KR20210034379A - Composition for prevention or treatment of atopic dermatitis with Sargentodoxa cuneata - Google Patents
Composition for prevention or treatment of atopic dermatitis with Sargentodoxa cuneata Download PDFInfo
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Abstract
Description
본 발명은 대혈등(大血藤) 추출물을 함유하는 아토피 피부염 개선 및 치료, 예방용 조성물에 관한 것이다.The present invention relates to a composition for improving, treating, and preventing atopic dermatitis containing an extract of Daehyeoldeung (大血藤).
아토피는 심한 가려움증을 가지는 재발성 만성피부염으로, 어린이의 약 10~15%가 아토피성 피부염을 가지고 있으며, 이중 90%는 5년 내에 자연적으로 호전되는 것으로 보고되어 있다. 아토피는 성인기에 대체로 호전되어 약 30~40%는 외관상으로 피부염이 재발되지 않으나, 나머지는 성인이 되어서도 피부 건조, 자극성 물질에 의한 피부자극, 주부습진 등의 피부염으로 어려움을 겪는 것으로 알려져 있다. 민감성 피부의 경우 수분 보유력 및 회복력이 낮고, 피부 각질화, 가려움증 등이 나타나기 쉽기 때문에 아토피성 피부염이 유발될 확률이 높다.Atopy is a recurrent chronic dermatitis with severe itching. About 10-15% of children have atopic dermatitis, and 90% of them are reported to improve spontaneously within 5 years. Atopy generally improves in adulthood, and about 30 to 40% of the dermatitis does not recur in appearance, but the rest are known to suffer from dermatitis such as dry skin, skin irritation by irritating substances, and housewife eczema even in adulthood. In the case of sensitive skin, moisture retention and resilience are low, and skin keratinization and itching are more likely to occur, so there is a high probability of causing atopic dermatitis.
아토피의 정확한 병리 생리는 아직까지 완전히 이해되고 있지 않지만 유전적 소인과 함께 면역학적, 비면역학적 기전이 관여한다고 보고 있다. 아토피의 대부분을 차지하는 외인성 아토피는 IgE와 연관된 면역 기전에 의해 발생되는데 특정 알레르겐(allergen)에 대한 즉시형 면역 반응보다는 T세포 이상에 의한 지연성 면역 반응이 관여한다는 보고들이 많다. 또한,요즘은 B세포로부터 IgE의 생성을 유도하는 IL-4 등 Th2 관련 사이토카인(cytokie)들이 아토피의 원인이라고 보고되고 있다(JS Kang et al., Ihhibition of atopic dermatitis by topical application of silymarin in NC/Nga mice. intl immunopharm. (2008) 8. 1475-1480).The exact pathophysiology of atopy is still not fully understood, but it is believed that immunological and non-immunological mechanisms are involved along with genetic predisposition. Exogenous atopy, which accounts for the majority of atopy, is caused by an immune mechanism associated with IgE, and there are many reports that a delayed immune response caused by T cell abnormalities is involved rather than an immediate immune response to a specific allergen. In addition, these days, Th2-related cytokines such as IL-4, which induce the production of IgE from B cells, are reported to be the cause of atopy (JS Kang et al., Ihhibition of atopic dermatitis by topical application of silymarin in NC. /Nga mice.intl immunopharm.(2008) 8. 1475-1480).
이러한 아토피를 치료하기 위하여, 종래 세라마이드, 리놀레산, 식물유 또는 광물성 오일 등의 성분들, 하이드로코티손(hydrocortisone) 등의 스테로이드(steroid) 제제 또는 이들에 항균 및 항염 기능을 강화한 물질, 자외선 요법을 통한 DNA 합성 억제게, 세포 과증식 억제제, 염증 및 가려움증 억제제 등이 제안된 바 있다. 그러나, 상기 스테로이드 제제는 표피의 성장억제나 부작용 등의 역효과를 유발할 수 있으며, 우레아 퍼록사이드 등은 피부의 과다 자극을 야기할 수 있고, 항히스타민제 등의 항생물질은 균의 내성(resistance) 및 광과민 등의 부작용을 유발할 가능성이 높은 문제점이 있다. 또한, 장기간 피부에 적용시 모세 혈관 확장증 또는 각질층의 두께 증가 내지 확장을 유발하는 등의 심각한 부작용을 발생시킬 염려가 있으며, 최근 아토피 완화에 많이 쓰이는 감마-리놀레산(한국특허공개 2000-0046633호)은 쉽게 산화되므로 안정성이 열등함과 아울러, 피부 자극성이 비교적 강하여 민감한 피부에는 사용하기 곤란한 등의 문제점이 있다.To treat such atopy, conventional ingredients such as ceramide, linoleic acid, vegetable oil or mineral oil, steroid preparations such as hydrocortisone, or substances enhancing antibacterial and anti-inflammatory functions thereof, DNA synthesis through ultraviolet therapy Inhibitors, cell hyperproliferation inhibitors, inflammation and itching inhibitors, and the like 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 antibiotics such as antihistamines are resistant to bacteria and photosensitivity. There is a problem that is highly likely to cause side effects such as. In addition, there is a concern that serious side effects such as telangiectasia or an increase in the thickness or expansion of the stratum corneum may occur when applied to the skin for a long period of time, and gamma-linoleic acid (Korean Patent Laid-Open No. 2000-0046633), which is widely used in relieving atopy in recent years, is Since it is easily oxidized, stability is inferior, and skin irritation is relatively strong, which makes it difficult to use for sensitive skin.
이에 따라 최근에는 아토피에 효과를 갖는 천연물질을 탐색하는데 초점을 맞추고 있다. 대표적으로, 쑥 추출물(대한민국 특허등록 제10-0377262호), 영지버섯, 유근피, 감초, 백봉령, 백지마 및 백년초의 추출물(대한민국 특허등록 제10-0517465호), 달맞이유, 알로에, 목초액, 제비꽃, 대추, 송이버섯, 두릅, 인삼, 녹차, 두충, 복분자, 오미자, 쑥, 포공영, 삼백초, 황기, 하고초, 해송, 황금 및 우슬의 추출물(대한민국 특허등록 제10-0451444호), 청자소엽과 적자소엽의 추출물(대한민국 특허등록 제10-0454752호), 건률, 의이인, 오미자, 길경, 나복자, 맥문동 및 석창포의 추출물(대한민국 특허등록 제10-0483539호), 라벤더, 유칼립투스 및 티트리 오일(대한민국 특허등록 제10-0597997호) 등이 있고, 그 외에도 상기생, 합환피, 창이자, 맥아 등이 효과가 있다고 알려져 있다. 그러나, 상기 천연물질들은 그 효능이 미미하거나, 효과는 있더라도 민감성 피부에 있어서는 알레르기 과민반응을 보여 그 사용이 제한되는 단점이 있었다. 따라서, 종래 알려진 아토피 개선 및 치료제에 비하여 보다 효과가 뛰어나고 안전한 물질을 개발하는 것이 요구되고 있는 실정이다.Accordingly, in recent years, the focus is on searching for natural substances that have an effect on atopy. Representatively, mugwort extract (Korean Patent Registration No. 10-0377262), Reishi Mushroom, Yugeun Peel, Licorice, Baekbongryeong, Baekjima and Baeknyeoncho extracts (Korea Patent Registration No. 10-0517465), evening primrose oil, aloe, wood herb juice, violet, Extracts of jujube, matsutake, arbor, ginseng, green tea, duchung, bokbunja, schisandra, mugwort, pogongyoung, sambaekcho, hwanggi, hagocho, haesong, golden and hyssop (Korean Patent Registration No. 10-0451444), celadon lobules and reds Leaflet extract (Korean Patent Registration No. 10-0454752), extracts of Kunyul, Uuiyiin, Schisandra chinensis, Gilkyung, Nabokja, Maekmundong and Seokchangpo (Korean patent registration No. 10-0483539), lavender, eucalyptus and tea tree oil (Korean patent) Registration No. 10-0597997), etc. In addition, it is known that above-mentioned saengseng, haphwanpi, changija, malt, etc. are effective. However, the natural substances have a disadvantage in that their efficacy is insignificant, or even if they are effective, they exhibit allergic hypersensitivity reactions in sensitive skin, limiting their use. Therefore, there is a need to develop a material that is more effective and safer than the conventionally known atopy improvement and therapeutic agents.
본 발명은 아토피 피부 개선 효과가 뛰어난 천연물의 추출물을 발굴하고, 이를 유효성분으로 함유하는 조성물을 개발하여 제공하는 것을 목적으로 한다.An object of the present invention is to discover an extract of a natural substance having excellent atopic skin improvement effect, and to develop and provide a composition containing it as an active ingredient.
본 발명은 대혈등 추출물을 함유하는 것을 특징으로 하는 아토피 피부염 예방 및 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of atopic dermatitis, characterized in that it contains an extract such as daeheyeol.
또한, 본 발명은 대혈등 추출물을 함유하는 것을 특징으로 하는 아토피 피부 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for improving atopic skin, characterized in that it contains an extract such as Daehyeol.
또한, 본 발명은 대혈등 추출물을 함유하는 것을 특징으로 하는 아토피 피부 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for improving atopic skin, characterized in that it contains an extract such as Daehyeol.
본 발명에 있어서, 상기 대혈등 추출물은, 바람직하게는 에탄올을 추출용매로 사용하는 것이 좋다.In the present invention, the extract of Daehyeol etc. is preferably used as an extraction solvent of ethanol.
본 발명은 대혈등 추출물을 함유하여 아토피 피부 개선, 예방 또는 치료 효과가 우수한 조성물을 제공한다.The present invention provides a composition having an excellent effect on improving, preventing or treating atopic skin by containing an extract such as Daehyeol.
도 1은 대혈등 추출물의 세포독성 실험 결과이다.
도 2는 대혈등 추출물의 염증유발인자인 산화질소 분비 억제 실험 결과이다.
도 3은 대혈등 추출물의 면역글로블린 E 분비 억제 실험 결과이다.
도 4는 대혈등 추출물의 히스타민 분비 억제 실험 결과이다.
도 5는 대혈등 추출물의 콜라겐 합성 실험 결과이다.1 is a cytotoxicity test result of the extract of Daehyeoldeung.
Figure 2 is a result of an experiment for inhibiting the secretion of nitric oxide, which is an inflammation-inducing factor, of an extract of Daehyeoldeung.
Figure 3 is a result of the immunoglobulin E secretion inhibition experiment of the extract of Daehyeol-eung.
Figure 4 is a result of histamine secretion inhibition experiment of the extract of Daehyeol-eung.
5 is a result of an experiment for collagen synthesis of an extract of Daehyeol-deung.
본 발명은 대혈등 추출물을 함유하는 아토피 피부염 예방 및 치료용 약학 조성물을 제공한다. 또한 본 발명은 대혈등 추출물을 함유하는 아토피 피부 개선용 화장료 조성물 및 식품 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of atopic dermatitis containing an extract such as daeheyeol. In addition, the present invention provides a cosmetic composition and a food composition for improving atopic skin containing an extract such as Daehyeol.
본 발명에서 사용하는 대혈등(大血藤, Sargentodoxa cuneata)은 으름덩굴과로, 원기둥 모양이며 약간 구부러진 형태이다. 길이 30~60cm이고 지름은 1~3cm이다. 바깥 면은 회갈색이나 갈색으로 거친 특징이 있다. 자른 면은 적갈색으로 여러 곳에서 안으로 향하여 목부로 끼어들어 방사상의 꽃무늬를 이룬다. 약간의 냄새가 있으며 쓴맛이 나는 특징이 있다.Daehyeoldeung (大血藤, Sargentodoxa cuneata ) used in the present invention is a vine, a cylindrical shape and a slightly curved shape. The length is 30~60cm and the diameter is 1~3cm. The outer surface is grayish brown or brown with rough characteristics. The cut side is reddish brown and faces inward from several places and intervenes into the neck to form a radial flower pattern. It has a slight odor and is characterized by a bitter taste.
본 발명에 있어서, 상기 대혈등 추출물은 본 발명이 속하는 기술 분야에서 통상적으로 사용되는 다양한 추출 방법을 이용할 수 있다. 예를 들면, 정제수, 탄소수 1-4의 무수 또는 함수 저급 알코올, 프로필렌글리콜, 부틸렌글리콜, 글리세린, 아세톤, 에틸 아세테이트, 클로로포름, 부틸 아세테이트, 디에틸에테르, 디클로로메탄, 헥산 또는 이들의 혼합물로 구성된 군으로부터 선택되는 추출용매를 이용할 수 있다. 바람직하게는 물 또는 유기용매를 가하여 추출하는 것이 좋다.In the present invention, the extract of Daehyeol etc. may use various extraction methods commonly used in the technical field to which the present invention belongs. For example, purified water, anhydrous or water-containing lower alcohol having 1-4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl ether, dichloromethane, hexane, or a mixture thereof. It is possible to use an extraction solvent selected from the group. It is preferable to extract by adding water or an organic solvent.
본 발명에 있어서, 상기 대혈등 추출물은 대혈등 전체 건조 중량의 5배 내지 20배를 첨가하는 것이 바람직하고, 전체 건조 중량의 20배를 첨가하는 것이 더욱 바람직하다.In the present invention, it is preferable to add 5 to 20 times the total dry weight of the Daehyeol etc. to the extract of Daehyeol etc., and it is more preferable to add 20 times the total dry weight.
본 발명에 있어서, 상기 대혈등 추출물의 추출시 온도는 10℃ 내지 120℃ 인 것이 바람직하며, 더욱 바람직하게는 40℃ 내지 100℃인 것일 수 있다. 상기 추출 시간은 1시간 내지 7시간인 것이 바람직하며, 더욱 바람직하게는 3시간 내지 5시간인 것일 수 있다. 상기 추출물을 제조하는 방법은 열수 추출, 침지 추출, 환류 냉각 추출, 초임계추출, 아임계추출, 고온추출, 고압추출 또는 초음파추출법 등의 추출장치를 이용한 방법 또는 XAD 및 HP-20을 포함한 흡착 수지를 이용하는 방법 등 당업계의 통상적인 추출방법을 사용할 수 있다.In the present invention, the temperature at the time of extraction of the extract such as Daehyeol etc. is preferably in the range of 10 ℃ to 120 ℃, more preferably it may be that of 40 ℃ to 100 ℃. The extraction time may be preferably 1 to 7 hours, more preferably 3 to 5 hours. The method of preparing the extract is a method using an extraction device such as hot water extraction, immersion extraction, reflux cooling extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, or ultrasonic extraction, or adsorption resin including XAD and HP-20. A conventional extraction method in the art, such as a method of using, can be used.
본 발명의 대혈등 추출물은 아토피에서 관찰되는 산화 질소 분비를 억제하여 항염 효과가 있는 것으로 확인되었으며, 면역글로불린 E(Immunoglobulin E)의 분비를 억제하고, 히스타민(histamine) 분비를 억제하여 아토피 개선 효과가 우수한 것으로 확인되었다. 또한, 본 발명의 대혈등 추출물은 콜라겐 합성 효과가 우수하여 피부세포 재생 효과를 발휘하는 것을 확인하였다. 이에 따라, 본 발명의 대혈등 추출물은 아토피 개선, 예방 또는 치료용 조성물로서 사용될 수 있다.The extract of the present invention was confirmed to have an anti-inflammatory effect by inhibiting the secretion of nitric oxide observed in atopy, inhibiting the secretion of immunoglobulin E, and suppressing the secretion of histamine, thereby improving atopy. It was found to be excellent. In addition, it was confirmed that the extract of Daehyeol etc. of the present invention exhibits a skin cell regeneration effect due to its excellent collagen synthesis effect. Accordingly, the extract of the present invention can be used as a composition for improving, preventing or treating atopy.
한편, 본 발명의 약학 조성물은 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 선택되는 하나 이상을 사용할 수 있다. 또한, 치료 및 예방제가 약제인 경우 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등이 추가적으로 포함될 수 있다.Meanwhile, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient. Examples of carriers, excipients or diluents that can be used include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and at least one selected from among them may be used. In addition, when the treatment and prophylactic agent is a drug, a filler, an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifying agent or a preservative may be additionally included.
한편, 본 발명의 약학 조성물의 제형은 사용 방법에 따라 바람직한 형태로 제조될 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LOTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPESIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다.On the other hand, the formulation of the pharmaceutical composition of the present invention may be prepared in a preferred form according to the method of use, and in particular, a method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal is used. It is better to adopt and formulate. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrup ( SYRUPS), OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsions ), Suspensions (SUSPESIONS), DECOCTIONS, Infusions (INFUSIONS), Eye Drops (OPHTHALMIC SOLUTIONS), Tablets (TABLETS), SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMAS ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pasta (PASTES), pills (PILLS), it may be any one selected from soft or hard gelatin capsules.
한편, 본 발명의 약학조성물에 있어서, 투여량은 투여방법, 복용자의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분인 대혈등 추출물을 기준 (건조중량)으로 하였을 때 1일 0.1 내지 100 mg/kg (체중)으로 1회 이상 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.Meanwhile, in the pharmaceutical composition of the present invention, the dosage is preferably determined in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. As an example, it can be administered once or more at 0.1 to 100 mg/kg (body weight) per day based on the active ingredient, Daehyeol etc. extract (dry weight). However, the above dosage is only an example for illustration, and may be changed according to the state of the taker and the prescription of the doctor.
한편, 본 발명의 화장료 조성물에 있어서, 상기 대혈등 추출물은, 총 중량 대비 0.001~30 중량% 첨가되는 것이 좋다. 0.001 중량% 미만으로 포함되는 경우에는 본 발명에서 확인한 여러 피부 기능성의 발휘 효과가 미미하고, 30.0 중량%를 초과하는 경우에는 재료 투입량 대비 효율성이 떨어져 경제적이지 못하다.On the other hand, in the cosmetic composition of the present invention, the extract, such as Daehyeol, is preferably added 0.001 to 30% by weight based on the total weight. If it is contained in an amount of less than 0.001% by weight, the effect of exerting various skin functions identified in the present invention is insignificant, and if it exceeds 30.0% by weight, it is not economical due to its efficiency compared to the amount of material input.
한편, 본 발명의 화장료 조성물은, 일 예로, 용액, 현탁액, 유탁액, 페이스트, 화장수, 젤, 수용성 리퀴드, 크림, 에센스, 계면활성제-함유 클렌징, 오일, 수중유(O/W)형 및 유중수(W/O)형 중 선택되는 어느 하나의 기초 화장료 제형; 스킨; 로션; 아이크림; 수딩젤; 연고; 마스크팩용 제형; 바디워시용 제형; 필링젤; 수중유형 및 유중수형 메이크업베이스; 파운데이션; 스킨커버; 립스틱, 립그로스, 페이스파우더, 투웨이케익, 아이새도, 치크칼라 및 아이브로우 펜슬류 중 선택되는 어느 하나의 색조화장료 제형; 두피용 제형; 중에서 선택되는 어느 하나인 것일 수 있다.On the other hand, the cosmetic composition of the present invention, for example, solution, suspension, emulsion, paste, lotion, gel, water-soluble liquid, cream, essence, surfactant-containing cleansing, oil, oil-in-water (O/W) type and oil Any one basic cosmetic formulation selected from heavy water (W/O) type; skin; Lotion; Eye cream; Soothing gel; Ointment; Formulations for mask packs; Formulation for body wash; Peeling gel; Water-in-water and water-in-oil makeup base; foundation; Skin cover; Any one color cosmetic formulation selected from lipstick, lip gloss, face powder, two-way cake, eye sado, cheek color, and eyebrow pencil; Formulation for scalp; It may be any one selected from among.
또한, 본 발명의 화장료 조성물은 화장 분야에서 통상적으로 사용되는 보조제 예컨대 친수성 또는 친유성 활성제, 보존제, 항산화제, 용매, 방향제, 충전제, 차단제, 안료, 흡취제, 염료 등을 함유할 수 있다. 이들 다양한 보조제의 양은 당해 분야에서 통상적으로 사용되는 양이며, 예컨대 조성물 총 중량에 대해 0.001 내지 30 중량% 이다. 다만, 어떠한 경우라도 보조제 및 그 비율은 본 발명에 따른 화장료 조성물의 바람직한 성질에 악영향을 미치지 않도록 선택될 것이다.In addition, the cosmetic composition of the present invention may contain auxiliary agents commonly used in the cosmetic field, such as hydrophilic or lipophilic activators, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants, dyes, and the like. The amounts of these various adjuvants are those commonly used in the art, such as 0.001 to 30% by weight based on the total weight of the composition. However, in any case, the adjuvant and its ratio will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.
한편, 본 발명의 화장료 조성물에 있어서, 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌글리콜, 실리콘, 벤토나이트, 실라카, 탈크 또는 산화아연 등이 이용될 수 있다.On the other hand, in the cosmetic composition of the present invention, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silaka as carrier components , Talc or zinc oxide may be used.
또한, 본 발명의 화장료 조성물에 있어서, 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 일례로 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤조에이트, 프로필렌글리콜, 1,3-부틸렌글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌글리콜 또는 소리비탄의 지방산 에스테르가 이용될 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of soribitan may be used.
또한 본 발명의 화장료 조성물에 있어서, 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라가칸트 등이 이용될 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester as carrier components. The same suspending agent, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like may be used.
한편, 본 발명의 화장료 조성물에 있어서, 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.Meanwhile, in the cosmetic composition of the present invention, when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally Propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.
한편, 본 발명의 화장료 조성물에 있어서, 제형이 계면활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.On the other hand, in the cosmetic composition of the present invention, when the formulation is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.
또한, 본 발명의 화장료 조성물에 있어서, 계면활성제 함유 클렌징 제형 또는 계면활성제 비함유 클렌징 제형 또는 비누일 경우에는, 피부에 도포한 후 닦아내거나 떼거나 물로 씻어낼 수도 있다. 일례로, 상기 비누는 액상비누, 가루비누, 고형비누 및 오일비누이며, 상기 계면활성제 함유 클렌징 제형은 클렌징 폼, 클렌징 워터, 클렌징 수건 및 클렌징 팩이며, 상기 계면활성제 비함유 클렌징 제형은 클렌징크림, 클렌징 로션, 클렌징 워터 및 클렌징 젤이며, 이에 한정되는 것은 아니다.In addition, in the cosmetic composition of the present invention, in the case of a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation or soap, it may be applied to the skin and then wiped off, removed, or washed with water. For example, the soap is liquid soap, powder soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream, Cleansing lotion, cleansing water, and cleansing gel, but are not limited thereto.
또한, 본 발명의 화장료 조성물은 본 발명 이외의 다른 화장료 조성물과 중복하여 사용할 수 있다. 또한 본 발명에 따른 화장료 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 피부 상태 또는 취향에 따라 그 사용횟수를 달리할 수 있다.In addition, the cosmetic composition of the present invention can be used in conjunction with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use thereof may be varied according to a user's skin condition or taste.
한편, 본 발명의 대혈등 추출물을 함유하는 약학 조성물 또는 화장료 조성물은 바람직하게는 나노리포좀 제형으로 만들어질 수 있다. 나노리포좀은 통상적인 리포좀의 형태를 갖는 것으로 평균 입자 지름이 1~100nm인 리포좀인 것이 바람직하며, 더욱 바람직하게는 30~70nm인 것이 좋다. 상기 리포좀은 수용성 머리와 불용성의 꼬리를 가진 양친매성 분자로 구성된 입자의 상호 자발적인 결합 및 정렬된 콜로이드 입자로, 세포막과 유사한 이중막 구조를 가지며, 이를 함유한 조성물은 피부 흡수력이 더욱 증진되어 아토피 피부 완화 효과가 우수하다.On the other hand, the pharmaceutical composition or cosmetic composition containing the extract of the present invention may preferably be made into a nano-liposomal formulation. Nanoliposomes are in the form of conventional liposomes, and are preferably liposomes having an average particle diameter of 1 to 100 nm, more preferably 30 to 70 nm. The liposome is a colloidal particle that is spontaneously bonded and aligned with each other of particles composed of amphiphilic molecules having a water-soluble head and an insoluble tail, and has a double-membrane structure similar to a cell membrane, and a composition containing it further enhances skin absorption and thus atopic skin. Excellent relief effect.
본 발명의 나노리포좀은 C12-22 알킬 체인의 포화 또는 불포화 지방산 및 세포간 지질을 포함한다. 본 발명의 나노 리포좀의 제조에 이용되는 C12-22 알킬 체인의 포화 또는 불포화 지방산은, 바람직하게는 라우린산, 미리스트산, 팔미트산, 스테아린산, 올레인산, 리놀레산 및 팔미트올레인산을 포함하며, 더욱 바람직하게는 스테아린산, 팔미트올레인산 및 이의 혼합물로 구성된 군으로부터 선택되는 어느 하나일 수 있다. 상기 나노 리포좀은 피부 외용제 총 중량에 대하여 0.05~5.0 중량%로 함유되는 것이 바람직하나, 더욱 바람직하게는 0.1~.0 중량% 함유되는 것이 좋다. 나노리포좀에 함유되는 대혈등 추출물의 함량은 나노리포좀의 총 중량에 대해서 1.0~20.0 중량%일 수 있으며, 더욱 바람직하게는 3.0~12.0 중량% 함유되는 것이 좋다.The nanoliposome of the present invention includes a saturated or unsaturated fatty acid of a C 12-22 alkyl chain and intercellular lipids. The saturated or unsaturated fatty acids of the C 12-22 alkyl chain used in the preparation of the nano-liposome of the present invention preferably include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and palmitoleic acid, , More preferably, it may be any one selected from the group consisting of stearic acid, palmitoleic acid, and mixtures thereof. The nano-liposome is preferably contained in an amount of 0.05 to 5.0% by weight based on the total weight of the external preparation for skin, more preferably 0.1 to .0% by weight. The content of the extract of Daehyeol etc. contained in the nanoliposome may be 1.0 to 20.0% by weight, more preferably 3.0 to 12.0% by weight based on the total weight of the nanoliposome.
한편, 본 발명의 식품 조성물은 일 예로 육류, 곡류, 카페인 음료, 일반음료, 초콜렛, 빵류, 스넥류, 과자류, 피자, 젤리, 면류, 껌류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나일 수 있으며, 반드시 이에 한정되는 것은 아니다.On the other hand, the food composition of the present invention is, for example, meat, grains, caffeine beverages, general beverages, chocolate, bread, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, vitamin complexes and other health It may be any one selected from supplements, but is not necessarily limited thereto.
한편, 본 발명에서 '유효성분으로 함유하는 것'의 의미는 본 발명에서 요구하는 아토피 피부의 개선, 치료, 예방의 효과가 본 발명의 성분인 대혈등 추출물로부터 발생함을 의미하고, 그 외에 다른 성분을 보조성분으로 포함할 수 있음을 의미한다.On the other hand, the meaning of'containing as an active ingredient' in the present invention means that the effect of improving, treating, and preventing atopic skin required by the present invention occurs from the extract of Daehyeol etc., which is a component of the present invention. It means that an ingredient can be included as an auxiliary ingredient.
이하, 본 발명에 대해 하기 실시예 및 실험예에서 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고, 이와 등가의 기술적 사상의 변형까지를 모두 포함한다.Hereinafter, the present invention will be described in more detail in the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes all modifications of the equivalent technical idea.
[실시예 1 : 대혈등 추출물의 제조][Example 1: Preparation of extracts of Daehyeol etc.]
대혈등(地楓皮)의 덩굴줄기를 정선하여 약전 통칙 중 절도에 따라 조절(4750 ㎛)로 한 뒤, 대혈등 분말 100g에 20배의 70 %(v/v) 에탄올을 넣어 침지하고, 80℃에서 3시간 추출한 후 농축 건조하여 대혈등 추출물을 제조하였다.After selecting the vines of Daehyeoldeung (地楓皮) and adjusting it according to theft (4750 ㎛) in the general rules of the Pharmacopoeia, 100g of Daehyeoldeung powder was immersed in 20 times 70% (v/v) ethanol. After extracting for 3 hours at °C, the extract was concentrated to dryness to prepare an extract such as Daehyeol.
[실험예 1 : 대혈등 추출물의 세포독성 측정][Experimental Example 1: Measurement of cytotoxicity of extracts of Daehyeol etc.]
본 실험예에서는 시험관 내(in vito)에서 본 발명에 따른 혼합 추출물의 아토피 개선 및 치료의 효능을 확인하기에 앞서, 세포에 대한 독성이 없으며 최적의 효과를 낼 수 있는 적합한 농도를 결정하기 위해 세포독성 검사를 하였다.In this experimental example, prior to confirming the efficacy of atopy improvement and treatment of the mixed extract according to the present invention in vitro (in vito), the cells were not toxic to the cells and the cells were Toxicology was tested.
MTT assay는 살아있는 세포 내의 미토콘드리아 탈수소효소에 의해 테트라졸리움(tetrazolium) 염이 포르마잔(formazan)색소로 변환되는 원리를 이용한다. 배지에 첨가한 테트라졸리움 염은 미토콘드리아의 호흡사슬에 존재하며 생존세포에만 활성이 있는 숙신산염 테트라졸리움 환원효소(succinate-tetrazolium-reductase)에 의해 포르마잔 색소로 변환한다. 살아있는 세포수가 증가하면 시료 중의 미토콘드리아 탈수소효소의 전체의 활성이 증가하여 이 효소활성의 증가가 포르마잔 색소의 생성증가를 유도하기 때문에 포르마잔 색소와 배지 중에서 대사활성이 있는 세포의 수는 직선적인 상관관계를 나타내게 된다. 즉, 농도가 높을수록 살아있는 세포수가 많다는 것을 확인할 수 있다.MTT assay uses the principle that tetrazolium salt is converted into formazan pigment by mitochondrial dehydrogenase in living cells. The tetrazolium salt added to the medium is converted into formazan pigment by succinate-tetrazolium-reductase, which is present in the mitochondrial respiratory chain and is active only in viable cells. As the number of living cells increases, the total activity of mitochondrial dehydrogenase in the sample increases, and the increase in this enzyme activity induces an increase in the production of formazan pigment.Therefore, the number of cells with metabolic activity in the medium and formazan pigment are linearly correlated. Relationship. In other words, it can be confirmed that the higher the concentration, the greater the number of living cells.
(1) 대식세포주인 RAW 264.7 배양(1) Macrophage cell line RAW 264.7 culture
쥐의 대식세포주인 RAW 264.7 세포는 10% 비활성화 소태아혈청(inactivated fetal bovine serum, GIBCO)과 1% 페니실린·스트렙토마이신(penicillin·streptomycin)이 포함된 DMEM(Dul-becco's Modified Eagle's Medicum)을 배양액으로 하여 37℃에서 5% CO2 조건으로 배양하였다.RAW 264.7 cells, a mouse macrophage cell line, were cultured with DMEM (Dul-becco's Modified Eagle's Medicum) containing 10% inactivated fetal bovine serum (GIBCO) and 1% penicillin/streptomycin. Then, it was cultured at 37° C. under 5% CO 2 conditions.
(2) 세포독성 실험(2) Cytotoxicity test
RAW 264.7 세포를 96 웰 플레이트(well plate)에 웰(well) 당 1×104개로 분주하고 24시간 배양한 후, 10, 50 및 100 ㎍/㎖ 농도로 상기 실시예 1의 대혈등 추출물을 처리하여 48시간동안 배양하였다. 배양액을 흡입하여 제거한 다음 PBS로 1회 세척하고, MTT 용액을 0.5㎎/㎖의 농도로 100㎕ 씩 세포에 첨가하여 4시간동안 37℃, 5% CO2에서 배양하였다. 배양액을 흡입하여 제거한 다음 DMSO(dimethyl sulfoxide) 200㎕를 첨가하고 쉐이커(shaker)에서 10분간 흔들어 준 후 엘라이저 리더(ELISA reader)로 540㎚에서 흡광도를 측정하였다.RAW 264.7 cells were dispensed into a 96-well plate at 1×10 4 per well, cultured for 24 hours, and then treated with the extract of the blood extract of Example 1 at concentrations of 10, 50 and 100 μg/ml. And incubated for 48 hours. The culture solution was removed by aspiration, washed once with PBS, and 100 μl of MTT solution was added to the cells at a concentration of 0.5 mg/ml, followed by incubation at 37° C. and 5% CO 2 for 4 hours. After the culture was removed by suction, 200 µl of dimethyl sulfoxide (DMSO) was added, and after shaking for 10 minutes on a shaker, the absorbance was measured at 540 nm with an ELISA reader.
(3) 실험 결과(3) Experiment result
도 1에서 보듯이, 본 발명의 대혈등 추출물이 10, 50 및 100 ㎍/㎖ 농도에서 세포독성이 낮게 나타났다.As shown in Figure 1, the extract of the present invention showed low cytotoxicity at concentrations of 10, 50 and 100 µg/ml.
[실험예 2 : 대혈등 추출물의 항염증 활성평가][Experimental Example 2: Evaluation of anti-inflammatory activity of extracts of Daehyeol etc.]
본 실험예에서는 대혈등 추출물의 산화질소 분비 억제에 따른 항염증 활성효과를 확인하고자 하였다.In this experimental example, it was attempted to confirm the anti-inflammatory effect of the extract of Daehyeol etc. by inhibiting nitric oxide secretion.
(1) 대식세포주인 RAW 264.7 배양(1) Macrophage cell line RAW 264.7 culture
쥐의 대식세포주인 RAW 264.7 세포는 10% 비활성화 소태아혈청(inactivated fetal bovine serum, GIBCO)과 1% 페니실린·스트렙토마이신(penicillin·streptomycin)이 포함된 DMEM(Dul-becco's Modified Eagle's Medicum)을 배양액으로 하여 37℃에서 5% CO2 조건으로 배양하였다.RAW 264.7 cells, a mouse macrophage cell line, were cultured with DMEM (Dul-becco's Modified Eagle's Medicum) containing 10% inactivated fetal bovine serum (GIBCO) and 1% penicillin/streptomycin. Then, it was cultured at 37° C. under 5% CO 2 conditions.
(2) 산화질소 측정(2) Nitric oxide measurement
24 웰 플레이트(well plate)에 쥐의 대식세포주인 RAW 264.7을 5×105 /well 정도로 분주하고 다음날 오전에 배지를 무혈청배양액(serum free media)으로 교체한 후 약 8시간 절식(starvation)시켰다. 배양 미디아(media)는 흡입한 후 새로운 무혈청배양액(fresh serum free media)을 500㎕/well을 넣고 상기 실시예 1의 대혈등 추출물을 농도별로 가한 후 LPS 스톡 솔루션(stock solution) (100㎎/㎖)을 2.5㎕/well 처리해주고 16시간 배양하였다. 미디아(media) 상층액을 96 웰 플레이트(well plate)에 100㎕를 옮기고 동량의 그리스 시약(Griess reagent)을 100㎕ 가하고 30분간 상온에서 배양한 다음 발색의 정도를 마이크로플레이트 리더(microplate reader)의 540㎚에서 흡광도를 측정하였다.RAW 264.7, a mouse macrophage cell line, was dispensed into a 24-well plate at about 5×10 5 /well, and the medium was replaced with serum free media the next morning, followed by starvation for about 8 hours. . After inhaling the culture media, 500 µl/well of fresh serum free media was added, and the extract of hematocrit of Example 1 was added by concentration, and then an LPS stock solution (100 mg/well) was added. Ml) was treated with 2.5µl/well and incubated for 16 hours.
(3) 실험 결과(3) Experiment result
도 2에서 보듯이, 본 발명의 대혈등 추출물은 LPS에 의해 유도된 산화질소 NO(nitric oxide) 분비를 현저하게 억제하여 항염증 활성 효과가 우수한 점을 확인하였다.As shown in Figure 2, it was confirmed that the extract of the present invention remarkably inhibits the secretion of nitric oxide (NO) induced by LPS, thereby having excellent anti-inflammatory activity.
[실험예 3 : 대혈등 추출물의 IgE 분비억제 효과 확인] [Experimental Example 3: Confirmation of IgE secretion inhibitory effect of Daehyeol etc. extract]
본 실험예에서는 대혈등 추출물의 아토피 개선 효과를 확인하고자 하였다. 아토피에 있어서 가장 큰 지표 타겟으로 알려진 물질은 면역글로불린 E(immunoglobulin E,IgE)이므로, 대혈등 추출물의 IgE의 분비 억제효과를 확인하였다. 여러 가지 항체 아이소타입(isotype) 중에서도 특별히 IgE를 많이 분비하는 사람에서 유래된 B세포주이다. B 세포가 분화가 되어 플라스마 세포가 되면 기본적으로 여러 가지 면역글로불린(immunoglobulin) 중에서 IgM을 분비하게 되고 클래스 스위칭 반응(class switching reaction)에 의해서 여러 가지 아이소타입(isotype)으로 전환된다고 알려져 있다. 이러한 아이소타입 결정에 있어서, T 세포에서 분비되는 다양한 사이토카인(cytokine)이 중요한 역할을 하며 각각의 환경에 많이 존재하는 사이토카인에 의해 IgA, IgE, IgE 등의 다양한 아이소타입으로 변화된다고 알려져 있다. 이 중에서 IgE로의 변환에 있어서는 지질다당류(polysaccharide, LPS)/ 안티-CD40 및 인터루킨-4(interleukin-4, IL-4)에 의한 자극이 필요하다는 것이 알려져 있으므로, B 세포가 IgE를 분비시키는 환경을 조성하기 위하여, B 세포의 배양시 LPS와 IL-4을 배양액에 첨가하였다.In this experimental example, it was attempted to confirm the atopy improvement effect of the extract of Daehyeol etc. Since the substance known as the largest indicator target for atopy is immunoglobulin E (IgE), the inhibitory effect of IgE secretion of the extract of Daehyeol etc. was confirmed. Among various antibody isotypes, it is a B cell line derived from humans that secrete a lot of IgE. When B cells differentiate into plasma cells, they basically secrete IgM from among various immunoglobulins and are known to be converted into various isotypes by a class switching reaction. In determining such isotypes, various cytokines secreted from T cells play an important role, and it is known that various isotypes such as IgA, IgE, and IgE are changed by cytokines present in many environments. Among these, it is known that stimulation by lipopolysaccharides (polysaccharide, LPS)/anti-CD40 and interleukin-4 (IL-4) is required for conversion to IgE, so that the environment in which B cells secrete IgE is established. For composition, LPS and IL-4 were added to the culture medium when B cells were cultured.
(1) B 세포주 배양(1) B cell line culture
사람의 다발성골수종세포(multiple myeloma cells)인 U266B1를 RPMI 1640 기본 배지에 10% FBS, 1mM 피브루산나트륨(sodium pyruvate), 2mML-글루타민(glutamine), 100U 페니실린(penicillin) 그리고 50mg/㎖ 스트렙토마이신(streptomycin)이 첨가되어 있는 B세포 배지에서 37℃, 5% CO2의 배양 조건에서 배양하였다. Human multiple myeloma cells, U266B1, were added to RPMI 1640 basic medium in 10% FBS, 1 mM sodium pyruvate, 2 mM-glutamine, 100 U penicillin, and 50 mg/ml streptomycin. It was cultured in a B cell medium to which (streptomycin) was added at 37° C. and 5% CO 2 .
(2) IgE 농도 측정(2) IgE concentration measurement
상기에서 배양한 B세포에 10㎍/㎖의 LPS와 5ng/㎖의 IL-4를 배양액에 첨가한 후, 세포를 72시간, 37℃ 배양기에서 배양하여 B세포를 자극하였다. 72시간 후, 상기 실시예 1의 대혈등 추출물을 10, 50 및 100 ㎍/㎖ 농도로 B세포에 처리한 후 3일을 배양하였다. 3일 배양 후, 세포를 수득하여 배양액에 존재하는 IgE의 농도를 엘라이자(ELISA)를 이용하여 측정하였다.After adding 10 µg/ml of LPS and 5 ng/ml of IL-4 to the cultured B cells, cells were cultured in an incubator at 37°C for 72 hours to stimulate B cells. After 72 hours, the extract of Daehyeol etc. of Example 1 was treated on B cells at concentrations of 10, 50 and 100 µg/ml, followed by culture for 3 days. After culturing for 3 days, cells were obtained and the concentration of IgE present in the culture medium was measured using ELISA.
(3) 실험 결과(3) Experiment result
도 3에서 보듯이, 본 발명의 대혈등 추출물은 B세포의 IgE 분비를 현저하게 억제하였다.As shown in Figure 3, the extract of the present invention remarkably suppressed IgE secretion of B cells.
[실험예 4 : 대혈등 추출물의 히스타민 분비억제 효과 확인][Experimental Example 4: Confirmation of the histamine secretion inhibitory effect of the extract of Daehyeol etc.]
본 실험예에서는 대혈등 추출물의 아토피 개선 효과를 확인하고자 하였다. 아토피의 또다른 특징은 아토피 및 알러지 환자의 비만세포(mast cell)나 호염세포(basophil)가 분비하는 히스타민 농도의 증가이다. 비만 세포의 경우 세포 표면에 IgE에 대한 강한 반응성의 수용체(high affinity receptor)를 발현하고 있어 외부의 알러젠(allergen)이 몸속으로 들어왔을 때 IgE가 여기에 결합하게 되고 이 IgE가 다시 알러젠을 붙잡기 때문에 비만 세포가 활성화되게 된다. 이때 활성화된 비만 세포는 탈과립(degranulation)에 의해 히스타민 및 염증성 매개 인자들을 분비하게 되고 아토피나 알러지 증상을 심화시킨다. 따라서 세포에 적정 농도의 혼합 추출물을 처리하여 자극을 준 후 배양액에 분비되는 히스타민의 농도를 히스타민 검출 키트(histamine assay kit)을 이용해 측정하여, 추출물의 항알러지 또는 항아토피 효과를 확인하고자 하였다. In this experimental example, it was attempted to confirm the atopy improvement effect of the extract of Daehyeol etc. Another characteristic of atopy is an increase in the concentration of histamine secreted by mast cells or basophils in patients with atopy and allergies. In the case of mast cells, a highly reactive receptor for IgE is expressed on the cell surface, so when an external allergen enters the body, IgE binds to it, and this IgE captures the allergen again. Mast cells are activated. At this time, the activated mast cells secrete histamine and inflammatory mediators by degranulation and intensify atopy or allergic symptoms. Therefore, the concentration of histamine secreted in the culture medium was measured using a histamine assay kit after stimulating the cells with an appropriate concentration of the mixed extract to confirm the anti-allergic or anti-atopic effect of the extract.
(1) HMC-1 세포주 배양(1) HMC-1 cell line culture
사람의 대식 세포주인 HMC-1 세포를 IMDM 기본 배지에 10% FBS와 1%의 ㅍ페페니실린/스트렙토마이신(penicillin/streptomycin)이 첨가된 배양액에서 37℃, 5% CO2 의 배양 조건에서 배양하였다.HMC-1 cells, a human macrophage cell line, were cultured in IMDM basal medium in a culture medium containing 10% FBS and 1% penicillin/streptomycin at 37° C. and 5% CO 2 .
(2) 히스타민 분비 측정(2) measurement of histamine secretion
HMC-1세포주에 상기 실시예 1의 대혈등 추출물을 10, 50 및 100 ㎍/㎖ 농도로 처리함과 동시에, 세포를 활성화시키기 위하여, 20nM의 PMA(Phorbol 12-myristate 13-acetate, sigma)와 2 mM의 아이오노마이신(ionomycin, sigma)을 24시간 처리한 후 세포 배양액에 분비된 히스타민의 양을 ELISA를 통해 확인하였다.HMC-1 cell line was treated with the extract of Daehyeol etc. of Example 1 at concentrations of 10, 50 and 100 μg/ml, and at the same time, in order to activate the cells, 20 nM of PMA (Phorbol 12-myristate 13-acetate, sigma) and After treatment with 2 mM of ionomycin (sigma) for 24 hours, the amount of histamine secreted in the cell culture was confirmed by ELISA.
(3) 실험 결과(3) Experiment result
도 4에서 보듯이, 세포배양엑에 아무것도 처리하지 않은 무처리 대조군에 비하여 대혈등 추출물이 히스타민의 분비를 감소시킴을 확인하였다.As shown in FIG. 4, it was confirmed that the extract of Daehyeol etc. reduced the secretion of histamine compared to the untreated control group in which nothing was treated in the cell culture extract.
[실험예 5 : 대혈등 추출물의 콜라겐 생성능 확인][Experimental Example 5: Confirmation of collagen production ability of extracts such as Daehyeol etc.]
본 실험예에서는 대혈등 추출물의 콜라겐 생성능 및 피부세포 재생 효과를 확인하고자 하였다.In this experimental example, it was attempted to confirm the collagen production ability and skin cell regeneration effect of the extract of Daehyeol etc.
(1) 피부아세포(HDFn)주 배양(1) cultivation of skin blast cells (HDFn)
사람의 피부아세포(HDFn, human Dermal fibroblast, neonatal)는 Gibco invitrogen cell culture에서 구입하여 실험에 사용하였다. 세포는 37℃, 5% CO2에서 10% FBS혈청, 페니실린(penicillin) 100 units/ml 과 스트렙토마이신(streptomycin) 100 ㎍/ml 그리고 2%의 LSGS (Low SErum Growth Supplement)가 첨가된 M106에 배지에서 배양하였다. 세포배양용기의 세포밀도가 80% 이상일 때 계대배양(subculture)을 실시하였으며 SFM(Serum Free Media)으로 일회세척하고 트립신(trypsin)/EDTA (0.05% trypsin, 0.53 mM EDTA)처리하여 37℃에서 세포를 탈리하였다. 이를 원심분리(1,300 rpm, 4℃)하여 포들을 회수하고 1 x 105 개로 계대하여 2-3일 간격으로 배지를 교체하면서 배양하였다.Human dermal fibroblast, neonatal (HDFn) was purchased from Gibco invitrogen cell culture and used in the experiment. Cells were cultured in M106 supplemented with 10% FBS serum, 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 2% LSGS (Low SErum Growth Supplement) at 37°C, 5% CO 2 Cultured in. When the cell density of the cell culture vessel was 80% or more, subculture was performed, and the cells were washed once with Serum Free Media (SFM) and treated with trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA). Was removed. This was centrifuged (1,300 rpm, 4° C.) to recover the cells, and passaged to 1×105 cells, and cultured while changing the medium at intervals of 2-3 days.
(2) 콜라겐 합성능 평가(2) Evaluation of collagen synthesis ability
콜라겐 합성능 평가는 콜라겐 전구체(PIP, Procollagen Type 1C-peptide)의 C-말단을 인식하는 항체 이용법을 활용하였다. 상기 실시예1의 대혈등 추출물 10, 50 및 100 ㎍/㎖ 농도의 추출물을 각각 세포에 처치하여 배양한 후 배양액을 모아서 enzymes-linked immunoassay kit (PIP-kit, Takara사)를 이용하여 콜라겐 전구체의 C-말단의 양을 측정하였다. The collagen synthesis ability was evaluated by using an antibody that recognizes the C-terminus of a collagen precursor (PIP, Procollagen Type 1C-peptide). The extracts of 10, 50, and 100 µg/ml of the extract of Example 1 were treated on cells and cultured, and then the culture solution was collected and the collagen precursor was prepared by using an enzymes-linked immunoassay kit (PIP-kit, Takara). The amount of the C-terminus was measured.
(3) 실험 결과(3) Experiment result
도 5에서 보듯이, 대혈등 추출물이 콜라겐 합성 및 피부세포 재생에 우수한 효과를 갖는 점을 확인하였다.As shown in FIG. 5, it was confirmed that the extract of Daehyeol etc. has an excellent effect on collagen synthesis and skin cell regeneration.
Claims (4)
A pharmaceutical composition for the prevention and treatment of atopic dermatitis, characterized in that it contains an extract such as Daehyeol.
A cosmetic composition for improving atopic skin, characterized in that it contains an extract such as Daehyeol.
A food composition for improving atopic skin, characterized in that it contains an extract such as Daehyeol.
상기 대혈등 추출물은,
에탄올을 추출용매로 사용하는 것을 특징으로 하는 조성물.
The method according to any one of claims 1 to 3,
The Daehyeol etc. extract,
Composition, characterized in that using ethanol as an extraction solvent.
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| JP2007099733A (en) * | 2005-10-07 | 2007-04-19 | Shiseido Co Ltd | Antioxidant and skin lotion |
| KR20180051647A (en) * | 2011-03-28 | 2018-05-16 | 마리 케이 인코포레이티드 | Topical skin care formulations comprising plant extracts |
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| JP2007099733A (en) * | 2005-10-07 | 2007-04-19 | Shiseido Co Ltd | Antioxidant and skin lotion |
| KR20180051647A (en) * | 2011-03-28 | 2018-05-16 | 마리 케이 인코포레이티드 | Topical skin care formulations comprising plant extracts |
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| CN114467981A (en) * | 2022-01-21 | 2022-05-13 | 湖南环境生物职业技术学院 | Black tiger fruit extract with antibacterial and antiviral effects, preparation method thereof and application thereof in preparing handmade soap or cleaning agent |
| CN114467981B (en) * | 2022-01-21 | 2023-03-21 | 湖南环境生物职业技术学院 | Black tiger fruit extract with antibacterial and antiviral effects, preparation method thereof and application thereof in preparing handmade soap or cleaning agent |
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