Application of Veratrine and Veratrum extract in the preparation of antiviral drugs

Abstract

This invention relates to a novel use of veratrine, specifically its application in the preparation of antiviral drugs using veratrine and veratrine extracts. Veratrine exhibits inhibitory effects against porcine deltacoronavirus, porcine epidemic diarrhea virus, pseudorabies virus, herpes simplex virus, influenza virus, and African swine fever virus. The veratrine extract and veratrine of this invention demonstrate highly significant antiviral effects. Furthermore, the veratrine extract and veratrine of this invention demonstrate highly significant broad-spectrum antiviral effects, are safe, have few toxic side effects, low drug residues, and cause no pollution.

Description

[0001] This invention is a divisional application of patent number 2023100718745, filed on February 7, 2023, concerning the use of veratrine and veratrine extract in the preparation of antiviral drugs. Technical Field

[0002] This invention relates to a new use of veratrine, specifically the application of veratrine and veratrine extracts in the preparation of antiviral drugs. Background Technology

[0003] African swine fever (ASF) is an acute, febrile, highly contagious, and deadly infectious disease of pigs caused by ASFV infection. Clinically, it is characterized by high fever, bleeding in the skin and internal organs, ataxia, and complete loss of appetite, with a mortality rate approaching 100%. ASFV belongs to the family Asfarviridae and the genus Asfivirus, and is the only member of this family. The ASFV genome is 170–190 kb in size; it is a large, enveloped, double-stranded DNA virus that replicates in the cytoplasm, encoding 160–175 genes. The virus particle has a diameter of 175–215 nm and exhibits icosahedral symmetry. Analysis of ASFV isolates from different regions using the B646L gene encoding the p72 protein reveals 22 major genotypes in prevalent ASFV strains, with genotype II being the most prevalent in China. In August 2018, my country reported its first outbreak of African swine fever (ASF). As of November 2021, my country had reported 203 ASF outbreaks, resulting in the culling of 1.193 million pigs and direct economic losses amounting to trillions of yuan. In the following year, ASF rapidly spread to the remaining 15 countries in Asia. In Vietnam, outbreaks were reported in 63 provinces / municipalities, with over 5 million pigs culled; in Laos, ASF outbreaks were reported in 17 of 18 provinces / municipalities. Globally, since January 2020, ASF outbreaks have been reported in 35 countries across five continents (Africa, the Americas, Asia, Europe, and Oceania) (OIE, 2022). The continued spread of ASF has had a significant impact on the global pork supply and on food security, animal health, and welfare. African swine fever has existed for a century since its first report, and to date, there is still no effective commercial vaccine or specific treatment. Studies have shown that inactivated vaccines offer almost no protection; vector or subunit vaccines provide only partial protection due to unclear protective antigens; and attenuated vaccines offer the best protection, but suffer from issues such as latent carrier infection and recombinant reversion. The ASFV genome is large and complex, encoding over 165 proteins, half of which have unknown functions, and 34% have completely unknown functions. Furthermore, it exhibits widespread immune evasion, posing a significant challenge to vaccine development. Currently, the only effective treatment for African swine fever infection is immediate culling and isolation to prevent further transmission. Developing effective preventative drugs will help uncover the viral infection mechanism and provide a reference for anti-ASFV drug research.

[0004] Porcine deltacoronavirus (PDCoV) belongs to the genus *δ-coronavirus* of the family Coronaviridae in the order Nidovirales. It is a newly emerging porcine coronavirus that primarily infects newborn piglets, causing diarrhea, vomiting, dehydration, and even death. The PDCoV genome is the smallest among coronaviruses, approximately 25.4 nt. The first two-thirds of the viral genome encodes two large replicase precursor polymers, pp1a and pp1ab, while the latter third primarily encodes four structural proteins and three accessory proteins: spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and accessory proteins NS6, NS7, and NS7a. Since its initial outbreak in the United States in 2014, PDCoV has rapidly spread globally. While PDCoV has relatively low clinical severity, infection rate, and mortality, it poses a potential risk of zoonotic transmission. In 2021, researchers detected PDCoV in plasma samples from three Haitian children with acute undifferentiated febrile illness, marking the first discovery of the virus in humans. Its rapid spread and potential for cross-species transmission pose a significant threat to animal and human health and safety. The pathogenic mechanism of PDCoV is unclear, and there is a lack of vaccines and specific drugs; therefore, diagnosis and prevention are still largely at the cognitive stage. Maintaining thorough cleaning and disinfection in pig farms, strengthening husbandry management, and improving biosecurity are crucial aspects of PDCoV prevention.

[0005] Porcine epidemic diarrhea virus (PEDV) is a severe and fatal intestinal infectious disease in piglets, with a mortality rate as high as 80%-100%, making it a key disease for prevention and control in pig farms. PEDV belongs to the alpha coronavirus family, is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 28kb, encoding four structural proteins (S, E, M, N), two polyprotein precursors (pp1a and pp1b), and one accessory protein, ORF3. PEDV includes five subgroups: GI-a / b, GII-a / b / c, with the GI strain being the predominantly circulating strain in the early stages, but the GII strain is currently dominant. Commercially available vaccines primarily target the PEDV GI strain. The GII variant, which emerged after 2010, has maintained a continuous prevalence for over a decade, severely impacting the pig industries in countries such as China, the United States, Canada, Mexico, South Korea, and Japan. Mutant strains that break through existing vaccine protection pose a significant challenge to disease control in pig farms. The development of effective preventive drugs is not only an important supplement to vaccination in controlling diseases, but also helps to discover viral infection mechanisms and provides a reference for research on anti-coronavirus drugs.

[0006] Pseudorabies virus (PRV) is a highly lethal infectious disease. In piglets, PRV infection is characterized by lethargy, vomiting, extreme lethargy, tremors, incoordination, convulsions, and diarrhea. Adult pigs are generally asymptomatic, and if symptoms are present, they are mild and easily resolved, mainly manifesting as fever, depression, and in some cases, vomiting and coughing. Pregnant sows may experience abortion, mummified fetuses, or stillbirths. PRV belongs to the alphaherpesvirus family, classified under the order Herpesvirales, family Herpesviridae, subfamily Alphaherpesvirinae, and genus Varicellavirus. The PRV virus particle is spherical, consisting of a core, capsid, envelope, and peritoneum from the inside out. The genome is 145kb in length, containing seven open reading frames that encode over 100 proteins, including capsid proteins, envelope proteins, peritoneum proteins, and Early Protein 0 (EP0). Currently, control of pseudorabies primarily relies on vaccination. The use of gI / gE gene-deleted attenuated vaccines has effectively controlled and gradually eradicated pseudorabies in my country. However, since 2011, outbreaks of pseudorabies have occurred in vaccinated pig herds in many parts of my country, with a PRV nucleic acid positivity rate of approximately 10% in surveyed pig herds nationwide, causing significant economic losses to the domestic pig farming industry. It is noteworthy that since 2018, there have been more than 20 cases of human infection with pseudorabies virus in my country, posing a significant threat to the health and safety of both animals and humans.

[0007] Herpes simplex virus (HSV-1) is a common pathogen that harms human health. Serological antibody testing shows a positive rate of over 80% in normal asymptomatic individuals. After infecting the human body through the skin or mucous membranes, HSV-1 often remains latent in sensory neurons, causing latent infection. In cases of weakened immunity, it can cause clinical symptoms. It primarily causes herpes on the lips and around the mouth and nose, and occasionally eye rashes. It rarely occurs near the genitals but can infect skin wounds; for example, on the fingers, it is called herpetic impetigo. HSV-1 is a double-stranded linear DNA virus with a genome length of 125–240 kb. It is a round virus composed of a membrane, capsid, core, and envelope. Currently, the most effective drugs for treating HSV-1 infection are acyclovir and its derivatives; however, acyclovir can only temporarily control HSV-1 infection and cannot completely eliminate the virus from the body. Currently, there is no effective vaccine to prevent HSV-1 infection. Prevention mainly focuses on cutting off transmission routes, avoiding close contact with patients, avoiding harmful factors that stimulate the body, and actively exercising to improve the body's immunity. There is currently no cure; only the symptoms can be alleviated.

[0008] Influenza virus (IAV) is a seasonal infectious disease that causes symptoms such as fever, cough, sore throat, headache, body aches, chills, fatigue, and even diarrhea or vomiting, muscle pain or tiredness, and red eyes. In some patients, the disease can progress rapidly, with a sudden onset of high fever exceeding 39°C, leading to severe pneumonia, acute respiratory distress syndrome, pulmonary hemorrhage, pleural effusion, pancytopenia, renal failure, sepsis, shock, Reye's syndrome, respiratory failure, and multiple organ damage, even death. It is estimated that seasonal influenza pandemics cause approximately 3 to 5 million severe cases and 290,000 to 650,000 respiratory deaths globally each year. Furthermore, influenza pandemics occur irregularly due to newly emerging or re-emerging variants. Influenza pandemic viruses can spread rapidly among the population, causing widespread global epidemics; for example, the 1918 H1N1 influenza pandemic caused more than 50 million deaths over two years. Influenza viruses are constantly evolving and mutating, continuously generating new viruses that threaten the health of animals and humans. Therefore, developing effective vaccines and antiviral drugs is an ongoing challenge.

[0009] Because the aforementioned viruses mutate rapidly, cause disease quickly, and have a high mortality rate, and existing drug treatments are limited or ineffective, finding new drugs with significant therapeutic effects has become a technical challenge in this field.

[0010] Veratrine is the main alkaloid of Veratrum nigrum L., with the chemical name (2S,3R,5S)-5-methyl-2-[(1S)-1-[(3S,6AR,11AS,11BR)-2,3,4,6,6A,11,11A,11B-octahydro-3-hydroxy-10,11B-dimethyl-1H-benzo[A]fluorene-9-yl]ethyl]-3-piperidinol, and has the following molecular structure:

[0011]

[0012] In the prior art, veratrine exhibits activities that lower blood pressure and inhibit tumors. However, there are no reports in the prior art regarding the use of veratrine as an antiviral drug. The object of this invention is to provide a broad-spectrum antiviral drug composition, as well as to provide the extraction of veratrine from Veratrum nigrum and the use of veratrine or its pharmaceutically acceptable salts in the preparation of a broad-spectrum antiviral drug. Summary of the Invention

[0013] This invention provides the extraction of veratrine from Veratrum nigrum and the application of Veratrum nigrum extract, veratrine or its pharmaceutically acceptable salt in the preparation of broad-spectrum antiviral drugs.

[0014] The viruses mentioned are porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), herpes simplex virus (HSV), influenza virus, and African swine fever virus (ASFV).

[0015] Optionally, the drug may further include a pharmaceutically acceptable carrier, excipient, or combination thereof.

[0016] Optionally, the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, gluconate, etc. The group consisting of glycosides, glycerol phosphates, gluconates, hemisulfates, heptanates, hexanoates, hydroiodates, 2-hydroxy-ethanesulfonates, lacturonates, lactates, laurates, lauryl sulfates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, palmitates, pectinates, persulfates, 3-phenylpropionates, picrates, p-valerates, propionates, stearates, thiocyanates, p-toluenesulfonates, undecanoates, and valerates.

[0017] Optionally, the excipient is selected from solid excipients or liquid and semi-solid excipients.

[0018] Optionally, the solid excipient is selected from the group consisting of starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, skim milk powder, or combinations thereof.

[0019] Optionally, the liquid and semi-solid excipients are selected from the group consisting of glycerol, propylene glycol, water, ethanol, oil, or combinations thereof.

[0020] Optionally, the dosage form of the drug is selected from the group consisting of tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, powders, or combinations thereof.

[0021] The present invention also provides a broad-spectrum antiviral pharmaceutical composition comprising veratrum extract, veratrine, its hydrate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

[0022] The "pharmaceutically acceptable salt" mentioned in this invention refers to the organic and inorganic salts of the veratrine of this invention, including, but not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, and organic acid salts, such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, gluconate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0023] To prepare the pharmaceutical compositions of the present invention, veratrum extract, veratrine, or a pharmaceutically acceptable salt may be mixed with a pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers that may be used in the compositions include any standard pharmaceutical carrier, such as phosphate-buffered aqueous solutions, water and emulsions, such as oil / water or water / oil emulsions, aqueous glucose, glycols, and various types of wetting agents.

[0024] The pharmaceutical compositions of the present invention may further comprise solid excipients, liquid excipients, and semi-solid excipients. Solid excipients may be selected from starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, skim milk powder, etc. Liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical compositions of the present invention may also comprise stabilizers and preservatives.

[0025] This composition may be administered by any method known in the art, including but not limited to intranasal, oral, percutaneous, ocular, intraperitoneal, inhalation, intravenous, intracisional injection or infusion, subcutaneous, implantation, intravaginal, sublingual, urethral, ​​subcutaneous, intramuscular, intravenous, rectal, sublingual, mucosal, ocular, spinal cord, intrathecal, intra-articular, intra-articular, subarachnoid, bronchial, and lymphatic administration. Topical formulations may be in the form of gels, ointments, creams, aerosols, etc.; intranasal formulations may be delivered as sprays or drops; percutaneous formulations may be administered via transdermal patches or iontophoresis; inhaled formulations may be delivered using a nebulizer or similar device. The composition may also be in the form of tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, powders, or any other suitable combination.

[0026] Beneficial effects

[0027] 1. The embodiments of this invention demonstrate that veratrine is not cytotoxic and does not exert its effects through cytotoxicity. Cells treated with different concentrations of veratrine showed a significant decrease in viral protein expression, proving that veratrine can significantly inhibit viral infection. Veratrine or its extracts exert antiviral effects against porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), herpes simplex virus (HSV-1), influenza virus (IAV), porcine deltacoronavirus (PDCoV), and African swine fever virus (ASFV) by inhibiting macropinocytosis and the PI3K / Akt pathway. It should be noted that the above viruses are not significantly or effectively treated with conventional antiviral drugs. This invention discovers that veratrine possesses a novel antiviral mechanism. The veratrine extract and veratrine of this invention exhibit highly significant antiviral effects against viruses (PEDV, PRV, HSV, IAV) at a concentration of 10 μM, demonstrating extremely strong antiviral infection activity. Furthermore, the veratrine extract and veratrine of this invention exhibit highly significant antiviral effects against ASFV at a concentration of 5 μM, demonstrating extremely strong anti-African swine fever virus infection activity.

[0028] 2. The veratrum extract and veratrine of the present invention have low residues, no pollution, are easily absorbed by animal bodies, have a high biological metabolism rate, and are excreted without pollution. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 The cytotoxicity of veratrine is indicated by A being PK15 and B being Vero-E6.

[0031] Figure 2 The inhibitory effect of Veratrum nigrum extract on the virus is shown in Figure 1. A represents PK15+PDCov; B represents Vero-E6+HLJBY; C represents Vero-E6+PRV; D represents Vero-E6+HSV; and E represents Vero-E6+IAV.

[0032] Figure 3 The inhibitory effect of veratrine on the virus is represented by A as PK15+PDCov; B as Vero-E6+HLJBY; C as Vero-E6+PRV; D as Vero-E6+HSV; and E as Vero-E6+IAV.

[0033] Figure 4 This study investigated the inhibitory effect of Veratrum nigrum extract on ASFV.

[0034] Figure 5 This is due to the cytotoxicity of vincaine.

[0035] Figure 6 This refers to the inhibitory effect of veratrine on ASFV.

[0036] Figure 7 The inhibitory effects of flavonoids and the PI3K / Akt pathway-specific inhibitor LY294002 on macropinocytosis were investigated.

[0037] Figure 8 This refers to the inhibitory effect of veratrine on the PI3K / Akt pathway.

[0038] Figure 9 The inhibitory effect of veratrine on porcine epidemic diarrhea virus depends on the PI3K / Akt pathway. Detailed Implementation

[0039] The claims of this disclosure are further described in detail below with reference to experiments and embodiments, but this does not constitute any limitation on this disclosure. Any limited modifications made within the scope of protection of the claims of this disclosure are still within the scope of protection of the claims of this disclosure. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] Unless otherwise defined herein, the scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials to those described herein may also be used in the practice or testing of this disclosure.

[0041] Test materials

[0042] Porcine Epidemic Diarrhea Virus (PEDV), Pseudorabies virus (PRV), Herpes simplex virus (HSV-1), influenza virus (IAV), porcine deltacoronavirus (PDCoV), African swine fever virus (ASFV), porcine kidney cells (PK15), African green monkey kidney cells (Vero-E6), and alveolar macrophages (PAM) were all obtained from the National African Swine Fever Reference Laboratory of the China Animal Health and Epidemiology Center (Qingdao, Shandong).

[0043] The CCK-8 kit was purchased from APExBio; veratrine (CAS No. 8051-02-3) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and Veratrum nigrum L. was purchased from Bozhou Jiayu Biotechnology Co., Ltd.

[0044] Example 1: Preparation of Veratrum nigrum extract

[0045] 100g of dried whole herb of Veratrum nigrum was pulverized, passed through an 80-mesh sieve, and 80% ethanol solution was added at a material-to-liquid ratio of 1:5 (g:ml). The mixture was ultrasonically extracted (100W, 50 degrees Celsius) three times, for 30 minutes each time. After filtration, the extract was concentrated and dried under reduced pressure and then freeze-dried to obtain 5.8g of Veratrum nigrum extract.

[0046] Example 2: Cytotoxicity of Veratrine

[0047] With 2×10 4PK15 (A) or Vero-E6 cells (B) were seeded into 96-well plates at a rate of 10 cells / well. After 12 hours, the medium was replaced with fresh medium containing different concentrations of veratrine, and the cells were incubated at 37°C for 48 hours (PK15) or 24 hours (Vero-E6). CCK-8 solution was then added at a rate of 10 μL / well, and the cells were incubated in the dark for another 3 hours. OD values ​​were then recorded. 450 The values ​​were calculated, and cell viability was determined. Each experiment was performed in triplicate, and the figures are shown as mean ± standard deviation (SD). Results are presented in... Figure 2 middle.

[0048] like Figure 2 As shown, cells treated with different concentrations of veratrine showed no significant difference in cell activity compared to untreated cells within a certain drug concentration range, demonstrating that veratrine at that concentration does not exhibit significant cytotoxicity.

[0049] Example 3: Inhibitory effects of Veratrum extract and veratrine on viruses (PDCoV, PEDV, PRV, HSV, IAV).

[0050] With 8×10 5 PK15(A) or Vero-E6(BE) cells were seeded into six-well plates at a cell / well ratio. After 12 h, the medium was replaced with fresh medium containing DMSO and different drug concentrations, and cultured at 37°C for 2 h. Then, the medium was replaced with the same drug concentration and 1 MOI of IPDCoV(A), PEDV(B), PRV(C), HSV(D), and IAV(E), and cultured at 37°C for 1 h. Afterward, the medium was replaced with the same drug concentration, and cultured at 37°C for another 18 h (PDCoV) or 12 h (PEDV, PRV, HSV, IAV), before collecting cell samples for Western blotting. The results are shown in Figures 1-3. Figure 1 and 3 middle.

[0051] like Figure 2 As shown, compared with untreated cells, cells treated with different concentrations of Veratrum nigrum extract showed significantly reduced viral protein expression, demonstrating that Veratrum nigrum extract can significantly inhibit viral infection.

[0052] like Figure 3 As shown, compared with untreated cells, cells treated with different concentrations of veratrine showed significantly reduced viral protein expression, demonstrating that veratrine can significantly inhibit viral infection.

[0053] Example 4: Cytotoxicity of Veratrine

[0054] With 2×10 4PAM cells were seeded into 96-well plates at a rate of 10 cells / well. After 12 hours, the medium was replaced with fresh medium containing different concentrations of quercetin and incubated at 37°C for 24 hours. Subsequently, CCK-8 solution was added at a rate of 10 μL / well, and the plates were incubated in the dark for another 3 hours. OD values ​​were then recorded. 450 The values ​​were calculated, and cell viability was determined. Each experiment was performed in triplicate, and the figures are shown as mean ± standard deviation (SD). Results are presented in... Figure 5 middle.

[0055] like Figure 5 As shown, cells treated with different concentrations of veratrine showed no significant difference in cell activity compared to untreated cells within a certain drug concentration range, demonstrating that veratrine at that concentration does not exhibit significant cytotoxicity.

[0056] Example 5: Determination of the antiviral activity of Veratrum nigrum extract and veratrine against Aspergillus flavus (ASFV)

[0057] With 8×10 5 PAM cells were seeded into six-well plates at a density of 10 cells / well. After 12 hours, the medium was replaced with fresh medium containing DMSO and different concentrations of the drug, and cultured at 37°C for 2 hours. Then, the medium was replaced with the same concentration of the drug and 1 MOI ASFV, and cultured at 37°C for 1 hour. Finally, the medium was replaced with the same concentration of the drug, and cultured at 37°C for another 24 hours. Cell samples were then collected and Western blotting was performed. The results are shown in [Figure number missing]. Figure 4 and 6 middle.

[0058] like Figure 4 As shown, compared with untreated cells, cells treated with different concentrations of Veratrum nigrum extract showed significantly reduced viral protein expression, demonstrating that Veratrum nigrum extract can significantly inhibit viral infection.

[0059] like Figure 6 As shown, compared with untreated cells, cells treated with different concentrations of veratrine showed significantly reduced viral protein expression, demonstrating that veratrine can significantly inhibit viral infection.

[0060] Example 6: Resveratrol and the PI3K / Akt pathway-specific inhibitor LY294002 inhibit macropinocytosis.

[0061] Vero-E6 cells were loaded at 4 × 10 5Cells / well were seeded into 6-well plates containing sterile cell crawling slides. After 12 hours, the culture medium was discarded, and the cells were washed three times with PBS. Then, 10 μM veratrine, or 10 μM LY294002 (a PI3K / Akt pathway-specific inhibitor), or LY294002 + 10 μM veratrine were added to 2 mL of incomplete culture medium (serum-free DMEM) to perform a Dextrocytosis (macropinocytosis pathway-specific marker) cell entry assay. The cells were first starved at 37°C for 2 hours to prevent serum interference with Dextrocytosis. Afterward, the culture medium was discarded, and the cells were washed three times with PBS. 2 mL of 10 μg / mL Dextro solution was added, and the cells were incubated at 37°C for 1 hour. The culture medium was discarded, and the cells were washed three times with cold citrate buffer (pH 3) and then three times with cold PBS to terminate cell entry and remove Dextro from the culture medium and cell membrane surface. Finally, fixation solution was added, and the cells were fixed at 37°C for 30 minutes. The cells were then rinsed twice with Glycine (0.02M) + PBS solution, followed by three washes with PBS, 5 min each time. Finally, the nuclei were stained with DAPI + DABCO solution and the slides were mounted. Indirect immunofluorescence detection was then performed. Results are shown in... Figure 7 middle.

[0062] like Figure 7 As shown, the fluorescence signal of Dextro, a marker of cell entry via macropinocytosis, completely disappeared in cells treated with 10 μM veratrine compared to untreated cells.

[0063] Example 7 illustrates the inhibitory effect of veratrine on the PI3K / Akt pathway.

[0064] With 8×10 5 Vero-E6 cells were seeded into six-well plates at a density of 10 cells / well. After 12 h, Vero-E6 cells were treated with DMSO, 10 μM veratrine, 1 MOI HLJBY, and 1 MOI HLJBY + 10 μM veratrine, respectively. Cell samples were collected at 10, 20, and 30 min for Western blotting. Results are shown in [Figure number missing]. Figure 8 middle.

[0065] like Figure 8 The results show that PEDV infection at early stages (10, 20, and 30 minutes) significantly activates the PI3K / Akt pathway. Compared to the untreated group, the pAkt protein level decreased in the 10 μM veratrine treatment group, indicating that veratrine significantly inhibits the activation of the PI3K / Akt pathway.

[0066] Example 8: The inhibitory effect of veratrine on porcine epidemic diarrhea virus depends on the PI3K / Akt pathway.

[0067] With 8×10 5Vero-E6 cells were seeded into six-well plates at a density of 10 cells / well. After 12 h, Vero-E6 cells were treated with DMSO, veratrine (10 μM), the PI3K / Akt pathway-specific inhibitor LY294002 (10 μM), and LY294002 + veratrine (10 μM), respectively, and cultured at 37°C for 2 h. The cells were then replaced with fresh DMEM medium containing the same drug concentration and 1 MOI of PEDV, and cultured at 37°C for 1 h. Afterward, the medium was replaced with DMEM containing the same drug concentration and cultured at 37°C for another 12 h. Samples were then collected for plaque formation assays. The results are shown in Figures 1-3. Figure 9 middle.

[0068] like Figure 9 As shown, compared with untreated cells, cells treated with LY294002, veratrine, and LY294002 + veratrine showed significantly reduced viral titers after treatment with either LY294002 or veratrine. Furthermore, the inhibitory effect of the combined treatment with veratrine and LY294002 on PEDV was the same as that of the drug-only treatments; the combined treatment did not produce any further inhibitory effect on PEDV. These results indicate that veratrine inhibits PEDV by inhibiting the PI3K / Akt pathway.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of veratrine and its pharmaceutically acceptable salts in the preparation of antiviral drugs, characterized in that, The virus in question is pseudorabies virus, herpes simplex virus, influenza virus, or African swine fever virus.

2. The application according to claim 1, characterized in that, It also includes pharmaceutically acceptable carriers.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable carrier is selected from solid excipients, liquid or semi-solid excipients.

4. The application according to claim 1, characterized in that, The dosage form of the drug is selected from tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, or powders.

Images