A traditional Chinese medicine composition for resisting mandarin fish double RNA virus, a preparation method and application thereof

Abstract

This invention discloses a traditional Chinese medicine composition for combating mandarin fish double RNA virus, comprising at least one of geniposide, baicalin, luteolin, magnolol, and mangiferin as active ingredients. The invention also discloses a method for preparing the traditional Chinese medicine composition and its applications. This invention can significantly inhibit the proliferation and replication of mandarin fish double RNA virus and significantly improve the survival rate of mandarin fish.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control technology, specifically relating to a traditional Chinese medicine composition for resisting mandarin fish double RNA virus, its preparation method and application. Background Technology

[0002] Mandarin fish ( Siniperca chuatsi Mandarin fish (Siniperca chuatsi) belongs to the genus *Siniperca* of the family Perciformes. It is a unique and valuable freshwater economic fish species endemic to my country, commonly known as mandarin fish or mandarin flower fish. Widely distributed in major water systems across my country, mandarin fish is highly favored by consumers due to its tender, smooth flesh, lack of intramuscular bones, and rich content of high-quality protein and unsaturated fatty acids. It has become one of the leading freshwater aquaculture species in my country. However, the healthy development of mandarin fish farming is facing serious challenges.

[0003] Mandarin fish double RNA virus ( Siniperca chuatsi Birnavirus (SCBV) is a novel non-enveloped icosahedral virus with a two-segmented stranded RNA genome. This virus primarily infects 2-5cm long mandarin fish fry, characterized by rapid onset and high mortality. Typical symptoms include darkening of body color and dragging feces. Autopsy reveals severe liver congestion and a large accumulation of yellow mucus in the intestines. Currently, mandarin fish farming is rapidly transitioning from traditional live feed to formulated feed, but SCBV infection leads to a decline in the health of fry, significantly reducing the success rate of feed adaptation and severely hindering the industry's upgrading process.

[0004] To date, there are no effective prevention and control technologies or products for mandarin fish double RNA virus disease, either domestically or internationally. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a safe, effective, and low-cost compound traditional Chinese medicine for the prevention and treatment of mandarin fish double RNA virus, thus providing an effective means of prevention and treatment for this viral disease.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A traditional Chinese medicine composition for resisting mandarin fish double RNA virus, wherein the traditional Chinese medicine composition comprises any one of geniposide, baicalin, luteolin, magnolol, and mangiferin as active ingredients.

[0008] Preferably, the traditional Chinese medicine composition includes geniposide, baicalin, luteolin, magnolol, and mangiferin.

[0009] Preferably, in the traditional Chinese medicine composition, the mass ratio of geniposide, baicalin, luteolin, magnolol, and mangiferin is 8-32:8-32:2-8:2-8:1.

[0010] Different mass ratios of the components in a compound traditional Chinese medicine composition can affect the anti-SCBV effect. Studies have found that when the mass ratio of geniposide, baicalin, luteolin, magnolol, and mangiferin is 32:32:8:8:1, it can better inhibit the proliferation and replication of SCBV and reduce the mortality rate of mandarin fish.

[0011] On the other hand, the present invention also provides a feed containing the aforementioned traditional Chinese medicine composition for resisting mandarin fish double RNA virus.

[0012] Preferably, each 1000g of feed contains 20g of the traditional Chinese medicine composition.

[0013] On the other hand, the present invention also provides the application of the traditional Chinese medicine composition in the preparation of drugs against mandarin fish double RNA virus.

[0014] Preferably, in the application, the safe in vitro cell antiviral concentrations of each component are: geniposide 50 μg / mL, baicalin 25 μg / mL, luteolin 0.78125 μg / mL, magnolol 6.25 μg / mL, and mangiferin 50 μg / mL.

[0015] Preferably, the cells are mandarin fish brain cell lines.

[0016] On the other hand, the present invention also provides the application of traditional Chinese medicine extracts in the preparation of drugs against mandarin fish double RNA virus, characterized in that the traditional Chinese medicine extracts are at least one of geniposide, baicalin, luteolin, magnolol, and mangiferin.

[0017] Compared with existing technologies, the beneficial effects of the present invention are as follows:

[0018] (1) At least one of geniposide, baicalin, luteolin, magnolol, and mangiferin can inhibit the proliferation and replication of SCBV in vitro.

[0019] (2) The compound Chinese medicine composed of geniposide, baicalin, luteolin, magnolol and mangiferin can significantly inhibit the proliferation and replication of SCBV in vivo and in vitro, and reduce the mortality rate of SCBV.

[0020] (3) It improved the antiviral ability and survival rate of mandarin fish, and increased the income of aquaculture. Attached Figure Description

[0021] Figure 1 This figure shows the copy number results of 11 Chinese herbal extracts against SCBV proliferation and replication in vitro. ** indicates the drug with better inhibitory effect on SCBV proliferation and replication, and **** indicates the drug with the best efficacy. 1 is geniposide, 2 is baicalin, 3 is luteolin, 4 is magnolol, 5 is mangiferin, 6 is arctigenin, 7 is limonene, 8 is quercetin, 9 is harbazoside, 10 is hyperoside, and 11 is chlorogenic acid.

[0022] Figure 2 This figure shows the copy number results of SCBV proliferation and replication in vitro by a compound traditional Chinese medicine composed of five herbal extracts. *** indicates the drug with better inhibitory effects on SCBV proliferation and replication. The drug ratios are: geniposide:baicalin:luteolin:magnoliol:mangiferin = 8:8:2:2:1 (Drug 1); geniposide:baicalin:luteolin:magnoliol:mangiferin = 16:16:4:4:1 (Drug 2); geniposide:baicalin:luteolin:magnoliol:mangiferin = 32:32:8:8:1 (Drug 3).

[0023] Figure 3 This is a graph showing the survival rate of fish at different time points after viral infection.

[0024] Figure 4 Clinical symptoms and anatomical images of fish in the control group and the drug group. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Unless otherwise specified, the reagents, instruments, etc. used in the embodiments of the present invention are all conventional commercially available products.

[0027] Example 1

[0028] 1. Materials and Methods

[0029] 1.1 Cells and Viruses

[0030] The Chinese perch brain cell (CPB) line was established and preserved in our laboratory. The Chinese perch double RNA virus (SCBV) was isolated and preserved in our laboratory.

[0031] 1.2 Main Reagents and Traditional Chinese Medicine Extracts

[0032] Phosphate-buffered saline (PBS) was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0033] L-15 medium was purchased from Biosharp.

[0034] Fetal bovine serum was purchased from Suzhou Ekosei Biotechnology Co., Ltd.

[0035] Eleven kinds of Chinese herbal extracts, including geniposide (CAS: 24512-62-7), baicalin (CAS: 21967-41-9), luteolin (CAS: 491-70-3), magnolol (CAS: 528-43-8), mangiferin (CAS: 4773-96-0), arctigenin (CAS: 7770-78-7), limonene (CAS: 1180-71-8), quercetin (CAS: 522-12-3), harbacoside (CAS: 6926-08-5), hyperoside (CAS: 548-04-9), and chlorogenic acid (CAS: 327-97-9), were purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0036] 1.3 Determination of the maximum safe concentration of 11 kinds of Chinese herbal extracts in CPB cells

[0037] Eleven Chinese herbal extracts—geniposide, baicalin, luteolin, magnolol, mangiferin, arctigenin, limonin, quercetin, harpaquinone, hypericin, and chlorogenic acid—were prepared to a concentration of 2 mg / mL using L-15 medium containing 2% dimethyl sulfoxide (DMSO). Before use, each of the eleven extracts was serially diluted 2-fold to five appropriate concentrations using L-15 medium containing 5% FBS. EPC cells were seeded into 96-well plates. After confluent monolayers were established, the culture medium was discarded, and 100 μL of each of the eleven extracts was added to each well, with four replicates for each dilution. A blank cell control was simultaneously set up. The cells were incubated at 28°C, and the morphology and growth of EPC cells were observed daily. After culturing for 72 hours, cell viability was assessed using the CellTiter96@AQueous One SolutionReagent kit. 20 μL of CellTiter 96@AQueous One SolutionReagent was added to each well, and the cells were incubated at 28°C for 4 hours. The D490nm value was then measured. The safe concentrations of the drug for different cell groups were calculated. The formula was: Cell viability = (D490nm value of the drug group / D490nm value of the control group).

[0038] 1.4 Inhibitory effect of 11 kinds of traditional Chinese medicine extracts on SCBV in vitro

[0039] Based on the determined safe concentrations of 11 traditional Chinese medicine extracts, 12-well plates confluent with monolayer CPB cells were used. L-15 medium was added to each well to dilute to the safe concentration of each drug, with L-15 medium serving as a control. 1 mL / well was used, with three replicates per drug. The plates were incubated at 28°C for 2 hours. The cell supernatant was discarded, and the cells were washed once with PBS. 1 mL of the diluted traditional Chinese medicine and virus mixture (TCID50) was then added. 50 = 10-7 ( / well), place in a 28℃ cell culture incubator for 2 hours, discard the cell supernatant, wash once with PBS, add 1mL of 2% FBS L-15 medium containing a safe concentration of traditional Chinese medicine extract, place in a 28℃ cell culture incubator, and when the virus control shows 80% lesions, collect the virus and traditional Chinese medicine extract samples. Repeat this experiment 3 times to determine the copy number of viral nucleic acid.

[0040] 1.5 In vitro inhibitory effect of a compound traditional Chinese medicine composed of extracts of five Chinese herbs—geniposide, baicalin, luteolin, magnolol, and mangiferin—on SCBV.

[0041] Based on the antiviral activity of five traditional Chinese medicine extracts against SCBV, compound traditional Chinese medicines were formulated according to the maximum safe concentrations of these five herbs, with L-15 culture medium serving as a blank control. The mass ratios of geniposide, baicalin, luteolin, magnolol, and mangiferin were 8:8:2:2:1 (Drug 1); 16:16:4:4:1 (Drug 2); and 32:32:8:8:1 (Drug 3). The combinations of the maximum safe concentrations of the five herbal extracts were then mixed.

[0042] Take 12-well plates confluent with a monolayer of CPB cells, using L-15 medium as a control, and add 1 mL of a traditional Chinese medicine mixture diluted to a safe concentration per well, with 3 replicates per well. Incubate at 28°C for 2 hours. Discard the cell supernatant, wash once with PBS, and add 1 mL of a traditional Chinese medicine and virus mixture diluted to a safe concentration (TCID55). 50 =10 -7 ( / well), place in a 28℃ cell culture incubator for 2 hours, discard the cell supernatant, wash once with PBS, add 1mL of 2% FBS L-15 medium containing a safe concentration of traditional Chinese medicine, place in a 28℃ cell culture incubator, and when the virus control lesions reach 80%, collect the virus and traditional Chinese medicine samples. Repeat this experiment 3 times to determine the copy number of viral nucleic acid.

[0043] 1.6 The inhibitory effect of a compound traditional Chinese medicine composed of five herbal extracts—geniposide, baicalin, luteolin, magnolol, and mangiferin—on SCBV in vivo.

[0044] Mandarin fish fry with a body length of 3-5 cm were selected and divided into a control group and an experimental group, with 30 fish in each group. Gardenin, baicalin, luteolin, magnolol, and mangiferin were formulated in a mass ratio of 32:32:8:8:1 to obtain a compound traditional Chinese medicine. The compound traditional Chinese medicine was mixed into the feed at a ratio of 20g of the compound traditional Chinese medicine per 1000g of feed (conventional mandarin fish feed). The control group was fed conventional mandarin fish feed without the added compound traditional Chinese medicine, 2g / time, twice a day, 3 tanks / group. After one week of continuous feeding, the fish were challenged by intraperitoneal injection at a dose of 1g TCID10. 50 =9.1, and the disease progression was observed and recorded for 14 consecutive days after the challenge.

[0045] 2. Results

[0046] 2.1 Maximum safe concentrations of 11 traditional Chinese medicine extracts in CPB cells

[0047] Table 1 shows the maximum safe concentrations of 11 Chinese herbal extracts for CPB cells.

[0048] Table 1: Maximum safe concentrations of 11 kinds of traditional Chinese medicine extracts in CPB cells

[0049]

[0050] 2.2 Inhibitory effect of 11 kinds of traditional Chinese medicine extracts on SCBV in vitro

[0051] The results of determining the SCBV viral copy number at the maximum safe concentration of 11 kinds of traditional Chinese medicine extracts are as follows: Figure 1 As shown. From Figure 1 It can be seen that among the 11 kinds of Chinese herbal extracts, the five kinds of Chinese herbal extracts with the best inhibitory effect on SCBV are geniposide (1), baicalin (2), luteolin (3), magnolol (4), and mangiferin (5), while the five kinds of Chinese herbal extracts with the best inhibitory effect on SCBV are arctigenin (6), limonene (7), quercetin (8), harbazoside (9), hyperoside (10), and chlorogenic acid (11).

[0052] 2.3 In vitro inhibitory effect of a compound traditional Chinese medicine consisting of five herbs—geniposide, baicalin, luteolin, magnolol, and mangiferin—on SCBV.

[0053] For compound traditional Chinese medicines composed of geniposide, baicalin, luteolin, magnolol, and mangiferin in different mass ratios:

[0054] (1) Drug 1: Gardenin: Baicalin: Luteolin: Magnolol: Mangiferin = 8:8:2:2:1 (mass);

[0055] (2) Drug 2: Gardenin: Baicalin: Luteolin: Magnolol: Mangiferin = 16:16:4:4:1 (mass);

[0056] (3) Drug 3: Gardenoside: Baicalin: Luteolin: Magnolol: Mangiferin = 32:32:8:8:1 (mass).

[0057] The SCBV viral copy number was determined under the action of the above compound Chinese medicines, and the results are as follows: Figure 2 As shown. By Figure 2 It is evident that the higher the content of geniposide, baicalin, luteolin, and magnolol, the more significant the effect of inhibiting SCBV in vitro.

[0058] 2.4 In vivo inhibitory effect of a compound traditional Chinese medicine formula consisting of five herbs—geniposide, baicalin, luteolin, magnolol, and mangiferin—on SCBV.

[0059] like Figure 3 As shown, animal experiments revealed that both the control and drug groups began dying 8 hours after infection. However, the mortality rate in the control group was explosive—the peak mortality period was concentrated between 8 and 21 hours, and the mortality rate reached 100% by 37 hours. Compared to the control group, the mortality rate in the drug group was significantly delayed, with death ceasing completely 37 hours after infection, and the final survival rate remaining at 30%. This result indicates that drug intervention can effectively delay the onset of death and significantly reduce the overall mortality rate. Furthermore, related experimental results showed that drugs 1 and 2 also exhibited similar survival rate results to drug 3.

[0060] like Figure 4 As shown, anatomical examination revealed that the liver of the control group fish was congested, the intestines contained yellow mucus, and the spleen was enlarged; no abnormalities were found in the drug group upon anatomical examination. These results indicate that the compound traditional Chinese medicine of this invention has a certain anti-SCBV effect, can delay the onset of disease, and reduce the mortality rate of SCBV infection.

Claims

1. A traditional Chinese medicine composition for resisting mandarin fish double RNA virus, wherein the traditional Chinese medicine composition is composed of geniposide, baicalin, luteolin, magnolol, and mangiferin, and the mass ratio of geniposide, baicalin, luteolin, magnolol, and mangiferin is 8-32:8-32:2-8:2-8:

1.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of geniposide, baicalin, luteolin, magnolol, and mangiferin is 32:32:8:8:

1.

3. A feed, characterized in that... The traditional Chinese medicine composition contains the one described in claim 1 or 2.

4. The feed according to claim 3, characterized in that, Each 1000g of feed contains 20g of traditional Chinese medicine composition.

5. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of drugs against mandarin fish double RNA virus.

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