JP7240732B2 - marine invertebrate repellent - Google Patents
marine invertebrate repellent Download PDFInfo
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- JP7240732B2 JP7240732B2 JP2019547547A JP2019547547A JP7240732B2 JP 7240732 B2 JP7240732 B2 JP 7240732B2 JP 2019547547 A JP2019547547 A JP 2019547547A JP 2019547547 A JP2019547547 A JP 2019547547A JP 7240732 B2 JP7240732 B2 JP 7240732B2
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- repellent
- extract
- marine
- seaweed
- macri
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
- A01M29/00—Scaring or repelling devices, e.g. bird-scaring apparatus
- A01M29/12—Scaring or repelling devices, e.g. bird-scaring apparatus using odoriferous substances, e.g. aromas, pheromones or chemical agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/03—Algae
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Toxicology (AREA)
- Birds (AREA)
- Insects & Arthropods (AREA)
- Pest Control & Pesticides (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Catching Or Destruction (AREA)
Description
本発明は、海産無脊椎動物忌避剤に関する。より詳細には、本発明は、マクリ、ヤハズグサ等の海藻抽出物を含有する海産無脊椎動物忌避剤に関する。 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.
自然界において生物多様性は生態系の機能を制御する役割を担うことが知られており、我々人類が自然界から受ける生態系サービスを持続的に利用し続けるために無くてはならないものである。しかしながら、地球上の生物は現在6回目の大量絶滅期を迎えており、少なくとも現存種の50%以上が絶滅する見込みであると言われるようになった。このことから、生物多様性の損失に伴う生態系の機能の変化や消失の影響を明らかし、生態系機能やサービスの変化に関して将来予測を行うことが早急の課題となり、生物多様性を操作してその検証実験を実施する必要性が生じた。その操作には薬剤等の投与が用いられることが多いが、その多くは農薬として開発された人工化合物であり、殺傷能力も高いことから、特に開放的な海洋で使用した際には自然界への広範囲な悪影響が強く懸念されている。そのため、より毒性が少なく、動物に忌避行動などを誘発することにより生物多様性を操作することができ、かつ安全な物質の開発が待たれている。 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.
水産利用加工や増養殖などの産業においても、水産物の洗浄や養殖魚の消毒等には、自然界や人体への悪影響が懸念される薬剤が使用された経緯があるため、より安全な薬剤の開発が進められている状況にある。 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.
そもそも自然界では、植物と動物間の生物間相互作用とその進化の過程において、植物には、動物からの摂食を阻害する化学防御物質を獲得した種もあり、それぞれの植物が種特有の化合物を持つことが多い。このような自然界に存在する化学的忌避物質は、人工化合物と比較して、環境にやさしい化学物質であると考えることができる。 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.
マクリ（Digenea simplex）は、フジマツモ科マクリ属の紅藻の一種である。マクリは回虫駆除のための生薬として古来より使用されている。その主成分はイミノ酸の一種であるカイニン酸である。カイニン酸は、カイニン酸型グルタミン酸受容体のアゴニストとして作用することが知られている。 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.
シワヤハズ（Dictyopteris undulata）は、アミジグサ科ヤハズグサ属の褐藻の一種である。シワヤハズに含まれるテルペノイドであるゾナロールが潰瘍性大腸炎を抑制することが報告されている（非特許文献1）。また、シワヤハズに含まれるクロマゾナロールがエゾアワビの摂食を阻害することが報告されている（非特許文献2）。 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).
しかしながら、マクリやシワヤハズ等海藻の抽出物を海産無脊椎動物の忌避剤として使用することは知られていない。 However, it is not known to use extracts of seaweeds such as Macri and Shiwayahazu as repellents for marine invertebrates.
本発明は、自然界に存在する化合物を使用した海産無脊椎動物の忌避剤を提供することを目的とする。 An object of the present invention is to provide a repellent for marine invertebrates using naturally occurring compounds.
本発明者らは、鋭意検討の結果、マクリ属及びヤハズグサ属の海藻の抽出物中に、海産無脊椎動物に対し忌避効果を有する成分が含まれていることを見出した。マクリ属海藻及びヤハズグサ属海藻は、薬効を有する化学物質を含有することが知られているが（マクリ属海藻：カイニン酸、ヤハズグサ属海藻：ゾナロール、クロマゾナロール）、成分分析によれば、これらのいずれの物質にも相当しない画分に海産無脊椎動物への忌避作用としての有効成分が含まれていた。マクリ属海藻抽出物とヤハズグサ属海藻抽出物は、忌避作用スペクトルが異なり、マクリ属海藻抽出物は、節足動物門に属する海産無脊椎動物に対する忌避効果に優れ、ヤハズグサ属海藻抽出物は、軟体動物門に属する海産無脊椎動物に対する忌避効果に優れていた。さらに、マクリ属海藻抽出物とヤハズグサ属海藻抽出物とを組み合わせることにより、広範な忌避作用スペクトルを達成することができた。 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.
発明者らは、上記知見に基づき更に検討を加え、本発明を完成するに至った。
即ち、本発明は下記の通りである：The inventors have further studied based on the above findings, and have completed the present invention.
Accordingly, the present invention is as follows:
［1］海藻抽出物を含有する、海産無脊椎動物忌避剤。
［2］海藻が、マクリ属に属する紅藻、又はヤハズグサ属に属する褐藻である、［1］記載の忌避剤。
［3］海藻が、マクリ属に属する紅藻であり、海産無脊椎動物が、節足動物門に属する海産無脊椎動物である、［2］記載の忌避剤。
［4］海藻が、ヤハズグサ属に属する褐藻であり、海産無脊椎動物が、軟体動物門に属する海産無脊椎動物である、［2］記載の忌避剤。
［5］海藻抽出物が、マクリ属に属する紅藻の抽出物、及びヤハズグサ属に属する褐藻の抽出物を含む、［1］記載の忌避剤。
［6］［1］～［5］の何れか記載の忌避剤を物品に塗布又は含浸する、あるいは物品を構成する基材に混練することを含む、物品の海産無脊椎動物忌避処理方法。
［7］［1］～［5］の何れか記載の忌避剤が、塗布又は含浸されている、あるいは物品を構成する基材に混練されていることを特徴とする、海産無脊椎動物忌避性物品。
［8］［1］～［5］の何れか記載の忌避剤を海水中に溶出させることを含む、海産無脊椎動物の忌避方法。[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.
本発明により、天然化合物を有効成分として含有する海産無脊椎動物忌避剤が提供される。本発明の忌避剤は、天然海藻抽出物又はその分画成分を有効成分としているので、人体や環境への安全性が高い。 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.
現在、タイラギやカキ等貝類の養殖で使用する飼育カゴなどの養殖施設では、フジツボ等の海産無脊椎動物の付着による貝類の成長阻害や斃死などが各地で深刻な問題となっており、その駆除に莫大なコストがかかっている。漁網防汚剤の活用が検討されているが、貝類への毒性が懸念されるため、その使用は限定的である。本発明の忌避剤は、それらの海産無脊椎動物の付着防止効果が期待でき、天然海藻由来の安全な物質であることから、安全性のニーズにもこたえることができる。また、本発明の忌避剤は、ワカメ、ヒジキ、ノリ等の海藻製品から海産無脊椎動物を除去する処理に使用できると期待でき、水産加工において有用である。貝類、海藻類、魚類等の養殖業に加え、発電所、工場等の海水パイプや、船底、バラスト等への海産無脊椎動物の付着の防止において、本発明の忌避剤は有効性が期待できる。このように、本発明の忌避剤は、海洋無脊椎動物やその付着による害を受ける産業分野において有用である。 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.
本発明は、海藻抽出物を含有する、海産無脊椎動物忌避剤（本明細書にて「本発明の忌避剤」と称することがある）を提供する。 The present invention provides a marine invertebrate repellent (sometimes referred to herein as a "repellent of the invention") containing a seaweed extract.
本発明において使用する海藻は、海産無脊椎動物を忌避する有効成分（以下、「海産無脊椎動物忌避成分」または「忌避成分」ということがある）を含有する海藻である。海産無脊椎動物忌避成分を含有する海藻としては、マクリ属（Digenea）に属する紅藻、ヤハズグサ属（Dictyopteris）に属する褐藻等を挙げることができるが、これらに限定されない。マクリ属に属する紅藻としては、マクリ（Digenea simplex）等を挙げることができるが、これに限定されない。ヤハズグサ属に属する褐藻としては、エゾヤハズ(Dictyopteris divaricata)、シワヤハズ(Dictyopteris undulata)、ヤハズグサ(Dictyopteris latiuscula)、ヘラヤハズ(Dictyopteris prolifera)、ウラボシヤハズ(Dictyopteris membranacea)等を挙げることができるが、これらに限定されない。 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.
抽出に付す前の海藻は、抽出作業の直前に採取してもよいし、予め採取し、乾燥保存したものであってもよい。また、海藻は、適度に細断又は粉砕した状態にして抽出に用いることが望ましい。あるいは、磨砕、擂潰、粉末化等した状態にして抽出に用いてもよい。 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.
抽出に用いる溶媒としては、水性溶媒、有機溶媒、又はその混合物を挙げることができる。水性溶媒としては、水、緩衝水溶液（リン酸緩衝生理食塩水（PBS）等）、食塩水、海水等が挙げられるが、これらに限定されない。有機溶媒としては、C1-6アルコール（メタノール、エタノール、ブタノール、プロパノール、イソプロパノール等）、C1-6飽和脂肪酸（酢酸等）等の親水性有機溶媒、C1-6アルキルエーテル（ジエチルエーテル、ジメチルエーテル、エチルメチルエーテル等）、ハロメタン（クロロメタン、ジクロロメタン、クロロホルム、四塩化炭素等）等の非親水性有機溶媒、又はその混合物を挙げることができるが、これらに限定されない。抽出に用いる溶媒は、好ましくは、水性溶媒、親水性有機溶媒、またはそれらの混合物であり、より好ましくは、水性溶媒、メタノール、エタノール、酢酸、又はそれらの混合物である。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.
抽出溶媒の使用量は、乾燥した海藻1重量部に対して、通常1～10重量部、好ましくは、1.5～5重量部、より好ましくは2～3重量部であるが、これらに限定されない。 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.
抽出時間は、通常0.5～48時間、好ましくは1～24時間であるが、これらに限定されない。 The extraction time is usually 0.5 to 48 hours, preferably 1 to 24 hours, but not limited thereto.
海藻に抽出溶媒を添加し、静置することによっても、抽出を行うことができるが、ホモジナイザー等により撹拌する方が、高い抽出効率が期待できる。 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.
抽出操作後、混合物を遠心分離やろ過等に付すことによって、不溶物を除去し、上清を回収することにより、海藻の抽出物を得ることができる。この抽出物をそのまま、あるいは必要に応じて濃縮又は乾燥して、本発明の海産無脊椎動物忌避剤として用いることができる。上記の溶媒で抽出された抽出液は、その純度においても、海産無脊椎動物忌避剤として有用であるが、更に、生化学分野において周知の精製工程（逆相クロマトグラフィー、疎水性相互作用クロマトグラフィー、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、アフィニティクロマトグラフィー、透析、限外濾過、液－液分配等）に付し、海産無脊椎動物忌避成分を分画してもよい。各画分中の海産無脊椎動物忌避活性は、後述の実施例に記載した方法により評価することができる。本発明において、海藻を抽出溶媒で直接抽出処理することにより得られる抽出物（一次抽出物）のみならず、一次抽出物を更なる精製工程に付して得られる産物も、海藻抽出物に包含される。 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.
以下に、マクリ属に属する紅藻の抽出物、及びヤハズグサ属に属する褐藻の抽出物からの忌避成分の精製の例を示すが、本発明はこれらに限定されるものではない。 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.
（マクリ属に属する紅藻の抽出液からの忌避成分の精製）
後述の実施例に記載したように、マクリ属に属する紅藻の抽出液を、分画分子量3500の透析膜を使用して透析すると、忌避成分は透析外液へと移行する。このことから、マクリ属に属する紅藻中に含まれる忌避成分の分子量は3500を下回ると見積もられる。即ち、マクリ属に属する紅藻の抽出物を、分子量サイズによって分離する精製手段に付し、分子量3500未満の成分を含む画分を分取することにより、忌避成分を精製することができる。本発明は、このような工程を含む、マクリ属に属する紅藻の海産無脊椎動物忌避成分の精製方法、および海産無脊椎動物忌避剤の製造方法をも提供する。分子量サイズによって分離する精製手段としては、透析、限外濾過、ゲル濾過クロマトグラフィー等を挙げることができるが、これらに限定されない。(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.
後述の実施例に記載したように、マクリ属に属する紅藻に含まれる忌避成分は、蒸留水によって抽出されるので、水溶性であるが、蒸留水で平衡化した活性炭カラムに吸着し、酢酸酸性メタノール（pH 3.0）によって溶出されるので、一定の疎水性を有する物質であると推測される。即ち、マクリ属に属する紅藻の抽出物（好ましくは、水性溶媒による抽出物）を、水性溶媒等により平衡化した疎水性カラムに付すことにより、忌避成分を疎水性カラムに吸着させ、その後、有機溶媒（例、酢酸酸性メタノール（pH 3.0））で吸着した忌避成分を疎水性カラムから溶出することにより、忌避成分を精製することができる。本発明は、このような工程を含む、マクリ属に属する紅藻の海産無脊椎動物忌避成分の精製方法、海産無脊椎動物忌避剤の製造方法をも提供する。精製に用いるカラムの種類としては、活性炭カラム、オクタデシルシリル基修飾シリカゲル（ODS）カラム、サイズ排除クロマトグラフィー（SEC）カラム、親水性相互作用クロマトグラフィー（HILIC）カラム等を挙げることができるが、これらに限定されない。 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
上述した2つの精製工程（分子量サイズによる分離、及び疎水性カラムによる分離）を組み合わせて行ってもよい。この場合、組み合わせる順番は特に限定されず、分子量サイズによる分離を行った後で、疎水性カラムによる分離を行ってもよいし、疎水性カラムによる分離を行った後で、分子量サイズによる分離を行ってもよい。2つの精製工程を組み合わせることにより、忌避成分のより高度な精製が期待できる。 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.
即ち、好ましい一態様において、
（A）マクリ属に属する紅藻の抽出物を、分子量サイズによって分離する精製手段に付し、分子量3500未満の成分を含む画分を分取し、
（B）（A）で得た分子量3500未満の成分を含む画分を、疎水性カラムに付し、忌避成分を疎水性カラムに吸着させ、その後、有機溶媒（例、酢酸酸性メタノール（pH 3.0））で吸着した忌避成分を疎水性カラムから溶出することにより、忌避成分を精製する。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.
また、別の好ましい一態様において、
（B’)マクリ属に属する紅藻の抽出物を、疎水性カラムに付し、忌避成分を疎水性カラムに吸着させ、その後、有機溶媒（例、酢酸酸性メタノール（pH 3.0））で吸着した忌避成分を疎水性カラムから溶出すること、
（A’）（B’)で得た、忌避成分を含有する画分を、分子量サイズによって分離する精製手段に付し、分子量3500未満の成分を含む画分を分取することにより、忌避成分を精製する。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.
なお、上記（A)または（B')の精製工程で得た画分を、直接（B)または（A')の精製工程に付してもよいし、濃縮や溶媒置換等の処理を加えた上で、（B)または（A')の精製工程に付してもよい。 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').
本発明において、最終工程で得られた画分について、節足動物門に属する海産無脊椎動物（例、ヨコエビ）に対する忌避作用を評価してもよい。海産無脊椎動物忌避活性は、後述の実施例に記載した方法により評価することができる。 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.
（ヤハズグサ属に属する褐藻の抽出液からの忌避成分の精製）
後述の実施例に記載したように、ヤハズグサ属に属する褐藻に含まれる忌避成分は、蒸留水によって抽出されるので、水溶性であるが、該忌避成分はメタノールによっても抽出されること、水およびメタノールの混合液（容積比50：50）／ジエチルエーテルによる液－液分配では主にエーテル層に分配されること、及びシリカゲルカラムに吸着し、ジエチルエーテルやメタノールで溶出されることから、一定の疎水性を有する物質であると推測される。即ち、ヤハズグサ属に属する褐藻の抽出物（好ましくは、水性溶媒、メタノール、又はその混合液による抽出物）を、水性溶媒又はメタノール濃度が50 %(v/v)以下である水性溶媒／メタノール混合液と、ジエチルエーテルと同等かそれよりも低い極性を有する有機溶媒との間で液－液分配を行い、有機溶媒層を回収することにより、忌避成分を精製することができる。好ましくは、水およびメタノールの混合液（容積比50：50）とジエチルエーテルによる液－液分配を行い、ジエチルエーテル層を回収する。また、ヤハズグサ属に属する褐藻の抽出物を、シリカゲルカラムに付し、忌避成分をシリカゲルカラムに吸着させ、吸着した忌避成分を有機溶媒で溶出することにより、忌避成分を精製することができる。忌避成分は、ヘキサンではシリカゲルカラムから溶出され難く、ジエチルエーテルやメタノールで溶出されるので、ヤハズグサ属に属する褐藻の抽出物は、ヘキサンと同等かそれよりも低い極性の有機溶媒に溶解した上で、シリカゲルカラムに付すことにより、忌避成分をシリカゲルカラムに吸着させることができる。忌避成分が吸着したシリカゲルカラムを、ヘキサンと同等かそれよりも低い極性の有機溶媒により洗浄した上で、溶媒の極性を漸進的に又は段階的に上昇させると、溶媒の極性がジエチルエーテルやメタノールと同等程度となった時点で、忌避成分がシリカゲルカラムから溶出される。本発明は、このような工程を含む、ヤハズグサ属に属する褐藻の海産無脊椎動物忌避成分の精製方法、および海産無脊椎動物忌避剤の製造方法をも提供する。(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.
上述した2つの精製工程（液－液分配、及びシリカゲルカラムによる分離）を組み合わせて行ってもよい。この場合、組み合わせる順番は特に限定されず、液－液分配を行った後で、シリカゲルカラムによる分離を行ってもよいし、シリカゲルカラムによる分離を行った後で、液－液分配を行ってもよい。2つの精製工程を組み合わせることにより、忌避成分のより高度な精製が期待できる。 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.
即ち、好ましい一態様において、
（A）ヤハズグサ属に属する褐藻の抽出物を、水性溶媒又はメタノール濃度が50 %(v/v)以下である水性溶媒／メタノール混合液と、ジエチルエーテルと同等かそれよりも低い極性を有する有機溶媒との間の液－液分配に付し、有機溶媒層を回収し、
（B）（A）で得た有機溶媒層画分を、ヘキサンと同等かそれよりも低い極性を有する有機溶媒に転溶し、シリカゲルカラムに付し、ヘキサンと同等かそれよりも低い極性を有する有機溶媒によりシリカゲルカラムを洗浄し、溶媒の極性を漸進的に又は段階的に上昇させて、忌避成分をシリカゲルカラムから溶出することにより、忌避成分を精製する。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.
また、別の好ましい態様において、
（B’)ヤハズグサ属に属する褐藻の抽出物を、ヘキサンと同等かそれよりも低い極性を有する有機溶媒に溶解し、シリカゲルカラムに付し、ヘキサンと同等かそれよりも低い極性を有する有機溶媒によりシリカゲルカラムを洗浄し、溶媒の極性を漸進的に又は段階的に上昇させて、忌避成分をシリカゲルカラムから溶出すること、
（A’）（B’)で得た、忌避成分を含有する画分を、水性溶媒又はメタノール濃度が50 %(v/v)以下である水性溶媒／メタノール混合液と、ジエチルエーテルと同等かそれよりも低い極性を有する有機溶媒との間の液－液分配に付し、有機溶媒層を回収することにより、忌避成分を精製する。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.
なお、上記（A)または（B')の精製工程で得た画分を、直接（B)または（A')の精製工程に付してもよいし、濃縮や溶媒置換等の処理を加えた上で、（B)または（A')の精製工程に付してもよい。 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').
本発明において、最終工程で得られた画分について、軟体動物門に属する海産無脊椎動物（例、チグサガイ等の巻貝）に対する忌避作用を評価してもよい。海産無脊椎動物忌避活性は、後述の実施例に記載した方法により評価することができる。 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.
上記海藻抽出物は、海産無脊椎動物に対して毒性を有し、海産無脊椎動物が該抽出物に対して忌避行動を取るため、海産無脊椎動物忌避剤として有用である。
ここで、「海産無脊椎動物」とは、無顎類、魚類、両生類、爬虫類、鳥類、哺乳類といった脊椎を有する動物以外の動物であって、海洋に棲息する動物をいい、たとえば、節足動物門（Arthropoda）、軟体動物門（Mollusca）、刺胞動物門（Cnidaria）、環形動物門（Annelida）等に属する海洋性の無脊椎動物が挙げられる。
マクリ属に属する紅藻の抽出物は、節足動物門に属する海産無脊椎動物に対する忌避効果に優れるため、節足動物門に属する海産無脊椎動物の忌避剤として有用である。節足動物門に属する海産無脊椎動物としては、甲殻亜門（Crustacea）（顎脚綱（Maxillopoda）（カイアシ亜綱（Copepoda）（カイアシ類）、貝虫亜綱（Ostracoda）（貝虫類）、鞘甲亜綱（Thecostraca）（フジツボ亜目（Balanomorpha）、エボシガイ亜目（Lepadomorpha）、カメノテ属（Capitulum）等））、軟甲綱（Malacostraca）（トゲエビ亜綱（Hoplocarida）（シャコ目（Stomatopoda）等）、真軟甲亜綱（Eumalacostraca）（Senticaudata亜目（ヨコエビ類）、タナイス目（Tanaidacea）、等脚（ワラジムシ）目（Isopoda）等））等）を挙げることができるが、これらに限定されない。
ヤハズグサ属に属する褐藻の抽出物は、軟体動物門に属する海産無脊椎動物に対する忌避効果に優れるため、軟体動物門に属する海産無脊椎動物の忌避剤として有用である。軟体動物門に属する海産無脊椎動物としては、多板綱（Polyplacophora）（ヒザラガイ類）、腹足綱（Gastropoda）（カサガイ目（Nacellidae）、チグサガイ（Cantharidus japonicus）等）等を挙げることができるが、これらに限定されない。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.
マクリ属に属する紅藻の抽出物は、節足動物門に属する海産無脊椎動物に対する忌避効果に優れ、ヤハズグサ属に属する褐藻の抽出物は、軟体動物門に属する海産無脊椎動物に対する忌避効果に優れるため、マクリ属海藻抽出物とヤハズグサ属海藻抽出物とを組み合わせることにより、海産無脊椎動物に対する広範な忌避作用スペクトル（節足動物門及び軟体動物門）を達成することができる。従って、一態様において、本発明の忌避剤は、マクリ属に属する紅藻の抽出物、及びヤハズグサ属に属する褐藻の抽出物を含有する。 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.
本発明の忌避剤の使用の一態様において、本発明の忌避剤を物品に塗布または含浸し、あるいは物品を構成する基材に混練することにより、当該物品を海産無脊椎動物忌避処理し、海産無脊椎動物忌避性物品を得ることができる。本発明の忌避剤により処理し得る対象物品としては、特に限定されないが、海産無脊椎動物の接近や付着を回避することが必要とされる物品、例えば、ワカメ、ヒジキ、ノリ、コンブ等の海藻、タイラギやカキ等の二枚貝、魚類等の養殖において海中に浸漬して使用する設備（飼育カゴ、隔離網、ロープ、ブイ、取水・排水管など）や、船舶の船底面や船側面、バラスト、海中に浸漬する建造物（発電所、工場等の海水パイプ、海中魚礁、港湾設備のコンクリート建造物など）等を挙げることができるが、これらに限定されない。ワカメ、ヒジキ、ノリ、コンブ等の海藻の養殖において、本発明の忌避剤で処理した設備を使用することにより、植食性動物プランクトン等の植食性無脊椎動物の接近を回避し、植食性無脊椎動物による食害を防ぐことが期待できる。また、タイラギやカキ等の養殖、あるいは魚類の養殖において、本発明の忌避剤で処理した設備を使用することにより、フジツボ等の海産無脊椎動物の付着による養殖水産物の成長阻害や斃死の防止や、魚類に付着する寄生性甲殻類の忌避などが期待できる。 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.
本発明の忌避剤の使用の一態様において、本発明の忌避剤を海水中に溶出させることにより、海産無脊椎動物を忌避する。本発明の忌避剤を海水中に溶出させる方法としては、本発明の忌避剤の散布、本発明の忌避剤を塗布、浸漬又は混練した物品の海水中への投入等が挙げられるがこれらに限定されない。海産無脊椎動物の接近を回避することが必要とされる海中の領域、例えば、ワカメ、ヒジキ、ノリ、コンブ等の海藻、タイラギやカキ等の二枚貝、魚類等の養殖場、海中に浸漬する建造物（発電所、工場、港湾設備等）の周辺海域、磯焼け海域等において、本発明の忌避剤を海水中に溶出させることにより、当該領域から海産無脊椎動物を除去し、当該領域への海産無脊椎動物の接近を防ぐことが期待できる。ワカメ、ヒジキ、ノリ、コンブ等の海藻の養殖場において、本発明の忌避剤を海水中に溶出させることにより、植食性無脊椎動物の接近を回避し、植食性無脊椎動物による食害を防ぐことが期待できる。また、海藻中に生息する海産無脊椎動物を除去することができるので、海藻への海産無脊椎動物の混入を抑制し、収穫される海藻の品質の向上が期待できる。また、磯焼け海域において、本発明の忌避剤を海水中に溶出させることにより、当該領域から植食性無脊椎動物を駆除する効果が期待できる。 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.
以下の実施例により本発明をより具体的に説明するが、実施例は本発明の単なる例示を示すものにすぎず、本発明の範囲を何ら限定するものではない。 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.
［試験例1］ 本発明の忌避剤の忌避効果の評価方法の構築
10t水槽で、流水環境を作成した。30cm四方のアマモの人工群落を作り、忌避剤溶解塔から1m間隔で3列（各列3個ずつ）設置した（図1）。各パッチ（群落）にヨコエビを50匹ずつ投入し、溶解塔から忌避剤（マクリ水抽出物、濃縮なし）を流布し、2時間後の各パッチにおけるヨコエビの残数を計測した。[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.
結果を図2に示す。忌避剤を流布した場合、溶解塔から距離が離れるほど、ヨコエビ残数が増加した。一方、忌避剤を流布しない場合は、このような相関関係は認められなかった。従って、この方法を用いて、忌避効果を評価し得ることが示された。 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.
［試験例2］ マクリ抽出液中の忌避成分の探索
カイニン酸はマクリに含まれるイミノ酸の一種で、回虫駆除薬として知られている。カイニン酸がマクリ抽出液に含まれる忌避成分の候補として考えられたため、マクリ抽出液の忌避効果を、カイニン酸純物質と比較した。
マクリ抽出液及びカイニン酸純物質の水溶液のそれぞれに、ヨコエビを72時間曝露し、死亡率を調べた。マクリ抽出液中のカイニン酸濃度が、カイニン酸純物質の水溶液の濃度と同一になるように、マクリ抽出液の濃度を調整し、マクリ抽出液とカイニン酸純物質の水溶液のヨコエビ死亡率への影響を比較した。[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.
結果を図3に示す。マクリ抽出液はヨコエビに対して強力な毒性効果を示した。一方、カイニン酸純物質によっては、ヨコエビは殺傷されなかった。従って、マクリ抽出液のヨコエビに対する毒性効果は、マクリに含まれるカイニン酸以外の物質による可能性が高いことが示された。 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.
［試験例3］ マクリ抽出液中の忌避成分の安定性の評価
マクリ抽出液（10 g/L）（乾燥藻体重量／体積）を、常温、乾燥、及び冷凍の各条件で30日間保存し、その忌避効果を保存前（保存0日）のものと比較した。忌避効果の評価は、試験例2と同様に、ヨコエビをマクリ抽出液に72時間曝露し、死亡率を調べることにより行った。[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.
結果を図4に示す。マクリ抽出液のヨコエビに対する毒性効果は、保存方法に関わらず安定であった。 The results are shown in FIG. The toxic effect of Macri extract on amphipods was stable regardless of the storage method.
［試験例4］ 抽出液の作成及び忌避活性の評価
（マクリ抽出液の作成）
乾燥マクリ100gを凍結粉砕し、超純水を加え、混合物をろ過した。ろ液を15000Gで10分間遠心分離後、上澄みを採取することによりマクリ抽出液（100 g/L）（乾燥藻体重量／体積）を得た。[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).
（ヤハズグサ抽出液の作成）
乾燥ヤハズグサ100gを凍結粉砕し、ろ過海水を加え、混合物をろ過した。ろ液を15000Gで10分間遠心分離後、上澄みを採取することによりヤハズグサ抽出液（100 g/L）（乾燥藻体重量／体積）を得た。(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).
（忌避活性の評価）
以下の表に記載した通り、マクリ抽出液及びヤハズグサ抽出液を段階的に希釈した抽出液を調製した。なお、表中「ＤＷ」は蒸留水を示す。(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.
暗所、20℃にて、ヨコエビ（n=12）及びチグサガイ（n=15）を、段階的に希釈した抽出液に24時間曝露し、生死をカウントした。ロジスティック回帰分析により、ヨコエビ及びチグサガイの各死亡率と抽出液濃度との関係を調べた。 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.
マクリ抽出液についての結果を表2及び図5に示す。 Table 2 and FIG. 5 show the results for the Macri extract.
表２中、上段にヨコエビに対する効果、下段にチグサガイに対する効果を示す。 In Table 2, the upper row shows the effect on the side shrimp, and the lower row shows the effect on the mussel.
ヨコエビ及びチグサガイの双方について、マクリ抽出液の濃度が高いほど、死亡率が高いことが示された。マクリ抽出液はヨコエビにもチグサガイにも毒性を示したが、両者の死亡曲線間には有意な差が認められ（F検定：p<0.0001）、その効果が種間で異なることが示された。 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. .
ヤハズグサ抽出液についての結果を表3及び図6に示す。 Table 3 and FIG. 6 show the results for the R. japonicus extract.
表３中、上段にヨコエビに対する効果、下段にチグサガイに対する効果を示す。 In Table 3, the upper row shows the effect on the side shrimp, and the lower row shows the effect on the mussel.
ヤハズグサ抽出液の濃度が高くなるほどチグサガイの死亡率は高くなるが（p<0.0001）、ヨコエビの死亡率とヤハズグサの濃度の間には関係性が認められなかった（p=0.53）。従って、ヤハズグサ抽出液の忌避成分はチグサガイには有効であるが、ヨコエビには効果がないことが示唆された。 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.
［試験例5］ マクリからの忌避成分の精製
乾燥マクリ（150g）を液体窒素で急速凍結し、ホモジナイザーを用いて粉末とした。粉末としたマクリに藻体重量の3倍量の蒸留水（450 ml）を加え、ホモジナイザーで十分に撹拌した。得られたホモジネートを、高速遠心分離器を用いて、3000rpmで5分間遠心分離し、上清と藻体残渣とに分けた。上清を1.5 mlチューブに移し、更に12000rpmで5分間遠心分離し、上清を回収した。藻体残渣に、再度蒸留水（300 ml）を加え、ホモジナイズして遠心分離し、この操作を2回繰り返し、合計3回藻体から有効成分の抽出を行い、抽出液を合わせた（抽出液1、600ml）。[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).
得られたマクリ抽出液（600 ml）を、300 mlずつ2つに分け、300 mlのマクリ抽出液を、2000 mlの蒸留水に対して、室温にて一晩透析した。分画分子量3500の透析膜を使用した。透析内液（700 ml）をヨコエビに対する毒性評価用に保存した（抽出液2）。透析外液（2000 ml x 2）を多検体濃縮装置（60℃、35 mber、260 rpm）を用いて、50 mlに濃縮した（抽出液3、毒性評価は1/10希釈で実施）。
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) (
活性炭カラムクロマトグラフィーにより、更なる精製を行った。よく水洗いした活性炭150 cm3をカラムに充填した。浮いた活性炭は除去した。450 mlの蒸留水でカラムを洗浄した。濃縮透析外液（50 ml）を70 mlに希釈した上で、カラムにアプライした。450 mlの蒸留水でカラムを洗浄した。非吸着画分（450 ml）をヨコエビに対する毒性評価用に保存した（抽出液4）。450 mlの酢酸酸性メタノール（pH 3.0）で溶出し、溶出画分（450 ml）を50 mlに減圧濃縮した（抽出液5、毒性評価は1/10希釈で実施）。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 (
得られた抽出液1～抽出液5について、濃度をそろえた上で、試験例4に準じ、ヨコエビに対する毒性試験を行った（n=7、20℃、暗条件）。ウェル内のヨコエビの全個体を死亡させたときの最大希釈率は以下の通りであった。
The
この結果から、マクリ抽出液中のヨコエビ忌避成分が、抽出液5に濃縮されたことが示唆された。
From this result, it was suggested that the
［試験例6］ シワヤハズからの忌避成分の精製
乾燥シワヤハズのメタノール抽出液155 ml（0.5 g/ml）にジエチルエーテル (155 ml)及び蒸留水（155 ml）を添加し、5分間振とう抽出を行った。上層（エーテル層）と下層（水、メタノール層）に分離し、下層（水、メタノール層）にジエチルエーテル (155 ml)を添加し、再抽出を行った。水、メタノール層は、活性評価用に保存した。[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.
エーテル層を合わせ、ロータリーエバポレーター及び窒素吹き付けで濃縮、乾固した。残渣をヘキサンに溶解し、15.5 mlにメスアップした（68 mg/ml）。該ヘキサン溶液を窒素吹き付けで更に濃縮し、5 mlにメスアップした（乾燥藻体 15 g/ml相当）（エーテル層A）。 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).
シリカゲルカラムクロマトグラフィーによる更なる精製を行った。Sep-Pak Silica 35cc Vac Cartridge, 10 g (Waters製)を3連で用いた。メタノール50 ml、ジクロロメタン50ml、ジエチルエーテル50 ml及びヘキサン50 mlをカラムに通して安定化させた。ヘキサンに転溶したエーテル層Aを0.33 ml添加し、ヘキサン50 ml、ジエチルエーテル50 ml、ジクロロメタン50 ml、及びメタノール50 mlで順次溶出し、以下の画分を得た。 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.
各画分を30 mlの蒸留水に転溶し、一部を活性測定に供した。これらの活性測定用溶液は乾燥藻体0.5 g/ml相当の濃度に統一した。 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.
［試験例7］ シワヤハズ抽出液のメタノール画分の巻貝に対する致死活性の評価
（材料）
・シワヤハズ抽出液のメタノール画分（A-4）
蒸留水に転溶し、乾燥藻体0.5 g/ml相当の濃度に調整した。
・チグサガイ
瀬戸内海区水産研究所の蓄養水槽より42個体を採取した。[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.
（方法）
（1）メタノール画分を、乾燥藻体10 g/L、7.5 g/L、5 g/L、2.5 g/L、1 g/L相当の濃度となるように、蒸留水（MilliQ）及びろ過海水で希釈した。海水の濃度は90%に統一した。
（2）試験管7本に（1）の溶液を7 mlずつ分注し、チグサガイを1個体ずつ入れて綿栓をした。
（3）コントロール（0 g/L）として、蒸留水5 mlとろ過海水45 mlを混合したものを試験管7本に7 mlずつ分注し、チグサガイを1個体ずつ入れて綿栓をした。
（4）20℃の暗所に24時間静置し、チグサガイの死亡個体数と生存個体数を計数した。試験管の壁面又は綿栓に足で付着している個体を生存個体、それ以外の個体（粘液のみでぶら下がっている個体、付着せずに底に転がっている個体）を死亡個体とした。(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.
（結果）
24時間後の死亡個体数及び生存個体数を以下の表に示す。(result)
The number of dead and surviving individuals after 24 hours is shown in the table below.
シワヤハズ抽出液のメタノール画分は、10 g/Lの濃度で高い致死活性を示し、7.5 g/Lの濃度でも活性を示したが、5 g/L以下の濃度では、致死活性が認められなかった。 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.
なお、データは示していないが、シワヤハズ抽出液の他の画分についてもチグサガイに対する致死活性を評価したところ、ジエチルエーテル画分（A-2）およびジクロロメタン画分（A-3）についてはやや低い致死活性が認められたが、ヘキサン画分（A-1）については、致死活性は認められなかった。 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).
［試験例8］ シワヤハズ抽出液の巻貝に対する致死活性と忌避効果との相関
曝露溶液の巻貝に対する致死活性と巻貝が曝露溶液から逃げる行動（忌避行動）との関係を調べるため、以下の試験を行った。[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.
チグサガイに対して高い致死活性が認められたシワヤハズのメタノール画分、やや低い活性が認められたジクロロメタン画分、ジエチルエーテル画分、活性が認められなかったヘキサン画分の溶液（それぞれ乾燥藻体10 g/L相当の濃度、海水90%になるように希釈したもの）を7 mlずつ7本の試験管に分注して、チグサガイを1個体ずつ入れ、1、3、5、10、60分後に溶液の外に移動している個体（忌避個体）を計数した。 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.
結果を図7に示す。致死活性が認められた溶液で顕著にチグサガイの忌避行動が認められた。ジエチルエーテル画分は、致死活性においてはメタノール画分より低かったものの、メタノール画分と同等以上の忌避効果を有する可能性が示された。 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.
［試験例9］ 野外散布による忌避効果の評価
マクリからの抽出物（乾燥藻体重量換算濃度で、100 g/L）とヤハズグサからの抽出物（乾燥藻体重量換算濃度で、100 g/L）とを1:1の体積比で混合した忌避物質液を調製し、野外のアマモ場において、忌避物質液を48時間連続的にマクリ及びヤハズグサのそれぞれを最大濃度:0.78 μg/L（乾燥藻体重量換算濃度）で添加した実験区(lm×lm)および海水を添加したコントロール区(lm×lm)でベントス個体数（海産無脊椎動物の個体数）を比較した。[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.
結果を図8-10に示す。コントロール区と実験区ごとに、各6つの複製区の平均値と標準偏差のエラーバーを示している。個体数について、試験開始時と48時間後で有意差があるか、尤度比検定（説明変数に時間の効果を入れたモデルと切片のみモデルを比較）により解析を行った。 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.
実験区で有意に海産無脊椎動物個体数が減少するデータが得られた。実験区において、節足動物門と軟体動物門の両方の個体数が有意に減少した。 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.
本発明により、海藻から抽出された天然化合物を有効成分として含有する海産無脊椎動物忌避剤が提供される。 The present invention provides a marine invertebrate repellent containing, as an active ingredient, a natural compound extracted from seaweed.
本願は、日本国で出願された特願２０１７－１９９５６２を基礎としており、その内容は本明細書にすべて包含されるものである。 This application is based on Japanese Patent Application No. 2017-199562 filed in Japan, the contents of which are all incorporated herein.
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| Application Number | Priority Date | Filing Date | Title |
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| JP2017199562 | 2017-10-13 | ||
| JP2017199562 | 2017-10-13 | ||
| PCT/JP2018/038204 WO2019074117A1 (en) | 2017-10-13 | 2018-10-12 | Marine invertebrate repellent |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009031637A1 (en) | 2007-09-07 | 2009-03-12 | National University Corporation Hokkaido University | Extract of red alga laurencia sp. with organic solvent, and agent for prevention of the settlement of barnacle comprising compound isolated from the extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS53113012A (en) * | 1977-03-10 | 1978-10-03 | Nippon Paint Co Ltd | Preparation of shellfish repellent |
| JPH04261109A (en) * | 1991-02-14 | 1992-09-17 | Chubu Electric Power Co Inc | Extraction of useful component of marine algae |
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| WO2009031637A1 (en) | 2007-09-07 | 2009-03-12 | National University Corporation Hokkaido University | Extract of red alga laurencia sp. with organic solvent, and agent for prevention of the settlement of barnacle comprising compound isolated from the extract |
Non-Patent Citations (3)
| Title |
|---|
| OHTA, Keiichi et al.，A Screening Procedure for Repellents against a Sea Snail，Agricultural and Biological Chemistry，1978年，Vol.42, No.8，pp.1491-1493 |
| POORE, A. G. et al.，Preference-performance relationships and effects of host plant choice in an herbivorous marine amphi，Ecological Monographs，1999年，Vol.69, No.4，pp.443-464 |
| 林 育夫 ほか，海藻より抽出された摂食阻害物質が小型植食性巻貝エゾサンショウガイ Homalopoma amussitatum の摂餌活動に，Venus the Japanese Journal of Malacology，1996年，Vol.55, No.4，pp.307-316 |
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