KR102013457B1 - Composition having activating effect for ampk - Google Patents
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- KR102013457B1 KR102013457B1 KR1020120088283A KR20120088283A KR102013457B1 KR 102013457 B1 KR102013457 B1 KR 102013457B1 KR 1020120088283 A KR1020120088283 A KR 1020120088283A KR 20120088283 A KR20120088283 A KR 20120088283A KR 102013457 B1 KR102013457 B1 KR 102013457B1
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- ampk
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- A23V2200/00—Function of food ingredients
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Abstract
본 발명은 AMPK 활성화 효과를 가지는 조성물에 관한 것으로서, 특히 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물로 이루어지는 군으로부터 1종 이상을 유효성분으로 포함하는 것을 특징으로 하는 AMPK 활성화 효과를 갖는 조성물로 비만, 2형 당뇨, 대사증후군, 알츠하이머형 치매 및 암 등의 예방 또는 치료에 효과적이다.The present invention relates to a composition having an AMPK activating effect, in particular bone clavicle, palsy, cerebral circumference, gukcheok, Bupyeongcho, Bleeding, tail light, erosion, Chungho, Hakseul, Geunpi, spectators, Kwanhwa, Journey room, cinnamon, etc. , Betel nut, smoke, sine, gall bladder, early morning, etc., a composition with AMPK activation effect, characterized in that it comprises at least one species from the group consisting of extracts and bark, obesity, type 2 diabetes, metabolic syndrome, Alzheimer's disease It is effective in preventing or treating dementia and cancer.
Description
본 발명은 AMPK 활성화 효과를 가지는 조성물에 관한 것으로서, 보다 상세하게는 비만, 2형 당뇨, 대사증후군의 치료 또는 예방에 효과적인 AMPK활성화제에 관한 것이다. The present invention relates to a composition having an AMPK activating effect, and more particularly to an AMPK activator effective for the treatment or prevention of obesity, type 2 diabetes, metabolic syndrome.
AMPK 활성화제는 AMPK(6'AMP-활성화 단백질 카이네이즈; 5‘ 아데노신 모노포스페이트-활성화 카이네이즈)의 활성을 증가시키는 물질로, AMPK는 세포 에너지 항상성에 중요한 역할을 하는 효소이다. AMPK를 활성화시킴으로써, 2형 당뇨, 비만, 대사증후군 등의 예방 또는 치료 효과를 나타낼 수 있다는 것이 알려져 있어, AMPK 활성화제에 대한 연구가 지속되고 있다. [AMPK and the biochemistry of exercise : Implications for human health and disease, Biochem J 418(2), 261-275(2009), Mechani는 linking obesity, chronic kidney disease, and fatty liver disease : the roles of fetuin-A, adiponectin, and AMPK, J Am Soc Nephrol 2010 Mar ;(21) 3 : 406-12]AMPK activators are substances that increase the activity of AMPK (6'AMP-activated protein kinase; 5 'adenosine monophosphate-activated kinase), and AMPK is an enzyme that plays an important role in cellular energy homeostasis. By activating AMPK, it is known that the prophylactic or therapeutic effect of type 2 diabetes, obesity, metabolic syndrome, etc. can be exhibited, and studies on AMPK activators continue. AMPK and the biochemistry of exercise: Implications for human health and disease, Biochem J 418 (2), 261-275 (2009), Mechani has linkeding obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK, J Am Soc Nephrol 2010 Mar; (21) 3: 406-12]
한편, 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물과 관련하여 AMPK 활성화 효과는 알려진 바 없다.
On the other hand, bone clavicle, scorpio, cerebral circumference, guchu, Bupyeongcho, suture, taillight, erosion, cheongho, school, hardship, spectator, Kwanhwahwa, journey room, cinnamon lamp, earlobe, betel nut, fellowship, autograph, bully, headlight, There is no known effect of AMPK activation with respect to the shrunken and bark extracts.
본 발명은 상기한 바와 같이 종래 AMPK 활성화 효과가 알려진 바 없는 다양한 추출물로부터 AMPK 활성화 효과가 우수한 추출물을 선발하여 비만, 2형 당뇨, 대사증후군 등의 예방 또는 치료에 효과적인 조성물을 제공하는 데 있다.The present invention is to provide an effective composition for the prevention or treatment of obesity, type 2 diabetes, metabolic syndrome and the like by selecting an extract having excellent AMPK activation effect from a variety of extracts that are not known conventional AMPK activation effect as described above.
상기와 같은 기술적 과제를 해결하기 위하여, 본 발명은 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물로 이루어지는 군으로부터 1종 이상을 유효성분으로 포함하는 것을 특징으로 하는 AMPK 활성화 효과를 같는 조성물을 제공한다.In order to solve the technical problems as described above, the present invention is a bone clam, palsy, cerebral circumference, gukcheok, Bupyeongcho, Byeonpyeong, taillights, Jero, Cheongho, Hakseul, Geunpi, spectators, Kwanhwahwa, Journey room, cinnamon, etc. It provides a composition having the same AMPK activation effect, characterized in that it comprises at least one species as an active ingredient from the group consisting of betel nut, pontoon, sine, gall bladder, head, etc., single axis and tree extract.
상기 AMPK 활성화제는 비만, 2형 당뇨, 알츠하이머형 치매, 대사증후군 및 암 중에서 선택된 하나 이상의 치료 또는 예방용일 수 있다.The AMPK activator may be for the treatment or prevention of one or more selected from obesity, type 2 diabetes, Alzheimer's dementia, metabolic syndrome and cancer.
상기 AMPK 활성화제는 약학 조성물 또는 식품조성물일 수 있다. 상기 식품조성물에 있어서, 치료 또는 예방은 개선 또는 방지를 포함하는 의미이다. 상기 식품조성물은 건강기능식품일 수 있으며, 제형은 통상의 방법에 따라 제조하며, 담체와 함께 건조한 후 갭슐화하거나 기타 정제, 과립, 분말, 음료, 죽 등의 형태로 제형화할 수 있으며, 상기 기재한 것 외에도 모든 식품 형태로 제조 가능하다.The AMPK activator may be a pharmaceutical composition or a food composition. In the food composition, the treatment or prevention is meant to include improvement or prevention. The food composition may be a health functional food, and the formulation may be prepared according to a conventional method, and then dried with a carrier and then encapsulated or formulated in the form of other tablets, granules, powders, beverages, porridges, and the like. In addition to one can be produced in all food forms.
상기 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물는 AMPK 활성화 효능을 발휘한다. 따라서, 본 발명은 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물의 AMPK 활성화 용도를 제공한다.The bone clasp, saengpunggi, cerebral circumference, gukcheok, Bupyeongcho, Byeonpyeong, taillights, erosion, Cheongho, Hakseul, hard blood, spectators, Kwanhwahwa, Journey room, cinnamon lamps, guinea pigs, betel nut, fellowships, autographs, offspring, head, etc. And the bark extract exhibits AMPK activation efficacy. Therefore, the present invention is a bone clavicle, scorpion, brain circumference, gukcheol, Bupyeongcho, suture, trailing light, erosion, cheongho, school chain, cinnabar, spectators, kantohwa, journey room, cinnamon lamp, ear woowoo, betel nut, fellowship, cause of death, gall bladder , Morninglight, uniaxial and bark extracts for AMPK activation.
또한, 본 발명은 약학적으로 유효한 양의 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물를 필요로 하는 포유류(인간을 포함함)에게 투여하는 단계를 포함하는 AMPK 활성화 방법을 제공한다.In addition, the present invention is a pharmaceutically effective amount of bone clavicle, saenggi, encephalopathy, gukjeol, Bupyeongcho, bongsu, trailing light, erosion, Chungho, Hakseul, Geunpi, spectators, Kwanhwahwa, journey room, cinnamon, ear cow, betel nut It provides a method for AMPK activation comprising the step of administering to mammals (including humans) in need, fragility, sine, gall bladder, head, etc., defecation and bark extract.
별도의 언급이 없는 한, 본 발명의 AMPK 활성화제에 관한 내용은 본 발명의 AMPK 활성화 용도, AMPK 활성화제 제조 용도, AMPK 활성화 방법에도 동일하게 적용된다.Unless otherwise stated, the content of the AMPK activator of the present invention is equally applicable to the AMPK activating use, AMPK activating agent preparation, and AMPK activating method of the present invention.
상기 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물과 그를 포함하는 AMPK 활성화제는 인간을 포함한 포유류에 경구 또는 비경구로 투여가능하며, 유효성분을 약학적으로 허용되는 담체와 함께 배합하여 제제화하여 투여할 수 있다. 제제화할 경우 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제 및 계면활성제 등의 희석제 또는 부형제를 상ㅇ할 수 있다. 경구투여를 위한 고형제제는 정제, 환제, 산제, 과립제, oqtbf제 등이 포함되며, 이러한 고형제제는 상기의 AMPK 활성화물질에 적어도 하나 이상의 부형제 예를 들며, 전분, 탄산칼슘, 수크로스, 락토오스, 및 젤라틴 등을 참가하여 제조한다. 또한, 마그네슘, 탈크 등 윤활제도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 및 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 주사가능한 액제, 현탁제, 유제, 동결건조제, 비강세척제 및 좌제가 포함된다.
The bone clasp, saengpunggi, cerebral circumference, gukcheok, Bupyeongcho, Byeonpyeong, taillights, erosion, Cheongho, Hakseul, hard blood, spectators, Kwanhwahwa, Journey room, cinnamon lamps, guinea pigs, betel nut, fellowships, autographs, offspring, head, etc. And AMPK activator comprising the extract and the AMPK activator thereof may be administered orally or parenterally to mammals, including humans, it can be administered in combination with a pharmaceutically acceptable carrier to formulate. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used may be employed. Solid preparations for oral administration include tablets, pills, powders, granules, oqtbf, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, And gelatin. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories.
본 발명의 조성물은 AMPK 활성화 효과가 우수하며, 그로 인해 비만, 2형 당뇨, 대사증후군 등의 예방 또는 치료에 효과적이다. The composition of the present invention is excellent in the AMPK activating effect, thereby effective in the prevention or treatment of obesity, type 2 diabetes, metabolic syndrome and the like.
도 1은 관중추출물의 AMPK 활성화 효능의 평가 결과이다.1 is an evaluation result of the AMPK activation efficacy of the spectral extract.
이하 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 AMPK 활성화 효과를 가지는 조성물은 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물로 이루어지는 군으로부터 1종 이상을 유효성분으로 포함하는 것을 특징으로 한다.The composition having the AMPK activating effect of the present invention is bone clavicle, saengpunggi, encephalopathy, gukjeol, Bupyeongcho, Bleeding, taillight, erosion, Cheongho, Hakseul, Geunpi, spectators, Kwanhwahwa, journey room, cinnamon, ear cow, betel nut, It is characterized in that it comprises at least one species as an active ingredient from the group consisting of edible, sine, gall bladder, head, etc., single axis and tree extract.
상기 골쇄보는 고란초과의 곡궐(Drynaria fortunei Smith)의 뿌리줄기이며, 대풍자는 산유자나무과의 대풍자(Hydnocarpus anthelmintica Pierre) 또는 동속 근연식무의 씨이며, 뇌환은 구멍장이버섯과의 뇌환(Omphalia lapidescens Schroet)의 균핵이며, 구척은 구척과의 금모구척( Cibotium barometz J. Smith)의 뿌리줄기이며, 부평초는 개구리밥과의 개구리밥(Spirodela polyrhiza Schleider) 또는 좀개구리밥(Lemna paucicostata Hegelm)의 전초이며, 봉출은 생강과의 아출(Curcuma zedoaria Roscoe)의 뿌리줄기이며, 미후등은 다래나무과의 다래나무(Actinidia arguta Planchon ex Miquel)의 덩굴이며, 여로는 백합과의 여로(Veratrum nigrum L. var. ussuriense Loes. fil.)의 뿌리줄기이며, 청호는 국화과의 개똥쑥(Artemisia apiacea Hance)의 지상부이며, 학슬은 국화과의 담배풀(Carpesium abrotanoides)의 과실이며, 고련피는 멀구슬나무(Melia azedarach L. var. japonica Makino)의 껍질이며, 관중은 면마과 관중(Dryopteris crassirhizoma Nakai)의 뿌리줄기이며, 관동화는 국화과 관동(Tussilago farfara L.)의 꽃이며, 여정실은 물푸레나무과의 광나무(japanes privet)의 열매이며 , 계혈등은 콩과의 밀화두(Spatholobus suberectus Dunn)의 작은 가지이며, 귀전우는 노박덩굴과의 화살나무(Euonymus alatus)의 가지부위이며, 빈랑자는 야자과 빈랑나무(Areca catechu Linne)의 종자이며, 연교는 물푸레나무과의 개나리(Forsythia koreana Nakai)의 과실이며, 사인은 생강과의 사인(Amomum xanthioides Wallich)의 과실이며, 오배자는 오배자(Chinensis Galla)이며, 조구등은 꼭두서니과의 조구등(Uncaria sinensis (Olv.) Havil)의 가지이며, 편축은 마디풀과의 마디풀(Polygonum aviculare Linne)의 전초이며 및 자목은 뽕나무과의 꾸지뽕나무(Cudrania tricuspidata)의 원줄기이다.The bone clasp is the root stem of Drynaria fortunei Smith, the sapling is the seed of Hydnocarpus anthelmintica Pierre, or the same plant, and the encephalopathy is the brain disease of Ophalia lapidescens Schroet. It is the fungal nucleus of, Gucheok is the root stem of Cibotium barometz J. Smith, Bupyeongcho is the outpost of Spirodela polyrhiza Schleider or Lemna paucicostata Hegelm, and the extract is ginger The root stem of Curcuma zedoaria Roscoe, the trailing tail is the vine of the Actinidia arguta Planchon ex Miquel, and the trail is the Veratrum nigrum L. var.ussuriense Loes.fil. It is the root of the tree, Cheongho is the ground part of Artemisia apiacea Hance of the Asteraceae, the crane is the fruit of the Carpesium abrotanoides of the Asteraceae, and the Melia azed Arach L. var. japonica Makino, the bark of which is the rhizome of Dryopteris crassirhizoma Nakai, the Kanto is the flower of the Tussilago farfara L., and the journey is the japanes privet It is the fruit of the fruit, the cinnamon is the small branch of Spatholobus suberectus Dunn, the deciduous tree is the branch of Euonymus alatus, and the betel is the tree of Areca catechu Linne. Seed, Falconus, Fruit of Forsythia koreana Nakai, Sign of Fruit of Amomum xanthioides Wallich, Sign of Ginja (Chinensis Galla), Head of Bulb (Uncaria sinensis) (Olv.) Havil, a short axis is the outpost of Polygonum aviculare Linne and the bark is the main stem of the Mulberry family Cudrania tricuspidata.
상기 약재는 통상의 방법에 따라 추출물로 제조할 수 있으며, 예를 들어 증류수, 탄소수 1~4개의 무수 또는 함수 저급알코올과 같은 유기용매로 추출한 후 감압 농축하는 통상의 방법으로 제조될 수 있다.The medicinal herb can be prepared as an extract according to a conventional method, for example, can be prepared by a conventional method of extracting with an organic solvent such as distilled water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms and concentrated under reduced pressure.
상기 약재들의 단일 또는 혼합추출물은 본 발명의 조성물에 각각 0.00000001 내지 20 중량%로 포함되는 것이 바람직하며, 더욱 바람직하게는 0.000001 내지 10 중량%로 포함되는 것이다. 그 함량이 0.000001 중량% 미만일 경우에는 뚜렷한 효과를 기대할 수 없다. Single or mixed extracts of the medicines are preferably included in the composition of the present invention in the amount of 0.00000001 to 20% by weight, more preferably 0.000001 to 10% by weight. If the content is less than 0.000001% by weight, no obvious effect can be expected.
상기와 같은 본 발명의 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물을 유효성분으로 함유하는 AMPK 활성화 효과를 가지는 조성물의 제형은 특별히 제한되지 않으며, 목적하는 바에 따라 적절히 선택할 수 있다. Bone clavicle of the present invention as described above, saenggihwan, cerebral circumference, gukcheol, Bupyeongcho, suture, taillights, erosion, cheongho, haengseul, pilgrimage, spectators, Kwanhwahwa, journey room, cinnamon lamp, ear woowoo, betel nut, fellowship, signature, The formulation of the composition having the AMPK activating effect containing the gall, mortar, etc., the short axis and the bark extract as an active ingredient is not particularly limited and may be appropriately selected as desired.
이하, 본 발명의 이해를 돕기위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.
Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.
[실시예]EXAMPLE
실시예Example 1 ~ 23 1 to 23
건조된 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목을 각각 잘게 세절하였다. 세절한 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 각 100g당 에탄올과 초순수 혼합액(혼합비율 에탄올/초순수 = 3/7) 500ml를 가한 후, 상온에서 7일간 숙성시켰다. 그 다음, 상기 추출물들을 여과지로 여과한 다음, 감압하에서 농축하여 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물을 각각 제조하였다.
Dried bone clavicle, Daegukgi, encephalopathy, Gucchi, Bupyeongcho, Bongchul, Taillight, Yeo, Cheongho, Hakseul, Geunpi, spectators, Kwanhwahwa, Journey room, Cinnamon lamp, guinea pig, betel nut, Fellowship, autograph, bully, headlight, The short axis and the bark were finely divided. Fine bone crest, scorpio, brain circumference, gukcheok, Bupyeongcho, suture, taillight, erosion, chungho, school, pilgrimage, spectators, Kanto, travel room, cinnamon lamp, ear cow, betel nut, fellowship, autograph, bully, head, 500 ml of a mixture of ethanol and ultrapure water (mixed ratio ethanol / ultra pure water = 3/7) was added to each 100 g of shrunk and hardwood, and aged at room temperature for 7 days. Then, the extracts were filtered through a filter paper, and then concentrated under reduced pressure, followed by bone chain bolster, saengpung, cerebral circumference, gukcheok, bupyeongcho, suture, taillight, erosion, cheongho, haengseul, kojipi, spectators, kantohwa, journey room, cinnamon, etc. Ear cow, betel nut, pontoon, sine, gall bladder, head bulb, etc., shrunk and tree extract were prepared, respectively.
[실험예]Experimental Example
실험예Experimental Example 1. One. AMPKAMPK 활성화 효과 확인 Check the activation effect
1-1. 시료의 준비1-1. Sample Preparation
상기 실시예 1 내지 23에서 제조한 골쇄보, 대풍자, 뇌환, 구척, 부평초, 봉출, 미후등, 여로, 청호, 학슬, 고련피, 관중, 관동화, 여정실, 계혈등, 귀전우, 빈랑자, 연교, 사인, 오배자, 조구등, 편축 및 자목 추출물을 세포실험의 경우 diemthyl sulfoxide(DMSO)에 용해시켜 사용하였고, 동물실험의 경우 sodium carboxymethyl cellulose에 용해시켜 30mg/kg의 농도로 투여하였다.
Bolchibo, prepared in Examples 1 to 23, saenggi, encephalopathy, gukcheol, Bupyeongcho, Bulgo, taillights, Yeo, Cheongho, Hakseul, Geunpi, spectators, Kwanhwahwa, Journey room, cinnamon lamp, ear cow, betel nut, poncho In the case of cell experiments, sine, gall bladder, crude bulbs, etc., extracts and extracts were used in the dissolution of diemthyl sulfoxide (DMSO) for cell experiments and in the concentration of 30mg / kg in sodium carboxymethyl cellulose.
1-2. 세포의 준비 및 처리1-2. Preparation and Processing of Cells
3T3-L1 지방전구세포를 ATCC(American Type Culture Collection)에서 구입하여 사용하였다. 세포배양은 10% FBS(fetal bovine serum), 페니실린(100U/ml), 스트렙토마이신(100U/ml)을 포함한 Dulbecco's modified Eagle's medium(DMEM) 배지(Gibco)를 사용하였고, CO2 배양기에 섭씨 37도, 5% CO2 조건에서 배양하였다. 3T3-L1 preadipocyte는 분화유도 배지 (5% fetal bovine serum, 0.5 mM 이소부틸메틸잔틴, 1mM 덱사메타존 및 10 μg/mL 인슐린이 포함된 DMEM)와 각각의 추출물(0.000005%, 0.00001%)를 함께 처리 하여 2일 간격으로 교환하고 총 4일 동안 배양한 후 10 μg/mL 인슐린이 포함된 DMEM으로 교환하여 2일을 더 배양하였다. 그 후 FBS만 포함된 DMEM으로 2일 배양하여 총 8일 동안 분화 유도하였다.
3T3-L1 adipocytes were purchased from the American Type Culture Collection (ATCC). Cell culture was performed using Dulbecco's modified Eagle's medium (DMEM) medium (Gibco), including 10% FBS (fetal bovine serum), penicillin (100 U / ml), streptomycin (100 U / ml), and 37 degrees Celsius in a CO2 incubator. Incubated at 5% CO2 conditions. 3T3-L1 preadipocytes were combined with differentiation induction medium (DMEM with 5% fetal bovine serum, 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone and 10 μg / mL insulin) and their respective extracts (0.000005%, 0.00001%) The treatment was exchanged at 2 days intervals and cultured for a total of 4 days, and then further exchanged with DMEM containing 10 μg / mL insulin for 2 more days. Thereafter, two days of incubation with DMEM containing only FBS induced differentiation for a total of 8 days.
1-3. AMPK 활성화 효과 확인1-3. AMPK activation effect confirmed
상기 1-2.에서 준비한 지방세포를 이용하여 웨스턴블롯 방법으로 AMPK 활성화 효과를 확인하기 위한 실험을 하였다.Using the adipocytes prepared in the above 1-2. Was tested to determine the effect of AMPK activation by Western blot method.
단백질 분석을 위해 지방세포를 라이시스버퍼(lysis buffer)로 균질화 하였다. 단백질 정량은 Bio Rad assay reagent (Bio-Rad, USA)를 이용하여 측정하였으며, 정량한 단백질 20 μg을 8% SDS-PAGE로 분리하였다. 이 후 젤을 멤브레인(Milipore, Cat. No: IPVH00010)에 옮기고 5% 스킴밀크로 상온에서 1시간 블로킹하였으며 1:1000 비율로 희석시킨 1차 항체(P-AMPK, Cell Signaling, 2531)와 4℃에서 밤샘반응하였다. Tris-buffered saline tween-20 (TBST)로 3번 세척한 후 1:2000 비율로 희석시킨 2차 항체(Santa Cruz, sc-2313)와 상온에서 1시간 반응시켰다. 이후 TBST로 3번 세척하고 ECL 용액(Amersham, Sweden)을 이용하여 X-ray 필름에 현상하였다. 그 결과를 하기 표 1에 나타내었다. Adipose cells were homogenized with lysis buffer for protein analysis. Protein quantification was measured using a Bio Rad assay reagent (Bio-Rad, USA), and 20 μg of the quantified protein was isolated by 8% SDS-PAGE. The gel was then transferred to a membrane (Milipore, Cat.No: IPVH00010) and blocked for 1 hour at room temperature with 5% skim milk and diluted to 1: 1000 with primary antibody (P-AMPK, Cell Signaling, 2531) and 4 ° C. Reaction overnight. After washing three times with Tris-buffered saline tween-20 (TBST) and reacting with a secondary antibody (Santa Cruz, sc-2313) diluted 1: 2000 at room temperature for 1 hour. It was then washed three times with TBST and developed on X-ray film using ECL solution (Amersham, Sweden). The results are shown in Table 1 below.
상기 표 1에서 보는 바와 같이, 본 발명의 실시예 1 내지 23은 매우 강력한 AMPK 활성화 효능을 나타내었으며, 이와 관련된 비만, 대사증후군, 2형 당뇨 등의 치료 또는 예방에 효과적임을 알 수 있다. As shown in Table 1, Examples 1 to 23 of the present invention showed a very strong AMPK activation effect, it can be seen that it is effective in the treatment or prevention of obesity, metabolic syndrome, type 2 diabetes and the like.
1-4. 동물의 준비 및 처리1-4. Preparation and Processing of Animals
5주령 수컷 ICR(imprinting control region) 마우스를 1주일간 적응시킨 후 실험에 사용하였다. 식사방법인 과식(overfeeding)과 과밀도 식사(energy dense diet)를 제공하여 비만을 유도하는 DIO(diet-induced obese) 모델로 변행시켰다. 실험군은 정상식이(10% Kcal% fat, Research Diets)를 제공한 대조군, 고지방식이(45% Kcal % fat, Research Diets)를 섭취시킨 고지방시이군, 고지방식이와 실시예 1~23)을 투여한 투여군으로 나누었다. 투여는 하루에 한번 2주간 경구로 투여하였으며, 체중과 혈당, 혈중 인슐린, 트리글리세라이드, 총 콜레스테롤, HDL-콜레스테롤, 혈장 nonesterified fatty acid(NEFA)는 실험 종료시 측정하였다. Five-week-old male imprinting control region (ICR) mice were acclimated for one week and used in the experiment. The dietary methods of overfeeding and energy dense diets were provided to make a diet-induced obese (DIO) model. The experimental group was a control group provided with a normal diet (10% Kcal% fat, Research Diets), a high fat diet group fed with a high fat diet (45% Kcal% fat, Research Diets), a high fat diet and Examples 1 to 23). The administration was divided into administration groups. Administration was orally administered once a week for two weeks. Body weight, blood sugar, blood insulin, triglycerides, total cholesterol, HDL-cholesterol, and plasma nonesterified fatty acid (NEFA) were measured at the end of the experiment.
체중은 체중계로 측정하고, 혈당은 SMARTLAB(Erba, USA)기기로 측정하였다. 혈중 인슐린, 트리글리세라이드 등의 함량은 각각 심장에서 혈액을 채취한 후 원심분리하여 혈청을 취하여 ELISA reader assay로 측정하였다.
Body weight was measured by a scale and blood glucose was measured by a SMARTLAB (Erba, USA) device. Insulin, triglyceride, etc. in the blood were measured by ELISA reader assay after taking blood from the heart and centrifuging serum.
1-5. 항비만 효과 확인1-5. Anti-obesity effect confirmed
상기 1-4.에서 처리한 마우스에 대한 체중 측정 결과를 다음 표 2에 정리하였다.Weight measurement results for the mice treated in 1-4. Are summarized in Table 2 below.
상기 표 2에서 보는 바와 같이, 고지방식이군은 대조군에 비해 체중 증가량이 현저하여 비만이 유도되었음을 확인할 수 있다. 고지방식이군과 동일한 조건에서 각 추출물들을 투여한 경우 체중 증가량이 감소함을 알 수 있다. 따라서, 상기의 추출물들은 항비만 효과를 나타냄을 알 수 있다.
As shown in Table 2, the high-fat diet group can be confirmed that the weight gain is significant compared to the control group obesity induced. When the extracts are administered under the same conditions as the high fat diet group, it can be seen that the weight gain decreases. Therefore, it can be seen that the extracts exhibit an anti-obesity effect.
1-6. 항당뇨 효과 확인1-6. Confirm antidiabetic effect
상기 1-4.에서 처리한 마우스에 대한 혈당과 혈중 인슐린 측정 결과를 다음 표 3에 정리하였다.Blood glucose and blood insulin measurement results for the mice treated in 1-4. Are summarized in Table 3 below.
상기 표 3에서 보는 바와 같이, 고지방식이군은 대조군에 비해 혈당과 혈중 인슐린이 대조군에 비해 높은 농도로 나타나지만, 약재추출물의 투여군은 농도가 감소함을 알 수 있다. 따라서, 본 발명의 추출물들은 혈당 감소, 혈중 인슐린 농도 감소 드에 의해 2형 당뇨, 대사증후군 등의 치료 또는 예방에 효과적임을 알 수 있다.As shown in Table 3, the high-fat diet group showed a higher concentration of blood glucose and blood insulin than the control group, but the concentration of the medicinal extract administration group can be seen to decrease. Therefore, it can be seen that the extracts of the present invention are effective for the treatment or prevention of type 2 diabetes, metabolic syndrome, etc. by reducing blood sugar and reducing blood insulin levels.
1-7. 혈액 지표 분석1-7. Blood indicator analysis
상기 1-4.에서 처리한 마우스에 대한 NEFA, 총콜레스테롤, 고밀도콜레스테롤, 트리글리세리드의 측정 결과를 하기 표 4에 정리하였다.Measurement results of NEFA, total cholesterol, high density cholesterol, and triglycerides for the mice treated in 1-4. Are summarized in Table 4 below.
(mg/dL)Total cholesterol
(mg / dL)
(mg/dL)HDL-cholesterol
(mg / dL)
(mg/dL)TG
(mg / dL)
상기 표에서 보는 바와 같이, 고지혈증, 동맥경화 등과 관련된 콜레스테롤의 경우 고지방식이군에서 대조구에 비해 증가하나, 본 발명의 추출물들의 투여에 의해 감소하는 경향을 나타내었다. 또한 중성지방과 관련된 혈중 유리지방산 NEFA의 경우 고지방식이군에서 대조군에 비해 증가하나, 본 발명의 물질 투여에 의해 감소함을 알 수 있다. 따라서 본 발명의 추출물들은 metabolic sensor에 해당하는 단백질인 AMPK의 활성화와 그로 인한 지방산 산화촉진 혈당치 및 지질 프로파일이 개선되는 활성 등을 나타내어, 이와 관련된 비만, 2형 당뇨, 대사증후군 등의 치료 또는 예방에 효과적임을 알 수 있다.As shown in the table, the cholesterol associated with hyperlipidemia, arteriosclerosis, etc. increased in the high fat diet group compared to the control group, but showed a tendency to decrease by administration of the extracts of the present invention. In addition, in the case of triglyceride-associated free fatty acid NEFA in the high fat diet group, compared to the control group, it can be seen that the decrease by the administration of the present invention. Therefore, the extracts of the present invention show the activation of AMPK, a protein corresponding to the metabolic sensor, and the resulting fatty acid oxidation-promoting blood glucose level and lipid profile. It can be seen that effective.
Claims (3)
At least one therapeutic or prophylactic pharmaceutical composition selected from obesity, type 2 diabetes mellitus, and metabolic syndrome, characterized in that it has an AMPK (5 'adenosine monophosphate-activated protein kinase) activating effect.
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