CN113082080B - Application of illicium plants or extracts thereof in preparation of anti-animal virus drugs - Google Patents

Abstract

The present invention relates to the application of the star anise plant or its extract in the preparation of anti-animal virus medicine, the said star anise plant is red poisonous fennel (I.lanceolatum ACSmith), red fennel (I.henryi Diels) or false maple bark (Illicium jiadifengpi BNChang). The plant extract of the genus Anise in the present invention can effectively and directly kill animal viruses in vitro, block the entry of animal viruses into cells, inhibit the replication of animal viruses in cells and play an antiviral effect. At the same time, it can inhibit the apoptosis of host cells induced by animal viruses, and inhibit the inflammatory response of host cells induced by animal viruses through the NF-κB and MAPKs pathways, thereby protecting host cells. It has significant preventive and therapeutic effects on animal virus-infected mice, can significantly reduce the viral load of mouse brain tissue, down-regulate the expression level of inflammatory factor genes, inhibit cytokine storm, alleviate the pathological changes of brain tissue, and improve the efficacy of animal virus-infected mice. survival rate, and prolong the survival time of animal virus-infected mice.

Description

Use of star anise plants or their extracts in the preparation of anti-animal virus drugs

technical field

Anti-animal virus effects and uses of plants of the genus Illicium and their extracts, characterized in that the plants of the genus Illicium include Illicium lanceolatum A.C.Smith, red fennel (I.henryi Diels) or False maple bark ( I. jiadifengpi B.N. Chang).

Background technique

With the intensification and large-scale development of livestock and poultry, the illegal use and abuse of vaccines and antibiotics in the breeding process will cause mutations in pathogens, the body is in a sub-healthy state, immune function is reduced, disease resistance is reduced, and new and re-emerged infectious diseases The outbreak and prevalence of the disease not only severely restrict the healthy development of the breeding industry, but also the cross-species transmission of some zoonotic disease pathogens poses a huge challenge to global public health.

At present, comprehensive prevention and control measures of immune prevention and drug prevention and control are generally adopted for animal viral diseases. Virus species and serotypes are complex and prone to mutation, and some viruses can also cause immune suppression in the body, which brings difficulties to the development and application of vaccines.

Viruses have no cell structure, only nucleic acid, mutate quickly, and are strictly intracellular parasitism. An ideal antiviral drug must selectively destroy or inhibit intracellular viruses without toxicity to uninfected cells. Clinically, there is no specific drug for viral diseases, and synthetic chemical antiviral drugs have high toxicity and side effects, are prone to drug resistance, and have very limited clinical efficacy. Since 2005, antiviral drugs such as amantadine, rimantadine, ribavirin, and acyclovir have been completely prohibited from being used in edible animals in China.

As a traditional medicine in my country, traditional Chinese medicine has been applied for more than 2,000 years. It has formed a complete theory of Wei Qi Ying Xue dialectics in the prevention and treatment of "plague", and it should play an important role in the prevention and control of epidemic diseases. The treatment of viral diseases by traditional Chinese medicine not only focuses on the direct antiviral effect, but also pays more attention to the relationship between "virus-body-Chinese medicine". Traditional Chinese medicine can not only directly kill the virus, prevent the adsorption, penetration, shelling, biosynthesis, assembly and release of the virus, but also enhance the immune function of the body, regulate the intestinal flora and its metabolites, indirectly play an antiviral role, and It can inhibit cytokine storm and improve the symptoms of body discomfort caused by virus infection. This multi-channel, multi-link antiviral comprehensive effect of traditional Chinese medicine can ensure a temporary and permanent cure, and prevent disease recurrence. At present, animal viral diseases mostly present atypical viral diseases, bacterial diseases, mixed infections, and secondary infections, and the advantages of traditional Chinese medicine can be fully utilized in areas where western medicine is weak. Traditional Chinese medicine has the advantages of pleiotropic effects, less toxic and side effects, no residue, less drug resistance, and a wide range of anti-virus, which is conducive to the prevention and control of animal viral diseases, reducing the use of antibiotics, ensuring the healthy breeding of livestock and poultry, and providing safe and high-quality livestock and poultry products. Therefore, the research and development of antiviral Chinese veterinary medicine is placed high expectations.

Illicium Linn. is the only genus in the family Illiciaceae, mainly distributed in eastern and southeastern Asia. This genus has a long history of medicinal use, which was recorded in the "Compendium of Materia Medica" in the Ming Dynasty. In traditional medicine, the roots, bark, bark, leaves, and fruits of these plants are used as medicines. They are mainly used to treat rheumatoid arthritis, bruises, abdominal distension, vomiting, and traumatic bleeding. They have certain medical value and broad development prospects. a group of . The plants of this genus mainly contain sesquiterpene lactones, flavonoids, lignans, phenolic acids, volatile oils and other components. Among them, sesquiterpene lactones are the characteristic components of this genus.

I.lanceolatum A.C.Smith, also known as lanceolatum and mangcao, is produced in southern Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, southeastern Henan, eastern Hubei, southern to eastern Hunan, and northern to eastern Guangdong. In moist evergreen broad-leaved forests such as hills or mountain valleys, streamsides, and streams at an altitude of 100-1600m. The roots, bark, and leaves are used as medicine, with pungent taste and warm nature; it has the effects of dispelling wind and dredging collaterals, relaxing tendons and promoting blood circulation, dissipating blood stasis and relieving pain, and can cure bruises, lumbar muscle strain, rheumatic arthralgia, carbuncle and swollen poison; leaf research Thin, oil-adjusted external application, can cure traumatic bleeding.

Red fennel I.henryi Diels, unique to China, is produced in southern Shaanxi, southern Gansu, western to southern Henan, western Hubei, northwestern to western Hunan, northern to eastern Guizhou, eastern to southeastern Chongqing and Sichuan, grown at an altitude of 300~ 2200m hills or mountain valleys, moist evergreen broad-leaved forests or forest margins by streams. The root and root bark are used as medicine, pungent in taste and warm in nature; it has the effects of promoting blood circulation and relieving pain, expelling wind and dehumidification, and can cure bruises, chest and abdomen pain, and wind-cold-damp arthralgia.

False ground maple skin I.jiadifengpi B.N.Chang, endemic to China, produced in southern Anhui, southwestern Zhejiang, Jiangxi, Fujian, southeastern Hubei, southern to eastern Hunan, Guangdong, Hong Kong and Guangxi, born in hills or hills at an altitude of 400-2100m In mountain valleys, ridges or hillsides in moist evergreen broad-leaved forests. The root bark is used as medicine, and externally used to treat rheumatic bone pain and bruises.

Red fennel injection is a crimson clear liquid obtained by extracting, refining, filtering and sterilizing the root bark or stem bark of I.lanceolatum A.C.Smith through alcohol extraction process. The pH value should be 6.8-8.5. , promoting blood circulation and relieving pain. It has been widely used clinically in the treatment of chronic low back pain, lumbar muscle strain, lumbar disc herniation, rheumatic joints, sciatica, frozen shoulder and other diseases, and has high safety.

Liu Jifeng tested the effect of red fennel (I.henryi Diels.) and wild star anise (I.simonsii Maxim.) compounds on hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) by using the liver cancer HepG2. ) secretion inhibition, anti-hepatitis B virus activity research, found that tashironin and tashironin A have better inhibitory effect on the secretion of HBsAg and HBeAg. However, this activity is an inhibitory effect on the proliferation of liver cancer cells, not an antiviral effect in the true sense.

The plant extract of the genus Anise in the present invention can effectively and directly kill animal viruses in vitro, block the entry of animal viruses into cells, inhibit the replication of animal viruses in cells and play an antiviral effect. At the same time, it can inhibit the apoptosis of host cells induced by animal viruses, and inhibit the inflammatory response of host cells induced by animal viruses through the NF-κB and MAPKs pathways, thereby protecting host cells. Anise plant extracts have significant preventive and therapeutic effects on mice infected with animal viruses, can significantly reduce the viral load of mouse brain tissue, down-regulate the expression level of inflammatory factor genes, inhibit cytokine storm, alleviate pathological changes in brain tissue, and improve Survival of animal virus-infected mice, and prolonging the survival time of animal virus-infected mice.

Contents of the invention

The primary purpose of the present invention is to provide a new class of medicines with anti-virus effects in animals. Specifically, the present invention includes plants of the genus Illicium lanceolatum A.C.Smith, red fennel (I.henryi Diels) or Water extract, alcohol extract or steam distillation volatile oil of maple bark (I.jiadifengpi B.N.Chang).

The second object of the present invention is to provide a preparation method of the above-mentioned star anise plant extract.

A further object of the present invention is to provide a pharmaceutical composition as an anti-animal virus.

Another object of the present invention is to provide the application of the above-mentioned star anise plant extract and composition in the prevention and treatment of animal viral diseases.

The star anise plant with anti-animal virus effect of the present invention includes red poisonous fennel (I.lanceolatum A.C.Smith), red fennel (I.henryi Diels) or false maple bark (Illicium jiadifengpi B.N.Chang).

The plants of the genus Anise in the present invention include the fresh or dry products of the above three plants; the medicinal parts can be the whole plant, root, root bark, stem, stem bark or any combination; the preferred medicinal parts are roots and root bark , stem, stem bark or any combination; the more preferred medicinal part is root bark or stem bark; the most preferred medicinal part is root bark.

The preparation method of the star anise plant extract of the present invention comprises the following steps:

a. Anise plants are added to solvent extraction;

b. filter the extract, combine the filtrates, and concentrate;

c. to dry.

The extraction method described in the above step a is a decoction method, a dipping method, a percolation method, a reflux method, a steam distillation method, and an ultrasonic extraction method.

The extraction solvent described in the above step a is water, 10%-95% ethanol aqueous solution or absolute ethanol.

The amount of the extraction solvent described in the above step a is 2 to 50 times that of the original medicinal material.

The extraction temperature described in the above step a is 0-100°C.

The number of extractions described in the above step a is 1 to 5 times.

The extraction time described in the above step a is 0.5-5 hours each time.

The concentration temperature described in the above step b is 25-100°C.

The relative density of the concentrate described in the above step a is 0.9-1.5.

The drying method described in the above step a includes normal pressure drying, reduced pressure drying, freeze drying or spray drying.

The anti-animal virus pharmaceutical composition of the present invention contains preventive or therapeutic effective dose of star anise plant extract and pharmaceutically acceptable carrier.

The effective dose of the star anise plant extract of the present invention is 0.01μg/kg-10g/kg body weight.

The star anise plant extract and the pharmaceutical composition of the invention can be used for preventing and treating animal viral diseases.

The pharmaceutically acceptable carrier mentioned above refers to the drug carrier in the field of pharmacy. For example: diluents, excipients such as water, saline, dextrose, mannitol, glycerin, ethanol and their mixtures, etc.; fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin and polystyrene Vinylpyrrolidone; humectants such as glycerin; disintegrants such as calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as Tween-80; lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycol etc. In addition, other auxiliary materials such as flavoring agents and sweetening agents can also be added to the composition.

The compounds of the present invention may be administered orally, nasally, rectally, parenterally or transdermally in the form of compositions to patients in need of such treatment or subjects in need of vaccination. For oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, pills, sustained-release pellets, solid dispersions, inclusions, etc., and prepared liquid preparations such as suspensions , emulsion, sol, syrup, mixture, solution, etc.; for parenteral administration, it can be made into solution for injection, water or oily suspension, emulsion, lyophilized powder, liposome, Microcapsules, microspheres, nanocapsules, nanospheres, etc. Preferred forms are tablets, coated tablets, capsules, pellets, suppositories, and injections, with site-specific targeted release formulations being particularly preferred.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the field of pharmacy. For example, the active ingredient is mixed with one or more carriers and brought into the desired dosage form.

The pharmaceutical composition of the present invention preferably contains the star anise plant extract in a total weight ratio of 0.01% to 99.9%. It is most preferred to contain the star anise plant extract in a total weight ratio of 0.5% to 95%.

The dosage of the star anise plant extract of the present invention can be based on various factors such as animal species, virus species, prevention/treatment purposes, etc., and its daily dosage can be 0.01 μ g/kg～10 g/kg body weight, preferably 0.1 μ g/kg～1g /kg body weight. Can be used one or more times.

The plant extract of the genus Anise in the present invention can effectively and directly kill animal viruses in vitro, block the entry of animal viruses into cells, inhibit the replication of animal viruses in cells and play an antiviral effect. At the same time, it can inhibit the apoptosis of host cells induced by animal viruses, and inhibit the inflammatory response of host cells induced by animal viruses through the NF-κB and MAPKs pathways, thereby protecting host cells. Anise plant extracts have significant preventive and therapeutic effects on mice infected with animal viruses, can significantly reduce the viral load of mouse brain tissue, down-regulate the expression level of inflammatory factor genes, inhibit cytokine storm, alleviate pathological changes in brain tissue, and improve Survival of animal virus-infected mice, and prolonging the survival time of animal virus-infected mice. Therefore, the plant extracts of the genus Anise have the potential value of developing new veterinary drugs against animal viruses.

Description of drawings

Figure 1 is the effect on the apoptosis and necrosis of Vero cells induced by pseudorabies virus (PRV).

Figure 2 is the effect on the apoptosis and necrosis of Marc-145 cells induced by porcine reproductive and respiratory syndrome virus (PRRSV).

Fig. 3 is the effect on PRV gD gene expression level of Vero cells infected by pseudorabies virus (PRV).

Fig. 4 shows the effect on the expression level of PRRSV N gene in Marc-145 cells infected by porcine reproductive and respiratory syndrome virus (PRRSV).

Fig. 5 is the impact on the number of virus-positive cells in Vero cells infected by pseudorabies virus (PRV).

Fig. 6 is the effect on the viral protein expression level of Vero cells infected by pseudorabies virus (PRV).

Fig. 7 is the effect on the positive cell number and expression level of PRRSV N protein in Marc-145 cells infected by porcine reproductive and respiratory syndrome virus (PRRSV).

Figure 8 is the effect on the mRNA expression level of inflammatory factors in BV2 cells infected by pseudorabies virus (PRV).

Figure 9 shows the effect on the phosphorylation levels of MAPK and NF-κB proteins in BV2 cells infected with pseudorabies virus (PRV).

Figure 10 is the preventive effect of intramuscular injection of HHXI on mice infected with pseudorabies virus (PRV).

Figure 11 is the therapeutic effect of intramuscular injection of HHXI on mice infected with pseudorabies virus (PRV).

Figure 12 is the preventive effect of EEIH intragastric administration on mice infected with pseudorabies virus (PRV).

Fig. 13 is the therapeutic effect of intragastric administration of EEIH on mice infected with pseudorabies virus (PRV).

Figure 14 is the effect of intramuscular injection of HHXI on the expression level of gD gene in brain tissue of mice infected with pseudorabies virus (PRV).

Figure 15 is the effect of HHXI intramuscular injection on the expression levels of inflammatory factor genes in the brain tissue of mice infected with pseudorabies virus (PRV).

Detailed ways

The present invention is further illustrated by examples below, without limiting its scope.

Embodiment 1: preparation of star anise plant extract

Take medicinal materials, pulverize them, put them in a round bottom flask, and add 75% ethanol to reflux for extraction. For the first time, 10-12 times of 75% ethanol was refluxed for 2 hours; for the second time, 8-10 times of 75% ethanol was extracted for 2 hours. The extract was filtered, and the filtrates were combined. Use a rotary evaporator to reclaim ethanol under reduced pressure in a 45°C water bath until there is no alcohol smell, and continue to concentrate and freeze-dry to obtain the ethanol extract EEIL (yield 21.51%) and ethanol alcohol extract EEIH (yield 9.16%) ) and EEIJ (yield 13.78%)

Embodiment 2: preparation of red fennel injection

Get the ethanol extract (equivalent to 50g of the original drug), add 800ml water for injection and 20ml polysorbate 80, stir well, adjust the pH value to about 9.7 with 10% sodium hydroxide solution, add water for injection to 1000ml, Stir well, filter, pot, and sterilize to obtain red fennel injection (HHXI). Each ml contains 50mg of the original drug of red poisonous fennel.

Embodiment 3: In vitro to the killing effect of pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV)

Taking the maximum safe concentration (maximum no-cytotoxic concentration, MNTC) for African green monkey kidney Vero cells and African green monkey embryonic kidney Marc-145 cells as the initial concentration, after doubling dilution with DMEM culture medium, the drug dilution Mix with equal volumes of 200TCID ₅₀ PRV or PRRSV virus dilution, and incubate for 2 hours at 37°C, 5% CO ₂ incubator. Add 100 μL per well to a 96-well plate that has grown into a monolayer of Vero or Marc-145 cells after 24 hours of adherent culture, with 8 replicate wells. At the same time, set blank control, normal cell control and virus control. Cultivate in a 37°C, 5% CO ₂ incubator, shaking once every 30 minutes. After incubation for 2 hours, the virus and drug mixture in the wells were discarded, 100 μL of maintenance solution was added to each well, and cultured in a 37° C., 5% CO ₂ incubator for 72 hours, and the cytopathic changes (CPE) were observed every day. 4 hours before the end of the culture, 50 μL of 2 mg/mL MTT solution was added to each well, and the shaker was shaken until the crystals were completely dissolved. Use a microplate reader to measure the OD value at a wavelength of 490nm, and calculate the killing rate of the virus according to the following formula.

Killing rate (Killing rate, %)=(OD value of test group-OD value of virus control group)/(OD value of cell control group-OD value of virus control group)/×100%.

EEIL, EEIH and EEIJ all have a certain killing effect on PRV (see Table 1), among which EEIL has the best anti-PRV effect in vitro. EEIL also has a significant killing effect on PRRSV. Because EEIL can significantly promote the proliferation of Marc-145 cells, the killing effect on PRRSV exceeds 100%, and the killing rate of PRRSV at a concentration of 0.10 mg/mL still reaches 98.99% (see Table 2).

Table 1 In vitro killing effect on pseudorabies virus (PRV) (killing rate, %)

Table 2 Killing effect of EEIL on porcine reproductive and respiratory syndrome virus (PRRSV) in vitro

Example 4: In vitro to the blocking effect of pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) adsorption cells

Take the MNTC for Vero cells and Marc-145 cells as the initial concentration, make doubling dilution with DMEM culture medium, add 100 μL per well to the 96-well plate of adherent culture for 24 hours, and grow into a single layer of Vero or Marc-145 cells Above, 8 replicate wells. At the same time, set blank control, normal cell control and virus control. Incubate for 2 hours at 37°C in a 5% CO ₂ incubator, and discard the drug solution. Except for the cell control, add 100 μL of 100 TCID ₅₀ PRV or PRRSV virus dilution to each well, and shake once every 30 minutes in a 37° C., 5% CO ₂ incubator. After incubation for 2 hours, the virus liquid in the wells was discarded, and 100 μL of maintenance solution was added to each well, and cultured in a 37° C., 5% CO ₂ incubator for 72 hours, and the cytopathic changes (CPE) were observed every day. 4 hours before the end of the culture, 50 μL of 2 mg/mL MTT solution was added to each well, and the shaker was shaken until the crystals were completely dissolved. Use a microplate reader to measure the OD value at a wavelength of 490 nm, and calculate the blocking rate of the virus-adsorbed cells according to the following formula.

Blocking rate (Blocking rate,%)=(OD value of test group-OD value of virus control group)/(OD value of cell control group-OD value of virus control group)/×100%.

EEIL, EEIH and EEIJ can all effectively block PRV from entering Vero cells, among which EEIL and EEIJ have better blocking effects (see Table 3). EEIL also significantly blocked PRRSV entry into Marc-145 cells. Since EEIL can significantly promote the proliferation of Marc-145 cells, the blocking rate of PRRSV exceeds 100% (see Table 4).

Table 3 In vitro blocking effect on PRV adsorption to Vero cells (blocking rate, %)

Table 4 Blocking effect of EEIL on PRRSV adsorption to Marc-145 cells in vitro

Example 5: Inhibition of Pseudorabies Virus (PRV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) proliferation in vitro

Take a 96-well plate that has grown into a monolayer of Vero or Marc-145 cells after 24 hours of adherent culture, add 100 μL of 100 TCID ₅₀ PRV or PRRSV virus dilution to each well, and shake at 37°C, 5% CO ₂ incubator at intervals of 30 minutes once. After incubation for 2 h, the virus liquid in the wells was discarded. Add 100 μL per well of Vero cells or Marc-145 cell MNTC with the highest drug concentration, and use DMEM culture solution to make drug dilutions, 8 replicate holes. At the same time, set blank control, normal cell control and virus control. Incubate for 2 hours at 37°C in a 5% CO ₂ incubator, and discard the drug solution. 100 μL of maintenance solution was added to each well, cultured in a 37° C., 5% CO ₂ incubator for 72 hours, and cytopathic changes (CPE) were observed every day. 4 hours before the end of the culture, 50 μL of 2 mg/mL MTT solution was added to each well, and the shaker was shaken until the crystals were completely dissolved. Use a microplate reader to measure the OD value at a wavelength of 490 nm, and calculate the inhibition rate of virus proliferation according to the following formula.

Inhibitory rate (Inhibitory rate, %)=(OD value of test group-OD value of virus control group)/(OD value of cell control group-OD value of virus control group)/×100%.

EEIL, EEIH and EEIJ all had significant inhibitory effects on PRV proliferation, among which EEIL and EEIJ had better inhibitory effects (see Table 5). EEIL can also significantly inhibit the proliferation of PRRSV, and the inhibition rate reaches 90.44% when the concentration is 1.56 mg/mL (see Table 6).

Table 5 Inhibitory effect on PRV proliferation in vitro (inhibition rate, %)

Table 6 Inhibitory effect of EEIL on PRRSV proliferation in vitro

Example 6: Effects on virus-induced apoptosis and necrosis of resident cells

Take the 24-well plates of Vero cells or Marc-145 cells adherently cultured for 24 hours, and set up the cell control group, virus control group, virus killing group, blocking virus adsorption cell group and virus proliferation inhibiting group. According to the methods of Examples 3, 4, and 5, the cells were treated with drugs and viruses, respectively. Remove the maintenance solution, wash each well twice with 0.5mL pre-cooled PBS, add 0.5mL EDTA-free trypsin to digest for 45s, add 0.5mL complete culture solution and blow gently, collect the cells into the corresponding flow tube, and centrifuge at 1500rpm 5min. Cells were collected, stained according to the instructions of the Annexin V-FITC/PI double-stained apoptosis detection kit, and detected by flow cytometry.

The apoptosis and necrosis of PRV-infected Vero cells and PRRSV-infected Marc-145 cells were detected by Annexin-V and PI double staining. Annexin-V single positive indicates early apoptosis, while Annexin-V and PI double positive indicates cell necrosis. It can be seen from Figures 1 and 2 that both PRV and PRRSV can induce apoptosis and necrosis in Vero cells and Marc-145 cells, respectively, and HHXI can significantly reduce PRV infection in Vero cells by killing, blocking and inhibiting the virus. Apoptosis rate and necrosis rate of Marc-145 cells infected with PRRSV (P<0.001).

Example 7: Influence on the expression level of gD gene in Vero cells infected by PRV

The Vero cells adhered to the wall and cultured for 24 hours were taken, and a cell control group, a virus control group, a virus killing group, a blocking virus adsorption cell group, and a virus proliferation inhibiting group were set up. According to the methods of Examples 3, 4, and 5, the cells were treated with drugs and viruses respectively, and maintenance solution was added, and the culture was continued for 48 hours. The cells were collected, the total DNA was extracted according to the instructions of the cultured cell DNA kit, and the expression level of gD gene was detected by real-time fluorescence quantitative PCR (RT-qPCR).

HHXI can significantly down-regulate the gD gene expression level of PRV-infected Vero cells by killing, blocking and inhibiting the virus (P<0.01 or P<0.001), and the relationship is concentration-dependent (see Figure 3).

Embodiment 8: Effect on PRRSV infection Marc-145 cell PRRSV N gene expression level

Get the 24-well plate of Marc-145 cells adhered to culture 24h, set cell control group, virus control group, ribavirin (Rib) control group and HHXI test group, press the method of embodiment 3,4,5 to cell respectively Carry out drug and virus treatment. The cells were collected, and the total RNA of TRIzol reagent was used for reverse transcription and PCR to detect the expression level of PRRSV N gene.

The results of routine PCR detection are shown in Figure 4A. PRRSV N was not detected in normal cells, but PRRSV significantly induced the expression of PRRSV N mRNA in Marc-145 cells. HHXI can significantly down-regulate the expression level of PRRSV N mRNA in Marc-145 cells infected with PRRSV by killing, blocking and inhibiting the virus. The results of RT-qPCR detection are shown in Figure 4B. HHXI and Rib can significantly down-regulate the expression level of N protein mRNA in Marc-145 cells infected with PRRSV by killing, blocking and inhibiting the virus (P<0.05 or P<0.001).

Embodiment 9: To the influence of PRV infection Vero cell virus-positive cell number

The Vero cells adhered to the wall and cultured for 24 hours were taken, and a cell control group, a virus control group, a virus killing group, a blocking virus adsorption cell group, and a virus proliferation inhibiting group were set up. According to the methods of Examples 3, 4, and 5, the cells were treated with drugs and viruses respectively, and maintenance solution was added, and the culture was continued for 48 hours. Cells were collected for immunofluorescence analysis (IFA), observed under a fluorescent inverted microscope, and photographed.

Vero cells infected with PRV showed obvious green fluorescence. In the case of killing effect, there was almost no positive fluorescence reaction of PRV protein at the three concentrations (Figure 5A); while in the case of blocking and inhibition, the positive rate of PRV gradually decreased in a drug concentration-dependent manner (Figure 5B and 5C), And the blocking effect is slightly better than the inhibitory effect. Semi-quantitative analysis showed that HHXI could significantly reduce the PRV-positive percentage of Vero cells by killing, blocking and inhibiting the virus (P<0.001, Figure 5D).

Example 10: Effects on the expression level of viral proteins in Vero cells infected by PRV

The Vero cells adhered to the wall and cultured for 24 hours were taken, and a cell control group, a virus control group, a virus killing group, a blocking virus adsorption cell group, and a virus proliferation inhibiting group were set up. According to the methods of Examples 3, 4, and 5, the cells were treated with drugs and viruses respectively, and maintenance solution was added, and the culture was continued for 48 hours. The cells were collected, and the expression level of viral protein was detected by Western blot method.

The experimental results are shown in Figure 6. HHXI can significantly down-regulate the expression level of virus protein in PRV-infected Vero cells in a concentration-dependent manner by killing, blocking and inhibiting the virus (P<0.001).

Example 11: Effects on the number of N protein-positive cells and expression levels of PRRSV infected Marc-145 cells

Get the 24-well plate of Marc-145 cells adhered to culture 24h, set cell control group, virus control group, ribavirin (Rib) control group and HHXI test group, press the method of embodiment 3,4,5 to cell respectively Carry out drug and virus treatment. The cells were collected for immunofluorescence analysis (IFA), observed under a fluorescent inverted microscope, and photographed; Western blot was used to detect the expression level of PRRSV N protein.

The results of IFA detection showed that: HHXI can significantly reduce the number of N protein-positive cells in PRRSV-infected Marc-145 cells in a concentration-dependent manner (Fig. 7A, B). HHXI and Rib can significantly reduce the number of PRRSV N protein-positive Marc-145 cells by killing, blocking and inhibiting the virus (Fig. 7C, D). The results of Western blot analysis showed that PRRSV infected Marc-145 cells could significantly express N protein, and HHXI could significantly down-regulate the expression level of PRRSV N protein in Marc-145 cells infected in a concentration-dependent manner (P<0.05 or P<0.001) (Figure 7E, F ). At the same time, HHXI can significantly inhibit the expression of PRRSV N protein in infected Marc-145 cells by killing, blocking and inhibiting the virus (Fig. 7G, H).

Example 12: Effects on the mRNA expression levels of inflammatory factors in PRV-infected BV2 cells

A 24-well plate of BV2 cells adhered to the culture for 24 hours was taken, and a cell control group, a virus control group, an acyclovir (ACV) control group, and a drug test group were set up. The BV2 cells were treated with drugs and viruses by way of killing, and cultured for 24 and 48 hours respectively. Collect cells, use TRIzol reagent for total RNA, and perform reverse transcription and RT-qPCR detection

PRV infection could significantly induce the mRNA expression levels of inflammatory factors such as IL-6, IL-1β, TNF-α, iNOS, MCP-1, COX-2 and IFN-β in BV2 cells (P<0.001). However, after PRV was treated with different concentrations of HHXI, the mRNA expression levels of these inflammatory factors in BV2 cells infected by PRV were significantly reduced, and showed a concentration-dependent relationship ( FIG. 8 ).

Example 13: Effects on the Phosphorylation of BV2 Cells MAPK and NF-κB Proteins Infected by PRV

A 24-well plate of BV2 cells adhered to the culture for 24 hours was taken, and a cell control group, a virus control group, an acyclovir (ACV) control group, and a drug test group were set up. BV2 cells were treated with drugs and viruses by killing. The cells were collected, and the expression levels of MAPK family (JNK, p38 and ERK) and NF-κB protein and their phosphorylation in PRV-infected BV2 cells were detected by Western-blot method.

The experimental results are shown in Figure 9. PRV infection could significantly induce phosphorylation of NF-κB p65, JNK, p38 and ERK in BV2 cells (P<0.001). After PRV was treated with different concentrations of HHXI, the phosphorylation levels of NF-κB p65, JNK, p38 and ERK in infected BV2 cells were significantly down-regulated (P<0.001).

Embodiment 14: The preventive effect of HHXI intramuscular injection on pseudorabies virus (PRV) infected mice

Clean grade ICR female mice were randomly divided into 6 groups: normal control group, model group, HHXI low, medium and high dose test groups (12.5mg/kg, 25mg/kg and 50mg/kg), acyclovir (ACV) Control group, 5 rats in each group. ACV control group and HHXI test group mice were intramuscularly injected with 0.2mL/10g of ACV dilution or different concentrations of HHXI dilution; normal control group and model group mice were intramuscularly injected with PBS at 0.2mL/10g, once a day , Continuous administration for 4 days. 2 hours after the last administration, except for the intramuscular injection of PBS in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). After infection, weigh every day, observe the symptoms and death situation, and observe continuously for 15 days. The protection rate and life extension rate of HHXI to PRV-infected mice were calculated.

Three doses of HHXI all had certain protective effects on PRV-infected mice ( FIG. 10 ). 100% of the mice in the PRV-infected model group died. The protection rates of low, medium and high doses of HHXI against PRV-induced death in mice were 20%, 40% and 40% respectively; the life extension rates were 35.29%, 64.53% and 52.94% respectively. The preventive effect of HHXI on PRV-infected mice was significantly better than that of ACV.

Embodiment 15: The therapeutic effect of HHXI intramuscular injection on mice infected with pseudorabies virus (PRV)

Clean grade ICR female mice were randomly divided into 6 groups: normal control group, infection model group, HHXI low, medium and high dose test groups (12.5mg/kg, 25mg/kg and 50mg/kg), acyclovir (ACV ) control group, 5 rats in each group. Except for the intramuscular injection of PBS to the mice in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). 2h after infection, mice in the ACV control group and HHXI test group were intramuscularly injected with ACV dilution or different concentrations of HHXI dilution at 0.2mL/10g; mice in the normal control group and infection model group were intramuscularly injected with PBS at 0.2mL/10g, 1 time a day, continuous administration for 4 days. Weigh every day, observe symptoms, death situation, and record, and observe continuously for 15 days. Calculate the cure rate and life extension rate of HHXI on PRV-infected mice.

Three doses of HHXI have certain therapeutic effects on PRV-infected mice ( FIG. 11 ). 100% of the mice in the PRV-infected model group died. The cure rates of low, medium and high doses of HHXI on PRV-infected mice were all 20%; the life extension rates of mice were 25.42%, 33.90% and 64.41% respectively. The therapeutic effect of HHXI on PRV-infected mice was significantly better than that of ACV.

Embodiment 16: The preventive effect of EEIH intragastric administration on mice infected with pseudorabies virus (PRV)

Clean grade ICR female mice were randomly divided into 5 groups: normal control group, virus infection model group, EEIH low, medium and high dose test groups (100mg/kg, 200mg/kg and 400mg/kg), 5 in each group. The mice in the EEIH test group were intragastrically administered EEIH dilutions of different concentrations at 0.2mL/10g; the mice in the normal control group and the model group were intragastrically administered PBS at 0.2mL/10g, once a day, for 4 consecutive days. 2 hours after the last administration, except for the intramuscular injection of PBS to the mice in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). After infection. Weigh every day, observe symptoms and death, and observe continuously for 15 days. The protection rate and life extension rate of EEIH on PRV-infected mice were calculated.

Three doses of EEIH all had a certain protective effect on PRV-infected mice ( FIG. 12 ). 100% of the mice in the PRV-infected model group died. The protection rates of low, medium and high doses of EEIH against PRV-induced death in mice were 40%, 20% and 20% respectively; the life extension rates were 77.42%, 41.94% and 33.87%, respectively.

Embodiment 17: The therapeutic effect of EEIH intragastric administration on mice infected with pseudorabies virus (PRV)

Clean-grade ICR female mice were randomly divided into 5 groups: normal control group, infection model group, EEIH low, medium and high dose test groups (100mg/kg, 200mg/kg and 400mg/kg), 5 in each group. Except for the intramuscular injection of PBS in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). 2h after infection, the mice in the HHXI test group were intramuscularly injected with different concentrations of EEIH dilutions at 0.2mL/10g; the mice in the normal control group and the infection model group were injected with PBS at 0.2mL/10g, once a day, continuously 4 days. Weigh every day, observe symptoms, death situation, and record, and observe continuously for 20 days. The cure rate and life extension rate of EEIH on PRV-infected mice were calculated.

Oral administration of three doses of EEIH has a certain therapeutic effect on PRV-infected mice ( FIG. 13 ). 100% of the mice in the model group died. The cure rate of PRV-infected mice by low, medium and high doses of EEIH was 20%, 40% and 20%; the life extension rate of mice was 57.53%, 79.45% and 42.67%, respectively.

Embodiment 18: The preventive effect of EEIJ intramuscular injection on mice infected with pseudorabies virus (PRV)

Clean grade ICR female mice were randomly divided into 5 groups: normal control group, model group, EEIJ low, medium and high dose test groups (100mg/kg, 200mg/kg and 400mg/kg), 5 in each group. The mice in the EEIJ test group were intramuscularly injected with different concentrations of EEIJ diluents at 0.2mL/10g; the mice in the normal control group and the model group were intramuscularly injected with PBS at 0.2mL/10g, once a day, for 4 consecutive days. 2 hours after the last administration, except for the intramuscular injection of PBS in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). After infection. Weigh every day, observe symptoms and death, and observe continuously for 20 days. The protection rate and life extension rate of EEIJ on PRV-infected mice were calculated.

Intramuscular injection of EEIJ has a certain protective effect on PRV-infected mice (Table 7). 100% of the mice in the PRV-infected model group died. EEIJ doses of 50, 100 and 200 mg/kg intramuscular injection had protection rates of 60%, 40% and 40% against death of PRV-infected mice, respectively; life extension rates were 89.16%, 68.68% and 66.27%.

Table 7 Preventive effect of intramuscular injection of EEIJ on mice infected with pseudorabies virus (PRV)

Note: Compared with the virus control group, ^*** P<0.001.

Embodiment 19: The therapeutic effect of EEIJ intramuscular injection on mice infected with pseudorabies virus (PRV)

Clean-grade ICR female mice were randomly divided into 5 groups: normal control group, infection model group, EEIJ low, medium and high dose test groups (50mg/kg, 100mg/kg and 20mg/kg), 5 in each group. Except for the intramuscular injection of PBS in the normal control group, the mice in the other groups were intramuscularly injected with 1/80 portion of porcine pseudorabies live vaccine (Bartha K61 strain). 2 hours after infection, the mice in the EEIJ test group were intramuscularly injected with different concentrations of EEIJ dilutions at 0.2mL/10g; the mice in the normal control group and the infection model group were intramuscularly injected with PBS at 0.2mL/10g, once a day, for continuous administration 4 days. Weigh every day, observe symptoms, death situation, and record, and observe continuously for 20 days. Calculate the cure rate and life extension rate of EEIJ on PRV-infected mice.

Intramuscular injection of EEIJ has a certain therapeutic effect on PRV-infected mice (Table 8). 100% of the mice in the PRV-infected model group died. EEIJ doses of 50, 100 and 200 mg/kg intramuscular injection have cure rates of 40%, 20% and 20% for PRV-infected mice respectively; the life extension rates of mice are 68.00%, 45.33% and 42.67% respectively.

Table 8 The therapeutic effect of intramuscular injection of EEIJ on mice infected with pseudorabies virus (PRV)

Note: Compared with the virus control group, ^** P<0.01 and ^*** P<0.001.

Embodiment 20: The influence of HHXI intramuscular injection on PRV viral load in brain tissue of pseudorabies virus (PRV) infection mouse

Clean grade ICR female mice were randomly divided into 5 groups: normal control group, infection model group, HHXI low, medium and high dose test groups (12.5mg/kg, 25mg/kg and 50mg/kg), 5 in each group. Except for the intramuscular injection of PBS in the normal control group, the mice in other groups were infected with PRV. 2 hours after infection, the mice in the HHXI test group were intramuscularly injected with different concentrations of HHXI dilutions at 0.2mL/10g; the mice in the normal control group and the infection model group were intramuscularly injected with PBS at 0.2mL/10g, once a day, continuously administered 4 days. On the 6th day after infection, the mouse brain tissue was dissected, DNA was extracted with an animal tissue DNA kit, and the viral load of PRV in the mouse brain tissue was detected by RT-qPCR.

The PRV gD gene can be detected in the brain tissue of PRV-infected mice. Intramuscular injection of three doses of HHXI can significantly down-regulate the gD gene expression level in the brain tissue of PRV-infected mice in a dose-dependent manner (P<0.001), indicating that HHXI can reduce the PRV viral load in the brain tissue of PRV-infected mice (Figure 14).

Example 21: Effect of HHXI Intramuscular Injection on the Expression Level of Inflammatory Factors mRNA in the Brain Tissue of Mice Infected with Pseudorabies Virus (PRV)

The brain tissue collected in Example 20 was taken, total RNA was extracted with TRIzol reagent, and after reverse transcription, the expression level of inflammatory factor mRNA was detected by RT-qPCR.

RT-qPCR detection results are shown in Figure 15. PRV infection could significantly induce the mRNA expression of inflammatory factors such as IL-6, IL-1β, TNF-α, iNOS, MCP-1, COX-2 and IFN-β in mouse brain tissue (P<0.001). Intramuscular injection of HHXI can significantly down-regulate the expression levels of these inflammatory mRNAs in the brain tissue of PRV-infected mice in a dose-dependent manner (P<0.001 or P<0.01), indicating that intramuscular injection of HHXI can significantly inhibit the cytokine storm induced by PRV infection in mice .

In summary, the plant extracts of the genus Anise in the present invention can effectively and directly kill animal viruses in vitro, block animal viruses from entering cells, inhibit animal viruses from replicating in cells, and play an antiviral role. At the same time, it can inhibit the apoptosis of host cells induced by animal viruses, and inhibit the inflammatory response of host cells induced by animal viruses through the NF-κB and MAPKs pathways, thereby protecting host cells. Anise plant extracts have significant preventive and therapeutic effects on mice infected with animal viruses, can significantly reduce the viral load of mouse brain tissue, down-regulate the expression level of inflammatory factor genes, inhibit cytokine storm, alleviate pathological changes in brain tissue, and improve Survival of animal virus-infected mice, and prolonging the survival time of animal virus-infected mice. Therefore, the plant extracts of the genus Anise have the potential value of developing new veterinary drugs against animal viruses.

Claims (3)

1. The application of star anise plants or their extracts in the preparation of anti-animal virus drugs, characterized in that the star anise plants are red poisonous fennel ( Illicium lanceolatum AC Smith), red fennel ( I. henryi Diels) or false Maple bark ( I. jiadifengpi BN Chang); the animal virus described is pseudorabies virus (PRV) or porcine reproductive and respiratory syndrome virus (PRRSV); the plant extract of the genus Anise can directly kill animal viruses in vitro, block Animal viruses enter cells, inhibit the replication of animal viruses in cells, and play an antiviral role. At the same time, they can inhibit the apoptosis of host cells induced by animal viruses, and inhibit the inflammatory response of host cells induced by animal viruses through the NF-κB and MAPKs pathways, thereby protecting host cells The preparation method of the star anise plant extract is as follows: take medicinal materials, pulverize them, place them in a round bottom flask, add 75% ethanol for reflux extraction, 10 to 12 times 75% ethanol for the first time, and reflux for 2 h; the second time 8 ~10 times 75% ethanol, extract for 2 hours; filter the extract, combine the filtrate, use a rotary evaporator to recover the ethanol under reduced pressure in a water bath at 45°C until there is no alcohol smell, continue to concentrate, and freeze-dry to obtain the ethanol extract EEIL , Alcohol extract of red fennel EEIH and alcohol extract of maple bark EEIJ. 2. the application of star anise plant according to claim 1 or its extract in the preparation of anti-animal virus medicine, is characterized in that, described medicine is the medicine of preventing and/or treating animal viral disease caused by animal virus . 3. An antiviral drug, which contains the star anise plant or its extract and a pharmaceutically acceptable carrier as claimed in any one of claims 1-2.

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