JPH054903A - Shellfish assimilation inhibitor and method for producing the same - Google Patents

Abstract

(57) [Summary] [Structure] A shellfish leg attachment inhibitor consisting of fat-soluble components of reef organisms, a method for producing a shellfish leg attachment inhibitor by extracting crushed reef organisms with an organic solvent, and a fatty acid. Shellfish assimilation inhibitor which is an active ingredient. Fatty acids have a large inhibitory activity on unsaturated fatty acids such as arachidonic acid, myristoleic acid and palmitoleic acid. [Effect] By using the inhibitor of the present invention in addition to, for example, a ship bottom paint, it is possible to prevent the adhesion of mussels and barnacles. The inhibitor of the present invention exists in the natural world and has no problem of marine pollution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for inhibiting the attachment of shellfish byssus and a method for producing the same.

[0002]

2. Description of the Related Art The attachment of shellfish to ships, marine structures, and various other equipment used in the ocean deteriorates its function, and therefore it is desired to take measures to prevent the attachment.

Conventionally, as a means for preventing the adhesion, a paint containing an organic tin compound such as tributyltin has been applied. In recent years, marine pollution due to this organotin compound has become a problem, and its use is being prohibited.

Therefore, a reaction product of dodecadienoic acid esters (Japanese Patent Laid-Open No. 50-31032) and a hydroxyl group-containing liquid diene polymer and maleic acid or maleic anhydride, which can be used in a synthetic reaction (Japanese Laid-Open Patent Publication No. 1-210475) An anti-adhesion agent for marine organisms and the like has been developed which contains gazette as an active ingredient.

[0005]

However, since these are also not present in the natural world, there has been a demand for the development of a shellfish byssus adhesion preventive agent which has no problem of environmental pollution.

[0006]

Means for Solving the Problems As a result of a broad search centered on marine organisms in order to solve such problems, the present inventor has found that fat-soluble components of reef organisms have an activity to inhibit shellfish byssus attachment. , Found that this active ingredient contains free fatty acids. Therefore, the activity of various free fatty acids to inhibit shellfish adhesion was investigated, and as a result, it was found that there are various free fatty acids having this activity widely, and the present invention has been completed based on these findings.

That is, the present invention comprises a shellfish byssus adhesion preventive agent comprising a fat-soluble component of a rock reef organism, a method for producing the same, which comprises extracting a crushed product of a reef organism with an organic solvent, and a fatty acid as an active ingredient. The present invention relates to a shellfish byssus adhesion inhibitor.

[0008] Reef organisms are living organisms attached to reefs, and include no shields such as Aplysia, shellfish such as barnacles such as barnacles, anemones, corals,
Examples include sponges, seaweeds, and microorganisms. Particularly, the fat-soluble fraction prepared from sponges, sea anemones, Aplysia and protein gland secretions of Aplysia showed strong activity.
In Aplysia, a considerable adhesion inhibitory effect was observed in the fat-soluble fractions of the midgut gland, body part, and glandular secretion.

It is desirable that the reef organisms be made as fine as possible in order to enhance the extraction effect. The crushing means is not critical and may be mere cutting in addition to grinding, freeze crushing and the like.

The organic solvent of the extraction solvent may be any one capable of extracting the fat-soluble component, such as alcohols such as methanol, ethanol and butanol, ketones such as acetone, ethers such as diethyl ether, chloroform, carbon tetrachloride and the like. Examples thereof include alkyl halides, hydrocarbons such as hexane and cyclohexane, and the like. Hexane, carbon tetrachloride and the like are particularly preferable. Extraction can be performed multiple times. The extraction may be performed at room temperature or may be heated.

The extract may be used as it is as a shellfish adhesion preventive agent, or may be concentrated to dryness before use.

The fatty acid preferably has about 8 to 35 carbon atoms, more preferably about 10 to 20 carbon atoms. Unsaturated fatty acids are better than saturated fatty acids, and those with a higher degree of unsaturation are more active. Examples of preferred fatty acids include saturated fatty acids such as capric acid, lauric acid, palmitic acid,
Examples of the unsaturated fatty acid include myristoleic acid, palmitoleic acid, oleic acid, ricinoleic acid, linoleic acid, linolenic acid, eleostearic acid, arachidonic acid and erucic acid.
Particularly, those having a large effect are myristoleic acid, palmitoleic acid, linoleic acid, linolenic acid, eleostearic acid and arachidonic acid, and arachidonic acid is the most active. Further, not only a single fatty acid but also a mixture of two or more fatty acids, for example, a fatty acid mixture such as a pig liver fatty acid mixture, shows synergistically large activity.

The shellfish leg attachment inhibitor of the present invention may be applied as it is, or may be added to a paint or the like and applied.

The shellfish footspin adhesion inhibitor of the present invention inhibits the footshoe sticking of various kinds of shellfish, and has a particularly high activity of inhibiting the sticking of the mussel footstripes and barnacles.

[0015]

【Example】

Example 1 Water-soluble components and fat-soluble components were extracted from various reef organisms. As a method for preparing the water-soluble component, 3 to 5 times amount of distilled water (only NK-1 and NK-2 physiological saline) was added to 20 g of the frozen sample, homogenized, and centrifuged at 15,000 rpm for 30 minutes at 4 ° C. The supernatant was used as the water-soluble component. Of this, 6 ml was freeze-dried and the weight was measured. The amount of protein was quantified by the BRADFORD method.

As a method for preparing the fat-soluble component, first, 250 ml of methanol was added to 50 g of the frozen sample, the mixture was homogenized and extracted twice. After the methanol was distilled off, 50 ml of distilled water was added and the mixture was extracted twice with 100 ml of diethyl ether. The aqueous layer was further extracted with 100 ml of n-butanol. The ether layer and the butanol layer were each concentrated, dried with a vacuum pump and weighed. The butanol layer was soluble in methanol, ether and acetone.
However, NK-1 was soluble in water, and NK-2 was soluble in hydrous ethanol.

The following table shows the extraction results for sponges and soft corals. These were collected on Kuchinoerabujima Island in Kagoshima Prefecture.

[0018]

[Table 1]

Next, the rate of inhibition of ascidian attachment of mussels was measured for ether extracts of various reef organisms extracted by the same method as above. The measuring method is as follows.

The mussel byssus adhesion test method (modified from the test plate method of Ina et al.) Application of the sample to a disc: A small amount of the test sample was applied to a waterproof paper (10 cm 2 area) processed into a grid with a diameter of 4 cm. Dissolve in ether and apply evenly, and adhere to a support plate stretched with waterproof paper on a plastic microtiter plate. Immobilization of mussels: Three mussels with a shell length of 20 to 25 mm are fixed around the sample disk with an adhesive. Adhesion of foot thread: Leave it in an outdoor water tank (flow rate 4 l / min) overnight or two nights (16 to 40 hours) and measure the number of adhering foot threads. Calculate the number of assassins that repelled the sample disc. For the bioassay, control only the solvent. Judgment: Judgment of repellent reaction is based on the proportion of repelled forefoot. ++ Proportion of repellent foot thread 100% + 80% or more and less than 100% ± 50% or more and less than 80% -50% or less The results are shown in the table below.

[0021]

[Table 2] a: Foot thread attachment inhibition rate, ++ 100%, +80 to 100%, ± 50 to
80%, -50% or less

Example 2 Extraction of fat-soluble components from each reef organism was carried out as follows. First, 320 ml of 90% methanol was added to 7.2 g of the ethanol extract of the methanol extract obtained in the same manner as in Example 1, and the mixture was extracted 4 times with 200 ml of n-hexane. The hexane layer obtained by liquid separation was dried and used as a hexane section. Add 90 ml of water to the 90% methanol layer and add 20 ml of carbon tetrachloride.
Extract 3 times with 0 ml. The carbon tetrachloride layer obtained by liquid separation was dried and used as a carbon tetrachloride group. The water-containing methanol layer was added with 102 ml of water and extracted with 200 ml of chloroform three times. The chloroform layer obtained by liquid separation was dried and used as a chloroform group.

The following table shows the results obtained by measuring the rate of inhibition of the attachment of the ascidian to the mussels of each of the obtained extracts in the same manner as in Example 1.

[0024]

[Table 3] a: Foot thread attachment inhibition rate, ++ 100%, +80 to 100%, ± 50 to
80%, -50% or less

All the solvents having no solvent name in the table were ether extracts of methanol extract obtained in the same manner as in Example 1.

Example 3 Adhesion test of blue mussel A water-resistant paper (10 × 15 cm) coated with the sample obtained in Example 1 and a control water-resistant paper were stuck together, and a boat-shaped adhesion test paper was float-bonded to the upper part. . Four mussels with a shell length of around 10 mm were placed in this, and left overnight in an aquarium.
The number of individuals adhering to the subject and the surface to which the sample was applied and the assimilation or the assimilation marks were measured.

[0027]

[Table 4]

Example 4 Sponge Phyllospongia papy collected on Kuchierabujima Island, Kagoshima Prefecture
200 g of racea sp was extracted with 500 ml of methanol, and the extracts were combined. Methanol was distilled off from the extract, and
% Methanol was added, and the mixture was extracted 4 times with n-hexane. The hexane fraction containing 807 mg of the component thus obtained was 30φ x 1
When it was applied to a 40 mm Kieselgel 60 (70-230 mesh) column and fractionated with a hexane-ethyl acetate system, the activity of inhibiting the attachment of the byssus was observed in the ethyl acetate-eluted fraction containing 11 mg of the component.
Therefore, the ethyl acetate-eluted fraction was further purified using a TOYOPEARL HW-40 column and a NUCLEOSIL 300-7C18 column to obtain 1 mg of active ingredient. When the purified product was methyl esterified and analyzed by gas chromatography, the main components of the purified fraction were C16: 0 (carbon number of fatty acid: unsaturated bond number) (59.9%), C16: 1 (11.8%), C18. : 1 (1
3.8%), C22: 6 (1.9%), and three unidentified components were observed. The method for testing the activity of inhibiting the attachment of the foreskin is as described above.

Example 5 Next, in the same manner as in Example 1 for various fatty acids, the results of measuring the activity of inhibiting adhesion of the mussel byssus are shown in Table 5.

[0030]

[Table 5]

The pig liver fatty acid extract is a commercially available product (Free
Fatty Acid ； Cat No. A-24 (pig liver) Serdary Res. L
aboratories; Ontario Canada) were used.

Example 6 The results of measuring the mussel byssus attachment inhibition activity of various fatty acids and mixtures thereof in the same manner as in Example 1 are shown in Table 6.

[0033]

[Table 6]

The above results show that by combining two or more fatty acids having high activity, a single fatty acid alone exhibits sufficient activity even if the concentration is insufficient.
On the other hand, the combination of fatty acids having low activity does not improve the activity.

Example 7 Table 7 shows the results of measuring the adhesion repellent activity of porcine liver fatty acid extract to Tadema barnacles.

[0036]

[Table 7]

The adhesion repellent activity test of Tatejima barnacle was carried out as follows. That is, a sample (0.01, 0.
1 or 1 mg / well). After completely evaporating the solvent, add 5 ml of seawater to each well. Next, 20 cypris larvae of Tadema barnacles are dropped into each well and incubated at 25 ° C. for 2 days. The number of adhered barnacles and cypris larvae is counted.

[0038]

EFFECTS OF THE INVENTION By using the inhibitor of the present invention in addition to, for example, a ship bottom paint, it is possible to prevent the adhesion of mussels and barnacles. The inhibitor of the present invention exists in the natural world and has no problem of marine pollution.

Claims (3)

[Claims] 1. A shellfish byssus attachment inhibitor comprising a fat-soluble component of reef organisms. 2. A method for producing a shellfish byssus attachment inhibitor, which comprises extracting crushed reef organisms with an organic solvent. 3. A shellfish byssus attachment inhibitor comprising a fatty acid as an active ingredient.