Application of Chinese medicine monomer, Borneol, in preparation of medicine for preventing or treating porcine pseudorabies

Abstract

The application belongs to the technical field of animal disease prevention and control, and discloses application of a traditional Chinese medicine monomer, i.e., a perillyl alcohol, in preparation of a medicine for preventing or treating a pseudorabies of pigs. The application verifies the role of the perillyl alcohol in inhibiting a pig pseudorabies virus infection process and explores the application value of the perillyl alcohol in resisting the pig pseudorabies virus, and provides a theoretical basis and practical guidance for prevention and control of the PRV. In addition, the perillyl alcohol is a natural source medicine, has the advantages of safety, few side effects, low metabolic residues and no pollution, and has good popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of animal disease prevention and control technology, and relates to the application of the traditional Chinese medicine monomer perilla alcohol in the preparation of drugs for the prevention or treatment of pseudorabies in pigs. Background Technology

[0002] Pseudorabies virus (PRV), belonging to the Herpesviridae family and Alphaherpesvirinae subfamily, is a DNA virus that can infect a wide range of hosts, including pigs, cattle, sheep, and mice. Pigs are the only viable host for PRV infection. Pseudorabies (PR) can occur in pigs of any age and breed, but the morbidity and mortality rates are highest in piglets under two weeks old, reaching 100%. The morbidity and mortality rates decrease with increasing age. The clinical manifestations of PR infection vary among pig herds of different ages. Newborn piglets may exhibit neurological damage, paddling convulsions, and a mortality rate as high as 100%. Finishing pigs may experience respiratory disorders and stunted growth. Pregnant sows may experience abortion, stillbirths, and mummified fetuses. Infected boars may become infertile and experience decreased semen quality. Mouse models are a viable alternative and feasible animal model for studying the pathogenicity and drug treatment of PRV.

[0003] Perillyl alcohol is a naturally occurring monoterpene compound widely found in citrus fruit peels, mint, sage, and many other plants. It boasts advantages such as safety, few side effects, low metabolic residue, and no pollution. Perillyl alcohol is widely used in the production of food flavorings and cosmetic flavorings, and is undergoing preclinical research as an anticancer drug. Oral capsules and skin cancer cream formulations have been developed.

[0004] There are currently no reports on perilla alcohol inhibiting porcine pseudorabies virus. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of the traditional Chinese medicine monomer perillyl alcohol in the preparation of drugs for the prevention or treatment of porcine pseudorabies (PRV). This invention demonstrates the significant inhibitory effect of perillyl alcohol on PRV infection, effectively regulating inflammation induced in mice after PRV infection. This provides a new potential target and treatment strategy for the prevention and control of PRV infection, offers a more theoretical basis for the development of novel antiviral drugs, and provides practical reference for the prevention and treatment of PRV.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention claims protection for the use of the traditional Chinese medicine monomer perillyl alcohol in the preparation of drugs for the prevention of pseudorabies infection. The CAS number for the aforementioned perillyl alcohol is 536-59-4.

[0008] Furthermore, its application in drugs for the prevention and / or treatment of pseudorabies infection in pigs.

[0009] Furthermore, the effective concentration of the perillaldehyde in BHK-21 cells is 31.25-125 μM, and the effective concentration in animals is 40-80 mg / kg.

[0010] Furthermore, perilla frutescens can reduce the expression of TNF-α and IL-6 in the serum of mice infected with porcine pseudorabies virus.

[0011] Furthermore, the drug comprises a pharmaceutically acceptable salt or carrier.

[0012] Furthermore, the drug is formulated in the form of a liquid preparation, granules, tablets, powders, capsules, sustained-release preparations, pellets, or injections.

[0013] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided herein apply to the group or term throughout the specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0014] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0015] The terms "salt" and "pharmaceutical salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds or their stereoisomers with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, hydrobroms, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0016] In some embodiments, one or more compounds of the present invention may be used in combination with each other. Alternatively, the compounds of the present invention may be used in combination with any other active agent to prepare a medicament or pharmaceutical composition for regulating cell function or treating pseudorabies infection. If a group of compounds is used, these compounds may be administered to the test subject simultaneously, separately, or sequentially.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention demonstrates the significant inhibitory effect of perillyl alcohol on PRV infection, effectively regulating inflammation induced by PRV infection in mice. This provides a new potential target and treatment strategy for the prevention and control of PRV infection, offers a stronger theoretical basis for the development of novel antiviral drugs, and provides practical reference for the prevention and treatment of PRV. Perillyl alcohol is a naturally derived drug with advantages such as safety, few side effects, low metabolic residue, and no pollution, making it highly valuable for widespread application. Attached Figure Description

[0019] Figure 1 The survival rate of BHK-21 cells provided in Example 1 under different concentrations of perillaldehyde is shown in the figure.

[0020] Figure 2 The graph shows the inhibitory effect of different concentrations of perillyl alcohol on PRV in BHK-21 cells, as provided in Example 2.

[0021] Figure 3 The graph shows the inhibitory effect of perillaldehyde on PRV under different modes of action, as provided in Example 3.

[0022] Figure 4 The effect of perillyl alcohol on the renal load of PRV-infected mice is shown in the figure for Example 4.

[0023] Figure 5 The figure shows the effect of perilla frutescens alcohol on serum TNF-α in PRV-infected mice, as shown in Example 5.

[0024] Figure 6 This is a graph showing the effect of perillyl alcohol on serum IL-6 in PRV-infected mice, as described in Example 5. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0026] Experimental strains and clinical samples: BHK-21 cells, PRV FJ-2012 strain, and 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, and CCK8 kit.

[0028] Main instruments: electronic analytical balance, autoclave, gradient PCR instrument, ultra-low temperature freezer, ice maker, benchtop multi-functional low-speed centrifuge, BSC series biosafety cabinet, LightCycler 96 fluorescence PCR detector, electric thermostatic water bath, vortex shaker, CO2 cell culture incubator, cryogenic grinder, fully automated enzyme reader, inverted fluorescence microscope, circulating water multi-purpose vacuum pump.

[0029] The herbal monomers mentioned in the following examples are all perillyl alcohol, CAS number: 536-59-4. Perillyl alcohol is abbreviated as "POH" in all the figures.

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

[0031] The perillaldehyde concentration gradient used was (μM): 0, 3.91, 7.81, 15.62, 31.25, 62.5, 125, 250, 500.

[0032] BHK-21 cells were seeded into 96-well plates, 100 μL per well (containing approximately 3 × 10⁻⁶ cells). 4 Cells were cultured in a 5% CO2, 37℃ incubator for 12 h. When the cell density reached 70%-80%, the old culture medium was discarded, and 100 μL of different concentrations of the traditional Chinese medicine monomer perillyl alcohol were added. The perillyl alcohol was prepared by serially diluting it twofold with 1% FBS maintenance medium, and blank and empty cell groups were set up. Cells were incubated with the drug for 24 h, the drug was discarded, and the cells were washed twice with pre-cooled PBS. 110 μL of a mixture of 1% FBS maintenance medium and CCK-8 at a ratio of 100:10 was added, and the cells were incubated in an incubator for 2 h. The absorbance at a wavelength of 450 nm was measured using a microplate reader, and the relative cell viability was calculated. The results showed (e.g.) Figure 1 When the concentration of perillyl alcohol is ≤125μM, it has no significant effect on the survival rate of BHK-21 cells.

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

[0034] The concentration gradient of perillaldehyde used was (μM): 0, 31.25, 62.5, 125

[0035] BHK-21 cells were seeded into 12-well plates. When the cell density reached 80%, the PRV stock solution was diluted with DMEM basal medium, and cells were infected with an MOI of 0.05 at a rate of 500 μL per well. The cells were incubated for 2 h in a 5% CO2, 37°C incubator. The virus incubation solution was discarded, and the cells were washed three times with PBS. 1 mL of maintenance medium containing 1% FBS with different concentrations of traditional Chinese medicine monomers was added to create PRV and different concentrations of traditional Chinese medicine monomer groups. The cells were then incubated for 24 h in a 5% CO2, 37°C incubator. Cells were then collected, and PRV-gE expression was detected by qRT-PCR. The results showed (e.g.) Figure 2 As the concentration of perillaldehyde increased, the viral load of PRV gradually decreased.

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

[0037] Cells were pre-seeded in 12-well plates, and groups were set up to inhibit viral adsorption and penetration, protect against viral infection, and directly kill viruses to study the mechanism of action of perilla frutescens.

[0038] Inhibition of viral adsorption and penetration group: When the cell density reaches 80%, discard the old culture medium, wash three times with PBS, add 500 μL of the mixture of Chinese medicine monomer and PRV, and incubate the Chinese medicine monomer and PRV together in a 5% CO2, 37℃ incubator for 2 h. Use PRV-incubated cells for 2 h as the virus control. Discard the incubation medium, wash three times with PBS, replace with maintenance medium containing 1% FBS and culture for 24 h. Collect cells for qRT-PCR detection.

[0039] Protective effect group: When the cell density reaches 80%, the old culture medium is discarded, the cells are washed 3 times with PBS, and 500 μL of diluted traditional Chinese medicine monomer is added. The cells are then incubated with the traditional Chinese medicine monomer in a 5% CO2 incubator at 37°C for 2 h. Cells incubated in DMEM basal medium for 2 h serve as a control. The incubation solution is then discarded, the cells are washed 3 times with PBS, and the diluted PRV virus solution is replaced. After incubation in an incubator for 2 h, the virus solution is discarded, the cells are washed 3 times with PBS, and the cells are cultured in a maintenance medium containing 1% FBS for 24 h. Cells are then harvested for qRT-PCR detection.

[0040] Direct killing group: When the cell density reached 80%, the mixture of PRV and traditional Chinese medicine monomer was placed in a 4°C refrigerator and incubated for 2 h. The PRV virus solution incubated at 4°C for 2 h served as a virus control. After incubation, the cells were removed from the incubator, the old culture medium was discarded, and the cells were washed 3 times with PBS. 500 μL of the pre-incubated mixture of traditional Chinese medicine monomer and PRV was added, and the cells were incubated in a 5% CO2, 37°C incubator for another 2 h. The incubation solution was discarded, the cells were washed 3 times with PBS, and the cells were cultured in a 1% FBS maintenance medium for 24 h. The cells were then harvested for qRT-PCR detection.

[0041] The results show (e.g.) Figure 3 Perillyl alcohol works by inhibiting the adsorption and penetration of PRV.

[0042] Example 4: Study on the protective effect of perillyl alcohol against PRV infection in mice.

[0043] The protective effect of perilla oil against PRV infection in mice was determined by detecting viral load in the kidneys.

[0044] Twenty-four BALB / c mice of similar body weight were randomly divided into four groups: a low-dose perillaldehyde group (L): administered 40 mg / kg perillaldehyde daily by gavage; a high-dose perillaldehyde group (H): administered 80 mg / kg perillaldehyde daily by gavage; a control group (C): administered 0.1 mL / 10 g perillaldehyde daily by gavage; and a PRV positive control group. After 7 days of continuous administration, the mice were infected with PRV FJ-2012. Three days later, the mice were sacrificed, and kidney tissue and serum were collected to evaluate the protective effect of perillaldehyde against PRV infection in mice. Results showed (e.g.) Figure 4 The protective effect of perillaldehyde against PRV infection in mice increased with increasing perillaldehyde concentration.

[0045] Example 5: Study on the effects of perillyl alcohol on inflammatory factors in mice

[0046] The levels of TNF-α and IL-6 in mouse serum were determined by ELISA.

[0047] The levels of TNF-α and IL-6 in serum were detected using TNF-α and IL-6 kits. After the reaction was terminated, the absorbance (OD value) of each well was read on a 450 nm wavelength microplate reader. The standard concentration was plotted on the x-axis, and the corresponding absorbance (OD value) on the y-axis. A standard curve equation was created using computer software with four-parameter logistic curve fitting (4-pl). The concentration of the sample was calculated using the sample absorbance (OD value) from the equation. The results showed ( Figure 5 The effect of perilla alcohol on serum TNF-α, Figure 6(The effect of perillyl alcohol on serum IL-6). Perillyl alcohol can reduce the expression of TNF-α and IL-6 after PRV infection. At the same time, the inhibition of TNF-α and IL-6 expression by perillyl alcohol is significantly enhanced with the increase of perillyl alcohol concentration.

[0048] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the traditional Chinese medicine monomer perillyl alcohol in the preparation of drugs for the prevention and / or treatment of pseudorabies infection in pigs.

2. The application according to claim 1, characterized in that, The concentration of perillyl alcohol used is 40-80 mg / kg.

3. The application according to claim 1, characterized in that, Perilla frutescens can reduce the expression of TNF-α and IL-6 in the serum of mice infected with porcine pseudorabies virus.

4. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable salts or carriers.

5. The application according to claim 1, characterized in that, The drug is formulated in the form of liquid, granules, tablets, powders, capsules, sustained-release preparations, drop pills, or injections.

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