KR101974145B1 - Composition for preventing, ameliorating or treating atopic dermatitis comprising Sagittaria aginashi extract as effective component - Google Patents
Composition for preventing, ameliorating or treating atopic dermatitis comprising Sagittaria aginashi extract as effective component Download PDFInfo
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- KR101974145B1 KR101974145B1 KR1020170178098A KR20170178098A KR101974145B1 KR 101974145 B1 KR101974145 B1 KR 101974145B1 KR 1020170178098 A KR1020170178098 A KR 1020170178098A KR 20170178098 A KR20170178098 A KR 20170178098A KR 101974145 B1 KR101974145 B1 KR 101974145B1
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- atopic dermatitis
- tslp
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Classifications
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/884—Alismataceae (Water-plantain family)
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- 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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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
Description
본 발명은 보풀 추출물을 유효성분으로 함유하는 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis, which contains a fluff extract as an active ingredient.
아토피 피부염은 주로 영아와 소아에서 발생하는 가장 흔한 피부 질환 중 하나로, 재발률이 높은 만성 피부염이며, 최근 전 세계적으로 증가하고 있는 대표적인 알레르기 피부과 질환으로 소아의 12%에서 15%까지 유병률을 보인다. 2015년 인구 10만 명당 질환별 연령대별 환자 수를 보면, 아토피 피부염은 12세 이하에서 7,685명으로 가장 진료를 많이 받았으며, 13~19세 2,868명, 20대 1,619명 순으로 나타났다. 아토피 피부염은 대개 영유아기부터 시작되는데, 약 90%가 5세 이내에 발병하며, 약 50%가 생후 1세 이내에 발병한다. 아토피 피부염 환자의 약 80% 정도는 성인까지 증상이 지속되거나, 피부병변이 손이나 발의 습진 같은 형태로 변형되어 나타난다. 연령대별 '아토피 피부염 진료현황'을 살펴보면 영유아기 뿐만 아니라 20대의 경우 매년 약 10만 명이 아토피 피부염으로 진료를 받고 있으며, 60대 환자들도 매년 3만 명 가까이 진료를 받는 것으로 나타나 성인 환자 수도 매년 유지되는 것을 확인하였다. 아토피 피부염은 유전적 요인, 면역학적 이상, 피부 장벽의 이상, 신경면역학적 이상, 환경적 요인 등 다양한 요인이 복합적으로 작용하여 발병하기 때문에 치료가 어렵다. Atopic dermatitis is one of the most common skin diseases that occur in infants and children. It is a chronic dermatitis with a high recurrence rate. It is a typical allergic skin disease that is increasing worldwide in recent years and it has a prevalence ranging from 12% to 15%. According to the number of patients by age group, the number of patients with atopic dermatitis was 7,685 at the age of 12 and under, and 2,868 at 13 ~ 19, and 1,619 at 20, respectively. Atopic dermatitis usually begins in infancy, with about 90% of cases occurring within 5 years of age and about 50% occurring within 1 year of age. Approximately 80% of patients with atopic dermatitis continue to have symptoms until adulthood, or skin lesions appear to deform to the form of eczema of the hands or feet. According to age, 'atopic dermatitis treatment' shows that not only infants, but also about 100,000 people in their 20s are treated with atopic dermatitis every year, and 60s are receiving about 30,000 people every year. . Atopic dermatitis is difficult to treat because of various factors such as genetic factors, immunological abnormalities, abnormal skin barrier, neuroimmunological abnormalities, and environmental factors.
피부 장벽이 손상되면 외부 항원 및 균의 침투를 막기 위하여 피부에서는 각질형성세포와 항원전달세포들을 통하여 즉각적인 선천면역반응이 일어나고, 후천면역반응도 이어지는데, 아토피 피부염 환자들에서는 이들 선천면역계와 후천면역계가 비정상적으로 반응하여서 염증반응이 일어나 피부염이 발생하는 것으로 알려져 있다. 그러나 아토피 피부염은 정확한 원인을 알 수 없어 특별한 치료제가 없고, 특히 대다수의 환자들이 가려움증으로 인해 병변부위가 쉽게 호전되지 않는다는 것이 가장 큰 문제이다. In order to prevent penetration of external antigens and bacteria in the skin barrier, skin barrier keratinocytes and antigen-presenting cells immediately undergo a congenital immune response and acquired immune response. In patients with atopic dermatitis, these innate and acquired immune systems are abnormal And it is known that inflammation reaction occurs to cause dermatitis. However, atopic dermatitis is the most serious problem because the exact cause is unknown and there is no special treatment. Especially, the majority of patients do not easily recover from lesions due to itching.
일반적으로, 알러지성 질환의 치료를 위하여 항히스타민, 스테로이드성 또는 비스테로이드성 항염증용 의약품 및 류코트리엔 길항제와 같은 의약품이 사용된다. 그러나 이러한 의약품들은 과도한 체액성 면역을 완화하거나 IgE 생산을 억제하는 것으로, 알러지성 질환의 근본적인 치유는 어렵다.Generally, medicines such as antihistamines, steroidal or nonsteroidal antiinflammatory drugs and leukotriene antagonists are used for the treatment of allergic diseases. However, these medicines relieve excessive humoral immunity or inhibit IgE production, and it is difficult to cure allergic diseases.
최근 다양한 연구결과들에서 유전 및 여러 환경 요인에 의해 피부 장벽이 손상되어 각종 알레르겐에 노출되면, 각질형성세포에서 IL-7과 유사한 사이토카인인 TSLP(Thymic Stromal Lymphopoietin)를 분비하여 면역반응을 일으킨다고 보고되고 있다. 특히, TSLP의 분비는 피부 내 뉴런세포의 TSLP 수용체에 결합하여 뇌하수체에 병변부위를 반복적으로 긁게 하는 신호를 주어 아토피 피부염을 더욱 악화시키는 것으로 보고되었고, TSLP를 차단함으로써 아토피 피부염을 현저히 감소시키는 결과가 보고됨으로써, 아토피 피부염의 시작과 병변증가에 TSLP가 마스터 스위치 역할을 하고 있음을 보여주었다.Recently, various studies have shown that when skin barrier is damaged by various genetic and environmental factors and exposed to various allergens, TSLP (Thymic Stromal Lymphopoietin), which is a cytokine similar to IL-7 in keratinocytes, Are reported. In particular, the secretion of TSLP has been reported to bind to the TSLP receptor of neuronal cells in the skin, causing the pituitary gland to repeatedly scratch the lesion area, thereby exacerbating atopic dermatitis and significantly reducing atopic dermatitis by blocking TSLP Reported that TSLP acts as a master switch in the onset of atopic dermatitis and increased lesions.
따라서 본 발명은 안정성이 입증된 천연물을 이용하여 TSLP 분비를 저해하여 아토피 피부염의 개시 및 병변증가를 개선하고자 하였다. Therefore, the present invention aims to improve initiation and lesion increase of atopic dermatitis by inhibiting TSLP secretion by using natural materials proved to be stable.
한편, 한국등록특허 제1114506호에는 빈랑자 및 정향수피의 열수 추출물을 유효성분으로 함유하는 TSLP 저해능에 의한 아토피 피부염 개선능을 가지는 한방 조성물이 개시되어 있으나, 본 발명의 보풀 추출물을 유효성분으로 함유하는 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관해 개시된 바 없다. Korean Patent No. 1114506 discloses a herbal composition having an ability to improve atopic dermatitis by inhibiting TSLP, which contains hot water extract of bingata and clove extract as an active ingredient. However, the herbal extract of the present invention contains as an active ingredient Or a composition for preventing, ameliorating or treating atopic dermatitis.
본 발명은 상기와 같은 요구에 의해 도출된 것으로, 보풀 추출물; 또는 보풀 추출물 및 쿠세놀 F의 혼합물을 유효성분으로 함유하는 아토피 피부염의 예방, 개선 또는 치료용 조성물을 제공하고, 상기 조성물이 TSLP의 발현을 억제하고, 아토피 피부염이 유발된 마우스에서 아토피 피부염을 개선하는 효과가 있다는 것을 확인함으로써, 본 발명을 완성하였다.SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, Or a mixture of fluff extract and xenol F as an active ingredient, which composition is capable of inhibiting the expression of TSLP and improving atopic dermatitis in mice induced with atopic dermatitis The present invention has been completed.
상기 과제를 해결하기 위해, 본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 치료용 약학 조성물을 제공한다. In order to solve the above-described problems, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising an extract of Lilac as an active ingredient.
또한, 본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 개선용 화장료 조성물을 제공한다. The present invention also provides a cosmetic composition for preventing or ameliorating atopic dermatitis comprising an extract of Lilac as an active ingredient.
또한, 본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for preventing or ameliorating atopic dermatitis comprising an extract of Lilac as an active ingredient.
본 발명은 보풀 추출물을 유효성분으로 함유하는 아토피 피부염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 보풀 추출물이 TSLP의 발현을 저해하고, 아토피 피부염이 유발된 동물모델에서 아토피 피부염을 개선하는 효과가 우수하며, 보풀 추출물 및 쿠세놀 F의 혼합물이 보풀 또는 쿠세놀 F에 비해 TSLP의 발현을 억제하는 효과가 더욱 증진되었으므로, 보풀 추출물; 또는 보풀 추출물 및 쿠세놀 F의 혼합물을 유효성분으로 함유하는 본 발명의 조성물은 아토피 피부염의 치료제, 아토피 피부염의 예방 또는 개선용 화장품; 또는 아토피 피부염의 예방 또는 개선용 건강기능식품의 소재로 사용할 수 있다.The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis containing an extract of Liliaceae as an active ingredient, wherein the Lilac extract inhibits the expression of TSLP and has the effect of improving atopic dermatitis in an animal model in which atopic dermatitis is induced And since the effect of the mixture of fluff extract and Cucinol F on the expression of TSLP in comparison with fluff or Cucinol F is further enhanced, the fluff extract; Or a mixture of fluff extract and xenol F as an active ingredient is used as a therapeutic agent for atopic dermatitis, a cosmetic for preventing or ameliorating atopic dermatitis; Or as a material for health functional foods for prevention or improvement of atopic dermatitis.
도 1은 보풀 추출물의 인간 각질형성세포에 대한 세포독성을 확인한 결과이다. N은 음성대조군이고, Dex는 덱사메타손 처리군이다.
도 2는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 인간 각질형성세포에 보풀 추출물을 농도별로 처리한 후 TSLP의 발현 변화를 ELISA로 확인한 결과이다. N은 음성대조군이고, P는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 군이며, D는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 후 아토피 치료제로 사용되는 덱사메타손을 처리한 군이다.
도 3은 집먼지 진드기 추출물 및 DNCB(2,4-dinitrochlorobenzene)를 처리하여 귀 부위에 아토피 피부염이 유발된 마우스에 보풀 추출물을 경구투여한 후 귀 두께 변화를 확인한 결과이다. Con은 아토피 피부염을 유발하지 않은 음성대조군이고, AD는 아토피 피부염을 유발한 군이며, AD+B1은 아토피 피부염 유발 후 보풀 추출물을 1mg/kg 경구투여한 군이고, AD+B5는 아토피 피부염 유발 후 보풀 추출물을 5mg/kg 경구투여한 군이고, AD+B25는 아토피 피부염 유발 후 보풀 추출물을 25mg/kg 경구투여한 군이며, AD+Dex는 아토피 피부염 유발 후 아토피 피부염 치료제인 덱사메타손을 1mg/kg 경구투여한 군이다.
도 4는 집먼지 진드기 추출물 및 DNCB(2,4-dinitrochlorobenzene)를 처리하여 귀 부위에 아토피 피부염이 유도된 마우스에 보풀 추출물을 경구투여한 후 귀의 피부 변화를 관찰한 사진이다. Con은 아토피 피부염을 유발하지 않은 음성대조군이고, AD는 아토피 피부염을 유발한 군이며, AD+2는 아토피 피부염 유발 후 보풀 추출물을 2mg/kg 경구투여한 군이고, AD+10은 아토피 피부염 유발 후 보풀 추출물을 10mg/kg 경구투여한 군이고, AD+50은 아토피 피부염 유발 후 보풀 추출물을 50mg/kg 경구투여한 군이며, AD+Dex1은 아토피 피부염 유발 후 아토피 피부염 치료제인 덱사메타손을 1mg/kg 경구투여한 군이다.
도 5는 마우스에서 보풀 추출물의 투여 후 시간에 따른 마우스 체중 변화를 확인한 결과이다. Con은 아토피 피부염을 유발하지 않은 음성대조군이고, AD는 아토피 피부염을 유발한 군이며, B2는 아토피 피부염 유발 후 보풀 추출물을 2mg/kg 경구투여한 군이고, B10은 아토피 피부염 유발 후 보풀 추출물을 10mg/kg 경구투여한 군이고, B50은 아토피 피부염 유발 후 보풀 추출물을 50mg/kg 경구투여한 군이며, Dex1은 아토피 피부염 유발 후 아토피 피부염 치료제인 덱사메타손을 1mg/kg 경구투여한 군이다.
도 6은 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 인간 각질형성세포에 쿠세놀 F; 보풀 추출물; 또는 보풀 추출물 및 쿠세놀 F 혼합물을 처리하여 TSLP 저해 효과를 확인한 결과이다. N은 음성대조군이고, P는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 군이고, Dex는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 후 아토피 치료제로 사용되는 덱사메타손을 처리한 군이고, KF는 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 후 쿠세놀 F를 처리한 군이고, 보풀은 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 후 보풀 추출물을 처리한 군이며, KF+보풀은 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도한 후 보풀 추출물 및 쿠세놀 F 혼합물을 처리한 군이다.
도 7은 보풀 추출물 및 쿠세놀 F 혼합물의 인간 각질형성세포에 대한 세포독성을 확인한 결과이다. None는 아무것도 처리하지 않은 음성대조군이고, DMSO는 보풀 추출물 및 쿠세놀 F 혼합물의 용매인 DMSO를 처리한 군이며, Ku+보풀은 보풀 추출물 및 쿠세놀 F의 혼합물을 100㎍/ml 처리한 군이다. Fig. 1 shows the results of confirming cytotoxicity of human extracts of lupus to human keratinocytes. N is a negative control, and Dex is a dexamethasone-treated group.
FIG. 2 shows the results of TSLP expression assay by ELISA after treatment of the follicular extract with concentration of human keratinocyte which induced the expression of TSLP by treating Th2 immunity-inducing cytokine. N is a negative control, P is a group that induces the expression of TSLP by treating a Th2 immunoreactive cytokine, D is a group treating Th2 immunoreactive cytokine to induce expression of TSLP, and then treated with dexamethasone It is a family.
FIG. 3 is a result of examining changes in ear thickness after orally administering the extracts of house dust mites and DNCB (2,4-dinitrochlorobenzene) to orally administered to a mouse in which atopic dermatitis was induced in ear area. AD + B1 is a group of oral administration of 1 mg / kg of foliar extract after induction of atopic dermatitis, AD + B5 is a group of atopic dermatitis-induced AD + D25 is a group of oral administration of 5 mg / kg of foliar extract, AD + B25 is a group of oral administration of 25 mg / kg of foliar extract after induction of atopic dermatitis, AD + Dex is a group of 1 mg / kg of oral dexamethasone .
FIG. 4 is a photograph showing changes in the skin of the ear after orally administering the extracts of house dust mite and DNCB (2,4-dinitrochlorobenzene) to a mouse in which atopic dermatitis was induced in the ear area. AD was the group that induced atopic dermatitis. AD + 2 was a group administered with 2 mg / kg of foliar extract after atopic dermatitis and AD + 10 was the group with atopic dermatitis AD + Dex1 is a group of oral administration of 10 mg / kg of foliar extract, AD + 50 is 50 mg / kg orally of foliar extract after induction of atopic dermatitis, AD + Dex1 is a group of 1 mg / kg of oral dexamethasone .
FIG. 5 shows the results of examining changes in mouse body weight with time after administration of the Lint extract. Con was a negative control group that did not induce atopic dermatitis. AD was a group causing atopic dermatitis. B2 was a group administered with 2 mg / kg of foliar extract after induction of atopic dermatitis. B10 showed 10 mg / kg of body weight, B50 is a group of 50 mg / kg orally administered lint extract after atopic dermatitis, and Dex 1 is a group of 1 mg / kg orally administered dexamethasone, a treatment for atopic dermatitis after atopic dermatitis.
FIG. 6 shows the expression of TSLP by treating Th2 immunity-inducing cytokine in human keratinocytes; Lint extract; Or a mixture of fluff extract and xenol F to confirm TSLP inhibitory effect. N is a negative control group, P is a group that induced the expression of TSLP by treating a Th2 immunity-inducing cytokine, and Dex treats Th2 immunity-inducing cytokine to induce TSLP expression and then treats dexamethasone KF is a group treated with Th2 immunoreactive cytokine to induce expression of TSLP and treated with Kucelin F. Lint fluids were treated with Th2 immunostimulatory cytokine to induce expression of TSLP, And KF + fluff was treated with Thl2 immunoreactive cytokine to induce the expression of TSLP and then treated with a mixture of fluff extract and xenol F. [
FIG. 7 shows the result of confirming the cytotoxicity of human follicle-forming cells to the mixture of Lint extract and Cucerol F. FIG. None is a negative control group treated with nothing, DMSO is a group treated with DMSO which is a solvent of a mixture of fluff extract and xenol F, and a group treated with 100 μg / ml of a mixture of fluff extract and cucinol F.
본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 치료용 약학 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising an extract of Lilac as an active ingredient.
상기 유효성분은 보풀 추출물에 쿠세놀 F를 추가로 더 포함할 수 있으며, 바람직하게는 보풀 추출물 1 중량부에 대하여 0.3~3 중량부의 쿠세놀 F를 추가로 더 포함하는 것이고, 더 바람직하게는 보풀 추출물 1 중량부에 대하여 1 중량부의 쿠세놀 F를 추가로 더 포함하는 것이지만, 이에 제한되지 않는다. The active ingredient may further contain Cucénol F in the fluff extract, preferably further comprising 0.3 to 3 parts by weight of Cucherin F per 1 part by weight of the fluff extract, But is not limited to, 1 part by weight of Cucerol F per 1 part by weight of the extract.
본 발명의 일 구현 예에서, 상기 보풀 추출물; 또는 보풀 추출물 및 쿠세놀 F의 혼합물은 TSLP(Thymic Stromal Lymphopoietin)의 발현을 억제하는 특징이 있다. In one embodiment of the present invention, said fluff extract; Or a mixture of fluff extract and xenol F is characterized by inhibiting the expression of TSLP (Thymic Stromal Lymphopoietin).
본 발명의 일 구현 예에서, 상기 보풀 추출물의 용매는 물, C1~C7의 알코올, 에틸아세테이트, 클로로포름, 디클로로메탄, 에텔, 헥산 또는 이들의 혼합물이고, 바람직하게는 물, C1~C4의 저급 알코올 또는 이들의 혼합물이며, 더 바람직하게는 메탄올이지만, 이에 제한되지 않는다. In one embodiment, the solvent of the magnetic brush extract is water, C 1 ~ C 7 a mixture of alcohol, ethyl acetate, chloroform, dichloromethane, ether, hexane or a, preferably water, C 1 ~ C 4 lower alcohol or a mixture thereof, more preferably methanol, but is not limited thereto.
본 발명에서 보풀 추출물은 전초, 지상부 또는 지하부를 사용하여 추출한 것일 수 있으며, 바람직하게는 전초를 사용하여 추출한 것이지만, 이에 제한하는 것은 아니다. In the present invention, the fowl extract may be an extract obtained by using an outpouring, a ground part, or a ground part, and is preferably extracted using a planting material, but the present invention is not limited thereto.
본 발명의 아토피 피부염의 예방 또는 치료용 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다. 본 발명에 따른 조성물의 약학적 투여 형태는 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 조합으로 사용될 수 있다.The pharmaceutical composition for preventing or treating atopic dermatitis of the present invention may further comprise a suitable carrier, excipient or diluent conventionally used in the production of a pharmaceutical composition. The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in suitable combinations.
본 발명에 따른 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제제, 외용제, 좌제 및 주사제의 형태로 제형화하여 사용될 수 있다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 보풀 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이 외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and injections according to conventional methods . Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 약학 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 약학 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내(intracerebroventricular) 주사에 의해 투여될 수 있다.The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
또한, 본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 개선용 화장료 조성물에 관한 것이다. The present invention also relates to a cosmetic composition for preventing or ameliorating atopic dermatitis comprising an extract of Lilac as an active ingredient.
상기 유효성분은 보풀 추출물에 쿠세놀 F를 추가로 더 포함할 수 있으며, 바람직하게는 보풀 추출물 1 중량부에 대하여 0.3~3 중량부의 쿠세놀 F를 추가로 더 포함하는 것이고, 더 바람직하게는 보풀 추출물 1 중량부에 대하여 1 중량부의 쿠세놀 F를 추가로 더 포함하는 것이지만, 이에 제한되지 않는다. The active ingredient may further contain Cucénol F in the fluff extract, preferably further comprising 0.3 to 3 parts by weight of Cucherin F per 1 part by weight of the fluff extract, But is not limited to, 1 part by weight of Cucerol F per 1 part by weight of the extract.
본 발명의 아토피 피부염 개선용 화장료 조성물에 있어서, 상기 아토피 피부염 개선용 화장료 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액, 파운데이션, 왁스 파운데이션 및 스프레이 중에서 선택된 어느 하나의 제형인 것이 바람직하며, 더 바람직하게는 피부외용 연고, 크림, 유연화장수, 영양화장수, 팩, 에센스, 헤어토닉, 샴푸, 린스, 헤어 컨디셔너, 헤어 트리트먼트, 젤, 스킨 로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 마사지 크림, 영양크림, 아이크림, 모이스처 크림, 핸드 크림, 파운데이션, 영양에센스, 선스크린, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디 로션 및 바디 클렌저로 이루어지는 군으로부터 선택된 어느 하나의 제형을 가질 수 있으나, 이에 제한되지 않는다. 이들 각 제형의 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.In the cosmetic composition for improving atopic dermatitis according to the present invention, the cosmetic composition for improving atopic dermatitis may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, A cream, a softener, a nutrient lotion, a pack, an essence, a hair tonic, a shampoo, a rinse, a hair conditioner, a hair conditioner, a hair conditioner, Lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, skin lotion, skin toner, , Soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers You can have a single dosage form slow, but not limited to this. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산아연 등이 이용될 수 있다. 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. 본 발명의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components . When 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. In the case of a spray, in particular, / Propane or dimethyl ether. In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 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 액상 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. 본 발명의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When 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. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from 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, linolenic derivatives or ethoxylated glycerol fatty acid esters.
또한, 본 발명은 보풀 추출물을 유효성분으로 포함하는 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다. The present invention also relates to a health functional food composition for preventing or ameliorating atopic dermatitis comprising an extract of Lilac as an active ingredient.
상기 유효성분은 보풀 추출물에 쿠세놀 F를 추가로 더 포함할 수 있으며, 바람직하게는 보풀 추출물 1 중량부에 대하여 0.3~3 중량부의 쿠세놀 F를 추가로 더 포함하는 것이고, 더 바람직하게는 보풀 추출물 1 중량부에 대하여 1 중량부의 쿠세놀 F를 추가로 더 포함하는 것이지만, 이에 제한되지 않는다. The active ingredient may further contain Cucénol F in the fluff extract, preferably further comprising 0.3 to 3 parts by weight of Cucherin F per 1 part by weight of the fluff extract, But is not limited to, 1 part by weight of Cucerol F per 1 part by weight of the extract.
본 발명의 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물은 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조될 수 있으나, 이에 제한되지 않는다. 상기 건강기능식품 조성물은 아토피 피부염을 예방하거나 개선하기 위해 섭취할 수 있는 것이면 특별히 제한되지 않는다. The health functional food composition for preventing or ameliorating atopic dermatitis of the present invention may be manufactured by any one of formulations selected from granules, rings, tablets, capsules, candies, syrups and beverages, but is not limited thereto. The health functional food composition is not particularly limited as long as it can be ingested to prevent or ameliorate atopic dermatitis.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 그러나 건강 조절을 목적으로 하는 장기간의 섭취인 경우에는 안전성 면에서 아무런 문제가 없는 범위의 양으로 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). However, in the case of long-term intake for the purpose of controlling health, it can be used in an amount that does not cause any safety problems. There is no particular limitation on the kind of the food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다. 상기 건강기능식품 조성물 외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.). In addition to the above-mentioned health functional food composition, it is possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like.
이하, 본 발명을 제조예 및 실시예에 의해 상세히 설명한다. 단, 하기 제조예 및 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 제조예 및 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples. However, the following Preparation Examples and Examples are illustrative of the present invention, and the content of the present invention is not limited to the following Production Examples and Examples.
제조예Manufacturing example 1. 보풀 추출물의 제조 1. Preparation of Lint Extract
보풀의 전초를 건조하여 분쇄한 가루 40g에 메탄올 400㎖를 첨가한 후, 초음파 추출기를 이용하여 8시간 동안 추출하였다. 추출된 용액을 여과지로 여과한 후 감압농축하여 보풀 추출물 3.5g을 획득하였다. 400 ml of methanol was added to 40 g of pulverized powder by drying the outline of the fluff, and then extracted with an ultrasonic wave extender for 8 hours. The extracted solution was filtered with a filter paper and concentrated under reduced pressure to obtain 3.5 g of a fluff extract.
제조예Manufacturing example 2. 2. 쿠세놀Kusselen F의 분리 및 정제 F separation and purification
건조한 고삼(Sophora flavescence) 뿌리 3kg을 분쇄한 후 메탄올 5ℓ를 첨가한 다음 초음파 추출기를 이용하여 8시간 동안 추출한 후 여과하는 과정을 총 2회 수행하였다. 그 후, 여과한 메탄올 추출액을 감압하여 약 500g의 메탄올 추출물을 획득하였다. 상기 메탄올 추출물을 증류수에 현탁한 후 증류수와 동량의 에틸아세테이트를 첨가하여 분배시키고 정치하여 에틸아세테이트 층을 획득하였다. 상기 증류수 및 에틸아세테이트를 첨가하고, 에틸아세테이트 층을 획득하는 과정을 2회 더 반복한 후, 획득한 에틸아세테이트 분획물을 감압 농축하여 약 200g의 고삼 에틸아세테이트 분획물을 획득하였다. 이 중 80g을 취해 1차 실리카겔 컬럼 크로마토그래피(실리카겔 약 2.5g)를 실시하였고, 클로로포름:메탄올이 99:1, 95:5, 90:10, 80:20, 70:30, 50:50 부피비로 혼합된 용출용매를 단계별로 흘려주면서 용출물 1.5ℓ마다 박층크로마토그래피를 실시하고, 그 결과에 따라 하위 분획으로 나누었다. 각각의 분획은 감압농축하였으며, 그 중 4번째 분획을 이용하여 2차 실리카겔 크로마토그래피를 실시하여 쿠세놀 F 약 15㎎을 획득하였다. 쿠세놀 F의 화학구조는 표준폼과 NMR, MS 등의 기기분석자료를 비교하여 동정하였고, 화학식 1로 나타내었다. Sophora flavescence roots were pulverized, and then 5 liters of methanol was added. Then, the mixture was extracted for 8 hours using an ultrasonic extractor and then filtered twice. Thereafter, the filtered methanol extract was decompressed to obtain about 500 g of methanol extract. The methanol extract was suspended in distilled water, and the same amount of ethyl acetate as that of distilled water was added thereto. The mixture was distributed and allowed to stand to obtain an ethyl acetate layer. The distilled water and ethyl acetate were added and the ethyl acetate layer was obtained twice. The obtained ethyl acetate fraction was concentrated under reduced pressure to obtain a high ethyl acetate fraction of about 200 g. 80 g of the obtained product was subjected to primary silica gel column chromatography (silica gel: about 2.5 g), and chloroform: methanol was used in a ratio of 99: 1, 95: 5, 90:10, 80:20, 70:30, Thin layer chromatography was performed every 1.5 l of the eluate while the mixed elution solvent was flowed step by step and divided into sub-fractions according to the result. Each fraction was concentrated under reduced pressure. Secondary silica gel chromatography was performed using the fourth fraction, thereby obtaining about 15 mg of Cucenol F. [ The chemical structure of Kusenol F was identified by comparing the instrumental analysis data such as NMR and MS with the standard form, and it was represented by the formula (1).
(MS m/z 424 [M]+; 1H-NMR (300 MHz, DMSO-d6) δ: 7.37(1H, d, J= 8.3 Hz, H-6'), 6.41(1H, dd, J = 8.3, 2.4 Hz, H-5'), 6.37(1H, d, J= 2.4 Hz, H-3'), 6.15(1H, s, H-8'), 5.60(1H, dd, J= 11.1, 5.0 Hz, H-2), 5.05(1H, t, J= 6.7 Hz, H-4"), 4.62(1H, d, J= 2.1 Hz, H-9"a), 4.60 (1H, d, J= 2.1 Hz, H-9"b), 1.61(3H, s, 10"-CH3), 1.56 (3H, s, 6"-CH3), 1.45 (3H, s, 7"-CH3); 13C-NMR (75 MHz, DMSO-d6) δ: 17.8(C-7"), 19.1(C-10"), 25.8(C-6"), 27.1(C-1"), 31.9(C-3"), 42.8(C-3), 47.8(C-2"), 75.3(C-2), 95.1(C-8), 102.7(C-10), 103.4(C-3'), 107.8(C-5'), 108.2(C-6), 111.2(C-9"), 117.8 (C-1'), 124.4(C-4"), 128.6(C-6'), 131.6(C-5"), 149.1(C-8"), 156.1(C-4'), 159.4 (C-2'), 162.1(C-5), 163.0(C-9), 165.3(C-7), 197.8(C-4))(1H, dd, J = 8.3 (1H, d, J = 8.3 Hz, H-6 '), , 2.4 Hz, H-5 '), 6.37 (1H, d, J = 2.4 Hz, H-3'), 6.15 (1H, d, J = 9 Hz), 5.05 (1H, t, J = 6.7 Hz, H- 13 C-NMR (
[화학식 1][Chemical Formula 1]
실시예Example 1. 보풀 추출물 처리에 의한 1. Treatment by Lint Extract Treatment TSLPTSLP 발현의 변화 Change in expression
제조예 1의 보풀 추출물의 세포독성을 확인하기 위해 96 웰 플레이트에 인간 각질형성세포(keratinocyte)를 3×104개 접종하고, 플레이트의 80%까지 자라도록 배양한 후, 덱사메타손 또는 제조예 1의 농도별(1, 10, 100 및 1000㎍/㎖) 보풀 추출물을 24시간 동안 처리하였다. 그 후, 20㎕의 MTT 용액(5mg/㎖)을 첨가한 후 2시간 동안 반응시켰다. 반응 후 배지는 제거하고 DMSO를 첨가하여 생성된 결정을 녹인 후, 570nm에서 흡광도를 측정하였다. In order to confirm the cytotoxicity of the fluff extract of Preparation Example 1, 3 × 10 4 human keratinocytes were inoculated in a 96-well plate and cultured to grow up to 80% of the plate. Then, dexamethasone or Lint extract (1, 10, 100 and 1000 μg / ml) was treated for 24 hours. Then, 20 μl of MTT solution (5 mg / ml) was added and reacted for 2 hours. After the reaction, the medium was removed, and the resulting crystals were dissolved by adding DMSO, and the absorbance was measured at 570 nm.
그 결과, 도 1에 나타난 바와 같이 덱사메타손 및 100㎍/㎖ 농도 이하의 보풀 추출물은 세포독성이 나타나지 않았으나, 1000㎍/㎖ 농도의 보풀 추출물은 세포독성이 나타나는 것을 확인하여 이후 실험에서는 100㎍/㎖ 농도 이하의 보풀 추출물을 사용하였다.As a result, as shown in FIG. 1, dexamethasone and the fluff extract below 100 μg / ml did not show cytotoxicity. However, it was confirmed that the fluff extract at a concentration of 1000 μg / ml showed cytotoxicity, Lint extract was used.
보풀 추출물의 TSLP 저해효과는 Th2 면역유도 사이토카인(IL-4 100ng/㎖, IL-13 100ng/㎖, TNF-α 20ng/㎖ cytokine mixture 및 Flagellin 100ng/㎖)을 처리하여 TSLP의 발현을 유도한 인간 각질형성세포(keratinocyte)에 상기 독성을 나타내지 않은 100㎍/㎖ 농도 이하의 보풀 추출물을 처리하여 확인하였다. TSLP inhibitory effect of Lilium extract was induced by the treatment of Th2 immunoreactive cytokine (IL-4 100 ng / ml, IL-13 100 ng / ml, TNF-
24 웰 플레이트에 2×105개의 세포를 접종하고, 플레이트의 80%까지 자라도록 배양한 다음 하이드로코르티손(hydrocortisone)이 들어있지 않은 배양액으로 교체한 후 Th2 면역유도 사이토카인을 처리하여 TSLP의 발현을 유도하였으며, 보풀 추출물을 농도별로 처리하여 24시간 동안 반응하였다. 2 × 10 5 cells were inoculated into a 24-well plate, cultured to grow up to 80% of the plate, replaced with a culture solution free of hydrocortisone, treated with Th2 immunity-inducing cytokine, and expressed with TSLP , And treated with Lint extract at different concentrations and reacted for 24 hours.
보풀 추출물의 TSLP 저해효과는 인간 TSLP 듀오세트 ELISA(Human TSLP DuoSet ELISA, R&D system)를 사용하였고, 포획 항체(capture antibody)를 실온에서 16시간 동안 코팅한 후 PBS에 1%(w/v) BSA를 첨가하여 만든 희석용 시약(reagent diluent) 200㎕로 교체한 후 2시간 동안 블로킹(blocking)하였다. 그 후 샘플(상기 보풀 추출물 처리 후 24시간 동안 반응시킨 배양 배지) 또는 표본(standard) 100㎕로 교체한 후 2시간 동안 실온에서 결합시킨 다음 검출용 항체(detection antibody) 100㎕로 교체하여 2시간 동안 실온에서 결합시켰다. 그 후 발색반응 측정을 위해 스트렙타비딘-HRP(streptavidin-HRP) 100㎕를 20분 동안 처리하고, 그 후 TMB 기질 100㎕를 20분 동안 반응시킨 후 450nm에서 흡광도를 측정하여 확인하였다. The TSLP inhibitory effect of the fluff extract was measured using a human TSLP DuoSet ELISA (R & D system), and the capture antibody was coated at room temperature for 16 hours. Then, 1% (w / v) BSA Was added to 200 쨉 l of a reagent diluent prepared in Example 1 and blocked for 2 hours. Thereafter, the sample was replaced with 100 μl of a sample (culture medium reacted for 24 hours after the fluvial extract treatment) or a standard (standard), bound at room temperature for 2 hours, replaced with 100 μl of detection antibody, Lt; / RTI > at room temperature. 100 μl of streptavidin-HRP was then treated for 20 minutes, 100 μl of TMB substrate was reacted for 20 minutes, and the absorbance at 450 nm was measured.
그 결과, 도 2에 나타난 바와 같이 Th2 면역유도 사이토카인을 처리하여 유도된 TSLP의 발현은 보풀 추출물을 처리하였을 때 감소하였고, 보풀 추출물의 농도가 증가될수록 TSLP 발현 감소효과가 우수하였다. As a result, as shown in FIG. 2, the expression of TSLP induced by treatment with Th2 immunity-inducing cytokine was decreased when treated with Liliaceae extract, and the effect of TSLP expression was better as the concentration of Liliaceae extract was increased.
실시예Example 2. 아토피 피부염 동물모델에서 보풀 추출물 처리에 의한 효과 확인 2. Identification of effects of lint extract treatment in atopic dermatitis animal model
집먼지 진드기 추출물(house dust mite extract, Dermatophagoides farinae extract, DFE) 및 DNCB(2,4-dinitrochlorobenzene)를 귀 부위에 처리하여 아토피 피부염이 유도된 마우스에 보풀 추출물을 경구투여한 후 귀 두께 변화를 확인하여 보풀 추출물의 아토피 피부염 개선효과를 확인하였다. House dust mite extract, Dermatophagoides farinae extract, DFE) and DNCB (2,4-dinitrochlorobenzene) were administered to the ear area to examine the change in ear thickness after oral administration of the foliar extract to the mice induced atopic dermatitis, thereby confirming the effect of improving the atopic dermatitis .
마우스 귀를 반창고로 3-4회 반복하여 스트리핑(striping)한 후, 10mg/㎖로 희석한 DFE를 매주 월요일에 마우스 귀의 앞·뒤 면에 20㎕씩 도포하였다. AOO(아세톤/올리브오일을 1:3 부피비로 혼합한 용액)에 녹인 1%(w/v) DNCB는 매주 금요일에 마우스 귀의 앞·뒤 면에 20㎕씩 도포하고, 매주 화요일 및 토요일마다 귀 두께를 측정하였다. The mouse ear was stripped repeatedly 3-4 times with a bandage, and 20 μl of DFE diluted to 10 mg / ml was applied to the front and back sides of the mouse ear every Monday. The 1% (w / v) DNCB dissolved in AOO (a mixture of acetone / olive oil in a volume ratio of 1: 3) was applied on the front and back sides of the mouse ear every Friday on a weekly basis, Were measured.
그 결과, 도 3에 나타난 바와 같이 DFE 및 DNCB를 마우스 피부에 도포하면 아토피 피부염이 유발되어 귀 두께가 증가하지만, DFE 및 DNCB 도포와 더불어 보풀 추출물의 경구투여를 시행하면 귀 두께의 증가가 감소하는 것을 확인하였다. 귀 두께 감소효과는 보풀 추출물의 농도가 증가할수록 크게 나타났고, 도 4에 개시한 바와 같이 귀 두께 감소효과뿐만 아니라 육안으로도 아토피 피부염이 유발되었던 귀 부위 피부의 증상을 완화시켰다. As a result, as shown in FIG. 3, when DFE and DNCB were applied to mouse skin, atopic dermatitis was induced and ear thickness was increased. However, oral administration of Luff extract together with DFE and DNCB application decreased the increase of ear thickness Respectively. As shown in FIG. 4, the effect of decreasing the thickness of ear thickness was increased as the concentration of the fluff extract was increased. In addition to the effect of decreasing the thickness of the ear as shown in FIG. 4, the symptoms of the ear skin area in which atopic dermatitis was induced visually were alleviated.
또한, 보풀 추출물이 마우스에 독성을 일으키지 않는다는 것을 마우스 체중변화로 확인한 결과, 도 5에 나타난 바와 같이 보풀 추출물을 투여한 경우에도 체중의 변화는 거의 없는 것으로 나타났다. In addition, the fact that the fluff extract did not cause toxicity to the mice was confirmed by the changes in the body weight of the mice. As a result, as shown in Fig. 5, even when the fluff extract was administered, there was almost no change in body weight.
이를 통해 보풀 추출물은 개체에 독성을 나타내지 않으며, 아토피 피부염 개선효과가 있다는 것을 확인하였다. It was confirmed that the Lint extract did not show any toxicity to the individual, and that atopic dermatitis was improved.
실시예Example 3. 보풀 추출물 및 3. Lint extract and 쿠세놀Kusselen F 혼합물 처리에 의한 F mixture treatment TSLPTSLP 발현의 변화 Change in expression
본 발명의 제조예 1 및 제조예 2의 보풀 추출물, 쿠세놀 F 또는 이들의 혼합물이 TSLP의 발현에 미치는 영향을 확인하였다. The effect of Liliaceae extract, Kucenol F, or a mixture thereof on the expression of TSLP of Production Example 1 and Production Example 2 of the present invention was confirmed.
24 웰 플레이트에 2×105개의 세포를 접종하고, 플레이트의 80%까지 자라도록 배양한 다음 하이드로코르티손(hydrocortisone)이 들어있지 않은 배양액으로 교체한 후 Th2 면역유도 사이토카인(IL-4 100ng/㎖, IL-13 100ng/㎖, TNF-α 20ng/㎖ cytokine mixture 및 Flagellin 100ng/㎖)을 처리하여 TSLP의 발현을 유도하였으며, 보풀 추출물 100㎍/㎖, 쿠세놀 F 100㎍/㎖ 또는 이들의 혼합물(보풀 추출물 50㎍/㎖+쿠세놀 F 50㎍/㎖) 100㎍/㎖을 1㎖의 배지에 분주하여 24시간 동안 반응하였다. 2 × 10 5 cells were inoculated in a 24-well plate and cultured to grow to 80% of the plate. Subsequently, the medium was replaced with a culture solution free of hydrocortisone, and then the Th2 immunoreactive cytokine (IL-4 100 ng / , 100 ng / ml of IL-13, 20 ng / ml cytokine mixture of TNF-α and 100 ng / ml of Flagellin) were induced to induce expression of TSLP. 100 μg / ml of fluff extract, 100 μg / (50 占 퐂 / ml of fluff extract + 50 占 퐂 / ml of Kuchenol F) was added to 1 ml of the medium and reacted for 24 hours.
보풀 추출물, 쿠세놀 F 또는 이들의 혼합물에 의한 TSLP 저해효과는 인간 TSLP 듀오세트 ELISA(Human TSLP DuoSet ELISA, R&D system)를 사용하였고, 포획 항체(capture antibody)를 실온에서 16시간 동안 코팅한 후 PBS에 1%(w/v) BSA를 첨가하여 만든 희석용 시약(reagent diluent) 200㎕로 교체한 후 2시간 동안 블로킹(blocking)하였다. 그 후 샘플(상기 24시간 동안 반응시킨 배양 배지) 또는 표본(standard) 100㎕로 교체한 후 2시간 동안 실온에서 결합시킨 다음 검출용 항체(detection antibody) 100㎕로 교체하여 2시간 동안 실온에서 결합시킨다. 그 후 발색반응 측정을 위해 스트렙타비딘-HRP(streptavidin-HRP) 100㎕를 20분 동안 처리하고, 그 후 TMB 기질 100㎕를 20분 동안 반응시킨 후 450nm에서 흡광도를 측정하여 확인하였다. Human TSLP DuoSet ELISA (R & D system) was used for the TSLP inhibition effect by the fluff extract, Cucinol F, or a mixture thereof. Capture antibody was coated at room temperature for 16 hours, Was replaced with 200 μl of a reagent diluent prepared by adding 1% (w / v) BSA to the wells and then blocked for 2 hours. Then, the sample was replaced with 100 μl of a sample (culture medium reacted for 24 hours) or a standard (standard), bound at room temperature for 2 hours, replaced with 100 μl of detection antibody, . 100 μl of streptavidin-HRP was then treated for 20 minutes, 100 μl of TMB substrate was reacted for 20 minutes, and the absorbance at 450 nm was measured.
그 결과, 도 6에 나타난 바와 같이 쿠세놀 F, 보풀 추출물 각각의 TSLP 저해효과를 확인하였으며, 이들을 혼합한 혼합물은 쿠세놀 F 또는 보풀 추출물을 단독으로 처리한 군에 비해 더욱 증진된 TSLP 저해효과를 나타냈다. As a result, as shown in FIG. 6, the TSLP inhibitory effect of each of the extracts of Cucuron F and Liliaceae was confirmed, and the mixture of them showed more enhanced TSLP inhibitory effect than that of Cucinol F or Lint extract alone .
또한, 도 7과 같이 본 발명에 사용된 보풀 추출물 및 쿠세놀 F 혼합물은 세포독성을 나타내지 않는 것을 확인함으로써 보풀 추출물 및 쿠세놀 F 혼합물이 세포독성없이 TSLP 저해효과를 나타내는 것을 확인하였다. In addition, as shown in FIG. 7, it was confirmed that the fluff extract and the mixture of Xenol F used in the present invention did not show cytotoxicity, so that the mixture of fluff extract and Xenol F showed TSLP inhibitory effect without cytotoxicity.
이를 통해 본 발명의 보풀 추출물, 쿠세놀 F 및 이들의 혼합물은 세포독성을 나타내지 않고, TSLP 저해효과가 우수하며, 특히 보풀 추출물 및 쿠세놀 F 혼합물의 TSLP 저해효과가 현저하다는 것을 확인하였다. Thus, it was confirmed that the foliar extract of the present invention, Kucelin F, and the mixture thereof did not show cytotoxicity, and that the TSLP inhibitory effect was excellent, and the TSC inhibitory effect of the mixture of Linalool extract and Cucinol F was remarkable.
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