CN116139155A - Application of Reedrine and Reed Extract in the Preparation of Antiviral Drugs - Google Patents

Abstract

The invention belongs to new application of veratrine, and in particular relates to the application of the veratrine and the veratrine extract in preparing antiviral drugs. Wherein the Li Phragmitis has inhibiting effect on pig delta coronavirus, pig epidemic diarrhea virus, pseudorabies virus, herpes simplex virus, influenza virus or African swine fever virus. The veratrine extract and the veratrine of the invention have very remarkable antiviral effect. The veratrine extract has very remarkable effect of broad-spectrum antiviral, is safe, has little toxic and side effects, has low drug residue and has no pollution.

Description

Application of reedrine and reed extract in the preparation of antiviral drugs

technical field

The invention belongs to the new use of saliva, and in particular relates to the application of saliva and its extract in the preparation of antiviral drugs.

Background technique

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

Porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) belongs to the genus deltacoronavirus of the Coronaviridae family of the Neveviridae. The genome of PDCoV is the smallest among coronaviruses, about 25.4nt. The first 2/3 of the viral genome is responsible for encoding two large replicase precursor polyproteins pp1a and pp1ab, and the latter 1/3 mainly encodes four structural proteins and three auxiliary proteins, which are the spike protein (S), package Membrane protein (E), membrane protein (M), nucleocapsid protein (N) and accessory proteins NS6, NS7, NS7a. Since the first outbreak of PDCoV in the United States in 2014, it has spread rapidly to all parts of the world. The clinical severity, infection rate, and mortality rate of PDCoV are low, however, it has the potential risk of zoonosis. In 2021, researchers detected PDCoV in plasma samples from three Haitian children with acute undifferentiated febrile illness, the first time the virus had been identified in humans. Its rapid spread and potential cross-species spread pose a huge threat to animal and human health and safety. The pathogenic mechanism of PDCoV is not clear, lacks vaccines and specific drugs, and is limited to the stage of awareness in terms of diagnosis and prevention. Doing a good job in cleaning and disinfecting pig farms, strengthening feeding management, and improving biological safety are important contents of PDCoV prevention work.

Porcine Epidemic Diarrhea Virus (Porcine Epidemic Diarrhea Virus, PEDV) can cause severe lethal intestinal infectious diseases in piglets, with a fatality rate as high as 80%-100%. It is a key disease prevention and control in pig farms. PEDV belongs to αcoronavirus, which is a single-stranded positive-sense RNA virus with an envelope. pp1b) and an accessory protein ORF3. PEDV includes five subgroups, GI-a/b and GII-a/b/c. In the early days, GI strains were mainly prevalent, and now GII strains are the main ones. The clinically available commercial vaccines are mainly aimed at PEDV GI strains, and the GII mutant strains that have emerged after 2010 have continued to be popular for more than a decade, and have provided great support to the pig industries in China, the United States, Canada, Mexico, South Korea, Japan and other countries. Both caused heavy damage. Mutant strains break through the protection of existing vaccines, which is a major challenge for the prevention and control of epidemic 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 virus infection and provide a reference for the research of anti-coronavirus drugs.

Pseudorabies virus (Pseudorabies virus, PRV) can cause a highly lethal infectious disease. The main characteristics of PRV-infected piglets are lethargy, vomiting, extreme mental fatigue, trembling, incoordination, convulsions, and diarrhea. Adult pigs are generally recessively infected, and if they have symptoms, they are mild and easy to recover. The main manifestations are fever, depression, and some sick pigs vomit and cough. Pregnant sows can suffer from miscarriage, mummification or stillbirth. PRV belongs to αherpesvirus (alphaherpesvirus), and is classified into Herpesviridae, Herpesviridae, Alphaherpesvirinae, and Varicellavirus genus. The PRV virion is spherical, and consists of core, capsid, envelope and capsule from inside to outside. The genome is 145kb in full length and contains seven open reading frames, which can encode more than 100 proteins, including capsid protein, envelope protein, envelope protein and Early Protein 0 (EPO) and other proteins. At present, the prevention and control of pseudorabies mainly relies on vaccine immunization. The use of gI/gE gene-deleted attenuated vaccine has effectively controlled and gradually purified pseudorabies in my country. However, since 2011, pseudorabies outbreaks have occurred in vaccinated pig herds in many places in my country, and the positive rate of PRV nucleic acid in the investigated pig herds nationwide is about 10%, which has caused great economic losses to the domestic pig breeding industry. It is worth noting that since 2018, there have been more than 20 cases of pseudorabies virus infection in my country, which has posed a huge threat to the health and safety of animals and humans.

Herpes simplex virus (HSV-1) is a common pathogen that endangers human health. It is found through serum antibody detection that the positive rate in normal asymptomatic individuals is over 80%. After HSV-1 infects the human body through the skin or mucous membranes, it often hides in the sensory neurons, causing latent infection. In the case of low immunity, it will cause clinical symptoms. Mainly cause herpes around the lips and mouth and nose, and occasionally cause eye rash. Wounds that rarely occur near the genitals but may infect the skin, such as on the fingers, are called impetigo herpetiformis. HSV-1 is a double-stranded linear DNA virus with a full-length genome of 125-240kb. It is a circular virus composed of envelope, capsid, core and envelope. At present, the most effective drug for treating HSV-1 infection is acyclovir and its derivatives. However, acyclovir can only temporarily control HSV-1 infection and cannot completely remove the virus in the body. And currently there is no effective vaccine to prevent the infection of HSV-1. The prevention is mainly to cut off the transmission route, try to avoid close contact with patients, avoid the stimulation of harmful factors to the body, and actively exercise to improve the body's immunity. At present, there is no cure, only clinical symptoms can be alleviated.

Influenza virus (IAV) can cause a seasonal infectious disease with fever, cough, sore throat, headache, body aches, chills and fatigue, or even diarrhea or vomiting, muscle pain or tiredness, and red eyes. The condition of some patients can progress rapidly, with fierce onset, sudden high fever, and body temperature exceeding 39°C, followed by severe pneumonia, acute respiratory distress syndrome, pulmonary hemorrhage, pleural effusion, pancytopenia, renal failure, sepsis, shock, and Wright's Syndrome (Reye's syndrome), respiratory failure and multiple organ damage, and even death. According to statistics, seasonal influenza pandemics cause approximately 3 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 mutated strains. Influenza pandemic viruses can spread rapidly among people, causing widespread epidemics around the world. For example, the 1918 H1N1 influenza pandemic virus caused more than 50 million deaths in two years. Influenza viruses continue to evolve and mutate, producing new viruses that threaten the health of animals and humans. Therefore, the development of effective vaccines and antiviral drugs is an ongoing challenge.

Due to the rapid mutation, rapid pathogenicity and high mortality of the above-mentioned viruses, and the limited or poor therapeutic effects of existing drugs, it has become a technical problem in the art to find new drugs with significant therapeutic effects.

Liregonine is the main alkaloid of Veratrum nigrum L., the chemical name is (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 has the following molecular structure:

In the prior art, reedrine has the activity of lowering blood pressure and inhibiting tumors. But in the prior art, there is no report that reedrine is used as an antiviral drug. The object of the present invention is to provide a broad-spectrum antiviral drug composition, as well as the extraction of salivaline from Lilu and the application of salivaline or a pharmaceutically acceptable salt thereof in the preparation of broad-spectrum antiviral drugs.

Contents of the invention

The invention provides the extraction of the reed reed and the application of the reed extract, the reedrine or a pharmaceutically acceptable salt thereof in the preparation of broad-spectrum antiviral drugs.

The virus is porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV), porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV), pseudorabies virus (Pseudorabies virus, PRV), herpes simplex virus (Herpes simplex virus, HSV) And influenza virus (Influenza virus), African swine fever virus (African swine fever virus, ASFV).

Optionally, the medicament further includes a pharmaceutically acceptable carrier, excipient or combination thereof.

Optionally, the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate, maleate, tartrate, Citrate, Succinate, Malonate, Adipate, Alginate, Ascorbate, Aspartate, Besylate, Benzoate, Bisulfate, Borate, Butyl salt, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptose salt, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, Lauryl Sulfate, Malate, Malonate, Methanesulfonate, 2-Naphthalenesulfonate, Nicotinate, Nitrate, Oleate, Palmitate, Pectate, Persulfate, Group consisting of 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate.

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

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, monostearate Glycerides, Sodium Chloride, Skimmed Milk Powder or combinations thereof.

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

Optionally, the dosage form of the drug is selected from tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, drop pills, granules, powders or combinations thereof Group.

The present invention also provides a broad-spectrum antiviral pharmaceutical composition, including the extract of Liludine, its hydrate, or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers and excipients or a combination thereof.

The "pharmaceutically acceptable salt" of the present invention refers to the organic and inorganic salts of pilodrine of the present invention, including but not limited to, inorganic acid salts formed by reacting 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, besylate, benzoate, bisulfate, borate, butyrate, camphor salt, camphorsulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerin Phosphate, Gluconate, Hemisulfate, Heptanoate, Hexanoate, Hydroiodide, 2-Hydroxy-ethanesulfonate, Lactobionate, Lactate, Laurate, Lauryl Sulfate , malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pectate, persulfate, 3-phenyl Propionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

In order to prepare the pharmaceutical composition of the present invention, the extract of the reed reed, the reedrine or a pharmaceutically acceptable salt can be mixed with a pharmaceutically acceptable carrier and/or excipient. Pharmaceutically acceptable carriers that can be used in this composition encompass any standard pharmaceutical carrier such as phosphate buffered saline, water and emulsions such as oil/water or water/oil emulsions, aqueous dextrose, glycols, and various types of moisturisers. agent.

The pharmaceutical composition of the present invention may additionally comprise solid excipients, liquid excipients and semi-solid excipients. The solid excipient may be selected from starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, chloride Sodium chloride, skimmed milk powder, etc. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical compositions of the present invention may also include stabilizers and preservatives.

The compositions may be administered by any method known in the art, including but not limited to intranasal, oral, transdermal, ophthalmic, intraperitoneal, inhalation, intravenous, intracisternal injection or infusion, subcutaneous, implant, vaginal Intra, sublingual, urethral, subcutaneous, intramuscular, intravenous, rectal, sublingual, mucosal, ophthalmic, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial, and lymphatic administration. Topical formulations can be in the form of gels, ointments, creams, aerosols, etc.; intranasal formulations can be delivered as a spray or droplets; transdermal formulations can be administered via a transdermal patch or iontophoresis; inhalation formulations can be administered using a nebulizer or similar device delivery. The composition can also be in the form of tablet, capsule, suspension, solution, emulsion, injection, ointment, gel, film, drop pill, granule or powder or any other suitable composition.

Beneficial effect

1. The embodiment of the present invention proves that reedrine does not have cytotoxicity, and does not produce its effect through cytotoxicity. After the cells were treated with different concentrations of reedrine, the expression of viral protein was significantly reduced, which proved that reedrine can significantly inhibit the infection of the virus. Phyllodrine or Phyllodrine extract can inhibit porcine epidemic diarrhea virus (Porcine Epidemic DiarrheaVirus, PEDV), pseudorabies virus (Pseudorabies virus, PRV), herpes simplex virus by inhibiting macropinocytosis and by inhibiting PI3K/Akt pathway. (Herpes simplex virus, HSV-1) and influenza virus (Influenza virus, IAV), porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV), African swine fever virus (African swine fever virus, ASFV) have antiviral effect, need to explain It is the above-mentioned virus that adopts general antiviral drug treatment effect is not obvious or invalid. The present invention finds that saliva has a new antiviral mechanism, and the saliva extract of the present invention and saliva have a very significant antiviral effect on viruses (PEDV, PRV, HSV, IAV) at a concentration of 10 μM, and can exhibit extremely high antiviral effects. Strong antiviral infection effect. The reed extract and reedrine of the present invention have a very significant antiviral effect on ASFV at a concentration of 5 μM, and can exhibit a very strong anti-African swine fever virus infection effect.

2. The reed extract and reedrine of the present invention have low residues of antiviral drugs, no pollution, are easily absorbed by animal bodies, have high biological metabolic rate, and are excreted without pollution.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following will briefly introduce the accompanying drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore are It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is the cytotoxicity of reedrine, wherein A is PK15, and B is Vero-E6.

Figure 2 shows the inhibitory effect of Lilu extract on viruses, A is PK15+PDCov; B is Vero-E6+HLJBY, C is Vero-E6+PRV, D is Vero-E6+HSV, E is Vero-E6+IAV .

Figure 3 shows the inhibitory effect of reedrine 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.

Figure 4 is the inhibitory effect of Li reed extract on ASFV.

Fig. 5 is the cytotoxicity of reedrine.

Fig. 6 is the inhibitory effect of pilodrine on ASFV.

Figure 7 shows the inhibitory effect of reedrine and LY294002, a specific inhibitor of the PI3K/Akt pathway, on macropinocytosis.

Fig. 8 shows the inhibitory effect of reedrine on PI3K/Akt pathway.

Fig. 9 shows that the inhibitory effect of reedrine on porcine epidemic diarrhea virus depends on the PI3K/Akt pathway.

Detailed ways

Below in conjunction with experiments and examples, the claims of the present disclosure will be further described in detail, but this does not constitute any limitation to the present disclosure. within the scope of protection. Those who do not indicate specific conditions in the implementation manner shall be carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Unless otherwise defined herein, scientific and technical terms used in connection 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.

experiment material

Porcine Epidemic Diarrhea Virus (PEDV), Pseudorabies virus (PRV), Herpes simplex virus (HSV-1) and Influenza virus (IAV), porcine deltacoronavirus Virus (Porcine deltacoronavirus, PDCoV), African swine fever virus (African swine fever virus, ASFV) and pig kidney cells (PK15), African green monkey kidney cells (Vero-E6) cells, alveolar macrophages (PAM) are all source In the National African Swine Fever Reference Laboratory of the China Center for Animal Health and Epidemiology (Qingdao, Shandong).

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

Embodiment 1 Li reed extract preparation

Grind 100g of the dried whole herb of Veratrum, a Chinese herbal medicine, pass through an 80-mesh sieve, add 80% ethanol solution according to the ratio of solid to liquid (1:5 (g:ml), and extract by ultrasonic (100W, 50°C) 3 times, each time for 30min , filtered, concentrated and dried under reduced pressure, and freeze-dried to obtain 5.8g of veratrum extract.

The cytotoxicity of embodiment 2 reedrine

PK15 (A) or Vero-E6 cells (B) were plated into 96-well plates at an amount of 2×10 ⁴ cells/well. After 12 hours, the culture medium was replaced with fresh medium containing different concentrations of reedline, and cultured at 37°C for 48 hours (PK15) or 24 hours (Vero-E6). CCK-8 solution was added in an amount of 10 μL/well, and culture was continued for 3 h in the dark. The value of _OD450 was then read and the cell viability was calculated. Three repetitions were set up for each group of experiments, and the graphs are shown as mean (Mean) ± standard deviation (SD). The results are shown in Figure 2.

As shown in Figure 2, the cells treated with different concentrations of pilostatine were compared with the cells without pilostatine treatment, and there was no significant difference in the cell viability within a certain range of drug concentrations, which proved that the concentration of pilostatine did not have Significant cytotoxicity.

Example 3 The inhibitory effect of Li reed extract and Li reedline on viruses (PDCoV, PEDV, PRV, HSV, IAV)

PK15 (A) or Vero-E6 (BE) cells were plated into six-well plates at an amount of 8×10 ⁵ cells/well. After 12 hours, replace with fresh medium containing DMSO and different concentrations of drugs, and incubate at 37°C for 2 hours. Then replace with the medium containing the same concentration of drug and 1MOI of PDCoV (A), PEDV (B), PRV (C), HSV (D), and IAV (E), and culture at 37°C for 1h. After the end, replace with the medium containing the same concentration of drugs, and continue to culture at 37°C for 18h (PDCoV) or 12h (PEDV, PRV, HSV, IAV) and collect cell samples for Western-Blot test. The results are shown in Figures 1 and 3, respectively.

As shown in Figure 2, compared with cells without drug treatment, the expression of viral proteins in cells treated with different concentrations of Li reed extracts was significantly reduced, which proved that Li reed extracts can significantly inhibit virus infection.

As shown in FIG. 3 , compared with cells without drug treatment, the expression of viral proteins in cells treated with different concentrations of sitrudline was significantly lower, which proves that sitrudline can significantly inhibit virus infection.

The cytotoxicity of embodiment 4 reedrine

Plate PAM cells into 96-well plates at an amount of 2× ¹⁰⁴ cells/well. After 12 hours, they were replaced with fresh medium containing different concentrations of reedline, and cultured at 37°C for 24 hours. Subsequently, CCK-8 solution was added in an amount of 10 μL/well, and culture was continued for 3 h in the dark, and the value of OD ₄₅₀ was read, and the cell viability was calculated. Three repetitions were set up for each group of experiments, and the graphs are shown as mean (Mean) ± standard deviation (SD). The results are shown in FIG. 5 .

As shown in Figure 5, compared with cells treated with different concentrations of pilostatine, there was no significant difference in cell viability within a certain drug concentration range, which proved that pilostatine at this concentration did not have Significant cytotoxicity.

Example 5 Determination of the Activity of Reed Extract and Reedrine Inhibiting Virus (ASFV)

PAM cells were plated into six-well plates at an amount of 8× ¹⁰⁵ cells/well. After 12 hours, replace with fresh medium containing DMSO and different concentrations of drugs, and incubate at 37°C for 2 hours. Then replace with the medium containing the same concentration of drug and 1MOI ASFV, and culture at 37°C for 1h. After the end, the culture medium containing the same concentration of drug was replaced, and the culture was continued at 37°C for 24 hours, and then the cell samples were collected for Western-Blot test. The results are shown in Figures 4 and 6, respectively.

As shown in Figure 4, compared with cells without drug treatment, the expression of viral proteins in cells treated with different concentrations of Li reed extracts was significantly reduced, which proves that Li reed extracts can significantly inhibit virus infection.

As shown in FIG. 6 , compared with cells without drug treatment, the expression of viral proteins in the cells treated with different concentrations of sitrudine was significantly lower, which proves that sitrudine can significantly inhibit the infection of the virus.

Example 6 Inhibition of macropinocytosis by reedrine and LY294002, a specific inhibitor of PI3K/Akt pathway

Vero-E6 cells were inoculated into 6-well plates with sterilized cell slides at 4× ¹⁰⁵ /well. After 12 hours, discard the medium, rinse with PBS three times, and then add reedrine (10 μM), or PI3K/Akt pathway-specific inhibitor LY294002 (10 μM), or LY294002 + redrine (10 μM) in 2 mL of incomplete medium ( Serum-free DMEM) was used for Dext (macropinocytosis pathway-specific marker) cell entry test, and starvation treatment was performed at 37°C for 2 hours to prevent serum from interfering with the endocytosis of Dext. Afterwards, the culture medium was discarded and rinsed three times with PBS. Add 2 mL of 10 μg/mL Dext solution and incubate at 37 °C for 1 h. The medium was discarded, rinsed 3 times with cold citrate buffer (pH 3) and rinsed 3 times with cold PBS to terminate its entry into the cells and remove the Dext in the medium and the surface of the cell membrane. Then add fixative and fix at 37°C for 30min. Then rinse twice with Glycine (0.02M)+PBS solution, and wash three times with PBS, 5min each time. Finally, the nuclei were stained with DAPI+DABCO solution and mounted, and finally indirect immunofluorescence detection was performed. The results are shown in FIG. 7 .

As shown in FIG. 7 , the fluorescent signal of Dext, a marker of macropinocytosis pathway, completely disappeared after treatment with 10 μM reedrine compared with cells without drug treatment.

Example 7 is the inhibitory effect of reedrine on PI3K/Akt pathway.

Spread Vero-E6 cells into six-well plates at an amount of 8× ¹⁰⁵ cells/well. After 12 hours, Vero-E6 cells were treated with DMSO, 10 μM salivaline, 1MOI HLJBY and 1MOI HLJBY+10 μM salivaline, and cell samples were collected at 10, 20 and 30 minutes, respectively, and Western-Blot test was performed. The results are shown in FIG. 8 .

As shown in FIG. 8 , the PI3K/Akt pathway can be obviously activated at the early stage of PEDV infection, that is, 10, 20 and 30 minutes. Compared with the untreated group, the level of pAkt protein decreased in the 10 μM sitrudline treatment group, which indicated that sichuanline significantly inhibited the activation of the PI3K/Akt pathway.

Example 8 The inhibitory effect of reedrine on porcine epidemic diarrhea virus depends on the PI3K/Akt pathway.

Spread Vero-E6 cells into six-well plates at an amount of 8× ¹⁰⁵ cells/well. After 12 hours, Vero-E6 cells were treated with DMSO, reedrine (10 μM), PI3K/Akt pathway-specific inhibitor LY294002 (10 μM) and LY294002+redrine (10 μM), respectively, and incubated at 37°C for 2 hours. Then it was replaced with fresh medium DMEM containing PEDV at the same drug concentration and 1 MOI, and after 1 hour of incubation at 37°C, it was replaced with DMEM at the same drug concentration. The results are shown in Fig. 9, respectively.

As shown in Figure 9, the cells treated with LY294002, lemtronine and LY294002 + lemtronine were compared with the cells without drug treatment, and the virus titer could be significantly reduced by LY294002 and lemtronine treatment, and lemtronine and LY294002 The inhibitory effect of the co-treatment group on PEDV is the same as that of the drug treatment group alone, and the co-treatment of the two will not produce further inhibitory effects on PEDV. The results showed that reedrine inhibited PEDV by inhibiting the PI3K/Akt pathway.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. The application of reedrine and pharmaceutically acceptable salts thereof in the preparation of antiviral drugs. 2. The application of Li reed extract in the preparation of antiviral drugs, wherein the Li reed extract is prepared by the following steps: Grind 100g of the dried whole herb of Veratrum, a traditional Chinese medicinal material, pass through a 80-mesh sieve, add 80% ethanol solution according to the ratio of 1:5g:ml to liquid, 100W, 50°C ultrasonic extraction 3 times, 30min each time, filter, reduce pressure Concentrate and dry, and freeze-dry to obtain the extract of veratrum. 3. The application according to claim 1 or 2, wherein the virus is porcine delta coronavirus, porcine epidemic diarrhea virus, pseudorabies virus, herpes simplex virus, influenza virus or African swine fever virus. 4. The application according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier. 5. The application according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate Acid, maleate, tartrate, citrate, succinate, malonate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoic acid Salt, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate , fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturaldehyde salt, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitic acid Salt, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecyl salt or valerate. 6. The use according to claim 4, wherein the pharmaceutically acceptable carrier is selected from solid excipients or liquid and semisolid excipients. 7. application according to claim 1, is characterized in that, the dosage form of described medicine is selected tablet, capsule, suspension, solution, emulsion, injection, ointment, gel, film, drop pill , granules or powders.

Images