KR102121573B1 - Composition for preventing, treating or improving obesity comprising Jaceosidin - Google Patents
Composition for preventing, treating or improving obesity comprising Jaceosidin Download PDFInfo
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- KR102121573B1 KR102121573B1 KR1020200004062A KR20200004062A KR102121573B1 KR 102121573 B1 KR102121573 B1 KR 102121573B1 KR 1020200004062 A KR1020200004062 A KR 1020200004062A KR 20200004062 A KR20200004062 A KR 20200004062A KR 102121573 B1 KR102121573 B1 KR 102121573B1
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Abstract
본 발명은 자세오시딘을 포함하는 비만 예방, 치료, 또는 개선용 조성물에 관한 것으로, 상기 자세오시딘은 지방세포의 분화를 억제하고 세포 내 지질 축적을 감소시키는 효과가 있다. 또한, 지방세포 형성 및 분화에 관여하는 전사인자인 퍼옥시좀 증식제 활성화 수용체 감마(PPARγ) 및 CCAAT-인핸서 결합 단백질 알파(C/EBPα)와 아디포넥틴의 발현을 억제시켜 뛰어난 항비만 효과를 나타내었다. 따라서, 본 발명의 자세오시딘은 비만 예방, 치료, 또는 개선용 약학적 조성물 및 건강기능식품 등으로 유용하게 사용될 수 있다.The present invention relates to a composition for preventing, treating, or improving obesity comprising posturetidine, wherein posturetidine suppresses the differentiation of adipocytes and reduces lipid accumulation in cells. In addition, it suppressed the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPα) and adiponectin, which are transcription factors involved in adipocyte formation and differentiation, and showed excellent anti-obesity effects. . Therefore, the posturesidine of the present invention can be usefully used as a pharmaceutical composition and health functional food for preventing, treating or improving obesity.
Description
본 발명은 자세오시딘을 유효성분으로 포함하는 비만 예방, 치료, 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for preventing, treating, or improving obesity, which includes posicydin as an active ingredient.
비만은 일반적으로 체내에 지방조직이 과다한 상태인 것을 의미하며, 음식물로 섭취한 에너지가 신체활동 등으로 소비한 에너지와 균형을 이루지 못하여 잉여의 에너지가 체지방으로 축적되는 현상이다. 비만은 유전적인 요인, 불규칙한 식습관이나 운동부족 또는 서구화되는 식생활에 의한 환경적인 영향, 우울감, 지루감, 과다한 스트레스에 의한 심리적인 영향, 갑상선이나 부신피질 호르몬 변화에 의한 병적인 요인 등 매우 다양한 원인에 의해 유발되는 것으로 알려져 있다. 최근에는 경제성장과 생활방식의 변화에 따라 식습관에도 많은 변화가 생겨 바쁜 현대인들은 패스트푸드 등의 고열량 식이와 적은 운동량으로 인하여 과체중 및 비만이 증가하고 있는 추세이다.Obesity generally means that the fat tissue in the body is excessive, and the energy consumed as food is not balanced with the energy consumed by physical activity, so excess energy is accumulated as body fat. Obesity is caused by a wide variety of causes, including genetic factors, irregular eating habits, lack of exercise, or environmental effects due to westernized diet, depression, boredom, and psychological effects of excessive stress, and pathological factors caused by thyroid or adrenal cortical hormone changes. It is known to be caused by. Recently, there have been many changes in eating habits due to economic growth and lifestyle changes, and busy modern people are increasing their overweight and obesity due to high calorie diet such as fast food and low exercise.
또한, 비만은 지방세포의 비대에 의해 유발되므로, 지방세포 분화의 억제는 비만 치료에 도움이 될 수 있다(Henninger, Eliasson, Jenndahl, & Hammarstedt, 2014). 종래에 지방세포 분화 및 지질 축적의 억제와 관련된 항비만 연구가 보고된 바 있으며(Kim & Lee, 2017; Li et al., 2017), 이 때 3T3-L1 마우스 섬유아세포는 지방 전구세포의 분화를 위한 잘 특성화된 모델로 일반적으로 시험관 내 연구에 사용된다(Kaestner et al., 1989; S. K. Kim & Kong, 2010).In addition, since obesity is caused by fat cell hypertrophy, inhibition of adipocyte differentiation may be helpful in treating obesity (Henninger, Eliasson, Jenndahl, & Hammarstedt, 2014). Anti-obesity studies related to the suppression of adipocyte differentiation and lipid accumulation have been reported in the past (Kim & Lee, 2017; Li et al., 2017), in which 3T3-L1 mouse fibroblasts differentiate adipocyte progenitor cells. Well-characterized model for general use in vitro studies (Kaestner et al., 1989; SK Kim & Kong, 2010).
비만은 그 자체의 위험성보다 비만으로 인해 유발될 수 있는 여러 합병증 때문에 그 심각성이 더욱 크게 인식되고 있는데, 오랜 시간에 걸쳐 에너지 불균형에 의해 체지방이 비정상적으로 많아지면 당뇨, 고지혈증, 심장병, 뇌졸중, 동맥경화증, 지방간 등의 각종 대사성 질환과 성인병이 유발된다. 더불어 비만은 신체적인 질환뿐만 아니라 사회적 고립감이나 소외, 자신감 결여, 우울감과 같은 정신적 질환을 유발할 수 있어, 이는 서구에서뿐만 아니라 우리나라에서도 심각한 사회문제로 대두되고 있으며 비만의 예방 및 치료에 대한 필요성이 매우 중요하게 인식되고 있다. Obesity is recognized more seriously due to various complications that can be caused by obesity than its own risk.If abnormal body fat increases due to energy imbalance over a long period of time, diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis , Fatty liver and various metabolic diseases and adult diseases are caused. In addition, obesity can cause not only physical illnesses but also psychological disorders such as social isolation, alienation, lack of confidence, and depression, which is emerging as a serious social problem not only in the West but also in Korea, and the need for prevention and treatment of obesity is very important Is recognized.
비만은 식이요법, 규칙적인 운동과 더불어 행동요법과 같은 생활습관의 개선, 및 식욕 억제제와 지방 흡수 억제제와 같은 약물을 통해 치료할 수 있다. 비만은 만성 질환이기 때문에 약물치료를 시도하는 경우 장기간의 사용이 필요하며, 현재 국내에서 3개월 이상 장기간 사용이 허가된 제품으로는 식욕억제제인 시부트라민(sibutramine)과 지방분해효소 억제제인 올리스타트(orlistat)가 있다. 그러나 이러한 비만 치료 약물들은 대부분 중추신경계에 작용하여 식욕을 조절하는 향정신성의약품들이므로 두통 및 구토 등의 부작용을 동반하며 남용 우려 등의 문제점이 있다. 따라서 상기 시판중인 항비만제의 부작용을 해결할 수 있는 안정성이 높고 항비만 효과가 우수한 소재를 개발하기 위한 연구가 활발히 이루어지고 있다.Obesity can be treated with diet, regular exercise, and lifestyle improvements such as behavioral therapy, and medications such as appetite suppressants and fat absorption inhibitors. Because obesity is a chronic disease, long-term use is required when attempting drug treatment. Currently, products that have been approved for long-term use for more than 3 months include appetite suppressant sibutramine and lipolytic inhibitor orlistat. ). However, most of these anti-obesity drugs are psychotropic drugs that act on the central nervous system to regulate appetite, and thus have side effects such as headache and vomiting, and there are problems of abuse. Accordingly, research has been actively conducted to develop a material having high stability and excellent anti-obesity effect that can solve the side effects of the commercially available anti-obesity agents.
한편, 자세오시딘(Jaceosidin)은 Artemisia 속의 주요 플라보노이드 계열의 화합물로, 들쑥 및 약쑥 등에 다량 함유되어 있는 것으로 알려져 있다. 자세오시딘은 항종양형성, 항산화, 및 항염증 작용을 포함한 여러 가지 생물학적 효과를 나타낸다고 보고 되었으나(Khan, Rasul, Yi, Zhong, & Ma, 2011; Min, Kim, Baek, & Kim, 2009), 항비만 효과에 대해서는 아직 보고된 바가 없다.Meanwhile, Postosidin is Artemisia It is a major flavonoid-based compound in the genus and is known to contain large amounts of wild wormwood and wormwood. Posiodine has been reported to exhibit several biological effects, including anti-tumor formation, antioxidant, and anti-inflammatory action (Khan, Rasul, Yi, Zhong, & Ma, 2011; Min, Kim, Baek, & Kim, 2009), No anti-obesity effects have been reported.
본 발명자들은 종래에 자세오시딘과 유사한 화학 구조를 갖는 히스피듈린(hispidulin)의 항지방형성 효과에 대해 입증함으로써(H.-S. Kim et al., 2017), 자세오시딘 역시 유사한 효과를 가질 것이라 가정하고 연구한 결과, 상기 자세오시딘이 뛰어난 항비만 효과를 나타냄을 확인하여 비만 예방, 치료, 또는 개선용 조성물로 사용될 수 있도록 하였다.The present inventors have demonstrated the anti-lipogenic effect of hispidulin, which has a chemical structure similar to post-osidin (H.-S. Kim et al., 2017), and post-osidin also has similar effects. As a result of the study, assuming that it would have, it was confirmed that the posturetidine exhibits an excellent anti-obesity effect so that it can be used as a composition for preventing, treating or improving obesity.
본 발명자들은 종래 항비만제의 문제점을 해결할 수 있는 안정성이 높고 항비만 효과가 우수한 비만 예방 또는 치료용 조성물을 개발하고자 노력한 결과, 본 발명의 자세오시딘이 뛰어난 항비만 효과를 가지는 것을 확인하고 본 발명을 완성하였다. The present inventors tried to develop a composition for preventing or treating obesity having high stability and excellent anti-obesity effect that can solve the problems of conventional anti-obesity agents, and confirmed that posture osdinin of the present invention has excellent anti-obesity effect. The invention was completed.
이에, 본 발명은 자세오시딘을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which includes postosidine as an active ingredient.
또한, 본 발명은 자세오시딘을 유효성분으로 포함하는 비만 예방 또는 개선용 건강기능식품 조성물을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a health functional food composition for preventing or improving obesity, which contains posturetidine as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 목적을 달성하기 위하여, 본 발명은 자세오시딘을 유효성분으로 포함하는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating obesity, which includes posturetidine as an active ingredient.
또한, 본 발명은 자세오시딘을 유효성분으로 포함하는, 비만 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving obesity, which includes posturetidine as an active ingredient.
본 발명의 일구현예로서, 상기 조성물은 지방세포의 분화를 억제할 수 있다.As an embodiment of the present invention, the composition can suppress the differentiation of adipocytes.
본 발명의 다른 일구현예로서, 상기 조성물은 세포 내 지질 축적을 감소시킬 수 있다.As another embodiment of the present invention, the composition may reduce lipid accumulation in cells.
본 발명의 또 다른 일구현예로서, 상기 조성물은 지방세포 형성 및 분화에 관여하는 전사인자 또는 아디포넥틴의 발현을 억제할 수 있다.As another embodiment of the present invention, the composition may suppress the expression of adiponectin or a transcription factor involved in adipocyte formation and differentiation.
본 발명의 또 다른 일구현예로서, 상기 전사인자는 퍼옥시좀 증식제 활성화 수용체 감마(PPARγ) 또는 CCAAT-인핸서 결합 단백질 알파(C/EBPα)일 수 있다.As another embodiment of the present invention, the transcription factor may be peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPα).
또한, 본 발명은 상기 조성물을 개체에 투여하는 단계를 포함하는, 비만 예방 또는 치료방법을 제공한다.In addition, the present invention provides a method for preventing or treating obesity, comprising administering the composition to an individual.
또한, 본 발명은 상기 조성물의 비만 예방 또는 치료용도를 제공한다.In addition, the present invention provides a composition for preventing or treating obesity.
본 발명에 따른 자세오시딘은 지방세포의 분화를 억제하고 세포 내 지질 축적을 감소시키는 효과가 있으며, 지방세포 형성 및 분화에 관여하는 전사인자인 퍼옥시좀 증식제 활성화 수용체 감마(PPARγ) 및 CCAAT-인핸서 결합 단백질 알파(C/EBPα)와 아디포넥틴의 발현을 억제시켜 항비만 효과가 뛰어남을 확인하였는바, 본 발명의 자세오시딘은 비만 예방, 치료, 또는 개선용 약학적 조성물 및 건강기능식품 등 다양한 방면으로 이용될 것으로 기대된다.Posture oxidin according to the present invention has the effect of inhibiting the differentiation of adipocytes and reducing the accumulation of lipids in the cell, and peroxisome proliferator activator gamma (PPARγ) and CCAAT, transcription factors involved in adipocyte formation and differentiation. -It was confirmed that the anti-obesity effect is excellent by inhibiting the expression of enhancer binding protein alpha (C/EBPα) and adiponectin, and the posturetidine of the present invention is a pharmaceutical composition for preventing, treating, or improving obesity, health functional food, etc. It is expected to be used in various ways.
도 1a은 본 발명의 일구현예에 따른 자세오시딘을 처리한 3T3-L1 지방전구세포에서의 세포 생존능 측정 결과를 MTT 분석에 의해 나타낸 도면이다.
도 1b는 본 발명의 일구현예에 따른 자세오시딘을 처리한 3T3-L1 지방전구세포, 음성 대조군(NC), 및 양성 대조군(PC)의 세포모양을 400x 배율의 현미경을 사용하여 관찰한 결과를 나타낸 도면이다.
도 2a는 본 발명의 일구현예에 따른 자세오시딘을 처리한 3T3-L1 지방전구세포에서 Oil Red O 염색한 결과를 나타낸 도면이다.
도 2b는 본 발명의 일구현예에 따른 자세오시딘을 처리한 3T3-L1 지방세포 내 Triglyceride(TG)의 함량을 측정하여 나타낸 도면이다.
도 3a는 본 발명의 일구현예에 따른 3T3-L1 지방세포에서 자세오시딘의 처리에 의한 PPARγ, C/EBPα, 및 아디포넥틴의 mRNA 발현량을 나타낸 도면이다.
도 3b는 본 발명의 일구현예에 따른 3T3-L1 지방세포에서 자세오시딘의 처리에 의한 PPARγ 및 아디포넥틴 단백질의 발현량을 나타낸 도면이다.1A is a view showing the results of measuring cell viability in 3T3-L1 adipocytes treated with posturetidine according to one embodiment of the present invention by MTT analysis.
Figure 1b is a result of observing the cell shape of 3T3-L1 adipocytes, negative control (NC), and positive control (PC) treated posture osdinin according to an embodiment of the present invention using a microscope of 400x magnification It is a figure showing.
Figure 2a is a diagram showing the results of Oil Red O staining in 3T3-L1 adipocytes treated with posturetidine according to one embodiment of the present invention.
Figure 2b is a view showing the measurement of the content of Triglyceride (TG) in 3T3-L1 adipocytes treated with posturetidine according to one embodiment of the present invention.
3A is a diagram showing mRNA expression levels of PPARγ, C/EBPα, and adiponectin by treatment of postosidin in 3T3-L1 adipocytes according to one embodiment of the present invention.
3B is a diagram showing the expression levels of PPARγ and adiponectin proteins by treatment of postosidin in 3T3-L1 adipocytes according to one embodiment of the present invention.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.The present invention can be applied to a variety of transformations and may have various embodiments, and specific embodiments will be illustrated in the drawings and described in detail. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In the description of the present invention, if it is determined that a detailed description of known technologies related to the present invention may obscure the subject matter of the present invention, the detailed description will be omitted.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 자세오시딘을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating obesity, which includes posicydine as an active ingredient.
본 발명의 예방 또는 치료 대상 질병인 "비만(obesity)"은, 대사 장애로 인하여 체내에 지방세포가 증식 분화하고 이로 인하여 지방이 과잉으로 축적된 상태를 의미하며, 고혈압, 당뇨, 및 이상지질혈증 등을 동반하는 대사증후군을 포함하는 관련 합병증을 유발할 수 있다. 에너지 흡수량이 소비량에 비해 상대적으로 증가하는 경우, 지방세포의 수와 부피가 증가되는 과정을 거쳐 결과적으로 지방조직의 질량이 증가된다.The disease to be prevented or treated according to the present invention, "obesity (obesity)" refers to a state in which fat cells proliferate and differentiate in the body due to metabolic disorders, and thus fat is accumulated excessively, and hypertension, diabetes, and dyslipidemia It can cause related complications, including metabolic syndrome with back. When the amount of energy absorbed increases relative to the amount consumed, the mass of adipose tissue increases as a result of the process of increasing the number and volume of fat cells.
본 발명에서 사용되는 용어 "예방"은, 본 발명에 따른 약학적 조성물의 투여에 의해 비만을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term "prevention" as used in the present invention means any action that suppresses obesity or delays the onset by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어 "치료"는, 본 발명에 따른 약학적 조성물의 투여에 의해 비만에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "treatment" as used in the present invention means any action in which symptoms of obesity are improved or beneficially altered by administration of the pharmaceutical composition according to the present invention.
또한, 본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 비만 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating obesity, comprising administering the pharmaceutical composition to an individual.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.As used herein, the term "administration" means providing the subject with the composition of the invention in any suitable way.
본 발명에서 사용되는 용어 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는, 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말, 및 소 등의 포유류를 의미한다.As used herein, the term "individual" refers to a subject in need of treatment of a disease, and more specifically, human or non-human primate, mouse, dog, cat, horse, and cow, etc. It means mammal.
또한, 본 발명은 상기 약학적 조성물의 비만 예방 또는 치료 용도를 제공한다.In addition, the present invention provides an obesity prevention or treatment use of the pharmaceutical composition.
본 발명에 따른 자세오시딘은 지방세포의 분화를 억제시키고, 지방세포 내 지질 축적을 감소시킬 수 있다. 또한, 지방세포 형성 및 분화에 관여하는 전사인자 또는 아디포넥틴의 발현을 억제할 수 있다. 이 때, 상기 전사인자는 퍼옥시좀 증식제 활성화 수용체 감마(PPARγ) 또는 CCAAT-인핸서 결합 단백질 알파(C/EBPα)일 수 있으나, 이에 제한되지 않는다. Posture oscidin according to the present invention can inhibit the differentiation of adipocytes and reduce lipid accumulation in adipocytes. In addition, it is possible to suppress the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation. At this time, the transcription factor may be a peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPα), but is not limited thereto.
본 발명의 일실험예에서는, 3T3-L1 지방세포의 분화기간 동안 자세오시딘을 처리 후 현미경을 통해 관찰한 결과, 자세오시딘이 농도의존적으로 3T3-L1 지방세포의 분화를 강력하게 억제하는 것을 확인하였다(실험예 2 및 도 1b 참조).In one experimental example of the present invention, as a result of observing posture osdinin during the differentiation period of 3T3-L1 adipocytes through a microscope, posture osidin strongly inhibits the differentiation of 3T3-L1 adipocytes in a concentration-dependent manner. It was confirmed (see Experimental Example 2 and Figure 1b).
본 발명의 다른 일실험예에서는, 3T3-L1 지방세포에서 Oil red O staining 실험기법을 이용하여 지질염색을 한 결과, 자세오시딘을 처리한 3T3-L1 지방세포에서 지질 축적이 농도의존적으로 감소하는 것을 확인하였으며, 자세오시딘을 처리한 3T3-L1 지방세포 내의 TG함량은 양성대조군에 비해 현저하게 감소하는 것을 확인하였다(실험예 3 및 도 2a~b 참조).In another experimental example of the present invention, as a result of lipid staining using the oil red O staining experiment technique on 3T3-L1 adipocytes, lipid accumulation in the 3T3-L1 adipocytes treated with postosidin decreases in a concentration-dependent manner. It was confirmed that the TG content in 3T3-L1 adipocytes treated with posturetidine was significantly reduced compared to the positive control group (see Experimental Example 3 and FIGS. 2A-B ).
본 발명의 또 다른 일실험예에서는, 자세오시딘을 처리한 3T3-L1 지방세포에서 분리한 RNA를 cDNA로 합성한 후 RT-PCR을 진행한 결과, 자세오시딘이 PPARγ, C/EBPα, 및 아디포넥틴의 mRNA 발현을 농도 의존적으로 억제하는 것을 확인하였다(실험예 4-1 및 도 3a 참조).In another experimental example of the present invention, RNA isolated from 3T3-L1 adipocytes treated with posturetidine was synthesized with cDNA and then subjected to RT-PCR. As a result, posturetidine was PPARγ, C/EBPα, and It was confirmed that the adiponectin mRNA expression was suppressed in a concentration-dependent manner (see Experimental Example 4-1 and FIG. 3A).
또한, 자세오시딘을 처리한 3T3-L1 지방세포에서 단백질 발현을 분석한 결과, 자세오시딘은 PPARγ2 및 아디포넥틴 단백질 발현을 억제시키는 것을 확인하였다(실험예 4-2 및 도 3b 참조).In addition, as a result of analyzing protein expression in 3T3-L1 adipocytes treated with posturetidine, it was confirmed that postureodine suppresses PPARγ2 and adiponectin protein expression (see Experimental Example 4-2 and FIG. 3B).
지방 세포의 분화는 PPARγ(peroxisome proliferator-activated receptor gamma) 및 C/EBPα(CCAAT-enhancer-binding protein alpha)와 같은 다양한 전사인자 발현의 변화에 의해 조절된다(Yoo, Seo, Shin, & Jeong, 2015).Differentiation of adipocytes is regulated by changes in expression of various transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha (C/EBPα) (Yoo, Seo, Shin, & Jeong, 2015). ).
이 때, 상기 PPARγ(peroxisome proliferator-activated receptor gamma)는 세포신호 경로에 중요한 요소이며 당대사와 지방세포형성과 관련된 전사인자를 조절한다고 알려져 있다. PPARγ는 지방세포에서 PPARγ1 및 PPARγ2 의 형태로 발현하는데, PPARγ2 는 PPARγ1 에 비해 N-말단에 30개의 아미노산이 더 있으나 나머지 아미노산 서열은 똑같고, PPARγ1 및 PPARγ2의 발현 수준은 조직마다 차이가 있다. PPARγ2 는 지방세포에서 특이적으로 발현되는 반면, PPARγ1은 대식세포, 결장상피세포, 및 내피세포와 같은 여러 세포 유형에서 낮은 수준으로 발현된다(Lehrke & Lazar, 2005; Sarjeant & Stephens, 2012). PPARγ2는 지방생성에 중대한 영향을 미칠 수 있으며, 비만 환자의 지방세포에서 발견되었다(Ren, Collingwood, Rebar, Wolffe, & Camp, 2002; Sarjeant & Stephens, 2012).At this time, the PPARγ (peroxisome proliferator-activated receptor gamma) is an important factor in the cell signaling pathway and is known to regulate transcription factors related to glycolysis and adipocyte formation. PPARγ is expressed in the form of PPARγ1 and PPARγ2 in adipocytes. PPARγ2 has 30 more amino acids at the N-terminus compared to PPARγ1, but the remaining amino acid sequences are the same, and the expression levels of PPARγ1 and PPARγ2 are different for each tissue. PPARγ2 is expressed specifically in adipocytes, while PPARγ1 is expressed at low levels in several cell types such as macrophages, colon epithelial cells, and endothelial cells (Lehrke & Lazar, 2005; Sarjeant & Stephens, 2012). PPARγ2 can have a significant effect on fat production and was found in fat cells of obese patients (Ren, Collingwood, Rebar, Wolffe, & Camp, 2002; Sarjeant & Stephens, 2012).
C/EBPα(CCAAT-enhancer-binding protein alpha)는 지방전구세포의 분화 조절에 PPARγ와 함께 중추적인 역할을 하는 단백질이다. 지방전구세포 분화 과정에 있어서 초기단계의 C/EBPδ 및 C/EBPβ는 PPARγ의 발현을 유도하며, PPARγ의 발현 증가는 C/EBPα의 발현에 의한 지방세포 분화 후기를 담당한다.C/EBPα (CCAAT-enhancer-binding protein alpha) is a protein that plays a central role with PPARγ in regulating the differentiation of adipocytes. In the process of adipocyte differentiation, C/EBPδ and C/EBPβ in the early stages induce the expression of PPARγ, and the increased expression of PPARγ is responsible for the late adipocyte differentiation by expression of C/EBPα.
또한, 아디포넥틴(Adiponectin)은 지방세포에 의해 분비된 아디포카인(adipokine)으로, 인슐린 저항 관련 비만을 조절하는 것으로 보고되었다(Lindsay et al., 2002).In addition, Adiponectin is adipokine secreted by adipocytes and has been reported to regulate insulin resistance-related obesity (Lindsay et al., 2002).
한편, 본 발명에서 "약학적 조성물"은 질병의 예방 또는 치료를 목적으로 제조된 것을 의미하며, 각각 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 예컨대, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽 등의 경구형 제형으로 제형화할 수 있고, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용할 수 있다.On the other hand, in the present invention, "pharmaceutical composition" means manufactured for the purpose of preventing or treating a disease, and can be used by formulating in various forms according to a conventional method. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and can be formulated and used in the form of external preparations, suppositories, and sterile injectable solutions.
본 발명에 따른 약학적 조성물은 유효성분 이외에 약제학적으로 허용되는 담체를 포함할 수 있다. 이때, 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피로리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 또한, 상기성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. may be further included in addition to the above components.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage may be adjusted according to the patient's condition, weight, and disease. It depends on the degree, drug type, route of administration, and time, but can be appropriately selected by those skilled in the art.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용 없이 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. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of discharge, duration of treatment, factors including co-drugs and other factors well known in the medical field can be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.
또한, 본 발명은 자세오시딘을 유효성분으로 포함하는 비만 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving obesity, which includes posturetidine as an active ingredient.
본 발명에서 사용되는 용어 "개선"은, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. 이 때 상기 건강기능식품 조성물은 비만의 예방 또는 개선을 위하여 해당 질환의 발병 단계 이전 또는 발병 후, 치료를 위한 약제와 동시에 또는 별개로서 사용될 수 있다.As used herein, the term “improvement” refers to any action that at least reduces the severity of parameters associated with the condition being treated, such as symptoms. In this case, the health functional food composition may be used simultaneously or separately with a drug for treatment before or after the onset stage of the disease in order to prevent or improve obesity.
본 발명에서 "건강기능식품 조성물"은, 담체, 희석제, 부형제 및 첨가제 중 하나 이상을 포함하여 정제, 환제, 산제, 과립제, 분말제, 캡슐제 및 액제 제형으로 이루어진 군에서 선택된 하나로 제형된 것을 특징으로 한다. In the present invention, "health functional food composition" is characterized by being formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, and additives. Is done.
본 발명의 자세오시딘에 첨가할 수 있는 식품으로는, 각종 식품류, 분말, 과립, 정제, 캡슐, 시럽제, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다. 상기 본 발명에 더 포함될 수 있는 첨가제로는, 천연 탄수화물, 향미제, 영양제, 비타민, 광물(전해질), 풍미제(합성 풍미제, 천연 풍미제 등), 착색제, 충진제, 팩트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH조절제, 안정화제, 방부제, 산화 방지제, 글리세린, 알코올, 탄산화제, 및 과육으로 이루어진 군으로부터 선택된 1종 이상의 성분을 사용할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토오스, 수크로오스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 상기 향미제로서 천연 향미제(타우마틴, 스테비아추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. Foods that can be added to the posturetidine of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), colorants, fillers, pactic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonic acid, and one or more components selected from the group consisting of flesh can be used. Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (Tau Martin, Stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
상기 외에 본 발명에 따른 조성물은 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제, 팩트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. In addition to the above, the composition according to the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring and neutralizing agents, pactic acid and salts thereof, alginic acid and salts thereof, organic acids, and protection It may contain sex colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like.
그밖에 본 발명에 다른 조성물은 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. Other compositions in the present invention may contain flesh for the preparation of natural fruit juices and vegetable drinks. These ingredients can be used independently or in combination.
상기 담체, 부형제, 희석제 및 첨가제의 구체적인 예로는 이에 한정하는 것은 아니나, 락토오스, 덱스트로즈, 수크로오스, 솔비톨, 만니톨, 에리스리톨, 전분, 아카시아 고무, 인산칼슘, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 미세결정성 셀룰로오스, 폴리비닐키롤리돈, 셀룰로오스, 폴리비닐피로리돈, 메틸셀룰로오스, 물, 설탕시럽, 메틸 하이드록시 벤조에이트, 프로필하이드록시 벤조에이트, 활석, 스테아르산 마그네슘, 및 미네랄 오일로 이루어진 그룹으로부터 선택된 1종 이상이 사용되는 것이 바람직하다.Specific examples of the carrier, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, Group consisting of microcrystalline cellulose, polyvinylkyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil It is preferred that at least one selected from is used.
이하, 본 발명의 바람직한 실시예 및 실험예를 첨부도면을 참조하여 상세히 설명하기로 한다. 다만, 이들 실시예 및 실험예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예 및 실험예에 의해 제한되는 것으로 해석되지는 않는다 할 것이다.Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings. However, these Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention will not be interpreted as being limited by these Examples and Experimental Examples.
실시예1. 시료 준비 및 세포 배양Example 1. Sample preparation and cell culture
자세오시딘(Jaceosidin)은 Chem Faces사 (Cas num. 18085-97-7)로부터 구입하였고, DMSO에 녹인 후, 실험에 사용하였다. 3T3-L1 지방세포는 한국세포주은행으로부터 구입하였고 Dulbecco's modified Eagle medium (DMEM) 배지에 10%NBCS를 추가한 배지에 배양하였다. 분화를 위해, 세포는 이틀간 post-confluence를 유지시킨 뒤, 지방세포분화 유도 물질(0.5 mM IBMX, 1 μM DEXA, 0.125 mM indomethacin, and 10 μg/mL insulin)을 처리하여 이틀간 자극시켰다. 이후로는, 10 μg/mL의 insulin 자극만으로 성숙한 지방세포로 분화가 완료될 때까지 배양하였다. 자세오시딘의 지방세포 분화 억제효과를 관찰하기 위해, 자세오시딘은 10, 20, 및 40μM의 농도별로 지방세포 분화기간 동안 지속적으로 처리하였다.Posture Osidin It was purchased from Chem Faces (Cas num. 18085-97-7), dissolved in DMSO, and used in experiments. 3T3-L1 adipocytes were purchased from the Korea Cell Line Bank and cultured in Dulbecco's modified Eagle medium (DMEM) medium with 10% NCSC added. For differentiation, cells were maintained for 2 days post-confluence and then stimulated for 2 days by treatment with adipocyte differentiation inducers (0.5 mM IBMX, 1 μM DEXA, 0.125 mM indomethacin, and 10 μg/mL insulin). Subsequently, the cells were cultured until the differentiation was completed with mature adipocytes only by stimulation of 10 μg/mL insulin. In order to observe the inhibitory effect of posturetidine on adipocyte differentiation, posturetidine was continuously treated during adipocyte differentiation periods at concentrations of 10, 20, and 40 μM.
실험예 1. 세포 생존능 측정Experimental Example 1. Cell viability measurement
세포 생존능에 자세오시딘이 미치는 영향을 조사하기 위해, 3T3-L1 지방전구세포를 96-well plate에 파종하고 이후, 자세오시딘을 처리하고 24시간 동안 배양하였다. 대조군에는 자세오시딘을 넣지 않고 동량의 DMSO를 처리하였다. 처리 시간 이후, MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) 용액을 각 well에 추가하고 3시간 반응시킨 뒤, 형성된 formazan을 용해하여 595nm 파장에서 흡광도를 측정하였다.Cell viability In order to investigate the effect of posturetidine, 3T3-L1 adipocytes were seeded in a 96-well plate, and then posturetidine was treated and cultured for 24 hours. The control group was treated with the same amount of DMSO without posturetidine. After the treatment time, MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) solution was added to each well and reacted for 3 hours, followed by dissolving the formed formazan to 595nm wavelength Absorbance was measured at.
그 결과, 도 1a에 나타낸 바와 같이, 자세오시딘은 0 내지 80μM의 농도에서 3T3-L1 지방전구세포의 생존력에 유의한 영향을 미치지 않았다. As a result, as shown in FIG. 1A, postosidine did not significantly affect the viability of 3T3-L1 adipocytes at a concentration of 0 to 80 μM.
실험예 2. 자세오시딘의 지방세포 분화 억제 효과 확인Experimental Example 2. Confirmation of the suppression effect of adipocyte differentiation
상기 실시예 1에서 배양한 3T3-L1 지방세포를 400x 배율의 현미경을 통해 관찰하여 도 1b에 나타내었다. 자세오시딘을 처리하여 배양한 3T3-L1 지방세포를 지방전구세포를 사용한 음성대조군(NC) 및 완전 분화된 지방세포를 사용한 양성대조군(PC)과 비교했을 때, 자세오시딘이 농도의존적으로 3T3-L1 지방세포 분화를 강력하게 억제하는 것을 확인하였다.3T3-L1 adipocytes cultured in Example 1 were observed through a microscope at 400x magnification and are shown in FIG. 1B. 3T3-L1 adipocytes cultured with posturesidine treatment compared to negative control (NC) using adipocytes and positive control (PC) using fully differentiated adipocytes, postureosidine concentration-dependently 3T3 It was confirmed that -L1 adipocyte differentiation was strongly inhibited.
실험예 3. 자세오시딘의 세포 내 지질 축적 감소 효과 확인Experimental Example 3. Confirmation of the effect of posturetidine on the reduction of lipid accumulation in cells
지질염색은 Oil Red O (ORO) staining 실험기법을 이용하였고 중성지방함량은 TG Quantification Kit (Bio Vision)를 이용하여 제조사의 매뉴얼에 따라 수행하였다. 지질염색을 위해, 상기 실시예 1에서 배양한 3T3-L1 지방세포를 0.6% glutaraldehyde를 이용하여 고정하고 0.4%의 ORO solution으로 염색 한 뒤, 증류수로 3번 세척하였다. 염색된 지질을 200x 배율의 현미경을 통해 관찰하고, 100% 이소프로필알콜 (isopropyl alcohol)을 이용하여 ORO 시약을 용해한 뒤, 495nm에서의 흡광도를 측정하고 정량화 하였다.Lipid staining was performed using Oil Red O (ORO) staining test method, and triglyceride content was performed according to the manufacturer's manual using the TG Quantification Kit (Bio Vision). For lipid staining, the 3T3-L1 adipocytes cultured in Example 1 were fixed using 0.6% glutaraldehyde, stained with 0.4% ORO solution, and washed 3 times with distilled water. The dyed lipid was observed through a microscope at 200x magnification, and after dissolving the ORO reagent using 100% isopropyl alcohol, the absorbance at 495 nm was measured and quantified.
그 결과, 도 2a에 나타낸 바와 같이, 3T3-L1 지방세포 내 지질 축적이 양성대조군에서 정상적으로 유도되었으나, 자세오시딘을 처리한 3T3-L1 지방세포에서는 지질 축적이 농도의존적으로 감소되었다.As a result, as shown in FIG. 2A, lipid accumulation in 3T3-L1 adipocytes was normally induced in the positive control, but lipid accumulation was decreased in a concentration-dependent manner in 3T3-L1 adipocytes treated with postosidin.
또한, 중성지방 함량 측정을 위해, 상기 실시예 1에서 배양한 3T3-L1 지방세포를 4% NP-40을 포함하는 용해액에 용해하고, 원심분리하여 상층액을 취한 뒤, 제조사의 매뉴얼에 따라 570nm에서의 흡광도를 측정하여 세포 내 TG함량을 분석하였다. In addition, for the measurement of triglyceride content, the 3T3-L1 adipocytes cultured in Example 1 were dissolved in a lysate containing 4% NP-40, centrifuged and the supernatant was taken, followed by the manufacturer's manual. The absorbance at 570 nm was measured to analyze the intracellular TG content.
그 결과, 도 2b에 나타낸 바와 같이, 10, 20, 및 40μM의 자세오시딘을 처리한 3T3-L1 지방세포 내의 TG함량은 각각 87.4, 81.3, 및 72.8%로 양성대조군에 비해 현저하게 감소하였다.As a result, as shown in FIG. 2B, the TG contents in 3T3-L1 adipocytes treated with 10, 20, and 40 μM of posturetidine were 87.4, 81.3, and 72.8%, respectively, and were significantly reduced compared to the positive control group.
따라서, 상기 결과로부터 자세오시딘이 3T3-L1 지방전구세포가 성숙한 지방세포로 분화하는 동안 세포 내 지질 축적을 감소시킨다는 것을 알 수 있었다. Therefore, it can be seen from the above results that postosidine decreases intracellular lipid accumulation while 3T3-L1 adipocytes differentiate into mature adipocytes.
실험예Experimental Example 4. 4. 자세오시딘의Posture 지방세포 형성 및 분화 관련 전사인자 및 Transcription factors related to adipocyte formation and differentiation, and 아디포넥틴Adiponectin 발현 억제 효과 확인 Confirmation of expression suppression effect
4-1. 4-1. mRNAmRNA 발현 분석 Expression analysis
상기 실시예 1에서 처리한 3T3-L1 지방세포 (mature adipocytes)에서 total RNA 추출시약(RNAiso Plus, TAKARA, Japan)을 사용하여 세포 용해 후, chloroform을 넣고 4℃, 12000rpm에서 15분간 centrifuge하여 상층액을 얻었다. 상층액을 isopropanol과 섞은 후 반응시켜 같은 조건에서 10분간 centrifuge하여 RNA를 침전시켜 75% Ethanol로 세척하여 RNA를 분리하였다. 추출한 RNA를 PrimeScript 1st strand cDNA Synthesis Kit(TAKARA)를 이용하여 cDNA로 합성하였다.After cell lysis using total RNA extraction reagent (RNAiso Plus, TAKARA, Japan) from 3T3-L1 adipocytes treated in Example 1 above, chloroform was added and centrifuge at 4℃, 12000rpm for 15 minutes to supernatant. Got The supernatant was mixed with isopropanol, reacted, and centrifuge for 10 minutes under the same conditions to precipitate RNA, and washed with 75% Ethanol to separate RNA. The extracted RNA was synthesized as cDNA using PrimeScript 1st strand cDNA Synthesis Kit (TAKARA).
이후, 합성된 cDNA를 이용하여 real-time PCR (RT-PCR)을 진행하였다. RT-PCR은 Real-Time PCR Detection System (Bio-Rad Laboratories, USA) 장비를 이용하여 SYBR Green 의 발광 정도를 측정하여 비교하였다. 검출한 유전자들의 Primer 종류 및 서열은 표 1에 표시한 바와 같다. Then, real-time PCR (RT-PCR) was performed using the synthesized cDNA. RT-PCR The luminous intensity of SYBR Green was measured and compared using a Real-Time PCR Detection System (Bio-Rad Laboratories, USA) equipment. Primer types and sequences of the detected genes are shown in Table 1.
그 결과, 도 3a에 나타낸 바와 같이, 자세오시딘은 PPARγ, C/EBPα, 및 아디포넥틴의 mRNA 발현을 농도 의존적으로 억제하는 것을 알 수 있었다.As a result, as shown in FIG. 3A, it was found that postosidin inhibits mRNA expression of PPARγ, C/EBPα, and adiponectin in a concentration-dependent manner.
4-2. 단백질 발현 분석4-2. Protein expression analysis
상기 실시예 1에서 처리한 3T3-L1세포를 lysis buffer를 이용하여 용해하고, 원심분리하여 상등액을 얻었다. 이후, Bradford 방식으로 총 단백질을 정량하여 동량의 단백질(30μg)을 10% SDS polyacrylamide gel에서 전기영동을 실시하였다. 전기영동 후 SDS polyacrylamide gel내의 단백질을 Nitrocellulose Blotting Membrane(Amersham Protran Premium 0.2μm NC, GE Healthcare Life Sciences, Germany)으로 transfer하였다. 단백질이 transfer된 Membrane에 5% skim milk를 처리하여 비특이적인 단백질에 대해 1시간 동안 blocking하였다. Membrane을 세척한 후 1차 항체를 반응시키고, 처리한 1차 항체에 알맞은 2차 항체를 사용하여 반응시켰다. 반응이 끝난 후 Enhanced Chemiluminoesence (ECL) solution으로 반응시켜 검출하였다.The 3T3-L1 cells treated in Example 1 were lysed using a lysis buffer, and centrifuged to obtain a supernatant. Subsequently, total protein was quantified by Bradford method, and the same amount of protein (30 μg) was electrophoresed on 10% SDS polyacrylamide gel. After electrophoresis, proteins in SDS polyacrylamide gel were transferred to Nitrocellulose Blotting Membrane (Amersham Protran Premium 0.2 μm NC, GE Healthcare Life Sciences, Germany). 5% skim milk was treated on the protein-transferred membrane to block non-specific proteins for 1 hour. After washing the membrane, the primary antibody was reacted, and the secondary antibody suitable for the treated primary antibody was reacted. After the reaction was completed, it was detected by reacting with Enhanced Chemiluminoesence (ECL) solution.
그 결과, 도 3b에 나타낸 바와 같이, 자세오시딘은 PPARγ2 및 아디포넥틴 단백질 발현을 억제시키는 것을 확인할 수 있었다.As a result, as shown in FIG. 3B, it was confirmed that postosidine inhibits PPARγ2 and adiponectin protein expression.
실시예 2. 통계분석Example 2. Statistical analysis
모든 실험은 3회 반복실험을 하였으며, 통계분석은 일원배치분산분석을 이용하였다. p<0.05는 통계학적 유의성을 나타낸다.All experiments were repeated 3 times, and one-way variance analysis was used for statistical analysis. p<0.05 indicates statistical significance.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Since the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
A composition for inhibiting adipocyte differentiation in vitro, comprising posidinine as an active ingredient.
상기 조성물은 세포 내 지질 축적을 감소시키는 것을 특징으로 하는, 시험관 내(in vitro) 지방세포 분화 억제용 조성물.
According to claim 1,
The composition is characterized in that to reduce the accumulation of lipids in cells, in vitro (in vitro) composition for inhibiting adipocyte differentiation.
상기 조성물은 지방세포 형성 및 분화에 관여하는 전사인자 또는 아디포넥틴의 발현을 억제하는 것을 특징으로 하는, 시험관 내(in vitro) 지방세포 분화 억제용 조성물.
According to claim 1,
The composition is characterized in that to inhibit the expression of adiponectin or transcription factors involved in adipocyte formation and differentiation, a composition for inhibiting adipocyte differentiation in vitro.
상기 전사인자는 퍼옥시좀 증식제 활성화 수용체 감마(PPARγ) 또는 CCAAT-인핸서 결합 단백질 알파(C/EBPα)인 것을 특징으로 하는, 시험관 내(in vitro) 지방세포 분화 억제용 조성물.The method of claim 4,
The transcription factor is a peroxisome proliferator-activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C/EBPα), in vitro adipocyte composition for inhibiting differentiation of adipocytes.
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