JP2007045811A - Anti-adhesive agent for adherent diatom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antisticking for attaching diatom, comprising a fat-soluble fraction of seaweed extracts or a compound discriminated therefrom. <P>SOLUTION: The antisticking for attaching diatom comprises at least one fat-soluble fraction selected from a group consisting of family Dictyotaceae brown algae, genus Laurencia red algae, Agarum clathratum, Sargassum miyabei and Sargassum thunbergii. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an adhesion preventive agent for adhering diatoms comprising a fat-soluble fraction of a seaweed extract or a compound identified therefrom.

  Marine adhering organisms such as barnacles and mussels attach to ships and reduce their navigation function, attach to fishing nets and cause damage, or adhere to power supply and drain pipes at power plants and reduce cooling efficiency. Giving economic losses. Conventionally, organotin compounds have been widely used as agents for preventing the attachment of marine organisms to fishnets, ship bottoms, and the like. However, the use of organotin compounds is regulated due to their strong toxicity and action as environmental hormones. Many alternative compounds also contain copper and zinc, and there are concerns about adverse effects on the environment, and development of safe drugs with less toxicity is awaited.

  When a structure is thrown into the sea, first a polymer film and then a microbial film are formed, and then attached diatoms adhere. It is known that this microbial film and attached diatom promote adhesion of large seaweed and adhesion of marine invertebrates (Non-patent Document 1). Therefore, formation of a microbial film or prevention of adhesion of attached diatoms can be a means for preventing attachment of large organisms. Furthermore, marine organisms with poor motility contain chemical defense substances, and chemical defense substances used in ecosystems can be considered environmentally friendly chemical substances.

  The present inventors have already reported an anti-adhesive agent against marine fouling organisms composed of a water-soluble extract of cricket (Tichocarpus crinitus) (Patent Document 1). However, this document does not disclose any seaweed extract other than the water-soluble extract of crickets or an adhesion preventive agent for adhering diatoms.

  The compound laurinterol has been isolated from the red alga Laurencia okamurae, antibacterial activity, photoaging prevention, antioxidant, barnacle adhesion inhibition, cytotoxicity, Na, K-ATPase inhibitor, etc. Is known (Non-Patent Documents 2, 3, and 4), but is not known for its adhesion-inhibiting activity against attached diatoms.

  In addition, the compound Debromolaurinterol has been isolated from the red alga Laurencia okamurae, Laurencia pacifica and Laurencia majuscula, as well as anti-aging and anti-aging. Biological activities such as oxidation (radical scavenging), cytotoxicity, antibacterial activity, and Na, K-ATPase inhibitor are known (Non-Patent Documents 2 and 3), but adhesion inhibition activity against attached diatoms is not known.

  In addition, the compound isorurointerol has been widely included in the red algae of the genus Laurencia, and it has been shown to inhibit feeding in fish such as larvae and gangaze, antibacterial activity, barnacle adhesion inhibition, Biological activity such as cytotoxicity is known (Non-Patent Document 3, Non-Patent Document 4), but it is not known about the adhesion-inhibiting activity against attached diatoms.

Further, the compound debromoisolaurinterol is isolated from the red alga Laurencia okamurae (Non-patent Document 3), and the prepacifenol is Laurencia nipponica, Patents from Laurencia filiformis, Laurencia johnstonii, Laurencia pacifica, Laurencia majuscula and Laurencia claviformi (non-patent from Laurencia claviformi) 5) Also, 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene is from Laurencia nipponica. Is known (Non-patent Document 6). However, the biological activity of these compounds is not known.
Cubenol is generally contained in seaweeds (Non-patent Document 7) belonging to the family Dictyotaceae and plants belonging to the genus Salvia and Pinus. Although known and antibacterial and antifungal activities are known (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12), there is no known anti-adhesion activity against attached diatoms.

The compound Chromazonarol has been isolated from Ezoyahaz (Dictyopteris divaricata) or Shiwayahaz (Dictyopteris undulata), and has been known to have an inhibitory activity on feeding to sea urchins and Ezo abalone (non-patent literature) 8, 9), It is not known about the adhesion inhibitory activity against adhered diatoms.
International Publication No. 2004/066751 Pamphlet Stephen C. Dexter, Biofouling, vol. 7, pp.97-127 (1993) Role of microfouling organisms in marine corrosion. Irie, T., Suzuki, M., Laurinterol and debromolaurinterol, constituents from Laurencia intermedia; Tetrahedron Lett. 1966: (No. 17) 1837-1840. T. Irie, M. Suzuki, E. Kurosawa and T. Masamune: Laurinterol, debromolaurinterol and isolaurinterol, constituents of Laurencia intermedia YAMADA. Tetrahedron, vol 26, pp.3271-3277 (1970)). Han'guk Hwankyong Uisaeng Hakhoechi (2003) 29 (3) 1-8 Abe, Tsuyoshi; Masuda, Michio; Suzuki, Teruaki; Suzuki, Minoru. Chemical races in the red alga Laurencia nipponica (Rhodomelaceae, Ceramiales) Suzuki, Minoru; Kawamoto, Tomotake; Vairappan, Charles Santhanaraju; Ishii, Takahiro; Abe, Tsuyoshi; Masuda, Michio. Halogenated metabolites from Japanese Laurencia spp. Phytochemistry (Elsevier) (2005), 66 (23), 2787-2793. Suzuki, M. 1981. Epicubenol and related sesquiterpenoids from the brown algae Dictyopteris divaricata; Bull. Chem. Soc. Jpn. 54: (No. 8) 2366-2368. Suzuki, M. 1991. Inhibitory effect of methanol extract of brown algae Ezoyahazu on sea urchin sea urchin and abalone Nippon Suisan Gakkaishi 57 (10), 1945-1948, Fenical, W .; McConnell, O. Chromazonarol and isochromazonarol, new chromanols from the brown seaweed Dictyopteris undulata (zonarioides). Experientia (1975), 31 (9), 1004-5 . Pitarokili, D., Tzakou, O., Couladis, M., Verykokidou, E .; J. Essential Oil Res. (1999) 11 (5) 655-659. Krauze-Baranowska, M., Mardarowicz, M., Wilwart, M., Poblocka, L., Dynowska, M .; Zeitschrift fuer Naturforschung, C (2002) 57 (5/6) 478-482. Solis, C., Becerra, J., Flores, C., Robledo, J., Silvia, M .: J. Chilean Chem. Soc. (2004) 49 (2) 157-161.

  An object of the present invention is to provide an adhesion preventive agent for adhering diatoms derived from seaweed.

As a result of intensive studies, the present inventors have found that an extract of seaweed and a compound isolated and identified therefrom have an anti-adhesion effect on adhering diatoms, and have completed the present invention. Accordingly, the present invention provides the following:
1. An anti-adhesive agent for adhering diatoms, containing a fat-soluble fraction of seaweed extract,
2. The seaweed is at least selected from the group consisting of brown algae belonging to the family Dictyotaceae, red algae belonging to the genus Laurencia, Agarum clathratum, Sargassum miyabei, and Sargassum thunbergii The anti-adhesive agent according to 1 above,
3. The brown alga belonging to the family Dictyotaceae is at least one selected from the group consisting of Ezoyahaz (Dictyopteris divaricata) and Shiwayahaz (Dictyopteris undulata), and the red alga belonging to the genus Laurencia is Laurencia okamurae, Laurencia nipponica, Laurencia pacifica, Laurencia majuscula, Laurencia filiformis, Laurencia johnstony (Laurencia johnston) the adhesion preventing agent according to 2 above, which is at least one selected from the group consisting of claviformi),
4). As an active ingredient, at least one selected from the group consisting of laurenterol, isolaurenterol, prepacylphenol, 2,10-dibromo-3-chloro-9-hydroxy-α-camiglen, cubenol and chromazonalol Including diatom adhesion inhibitor,
It is.

  The fat-soluble fraction or the identified compound of the seaweed extract of the present invention can be used as an adhesion antifouling agent, for example, by incorporating them into a ship bottom paint.

As described above, the present invention relates to an adhesion preventive agent for adhering diatoms comprising a fat-soluble fraction of a seaweed extract or an identified compound.
In the present invention, seaweed means a large macroalgae from the algae that does not contain unicellular algae such as chlorella, green algae having chlorophyll a and b as photosynthetic pigments, red algae having chlorophyll a as photosynthetic pigments, Brown algae with chlorophyll a and c as photosynthetic pigments are included. Examples of seaweed include Laurencia nipponica, L. okamurae, L. pinnata, L. intermedia, L. venusta, Magilezo (L. saitoi), Kobusozo (L undulata, Laurencia pacifica, Laurencia majuscula, Laurencia filiformis, Laurencia johnstonii, Laurencia johnstonii, gun Red algae belonging to the genus Laurencia such as Laurencia claviformi; Ezoyahaz (Dictyopteris divaricata), Shiwayahaz (Dictyopteris undulata), Amygusa (Dictyota dichotoma), Dophtyogogari (Dictyota linearis), Diltyus ogum (Dictyota pacificum), Sanada Brown algae belonging to the family Dictyotaceae such as Pachydictyon coriaceum (Holmes) Okamura, Padina arborescens Holmes, Padina minor, Padina crassa; Agarum clathratum ), Brown algae belonging to Laminariaceae such as Ecklonia stolonifera; Sargassum thunbergii, Sargassum miyabei, Cystoseira hakodatensis, Myagropsum mygas Examples include brown algae belonging to the Sargassaceae family such as Sargassum yezoense. The seaweed used by this invention may combine not only 1 type but 2 or more types. In the present invention, the attached diatom refers to a diatom that grows on the surface of an underwater structure such as a rock, a pile, or a ship bottom. Examples of the attached diatom include, but are not limited to, Nitzchia sp., Cylindrotheca closterium, Navicula sp., Amphora sp., Achnanthes sp.

  As the seaweed used in the present invention, the collected seaweed can be drained, or the collected seaweed can be dried or powdered. This seaweed is extracted by dipping in, for example, methanol. The extraction temperature is preferably room temperature (about 20 ° C.), and the extraction time is 1 hour to 1 week, preferably 24 hours. The extraction operation may be performed once, but more extracts can be obtained when the extraction operation is performed twice or more. Concentration of the methanol extract can be performed under reduced pressure by setting the temperature of the hot water bath to 20 to 25 ° C. After concentration of the extract under reduced pressure, the active ingredient of the present invention can be separated and purified from a fat-soluble fraction obtained by partitioning the residue with, for example, diethyl ether / water.

  The compound laurinterol used in the present invention is:

For example, it can be extracted from red algae belonging to the genus Laurencia, preferably Laurencia okamurae, using a conventionally known method (Non-Patent Documents 2, 3, and 4). The organism for extraction is not particularly limited as long as it is an organism containing lorinterol. Moreover, laurenterol may be chemically synthesized or obtained from the market. A preferred concentration of Rollin Te role as an adhesion diatomaceous inhibitor, 1.5 [mu] g / cm ² or more, more preferably 2.0 [mu] g / cm ² or more, more preferred is 3.0 [mu] g / cm ² or more.

The compound debromolaurinterol used in the present invention is:

For example, a red algae belonging to the genus Laurencia, preferably Laurencia okamurae, Laurencia pacifica or Laurencia pacifica, using a conventionally known method (Non-Patent Documents 2 and 3). Although it can be extracted from Laurencia majuscula, the organism for extraction is not particularly limited as long as it is an organism containing debromolaurinterol. Further, debromolorine terol may be chemically synthesized or obtained from the market. A preferred concentration of de bromo Rollin Te role as an adhesion diatomaceous inhibitor, 5.0 [mu] g / cm ² or more, more preferably 10.0 [mu] g / cm ² or more, more preferred is 20.0μg / cm ² or more.

  The compound isolaurinterol used in the present invention is:

And can be extracted from, for example, red algae belonging to the genus Laurencia, preferably Laurencia okamurae, using a conventionally known method (Non-patent Document 3). The organism for extraction is not particularly limited as long as it contains an organism. Further, isoruroterol may be chemically synthesized or obtained from the market. The concentration of isoruroline terol preferred as an adhesion diatom inhibitor is 0.2 μg / cm ² or more, more preferably 0.5 μg / cm ² or more, and still more preferably 1.0 μg / cm ² or more.

The compound debromoisolaurinterol used in the present invention is:

And can be extracted from, for example, red algae belonging to the genus Laurencia (preferably Laurencia okamurae) using a conventionally known method (Non-patent Document 3). The organism for extraction is not particularly limited as long as it is an organism containing isoruroline terol. Moreover, debromoisolorine terol may be chemically synthesized or obtained from the market. A preferred concentration of de-bromoisoquinolin Rollin Te role as an adhesion diatomaceous inhibitor, 5.0 [mu] g / cm ² or more, more preferably 10.0 [mu] g / cm ² or more, more preferred is 20.0μg / cm ² or more.

  The compound prepacifenol used in the present invention is:

For example, a red alga belonging to the genus Laurencia, preferably Laurencia nipponica, Laurencia filiformis, Lauren using a conventionally known method (Non-patent Document 5). Can be extracted from Laurencia johnstonii, Laurencia pacifica, Laurencia majuscula, Laurencia claviformi, but any organism that contains prepasiphenol For example, the organism for extraction is not particularly limited. In addition, prepasiphenol may be chemically synthesized or obtained from the market. A preferred concentration of the pre-Pasi phenol as an adhesion diatomaceous inhibitor, 4.0 [mu] g / cm ² or more, more preferably 8.0μg / cm ² or more, more preferred is 10.0 [mu] g / cm ² or more.

  The compound 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene used in the present invention is as follows:

For example, it can be extracted from red algae belonging to the genus Laurencia, preferably Laurencia nipponica, using a conventionally known method (Non-patent Document 6). The organism for extraction is not particularly limited as long as it is an organism containing dibromo-3-chloro-9-hydroxy-α-camigrene. Moreover, 2,10-dibromo-3-chloro-9-hydroxy-α-camigrene may be chemically synthesized or obtained from the market. A preferred concentration of 2,10-dibromo-3-chloro-9-hydroxy -α- Kamiguren as adhesion diatomaceous inhibitor, 4.0 [mu] g / cm ² or more, more preferably 8.0μg / cm ² or more, more preferred is 10.0 μg / cm ² or more.

  The compound cubenol used in the present invention is:

For example, a brown alga belonging to the family Dictyotaceae (preferably Dictyopteris divaricata or Dictyopteris undulata) or Salvia (Salvia) using a conventionally known method (Non-patent Document 7). ) And plants belonging to the genus Pinus (Pinus), the organism for extraction is not particularly limited as long as it is an organism containing cuvenol. Cubenol may be chemically synthesized or obtained from the market. The preferred concentration of Kyubenoru as adhesion diatomaceous inhibitor, 4.0 [mu] g / cm ² or more, more preferably 8.0μg / cm ² or more, more preferred is 10.0 [mu] g / cm ² or more.

  The compound Chromazonarol used in the present invention is:

For example, extracted from brown algae belonging to the family Dictyotaceae, preferably Dictyopteris divaricata or Dictyopteris undulata, using a conventionally known method (Non-Patent Documents 8 and 9). However, the organism for extraction is not particularly limited as long as the organism contains chromazonalol. Chromazonalol may be chemically synthesized or obtained from the market. A preferred concentration of chroma Zona role as an adhesion diatomaceous inhibitor, 2.0 [mu] g / cm ² or more, more preferably 3.0 [mu] g / cm ² or more, more preferred is 10. 0 μg / cm ² or more.

  The compounds used in the present invention, low rosterol, isolouroterol, prepasiphenol, 2,10-dibromo-3-chloro-9-hydroxy-α-camiglen, cubenol or chromazonalol are one kind as an anti-adhesive agent for attached diatoms. May be used alone or in combination of two or more.

  The anti-adhesion agent for marine fouling organisms of the present invention can be prepared as it is, or in the form of a gel such as a hydrogel, a paint, a solution, or an emulsion. Moreover, a thickener, a plasticizer, a coloring agent, etc. can be mix | blended with the adhesion preventive agent of the adhesion diatom of this invention as needed.

Lorinterol, an inhibitor of Mitsodezo's adhesion, Red Alga's Mitsodezo (232 g, wet weight) collected in Otaru City was immersed and extracted with 1.2 L of methanol for 24 hours. The extract was concentrated under reduced pressure using an evaporator, and then divided into two layers with water and diethyl ether (125 mL each) three times. The resulting fat-soluble fraction was dehydrated by adding sodium sulfate, and concentrated under reduced pressure. After filtration, the residue was eluted with hexane / ethyl acetate (95: 5) by flash chromatography on silica gel (2.5 × 38 cm), and further eluted with hexane / ethyl acetate (9: 1). The latter fraction was developed with toluene by large-scale preparative thin-layer chromatography on silica gel to isolate 16.7 mg of lorinterol. ¹ H NMR data was consistent with literature values (Non-Patent Documents 2 and 3).

Diatom Adhesion Test This test is an original test that was used as a feeding inhibition test method for sea urchins and abalone (Monthly Ocean vol27 (1) 13-21 (1995) Suzuki Kaoru, diversity of bioactive substances in algae). Modified for the test of attached diatoms.
The diatoms used were Cylindrotheca closterium and Nitzchia sp. Using filtered natural seawater, a Yoganson medium was prepared and autoclaved at 121 ° C. for 20 minutes. 25 mL of the culture solution was added to a shallow sterile petri dish (90 × 15 mm). A substance to be tested or an extract was adsorbed in a circle having a diameter of 1.5 cm on a TLC aluminum sheet of cellulose cut to a 5.9 cm square, and then allowed to stand in a petri dish. Diatoms were planted and evaluated by visual observation.
Lorinterol was adsorbed on TLC aluminum sheet of cellulose 180μg / cm ² , left in the aquarium, filtered natural seawater (naturally pumped directly with a pump in the facility of the National Fisheries Research Center, Hokkaido Regional Fisheries Research Institute) Seawater was sand-filtered. Conducted from December to March.) After 2 weeks, no adsorbed diatom was observed in the adsorbed part, but diatom adhering was observed in the non-adsorbed part. . Furthermore, Lorintelol showed strong adhesion inhibitory activity at 3 μg / cm ² against the adhering diatom Nitzchia sp., And the area where the diatom adhered in the circle of the test area was 1% or less. Moreover, it showed strong adhesion inhibitory activity at 3 μg / cm ² against the adhering diatom Cylindrotheca closterium, and the area where the diatom adhered in the circle of the test area was 1% or less.

Adhesion inhibitory activity of sharks Brown algae sharks (58 g, wet weight) collected in Nemuro City were extracted three times with 300 mL of methanol. The extract was concentrated under reduced pressure and then partitioned three times with water and diethyl ether (50 mL each), and the resulting fat-soluble fraction was subjected to flash chromatography on silica gel with hexane / ethyl acetate 95: 5 and hexane / ethyl acetate 9: 1. And then eluted with hexane / ethyl acetate 4: 1 (21 LC). The last fraction 47 mg (dry weight) was separated by ODS flash chromatography. After eluting with 80% methanol, 90% methanol and 100% methanol, 11 mg (dry weight) of a fraction (21LC-D) eluted with 100% chloroform was obtained.

Fraction 21LC (concentration adjusted to 10 mg / mL with ethanol) was adsorbed on a TLC aluminum sheet of cellulose at 140 μg / cm ² , allowed to stand in a water tank, and the above filtered natural seawater was allowed to flow. There was no diatom adhesion on the area, and diatom adhesion on the non-adsorbed area. Fraction 21LC-D (concentration adjusted to 10 mg / mL with ethanol) was adsorbed on a cellulose TLC aluminum sheet at 140 μg / cm ² and allowed to stand in a petri dish in which diatom Cylindrotheca closterium was cultured. Although no diatom was attached to the part, diatom was attached to the non-adsorbed part. From the result of ¹ H NMR of fraction 21LC-D, it was estimated that glyceroglycolipid was the main component.

Miyabemoku adhesion inhibitory activity The brown alga Miyabemoku (365 g, wet weight) collected in Otaru City was extracted three times with 1.5 L of methanol. The extract was concentrated under reduced pressure, and then partitioned three times with water and diethyl ether (250 mL each). The resulting fat-soluble fraction was subjected to silica gel flash chromatography using hexane / ethyl acetate 95: 5 and hexane / ethyl acetate 9: 1. Elution with hexane / ethyl acetate 4: 1, hexane / ethyl acetate 1: 1, ethyl acetate 100%, ethyl acetate / methanol 9: 1, ethyl acetate / methanol 4: 1, ethyl acetate / methanol 1: 1, Elution with ethyl acetate / methanol 1: 3. Hexane / ethyl acetate 4: 1 fraction (473 mg dry product prepared to a concentration of 10 mg / mL with ethanol) and ethyl acetate / methanol 1: 3 fraction (10.8 mg dry product to 10 mg / mL with ethanol) (Adjusted concentration) was adsorbed on a TLC aluminum sheet of cellulose at 140 μg / cm ² and allowed to stand in a petri dish where the diatom Cylindrotheca closterium was cultured. After 2 weeks, no adsorbed diatom was observed on the adsorbed part. In the non-adsorbed part, diatom adhesion was observed.

Umitrano's adhesion inhibitory activity The brown alga Umitorano (10 g, wet weight) collected in Muroran was extracted three times with methanol. The extract was concentrated under reduced pressure, then partitioned three times with water and diethyl ether (25 mL each), and the resulting fat-soluble fraction (dried product prepared with ethanol at a concentration of 10 mg / mL) was added to a cellulose TLC aluminum sheet at 140 μg. / cm ^{2 was} adsorbed and allowed to stand in a petri dish where the diatom Cylindrotheca closterium was cultured. After 2 weeks, no adsorbed diatom was observed in the adsorbed part, but diatom adhering was observed in the non-adsorbed part. .

Adhesion inhibitory activity of urazozo Red alga urasozo (40 g, wet weight) collected in Muroran city was extracted three times with methanol. The extract was concentrated under reduced pressure, then partitioned three times with water and diethyl ether (25 mL each), and the resulting fat-soluble fraction (340 mg of dried product adjusted to a concentration of 10 mg / mL with ethanol) was added to the TLC aluminum of cellulose. When 140 μg / cm ^{2 was} adsorbed on the sheet and allowed to stand in a petri dish in which the diatom Cylindrotheca closterium was cultured, diatom did not adhere to the adsorbed portion after 2 weeks, and diatom adhered to the non-adsorbed portion. The diatom Nitzchia sp. Also showed an adhesion inhibitory effect.

Prepacifenol and 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene are adhesion inhibitors of urazozo
In the same manner as described in Example 5, 400 g (Frozen wet weight) of Chrysomelidae / Familyceae, collected in Muroran, was extracted three times with 500 ml of methanol. The extract was concentrated, and two-layer partitioning was performed with water and diethyl ether. The diethyl ether layer showing activity at 12.5 μg / cm ² was fractionated by silica gel column flash chromatography using n-hexane and ethyl acetate. After fractionation, fractions of n-hexane / ethyl acetate = 4: 1 and n-hexane / ethyl acetate = 1: 1 were subjected to PTLC. As the solvent, 100% benzene and chloroform: methanol = 97: 3 were used, and two kinds of compounds showing the adhesion diatom inhibitory activity were obtained. Each of the two compounds was repurified by PTLC to obtain Compound A (2.8 mg) and Compound B (4 mg).
From the NMR measurement, it was found from the NMR measurement that Compound A was Prepaciphenol (Non-patent Document 5) Phycological Research (1999), 47 (2), 87-95.). Compound B was found to be 2,10-dibromo-3-chloro-9-hydroxy-α-camigrene (Non-patent Document 6.).

As described in Example 1, the adhesion inhibitory activity test for the adhered diatom was performed using a TLC cellulose sheet. Using the attached diatom Nitzchia sp. Or Cylindrotheca closterium cultivated in an incubator at 25 ° C., the adhesion inhibitory activity of isolated and identified prepasiphenol and 2,10-dibromo-3-chloro-9-hydroxy-α-camigrene was observed. Tested. For the inhibitory activity test, a TLC aluminum sheet manufactured by MERCK was used, and 10 μl of a solution obtained by dissolving these compounds in ethanol was poured into each test section, dried, and then left to stand in a circular petri dish, and 25 ml of modified yogansun medium was injected. . The size of the test plot is 1.6 cm in diameter. The concentration of the compound to be tested was 10 μg / cm ² . Diatoms were planted in the petri dish and the state of adhesion after 7 days was observed with the naked eye.

Both prepasiphenol and 2,10-dibromo-3-chloro-9-hydroxy-α-camigrene showed strong adhesion-inhibiting activity at 10 μg / cm ² against the attached diatom Nitzchia sp. The area where diatoms were attached to was 1% or less. Moreover, it showed strong adhesion inhibitory activity at 10 μg / cm ² against Cylindrotheca closterium, and the area where diatoms adhered within the circle of the test area was 1% or less.

Mitsudezo's adhesion inhibitory activity and its adhesion inhibitors debromolaurinterol, isolaurinterol, debromoisolaurinterol
In order to further isolate and identify an adhesion-inhibiting substance different from Laurinterol in Example 1 from Mitsodezo, 300 g (Frozen and wet weight) of the squirrel family, Odonata, was collected three times with 500 ml of methanol. The extract was concentrated, and two-layer partitioning was performed with water and diethyl ether. The diethyl ether layer in which 50 μg / cm ² activity was observed was fractionated by silica gel column flash chromatography using n-hexane and ethyl acetate. After fractionation, the fraction of n-hexane / ethyl acetate = 9: 1 was subjected to PTLC. As the solvent, 100% toluene and hexane: benzene = 1: 1 were used, and four kinds of compounds exhibiting attached diatom inhibitory activity were obtained. Each of the four compounds was re-purified by PTLC to obtain Compound A (11.6 mg) (yield about 13%), Compound B (3.8 mg), and a mixture of Compounds C and D (0.5 mg).
From the NMR measurement, it was found that Compound A was Laurinterol described in Example 1. Compound B was found to be debromolaurinterol. It was found that the mixture of compounds C and D was a mixture (1: 1) of isolaurinterol and debromoisolaurinterol (Non-patent Documents 2 and 3).
As described in Example 1, the adhesion inhibitory activity test for the adhered diatom was performed using a TLC cellulose sheet.
Inhibitory activity of isolated lorinterol, debromololineterol, and a mixture of isolaurinterol and debromoisololineterol (1: 1) using adherent diatoms Nitzchia sp. And Cylindrotheca closterium bred in an incubator at 25 ° C Was tested. For the inhibitory activity test, a TLC aluminum sheet manufactured by MERCK was used, and 10 μl of a solution obtained by dissolving these compounds in ethanol was poured into each test section, dried, and then left to stand in a circular petri dish, and 25 ml of modified yogansun medium was injected. . The size of the test plot is 1.6 cm in diameter. The concentration of the compound to be tested was 3 μg / cm ² . Diatoms were planted in the petri dish and the state of adhesion after 7 days was observed with the naked eye.
Debromololineterol showed weak adhesion inhibitory activity against the attached diatom Nitzchia sp. At 10 μg / cm ² . On the other hand, no activity was shown at 10 μg / cm ² against Cylindrotheca closterium. This was considered because the concentration was too low.
A mixture of isolaurinterol and debromoisolorinterol (1: 1) showed strong adhesion inhibitory activity against adhering diatom Nitzchia sp. At 1 μg / cm ² , and diatoms adhered to the circle in the test area. The area occupied was 1% or less. Moreover, it showed strong adhesion inhibitory activity at 1 μg / cm ² against the attached diatom Cylindrotheca closterium, and the area where the diatom adhered in the circle of the test area was 1% or less.
Although each product of isolaurinterol and debromoisolaurinterol could not be separated from each other, the activity of laurotererol was strong, whereas the activity of debromololineterol was weak. It was speculated that the activity was greatly affected. From this, it was speculated that the activity of the mixture of isolaurin terol and debromoisolaurin terol (1: 1) is almost all in the isolaurin terol and not so much in the debromoisolaurin terol. Therefore, it was estimated that isorurolineterol exhibits adhesion inhibitory activity at about 0.5 μg / cm ² .

Adhesion inhibitory activity of Ezoyahaz and its attachment inhibitors Cubenol and Chromazonarol
450 g (frozen wet weight) of Amythidae / Aphididae collected in Muroran was extracted three times with 500 ml of methanol. The extract was concentrated, and two-layer partitioning was performed with water and diethyl ether. The diethyl ether layer showing activity at 12.5 μg / cm ² was fractionated by silica gel column flash chromatography using n-hexane and ethyl acetate. After fractionation, PTLC was repeated twice for fractions of n-hexane / ethyl acetate = 9: 1 and n-hexane / ethyl acetate = 9: 3. From the fraction of n-hexane / ethyl acetate = 9: 1, Compound A (10 mg in dry weight) was obtained using 100% benzene as a solvent. From the fraction of n-hexane / ethyl acetate = 9: 3, chloroform: methanol = 99: 1 was used as a solvent to obtain Compound B (1 mg by dry weight).
From the NMR and MS measurements, Compound A was found to be cubenol (Non-Patent Document 7). Further, NMR and MS measurements revealed that Compound B was chromazonalol (Non-patent Documents 8 and 9).

As described in Example 1, the adhesion inhibitory activity test for the adhered diatom was performed using a TLC cellulose sheet. The inhibitory activity of cuvenol and chromazonalol isolated and identified was tested using the adherent diatoms Nitzchia sp. And Cylindrotheca closterium bred in an incubator at 25 ° C. For the inhibitory activity test, a TLC aluminum sheet manufactured by MERCK was used, and 10 μl of a solution obtained by dissolving these compounds in ethanol was poured into each test section, dried, and then left to stand in a circular petri dish, and 25 ml of modified yogansun medium was injected. . The size of the test plot is 1.6 cm in diameter. The concentration of the compound to be tested was 10 μg / cm ² . Diatoms were planted in the petri dish and the state of adhesion after 7 days was observed with the naked eye.

Cubenol exhibited strong adhesion diatom inhibitory activity at 10 μg / cm ² against the adhering diatom Nitzchia sp. The area where the diatom adhered in the circle of the test area was 1% or less. Moreover, it showed strong adhesion diatom inhibitory activity at 10 μg / cm ² against the adherent diatom Cylindrochetaca closterium, and the area where the diatom adheres within the circle of the test area was 1% or less.
Chromazonalol showed strong adhesion inhibitory activity at 10 μg / cm ² against the adhering diatom Nitzchia sp., And the area where the diatom adhered in the circle of the test area was 1% or less. Moreover, it showed strong adhesion inhibitory activity at 3 μg / cm ² against the adhering diatom Cylindrotheca closterium, and the area where the diatom adhered in the circle of the test area was 1% or less.

  The present invention provides an adhesion preventive agent for adhering diatoms containing a fat-soluble fraction of a seaweed extract or a compound isolated therefrom, which is highly safe for seafood and human bodies.

Claims (4)

An anti-adhesive agent for adhering diatoms containing a fat-soluble fraction of seaweed extract. The seaweed is at least selected from the group consisting of brown algae belonging to the family Dictyotaceae, red algae belonging to the genus Laurencia, Agarum clathratum, Sargassum miyabei, and Sargassum thunbergii The adhesion preventing agent according to claim 1, which is one. The brown alga belonging to the family Dictyotaceae is at least one selected from the group consisting of Ezoyahaz (Dictyopteris divaricata) and Shiwayahaz (Dictyopteris undulata), and the red alga belonging to the genus Laurencia is Laurencia okamurae, Laurencia nipponica, Laurencia pacifica, Laurencia majuscula, Laurencia filiformis, Laurencia johnstony (Laurencia johnston) The adhesion preventing agent according to claim 2, which is at least one selected from the group consisting of claviformi). As an active ingredient, at least one selected from the group consisting of laurenterol, isolaurenterol, prepacylphenol, 2,10-dibromo-3-chloro-9-hydroxy-α-camiglen, cubenol and chromazonalol Including anti-adhesion agent for adhering diatoms.