JP7240732B2 - marine invertebrate repellent - Google Patents

Description

The present invention relates to marine invertebrate repellents. More particularly, the present invention relates to marine invertebrate repellents containing seaweed extracts such as Macri, Duckweed, and the like.

Biodiversity is known to play a role in regulating ecosystem functions in the natural world, and is indispensable for us humans to continue to use the ecosystem services we receive from the natural world in a sustainable manner. However, organisms on the earth are now facing the sixth mass extinction period, and it is said that at least 50% or more of the existing species are expected to become extinct. For this reason, it is an urgent task to clarify the effects of changes in ecosystem functions and loss associated with biodiversity loss, and to make future predictions about changes in ecosystem functions and services. Therefore, it became necessary to conduct verification experiments. Administration of drugs etc. is often used for its operation, but many of them are artificial compounds developed as pesticides, and since they have high killing power, especially when used in open oceans, they are harmful to the natural world. Widespread adverse effects are of great concern. Therefore, the development of a safe substance that is less toxic and that can manipulate biodiversity by inducing aversion behavior in animals is awaited.

In industries such as marine products processing and aquaculture, there is a history of using chemicals that are feared to have an adverse effect on the natural world and the human body for cleaning marine products and disinfecting farmed fish. It is in progress.

To begin with, in the natural world, in the process of biological interactions between plants and animals and their evolution, some species of plants have acquired chemoprotective substances that inhibit feeding from animals, and each plant has species-specific compounds. often have Such naturally occurring chemical repellent substances can be considered environmentally friendly chemical substances compared to artificial compounds.

Macri (Digenea simplex) is a kind of red algae belonging to the genus Macri of the family Lacqueraceae. Macri has been used since ancient times as a crude drug for exterminating roundworms. Its main component is kainic acid, which is a kind of imino acid. Kainate is known to act as an agonist of kainate-type glutamate receptors.

Dictyoopteris undulata is a kind of brown alga belonging to the family Myrthyraceae. Zonalol, a terpenoid contained in Shiwayahazu, has been reported to suppress ulcerative colitis (Non-Patent Document 1). In addition, it has been reported that clomazonalol contained in Shiwayahazu inhibits the feeding of Ezo abalone (Non-Patent Document 2).

However, it is not known to use extracts of seaweeds such as Macri and Shiwayahazu as repellents for marine invertebrates.

Yamada, S. et al. (2014) Marine hydroquinone zonarol prevents inflammation and apoptosis in dextran sulfate sodium-induced mice ulcerative colitis” PloS one 9.11: e113509 Taniguchi, K. et al. (1993) Antifeedant substance for Ezo abalone of brown alga Shiwayahazu, Nihon Suisan Gakkaishi 59: 339-343

An object of the present invention is to provide a repellent for marine invertebrates using naturally occurring compounds.

As a result of intensive studies, the present inventors have found that extracts of seaweeds of the genus Macuri and the genus Dracula contain components that have a repelling effect on marine invertebrates. Seaweeds of the genus Macuri and seaweeds of the genus Latvia are known to contain chemical substances with medicinal properties (seaweeds of the Macri genus: kainic acid, seaweeds of the genus Latvia: zonalol, clomazonalol), but according to component analysis, these A fraction corresponding to none of the substances contained an active ingredient as a repellent action against marine invertebrates. The genus Macri seaweed extract and the genus Seaweed extract have different repellent action spectra. It was excellent in repelling effect against marine invertebrates belonging to the animal phylum. Furthermore, by combining the Macri algae extract and the Dracula algae extract, a broad repellent spectrum could be achieved.

The inventors have further studied based on the above findings, and have completed the present invention.
Accordingly, the present invention is as follows:

[1] A marine invertebrate repellent containing a seaweed extract.
[2] The repellent according to [1], wherein the seaweed is a red alga belonging to the genus Macri or a brown alga belonging to the genus Dracula.
[3] The repellent according to [2], wherein the seaweed is red algae belonging to the genus Macri, and the marine invertebrates are marine invertebrates belonging to the phylum Arthropoda.
[4] The repellent according to [2], wherein the seaweed is a brown algae belonging to the genus Plasmodium, and the marine invertebrates are marine invertebrates belonging to the phylum Mollusca.
[5] The repellent agent according to [1], wherein the seaweed extract includes an extract of red algae belonging to the genus Macri and an extract of brown algae belonging to the genus Dracula.
[6] A method for treating an article to repel marine invertebrates, comprising applying or impregnating the article with the repellent according to any one of [1] to [5], or kneading the article with a base material constituting the article.
[7] Marine invertebrate repellency, characterized in that the repellent according to any one of [1] to [5] is applied, impregnated, or kneaded into a base material constituting an article. Goods.
[8] A method for repelling marine invertebrates, comprising dissolving the repellent according to any one of [1] to [5] into seawater.

The present invention provides a marine invertebrate repellent containing a natural compound as an active ingredient. Since the repellent of the present invention contains a natural seaweed extract or a fractionated component thereof as an active ingredient, it is highly safe to the human body and the environment.

At present, in aquaculture facilities such as breeding cages used for cultivating shellfish such as oysters and oysters, growth inhibition and death of shellfish due to the adhesion of marine invertebrates such as barnacles have become serious problems in various places. incurs enormous costs. The use of antifouling agents for fishing nets is being considered, but its use is limited due to concerns about toxicity to shellfish. The repellent of the present invention can be expected to have an effect of preventing adhesion of these marine invertebrates, and since it is a safe substance derived from natural seaweed, it can also meet the need for safety. In addition, the repellent of the present invention can be expected to be used for removing marine invertebrates from seaweed products such as wakame seaweed, hijiki seaweed, laver, etc., and is useful in marine product processing. In addition to the aquaculture of shellfish, seaweed, fish, etc., the repellent of the present invention can be expected to be effective in preventing adhesion of marine invertebrates to seawater pipes of power plants, factories, etc., ship bottoms, ballasts, etc. . Thus, the repellent of the present invention is useful in industrial fields that are harmed by marine invertebrates and their attachment.

FIG. 1 is a schematic diagram showing a method for evaluating the repellent effect of a marine invertebrate repellent. FIG. 2 is a diagram showing the repelling effect of the Macri extract on the side shrimp. The vertical axis indicates the remaining number of amphipods in each patch, and the horizontal axis indicates the distance from the dissolution tower. FIG. 3 is a diagram showing a comparison of the toxic effects of the Macri extract and the kainic acid aqueous solution on the side shrimp. FIG. 4 is a diagram showing the toxic effects of Macri extracts preserved by various methods on the leaf shrimp. FIG. 5 is a graph showing the correlation between Macri extract concentration and mortality. FIG. 6 is a diagram showing the correlation between the concentration of the ragweed extract and the mortality rate. FIG. 7 is a diagram showing the ratio of mussels in which repellent behavior was induced by exposure to each fraction derived from the extract of the sedge. FIG. 8 is a diagram showing changes in the population of benthos (arthropods, mollusks and annelids that have emerged) in eelgrass beds before and after application of the marine invertebrate repellent of the present invention. The symbols in the figure have the following meanings. *: Significant difference p<0.05, **: Significant difference p<0.01, n.s.: No significant difference. FIG. 9 is a diagram showing changes in the number of benthos (arthropods that have emerged) in eelgrass beds before and after application of the marine invertebrate repellent of the present invention. The symbols in the figure have the following meanings. *: Significant difference p<0.05, **: Significant difference p<0.01, n.s.: No significant difference. FIG. 10 is a diagram showing changes in the population of benthos (emerged mollusks and annelids) in eelgrass beds before and after application of the marine invertebrate repellent of the present invention. The symbols in the figure have the following meanings. *: Significant difference p<0.05, **: Significant difference p<0.01, n.s.: No significant difference.

The present invention provides a marine invertebrate repellent (sometimes referred to herein as a "repellent of the invention") containing a seaweed extract.

The seaweed used in the present invention is seaweed containing an active ingredient that repels marine invertebrates (hereinafter sometimes referred to as "marine invertebrate repellent ingredient" or "repellent ingredient"). Seaweeds containing marine invertebrate repellent components include, but are not limited to, red algae belonging to the genus Digenea, brown algae belonging to the genus Dictyopteris, and the like. Red algae belonging to the genus Macri include, but are not limited to, Macri (Digenea simplex) and the like. Examples of brown algae belonging to the genus Dictyopteris include, but are not limited to, Dictyopteris divaricata, Dictyopteris undulata, Dictyopteris latiuscula, Dictyopteris prolifera, and Dictyopteris membranacea.

The seaweed before being subjected to extraction may be collected immediately before the extraction operation, or may be collected in advance and dried and stored. Moreover, it is desirable to use seaweed for extraction in a state of being appropriately shredded or pulverized. Alternatively, it may be used for extraction after being ground, ground, pulverized, or the like.

Solvents used for extraction can include aqueous solvents, organic solvents, or mixtures thereof. Aqueous solvents include, but are not limited to, water, buffered aqueous solutions (such as phosphate buffered saline (PBS)), saline, seawater, and the like. Organic solvents include _C1-6 alcohols (methanol, ethanol, butanol, propanol, isopropanol, etc.), hydrophilic organic solvents such as _C1-6 saturated fatty acids (acetic acid, etc.), _C1-6 alkyl ethers (diethyl ether, dimethyl ether, ethyl methyl ether, etc.), non-hydrophilic organic solvents such as halomethanes (chloromethane, dichloromethane, chloroform, carbon tetrachloride, etc.), or mixtures thereof. The solvent used for extraction is preferably an aqueous solvent, a hydrophilic organic solvent, or a mixture thereof, more preferably an aqueous solvent, methanol, ethanol, acetic acid, or a mixture thereof.

The amount of the extraction solvent used is generally 1 to 10 parts by weight, preferably 1.5 to 5 parts by weight, more preferably 2 to 3 parts by weight, per 1 part by weight of dried seaweed, but is not limited thereto.

The extraction time is usually 0.5 to 48 hours, preferably 1 to 24 hours, but not limited thereto.

Extraction can also be performed by adding an extraction solvent to seaweed and allowing it to stand still, but higher extraction efficiency can be expected by stirring with a homogenizer or the like.

After the extraction operation, the mixture is subjected to centrifugation, filtration, or the like to remove insoluble matter, and the supernatant is collected to obtain a seaweed extract. This extract can be used as it is, or after being concentrated or dried as necessary, as the marine invertebrate repellent of the present invention. The extract extracted with the above solvent is useful as a marine invertebrate repellent due to its purity, and further purification processes well known in the biochemical field (reverse phase chromatography, hydrophobic interaction chromatography , ion exchange chromatography, gel filtration chromatography, affinity chromatography, dialysis, ultrafiltration, liquid-liquid partition, etc.) to fractionate the marine invertebrate repellent components. The marine invertebrate repellent activity in each fraction can be evaluated by the method described in Examples below. In the present invention, seaweed extracts include not only extracts (primary extracts) obtained by directly extracting seaweed with an extraction solvent, but also products obtained by subjecting the primary extracts to further purification steps. be done.

Examples of the purification of repellent components from extracts of red algae belonging to the genus Macri and extracts of brown algae belonging to the genus Possum are shown below, but the present invention is not limited thereto.

(Purification of Repellent Component from Extract of Red Algae Belonging to Macri)
As described in Examples below, when the extract of red algae belonging to the genus Macri is dialyzed using a dialysis membrane with a cutoff molecular weight of 3500, the repellent components are transferred to the dialysate. From this, it is estimated that the molecular weight of the repellent component contained in red algae belonging to the genus Macri is less than 3,500. That is, the repellent component can be purified by subjecting an extract of red algae belonging to the genus Macri to purification means for separating according to molecular weight size, and fractionating a fraction containing components with a molecular weight of less than 3,500. The present invention also provides a method for purifying a marine invertebrate repellent component of red algae belonging to the genus Macri, and a method for producing a marine invertebrate repellent, comprising such steps. Purification means for separating by molecular weight size include, but are not limited to, dialysis, ultrafiltration, gel filtration chromatography, and the like.

As described in the examples below, the repellent component contained in red algae belonging to the genus Macri is extracted with distilled water, so it is water-soluble, but it is adsorbed on an activated carbon column equilibrated with distilled water, and acetic acid Since it is eluted with acidic methanol (pH 3.0), it is presumed to be a substance with certain hydrophobicity. That is, an extract of red algae belonging to the genus Macri (preferably an extract with an aqueous solvent) is subjected to a hydrophobic column equilibrated with an aqueous solvent or the like to adsorb the repellent component to the hydrophobic column, and then, The repellent component can be purified by eluting the adsorbed repellent component from the hydrophobic column with an organic solvent (eg methanol acidified with acetic acid (pH 3.0)). The present invention also provides a method for purifying a marine invertebrate repellent component of red algae belonging to the genus Macri and a method for producing a marine invertebrate repellent, including such steps. Types of columns used for purification include activated carbon columns, octadecylsilyl group-modified silica gel (ODS) columns, size exclusion chromatography (SEC) columns, hydrophilic interaction chromatography (HILIC) columns, and the like. is not limited to

The two purification steps described above (separation by molecular weight size and separation by hydrophobic column) may be combined. In this case, the order of combination is not particularly limited, and after separation by molecular weight size, separation by a hydrophobic column may be performed, or after separation by a hydrophobic column, separation by molecular weight size is performed. may By combining the two purification processes, a higher degree of purification of the repellent component can be expected.

That is, in a preferred embodiment,
(A) subjecting an extract of red algae belonging to the genus Macri to purification means for separating by molecular weight size, fractionating a fraction containing components with a molecular weight of less than 3500,
(B) The fraction containing components with a molecular weight of less than 3500 obtained in (A) is applied to a hydrophobic column to adsorb repellent components to the hydrophobic column, and then an organic solvent (e.g., methanol acidified with acetic acid (pH 3.0 ))), the repellent component is purified by eluting the adsorbed repellent component from the hydrophobic column.

Also, in another preferred embodiment,
(B') An extract of red algae belonging to the genus Macri was applied to a hydrophobic column, and repellent components were adsorbed on the hydrophobic column, followed by adsorption with an organic solvent (eg, methanol acidified with acetic acid (pH 3.0)). eluting the repellent component from the hydrophobic column;
(A') The fraction containing the repellent component obtained in (B') is subjected to purification means for separating according to the molecular weight size, and the fraction containing the component with a molecular weight of less than 3500 is fractionated to obtain the repellent component. to purify.

The fraction obtained in the purification step (A) or (B') above may be directly subjected to the purification step (B) or (A'), or may be subjected to a treatment such as concentration or solvent replacement. After that, it may be subjected to the purification step (B) or (A').

In the present invention, the fraction obtained in the final step may be evaluated for its repellent effect on marine invertebrates belonging to the phylum Arthropoda (eg, amphipod). The marine invertebrate repellent activity can be evaluated by the method described in the Examples below.

(Purification of Repellent Component from Extract of Brown Algae Belonging to the Genus Poison)
As described in the examples below, the repellent component contained in the brown algae belonging to the genus Sargassum is water-soluble because it is extracted with distilled water, but the repellent component can also be extracted with methanol. Liquid-liquid partitioning with a mixture of methanol (volume ratio 50:50)/diethyl ether is mainly partitioned in the ether layer, and it is adsorbed on a silica gel column and eluted with diethyl ether or methanol. It is presumed to be a substance having hydrophobic properties. That is, an extract of brown algae belonging to the genus Dermatology (preferably an extract with an aqueous solvent, methanol, or a mixture thereof) is added to an aqueous solvent or an aqueous solvent/methanol mixture having a methanol concentration of 50% (v / v) or less The repellent component can be purified by liquid-liquid partitioning between the liquid and an organic solvent having a polarity equal to or lower than that of diethyl ether and recovering the organic solvent layer. Preferably, a mixture of water and methanol (volume ratio 50:50) and diethyl ether are used for liquid-liquid partitioning, and the diethyl ether layer is recovered. Alternatively, the repellent component can be purified by subjecting the extract of brown algae belonging to the genus Pygma to a silica gel column, allowing the repellent component to be adsorbed on the silica gel column, and eluting the adsorbed repellent component with an organic solvent. The repellent components are difficult to elute from the silica gel column with hexane, but are eluted with diethyl ether or methanol. , the repellent component can be adsorbed on the silica gel column by applying it to the silica gel column. After washing the silica gel column on which the repellent component was adsorbed with an organic solvent having a polarity equal to or lower than that of hexane, the polarity of the solvent was gradually increased or stepwise increased to diethyl ether or methanol. , the repellent component is eluted from the silica gel column. The present invention also provides a method for purifying a marine invertebrate repellent component of brown algae belonging to the genus Sargassum, and a method for producing a marine invertebrate repellent, comprising such a step.

The two purification steps described above (liquid-liquid partitioning and silica gel column separation) may be combined. In this case, the order of combination is not particularly limited, and separation by a silica gel column may be performed after liquid-liquid distribution, or liquid-liquid distribution may be performed after separation by a silica gel column. good. By combining the two purification processes, a higher degree of purification of the repellent component can be expected.

That is, in a preferred embodiment,
(A) an extract of brown algae belonging to the genus Porphyra, an aqueous solvent or an aqueous solvent/methanol mixture having a methanol concentration of 50% (v/v) or less, and an organic solvent having a polarity equal to or lower than that of diethyl ether; subjecting to liquid-liquid partitioning with a solvent and recovering the organic solvent layer;
(B) The organic solvent layer fraction obtained in (A) was transferred to an organic solvent having a polarity equal to or lower than that of hexane, applied to a silica gel column, and subjected to The repellent component is purified by washing the silica gel column with an organic solvent having the organic solvent and gradually or stepwise increasing the polarity of the solvent to elute the repellent component from the silica gel column.

Also, in another preferred embodiment,
(B') An extract of brown algae belonging to the genus Pseudomonas is dissolved in an organic solvent having a polarity equal to or lower than that of hexane, subjected to a silica gel column, and an organic solvent having a polarity equal to or lower than hexane. washing the silica gel column with and gradually or stepwise increasing the polarity of the solvent to elute the repellent components from the silica gel column;
(A') The fraction containing the repellent component obtained in (B') was combined with an aqueous solvent or an aqueous solvent/methanol mixture with a methanol concentration of 50% (v/v) or less, and diethyl ether or less. The repellent component is purified by subjecting it to liquid-liquid partitioning with an organic solvent of lower polarity and recovering the organic solvent layer.

The fraction obtained in the purification step (A) or (B') above may be directly subjected to the purification step (B) or (A'), or may be subjected to a treatment such as concentration or solvent replacement. After that, it may be subjected to the purification step (B) or (A').

In the present invention, the fraction obtained in the final step may be evaluated for its repellent effect on marine invertebrates belonging to the phylum Mollusca (eg, snails such as mussels). The marine invertebrate repellent activity can be evaluated by the method described in the Examples below.

The above seaweed extract is useful as a marine invertebrate repellent because it is toxic to marine invertebrates and the marine invertebrates act repellent to the extract.
Here, the term "marine invertebrates" refers to animals other than animals having vertebrates such as angnathia, fish, amphibians, reptiles, birds, and mammals, and animals living in the sea, such as arthropods. Examples include marine invertebrates belonging to phylum Arthropoda, phylum Mollusca, phylum Cnidaria, phylum Annelida, and the like.
The extract of red algae belonging to the genus Macri has an excellent repelling effect on marine invertebrates belonging to the phylum Arthropoda, and is therefore useful as a repellent for marine invertebrates belonging to the phylum Arthropoda. Marine invertebrates belonging to the phylum Arthropoda include Crustacea (Maxillopoda) (Copepoda (copepods), Ostracoda (Ostracoda) , Thecostraca (Balanomorpha, Lepadomorpha, Capitulum, etc.), Malacostraca (Hoplocarida) (Stomatopoda ), etc.), Eumalacostraca (Senticaudata suborder (spotted shrimp), Tanaidacea, Isopoda, etc.)), etc.), but these include Not limited.
The extract of brown algae belonging to the genus Sargassum is useful as a repellent for marine invertebrates belonging to the phylum Mollusca, since it has an excellent repelling effect against marine invertebrates belonging to the phylum Mollusca. Examples of marine invertebrates belonging to the phylum Mollusca include Polyplacophora (snails), Gastropoda (Nacellidae, Cantharidus japonicus, etc.), and the like. , but not limited to.

Extracts of red algae belonging to the genus Macri are highly effective in repelling marine invertebrates belonging to the phylum Arthropods, and extracts of brown algae belonging to the genus Sargassum are effective in repelling marine invertebrates belonging to the phylum Mollusca. Due to its superiority, a broad spectrum of repellency against marine invertebrates (phylum Arthropods and Mollusca) can be achieved by combining Macri and Dracula seaweed extracts. Accordingly, in one embodiment, the repellent of the present invention contains an extract of red algae belonging to the genus Macri and an extract of brown algae belonging to the genus Scrophulari.

In one aspect of the use of the repellent of the present invention, the article is treated to repel marine invertebrates by coating or impregnating the article with the repellent of the present invention, or kneading it into a base material that constitutes the article. An invertebrate repellent article can be obtained. Objects that can be treated with the repellent of the present invention are not particularly limited, but articles that need to be avoided from approaching or adhering to marine invertebrates, such as seaweed such as wakame seaweed, hijiki seaweed, seaweed, and kelp. , bivalves such as sea bream and oysters, facilities used by immersing them in the sea for aquaculture of fish (breeding cages, isolation nets, ropes, buoys, water intake/drainage pipes, etc.), ship bottoms and sides, ballast, Examples include, but are not limited to, structures immersed in the sea (sea water pipes for power plants, factories, etc., underwater fish reefs, concrete structures for harbor facilities, etc.). By using the equipment treated with the repellent of the present invention in the cultivation of seaweed such as wakame seaweed, hijiki seaweed, laver, and kelp, the approach of herbivorous invertebrates such as herbivorous zooplankton is avoided and herbivorous invertebrates are grown. It can be expected to prevent feeding damage by animals. In addition, in the aquaculture of sea bream, oysters, etc., or the aquaculture of fish, by using the equipment treated with the repellent of the present invention, it is possible to prevent the growth inhibition and death of aquaculture products due to the adhesion of marine invertebrates such as barnacles. , repelling of parasitic crustaceans adhering to fish, etc. can be expected.

In one embodiment of the use of the repellent of the present invention, marine invertebrates are repelled by dissolving the repellent of the present invention into seawater. Methods for eluting the repellent of the present invention into seawater include, but are not limited to, spraying the repellent of the present invention, applying, soaking, or kneading an article coated with the repellent of the present invention into seawater. not. Undersea areas where it is necessary to avoid the approach of marine invertebrates, such as seaweed such as wakame seaweed, hijiki seaweed, laver, and kelp; bivalves such as tiragi and oysters; By eluting the repellent of the present invention into seawater in the surrounding sea area of things (power plants, factories, port facilities, etc.), the sea area, etc., marine invertebrates are removed from the area, It can be expected to prevent the approach of marine invertebrates. To avoid the approach of herbivorous invertebrates and prevent feeding damage by herbivorous invertebrates in seaweed farms such as wakame seaweed, hijiki seaweed, nori seaweed, and kelp, by eluting the repellent of the present invention into seawater. can be expected. In addition, since marine invertebrates living in seaweed can be removed, contamination of the seaweed with marine invertebrates can be suppressed, and an improvement in the quality of harvested seaweed can be expected. In addition, by eluting the repellent of the present invention into seawater in rocky sea areas, the effect of exterminating herbivorous invertebrates from the area can be expected.

The present invention will be described in more detail with reference to the following examples, but the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[Test Example 1] Establishment of a method for evaluating the repellent effect of the repellent of the present invention
A running water environment was created in a 10t water tank. An artificial colony of eelgrass of 30 cm square was created, and placed in 3 rows (3 rows in each row) at 1 m intervals from the repellent dissolution tower (Fig. 1). 50 lobsters were put into each patch (community), a repellent (Macri water extract, not concentrated) was spread from the dissolution tower, and the number of lobsters remaining in each patch after 2 hours was measured.

The result is shown in figure 2. When the repellent was spread, the number of remaining lobsters increased as the distance from the dissolution tower increased. On the other hand, no such correlation was observed when no repellent was distributed. Therefore, it was shown that the repellent effect can be evaluated using this method.

[Test Example 2] Search for Repellent Component in Macri Extract Kainate is a type of imino acid contained in Macri, and is known as a roundworm extermination agent. Since kainic acid was considered as a candidate for the repellent component contained in Macri extract, the repellent effect of Macri extract was compared with pure kainate.
The mortality rate was examined by exposing the lobster to each of the Macri extract and the aqueous solution of pure kainate for 72 hours. The concentration of kainic acid in the Macri extract was adjusted to be the same as that of the aqueous solution of pure kainic acid. Compare the impact.

The results are shown in Figure 3. Macri extract showed a strong toxic effect on the amphipod. On the other hand, the pure kainic acid did not kill the amphipods. Therefore, it was shown that the toxic effect of the Macri extract on the amphipod is likely due to substances other than kainate contained in the Macri.

[Test Example 3] Evaluation of the stability of the repellent components in the Macri extract The Macri extract (10 g/L) (weight/volume of dry algae) was stored under normal temperature, dry, and frozen conditions for 30 days. , the repellent effect was compared with that before storage (0 days of storage). Similar to Test Example 2, the repellent effect was evaluated by exposing the shrimp to the Macri extract for 72 hours and examining the mortality rate.

The results are shown in FIG. The toxic effect of Macri extract on amphipods was stable regardless of the storage method.

[Test Example 4] Preparation of extract and evaluation of repellent activity (preparation of Macri extract)
100 g of dried makuri was freeze ground, ultrapure water was added and the mixture was filtered. After centrifuging the filtrate at 15000 G for 10 minutes, the supernatant was collected to obtain a Macri extract (100 g/L) (dry alga body weight/volume).

(Preparation of sedge extract)
100 g of dry prickly pear was freeze-milled, filtered seawater was added, and the mixture was filtered. After centrifuging the filtrate at 15000 G for 10 minutes, the supernatant was collected to obtain an extract (100 g/L) (dry alga body weight/volume).

(Evaluation of repellent activity)
Extracts were prepared by serially diluting the Macri extract and the Rhododendron extract as described in the table below. "DW" in the table indicates distilled water.

In the dark at 20° C., lobsters (n=12) and snails (n=15) were exposed to serially diluted extracts for 24 h and live and dead counts were made. A logistic regression analysis was performed to examine the relationship between the mortality rate and the concentration of the extract of the mussel and the mussel.

Table 2 and FIG. 5 show the results for the Macri extract.

In Table 2, the upper row shows the effect on the side shrimp, and the lower row shows the effect on the mussel.

Higher concentration of Macri extract was shown to be associated with higher mortality rates for both lobsters and mussels. Macri extract was toxic to both lobsters and mussels, but there was a significant difference between the mortality curves (F-test: p<0.0001), indicating that the effects differed between species. .

Table 3 and FIG. 6 show the results for the R. japonicus extract.

In Table 3, the upper row shows the effect on the side shrimp, and the lower row shows the effect on the mussel.

Mortality of the mussel increased as the concentration of the extract of the ragweed extract increased (p<0.0001), but there was no relationship between the mortality rate of the amphipod and the concentration of the ragweed extract (p=0.53). Therefore, it was suggested that the repellent component of the ragweed extract is effective against the mussel, but not against the side shrimp.

[Test Example 5] Purification of Repellent Component from Macri Dried Macri (150 g) was rapidly frozen with liquid nitrogen and powdered using a homogenizer. Distilled water (450 ml) in an amount three times the weight of the algal body was added to the pulverized Macri, and the mixture was sufficiently stirred with a homogenizer. The resulting homogenate was centrifuged at 3000 rpm for 5 minutes using a high-speed centrifuge to separate the supernatant and alga body debris. The supernatant was transferred to a 1.5 ml tube and centrifuged at 12000 rpm for 5 minutes to collect the supernatant. Distilled water (300 ml) was again added to the alga body residue, followed by homogenization and centrifugal separation. This procedure was repeated twice to extract the active ingredient from the alga body three times in total. 1, 600ml).

The resulting Macri extract (600 ml) was divided into two 300 ml portions, and 300 ml of the Macri extract was dialyzed against 2000 ml of distilled water overnight at room temperature. A dialysis membrane with a molecular weight cut off of 3500 was used. The dialysis fluid (700 ml) was saved for toxicity evaluation to the amphipod (extract 2). The dialysate (2000 ml x 2) was concentrated to 50 ml using a multi-analyte concentrator (60°C, 35 mber, 260 rpm) (extract 3, toxicity evaluation was performed at 1/10 dilution).

Further purification was performed by activated carbon column chromatography. A column was packed with 150 cm ³ of activated carbon that had been thoroughly washed with water. Floating activated carbon was removed. The column was washed with 450 ml distilled water. The concentrated dialysate (50 ml) was diluted to 70 ml and applied to the column. The column was washed with 450 ml distilled water. The non-adsorbed fraction (450 ml) was saved for toxicity evaluation to the amphipod (extract 4). Elution was performed with 450 ml of methanol acidified with acetic acid (pH 3.0), and the eluted fraction (450 ml) was concentrated under reduced pressure to 50 ml (Extract 5, toxicity evaluation was performed at 1/10 dilution).

The extracts 1 to 5 thus obtained were adjusted in concentration and subjected to a toxicity test for side shrimp according to Test Example 4 (n=7, 20° C., dark conditions). The maximum dilution rate when all the individuals of the amphipod in the well were killed was as follows.

From this result, it was suggested that the extract 5 was concentrated with the amphipod repellent component in the Macri extract.

[Test Example 6] Purification of repellent components from shiwayahazu Diethyl ether (155 ml) and distilled water (155 ml) were added to 155 ml (0.5 g/ml) of methanol extract of dried shiwayahazu and extracted with shaking for 5 minutes. gone. The upper layer (ether layer) and lower layer (water/methanol layer) were separated, and diethyl ether (155 ml) was added to the lower layer (water/methanol layer) for re-extraction. The water and methanol layers were saved for activity evaluation.

The ether layers were combined and concentrated to dryness using a rotary evaporator and nitrogen blower. The residue was dissolved in hexane and made up to 15.5 ml (68 mg/ml). The hexane solution was further concentrated by blowing nitrogen and diluted to 5 ml (equivalent to 15 g/ml of dry algae) (ether layer A).

Further purification was performed by silica gel column chromatography. Sep-Pak Silica 35cc Vac Cartridge, 10 g (manufactured by Waters) was used in triplicate. 50 ml of methanol, 50 ml of dichloromethane, 50 ml of diethyl ether and 50 ml of hexane were passed through the column and stabilized. 0.33 ml of ether layer A dissolved in hexane was added and eluted with 50 ml of hexane, 50 ml of diethyl ether, 50 ml of dichloromethane and 50 ml of methanol in order to obtain the following fractions.

Each fraction was redissolved in 30 ml of distilled water and a portion thereof was subjected to activity measurement. These activity measurement solutions were standardized to a concentration equivalent to 0.5 g/ml of dry alga bodies.

[Test Example 7] Evaluation of the lethal activity of the methanol fraction of the extract of Shiwayahazu to snails (material)
・Methanol fraction of Shiwayahazu extract (A-4)
It was dissolved in distilled water and adjusted to a concentration equivalent to 0.5 g/ml of dry algal bodies.
・Chigusagai 42 individuals were collected from the farming tank of the Seto Inland Sea Area Fisheries Research Institute.

(Method)
(1) Distilled water (MilliQ) and filtration of the methanol fraction to concentrations equivalent to 10 g/L, 7.5 g/L, 5 g/L, 2.5 g/L, and 1 g/L of dried algal cells Diluted with seawater. The concentration of sea water was standardized to 90%.
(2) 7 ml of the solution in (1) was dispensed into 7 test tubes, each of which was filled with snails and plugged with cotton.
(3) As a control (0 g/L), 7 ml of a mixture of 5 ml of distilled water and 45 ml of filtered seawater was dispensed into 7 test tubes, each of which was filled with a snail and was plugged with a cotton plug.
(4) Leave the specimens in a dark place at 20°C for 24 hours, and count the number of dead and surviving snails. Individuals that adhered to the wall surface or cotton plug of the test tube with their feet were considered alive, and other individuals (individuals that were hanging only with mucus, individuals that were not attached and were lying on the bottom) were defined as dead individuals.

(result)
The number of dead and surviving individuals after 24 hours is shown in the table below.

The methanol fraction of Shiwayahazu extract showed high lethal activity at a concentration of 10 g/L and activity at a concentration of 7.5 g/L, but no lethal activity was observed at concentrations of 5 g/L or less. rice field.

Although the data are not shown, when we evaluated the lethal activity against the mussels of the other fractions of the extract of the mussel, the diethyl ether fraction (A-2) and the dichloromethane fraction (A-3) were slightly lower. A lethal activity was observed, but no lethal activity was observed for the hexane fraction (A-1).

[Test Example 8] Correlation between lethal activity against snails and repellent effect of snail extract The following test was conducted in order to examine the relationship between the lethal activity against snails of the exposed solution and the behavior of the snails to escape from the exposed solution (repellent behavior). rice field.

A solution of the methanol fraction, diethyl ether fraction, and hexane fraction, which showed a slightly low activity, and a hexane fraction, which had no activity (10 dried algae each). concentration equivalent to g/L, diluted to 90% seawater), dispense 7 ml each into 7 test tubes, add snails one by one, and leave for 1, 3, 5, 10, 60 minutes. Individuals moving out of the solution later (repellent individuals) were counted.

The results are shown in FIG. The repellent behavior of the mussel was remarkably observed in the solution in which the lethal activity was confirmed. Although the lethal activity of the diethyl ether fraction was lower than that of the methanol fraction, it was suggested that the diethyl ether fraction may have a repellent effect equal to or greater than that of the methanol fraction.

[Test Example 9] Evaluation of repellent effect by outdoor application ) was mixed at a volume ratio of 1:1, and the repellent substance solution was continuously placed in an outdoor eelgrass field for 48 hours. The number of benthos populations (number of marine invertebrates) was compared between the experimental group (lm×lm) in which seawater was added (concentration in terms of body weight) and the control group (lm×lm) in which seawater was added.

The results are shown in Figures 8-10. Mean values and standard deviation error bars for each of six replicates are shown for each control plot and experimental plot. The number of individuals was analyzed by the likelihood ratio test (comparing the model with the effect of time as an explanatory variable and the intercept only model) to determine whether there was a significant difference between the number of individuals at the start of the test and 48 hours later.

Data were obtained that the population of marine invertebrates decreased significantly in the experimental plot. Populations of both Arthropoda and Mollusca were significantly reduced in experimental plots.

The present invention provides a marine invertebrate repellent containing, as an active ingredient, a natural compound extracted from seaweed.

This application is based on Japanese Patent Application No. 2017-199562 filed in Japan, the contents of which are all incorporated herein.

Claims (6)

Marine invertebrate repellent containing an extract of one or more selected from the group consisting of seaweed water, buffer solution, saline solution, seawater, alcohol having 1 to 6 carbon atoms and saturated fatty acid having 1 to 6 carbon atoms A marine invertebrate repellent agent, wherein the seaweed extract is a Macri extract and the marine invertebrate is a Senticaudata suborder (shrimp) or a mussel. Marine invertebrate repellent containing an extract of one or more selected from the group consisting of seaweed water, buffer solution, saline solution, seawater, alcohol having 1 to 6 carbon atoms and saturated fatty acid having 1 to 6 carbon atoms marine invertebrate repellent, wherein the seaweed extract is one or more extracts selected from the group consisting of sedges and sedges, and the marine invertebrates are mussels. Marine invertebrate repellent containing an extract of one or more selected from the group consisting of seaweed water, buffer solution, saline solution, seawater, alcohol having 1 to 6 carbon atoms and saturated fatty acid having 1 to 6 carbon atoms wherein the seaweed extract is a mixture of Macri extract and sagebrush extract, and the marine invertebrates are mild carapaceous, gastropodal, polychaetous, amphipoda, warecara, tanais and etc. A marine invertebrate repellent which is one or more selected from the group consisting of invertebrates belonging to the order Pedoda. A method for treating an article to repel marine invertebrates, which comprises coating or impregnating an article with the repellent according to any one of claims 1 to 3 , or kneading it into a base material constituting the article. An article for repelling marine invertebrates, characterized in that the repellent according to any one of claims 1 to 3 is applied, impregnated, or kneaded into a base material constituting the article. A method for repelling marine invertebrates, comprising dissolving the repellent according to any one of claims 1 to 3 into seawater.

Images