KR20240125144A - Composition for preventing or treating obesity containing ascochlorin as an active ingredient - Google Patents
Composition for preventing or treating obesity containing ascochlorin as an active ingredient Download PDFInfo
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- KR20240125144A KR20240125144A KR1020230017823A KR20230017823A KR20240125144A KR 20240125144 A KR20240125144 A KR 20240125144A KR 1020230017823 A KR1020230017823 A KR 1020230017823A KR 20230017823 A KR20230017823 A KR 20230017823A KR 20240125144 A KR20240125144 A KR 20240125144A
- Authority
- KR
- South Korea
- Prior art keywords
- preventing
- treating obesity
- pharmaceutical composition
- ascochlorin
- expression
- Prior art date
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Classifications
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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
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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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
- A23V2200/332—Promoters of weight control and weight loss
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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
- A23V2250/00—Food ingredients
- A23V2250/30—Other Organic compounds
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- Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
본 발명은 아스코클로린 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물 및 건강기능식품 조성물에 관한 것으로, 보다 구체적으로는 지방전구세포의 분화를 억제하고, 지방세포 분화 관련 유전자와 지방세포 생성 억제 유전자의 발현을 조절하고, 지방세포의 세포주기중지를 유발하여 인체에 부작용이 없는 비만 예방 또는 치료용 약학적 조성물 및 건강기능식품 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating obesity, which contain ascochlorine or a pharmaceutically acceptable salt thereof as an active ingredient, and more specifically, to a pharmaceutical composition and a health functional food composition for preventing or treating obesity, which suppress the differentiation of preadipocytes, regulate the expression of genes related to adipocyte differentiation and genes inhibiting adipocyte production, and induce cell cycle arrest of adipocytes, thereby having no side effects on the human body.
Description
본 발명은 아스코클로린을 유효성분으로 포함하는 비만 예방 또는 치료용 조성물에 관한 것으로, 보다 구체적으로는 아스코클로린 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물 및 건강기능식품 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating obesity, which comprises ascochlorine as an active ingredient, and more specifically, to a pharmaceutical composition and a health functional food composition for preventing or treating obesity, which comprises ascochlorine or a pharmaceutically acceptable salt thereof as an active ingredient.
비만은 열량의 섭취와 소비의 불균형으로 발생하는 대사성 질환으로, 상기 과잉된 열량으로 인해 지방 조직이 비정상적으로 증가된 상태를 의미한다. 한국인의 경우 체질량지수(BMI)가 25 이상이면 비만에 해당한다. 비만은 고혈압, 혈중 콜레스테롤 상승, 당뇨병, 신장 질환, 뇌졸중, 동맥경화증, 지방간, 관절염, 암, 수면 무호흡증 등의 여러 질환의 원인으로 작용한다. 이러한 비만을 치료하기 위한 다양한 비만 치료제가 판매되고 있다.Obesity is a metabolic disease caused by an imbalance between calorie intake and consumption, and refers to a state in which fat tissue abnormally increases due to the excess calories. In the case of Koreans, a body mass index (BMI) of 25 or higher is considered obesity. Obesity acts as a cause of various diseases such as high blood pressure, elevated blood cholesterol, diabetes, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, and sleep apnea. Various anti-obesity drugs are sold to treat this obesity.
비만 치료제는 식욕을 억제하여 음식을 적게 섭취하도록 하는 식욕억제제, 지방이 흡수되지 않도록 하는 소화흡수 억제제, 체내 에너지 대사를 촉진하는 체내 에너지 대사 촉진제로 분류할 수 있다. 종래의 식욕억제제 중 대표적인 치료제는 리덕틸(ReductilTM, 애보트사, 미국)이 있으며, 체내 에너지 대사 촉진제 중 대표적인 치료제는 엑소리제(ExoriseTM, 아코파마사, 프랑스)가 있고, 소화흡수 억제제 중 대표적인 치료제는 제니칼(XenicalTM, 로슈제약회사, 스위스)가 있다.Anti-obesity drugs can be categorized into appetite suppressants that suppress appetite and cause less food intake, digestion and absorption inhibitors that prevent fat absorption, and energy metabolism accelerators that promote energy metabolism in the body. The representative treatment among conventional appetite suppressants is Reductil (Reductil TM , Abbott, USA), the representative treatment among energy metabolism accelerators is Exorise TM , Acopharma, France, and the representative treatment among digestion and absorption inhibitors is Xenical TM , Roche Pharmaceuticals, Switzerland.
하지만 종래의 비만 치료제는 중추신경계를 자극하여 불면증, 어지러움, 두통 등의 부작용이 나타날 수 있으며 오심, 구토, 심계항진, 혈압 상승, 불안, 우울 등의 부작용이 나타날 수 있다. 특히, 상기 리덕틸은 신경계에 작용하는 약제로 잦은 두통, 불면증 및 어지럼증과 같은 부작용이 있으며, 상기 제니칼은 소장에서 리파아제의 작용을 억제하여 지방변, 가스생성, 지용성 비타민 흡수 저하 등의 위장계 부작용을 수반하고, 상기 엑소리제는 구역질 및 구토감과 같은 부작용이 존재한다.However, conventional obesity treatment drugs can cause side effects such as insomnia, dizziness, and headache by stimulating the central nervous system, and can cause side effects such as nausea, vomiting, tachycardia, increased blood pressure, anxiety, and depression. In particular, the above-mentioned Reductil is a drug that acts on the nervous system and has side effects such as frequent headaches, insomnia, and dizziness, and the above-mentioned Xenical inhibits the action of lipase in the small intestine and causes gastrointestinal side effects such as steatorrhea, gas production, and decreased absorption of fat-soluble vitamins, and the above-mentioned Exolyzer has side effects such as nausea and vomiting.
이에, 인체에 부작용이 없으면서 비만을 치료할 수 있는 새로운 비만 치료제에 대한 필요성이 커지고 있는 실정이다.Accordingly, there is a growing need for new obesity treatments that can treat obesity without causing side effects on the human body.
본 발명의 목적은 인체 부작용 없이 비만을 예방 및 치료할 수 있는 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물을 제공하는 것이다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which contains ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, which can prevent and treat obesity without side effects on the human body.
본 발명의 다른 목적은 인체 부작용 없이 비만을 예방 및 치료할 수 있는 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or treating obesity, which contains ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, which can prevent and treat obesity without side effects on the human body.
발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있다.The technical problems to be solved by the invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the art from the description of the present invention.
본 발명은 하기 화학식 1로 표시되는 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
본 발명에 있어서, 상기 비만 예방 또는 치료용 약학적 조성물은 Pref1(Preadipocyte factor 1), GATA2(GATA-binding factor 2) 및 KLF2(Kruppel-like Factor 2)으로 이루어진 군에서 선택된 1종 이상의 유전자 발현을 증가시키는 것을 특징으로 한다.In the present invention, the pharmaceutical composition for preventing or treating obesity is characterized by increasing the expression of at least one gene selected from the group consisting of Pref1 (Preadipocyte factor 1), GATA2 (GATA-binding factor 2), and KLF2 (Kruppel-like Factor 2).
본 발명에 있어서, 상기 비만 예방 또는 치료용 약학적 조성물은 Peroxisome proliferator-activated receptor γ(PPARγ), CCAAT/enhancer-binding protein α(C/EBPα), Fatty acid synthase(FAS), Perilipin 및 Hormone-sensitive lipase(HSL)으로 이루어진 군에서 선택된 1종 이상의 유전자 발현을 감소시키는 것을 특징으로 한다.In the present invention, the pharmaceutical composition for preventing or treating obesity is characterized by reducing the expression of one or more genes selected from the group consisting of Peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), Fatty acid synthase (FAS), Perilipin, and Hormone-sensitive lipase (HSL).
본 발명에 있어서, 상기 비만 예방 또는 치료용 약학적 조성물은 Cyclin A의 단백질 발현을 억제하는 것을 특징으로 하고, 지방세포의 세포주기중지(Cell cycle arrest)를 유도하는 것을 특징으로 한다.In the present invention, the pharmaceutical composition for preventing or treating obesity is characterized by inhibiting the protein expression of Cyclin A and inducing cell cycle arrest of adipocytes.
본 발명은 하기 화학식 1로 표시되는 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물을 제공한다.The present invention provides a health functional food composition for preventing or treating obesity, which contains ascochlorin represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Chemical Formula 1]
본 발명에 따른 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 인체에 부작용 없이 비만을 예방 및 치료할 수 있다.A pharmaceutical composition for preventing or treating obesity comprising ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can prevent and treat obesity without side effects on the human body.
또한, 본 발명에 따른 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 인체에 부작용 없이 비만을 예방 및 치료할 수 있다.In addition, a health functional food composition for preventing or treating obesity containing ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can prevent and treat obesity without side effects on the human body.
본 발명의 효과들은 이상에서 언급한 효과들로 제한되지 않으며, 언급되지 않은 또 다른 효과들은 청구범위의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.
도 1은 아스코클로린의 지방전구세포 분화 억제 효과를 확인한 Oil red O 염색 결과이다.
도 2는 타 화합물에 비해 우수한 아스코클로린의 지방전구세포 분화 억제 효과를 나타낸 Oil red O 염색 결과이다.
도 3은 타 화합물에 비해 우수한 아스코클로린의 지방전구세포 분화 억제 효과를 나타낸 Oil red O 염색 결과이다.
도 4는 아스코클로린의 세포독성 평가 결과를 나타낸 그래프이다.
도 5는 아스코클로린의 PPARγ, C/EBPα 및 FAS 유전자의 mRNA 발현 억제 효과를 나타낸 그래프이다.
도 6은 아스코클로린의 PPARγ 및 C/EBPα 유전자의 mRNA 발현을 분화 유도 일수에 따라 나타낸 그래프이다.
도 7은 아스코클로린의 KLF2, Pref1 및 GATA2 유전자의 mRNA 발현 증가 효과를 나타낸 그래프이다.
도 8은 아스코클로린의 Perilipin, FAS 및 HSL 유전자의 mRNA 발현 억제 효과를 나타낸 그래프이다.
도 9는 아스코클로린의 Pref1, c/EBPβ, PPARγ, c/EBPα 및 FABP4 단백질 발현 조절 효과를 나타낸 그림이다.
도 10은 아스코클로린의 지방세포 세포주기중지 효과를 나타낸 그래프이다.
도 11은 아스코클로린의 Cyclin A 단백질 발현 억제 효과를 나타낸 그림이다.Figure 1 shows the results of Oil red O staining confirming the inhibitory effect of ascochlorine on adipocyte differentiation.
Figure 2 shows the results of Oil red O staining showing that ascochlorine has a superior effect on preadipocyte differentiation compared to other compounds.
Figure 3 shows the results of Oil red O staining showing that ascochlorine has a superior effect on preadipocyte differentiation compared to other compounds.
Figure 4 is a graph showing the results of the cytotoxicity evaluation of ascochlorine.
Figure 5 is a graph showing the inhibitory effect of ascochlorine on the mRNA expression of PPARγ, C/EBPα, and FAS genes.
Figure 6 is a graph showing the mRNA expression of PPARγ and C/EBPα genes by ascochlorin according to the number of days of differentiation induction.
Figure 7 is a graph showing the effect of ascochlorine on increasing mRNA expression of KLF2, Pref1, and GATA2 genes.
Figure 8 is a graph showing the inhibitory effect of ascochlorine on the mRNA expression of Perilipin, FAS, and HSL genes.
Figure 9 is a drawing showing the regulatory effect of ascochlorine on the expression of Pref1, c/EBPβ, PPARγ, c/EBPα, and FABP4 proteins.
Figure 10 is a graph showing the adipocyte cell cycle arrest effect of ascochlorine.
Figure 11 is a drawing showing the inhibitory effect of ascochlorine on Cyclin A protein expression.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다. The terms used in this specification are selected from the most widely used general terms possible while considering the functions in the present invention, but they may vary depending on the intention of engineers working in the field, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meanings thereof will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the overall contents of the present invention, rather than simply the names of the terms.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in commonly used dictionaries, such as those defined in common usage, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense unless expressly defined in this application.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
비만 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating obesity
본 발명은 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에서 “아스코클로린(Ascochlorin)”은 하기 화학식 1로 표시되는 화합물을 의미할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, “ascochlorin” may mean a compound represented by the following chemical formula 1, but is not limited thereto.
[화학식 1][Chemical Formula 1]
본 발명에서 “약학적 조성물”은 각각 통상의 방법에 따라 산제, 과립제, 서방형 과립제, 장용과립제, 액제, 점안제, 엘실릭제, 유제, 현탁액제, 주정제, 트로키제, 방향수제, 리모나아데제, 정제, 서방형정제, 장용정제, 설하정, 경질캅셀제, 연질캅셀제, 서방캅셀제, 장용캅셀제, 환제, 틴크제, 연조엑스제, 건조엑스제, 유동엑스제, 주사제, 캡슐제, 관류액, 경고제, 로션제, 파스타제, 분무제, 흡입제, 패취제, 멸균주사용액, 또는 에어로졸 등의 외용제 등의 형태로 제형화하여 사용될 수 있으며, 상기 외용제는 크림, 젤, 패치, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제 등의 제형을 가질 수 있다.In the present invention, the “pharmaceutical composition” may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipses, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigants, ointments, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.
본 발명에서 “약학적으로 허용가능한 염”이란 환자에게 비교적 비독성이고 무해한 유효작용을 갖는 농도로서, 이 염에 기인한 부작용이 아스코클로린(Ascochlorin)의 비만 예방 또는 치료 효과을 저하시키지 않는 임의의 모든 유기 또는 무기 부가염을 말하며, 예컨대 유리산으로는 유기산과 무기산 또는 무독성 염류 등을 사용할 수 있으며, 무기산으로는 염산, 인산, 황산, 질산, 주석산 등을 사용할 수 있고, 유기산으로는 메탄설폰산, p-톨루엔설폰산, 아세트산, 트라이플루오로아세트산, 말레인산(maleic acid), 석신산, 옥살산, 벤조산, 타르타르산, 푸마르산(fumaric acid), 만데르산, 프로피온산 (propionic acid), 구연산(citric acid), 젖산(lactic acid), 글리콜산(glycollic acid), 글루콘산(gluconic acid), 갈락투론산, 글루탐산, 글루타르산(glutaric acid), 글루쿠론산(glucuronic acid), 아스파르트산, 아스코르브산, 카본산, 바닐릭산, 요오드화수소산(hydroiodic acid) 등을 사용할 수 있다.In the present invention, the term “pharmaceutically acceptable salt” refers to any organic or inorganic addition salt having a relatively non-toxic and harmless effective effect for patients, and side effects caused by the salt do not reduce the obesity prevention or treatment effect of ascochlorin. For example, organic acids and inorganic acids or non-toxic salts can be used as free acids, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, Glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방전구세포의 분화를 억제할 수 있다. In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient can inhibit differentiation of preadipocytes.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방전구세포가 지방세포로 분화하는 데 관여하는 유전자 발현을 조절할 수 있으며, 보다 구체적으로는 PPARγ, C/EBPα 및 FAS 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, can regulate the expression of genes involved in the differentiation of preadipocytes into adipocytes, and more specifically, can inhibit the mRNA and protein expression of PPARγ, C/EBPα, and FAS genes, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방세포 생성을 억제하는 유전자(anti-adipogenic gene)의 발현을 조절할 수 있으며, 보다 구체적으로는 KLF2, Pref1 및 GATA2 유전자의 mRNA 및 단백질 발현을 증가시킬 수 있다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, can regulate the expression of an anti-adipogenic gene that inhibits adipocyte production, and more specifically, can increase the mRNA and protein expression of KLF2, Pref1, and GATA2 genes.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방생성 타겟 유전자(adipogenic target gene)의 발현을 조절할 수 있으며, 보다 구체적으로는 Perilipin, FAS 및 HSL 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient can regulate the expression of adipogenic target genes, and more specifically, can inhibit mRNA and protein expression of Perilipin, FAS, and HSL genes, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방세포 분화와 관련된 유전자인 c/EBPβ, PPARγ, c/EBPα 및 FABP4 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, can inhibit mRNA and protein expression of c/EBPβ, PPARγ, c/EBPα and FABP4 genes, which are genes related to adipocyte differentiation, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 지방세포의 세포주기중지(Cell cycle arrest)를 유발할 수 있으며, 상기 세포주기중지는 세포주기가 S기로의 이행이 이루어지지 않고 G0/G1에서 멈추는 것을 의미할 수 있다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient, may induce cell cycle arrest of adipocytes, and the cell cycle arrest may mean that the cell cycle does not transition to the S phase but stops at G0/G1.
본 발명의 일 실시예에 있어서, 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 약학적 조성물은 상기 세포주기중지(Cell cycle arrest)에 관여하는 유전자의 mRNA 및 단백질 발현을 억제할 수 있으며, 바람직하게는 Cyclin A 유전자의 mRNA 및 단백질 발현을 억제할 수 있다.In one embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient can inhibit mRNA and protein expression of genes involved in cell cycle arrest, and preferably can inhibit mRNA and protein expression of Cyclin A gene.
비만 예방 또는 치료용 건강기능식품 조성물Health functional food composition for preventing or treating obesity
본 발명은 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물을 제공한다.The present invention provides a health functional food composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에서 “아스코클로린(Ascochlorin)”은 하기 화학식 1로 표시되는 화합물을 의미할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, “ascochlorin” may mean a compound represented by the following chemical formula 1, but is not limited thereto.
[화학식 1][Chemical formula 1]
본 발명에서 “건강기능식품”은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 또는 음료의 형태로 제형화되어 사용될 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the “health functional food” may be formulated and used in the form of powder, granules, pills, tablets, capsules, candy, syrup, or beverage, but is not limited thereto.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방전구세포의 분화를 억제할 수 있다. The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can inhibit the differentiation of preadipocytes.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방전구세포가 지방세포로 분화하는 데 관여하는 유전자 발현을 조절할 수 있으며, 보다 구체적으로는 PPARγ, C/EBPα 및 FAS 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.The health functional food composition for preventing or treating obesity, comprising ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can regulate the expression of genes involved in the differentiation of preadipocytes into adipocytes, and more specifically, can inhibit the mRNA and protein expression of PPARγ, C/EBPα, and FAS genes, but is not limited thereto.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방세포 생성을 억제하는 유전자(anti-adipogenic gene)의 발현을 조절할 수 있으며, 보다 구체적으로는 KLF2, Pref1 및 GATA2 유전자의 mRNA 및 단백질 발현을 증가시킬 수 있다.The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can regulate the expression of an anti-adipogenic gene that inhibits the production of adipocytes, and more specifically, can increase the mRNA and protein expression of KLF2, Pref1 and GATA2 genes.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방생성 타겟 유전자(adipogenic target gene)의 발현을 조절할 수 있으며, 보다 구체적으로는 Perilipin, FAS 및 HSL 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can regulate the expression of adipogenic target genes, and more specifically, can suppress mRNA and protein expression of Perilipin, FAS, and HSL genes, but is not limited thereto.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방세포 분화와 관련된 유전자인 c/EBPβ, PPARγ, c/EBPα 및 FABP4 유전자의 mRNA 및 단백질 발현을 억제할 수 있으나, 이에 한정되는 것은 아니다.The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can suppress mRNA and protein expression of c/EBPβ, PPARγ, c/EBPα and FABP4 genes, which are genes related to adipocyte differentiation, but is not limited thereto.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 지방세포의 세포주기중지(Cell cycle arrest)를 유발할 수 있으며, 상기 세포주기중지는 세포주기가 S기로의 이행이 이루어지지 않고 G0/G1에서 멈추는 것을 의미할 수 있다.The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an effective ingredient, can induce cell cycle arrest of adipocytes, and the cell cycle arrest can mean that the cell cycle does not transition to the S phase but stops at G0/G1.
본 발명에 따른 상기 아스코클로린(Ascochlorin) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 비만 예방 또는 치료용 건강기능식품 조성물은 상기 세포주기중지(Cell cycle arrest)에 관여하는 유전자의 mRNA 및 단백질 발현을 억제할 수 있으며, 바람직하게는 Cyclin A 유전자의 mRNA 및 단백질 발현을 억제할 수 있다.The health functional food composition for preventing or treating obesity, which comprises ascochlorin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, can inhibit mRNA and protein expression of genes involved in cell cycle arrest, and preferably can inhibit mRNA and protein expression of Cyclin A gene.
이하, 본 발명의 실시예를 상세히 기술하나, 하기 실시예에 의해 본 발명이 한정되지 아니함은 자명하다.Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited to the following examples.
실시예 1. 아스코클로린의 지방전구세포 분화 억제 효과Example 1. Inhibitory effect of ascochlorine on preadipocyte differentiation
1-1. 지방전구세포 분화 유도1-1. Induction of preadipocyte differentiation
아스코클로린(Ascochlorin)의 지방전구세포 분화 억제 효과를 확인하기 위해 실시예 1을 실시하였다. 상기 실시예 1에서 사용한 하기 화학식 1로 표시되는 아스코클로린은 Chugai Pharmaceutical(Tokyo, Japan)으로부터 제공받아 사용하였으며, 지방전구세포는 3T3-L1 세포를 ATCC(Manassas, VA, USA)에서 분양받아 사용하였다.Example 1 was conducted to confirm the inhibitory effect of ascochlorin on adipocyte differentiation. Ascochlorin represented by the following chemical formula 1 used in Example 1 was provided by Chugai Pharmaceutical (Tokyo, Japan), and 3T3-L1 cells were provided by ATCC (Manassas, VA, USA) as adipocyte precursor cells.
[화학식 1][Chemical formula 1]
상기 3T3-L1 세포는 DMEM 배지(Dulbeccos modified Eagles medium high glucose; Invitrogen, Carlsbad, CA, USA)에 10% 우혈청(bovine serum; BS) 및 항생제를 첨가하여 배양 및 분화 배지로 사용하고, 5% CO2 및 37℃ 인큐베이터에서 배양하였다. 상기 배양한 세포가 confluent 상태가 되면 트립신/EDTA를 처리하였으며, 상기 트립신 처리한 세포를 원심분리하여 세포를 수집하고 3×105 cells/well의 밀도로 6-well에 분주한 후 100% confluency 상태에서 48시간 동안 배양한 후, 10% FBS, 23 mg/mL IBMX(Sigma-Aldrich, Chem. Co., St. Louis, MO, USA), 5 mg/mL 인슐린(Sigma-Aldrich, Chem. Co.) 및 1 mM dexamethasone(Sigma-Aldrich, Chem. Co.)이 첨가된 DMEM 배지(MDI)를 처리하였으며, 상기 MDI 배지를 처리함과 동시에 상기 아스코클로린을 0, 0.5, 1, 5 또는 10 μM 농도로 함께 처리하고, 48시간 동안 분화를 유도하였다. 상기 분화를 유도한 세포는 2일 마다 10% FBS, 5 mg/mL 인슐린이 첨가된 배지로 교체하면서 8일 동안 분화를 유도하였다.The above 3T3-L1 cells were cultured and differentiated in DMEM medium (Dulbeccos modified Eagles medium high glucose; Invitrogen, Carlsbad, CA, USA) containing 10% bovine serum (BS) and antibiotics, and cultured in an incubator at 5% CO 2 and 37°C. When the cultured cells became confluent, they were treated with trypsin/EDTA, and the trypsin-treated cells were collected by centrifugation and seeded into 6-wells at a density of 3× 105 cells/well. After culturing for 48 hours at 100% confluency, they were treated with DMEM medium (MDI) supplemented with 10% FBS, 23 mg/mL IBMX (Sigma-Aldrich, Chem. Co., St. Louis, MO, USA), 5 mg/mL insulin (Sigma-Aldrich, Chem. Co.), and 1 mM dexamethasone (Sigma-Aldrich, Chem. Co.), and at the same time as the MDI medium was treated, the ascochlorine was treated together at a concentration of 0, 0.5, 1, 5, or 10 μM, and differentiation was induced for 48 hours. The cells that were induced to differentiate above were induced to differentiate for 8 days, with the medium supplemented with 10% FBS and 5 mg/mL insulin replaced every 2 days.
1-2. Oil red O 염색을 통한 아스코클로린의 지방전구세포 분화 억제 효과 확인1-2. Confirmation of the effect of ascochlorine on preadipocyte differentiation through oil red O staining
상기 8일 동안 분화를 유도한 세포의 배지를 제거하였으며, 이후 PBS(phosphate buffered saline)으로 세척하고, 10% 포름알데하이드 용액으로 세포를 고정하였다. 상기 고정한 세포를 다시 PBS로 세척하고 Oil red O 용액을 첨가하여 30분 동안 실온에서 염색하였으며, 30분 후에 Oil red O 용액을 제거하고 증류수로 세척하고 위상차 현미경으로 상기 세포를 관찰하였다. The medium of the cells that had been differentiated for the above 8 days was removed, and then the cells were washed with phosphate buffered saline (PBS), and fixed with a 10% formaldehyde solution. The fixed cells were washed again with PBS, and Oil red O solution was added, and the cells were stained for 30 minutes at room temperature. After 30 minutes, the Oil red O solution was removed, washed with distilled water, and the cells were observed under a phase contrast microscope.
또한, 상기 현미경으로 관찰한 세포를 100% 이소프로필 알콜(isopropyl alcohol)로 염색된 지방구를 용해시켜 510 nm에서 흡광도를 측정하였다. 상기 현미경으로 관찰한 세포 사진과 상기 측정한 흡광도를 도 1에 나타내었다.In addition, the cells observed under the microscope were stained with 100% isopropyl alcohol to dissolve the fat globules and measure the absorbance at 510 nm. The cell images observed under the microscope and the measured absorbance are shown in Figure 1.
본 실시예 1-2의 결과, 상기 아스코클로린을 처리하지 않고 상기 MDI 배지로 분화를 유도한 지방전구세포는 lipid droplet이 빨갛게 염색되어 분화가 유도된 것을 확인하였으며, 상기 MDI 배지로 분화를 유도한 지방전구세포는 상기 아스코클로린을 0, 0.5, 1, 5 또는 10 μM 농도별로 처리함에 따라 지방전구세포 분화가 억제되는 것을 확인하였고, 상기 아스코클로린을 10 μM 농도로 처리하였을 때, 가장 분화가 많이 억제되는 것을 확인하였다(도 1).As a result of Example 1-2, it was confirmed that preadipocytes that were differentiated with the MDI medium without treating the ascochlorine were differentiated as shown by red staining of lipid droplets, and it was confirmed that preadipocyte differentiation was inhibited when preadipocytes that were differentiated with the MDI medium were treated with the ascochlorine at concentrations of 0, 0.5, 1, 5, or 10 μM, and it was confirmed that differentiation was inhibited the most when the ascochlorine was treated at a concentration of 10 μM (Fig. 1).
1-3. 타 화합물에 비해 뛰어난 아스코클로린의 지방전구세포 분화 억제 효과1-3. Ascochlorine's superior effect on preadipocyte differentiation compared to other compounds
하기 화학식 1로 표시되는 아스코클로린(Ascochlorin; ASC), 하기 화학식 2로 표시되는 메틸아스코클로린(Methylascochlorin; MAC), 하기 화학식 3으로 표시되는 4-o-카복실메틸아스코클로린(4-o-carboxymethylascochlorin; AS-6) 및 하기 화학식 4로 표시되는 아스코푸라논(Ascofuranone; AF)의 지방전구세포 분화 억제 효과를 비교하기 위해 실시예 1-3을 실시하였다. 상기 실시예 1-3에서 실시한 Oil red O 염색 방법은 상기 실시예 1-1 및 1-2와 동일한 방법으로 실시하였으며, 상기 화학식 1 내지 4로 표시되는 화합물은 각각 10 μM 농도로 처리하였다. 단, 체중 감소와 제2형 당뇨병 치료제로 알려진 Metformin(Met)은 10 mM 농도로 처리하여 양성대조구로 사용하였다. 상기 실시예 1-3의 Oil red O 염색 결과를 현미경으로 관찰한 사진을 도 2에 나타내었으며, 상기 Oil red O 염색 결과를 흡광도 측정한 결과를 도 3에 그래프로 나타내었다.In order to compare the effects of ascochlorin (ASC) represented by the following chemical formula 1, methylascochlorin (MAC) represented by the following chemical formula 2, 4-o-carboxymethylascochlorin (AS-6) represented by the following chemical formula 3, and ascofuranone (AF) represented by the following chemical formula 4 on the inhibition of preadipocyte differentiation, Example 1-3 was performed. The Oil red O staining method performed in Example 1-3 was performed in the same manner as in Examples 1-1 and 1-2, and the compounds represented by the above chemical formulas 1 to 4 were each treated at a concentration of 10 μM. However, Metformin (Met), known as a weight loss and type 2 diabetes treatment agent, was treated at a concentration of 10 mM and used as a positive control. The results of the Oil red O staining of Example 1-3 above are shown in a photograph observed under a microscope in Fig. 2, and the results of measuring the absorbance of the Oil red O staining results above are shown graphically in Fig. 3.
[화학식 1][Chemical Formula 1]
[화학식 2][Chemical formula 2]
[화학식 3][Chemical Formula 3]
[화학식 4][Chemical Formula 4]
본 실시예 1-3의 결과, MAC, AS-6 및 AF 처리군의 경우에는 MDI 배지에 의해 분화 유도된 지방세포(Differentiated control; DC)와 동일하게 lipid droplet이 빨갛게 염색되었지만, 아스코클로린(ASC) 처리군는 lipid droplet 형성이 거의 되지 않았으며, 분화를 유도하지 않은 세포(Undifferentiated control; UD)와 동일한 양상을 확인하였다. 즉, 아스코클로린과 구조가 비슷한 MAC, AS-6 및 AF는 지방세포 분화 유도 억제 효과가 없지만, 아스코클로린은 지방세포 분화 유도 억제 효과가 뛰어난 것을 확인하였다(도 2 및 도 3).As a result of Example 1-3, in the MAC, AS-6, and AF treatment groups, lipid droplets were stained red, similar to adipocytes (Differentiated control; DC) differentiated by MDI medium, but in the ascochlorine (ASC) treatment group, lipid droplet formation was hardly observed, and the same pattern as the cells that were not differentiated (Undifferentiated control; UD) was confirmed. That is, MAC, AS-6, and AF, which have a similar structure to ascochlorine, did not have an inhibitory effect on inducing adipocyte differentiation, but ascochlorine was confirmed to have an excellent inhibitory effect on inducing adipocyte differentiation (Figs. 2 and 3).
실험예 1. 아스코클로린의 세포독성 평가Experimental Example 1. Evaluation of cytotoxicity of ascochlorine
상기 화학식 1로 표시되는 아스코클로린(Ascochlorin; ASC), 상기 화학식 2로 표시되는 메틸아스코클로린(Methylascochlorin; MAC), 상기 화학식 3으로 표시되는 4-o-카복실메틸아스코클로린(4-o-carboxymethylascochlorin; AS-6) 및 상기 화학식 4로 표시되는 아스코푸라논(Ascofuranone; AF)의 세포독성을 평가하기 위해 실험예 1을 실시하였다.Experimental Example 1 was conducted to evaluate the cytotoxicity of ascochlorin (ASC) represented by the chemical formula 1, methylascochlorin (MAC) represented by the chemical formula 2, 4-o-carboxymethylascochlorin (AS-6) represented by the chemical formula 3, and ascofuranone (AF) represented by the chemical formula 4.
각 시료의 세포증식과 독성을 측정하기 위해 Green 등의 방법에 따라 3-(3,4-dimethyl- thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay를 실시하였다. 상기 실시예 1-1과 동일한 방법으로 준비한 3T3-L1 preadipocyte 세포를 1 × 104 cells/well의 농도가 되도록 96 well plate에 분주하고, 37℃, 5% CO2 incubator에서 24시간 동안 배양한 후, 시료를 처리하고 48 시간 배양하였다. 이후 5 mg/mL의 MTT 용액을 처리하여 4시간 동안 배양한 후 상등액을 제거하고 각 well에 100 μL의 DMSO를 첨가하여 생성된 formazan 결정을 용해시켜 microplate reader로 550 nm에서 흡광도를 측정하였고, 세포독성은 시료의 흡광도를 대조군의 흡광도에 대한 백분율로 나타내었다. 상기 실험예 1의 결과는 도 4에 그래프로 나타내었다. To measure cell proliferation and toxicity of each sample, 3-(3,4-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed according to the method of Green et al. 3T3-L1 preadipocyte cells prepared in the same manner as in Example 1-1 were dispensed into a 96-well plate at a concentration of 1 × 10 4 cells/well and cultured for 24 hours in a 37°C, 5% CO 2 incubator. Then, the samples were treated and cultured for 48 hours. After treating with 5 mg/mL MTT solution and culturing for 4 hours, the supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the formed formazan crystals. The absorbance was measured at 550 nm using a microplate reader, and the cytotoxicity was expressed as a percentage of the absorbance of the sample to the absorbance of the control group. The results of Experimental Example 1 are shown graphically in Fig. 4.
본 실험예 1의 결과, 상기 화학식 1 내지 4로 표시되는 ASC, MAC, AS-6 및 AF 모두 세포독성이 없는 것으로 확인되었다(도 4).As a result of Experimental Example 1, it was confirmed that ASC, MAC, AS-6, and AF represented by the chemical formulas 1 to 4 were all non-cytotoxic (Fig. 4).
실시예 2. 아스코클로린의 지방세포 분화 관련 유전자 발현 조절 효과Example 2. Effect of ascochlorine on the regulation of gene expression related to adipocyte differentiation
아스코클로린의 지방세포 분화와 관련된 유전자의 발현 조절 효과를 확인하기 위해 실시예 2를 실시하였다. 상기 실시예 1-1과 동일한 방법으로 8일 동안 지방세포 분화를 유도하였으며, 분화 유도 후 상기 세포의 배지를 제거하고 PBS 10 mL로 2번 세척하였다. 상기 PBS로 세척한 세포의 RNA를 추출하기 위해 TRI 시약을 처리하고 1.5 mL 튜브에 1 mL씩 분주한 후, 1-브로모-3-클로로프로판을 200 μL씩 첨가하여 30초 동안 흔들고 3000 rpm, 4℃에서 15분 동안 원심분리한 후 상등액을 새로운 튜브로 옮겼다. 상기 새 튜브에 옮긴 상등액에 상등액과 동량의 이소프로판올(isopropanol)을 첨가하여 -20℃에서 2시간 동안 방치한 후, 13,000 rpm, 4℃에서 15분 동안 원심분리하여 상등액을 제거하였다. 상기 상등액 제거 후 남은 침전물에 75% 에탄올을 첨가하여 세척한 후, 제거하고 DEPC가 처리된 증류수를 첨가하여 65℃ 수조에서 10분 동안 반응시켜 침전물을 녹였다. 상기 과정으로 추출된 RNA를 이용하여 cDNA를 합성한 후, SYBER Green(Takara, Ohtsu, Japan)을 사용하여 지방세포 분화 관련 유전자의 발현량을 확인하였으며, 상기 지방세포 분화 관련 유전자로는 PPARγ, C/EBPα 및 FAS를 확인하였다. Example 2 was performed to confirm the effect of ascochlorine on the regulation of gene expression related to adipocyte differentiation. Adipocyte differentiation was induced for 8 days using the same method as Example 1-1, and after differentiation induction, the medium of the cells was removed and washed twice with 10 mL of PBS. To extract RNA of the cells washed with PBS, TRI reagent was treated, 1 mL was dispensed into 1.5 mL tubes, 200 μL of 1-bromo-3-chloropropane was added, shaken for 30 seconds, centrifuged at 3,000 rpm, 4°C for 15 minutes, and the supernatant was transferred to a new tube. An equal amount of isopropanol was added to the supernatant transferred to the new tube, left at -20°C for 2 hours, and centrifuged at 13,000 rpm, 4°C for 15 minutes to remove the supernatant. After removing the supernatant, the remaining precipitate was washed with 75% ethanol, removed, and distilled water treated with DEPC was added. The precipitate was dissolved by reacting in a 65°C water bath for 10 minutes. Using the RNA extracted through the above process, cDNA was synthesized, and the expression level of genes related to adipocyte differentiation was confirmed using SYBER Green (Takara, Ohtsu, Japan). PPARγ, C/EBPα, and FAS were confirmed as the genes related to adipocyte differentiation.
상기 PPARγ, C/EBPα 및 FAS 유전자의 발현량은 실시간 PCR(Real-time PCR)을 통해 확인하였으며, 상기 실시간 PCR에서 사용된 프라이머의 염기서열은 표 1에 기재하였다. 상기 Real-time PCR 과정은 초기변성(pre-denaturation) 95℃ 30초, 변성(denaturation)은 95℃ 5초, annealing은 60℃ 15초, 신장반응(elongation)은 72℃ 10초로 하여 40 cycle을 수행하였다. 용해 곡선(Melting curve)은 55℃에서 시작하여 95℃를 종말점으로 0.5℃씩 상승시키면서 80번 반응시켜 원하는 형광 값을 검출하였다. 상기 실시예 2에서 8일 이후의 PPARγ, C/EBPα 및 FAS 유전자 발현 변화 결과를 도 5에 그래프로 나타내었다. 또한, 상기와 동일한 방법으로 아스코클로린을 10 μM 농도로 처리하고, MDI 배지로 분화 유도를 시작하고 2, 4, 6 및 8일 이후의 PPARγ 및 C/EBPα 유전자 발현 변화 결과를 도 6에 그래프로 나타내었다.The expression levels of the above PPARγ, C/EBPα, and FAS genes were confirmed through real-time PCR, and the base sequences of the primers used in the real-time PCR are listed in Table 1. The real-time PCR process was performed 40 cycles with pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing at 60°C for 15 seconds, and elongation at 72°C for 10 seconds. The melting curve was performed 80 times starting at 55°C and increasing by 0.5°C each time to 95°C as the endpoint, and the desired fluorescence value was detected. The results of changes in the expression of the PPARγ, C/EBPα, and FAS genes after 8 days in Example 2 are shown graphically in Fig. 5. In addition, ascochlorine was treated at a concentration of 10 μM in the same manner as above, differentiation was induced with MDI medium, and the results of changes in PPARγ and C/EBPα gene expression after 2, 4, 6, and 8 days were shown graphically in Figure 6.
본 실시예 2의 결과, 상기 아스코클로린이 처리된 세포에서는 지방세포의 분화에 관여하는 유전자인 PPARγ, C/EBPα 및 FAS의 유전자 발현이 유의적으로 억제되었다. 이에, 아스코클로린은 지방전구세포가 지방세포로 분화하는 데 관여하는 유전자들의 발현을 억제시켜 지방 축적 및 지방세포로의 분화를 억제하는 것을 확인하였다(도 5 및 도 6).As a result of Example 2, in cells treated with ascochlorine, the gene expression of PPARγ, C/EBPα, and FAS, which are genes involved in the differentiation of adipocytes, was significantly suppressed. Accordingly, it was confirmed that ascochlorine suppresses the expression of genes involved in the differentiation of preadipocytes into adipocytes, thereby suppressing fat accumulation and differentiation into adipocytes (Figs. 5 and 6).
실시예 3. 아스코클로린의 지방세포 생성 억제 유전자 발현 조절 효과Example 3. Effect of Ascochlorine on Regulating Expression of Adipocyte Production Inhibitory Genes
아스코클로린의 지방세포 생성 억제 유전자(anti-adipogenic gene)인 KLF2, Pref1 및 GATA2 유전자의 발현 조절 효과를 확인하기 위해 실시예 3을 실시하였다. 상기 실시예 3에서 사용한 프라이머 서열은 하기 표 2에 기재하였으며, 상기 아스코클로린은 10 μM의 농도로 처리하였고, 이외에는 상기 실시예 2와 동일한 방법으로 진행하였다. 상기 실시예 3의 결과는 도 7에 그래프로 나타내었다.Example 3 was conducted to confirm the expression regulation effect of ascochlorine on the anti-adipogenic genes KLF2, Pref1 and GATA2 genes. The primer sequences used in Example 3 are listed in Table 2 below, ascochlorine was treated at a concentration of 10 μM, and other than that, the same method as Example 2 was conducted. The results of Example 3 are shown graphically in Fig. 7.
본 실시예 3의 결과, 상기 지방세포 생성을 억제하는 유전자인 KLF2, Pref1 및 GATA2 유전자는 상기 아스코클로린 10 μM을 처리함으로써 유전자 발현량이 유의적으로 증가하였다(도 7).As a result of Example 3, the gene expression levels of KLF2, Pref1, and GATA2 genes, which are genes that suppress adipocyte production, significantly increased by treatment with 10 μM of ascochlorine (Fig. 7).
실시예 4. 아스코클로린의 지방생성 타겟 유전자 발현 조절 효과Example 4. Effect of ascochlorine on the regulation of lipogenesis target gene expression
아스코클로린의 지방생성 타겟 유전자(adipogenic target gene)인 Perilipin, FAS 및 HSL 유전자 발현 조절 효과를 확인하기 위해 실시예 4를 실시하였다. 상기 실시예 4에서 사용한 프라이머 서열은 하기 표 3에 기재하였으며, 상기 아스코클로린은 10 μM의 농도로 처리하였고, 이외에는 상기 실시예 2와 동일한 방법으로 진행하였다. 상기 실시예 4의 결과는 도 8에 그래프로 나타내었다.Example 4 was conducted to confirm the effect of ascochlorine on the expression of adipogenic target genes, Perilipin, FAS, and HSL. The primer sequences used in Example 4 are listed in Table 3 below, ascochlorine was treated at a concentration of 10 μM, and other than that, the same method as Example 2 was conducted. The results of Example 4 are shown graphically in Fig. 8.
본 실시예 4의 결과, 상기 Perilipin, FAS 및 HSL 유전자는 상기 아스코클로린 10 μM을 처리함으로써 유전자 발현량이 유의적으로 감소하였다(도 8).As a result of Example 4, the gene expression levels of the Perilipin, FAS, and HSL genes were significantly reduced by treatment with 10 μM of ascochlorine (Fig. 8).
실시예 5. 아스코클로린의 지방세포 생성 억제 유전자 및 지방세포 분화 관련 단백질 발현 조절 효과Example 5. Effect of Ascochlorine on Regulating Expression of Adipocyte Production Inhibitory Genes and Adipocyte Differentiation-Related Proteins
아스코클로린의 지방세포 생성 억제 유전자 및 지방세포 분화 관련 단백질 발현 조절 효과를 확인하기 위해 실시예 5를 실시하였으며, 상기 지방세포 생성 억제 유전자(Anti-adipogenesis gene)은 Pref1 유전자를 사용하였고, 상기 지방세포 분화 관련 유전자는 c/EBPβ, PPARγ, c/EBPα 및 FABP4 유전자를 사용하였다. Example 5 was conducted to confirm the effect of ascochlorine on regulating the expression of anti-adipogenesis genes and proteins related to adipocyte differentiation. The Pref1 gene was used as the anti-adipogenesis gene, and the c/EBPβ, PPARγ, c/EBPα, and FABP4 genes were used as the genes related to adipocyte differentiation.
상기 실시예 1-1과 동일한 방법으로 8일 동안 지방세포 분화를 유도하였으며, 상기 분화 유도한 세포의 단백질을 추출 및 정량한 후 10% running gel과 4.5% stacking gel을 이용하여 125V에서 SDS-PAGE를 실시하였다. 상기 SDS-PAGE로 크기별로 분리한 단백질은 Transfer buffer(20% methanol, 25 mM Tris-HCl 및 192 mM Glycine)을 이용하여 350 mA에서 120분 동안 PVDF membrane으로 이동시켰다. 상기 단백질이 이동된 PVDF membrane은 5% 탈지유(non-fat dry milk)로 블로킹(blocking)하였다. 상기 블로킹한 membrane에 1차 항체를 TBST(Tris buffered saline and tween 20) 용액에 1:1000으로 희석하여 24시간 동안 반응시킨 후 TBST로 3회 세척하였다. 상기 1차 항체를 반응시킨 membrane에 2차 항체를 TBST 용액에 1:2000으로 희석하여 2시간 동안 반응시키고 다시 TBST로 3회 세척하였다. 이후, membrane에 ECL detection kit(WSE-7120s, ATTO Co., Tokyo, Japan)의 발색시약 Ⅰ과 Ⅱ를 1:1로 섞은 혼합액을 도포하고, X-선 필름(X-ray film; CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA)에 노출하여 현상한 다음 필름 상에서 단백질의 발현 정도를 관찰하였다. 상기 Western blot 실험 결과를 도 9에 그림으로 나타내었다.In the same manner as in Example 1-1, adipocyte differentiation was induced for 8 days, and the proteins of the differentiation-induced cells were extracted and quantified, followed by SDS-PAGE at 125 V using a 10% running gel and a 4.5% stacking gel. The proteins separated by size by the SDS-PAGE were transferred to a PVDF membrane using a transfer buffer (20% methanol, 25 mM Tris-HCl, and 192 mM Glycine) at 350 mA for 120 minutes. The PVDF membrane onto which the proteins were transferred was blocked with 5% non-fat dry milk. The blocked membrane was incubated with a primary antibody diluted 1:1000 in TBST (Tris buffered saline and tween 20) solution, reacted for 24 hours, and then washed three times with TBST. The membrane reacted with the primary antibody was diluted 1:2000 with a secondary antibody in TBST solution, reacted for 2 hours, and washed three times with TBST. After that, a mixture of chromogenic reagents I and II of the ECL detection kit (WSE-7120s, ATTO Co., Tokyo, Japan) in a 1:1 ratio was applied to the membrane, and exposed to an X-ray film (X-ray film; CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA), developed, and the degree of protein expression was observed on the film. The results of the Western blot experiment are shown in Fig. 9.
본 실시예 5의 결과, 지방세포 생성 억제 유전자(Anti-adipogenic gene)인 Pref1 단백질 발현은 분화 유도 4일 후부터 증가하였으며, 지방 세포 분화 관련 유전자인 c/EBPβ, PPARγ, c/EBPα 및 FABP4 단백질 발현은 상기 아스코클로린을 처리함으로써 유의미하게 억제되는 것을 확인하였다(도 9).As a result of Example 5, it was confirmed that the expression of Pref1 protein, an anti-adipogenic gene, increased from 4 days after differentiation induction, and that the expression of c/EBPβ, PPARγ, c/EBPα, and FABP4 protein, which are adipocyte differentiation-related genes, were significantly suppressed by treatment with ascochlorine (Fig. 9).
실시예 6. 아스코클로린의 세포주기중지 효과Example 6. Cell cycle arrest effect of ascochlorine
6-1. 세포주기중지6-1. Cell cycle arrest
아스코클로린의 세포주기중지(Cell cycle arrest) 효과를 확인하기 위해 실시예 6을 실시하였다. Example 6 was conducted to confirm the cell cycle arrest effect of ascochlorine.
상기 실시예 1-1과 동일한 방법으로 배양한 3T3-L1 preadipocytes를 6 well plate에 2 × 105 내지 3 × 105 cells/well의 밀도로 분주한 후, 세포가 꽉 찬 상태가 되면 배지를 교환하여 48 시간 더 배양하였다. 그 후 MDI가 첨가된 분화 배지로 교환하고, ASC를 첨가하여 18시간 후 상등액을 제거하고 70% ethanol에 세포를 분산시킨 후, 4℃에서 1시간 동안 세포를 고정하였다. 고정된 세포를 PI 용액과 RNase가 함유된 PBS에 현탁 시킨 후 암소에서 10분간 방치하고, flow cytometer를 이용해 세포주기 분석을 실시하였다. 상기 실시예 6의 결과는 도 10에 그래프로 나타내었다.3T3-L1 preadipocytes cultured in the same manner as in Example 1-1 were seeded at a density of 2 × 10 5 to 3 × 10 5 cells/well in a 6-well plate. When the cells became confluent, the medium was replaced and cultured for another 48 hours. Thereafter, the medium was replaced with differentiation medium containing MDI, ASC was added, and after 18 hours, the supernatant was removed, the cells were dispersed in 70% ethanol, and the cells were fixed at 4°C for 1 hour. The fixed cells were suspended in PBS containing PI solution and RNase, left in the dark for 10 minutes, and cell cycle analysis was performed using a flow cytometer. The results of Example 6 are shown graphically in Fig. 10.
본 실시예 6의 결과, 분화 중인 세포는 분화 유도를 시작하고 18시간 이후에 S기로 들어간다고 알려져 있지만, 상기 아스코클로린을 처리함으로써 세포주기가 S기로의 이행이 이루어지지 않고, G0/G1에서 세포주기중지(cell cycle arrest)가 일어났다(도 10). As a result of this Example 6, it is known that differentiating cells enter the S phase 18 hours after differentiation induction begins, but by treating with the ascochlorine, the cell cycle did not transition to the S phase, and cell cycle arrest occurred in G0/G1 (Fig. 10).
6-2. 세포주기중지에 관여하는 단백질 발현 변화6-2. Changes in protein expression involved in cell cycle arrest
상기 실시예 6-1에서 확인한 아스코클로린의 세포주기중지(Cell cycle arrest)에 관여하는 Cyclin A 단백질 발현 변화를 확인하기 위해 실시예 6-2를 실시하였다. 상기 실시예 6-2에서 사용한 western blot 실험방법은 상기 실시예 5와 동일하게 진행하였다.In order to confirm the change in the expression of Cyclin A protein involved in the cell cycle arrest of ascochlorine confirmed in the above Example 6-1, Example 6-2 was conducted. The western blot experiment method used in the above Example 6-2 was the same as in the above Example 5.
본 실시예 6-2의 결과, 상기 아스코클로린 10 μM 처리시 Cyclin A 단백질 발현이 현저하게 감소하는 것을 확인하였다(도 11). 이는 상기 감소한 Cyclin A 발현이 정상적인 세포주기에 영향을 미쳐서 지방세포의 분화가 억제되고 지방세포내 지방 축적이 감소할 수 있음을 의미한다.As a result of this Example 6-2, it was confirmed that Cyclin A protein expression was significantly reduced when treated with 10 μM ascochlorine (Fig. 11). This means that the reduced Cyclin A expression can affect the normal cell cycle, thereby inhibiting adipocyte differentiation and reducing fat accumulation in adipocytes.
이상 설명으로부터, 본 발명에 속하는 기술 분야의 당업자는 본 발명의 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며, 한정적인 것이 아닌 것으로서 이해해야만 한다.From the above description, those skilled in the art will be able to understand that the present invention can be implemented in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
Claims (5)
[화학식 1]
A pharmaceutical composition for preventing or treating obesity, comprising ascochlorin represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]
상기 비만 예방 또는 치료용 약학적 조성물은
Pref1(Preadipocyte factor 1), GATA2(GATA-binding factor 2) 및 KLF2(Kruppel-like Factor 2)으로 이루어진 군에서 선택된 1종 이상의 유전자 발현을 증가시키는 것을 특징으로 하는 비만 예방 또는 치료용 약학적 조성물.
In the first paragraph,
The pharmaceutical composition for preventing or treating obesity is
A pharmaceutical composition for preventing or treating obesity, characterized by increasing the expression of at least one gene selected from the group consisting of Pref1 (Preadipocyte factor 1), GATA2 (GATA-binding factor 2), and KLF2 (Kruppel-like Factor 2).
상기 비만 예방 또는 치료용 약학적 조성물은
Peroxisome proliferator-activated receptor γ(PPARγ), CCAAT/enhancer-binding protein α(C/EBPα), Fatty acid synthase(FAS), Perilipin 및 Hormone-sensitive lipase(HSL)으로 이루어진 군에서 선택된 1종 이상의 유전자 발현을 감소시키는 것을 특징으로 하는 비만 예방 또는 치료용 약학적 조성물.
In the first paragraph,
The pharmaceutical composition for preventing or treating obesity is
A pharmaceutical composition for preventing or treating obesity, characterized in that it reduces the expression of one or more genes selected from the group consisting of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), perilipin, and hormone-sensitive lipase (HSL).
상기 비만 예방 또는 치료용 약학적 조성물은
Cyclin A의 단백질 발현을 억제하는 것을 특징으로 하고, 지방세포의 세포주기중지(Cell cycle arrest)를 유도하는 것을 특징으로 하는 비만 예방 또는 치료용 약학적 조성물.
In the first paragraph,
The pharmaceutical composition for preventing or treating obesity is
A pharmaceutical composition for preventing or treating obesity, characterized by inhibiting the protein expression of cyclin A and inducing cell cycle arrest of adipocytes.
[화학식 1]
A health functional food composition for preventing or treating obesity, comprising ascochlorin represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]
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| KR1020230017823A KR20240125144A (en) | 2023-02-10 | 2023-02-10 | Composition for preventing or treating obesity containing ascochlorin as an active ingredient |
| PCT/KR2024/001681 WO2024167254A1 (en) | 2023-02-10 | 2024-02-06 | Composition for preventing or treating obesity comprising ascochlorin as active ingredient |
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| KR102486633B1 (en) | 2020-02-07 | 2023-01-10 | 건국대학교 글로컬산학협력단 | Lipid accumulation inhibitory or anti-obesity composition comprising ergosterol peroxide as an active ingredient |
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| JPWO2003063849A1 (en) * | 2002-01-31 | 2005-05-26 | アリジェン株式会社 | Pharmaceutical composition for diagnosis, prevention or treatment of multiple risk factor syndrome |
| AU2017205795A1 (en) * | 2016-01-05 | 2018-07-05 | Nrl Pharma, Inc. | Ascochlorin derivative and use thereof as AMPK activator |
| WO2018216821A1 (en) * | 2017-05-23 | 2018-11-29 | Nrl Pharma, Inc. | Use of ascochlorin derivative for combination therapy |
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| KR102486633B1 (en) | 2020-02-07 | 2023-01-10 | 건국대학교 글로컬산학협력단 | Lipid accumulation inhibitory or anti-obesity composition comprising ergosterol peroxide as an active ingredient |
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