KR20200129670A - Composition comprising Fritillariae Thunbergii Bulbs extract for preventing or treating atopic dermatitis - Google Patents
Composition comprising Fritillariae Thunbergii Bulbs extract for preventing or treating atopic dermatitis Download PDFInfo
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- KR20200129670A KR20200129670A KR1020190054426A KR20190054426A KR20200129670A KR 20200129670 A KR20200129670 A KR 20200129670A KR 1020190054426 A KR1020190054426 A KR 1020190054426A KR 20190054426 A KR20190054426 A KR 20190054426A KR 20200129670 A KR20200129670 A KR 20200129670A
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- atopic dermatitis
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Abstract
Description
본 발명은 절패모 추출물을 함유하는 아토피 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating atopy containing an extract of Jeolpaemo.
아토피(Atopy) 또는 아토피 증후군은 알레르기 항원에 대한 직접 접촉 없이 신체가 극도로 민감해지는 알러지 반응을 말하며 주로 유아기 혹은 소아기에 시작되는 만성적이고 재발성의 염증성 피부질환으로 소양증(가려움증)과 피부건조증 및 특징적인 습진을 동반한다. 유아기에는 얼굴과 팔다리의 펼쳐진 쪽 부분에 습진으로 시작되지만, 성장하면서 특징적으로 팔이 굽혀지는 부분과 무릎 뒤의 굽혀지는 부위에 습진의 형태로 나타나며, 많은 경우에 성장하면서 자연히 호전되는 경향을 보인다. 그러나 어른의 경우에는 접히는 부위의 피부가 두꺼워지는 태선화(lichenification)가 나타나고, 유소아기에 비해 얼굴에 습진이 생기는 경우가 많다. Atopy or atopic syndrome refers to an allergic reaction in which the body becomes extremely sensitive without direct contact with an allergen. It is a chronic and recurrent inflammatory skin disease that begins mainly in infancy or childhood.Itching (itch), dry skin, and characteristic Accompanying eczema. In infancy, it starts with eczema on the spread side of the face and limbs, but as it grows, it appears in the form of eczema on the bending part of the arm and the bend behind the knee, and in many cases it tends to improve naturally as it grows. However, in the case of adults, lichenification, in which the skin of the fold area becomes thicker, appears, and eczema on the face is more common than in childhood.
아토피성 피부염은 세계적으로 증가하는 추세이며 유병률이 인구의 20%라는 보고도 있으며 국민건강보험공단 2014년 3월 보도 자료에 의하면 '아토피 피부염' 질환의 건강보험 진료비 지급자료를 분석한 결과 2008년~2012년 연평균 진료인원은 104만 명이었고, 이중 남성은 49만 명, 여성은 55만 명으로 여성이 남성보다 더 많이 진료받는 것으로 조사되었다.Atopic dermatitis is on the rise worldwide, and there are reports that the prevalence rate is 20% of the population.According to a press release from March 2014 by the National Health Insurance Service, the results of an analysis of health insurance medical expenses payment data for atopic dermatitis disease from 2008~ In 2012, the annual average number of people to be treated was 1,040,000, of which 490,000 were male and 550,000 were female, and it was surveyed that women received more treatment than men.
아토피성 피부염의 발병 원인은 아직 확실하게 알려져 있지 않은 상태이며, 발생원인은 구체적으로 밝혀진 것이 없고, 가족력, 성별, 신체비만지수(Body mass index), 거주 지역, 애완동물, 출생주수, 사회경제적 요인 등 복합적 요인으로 발생하는 다인자성 질환으로 알려져 있다. 또한 환자의 연령에 따라 나타나는 증상이 다양한 것으로 알려져 있고 임상 증상도 피부 건조증, 습진 등으로 다양하게 나타나기 때문에 발병 원인이 어느 한 가지로만 설명될 수는 없지만, 환경적인 요인과 유전적인 소인, 면역학적 반응 및 피부보호막의 이상 등이 주요 원인으로 여겨지고 있다. 환경적인 요인으로는 산업화로 인한 매연 등 환경 공해, 식품 첨가물 사용의 증가, 서구식 주거 형태로 인한 카펫, 침대, 소파의 사용 증가, 실내 온도 상승으로 인한 집먼지 진드기 등의 알레르기를 일으키는 원인 물질(알레르겐)의 증가 등이 있다. 그러나 아토피 피부염이 발생하게 되는 직접적인 원인은 아직까지 명확하지 않기 때문에 이에 대한 연구가 계속적으로 진행되고 있다.The cause of the onset of atopic dermatitis is still unknown, and the cause of its occurrence has not been specifically identified, and the family history, sex, body mass index, area of residence, pet, age of birth, socioeconomic factors It is known as a multifactorial disease caused by multiple factors such as. In addition, it is known that the symptoms appearing vary according to the patient's age, and clinical symptoms are also variously manifested, such as dry skin and eczema, so the cause of the onset cannot be explained by only one, but environmental factors, genetic predisposition, and immunological reactions. And abnormal skin barriers are considered to be the main causes. Environmental factors include environmental pollution such as smoke caused by industrialization, increased use of food additives, increased use of carpets, beds, and sofas due to Western-style housing, and substances that cause allergies such as house dust mites due to an increase in indoor temperature (allergens). ), etc. However, since the direct cause of the occurrence of atopic dermatitis is not yet clear, research on this is ongoing.
아토피 알레르기의 메카니즘을 보면 알레르겐이 각질층을 통해 침입해서 표피세포에까지 이르게 되면 백혈구의 일종인 대식세포가 이를 포식하여 조각조각 분해시키게 된다. 침입한 알레르겐의 양이 많아 대식세포가 다 처리하지 못할 경우에는 미처 처리하지 못한 알레르겐의 존재에 대한 정보를 같은 백혈구의 일종인 T세포(면역촉진과 억제의 조절 기능을 말고 있다)에 보내주고 T세포는 그 정보를 다시 백혈구의 일종인 호산구(호산성 백혈구)에 전하여 혈중에 널리 분포된 호산구를 알레르겐이 침입한 장소로 불러 모으게 된다. 이때 환부에 집적(모려 쌓임)된 호산구가 화학전달물질(활성산소)을 과잉으로 방출시켜 염증이 일어나게 되는데 이것이 바로 알레르기 반응의 원인이 된다.Looking at the mechanism of atopic allergy, when the allergen invades through the stratum corneum and reaches the epidermal cells, macrophages, a type of white blood cells, prey on them and break them down into pieces. If the amount of invading allergen is high and macrophages cannot process all of the invading allergens, information about the presence of allergens that have not been processed is sent to T cells, which are a type of white blood cell (excluding the function of regulating immunity promotion and inhibition), and T The cells pass the information back to eosinophils (eosinophils), which are a type of white blood cells, and collect eosinophils, which are widely distributed in the blood, to the place where the allergen has invaded. At this time, the eosinophils accumulated (stacked) in the affected area release chemical transport substances (active oxygen) in excess, causing inflammation, which is the cause of the allergic reaction.
아토피를 치료하기 위하여, 종래 세라마이드, 리놀레산, 식물유 또는 광물성 오일 등의 성분, 하이드로코티손(hydrocortisone) 등의 스테로이드(steroid) 제제 또는 이들에 항균 및 항염 기능을 강화한 물질들이 제안된 바 있다. 그러나, 상기 스테로이드 제제는 표피의 성장억제나 부작용 등의 역효과를 유발할 수 있으며, 우레아 퍼록사이드 등은 피부의 과다 자극을 야기할 수 있고, 균의 내성(resistance) 및 광과민 등의 부작용을 유발할 가능성이 높은 문제점이 있다. 또한, 장기간 피부에 적용시 모세 혈관 확장증 및/또는 각질층의 두께 증가 내지 확장을 유발하는 등의 심각한 부작용을 발생시킬 염려가 있으며, 최근 아토피 완화에 많이 쓰이는 감마-리놀레산은 쉽게 산화되므로 안정성이 낮을 뿐만 아니라, 피부 자극성이 비교적 강하여 민감한 피부에는 사용하기 곤란한 문제점이 있다.In order to treat atopy, conventional ingredients such as ceramide, linoleic acid, vegetable oil or mineral oil, steroid preparations such as hydrocortisone, or substances that enhance antibacterial and anti-inflammatory functions have been proposed. However, the steroid preparation may cause adverse effects such as epidermal growth inhibition or side effects, and urea peroxide may cause excessive skin irritation, and may cause side effects such as resistance of bacteria and photosensitivity. There is a high problem. In addition, when applied to the skin for a long period of time, there is a concern that serious side effects such as telangiectasia and/or an increase in the thickness or expansion of the stratum corneum may occur, and gamma-linoleic acid, which is widely used for relieving atopy in recent years, is easily oxidized and thus has low stability. In addition, there is a problem that it is difficult to use for sensitive skin due to relatively strong skin irritation.
이러한 종래의 화학 조성물에 의한 아토피 증상 완화제의 단점을 보완하여 장기적으로 사용하더라도 부작용이 없고, 효과적으로 아토피 증상을 예방, 완화 또는 치료할 수 있는 것으로 자연(특히, 식물)에서 추출한 천연물질을 이용한 아토피성 피부질환 치료제가 각광받고 있다.Atopic skin using natural substances extracted from nature (especially plants) without side effects even when used for a long period of time by supplementing the shortcomings of such conventional chemical compositions, and can effectively prevent, alleviate or treat atopic symptoms. Disease treatments are in the spotlight.
한편, 절패모(Fritillariae Thunbergii Bulbs)는 다년생초본으로 우리나라 북부지방의 심산지역, 함경남도 갑산, 백두산의 산지초원에 자생한다. 높이는 25cm 안팎이며 인경은 백색이고 5~6개의 육질 인편으로 되어 있으며 둥글고 밑부분에 수염뿌리가 달린다. 원줄기는 곧게 자라며 잎은 대생 또는 3개씩 윤생하고 선형이며 길이 10cm로서 엽병이 없고 끝이 뾰족하며 윗부분의 잎은 덩굴손처럼 말린다. On the other hand, Fritillariae Thunbergii Bulbs are perennial plants that grow wild in the mountainous regions of the northern regions of Korea, Gapsan, and Baekdusan, South Hamgyeong Province. The height is around 25cm, the diameter is white, consists of 5~6 flesh scales, round, with a beard root at the bottom. The main stem grows straight, the leaves are coarse or three by one, linear, 10cm long, without petioles, and the tip is sharp, and the upper leaves are dried like tendrils.
절패모에는 페이민(베르티신), 페이미닌, 이소베르티신 등이 함유되어 있으며 그 외에도 티미딘, 아데노신 등이 포함되어 있다. 패모는 전통적으로 진해거담제와 항고혈압제로 사용되었다. 패모에 포함된 페이민(베르티신), 페이민닌을 투여하면 기침의 발작 간격이 줄어들고 강도도 약해지는 것으로 보고되었다. 패모의 추출물은 동맥의 혈압을 현저히 저하시키는데 그 기전은 혈압 상승 효소인 ACE (안지오텐신 전환 효소)의 활성을 억제하는 것으로 보고되고 있다. 하지만, 현재까지 절패모 추출물 또는 분획물의 아토피 예방 또는 개선에 대한 연구내용은 알려지지 않았다. Jeopaemo contains faymin (bertisin), faiminin, isoverticin, and other thymidine and adenosine. Pamo has traditionally been used as an antitussive expectorant and antihypertensive agent. It has been reported that the administration of feymin (verticin) and feminin, which are included in Femo, decreases the seizure interval of cough and decreases the intensity. The extract of Famo remarkably lowers arterial blood pressure, and its mechanism is reported to inhibit the activity of ACE (angiotensin converting enzyme), an enzyme that raises blood pressure. However, until now, the contents of research on the prevention or improvement of atopy of Jeolpaemo extract or fraction were not known.
이에 본 발명자들은, 인체에 부작용이 없는 천연물 유래의 아토피 피부염 치료제를 개발하기 위하여 노력한 결과, 절패모 추출물 또는 분획물이 아토피 피부염의 예방 또는 치료에 유용하게 사용될 수 있음을 확인하고 본 발명을 완성하였다. Accordingly, the present inventors have endeavored to develop a therapeutic agent for atopic dermatitis derived from a natural product that does not have side effects on the human body, and as a result, it has been confirmed that the extract or fraction of Jeolpaemo can be usefully used for the prevention or treatment of atopic dermatitis and completed the present invention.
본 발명의 목적은 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학적 조성물을 제공하는 것이다. It is an object of the present invention to provide a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an extract or a fraction thereof as an active ingredient.
또한 본 발명의 목적은, 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다. In addition, an object of the present invention is to provide a health functional food composition for preventing or improving atopic dermatitis, comprising an extract of Jeolpaemo or a fraction thereof as an active ingredient.
또한 본 발명의 목적은, 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물을 제공하는 것이다. In addition, it is an object of the present invention to provide a cosmetic composition for preventing or improving atopic dermatitis, comprising an extract of Jeolpaemo or a fraction thereof as an active ingredient.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학적 조성물을 제공한다. As an aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an extract of Jeolpaemo or a fraction thereof as an active ingredient.
또한, 본 발명은 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving atopic dermatitis, comprising an extract of Jeolpaemo or a fraction thereof as an active ingredient.
아울러, 본 발명은 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물을 제공한다. In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, including the extract or a fraction thereof as an active ingredient.
본 발명의 절패모 추출물 및 이의 분획물을 유효성분으로 이용 시, in vitro 상에서 염증성 사이토 카인인 인터루킨-4(IL-4), TARC, MDC의 분비를 억제하는 효과가 있으며 피부 보호 단백질인 필라그린(filaggrin), 인볼루크린(Involucrin), 아쿠아포린(aquaporin), 트랜스글루타미나아제(transglutaminase)의 발현을 유의하게 증가시키는 효과가 있다.When using the Jeolpaemo extract and its fractions of the present invention as an active ingredient, it has the effect of inhibiting the secretion of inflammatory cytokines interleukin-4 (IL-4), TARC, and MDC in vitro, and is effective in inhibiting the secretion of the inflammatory cytokines, filaggrin ( filaggrin), Involucrin, aquaporin, and transglutaminase.
또한, in vivo 상에서 조직 내 면역세포인 CD4 세포 및 CD8 세포의 침윤을 억제시키고 피부 두께 등을 유의하게 감소시키는 효과가 있다. In addition, there is an effect of inhibiting the invasion of immune cells, CD4 cells and CD8 cells in the tissue in vivo, and significantly reducing skin thickness.
따라서, 아토피 피부염을 예방, 치료 또는 개선할 수 있는 효능을 가지므로, 아토피 피부염을 예방, 치료 또는 개선하기 위한 의약품, 건강기능식품 또는 화장료 등의 다양한 방면에서 유용하게 사용될 수 있다.Therefore, since it has an effect of preventing, treating or improving atopic dermatitis, it can be usefully used in various fields, such as pharmaceuticals, health functional foods, or cosmetics for preventing, treating or improving atopic dermatitis.
도 1은 절패모 클로로포름 분획물의 분획 분리 방법을 나타낸 도이다.
도 2는 절패모 알코올 추출물과 절패모 클로로포름 분획물의 비만세포인 RBL2H3에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 세포 배양액 내 인터루킨-4(IL-4) 농도 감소 효과를 나타낸 도이다.
도 3은 절패모 알코올 추출물과 절패모 클로로포름 분획물의 비만세포인 RBL2H3에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 세포 배양액 내 베타-헥소아미다제(β-hexosaminidase)의 분비 억제 효과를 나타낸 도이다.
도 4는 절패모 알코올 추출물과 절패모 클로로포름 분획물의 복강 비만세포인 RPMC에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 히스타민(histamine)의 분비 억제 효과를 나타낸 도이다.
도 5는 절패모 알코올 추출물과 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 세포 배양액 내 TARC 농도 감소 효과를 나타낸 도이다.
도 6은 절패모 알코올 추출물과 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 세포 배양액 내 MDC 농도 감소 효과를 나타낸 도이다.
도 7은 절패모 알코올 추출물과 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 세포 배양액 내 IL-4 농도 감소 효과를 나타낸 도이다.
도 8은 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, MAPK 기전 분석 결과를 나타낸 도이다.
도 9는 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, NF-kB 기전 분석 결과를 나타낸 도이다.
도 10은 절패모 클로로포름 분획물의 인간 각질세포인 HaCaT에서 피부 장벽 조절 인자에 미치는 영향을 알아보기 위한 실험 결과를 나타낸 것으로, 보습 지표 분석 결과를 나타낸 도이다.
도 11은 절패모 알코올 추출물과 절패모 클로로포름 분획물의 아토피 동물 모델에서의 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 귀의 두께 평가, 행동평가 및 관능평가 결과를 나타낸 도이다.
도 12는 절패모 알코올 추출물과 절패모 클로로포름 분획물의 아토피 동물 모델에서의 항아토피 효과를 설명하기 위하여 실험한 결과를 나타낸 것으로, 표피의 피부 두께 평가, 피부조직내 비만세포의 수 및 CD4 세포와 CD8 세포의 조직 내 침윤을 분석한 결과를 나타낸 도이다. 1 is a diagram showing a method for separating a fraction of a chloroform fraction from Jeolpaemo.
FIG. 2 is a diagram showing the results of an experiment to explain the anti-atopic effect in RBL2H3, which is a mast cell of Jeolpaemo alcohol extract and Jeolpamo chloroform fraction, showing the effect of reducing the concentration of interleukin-4 (IL-4) in cell culture. .
Figure 3 shows the results of an experiment to explain the anti-atopic effect in the mast cells of RBL2H3, which is a mast cell of the alcohol extract and the chloroform fraction of Jeopaemo, and the effect of inhibiting the secretion of beta-hexosaminidase in cell culture. Is a diagram showing.
FIG. 4 is a diagram showing the results of an experiment to explain the anti-atopic effect in RPMC, which is a peritoneal mast cell of Jeolpaemo alcohol extract and Jeolpamo chloroform fraction, and is a diagram showing the secretion inhibitory effect of histamine.
5 is a diagram showing the experimental results to explain the anti-atopic effect in the human keratinocytes HaCaT of the alcohol extract and the chloroform fraction of Jeolpaemo, showing the effect of reducing the TARC concentration in the cell culture.
6 is a diagram showing the experimental results to explain the anti-atopic effect in human keratinocytes, HaCaT, of an alcohol extract from Jeolpamo and a chloroform fraction from Jeolpamo, and is a diagram showing the effect of reducing MDC concentration in a cell culture.
7 is a diagram showing the experimental results to explain the anti-atopic effect in the human keratinocytes HaCaT of the alcohol extract and the chloroform fraction of Jeolpaemo, showing the effect of reducing the concentration of IL-4 in the cell culture.
8 is a diagram showing the results of an experiment to explain the anti-atopic effect in HaCaT, which is human keratinocytes, of a chloroform fraction of Jeolpaemo, and is a diagram showing the results of MAPK mechanism analysis.
9 is a diagram showing the results of an experiment to explain the anti-atopic effect in HaCaT, which is a human keratinocyte, of a chloroform fraction of Jeolpaemo, and is a diagram showing the results of NF-kB mechanism analysis.
FIG. 10 is a diagram showing an experimental result for examining the effect of a chloroform fraction of Jeolpaemo chloroform on a skin barrier modulator in HaCaT, a human keratinocyte, and a view showing the result of analyzing a moisturizing index.
11 is a diagram showing the results of an experiment to explain the anti-atopic effect in an atopic animal model of an alcohol extract from Jeolpaemo and a chloroform fraction from Jeolpamo, and is a diagram showing the results of ear thickness evaluation, behavior evaluation, and sensory evaluation.
12 shows the results of an experiment to explain the anti-atopic effect in an atopic animal model of an alcohol extract and a chloroform fraction of Jeopaemo, and evaluation of the skin thickness of the epidermis, the number of mast cells in the skin tissue, and CD4 cells and CD8 It is a diagram showing the results of analyzing the infiltration of cells in the tissue.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.
본 발명은 절패모 추출물, 또는 이의 분획물을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an extract of Jeolpaemo, or a fraction thereof as an active ingredient.
본 발명에서 용어, "절패모(Fritillariae Thunbergii Bulbs)"는 우리나라 북부지방의 심산지역, 함경남도 갑산, 백두산의 산지초원에 자생하는 다년생초본이다. 주요성분으로는 페이민(베르티신), 페이미닌, 이소베르티신 등이 함유되어 있으며 그 외에도 티미딘, 아데노신 등이 포함되어 있다. 한방에서는 혈압강하, 거담, 진정 등에 약재로 사용된다.In the present invention, the term "jeolpaemo ( Fritillariae Thunbergii Bulbs )" is a perennial herb that grows naturally in the mountainous grasslands of Shimsan region, Gapsan, South Hamgyeong Province, and Mt. The main ingredients include Feymin (Berticin), Fayminin, Isoverticin, and other ingredients such as thymidine and adenosine. In oriental medicine, it is used as a medicinal for lowering blood pressure, geodam, and soothing.
본 발명에서 용어, "절패모 추출물"은 절패모를 적절한 침출액으로 짜내고 침출액을 증발시켜 농축한 제제를 총칭하는 것으로서, 상기 절패모 추출물은 절패모 분쇄물을 물, 유기용매, 또는 이들의 혼합용매를 이용하는 추출과정으로 획득할 수 있으며, 추출액, 이의 희석액 또는 농축액, 또는 추출액의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 포함할 수 있다. 또한 추출 방법에 있어서, 열탕 추출, 열수 추출, 냉침 추출, 온침 추출, 가압 추출, 환류 냉각 추출 또는 초음파 추출 등의 방법을 사용할 수 있으며, 이에 제한되지 않는다.In the present invention, the term "jeolpamo extract" is a generic term for a formulation obtained by squeezing Jeolpamo into an appropriate leach solution and evaporating the leachate to concentrate, wherein the Jeolpamo extract is a pulverized product of Jeolpamo in water, an organic solvent, or a mixed solvent thereof It can be obtained by an extraction process using the extract, a diluted solution or concentrate thereof, or a dry powder of the extract or any form formulated using the extract. In addition, in the extraction method, methods such as hot water extraction, hot water extraction, cold needle extraction, hot needle extraction, pressure extraction, reflux cooling extraction, or ultrasonic extraction may be used, but are not limited thereto.
절패모 추출물을 수득함에 있어, 바람직하게는 물, 유기용매 또는 이들의 혼합용매를 사용하여 추출할 수 있다. 유기용매를 사용하여 추출하는 경우, 메탄올, 에탄올, 이소프로판올, 부탄올, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, N, N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매인 유기용매를 사용할 수 있으며, 생약의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있다. 추출하는 유기용매에 따라 약제의 유효성분의 추출정도와 손실 정도가 차이가 날 수 있으므로, 적절한 유기용매를 선택하여 사용하도록 한다.In obtaining the Jeolpaemo extract, it can be extracted preferably using water, an organic solvent, or a mixed solvent thereof. When extracted using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or an organic solvent that is a mixed solvent thereof may be used, and the active ingredient of the herbal medicine may be extracted at room temperature or heated under conditions that are not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may be different, so select and use an appropriate organic solvent.
상기 용매 추출물은 부유하는 고체 입자를 제거하기 위하여 추출물을 여과시키는 단계를 추가로 포함할 수 있다. 면, 나일론 등을 이용하여 입자를 걸러내거나 한외여과, 냉동여과법, 원심분리법 등을 사용할 수 있다.The solvent extract may further include filtering the extract to remove suspended solid particles. The particles can be filtered using cotton or nylon, or ultrafiltration, cryofiltration, centrifugation, etc. can be used.
추출액의 농축에는 감압농축, 역삼투압 농축 등의 방법이 사용될 수 있다.For the concentration of the extract, a method such as vacuum concentration or reverse osmosis concentration may be used.
농축 후 건조 단계는 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 포말건조, 고주파건조, 또는 적외선건조 등을 포함한다. 경우에 따라, 최종 건조된 추출물을 분쇄하는 공정을 추가로 포함할 수 있다.The drying step after concentration includes freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, or infrared drying. In some cases, it may further include a process of grinding the final dried extract.
또한, 상기 추출물은 추가의 분획 공정을 수행할 수 있다. 예를 들어, 상기 추출물을 증류수에 현탁시켜 비극성 유기용매, 예를 들어, 헥산, 에테르, 디클로로메탄, 클로로포름, 에틸아세테이트 또는 이들의 혼합 용매로 비극성용매 가용층을 추출, 분리하여 수득하도록 하고, 이를 농축 및/또는 건조하여 사용할 수 있다.In addition, the extract may be subjected to an additional fractionation process. For example, the extract is suspended in distilled water to obtain a non-polar solvent soluble layer by extraction and separation with a non-polar organic solvent, such as hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. It can be used after concentration and/or drying.
본 발명에서 용어, "예방"은 본 발명의 조성물의 투여로 아토피 피부염의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, "치료"는 본 발명의 조성물에 의해 아토피 피부염에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to all actions of inhibiting or delaying the onset of atopic dermatitis by administration of the composition of the present invention, and "treatment" is improved or beneficial by the composition of the present invention. It means all actions that change.
본 발명에서 용어, "아토피 피부염"은 만성적이고 재발성의 염증성 피부질환으로 가려움증(가려움증)과 피부건조증, 특징적인 습진을 동반하는 질병을 의미한다. 아토피 피부염의 급성 병소에서는 혈청 면역글로불린 E(IgE)의 유의한 증가현상이 특징으로 나타나며, 이에 부가하여 병소의 조직병리학적인 변화 및 피부염 병변에 대한 관능평가 등을 행하여 아토피 피부염의 진단 및 심각도를 판정하기도 한다.In the present invention, the term "atopic dermatitis" is a chronic and recurrent inflammatory skin disease and refers to a disease accompanied by itching (itching), dry skin, and characteristic eczema. Atopic dermatitis acute lesions are characterized by a significant increase in serum immunoglobulin E (IgE).In addition, histopathological changes in the lesion and sensory evaluation for dermatitis lesions are performed to determine the diagnosis and severity of atopic dermatitis. Sometimes it does.
아토피 피부염의 정확한 원인은 아직까지 완전히 이해되고 있지 않지만 유전적 소인과 함께 면역학적, 비면역학적 기전이 관여한다고 보고 있다.The exact cause of atopic dermatitis is still not fully understood, but it is believed that immunological and non-immunological mechanisms are involved along with genetic predisposition.
아토피 피부염은 발병 대상의 연령에 따라, 영·유아 아토피(0~3세), 유·소아 아토피(3세~12세), 성인 아토피(13세~50세)으로 구분될 수 있다. 구체적으로 영·유아 아토피는 습진형으로 발병하는 것이 일반적이며, 자극성 피부염이 반복적으로 나타나거나, 계란, 우유, 대두 등의 음식에 의해 발병하는 것이 특징이다. 유·소아 아토피는 영·유아 아토피 피부염보다는 건조한 것이 특징이고, 가려움증이 심해 계속적으로 피부를 긁게 되어 딱딱하게 되며 색소 침착이 일어날 수 있다. 환절기에 증상이 더욱 악화되는 경향이 있으며, 주로 주변 환경의 먼지, 진드기, 곰팡이균, 섬유, 쓰레기, 꽃가루 등이 피부 또는 호흡기관을 통해 체내로 들어오는 것에 기인한다. 마지막으로, 성인 아토피의 경우 피부가 건조하고 거친 증상이 악화되는 것이 특징이며, 흔히 천식 또는 알러지성 비염을 동반하기도 한다. 주로 생활방식, 환경, 스트레스가 주요 악화요인이다. 본 발명에서의 아토피 피부염은 바람직하게는 성인 아토피 피부염일 수 있다.Atopic dermatitis can be classified into infant/infant atopy (0-3 years old), infant/child atopy (3-12 years old), and adult atopy (13-50 years old), depending on the age of the target. Specifically, it is common for infants and toddlers to develop eczema-type, and is characterized by repeated irritation dermatitis or by foods such as eggs, milk, and soybeans. Infant/child atopy is characterized by dryness rather than infant/infant atopic dermatitis, and itching is severe, causing continuous scratching of the skin to become hard and pigmentation may occur. Symptoms tend to worsen during the change of seasons, and it is mainly caused by dust, mites, fungi, fibers, garbage, and pollen from the surrounding environment entering the body through the skin or respiratory tract. Finally, in the case of adult atopy, dry and rough skin is characterized by worsening symptoms, and is often accompanied by asthma or allergic rhinitis. Mainly, lifestyle, environment, and stress are major deteriorating factors. Atopic dermatitis in the present invention may be preferably adult atopic dermatitis.
또한 아토피 피부염은 증상에 따라, 열성아토피, 만성아토피, 건성아토피, 스트레스성 아토피로 구분될 수 있다. 구체적으로 열성아토피는 주로 세포 기능 이상으로 인한 열과 독소의 과잉에 의해 발병하며, 이에 따라 심폐기능 저하, 해독기능 및 면역이 불안정하게 된다. 이것이 피부 열사화로 이어져 아토피 피부염이 발생하게 되며, 특히 열이 과잉 발생할 수 있는 상황이나 환경에서 악화될 수 있다. 만성아토피는 5~10년 이상의 기간 동안 아토피 피부염이 호전악화를 반복하며 지속된 경우를 말하며, 피부 변형 정도가 심하고 피부가 태선화되어 있거나 반복적인 피부염으로 피부착색이 될 수 있다. 건성아토피는 피부가 매우 건조하고 마른 각질이 일어나는 경우가 많은 아토피 피부염으로서, 피부 건조로 인한 가려움증으로 고통을 겪으며, 환절기, 겨울 등 습도가 낮은 계절에 특히 악화된다. 마지막으로 스트레스성 아토피는 스트레스 정도나 심리 상태에 따라 피부염의 증상이 악화 혹은 호전되는 아토피 피부염으로서, 심리적 원인에 의해 아토피 증성이 갑자기 악화되며 병변부위가 붉어지고 가려움증이나 진물이 심해질 수 있다. In addition, atopic dermatitis can be classified into febrile atopy, chronic atopy, dry atopy, and stress atopy according to symptoms. Specifically, febrile atopy is mainly caused by fever and an excess of toxins due to abnormal cellular functions, and thus cardiopulmonary function decreases, detoxification function and immunity become unstable. This leads to skin heat death, leading to atopic dermatitis, which can be exacerbated especially in situations or environments where excessive heat may occur. Chronic atopy refers to a case in which atopic dermatitis continues to improve and worsen for a period of 5 to 10 years or more, and the degree of skin deformation is severe and the skin is lichenified, or skin pigmentation may occur due to repetitive dermatitis. Dry atopy is an atopic dermatitis in which the skin is very dry and dry dead skin cells often occur. It suffers from itchiness caused by dry skin, and is particularly deteriorated in low humidity seasons such as the change of seasons and winter. Lastly, stress-related atopy is atopic dermatitis in which symptoms of dermatitis worsen or improve depending on the level of stress or psychological state, and atopic sensation suddenly worsens due to psychological causes, and the lesion area may become red and itching or soreness may become severe.
본 발명에서 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propyleneglycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In the present invention, the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, and suppositories. It may have any one dosage form, and may be various oral or parenteral dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 아토피 피부염 의심 개체에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 구체적으로, 국소도포 등을 통한 경피투여 방식으로 투여될 수 있으나 이에 제한되지 않는다.In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention to an individual suspected of atopic dermatitis by any suitable method, and the route of administration is oral or parenteral through various routes as long as it can reach the target tissue. Can be administered. Specifically, it may be administered by transdermal administration through topical application, but is not limited thereto.
본 발명의 치료 방법은 절패모 추출물을 포함하는 약학적 조성물을 약학적으로 유효한 양으로 투여하는 것을 포함할 수 있다.The treatment method of the present invention may include administering a pharmaceutical composition containing the extract of Jeolpaemo in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and disease. It can be determined by the type, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.
본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르며, 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다.The preferred dosage of the composition of the present invention depends on the condition and weight of the patient, the degree of the disease, the form of the drug, the route and duration of administration, and the total amount of daily dosage can be determined by the treating physician within the correct medical judgment range.
또 하나의 양태로서, 본 발명은 절패모 추출물을 유효성분으로 함유하는, 아토피 피부염의 예방 또는 개선용 건강기능식품 조성물을 제공한다.As another aspect, the present invention provides a health functional food composition for the prevention or improvement of atopic dermatitis, containing a jeolpaemo extract as an active ingredient.
구체적으로, 본 발명의 절패모 추출물을 아토피 피부염의 예방 또는 개선을 목적으로 건강기능식품 조성물에 포함시킬 수 있다.Specifically, the Jeolpaemo extract of the present invention may be included in a health functional food composition for the purpose of preventing or improving atopic dermatitis.
상기 절패모 추출물 및 아토피 피부염에 대해서는 상기에서 설명한 바와 같다.The Jeolpamo extract and atopic dermatitis are as described above.
본 발명에서 용어, "개선"은 상기 조성물을 이용하여 아토피 피부염의 의심 및 발병 개체의 증상이 호전되거나 이롭게 되는 모든 행위를 말한다.In the present invention, the term "improvement" refers to any action that improves or benefits the symptoms of an individual with suspicion of atopic dermatitis using the composition.
본 발명의 조성물을 건강기능식품에 포함하여 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 건강기능식품 또는 건강기능식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정될 수 있다. When the composition of the present invention is included in a health functional food, the composition may be added as it is or used with other health functional food or health functional food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.
본 발명의 조성물을 포함할 수 있는 건강기능식품의 종류에는 특별한 제한은 없으며, 구체적인 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있고, 통상적인 의미에서의 건강기능식품을 모두 포함할 수 있으며, 동물을 위한 사료로 이용되는 식품을 포함할 수 있다.There is no particular limitation on the types of health functional foods that may contain the composition of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream. There are dairy products, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in a conventional sense, and foods used as feed for animals.
본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.The health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , Carbonated beverages used in carbonated beverages, and the like. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.
또 하나의 양태로서, 본 발명은 절패모 추출물을 유효성분으로 함유하는, 아토피 피부염의 예방 또는 개선용 의약외품 조성물을 제공한다.As another aspect, the present invention provides a quasi-drug composition for preventing or improving atopic dermatitis, containing an extract of Jeolpaemo as an active ingredient.
구체적으로, 본 발명의 절패모 추출물을 아토피 피부염의 예방 또는 개선을 목적으로 의약외품 조성물에 포함시킬 수 있다.Specifically, the Jeolpaemo extract of the present invention may be included in a quasi-drug composition for the purpose of preventing or improving atopic dermatitis.
상기 절패모 추출물 및 아토피 피부염에 대해서는 상기에서 설명한 바와 같다.The Jeolpamo extract and atopic dermatitis are as described above.
본 발명에서 용어, "의약외품"은 사람이나 동물의 질병을 치료, 경감, 처치 또는 예방할 목적으로 사용되는 섬유, 고무제품 또는 이와 유사한 것, 인체에 대한 작용이 약하거나 인체에 직접 작용하지 않으며, 기구 또는 기계가 아닌 것과 이와 유사한 것, 감염 예방을 위하여 살균, 살충 및 이와 유사한 용도로 사용되는 제제 중 하나에 해당하는 물품으로서, 사람이나 동물의 질병을 진단, 치료, 경감, 처치 또는 예방할 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것 및 사람이나 동물의 구조와 기능에 약리학적 영향을 줄 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것을 제외한 물품을 총칭하며, 여기에는 피부 외용제 및 개인위생용품도 포함한다.In the present invention, the term "quasi-drug" refers to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, weakly acting on the human body or not directly acting on the human body, and Or non-machine or similar, as an item that is one of the preparations used for sterilization, insecticide and similar purposes to prevent infection, and used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. A generic term refers to items other than instruments, machines, or devices among the items that are not apparatus, machines, or devices, and those that are not instruments, machines, or devices among items used for the purpose of pharmacologically affecting the structure and function of humans or animals. It also includes personal hygiene products.
본 발명의 조성물을 아토피 피부염의 예방 또는 개선을 목적으로 의약외품에 포함시킬 경우, 상기 조성물을 그대로 포함하여 사용하거나 다른 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합량은 사용 목적에 따라 적합하게 결정할 수 있다.When the composition of the present invention is included in a quasi-drug for the purpose of preventing or improving atopic dermatitis, the composition may be used as it is or may be used together with other quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the intended use.
상기 피부외용제는 특별히 이에 제한되지 않으나, 예를 들어 연고제, 로션제, 스프레이제, 패치제, 크림제, 산제, 현탁제 또는 젤제의 형태로 제조되어 사용될 수 있다.The external preparation for skin is not particularly limited thereto, but may be prepared and used in the form of, for example, an ointment, lotion, spray, patch, cream, powder, suspension or gel.
또 하나의 양태로서, 본 발명은 절패모 추출물을 유효성분으로 함유하는, 아토피 피부염의 예방 또는 개선용 화장료 조성물을 제공한다.As another aspect, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, containing the extract of Jeolpaemo as an active ingredient.
구체적으로, 본 발명의 절패모 추출물을 아토피 피부염의 예방 또는 개선을 목적으로 화장료 조성물에 포함시킬 수 있다.Specifically, it may be included in the cosmetic composition for the purpose of preventing or improving atopic dermatitis the Jeolpaemo extract of the present invention.
상기 절패모 추출물 및 아토피 피부염에 대해서는 상기에서 설명한 바와 같다.The Jeolpamo extract and atopic dermatitis are as described above.
본 발명의 화장료 조성물은 상기 유효성분 이외에 통상적으로 허용되는 성분들을 제한 없이 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition of the present invention may include, without limitation, ingredients that are generally permitted in addition to the active ingredients, and may include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. have.
본 발명에 따른 화장료 조성물은 용액, 외용연고, 크림, 폼, 영양화장수, 유연화장수, 팩, 유연수, 유액, 메이크업베이스, 에센스, 비누, 액체 세정료, 입욕제, 선 스크린크림, 선오일, 현탁액, 유탁액,페이스트, 겔, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패치 및 스프레이 등의 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nutrient lotion, soft lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleaning agent, bathing agent, sunscreen cream, sun oil, suspension, Emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, can be prepared in a formulation such as a patch and spray, but is not limited thereto.
또한, 본 발명의 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한담체를 1 종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면활성제, 보습제, 저급 알콜, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 제형에 따라 다양하다.In addition, the cosmetic composition of the present invention may additionally contain one or more cosmetically acceptable carriers that are blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, moisturizer, lower alcohol, A thickener, a chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately mixed, but the present invention is not limited thereto. The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation.
본 발명의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화아연 또는 이들의 혼합물이 이용될 수 있다.When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.
본 발명의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카,알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 또는 이들의 혼합물이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component. In particular, in the case of a spray, additional chloro Propellants such as fluorohydrocarbon, propane/butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알콜, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일이 이용될 수 있으며, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브 오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil may be used, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan may be used. have.
본 발명의 제형이 현탁액인 경우에는, 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알콜, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
본 발명의 제형이 비누인 경우에는 담체 성분으로서 지방산의 알칼리 금속 염, 지방산 헤미에스테르 염, 지방산 단백질 히드롤리제이트, 이세티오네이트, 라놀린 유도체, 지방족 알콜, 식물성 유, 글리세롤, 당 등이 이용될 수 있다.When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. may be used as a carrier component. I can.
본 발명의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시테이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 오일, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcositate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
실시예 1: 절패모 추출물의 제조 및 생체 외 세포주 모델 제작Example 1: Preparation of Jeolpaemo extract and preparation of in vitro cell line model
실시예 1-1: 절패모 추출물의 제조Example 1-1: Preparation of Jeolpaemo extract
절패모 에탄올 추출물의 경우, 에탄올 80%에 침지하여 121℃에서 15분간 추출하여 제조하였다. For the ethanol extract of Jeolpaemo, it was prepared by immersing in 80% ethanol and extracting at 121°C for 15 minutes.
절패모 클로로포름 분획물의 경우, 절패모 건조약재 800g을 80% 메탄올 3L에 침지하여 1주일간 추출하여 얻어진 추출물을 여과(Whatman, No2)하여 여액을 회수한 다음 회전증발농축기를 이용하여 농축하여 약 500 ㎖까지 농축한다. 농축물은 헥산(Hexane), 클로로포름(Chloroform), 에틸아세테이트(Ethylacetate)를 이용하여 도 1과 같이 용매의 극성도에 따라 순차적으로 분획한다. 각 용매별 분획은 수용액층(농축액) : 각 용매별 = 2:1 비율, 즉, 분액깔때기에 수층 500 ㎖에 헥산 250 ㎖를 가하고 충분히 섞어준 후 수층과 헥산층을 분리하고, 수층에 다시 헥산 250 ㎖를 가한 다음 동일한 방법으로 총 3회 실시하여 각 층을 분리하였다. 헥산을 분리한 뒤 헥산 분리와 동일한 방법으로 수용액층에 순차적으로 클로로포름, 에칠아세테이트를 가하여 각 층을 분리, 회수한 다음 농축, 건조하여 활성 측정을 위한 시료로 사용하였다. In the case of Jeolpaemo chloroform fraction, 800g of Jeolpaemo dried medicinal material was immersed in 3L of 80% methanol and extracted for 1 week. The extract obtained was filtered (Whatman, No2) to collect the filtrate, and then concentrated using a rotary evaporator to about 500 ml. To concentrate. The concentrate is sequentially fractionated according to the polarity of the solvent as shown in FIG. 1 using hexane, chloroform, and ethylacetate. For each solvent fraction, the aqueous layer (concentrate): for each solvent = 2:1 ratio, i.e., 250 ml of hexane was added to 500 ml of the aqueous layer in the separating funnel and sufficiently mixed, the aqueous layer and the hexane layer were separated, and the aqueous layer was re-hexane with hexane. After adding 250 ml, each layer was separated by performing the same method three times. After separating hexane, chloroform and ethyl acetate were sequentially added to the aqueous solution layer in the same manner as for hexane separation, and each layer was separated, recovered, concentrated, dried, and used as a sample for activity measurement.
상기 절패모 에탄올 추출물을 FTB, 절패모 클로로포름 분획물은 CL로 나타내었다. The ethanol extract of Jeopaemo was denoted by FTB, and the chloroform fraction of Jeopaemo was expressed by CL.
실시예 1-2: 생체 외 세포주 모델 제작Example 1-2: In vitro cell line model construction
아토피 피부염을 표방하는 생체 외(in vitro) 세포주(cell line) 모델을 구축하기 위하여, 인간 각질세포인 HaCaT 세포와 비만세포인 RBL2H3 세포를 10% FBS와 1% PS가 포함된 DMEM 배지를 사용하여 5% CO2, 37℃ 조건에서 배양하였다. 세포는 3일 간격으로 세포의 밀집도가 80~90%에 도달했을 때 trypsin을 이용하여 계대 배양하여 세포주 모델을 구축하였다. To construct an in vitro cell line model for atopic dermatitis, human keratinocytes, HaCaT cells and mast cells, RBL2H3 cells were used in DMEM medium containing 10% FBS and 1% PS. Incubated under conditions of 5% CO 2 and 37°C. Cells were subcultured using trypsin when the cell density reached 80-90% at 3 day intervals to construct a cell line model.
RPMC(Rat Peritoneal Mast Cells)는 300g의 SD-rat을 에틸에테르로 마취 한 후 HEPES-tyrode 버퍼를 복강에 주입한 후 90초간 복강을 마사지한 후 스포이드로 복강 내 버퍼를 채취한 후 800rpm, 4℃에서 10분 동안 원심분리한 후 상층액은 버리고, 침전된 세포를 1ml의 HEPES-tyrode로 재부유 시켰다. 그 후 15ml 튜브에 45% percoll 3.5ml 을 넣은 후 재부유 시킨 1ml의 세포를 흘려 넣은 후 190g, 4℃에서 10분간 원심분리하여 상층액을 버리고 남은 세포를 HEPES-tyrode로 3회 수세 하여 트리판 블루(Trypan blue)로 염색하여 비만세포가 90%이상일 경우 실험에 사용하는 방법으로 세포주 모델을 구축하였다. RPMC (Rat Peritoneal Mast Cells) anesthetizes 300g of SD-rat with ethyl ether, injects HEPES-tyrode buffer into the abdominal cavity, massages the abdominal cavity for 90 seconds, collects the intraperitoneal buffer with a dropper, and then collects the intraperitoneal buffer at 800 rpm, 4℃. After centrifugation at for 10 minutes, the supernatant was discarded, and the precipitated cells were resuspended with 1 ml of HEPES-tyrode. After that, put 3.5ml of 45% percoll in a 15ml tube, pour 1ml of resuspended cells, centrifuge at 190g, 4℃ for 10 minutes, discard the supernatant, and wash the remaining cells with HEPES-
실시예 2: 비만세포인 RBL2H3에서의 절패모 추출물에 의한 인터루킨(IL-4), 베타-헥소아미다제(β-Hexosaminidase) 분비 억제 효과 확인Example 2: Confirmation of the effect of inhibiting the secretion of interleukin (IL-4) and beta-hexoamidase (β-Hexosaminidase) by the extract of scabbard hair in mast cells RBL2H3
아토피 피부염은 염증반응을 일으키는 인터루킨-4(IL-4)가 증가되고, 알레르기 주요인자인 베타-헥사아미다제(β-Hexosaminidase)가 증가되는바, 본 발명의 FTB, CL 처리에 따른 이의 분비억제 효과를 확인하였다. In atopic dermatitis, interleukin-4 (IL-4) causing an inflammatory reaction is increased, and beta-hexaamidase, which is a major allergy factor, is increased, and its secretion is suppressed according to FTB and CL treatment of the present invention. The effect was confirmed.
RBL2H3 세포를 24-well plate에 2 x 105cells/well에 분주한 뒤 24시간 동안 배양하였다. 상층액을 제거한 뒤 piperazine-N,N-bis(2-ethanesulfonic acid) PIPES buffer로 수세한 뒤 PIPES buffer로 FTB 및 CL을 농도별로 전처리 하였다. 1시간 후 A23187 10μM로 자극한 후 30분 동안 반응시켰다. 배지를 걷어내어 2,000 rpm에 5분간 원심분리한 후 상층액을 96- well plate에 옮긴 후 1 mM p-NAG 50ml를 추가하여 37℃에서 1시간 동안 반응시켰다. 1시간 후 carbonate stop buffer를 이용하여 반응을 종료시킨 후 405nm에서 흡광도를 측정하였다.RBL2H3 cells were dispensed into a 24-well plate at 2 x 10 5 cells/well and cultured for 24 hours. After removing the supernatant, it was washed with piperazine-N,N-bis (2-ethanesulfonic acid) PIPES buffer, and then FTB and CL were pretreated by concentration with PIPES buffer. After 1 hour, A23187 was stimulated with 10 μM and reacted for 30 minutes. After removing the medium and centrifuging at 2,000 rpm for 5 minutes, the supernatant was transferred to a 96-well plate, and 50 ml of 1 mM p-NAG was added and reacted at 37° C. for 1 hour. After 1 hour, the reaction was terminated using a carbonate stop buffer, and the absorbance was measured at 405 nm.
RBL2H3 세포에서 염증성 사이토카인인 IL-4에 대한 절패모 추출물의 효과를 분석하기 위해 6-well plate에 1×106 cells/well을 분주한 후 24시간 동안 부착시키고 FTB 및 CL을 농도별로 처리하였다. 1시간 후 A23187 10μM로 자극한 후 30분후 배지를 4,000 rpm, 4℃에서 원심분리하여, 세포 배양액에 존재하는 IL-4의 농도를 ELISA kit를 이용하여 측정하였다. In order to analyze the effect of Jeolpamo extract on IL-4, an inflammatory cytokine in RBL2H3 cells, 1×10 6 cells/well was dispensed into a 6-well plate, and then adhered for 24 hours, and FTB and CL were treated by concentration. . After 1 hour, after stimulation with 10 μM of A23187, the medium was centrifuged at 4,000 rpm and 4° C. 30 minutes later, and the concentration of IL-4 present in the cell culture was measured using an ELISA kit.
그 결과, 도 2에 나타낸 바와 같이, A23187을 처리한 세포의 세포 배양액 내 인터루킨-4(IL-4)의 생성이 유의하게 증가하였으며, FTB 처리군에서는 인터루킨-4(IL-4)의 분비에 차이를 보이지 않았으나, CL 25 μg/ml과 50 μg/ml의 처리한 군 인터루킨-4(IL-4) 분비는 Con군에 비해 유의하게 감소하였다.As a result, as shown in Fig. 2, the production of interleukin-4 (IL-4) in the cell culture medium of the cells treated with A23187 was significantly increased, and the secretion of interleukin-4 (IL-4) in the FTB-treated group was significantly increased. Although there was no difference, the secretion of interleukin-4 (IL-4) in the group treated with
또한, 도 3에 나타낸 바와 같이, A23187을 처리한 Con군이 Nor군에 비해 베타-헥소아미다제(β-hexosaminidase)가 유의하게 증가하였으며, FTB 400 μg/ml 처리한 군이 Con군에 비해 베타-헥소아미다제(β-hexosaminidase)의 분비가 유의하게 억제 되었다. 또한 CL처리군은 25 μg/ml과 50 μg/ml의 농도에서 베타-헥소아미다제(β-hexosaminidase)의 분비를 유의하게 억제함을 확인하였다. In addition, as shown in Fig. 3, the A23187-treated Con group significantly increased beta-hexosaminidase compared to the Nor group, and the
실시예 3: 복강비만세포 패모 추출물에 의한 histamine 분비 억제 효과 확인Example 3: Confirmation of the effect of inhibiting histamine secretion by extract of peritoneal mast cells
아토피 피부염은 알레르기 주요인자인 히스타민의 분비가 증가되는 바, 본 발명의 FTB, CL처리에 따른 이의 분비억제효과를 확인하였다. In atopic dermatitis, the secretion of histamine, which is a major allergy factor, is increased, and its secretion inhibitory effect according to the FTB and CL treatment of the present invention was confirmed.
분리한 RPMC를 1 x 106cells/ml로 부유한 후, 200μl를 취하여 1.5ml 튜브에 옮긴 후, FTB 및 CL을 농도별로 처리한 후 30℃에서 10분간 배양하였다. 10 분후 자극제로서 compound 48/80 0.5μg/ml을 처리 한 후 37℃에서 20분간 반응시켰다. 반응이 종료된 튜브를 3,000rpm, 4℃, 15분간 원심분리한 후 상층액을 히스타민(histamine) 정량에 사용하였다. 히스타민(histamine)정량은 o-phthalaldehyde spectrofluorometric procedure를 이용하여 측정하였다.After the isolated RPMC was suspended at 1 x 10 6 cells/ml, 200 μl was taken and transferred to a 1.5 ml tube, and FTB and CL were treated by concentration, followed by incubation at 30° C. for 10 minutes. After 10 minutes, 0.5 μg/ml of compound 48/80 was treated as an irritant and reacted at 37° C. for 20 minutes. After the reaction was completed, the tube was centrifuged for 15 minutes at 3,000 rpm, 4° C., and the supernatant was used for histamine quantification. Histamine was measured using an o-phthalaldehyde spectrofluorometric procedure.
그 결과, 도 4에 나타낸 바와 같이, Nor군에 비해 자극제만 처리한 Con군의 히스타민(histamine)분비량이 유의하게 증가하였으며, FTB 400μg/ml 처리군의 히스타민(histamine)분비량이 유의하게 감소하였다. 또한 CL 25 μg/ml과 50 μg/ml처리한 군의 히스타민(histamine) 분비량은 Con군에 비해 유의하게 감소함을 확인하였다. As a result, as shown in FIG. 4, the histamine secretion of the Con group treated with only the stimulant was significantly increased compared to the Nor group, and the histamine secretion of the
실시예 4: 인간 각질세포 HaCaT 세포에서의 절패모 추출물에 의한 항아토피 효과 확인 Example 4: Confirmation of anti-atopic effect by extract of scabbard hair in human keratinocyte HaCaT cells
실시예 4-1: 염증성 사이토카인의 분비 억제 확인Example 4-1: Confirmation of inhibition of secretion of inflammatory cytokines
HaCaT 1x105 cell을 6well에 분주 한 후 24시간 동안 안정화함. 24시간 후 약물을 농도별로 처리한 후 1시간 배양한 후, TNF-α와 IFN-γ를 10ng/ml로 처리한 후 24시간 반응시켰다. 24시간 후 media를 수확하여 원심분리 한 후 ELISA kit를 이용하여 TARC를 정량하였다.After dispensing HaCaT 1x10 5 cells into 6 wells, stabilized for 24 hours. After 24 hours, the drug was treated for each concentration and incubated for 1 hour, and then TNF-α and IFN-γ were treated at 10 ng/ml and then reacted for 24 hours. After 24 hours, the media was harvested, centrifuged, and TARC was quantified using an ELISA kit.
HaCaT 세포에서 FTB, CL의 효과를 분석하기 위해 6-well plate에 1x106/well을 분주한 후 24시간 동안 부착시킨 후, 약물을 농도별로 처리하였다. 1시간 후 TNF-α/IFN-γ를 10 ng/ml를 처리한 후 24시간 동안 자극하였다. 24시간 후 배지를 4,000 rpm, 4°C 에서 원심분리하여, 세포 배양액에 존재하는 TARC, MDC와 IL-γ의 농도를 ELISA kit를 이용하여 측정하였다. 실험 방법은 제조사의 매뉴얼에 따라 진행하였다.In order to analyze the effects of FTB and CL in HaCaT cells, 1×10 6 /well was dispensed on a 6-well plate and attached for 24 hours, and then the drugs were treated by concentration. After 1 hour, TNF-α/IFN-γ was treated with 10 ng/ml and then stimulated for 24 hours. After 24 hours, the medium was centrifuged at 4,000 rpm and 4 °C, and the concentrations of TARC, MDC, and IL-γ in the cell culture were measured using an ELISA kit. The experimental method was carried out according to the manufacturer's manual.
그 결과, TARC농도의 경우, 도 5에 나타낸 바와 같이, HaCaT에 TNF-α와 IFN-γ를 처리한 Con군이 Nor군에 비해 TARC가 유의하게 증가하였으며, FTB 200μg/ml, 400μg/ml를 처리한 군은 TARC의 분비를 유의하게 억제하였으며, CL 25μg/ml, 50μg/ml를 처리한 군도 Con에 비해 TARC의 분비가 유의하게 억제되었다. CL의 처리는 FTB보다 더 낮은 농도에서 히스타민(histamine)의 분비를 억제하는 것을 확인하였다.As a result, in the case of TARC concentration, as shown in FIG. 5, the Con group treated with TNF-α and IFN-γ in HaCaT significantly increased TARC compared to the Nor group, and
또한, MDC농도의 경우, 도 6에 나타낸 바와 같이, Nor군에 비해 Con군의 MDC의 분비가 유의하게 증가하였으며, FTB 400μg/ml을 처리한 군의 MDC분비가 유의하게 감소하였으며, CL 50μg/ml을 처리한 군의 MDC분비가 Con군에 비해 유의하게 감소하였다.In addition, in the case of the MDC concentration, as shown in FIG. 6, the secretion of MDC in the Con group was significantly increased compared to the Nor group, the MDC secretion in the group treated with
인터루킨-4(IL-4) 농도의 경우, 도 7에 나타낸 바와 같이, Nor군에 비해 Con군의 인터루킨-4(IL-4)의 분비가 유의하게 증가하였으며, FTB를 처리한 군의 인터루킨-4(IL-4)의 분비양은 Con군과 차이를 보이지 않았으며, CL 50μg/ml을 처리한 군의 인터루킨-4(IL-4)의 분비가 Con에 비해 유의하게 감소하였다.In the case of interleukin-4 (IL-4) concentration, as shown in FIG. 7, the secretion of interleukin-4 (IL-4) in the Con group was significantly increased compared to the Nor group, and interleukin in the FTB-treated group- The amount of secretion of 4(IL-4) was not different from that of the Con group, and the secretion of interleukin-4 (IL-4) in the group treated with
실시예 4-2: MAPK 기전 분석Example 4-2: MAPK mechanism analysis
HaCaT 1x105 cell을 6well에 분주한 후 24시간동안 안정화 하였다. 24시간 후 약물을 농도별로 처리한 후 1시간 배양한 후, TNF-α와 IFN-γ를 10ng/ml로 처리하여 자극하였다. 자극 5분, 15분, 30분에 cell을 RIPA buffer를 이용하여 lysis한 후 30 ug의 단백질을 10% SDS-polyacrylamide gel에서 분리한 후 transfer kit를 이용하여 단백질을 nictrocellulose membrane로 이동시킨 후, 각각에 해당하는 Ab를 이용하여 발현 양을 측정하였다. HaCaT 1x10 5 cells were dispensed into 6 wells and stabilized for 24 hours. After 24 hours, the drug was treated by concentration and incubated for 1 hour, followed by stimulation with TNF-α and IFN-γ at 10 ng/ml. After lysis of the cells using RIPA buffer at 5, 15, and 30 minutes of stimulation, 30 ug of protein was separated from 10% SDS-polyacrylamide gel, and then the protein was transferred to the nictrocellulose membrane using a transfer kit. The amount of expression was measured using the Ab corresponding to.
그 결과, 도 8에 나타낸 바와 같이, HaCaT 세포에서의 p-ERK군의 발현은 Nor과 Con의 차이는 보이지 않으며, CL을 처리한 군에서의 차이를 보이지 않았다. P-P38은 Con군 5분에서만 Nor과 Con 발현 차이가 났으며, CL추출물은 5분에서 억제 효과를 보였다.As a result, as shown in Fig. 8, the expression of the p-ERK group in HaCaT cells did not show a difference between Nor and Con, and did not show a difference in the group treated with CL. P-P38 showed a difference in expression of Nor and Con only at 5 minutes in the Con group, and CL extract showed inhibitory effect at 5 minutes.
실시예 4-3: NF-kB 기전 분석Example 4-3: NF-kB mechanism analysis
HaCaT 1x105 cell을 6well에 분주 한 후 24시간동안 안정화하고 24시간 후 약물을 농도별로 처리한 후 1시간 배양한 후, TNF-α와 IFN-γ를 10 ng/ml로 처리하여 자극하였다. 자극을 5분, 15분, 30분으로 주었으며 cell을 lysis한 후 50 ug의 단백질을 10% SDS-polyacrylamide gel에서 분리한 후 transfer kit를 이용하여 단백질을 nictrocellulose membrane로 이동시킨 후, 각각에 해당하는 Ab를 이용하여 발현양을 측정하였다. HaCaT 1x10 5 cells were dispensed into 6 wells, stabilized for 24 hours, and after 24 hours, the drug was treated by concentration and incubated for 1 hour, and then TNF-α and IFN-γ were treated with 10 ng/ml to stimulate. Stimulation was given for 5 minutes, 15 minutes, and 30 minutes. After cell lysis, 50 ug of protein was separated from 10% SDS-polyacrylamide gel, and the protein was transferred to the nictrocellulose membrane using a transfer kit. The expression level was measured using Ab.
그 결과, 도 9에 나타낸 바와 같이, P-IkB는 Con군 5, 30분에서만 Nor과 Con 발현 차이가 났으며, 약물을 처리한 군에서는 억제 효과를 보이지 않았다. P-NF-kb는 Nor군에 비해 Con군의 발현이 증가하였으며, CL은 15분에서 억제 효과를 보였다.As a result, as shown in FIG. 9, P-IkB showed a difference in expression of Nor and Con only at 5 and 30 minutes in the Con group, and did not show an inhibitory effect in the drug-treated group. P-NF-kb increased the expression of Con group compared to Nor group, and CL showed inhibitory effect at 15 minutes.
실시예 4-4: 피부 보습인자 분석 Example 4-4: Analysis of skin moisturizing factors
피부 장벽 보호 단백질인 필라그린(filaggrin), 인볼루크린(Involucrin), 아쿠아포린(aquaporin), 트랜스글루타미나아제(transglutaminase)에 대하여 FTB, CL이 미치는 영향을 분석하기 위해서, HaCaT 세포를 6-well plate에 1x106cells/well을 분주하고 24시간 후 절패모 추출물을 농도별로 처리하였다. 24시간 후 cell을 RNA iso plus를 사용하여 RNA를 추출하고, SuperScript II Reverse transcription kit를 이용하여 cDNA를 합성하였다. PCR은 Kapa Taq PCR kit를 이용하여 제조사 매뉴얼에 따라 수행하였다. PCR은 95℃에서 10분 동안 초기 변성하였으며, 95℃ 염기열은 표 1에 나타내었다.To analyze the effects of FTB and CL on filaggrin, Involucrin, aquaporin, and transglutaminase, which are skin barrier protective proteins, HaCaT cells were 6- 1x10 6 cells/well were dispensed into a well plate, and after 24 hours, the extract of Jeolpaemo was treated by concentration. After 24 hours, RNA was extracted from the cell using RNA iso plus, and cDNA was synthesized using SuperScript II Reverse transcription kit. PCR was performed according to the manufacturer's manual using a Kapa Taq PCR kit. PCR was initially denatured at 95°C for 10 minutes, and the base sequence at 95°C is shown in Table 1.
R 5’-CTGGACACTCAGGTTCCCAT-3’F 5'- TTTCGTGTTTGTCTGCTTGC- 3'
R 5'-CTGGACACTCAGGTTCCCAT-3'
R 5′-AGCTGCTGATCCCTTTGTGTF 5′-TGCCTGAGCAAGAATGTGAG
R 5′-
R 5'-GGGACCCTCATCCTGGTG-3'F 5'-GGTTGATGGTGAGGAAACCA-3'
R 5'-GGGACCCTCATCCTGGTG-3'
R 5′-TCCACTTCCTTCTTGGTCTCCTTF 5′-TCTGATGGTCTCTGTGATGCTGAT
R 5′-TCCACTTCCTTCTTGGTCTCCTT
그 결과, 도 10에 나타낸 바와 같이, 각 피부 장벽 단백질의 mRNA 발현을 확인한 결과, FTB 처리군은 아무것도 처리하지 않은 정상군에 비해 filaggrin, Involucrin, aquaporin의 발현을 유의하게 증가시켰으며, CL을 처리한 군은 filaggrin, Involucrin, aquaporin, transglutaminase의 발현을 유의하게 증가시켰다. As a result, as shown in FIG. 10, as a result of confirming the mRNA expression of each skin barrier protein, the FTB-treated group significantly increased the expression of filaggrin, Involucrin, and aquaporin compared to the normal group without treatment, and treated CL One group significantly increased the expression of filaggrin, Involucrin, aquaporin, and transglutaminase.
실시예 5: 동물 모델에서의 절패모 추출물의 항아토피 효과 확인Example 5: Confirmation of the anti-atopic effect of Jeolpaemo extract in an animal model
실시예 5-1: 동물 모델의 구축Example 5-1: Construction of an animal model
7주령 balb/c mouse를 깨끗이 제모하고 24시간 후, 1% DNCB (아세톤:올리브 오일=3:1)를 등 및 귀에 3일간 연속 도포한 후, 4일 후 0.5% DNCB (PBS:올리브 오일=9:1)를 등 및 귀에 일주일에 2~3회 4주간 도포하였다. FTB와 CL은 DNCB가 충분히 흡수된 2시간 이후 도포하였으며, 매일 4주간 도포하였다. 약물도포는 저농도는 1mg/ml, 고농도는 10mg/ml 현탁하여 마우스 한 마리당 200μl씩 도포하였다. After 24 hours of cleanly depilating a 7-week-old balb/c mouse, 1% DNCB (acetone: olive oil = 3:1) was applied to the back and ears for 3 consecutive days, after 4 days 0.5% DNCB (PBS: olive oil = 9:1) was applied to the back and ears 2-3 times a week for 4 weeks. FTB and CL were applied 2 hours after DNCB was sufficiently absorbed, and applied every day for 4 weeks. The drug was applied at a low concentration of 1 mg/ml, and a high concentration of 10 mg/ml was suspended, and 200 μl per mouse was applied.
실시예 5-2: 동물 모델에서의 절패모 추출물의 아토피 피부염 치료 효과 확인Example 5-2: Confirmation of the treatment effect of atopic dermatitis of the extract of Jeolpaemo in animal model
동물모델에서의 FTB, CL의 아토피 피부염의 예방 또는 치료활성을 관찰하기 위하여, 상기 실시예 5-1에서 구축한 아토피 질환 마우스모델의 귀의 부종 측정, 행동 평가, 귀의 두께 측정, 피보 조직의 표피 두께 및 비만세포의 침윤 관찰 및 피부 조직내 면역 세포인 CD4 양성세포와 CD8 양성세포의 침윤분석을 하였다. In order to observe the prophylactic or therapeutic activity of FTB and CL in an animal model of atopic dermatitis, the ear swelling measurement, behavior evaluation, ear thickness measurement, epidermal thickness of the atopic disease mouse model constructed in Example 5-1 above And invasion of mast cells were observed, and invasion analysis of CD4 positive cells and CD8 positive cells, which are immune cells in skin tissue, were performed.
귀의 부종은 아토피 피부염 유발 후 FTB와 CL분획물 도포 후 2주와 4주 후 귀의 두께 변화를 마이크로 캘리퍼를 이용하여 측정하였다. 그 결과, 도 11에 나타낸 바와 같이, FTB를 처리한 군은 4주차에 감소하는 경향을 보였으며, CL고농도 처리군은 2주차와 4주차에 귀의 부종을 유의하게 억제하였다.Ear swelling was measured 2 weeks and 4 weeks after application of FTB and CL fractions after induction of atopic dermatitis, using a micro caliper. As a result, as shown in Fig. 11, the group treated with FTB showed a tendency to decrease at
행동 평가는 희생 전날 마우스를 행동케이지에 10분간 적응 시킨 후 30분간 뒷발을 이용하여 긁는 행동을 관찰하였다. 그 결과, FTB 고농도 처리군, CL 저농도와 고농도 처리군의 긁는 행동이 Con군에 비해 유의하게 감소하였다. For behavioral evaluation, the day before sacrifice, the mice were acclimated to the behavioral cage for 10 minutes and then the scratching behavior was observed using the hind paws for 30 minutes. As a result, the scratching behavior of the high FTB concentration treatment group, the low CL concentration and the high concentration treatment group was significantly reduced compared to the Con group.
관능 평가는 희생 전날 등의 상태를 홍반, 가려움, 건조, 부종, 짓무름, 태선화로 나누어, 증상 없음 (0), 약함(1), 중증(2), 심함(3)으로 나누어 채점하였다. 그 결과, 도 11에 나타낸 바와 같이, FTB를 처리한 군은 저농도 처리한 군에서 아토피 상태를 호전시키는 경향을 보였으며, CL 고농도 처리군은 대조군에 비해 유의하게 호전시키는 것을 확인 할 수 있었다.The sensory evaluation was evaluated by dividing conditions such as the day before sacrifice into erythema, itchiness, dryness, swelling, soreness, and lichenification, with no symptoms (0), weak (1), severe (2), and severe (3). As a result, as shown in Figure 11, the group treated with FTB showed a tendency to improve the atopic state in the group treated with the low concentration, and it was confirmed that the group treated with the high CL concentration significantly improved compared to the control group.
피부 조직의 표피 두께 및 비만세포의 침윤 관찰의 경우, 실험 종료 후 희생한 마우스의 등을 채취하여 NBF로 고정 후 파라핀 포매 후 절편하였다. 절편한 조직을 H&E염색을 통해 표피의 두께를 측정하였으며, 톨루이딘 블루로 염색하여 조직내 비만세포의 침윤을 관찰하였다. In the case of observation of the epidermal thickness of the skin tissue and the infiltration of mast cells, the back of the sacrificed mouse was collected after the experiment was completed, fixed with NBF, embedded with paraffin, and sectioned. The sliced tissue was measured for the thickness of the epidermis through H&E staining, and stained with toluidine blue to observe the invasion of mast cells in the tissue.
피부 조직의 표피 두께의 경우, 도 12에 나타낸 바와 같이, FTB는 아토피 피부염으로 인한 표피의 두께는 감소시키는 경향을 보였으며, CL 고농도 처리군은 표피의 두께를 유의하게 감소시켰다.In the case of the epidermal thickness of the skin tissue, as shown in FIG. 12, FTB showed a tendency to decrease the thickness of the epidermis due to atopic dermatitis, and the high CL concentration treatment group significantly reduced the thickness of the epidermis.
비만세포의 침윤 관찰의 경우, 도 12a에 나타낸 바와 같이, FTB 고농도 처리는 아토피 피부염으로 인한 비만세포의 조직내 침윤을 유의하게 감소시켰으며, CL은 저농도군과 고농도 처리군 모두에서 조직내 비만세포의 침윤을 유의하게 감소시켰다.In the case of observation of mast cell invasion, as shown in FIG. 12A, treatment with high FTB concentration significantly reduced the invasion of mast cells due to atopic dermatitis, and CL is intra-tissue mast cells in both the low concentration group and the high concentration treatment group. Significantly reduced the infiltration of.
피부 조직내 면역 세포인 CD4세포와 CD8세포 침윤 분석의 경우, 절편한 조직을 citrate buffer에 10분간 끓여 준 후 충분히 식으면, TBS로 수세한후 3% H2O2에 10분간 담가 peroxidase의 활성을 억제하였다. 10분 후 조직을 10% normal serum에 반응 시킨 후 APAAP(alkaline phosphatase antialkaline phosphatase)kit를 사용하여 CD4와 CD8세포를 염색하여 분석하였다. For the analysis of invasion of CD4 cells and CD8 cells, which are immune cells in skin tissue, boil the sectioned tissue in citrate buffer for 10 minutes, and when cooled sufficiently, rinse with TBS and soak in 3% H 2 O 2 for 10 minutes to activate peroxidase. Was suppressed. After 10 minutes, the tissues were reacted with 10% normal serum, and then CD4 and CD8 cells were stained and analyzed using an APAAP (alkaline phosphatase antialkaline phosphatase) kit.
그 결과, 도 12b에 나타낸 바와 같이, CD4 양성 세포의 조직내 침윤을 분석한 결과, FTB는 Con군에 비해 별다른 차이를 보이지 않았으나, CL 저농도 처리군은 Con에 비해 CD4의 조직내 침윤을 유의하게 감소시켰다. CD8 양성 세포의 조직내 침윤을 분석한 결과, FTB 저농도 군과 고농도군, CL저농도군과 고농도 군 모두에서 Con에 비해 CD8의 조직내 침윤을 유의하게 감소시켰다.As a result, as shown in FIG. 12B, as a result of analyzing the tissue infiltration of CD4-positive cells, FTB showed no significant difference compared to the Con group, but the CL low concentration treatment group significantly increased the infiltration of CD4 tissues compared to Con. Reduced. As a result of analyzing the tissue invasion of CD8-positive cells, in both the FTB low and high concentration groups, the CL low and high concentration groups, the invasion of CD8 was significantly reduced compared to Con.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 예들은 모든 면에서 예시적인 것이며, 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허청구범위의 의미 및 범위, 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the examples described above are illustrative in all respects, and should be understood as non-limiting. The scope of the present invention should be construed as including the meaning and scope of the claims to be described later rather than the above detailed description, and all changes or modifications derived from the equivalent concept within the scope of the present invention.
Claims (8)
A cosmetic composition for preventing or improving atopic dermatitis, comprising the extract of Jeolpaemo, or a fraction thereof as an active ingredient.
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