KR20220085244A - Composition for Prophylaxis and Treatment of Osteoporosis Comprising Sparganium Rhizoma Extract - Google Patents
Composition for Prophylaxis and Treatment of Osteoporosis Comprising Sparganium Rhizoma Extract Download PDFInfo
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Abstract
본 발명은 삼릉 추출물의 골다공증 예방 또는 치료 효과와 골 손실, 골 흡수 및 파골세포 분화 억제 효과에 관한 것으로, 상기 삼릉 추출물은 세포독성이 없으면서, 파골세포의 분화, 골 흡수 및 액틴 고리 형성을 억제하고, 파골세포의 분화 전사인자, 파골세포의 분화 관련 유전자, 골 흡수 관련 유전자 및 파골세포의 융합 관련 유전자의 발현을 억제하므로, 이를 골 손실 억제 용도 또는 골 질환 예방 또는 치료 용도로 유용하게 이용할 수 있다.The present invention relates to the osteoporosis preventive or therapeutic effect of Samreung extract, and the effect of inhibiting bone loss, bone resorption and osteoclast differentiation, wherein the Samneung extract inhibits osteoclast differentiation, bone resorption and actin ring formation without cytotoxicity, , because it suppresses the expression of osteoclast differentiation transcription factors, osteoclast differentiation-related genes, bone resorption-related genes, and osteoclast fusion-related genes, it can be usefully used for inhibiting bone loss or preventing or treating bone diseases .
Description
본 발명은 삼릉 추출물의 골다공증 예방 또는 치료 효과와 골 손실, 골 흡수 및 파골세포 분화 억제 효과에 관한 것이다.The present invention relates to the osteoporosis prevention or treatment effect of Samneung extract, and to the effect of inhibiting bone loss, bone resorption, and osteoclast differentiation.
골은 역동적인 조직으로 골을 흡수하는 파골세포와 골을 형성하는 조골세포의 균형으로 양질의 골이 지속적으로 재형성(remodeling)된다. 하지만 폐경기 이후 여성은 에스트로겐 결핍으로 파골세포의 수 그리고 활성이 급격하게 증가하게 되어 골량이 감소하고 골다공증 및 류마티스 관절염과 같은 골 관련 질환의 발병위험이 높아진다. 골다공증은 뼈가 약해져 골절의 위험이 높아지는 질환이며, 나이와 깊은 연관이 있다. 따라서 최근 평균 수명이 연장됨에 따라 골다공증에 대한 관심은 증가하고 있다.Bone is a dynamic tissue, and high-quality bone is continuously remodeled by the balance of osteoclasts that absorb bone and osteoblasts that form bone. However, in postmenopausal women, the number and activity of osteoclasts rapidly increase due to estrogen deficiency, leading to a decrease in bone mass and an increased risk of bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoporosis is a disease that increases the risk of fractures due to weak bones, and is closely related to age. Therefore, interest in osteoporosis is increasing as the average lifespan is recently extended.
파골세포는 조혈모세포에서 유래되는 다핵형 세포이다. 파골세포로의 분화는 골흡수에 매우 중요한 과정이며 파골세포로 분화를 담당하는 주요 인자는 RANKL (receptor activator of NF-κB ligand)이다. RANKL은 수용체인 RANK와 결합하여 파골세포의 형성에 중요한 세포 내 여러 신호전달 물질인 NF-κB, c-Fos 그리고 NFATc1 (nuclear factor of activated T cell c1)을 활성화 시킨다고 보고되었다. 활성화된 c-Fos는 RANKL에 의해 유도 되며, 또 다른 전사 인자인 NFATc1을 유도한다. NFATc1은 파골세포의 분화에 필수적인 중요한 전사인자로 파골세포 지표인 TRAP(tartrate-resistant acid phosphatase), OSCAR(osteoclast-associated receptor), CtsK(cathepsin K), MMP-9(matrix methalloproteinase 9) 및 DC-STAMP(dendritic cell-specific transmembrane protein) 등의 발현을 촉진하여 파골세포의 분화 및 기능을 활성화시켜, 골 흡수, 활성화, 생존 및 분화를 조절한다.Osteoclasts are multinucleated cells derived from hematopoietic stem cells. Differentiation into osteoclasts is a very important process for bone resorption, and the major factor responsible for differentiation into osteoclasts is RANKL (receptor activator of NF-κB ligand). It has been reported that RANKL binds to the receptor RANK and activates NF-κB, c-Fos, and NFATc1 (nuclear factor of activated T cell c1), which are several intracellular signaling substances important for the formation of osteoclasts. Activated c-Fos is induced by RANKL and another transcription factor, NFATc1. NFATc1 is an important transcription factor essential for osteoclast differentiation, osteoclast markers tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), cathepsin K (CtsK), matrix methalloproteinase 9 (MMP-9) and DC- It promotes the expression of STAMP (dendritic cell-specific transmembrane protein), etc. to activate the differentiation and function of osteoclasts, thereby regulating bone resorption, activation, survival and differentiation.
현재 골다공증 치료에 가장 많이 사용되고 있는 치료제는 비스포스포네이트(bisphosphonates) 제제이다. 하지만, 비스포스포네이트 제제로 골다공증 치료시 골 괴사 그리고 비 전형적 골절 등과 같은 부작용을 동반시킬 수 있는 것으로 알려져 있다. 따라서 부작용이 없는 천연물 유래의 치료제 개발이 필요한 실정이다. Currently, the most widely used treatment for osteoporosis is bisphosphonates. However, it is known that side effects such as bone necrosis and atypical fractures may accompany the treatment of osteoporosis with bisphosphonates. Therefore, there is a need to develop a therapeutic agent derived from a natural product without side effects.
한편, 삼릉(三稜, Sparganium Rhizoma; 이하 SR)은 흑삼릉과 (Sparganiaceae)의 흑삼릉(Sparganium stoloniferum Buchanan-Hamilton)의 덩이뿌리를 말린 약재로서 뿌리와 줄기부분을 약용한다. 삼릉은 혈소판 응고, 혈전제거, 어혈을 풀어주는 약리작용을 하며, 항균 활성 작용 및 항산화 효과가 있는 것으로 알려져 있으나, 골대사 치료 효과에 대해서는 알려진 바가 없다.On the other hand, Samneung (Sparganium Rhizoma; hereafter referred to as SR) is a medicinal herb obtained by drying the tuberous roots of Sparganium stoloniferum Buchanan-Hamilton of the Sparganiaceae family, and its roots and stems are medicinal. Samneung has pharmacological actions for platelet coagulation, thrombus removal and eohyeol release, and is known to have antibacterial and antioxidant effects, but nothing is known about its effect on bone metabolism.
이에, 본 발명자들은 삼릉이 파골세포 분화 관련 인자에 미치는 영향을 확인한 결과, 삼릉 추출물이 폐경기 골다공증의 주요 원인으로 알려진, 파골세포의 분화를 효과적으로 억제할 수 있는 효과가 있음을 확인함으로써, 상기 삼릉을 골다공증을 포함한 다양한 골 질환 개선에 응용되어 사용될 수 있음을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors confirmed the effect of Samreung on osteoclast differentiation-related factors, and as a result of confirming that Samneung extract has the effect of effectively inhibiting the differentiation of osteoclasts, known as the main cause of postmenopausal osteoporosis, The present invention was completed by confirming that it can be applied and used to improve various bone diseases including osteoporosis.
본 발명의 목적은 골 손실 억제용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for inhibiting bone loss.
본 발명의 다른 목적은 골 질환 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease.
본 발명의 또 다른 목적은 골 질환 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving bone disease.
본 발명의 또 다른 목적은 파골세포 분화 또는 골 흡수 억제용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for inhibiting osteoclast differentiation or bone resorption.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명은 삼릉(Sparganium Rhizoma) 추출물을 유효성분으로 함유하는 골 손실 억제용 조성물을 제공한다.The present invention provides a composition for inhibiting bone loss containing an extract of Sparganium Rhizoma as an active ingredient.
또한, 본 발명은 삼릉 추출물을 유효성분으로 함유하는 골 질환 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease containing Samreung extract as an active ingredient.
나아가, 본 발명은 삼릉 추출물을 유효성분으로 함유하는 골 질환 예방 또는 개선용 식품 조성물을 제공한다.Furthermore, the present invention provides a food composition for preventing or improving bone disease containing Samreung extract as an active ingredient.
더 나아가, 본 발명은 삼릉 추출물을 유효성분으로 함유하는 파골세포 분화 또는 골 흡수 억제용 조성물을 제공한다.Furthermore, the present invention provides a composition for inhibiting osteoclast differentiation or bone resorption containing Samneung extract as an active ingredient.
본 발명에 따른 삼릉 추출물은 세포독성이 없으면서, 파골세포의 분화 및 골 흡수를 억제하고, 파골세포의 액틴 고리 형성을 억제하며, 파골세포의 분화 전사인자, 파골세포의 분화 관련 유전자, 골 흡수 관련 유전자 및 파골세포의 융합 관련 유전자의 발현을 억제하므로, 이를 골 손실 억제 용도 또는 골 질환 예방 또는 치료 용도로 유용하게 이용할 수 있다.Samneung extract according to the present invention has no cytotoxicity, inhibits osteoclast differentiation and bone resorption, inhibits osteoclast actin ring formation, osteoclast differentiation transcription factor, osteoclast differentiation-related gene, bone resorption-related Since it suppresses the expression of genes and osteoclast fusion-related genes, it can be usefully used for inhibiting bone loss or for preventing or treating bone diseases.
도 1은 삼릉 에탄올 추출물(SR)의 세포독성을 확인한 결과를 나타낸 그래프이다.
도 2는 삼릉 에탄올 추출물(SR) 처리에 의한 파골세포 분화능을 확인한 도이다.
도 3은 삼릉 에탄올 추출물(SR)에 의한 골 흡수 억제 효과를 확인한 도이다.
도 4는 삼릉 에탄올 추출물(SR)에 의한 파골세포 액틴 고리 형성 억제 효과를 확인한 도이다.
도 5는 삼릉 에탄올 추출물(SR) 처리에 의한 파골세포의 분화 전사인자인 NFATc1 및 c-Fos의 단백질 발현 억제 효과를 확인한 도이다.
도 6은 삼릉 에탄올 추출물(SR) 처리에 의한 파골세포 분화 관련 TRAP, NFATc1, c-Fos 및 RANK와, 골 흡수 관련 CTK, MMP-9 및 CA2, 및 파골세포 융합 관련 ATP6v0d2, OSCAR 및 DC-STAMP의 유전자 발현 억제 효과를 확인한 도이다.1 is a graph showing the results of confirming the cytotoxicity of Samneung ethanol extract (SR).
Figure 2 is a view confirming the osteoclast differentiation ability by Samneung ethanol extract (SR) treatment.
3 is a view confirming the effect of inhibiting bone resorption by Samneung ethanol extract (SR).
Figure 4 is a view confirming the inhibitory effect on osteoclast actin ring formation by Samneung ethanol extract (SR).
5 is a diagram confirming the effect of inhibiting protein expression of NFATc1 and c-Fos, which are transcription factors for differentiation of osteoclasts, by treatment with Samneung ethanol extract (SR).
6 shows osteoclast differentiation-related TRAP, NFATc1, c-Fos and RANK, bone resorption-related CTK, MMP-9 and CA2, and osteoclast fusion-related ATP6v0d2, OSCAR and DC-STAMP by Samneung ethanol extract (SR) treatment It is a diagram confirming the effect of suppressing gene expression of
이하 첨부된 도면을 참조하여 본 발명의 실시예들을 상세히 설명한다. 이하의 설명에 있어, 당업자에게 주지 저명한 기술에 대해서는 그 상세한 설명을 생략할 수 있다. 또한, 본 발명을 설명함에 있어서, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있다. 또한, 본 명세서에서 사용되는 용어 (terminology)들은 본 발명의 바람직한 실시 예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, terms used in this specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.
따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.
본 발명은 삼릉(Sparganium Rhizoma) 추출물을 유효성분으로 함유하는 골 손실 억제용 조성물에 관한 것이다.The present invention relates to a composition for inhibiting bone loss containing an extract of Sparganium Rhizoma as an active ingredient.
본 발명에서 사용되는 용어 "추출물(extract)"은 생약을 적절한 침출액으로 짜내고 침출액을 증발시켜 농축한 제제를 의미하는 것으로, 이에 제한되지는 않으나, 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 이들의 조정제물 또는 정제물일 수 있다. 상기 우슬 추출물 또는 왕불류행 추출물은 통상의 기술분야에 공지된 일반적인 추출방법, 분리 및 정제방법을 이용하여 제조할 수 있다. 상기 추출방법으로는, 이에 제한되지는 않으나, 바람직하게 열탕 추출, 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등의 방법을 사용할 수 있다.As used herein, the term "extract" refers to a preparation concentrated by squeezing a crude drug with an appropriate leaching solution and evaporating the leachate, but is not limited thereto, but includes an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, It may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The hyssop extract or Wangbulryuhaeng extract can be prepared using general extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably, methods such as hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction may be used.
본 발명에 있어서, 상기 추출물은 추출용매로 추출하거나 추출용매로 추출하여 제조한 추출물에 분획용매를 가하여 분획함으로써 제조할 수 있다. 상기 추출용매는 이에 제한되지 않으나, 물, 유기용매 또는 이들의 혼합용매 등을 사용할 수 있으며, 상기 유기용매는 탄소수 1 내지 4의 알코올이나, 에틸아세테이트 또는 아세톤 등의 극성용매, 헥산 또는 디크로로메탄의 비극성용매 또는 이들의 혼합용매를 사용할 수 있다. 또한, 바람직하게는 물, 탄소수 1 내지 4의 알코올 또는 이들의 혼합용매를 사용할 수 있으며, 보다 바람직하게는 물 및 에탄올의 혼합용매를 사용할 수 있다. 본 발명의 일 실시예에서는 상기 용매로서 80%(v/v) 에탄올 수용액을 이용하여 추출한 뒤 동결 건조하여 '삼릉 에탄올 추출물(SR)'을 제조하였다.In the present invention, the extract can be prepared by fractionation by adding a fractionation solvent to the extract prepared by extraction with an extraction solvent or extraction with an extraction solvent. The extraction solvent is not limited thereto, but water, an organic solvent, or a mixture thereof may be used, and the organic solvent is an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloro. A non-polar solvent of methane or a mixed solvent thereof may be used. In addition, preferably water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be used, and more preferably a mixed solvent of water and ethanol may be used. In an embodiment of the present invention, extraction was performed using an 80% (v/v) aqueous ethanol solution as the solvent and then freeze-dried to prepare a 'Samneung ethanol extract (SR)'.
일 구현예에서, 본 발명의 추출물은 물, 탄소수 1 내지 4의 무수 또는 함수알코올, 에틸아세테이트, 아세톤, 글리세린, 에틸렌글리콜, 프로필렌글리콜 및 부틸렌글리콜로 이루어진 군에서 선택된 적어도 어느 하나 이상의 추출용매로 추출될 수 있으며, 물 및 에탄올을 혼합한 추출용매로 추출된 것이 더욱 바람직하다.In one embodiment, the extract of the present invention is at least one selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol and butylene glycol as an extraction solvent. It can be extracted, more preferably extracted with an extraction solvent mixed with water and ethanol.
일 구현예에서, 본 발명의 조성물은 파골세포의 분화 또는 골 흡수를 억제할 수 있다.In one embodiment, the composition of the present invention may inhibit osteoclast differentiation or bone resorption.
일 구현예에서, 본 발명의 조성물은 왕불류행 열수 추출물을 1 내지 2000 μg/ml의 농도로 포함할 수 있고, 바람직하게는 125 내지 1000μg/ml의 농도로 포함할 수 있으나, 이에 제한되지는 않는다.In one embodiment, the composition of the present invention may include the Wangbulryuhaeng hot water extract at a concentration of 1 to 2000 μg/ml, preferably at a concentration of 125 to 1000 μg/ml, but is not limited thereto. .
또한 본 발명은 삼릉(Sparganium Rhizoma) 추출물을 유효성분으로 함유하는 골 질환 예방 또는 치료용 약학 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition for preventing or treating bone disease containing an extract of Sparganium Rhizoma as an active ingredient.
일 구현예에서, 상기 골 질환은 골다공증(osteoporosis), 골 감소증(osteopenia), 골 융해성 전이(osteolytic metastasis), 노인성척추후만증(senile kyphosis) 및 파제트병(paget disease)으로 이루어진 군에서 선택되는 하나 이상일 수 있으며, 바람직하게는 골다공증 또는 골 감소증일 수 있고, 골다공증인 것이 더욱 바람직하다.In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. It may be one or more, preferably osteoporosis or osteopenia, and more preferably osteoporosis.
일 구현예에서, 본 발명의 조성물은 RANK 신호 경로(RANK signaling pathway)를 억제할 수 있다.In one embodiment, the composition of the present invention may inhibit the RANK signaling pathway.
일 구현예에서, 본 발명의 조성물은 파골세포 분화와 관련된 단백질 또는 유전자의 발현을 억제할 수 있다. 상기 파골세포 분화와 관련된 단백질 또는 유전자는 NFATc1(nuclear factor of activated T cell c1), c-Fos, TRAP(tartrate-resistant acid phosphatase) 및 OSCAR(osteoclast-associated receptor)로 이루어진 군으로부터 선택된 어느 하나일 수 있다.In one embodiment, the composition of the present invention can inhibit the expression of a protein or gene associated with osteoclast differentiation. The protein or gene related to osteoclast differentiation may be any one selected from the group consisting of NFATc1 (nuclear factor of activated T cell c1), c-Fos, tartrate-resistant acid phosphatase (TRAP) and OSCAR (osteoclast-associated receptor). have.
일 구현예에서, 본 발명의 조성물은 골 흡수와 관련된 유전자의 발현을 억제할 수 있다. 상기 골 흡수와 관련된 유전자는 MMP-9(matrix methalloproteinase 9), CTK(Cathepsin K) 및 CA2(carbonic anhydrase Ⅱ로 이루어진 군으로부터 선택된 어느 하나일 수 있다.In one embodiment, the composition of the present invention can inhibit the expression of genes related to bone resorption. The gene related to bone resorption may be any one selected from the group consisting of matrix methalloproteinase 9 (MMP-9), cathepsin K (CTK), and carbonic anhydrase II (CA2).
일 구현예에서, 본 발명의 조성물은 파골세포 융합과 관련된 유전자의 발현을 억제할 수 있다. 상기 파골세포 융합과 관련된 유전자는 ATP6v0d2(vacuolar H+-ATPase V0 domain의 d2isoform) 및 DC-STAMP(dendritic cell-specific transmembrane protein)로 이루어진 군으로부터 선택된 어느 하나일 수 있다.In one embodiment, the composition of the present invention can inhibit the expression of genes associated with osteoclast fusion. The osteoclast fusion-related gene may be any one selected from the group consisting of ATP6v0d2 (d2isoform of vacuolar H+-ATPase V0 domain) and DC-STAMP (dendritic cell-specific transmembrane protein).
일 구현예에서, 본 발명의 상기 골 질환은 파골세포 과다 분화 또는 과다 활성화에 의해 유발되는 것일 수 있다. 상기 파골세포의 분화 또는 활성화는 RANKL에 의해 유도되는 것일 수 있다.In one embodiment, the bone disease of the present invention may be caused by hyper-differentiation or hyper-activation of osteoclasts. The differentiation or activation of osteoclasts may be induced by RANKL.
일 구현예에서, 본 발명의 상기 골 질환은 파골세포 분화와 관련된 단백질 또는 유전자의 활성 증가 또는 발현 증가가 나타나는 것일 수 있고, 상기 파골세포 분화와 관련된 단백질 또는 유전자는 TRAP, NFATc1, c-Fos, RANK 및 OSCAR로 이루어진 군으로부터 선택되는 것일 수 있다.In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to osteoclast differentiation, and the protein or gene related to osteoclast differentiation is TRAP, NFATc1, c-Fos, It may be selected from the group consisting of RANK and OSCAR.
일 구현예에서, 본 발명의 상기 골 질환은 골 흡수와 관련된 단백질 또는 유전자의 활성 증가 또는 발현 증가가 나타나는 것일 수 있고, 상기 파골세포 분화와 관련된 단백질 또는 유전자는 CTK, MMP-9 및 CA2로 이루어진 군으로부터 선택되는 것일 수 있다.In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to bone resorption, and the protein or gene related to osteoclast differentiation is composed of CTK, MMP-9 and CA2 It may be selected from the group.
일 구현예에서, 본 발명의 상기 골 질환은 파골세포 융합과 관련된 단백질 또는 유전자의 활성 증가 또는 발현 증가가 나타나는 것일 수 있고, 상기 파골세포 분화와 관련된 단백질 또는 유전자는 ATP6v0d2 및 DC-STAMP로 이루어진 군으로부터 선택되는 것일 수 있다.In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to osteoclast fusion, and the protein or gene related to osteoclast differentiation is a group consisting of ATP6v0d2 and DC-STAMP It may be selected from
본 발명의 약학 조성물에는 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 면역성을 증가시킬 수 있다.The pharmaceutical composition of the present invention may further include an adjuvant. The adjuvant may be used without limitation as long as it is known in the art, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the immunity thereof.
본 발명의 용어, "치료"란, 달리 언급되지 않는 한, 상기 용어가 적용되는 질환 또는 질병, 또는 상기 질환 또는 질병의 하나 이상의 증상을 역전시키거나, 완화시키거나, 그 진행을 억제하거나, 또는 예방하는 것을 의미하며, 본원에서 사용된 상기 치료란 용어는 치료하는 행위를 말한다. 따라서 포유동물에 있어서 골 질환의 치료 또는 치료요법은 하기의 하나 이상을 포함할 수 있다:As used herein, the term "treatment" means, unless otherwise stated, the disease or condition to which the term applies, or one or more symptoms of the disease or condition, which reverses, ameliorates, inhibits the progression, or It means to prevent, and the term treatment as used herein refers to the act of treating. Accordingly, treatment or therapy for bone disease in a mammal may include one or more of the following:
(1) 골 질환의 성장을 저해함, 즉, 그 발달을 저지시킴;(1) inhibit the growth of, ie, arrest the development of, bone disease;
(2) 골 질환의 확산을 예방함, 즉, 전이를 예방함; (2) preventing the spread of bone disease, ie, preventing metastasis;
(3) 골 질환을 경감시킴; (3) alleviating bone disease;
(4) 골 질환의 재발을 예방함; 및 (4) prevent recurrence of bone disease; and
(5) 골 질환의 증상을 완화함(palliating) (5) alleviating the symptoms of bone disease (palliating)
여기에서 사용된 용어 "포유동물"은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다.As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.
만약, 수혜동물이 조성물의 투여에 견딜 수 있거나, 조성물의 그 동물에의 투여가 적합한 경우라면, 조성물은 "약학적으로 또는 생리학적으로 허용가능함"을 나타낸다. 투여된 양이 생리학적으로 중요한 경우에는 상기 제제는 "치료학적으로 유효량"으로 투여되었다고 말할 수 있다. 상기 제제의 존재가 수혜 환자의 생리학적으로 검출가능한 변화를 초래한 경우라면 상기 제제는 생리학적으로 의미가 있다.A composition is indicated to be "pharmaceutically or physiologically acceptable" if the recipient animal can tolerate administration of the composition, or if administration of the composition to that animal is suitable. When the amount administered is physiologically important, the agent can be said to have been administered in a "therapeutically effective amount". An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.
본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어있다.The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질병의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 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 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. .
본 발명에서 사용되는 용어, "약학적으로 허용가능한"이란 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다. As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.
본 발명의 약학 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
본 발명에서 사용되는 용어, "투여"란, 임의의 적절한 방법으로 환자에게 소정의 물질을 제공하는 것을 의미하며, 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 주사 제형으로 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal or topical administration according to a desired method) ) or oral administration, and the dosage varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 약학 조성물 중 포함되는 유효성분의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 약학적 조성물에 0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 인체에 독성을 나타낼 수 있다.The composition of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the age, weight, sex, physical condition, etc. of the subject. is selected taking into account. It is self-evident that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.
본 발명의 약학 조성물은 다양한 경구 또는 비경구 투여 형태로 제형화될 수 있다. 경구 투여용 제형으로는 예를 들면 정제, 환제, 경질, 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/ 또는 폴리에틸렌 글리콜)를 추가로 포함할 수 있다. 또한, 상기 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유할 수 있다. 상기 제형은 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다. 또한, 비경구 투여용 제형의 대표적인 것은 주사용 제제이며, 주사용 제제의 용매로서 물, 링거액, 등장성 생리식염수 또는 현탁액을 들 수 있다. 상기 주사용 제제의 멸균 고정 오일은 용매 또는 현탁 매질로서 사용할 수 있으며 모노-, 디-글리세라이드를 포함하여 어떠한 무자극성 고정오일도 이러한 목적으로 사용될 수 있다. 또한, 상기 주사용 제제는 올레산과 같은 지방산을 사용할 수 있다. The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid. or a disintegrant such as its sodium salt or a boiling mixture and/or absorbent, coloring, flavoring and sweetening agent. The formulation may be prepared by conventional mixing, granulating or coating methods. In addition, a representative formulation for parenteral administration is an injection formulation, and examples of the solvent for the injection formulation include water, Ringer's solution, isotonic saline, or suspension. The sterile, fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose. In addition, the injection preparation may use a fatty acid such as oleic acid.
본 발명에서, 용어 "예방"이란 본 발명에 따른 약학 조성물의 투여에 의해 골 질환 또는 골다공증의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of bone disease or osteoporosis by administration of the pharmaceutical composition according to the present invention.
나아가, 본 발명은 삼릉(Sparganium Rhizoma) 추출물을 유효성분으로 함유하는 골 질환 예방 또는 개선용 식품 조성물에 관한 것이다.Furthermore, the present invention relates to a food composition for preventing or improving bone disease containing an extract of Sparganium Rhizoma as an active ingredient.
일 구현예에서, 상기 골 질환은 골다공증(osteoporosis), 골 감소증(osteopenia), 골 융해성 전이(osteolytic metastasis), 노인성척추후만증(senile kyphosis) 및 파제트병(paget disease)으로 이루어진 군에서 선택되는 하나 이상일 수 있으며, 바람직하게는 골다공증 또는 골 감소증일 수 있고, 골다공증인 것이 더욱 바람직하다.In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. It may be one or more, preferably osteoporosis or osteopenia, and more preferably osteoporosis.
본 발명의 조성물을 식품 조성물로 사용하는 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 상기 조성물은 유효성분 이외에 식품학적으로 허용가능한 식품보조첨가제를 포함할 수 있으며, 유효성분의 혼합량은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the composition of the present invention is used as a food composition, the composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may include a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).
본 발명에서 사용되는 용어 "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.As used in the present invention, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에서 사용되는 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 '기능성'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 통상의 기술분야에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 통상의 기술분야에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한없이 제조될 수 있다. 본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 관절염 또는 골다공증 치료제의 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method commonly used in the ordinary technical field, and at the time of the preparation, it can be prepared by adding raw materials and components commonly added in the conventional technical field. In addition, if the formulation of the health functional food is also recognized as a health functional food, it may be manufactured without limitation. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and it is excellent in portability, and the present invention health functional food can be taken as an adjuvant to enhance the effect of arthritis or osteoporosis treatment.
또한, 본 발명의 조성물이 사용될 수 있는 건강식품의 종류에는 제한이 없다. 아울러 본 발명의 추출물 또는 이의 분획물을 활성성분으로 포함하는 조성물은 당업자의 선택에 따라 건강기능식품에 함유될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림 류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다.In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition comprising the extract of the present invention or a fraction thereof as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.
더 나아가, 본 발명은 삼릉(Sparganium Rhizoma) 추출물을 유효성분으로 함유하는 파골세포 분화 또는 골 흡수 억제용에 관한 것이다.Furthermore, the present invention relates to a use for inhibiting osteoclast differentiation or bone resorption containing an extract of Sparganium Rhizoma as an active ingredient.
일 구현예에서, 상기 파골세포 분화 또는 골 흡수는 RANKL에 의해 유도되는 것일 수 있다.In one embodiment, the osteoclast differentiation or bone resorption may be induced by RANKL.
본 발명의 조성물은 천연 재료를 원료로 하므로 약학 조성물 또는 식품 조성물로 사용할 경우에도 일반적인 합성 화합물에 비하여 부작용이 덜할 수 있으므로, 안전하게 약학 조성물 및 건강기능식품에 포함되어 유용하게 사용될 수 있다.Since the composition of the present invention is made from natural materials, side effects may be less than that of general synthetic compounds even when used as a pharmaceutical composition or a food composition, so it can be safely included in pharmaceutical compositions and health functional foods to be usefully used.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.
<실시예 1> 삼릉 추출물의 제조<Example 1> Preparation of Samreung extract
삼릉(三稜, 중국)은 옴니허브를 통하여 구입하였다. 삼릉 150g에 80%(v/v) 에탄올 수용액 1500ml을 첨가하여 2주간 침지 추출하였다. 추출액은 여과지(Whatman, No 2)로 여과하였다. 이후 감압농축기를 이용하여 농축 후 동결건조하여 분말화 하였다. 최종적으로 수득된 건조된 삼릉 에탄올 추출물(Ethanol extract of Sparganium Rhizoma, SR)은 5.5 g이었으며, 수득률은 3.7%였다. 상기 삼릉 에탄올 추출물(SR)은 사용하기 전까지 냉장 보관하였다.Samneung (三稜, China) was purchased through Omni Hub. To 150 g of Samneung, 1500 ml of 80% (v/v) ethanol aqueous solution was added, followed by immersion extraction for 2 weeks. The extract was filtered with filter paper (Whatman, No 2). After concentration using a vacuum concentrator, it was freeze-dried and powdered. The finally obtained dried ethanol extract of Sparganium Rhizoma (SR) was 5.5 g, and the yield was 3.7%. The Samneung ethanol extract (SR) was refrigerated until use.
<실시예 2> 세포 독성 확인<Example 2> Confirmation of cytotoxicity
RAW 264.7 세포는 100ø 디쉬(dish)에서 10% FBS 및 1% P/S가 혼합된 DMEM 배지를 사용하여, 95%의 습도, 37℃의 온도 및 5% CO2 환경조건의 배양기에서 배양하였다. RAW 264.7 세포를 96-웰 플레이트(96-well plate)에 분주하여 24시간 동안 배양하였다. 안정화 후 배지를 제거하고 새 배지를 첨가하면서, 상기 실시예 1에서 제조한 SR 분말을 멸균 필터 (포어 사이즈 0.22 μm)로 여과하여 각각의 배양액에 125, 250, 500 또는 1000 μg/ml 농도로 첨가한 뒤 24시간 동안 배양하였다. 그 후 MTS 분석 용액을 처리한 후 405 nm에서 흡광도를 측정하여 세포 생존율을 확인하였다. 세포 독성은 아무것도 처리하지 않은 대조군에 대한 백분율로 표기하였다. RAW 264.7 cells were cultured in a 100° dish using DMEM medium mixed with 10% FBS and 1% P/S, in an incubator of 95% humidity, 37° C., and 5% CO 2 environmental conditions. RAW 264.7 cells were seeded in a 96-well plate and cultured for 24 hours. After stabilization, the medium was removed and a fresh medium was added, and the SR powder prepared in Example 1 was filtered through a sterile filter (pore size 0.22 μm) and added to each culture medium at a concentration of 125, 250, 500 or 1000 μg/ml. and then incubated for 24 hours. Thereafter, the cell viability was confirmed by measuring the absorbance at 405 nm after treatment with the MTS assay solution. Cytotoxicity was expressed as a percentage relative to the untreated control group.
그 결과, 삼릉 에탄올 추출물(SR)은 모든 농도에서 세포독성을 나타내지 않았다 (도 1).As a result, Samneung ethanol extract (SR) did not show cytotoxicity at any concentration (Fig. 1).
<실시예 3> 파골세포 분화 억제능 확인<Example 3> Confirmation of osteoclast differentiation inhibitory ability
본 발명에 따른 삼릉 에탄올 추출물(실시예 1의 SR)의 파골세포 분화 억제능을 평가하기 위하여, TRAP (tartrateresistant acid phosphatase) 염색 및 TRAP activity 측정을 실시하였다.In order to evaluate the osteoclast differentiation inhibitory ability of the Samneung ethanol extract (SR of Example 1) according to the present invention, TRAP (tartrateresistant acid phosphatase) staining and TRAP activity were measured.
파골세포의 분화를 유도하기 위하여, 상기 실시예에서와 같이 RAW 264.7 세포를 96-웰 플레이트에 분주하여 24시간 배양한 후에 RANKL (100 ng/ml)를 첨가한 배지로 교환하였으며, 이때, 삼릉 실험군은 상기 배지에 SR을 125, 250, 500 또는 1000 μg/ml의 농도로 첨가하였고, 2일마다 동일한 배지로 교환하면서 5일 동안 배양하였다. 분화된 파골세포는 TRAP 분석 키트(assay kit)를 사용하여 염색하였다. 이후, 염색된 세포는 도립현미경을 사용하여 관찰하고, 핵이 3개 이상인 세포를 다핵의 파골세포로 계수하였다. 여기서, 세포에 RANKL을 처리하지 않은 세포를 정상군으로 확인하였다.In order to induce osteoclast differentiation, RAW 264.7 cells were aliquoted into 96-well plates and cultured for 24 hours as in the above example, and then exchanged with a medium to which RANKL (100 ng/ml) was added. SR was added to the medium at a concentration of 125, 250, 500 or 1000 μg/ml, and cultured for 5 days while replacing the same medium every 2 days. Differentiated osteoclasts were stained using a TRAP assay kit. Thereafter, the stained cells were observed using an inverted microscope, and cells having three or more nuclei were counted as multinucleated osteoclasts. Here, cells that were not treated with RANKL were identified as the normal group.
그 후, TRAP activity 측정을 위해 4-Nitrophenyl phosphate disodium salt hexahydrate (Sigma aldrich, MO, USA) 4.93 mg, 0.5 M acetate 850 μL 및 tartrate acid solution 150 μL를 섞어서 만든 시약과 상기 배양된 세포의 배지(배양액) 각 50μ를 새로운 96-웰 플레이트에 분주하고, 60분 동안 37℃에서 반응시켰다. 이 후 0.5M NaOH를 50 μL 처리하여 반응을 종료시킨 후 ELISA 기기로 405nm에서의 흡광도를 측정하여 TRAP 활성을 확인하였다.Then, for the measurement of TRAP activity, a reagent prepared by mixing 4.93 mg of 4-Nitrophenyl phosphate disodium salt hexahydrate (Sigma aldrich, MO, USA), 850 μL of 0.5 M acetate, and 150 μL of tartrate acid solution, and the culture medium (culture solution) ) 50 μ each was aliquoted into a new 96-well plate, and reacted at 37° C. for 60 minutes. After the reaction was terminated by treatment with 50 μL of 0.5M NaOH, the TRAP activity was confirmed by measuring the absorbance at 405 nm with an ELISA instrument.
그 결과, 대조군(RANKL만 처리한 군)은 정상군(RANKL 무처리군)에 비해 TRAP-positive 세포, 즉, 다핵의 파골세포가 많이 형성되었으나, SR을 처리한 실험군에서는 파골세포 형성이 감소하는 것을 확인하였다. 특히, TRAP 효소 활성(TRAP activity)을 측정한 결과, 대조군은 정상군에 비해 TRAP 효소 활성이 증가한 반면, SR을 처리한 군에서는 대조군에 비해 TRAP 효소 활성이 감소하는 것으로 나타났다(도 2).As a result, more TRAP-positive cells, that is, multinucleated osteoclasts, were formed in the control group (group treated with RANKL only) compared to the normal group (group not treated with RANKL), but osteoclast formation was decreased in the experimental group treated with SR. confirmed that. In particular, as a result of measuring TRAP enzyme activity (TRAP activity), the control group showed an increase in TRAP enzyme activity compared to the normal group, whereas the SR-treated group showed a decrease in TRAP enzyme activity compared to the control group (FIG. 2).
<실시예 4> 골 흡수 억제 효과 확인<Example 4> Confirmation of the effect of inhibiting bone resorption
본 발명에 따른 삼릉 에탄올 추출물(실시예 1의 SR)의 골 흡수 억제 효과를 평가하기 위하여, Pit 형성 억제능 분석을 실시하였다.In order to evaluate the bone resorption inhibitory effect of the Samneung ethanol extract (SR of Example 1) according to the present invention, a pit formation inhibitory ability analysis was performed.
상기 실시예에서와 같이 배양하되, RAW 264.7 세포를 osteo assay surface multiple well plate에 분주한 후 24시간 뒤에, RANKL (100 ng/ml)와 SR 125, 250, 500 또는 1000 μg/ml가 첨가된 배양액으로 교환하여 5일간 배양하였다. 첨가한 배양액으로 교환하여 안정화시켰다. 이후 분화 종료 후 4% NaCIO를 처리하여 세포를 떼어냈다. 플레이트는 증류수로 수세하고 도립현미경을 이용하여 파골세포가 형성한 구멍(pit)을 관찰하여 흡수된 pit 면적을 분석하였다. Pit 면적(pit area)은 Image J software Ver. 1.46 (National Institutes of Health, Washington D.C, USA)을 사용하여 측정하였고, 전체 면적에 대한 pit 면적을 백분율로 나타내었다.Cultivated as in the above example, but 24 hours after the RAW 264.7 cells were dispensed on the osteo assay surface multiple well plate, RANKL (100 ng/ml) and
그 결과, RANKL만 처리한 대조군은 정상군에 비해 pit 면적이 증가하였으나, SR을 처리한 실험군에서는 농도 의존적으로 pit 면적이 감소하는 것으로 나타나, SR이 골 흡수를 나타내는 pit의 형성을 억제함을 확인하였다(도 3).As a result, the pit area was increased in the control group treated only with RANKL compared to the normal group, but the pit area decreased in a concentration-dependent manner in the experimental group treated with SR, confirming that SR suppressed the formation of pits indicating bone resorption. (FIG. 3).
<실시예 5> 파골세포 액틴 고리 억제 효과 확인<Example 5> Confirmation of osteoclast actin ring inhibitory effect
본 발명에 따른 삼릉 에탄올 추출물(실시예 1의 SR)의 파골세포 액틴 고리(actin ring) 억제능을 평가하기 위하여, 면역형광분석을 실시하였다.In order to evaluate the osteoclast actin ring inhibitory ability of Samneung ethanol extract (SR of Example 1) according to the present invention, immunofluorescence analysis was performed.
상기 실시예에서와 같이 RAW 264.7 세포를 96-웰 플레이트에 분주하고 24시간 후에 RANKL (100 ng/ml)과 SR 125, 250, 500 또는 1000 μg/ml를 첨가한 배지로 교환하여 5일 동안 배양하였다. 그 후, 동일한 배지로 2일마다 교환하여 성숙한 파골세포를 형성할 때까지 배양하여 안정화시켰다. 그 후, 배지를 제거하고 4% 포름알데하이드로 고정한 뒤 0.1% 트리톤 X-100(in PBS)을 1분 동안 처리하여 세포의 투과성을 높였다. 그 후, 액틴 고리를 시각화하기 위하여, Acti-stain™ 488 Fluorescent Phalloidin으로 F-액틴 고리를 염색한 다음, 액틴 고리와 DAPI 염색을 면역현광현미경을 사용하여 관찰하였다.As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, exchanged with a medium containing RANKL (100 ng/ml) and
그 결과, 대조군 (RANKL 처리군)은 정상군 (RANKL 무처리군)에 비해 성숙한 액틴 고리가 형성된 반면, SR을 처리한 실험군에서는 액틴 고리 형성이 억제되는 것으로 나타났다 (도 4).As a result, it was found that the control group (RANKL-treated group) formed a mature actin ring compared to the normal group (RANKL untreated group), whereas the SR-treated experimental group suppressed the actin ring formation ( FIG. 4 ).
<실시예 6> 파골세포 분화 전사인자 발현 억제 효과 확인<Example 6> Confirmation of the inhibitory effect of osteoclast differentiation transcription factor expression
본 발명에 따른 삼릉 에탄올 추출물(실시예 1의 SR)이 파골세포 분화의 핵심기전 인자인 NFATc1 및 c-Fos의 단백질 발현에 미치는 영향을 하기의 방법으로 확인하였다.The effect of the Samneung ethanol extract (SR of Example 1) according to the present invention on the protein expression of NFATc1 and c-Fos, which are key mechanisms of osteoclast differentiation, was confirmed by the following method.
상기 실시예에서와 같이 RAW 264.7 세포를 96-웰 플레이트에 분주하고 24시간 후에 RANKL (100 ng/ml)과 SR 125, 250, 500 또는 1000 μg/ml를 첨가한 배지로 교환하여 1일 동안 배양하였다. 그 후 배양된 세포를 PBS로 세척한 다음, 프로테이즈 억제제(protease inhibitor) 및 포스파테이즈 억제제(phosphatase inhibitor)가 들어있는 RIPA 버퍼 (50 mM Tris.Cl, 150 mM NaCl, 1% NP-40, 0.5% Na.deoxycholate 및 0.1% SDS)를 사용하여 단백질을 용해하여, 세포에서 단백질을 추출하였다.As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, exchanged with a medium containing RANKL (100 ng/ml) and
추출한 단백질을 BCA protein assay로 정량한 후, western blot assay를 통해 1, 2차 항체 반응으로 검출하기 위하여 10% polyacrylamide/SDS 젤에서 전기영동한 뒤, nitrocellulose transfer 멤브레인에 트랜스퍼하였다. 그 후 멤브레인을 TBST(0.05% Tween-20 in Tris-buffered saline, pH 7.4)에 녹인 5% 스킴밀크로 블로킹하고 항-NFATc1 항체 및 항-c-Fos 항체로 반응시킨 뒤, 2차 항체로 반응시켰다. 그 다음, ECL 용액(Santacruz, California USA)을 1:1로 잘 섞어서 멤브레인 위에 가하여 발광(발색)시키고, X-ray 필름에 현상하여, 단백질 발현 정도를 확인하였다. NFATc1 및 c-Fos의 단백질 발현량은 Actin을 통해 정량화 하였다.The extracted protein was quantified by BCA protein assay, and then electrophoresed on a 10% polyacrylamide/SDS gel to detect primary and secondary antibody reactions through western blot assay, and then transferred to a nitrocellulose transfer membrane. After that, the membrane was blocked with 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4) and reacted with anti-NFATc1 antibody and anti-c-Fos antibody, followed by reaction with a secondary antibody. made it Then, the ECL solution (Santacruz, California USA) was mixed well at a 1:1 ratio, added to the membrane to emit light (color development), and developed on an X-ray film to check the protein expression level. Protein expression levels of NFATc1 and c-Fos were quantified through Actin.
그 결과, RANKL 을 처리한 대조군은 정상군에 비해 NFATc1 및 c-Fos 단백질의 발현이 증가하였으며, SR을 처리한 실험군은 NFATc1 및 c-Fos의 발현이 대조군에 비해 유의하게 감소하는 것을 확인하였다(도 5). 이를 Actin을 통해 정량하였을 때도, SR을 처리한 실험군은 NFATc1/c-Fos의 발현이 500 및 1000 μg/ml 농도에서 유의하게 억제되는 것으로 나타났다(p<0.05, p<0.01).As a result, the control group treated with RANKL increased the expression of NFATc1 and c-Fos compared to the normal group, and it was confirmed that the expression of NFATc1 and c-Fos in the experimental group treated with SR was significantly decreased compared to the control group ( 5). When this was quantified through Actin, the SR-treated experimental group showed that the expression of NFATc1/c-Fos was significantly inhibited at concentrations of 500 and 1000 μg/ml (p<0.05, p<0.01).
<실시예 7> 파골세포 분화 및 골흡수 관련 유전자 발현 확인<Example 7> Confirmation of expression of genes related to osteoclast differentiation and bone resorption
본 발명에 따른 삼릉 에탄올 추출물(실시예 1의 SR)이 파골세포의 핵심기전과 관련된 유전자(mRNA)의 발현에 미치는 영향을 하기의 방법으로 확인하였다.The effect of the Samneung ethanol extract (SR of Example 1) according to the present invention on the expression of genes (mRNA) related to the core mechanism of osteoclasts was confirmed by the following method.
상기 실시예에서와 같이 RAW 264.7 세포를 96-웰 플레이트에 분주하고 24시간 후에 RANKL (100 ng/ml)과 SR 125, 250, 500 또는 1000 μg/ml를 첨가한 배지로 교환하여 4일 동안 배양하였다. 배양된 세포에 Trizol 용액 (Invitrogen, USA)을 처리하여 전체 RNA를 추출하였다. 추출한 RNA를 후 nanodrop 기기를 통해 2 μg으로 정량한 뒤, 역전사효소(reverse transcriptase)를 사용하여 cDNA(complementary DNA)로 합성하였다. 합성된 cDNA는 Taq 폴리머레이즈(polymerase)를 사용하여 PCR으로 증폭하였으며, 하기 표 1의 프라이머들을 사용하여 real-time PCR을 수행하였다. 이 후, 결과물을 1.2% 아가로스 젤에서 전기영동하여 SYBR 그린으로 염색하였다. 각 유전자의 발현량은 GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)를 이용하여 mRNA 농도를 표준화하였고, Image J software (Ver. 1.46, National Institutes of Health)를 사용하여 각 밴드의 밀도(band density)를 분석하였다.As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, exchanged with a medium containing RANKL (100 ng/ml) and
GR: TCA GCA CAT AGC CCA CAC CGF: ACT TCC CCA GCC CTT ACT ACC
GR: TCA GCA CAT AGC CCA CAC CG
R: CGT CTT CCA CCT CCA CGT CGF: TGC TCC TCC TCC TGC TGC TC
R: CGT CTT CCA CCT CCA CGT CG
R: GGC TGC CAA AAT AAA CTC CAF: ATG GGC TCT CCT GTC AAC AC
R: GGC TGC CAA AAT AAA CTC CA
R: ACC ATC TTC TCC TCC CHA GTF: AAA CCT TGG ACC AAC TGC AC
R: ACC ATC TTC TCC TCC CHA GT
R: CCG AGC CAA GAG AGC ATA TCF: AGG CGG CTA TAT GAC CAC TG
R: CCG AGC CAA GAG AGC ATA TC
R: TGA AGG TTT GGA ATC GAC CCF: CGA CTT TTG TGG TCT TCC CC
R: TGA AGG TTT GGA ATC GAC CC
R: ATC CAG GTC ACA CAT TCC AGC AF: CTC TCA GGA CAA TGC AGT GCT GA
R: ATC CAG GTC ACA CAT TCC AGC A
R: CCA AGG AGC CAG AAC CTT CGA AAC TF: CTG CTG GTA ACG GAT CAG CTC CCC AGA
R: CCA AGG AGC CAG AAC CTT CGA AAC T
R: CGA CAG CGT CAA ACA AAG GCT TGT AF: ATG GGGCCTTGCAAAAGAAATCTG
R: CGA CAG CGT CAA ACA AAG GCT TGT A
R: CTC GGT TTC CCG TCA GCC TCT CTCF: TGG AAG TTC ACT TGA AAC TAC GTG
R: CTC GGT TTC CCG TCA GCC TCT CTC
R: TGC AGC GAA CTT TAT TGA TGF: ACT TTG TCA AGC TCA TTT CC
R: TGC AGC GAA CTT TAT TGA TG
SR이 파골세포 분화의 주된 기전의 지표인 TRAP, NFATc1, c-Fos, RANK 및 OSCAR와 골 흡수 관련 기전의 지표인 CTK, MMP-9 및 CA2 및 파골세포 융합 관련 기전의 지표인 ATP6v0d2 및 DC-STAMP의 mRNA 발현에 미치는 영향을 분석한 결과, RANKL만 처리한 대조군에서는 TRAP, NFATc1, c-Fos, RANK, MMP-9, CA2, OSCAR, ATPv06d2 및 DC-STAMP의 mRNA 발현이 정상군에 비해 유의하게 증가한 반면, SR 처리군에서는 TRAP, NFATc1, c-Fos, RANK, MMP-9, CA2, OSCAR, ATPv06d2 및 DC-STAMP의 발현이 농도 의존적으로 억제되는 것으로 나타났다 (도 6).SR is TRAP, NFATc1, c-Fos, RANK and OSCAR, which are indicators of the main mechanisms of osteoclast differentiation, CTK, MMP-9 and CA2, which are indicators of mechanisms related to bone resorption, and ATP6v0d2 and DC-, which are indicators of mechanisms related to osteoclast fusion. As a result of analyzing the effect on the mRNA expression of STAMP, the mRNA expression of TRAP, NFATc1, c-Fos, RANK, MMP-9, CA2, OSCAR, ATPv06d2 and DC-STAMP was significantly higher in the control group treated with only RANKL than in the normal group. On the other hand, in the SR treatment group, the expression of TRAP, NFATc1, c-Fos, RANK, MMP-9, CA2, OSCAR, ATPv06d2 and DC-STAMP was concentration-dependently inhibited (Fig. 6).
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