KR20210075053A - A novel antifungal composition comprising aminopyrrolnitrin as an effective substance - Google Patents
A novel antifungal composition comprising aminopyrrolnitrin as an effective substance Download PDFInfo
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Abstract
Description
본 발명은 신규 항진균 조성물에 관한 것으로서, 더 상세하게는 아미노피롤니트론을 유효성분으로 함유하는 신규 항진균 조성물에 관한 것이다. The present invention relates to a novel antifungal composition, and more particularly, to a novel antifungal composition containing aminopyrrolenitrone as an active ingredient.
하기 화학식 I의 구조를 가지고 있는 화합물 피롤니트린(pyrrolnitrin)은 Burkholderia pyrrocinia와 Pseudomonas fluorescens 등에서 분리된 물질로 다양한 진균과 세균에 대한 폭넓은 항균활성을 나타내는 것으로 보고된 바 있다(Z. A. Siddiqui(ed.), PGPR: Biocontrol and Biofertilization, 67-109, 2005). 피롤니트린은 일부 Serratia 속 세균에서 보고된 바 있으나(Kalbe et al., Microbiol. Res., 151: 433-439, 1996), Serratia grimesii에서 보고된 바는 없다. Pseudomonas fluorescens Pf-5에서 피롤니트린 합성 대사경로를 분석한 결과, 유전체 상에 prn 클러스터(prnA, prnB, prnC, prnD, prnE, prnF, prnS 및 prnR)가 관여하는 것으로 밝혀졌다(Lee and Zhao, J. Bacteriol., 189(23): 8556-8563, 2007).The compound pyrrolnitrin having the structure of the following formula (I) is a material isolated from Burkholderia pyrrocinia and Pseudomonas fluorescens, etc. It has been reported to exhibit a wide range of antibacterial activity against various fungi and bacteria (ZA Siddiqui (ed.) , PGPR: Biocontrol and Biofertilization, 67-109, 2005). Pyrrolnitrine has been reported in some bacteria of the genus Serratia (Kalbe et al ., Microbiol. Res. , 151: 433-439, 1996), but has not been reported in Serratia grimesii. As a result of analyzing the pyrronitrine synthesis metabolic pathway in Pseudomonas fluorescens Pf-5, it was found that prn clusters ( prnA , prnB , prnC , prnD , prnE , prnF , prnS and prnR ) are involved in the genome (Lee and Zhao, J. Bacteriol ., 189(23): 8556-8563, 2007).
항진균 활성을 갖는 매우 다양한 피롤니트린 유사체가 보고된 바 있으나(van Pee and Ligon, Nat. Prod. Rep., 17: 157-164, 2000), 이들의 항세균 및 항진균 스펙트럼은 일관된 경향이 없기 때문에, 특정 세균 및 진균에 대한 항균활성에 대하여 예측하기 어렵다. A wide variety of pyrronitrine analogs with antifungal activity have been reported (van Pee and Ligon, Nat. Prod. Rep ., 17: 157-164, 2000), but their antibacterial and antifungal spectra do not tend to be consistent. , it is difficult to predict the antibacterial activity against specific bacteria and fungi.
상기 피롤니트린 유사체 중 하기 화학식 II의 구조를 갖는 아미노피롤니트린은 피롤니트린의 전구체로서 최초 출발물질인 트립토판으로부터 트립토판 할로게네이즈(PrnA), 피롤니트린 생합성 효소(PrnB) 및 할로게네이즈(PrnB)에 의해 생성되나, 곧바로 아미노피롤니트린 산화효소(PrnD)에 의해 아미노피롤니트린의 아미노기가 니트로기로 산화되면서, 피롤니트린이 생성이 된다. 이때 PrnD의 조효소로 FADH2가 사용되어 FAD로 산화되는데, 산화된 FAD는 플라빈 환원효소(PrnF)의 작용으로 다시 FADH2로 환원이 되어, 아미노피롤니트린의 피롤니트린으로의 산화 반응이 지속적으로 일어나게 된다.Among the pyrronitrine analogs, aminopyrrolenitrine having the structure of the following formula (II) is a precursor of pyrronitrine, and is a tryptophan halogenase (PrnA), a pyrronitrine biosynthetic enzyme (PrnB) and a halogenase from tryptophan, the initial starting material. (PrnB), but as the amino group of aminopyrronitrine is oxidized to a nitro group by aminopyrrolenitrine oxidase (PrnD), pyrrolenitrine is produced. At this time, FADH 2 is used as a coenzyme of PrnD and oxidized to FAD. The oxidized FAD is reduced back to FADH 2 by the action of flavin reductase (PrnF). will happen continuously.
아미노피롤니트린은 이와 같이 중간물질로서 세포 내에 대량으로 존재하지 않기 때문에, 그 기능에 대하여는 거의 알려진 바 없는 물질이다.Since aminopyrrolenitrine does not exist in a large amount in cells as an intermediate as described above, little is known about its function.
그러나 피롤니트린의 경우, 현재 백선균에 대한 외용제로만 사용되고 있을 뿐 식물 병원성 진균 방제 용도로는 사용되지 않고 있는 문제점이 있다.However, in the case of pyrrolenitrine, there is a problem that it is currently used only as an external preparation for ringworm, but is not used for controlling plant pathogenic fungi.
본 발명은 상기와 같은 문제점을 포함하여 여러 문제점들을 해결하기 위한 것으로서, 항균활성이 더 우수하고 더 폭넓은 항진균 스펙트럼을 가질 뿐만 아니라, 원생동물인 스쿠치카충에 대한 방제활성이 우수하여 수산분야에서 활용이 가능한 아미노피롤니트린을 유효성분으로 포함하는 신규 항진균 조성물을 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.The present invention is intended to solve various problems including the above problems, and has excellent antibacterial activity and a broader antifungal spectrum, as well as excellent control activity against the protozoan Scotchka spp. An object of the present invention is to provide a novel antifungal composition comprising available aminopyrrolenitrine as an active ingredient. However, these problems are exemplary, and the scope of the present invention is not limited thereto.
본 발명의 일 관점에 따르면, 하기 화학식 II의 구조를 갖는 아미노피롤니트린을 유효성분으로 함유하는, 항진균 조성물이 제공된다:According to one aspect of the present invention, there is provided an antifungal composition containing aminopyrrolenitrine having the structure of the following formula (II) as an active ingredient:
본 발명의 일 관점에 따르면, 하기 화학식 II의 구조를 갖는 아미노피롤니트릴을 식물 개체에 처리하는 단계를 포함하는 식물 병원성 진균 방제방법이 제공된다:According to one aspect of the present invention, there is provided a method for controlling plant pathogenic fungi comprising the step of treating a plant subject with aminopyrrolenitrile having the structure of the following formula (II):
상기한 바와 같이 이루어진 본 발명의 아미노피롤니트린을 유효성분으로 포함하는 신규 항진균 조성물은 다양한 병원성 진균에 대한 방제효과는 물론 다양한 식물 병원성 진균, 특히 딸기 잿빛곰팡이병을 유발하는 Botrytis sp.의 방제와 어류의 감염성 원생생물인 스쿠치카충에 대한 매우 효율적인 방제효과를 구현할 수 있다. 물론 이러한 효과에 의해 본 발명의 범위가 한정되는 것은 아니다.The novel antifungal composition comprising the aminopyrrolenitrine of the present invention as an active ingredient, as described above, has a control effect on various pathogenic fungi as well as various plant pathogenic fungi, particularly Botrytis sp., which causes strawberry gray mold disease. It is possible to implement a very effective control effect against Scotchka, an infectious protozoa of fish. Of course, the scope of the present invention is not limited by these effects.
도 1은 미생물 유전체 정보 기반 피롤니트린 및 아미노피롤니트린의 생합성 경로에 대한 분석 과정을 개략적으로 도시한 개요도이다.
도 2는 본 발명의 일 실시예에 따른 아미노피롤니트린의 생산에 이용된 Seratia grimessi MRS-1 균주의 평판배지에서의 배양 양상을 촬영한 사진(A) 및 16S rDNA 염기서열 분석에 따른 계통학적 트리(B)를 나타낸다.
도 3은 본 발명의 일 실시예에 따라 분리된 배양된 Seratia grimessi MRS-1 균주를 트립토판 포함 배지에서 배양시 시간의 경과에 따라 트립토판의 소모 및 아미노피롤니트릴이 생성되었음을 보여주는 액체 크로마토그래피(LC) 분석결과를 나타내는 크로마토그램이다.
도 4는 유기합성법으로 합성된 아미노피롤니트린의 구조를 확인하기 위한 NMR 분석결과(A) 및 LC-MS 분석결과(B)를 나타낸다.
도 5는 본 발명의 일 실시예에 따라 합성된 아미노피롤니트린의 다양한 진균에 대한 항균활성을 디스크 확산 실험을 통해 확인한 결과를 나타내는 일련의 사진이다.
도 6은 본 발명의 일 실시예에 따라 합성된 아미노피롤니트린의 다양한 원예작물 병원성 진균에 대한 항균활성을 분석한 결과를 나타내는 일련의 사진이다.
도 7은 본 발명의 일 실시예에 따라 합성된 아미노피롤니트린의 딸기 잿빛곰팡이병 방제효과를 확인한 결과를 나타내는 사진이다.
도 8은 본 발명의 일 실시예에 따라 합성된 아미노피롤니트린의 항스쿠치카충 활성을 분석한 결과로, 다양한 농도의 아미노피롤니트린 존재하에 항스쿠치카충을 배양시 광학현미경으로 촬영한 사진(A) 및 다양한 농도의 아미노피롤니트린 처리시 스쿠치카충의 생존도를 기록한 그래프(B)이다.1 is a schematic diagram schematically illustrating an analysis process for the biosynthetic pathway of pyrrolenitrine and aminopyrrolenitrine based on microbial genome information.
2 is a photograph (A) of the culture pattern of the Seratia grimessi MRS-1 strain used for the production of aminopyrrolenitrine according to an embodiment of the present invention in a plate medium and a phylogenetic according to 16S rDNA sequencing analysis. A tree (B) is shown.
3 is a liquid chromatography (LC) showing consumption of tryptophan and the generation of aminopyrrolenitrile over time when the cultured Seratia grimessi MRS-1 strain isolated according to an embodiment of the present invention is cultured in a tryptophan-containing medium. It is a chromatogram showing the analysis result.
4 shows the results of NMR analysis (A) and LC-MS analysis (B) for confirming the structure of aminopyrrolenitrine synthesized by organic synthesis.
5 is a series of photographs showing the results of confirming the antimicrobial activity of aminopyrrolenitrine synthesized according to an embodiment of the present invention against various fungi through a disk diffusion experiment.
6 is a series of photographs showing the results of analyzing the antimicrobial activity of aminopyrrolenitrine synthesized according to an embodiment of the present invention against pathogenic fungi of various horticultural crops.
7 is a photograph showing the results of confirming the strawberry gray mold control effect of aminopyrrolenitrine synthesized according to an embodiment of the present invention.
Figure 8 is the result of analyzing the anti-Scotchika activity of aminopyrrolenitrine synthesized according to an embodiment of the present invention, taken with an optical microscope when culturing anti-Scotchika parasite in the presence of various concentrations of aminopyrrolenitrine; It is a photograph (A) and a graph (B) recording the viability of Scotchka worms when treated with aminopyrrolenitrine at various concentrations.
본 발명의 일 관점에 따르면, 하기 화학식 II의 구조를 갖는 아미노피롤니트린을 유효성분으로 함유하는, 항진균 조성물이 제공된다:According to one aspect of the present invention, there is provided an antifungal composition containing aminopyrrolenitrine having the structure of the following formula (II) as an active ingredient:
상기 조성물은 식물 병원성 진균에 대해 항진균 활성을 가질 수 있고 상기 식물 병원성 진균은 양파 잿빛곰팡이병 병원균, 복숭아 잿빛무늬병 병원균, 장미 잿빛곰팡병 병원균, 딸기 잿빛곰팡이병 병원균 또는 토마토 분리병 병원균일 수 있다. The composition may have antifungal activity against phytopathogenic fungi and the phytopathogenic fungi may be onion gray mold pathogens, peach gray mold pathogens, rose gray mold pathogens, strawberry gray mold pathogens or tomato isolate pathogens. .
본 발명의 다른 일 관점에 따르면, 하기 화학식 II의 구조를 갖는 아미노피롤니트릴을 식물 개체에 처리하는 단계를 포함하는 식물 병원성 진균 방제방법이 제공된다: According to another aspect of the present invention, there is provided a method for controlling plant pathogenic fungi comprising the step of treating a plant subject with aminopyrrolenitrile having the structure of Formula II:
이하, 실시예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, and the following embodiments allow the disclosure of the present invention to be complete, and the scope of the invention to those of ordinary skill in the art It is provided to fully inform
실시예 1: Example 1: Serratia grmessiSerratia grmessi 로부터 아미노피롤니트린 생산 가능성 확인Confirmation of the possibility of aminopyrrolenitrine production from
본 발명자들은 미국 생명공학 정보센터(NCBI) DB에서 유전체 서열이 밝혀진 Pseudomonas fluorescens Pf-5의 피롤니트리닌 합성 대사경로의 분석을 통해서 P. fluorescence Pf-5의 유전체 상에 존재하는 prn 클러스터(prnA, prnB, prnC, prnD, prnE, prnF, prnS 및 prnR)를 확인하였다. 이를 바탕으로 본 발명자들은, NCBI에 보고된 2개의 S. grimesii 유전체 정보를 분석 결과, prn 클러스터에서 플라빈 환원효소(flavin reductase)를 암호화하는 prnF 유전자의 결손으로 피롤니트린이 생성되지 않고 피롤니트린의 전구체인 아미노피롤니트린이 생성될 것으로 예측하였다(도 1). The present inventors analyzed the pyrronitrine synthesis metabolic pathway of Pseudomonas fluorescens Pf-5 whose genome sequence was revealed in the US Center for Biotechnology Information (NCBI) DB, and the prn cluster ( prnA) present on the genome of P. fluorescence Pf-5. prnB , prnC , prnD , prnE , prnF , prnS and prnR ) were identified. Based on this, the present inventors analyzed two S. grimesii genome information reported to NCBI, as a result of the deletion of the prnF gene encoding flavin reductase in the prn cluster. It was predicted that aminopyrrolenitrine, a precursor of , would be produced (FIG. 1).
실시예 2: Example 2: Serratia grimessiSerratia grimessi 로부터 아미노피롤니트린의 생산production of aminopyrrolenitrine from
상기 실시예 1의 결과로부터 본 발명자들은 0.1% L-tryptophan를 첨가하거나 하지 않은 LB 배지를 제조한 후, 각 배지에 Seratia grimessi MRS-1 균주를 접종하여 28℃, 200 rpm으로 24시간~ 84시간 동안 배양 후 배양액을 원심분리 후 상등액을 필터링하여 aminopyrrolnitrin 생산여부를 분석하였다. S. grimessi MRS-1 균주는 25℃에서 MRS(Difco) 배지를 이용하여 곤쟁이 내장에서 분리하였으며, 16S rDNA 염기서열 분석결과 S. grimessi에 속하는 신규 균주로 파악되어(서열번호 1), 2019년 4월 2일자로 한국미생물보존센터(KCCM)에 기탁하여, 수탁번호 KCCM12480PP를 부여받았다(도 2). 배양시 20시간 까지는 L-tryptophan이 소모되지 않았으나, 84시간에서 모두 소모가 되었고 HPLC 분석결과 아미노피롤니트린이 생성되는 것을 확인할 수 있었다(도 3).From the results of Example 1, the present inventors prepared LB medium with or without the addition of 0.1% L-tryptophan, and then inoculated each medium with Seratia grimessi MRS-1 strain at 28° C., 200 rpm for 24 hours to 84 hours. After culturing for a while, the culture medium was centrifuged and the supernatant was filtered to analyze whether aminopyrrolnitrin was produced. The S. grimessi MRS-1 strain was isolated from the intestines of the crayfish using MRS (Difco) medium at 25 ° C. As a result of 16S rDNA sequencing, it was identified as a new strain belonging to S. grimessi (SEQ ID NO: 1), 4, 2019 It was deposited with the Korea Microorganism Conservation Center (KCCM) on the 2nd of the month and was given an accession number KCCM12480PP (FIG. 2). L-tryptophan was not consumed until 20 hours during incubation, but was consumed at 84 hours, and it was confirmed that aminopyrrolenitrine was produced as a result of HPLC analysis (FIG. 3).
실시예 3: 아미노피롤니트린의 화학합성Example 3: Chemical synthesis of aminopyrrolenitrine
본 발명자들은 중국 WuXi AppTech(Wuhan) Co., Ltd.사에 아미노피롤니트린의 유기합성을 의뢰하였으며, LC-MS 및 NMR 분석을 통해서 아미노피롤니트린의 분자량과 구조를 확인하였다(도 4). The present inventors requested the organic synthesis of aminopyrrolenitrine from WuXi AppTech (Wuhan) Co., Ltd. of China, and confirmed the molecular weight and structure of aminopyrrolenitrine through LC-MS and NMR analysis (FIG. 4) .
실시예 4: 아미노피롤니틴의 병원성 진균에 대한 항진균 활성 분석Example 4: Analysis of antifungal activity of aminopyrrolnitine against pathogenic fungi
본 발명자들은 상기 실시예 3에서 합성된 아미노피롤리틴을 이용하여 다양한 병원성 진균에 대한 항균활성을 분석하였다. 이를 위해 구체적으로 Paik et al.(1998)의 방법을 응용하여 고압 멸균한 PDA 배지 100 ml에 각 병원성 진균의 포자 또는 균사 현탁액 1 ml을 넣고 섞어준 후 20 ml을 페트리 디쉬에 분주하여 굳힌 후, 페이퍼 디스크(ø8 mm)에 1 mg/ml의 아미노피롤니틴 50 μl를 점적하였다. 이어 25℃에서 각각의 시험균주가 다 자랄 때까지 배양하였다. 균이 다 자라면 투명환(clear zone)의 형성 유무를 확인함으로써 항균활성 여부를 조사하였다.The present inventors analyzed the antibacterial activity against various pathogenic fungi using the aminopyrrolitin synthesized in Example 3. For this, 1 ml of the spore or mycelium suspension of each pathogenic fungus is added to 100 ml of high-pressure sterilized PDA medium by applying the method of Paik et al. 50 μl of aminopyrrolnitine at 1 mg/ml was dripped onto a paper disc (ø8 mm). Then, each test strain was incubated at 25°C until fully grown. Antibacterial activity was investigated by checking whether or not a clear zone was formed when the bacteria were fully grown.
그 결과, 아미노피롤니트린은 다양한 병원성 세균에 대하여는 아주 약한 항균활성을 나타냈으나(데이터 미제시), 병원성 진균류인 Candida albicans, Candida palmioleophila, Candida zeylaroides, Barnettozyma californica, Fusarium poliferatum, Hortaea wermeckii, Trichosporon cavernicola, 및 Wickerhamomyces anomalus에 대하여는 강한 항균활성을 나타냈다(도 5). 아울러, 상기 진균들에 대한 항진균 활성을 정량화하기 위해 MEC를 측정하였다. 그 결과, 본 발명의 아미노피롤니트린은 그 정도는 상이하지만 시험에 사용된 진균 모두에 대하여 항진균 활성을 나타냈으며, 특히 Candida albicans, Barnettozyma californica, Candidida palmioleophil, Fusaruium proliferatum 및 Wickerhamomyces anomalus에 대하여 매우 우수한 항진균 활성을 나타냈다(표 1).As a result, the amino pyrrole knitted Lin, but did show a very weak antibacterial activity with respect to a variety of pathogenic bacteria (the data shown), pathogenic fungi of Candida albicans, Candida palmioleophila, Candida zeylaroides, Barnettozyma californica, Fusarium poliferatum, Hortaea wermeckii, Trichosporon cavernicola , and Wickerhamomyces anomalus showed strong antibacterial activity (FIG. 5). In addition, MEC was measured to quantify the antifungal activity against the fungi. As a result, the aminopyrrolenitrine of the present invention exhibited antifungal activity against all the fungi used in the test, although to a different degree, in particular, against Candida albicans , Barnettozyma californica , Candidida palmioleophil , Fusaruium proliferatum and Wickerhamomyces anomalus . activity (Table 1).
실시예 5: 식물병원성 진균에 대한 방제효과 확인Example 5: Confirmation of control effect on phytopathogenic fungi
이어 본 발명자들은 본 발명의 일 실시예에 따른 아미노피롤니트린이 원예작물의 질병을 야기하는 식물 병원성 진균에 대한 방제효과가 있는지 여부를 상기 실시예 5에 기재된 바와 같이 디스크 확산법을 이용하여 조사하였다(표 2 및 도 6).Next, the present inventors investigated whether aminopyrrolenitrine according to an embodiment of the present invention has a control effect on phytopathogenic fungi causing diseases of horticultural crops using the disk diffusion method as described in Example 5 above ( Table 2 and Figure 6).
그 결과, 도 6에서 확인되는 바와 같이, 토마토 시들음병의 병원균인 Fusarium oxysporum에 대하여만 보통 정도의 항균활성을 나타내었을 뿐, 잿빛곰팡이병 유발균인 Botrytis sp. 균이나 복숭아 잿빛무늬병 유발균인 Monillinia sp. 균 및 토마토 분리병 유발균인 Rhizoctonia sp. 균에 대하여는 매우 높은 항균활성을 나타냈다.As a result, as confirmed in FIG. 6, only showed moderate antibacterial activity against Fusarium oxysporum , a pathogen of tomato wilt, and Botrytis sp. Fungi or peach gray blotch-causing bacterium, Monillinia sp. Rhizoctonia sp. It showed very high antibacterial activity against bacteria.
실시예 6: 딸기 잿빛곰팡이병에 대한 방제 효과 검증Example 6: Verification of control effect on strawberry gray mold disease
딸기 잿빛곰팡이병의 병원균은 Botrytis cinerea로 병환부에 분생포자를 무수히 많이 생성하는 것이 특징인데, 이포자가 공기를 통하여 전염되며, 저온기의 시설재배시 발생하는 경향이 있어서, 딸기 시설재배에 큰 타격을 주는 질병이다. The pathogen of strawberry gray mold disease is Botrytis cinerea , which is characterized by the formation of innumerable conidia in the affected area. giving is a disease
이에 본 발명자들은 본 발명의 일 실시예에 따른 아미노피롤니트린이 딸기 잿빛곰팡이병에 대한 방제효과를 가지고 있는지 확인하기 위해 포장 시험을 수행하였다.Accordingly, the present inventors conducted a field test to confirm whether aminopyrrolenitrine according to an embodiment of the present invention has a control effect on strawberry gray mold disease.
구체적으로 딸기 개체에 Botrytis cinerea 균주를 접종한 후 두 그룹으로 나누어 실험군에는 70% 에탄올 수용액에 1 mg/ml로 용해시킨 아미노피롤니트린 5 ml를 대조군에는 70% 에탄올 5 ml를 딸기 개체 당 3-4회 스프레이로 뿌리고 이틀 후 2차로 각각 딸기 개체 당 5회 뿌린 후 잿빛곰팡이병 발생 및 정도를 분석하였다. Specifically, after inoculating the strawberry individual with the Botrytis cinerea strain, the group was divided into two groups, 5 ml of aminopyrrolenitrine dissolved at 1 mg/ml in 70% ethanol aqueous solution for the experimental group, and 5 ml of 70% ethanol for the control group. After spraying 4 times and 2 days after spraying each
그 결과, 도 7에서 확인되는 바와 같이, 아미노피롤니트린 처리군의 경우 대조군에 비해서 딸기 잿빛곰팡이병에 대한 방제효과가 나타났다.As a result, as confirmed in FIG. 7 , in the case of the aminopyrrolenitrine treatment group, the control effect on strawberry gray mold was shown compared to the control group.
실시예 7: 스쿠치카충에 대한 방제효과 분석Example 7: Analysis of the control effect on Scotchka worms
본 발명의 일 실시예에 따른 아미노피롤니트린의 항-스쿠치카 활성을 분석하기 위해 스쿠치카(scutica, Uronema marinum) 충을 HINAE 세포에 1/10로 감염시킨 후 96웰 플레이트에 100 ㎕씩 분주한 후 아미노피롤니트린을 농도별(0, 55, 110 및 220 μM)로 100 ㎕씩 첨가하였고 대조구는 DMSO(100 ㎕)를 처리한 다음 20℃에서 1~2 시간 배양하였다. 상기 배양 후 역상현미경(Carl zeiss, DE/Axio vert. A1)의 배율을 X200 배율로 스쿠치카 충의 형태적 변화와 운동성 변화를 관찰하였다. 그 결과, 대조구와 비교하여 아미노피롤니트린을 55 μM 이상 처리한 실험군에서 스쿠치카 충이 타원형으로 부풀었으며 운동성도 저하되는 것을 확인하였다(도 8A). 본 발명의 아미노피롤니트린의 항충(anti-parasitic activity) 효과를 정량화하기 위해 WST-1 세포 증식 분석 시스템을 사용하였다. 먼저, 스쿠치카 충에 아미노피롤니트린을 처리한 후 1~2 시간 경과 시점에서 Primx WST-1(Takara, Japan)을 웰당 10 ㎕씩 첨가하였고 ELISA 판독기로 흡광도(450nm)를 측정하였다. 그 결과, 아미노피롤니트린 110 μM 처리시 80% 이상의 항충 효능(생존율 17%)을 나타내었다(도 8B). 상기 결과는 실시예 1의 현미경 관찰 결과와 거의 동일한 결과로 우수한 항충 효과가 있음을 확인하였다.In order to analyze the anti-Scuchika activity of aminopyrrolenitrine according to an embodiment of the present invention , 100 μl each of scutica (Uronema marinum ) was infected with HINAE cells at 1/10 and then dispensed in a 96-well plate. Then, 100 μl of aminopyrrolenitrine was added at each concentration (0, 55, 110 and 220 μM), and the control was treated with DMSO (100 μl) and then incubated at 20° C. for 1-2 hours. After the incubation, morphological changes and motility changes of Scotchka were observed at a magnification of X200 using an inverted microscope (Carl zeiss, DE/Axio vert. A1). As a result, compared with the control group, in the experimental group treated with
본 발명은 상술한 실시예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.Although the present invention has been described with reference to the above-described embodiment, it will be understood that this is merely exemplary, and that those skilled in the art may make various modifications and equivalent other embodiments therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.
Claims (4)
식물 병원성 진균에 대해 항진균 활성을 갖는, 조성물.According to claim 1,
A composition having antifungal activity against phytopathogenic fungi.
상기 식물 병원성 진균은 양파 잿빛곰팡이병 병원균, 복숭아 잿빛무늬병 병원균, 장미 잿빛곰팡병 병원균, 딸기 잿빛곰팡이병 병원균 또는 토마토 분리병 병원균인, 조성물.3. The method of claim 2,
The phytopathogenic fungus is an onion gray mold pathogen, a peach gray mold pathogen, a rose gray mold pathogen, a strawberry gray mold pathogen or a tomato isolate pathogen.
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| KR20220070406A (en) | 2022-05-31 |
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