Application of salidroside in preparation of preparation for resisting virus infection of macrobrachium rosenbergii

Abstract

The invention discloses application of salidroside in preparation of a preparation for resisting virus infection of macrobrachium rosenbergii, and relates to the field of antiviral application. The salidroside is injected into the macrobrachium rosenbergii infected with the DIV1 virus, so that the accumulative survival rate of the macrobrachium rosenbergii infected with the DIV1 virus can be remarkably increased, the replication capacity of the DIV1 virus is inhibited, and the relative expression quantity of antiviral genes is increased; the salidroside can be used as a medicine or a feed additive for preventing or treating antiviral infection of macrobrachium rosenbergii.

Description

Technical Field

[0001] The present invention relates to the field of antiviral applications, and particularly to the application of salidroside in the preparation of a preparation for antiviral infection of Macrobrachium rosenbergii. Background Art

[0002] Decapod iridescent virus 1 (DIV1) is a virus that can cause a 100% mortality rate in shrimp. It spreads rapidly and often causes symptoms such as white spots, empty intestines, and atrophy of the hepatopancreas after infecting shrimp. Especially during the cultivation of Macrobrachium rosenbergii, the incidence rate is high, resulting in serious economic losses. Currently, some chemical drugs are often used to prevent and treat the outbreak of DIV1 virus, but long-term use will lead to drug resistance of pathogens and the effect is not significant.

[0003] Chinese herbal medicines have good disease resistance effects, are not prone to drug resistance, have little impact on the ecological environment, and have the dual effects of individual treatment and group prevention, and can effectively promote the health and sustainable development of the aquaculture industry.

[0004] For example, the patent application with the publication number CN110367399A discloses an expanded feed for juvenile Micropterus salmonides to improve the survival rate of fry and its preparation method. The expanded feed for juvenile Micropterus salmonides includes the following components in parts by weight: 35 - 45 parts of Japanese grade fish meal, 3 - 12 parts of fermented soybean meal, 20 - 25 parts of high-gluten flour, 2 - 5 parts of fish oil, 2 - 7 parts of seaweed extract, 1 - 3 parts of fish solubles, 5 - 15 parts of krill meal, 2 parts of vitamin premix for Micropterus salmonides, 3 parts of mineral and other premixes for Micropterus salmonides, 1 - 2 parts of a Chinese herbal medicine immune preparation mixture, and 1 part of a live fly maggot antibacterial peptide original pulp preparation. The feed of this invention adds a Chinese herbal medicine immune preparation mixture. Flavonoids, organic acids, flavans and their polymers contained in hawthorn in the Chinese herbal medicine immune preparation mixture play an important role in improving the feeding and appetizing of fish; in addition, it is more effective when combined with Andrographis paniculata, Isatis tinctoria, Eucommia ulmoides and Lysimachia christinae with antiviral effects.

[0005] The patent application with the publication number CN111227137A discloses a shrimp feed and its preparation method, which is made of the following components in parts by weight: including 20-25 parts of fish meal, 20-25 parts of corn flour, 10-15 parts of wheat middlings, 15-20 parts of oyster shell powder, 4-6 parts of algal powder, 1-3 parts of compound vitamins, 3-5 parts of traditional Chinese medicine preparation, and 8-10 parts of mineral additive; among them, the traditional Chinese medicine preparation is made of the following components in parts by weight: 1-3 parts of Houttuynia cordata, 1-3 parts of forsythia and honeysuckle, 0.5-1.5 parts of dandelion, 0.5-1.5 parts of isatis root, 0.5-1.5 parts of bupleurum, and 0.5-1.5 parts of liquorice. This shrimp feed contains rich starch, protein, minerals and vitamins required for the growth of grass shrimp, and also contains Chinese herbal medicine components, has an antiviral effect, can improve the immune ability of grass shrimp, has no antibiotic residue, and is safe and healthy.

[0006] Salidroside (SAL) is a phenolic glycoside compound extracted from Chinese herbal medicine Rhodiola plants, and can also be artificially synthesized through other ways. It has significant advantages such as a wide range of applications, remarkable curative effects, small side effects, and low prices. A large number of studies have shown that salidroside has a variety of effects. It not only has anti-fatigue and antioxidant effects, but also can play the effects of anti-cancer, anti-cell apoptosis and anti-inflammatory. However, there is currently no relevant report on the research of salidroside in shrimp antiviral. Summary of the Invention

[0007] The purpose of the present invention is to provide the use of salidroside in the preparation of a drug or feed additive for preventing or treating antiviral infection of Macrobrachium rosenbergii, improve the survival rate of Macrobrachium rosenbergii infected with DIV1, and prevent the outbreak of diseases of Macrobrachium rosenbergii caused by DIV1.

[0008] In order to achieve the above purpose, the specific technical solution of the present invention is as follows: The present invention provides the application of salidroside in the preparation of a preparation for antiviral infection of Macrobrachium rosenbergii.

[0009] Preferably, the virus is Decapod iridovirus 1.

[0010] Preferably, the preparation is a drug or a feed additive.

[0011] Preferably, when applied, the dose of salidroside in the preparation is 50-400 mg / kg.

[0012] More preferably, the dose of salidroside in the preparation is 200 mg / kg. When the concentration of salidroside is 200 mg / kg, it can significantly improve the ability of Macrobrachium rosenbergii to resist DIV1 virus, inhibit the replication ability of DIV1 virus, and can extremely significantly improve the relative expression of antiviral genes at 48 hours and 72 hours. irf4 Relative expression.

[0013] Preferably, the salidroside plays an antiviral infection role by inhibiting the virus replication ability.

[0014] Preferably, the salidroside plays an antiviral infection role by enhancing the expression of antiviral genes. irf4

[0015] The present invention also provides a cultivation method for improving the survival rate of Macrobrachium rosenbergii. After Macrobrachium rosenbergii is infected with Decapod iridescent virus 1, salidroside or a biological preparation containing salidroside is injected or fed.

[0016] Preferably, the dosage of the salidroside is 50 - 400 mg / kg.

[0017] More preferably, the dosage of the salidroside is 200 mg / kg.

[0018] Salidroside at dosages of 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg is not toxic to Macrobrachium rosenbergii. Moreover, salidroside at 200 mg / kg can significantly improve the cumulative survival rate of Macrobrachium rosenbergii after infection with DIV1 virus, inhibit the replication ability of DIV1 virus, and increase the relative expression level of antiviral genes.

[0019] Advantages of the present invention: In the present invention, by injecting salidroside into Macrobrachium rosenbergii infected with DIV1 virus, the cumulative survival rate of Macrobrachium rosenbergii after infection with DIV1 virus can be significantly improved, the replication ability of DIV1 virus can be inhibited, and the relative expression level of antiviral genes can be increased; salidroside can be used as a drug or feed additive for preventing or treating antiviral infection of Macrobrachium rosenbergii. Description of the drawings

[0020] Figure 1 shows the effect of salidroside on the cumulative survival rate of Macrobrachium rosenbergii after infection with DIV1; * indicates P <0.05.

[0021] Figure 2 shows the effect of salidroside on the amount of DIV1 virus in the hepatopancreas of Macrobrachium rosenbergii; * indicates P <0.05, ** indicates P <0.01.

[0022] Figure 3 shows the effect of salidroside on the irf4 gene expression in the hepatopancreas of Macrobrachium rosenbergii; among them, Figure 3 A in Figure 3 B in Figure 3 C in respectively represent the effects of salidroside on the hepatopancreas of Macrobrachium rosenbergii 24 hours, 48 hours, and 72 hours after DIV1 stimulation.irf4 Effect on gene expression; ** indicates P <0.01. Specific implementation mode

[0023] Example 1 Safety experiment of salidroside

[0024] Dissolve salidroside (purchased from Yuanye Bio-Technology Co., Ltd., CAS No.: 10338-51-9) in PBS buffer to prepare a stock solution with a concentration of 48 mg / ml for standby. Divide 120 healthy and evenly sized Macrobrachium rosenbergii (body weight: 0.5 ± 0.01 g) into 4 groups, with 30 shrimps in each group. Place them in 40 L square water tanks at a water temperature of 20 °C, and continuously aerate with oxygen. Dilute the salidroside stock solution to 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg respectively, and inject Macrobrachium rosenbergii, with each shrimp injected with 50 μL. Another 30 Macrobrachium rosenbergii of the same specification are injected with the same volume of PBS buffer as the control group, and observe the cumulative survival rate of Macrobrachium rosenbergii in each treatment group.

[0025] As Figure 1 shown, observe the Macrobrachium rosenbergii in each treatment group for 168 hours in total. The results show that there is no death in the experimental shrimps of each treatment group, and the survival rate is 100%, indicating that salidroside has no significant toxic effect on Macrobrachium rosenbergii at this experimental concentration.

[0026] Example 2 Antiviral experiment of salidroside

[0027] Dissolve salidroside (purchased from Yuanye Bio-Technology Co., Ltd., CAS No.: 10338-51-9) in PBS buffer to prepare a stock solution with a concentration of 48 mg / ml for standby. Divide 150 healthy and evenly sized Macrobrachium rosenbergii (body weight: 0.5 ± 0.01 g) into 5 groups, with 30 shrimps in each group. Place them in 40 L square water tanks at a water temperature of 20 °C, and continuously aerate with oxygen. Dilute the salidroside stock solution to 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg respectively for standby. Take the muscle of DIV1 positive Macrobrachium rosenbergii and PBS buffer in a ratio of 1:10 in a sterile and enzyme-free glass grinding tube. Place the grinding tube in an ice-water bath and slowly grind it into a homogenate. Then transfer the homogenate into a centrifuge tube and centrifuge it at 3000 r / min for 20 min at 4 °C in a low-temperature centrifuge. Take the supernatant, and then centrifuge it at 8000 r / min for 25 min at 4 °C. Take the supernatant again and filter it through a filter membrane with a pore size of 0.22 μm, and store it at 4 °C to prepare the crude virus extract. Dilute the DIV1 crude extract with PBS buffer, and the concentration is 6.1×10 4.26Copy number / μl was used for the challenge test. Different concentrations of salidroside solutions were mixed with the DIV1 suspension in a volume ratio of 1:1 to form drug groups, namely DIV1 + SAL50mg / kg, DIV1 + SAL100mg / kg, DIV1 + SAL200mg / kg, and DIV1 + SAL400mg / kg. These were injected into Macrobrachium rosenbergii, with 50 μL injected per shrimp. The control group was injected with the same dose of DIV1 virus suspension without salidroside. After the challenge test, the cumulative survival rate of Macrobrachium rosenbergii in each treatment group was observed. The Kaplan-Meier method of survival analysis was used to calculate the cumulative survival rate of the experimental shrimp in different treatment groups, and the log-rank method was used to test the differences in cumulative survival rates.

[0028] As Figure 1 shown, when the salidroside content was 200 mg / kg, after 120 hours and 144 hours of DIV1 infection, the cumulative survival rate of Macrobrachium rosenbergii was significantly higher than that of the DIV1-infected control group ( P <0.05), indicating that when the salidroside concentration was 200 mg / kg, it could significantly improve the ability of Macrobrachium rosenbergii to resist DIV1 virus.

[0029] Example 3 Inhibition of virus replication and increase in antiviral gene expression by salidroside

[0030] Salidroside (purchased from Yuanye Bio-Technology Co., Ltd., CAS No.: 10338-51-9) was dissolved in PBS buffer to prepare a stock solution with a concentration of 48 mg / ml for standby. 120 healthy and uniformly sized Macrobrachium rosenbergii (weight 0.5 ± 0.01 g) were evenly divided into 4 groups, with 30 shrimp in each group. They were placed in 40-L square water tanks at a water temperature of 20 °C with continuous oxygen aeration. According to the antiviral experimental results of salidroside in Example 2, the best disease resistance effect was achieved when the salidroside concentration was 200 mg / kg. The salidroside stock solution was diluted to 200 mg / kg, and the DIV1 suspension was prepared by dilution with PBS buffer at a concentration of 6.1×10 4.26 copy number / μl. The salidroside solution was mixed with the DIV1 suspension in a volume ratio of 1:1 for standby.

[0031] The experiment was divided into a DIV1 stimulation group and a non-stimulation group. The DIV1 stimulation group had 2 treatment groups, namely DIV1 injection (DIV1) and injection of a mixture of DIV1 and salidroside (DIV1 + SAL200mg / kg). The non-stimulation group also had 2 treatment groups, namely PBS buffer injection (PBS) and salidroside solution injection (SAL200mg / kg). 60 Macrobrachium rosenbergii were injected in each treatment group, with 50 μL injected per shrimp. Then, the hepatopancreas tissues of Macrobrachium rosenbergii were collected at 24 hours, 48 hours, and 72 hours after injection for analysis of DIV1 virus load and antiviral gene expression.

[0032] Extract the DNA of the hepatopancreas using a kit, perform real-time fluorescence PCR to detect the expression of DIV1 virus genes, and the primer sequences for quantitative PCR are shown in Table 1. Extract the total RNA of the hepatopancreas using the Trizol method, then obtain cDNA using a reverse transcription kit, and perform real-time fluorescence quantitative PCR to detect the antiviral-related genes of the hepatopancreas irf4 The expression was analyzed using SPSS 19.0 for the homogeneity of variance test, one-way ANOVA, Duncan's test, and independent samples T-test of the experimental data, P and <0.05 was considered significantly different.

[0033] Table 1 Primer sequences for fluorescence quantitative PCR

[0034] Among them, FAM is 6-carboxyfluorescein and TAMRA is tetramethylrhodamine.

[0035] Analyze the viral loads in the hepatopancreas of Macrobrachium rosenbergii at different time points in two treatment groups stimulated by DIV1. As Figure 2 shown, at 24 hours, 48 hours, and 72 hours, the viral load in the hepatopancreas of Macrobrachium rosenbergii in the treatment group injected with a mixture of DIV1 and 200 mg / kg salidroside was significantly lower than that in the DIV1 injection treatment group ( P <0.05), indicating that when the concentration of salidroside is 200 mg / kg, it can significantly inhibit the replication ability of DIV1 virus.

[0036] Interferon regulatory factor 4 (IRF4) is important in regulating the interferon response to virus infection and regulating interferon-induced genes, and is involved in the antiviral immune response of the body. Quantitatively analyze the antiviral genes in the hepatopancreas of Macrobrachium rosenbergii at different time points in the DIV1 virus-stimulated group and the non-stimulated group. irf4 As Figure 3 shown, after stimulation with DIV1 virus, the relative expression of antiviral genes in the hepatopancreas of Macrobrachium rosenbergii increased, and at the same time, 200 mg / kg salidroside could extremely significantly increase the relative expression of antiviral genes irf4 at 48 hours and 72 hours ( irf4 <0.01). P <0.01).

[0037] In summary, the research results of the present invention show that salidroside at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg is not toxic to Macrobrachium rosenbergii, and 200 mg / kg salidroside can significantly increase the cumulative survival rate of Macrobrachium rosenbergii after infection with DIV1 virus, inhibit the replication ability of DIV1 virus, and increase the relative expression level of antiviral genes.

Claims

1. Application of salidroside in preparing a preparation for anti-viral infection of Macrobrachium rosenbergii.

2. The application according to claim 1, characterized in that The virus is Decapod iridovirus 1.

3. The application according to claim 1, wherein The preparation is a drug or a feed additive.

4. The application according to claim 1, wherein When applied, the dosage of salidroside in the preparation is 50 - 400 mg / kg.

5. The application according to claim 4, wherein The dosage of salidroside in the preparation is 200 mg / kg.

6. The application according to claim 1, wherein The salidroside plays a role in anti-viral infection by inhibiting the virus replication ability.

7. The application according to claim 1, wherein The salidroside plays a role in antiviral infection by enhancing the expression of antiviral genes irf4 ​

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