KR102392744B1 - Composition for Preventing Biofilm Formation Comprising Extract of Leaves of Diospyros kaki - Google Patents
Composition for Preventing Biofilm Formation Comprising Extract of Leaves of Diospyros kaki Download PDFInfo
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- KR102392744B1 KR102392744B1 KR1020170132727A KR20170132727A KR102392744B1 KR 102392744 B1 KR102392744 B1 KR 102392744B1 KR 1020170132727 A KR1020170132727 A KR 1020170132727A KR 20170132727 A KR20170132727 A KR 20170132727A KR 102392744 B1 KR102392744 B1 KR 102392744B1
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/44—Ebenaceae (Ebony family), e.g. persimmon
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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Abstract
시엽 추출물을 포함하는 바이오필름 형성 억제용 조성물 및 바이오필름 형성과 관련된 질환의 예방 및 개선용 조성물에 관한 것이다.It relates to a composition for inhibiting biofilm formation and a composition for preventing and ameliorating diseases related to biofilm formation, including an extract of the leaf leaf.
Description
본 발명은 크산토모나스 속(Xanthomonas sp.) 또는 예르시니아 속(Yersinia sp.) 미생물에 의한 바이오필름 형성을 억제하는, 시엽 추출물을 유효성분으로 함유하는 바이오필름 형성 억제용 조성물 및 바이오필름 형성과 관련된 질환의 예방 및 치료용 조성물에 관한 것이다.The present invention is Xanthomonas genus ( Xanthomonas sp.) or Yersinia genus ( Yersinia sp.) Inhibiting biofilm formation by microorganisms, a composition for inhibiting biofilm formation containing a leaf extract as an active ingredient, and a composition for preventing and treating diseases related to biofilm formation is about
바이오필름(biofilm)은 생물막이라고도 불리우며, 미생물이 세포 주위에 다당체를 생산하고 이것을 매개로 인접한 미생물과 응집하여 고체나 생체 표면에 막(film)을 형성하고 있는 상태를 의미한다. 바이오필름을 형성하는 미생물은 부유 상태의 미생물보다 항생제에 대한 저항성이 1000배 이상 높고, 바이오필름 형성은 세균성 심내막염(bacterial endocarditis), 치아 우식증(dental caries), 만성 부비동염(chronic sinusitis), 식중독, 폐렴 등과 같이 여러 질병의 발병(pathogenesis)에 중요한 역할을 하는 것으로 알려져 있다(비특허문헌 1).Biofilm, also called biofilm, refers to a state in which microorganisms produce polysaccharides around cells and aggregate with adjacent microorganisms as a medium to form a film on a solid or living body surface. Microorganisms that form biofilms are more than 1000 times more resistant to antibiotics than microorganisms in a suspended state, and biofilm formation is associated with bacterial endocarditis, dental caries, chronic sinusitis, food poisoning, and pneumonia. It is known to play an important role in the pathogenesis of various diseases, such as (Non-Patent Document 1).
임상에서는 카테터(catheter), 스텐트(stent)와 같은 의료기구에 대한 바이오필름 오염이 문제가 되고 있다. 의료기구 오염을 유발하는 미생물에는 엔테로코커스 패칼리스(Enterococcus faecalis) 및 스태필로코커스 아우레우스 (Staphylococcus aureus) 등이 있으며, 상기 미생물의 오염으로 인하여 골수염, 독소충격증후군, 식중독 및 피부 감염증 등이 유발될 수 있다.In clinical practice, biofilm contamination on medical devices such as catheters and stents is a problem. Microorganisms that cause contamination of medical devices include Enterococcus faecalis . faecalis ) and Staphylococcus aureus , and the like, and may cause osteomyelitis, toxin shock syndrome, food poisoning and skin infections due to contamination of the microorganisms.
따라서 미생물 감염에 의한 질병을 예방하기 위해서는 미생물의 바이오필름 형성을 효과적으로 억제하는 것이 필요하다. 이러한 바이오필름의 형성을 억제하기 위하여 기존에는 바이오필름을 형성하는 미생물의 생장을 저해하는 항균 활성을 이용하였다. 그러나 항균 활성을 갖는 물질로 바이오필름을 형성하는 미생물의 생장을 억제하면 내성을 갖는 미생물만 남게 되며, 결과적으로 바이오필름의 제거나 형성 억제를 어렵게 할 수 있다. 따라서, 미생물의 생장에 미치는 영향은 줄이면서 바이오필름의 형성을 억제할 수 있는 물질을 개발하는 것이 필요하다.Therefore, in order to prevent diseases caused by microbial infection, it is necessary to effectively inhibit the formation of biofilms of microorganisms. In order to suppress the formation of such a biofilm, an antibacterial activity that inhibits the growth of microorganisms forming a biofilm was used in the past. However, when the growth of microorganisms forming a biofilm is inhibited with a material having antibacterial activity, only resistant microorganisms remain, and as a result, it may be difficult to remove or inhibit the formation of the biofilm. Therefore, it is necessary to develop a material capable of inhibiting the formation of a biofilm while reducing the effect on the growth of microorganisms.
본 발명자들은 천연물질인 시엽 추출물이 바이오필름 형성을 억제하는 능력이 우수한 것을 확인하여 본 발명을 완성하였다.The present inventors have completed the present invention by confirming that the natural material, leaf leaf extract, has excellent ability to inhibit biofilm formation.
본 발명의 일 목적은 크산토모나스 속(Xanthomonas sp.) 또는 예르시니아 속(Yersinia sp.) 미생물에 의한 바이오필름 형성을 억제하는, 시엽 추출물을 유효성분으로 함유하는 바이오필름 형성 억제용 조성물을 제공하는 것이다.One object of the present invention is Xanthomonas genus ( Xanthomonas sp.) or Yersinia genus ( Yersinia sp.) to provide a composition for inhibiting the formation of a biofilm containing a leaf extract as an active ingredient, which inhibits the formation of a biofilm by microorganisms.
본 발명의 다른 목적은 상기 바이오필름 형성 억제용 조성물을 유효성분으로 함유하는 바이오필름 형성과 관련된 질환의 예방 또는 치료용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for preventing or treating diseases related to biofilm formation containing the composition for inhibiting biofilm formation as an active ingredient.
본 발명의 일 양상은 크산토모나스 속(Xanthomonas sp.) 또는 예르시니아 속(Yersinia sp.) 미생물에 의한 바이오필름 형성을 억제하는, 시엽 추출물을 유효성분으로 함유하는 바이오필름 형성 억제용 조성물을 제공한다.One aspect of the present invention is Xanthomonas genus ( Xanthomonas sp.) or Yersinia genus ( Yersinia sp.) provides a composition for inhibiting biofilm formation containing a leaf extract as an active ingredient, which inhibits biofilm formation by microorganisms.
본 명세서에서 사용되는 용어, '바이오필름(biofilm, 생물막)'은 미생물에 의하여 형성된 막 모양의 구조체를 의미하며, 상호 연결된 미생물 세포층 및 미생물 분비물로 이루어진 구조적 특징을 가진다. 미생물은 다당류를 생성하는 효소를 세포 외부로 분비하여 외부 환경에 존재하는 당물질로부터 수용성 또는 불용성 다당류를 형성하며, 이러한 불용성 다당류가 지속적으로 축적되면 필름 형태의 얇은 바이오필름이 형성된다. 형성된 바이오필름은 미생물 사이의 응집에 도움을 주어 결과적으로 낭포성 섬유종(cystic fibrosis), 패혈증과 같은 난치성 감염증 등의 질환을 유발할 수 있다. 또한, 기관이나 구조물의 손상을 일으킬 수 있으며, 카테터와 같은 의료 기구를 오염시킬 수 있다.As used herein, the term 'biofilm (biofilm)' refers to a membrane-like structure formed by microorganisms, and has structural features composed of interconnected microbial cell layers and microbial secretions. Microorganisms secrete enzymes that produce polysaccharides to the outside of cells to form water-soluble or insoluble polysaccharides from sugar substances present in the external environment, and when these insoluble polysaccharides are continuously accumulated, a thin biofilm in the form of a film is formed. The formed biofilm helps aggregation between microorganisms, and as a result, cystic fibrosis and intractable infections such as sepsis can be caused. It can also cause damage to organs or structures and contaminate medical devices such as catheters.
본 발명의 바이오필름 형성 억제용 조성물의 유효성분으로 사용되는 시엽은 감나무과 식물인 감나무(Diospyros kaki L. fil.)의 잎을 말하며, 여름에 잎을 따서 뜨거운 물에 잠깐 담그거나 증기에 쪄서 말린 후 약재로 이용한다. 혈압강하, 혈중 콜레스테롤 강하, 혈액순환 촉진 및 지혈 작용 등의 효능이 알려져 있으나 미생물에 의한 바이오필름의 형성을 억제하는 효과에 대해서는 알려진 바가 없다.The leaf used as an active ingredient of the composition for inhibiting biofilm formation of the present invention is a persimmon tree ( Diospyros ) kaki L. fil.), which are picked in summer and soaked in hot water for a while or steamed and dried before use as medicine. Efficacy of lowering blood pressure, lowering blood cholesterol, promoting blood circulation and hemostasis is known, but the effect of inhibiting the formation of biofilm by microorganisms is not known.
본 발명의 일 구체예에 따르면 상기 시엽 추출물은 당 업계에 공지된 통상의 방법에 따라 제조될 수 있다. 예를 들어, 시엽을 분쇄한 후 추출에 통상적으로 사용되는 용매를 첨가하고, 적절한 온도와 압력으로 추출하여 시엽 추출물을 제조할 수 있다.According to one embodiment of the present invention, the leaf extract may be prepared according to a conventional method known in the art. For example, after pulverizing the leaf, a solvent commonly used for extraction may be added, followed by extraction at an appropriate temperature and pressure to prepare the leaf extract.
본 발명의 일 구체예에 따르면 상기 용매는 정제수, 헥산, 1,3-부틸렌글리콜, 탄소수 1 내지 4의 알코올, 에틸아세테이트, 디에틸에테르, 디클로로메탄, 아세톤 및 이들의 혼합물로 이루어진 군에서 선택될 수 있다.According to one embodiment of the present invention, the solvent is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, alcohols having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, and mixtures thereof. can be
본 발명의 일 구체예에 따르면 상기 용매는 정제수 또는 탄소수 1 내지 4의 알코올일 수 있다.According to one embodiment of the present invention, the solvent may be purified water or an alcohol having 1 to 4 carbon atoms.
한편, 상기 시엽 추출물은 상술한 용매 추출법에 의한 추출물과 통상적인 정제 과정을 거친 추출물을 모두 포함한다. 예를 들어, 일정한 분자량 컷-오프(cut-off) 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시한 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 시엽 추출물에 포함될 수 있다. 또한, 상기 시엽 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.On the other hand, the leaf extract includes both the extract by the solvent extraction method described above and the extract that has undergone a conventional purification process. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the leaf extract of the present invention. In addition, the leaf extract may be prepared in a powder state by an additional process such as distillation under reduced pressure, freeze-drying, or spray-drying.
본 발명의 바이오필름 형성 억제용 조성물은 조성물 전체 중량에 대하여 상기 시엽 추출물을 0.1 내지 20 중량%, 또는 1 내지 10 중량%로 포함할 수 있으며, 조성물의 사용방법 및 사용목적에 따라 유효성분의 함량을 적절히 조절할 수 있다.The composition for inhibiting biofilm formation of the present invention may contain the leaf extract in an amount of 0.1 to 20% by weight, or 1 to 10% by weight, based on the total weight of the composition, and the content of the active ingredient depends on the method and purpose of use of the composition. can be appropriately adjusted.
본 발명의 일 구체예에 따르면 상기 미생물은 크산토모나스 오리재(Xanthomonas oryzae) 및 예르시니아 엔테로콜리티카(Yersinia enterocolitica)로 이루어진 군에서 선택될 수 있으나, 이에 제한되지 아니한다.According to one embodiment of the present invention, the microorganism is Xanthomonas duck material ( Xanthomonas oryzae ) and Yersinia enterocolica ( Yersinia ) enterocolitica ) may be selected from the group consisting of, but is not limited thereto.
본 발명의 일 구체예에 따르면 크산토모나스 오리재가 벼의 도관에서 바이오필름을 형성하면 벼의 흰잎마름병을 유발시킬 수 있다. 또한, 예르시니아 엔테로콜리티카가 형성하는 바이오필름에 의하여 식중독, 장염 등이 유발될 수 있다.According to one embodiment of the present invention, when Xanthomonas duck material forms a biofilm in the conduit of rice, it may cause white leaf blight in rice. In addition, food poisoning, enteritis, etc. may be induced by the biofilm formed by Yersinia enterocolica.
본 발명의 일 구체예에 따르면 바이오필름은 식품의 표면 등과 같이 미생물의 생장 또는 증식이 가능한 곳에서 형성되기 때문에 이를 방지하기 위하여 상기 바이오필름 형성 억제용 조성물을 식품 또는 화장료 조성물로 이용할 수 있다. 또한, 카테터, 스텐트와 같은 의료기구에 대한 바이오필름 형성을 방지하기 위하여 본 발명에 따른 바이오필름 형성 억제용 조성물을 코팅용 조성물로 이용할 수 있다.According to one embodiment of the present invention, since the biofilm is formed in a place where the growth or proliferation of microorganisms is possible, such as on the surface of food, the composition for inhibiting biofilm formation can be used as a food or cosmetic composition to prevent this. In addition, in order to prevent biofilm formation on medical devices such as catheters and stents, the composition for inhibiting biofilm formation according to the present invention may be used as a coating composition.
본 발명의 다른 양상은 상기 바이오필름 형성 억제용 조성물을 유효성분으로 함유하는 벼의 흰잎마름병(bacterial leaf blight of rice) 방제용 조성물을 제공한다.Another aspect of the present invention provides a composition for controlling bacterial leaf blight of rice containing the composition for inhibiting biofilm formation as an active ingredient.
벼의 흰잎마름병은 쌀 생산에 가장 많은 경제적 손실을 야기하는 질병으로 세계적으로 광범위하게 발생한다. 벼의 도관에 크산토모나스 오리재(Xanthomonas oryzae)가 흡착하여 바이오 필름을 형성하면 도관의 통로를 막게 되고, 이를 통해 영양분이나 물의 전달이 원활하지 않게 된다. 이에 따라 잎이 죽기 시작하며, 벼가 죽거나 살아남더라도 쌀을 정상적으로 생산할 수 없게 된다. 따라서 크산토모나스 오리재에 의한 바이오필름의 형성을 억제하면 벼 도관의 막힘 현상을 감소시킬 수 있으며, 크산토모나스 오리재에 감염되어도 벼가 정상적으로 자랄 수 있어 벼의 수확량을 정상적으로 유지할 수 있다.Rice blight is the disease that causes the greatest economic loss in rice production and occurs widely around the world. When Xanthomonas oryzae adsorbs to the conduit of rice to form a biofilm, the passage of the conduit is blocked, thereby preventing the smooth delivery of nutrients or water. As a result, the leaves begin to die, and even if the rice dies or survives, normal rice production is not possible. Therefore, suppressing the formation of a biofilm by Xanthomonas duckweed can reduce the clogging of rice ducts, and even if infected with Xanthomonas duckweed, rice can grow normally, thereby maintaining normal rice yield.
상기 벼의 흰잎마름병 방제용 조성물은 직접 분사 가능한 용액, 분말 및 현탁액의 형태 또는 고농축 수성, 유성 또는 다른 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 흩뿌림 물질 또는 과립제로 제조될 수 있으나, 이에 제한되지 아니한다. 또한, 상기 방제용 조성물은 분사, 분무, 살포, 흩뿌림 또는 붓기에 의해 사용될 수 있다. The composition for controlling white leaf blight of rice may be prepared in the form of solutions, powders and suspensions that can be directly sprayed, or highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering materials or granules, It is not limited thereto. In addition, the control composition may be used by spraying, spraying, spraying, scattering or pouring.
또한, 상기 흰잎마름병 방제용 조성물은 다양한 형태로 제제화될 수 있으며, 예를 들어 용매 및/또는 담체를 첨가함으로서 제조될 수 있다. 적절한 비활성 첨가제 및 표면-활성 물질, 예를 들어 유화제 또는 분산제를 제제에 혼합할 수 있다.In addition, the composition for controlling white leaf blight may be formulated in various forms, for example, it may be prepared by adding a solvent and/or a carrier. Suitable inert additives and surface-active substances, for example emulsifying or dispersing agents, may be admixed into the formulation.
본 발명의 일 구체예에 따르면 상기 벼의 흰잎마름병 방제용 조성물은 시엽 메탄올 추출물을 포함하는 바이오필름 형성 억제용 조성물을 포함할 수 있다.According to one embodiment of the present invention, the composition for controlling white leaf blight of rice may include a composition for inhibiting biofilm formation including a methanol extract of leaf leaves.
본 발명의 또 다른 양상은 상기 바이오필름 형성 억제용 조성물을 유효성분으로 함유하는 예르시니아 엔테로콜리티카에 의해 발생하는 질병의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by Yersinia enterocolica containing the composition for inhibiting biofilm formation as an active ingredient.
본 발명의 일 구체예에 따르면 상기 질병은 급성위장염, 종말회장염, 패혈증, 결핵성 홍반, 식중독 및 장염으로 이루어진 군에서 선택될 수 있으나, 이제 제한되지 아니한다.According to one embodiment of the present invention, the disease may be selected from the group consisting of acute gastroenteritis, terminal ileitis, sepsis, tuberculous erythema, food poisoning and enteritis, but is not limited thereto.
예르시니아 엔테로콜리티카(Yersinia enterocolitica)는 장내세균과에 속하는 그람 음성(Gram-negative) 세균이며, 위장염(gastroenteritis; 식중독 및 장염)을 유발한다. 사람에서는 급성 위장염, 종말회장염, 패혈증, 결핵성 홍반 등 다양한 질병을 발생시키며, 바이오필름을 형성함으로써 외부의 자극이나 항생제에 대하여 저항성을 가지는 것으로 알려져 있다. Yersinia Enterocolica enterocolitica ) is a Gram-negative bacteria belonging to the Enterobacteriaceae family, and causes gastroenteritis (food poisoning and enteritis). In humans, it causes various diseases such as acute gastroenteritis, terminal ileitis, sepsis, tuberculous erythema, and is known to have resistance to external stimuli or antibiotics by forming a biofilm.
본 발명의 일 구체예에 따르면 시엽 추출물이 예르시니아 엔테로콜리티카에 의한 바이오필름 형성을 효과적으로 억제할 수 있으므로 바이오필름 형성 억제용 조성물을 예르시니아 엔테로콜리티카에 의해 발생하는 질병의 예방 또는 치료 용도로 유용하게 이용할 수 있다.According to one embodiment of the present invention, since the leaf extract can effectively inhibit biofilm formation by Yersinia enterocolytica, the composition for inhibiting biofilm formation is used for preventing or treating diseases caused by Yersinia enterocolitica It can be usefully used for
본 발명의 약학적 조성물은 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기에서 '약학적으로 허용되는'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.
본 발명에 따른 약학적 조성물은 상술한 바와 같은 약학적으로 허용되는 담체와 함께 당 업계에 공지된 방법에 따라 적합한 형태로 제형화 될 수 있다. 즉, 본 발명의 약학적 조성물은 공지의 방법에 따라 다양한 비경구 또는 경구 투여용 형태로 제조될 수 있다. 비경구 투여용 제형의 대표적인 것으로는 주사용 제형으로 등장성 수용액 또는 현탁액이 바람직하다. 주사용 제형은 적합한 분산제 또는 습윤제 및 현탁화제를 사용하여 당 업계에 공지된 기술에 따라 제조할 수 있다. 예를 들면, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화될 수 있다. 또한, 경구 투여용 제형으로는, 이에 한정되지는 않으나, 분말, 과립, 정제, 환약 및 캡슐 등이 있다.The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods. A typical example of a formulation for parenteral administration is an isotonic aqueous solution or suspension as an injection formulation. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.
상기와 같은 방법으로 제형화된 약학적 조성물은 유효량으로 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 상기에서 '유효량'이란 환자에게 투여하였을 때, 예방 또는 치료 효과를 나타내는 양을 말한다. 본 발명에 따른 약학적 조성물의 투여량은 투여 경로, 투여 대상, 연령, 성별 체중, 개인차 및 질병 상태에 따라 적절히 선택할 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있으며, 비경구 투여의 형태일 때 1 내지 100 mg/kg/day의 유효량으로 투여할 수 있다.The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, administration, age, sex and weight, individual differences and disease state. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, and may be administered in an effective amount of 1 to 100 mg/kg/day in the form of parenteral administration.
시엽 추출물은 미생물이 생성하는 바이오필름의 형성을 효과적으로 억제할 수 있으므로 바이오필름 형성 억제용 조성물 및 바이오필름 형성과 관련된 질환의 예방 또는 치료용 조성물로 유용하게 이용될 수 있다.Since the leaf leaf extract can effectively inhibit the formation of biofilms generated by microorganisms, it can be usefully used as a composition for inhibiting biofilm formation and a composition for preventing or treating diseases related to biofilm formation.
도 1은 시엽 추출물이 예르시니아 엔테로콜리티카(Yersinia enterocolitica)의 바이오필름 형성에 미치는 효과를 확인한 결과를 보여주는 그래프이다: A는 시엽 메탄올 추출물, B는 시엽 물 추출물, C는 시엽 50% 에탄올 추출물 및 D는 시엽 95% 에탄올 추출물 결과이며, X축의 '희석 비율'은 시엽 추출물의 희석 비율을 의미한다.
도 2는 시엽 추출물이 예르시니아 엔테로콜리티카의 생장에 미치는 효과를 확인한 결과를 보여주는 그래프이다: A는 시엽 메탄올 추출물, B는 시엽 물 추출물, C는 시엽 50% 에탄올 추출물 및 D는 시엽 95% 에탄올 추출물 결과이며, X축의 '희석 비율'은 시엽 추출물의 희석 비율을 의미한다.
도 3은 시엽 메탄올 추출물이 크산토모나스 오리재(Xanthomonas oryzae)에 의한 바이오필름 형성과 크산토모나스 오리재의 생장에 미치는 효과를 확인한 결과를 보여주는 그래프이다: A는 바이오필름 생성량, B는 세포 밀도 측정 결과이며, X축의 '희석 비율'은 시엽 추출물의 희석 비율을 의미한다.1 shows that the leaf extract is Yersinia enterocolica ( Yersinia enterocolitica ) is a graph showing the results of confirming the effect on biofilm formation: A is a methanol extract of young leaves, B is a water extract of young leaves, C is a result of 50% ethanol extract of young leaves, and D is a result of 95% ethanol extract of young leaves, and the ' The dilution ratio' means the dilution ratio of the leaf extract.
2 is a graph showing the results of confirming the effect of the leaf extract on the growth of Yersinia enterocolica: A is a methanol extract of the leaves, B is a water extract of the leaves, C is a 50% ethanol extract of the leaves, and D is a 95% of the leaves. It is the result of ethanol extract, and the 'dilution ratio' on the X-axis means the dilution ratio of the leaf extract.
3 is a graph showing the results of confirming the effect of the methanol extract on the growth of Xanthomonas duckjae and biofilm formation by Xanthomonas oryzae : A is biofilm production amount, B is cell density measurement As a result, the 'dilution ratio' on the X-axis means the dilution ratio of the leaf extract.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
실시예Example 1: One: 시엽early leaf 추출물의 제조 Preparation of extracts
시엽 10g에 물, 메탄올, 50% 에탄올 및 95% 에탄올 100㎖를 각각 첨가하고, 50℃ 항온 수조에서 3시간 동안 추출하였다. 이후 여과지(filter paper)를 사용하여 추출물을 1차 여과하고, 여과된 1차 추출물의 부피를 측정하였다.Water, methanol, 50% ethanol, and 100 ml of 95% ethanol were each added to 10 g of the leaf, and extraction was performed in a water bath at 50° C. for 3 hours. Then, the extract was first filtered using filter paper, and the volume of the filtered primary extract was measured.
물 추출물은 셀룰로스 아세테이트 필터(cellulose acetate filter; 0.2 ㎛ pore size)로 추가로 여과한 후 실험에 이용하였다. 메탄올 추출물, 50% 및 95% 에탄올 추출물은 감압 농축기(rotary evaporator)로 추출물 전체 부피의 10%까지 농축한 후 다시 여과하였다. 여과액에 1차 추출물의 부피만큼 물을 첨가하고, 셀룰로스 아세테이트 필터로 추가로 여과한 후 실험에 이용하였다.The water extract was further filtered with a cellulose acetate filter (0.2 μm pore size) and then used for the experiment. The methanol extract, 50% and 95% ethanol extract were concentrated to 10% of the total volume of the extract with a rotary evaporator, and then filtered again. Water was added as much as the volume of the primary extract to the filtrate, and it was further filtered through a cellulose acetate filter and used in the experiment.
실시예Example 2: 2: 시엽early leaf 추출물의 바이오필름 형성 억제 효과 Inhibitory effect of extracts on biofilm formation
2-1. 2-1. 예르시니아Yersinia 엔테로콜리티카에in Enterocolica 대한 바이오필름 형성 억제 효과 Inhibitory effect on biofilm formation
예르시니아 엔테로콜리티카(Yersinia enterocolitica; 이하, Y. enterocolitica로 기재함)는 TYE 한천 플레이트(1% tryptone, 0.5% yeast extract, 1.5% agar)에 도말(streaking)한 후, 26℃에서 2일 동안 배양하였다. 플레이트에서 단일 군집(single colony)을 하나 취하여 TYE 액체배지 5㎖에 접종하고, 26℃에서 1일 동안 250rpm으로 진탕배양하였다. Yersinia Enterocolica enterocolitica ; Hereinafter, Y. enterocolitica ) was streaked on a TYE agar plate (1% tryptone, 0.5% yeast extract, 1.5% agar), and then cultured at 26°C for 2 days. A single colony was taken from the plate, inoculated into 5 ml of TYE broth, and cultured with shaking at 26° C. for 1 day at 250 rpm.
96-웰 폴리비닐 클로라이드(polyvinyl chloride, PVC) 마이크로플레이트에 M63 액체배지(3g/L KH2PO4, 7g/L K2HPO4, 2g/L (NH4)2SO4, 0.5 mg/L FeSO4, 0.2% glucose, 0.02% MgSO4)를 100 ㎕씩 분주하였다. 여기에 실시예 1에서 제조한 메탄올 추출물을 1㎕ 분주하고, 물 추출물과 50% 에탄올 및 95% 에탄올 추출물은 각각 5㎕를 분주하였다. 다음으로 각각의 웰에 상기 배양한 Y. enterocolitica를 OD600이 0.05가 되도록 접종하여 26℃에서 24시간 동안 배양하였다. 24시간 후 OD595에서 흡광도(optical density, OD; absorbance)를 측정하여 Y. enterocolitica의 세포 밀도를 확인하고, 플레이트에서 배양액을 제거한 후 증류수로 3번 부드럽게 세척하였다. 각각의 웰에 1% 크리스탈 바이올렛(crystal violet) 용액 100㎕를 분주하여 15분 동안 상온에서 바이오필름을 염색하였다. 15분 후 증류수로 3번 세척하고, 크리스탈 바이올렛을 용출시키기 위하여 95% 에탄올 100㎕를 첨가하여 15분 동안 방치하였다. 이후 마이크로플레이트 리더(Microplate reader)로 OD595에서 흡광도를 측정하여 형성된 바이오필름의 양을 정량하였다.M63 broth (3 g/L KH 2 PO 4 , 7 g/LK 2 HPO 4 , 2 g/L (NH 4 ) 2 SO 4 , 0.5 mg/L FeSO in 96-well polyvinyl chloride (PVC) microplates 4 , 0.2% glucose, 0.02% MgSO 4 ) was dispensed in 100 μl each. Here, 1 μl of the methanol extract prepared in Example 1 was dispensed, and 5 μl of each of the water extract, 50% ethanol and 95% ethanol extract was dispensed. Next, the cultured Y. enterocolitica was inoculated into each well to have an OD 600 of 0.05 and cultured at 26° C. for 24 hours. After 24 hours, the absorbance (optical density, OD; absorbance) was measured at OD 595 to confirm the cell density of Y. enterocolitica , and after removing the culture medium from the plate, it was gently washed 3 times with distilled water. 100 μl of a 1% crystal violet solution was dispensed into each well and the biofilm was stained at room temperature for 15 minutes. After 15 minutes, it was washed 3 times with distilled water, and 100 μl of 95% ethanol was added to elute the crystal violet and left for 15 minutes. Thereafter, the amount of the biofilm formed was quantified by measuring the absorbance at OD 595 with a microplate reader.
확인 결과, 도 1에 나타난 바와 같이 시엽 추출물에 의하여 바이오필름 형성이 억제되는 것을 확인할 수 있었으며, 특히 시엽 메탄올 추출물이 현저한 바이오필름 형성 억제 효과를 나타내었다.As a result of the confirmation, as shown in FIG. 1 , it was confirmed that the biofilm formation was inhibited by the leaf extract, and in particular, the methanol extract of the leaf leaf showed a remarkable biofilm formation inhibitory effect.
2-2. 크산토모나스 오리재에 대한 바이오필름 형성 억제 효과2-2. Inhibitory effect of biofilm formation on Xanthomonas duck material
크산토모나스 오리재(Xanthomonas oryzae; 이하, X. oryzae로 기재함)는 YGC 한천 플레이트(5% glucose, 0.5% yeast extracts, 1.25% CaCO3, 1.5% agar)에 도말한 후, 28℃에서 2일 동안 배양하였다. 플레이트에서 단일 군집을 하나 취하여 210 액체배지(0.5% sucrose, 0.8% casein enzymatic hydrolysate, 0.4% yeast extract, 0.3% K2HPO4, 0.03% MgSO47H2O) 5㎖에 접종하고, 28℃에서 2일 동안 250rpm으로 진탕배양하였다.Xanthomonas duck material ( Xanthomonas oryzae ; hereinafter referred to as X. oryzae ) was smeared on YGC agar plates (5% glucose, 0.5% yeast extracts, 1.25% CaCO 3 , 1.5% agar), followed by 2 at 28 ° C. incubated for one day. A single colony is taken from the plate and inoculated into 5 ml of 210 liquid medium (0.5% sucrose, 0.8% casein enzymatic hydrolysate, 0.4% yeast extract, 0.3% K 2 HPO 4 , 0.03% MgSO 4 7H 2 O), and inoculated at 28°C. Shaking culture was performed at 250 rpm for 2 days.
96-웰 폴리비닐 클로라이드 마이크로플레이트에 XOM2 액체배지(670μM L-methionine, 10 mM sodium L-(+)-glutamate monohydrate, 14.7mM KH2PO4, 40μM MnSO4, 240μ Fe(III)-EDTA, 5 mM MgCl2, 0.18% D-xylose, pH6.5)를 사용한 것을 제외하고, 상기 2-1과 동일한 방법으로 시엽 메탄올 추출물의 바이오필름 형성 억제 효과를 확인하였다.XOM2 broth (670 μM L-methionine, 10 mM sodium L-(+)-glutamate monohydrate, 14.7 mM KH 2 PO 4 , 40 μM MnSO 4 , 240 μ Fe(III)-EDTA, 5 mM MgCl 2 , 0.18% D-xylose, pH6.5) was confirmed, but the biofilm formation inhibitory effect of the methanol extract of the leaf leaves was confirmed in the same manner as in 2-1.
확인 결과, 도 3에 나타난 바와 같이 시엽 메탄올 추출물에 의하여 바이오필름 형성이 현저하게 억제되는 것을 알 수 있었다.As a result of the confirmation, as shown in FIG. 3 , it was found that the biofilm formation was significantly inhibited by the methanol extract.
본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 구체예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.
Claims (6)
Xanthomonas genus ( Xanthomonas sp.) or Yersinia genus ( Yersinia sp.) Inhibits biofilm formation by microorganisms, containing the leaf extract as an active ingredient, Xanthomonas genus ( Xanthomonas sp.) or Yersi A composition for inhibiting biofilm formation by Yersinia sp. microorganisms.
The method of claim 1, wherein the leaf extract is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, alcohols having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, and mixtures thereof. A composition that is extracted with a solvent.
According to claim 1, wherein the microorganism is Xanthomonas duck material ( Xanthomonas oryzae ) and Yersinia enterocolica ( Yersinia ) enterocolitica ) a composition selected from the group consisting of.
A composition for controlling bacterial leaf blight of rice, containing the composition of claim 1 as an active ingredient.
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