anti-pseudorabies virus drug of the present invention.

[0044] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention herein is not limited to technical solutions formed by a specific combination of the aforementioned technical features. It also encompasses other technical solutions formed by any combination of the aforementioned technical features or their equivalents, without departing from the inventive concept. For example, a technical solution formed by replacing the aforementioned features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. Use of cepharanthine in the preparation of anti-pseudorabies virus drugs.

2. The use according to claim 1, characterized in that In the anti-pseudorabies virus drug, the concentration of cepharanthine that is non-toxic to cells is 0-8 μM.

3. The use according to claim 1, characterized in that In the anti-pseudorabies virus drug, the concentration of the stephanothin to inhibit the proliferation of pseudorabies virus is 2-4 μM.

4. The use according to claim 1, characterized in that The stephanothine is used for preparing a medicine for inhibiting the proliferation of the pseudorabies virus.

5. The use according to claim 1, characterized in that The anti-pseudorabies virus drug comprises a pharmaceutical composition or compound preparation with cepharanthine as an effective ingredient.

6. The use according to claim 5, characterized in that The concentration of stephania pine in the pharmaceutical composition or compound preparation is 2-4 μM.

7. The use according to claim 5, characterized in that The pharmaceutical composition or compound preparation also includes pharmaceutically acceptable excipients or auxiliary ingredients.

8. The use according to claim 5, characterized in that The dosage forms of the anti-pseudorabies virus drug include tablets, powders, granules, suspensions, emulsions, capsules, oral solutions, injections or sustained-release preparations.

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