阅读说明：本技术 一种抗鱼类弹状病毒的复方中草药制剂 (Compound Chinese herbal medicine preparation for resisting fish rhabdovirus ) 是由 牛银杰 李宁求 付小哲 林强 罗霞 刘礼辉 梁红茹 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种抗鱼类弹状病毒的复方中草药制剂。本发明的复方中草药制剂包括以下重量份数计的组分：连翘苷提取物1-2份、柠檬苦素提取物0.5-1份、香豆素提取物1-2份、甘草酸提取物3-4份。本发明的复方中草药制剂可显著抑制鱼类弹状病毒复制,延缓其发病及降低死亡率；为鱼弹状病毒防治提供了新的方法,为鱼药的开发提供了新的思路。(The invention discloses a compound Chinese herbal medicine preparation for resisting fish rhabdovirus. The compound Chinese herbal medicine preparation comprises the following components in parts by weight: 1-2 parts of phillyrin extract, 0.5-1 part of limonin extract, 1-2 parts of coumarin extract and 3-4 parts of glycyrrhizic acid extract. The compound Chinese herbal medicine preparation can obviously inhibit the replication of the fish rhabdovirus, delay the morbidity of the fish rhabdovirus and reduce the mortality; provides a new method for preventing and treating the fish rhabdovirus and provides a new idea for developing fish drugs.)

1. The compound Chinese herbal medicine preparation for resisting the fish rhabdovirus is characterized by comprising the following components in parts by weight: 1-2 parts of phillyrin extract, 0.5-1 part of limonin extract, 1-2 parts of coumarin extract and 3-4 parts of glycyrrhizic acid extract.

2. The fish rhabdovirus-resistant compound Chinese herbal medicine preparation as claimed in claim 1, comprising the following components in parts by weight: 2 parts of phillyrin extract, 1 part of limonin extract, 2 parts of coumarin extract and 4 parts of glycyrrhizic acid extract.

3. The fish rhabdovirus-resistant compound Chinese herbal medicine formulation of claim 1 or 2, wherein said fish rhabdovirus is a mandarin fish rhabdovirus.

4. The fish rhabdovirus-resistant compound Chinese herbal medicine preparation as claimed in claim 1 or 2, wherein said compound Chinese herbal medicine preparation is applied in a manner that: the compound Chinese herbal medicine preparation is added into feed.

5. The fish rhabdovirus resistant compound Chinese herbal preparation according to claim 4, wherein said compound Chinese herbal preparation is added in an amount of 10g of preparation per kg of feed.

6. The fish rhabdovirus-resistant compound Chinese herbal medicine preparation as claimed in claim 1 or 2, wherein said compound Chinese herbal medicine preparation has an in vitro cell antiviral safety concentration of: phillyrin extract 50 μ g/mL, limonin extract 25 μ g/mL, coumarin extract 50 μ g/mL, glycyrrhizic acid extract 100 μ g/mL.

7. The fish rhabdovirus-resistant compound Chinese herbal medicine formulation of claim 6, wherein said cells are carp epithelial cells.

Technical Field

The invention relates to the technical field of Chinese herbal medicines, in particular to a compound Chinese herbal medicine preparation for resisting fish rhabdovirus.

Background

Fish rhabdovirus disease is a highly contagious viral disease caused by fish rhabdovirus. In 1999, Siniperca Chuatsi Rhabdovirus (SCRV) was first observed by Chinese scholaria in diseased mandarin fish tissue in the Guangdong. SCRV is one of the members of the Rhabdoviridae (Rhabdoviridae) family, and is a single-stranded, negative-strand, non-segmented RNA virus. The virus can infect mandarin fish, perch and marine fishes, and causes huge economic loss to fishery culture. At present, for diseases caused by fish rhabdovirus, the prevention and control mainly depends on vaccine prevention, no effective medicine exists, and the comprehensive prevention and control is difficult. Therefore, the search for safe, highly effective and low-toxic anti-rhabdovirus drugs is a problem to be solved urgently.

Chinese herbal medicines are natural plants, have wide sources and the characteristics of high efficiency and low toxicity, and increasingly become a hotspot for researching and developing antiviral medicines. At present, the anti-rhabdovirus compound Chinese herbal medicine is rarely reported.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a compound Chinese herbal medicine preparation for resisting fish rhabdovirus.

The purpose of the invention is realized by the following technical scheme: a compound Chinese herbal medicine preparation for resisting fish rhabdovirus comprises the following components in parts by weight: 1-2 parts of phillyrin extract, 0.5-1 part of limonin extract, 1-2 parts of coumarin extract and 3-4 parts of glycyrrhizic acid extract.

Preferably, the compound Chinese herbal medicine preparation for resisting the fish rhabdovirus comprises the following components in parts by weight: 2 parts of phillyrin extract, 1 part of limonin extract, 2 parts of coumarin extract and 4 parts of glycyrrhizic acid extract.

Preferably, the fish rhabdovirus is mandarin fish rhabdovirus (SCRV).

Preferably, the application mode of the compound Chinese herbal medicine preparation is as follows: the compound Chinese herbal medicine preparation is added into feed.

Preferably, the addition amount of the compound Chinese herbal medicine preparation is 10g of preparation per kg of feed.

The in vitro cell antiviral safe concentration of the compound Chinese herbal medicine preparation is as follows: phillyrin extract 50 μ g/mL, limonin extract 25 μ g/mL, coumarin extract 50 μ g/mL, glycyrrhizic acid extract 100 μ g/mL.

Preferably, the cell is a carp epithelial cell (EPC).

Compared with the prior art, the invention has the following beneficial effects:

1. the inventor selects the mandarin rhabdovirus as a model virus and relies on a carp epithelial cell line, and finds that the compound Chinese herbal medicine preparation combined by phillyrin, limonin, coumarin and glycyrrhizic acid extracts can obviously inhibit SCRV replication in vivo and in vitro, delay morbidity and reduce mortality.

2. The compound Chinese herbal medicine preparation provides a new method for preventing and controlling the fish rhabdovirus and provides a new idea for developing the fishing medicine.

Drawings

FIG. 1 is a graph showing the results of measurement of the maximum safe concentration of EPC cells by herbal extracts; wherein A is phillyrin extract, B is limonin extract, C is coumarin extract, and C is glycyrrhizic acid extract.

FIG. 2 is a graph showing the results of testing the effect of four herbal extracts on SCRV replication; wherein A is phillyrin extract, B is limonin extract, C is coumarin extract, and D is glycyrrhizic acid extract.

FIG. 3 is a diagram showing the results of testing the effect of a compound Chinese herbal medicine preparation prepared from four Chinese herbal medicine extracts on the replication of SCRV.

FIG. 4 is a diagram showing the results of the measurement of the effect of the compound Chinese herbal medicine preparation prepared from the four Chinese herbal medicine extracts on the SCRV in fish bodies.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1 materials and methods

1.1 cells and viruses

Carp epithelial cells (Epithelioma Papulosum Cyprini, EPC) were purchased from co-located biotechnology limited. EPC was propagated in M199 medium containing 10% Fetal Bovine Serum (FBS) and cultured at 28 ℃. Mandarin rhabdovirus (SCRV-SS) was isolated and stored by this experiment (published in "Fu X, Lin Q, Liang H, Liu L, Huang Z, Li N, Su J. the biological diseases and genetic diversity of novel fish rhabdoviruses in China. Arch virol.2017Sep 162; 9: 2829. Su 2834.). And (3) proliferating the SCRV-SS in an M199 culture medium containing 5% FBS, culturing at 28 ℃, repeatedly freezing and thawing for 2 times after complete lesion, and subpackaging and storing at-80 ℃ for later use.

1.2 Main reagents and Chinese medicinal extracts

M199 medium, fetal bovine serum and pancreatin were purchased from Gibco. CellTiter 96^@The AQueous one solution Cell promotion Assay kit was purchased from Promega corporation. The four extracts of phillyrin, limonin, coumarin, and glycyrrhizic acid are available from Goodpastel Biotech, Inc.

1.3 determination of maximum safe concentration of EPC cells by Chinese medicinal extract

The extracts of the four Chinese medicinal materials of phillyrin, limonin, coumarin and glycyrrhizic acid are prepared into 2mg/mL by using an M199 minimal medium containing 2% dimethyl sulfoxide (DMSO). Before use, the four traditional Chinese medicine extracts are diluted into 5 concentrations of 200 mu g/mL, 100 mu g/mL, 50 mu g/mL, 25 mu g/mL and 12.5 mu g/mL in L15 complete medium containing 10% FBS. EPC cellsInoculating to 96-well plate, removing culture solution when the single layer grows, adding four Chinese medicinal extracts respectively, each well is 100 μ L, and each dilution is repeated 3 times; synchronously setting blank cell control; culturing at 28 deg.C in incubator, and observing morphology and growth of EPC cells every day. After 72h of culture, CellTiter 96 is adopted^@The AQueous one solution Cell promotion Assay kit is used for detecting Cell viability, namely 20 mu L of CellTiter 96 is added into each hole^@AQueous One Solution Reagent at 28 ℃ with 5% CO₂Incubate in incubator for 4h, determine D_490nmThe value is obtained. The viability of the cells was calculated. The calculation formula is as follows: drug group D_490nmValue/cell control group D_490nmThe value is obtained.

1.4 Effect of four herbal extracts on SCRV replication

According to the determined safe concentrations of the four traditional Chinese medicine extracts, taking a 24-pore plate with a single layer of EPC cells, respectively adding 4 Chinese herbal medicine extracts according to the maximum safe concentration, culturing at 400 mu L/pore in an incubator at 28 ℃ for 2 h; each drug was provided with 4 replicates; a blank with DMSO addition was set simultaneously and allowed to act for 4 h. Discarding the liquid medicine, washing with Hank's buffer solution for 3 times, adding SCRV virus liquid into the well containing each medicine at safe concentration to 4 × 10³TCID₅₀A hole. After further incubation in an incubator at 28 ℃ for 2h, the virus solution was discarded, washed 3 times with Hank's buffer, and further incubated with 5% FBS-containing M199 medium, and the cells were observed daily for statistical effect (CPE). When all positive control of the SCRV are diseased, virus liquid is collected to determine half of Tissue Culture Infection Dose (TCID)₅₀)。

1.5 Effect of Compound Chinese herbal medicine preparation prepared from four Chinese medicinal extracts on SCRV replication

On the basis that the four traditional Chinese medicine extracts resist the SCRV, the medicines are prepared according to the maximum safe concentration of each medicine. In 12-well culture plates with EPC cells grown to a monolayer, DMSO was used as a blank; phillyrin extract, limonin extract and coumarin extract (A + B + C); limonin extract, coumarin extract and glycyrrhizic acid extract (B + C + D); phillyrin extract, limonin extract and glycyrrhizic acid extract (A + B + D); forsythiaside extract, coumarin extract and glycyrrhizic acid extractFetching (A + C + D); phillyrin extract, limonin extract, coumarin extract and glycyrrhizic acid extract (A + B + C + D) are combined at the maximum safe concentration and mixed for incubation of cells for 4 h. Discarding the liquid medicine, washing with Hank's buffer solution for 3 times, adding 4 × 10 of SCRV virus solution containing different combination mixed medicines³TCID₅₀The cells were incubated for 2h, washed 3 times with Hank's buffer, and incubated in M199 medium containing 5% FBS with mixed drug for further observation for CPE. Collecting each dilution of virus solution to determine TCID when all positive control lesions of SCRV are detected₅₀。

1.6 action of Compound Chinese herbal preparation on SCRV in Fish

100 micropterus salmoides with the length of 4-5 cm are selected and divided into 50 pieces for a control group (Con) and a drug test group (Experimental). Mixing forsythin extract, limonin extract, coumarin extract and glycyrrhizic acid extract according to the weight ratio of 2: 1: 2: 4 to obtain the compound Chinese herbal medicine preparation, mixing the preparation into the feed according to the proportion of 10g of the compound Chinese herbal medicine preparation per kg of the feed, feeding for 4g per day, and continuously feeding for 3 days. The control group was fed with normal diet. Then at 10⁴TCID₅₀The tail was challenged by intraperitoneal injection, observed for 10 days, and the death time and number of fish were recorded.

1.7 statistical analysis of data

The data obtained in the experiment are analyzed by all data in the experiment, and are analyzed and drawn by Graphpad prism 5.0.

2 results

2.1 maximum safe concentration determination of herbal extracts on EPC cells

The high concentration Chinese medicinal extract has toxicity to EPC cells, and the higher the concentration of the Chinese medicinal extract is, the higher the toxicity to cells is, D_490nmThe smaller the value. In order to determine the in vitro antiviral effect of the traditional Chinese medicine extract, the maximum safe concentration of the traditional Chinese medicine extract on EPC cells needs to be determined in advance, so that the influence of the toxicity of the medicine on the growth of the EPC cells is avoided. As shown in fig. 1, the maximum safe concentrations of forsythin, limonin, coumarin, glycyrrhizic acid extract are: 50. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL.

2.2 Effect of Each herb extract on SCRV replication

Collecting virus liquid when all the SCRV positive controls are diseased, and determining TCID₅₀. As shown in figure 2, the four herbal extracts all have a certain inhibitory effect on SCRV replication.

2.3 inhibition of SCRV replication by Compound herbal formulation

Treating EPC cells with phillyrin extract, limonin extract, coumarin extract, and glycyrrhizic acid extract (the concentrations of the extracts in the system are 50 μ g/mL, 25 μ g/mL, 50 μ g/mL, and 100 μ g/mL in sequence), and determining titer when SCRV positive control is completely diseased. From the results in fig. 3, it can be seen that the combination of the four drugs can significantly inhibit the replication of SCRV, and the inhibition effect is greater than that of any single drug.

2.4 Effect of Compound Chinese herbal medicine preparation on SCRV on Fish body

Animal experiment results show that the compound Chinese herbal medicine preparation can delay the incidence of the SCRV and improve the survival rate of the micropterus salmoides by 30 percent.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.