KR101929388B1 - Composition for prevention, treatment or improvement of liver disease comprising italian ryegrass extraction fraction as an active ingredient - Google Patents
Composition for prevention, treatment or improvement of liver disease comprising italian ryegrass extraction fraction as an active ingredient Download PDFInfo
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- KR101929388B1 KR101929388B1 KR1020170149216A KR20170149216A KR101929388B1 KR 101929388 B1 KR101929388 B1 KR 101929388B1 KR 1020170149216 A KR1020170149216 A KR 1020170149216A KR 20170149216 A KR20170149216 A KR 20170149216A KR 101929388 B1 KR101929388 B1 KR 101929388B1
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- methanol
- chloroform
- aqueous solution
- residue
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- 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
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- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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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
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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
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- Y10S514/893—
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Abstract
Description
본 발명은 이탈리안라이그라스 추출 분획물을 유효성분으로 함유하는 간질환의 예방, 치료 또는 개선용 조성물에 관한 것으로, 구체적으로 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득한 용출물을 유효성분으로 함유하는 간질환의 예방, 치료 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for the prevention, treatment or amelioration of liver disease, which comprises an extract of Italian rice glaze as an active ingredient. Specifically, a composition comprising 60 to 80% (v / v) And the residue was extracted with chloroform to obtain a chloroform-soluble extract. The chloroform-soluble extract was passed through a sephadex LH20 resin and eluted with a 30% (v / v) aqueous methanol solution And a composition for preventing, treating or ameliorating liver disease containing the eluted product as an active ingredient.
일반적으로 이탈리안라이그라스는 겨울철 휴경 논을 이용하는 이점이 있고 우리나라와 같이 조사료 생산이 어려운 여건에서 아주 중요한 사료작물로 재배되고 있다. 특히 국가적 차원에서 맞춤형 조사료 신품종 개발 및 안정생산 기술개발에 많은 노력을 기울이고 있는 가운데 이탈리안라이그라스는 가장 중요한 품종으로 인식되고 있고, 농진청에서도 안정적 재배지역의 확대, 이탈리안라이그라스 신품종 개발, 품질규격화 및 종자 보급률 확대에 중점을 두고 있다.Generally, Italian raglas have the advantage of using winter rice fields in winter and they are cultivated as very important forage crops in difficult conditions such as Korea. Especially, we are making efforts to develop new kinds of customized forage and stable production technology at the national level. Among them, Italian rice is recognized as the most important varieties. In addition, the RDA is expanding the stable cultivation area, developing new varieties of Italian rice, It focuses on expanding penetration rate.
한편 조사료는 가축의 생리활성기능 향상 및 고급육 생산능력 향상에 중요한 생리활성물질이 많이 함유되어 있는 것으로 추정되었으며, 이러한 생리활성물질들은 가축의 면역력 증강을 통한 생산성 향상과 더불어 인간에게도 유용한 효과를 줄 것으로 기대되고 있다. 또한 유럽에서는 조사료에 많이 함유된 섬유소 성분이 임신돈의 포만감을 형성하고, 포유돈에는 변비 방지 효과가 있다고 보고하였으며, 여러 축산 선진국에서는 조사료 유래 생리활성물질을 사람에게도 유용하게 이용해보고자 하는 연구를 수행하고 있다. 예로서, 귀리 추출물은 당대사에 생리적 활성을 나타내어 당뇨치료제로서의 효용성이 기대되고 있으며, 항암 및 면역질환 억제에 대한 기능도 보고되었다. 청보리와 같은 조사료원에 대한 연구에서도 특정 활성분획물에 의한 면역세포 활성, 항산화, 고지혈증 억제, 지질대사 활성 및 당대사 촉진 등과 같은 생리활성 기능이 보고되는 등 조사료 유래 활성성분에 대한 다양한 이용성 증진 연구가 수행되었다.On the other hand, it is estimated that the forage contains a large amount of physiologically active substances that are important for improving the physiological activity of livestock and improving the quality of high-quality meat production. Such physiologically active substances will have a beneficial effect on humans, It is expected. In Europe, it has been reported that the fibrin components contained in the forage feed form the feeling of satiety of the pregnant donor, the inhibitory effect on the spermatozoa, and the research on the use of the physiologically active substances derived from the fodder . As an example, oat extracts exhibit physiological activity in glucose metabolism and are expected to be useful as a diabetic therapeutic agent, and have also been reported to have anticancer and immunosuppressive functions. Studies on forage crops such as blueberries have also reported various physiological activities such as immune cell activity, antioxidation, hyperlipidemia inhibition, lipid metabolism activity and sugar metabolism promotion by specific active fractions, .
천연자원으로부터 생리활성소재의 탐색은 대부분 약용식물을 주요 소재로 연구해왔으나 이탈리안라이그라스와 같은 사료 작물을 대상으로 하는 활성성분의 분리·정제에 관한 연구는 미비한 실정이다.Research on bioactive materials from natural resources has been mainly used for medicinal plants, but studies on the separation and purification of active ingredients in forage crops such as Italian Ryegrass are insufficient.
이에, 본 발명자들은 이탈리안라이그라스의 생리활성에 관해 연구한 결과, 특정한 방법으로 추출 및 분획하여 수득한 추출분획물이 간질환의 예방, 치료 또는 개선에 우수한 효과가 있음을 확인하고 본 발명을 완성하게 되었다.Accordingly, the inventors of the present invention have studied the physiological activities of Italian Ryegrass. As a result, they have found that the extract fractions obtained by extracting and fractionating by a specific method have excellent effects on the prevention, treatment or improvement of liver disease, .
따라서 본 발명의 주된 목적은 이탈리안라이그라스로부터 추출 및 분획하여 수득한 추출분획물을 유효성분으로 함유하여 간질환의 예방, 치료 또는 개선에 우수한 효과가 있는 조성물을 제공하는데 있다.Accordingly, a main object of the present invention is to provide a composition having an excellent effect for prevention, treatment or improvement of liver disease by containing an extract fraction obtained by extracting and fractionating from Italian raglas as an active ingredient.
본 발명의 한 양태에 따르면, 본 발명은 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득한 용출물을 유효성분으로 함유하는 간질환의 예방 또는 치료용 약학적 조성물을 제공한다.According to one aspect of the present invention, the present invention relates to a process for the preparation of a compound of formula (I) in which 60-80% (v / v) methanol extracts of Italian ryegrass are extracted with petroleum ether and filtered to obtain a residue, the residue is extracted with chloroform, The pharmaceutical composition for preventing or treating liver diseases containing the eluted product obtained by eluting the chloroform-soluble extract with Sephadex LH20 resin and eluting it with 30% (v / v) methanol aqueous solution Gt;
본 발명의 다른 양태에 따르면, 본 발명은 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득한 용출물을 유효성분으로 함유하는 간질환의 예방 또는 개선용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention relates to a process for the preparation of a compound of formula (I), which comprises extracting and filtering 60-80% (v / v) methanol extracts of Italian ryegrass with petroleum ether and extracting the residue, extracting the residue with chloroform, Extract, and said chloroform-soluble extract is passed through a sephadex LH20 resin and eluted with an aqueous 30% (v / v) methanol solution, as an active ingredient, to prevent or ameliorate liver disease Lt; / RTI >
본 발명의 또 다른 양태에 따르면, 본 발명은 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득한 용출물을 유효성분으로 함유하는 간질환의 예방 또는 개선용 사료 조성물을 제공한다.According to another aspect of the present invention, the present invention relates to a process for the preparation of a compound of formula (I) wherein 60-80% (v / v) methanol extracts of Italian Ryegrass are extracted with petroleum ether and filtered to obtain a residue, the residue is extracted with chloroform and washed with chloroform Soluble extract is obtained and the chloroform-soluble extract is passed through a sephadex LH20 resin and eluted with an aqueous 30% (v / v) methanol solution to prevent or ameliorate liver disease containing the effluent as an active ingredient Feed composition.
본 발명의 또 다른 양태에 따르면, 본 발명은 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득한 용출물을 유효성분으로 함유하는 간질환의 예방 또는 개선용 사료 첨가제를 제공한다.According to another aspect of the present invention, the present invention relates to a process for the preparation of a compound of formula (I) wherein 60-80% (v / v) methanol extracts of Italian Ryegrass are extracted with petroleum ether and filtered to obtain a residue, the residue is extracted with chloroform and washed with chloroform Soluble extract is obtained and the chloroform-soluble extract is passed through a sephadex LH20 resin and eluted with an aqueous 30% (v / v) methanol solution to prevent or ameliorate liver disease containing the effluent as an active ingredient Feed additives.
본 발명의 약학적 조성물, 식품 조성물, 사료 조성물 또는 사료 첨가제에 있어서, 상기 용출물은 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 얻은 용출물 중 세 번째로 용출된 용출물인 것이 바람직하다.In the pharmaceutical composition, the food composition, the feed composition or the feed additive of the present invention, the eluted product is obtained by passing the chloroform-soluble extract through a sephadex LH20 resin, adding water, a 10% (v / v) It is preferable that the eluate is the third one eluted from the eluate obtained by sequentially eluting with 30% (v / v) aqueous methanol solution and fractionating into three regions using a 30% (v / v) methanol aqueous solution.
본 발명의 약학적 조성물, 식품 조성물, 사료 조성물 또는 사료 첨가제는 간독성 물질로 인한 간 무게 증가, 간조직의 손상 및 간조직 내 항산화효소의 활성저하를 억제함으로써 간질환을 효과적으로 예방, 치료 또는 개선할 수 있다.The pharmaceutical composition, food composition, feed composition or feed additive of the present invention can effectively prevent, treat or ameliorate liver disease by inhibiting liver weight increase caused by hepatotoxic substances, damage of liver tissue and degradation of antioxidant enzyme activity in liver tissue .
도 1은 본 발명의 일실시예에 따른 이탈리안라이그라스 추출분획물을 수득하는 과정을 나타낸 블록도이다.
도 2는 본 발명 이탈리안라이그라스 추출분획물의 간독성물질 유도성 간조직 무게 증가에 대한 억제 효과 실험결과를 나타낸 것이다.
도 3은 본 발명 이탈리안라이그라스 추출분획물의 간독성물질 유도성 간손상에 대한 억제 효과 실험결과를 나타낸 것이다.
도 4는 본 발명 이탈리안라이그라스 추출분획물의 간독성물질 유도성 간조직 내 항산화효소 활성저하 및 지방과산화 물질 생성 증가에 대한 억제 효과 실험결과를 나타낸 것이다.
도 5는 본 발명 이탈리안라이그라스 추출분획물의 간독성물질 유도성 간조직 손상에 대한 억제 효과 실험결과를 나타낸 것이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram showing a process for obtaining an extract of Italian rice straw according to an embodiment of the present invention. FIG.
FIG. 2 is a graph showing the results of an experiment for inhibiting the hepatic toxicant-induced liver weight increase of the extract of Italian Ryegrass of the present invention.
FIG. 3 is a graph showing the results of experiments for inhibiting hepatic toxicity-induced liver damage of the extract of Italian Ryegrass of the present invention.
4 is a graph showing the results of experiments for inhibiting the antioxidant enzyme activity and the production of lipid peroxidation in the hepatic toxin-induced hepatic tissue of the extract of the inventive Italian ryegrass.
FIG. 5 is a graph showing the results of experiments for inhibiting hepatic toxicity-induced hepatic tissue damage of the extract of Italian Ryegrass of the present invention.
본 발명의 약학적 조성물, 식품 조성물, 사료 조성물 또는 사료 첨가제는 이탈리안라이그라스의 특정한 방법으로 추출 및 분획하여 얻은 추출분획물을 유효성분으로 함유하는 것을 특징으로 한다.The pharmaceutical composition, food composition, feed composition or feed additive of the present invention is characterized by containing an extract fraction obtained by extracting and fractionating by a specific method of Italian Ryegrass as an active ingredient.
본 발명의 이탈리안라이그라스 추출분획물은 이탈리안라이그라스의 60 내지 80%(v/v) 메탄올 추출 건고물을 석유에테르로 추출 및 여과하여 잔사를 수득하고, 상기 잔사를 클로로포름으로 추출하여 클로로포름 용해성 추출물을 수득하고, 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고 30%(v/v) 메탄올 수용액으로 용출하여 수득할 수 있다.The extract of the present invention is obtained by extracting 60-80% (v / v) methanol extracts of Italian Ryegrass with petroleum ether and filtering to obtain a residue. The residue is extracted with chloroform to obtain a chloroform-soluble extract , And eluting the chloroform-soluble extract with a 30% (v / v) methanol aqueous solution through a sephadex LH20 resin.
바람직하게는 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 세 번째로 용출되는 것을 수득하는 것이 좋다.Preferably, the chloroform-soluble extract is passed through a Sephadex LH20 resin and fractionated by elution with water, 10% (v / v) methanol aqueous solution and 30% (v / v) methanol aqueous solution, (v / v) methanol aqueous solution to obtain a third elution.
본 발명은 상기와 같은 이탈리안라이그라스 추출분획물이 간독성 물질로 인한 간 무게 증가, 간조직의 손상 및 간조직 내 항산화효소의 활성저하를 억제한다는 새로운 발견을 통해 안출되었다.The present invention is based on the discovery that the above-mentioned extract of the Italian raglass inhibits hepatic toxicity caused by liver toxicity, damage of liver tissue and decrease of antioxidant enzyme activity in liver tissue.
본 발명의 약학적 조성물은 전체 조성물의 중량을 기준으로 본 발명의 추출분획물을 0.1 내지 90중량%로 함유할 수 있을 것으로 판단된다.The pharmaceutical composition of the present invention may contain 0.1 to 90% by weight of the extract fraction of the present invention based on the weight of the whole composition.
본 발명의 약학적 조성물은 임상 투여 시에 경구 또는 비경구로 투여가 가능하며 비경구 투여시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고, 일반적인 의약품 제제의 형태로 사용될 수 있을 것이다.The pharmaceutical composition of the present invention can be administered orally or parenterally at the time of clinical administration, and can be administered orally or parenterally at the time of parenteral administration, and can be administered intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, Or by intramuscular injection, and may be used in the form of a conventional pharmaceutical preparation.
본 발명의 약학적 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있을 것으로 판단된다.The pharmaceutical compositions of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
본 발명 약학적 조성물의 일일 투여량은 조성물에 함유된 본 발명의 추출분획물을 기준으로 하루에 약 1㎎ 내지 1g/㎏, 바람직하게는 50 내지 700㎎/㎏일 수 있으며, 하루 1회 내지 수회 나누어 투여될 수 있으나 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양할 것이다.The daily dosage of the pharmaceutical composition of the present invention may be about 1 mg to 1 g / kg, preferably 50 to 700 mg / kg per day based on the extract fraction of the present invention contained in the composition, However, the range may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
실제 임상 투여 시에 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있을 것이다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있을 것이다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있을 것이다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있을 것이다.In practical administration, it may be administered in various formulations of parenteral administration. In the case of formulation, it may be prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension agent. The base of suppositories may be witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 약학적 조성물은 본 발명의 추출분획물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있을 것이다.The pharmaceutical composition of the present invention may contain, in addition to the extract fraction of the present invention, one or more active ingredients showing the same or similar functions.
본 발명의 식품 조성물은 상기 유효량을 기준으로 본 발명의 추출분획물 그 자체 또는 식품학적으로 허용된 담체와 혼합한 조성물일 수 있다. 본 발명의 식품 조성물은 식육가공품, 어육제품, 두부, 묵, 죽, 라면이나 국수 등의 면류, 간장, 된장, 고추장, 혼합장 등의 조미식품, 소스, 과자, 발효유나 치즈 등의 유가공품, 김치나 장아찌 등의 절임식품, 과실, 채소, 두유, 발효음료 등의 음료수의 식품 형태가 가능할 것으로 판단된다. 또한 식품학적으로 허용된 담체는 상기 약학적으로 허용된 담체도 사용할 수 있을 것이다.The food composition of the present invention may be an extract fraction of the present invention or a composition mixed with a pharmaceutically acceptable carrier on the basis of the effective amount. The food composition of the present invention can be applied to food products such as meat products, fish products, tofu, mushrooms, porridge, noodles such as noodles or noodles, seasonings such as soy sauce, miso, It is considered that food types such as pickles such as pickles, pickles, fruits, vegetables, soymilk, and fermented beverages are possible. The pharmacologically acceptable carrier may also be the pharmaceutically acceptable carrier.
본 발명의 사료 첨가제는 상기 유효량을 기준으로 본 발명의 추출분획물 그 자체 또는 사료학적으로 허용된 담체와 혼합한 것일 수 있다.The feed additive of the present invention may be a mixture of the extract fraction of the present invention or the dietary allowance carrier based on the effective amount.
본 발명의 사료 조성물은 본 발명의 추출분획물 또는 사료 첨가제를 사료와 혼합하여 제조될 수 있다. 배합사료, 펠렛형태 및 사일레지 등의 형태가 가능할 것으로 판단된다. 상기 배합사료는 여러 종류의 일반사료와 상기 본 발명의 추출분획물 또는 사료 첨가제와 혼합하여 제조될 수 있다. 펠렛 형태의 사료는 상기 배합사료를 펠렛기로 제형화하여 제조될 수 있으며, 사일레지는 청예사료를 발효한 다음 상기 본 발명의 추출분획물 또는 사료 첨가제를 첨가하는 방법으로 제조될 수 있을 것이다.The feed composition of the present invention can be prepared by mixing the extract fraction or the feed additive of the present invention with the feed. It is considered that form of feed, pellet and silage can be used. The compound feed can be prepared by mixing various kinds of general feeds with the extract fraction or feed additive of the present invention. The pellet-shaped feed can be prepared by formulating the compound feed into a pelletizer, and the silage can be prepared by fermenting a livestock feed, followed by adding the extract fraction or the feed additive of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 이탈리안라이그라스 추출분획물 제조Example 1. Preparation of Italian Ryegrass Extracted Fraction
본 실시예에서 사용된 사료작물인 이탈리인라이그라스는 천안 국립축산과학원 축산기술개발원에서 직접 재배 및 수확(출수일 5월 5일부터 이를 기준으로 5일째되는 날 사이에 수확)하여 사일리지로 만들고 60일간 발효시킨 뒤 건조시켜 분말형태로 만든 것을 사용하였다.The Italian ragras used in this example were cultivated and harvested directly from the Livestock Technology Development Institute of Cheonan National Livestock Research Institute (harvested between May 5 and 5 on the 5th day) After fermentation for one day, it was dried and used in powder form.
이탈리안라이그라스 사일리지 건조물 771g에 70%(v/v) 메탄올 5ℓ를 첨가하고 12 ~ 24시간 동안 추출한 다음 여과지(Whatman No. 1)로 여과하여 추출액을 수득하고, 다시 추출박에 5ℓ의 70%(v/v) 메탄올을 첨가하고 동일한 방법으로 추출액을 얻었다. 여과된 추출액을 합한 다음 35℃의 수욕상에서 감압 농축 건고하였다.To 5 g of 70% (v / v) methanol was added to 771 g of dried Italian ryegrass silage and 5 l of methanol was added thereto. The mixture was extracted for 12 to 24 hours and filtered through a filter paper (Whatman No. 1) v / v) methanol was added and an extract was obtained in the same manner. The filtered extracts were combined and concentrated in vacuo on a water bath at 35 < 0 > C to dry.
이 건고물에 1ℓ 석유에테르(petroleum ether)를 첨가하고 12 ~ 24시간 동안 추출한 다음 여과하여 잔사를 수득하고, 이 잔사에 1ℓ의 클로로포름을 첨가하고 12 ~ 24시간 동안 추출한 다음 여과하여 클로로포름 추출액을 수득하였다.1 liter of petroleum ether was added to the mixture, and the mixture was extracted for 12 to 24 hours. After filtration, a residue was obtained. 1 liter of chloroform was added to the residue, which was then extracted for 12 to 24 hours, followed by filtration to obtain a chloroform extract Respectively.
이후 이 클로로포름 추출액을 35℃의 수욕상에서 감압 농축 건고하여 3.936g의 클로로포름 용해성 조추출물(chloroform-soluble crude extract)을 확보하였다.The chloroform extract solution was concentrated under reduced pressure on a water bath at 35 ° C to obtain 3.936 g of chloroform-soluble crude extract.
상기 클로로포름 용해성 조추출물을 소량의 70%(v/v) 메탄올에 용해하고 Sephadex LH-20 수지(Sigma)가 충진된 open column(수지 50g이 담긴 직경 2.8cm, 길이 45cm의 유리컬럼)을 이용하여 분획하였다. 이때 전개용매로 물(수지 충진부피의 1/3배 부피), 10%(v/v) 메탄올 수용액(수지 충진부피의 2배 부피), 30%(v/v) 메탄올 수용액(수지 충진부피의 2배 부피), 50%(v/v) 메탄올 수용액(수지 충진부피의 2배 부피), 70%(v/v) 메탄올 수용액(수지 충진부피의 2배 부피), 100%(v/v) 메탄올(수지 충진부피의 2배 부피)를 사용하였으며, 전개용매가 물일 때는 1개의 분획물(fraction)을 얻어내었고, 메탄올 수용액과 100% 메탄올을 전개용매로 사용했을 때는 각각 동일한 양의 3개의 분획물로 나누어 총 16개의 분획물을 얻었다. 이때 각 분획물은 물 분획물부터 먼저 얻은 순서대로 분획물 1부터 16번으로 정했다.The chloroform-soluble crude extract was dissolved in a small amount of 70% (v / v) methanol, and an open column (glass column with a diameter of 2.8 cm and a length of 45 cm containing 50 g of resin) filled with Sephadex LH-20 resin Lt; / RTI > At this time, as a developing solvent, water (1/3 volume of the resin filling volume), 10% (v / v) methanol aqueous solution (twice the volume of the resin filling volume), 30% (v / v) methanol aqueous solution (V / v) aqueous methanol solution (two times the volume of the resin filling volume), 50% (v / v) aqueous methanol solution (twice volume of the resin filling volume) One fraction of methanol was used when the developing solvent was water. When methanol solution and 100% methanol were used as developing solvent, three fractions of the same amount were obtained. And total 16 fractions were obtained. At this time,
이후 각 분획물을 35℃의 수욕상에서 감압 농축 건고하였다. 각 분획물의 제조과정 및 수득한 최종 건고물의 양을 도 1에 나타내었다.The fractions were then concentrated under reduced pressure on a water bath at 35 캜 for drying. The production process of each fraction and the amount of the final dry matter obtained are shown in Fig.
실험예 1. 이탈라안라이그라스 추출분획물의 간독성 유도물질의 투여에 따른 간조직 무게 증가, 간독성 지표물질 증가, 간 항산화효소 활성 감소 등에 대한 억제효과 검증EXPERIMENTAL EXAMPLE 1 Inhibitory effect on hepatic tissue weight increase, hepatotoxicity index increase and reduction of hepatic antioxidant enzyme activity by administering the hepatotoxic inducer of the extract fraction of the R. raglas extract
본 실험예서는 이탈리안라이그라스 추출분획물의 간독성 물질인 D-galactosamine (GalN)의 처리에 의해 유도되는 간 손상에 대한 보호효과를 확인하고자 수행되었다.This experiment was carried out to confirm the protective effect of hepatic injury induced by the treatment of D-galactosamine (GalN), which is a hepatotoxic substance, in the extract of Italian Ryegrass.
본 실험예에서 사용한 실험동물은 서울소재 오리엔트사에서 구입한 수컷 ICR 생쥐이다. 생쥐는 구입 일주일 후 각 그룹별 10수로 총 6그룹으로 나누어 실험에 사용하였다. GalN은 사용 전 PBS에 녹여 사용하였으며, 분획물 7(Fraction 7; F7)은 DMSO에 녹여 투여하였다. 이때 투여된 DMSO의 농도는 10%를 넘지 않았다. 그룹 1 생쥐는 대조군으로서 10% DMSO를 포함한 200μl PBS로 매 2일마다 총 7회 경구 투여되었으며, 그룹 2 생쥐는(GalN 단독처리군) 대조군과 동일한 기간 동안 동일한 횟수로 PBS만으로 처리되었으며, PBS 마지막 투여 후 800mg/kg GalN이 들어있는 PBS를 복강투여하였다. 그룹 3, 4 및 5 생쥐들은 분획물 7이 각각 50, 100, 200mg/kg body weight로 함유된 PBS로 동일한 기간과 방법으로 처리하였으며, 마지막 처리 후 800mg/kg GalN이 들어있는 PBS를 복강투여하였다. 그룹 6 생쥐들은 GalN 처리 없이 200mg/kg의 분획물 7이 들어있는 PBS를 동일한 기간과 방법으로 투여하였다.The experimental animal used in this experiment is a male ICR mouse purchased from Orient, Seoul, Korea. The mice were divided into 6 groups, 10 groups for each group one week after purchase. GalN was dissolved in PBS before use, and fraction 7 (Fraction 7; F7) was dissolved in DMSO. At this time, the concentration of DMSO administered did not exceed 10%.
GalN 처리 후 생쥐로부터 심장 천공법을 이용하여 주사기로 혈액을 채취한 후 혈청을 확보하였으며, 확보된 혈청은 주요한 간독성 지표물질로 알려진 alkaline phosphate(ALP), alanine aminotransferase(ALT), aspartate aminotransferase(AST) 및 lactate dehydrogenase(LDH) 등과 같은 효소의 활성 측정에 사용하였다. 또한 혈액 채취 후 생쥐의 간과 비장 조직을 분리하여 무게를 측정하였다. 간 조직의 경우에는 무게 측정 후 차가운 isotonic saline으로 세척한 후 3개의 조각으로 분리하였다. 각 그룹별 간조직을 일정한 무게로 자른 후 차가운 용매(50mM ice-cold KH2PO4 solution (1:5 w/v) 또는 BioAssay Systems(Hayward, CA, USA)사에서 제공한 균질 용매 200μl에 넣고 균질기(homogenizer; PRO Scientific Inc., Oxford, CT, USA)를 이용하여 균질하였다. 그 후 원심분리기로 10분간 10,000g의 중력으로 원심 분리하여 맑은 상층액을 확보하였다. 이 상층액들은 malondialdehyde(MDA) 함량과 항산화효소들인 glutathione(GSH), glutathione peroxidase(GPx), superoxide dismutase(SOD), catalase(CAT) 등의 활성 측정에 사용하였다.(AST), alanine aminotransferase (ALT), alanine aminotransferase (ALT), and alanine aminotransferase (ALT), which are known to be major hepatotoxicity indicators, were obtained from the mice after cardiac puncture by GalN treatment. And lactate dehydrogenase (LDH). The liver and spleen tissues of mice were separated and weighed. In the case of liver tissue, after weighing, it was washed with cold isotonic saline and then separated into three pieces. The liver tissues of each group were cut into a uniform weight and placed in 200 μl of a homogeneous solvent (50 mM ice-cold KH 2 PO 4 solution (1: 5 w / v) or BioAssay Systems (Hayward, CA, USA) Homogenizer (PRO Scientific Inc., Oxford, CT, USA) was homogenized and centrifuged for 10 min at 10,000g gravity to obtain a clear supernatant. MDA) and antioxidant enzymes such as glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT)
분획물 7이 GalN 유도성 간조직의 지질과산화(lipid peroxidation)에 미치는 효과를 확인하기 위해 lipid peroxidation의 최종산물인 MDA의 수준을 측정하였다. 보다 상세히 설명하면 각 그룹별 간 조직 균질화 상층액 100μl를 8% sodium dodecyl sulfate(SDS) 함유된 용액 50μl와 혼합하여 10분간 상온에 정치시켰다. 그 후 375μl의 20% acetic acid(pH 3.5)와 375μl의 0.6% thiobarbituric acid를 넣은 후 80℃에서 60분간 반응시켰다. 그 다음 250μl의 정제수와 1.25ml의 butanol : pyridine 혼합물(15:1, v/v)을 반응용액에 넣고 강하게 흔들어 준 다음 1,000g의 중력으로 5분간 원심 분리 하였다. 최종적으로는 상층액만을 취하여 1,1,3,3-tetraethoxypropane를 표지물질로 사용하여 532nm에서 흡광도를 측정하여 각 그룹의 간조직 샘플별 MDA의 함량을 측정하였다.To determine the effect of fraction 7 on the lipid peroxidation of GalN-induced liver tissue, the level of MDA, the final product of lipid peroxidation, was measured. More specifically, 100 μl of the liver tissue homogenization supernatant of each group was mixed with 50 μl of a solution containing 8% sodium dodecyl sulfate (SDS) and allowed to stand at room temperature for 10 minutes. Then, 375 μl of 20% acetic acid (pH 3.5) and 375 μl of 0.6% thiobarbituric acid were added and reacted at 80 ° C for 60 minutes. Then, 250 μl of purified water and 1.25 ml of butanol: pyridine mixture (15: 1, v / v) were added to the reaction solution and vigorously shaken. Then, the mixture was centrifuged at 1,000 g for 5 minutes. Finally, only the supernatant was taken and absorbance was measured at 532 nm using 1,1,3,3-tetraethoxypropane as a labeling substance, and the content of MDA in each group of liver tissue samples was measured.
한편 간조직 내 항산화효소들의 측정에 있어서 GSH와 SOD의 함량은 BioVision Research Products(Milpitas, CA, USA)사에서 제공한 측정키트인 각각 #K264-100과 #K335-100를 사용하여 측정하였으며, CAT(EC 1.11.1.6)와 GPx(EC 1.11.1.9)의 활성은 각각 Amplex® Red Catalase Assay Kit(A22180, Molecular Probes, Inc., Eugene, OR, USA)와 GPx assay kit(EGPX-100; BioAssay Systems)를 사용하여 측정하였다. 모든 측정은 각 제조사에 제공한 매뉴얼에 따라 수행되었다.The contents of GSH and SOD were measured by using # K264-100 and # K335-100, which were provided by BioVision Research Products (Milpitas, CA, USA) (EC 1.11.1.6) and GPx (EC 1.11.1.9) of the activity of each Amplex ® Red Catalase assay Kit (A22180 , Molecular Probes, Inc., Eugene, OR, USA) and GPx assay kit (EGPX-100; BioAssay Systems ). All measurements were performed according to the manual provided to each manufacturer.
다른 한편 혈청 내 간독성 관련 효소인 ALP(EC 3.1.3.1), ALT(E.C 2.6.1.2), AST(EC 2.6.1.1), LDH(EC 1.1.1.27) 등의 함량은 Bayer사(ADVIA 1650, Bayer, Japan)에서 제공한 분석키트와 평가방법에 의거하여 측정하였다. 또한 GalN 처리에 따른 간조직의 손상여부를 직접적으로 확인하기 위해 각 그룹별 간조직을 4% paraformaldehyde 용액에 24시간 동안 정치시킨 후 조직 내 수분을 제거하고 paraffin 용액으로 처리하였다. 파라핀이 처리된 각 그룹별 간조직 샘플을 절단기를 이용하여 5.0㎛의 두께로 절편하고 각 절편 조직을 glass 슬라이드에 올려 고정시킨 후 hematoxylin과 eosin 용액으로 염색하였다. 염색된 각 조직 샘플들은 위상차 현미경(EL-Einsatz 451888, Carl Zeiss, Ostalbkreis, Germany)으로 관찰하였다.The content of ALP (EC 3.1.3.1), ALT (EC 2.6.1.2), AST (EC 2.6.1.1) and LDH (EC 1.1.1.27), which are hepatotoxicity related enzymes in serum, were measured by Bayer (ADVIA 1650, Bayer , Japan) and the evaluation method. In order to directly determine whether the liver tissue was damaged by GalN treatment, the liver tissue of each group was left in a 4% paraformaldehyde solution for 24 hours, and the tissue water was removed and treated with paraffin solution. The tissue samples of each group treated with paraffin were sectioned to a thickness of 5.0 μm using a cutter, and each section was fixed on a glass slide and stained with hematoxylin and eosin solution. Each stained tissue sample was observed with a phase contrast microscope (EL-Einsatz 451888, Carl Zeiss, Ostalbkreis, Germany).
도 2은 간독성 유도물질 GalN 처리에 따른 간과 비장 조직 무게의 변화에 이에 대한 분획물 7의 효과에 대한 표이다. 비장의 무게는 GalN 처리 또는 GalN 처리 전 분획물 7의 처리 유무와 관계없이 무게의 변화는 관찰되지 않았다. 반면 간조직은 GalN 단독 처리구에서는 대조군에 비해 유의적인 증가가 나타났으며, GalN 처리전 분획물 7 처리군에서는 농도의존적인 간 무게 감소효과가 나타났다.FIG. 2 is a table for the effect of fraction 7 on changes in liver and spleen tissue weights following treatment with hepatotoxic inducer GalN. The weight of the spleen did not change in weight regardless of whether the treatment with the fraction 7 before GalN treatment or GalN treatment was carried out. On the other hand, liver tissue showed a significant increase in the treatment with GalN alone compared to the control group, and the concentration-dependent liver weight reduction effect was shown in the fraction 7 treated group before treatment with GalN.
도 3은 간독성 유도물질의 투여에 따른 간 조직 손상 특이적 지표물질의 증가에 대한 분획물 7의 억제효과에 대한 표이다. GalN 단독 처리군에서는 혈액 내 ALT, ALP, AST, LDH 농도가 대조군에 비해 모두 유의적 수준으로 증가하였으며, 이러한 증가는 GalN 처리에 따른 간독성 유발을 의미한다. 반면 분획물 7을 처리한 생쥐들에서는 측정된 모든 간독성 지표물질의 활성이 농도의존적으로 감소되었으며, GalN과 200mg/kg 분획물 7을 처리한 군에서는 대조군과 거의 유사한 수준까지 감소된다는 것을 확인하였다. 이러한 결과들은 GalN 처리에 따른 간독성에 대한 분획물 7의 억제 효능을 보여준다.FIG. 3 is a table showing the inhibitory effect of the fraction 7 on the increase of the liver tissue-specific index substance upon administration of the hepatotoxic inducer. In the GalN monotherapy group, ALT, ALP, AST and LDH concentrations in the blood were significantly increased compared to the control group, and this increase was indicative of hepatotoxicity induced by GalN treatment. On the other hand, in the fraction 7 treated mice, the activity of all of the hepatic toxicities measured was decreased in a concentration-dependent manner, and it was confirmed that in the group treated with GalN and 200 mg / kg fraction 7, These results show that fraction 7 inhibits hepatotoxicity due to GalN treatment.
도 4는 간독성 유도물질의 투여에 따른 간조직 내 항산화효소 활성저하와 산화물질 증가에 대한 분획물 7의 억제효과 검증결과이다. 표에서 보는 바와 같이 GalN 단독 처리군에서는 대조군에 비해 GSH, CAT, SOD, GPx와 같은 항산화효소 활성이 유의적으로 감소하였으며, 반면 지방과산화 산물인 MDA의 함량은 현저하게 증가한다는 것을 확인하였다. 분획물 7 혼용 처리군에서는 모든 항산화효소의 활성을 농도의존적으로 증가시켰으며, 200mg/kg의 분획물 7이 처리된 생쥐에서는 거의 대조군과 같은 수준까지 증가됨을 확인하였다. 한편 분획물 7 단독 처리군에서는 대조군에 비해 약간의 항산화효소 활성 증가가 나타났으나 유의적 수준은 아니었다.FIG. 4 shows the result of the inhibition of the antioxidant enzyme activity and the inhibitory effect of the fraction 7 on the increase of the oxidizing substance in the liver tissue upon administration of the hepatotoxic inducer. As shown in the table, the activities of antioxidant enzymes such as GSH, CAT, SOD and GPx were significantly decreased in the GalN-treated group, while the content of MDA, the fatty peroxide, was significantly increased. In the fraction 7 mixed treatment group, the activity of all antioxidant enzymes was increased in a concentration-dependent manner, and it was confirmed that the fraction treated with 200 mg / kg of fraction 7 increased to almost the same level as that of the control group. On the other hand, there was a slight increase in the activity of antioxidant enzymes in the fraction 7 treated group, but not significant.
도 5는 간독성 유도물질의 투여에 따른 간조직 손상에 대한 분획물 7의 억제효과를 조직염색을 통해 현미경으로 확인한 결과이다. 도 5의 A는 대조군의 간조직이며, 도 5의 B는 GalN 단독 처리군, 도 5의 C와 D는 각각 100mg/kg과 200mg/kg의 분획물 7을 GalN 처리 전 투여한 실험군의 간조직 형상을 보여주고 있다. 사진에서 볼 수 있듯이 GalN 처리에 의한 간조직 손상이 나타났으며, 분획물 7 처리군에서는 현저하게 감소되었다.FIG. 5 shows the result of microscopic examination of the inhibitory effect of the fraction 7 on hepatic tissue damage upon administration of the hepatotoxicity inducing substance through tissue staining. FIG. 5A shows the liver tissue of the control group, FIG. 5B shows the group treated with GalN alone, FIG. 5C and D show the liver tissue form of the experimental group administered with 100 mg / kg and 200 mg / Respectively. As shown in the photograph, liver injury was observed by GalN treatment, and it was remarkably decreased in the fraction 7 treated group.
Claims (8)
상기 용출물은 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 얻은 용출물 중 세 번째로 용출된 용출물인 것을 특징으로 하는 약학적 조성물.The method according to claim 1,
The eluate was fractionated by eluting with chloroform-soluble extracts through a sephadex LH20 resin, water, 10% (v / v) methanol aqueous solution and 30% (v / v) methanol aqueous solution, wherein the eluted product is a third eluted fraction of the eluate obtained by fractionation into three regions using an aqueous solution of methanol (% (v / v) methanol).
상기 용출물은 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 얻은 용출물 중 세 번째로 용출된 용출물인 것을 특징으로 하는 식품 조성물.The method of claim 3,
The eluate was fractionated by eluting with chloroform-soluble extracts through a sephadex LH20 resin, water, 10% (v / v) methanol aqueous solution and 30% (v / v) methanol aqueous solution, wherein the eluted product is a third eluted fraction of the eluate obtained by fractionation into three regions using an aqueous solution of 1% (v / v) methanol.
상기 용출물은 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 얻은 용출물 중 세 번째로 용출된 용출물인 것을 특징으로 하는 사료 조성물.6. The method of claim 5,
The eluate was fractionated by eluting with chloroform-soluble extracts through a sephadex LH20 resin, water, 10% (v / v) methanol aqueous solution and 30% (v / v) methanol aqueous solution, wherein the eluate is a third eluted fraction of the eluate obtained by fractionation into three regions using an aqueous solution of methanol (% / v) methanol.
상기 용출물은 상기 클로로포름 용해성 추출물을 세파덱스(sephadex) LH20 수지에 통과시키고, 물, 10%(v/v) 메탄올 수용액, 30%(v/v) 메탄올 수용액으로 순서대로 용출하여 분획하되, 30%(v/v) 메탄올 수용액을 사용하여 세 개의 영역으로 분획하여 얻은 용출물 중 세 번째로 용출된 용출물인 것을 특징으로 하는 사료 첨가제.
8. The method of claim 7,
The eluate was fractionated by eluting with chloroform-soluble extracts through a sephadex LH20 resin, water, 10% (v / v) methanol aqueous solution and 30% (v / v) methanol aqueous solution, wherein the eluate is a third eluted fraction of the eluate obtained by fractionation into three regions using an aqueous solution of methanol (% / v) methanol.
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KR100263393B1 (en) | 1991-08-16 | 2000-08-01 | 제이. 비. 포터 | Ryegrass pollen allergen |
WO2007006079A1 (en) | 2005-07-08 | 2007-01-18 | Hexima Limited | Management of plant pathogens |
JP2008193916A (en) | 2007-02-09 | 2008-08-28 | Hokkaido | Weed preventing method using italian ryegrass |
JP2008237180A (en) | 2007-03-28 | 2008-10-09 | Japan Grassland Farming Forage Seed Association | Ssr primer pair useful for determination of species of italian ryegrass and use thereof |
US20130108618A1 (en) | 2011-10-28 | 2013-05-02 | University Of Maryland | METHODS AND COMPOSITIONS RELATED TO INTRACELLULAR NEUTRALIZATION BY IgG |
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KR100263393B1 (en) | 1991-08-16 | 2000-08-01 | 제이. 비. 포터 | Ryegrass pollen allergen |
WO2007006079A1 (en) | 2005-07-08 | 2007-01-18 | Hexima Limited | Management of plant pathogens |
JP2008193916A (en) | 2007-02-09 | 2008-08-28 | Hokkaido | Weed preventing method using italian ryegrass |
JP2008237180A (en) | 2007-03-28 | 2008-10-09 | Japan Grassland Farming Forage Seed Association | Ssr primer pair useful for determination of species of italian ryegrass and use thereof |
US20130108618A1 (en) | 2011-10-28 | 2013-05-02 | University Of Maryland | METHODS AND COMPOSITIONS RELATED TO INTRACELLULAR NEUTRALIZATION BY IgG |
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