KR101649707B1 - Composition for Preventing Improving or Treating of 2-Mediated Immune Disease Comprising Extracts from Chrysosplenium grayanum - Google Patents
Composition for Preventing Improving or Treating of 2-Mediated Immune Disease Comprising Extracts from Chrysosplenium grayanum Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
본 발명은 갈매나무(Rhamnus davurica) 추출물, 괭이눈(Chrysosplenium grayanum) 추출물, 나한송(Podocarpus macrophyllus) 추출물, 조록나무(Distylium racemosom) 추출물 및 진달래(Rhododendron mucronulatum) 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물을 유효성분으로 포함하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. 본 발명에 따르면, 상기 추출물은 IL-4 생성 억제, 알레르겐의 장상피세포 통과 억제 및 비만세포의 탈과립 억제 활성을 가짐으로 Th2-매개 면역 질환을 예방, 개선 또는 치료할 수 있다.The present invention relates to a method for the production of an extract of Rhamnus davurica , Chrysosplenium grayanum extract, Podocarpus macrophyllus extract, Distylium racemosom extract and Rhododendron mucronulatum extract, As an active ingredient, a composition for preventing, ameliorating or treating a Th2-mediated immune disease. According to the present invention, the above extract can prevent, ameliorate, or treat a Th2-mediated immune disease by inhibiting IL-4 production, suppressing the passage of intestinal epithelial cells through allergens and inhibiting degranulation of mast cells.
Description
본 발명은 괭이눈(Chrysosplenium grayanum) 추출물을 포함하는 알레르기 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, ameliorating or treating an allergic disease comprising a chrysosplenium grayanum extract.
근년 소득수준의 향상과 더불어 식습관의 변화, 위생의 청결화, 공해의 심화 등으로 인하여 알레르기는 날로 증가하는 추세를 보이고 있어 전 국민의 20-25%가 아토피피부염(AD), 천식, 비염 등의 증상을 가지고 있다. 이는 삶의 질을 현격히 저하시킴으로써 심각한 사회문제를 여기하고 있다. 또한, 식품 알레르기는 영유아의 6-8%에서 확인되고 있고, 소아의 AD는 35%가 식품알레르기에 의한 것으로 결코 무시할 수 없다.Allergy is increasing day by day due to changes in diet, cleanliness of hygiene and intensification of pollution, and 20-25% of the whole population are suffering from atopic dermatitis (AD), asthma and rhinitis I have symptoms. This is a serious social problem by significantly reducing the quality of life. In addition, food allergies are identified in 6-8% of infants and 35% of children in AD are due to food allergies.
아직까지 여러 알레르기에 대하여 확실한 효과를 보이는 치료제가 거의 없으며, 소아 아토피피부염이 증가하고 있으나 소아의 특성상 치료에 어려움을 겪고 있다. 특히 스테로이드 계통의 약물은 부작용이 심하여 세계적으로 그 대안을 마련하기위한 시도가 활발하게 이뤄지고 있다. 이러한 알레르기에 대응하는 방안으로써 독성과 부작용이 없으면서 확실한 효능이 기대되는 소재를 천연물과 식품으로부터 발굴하여 적극적으로 활용할 필요가 있다. 또한, 활성소재 및 성분의 항알레르기 작용기전도 함께 구명되어야 할 것이다.Although there are few therapeutic agents that have a definite effect on various allergies and the increase of pediatric atopic dermatitis, In particular, steroid drugs have serious side effects, and attempts have been actively made to provide alternatives to the world. As a countermeasure against such allergies, it is necessary to excavate and proactively utilize natural materials and foods in which a certain efficacy is expected without toxicity and side effects. In addition, the antiallergic action of active ingredients and ingredients must also be studied.
21세기에 주류를 이루는 치료제는 알레르기 및 당뇨에 대한 것이다(일본 닛케이산쿄신문). 미국에서는 5,000만 명이 다양한 알레르기 질환을 갖고 있다(의약품시장, 연 52억불). 일본에서는 연간 1,560억 엔의 알레르기 관련 의약품시장이 형성되어 있으며, 기능성식품시장은 약 600억 엔이다. 국내의 경우 이 분야 의약품시장은 약 500억 원이다. 최근 일본 기능성식품의 특허 동향에서 “항비만” 다음으로 출원건수가 많은 분야가 “항알레르기”이다. 향후 높은 부가가치가 기대되며 이에 대한 대비가 요망된다.The main cure for the 21st century is allergy and diabetes (Nikkei Sankyo Shimbun, Japan). In the United States, 50 million people have various allergic diseases (drug market, $ 5.2 billion annually). In Japan, an allergic-related drug market of ¥ 156 billion annually is formed, and the functional food market is about ¥ 60 billion. In Korea, the drug market in this field is about 50 billion won. In recent trends in Japanese patented functional foods, "anti-allergy" is the field with the largest number of applications next to "anti-obesity". High value-added is expected in the future and it is expected to be prepared.
최근에 면역기능조절이나 알레르기억제 활성을 강조하는 건강기능식품의 개발요구가 높아지고 있으며, 그 인정을 위하여 식품의약품안전청에 많은 건수가 신청되고 있다. 또한, 근년 일본의 특허분석을 통하여 조사한 미래 기능성식품의 잠재력은 “항비만” 다음으로 “항알레르기” 활성으로 보고되었다.Recently, there is a growing demand for the development of health functional foods that emphasize immune function regulation and allergen inhibitory activity, and many cases have been filed with the Korea Food & Drug Administration for recognition. In addition, the potential of future functional foods investigated through patent analysis in recent years has been reported as "antiallergic" activity after "anti-obesity".
알레르기는 “과민반응”이라는 뜻으로 그리스어인 “allos”가 어원이며, 이는 “변형된 것”을 뜻한다. 이처럼 알레르기란 면역시스템의 오작동으로 보통 사람에게는 별 영향이 없는 물질이 어떤 사람에게만 두드러기, 가려움, 콧물, 기침 등의 이상 과민 반응을 일으키는 것을 말한다. 최근 20 년 동안 이와 같은 알레르기성 질병의 발병율이 세계적으로 증가하고 있는 추세이다. 주된 증가요인으로는 실내생활의 증가, 신소재의 개발, 외국으로부터 이물질 유입등과 같은 알레르기 유발 물질의 급증과 환경오염 및 스트레스로 인한 면역상태의 불안정, 그리고 식생활의 변화 등이 있다. 이처럼 현대인들의 알레르기 질병 발병율은 점차 증가하나 그 치료방법은 대부분 항히스타민제, 스테로이드제 등의 대중적 요법에 의존하고 있는 실정이며, 근본적인 치료방법은 찾지 못하고 있다.Allergy means "hypersensitive", the Greek word "allos" is etymology, which means "altered". Allergy is a malfunction of the immune system, which means that substances that do not have a significant effect on normal people cause irritable reactions such as urticaria, itching, runny nose, and coughing in some people. In recent 20 years, the incidence of such allergic diseases has been increasing worldwide. The main reasons for the increase include the increase of indoor living, the development of new materials, the surge of allergenic substances such as foreign matter from foreign countries, instability of immune status due to environmental pollution and stress, and change of diet. In this way, the incidence of allergic diseases in modern people gradually increases, but most of the treatment methods depend on popular therapy such as antihistamines and steroids.
Gell 및 Coombes(1963)는 알레르기반응을 발현시간과 타입에 기초하여 전통적으로 4 개의 부류로 나누었다. I - III 형은 항체가 관여하는 체액성 면역 반응(즉시형)이고, IV 형은 세포가 직접 관여하는 세포성 면역 반응(지연형)이다. 그리고 면역반응에 관여하는 세포와 매체의 종류에 따라 수분내에 증상이 나타나는 즉시 또는 조기 반응과 몇 시간 후에 나타나는 후기단계 반응으로 나뉘는데 제 I 형은 즉시형 과민반응, 제 IV 형은 지연형 과민반응의 형태로 나타난다. 대부분의 알레르기 반응은 제 I 형으로 천식, 비염, 결막염, 식품 및 약물 알레르기, 아토피성 피부염 등이 대표적인 질환으로 나타나고 심한 경우 아나필락시스(anaphylaxis)가 일어나 생명을 위협하기도 한다.Gell and Coombes (1963) traditionally divided allergic reactions into four classes based on expression time and type. Type I - III is a humoral immune response (immediate type) involving antibodies, and Type IV is a cell - mediated immune response (delayed type). In addition, there are two types of immune response: type I, type II, type III, type IV, type IV, type IV, . Most allergic reactions are Type I, which are typical diseases such as asthma, rhinitis, conjunctivitis, food and drug allergies, and atopic dermatitis. In severe cases, anaphylaxis may cause life-threatening reactions.
제 I 형 즉시형 과민반응은 2 단계로 나뉘는데 제 1 단계는 알레르겐의 체내 침입에 의하여 IgE 및 IgG1의 분비를 억제하고, IgG2a의 분비를 증가시키는 IL-12 및 IFN-r을 생산하는 Th1 세포반응과 IL-4, IL-5 및 IL-13등을 생산하는 Th2 세포반응의 균형이 Th2 쪽으로 기울어지게 되면 Th2의 과도한 면역반응으로 IL-4, IL-13 등이 분비되고 그 영향으로 B 세포가 생산한 IgE 특이항체들이 비만세포(mast cell)나 호염기구(basophil)의 표면에 부착됨으로써 알레르기 발증이 준비된 단계이다. 이를 알레르겐에 감작되었다고 한다.The type I immediate-type hypersensitivity reaction is divided into two stages. The first stage is the Th1 cell reaction which produces IL-12 and IFN- r which inhibits the secretion of IgE and IgG1 by invasion of the allergen and increases the secretion of IgG2a IL-4, and IL-13 are secreted by excessive immune response of Th2 when the balance of Th2 cells producing IL-4, IL-5 and IL-13 is shifted toward Th2. The IgE-specific antibodies produced are attached to the surface of mast cells or basophils, so that they are ready for allergy development. It has been sensitized to allergens.
알레르기 발증의 제 2 단계는 초기반응과 후기반응으로 나뉘며, 초기반응은 알레르겐이 체내에 재침입하여 비만세포를 자극하고 탈과립 반응을 유발하여 이 때 방출된 히스타민, 지질대사물, 사이토카인 등에 의한 혈관확장 등이 일어나는 것이고, 후기반응은 해당조직에 호중구, 호산구, 대식세포, Th2 세포, 호염기구 등이 침윤하여 활성화됨으로써 염증이 유발되어 아토피피부염, 비염, 천식 등을 일으키는 것이다. 이 같은 탈과립 분비 물질 중 히스타민은 가장 잘 알려진 인자이며, 즉각형 과민반응과도 관련되어 알레르기 증상의 중요한 지표로 사용되고 있다.The second stage of the allergic manifestation is divided into an initial reaction and a late reaction. The allergen is re-invaded into the body to stimulate mast cells and triggers the degranulation reaction, and blood vessels caused by released histamine, lipid metabolites, cytokines, And eosinophil, eosinophil, macrophage, Th2 cell, and basophil are infiltrated into the affected tissue to induce inflammation, resulting in atopic dermatitis, rhinitis, asthma, and the like. Among these degranulation secretions, histamine is the most well-known factor and is also used as an important indicator of allergic symptoms in relation to immediate hypersensitivity reaction.
한편 식품알레르기는 최근 10년 동안 선진국에서는 약 3-6%의 어린이들에게서 발병되며 증가하는 추세를 보이고 있다. 식품알레르기는 소화관을 통해 체내에 들어 온 식품에 대하여 발생하는 것으로 주로 제 I 형 과민반응에 해당한다. 알레르기 환자의 경우 장관을 통하여 식품알레르겐이 소화되지 않은 상태로 침투하게 되며, 이러한 알레르겐의 장내 흡수가 식품알레르기 발증의 첫 단계라 할 수 있다. 장관은 영양소의 소화흡수를 담당하는 기관일 뿐만 아니라 여러 가지 생체 조절에 관여하고 있다. 장상피 세포는 소화흡수, 장벽(barrier), 식신호의 전달/ 변환 등의 주요 기능을 가지고 있을 뿐만 아니라 그 밖에도 독특한 면역시스템(mucosal immunity)이나 신경조직이 발달되어 있는 등 소화관/장관의 역할을 결코 과소평가할 수 없다. 이렇듯 장상피세포는 장벽으로써 밀착연접(tight junction)에 의해 거대 분자의 투과를 제한하나, 밀착연접이 손상된 경우에는 장벽의 기능 장애로 인해 투과성이 증가되어 식품알레르기뿐만 아니라 소아지방변증, 급성 췌장염과 같은 질환이 발생된다. 즉, 밀착연접의 안정화를 통해 장관의 장벽 기능을 강화시킨다면 식품 알레르기를 예방할 수 있을 것이다.On the other hand, food allergy has been increasing in the developed countries for the last 10 years in about 3-6% of children. Food allergy is a type I hypersensitivity reaction that occurs in food entering the body through the digestive tract. In the case of allergy patients, the food allergen penetrates into the digestive tract through the intestinal tract, and absorption of such allergens into the intestines is the first step in the development of food allergies. The minister is involved not only in the digestion and absorption of nutrients but also in various biological control. The intestinal epithelial cells have major functions such as digestion and absorption, barrier, and signal transduction / transformation, as well as a unique immune system (mucosal immunity) and development of nervous tissue. It can never be underestimated. In this study, we investigated the effect of intestinal epithelial cells on the permeability of intestinal epithelium. The intestinal epithelium is a barrier that restricts the permeation of macromolecules by tight junctions. However, The same disease occurs. In other words, if the barrier function of the intestinal tract is strengthened by stabilizing the tight junction, food allergy can be prevented.
이미 앞선 논문들에서는 식품 및 천연물의 알레르기 억제 활성이 상당수 보고된 바 있으며, 특히 우리나라에서는 알레르기의 치료 및 예방에 식품과 천연물이 한방과 민간요법으로 사용되어 왔다. 이러한 연구들은 주로 빈랑자, 지모, 호로파 등의 한약재 및 약초들의 탈과립 억제 활성을 평가한 결과이며 , 퀘르세틴(quercetin), 다래, 마늘, 들깨 기름, 곤약, 소엽, 맥문동의 다당류성분, 영지버섯, 일본간장의 다당체 성분, 로얄 젤리, 동충하초 등에서의 Th1/Th2 면역반응 조절에 관한 연구도 다수 보고되었다.
In previous papers, a lot of allergen suppressing activity of food and natural materials has been reported, and in Korea, food and natural products have been used as herbal medicine and folk remedy for the treatment and prevention of allergy. These studies were conducted to evaluate the inhibitory activities of herbicides and medicinal herbs such as quinquinone, quercetin, garlic, perilla oil, konjac, lobule, Many studies have been reported on the regulation of Th1 / Th2 immune response in polysaccharide components of Japanese soy sauce, royal jelly, and Chinese caterpillar fungus.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 알레르기와 같은 Th2 면역 반응에 의해 유발되는 질병, 질환 또는 상태를 예방 또는 치료할 수 있는 천연물을 개발하고자 노력하였다. 그 결과, 본 발명자들은 갈매나무(Rhamnus davurica) 추출물, 괭이눈(Chrysosplenium grayanum) 추출물, 나한송(Podocarpus macrophyllus) 추출물, 조록나무(Distylium racemosom) 추출물 및 진달래(Rhododendron mucronulatum) 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물이 IL-4 생성 억제, 알레르겐의 장상피세포 통과 억제 및 비만세포의 탈과립 억제 활성을 가짐을 확인함으로써, 본 발명을 완성하였다.The present inventors have sought to develop natural products capable of preventing or treating diseases, diseases or conditions caused by the Th2 immune response such as allergy. As a result, the present inventors have found that sea buckthorn (Rhamnus davurica) extract, gwaengyinun (Chrysosplenium grayanum extract, Podocarpus macrophyllus extract, Distalum racemosom ) and Rhododendron mucronulatum extract of the present invention has inhibitory effect on IL-4 production, suppression of passage of intestinal epithelial cells of allergens and inhibition of degranulation of mast cells, thus completing the present invention.
따라서, 본 발명의 목적은 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물을 제공하는 데 있다.
Accordingly, it is an object of the present invention to provide a composition for preventing, ameliorating or treating a Th2-mediated immune disorder.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 갈매나무(Rhamnus davurica) 추출물, 괭이눈(Chrysosplenium grayanum) 추출물, 나한송(Podocarpus macrophyllus) 추출물, 조록나무(Distylium racemosom) 추출물 및 진달래(Rhododendron mucronulatum) 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물을 유효성분으로 포함하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물을 제공한다.
According to one aspect of the present invention, the present invention provides a composition comprising a Rhamnus davurica extract, a Chrysosplenium grayanum extract, a Podocarpus macrophyllus extract, a Distylium racemosom extract and a Rhododendron mucronulatum extract As an active ingredient, at least one extract selected from the group consisting of:
본 발명자들은 알레르기와 같은 Th2 면역 반응에 의해 유발되는 질병, 질환 또는 상태를 예방 또는 치료할 수 있는 천연물을 개발하고자 노력하였다. 그 결과, 본 발명자들은 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물이 IL-4 생성 억제, 알레르겐의 장상피세포 통과 억제 및 비만세포의 탈과립 억제 활성을 가짐을 확인하였다.
The present inventors have sought to develop natural products capable of preventing or treating diseases, diseases or conditions caused by the Th2 immune response such as allergy. As a result, the present inventors have found that at least one extract selected from the group consisting of Seagrass extract, Hawthorn extract, Isoflavone extract, Alnus japonica extract and Azalea extract inhibits IL-4 production, inhibits passage of intestinal epithelial cells of allergens, It was confirmed that it had degranulation inhibitory activity.
본 발명의 조성물에서 이용되는 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물은 갈매나무, 괭이눈, 나한송, 조록나무 또는 진달래에 추출용매를 처리하여 수득하는 경우에는, 다양한 추출용매가 이용될 수 있다. 바람직하게는, 극성 용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물, (ii) 알코올(바람직하게는, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올, 1-펜탄올, 2-부톡시에탄올 또는 에틸렌글리콜), (iii) 아세트산, (iv) DMFO(dimethyl-formamide) 및 (v) DMSO(dimethyl sulfoxide)를 포함한다. 비극성 용매로서 적합한 것은, 아세톤, 아세토나이트릴, 에틸 아세테이트, 메틸 아세테이트, 플루오로알칸, 펜탄, 헥산, 2,2,4-트리메틸펜탄, 데칸, 사이클로헥산, 사이클로펜탄, 디이소부틸렌, 1-펜텐, 1-클로로부탄, 1-클로로펜탄, o-자일렌, 디이소프로필 에테르, 2-클로로프로판, 톨루엔, 1-클로로프로판, 클로로벤젠, 벤젠, 디에틸 에테르, 디에틸 설파이드, 클로로포름, 디클로로메탄, 1,2-디클로로에탄, 어닐린, 디에틸아민, 에테르, 사염화탄소 및 THF를 포함한다.When the extract of Solanum tuberosus, Hawthorne extract, Miscanthus sinensis extract, Rhododendron japonica and Azalea extract, which are used in the composition of the present invention, A solvent may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.
보다 바람직하게는, 본 발명에서 이용되는 추출용매는 (a) 물, (b) 탄소수 1-4의 무수 또는 함수 저급 알코올 (메탄올, 에탄올, 프로판올, 부탄올 등), (c) 상기 저급 알코올과 물과의 혼합용매, (d) 아세톤, (e) 에틸 아세테이트, (f) 클로로포름, (g) 부틸아세테이트, (h) 1,3-부틸렌글리콜, (i) 헥산 및 (j) 디에틸에테르를 포함한다. 가장 바람직하게는, 본 발명의 추출물은 물, 에탄올 또는 이의 조합을 갈매나무, 괭이눈, 나한송, 조록나무 및 진달래에 각각 처리하여 수득한 것이다.More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Most preferably, the extract of the present invention is obtained by treating water, ethanol, or a combination thereof with each of the following: Seagrass, Honeysuckle, Isofusa, Alaskan, and Azalea.
본 발명의 바람직한 구현예에 따르면, 본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물은 각각 에탄올 또는 물로 추출한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물이다.According to a preferred embodiment of the present invention, the extracts of Seagulls, Horns, Extracts, Rhododendron and Rhododendron extract of the present invention can be obtained by extracting the extracts of Seaweed, It is an azalea extract.
본 명세서에서 사용되는 용어 ‘추출물’은 상술한 바와 같이 당업계에서 조추출물(crude extract)로 통용되는 의미를 갖지만, 광의적으로는 추출물을 추가적으로 분획(fractionation)한 분획물도 포함한다. 즉, 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물은 상술한 추출용매를 이용하여 얻은 것뿐만 아니라, 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대, 상기 추출물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 추출물에 포함되는 것이다.As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, not only the extract of Seagrass, the extract of the hoe, the extract of Aspergillus, the extract of Aspergillus oryzae, and the Azalea extract are obtained not only by using the above-mentioned extraction solvent but also by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the extract of the present invention.
본 발명에서 이용되는 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.The ginkgo tree extract, horn extract, bamboo extract, bark extract and azalea extract used in the present invention can be prepared in powder form by an additional process such as vacuum distillation, freeze drying, spray drying and the like.
본 명세서에서 용어 ‘유효성분으로 포함하는’이란 하기의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물의 효능 또는 활성을 달성하는 데 충분한 양을 포함하는 것을 의미한다. 본 발명은 천연식물재료인 갈매나무, 괭이눈, 나한송, 조록나무 및 진달래로 구성된 군으로부터 선택되는 하나 이상의 천연식물재료로부터 추출한 조성물로서 과량 투여하여도 인체에 부작용이 없으므로 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물이 본 발명의 조성물에 포함된 양적 상한은 당업자가 적절한 범위 내에서 선택하여 실시할 수 있다.As used herein, the term "comprising as an active ingredient" means an amount sufficient to achieve the efficacy or activity of one or more of the extracts selected from the group consisting of the following extracts of Seagull, horn extract, bamboo extract, ≪ / RTI > The present invention is a composition extracted from at least one natural plant material selected from the group consisting of natural plant materials such as a ginkgo tree, a hoe's eye, a bamboo shoot, a Japanese apricot tree and a rhododendron. The quantitative upper limit in which the composition of the present invention is contained in one or more extracts selected from the group consisting of a herbal extract, a timber extract and an azalea extract can be selected by a person skilled in the art within a suitable range.
본 발명의 조성물은 IL-4의 생성을 억제하여 Th2 면역 반응을 감소시킨다.The composition of the present invention inhibits the production of IL-4 and reduces the Th2 immune response.
본 발명의 일 구현예에 따르면, 상기 갈매나무 추출물은 대조군과 비교하여 IL-4의 생성을 20-40% 감소시키고, 상기 괭이눈 추출물은 대조군과 비교하여 IL-4의 생성을 10-100% 감소시키며, 상기 나한송 추출물은 대조군과 비교하여 IL-4의 생성을 20-70% 감소시키고, 상기 조록나무 추출물은 대조군과 비교하여 IL-4의 생성을 10-95% 감소시키며, 상기 진달래 추출물은 대조군과 비교하여 IL-4의 생성을 50-99% 감소시킨다.According to one embodiment of the present invention, the Seagrass extract reduces the production of IL-4 by 20-40% as compared with the control, and the extract of the hornet has a 10-100% reduction in the production of IL-4 . The extracts of the herbal extracts reduced the production of IL-4 by 20-70% as compared with the control, and the extracts of the Alaska pollack decreased the production of IL-4 by 10-95% Compared with the control, the production of IL-4 is reduced by 50-99%.
본 발명의 조성물은 알레르겐의 장상피세포 통과를 억제한다.The composition of the present invention inhibits the passage of intestinal epithelial cells of allergens.
본 발명의 일 구현예에 따르면, 상기 갈매나무 추출물은 알레르겐의 장상피세포 통과를 60-80% 억제하고, 상기 괭이눈 추출물은 알레르겐의 장상피세포 통과를 55-75% 억제하며, 상기 나한송 추출물은 알레르겐의 장상피세포 통과를 75-95% 억제하고, 상기 조록나무 추출물은 알레르겐의 장상피세포 통과를 5-25% 억제하며, 상기 진달래 추출물은 알레르겐의 장상피세포 통과를 35-55% 억제한다.According to one embodiment of the present invention, the Seagrass extract inhibits the passage of allergen through the intestinal epithelial cells by 60-80%, the extract of the hoe extract inhibits the passage of intestinal epithelial cells through allergens by 55-75% Inhibits the passage of allergen through the intestinal epithelial cells by 75-95%, the extract of the Alaska pollack inhibits the passage of allergens through the intestinal epithelial cells by 5-25%, and the Azalea extract inhibits the passage of allergens through the intestinal epithelium by 35-55% do.
본 발명의 조성물은 비만세포(mast cell)의 탈과립을 억제한다.The composition of the present invention inhibits degranulation of mast cells.
본 발명의 일 구현예에 따르면, 상기 갈매나무 추출물은 비만세포의 탈과립을 50-80% 억제하고, 상기 괭이눈 추출물은 비만세포의 탈과립을 70-90% 억제하며, 상기 나한송 추출물은 비만세포의 탈과립을 50-80% 억제하고, 상기 조록나무 추출물은 비만세포의 탈과립을 60-80% 억제하며, 상기 진달래 추출물은 비만세포의 탈과립을 30-60% 억제한다.According to an embodiment of the present invention, the extract of Seagrass suppresses the degranulation of mast cells by 50-80%, the extract of the mussel suppresses degranulation of mast cells by 70-90% Degreasing is inhibited by 50-80%, and the extract of the Allium cepaensis inhibits degranulation of mast cells by 60-80%, and the Azalea extract inhibits degranulation of mast cells by 30-60%.
본 발명의 조성물은 다양한 Th2-매개 면역 질환, 질병 또는 상태의 예방 또는 치료에 이용될 수 있다.The compositions of the present invention may be used for the prevention or treatment of various Th2-mediated immune diseases, diseases or conditions.
본 명세서에서, 용어 “Th2-매개 면역 질환”은 알레르겐-특이 Th2 세포의 생성 및 활성에 의한 IgE 및 비만세포가 관여하는 질환을 의미한다.As used herein, the term " Th2-mediated immune disease " refers to diseases in which IgE and mast cells are involved by the production and activity of allergen-specific Th2 cells.
본 명세서에서 용어 “Th2 세포”는 유전자 발현, 단백질 분비 및 기능적 활성 측면에서 특정되는 헬퍼 T 세포 림포사이트의 서브세트를 의미한다. 예를 들어, Th2 세포는 IL-4, IL-5, IL-10, 및 IL-13 사이토카인 발현 패턴을 나타낸다. Th2 세포는 체액성 면역반응에 관여한다.As used herein, the term " Th2 cell " refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th2 cells exhibit IL-4, IL-5, IL-10, and IL-13 cytokine expression patterns. Th2 cells are involved in the humoral immune response.
본 발명의 조성물이 적용되는 Th2-매개 면역 질환은 특별하게 제한되지 않으며, 바람직하게는 알레르기 질환에 적용된다.The Th2-mediated immune diseases to which the composition of the present invention is applied are not particularly limited, and preferably apply to allergic diseases.
본 발명의 조성물이 적용되는 Th2-매개 면역 질환은 아토피 피부염, 아토피와 관련된 다른 피부학적 질환, 알레르기 비염(급성 또는 만성), 고초열 및 알레르기 기관지 천식을 포함하나, 이에 한정되는 것은 아니다.Th2-mediated immune diseases to which the composition of the present invention is applied include, but are not limited to, atopic dermatitis, other skin diseases associated with atopy, allergic rhinitis (acute or chronic), alopecia and allergic bronchial asthma.
본 발명의 조성물은 Th2 면역 반응을 감소시키는 효과가 있다.
The composition of the present invention has an effect of reducing the Th2 immune response.
본 발명의 조성물은 약제학적 조성물, 식품 조성물, 기능성 식품 조성물 또는 화장료 조성물이다.The composition of the present invention is a pharmaceutical composition, a food composition, a functional food composition or a cosmetic composition.
본 발명의 조성물은 약제학적 조성물로 제조될 수 있다.The compositions of the present invention may be prepared with pharmaceutical compositions.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 (a) 상술한 본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물이다. 본 명세서에서 용어 “약제학적 유효량”은 상술한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물의 효능 또는 활성을 달성하는 데 충분한 양을 의미한다.According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) at least one extract chemical selected from the group consisting of the above-mentioned Seaweed extract of the present invention, a hoe extract, a hansan extract, Effective amount; And (b) a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of one or more of the extracts selected from the group consisting of the above-mentioned Seaweed extracts, Hawthorn extracts, Hanson extracts, do.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 비경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably parenterally administered.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.0001-100 ㎎/kg 범위 내이다.The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.0001-100 mg / kg on an adult basis.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.
The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 조성물은 식품 조성물로 제공될 수 있다. 본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물을 유효성분 포함하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물뿐 만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다.
The composition of the present invention can be provided as a food composition. A composition for preventing, improving or treating a Th2-mediated immune disease comprising the active ingredient of at least one extract selected from the group consisting of the extracts of Seagulls, horns, extracts, The present invention includes not only one or more extracts selected from the group consisting of Seagull's tree extract, horn extract, bamboo extract, trefoil extract and azalea extract as an active ingredient, but also components that are ordinarily added in food production , For example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, it may contain one or more extracts selected from the group consisting of the extracts of Seagulls, horns, mushrooms, , Glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, and the like.
본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물을 유효성분으로 포함하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물은 기능성 식품 조성물로 제조될 수 있다. 본 발명의 조성물이 기능성 식품 조성물로 제조되는 경우, 식품 제조 시 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분으로서 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물 이외에 향미제 또는 천연 탄수화물을 추가 성분으로 포함시킬 수 있다. 예를 들어, 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등); 디사카라이드(예컨대, 말토스, 수크로오스 등); 올리고당; 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등); 및 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)을 포함한다. 향미제로서 천연 향미제(예컨대, 타우마린, 스테비아 추출물 등) 및 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.
The composition for preventing, improving or treating a Th2-mediated immune disease comprising the extract of the present invention as an active ingredient, at least one extract selected from the group consisting of the extracts of the scarabs, the hoe's eye extract, the bamboo shark extract, Food composition. When the composition of the present invention is prepared with a functional food composition, it includes components that are conventionally added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when it is made of a drink, as an active ingredient, a flavoring agent or a natural carbohydrate may be included as an additional ingredient in addition to one or more extracts selected from the group consisting of the extracts of Seagull, horn extract, Iris extract, have. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); oligosaccharide; Polysaccharides (e.g., dextrin, cyclodextrin and the like); And sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). Natural flavoring agents (e.g., tau marin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be used as flavorings.
본 발명의 조성물은 화장료 조성물로 제조될 수 있다. 본 발명의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물을 유효성분으로 포함하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물이 화장료 조성물로 제조되는 경우, 유효 성분으로서의 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물로 구성된 군으로부터 선택되는 하나 이상의 추출물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다.The composition of the present invention can be prepared with a cosmetic composition. A composition for preventing, improving or treating a Th2-mediated immune disease comprising at least one extract selected from the group consisting of Seagull's extract, hoe's eye extract, bamboo shark extract, Composition comprises components commonly used in cosmetic compositions in addition to one or more extracts selected from the group consisting of ginkgo tree extract, horn extract, bamboo extract, trefoil extract and azalea extract as active ingredients, Conventional adjuvants such as chelating agents, solubilizers, vitamins, pigments and flavoring agents, and carriers.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.
When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 천연물을 소재로 하는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물을 제공한다.(a) The present invention provides a composition for preventing, ameliorating or treating a Th2-mediated immune disorder comprising a natural substance.
(b) 본 발명의 조성물은 IL-4 생성 억제, 알레르겐의 장상피세포 통과 억제 및 비만세포의 탈과립 억제 활성을 제공한다.(b) The composition of the present invention provides inhibition of IL-4 production, inhibition of passage of intestinal epithelial cells of allergens and inhibition of degranulation of mast cells.
(c) 본 발명은 천연물을 소재로 이용하여 저렴한 생산비용과 사용의 편이성을 제공할 수 있다.
(c) The present invention can provide an inexpensive production cost and ease of use by using a natural material as a material.
도 1은 알레르겐의 장상피세포 통과를 조사한 실험에 대한 모식도를 나타낸다.
도 2는 마우스 비만세포(mast cell)의 탈과립에 대한 갈매나무(Rhamnus davurica Pall .) 추출물의 영향을 확인한 결과를 보여준다.
도 3은 마우스 비만세포의 탈과립에 대한 괭이눈(Chrysosplenium grayanum) 추출물의 영향을 확인한 결과를 보여준다.
도 4는 마우스 비만세포의 탈과립에 대한 나한송(Podocarpus macrophyllus) 추출물의 영향을 확인한 결과를 보여준다.
도 5는 마우스 비만세포의 탈과립에 대한 조록나무(Distylium racemosom) 추출물의 영향을 확인한 결과를 보여준다.
도 6은 마우스 비만세포의 탈과립에 대한 진달래(Rhododendron mucronulatum) 추출물의 영향을 확인한 결과를 보여준다.Figure 1 shows a schematic diagram of an experiment in which intestinal epithelial cell passage of allergens is examined.
FIG. 2 is a graph showing the effect of Rhamnus ( Rhamnus ) on the degranulation of mouse mast cells davurica Pall . ) Extracts.
FIG. 3 is a graph showing the number of horseshoe chrysosporium grayanum ) extracts.
FIG. 4 is a graph showing the effect of Podocarpus macrophyllus ) extracts.
FIG. 5 is a graph showing the effect of the Distylium racemosom ) extracts.
FIG. 6 shows the results of confirming the effect of Rhododendron mucronulatum extract on degranulation of mouse mast cells.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예 1: 시료의 확보 및 용매 추출물의 준비Example 1: Preparation of sample and preparation of solvent extract
갈매나무(Rhamnus davurica Pall., 잎), 괭이눈(Chrysosplenium grayanum, 전초), 나한송(Podocarpus macrophyllus, 잎 및 꽃), 조록나무(Distylium racemosom, 잎, 줄기 및 열매) 추출물 및 진달래(Rhododendron mucronulatum, 줄기 및 꽃) 추출물을 한국식물추출물은행에서 구입하여 시료로 사용하였으며, 각 시료는 95% 에탄올 또는 물로 추출한 추출물이다.
Buckthorn (Rhamnus davurica Pall., Leaves), gwaengyinun (Chrysosplenium grayanum, Outpost), or Hansong (Podocarpus macrophyllus, leaves and flowers), jorok trees (Distylium racemosom, leaves, stems and fruit) extract and rhododendrons (Rhododendron mucronulatum , stem and flower) were purchased from the Korean plant extract bank and used as samples. Each sample was extracted with 95% ethanol or water.
실시예 2: 추출물에 의한 Th1/Th2 사이토카인 균형조절 활성시험(Example 2: Th1 / Th2 cytokine balance control activity test by extract ( ex vivoex vivo ))
OVA로 면역한 마우스 비장세포의 배양Culture of mouse splenocytes immunized with OVA
5주령된 자성 BALB/c 마우스에 1주 간격으로 2차례 알레르겐(allergen)을 복강면역하여 알레르기를 유발하였다. 알레르겐은 OVA(20 ㎍) 및 알럼(alum, 2 ㎎)의 혼합액으로, 이를 30분 동안 혼합한 다음 한 마리당 100 ㎕씩 복강 주사하였다(n=5). 면역 1주후 마우스를 경추탈골 시키고 무균상태에서 비장을 적출하여 1 ㎖의 기본배지(2-머캅토에탄올 및 항생제를 첨가한 RPMI1640, WelGene, 대구, 대한민국)를 첨가한 작은 페트리 접시에 옮겨 아이스박스 상에 보관하였다. 페트리 접시에 담긴 비장은 메쉬를 이용하여 단세포화 시킨 뒤 5 ㎖의 기본배지로 2회 세척하고 1500 rpm에서 5분 동안 원심분리를 실시하였다. 상층액 제거 후 1 ㎖의 RBC(Red Blood Cell) 용해 완충액(SIGMA R7757, 미국)을 첨가하여 손으로 잘 흔들어 현탁하고, 3분 동안 아이스 위에서 반응시킨 뒤 10 ㎖의 기본배지 첨가 후 현탁한 뒤 1500 rpm에서 5분 동안 원심분리를 2회 실시 하였다. 96-웰 플레이트에 항원(OVA, 100 ㎍/㎖)을 처리하고 각각의 농도로 준비된 추출물을 첨가하였으며, 비장세포의 농도는 5×106 세포/웰로 처리하였다. 다음으로 37℃, CO2 항온반응기에서 72시간 동안 배양한 후 상층액을 회수하였다.
Allergen was induced by immunization of allergen twice weekly at 5 weeks of age in magnetic BALB / c mice. The allergen was mixed with OVA (20 μg) and alum (2 mg), and the mixture was mixed for 30 minutes and then 100 μl per mouse was intraperitoneally injected (n = 5). After 1 week of immunization, the mice were disassociated from the cervical vertebrae, and the spleen was removed from the aseptic state and transferred to a small Petri dish supplemented with 1 ml of a basic medium (RPMI1640 with 2-mercaptoethanol and antibiotics, WelGene, Daegu, Korea) Lt; / RTI > The spleen in the Petri dish was single-celled using a mesh, washed twice with 5 ml of the basic medium, and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, 1 ml of Red Blood Cell (RBC) Lysis Buffer (SIGMA R7757, USA) was added, and the mixture was shaken by hand and allowed to react on ice for 3 minutes. After 10 ml of the basic medium was added, centrifugation was performed twice at rpm for 5 minutes. Process the antigen (OVA, 100 ㎍ / ㎖) in a 96-well plate, and was added to the prepared extract at various concentrations, the concentration of the spleen cells were treated with 5 × 10 6 cells / well. After incubation at 37 ° C in a CO 2 incubator for 72 h, the supernatant was recovered.
비장세포 배양상층액의 사이토카인 분석 Cytokine analysis of spleen cell culture supernatant
항원(OVA, 100 ㎍/㎖), 및 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 또는 진달래 추출물이 처리된 비장세포 배양 상층액을 72시간 후 회수하여 IL-4 분석에 사용하였다. 사이토카인 분석을 위하여 BD OptEIATM MOUSE ELISA(Enzyme-linked Immunosorbent Assay) 키트를 사용하였으며, 실험방법은 키트에 명시된 실험방법에 준하여 실험을 실시하였다. 간단히 설명하면 96웰 플레이트에 포획항체를 4℃에서 하룻밤 정치하였다. 코팅된 96웰 플레이트를 세척 완충액(PBST)으로 세척한 후 분석용 희석제(10% FBS가 첨가된 PBS)를 200 ㎕씩 각 웰에 첨가하고 상온에서 1시간 동안 블로킹(blocking)하였다. 다음으로 표준 시료와 샘플 시료를 100 ㎕씩 각 웰에 분주하여 상온에서 2시간 반응시킨 다음, 검출항체와 스트렙타비딘(streptoavidin)-HRP를 100 ㎕씩 분주한 후 상온에서 1시간 동안 반응시켰다. 반응시간 후 100 ㎕의 기질용액(0.01% TMB가 첨가된 인산염-구연산염 완충액, pH 5.0, 0.001% H2O2)으로 상온에서 30분 동안 발색시킨 다음, 2 M H2SO4를 50 ㎕씩 각 웰에 첨가하여 발색반응을 중지시켰다. 발색 정도는 450 ㎚에서 마이크로플레이트리더(THERMOmax, Molecular Devices, 미국)로 흡광도를 측정하였다.The supernatant of spleen cells treated with antigens (OVA, 100 ㎍ / ㎖) and the extracts of Seagrass, hoe's ointment, Nanso sp., Oryza sativa or Azalea were collected after 72 hours and used for IL-4 analysis. For the cytokine analysis, BD OptEIATM MOUSE ELISA (Enzyme-linked Immunosorbent Assay) kit was used. Experiments were performed according to the experimental methods specified in the kit. Briefly, the captured antibody was allowed to stand at 4 ° C overnight in a 96-well plate. The coated 96 well plate was washed with wash buffer (PBST), and 200 [mu] l of assay diluent (PBS supplemented with 10% FBS) was added to each well and blocked at room temperature for 1 hour. Next, 100 μl of the standard sample and the sample were dispensed into each well, reacted at room temperature for 2 hours, 100 μl of the detection antibody and streptoavidin-HRP were dispensed, and reacted at room temperature for 1 hour. After the reaction time, 100 μl of substrate solution (phosphate-citrate buffer with 0.01% TMB, pH 5.0, 0.001% H 2 O 2 ) was developed at room temperature for 30 minutes and then 2 μl of 2M H 2 SO 4 Well to stop the color reaction. The degree of color development was measured with a microplate reader (THERMOmax, Molecular Devices, USA) at 450 nm.
비장세포의 IL-4 생산에 대한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물의 영향을 조사한 결과, 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 또는 진달래 추출물을 첨가한 경우에 대조군과 비교하여 비장세포의 IL-4 생산을 각각 20-40%(30 ㎕/㎖ 갈매나무 추출물), 10-100%(10-100 ㎕/㎖ 괭이눈 추출물), 20-70%(50-100 ㎕/㎖ 나한송 추출물), 10-95%(50-100 ㎕/㎖ 조록나무 추출물) 및 50-99%(75-100 ㎕/㎖ 진달래 추출물) 감소시켰다(표 1 내지 5). 이러한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물의 IL-4 생산 감소 효과는 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물이 Th2 면역반응을 감소시킨다는 것을 보여준다.In order to investigate IL-4 production of splenocytes, we examined the effects of Seagrass, Horn, Extract, and Azalea extracts on the production of IL-4. IL-4 production of splenocytes was 20-40% (30 μl / ml of Seaweed extract), 10-100% (10-100 μl / ml of a pod extract), 20-70% (50-100 [mu] l / ml Angelicae extract), 10-95% (50-100 [mu] / ml conidia extract) and 50-99% (75-100 [ . The reduction of IL-4 production by the gill tree extract, hoe extract, hansoon extract, acacia var. Umbraculifera and azalea extracts was found to be due to the decrease of Th2 immune response by the extracts of Seagrass, hoe extract, .
실시예 3: Caco-2 단세포층을 이용한 OVA 통과억제 활성시험Example 3: Test for inhibiting the passage of OVA using Caco-2 single cell layer
CacoCaco -- 2세포배양2 cell culture 및 And OVAOVA 통과억제 실험 Passage inhibition experiment
ATCC 유래의 Caco-2세포주(HTB-37)는 DMEM(Dulbeco's Modified Eagle's Medium)에 10% FBS(Fetal Bovine Serum), 1% 100 U/㎖ 페니실린, 100 ㎍/㎖ 스트렙토마이신 및 0.1 mM 비필수 아미노산을 첨가하여 37℃, 5% CO2 조건하에서 계대배양 하였다. Caco-2 단세포층을 이용한 식품 알레르겐 통과시험을 하기 위하여 상기 배양배지를 사용하여 Caco-2 세포를 12 웰 플레이트 트랜스웰 (transwell, 도 1)의 상층부(apical side)인 트랜스멤브레인(transmembrane) 위에 2×105 세포/㎖의 밀도로 0.5 ㎖ 분주하였다. 웰의 하층부(basolateral side)에는 배양배지 1.5 ㎖을 첨가하고 37℃, 5% CO2 항온반응기에서 배양하였다. 배지교환은 매 2-3일에 한 번씩 실시하였다. 분주 후 약 2-3주가 지나 단세포층을 형성한 Caco-2 세포의 하층부와 상층부간의 전기저항치(TEER, transepitherial electrical resistance, Ω×㎠)를 Millipore사의 Millicell-ERS (Electrical Resistance System)로 측정하여 수치가 300 Ω×㎠ 초과인 경우 실험에 사용하였다. Caco-2 단세포층은 HBSS(Hank's Balanced Salt Solution)로 3회 세척 한 후 갈매나무 추출물(최종농도: 400 ㎍/㎖), 괭이눈 추출물(최종농도: 400 ㎍/㎖), 나한송 추출물(최종농도: 400 ㎍/㎖), 조록나무 추출물(최종농도: 400 ㎍/㎖) 또는 진달래 추출물(최종농도: 400 ㎍/㎖)을 담즙산염(bile salts, 최종농도: 100-150 ㎍/㎖)과 함께 상층부에 첨가한 후 37℃, 5% CO2 항온반응기에서 1시간 동안 처리하였다. 다음으로 OVA(Ovalbumin, 최종농도: 400 ㎍/㎖)를 상층부에 첨가 한 후 37℃, 5% CO2 항온반응기에서 3시간 동안 반응시켰다. 여기에서 하층부의 액을 회수하여 통과된 OVA의 농도를 sELISA(sandwich Enzyme-Linked Immunosorbent Assay)로 측정하였다. 하층부 내의 통과된 OVA량은 다음과 같이 구하였다:The ATCC-derived Caco-2 cell line (HTB-37) was cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine Serum), 1% 100 U / ml penicillin, 100 ug / ml streptomycin, And the cells were subcultured under the condition of 37 ° C and 5% CO 2 . Caco-2 cells were cultured on a transmembrane, which is the apical side of a 12-well plate transwell (Fig. 1), using the culture medium described above for the food allergen passage test using the Caco-2 monocytic layer And then 0.5 ml of the solution was dispensed at a density of 10 5 cells / ml. 1.5 ml of the culture medium was added to the basolateral side of the well and cultured in a 5% CO 2 incubator at 37 ° C. The medium was changed every 2-3 days. The electrical resistance (TEER, transepitiorial electrical resistance, Ω × ㎠) between the lower and upper layers of Caco-2 cells forming the unicellular layer was measured by Millipore's Millicell-ERS (Electrical Resistance System) Was used in the experiment when it exceeded 300 Ω × ㎠. The Caco-2 monocyte layer was washed three times with HBSS (Hank's Balanced Salt Solution), and then extracted with Seagrass (final concentration: 400 ㎍ / ㎖), hoe extract (final concentration: 400 ㎍ / ㎖) (Final concentration: 400 ㎍ / ㎖) or Azalea extract (final concentration: 400 / / ㎖) with bile salts (final concentration: 100-150 / / ㎖) And then treated in a 5% CO 2 incubator at 37 ° C for 1 hour. Next, OVA (Ovalbumin, final concentration: 400 μg / ml) was added to the upper layer and reacted for 3 hours in a 5% CO 2 incubator at 37 ° C. Here, the concentration of the OVA passed through the lower layer was measured by sandwich enzyme-linked immunosorbent assay (sELISA). The amount of OVA passed through the lower layer was determined as follows:
Flux = C ng/ H hr/ S cm2 Flux = C ng / H hr / S cm 2
여기에서 C는 ELISA에 의하여 구한 OVA의 통과량이고, H는 Caco-2 단세포층 상층부에 OVA 첨가 후 반응시간(3h)을 나타내며, S는 트랜스웰 플레이트의 상층부 표면적(1.12 cm2)을 나타낸다.
Here, C represents the passage amount of OVA obtained by ELISA, H represents the reaction time (3 h) after the OVA addition at the upper part of the Caco-2 monolayer, and S represents the upper surface area of the transwell plate (1.12 cm 2 ).
ELISAELISA 에 의한 On by OVAOVA 분석 analysis
코팅 완충액((tris hydroxymethyl) aminomethane 0.05 M, pH 9.0)에 2 ㎍/㎖의 농도로 희석한 항-래빗-OVA 항체를 96웰 플레이트에 각 웰 당 100 ㎕씩 분주하여 4℃에서 하룻밤 정치하였다. 코팅된 96웰 플레이트를 세척 완충액버퍼[Tween 20을 포함하는 PBS(Phosphate Buffered Saline); PBST, 0.138 M NaCl, 0.0027 M KCl 및 0.05% Tween 20을 포함하는 0.01 M 인산염 완충액)로 3회 세척하였다. HBSS에 일정한 배수로 희석한 표준 OVA와 회수액을 각 웰 당 100 ㎕씩 첨가한 후 1시간 동안 상온에서 반응시켰다. 다시 세척 완충액으로 앞에서와 같이 세척한 다음 항-OVA 항체가 결합된 바이오틴(biotin)을 PBST로 일정농도 희석하고 웰 당 100 ㎕씩 처리하여 1시간 동안 상온에서 반응시켰다. 다음으로 상기와 같이 세척 완충액으로 세척한 후 아비딘(avidin)-HRP를 PBST로 일정농도 희석하여 웰 당 100 ㎕씩 처리하여 1시간 동안 상온에서 반응시켰다. 이후, 세척 완충액으로 앞서 언급한 바와 같이 세척하였다. 다음으로 기질용액으로써 페닐 프로피온산(phenyl propionic acid)/인산염 버퍼(36 mM, pH 7.0)에 H2O₂ 0.002%를 첨가한 것을 사용하고 반응정지액으로 1 M NaOH-글라이신(glycine)(1 M 글라이신/1 M NaOH)을 각 웰 당 100 ㎕씩 사용하고 형광 마이크로플레이트 리더(microplate reader)로 320/405 ㎚(Ex./Em.파장)에서의 형광을 측정하였다.Rabbit-OVA antibody diluted to a concentration of 2 μg / ml in a coating buffer (0.05 M tris hydroxymethyl aminomethane, pH 9.0) was dispensed into a 96-well plate at a rate of 100 μl per well and allowed to stand overnight at 4 ° C. Coated 96-well plates were washed with wash buffer (PBS (Phosphate Buffered Saline) containing Tween 20; PBST, 0.138 M NaCl, 0.0027 M KCl, and 0.05% Tween 20). 100 ㎕ of standard OVA diluted with HBSS and diluted with PBS was added to each well, and reacted at room temperature for 1 hour. After washing with the washing buffer as described above, biotin conjugated with anti-OVA antibody was diluted with PBST at a constant concentration and treated with 100 μl per well, followed by reaction at room temperature for 1 hour. After washing with washing buffer as described above, avidin-HRP was diluted with PBST at a constant concentration and treated with 100 μl per well, followed by reaction at room temperature for 1 hour. Thereafter, it was washed with the washing buffer as mentioned above. Next, 0.002% of H 2 O 2 was added to the phenylpropionic acid / phosphate buffer (36 mM, pH 7.0) as the substrate solution, and 1 M NaOH-glycine (1 M glycine / 1 M NaOH) was used at a rate of 320/405 nm (Ex./Em. Wavelength) using a fluorescent microplate reader.
알레르겐의 장상피세포 통과에 대한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물의 영향에 대하여 조사한 결과, OVA 단독으로 처리한 양성대조군과 비교하여, 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 또는 진달래 추출물을 처리한 경우 OVA의 통과량이 감소되는 것을 확인하였다. 이를 백분율로 환산한 결과, 알레르겐의 통과율을 각각 60-80%, 55-75%, 75-95%, 5-25% 및 35-55% 감소시키는 효과를 보여주었다(표 6 내지 10).As a result of investigating the effects of Seagrass, Horn, Extract, Allium japonica and Azalea extracts on the passage of intestinal epithelial cells of allergens, compared with the positive control treated with OVA alone, the extracts of Seagrass, It was confirmed that the amount of OVA was decreased when Hanson extract, Alnus japonica extract or Azalea extract were treated. As a result, the percentage of allergens was reduced by 60-80%, 55-75%, 75-95%, 5-25%, and 35-55%, respectively (Tables 6-10).
실시예Example 4: 4: 탈과립Degranulation 억제활성 시험 Inhibitory activity test
복강 비만세포의 수확Harvesting abdominal mast cells
마우스를 에테르로 마취시키고 머리의 뒤통수를 강타하여 치사시킨 후 약 10 ㎖의 배지(pH 7.4)를 마우스의 복강 내에 주입하고 90초 동안 복벽을 가볍게 마사지하였다. 복벽 중앙선을 절개하고 복강 세척액을 스포이드로 채취하여 100×g에서 10분 동안 원심 시킨 후 상층액을 버리고, 동일 배지로 비만세포(mast cells) 수가 1×106 세포/㎖ 되도록 복강 부유액을 만들어 실험에 이용하였다.
The mice were anesthetized with ether and the head backs were struck to death. Approximately 10 ml of medium (pH 7.4) was injected into the abdominal cavity of the mice, and the abdominal wall was lightly massaged for 90 seconds. The abdominal wall was cut and the abdominal cavity washing solution was collected with a syringe and centrifuged at 100 xg for 10 minutes. The supernatant was discarded and an abdominal suspension was made so that the number of mast cells was 1 x 10 6 cells / Lt; / RTI >
비만세포의 형태변화 관찰Observation of morphological changes of mast cells
재부유시킨 비만세포 부유액 200 ㎕에 살린(saline), 갈매나무 추출물(1 ㎎/㎖, 0.1 ㎎/㎖ 또는 0.01 ㎎/㎖), 괭이눈 추출물(0.1 ㎎/㎖ 또는 0.01 ㎎/㎖), 나한송 추출물(0.1 ㎎/㎖ 또는 0.01 ㎎/㎖), 조록나무 추출물((0.1 ㎎/㎖ 또는 0.01 ㎎/㎖) 또는 진달래 추출물(1 ㎎/㎖, 0.1 ㎎/㎖ 또는 0.01 ㎎/㎖) 25 ㎕를 각각 넣어 37℃ 배양기에서 10분 동안 반응시켰다. 반응 후 컴파운드 48/80(Sigma Chemical Co., St. Louis, Mo, 미국) 용액 25 ㎕를 첨가하여 20분 동안 반응시켰다.(1 ㎎ / ㎖, 0.1 ㎎ / ㎖ or 0.01 ㎎ / ㎖), horn extract (0.1 ㎎ / ㎖ or 0.01 ㎎ / ㎖) and saline, saline or saline in 200 ㎕ of resuspended mast cell suspension. (0.1 mg / ml or 0.01 mg / ml) of the extract (0.1 mg / ml or 0.01 mg / ml) (Sigma Chemical Co., St. Louis, Mo., USA) solution was added and reacted for 20 minutes.
광학현미경 상으로 비만세포의 형태를 관찰하기 위하여 반응이 끝난 비만세포 부유액 200 ㎕를 도립현미경 재물대 위에 놓여진 슬라이드글라스(slide glass, 22×60 ㎜) 위에 떨어뜨려 비만세포들이 침전될 수 있도록 실온에서 10분 동안 정치시켰다. 400배의 배율 하에서 헤마사이토메타(hemacytometer)를 이용하여 비만세포를 도립현미경(Olympus, Japan)으로 관찰하였다. 정상 비만세포의 형태는 대부분 원형 또는 난원형으로 세포윤곽이 뚜렷하고, 세포질 내에는 많은 과립들이 충만 되어 있었다. 비만세포의 직경은 대략 10-20 ㎛ 정도로 복강 부유액의 다른 세포들(림프구 또는 호중성백혈구)에 비해 2배 이상 컸다. 비만세포의 형태가 원형 또는 난원형으로 세포윤곽이 뚜렷하고, 세포질 내에 광굴절율이 높은 과립들로 충만된 상태를 정상형 비만세포로 구분하였다. 반면에 세포윤곽이 불분명하고 세포질 내 과립들이 세포표면으로 돌출 되거나 세포주위에 흩어져 있는 경우를 탈과립형으로 구분하였다. 탈과립율은 다음 공식에 의하여 산출되었다:To observe the morphology of the mast cells on an optical microscope, 200 μl of the suspended mast cell suspension was placed on a slide glass (22 × 60 mm) placed on an inverted microscope platform and incubated at room temperature Min. Mice were observed under an inverted microscope (Olympus, Japan) using a hemacytometer under a magnification of 400x. Normal mast cells were mostly round or ovoid, and cell contours were prominent and many granules were filled in cytoplasm. The diameter of mast cells was about 10-20 ㎛, which was more than twice as large as that of other cells of the abdominal suspension (lymphocytes or neutrophilic leukocytes). The mast cells were classified into normal mast cells in the form of round or ovoid cell with clear cell contours and filled with granules with high photorefractive index in cytoplasm. On the other hand, the cell contours were unclear, and the intracellular granules protruding from the cell surface or scattered around the cells were classified as degranulation type. The degranulation rate was calculated by the following formula:
비만세포 탈과립율(%)=(탈과립된 비만세포 수/비만세포 총수)×100 Mast cell degranulation ratio (%) = (degranulated mast cell number / total mast cell number) × 100
컴파운드 48/80에 의한 비만세포 탈과립율에 대한 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물의 영향을 조사한 결과, 컴파운드 48/80을 단독 처리한 대조군과 비교하여, 갈매나무 추출물, 괭이눈 추출물, 나한송 추출물, 조록나무 추출물 및 진달래 추출물 처리 후 컴파운드 48/80를 처리한 실험군에서 각각 50-80%, 70-90%, 50-80%, 60-80% 및 30-60%의 탈과립 억제 효과를 확인하였다(도 2 내지 6).
Compared with the control group treated with compound 48/80 alone, the effects of compound 48/80 on the manganese deaggregation rate of the mangrove, hoe, hanson, 50-80%, 50-80%, 60-80%, and 30-60% in the experimental group treated with compound 48/80 after treatment with the extracts, hoe's oak extract, % Of degranulation inhibitory effect (Figs. 2 to 6).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
Claims (7)
A pharmaceutical composition for preventing, ameliorating or treating an allergic disease comprising Chrysosplenium grayanum extract as an active ingredient.
2. The composition of claim 1, wherein the extract of the pod is inhibiting the production of IL-4.
2. The composition of claim 1, wherein the extract of the hoe extract inhibits passage of intestinal epithelial cells of the allergen.
The composition according to claim 1, wherein the extract of the pony extract inhibits degranulation of a mast cell.
A food composition for preventing or ameliorating an allergic disease comprising an extract of Chrysosplenium grayanum as an active ingredient.
A functional food composition for preventing or ameliorating an allergic disease comprising an extract of Chrysosplenium grayanum as an active ingredient.
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