Natural composition for resisting fish viruses

Abstract

The invention relates to the technical field of anti-fish virus natural compositions, and discloses a natural composition with an anti-fish virus effect, the natural composition comprises a sandalwood leaf extract, a coptis chinensis extract, a gardenia extract and a radix tinosporae cape extract, the sandalwood leaf extract is 50-90% of sandalwood leaf acetone extract, and the coptis chinensis extract is 50-90% of radix tinosporae cape extract, and the gardenia cape extract is 50-90% of radix tinosporae cape extract. The rhizoma coptidis extract is 50%-90% acetone extract of rhizoma coptidis, the fructus gardeniae extract is 50%-90% acetone extract of fructus gardeniae, and the radix stephaniae tetrandrae extract is 50%-90% acetone extract of radix stephaniae tetrandrae. The antiviral activity of the natural crude drug on salmon cells infected with IPNV can be determined by combining the mutual cooperation of all the extracted substances in order to determine the antiviral activity of the natural crude drug on the salmon cells infected with IPNV.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-fish viral natural compositions, and specifically relates to a natural composition for combating fish viruses. Background Art

[0002] Infectious pancreatic necrosis virus (IPNV) is a viral disease of salmon, and is the pathogen of an infectious and highly lethal disease of young salmon hatched and reared in many places in the world, especially in cultured populations.

[0003] IPNV belongs to the genus Aquabirnavirus of the family Birnaviridae of double-stranded RNA viruses. The virus particles are icosahedral and non-enveloped. There is double-stranded RNA gene composed of 2 segments in the capsid. Segment A encodes a polyprotein, including VP2, VP3, VP4, and encodes a non-structural protein VP5 through an intervening ORF; segment B encodes an RNA-dependent RNA polymerase (VP1). Among them, VP2 is the main structural protein of IPNV, containing the main antigenic epitopes and neutralizing antibody epitopes of the virus.

[0004] Therefore, generally, IPNV can grow in various fish cells at temperatures below 24°C, thereby producing characteristic cells that act on various fish and cause pathogenic phenomena. The infection is widespread and has an economic impact on the salmon fishery. The mortality rate of salmon under 4 months old and old salmon is usually very high, causing serious economic losses. In order to develop and determine the antiviral effect of compounds in vitro, the plaque reduction method or the cytopathic effect (CPE) inhibition method has been adopted on a microtiter plate with 96 flat-bottomed wells in the prior art of Sidwell and Huffman to determine the antiviral effect of compounds in vitro.

[0005] Although the prior art records the development of antiviral chemotherapy using nucleoside analogs (such as Virazole and Acyclovir), and some compounds are found to be effective against the virus, they are too toxic to fish cells in salmon and still affect the growth of salmon; since 1978, the World Health Organization has been encouraging research on natural resources. Therefore, traditional Chinese medicine or natural crude drugs have attracted more and more attention as a natural alternative to antibiotics. However, which traditional Chinese medicine or natural crude drug can combat IPNV virus and will not be toxic to fish cells in salmon during the treatment process, and at the same time, how to determine the antiviral activity of traditional Chinese medicine or natural crude drugs against fish cells of salmon infected with IPNV is a difficult problem that has not been overcome so far. Summary of the Invention

[0006] The present invention aims to provide a natural composition against fish viruses. The inventors extracted natural medicinal herbs to obtain their natural extracts and conducted antiviral experiments on IPNV virus through multiple tests to determine the antiviral activity of natural medicinal herbs against fish cells of salmon infected with IPNV.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] 1) A natural composition against fish viruses, including extracts of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras. Among them, the extract of sandalwood leaves is a 50%-90% acetone extract of sandalwood leaves, the extract of Coptis chinensis is a 50%-90% acetone extract of Coptis chinensis, the extract of Gardenia jasminoides is a 50%-90% acetone extract of Gardenia jasminoides, and the extract of Bucida buceras is a 50%-90% acetone extract of Bucida buceras.

[0009] In the above technical solution, the extract of sandalwood leaves is a 50%-90% acetone extract of sandalwood leaves, which contains taraxerol, taraxerone, and lupenone. Taraxerol, taraxerone, and lupenone cooperate synergistically with the iridoid glycosides contained in the 50%-90% acetone extract of Gardenia jasminoides to inhibit the cascade amplification of covalently regulated enzymes in salmon on lipid peroxidation reaction, playing an antiviral role, and then helping salmon against the damage caused by the IPNV virus to its fish cell oxidation reaction.

[0010] In addition, the 50%-90% acetone extract of sandalwood leaves also contains methyl 3-oxoolean-18-ene-28-oate. Methyl 3-oxoolean-18-ene-28-oate cooperates synergistically with berberine in the 50%-90% acetone extract of Coptis chinensis to limit the influence of IPNV virus on other fish cells in salmon by stabilizing the ATF4 transcription factor in cells. Therefore, the antiviral activity against fish cells of salmon infected with IPNV is achieved.

[0011] Meanwhile, the natural cross-linking agent genipin in the 50%-90% acetone extract of Gardenia jasminoides can promote the regeneration of neurons in the central nervous system of salmon after injury. Therefore, genipin in the natural composition against fish viruses can treat the neuronal injury of salmon after being infected with IPNV, thereby improving the antiviral activity of other fish cells in salmon against IPNV virus. Combining with the rich plant latex in the 50%-90% acetone extract of Bucida buceras, the plant latex contains chemical substances such as alkaloids, terpenes, and glycosides. At the same time, the 50%-90% acetone extract of Bucida buceras and the 50%-90% acetone extract of Gardenia jasminoides interact to provide sufficient nutrition and antiviral activity for salmon.

[0012] Therefore, the inventor selected sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras as natural raw medicines, extracted the natural raw medicine extracts from the above natural organisms, conducted antiviral experiments on IPNV virus through multiple tests, and determined the antiviral activity of the natural raw medicine against the fish cells of salmon infected with IPNV by the mutual synergy among the above various extracts.

[0013] 2) A natural composition for combating fish viruses according to 1), wherein:

[0014] The sandalwood leaf extract is a 50% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 50% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 50% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 50% acetone extract of Bucida buceras.

[0015] Since the Gardenia jasminoides extract contains geniposide, and excessive geniposide content is likely to cause toxic reactions in salmon, therefore, the Gardenia jasminoides extract in the present invention is a 50% acetone extract of Gardenia jasminoides, which jointly synergistically cooperates with the 50% acetone extract of sandalwood leaves and the 50% acetone extract of Bucida buceras, so that the content of the 50% acetone extract of Gardenia jasminoides can be completely fused with the 50% acetone extract of sandalwood leaves and the 50% acetone extract of Bucida buceras, thereby ensuring the antiviral activity of other fish cells in salmon against IPNV virus.

[0016] 3) A natural composition for combating fish viruses according to 1), wherein:

[0017] The sandalwood leaf extract is a 90% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 90% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 90% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 90% acetone extract of Bucida buceras.

[0018] Since the Gardenia jasminoides extract contains geniposide, and excessive geniposide content is likely to cause toxic reactions in salmon, therefore, the Gardenia jasminoides extract in the present invention is a 90% acetone extract of Gardenia jasminoides, which jointly synergistically cooperates with the 90% acetone extract of sandalwood leaves and the 90% acetone extract of Bucida buceras, so that the content of the 90% acetone extract of Gardenia jasminoides can be completely fused with the 90% acetone extract of sandalwood leaves and the 90% acetone extract of Bucida buceras, thereby ensuring the antiviral activity of other fish cells in salmon against IPNV virus.

[0019] 4) A natural composition for combating fish viruses according to 1), wherein:

[0020] The sandalwood leaf extract is a 70% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 70% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 70% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 70% acetone extract of Bucida buceras.

[0021] The sandalwood leaf extract of the present invention is a 70% acetone extract of sandalwood leaves, which can extract a large amount of taraxerol, taraxerone and lupenone in sandalwood leaves with 70% acetone to obtain a 70% acetone extract of sandalwood leaves; the coptis extract is a 70% acetone extract of coptis, which can extract berberine in coptis with 70% acetone to obtain a 70% acetone extract of coptis; the gardenia extract is a 70% acetone extract of gardenia, which can extract iridoid glycosides in gardenia with 70% acetone to obtain a 70% acetone extract of gardenia; the palandra extract is a 70% acetone extract of palandra, which can extract plant latex in palandra with 70% acetone to obtain a 70% acetone extract of palandra, so that the 70% acetone extract of sandalwood leaves, the 70% acetone extract of coptis, the 70% acetone extract of gardenia and the 70% acetone extract of palandra are optimized ratios. It can not only inhibit the cascade amplification of covalently regulated enzymes in salmon on lipid peroxidation reaction, play an antioxidant role, and then help salmon against the damage caused by the influence of IPNV virus on the oxidation reaction of its fish cells, but also achieve the antiviral activity of the fish cells of salmon infected with IPNV.

[0022] 5) A natural composition for combating fish viruses according to 4), wherein the extraction steps of the 70% acetone extract of sandalwood leaves include:

[0023] Step 1: Take 50 grams of sandalwood leaves, cut them into pieces, and dry them in a cool place to form dry sandalwood leaf blocks;

[0024] Step 2: Mix the dry sandalwood leaf blocks in Step 1 with 10 times distilled water, methanol, 70% acetone and alcohol and extract twice, refluxing for 2 hours each time to obtain a sandalwood leaf solution;

[0025] Step 3: Filter the sandalwood leaf solution in Step 2 to obtain a sandalwood leaf filtrate, concentrate the sandalwood leaf filtrate under vacuum and then freeze-dry it to obtain a powdery 70% acetone extract of sandalwood leaves.

[0026] 50 grams of sandalwood leaves of the present invention are chopped and evenly dried naturally to obtain dried sandalwood leaf blocks. Natural drying can ensure that the components of taraxerol, taraxerone, and lupenone in the sandalwood leaf blocks will not be lost. Then, it is extracted twice with a mixture of 10 times distilled water, methanol, 70% acetone, and alcohol. The 70% acetone used here can extract different substance components in the sandalwood leaf blocks. Similarly, during the reflux extraction for 2 hours each time, the components of taraxerol, taraxerone, and lupenone can be fully extracted. During this process, not only the components of taraxerol, taraxerone, and lupenone in the sandalwood leaves are extracted, but also other substance components in the sandalwood leaves are extracted. Through the steps of filtration, concentration, and freeze-drying, all the substance components in the sandalwood leaves are extracted to obtain a powdery 70% acetone extract of sandalwood leaves.

[0027] 6) According to the natural composition for combating fish viruses described in 4), the extraction steps of the 70% acetone extract of Coptis chinensis include:

[0028] Step 1: Take 50 grams of Coptis chinensis, chop it, and place it in a cool place to dry to form dried Coptis chinensis blocks;

[0029] Step 2: Mix the dried Coptis chinensis blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol and extract twice. Each extraction is refluxed for 2 hours to obtain a Coptis chinensis solution;

[0030] Step 3: Filter the Coptis chinensis solution in Step 2 to obtain a Coptis chinensis filtrate. Concentrate the Coptis chinensis filtrate under vacuum and then perform freeze-drying to obtain a powdery 70% acetone extract of Coptis chinensis.

[0031] 50 grams of Coptis chinensis of the present invention are chopped and evenly dried naturally to obtain dried Coptis chinensis blocks. Natural drying can ensure that the coptisine component in the Coptis chinensis blocks will not be lost. Then, it is extracted twice with a mixture of 10 times distilled water, methanol, 70% acetone, and alcohol. The 70% acetone used here can extract different substance components in the Coptis chinensis blocks. Similarly, during the reflux extraction for 2 hours each time, the coptisine component can be fully extracted. During this process, not only the coptisine component in the Coptis chinensis is extracted, but also other substance components in the Coptis chinensis are extracted. Through the steps of filtration, concentration, and freeze-drying, all the substance components in the Coptis chinensis are extracted to obtain a powdery 70% acetone extract of Coptis chinensis.

[0032] 7) According to the natural composition for combating fish viruses described in 4), the extraction steps of the 70% acetone extract of Gardenia jasminoides Ellis include:

[0033] Step 1: Take 50 grams of Gardenia jasminoides Ellis, chop it, and place it in a cool place to dry to form dried Gardenia jasminoides Ellis blocks;

[0034] Step 2: Mix the dried gardenia pieces from Step 1 with 10 times the amount of distilled water, methanol, 70% acetone, and alcohol and perform extraction twice. Reflux each extraction for 2 hours to obtain a gardenia solution.

[0035] Step 3: Filter the gardenia solution from Step 2 to obtain a filtered gardenia solution. Concentrate the filtered gardenia solution under vacuum and then perform lyophilization to obtain a powdery 70% acetone extract of gardenia.

[0036] 50 grams of gardenia in the present invention are chopped and evenly air-dried naturally to obtain dried gardenia pieces. Natural air-drying can ensure that the iridoid glycoside compounds in the gardenia pieces will not be lost. Then, it is mixed with 10 times the amount of distilled water, methanol, 70% acetone, and alcohol for extraction twice. The 70% acetone used here can extract different substance components in the gardenia pieces. Also, during each 2-hour reflux extraction, the extraction of iridoid glycoside compounds can be sufficient. During this process, not only the iridoid glycoside compounds in the gardenia are extracted, but also other substance components in the gardenia are extracted. Through the steps of filtration, concentration, and lyophilization, all the substance components in the gardenia are extracted to obtain a powdery 70% acetone extract of gardenia.

[0037] 8) The natural composition for combating fish viruses according to 4), wherein the extraction steps of the 70% acetone extract of Bucida buceras include:

[0038] Step 1: Take 50 grams of Bucida buceras, chop it, and place it in a cool place to dry to form dried Bucida buceras pieces.

[0039] Step 2: Mix the dried Bucida buceras pieces from Step 1 with 10 times the amount of distilled water, methanol, 70% acetone, and alcohol and perform extraction twice. Reflux each extraction for 2 hours to obtain a Bucida buceras solution.

[0040] Step 3: Filter the Bucida buceras solution from Step 2 to obtain a filtered Bucida buceras solution. Concentrate the filtered Bucida buceras solution under vacuum and then perform lyophilization to obtain a powdery 70% acetone extract of Bucida buceras.

[0041] 50 grams of Pouteria grandifolia of the present invention are chopped and evenly air-dried naturally to obtain dried Pouteria grandifolia blocks. Natural air-drying can ensure that the plant latex components in the Pouteria grandifolia blocks will not be lost; then, it is extracted twice with a mixture of 10 times distilled water, methanol, 70% acetone and alcohol. Here, 70% acetone can extract different substance components in the Pouteria grandifolia blocks. Similarly, during each extraction and reflux for 2 hours, the plant latex components can be fully extracted. During this process, not only the plant latex components in the Pouteria grandifolia are extracted, but also other substance components in the Pouteria grandifolia are extracted, and all the substance components in the Pouteria grandifolia are extracted into a powdery 70% acetone extract of Pouteria grandifolia through the steps of filtration, concentration and freeze-drying.

[0042] Compared with the prior art, the present invention also has the following technical effects:

[0043] The present invention uses 50%-90% acetone extracts of Santalum album leaves, 50%-90% acetone extracts of Coptis chinensis, 50%-90% acetone extracts of Gardenia jasminoides and 50%-90% acetone extracts of Pouteria grandifolia. During the process of obtaining the above extracts, acetone is used as the main extraction agent, which can extract a large amount of taraxerol, taraxerone and lupenone in the Santalum album leaves. Taraxerol, taraxerone and lupenone cooperate synergistically with the iridoid glycosides in the 50%-90% acetone extract of Gardenia jasminoides to inhibit the cascade amplification of covalently regulated enzymes in salmon on lipid peroxidation reaction, playing an antiviral role, and then helping salmon against the damage caused by the IPNV virus to the oxidation reaction of its fish cells.

[0044] At the same time, the 50%-90% acetone extract of Santalum album leaves also contains methyl 3-oxo-olean-18-ene-28-oate. Methyl 3-oxo-olean-18-ene-28-oate cooperates synergistically with coptisine in the 50%-90% acetone extract of Coptis chinensis to limit the influence of IPNV virus on other fish cells in salmon by stabilizing the ATF4 transcription factor in cells, thus achieving the antiviral activity of the fish cells of salmon infected with IPNV. In addition, the natural cross-linking agent genipin in the 50%-90% acetone extract of Gardenia jasminoides can promote the regeneration of neurons in the central nervous system of salmon after injury. Therefore, using genipin in the natural composition against fish viruses can treat the neuronal damage of salmon after being infected with IPNV, thereby improving the antiviral activity of other fish cells in salmon against the IPNV virus. Combining with the rich plant latex in the 50%-90% acetone extract of Pouteria grandifolia, the plant latex contains chemical substances such as alkaloids, terpenes and glycosides. At the same time, the 50%-90% acetone extract of Pouteria grandifolia and the 50%-90% acetone extract of Gardenia jasminoides interact to provide sufficient nutrition and antiviral activity for salmon. Detailed implementation mode

[0045] The following is a further detailed description through specific embodiments:

[0046] Now, reference will be made in detail to the embodiments disclosed in the present invention. Although the present disclosure will be described in conjunction with embodiments and / or examples, they do not limit the present disclosure to these embodiments and / or examples. On the contrary, the present invention disclosure covers alternatives, modifications, and equivalents.

[0047] Example 1

[0048] A natural composition for combating fish viruses, comprising extracts of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras, wherein the extract of sandalwood leaves is a 50% acetone extract of sandalwood leaves, the extract of Coptis chinensis is a 50% acetone extract of Coptis chinensis, the extract of Gardenia jasminoides is a 50% acetone extract of Gardenia jasminoides, and the extract of Bucida buceras is a 50% acetone extract of Bucida buceras. [[ID=I1]]

[0049] The extraction steps of the above-mentioned extracts of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras include:

[0050] Step 1: Take 50 grams of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras, chop them up, and place them in a cool place to dry to form dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks respectively;

[0051] Step 2: Mix the dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol, and perform extraction twice. Each extraction is refluxed for 2 hours to obtain sandalwood leaf solution, Coptis chinensis solution, Gardenia jasminoides solution, and Bucida buceras solution respectively;

[0052] Step 3: Filter the sandalwood leaf solution, Coptis chinensis solution, Gardenia jasminoides solution, and Bucida buceras solution in Step 2 respectively to obtain sandalwood leaf filtrate, Coptis chinensis filtrate, Gardenia jasminoides filtrate, and Bucida buceras filtrate. Concentrate the sandalwood leaf filtrate, Coptis chinensis filtrate, Gardenia jasminoides filtrate, and Bucida buceras filtrate under vacuum and then perform lyophilization to obtain powdery 50% acetone extracts of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras respectively.

[0053] Example 2

[0054] A natural composition for combating fish viruses, comprising extracts of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras, wherein the extract of sandalwood leaves is a 60% acetone extract of sandalwood leaves, the extract of Coptis chinensis is a 60% acetone extract of Coptis chinensis, the extract of Gardenia jasminoides is a 60% acetone extract of Gardenia jasminoides, and the extract of Bucida buceras is a 60% acetone extract of Bucida buceras.

[0055] The extraction steps of the above-mentioned sandalwood leaf extract, Coptis chinensis extract, Gardenia jasminoides extract, and Bucida buceras extract include:

[0056] Step 1: Take 60 grams of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras, chop them up, and place them in a cool place to dry, respectively forming dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks;

[0057] Step 2: Mix the dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol, and perform extraction twice. Each extraction is refluxed for 2 hours to obtain sandalwood leaf solution, Coptis chinensis solution, Gardenia jasminoides solution, and Bucida buceras solution respectively;

[0058] Step 3: Filter the sandalwood leaf solution, Coptis chinensis solution, Gardenia jasminoides solution, and Bucida buceras solution in Step 2 respectively to obtain sandalwood leaf filtrate, Coptis chinensis filtrate, Gardenia jasminoides filtrate, and Bucida buceras filtrate. Concentrate the sandalwood leaf filtrate, Coptis chinensis filtrate, Gardenia jasminoides filtrate, and Bucida buceras filtrate under vacuum and then perform freeze-drying to obtain powdery 60% acetone extracts of sandalwood leaf, Coptis chinensis, Gardenia jasminoides, and Bucida buceras respectively.

[0059] Example 3

[0060] A natural composition for combating fish viruses, comprising sandalwood leaf extract, Coptis chinensis extract, Gardenia jasminoides extract, and Bucida buceras extract. Among them, the sandalwood leaf extract is 70% acetone extract of sandalwood leaf, the Coptis chinensis extract is 70% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is 70% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is 70% acetone extract of Bucida buceras.

[0061] The extraction steps of the above-mentioned sandalwood leaf extract, Coptis chinensis extract, Gardenia jasminoides extract, and Bucida buceras extract include:

[0062] Step 1: Take 70 grams of sandalwood leaves, Coptis chinensis, Gardenia jasminoides, and Bucida buceras, chop them up, and place them in a cool place to dry, respectively forming dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks;

[0063] Step 2: Mix the dry sandalwood leaf blocks, Coptis chinensis blocks, Gardenia jasminoides blocks, and Bucida buceras blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol, and perform extraction twice. Each extraction is refluxed for 2 hours to obtain sandalwood leaf solution, Coptis chinensis solution, Gardenia jasminoides solution, and Bucida buceras solution respectively;

[0064] Step 3: Filter the sandalwood leaf solution, coptis solution, gardenia solution, and bocote solution in Step 2 to obtain the sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate respectively. Concentrate the sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate under vacuum and then perform lyophilization to obtain powdery 70% acetone extracts of sandalwood leaf, coptis, gardenia, and bocote respectively.

[0065] Example 4

[0066] A natural composition for combating fish viruses, comprising sandalwood leaf extract, coptis extract, gardenia extract, and bocote extract. Among them, the sandalwood leaf extract is 80% acetone extract of sandalwood leaf, the coptis extract is 80% acetone extract of coptis, the gardenia extract is 80% acetone extract of gardenia, and the bocote extract is 80% acetone extract of bocote.

[0067] The extraction steps of the above-mentioned sandalwood leaf extract, coptis extract, gardenia extract, and bocote extract include:

[0068] Step 1: Take 80 grams of sandalwood leaf, coptis, gardenia, and bocote, chop them up, and place them in a cool place to dry to form dry sandalwood leaf blocks, coptis blocks, gardenia blocks, and bocote blocks respectively;

[0069] Step 2: Mix the dry sandalwood leaf blocks, coptis blocks, gardenia blocks, and bocote blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol and perform extraction twice. Each extraction is refluxed for 2 hours to obtain sandalwood leaf solution, coptis solution, gardenia solution, and bocote solution respectively;

[0070] Step 3: Filter the sandalwood leaf solution, coptis solution, gardenia solution, and bocote solution in Step 2 to obtain the sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate respectively. Concentrate the sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate under vacuum and then perform lyophilization to obtain powdery 80% acetone extracts of sandalwood leaf, coptis, gardenia, and bocote respectively.

[0071] Example 5

[0072] A natural composition for combating fish viruses, comprising sandalwood leaf extract, coptis extract, gardenia extract, and bocote extract. Among them, the sandalwood leaf extract is 90% acetone extract of sandalwood leaf, the coptis extract is 90% acetone extract of coptis, the gardenia extract is 90% acetone extract of gardenia, and the bocote extract is 90% acetone extract of bocote.

[0073] The extraction steps of the above-mentioned sandalwood leaf extract, coptis extract, gardenia extract, and bocote extract include:

[0074] Step 1: Take 90 grams of sandalwood leaves, coptis, gardenia, and bocote, chop them up, and place them in a cool place to dry, respectively forming dry sandalwood leaf blocks, coptis blocks, gardenia blocks, and bocote blocks;

[0075] Step 2: Mix the dry sandalwood leaf blocks, coptis blocks, gardenia blocks, and bocote blocks in Step 1 with 10 times distilled water, methanol, 70% acetone, and alcohol, and conduct extraction twice. Each extraction is refluxed for 2 hours to obtain sandalwood leaf solution, coptis solution, gardenia solution, and bocote solution respectively;

[0076] Step 3: Filter the sandalwood leaf solution, coptis solution, gardenia solution, and bocote solution in Step 2 respectively to obtain sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate. Concentrate the sandalwood leaf filtrate, coptis filtrate, gardenia filtrate, and bocote filtrate under vacuum and then conduct freeze-drying to obtain powdery 90% acetone extract of sandalwood leaf, 90% acetone extract of coptis, 90% acetone extract of gardenia, and 90% acetone extract of bocote respectively.

[0077] Comparative Example 1

[0078] A natural composition, including sandalwood leaf extract.

[0079] Comparative Example 2

[0080] A natural composition, including coptis extract.

[0081] Comparative Example 3

[0082] A natural composition, including gardenia extract.

[0083] Comparative Example 4

[0084] A natural composition, including bocote extract.

[0085] Comparative Example 5

[0086] A natural composition, including sandalwood leaf extract and coptis extract.

[0087] Comparative Example 6

[0088] A natural composition, including sandalwood leaf extract and gardenia extract.

[0089] Comparative Example 7

[0090] A natural composition, including sandalwood leaf extract and bocote extract.

[0091] Comparative Example 8

[0092] A natural composition comprising Coptis chinensis extract and Gardenia jasminoides extract.

[0093] Comparative Example 9

[0094] A natural composition comprising Coptis chinensis extract and Bucida buceras extract.

[0095] Comparative Example 10

[0096] A natural composition comprising Gardenia jasminoides extract and Bucida buceras extract.

[0097] Comparative Example 11

[0098] A natural composition comprising Santalum album leaf extract, Coptis chinensis extract and Gardenia jasminoides extract.

[0099] Comparative Example 12

[0100] A natural composition comprising Santalum album leaf extract, Coptis chinensis extract and Bucida buceras extract.

[0101] Comparative Example 13

[0102] A natural composition comprising Santalum album leaf extract, Gardenia jasminoides extract and Bucida buceras extract.

[0103] Comparative Example 14

[0104] A natural composition comprising Coptis chinensis extract, Gardenia jasminoides extract and Bucida buceras extract.

[0105] To determine the technical effects of the natural compositions of Examples 1 - 5 against fish viruses and the natural compositions of Comparative Examples 1 - 14, the natural compositions of Examples 1 - 5 against fish viruses and the natural compositions of Comparative Examples 1 - 14 were tested in cell cultures.

[0106] Specific test: The distilled water extract was dissolved in serum-free medium, and the methanol, 70% acetone and alcohol solvent extracts were dissolved in EDTA, and then added to a medium with a final concentration of EDTA not exceeding 1%. The natural compositions of Examples 1 - 5 against fish viruses and the natural compositions of Comparative Examples 1 - 14 were placed in the medium and sterilized by filtration through a 0.2 m filter, and the stock solution concentration was 2 mg / ml.

[0107] Cells taken from CHSE-214 (Chinhook Salmon Embryo) cells of the Institute of Animal Science were used in 125 cm 2Maintained at 18 °C in MEM medium in plastic culture flasks; Eagle's Minimum Essential Medium (Boehringer Mannheim) supplemented with 10% fetal bovine serum (M-10), gentamicin 5 mg / ml, Fungizone 250 μg / ml, penicillin 2.994 mg / (5000 units / ml), streptomycin 6.756 mg / (5000 units / ml) and sodium bicarbonate 7.5%; M-0; without fetal bovine serum).

[0108] The virus used was infectious pancreatic necrosis virus; IPNV-T42G from the Institute of Animal Science. In MEM medium, continuous CHSE-214 cells were infected with IPNV, and the fetal bovine serum culture infectious dose (TCID50) was 0.01 / cell. Then the virus suspension was collected and filtered through a 0.45 μm filter after complete cytopathic effect (CPE).

[0109] 1. Cytotoxicity assay:

[0110] CHSE-214 cells (5000 cells / well) were cultured and grown until a monolayer was formed in a 96-well culture microplate (2 - 3 days). Then, after discarding the old medium, fresh medium (100 μg / ml) containing the natural compositions against fish viruses of Examples 1 - 5 and the natural compositions of Comparative Examples 1 - 14 was added. On the second day, the test was examined by an optical inverted microscope, and cell growth was observed to give cytotoxicity.

[0111] 2. Preliminary screening for anti-IPNV activity:

[0112] CHSE-214 cells (5000 cells / well) were inoculated and cultured until a monolayer was formed in a 96-well culture microplate (2 - 3 days). Then, after discarding the old medium, fresh medium containing viruses at various titers (10-1 - 10-11 / ml), the natural compositions against fish viruses of Examples 1 - 5 (100 μg / ml) and the natural compositions of Comparative Examples 1 - 14 (100 μg / ml) was added. At the same time, control assays were performed on IPNV-infected and non-infected cultures without the natural composition. The cytopathic effect (CPE) was examined by an optical inverted microscope, and the virus inhibition rate (VIR%) was counted after 84 hours of incubation. Yuqiding was used as a positive control, and amantadine was used as a negative control.

[0113] 3. Selectivity index (SI):

[0114] In the preliminary screening test, the natural compositions against fish viruses from Examples 1-5 and the natural compositions of Comparative Examples 1-14 were used to inhibit the cytopathogenicity induced by IPNV. Then, the anti-IPNV activity test and cytotoxicity test at various concentrations were repeated. The ratio of SI = CI%50 / VIR%50 was calculated as the selectivity index (SI).

[0115] The natural compositions against fish viruses from Examples 1-5 (1000 gm) were homogenized and extracted with 70% acetone (2 × 6 L). The mixed 70% acetone extract was concentrated under reduced pressure. The residue was dissolved in 1 L of H2O and continuously shaken in a separatory funnel containing petroleum ether (6 × 0.5 L), EtOAc (8 × 0.5 L), and n-BuOH (4 × 0.5 L). The fractions combined with each solvent and the remaining aqueous solution were evaporated to dryness under reduced pressure and freeze-dried, and then stored at 4°C until use. The water-soluble fraction was chromatographed on a polar diion gel (water - 50% EtOH) to obtain different fractions. These fractions were monitored by in vitro bioassay, leading to the isolation of the active ingredient.

[0116] Determination of virus adsorption and penetration into cells. CHSE-214 cells (5000 cells / well) were inoculated and cultured to form a monolayer (2 - 3 days) in a 96-well culture microplate. After discarding the old medium, a new medium containing viruses at various titers (10-1 - 10-11 / ml), the natural compositions against fish viruses from Examples 1-5 (100 μg / ml), and the natural compositions of Comparative Examples 1-14 (100 μg / ml) was added. After 1 hour of adsorption, the inoculum was replaced with 200 μl of M-0 medium at 18°C. The cultures were examined using an abincerted optical microscope to calculate the virus inhibition rate.

[0117] Preventive effect. The CHSE cell monolayer was pretreated with the drug at 18°C for 1 - 3 h, and then a new medium containing viruses at various titers (10-1 - 10-11 / ml), the natural compositions against fish viruses from Examples 1-5 (100 μg / ml), and the natural compositions of Comparative Examples 1-14 (100 μg / ml) was added after discarding the old medium. The cultures were examined using an abincerted optical microscope to calculate the virus inhibition rate.

[0118] CHSE-214 cells (5000 cells / well) were inoculated and cultured to allow growth until confluent monolayer growth (2 - 3 days) in a 96-well culture microplate. Then, after discarding the old medium, fresh medium containing virus at various titers (10-1 - 10-11 / ml) was added. After 1 hour of adsorption, the inoculum was replaced with 100 μl of M-0 medium. The cultures were incubated at 18 °C for different times, and 100 μl of medium containing 200 μg / ml of the SA1 fraction of Santalum album L. extract was added. The cultures were examined using an inverted optical microscope to calculate the virus inhibition rate.

[0119] Inhibitory effect of the extract on IPNV. An in vitro bioassay system using the cytopathic effect (CPE) inhibition method for microtiter is very convenient, which enables us to evaluate many crude drug extracts and a large number of compounds isolated from active drugs. In preliminary studies, IPNV was used to screen the effects of various extracts obtained from Chinese herbs or natural plants. Results of the antiviral activities of various extracts from this screening system against infectious pancreatic necrosis virus.

[0120] The 70% acetone extract of sandalwood leaves showed higher potential antiviral activity and very low cytotoxicity against CHSE monolayer cells (virus inhibition rate = 24%, cytotoxicity = 8% at 100 μg / ml; SI = 2.23).

[0121] Bioassay-directed isolation was performed from the 70% acetone extracts of Santalum album; Coptis chinensis; Gardenia jasminoides; and Bucida buceras and isolated. The aqueous fraction (SA1 fraction) was found to have higher antiviral activity against IPNV and had the same effect in a medium containing 1% fetal bovine serum. The effect of the extract on IPNV inactivation was concentration-dependent. At a higher concentration (200 μg / ml), the VIR% was 72% (see Table 1). While the natural compositions of Comparative Examples 1 - 14 did not have the above technical effects.

[0122] Table 1 Selectivity index (SI) of natural compositions with potential anti-fish virus effects against IPNV

[0123]

[0124]

[0125] Plants are a vast but largely untapped potential source of antiviral drugs. We have been researching the search for antiviral agents from plants, but these studies have shown that the occurrence of antiviral activity in plants is unexpectedly frequent. It is commonly observed that 20 - 30% of tropical or temperate plants possess antiviral drugs. The antiviral activity of Chinese herbal medicines or natural plants has been studied and first applied to other animals such as fish, etc. We have determined the antiviral activity against infectious pancreatic necrosis virus. It was selected from these plants for detailed study because it showed a much higher level of antiviral activity than the extracts of any other plant in the study. In vitro anti - plant containing infectious pancreatic necrosis virus (IPNV virus, in CHSE cells, where cytotoxicity was observed at 100 μg / ml) has been extensively bioassayed for the 70% acetone fraction = guided (anti - IPNV - activity) fractionation studies, which retained most of the antiviral activity in the plant. These studies yielded highly active components. However, the SA1 part of the 70% acetone extract showed no activity against IPNV in the CHSE cell screening system, resulting in a component that was easy to purify and had antiviral activity against the IPNV virus.

[0126] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics well - known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and applications can be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A natural composition for counteracting the effect of fish viruses, characterized in that, It includes sandalwood leaf extract, Coptis chinensis extract, Gardenia jasminoides extract and Bucida buceras extract. Among them, the sandalwood leaf extract is a 50%-90% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 50%-90% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 50%-90% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 50%-90% acetone extract of Bucida buceras.

2. A natural composition for counteracting fish viruses according to claim 1, characterized in that: The sandalwood leaf extract is a 50% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 50% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 50% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 50% acetone extract of Bucida buceras.

3. A natural composition for combating fish viruses according to claim 1, characterized in that: The sandalwood leaf extract is a 90% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 90% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 90% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 90% acetone extract of Bucida buceras.

4. A natural composition for combating fish viruses according to claim 1, characterized in that: The sandalwood leaf extract is a 70% acetone extract of sandalwood leaves, the Coptis chinensis extract is a 70% acetone extract of Coptis chinensis, the Gardenia jasminoides extract is a 70% acetone extract of Gardenia jasminoides, and the Bucida buceras extract is a 70% acetone extract of Bucida buceras.

5. A natural composition for combating fish viruses according to claim 4, characterized in that: The extraction steps of the 70% acetone extract of sandalwood leaves include: Step 1: Take 50 grams of sandalwood leaves, cut them into pieces, and place them in a cool place to dry to form dry sandalwood leaf blocks; Step 2: Mix the dry sandalwood leaf blocks in Step 1 with 10 times distilled water, methanol, 70% acetone and alcohol and extract twice, with each extraction refluxing for 2 hours to obtain a sandalwood leaf solution; Step 3: Filter the sandalwood leaf solution in Step 2 to obtain a sandalwood leaf filtrate, concentrate the sandalwood leaf filtrate under vacuum and then freeze-dry it to obtain a powdery 70% acetone extract of sandalwood leaves.

6. A natural composition against fish viruses according to claim 4, characterized in that: Among them, Coptis chinensis The extraction steps of the 70% acetone extract include: Step 1: Take 50 grams of Coptis chinensis, cut it into pieces, and place it in a cool place to dry to form dry Coptis chinensis blocks; Step 2: Mix the dry Coptis chinensis blocks in Step 1 with 10 times distilled water, methanol, 70% acetone and alcohol and extract twice, with each extraction refluxing for 2 hours to obtain a Coptis chinensis solution; Step 3: Filter the Coptis chinensis solution in Step 2 to obtain a Coptis chinensis filtrate, concentrate the Coptis chinensis filtrate under vacuum and then freeze-dry it to obtain a powdery 70% acetone extract of Coptis chinensis.

7. A natural composition for combating fish viruses according to claim 4, characterized in that: Among them, Gardenia jasminoides The extraction steps of the 70% acetone extract include: Step 1: Take 50 grams of Gardenia jasminoides, cut it into pieces, and place it in a cool place to dry to form dry Gardenia jasminoides blocks; Step 2: Mix the dry Gardenia jasminoides blocks in Step 1 with 10 times distilled water, methanol, 70% acetone and alcohol and extract twice, with each extraction refluxing for 2 hours to obtain a Gardenia jasminoides solution; Step 3: Filter the Gardenia jasminoides solution in Step 2 to obtain a Gardenia jasminoides filtrate, concentrate the Gardenia jasminoides filtrate under vacuum and then freeze-dry it to obtain a powdery 70% acetone extract of Gardenia jasminoides.

8. A natural composition for counteracting fish viruses according to claim 4, characterized in that: Among them, Bucida buceras The extraction steps of the 70% acetone extract include: Step 1: Take 50 grams of Bucida buceras, cut it into pieces, and place it in a cool place to dry to form dry Bucida buceras blocks; Step 2: Mix the dried Palaquium gutta pieces in Step 1 with distilled water, methanol, 70% acetone, and alcohol in a ratio of 1:10 and perform extraction twice. Each extraction is refluxed for 2 hours to obtain a Palaquium gutta solution. Step 3: Filter the Palaquium gutta solution in Step 2 to obtain a Palaquium gutta filtrate. Concentrate the Palaquium gutta filtrate under vacuum and then perform lyophilization to obtain a 70% acetone extract of Palaquium gutta in powder form.

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