KR20160086310A - Composition for preventing or improving atopic dermatitis comprising ethyl acetate fraction of methanol extract from Diospyros lotus leaf as effective component - Google Patents
Composition for preventing or improving atopic dermatitis comprising ethyl acetate fraction of methanol extract from Diospyros lotus leaf as effective component Download PDFInfo
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- KR20160086310A KR20160086310A KR1020160087245A KR20160087245A KR20160086310A KR 20160086310 A KR20160086310 A KR 20160086310A KR 1020160087245 A KR1020160087245 A KR 1020160087245A KR 20160087245 A KR20160087245 A KR 20160087245A KR 20160086310 A KR20160086310 A KR 20160086310A
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- Prior art keywords
- ethyl acetate
- fraction
- leaf
- composition
- atopic dermatitis
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- 239000002038 ethyl acetate fraction Substances 0.000 title claims abstract description 61
- 239000000203 mixture Substances 0.000 title claims abstract description 56
- 239000000401 methanolic extract Substances 0.000 title claims abstract description 54
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- 201000008937 atopic dermatitis Diseases 0.000 title claims abstract description 29
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
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- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
Abstract
Description
본 발명은 고욤나무 잎 메탄올 추출물의 에틸아세테이트 분획물을 유효성분으로 함유하는 아토피 피부염 개선용 조성물에 관한 것으로, 더욱 상세하게는 고욤나무 (Diospyros lotus L.) 잎 메탄올 추출물의 에틸아세테이트 분획물을 유효성분으로 함유하는 아토피 피부염 예방 또는 개선용 건강식품 조성물, 약학 조성물 및 화장료 조성물에 관한 것이다.The present invention relates to a composition for improving atopic dermatitis comprising an ethyl acetate fraction of a methanol extract of Gomatomivorus leaf as an active ingredient, and more particularly to a composition for improving atopic dermatitis comprising an ethyl acetate fraction of a methanol extract of a leaf of Dioscoros lotus L. leaf, To a health food composition, a pharmaceutical composition and a cosmetic composition for preventing or ameliorating atopic dermatitis.
아토피 피부염 (atopic dermatitis)은 만성 피부염증과 함께 심한 가려움증 (pruritus)을 동반한다. 긁고 싶은 감정을 불러일으키는 가려움증은 히스타민과 같은 가려움 매개 물질에 의해서 유도되는데, 정상인의 경우 긁으면 그 증상이 쉽게 완화되지만, 아토피 환자의 경우 긁으면 긁을수록 더욱 긁고 싶은 감정이 형성되어 심하게 긁게 된다. 아토피 환자의 이러한 긁는 행동으로 인해서 피부장벽이 붕괴되고 2차 감염을 유발하여 염증이 더욱 악화된다. 아토피 피부염은 일시적으로 완화될 수 있지만, 환경 및 식품 등 자극원에 의해서 재발되어 악화되며, 악화와 완화가 반복되는 현상으로 그 원인은 아직까지 명확하게 알려지지 않았다. 이와 같이 아토피 피부염에 동반되는 염증은 과도한 면역세포 작용으로 인해 종양괴사인자-알파 (TNF-α, tumor necrosis factor-α)와 인터루킨-1 (IL-1, interleukin-l) 등의 염증성 사이토카인과 하이드록시 라디칼 (·OH), 슈퍼옥사이드 라디칼 (·O2 -), 과산화수소 (H2O2) 등과 같은 활성산소류 (ROS, reactive oxygen species)를 대량생산하기 때문에 주변 조직의 손상을 야기한다. 그러므로 아토피 피부염과 같은 피부질환을 개선시키기 위해서는 가려움증을 완화시키고 활성산소류를 효과적으로 제거할 수 있는 소재가 필요하다.Atopic dermatitis is accompanied by severe itching (pruritus) with chronic skin inflammation. The itching that causes feelings of scratching is induced by an itch medication such as histamine. In the case of a normal person, the symptoms are easily alleviated by scratching. However, in the case of atopy, These scratching behaviors of atopic patients may cause skin barriers to collapse and secondary infections leading to further deterioration of inflammation. Atopic dermatitis can be temporarily alleviated, but it recurs and is aggravated by stimulants such as the environment and food, and deterioration and mitigation are repeated, and the cause is not clearly known yet. The inflammation associated with atopic dermatitis is associated with inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1, interleukin-1) (ROS), such as hydroxyl radicals (OH), superoxide radicals (O 2 - ), hydrogen peroxide (H 2 O 2 ) and the like. Therefore, in order to ameliorate skin diseases such as atopic dermatitis, materials that can relieve itching and effectively remove active oxygen species are needed.
고욤나무(Diospyros lotus L.)는 우리나라와 중국을 비롯한 아시아에 분포하는 낙엽성 식물로 성숙한 과일을 고욤이라 하며 직접 섭취할 수 있다. 고욤은 전통의학에서 진정제(sedative), 수렴제(astringent), 항암(anti-tumor), 항당뇨(anti-diabetic), 해열제(febrifuge) 및 변비완화제(laxative) 등으로 사용되어 왔다. 최근에는 지방산, 당, 플라보노이드 및 비휘발성 성분이 알려지면서 혈액의 항응고, 뇌세포 보호 작용, 항산화 및 항암효과에 대한 보고가 있다.Goomae ( Diospyros lotus L.) is a deciduous plant distributed in Korea including Korea and China, and can be directly consumed as mature fruit. Goyom has been used in traditional medicine as a sedative, astringent, anti-tumor, anti-diabetic, febrifuge and laxative. Recently, fatty acids, sugars, flavonoids and nonvolatile components have been known, and anticoagulation, brain cell protection, antioxidant and anticancer effects of blood have been reported.
이에 본 발명자들은 고욤나무 잎으로부터 수용성 추출물과 메탄올 추출물을 얻고, 메탄올 추출물에서 에틸아세테이트(ethyl acetate, EA) 분획물을 얻어 각 추출물 및 분획물을 대상으로 DPPH (2,2-diphenyl-1-picrylhydrazyl) 및 ABTS (2,2(3-ethylbenzthiazoline- 6-sulfonic acid)) 라디칼 소거 활성을 조사하였다. 또한, 각 추출물 및 분획물을 대상으로 PMA (phorbol 12-myristate 13-acetate)와 칼슘 운반체 (calcium ionophore) A23187로 활성화된 랫트 복강 비만세포 (RPMCs, rat peritoneal mast cells)에서 TNF-α 생성 및 히스타민 방출량을 조사한 후 compound 48/80으로 가려움증을 유도한 동물모델에서 각 추출물과 분획물을 대상으로 가려움증 억제 효과를 조사한 결과 매우 유의한 결과를 얻었다.Thus, the present inventors obtained water-soluble extracts and methanol extracts from Gomam tree leaves and obtained ethyl acetate (EA) fractions from methanol extracts. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2 (3-ethylbenzthiazoline-6-sulfonic acid)) radical scavenging activity was investigated. In addition, TNF-α production and histamine release in rats peritoneal mast cells (RPMCs) activated with PMA (phorbol 12-myristate 13-acetate) and calcium ionophore A23187 in each extract and fraction , And the inhibitory effect of the extracts and fractions on the itchiness in the animal model induced by the compound 48/80 was very significant.
한편, 한국공개특허 제2013-0032099호에는 '카페오일알파네오엔돌핀 펩타이드 유도체 및 이의 항가려움 및 항아토피 소재로의 활용'이 개시되어 있고, 한국등록특허 제1231590호에는 '항바이러스, 항산화 및 항균 효과를 가지는 비파 추출물 및 이의 분획물'이 개시되어 있으나, 본 발명의 고욤나무 잎 메탄올 추출물의 에틸아세테이트 분획물을 유효성분으로 함유하는 아토피 피부염 개선용 조성물에 대해서는 기재된 바가 없다.Korean Patent Laid-Open Publication No. 2013-0032099 discloses a caffeoyl alpha neoendolphin peptide derivative and its use as anti-itching and anti-atopic substances, and Korea Patent No. 1231590 discloses an antiviral, antioxidant and antimicrobial Has been disclosed, but there is no description of a composition for improving atopic dermatitis containing the ethyl acetate fraction of the methanol extract of Gomam leaf of the present invention as an active ingredient.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 고욤나무 잎의 메탄올 추출물로부터 얻은 에틸아세테이트 분획물이 항산화 및 항가려움 효과가 있음을 확인함으로써, 본 발명을 완성하였다.The present invention has been made in view of the above-mentioned needs. The present inventors have completed the present invention by confirming that the ethyl acetate fraction obtained from the methanol extract of Gomamine leaf has an antioxidant and anti-itch effect.
상기 과제를 해결하기 위해, 본 발명은 고욤나무 (Diospyros lotus L.) 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 예방 또는 개선용 건강식품 조성물을 제공한다.In order to solve the above problems, the present invention provides a health food composition for preventing or ameliorating atopic dermatitis, which comprises a fraction of Dioscorea lotus L. leaf methanol extract as an active ingredient.
또한, 본 발명은 고욤나무 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, which comprises a fraction of methanol extract of Gomam leaf as an active ingredient.
또한, 본 발명은 고욤나무 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for improving atopic dermatitis containing a fraction of methanol extract of Gomam leaf as an active ingredient.
또한, 본 발명은 고욤나무 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 항산화용 조성물을 제공한다.In addition, the present invention provides a composition for antioxidant containing a fraction of methanol extract of Gomam leaf as an active ingredient.
본 발명에 따르면, 고욤나무 (Diospyros lotus L.) 잎 메탄올 추출물의 에틸아세테이트 분획물은 식물로부터 유래된 물질이므로 세포 독성이 적고, 라디칼 소거 활성을 보임과 동시에, 염증성 물질인 TNF-α의 생성 억제 및 히스타민의 방출 억제 효과 및 가려움 억제 효과가 우수하여, 항산화 활성 증가용 또는 가려움증 경감용 식품, 의약품 또는 화장품으로 유용하게 사용될 수 있으므로, 아토피 피부염과 같은 피부질환을 개선시키는데 유용하게 사용될 수 있다.According to the present invention, the ethylacetate fraction of Diospyros lotus L. leaf methanol extract is a plant-derived substance and thus has low cytotoxicity and shows radical scavenging activity, and inhibits the production of TNF-α, an inflammatory substance, Histamine release inhibitory effect and itching inhibitory effect and can be usefully used as a food for increasing antioxidant activity or for alleviating itching, medicines or cosmetics, and thus can be usefully used for improving skin diseases such as atopic dermatitis.
도 1은 고욤나무 잎의 수용성 추출물(water soluble extract), 메탄올 추출물(MeOH extract) 및 에틸아세테이트 분획물(EA fraction)의 DPPH(A)와 ABTS(B) 라디칼 소거 활성을 확인한 결과이다. 각 수치는 3회 반복 수행한 결과의 평균±표준오차를 나타낸다.
도 2는 고욤나무 잎의 수용성 추출물, 메탄올 추출물 및 에틸아세테이트 분획물의 세포 생존율에 미치는 영향을 확인한 결과로, (A)는 다양한 농도의 에틸아세테이트 분획물(EA)에 의한 랫트 복강 비만세포 (RPMCs)의 생존율에 미치는 영향을 분석한 결과이고, (B)는 동일 농도의 수용성 추출물(WS), 메탄올 추출물(MeOH) 및 에틸아세테이트 분획물(EA)에 의한 랫트 복강 비만세포의 세포 생존율을 분석한 결과이다. 정상군은 PMA와 A23187, 추출물 또는 분획물 등 무처리 세포군이고, 대조군은 추출물 또는 분획물 처리 없이 PMA와 A23187만 처리한 그룹이다. #p<0.001는 정상군에 대한 대조군의 유의성을 분석한 것이고, *p<0.01은 대조군에 대한 실험군의 유의성을 분석한 것이다.
도 3은 compound 48/80 처리에 의한 랫트 복강 비만세포로부터 분비되는 히스타민의 양에 대한 고욤나무 잎의 수용성 추출물, 메탄올 추출물 및 에틸아세테이트 분획물의 영향을 분석한 결과이다. (A)는 다양한 농도의 에틸아세테이트 분획물(EA) 처리에 따른 히스타민 분비량을, (B)는 동일한 농도의 수용성 추출물(WS), 메탄올 추출물(MeOH) 및 에틸아세테이트 분획물(EA) 처리에 따른 히스타민 분비량을 분석한 결과이다. 정상군은 compound 48/80, 추출물 또는 분획물 등 무처리 세포군이고, 대조군은 추출물 또는 분획물 처리 없이 compound 48/80만 처리한 그룹이다. #p<0.001는 정상군에 대한 대조군의 유의성을 분석한 것이고, *p<0.05, **p<0.01과 ***p<0.001은 대조군에 대한 실험군의 유의성을 분석한 것이다.
도 4는 PMA와 A23187 처리에 의한 랫트 복강 비만세포로부터 생산되는 TNF-α의 양에 대한 고욤나무 잎의 수용성 추출물, 메탄올 추출물 및 에틸아세테이트 분획물의 영향을 분석한 결과이다. (A)는 다양한 농도의 에틸아세테이트 분획물(EA) 처리에 따른 TNF-α 생산량을, (B)는 동일한 농도의 수용성 추출물(WS), 메탄올 추출물(MeOH) 및 에틸아세테이트 분획물(EA) 처리에 따른 TNF-α 생산량을 분석한 결과이다. 정상군은 PMA와 A23187, 추출물 또는 분획물 등 무처리 세포군이고, 대조군은 추출물 또는 분획물 처리 없이 PMA와 A23187만 처리한 그룹이다. #p<0.001는 정상군에 대한 대조군의 유의성을 분석한 것이고, *p<0.05, **p<0.01과 ***p<0.001은 대조군에 대한 실험군의 유의성을 분석한 것이다.
도 5는 compound 48/80에 의해 가려움증이 유도된 Balb/c 마우스에 대한 고욤나무 잎의 수용성 추출물, 메탄올 추출물 및 에틸아세테이트 분획물의 항가려움 효과를 분석한 결과이다. (A)는 다양한 농도의 에틸아세테이트 분획물(EA) 처리에 따른 마우스의 긁는 횟수를, (B)는 동일한 농도의 수용성 추출물(WS), 메탄올 추출물(MeOH) 및 에틸아세테이트 분획물(EA) 처리에 따른 마우스의 긁는 횟수를 분석한 결과이다. 정상군은 compound 48/80, 추출물 또는 분획물 등 무처리 세포군이고, 대조군은 추출물 또는 분획물 처리 없이 compound 48/80만 처리한 그룹이고, 아젤라스틴(azelastine) 또는 메티세르지드(methysergide)는 양성 대조군이다. #p<0.001는 정상군에 대한 대조군의 유의성을 분석한 것이고, *p<0.05, **p<0.01과 ***p<0.001은 대조군에 대한 실험군의 유의성을 분석한 것이다.FIG. 1 shows the results of DPPH (A) and ABTS (B) radical scavenging activities of water soluble extract, methanol extract and ethyl acetate fraction (EA fraction) Each value represents the mean ± standard error of the results that were repeated three times.
FIG. 2 shows the effect of various concentrations of ethyl acetate fraction (EA) on rat peritoneal mast cells (RPMCs) as a result of examining the effect of water extract, methanol extract and ethylacetate fraction on the cell viability (B) is the result of analysis of cell viability of rat peritoneal mast cells by water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction (EA) at the same concentration. The normal group was the untreated cell group such as PMA and A23187, the extract or fraction, and the control group was the group treated only with PMA and A23187 without treatment with the extract or fraction. #p <0.001 was the significance of the control group for the normal group, and * p <0.01 was the significance of the test group for the control group.
FIG. 3 shows the results of analysis of the effects of water-soluble extracts, methanol extracts and ethyl acetate fractions on the amount of histamine secreted from rat peritoneal mast cells by compound 48/80 treatment. (A) shows histamine secretion amount according to the treatment with various concentrations of ethyl acetate fraction (EA), (B) shows the amount of histamine secretion according to the treatment of water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction . The normal group was the compound 48/80, the untreated cell group such as the extract or the fraction, and the control group was the compound 48/80 only group treated without the extract or fraction. * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.
FIG. 4 shows the results of analysis of the effects of water-soluble extracts, methanol extracts and ethylacetate fractions on the amount of TNF-α produced from rat peritoneal mast cells by PMA and A23187 treatment. (A) shows the production of TNF-α by treatment with various concentrations of ethyl acetate fraction (EA) and (B) shows the production of TNF-α by treatment with aqueous extracts (WS), methanol extracts (MeOH) and ethyl acetate fractions TNF-α production. The normal group was the untreated cell group such as PMA and A23187, the extract or fraction, and the control group was the group treated only with PMA and A23187 without treatment with the extract or fraction. * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.
FIG. 5 shows the anti-itching effect of water-soluble extracts, methanol extracts and ethyl acetate fractions of Gomam leaf on Balb / c mice induced by itching by compound 48/80. (A) shows the number of scratching of the mouse according to the treatment with various concentrations of ethyl acetate fraction (EA), and (B) shows the number of scratching of the mouse according to the treatment of water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction It is the result of analyzing the number of scraping of the mouse. The normal group was the compound 48/80, the untreated group such as the extract or the fraction, the control group was the compound 48/80 only group treated without the extract or fraction, and the azelastine or methysergide was the positive control group . * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.
본 발명의 목적을 달성하기 위하여, 본 발명은 고욤나무 (Diospyros lotus L.) 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 예방 또는 개선용 건강식품 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides a health food composition for preventing or ameliorating atopic dermatitis, which comprises a fraction of Dioscorea lotus L. leaf methanol extract as an active ingredient.
본 발명의 일 구현 예에 따른 아토피 피부염 예방 또는 개선용 건강식품 조성물에서, 상기 고욤나무 잎 메탄올 추출물은 바람직하게는 채취한 고욤나무 잎을 물로 세척한 후 4~10분 동안 증기로 찌고, 실온에서 수분을 제거하고 건조기에서 35~45℃로 10~14시간 동안 건조한 후 건조된 고욤나무 잎 500~700 g에 3,000~5,000 ㎖의 메탄올을 넣고 5~10일간 반응시킨 후 추출물을 여과, 감압 및 동결 건조하여 제조될 수 있고, 더욱 바람직하게는 채취한 고욤나무 잎을 물로 세척한 후 5분 동안 증기로 찌고, 실온에서 수분을 제거하고 건조기에서 40℃로 12시간 동안 건조한 후 건조된 고욤나무 잎 600 g에 4,000 ㎖의 메탄올을 넣고 7일간 반응시킨 후 추출물을 여과, 감압 및 동결 건조하여 제조될 수 있으나, 이에 제한되는 것은 아니다.In the health food composition for preventing or ameliorating atopic dermatitis according to an embodiment of the present invention, the gomam leaf methanol extract is preferably prepared by washing collected gomam leaf with water, steaming the gomam leaf for 4 to 10 minutes, After removing water and drying at 35 ~ 45 ℃ for 10 ~ 14 hours, 3 ~ 5,000ml methanol was added to 500 ~ 700g of dried gomam leaf and allowed to react for 5-10 days. The extract was filtered, Dried, and more preferably, the collected gomam leaf is washed with water, steamed for 5 minutes, removed at room temperature, dried in a dryer at 40 ° C for 12 hours, g, 4,000 ml of methanol, reacted for 7 days, and then the extract is filtered, reduced in pressure and lyophilized, but is not limited thereto.
또한, 본 발명의 일 구현 예에 따른 조성물에서, 상기 고욤나무 잎 메탄올 추출물의 분획물은 바람직하게는 고욤나무 잎 메탄올 추출물에 에틸아세테이트(ethyl acetate)를 가하여 얻어진 에틸아세테이트 분획물일 수 있으나, 이에 제한되지 않는다.In addition, in the composition according to an embodiment of the present invention, the fraction of the methanol extract of Gomam leaf may be an ethyl acetate fraction obtained by adding ethyl acetate to the methanol extract of Gomam leaf, Do not.
본 발명의 상기 고욤나무 잎 메탄올 추출물의 에틸아세테이트 분획물은 DPPH 또는 ABTS 라디칼 소거 활성이 우수하고, 세포 독성이 낮으며, 알레르기 반응이나 염증에 관여하는 히스타민의 방출억제 효과가 우수하고, 전염증성 사이토카인(pro-inflammatory cytokine)인 TNF-α의 생성 억제 효과 또한 우수하며, 가려움증 경감 효과도 우수한 것으로 확인되어, 항산화 및/또는 항가려움증 효과를 통해 아토피 피부염을 예방 또는 개선할 수 있다.The ethyl acetate fraction of the methanol extract of Gomind tree leaf of the present invention is excellent in DPPH or ABTS radical scavenging activity, low in cytotoxicity, excellent in the inhibitory effect of histamine release which is involved in allergic reaction or inflammation, and proinflammatory cytokine the pro-inflammatory cytokine TNF-alpha is also excellently suppressed and the itching-relieving effect is also excellent. Thus, atopic dermatitis can be prevented or improved through antioxidant and / or anti-itching effects.
상기 건강식품은 아토피 피부염을 예방하거나 개선하기 위해 섭취할 수 있는 것이면 특별히 제한되지 않는다.The health food is not particularly limited as long as it can be ingested to prevent or ameliorate atopic dermatitis.
본 발명의 상기 분획물을 식품첨가물로 사용하는 경우, 상기 분획물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 분획물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the fraction of the present invention is used as a food additive, the fraction may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the fraction of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.
본 발명의 건강 음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 추출물 100 ㎖당 일반적으로 약 0.01~0.04 g, 바람직하게는 약 0.02~0.03 g일 수 있으나, 이에 제한되는 것은 아니다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention, but is not limited thereto.
상기 외에 본 발명의 분획물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 분획물은 천연 과일 주스, 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01~0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the fraction of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the fractions of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명은 고욤나무 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 예방 또는 치료용 약학 조성물을 제공한다. 본 발명의 약학 조성물에서, 고욤나무 잎 메탄올 추출물의 분획물은 상기 건강식품 조성물에서 기재한 바와 같다.In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, which comprises a fraction of methanol extract of Gomam leaf as an active ingredient. In the pharmaceutical composition of the present invention, the fractions of methanol extract of Gomam leaf are as described in the above-mentioned health food composition.
본 발명의 약학 조성물은 피부 외용제 조성물, 경구용 조성물 또는 주사제와 같은 비경구용 조성물일 수 있으며, 그 제형이 특별히 한정되지는 않지만, 피부 외용제 조성물의 경우에는 연고, 크림, 페이스트, 로션, 도찰제(liniment), 외용액제, 틴크제, 에어로졸, 경고제(plaster) 등의 형태일 수 있고, 경구용 조성물의 경우에는 환제, 캡슐제, 단제, 과립제, 드링크제 등의 형태로 제형화할 수 있다.The pharmaceutical composition of the present invention may be a composition for parenteral use such as a composition for external application for skin, an oral composition or an injection, and the formulation thereof is not particularly limited. In the case of composition for external application for skin, ointment, cream, paste, lotion, ), A liquid for external use, a tincture, an aerosol, and a plaster, and in the case of an oral composition, it may be formulated into a form such as a pill, a capsule, a single agent, a granule or a drink.
본 발명의 분획물을 포함하는 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용하여 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 분획물을 포함하는 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로오스 (sucrose) 또는 락토오스 (lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 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.The pharmaceutical compositions comprising the fractions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition including the fraction include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium A variety of compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 분획물의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage forms of the fractions of the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.
본 발명의 약학 조성물은 경구, 경피, 피하, 정맥, 복강, 근육, 국소도포, 첩포 및 이온토포레시스 (iontophoresis)를 포함한 여러 경로를 통해 투여될 수 있고, 이 중에서 국소 적용 및 경구투여가 바람직하다.The pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, intraperitoneal, muscle, topical application, patch and iontophoresis, and local application and oral administration are preferred Do.
또한, 본 발명에 의한 조성물에서 아토피 피부염에 대한 예방 또는 치료 용량 범위는 중증도 및 제형에 따라 변할 수 있으며, 적용 횟수도 환자의 연령, 체중 및 체질에 따라 변할 수 있다. 예를 들어, 본 발명의 피부 외용제 조성물의 투여량은 연령, 성별, 체중, 증상, 투여 방법에 의해 상이하나, 1일당 1.0 내지 3.0 ㎖로 이를 1일 1 내지 5회 도포하여 1개월 이상 계속하는 것이 좋다. 또한, 예를 들어, 본 발명의 경구용 조성물의 통상적인 1일 투여량은 1 내지 100 mg/kg체중, 바람직하게는 5 내지 70 mg/kg체중의 범위일 수 있고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 상기 투여량은 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.In addition, the dosage for preventing or treating atopic dermatitis in the composition according to the present invention may vary depending on the severity and formulation, and the number of application times may vary depending on the age, body weight, and constitution of the patient. For example, the dose of the composition for external application for skin of the present invention varies depending on age, sex, weight, symptom and administration method, but it is 1.0 to 3.0 ml per day, which is applied 1 to 5 times a day, It is good. In addition, for example, a typical daily dose of the oral composition of the present invention may range from 1 to 100 mg / kg body weight, preferably from 5 to 70 mg / kg body weight, divided once or several times Lt; / RTI > However, the dose does not limit the scope of the present invention in any way.
또한, 본 발명은 고욤나무 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 아토피 피부염 개선용 화장료 조성물을 제공한다. 본 발명의 화장료 조성물에서, 고욤나무 잎 메탄올 추출물의 분획물은 상기 건강식품 조성물에서 기재한 바와 같다.In addition, the present invention provides a cosmetic composition for improving atopic dermatitis containing a fraction of methanol extract of Gomam leaf as an active ingredient. In the cosmetic composition of the present invention, the fraction of the methanol extract of Gomam tree leaves is as described in the above health food composition.
본 발명의 일 구현에 따른 화장료 조성물에 있어서, 상기 아토피 피부염 개선용 화장료 조성물은 피부외용연고, 크림, 유연화장수, 영양화장수, 팩, 에센스, 헤어토닉, 샴푸, 린스, 헤어 컨디셔너, 헤어 트리트먼트, 젤, 스킨로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 마사지 크림, 영양크림, 아이크림, 모이스처 크림, 핸드 크림, 파운데이션, 영양에센스, 선스크린, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디 로션 및 바디 클렌저로 이루어지는 군으로부터 선택된 제형을 가질 수 있으나, 이에 제한되지 않는다. 이들 각 제형의 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for improving atopic dermatitis may be at least one selected from the group consisting of ointment for external use, cream, softener, nutritional lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, Skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing But are not limited to, a formulation selected from the group consisting of a foam, a cleansing lotion, a cleansing cream, a body lotion and a body cleanser. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산아연 등이 이용될 수 있다. When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components .
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.
본 발명의 일 구현예에 따른 화장료 조성물에 있어서, 상기 아토피 피부염 개선용 화장료 조성물은 상기 고욤나무 잎 메탄올 추출물의 에틸아세테이트 분획물을 상기 조성물의 총 중량을 기준으로 0.0001 내지 10% 함유되어 있을 수 있고, 바람직하게는 0.005 내지 1% 함유되어 있을 수 있으나, 바람직한 아토피 피부염의 개선 효과를 제공할 수 있는 유효량을 포함한다면 이에 제한되지 않는다.In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for improving atopic dermatitis may contain 0.0001% to 10% by weight of the ethyl acetate fraction of the methanol extract of Gomam tree leaf, based on the total weight of the composition, Preferably 0.005 to 1%, but is not limited thereto, including an effective amount that can provide a desired improvement effect of atopic dermatitis.
본 발명은 또한, 고욤나무 (Diospyros lotus L.) 잎 메탄올 추출물의 분획물을 유효성분으로 함유하는 항산화용 조성물을 제공한다. 본 발명의 항산화용 조성물에서, 상기 분획물은 바람직하게는 에틸아세테이트 분획물일 수 있으나, 이에 제한되지 않는다. 상기 분획물은 DPPH 및 ABTS 라디칼 소거 활성이 우수하였으므로, 우수한 항산화용 조성물로 이용될 수 있다.The present invention also provides a composition for antioxidation comprising as an active ingredient, a fraction of Leuconostoc spider ( Diospyros lotus L.) leaf methanol extract. In the antioxidant composition of the present invention, the fraction may be, but is not limited to, the ethyl acetate fraction. Since the fractions have excellent DPPH and ABTS radical scavenging activity, they can be used as an excellent antioxidant composition.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
시약 재료Reagent material
TNF-α 및 히스타민 측정을 위한 ELISA 키트는 R&D Systems사 (Minneapolis, MN, USA)로부터 구입하였으며, 퍼콜 (percoll), compound 48/80, PMA (phorbol 12-myristate 13-acetate), 칼슘운반체 (calcium ionophore) A23187, 페니실린/스트렙토마이신, 메티세르지드 말레이트 (MM, methysergide maleate), 부틸레이트 히드록시톨루엔 (BHT, butylated hydroxytoluene), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), 과황화칼륨 (potassium persulfate), 탄산수소나트륨 (sodium bicarbonate), 아젤라스틴 (azelastine), LPS (lipopolysaccharide), 메티세르지드 (methysergide), Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) 및 기타 시약은 reagent grade로 Sigma-Aldrich사 (Louis, MO, USA)로부터 구입하였다.An ELISA kit for the measurement of TNF-α and histamine was purchased from R & D Systems (Minneapolis, Minn., USA), and the percoll, compound 48/80, phorbol 12-myristate 13- acetate, calcium ionophore A23187, penicillin / streptomycin, methysergide maleate, butylated hydroxytoluene, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), potassium persulfate, sodium bicarbonate, azelastine, lipopolysaccharide, methysergide, , Trolox ((±) -6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and other reagents were purchased from Sigma-Aldrich (Louis, MO, USA) as a reagent grade.
고욤잎 추출Guillaume Leaf Extract
고욤 (Diospyros lotus L.) 잎은 전라북도 진안군 부귀면 수항리 신기마을에서 2013년 7월 10일에 채취한 후 우석대학교 한의과대학 본초방제학교실 김홍준 교수님으로부터 동정을 받았다. 고욤잎 표본(#2013-07-10)은 전주대학교 의과학대학 보건관리 연구실에 보관하고 있다. 채취된 고욤잎은 즉시 증류수로 세척한 후 5분간 증기찜을 한 후 실온에서 선풍기를 활용하여 건조한 후 최종적으로 건조기에서 40℃로 12시간 동안 건조하였다. 건조된 고욤잎은 500 g으로 정량하여 증류수 3,000 ㎖를 주입하고 10분간 끓인 후 추출물은 0.45 ㎛ 필터를 사용하여 여과하고 동결건조기 (EYELA FDU-2100, Japan)에서 건조하여 45.3 g을 얻었다. 또한 건조된 고욤잎을 600 g으로 정량하여 메탄올 4,000 ㎖을 주입하여 7일간 방치한 다음 추출물은 0.45 ㎛ 필터를 사용하여 여과하고 감압에 의해 유기용매를 제거하고 최종적으로 동결건조하여 70.3 g을 얻은 후 50 g을 취하여 에틸아세테이트(EA)를 충분히 주입하여(500 ㎖) 약 650 mg을 얻고 -20℃에서 보관하면서 실험에 사용하였다.The gourd ( Diospyros lotus L.) leaf was collected on July 10, 2013 in Shigi Village, Suwang-ri, Buyeon-myeon, Jinan-gun, Jeollabuk-do, and received sympathy from Professor Kim Hong-joon of Woosuk University School of Oriental Medicine. The goat leaf specimen (# 2013-07-10) is kept in the Health Management Laboratory of Chonju University Medical School. The collected gomilla leaf was immediately washed with distilled water, steamed for 5 minutes, dried using a fan at room temperature, and finally dried at 40 ° C for 12 hours in a dryer. The dried goat leaves were quantified as 500 g, 3,000 ml of distilled water was added and boiled for 10 minutes. The extract was filtered using a 0.45 ㎛ filter and dried in a freeze dryer (EYELA FDU-2100, Japan) to obtain 45.3 g. The dried goat leaves were weighed to 600 g, and 4,000 ml of methanol was added. After 7 days, the extracts were filtered using a 0.45 ㎛ filter, and the organic solvent was removed by reduced pressure. Finally, the filtrate was lyophilized to obtain 70.3 g 50 g of ethyl acetate (EA) was sufficiently injected (500 ml) to obtain about 650 mg of the compound, which was stored at -20 ° C.
DPPHDPPH 라디칼Radical 소거 활성 측정 Measurement of scavenging activity
DPPH 라디칼 소거 활성은 Blois (Nature, 1958, 181:1199-1200)의 방법으로 측정하였다. 시료를 메탄올로 녹여 최종 농도가 3.9-1,000 ㎍/㎖이 되도록 정량하여 96-웰 플레이트에 각 시료를 100 ㎕씩 주입하고, 동시에 0.3 mM DPPH 100 ㎕를 넣어 총량이 200 ㎕가 되도록 하였다. 실온에서 10분간 반응시킨 후 ELISA reader (Tecan Group Ltd., Mannedorf, Swizerland)로 540 nm 파장에서 흡광도를 측정하였다. DPPH 라디칼 소거 활성은 시료 용액의 첨가군과 무첨가군 사이의 흡광도의 차이를 백분율로 나타내었다.DPPH radical scavenging activity was measured by the method of Blois ( Nature , 1958, 181: 1199-1200). The sample was dissolved in methanol to give a final concentration of 3.9-1,000 .mu.g / ml. 100 .mu.l of each sample was injected into a 96-well plate, and 100 .mu.l of 0.3 mM DPPH was added to make a total amount of 200 .mu.l. After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm with an ELISA reader (Tecan Group Ltd., Mannedorf, Swizerland). The DPPH radical scavenging activity was expressed as a percentage of absorbance difference between the addition group and the no addition group of the sample solution.
DPPH 라디칼 소거 활성(%) = {1-(시료 첨가군 흡광도/시료 무첨가군 흡광도)} x 100DPPH radical scavenging activity (%) = {1- (absorbance of sample addition group / absorbance of sample addition group)} x 100
ABTSABTS 라디칼Radical 소거 활성 측정 Measurement of scavenging activity
ABTS 라디칼 소거능은 Re 등 (Free Radical Biol . Med . 1999, 26:1231-1237)의 방법에 의하여 측정하였다. 7.4 mM ABTS와 2.6 mM potassium persulfate를 혼합한 다음 빛이 차단된 실온에서 24시간 동안 방치하여 라디칼을 형성한 다음 ABTS 용액을 실험직전에 732 nm에서 흡광도가 0.70±0.03이 되도록 메탄올로 희석하여 사용하였다. 각 추출물과 Trolox의 최종농도가 3.9-1,000 ㎍/㎖이 되도록 정량하여 50 ㎕에 준비된 ABTS 용액 950 ㎕를 첨가하여 암소에서 30분간 반응시킨 후 732 nm에서 흡광도를 측정하였다. ABTS의 소거활성은 시료 용액의 첨가군과 무첨가군 사이의 흡광도의 차이를 백분율로 나타내었다.The ABTS radical scavenging activity was measured by the method of Re et al. ( Free Radical Biol . Med . 1999, 26: 1231-1237). 7.4 mM ABTS and 2.6 mM potassium persulfate were mixed and allowed to stand at room temperature for 24 hours to form radicals. The ABTS solution was diluted with methanol to an absorbance of 0.70 ± 0.03 at 732 nm immediately before the experiment . The final concentration of each extract and Trolox was determined to be 3.9-1,000 ㎍ / ㎖. After adding 950 ㎕ of ABTS solution to 50 ㎕, the reaction was allowed to proceed in the dark for 30 minutes, and the absorbance was measured at 732 nm. The scavenging activity of ABTS was expressed as a percentage of absorbance difference between the addition group and the no addition group of the sample solution.
ABTS 라디칼 소거 활성(%) = {1-(시료 첨가군 흡광도/시료 무첨가군 흡광도)} x 100ABTS radical scavenging activity (%) = {1- (absorbance of sample addition group / absorbance of sample addition group)} x 100
실험동물Experimental animal
무균환경에서 사육된 7주령의 수컷 Balb/c 마우스와 10주령의 Sprague-Dawley 랫트는 중앙실험동물(주)(서울시, 대한민국)에서 구입하였고, 사료와 물을 충분히 공급하면서 1주일간 순화시킨 후 실험에 사용하였다. 사육환경은 낮과 밤의 주기를 12시간씩 하였고, 온도(20-22℃)와 습도(50-60%)는 일정하게 유지하였으며, 실험동물위원회의 규정에 준하여 실험하였다.Seven weeks old male Balb / c mice and 10-week old Sprague-Dawley rats raised in sterile environment were purchased from Central Laboratory Animals (Seoul City, Korea), purified and fed for a week with sufficient feed and water, Lt; / RTI > The incubation environment consisted of day and night cycles for 12 hours, and the temperature (20-22 ℃) and humidity (50-60%) were kept constant and tested according to the provisions of the Laboratory Animal Committee.
RPMCsRPMCs 분리 및 고욤잎 Separation and Grouse leaf 분획물Fraction 처리 process
랫트 복강 비만세포 (RPMCs, rat peritoneal mast cells)의 분리는 Martynova 등 (Cell Res. 2005, 15:811-816)의 방법에 따라 분리하였다. 요약하면, 에테르로 랫트를 마취시킨 다음 칼슘 프리 HEPES-Tyrode 버퍼 (10 mM HEPES, 113 mM NaCl, 4.7 mM KCl, 2.13 mM MgCl2, 0.6 mM NaH2PO4, 10 mM glucose, pH 7.4) 10 ㎖을 복강에 주입시켜 약 90초간 복강을 부드럽게 맛사지 한 후 복강을 주의 깊게 열어 파스테르 피펫을 사용하여 세포부유액을 얻어 퍼콜 (percoll) 농도 구배법으로 RPMCs를 얻었다. 본 실험의 목적에 따라 충분한 세포를 확보하기 위해서 5~10마리 랫트에서 얻은 RPMCs를 혼합하여 사용하였다. 에틸아세테이트(EA) 분획물(5~20 ㎍/㎖)을 RPMCs (5x105)에 10분 또는 2시간 동안 전처리한 후, compound 48/80이나 PMA와 A23187로 자극하여 히스타민과 전염증성 사이토카인 (pro-inflammatory cyotokine) 측정에 사용하였다.Separation of rat peritoneal mast cells (RPMCs) was performed according to the method of Martynova et al. ( Cell Res . 2005, 15: 811-816). Briefly, ether rats were anesthetized and then anesthetized with 10 ml of calcium free HEPES-Tyrode buffer (10 mM HEPES, 113 mM NaCl, 4.7 mM KCl, 2.13 mM MgCl 2 , 0.6 mM NaH 2 PO 4 , 10 mM glucose, pH 7.4) Was infused into the abdominal cavity and gently massaged the abdominal cavity for about 90 seconds. The abdominal cavity was carefully opened, and a cell suspension was obtained using a pastel pipette to obtain RPMCs by a percoll concentration gradient method. For the purpose of this experiment, RPMCs obtained from 5 to 10 rats were mixed and used to obtain sufficient cells. Ethyl acetate (EA) fractions (5-20 μg / ml) were pretreated with RPMCs ( 5 × 10 5 ) for 10 min or 2 h and then stimulated with compound 48/80 or PMA and A23187 to induce histamine and proinflammatory cytokines -inflammatory cyotokine).
세포 생존율 측정Cell viability measurement
RPMCs의 세포 생존율은 MTT 어세이 방법에 의해 측정하였다. 요약하면, RPMCs (5x105/㎖) 세포를 24-웰 배양용기에 접종하고 상기와 같이 여러 가지 농도로 에틸아세테이트 분획물을 처리한 후에 PMA 및 A23187로 자극하고, 12시간 후에 MTT 용액 (5 ㎎/㎖)을 주입하고 4시간동안 37℃에서 방치한 후 상층액을 제거하고 포마잔 산물을 DMSO로 용해하여 96-웰 플레이트로 옮겨 540 nm에서 흡광도를 측정하였다.Cell viability of RPMCs was measured by MTT assay. In summary, RPMCs (5x10 5 / ㎖) inoculated with the cells in 24-well culture plate, and then treated with ethyl acetate fraction in various concentrations as described above and stimulated with PMA and A23187, after 12 hours MTT solution (5 ㎎ / Ml) was added, and the mixture was allowed to stand at 37 ° C for 4 hours. The supernatant was removed, and the formazan product was dissolved in DMSO, transferred to a 96-well plate, and absorbance was measured at 540 nm.
히스타민 방출량 측정Histamine release measurement
RPMCs (5x105/㎖)는 여러 가지 농도 (50-200 ㎍/㎖)의 고욤잎 유래 에틸아세테이트 분획물을 37℃에서 10분간 전처리한 다음 0.5 ㎍/㎖의 compound 48/80으로 자극하여 30분간 방치시키고, 반응을 중지시키기 위하여 배양튜브를 빙수에 넣어 충분히 냉각시킨 후 1,200 rpm으로 15분간 원심침전시키고 상층액을 얻고 히스타민을 측정하였다. 요약하면, 항-히스타민 항체를 활용하여 상기와 같이 얻은 상층액 시료에 반응시키고, 항-히스타민 항체에 특이적으로 작용하는 기질효소가 부착된 2차 항체를 주입한 후 발색시켜 R&D Systems사가 제공하는 방법에 따라 ELISA reader (Molecular Devices, USA)로 측정하였으며, 히스타민의 정량은 농도별로 히스타민을 처리하여 반응시킨 후 표준곡선을 정하여 계산하였다. RPMCs ( 5 × 10 5 / ml) were pretreated with ethylacetate fractions of various concentrations (50-200 μg / ml) at 37 ° C. for 10 minutes, stimulated with 0.5 μg / ml of compound 48/80 and left for 30 minutes To stop the reaction, the culture tube was placed in ice water and sufficiently cooled. After centrifugation at 1,200 rpm for 15 minutes, supernatant was obtained and histamine was measured. To summarize, an anti-histamine antibody is used to react with the supernatant obtained as described above, and a secondary antibody with a substrate enzyme specifically acting on the anti-histamine antibody is injected thereinto, followed by color development. The concentration of histamine was determined by histamine treatment and the standard curve was determined. The histamine concentration was measured by ELISA reader (Molecular Devices, USA).
TNFTNF -α 측정-α measurement
RPMCs로부터 사이토카인의 측정은 에틸아세테이트(EA) 분획물 (2.5-10 ㎍/㎖)를 2시간동안 전처리한 후 20 nM의 PMA와 1 μM의 A23187로 동시에 자극한 다음 12시간 후에 세포 상층액으로부터 TNF-α를 측정하였다. TNF-α는 항-마우스 TNF-α 항체를 사용하여 ELISA 키트를 사용하여 제조사가 제공하는 방법에 준하여 측정하고 정량하였다. 요약하면, 세포상층액 또는 5배 희석 혈청 100 ㎕를 항체가 코팅된 플레이트에 주입하고 반응시킨 후 잘 세척한 다음 HRP (horseradish peroxidase)가 부착된 2차 항체를 주입하고 반응시킨 후 발색 기질을 주입하고 반응시켜 ELISA reader로 측정하였으며, 각 물질에 대한 정량은 각각의 물질을 농도별로 처리하여 반응시켜 표준곡선을 정하고 세포상층액 또는 혈청에 함유된 물질의 량을 계산하였다.Cytokines from RPMCs were pretreated with an ethyl acetate (EA) fraction (2.5-10 μg / ml) for 2 hours, stimulated simultaneously with 20 nM PMA and 1 μM A23187, and then 12 hours later, TNF -α was measured. TNF-α was measured and quantified using an ELISA kit using the anti-mouse TNF-α antibody according to the method provided by the manufacturer. In summary, 100 μl of the supernatant or 5-fold diluted serum was injected into the plate coated with antibody, reacted, washed well, injected with secondary antibody with HRP (horseradish peroxidase), reacted, And the reaction was measured with an ELISA reader. The quantification of each substance was performed by treating each substance with a concentration to determine a standard curve, and the amount of substance contained in the supernatant or serum was calculated.
가려움증 유도 및 긁는 행동 측정Measures itching and scratching behavior
일주일간 순화시킨 Balb/c 마우스는 실험군당 5마리의 마우스를 각각 투명 아크릴 케이지(20cm)에 한 마리씩 넣고, 안정을 위해 30분 동안 동일한 실험환경에 방치하였다. 그 후 대조군은 생리식염수를 경구투여 하였고, 실험군은 에틸아세테이트(EA) 분획물(2.5-10 mg/kg)과 양성대조군인 메티세르지드(methysergide) 또는 아젤라스틴(azelastine)을 각각 10 mg/kg를 경구투여하고 60분 후에 compound 48/80 (50 ㎍/site) 또는 히스타민 (100 ㎍/site)의 농도로 100 ㎕씩 마우스 등의 양쪽 어깨 사이 높이에 피하주사 하였다. 가려움증 유발물질을 주사한 마우스는 곧바로 Mihara (Br. J. Dermatol. 2004, 151:335-345)의 방법을 따라 마이크로 카메라 (ONCCTV, 서울, 대한민국)를 사용하여 60분 동안 녹화하였으며, 뒷발로 가려움증 유발물질이 주입된 부위를 긁는 횟수를 이중맹검법으로 계수하여 평가하였다. 각 유발물질에 따른 실험은 각각 다른 날에 진행되었으며 매 실험에 사용된 마우스는 1회 사용되었다.Five weekly Balb / c mice were inoculated into a transparent acrylic cage (20 cm) each of five mice per experimental group and left in the same experimental environment for 30 minutes for stability. The control group was then administered orally with physiological saline and the experimental group was treated with 10 mg / kg of the ethyl acetate (EA) fraction (2.5-10 mg / kg) and the positive control methysergide or azelastine After 60 minutes of oral administration, 100 μl of compound 48/80 (50 μg / site) or histamine (100 μg / site) was subcutaneously injected at the level between both shoulders such as mice. Mice injected with the itch inducer were immediately recorded for 60 minutes using a micro camera (ONCCTV, Seoul, Korea) according to the method of Mihara ( Br. J. Dermatol . 2004, 151: 335-345) The number of times scratching the injected area was assessed by counting with a double blind method. Experiments on each inducer were conducted on different days and the mice used in each experiment were used once.
통계처리Statistical processing
모든 실험값은 평균±표준오차로 표시하였으며, 통계분석은 ANOVA와 Student's t-test로 처리하였으며, 유의성 한계는 p<0.05로 정하였다.Statistical analysis was performed with ANOVA and Student's t-test. The significance level was set at p <0.05.
실시예 1. 고욤잎 유래 에틸아세테이트 분획물의 DPPH 및 ABTS 라디칼 소거 활성 Example 1: Preparation of ethyl acetate fraction of goat's leaf DPPH and ABTS Radical scavenging activity
고욤잎 메탄올 추출물의 에틸아세테이트 분획물(EA)(이하, 고욤잎 유래 에틸아세테이트 분획물 또는 에틸아세테이트 분획물이라 칭함)의 DPPH와 ABTS 라디칼 소거 활성을 알아보기 위하여, 상기 에틸아세테이트 분획물과 고욤잎의 수용성 추출물 및 메탄올 추출물 그리고 합성 항산화제로 알려진 BHT(Butylated hydroxytoluene) 및 Trolox의 라디칼 소거 활성을 비교하였다. 그 결과 고욤잎 유래 에틸아세테이트 분획물은 모든 농도에서 수용성 추출물과 메탄올 추출물뿐만 아니라 BHT 보다도 DPPH 라디칼 소거 활성이 우수하였다(도 1A). 수용성 추출물과 메탄올 추출물의 경우도 31.3 ㎍/㎖ 이하의 저농도에서는 BHT와 유사한 수준의 DPPH 라디칼 소거활성을 보였지만 그 이상의 농도에서는 BHT 보다 DPPH 라디칼 소거 활성이 우수하게 확인되었다. 더불어 에틸아세테이트 분획물의 DPPH 라디칼 소거 활성에 대한 IC50은 5.3 ㎍/㎖로 수용성 수출물(37.9 ㎍/㎖), 메탄올 추출물(41.2 ㎍/㎖) 및 BHT(50.3 ㎍/㎖) 보다 약 5-7배 낮은 농도임이 확인되었다. 또한 ABTS 라다칼 소거 활성의 경우에서도, 고욤잎 유래 에틸아세테이트 분획물은 모든 농도에서 수용성 추출물과 메탄올 추출물뿐만 아니라 Trolox 보다도 ABTS 라디칼 소거 활성이 우수하였다. 수용성 추출물과 메탄올 추출물은 Trolox 보다 각 농도에서 ABTS 라디칼 소거 활성이 낮았지만 농도가 증가할수록 그 활성이 증가하는 경향을 보여주었다. 에틸아세테이트 분획물의 ABTS 라디칼 소거 활성에 대한 IC50은 53.8 ㎍/㎖로 수용성 수출물(311.2 ㎍/㎖), 메탄올 추출물(402.5 ㎍/㎖) 및 Trolox(178.6 ㎍/㎖) 보다 약 3-7배 낮은 농도에서 그 활성을 보여주었다(도 1B).To investigate the DPPH and ABTS radical scavenging activities of ethyl acetate fraction (EA) (hereinafter referred to as ethyl acetate fraction or ethylacetate fraction derived from gomule leaf) of methanol extract of Goat Leaf, the ethyl acetate fraction, Methanol extracts, and radical antioxidants BHT (butylated hydroxytoluene) and Trolox. As a result, the ethyl acetate fraction derived from goat leaf showed better DPPH radical scavenging activity than BHT, as well as water soluble extract and methanol extract at all concentrations (FIG. 1A). In the case of water - soluble extracts and methanol extracts, DPPH radical scavenging activity was similar to that of BHT at a low concentration of less than 31.3 ㎍ / ㎖, but DPPH radical scavenging activity was better than BHT at higher concentrations. In addition, the IC 50 for the DPPH radical scavenging activity of the ethyl acetate fractions was 5.3 ㎍ / ㎖, about 5-7 (w / w) more soluble than water soluble exports (37.9 ㎍ / ㎖), methanol extract (41.2 ㎍ / It was confirmed that the concentration was low. Also, in the case of ABTS radical scavenging activity, the ethyl acetate fraction derived from gomoma leaf showed better ABTS radical scavenging activity than Trolox as well as water soluble extract and methanol extract at all concentrations. Water soluble extracts and methanol extracts showed lower ABTS radical scavenging activity at each concentration than Trolox but increased activity as concentration increased. The IC 50 for the ABTS radical scavenging activity of the ethyl acetate fractions was 53.8 ug / ml, about 3-7 fold higher than the aqueous extract (311.2 / / ml), methanol extract (402.5 / / ml) and Trolox (178.6 / / And showed its activity at low concentrations (FIG. 1B).
이러한 고욤잎 유래 에틸 아세테이트 분획물의 DPPH 라디칼 소거 활성(IC50: 5.3 ㎍/㎖)은 고욤(D. lotus fruit) 추출물(72.6 ㎍/㎖)보다 높았으며 (Loizzo et al., 2009, Plant Foods Hum Nutr. 64:264-270), 고욤씨 유래 퀘세틴(5.8 ㎍/㎖)과 유사하였고(Moghaddam et al., 2012, Act Po Phar 69: 687-692), Zadra 등 (Molecules, 2012, 17:12560-12574)이 보고한 가지과에 속하는 Solanum guaraniticum (9.11 ㎍/㎖)보다 우수한 효과임이 확인되었다. 따라서, 고욤잎 유래 에틸아세테이트 분획물은 강력한 항산화 효과를 발휘할 수 있는 소재로 사료된다.The DPPH radical scavenging activity (IC 50 : 5.3 / / ㎖) of the ethyl acetate fraction derived from goat leaf was higher than that of D. lotus fruit (72.6 / / ㎖) (Loizzo et al., 2009, Plant Foods Hum Nutr 64:. 264-270), was similar to the seed derived goyom Quebec paroxetine (5.8 ㎍ / ㎖) (Moghaddam et al, 2012, Act Po Phar 69:. 687-692), Zadra etc. (Molecules, 2012, 17: 12560-12574) was found to be superior to Solanum guaraniticum (9.11 ㎍ / ㎖) belonging to the above group. Therefore, ethyl acetate fraction derived from goat 's leaf is considered to be able to exert a strong antioxidative effect.
실시예Example 2. 고욤잎 유래 에틸아세테이트 2. Ethyl acetate derived from goat leaf 분획물이The fraction 세포 생존율에 미치는 영향 Effect on cell survival rate
고욤잎 유래 에틸아세테이트 분획물의 농도에 따른 세포 생존율을 조사하기 위하여, 랫트 복강 비만세포 (RPMCs)를 활성화하기 위해 사용된 compound 48/80 (0.5 ㎍/㎖)을 처리하기 전에, 고욤잎 유래 에틸아세테이트 분획물 (25-100 ㎍/㎖), 수용성 추출물 또는 메탄올 추출물을 2시간동안 처리하여 MTT 어세이로 세포 생존율을 비교 조사하였다. 그 결과 도 2에서 보여지는 바와 같이 compound 48/80을 처리한 대조군은 정상군에 비해 세포독성이 있었지만, 에틸아세테이트 분획물을 처리한 모든 농도 구간에서는 정상군과 유사한 세포 생존율을 보였고, 더불어 수용성 추출물과 메탄올 추출물을 처리한 군에서도 정상군과 유사한 결과를 얻었다. 따라서 본 발명에서 사용한 모든 에틸아세테이트 분획물과 수용성 추출물 및 메탄올 추출물은 세포독성이 없음을 확인할 수 있었다.To investigate the cell survival rate according to the concentration of ethyl acetate fraction derived from goat leaf, before treatment of compound 48/80 (0.5 / / ml) used to activate rat peritoneal mast cells (RPMCs), ethyl acetate Fractions (25-100 ㎍ / ㎖), aqueous extracts or methanol extracts were treated for 2 hours to compare cell viability with MTT assays. As a result, as shown in FIG. 2, the compound 48/80-treated control group was more cytotoxic than the normal control group. However, the cell survival rate was similar to that of the normal control group at all concentration levels of the ethyl acetate fraction, Similar results were obtained in the methanol extract treated group. Therefore, it was confirmed that all ethyl acetate fractions, water-soluble extracts and methanol extracts used in the present invention had no cytotoxicity.
실시예Example 3. 고욤잎 유래 에틸아세테이트 3. Ethyl acetate from gomum leaf 분획물의Fraction 히스타민 방출 억제 효과 Histamine release inhibitory effect
고욤잎 유래 에틸아세테이트 분획물의 가려움증 유발의 핵심 물질인 히스타민 방출에 대한 억제 효과를 알아보기 위하여 랫트 복강 비만세포 (RPMCs)를 접종하고 에틸아세테이트 분획물 (25-100 ㎍/㎖)를 주입하고 37℃에서 10분간 전처리한 다음 compound 48/80 (0.5 ㎍/㎖)으로 자극하여 히스타민의 방출량을 측정하였다. 그 결과 도 3과 같이 RPMCs를 compound 48/80으로 자극할 경우 히스타민 방출량은 63.49±5.67 ng/㎖으로 정상 대조군(10.17±1.40 ng/㎖)에 비해서 현저히 증가하였다(p<0.001). 그러나 에틸아세테이트 분획물을 처리하였을 경우에는, 농도 의존적으로 히스타민의 방출을 억제하는 결과가 확인되었다. 특히 100 ㎍/㎖ 처리군에서는 히스타민 방출이 31.57±1.40 ng/㎖로 대조군에 비해서 약 47.6%가 억제되는 우수한 효과가 있었다(p<0.001). 또한 에틸아세테이트 분획물과 고욤잎 수용성 추출물 및 메탄올 추출물의 히스타민 방출 억제에 대한 효과를 비교하기 위해서 상기 분획물 및 각 추출물을 100 ㎍/㎖ 농도로 고정하여 실험한 결과, 도 3B에 나타난 바와 같이 고욤잎 수용성 추출물과 메탄올 추출물은 각각 48.97±3.70 ng/㎖과 46.87±3.54 ng/㎖로 유사하게 히스타민 방출억제 효과가 있었고(p<0.05), 에틸아세테이트 분획물은 고욤잎 수용성 추출물 및 메탄올 추출물 보다 히스타민 방출억제 효과가 우수한 것으로 확인되었다.(RPMCs) were inoculated and ethylacetate fractions (25-100 ㎍ / ㎖) were injected and incubated at 37 ℃ for 30 min at 37 ℃. The inhibitory effect of ethyl acetate fraction on the inhibition of histamine release, After pretreatment for 10 minutes, histamine release was measured by stimulation with compound 48/80 (0.5 / / ml). As a result, when the RPMCs were stimulated with compound 48/80, the histamine release was 63.49 ± 5.67 ng / ml, which was significantly higher than that of the normal control (10.17 ± 1.40 ng / ml) ( p <0.001). However, when the ethyl acetate fraction was treated, it was confirmed that the release of histamine was inhibited in a concentration-dependent manner. Especially, 100 ㎍ / ㎖ treated group had histamine release of 31.57 ± 1.40 ng / ㎖, which was about 47.6% lower than that of the control group ( p <0.001). In order to compare histamine release inhibition effects of the ethyl acetate fraction, the water soluble extract of methanol extract and methanol extract, the fraction and each extract were fixed at a concentration of 100 μg / ml. As a result, as shown in FIG. 3B, The extracts and methanol extracts showed 48.97 ± 3.70 ng / ㎖ and 46.87 ± 3.54 ng / ㎖ inhibition of histamine release (p <0.05), respectively. The ethyl acetate fraction showed higher histamine release inhibitory effect Respectively.
Compound 48/80은 비만세포를 활성화시키는 물질로 알려져 있는데, 활성화된 비만세포는 히스타민, 세로토닌 또는 substance P와 같은 가려움증 매개물질을 분비한다. 이러한 비만세포에서 분비되는 물질로 인해 유발되는 가려움증은 아토피 피부염 등 알레르기성 피부질환 환자에서 흔히 발견되는 가려움증과 유사한 증상을 유발한다. 따라서, 본 발명의 에틸아세테이트 분획물은 히스타민 방출억제 효과를 가지는, 알레르기성 피부질환에 적용될 수 있는 우수한 소재로 확인되었다.Compound 48/80 is known to activate mast cells, and activated mast cells secrete itching mediators such as histamine, serotonin or substance P. Itching caused by substances secreted from these mast cells leads to symptoms similar to itching, which is common in allergic skin diseases such as atopic dermatitis. Therefore, the ethyl acetate fraction of the present invention was confirmed to be an excellent material that can be applied to allergic skin diseases, which has an effect of inhibiting histamine release.
실시예Example 4. 고욤잎 유래 에틸아세테이트 4. Ethyl acetate derived from goom leaf 분획물의Fraction TNFTNF -α 생성 억제 효과-α generation inhibitory effect
고욤잎 유래 에틸아세테이트 분획물이 활성화된 랫트 복강 비만세포 (RPMCs)의 전염증성 사이토카인인 TNF-α의 생성 억제에 미치는 영향을 알아보기 위하여 RPMCs를 접종하고 에틸아세테이트 분획물 (25-100 ㎍/㎖)을 2시간 전처리한 다음 PMA (30 ng/㎖)와 A23187 (10 μM)을 동시에 처리하고 12시간 후 배양액에 축척된 TNF-α양을 측정하였다. 그 결과 도 4A와 같이 PMA와 A23187을 처리한 대조군은 정상군에 비해서 TNF-α의 생성이 594.51±28.99 pg/㎖로 현저히 증가(p<0.001)한 반면, 에틸아세테이트 분획물을 처리한 실험군는 농도 의존적으로 TNF-α 생성이 억제되었으며, 모든 농도에서 유의한 억제 효과가 확인되었다(p<0.05, p<0.001). 또한, 에틸아세테이트 분획물과 고욤잎 수용성 추출물 및 메탄올 추출물의 TNF-α 생성 억제에 대한 효과를 비교하기 위해서 100 ㎍/㎖ 농도로 고정하여 실험한 결과, 도 4B에 나타난 바와 같이 고욤잎 수용성 추출물과 메탄올 추출물은 각각 430.45±31.87 pg/㎖과 450.45±27.74 pg/㎖로 유사하게 TNF-α 생성 억제 효과가 있었고(p<0.05), 에틸아세테이트 분획물은 고욤잎 수용성 추출물 및 메탄올 추출물 보다 TNF-α 생성 억제 효과가 우수하였다.In order to investigate the effect of acetoacetate fraction derived from goat's leaf on the inhibition of the production of TNF-α, a proinflammatory cytokine of activated rat peritoneal mast cells (RPMCs), RPMCs were inoculated and fractionated with ethyl acetate (25-100 ㎍ / Was pretreated for 2 hours, and PMA (30 ng / ml) and A23187 (10 μM) were simultaneously treated. After 12 hours, the amount of TNF-α accumulated in the culture solution was measured. As shown in FIG. 4A, the production of TNF-α was significantly increased in the control group treated with PMA and A23187 (594.51 ± 28.99 pg / ㎖) ( p <0.001) TNF-α production was inhibited, and significant inhibition was observed at all concentrations (p <0.05, p <0.001). In addition, in order to compare the effect of the ethyl acetate fraction on the inhibition of TNF-α production of the water extract of Guoyom leaf and the methanol extract, it was fixed at a concentration of 100 μg / ml. As a result, as shown in FIG. 4B, (P <0.05), whereas the ethyl acetate fraction inhibited TNF-α production more than watery extracts and methanol extracts of gom- yle leaves (p <0.05) The effect was excellent.
TNF-α는 생체의 염증반응을 야기하는 대표적인 사이토카인으로 주로 활성화된 대식세포 (macrophages)에서 대량 생산되지만, 림포이드계의 세포 (lymphoid cells), 비만세포, 내피세포 (endothelial cells) 등을 비롯하여 생체에 존재하는 다양한 세포에서도 생산된다. 특히 비만세포는 PMA와 A23187로 동시에 자극할 경우 TNF-α가 대량 생산되는 것으로 잘 알려져 있다. 그러므로 염증반응을 완화하기 위해서는 TNF-α를 효과적으로 제어할 수 있는 물질이 필요한 바, 본 발명의 고욤잎 유래 에틸아세테이트 분획물은 이런 관점에서 염증반응을 제어할 수 있는 효과적인 물질이라 사료된다.TNF-α is a typical cytokine that causes inflammatory responses in vivo, and is mainly produced in macrophages, which are mainly activated. However, lymphoid cells such as lymphoid cells, mast cells, endothelial cells and the like It is also produced in various cells present in the body. In particular, mast cells are known to be produced in large quantities when TNF-α is stimulated simultaneously with PMA and A23187. Therefore, in order to alleviate the inflammatory reaction, a substance capable of effectively controlling TNF-a is required. Thus, the ethyl acetate fraction derived from the gomoma leaf of the present invention is considered to be an effective substance capable of controlling the inflammatory reaction in this respect.
실시예Example 5. 고욤잎 유래 에틸아세테이트 5. Ethyl acetate derived from goom leaf 분획물의Fraction 가려움 억제 효과 Itching inhibitory effect
고욤잎 유래 에틸아세테이트 분획물이 피부 가려움증 억제에 미치는 영향을 알아보기 위하여 에틸아세테이트 분획물 (2.5-10 mg/kg)과 수용성 추출물 및 메탄올 추출물 (각각 10 mg/kg)을 Balb/c 마우스에 경구투여하고 1시간 후에 가려움 유발물질 compound 48/80을 마우스의 등 양쪽 어깨 사이 높이에 피하주사하였다. 그 결과 compound 48/80을 피하에 주입한 마우스는 긁는 횟수가 255.3±39.5회/60분으로 정상 대조군 (32.3±4.9회/60분)에 비해서 긁는 횟수가 현저히 증가하였다(도 5A, p<0.001). 그러나 에틸아세테이트 분획물 처리 실험군은 농도 의존적으로 가려움증을 억제하는 효과가 확인되었다. 특히 고욤잎 유래 에틸아세테이트 분획물 10 mg/kg 투여군에서는 긁는 횟수가 120.7±24.2회/60분으로 약 53% 억제된 효과가 있었다(p<0.001). 항가려움증 효과가 우수한 약물로 알려진 아젤라스틴 (azelastine)과 메티세르지드 (methysergide)와 같은 농도에서 비교하였을 때 에틸아세테이트 분획물의 항가려움증 효과는 아젤라스틴 보다는 낮게 확인되었지만, 메티세르지드 보다는 높게 확인되었다(도 5B).(2.5-10 mg / kg), aqueous extracts and methanol extracts (10 mg / kg, respectively) were orally administered to Balb / c mice in order to investigate the effect of ethyl acetate fraction derived from goat leaf on skin itching inhibition After 1 hour, the itch inducer compound 48/80 was injected subcutaneously at the level between the shoulders of the back of the mouse. As a result, mice injected with compound 48/80 subcutaneously showed a greater number of scratches than those of the normal control group (32.3 ± 4.9 times / 60 minutes) with 255.3 ± 39.5 times / 60 minutes of scratching (FIG. 5A, p <0.001 ). However, the experimental group treated with ethyl acetate fraction showed inhibitory effects on the concentration - dependent manner. Especially, in the group treated with 10 mg / kg ethyl acetate fraction derived from goat leaf, the number of scratching was 120.7 ± 24.2 times / 60 minutes, which was about 53% (p <0.001). The anti-itching effect of the ethyl acetate fraction was found to be lower than that of azelastine, but was found to be higher than that of methicillinide at concentrations such as azelastine and methysergide, which are known to be excellent anti-itching drugs 5B).
아젤라스틴은 H1 히스타민 수용체에 대한 길항제로로 알려진 항가려움증 약물로 히스타민과 substance P 뿐만 아니라 compound 48/80과 같은 가려움 유발물질로 유도된 가려움증을 효과적으로 완화하는 것으로 알려졌다. 또한 메티세르지드도 compound 48/80 유도 가려움증을 완화시킬 수 있는 약물로 잘 알려져 있다. 추후 에틸아세테이트 분획물을 대상으로 가려움 억제에 대한 분자적 기전을 규명할 필요가 있겠지만, 상기의 결과들을 토대로, 본 발명의 고욤잎 유래 에틸아세테이트 분획물의 우수한 항가려움증 효과는 아토피 피부질환과 같은 피부가려움증에 사용될 수 있는 매우 우수한 소재라 사료된다.Azelastine is an anti-itching drug known as an antagonist to the H1 histamine receptor and is known to effectively alleviate itching induced by itch inducers such as compound 48/80 as well as histamine and substance P. Metisergide is also well known as a drug that can relieve compound 48/80 induced itching. Although it may be necessary to elucidate the molecular mechanism of the inhibition of itching in the later ethyl acetate fraction, based on the above results, the excellent anti-itching effect of the ethyl acetate fraction derived from the goat leaf of the present invention is effective for skin itching such as atopic skin disease It is considered to be a very good material that can be used.
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