KR101332074B1 - Composition Comprising Esculetin for Inhibition of Bone Loss - Google Patents
Composition Comprising Esculetin for Inhibition of Bone Loss Download PDFInfo
- Publication number
- KR101332074B1 KR101332074B1 KR1020120076532A KR20120076532A KR101332074B1 KR 101332074 B1 KR101332074 B1 KR 101332074B1 KR 1020120076532 A KR1020120076532 A KR 1020120076532A KR 20120076532 A KR20120076532 A KR 20120076532A KR 101332074 B1 KR101332074 B1 KR 101332074B1
- Authority
- KR
- South Korea
- Prior art keywords
- present
- inhibiting
- bone
- bone loss
- esculletin
- Prior art date
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
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- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
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- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
Abstract
본 발명은 에스큘레틴을 유효성분으로 함유하는 골 손실 억제용 조성물에 관한 것이다. 본 발명에 따른 에스큘레틴은 RANKL의 자극에 인해 유발되는 파골세포 형성을 억제하는 효과가 있으며, 이렇게 파골세포 형성을 억제시킴으로써 궁극적으로 파골세포에 의한 골 흡수를 억제하는 효과를 갖게 된다. 더욱 자세하게는, 본 발명에 따른 에스큘레틴은 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT의 발현을 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 갖는다. 따라서 상기와 같은 효과를 갖는 에스큘레틴을 유효성분으로 포함하는 본 발명의 조성물은 골 손실로 일어나는 골대사 질환 예방 또는 치료에서 유용하게 사용될 수 있으며, 특히 골 손실에 따른 골다공증 개선 및 치료하기 위한 용도로 널리 사용할 수 있다.The present invention relates to a composition for inhibiting bone loss containing esculin as an active ingredient. Esculletin according to the present invention has the effect of inhibiting the osteoclast formation caused by the stimulation of RANKL, thereby suppressing the osteoclast formation will ultimately have the effect of inhibiting bone resorption by the osteoclasts. More specifically, esculletin according to the present invention, osteoclasts through the mechanism of inhibiting the expression of NFAT, a key transcription factor that can regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation Has the effect of suppressing differentiation. Therefore, the composition of the present invention containing an esculinine having the above effects as an active ingredient may be usefully used in the prevention or treatment of bone metabolic diseases caused by bone loss, and particularly for the purpose of improving and treating osteoporosis due to bone loss. It can be widely used.
Description
본 발명은 골 손실을 효과적으로 개선시킬 수 있는 조성물에 관한 것으로서, 에스큘레틴(esculetin)을 유효성분으로 포함하는 골 손실 억제용 조성물에 관한 것이다.The present invention relates to a composition capable of effectively improving bone loss, and relates to a composition for inhibiting bone loss, including esculetin as an active ingredient.
뼈(bone)는 인체의 연조직과 체중을 지탱해주고 내부기관을 둘러싸서 내부 장기를 외부의 충격으로부터 보호한다. 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 뼈의 재형성(bone remodeling)이라고 한다. 이러한 뼈의 재형성은 성장과 스트레스에 의해서 일어나는 뼈의 미세한 손상을 회복시키고 적절히 뼈의 기능을 유지하는데 필수적이다.Bones support the soft tissues and weight of the body and surround internal organs to protect internal organs from external shocks. It is also an important part of the body that not only supports the muscles or organs but also stores calcium or other essential minerals, such as phosphorus or magnesium, in the body. Therefore, the bones of grown adults do not stop, and until the day of their death, the bones of the old bones are removed and replaced with new bones. This is called bone remodeling. This bone remodeling is essential to repair the bone's microscopic damage caused by growth and stress and to maintain proper bone function.
뼈의 재형성에는 크게 두 종류의 세포가 관여하는 것으로 알려져 있다. 두 세포 중 하나는 뼈를 생성하는 조골세포(osteoblast)이고, 다른 하나는 뼈를 파괴하는 파골세포(osteoclast)이다. 조골세포는 RANKL(receptor activator of NF-κB ligand: 이하 간략하게 ‘RANKL’이라 함)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: 이하 간략하게 ‘RANK'라 함)에 결합하면 파골 전구세포가 파골세포로 분화되어 골 흡수(resorption)가 일어난다. 자세하게는 RANKL 자극에 의한 세포내 칼슘 신호 전달로 파골 전구세포에서 파골세포의 분화를 조절하는 핵심 전사인자로 알려져 있는 NFAT(nuclear factor of activated T cells: 이하 간단하게 ‘NFAT'라 함)의 발현이 유도되고, 그 후 세포외 신호조절인산화효소(extracellular signal-regulated kinase: 이하 간단하게 ‘ERK'라 함), NF-κB, p38 MAP kinase 등의 활성화가 NFAT의 자가증폭(autoamplification)을 유발하여 활성이 지속되는 것이다. Two types of cells are known to be involved in bone remodeling. One of the two cells is an osteoblast that produces bone, and the other is an osteoblast that destroys bone. Osteoblasts produce RANKL (receptor activator of NF-κB ligand (hereinafter simply referred to as "RANKL") and its degenerate receptor, OPG (osteoprotegerin). When RANKL binds to the receptor activator of NF-κB (hereinafter referred to simply as 'RANK'), a receptor on the surface of osteoclast progenitor cells, osteoclast progenitor cells differentiate into osteoclasts, resulting in bone resorption. Happens. Specifically, expression of nuclear factor of activated T cells (hereinafter simply referred to as 'NFAT'), a key transcription factor that regulates osteoclast differentiation in osteoclast progenitor cells by intracellular calcium signaling by RANKL stimulation. After activation, activation of extracellular signal-regulated kinase (hereinafter simply referred to as 'ERK'), NF-κB, and p38 MAP kinase triggers autoamplification of NFAT. This will last.
따라서 오래된 뼈의 흡수 또는 파괴는 상기와 같은 기작을 통해 파골세포에 의해 이루어지며, 이는 뼈에 구멍을 내어 적은 양의 칼슘이 혈류로 방출되어 신체기능을 유지하는데 사용된다(William J. et al., NATURE., 423, pp.337342, 2033). 파골세포는 실제적인 뼈의 흡수작용을 할 때는 다핵세포로 융합된 후 그 구조가 변하여 주름진 테두리를 형성하며, 골 기질 표면에 흡착하여 산성화 환경에서 카텝신(cathepsin) K 나 H+-ATPase 등 많은 효소의 작용으로 뼈를 흡수한다. 이에 반해, 조골세포는 교원질로 구멍을 채우고 칼슘과 인의 침적물(hydroxyapatite)을 덮어서 단단한 새로운 뼈를 만들어 골격을 재건하게 된다.Thus, the absorption or destruction of old bone is carried out by the osteoclast through the above mechanism, which is used to keep the body function by piercing the bone and releasing a small amount of calcium into the bloodstream (William J. et al. , NATURE., 423, pp. 3737342, 2033). When osteoclasts are actually absorbed by bone, they are fused to polynuclear cells, and their structures are changed to form a corrugated border. They are adsorbed on the surface of bone matrix, and in the acidified environment, many enzymes such as cathepsin K and H + -ATPase To absorb the bone. Osteoblasts, on the other hand, fill the pores with collagen and cover the deposits of calcium and phosphorus (hydroxyapatite) to form new bones that rebuild the skeleton.
성인의 경우 매년 골격의 약 10-30%가 이런 과정을 통하여 재형성되며, 파골속도와 조골속도가 동일해야만 전과 같은 골밀도를 유지할 수 있다. 정상 성인에서는 골흡수 양과 골형성 양 사이에는 항상 균형이 유지되고 있다. 이와 같이 중요한 뼈에 균형이 깨질 경우 많은 질병이 야기될 수 있는데, 이러한 질환을 하기에서는 ‘골대사 질환’이라 하였다.In adults, about 10-30% of the skeleton is remodeled through this process every year, and the bone density is the same as before when the osteoclast and osteoblast speeds are the same. There is always a balance between bone resorption and bone formation in normal adults. When the balance of such important bones can be broken, many diseases can be caused. Such a disease is referred to as 'bone metabolism disease' in the following.
골대사 질환 중 고령사회에서 큰 문제로 대두되고 있는 골다공증은 골의 화학적 조성에는 큰 변화 없이 단위 용적내의 골량이 감소하여 경미한 충격에도 쉽게 골절을 일으킬 수 있는 질환이다. 노인 특히 폐경 후 여성들의 경우 에스트로겐의 분비부족으로 인하여 가장 그 발생빈도가 높게 나타난다고 알려져 있다. 현재 에스트로겐이나 천연에 존재하면서 약한 에스트로겐 활성을 나타내는 식물 유래의 피토에스트로겐이 폐경 후 골다공증 예방에 매우 유용하다는 연구가 보고되었다. 그러나 이러한 방법은 폐경성 골다공증 이외의 원인에는 제한적으로 사용된다는 한계점을 가진다. 이 밖에 칼슘염, 우골분 등의 칼슘 보강제, 비타민 D 대사산물 요법 등이 비 폐경성 골다공증 치료에 이용되고 있으나, 이들은 용해성이나 흡수성, 미각적 측면에서 문제점을 가지고 있다. 또한 약물 투여에는 비스포스포네이트 (bisphosphonates)나 칼시토닌 제제 등이 사용되며 골다공증의 치료에 유효하다고 알려져 있으나, 이러한 약물은 전문의약품이고, 식도에 협착될 수 있는 등의 부작용이 있어 복용에 매우 주의를 요하며, 이명, 두통, 식욕 부진 등의 부작용도 나타낼 수 있다고 보고된 바 있다. Osteoporosis, which has emerged as a major problem in the elderly society among bone metabolic diseases, is a disease that can easily cause fractures even with a slight impact due to a decrease in the amount of bone in the unit volume without a great change in the chemical composition of the bone. The elderly, especially postmenopausal women, are known to have the highest incidence due to lack of estrogen. Currently, research has been reported that phytoestrogens derived from estrogens or plants present in nature and exhibiting weak estrogen activity are very useful for preventing postmenopausal osteoporosis. However, this method has a limitation that it is used for a cause other than menopausal osteoporosis. In addition, calcium adjuvant such as calcium salt, right bone meal, and vitamin D metabolite therapy are used for the treatment of non-menopausal osteoporosis, but they have problems in terms of solubility, absorption, and taste. In addition, bisphosphonates or calcitonin preparations are used for drug administration and are known to be effective in the treatment of osteoporosis. However, these drugs are specialty medicines and have serious side effects such as narrowing to the esophagus. It has also been reported to have side effects such as tinnitus, headache, and loss of appetite.
한편 매실, 레몬, 인진쑥 등을 포함한 다양한 식물에 많이 함유되어 있는 쿠마린(coumarin) 유도체인 에스큘레틴(6,7-dihydroxycoumarin)은 혈소판응집과 잔틴 옥시데이스(Xanthin oxidase) 활성 억제, N-메틸-N-니트로소유레아(N-methyl-N-nitrosourea)에 의해 의하여 유도된 유방암 및 benzo[a]pyrene plus 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone에 의하여 유도된 폐암의 억제효과를 가지는 등 약리학 및 생화학적 활성이 뛰어난 것으로 보고되어지고 있다. 그러나 이러한 에스큘레틴 화합물이 골 손실과 관련한 질환 치료에 효과를 가진다는 내용에 대해서는 전혀 보고된 바가 없다. On the other hand, esculin (6,7-dihydroxycoumarin), a coumarin derivative that is found in various plants including plum, lemon, and ginko wormwood, inhibits platelet aggregation and xanthin oxidase activity, N-methyl- Of breast cancer induced by N-methyl-N-nitrosourea and lung cancer induced by benzo [a] pyrene plus 4- (methylnitrosoamino) -1- (3-pyridyl) -1-butanone It has been reported to have excellent pharmacological and biochemical activity, such as having an inhibitory effect. However, there have been no reports about the effects of these esculletin compounds in treating diseases related to bone loss.
이에 본 발명자들은 에스큘레틴 화합물이 골 손실을 억제함으로써 골 손실에 따른 질환 치료에 효과가 있는지 연구하던 중, 본 화합물이 RANKL에 의한 파골세포 분화를 억제하는 효과가 있음을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors completed the present invention by confirming that the esculin compound is effective in treating diseases caused by bone loss by inhibiting bone loss, and confirming that the compound has an effect of inhibiting osteoclast differentiation by RANKL. Was done.
따라서 본 발명의 목적은 파골세포 형성 억제효과가 우수한 에스큘레틴 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 억제용 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a composition for inhibiting bone loss, which contains an esculin compound having excellent osteoclast formation inhibitory effect or a salt thereof as an active ingredient.
또한, 본 발명의 다른 목적은 파골세포 형성 억제효과가 우수한 에스큘레틴 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 개선용 건강기능식품을 제공하는 것이다.Another object of the present invention is to provide a health functional food for improving bone loss containing an esculin compound having excellent osteoclast formation inhibitory effect or a salt thereof as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 에스큘레틴(esculetin) 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 억제용 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a composition for inhibiting bone loss containing an esculetin compound or a salt thereof as an active ingredient.
본 발명의 일실시예에 있어서, 상기 화합물은 파골세포 형성 억제 효능을 가질 수 있다.In one embodiment of the present invention, the compound may have an inhibitory effect on osteoclast formation.
본 발명의 일실시예에 있어서, 상기 파골세포 형성 억제는 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT의 발현을 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 가질 수 있다.In one embodiment of the present invention, the inhibition of osteoclast formation may have the effect of inhibiting the differentiation of osteoclasts through a mechanism of inhibiting the expression of NFAT, a key transcription factor that regulates the differentiation of osteoclasts.
본 발명의 일실시예에 있어서, 상기 골 손실 억제를 통해 치료할 수 있는 질환은 골다공증, 파제트 골질환, 구루병, 골연화증 및 퇴행성 골질환으로 이루어진 군으로부터 선택된 1종일 수 있다.In one embodiment of the present invention, the disease that can be treated by inhibiting bone loss may be one selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia and degenerative bone disease.
또한, 본 발명은 에스큘레틴(esculetin) 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 개선용 건강기능식품을 제공한다.In another aspect, the present invention provides a dietary supplement for improving bone loss containing an esculetin compound or a salt thereof as an active ingredient.
본 발명의 일실시예에 있어서, 상기 화합물은 파골세포 형성 억제 효능을 가질 수 있다.In one embodiment of the present invention, the compound may have an inhibitory effect on osteoclast formation.
본 발명의 일실시예에 있어서, 상기 식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류로 이루어진 군으로부터 선택된 1종일 수 있다.In one embodiment of the invention, the food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements It may be one kind.
본 발명에 따른 에스큘레틴은 RANKL의 자극에 인해 유발되는 파골세포 형성을 억제하는 효과가 있으며, 이렇게 파골세포 형성을 억제시킴으로써 궁극적으로 파골세포에 의한 골 흡수를 억제하는 효과를 갖게 된다. 더욱 자세하게는, 본 발명에 따른 에스큘레틴은 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT의 발현을 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 갖는다. 따라서 상기와 같은 효과를 갖는 에스큘레틴을 유효성분으로 포함하는 본 발명의 조성물은 골 손실로 일어나는 골대사 질환 예방 또는 치료에서 유용하게 사용될 수 있으며, 특히 골 손실에 따른 골다공증 개선 및 치료하기 위한 용도로 널리 사용할 수 있다.Esculletin according to the present invention has the effect of inhibiting the osteoclast formation caused by the stimulation of RANKL, thereby suppressing the osteoclast formation will ultimately have the effect of inhibiting bone resorption by the osteoclasts. More specifically, esculletin according to the present invention, osteoclasts through the mechanism of inhibiting the expression of NFAT, a key transcription factor that can regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation Has the effect of suppressing differentiation. Therefore, the composition of the present invention containing an esculinine having the above effects as an active ingredient may be usefully used in the prevention or treatment of bone metabolic diseases caused by bone loss, and particularly for the purpose of improving and treating osteoporosis due to bone loss. It can be widely used.
도 1은 본 발명의 에스큘레틴 화합물의 처리에 따른 BMM 세포 생성 억제(파골세포 형성 억제)정도를 TRAP 염색을 통해 나타낸 사진이다.
도 2는 본 발명의 에스큘레틴 화합물의 처리에 따른 BMM 세포 생성 억제(파골세포 형성 억제)정도를 TRAP 활성을 측정하여 그래프로 나타낸 것이다.
도 3은 본 발명의 에스큘레틴 화합물의 처리에 따른 세포 생존률을 측정하여 나타낸 그래프이다.
도 4는 본 발명의 에스큘레틴 화합물의 처리에 따른 파골세포 특이적 마커 유전자(CathepsinK, MMP9, TRAP, V-ATPase, C-Fos, NEATc1, c-SRC)의 발현량을 측정하기 위하여 역전사-중합효소연쇄반응법(RT-PCR)을 수행한 결과이다.
도 5는 본 발명의 에스큘레틴 화합물의 처리에 따른 파골세포 발생의 전사 인자의 발현에 미치는 영향을 나타내는 도면이다.
도 6은 본 발명의 에스큘레틴 화합물 처리에 따른 파골세포 발생의 신호 분자의 활성화에 미치는 영향을 나타내는 도면이다.
도 7은 성숙한 파골세포의 뼈 흡수 활성에 미치는 본 발명의 에스큘레틴 화합물의 영향을 나타내는 도면이다.1 is a photograph showing the degree of inhibition of BMM cell production (inhibition of osteoclast formation) according to the treatment of the esculin compound of the present invention through TRAP staining.
Figure 2 is a graph showing the degree of inhibition of BMM cell production (inhibiting osteoclast formation) according to the treatment of the esculin compound of the present invention by measuring TRAP activity.
Figure 3 is a graph showing the measurement of cell viability according to the treatment of the esculletin compound of the present invention.
Figure 4 is a reverse transcription-in order to measure the expression level of osteoclast specific marker genes (CathepsinK, MMP9, TRAP, V-ATPase, C-Fos, NEATc1, c-SRC) according to the treatment of the esculin compound of the present invention This is the result of polymerase chain reaction (RT-PCR).
Figure 5 is a diagram showing the effect on the expression of transcription factors of osteoclast development according to the treatment of the esculletin compound of the present invention.
Figure 6 is a view showing the effect on the activation of the signaling molecules of osteoclasts generated by the treatment of the esculletin compound of the present invention.
7 is a diagram showing the effect of the esculletin compound of the present invention on the bone resorption activity of mature osteoclasts.
본 발명은 하기 화학식 1로 표시되는 에스큘레틴(esculetin) 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 억제용 조성물을 제공함에 그 특징으로 한다. The present invention is characterized by providing a composition for inhibiting bone loss containing an esculetin compound represented by the following formula (1) or a salt thereof as an active ingredient.
<화학식 1>≪ Formula 1 >
본 발명의 에스큘레틴 화합물은 매실, 레몬, 인진쑥 등을 포함한 다양한 식물에 많이 함유되어 있는 쿠마린(coumarin) 유도체로서, 6,7-Dihydroxycoumarin으로 명명되며, 이 물질의 분자식은 C9H6O4이고, 분자량은 178.14이다. The esculletin compound of the present invention is a coumarin derivative contained in a variety of plants, including plum, lemon, zinnia, etc., is named 6,7-Dihydroxycoumarin, the molecular formula of the material is C 9 H 6 O 4 And a molecular weight is 178.14.
본 발명에 따른 상기 에스큘레틴 화합물은 염, 바람직하게는 약학적으로 허용 가능한 염의 형태로 사용될 수 있는데, 상기 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의하여 형성된 산 부가염이 바람직하며, 상기 유리산으로는 유기산과 무기산을 사용할 수 있다. 상기 유기산은 이에 제한되는 것은 아니나, 구연산, 초산, 젖산, 주석산, 말레인산, 푸마르산, 포름산, 프로피온산, 옥살산, 트리플로오로아세트산, 벤조산, 글루콘산, 메타술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 글루탐산 및 아스파르트산을 포함한다. 또한 상기 무기산은 이에 제한되는 것은 아니나, 염산, 브롬산, 황산 및 인산을 포함한다. The esculletin compound according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt, wherein the salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.
또한, 본 발명에 따른 에스큘레틴 화합물은 천연으로부터 분리되거나 또는 당업계에 공지된 화학적 합성법으로 제조하여 사용할 수 있으며, 시중에서 판매되고 있는 에스큘레틴 화합물이라면 모두 사용 가능하다. In addition, the esculletin compound according to the present invention may be isolated from nature or manufactured and used by a chemical synthesis method known in the art, and may be used as long as it is a commercially available esculletin compound.
본 발명의 에스큘레틴 화합물을 천연으로부터 분리할 경우, 종래의 물질을 추출 및 분리하는 방법을 사용하여 에스큘레틴을 함유하고 있는 모든 종류의 식물로부터 수득할 수 있는데, 즉, 적절한 용매를 사용하여 식물의 추출물을 수득한 다음, 본 발명이 속하는 기술분야의 당업자에게 알려진 정제 방법을 이용하여 상기 추출물로부터 에스큘레틴을 정제할 수 있다. When the esculletin compound of the present invention is separated from nature, it can be obtained from all kinds of plants containing esculletin by using a method of extracting and isolating conventional substances, that is, using an appropriate solvent After obtaining an extract of the plant, it is possible to purify the esculletin from the extract using purification methods known to those skilled in the art.
또한, 상기 추출을 위한 적절한 용매로는 물 또는 유기용매를 사용할 수 있으며, 바람직하게는 정제수, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol), 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다. Examples of suitable solvents for the extraction include water or an organic solvent. Preferred examples of the solvent include purified water, methanol, ethanol, propanol, isopropanol, butanol, acetone it is possible to use various solvents such as acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane, Or may be used in combination.
또한, 상기 식물 추출물로부터 에스큘레틴 화합물의 분리 및 정제는 실리카겔(silica gel)이나 활성 알루미나(alumina)등의 각종 합성수지를 충진한 컬럼 크로마토그래피(column chromatography) 및 고속액체크로마토그래피(HPLC) 등을 단독으로 혹은 병행하여 사용할 수 있다. 그러나 유효성분의 추출 및 분리정제 방법은 반드시 상기한 방법에 한정되는 것은 아니다. In addition, the separation and purification of the esculin compound from the plant extracts include column chromatography and high-performance liquid chromatography (HPLC) filled with various synthetic resins such as silica gel or activated alumina. It may be used alone or in combination. However, the method of extracting and separating and purifying the active ingredient is not necessarily limited to the above-mentioned method.
본 발명의 에스큘레틴 화합물은 순수하게 분리·정제된 화합물의 형태로 사용될 수 있으며, 또한 이를 함유하는 추출물의 형태로도 사용될 수 있다. The esculletin compound of the present invention may be used in the form of a purely isolated and purified compound, and may also be used in the form of an extract containing the same.
본 발명의 하기 실시예에서는 Sigma-Aldrich에서 판매하고 있는 에스큘레틴 화합물을 구입하여 사용하였다.In the following examples of the present invention, an esculin compound sold by Sigma-Aldrich was purchased and used.
한편, 쿠마린(coumarin) 유도체인 에스큘레틴 화합물은 혈소판응집과 잔틴 옥시데이스(Xanthin oxidase) 활성 억제, N-메틸-N-니트로소유레아(N-methyl-N-nitrosourea)에 의해 의하여 유도된 유방암 및 benzo[a]pyrene plus 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone에 의하여 유도된 폐암의 억제효과를 가지 는 등 약리학 및 생화학적 활성이 뛰어난 것으로 알려져 있다. 그러나 이러한 에스큘레틴 화합물이 골 손실과 관련한 질환 치료에 효과를 가진다는 내용에 대해서는 전혀 보고된 바가 없다. Meanwhile, the coumarin derivative, the esculletin compound, is a breast cancer induced by platelet aggregation, inhibition of Xanthin oxidase activity, and N-methyl-N-nitrosourea. And benzo [a] pyrene plus 4- (methylnitrosoamino) -1- (3-pyridyl) -1-butanone, which is known to be excellent in pharmacological and biochemical activity. However, there have been no reports about the effects of these esculletin compounds in treating diseases related to bone loss.
본 발명에서는 상기 에스큘레틴 화합물이 우수한 파골세포의 형성 억제 활성을 가지고 있다는 사실을 확인함으로써, 파골세포의 형성에 의해 유발될 수 있는 골 손실 질환 개선 및 치료에 유용하다는 사실을 최초로 규명하였다.In the present invention, by confirming that the esculletin compound has an excellent inhibitory activity on the formation of osteoclasts, it was first identified that it is useful for improving and treating bone loss diseases that may be caused by the formation of osteoclasts.
보다 구체적으로, 본 발명에 따른 에스큘레틴 화합물은 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT의 발현을 억제하는 기전을 통하여 파골세포의 분화를 효과적으로 억제할 수 있는 특징이 있다.More specifically, the esculletin compound according to the present invention is characterized by effectively inhibiting the differentiation of osteoclasts through a mechanism of inhibiting the expression of NFAT, a key transcription factor that regulates the differentiation of osteoclasts.
하기 실시예<1-2>에서는 파골세포 분화인자인 RANKL를 마우스 골수세포에 처리하여 파골세포 형성을 유도하는 과정에서, 본 발명의 에스큘레틴 화합물을 첨가한 처리군의 경우 에스큘레틴 농도 의존적으로 파골세포 형성이 억제(BMM 세포 생성 억제)되는 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명자들은 에스큘레틴 화합물이 파골세포 형성을 억제하는 효과가 있음을 알 수 있었다.In the following Example <1-2> in the process of inducing osteoclast formation by treating RANKL, an osteoclast differentiation factor to mouse bone marrow cells, the concentration of esculletin in the treatment group to which the esculletin compound of the present invention was added It has been shown that osteoclast formation is inhibited (inhibition of BMM cell production). Therefore, the present inventors have found that the esculletin compound has an effect of inhibiting osteoclast formation.
또한, 하기 실시예<1-4>에서는 파골세포 분화인자인 RANKL를 마우스 골수세포에 처리하여 파골세포 분화를 유도하는 과정에서, 본 발명의 에스큘레틴 화합물을 첨가한 처리군의 경우 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT의 발현을 억제할 수 있는지 실험하였는데, 그 결과 본 발명의 에스큘레틴 화합물을 처리한 처리군에서 NFATc1의 발현량이 확실히 감소하는 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명자들은 에스큘레틴 화합물이 NFAT의 발현을 억제하는 기작을 가지며, 이러한 기작으로 파골세포 분화가 억제되는 것을 유추할 수 있었다.In addition, in the following Example <1-4> in the process of inducing osteoclast differentiation by treating RANKL which is an osteoclast differentiation factor to mouse bone marrow cells, the osteoclasts of the treated group to which the esculin compound of the present invention was added It was tested whether the expression of NFAT, a key transcription factor that regulates differentiation, could be suppressed. As a result, the expression level of NFATc1 was clearly reduced in the treatment group treated with the esculin compound of the present invention. Therefore, the present inventors could infer that the esculin compound has a mechanism of inhibiting the expression of NFAT, and the osteoclast differentiation is suppressed by this mechanism.
따라서 상기와 같은 특징을 갖는 에스큘레틴 화합물 또는 그의 염을 유효성분으로 포함하는 본 발명의 조성물은 파골세포의 형성(분화) 억제를 통하여 골 손실로 인해 발생되는 골대사 질환 예방 또는 치료에 유용하게 사용될 수 있다.Therefore, the composition of the present invention containing an esculin compound having a characteristic as described above or a salt thereof as an active ingredient can be usefully used for preventing or treating bone metabolic diseases caused by bone loss through inhibition of osteoclast formation (differentiation). Can be.
본 발명의 조성물로 예방 또는 치료할 수 있는 골대사 질환으로는 골다공증, 파제트 골질환, 구루병, 골연화증, 퇴행성 골질환등과 같은 질환일 수 있으나, 가장 바람직하게는 골 손실에 따른 골다공증일 수 있다.Bone metabolism diseases that can be prevented or treated with the composition of the present invention may be diseases such as osteoporosis, Paget's bone disease, rickets disease, osteomalacia, degenerative bone disease, etc., but most preferably osteoporosis due to bone loss.
그러므로 본 발명은 에스큘레틴을 유효성분으로 포함하는 골 손실 억제용 조성물을 제공할 수 있다.Therefore, the present invention can provide a composition for inhibiting bone loss, including esculin as an active ingredient.
본 발명에 따른 조성물은 약제학적 조성물이나 식품 조성물로 사용될 수 있다.The composition according to the present invention can be used as a pharmaceutical composition or a food composition.
본 발명의 에스큘레틴 화합물을 유효성분으로 포함하는 약제학적 조성물은 상기 유효성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다.A pharmaceutical composition comprising the esculintine compound of the present invention as an active ingredient may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include an excipient, a disintegrant, and a sweetener. , Binders, coatings, expanding agents, lubricants, lubricants, or flavoring agents may be used.
상기 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다. The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
상기 약제학적 조성물의 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효 성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 해당분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화 할 수 있다. The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
본 발명의 에스큘레틴 화합물을 유효성분으로 포함하는 식품 조성물은 상기 약제학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.The food composition comprising the esculletin compound of the present invention as an active ingredient may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .
본 발명의 식품 조성물은 유효성분인 에스큘레틴 화합물을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. The food composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, in addition to containing the esculletin compound, which is an active ingredient, as in general food compositions. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).
또한 상기 식품 조성물은 유효성분인 에스큘레틴 화합물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. In addition to the esculletin compound as an active ingredient, a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and Salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
본 발명의 유효성분인 에스큘레틴 화합물은 천연유래물질로서 독성 및 부작용은 거의 없으므로 골 손실로 인해 발생되는 골대사 질환 예방 또는 치료 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.As an active ingredient of the present invention, the esculletin compound is a natural derivative, and thus has little toxicity and side effects, so that it can be used safely for long-term administration for the purpose of preventing or treating bone metabolic diseases caused by bone loss.
본 발명은 또한 에스큘레틴 화합물 또는 그의 염을 유효성분으로 함유하는 골 손실 개선용 건강기능식품을 제공한다.The present invention also provides a dietary supplement for improving bone loss containing an esculin compound or a salt thereof as an active ingredient.
본 발명의 건강기능식품은 골강화 또는 골 손실 개선을 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.The health functional food of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of strengthening bones or improving bone loss.
본 발명에서 “건강기능식품”이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.
본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.
상기 “식품 첨가물 공전”에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다.Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.
예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분인 에스큘레틴 화합물(또는 그의 염)을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다.For example, a dietary supplement in a tablet form may be granulated in a conventional manner by mixing a mixture of an esculletin compound (or a salt thereof), an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives, The lubricant may be added by compression molding or the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent and the like as necessary.
캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분인 에스큘레틴 화합물(또는 그의 염)을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 에스큘레틴 화합물(또는 그의 염)을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Hard capsules of the health functional foods in the form of capsules may be prepared by filling a mixture of an esculin compound (or a salt thereof), which is an active ingredient of the present invention, with an additive, such as an excipient, in a conventional hard capsule. A mixture obtained by mixing an esculin compound (or a salt thereof) with an additive such as an excipient may be prepared by filling a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.
환 형태의 건강기능식품은 본 발명의 유효성분인 에스큘레틴 화합물(또는 그의 염)과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다.The health functional food in cyclic form may be prepared by molding a mixture of an esculin compound (or a salt thereof), an excipient, a binder, a disintegrant, and the like, which is an active ingredient of the present invention, and is prepared by a known method. It can therefore be stripped with sugar or other coatings, or it can be coated with a substance such as starch or talc.
과립 형태의 건강기능식품은 본 발명의 유효성분인 에스큘레틴 화합물과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The health functional food in the form of granules can be prepared by granulation of a mixture of an esculin compound, an excipient, a binder, a disintegrant, and the like, which is an active ingredient of the present invention, and may be flavored and copulated as necessary. It may contain an agent and the like.
본 발명의 에스큘레틴 화합물 또는 그의 염을 유효성분으로 포함하는 건강기능식품은 골 손실을 억제하므로 골 손실로 야기되는 골대사 질환 예방 또는 개선에 효과적이다. 본 발명의 골대사 질환은 골대사와 관련된 질환이면 이의 종류를 특별히 한정하는 것은 아니나, 본 발명의 일실시예에 따르면 골다공증, 파제트 골질환, 구루병, 골연화증, 퇴행성 골질환등과 같은 질환일 수 있으나, 가장 바람직하게는 골 손실에 따른 골다공증일 수 있다.The health functional food containing the esculintin compound of the present invention or a salt thereof as an active ingredient is effective in preventing or improving bone metabolic diseases caused by bone loss since it suppresses bone loss. If the bone metabolic disease of the present invention is a disease associated with bone metabolism is not particularly limited, but according to an embodiment of the present invention may be a disease such as osteoporosis, Paget's bone disease, rickets, osteomalacia, degenerative bone disease, etc. Most preferably, osteoporosis following bone loss.
상기 건강기능식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등일 수 있다.The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.
본 발명은 또한 골 손실 억제용 의약 또는 식품의 제조를 위한 에스큘레틴을 유효성분으로 포함하는 조성물의 용도를 제공한다. 상기한 에스큘레틴을 유효성분으로 포함하는 본 발명의 조성물은 골 손실과 관련된 질환의 예방 또는 치료용 의약 또는 식품의 제조를 위한 용도로 이용될 수 있다.The present invention also provides the use of a composition comprising esculintin as an active ingredient for the manufacture of a medicament or food for inhibiting bone loss. The composition of the present invention comprising the above esculletin as an active ingredient can be used for the manufacture of a medicament or food for the prevention or treatment of diseases related to bone loss.
또한 본 발명은 포유동물에게 에스큘레틴 화합물을 투여하는 것을 포함하는 골 손실에 의한 골대사 질환 예방 또는 치료방법을 제공한다.The present invention also provides a method for preventing or treating bone metabolic diseases caused by bone loss, comprising administering an esculin compound to a mammal.
여기에서 사용된 용어 "포유동물"은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다. The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.
여기에서 사용된 용어 "치료상 유효량"은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. 본 발명의 유효 성분에 대한 치료상 유효 투여량 및 투여횟수는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 본 발명의 치료방법에 있어서, 성인의 경우, 본 발명의 에스큘레틴 화합물을 1일 1회 내지 수회 투여시, 1㎎/kg~250㎎/kg의 용량으로 투여하는 것이 바람직하다. The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the esculletin compound of the present invention at a dose of 1 mg / kg to 250 mg / kg once or several times a day.
본 발명의 치료방법에서 본 발명의 에스큘레틴 화합물을 유효성분으로 포함하는 조성물은 경구, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.
In the treatment method of the present invention, the composition comprising the esculletin compound of the present invention as an active ingredient may be administered by oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. Can be administered in a conventional manner.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.
<< 실시예Example > >
본 발명의 실시예를 위해 사용한 시약 및 이들의 구입처는 다음과 같다.Reagents used for the examples of the present invention and where they are purchased are as follows.
대식세포증식자극인자(macrophage colony stimulating factor : 이하 간략하게 ‘M-CSF’이라 함)는 R&D system (Minneapolis, MN, USA)로부터 구입하였다. RANKL은 Dr. Lee(University of Ewha Women's University, Korea)로부터 제공받았다. 에스큘레틴은 sigma Aldrich로 부터 구입하였으며, DMSO(dimethylsulfoxide)에 녹여 최종 DMSO 농도가 0.1%가 되게 하였다.
Macrophage colony stimulating factor (hereinafter referred to as 'M-CSF') was purchased from an R & D system (Minneapolis, MN, USA). RANKL is Dr. It was provided by Lee (University of Ewha Women's University, Korea). Esculletin was purchased from sigma Aldrich and dissolved in DMSO (dimethylsulfoxide) to give a final DMSO concentration of 0.1%.
<< 실시예Example 1> 1>
에스큘레틴에To esculletin 의한 파골세포 분화 억제 효과 ( Inhibitory Effect of Osteoclast Differentiation by inin vitrovitro ))
<1-1> 마우스 골수 <1-1> mouse bone marrow 단핵세포의Mononuclear 배양 및 파골세포 형성 유도 Induction of culture and osteoclast formation
골수세포는 4주령 C57BL/6 수컷 마우스(Sam tako, Korea)의 장골(long bone)로부터 분리되었으며, 37℃ 인큐베이터(humidified incubator: 5% CO2 in air) 에서 10% α-minimal essential medium(MEM), % 소태아혈청(FBS), 항생제 및 M-CSF(10ng/ml)가 첨가된 배지에서 배양하였다. 배양 후 24시간이 지난 다음, 부유 세포들을 수집한 후 10% FBS 및 10ng/ml M-CSF가 첨가된 α-MEM 배지에서 3일 동안 배양한 다음, 부착된 세포들을 BMMs(Bone marrow Macrophages)로 사용하였다. 파골세포 분화 실험을 위하여 BMM 세포를 24-웰 플레이트(1×105/well)에서 배양한 후, 96-웰 플레이트(3×104/well)로 옮겨 M-CSF 20ng/ml가 첨가된 배지로 배양하였고, 파골세포분화인자인 RANKL 200ng/ml로 6일 동안 자극시켜 파골세포 형성을 유도하였다. 이때 에스큘레틴은 RANKL과 동시에 처리하여 배양하였다.
Bone marrow cells were isolated from the long bones of four-week-old C57BL / 6 male mice (Sam tako, Korea) and 10% α-minimal essential medium (MEM) in a 37 ° C incubator (5% CO 2 in air). ),% Fetal bovine serum (FBS), antibiotics and M-CSF (10 ng / ml) was added to the culture medium. After 24 hours of incubation, the suspended cells were collected and incubated for 3 days in α-MEM medium to which 10% FBS and 10ng / ml M-CSF were added, and then the attached cells were transferred to BMMs (Bone marrow Macrophages). Used. For osteoclast differentiation experiments, BMM cells were cultured in 24-well plates (1 × 10 5 / well), and then transferred to 96-well plates (3 × 10 4 / well) to which M-CSF 20ng / ml was added. The cells were stimulated with osteoclast differentiation factor RANKL 200ng / ml for 6 days to induce osteoclast formation. At this time, esculintin was incubated with treatment with RANKL.
<1-2> <1-2> TRAPTRAP 염색 및 Dyeing and TRAPTRAP 활성 측정 Active measurement
상기 실시예<1-1>에 따라 분화 유도가 끝난 세포들은 3.7% 포르말린으로 10분 동안 고정한 후 PBS로 두 번 세척하였다. 세포들은 제조자의 지시에 따라 키트(sigma)를 이용하여 TARP(tartrate-resistant acid phosphatase) 염색을 수행하였다. TRAP 활성의 측정을 위해, 세포들을 3.7% 포르말린에서 10분 동안 고정시켰으며, 그 후 아세톤 및 포르말린(1:1, v/v) 혼합물에서 1분 동안 침지시킨 후, 건조시켰다. 세포들은 37℃ 온도 조건에서 10분 동안 100μl 포스포타아제 기질 용액(3.7mM ρ-니트로페닐 포스페이트 및 10mM 주석산나트륨[sodium tartrate]을 포함하는 50mM 시트레이트 버퍼[pH4.6]) 내에서 배양되었다. 배양 후 효소반응 혼합물을 다른 플레이트로 옮긴 후 100μL의 0.1N NaOH로 반응을 정지시켰다. ELISA reader를 이용하여 405nm에서 흡광도를 측정하였다.After the differentiation induction according to Example <1-1>, the cells were fixed with 3.7% formalin for 10 minutes and washed twice with PBS. The cells were stained with TARP (tartrate-resistant acid phosphatase) using a kit (sigma) according to the manufacturer's instructions. For the measurement of TRAP activity, cells were fixed for 10 minutes in 3.7% formalin, then immersed in acetone and formalin (1: 1, v / v) mixture for 1 minute and then dried. Cells were incubated in 100 μl phosphatase substrate solution (50 mM citrate buffer [pH4.6] containing 3.7 mM ρ-nitrophenyl phosphate and 10 mM sodium tartrate) at 37 ° C. temperature conditions for 10 minutes. After incubation, the enzyme reaction mixture was transferred to another plate and the reaction was stopped with 100 μL of 0.1N NaOH. Absorbance was measured at 405 nm using an ELISA reader.
그 결과 도 1 및 도 2에서 보이는 바와 같이, 본 발명의 에스큘레틴 화합물을 처리한 실험군에서 농도 의존적으로 파골세포 형성이 억제되는 것(BMM세포의 생성 억제)을 확인할 수 있었으며, 특히 40μM 농도에서는 파골세포로의 분화가 이루어지지 않는 것으로 나타나, 에스큘레틴의 파골세포 형성 억제 효능이 뛰어남을 알 수 있었다.
As a result, as shown in Figs. 1 and 2, it was confirmed that osteoclast formation was inhibited in a concentration-dependent manner (inhibition of production of BMM cells) in the experimental group treated with the esculletin compound of the present invention. The differentiation into osteoclasts did not appear, indicating that esculletin had an excellent effect on inhibiting osteoclast formation.
<1-3> <1-3> 에스큘레틴에To esculletin 대한 골수세포의 세포 Myeloid cells 생존률Survival rate 측정 Measure
파골세포 형성 억제 효과가 에스큘레틴 화합물의 세포 독성에 의한 것인지를 확인하기 위하여 세포 생존율을 측정하였다. Cell viability was measured to determine whether the osteoclast formation inhibitory effect was due to the cytotoxicity of the esculletin compound.
에스큘레틴의 세포독성은 CyQUANT Cell Proliferation Assay Kit(Invitrogen, Carlsbad, CA, USA)를 이용하여 평가하였다. M-CSF 및 RANKL 존재 하에서 3×104cells/well 농도로 96 웰 플레이트에 BMM세포를 분주 한 후, 5μM, 10μM 및 20μM의 에스큘레틴을 처리하고 일정 시간 동안 배양하였다.Cytotoxicity of esculintin was assessed using CyQUANT Cell Proliferation Assay Kit (Invitrogen, Carlsbad, Calif., USA). After dispensing BMM cells into 96 well plates at a concentration of 3 × 10 4 cells / well in the presence of M-CSF and RANKL, 5 μM, 10 μM, and 20 μM of esculinate were treated and incubated for a predetermined time.
그 결과 도 3에서 나타난 바와 같이, 에스큘레틴 미처리 군(대조군)과 비교하여 에스큘레틴을 처리한 실험군에서 세포 생존률이 거의 차이를 보이지 않는 것을 통해, 본 발명의 에스큘레틴 화합물은 세포 독성을 유발하지 않는 것을 알 수 있었으며, 따라서 파골세포의 형성 억제는 에스큘레틴의 세포 분화 조절에 의한 것이지 세포 독성으로 인한 억제가 아님을 알 수 있었다.
As a result, as shown in FIG. 3, the cell survival rate in the experimental group treated with esculletin compared to the untreated esculletin (control) showed little difference, the esculletin compound of the present invention is cytotoxic It can be seen that it is not induced, and therefore, the inhibition of osteoclast formation is due to the regulation of cellular differentiation of esculletin, not inhibition of cytotoxicity.
<1-4> <1-4> 에스큘레틴Esculetin 처리에 따른 파골세포 특이적 Osteoclast specificity following treatment 마커Marker 유전자의 발현에 미치는 영향 Effect on gene expression
앞서 수행한 실시예들을 통해 에스큘레틴이 파골세포 형성을 억제한다는 사실을 확인하였으므로, 본 발명자들은 에스큘레틴이 파골 세포 특이적 마커 유전자의 발현에 영향을 미치는 지를 조사하기 위해 역전사-중합효소연쇄반응법(RT-PCR)을 수행하였다. As a result of confirming the fact that esculin inhibits osteoclast formation through the previous examples, the inventors of the present invention have investigated reverse transcriptase-polymerase chain to investigate whether esculin affects the expression of osteoclast specific marker genes. Reaction method (RT-PCR) was performed.
각 세포로부터 총 RNA의 수득은 TRIzol 시약(Invitrogen, Carlsbad, CA)을 사용하여 분리하였으며, 제조자의 지시에 따라 ImPROM-Ⅱ Reverse Transcription System(Promega)로 역전사하였다. PCR 프라이머 서열은 하기와 같은 것을 사용하였다.
Total RNA from each cell was isolated using TRIzol reagent (Invitrogen, Carlsbad, Calif.) And reverse transcribed with ImPROM-II Reverse Transcription System (Promega) according to the manufacturer's instructions. PCR primer sequences were used as follows.
그 결과 도 4에서 나타난 바와 같이, RANKL에 의해 파골세포로 분화된 세포는 마커 유전자로 알려진 칼시토닌 카셉신 K(cathepsin K), MMP-9(Matrix metalloproteinase-9), TRAP, V-ATPase, C-Fos, NFATc1, c-SRC 유전자 발현이 모두 증가되는 것을 알 수 있었다. 그러나 에스큘레틴을 첨가한 실험군에서는 cathepsin K, TRAP, NFATc1 및 c-SRC의 유전자 발현이 현저하게 억제되는 것을 확인할 수 있었다.As a result, as shown in Figure 4, cells differentiated into osteoclasts by RANKL is known as a marker gene calcitonin cathepsin K, matrix metalloproteinase-9 (MMP-9), TRAP, V-ATPase, C- Fos, NFATc1, c-SRC gene expression was all increased. However, it was confirmed that the expression of cathepsin K, TRAP, NFATc1, and c-SRC was significantly suppressed in the experimental group to which esculletin was added.
파골세포 분화에 관한 RANKL 자극을 매개하는 다양한 신호전달 경로가 밝혀진바 있다. 특히 RANKL의 자극은 파골전구세포에서 파골세포의 분화를 조절하는 핵심 전사인자로 알려져 있는 NFAT(nuclear factor of activated T cells)의 발현을 유도한다. NFAT는 RANKL 자극에 의한 세포내 칼슘 신호 전달로 초기 유도되며, 그 후 ERK(extracellular signal-regulated kinase), NF-κB, MAPK(p38 MAP kinase), c-Fos 등의 활성화가 NFAT의 자가증폭(autoamplification)을 유발하여 활성이 지속된다. 핵심 전사인자인 NFAT 단백질은 현재까지 5 종류가 확인되었다. 즉, NFAT family는 NFAT1 (NFATp 또는 NFATc2), NFAT2 (NFATc 또는 NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx 또는 NFATc3) 및 NFAT5으로 구성되어 있다.Various signaling pathways mediating RANKL stimulation on osteoclast differentiation have been identified. In particular, RANKL stimulation induces the expression of NFAT (nuclear factor of activated T cells), a key transcription factor that regulates osteoclast differentiation in osteoclast precursor cells. NFAT was initially induced by intracellular calcium signaling by RANKL stimulation and then activation of ERK (extracellular signal-regulated kinase), NF-κB, MAPK (p38 MAP kinase) autoamplification) and the activity continues. NFAT protein, a key transcription factor, has been identified to date. That is, the NFAT family consists of NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3) and NFAT5.
활성화된 NFAT에 의해 NF-κB 등이 핵 내에서 전사인자로 작용하게 되면 파골세포 분화와 관련된 c-SRC 티로신 키나아제 등의 발현이 증가되게 된다. 또한 골 기질을 흡수하기 위한 탈산수소효소 활성을 가지게 되는 TRAP의 활성을 높아지게 되며 활성화된 파골세포의 주름진 막의 프로톤 펌프를 통해 프로톤이 세포 밖으로 보내 파골세포 둘레가 산성 조건으로 유지되게 되며 라이소조말 단백질분해효소인 cathepsin K가 발현되게 되어 유기성 뼈 기질층이 분해, 골 기질이 흡수되게 된다.When NF-κB acts as a transcription factor in the nucleus by activated NFAT, expression of c-SRC tyrosine kinase related to osteoclast differentiation is increased. It also increases the activity of TRAP, which has dehydrogenase activity to absorb bone matrix, and protons are sent out of the cell through the proton pump of the corrugated membrane of activated osteoclasts to keep the osteoclast around acidic conditions. Cathepsin K, a degrading enzyme, is expressed and organic bone matrix is degraded and bone matrix is absorbed.
본 발명의 에스큘레틴의 처리는 RANKL에 의한 cathepsin K, TRAP, NFATc1 및 c-SRC의 발현을 유의성 있게 감소시키는 것으로 나타났으며, 이러한 결과는 에스큘레틴이 NFATc1의 발현의 감소를 통하여 파골세포 분화를 억제하는 것을 시사한다.
Treatment of esculletin of the present invention has been shown to significantly reduce the expression of cathepsin K, TRAP, NFATc1 and c-SRC by RANKL, these results indicate that esculletin osteoclasts through a decrease in the expression of NFATc1 Suppresses differentiation.
<1-5> <1-5> 에스큘레틴Esculetin 처리에 따른 파골세포 발생의 전사 인자의 발현에 미치는 영향 Effect of Osteocell Developmental Transcription Factor on Treatment
파골세포 발생의 전사 인자인 NFATc1과 c-Fos의 발현에 대한 본 발명의 에스큘레틴의 영향을 평가하기 위하여 하기와 같은 western blot analysis를 실행하였다.Western blot analysis was performed to evaluate the effect of the esculletin of the present invention on the expression of the transcription factors NFATc1 and c-Fos of osteoclasts.
우선, RIPA 버퍼(50mM Tris pH 7.4, 1% NP-40, 0.1% SDS, 150mM NaCl, 1mM EDTA, 1mM PMSF, 1mM Na3VO4, 1mM NaF, 1㎍/㎖ aprotinin, 1㎍/㎖ leupeptin)를 이용하여 세포를 용해한 후, 4℃ 13,000 rpm에서 20분 동안 원심분리하여 단백질을 추출하였다. 추출한 단백질은 DC Protein Assay Kit(Bio-Rad)방법으로 정량하였다.First, RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, 1 µg / ml aprotinin, 1 µg / ml leupeptin) After lysing the cells, the proteins were extracted by centrifugation at 4 ° C. 13,000 rpm for 20 minutes. The extracted protein was quantified by DC Protein Assay Kit (Bio-Rad) method.
동일한 양의 단백질을 10% SDS-polyacrylamide gel electrophoresis(SDS-PAGE)에서 전기영동하여 gel상의 단백질을 semi-dry transfer unit-TE 70(Amersham Biosciences)을 이용하여 PVDF membrane(Millipore)에 단백질을 이동시켰다. 상기 PVDF membrane을 5% BSA로 블로킹한 후, 1차 항체와 반응시키고 이와 반응한 2차 항체를 ELC reagent를 이용하여 형광 표지한 것을 LAS-3000(FUJI FILM)을 이용하여 단백질의 발현을 확인하였다. The same amount of protein was electrophoresed in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to transfer the protein on the gel to the PVDF membrane (Millipore) using a semi-dry transfer unit-TE 70 (Amersham Biosciences). . After blocking the PVDF membrane with 5% BSA, reacted with the primary antibody and fluorescently labeled the reacted secondary antibody with ELC reagent to confirm protein expression using LAS-3000 (FUJI FILM). .
즉, BMMs을 본 발명에 따른 에스큘레틴(20μM)으로 처리한 군과 미처리한 군으로 나누어 각각 M-CSF 및 RANKL로 일정시간(1일, 2일, 3일) 배양하였다. 그런 다음, 모든 세포 용해물을 NFATc1, c-Fos 및 β-actin 항체들을 각각 사용하여 상기한 western blot 분석을 실시하였다. That is, BMMs were divided into groups treated with esculinine (20 μM) and untreated groups according to the present invention, and then cultured with M-CSF and RANKL for a predetermined time (1 day, 2 days, 3 days). All cell lysates were then subjected to the western blot assay described above using NFATc1, c-Fos and β-actin antibodies, respectively.
그 결과 도 5에서 나타난 바와 같이, 본 발명의 에스큘레틴에 의해 파골세포 발생의 전사 인자인 NFATc1과 c-Fos의 발현이 대조군에 비해 감소되는 것을 확인할 수 있었다. 즉, 본 발명의 에스큘레틴은 RANKL에 의해 발현이 유도되는 NFATc1 및 c-Fos를 억제함을 알 수 있다.
As a result, as shown in Figure 5, it was confirmed that the expression of the transcription factors NFATc1 and c-Fos of osteoclast generation by the escullet of the present invention compared to the control. In other words, it can be seen that the esculinin of the present invention inhibits NFATc1 and c-Fos induced expression by RANKL.
<1-6> <1-6> 에스큘레틴Esculetin 처리에 따른 파골세포 발생의 신호 분자의 활성화에 미치는 영향 Effect of Signaling Molecules on Osteoblastogenesis Following Treatment
본 발명에 따른 에스큘레틴(20μM)으로 2시간 동안 예비처리하거나 그렇지 않은 BMMs을 3시간 동안 혈청이 결핍된 상태로 방치한 다음, 일정시간(0분, 5분, 15분, 30분) RANKL(500 ng/㎖)로 자극하였다. 그런 다음 세포 용해물을 p38, JNK 및 ERK 항체들을 각각 사용하여 상기한 <1-5>에서처럼 western blot 분석을 실시하였다. Pretreatment with Esculletine (20 μM) according to the present invention for 2 hours or BMMs that do not otherwise leave serum-free for 3 hours, followed by a certain time (0 minutes, 5 minutes, 15 minutes, 30 minutes) RANKL Stimulated (500 ng / ml). Cell lysates were then subjected to western blot analysis using p38, JNK and ERK antibodies, respectively, as described above in <1-5>.
그 결과 도 6에서 나타난 바와 같이, 본 발명의 에스큘레틴에 의해 파골세포 발생의 신호 분자 중 p38의 인산화가 감소되는 것을 확인할 수 있었다. 즉, 본 발명의 에스큘레틴은 RANKL에 의해 활성화되는 p38을 억제함을 알 수 있다.
As a result, as shown in Figure 6, it was confirmed that the phosphorylation of p38 in the signal molecule of osteoclast generation by the esculletin of the present invention. That is, it can be seen that the esculletin of the present invention inhibits p38 activated by RANKL.
<1-7> 성숙한 파골세포의 뼈 흡수 활성에 미치는 <1-7> Effect on bone resorption activity of mature osteoclasts 에스큘레틴의Esculletin 영향 effect
성숙한 파골세포의 뼈 흡수 활성에 대한 본 발명의 에스큘레틴의 영향을 평가하기 위하여, BMMs을 BioCoat plate(BD, bone culture system)에 깔고, M-CSF(20 ng/㎖)와 RANKL(200 ng/㎖), 그리고 에스큘레틴(20μM)을 처리하여 10일 동안 분화를 유도하였다. 그 후, BioCoat plate를 5% sodium hypochlorite로 5분 동안 처리하여 세포를 처리한 후 흡수된 영역을 현미경 하에서 촬영하였다.In order to evaluate the effect of the esculletin of the present invention on bone resorption activity of mature osteoclasts, BMMs were placed on a BioCoat plate (BD, bone culture system), and M-CSF (20 ng / ml) and RANKL (200 ng). / Ml), and esculletin (20 μM) to induce differentiation for 10 days. Thereafter, the BioCoat plate was treated with 5% sodium hypochlorite for 5 minutes to treat the cells, and the absorbed area was photographed under a microscope.
그 결과 도 7에서 나타난 바와 같이, 본 발명의 에스큘레틴에 의해 파골세포 활성을 나타낼 수 있는 뼈 흡수 활성이 억제되는 것을 확인할 수 있었다. As a result, as shown in Figure 7, it was confirmed that the bone resorption activity that can exhibit the osteoclast activity by the esculletin of the present invention.
즉, 도 7에서 성숙한 파골세포에 의해 흡수된 영역은 화살표로 표기되었는데, 본 발명의 에스큘레틴이 처리되지 않은 경우 다수의 뼈 흡수 영역이 존재함을 명백히 알 수 있다. 반면 본 발명의 에스큘레틴이 처리된 경우 뼈 흡수 영역이 발생하지 않았다.That is, the region absorbed by the mature osteoclast in Figure 7 is indicated by the arrow, it can be clearly seen that there is a large number of bone absorption region when the esculletin of the present invention is not treated. On the other hand, when the esculletin of the present invention was treated, no bone absorption region occurred.
상기의 실시예를 종합해 보면, 파골세포로 분화 과정 중인 세포에 본 발명의 에스큘레틴을 처리한 경우 파골세포로의 형성이 억제되는 것이 증명되었으며; 이러한 파골세포의 형성을 억제하는 기작은 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT의 발현 억제와 연계되어 있는 것을 확인할 수 있었다. 따라서 상기와 같은 특징을 가진 에스큘레틴을 이용하는 경우 파골세포로의 형성을 억제하여 골 손실 개선에 효과적일 것으로 판단되었다.
Comprehensive examples of the above, it was proved that the formation of osteoclasts is suppressed when the esculinin of the present invention is treated to cells in the process of differentiation into osteoclasts; The mechanism of inhibiting the formation of osteoclasts was confirmed to be associated with the inhibition of the expression of NFAT, a key transcription factor that can regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation. Therefore, it was determined that the use of escullet having the above characteristics would be effective in improving bone loss by inhibiting the formation of osteoclasts.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (7)
<화학식 1>
≪ Formula 1 >
상기 화합물은 파골세포 형성 억제 효능을 갖는 것을 특징으로 하는 골 손실 억제용 조성물.The method of claim 1,
The compound has a composition for inhibiting bone loss, characterized in that it has the effect of inhibiting osteoclast formation.
상기 파골세포 형성 억제는 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT의 발현을 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 갖는 것을 특징으로 하는 골 손실 억제용 조성물.3. The method of claim 2,
The inhibition of osteoclast formation is a composition for inhibiting bone loss, characterized in that it has an effect of inhibiting the differentiation of osteoclasts through a mechanism of inhibiting the expression of NFAT which is a key transcription factor that controls the differentiation of osteoclasts.
상기 골 손실 억제를 통해 치료할 수 있는 질환은 골다공증, 파제트 골질환, 구루병, 골연화증 및 퇴행성 골질환으로 이루어진 군으로부터 선택된 1종인 것을 특징으로 하는 골 손실 억제용 조성물.4. The method according to any one of claims 1 to 3,
The disease that can be treated through the inhibition of bone loss is a composition for inhibiting bone loss, characterized in that one selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia and degenerative bone disease.
<화학식 1>
≪ Formula 1 >
상기 화합물은 파골세포 형성 억제 효능을 갖는 것을 특징으로 하는 골 손실 개선용 건강기능식품.The method of claim 5,
The compound is a health functional food for bone loss improvement, characterized in that it has the effect of inhibiting osteoclast formation.
상기 식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류로 이루어진 군으로부터 선택된 1종인 것을 특징으로 하는 골 손실 개선용 건강기능식품.The method according to claim 5 or 6,
The food is bone loss improvement, characterized in that the one selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and dietary supplements Dietary supplements.
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| JP2009504776A (en) | 2005-08-18 | 2009-02-05 | アクセラロクス， インコーポレイテッド | Methods for treating bone by modulating arachidonic acid metabolic or signaling pathways |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200016640A (en) | 2018-08-07 | 2020-02-17 | 서울대학교산학협력단 | Composition for inhibiting osteoclast differentiation comprising gold compound as an active ingredient |
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