Application of folium artemisiae argyi essential oil in preparation of virus inhibition products

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of folium artemisiae argyi essential oil in preparation of a product for inhibiting viruses, the viruses are Marek's disease viruses, and the folium artemisiae argyi essential oil is used for inhibiting replication of the Marek's disease viruses in cells and has the effect of resisting the Marek's disease viruses.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of wormwood essential oil in preparing a virus-inhibiting product. Background Art

[0002] Marek's disease virus (MDV) belongs to the herpesvirus family. It can destroy the immune system of chickens and cause tumors in multiple internal organs of chickens. It is highly contagious.

[0003] Mugwort, called "herbal medicine" by "Medicine Saint" Li Shizhen, is known as the "diamond among herbs". It has the effects of warming the meridians and stopping bleeding, removing dampness and cold, inhibiting bacteria and resisting insects. Mugwort leaves are the dried leaves of mugwort, which mainly contain volatile oils, terpenes, flavonoids, alkaloids and polysaccharides. Recent studies have shown that the essential oil and other active ingredients in mugwort leaves have the effects of scavenging free radicals and anti-oxidation. It can be used as a new antioxidant in food, beauty and skin care, and industrial production. However, the effect of mugwort essential oil on Marek's virus is still unclear. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of wormwood essential oil in the preparation of virus-inhibiting products.

[0005] Application of wormwood essential oil in preparing a product for inhibiting viruses, wherein the virus is Marek's disease virus.

[0006] Preferably, the wormwood essential oil inhibits the replication of Marek's disease virus in chicken cells.

[0007] Preferably, the concentration of the wormwood essential oil is 30 μg / mL to 960 μg / mL.

[0008] Preferably, the concentration of the wormwood essential oil is 60 μg / mL to 360 μg / mL.

[0009] Preferably, the wormwood essential oil reduces the expression level of the viral gene pp38.

[0010] Preferably, the wormwood essential oil reduces the expression level of viral protein VP22.

[0011] Preferably, the wormwood essential oil reduces Marek's disease virus plaques.

[0012] Preferably, the product is a medicine comprising Artemisia argyi essential oil.

[0013] Preferably, the drug is used to prevent or combat Marek's disease in chickens, and to inhibit the formation of Marek's disease tumors in chickens infected with Marek's disease virus.

[0014] Preferably, the product is chicken feed, and the chicken feed consists of wormwood essential oil and basic feed.

[0015] Preferably, the basic feed is protein feed, carbohydrate feed, vitamin feed or mineral feed.

[0016] Preferably, the wormwood essential oil is purchased from Jishui Zhongnan Natural Spice Oil Factory in Jiangxi Province, model number ZN0139.

[0017] Md5 is a strong strain of MDV, belonging to serotype 1 MDV. It is more likely to cause lymphoid organ atrophy and early death syndrome than other low pathogenic strains. By studying the effect of AAEO on Md5 infection of chicken embryo fibroblasts, it was found that AAEO has a certain anti-MDV effect. Although vaccines have been developed for Marek's virus, Marek's virus is still widely spread, causing huge economic losses. Taking advantage of the anti-MDV property of AAEO and the strong antioxidant properties of AAEO, AAEO has a very broad prospect for the prevention and treatment of Marek's disease.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Mugwort essential oil can effectively inhibit the replication of Marek's virus. Mugwort essential oil can be used together with Marek's virus vaccine to protect chickens from Marek's virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of AAEO's toxicity test on CEF are shown;

[0021] Figure 2 The effect of different concentration gradients of AAEO on the gene expression of Md5 virus;

[0022] Figure 3 The changes of virus plaques in Md5 infected cells after AAEO treatment;

[0023] Figure 4 This is the effect of AAEO on the expression of Md5 viral protein. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0025] In the present invention, AAEO was purchased from Zhongnan Natural Fragrance Oil Factory, Jishui, Jiangxi Province, with model number ZN0139.

[0026] In the present invention, wormwood essential oil and Tween 80 are prepared in a mass ratio of 1:4, with a final concentration of 60 mg / mL, and a mixture of 60 mg / mL wormwood essential oil and Tween 80 is diluted to form AAEO of different concentrations in the embodiment, and DMEM medium is used during dilution, and DMEM medium information: brand is gibco, REF is C11995500BT, and lot is 6124171. Among them, Tween 80 and DMEM medium have no inhibitory effect on Marek's disease virus.

[0027] 1. Determination of AAEO toxicity to chicken embryo fibroblasts

[0028] After chicken embryo fibroblasts (CEF) adhered for 12 hours, different concentrations of AAEO were added, including 30 μg / mL, 60 μg / mL, 120 μg / mL, 240 μg / mL, 360 μg / mL, 480 μg / mL and 960 μg / mL. After incubation for 24 hours, 10% CCK8 was added. After incubation for a period of time, the OD value was measured at a wavelength of 450 mm using a microplate reader.

[0029] The results are as follows Figure 1 As shown, the cell viability of the treatment groups with different concentrations of AAEO was close to 100% compared with the control group without AAEO, indicating that AAEO has basically no toxicity to CEF.

[0030] 2. Effects of AAEO on MDV gene transcription

[0031] pp38 is a unique gene of Md5. The effects of different concentrations of AAEO on Md5 virus were determined by amplifying the viral gene pp38. The different concentrations were 0μg / mL, 60μg / mL, 120μg / mL, 240μg / mL, and 360μg / mL.

[0032] Chicken embryo fibroblasts were inoculated into a 12-well plate and cultured in a 37°C incubator for 12 hours. After the cells adhered to the wall, wormwood essential oil was added at concentrations of 0 μg / mL, 60 μg / mL, 120 μg / mL, 240 μg / mL, and 360 μg / mL. After pretreatment for 2 hours, Md5 was inoculated. RNA samples were collected 24 hours and 48 hours after virus infection, and the expression of viral gene pp38 was detected by reverse transcription and fluorescence quantitative PCR.

[0033] RT-qPCR results showed that AAEO could inhibit the expression of Md5 virus pp38 gene. Figure 2As shown, 24 hours after infection, the wormwood essential oil treatment group was compared with the control group. The viral RNA in the 60μg / mL wormwood concentration treatment group did not decrease significantly, P < 0.05, the viral gene pp38 in the 120μg / mL and 240μg / mL wormwood essential oil treatment groups decreased, P < 0.01, and the viral gene pp38 in the 360μg / mL wormwood essential oil treatment group decreased significantly, P < 0.001; the wormwood essential oil treatment group for 48 hours of virus infection, compared with the control group, did not significantly decrease the relative expression of the viral gene pp38 at a concentration of 60μg / mL. The relative expression of the viral gene pp38 in the wormwood essential oil treatment groups at concentrations of 120μg / mL (P < 0.05), 240μg / mL (P < 0.01) and 360μg / mL (P < 0.001) was significantly decreased compared with the control group, indicating that wormwood essential oil inhibited viral growth.

[0034] 3. Effect of AAEO on viral protein expression levels

[0035] Chicken embryo fibroblasts were inoculated into a 6-well plate and cultured in a 37°C incubator for 12 h. After the cells adhered to the wall, mugwort essential oil was added at concentrations of 0 μg / mL, 60 μg / mL, 120 μg / mL, 240 μg / mL, and 360 μg / mL, respectively. Md5 was inoculated after pretreatment for 2 h.

[0036] By collecting cell samples 72 hours after infection and observing them under a microscope, it was found that compared with the control group, the virus plaques in the test group treated with wormwood essential oil gradually became smaller and the number decreased. AAEO had a significant inhibitory effect on MDV. The results are as follows Figure 3 shown.

[0037] Through Western-blot experiments, β-actin was used as the internal reference gene to explore the effect of adding wormwood essential oil on VP22 virus protein after 72 hours. The results are as follows Figure 4 As shown in the figure, compared with the control group with only virus added, the band of viral protein VP22 in the experimental group with wormwood essential oil and virus gradually weakened, and the viral protein showed a downward trend, indicating that AAEO has an inhibitory effect on the replication of Md5 virus.

[0038] Add mugwort essential oil as a beneficial additive to the feed consumed by chickens to improve the immunity of the flock.

[0039] The feed consumed by chickens includes protein feed, carbohydrate feed, vitamin feed and mineral feed.

[0040] Protein feed includes beans, cakes and fish meal.

[0041] Carbohydrate feed includes grains, brans and potatoes. The grains include corn, sorghum, barley and wheat. The brans include wheat bran and rice bran.

[0042] The vitamin feed includes forage, vegetables, melons and leaves. The forage includes clover and comfrey, the vegetables include carrot, cabbage and Chinese cabbage, and the leaves include elm leaves and acacia leaves.

[0043] Mineral feed includes bone meal, shell meal, eggshell meal, calcium carbonate and salt.

[0044] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0045] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0046] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The application of wormwood essential oil in the preparation of antiviral products, characterized in that: The virus is Marek's disease virus.

2. The use of the wormwood essential oil according to claim 1 in the preparation of a virus-inhibiting product, characterized in that: The wormwood essential oil inhibits the replication of Marek's disease virus in chicken cells.

3. The use of the wormwood essential oil according to claim 1 in the preparation of antiviral products, characterized in that: The concentration of the wormwood essential oil is 30 μg / mL to 960 μg / mL.

4. The use of the wormwood essential oil according to claim 1 in the preparation of antiviral products, characterized in that: The concentration of the wormwood essential oil is 60 μg / mL to 360 μg / mL.

5. The use of the wormwood essential oil according to claim 1 in the preparation of antiviral products, characterized in that: The wormwood essential oil reduces Marek's disease virus plaques.

6. The use of the wormwood essential oil according to claim 1 in preparing a virus-inhibiting product, characterized in that: The product is a medicine comprising wormwood essential oil.

7. The use of the wormwood essential oil according to claim 6 in preparing a virus-inhibiting product, characterized in that: The medicine is used for preventing or resisting Marek's disease in chickens and inhibiting the generation of Marek's disease tumors in chickens infected with Marek's disease virus.

8. The use of the wormwood essential oil according to claim 1 in preparing a virus-inhibiting product, characterized in that: The product is chicken feed, which consists of wormwood essential oil and basic feed.

9. The use of the wormwood essential oil according to claim 8 in preparing a virus-inhibiting product, characterized in that: The basic feed is protein feed, carbohydrate feed, vitamin feed or mineral feed.

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