Application of veratrine and veratrine extract in preparation of antiviral drugs

Abstract

The invention belongs to novel application of veratrine, and particularly relates to application of veratrine and veratrine extract in preparation of antiviral drugs. Wherein the veratrine has an inhibition effect on porcine deltacoronavirus, porcine epidemic diarrhea virus, pseudorabies virus, herpes simplex virus, influenza virus or African swine fever virus. The anti-virus effect of the veratrum nigrum extract and the veratrum nigrum alkali is very remarkable. The veratrum nigrum extract and the veratrum nigrum alkali have very remarkable broad-spectrum antiviral effects, and are safe, low in toxic and side effects, low in drug residue and free of pollution.

Description

[0001] This invention is a divisional application of 2023100718745, 2023.02.07, for the use of lycopodine and lycopodine extract in the preparation of antiviral drugs Technical Field

[0002] The invention belongs to a new use of veratridine, and particularly relates to the use of veratridine and veratridine extract in the preparation of antiviral drugs. Background Art

[0003] ASF is an acute, febrile, highly contagious disease of pigs caused by ASFV infection. Clinically, it is mainly characterized by high fever, bleeding in the skin and internal organs, ataxia and loss of appetite, with a mortality rate close to 100%. ASFV belongs to the Asfarviridae family and the Asfivirus genus, and is the only member of this family. The ASFV genome size is 170-190kb. It is a large double-stranded DNA virus with an envelope that replicates in the cytoplasm and encodes 160-175 genes. The diameter of the virus particle is 175-215nm and is icosahedral symmetric. The B646L gene encoding the p72 protein was used to analyze ASFV isolates from different regions, and the ASFV epidemic strains can be divided into 22 major genotypes, of which the main genotype prevalent in China is genotype II. In August 2018, the first ASF outbreak occurred in my country. As of November 2021, a total of 203 ASF outbreaks were reported in my country, 1.193 million pigs were culled, and direct economic losses amounted to one trillion yuan. In the more than a year since then, ASF has spread rapidly to the remaining 15 countries in Asia. In Vietnam, 63 provinces / cities have reported outbreaks, and more than 5 million pigs have been culled; in Laos, 17 of 18 provinces / cities have reported ASF outbreaks. Globally, since January 2020, ASF outbreaks have been reported in 35 countries and five continents (Africa, America, Asia, Europe and Oceania) (OIE, 2022). The continued spread of ASF has had a huge impact on the global supply of pork products, as well as on food security, animal health and welfare. It has been a hundred years since African swine fever was first reported, and there is still no effective commercial vaccine or special medicine. Studies have shown that inactivated vaccines can provide almost no protection; vector vaccines or subunit vaccines can only provide partial protection due to unclear protective antigens; attenuated vaccines have the best protection effect, but there are problems such as latent virus transmission and recombinant strong. Due to the large size and complex structure of the ASFV genome, it encodes more than 165 proteins, half of which have unknown functions, 34% of which are completely unknown, and there is a widespread immune escape phenomenon, which poses a huge challenge to vaccine development. At present, after being infected with African swine fever, the only way to prevent infection is to kill and isolate immediately. The development of effective preventive drugs will help discover the mechanism of viral infection and provide a reference for anti-ASFV drug research.

[0004] Porcine deltacoronavirus (PDCoV) belongs to the genus Deltacoronavirus of the family Coronaviridae of the order Nidovirales. It is a newly emerged pig-origin coronavirus that mainly infects newborn piglets, causing diarrhea, vomiting, dehydration and even death in piglets. The genome of PDCoV is the smallest among coronaviruses, about 25.4 nt. The first two-thirds of the viral genome is responsible for encoding two large replicase precursor polyproteins pp1a and pp1ab, and the last one-third mainly encodes four structural proteins and three auxiliary proteins, namely spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N) and auxiliary proteins NS6, NS7, and NS7a. Since the first outbreak of PDCoV in the United States in 2014, it has spread rapidly around the world. The clinical severity, infection rate and mortality rate of PDCoV are low, however, it has the potential risk of zoonotic transmission. In 2021, researchers detected PDCoV in plasma samples from three Haitian children with acute undifferentiated febrile illness, which was the first time the virus was found in humans. Its rapid spread and potential cross-species transmission characteristics pose a huge threat to the health and safety of animals and humans. The pathogenic mechanism of PDCoV is unclear, there is a lack of vaccines and effective drugs, and the diagnosis and prevention are limited to the awareness stage. Cleaning and disinfecting pig farms, strengthening feeding management, and improving biosafety are important aspects of PDCoV prevention.

[0005] Porcine Epidemic Diarrhea Virus (PEDV) can cause serious fatal intestinal infectious diseases in piglets, with a mortality rate of up to 80%-100%. It is a key disease to be prevented and controlled in pig farms. PEDV belongs to alpha coronavirus, a single-stranded positive-strand RNA virus with an envelope. The genome is about 28kb long and can encode 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. The GI strain was the main one in the early stage, and the GII strain is the main one at present. The commercial vaccines available for clinical use are mainly for PEDV GI strains. The GII mutant strains that appeared after 2010 have been on a continuous prevalence trend for more than a decade, causing heavy damage to the pig farming industry in China, the United States, Canada, Mexico, South Korea and Japan. The mutant strains break through the protection of existing vaccines, which is a major challenge for the prevention and control of diseases in pig farms. The development of effective preventive drugs is not only an important supplement to vaccination to control the epidemic, but also helps to discover the mechanism of viral infection and provide a reference for the research of anti-coronavirus drugs.

[0006] Pseudorabies virus (PRV) is a highly lethal infectious disease. PRV-infected piglets are mainly characterized by lethargy, vomiting, extreme mental fatigue, trembling, uncoordinated movements, convulsions, and diarrhea. Adult pigs are generally latently infected, and if there are symptoms, they are also mild and easy to recover. The main manifestations are fever, depression, vomiting, and coughing in some sick pigs. Pregnant sows may have miscarriages, mummified fetuses, or stillbirths. PRV belongs to the alphaherpesvirus order, herpesviridae family, alphaherpesvirinae subfamily, and varicellavirus genus. PRV virus particles are spherical, and from the inside to the outside are the core, capsid, capsule, and envelope. The genome is 145kb long and contains seven open reading frames, which can encode more than 100 proteins, including capsid proteins, envelope proteins, envelope proteins, and Early Protein 0 (EP0) and other proteins. At present, the prevention and control of pseudorabies mainly relies on vaccine immunity. The use of gI / gE gene-deficient attenuated vaccines has effectively controlled and gradually eliminated pseudorabies in my country. However, since 2011, pseudorabies has broken out in vaccinated pigs in many parts of my country. The PRV nucleic acid positivity rate in the surveyed pigs nationwide is about 10%, causing great economic losses to the domestic pig farming industry. It is worth noting that since 2018, more than 20 cases of pseudorabies virus infection have occurred in my country, posing a huge threat to the health and safety of animals and humans.

[0007] Herpes simplex virus (HSV-1) is a common pathogen that endangers human health. Through serum antibody detection, it is found that the positive rate in normal asymptomatic individuals is more than 80%. After HSV-1 infects the human body through the skin or skin mucosa, it often lurks in sensory neurons, causing latent infection. In the case of low immunity, it will cause clinical symptoms. It mainly causes herpes around the lips and mouth and nose, and occasionally causes eye rash. It is not common to occur in wounds near the genitals but may infect the skin, such as on the fingers, which is called herpetic purulent finger disease. HSV-1 is a double-stranded linear DNA virus with a genome length of 125 to 240kb. It is a round virus synthesized by a membrane, capsid, core, and capsule. At present, the most effective drug for treating HSV-1 infection is acyclovir and its derivatives, but acyclovir can only temporarily control HSV-1 infection and cannot completely eliminate the virus in the body. There is no effective vaccine to prevent HSV-1 infection. Prevention mainly focuses on cutting off the transmission route, avoiding close contact with patients, avoiding the stimulation of harmful factors on the body, and actively exercising to improve the body's immunity. It cannot be cured at present, but can only alleviate clinical symptoms.

[0008] Influenza virus (IAV) can cause seasonal infectious diseases with fever, cough, sore throat, headache, body pain, chills and fatigue, even diarrhea or vomiting, muscle pain or fatigue, red eyes and other symptoms. Some patients may develop a rapid progression of the disease, with a fierce onset, sudden high fever, body temperature exceeding 39°C, followed by severe pneumonia, acute respiratory distress syndrome, pulmonary hemorrhage, pleural effusion, pancytopenia, renal failure, sepsis, shock and Reye's syndrome, respiratory failure and multiple organ damage, and even death. According to statistics, seasonal influenza pandemics cause about 3 million to 5 million severe cases and 290,000 to 650,000 respiratory deaths worldwide each year. In addition, influenza pandemics occur irregularly due to new or re-emerging mutant strains. Pandemic influenza viruses can spread rapidly among the population, causing widespread global epidemics, such as the 1918 H1N1 influenza pandemic virus that caused more than 50 million deaths in two years. Influenza viruses continue to evolve and mutate, producing new viruses that threaten animal and human health, so the development of effective vaccines and antiviral drugs is an ongoing challenge.

[0009] The above-mentioned viruses mutate quickly, cause disease quickly, have a high mortality rate, and the existing drug treatment effects are limited or poor. Therefore, finding new drugs with significant therapeutic effects has become a technical challenge in this field.

[0010] Veratrum nigrum L. 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]fluoren-9-yl]ethyl]-3-piperidinol, and has the following molecular structure:

[0011]

[0012] In the prior art, verrucine has the activity of lowering blood pressure and inhibiting tumors. However, there is no report in the prior art that verrucine is used as an antiviral drug. The purpose of the present invention is to provide a broad-spectrum antiviral drug composition, as well as to provide the extraction of verrucine from verrucine and the use of verrucine or its pharmaceutically acceptable salt in the preparation of a broad-spectrum antiviral drug. Summary of the invention

[0013] The invention provides the extraction of verrucine from verrucine and the application of the verrucine extract, verrucine or a pharmaceutically acceptable salt thereof in the preparation of a broad-spectrum antiviral drug.

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

[0015] Optionally, the drug further comprises a pharmaceutically acceptable carrier, excipient or a 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, fumarate, glucoheptate, The group consisting of saccharate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0017] Optionally, the excipient is selected from solid excipients or liquid and semisolid 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, skimmed milk powder or a combination thereof.

[0019] Optionally, the liquid and semisolid excipients are selected from the group consisting of glycerol, propylene glycol, water, ethanol, oil or a combination 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, pills, granules, powders or a combination thereof.

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

[0022] The "pharmaceutically acceptable salts" of the present invention refer to organic and inorganic salts of veratridine of the present invention, including but not limited to inorganic acid salts formed by reaction with an amino group, such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, and organic acid salts, such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like.

[0023] To prepare the pharmaceutical composition of the present invention, the extract of Verriculum chinense, Verriculum alkaloids or pharmaceutically acceptable salts can be mixed with pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers that can be used in the present composition include any standard pharmaceutical carriers, such as phosphate buffered aqueous solution, water and emulsions, such as oil / water or water / oil emulsions, aqueous glucose, glycols and various types of wetting agents.

[0024] Pharmaceutical composition of the present invention can comprise solid excipient, liquid excipient and semisolid excipient in addition.Solid excipient can be selected from starch, cellulose, talcum, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, skimmed milk powder etc.Liquid and semisolid excipient can be selected from glycerine, propylene glycol, water, ethanol and various oils, comprise oil of petroleum, animal, plant or synthetic origin, for example peanut oil, soybean oil, mineral oil, sesame oil etc.Pharmaceutical composition of the present invention can also comprise stabilizer and preservative.

[0025] The present composition can be administered by any method known in the art, including but not limited to intranasal, oral, transdermal, ocular, intraperitoneal, inhalation, intravenous, intracisternal injection or infusion, subcutaneous, implanted, intravaginal, sublingual, urethral, ​​subcutaneous, intramuscular, intravenous, rectal, sublingual, mucosal, ocular, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial and lymphatic administration. External preparations can be in the form of gels, ointments, creams, aerosols, etc.; intranasal preparations can be delivered in the form of sprays or droplets; transdermal preparations can be administered via transdermal patches or ion permeation therapy; inhaled preparations can be delivered using a sprayer or similar device. The composition can also be in the form of tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules or powders or any other suitable composition.

[0026] Beneficial Effects

[0027] 1. The embodiment of the present invention proves that verrucine is not cytotoxic and does not work through cytotoxicity. After cells treated with verrucine at different concentrations, the expression of viral proteins was significantly reduced, proving that verrucine can significantly inhibit viral infection. Verrucine or verrucine extracts produce antiviral effects on 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 inhibiting the PI3K / Akt pathway. It should be noted that the above-mentioned viruses are not significantly or ineffectively treated with general antiviral drugs. The present invention finds that verrucine has a new antiviral mechanism. The verrucine extract and verrucine of the present invention have a very significant antiviral effect on viruses (PEDV, PRV, HSV, IAV) at a concentration of 10 μM, which can show a very strong antiviral infection effect. The verrucine extract and verrucine of the present invention have a very significant antiviral effect on ASFV at a concentration of 5 μM, which can show a very strong anti-African swine fever virus infection effect.

[0028] 2. The Veratrum extract and Veratrum alkaloids of the present invention have low antiviral drug residues, are pollution-free, are easily absorbed by animal bodies, have high biological metabolism rates, and are excreted without pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The cytotoxicity of veratridine, where A is PK15 and B is Vero-E6.

[0031] Figure 2 The inhibitory effect of Verrox versicolor extract on viruses, A is PK15+PDCov; B is Vero-E6+HLJBY, C is Vero-E6+PRV, D is Vero-E6+HSV, and E is Vero-E6+IAV.

[0032] Figure 3 The inhibitory effect of Veratridine on viruses, A is PK15+PDCov; B is Vero-E6+HLJBY, C is Vero-E6+PRV, D is Vero-E6+HSV, and E is Vero-E6+IAV.

[0033] Figure 4 The inhibitory effect of Li Lu extract on ASFV.

[0034] Figure 5 The cytotoxicity of veratridine.

[0035] Figure 6 The inhibitory effect of veratridine on ASFV.

[0036] Figure 7 The inhibitory effects of veratridine and LY294002, a specific inhibitor of the PI3K / Akt pathway, on macropinocytosis.

[0037] Figure 8 The inhibitory effect of veratridine on PI3K / Akt pathway.

[0038] Fig. 9 The inhibitory effect of veratridine on porcine epidemic diarrhea virus depends on the PI3K / Akt pathway. DETAILED DESCRIPTION

[0039] The following is a further detailed description of the claims of the present disclosure in conjunction with experiments and examples, but does not constitute any limitation to the present disclosure. Any limited modifications made within the scope of protection of the claims of the present disclosure are still within the scope of protection of the claims of the present disclosure. If no specific conditions are specified in the implementation method, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0040] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present 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 pulmonary alveolar macrophages (PAM) were all from the National African Swine Fever Reference Laboratory of China Animal Health and Epidemiology Center (Qingdao, Shandong).

[0043] CCK-8 kit was purchased from APExBio; Veratrum nigrum L. was purchased from Bozhou Jiayu Biotechnology Co., Ltd.

[0044] Example 1 Preparation of Ligusticum chuanxiong Extract

[0045] 100 g of the dried whole herb of Veratrum, a traditional Chinese medicinal material, was crushed and passed through an 80-mesh sieve. An 80% ethanol solution was added according to a solid-liquid ratio of 1:5 (g:ml). Ultrasonic extraction (100 W, 50 degrees) was performed 3 times, each time for 30 min. The extract was filtered, concentrated and dried under reduced pressure, and freeze-dried to obtain 5.8 g of Veratrum extract.

[0046] Example 2 Cytotoxicity of Veratridine

[0047] 2×10 4PK15 (A) or Vero-E6 cells (B) were plated in 96-well plates at a volume of 10 cells / well. After 12 hours, fresh culture medium containing different concentrations of veratridine was used and cultured at 37°C for 48 hours (PK15) or 24 hours (Vero-E6). CCK-8 solution was added at a volume of 10 μL / well and cultured for another 3 hours in the dark. OD was then read. 450 The values ​​were calculated and the cell viability was calculated. Three replicates were set for each group of experiments, and the graphs show the mean ± standard deviation. The results are shown in Figure 2 middle.

[0048] like Figure 2 As shown, there is no significant difference in cell activity between cells treated with different concentrations of veratridine and cells not treated with veratridine within a certain drug concentration range, which proves that veratridine at this concentration has no significant cytotoxicity.

[0049] Example 3 Inhibitory Effects of Ligusticum chuanxiong Extract and Ligusticum chuanxiong Alpine on Viruses (PDCoV, PEDV, PRV, HSV, IAV)

[0050] 8×10 5 PK15 (A) or Vero-E6 (BE) cells were plated into six-well plates at a volume of 1 cell / well. After 12 hours, the culture medium was replaced with fresh culture medium containing DMSO and different concentrations of drugs and cultured at 37°C for 2 hours. Then, the culture medium was replaced with culture medium containing the same concentration of drugs and 1MOIPDCoV (A), PEDV (B), PRV (C), HSV (D), and IAV (E), and cultured at 37°C for 1 hour. After the end, the culture medium was replaced with culture medium containing the same concentration of drugs, and cultured at 37°C for 18 hours (PDCoV) or 12 hours (PEDV, PRV, HSV, IAV), and then cell samples were collected and subjected to Western-Blot experiments. The results are shown in Figure 1 and 3 middle.

[0051] like Figure 2 As shown, the expression of viral proteins in cells treated with different concentrations of Li Lu extract was significantly reduced compared with cells not treated with drugs, proving that Li Lu extract can significantly inhibit viral infection.

[0052] like Figure 3 As shown, the expression of viral proteins in cells treated with different concentrations of leucine was significantly reduced compared with cells not treated with the drug, proving that leucine can significantly inhibit viral infection.

[0053] Example 4 Cytotoxicity of Veratridine

[0054] 2×10 4PAM cells were plated in 96-well plates at a volume of 10 cells / well. After 12 hours, fresh culture medium containing different concentrations of verrucine was used and cultured at 37°C for 24 hours. CCK-8 solution was then added at a volume of 10 μL / well and cultured for another 3 hours in the dark. The OD was read. 450 The values ​​were calculated and the cell viability was calculated. Three replicates were set for each group of experiments, and the graphs show the mean ± standard deviation. The results are shown in Figure 5 middle.

[0055] like Figure 5 As shown, there is no significant difference in cell activity between cells treated with different concentrations of veratridine and cells not treated with veratridine within a certain drug concentration range, which proves that veratridine at this concentration has no significant cytotoxicity.

[0056] Example 5 Determination of the activity of lycopodii extract and lycopodine in inhibiting virus (ASFV)

[0057] 8×10 5 PAM cells were plated in six-well plates at the amount of cells / well. After 12 hours, fresh culture medium containing DMSO and different concentrations of drugs was replaced and cultured at 37°C for 2 hours. Then, culture medium containing the same concentration of drugs and 1MOI ASFV was replaced and cultured at 37°C for 1 hour. After the end, culture medium containing the same concentration of drugs was replaced and cultured at 37°C for another 24 hours. Cell samples were collected and subjected to Western-Blot experiments. The results are shown in Figure 4 and 6 middle.

[0058] like Figure 4 As shown, the expression of viral proteins in cells treated with different concentrations of Li Lu extract was significantly reduced compared with cells not treated with drugs, proving that Li Lu extract can significantly inhibit viral infection.

[0059] like Figure 6 As shown, the expression of viral proteins in cells treated with different concentrations of leucine was significantly reduced compared with cells not treated with the drug, proving that leucine can significantly inhibit viral infection.

[0060] Example 6 Veratridine and PI3K / Akt pathway specific inhibitor LY294002 inhibit macropinocytosis

[0061] Vero-E6 cells were cultured at 4 × 10 5100 cells / well were inoculated into a 6-well plate with a sterilized cell slide. After 12 hours, the culture medium was discarded, and the cells were rinsed three times with PBS. Then, veratridine (10 μM), PI3K / Akt pathway specific inhibitor LY294002 (10 μM), or LY294002+veratridine (10 μM) were added to 2 mL of incomplete culture medium (serum-free DMEM) for Dext (macropinocytosis pathway specific marker) endocytosis test. The cells were starved for 2 hours at 37°C to prevent serum from interfering with Dext endocytosis. After that, the culture medium was discarded and the cells were rinsed three times with PBS. 2 mL of 10 μg / mL Dext solution was added and cultured at 37°C for 1 hour. The culture medium was discarded, and the cells were rinsed three times with cold citrate buffer (pH 3) and three times with cold PBS to terminate their endocytosis and remove Dext from the culture medium and cell membrane surface. Then, the fixative was added and fixed at 37°C for 30 minutes. Then, the cells were rinsed twice with Glycine (0.02M) + PBS solution and washed three times with PBS, 5 min / time. Finally, the cell nuclei were stained with DAPI + DABCO solution and the slides were sealed, and indirect immunofluorescence detection was performed. The results are shown in Figure 7 middle.

[0062] like Figure 7 As shown, the fluorescence signal of Dext, a marker for macropinocytosis, completely disappeared in cells treated with 10 μM veratridine compared with cells not treated with the drug.

[0063] Example 7 is the inhibitory effect of veratridine on the PI3K / Akt pathway.

[0064] 8×10 5 Vero-E6 cells were plated in a six-well plate at a concentration of 10 cells / well. After 12 hours, Vero-E6 cells were treated with DMSO, 10 μM veratridine, 1 MOI HLJBY, and 1 MOI HLJBY + 10 μM veratridine, respectively. Cell samples were collected at 10, 20, and 30 minutes, respectively, and Western-Blot assay was performed. The results are shown in Figure 8 middle.

[0065] like Figure 8 The results show that the PI3K / Akt pathway can be significantly activated at the early stage of PEDV infection, i.e., 10, 20, and 30 minutes. The pAkt protein level in the 10 μM veratridine-treated group was decreased compared with the untreated group, indicating that veratridine significantly inhibited the activation of the PI3K / Akt pathway.

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

[0067] 8×10 5Vero-E6 cells were plated in six-well plates at a volume of 10 cells / well. After 12 hours, Vero-E6 cells were treated with DMSO, veracrucine (10 μM), PI3K / Akt pathway specific inhibitor LY294002 (10 μM) and LY294002 + veracrucine (10 μM), respectively, and cultured at 37°C for 2 hours. Then, fresh medium DMEM containing the same drug concentration and 1 MOI of PEDV was replaced, and after culturing at 37°C for 1 hour, DMEM with the same drug concentration was replaced. After continuing to culture at 37°C for 12 hours, samples were collected for plaque formation assay. The results are shown in Fig. 9 middle.

[0068] like Fig. 9 The results show that compared with cells not treated with drugs, cells treated with LY294002, veratridine, and LY294002+veratridine can significantly reduce the viral titer after treatment with LY294002 and veratridine, and the inhibitory effect of the group treated with veratridine and LY294002 on PEDV is the same as that of the group treated with drugs alone, and the co-treatment of the two will not produce further inhibitory effects on PEDV. The results show that veratridine 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: Also included are pharmaceutically acceptable carriers.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable carrier is selected from solid excipients, liquid or semisolid excipients.

4. The use 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, pills, granules or powders.

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