KR20190046016A - Pharmaceutical composition for use in preventing or treating osteoporosis containing stigmasterol as an active ingredient - Google Patents
Pharmaceutical composition for use in preventing or treating osteoporosis containing stigmasterol as an active ingredient Download PDFInfo
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- KR20190046016A KR20190046016A KR1020170139075A KR20170139075A KR20190046016A KR 20190046016 A KR20190046016 A KR 20190046016A KR 1020170139075 A KR1020170139075 A KR 1020170139075A KR 20170139075 A KR20170139075 A KR 20170139075A KR 20190046016 A KR20190046016 A KR 20190046016A
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- Life Sciences & Earth Sciences (AREA)
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Abstract
Description
본 발명은 스티그마스테롤 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising stigmasterol or a pharmaceutically acceptable derivative thereof as an active ingredient.
최근 각종 산업재해 및 교통사고가 증가하고 고령화 사회로 접어들면서 골다공증 및 그에 따른 골절등에 의한 근골격계 질환이 증가하고 있다. 이와 같은 골질환등의 예방과 치료를 위해 골 재생 능력유도를 위한 다양한 형태의 뼈이식제와 조골세포의 활성향상 및 파골세포의 활성을 저하시키기 위한 새로운 형태의 관련질환 치료제들이 개발되고 있다. 또한, 골종양, 골결손이 심한 부위를 채울 수 있는 신생골을 능동적으로 유도하기 위하여 줄기세포와 조골세포를 이용하여 다양한 연구들이 시도되고 있다.BACKGROUND ART [0002] As various industrial accidents and traffic accidents have increased and an aging society has recently been developed, musculoskeletal diseases caused by osteoporosis and fractures resulting from such diseases have been increasing. In order to prevent and treat bone diseases and the like, there have been developed various types of bone grafting agents for inducing bone regeneration ability, new types of related diseases for improving osteoclast activity and osteoclast activity. In addition, various studies using stem cells and osteoblasts have been attempted to actively induce new bone that can fill a bone tumor or bone defect.
골의 재형성 과정은 오래된 골이 주기적으로 새로운 골로 전환되는 과정으로, 이 과정은 증식, 분화 및 세포외기질의 석회화유도 등의 단계를 거쳐 진행된다. 일반적으로 성인의 골 형성과 흡수 과정은 중간엽줄기세포 유래의 조골세포와 조혈모세포 유래의 파골세포의 상호작용에 의해 균형을 이루면서 골의 건강을 유지하는데, 조골세포의 활성도가 낮아져 골 형성이 감소 되거나 파골세포의 활성도가 강해져 골 흡수가 증가 되는 등의 조골세포와 파골세포 간의 활성 불균형이 일어나게 되면 조직 내 화학조성에는 큰 변화가 없지만 골 질량이 감소하여 골다공증(osteoporosis)과 같은 골 대사질환이 유발될 수 있다.The bone remodeling process is a process in which old bone is periodically converted into a new bone, and this process proceeds through steps such as proliferation, differentiation, and induction of calcification of the extracellular matrix. In general, the bone formation and absorption process of adult is balanced by the interaction of osteoblasts derived from mesenchymal stem cells with osteoblast-derived osteoblasts, maintaining bone health, lowering osteoblast activity and reducing bone formation Or osteoclast activity increases osteoclast activity and increases osteoclast activity, there is no significant change in the chemical composition of the osteoblast and osteoclast. However, osteopenia such as osteoporosis is caused by decreased bone mass. .
골다공증은 유전적 요인이나 식이, 생활 방식과 같은 환경적 요인에 의해서도 영향을 받는 복합적인 질병으로, 특히 여성의 경우 폐경기에 이르면 호르몬의 변화에 의해 골 감소가 급격히 진행된다.Osteoporosis is a complex disease that is also influenced by genetic factors, environmental factors such as diet and lifestyle, and especially in women, bone turnover is dramatically accelerated by hormonal changes in menopause.
현재 시판되고 있는 골다공증 치료제는 비타민 D, 여성호르몬제, 비스포스포네이트제제, 선택적 에스트로겐 수용체 조절제, 칼시토닌 제제 등이 있는데 대부분 파골세포의 활성을 감소시켜 골 흡수를 조절함으로써 뼈의 소실을 막아주는 방식에 의하고 있다. 그러나 파골세포의 활성억제를 통한 골다공증 치료법들은 근본적으로 골다공증을 치료할 수 있는 치료법이 아니기 때문에 완치에 한계가 있으며 실질적인 골밀도 증가 및 골 강화가 이루어질 수 있는 조골세포 활성의 증가가 필수적으로 요구된다.Currently available osteoporosis drugs are vitamin D, female hormones, bisphosphonates, selective estrogen receptor modulators, and calcitonin preparations. Most of them are based on the way of preventing loss of bones by decreasing the activity of osteoclasts and regulating bone resorption . However, since osteoporosis treatments through inhibition of osteoclast activity are not fundamentally therapeutic methods for treating osteoporosis, there is a limit to cure and it is essential to increase osteoblast activity which can increase bone mass and bone strength.
또한, 골다공증은 약물의 단기 투여만으로는 치료할 수 없고 약물의 장기 투여가 필수적인 질환이므로, 약물을 장기 투여할 때에도 부작용이 없으면서 우수한 약효를 갖는 새로운 물질들의 개발이 요구되고 있다.In addition, osteoporosis is a disease that can not be treated only by short-term administration of a drug, and long-term administration of the drug is essential. Therefore, there is a need to develop new substances with superior drug efficacy without side effects when the drug is administered for a long period of time.
이에 따라, 최근에는 기존 치료법의 부작용 최소화와 골 소실을 최소화하면서 골 형성을 촉진할 수 있는 한약재 및 식품 등의 천연물 유래 활성성분을 이용한 대체요법 연구들이 활발히 진행되고 있다.Recently, alternative therapies using active ingredients derived from natural products such as herbal medicines and foods that can promote bone formation with minimal side effects and minimizing bone loss have been actively conducted.
한편, 스티그마스테롤은 곁사슬에 이중 결합을 갖는 식물성 스테롤(phytosterol)의 일종으로, 22번과 23번 탄소사이에 이중결합을 가진다는 점에서 콜레스테롤과 구별된다. 식물계에 널리 분포하고 있으며, 각종 스테로이드호르몬의 합성재제로서도 사용된다. 대두 스테롤(sterol)의 약 25%, 강낭콩, 야자기름이나 채종유, 맥문동(Ophiopogon japonicus) 등 기타 식물의 종자나 두류, 견과류, 채소류, 생우유에 널리 들어 있고, 특히 대두의 불검화물에서부터 추출, 정제되는 스테롤로서 호르몬 합성의 원료로서 이용되어져 왔다.On the other hand, stigmasterol is a kind of phytosterol having a double bond in the side chain and is distinguished from cholesterol in that it has a double bond between carbon 22 and carbon 23. It is widely distributed in plants and is also used as a synthesis agent for various steroid hormones. It is widely distributed in seeds, beans, nuts, vegetables, and raw milk of other plants such as kidney beans, palm oil, seed oil and Ophiopogon japonicus, and is extracted and purified from unsaponifiables of soybean, about 25% Sterols have been used as raw materials for hormone synthesis.
최근 연구보고에 따르면, 암을 억제하는 효과가 우수한 것으로 보고되고 있는데 유방암, 난소암, 전립선암, 결장암 등에 효과적이며, 콜레스테롤 수치를 저하시키는 작용이 있고 골격의 동통, 견비통, 신경통 및 해열, 진통, 협통 등에도 유효한 것이 밝혀진바 있다. 이와 관련하여, 한국등록특허공보 제10-1745504호에는 잠분추출물로부터 분리된 스티그마스테롤을 이용한 천연물 유래의 항암 조성물이 개시된 바 있다.It has been reported that a cancer suppressing effect is excellent in breast cancer, ovarian cancer, prostate cancer, colon cancer, etc. It has a function of lowering cholesterol level, and has a function of skeletal pain, shoulder pain, neuralgia and fever, It has been found that it is effective also for interlocking. In this regard, Korean Patent Registration No. 10-1745504 discloses a natural cancer-derived anticancer composition using stigmasterol isolated from a sparing extract.
그러나 스티그마스테롤에 대한 조골세포의 증식 및 분화에 미치는 영향에 대한 연구결과는 아직 보고된 바 없다.However, no study on the effect of stigmasterol on osteoblast proliferation and differentiation has been reported.
본 발명자들은 근본적으로 골다공증을 치료하기 위해 실질적인 골밀도 증가 및 골 강화를 위해 조골세포의 활성을 증가시키고, 장기간 복용하여도 부작용이 적은 새로운 약학적 조성물을 개발하기 위해 노력하던 중, 스티그마스테롤 또는 이의 약학적으로 허용 가능한 유도체들이 조골세포의 증식 및 분화를 촉진함을 확인함으로써 본 발명을 완성하였다.The present inventors intend to develop a new pharmaceutical composition which is basically effective to increase osteoblast activity and increase the osteoblast activity for bone strengthening and bone strengthening in order to treat osteoporosis, The present inventors have accomplished the present invention by confirming that allowable derivatives promote the proliferation and differentiation of osteoblasts.
따라서, 본 발명의 목적은 골다공증 예방 및 치료 효능이 우수하고 장기간 복용하여도 부작용이 적은 약학적 조성물 및 건강기능식품을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition and a health functional food having excellent osteoporosis prevention and treatment efficacy and having little side effects even when taken for a long time.
상기 목적을 달성하기 위해,In order to achieve the above object,
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
상기 화학식 1로 표시되는 화합물은 스티그마스테롤(stigmasterol)로 명명될 수 있다.The compound represented by Formula 1 may be named stigmasterol.
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다. The pharmaceutical composition may be characterized in promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be 0.01 to 10 nM.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 할 수 있다.The compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be used in combination with any one or more of osteolysis (Runx-related transcription factor 2), Dlx5 (distal-less homeobox 5), osteocalcin (OC), and alkaline phosphatase The expression level of the cell differentiation marker gene is increased.
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화되는 것을 특징으로 할 수 있다.The pharmaceutical composition may be formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 스티그마스테롤(stigmasterol)로 명명될 수The compound represented by the above formula (1) may be named stigmasterol
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다.The health functional food may be characterized by promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be 0.01 to 10 nM.
본 발명에 따른 약학적 조성물은 조골세포 분화 마커 유전자인 Dlx5, Runx2, ALP, OC의 발현량을 증가시킴으로써 조골세포의 분화를 촉진하는 효과가 있어 골다공증의 예방 또는 치료용 의약품 또는 건강식품으로 널리 이용될 수 있다.The pharmaceutical composition according to the present invention promotes the differentiation of osteoblast by increasing the expression level of Dlx5, Runx2, ALP, and OC, which are osteoblast differentiation marker genes, and is widely used as a medicine or a health food for prevention or treatment of osteoporosis .
또한, 파골세포를 표적으로 하기보다는 조골세포의 활성을 증가시킴으로써 골형성을 촉진시켜 근본적으로 골다공증을 치료할 수 있으며, 2차 골절 예방효과를 나타낼 수 있는 장점이 있다.In addition, rather than targeting osteoclasts, osteoblast activity can be increased to promote osteogenesis, thereby osteoporosis can be fundamentally treated, and secondary osteoporosis can be effectively prevented.
도 1은 스티그마스테롤(stigmasterol)의 농도에 따른 MTT 분석의 결과 그래프이다.
도 2는 스티그마스테롤(stigmasterol)에 의한 조골세포 분화 마커의 발현정도를 농도에 따라 비교한 <실험예 2>에 따른 결과이다.
도 3은 스티그마스테롤(stigmasterol)에 의한 조골세포 분화 마커의 발현정도를 처리 시간에 따라 비교한 <실험예 2>에 따른 결과이다.Figure 1 is a graph of MTT analysis according to the concentration of stigmasterol.
FIG. 2 shows results of Experimental Example 2 in which the degree of expression of osteoblast differentiation markers by stigmasterol was compared according to concentration.
FIG. 3 shows the results of Experimental Example 2 in which the degree of expression of osteoblast differentiation markers by stigmasterol was compared according to treatment time.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 명세서 및 청구범위에 사용되는 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and the inventor may properly define the concept of the term to describe its invention in the best possible way It should be construed as meaning and concept consistent with the technical idea of the present invention.
본 발명은 골다공증의 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating osteoporosis.
본 명세서에서 용어 골다공증은 뼈의 양이 감소하고 질적인 변화로 인하여 뼈의 강도가 약해진 상태를 의미하고, 골다공증의 예방, 개선 및 치료라 함은 골 밀도 저하, 골 손실로 인한 각종 질병을 모두 포함하는 것으로 해석된다.As used herein, the term osteoporosis refers to a state in which the amount of bone is reduced and the strength of the bone is weakened due to a qualitative change, and the prevention, improvement and treatment of osteoporosis includes all kinds of diseases caused by decreased bone density and bone loss .
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 스티그마스테롤 (stigmasterol)로 명명될 수 있다.The compound represented by Formula 1 may be named stigmasterol.
상기 화학식 1로 표시되는 화합물은 약학적으로 허용 가능한 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산, 아인산 등과 같은 무기산류, 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류 등과 같은 무독성 유기산, 아세트산, 안식향산, 구연산, 젖산, 말레인산, 글루콘산, 메탄설폰산, 4-톨루엔설폰산, 주석산, 푸마르산 등과 같은 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염의 종류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 다이하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트, 만델레이트 등을 포함한다.The compound represented by the formula (1) can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Examples of such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chloro Such as benzenesulfonate, benzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1-sulfonate, naphthalene-2-sulfonate, mandelate and the like.
본 발명에 따른 산 부가염은 통상의 방법으로 제조할 수 있으며, 예를 들면 화학식 1의 유도체를 메탄올, 에탄올, 아세톤, 디클로로메탄, 아세토니트릴 등과 같은 유기용매에 녹이고 유기산 또는 무기산을 가하여 생성된 침전물을 여과, 건조시켜 제조하거나, 용매와 과량의 산을 감압 증류한 후 건조시켜 유기용매 하에서 결정화시켜서 제조할 수 있다.The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the derivative of Chemical Formula 1 in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile and the like, Followed by filtration and drying, or by distillation of the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.
또한, 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 은염(예, 질산은)과 반응시켜 얻는다.In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).
상기 화학식 1로 표시되는 화합물인 스티그마스테롤은 공지된 방법에 의해 천연물로부터 추출된 것이나 합성된 것을 사용할 수 있으며, 그 제조방법이나 기원은 한정되지 않는다.The stigmasterol, which is a compound represented by the above formula (1), can be extracted from natural products or synthesized by a known method, and the production method and origin are not limited.
스티그마스테롤은 대두 스테롤(sterol)의 약 25%, 강낭콩, 야자기름이나 채종유, 맥문동(Ophiopogon japonicus)등 기타 식물의 종자나 두류, 견과류, 채소류, 생우유에 널리 들어 있고, 특히 대두의 불검화물에서부터 추출, 정제되는 스테롤로서 호르몬 합성의 원료로서 이용되어져 왔다고 알려져 있다.Stigmasterol is widely distributed in seeds, pulses, nuts, vegetables, and raw milk of other plants such as kidney beans, palm oil, seed oil, and Ophiopogon japonicus, about 25% of the sterol, , It has been known that it has been used as a raw material for hormone synthesis as a sterol to be purified.
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다. The pharmaceutical composition may be characterized in promoting osteoblast differentiation.
조골세포의 분화를 촉진시키는 화합물은 골다공증 및 골절 등과 같은 질환 및 증상의 예방 또는 치료에 사용될 수 있을 뿐만 아니라, 골 강화를 목적으로 하는 치주질환 치료에도 사용될 수 있으며(Zhang et al., ShanghaiKou Qiang Yi Xue 7(2), pp99-103, 1998; Cahill et al, Biol Blood Marrow Transplant 10(10), pp709-717, 2004), 화상으로 비롯되는 골 성장 장애 치료에 유용하게 사용될 수 있다는 연구도 보고된바 있다(Klein et al.,Osteoporos Int. 16(6), pp631-635, 2005).Compounds that promote osteoclast differentiation can be used not only for the prevention or treatment of diseases and symptoms such as osteoporosis and fracture but also for the treatment of periodontal disease for the purpose of bone strengthening (Zhang et al., Shanghai Kou Qiang Yi It has also been reported that it may be useful for the treatment of bone growth disorders resulting from burns, as reported by Xue 7 (2), pp 99-103, 1998; Cahill et al, Biol Blood Marrow Transplant 10 (10), pp709-717, (Klein et al., Osteoporos Int. 16 (6), pp. 631-635, 2005).
상기 약학적 조성물은 조골세포 등의 부족으로 인한 골 형성에 문제가 있는 질환에 제한 없이 사용될 수 있으며, 구체적으로 에스트로겐의 부족으로 인한 인터루킨-1(IL-1)에 의한 골 파괴와 염증성 질환 등이 포함될 수 있고, 과도한 파골 세포의 골 흡수에 의한 골다공증, 뼈전이암 병소(bone metastatic lesion), 원발성으로 뼈에 생성된 종양, 류마티스성 또는 퇴행성 관절염, 치주질환, 염증성 치조골 흡수질환, 염증성 뼈 흡수 질환 및 파제트병(Paget's disease) 등과 같은 병리학적 골 질환으로 골 파괴를 촉진하는 질환이 포함될 수 있다.The pharmaceutical composition can be used without limitation in diseases causing bone formation due to deficiency of osteoblasts and the like. More specifically, the pharmaceutical composition can be used for the treatment of bone destruction and inflammatory diseases caused by interleukin-1 (IL-1) And can be used to treat osteoporosis, bone metastatic lesion, primary bone tumor, rheumatic or degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease And Paget's < Desc / Clms Page number 2 > disease, and the like.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be 0.01 to 10 nM.
만일 농도가 0.01 nM 미만일 경우 약학적인 효과가 미미하게 나타날 수 있고, 10 nM를 초과할 경우 세포독성이 나타날 수 있는 문제점이 있다.If the concentration is less than 0.01 nM, the pharmacological effect may be insignificant, and if it is more than 10 nM, the cytotoxicity may occur.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 할 수 있다.The compound represented by
조골세포 분화는 호르몬, 뼈형성단백질(bone morphogenetic proteins:BMPs)과 같은 사이토카인, Runx2(runt-related transcription factor 2), Id1(inhibitor of DNA binding 1), Dlx5(distal-less homeobox 5), OC(osteocalcin), ALP(alkaline phosphatase)과 같은 다양한 전사인자에 의해서 조절된다. 그 중에서도 Id1, Dlx5, Runx2는 조골세포 분화 초기에 발현되는 필수적인 유전자로 이들 유전자의 발현은 조골세포 분화가 가속화됨을 의미한다.Osteoblast differentiation can be induced by a combination of hormones, cytokines such as bone morphogenetic proteins (BMPs), runt-related transcription factor 2 (Runx2), inhibitor of DNA binding 1, Dlx5 (distal-less homeobox 5) (osteocalcin), and alkaline phosphatase (ALP). Among them, Id1, Dlx5, and Runx2 are essential genes that are expressed at the early stage of osteoblast differentiation. Expression of these genes means that osteoblast differentiation is accelerated.
구체적으로, Runx2(runt-related transcription factor 2)는 조골세포 분화에 있어 중요한 조절자로 많이 알려져 있으며 다분화능세포 또는 조골전구세포에서 ALP(alkaline phosphatase), OC(osteocalcin) 그리고 골격시알로단백질(bone sialoprotein)과 같은 유전자를 조절함으로써 조골세포 분화를 촉진하는 유전자이다.Runx-related transcription factor 2 (Runx2) is known to be an important regulator of osteoblast differentiation, and it is known that ALP (alkaline phosphatase), OC (osteocalcin) and bone sialoprotein ) To control osteoblast differentiation.
Id1(inhibitor of DNA binding 1)은 조골세포에서 세포의 성장과 분화 사이의 균형에 영향을 미치는 전사인자이고, 조골세포 특이적 분화에 있어서 초기 단계에 중요한 역할을 하는 것으로 알려져 있다. 뿐만 아니라 근원세포, 중간엽 줄기세포에서 조골세포 분화로 전환될 때 Id1(inhibitor of DNA binding 1) 유전자 발현의 조절에 따라 분화가 촉진된다. Id1 (inhibitor of DNA binding 1) is a transcription factor that affects the balance between cell growth and differentiation in osteoblasts and is known to play an important role in the early stages of osteoblast-specific differentiation. In addition, differentiation is promoted by the regulation of Id1 (inhibitor of DNA binding 1) gene expression when transformed into osteoblast differentiation from myoblast and mesenchymal stem cells.
Dlx5(distal-less homeobox 5) 또한 뼈에 의해 유도되며 Runx2(runt-related transcription factor 2)의 프로모터에 직접적으로 결합하여 전사를 조절하고, 조골세포 분화의 후기 표지 유전자인 OC(osteocalcin)의 유전자 발현을 조절함으로써 조골세포 분화를 조절한다.Dlx5 (distal-less homeobox 5) is also induced by bone and directly binds to the promoter of Runx2 (runt-related transcription factor 2) to regulate transcription, and gene expression of OC (osteocalcin), the late marker gene of osteoblast differentiation To control osteoblast differentiation.
상기 약학적 조성물은 임상 투여시에 경구 또는 비경구로 투여가 가능하며, 일반적인 의약품 제제의 형태로 사용될 수 있다.The pharmaceutical composition can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화되는 것을 특징으로 할 수 있다.The pharmaceutical composition may be formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
보다 상세하게는, 각각 통상적인 방법에 따라 산제, 과립제, 정제, 캡슐, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 멸균 주사용액, 사전 충전식 주사 용액제의 형태 또는 동결건조된 형태로 제형화할 수 있으나, 이에 제한되는 것은 아니다.More specifically, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppository sterilized injection solutions, But the present invention is not limited thereto.
제형화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조될 수 있다.In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.
경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 유효 성분에 적어도 하나 이상의 부형제, 예컨대, 전분, 칼슘 카르보네이트, 수크로오스, 락토오스, 젤라틴 등을 혼합하여 제조할 수 있다. 또한, 단순한 부형제 이외에도 마그네슘 스테아레이트, 탈크와 같은 윤활제도 사용될 수 있다.Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included in addition to water and liquid paraffin which are simple diluents commonly used have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
본 발명의 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 증상의 정도, 약물 형태, 투여 경로 및 기간에 따라 적절하게 선택될 수 있다.A preferable dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the condition and the weight of the patient, the degree of symptoms, the drug form, the administration route and the period.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체를 유효성분으로 함유하는 골다공증의 예방 또는 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
상기 화학식 1로 표시되는 화합물은 스티그마스테롤 (stigmasterol)로 명명될 수 있다.The compound represented by
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 할 수 있다.The health functional food may be characterized by promoting osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 할 수 있다.The concentration of the compound represented by
만일 농도가 0.01 nM 미만일 경우 약학적인 효과가 미미하게 나타날 수 있고, 10 nM를 초과할 경우 세포독성이 나타날 수 있는 문제점이 있다.If the concentration is less than 0.01 nM, the pharmacological effect may be insignificant, and if it is more than 10 nM, the cytotoxicity may occur.
상기 건강기능식품은, 비제한적으로 각종 음료, 껌, 차, 과자, 비타민 복합체, 건강 보조식품 등의 형태로 제조될 수 있다.The health functional food may be manufactured in the form of, but not limited to, various drinks, gum, tea, confectionery, vitamin complex, health supplement, and the like.
또한, 상기 건강기능식품은 골다공증 개선을 목적으로 건강식품에 첨가되는 경우도 포함하며, 식품의 종류에 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.In addition, the health functional food includes a case where it is added to health food for the purpose of improving osteoporosis, and there is no particular limitation on the kind of food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.
상기 건강기능식품의 바람직한 섭취량은 섭취자의 상태 및 체중, 증상의 정도, 식품 형태, 섭취 기간에 따라 다르며 적절하게 선택될 수 있다.The preferred amount of the health functional food to be consumed depends on the condition and the weight of the recipient, the degree of symptoms, the type of food, the period of consumption, and may be appropriately selected.
골의 형성과정에 조골세포의 분화는 유전형질의 발현에 의해 조절되며, 배양방법에 따라 고유의 특성을 가진다. 골세포의 분화 및 골 형성에 관여하는 중요한 신호전달체계는 대표적으로 변화 성장 인자-β(transforming growth factor-β: TGF-β)와 뼈형성단백질(bone morphogenetic protein: BMP), 윈트/베타-카테닌(Wnt/β-catenin), 섬유아세포 증식인자(fibroblast growth factor: FGF), 헤지호그(hedgehog), 노치(notch) 등이 알려져 있다.The differentiation of osteoblasts during bone formation is controlled by the expression of genotypes and has inherent characteristics depending on the culture method. Important signaling pathways involved in osteocyte differentiation and osteogenesis are transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and beta-catenin , Fibroblast growth factor (FGF), hedgehog, and notch are known.
이러한 신호전달체계는 골세포 분화과정 동안 Runx2(Runt-related transcription factor 2) 및 Dlx5(Distalless-related homeobox 5) 등과 같은 골 형성과 관련한 다양한 전사인자의 발현과 활성화를 유도하며, ALP(alkaline phosphatase), Id1(inhibitor of DNA binding 1), OC(Osteocalcin) 등의 골 분화 관련 유전자를 발현시킨다. 이러한 조골세포에서의 분화 유도 물질로는 아스코르브산(ascorbic acid:AA), 베타-글리세로포스페이트(β-glycerophosphate:β-GP), 덱사메타손(dexamethasone) 등이 알려져 있다.These signaling pathways induce the expression and activation of various transcription factors related to osteogenesis such as Runx2 (Runt-related transcription factor 2) and Dlx5 (Distalless-related homeobox 5) during osteoclast differentiation, and ALP (alkaline phosphatase) , Id1 (inhibitor of DNA binding 1), and OC (osteocalcin). As a differentiation inducer in such osteoblasts, ascorbic acid (AA), beta-glycerophosphate (beta -GP), dexamethasone and the like are known.
본 발명에서는 골조직에 존재하는 조골세포와 유사한 MC3T3-E1 전조골세포(preosteoblasts)를 이용하여 스티그마스테롤이 조골세포의 증식 및 분화에 미치는 영향을 알아보기 위한 실험을 진행하였으며, 그 결과 스티그마스테롤은 조골세포의 증식 및 분화에 긍정적인 효과를 나타냄을 확인할 수 있었다.In the present invention, experiments were conducted to investigate the effect of stigmasterol on the proliferation and differentiation of osteoblasts using MC3T3-E1 preosteoblasts similar to osteoblasts present in bone tissue. As a result, It was confirmed that the cell proliferation and differentiation were positively effected.
이하, 본 발명을 구체적으로 설명하기 위해 실시예 및 실험예를 들어 상세하게 설명하기로 한다. 그러나 본 발명에 따른 실시예 및 실험예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예 및 실험예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and thus are not limitative of the present invention, It is provided for a complete description.
<< 실험예Experimental Example 1> 세포독성실험 1> Cytotoxicity experiment
1-1. 실험방법1-1. Experimental Method
(1) 실험재료 및 세포배양(1) Experimental materials and cell culture
마우스 섬유아세포(Mouse fibroblast cell)에서 유래된 C3H10T1/2 cell(ATCC, Manassas, VA, USA)및 마우스 두개관(Mouse calvaria)에서 유래된 MC3T3-E1 cell(ATCC, Manassas, VA, USA)과 스티그마스테롤(Sigma-Aldrich Co., St. Louis, MO, USA)을 사용하여 실험을 진행하였다.MC3T3-E1 cells (ATCC, Manassas, Va., USA) derived from mouse fibroblast cell-derived C3H10T1 / 2 cells (ATCC, Manassas, Va., USA) and mouse calvaria Sterol (Sigma-Aldrich Co., St. Louis, MO, USA).
세포 배양에는 Dulbeco’s Modified Eagle’s Medium-high glucose(DMEM, Gibco, Grand Island, NY, USA), 포스페이트 버퍼 살린(PBS, Gibco, Grand Island, NY, USA)을 사용하였다.Cell culture was performed with Dulbecco's Modified Eagle's medium-high glucose (DMEM, Gibco, Grand Island, NY, USA) and phosphate buffer saline (PBS, Gibco, Grand Island, NY, USA).
계대 배양은 우선 배양액을 석션한 뒤 포스페이트 버퍼살로 워싱하였다. 그리고 0.25% 트립신EDTA(trypsin EDTA) 500μl를 처리 한 다음, 인큐베이터(37℃, 5min)에서 반응하여 세포가 하얗게 보일 때 새로운 배양액을 1ml 첨가하고 원심분리(1200rpm, 4℃, 5min)를 진행하였다. 펠렛을 제외한 1ml의 배양액을 석션한 뒤 다시 1ml의 배양액을 첨가하여 인버팅 후 10μl을 혈구계(hemacytometer)를 이용해 세포를 계수하였다. 세포 계수가 끝나면 새로운 접시에 배양액 7ml 첨가 후 세포를 접종하였고, 배양에 사용된 배양액은 2일에 한번씩 갈아 주었다. For the subculture, the culture solution was first sucked and washed with phosphate buffer sal. Then, 500 μl of 0.25% trypsin EDTA was treated and reacted at an incubator (37 ° C., 5 min). When the cells appeared whiter, 1 ml of a new culture was added and centrifuged (1200 rpm, 4 ° C., 5 min). 1 ml of the culture solution except for the pellet was sucked, and then 1 ml of the culture solution was added. After inversion, 10 μl of the culture solution was counted using a hemacytometer. After the cell count was completed, the cells were inoculated with 7 ml of the culture solution in a new dish, and the culture medium used for the culture was changed every 2 days.
본 실험에서는 MC3T3-E1 cell과 C3H10T1/2 cell을 조골세포로 분화시키기 위해 또한 스티그마스테롤의 농도변화와 처리 시간변화에 따른 분화정도를 파악하기 위해 조건을 주어 배양하였다. In order to differentiate MC3T3-E1 cells and C3H10T1 / 2 cells into osteoblasts, we also cultured them in order to determine the level of stigmasterol concentration and the differentiation according to the treatment time.
(2) MTT 분석(2) MTT analysis
세포독성을 측정하기 위하여 3-(4,5-디메틸-티아졸-2-일)-2,5-디페닐테트라졸리움-브로마이드(MTT)(Sigma-Aldrich Co., St. Louis, MO, USA)를 사용한 MTT assay로 살아있는 세포의 생존율을 측정하였다. (MTT) (Sigma-Aldrich Co., St. Louis, Mo., USA) was used to measure cytotoxicity ) Was used to measure the survival rate of living cells.
사용된 C3H10T1/2 cell은 48 웰에서 계대배양 하여 바닥에 부착할 때 까지 하루동안 안정화 시킨 뒤, 스티그마스테롤을 조건에 맞게 1, 10, 20, 50, 100 nM/ml의 농도로 각각 처리하여 추가 배양하였다. DMEM 900μl, MTT 100μl를 1시간 30분 처리하고, 여기에 디메틸설폭사이드(DMSO)를 200μl 처리하면 보라색으로 변하게 되는데, 이를 96 웰에 100μl씩 두 번 분주하고 흡광도 540㎚로 측정하였다.The C3H10T1 / 2 cells used were subcultured in 48 wells and stabilized for one day until they adhered to the bottom. Stigmasterol was added to the cells at concentrations of 1, 10, 20, 50 and 100 nM / ml Lt; / RTI > 900 μl of DMEM and 100 μl of MTT were treated for 1 hour and 30 minutes, and treated with 200 μl of dimethylsulfoxide (DMSO) to change to purple. The resultant was dispensed twice in 96 wells twice, and the absorbance was measured at 540 nm.
1-2. 실험결과1-2. Experiment result
스티그마스테롤의 세포에 미치는 독성을 파악하기 위해 C3H10T1/2 cell의 생존능을 측정하였으며, 그 결과를 도 1에 나타내었다. The viability of C3H10T1 / 2 cells was measured to determine the toxicity to stigmasterol cells. The results are shown in Fig.
나타낸 바와 같이, 10 nM의 농도까지는 세포에 큰 독성을 미치지 않았지만, 20 nM의 농도부터는 세포에 독성을 나타냄을 확인할 수 있었다. 이후, 실험은 독성을 나타내지 않는 1 nM와 10 nM의 조건에서 진행하였다.As shown, up to a concentration of 10 nM did not lead to significant toxicity to the cells, but from a concentration of 20 nM, the cells were toxic. Subsequently, the experiment was conducted under the conditions of 1 nM and 10 nM, which do not exhibit toxicity.
<< 실험예Experimental Example 2> 2> 스티그마스테롤Stigma sterol (stigmasterol) 처리에 따른 조골세포 분화 osteoblast differentiation by treatment with stigmasterol 마커의Marker 발현 분석 Expression analysis
2-1. 실험방법: 실시간 중합효소 연쇄반응(RT-PCR, Real-time polymerase chain reaction)2-1. Real-time polymerase chain reaction (RT-PCR)
C3H10T1/2 cell과 MC3T3-E1 cell에 DMEM, 스티그마스테롤을 분화 조건에 맞게 처리하여 하루동안 배양 한하였다. 그리고 6 웰에 300㎕의 트리졸을 처리하고 스크래퍼를 이용해 cell을 수거하여 Eppendorf Tube로 옮긴 후, 볼텍스(vortex)하여 실온에서 10분간 반응시켰다. 다음으로 클로로폼을 60㎕ 처리 후 볼텍스(vortex)하여 다시 실온에서 10분간 반응시켰다. 그 후 원심분리 (14500rpm, 4℃, 15min) 처리한 후, RNA층만 회수하고, 회수한 RNA층에 이소프로판올 150㎕ 첨가 후 인버팅, 4℃에서 1시간 보관하였다. 다음으로, 원심분리 (14500rpm, 4℃, 10min) 처리한 후, 상층액을 제거하고 70% 에탄올 150㎕로 워싱하였다. 이후 또다시 원심분리 (13000rpm, 4℃, 10min) 처리한 후, 상층액의 에탄올을 제거하고 완전히 건조시켰다. 마지막으로 3차 증류수 20㎕에 녹인 후 정량하였다. 정량수치만큼 cDNA 키트에 넣고, 나머지를 증류수로 채운 후 cDNA를 합성하였다. (cDNA합성 조건 : 50℃ 1hr, 95℃ 5min)The C3H10T1 / 2 cells and MC3T3-E1 cells were treated with DMEM and stigmasterol for one day. Cells were collected by scrapers, transferred to Eppendorf tubes, vortexed, and reacted at room temperature for 10 minutes. Next, 60 μl of chloroform was treated, vortexed, and reacted at room temperature for 10 minutes. Thereafter, after centrifugation (14500 rpm, 4 ° C, 15 min), only the RNA layer was recovered and 150 μl of isopropanol was added to the recovered RNA layer, followed by inverting and storing at 4 ° C for 1 hour. Next, after centrifugation (14500 rpm, 4 DEG C, 10 min), the supernatant was removed and washed with 150 mu l of 70% ethanol. Then, after centrifugation (13000 rpm, 4 ° C, 10 min), ethanol was removed from the supernatant and completely dried. Finally, 20 μl of tertiary distilled water was dissolved and quantified. The cDNA was synthesized by filling the cDNA kit with quantitative values and filling the rest with distilled water. (cDNA synthesis conditions: 50 DEG C for 1 hour, 95 DEG C for 5 minutes)
이 과정에서 얻은 cDNA를 토대로 조골세포 분화 유전자를 타겟으로 RT-PCR을 진행하였다. RT-PCR에 사용되는 혼합물로 emerald(Takara Bio Inc, Japen)가 사용되었다. 분석에 사용한 각각의 프라이머의 염기서열 및 반응 조건을 표 1 및 표 2에 나타내었다.RT-PCR was performed using the osteogenic differentiation gene as a target based on the cDNA obtained in this process. An emerald (Takara Bio Inc, Japan) was used as a mixture for RT-PCR. The nucleotide sequences of the respective primers used in the analysis and the reaction conditions are shown in Tables 1 and 2.
gene
gene
DenaturationPre-
Denaturation
ExtensionPost-
Extension
2-2. 실험결과2-2. Experiment result
(1) 스티그마스테롤의 농도에 따른 조골세포 분화 유전자의 mRNA 발현(1) mRNA expression of osteoblast differentiation gene according to the concentration of stigmasterol
세포독성실험 결과에 따라 스티그마스테롤 1 nM, 10 nM 농도에서 조골세포 분화 유전자의 mRNA 발현정도를 나타내는 실험을 Runx2, Dlx5, OC를 이용하여 진행하였다.Based on the results of the cytotoxicity test, experiments were carried out using Runx2, Dlx5 and OC to show the mRNA expression level of osteoblast differentiation gene at 1 nM and 10 nM of stigmasterol.
C3H10T1/2 cell에 DEME, 1 nM 및 10 nM 농도의 스티그마스테롤을 각각 처리하여 24시간동안 배양하였으며, 그 결과를 도 2에 나타내었다. C3H10T1 / 2 cells were treated with DEME, 1 nM and 10 nM stigmasterol, respectively, and cultured for 24 hours. The results are shown in Fig.
나타낸 바와 같이, Dlx5, Runx2 모두 스티그마그테롤의 농도가 증가함에 따라 발현 정도가 증가하는 것으로 나타났고, OC의 경우 C3H10T1/2 cell에 배양액으로 DMEM만을 첨가한 대조군(Control)에 비해 1 nM에서는 증가하는 게 확연히 보이지만, 10 nM 보다 1 nM에서 발현정도가 더 증가된 건을 확인할 수 있었다. 따라서, 스티그마스테롤은 조골세포 분화를 유도하는 것을 알 수 있었으며, 농도에 따른 발현정도에 큰 차이가 없는 유전자도 있기 때문에 시간에 따른 발현 정도를 확인해보았다. As shown in the figure, the expression level of Dlx5 and Runx2 increased with increasing concentration of stigma gestorol. In the case of OC, C3H10T1 / 2 cell was increased by 1 nM compared with control (control) , But it was confirmed that the expression level was further increased at 1 nM than 10 nM. Therefore, it was found that stigmasterol induces osteoblast differentiation, and since there are some genes that do not significantly differ in the degree of expression depending on the concentration, the degree of expression by time was confirmed.
(2) 스티그마스테롤의 처리시간에 따른 조골세포 분화 유전자의 mRNA 발현(2) mRNA expression of the osteoblast differentiation gene according to the treatment time of stigmasterol
스티그마스테롤 10 nM를 사용하여, 처리 시간에 따른 mRNA 발현정도를 Dlx5, Runx2, ALP를 이용하여 진행하였으며, 그 결과를 도 3에 나타내었다.Using
나타낸 바와 같이, Dlx5와 ALP는 시간이 지남에 따라서 시간의존적으로 발현이 증가하였으며, Runx2는 대조군(control)에 비해 증가하다가 24시간에서 확연히 감소하였고, 48시간에서는 다시 증가하는 결과를 나타내었다.As shown, expression of Dlx5 and ALP increased in time dependent manner, and Runx2 was increased compared to control (control), but decreased significantly at 24 hours and increased again at 48 hours.
한편, 본 발명에 따른 상기 화학식 1로 표시되는 화합물은 목적에 따라 여러 형태로 제제화가 가능하다. 하기는 본 발명에 따른 상기 화학식 1로 표시되는 화합물을 활성성분으로 함유시킨 몇몇 제제화 방법을 예시한 것으로 본 발명이 이에 한정되는 것은 아니다.Meanwhile, the compound represented by
<제제예> <Formulation example>
1-1. 산제의 제조1-1. Manufacture of Powder
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
유당 100 ㎎
탈크 10 ㎎ 10 mg of talc
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다. The above components are mixed and filled in airtight bags to prepare powders.
1-2. 정제의 제조1-2. Manufacture of tablets
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎ 2 mg of magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다. After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
1-3. 캅셀제의 제조1-3. Manufacture of capsules
화학식 1의 화합물 500 ㎎ 500 mg of the compound of formula (1)
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다. The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
1-4. 주사제의 제조1-4. Injection preparation
화학식 1의 화합물 500 ㎎500 mg of the compound of formula (1)
주사용 멸균 증류수 적량 Sterile sterilized water for injection
pH 조절제 적량pH adjuster
통상의 주사제의 제조방법에 따라 1 앰플당(2 ㎖) 상기의 성분 함량으로 제조한다. (2 ml) per ampoule in accordance with the usual injection method.
1-5. 액제의 제조1-5. Manufacture of liquid agent
화학식 1의 화합물 100 ㎎ 100 mg of the compound of formula (1)
이성화당 10 g 10 g per isomer
만니톨 5 g 5 g mannitol
정제수 적량Purified water quantity
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬 향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ㎖로 조절한 후 갈색 병에 충진하여 멸균시켜 액체를 제조한다.Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, The liquid is prepared by sterilization.
Claims (8)
[화학식 1]
1. A pharmaceutical composition for preventing or treating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[Chemical Formula 1]
상기 약학적 조성물은 조골세포 분화를 촉진시키는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
A pharmaceutical composition for preventing or treating osteoporosis, wherein the pharmaceutical composition promotes osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
The pharmaceutical composition for preventing or treating osteoporosis, wherein the concentration of the compound represented by the formula (1) or a pharmaceutically acceptable derivative thereof is 0.01 to 10 nM.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체는 Runx2(runt-related transcription factor 2), Dlx5(distal-less homeobox 5), OC(osteocalcin) 및 ALP(alkaline phosphatase) 중 어느 하나 이상의 조골세포 분화 마커 유전자의 발현량을 증가시키는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.
The method according to claim 1,
The compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof may be used in combination with any one or more of osteolysis (Runx-related transcription factor 2), Dlx5 (distal-less homeobox 5), osteocalcin (OC), and alkaline phosphatase Wherein the expression level of the cell differentiation marker gene is increased.
상기 약학적 조성물은 산제, 과립제, 정제, 경질 캡슐제, 연질 캐슐제 또는 주사제의 형태로 제형화되는 것을 특징으로 하는 약학적 조성물.
The method according to claim 1,
Wherein said pharmaceutical composition is formulated in the form of powders, granules, tablets, hard capsules, soft capsules or injections.
[화학식 1]
A health functional food for preventing or ameliorating osteoporosis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable derivative thereof as an active ingredient.
[Chemical Formula 1]
상기 건강기능식품은 조골세포 분화를 촉진시키는 것을 특징으로 하는 건강기능식품.
The method according to claim 6,
Wherein said health functional food promotes osteoblast differentiation.
상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 유도체의 농도는 0.01 내지 10 nM 인 것을 특징으로 하는 건강기능식품.The method according to claim 6,
Wherein the concentration of the compound represented by Formula 1 or a pharmaceutically acceptable derivative thereof is 0.01 to 10 nM.
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| KR1020170139075A KR102139994B1 (en) | 2017-10-25 | 2017-10-25 | Pharmaceutical composition for use in preventing or treating osteoporosis containing stigmasterol as an active ingredient |
| PCT/KR2018/012487 WO2019083236A1 (en) | 2017-10-25 | 2018-10-22 | Pharmaceutical composition containing stigmasterol as active ingredient for preventing or treating osteoporosis |
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| JP2005139150A (en) * | 2003-11-10 | 2005-06-02 | Api Co Ltd | Estrogenic agent, method for producing the same, prophylactic agent for osteoporosis and food and beverage |
| KR101745504B1 (en) | 2014-10-13 | 2017-06-12 | 서울대학교산학협력단 | Method for Isolating of Vomifoliol and Stigmasterol Having Anti-cancer Activity from Silkworm Feces |
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| KR100380863B1 (en) * | 2000-12-06 | 2003-04-18 | 한국 한의학 연구원 | Extract of Spatholobi Caulis for the prevention and treatment of osteoporosis |
| EP1644399A2 (en) * | 2003-07-09 | 2006-04-12 | Forbes Medi-Tech Inc. | Novel compounds and compositions comprising sterols and/or stanols and cholesterol biosynthesis inhibitors and use thereof in treating or preventing a variety of diseases and conditions. |
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| JP2005139150A (en) * | 2003-11-10 | 2005-06-02 | Api Co Ltd | Estrogenic agent, method for producing the same, prophylactic agent for osteoporosis and food and beverage |
| KR101745504B1 (en) | 2014-10-13 | 2017-06-12 | 서울대학교산학협력단 | Method for Isolating of Vomifoliol and Stigmasterol Having Anti-cancer Activity from Silkworm Feces |
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| Triutomo, et al., Indones. J. Cancer Chemoprevent., 7(3), 104-109 (2017.02.03)* * |
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