JP2006076960A - Garlic-derived antifungal substance and its production method - Google Patents
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Abstract
Description
本発明は,ニンニク、特にニンニクの皮に由来する抗菌性物質、及びその製造方法に関する。 The present invention relates to an antibacterial substance derived from garlic, particularly garlic skin, and a method for producing the same.
青森県産のニンニク（Allium sativum L.）は全国シェアの約８割を占め、青森県の産業の重要な部分を構成している。ニンニクの可食部分を利用するために除去するニンニクの皮は膨大な量に昇り、現在、産業廃棄物として処理されており、その有効利用が望まれている。ニンニクの皮はニンニクの可食部に対する保護作用を有すると考えられ、ニンニクの皮には何らかの抗菌性物質の存在が予想されるが、そのような物質の存在は確認されていない。 Garlic (Allium sativum L.) from Aomori Prefecture occupies about 80% of the national market share and constitutes an important part of Aomori Prefecture's industry. The garlic skin to be removed to use the edible portion of garlic has risen to an enormous amount and is currently treated as industrial waste, and its effective use is desired. Garlic skin is thought to have a protective effect on the edible part of garlic, and some antibacterial substances are expected to exist in garlic skin, but the presence of such substances has not been confirmed.
植物炭素病の一種であるリンゴ炭素病は、植物炭素病原菌であるコレトトリカム・アクテイタム（Colletotrichum acutatum）がリンゴの果実の表面に繁殖する植物病であり、このリンゴ炭素病に対しては、べノミル（Venomyl）、ジエトフェンカルブ（Diethofencarb）等の合成農薬使用されている。コレトトリカム・アクテイタム（Colletotrichum acutatum）に対するべノミル（Venomyl）の最少阻止濃度は1250μmであり、ジエトフェンカルブ（Diethofencarb）のそれは625μmであり、植物炭素病原菌に対する、より強力な農薬の開発が望まれている。 Apple carbon disease, a type of plant carbon disease, is a plant disease in which the plant carbon pathogen, Colletotrichum acutatum, propagates on the fruit surface of apples. Synthetic pesticides such as Venomyl and Dietophencarb are used. The minimum inhibitory concentration of Venomyl against Colletotrichum acutatum is 1250 μm and that of Dietofencarb is 625 μm, and the development of a more powerful pesticide against plant carbon pathogens is desired.
本発明は、青森県の重要な農産物であるニンニクの皮から、同じく青森県の重要な農産物であるリンゴの炭素病に対する農薬を得ようとするものであり、より具体的にはニンニクの皮から、植物炭素病などに有効な抗菌性物質を製造する方法、及びその新規な抗菌性物質を提供しようとするものである。 The present invention seeks to obtain a pesticide for apple carbon disease, which is also an important agricultural product of Aomori Prefecture, from garlic skin, which is an important agricultural product of Aomori Prefecture, and more specifically, from garlic skin. An object of the present invention is to provide a method for producing an antibacterial substance effective for plant carbon disease and the like, and the novel antibacterial substance.
本発明者らは、上記の課題を解決すべく種々検討した結果、ニンニクの皮から、白色結晶性の新規な抗菌性物質を抽出することに成功し、本発明を完成した。
従って本発明は、ニンニク皮由来の抗菌性物質の製造方法において、下記の工程：
（１）ニンニクの皮を用意する；
（２）ニンニクの皮を、所望により粉砕した後、酢酸エチルで抽出して、酢酸エチル抽出物を得る；
（３）酢酸エチル抽出物を、ｎ−ヘキサンと酢酸エチルとから成る展開溶媒を用いてフラッシュカラムクロマトグラフィー処理して、３番目に溶出する画分III （Fr.III ）を得る；
（４）画分III 中の成分をシリカゲル箔層クロマトグラフィーにかけ、展開溶媒として塩化メチレン／エチルエーテル／ｎ−ヘキサン（10：1：7）を用いるシリカゲル箔層クロマトグラフィーにおいて、およそ0.44のRf値を示す物質を採取する；
を含んで成る方法を提供する。
As a result of various studies to solve the above-mentioned problems, the present inventors have succeeded in extracting a novel white crystalline antibacterial substance from garlic skin and completed the present invention.
Therefore, the present invention provides a method for producing an antibacterial substance derived from garlic skin, comprising the following steps:
(1) Prepare garlic skin;
(2) The garlic crust is ground if necessary and then extracted with ethyl acetate to obtain an ethyl acetate extract;
(3) The ethyl acetate extract is subjected to flash column chromatography using a developing solvent consisting of n-hexane and ethyl acetate to obtain the third eluting fraction III (Fr.III);
(4) Rf value of about 0.44 in silica gel foil layer chromatography using methylene chloride / ethyl ether / n-hexane (10: 1: 7) as developing solvent. Collect substances that indicate
A method comprising the steps of:
本発明に使用するニンニクとしては、暖地種である佐賀大ニンニク（佐賀県）、 定種（熊本、阿蘇）、壱州ニンニク（壱岐）、寒地種である福地ホワイト（青森県）、千葉大球（千葉県）などが挙げられ、いずれも使用することができるが、原料が大量に確保できるなどの実用的観点から、福地ホワイトなどが好ましい。
ニンニクの皮は、ニンニクの可食部を得た後の副産物として入手できる。抽出に先立ち、粉砕することが望ましい。粉砕は、常用の粉砕機を用いて行うことが出来る。
The garlic used in the present invention includes Saga University garlic (Saga Prefecture), which is a warm species, Kumamoto (Aso), Zhengzhou Garlic (Iki), Fukuchi White (Aomori Prefecture), which is a cold region species, and Chiba University. Sphere (Chiba Prefecture) and the like can be used, and any of them can be used, but Fukuchi White is preferred from a practical viewpoint such as securing a large amount of raw materials.
Garlic peel is available as a by-product after obtaining the edible portion of garlic. It is desirable to grind prior to extraction. The pulverization can be performed using a conventional pulverizer.
ニンニクの皮の、酢酸エチルによる抽出は、例えば、ニンニクの皮１重量部に対して、１〜10重量部の酢酸エチルを用い、例えば10℃〜40℃の温度にて、１時間〜120時間、例えば12時間〜48時間、行なうことが出来る。抽出機としては、固形物の溶剤抽出に常用される抽出機を用いて行なうことが出来る。抽出後、濾過などの常用の固−液分離手段を用いて固−液分離して溶剤層を得る。次に、抽出液から溶剤を例えば蒸発により除去し、抽出物を得る。 Extraction of garlic crust with ethyl acetate uses, for example, 1 to 10 parts by weight of ethyl acetate per 1 part by weight of garlic crust, for example, at a temperature of 10 ° C. to 40 ° C. for 1 hour to 120 hours. For example, it can be performed for 12 hours to 48 hours. As an extractor, it can carry out using the extractor normally used for the solvent extraction of a solid substance. After the extraction, the solvent layer is obtained by solid-liquid separation using conventional solid-liquid separation means such as filtration. Next, the solvent is removed from the extract by, for example, evaporation to obtain an extract.
次に、上記の抽出物を、フラッシュカラムクロマトグラフィーにより分画する。カラム充填剤としては、Silica Gel 60N (Spherical Neutra) 40−100μmなどを使用することが出来る。抽出溶媒としては、例えば下記の溶媒を順次使用することが出来る。
（１）ｎ−ヘキサン／酢酸エチル＝2:1 900体積
（２）ｎ−ヘキサン／酢酸エチル＝1:1 200体積
（３）ｎ−ヘキサン／酢酸エチル＝1:2 420体積
（４）メタノール 500体積
上記の条件での分画により、３番目の画分（Fr.III ）及び４番目の画分（Fr.IV）に抗菌活性化認められる。
Next, the above extract is fractionated by flash column chromatography. As the column filler, Silica Gel 60N (Spherical Neutra) 40-100 μm or the like can be used. As the extraction solvent, for example, the following solvents can be used sequentially.
(1) n-hexane / ethyl acetate = 2: 1 900 volume (2) n-hexane / ethyl acetate = 1: 1 200 volume (3) n-hexane / ethyl acetate = 1: 2 420 volume (4) methanol 500 Volume Antibacterial activation is observed in the third fraction (Fr.III) and the fourth fraction (Fr.IV) by fractionation under the above conditions.
次に、上記のようにして得た画分III を、中圧フラッシュカラムクロマトグラフィーにより更に分画することが出来る。例えば、下記の溶媒を使用することが出来る。
（１）ｎ−ヘキサン／酢酸エチル＝1:1 100体積
（２）メタノール 100体積
上記の条件での操作により、第一の画分（Fr.III -１）に活性が回収される。
Next, fraction III obtained as described above can be further fractionated by medium pressure flash column chromatography. For example, the following solvents can be used.
(1) n-hexane / ethyl acetate = 1: 1 100 volume (2) 100 volume of methanol Activity is recovered in the first fraction (Fr.III-1) by the operation under the above conditions.
上記のようにして得た抗菌性物質を更に精製するには、シリカゲル薄層クロマトグラフィーを用いることが出来る。得られた結晶は下記の理化学的性質を有する。NMR：7.6323（2H.d,T＝ ）；4.3135（1H,m）；4.2834-4.0（1h,m）；3.4829（1H,d,T＝ ）。 Silica gel thin layer chromatography can be used to further purify the antibacterial substance obtained as described above. The obtained crystal has the following physicochemical properties. NMR: 7.6323 (2H.d, T =); 4.3135 (1H, m); 4.2834-4.0 (1h, m); 3.4829 (1H, d, T =).
生物学的性質
この抗菌物質は、コレトトリカム・アクタツム（Colletotrichum acutatum）に対して、６μg／mlの最少阻止濃度を有する。この抗菌力は、植物炭素病に対する合成農薬であるべノミル（Venomyl）の最少阻止濃度は1250μm、及びジエトフェンカルブ（Diethofencarb）の最少阻止濃度625μmに比べて100倍以上強力である。
Biological properties This antibacterial substance has a minimum inhibitory concentration of 6 μg / ml against Colletotrichum acutatum. This antibacterial activity is more than 100 times more potent than the minimum inhibitory concentration of Venomyl, a synthetic pesticide against plant carbon disease, of 1250 μm and the minimum inhibitory concentration of Diethofencarb, 625 μm.
次に、本発明を実施例により更に具体的に記載する。
実施例１．
ニンニクの皮150gを粉砕し、酢酸エチル2000mlを加え、室温にて24時間かき混ぜた後、吸引濾過を行い、抽出液と沈殿物を得た。抽出液は、ロータリーエバポレーターを用いて溶媒を留去し、抽出物628.1mgを得た。さらに、この沈殿物に酢酸エチル1500mlを加え、先程と同様の操作を行い抽出物282.8mgと沈殿物を得た。
Next, the present invention will be described more specifically with reference to examples.
Example 1.
150 g of garlic crust was pulverized, 2000 ml of ethyl acetate was added, and the mixture was stirred at room temperature for 24 hours, followed by suction filtration to obtain an extract and a precipitate. In the extract, the solvent was distilled off using a rotary evaporator to obtain 628.1 mg of an extract. Further, 1500 ml of ethyl acetate was added to this precipitate, and the same operation as above was performed to obtain 282.8 mg of an extract and a precipitate.
２回の抽出操作で得られた抽出物910.9mgを、カラム管（3.4cm×45cm）でフラッシュカラムクロマトグラフィー（Silica gel 60N spherical,neutral 40-100μm Cica-Reagent 関東化学（株）Cat.No.37561-79,181.8g）、展開溶媒として（１）ｎ−ヘキサン／酢酸エチル＝2:1 900ml、（２）ｎ−ヘキサン／酢酸エチル＝1:1 200ml、（３）ｎ−ヘキサン／酢酸エチル＝1:2 420ml、（４）メタノール 500ml、を用いて展開して分画を行い、３番目の画分（Fr.III, 35.9mg）及び４番目の画分（Fr.IV, 24.0mg）に抗菌活性が認められた。 910.9 mg of the extract obtained by the two extraction operations was subjected to flash column chromatography (Silica gel 60N spherical, neutral 40-100 μm Cica-Reagent Kanto Chemical Co., Ltd. Cat. No.) using a column tube (3.4 cm × 45 cm). 37561-79,181.8g), (1) n-hexane / ethyl acetate = 2: 1 900 ml, (2) n-hexane / ethyl acetate = 1: 1 200 ml, (3) n-hexane / ethyl acetate = 1 : 2 420ml, (4) 500ml of methanol, and fractionate, antibacterial in the 3rd fraction (Fr.III, 35.9mg) and 4th fraction (Fr.IV, 24.0mg) Activity was observed.
次に、上記のようにして得た画分Fr.IIIを、中圧フラッシュクロマトグラフィー装置（EYELA CERAMIC PUMP VSP-3050）で、カラム管（外径1.5cm×長さ22cm、シリカゲルMERCK Silica gel 60 0.040-0.063mm，1.09385.1000）を用いて分画を行った。展開溶媒として（１）ｎ−ヘキサン／酢酸エチル＝1:1 100ml、（２）メタノール 100mlを用いて分画し、第一の画分（Fr.III-1, 13.2mg）に活性が見られた。 Next, the fraction Fr.III obtained as described above was subjected to a column tube (outer diameter 1.5 cm × length 22 cm, silica gel MERCK Silica gel 60 using an intermediate pressure flash chromatography apparatus (EYELA CERAMIC PUMP VSP-3050). Fractionation was carried out using 0.040-0.063mm, 1.09385.1000). Fractionation was performed using (1) n-hexane / ethyl acetate = 1: 1 100 ml as a developing solvent and (2) 100 ml of methanol, and activity was observed in the first fraction (Fr.III-1, 13.2 mg). It was.
上記のようにして得た抗菌性物質（Fr.III-1, 11.4mg）をさらに精製するために、シリカゲル薄層クロマトグラフィー（20×20cm，MERCK Silica gel 60 F254,1.05715.0009）１枚、展開溶媒（ｎ−ヘキサン／酢酸エチル＝1:1）で分画し、Fr.III-1-iiを4.1mg得た。
さらに、Fr.III-1-ii（3.1mg）を上記と同様にしてシリカゲル薄層クロマトグラフィー１枚（展開溶媒は同じ）で分画し、Fr.III-1-ii-Cを2.0mg得た。
さらに、Fr.III-1-ii-C（1.0mg）を上記と同様にシリカゲル薄層クロマトグラフィー１枚（展開溶媒CH2Cl2/Et2O/n-ヘキサン＝10:1:5）で分画し、Fr.III-1-ii-C-bを0.5mg得た。Fr.III-1-ii-C-bは分析用の薄層クロマトグラフィー（2×5cm、展開溶媒CH2Cl2/Et2O/n-ヘキサン＝10:1:7）においてRf値は0.44であった。
In order to further purify the antibacterial substance (Fr.III-1, 11.4 mg) obtained as described above, one piece of silica gel thin layer chromatography (20 × 20 cm, MERCK Silica gel 60 F 254 , 1.05715.0009) And fractionation with a developing solvent (n-hexane / ethyl acetate = 1: 1) to obtain 4.1 mg of Fr.III-1-ii.
Further, Fr.III-1-ii (3.1 mg) was fractionated by one silica gel thin layer chromatography (the same developing solvent) in the same manner as above to obtain 2.0 mg of Fr.III-1-ii-C. It was.
In addition, Fr.III-1-ii-C (1.0 mg) was subjected to one silica gel thin layer chromatography (developing solvent CH 2 Cl 2 / Et 2 O / n-hexane = 10: 1: 5) in the same manner as above. Fr. III-1-ii-Cb 0.5 mg was obtained by fractionation. Fr.III-1-ii-Cb had an Rf value of 0.44 in thin-layer chromatography for analysis (2 × 5 cm, developing solvent CH 2 Cl 2 / Et 2 O / n-hexane = 10: 1: 7). It was.
Claims (4)
（１）ニンニクの皮を用意する；
（２）ニンニクの皮を、所望により粉砕した後、酢酸エチルで抽出して、酢酸エチル抽出物を得る；
（３）酢酸エチル抽出物を、ｎ−ヘキサンと酢酸エチルとから成る展開溶媒を用いてフラッシュカラムクロマトグラフィー処理して、３番目に溶出する画分III （Fr.III ）を得る；
（４）画分III 中の成分をシリカゲル薄層クロマトグラフィーにかけ、展開溶媒として塩化メチレン／エチルエーテル／ｎ−ヘキサン（10：1：7）を用いるシリカゲル箔層クロマトグラフィーにおいて、およそ0.44のRf値を示す物質を採取する；
を含んで成る方法。 In the method for producing an antibacterial substance derived from garlic peel (protective leaf, the same applies hereinafter), the following steps:
(1) Prepare garlic skin;
(2) The garlic crust is ground if necessary and then extracted with ethyl acetate to obtain an ethyl acetate extract;
(3) The ethyl acetate extract is subjected to flash column chromatography using a developing solvent consisting of n-hexane and ethyl acetate to obtain the third eluting fraction III (Fr.III);
(4) Rf value of about 0.44 in silica gel thin layer chromatography using silica gel thin layer chromatography with methylene chloride / ethyl ether / n-hexane (10: 1: 7) as developing solvent. Collect substances that indicate
Comprising a method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100949148B1 (en) * | 2007-06-28 | 2010-03-25 | 이진화 | Adhesive using garlic and fabricating method the same |
| JP2012051834A (en) * | 2010-09-01 | 2012-03-15 | Yamaguchi Univ | Antibacterial activator containing saponin derived from allium fistulosum. l as active ingredient |
| KR101242580B1 (en) | 2011-03-21 | 2013-03-19 | 숙명여자대학교산학협력단 | Preparation method for ajoene-enriched garlic extract |
| KR20220106605A (en) * | 2021-01-22 | 2022-07-29 | 주식회사 상상구르메 | Method for preparing vegetable culture solution using garlic bark |
-
2004
- 2004-09-10 JP JP2004264415A patent/JP4437225B2/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100949148B1 (en) * | 2007-06-28 | 2010-03-25 | 이진화 | Adhesive using garlic and fabricating method the same |
| US8262792B2 (en) | 2007-06-28 | 2012-09-11 | Jin Hwa Lee | Natural adhesive using garlic and fabricating method of the same |
| JP2012051834A (en) * | 2010-09-01 | 2012-03-15 | Yamaguchi Univ | Antibacterial activator containing saponin derived from allium fistulosum. l as active ingredient |
| KR101242580B1 (en) | 2011-03-21 | 2013-03-19 | 숙명여자대학교산학협력단 | Preparation method for ajoene-enriched garlic extract |
| KR20220106605A (en) * | 2021-01-22 | 2022-07-29 | 주식회사 상상구르메 | Method for preparing vegetable culture solution using garlic bark |
| KR102504851B1 (en) | 2021-01-22 | 2023-03-03 | 주식회사 상상구르메 | Method for preparing vegetable culture solution using garlic bark |
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| JP4437225B2 (en) | 2010-03-24 |
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