JP6635524B2 - Plant disease control agent - Google Patents
Plant disease control agent Download PDFInfo
- Publication number
- JP6635524B2 JP6635524B2 JP2018120010A JP2018120010A JP6635524B2 JP 6635524 B2 JP6635524 B2 JP 6635524B2 JP 2018120010 A JP2018120010 A JP 2018120010A JP 2018120010 A JP2018120010 A JP 2018120010A JP 6635524 B2 JP6635524 B2 JP 6635524B2
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- plant
- virus
- infection
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- extract
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- Cultivation Of Plants (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Description
本発明は、プロアントシアニジンを有効成分とする植物病害の防除剤、および当該防除剤を植物に施用することを含む植物病害の防除方法に関する。 The present invention relates to a plant disease controlling agent comprising proanthocyanidin as an active ingredient, and a method for controlling a plant disease comprising applying the controlling agent to a plant.
糸状菌病、細菌病、植物ウイルス病等により引き起こされる植物病害による世界の農業生産量の損失は15%にもおよび、そのうち植物ウイルス病による作物生産量の損失額は年間6兆円を超えると予想されている。植物ウイルス病は農作物に様々な異常を引き起こし、収量および品質に大きな被害を与えている。 If plant disease caused by fungal disease, bacterial disease, plant virus disease, etc., causes loss of world agricultural production by as much as 15%, of which crop production loss due to plant virus disease exceeds 6 trillion yen annually Is expected. Plant virus diseases cause a variety of abnormalities in crops, severely affecting yield and quality.
植物ウイルス病の防除剤としては、日本においてレンテミンが農薬登録されているものの、特効薬となる化学農薬は存在しない(レンテミンは、野田食菌工業(株)の登録商標。農薬登録第15584号、第17774号、第19439号、第19440号)。そこで、媒介生物の防除や弱毒ウイルスの接種など時間と手間をかけた耕種的防除を行って、植物ウイルス病の被害を軽減させているのが実情である。 As a plant virus control agent, lentemin is registered as an agricultural chemical in Japan, but there is no chemical pesticide that is a specific medicine (lentemin is a registered trademark of Noda Shokubai Kogyo Co., Ltd. 17774, 19439 and 19440). Therefore, it is a fact that the time-consuming and time-consuming cultivated pest control, such as vector control and attenuated virus inoculation, is carried out to reduce the damage of plant virus diseases.
このため、植物ウイルス病に対して優れた効果を示す防除剤の開発が切望されている。 For this reason, development of a controlling agent exhibiting an excellent effect on plant virus diseases is eagerly desired.
なお、植物ウイルス病の防除剤に関しては、例えば、アミノ酸発酵副生物、デヒドロアスコルビン酸、アスコルビン酸(ビタミンC)、リグニン抽出物などが有効であるとの報告がある(特許文献1〜4)。 As a control agent for plant virus diseases, for example, there are reports that amino acid fermentation by-products, dehydroascorbic acid, ascorbic acid (vitamin C), lignin extract, and the like are effective (Patent Documents 1 to 4).
本発明は、このような状況に鑑みてなされたものであり、その目的は、植物病害に対して優れた防除効果を有する分子を同定し、当該分子を利用した植物病害の防除剤および防除方法を提供することにある。 The present invention has been made in view of such circumstances, and an object of the present invention is to identify a molecule having an excellent control effect on a plant disease, and to control a plant disease control agent and a control method using the molecule. Is to provide.
本発明者は、上記課題を解決すべく鋭意検討を行った結果、植物からの特定の抽出画分が、様々な植物ウイルスや植物病原菌に対して優れた防除効果を有することを見出した。本発明者は、さらに、当該抽出画分に含まれる活性成分の同定を試みた結果、遂に、プロアントシアニジンが活性成分であることを突き止め、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that a specific extract fraction from a plant has an excellent control effect on various plant viruses and plant pathogens. The present inventor further tried to identify the active ingredient contained in the extracted fraction, and finally found out that proanthocyanidin was the active ingredient, thereby completing the present invention.
すなわち、本発明は、植物精製物を有効成分とする植物病害の防除剤、および当該防除剤を植物に施用することを含む植物病害の防除方法に関し、より詳しくは、以下の発明を提供するものである。 That is, the present invention relates to a plant disease controlling agent comprising a purified plant product as an active ingredient, and a method for controlling a plant disease including applying the controlling agent to a plant, and more specifically, the following inventions are provided. It is.
(1)発酵過程を経ない精製により得られる植物精製物を含む、植物病害の防除剤。 (1) An agent for controlling plant diseases, including a purified plant product obtained by purification without going through a fermentation process.
(2)精製が、エタノールによる抽出またはアセトニトリルによる溶出を含む、(1)に記載の防除剤。 (2) The control agent according to (1), wherein the purification comprises extraction with ethanol or elution with acetonitrile.
(3)精製が、少なくとも3500の分子量での分画を含む、(1)または(2)に記載の防除剤。 (3) The control agent according to (1) or (2), wherein the purification comprises fractionation at a molecular weight of at least 3500.
(4)プロアントシアニジンを有効成分とする、植物病害の防除剤。 (4) A plant disease controlling agent comprising proanthocyanidin as an active ingredient.
(5)プロアントシアニジンを含む植物精製物を含む、(4)に記載の防除剤。 (5) The control agent according to (4), comprising a purified plant product containing proanthocyanidin.
(6)プロアントシアニジンを乾燥重量で1%以上含む、(4)または(5)に記載の防除剤。 (6) The control agent according to (4) or (5), which contains 1% or more by weight of proanthocyanidin on a dry basis.
(7)植物病害が、植物ウイルス病、植物糸状菌病、または植物細菌病である、(1)から(6)のいずれかに記載の防除剤。 (7) The control agent according to any one of (1) to (6), wherein the plant disease is a plant virus disease, a plant fungal disease, or a plant bacterial disease.
(8)植物病害が、モザイク病である、(7)に記載の防除剤。 (8) The control agent according to (7), wherein the plant disease is mosaic disease.
(9)植物病害が、炭疽病または黒斑細菌病である、(7)に記載の防除剤。 (9) The control agent according to (7), wherein the plant disease is anthrax or black spot bacterial disease.
(10)(1)〜(9)のいずれかに記載の防除剤を植物に施用することを含む、植物病害の防除方法。 (10) A method for controlling plant diseases, which comprises applying the control agent according to any one of (1) to (9) to plants.
本発明の防除剤を用いれば、植物に含まれる成分によって、葉の萎縮や斑点の発生などの薬害を生じさせることなく、安全に植物病害を防除することが可能である。本発明の防除剤による防除効果は、それを直接散布した葉のみならず、その上位葉にも認められたことから、植物の免疫力(病原に対する抵抗性)の向上を引き起こすことが考えられる。従って、本発明の防除剤によれば、非常に効果的に防除効果をもたらすことが可能である。 The use of the control agent of the present invention makes it possible to safely control plant diseases without causing phytotoxicity such as leaf atrophy and spots due to components contained in plants. Since the controlling effect of the controlling agent of the present invention was observed not only on the leaves directly sprayed but also on the upper leaves thereof, it is considered that the controlling effect of the controlling agent causes an improvement in plant immunity (resistance to pathogens). Therefore, according to the controlling agent of the present invention, a controlling effect can be provided very effectively.
本発明は、発酵過程を経ない精製により得られる植物精製物を含む、植物病害の防除剤に関する。 The present invention relates to an agent for controlling plant diseases, including a purified plant product obtained by purification without going through a fermentation process.
国際公開第2017/125993号公報に記載の方法では、活性成分であるリグニン分解物を得るために、精製工程において発酵を行っているが、本発明では精製工程において発酵は必要ない。また、同公報の精製工程においては、リグニン分解物を得るために鉄イオンとの接触(鉄製破砕機の利用)を要するが、本発明においてプロアントシアニジンの精製物を得るためには、鉄イオンとの接触は必要なく、例えば、ステンレス製破砕機を利用してもよい。 In the method described in WO 2017/125993, fermentation is performed in the purification step in order to obtain a lignin hydrolyzate as an active ingredient, but the present invention does not require fermentation in the purification step. Further, in the purification step of the publication, contact with iron ions (use of an iron crusher) is required to obtain a lignin degradation product, but in order to obtain a purified proanthocyanidin in the present invention, iron ion No contact is required, for example, a crusher made of stainless steel may be used.
本発明における精製工程は、エタノールによる抽出またはアセトニトリルによる溶出を含むことができる。エタノールによる抽出を行う場合、エタノール濃度は、通常、40%〜80%であり、好ましくは、60%〜80%である。一方、アセトニトリルによる溶出を行う場合、アセトニトリル濃度は、通常、5%以上、好ましくは5%〜30%であり、より好ましくは5%〜20%である。40%以上の濃度のアセトニトリルを用いてもよいが、抽出物において非活性成分の割合が高まることが考えられる。 The purification step in the present invention can include extraction with ethanol or elution with acetonitrile. When performing extraction with ethanol, the ethanol concentration is usually 40% to 80%, preferably 60% to 80%. On the other hand, when performing elution with acetonitrile, the acetonitrile concentration is usually 5% or more, preferably 5% to 30%, and more preferably 5% to 20%. Acetonitrile at a concentration of 40% or more may be used, but it is conceivable that the proportion of inactive ingredients in the extract will increase.
また、例えば、3500の分子量での分画(例えば、4000以上、5000以上、6000以上、7000以上、8000以上、9000以上、10000以上の分子量での分画)を含むことができる。これにより植物病害の防除活性に寄与しない低分子量の成分を除去することができる。 Further, for example, fractionation at a molecular weight of 3500 (eg, fractionation at a molecular weight of 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10,000 or more) can be included. This makes it possible to remove low molecular weight components that do not contribute to the activity of controlling plant diseases.
その他、本発明においては、必要に応じて、他の精製工程を組み合わせてもよい。 In addition, in the present invention, other purification steps may be combined as necessary.
本発明においては、プロアントシアニジンが活性成分(若しくはその一つ)であることが見出された。従って、本発明は、プロアントシアニジンを有効成分とする、植物病害の防除剤を提供する。 In the present invention, it has been found that proanthocyanidins are the active ingredient (or one of them). Therefore, the present invention provides a plant disease controlling agent comprising proanthocyanidin as an active ingredient.
本発明において「プロアントシアニジン」とは、カテキンやエピカテキンといったフラバン-3-オール類が複数結合した化合物である。本発明の防除剤に用いるプロアントシアニジンは、植物病害の防除効果を有するものであれば特に制限はなく、天然由来の精製物であっても、人工的に合成された化合物であってもよい。精製物は、完全な精製物であっても、粗精製物であってもよい。 In the present invention, “proanthocyanidins” are compounds in which a plurality of flavan-3-ols such as catechin and epicatechin are bonded. The proanthocyanidin used in the control agent of the present invention is not particularly limited as long as it has a plant disease control effect, and may be a naturally-derived purified product or an artificially synthesized compound. The purified product may be a completely purified product or a crude product.
本発明におけるプロアントシアニジンの好ましい形態は、植物精製物である。プロアントシアニジンの精製物(抽出液)は、通常、プロアントシアニジンを0.1mg/ml以上、好ましくは0.3mg/ml以上、より好ましくは0.5mg/ml以上(例えば、1mg/ml以上、5mg/ml以上、10mg/ml以上、20mg/ml以上)含む。薬害などの負の影響を生じさせないためには、通常、50mg/ml以下が好ましいと考えられる。また、プロアントシアニジンの精製物は、通常、プロアントシアニジンを、乾燥重量で1%以上、好ましくは5%以上、より好ましくは10%(例えば、20%以上、30%以上、50%以上)含む。従って、本発明は、プロアントシアニジンを上記濃度または割合で含む、植物病害の防除剤を提供する。プロアントシアニジンは、1種単独でまたは2種以上組み合わせて使用することができる。 The preferred form of proanthocyanidins in the present invention is a purified plant. A purified product of proanthocyanidin (extract) is usually 0.1 mg / ml or more, preferably 0.3 mg / ml or more, more preferably 0.5 mg / ml or more (for example, 1 mg / ml or more, 5 mg / ml or more). , 10 mg / ml or more, 20 mg / ml or more). In order not to cause adverse effects such as phytotoxicity, it is usually considered that the dose is preferably 50 mg / ml or less. Further, the purified proanthocyanidin usually contains proanthocyanidin in a dry weight of 1% or more, preferably 5% or more, more preferably 10% (eg, 20% or more, 30% or more, 50% or more). Accordingly, the present invention provides a plant disease controlling agent comprising proanthocyanidin at the above-mentioned concentration or ratio. Proanthocyanidins can be used alone or in combination of two or more.
本発明の防除剤においては、植物病害の防除効果が阻害されない限り、他の有効成分を含有していてもよい。例えば、植物病害に対する防除効果をさらに高めるため、あるいは、適用対象とする植物病害の範囲を広げるため、レンテミンなどの既知の薬剤と組み合わせて用いることができる。また、植物ウイルスは、虫媒感染する例もあることから、既知の殺虫剤、殺ダニ剤、抗菌剤などと組み合わせて用いることもできる。 The control agent of the present invention may contain other active ingredients as long as the control effect on plant diseases is not inhibited. For example, it can be used in combination with a known drug such as lentemin in order to further enhance the control effect against plant diseases or to widen the range of plant diseases to be applied. In addition, plant viruses can be used in combination with known insecticides, acaricides, antibacterial agents, and the like, because there are cases in which plant viruses are transmitted by insect vectors.
本発明の防除剤においては、植物病害の防除効果が阻害されない限り、他の任意成分を含んでもよい。他の任意成分としては、例えば、展着剤、充填剤、増量剤、結合剤、付湿剤、崩壊剤、滑沢剤、稀釈剤、賦形剤等を挙げることができる。 The control agent of the present invention may contain other optional components as long as the effect of controlling plant diseases is not inhibited. Other optional components include, for example, spreading agents, fillers, extenders, binders, humectants, disintegrants, lubricants, diluents, excipients, and the like.
本発明の防除剤の植物への施用方法は特に制限されず、例えば、散布、塗布、浸漬などが挙げられる。また、植物組織培養を行っている場合には、その培地への添加なども考えられる。本発明の防除剤が施用されている領域では非常に高い感染防除効果が認められることから、施用方法としては、散布や塗布などによる植物体の全体や葉への施用、種子や球根などへの浸漬が好ましい。本発明の防除剤は、施用された部位の周辺部位においても、植物病害に対する防除効果を示すことができる点で有利である。 The method for applying the control agent of the present invention to plants is not particularly limited, and examples thereof include spraying, coating, and dipping. When plant tissue culture is performed, addition to the culture medium may be considered. Since a very high infection control effect is observed in the area where the control agent of the present invention is applied, application methods include application to whole plants and leaves by spraying or application, and application to seeds and bulbs. Immersion is preferred. The control agent of the present invention is advantageous in that it can exhibit a control effect on plant diseases even in a site surrounding the applied site.
本発明の防除剤の剤型は、施用方法などに応じて各種の形態を採ることができる。例えば、乳剤、油剤、エアゾール、フロアブル剤などの液剤の他、水和剤、水溶剤、粉剤、粒剤、錠剤などが挙げられる。散布などにより植物体へ施用する場合には、液剤、または施用時に液状にすることができる剤型が好ましい。 The dosage form of the control agent of the present invention can take various forms depending on the application method and the like. Examples include liquid preparations such as emulsions, oils, aerosols and flowables, as well as wettable powders, aqueous solvents, powders, granules and tablets. When applied to a plant by spraying or the like, a liquid formulation or a dosage form that can be made liquid at the time of application is preferable.
施用量は、植物病害に対して防除効果を有する量であれば特に制限はなく、当業者であれば、適宜調整することができる。 The application rate is not particularly limited as long as it has an effect of controlling plant diseases, and can be appropriately adjusted by those skilled in the art.
本発明の防除剤の植物への施用時期は特に制限されないが、予防的な防除が最も有効である。具体的には、育苗期から収穫前にかけての施用が有効である。また、防除剤の施用回数に、特に制限はない。 The application time of the control agent of the present invention to plants is not particularly limited, but preventive control is most effective. Specifically, application from the seedling raising stage to before harvest is effective. There is no particular limitation on the number of times the pesticide is applied.
本発明の防除剤を適用する植物ウイルス病としては、例えば、トバモウイルス属ウイルス、ポテックスウイルス属ウイルス、カルラウイルス属ウイルス、ククモウイルス属ウイルス、カルモウイルス属ウイルス、またはベゴモウイルス属ウイルスの感染により発症する病気が挙げられる。 Plant virus diseases to which the control agent of the present invention is applied include, for example, those caused by infection with a tobamovirus, a potexvirus, a carlavirus, a cucumovirus, a carmovirus, or a begomovirus. Illness.
トバモウイルス属ウイルスとしては、例えば、タバコモザイクウイルス(TMV)、トマトモザイクウイルス(ToMV)、キュウリ緑斑モザイクウイルス(KGMMV)、トウガラシマイルドモットルウイルス(PMMoV)、スイカ緑斑モザイクウイルス(CGMMV)などが挙げられ、ポテックスウイルス属ウイルスとしては、例えば、オオバコモザイクウイルス(P1AMV)、ジャガイモXウイルス(PVX)などが挙げられ、カルラウイルス属ウイルスとしては、例えば、ジャガイモモザイクウイルス(PVM)が挙げられ、ククモウイルス属ウイルスとしては、例えば、キュウリモザイクウイルス(CMV)などが挙げられ、カルモウイルス属ウイルスとしては、例えば、メロンえそ斑点ウイルス(MNSV)などが挙げられ、ベゴモウイルス属ウイルスとしては、例えば、トマト黄化葉巻ウイルス(TYLCV)などが挙げられるが、これらに制限されない。 Examples of tobamoviruses include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), cucumber green mottle mosaic virus (KGMMV), capsicum mild mottle virus (PMMoV), watermelon green mottle mosaic virus (CGMMV), and the like. Examples of the virus of the genus Potex virus include psyllium mosaic virus (P1AMV) and potato X virus (PVX). Examples of the virus of the genus Carlavirus include, for example, potato mosaic virus (PVM), Examples of the cucumber virus include, for example, cucumber mosaic virus (CMV), and examples of the carmovirus include, for example, melon necrotic spot virus (MNSV), and examples of the begomovirus include, for example, Yellow tomato Such as wound Virus (TYLCV) include, but are not limited thereto.
本発明の防除剤の対象とする植物ウイルス病は、好ましくは、各種モザイクウイルスやジャガイモXウイルスなどの感染により発症するモザイク病である。 The plant virus disease targeted by the control agent of the present invention is preferably a mosaic disease caused by infection with various mosaic viruses or potato X virus.
本発明の防除剤を適用する糸状菌としては、例えば、うどんこ病菌(Erysiphe属菌、Sphaerotheca属菌、Leveillula属菌)、ボトリチス(Botrytis)属菌、フルビア (Fulvia fulva)属菌、コリネスポラ(Corynespora)属菌、アルブゴ(Albugo)属菌、べと病菌(プシウドペロノスポラ(Pseudoperonospora)属菌、Peronospora属菌、Plasmopara属菌、Bremia属菌)、ピリキュラリア(Pyricularia)属菌、いもち病菌(Pyricularia grisea)、ごま葉枯病菌(Cochliobolus miyabeanus)、サーコスポラ類縁菌(Cercospora属菌、Cercosporella属菌、Pseudocercospora属菌、Paracercospora属菌 Mycovellosiella属菌)、炭疽病菌(コレトトリカム(Colletotrichum)属菌、Glomerella属菌)、さび病菌(Puccinia属菌)、アルタナリア(Alternaria)属菌、スクレロチニア(Sclerotinia)属菌、土壌伝染性糸状菌の根こぶ病菌(Plasmodiophora brassicae)、フィトフィトラ(Phytophthora)属菌、フザリウム(Fusarium)属菌、バーティシリウム(Verticillium)属菌、ピシウム(Pythium)属菌、リゾクトニア(Rhizoctonia)属菌が挙げられる。好ましくは、炭疽病菌である。 Examples of the filamentous fungi to which the control agent of the present invention is applied include powdery mildew (Erysiphe spp., Sphaerotheca spp., Leveillula spp.), Botrytis spp., Fulvia fulva spp., Corynespora. ), Genus Albugo, downy mildew (Pseudoperonospora, Peronospora, Plasmopara, Bremia), pyricularia (Pyricularia), blast (Pyricularia) grisea), sesame leaf blight (Cochliobolus miyabeanus), circospora related fungi (Cercospora, Cercosporella, Pseudocercospora, Paracercospora Mycovellosiella), anthrax (Colletotrichum), Glomer , Rust fungus (Puccinia genus), Alternaria (Alternaria) genus, Sclerotinia (Sclerotinia) genus, soil-borne fungal root-knot fungus (Plasmodiophora brassicae), Phytophthora (Phytophthora) genus, Fusarium (Fusarium) genus, Verticillium (Pythium) genus, Rhizoctonia (Rhizoctonia) genus. Preferably, it is an anthrax fungus.
本発明の防除剤を適用する細菌としては、例えば、アグロバクテリウム(Agrobacterium)属、クラビバクター(Clavibacter)属、エルウィニア(Erwinia)属、シュードモナス(Pseudomonas)属、ラルストニア(Ralstonia)属、コリネバクテリウム(Corynebacterium)属、クルトバクテリウム(Curtobacterium)属、バークホルデリア(Burkholderia)属、キサントモナス(Xanthomonas)属、リゾバクター(Rhizobacter)属、クロストリジウム(Clostridium)属に属する細菌が挙げられる。好ましくは、黒斑細菌病を引き起こす、シュードモナス属のシュードモナス・シリンゲ・パソバー・マクリコラ(Pseudomonas syringae pv. maculicola)である。 Examples of bacteria to which the control agent of the present invention is applied include, for example, Agrobacterium (Agrobacterium), Clavibacter (Clavibacter), Erwinia (Erwinia), Pseudomonas (Pseudomonas), Ralstonia (Ralstonia), Corynebacterium Examples include bacteria belonging to the genus (Corynebacterium), the genus Curtobacterium, the genus Burkholderia, the genus Xanthomonas, the genus Rhizobacter, and the genus Clostridium. Preferred is Pseudomonas syringae pv. Maculicola, which causes black spot bacterial disease.
本発明の防除剤を施用する植物は、上記病原が感染するものであれば特に制限はないが、例えば、タバコ、トマト、トウガラシ、キュウリ、メロン、スイカ、リンゴ、ネギ、タマネギ、ニラ、ピーマン、ジャガイモ、イネ、オオムギ、カボチャ、ハクサイ、キャベツ、チンゲンサイ、レタス、イチゴ、花卉類(キク、バラ、カーネーション、チューリップ、カトレア、シンビジウム、トルコギキョウ、スターチス、ガーベラなど)等が挙げられる。このように本発明の防除剤は、ナス科植物、ウリ科植物、アブラナ科植物、バラ科、イネ科植物など広範囲に適用することができる。植物ウイルスと宿主植物との関係については、日本植物病名データベース(農業生物資源ジーンバンク)を参照のこと。 Plants to which the control agent of the present invention is applied are not particularly limited as long as the above-mentioned pathogens are infected.For example, tobacco, tomato, capsicum, cucumber, melon, watermelon, apple, leek, onion, leek, pepper, Potato, rice, barley, pumpkin, Chinese cabbage, cabbage, bok choy, lettuce, strawberry, flowers (chrysanthemum, rose, carnation, tulip, cattleya, cymbidium, eustoma, starches, gerbera, etc.). As described above, the control agent of the present invention can be applied to a wide range of plants such as Solanaceous plants, Cucurbitaceae plants, Brassicaceae plants, Rosaceae, and Poaceae plants. For the relationship between plant viruses and host plants, refer to the Japanese Plant Disease Database (Agricultural Biological Resources Genebank).
以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.
[実施例1] 発酵過程を経ない月桃抽出物の精製
草本系の事例として、ショウガ科ハナミョウガ属の月桃(ゲットウ(サンニン))の茎葉部分を搾汁機に投入し、水を添加することなく、植物から抽出される液を回収し、遠心機(6000rpm、5分間)により夾雑物を除去し、上清を濾紙及び孔径0.22μmのフィルター(TPP社製)によって濾過した。
[Example 1] Purification of moon peach extract without fermentation process As a herbaceous case, the foliage portion of moon peach (getto (Sannin)) of the genus Ganaceae is added to a juicer and water is added. Without extracting the liquid extracted from the plant, the contaminants were removed by a centrifuge (6000 rpm, 5 minutes), and the supernatant was filtered with a filter paper and a filter having a pore size of 0.22 μm (manufactured by TPP).
なお、国際公開第2017/125993号公報に記載の方法では、精製過程において、搾汁液の発酵を行っているが、本実施例による方法は、発酵は行っていないため、得られる抽出物に含まれる成分が異なると考えられる。 In the method described in WO 2017/125993, in the purification process, fermentation of the squeezed liquid is performed, but the method according to the present example does not perform fermentation, and thus is included in the obtained extract. Components are considered to be different.
[実施例2] 月桃亜種抽出物のToMVに対する感染抑制効果
実施例1で調製した植物抽出液を、ToMV-ベンサミアーナタバコ評価系を用いて評価した。すなわち、終濃度0.01%のマイリノーを添加した植物抽出液(希釈[10%、20%、50%、無希釈])を、ベンサミアーナタバコの葉に噴霧処理を行い、その3日後に、ToMVウイルスを接種した。ウイルスの接種は、ToMV-GFP(緑色蛍光蛋白質GFPを付加したウイルス)のプラスミド(pTLBN.G3)2μgについて、AmpliCap-MaxTM T7 High Yield Message Maker Kitsを用いてRNAを転写合成し、RNA転写物を40倍に希釈して、カーボランダムを用いてベンサミアーナタバコ葉に10μlを2ヶ所に塗布することにより行った。ウイルスの接種から3日後に、GFPの蛍光斑点(ToMVの感染、増殖部位に一致する)をカウントし、ウイルスの感染抑制率を検定して植物抽出液の評価を行った。その結果、いずれの希釈率でも強いウイルス感染抑制効果が認められた(図1)。
[Example 2] Infection-suppressing effect of Toro peach subspecies extract on ToMV The plant extract prepared in Example 1 was evaluated using a ToMV-Bensamiana tobacco evaluation system. That is, a plant extract solution (diluted [10%, 20%, 50%, undiluted]) containing 0.01% final concentration of mylino was spray-treated on Bensamiana tobacco leaves. The virus was inoculated. The virus was inoculated by transcribing and synthesizing RNA using 2 μg of ToMV-GFP (virus to which green fluorescent protein GFP was added) plasmid (pTLBN.G3) using AmpliCap-Max ™ T7 High Yield Message Maker Kits. Was diluted 40-fold, and 10 μl was applied to two places of Bensamiana tobacco leaves using carborundum. Three days after the inoculation of the virus, fluorescent spots of GFP (corresponding to the infection site of ToMV and the growth site) were counted, and the rate of inhibition of virus infection was evaluated to evaluate the plant extract. As a result, a strong virus infection inhibitory effect was observed at all dilution ratios (FIG. 1).
なお、感染抑制率は、以下の式から算出した。
感染抑制率(防除価)%=100-{(処理植物の蛍光斑点数の平均)/(未処理植物の蛍光斑点数の平均)}×100
[実施例3] 原材料の違う搾汁機の刃を用いて得た月桃抽出物のToMVおよびTMVに対する感染抑制効果
草本系植物の事例として、ショウガ科ハナミョウガ属の月桃(ゲットウ(サンニン))の茎葉部分を鉄製またはステンレス製の刃を備えた搾汁機に投入し、水を添加することなく、植物から抽出される液を回収して、遠心機(6000rpm、5分間)により夾雑物を除去し、上清を濾紙及び孔径0.22μmのフィルター(TPP社製)によって濾過した。
The infection control rate was calculated from the following equation.
Infection control rate (control value)% = 100-{(average number of fluorescent spots of treated plants) / (average number of fluorescent spots of untreated plants)} x 100
[Example 3] Infection suppression effect on ToMV and TMV of moon peach extract obtained using blades of juice extractors with different raw materials As an example of a herbaceous plant, moon peach (Ganto (Sannin)) of the genus Gingeraceae The stem and leaves are put into a juicer equipped with an iron or stainless steel blade, and the liquid extracted from the plant is collected without adding water, and the contaminants are removed by a centrifuge (6000 rpm, 5 minutes). After removal, the supernatant was filtered through filter paper and a filter having a pore size of 0.22 μm (manufactured by TPP).
ToMVに関しては、0.01%マイリノーを添加した10%溶液を噴霧処理し、3日後に2μgプラスミドを転写後、40倍希釈したToMV-GFP(緑色蛍光蛋白質GFPを付加したウイルス)を各植物体に10μlずつ第3葉にカーボランダムを用いて接種し、3日後に接種葉のGFP蛍光のスポットを測定した。コントロールに対する感染抑制率を算出し、その平均値を示した。1処理あたり、ベンサミアーナタバコ3個体を用いた。 As for ToMV, a 10% solution containing 0.01% mylino was sprayed, and after 3 days, 2 μg of the plasmid was transcribed. Then, 40 μl of diluted ToMV-GFP (a virus to which green fluorescent protein GFP was added) was added to each plant in an amount of 10 μl. Each third leaf was inoculated with carborundum, and three days later, GFP fluorescence spots on the inoculated leaves were measured. The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.
TMVに関しては、0.01%マイリノーを添加した10%溶液を噴霧処理し、3日後に2μgプラスミドを転写後、6倍希釈したTMV-GFP(緑色蛍光蛋白質GFPを付加したウイルス)を各植物体に10μlずつ第3葉にカーボランダムを用いて接種し、3日後に接種葉のGFP蛍光のスポットを測定した。コントロールに対する感染抑制率を算出し、その平均値を示した。1処理あたり、ベンサミアーナタバコ3個体を用いた。 Regarding TMV, a 10% solution containing 0.01% mylinol was sprayed, and 3 days later, 2 μg of the plasmid was transcribed, and 6-fold diluted TMV-GFP (virus to which green fluorescent protein GFP was added) was added to each plant at 10 μl. Each third leaf was inoculated with carborundum, and three days later, GFP fluorescence spots on the inoculated leaves were measured. The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.
その結果、鉄製またはステンレス製の刃を備えた搾汁機のいずれで搾汁しても、ToMVおよびTMVに対して、強いウイルス感染抑制効果が認められた(図2)。 As a result, a strong virus infection-suppressing effect on ToMV and TMV was observed, regardless of whether the juice was extracted using an iron or stainless steel blade equipped with a blade (FIG. 2).
国際公開第2017/125993号公報に記載の方法では、抗ウイルス効果に鉄および鉄イオンは関係しているとされていたが、本実施例1の精製方法で得た抽出物においては、抗ウイルス効果に鉄および鉄イオンは関係なかった。 In the method described in WO 2017/125993, iron and iron ions were considered to be related to the antiviral effect, but in the extract obtained by the purification method of Example 1, the antiviral effect Iron and iron ions were not related to the effect.
[実施例4] 月桃のエタノール抽出物のToMVに対する防除効果
ショウガ科ハナミョウガ属の月桃(ゲットウ(サンニン))の茎葉部分をステンレス製の刃を備えたハサミで裁断し、当該裁断物20gに対して40mlの0〜100%エタノール溶液につけ込んだ。25℃で一晩往復振盪(180rpm)し、植物から抽出される液を回収した。次いで、遠心機(6000rpm、5分間)により夾雑物を除去し、上清を濾紙で濾過した。
[Example 4] Pest control effect on ToMV of ethanol extract of moon peach against ToMV The stem and leaves of moon peach (Gettou (Sannin)) of the genus Gingeraceae are cut with scissors equipped with stainless steel blades, and 20 g of the cut material is cut. On the other hand, it was immersed in 40 ml of a 0-100% ethanol solution. The mixture was reciprocally shaken (180 rpm) at 25 ° C. overnight to collect a liquid extracted from the plant. Next, impurities were removed by a centrifuge (6000 rpm, 5 minutes), and the supernatant was filtered with filter paper.
0.01%マイリノーを添加した各植物抽出液の20%溶液をベンサミアーナタバコに噴霧処理し3日間静置した。実施例2の方法で2μgプラスミドを転写後、40倍希釈したToMV-GFPを10μlずつ2ヶ所に第3葉にカーボランダムを用いて接種し、3日後に接種葉のGFP蛍光のスポットを測定した。コントロールに対する感染抑制率を算出し、その平均値を示した。1処理あたり、ベンサミアーナタバコ3個体を用いた。 A 20% solution of each plant extract to which 0.01% mylinol was added was spray-treated on Bensamiana tobacco and allowed to stand for 3 days. After transferring 2 μg of the plasmid by the method of Example 2, 10 μl of ToMV-GFP diluted 40-fold was inoculated into the third leaf at two locations using carborundum, and the spot of GFP fluorescence of the inoculated leaf was measured three days later. . The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.
その結果、60-80%エタノール溶液での抽出物に強いウイルス感染抑制効果が認められた(表1)。なお、防除価における括弧内の数値はマイナスを意味する。 As a result, a strong virus infection-suppressing effect was observed in the extract with the 60-80% ethanol solution (Table 1). In addition, the numerical value in parenthesis in a control value means minus.
[実施例5] 上位葉におけるウイルス感染抑制効果
(1)ベンサミアーナタバコ
ベンサミアーナタバコに対して、実施例1で調製した植物抽出液(0.01%マイリノーを添加した10%溶液と50%溶液)を噴霧処理した。陽性対照として、アスコルビン酸とレンテミンを用いた。各資材処理3日後に2μgプラスミドをRNA転写し、20倍希釈したToMV-GFPを各植物体に10μlずつ第3葉にカーボランダムを用いて接種した。3日後にリアルタイムPCRを用いて、ToMVの感染量を定量した。1処理あたり、ベンサミアーナタバコ3個体以上を用い、平均値および標準誤差を求めた。その結果、上位葉においても、ToMVに対して、強いウイルス感染抑制効果が認められた(図3)。
[Example 5] Inhibitory effect of viral infection on upper leaves (1) Bensamiana tobacco To the Bensamiana tobacco, the plant extract prepared in Example 1 (10% solution and 0.01% solution containing 0.01% mylino) ) Was sprayed. Ascorbic acid and lentemin were used as positive controls. Three days after the treatment with each material, 2 μg of the plasmid was transcribed by RNA, and 20 μl-diluted ToMV-GFP was inoculated to the third leaf of each plant by 10 μl using a carborundum. Three days later, the amount of ToMV infection was quantified using real-time PCR. The average value and the standard error were determined using three or more Bensamiana tobacco plants per treatment. As a result, even in the upper leaves, a strong virus infection inhibitory effect on ToMV was observed (FIG. 3).
(2)トマト
14日齢トマト(品種レジナ)に対して、実施例1で調製した植物抽出液(0.01%マイリノーを添加した50%溶液)を噴霧処理した。陽性対照として、アスコルビン酸とレンテミンを用いた。各資材処理3日後に2μgプラスミドをRNA転写して5倍希釈したToMV-GFPを各植物体に10μlずつ第1葉にカーボランダムを用いて接種した。6日後にリアルタイムPCRを用いて、接種葉および上位葉のToMVの感染量を定量した。1処理あたり、トマト3個体以上を用い、平均値および標準誤差を求めた。その結果、上位葉においても、ToMVに対して、強いウイルス感染抑制効果が認められた(図4)。
(2) Tomato
A 14-day-old tomato (cultivar Regina) was spray-treated with the plant extract prepared in Example 1 (50% solution containing 0.01% mylino). Ascorbic acid and lentemin were used as positive controls. Three days after each material treatment, 2 μg of plasmid was transcribed with RNA, and ToMV-GFP diluted 5-fold was inoculated to the first leaf of each plant by 10 μl using a carborundum. Six days later, the amount of ToMV infection in the inoculated leaves and upper leaves was quantified using real-time PCR. The average value and the standard error were determined using three or more tomatoes per treatment. As a result, even in the upper leaves, a strong virus infection inhibitory effect on ToMV was observed (FIG. 4).
[実施例6] 病原菌に対する感染抑制効果
(1)炭疽病菌
実施例1で調製した月桃由来の植物抽出液を用いて、以下の方法によりアブラナ科野菜類炭疽病菌に対する感染抑制効果を評価した。アブラナ科植物のシロイヌナズナに、50%植物抽出液(0.01%マイリノー添加)を茎葉散布した。植物抽出液の散布から2日後、シロイヌナズナにアブラナ科野菜類炭疽病菌(5×105胞子/ml)を噴霧接種した。接種から5日後、接種葉をサンプリングし、qRT-PCR法により、アブラナ科野菜類炭疽病菌のシロイヌナズナへの感染量を定量した。なお、コントロールとして、植物抽出液を含まない展着剤(0.01%マイリノー添加)のみを散布し、同様の定量を行った。
Example 6 Inhibitory Effect on Infection with Pathogenic Bacteria (1) Anthrax Bacteria Using the plant extract derived from moon peach prepared in Example 1, the effect of inhibiting infection with cruciferous vegetables on anthrax was evaluated by the following method. A 50% plant extract (with 0.01% myrine) was sprayed on foliage of Arabidopsis thaliana. Two days after the application of the plant extract, Arabidopsis was spray-inoculated with a cruciferous vegetable anthrax (5 × 10 5 spores / ml). Five days after the inoculation, the inoculated leaves were sampled, and the amount of infection of Arabidopsis thaliana by Brassica anthracnose was determined by qRT-PCR. As a control, the same quantification was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added).
その結果、コントロールと比較して、植物抽出液において有意に感染抑制効果が認められた(図5)。 As a result, a significant effect of suppressing infection was observed in the plant extract as compared with the control (FIG. 5).
また、実施例1で調製した月桃由来の植物抽出液を用いて、以下の方法によりイチゴ炭疽病菌(Colletotrichum gloeosporioides )に対する感染抑制効果を評価した。バラ科のイチゴに、50%植物抽出液(0.01%マイリノー添加)を茎葉散布した。植物抽出液の散布から3日後、イチゴにイチゴ炭疽病菌(5×105胞子/ml)を噴霧接種した。接種から6日後、病徴の調査によりイチゴ炭疽病菌のイチゴへの感染量を評価した。なお、コントロールとして、植物抽出液を含まない展着剤(0.01%マイリノー添加)のみを散布し、同様の評価を行った。発病度は以下の式で表される。
発病度={(1n1+2n2+3n3+4n4+5n5)/(5×調査数)}×100
発病調査は発病程度を以下の5つに区分して調査した。
0:病徴なし、1:微小斑点、2:葉の25%以下の面積に病斑が認められる、3:葉の25〜50%の面積に病斑が認められる、4:葉の50%以上の面積に病斑が認められるまたは葉柄が折れる、5:枯死
n1からn5は個体数
防除価は以下の図式で表される。
防除価={1-処理区の発病度/無処理区の発病度)}×100
その結果、コントロールと比較して、植物抽出液において有意に感染抑制効果が認められた(表2)。
In addition, using the plant extract derived from moon peach prepared in Example 1, the effect of inhibiting infection with strawberry anthrax (Colletotrichum gloeosporioides) was evaluated by the following method. A 50% plant extract (with 0.01% myrinoux) was sprayed onto foliage strawberry. Three days after the application of the plant extract, strawberries were inoculated by spraying with strawberry anthrax (5 × 10 5 spores / ml). Six days after the inoculation, the amount of strawberry anthracnose infecting strawberry was evaluated by examining the symptoms. As a control, the same evaluation was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added). The severity is expressed by the following equation.
Severity = {(1n 1 + 2n 2 + 3n 3 + 4n 4 + 5n 5 ) / (5 × number of surveys)} × 100
The disease incidence was surveyed by dividing the disease severity into the following five categories.
0: No symptom, 1: Microscopic spot, 2: Lesion is found in area of 25% or less of leaf 3: Lesion is found in area of 25 to 50% of leaf, 4: 50% of leaf Lesion is observed in the above area or petiole is broken. 5: Withering
n 5 from n 1 is the number of individuals controlling value is expressed by the following scheme.
Control value = {1-degree of disease in treated area / degree of disease in untreated area) x 100
As a result, a significant effect of suppressing infection was observed in the plant extract as compared with the control (Table 2).
(2)黒斑細菌病菌
実施例1で調製した月桃由来の植物抽出液を用いて、以下の方法で、黒斑細菌病菌に対するに対する感染抑制効果を評価した。アブラナ科植物のシロイヌナズナおよびチンゲンサイ(品種:シャパオ)に、50%植物抽出液(0.01%マイリノー添加)を茎葉散布した。植物抽出液の散布から2日後、シロイヌナズナおよびチンゲンサイにそれぞれ黒斑細菌病菌(1×108cfu/ml)を噴霧接種した。接種から3日後、接種葉をサンプリングし、qRT-PCR法により、黒斑細菌病菌のシロイヌナズナおよびチンゲンサイへの感染量を定量した。なお、コントロールとして、植物抽出液を含まない展着剤(0.01%マイリノー添加)のみを散布し、同様の定量を行った。
(2) Black Spot Bacterial Bacteria Using the plant extract derived from moon peach prepared in Example 1, the effect of suppressing infection against black spot bacilli was evaluated by the following method. A 50% plant extract (with 0.01% myrineau) was sprayed onto foliage of Arabidopsis thaliana and bok choy (variety: Shapao). Two days after the application of the plant extract, Arabidopsis thaliana and bok choy were each spray-inoculated with black spot bacterial pathogen (1 × 10 8 cfu / ml). Three days after inoculation, the inoculated leaves were sampled, and the amount of infection of Arabidopsis thaliana and bok choy by the bacterial spot of black spot was quantified by qRT-PCR. As a control, the same quantification was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added).
その結果、コントロールと比較して、植物抽出液において有意に感染抑制効果が認められた(図6、図7)。 As a result, a significant infection-suppressing effect was observed in the plant extract as compared with the control (FIGS. 6 and 7).
[実施例7] 月桃のアセトニトリル抽出物のToMVに対する防除効果
沖縄月桃の搾汁液を、逆相担体(Sep-Pak C18)にかけ、水で洗浄した後、5%、10%、15%、20%、40%アセトニトリルで、段階的に溶出した画分を得た。得られた画分に含まれるアセトニトリルを、エバポレーターにて留去したのち、残った溶液を凍結乾燥した。得られた各画分の凍結乾燥物を、水で溶解し、ウイルス感染抑制効果を評価した。
[Example 7] Control effect of acetonitrile extract of moon peach on ToMV Okinawa peach squeezed solution was applied to a reverse phase carrier (Sep-Pak C18), washed with water, and then 5%, 10%, 15%, Stepwise eluted fractions were obtained with 20% and 40% acetonitrile. Acetonitrile contained in the obtained fraction was distilled off by an evaporator, and the remaining solution was freeze-dried. The freeze-dried product of each of the obtained fractions was dissolved in water, and the effect of suppressing virus infection was evaluated.
ウイルス感染抑制効果の評価においては、ベンサミアーナタバコに月桃抽出物を処理して3日後に、ウイルスを接種(すりこみ接種)した。この評価においては、ToMVに、蛍光を発するタンパク質GFP遺伝子を導入し、感染すると蛍光を発するようにしたウイルスを用いた。 In the evaluation of the virus infection-suppressing effect, the virus was inoculated (recruited inoculation) 3 days after treating the Bensamiana tobacco with the moon peach extract. In this evaluation, a virus in which a protein GFP gene that emits fluorescence was introduced into ToMV, and which emitted fluorescence upon infection was used.
その結果、5%、10%、15%、20%アセトニトリル溶出画分に、ウイルス感染抑制効果が認められた(図8)。 As a result, a virus infection inhibitory effect was observed in the fractions eluted with 5%, 10%, 15%, and 20% acetonitrile (FIG. 8).
なお、上記アセトニトリル溶出画分の乾燥重量を図9に示した。また、以降の実験におけるウイルス感染抑制効果の評価は、全て、このGFP挿入ToMVとベンサミアーナタバコを用いた系で行った。 The dry weight of the acetonitrile-eluted fraction is shown in FIG. In addition, all the evaluations of the virus infection suppressing effect in the subsequent experiments were performed using a system using this GFP-inserted ToMV and Bensamiana tobacco.
[実施例8] 透析膜による活性画分の分離
沖縄月桃の搾汁液(20ml)を、蒸留水に対して透析して得られた透析内液のウイルス感染抑制効果を評価した。透析膜として分画分子量の異なる3種(100-500、1,000、3,500)を用いた。その結果、3種の透析膜による透析内液のいずれにも、ウイルス感染抑制効果が認められたことから、活性成分は、分子量3,500以上のものであると推測された。
Example 8 Separation of Active Fraction by Dialysis Membrane The virus infection-suppressing effect of the inner solution of dialysis obtained by dialyzing a squeezed solution of Okinawa Tsukipeach (20 ml) against distilled water was evaluated. Three types (100-500, 1,000, 3,500) having different molecular weight cutoffs were used as dialysis membranes. As a result, the effect of suppressing virus infection was observed in any of the inner dialysis solutions obtained by the three types of dialysis membranes, indicating that the active ingredient had a molecular weight of 3,500 or more.
[実施例9] 限外濾過による活性画分の分離
上記の5%、10%、15%、20%アセトニトリル溶出画分を合わせて、蒸留水に対して透析した。透析膜として分画分子量3,500のものを用いた。得られた透析内液を、凍結乾燥した後、再度水に溶解したものを、遠心限外ろ過(分画分子量:10,000)にかけた。通過したもの(すなわち、分子量10,000以下)と、残存したもの(分子量:10,000以上)の画分を凍結乾燥し、抗ウイルス活性を評価した。
Example 9 Separation of Active Fraction by Ultrafiltration The above 5%, 10%, 15%, and 20% acetonitrile-eluted fractions were combined and dialyzed against distilled water. A dialysis membrane having a molecular weight cutoff of 3,500 was used. The obtained dialysis solution was freeze-dried and then redissolved in water and subjected to centrifugal ultrafiltration (fraction molecular weight: 10,000). The fractions that passed (that is, molecular weight of 10,000 or less) and those that remained (molecular weight: 10,000 or more) were freeze-dried and evaluated for antiviral activity.
その結果、限外ろ過の残存画分に活性が存在していたことから、活性成分は、分子量10,000以上であると推測された。 As a result, it was inferred that the active component had a molecular weight of 10,000 or more, because the activity was present in the remaining fraction of the ultrafiltration.
[実施例10] アセトニトリル溶出画分における無機金属の分析
実施例9と同様にして、アセトニトリル溶出画分の透析内液の凍結乾燥物を得た。得られた凍結乾燥物に含まれる無機金属をICP発光で、分析した。
[Example 10] Analysis of inorganic metal in acetonitrile-eluting fraction In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluting fraction in dialysis solution was obtained. The inorganic metal contained in the obtained freeze-dried product was analyzed by ICP emission.
その結果、凍結乾燥物1,000mgに対して、マグネシウムが2.6mg、鉄が2.1mg、カルシウムが1.0mg、ナトリウムが1.0mg含量していた。 As a result, 2.6 mg of magnesium, 2.1 mg of iron, 1.0 mg of calcium, and 1.0 mg of sodium were contained in 1,000 mg of the freeze-dried product.
また、得られた凍結乾燥物の溶媒に対する溶解性を検討したところ、水およびジメチルホルムアミド(DMF)には可溶であったが、テトラヒドロフラン(THF)およびクロロホルムには不溶であった。 When the solubility of the obtained lyophilized product in a solvent was examined, it was soluble in water and dimethylformamide (DMF), but insoluble in tetrahydrofuran (THF) and chloroform.
[実施例11] ゲル浸透クロマトグラフフィーによる分子量分布の分析
実施例9と同様にして、アセトニトリル溶出画分の透析内液の凍結乾燥物を得た。得られた凍結乾燥物の分子量分布および吸収スペクトルを、DMFを溶媒に用いたゲルパー浸透クロマトグラフフィーで検討した。
Example 11 Analysis of Molecular Weight Distribution by Gel Permeation Chromatography In the same manner as in Example 9, a freeze-dried product of the acetonitrile-eluting fraction in the dialyzed solution was obtained. The molecular weight distribution and absorption spectrum of the obtained lyophilized product were examined by gel permeation chromatography using DMF as a solvent.
その結果、サンプルは、283nmと263nmに、強い吸収極大をもつことが判明し、その分子量は10,000以上と見積もられた(図10)。 As a result, the sample was found to have strong absorption maximums at 283 nm and 263 nm, and its molecular weight was estimated to be 10,000 or more (FIG. 10).
なお、重合度の異なるポリスチレンを検量線に用いて換算した、吸収極大波長における重量平均分子量を図の右上に示した。 The weight average molecular weight at the absorption maximum wavelength, which was obtained by using polystyrene having different degrees of polymerization as a calibration curve, is shown in the upper right of the figure.
[実施例12] 質量分析による分子量分布の分析
実施例9と同様にして、アセトニトリル溶出画分の透析内液の凍結乾燥物を得た。得られた凍結乾燥物の分子量分布を、MALDI-TOF質量分析にて推定した。その結果、サンプルは、分子量288ユニットがいくつか重合したポリマーであることが示唆された(図11)。既報文献(Xiaolan Jiang et al., SCIENTIFIC REPORTS 5:8742, DOI:10.1038/srep08742、Sauro Bianchi et al., Phytochemistry 120(2015)53-61)で、分子量288は、カテキンあるいはエピカテキンであることが報告されている。この事から、活性本体が、そのポリマーであるプロアントシアニジンであることが推測された。
[Example 12] Analysis of molecular weight distribution by mass spectrometry In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluted fraction in the dialyzed solution was obtained. The molecular weight distribution of the obtained freeze-dried product was estimated by MALDI-TOF mass spectrometry. As a result, it was suggested that the sample was a polymer in which some 288 units of molecular weight were polymerized (FIG. 11). In published literature (Xiaolan Jiang et al., SCIENTIFIC REPORTS 5: 8742, DOI: 10.1038 / srep08742, Sauro Bianchi et al., Phytochemistry 120 (2015) 53-61), molecular weight 288 may be catechin or epicatechin. It has been reported. From this, it was inferred that the active substance was the polymer, proanthocyanidins.
なお、ベンサミアーナタバコに500ppmのエピカテキンを噴霧処理して2時間後にToMVを接種したところ、若干の感染抑制効果が認められたが、噴霧処理して3日後にToMVを接種した場合は、感染抑制効果が認められなかった。 In addition, when Bensamiana tobacco was sprayed with 500 ppm epicatechin and inoculated with ToMV 2 hours later, a slight infection-suppressing effect was observed.However, when ToMV was inoculated 3 days after spraying, No infection control effect was observed.
[実施例13] 部分構造の推定
実施例9と同様にして、アセトニトリル溶出画分の透析内液の凍結乾燥物を得た。得られた凍結乾燥物の部分構造をプロトンNMRおよび赤外吸収スペクトルにて推定した(図12、図13)。その結果、部分構造として、芳香族環や水酸基などが多く含まれることが推測された。さらに、得られた凍結乾燥物の部分構造につき、熱分析GC-MSによる解析を行った結果、ポリフェノール類が多く含まれていることが推測された(図14)。以上の結果は、活性本体がプロアントシアニジンであることをさらに裏付けた。
Example 13 Estimation of Partial Structure In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluted fraction in the dialysate was obtained. The partial structure of the obtained lyophilized product was estimated by proton NMR and infrared absorption spectrum (FIGS. 12 and 13). As a result, it was speculated that the partial structure contained many aromatic rings, hydroxyl groups, and the like. Further, the partial structure of the obtained freeze-dried product was analyzed by thermal analysis GC-MS, and as a result, it was estimated that a large amount of polyphenols was contained (FIG. 14). The above results further confirmed that the active substance was proanthocyanidins.
実施例9で得られた透析内液の凍結乾燥物を、蒸留水に再溶解し、分画分子量(10,000)の透析膜を用いて、蒸留水に対して透析し、透析内液の凍結乾燥物を得た。これをアセトンに溶解し、C13カーボンNMRにて構造を推定した(図15)。その結果、文献(Thomas L. Eberhardt et al., J. Agric. Food Chem. 42(1994)1704-1708)に報告されているプロアントシアニジンのC13カーボンNMRデータとよく一致した。以上の結果は、活性本体がプロアントシアニジンであることをさらに裏付けた。 The freeze-dried product of the dialysis solution obtained in Example 9 was redissolved in distilled water, dialyzed against distilled water using a dialysis membrane having a molecular weight cut-off (10,000), and lyophilized of the dialysis solution. I got something. This was dissolved in acetone and the structure was estimated by C13 carbon NMR (FIG. 15). As a result, it was in good agreement with the C13 carbon NMR data of proanthocyanidin reported in the literature (Thomas L. Eberhardt et al., J. Agric. Food Chem. 42 (1994) 1704-1708). The above results further confirmed that the active substance was proanthocyanidins.
[実施例14] プロアントシアニジン濃度の測定
実施例9で得られた透析内液(分画分子量3,500)の凍結乾燥物を、蒸留水に再溶解し、分画分子量(10,000)の透析膜を用いて、蒸留水に対して透析し、透析内液の凍結乾燥物を得た。これを蒸留水に溶解し、月桃由来検量線用精製アントシアニジンとした。
Example 14 Measurement of Proanthocyanidin Concentration The freeze-dried product of the inner dialysis solution (fraction molecular weight: 3,500) obtained in Example 9 was redissolved in distilled water, and a dialysis membrane having a fraction molecular weight (10,000) was used. Then, the solution was dialyzed against distilled water to obtain a lyophilized product of the inner solution of the dialysis. This was dissolved in distilled water to obtain purified anthocyanidins for a calibration curve derived from moon peach.
プロアントシアニジンの定量については、沖らの報告(沖智之ら、Nippon Shkuhin Kagaku Kogaku Kaishi Vol.60, No6, 301-309(2013))に記載された4-ジメチルアミノシンナムアルデヒド(DMAC)を用いる方法におおむね従った。すなわち、検量線用プロアントシアニジン水溶液(10mg/ml)を、アッセイ溶液A(エタノール:メタノール:2-プロパノール=90:5:5)で、20倍希釈したものを、さらにアッセイ溶液B(アッセイ溶液A:水=95:5)で、2倍希釈系列を作成し、検量線用サンプルとした。月桃搾汁液は、アッセイ溶液A(エタノール:メタノール:2-プロパノール=90:5:5)で、20倍希釈したものを、測定用サンプルとした。また、対称サンプルとして、ショウガ搾汁液および実施例9に記載した透析内液(分画分子量3,500)の凍結乾燥物についても、月桃搾汁液と同様に調整した。 For the determination of proanthocyanidins, a method using 4-dimethylaminocinnamaldehyde (DMAC) described in the report by Oki et al. (Tomoyuki Oki et al., Nippon Shkuhin Kagaku Kogaku Kaishi Vol. 60, No. 6, 301-309 (2013)) I almost followed. That is, the proanthocyanidin aqueous solution (10 mg / ml) for a calibration curve was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90: 5: 5), and further diluted with assay solution B (assay solution A). : Water = 95: 5) to prepare a two-fold dilution series, which was used as a sample for a calibration curve. The moon peach juice was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90: 5: 5) to obtain a measurement sample. In addition, as a symmetrical sample, a ginger juice and a freeze-dried product of the internal dialysis solution (fraction molecular weight: 3,500) described in Example 9 were also prepared in the same manner as the moon peach juice.
DMAC溶液は、濃塩酸(3ml)を、上記アッセイ溶液A(27ml)に添加後、氷上で15分冷却した溶液にDMAC(30mg)を添加し、攪拌により溶解し、使用直前まで氷上で保管し、DMAC溶液とした。 The DMAC solution was prepared by adding concentrated hydrochloric acid (3 ml) to the above-mentioned assay solution A (27 ml), then adding DMAC (30 mg) to a solution cooled on ice for 15 minutes, dissolving by stirring, and storing on ice until just before use. And a DMAC solution.
96穴マイクロプレートのウェルに、検量線用および測定用サンプルを、それぞれ40μlづつ分注し、8連マイクロピペッターを用いて、サンプルを添加したウェルに、DMAC溶液を200μlづつ添加し、プレート上面にプレートシールを貼付し、庫内温度を30℃に設定したプレートリーダー中で、攪拌後、20分間放置後、各ウェルの640nmにおける吸光度を測定した。検量線用サンプルから得られた吸光度値と濃度から検量線を作成し、月桃搾汁液に含まれるプロアントシアニジンを定量した。その結果、月桃搾汁液に含まれるプロアントシアニジンは、5〜20mg/mlの範囲であると見積もることができた(表3)。 To the wells of a 96-well microplate, dispense 40 μl each of the sample for the calibration curve and the sample for measurement, and add 200 μl of the DMAC solution to the wells to which the sample was added using an 8-well micropipette. A plate seal was attached, and the plate was stirred and left for 20 minutes in a plate reader set at 30 ° C., and the absorbance of each well at 640 nm was measured. A calibration curve was prepared from the absorbance values and concentrations obtained from the calibration curve samples, and proanthocyanidins contained in the peach juice were quantified. As a result, the amount of proanthocyanidins contained in the peach juice was estimated to be in the range of 5 to 20 mg / ml (Table 3).
なお、表2に記載した月桃搾汁液2の10mlを凍結乾燥したところ、得られた乾燥物の重量は、431.2mgであった。これから換算すると、プロアントシアニジンの乾燥物に占める割合は、43.0%となる。さらに、搾汁液に含まれるプロアントシアニジンが、5〜20mg/mlであるとすると、搾汁液乾燥物に占めるプロアントシアニジンの割合は、10〜50%程度と見積もることができる。 In addition, when 10 ml of the moon peach juice 2 shown in Table 2 was freeze-dried, the weight of the obtained dried product was 431.2 mg. When converted from this, the ratio of proanthocyanidins to dry matter is 43.0%. Furthermore, assuming that proanthocyanidin contained in the juice is 5 to 20 mg / ml, the ratio of proanthocyanidin in the dried juice can be estimated to be about 10 to 50%.
上記の通り、本発明の防除剤は、植物に含まれる成分を利用して、植物病害に対して優れた防除効果を示すことができる。このため、安全性と効果を兼ね備えた農薬として、農業分野において大きく貢献しうるものである。 As described above, the control agent of the present invention can exhibit an excellent control effect on plant diseases by utilizing components contained in plants. For this reason, it can greatly contribute to the agricultural field as an agricultural chemical having both safety and effect.
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