KR20120044450A - Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba - Google Patents

Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba Download PDF

Info

Publication number
KR20120044450A
KR20120044450A KR1020100105726A KR20100105726A KR20120044450A KR 20120044450 A KR20120044450 A KR 20120044450A KR 1020100105726 A KR1020100105726 A KR 1020100105726A KR 20100105726 A KR20100105726 A KR 20100105726A KR 20120044450 A KR20120044450 A KR 20120044450A
Authority
KR
South Korea
Prior art keywords
extract
osteoporosis
rankl
treatment
water
Prior art date
Application number
KR1020100105726A
Other languages
Korean (ko)
Inventor
마진열
심기석
이윤희
최성업
엄영란
이재훈
Original Assignee
한국 한의학 연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국 한의학 연구원 filed Critical 한국 한의학 연구원
Priority to KR1020100105726A priority Critical patent/KR20120044450A/en
Publication of KR20120044450A publication Critical patent/KR20120044450A/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • 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
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • 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
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Chemical & Material Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medical Informatics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Nutrition Science (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

PURPOSE: A composition containing Cirsii Herba extract is provided to suppress osteoclast differentiation and relating gene expression and to prevent and/or treat osteoporosis. CONSTITUTION: A pharmaceutical composition for preventing or treating osteoporosis contains Cirsii Herba extract prepared using water or organic solvent as an active ingredient. The organic solvent includes C1-C4 alcohol, acetone, or solution thereof. A health food for preventing or treating Cirsii Herba contains the extract as an active ingredient. The food includes meat, sausage, bread, chocolate, candy, pizza, ramen, other noodles, gum, soup, beverage, tea, alcoholic beverage, or vitamin complex.

Description

대계 추출물을 포함하는 골다공증 예방 또는 치료용 조성물{Composition for Prevention or Treatment of Osteoporosis Comprising Extract of Cirsii Herba}Composition for Prevention or Treatment of Osteoporosis Comprising Extract of Cirsii Herba}

본 발명은 골다공증 예방 또는 치료용 조성물에 관한 것으로서, 보다 상세하게는 대계 추출물을 유효성분으로 함유하며, 골다공증 예방 및/또는 치료용으로 유용하게 사용될 수 있는 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating osteoporosis, and more particularly, to a composition containing the extract as an active ingredient, which can be usefully used for the prevention and / or treatment of osteoporosis.

생물의 뼈조직은 파골세포에 의한 뼈흡수와 조골세포에 의한 뼈형성이 계속적으로 반복되어 동적인 평형 상태를 유지하고 있으며, 두 작용의 상대적인 균형에 의해 뼈의 항상성이 유지된다. 하지만, 비정상정인 파골세포의 생성이나 활성의 증가는 뼈의 리모델링에 불균형을 초래하여 골다공증과 같은 여러 가지 뼈 관련 질환을 유발한다. 따라서, 파골세포의 분화와 활성을 조절하는 연구가 골다공증 치료제 개발을 위한 연구로 주목받고 있다(Rodan and Martin, 2000, Science, 289:1508-1514; Boyle et al., 2003, Nature, 423:337-342). 파골세포는 단핵백혈구/대식세포로 단핵구 전구 세포에서 파골세포로 분화되어 뼈를 흡수하는 기능을 한다. RANKL (Receptor activator for nuclear factor κB (NF-κB) ligand)은 TNF-수퍼패밀리의 일원으로서 파골세포 분화 과정의 조절과 분화된 파골세포의 성장에 중요한 인자이다. RANKL이 이것의 수용체인 RANK에 결합하면 TRAF 6 (tumor necrosis factor receptor-associated factor 6)을 활성화시키고, TRAF 6는 다시 MAP (mitogen-activated protein) 키나제, 전사 인자인 NF-kB, AP-1, 및 NFATc1의 활성을 연속적으로 유도한다. 이 전사인자들은 파골세포 분화 및 파골세포의 골흡수 기능과 연관된 유전자들의 조절에 필수적이며 중요한 역할을 한다(Takayanagi et al., 2002, Dev Cell, 3:889-901; Boyle et al., 2003, Nature, 423:337-342).Bone tissues of organisms maintain a dynamic equilibrium by continuously repeating bone resorption by osteoclasts and bone formation by osteoblasts, and maintaining homeostasis of bones by the relative balance of the two functions. However, an increase in the production or activity of abnormal osteoclasts leads to an imbalance in bone remodeling, leading to various bone-related diseases such as osteoporosis. Therefore, researches that control the differentiation and activity of osteoclasts have attracted attention as a research for the development of therapeutic agents for osteoporosis (Rodan and Martin, 2000, Science, 289: 1508-1514; Boyle et al., 2003, Nature, 423: 337). -342). Osteoclasts are mononuclear leukocytes / macrophages that differentiate from monocyte progenitor cells into osteoclasts and take up bones. RANKL (Receptor activator for nuclear factor κB (NF-κB) ligand) is a member of the TNF-superfamily and is important for the regulation of osteoclast differentiation and growth of differentiated osteoclasts. When RANKL binds to its receptor, RANK, it activates tumor necrosis factor receptor-associated factor 6 (TRAF 6), which in turn triggers mito-activated protein (MAP) kinases, transcription factors NF-kB, AP-1, And continuously induce the activity of NFATc1. These transcription factors play an essential and important role in the regulation of osteoclast differentiation and the genes associated with osteoclast function of osteoclasts (Takayanagi et al., 2002, Dev Cell, 3: 889-901; Boyle et al., 2003, Nature, 423: 337-342.

골다공증은 골 흡수가 골 형성보다 항진됨으로서 골리모델링의 평형이 무너져 유발되는 질병으로, 골 조직의 석회가 감소되어 뼈의 치밀질이 엷어지고 그로 인해 골수강(骨髓腔)이 넓어지게 되며, 증세가 진전됨에 따라 뼈가 약해지기 때문에 작은 충격에도 골절되기 쉽다. 골다공증의 원인으로는 노령, 운동 부족, 저체중, 흡연, 저칼슘 식이, 폐경, 난소 절제 등이 알려져 있는데, 특히 여성의 경우 30세 이후부터 골 감소가 지속적으로 진행되며, 폐경기에 이르면 호르몬의 변화에 의해 골 감소가 급격히 진행된다. 즉, 골다공증은 정도의 차이는 있으나 노년층, 특히 폐경기 이후의 여성에게 있어서는 피할 수 없이 나타나는 증상으로, 선진국에서는 인구가 노령화됨에 따라 골다공증 및 그 치료제에 대한 관심이 점차 증가되고 있다. 골다공증은 크게 3가지 유형으로 나눌 수 있는데, 첫째는 원인불명(idiopathic)의 골다공증, 둘째는 폐경기 이후 여성호르몬 부족에 의해 나타나는 타입 Ⅰ 골다공증, 셋째는 고령화된 노인층의 남녀에게 발견되는 타입 Ⅱ 골다공증이다.Osteoporosis is a disease caused by the loss of bone remodeling than bone formation, resulting in a reduction in the balance of bone modeling, resulting in a decrease in lime in bone tissue, resulting in thinning of the bone's densities and widening of the bone marrow cavity. As the bones weaken as they progress, they are more likely to fracture even with a small impact. The causes of osteoporosis are known to be old age, lack of exercise, low weight, smoking, low calcium diet, menopause, ovarian resection, and especially in women, bone loss continues after age 30, and hormone changes in menopause. As a result, bone reduction progresses rapidly. In other words, osteoporosis is an unavoidable symptom in elderly people, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. Osteoporosis can be divided into three types: first, idiopathic osteoporosis, second, type I osteoporosis caused by post-menopausal female hormone deficiency, and third, type II osteoporosis found in older men and women.

현재 사용되고 있는 골다공증 치료제는 대부분 에스트로겐 계통의 물질로서, 이들은 장기 투여할 경우 암, 담석, 혈전증 등의 부작용이 나타나는 문제점이 있다. 골다공증은 약물의 단기 투여만으로는 치료할 수 없고 장기 투여가 필수적인 질환이므로, 약물을 장기 투여할 때에도 상기와 같은 부작용이 없고 에스트로겐을 대체할 수 있을 만큼 우수한 약효를 갖는 새로운 물질의 개발이 요구되고 있다. 따라서, 콩 및 콩 가공품 등 식품에 함유된 제니스테인(genistein)이나 다이드제인(daidzein) 등의 피토에스트로겐(phytoestrogen)을 응용하여 장기간 복용할 수 있고 상대적으로 부작용이 적은 골다공증 예방 및 치료제를 개발하고자 많은 연구가 진행 중이다.Currently used osteoporosis therapeutic agents are mostly estrogen-based substances, these have the problem that side effects such as cancer, gallstones, thrombosis when long-term administration. Osteoporosis is a disease that can not be treated only by short-term administration of drugs, and long-term administration is essential, and thus, there is a need for development of a new substance having long-term administration of drugs and excellent efficacy to replace estrogen. Therefore, many people are trying to develop phytoestrogens such as genistein or daidzein in foods such as soybeans and soybean processed products that can be taken for a long time and have relatively low side effects. Research is ongoing.

식물성 호르몬인 피토에스토로겐은 생체이용률은 비교적 낮으나 에스트로겐 수용체-α (ER-α) 및 에스트로겐 수용체-β (ER-β)에 대한 친화력이 좋고 유방암 등의 부작용이 적어 골다공증 치료제인 Estradiol의 대체 의약품으로 각광받고 있다. 피토에스트로겐으로 알려진 물질로는 다이드제인, 제니스테인, 포르모노네틴(formononetin), 비오카닌 A (biochanin A) 등의 이소플라본(isoflavone)류 화합물, 쿠메스트롤(coumestrol) 등의 쿠메스탄(coumestan)류 화합물, 엔테로락톤(enterolactone) 등의 리그난(lignan)계 화합물 및 엔테로디올(enterodiol) 등의 페놀(phenol)계 화합물이 있다. 상기 화합물들 중 당 화합물은 장내 박테리아의 β-글루코시다제(glucosidase) 또는 위산에 의해 가수분해되어 결국 비배당체 형태인 유리(free) 이소플라본으로 흡수된다.Phytoestrogogen, a plant hormone, has a relatively low bioavailability but has a good affinity for estrogen receptor-α (ER-α) and estrogen receptor-β (ER-β) and has less side effects such as breast cancer. I am getting it. Substances known as phytoestrogens include isoflavone compounds such as dydzein, genistein, formononetin, and biochanin A, and coumestan, such as coumestrol. ) Compounds, lignan compounds such as enterolactone, and phenol compounds such as enterodiol. Of these compounds, sugar compounds are hydrolyzed by the intestinal bacteria β-glucosidase or gastric acid and are eventually absorbed into free isoflavones in the nonglycoside form.

한편, 대계는 자궁출혈 억제 및 혈액 순환을 증진시키는 효과가 있는 전통 허브 치료제이다. 현재까지 밝혀진 대계의 다양한 약리 효과로는 H22 간암 및 S180 육종의 암세포 성장을 억제하는 효과(Liu et al., 2006, Int. Immunopharmacol, 6(9):1387-1393), 내피조직 내 H1 수용체 자극을 통해 대동맥 이완을 유도하는 효과(Kim et al., 2008, J. Ethnopharmacol, 116(2):223-227), 지질 과산화를 억제시키고 알코올 산화를 억제하여 알코올로 인한 간독성을 억제하는 효과(Park et al., 2004, Phytother Res, 18(1):19-24) 등이 보고된 바가 있다. 하지만, 아직까지 대계가 파골세포 분화를 억제하여 골다공증의 완화 또는 치료에 사용될 수 있음을 보여주는 종래문헌은 알려져 있지 않다.On the other hand, the large system is a traditional herbal treatment that has the effect of inhibiting uterine bleeding and promoting blood circulation. Various pharmacological effects of the current system have been found to inhibit cancer cell growth of H22 liver cancer and S180 sarcoma (Liu et al., 2006, Int. Immunopharmacol, 6 (9): 1387-1393), stimulating H1 receptors in endothelial tissues. To induce aortic relaxation (Kim et al., 2008, J. Ethnopharmacol, 116 (2): 223-227), to inhibit lipid peroxidation and to inhibit alcohol oxidation to inhibit hepatotoxicity caused by alcohol (Park et al., 2004, Phytother Res, 18 (1): 19-24). However, there is no known literature that shows that the larvae can be used for alleviating or treating osteoporosis by inhibiting osteoclast differentiation.

본 발명은 대계 추출물을 함유하는 골다공증 예방 또는 치료용 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a composition for the prevention or treatment of osteoporosis containing the extract.

상기 목적을 달성하기 위하여, 본 발명은 대계 추출물을 유효성분으로 함유하는 골다공증 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which contains the extract as an active ingredient.

상기 대계 추출물을 제조함에 있어서, 추출용매로는 물을 사용하는 것이 일반적이지만, 필요에 따라 물 이외에 다른 유기용매를 사용해도 무방하다. 사용될 수 있는 유기용매로는 C1 내지 C4의 저급 알코올, 아세톤, 또는 이의 수용액 등을 들 수 있지만 이에 한정되는 것은 아니다.In the preparation of the base extract, it is common to use water as the extraction solvent, but other organic solvents other than water may be used if necessary. Organic solvents that can be used include, but are not limited to, C 1 to C 4 lower alcohols, acetone, or aqueous solutions thereof.

본 발명의 바람직한 구현예에 따르면, 상기 대계 추출물을 제조하기 위해서는 대계에 2?20배량의 추출용매, 바람직하게는 물을 가한 후 70 내지 100℃에서 열수 추출한 수추출물을 제조한다. 이때, 상기 대계를 0.5 내지 5시간 동안 추출용매에 침적시킨 후 추출 과정을 수행하는 것이 바람직하지만, 이에 한정되는 것은 아니다.According to a preferred embodiment of the present invention, in order to prepare the base extract, a water extract of hot water extracted at 70 to 100 ° C. is prepared after adding 2-20 times the amount of an extraction solvent, preferably water. At this time, it is preferable to perform the extraction process after immersing the base in the extraction solvent for 0.5 to 5 hours, but is not limited thereto.

본 발명에 따른 대계 추출물은 임상 투여시에 경구 또는 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다. 즉, 본 발명의 대계 추출물은 실제 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 대계 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제, 연고, 크림제 및 경피제제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다. 또한, 골다공증 예방 또는 치료 효능 증진을 위해 칼슘이나 비타민 D3를 추가로 첨가할 수 있다.The alternative extract according to the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. That is, the base extract of the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the extract. Mix is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, ointments, creams and transdermal preparations. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, uthepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. In addition, calcium or vitamin D 3 may be additionally added to prevent or improve the efficacy of osteoporosis.

또한, 상기 대계 추출물은 여러 한의학 처방들, 예를 들면, 가감속명탕(加減續命湯), 갈근(葛根), 갈근귤피탕(葛根橘皮湯), 갈근맥문동산(葛根麥門冬散), 갈근죽여탕(葛根竹茹湯), 갈근탕(葛根湯), 갈근해기탕(葛根解肌湯), 갈출탕(葛朮湯), 갈황환(葛黃丸), 지각자산(枳殼煮散), 당귀백출탕(當歸白朮湯), 맥문동음자(麥門冬飮子), 발운탕(撥雲湯), 방풍탕(防風湯), 백출산(白朮散), 사물해기탕(四物解肌湯), 사위탕(瀉胃湯), 산종궤견탕(散腫潰堅湯), 삼두해정탕(三豆解湯), 삼양탕(三陽湯), 생갈근즙(生葛根汁), 소풍순기탕(疏風順氣湯), 승마갈근탕(升麻葛根湯), 승마부자탕 (升麻附子湯), 승마위풍탕(升麻胃風湯), 승양보위탕(升陽補胃湯), 시호승마탕(柴胡升麻湯), 시호조경탕(柴胡調經湯), 신달탕(腎疸湯), 신선불취단(神仙不醉丹), 신성산(神聖散), 신효명목탕(神效明目湯), 십신탕(十神湯), 여택통기탕(麗澤通氣湯), 연교승마탕(連翹升麻湯), 영선제통음(靈仙除痛飮), 옥설탕(沃雪湯), 온폐탕(溫肺湯), 익기총명탕(益氣聰明湯), 익지화중탕(益智和中湯), 인삼청기산(人蔘淸肌散), 인진사황탕(茵蔯瀉黃湯), 일보일발단(一補一發丹), 자음청위환(滋陰淸胃丸), 전생활혈탕(全生活血湯), 정력산(散), 조중탕(調中湯), 지갈탕(止渴湯), 지유산(地楡散), 진교승마탕(秦교升麻湯), 청양탕(淸陽湯), 청열해기탕(淸熱解肌湯), 충화순기탕(沖和順氣湯), 취향보설(醉鄕寶屑), 통치백물독일방(通治百物毒一方), 평위지유탕(平胃地楡湯), 평혈음(平血飮), 해백물독일방(解百物毒一方), 해소주독일방(解燒酒毒一方), 해약중독(解藥中毒), 해주화독산(解酒化毒散), 해채소독일방(解菜蔬毒一方), 해파두독방(解巴豆毒方), 홍설통중산(紅雪通中散) 등에도 첨가되어 사용될 수 있다.In addition, the Daegye extract is a variety of herbal medicine prescription, for example, gagamsokmyeongtang (加減 續 命 湯), brown root (葛根), galgun gulpipitang (葛根 橘皮 湯), Galgeummunmunsan (葛根 麥 門冬 散) , Gal Geunjukyeotang, Gal Geun-tang, Gal Geunhaegi-tang, Galchultang, Gal Hwanghwan, Crustal Assets ), Danggui Baekchultang (맥 白 朮 湯), Macmundongumja (자 子), Baluntang (방), Bangpungtang (防風 湯), Baekchulsan (白 朮 散), Samhaehaegitang (四 物 解肌) ,, Sawi-tang, Sanjong Gwagtang-tang, Samduhaejeong-tang, Samyang-tang, San-Geun-Geun juice, Excursion Soongi-tang, Horse-riding Gal Geun-tang, Horse Riding Budang-tang, Horse-riding Yueung-tang, Seungyang-bo-wi-tang, Shiho Seungmatang, Shiho Jogyeongtang, Sindaltang, Sinseonbuldandan, Sinseongsan, Shinhyo神 신 明目 湯, Shissintang, Yeogtongtonggitang, Yeongyo Horseriding Bath, Yeongseonjetong Yintong, Jade Sugar雪 湯, Onbungtang, Ikgi Chongmyeongtang, Ikjihwajungtang, Ginseng Cheonggisan, Injinsawangtang Huangshan, Ilboil Independence (一 補 一發 丹), Jyum-Cheng Yu-Whan (滋陰 淸 胃 丸), Jeon-Sang Hyeol-Tang (全 生活 血 湯), Jeong-Jung-San, Jo-Jung-Tang, Ji Galtang, Jiyusan, Jingyo Seungmatang, Cheongyangtang, Cheongyeolhaegitang, Chunghwasungitang和順 氣 湯, taste-preservation, governing white water german room, pyeongwijiyutang, flat blood sound, sea white water german room解 百物 毒 一方, Haejuju German Bang, Hae poisoning poisoning, Haeju Hwadoksan, Haechae Soebangbang, Haepado Dokbang Hongseoltong Zhongshan (산 雪)通 中 散) and the like can also be used.

투약 단위는, 예를 들면 개별 투약량의 1, 2, 3 또는 4배로, 또는 1/2, 1/3 또는 1/4배로 함유할 수 있다. 개별 투약량은 유효 약물이 1회에 투여되는 양을 함유하며, 이는 통상 1일 투여량의 전부, 1/2, 1/3 또는 1/4배에 해당한다.Dosage units may, for example, contain 1, 2, 3 or 4 times the individual dose, or 1/2, 1/3 or 1/4 times. Individual dosages contain amounts in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

본 발명의 대계 추출물의 인체 투여량은 체내에서의 활성성분의 흡수도, 불활성화율 및 배설속도, 환자의 연령, 성별, 상태, 질병의 정도 등에 따라 적절히 선택되며, 성인에게 (10?300 ㎎/㎏)이고, 바람직하기로는 (20?100 ㎎/㎏)이며, 하루 1 내지 6회 나누어 투여될 수 있다.The human dosage of the extract of the present invention is appropriately selected according to the absorbency, inactivation rate and excretion rate of the active ingredient in the body, the age, sex, condition, degree of disease, etc. of the patient, and the adult (10-300 mg / Kg), preferably (20-100 mg / kg), and may be administered in 1 to 6 divided doses per day.

본 발명의 조성물을 마우스에 경구 투여시 및 복강내 투여시의 독성 실험을 수행한 결과, 경구 독성시험에 의한 50% 치사량(LD50)은 적어도 (5 g/㎏) 이상인 안전한 물질로 판명되었다.Toxicity experiments during oral and intraperitoneal administration of the compositions of the present invention to the mice, 50% lethal dose (LD50) by oral toxicity test was found to be a safe substance at least (5 g / kg) or more.

본 발명의 한 구현예에 따르면, 대계 추출물은 세포 독성을 나타내지 않을 뿐만 아니라(도 1 참조), 대계 추출물을 처리하면 파골세포 분화 관련 마커인 TRAP 활성과, 다핵성 파골세포의 형성이 유의성 있게 감소한다(도 2 참조).According to one embodiment of the invention, not only does the larvae extract exhibit no cytotoxicity (see FIG. 1), but the treatment of the larvae extract significantly reduces TRAP activity, a marker for osteoclast differentiation, and the formation of multinuclear osteoclasts. (See FIG. 2).

또한, 본 발명의 다른 구현예에 따르면, 대계 추출물 처리시 대표적인 파골세포 분화 관련 유전자들의 발현 및 이들의 발현을 조절하는 전사 인자의 발현도 유의성 있게 감소하는 효과를 얻을 수 있다(표 2 및 표 3 참조).In addition, according to another embodiment of the present invention, the expression of representative osteoclast differentiation-related genes and the expression of transcription factors that regulate their expression can also be significantly reduced when the extract is treated (Table 2 and Table 3). Reference).

또한, 본 발명의 다른 구현예에 따르면, 대계 추출물은 파골 세포 분화의 신호전달에 참여하는 것으로 알려져 있는 ERK, JNK, p38의 활성 증가를 억제시킨다(도 3 참조)(Lee and Kim, 2003, BBRC, 305:211-214).In addition, according to another embodiment of the present invention, the larvae extract inhibits the increase in activity of ERK, JNK, p38, which is known to participate in signaling of osteoclast differentiation (see FIG. 3) (Lee and Kim, 2003, BBRC). , 305: 211-214).

또한, 본 발명은 대계 추출물을 유효성분으로 함유하는 골다공증 예방 또는 치료용 건강식품을 제공한다.The present invention also provides a health food for preventing or treating osteoporosis, which contains the extract as an active ingredient.

본 발명의 대계 추출물은 골다공증 개선을 목적으로 건강식품에 첨가될 수 있다. 상기 대계 추출물을 식품 첨가물로 사용할 경우, 대계 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에는 대계 추출물이 원료에 대하여 30 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.Greater extract of the present invention may be added to health food for the purpose of improving osteoporosis. When the base extract is used as a food additive, the base extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, when producing foods or beverages, the base extract is added in an amount of up to 30% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물에는 포도당, 과당과 같은 모노사카라이드, 말토오스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이 있다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 대계 추출물 100 ㎖당 일반적으로 약 0.01?0.04 g, 바람직하게는 약 0.02?0.03 g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Natural carbohydrates described above include glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the base extract.

상기 외에도 대계 추출물은 여러가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 대계 추출물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 대계 추출물 100 중량부 당 0.01?0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract is used in various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonated drinks The carbonation agent used etc. can be contained. Other extracts may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the extract.

상기에서 살펴본 바와 같이, 대계 추출물은 파골세포 분화 및 관련 유전자 발현의 억제 효과가 뛰어나므로 골다공증의 예방 및/또는 치료용으로 유용하게 사용될 수 있다.As discussed above, the larvae extract may be useful for the prevention and / or treatment of osteoporosis because it has an excellent inhibitory effect on osteoclast differentiation and related gene expression.

도 1은 대계 추출물(CH)의 세포 독성을 보여주는 그래프이다.
도 2는 대계 추출물(CH)이 각각 RAW264.7 세포에서 RANKL에 의해 유도된 TRAP 활성(A)과 TRAP-양성 다핵성 파골세포의 형성(B)에 미치는 효과를 보여주는 그래프 및 사진이다.
도 3은 대계 추출물(CH)이 RAW264.7 세포에서 RANKL에 의해 유도된 MAP 키나제의 활성에 미치는 영향을 보여주는 웨스턴 블롯 전기영동 사진이다.Figure 1 is a graph showing the cytotoxicity of the far field extract (CH).
Figure 2 is a graph and photograph showing the effect of the extract (CH) on the effects of RANKL-induced TRAP activity (A) and the formation of TRAP-positive multinuclear osteoclasts (B) in RAW264.7 cells, respectively.
Figure 3 is a Western blot electrophoresis picture showing the effect of the extract (CH) on the activity of MAP kinase induced by RANKL in RAW264.7 cells.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 하기 실시예에 의해 한정되는 것은 아니다.
However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

실시예 1: 대계 추출물의 제조Example 1 Preparation of Large Extract

대계(영천현대약업사, YeongCheon, Korea) 50 g을 생수 1 ℓ에 넣고 1시간 동안 침적한 후, 대웅약탕기(Daewoong Extractor DWP-5000M, Incheon, Korea)를 이용하여 4시간 동안 추출하였다. 이후, 상기 추출물을 표준 시험용 체(106 ㎛, Retsch, Haan, Germany)를 이용하여 여과한 후 최종 100 ㎖이 되도록 하였다. 이를 동결 건조하여 8.7820 g(수율: 17.55%)의 대계 추출물을 얻었고, 사용하기 전까지 4℃에 보관하였다.
50 g of Daegye (Youngcheon Hyundai Pharmaceutical Co., Ltd., YeongCheon, Korea) was added to 1 liter of bottled water and soaked for 1 hour, and then extracted for 4 hours using Daewoong Extractor DWP-5000M, Incheon, Korea. The extract was then filtered using a standard test sieve (106 μm, Retsch, Haan, Germany) to a final 100 mL. It was freeze-dried to obtain 8.7820 g (yield: 17.55%) of the base extract, which was stored at 4 ° C. until use.

실시예 2. 세포 독성 측정Example 2. Cytotoxicity Measurement

실시예 1에서 제조한 대계 추출물의 세포 독성을 측정하기 위하여, 96-웰 플레이트에 1,000 세포/웰이 되도록 RAW264.7 세포(TB-71, ATCC, UT, USA)를 분주하여 24시간 동안 배양한 후, 대계 추출물을 농도별(50, 100, 200, 400 ㎍/㎖)로 처리하고 3일 동안 배양하였다. 대계 추출물의 세포 독성은 CCK-8 키트(Dojindo Molecular Technologies, Rockville, MD, USA)를 사용하여 측정하였다.In order to measure the cytotoxicity of the base extract prepared in Example 1, RAW264.7 cells (TB-71, ATCC, UT, USA) were aliquoted to 1,000 cells / well in 96-well plates and cultured for 24 hours. After, the extract was treated by concentration (50, 100, 200, 400 ㎍ / ㎖) and incubated for 3 days. Cytotoxicity of the extract was determined using a CCK-8 kit (Dojindo Molecular Technologies, Rockville, MD, USA).

4가지 농도(50, 100, 200, 400 ㎍/㎖)의 대계 추출물을 RAW264.7 세포에 처리한 후 3일 동안 배양한 결과, 최고 농도(400 ㎍/㎖)까지 RAW264.7 세포 성장에 영향을 주지 않았다(도 1). 따라서, 상기 농도 범위 내에서 TRAP 활성과 파골세포 분화 관련 유전자의 발현 변화를 조사하였다.
Treatment of RAW264.7 cells with four different concentrations (50, 100, 200, 400 μg / mL) of extracts was performed on RAW264.7 cells and cultured for 3 days, which affected RAW264.7 cell growth up to the highest concentration (400 μg / mL). Not given (FIG. 1). Therefore, the changes of TRAP activity and osteoclast differentiation related gene expression within the concentration range were investigated.

실시예 3. 대계 추출물이 파골세포 분화에 미치는 영향Example 3. Effect of Greater Extracts on Osteoclast Differentiation

단핵구 세포인 RAW264.7 세포(TB-71, ATCC, UT, USA)는 RANKL(R&D Systems Inc., Minneapolis, MN, USA) 처리시 파골세포 분화의 마커인 TRAP 활성의 증가를 보이며 파골세포로 분화한다(Minkin, 1982, Calcif Tissue Int.,34(3):285-90; Hsu et al., 1999, Proc.Nat.Acad.Sci. USA, 96:35403545). RAW264.7 세포의 성장 배지로 10% FBS가 포함된 DMEM(Invitrogen Inc., NY, USA) 배지를 사용하였으며, 2일 간격으로 새로운 배지로 교체하며 배양하였다. 파골 세포 분화를 유도하기 위하여 RAW264.7 세포가 70% 정도 자라면 스크랩퍼로 세포를 긁어 배양 플레이트에서 분리하고, 이를 96-웰 플레이트에 1,000 세포/웰이 되도록 분주하였다. 이 때 세포를 부유시키는 세포 분화 배지로 100 ng/㎖의 RANKL과 10% FBS가 포함된 α-MEM (Invitrogen Inc., NY, USA)을 사용하였으며, RANKL이 포함된 분화 배지에서 세포를 4일 동안 배양하였다.Monocytes RAW264.7 (TB-71, ATCC, UT, USA) differentiated into osteoclasts, showing an increase in TRAP activity, a marker of osteoclast differentiation when RANKL (R & D Systems Inc., Minneapolis, MN, USA) (Minkin, 1982, Calcif Tissue Int., 34 (3): 285-90; Hsu et al., 1999, Proc. Nat. Acad. Sci. USA, 96: 35403545). DMEM (Invitrogen Inc., NY, USA) medium containing 10% FBS was used as a growth medium of RAW264.7 cells, and cultured with fresh medium every two days. In order to induce osteoclast differentiation, when RAW264.7 cells were grown to about 70%, the cells were scraped with a scraper and separated from the culture plate, and the cells were divided into 96-well plates at 1,000 cells / well. At this time, α-MEM (Invitrogen Inc., NY, USA) containing 100 ng / ml of RANKL and 10% FBS was used as a cell differentiation medium for suspending the cells. Incubated for

시험 물질 처리 4일째에 TRAP 활성을 측정하였고, TRAP 양성의 다핵성 파골세포 형성을 확인하는 TRAP 염색을 실시한 후, 현미경으로 다핵성 파골세포를 확인하였다. 이를 위하여, RANKL로 분화된 파골세포를 10% 포르말린에서 10분 동안 고정시킨 후, 95% 에탄올로 탈수시키고, 상온에서 건조시켰다. 이후, 염색 키트(Leukocyte Acid Phosphatase Kit 387-A, Sigma, MO, USA)를 사용하여 분화배지에서 배양한 RAW264.7 세포를 TRAP 염색하였다. 분화된 다핵성 파골세포는 eXcope(DIXI Optics, Seoul, Korea)가 장착된 현미경(Olympus Optical Co. Ltd., Tokyo, Japan)으로 관찰하였다. TRAP 활성 측정을 위하여, 상기에서 고정한 파골세포가 든 플레이트에 10 mM 나트륨 타르트레이트, 5 mM p-니트로페닐포스페이트(Sigma, MO)가 들어간 100 ㎕의 시트레이트 버퍼(50 mM, pH 4.6)를 첨가한 후 1시간 동안 37℃에서 배양하였다. 이후, 새로운 96-웰 플레이트에 90 ㎕의 효소 반응액을 옮기고, 이 효소 반응액을 동일량의 0.1 N NaOH와 혼합한 후, 410 ㎚에서 흡광도를 측정하였다. TRAP 활성은 대조군 대비 %로 환산하여 나타내었다.TRAP activity was measured on day 4 of treatment of the test substance, and after performing TRAP staining to confirm TRAP-positive multinucleated osteoclast formation, multinucleated osteoclasts were identified under a microscope. For this purpose, osteoclasts differentiated with RANKL were fixed in 10% formalin for 10 minutes, dehydrated with 95% ethanol, and dried at room temperature. Thereafter, using a staining kit (Leukocyte Acid Phosphatase Kit 387-A, Sigma, MO, USA) was used to TRAP staining RAW264.7 cells cultured in differentiation medium. Differentiated multinucleated osteoclasts were observed under a microscope (Olympus Optical Co. Ltd., Tokyo, Japan) equipped with eXcope (DIXI Optics, Seoul, Korea). To measure TRAP activity, 100 μl of citrate buffer (50 mM, pH 4.6) containing 10 mM sodium tartrate and 5 mM p-nitrophenylphosphate (Sigma, MO) was added to the plate containing the osteoclasts fixed above. After incubation at 37 ℃ for 1 hour. Thereafter, 90 µl of the enzyme reaction solution was transferred to a new 96-well plate, and the enzyme reaction solution was mixed with the same amount of 0.1 N NaOH, and the absorbance was measured at 410 nm. TRAP activity was expressed in% compared to the control.

RAW264.7 세포에 대계 추출물을 농도별로 4일 동안 첨가하여 배양한 결과, 대계 추출물은 100 ㎍/㎖ 농도에서부터 RANKL에 의해 유도되는 TRAP 활성(도 2의 A) 및 다핵성 파골 세포의 형성(도 2의 B)을 유의성 있게 억제하였다. 파골세포의 분화를 억제한다고 알려진 phosphatidylinositol 3-kinase 억제제 LY294002(Lee et al., 2002, Bone, 30:717-717)를 양성대조물질로 사용하였고, LY294002를 RAW264.7 세포에 4일 동안 첨가하여 배양한 결과, RANKL에 의해 유도되는 TRAP 활성(도 2의 A)을 유의성 있게 억제하였다.
As a result of cultivation by adding the extract to the RAW264.7 cells by concentration for 4 days, the extract was obtained from RANKL-induced TRAP activity (Fig. 2A) and the formation of multinuclear osteoclasts (Fig. 2A). 2) B) was significantly inhibited. The phosphatidylinositol 3-kinase inhibitor LY294002 (Lee et al., 2002, Bone, 30: 717-717), known to inhibit osteoclast differentiation, was used as a positive control and LY294002 was added to RAW264.7 cells for 4 days. As a result of the culture, the TRAP activity induced by RANKL (A of FIG. 2) was significantly inhibited.

실시예Example 4. 대계 추출물이 전사 인자 및 파골세포 분화 관련 유전자에 미치는 영향 4. Effects of Daekyo Extract on Transcription Factors and Osteoclast Differentiation Related Genes

TRAP 염색과 활성을 억제하는 대계 추출물이 파골세포 분화 관련 유전자인 TRAP, c-Src, Cathepsin K, MMP-9 및 ATP6v0d2의 발현과 이들의 발현 조절에 관여하는 전사 인자인 c-fos, Fra-1, Fra-2 및 NFATc1의 발현에 미치는 효과를 확인하기 위하여 실시간 정량 PCR (real time quantitative PCR)을 수행하였다. 전사인자의 발현 확인을 위한 RNA는 대계 추출물을 2시간 동안 전처리하고 RANKL을 처리하고 24시간 후에 분리하였다. 파골세포 분화 관련 유전자 발현 확인을 위한 RNA는 RANKL을 처리하고 24시간 후에 대계 추출물을 3일 동안 처리하고 분리하였다. 총 RNA는 RNeasy 미니 키트(Qiagen, Valencia, CA, USA)를 사용하여 분리하였다. 파골세포 분화 관련 유전자 및 전사 인자에 특이적인 프라이머는 온라인 프라이머 디자인 프로그램을 사용하여 디자인하였으며(Rozen and Skaletsky, 2000, Methods Mol. Biol., 132:365-386), 각 유전자에 대한 프라이머로는 하기 표 1에 나타낸 전방(forward) 및 후방(reverse) 프라이머쌍을 사용하였다. cDNA의 합성은 2 ㎍의 총 RNA, 1 μM의 oligo-dT18 primer 및 10 unit의 RNase inhibitor RNasin (Promega, WI, USA)과 4 Unit의 Omniscript Reverse Transcriptase (Qiagen, CA)를 이용하여 합성하였다. SYBR 그린계 정량 PCR 증폭은 50배 희석한 cDNA 와 20 p㏖의 프라이머를 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA)와 혼합한 후, Applied Biosystems 7500 Real-Time PCR system을 이용하여 수행하였다. PCR 프로그램은 다음과 같다. 폴리머라제 활성을 위해 95℃에서 10분간 유지한 후, 94℃에서 40초(변성), 60℃에서 40초(어닐링) 및 72℃에서 1분(연장)을 40회 반복 수행하였다. 이후, 95℃에서 1분, 55℃에서 30초 및 95℃에서 30초간 유지하여 생성된 PCR 산물의 온도 해리 곡선(temperature dissociation curves, 'melting curves'라 하기도 함)을 작성하였다. 모든 실험은 3회 반복 실시하였으며, PCR 데이터는 2-△△ CT 방법에 따라 분석하였다(Livak and Schmittgen, 2001, Methods 25:402-408). 이때, GAPDH를 PCR의 내부 대조군(internal control)으로 사용하였고, GAPDH-normalized 2-△△ CT 데이터를 Student's t-test로 분석한 후, p < 0.05을 유의성 있는 차이로 판정하였다.The extracts that inhibit TRAP staining and activity are expressed in osteoclast differentiation related genes TRAP, c-Src, Cathepsin K, MMP-9 and ATP6v0d2 and c-fos, Fra-1, which are transcription factors involved in their expression regulation. Real time quantitative PCR (real time quantitative PCR) was performed to confirm the effect on the expression of Fra-2 and NFATc1. RNA for confirming the expression of the transcription factor was pretreated for 2 hours and separated for 24 hours after treatment with RANKL. RNA for identifying osteoclast differentiation-related gene expression was treated with RANKL and separated and treated for 3 days after the extract was processed for 24 days. Total RNA was isolated using RNeasy mini kit (Qiagen, Valencia, CA, USA). Primers specific for osteoclast differentiation related genes and transcription factors were designed using an online primer design program (Rozen and Skaletsky, 2000, Methods Mol. Biol., 132: 365-386). The forward and reverse primer pairs shown in Table 1 were used. cDNA was synthesized using 2 μg total RNA, 1 μM oligo-dT 18 primer, 10 units of RNase inhibitor RNasin (Promega, WI, USA) and 4 units of Omniscript Reverse Transcriptase (Qiagen, CA). SYBR green quantitative PCR amplification was performed by mixing 50-fold diluted cDNA and 20 mmol of the primer with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and then using the Applied Biosystems 7500 Real-Time PCR system. It was performed by. The PCR program is as follows. After holding for 10 minutes at 95 ° C. for polymerase activity, 40 times at 94 ° C. (denature), 40 seconds at 60 ° C. (annealing) and 1 minute at 72 ° C. (extension) were performed 40 times. Subsequently, temperature dissociation curves (also referred to as 'melting curves') of the PCR products generated by holding at 95 ° C for 1 minute, 55 ° C for 30 seconds, and 95 ° C for 30 seconds were prepared. All experiments were repeated three times, and PCR data were analyzed according to the 2 -ΔΔ CT method (Livak and Schmittgen, 2001, Methods 25: 402-408). At this time, GAPDH was used as an internal control of PCR, and after analyzing GAPDH-normalized 2 -DELTA CT data by Student's t-test, p <0.05 was determined as a significant difference.

그 결과, RANKL 미처리한 세포와 비교하여, RANKL 처리한 세포에서 TRAP, c-Src, Cathepsin K, MMP-9, 및 ATP6v0d2 유전자의 mRNA 발현양이 유의성 있게 증가하였지만, 대계 추출물 처리군(400 ㎍/㎖)의 경우에는 TRAP, c-Src, Cathepsin K, 및 MMP-9 유전자의 mRNA 발현이 모두 유의성 있게 감소되었다(표 2). 또한, 대계 추출물은 50 및 400 ㎍/㎖ 농도에서 RANKL로 유도되는 c-fos 및 NFATc1 유전자의 발현양을 유의성 있게 감소시켰다(표 3).As a result, the mRNA expression levels of TRAP, c-Src, Cathepsin K, MMP-9, and ATP6v0d2 genes in the RANKL treated cells were significantly increased compared to those in the RANKL untreated cells, but the control extract treatment group (400 ㎍ / Ml), mRNA expressions of TRAP, c-Src, Cathepsin K, and MMP-9 genes were all significantly reduced (Table 2). In addition, the extracts significantly reduced the expression of RANKL-induced c-fos and NFATc1 genes at 50 and 400 μg / ml concentrations (Table 3).

유전자gene 전방 프라이머(5'->3')Forward primer (5 '-> 3') 서열
번호order
number 후방 프라이머(5'->3')Back primer (5 '-> 3') 서열
번호order
number c-c- fosfos CCAGTCAAGAGCATCAGCAACCAGTCAAGAGCATCAGCAA 1One AAGTAGTGCAGCCCGGAGTAAAGTAGTGCAGCCCGGAGTA 22 FraFra -1-One CAGCCTCATTTCCTGGGACCCAGCCTCATTTCCTGGGACC 33 CCTTTCTTCGGTTTCTGCACTCCTTTCTTCGGTTTCTGCACT 44 FraFra -2-2 ATCCACGCTCACATCCCTACATCCACGCTCACATCCCTAC 55 GTTTCTCTCCCTCCGGATTCGTTTCTCTCCCTCCGGATTC 66 NFATc1NFATc1 GGGTCAGTGTGACCGAAGATGGGTCAGTGTGACCGAAGAT 77 GGAAGTCAGAAGTGGGTGGAGGAAGTCAGAAGTGGGTGGA 88 TRAPTRAP ACACAGTGATGCTGTGTGGCAACTCACACAGTGATGCTGTGTGGCAACTC 99 CCAGAGGCTTCCACATATATGATGGCCAGAGGCTTCCACATATATGATGG 1010 c-c- SrcSrc CCAGGCTGAGGAGTGGTACTCCAGGCTGAGGAGTGGTACT 1111 CAGCTTGCGGATCTTGTAGTCAGCTTGCGGATCTTGTAGT 1212 MMPMMP -9-9 GCCCACCGTCCTTTCTTGTTGCCCACCGTCCTTTCTTGTT 1313 CGGTGAAGTGCCTGTCACAACGGTGAAGTGCCTGTCACAA 1414 CathepsinCathepsin K K GGCCAACTCAAGAAGAAAACGGCCAACTCAAGAAGAAAAC 1515 GTGCTTGCTTCCCTTCTGGGTGCTTGCTTCCCTTCTGG 1616 ATP6v0d2ATP6v0d2 AGACCACGGACTATGGCAACAGACCACGGACTATGGCAAC 1717 CAGTGGGTGACACTTGGCTACAGTGGGTGACACTTGGCTA 1818 GAPDHGAPDH AACTTTGGCATTGTGGAAGGAACTTTGGCATTGTGGAAGG 1919 ACACATTGGGGGTAGGAACAACACATTGGGGGTAGGAACA 2020

RAW264.7 세포에서 RANKL에 의해 유도된 파골세포 특이적 유전자의 mRNA 발현에 대계 추출물이 미치는 영향Effect of Cauliflower Extract on mRNA Expression of RANKL-induced Osteoclast-Specific Genes in RAW264.7 Cells TRAPTRAP c-c- SrcSrc CathepsinCathepsin K K ATP6v0d2ATP6v0d2 MMPMMP -9-9 RANKL 미처리RANKL untreated 1.01±0.201.01 ± 0.20 1.04±0.381.04 ± 0.38 1.02±0.281.02 ± 0.28 1.04±0.361.04 ± 0.36 1.15±0.781.15 ± 0.78 RANKL 처리RANKL treatment 1076.70±211.79* 1076.70 ± 211.79 * 37.00±5.34* 37.00 ± 5.34 * 2002.69±49.23* 2002.69 ± 49.23 * 99.43±26.35* 99.43 ± 26.35 * 1125.63±77.95* 1125.63 ± 77.95 * RANKL+추출물
(50 ㎍/㎖)RANKL + Extract
(50 μg / ml) 963.46±280.34* 963.46 ± 280.34 * 41.98±13.43* 41.98 ± 13.43 * 1890.46±552.91* 1890.46 ± 552.91 * 87.39±27.11* 87.39 ± 27.11 * 1349.52±287.63* 1349.52 ± 287.63 * RANKL+추출물
(400 ㎍/㎖)RANKL + Extract
(400 μg / ml) 525.03±70.26# 525.03 ± 70.26 # 26.70±2.81*# 26.70 ± 2.81 * # 978.71±144.59*# 978.71 ± 144.59 * # 74.35±21.42* 74.35 ± 21.42 * 863.13±34.01*# 863.13 ± 34.01 * #

* p < 0.05, RANKL 미처리 군과 비교하여 현저하게 차이가 남.* p <0.05, significantly different from RANKL-treated group.

# p < 0.05, RANKL 처리 군과 비교하여 현저하게 차이가 남. # p <0.05, significantly different from RANKL treated group.

RAW264.7 세포에서 RANKL에 의해 유도된 전사 인자의 mRNA 발현에 대계 추출물이 미치는 영향Effect of Cauliflower Extract on mRNA Expression of RANKL-induced Transcription Factors in RAW264.7 Cells c-c- fosfos FraFra -1-One FraFra -2-2 NFATc1NFATc1 RANKL 미처리RANKL untreated 1.00±0.041.00 ± 0.04 1.01±0.111.01 ± 0.11 1.01±0.191.01 ± 0.19 1.01±0.201.01 ± 0.20 RANKL 처리RANKL treatment 1.27±0.03* 1.27 ± 0.03 * 2.06±0.34* 2.06 ± 0.34 * 5.49±1.92* 5.49 ± 1.92 * 8.26±1.47* 8.26 ± 1.47 * RANKL+추출물
(50 ㎍/㎖)RANKL + Extract
(50 μg / ml) 0.89±0.09*# 0.89 ± 0.09 * # 1.75±0.28* 1.75 ± 0.28 * 4.29±0.25* 4.29 ± 0.25 * 5.55±1.27*# 5.55 ± 1.27 * # RANKL+추출물
(400 ㎍/㎖)RANKL + Extract
(400 μg / ml) 0.85±0.68*# 0.85 ± 0.68 * # 2.32±0.26* 2.32 ± 0.26 * 3.36±0.41* 3.36 ± 0.41 * 3.50±0.70*# 3.50 ± 0.70 * #

* p < 0.05, RANKL 미처리 군과 비교하여 현저하게 차이가 남. * p <0.05, significantly different from RANKL-treated group.

# p < 0.05, RANKL 처리 군과 비교하여 현저하게 차이가 남.
 # p <0.05, significantly different from RANKL treated group.

실시예Example 5.  5. 웨스턴Weston 블롯Blot 분석 analysis

단백질 추출 버퍼(50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, and one protease inhibitor cocktail tablet (Roche, Germany)를 이용하여 세포내 단백질을 추출한 후 원심 분리(10,000×g, 15분, 4℃)를 하여 단백질 추출물을 얻었다. 단백질 농도는 BCA 단백질 분석 키트(Pierce, IL)를 사용하여 정량하였다. 10 ㎍ 단백질 샘플을 SDS-PAGE용 샘플 버퍼(100 mM Tris-HCl, 2% SDS, 1% 2-mercaptoethanol, 2% glycerol, 0.01% bromophenol blue, pH 7.6)와 섞은 후 가열(100℃, 5분)하여 변성시켰고, 10% 폴리아크릴 아마이드 겔로 전기영동하였다. 전기영동에는 Mini protean 3 Cell (Bio-Rad, CA)을 사용하였다. 겔에 분리된 단백질은 나이트로 셀롤로오스 막(Whatman, Germany)에 전달하였고 Ponceau S 염색으로 단백질 전달과 점적된 단백질 양을 확인하였다. 이 후 나이트로 셀롤로오스 막을 블록킹 버퍼(10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 3% nonfat dry milk)로 블록킹하고 2시간 동안 1차 항체(희석 배수, 1:1000; Cell Signaling Technology, Inc., MA)와 배양시켰다. 이때, 상기 1차 항체는 RANKL에 의해 활성이 증가되며 파골 세포 분화의 시그널에 참여하는 것으로 알려져 있는 ERK, JNK 및 p38에 특이적으로 결합하는 항체(Cell Signaling Technology, Inc., MA)를 사용하였다(Lee and Kim, 2003, BBRC, 305:211-214). 1차 항체와 반응시킨 막을 블록킹 버퍼로 10분간 3회 세척한 후 2차 항체(1:2000; Cell Signaling Technology, Inc., MA)와 1시간 동안 배양시켰다. 이 후, 블록킹 버퍼로 10분간 3회 세척한 후 SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, IL)로 현상하였다. 형광 시그널은 LAS-3000 발광 이미지 분석기(Fuji Photo Film Co., Japan)를 사용하여 검출하였고, 밴드 밀도는 Multi Gauge software version 3.0 (Fuji Photo Film Co.)을 사용하여 측정하였다.Using protein extraction buffer (50 mM Tris-HCl, pH 8.0), 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, and one protease inhibitor cocktail tablet (Roche, Germany) Extraction of intracellular proteins was followed by centrifugation (10,000 × g, 15 min, 4 ° C.) to obtain protein extracts Protein concentrations were quantified using the BCA Protein Assay Kit (Pierce, IL) 10 μg protein samples were SDS Denatured by mixing with -PAGE buffer (100 mM Tris-HCl, 2% SDS, 1% 2-mercaptoethanol, 2% glycerol, 0.01% bromophenol blue, pH 7.6) and heating (100 ℃, 5 minutes), 10 Electrophoresis was carried out with% polyacrylamide gel Mini electrote 3 Cell (Bio-Rad, CA) was used for electrophoresis The proteins separated on the gel were transferred to nitro cellulose membrane (Whatman, Germany) and stained with Ponceau S The protein delivery and the amount of instilled protein were confirmed by using a nitro cellulose membrane, followed by blocking buffer (10 mM Tris-HCl, Blocked with pH 7.5, 150 mM NaCl, 0.1% Tween 20, 3% nonfat dry milk) and incubated with primary antibody (dilution multiple, 1: 1000; Cell Signaling Technology, Inc., MA) for 2 hours. The primary antibody used was an antibody (Cell Signaling Technology, Inc., MA) that specifically binds to ERK, JNK, and p38, which is increased in activity by RANKL and is known to participate in signals of osteoclast differentiation (Lee). and Kim, 2003, BBRC, 305: 211-214) After washing the membrane reacted with the primary antibody three times with blocking buffer for 10 minutes, the secondary antibody (1: 2000; Cell Signaling Technology, Inc., MA) and 1 Incubated for hours. After washing 3 times with blocking buffer for 10 minutes, it was developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, IL). Fluorescence signals were detected using a LAS-3000 luminescence image analyzer (Fuji Photo Film Co., Japan), and the band density was measured using Multi Gauge software version 3.0 (Fuji Photo Film Co.).

그 결과, RANKL 처리 15분부터 ERK, JNK 및 p38의 활성이 증가하였다. 대계 추출물을 처리할 경우에는 15분부터 나타나는 ERK, JNK 및 p38의 활성 증가가 현저하게 감소하였다(도 3).
As a result, the activity of ERK, JNK and p38 increased from 15 minutes of RANKL treatment. When the extract was treated, the increased activity of ERK, JNK, and p38, which appeared from 15 minutes, was markedly decreased (FIG. 3).

제조예Manufacturing example 1: 대계 추출물을 포함하는 약학적 조성물의 제조 1: Preparation of a pharmaceutical composition comprising the extract

<1-1> 시럽제의 제조<1-1> Preparation of Syrup

대계 추출물을 유효성분 2%(중량/부피)로 함유하는 시럽은 다음과 같은 방법으로 제조하였다. 먼저, 실시예 1에서 제조한 대계 추출물 분말, 사카린, 당을 온수 80 g에 용해시켰다. 상기 용액을 냉각시킨 후, 여기에 글리세린, 사카린, 향미료, 에탄올, 소르브산 및 증류수로 이루어진 용액을 제조하여 혼합하였다. 이 혼합물에 물을 첨가하여 100 ㎖가 되게 하였다.Syrup containing the extract from the active ingredient 2% (weight / volume) was prepared by the following method. First, the base extract powder, saccharin and sugar prepared in Example 1 were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml.

상기 시럽제의 구성 성분은 다음과 같다.The components of the syrup are as follows.

대계 추출물????????????????????????? 2 gGrandfather Extract ????????????????????????? 2 g

사카린 ?????????????????????????? 0.8 gSaccharin ?????????????????????????? 0.8 g

당???????????????????????????? 25.4 gParty???????????????????????????? 25.4 g

글리세린 ????????????????????????? 8.0 gGlycerin ????????????????????????? 8.0 g

향미료?????????????????????????? 0.04 gSpices ?????????????????????????? 0.04 g

에탄올 ?????????????????????????? 4.0 gethanol ?????????????????????????? 4.0 g

소르브산 ????????????????????????? 0.4 gSorbic acid ????????????????????????? 0.4 g

증류수??????????????????????????? 정량Distilled water??????????????????????????? dose

<1-2> 정제의 제조<1-2> Preparation of Tablet

유효성분 15 ㎎이 함유된 정제를 다음과 같은 방법으로 제조하였다.A tablet containing 15 mg of active ingredient was prepared by the following method.

대계 추출물 250 g을 락토오스 175.9 g, 감자전분 180 g 및 콜로이드성 규산 32 g과 혼합하였다. 상기 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160 g, 활석 50 g 및 스테아린산 마그네슘 5 g을 첨가해서 얻은 혼합물을 정제로 만들었다.250 g of the extract was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

대계 추출물???????????????????????? 250 gDaikon extract ???????????????????????? 250 g

락토오스 ???????????????????????? 175.9 gLactose ???????????????????????? 175.9 g

감자전분 ????????????????????????? 180 gPotato starch ????????????????????????? 180 g

콜로이드성 규산 ?????????????????????? 32 gColloidal silicate ?????????????????????? 32 g

10% 젤라틴 용액10% gelatin solution

감자전분 ????????????????????????? 160 gPotato starch ????????????????????????? 160 g

활석???????????????????????????? 50 gtalc???????????????????????????? 50 g

스테아르산 마그네슘????????????????????? 5 g
Magnesium Stearate ????????????????????? 5 g

제조예Manufacturing example 2: 대계 추출물을 함유하는 건강식품의 제조 2: Preparation of Health Foods Containing Daikon Extract

<2-1> 식품의 제조<2-1> Preparation of food

대계 추출물을 포함하는 식품들을 다음과 같이 제조하였다.Foods containing the extract were prepared as follows.

1. 조리용 양념의 제조1. Preparation of Cooking Seasonings

대계 추출물 20?95 중량%로 건강 증진용 조리용 양념을 제조하였다.A health promotion cooking seasoning was prepared with 20-95% by weight of extract.

2. 토마토 케찹 및 소스의 제조2. Preparation of Tomato Ketchup and Sauce

대계 추출물 0.2?1.0 중량%를 토마토 케찹 또는 소스에 첨가하여 건강 증진용 토마토 케찹 또는 소스를 제조하였다.Health-promoting tomato ketchup or sauce was prepared by adding 0.2-1.0% by weight of extract from tomato ketchup or sauce.

3. 밀가루 식품의 제조3. Manufacturing of Flour Foods

대계 추출물 0.5?5.0 중량%를 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.0.5 to 5.0% by weight of the extract was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

4. 스프 및 육즙(gravies)의 제조4. Preparation of soups and gravy

대계 추출물 0.1?5.0 중량%를 스프 및 육즙에 첨가하여 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.0.1-5.0% by weight of the extract was added to soups and broths to prepare meat products for health promotion, soups and noodles of noodles.

5. 그라운드 비프(ground beef)의 제조5. Preparation of Ground Beef

대계 추출물 10 중량%를 그라운드 비프에 첨가하여 건강 증진용 그라운드 비프를 제조하였다.10% by weight of the extract was added to the ground beef to prepare a ground beef for health promotion.

6. 유제품(dairy products)의 제조6. Manufacture of Dairy Products

대계 추출물 5?10 중량%를 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.5-10% by weight of the extract was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

7. 선식의 제조7. Manufacture of wire

현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60메쉬의 분말로 제조하였다. 검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60메쉬의 분말로 제조하였다. 대계 추출물을 진공 농축기에서 감압?농축하고, 분무, 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60메쉬로 분쇄하여 건조분말을 얻었다. 상기에서 제조한 곡물류, 종실류 및 대계 추출물의 건조분말을 다음의 비율로 배합하여 제조하였다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder. Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh. The dried extract was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and a hot air dryer, and ground to a mesh size of 60 mesh with a grinder to obtain a dry powder. It was prepared by combining the dry powder of the grains, seeds and grand extracts prepared in the following ratio.

곡물류(현미 30 중량%, 율무 15 중량%, 보리 20 중량%),Cereals (30% by weight brown rice, 15% by weight barley, 20% by weight barley),

종실류(들깨 7 중량%, 검정콩 8 중량%, 검정깨 7 중량%),Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

대계 추출물의 건조분말(3 중량%),Dry powder (3% by weight) of the base extract,

영지(0.5 중량%),Ganoderma lucidum (0.5% by weight),

지황(0.5 중량%)Sulfur (0.5 wt%)

<2-2> 음료의 제조<2-2> Preparation of Drink

1. 탄산음료의 제조1. Preparation of Carbonated Drinks

설탕 5?10%, 구연산 0.05?0.3%, 카라멜 0.005?0.02%, 비타민 C 0.1?1%의 첨가물을 혼합하고, 여기에 79?94%의 정제수를 섞어서 시럽을 만들고, 상기 시럽을 85?98℃에서 20?180초간 살균하여 냉각수와 1:4의 비율로 혼합한 다음 탄산가스를 0.5?0.82%를 주입하여 대계 추출물을 함유하는 탄산음료를 제조하였다.5 to 10% sugar, 0.05 to 0.3% citric acid, 0.005 to 0.02% caramel, and 0.1 to 1% vitamin C are mixed, and 79 to 94% purified water is mixed to make syrup, and the syrup is 85 to 98 Sterilizing at 20 ℃ for 180 seconds, mixed with cooling water in a ratio of 1: 4, and then injected carbon dioxide gas 0.5 to 0.82% to prepare a carbonated beverage containing a large extract.

2. 건강음료의 제조2. Manufacture of health drinks

액상과당(0.5%), 올리고당(2%), 설탕(2%), 식염(0.5%), 물(75%)과 같은 부재료와 대계 추출물을 균질하게 배합하여 순간 살균을 한 후 이를 유리병, 페트병 등 소포장 용기에 포장하여 건강음료를 제조하였다.Homogeneous blending of subsidiary ingredients such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), water (75%) and extracts, and after sterilization Healthy drinks were prepared by packaging in small packaging containers such as PET bottles.

3. 야채주스의 제조3. Preparation of Vegetable Juice

대계 추출물 5 g을 토마토 또는 당근주스 1,000 ㎖에 가하여 건강 증진용 야채주스를 제조하였다.5 g of the extract was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. 과일주스의 제조4. Preparation of Fruit Juice

대계 추출물 1 g을 사과 또는 포도주스 1,000 ㎖에 가하여 건강 증진용 과일주스를 제조하였다.1 g of the extract was added to 1,000 ml of apple or wine to prepare fruit juice for health promotion.

<110> Korea Institute of Oriental Medicine <120> Composition for Prevention or Treatment of Osteoporosis Comprising Extract of Cirsii Herba <130> 2010-dpa-0121 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for c-fos <400> 1 ccagtcaaga gcatcagcaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for c-fos <400> 2 aagtagtgca gcccggagta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Fra-1 <400> 3 cagcctcatt tcctgggacc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Fra-1 <400> 4 cctttcttcg gtttctgcac t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Fra-2 <400> 5 atccacgctc acatccctac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Fra-2 <400> 6 gtttctctcc ctccggattc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NFATc1 <400> 7 gggtcagtgt gaccgaagat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NFATc1 <400> 8 ggaagtcaga agtgggtgga 20 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TRAP <400> 9 acacagtgat gctgtgtggc aactc 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRAP <400> 10 ccagaggctt ccacatatat gatgg 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for c-Src <400> 11 ccaggctgag gagtggtact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for c-Src <400> 12 cagcttgcgg atcttgtagt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MMP-9 <400> 13 gcccaccgtc ctttcttgtt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MMP-9 <400> 14 cggtgaagtg cctgtcacaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Cathepsin K <400> 15 ggccaactca agaagaaaac 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Cathepsin K <400> 16 gtgcttgctt cccttctgg 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ATP6v0d2 <400> 17 agaccacgga ctatggcaac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ATP6v0d2 <400> 18 cagtgggtga cacttggcta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 19 aactttggca ttgtggaagg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 20 acacattggg ggtaggaaca 20 <110> Korea Institute of Oriental Medicine <120> Composition for Prevention or Treatment of Osteoporosis          Comprising Extract of Cirsii Herba <130> 2010-dpa-0121 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for c-fos <400> 1 ccagtcaaga gcatcagcaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for c-fos <400> 2 aagtagtgca gcccggagta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Fra-1 <400> 3 cagcctcatt tcctgggacc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Fra-1 <400> 4 cctttcttcg gtttctgcac t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Fra-2 <400> 5 atccacgctc acatccctac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Fra-2 <400> 6 gtttctctcc ctccggattc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NFATc1 <400> 7 gggtcagtgt gaccgaagat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NFATc1 <400> 8 ggaagtcaga agtgggtgga 20 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TRAP <400> 9 acacagtgat gctgtgtggc aactc 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRAP <400> 10 ccagaggctt ccacatatat gatgg 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for c-Src <400> 11 ccaggctgag gagtggtact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for c-Src <400> 12 cagcttgcgg atcttgtagt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MMP-9 <400> 13 gcccaccgtc ctttcttgtt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MMP-9 <400> 14 cggtgaagtg cctgtcacaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Cathepsin K <400> 15 ggccaactca agaagaaaac 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Cathepsin K <400> 16 gtgcttgctt cccttctgg 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ATP6v0d2 <400> 17 agaccacgga ctatggcaac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ATP6v0d2 <400> 18 cagtgggtga cacttggcta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 19 aactttggca ttgtggaagg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 20 acacattggg ggtaggaaca 20

Claims (6)

대계에 물 또는 유기용매를 가하여 추출한 대계 추출물을 유효성분으로 함유하는 골다공증 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of osteoporosis, which comprises the extract of Daegyu by adding water or an organic solvent as an active ingredient. 청구항 1에 있어서,
상기 대계 추출물은 대계에 2?20배량의 물을 가한 후 70 내지 100℃에서 열수 추출하여 제조되는 것을 특징으로 하는 약학적 조성물.The method according to claim 1,
The base extract is a pharmaceutical composition, which is prepared by adding hot water at 70 to 100 ℃ after adding 2 ~ 20 times the amount of water to the base. 청구항 1에 있어서,
상기 유기용매는 C1 내지 C4의 알코올, 아세톤 및 이의 수용액으로 구성된 군으로부터 선택되는 것을 특징으로 하는 약학적 조성물.The method according to claim 1,
The organic solvent is a pharmaceutical composition, characterized in that selected from the group consisting of C 1 to C 4 alcohol, acetone and an aqueous solution thereof. 대계에 물 또는 유기용매를 가하여 추출한 대계 추출물을 유효성분으로 함유하는 골다공증 예방 또는 개선용 건강식품.Health food for the prevention or improvement of osteoporosis, which contains the extract of Daegye as an active ingredient extracted by adding water or organic solvent. 청구항 4에 있어서,
상기 대계 추출물은 대계에 2?20배량의 물을 가한 후 70 내지 100℃에서 열수 추출하여 제조되는 것을 특징으로 하는 건강식품.The method of claim 4,
The base extract is a health food, characterized in that produced by hot water extraction at 70 to 100 ℃ after adding 2 ~ 20 times the amount of water to the base. 청구항 4에 있어서,
상기 유기용매는 C1 내지 C4의 알코올, 아세톤 및 이의 수용액으로 구성된 군으로부터 선택되는 것을 특징으로 하는 건강식품.The method of claim 4,
The organic solvent is a health food, characterized in that selected from the group consisting of C 1 to C 4 alcohol, acetone and an aqueous solution thereof.
KR1020100105726A 2010-10-28 2010-10-28 Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba KR20120044450A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020100105726A KR20120044450A (en) 2010-10-28 2010-10-28 Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020100105726A KR20120044450A (en) 2010-10-28 2010-10-28 Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba

Publications (1)

Publication Number Publication Date
KR20120044450A true KR20120044450A (en) 2012-05-08

Family

ID=46264221

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020100105726A KR20120044450A (en) 2010-10-28 2010-10-28 Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba

Country Status (1)

Country Link
KR (1) KR20120044450A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101318055B1 (en) * 2011-12-13 2013-10-14 서림바이오 주식회사 The composition of milk thistle and isoflavones for treatment and prevention of osteoporosis
KR20150145605A (en) 2014-06-20 2015-12-30 한국과학기술연구원 Antibacterial composition containg peucedani radix exctract
KR20150146046A (en) 2014-06-20 2015-12-31 한국과학기술연구원 Antibacterial composition containg euscaphis japonica exctract

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101318055B1 (en) * 2011-12-13 2013-10-14 서림바이오 주식회사 The composition of milk thistle and isoflavones for treatment and prevention of osteoporosis
KR20150145605A (en) 2014-06-20 2015-12-30 한국과학기술연구원 Antibacterial composition containg peucedani radix exctract
KR20150146046A (en) 2014-06-20 2015-12-31 한국과학기술연구원 Antibacterial composition containg euscaphis japonica exctract

Similar Documents

Publication Publication Date Title
KR101252639B1 (en) Composition for Prevention or Treatment of Osteoporosis Comprising Ssanghwatang and Fermentation Product Thereof with Lactic Acid Bacteria
KR101997060B1 (en) Composition for prevention or treatment of muscular disorder, or improvement of muscular functions comprising fermented deer antler
CN108697685A (en) Containing moracin, Phellinus copper G or the root bark of white mulberry, the prevention of muscle disease and treatment is used or muscular function improvement composition
KR101242635B1 (en) Composition for Prevention or Treatment of Osteoporosis Comprising Extract of Selaginellae Herba
KR20120001283A (en) Composition for prevention or treatment of osteoporosis comprising herbal extract and fermentation product thereof with lactic acid bacteria
KR101199961B1 (en) Composition for Prevention or Treatment of Osteoporosis Comprising Bangpungtongseungsan and Fermentation Product Thereof with Lactic Acid Bacteria
KR101466443B1 (en) Composition comprising herbal mixture extract for treating or preventing cancer
CN102186488B (en) Pharmaceutical composition and health food composition containing youngia denticulata extract, fraction thereof, or compound isolated therefrom as active ingredient for improving liver function
KR20120044450A (en) Composition for prevention or treatment of osteoporosis comprising extract of cirsii herba
KR100700480B1 (en) Composition comprising the extract of Phellodendron amurensis for preventing and treating fracture
KR102230883B1 (en) Anticancer composition comprising herbal extract
KR100874778B1 (en) Composition for the prevention and treatment of menopausal diseases comprising fermented Cheonggukjang extract
KR100733335B1 (en) Fermentation Product of Puerariae Radix Extract and Composition for Prevention or Treatment of Osteoporosis Comprising the Same
KR20100009151A (en) Composition containing mixture extract of saururus chinesis and scutellaria baicalensis for prevention or treatment of osteoporosis
KR101903905B1 (en) Composition for anti-cancer or anti-oxidation comprising the extracts of Sedeveria Pink Ruby as active ingredient
KR20050110809A (en) A herbal mixture extract of pleurotus eryngii and acanthopanacis cortex and composition comprising the same for prevention and treatment of osteoporosis
JP2002363086A (en) Bone metabolism-improving agent, and food and drink for preventing or curing osteoporosis
KR101457117B1 (en) Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the dryopteris crassithizoma extract
JP6044944B2 (en) Process for producing new processed ume products and functional composition, food composition, and pharmaceutical composition using the same
JP2021024858A (en) Hypotensive composition
KR102448274B1 (en) Composition for preventing, ameliorating or treating bone disease comprising crude polysaccharide fraction of Psoralea corylifolia extract as effective component
KR20170014332A (en) Composition for preventing and treatment of osteoporosis
KR101850838B1 (en) Composition for prevention and treatment bone disease comprising extracts of Stryphnodendron barbatiman as an active ingredient
JP2021525222A (en) Compositions for the prevention or treatment of osteoporosis
EP4321167A1 (en) Sanguisorba officinalis linne extract composition inhibiting 3cl protease and rdrp activity of sars-cov-2

Legal Events

Date Code Title Description
A201 Request for examination
AMND Amendment
E601 Decision to refuse application
AMND Amendment