Application of traditional Chinese medicine monomer perillyl alcohol in preparation of medicine for preventing or treating porcine pseudorabies

Abstract

The invention belongs to the technical field of animal disease prevention and control, and discloses application of a traditional Chinese medicine monomer perillyl alcohol in preparation of a medicine for preventing or treating porcine pseudorabies. According to the present invention, the influence of the perillyl alcohol with different concentrations on the activity of BHK-21 cells, the inhibition effect of the perillyl alcohol on the PRV on the BHK-21 cells, the inhibition effect of the perillyl alcohol on the PRV under different action modes, the protection effect of the perillyl alcohol on the PRV infection of mice, and the influence of the perillyl alcohol on inflammatory factors in the serum of the mice are determined; the effect of the perillyl alcohol in the process of inhibiting porcine pseudorabies virus infection is verified, the application value of the perillyl alcohol in resisting the porcine pseudorabies virus is explored, and a theoretical basis and practical guidance are provided for prevention and control of the PRV. Besides, the perillyl alcohol is a medicine with a natural source, has the advantages of safety, few side effects, low metabolic residue and no pollution, and has relatively good popularization and application values.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal disease prevention and control, and relates to application of a traditional Chinese medicine monomer perillyl alcohol in preparation of a medicine for preventing or treating pseudorabies of pigs. BACKGROUND

[0002] Pseudorabies virus (PRV) belongs to the Herpesviridae and Alphaherpesvirinae, and is a DNA virus that can infect a wide range of hosts including pigs, cattle, sheep, mice, etc. Pigs are the only surviving host of PRV infection, and pseudorabies (PR) can occur in pigs of any age and breed, especially in piglets under 2 weeks old, with a morbidity rate of 100%. The older the piglets are, the lower the morbidity rate and mortality rate are. The clinical manifestations of PR infection in pigs of different ages are also different. Newborn piglets infected with PR can exhibit damage to the nervous system, paddling-like convulsions of the limbs, and a mortality rate of up to 100%. Growing pigs infected with PR can exhibit respiratory system disorders and growth and development inhibition. Pregnant sows infected with PR can exhibit abortion, stillbirth and mummified fetus. Breeding boars infected with PR can cause infertility and semen quality decline. Mouse models are one of the animal models for studying the pathogenicity of PRV and drug prevention and treatment, and have good substitutability and feasibility.

[0003] Perillyl alcohol is a naturally occurring monoterpene compound that widely exists in the peels of citrus fruits, peppermint, sage and many other plants, and has the advantages of safety, few side effects, low metabolic residues and no pollution. Perillyl alcohol is widely used in the production of edible and daily-use fragrances, and is used as an anticancer drug for preclinical research, and oral capsules and skin cancer cream formulations have been developed.

[0004] There is no related report on perillyl alcohol inhibiting pseudorabies virus of pigs. SUMMARY

[0005] In view of the deficiencies in the prior art, the application provides application of a traditional Chinese medicine monomer perillyl alcohol in preparation of a medicine for preventing or treating pseudorabies (PRV) of pigs. The application proves that perillyl alcohol has a significant inhibitory effect on PRV infection, can effectively regulate inflammation caused after PRV infection in mice, provides a new potential target and treatment strategy for prevention and control of PRV infection, provides more theoretical basis for development of a new type of antiviral drug, and provides a practical reference for prevention and treatment of PRV.

[0006] To achieve the above object, the application provides the following technical solutions.

[0007] The application claims protection of application of a traditional Chinese medicine monomer perillyl alcohol in preparation of a medicine for preventing pseudorabies infection. The CAS number of the perillyl alcohol is 536-59-4.

[0008] Further, the application is used in the medicine for preventing and / or treating the infection of pseudorabies virus in pigs.

[0009] Further, the effective concentration of perillyl alcohol on BHK-21 cells is 31.25-125 μM, and the effective concentration of perillyl alcohol on animals is 40-80 mg / kg.

[0010] Further, perillyl alcohol can reduce the expression of TNF-α and IL-6 in the serum of mice infected with pseudorabies virus.

[0011] Further, the medicine comprises a pharmaceutically acceptable salt or carrier.

[0012] Further, the medicine is in the form of liquid preparation, granule, tablet, powder, capsule, sustained release preparation, dripping pill or injection.

[0013] Definitions of terms used in the application: unless otherwise stated, the initial definition of a term provided herein applies throughout this specification to that group or term; for terms not specifically defined herein, the meaning of such terms should be clear from the context in which they are used and / or can be given their meaning as understood by one of skill in the art.

[0014] The term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, adjuvant, and / or formed salt is generally chemically and / or physically compatible with the other ingredients constituting a pharmaceutical formulation and physiologically compatible with the recipient thereof.

[0015] The terms "salt" and "pharmaceutically acceptable salt" refer to the acid and / or base salts of the compounds described above, or stereoisomers thereof, formed with inorganic and / or organic acids and bases. Also included are zwitterions (inner salts), and quaternary ammonium salts such as alkyl ammonium salts. These salts can be formed during the final isolation and purification of the compounds. They can also be formed by treating the compounds described above, or stereoisomers thereof, with an appropriate acid or base, for example, an equivalent amount of the acid or base. These salts can be formed in solution, filtration, or upon evaporation of the solvent, or by reaction in aqueous medium followed by lyophilization. The salts of the present application can be hydrochloric, sulfuric, hydrobromic, hydrofluoric, phosphoric, acetic, propionic, succinic, oxalic, malic, succinic, fumaric, maleic, tartaric, or trifluoroacetic acid salts of the compounds.

[0016] In certain embodiments, one or more compounds of the present application can be used in combination with each other. The compounds of the present application can also be used in combination with any other active agents for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease associated with a pseudorabies virus infection. If a combination of compounds is used, the compounds can be administered simultaneously, separately or sequentially to the subject.

[0017] Compared with the prior art, the present application has the beneficial effects that:

[0018] The present application proves the significant inhibitory effect of perillyl alcohol on PRV infection, can effectively regulate the inflammation produced after PRV infection in mice, provides a new potential target and treatment strategy for the prevention and control of PRV infection, provides more theoretical basis for the development of new antiviral drugs, and provides practical reference for the prevention and treatment of PRV. Perillyl alcohol is a natural source of medicine, has the advantages of safety, few side effects, low metabolic residues and no pollution, and has good popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Survival rate diagram of BHK-21 cells provided for Example 1 under the action of different concentrations of perillyl alcohol.

[0020] Figure 2 Inhibition diagram of PRV on BHK-21 cells by different concentrations of perillyl alcohol provided for Example 2.

[0021] Figure 3 Inhibition diagram of PRV by perillyl alcohol under different action modes provided for Example 3.

[0022] Figure 4 Influence diagram of perillyl alcohol on the kidney load of PRV infected mice provided for Example 4.

[0023] Figure 5 Influence diagram of perillyl alcohol on TNF-α in the serum of PRV infected mice provided for Example 5.

[0024] Figure 6 Influence diagram of perillyl alcohol on IL-6 in the serum of PRV infected mice provided for Example 5. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The test methods used in the embodiments of the present application are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0026] Experimental strain and clinical sample: BHK-21 cells, PRV FJ-2012 strain, 6-week-old BALB / c mice.

[0027] Main materials: fetal bovine serum FBS, cell culture medium DMEM, 0.25% trypsin (containing EDTA), PBS buffer, penicillin-streptomycin mixture, viral DNA extraction kit, one-step reverse transcription kit, Probe qPCR SuperMix, SYBR Green qPCR Mix, total RNA extraction kit, anhydrous ethanol, CCK8 kit.

[0028] Main instruments: electronic analytical balance, high-pressure sterilization pot, gradient PCR instrument, ultra-low temperature refrigerator, ice maker, table-top multifunctional low-speed centrifuge, BSC series biological safety cabinet, LightCycler 96 fluorescent PCR detector, electric heating constant-temperature water bath, vortex shaker, CO2 cell incubator, refrigerated grinder, full-automatic enzyme label instrument, inverted fluorescence microscope, circulating water type multi-purpose vacuum pump.

[0029] The Chinese medicine monomer mentioned in the following examples is all perillyl alcohol, CAS No.: 536-59-4. Perillyl alcohol is represented by the abbreviation “POH” in each figure.

[0030] Example 1: Study on the effect of different concentrations of perillyl alcohol on BHK-21 cells

[0031] The concentration gradient of perillyl alcohol used is (μM): 0, 3.91, 7.81, 15.62, 31.25, 62.5, 125, 250, 500

[0032] BHK-21 cells were plated into 96-well plates, 100 μL per well (about 3×10 4 cells), and cultured in a 5% CO2, 37°C incubator for 12 h. When the cell density reached 70%-80%, the old culture medium in the plate was discarded, and 100 μL of different concentrations of Chinese medicine monomer perillyl alcohol was added. The Chinese medicine monomer was prepared by continuous two-fold gradient dilution with 1% FBS maintenance solution, and a blank group and a blank cell group were set. The cells were incubated with the drug for 24 h, the drug was discarded, and the cells were washed twice with pre-cooled PBS. Then, 110 μL of a mixture of 1% FBS maintenance solution and CCK-8 in a ratio of 100:10 was added. After incubation in the incubator for 2 h, the absorbance at 450 nm was measured on an enzyme label instrument, and the relative viability of the cells was calculated. The results showed (as shown in Figure 1 ) that when the concentration of perillyl alcohol was ≤125 μM, it had no significant effect on the survival rate of BHK-21 cells.

[0033] Example 2: Study on the inhibitory effect of perillyl alcohol on PRV on BHK-21 cells

[0034] The concentration gradient of perillyl alcohol used is (μM): 0, 31.25, 62.5, 125

[0035] BHK-21 cells were plated in 12-well plates, and when the cell density reached 80%, the PRV stock solution was diluted with DMEM base medium, and the cells were infected with a dose of MOI = 0.05, 500 μL per well, and incubated in a 5% CO2, 37°C incubator for 2 h. The virus incubation solution was discarded, the cells were washed with PBS 3 times, 1 mL of 1% FBS-containing medium containing different concentrations of Chinese medicine monomers was added, and PRV groups and different concentrations of Chinese medicine monomer groups were set. After incubation in a 5% CO2, 37°C incubator for 24 h, the cells were collected, and the expression of PRV-gE was detected by qRT-PCR. The results showed (as shown in Figure 2 ), and as the concentration of perilla alcohol increased, the viral load of PRV gradually decreased.

[0036] Example 3: Study of the inhibitory effect of perilla alcohol on PRV under different modes of action

[0037] The cells were inoculated in 12-well plates in advance, and the inhibition of viral adsorption and penetration, protection, and direct killing groups were set up to study the mode of action of perilla alcohol.

[0038] Inhibition of viral adsorption and penetration group: when the cell density reached 80%, the old culture medium was discarded, washed with PBS 3 times, and 500 μL of a mixture of Chinese medicine monomers and PRV was added. The Chinese medicine monomers and PRV were incubated in a 5% CO2, 37°C incubator for 2 h. The PRV incubated cells for 2 h were used as the virus control, the incubation solution was discarded, washed with PBS 3 times, and replaced with 1% FBS-containing medium for 24 h. The cells were collected for qRT-PCR detection.

[0039] Protection group: when the cell density reached 80%, the old culture medium was discarded, washed with PBS 3 times, and 500 μL of the diluted Chinese medicine monomers was added. The Chinese medicine monomers were incubated in a 5% CO2, 37°C incubator for 2 h. The DMEM base medium incubated cells for 2 h were used as the control. The incubation solution was discarded, washed with PBS 3 times, and replaced with diluted PRV virus solution. After incubation in an incubator for 2 h, the virus solution was discarded, washed with PBS 3 times, and replaced with 1% FBS-containing medium for 24 h. The cells were collected for qRT-PCR detection.

[0040] Direct killing group: when the cell density reaches 80%, the mixed solution of PRV and traditional Chinese medicine monomer is placed in a 4°C refrigerator for 2 hours, and PRV virus liquid is incubated at 4°C for 2 hours as a virus control. After incubation, the cells are taken out of the incubator, the old culture medium is discarded, and the cells are washed with PBS for 3 times. Then 500 μL of the mixed solution of traditional Chinese medicine monomer and PRV which has been incubated in advance is added, and the cells are incubated in a 5% CO2, 37°C incubator for 2 hours. Then the incubation solution is discarded, the cells are washed with PBS for 3 times, and the cells are cultured in a medium containing 1% FBS for 24 hours. Then the cells are collected for qRT-PCR detection.

[0041] The results show (as shown in Figure 3 ), the pathway through which linalool exerts its effect is by inhibiting the adsorption and penetration of PRV.

[0042] Example 4: Study on the protective effect of linalool on mice infected with PRV

[0043] The protective effect of linalool on mice infected with PRV is determined by detecting the viral load in the kidney

[0044] Twenty-four BALB / c mice with similar body weight are randomly divided into a low-dose linalool group (L): linalool 40 mg / kg is administered by gavage daily, a high-dose linalool group (H): linalool 80 mg / kg is administered by gavage daily, a control group (C): solvent (0.1 mL / 10 g) is administered by gavage daily, and a PRV positive control group. After 7 days of continuous administration, the mice are infected with PRV FJ-2012. The mice are sacrificed after 3 days, and kidney tissue and serum are collected to evaluate the protective effect of linalool on mice infected with PRV. The results show (as shown in Figure 4 ), the protective effect of linalool on mice infected with PRV increases with the increase of linalool concentration.

[0045] Example 5: Study on the effect of linalool on inflammatory factors in mice

[0046] The effect of linalool on inflammatory factors in mice is determined by detecting TNF-α and IL-6 in mouse serum by ELISA

[0047] The content of TNF-α and IL-6 in serum is detected using TNF-α kit and IL-6 kit. After the reaction is terminated, the absorbance (OD value) of each well is read on an enzyme marker at a wavelength of 450 nm. The concentration of the standard is used as the abscissa, and the absorbance (OD value) is used as the ordinate. The computer software is used to create a standard curve equation by four-parameter Logistic curve fitting (4-pl). The concentration value of the sample is calculated by the absorbance (OD value) of the sample using the equation. The results show (as shown in Figure 5 the effect of linalool on TNF-α in serum, Figure 6For the influence of perillyl alcohol on IL-6 in serum, perillyl alcohol can reduce the expression of TNF-α and IL-6 after PRV infection, and the inhibition of perillyl alcohol on the expression of TNF-α and IL-6 is significantly enhanced with the increase of perillyl alcohol concentration.

[0048] Obviously, the above specific embodiments only further illustrate the purposes, technical solutions and beneficial effects of the present application, and it should be understood that the above only serves as specific examples of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. The application of Chinese medicine monomer, eucalyptol, in the preparation of a medicine for preventing pseudorabies infection.

2. Use according to claim 1, characterized in that, The application is in the preparation of a medicine for preventing and / or treating pseudorabies infection in pigs.

3. Use according to claim 1, characterized in that, The concentration of the eucalyptol is 40-80 mg / kg.

4. Use according to claim 1, characterized in that, The eucalyptol can reduce the expression of TNF-α and IL-6 in the serum of mice infected with pseudorabies virus.

5. The use according to claim 1, characterized in that, The medicine comprises a pharmaceutically acceptable salt or carrier.

6. Use according to claim 1, characterized in that, The preparation form of the medicine is liquid preparation, granules, tablets, powder, capsules, sustained-release preparation, dripping pills or injection.

Images