KR101106052B1 - Antimicrobial composition for pathogenic microorganism comprising plant or extract thereof - Google Patents
Antimicrobial composition for pathogenic microorganism comprising plant or extract thereof Download PDFInfo
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- KR101106052B1 KR101106052B1 KR1020080111792A KR20080111792A KR101106052B1 KR 101106052 B1 KR101106052 B1 KR 101106052B1 KR 1020080111792 A KR1020080111792 A KR 1020080111792A KR 20080111792 A KR20080111792 A KR 20080111792A KR 101106052 B1 KR101106052 B1 KR 101106052B1
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- extract
- plant
- haemopillus
- leaf
- lime
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/752—Citrus, e.g. lime, orange or lemon
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/314—Foods, ingredients or supplements having a functional effect on health having an effect on lung or respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
Abstract
본 발명은 식물 또는 그 추출물을 유효성분으로 포함하는 병원성 미생물에 대한 항생용 조성물, 그 제조방법 및 상기 조성물의 사료에 응용에 관한 것이다.The present invention relates to a composition for antibiotic against pathogenic microorganisms comprising a plant or an extract thereof as an active ingredient, a method for preparing the same, and an application of the composition to feed.
열대 식물, 항생, 사료 Tropical plants, antibiotics, feed
Description
본 발명은 식물의 추출물을 유효성분으로 포함하는 병원성 미생물에 대한 항생용 조성물에 관한 것으로 더욱 상세하게는 사이트러스 아우란티롤리아 스윙글(Citrus aurantifolia swingle;Lime), 사이트러스 아우란티롤리아 잎(Citrus aurantifolia swingle leaf;Lime leaf), 쿠르쿠마 도메스티가 바렌톤(Curcuma domestica Valeton;Turmeric), 세스바니아 그랜디프로라 (Sesbania grandiflora L). 및 파이퍼 사르멘토섬 로스브(Piper sarmentosum Roxb)로 구성된 군으로부터 선택된 하나 이상의 식물 및/또는 그 추출물을 유효성분으로 포함하는 병원성 미생물에 대한 항생용 조성물에 관한 것이다.The present invention relates to a composition for antibiotics against pathogenic microorganisms comprising an extract of a plant as an active ingredient, and in more detail, Citrus aurantifolia swingle (Lime), Citrus aurantirolia leaf (Citrus aurantifolia swingle leaf; Lime leaf), Curcuma domestica Valeton (Turmeric), Sesbania grandiflora L. And Piper Sarmentosum Roxb (Piper sarmentosum Roxb) relates to a composition for antibiotics against pathogenic microorganisms comprising one or more plants and/or extracts thereof selected from the group consisting of as an active ingredient.
일반적으로 항생제는 인간뿐 아니라 축산업에 있어서 매우 중요한 역할을 한다. 과거 축산의 발전에 따라서 단위 면적당 사육두수가 증가되고 생산성을 향상시키기 위하여 질병의 예방 및 치료는 성공적인 축산의 중요한 요인으로 작용하였다. 이에 다양한 항생제와 성장촉진제들이 개발되었고, 현장에서 사용되어져 왔다. 이러한 항생제 사용량의 증가는 축산물에 대한 오염을 초래하였고, 더 나아가 생물농축현상을 통하여 축산물을 소비하는 인간의 생명을 위협하는 상황까지도 초래하였다.In general, antibiotics play a very important role not only in humans but also in the livestock industry. With the development of livestock in the past, the number of breeding heads per unit area increases, and in order to improve productivity, the prevention and treatment of diseases served as an important factor in successful livestock production. Accordingly, various antibiotics and growth promoters have been developed and have been used in the field. Such an increase in the amount of antibiotics used has resulted in contamination of livestock products, and further, a situation that threatens the lives of humans who consume livestock products through bioaccumulation.
또 항생제의 사용은 축산업의 발전에 있어 중요한 역할을 하나 내성균의 발생 등 다양한 부작용을 초래하였고, 또한 최근 친환경 농산물의 소비량 증가 및 요구도 증가로 인하여 항생제의 사용에 대한 부정적인 인식이 증가하였다. 실제로 EU(European Union)에서는 avilamycin, flavophopholipidol, salinomycin, monensin 4 종류의 항생제만을 허용하는 등 항생제에 대한 사용 규제를 강화하고 있는 실정이다. 또한 현재와 같이 급변하는 자유무역 체계에서 축산업의 생존을 위해서는 항생제의 사용을 원천적으로 배제하고 환경친화적으로 가축을 사육하는 것이 축산업 발전 및 국가 농업 발전에 있어 매우 중요한 과제이다. In addition, although the use of antibiotics plays an important role in the development of the livestock industry, it has caused various side effects such as the occurrence of resistant bacteria. Also, negative perceptions about the use of antibiotics have increased due to the increase in consumption and demand of eco-friendly agricultural products. In fact, the EU (European Union) is tightening restrictions on the use of antibiotics, such as allowing only four types of antibiotics: avilamycin, flavophopholipidol, salinomycin, and monensin. In addition, for the survival of the livestock industry in the current rapidly changing free trade system, the use of antibiotics is fundamentally excluded and the raising of livestock in an environmentally friendly manner is a very important task for the development of the livestock industry and the development of national agriculture.
친환경 축산의 구축을 위하여 항생제의 기능을 대체할 수 있는 환경친화적인 물질의 개발이 중요한 과제이다. 친환경 항생제 대체제는 다양한 분야에서 접근되어져 왔으며, 크게 병원성 미생물에 대하여 직접적인 길항작용을 나타내는 유용 미생물들을 중심으로한 생균제 (Probiotics)분야와 식물 및 기타 천연물질로부터 항균활성을 나타내는 물질을 개발하여 상용화하는 추출물 분야로 나뉠 수 있다 (김수 기. 2005. 사료첨가용 식물추출물의 이용과 기능, 월간 피드저널).For the establishment of eco-friendly livestock, the development of eco-friendly materials that can replace the function of antibiotics is an important task. Eco-friendly antibiotic substitutes have been approached in various fields, and extracts that develop and commercialize substances that exhibit antibacterial activity from plants and other natural substances, mainly in the field of probiotics, mainly for useful microorganisms that exhibit direct antagonism against pathogenic microorganisms. It can be divided into fields (Soogi Kim. 2005. Use and Function of Plant Extracts for Feed Addition, Monthly Feed Journal).
향료식물(herbs)과 같은 유용한 식물자원들은 직접적인 항균활성 기능 뿐만 아니라 수많은 생리활성물질 (bio-active chemical compounds)과 비타민, 칼슘, 철분 등의 다양한 영양소를 가지고 있어 질병예방 및 치료에 적용되어져 왔다. Useful plant resources such as herbs have not only direct antibacterial activity, but also numerous bio-active chemical compounds and various nutrients such as vitamins, calcium, and iron, so they have been applied to disease prevention and treatment.
다양한 식물자원중 향료 식물은 일반적으로 향기가 있는 다년생 식물로서 식용, 향료, 의약 등으로 사용되어져 왔다. 특히 마늘, 겨자무(horseradish), 겨자, oregano 등은 일반 식품으로 널리 사용하여 왔고, 인삼, 향 아카시아, 쑥, 국화, 꽃창포, 구절초, 장미, 백일초, 천일홍, 계피나무, 향나무, 유자, 탱자 등의 향료식물들은 생리활성 기능이 우수하여 널리 이용되어져오고 있다.Among various plant resources, perfume plants are generally fragrant perennial plants and have been used for food, flavoring, medicine, and the like. In particular, garlic, horseradish, mustard, oregano have been widely used as general foods, and ginseng, incense acacia, mugwort, chrysanthemum, iris, Gujeolcho, rose, zinnia, cheonilhong, cinnamon, juniper, yuzu, tanja Perfume plants, such as, have been widely used because of their excellent physiological activity.
식물자원에는 생리활성 기능 외에 다양한 병원성 미생물에 대한 항균활성을 나타내는 물질을 함유하고 있고, 식물자원에 존재하는 항균성 물질을 항균소재로 이용하고자 하는 연구는 식품, 의약 및 생물공학산업 등에서 오래전부터 활발하게 연구되어져 오고 있다. (Shin. D.H, Kim. M.S, Han. J.S. 1997. Korean J. Food SCI. TECHNOL. Vol. 29, pp. 808~816; Yang. M.S, Ha. Y.L, Nam. S.H, Choi. S.U, Jang. D.S. 1995. Korean SOC. ApplI. Bio. Chem. Vol. 38, pp. 584~589). Plant resources contain substances that exhibit antimicrobial activity against various pathogenic microorganisms in addition to their physiological functions, and research to use antimicrobial substances present in plant resources as antibacterial materials has been active since long ago in the food, medicine and biotechnology industries. It has been studied. (Shin. DH, Kim. MS, Han. JS 1997. Korean J. Food SCI. TECHNOL. Vol. 29, pp. 808~816; Yang. MS, Ha. YL, Nam. SH, Choi. SU, Jang. DS 1995. Korean SOC. ApplI. Bio. Chem. Vol. 38, pp. 584~589).
항균활성 및 약리활성을 지닌 식물자원은 지구상에 약 250,000~500,000종이 존재하고 있으나 이중 단지 1%만이 항균활성 및 생리활성에 대한 가능성을 평가받은 실정이다 (Cowan. M.M. 1999. Clin. Microbiol. Rev. 14, 564.584.). 이에 다양한 식물자원의 탐색 및 항균활성 스크리닝 연구는 매우 가치가 있는 분야로서 항생제 대체물질 개발에 있어 중요한 역할을 할 수 있다.Plant resources with antimicrobial and pharmacological activity exist on the earth of about 250,000-500,000 species, but only 1% of them have been evaluated for their potential for antimicrobial and physiological activities (Cowan. MM 1999. Clin. Microbiol. Rev. 14, 564.584.). Accordingly, the search for various plant resources and the study of screening for antimicrobial activity are very valuable fields and can play an important role in the development of antibiotic substitutes.
본 발명은 상기의 문제점을 해결하고, 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 항생제의 사용을 대체할 수 있는 식물 추출물을 제공하는 것이다. The present invention solves the above problems and has been conceived by the necessity of the above, and an object of the present invention is to provide a plant extract that can replace the use of antibiotics.
본 발명은 또 다른 목적은 상기 식물 또는 그 식물 추출물의 사료의 용도를 제공하는 것이다. Another object of the present invention is to provide the use of the plant or the plant extract as a feed.
상기의 목적을 달성하기 위하여 본 발명은 사이트러스 아우란티롤리아 스윙글(Citrus aurantifolia swingle;Lime), 사이트러스 아우란티롤리아 잎(Citrus aurantifolia swingle leaf;Lime leaf), 쿠르쿠마 도메스티가 바렌톤(Curcuma domestica Valeton;Turmeric), 세스바니아 그랜디프로라 (Sesbania grandiflora L), 및 파이퍼 사르멘토섬 로스브(Piper sarmentosum Roxb)로 구성된 군으로부터 선택된 하나 이상의 식물 또는 그 식물의 추출물을 유효성분으로 포함하는 병원성 미생물에 대한 항생용 조성물을 제공한다.In order to achieve the above object, the present invention is a citrus aurantifolia swingle (Citrus aurantifolia swingle; Lime), a citrus aurantifolia swingle leaf (Lime leaf), Curcuma domestiga barrenton (Curcuma domestica Valeton; Turmeric), Sesbania grandiflora (Sesbania grandiflora L), and Piper sarmentosum Roxb (Piper sarmentosum Roxb) containing at least one plant selected from the group consisting of or an extract of the plant as an active ingredient It provides a composition for antibiotics against pathogenic microorganisms.
또한 본 발명은 사이트러스 아우란티롤리아 스윙글(Citrus aurantifolia swingle;Lime), 사이트러스 아우란티롤리아 잎(Citrus aurantifolia swingle leaf;Lime leaf), 쿠르쿠마 도메스티가 바렌톤(Curcuma domestica Valeton;Turmeric), 세스바니아 그랜디프로라 (Sesbania grandiflora L), 및 파이퍼 사르멘토섬 로스브(Piper sarmentosum Roxb) 으로 구성된 군으로부터 선택된 하나 이상의 식물을 물 또는 유기용매로 추출하고 정제하는 단계를 포함하는 병원성 미생물에 대한 항생용 조성물 제조방법을 제공한다.In addition, the present invention is Citrus aurantifolia swingle (Citrus aurantifolia swingle; Lime), Citrus aurantifolia swingle leaf (Lime leaf), Curcuma domestica Valeton; Turmeric ), Sesbania grandiflora (Sesbania grandiflora L), and Piper sarmentosum Roxb (Piper sarmentosum Roxb) to pathogenic microorganisms comprising the step of extracting and purifying one or more plants selected from the group consisting of water or an organic solvent It provides a method for preparing a composition for antibiotics.
또한 본 발명은 사이트러스 아우란티롤리아 스윙글(Citrus aurantifolia swingle;Lime), 사이트러스 아우란티롤리아 잎(Citrus aurantifolia swingle leaf;Lime leaf), 쿠르쿠마 도메스티가 바렌톤(Curcuma domestica Valeton;Turmeric), 세스바니아 그랜디프로라 (Sesbania grandiflora L), 및 파이퍼 사르멘토섬 로스브(Piper sarmentosum Roxb)로 구성된 군으로부터 선택된 하나 이상의 식물 또는 그 식물의 추출물을 포함하는 사료용 조성물을 제공한다.In addition, the present invention is Citrus aurantifolia swingle (Citrus aurantifolia swingle; Lime), Citrus aurantifolia swingle leaf (Lime leaf), Curcuma domestica Valeton; Turmeric ), Sesbania grandiflora (Sesbania grandiflora L), and Piper sarmentosum Roxb (Piper sarmentosum Roxb) provides a composition for feed comprising at least one plant selected from the group consisting of or an extract of the plant.
이하, 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명은 적합한 담체 또는 사료 보충물의 형태에 혼합된 상기 본 발명의 식물 또는 그 추출물물을 활성성분으로 구성된 수의 또는 축산 용도의 조성물에 관한 것이다.The present invention relates to a composition for veterinary or livestock use, comprising an active ingredient of the plant of the present invention or an extract thereof mixed in the form of a suitable carrier or feed supplement.
언급된 조성물은 항생제의 결점과 부작용의 유발 없이, 동물 특히 가축 예를 들어 돼지(swine), 소(bovine), 양(ovine), 닭, 또는 말(equine) 등에 유용하게 사용될 수 있다.The mentioned composition can be usefully used in animals, especially livestock, such as pigs, bovines, ovines, chickens, or horses, without causing defects and side effects of antibiotics.
복용량은 동물의 종(species), 크기(size), 무게(weight), 나이(age)와 같은 다수의 요인들에 좌우될 것이다. 원칙적으로, 전형적인 복용량은 동물/일 당 0.1 - 10 g의 추출물 또는 식물 범위일 것이다.Dosage will depend on a number of factors such as the animal's species, size, weight, and age. In principle, a typical dosage will range from 0.1-10 g of extract or plant per animal/day.
본 발명에 따른 사용 가능한 추출물은 다음과 같이 구성된 공정에 의해 제조될 수 있다 :a) 용매로 상기 식물을 추출하고, 그리고 선택적으로 b) 물 불용성으로부터 정제한다.Usable extracts according to the invention can be prepared by a process consisting of: a) extracting the plant with a solvent, and optionally b) purifying from water insoluble.
본 발명의 식물 또는 식물의 일부의 추출물을 생성하는 경우, 물, 유기 용매 또는 이의 혼합물은 전형적인 방법에 따라 제조될 수 있지만, 무수 증류물 또한 적용될 수 있다. 적합한 유기 용매의 예는 메탄올, 에탄올, 프로판올, 부탄올, 메틸 아세테이트, 에틸 아세테이트, 메틸 에틸 에테르, 다이에틸 에테르, 메틸렌 클로라이드, 클로로폼, 사염화탄소, 벤젠, 톨루엔, 헥산, 바셀린 또는 아세톤이다. 본 발명의 추출은 또한 상기의 용매 추출 및/또는 기계적인 추출의 사용을 수반한다. 추출 온도는 5 ℃에서 선택된 용매의 끊는 온도까지의 범위가 가능하다.When producing an extract of the plant or plant part of the present invention, water, organic solvents or mixtures thereof may be prepared according to typical methods, but anhydrous distillate may also be applied. Examples of suitable organic solvents are methanol, ethanol, propanol, butanol, methyl acetate, ethyl acetate, methyl ethyl ether, diethyl ether, methylene chloride, chloroform, carbon tetrachloride, benzene, toluene, hexane, petrolatum or acetone. The extraction of the present invention also entails the use of the above solvent extraction and/or mechanical extraction. The extraction temperature can range from 5 °C to the boiling temperature of the selected solvent.
천연 추출물은 물에 녹지 않는 것을 제거하여 정제시킬 수 있다(단계(b)).Natural extracts can be purified by removing those insoluble in water (step (b)).
단계(a)에서 천연 추출물은 용액에서 알콜(또는 위에 인용된 것들중 사용된 어떤 다른 용매)을 완전히 제거하는, 60 ℃의 감압 하에서 농축시키고, 다음으로 추출물은 5 % w/w - 55 % w/w, 바람직하게는 25 % w/w 건조 잔사로 농축시키고, 온도를 1 ℃ - 30 ℃, 바람직하게는 4 ℃로, 1 시간 - 32 시간, 바람직하게는 16 시간 동안 교반 없이 유지시켰다.In step (a) the natural extract is concentrated under reduced pressure at 60° C., which completely removes the alcohol (or any other solvent used among those cited above) from the solution, then the extract is 5% w/w-55% w /w, preferably 25% w/w, was concentrated to a dry residue, and the temperature was maintained at 1°C-30°C, preferably 4°C, for 1 hour-32 hours, preferably 16 hours without stirring.
정제된 추출물로 구성하는, 얻어진 맑은 용액을 원심분리(centrifugation)시켜 불용물로부터 분리시켰다.The resulting clear solution, consisting of a purified extract, was separated from insoluble matter by centrifugation.
동물에 투여되는 성분의 총 양은 성분이 필수 화합물인지, 식물 물질의 추출물인지 식물 물질인지에 따른다. 성분이 단지 식물 물질로 구성되는 경우, 투여되는 양은 일반적으로 하루에 체중 1kg당 0.02 내지 10g이다.The total amount of an ingredient administered to an animal depends on whether the ingredient is an essential compound, an extract of a plant substance or a plant substance. When the component consists only of plant material, the amount administered is generally 0.02 to 10 g/kg body weight per day.
본 발명의 조성물은 본 발명의 성분이 아닌 다른 성분을 함유할 수 있다. 적합하게는 첨가제는 성장 촉진제, 향료, 흡수 지지체 및/또는 다른 사료 성분 뿐만 아니라, 0.1 내지 100중량%, 바람직하게는 0.2 내지 90중량%의 성분을 함유한다. 바람직하게는, 성장 촉진제, 향료, 흡수 지지체 및/또는 다른 사료 성분의 총 양은 첨가제의 10 내지 75중량%이다. 임의의 성분이 식물 물질인 경우, 이들은 첨가제 중의 임의의 다른 성분과 혼합되기 전에 적합하게 건조되고, 미립자 또는 분말로 분쇄된다.The compositions of the present invention may contain components other than those of the present invention. Suitably the additive contains 0.1 to 100% by weight, preferably 0.2 to 90% by weight of ingredients, as well as growth promoters, flavors, absorbent supports and/or other feed ingredients. Preferably, the total amount of growth promoters, flavors, absorbent supports and/or other feed ingredients is 10 to 75% by weight of the additive. If any of the ingredients are plant materials, they are suitably dried and ground into fines or powders before being mixed with any other ingredients in the additive.
적합한 성장 촉진 첨가제 및 향료의 예는 크레졸, 아네톨, 데카락톤, 운데카락톤 및/또는 도데카락톤, 이오논(ionone), 아이론(irone), 진저롤, 피페리딘, 프로필리덴 프탈라이드, 부틸리덴 프탈라이드, 캡사이신 및/또는 타닌산이다. 지지체는 예를 들면 40 내지 50중량%의 목재 섬유, 8 내지 10중량%의 스테아린, 4 내지 5중량%의 강황 분말, 4 내지 5중량%의 로즈마리 분말, 22 내지 28중량%의 석회석, 1 내지 3중량%의 고무, 예를 들면 아라비아 고무, 5 내지 50중량%의 당 및/또는 전분 및 5 내지 15중량%의 물을 함유할 수 있다. 다른 사료 성분은 적합하게는 비타민, 효소, 무기 염, 분쇄된 곡물, 단백질함유 성분, 탄수화물-함유 성분, 거친 밀가루 및/또는 밀기울로 구성된 군에서 선택된다. 첨가제는 적합하게는 사료가 0.4ppm 내지 80중량%의 성분을 함유하게 하는 양으로 본 발명에 따른 사료 조성물에 첨가된다.Examples of suitable growth promoting additives and flavorings include cresol, anetol, decalactone, undecalactone and/or dodecalactone, ionone, ironone, gingerol, piperidine, propylidene phthalide, bu Thylidene phthalide, capsaicin and/or tannic acid. The support is, for example, 40 to 50% by weight of wood fibers, 8 to 10% by weight of stearin, 4 to 5% by weight of turmeric powder, 4 to 5% by weight of rosemary powder, 22 to 28% by weight of limestone, 1 to It may contain 3% by weight of rubber, such as gum arabic, 5 to 50% by weight of sugar and/or starch and 5 to 15% by weight of water. The other feed ingredients are suitably selected from the group consisting of vitamins, enzymes, inorganic salts, ground grains, protein-containing ingredients, carbohydrate-containing ingredients, coarse flour and/or bran. The additives are suitably added to the feed composition according to the invention in an amount such that the feed contains 0.4 ppm to 80% by weight of the ingredients.
본 발명에 따른 사료 조성물은 일반적으로, 사료의 건조 중량을 기준으로 계산하였을 때 다음과 같은 성분을 함유한다:The feed composition according to the present invention generally contains the following components as calculated based on the dry weight of the feed:
a) 0 내지 80 무수 중량%의 곡류,b) 0 내지 30 무수 중량%의 지방, c) 0 내지 85 무수 중량%의, 곡류 이외의 유형의 단백질-함유 영양 물질, 및 d) 10ppm 내지 40 무수 중량%의 본 발명의 성분. a) 내지 d)의 총 양은 바람직하게는 최소한 50무수중량%이다.a) 0 to 80 anhydrous weight percent cereals, b) 0 to 30 anhydrous weight percent fat, c) 0 to 85 anhydrous weight percent, protein-containing nutritional substances of types other than cereals, and d) 10 ppm to 40 anhydrous % By weight of the ingredients of the invention. The total amount of a) to d) is preferably at least 50 anhydrous weight percent.
사료 조성물을 제조하는 경우, 첨가제는 곡류, 예를 들면 연마되거나 분쇄된 밀, 귀리, 보리, 옥수수 및 쌀; 평지씨, 대두콩 및 해바라기 씨에 근거한 식물성 단백질 공급원; 동물성 단백질 공급원; 당밀; 및 우유 제품, 예를 들면 다양한 우유 분말 및 유장 분말로 구성된 건조 성분과 혼합될 수 있다. 모든 건조 성분과 혼합된 후, 액체 성분 및 가열후 액체가 된 성분을 첨가할 수 있다. 액체 성분은, 선택적으로 가열에 의해 액화된 지질, 예를 들면 지방, 예를 들면 식물성 지방, 및/또는 카복실산,예를 들면 지방산으로 구성될 수 있다. 완전히 혼합한 후, 성분의 연마도에 따라 가루 또는 입자상 농도가 수득된다. 저장하는 동안 분리되는 것을 방지하기 위해서, 물을 바람직하게는 동물 사료에 첨가해야만 하고, 이 사료는 후속적으로 종래의 펠렛화, 증량 또는 압출 공정으로 처리된다. 임의의 추가의 물을 건조에 의해 제거할 수 있다. 원하는 경우, 생성된 입자상 동물 사료를 더 작은 입자 크기로 분쇄할 수 있다. 개시된 사료 조성물은 일반적으로 건조된 녹색 꼴 및/또는 목초와 조합되어 투여된다.When preparing the feed composition, the additives include cereals, such as ground or pulverized wheat, oats, barley, corn and rice; Vegetable protein sources based on rapeseed, soybean and sunflower seeds; Animal protein sources; molasses; And milk products, such as dry ingredients consisting of various milk powders and whey powders. After mixing with all the dry ingredients, the liquid ingredient and the ingredient which became liquid after heating can be added. The liquid component may consist of lipids, such as fats, such as vegetable fats, and/or carboxylic acids, such as fatty acids, optionally liquefied by heating. After thoroughly mixing, a powdery or particulate concentration is obtained depending on the degree of polishing of the components. In order to prevent segregation during storage, water should preferably be added to the animal feed, which feed is subsequently subjected to conventional pelleting, bulking or extrusion processes. Any additional water can be removed by drying. If desired, the resulting particulate animal feed can be ground into smaller particle sizes. The disclosed feed compositions are generally administered in combination with dried green grasses and/or grasses.
식수 보충물은 1 무수 중량% 이상, 적합하게는 1 내지 99 무수 중량%, 바람직하게는 10 내지 50 무수 중량%의 성분을 함유할 수 있다. 하나 이상의 성분에 추가하여, 보충물은 더 많은 수의 다른 성분을 1 내지 99 무수 중량%로 함유할 수 있다.The drinking water supplement may contain at least 1 anhydrous weight percent, suitably 1 to 99 anhydrous weight percent, preferably 10 to 50 anhydrous weight percent of ingredients. In addition to the one or more ingredients, the supplement may contain 1 to 99 dry weight percent of a greater number of other ingredients.
다른 성분의 적합한 예는 광물성 염, 비타민, 건강 및 성장 개선 첨가제, 향료, 수용성 또는 수-분산성 담체, 예를 들면 당,분말 우유, 우유 부산물 및 셀룰로즈 유도체, 분산제 및 안정화제, 예를 들면 수용성 또는 수-분산성 중합체 및 이의 혼합물이다. 식수를 제조하는 경우, 보충물은 일반적으로 성분의 농도가 1ppm 내지 10중량%가 되는 양으로 물에 첨가된다.Suitable examples of other ingredients are mineral salts, vitamins, health and growth improving additives, flavors, water-soluble or water-dispersible carriers, such as sugars, powdered milk, milk by-products and cellulose derivatives, dispersants and stabilizers, such as water-soluble. Or water-dispersible polymers and mixtures thereof. When preparing drinking water, the supplement is generally added to the water in an amount such that the concentration of the ingredients is 1 ppm to 10% by weight.
본 발명의 범위 이내에서, 사료 조성물의 현탁액을 제조하는 것 또한 가능하다. 사료가 즉각적인 소비를 위해 제조되는 경우 이는 특히 편리하다.Within the scope of the present invention, it is also possible to prepare suspensions of feed compositions. This is particularly convenient if the feed is prepared for immediate consumption.
본 발명에서는 가축에게 사용되는 항생제중 병원성 세균에 의한 질병을 예방 및 치료를 목적으로하는 항생제를 대체하기 위하여 잠재력이 있는 유용 식물자원을 탐색하고 항균활성 스펙트럼을 조사하기 위하여 수행되었다. 특히 약리기능이 우수한 것으로 잘 알려진 야생 식물 중, 비교적 서식지가 넓고 대량으로 자생하고 있는 식물들을 대상으로 가축의 소화기성 질병 및 호흡기 질병에 관여하는 주요 병원성 미생물에 대한 항균활성 스펙트럼을 조사하였다. 또한 항균활성 물질의 규명을 위 하여 각 식물체에 대한 다양한 용매 추출 분획을 확보하고, 항균활성 스펙트럼을 조사하였다. In the present invention, it was carried out to search for useful plant resources and to investigate the antimicrobial activity spectrum in order to replace antibiotics for the purpose of preventing and treating diseases caused by pathogenic bacteria among antibiotics used in livestock. Particularly, among wild plants known to have excellent pharmacological functions, the antimicrobial activity spectrum of major pathogenic microorganisms involved in gastrointestinal diseases and respiratory diseases of livestock was investigated for plants with relatively wide habitats and large amounts of wild plants. In addition, to identify the antimicrobial active substances, various solvent-extracted fractions were obtained for each plant, and the antimicrobial activity spectrum was investigated.
보다 세밀한 식물 추출물의 항균활성을 평가하기 위하여 열수추출 방법과 methanol, n-hexane, chloroform, ethtyl acetate 그리고 butanol 등 다양한 용매를 이용한 분획을 확보하였고, Clostridium perfringens Type E, Enterotoxigenic E. coli, Burkholderia sp, Pantoea agglomerans, Haemopillus parsuis, 그리고 Haemopillus somnus 등 가축의 질병을 유발하는 6종의 주요 병원성 세균에 대한 항균활성을 paper disc methods를 이용하여 평가하였다. In order to evaluate the antimicrobial activity of plant extracts in more detail, fractions using a hot water extraction method and various solvents such as methanol, n-hexane, chloroform, ethtyl acetate and butanol were obtained. Clostridium perfringens Type E, Enterotoxigenic E. coli, Burkholderia sp, and Antimicrobial activity against six major pathogenic bacteria causing diseases in livestock such as Pantoea agglomerans, Haemopillus parsuis, and Haemopillus somnus was evaluated using paper disc methods.
Piper sarmentosum Roxb(Wild Betal)의 경우 methanol 추출 분획에서 미약한 항균활성을 나타내었으며, Citrus aurantifolia swingle leaf(Lime leaf)의 경우 methanol, hexane 그리고 chloroform 추출분획에서 미약한 항균활성을 나타내었으며, Burkholderia sp에 특이적인 항균활성을 나타내었다. Curcuma domestica Valeton(Turmeric)의 경우 모든 용매 추출 분획에서 항균활성을 나타내지 않았으나 열수 추출 분획에서 Clostridium perfringens에 대하여 특이적인 항균활성을 나타내었으며, 항균력 또한 매우 우수하였다. Sesbania grandiflora L.경우 대부분의 추출분획에서 항균활성이 나타나지 않았으나, butanol fraction에서 Burkholderia sp.에 대한 항균활성이 나타났다. Citrus aurantifolia swingle(Lime)의 경우 열수추출분획과 methanol 추출 분획에서 매우 우수한 항균력을 나타내었으며, 다른 식물 추출물에 비하여 매우 우수한 항균력을 나타내었다.Piper sarmentosum Roxb (Wild Betal) showed weak antimicrobial activity in the methanol extracted fraction, and Citrus aurantifolia swingle leaf (Lime leaf) showed weak antimicrobial activity in methanol, hexane and chloroform extracted fractions. It showed specific antibacterial activity. Curcuma domestica Valeton (Turmeric) did not show antimicrobial activity in all solvent-extracted fractions, but showed specific antimicrobial activity against Clostridium perfringens in hot water-extracted fractions, and the antimicrobial activity was also very excellent. In the case of Sesbania grandiflora L., most of the extracted fractions did not show antimicrobial activity, but the butanol fraction showed antimicrobial activity against Burkholderia sp. Citrus aurantifolia swingle(Lime) showed very good antibacterial activity in hot water extract fraction and methanol extract fraction, and showed very excellent antibacterial activity compared to other plant extracts.
항균활성이 확인된 물질들을 대상으로 MIC (minimum inhibition concentration)를 조사한 결과 Sesbania grandiflora L(Agati sesbania,Bai Khae)의 buthanol 추출물에서 호흡기 병원균인 Burkholderia sp에 대한 MIC 농도가 135 ㎍/㎖로 가장 강한 항균 활성을 나타내었다.As a result of examining the MIC (minimum inhibition concentration) of substances with confirmed antimicrobial activity, Sesbania grandiflora L (Agati sesbania, Bai Khae) buthanol extract against the respiratory pathogen Burkholderia sp The MIC concentration was 135 µg/ml, showing the strongest antibacterial activity.
항산화활성 평가에서는 대부분의 식물들이 비교적 높은 항산화 활성을 나타내었고, 특히 Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf), Morinda citrifolia L. leaf(Beach mulberry leaf), Piper sarmentosum Roxb(Wild Betal), Curcuma domestica Valeton(Turmeric), Cassia siamea britt(Siamea Cassia) 등의 methnol 추출물이 높은 활성을 나타내었다.In the evaluation of antioxidant activity, most plants showed relatively high antioxidant activity, especially Citrus aurantifolia swingle (Lime), Citrus aurantifolia swingle leaf (Lime leaf), Morinda citrifolia L. leaf (Beach mulberry leaf), Piper sarmentosum Roxb (Wild Betal). ), Curcuma domestica Valeton(Turmeric), Cassia siamea britt(Siamea Cassia) And methnol extract showed high activity.
일련의 본 발명의 결과를 종합한 결과 Sesbania grandiflora L(Agati sesbania,Bai Khae)의 buthanol 추출물에서 호흡기 병원균인 Burkholderia sp.에 대한 가장 강한 항균 활성을 나타내었고, 항산화활성 평가 대부분의 식물에서 비교적 높은 항산화 활성을 나타내었고, 특히 Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf), Piper sarmentosum Roxb(Wild Betal), Curcuma domestica Valeton(Turmeric) 등에서 유래한 추출물이 높은 활성을 나타내어 항생제 대체제로서 개발 가능성이 높다. As a result of synthesizing the results of a series of the present invention, Sesbania grandiflora L (Agati sesbania, Bai Khae) Burkholderia sp. for It showed the strongest antibacterial activity, and the antioxidant activity was evaluated relatively high in most plants. In particular, Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf), Piper sarmentosum Roxb(Wild Betal), Curcuma domestica Valeton(Turmeric) Since the extract derived from the back shows high activity, it is highly likely to be developed as an antibiotic substitute.
이하, 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기의 실시예는 본 발명을 설명하는 것이지 하기 실시예에 의하여 본 발명의 범위를 한정하려는 것은 아니다.Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples illustrate the present invention, but are not intended to limit the scope of the present invention by the following examples.
실시예 1:후보 식물Example 1: Candidate Plant
다양한 병원균에 대한 항균활성이 우수한 천연 식물자원을 탐색하기 위하여 본 실험에서는 현재 열대지방에서 자생하고 있는 열대 식물들을 대상으로 항균활성을 평가하였다.In order to search for natural plant resources with excellent antibacterial activity against various pathogens, in this experiment, antibacterial activity was evaluated for tropical plants that are currently growing in the tropical regions.
열대지방에서 자생하고 있는 Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf,), Morinda citrifolia L. leaf(Beach mulberry leaf), Sesbania grandiflora L.(Agati sesbania,), Piper sarmentosum Roxb(Wild Betal), Curcuma domestica Valeton(Turmeric), Cassia siamea britt(Siamea Cassia) 그리고 Cocos nucifera L.(Coconut)을 식물자원으로 사용하였고, 본 발명에 사용한 각 식물은 도 1 또는 표 1에서 보는 것과 같다. Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf,), Morinda citrifolia L. leaf(Beach mulberry leaf), Sesbania grandiflora L.(Agati sesbania,), Piper sarmentosum Roxb(Wild Betal) growing wild in tropical regions ), Curcuma domestica Valeton (Turmeric), Cassia siamea britt (Siamea Cassia) and Cocos nucifera L. (Coconut) were used as plant resources, and each plant used in the present invention is as shown in FIG. 1 or Table 1.
각 식물체는 수집 후에 증류수를 이용하여 2~3회 세척하였고, 킴와이프 티슈를 이용하여 물기를 제거한 후 48시간 동안 음건하였고, 건조된 시료는 분쇄기를 이용하여 분쇄한 후 추출시료로 사용하였다. After collection, each plant was washed 2-3 times with distilled water, dried with Kimwipe tissue and shaded for 48 hours, and the dried sample was pulverized using a grinder and used as an extraction sample.
상기 표 1은 본 발명에 사용된 열대식물의 이름이다.Table 1 is the names of tropical plants used in the present invention.
실시예 2: 항균활성 평가를 위한 추출물 및 분획물의 조제Example 2: Preparation of extracts and fractions for evaluation of antibacterial activity
각 식물체의 추출물 제조는 한 (2005)의 방법에 기초하여 단계별 추출 분획을 확보하였다. 상세한 추출 분획 회수 방법은 다음과 같다. 건조 후 분쇄된 각 식물체 3g을 메탄올 27 ml와 혼합한 후에 실온에서 약 6시간 동안 150rpm으로 교반 추출하였다. methanol 추출물은 여과지 (Whatman filter paper No 2)를 이용하여 잔사를 제거하고 여과액을 회수하였다. 여과액은 감압 농축하여 최종 methanol 분획으로 회수하였다. 회수된 methanol 분획은 일부 항균활성 평가에 사용하였고 나머지는 극성에 따라 n-hexane, chloroform, ethylacetate, butanol등 순차적으로 용매 분획하였다 (도 2).For the preparation of extracts of each plant, a step-by-step extraction fraction was obtained based on the method of Han (2005). The detailed extraction fraction recovery method is as follows. After drying, 3 g of each pulverized plant was mixed with 27 ml of methanol, and extracted with stirring at room temperature for about 6 hours at 150 rpm. The methanol extract was removed from the residue using a filter paper (Whatman filter paper No 2) and the filtrate was recovered. The filtrate was concentrated under reduced pressure and recovered as a final methanol fraction. The recovered methanol fraction was used for evaluation of some antibacterial activity, and the rest were solvent fractionated sequentially, such as n-hexane, chloroform, ethylacetate, and butanol, depending on the polarity (FIG. 2).
감압 농축된 methanol 분획과 3차 증류수, 그리고 n-hexane을 1:1의 비율로 혼합한 후, voltex mixer를 이용하여 교반하였다. 용매 혼합액은 상온에서 정치시켜 tube내 층분리를 유도하였고, pasteur pippet를 이용하여 n-hexane 층을 회수하였다. 회수된 n-hexane 층은 감압 농축하여 항균활성 평가에 사용하였고, 나머지 물층은 3차증류수와 chloroform을 1:1의 비율로 혼합한 후 교반하였다. 교반 후에 정치시켜 층분리를 유도하였고 pasteur pippet를 이용하여 chloroform 층을 회수하였다. 회수된 chloroform 분획은 감압 농축하여 항균활성을 평가하였고 나머지 물층은 3차 증류수와 ethylacetate (1:1)와 혼합한 후에 chloroform 분획 회수 방법과 동일하게 ethylacetate 층과 물층을 분리하였고, ethylacetate 분획은 감압 농축하여 항균활성 평가에 사용하였고, 나머지 물층은 3차 증류수와 butanol (1:1)과 혼합한 후, 위와 동일한 방법으로 최종 butanol 분획을 회수하였다. 일련의 방법은 도 2에서 보는 것과 같다. 감압농축을 통하여 회수한 각 분획물은 해당 용매로 다시 용해시켜 4℃에서 보관하며 항균활성 평가에 사용하였다.After the methanol fraction concentrated under reduced pressure, tertiary distilled water, and n-hexane were mixed at a ratio of 1:1, the mixture was stirred using a voltex mixer. The solvent mixture was allowed to stand at room temperature to induce layer separation in the tube, and the n-hexane layer was recovered using a pasteur pipette. The recovered n-hexane layer was concentrated under reduced pressure and used for antibacterial activity evaluation, and the remaining water layer was stirred after mixing tertiary distilled water and chloroform in a ratio of 1:1. After stirring, the mixture was allowed to stand to induce layer separation, and the chloroform layer was recovered using a pasteur pippet. The recovered chloroform fraction was concentrated under reduced pressure to evaluate its antimicrobial activity. The remaining water layer was mixed with tertiary distilled water and ethylacetate (1:1), and the ethylacetate layer and the aqueous layer were separated in the same manner as the chloroform fraction recovery method, and the ethylacetate fraction was concentrated under reduced pressure. Thus, it was used for the evaluation of antibacterial activity, and the remaining water layer was mixed with tertiary distilled water and butanol (1:1), and the final butanol fraction was recovered in the same manner as above. The series of methods is as shown in FIG. 2. Each fraction recovered through concentration under reduced pressure was re-dissolved with the corresponding solvent, stored at 4°C, and used for antibacterial activity evaluation.
실시예 3: 가축 병원성 세균에 대한 식물 추출물의 항균활성 평가Example 3: Evaluation of antibacterial activity of plant extracts against livestock pathogenic bacteria
다양한 용매로 분획된 추출물은 주요 가축병원성 세균에 대하여 항균활성을 평가하여, 우수한 식물 추출물을 선별하였다.Extracts fractionated with various solvents were evaluated for antibacterial activity against major animal pathogenic bacteria, and excellent plant extracts were selected.
실시예 4: 병원균의 준비Example 4: Preparation of pathogens
본 발명에서 사용된 병원균은 가축의 소화기성 질병과 호흡기 질병 등을 유발하는 주요 병원성 세균으로 수의과학검역원에서 분양받아 사용하였고, 각 균주는 표 2에서 보는 것과 같다.The pathogens used in the present invention are major pathogenic bacteria that cause gastrointestinal diseases and respiratory diseases of livestock, and were sold and used by the Veterinary Science and Quarantine Service, and each strain is as shown in Table 2.
-70℃에 보관된 각 병원균은 멸균된 백금이를 이용하여 LB agar에 streak 한 후, 37℃에서 24시간 동안 배양하였다. LB agar 표면에 나타난 각 병원성 세균의 colony 중 하나를 백금이를 이용하여 취한 후, LB broth에 옮겨 37℃ 진탕배양기에서 200rpm으로 24시간 배양한 후 피검균으로 사용하였다.Each pathogen stored at -70°C was streaked on LB agar using sterilized platinum ear, and then incubated at 37°C for 24 hours. One of the colony of each pathogenic bacteria that appeared on the surface of the LB agar was taken with a platinum ear, transferred to LB broth, and cultured for 24 hours at 200 rpm in a shaking incubator at 37°C, and used as the test bacteria.
Type E Clostridium perfringens
Type E
partial partial
표 2는 본 발명에 사용된 병원성 세균에 대한 리스트이고, *National Veterinary Research Quarantine Service(수의과학검역원)을 나타낸다.Table 2 is a list of pathogenic bacteria used in the present invention, and shows * National Veterinary Research Quarantine Service (Veterinary Science and Quarantine Service).
실시예 5: 항균활성 평가를 위한 중층 배지의 제조Example 5: Preparation of intermediate layer medium for evaluation of antibacterial activity
각 식물 추출물 분획의 항균활성을 평가하기 위하여, 병원균의 준비는 중층 배지 방법을 사용하였다. 항균활성 시험에 사용된 중층 배지는 0.8% agar (Bacto agar, Difco) 용액을 15ml screw cap tube 에 10ml 씩 나누어 담고, 121℃에서 15분간 멸균하여 준비하였고, tube의 agar가 굳지 않게 하기 위하여 50℃ water bath에서 보관하였다. In order to evaluate the antimicrobial activity of each plant extract fraction, the preparation of pathogens was performed using a medium-layer medium method. The middle layer medium used in the antimicrobial activity test was prepared by dividing 0.8% agar (Bacto agar, Difco) solution into a 15ml screw cap tube by 10ml, sterilizing at 121℃ for 15 minutes, and 50℃ to prevent the agar of the tube from hardening. Stored in a water bath.
배양된 병원균은 최종 농도를 1×107 ~ 1×108 cfu/ml가 되게 희석한 후에 50℃ water bath 보관된 멸균 0.8% agar 용액 tube에 100㎕를 접종하였고, voltex mixer를 이용하여 교반한 후에 LB agar plate에 붓고 상온에서 식히면서 고형화시켰다.Cultured pathogens were diluted to a final concentration of 1×10 7 ~ 1×10 8 cfu/ml, and then 100 μl was inoculated into a sterile 0.8% agar solution tube stored in a 50° C. water bath, and stirred using a voltex mixer. Then, it was poured onto an LB agar plate and allowed to solidify while cooling at room temperature.
실시예 6: 식물 추출물 분획의 항균활성 평가Example 6: Evaluation of antibacterial activity of plant extract fractions
일련의 과정을 통하여 확보된 식물 추출물 분획의 항균활성은 윤 등(2005)의 방법에 따라서 paper disc method를 통하여 평가하였다. 용매별로 추출된 분획을 멸균된 6mm filter paper disk (Whatman AA Discs)에 25㎕씩 흡수시키고 상온에서 각 용매가 휘발되도록 30분간 방치하였다. 각 용매가 휘발된 후에 병원성 세균이 도말된 중층배지 위에 올려놓은 후, 37℃ incubator에서 24~48시간 동안 배양하였다. 배양 후 paper disc 주변으로 형성된 생육저지 환을 관찰하여 항균활성의 유무와 정도를 평가하였다. The antimicrobial activity of the plant extract fraction obtained through a series of processes was evaluated by the paper disc method according to the method of Yun et al. (2005). The fractions extracted for each solvent were absorbed into a sterilized 6mm filter paper disk (Whatman AA Discs) by 25 µl, and left at room temperature for 30 minutes to volatilize each solvent. After each solvent was volatilized, it was placed on a layered medium smeared with pathogenic bacteria, and incubated for 24 to 48 hours in an incubator at 37°C. After cultivation, the presence and degree of antimicrobial activity were evaluated by observing the growth inhibition ring formed around the paper disc.
실시예 7: 식물 추출물 분획의 최소저해농도(MIC) 평가 Example 7: Evaluation of the minimum inhibitory concentration (MIC) of the plant extract fraction
각 균주의 최소저해농도(MIC)는 broth microdilution method에 의해 다음과 같이 측정하였다. 즉, 96웰 플레이트에 LB를 50㎕씩 분주하고 50㎕의 식물추출물을 two-fold dilution하여 농도를 조절한 후 균의 농도를 104~105 CFU/ml이 되도록 희석시켜 50㎕씩 첨가하였다. 그 후 37℃에서 16시간 이상 배양한 뒤, 600nm에서 microplate reader로 흡광도를 측정하였다. Turbidity가 나타나지 않은 well의 해당 시료 농도를 MIC(minimum inhibition concentration)값으로 결정하였다. The minimum inhibitory concentration (MIC) of each strain was measured by the broth microdilution method as follows. That is, 50 µl of LB was dispensed into a 96-well plate by two-fold dilution of 50 µl of plant extract to adjust the concentration, and then the concentration of the bacteria was diluted to 10 4 ~10 5 CFU/ml, and 50 µl was added each. . Then, after incubation at 37° C. for 16 hours or more, absorbance was measured at 600 nm with a microplate reader. The concentration of the sample in the well without turbidity was determined as the minimum inhibition concentration (MIC) value.
실시예 8: 식물 추출물 분획의 항산화활성 평가 Example 8: Evaluation of antioxidant activity of plant extract fraction
항산화 활성은 DPPH photometric assay를 사용하였다. Ethanal 990 ㎕에 식물추출액 10 ㎕를 첨가하여 1/100로 희석한 후 0.3 mM DPPH ethanal solution을 사용하여 실온에서 30 min 반응 후 518 nm에서 흡광도를 측정하고 다음의 식을 이용 항산화활성을 측정하였다. For antioxidant activity, DPPH photometric assay was used. After adding 10 µl of plant extract to 990 µl of ethanol, diluting it to 1/100, and then reacting for 30 min at room temperature with 0.3 mM DPPH ethanal solution, absorbance was measured at 518 nm, and antioxidant activity was measured using the following equation.
항산화 활성 % = 100-[(샘플의 흡광도 - 브랭크의 흡광도) x 100/ 대조군의 흡광도]Antioxidant activity% = 100-[(absorbance of sample-absorbance of blank) x 100/ absorbance of control]
상기의 실시예의 결과는 다음과 같다.The results of the above examples are as follows.
열대에서 자생하는 식물 8종에 대한 용매별 추출 분획의 항균활성을 평가한 결과 Piper sarmentosum Roxb(Wild Betal), Citrus aurantifolia swingle leaf(Lime leaf), Curcuma domestica Valeton(Turmeric), Sesbania grandiflora L.(Agati sesbania) 그리고 Citrus aurantifolia swingle(Lime)에서 항균활성이 검출되었고 특히 Citrus aurantifolia swingle(Lime)에서 매우 우수한 항균활성이 검출되었다. 그러나 Morinda citrifolia L. leaf(Beach mulberry leaf), Cassia siamea britt(Siamea Cassia) 그리고 Cocos nucifera L.(Coconut)에서는 다양한 용매에서 추출하였음에도 불구하고 항균활성이 관찰되지 않았다(표 3).As a result of evaluating the antimicrobial activity of extract fractions by solvent against 8 species of tropical plants, Piper sarmentosum Roxb(Wild Betal), Citrus aurantifolia swingle leaf(Lime leaf), Curcuma domestica Valeton(Turmeric), Sesbania grandiflora L.(Agati sesbania) and Citrus aurantifolia swingle (Lime), and especially Citrus aurantifolia swingle (Lime), showed excellent antimicrobial activity. However, in Morinda citrifolia L. leaf (Beach mulberry leaf), Cassia siamea britt (Siamea Cassia) and Cocos nucifera L. (Coconut), antibacterial activity was not observed despite extraction from various solvents (Table 3).
표 3은 병원성 세균에 대한 열대식물의 용매 추출물의 항균활성을 나타낸 표로 1용매 분획: A. 물; B. 메탄올; C. 헥산; D. 클로로포름; E. 에틸 아세테이트 F. 부탄올을 나타내고, 항균 활성: -, no inhibition (8mm); +, very slight inhibition (9mm~11mm); ++, moderate inhibition (11mm~13mm); +++, heavy inhibition (13~17mm)으로 나타내었다. Table 3 is a table showing the antibacterial activity of solvent extracts of tropical plants against pathogenic bacteria. 1 Solvent fraction: A. Water; B. methanol; C. hexane; D. chloroform; E. Ethyl acetate F. Represents butanol, antibacterial activity: -, no inhibition (8mm); +, very slight inhibition (9mm~11mm); ++, moderate inhibition (11mm~13mm); +++, heavy inhibition (13~17mm).
1. 추출물과 용매별 분획물의 항균력1. Antibacterial activity of fractions by extract and solvent
비교적 단단한 wax 층의 엽을 가진 향료 식물인 Citrus aurantifolia swingle leaf(Lime leaf)의 용매 추출 분획 중 methanol, n-hexane, Chloroform 분획에서 항균활성을 나타내었다. 그러나 각 항균활성 추출분획은 호흡기 질병의 원인균인 Burkholderia sp.과 Haemopillus somnus에 대하여 특이적인 항균활성을 나타내었고 다른 병원성 세균에 대하여서는 항균활성을 나타내지 않았다. Among the solvent-extracted fractions of Citrus aurantifolia swingle leaf (Lime leaf), a fragrance plant with a relatively hard wax layer, the methanol, n-hexane, and chloroform fractions exhibited antimicrobial activity. However, each of the extracted fractions of antimicrobial activity was produced by Burkholderia sp. And Haemopillus somnus showed specific antibacterial activity, but not against other pathogenic bacteria.
넓은 엽의 특성을 보이는 Piper sarmentosum Roxb(Wild Betal)에 대하여 다양한 용매로 추출한 분획의 항균력을 평가한 결과 methanol 추출물에서 미약한 항균활성이 검출되었고 착즙 및 기타 다른 용매에서는 항균활성이 검출되지 않았다. 특히 돼지 글래서병의 원인 균인 Haemopillus parasuis 대한 항균활성이 우수하였다. As a result of evaluating the antimicrobial activity of the fractions extracted with various solvents for Piper sarmentosum Roxb (Wild Betal), which exhibits broad lobe characteristics, weak antimicrobial activity was detected in methanol extract, and no antibacterial activity was detected in juice and other solvents. In particular, the antimicrobial activity was excellent against Haemopillus parasuis, the causative agent of porcine glasser disease.
근채류에 속하는 Curcuma domestica Valeton(Turmeric)은 Ethylacetate 분획에서 미약한 항균활성을 나타내었고 다른 용매 분획에서는 항균활성을 보이지 않았다. 그러나 가축의 장독혈증을 일으키는 Clostridium perfringens Type E 균주에 대하여서만 특이적인 항균활성을 나타내었고 항균활성 정도도 매우 우수하였다. Curcuma domestica Valeton (Turmeric) belonging to the root vegetable showed weak antimicrobial activity in the Ethylacetate fraction and no antibacterial activity in other solvent fractions. However, it showed specific antimicrobial activity only against Clostridium perfringens Type E strain, which causes enterotoxemia in livestock, and the degree of antimicrobial activity was also very good.
Sesbania grandiflora L.(Agati sesbania) 추출 분획은 Burkholderia sp에 대하여서만 항균활성을 나타내었다. 그러나 매우 미약한 활성만을 나타내었다. The fraction extracted from Sesbania grandiflora L. (Agati sesbania) showed antimicrobial activity only against Burkholderia sp. However, it showed only very weak activity.
과실류에 속하는 Citrus aurantifolia swingle(Lime)는 다른 식물 및 추출분획에 비하여 매우 넓은 항균활성 스팩트럼을 나타내었으며, 항균활성 정도도 매우 우수하였다. 특히 열수 추출한 분획은 Haemopillus somnus에 대하여 높은 항균활성을 보였으며, methanol 추출 분획은 다양한 병원균에 대하여 높은 항균활성을 나타내었다. 그러나 가축의 장독혈증을 유발하는 Clostridium perfringens Type E에 대하여서는 항균활성을 나타내지 않았다. 그 외 Morinda citrifola L. leaf, Cassia siamea britt 그리고 Cocos mucifera L.의 모든 추출분획들에서는 항균활성이 나타나지 않았다. Citrus aurantifolia swingle (Lime), which belongs to the fruit family, showed a very broad spectrum of antimicrobial activity compared to other plants and extract fractions, and the degree of antimicrobial activity was also very good. In particular, the fraction extracted with hot water showed high antibacterial activity against Haemopillus somnus , and the fraction extracted with methanol showed high antibacterial activity against various pathogens. However, it did not show antimicrobial activity against Clostridium perfringens Type E, which causes enterotoxemia in livestock. In addition , all extracted fractions of Morinda citrifola L. leaf, Cassia siamea britt and Cocos mucifera L. showed no antimicrobial activity.
2. 식물 추출물 분획의 최소저해농도(MIC) 평가 2. Evaluation of the minimum inhibitory concentration (MIC) of the plant extract fraction
항균활성이 확인된 물질들을 대상으로 MIC (minimum inhibition concentration)를 조사한 결과 Sesbania grandiflora L(Agati sesbania, BaiKhae)의 buthanol 추출물에서 호흡기 병원균인 Burkholderia sp에 대한 MIC 농도가 135 ㎍/㎖로 가장 강한 항균 활성을 나타내었다 (표 4).As a result of examining the MIC (minimum inhibition concentration) of substances with confirmed antimicrobial activity, Sesbania grandiflora L (Agati sesbania, BaiKhae) buthanol extract against the respiratory pathogen Burkholderia sp MIC concentration is 135 ㎍ / ㎖ It showed the strongest antibacterial activity (Table 4).
agglomerans agglomerans
E. coli Enterotoxigenic
E. coli
sp.sp.
somnus somnus
parsuis parsuis
perfringens perfringens
표 4는 미생물에 대한 열대 식물의 분획 및 추출물의 최소 저해 농도(Minimum inhibitory concentration)를 나타내고, 표에서 ND : not detected를 나타낸다.Table 4 shows the minimum inhibitory concentration of fractions and extracts of tropical plants against microorganisms, and ND: not detected in the table.
3. 식물 추출물 분획의 항산화활성 평가3. Evaluation of antioxidant activity of plant extract fractions
대부분의 식물에서 비교적 높은 항산화 활성을 나타내었고, 특히 Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf), Morinda citrifolia L. leaf(Beach mulberry leaf), Curcuma domestica Valeton(Turmeric), Cassia siamea britt(Siamea Cassia) 등에서 methnol 추출물이 높은 활성을 나타내었다 (표 5).It showed relatively high antioxidant activity in most plants, especially Citrus aurantifolia swingle(Lime), Citrus aurantifolia swingle leaf(Lime leaf), Morinda citrifolia L. leaf(Beach mulberry leaf), Curcuma domestica Valeton(Turmeric), Cassia siamea britt (Siamea Cassia) In the same, methnol extract showed high activity (Table 5).
표 5는 열대 식물의 용매 추출물의 항산화활성을 나타낸다Table 5 shows the antioxidant activity of the solvent extract of tropical plants.
도 1은 본 발명에 사용된 열대식물의 사진이다. 사진에서 A.Citrus aurantifolia swingle; B.Citrus aurantifolia swingle leaf; C.Cassia siamea britt; D.Cocos nucifera L.; E.Morinda citrifolia L. leaf.; F.Sesbania grandiflora L.; G,Piper sarmentosum Roxb; H.Curcuma domestica Valeton를 나타낸다.1 is a photograph of a tropical plant used in the present invention. In the photo A.Citrus aurantifolia swingle; B. Citrus aurantifolia swingle leaf; C. Cassia siamea britt; D.Cocos nucifera L.; E. Morinda citrifolia L. leaf.; F. Sesbania grandiflora L.; G, Piper sarmentosum Roxb; Represents H. Curcuma domestica Valeton.
도 2는 열대 식물의 여러 추출단계를 나타낸다.2 shows several extraction steps of tropical plants.
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