KR100820247B1 - The insecticide composition comprising effective extract of pine gnarl for scuticociliates - Google Patents
The insecticide composition comprising effective extract of pine gnarl for scuticociliates Download PDFInfo
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- KR100820247B1 KR100820247B1 KR1020060119433A KR20060119433A KR100820247B1 KR 100820247 B1 KR100820247 B1 KR 100820247B1 KR 1020060119433 A KR1020060119433 A KR 1020060119433A KR 20060119433 A KR20060119433 A KR 20060119433A KR 100820247 B1 KR100820247 B1 KR 100820247B1
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Abstract
Description
도 1은 알파-피넨(α-pinene)의 구조식.1 is a structural formula of alpha -pinene (α-pinene).
도 2는 스쿠티카충 배양 후 현미경 사진(배율 400 배).Figure 2 is a micrograph (400-fold magnification) after Scutica cultivation.
도 3은 스쿠티카충에 알파-피넨을 10 mg/ℓ농도로 처리할 시, 세포질이 공동화되며 파괴되는(lysis) 모습을 나타내는 사진.Figure 3 is a photograph showing the appearance of the cytoplasm is cavitation (lysis) when treated with a 10 mg / ℓ of alpha-pinene in Scutika insects.
도 4는 용매 분획별 추출물 획득 순서도.Figure 4 is a flow chart obtained by extracting solvent fractions.
도 5는 2차 크로마토그래피과정을 나타내는 순서도5 is a flow chart showing a secondary chromatography process
본 발명은 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물에 관한 것이다.The present invention relates to a composition for insecticides Scutika insects containing the extract of Songjeol as an active ingredient.
양식어류에 기생하는 섬모충으로는 백점충(Ichthyophthirius multifillis), 크립토카련 이리탄스(Cryptocaryon irritans), 트리코디나(Trichodina), 킬로도넬라(Chilodonella cyprini), 엠비퓨라(Ambiphrya), 엠피오소마(Ampiosoma), 에피시티스(Epistylis), 칼케시움(Carchesium), 스쿠티카충(Scuticociliads) 등이 있다.Parasites of farmed fish include Ichthyophthirius multifillis, Cryptocaryon irritans, Trichodina, Chilodonella cyprini, Ambiphrya, Amphiphroma, and Epimpoma. Epistylis, Carchesium, and Scuticociliads.
그 중 스쿠티카충은 30 ~ 40 ㎛의 섬모충으로써, 흔히 넙치의 인공종묘 및 양성어에 많이 발생한다.Among them, Scutika is a ciliated insect of 30 to 40 μm, and frequently occurs in artificial seedlings and fish of the flounder.
스쿠티카충은 어류의 체표와 지느러미 그리고 신장, 혈관 및 뇌조직까지 다양하게 유발되고, 대부분 소성결합 조직의 파괴를 통하여 넙치를 대량 폐사시킨다고 보고되어 있다.Scutika insects have been reported to cause a variety of fish body surface, fins and kidneys, blood vessels and brain tissues, and most of them have been reported to kill flounder through destruction of plastic connective tissue.
또한, 다른 어류의 기생충과의 달리 혈관을 통하여 뇌조직까지 침입하여 기생하기 때문에 감염시 치료가 어려운 실정이다.In addition, unlike the parasites of other fish, it is difficult to treat during infection because the parasite is invaded through the blood vessels through the blood vessels.
스쿠티카충은 넙치의 먹이생물인 로티퍼에도 검출되고 있으며, 넙치의 치어에서 성어에 이르기까지 기생하고 있어서 조기에 발견하지 못하면 치료하는데 어려울뿐만 아니라 대량 폐사를 초래한다.Scotic worms are also detected in rotifers, the prey organisms for flounder, and parasitic from fry to sea urchins, which can be difficult to treat if not detected early, resulting in massive mortality.
이러한 스쿠티카충의 살충을 위해 넙치 양어장에서 최근까지 포르말린을 사용하였다.Formalin was used until recently in flounder fish farms for the insecticide of Scutika.
그러나, 포르말린의 독성 성분으로 인해 사용이 금지되었고 이를 대체하기 위해, 포르말린 외에 과산화수소, 안정화 염소 등이 체외 기생충 구제에 사용되고 있으나 효과를 거의 보지 못하고 있는 실정이다.However, due to the toxic component of formalin is prohibited, and to replace it, hydrogen peroxide, stabilized chlorine, etc., in addition to formalin, are used for in vitro parasite control, but have little effect.
따라서, 최근 이러한 문제를 해결하기 위한 방안으로 독성이 약한 성분들을 이용한 스쿠티카충 살충제에 관한 연구가 이루어지고 있다.Therefore, in recent years, as a way to solve this problem, a study on the insecticide Skutika insects using weak toxic components have been made.
한국등록특허공보 제10-0563273호(염산 터피나핀을 유효성분으로 함유하는 양식어류의 스쿠티카병 예방치료제)에는, 염산 터피나핀이 함유된 약제를 이용하여 양식어류의 스쿠티카병 예방 및 치료효과에 관한 것이 공개되어 있다.Korean Patent Publication No. 10-0563273 (Scutica disease preventive treatment of aquaculture fish containing terpena hydrochloride as an active ingredient) is used to prevent squatic disease of cultured fish by using a drug containing terpena hydrochloride. Therapeutic effects are publicly available.
한국등록특허공보 제10-0279281호(알긴산염 올리고당 유도체를 유효성분으로 하는 어류기생충 스쿠티카살충제)에는, 고분자인 알긴산염의 저분자화 및 알긴산올리고당의 제조와 어류 기생충 스쿠티카 살충제의 개발 및 전기 살충제의 제조방법에 관한 것이 공개되어 있다.Korean Registered Patent Publication No. 10-0279281 (Fish Parasite Scutica Insecticide with Alginate Oligosaccharide Derivatives) includes low molecular weight of alginate as a polymer, preparation of oligosaccharide alginate, development of fish parasite Scutica insecticide and electric insecticide. Disclosed is a manufacturing method.
한국등록특허공보 제10-0379026호(양식 넙치의 스쿠티카충 구제방법)에는, 케토코나졸을 사용하여 양식 넙치의 체내 및 외부에 감염된 스쿠티카충을 구제하는 방법에 관한 것이 공개되어 있다.Korean Patent Laid-Open No. 10-0379026 (Method for Controlling Scutika Insects of Cultured Flounder) discloses a method for controlling Scutica insects infected inside and outside of cultured flounder using ketoconazole.
그러나, 상기의 발명들은 살충제 제조방법이 어려워, 경제적인 면에서 적합하지 않으며, 스쿠티카충 구제방법으로 이용한 경구투여방법은 실질적으로 이용하기 어려운 방법이다. However, the above inventions are difficult to manufacture insecticides, and are not suitable in terms of economics, and the oral administration method used as a method for controlling Scutica insects is practically difficult to use.
또한, 살충제로 이용한 성분들의 독성으로 인해 어류패사 및 어독성이 발생하는 문제점이 있다.In addition, due to the toxicity of the components used as pesticides there is a problem that the fish pass and fish toxicity occurs.
한편, 본 발명은 본 발명의 출원인이 선출원하고 미공개 상태인 특허출원 제10-2005-0124329호(부틸알데하이드를 이용한 스쿠티카충 살충용 조성물)의 개량발명이다.On the other hand, the present invention is an improved invention of the patent application No. 10-2005-0124329 (Skutika insecticidal composition using butyl aldehyde) that is filed by the applicant of the present invention and is unpublished.
본 발명은 상기와 같은 문제점을 해소하기 위한 것으로, 종래에는 스쿠티카충 살충용으로 사용되지 않았던 송절추출물을 스쿠티카충 살충용 조성물로 적용하려는 목적이 있다.The present invention is to solve the above problems, the object of the present invention is to apply the extract extract that was not previously used for the insecticide insecticide Skutika insects as a composition for insecticide insecticides.
특히, 송절추출물을 스쿠티카충 살충용 조성물로 적용함으로써, 스쿠티카충 살충효과를 향상시키려는 목적도 있다.In particular, there is also an object to improve the insecticidal insecticidal effect by applying the extract of the extract in the insecticidal insecticidal composition.
또한, 간단한 약욕방법으로도 감염된 어류를 구제할 수 있는 스쿠티카충 살충용 조성물을 제공하려는 목적도 있다.In addition, there is also an object to provide a composition for insecticide insecticide Scutika that can control the infected fish by a simple bathing method.
본 발명은 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물에 관한 것이다.The present invention relates to a composition for insecticides Scutika insects containing the extract of Songjeol as an active ingredient.
본 발명의 살충용 조성물은, 송절을 분쇄하여 송절분쇄물을 제조한 후, 송절분쇄물에 메탄올을 송절분쇄물 1 kg 당 2 ~ 4 ℓ를 넣고, 20 ~ 25 ℃에서 2 회 추출하고 여과한 다음, 건조시켜 메탄올추출물을 제조한 후, 메탄올추출물에 물과 헥산을 넣고 교반한 다음, 분별깔대기로 물층을 분리한 후 제거하고, 남은 헥산층을 획득한 후, 이를 건조시켜 제조된 스쿠티카충 살충용 조성물이다.In the insecticidal composition of the present invention, after pulverizing the pine seedling to prepare a truncated powder, methanol is added to the truncated powder per 2 kg per kg of the truncated powder, and extracted twice and filtered at 20-25 ° C. Next, after drying to prepare a methanol extract, water and hexane was added to the methanol extract, the mixture was stirred, the water layer was separated with a separatory funnel, and then removed, and the remaining hexane layer was obtained. It is an insecticidal composition.
본 발명의 발명자들은 스쿠티카충을 대상으로 살충용 조성물을 개발하기 위하여, 약 360여 가지의 천연물을 이용하여 스쿠티카충에 대한 살충효과를 관찰하였다.The inventors of the present invention observed the insecticidal effect against Scutika insects using about 360 natural products in order to develop insecticidal compositions for Scutika insects.
그 결과, 살충효과가 우수하면서 어독성이 없으며, 경제적으로 적합한 재료 로 송절을 선별하여 본 발명의 살충용 조성물 제조에 이용하게 되었다.As a result, the insecticidal effect is excellent, there is no fish toxicity, and it is used to prepare the insecticidal composition of the present invention by selecting the songjeol as an economically suitable material.
송절은 소나무의 마디로서, 본초강목과 황실동의대보감에 "백절풍과 각비, 골절통을 낫게 하며 술을 빚어 먹으면 다리를 튼튼하게 한다"라고 기록되어 있다.Songjeol is a node of pine tree, and it is written in the bodhisattva of the main tree and the imperial palace, "It relieves white cramps, shells, and fracture pains, and makes liquor strong when you make alcohol."
송절에는 송진이 함유되어 있는데, 여기서 송진의 맛은 달고 쓰며 독이 없고 오장을 편하게 해주며 열을 없애고 풍미를 낫게 하며, 모든 약창, 두양, 백독을 치료하고 사기를 없애며 귀가 먹은 것과 충치를 치료하고 모든 창에 부착하면 통증과 살균을 시키는 효과가 있다고 본초강목과 황실동의대보감에 기록되어 있다.Songjeol contains rosin, where the taste of rosin is sweet and bitter, it has no poison, relaxes the five intestines, removes heat, improves flavor, heals all medicines, duyang and white poison, removes fraud, cures deafness and tooth decay, Attached to the window is said to have the effect of pain and sterilization, it is recorded in the herbal wood and imperial dongbom.
본 발명의 발명자들은 상기와 같은 효과를 지닌 송절을 이용하여 독성으로 인한 어류패사를 방지함과 동시에 스쿠티카충에 대한 높은 살충효과를 나타내는 방법을 찾기 위해 많은 연구를 해오던 결과, 송절을 메탄올로 추출한 후, 여러 유기용매를 이용하여 분획하였으며, 그 중 헥산을 이용하여 분획된 헥산분획층에서 독성이 없으면서, 스쿠티카충에 대한 살충효과가 높게 나타냄을 알 수 있었다.The inventors of the present invention have been doing a lot of research to find a method that exhibits a high insecticidal effect against Scutika insects at the same time to prevent fish death due to toxicity by using a songjeol with the above effects, the songjeol to methanol After extraction, it was fractionated using several organic solvents, and among them, it was found that the insecticidal effect against Scutika insects was high without being toxic in the hexane fractionation layer.
여기서 유기용매 자체에도 독성이 있으므로, 적정량을 이용해야되므로, 본 발명에서는 메탄올추출물과 증류수와 헥산을 1 : 20 ~ 50 : 20 ~ 50 부피비로 교반한 후, 분별깔대기로 분리하여 획득된 헥산분획층을 스쿠티카충 살충제로 이용할 시, 어류가 폐사되지 않으면서, 스쿠티카충에 대한 살충효과가 가장 높게 나타냄을 알 수 있었다.Since the organic solvent itself is toxic, since an appropriate amount must be used, in the present invention, the methanol extract, distilled water, and hexane are stirred at a volume ratio of 1:20 to 50:20 to 50, and then separated by a separatory funnel. When used as a insecticide insecticide, it was found that the highest insecticidal effect on the insect insects without killing the fish.
또한, 상기 살충용 조성물을 양식장해수 1 ton 당 0.2 ~ 1.0 kg을 사용할 시, 천적, 인축에 대한 무독성으로 환경보존 및 정화작용이 있는 환경배려형 위생 해충제로도 유용하게 사용될 수 있다.In addition, when using the insecticidal composition 0.2 ~ 1.0 kg per 1 ton aquaculture water, it can be usefully used as an environmentally-friendly sanitary pesticide with environmental preservation and purification as non-toxic to natural enemies, shrinkage.
본 발명의 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물의 제조방법에 대하여 자세히 설명하면 다음과 같다.Referring to the method for producing a composition for insecticide insecticide Scutika including the extract of the present invention as an active ingredient in detail.
<송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물의 제조방법><Method of producing Scutika insecticidal insect composition comprising pine extract as an active ingredient>
1. 제1단계 : 메탄올추출물 제조1. First Step: Methanol Extract Preparation
송절을 분쇄하여 송절분쇄물을 제조한 후, 송절분쇄물에 메탄올을 송절분쇄물 1 kg 당 2 ~ 4 ℓ를 넣고, 20 ~ 25 ℃에서 2 회 추출하고 여과한 다음, 건조시켜 메탄올추출물을 제조한다.After pulverizing the Songjeol to prepare the Songjeol crushed product, Methanol was added to 2 ~ 4 L per 1 kg of Songjeol crushed product, extracted twice at 20 ~ 25 ℃, filtered, and dried to prepare methanol extract. do.
2. 제2단계 : 스쿠티카충 살충용 조성물 제조2. Second Step: Preparation of Scutika insecticidal insect composition
메탄올추출물에 물과 헥산을 넣고 교반한 다음, 분별깔대기로 물층을 분리한 후 제거하고, 남은 헥산층을 획득한 후, 이를 건조시켜 스쿠티카충 살충용 조성물을 제조한다.Water and hexane were added to the methanol extract, followed by stirring. After separating the water layer with a separatory funnel, the remaining hexane layer was obtained and dried to prepare a composition for the insecticidal insecticide.
이때, 메탄올추출물과 증류수와 헥산을 1 : 20 ~ 50 : 20 ~ 50 부피비로 넣고 교반하는 것이 바람직하다.At this time, it is preferable to add the methanol extract, distilled water and hexane in a volume ratio of 1:20 to 50:20 to 50 and stirred.
또, 알파-피넨(α-pinene)성분(도 1)이 함유되는 것이 특징이다.In addition, it is characterized by containing an alpha-pinene component (FIG. 1).
이때, 알파-피넨 성분은 물에 불용성이기 때문에 알파피넨, 디메틸설폭사이드, 계면활성제를 20 ~ 50 : 50 ~ 80 : 1 부피비로 혼합된 혼합물을 사용할 수도 있다.In this case, since the alpha-pinene component is insoluble in water, a mixture of alpha pinene, dimethyl sulfoxide, and a surfactant in a volume ratio of 20 to 50:50 to 80: 1 may be used.
이하, 본 발명의 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물에 대하여 실시예와 실험예를 통해 상세히 설명하나, 이들이 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention is described in detail through Examples and Experimental Examples for the insecticidal insecticidal composition comprising the extract of Songjeol as an active ingredient, but they are not intended to limit the scope of the present invention.
<실시예 1> 본 발명의 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물 제조1<Example 1> Preparation of Scutika
송절을 시중에서 구입하여 준비하였다.Songjeol was commercially available and prepared.
준비된 송절을 분쇄기로 분쇄하여 송절분쇄물을 제조하였다.The prepared Songjeol was pulverized with a grinder to prepare Songjeol.
송절분쇄물 1.2 kg에 메탄올 3 ℓ를 넣고, 20 ℃에서 총 2 회 추출하고 여과한 다음, 건조기에서 건조시켜 메탄올추출물을 제조하였다.Methanol extract was prepared by adding 3 L of methanol to 1.2 kg of pulverized powder, extracting twice at 20 ° C., filtering, and drying in a drier.
메탄올추출물 20 g에 증류수 800 ㎖와 헥산 800 ㎖를 넣고 교반한 다음, 분별깔대기로 물층을 분리한 후 제거하고, 남은 헥산층을 획득한 후, 이를 건조시켜 스쿠티카충 살충용 조성물을 제조하였다.After distilled water 800 ml and hexane 800 ml were added to 20 g of methanol extract and stirred, the water layer was separated and removed with a separatory funnel, and the remaining hexane layer was obtained.
<실시예 2> 본 발명의 송절추출물을 유효성분으로 포함하는 스쿠티카충 살충용 조성물 제조2Example 2 Preparation of Scutika insecticidal composition 2 comprising the extract of Songjeol of the present invention as an active ingredient
송절을 시중에서 구입하여 준비하였다.Songjeol was commercially available and prepared.
준비된 송절을 분쇄기로 분쇄하여 송절분쇄물을 제조하였다.The prepared Songjeol was pulverized with a grinder to prepare Songjeol.
송절분쇄물 1.2 kg에 메탄올 3 ℓ를 넣고, 20 ℃에서 총 2 회 추출하고 여과한 다음, 건조기에서 건조시켜 메탄올추출물을 제조하였다.Methanol extract was prepared by adding 3 L of methanol to 1.2 kg of pulverized powder, extracting twice at 20 ° C., filtering, and drying in a drier.
메탄올추출물 20 g에 증류수 1000 ㎖와 헥산 1000 ㎖를 넣고 교반한 다음, 분별깔대기로 물층을 분리한 후 제거하고, 남은 헥산층을 획득한 후, 이를 건조시켜 스쿠티카충 살충용 조성물을 제조하였다.1000 g of distilled water and 1000 mL of hexane were added to 20 g of methanol extract, followed by stirring. After separating the water layer with a separatory funnel, the remaining hexane layer was obtained and dried to prepare a composition for insecticidal insecticides.
<실험예 1> 추출최적화 실험Experimental Example 1 Extraction Optimization Experiment
도 4에 제시되어 있는 추출순서도에 따라 추출물을 용매별로 분획을 실시하였다.The extract was fractionated for each solvent according to the extraction flow chart shown in FIG.
실시예 1에 제시된 바와 같이, 송절을 분쇄기로 분쇄하여 송절분쇄물을 제조한 후, 송절분쇄물 1.2 kg에 메탄올 3 ℓ를 넣고, 20 ℃에서 총 2 회 추출하고 여과한 다음, 건조기에서 건조시켜 메탄올추출물 20 g을 제조한 후, 증류수 800 ㎖와 헥산(n-Hexane) 800 ㎖에 녹여 2 ℓ분액여두에 넣은 후, 마개를 닫고 잘 혼합한 다음 물층과 헥산층으로 분리하였다.As shown in Example 1, the Songjeol was pulverized with a grinder to prepare Songjeol, and then 1.2 liters of Songgeol was added to 3 liters of methanol, extracted twice at 20 ° C., filtered, and then dried in a drier. After preparing 20 g of methanol extract, it was dissolved in 800 ml of distilled water and 800 ml of hexane (n-Hexane) and placed in a 2 L separation filter. The cap was closed and mixed well, and then separated into a water layer and a hexane layer.
30 분 후에 다른 분액여두를 이용하여 1차 헥산층을 분리시키고, 물층에 새로운 헥산 800 ㎖를 혼합하고 마개를 닫은 후 충분히 흔들어 물층과 헥산층을 2차로 분리하였다.After 30 minutes, the primary hexane layer was separated using another separatory filter, 800 ml of fresh hexane was mixed with the water layer, the stopper was closed, and the mixture was shaken sufficiently to separate the water layer and the hexane layer.
남아있는 물층에 클로로포름 800 ㎖를 혼합하고 마개를 닫은 후 충분히 흔들어 물층과 클로로포름층을 분리시키고 30 분 후에 1차 클로로포름층을 분리하였다.800 ml of chloroform was mixed with the remaining water layer, and the stopper was sufficiently shaken to separate the water layer and the chloroform layer, and after 30 minutes, the primary chloroform layer was separated.
다시 물층에 새로운 클로로포름 800 ㎖를 혼합하고 마개를 닫은 후 충분히 흔들어 물층과 클로로포름층을 2차로 분리하였다.Again, 800 ml of fresh chloroform was mixed into the water layer, and the stopper was closed. Then, the mixture was shaken sufficiently to separate the water layer and the chloroform layer secondarily.
남아있는 물층에 에틸아세테이트 800 ㎖를 부어 헥산 분배시와 동일한 방법 으로 에틸아세테이트층을 2 회에 걸쳐 분리하고, 남은 물층에 부탄올 800 ㎖를 부어 동일한 방법으로 부탄올층을 분리하였다.800 ml of ethyl acetate was poured into the remaining water layer, and the ethyl acetate layer was separated twice in the same manner as in the hexane distribution, and 800 ml of butanol was poured into the remaining water layer to separate the butanol layer.
위와 같은 용매 분획법으로 헥산층, 클로로포름층, 에틸아세테이트층, 부탄올층, 물층의 5 분획을 수득하였으며, 물층은 동결건조기로 농축하고, 헥산층, 클로로포름층, 에틸아세테이트층 및 부탄올층은 각각 회전진공농축기에서 농축시켰다.5 fractions of hexane layer, chloroform layer, ethyl acetate layer, butanol layer, and water layer were obtained by the solvent fractionation method as described above. The water layer was concentrated by lyophilizer, and the hexane layer, chloroform layer, ethyl acetate layer and butanol layer were rotated, respectively. Concentrated in a vacuum concentrator.
분획별 중량은 헥산 분획(6.7 g), 클로로포름분획(5.5 g), 에틸아세테이트 분획(1.3 g), 부탄올분획(4.0 g), 수용성분획(2.5 g)이였다.The weight of each fraction was hexane fraction (6.7 g), chloroform fraction (5.5 g), ethyl acetate fraction (1.3 g), butanol fraction (4.0 g) and water soluble fraction (2.5 g).
각 분획별 스쿠티카충에 대한 살충능력은 아래 표 1과 같이 나타났다.Insecticidal capacity for Scutika insects by each fraction is shown in Table 1 below.
<표 1> 분획별 스쿠티카충 살충능력Table 1 Insecticidal Insecticidal Capacity
상기 표 1에 나타나 있듯이, 본원 발명인 송절을 이용한 헥산 분획층에서 가장 높은 스쿠티카충 살충능력을 나타냄을 알 수 있었다.As shown in Table 1, it was found that exhibited the highest insecticidal insecticidal ability in the hexane fractionation layer using the present invention Songjeol.
<실험예 2> 물질분석Experimental Example 2 Material Analysis
1) 1차 활성 분획분리 실험1) First active fractionation experiment
물질 분석은 분획별로 분리한 후 스쿠티카 살충력을 측정한 후 활성 분획에 대해 HPLC와 G.C-MASS로 물질 분석을 실시하였다.Material analysis was carried out by fractionation and after measuring the Scutica insecticidal activity was analyzed by HPLC and G.C-MASS for the active fraction.
헥산분획 10g을 실리카겔 컬럼(berck 70-230 mesh, 600g, 5.5*70 cm)에 흡착시킨 후 헥산과 에틸아세테이트의 단계적 구배(stepwise gradient)로 1차 색층분석을 하였다.10 g of hexane fractions were adsorbed onto silica gel columns (berck 70-230 mesh, 600 g, 5.5 * 70 cm) and subjected to primary chromatographic analysis with a stepwise gradient of hexane and ethyl acetate.
사용한 단계적 구배는 헥산 : 에틸아세테이트 비율을 90:10, 85:15, 70:30, 50:50, 0:100 순으로 진행하였고, 각 비율에 따른 분획을 H1, H2, H3, H4, H5로 용리하였다.The gradient used was hexane: ethyl acetate ratio in the order of 90:10, 85:15, 70:30, 50:50, 0: 100, and the fraction according to each ratio was H1, H2, H3, H4, H5. Eluted.
5 개 분획에 대해 스쿠티카충 살충 test를 실시하여 H1이 활성을 가진 분획임을 알 수 있었다.Scutica insecticidal test was performed on the five fractions, indicating that H1 was an active fraction.
2) 2차 크로마토그라피 실험2) Second Chromatography Experiment
1차에서 분리한 활성 분획에 대한 실리카겔 크로마토그래피를 실시하였다(도 5).Silica gel chromatography was performed on the active fraction separated from the primary (FIG. 5).
H1분획은 고속액체크로마토그라피를 이용하여 더욱 정제하였다.The H1 fraction was further purified using high performance liquid chromatography.
H1은 유속 4.0 ㎖/min, 헥산-에틸아세테이트 (9:1), μ porasil silica(19*300mm, Waters)칼럼, 흡광도 260 nm로 실시하여 H21, H22, H23분획을 획득하였고, 도 5에서와 같은 정제 과정을 거쳐 물질 A(알파-피넨(α-pinene), 도 1)를 얻을 수 있었다. H1 was carried out at a flow rate of 4.0 ml / min, hexane-ethyl acetate (9: 1), μ porasil silica (19 * 300 mm, Waters) column, absorbance 260 nm to obtain H21, H22 and H23 fractions. Through the same purification process, substance A (alpha-pinene, FIG. 1) was obtained.
최종 물질은 GC-mass를 통해 물질을 확인하였다.The final material was identified through GC-mass.
<실험예 3> 스쿠티카충에 대한 효과조사Experimental Example 3 Investigation of the Effect on Scutika Insects
1. 실험재료 준비1. Preparation of experimental materials
본 발명인 실시예 1의 스쿠티카충 살충용 조성물을 수용액으로 제조하여 준비하였다(표 2).The present invention was prepared by preparing an insecticidal insecticidal composition of Example 1 in aqueous solution (Table 2).
실험예 2를 토대로 본 발명에 함유되어 있는 성분인 알파-피넨 성분을 아래와 같은 방법으로 제조하여 알파-피넨 수용액을 준비하였다(표 2).Based on Experimental Example 2, an alpha-pinene component, which is a component contained in the present invention, was prepared by the following method to prepare an aqueous alpha-pinene solution (Table 2).
<알파-피넨(α-pinene) 수용액의 제조><Preparation of alpha -pinene aqueous solution>
알파-피넨(α-pinene, <aldrich, USA, 98%>)은 소수성으로 물에 소량이 녹기 때문에 원액을 약욕에 사용하기는 곤란하다.Alpha-pinene (α-pinene, <aldrich, USA, 98%>) is hydrophobic and is dissolved in water in small amounts, making it difficult to use the stock solution for bathing.
따라서, 알파-피넨(α-pinene)이 잘 녹는 알콜, 디메틸설폭사이드(dimethylsulfoxide)에 녹여서 사용하여야 한다.Therefore, alpha-pinene should be used after dissolving in soluble alcohol, dimethylsulfoxide.
분산제로는 tween 80과 span 80을 사용하였다.Tween 80 and span 80 were used as the dispersant.
본 발명에서는 그 중 독성이 거의 없는 디메틸설폭사이드(dimethylsulfoxide)에 분산제로 tween 80(junsei, japan)+span 80((junsei, japan)중량부2.5:1)혼합액을 첨가하여 수용액을 제조하였다.In the present invention, an aqueous solution was prepared by adding a mixture of tween 80 (junsei, japan) + span 80 ((junsei, japan) by weight 2.5: 1) as a dispersant to dimethyl sulfoxide (dimethylsulfoxide) having little toxicity.
알파-피넨(α-pinene)중량부로 20 %와 디메틸설폭사이드(dimethylsulfoxide) 중량부 74%, tween 80+span 80 중량부 1 %를 혼합한 용액을 제조하고 이것을 실험에 사용하였다.A solution was prepared by mixing 20% by weight of alpha-pinene by weight, 74% by weight of dimethylsulfoxide, and 80% by weight of tween 80 + span.
비교 독성 실험을 위해 이젠 금지되었지만 기존에 스쿠티카충 구제제로 많이 사용되던 포름알데하이드 수용액(formaldehyde solution, 36.0 ~ 38.0 % ; JUNSEI, japan)도 준비하였다.Formaldehyde solution (36.0 ~ 38.0%; JUNSEI, Japan) was also prepared for the comparative toxicity experiments, which have been banned, but have been widely used as a control agent for Scutica.
<표 2> 각 재료별 수용액 제조시, 농도에 따른 조성비(100 ℓ의 해수처리기준)<Table 2> Composition ratio according to concentration in preparing aqueous solution for each material (100 ℓ of seawater treatment standard)
2. 실험방법2. Experimental method
1) 스쿠티카충 배양1) Scutika insect culture
스쿠티카섬모충에 감염된 넙치 병어에서 직접 분리하여 배양하였다.The isolates were cultured directly from flounder diseased infected Scutica ciliated caterpillars.
넙치 병어로부터 감염부위의 조직을 일부 절제하여 슬라이드 글라스에 올려놓고 현미경 관찰을 한 결과, 스쿠티카 충에 감염되어 있음을 확인하였다.The tissues of the infected area were partially removed from the flounder diseased fish, placed on a slide glass, and examined under a microscope. As a result, it was confirmed that Scutica was infected.
확인된 스쿠티카 충체를 멸균 해수에 P1Y1(1% proteose peptone, 1% yeast extract) 배지에 충체를 접종하여 15 ℃에서 1차 배양을 수행하였다.The identified Scutica locus was inoculated with P1Y1 (1% proteose peptone, 1% yeast extract) medium in sterile seawater to perform primary culture at 15 ° C.
1차 배양된 스쿠티카 배양액에는 많은 박테리아가 존재하고 있어 박테리아의 오염과 증식수의 한계로 어류 주화세포주를 이용한 순수배양을 수행하였다.In the primary culture of Scutica culture, many bacteria were present. Therefore, pure cultures using fish coin cell lines were performed due to the contamination of bacteria and the limitation of the number of growth.
스쿠티카 순수배양을 위하여 어류주화세포 CHSE-214 (Chinook salmon embryo)를 7.5 % sodium bicarbonate로 pH 7.3로 적정된 MEM(Modified Eagle's Medium)배지에 10 % FBS와 penicillin-streptomycin을 첨가시켜 20 ℃에서 3 일간 배양하였다.For pure cultivation, 10% FBS and penicillin-streptomycin were added to MEM (Modified Eagle's Medium) medium titrated with CHSE-214 (Chinook salmon embryo) with 7.5% sodium bicarbonate at pH 7.3 for pure culturing. Incubated daily.
상기 배양된 어류주화 세포주에 1차 배양된 스쿠티카충배양액을 접종한 후 20 ℃에서 5 일동안 순수배양하였다.The cultured fish-coated cell line was inoculated with the first cultured Scutica insect culture solution, and then purely cultured at 20 ° C. for 5 days.
2) 스쿠티카충 살충율 조사2) Insecticide Insect Investigation
본 실험을 위해 스쿠티카충은 대수성장기 말기나 정지기 초기에 상태에 있는 스쿠티카충을 사용하여 650×g에서 5 분간 원심분리한 다음 PBS 또는 필터된 해수에 재부양시켰다.For this experiment, Scutica was centrifuged at 650 × g for 5 minutes using Scutica at the end of the logarithmic phase or at the beginning of the phase of suspension and resuspended in PBS or filtered seawater.
헤마토크리트(haematocrit)로 카운팅 한 후 0.1×104 ㎖의 스쿠티카충을 함유하는 액에 표 2의 조성물인 본 발명인 실시예 1의 스쿠티카충 살충용 조성물 수용액, 알파-피넨 수용액, 포름알데하이드 수용액을 0.9 ㎖씩 각각 24 wellplate에 넣은 후 혼합하고 20 ℃ incubator에서 shaking 시켰으며, 1 시간이 지난 후 현미경으로 관찰하였다.A pinene aqueous formaldehyde solution - hematocrit after counting to (haematocrit) 0.1 × 10 4 ㎖ of Surgical urticae The composition of Table 2, the liquid present inventors carried containing charge Example 1 Surgical urticae charge insecticidal composition aqueous solution of alpha 0.9 ml of each was added to 24 wellplates, mixed and shaken at 20 ° C. incubator, and observed after 1 hour.
본 발명인 실시예 1의 스쿠티카충 살충용 조성물 수용액, 알파-피넨 수용액, 포름알데하이드 수용액은 100 ℓ를 기준으로 만들어졌기에 1 ℓ에 맞게 1/100으로 사용량을 줄인 후 1 ℓ의 필터된 해수에 분산 시켰다.The aqueous solution for the insecticidal composition of the present invention Example 1, aqueous alpha-pinene solution, and aqueous formaldehyde solution were made on the basis of 100 L, and the amount was reduced to 1/100 to 1 L, and then to 1 L of filtered seawater. Dispersed.
최종 24 wellplate에서의 농도가 해당 농도가 되도록 조정하여 사용하였다. The concentration in the final 24 wellplate was used to adjust the concentration.
교반 후, 1 시간과 2 시간에 스쿠티카충체수를 측정하였다.After stirring, the number of Scutica insects was measured for 1 hour and 2 hours.
측정시, 배양액을 슬라이드 글라스(slide glass)위에 떨어뜨린 후 현미경하에서 측정하여 lysis된 개체를 제외한 정상적인 개체의 수를 측정하였다.In the measurement, the culture solution was dropped on a slide glass, and then measured under a microscope to determine the number of normal individuals except the lysed individuals.
정확한 개체의 수는 헤마토크리트(haematocrit)로 카운팅하였다.The exact number of individuals was counted with hematocrit.
3. 실험결과3. Experimental Results
그 결과, 아래의 표 3에 나타내었다.As a result, it is shown in Table 3 below.
<표 3> 본 발명의 살충용 조성물을 이용한 스쿠티카충 살충율<Table 3> Insecticidal insecticidal rate using the insecticidal composition of the present invention
상기 표 3에 나타나 있듯이, 본 발명인 실시예 1의 수용액과 이를 통해 분리된 알파-피넨 성분의 수용액에서는 저농도(50, 75, 100 ppm)에서도 스쿠티카충에 대해 높은 살충효과를 나타냄을 알 수 있었으며, 대조구로 사용한 포름알데하이드 수용액은 저농도(50, 75, 100 ppm)로 처리할 시에는 살충효과를 나타내지 않고 스쿠티카충의 공포화가 진행되었으며, 150 ppm 이상으로 처리할 시 살충효과를 나타냄을 알 수 있었다.As shown in Table 3, it was found that the aqueous solution of Example 1 and the alpha-pinene component separated therefrom exhibited a high insecticidal effect against Scutica at low concentrations (50, 75, 100 ppm). , Formaldehyde aqueous solution used as a control did not show a pesticidal effect when treated at low concentrations (50, 75, 100 ppm), and the fear of Scutica insects progressed, and when treated at 150 ppm or more, it showed an insecticidal effect. .
<실험예 4> 넙치 치어에 대한 독성조사Experimental Example 4 Investigation of Toxicity of Flounder
1. 실험재료준비1. Preparation of experimental materials
상기 실험예 3에 준비된 본 발명인 실시예 1의 스쿠티카충 살충용 조성물 수용액, 알파-피넨 수용액, 포름알데하이드 수용액을 준비하였다.The aqueous solution of the composition for insecticide insecticide Scutica of Example 1 prepared in Experimental Example 3, an aqueous solution of alpha-pinene, and an aqueous solution of formaldehyde were prepared.
실험어로로는 넙치 치어(5 cm)를 각 수조당 100 마리씩 준비하였다.As an experimental fish, flounder fry (5 cm) were prepared for each 100 tanks.
2. 실험방법2. Experimental method
각각 준비된 5 개의 원형 수조에 100 ℓ의 해수를 채운 후 각 수조에 준비한 넙치 치어를 넣은 후, 상기 준비된 본 발명인 실시예 1의 스쿠티카충 살충용 조성물 수용액, 알파-피넨 수용액, 포름알데하이드 수용액을 각각 500 ㎖씩 넣어 넙치 치어에 대한 독성을 확인하였다.After filling 100 l of seawater in each of the five prepared round tanks, the flounder fry prepared in each tank was prepared, and the aqueous solution of the insecticidal composition of Scutica insect insecticide of Example 1, the aqueous alpha-pinene solution and the aqueous formaldehyde solution of Example 1 were prepared. 500 ml each was checked for toxicity to flounder fry.
약욕 1 시간, 2 시간 후 독성을 확인하였다Toxicity was confirmed after 1 and 2 hours of bathing.
3. 실험결과3. Experimental Results
그 결과, 아래의 표 4에 나타내었다.As a result, it is shown in Table 4 below.
<표 4> 안전성 실험Table 4 Safety Experiments
즉, 통상적으로 쓰이는 포름알데하이드 수용액은 적은 농도에서도 그 독성이 강하여 실험어류의 일부 개체 형태가 변화됨을 볼 수 있었으며, 1 시간 뒤에는 전 개체가 사망함을 볼 수 있었다.That is, the commonly used aqueous formaldehyde solution was found to have a strong toxicity even at low concentrations, so that some individual fish species were changed, and one hour later, all individuals died.
그러나, 본 발명인 실시예 1의 수용액과 본 발명에서 분리된 알파-피넨 물질을 이용시에는 어류에 대해 독성이 나타나지 않음을 알 수 있었다.However, when using the aqueous solution of Example 1 of the present invention and the alpha-pinene material separated from the present invention it can be seen that no toxicity to fish.
특히 본 발명에서 분리된 알파-피넨 물질의 경우는 200 ppm까지 안전하였다. In particular, the alpha-pinene material isolated in the present invention was safe to 200 ppm.
<실험예 5> 넙치 치어에 남아 있는 잔류량 테스트 실험<Experiment 5> Residual amount test experiment remaining on flounder fry
1. 실험재료1. Experimental Materials
실험예 4에서 사용된 실험어를 대상으로 잔류량 테스트를 실시하였다.Residual amount tests were performed on the experimental words used in Experimental Example 4.
알파-피넨 수용액 200 ppm 과 포름알데하이드 수용액 200 ppm으로 2 시간 약욕한 후, 넙치 근육내의 잔류량을 측정하였다.After bathing with 200 ppm of alpha-pinene aqueous solution and 200 ppm of formaldehyde aqueous solution for 2 hours, the residual amount in the flounder muscle was measured.
2. 실험방법2. Experimental method
1) 포름알데하이드 잔류량 측정1) Formaldehyde residual amount measurement
잘게 다진 근육 10 g을 원심분리용 튜브에 넣고 10 ㎖ 0.5 N NaOH를 첨가하여 호모게나이저로 잘 균질화 시킨 후, 10 ㎖ 12 % ZnSO4·7H2O를 첨가하여 다시 호모게나이저로 균질화 한 후, 30 분동안 실온에서 정치하였다.10 g finely chopped muscle was placed in a centrifuge tube and homogenized well by adding 10 ml 0.5 N NaOH to homogenizer, and then homogenized again by adding 10 ml 12% ZnSO 4 · 7H 2 O. The mixture was allowed to stand at room temperature for 30 minutes.
800 rpm에서 5 분간 원심분리한 후 상층액을 와트만 NO1(whatman NO1)여과지로 여과하였다.After centrifugation at 800 rpm for 5 minutes, the supernatant was filtered with Whatman NO1 filter paper.
잔사물은 10 ㎖의 증류수를 가하여 볼텍스 믹서기(vortex mixer)로 혼합하여 세척한 다음 8,000 rpm에서 5 분간 원심분리하였다.The residue was mixed with a vortex mixer, washed with 10 ml of distilled water, and then centrifuged at 8,000 rpm for 5 minutes.
세척액을 여과액으로 모으고 이 과정을 2번 반복하였다.The washes were combined with filtrate and this process was repeated twice.
전부 모아진 추출액을 최종 증류수 100 ㎖로 조정하는데 이때 가장 회수율이 좋은 pH 4.5의 상태를 위해 0.5 N HCl을 첨가하였다.The combined extracts were adjusted to 100 mL of final distilled water with 0.5 N HCl added for the best recovery pH 4.5.
최종추출액 2 ㎖를 시험관에 취해 포름알데히드 측정법(AHMT method)에 따라서 포름알데히드 농도를 구하였다.2 ml of the final extract was taken in a test tube to determine the formaldehyde concentration according to the formaldehyde measurement method (AHMT method).
이때, 검출 한계는 0.05 μg/㎖이다.At this time, the detection limit is 0.05 μg / ml.
2) 알파-피넨 정량분석2) Alpha-pinene quantitative analysis
잘게 다진 근육 10 g을 원심분리용 튜브에 넣고 15 ㎖의 에틸 아세테이트(Ethyl acetate)를 첨가하여 호모게나이저로 잘 균질화 시킨 다음, 0.5 N NaOH를 첨가하여 다시 호모게나이저로 잘 균질화 시킨 후 30 분간 실온에서 정치하였다.10 g of finely chopped muscle was placed in a centrifuge tube, homogenized well with a homogenizer by adding 15 ml of ethyl acetate, and then homogenized again with a homogenizer by adding 0.5 N NaOH, followed by 30 minutes. It was left at room temperature.
800 rpm에서 5 분간 원심분리한 후 상층액을 와트만 NO1(whatman NO1)여과지로 여과하였다.After centrifugation at 800 rpm for 5 minutes, the supernatant was filtered with Whatman NO1 filter paper.
여과액은 50 ㎖ 메스플라스크에 넣고, 잔사물은 15 ㎖의 에틸아세테이트(Ethyl acetate)를 가하여 볼텍스 믹서기(vortex mixer)로 혼합하여 세척한 다음 8,000 rpm에서 5 분간 원심분리하였다.The filtrate was placed in a 50 ml volumetric flask, and the residue was mixed with a vortex mixer by adding 15 ml of ethyl acetate (Ethyl acetate), and then centrifuged at 8,000 rpm for 5 minutes.
800 rpm에서 5 분간 원심분리한 후 상층액을 와트만 NO1(whatman NO1)여과지로 여과하였다.After centrifugation at 800 rpm for 5 minutes, the supernatant was filtered with Whatman NO1 filter paper.
세척액을 여과액으로 모으고 이 과정을 2번 반복하였다.The washes were combined with filtrate and this process was repeated twice.
모아진 여과액은 50 ㎖ 메스플라스크에 넣은 후, 에틸아세테이트(ethyl acetate)를 가하여 총 50 ㎖로 만들었다. The collected filtrate was placed in a 50 ml volumetric flask, and ethyl acetate was added to make 50 ml in total.
3) 분석3) analysis
GC는 HP사 GC 5890 model을 이용하였으며 칼럼(column)은 HP-5MS capillary column을 사용하였으며 헬륨가스를 1.8 ㎖/min에 속도를 흘려주었다.GC used HP GC 5890 model, and the column (column) was used HP-5MS capillary column and helium gas was flowed at 1.8 ㎖ / min.
Injector 온도는 220 ℃, detector 온도는 320 ℃, oven 온도는 37 ℃에서 5분 유지 후 180 ℃까지 5 ℃/min의 속도로 온도를 상승 시킨 후 300 ℃까지는 20 ℃/min의 속도로 상승시켰다.The injector temperature was maintained at 220 ℃, the detector temperature was 320 ℃, the oven temperature was maintained at 37 ℃ for 5 minutes, and then the temperature was increased at a rate of 5 ℃ / min to 180 ℃ and then at a rate of 20 ℃ / min up to 300 ℃.
detector로는 mass를 이용하였다.Mass was used as a detector.
3. 실험결과3. Experimental Results
상기 실험결과 , 아래의 표 5와 표 6에 나타냈다.The experimental results, shown in Table 5 and Table 6 below.
<표 5> 2 시간 약욕 후 넙치 치어에 남아 있는 앞파-피넨 및 포르말린 잔류량 측정Table 5 Measurement of the remaining amount of eruption-pinene and formalin remaining in halibut fry after 2-hour bathing
<표 6> 알파-피넨과 포름알데하이드의 물질안전보건자료상의 안전성 Table 6 Safety of material safety data sheets for alpha-pinene and formaldehyde
상기 실험결과, 표 5, 6에 나타나 있듯이 본 발명의 송절 추출물에 함유되어 있는 성분인 알파-피넨은 시간이 지날수록 넙치 치어에 남아 있는 잔류량이 줄어들면서 48 시간째는 모두 소멸됨을 알 수 있었다.As a result of the experiment, as shown in Tables 5 and 6, alpha-pinene, a component contained in the Songjeol extract of the present invention, was found to disappear after 48 hours as the residual amount of the flounder fry decreased.
그러나, 포름알데하이드는 시간이 지날수록 그 잔류량 계속 남아 있음을알 수 있었으며, 이러한 포름알데하이드는 환경에도 악영향을 미침을 알 수 있었다.However, it was found that formaldehyde remained its residual amount over time, and such formaldehyde had an adverse effect on the environment.
<실험예 6> 양식장에서 질병 발생 상황에서의 질병 치료 예 조사Experimental Example 6 Investigation of Disease Treatment in Disease Occurrence Conditions
넙치 양어장에서 스쿠티카충의 감염이 발생되어 폐사가 진행되고 있는 양어장에 본 발명에서 분리된 알파-피넨의 수용액을 처리하였다.An aqueous solution of alpha-pinene isolated from the present invention was treated to a fish farm in which Scutika insects were infected in the flounder fish farm, where death occurred.
선별을 통해 감염어만 모아진 병어용 10 평 수조에 해수를 30 cm높이로 채웠다.Through screening, seawater was filled to a height of 30 cm in a 10 pyeong tank for bottled fish collected only from infected fish.
샘플링을 통해 포피와 아가미 쪽에 스쿠티카 충의 감염을 확인하였다.Sampling confirmed infection of Scutica on the foreskin and gills.
그 후 수조에 물량은 9.9 ton에 달하였고 여기에 α-pinene 수용액을 골고루 수조에 뿌려 주었다.After that, the water volume reached 9.9 ton, and the α-pinene aqueous solution was evenly sprayed on the tank.
α-pinene의 농도로는 50 mg/ℓ였다.The concentration of α-pinene was 50 mg / l.
처리한 후 2 시간 후 환수를 시키고 5 마리를 샘플링하여 표피 부분과 아가미 부분을 현미경으로 확인하니 스쿠티카 충이 표피 부분에서는 제거가 되었으며 아가미 부분에도 스쿠티카 충이 완전히 제거되었다.After 2 hours after treatment, the cells were collected and sampled with 5 mice, and the epidermis and gills were examined under a microscope. Scoutica was removed from the epidermis and Scutica was completely removed from the gills.
그 후 일주일간 관찰한 결과 더 이상의 폐사는 발생하지 않았고 넙치를 정상적으로 사육할 수 있었다.After a week of observation, no further mortality occurred and the flounder could be raised normally.
본 발명에 의해, 종래에는 스쿠티카충 살충용으로 사용되지 않았던 송절추출 물을 스쿠티카충 살충용 조성물로 적용함으로써, 스쿠티카충 살충효과가 향상된 살충용 조성물이 제공된다.According to the present invention, the insecticidal composition with improved insecticidal insecticidal effect is provided by applying the extract of S. aeruginosa insecticide, which has not been conventionally used for the insecticidal insecticidal insecticide, as the composition for the insecticidal insecticidal insecticide.
또한, 간단한 약욕방법으로도 감염된 어류를 구제할 수 있는 스쿠티카충 살충용 조성물이 제공된다.In addition, there is provided a composition for insecticide insecticide Skutika which can control the infected fish by a simple bathing method.
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KR20190088804A (en) | 2018-01-19 | 2019-07-29 | (주)창조바이오텍 | Composition comprising fermented extract of pine gnarl for preventing or treating scuticociliatosis |
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