KR100934022B1 - Composition comprising an active ingredient isolated from Bogolgol extract or Bogolgol extract excellent in estrogen activity - Google Patents
Composition comprising an active ingredient isolated from Bogolgol extract or Bogolgol extract excellent in estrogen activity Download PDFInfo
- Publication number
- KR100934022B1 KR100934022B1 KR1020070102066A KR20070102066A KR100934022B1 KR 100934022 B1 KR100934022 B1 KR 100934022B1 KR 1020070102066 A KR1020070102066 A KR 1020070102066A KR 20070102066 A KR20070102066 A KR 20070102066A KR 100934022 B1 KR100934022 B1 KR 100934022B1
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- KR
- South Korea
- Prior art keywords
- fractions
- extract
- dichloromethane
- column chromatography
- bakuchiol
- Prior art date
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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)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/487—Psoralea
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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
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
본 발명은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물에 관한 것이다.The present invention relates to a composition comprising an active ingredient isolated from the Bogolji extract or Bogolji extract excellent in estrogen activity.
본 발명은 인위적으로 에스트로겐을 투여하는 호르몬 보충요법을 대체할 수 있는 천연물 유래의 보다 안전하고 효과가 우수하며 피토에스트로겐(phytoestrogen)으로 사용할 수 있는 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물을 제공하는데 목적이 있다.The present invention is safer and more effective from natural products that can replace hormonal supplementation for artificially administering estrogens, and isolated from Bogolji extract or Bogolji extract with excellent estrogen activity that can be used as phytoestrogen. It is an object to provide a composition comprising one active ingredient.
보골지, 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone) Bongol, bakuchiol, babachromene, isobavachromene, babachalcone, isobavachalcone
Description
본 발명은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물에 관한 것이다.The present invention relates to a composition comprising an active ingredient isolated from the Bogolji extract or Bogolji extract excellent in estrogen activity.
여성은 난소기능이 점차 감소하는 갱년기가 40세 이후부터 서서히 시작되며 여성의 평균수명이 점차 연장됨에 따라 호르몬 결핍기간은 수명의 약 1/3 내지 1/2을 차지하게 되었다. 난소로부터의 여성호르몬의 생성이 줄어들면서 폐경의 유발되고 여러 가지 갱년기 여성호르몬 결핍 증상을 경험하는 여성은 전체 여성의 20％에 달한다. 갱년기 여성의 대부분이 안면홍조, 빈맥, 발한, 불안 등의 여성호르몬 급성결핍증상을 경험하고 만성적인 결핍으로 인하여 심혈관질환, 골다공증 등의 만성 질환을 초래하기 때문에 폐경기 여성에 대한 치료의 필요성이 점차 부각되고 있다. 특히 폐경기 이후의 여성에게 심장질환은 가장 큰 사망원인 중 하나이며 폐경 후의 에스트로겐 감소는 급속한 골 손실을 초래하게 된다.In women, menopausal age gradually decreases after the age of 40, and the hormonal deficiency period accounts for about one third to one half of life expectancy. As women produce less hormones from the ovaries, 20% of all women experience menopause-induced menopausal female hormone deficiency. Most menopausal women experience acute deficiency symptoms of female hormones such as hot flashes, tachycardia, sweating, and anxiety, and chronic deficiencies cause chronic diseases such as cardiovascular disease and osteoporosis. It is becoming. Especially in postmenopausal women, heart disease is one of the leading causes of death, and postmenopausal estrogen reduction causes rapid bone loss.
여성의 갱년기 증상의 치료를 위한 방법으로 인위적으로 에스트로겐을 투여하는 호르몬 보충요법(Hormone Replacement Therapy: HRT)이 이용되고 있다. 구체적으로는 에스트로겐 단독투여, 에스트로겐과 프로게스트론의 병용 투여 방법이 주로 이용된다. 그러나 에스트로겐의 투여는 갱년기 증상 개선에는 효과적이나 에스트로겐 투여시 자궁암 또는 유방암의 발생률을 높인다는 연구결과가 발표되고, 오심, 신체 통증, 복부 팽만감, 부종 등 여러 부작용이 따르며 특히 장기간 사용시 유방암, 정맥혈전증, 담낭질환 발생의 위험률이 높아진다고 밝혀졌다. 따라서 보다 안전하고 효과있는 에스트로겐 대체 물질이나 선택적 에스트로겐 결합조절제(SERM: selective estrogen receptor modulater)의 개발이 활발히 연구되어 왔다.Hormone Replacement Therapy (HRT), which is an artificially administered estrogen, is used as a method for treating women's menopausal symptoms. Specifically, the administration of estrogen alone and the combined administration method of estrogen and progesterone are mainly used. However, estrogen administration is effective for improving menopausal symptoms, but studies have shown that estrogen increases the incidence of uterine cancer or breast cancer, and there are many side effects such as nausea, physical pain, bloating, and edema, especially breast cancer, venous thrombosis, The risk of developing gallbladder disease has been shown to increase. Therefore, the development of safer and more effective estrogen substitute or selective estrogen receptor modulator (SERM) has been actively studied.
대표적인 SERM의 화합물인 랄록시펜(raloxifen)은 현재 골다공증 치료제로 인정되어 사용되고 있는 물질로서(Mark et al., Am. J. Physiol. Cell Physiol., 279:C1550-1557, 2000), 뼈에 선택적으로 에스트로겐 작용을 나타내면서 자궁에 미치는 영향은 거의 없는 것으로 보고되었다. 그러나 랄록시펜은 안면홍조 악화 및 혈전증이 발생될 가능성이 높은 것으로 보고되었고, 현재 합성에스트로겐 물질들을 대체할 수 있는 천연물 유래의 보다 안전하고 효과있는 피토에스트로겐(phytoestrogen) 물질의 개발이 요구되고 있다.Raloxifen, a representative compound of SERM, is a substance currently recognized and used as a therapeutic agent for osteoporosis (Mark et al., Am. J. Physiol. Cell Physiol., 279: C1550-1557, 2000) It has been reported that there is little effect on the uterus with its effect. However, raloxifene has been reported to have a high possibility of developing hot flashes and thrombosis, and there is a need for developing a safer and more effective phytoestrogen substance derived from natural products that can replace synthetic estrogen substances.
최근 미국 FDA는 호르몬 대체요법(HRT)에 쓰이는 에스트로겐, 프로게스틴 등 모든 호르몬 제제에 '복용 후 심장병, 심장마비, 뇌졸중, 유방암에 걸릴 위험이 높 아질 수 있다'는 내용의 강력한 경고문을 부착하도록 하였는데 이는 국립보건연구원(NIH)이 2002년 7월 폐경 여성들이 자주 복용하는 호르몬제제인 '프렘프로'가 심장병, 심장마비, 뇌졸중, 유방암의 위험을 증가시킨다는 발표를 한 데 이어 취해진 것이다. 따라서 갱년기 장애 및 호르몬 결핍성 만성질환 예방을 위하여 호르몬 치료를 받아야 하는 환자 및 여성들은 큰 혼란에 빠져 있는 상태이다. 호르몬 대체요법의 대안으로 여러 치료법이 제시되고 있으며 특히 NIH는 여성호르몬 활성을 나타내는 식물유래의 이소플라본, 피토에스트로겐을 함유하고 있는 승마, 레드 클로버의 시험사용을 허가하고 있다. Recently, the U.S. Food and Drug Administration (FDA) has issued a strong warning to all hormone products, including estrogen and progestin, used in hormone replacement therapy (HRT), which may increase the risk of heart disease, heart attack, stroke, and breast cancer after use. It was taken after the National Institutes of Health (NIH) announced in July 2002 that prempro, a hormone product often taken by menopausal women, increases the risk of heart disease, heart attack, stroke and breast cancer. Therefore, patients and women who need to undergo hormonal therapy to prevent menopausal disorders and hormonal deficiency chronic diseases are in a state of great confusion. As an alternative to hormone replacement therapy, several therapies have been proposed, and in particular, NIH authorizes the use of plant-derived isoflavones, phytoestrogens, horse riding, and red clover, which show female hormone activity.
피토에스트로겐은 여성호르몬 에스트로겐과 기능과 구조가 유사한 식물유래의 화합물로서, 식물의 줄기, 뿌리, 꽃이나 종자에서 발견된다. 피토에스트로겐으로 알려진 물질로는 콩과 칡의 이소플라본 류(isoflavone), 클로버와 알팔파의 쿠메스탄 류(coumestan), 아마종자의 리그난 류(lignan)가 있다. 이소플라본 중에서는 몇 종류만이 에스트로겐 활성을 나타내며 다이드제인(daidzein), 제니스테인(genistein)과 함께 그들의 배당체인 다이드진(daidzin), 제니스틴(genistin), 그들의 methyl ethyl 유도체인 포모노네틴(formononetin)과 바이오카닌 A(biochanin A)가 대표적이다. 클로버(clover)도 다이드제인, 바이오카닌 A 및 포모노네틴을 높은 수준으로 함유하고 있다. 인도 등에서 즐겨 먹는 콩류(chick pea)에는 포모노네틴 또는 바이오카닌이, 태국에서 많이 소비되는 칡에는 푸에라린(pueralin)과 디옥시미로에스트롤이 들어 있다. 이 외에도 식물 유래의 사포닌(saponin)이나 터페노이드(terpenoid) 계열 물질도 일부 에스트로겐 효과를 갖고 있어 승마(black cohosh)나 감초(licorice)의 에스트로겐 효과를 나타내는 주요 성분이다. 그 외 플라바논(flavanones), 플라본(flavones), 디하이드로칼콘(dihydrocalchones) 등 다양한 화합물에서 활성이 기대되나 관련 연구가 부족한 실정이다.Phytoestrogens are plant-derived compounds with similar functions and structures to female hormone estrogens, found in the stems, roots, flowers and seeds of plants. Known phytoestrogens include isoflavones of legumes, coumestans of clover and alfalfa, and lignans of flax seeds. Only a few of the isoflavones show estrogen activity and formononetin, which is their glycosides such as daidzin, genistin and their methyl ethyl derivatives, together with diedzein and genistein. ) And biochanin A. Clover also contains high levels of dydze, biocanin A and pomononetin. Commonly eaten in chick peas, such as pomononetin or biocanine, and Thailand, which consumes a lot of puerin (pueralin) and deoxymyroestrol. In addition, saponins or terpenoids derived from plants also have some estrogen effects, which is a major component of the estrogen effect of horse riding (black cohosh) or licorice (licorice). In addition, the activity is expected in a variety of compounds, such as flavanone (flavanones), flavones (flavones), dihydrocalchones (dihydrocalchones), but there is a lack of research.
보골지(補骨脂, Psoralea fructus)는 콩과(Leguminosae)에 속하는 일년생 초본인 보골지(개암풀, Psoralea coylifolia L.)의 씨(seed)를 일컫는 것으로 그 형태가 콩팥 모양으로 납작하다. 한방에서는 비신허(??腎虛)에 사용되며, 관상동맥 확장제, 자궁출혈, 백반병, 탈모, 조갑진균증 등에 사용된다고 알려져 있다. 보골지에 함유되어 있는 주요화학성분은 지질, 정유성분 이외에 프소라렌(psoralene), 이소프소라렌(isopsoralene) 및 바쿠치올(bakuchiol)이 있으며 이중 모노테르펜 페놀(monoterpene phenol) 구조를 갖는 바쿠치올은 다음과 같은 많은 약리활성이 보고되어 주목을 받아왔다.Bogolgol (補骨脂, Psoralea fructus) refers to the seed of the annual herb, Bogolji (Hazelweed, Psoralea coylifolia L.) belonging to the legume (Leguminosae), its shape is flat. In oriental medicine, it is used for non-belief, and it is known to be used for coronary artery dilator, uterine bleeding, vitiligo, hair loss, and fungal fungal disease. The main chemicals contained in golgol are lipids and essential oils. In addition to psoralene, isopsoralene and bakuchiol, bakuchiol having a monoterpene phenol structure is as follows. Many of the same pharmacological activities have been reported and attracted attention.
일본 특허공고 제11-71231호는 바쿠치올이 티로시나아제(tyrosinase)를 저해할 수 있어 피부 미백효과를 가지는 화장품에 유용하다고 기술하고 있다. Japanese Patent Publication No. 11-71231 describes that bakuchiol can inhibit tyrosinase and is useful for cosmetics having a skin whitening effect.
대한민국 특허 KR2000-0007648호에 의하면 보골지 추출물 또는 보골지 유효성분이라고 알려져 있는 바쿠치올(bakuchiol)을 함유하는 항염증 효능을 갖는 화장료에 대한 발명이 개시되어 있다. Korean Patent KR2000-0007648 discloses a cosmetic having an anti-inflammatory effect containing bakuchiol known as golgogi extract or golgogi active ingredient.
대한민국 특허공보 KR2003-0081810호에 의하면 보골지 추출물을 함유하는 뇌세포 보호 및 뇌졸중 치료를 위한 조성물에 대한 발명이 개시되어 있다. Korean Patent Publication No. KR2003-0081810 discloses a composition for protecting the brain cells and the treatment of stroke containing golgol extract.
바쿠치올은 여성 골다공증 치료용 약제학적 조성물(US 2005/0256208, 대한민 국 특허 공개 10-2006-0047787), 유방암 치료용 약제학적 조성물(US 2005/0256208)이 보고된바 있다. 바쿠치올외 보골지의 유효성분들의 효능에 관한 연구는 미약한 실정이다. Bakuchiol has been reported a pharmaceutical composition for treating female osteoporosis (US 2005/0256208, Korean Patent Publication No. 10-2006-0047787), a pharmaceutical composition for treating breast cancer (US 2005/0256208). Studies on the efficacy of the active ingredients of bakuchiol et al bones are weak.
본 발명은 인위적으로 에스트로겐을 투여하는 호르몬 보충요법을 대체할 수 있는 천연물 유래의 보다 안전하고 효과가 우수하며 피토에스트로겐(phytoestrogen)으로 사용할 수 있는 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물을 제공하는데 목적이 있다. The present invention is safer and more effective from natural products that can replace hormonal supplementation for artificially administering estrogens, and isolated from Bogolji extract or Bogolji extract with excellent estrogen activity that can be used as phytoestrogen. It is an object to provide a composition comprising one active ingredient.
본 발명자들은 새로운 피토에스트로겐을 찾아 그 활성을 조사한 결과, 보골지(Psoralea corylifolia L.) 종자인 보골지 추출물이 에스트로겐 활성이 탁월함을 확인하였고, 보골지 추출물로부터 분리한 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone) 등 에스트로겐 활성이 우수한 활성성분을 분리함에 따라 호르몬 대체요법으로 사용할 수 있는 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물에 대한 본 발명을 완성하였다. The present inventors found new phytoestrogens and investigated their activity. As a result, Bogolgi extract, which is a seed of Psoralea corylifolia L., was found to have excellent estrogen activity, and bakuchiol (bakuchiol) and Babacro were isolated from Bogolji extract. Bogolgi extract with excellent estrogen activity, which can be used as a hormone replacement therapy, by separating active ingredients with excellent estrogen activity, such as min (bavachromene), isobavachromene, isavavachalcone, and isobavachalcone Or completed the present invention for a composition comprising the active ingredient isolated from the Bogolji extract.
본 발명에 보골지(Psoralea corylifolia L.) 종자인 보골지 추출물, 특히 보 골지 추출물로부터 분리한 바쿠치올은 에스트로젠 활성에 미치는 효과를 탐색한바, 에스트로젠 수용체와의 결합능이 뛰어나고 난소절제 갱년기 모델에서 갱년기 호르몬 결핍으로 인한 바이오마커(biomarker)에 미치는 효과가 우수하다.In the present invention, the Bogolgol extract (Psoralea corylifolia L.) seed, especially bakuchiol isolated from Bogolgol extract, explores the effect on estrogen activity, and has excellent binding ability with estrogen receptor and menopausal hormone in ovarian abdominal menopausal model. The effect on biomarkers due to deficiency is excellent.
따라서 본 발명의 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물은 이를 유효성분으로 함유하는 갱년기 증후군 개선 및/또는 치료용 제제로 사용할 수 있다. Therefore, the composition comprising the Bogolji extract excellent in the estrogen activity of the present invention or the active ingredient isolated from the Bogolji extract can be used as an agent for improving and / or treating menopausal syndrome containing it as an active ingredient.
본 발명은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물을 나타낸다.The present invention shows a composition comprising an active ingredient separated from the Bogolji extract or Bogolji extract excellent in estrogen activity.
본 발명의 에스트로겐 활성이 우수한 보골지 추출물은 탄소수 1 내지 10개인 알코올 용매로 보골지를 추출할 수 있다. Bogolji extract excellent in the estrogen activity of the present invention can be extracted Bogolji with an alcohol solvent having 1 to 10 carbon atoms.
본 발명의 에스트로겐 활성이 우수한 보골지 추출물은 메탄올, 에탄올, 프로판올, 부탄올, 헥산올, 헵탄올 중에서 선택된 어느 하나의 알코올 용매로 보골지를 추출할 수 있다. Bogolji extract excellent in estrogen activity of the present invention can be extracted from Bogolji with any alcohol solvent selected from methanol, ethanol, propanol, butanol, hexanol, heptanol.
본 발명의 에스트로겐 활성이 우수한 보골지 추출물은 에탄올로 보골지를 추출할 수 있다. Bogolji extract excellent in the estrogen activity of the present invention can be extracted golgoji with ethanol.
본 발명은 에스트로겐 활성이 우수한 보골지 추출물로부터 분리한 활성성분은 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone) 중 에서 선택된 어느 하나 이상을 포함한다.In the present invention, the active ingredient isolated from the extract of Bogolgol with excellent estrogen activity is bakuchiol (bakuchiol), babachromine (bavachromene), isobavachromene (isobavachromene), babachalcone, isobavachalcone (isobavachalcone) It includes any one or more selected from.
상기에서 바쿠치올은 하기의 (A-1)단계 내지 (A-3)단계를 포함하는 분리방법을 이용하여 보골지 추출물로부터 분리할 수 있다. In the above bakuchiol can be separated from Bogolgol extract using a separation method comprising the following steps (A-1) to (A-3).
(A-1)보골지 알코올 용매 추출물을 다이클로로메탄으로 추출하는 단계,(A-1) extracting Bogolgi alcohol solvent extract with dichloromethane,
(A-2)보골지 다이클로로메탄 추출물을 다이클로로메탄/메탄올의 혼합용액을 부피비 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1의 순차적으로 올려주는 농도 구배 용출방식의 실리카겔 컬럼크로마토그래피를 하여 제1분획 내지 제11분획의 11개 분획을 얻는 단계,(A-2) Boholgi dichloromethane extract was mixed with dichloromethane / methanol in a volume ratio of 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, Obtaining eleven fractions of the first to eleventh fractions by silica gel column chromatography in a concentration gradient elution method of sequentially raising 30: 1, 20: 1, 10: 1, 1: 1,
(A-3)상기 (2)단계에서 얻은 11개의 분획 중 제3분획(80:1)을 노말헥산/에틸아세테이트가 혼합된 혼합용액을 이동상으로 하는 실리카겔 컬럼크로마토 그래피를 실시하여 3-1분획 내지 3-10분획의 10개 분획을 얻고, 상기 3-2분획을 진공농축하여 바쿠치올(bakuchiol)을 분리할 수 있다.(A-3) Third fraction (80: 1) of the 11 fractions obtained in step (2) was subjected to silica gel column chromatography using a mixed solution of normal hexane / ethyl acetate as a mobile phase, and then fraction 3-1. 10 fractions of 3 to 10 fractions are obtained, and the 3-2 fractions are concentrated in vacuo to separate bakuchiol.
상기 (A-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 탄소수 1 내지 10개인 알코올 용매를 사용하여 추출한 것을 사용할 수 있다. Bogolji alcohol solvent extract of the (A-1) step can be used to extract Bogolji powder using an alcohol solvent having 1 to 10 carbon atoms.
상기 (A-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 메탄올, 에탄올, 프로판올, 부탄올, 헥산올, 헵탄올 중에서 선택된 어느 하나의 알코올 용매를 사용하여 추출한 것을 사용할 수 있다. Bogolji alcohol solvent extract of the (A-1) step can be used to extract Bogolji powder using any one alcohol solvent selected from methanol, ethanol, propanol, butanol, hexanol, heptanol.
상기 (A-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 에탄올를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (A-1) step can be used to extract Bogolji powder using ethanol.
상기 (A-3)단계에서 제3분획을 이동상인 노말헥산/에틸아세테이트는 1∼20 : 20∼1의 부피비로 혼합된 혼합용액을 1∼5ml/min의 유속으로 용출하는 실리카겔 컬럼크로마토 그래피를 실시하여 3-1분획 내지 3-10분획의 10개 분획을 얻을 수 있다.In the step (A-3), the normal fraction of hexane / ethyl acetate as a mobile phase is subjected to silica gel column chromatography, which elutes a mixed solution mixed at a volume ratio of 1 to 20:20 to 1 at a flow rate of 1 to 5 ml / min. 10 fractions from 3-1 fractions to 3-10 fractions can be obtained.
상기 (A-3)단계에서 제3분획을 이동상인 노말헥산/에틸아세테이트는 20:1의 부피비 혼합된 혼합용액을 3ml/min의 유속으로 용출하는 실리카겔 컬럼크로마토 그래피를 실시하여 3-1분획 내지 3-10분획의 10개 분획을 얻을 수 있다.In the step (A-3), the third fraction is a mobile phase of normal hexane / ethyl acetate, which is subjected to silica gel column chromatography eluting a mixed solution having a volume ratio of 20: 1 at a flow rate of 3 ml / min. Ten fractions of 3-10 fractions can be obtained.
상기 (A-3)단계에서 제3분획을 이동상인 노말헥산/에틸아세테이트는 1:1의 부피비 혼합된 혼합용액을 3ml/min의 유속으로 용출하는 실리카겔 컬럼크로마토 그래피를 실시하여 3-1분획 내지 3-10분획의 10개 분획을 얻을 수 있다.In the step (A-3), the third fraction is a mobile phase of normal hexane / ethyl acetate, which is subjected to silica gel column chromatography eluting a mixed solution having a volume ratio of 1: 1 at a flow rate of 3 ml / min. Ten fractions of 3-10 fractions can be obtained.
상기에서 바바크로민, 이소바바크로민 또는 바바찰콘은 하기의 (B-1)단계 내지 (B-3)단계를 포함하는 분리방법을 이용하여 보골지 추출물로부터 분리할 수 있다. In the above barbacromine, isobaba cromine or barbachalcone can be separated from Bogolgol extract using a separation method comprising the following steps (B-1) to (B-3).
(B-1)보골지 알코올 용매 추출물을 다이클로로메탄으로 추출하는 단계,(B-1) extracting the golgol alcohol solvent extract with dichloromethane,
(B-2)보골지 다이클로로메탄 추출물을 다이클로로메탄/메탄올의 혼합용액을 부비피 100:1에서부터 1:1까지(100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1) 순차적으로 올려주는 농도 구배 용출방식의 실리카겔 컬럼크로마토 그래피를 하여 제1분획 내지 제11분획의 11개 분획을 얻는 단계,(B-2) Bogolgi dichloromethane extract from dichloromethane / methanol mixed solution from buoyant 100: 1 to 1: 1 (100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1: 1) Concentration gradient elution silica gel column chromatography Obtaining the dog fraction,
(B-3)상기 (B-2)단계에서 얻은 11개의 분획 중 제6분획(50:1)을 메탄올을 이동상으로 하는 역상 컬럼크로마토 그래피를 실시하여 6-1분획 내지 6-9분획의 9개 분획을 얻고, 상기 6-1분획 내지 6-9분획 중에서 6-5분획을 진공농축하는 방법으로 바바크로민(bavachromene)을, 6-7분획을 진공농축하여 이소바바크로민(isobavachromene)을, 또는 6-9분획을 진공농축하여 바바찰콘(bavachalcone)을 분리할 수 있다.(B-3) The sixth fraction (50: 1) of the eleven fractions obtained in step (B-2) was subjected to reverse phase column chromatography using methanol as a mobile phase, and thus the fraction of 6-1 to 6-9 was collected. Dog fractions were obtained, and 6-5 fractions were vacuum concentrated in 6-6 fractions, and 6-7 fractions were vacuum-concentrated to obtain isobavachromene. Alternatively, 6-9 fractions can be concentrated in vacuo to separate bacavachalcone.
상기 (B-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 탄소수 1 내지 10개인 알코올 용매를 사용하여 추출한 것을 사용할 수 있다. Bogolji alcohol solvent extract of the (B-1) step can be used to extract Bogolji powder using an alcohol solvent having 1 to 10 carbon atoms.
상기 (B-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 메탄올, 에탄올, 프로판올, 부탄올, 헥산올, 헵탄올 중에서 선택된 어느 하나의 알코올 용매를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (B-1) step can be used to extract Bogolji powder using any one alcohol solvent selected from methanol, ethanol, propanol, butanol, hexanol, heptanol.
상기 (B-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 에탄올를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (B-1) step can be used to extract Bogolji powder using ethanol.
상기 (B-3)단계에서 제6분획을 메탄올을 이동상으로 하는 역상 컬럼크로마토 그래피를 실시함에 있어서 이동상인 메탄올은 농도가 60％ 메탄올을 사용하되, 이동상인 메탄올 농도를 5％씩 증가시키는 역상 컬럼크로마토 그래피를 실시하여 6-1분획 내지 6-9분획의 9개 분획을 얻을 수 있다.In performing the reverse phase column chromatography using methanol as the mobile phase in step (B-3), the mobile phase methanol has a concentration of 60% methanol, but the reverse phase column increases the methanol concentration of the mobile phase by 5%. Chromatography can yield 9 fractions from 6-1 fractions to 6-9 fractions.
상기에서 이소바바찰콘은 하기의 (C-1)단계 내지 (C-3)단계를 포함하는 분리방법을 이용하여 보골지 추출물로부터 분리할 수 있다. Isobabasalcon in the above can be separated from Bogolgol extract using a separation method comprising the following (C-1) to (C-3) step.
(C-1)보골지 알코올 용매 추출물을 다이클로로메탄으로 추출하는 단계,(C-1) extracting the golgol alcohol solvent extract with dichloromethane,
(C-2)보골지 다이클로로메탄 추출물을 다이클로로메탄/메탄올의 혼합용액을 부비피 100:1에서부터 1:1까지(100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1) 순차적으로 올려주는 농도 구배 용출방식의 실리카겔 컬럼크로마 토 그래피를 하여 제1분획 내지 제11분획의 11개 분획을 얻는 단계,(C-2) Bolgolgi dichloromethane extract from dichloromethane / methanol mixed solution from buoyant 100: 1 to 1: 1 (100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1: 1) Concentration gradient elution silica gel column chromatography Obtaining 11 fractions,
(C-3)상기 (C-2)단계에서 얻은 11개의 분획 중 제7분획(40:1)을 메탄올을 이동상으로 하는 역상 컬럼크로마토 그래피를 실시하여 7-1분획 내지 7-9분획의 9개 분획을 얻고, 상기 7-1분획 내지 7-9분획 중에서 7-3분획을 노말헥산/에틸아세테이트를 이동상으로 하는 실리카겔 컬럼크로마토 그래피를 실시하여 7-3-1분획 내지 7-3-5분획의 5개 분획을 얻고, 상기 7-3-5 분획을 진공농축하여 이소바바찰콘(isobavachalcone)을 분리할 수 있다.(C-3) The seventh fraction (40: 1) of the eleven fractions obtained in the above (C-2) step was subjected to reverse phase column chromatography using methanol as a mobile phase. 7 fractions were obtained from 7-1 fractions and 7-9 fractions were subjected to silica gel column chromatography using normal hexane / ethyl acetate as a mobile phase, to yield 7-3-1 fractions to 7-3-5 fractions. Five fractions of are obtained, and the 7-3-5 fractions are concentrated in vacuo to separate isobavachalcone.
상기 (C-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 탄소수 1 내지 10개인 알코올 용매를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (C-1) step can be used to extract Bogolji powder using an alcohol solvent having 1 to 10 carbon atoms.
상기 (C-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 메탄올, 에탄올, 프로판올, 부탄올, 헥산올, 헵탄올 중에서 선택된 어느 하나의 알코올 용매를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (C-1) step can be used to extract Bogolji powder using any one of the alcohol solvent selected from methanol, ethanol, propanol, butanol, hexanol, heptanol.
상기 (C-1)단계의 보골지 알코올 용매 추출물은 보골지 분말을 에탄올를 사용하여 추출한 것을 사용할수 있다. Bogolji alcohol solvent extract of the (C-1) step can be used to extract Bogolji powder using ethanol.
상기 (C-3)단계에서 제7분획은 이동상인 메탄올을 이동상으로 하는 역상 컬럼크로마토 그래피를 실시함에 있어서 이동상인 메탄올은 농도가 60％ 메탄올을 사용하되, 이동상인 메탄올 농도를 5％씩 증가시키는 역상 컬럼크로마토 그래피를 실시하여 7-1분획 내지 7-9분획의 9개 분획을 얻을 수 있다.In the step (C-3), the seventh fraction uses 60% methanol as the mobile phase in performing reverse phase column chromatography using methanol as the mobile phase to increase the methanol concentration by 5%. Reverse phase column chromatography can be performed to obtain 9 fractions from 7-1 fractions to 7-9 fractions.
상기 (C-3)단계에서 제7-3분획은 이동상인 노말헥산/에틸아세테이트는 1∼10 : 10∼1의 부피비로 혼합된 혼합용액을 1∼5ml/min의 유속으로 하여 실리카겔 컬럼크로마토 그래피를 실시할 수 있다.In the step (C-3), the silica gel column chromatography uses the mixed solution of normal hexane / ethyl acetate, which is the mobile phase, in a volume ratio of 1 to 10: 10 to 1 at a flow rate of 1 to 5 ml / min. Can be carried out.
상기 (C-3)단계에서 제7-3분획은 이동상인 노말헥산/에틸아세테이트를 7 : 1의 부피비로 혼합된 혼합용액을 3ml/min의 유속으로 하여 실리카겔 컬럼크로마토 그래피를 실시할 수 있다.In the step (C-3), the 7-3 fraction may be subjected to silica gel column chromatography using a mixed solution of normal hexane / ethyl acetate in a volume ratio of 7: 1 at a flow rate of 3 ml / min.
본 발명은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물이 함유된 갱년기 증후군 개선 및/또는 치료용 제제를 포함한다.The present invention includes an agent for improving and / or treating menopausal syndrome, which contains a composition containing an active ingredient separated from the Bogolge extract or Bogolge extract excellent in estrogen activity.
상기에서 에스트로겐 활성이 우수한 보골지 추출물로부터 분리한 활성성분은 보고지 추출물로부터 분리한 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone) 중에서 선택된 어느 하나 이상을 나타낸다.The active ingredient isolated from Bogolgol extract with excellent estrogen activity is bakuchiol, babachromene, isobavachromene, isavavachromene, babachalcone and isobaba Any one or more selected from isobavachalcone.
상기에서 갱년기 증후군 개선 및 치료용 제제는 약제학적으로 허용되는 부형제, 담체 중에서 선택된 어느 하나 이상를 포함한다. The agent for improving and treating menopausal syndrome includes any one or more selected from pharmaceutically acceptable excipients and carriers.
상기에서 갱년기 증후군 개선 및 치료용 제제는 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분과 약제학적으로 허용되는 부형제, 담체 중에서 선택된 어느 하나 이상을 포함하여 정제, 산제, 과립제, 환제, 캡슐제, 현탁제, 주사용 제제, 유화액, 비경구 투여용 제제, 또는 경구용 제제의 형태로 제형화될 수 있다.The agent for improving and treating menopausal syndrome is a tablet, powder, granules, including any one or more selected from the golgogi extract with excellent estrogen activity or the active ingredient isolated from the golgogi extract, a pharmaceutically acceptable excipient, a carrier, It may be formulated in the form of pills, capsules, suspensions, injectable preparations, emulsions, preparations for parenteral administration, or oral preparations.
상기의 갱년기 증후군 개선 및 치료용 제제에 있어서 에스트로겐 활성이 우 수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분은 갱년기 증후군 개선 및 치료용 제제 전체 중량 대비 0.1∼10중량％를 포함될 수 있다. In the above-mentioned preparations for improving and treating menopausal syndrome, the bologous extract having excellent estrogen activity or the active ingredient isolated from the bogolji extract may include 0.1 to 10% by weight relative to the total weight of the preparation for improving and treating menopausal syndrome.
상기의 갱년기 증후군 개선 및 치료용 제제에 있어서 약제학적으로 허용되는 부형제, 담체 중에서 선택된 어느 하나 이상이 갱년기 증후군 개선 및 치료용 제제 전체 중량 대비 90∼99.9중량％를 포함될 수 있다. Any one or more selected from pharmaceutically acceptable excipients and carriers in the above-mentioned menopausal syndrome improvement and treatment agent may include 90 to 99.9 wt% of the total weight of the menopausal syndrome improvement and treatment agent.
상기 약제학적으로 허용되는 부형제, 담체는 당해 기술분야에서 통상의 지식을 가진 당업자가 적의 선택하여 실시할 수 있으므로 이들에 대한 자세한 내용은 생략하기로 한다.Since the pharmaceutically acceptable excipients and carriers may be appropriately selected by those skilled in the art, details thereof will be omitted.
본 발명은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물이 함유된을 포함하는 갱년기 증후군 개선 및 치료용 식품을 포함한다.The present invention includes foods for improving and treating menopausal syndrome, which contain a composition containing an active ingredient separated from the Bogolgo extract or Bogolji extract excellent in estrogen activity.
상기에서 에스트로겐 활성이 우수한 보골지 추출물로부터 분리한 활성성분은 보고지 추출물로부터 분리한 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone) 중에서 선택된 어느 하나 이상을 나타낸다.The active ingredient isolated from Bogolgol extract with excellent estrogen activity is bakuchiol, babachromene, isobavachromene, isavavachromene, babachalcone and isobaba Any one or more selected from isobavachalcone.
상기에서 갱년기 증후군 개선 및 치료용 건강보조식품은 식품학적으로 허용되는 감미료, 당류, 식품첨가물 중에서 선택된 어느 하나 이상의 부재료를 포함한다. The menopausal syndrome improvement and treatment health supplements include any one or more of the ingredients selected from the food-acceptable sweeteners, sugars, food additives.
상기에서 갱년기 증후군 개선 및 치료용 건강보조식품은 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분과 식품학적으로 허용되는 감미료, 당류, 식품첨가물 중에서 선택된 어느 하나 이상의 부재료를 포함하여 정제, 산제, 과립제, 환제, 캡슐제의 형태로 제형화될 수 있다.The dietary supplement for improving and treating menopausal syndrome includes a bogolgol extract excellent in estrogen activity or an active ingredient isolated from the bogolgol extract and any one or more subsidiary materials selected from food-acceptable sweeteners, sugars, food additives It may be formulated in the form of tablets, powders, granules, pills, capsules.
상기의 갱년기 증후군 개선 및 치료용 건강보조식품에 있어서 에스트로겐 활성이 우수한 보골지 추출물 또는 상기 보골지 추출물로부터 분리한 활성성분을 포함하는 조성물이 함유된은 갱년기 증후군 개선 및 치료용 건강보조식품 전체 중량 대비 0.1∼10중량％를 포함될 수 있다. Compared with the total weight of silver menopausal syndrome improvement and treatment health supplements containing the composition containing the active ingredient isolated from the Bogolge extract or the bogolgol extract excellent in estrogen activity in the above-mentioned menopausal syndrome improvement and treatment health supplement 0.1 to 10% by weight may be included.
상기의 갱년기 증후군 개선 및 치료용 건강보조식품에 있어서 식품학적으로 허용되는 감미료, 당류, 식품첨가물 중에서 선택된 어느 하나 이상의 부재료는 갱년기 증후군 개선 및 치료용 건강보조식품 전체 중량 대비 90∼99.9중량％를 포함될 수 있다. In the above-mentioned menopausal syndrome improvement and therapeutic dietary supplements, any one or more ingredients selected from food-acceptable sweeteners, sugars, and food additives may include 90 to 99.9 wt% of the total weight of the menopausal syndrome improvement and dietary supplements. Can be.
상기 식품학적으로 허용되는 감미료, 당류, 식품첨가물 중에서 선택된 어느 하나 이상의 부재료는 당해 기술분야에서 통상의 지식을 가진 당업자가 적의 선택하여 실시할 수 있으므로 이들에 대한 자세한 내용은 생략하기로 한다.Any one or more subsidiary materials selected from the food-acceptable sweeteners, sugars, food additives can be appropriately selected by those skilled in the art and will not be described in detail.
상기에서 식품은 선식, 껌, 캔디, 비스켓, 음료, 건강보조식품 중에서 선택된 어느 하나이다.The food is any one selected from the wire type, gum, candy, biscuits, beverages, health supplements.
이하 본 발명의 내용을 실시예 및 시험예를 통하여 구체적으로 설명한다. 그러나, 이들은 본 발명을 보다 상세하게 설명하기 위한 것으로 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the content of the present invention will be described in detail through examples and test examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.
<실시예 1> 보골지 추출물의 제조 및 활성성분의 분리 Example 1 Preparation of Bogolji Extract and Isolation of Active Ingredients
보골지 표면에 잔류할 수 있는 농약류 등에 의한 에스트로젠 활성 간섭을 배제시키기 위해 보골지를 3회 초음파 세척(27℃, 1시간 초음파 세척, 3회 반복) 후 건조시켰다. 건조된 시료를 40메시(mesh) 크기로 분쇄 후 보골지 건조 시료 중량 10배량의 80％ 에탄올을 사용하여 활성물질이 용출될 수 있도록 90℃에서 5시간 동안 3회 반복하여 환류추출 한 후 용매추출액을 감압농축하였다. 보골지 에탄올 추출물은 여과지로 여과하였고, 감압 농축기를 이용하여 농축한 뒤 동결 건조하여 분말화 시켰다. To eliminate estrogen activity interference by pesticides, etc., which may remain on the surface of the bones, the bones were dried after three ultrasonic washes (27 ° C, 1 hour ultrasonic wash, 3 repetitions). After crushing the dried sample to 40 mesh size, using a 80% ethanol of 10 times the weight of dry bone sample, reflux extraction was repeated three times at 90 ° C. for 5 hours so that the active substance was eluted. Was concentrated under reduced pressure. Bogolgol ethanol extract was filtered through a filter paper, concentrated using a vacuum concentrator and then lyophilized to powder.
상기 공정을 이용하여 얻은 보골지 에탄올 추출물 1.8kg을 증류수 20리터(L)에 현탁시킨 후, 20리터의 다이클로로메탄으로 분획 및 회수 후 감압농축(50℃) 하여 다이클로로메탄 추출물 1.57kg을 얻었으며, 남은 물층을 동결 건조하였다.1.8 kg of Bogolgol ethanol extract obtained using the above process was suspended in 20 liters (L) of distilled water, fractionated and recovered with 20 liters of dichloromethane, and concentrated under reduced pressure (50 ° C.) to obtain 1.57 kg of dichloromethane extract. The remaining water layer was lyophilized.
상기에서 얻은 보골지 다이클로로메탄 추출물 200g을, 다이클로로메탄/메탄올 혼합용액을 이동상으로서 3ml/분의 유속으로 흘려주면서 다이클로로메탄/메탄올의 혼합비를 100 : 1에서부터 1 : 1까지(100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1) 순차적으로 올려주는 농도 구배 용출방식으로 실리카겔 컬럼크로마토그래피(silica gel column chromatography)를 실시하여 총 11개의 분획으로 나누었다. Mixing ratio of dichloromethane / methanol from 200: 1 to 1: 1 while distilling 200 g of bolgonggi dichloromethane extract obtained above, dichloromethane / methanol mixed solution as a mobile phase at a flow rate of 3 ml / min (100: 1 , 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 1: 1) Silica gel column chromatography was performed and divided into a total of 11 fractions.
상기의 11개 분획 중 제3분획(80:1)을 이동상으로서 노말헥산/에틸아세테이트가 20:1의 부피비로 혼합된 혼합용액을 이동상으로 하여 3ml/min으로 각각 용출 하는 실리카겔 컬럼 크로마토그래피를 실시하여 3-1분획 내지 3-10분획의 10개 분획을 얻은 후 상기 10개 분획 중 3-2분획을 진공농축하여 15.7g의 바쿠치올(bakuchiol)을 분리하였다.A silica gel column chromatography was eluted at 3 ml / min using the third solution (80: 1) of the above 11 fractions as a mobile phase and a mixed solution containing normal hexane / ethyl acetate in a volume ratio of 20: 1 as a mobile phase. After obtaining 10 fractions from 3-1 fractions to 3-10 fractions, 35.7 fractions of the 10 fractions were concentrated in vacuo to separate 15.7 g of bakuchiol.
상기의 11개 분획 중 6분획(50:1)은 이동상 60％ 메탄올을 사용하고 상기 이동상의 메탄올 농도를 5％씩 증가시켜 5ml로 분획하는 역상 컬럼 크로마토그래피를 실시하여 6-1분획 내지 6-9분획의 9개 분획을 얻고, 상기 6-1분획 내지 6-9분획 중 6-5분획을 진공농축하여 200mg의 바바크로민(bavachromene)을 분리 하였고, 상기 상기 6-1분획 내지 6-9분획 중 6-7분획을 진공농축하여 140mg의 이소바바크로민(isobavachromene)을 분리하였고, 상기 6-1분획 내지 6-9분획 중 6-9분획을 진공농축하여 600mg의 바바찰콘(bavachalcone)을 분리하였다. Six fractions (50: 1) of the eleven fractions were subjected to reverse phase column chromatography using 60% methanol in mobile phase and fractionating to 5 ml by increasing the methanol concentration of the mobile phase by 5%. Nine fractions of nine fractions were obtained, 200 mg of baravachromene was isolated by vacuum concentrating 6-5 fractions of the 6-1 fractions to 6-9 fractions, and the 6-1 fractions to 6-9. 140 mg of isobavachromene was isolated by vacuum concentrating 6-7 fractions of the fraction, and 6-9 fractions of the 6-1 to 6-9 fractions were vacuum concentrated to 600 mg of barvachalcone. Separated.
상기의 10개 분획 중 7분획은 이동상 60％ 메탄올을 사용하고 상기 이동상의 메탄올 농도를 5％씩 증가시켜 5ml로 분획하는 역상 컬럼 크로마토그래피를 실시하여 7-1분획 내지 7-9분획의 9개 분획을 얻고, 상기 7-1분획 내지 7-9분획 중 제 7-3분획은 노말헥산/에틸아세테이트가 7:1의 부피비로 혼합된 혼합용액을 이동상으로 하고, 상기 이동상을 3ml/min으로 용출하는 실리카겔 컬럼 크로마토그래피를 실시하여 7-3-1분획 내지 7-3-5분획의 5개 분획으로 나누고 이중 7-3-5분획을 진공농축하여 90mg의 이소바바찰콘(isobavachalcone)을 분리하였다. Seven fractions out of the ten fractions were subjected to reverse phase column chromatography using 60% methanol in mobile phase and fractionating to 5 ml by increasing the methanol concentration of the mobile phase by 5%. A fraction was obtained, and the 7-3 fractions of the 7-1 fractions 7-9 fractions used as a mobile phase a mixed solution of normal hexane / ethyl acetate in a volume ratio of 7: 1, and the mobile phase was eluted at 3 ml / min. Silica gel column chromatography was performed, and the fraction was divided into 5 fractions of 7-3-1 fractions to 7-3-5 fractions, and the 7-3-5 fractions were concentrated in vacuo to separate 90 mg of isobavachalcone.
상기에서 보골지 다이클로로메탄 추출물로부터 분리한 바쿠치올(bakuchiol), 바바크로민(bavachromene), 이소바바크로민(isobavachromene), 바바찰콘(bavachalcone), 이소바바찰콘(isobavachalcone)의 화합물들은 질량분석 및 핵자 기 공명 분석스펙트럼을 통하여 확인하였다. Compounds of bakuchiol, babachromene, isobavachromene, babachalcone, and isobavachalcone isolated from Bogolji dichloromethane extracts were analyzed by mass spectrometry and Nuclear magnetic resonance analysis was confirmed through the spectrum.
bakuchiol bakuchiol
1H-NMR (300 MHz, CDCl3) : δ 7.23(2H, m, H-2,6), 6.76(2H, m, H-3,5), 6.25(1H, d, J=16.2Hz, H-7), 6.05(1H, d, J=16.2Hz, H-8), 5.87(1H, dd, J=10.8, 17.3Hz, H-17), 5.10(1H, t-like, H-12), 5.01(2H, m, H-18), 1.96(2H, m, H-10), 1.67(3H, s, H-14), 1.48(2H, m, H-11), 1.18(3H, s, H-16); 1 H-NMR (300 MHz, CDCl 3 ): δ 7.23 (2H, m, H-2,6), 6.76 (2H, m, H-3,5), 6.25 (1H, d, J = 16.2 Hz, H-7), 6.05 (1H, d, J = 16.2 Hz, H-8), 5.87 (1H, dd, J = 10.8, 17.3 Hz, H-17), 5.10 (1H, t-like, H-12 ), 5.01 (2H, m, H-18), 1.96 (2H, m, H-10), 1.67 (3H, s, H-14), 1.48 (2H, m, H-11), 1.18 (3H, s, H-16);
13C-NMR (125 MHz, CDCl3) : δ 154.7(C-4), 146.1(C-17), 136.0(C-7), 131.4(C-1), 131.0(C-13), 127.5(C-2,6), 126.6(C-8), 124.9(C-12), 115.5(C-3,5), 112.0(C-18), 42.6(C-9), 41.4(C-10), 25.8(C-15), 23.4(C-16), 23.3(C-11), 17.8(C-14). 13 C-NMR (125 MHz, CDCl 3 ): δ 154.7 (C-4), 146.1 (C-17), 136.0 (C-7), 131.4 (C-1), 131.0 (C-13), 127.5 ( C-2,6), 126.6 (C-8), 124.9 (C-12), 115.5 (C-3,5), 112.0 (C-18), 42.6 (C-9), 41.4 (C-10) , 25.8 (C-15), 23.4 (C-16), 23.3 (C-11), 17.8 (C-14).
bavachromenebavachromene
1H-NMR (300 MHz, DMSO-d 6) : δ 8.09(1H, s, H-b), 7.76(4H, m, H-2',6',5,a), 6.87(2H, d, J=8.7Hz, H-3',5'), 6.45(1H, d, J=9.9Hz, H-4), 6.28(1H, s, H-8), 5.75(1H, d, J=9.9Hz, H-3), 1.41(6H, s, -CH3ㅧ2); 1 H-NMR (300 MHz, DMSO- d 6 ): δ 8.09 (1H, s, Hb), 7.76 (4H, m, H-2 ', 6', 5, a), 6.87 (2H, d, J = 8.7 Hz, H-3 ', 5'), 6.45 (1H, d, J = 9.9 Hz, H-4), 6.28 (1H, s, H-8), 5.75 (1H, d, J = 9.9 Hz , H-3), 1.41 (6H, s, -CH 3 ㅧ 2);
13C-NMR (125 MHz, DMSO-d 6) : δ 191.7(C=O), 165.4, 160.4, 159.7, 144.7(C-b), 131.3, 128.8, 128.7, 125.6(ㅧ2), 120.6(C-a), 117.3, 115.8(ㅧ2), 114.0, 113.3, 103.5, 78.0(C-2), 28.2(ㅧ2). 13 C-NMR (125 MHz, DMSO- d 6 ): δ 191.7 (C = O), 165.4, 160.4, 159.7, 144.7 (Cb), 131.3, 128.8, 128.7, 125.6 (ㅧ 2), 120.6 (Ca), 117.3, 115.8 (X2), 114.0, 113.3, 103.5, 78.0 (C-2), 28.2 (X2).
isobavachromene isobavachromene
1H-NMR (300 MHz, CDCl3) : δ 7.80(1H, d, J=15.6Hz, H-b), 7.69(1H, d, J=9.0Hz, H-7), 7.50(2H, d, J=8.1Hz, H-2',6'), 7.40(1H, d, J=7.8Hz, H-a), 6.86(1H, d, J=7.8Hz, H-3',5'), 6.74(1H, d, J=10.2Hz, H-4), 6.38(1H, d, J=9.0Hz, H-8), 5.58(1H, d, J=9.9Hz, H-3), 1.46(6H, s, -CH3ㅧ2); 1 H-NMR (300 MHz, CDCl 3 ): δ 7.80 (1H, d, J = 15.6 Hz, Hb), 7.69 (1H, d, J = 9.0 Hz, H-7), 7.50 (2H, d, J = 8.1 Hz, H-2 ', 6'), 7.40 (1H, d, J = 7.8 Hz, Ha), 6.86 (1H, d, J = 7.8 Hz, H-3 ', 5'), 6.74 (1H , d, J = 10.2 Hz, H-4), 6.38 (1H, d, J = 9.0 Hz, H-8), 5.58 (1H, d, J = 9.9 Hz, H-3), 1.46 (6H, s , -CH 3 ㅧ 2);
13C-NMR (125 MHz, CDCl3) : δ 192.4(C=O), 160.9, 159.9, 158.6, 144.7, 130.9(ㅧ2), 128.3, 127.4, 117.6, 116.2(ㅧ2), 115.9, 114.2, 109.5, 108.5, 78.0(C-2), 28.5(ㅧ2). 13 C-NMR (125 MHz, CDCl 3 ): δ 192.4 (C = O), 160.9, 159.9, 158.6, 144.7, 130.9 (ㅧ 2), 128.3, 127.4, 117.6, 116.2 (ㅧ 2), 115.9, 114.2, 109.5, 108.5, 78.0 (C-2), 28.5 (ㅧ 2).
bavachalconebavachalcone
1H-NMR (300 MHz, DMSO-d 6) : δ 8.01(1H, s, H-a), 7.76(4H, m, H-2',6',6,b), 6.86(2H, d, J=8.6H, H-3',5'), 6.51(1H, s, H-3), 5.22(1H, t-like, H-2''), 3.85(3H, s, -OCH3), 3.24(2H, d, J=6.8Hz, H-1''), 1.70(3H, s, H-4''), 1.62(3H, s, H-5''); 1 H-NMR (300 MHz, DMSO- d 6 ): δ 8.01 (1H, s, Ha), 7.76 (4H, m, H-2 ', 6', 6, b), 6.86 (2H, d, J = 8.6H, H-3 ', 5'), 6.51 (1H, s, H-3), 5.22 (1H, t-like, H-2 ''), 3.85 (3H, s, -OCH 3 ), 3.24 (2H, d, J = 6.8 Hz, H-1 "), 1.70 (3H, s, H-4 "), 1.62 (3H, s, H-5 ");
13C-NMR (125 MHz, DMSO-d 6) : δ 191.6(C=O), 164.5(C-2), 163.7(C-4), 160.4(C-4'), 144.5(C-a), 131.3(C-3''), 131.0(C-2',6'), 130.8(C-6), 125.6(C-1'), 123.1(C-2''), 121.2(C-5), 117.3(C-b), 115.8(C-3',5'), 113.0(C-1), 99.3(C-3), 55.9(-OCH3), 28.1(C-1''), 25.4(C-4''), 17.7(C-5''). 13 C-NMR (125 MHz, DMSO- d 6 ): δ 191.6 (C = O), 164.5 (C-2), 163.7 (C-4), 160.4 (C-4 '), 144.5 (Ca), 131.3 (C-3``), 131.0 (C-2 ', 6'), 130.8 (C-6), 125.6 (C-1 '), 123.1 (C-2''), 121.2 (C-5), 117.3 (Cb), 115.8 (C-3 ', 5'), 113.0 (C-1), 99.3 (C-3), 55.9 (-OCH 3 ), 28.1 (C-1 ''), 25.4 (C- 4 ''), 17.7 (C-5 '').
isobavachalconeisobavachalcone
1H-NMR (300 MHz, acetone-d 6) : δ 7.97(1H, d, J=9.0Hz, H-6'), 7.84(1H, d, J=15.9, H-b), 7.74(3H, m, H-2,6,a), 6.93(2H, d, J=8.7Hz, H-3,5), 6.54(1H, d, J=8.7Hz, H-5'), 5.30(1H, t-like, H-2''), 3.39(2H, d, J=7.2Hz, H-1''), 1.79(3H, s, H-4''), 1.65(3H, s, H-5''); 1 H-NMR (300 MHz, acetone- d 6 ): δ 7.97 (1H, d, J = 9.0 Hz, H-6 ′), 7.84 (1H, d, J = 15.9, Hb), 7.74 (3H, m , H-2,6, a), 6.93 (2H, d, J = 8.7 Hz, H-3,5), 6.54 (1H, d, J = 8.7 Hz, H-5 '), 5.30 (1H, t -like, H-2 ''), 3.39 (2H, d, J = 7.2 Hz, H-1 ''), 1.79 (3H, s, H-4 ''), 1.65 (3H, s, H-5 '');
13C-NMR (125 MHz, acetone-d 6) : δ 193.0(C=0), 165.1(C-4'), 162.7(C-2'), 160.9(C-4), 144.9(C-a), 131.7(C-2,6), 131.5(C-3''), 130.2(C-6'), 127.6(C-1), 123.3(C-2''), 118.4(C-b), 116.8(C-3,5), 116.1(C-3'), 114.4(C-1'), 108.0(C-5'), 25.9(C-5''), 22.3(C-1''), 18.0(C-4''). 13 C-NMR (125 MHz, acetone- d 6 ): δ 193.0 (C = 0), 165.1 (C-4 '), 162.7 (C-2'), 160.9 (C-4), 144.9 (Ca), 131.7 (C-2,6), 131.5 (C-3``), 130.2 (C-6 '), 127.6 (C-1), 123.3 (C-2''), 118.4 (Cb), 116.8 (C -3,5), 116.1 (C-3 '), 114.4 (C-1'), 108.0 (C-5 '), 25.9 (C-5''), 22.3 (C-1''), 18.0 ( C-4 '').
bavachininbavachinin
1H-NMR (300 MHz, DMSO-d 6) : δ 7.48(1H, s, H-5), 7.33(2H, d, J=8.3Hz, H-2',6'), 6.79(2H, d, J=8.3Hz, H-3',5'), 6.60(1H, s, H-8), 5.45(1H, dd, J=2.4, 12.9Hz, H-2), 5.23(1H, m, H-2''), 3.83(3H, s, -OCH3), 3.18(2H, d, J=6.7Hz, H-1''), 3.10(1H, m, H-3b), 2.64(1H, m, H-3a), 1.70(3H, s, H-4''), 1.65(3H, s, H-5''); 1 H-NMR (300 MHz, DMSO- d 6 ): δ 7.48 (1H, s, H-5), 7.33 (2H, d, J = 8.3 Hz, H-2 ', 6'), 6.79 (2H, d, J = 8.3 Hz, H-3 ', 5'), 6.60 (1H, s, H-8), 5.45 (1H, dd, J = 2.4, 12.9 Hz, H-2), 5.23 (1H, m , H-2 ''), 3.83 (3H, s, -OCH 3 ), 3.18 (2H, d, J = 6.7 Hz, H-1 ''), 3.10 (1H, m, H-3b), 2.64 ( 1H, m, H-3a), 1.70 (3H, s, H-4 ''), 1.65 (3H, s, H-5 '');
13C-NMR (125 MHz, DMSO-d 6) : δ 190.4(C-4), 163.4(C-7), 161.9(C-9), 157.6(C-4'), 132.2(C-3''), 129.2(C-1'), 128.2(C-2',6'), 126.0(C-5), 123.4(C-6), 121.9(C-2''), 115.1(C-3',5'), 113.5(C-10), 99.3(C-8), 79.2(C-2), 56.1(-OCH3), 43.1(C-3), 27.2(C-1'), 25.5(C-4''), 17.5(C-5''). 13 C-NMR (125 MHz, DMSO- d 6 ): δ 190.4 (C-4), 163.4 (C-7), 161.9 (C-9), 157.6 (C-4 '), 132.2 (C-3''), 129.2 (C-1'), 128.2 (C-2 ', 6'), 126.0 (C-5), 123.4 (C-6), 121.9 (C-2 ''), 115.1 (C-3 ', 5'), 113.5 (C-10), 99.3 (C-8), 79.2 (C-2), 56.1 (-OCH 3 ), 43.1 (C-3), 27.2 (C-1 '), 25.5 (C-4 ''), 17.5 (C-5 '').
<실시예 2> 보골지 추출물 및 분리 화합물의 에스트로겐 활성 측정 Example 2 Determination of Estrogen Activity of Bogolji Extract and Isolated Compound
재조합 효모 분석법(recombinant yeast assay)은 인간 에스트로젠 수용체[Saccharomyces cerevisiae ER+ LYS 8127(YER)]를 발현하는 재조합 효모를 사용하며, 베타 갈락토시데이즈(β-galactosidase)활성의 유도 유무로 여러 화합물의 호르몬 활성을 평가할 수 있는 방법이다. Recombinant yeast assay uses recombinant yeast expressing the human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127 (YER)], and the induction of beta galactosidase activity of various compounds It is a way to assess activity.
17β-에스트라디올(17β-estradiol, E2) 및 에스트로젠성 물질은 재조합 효모(Saccharomyces cerevisiae ER + Lys 8127) 내로 유입되어 발현벡터(expression vector)에 의해 발현된 호르몬 수용체와 결합하고 이는 다시 수용벡터(receptor vector)에서 β-galactosidase의 발현을 유도하게 된다. 에스트로젠 활성은 β-galactosidase의 발현되었는지를 β-galactosidase에 의해 전환되는 o-니트로페놀(o-nitrophenol)의 황색 발색을 측정함으로서 검출된다. 17β-estradiol (E2) and estrogens enter the recombinant yeast (Saccharomyces cerevisiae ER + Lys 8127) and bind to the hormone receptor expressed by the expression vector, which in turn is the receptor vector. vector) induces β-galactosidase expression. Estrogen activity is detected by measuring the yellow color development of o-nitrophenol, which is converted by β-galactosidase to see if β-galactosidase is expressed.
양성 대조군으로 사용한 17β-estradiol(E2)는 10-9 M의 β-galactosidase 유도 활성을 100으로 할 때 농도별 추출물과 분리화합물의 에스트로젠 활성을 측정한 결과는 도 1 및 도 2와 같다.17β-estradiol (E2) used as a positive control, when the β-galactosidase-induced activity of 10 -9 M to 100, the results of measuring the estrogen activity of the extracts and the separated compounds according to the concentration are shown in Figs.
도 1은 재조합 효모 분석법에 의한 보골지 에탄올 추출물의 농도별 에스트로 겐 활성에 대한 그래프이고, 도 2는 재조합 효모 분석법에 의한 바쿠치올, 바바치닌, 바바찰콘, 이소바바찰콘, 바바크로민, 이소바바크로민의 농도별 에스트로겐 활성에 대한 그래프이다.1 is a graph of the estrogen activity by concentration of golgol ethanol extract by the recombinant yeast assay, Figure 2 is bakuchiol, babachinin, babachalcon, isovabachalcon, barbacromine, by recombinant yeast assay, It is a graph of estrogen activity according to the concentration of isobavacromine.
<실시예 3> 보골지 추출물 및 분리 화합물의 에스트로젠 수용체 결합능Example 3 Estrogen Receptor Binding Ability of Bogolji Extract and Separation Compound
에스트로겐수용체 알파(ER α)와 에스트로겐수용체 베타(ERβ)는 시퀀스가 53％ 유사성을 가짐에도 불구하고 수용체 결합능에 있어 차이를 보인다. 즉, ERα가 난소나 자궁, 질, 유선과 같은 주요 여성생식기관에 발현되는 반면에, ERβ는 고환이나 전립선과 같은 남성의 생식 기관과 난소에서 발현된다고 알려져 있다(Mosselman et al., 1996; Muramatsu and Inoue, 2000). ERα와 β는 유전자 조절에 있어서도 다른 역할을 하는 것으로 알려져 있는데 ERβ는 에스트로젠 작용 메커니즘에서 중요한 인자이며, 중추신경계, 심혈관계, 비뇨생식기계, 소화기계, 신장, 간을 포함한 다양한 조직에서 발현되는 것으로 알려져 있다(Arts et al., Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology, 138, 5067∼70, 1997; Enmark et al., Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab, 82, 4258∼65, 1997; Kuiper et al., The novel estrogen receptor-beta subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS Lett, 410, 87∼90, 1997, The estrogen receptor beta subtype: a novel mediator of estrogen action in neuroendocrine systems. Front Neuroendocrinol, 19, 253∼86, 1998). 폐경기 여성에게 호르몬 대체 요법(hormone replacement therapy, HRT)를 실시하는 점에서 이러한 조직 분포의 차이는 매우 중요하게 인식되고 있다(Gustafsson, Estrogen receptor beta - a new dimension in estrogen mechanism of action. J Endocrinol, 163, 379∼83, 1999).Estrogen receptor alpha (ER α) and estrogen receptor beta (ERβ) show differences in receptor binding capacity despite 53% similarity in sequence. In other words, while ERα is expressed in major female reproductive organs such as the ovary, uterus, vagina and mammary gland, ERβ is known to be expressed in male reproductive organs and ovaries such as the testicle or prostate (Mosselman et al ., 1996; Muramatsu and Inoue, 2000). ERα and β are known to play a different role in gene regulation. ERβ is an important factor in the estrogen action mechanism and is expressed in various tissues including the central nervous system, cardiovascular system, genitourinary system, digestive system, kidney and liver. (Arts et al ., Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells.Endocrinology, 138, 5067-70, 1997; Enmark et al ., Human estrogen receptor beta-gene structure, chromosomal localization , and expression pattern.J Clin Endocrinol Metab, 82, 4258-65, 1997; Kuiper et al ., The novel estrogen receptor-beta subtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens.FEBS Lett, 410, 87-90, 1997, The estrogen receptor beta subtype: a novel mediator of estrogen action in neuroendocrine systems.Front Neuroendocrinol, 19, 253-86, 1998). Differences in this tissue distribution are very important in the practice of hormone replacement therapy (HRT) in postmenopausal women (Gustafsson, Estrogen receptor beta-a new dimension in estrogen mechanism of action.J Endocrinol, 163 , 379-83, 1999).
ERα에 대한 에스트로젠 수용체 결합능 측정법은 ER competitor 탐색용 킷트(Wako Pure Chemical Industries, Ltd., Japan)를 사용하여 측정하였다. Fluorescence labeled 17β estradiol(E2)과 실시예 1의 보골지 에탄올 추출물 및 보골지 에탄올 추출물로부터 분리한 바쿠치올, 바바크로민, 이소바바크로민, 바바찰콘, 이소바바찰콘 분말을 DMSO에 녹인 후 human ERα(hERα)가 코팅된 well에 첨가하고 상온에서 2시간 경쟁하도록 한후, 세척한 후 assay solution을 각 well에 첨가하였으며 형광발현강도는 microplate-Fluoroskan Ascent (Lab systems, USA)를 이용하여 excitation 485nm, emission 538nm에서 측정하였다.Estrogen receptor binding capacity for ERα was measured using a kit for ER competitor search (Wako Pure Chemical Industries, Ltd., Japan). Bacchiol, babacromine, isobavacromine, babachalcon, and isobavacchalcon powder isolated from fluorescence labeled 17β estradiol (E2) and Bogolgol ethanol extract and Bogolgol ethanol extract of Example 1 were dissolved in DMSO and then human ERα. (hERα) was added to the wells coated and allowed to compete for 2 hours at room temperature. After washing, assay solution was added to each well. The fluorescence expression intensity was excitation 485 nm, emission using microplate-Fluoroskan Ascent (Lab systems, USA). Measured at 538 nm.
ERβ에 대한 에스트로젠 수용체 결합능 측정법은 GENios ProTM(Tecan, USA)과 에스트로젠 수용체 competitor kit(Invitrogen, USA)을 사용하여 protocol에 따라 측정하였다. 모든 분석은 black round bottom인 96-well plate(Thermolabsystems, USA)를 사용하였다. 2nM fluoromone ES2(fluorescein-tagged estradiol derivative)와 20nM ERβ를 반응 완충액에 녹여 결합체를 형성시키고 실시예 1의 보골지 에탄올 추출물 및 보골지 에탄올 추출물로부터 분리한 바 쿠치올, 바바크로민, 이소바바크로민, 바바찰콘, 이소바바찰콘을 DMSO에 녹여 50㎕를 웰(well)에 분주한 후, 반응 버퍼(reaction buffer)에 녹인 ERβ/Fluormone ES2 complex 50㎕를 well에 첨가하여 알루미늄 호일로 잘 싼 후, shaker로 2시간 동안 반응시키고, excitation 485nm, emission 530nm에서 polarization value를 측정하였다. human ERβ에 대한 결합능은 fluorescence labeled E2(ES1)의 polarization 변화를 측정하였다. 데이터는 ES1 bound와 시료의 molar concentration의 percent로 나타내었다. Estrogen receptor binding capacity for ERβ was measured according to protocol using GENios Pro ™ (Tecan, USA) and estrogen receptor competitor kit (Invitrogen, USA). All analyzes used a 96-well plate (Thermolabsystems, USA) with a black round bottom. 2 nM fluoromone ES2 (fluorescein-tagged estradiol derivative) and 20 nM ERβ were dissolved in the reaction buffer to form a conjugate, and bakuchiol, babacromine, and isobaba cromine separated from the golgol ethanol extract and golgol ethanol extract of Example 1 After dissolving barbachalcone and isobabachalcone in DMSO, 50µl was dispensed into the wells, and then 50µl of ERβ / Fluormone ES2 complex dissolved in the reaction buffer was added to the wells and wrapped in aluminum foil. The reaction was performed for 2 hours with a shaker, and the polarization value was measured at excitation 485 nm and emission 530 nm. The binding capacity to human ERβ was measured by the polarization change of fluorescence labeled E2 (ES1). Data are expressed as ES1 bound and percent of molar concentration of the sample.
보골지에서 추출한 단일 화합물의 데스트로젠 수용체 결합능을 측정한 결과 에스트로젠 알파와 에스트로젠 베타 모두 바쿠치올이 가장 높았다. Destrogen receptor binding ability of single compound extracted from Bogolgol was the highest for both estrogen alpha and estrogen beta.
실험 결과, 에스트로젠 수용체 결합능은 바쿠치올이 ERα에 대해 IC50=1.01×10-6, ERβ에 대해 IC50=1.20×10-6 로 가장 높은 것으로 나타나 바쿠치올의 에스트로젠 수용체 결합능이 가장 높은 것으로 나타났다(표 1 참조). As a result, the estrogen receptor binding capacity was highest as bakuchiol with IC 50 = 1.01 × 10 -6 for ERα and IC 50 = 1.20 × 10 -6 for ERβ, indicating the highest estrogen receptor binding capacity of bakuchiol ( See Table 1).
표 1.Table 1.
a) 에스트로겐 수용체(ER)와의 50％ 결합농도a) 50% binding concentration with estrogen receptor (ER)
b)ND : not detected(결합 불검출)b) ND: not detected
<실시예 4> 난소절제 갱년기 모델에서 보골지 추출물 및 바쿠치올의 효능Example 4 Efficacy of Bone Golgi Extract and Bakuchiol in Ovarian Climacteric Model
보골지 에탄올 추출물과 보골지에서 분리한 바쿠치올을 대상으로 난소절제 동물 모델에서 에스트로겐 활성에 대한 동물실험(SD female rats, 4주령)을 수행하였으며 완전한 난소제거를 체외에서 확인하기 위해서 수술 후 회복기 동안 질구검사(vaginal smear)를 실시하였고, 발정기(estrus cycle)를 보이지 않는 개체만을 선별하여 실험에 사용하였다. Animal experiments on oestrogen activity (SD female rats, 4 weeks old) in ovarian ablation animal model were performed on ethanol extracts from golgol and bakuchiol isolated from golgol. Vaginal smears were performed and only individuals that did not show an estrus cycle were selected and used in the experiment.
실험 초기의 체중은 78g±2.6(4주령)이였으나, final weight(10주령) 6주간 투여를 한 결과, 난소적출에 의해 체중증가량과 식이효율이 증가하였다. 난소절제 전부터 보골지 에탄올 추출물 식이를 급여한 군(PL, PH군)은 난소절제(OVX)군에 비하여 유의적으로 체중이 낮았고 난소절제 후 3주간의 바쿠치올 투여군(BL, BH군)에서는 OVX군의 체중과 유의적인 차이는 보이지 않았다. The initial body weight was 78g ± 2.6 (4 weeks old), but after 6 weeks of final weight (10 weeks old), weight gain and dietary efficiency were increased by ovarian extraction. The group fed the dietary bone ethanol extract before ovarian resection (PL, PH) had significantly lower body weight than the ovarian resection (OVX) group, and OVX in the bakuchiol-administered group (BL, BH group) for 3 weeks after ovarian resection. There was no significant difference from the weight of the group.
난소절제로 인한 자궁위축에 미치는 영향을 살펴보면, OVX군에서는 자궁의 무게가 크게 감소되었는데 반면 난소절제 후 17β-에스트라디올(17β-estradiol, E2)을 주사한 OE군에서는 자궁위축이 심하게 일어나지 않아 자궁의 무게가 다른 OVX군들과 유의적으로 차이가 났으며 난소를 적출하지 않은 Sham군보다는 낮으나 난소적출에도 불구하고 자궁위축이 상당히 억제된 것으로 나타났다. 바쿠치올 투여군(BL군, BH군)에서는 OVX군과 비교하였을 때 일부 자궁위축 억제 효과를 보였으며 OVX군보다 무게가 증가하였고 총길이도 차이가 있었으나, 전체 군을 통계처리 했을 때 OVX군과 유의적인 차이는 보이지 않았다. 보골지추출물 투여군(PL군, PH군)은 BL군과 BH군 보다 자궁위축 억제효과가 더 높았다. 간과 신장의 무게는 각 군별 차이가 없었으나, 체중 100g 당 무게는 OE군이 대체적으로 높게 나타났다. In the OVX group, the weight of the uterus was significantly reduced, whereas in the OE group injected with 17β-estradiol (E2) after ovarian resection, the uterine atrophy did not occur severely. WT was significantly different from other OVX groups and was significantly lower than Sham group without ovarian extraction but significantly suppressed uterine atrophy despite ovarian extraction. The bakuchiol-administered group (BL group, BH group) showed some suppression of uterine atrophy compared to OVX group, increased weight and difference in total length than OVX group, but statistically significant compared with OVX group. No difference was seen. The golgol extract group (PL group, PH group) had a higher inhibitory effect on uterine atrophy than the BL group and BH group. The weight of liver and kidney was not different in each group, but the weight per 100g body weight was higher in OE group.
혈중 중성지방, 총 콜레스테롤, HDL-콜레스테롤, LDL-콜레스테롤, 총콜레스테롤 및 유리지방산을 분석한 결과, 난소 적출에 의해 중성지방과 지방산이 증가하고 HDL 농도가 감소되는 경향을 보였다. OE군에서는 HDL, fatty acid 수치는 개선되나 TG 수치는 개선효과가 관찰되지 않고 오히려 증가되었으며, LDL, free cholesterol과 총 콜레스테롤 함량이 크게 증가되었다. 한편, 바쿠치올 투여군과 보골지 추출물 투여군 모두 TG, TC가 뚜렷하게 감소하였고(표 2 참조) LDL, FFA, FC 저하에도 효과적이었다. Analysis of blood triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, total cholesterol and free fatty acids showed that ovarian extraction increased triglycerides and fatty acids and decreased HDL levels. In the OE group, HDL and fatty acid levels were improved, but TG levels were not improved but rather increased, and LDL, free cholesterol and total cholesterol contents were significantly increased. On the other hand, TG and TC were significantly decreased in the bakuchiol-administered and golgol extract-treated groups (see Table 2), and were also effective in reducing LDL, FFA, and FC.
혈중 estradiol(E2)의 농도는 난소적출에 의해 혈중 Estradiol level이 현저하게 낮아졌으며(OVX군), 이러한 혈중 Estradiol level 수치는 17β-estradiol과 bakuchiol 투여, 보골지 추출물 투여에 의해 유의적으로 증가하였다. 혈청 alkaline phsphatase(ALP)농도는 난소적출에 의해 현저하게 상승되었으나(OVX군), bakuchiol 투여, 보골지 추출물 투여에 의해 SHAM군 수준으로 낮아지는 효과가 나타났다. Serum estradiol (E2) levels were significantly lowered by ovarian extraction (OVX group), and these levels were significantly increased by 17β-estradiol, bakuchiol, and Bone Golgi extracts. Serum alkaline phsphatase (ALP) concentration was significantly increased by ovarian extraction (OVX group), but decreased to SHAM group level by bakuchiol and golgol extract.
간 중 지질함량을 분석한 결과, 난소적출에 의해 지질의 함량, 중성지방 및 총 콜레스테롤의 증가를 보였으며, 이러한 증가는 bakuchiol 투여에 의해 개선되는 경향을 보였고 특히 콜레스테롤 함량의 저하효과가 컸다. PL과 PH군에서는 bakuchol보다 더 효과적이었다. 이는 투여기간의 차이(bakuchiol 3주, PL, PH 6주)와 bakuchol외에 보골지 추출물에 함유된 성분에 기인한 것으로 사료되었다. As a result of analyzing the lipid content in liver, lipid content, triglyceride, and total cholesterol were increased by ovarian extraction, and this increase tended to be improved by bakuchiol administration. PL and PH groups were more effective than bakuchol. This may be due to the difference in administration period (3 weeks bakuchiol, PL, 6 weeks PH) and the components contained in the extract of Bogolgo besides bakuchol.
간 중 항산화효소계 활성을 측정한 결과, 난소를 적출한 OVX군에서 MDA 수치의 증가와 catalase, SOD 및 GPx 수치의 감소를 나타내었다. Bakuchiol 및 보골지 추출물의 투여에 의해 MDA 수치가 감소하였으며, 그 외 catalase, SOD, GPx 등의 항산화 활성은 유의적인 증가를 보였으며, 특히 catalase와 GPx 활성면에서 더 큰 효과를 보이는 것으로 나타났다(표 3 참조). Antioxidant enzyme activity in the liver was shown to increase MDA levels and decrease catalase, SOD and GPx levels in the OVX group. The administration of Bakuchiol and Bogolgol extracts decreased MDA levels, and other antioxidant activities such as catalase, SOD and GPx showed a significant increase, especially in terms of catalase and GPx activity. 3).
표 2. Table 2.
표 3.Table 3.
골밀도 측정기로 대퇴골의 골밀도를 측정한 결과, OVX군에서 유의적으로 골밀도가 낮아져 Sham군의 50％ 이하 수준을 보였으나 OE군의 경우 Sham군보다도 오히려 높은 골밀도를 나타내어 골대사에 에스트로겐 활성이 미치는 영향을 확인할 수 있었다. 바쿠치올투여군은 OVX로 인한 골손실 진행에 억제 효과가 나타났으며 특히 고농도 투여군(BH군)의 경우 OE 수준으로 골밀도가 높았다. PL과 PH 투여군의 경우도 골손실 억제 효과가 뚜렷하였고 농도의존적인 효과를 보였다.As a result of measuring the BMD of the femur with BMD, BMD was significantly lower in the OVX group, which was below 50% of the Sham group. I could confirm it. The bakuchiol-administered group had an inhibitory effect on the progression of bone loss due to OVX, especially in the high-concentration group (BH group), which had high bone density. In the PL and PH groups, bone loss was also suppressed and concentration-dependent.
혈중 무기질 농도에서는 바쿠치올투여군(BL. BH)과 보골지추출물투여군(PL, PH)의 경우 OE군만큼 현저한 칼슘(Ca), 인(P) 수준의 변화는 관찰되지 않았으나 칼슘농도는 Sham군의 수준을 유지하는 것으로 나타났다. In the case of serum mineral concentrations, no significant changes in calcium (Ca) and phosphorus (P) levels were observed in the bakuchiol administration group (BL. BH) and the golgol extract administration group (PL, PH). Appeared to maintain levels.
이상의 결과와 같이 난소절제로 유도한 갱년기 동물모델을 대상으로 혈청 및 간의 지질 대사계 변화, 체내 항산화 효소계 변화를 살펴본 결과 체중증가, 자궁 중량, 혈청의 estradiol 농도, 간의 지질, TG, TC, 골밀도 등 갱년기 호르몬 결핍 으로 인한 biomarker들에 바쿠치올과 보골지 추출물의 효과가 확인되었고, 특히 혈청 에스트라디올 농도, 골밀도, 간의 TC 저하에 효과가 우수하였다. As described above, the changes in serum metabolism and hepatic lipid metabolism and changes in the antioxidant enzyme system in the menopausal animal model induced by ovarian resection showed weight gain, uterine weight, serum estradiol concentration, hepatic lipids, TG, TC, bone density, etc. The effects of bakuchiol and golgol extracts on biomarkers due to deficiency of menopausal hormones were confirmed.
<실시예 5> 보골지 추출물과 및 바쿠치올이 유방암 세포증식에 미치는 영향Example 5 Effects of Bogolgol Extract and Bakuchiol on Breast Cancer Cell Proliferation
보골지 추출물이 유방암 세포증식에 미치는 영향을 측정하기 위해 E-screen assay를 이용하였다. 실험방법은 세포배양과 시료에 의한 세포증식으로 구분된다. 실험에 이용된 세포주 및 세포배양방법은 인간 유선(human mammary gland) 유래의 상피세포로 ER을 가진 MCF-7cell과 ER을 가지지 않은 MDA-MB231 cell(KCLB, Korea)을 CO2 incubator(5％ CO2, 37℃)에서 10％ FBS와 0.01mg/mL의 bovine insulin, 항생제(100 unit/mL penicillin, 100㎍/mLstreptomycin)를 첨가한 RPMI 1640 배지로 3∼4일마다 배지를 교환하고 7일마다 계대 배양을 통하여 사용하였다. MDA-MB231 cell은 10％ FBS와 항생제(100 unit/mL penicillin, 100㎍/mL streptomycin)을 첨가한 RPMI 1640 배지로 4∼5일마다 배지를 교환하고 6∼7일마다 계대 배양을 통하여 사용하였다. 세포증식 효과의 측정은 Soto et al.(1995)의 방법(Soto et al., The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic enviromental pollutants. Envirom Health Pers, 103, 113∼22, 1995).을 일부 변형하여 적용하였다. MCF-7과 MDA-MB 231 cell(KCLB, Korea)은 배지 중의 에스트로겐 활성을 나타내는 성분을 배제하기 위하여, 10％ charcoal treated FBS을 함유한 phenol red를 포함하지 않는 RPMI 1640 medium으로 96 well plate에 well당 2×103 개를 seeding하고 24시간 후 test medium (5％ charcoal dextran treated FBS를 함유한 phenol-red를 포함하지 않는 RPMI 1640)으로 교환한 후, 각 플라보노이드 화합물을 농도별로 144시간 노출시켰다. 각 물질은 DMSO로 용해시켜 사용하였으며, 세포에 처치하는 DMSO의 최종 농도는 0.1％가 넘지 않게 하였다. 대조군은 용매로 사용한 DMSO만을 처치한 군으로 하였다. 세포 증식 효과를 측정하기 위하여 MTT 용액을 well 당 0.1mg 가해주고 37℃에서 4시간 더 배양하여 생긴insoluble purple formazan crystal에 DMSO와 glycine buffer(0.1M glycine, 0.1M NaCl, adjusted to pH 10.5 with 1M NaOH)로 solubilization을 유도한 후 microplate ELISA reader로 540 nm에서 흡광도를 측정하였다.E-screen assay was used to determine the effect of Bogolgol extract on breast cancer cell proliferation. Experimental methods are divided into cell culture and cell proliferation by sample. The cell lines and cell culture methods used in the experiment were epithelial cells derived from human mammary gland, and MC 2-7 cells with ER and MDA-MB231 cells without ER (KCLB, Korea) were CO 2 incubator (5% CO 2 , 37 ° C), medium-changed every 3-4 days with RPMI 1640 medium supplemented with 10% FBS, 0.01 mg / mL bovine insulin and antibiotics (100 unit / mL penicillin, 100 μg / mL streptomycin) It was used through passage culture. MDA-MB231 cells were replaced with RPMI 1640 medium supplemented with 10% FBS and antibiotics (100 unit / mL penicillin, 100µg / mL streptomycin) every 4 to 5 days, and then subcultured every 6 to 7 days. . Measurement of cell proliferation effects is described in Soto et al. (1995) (Soto et al ., The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic enviromental pollutants. Envirom Health Pers, 103, 113-22, 1995). MCF-7 and MDA-MB 231 cells (KCLB, Korea) are well prepared in 96 well plates with RPMI 1640 medium that does not contain phenol red containing 10% charcoal treated FBS in order to exclude components showing estrogen activity in the medium. After 2 x 10 3 sugars were seeded and replaced with test medium (RPMI 1640 without phenol-red containing 5% charcoal dextran treated FBS) after 24 hours, each flavonoid compound was exposed for 144 hours by concentration. Each material was used by lysing with DMSO and the final concentration of DMSO treated in the cells was no more than 0.1%. The control group was treated with DMSO used as a solvent. In order to measure the effect of cell proliferation, 0.1mg of MTT solution was added per well, followed by incubation for 4 hours at 37 ° C. DMSO and glycine buffer (0.1M glycine, 0.1M NaCl, adjusted to pH 10.5 with 1M NaOH) Induction of solubilization with) was measured at 540 nm with a microplate ELISA reader.
실험결과, 10-9M의 17β-estradiol(E2)을 positive control로, DMSO 처리군(100％)과 비교했을 때 보골지 에탄올 추출물은 0.1㎍/ml의 농도에서 139％의 세포증식효과를 보였다. 특히 1㎍/ml의 농도에서는 10-9 M의 E2 처리군의 효과(228％)보다도 높은 243％의 높은 세포증식효과를 보여 매우 효과적이었다. 보골지로부터 분리한 바쿠치올은 10-7∼10-6M의 농도에서 가장 높은 세포증식 효과를 보였으며 이는 대조군과 비교했을 때 약 2.3배에 이르는 것으로 세포증식효과가 큰 것으로 나타났다. Experimental results showed that ethanol extract of Bulgogi ethanol showed 139% cell proliferation effect at the concentration of 0.1 µg / ml when 10 -9 M of 17β-estradiol (E2) was positive control. . In particular, at a concentration of 1 µg / ml, the cell proliferation effect of 243% was higher than that of the 10 -9 M E2 treatment group (228%), which was very effective. The bakuchiol isolated from the bones showed the highest cell proliferation effect at the concentration of 10 -7 ~ 10 -6 M, which was about 2.3 times higher than that of the control group.
<제조예 1> 정제의 제조Preparation Example 1 Preparation of Tablet
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 10g10 g of bakuchiol isolated from Bogolgol ethanol extract of Example 1
락토스 70gLactose 70g
결정성 셀룰로오스 15g15 g of crystalline cellulose
마그네슘 스테아레이트 5gMagnesium Stearate 5g
총량 100g100 g
상기에서 나열된 성분들을 잘게 부숴 혼합한 후 직타법에 의해 정제를 제조하였다. The tablets were prepared by direct stroke after mixing the ingredients listed above finely.
<제조예 2> 산제Production Example 2 Powder
하기 성분을 혼합한 후 통상의 산제 제조방법에 따라서 기밀포에 충진하여 산제를 제조하였다:The powders were prepared by mixing the following ingredients and then filling the airtight cloth according to a conventional powder preparation method:
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 2g2 g of bakuchiol isolated from Bogolgol ethanol extract of Example 1
유당 1g1g lactose
<제조예 3> 캡슐제Preparation Example 3 Capsule
하기 성분을 혼합한 후 통상의 캡슐제 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다:The capsules were prepared by mixing the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method:
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 100㎎100 mg of bakuchiol isolated from Bogolchi ethanol extract of Example 1
옥수수 전분 100㎎Corn Starch 100mg
유당 100㎎Lactose 100mg
스테아린산 마그네슘 2㎎2 mg magnesium stearate
<제조예 4> 주사제Production Example 4 Injection
통상의 주사제 제조방법에 따라 활성성분을 주사용 증류수에 용해하고 pH 조절제로 pH를 7.5로 조절한 다음 하기 나머지 성분 전체를 주사용 증류수로 2㎖ 용량의 앰플에 충진하고 멸균시켜서 주사제를 제조하였다:Injectables were prepared by dissolving the active ingredient in distilled water for injection according to a conventional injection method, adjusting the pH to 7.5 with a pH adjusting agent, and then filling the 2 ml volume of the ampoule with distilled water for injection and sterilizing the following remaining ingredients:
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 100㎎100 mg of bakuchiol isolated from Bogolchi ethanol extract of Example 1
주사용수 10ml10ml water for injection
pH 조절제 5mgpH adjuster 5mg
<제조예 5> 선식<Manufacture example 5> Wire type
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 만들었다. 검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 만들었다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.
본 발명의 보골지 추출물(바쿠치올함유)을 진공 농축기에서 감압 농축하고, 분무 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60 메쉬로 분쇄하여 대황 추출물 건조 분말을 얻었다.Bogolji extract (containing bakuchiol) of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray hot air dryer was pulverized to a particle size of 60 mesh with a grinder to obtain a rhubarb extract dry powder.
상기에서 제조한 곡물류, 종실류 및 포도나무 수피 추출물의 건조 분말을 다음의 비율로 배합하여 과립을 만들었다.Granules were prepared by combining the dry powders of the grains, seeds and vine bark extract prepared above in the following proportions.
곡물류 : 현미 30 중량％, 율무 15 중량％, 보리 20 중량％, 찹쌀 9 중량％,Cereals: 30% by weight brown rice, 15% by weight barley, 20% by weight barley, 9% by weight glutinous rice,
종실류 : 들깨 7 중량％, 검정콩 8 중량％, 검정깨 7 중량％,Seeds: perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight,
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 건조 분말 3 중량％, 영지 0.5 중량％, 지황 0.5 중량％3% by weight of bakuchiol dry powder, 0.5% by weight of Ganoderma lucidum, and 0.5% by weight of sulfuric acid
<제조예 6> 츄잉껌Preparation Example 6 Chewing Gum
껌 베이스 20 중량％, 설탕 76.9 중량％, 향료 1 중량％ 및 물 2 중량％와 실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 0.1 중량％를 배합하여 통상의 방법으로 츄잉껌을 제조하였다.Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 0.1% by weight of bakuchiol isolated from the Bogolgo ethanol extract of Example 1.
<제조예 7> 캔디Production Example 7 Candy
설탕 60중량％, 물엿 39.8중량％ 및 향료 0.1중량％와 실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 0.1중량％를 배합하여 통상의 방법으로 캔디를 제조하였다.Candy was prepared in a conventional manner by combining 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of fragrance, and 0.1% by weight of bakuchiol isolated from the Bogolji ethanol extract of Example 1.
<제조예 8> 비스켓Production Example 8 Biscuits
박력 1급 25.59중량％, 중력 1급 22.22중량％, 정백당 4.80중량％, 식염 0.73중량％, 포도당 0.78중량％, 팜쇼트닝 11.78중량％, 암모늄 1.54중량％, 중조 0.17중량％, 중아황산나트륨 0.16중량％, 쌀가루 1.45중량％, 비타민 B₁0.0001중량％, 비타민 B₂0.0001중량％, 밀크향 0.04중량％, 물 20.6998중량％, 전지분유 1.16중량％, 대용분유 0.29중량％, 제1인산칼슘 0.03중량％, 살포염 0.29중량％ 및 분무유 7.27중량％와 실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 1중량％를 배합하여 통상의 방법으로 비스켓을 제조하였다. Force primary 25.59% by weight, gravity primary 22.22% by weight, white sugar 4.80% by weight, salt 0.73% by weight, glucose 0.78% by weight, palm shortening 11.78% by weight, ammonium 1.54% by weight, 0.17% by weight sodium bisulfite 0.16% by weight , Rice flour 1.45%, Vitamin B₁0.0001%, Vitamin B20.0001%, Milk flavor 0.04%, Water 20.6998%, Whole milk powder 1.16%, Substitute milk powder 0.29%, Monobasic calcium phosphate 0.03% , Biscuits were prepared in a conventional manner by combining 0.29% by weight of spraying salt, 7.27% by weight of spray oil, and 1% by weight of bakuchiol separated from the Bogolji ethanol extract of Example 1.
<제조예 9> 건강 음료 <Manufacture example 9> Healthy drink
꿀 0.26중량％, 치옥토산아미드 0.0002중량％, 니코틴산아미드 0.0004중량％, 염산리보플라빈나트륨 0.0001중량％, 염산피리독신 0.0001중량％, 이노시톨 0.001중량％, 오르트산 0.002 중량％ 및 물 98.7362중량％와 실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 1중량％를 배합하여 통상의 방법으로 건강 음료를 제조하였다.0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of sodium riboflavinate, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 98.7362% by weight of water. 1 wt% of bakuchiol isolated from Bogolgol ethanol extract of 1 was blended to prepare a health beverage in a conventional manner.
<제조예 10> 건강보조식품Preparation Example 10 Health Supplement
스피루리나 55중량％, 구아검효소 분해물 10중량％, 비타민 B₁염산염 0.01중량％, 비타민 B6 염산염 0.01중량％, DL-메티오닌 0.23중량％, 스테아린산 마그네슘 0.7중량％, 유당 22.2중량％ 및 옥수수전분 1.85중량％와 실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 10중량％를 배합하여 통상의 방법으로 정제형 건강보조식품을 제조하였다.55% by weight of spirulina, 10% by weight of guar gum enzyme digestion, 0.01% by weight of vitamin B ₁ hydrochloride, 0.01% by weight of vitamin B 6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch % And 10% by weight of bakuchiol isolated from the Bogolgo ethanol extract of Example 1 were prepared to prepare a tablet health supplement in a conventional manner.
<제조예 11-20>Production Example 11-20
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 대신 실시예 1의 보골지 에탄올 추출물로부터 분리한 바바크로민을 사용하는 것을 제외하고는 상기 제조예 1 내지 제조예 10과 동일한 방법을 사용하여 바바크로민을 포함하는 정젯, 바바크로민을 포함하는 산제, 바바크로민을 포함하는 과립제, 바바크로민을 포함하는 주사제, 바바크로민을 포함하는 선식, 바바크로민을 포함하는 츄잉껌, 바바크로민을 포함하는 캔디, 바바크로민을 포함하는 비스켓, 바바크로민을 포함하는 건강음료, 바바크로민을 포함하는 건강보조식품을 제조하였다.Baba was used in the same manner as in Preparation Examples 1 to 10 except for using Babacromine isolated from Bogolji ethanol extract of Example 1 instead of Bakuchiol isolated from Bogolgi ethanol extract of Example 1 Judges with chromin, powders with babacromine, granules with babacromine, injections with babacromine, wire with babacromine, chewing gum with babacromine, babacromine Candy, including a biscuit containing babacromine, a health drink containing babacromine, a health supplement containing barbacromine was prepared.
<제조예 21-30>Production Example 21-30
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 대신 실시예 1의 보골지 에탄올 추출물로부터 분리한 이소바바크로민을 사용하는 것을 제외하고는 상기 제조예 1 내지 제조예 10과 동일한 방법을 사용하여 이소바바크로민을 포함하는 정젯, 이소바바크로민을 포함하는 산제, 이소바바크로민을 포함하는 과립제, 이소바바크로민을 포함하는 주사제, 이소바바크로민을 포함하는 선식, 이소바바크로민을 포함하는 츄잉껌, 이소바바크로민을 포함하는 캔디, 이소바바크로민을 포함하는 비스켓, 이소바바크로민을 포함하는 건강음료, 이소바바크로민을 포함하는 건강보조식품을 제조하였다.Using the same method as Preparation Examples 1 to 10 except for using isobavaccromin isolated from Bogolgol ethanol extract of Example 1 instead of bakuchiol separated from Bogolgol ethanol extract of Example 1 Isobarbacromine, powder containing isovabacromine, granules containing isobabacromine, injectables containing isovaba cromine, wire containing isovaba cromine, isovaba cromine Chewing gum containing, candy containing isobavacromine, biscuits containing isobavacromine, health beverages containing isobavacromine, and health supplements containing isobavacromine were prepared.
<제조예 31-40>Production Example 31-40
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 대신 실시예 1의 보골지 에탄올 추출물로부터 분리한 바바찰톤을 사용하는 것을 제외하고는 상기 제 조예 1 내지 제조예 10과 동일한 방법을 사용하여 바바찰톤을 포함하는 정젯, 바바찰톤을 포함하는 산제, 바바찰톤을 포함하는 과립제, 바바찰톤을 포함하는 주사제, 바바찰톤을 포함하는 선식, 바바찰톤을 포함하는 츄잉껌, 바바찰톤을 포함하는 캔디, 바바찰톤을 포함하는 비스켓, 바바찰톤을 포함하는 건강음료, 바바찰톤을 포함하는 건강보조식품을 제조하였다.Babachalton using the same method as in Preparation Example 1 to Preparation Example 10, except that bachalol separated from the golgoji ethanol extract of Example 1 was used instead of bakuchiol isolated from the golgoji ethanol extract of Example 1 Containing a tablet, a powder containing barbachaltone, granules containing barbachalton, injection containing barbachalton, a wire containing barbachalton, chewing gum containing barbachaltone, chewing gum including barbachaltone, barbachalton Biscuits containing, a health drink containing barbachalton, a health supplement containing barbachalton was prepared.
<제조예 41-50>Production Example 41-50
실시예 1의 보골지 에탄올 추출물로부터 분리한 바쿠치올 대신 실시예 1의 보골지 에탄올 추출물로부터 분리한 바바찰톤을 사용하는 것을 제외하고는 상기 제조예 1 내지 제조예 10과 동일한 방법을 사용하여 이소바바찰톤을 포함하는 정젯, 이소바바찰톤을 포함하는 산제, 이소바바찰톤을 포함하는 과립제, 이소바바찰톤을 포함하는 주사제, 이소바바찰톤을 포함하는 선식, 이소바바찰톤을 포함하는 츄잉껌, 이소바바찰톤을 포함하는 캔디, 이소바바찰톤을 포함하는 비스켓, 이소바바찰톤을 포함하는 건강음료, 이소바바찰톤을 포함하는 건강보조식품을 제조하였다.Isobaba using the same method as Preparation Examples 1 to 10 except for using Babachaltone separated from Bogolji ethanol extract of Example 1 instead of Bakuchiol isolated from Bogolgi ethanol extract of Example 1 A jet containing Charlton, a powder containing Isobarchalton, a granule containing Isobarchalton, an injection containing Isobarchalton, a wire containing Isobarchalton, a chewing gum containing Isobarchalton, a chewing gum with Isobarchalton Candy containing, biscuits containing isobavacalton, health beverages containing isobavacalton, and health supplements including isobavacalton were prepared.
상술한 바와 같이, 본 발명의 바람직한 실시예를 참조하여 설명하였지만 해당 기술 분야의 숙련된 당업자라면 하기의 특허청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다.As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.
도 1은 재조합 효모 분석법에 의한 보골지 에탄올 추출물의 농도별 에스트로겐 활성에 대한 그래프이다.Figure 1 is a graph of the estrogen activity by concentration of golgol ethanol extract by recombinant yeast assay.
도 2는 재조합 효모 분석법에 의한 바쿠치올, 바바치닌, 바바찰콘, 이소바바찰콘, 바바크로민, 이소바바크로민의 농도별 에스트로겐 활성에 대한 그래프이다.Figure 2 is a graph of the estrogen activity according to the concentration of bakuchiol, babachinin, babachalcon, isobavacchalcon, babacromine, isobavacromine by recombinant yeast assay.
도 3은 난소절제 갱년기 모델에서 에스트라디올(OE군), 보골지추출물(PL, PH군), 바쿠치올(BL, BH) 투여에 따른 자궁의 변화에 대한 사진이다.Figure 3 is a photograph of the change of the uterus according to the administration of estradiol (OE group), golgol extract (PL, PH group), bakuchiol (BL, BH) in the ovarian resection menopausal model.
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| KR20190020874A (en) | 2017-08-21 | 2019-03-05 | 주식회사 미즈바이오 헬스케어 | Composition for treating or preventing postmenopausal syndrome containing glycyrrizae radix extract, astragali radix extract and spatholobus suberectus extract |
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| CN104496783B (en) * | 2014-12-30 | 2016-09-21 | 成都普思生物科技有限公司 | A kind of isolation and purification method of 4 '-O-methyl Bavachalcone B monomer |
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| KR20190020874A (en) | 2017-08-21 | 2019-03-05 | 주식회사 미즈바이오 헬스케어 | Composition for treating or preventing postmenopausal syndrome containing glycyrrizae radix extract, astragali radix extract and spatholobus suberectus extract |
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