Application of salidroside in preparing preparation for antiviral infection of Macrobrachium rosenbergii

Abstract

The present invention discloses the use of salidroside in preparing a preparation for antiviral infection of Macrobrachium rosenbergii, relating to the field of antiviral applications. In the present invention, by injecting salidroside into Macrobrachium rosenbergii infected with the DIV1 virus, the cumulative survival rate of Macrobrachium rosenbergii after infection with the DIV1 virus can be significantly improved, the replication ability of the DIV1 virus can be inhibited, and the relative expression of antiviral genes can be increased. Salidroside can be used as a drug or feed additive for preventing or treating viral infection in Macrobrachium rosenbergii.

Description

Technical Field

[0001] The present invention relates to the field of antiviral applications, and in particular 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 shrimp virus that can cause up to 100% mortality. It spreads rapidly, often causing symptoms such as white spots, jejunal atrophy, and hepatopancreatic atrophy. The disease is particularly prevalent in Macrobrachium rosenbergii aquaculture, causing severe economic losses. Currently, chemical drugs are commonly used to prevent and treat DIV1 outbreaks, but long-term use can lead to drug resistance in the pathogen and is ineffective.

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

[0004] For example, patent application CN110367399A discloses an extruded feed for California sea bass juveniles that improves the survival rate of standard seedlings and its preparation method. The feed comprises the following components by weight: 35-45 parts Japanese-grade fish meal, 3-12 parts fermented soybean meal, 20-25 parts high-gluten flour, 2-5 parts fish oil, 2-7 parts seaweed extract, 1-3 parts fish pulp, 5-15 parts krill meal, 2 parts California sea bass vitamin premix, 3 parts California sea bass mineral and other premix, 1-2 parts Chinese herbal immune preparation mixture, and 1 part live fly larvae antimicrobial peptide puree preparation. The feed in this invention incorporates the Chinese herbal immune preparation mixture, which contains flavonoids and their glycosides, organic acids, flavans, and their polymers found in hawthorn, which play an important role in improving fish feeding and stimulating appetite. Furthermore, the feed is supplemented with antiviral ingredients such as Andrographis paniculata, Isatis indigotica, Eucommia ulmoides, and Lysimachia chinensis, further enhancing the effect.

[0005] Patent application CN111227137A discloses a shrimp feed and its preparation method. The feed is made from the following components by weight: 20-25 parts fish meal, 20-25 parts corn flour, 10-15 parts cornstarch, 15-20 parts shell powder, 4-6 parts algae powder, 1-3 parts multivitamins, 3-5 parts Chinese herbal medicine, and 8-10 parts mineral additives. The Chinese herbal medicine is made from the following components by weight: 1-3 parts houttuynia cordata, 1-3 parts sterculia ginseng, 0.5-1.5 parts dandelion, 0.5-1.5 parts isatis root, 0.5-1.5 parts bupleurum root, and 0.5-1.5 parts liquorice. This shrimp feed contains abundant starch, protein, minerals, and vitamins necessary for the growth of grass shrimp. It also contains Chinese herbal ingredients, which have antiviral effects, can enhance the shrimp's immunity, and are free of antibiotic residues, making it safe and healthy.

[0006] Salidroside (SAL) is a phenolic glycoside compound extracted from the traditional Chinese medicine Rhodiola rosea. It can also be synthesized through other routes. It has significant advantages, including a wide range of applications, significant efficacy, minimal side effects, and low cost. Numerous studies have shown that salidroside has multiple benefits, including anti-fatigue and antioxidant properties, as well as anti-cancer, anti-apoptosis, and anti-inflammatory effects. However, there are currently no reports on the antiviral effects of salidroside in shrimp. Summary of the Invention

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

[0008] In order to achieve the above-mentioned purpose, the specific technical solutions of the present invention are as follows:

[0009] The present invention provides the use of salidroside in preparing a preparation for Macrobrachium rosenbergii to resist viral infection.

[0010] Preferably, the virus is Decapoda iridescent virus 1.

[0011] Preferably, the preparation is a medicine or a feed additive.

[0012] Preferably, when used, the dosage of salidroside in the preparation is 50-400 mg / kg.

[0013] More preferably, the dosage 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 of DIV1 virus, and significantly improve the antiviral gene expression at 48 hours and 72 hours. irf4Relative expression.

[0014] Preferably, the salidroside exerts its anti-viral infection effect by inhibiting viral replication ability.

[0015] As a preference, the salidroside can enhance the antiviral gene irf4 expression, and play an antiviral infection role.

[0016] The present invention also provides a breeding method for improving the survival rate of Macrobrachium rosenbergii. After Macrobrachium rosenbergii is infected with Decapoda iridescent virus 1, salidroside or a biological preparation containing salidroside is injected or fed.

[0017] Preferably, the dosage of salidroside is 50-400 mg / kg.

[0018] More preferably, the dosage of salidroside is 200 mg / kg.

[0019] Salidroside at doses of 50 mg / kg, 100 mg / kg, 200 mg / kg and 400 mg / kg is not toxic to Macrobrachium rosenbergii, and 200 mg / kg of salidroside 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 of antiviral genes.

[0020] Beneficial effects of the present invention:

[0021] In the present invention, by injecting salidroside into Macrobrachium rosenbergii infected with the DIV1 virus, the cumulative survival rate of Macrobrachium rosenbergii after infection with the DIV1 virus can be significantly improved, the replication ability of the 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 in Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of salidroside on the cumulative survival rate of Macrobrachium rosenbergii infected with DIV1; * indicates P <0.05.

[0023] Figure 2 The effect of salidroside on the viral load of DIV1 in the hepatopancreas of Macrobrachium rosenbergii; * indicates P <0.05, ** indicates P <0.01.

[0024] Figure 3 Effects of salidroside on the hepatopancreas of Macrobrachium rosenbergii irf4 The effects of gene expression; Figure 3 A in Figure 3 B in Figure 3C in the figure indicates the effects of salidroside on the hepatopancreas of Macrobrachium rosenbergii after stimulation for 24 hours, 48 ​​hours and 72 hours at DIV1. irf4 Effects on gene expression; ** indicates P <0.01. DETAILED DESCRIPTION

[0025] Example 1 Safety Experiment of Salidroside

[0026] Salidroside (purchased from Yuanye Bio, CAS number: 10338-51-9) was dissolved in PBS buffer to prepare a stock solution at a concentration of 48 mg / ml. One hundred and twenty healthy, uniformly sized Macrobrachium rosenbergii (weighing 0.5 ± 0.01 g) were divided equally into four groups of 30 shrimp each and placed in 40-liter square tanks at 20°C with continuous aeration. The salidroside stock solution was diluted to 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg, respectively, and injected into Macrobrachium rosenbergii, with 50 μL injected per shrimp. Thirty shrimp of the same size were injected with the same volume of PBS buffer as a control group. The cumulative survival rate of each treatment group was measured.

[0027] like Figure 1 As shown in the results, the Macrobrachium rosenbergii in each treatment group was observed for a total of 168 hours. The results showed that there was no death in the experimental shrimp in each treatment group, and the survival rate was 100%, indicating that salidroside had no significant toxic effect on Macrobrachium rosenbergii at this experimental concentration.

[0028] Example 2 Salidroside antiviral experiment

[0029] Salidroside (purchased from Yuanye Bio, CAS number: 10338-51-9) was dissolved in PBS buffer to a stock solution at a concentration of 48 mg / ml. One hundred fifty healthy, uniformly sized Macrobrachium rosenbergii (weighing 0.5 ± 0.01 g) were divided equally into five groups of 30 shrimp each and placed in 40-liter square tanks at 20°C with continuous aeration. The salidroside stock solution was diluted to 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg for later use. The DIV1 positive Macrobrachium rosenbergii muscle and PBS buffer were taken in a sterile enzyme-free glass grinding tube at a ratio of 1:10. The grinding tube was placed in an ice water bath and slowly ground into a homogenate. The homogenate was then transferred to a centrifuge tube and centrifuged in a low-temperature centrifuge at 4°C, 3000 r / min for 20 min. The supernatant was taken and centrifuged again at 4°C, 8000 r / min for 25 min. The supernatant was filtered through a 0.22 μm pore size filter membrane and stored at 4°C to prepare the crude virus extract. The DIV1 crude extract was diluted with PBS buffer to a concentration of 6.1×10 4.26The number of copies / μl was used in the challenge experiment. Salidroside solutions of varying concentrations were mixed with DIV1 suspension at a volume ratio of 1:1 to form the drug groups: DIV1+SAL 50 mg / kg, DIV1+SAL 100 mg / kg, DIV1+SAL 200 mg / kg, and DIV1+SAL 400 mg / kg. Each group was injected with 50 μL of the DIV1 virus suspension without salidroside. The control group was injected with the same dose of DIV1 virus suspension without salidroside. After the challenge experiment, the cumulative survival rate of each treatment group was observed. The Kaplan-Meier survival analysis was used to calculate the cumulative survival rate of the experimental shrimp in the different treatment groups, and the log-rank method was used to test the differences in cumulative survival rate.

[0030] like Figure 1 As shown, when the salidroside content was 200 mg / kg, the cumulative survival rate of Macrobrachium rosenbergii was significantly higher than that of the DIV1 infection control group at 120 hours and 144 hours after DIV1 infection ( P <0.05), indicating that salidroside at a concentration of 200 mg / kg can significantly improve the ability of Macrobrachium rosenbergii to resist DIV1 virus.

[0031] Example 3 Salidroside inhibits viral replication and enhances antiviral gene expression

[0032] Salidroside (purchased from Yuanye Bio, CAS No. 10338-51-9) was dissolved in PBS buffer to prepare a stock solution at a concentration of 48 mg / ml for later use. 120 healthy and uniformly sized Macrobrachium rosenbergii (weighing 0.5 ± 0.01 g) were divided equally into four groups, 30 shrimp per group, and placed in a 40 L square water tank at 20°C with continuous aeration. Based on the antiviral test results of salidroside in Example 2, a salidroside concentration of 200 mg / kg showed the best antiviral effect. The salidroside stock solution was diluted to 200 mg / kg and diluted with PBS buffer to prepare a DIV1 suspension with a concentration of 6.1 × 10 4.26 The salidroside solution was mixed with the DIV1 suspension at a volume of 1:1 for later use.

[0033] The experiment involved two groups: a DIV1 stimulation group and a non-stimulation group. The DIV1 stimulation group had two treatment groups: DIV1 injection (DIV1) and DIV1 injection of a salidroside solution (DIV1+SAL 200 mg / kg). The non-stimulation group also had two treatment groups: PBS injection (PBS) and salidroside solution (SAL 200 mg / kg). Sixty Macrobrachium rosenbergii were injected into each treatment group, with 50 μL injected per fish. Hepatopancreatic tissue was collected 24, 48, and 72 hours after injection for analysis of viral load and antiviral gene expression at DIV1.

[0034] DNA from the hepatopancreas was extracted using a kit and real-time fluorescence PCR was performed to detect DIV1 viral gene expression. The primer sequences for quantitative PCR are shown in Table 1. Total RNA from the hepatopancreas was extracted using the Trizol method, and then cDNA was obtained using a reverse transcription kit. Real-time fluorescence quantitative PCR was performed to detect hepatopancreas antiviral related genes. irf4 The expression of 147 genes was detected by fluorescence quantitative PCR. The primer sequences of fluorescence quantitative PCR are shown in Table 1. Experimental data were analyzed by SPSS 19.0 using homogeneity of variance test, one-way ANOVA, Duncan's test and independent sample T test. P The difference was significant when the value was <0.05.

[0035] Table 1 Fluorescence quantitative PCR primer sequences

[0036]

[0037] Among them, FAM is 6-carboxyfluorescein and TAMRA is tetramethylrhodamine.

[0038] The viral load of the hepatopancreas of the two treatment groups stimulated at DIV1 at different time points was analyzed. Figure 2 As shown, at 24 hours, 48 ​​hours and 72 hours, the viral load in the hepatopancreas of the group injected with DIV1 and 200 mg / kg salidroside was significantly lower than that of the group injected with DIV1 ( P <0.05), indicating that salidroside at a concentration of 200 mg / kg can significantly inhibit the replication ability of DIV1 virus.

[0039] Interferon regulatory factor 4 (IRF4) is important in regulating interferon response to viral infection and regulating interferon-induced genes, and is involved in the body's antiviral immune response. irf4 Perform quantitative analysis. Figure 3 As shown, after DIV1 virus stimulation, the hepatopancreas antiviral gene of Macrobrachium rosenbergii irf4 The relative expression increased, and 200mg / kg salidroside could significantly increase the antiviral gene expression at 48 hours and 72 hours. irf4 Relative expression ( P <0.01).

[0040] In summary, the research results of the present invention show that 50 mg / kg, 100 mg / kg, 200 mg / kg and 400 mg / kg of salidroside are not toxic to Macrobrachium rosenbergii, and 200 mg / kg of salidroside 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 of antiviral genes.

Claims

1. Use of salidroside in the preparation of a preparation for antiviral infection of Macrobrachium rosenbergii; the virus is Decapoda iridescent virus 1.

2. The use according to claim 1, characterized in that The preparation is a medicine or a feed additive.

3. The use according to claim 1, characterized in that The dosage of salidroside in the preparation is 200 mg / kg.

4. The use according to claim 1, characterized in that The salidroside exerts an anti-viral infection effect by inhibiting the viral replication ability.

5. The use according to claim 1, characterized in that Salidroside increases antiviral genes irf4 expression, and play an antiviral infection role.

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