KR101158503B1 - Compound isolated from Phomopsis longicolla and method for preparing thereof - Google Patents
Compound isolated from Phomopsis longicolla and method for preparing thereof Download PDFInfo
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- KR101158503B1 KR101158503B1 KR1020090078943A KR20090078943A KR101158503B1 KR 101158503 B1 KR101158503 B1 KR 101158503B1 KR 1020090078943 A KR1020090078943 A KR 1020090078943A KR 20090078943 A KR20090078943 A KR 20090078943A KR 101158503 B1 KR101158503 B1 KR 101158503B1
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Abstract
본 발명은 식물내생 곰팡이인 포몹시스 롱지콜라(Phomopsis lingicolla ssp.)로부터 분리한 화학식 1로 표시되는 신규 화합물 및 그의 제조방법에 관한 것이다.
본 발명에 따른 화학식 1의 화합물을 항균 활성이 우수하므로 항균제로서 유용하게 사용될 수 있다.
[화학식 1]
포몹시스 롱지콜라, 항균 활성, 항균제, 벼흰잎마름병
The present invention relates to a novel compound represented by Chemical Formula 1 isolated from Phomopsis lingicolla ssp., A plant endogenous fungus, and a preparation method thereof.
Since the compound of Formula 1 according to the present invention has excellent antibacterial activity, it may be usefully used as an antimicrobial agent.
[Formula 1]
Pomopsis longji cola, antibacterial activity, antimicrobial agent, rice leaf blight
Description
본 발명은 포몹시스 롱지콜라(Phomopsis lingicolla ssp.)로부터 분리한 신규 화합물 및 그의 제조방법에 관한 것으로, 더욱 상세하게는 식물내생 곰팡이인 포몹시스 롱지콜라(Phomopsis lingicolla ssp.)로부터 분리한 화학식 1로 표시되는 화합물 또는 그의 유도체와 상기 화합물의 제조방법, 및 상기 화합물의 항균제로서의 용도에 관한 것이다.The present invention relates to a novel compound isolated from Phomopsis lingicolla ssp. And a method for preparing the same, and more particularly, to a chemical formula isolated from phomopsis lingicolla ssp. The compound represented by 1, its derivative (s), the manufacturing method of the said compound, and its use as an antimicrobial agent.
[화학식 1][Formula 1]
병자각을 형성하는 포몹시스 롱지콜라(Phomopsis longicolla ssp.)는 병자각 내에 α와 β 포자를 형성하는데, 방추형의 α 포자는 무색, 단세포로 크기가 5~9 ×1.5~3 ㎛이고, 낚시모양인 β 세포의 크기는 17~36×0.7~1.5 ㎛이다.Phomopsis longicolla ssp., Which forms sickle shell , forms α and β spores in the sickle shell . The fusiform α spores are colorless, single cells, 5-9 × 1.5-3 μm in size, and are fishing-like. β cells have a size of 17-36 × 0.7-1.5 μm.
최근에는 포몹시스 롱지콜라(Phomopsis longicolla ssp.)의 2차 대사사물에서 마이크로튜블, 항말라리아, 결핵치료 등의 활성들이 보고되고 있다.Recently, secondary metabolites of Phomopsis longicolla ssp. Have been reported to have activities such as microtubules, antimalaria, and tuberculosis treatment.
본 발명에 따른 화합물은 포몹시스 속에서 분리된 포모잔톤 B(Masahiko Isaka, Amonlaya Jaturapat, Kamolchanok Rekseree, Kannawat Danwisetkanjana, Morakot Tanticharoen, and Yodhathai Thebtrarnonth, J. Nat. Prod., 64, 1015-1018, 2001)와 디아세틸포모잔톤 B(Vatcharin Rukachaisirkikul, Ubont Sommart, Souwalak Phongpaichit, Jariya Sakayaroj, and Kanyawim Kirtikara, Phytochemistry, 69, 783-787, 2008)의 유사계열이나, 현재까지 보고된 바는 없다.Compounds according to the present invention can be prepared in the form of pomoxanthone B (Masahiko Isaka, Amonlaya Jaturapat, Kamolchanok Rekseree, Kannawat Danwisetkanjana, Morakot Tanticharoen, and Yodhathai Thebtrarnonth, J. Nat. Prod., 64, 1015-1018, 2001 ) And diacetylformomoxanthone B (Vatcharin Rukachaisirkikul, Ubont Sommart, Souwalak Phongpaichit, Jariya Sakayaroj, and Kanyawim Kirtikara, Phytochemistry, 69, 783-787, 2008), but have not been reported to date.
이에 본 발명자들은 천연물로부터 새로운 생리활성 물질을 발굴하고자 예의 연구한 결과, 포몹시스 롱지콜라 균주로부터 항균 활성을 갖는 신규 생리활성 물질을 분리하고 본 발명을 완성하였다.Accordingly, the present inventors earnestly researched to discover new bioactive substances from natural products, and have separated the novel bioactive substances having antimicrobial activity from the pomopsis longjicola strain and completed the present invention.
결국, 본 발명의 주된 목적은 포몹시스 롱지콜라(Phomopsis longicolla ssp.)로부터 분리한 항균 활성을 갖는 신규 화합물 또는 그의 유도체를 제공하는데 있다.After all, the main object of the present invention is to provide a novel compound or derivative thereof having antibacterial activity isolated from Phomopsis longicolla ssp.
또한, 본 발명은 상기 화합물 또는 그의 유도체의 제조방법을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide a method for preparing the compound or derivative thereof.
또한, 본 발명은 상기 화합물 또는 그의 유도체의 항균제로서의 용도를 제공하는데 또 다른 목적이 있다. It is another object of the present invention to provide a use of the compound or its derivatives as an antimicrobial agent.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 신규 화합물 또는 그의 유도체를 제공한다.In order to achieve the above object, the present invention provides a novel compound or a derivative thereof represented by the following formula (1).
본 발명은 또한, (1) 포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주를 배양한 후 배양액으로부터 균사 덩어리를 분리하는 단계; (2) 상기 분리된 균사 덩어리에 극성용매를 가하여 추출한 후 감압 여과하여 조추출물을 수득하는 단계; (3) 상기 조추출물을 비극성용매로 추출하여 액상의 활성분획을 얻은 후 상기 활성분획을 감압 농축하는 단계; 및 (4) 상기 농축된 활성분획을 겔 여과 크로마토그래피(Gel Filtration Chromatography) 및 고성능 액체 크로마토그래피(High Performance Liquid Chromatography)를 수행하여 순수한 단일 화합물로 분리하는 단계;를 포함하는 상기 화학식 1로 표시되는 화합물 또는 그의 유도체의 제조방법을 제공한다.The present invention also relates to (1) phomopsis longjicola ( Phomopsis) longicolla ssp.) separating the mycelium mass from the culture after culturing the strain; (2) extracting by adding a polar solvent to the separated mycelial mass and filtering under reduced pressure to obtain a crude extract; (3) extracting the crude extract with a nonpolar solvent to obtain an active fraction of a liquid phase, and then concentrating the active fraction under reduced pressure; And (4) separating the concentrated active fraction into a single pure compound by performing gel filtration chromatography (Gel Filtration Chromatography) and high performance liquid chromatography (High Performance Liquid Chromatography). Provided are methods for preparing a compound or derivative thereof.
본 발명은 또한, 약제학적으로 허용되는 담체와 혼합한 상기 화학식 1로 표시되는 화합물 또는 약제학적으로 허용 가능한 이의 염을 유효성분으로 하는 약학적 제제를 제공한다. The present invention also provides a pharmaceutical formulation comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof mixed with a pharmaceutically acceptable carrier as an active ingredient.
본 발명에 있어서, 상기 화학식 1의 화합물은 포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주의 배양액으로부터 분리되거나 화학합성 또는 반합성되는 것이 특징이며, 상기 약학적 제제는 항균제 또는 벼흰잎마름병 방제약제인 것을 특징으로 한다.In the present invention, the compound of Formula 1 is characterized in that it is isolated from the culture medium of Phomopsis longicolla ssp. Strain or is chemically synthesized or semi-synthesized, wherein the pharmaceutical agent is an antimicrobial agent or rice leaf blight control agent It is characterized by.
본 발명은 포몹시스 롱지콜라(Phomopsis longicolla ssp.)로부터 분리한 항균 활성을 갖는 신규 화합물 및 그 제조방법을 제공하는 효과가 있다.The present invention has the effect of providing a novel compound having an antimicrobial activity isolated from Phomopsis longicolla ssp. And a method for producing the same.
본 발명에 따른 화합물은 항균 활성이 우수하여 항균제의 개발에 유용하게 이용될 수 있으며, 특히 벼흰잎마름병원균의 활성을 현저하게 억제하므로 벼흰잎마 름병의 방제약제로 사용될 수 있다. The compound according to the present invention is excellent in antimicrobial activity can be usefully used in the development of antimicrobial agents, in particular it can be used as a control agent of rice white leaf blight because it significantly inhibits the activity of the rice leaf blight pathogen.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기 화학식 1로 표시되는 신규 화합물 또는 그의 유도체를 제공한다.The present invention provides a novel compound represented by the following formula (1) or a derivative thereof.
[화학식 1][Formula 1]
본 발명의 상기 화학식 1의 화합물은 식물내생 곰팡이인 포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주의 배양액으로부터 분리하여 얻을 수 있다.The compound of formula 1 of the present invention can be obtained by separating from the culture solution of Phomopsis longicolla ssp.
본 발명에 따른 포몹시스 롱지콜라 균주의 배양액으로부터 상기 화합물 1을 제조하는 방법은, (1) 포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주를 배양한 후 배양액으로부터 균사 덩어리를 분리하는 단계; (2) 상기 분리된 균사 덩어리에 극성용매를 가하여 추출한 후 감압 여과하여 조추출물을 수득하는 단계; (3) 상기 조추출물을 비극성용매로 추출하여 액상의 활성분획을 얻은 후 상기 활성분획을 감압 농축하는 단계; 및 (4) 상기 농축된 활성분획물을 겔 여과 크로마토그래 피(Gel Filtration Chromatography) 및 고성능 액체 크로마토그래피(High Performance Liquid Chromatography)를 수행하여 순수한 단일 화합물로 분리하는 단계;를 포함할 수 있다.The method for preparing Compound 1 from the culture medium of the pomopsis longjicola strain according to the present invention comprises the steps of: (1) culturing a phomopsis longicolla ssp. (2) extracting by adding a polar solvent to the separated mycelial mass and filtering under reduced pressure to obtain a crude extract; (3) extracting the crude extract with a nonpolar solvent to obtain an active fraction of a liquid phase, and then concentrating the active fraction under reduced pressure; And (4) separating the concentrated active fraction into a single pure compound by performing gel filtration chromatography (Gel Filtration Chromatography) and high performance liquid chromatography (High Performance Liquid Chromatography).
본 발명에 있어서, 상기 (1) 단계는 포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주를 PDA(potato dextrose agar) 플레이트에 배양 후 배양된 균주의 아가 플러그를 PDB(potato dextrose broth) 배지에 접종하여 25℃에서 진탕배양하는 것이 바람직하며, 배양 후에는 그 배양액을 여과기와 원심분리기를 이용하여 균사 덩어리는 분리한다.In the present invention, the step (1) is a culture of Phomopsis longicolla ssp. ( Pomopsis longicolla ssp.) Strain incubated on a PDA (potato dextrose agar) plate inoculated agar plugs of the cultured strain in PDB (potato dextrose broth) medium The shaker is preferably cultured at 25 ° C., and after the incubation, the mycelium mass is separated using a filter and a centrifuge.
상기 분리된 균사 덩어리는 상기 (2) 단계에서 80% 아세톤에 용해시키고, 용해된 균사 덩어리를 여과 후 그 여액을 채취하여 40~50℃에서 감압농축하는 것이 바람직하며, 상기 (3) 단계에서는 상기 농축된 조추출물을 과량의 물에 현탁하여 분액여두에 넣은 후 과량의 에틸아세테이트를 넣고 진탕추출한 다음 얻은 상등액을 회수하여 40~50℃에서 감압농축하는 것이 바람직하다.The separated mycelial mass is dissolved in 80% acetone in the step (2), the dissolved mycelial mass is filtered and the filtrate is collected and concentrated under reduced pressure at 40 ~ 50 ℃, in the step (3) It is preferable to suspend the concentrated crude extract in excess water, add the excess ethyl acetate, shake extract, and recover the supernatant obtained at 40 to 50 ° C.
본 발명에서 사용되는 용매는 화학식 1의 화합물 또는 그의 유도체를 효과적으로 추출할 수 있는 용매라면 특별히 제한되지 아니하며, 그 예로는 물; 메탄올, 에탄올, 이소프로판올, 에틸렌글리콜을 포함하는 알코올; 염화메틸렌, 클로로포름, 사염화탄소 등을 포함하는 할로겐 원자로 치환된 탄화수소류; THF, DMF, DMSO, 에틸아세테이트 등을 들 수 있다. 본 발명에서는 80% 아세톤과 에틸아세테이트를 사용하였으나 이는 예시에 불과할 뿐 이들을 사용하는 것을 제한하는 것은 아니다.The solvent used in the present invention is not particularly limited as long as it is a solvent capable of effectively extracting the compound of Formula 1 or a derivative thereof, and examples thereof include water; Alcohols including methanol, ethanol, isopropanol, ethylene glycol; Hydrocarbons substituted with halogen atoms including methylene chloride, chloroform, carbon tetrachloride and the like; THF, DMF, DMSO, ethyl acetate, etc. are mentioned. In the present invention, 80% acetone and ethyl acetate were used, but this is only an example and does not limit the use thereof.
또한, 상기 (4) 단계에서는 상기 농축된 활성분획물을 소량의 메탄올에 용해 한 후 세파덱스 LH-20 겔 컬럼 크로마토그래피(Sephadex LH-20 gel column chromatography)를 수행하여 활성 분획을 모은 후 모아진 활성분확들을 40~50℃에서 감압농축하는 것이 바람직하다. 상기 농축된 활성분획들은 다시 소량의 메탄올에 용해하여 고성능 액체 크로마토그래피를 이용하여 단일 순수 화합물로 얻어낸다.In the step (4), the concentrated active fraction is dissolved in a small amount of methanol, and then Sepadex LH-20 gel column chromatography is performed to collect the active fractions and collect the active fractions. It is preferable to concentrate them under reduced pressure at 40 ~ 50 ℃. The concentrated active fractions are again dissolved in a small amount of methanol to obtain a single pure compound using high performance liquid chromatography.
또한, 본 발명은 상기의 과정을 포함하는 것을 특징으로 하는 상기 화학식 1로 표시되는 화합물 또는 그의 유도체의 제조방법을 제공한다.In another aspect, the present invention provides a method for producing a compound represented by the formula (1) or derivatives thereof comprising the above process.
본 발명의 화학식 1의 화합물 또는 그의 유도체는 상기와 같이 포몹시스 롱지콜라 균주의 배양액으로부터 분리되거나 통상의 방법에 의한 화학합성 또는 반합성에 의해 인공적으로 제조될 수도 있다.The compound of formula 1 or a derivative thereof of the present invention may be isolated from the culture medium of the pomopsis longjicola strain as described above or artificially prepared by chemical synthesis or semisynthesis by a conventional method.
본 발명의 제조방법에 의해 수득된 상기 화학식 1의 화합물에 대하여 ESI-MS(Electron Spray Ionization Mass Spectrometer)를 이용하여 이화학적 특성을 분석한 결과, 하기와 같은 이화학적 특성을 갖는 것으로 확인되었다.As a result of analyzing physicochemical properties of the compound of Formula 1 obtained by the preparation method of the present invention using an ESI-MS (Electron Spray Ionization Mass Spectrometer), it was confirmed to have the following physicochemical properties.
ⅰ) 물질 성상: 노란 분말Iii) Material Appearance: Yellow Powder
ⅱ) 분자량: 708Ii) molecular weight: 708
ⅲ) 분자식: C36H36O15 Iii) molecular formula: C 36 H 36 O 15
ⅳ) 질량 분석치([M+H]+): 709(m/z) V) mass spectrometry ([M + H] + ): 709 ( m / z )
또한, 듀테륨 아세토니트릴을 용매로 이용하여 특정한 수소 핵자기공명 스펙 트럼 및 탄소 핵자기공명 스펙트럼 분석 결과, 본 발명의 화합물은 상기 화학식 1의 구조를 갖는 것으로 확인되었다.In addition, specific hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectrum analysis using deuterium acetonitrile as a solvent, it was confirmed that the compound of the present invention has the structure of formula (1).
본 발명에 따른 포몹시스 롱지콜라로부터 분리된 상기 화학식 1의 화합물은 현재까지 발견된 유사한 계열인 디세란드롤 시리즈 등과 마찬가지로 항균활성, 항암활성을 나타낼 것으로 예상된다.The compound of Formula 1 isolated from pomopsis longjicola according to the present invention is expected to exhibit antimicrobial activity and anticancer activity similarly to the disserlandol series, which is a similar series found so far.
따라서 본 발명은 또한, 약제학적으로 허용되는 담체와 혼합한 상기 화학식 1로 표시되는 화합물 또는 약제학적으로 허용 가능한 이의 염을 유효성분으로 하는 약학적 제제를 제공할 수 있다.Therefore, the present invention can also provide a pharmaceutical formulation comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof mixed with a pharmaceutically acceptable carrier as an active ingredient.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 포몹시스 롱지콜라에서 활성물질의 분리Example 1 Isolation of Active Substance from Formomsis Longji Cola
283 개의 식물내생 곰팡이 중에서 잔토모나스 오라이제(Xanthomonas oryzae)에 활성을 가지는 균주를 선택하여 동정하였으며, 동정결과 가장 높은 활성을 가지는 균주는 포몹시스 롱지콜라인 것으로 확인되었다.Among 283 endophytic fungi, strains having activity against Xanthomonas oryzae were selected and identified, and the strain having the highest activity was identified as pomopsis longjicoline.
포몹시스 롱지콜라(Phomopsis longicolla ssp.) 균주는 PDB(potato dextrose broth) 배지 10ℓ를 이용하여 25℃에서 18일간 배양하였다. 배양이 끝나면 여과기 와 원심분리기를 사용하여 균사 덩어리를 얻었다. 균사 덩어리는 2ℓ의 80% 아세톤(w/w)을 사용하여 용해시키고, 여과 후 그 여액을 채취, 40~50℃의 수조에서 감압농축하여 추출물을 얻었다. Phomopsis longicolla ssp. Strain was incubated at 25 ° C. for 18 days using 10 l of PDB (potato dextrose broth) medium. After incubation, mycelial mass was obtained using a filter and a centrifuge. The mycelial mass was dissolved using 2 L of 80% acetone (w / w), the filtrate was collected after filtration, and concentrated under reduced pressure in a water bath at 40 to 50 ° C to obtain an extract.
상기 농축된 추출물은 100㎖의 증류수에 현탁시키고, 분액여두에서 동량의 에틸아세테이트로 진탕추출하여 에틸아세테이트 가용부를 추출하여 얻은 다음, 다시 추출액을 40℃의 수조에서 감압 농축하여 에틸아세테이트 추출물을 얻었다.The concentrated extract was suspended in 100 ml of distilled water, shaken with an equal amount of ethyl acetate in a separatory extract to obtain an ethyl acetate soluble part, and the extract was concentrated under reduced pressure in a 40 ° C. water bath to obtain an ethyl acetate extract.
실시예 2. 포몹시스 롱지콜라 균사 덩어리 추출물로부터 활성화합물의 단리Example 2. Isolation of Active Compounds from Formomsis Longjicola Mycelia Clump Extract
상기 실시예 1에서 얻은 에틸아세테이트 추출물 749㎎을 메탄올 2㎖에 용해시킨 후 80% 메탄올을 용출용매로 하여 세파덱스 LH-20 겔 컬럼 크로마토그래피(SephadexTM, GE Healthcare Bio-Sciences AB, Sweden)를 수행하여 활성분획을 분리하였으며, 감압건조기로 용매를 제거한 후 얻은 잔사(residue)는 고급 메탄올에 용해하여 YMC-Pack pro C18 (250×10㎜) 컬럼을 사용한 고성능 액체 크로마토그래피(Model L-2000 Series Organizer Box, HITACHI, Japan)를 수행하고, 활성물질을 얻었다. 고성능 액체 크로마토그래피의 용리조건은 이동상으로 70% 아세토니트릴을 사용하였으며, 80분 동안 3 ㎖/분의 속도로 흘려보내 주었으며 본 발명의 활성화합물은 30분에서 35분 사이에서 얻을 수 있었다.After dissolving 749 mg of ethyl acetate extract obtained in Example 1 in 2 ml of methanol, Sephadex LH-20 gel column chromatography (Sephadex ™ , GE Healthcare Bio-Sciences AB, Sweden) was prepared using 80% methanol as the eluent. The active fraction was separated, and the residue obtained after removing the solvent by a vacuum dryer was dissolved in high-quality methanol and subjected to high performance liquid chromatography using a YMC-Pack pro C 18 (250 × 10 mm) column (Model L-2000). Series Organizer Box, HITACHI, Japan) was carried out to obtain the active material. Elution conditions of high performance liquid chromatography used 70% acetonitrile as the mobile phase, flowed at a rate of 3 ml / min for 80 minutes and the active compound of the present invention was obtained between 30 and 35 minutes.
실시예 3. 구조분석Example 3. Structural Analysis
상기 실시예 2에서 얻은 활성물질의 구조를 확인하기 위하여, LC-ESI-MS(Liquid chromatography electron spray ionization mass spectrometer; Varian 500-MS, VARIAN, USA)를 사용하여 분자량, 분자식, 및 질량을 분석하고, 핵자기공명 스펙트럼(Varian-500㎒ NMR, VARIAN, USA)을 통해 분자구조를 결정하였다.In order to confirm the structure of the active material obtained in Example 2, the molecular weight, molecular formula, and mass were analyzed using LC-ESI-MS (Liquid chromatography electron spray ionization mass spectrometer; Varian 500-MS, VARIAN, USA) The molecular structure was determined by nuclear magnetic resonance spectra (Varian-500MHz NMR, VARIAN, USA).
그 결과, 상기 활성물질은 노란색 분말의 성상을 가지며, 분자량은 708이고, 분자식은 C36H36O15, 질량 분석치([M+H]+)는 709(m/z)로 확인되었다. As a result, the active substance had the properties of a yellow powder, a molecular weight of 708, a molecular formula of C 36 H 36 O 15 , a mass spectrometry ([M + H] + ) was found to be 709 ( m / z ).
또한, 듀테륨 아세토니트릴을 용매로 측정한 1H NMR 및 13C NMR 스펙트럼(도 1 및 도 2 참조)을 분석한 결과, 본 발명의 활성물질은 하기의 화학식 1로 표시되는 화합물인 것으로 판명되었다.In addition, as a result of analyzing 1 H NMR and 13 C NMR spectra (see FIGS. 1 and 2) measured with deuterium acetonitrile as a solvent, it was found that the active substance of the present invention was a compound represented by the following Chemical Formula 1.
[화학식 1][Formula 1]
실험예. 활성물질의 항균 활성 확인Experimental example. Confirmation of antimicrobial activity of active substance
본 발명에 따른 포몹시스 롱지콜라로부터 분리한 화학식 1의 화합물의 항균 활성을 확인하기 위하여, 상기 실시예 2에서 얻은 활성물질을 128 ㎍/㎖에서 0.25 ㎍/㎖ 농도까지 2배 희석 방법을 사용하였으며, 하기 표 1의 조성으로 구성된 배양용 배지에 한국농업미생물자원센터로부터 분양받은 잔토모나스 오라이제(Xanthomonas oryzae, KACC 10331)를 접종하여 보존하였다. In order to confirm the antimicrobial activity of the compound of Formula 1 isolated from pomopsis longji cola according to the present invention, a 2-fold dilution method of the active material obtained in Example 2 from 128 μg / ml to 0.25 μg / ml was used. The culture medium consisting of the composition of Table 1 was inoculated and preserved by Xanthomonas oryzae (KACC 10331) distributed from the Korea Agricultural Microbial Resources Center.
구체적으로, 잔토모나스 오라이제를 상기 배지를 사용하여 28℃에서 1일간 배양하였으며, 최종 균농도가 2.5×103개/mol이 되도록 상기 활성물질을 함유하고 있는 배지에 접종하여 1일간 보존 후 마이크로플레이트 리더(EL 808 Ultra Microplate reader, BIO-TEK instruments, USA)를 이용하여 600 ㎚에서 흡광도를 측정하였다.Specifically, xanthomonas olease was incubated at 28 ° C. for 1 day using the medium, and then inoculated into a medium containing the active material so that the final bacterial concentration was 2.5 × 10 3 / mol, and then stored for 1 day. Absorbance was measured at 600 nm using a plate reader (EL 808 Ultra Microplate reader, BIO-TEK instruments, USA).
이때, 항균활성은 최소생육저해농도(Minimum Inhibitory Concentration, MIC)로 나타내었으며, MIC는 잔토모나스 오라이제균이 100% 생육되지 않은 시료의 최소 농도로 결정하였다. 또한, 대조군으로는 DAPG(2,4-diacetylphloroglucinol)를 사용하였으며, 모든 항균활성은 6회 반복하여 측정하였다.At this time, the antimicrobial activity was expressed as Minimum Inhibitory Concentration (MIC), and MIC was determined as the minimum concentration of the sample in which 100% of Xanthomonas lyase bacteria were not grown. In addition, DAPG (2,4-diacetylphloroglucinol) was used as a control, and all antibacterial activity was measured 6 times.
그 결과, 본 발명의 화학식 1의 화합물이 기존에 알려진 항생제(DAPG)와 비교하여 잔토모나스 오라이제균에 대해 높은 활성을 나타내는 것을 확인하였다(표 2 참조). As a result, it was confirmed that the compound of the formula (1) of the present invention exhibits high activity against Xanthomonas lyase bacteria compared to the known antibiotic (DAPG) (see Table 2).
이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
도 1은 본 발명에 따른 포몹시스 롱지콜라로부터 분리한 신규 화합물의 수소 핵자기공명(1H-NMR) 스펙트럼이다. 1 is a hydrogen nuclear magnetic resonance ( 1 H-NMR) spectrum of a novel compound isolated from pomopsis longji cola according to the present invention.
도 2는 본 발명에 따른 포몹시스 롱지콜라로부터 분리한 신규 화합물의 탄소 핵자기공명(13C-NMR) 스펙트럼이다.2 is a carbon nuclear magnetic resonance ( 13 C-NMR) spectrum of a novel compound isolated from pomopsis longji cola according to the present invention.
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