KR101529493B1 - Perillae semen extracts for enhancing differentiation of osteoblast and inhibiting differentiation of osteoclast and use of thereof as bone formation promoting products and bone resorption inhibitory products - Google Patents
Perillae semen extracts for enhancing differentiation of osteoblast and inhibiting differentiation of osteoclast and use of thereof as bone formation promoting products and bone resorption inhibitory products Download PDFInfo
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- KR101529493B1 KR101529493B1 KR1020120128924A KR20120128924A KR101529493B1 KR 101529493 B1 KR101529493 B1 KR 101529493B1 KR 1020120128924 A KR1020120128924 A KR 1020120128924A KR 20120128924 A KR20120128924 A KR 20120128924A KR 101529493 B1 KR101529493 B1 KR 101529493B1
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- differentiation
- bone
- extract
- osteoblast
- osteoclast
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Abstract
본 발명은 자소자 추출물 및 이를 유효성분으로 함유하는 제품에 관한 것으로서, 골개형(bone remodeling)에 있어서 골생성(bone formation)에 대한 골흡수(bone resorption)의 증가로 인한 불균형에 따라 초래되는 골질환의 주된 원인이 되는 조골세포의 기능 및 분화 감소와 파골세포의 활성 증가를 효과적으로 억제할 수 있는 자소자 추출물을 이용하여 노인들의 골질환이나 여성들의 폐경기로 인해 발생하는 골질환 개선에 도움을 줄 수 있는 약학조성물, 발효유, 건강기능식품에 유용하게 적용될 수 있다.The present invention relates to a self-component extract and a product containing the same as an active ingredient, and more particularly, to a bone remodeling method and a bone remodeling method, It helps to improve osteopathy of elderly people and osteoporosis caused by menopause of women by using self-extracting substance which can effectively suppress osteoclast function and differentiation and increase osteoclast activity which is the main cause of disease And can be usefully applied to pharmaceutical compositions, fermented milk, and health functional foods.
Description
본 발명은 조골세포 분화 촉진과 파골세포 분화 억제 활성을 갖는 자소자 추출물 및 상기 자소자 추출물을 유효성분으로 포함하는 제품에 관한 것으로서, 더욱 상세하게는 골개형(bone remodeling)에 있어서 골생성(bone formation)의 감소와 골흡수(bone resorption)의 증가에 대하여 조골세포 활성 및 분화를 효과적으로 촉진하며 파골세포 분화를 억제하는 활성을 갖는 자소자 추출물 및 상기 자소자 추출물을 유효성분으로 함유하는 골강화 또는 성장발육 촉진용 약제학적 조성물, 식품조성물 등에 관한 것이다.
The present invention relates to a self-extracting substance having an osteoblast differentiation promotion and an osteoclast differentiation inhibiting activity and a product containing the self-extracting substance as an active ingredient, and more particularly, to a bone remodeling product, The present invention relates to a self-component extract having an activity of effectively promoting osteoblast activation and differentiation and inhibiting osteoclast differentiation from a decrease in bone formation and bone resorption, A pharmaceutical composition for promoting growth and development, a food composition, and the like.
체내에는 인체를 지지하는 조직으로 200여개의 뼈(bone)와 각기 다른 뼈를 연결하는 조직인 연골(cartilage)로 구성되어 있다. 이들은 중요한 장기의 보호, 조혈화에 필요한 미세 환경제공과 같은 기본적이며 다양한 기능을 제공하여 준다. 또한 근육이나 장기를 구조적으로 지탱할 뿐만 아니라 체내의 칼슘이나 다른 필수 무기질 즉, 인이나 마그네슘과 같은 물질을 저장하는 인체의 중요한 부분 중 하나이다. 따라서 성장이 끝난 성인의 뼈는 멈추지 않고 죽는 날까지 오래된 뼈는 제거하고 새로운 뼈로 대체하는 생성과 흡수과정을 매우 역동적· 지속적으로 반복 재생하면서 균형을 유지하게 된다. 이를 골재형성(bone remodeling) 이라고 한다(Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50(6Suppl), pp.664-669, 2005). 오래된 뼈는 제거하고 새로운 뼈로 대체하는 순환(turnover)은 성장과 스트레스에 의해서 일어나는 뼈의 미세한 손상을 회복시키고 적절히 뼈의 기능을 유지하는데 필수적이다(Cohen-Solal M. et al., Therapie., 58(5), pp.391-393, 2003).
The body supports the human body and is composed of cartilage, which is a tissue that connects 200 different bones and different bones. They provide basic and versatile functions such as protecting important organs, providing a microenvironment for hematopoiesis. It is also an important part of the human body that not only supports the muscles or organs but also stores calcium or other essential minerals in the body, such as phosphorus or magnesium. Thus, adult bones do not stop growing until they die, and they maintain a balanced balance by continuously dynamically and continuously repeating the generation and absorption processes that remove old bones and replace them with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50 (6Suppl), pp.664-669, 2005). The turnover, which removes the old bone and replaces it with the new bone, is essential for restoring the fine damage of the bone caused by growth and stress and maintaining proper bone function (Cohen-Solal M. et al., Therapie., 58 (5), pp. 391-393, 2003).
골재형성에는 크게 두 종류의 세포가 관여하는 것으로 알려져 있다. 두 세포 중 하나는 뼈를 생성하는 조골세포(osteoblast)이고, 다른 하나는 뼈를 파괴하는 파골세포(osteoclast)이다. 이들은 서로 다른 세포에서 유래하는데 간엽줄기세포(mesenchymal stem cell)가 분화되어 조골세포(osteoblast), 근원세포(myoblast), 지방세포(adipocyte), 섬유아세포(fibroblast), 연골세포(chondrocyte) 등의 세포로 분화되고, 조혈모세포(hematopoietic stem cell)가 분화되어 파골세포(osteoclast), 대식세포(macrophage), T 세포(T cell), B 세포(B cell), 수지상세포(dentritic cell), 호염구(basophile), 호산구(eosinophile), 호중구(neutrophile), 혈소판(platelet), 적혈구(Red blood cell) 등의 세포로 분화된다. 조골세포 주요 분화인자인 Runx2(Runt-related transcription factor 2), 근원세포 주요 분화인자인 MyoD, 지방세포 주요 분화인자인 PPARγ(peroxisome proliferator-activated receptors γ), 연골세포 주요 분화인자인 Sox9 등은 서로의 발현을 억제조절하며 분화를 유도한다(Yamashita S. et al., Exp Cell Res., 315(13), pp.2231-40, 2009, Charles A. et al., Exp Cell Res., 300(2), pp.406-17, 2004, David V. et al., Endocrinology., 148(5), pp.2553-62, 2007, Hill TP. et al., Dev Cell., 8(5), pp.727-38, 2005, Wang H. et al., J Bone Miner Res., 23(6), pp.939-48, 2008, Lengner CJ. et al., J Biol Chem., 280(16), pp.15872-9, 2005). 또한 연골세포 분화의 주요 인자인 Sox9은 조골세포의 성숙과 분화를 억제시키고, 조골세포의 분화인자인 Wnt는 Runx2를 촉진시키나 Sox9은 분해시켜 억제시키며 조골세포의 분화인자인 osterix는 연골세포의 분화를 억제한다(Dy P. et al., Dev Cell., 13;22(3), pp.597-609, 2012, Gunil I. et al., 대한류마티스학회지, 15(1), 2008, Tominaga H. et al., J Bone Miner Metab., 27(1), pp.36-45, 2009).
It is known that two types of cells are involved in aggregate formation. One of the cells is osteoblast that produces bone, and the other is osteoclast that destroys bone. These cells are derived from different cells, and mesenchymal stem cells are differentiated into cells of osteoblast, myoblast, adipocyte, fibroblast, chondrocyte, etc. And hematopoietic stem cells are differentiated into osteoclasts, macrophages, T cells, B cells, dentritic cells, basophiles, ), Eosinophile, neutrophile, platelet, red blood cell and the like. Run-2 (Runt-related transcription factor 2), MyoD (myoepithelial cell differentiation factor), PPARγ (peroxisome proliferator-activated receptors γ), and Sox9 the control of inhibiting the expression and induce differentiation (Yamashita S. et al., Exp Cell Res., 315 (13), pp.2231-40, 2009, Charles A. et al., Exp Cell Res., 300 ( Et al. , Dev Cell., 8 (5), pp. 406-17, 2004, David V. et al. , Endocrinology., 148 (5), pp.2553-62, pp.727-38, 2005, Wang H. et al ., J Bone Miner Res., 23 (6), pp.939-48, 2008, Lengner CJ. et al., J Biol Chem., 280 (16) , pp. 15872-9, 2005). In addition, Sox9, which is a major factor of chondrocyte differentiation, inhibits the maturation and differentiation of osteoblasts. Wnt, which is a differentiation factor of osteoblast, promotes Runx2 but inhibits Sox9 by degrading osteoblast. Osterix, inhibit (Dy P. et al, Dev Cell , 13;... 22 (3), pp.597-609, 2012, Gunil I. et al, Journal of the Society of rheumatism, 15 (1), 2008, Tominaga H et al ., J Bone Miner Metab., 27 (1), pp. 36-45, 2009).
조골세포는 RANKL(Receptor activator of NF-κB ligand)과 이것의 유도 수용체(decoy receptor)인 OPG(Osteoprotegerin)를 생성한다. RANKL이 파골 전구세포(osteoclast progenitor cells) 표면에 있는 수용체인 RANK(Receptor activator of NF-κB)에 결합하면 파골 전구 세포가 거대 다핵 파골세포로 성숙화(maturation)되어 골 흡수(bone resorption)가 일어난다. 그러나 OPG가 RANKL과 결합하면 RANKL과 RANK간 결합이 차단되어 파골세포의 형성이 억제되고 필요 이상의 골 흡수가 일어나지 않게 된다(Theill LE. et al., Annu Rev Immunol., 20, pp.795-823, 2002; Wagner EF. et al., Curr Opin Genet Dev., 11, pp.527-532, 2001). 이러한 파골세포의 활성으로 오래된 뼈의 흡수 또는 파괴가 이루어지며 이는 뼈에 구멍을 내어 적은 양의 칼슘이 혈류로 방출되어 신체기능을 유지하는데 사용된다(William J. et al., Nature., 423, pp.337342, 2003).
Osteoblasts produce RANKL (Receptor activator of NF-κB ligand) and its inducible receptor (decoy receptor), OPG (Osteoprotegerin). When RANKL binds to the receptors RANK (Receptor activator of NF-κB) on the surface of osteoclast progenitor cells, osteoclast precursor cells are maturated into large polynuclear osteoclasts and bone resorption occurs. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, resulting in inhibition of osteoclast formation and no more bone resorption than necessary (Theill LE et al ., Annu Rev Immunol., 20, pp. 795-823 , 2002; Wagner EF. Et al ., Curr Opin Genet Dev., 11, pp. 527-532, 2001). The activity of these osteoclasts results in the absorption or destruction of old bone, which is used to maintain bodily function by piercing the bone and releasing a small amount of calcium into the bloodstream (William J. et al., Nature 423, pp. 3737342, 2003).
한편, 뼈세포에서 생성된 조골세포는 교원질로 구멍을 채우고 칼슘과 인의 침척물(hydroxyapatite)을 덮어서 단단한 새로운 뼈를 만들어 골격을 재건한다(Stains JP. et al., Birth Defects Res C Embryo Today., 75(1), pp.72-80, 2005). 뼈가 파괴되기 시작하여 다시 새로운 뼈로 재형성 되기까지는 약 100일 정도 걸린다(Schwarz EM. et al., Curr Opin Orthop., 11, pp.329-335, 2000). 유아에서는 1년 내에 뼈의 칼슘이 100% 바뀌지만 성인에서는 매년 골격의 약 10~30%가 이런 과정을 통하여 재형성되며, 파골속도와 조골속도가 동일해야만 전과 같은 골밀도를 유지할 수 있다. On the other hand, the osteoblasts generated from bone cells fill holes with collagen and cover the hydroxyapatite of calcium and phosphorus to form new hard bones (Stains JP et al ., Birth Defects Res C Embryo Today. 75 (1), pp. 72-80, 2005). It takes about 100 days for the bones to begin to break down and rebuild into new bones (Schwarz EM. Et al ., Curr Opin Orthop., 11, pp. 329-335, 2000). In infants, bone calcium is 100% changed within a year, but about 10-30% of the bone is reshaped every year in adults, and the osteopenic rate and osteotomy speed are the same so that bone density can be maintained as before.
이러한 두 과정 즉, 골흡수와 골형성은 서로 밀접하게 연결되어 이루어지고 골의 정상적인 구조를 유지하는 데 중요하다. 한편, 고령화 사회에서 큰 문제로 대두되고 있는 골다공증(Osteoporosis)은 여러 가지 원인에 의하여 뼈의 질량이 감소하고 뼈 조직의 미세구조의 퇴화로 골절 위험이 지속적으로 증가하는 질환으로 뼈를 구성하는 미네랄(특히, 칼슘)과 기질이 감소한 상태이며, 골재형성의 균형이 깨져서 파골작용이 조골작용보다 증가된 상태에서 발생한다(Iqbal MM., South Med J., 93(1),pp.2-18, 2000). 정상적인 뼈 내부는 그물망처럼 치밀한 구조를 이루고 있으나, 골다공증의 경우에는 구조 사이의 간격이 넓어지고 미세구조가 얇아져 약해짐으로써 조그만 충격에도 뼈가 쉽게 골절될 수 있는 상태로 진행된다(Stepan JJ. et al., Endocr Regul., 37(4), pp.225-238, 2003). 골다공증은 폐경기의 시작과 동시에 급속한 골 손실(년간 2~3%)이 나타나며 척추의 압박 및 손목뼈가 쉽게 골절될 위험이 증가하는 폐경기 이후의 골다공증(Postmenopausal osteoporosis), 70세 이상의 남녀 노인에게 서서히 발생하며(연간 0.5~1%) 골반골(hip bone)과 척추뼈의 점진적인 골손실을 가져오는 노년기의 골다공증(Senile osteoporosis), 그리고 연령에 상관없이 질병(내분비질환, 위장관질환, 악성종양)이나 약물(부신피질호르몬, 항암화학요법, 갑상선호르몬, 항경련제, 항응고제, methotexate, cyclosporine, GnRH 등), 알코올, 흡연, 사고로 인해 발생하는 2차 골다공증(Secondary osteoporosis)으로 분류된다(Rosen CJ., N Engl J Med., 353(6), pp.595-603, 2005; Davidson M., Clinicain Reviews., 12(4), pp.75-82, 2002).These two processes, namely, bone resorption and bone formation, are closely linked to each other and are important for maintaining the normal structure of the bone. On the other hand, osteoporosis, which is becoming a big problem in aging society, is a disease in which bone mass is decreased due to various causes and the risk of fracture is continuously increased due to degeneration of microstructure of bone tissue. (Iqbal MM., South Med. J., 93 (1), pp. 2-18, 1999). In addition, 2000). Inside the normal bone is a dense structure like a net, but in the case of osteoporosis, the gap between the structures is widened and the microstructure is thinned and weakened, so that the bone is easily broken even in a small impact (Stepan JJ et al , Endocr Regul., 37 (4), pp. 225-238, 2003). Postmenopausal osteoporosis (postmenopausal osteoporosis), which is characterized by rapid bone loss (2-3% per year) at the onset of menopause and slowly increasing risk of fracture of the spine Senile osteoporosis, which causes progressive bone loss in the hip bone and vertebrae, and age-related diseases (endocrine disease, gastrointestinal diseases, malignant tumors) or drugs Secondary osteoporosis resulting from alcoholism, smoking, and accident (Rosen CJ, N., et al.) Are classified into two types: osteoporosis (corticosteroids, chemotherapy, thyroid hormones, anticonvulsants, anticoagulants, methotexate, cyclosporine and GnRH) Engl J Med., 353 (6), pp. 595-603, 2005; Davidson M., Clinicain Reviews., 12 (4), pp.75-82, 2002).
현재 골다공증 예방 및 치료에 사용되고 있는 약제는 대부분 골흡수를 억제하는 작용을 하기 때문에 이미 진행된 골소실을 완전히 회복할 수 없으며, 따라서 궁극적인 목표인 골다공증의 발생을 완전히 예방할 수 없는 것이 현실이다. 이에 골다공증의 예방과 치료를 위해 골형성 증가에 관한 연구가 최근 주목받고 있으며 골조직 재생능력에 미치는 영향 등에 대한 과학적인 접근이 필요한 실정이다 (Chp SH et al., Korean J Obstet Gynecol. 39:1497-1506, 1996, Boonen A et al., J Internal Medicine., 242:285-290, 1997)
Currently, medicines used for the prevention and treatment of osteoporosis mostly inhibit bone resorption, so that it is impossible to completely recover the already advanced bone loss, and thus it is a reality that the ultimate goal of osteoporosis can not be completely prevented. However, there is a need for a scientific approach to osteoporosis prevention and treatment, and the effect of osteogenesis on bone regeneration (Chp SH et al ., Korean J Obstet Gynecol. 39: 1497- 1506, 1996, Boonene et al ., J Internal Medicine., 242: 285-290, 1997)
또한 치주질환은 병의 정도에 따라 치은염과 치주염으로 나눌 수 있으며, 박테리아에 의한 염증으로 잇몸과 잇몸뼈 주변까지 진행된 경우를 치주염이라고 한다. 치주염이 심할수록 치주낭의 깊이가 깊어지게 되며, 치주낭이 깊어지면서 치주인대에 염증이 생기게 되고 골소실이 일어나게 되는 것이 치주질환이다. 치주조직을 치유시키기 위하여 치아와 치조골을 연결하는 치주인대 재형성이 필요하며, 이러한 치유과정에서 가장 중요한 것은 조골세포의 증식이라고 알려져 있다. 치주인대 세포는 조골세포와 유사한 특성을 가지며 분화되어 경조직을 형성한다. 현재 국내외에서 치주치료제로서 개발된 천연물 유래 약물로는 옥수수(Zea Mays L.) 추출물이 유일하며, 인사돌이라는 상품명으로 시판되고 있으나 약리기전이 밝혀지지 않았다. 현재 치주질환에 탁월한 효과를 나타내는 약물로서 개발된 것은 없으며 특히 조골세포의 활성을 증가시킴으로써 치주질환을 치료하고자 하는 시도는 없었다.
Periodontal disease can be divided into gingivitis and periodontal disease according to the severity of the disease. Bacterial inflammation is called gingivitis when it progresses to the periphery of the gums and gums. The more periodontal disease, the deeper the depth of the periodontal pouch, the periodontal ligament becomes deeper, the periodontal ligament is inflamed, and the bone loss is periodontal disease. To heal periodontal tissue, it is necessary to regenerate periodontal ligament connecting tooth and alveolar bone. It is known that proliferation of osteoblast is most important in this healing process. Periodontal ligament cells have similar characteristics to osteoblasts and differentiate to form hard tissues. Currently, the extract of Zea Mays L. is the only drug derived from natural products developed as a treatment for periodontal disease in Korea and abroad. It is commercially available under the trade name INVENDOL, but its pharmacological mechanism has not been elucidated. There has been no development as a drug showing excellent effects on periodontal disease. In particular, there has been no attempt to treat periodontal disease by increasing osteoblast activity.
한편, 자소자(Perillae semen; PS)는 차조기(Perilla frutescens L. Britton var. acuta KUDO.)의 열매로써 난원형 또는 구형에 가까우며 지름 0.6~2mm이다. 바깥면은 회갈색 또는 진한 회갈색이고 약간 융기된 그물무늬가 있다. 기부는 약간 뾰족하고 흰색의 점 모양인 열매꼭지 자국이 있다. 열매껍질은 얇고 누르면 쉽게 부서진다. 씨껍질은 막질이고 떡잎은 기름이 풍부하다. 거의 냄새가 없으나 씹으면 특유한 향기가 있고 맛은 텁텁하고 약간 맵다. 외용약으로 쓰는 경우 가루를 내어 뿌리거나 기초제에 개어 바른다. 약리 실험 결과 평활근 진경 작용, 혈압 강하 작용, 호흡 중추에 대한 흥분 작용이 밝혀졌으며, 항암 효과, 항균 효과, 미백 효과, 항 HIV효과 등의 활성이 보고되었다. Perillae semen (PS) is a fruit of Perilla frutescens L. Britton var. Acuta KUDO. It is near oval or spherical shape and has a diameter of 0.6 ~ 2mm. Outer surface is grayish brown or dark grayish brown with slightly raised net pattern. The donation has a pointed, white dotted fruit stalk. The fruit skin is thin and easily broken when pressed. Seed shells are membranous and cotyledons are rich in oil. There is almost no smell, but if you chew, there is a peculiar smell. The taste is tough and slightly spicy. If you use it as an external medicine, put out the powder and apply it to the foundation. Pharmacological experiments revealed smooth muscle rooting activity, hypotensive action, and excitatory action on the respiratory center, and anticancer activity, antimicrobial activity, whitening effect, and anti-HIV activity were reported.
강활, 인진, 소엽 혼합 추출물의 연골세포 재생에 관하여 등록된 특허(등록번호 10-0595735)는 있으나 그러나 자소자 추출물의 조골세포 분화 촉진 활성과 파골세포 분화억제에 의한 골질환 예방 또는 치료 효과에 대해서는 개시된 바 없다. 연골세포와 조골세포는 간엽줄기세포에서 유래하여 조골세포, 근원세포, 지방세포, 섬유아세포, 연골세포 등의 각기 다른 세포로 분화되며 조골세포 촉진 인자의 연골세포 억제기작 및 연골세포 촉진 인자의 조골세포 억제기작 등은 이미 알려져 있으므로 전혀 다른 세포이다(Dy P. et al., Dev Cell., 13;22(3), pp.597-609, 2012, Gunil I. et al., 대한류마티스학회지, 15(1), 2008, Tominaga H. et al., J Bone Miner Metab., 27(1), pp.36-45, 2009).
There is a registered patent (Registration No. 10-0595735) regarding regeneration of chondrocyte of ganghwam, prolene, and lobular mixed extract. However, the effect of prodrug extract on the osteoblast differentiation promoting activity and osteoclast differentiation inhibition effect It has not been disclosed. Chondrocytes and osteoblasts are derived from mesenchymal stem cells and differentiated into different cells such as osteoblasts, myoblasts, adipocytes, fibroblasts, and chondrocytes, and the osteoblast-promoting factor's cartilage cell suppression mechanism and osteoblast- since cell suppression mechanism such as is already known for a different cell (Dy P. et al, Dev cell , 13;... 22 (3), pp.597-609, 2012, Gunil I. et al, Journal of the Society of rheumatism, 15 (1), 2008, Tominaga H. et al ., J Bone Miner Metab., 27 (1), pp. 36-45, 2009).
이에 본 발명자들은 일상생활에서 약용 또는 식용으로 이용되고 있는 천연물에 대한 생리활성 물질을 규명하기 위해 조골세포 분화 촉진과 파골세포 분화 억제 효과를 시험하던 중 자소자 추출물이 이러한 활성이 있음을 발견하여 본 발명을 완성하게 되었다.
Accordingly, the inventors of the present invention have found that the extract of Z-element extract has an activity to promote osteoblast differentiation and inhibit osteoclast differentiation in order to identify a physiologically active substance for natural or medicinal use in daily life. Thereby completing the invention.
본 발명은 조골세포 분화 촉진 및 파골세포 분화 억제 활성을 갖는 자소자 추출물 및 상기의 자소자 추출물을 유효성분으로 함유하는 약학적 조성물, 식품조성물, 발효유, 건강기능식품 등을 제공하는 것을 목적으로 한다.
It is an object of the present invention to provide a pharmaceutical composition, a food composition, a fermented milk, a health functional food, and the like containing a self-component extract having the osteoblast differentiation promotion and osteoclast differentiation inhibiting activity and the self-component extract as an active ingredient .
상기와 같은 목적을 달성하기 위하여 본 발명은 조골세포 분화 촉진 및 파골세포 분화 억제 활성을 갖는 자소자 추출물 및 상기의 자소자 추출물을 유효성분으로 함유하는 약학적 조성물, 식품 조성물, 발효유, 건강기능식품을 제공하는 것을 특징으로 한다.
In order to accomplish the above object, the present invention provides a pharmaceutical composition, a food composition, a fermented milk, a health functional food, a pharmaceutical composition, a cosmetic composition, a cosmetic composition, .
이하, 본 발명을 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명의 유효성분으로 이용되는 추출물의 소스(source)로서의 "차조기"( Perilla frutescens L. Britton var. acuta Kudo)는 꿀풀과의 일년생 초본으로 한방에서는 잎을 소엽, 종자를 자소자라고하며, 줄기는 곧게 서고 높이가 20~80cm이며 단면이 사각형이고 자주빛이 돌며 향기가 있다. 잎은 마주나고 넓은 달걀모양이며 끝이 뾰족하고 밑 부분이 둥글며 가장자리에 톱니가 있다. 잎 양면에는 털이 있고, 뒷면 맨 위에는 긴 털이 있으며 잎자루가 길다. 차조기 전체의 정유 성분에는 페릴알데하이드 약 55%, l-리몬넨(l-limonene) 20~30% 및 α-피넨(pinene) 소량 외에 아르기닌, 쿠민산(cumic acid), 시아니딘-3-(6-p-쿠마로일-β-D-글루코사이드)-5-β-D-글루코사이드를 함유하고 있고, 잎의 정제된 유지성분에는 이소에고마 케톤(isoegoma ketone)등이 함유되어 있다. 발한, 진해, 건위, 이뇨, 진정 및 진통제로 사용한다. 소두(蘇頭)는 차조기의 뿌리 및 뿌리에 가까운 부분의 오래된 줄기로, 통풍을 물리치고, 발한, 거담에 효과가 있으며 기(氣)를 내리는데 효과가 있으며, 현기증과 신체의 통증 및 코막힘, 콧물을 치료하는데 효과가 있다(정보섭 및 신민교; 도해 향약(생약)대사전, 영림사, pp855-856, 1998).
" Perilla frutescens L. Britton var. Acuta Kudo" as a source of the extract used as an active ingredient of the present invention is an annual herb of Lamiaceae. It is called leaflet, It stands straight and has a height of 20 ~ 80cm. It has a square cross section, mauve light and scent. Leaves are opposite, wide ovate, pointed end, round bottom, with sawtooth on edge. There are hairs on both sides of the leaf, there is long hairs on the top of the back side, and petiole is long. The essential oil constituents of the whole plant are arginine, cumic acid, cyanidin-3- (6 (meth) acrylamide) in addition to a small amount of about 55% of perylaldehyde, 20 to 30% of 1-limonene and a- p-coumaroyl- beta -D-glucoside) -5- [beta] -D-glucoside, and the purified fat component of the leaves contains isoegoma ketone. It is used as sweating, calming, dryness, diuretic, soothing and analgesic.頭 head is an old stem near the roots and roots of the chrysanthemum. It is effective in reducing gout, sweating, and germinating, and is effective in lowering 气, It is effective in treating runny nose (Ji-Sup and Shin Min-gyo;
본 발명의 자소자 추출물은 차조기의 다양한 기관(예컨대, 잎, 줄기, 뿌리 또는 종자)로부터 추출하여 얻는 것이 바람직하며, 가장 바람직하게는 차조기의 종자로부터 추출하여 얻은 것이다. 본 발명에 따른 자소자 추출물은 당업계에 공지된 통상적인 추출용매, 바람직하게는, (a)물, (b)탄소수 1 내지 4의 무수 또는 함수 저급 알코올(예: 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-이로판올 및 노말-부탄올 등), (c)상기 저급 알코올과 물과의 혼합용매, (d)아세톤, (e)에틸 아세테이트, (f)클로로포름, (g)1,3-부틸렌글리콜, (h)헥산, (i)디에틸에테르, (j)부틸아세테이트 등을 이용하여 얻을 수 있다. 바람직하게는 본 발명의 자소자 추출물은 물, 메탄올 또는 에탄올, 가장 바람직하게는 에탄올이 사용될 수 있으며, 특히 바람직하게는 70% 에탄올이 사용될 수 있다. 그러나 이에 한정되는 것이 아니다.
The extract of the present invention is preferably obtained by extracting from various organs (for example, leaves, stems, roots or seeds) of a garnish, and most preferably, extracted from seeds of a garlic. (A) water, (b) an anhydrous or hydro-lower alcohol having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol, butanol (D) acetone, (e) ethyl acetate, (f) chloroform, (g) 1, 2-propanol, 3-butylene glycol, (h) hexane, (i) diethyl ether, (j) butyl acetate and the like. Preferably, the self-component extract of the present invention may be water, methanol or ethanol, most preferably ethanol, and particularly preferably 70% ethanol may be used. However, it is not limited thereto.
상기 자소자 추출물은 추출에 의해 수득되는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 수득되는 건조물 또는 추출액의 조정제물 또는 정제물일 수 있다.
The child element extract may be a diluent or concentrate of the extract obtained by extraction, a dried product obtained by drying the extract, or a preparation or purified product of the extract.
특히, 물을 이용하여 자소자 추출물을 얻는 방법을 좀 더 구체적으로 살펴보면, 시료인 자소자를 5~10배 가량의 물을 이용하여 100℃ 2회 반복 추출한다. 추출액을 여과지를 사용하여 1회 이상 여과하고 동결건조한 후 분말화하여 이를 시료로 사용하는 방법으로 제조될 수 있다.More specifically, a method of obtaining a magnetic element extract using water is repeatedly performed at a temperature of 100 ° C two times using 5 to 10 times of water as a sample magnetic element. The extract can be prepared by filtration using filter paper at least once, lyophilized and powdered, and used as a sample.
또한, 물과 에탄올의 혼합용매(70% 에탄올)를 이용하여 자소자 추출물을 얻는 방법을 구체적으로 살펴보면, 시료인 자소자를 5~10배 가량의 물과 에탄올의 혼합용매(70% 에탄올)를 가하여 30 내지 80℃에서 2 내지 5시간 동안 2회 반복하여 환류 추출한 다음, 추출액을 와트만 No.3. 여과지를 사용하여 2회 여과하고 회전감압 농축기로 농축하여 동결 건조하여 분말화하여 시료로서 사용하는 방법으로 제조될 수 있다.
In detail, a method of obtaining a magnetic element extract using a mixed solvent of water and ethanol (70% ethanol) is as follows. A magnetic element as a sample is mixed with 5 to 10 times of a mixed solvent of water and ethanol (70% ethanol) Followed by refluxing twice at 30 to 80 ° C for 2 to 5 hours, and then the extract was added to Watman No. 3. Filtered twice using a filter paper, concentrated using a rotary evaporator, lyophilized and powdered, and used as a sample.
본 발명에 따른 조골세포 분화 촉진 및 파골세포 분화 억제 효능을 갖는 자소자 추출물을 유효성분으로 함유하는 골질환의 예방 및/또는 치료용 약제학적 조성물은 약제학적으로 허용 가능한 담체를 포함하여 약학 조성물로 제제화될 수 있으며, 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화될 수 있다.The pharmaceutical composition for preventing and / or treating osteoporosis comprising an extract of a self-component having the osteoblast differentiation promoting activity and osteoclast differentiation inhibiting activity according to the present invention as an active ingredient can be used as a pharmaceutical composition including a pharmaceutically acceptable carrier And may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method.
상기 골질환은 골다공증, 파제트 골질환, 구루병, 신부전 환자의 신성골이영양증, 류마티스성 또는 퇴행성 골질환, 뼈전이암 병소(bone metastatic lesion), 원발성으로 뼈에 생성된 종양, 치주질환, 염증성 치조골 흡수질환 및 염증성 뼈 흡수질환으로 구성된 그룹에서 선택될 수 있으나 이에 한정되는 것은 아니다.
The bone diseases include osteoporosis, Paget's bone disease, rickets, nephrolithiasis in renal failure patients, rheumatic or degenerative bone disease, bone metastatic lesion, primary bone-derived tumors, periodontal disease, inflammatory alveolar bone An inflammatory disease, an inflammatory disease, an inflammatory disease, an inflammatory disease and an inflammatory bone resorption disease.
본 발명의 조골세포 분화 촉진 및 파골세포 분화 억제 활성을 갖는 자소자 추출물을 유효성분으로 함유하는 골강화 또는 성장발육 촉진용 식품 조성물은 발효유, 기능성 음료, 건강기능식품 등의 형태로 제공될 수 있으며, 식품 조성물에는 식품학적으로 허용 가능한 식품 보조 첨가제가 포함될 수 있다.
The composition for promoting bone strengthening or growth promotion, which contains the self-extracting ingredient having the osteoblast differentiation promotion activity and osteoclast differentiation inhibiting activity as an active ingredient of the present invention, can be provided in the form of fermented milk, functional beverage, , The food composition may include a foodstuff acceptable food supplement additive.
상기 본 발명의 조골세포의 분화촉진 및 파골세포의 분화억제 활성을 갖는 것을 특징으로 하는 자소자 추출물을 유효성분으로 함유하는 발효유는 유산균 배양액, 혼합과즙시럽 및 본 발명의 자소자 추출물을 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 제조한다.
The fermented milk containing the self-extracting ingredient having the activity of promoting osteoblast differentiation and osteoclast differentiation according to the present invention as an active ingredient can be prepared by combining the lactic acid bacteria culture broth, the mixed juice syrup and the self- And then cooled to < RTI ID = 0.0 > 10 C < / RTI >
상기 본 발명의 조골세포의 분화촉진 및 파골세포의 분화억제 활성을 갖는 것을 특징으로 하는 자소자 추출물을 유효성분으로 함유하는 건강기능식품은 본 발명의 자소자 추출물에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 첨가하여 고속회전 혼합기에서 혼합한 후 상기 혼합물에 멸균 정제수를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형한다. 상기 성형된 과립은 40~50℃의 진공건조기에서 건조시킨 후 12~14메쉬(mesh)를 통과시켜 균일하게 과립을 제조하여 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조한다.
The health functional food containing the self-extracting ingredient having the activity of promoting differentiation of osteoblasts and inhibiting osteoclast differentiation according to the present invention is characterized in that nutritional supplement components (vitamin B 1 , B 2 , B 5 , B 6 , E and acetic acid ester, nicotinic acid amide) and oligosaccharide are added and mixed in a high-speed rotary mixer. Sterile purified water is added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules are dried in a vacuum drier at 40 to 50 ° C. and then passed through 12 to 14 mesh to uniformly produce granules. The granules are extruded in appropriate amounts to be purified or powdered or filled into hard capsules, .
본 발명의 자소자 추출물은 조골세포의 분화를 촉진시키고 파골세포의 분화를 억제함으로써 골개형(bone remodeling)에 있어서 골생성(bone formation)에 대한 골흡수(bone resorption)의 증가로 인한 불균형에 따라 초래되는 골질환, 예를 들어 노인들의 골질환이나 여성들의 폐경기로 인해 발생하는 골 질환을 개선할 수 있는 약학적 조성물, 기능성 식품 등에 유용하게 적용될 수 있다.
The self-component extract of the present invention promotes the differentiation of osteoblasts and inhibits the differentiation of osteoclasts, resulting in an imbalance due to an increase in bone resorption for bone formation in bone remodeling The present invention can be applied to pharmaceutical compositions and functional foods that can improve bone diseases caused by osteopathy of the elderly or menopause of women, for example.
도 1은 본 발명의 자소자 추출물의 마우스 두개골 골아세포의 세포독성에 대한 영향을 그래프로 나타낸 것이다.
도 2는 본 발명의 자소자 추출물의 마우스 두개골 골아세포주인 MC3T3-E1 세포주의 세포 독성에 대한 영향을 그래프로 나타낸 것이다.
도 3은 본 발명의 자소자 추출물의 인간 조골세포주인 HOS 세포주의 세포 독성에 대한 영향을 그래프로 나타낸 것이다.
도 4는 본 발명의 자소자 추출물의 마우스 두개골 골아세포의 염기성 인산 분해효소에 대한 영향을 그래프로 나타낸 것이다.
도 5는 본 발명의 자소자 70% 에탄올 추출물의 마우스 두개골 골아세포의 염기성 인산 분해 효소에 대한 영향을 그래프로 나타낸 것이다.
도 6는 본 발명의 자소자 추출물의 마우스 두개골 골아세포의 골석회화에 대한 영향을 알리자린-레드로 염색하여 나타낸 것이다.
도 7은 본 발명의 자소자 추출물의 마우스 두개골 골아세포의 골석회화에 대한 영향을 흡광도로 측정하여 그래프로 나타낸 것이다.
도 8은 본 발명의 자소자 열수추출물의 RANKL 유도 파골세포 분화억제 영향을 사진으로 나타낸 것이다.
도 9는 본 발명의 물과 에탄올의 혼합용매에 의한 자소자 추출물의 RANKL 유도 파골세포 분화억제 영향을 사진으로 나타낸 것이다.
도 10은 본 발명의 자소자 열수추출물의 RANKL 유도 파골세포 분화 억제된 거대 다핵 파골세포 수를 그래프로 나타낸 것이다.
도 11은 본 발명의 자소자 70% 에탄올 추출물의 RANKL 유도 파골세포 분화 억제된 거대 다핵 파골세포 수를 그래프로 나타낸 것이다.FIG. 1 is a graph showing the effect of the self-extract of the present invention on cytotoxicity of mouse skull osteoblasts.
Fig. 2 is a graph showing the effect of the self-extract of the present invention on the cytotoxicity of the mouse skull osteoblast cell line MC3T3-E1.
FIG. 3 is a graph showing the effect of the self-extract of the present invention on cytotoxicity of human osteoblast cell line HOS cell line.
FIG. 4 is a graph showing the effect of the self-extract of the present invention on the basic phosphatase of the mouse skull osteoblast.
FIG. 5 is a graph showing the effect of the magnetic element 70% ethanol extract of the present invention on the basic phosphatase of the mouse skull osteoblast.
FIG. 6 shows the effect of the self-extract of the present invention on alveolar-red staining of the effect of osteocarcinosis on mouse skull osteoblasts.
FIG. 7 is a graph showing the effect of the magnetic element extract of the present invention on bone calcification of mouse skull osteoblasts measured by absorbance.
FIG. 8 is a photograph showing the inhibitory effect of RANKL induced osteoclast differentiation of the hot-water extract of the magnetic element of the present invention.
FIG. 9 is a photograph showing the effect of inhibition of RANKL-induced osteoclast differentiation by the self-extract of the present invention with a mixed solvent of water and ethanol.
FIG. 10 is a graph showing the number of large polynuclear osteoclasts inhibited by RANKL-induced osteoclast differentiation of the magnetic element hydrothermal extract of the present invention.
FIG. 11 is a graph showing the number of large polynuclear osteoclasts inhibited by RANKL-induced osteoclast differentiation of the 70% ethanol extract of the magnetic element of the present invention.
이하, 본 발명을 다음과 같은 실시예에 의하여 더욱 상세하게 설명하고자 한다. 다만, 다음의 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
<실시예 1>≪ Example 1 >
자소자 추출물의 제조Production of magnetic element extract
1-1. 자소자의 열수추출물의 제조 1-1. Production of hot water extract of magnetic element
자소자에 5~10배의 물을 가하여 100℃에서 2시간 가열하는 과정을 2회 반복하여 추출한 다음, 상기 추출액을 와트만 No.3. 여과지를 사용하여 2회 여과하고 동결 건조하여 분말화하여 시료로서 사용하였다.
The process of adding 5 to 10 times of water to the magnetic element and heating it at 100 占 폚 for 2 hours was repeated twice, and the extract was added to Watman No. 3. Filtered twice using filter paper, lyophilized and powdered, and used as a sample.
1-2. 물과 에탄올의 혼합용매(70% 에탄올)에 의한 자소자의 추출물의 제조 1-2. Preparation of extract of magnetic element by mixed solvent of water and ethanol (70% ethanol)
자소자에 5~10배의 물과 에탄올의 혼합액(70% 에탄올)을 가하여 70~80℃에서 2~5시간 동안 2회 반복하여 추출한 다음, 상기 추출액을 와트만 No.3. 여과지를 사용하여 2회 여과하고 회전감압 농축기(EYELA,NE-1001, Japan)로 농축하여 동결 건조하여 분말화하여 시료로서 사용하였다.
(70% ethanol) of 5 to 10 times of water and ethanol was added to the sample, and the mixture was repeatedly extracted twice at 70 to 80 ° C for 2 to 5 hours. The mixture was filtered twice using a filter paper, concentrated using a rotary evaporator (EYELA, NE-1001, Japan), lyophilized and powdered, and used as a sample.
<실시예 2>≪ Example 2 >
자소자 추출물을 유효성분으로 함유하는 약학적 조성물의 제조Of a pharmaceutical composition containing a self-extracting ingredient as an active ingredient
액제의 제조Manufacture of liquid agent
상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말 100㎎, 이성화당 10g, 만니톨 5g을 통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조하였다.
100 mg of lyophilized powder of a hot-water extract of the magnetic element of Example 1 or lyophilized powder of a 70% ethanol extract of a magnetic element, 10 g of isomerized sugar, and 5 g of mannitol were dissolved and dissolved in purified water in accordance with a conventional liquid preparation method After adding proper amount of lemon flavor, purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.
캡슐제의 제조Preparation of capsules
상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말 100㎎에 옥수수 전분 100㎎, 유당 100㎎, 스테아린산 마그네슘 2㎎을 완전히 혼합한 후 통상의 캡슐제의 제조방법에 따라 경질 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed thoroughly with 100 mg of the lyophilized powder or the lyophilized powder of the magnetic element 70% ethanol extract of Example 1, The capsules were filled in hard gelatin capsules according to the manufacturing method.
<실시예 3> ≪ Example 3 >
자소자 추출물을 유효성분으로 함유하는 발효유의 제조Production of fermented milk containing fermented tea extract as an active ingredient
본 발명의 자소자 추출물을 유효성분으로 함유하는 발효유를 제조하는 방법은 다음과 같다.A method for producing a fermented milk containing the present invention's extract as an active ingredient is as follows.
먼저, 유산균 배양액은 원유 95.36중량%와 탈지분유(또는 혼합분유) 4.6중량%를 교반하여 15℃에서의 비중은 1.0473~1.0475, 적정산도는 0.200~0.220%, pH는 6.65~6.70, 20℃에서의 브릭스(Brixo)는 16.3~16.5%정도가 되도록 혼합하였다. 혼합 후에 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 스트렙토코커스 써모필러스균과 유당분해효소(Valley laboratory, USA)를 각기 0.02중량%씩 첨가하고 6시간 동안 배양하여 BCP 배지에서의 총 유산균수가 1.0 × 109 cfu/㎖ 이상, 적정산도가 0.89~0.91%, pH는 4.55~4.65가 되도록 하여 제조하였다.The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 × 10 9 cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.
그런 다음, 혼합과즙시럽은 액상과당 13중량%, 백설탕 5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Then, mixed juice syrup, liquid fructose, 13 wt.%, Sugar 5 wt.%, Mixed juice concentrate 56Brix o 10.9% by weight of pectin, 1.0% by weight, fresh fruit mix Essence 0.1% and purified water 70% by weight at 30 ~ 35 ℃ Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.
그런 다음, 상기 유산균배양액 69.5중량%와 상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말 0.1중량% 및 상기 혼합과즙시럽 30.4중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 발효유를 제조하였다.
Then, a mixture of 69.5% by weight of the above-mentioned lactic acid bacteria culture solution and 0.1% by weight of freeze-dried powder of a lyophilized powder or a 70% ethanol extract of a magnetic element hot water extract of Example 1 and 30.4% Homogenized and cooled to below 10 캜 to produce fermented milk.
<실시예 4> <Example 4>
자소자 추출물을 유효성분으로 함유하는 건강기능식품의 제조Production of Health Functional Foods Containing Self-extracting Ingredients as Active Ingredients
상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말 0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 상기의 실시예 1의 자소자 추출물 동결건조분말 100중량부에 대하여 10중량부가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물에 멸균 정제수 10중량부를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형하였다. 상기 성형된 과립은 40~50℃의 진공건조기에서 건조시킨 후 12~14메쉬(mesh)를 통과시켜 균일하게 과립을 제조하였다. 상기와 같이 제조된 과립은 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조하였다.
B vitamins B 1 , B 2 , B 5 , B 6 , E and acetic acid esters were added to 0.1% by weight of the lyophilized powder of the magnetic element hot-water extract of Example 1 or the lyophilized powder of the magnetic element 70% Nicotinic acid amide) and oligosaccharide were added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder obtained in Example 1, followed by mixing in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.
<시험예 1>≪ Test Example 1 >
자소자 추출물에 대한 조골세포의 세포독성 측정Cytotoxic measurement of osteoblast cell extracts
1-1. 마우스 두개골 골아세포의 세포독성 측정 1-1. Cytotoxicity measurement of mouse skull osteoblast
가. 골아세포의 준비end. Preparation of osteoblasts
1 내지 2일령 된 신생 ICR 마우스로부터 전구골과 두정골을 무균적으로 적출하여 인산염완충용액(PBS)으로 씻어준 후, 0.1% 콜라게나제(collagenase, Gibco), 0.1% 디스파제(dispase, Gibco)가 포함된 효소용액에 연속적으로 6회 처리하고 조골세포의 특성을 지닌 세포들이 많이 존재하는 3 내지 6회군의 세포를 집중적으로 수집하여 배양액(serum free α-MEM)으로 세척하였다. 세척된 세포들을 10% FBS가 포함된 α-MEM에서 2일 정도 배양한 후 1차 계대 배양하여 모은 세포들을 실험에 사용하였고, 회수한 세포들을 1X106세포/㎖의 농도가 되도록 희석하여 영하 70℃에서 보관하였다.
Bone marrow cells were treated with 0.1% collagenase (Gibco), 0.1% dispase (Gibco), and 0.1% collagenase (PBS) in phosphate buffered saline (PBS) Were sequentially treated 6 times with an enzyme solution containing 3 to 6 times of cells in which osteoblast-specific cells were intensively collected and washed with serum free α-MEM. The washed cells were cultured in α-MEM containing 10% FBS for 2 days and then cultured for the first time. The collected cells were used for the experiment, and the recovered cells were diluted to a concentration of 1 × 10 6 cells / ≪ / RTI >
나. 세포독성 측정I. Cytotoxicity measurement
시료의 세포독성을 측정하기 위해 Green 등의 방법(Green LM, Reade JL, Ware CF. Rapid colometric assay for cell viability: Application to the quantitation of cytotoxic and growthinhibitory lympokines. J. Immunal. Meth. 70:257(1984))에 준하여 3-(4,5-디메틸-티아졸-2-일)-2,5-디페닐테트라졸륨브로마이드[3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide, MTT]분석을 실시하여 측정하였다.
To measure the cytotoxicity of the sample, the method of Green et al. (Green LM, Reade JL, Ware CF. Rapid colometric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lympocines. J. Immun. Meth. 70: 257 ), Was prepared 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide [3- -diphenyltetrazoliumbromide, MTT] analysis.
구체적으로는 먼저 상기 배양한 마우스의 두개골 골아세포를 96 웰 플레이트(Well Plate)에 각 웰(well)당 1X104세포/200㎕ 농도로 분주하고 24시간 배양 후 배지를 제거하였다.Specifically, the skull osteoblast of the cultured mouse was dispensed into a 96-well plate at a concentration of 1 × 10 4 cells / 200 μl per well, cultured for 24 hours, and then the medium was removed.
이와는 별도로 새로운 MEM 배지 200㎕에 상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말을 각각 3.12, 6.25, 12.5, 25, 50, 100㎍/㎖의 농도로 녹이고, 이들을 상기 준비한 각 웰(Well)에 첨가하였다. 시료가 첨가된 배양액에서 48시간 배양한 후, MTT(5㎎/㎖) 용액 10㎕를 각 웰(Well)에 가하고 4시간 동안 배양하였다. 배양 종료 후 상등액을 제거하고 각 웰(Well)에 100㎕의 DMSO를 첨가하여 생성된 포마잔(formazan) 결정을 용해시켜 마이크로플레이트 리더(microplate reader)로 570nm에서 흡광도를 측정하였다. 세포독성은 시료의 흡광도를 대조군의 흡광도에 대한 백분율로 나타내어 그 결과를 도 1에 나타내었다. 대조군(Con)으로는 본 발명의 자소자 추출물을 첨가하지 않은 것을 사용하였다.
Separately, the lyophilized powder of the hot-water extract of Example 1 or the lyophilized powder of the 70% ethanol extract of the magnetic element was added to 200 μl of the fresh MEM medium at a concentration of 3.12, 6.25, 12.5, 25, 50 and 100 μg / , And these were added to each well prepared above. After culturing for 48 hours in the culture medium to which the sample had been added, 10 μl of MTT (5 mg / ml) solution was added to each well and cultured for 4 hours. After the completion of the incubation, the supernatant was removed, 100 μl of DMSO was added to each well, and the resulting formazan crystals were dissolved. The absorbance was measured at 570 nm using a microplate reader. The cytotoxicity was expressed as a percentage of the absorbance of the test sample in the absorbance of the sample, and the results are shown in FIG. As the control (Con), those without the self-extract of the present invention were used.
1-2. 마우스의 두개골 골아세포주인 MC3T3-E1 세포주의 세포독성 측정 1-2. Cytotoxicity of mouse osteoblast cell line MC3T3-E1 cell line
가. 세포주의 준비end. Preparation of cell line
세포주로 사용된 MC3T3-E1(mouse pre-osteoblast)은 ATCC(CRL-2594)에서 분양받아 10% FBS(fetal bovine serum)와 1X 항생물질(antibiotics, Anti-Anti, Gibco 15240)을 첨가한 α-MEM(alpha-minimum essential medium, Gibco A10490) 배지를 이용하여 5% 이산화탄소(CO2)가 존재하는 인큐베이터에서 1주일에 2~3회 계대 배양하였다.
MC3T3-E1 (mouse pre-osteoblast), which was used as a cell line, was purchased from ATCC (CRL-2594) and mixed with 10% fetal bovine serum (FBS) and 1X antibiotic (antibiotics, Anti-Anti, Gibco 15240) MEM (alpha-minimum essential medium, Gibco A10490) culture medium was a 5% carbon dioxide (CO 2) incubator for one passage is present in two or three times a week in culturing using.
나. 세포독성 측정I. Cytotoxicity measurement
마우스의 두개골 골아세포 대신 마우스의 두개골 골아세포주인 MC3T3-E1 세포주를 사용한 것을 제외하고는 상기 시험예 1-1의 세포독성 측정방법과 동일한 방법으로 측정하였다.Except that MC3T3-E1 cell line, a skull osteoblast cell of mouse, was used instead of mouse osteoblast cell.
그 결과를 도 2에 나타내었다.
The results are shown in Fig.
1-3. 인간 조골세포주인 HOS 세포주의 세포독성 측정 1-3. Cytotoxicity measurement of human osteoblast cell line HOS cell line
가. 세포주의 준비end. Preparation of cell line
세포주로 사용된 Hos cell(Human osteosarcoma cell)은 한국세포주 은행(KCLB-21543)으로부터 분양받아 10% FBS(fetal bovine serum)와 1X 항생물질(antibiotics, Anti-Anti, Gibco 15240)을 첨가한 α-MEM(alpha-minimum essential medium, Gibco A10490) 배지를 이용하여 5% 이산화탄소(CO2)가 존재하는 인큐베이터에서 1주일에 2~3회 계대 배양하였다.
Hos cell (Human osteosarcoma cell) used as a cell line was purchased from Korean Cell Line Bank (KCLB-21543) and cultured with α-fetoprotein (FBS) supplemented with 10% fetal bovine serum and 1 × antibiotic MEM (alpha-minimum essential medium, Gibco A10490) culture medium was a 5% carbon dioxide (CO 2) incubator for one passage is present in two or three times a week in culturing using.
나. 세포독성 측정I. Cytotoxicity measurement
마우스의 두개골 골아세포 대신 인간 조골세포주인 HOS 세포주를 사용한 것을 제외하고는 상기 실시예 1-1의 세포독성 측정방법과 동일한 방법으로 측정하였다.Except that human osteoblast cell line HOS cell line was used instead of mouse osteoblast cell line.
그 결과를 도 3에 나타내었다.
The results are shown in Fig.
도 1 내지 도 3에서 확인할 수 있는 바와 같이, 본 발명의 자소자 열수 추출물 및 자소자 70% 에탄올 추출물은 높은 농도로 처리하였을 때도 세포독성이 없음을 알 수 있었다.
As can be seen from FIGS. 1 to 3, the hot-water extract of the magnetic element and the 70% ethanol extract of the magnetic element of the present invention were not cytotoxic even when treated at a high concentration.
<시험예 2>≪ Test Example 2 &
자소자 추출물에 대한 조골세포의 염기성 인산분해 효소 활성 측정Measurement of basic phosphatase activity of osteoblast cells in a rat
염기성 인산분해 효소(Alkaline phosphatase; ALP)활성 검사는 p-니트로페닐 포스페이트(p-nitrophenyl phosphate)의 가수분해 반응에 ALP가 촉매로 작용하는 것을 이용하여, 가수분해 산물인 p-니트로페놀의 양을 측정함으로써 ALP의 농도를 산출하는 방법이다.ALP (Alkaline phosphatase; ALP) activity test p - the amount of nitrophenol-nitrophenylphosphate ALP p is the hydrolysis product using that acts as a catalyst in the hydrolysis reaction of the (p -nitrophenyl phosphate) And the concentration of ALP is calculated by measurement.
상기 시험예 1-1의 마우스 두개골 골아세포를 24 웰 플레이트(Well Plate)에 각 5X104 세포/well 농도로 분주한 후, 37℃, 5% CO2 인큐베이터에서 24시간 배양 후, 배지를 제거하였다.The mouse skull osteoblasts of Test Example 1-1 were divided into well plates at a concentration of 5 × 10 4 cells / well, and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours. Then, the medium was removed .
이와는 별도로 10% FBS, 1% 항생제(penicillin-streptomycin)가 첨가된 배지에 상기 실시예 1의 자소자 열수 추출물의 동결건조분말 혹은 자소자 70% 에탄올 추출물의 동결건조분말을 각각 100, 50, 25, 12.5㎍/㎖의 농도로 녹인 후 상기 준비한 각 웰(Well)에 분주하였다. 7일간 배양 후 배양액을 제거하고 0.1% 트리톤(Triton) X-100을 첨가해 세포를 용해시킨 후, 50㎕을 꺼내 여기에 p-니트로페닐포스페이트(1.21mM)를 100㎕ 첨가하였다. 37℃에서 30분간 반응시키고, 0.2N 수산화나트륨을 50㎕ 첨가하여 반응을 정지시켰다.Separately, the lyophilized powder of the hot-water extract of Example 1 or the lyophilized powder of the 70% ethanol extract of the magnetic element was added to the medium supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin) , And 12.5 占 퐂 / ml, and then dispensed into each well prepared above. After 7 days of culture, the culture medium was removed, and 0.1% Triton X-100 was added to dissolve the cells. 50 μl of the solution was taken out, and 100 μl of p -nitrophenyl phosphate (1.21 mM) was added thereto. The reaction was carried out at 37 캜 for 30 minutes, and 50 ㎕ of 0.2 N sodium hydroxide was added to stop the reaction.
이러한 과정을 통하여 p-니트로페닐포스페이트로부터 가수분해된 p-니트로페놀의 생성량을 측정하여 활성도를 도출하였고, 단백질량으로 나누어 단위단백질 함량당 효소 활성도를 산출하였다. 마이크로플레이트 리더(Bio-tek, Synergy HT, USA)를 이용하여 405nm에서 흡광도를 측정하였다. 단백질 양은 우혈청 알부민 단백질 어세이 시약(Bovine serum albumin protein assay reagent)을 이용하여 측정하였으며 효소 활성은 대조군에 대한 백분율로 나타내었다. 대조군은 본 발명의 자소자 추출물을 첨가하지 않은 것을 사용하였다.Hydrolyzed from p-nitrophenyl phosphate - - p Through this process by measuring the amount of the nitrophenol was obtained the activity was calculated for the enzyme activity per unit protein content divided by the protein content. Absorbance was measured at 405 nm using a microplate reader (Bio-tek, Synergy HT, USA). Protein content was measured using a bovine serum albumin protein assay reagent. Enzyme activity was expressed as a percentage of the control. As the control, those without the self-extract of the present invention were used.
그 결과를 도 4 및 도 5에 나타내었다.
The results are shown in Fig. 4 and Fig.
도 4 및 도 5에서 확인할 수 있는 바와 같이, 본 발명의 자소자 추출물의 ALP 활성을 측정한 결과, 자소자 추출물은 농도 의존적이고 유의적으로 ALP 활성이 증가하였으며 자소자 열수 추출물 100㎍/㎖ 농도에서 약 70% 이상의 ALP 활성 증가효과를 나타내었다. ALP는 조골세포의 분화에 발현되는 표식인자(Biomarker)이므로 상기 결과를 통해 본 발명의 자소자 추출물이 조골세포의 분화를 촉진시킴을 알 수 있었다.
As can be seen from FIGS. 4 and 5, the ALP activities of the self-extracts of the present invention were determined to be concentration-dependent and significantly increased in ALP activity, Showed about 70% or more increase in ALP activity. ALP is a biomarker expressed in the differentiation of osteoblasts. Thus, it can be seen that the self-extract of the present invention promotes differentiation of osteoblasts.
<시험예 3>≪ Test Example 3 >
알리자린-레드(Alizarin-red) 염색법에 의한 자소자 추출물의 조골세포의 골석회화 형성도 측정Measurement of bone calcification formation of osteoblast-derived cell extracts by Alizarin-red staining
골 석회화 형성능은 조골세포의 분화에 중요한 표식인자로서, 알리자린은 무기질화된 세포의 기질에 염색되므로 석회화된 양과 염색 정도가 상호 비례한다.
Bone calcification ability is an important marker for osteoblast differentiation, and alizarin is stained in the matrix of mineralized cells, so that the amount of calcified and the degree of staining are proportional to each other.
상기 시험예 1-1의 마우스 두개골 골아세포를 24 웰 플레이트(Well Plate)에 5X104 세포/well 농도로 분주한 후, 37℃, 5% CO2 인큐베이터에서 24시간 배양 후, 배지를 제거하였다.The mouse skull osteoblast cells of Test Example 1-1 were divided into well plates at a concentration of 5 × 10 4 cells / well and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours. Then, the medium was removed.
석회화 유도를 위해 50㎍/㎖ 아스코르브산(L-ascorbic acid), 10mM β-글리세로 포스페이트(β-glycero phosphate)가 첨가된 배지에 상기 실시예 1의 자소자 열수 추출물 시료를 각각 100, 50, 25, 12.5㎍/㎖의 농도로 녹인 후 상기 준비한 각 웰(Well)에 분주하고 14일까지 인큐베이터에서 배양하였다. 배양 후, 배지를 제거하고 PBS로 세척한 뒤 포르말린으로 고정시켰다.For the induction of calcification, samples of the hot-water extracts of the magnetic element of Example 1 were added to the medium to which 50 μg / ml of ascorbic acid and 10 mM of β-glycerophosphate were added, 25, and 12.5 μg / ml, and the cells were dispensed into each of the prepared wells and cultured in an incubator until 14 days. After incubation, the medium was removed, washed with PBS and fixed with formalin.
알리자린 레드(AR) 용액은 알리자린 레드(Sigma)를 증류수에 녹여 40mM로 농도를 맞추고 pH4.2로 조정하였다. 세포를 고정시킨 후, AR 용액(0.5㎖/well)으로 10분간 염색하고 증류수로 5번 세척한 뒤 염색되지 않은 부분은 PBS로 씻어주면서 제거하고 표면이 너무 마르지 않게 PBS로 조금 적셔주면서 현미경으로 석회화 형성 정도를 관찰하였다.The alizarin red (AR) solution was prepared by dissolving alizarin red (Sigma) in distilled water, adjusting the concentration to 40 mM and adjusting the pH to 4.2. After staining the cells, the cells were stained with AR solution (0.5 ml / well) for 10 minutes, washed 5 times with distilled water, and the unstained portions were washed with PBS and removed with a little wetness with PBS. The degree of formation was observed.
석회화 형성 확인 후 10% 세틸피리니디움 클로라이드(Cetylpyridinium chloride)를 첨가한 10mM 소디움 포스페이트(Sodium phosphate)(pH 7.0) 용액을 웰(Well)에 0.5㎖씩 첨가하여 15분간 녹이고 염색된 정도를 마이크로플레이트 리더를 이용하여 562nm에서 흡광도를 측정하였고, 대조군의 흡광도를 기준으로 하여 골 석회화 형성능을 계산하였다. 대조군은 본 발명의 자소자 추출물을 첨가하지 않은 것을 사용하였다.After confirming the formation of calcification, 0.5 ml of a 10 mM sodium phosphate (pH 7.0) solution to which 10% cetylpyridinium chloride was added was added to each well and the mixture was dissolved for 15 minutes. Absorbance was measured at 562 nm using a reader and the bone formation capacity was calculated based on the absorbance of the control group. As the control, those without the self-extract of the present invention were used.
그 결과를 도 6 및 도 7에 나타내었다.
The results are shown in Fig. 6 and Fig.
도 6 및 도 7에서 확인할 수 있는 바와 같이, 자소자 추출물의 농도 의존적으로 조골세포의 석회화 증가 효과를 뚜렷하게 관찰할 수 있었다. 따라서 본 발명의 자소자 추출물은 조골세포의 분화를 촉진하고, 석회화 형성능이 있음을 알 수 있다.
As can be seen from FIGS. 6 and 7, the effect of increasing the calcification of the osteoblasts was observed in a concentration-dependent manner. Therefore, it can be seen that the magnetic element extract of the present invention promotes differentiation of osteoblasts and has calcification-forming ability.
<시험예 4><Test Example 4>
자소자 추출물에 의한 파골세포 분화 억제 측정Inhibition of osteoclast differentiation by cell extract
본 발명의 자소자 추출물이 파골세포의 성장 및 활성에 어떠한 영향을 미치는지 알아보기 위하여 파골세포 전구세포를 배양하여 파골세포의 표지효소인 TRAP(Tartrate resistance acid phosphatase) 염색 및 활성을 분석하였다.To examine the effect of the extract of the present invention on the growth and activity of osteoclast, osteoclast precursor cells were cultured and their expression and activity of TRAP (Tartrate resistance acid phosphatase), which is a labeling enzyme of osteoclast, were analyzed.
TRAP 효소는 파골세포가 골 흡수작용을 할 때 효소의 분비가 증가되고 ATP, 니트로페닐 포스페이트(Nitrophenyl phosphate)가 존재할 때 높은 활성을 가지는 효소로서 파골분화 정도를 측정할 수 있는 파골세포의 세포 화학적 표지효소(marker enzyme)이다.The TRAP enzyme is an enzyme with high activity when the osteoclast is osteoclastic and the enzyme secretion is increased and ATP, Nitrophenyl phosphate is present. The osteoclast cell cytochemical mark It is a marker enzyme.
한편, 파골세포는 골 내막에 위치하며 골 조직에 존재하는 유일한 다핵세포로 RANKL에 의해 분화 초기에는 단핵의 전 파골세포를 형성하지만 세포가 융합되어 다핵의 성숙 파골세포로 분화되면 골 표면에 흡착하여 주름막을 형성하는 특징을 가진다.
On the other hand, osteoclasts are located in the lining of the bone and are the only polynuclear cells present in the bone tissue. RANKL forms osteoclast cells at the early stage of differentiation. However, when cells are fused and differentiated into polynuclear mature osteoclasts, And has a feature of forming a wrinkle film.
4-1. 자소자 열수 추출물에 의한 RANKL을 이용한 성숙한 파골세포로의 분화 억제 측정 4-1. Inhibition of differentiation into mature osteoclasts using RANKL by hydrothermal extract
파골세포의 전구세포를 96 웰 플레이트(Well Plate)에 웰(Well)당 1X105개의 세포가 되도록 분주하였다.The osteoclast precursor cells were dispensed into 96 well plates (Well Plate) to give 1 x 10 5 cells per well.
이와는 별도로 30ng/㎖ 대식세포집락자극인자(Macrophage-colony stimulating factor; M-CSF, sigma)와 100ng/㎖ RANKL(Sigma)이 포함된 α-MEM에 상기 실시예 1의 자소자 열수추출물 시료를 각각 100, 50, 25, 12.5, 6.25, 3.12㎍/㎖의 농도로 녹인 후 상기 준비한 각 웰(Well)에 분주하여 5일간 배양하였다. 배양이 끝난 후 파골세포의 생성을 검사하기 위하여 세포를 고정하여 TRAP 염색을 시행하였다.Separately, samples of the hot-water extract of the ruled element of Example 1 were added to α-MEM containing 30ng / ml Macrophage-colony stimulating factor (M-CSF, sigma) and 100ng / ml RANKL (Sigma) 100, 50, 25, 12.5, 6.25, and 3.12 占 퐂 / ml, and the wells were divided into the wells and cultured for 5 days. After cultivation, cells were fixed and TRAP staining was performed to examine osteoclast formation.
구체적으로는 10% 포름알데히드로 고정한 상기 세포에 기질 나프톨 AS-MX 포스페이트(naphtol AS-MX phosphate, sigma) 5㎎ 및 색소(Fast Red Violet LB salt) 25㎎을 N,N-디메틸포름아미드(N,N-dimethylformamide) 0.5㎖에 녹인 용액을 50mM 타르타르산(Tartaric acid)을 포함하는 0.1N NaHCO3 완충액 50㎖(pH 5.0)에 첨가한 TRAP 염색액을 70㎕씩 분주하고 현미경으로 관찰하였다. 대조군(Control)은 본 발명의 자소자 추출물을 첨가하지 않은 것을 사용하였다.Specifically, 5 mg of substrate naphthol AS-MX phosphate (Sigma) and 25 mg of a pigment (Fast Red Violet LB salt) were dissolved in N, N-dimethylformamide (N , N-dimethylformamide) was added to 50 ml (pH 5.0) of 0.1N NaHCO 3 buffer containing 50 mM Tartaric acid, and 70 μl of the TRAP staining solution was dispensed and observed under a microscope. As a control (Control), those without the self-extract of the present invention were used.
그 결과를 도 8에 나타내었다.
The results are shown in Fig.
4-2. 자소자 70% 에탄올 추출물에 의한 RANKL을 이용한 성숙한 파골세포로의 분화 억제 측정 4-2. Measurement of inhibition of mature osteoclast differentiation using RANKL by ethanol extract of 70% ethanol
자소자 70% 에탄올추출물을 사용한 것을 제외하고는 상기 실시예 4-1의 RANKL을 이용한 성숙한 파골세포로의 분화억제 측정과 동일한 방법으로 측정하였다.The measurement was carried out in the same manner as in the measurement of inhibition of differentiation into mature osteoclasts using the RANKL of Example 4-1, except that a 70% ethanol extract was used.
그 결과를 도 9에 나타내었다.
The results are shown in Fig.
도 8 및 도 9에서 확인할 수 있는 바와 같이, 파골세포의 직접적인 분화인자인 RANKL을 처리한 대조군에 비하여 자소자 열수추출물 50, 100㎍/㎖을 처리한 경우 분화 억제 효과를 확인할 수 있었고, 자소자 70% 에탄올 추출물은 12.5, 25, 50, 100㎍/㎖을 처리한 경우 농도의존적인 분화 억제 효과를 확인할 수 있었다.
As can be seen from FIGS. 8 and 9, the inhibition effect of differentiation inhibition was confirmed when 50 or 100 μg / ml of hot-water extract of magnetic element was treated, compared with the control group treated with RANKL, which is a direct differentiation factor of osteoclast, The 70% ethanol extract showed 12.5, 25, 50, and 100 μg / ㎖ of inhibitory effect on the concentration-dependent differentiation.
4-3. 자소자 열수 추출물에 의한 RANKL 유도 파골세포 분화 억제된 거대 다핵 파골세포 수 측정 4-3. RANKL-Induced Osteoclast Differentiation Suppressed by Massive Hydrothermal Extract
상기 시험예 4-1의 TRAP 염색 결과, TRAP에 양성(+)이며, 현미경 관찰 시 다핵(3개 이상의 핵)을 갖는 TRAP(+) 다핵세포들을 파골세포로 보아 TRAP(+) 다핵세포수를 육안으로 세어 측정하였다. 정확한 측정을 위하여 각 웰을 4등분하여 계수하여 측정하였다.As a result of TRAP staining of Test Example 4-1, TRAP (+) polynuclear cells positive to TRAP (+) and having a polynuclear (more than 3 nuclei) And then counted by naked eyes. For accurate measurement, each well was divided into quarters and counted.
그 결과를 도 10에 나타내었다.
The results are shown in Fig.
4-4. 자소자 70% 에탄올 추출물에 의한 RANKL 유도 파골세포 분화 억제된 거대 다핵 파골세포 수 측정 4-4. RANKL-induced osteoclast differentiation inhibited by large-sized 70% ethanol extract
상기 시험예 4-2의 TRAP 염색 결과, TRAP에 양성(+)이며, 현미경 관찰 시 다핵(3개 이상의 핵)을 갖는 TRAP(+) 다핵세포들을 파골세포로 보아 TRAP(+) 다핵세포수를 육안으로 세어 측정하였다. 정확한 측정을 위하여 각 웰을 4등분하여 계수하여 측정하였다.As a result of TRAP staining of Test Example 4-2, TRAP (+) polynuclear cells positive to TRAP (+) and having polynuclear (more than 3 nuclei) And then counted by naked eyes. For accurate measurement, each well was divided into quarters and counted.
그 결과를 도 11에 나타내었다.
The results are shown in Fig.
도 10 및 도 11에서 확인할 수 있는 바와 같이, 파골세포의 직접적인 분화인자인 RANKL을 처리한 대조군에 비하여 자소자 열수추출물 50, 100㎍/㎖을 처리한 경우와 자소자의 70% 에탄올 추출물 12.5, 25, 50, 100㎍/㎖를 처리한 경우에 파골세포의 수가 감소한 것을 확인할 수 있었다.10 and 11, compared with the control group treated with RANKL, which is a direct differentiation factor of osteoclast, the case of treatment with
Claims (7)
A pharmaceutical composition for the prevention and treatment of osteoporosis by promoting differentiation of osteoblasts and inhibiting osteoclast differentiation containing hot water extracts of the present invention as active ingredients.
A composition for improving osteoporosis by promoting differentiation of osteoblasts and inhibiting osteoclast differentiation containing hot water extract of eggplant component as an active ingredient.
상기 식품 조성물은 발효유, 기능성 음료, 건강기능식품 중 어느 하나인 것을 특징으로 하는 자소자 열수추출물을 유효성분으로 함유하는 조골세포의 분화촉진 및 파골세포의 분화억제 활성에 의한 골다공증 개선용 식품 조성물.The method according to claim 6,
Wherein the food composition is any one of fermented milk, functional beverage, and health functional food. The food composition for improving osteoporosis by promoting differentiation of osteoblasts and inhibiting osteoclast differentiation containing the hydrothermal extract as an active ingredient.
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KR102207872B1 (en) * | 2019-08-30 | 2021-01-26 | 국민대학교산학협력단 | Oral composition for preventation or treatment of oral disease |
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KR20030006736A (en) * | 2001-07-14 | 2003-01-23 | (주) 메드빌 | Composition for preventing or treating osteoporosis comprising plant extracts and method of preparing the same |
KR20030031418A (en) * | 2001-10-10 | 2003-04-21 | 한국 한의학 연구원 | Extract of herbal mixture and pharmaceutical compositions for prevention or treatment of osteoporosis |
KR100595735B1 (en) * | 2005-07-08 | 2006-06-30 | 주식회사 케이엠에스아이 | Herbal pharmaceutical composition for regenerative agent of cartilaginous tissue and treatment of osteoarthritis |
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KR20030006736A (en) * | 2001-07-14 | 2003-01-23 | (주) 메드빌 | Composition for preventing or treating osteoporosis comprising plant extracts and method of preparing the same |
KR20030031418A (en) * | 2001-10-10 | 2003-04-21 | 한국 한의학 연구원 | Extract of herbal mixture and pharmaceutical compositions for prevention or treatment of osteoporosis |
KR100595735B1 (en) * | 2005-07-08 | 2006-06-30 | 주식회사 케이엠에스아이 | Herbal pharmaceutical composition for regenerative agent of cartilaginous tissue and treatment of osteoarthritis |
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Title |
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임남경 외., ‘한약재 추출물의 조골세포 분화 및 파골세포 형성에 미치는 영향’, 한국식품과학회지 Vol.42, No.5, kpp. 637-642, 2010* |
임남경 외., '한약재 추출물의 조골세포 분화 및 파골세포 형성에 미치는 영향', 한국식품과학회지 Vol.42, No.5, kpp. 637-642, 2010 * |
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