KR101712450B1 - Food composition, pharmaceutical composition, animal medicine and feed composition against obesity with genipin - Google Patents
Food composition, pharmaceutical composition, animal medicine and feed composition against obesity with genipin Download PDFInfo
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- KR101712450B1 KR101712450B1 KR1020150045020A KR20150045020A KR101712450B1 KR 101712450 B1 KR101712450 B1 KR 101712450B1 KR 1020150045020 A KR1020150045020 A KR 1020150045020A KR 20150045020 A KR20150045020 A KR 20150045020A KR 101712450 B1 KR101712450 B1 KR 101712450B1
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- genipin
- obesity
- composition
- weight
- adipocytes
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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- C—CHEMISTRY; METALLURGY
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- 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
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Abstract
본 발명은 게니핀(genipin)을 유효성분으로 함유하는 것을 특징으로 하는 비만 개선용 식품 조성물, 비만 예방 및 치료용 약학 조성물, 비만 예방 및 치료용 동물용 의약품, 비만 개선용 동물 사료 첨가제, 비만 개선용 동물 사료 조성물에 관한 것으로, 지방전구세포의 지방세포분화 억제 및 지방세포의 지방 저장능력 억제 효과를 발휘하는 게니핀을 유효성분으로 함유함으로써 비만을 예방 또는 치료할 수 있는 효과를 발휘할 수 있다.The present invention relates to a food composition for improving obesity, which comprises genipin as an active ingredient, a pharmaceutical composition for preventing and treating obesity, an animal medicine for preventing and treating obesity, an animal feed additive for improving obesity, It is possible to prevent or treat obesity by containing genipin as an active ingredient that exhibits an effect of inhibiting adipocyte differentiation of fat precursor cells and inhibiting lipid storage capacity of adipocytes.
Description
본 발명은 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 식품 조성물, 비만 예방 및 치료용 약학 조성물, 비만 예방 및 치료용 동물용 의약품 및 비만 개선용 동물 사료 첨가제 및 비만 개선용 동물 사료 조성물에 관한 것이다.
The present invention relates to a composition for improving obesity containing genipin as an active ingredient, a pharmaceutical composition for preventing and treating obesity, an animal drug for preventing and treating obesity, an animal feed additive for improving obesity, and an animal feed composition for improving obesity .
서구화된 식습관과 운동량 감소로 인해 현재 비만 인구 비율은 전 세계적으로 급증하고 있는 추세이다. 또한 비만은 제 2형 당뇨, 심혈관질환, 고지혈증, 고혈압 등 다른 여러 합병증을 유발하기 때문에 단순한 미용상의 문제가 아니라 질병으로 인식되고 있다. 또한 비만인 사람은 성호르몬 분비가 자극되어 성장장애를 겪게 된다는 보고도 이루어져 있어, 비만은 시급히 해결되어야 할 사회적인 문제로 인식되고 있다.Due to westernized eating habits and a decrease in exercise, the proportion of obese people is rapidly increasing worldwide. Obesity is also recognized as a disease, not merely a cosmetic problem, because it causes other complications such as type 2 diabetes, cardiovascular disease, hyperlipemia and hypertension. Obesity has also been reported to be caused by growth hormone secretion stimulated by the growth of obesity is recognized as a social problem urgently need to be resolved.
현재까지 개발된 비만 치료제들은 소화계 또는 신경계에 작용을 하는 기전을 가지고 있다. 시부트라민의 경우는 신경계에서 식욕을 억제하는 활성을 나타내며, 현재 가장 널리 사용되고 있는 비만 치료제인 올리스타트의 경우는 소화계에서 지방 흡수를 억제하는 활성을 나타내고 있다. 이러한 방법은 우울증 및 지방변과 같은 부작용을 나타낸다는 것이 보고되어 있다. 최근에는 지방세포에서 직접적으로 작용하여 비만을 예방 및 치료하고자 하는 연구가 진행되고 있는데, 이러한 기작에는 지방전구세포의 지방세포분화를 억제하는 방법이 있고, 지방세포의 지방 축적을 저해하는 방법이 있다. 이러한 기작들은 기존에 알려진 식욕 억제제나 지방 흡수 억제제와는 다른 기작을 가지고 있기 때문에 새로운 개념의 비만 치료제를 개발하는데 있어서 중요한 역할을 할 수 있을 것으로 생각된다.Obesity drugs developed until now have a mechanism that acts on digestive system or nervous system. In the case of sibutramine, the activity of inhibiting appetite in the nervous system is shown. In the case of orlistat, the most widely used treatment for obesity, the activity of suppressing fat absorption in the digestive system is shown. This method has been reported to exhibit adverse effects such as depression and fatty liver. In recent years, studies have been conducted to prevent and treat obesity directly by acting on adipocytes. Such a mechanism includes a method of inhibiting adipocyte differentiation of adipose precursor cells and a method of inhibiting adipocyte fat accumulation . These mechanisms may play an important role in the development of a new concept of obesity treatment because they have mechanisms that are different from previously known appetite suppressants or fat absorption inhibitors.
한편, 게니핀(Genipin)은 치자나무(Gardenia jasminoides)에 존재하는 게니포사이드(geniposide)로 알려진 이리도이드 배당체(iridoid glycoside)로부터 유래되는 비당질 배당체(aglycone)의 일종으로서, 일반적으로 사용되고 있는 합성 가교제보다 독성이 낮기 때문에 우수한 천연 단백질 가교제로 많이 사용되고 있다. 게니핀은 간 질환에서는 이담작용에 도움을 주고, 항염증 및 항혈관형성을 억제하며, 항암 효과, 폐혈증 예방 및 치료 효과, 간암, 전립 유방암 및 자궁경부암 세포에서 아폽토시스(apoptosis)를 유도한다고 보고되어 있다. On the other hand, Genipin is a kind of non-saccharide aglycone derived from iridoid glycoside known as geniposide present in Gardenia jasminoides , and is a synthetic crosslinking agent Is less toxic and is thus widely used as an excellent natural protein crosslinking agent. It has been reported that genipin induces anti-inflammatory and anti-angiogenic effects, induces anti-cancer effects, prophylactic and therapeutic effects of sepsis, hepatocellular carcinoma, prostate breast cancer and apoptosis in cervical cancer cells have.
그러나, 아직까지 게니핀의 비만 예방 및 치료 효과에 대한 기술은 보고된 바 없다.
However, there has not yet been reported a technique for preventing and treating obesity in genipin.
본 발명은 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 식품 조성물, 비만 예방 및 치료용 약학 조성물, 비만 예방 및 치료용 동물 의약품, 비만 개선용 동물 사료 첨가제 및 비만 개선용 동물 사료 조성물을 제공하는 것을 목적으로 한다.
The present invention relates to a food composition for improving obesity containing genipin as an active ingredient, a pharmaceutical composition for preventing and treating obesity, an animal medicine for preventing and treating obesity, an animal feed additive for improving obesity, and an animal feed composition for improving obesity The purpose is to provide.
상기 목적을 달성하기 위하여, 본 발명은 제1형태로 하기의 화학식 1의 구조를 가지는 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 식품 조성물을 제공한다.In order to achieve the above object, the present invention provides, as a first aspect, a food composition for improving obesity containing genipin having the structure represented by the following general formula (1) as an active ingredient.
[화학식 1][Chemical Formula 1]
본 발명에서는 게니핀이 지방전구세포의 지방세포분화를 억제하고, 지방세포의 지방 저장능력을 감소시킨다는 것을 밝혔다. 게니핀은 지방전구세포의 지방세포분화 억제 효능은 체내에서 새로운 지방세포가 생기는 것을 억제하여 지방조직이 커지는 것을 막을 수 있으며, 게니핀의 지방세포의 지방 저장능력 감소 효능은 지방세포 내에 저장되는 지방의 양을 감소시켜 지방조직의 크기가 커지는 것을 막을 수 있다.In the present invention, it has been revealed that genipin inhibits adipocyte differentiation of lipid precursor cells and decreases lipid storage capacity of adipocytes. Genipin can inhibit the adipocyte differentiation inhibition effect of the fat precursor cells to prevent the adipose tissue from being enlarged by inhibiting the generation of new adipocytes in the body, and the effect of reducing the fat storage capacity of the adipocyte lipoprotein The size of the adipose tissue can be prevented from increasing.
본 발명의 비만 개선용 식품 조성물에 있어서, 상기 게니핀은 바람직하게 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것이 좋다. 상기 게니핀의 함량이 0.000001중량% 미만인 경우에는 비만 개선 효과가 미미하며, 50중량%를 초과하는 경우에는 사용량 대비 비만 개선 효과 증가가 미미하여 비경제적이기 때문이다. In the food composition for improving obesity of the present invention, the genipin is preferably contained in an amount of 0.000001 to 50% by weight based on the total weight of the composition. When the content of geniprin is less than 0.000001 wt%, the effect of improving obesity is insignificant. When the content of genipin is more than 50 wt%, the effect of improving obesity relative to the amount of usage is insignificant.
또한, 본 발명의 비만 개선용 식품 조성물에 있어서, 상기 게니핀의 농도는 바람직하게 10 μM~1 mM 인 것이 좋다.In the food composition for improving obesity of the present invention, the concentration of the genipin is preferably 10 μM to 1 mM.
또한, 본 발명의 비만 개선용 식품 조성물은 일 예로, 육류, 곡류, 카페인 음료, 일반음료, 초콜렛, 빵류, 스넥류, 과자류, 피자, 젤리, 면류, 껌류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나일 수 있으며, 반드시 이에 한정되는 것은 아니다.
In addition, the food composition for obesity improvement of the present invention may be, for example, meat, cereal, caffeinated beverages, general beverages, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic drinks, And other health supplement foods, and the present invention is not limited thereto.
한편, 본 발명은 제2형태로 상기 화학식 1의 구조를 가지는 게니핀(genipin)을 유효성분으로 함유하는 비만 예방 및 치료용 약학 조성물을 제공한다. The present invention also provides, as a second aspect, a pharmaceutical composition for preventing and treating obesity, which comprises genipin having the structure of Formula 1 as an active ingredient.
본 발명의 비만 예방 및 치료용 약학 조성물에 있어서, 상기 게니핀은 바람직하게 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것이 좋은데, 예방 및 치료제의 사용방법, 복용자의 상태, 질환의 종류 및 질환의 중증 정도에 따라 조절하는 것이 좋다. 상기 게니핀의 함량이 0.000001중량% 미만인 경우에는 비만 예방 및 치료 효과가 미미하며, 50중량%를 초과하는 경우에는 사용량 대비 비만 예방 및 치료 효과 증가가 미미하여 비경제적이다. In the pharmaceutical composition for preventing and treating obesity of the present invention, it is preferable that the genipin is contained in an amount of 0.000001 to 50% by weight based on the total weight of the composition. The method of using the preventive and therapeutic agent, the condition of the recipient, It is advisable to adjust according to the severity of the disease. When the content of the geniprin is less than 0.000001% by weight, the effect of preventing and treating obesity is insignificant. When the content exceeds 50% by weight, prevention of obesity and increase of therapeutic effect are less economical.
또한, 본 발명의 비만 예방 및 치료용 약학 조성물에 있어서, 상기 게니핀의 농도는 바람직하게 10 μM~1 mM인 것이 좋다.In the pharmaceutical composition for preventing and treating obesity of the present invention, the concentration of the genipin is preferably 10 μM to 1 mM.
또한, 본 발명의 비만 예방 및 치료용 약학 조성물은 유효성분 이외에 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용 가능한 담체, 부형제 또는 희석제로는 일 예로, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 1종 이상 사용될 수 있다. 또한, 예방 및 치료제가 약제인 경우 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등이 추가적으로 포함될 수 있다.
In addition, the pharmaceutical composition for preventing and treating obesity of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. One or more of these may be used. When the preventive and therapeutic agent is a pharmaceutical agent, a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent may be additionally included.
한편, 본 발명은 제3형태로 상기 화학식 1의 구조를 가지는 게니핀(genipin)을 유효성분으로 함유하는 비만 예방 및 치료용 동물 의약품 조성물을 제공한다. According to a third aspect of the present invention, there is provided an animal pharmaceutical composition for preventing and treating obesity comprising genipin having the structure of Formula 1 as an active ingredient.
본 발명의 비만 예방 및 치료용 동물 의약품 조성물에 있어서, 상기 게니핀은 바람직하게 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것이 좋다. 상기 게니핀의 함량이 0.000001중량% 미만인 경우에는 비만 예방 및 치료 효과가 미미하며, 50중량%를 초과하는 경우에는 사용량 대비 비만 예방 및 치료 효과 증가가 미미하여 비경제적이다. In the animal pharmaceutical composition for preventing and treating obesity of the present invention, it is preferable that the genipin is contained in an amount of 0.000001 to 50% by weight based on the total weight of the composition. When the content of the geniprin is less than 0.000001% by weight, the effect of preventing and treating obesity is insignificant. When the content exceeds 50% by weight, prevention of obesity and increase of therapeutic effect are less economical.
또한, 본 발명의 비만 예방 및 치료용 동물 의약품 조성물에 있어서, 상기 게니핀의 농도는 바람직하게 10 μM~1 mM인 것이 좋다. In the animal pharmaceutical composition for preventing and treating obesity of the present invention, the concentration of the genipin is preferably 10 μM to 1 mM.
한편, 본 발명의 비만 예방 및 치료용 약학 조성물 및 동물 의약품 조성물의 제형은 사용방법에 따라 바람직한 형태로 제조될 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LPTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPENSIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다. Meanwhile, the pharmaceutical composition for preventing and treating obesity of the present invention and the pharmaceutical composition for veterinary medicine can be prepared in a desired form depending on the method of use, and in particular, provide rapid, sustained or delayed release of the active ingredient after administration to a mammal It is preferable to employ a method known in the art to formulate it. Examples of specific formulations include PLASTERS, GRANULES, LPTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrups SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.
한편, 본 발명의 비만 예방 및 치료용 약학 조성물 및 동물 의약품 조성물의 투여량은 투여방법, 복용자(또는 복용 동물)의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분인 게니핀을 기준으로 하였을 때 1일 0.1 내지 100 mg/kg (체중)으로 1회 이상 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.
On the other hand, the dosage of the pharmaceutical composition for preventing and treating obesity and the pharmaceutical composition for an obesity according to the present invention is determined in consideration of the administration method, the age, sex, and weight of the recipient (or animal), and the severity of the disease. For example, it can be administered at least once at a dose of 0.1 to 100 mg / kg (body weight) per day based on the effective component of genipin. However, the dose is merely an example, and may be changed depending on the condition of the recipient and the doctor's prescription.
한편, 본 발명은 제4형태로 상기 화학식 1의 구조를 가지는 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 동물 사료 첨가제를 제공한다.According to a fourth aspect of the present invention, there is provided an animal feed additive for improving obesity containing genipin having the structure of Formula 1 as an active ingredient.
본 발명의 비만 개선용 동물 사료 첨가제에 있어서, 상기 게니핀은 바람직하게 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것이 좋다. 상기 게니핀의 함량이 0.000001중량% 미만인 경우에는 비만 개선 효과가 미미하며, 50중량%를 초과하는 경우에는 사용량 대비 비만 개선 효과 증가가 미미하여 비경제적이다. In the animal feed additive for improving obesity of the present invention, it is preferable that the genipin is contained in an amount of 0.000001 to 50% by weight based on the total weight of the composition. When the content of the geniprin is less than 0.000001% by weight, the effect of improving obesity is insignificant. When the content of geniprin is more than 50% by weight, the effect of improving obesity relative to the amount of usage is insignificant, which is uneconomical.
또한, 본 발명의 비만 개선용 동물 사료 첨가제에 있어서, 상기 게니핀의 농도는 바람직하게 10 μM~1 mM인 것이 좋다.
In addition, in the animal feed additive for improving obesity of the present invention, the concentration of the genipin is preferably 10 μM to 1 mM.
한편, 본 발명은 제5형태로 상기 화학식 1의 구조를 가지는 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 동물 사료 조성물을 제공한다.According to a fifth aspect of the present invention, there is provided an animal feed composition for improving obesity containing genipin having the structure of Formula 1 as an active ingredient.
본 발명의 비만 개선용 동물 사료 조성물에 있어서, 상기 게니핀은 바람직하게 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것이 좋다. 상기 게니핀의 함량이 0.000001중량% 미만인 경우에는 비만 개선 효과가 미미하며, 50중량%를 초과하는 경우에는 사용량 대비 비만 개선 효과 증가가 미미하여 비경제적이다. In the animal feed composition for improving obesity of the present invention, it is preferable that the genipin is contained in an amount of 0.000001 to 50% by weight based on the total weight of the composition. When the content of the geniprin is less than 0.000001% by weight, the effect of improving obesity is insignificant. When the content of geniprin is more than 50% by weight, the effect of improving obesity relative to the amount of usage is insignificant, which is uneconomical.
또한, 본 발명의 비만 개선용 동물 사료 조성물에 있어서, 상기 게니핀의 농도는 바람직하게 10 μM~1 mM인 것이 좋다. In the animal feed composition for improving obesity of the present invention, the concentration of the genipin is preferably 10 μM to 1 mM.
한편, 본 발명에서 '유효성분으로 함유하는 것'의 의미는 본 발명에서 요구하는 비만 개선, 예방 또는 치료의 효과가 본 발명의 유효성분인 게니핀으로부터 발생함을 의미하고, 그 외에 다른 성분을 보조성분으로 포함할 수 있음을 의미한다.
In the present invention, the term " containing as an active ingredient " means that the effect of improving, preventing or treating obesity required by the present invention arises from genipin, an active ingredient of the present invention. It can be included as an auxiliary component.
본 발명은 게니핀(genipin)을 유효성분으로 함유하는 비만 개선용 식품 조성물, 비만 예방 및 치료용 약학 조성물, 비만 예방 및 치료용 동물용 의약품, 비만 개선용 동물 사료 첨가제 및 비만 개선용 동물 사료 조성물을 제공할 수 있다.
The present invention relates to a composition for improving obesity comprising genipin as an active ingredient, a pharmaceutical composition for preventing and treating obesity, an animal drug for preventing and treating obesity, an animal feed additive for improving obesity, and an animal feed composition for improving obesity Can be provided.
도 1은 게니핀(genipin)과 게니포사이드(geniposide) 처리에 대한 지방전구세포의 생존도를 확인한 결과이다. A는 게니핀에 관한 결과이고, B는 게니포사이드에 관한 결과이다.
도 2는 MDI(지방세포분화 유도제)로 유도되는 지방전구세포 분화에 게니핀(genipin)과 게니포사이드(geniposide) 처리가 미치는 영향을 확인한 결과이다. 레인 1은 지방세포로 분화시키지 않은 음성 대조군, 레인 2는 MDI 처리로 분화된 대조군, 레인 3은 MDI와 게니핀 25 μM 처리군, 레인 4는 MDI와 게니핀 50 μM 처리군, 레인 5는 MDI와 게니핀 100 μM처리군, 레인 6은 MDI와 게니포사이드 25 μM 처리군, 레인 7은 MDI와 게니포사이드 50 μM 처리군, 레인 8은 MDI와 게니포사이드 100 μM 처리군이다.
도 3은 게니핀과 게니포사이드의 지방세포분화조절 마커인 C/EBPα와 PPARγ 단백질 발현 억제 효과를 확인한 결과이다. 레인 1은 분화시키지 않은 음성 대조군, 레인 2는 MDI 처리로 분화된 대조군, 레인 3은 MDI와 게니핀 25 μM 처리군, 레인 4는 MDI와 게니핀 50 μM 처리군, 레인 5는 MDI와 게니핀 100 μM 처리군, 레인 6은 MDI와 게니포사이드 100 μM 처리군이다. PPARγ는 isoform이 2개라서 밴드가 2개로 나타나게 되고, C/EBPα도 isoform이라서 밴드가 2개 나타나게 되는데 p42는 42kDa, p28은 28kDa의 C/EBPα를 의미한다.
도 4는 게니핀과 게니포사이드가 지질축적 마커인 FAS의 단백질 발현에 미치는 영향을 확인한 결과이다. 레인 1은 분화시키지 않은 음성 대조군, 레인 2는 MDI 처리로 분화된 대조군, 레인 3은 MDI와 게니핀 25 μM 처리군, 레인 4는 MDI와 게니핀 50 μM 처리군, 레인 5는 MDI와 게니핀 100 μM 처리군, 레인 6은 MDI와 게니포사이드 100 μM 처리군이다.
도 5는 게니핀과 게니포사이드가 성숙한 지방세포 내 지방 축적을 저해하는지를 확인한 것이다. 레인 1은 성숙한 지방세포 대조군, 레인 2는 게니핀 25 μM 처리군, 레인 3은 게니핀 50 μM 처리군, 레인 4는 게니핀 100 μM 처리군, 레인 5는 게니포사이드 100 μM 처리군이다.FIG. 1 shows the results of confirming the survival of adipose precursor cells against genipin and geniposide treatment. A is the result of genipin and B is the result of geniposide.
FIG. 2 shows the effect of genipin and geniposide treatment on lipid precursor differentiation induced by MDI (adipocyte differentiation inducer).
Fig. 3 shows the results of confirming the inhibitory effect of genipin and geniposide on the expression of C / EBP? And PPAR?
FIG. 4 shows the results of confirming the effect of genipin and geniposide on protein expression of lipid accumulation marker FAS.
FIG. 5 confirms whether genipin and geniposide inhibit mature lipid accumulation in adipocytes.
이하, 본 발명의 내용을 하기 실시예 또는 실험예를 들어 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.
Hereinafter, the present invention will be described in more detail with reference to the following examples or experimental examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.
[실험예 1: 게니핀 및 게니포사이드가 3T3-L1 지방전구세포주의 생존도에 미치는 영향 확인][Experimental Example 1: Determination of the effect of genipin and geniposide on the survival rate of 3T3-L1 adipose precursor cell line]
본 실험예에서는 게니핀과 게니포사이드가 3T3-L1 지방전구세포주에 미치는 독성 정도를 측정하기 위하여, MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) 분석법을 실시하였다. In this experiment, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to measure the degree of toxicity of geniprin and geniposide on 3T3-L1 adipocyte precursor cell line .
먼저 3T3-L1 세포를 96-웰 플레이트(96-well plate)에 접종한 뒤 웰에 세포가 꽉 차도록 배양하고 게니핀과 게니포사이드를 각각 40, 60, 80, 100 μM 농도로 처리하였다. 각각 72시간 동안 배양한 뒤에 MTT를 0.5 mg/㎖의 농도가 되도록 처리하고 30분 동안 37℃, 5% 이산화탄소 배양기 안에서 배양하였다. 그 후, 배양액을 제거하고 DMSO(dimethylsulfoxide)를 200 ㎕ 처리한 뒤 면역 흡광도 분석기(Versa Max, Moleular device, Califonia, USA)를 이용하여 570 nm의 파장에서 흡광도를 측정하였다. 세포 생존도는 샘플을 처리하지 않은 대조구를 100%로 계산하였다.First, 3T3-L1 cells were inoculated on a 96-well plate, and then cultured in wells so that the cells were filled, and then the genipin and geniposide were treated at 40, 60, 80, and 100 μM concentrations, respectively. After culturing for 72 hours each, MTT was treated to a concentration of 0.5 mg / ml and cultured in a 5% CO 2 incubator at 37 ° C for 30 minutes. Thereafter, the culture medium was removed and 200 μl of DMSO (dimethylsulfoxide) was treated. Absorbance was measured at a wavelength of 570 nm using an immunoabsorbant analyzer (Versa Max, Molecular device, Califonia, USA). Cell viability was calculated as 100% of the control without sample treatment.
실험결과, 게니핀과 게니포사이드 처리 농도(40, 60, 80, 100 μM)가 3T3-L1 지방전구세포 생존도에 영향을 미치지 않음을 확인할 수 있었다 (도 1).
As a result, it was confirmed that the concentration of genipin and geniposide treatment (40, 60, 80, 100 μM) did not affect the survival of 3T3-L1 lipoprotein (FIG. 1).
[실험예 2: 게니핀 및 게니포사이드가 3T3-L1 지방전구세포의 분화에 미치는 영향 확인][Experimental Example 2: Confirmation of the effect of genipin and geniposide on the differentiation of 3T3-L1 adipocyte precursor cells]
본 실험예에서는 게니핀, 게니포사이드가 지방전구세포의 지방세포분화에 미치는 영향을 확인하고자 지방전구세포를 지방세포로 분화시키면서 게니핀과 게니포사이드를 처리하였다. In this experiment, to examine the effect of genipin and geniposide on the adipocyte differentiation of adipocyte precursor cells, genipin and geniposide were treated while adipocyte precursor cells were differentiated into adipocytes.
지방전구세포를 지방세포로 분화시키는 과정은 다음과 같았다. 3T3-L1 지방전구세포 (입수처: 미국세포주은행 (ATCC))를 24-웰 플레이트(24-well plate)에 접종한 후 10% 우아 혈청(bovine calf serum; BCS)을 넣은 DMEM (DulbeccoModified EagleMedium) 배지를 사용하여 5% 이산화탄소 배양기 (Forma Scientific Co., Marjetta, OH, USA)에서 세포가 꽉 찰 때까지 배양하였다. 세포가 꽉 찼을 때 MDI {0.5 mM 이소부틸메틸잔틴 (isobutylmethylxanthine), 1 μM 덱사메타손 (dexamethasone) 및 5 ㎍/㎖ 인슐린(insulin)의 혼합물}와 10% 우태아 혈청(Fetal Bovine Serum; FBS)이 함유된 DMEM 배지를 넣고 이틀간 배양하였다. 그 후 10% 우태아 혈청과 5 ㎍/㎖ 인슐린이 첨가된 DMEM 배지를 넣고 이틀간 배양하였고, 그 후 10% 우태아 혈청만 첨가된 DMEM 배지를 넣고 이틀간 추가 배앙하여 완전히 분화된 지방세포를 만들었다. 상기와 같은 과정을 게니핀과 게니포사이드가 있거나 없는 상태에서 수행하였고, 게니핀과 게니포사이드는 DMSO에 녹여서 사용하였으며, 처리농도는 25, 50, 100 μM였다. The process of differentiating the precursor cells into adipocytes was as follows. (Dulbecco Modified Eagle Medium) supplemented with 10% bovine calf serum (BCS) after inoculating a 24-well plate with 3T3-L1 adipose precursor cells (available from American Cell Line Bank (ATCC) The cells were cultured in a 5% carbon dioxide incubator (Forma Scientific Co., Marjetta, OH, USA) using the medium until the cells were full. When the cells are full, a mixture of MDI {0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 5 μg / ml insulin) and 10% Fetal Bovine Serum (FBS) DMEM medium was added and cultured for two days. Then, DMEM medium supplemented with 10% fetal bovine serum and 5 μg / ml insulin was added and incubated for 2 days. Then, DMEM supplemented with 10% fetal bovine serum alone was added to the wells for additional two days to produce fully differentiated adipocytes. The above procedure was carried out with or without genipin and geniposide. Gunipin and geniposide were dissolved in DMSO, and the treatment concentrations were 25, 50 and 100 μM.
3T3-L1 지방전구세포가 지방세포로 완전히 분화된 후 세포 내에 함유된 지방을 오일 레드 오(Oil red O) 용액으로 염색하였다. 오일 레드 오 염색은 다음과 같이 실시하였다. 먼저 배지를 제거한 후 4% 포름알데히드(formaldehyde) 500 ㎕를 넣고 5분간 둔 후 제거하고, 다시 4% 포름알데히드 500 ㎕를 넣고 15분간 두어서 세포를 고정시켰다. 그 후 4% 포름알데히드를 제거하고 인산 완충 식염수 (PBS) 500 ㎕로 세척하고, 오일 레드 오 용액을 500 ㎕씩 넣고 15분간 두어서 지방을 염색하였다. 그 후 여분의 오일 레드 오 용액을 제거하고 PBS 500 ㎕로 두번 세척한 후 동량의 2-프로판올(2-propanol)을 넣고 5분간 흔들어서 지방에 염색되어 있던 오일 레드 오를 녹여낸 후 515 nm에서 흡광도를 측정하였다.After the 3T3-L1 adipose precursor cells were completely differentiated into adipocytes, the fat contained in the cells was stained with an oil red O solution. The oil red ocher was performed as follows. After removing the medium, 500 μl of 4% formaldehyde was added, and the mixture was allowed to stand for 5 minutes and then removed. 500 μl of 4% formaldehyde was added thereto and the cells were fixed for 15 minutes. Then, 4% formaldehyde was removed, washed with 500 μl of phosphate buffered saline (PBS), 500 μl of Oiledo solution was added and the fat was stained for 15 minutes. After removing the excess oil red oozing solution, wash twice with 500 μl of PBS, add the same amount of 2-propanol and shake for 5 minutes to dissolve the oil-red ocher stained in fat and measure the absorbance at 515 nm Respectively.
실험결과, 게니핀의 농도 의존적으로 세포 내 지방 축적량이 감소함을 확인할 수 있었다. 또한, 게니포사이드는 지방전구세포의 분화에 아무런 영향을 끼치지 않음을 확인할 수 있었다 (도 2).As a result, it was confirmed that intracellular fat accumulation was decreased in a concentration dependent manner of genipin. In addition, it was confirmed that the geniposide had no effect on the differentiation of lipid precursor cells (Fig. 2).
[실험예 3: 게니핀 및 게니포사이드의 지방세포분화조절 마커(C/EBPα, PPARγ) 발현 저해 효과 측정][Experimental Example 3: Measurement of Inhibitory Effect of Genipin and Geniposide on Adipocyte Differentiation Regulation Markers (C / EBP ?, PPAR?)]
본 실험예에서는 게니핀과 게니포사이드가 지방세포로의 분화 시 발현되는 가장 중요한 전사인자들 중 하나인 지방세포분화조절 마커(C/EBPα, PPARγ)의 단백질 발현에 미치는 영향을 확인하고자 웨스턴 블랏팅(Western Blotting) (B.L. Upham et al., 1997, Carcinogenesis. 18:37-42.)을 수행하였다. In order to examine the effect of genipin and geniposide on protein expression of adipocyte differentiation regulatory markers (C / EBPα, PPARγ), which is one of the most important transcription factors expressed upon differentiation into adipocytes, Western blotting Western Blotting) (BL Uphamet al, 1997, Carcinogenesis. 18: 37-42).
상기 실험예 2와 같이 배양된 세포로부터 단백질을 추출하기 위하여 차가운 인산완충식염수(phosphate buffered saline, PBS)로 두번 세척하고 RIPA 완충액 (50 mM, Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF(Phenylmethylsulfonyl fluoride))에 단백질 분해효소 저해제 (시그마알드리치) 0.5%와 인산 분해효소 저해제 {250 mM 플루오르화나트륨(Sodium Fluoride), 5 mM 오르토바나듐산나트륨(Sodium Orthovanadate), 50 mM 피로인산나트륨(Sodium Pyrophosphate), 50 mM 글리세로인산(Glycerophosphate)} 0.5%를 함유하는 단백질 추출 용액으로 세포를 용해하였다. 그 후, 상기 세포 용해물을 4℃에서 12,000 rpm으로 20분간 원심분리하여 수득한 상등액을 새로운 1.5 ㎖ 에펜도르프 튜브(eppendorf tube)에 옮겼다. To extract proteins from the cultured cells as in Experimental Example 2, the cells were washed twice with cold phosphate buffered saline (PBS), washed with RIPA buffer (50 mM, Tris pH 8.0, 150 mM NaCl, 1% NP- (Sigma Aldrich) 0.5% and a phosphatase inhibitor (250 mM Sodium Fluoride, 5 mM Sodium Orthovanadate, 5 mM EDTA, and 1 mM PMSF (phenylmethylsulfonyl fluoride) 50 mM sodium pyrophosphate, 50 mM glycerophosphate} 0.5%. Thereafter, the cell lysate was centrifuged at 12,000 rpm for 20 minutes at 4 DEG C, and the resulting supernatant was transferred to a new 1.5 mL eppendorf tube.
단백질 함량은 소 혈청 알부민(bovine serum albumin, BSA)을 표준용액으로 한 'DC assay kit [Bio-Rad Corp., Richmond, CA, USA]'를 이용하여 결정하였다. 20~50 ㎍의 단백질을 브로모페놀 블루를 함유하는 시료 완충액에 섞은 후 100℃에서 5분간 반응하여 변성을 가한 뒤 10% SDS-폴리아크릴아미드 겔에 로딩하고, 전류를 가하여 크기 별로 분리하였다. 상기 분리된 단백질을 겔에서 폴리비닐리덴 플루로라이드(Polyvinylidene fluoride, PVDF) 막으로 옮긴 후 차단 완충액 {탈지유 5%, 세척 완충액 94.8%, 아지드화나트륨(sodium azide) 0.2%)}으로 실온에서 1시간 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. Protein content was determined using a DC assay kit (Bio-Rad Corp., Richmond, CA, USA) with bovine serum albumin (BSA) as a standard solution. 20 to 50 μg of the protein was mixed with a buffer solution containing bromophenol blue and denatured by reacting at 100 ° C. for 5 minutes. Then, the protein was loaded on a 10% SDS-polyacrylamide gel and subjected to electric current to separate by size. The separated proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane and incubated with blocking buffer (5% skim milk, 94.8% washing buffer, 0.2% sodium azide) at room temperature After reacting for 1 hour, it was washed three times with washing buffer for 10 minutes.
그 후, 'Santacruz'에서 구입한 PPARγ 항체와 'Cell signal'에서 구입한 C/EBPα, 'Sigma'에서 구입한 β-actin 항체를 상온에서 2시간 동안 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. 그 후, 양고추냉이 과산화효소(horseradish peroxidase, HRP)와 공유결합한 2차 항체(anti-rabbit IgG-HRP 또는 anti-mouse IgG, Santa cruz)를 1시간 동안 상온에서 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. 그 후, 화학 발광액 (EZ-Western Detection Kit, Daeillab Service Co, Ltd)으로 5분간 반응시킨 뒤 엑스레이 필름에 노출시켜 엑스레이 필름을 인화하였다.Subsequently, PPARγ antibody purchased from Santacruz, C / EBPα purchased from 'Cell signal' and β-actin antibody purchased from 'Sigma' were reacted at room temperature for 2 hours and then washed three times with washing buffer for 10 minutes . Then, secondary antibody (anti-rabbit IgG-HRP or anti-mouse IgG, Santa Cruz) covalently bound to horseradish peroxidase (HRP) was reacted at room temperature for 1 hour and then washed with washing buffer for 10 minutes Washed three times. Thereafter, the cells were reacted with a chemiluminescent solution (EZ-Western Detection Kit, Daeillab Service Co., Ltd.) for 5 minutes, and exposed to an X-ray film to print an X-ray film.
실험결과, 게니핀의 농도 의존적으로 C/EBPα, PPARγ의 단백질 수준이 감소함을 확인할 수 있었다. 또한, 게니포사이드는 100 μM 처리에도 C/EBPα 단백질 수준에 대한 변화가 없음을 확인할 수 있었다 (도 3).
As a result of the experiment, it was confirmed that the protein level of C / EBPα and PPARγ was decreased in a dose dependent manner of genipin. In addition, it was confirmed that the geniposide did not change the C / EBPα protein level even at 100 μM treatment (FIG. 3).
[실험예 4: 게니핀 및 게니포사이드의 지질 축적 마커(FAS) 발현 저해 효과 측정][Experimental Example 4: Measurement of inhibitory effect of genipin and geniposide on lipid accumulation marker (FAS)] [
본 실험예에서는 지방세포분화의 주요 전사인자인 C/EBPα와 PPARγ에 의해 조절되는 지질 축적 마커인 FAS의 단백질 발현에 게니핀과 게니포사이드가 미치는 영향을 살펴보기 위하여 웨스턴 블랏팅(Western Blotting) (B.L. Upham et al., 1997, Carcinogenesis. 18:37-42.)을 수행하였다. In this experiment, to examine the effect of geniprin and geniposide on the protein expression of lipid accumulation marker FAS regulated by C / EBPα and PPARγ, major transcription factors of adipocyte differentiation, Western blotting (Western blotting BL Uphamet al, 1997, Carcinogenesis. 18: 37-42).
상기 실험예 2에 배양된 세포로부터 단백질을 추출하기 위하여 차가운 인산완충식염수(phosphate buffered saline, PBS)로 두번 세척하고 RIPA 완충액 {50 mM, Tris pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF(Phenylmethylsulfonyl fluoride)}에 단백질 분해효소 저해제 (시그마알드리치) 0.5%와 인산 분해효소 저해제 {250 mM 플루오르화나트륨(Sodium Fluoride), 5 mM 오르토바나듐산나트륨(Sodium Orthovanadate), 50 mM 피로인산나트륨(Sodium Pyrophosphate), 50 mM 글리세로인산(Glycerophosphate)} 0.5%를 함유하는 단백질 추출 용액으로 세포를 용해하였다. 그후, 상기 세포 용해물을 4℃에서 12,000 rpm으로 20분간 원심분리하여 수득한 상등액을 새로운 1.5 ㎖ 에펜도르프 튜브(eppendorf tube)에 옮겼다. In order to extract proteins from the cells cultured in Experimental Example 2, the cells were washed twice with cold phosphate buffered saline (PBS), and washed with RIPA buffer (50 mM, Tris pH 8.0, 150 mM NaCl, 1% (250 mM Sodium Fluoride, 5 mM Sodium Orthovanadate, 50 mM) were added to a solution containing 0.5% of a protease inhibitor (Sigma Aldrich), 1 mM PEG, 0.5 mM EDTA, 1 mM Phenylmethylsulfonyl fluoride) The cells were lysed with a protein extraction solution containing 0.5% sodium pyrophosphate, 50 mM glycerophosphate}. Then, the supernatant obtained by centrifuging the cell lysate at 12,000 rpm for 20 minutes at 4 DEG C was transferred to a new 1.5 mL eppendorf tube.
단백질 함량은 소 혈청 알부민(bovine serum albumin, BSA)을 표준용액으로 한 'DC assay kit [Bio-Rad Corp., Richmond, CA, USA]'를 이용하여 결정하였다. 20~50 ㎍의 단백질을 브로모페놀 블루를 함유하는 시료 완충액에 섞은 후 100℃에서 5분간 반응하여 변성을 가한 뒤 10% SDS-폴리아크릴아미드 겔에 로딩하고, 전류를 가하여 크기 별로 분리하였다. 상기 분리된 단백질을 겔에서 폴리비닐리덴 플루로라이드(Polyvinylidene fluoride, PVDF) 막으로 옮긴 후 차단 완충액 (탈지유 5%, 세척완충액 94.8%, 아지드화나트륨(sodium azide) 0.2%)으로 실온에서 1시간 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. Protein content was determined using a DC assay kit (Bio-Rad Corp., Richmond, CA, USA) with bovine serum albumin (BSA) as a standard solution. 20 to 50 μg of the protein was mixed with a buffer solution containing bromophenol blue and denatured by reacting at 100 ° C. for 5 minutes. Then, the protein was loaded on a 10% SDS-polyacrylamide gel and subjected to electric current to separate by size. The separated proteins were transferred from the gel to polyvinylidene fluoride (PVDF) membrane and incubated with blocking buffer (5% skim milk, 94.8% washing buffer, 0.2% sodium azide) at room temperature for 1 After reacting for a time, the cells were washed three times with washing buffer for 10 minutes.
그 후, 'Cell signaling'에서 구입한 FAS 항체와 'Sigma'에서 구입한 β-actin 항체를 상온에서 2시간 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. 그 후, 양고추냉이 과산화효소(horseradish peroxidase, HRP)와 공유결합한 2차 항체(anti-rabbit IgG-HRP 또는 anti-mouse IgG, Santa cruz)를 1시간 동안 상온에서 반응시킨 뒤 세척 완충액으로 10분간 세번 세척하였다. 그 후, 화학 발광액 (EZ-Western Detection Kit, Daeillab Service Co, Ltd)으로 5분간 반응시킨 뒤 엑스레이 필름에 노출시킨 뒤에 엑스레이 필름을 인화하였다.Then, the FAS antibody purchased from 'Cell signaling' and the β-actin antibody purchased from 'Sigma' were reacted at room temperature for 2 hours and then washed three times with washing buffer for 10 minutes. Then, secondary antibody (anti-rabbit IgG-HRP or anti-mouse IgG, Santa Cruz) covalently bound to horseradish peroxidase (HRP) was reacted at room temperature for 1 hour and then washed with washing buffer for 10 minutes Washed three times. Thereafter, the cells were reacted with a chemiluminescent solution (EZ-Western Detection Kit, Daeillab Service Co., Ltd.) for 5 minutes, exposed to an X-ray film, and then an X-ray film was printed.
실험결과, 게니핀의 농도 의존적으로 FAS의 단백질 수준이 감소함을 확인할 수 있었다. 또한, 게니포사이드는 100 μM 처리에도 게니핀 50 μM 처리시보다 낮은 FAS 단백질 감소 수준을 보임을 확인할 수 있었다 (도 4).
As a result of the experiment, it was confirmed that the protein level of FAS was decreased in a concentration-dependent manner of genipin. In addition, it was confirmed that the geniposide treatment showed a lower level of FAS protein than 100 μM treatment with 50 μM of genipin (FIG. 4).
[실험예 5: 게니핀 및 게니포사이드의 성숙한 지방세포 내 지방 축적 저해 효과 측정][Experimental Example 5: Measurement of inhibitory effects of genipin and geniposide on fat accumulation in mature adipocytes]
본 실험예에서는 게니핀 및 게니포사이드의 성숙한 지방세포 내 지방 축적 저해 효과를 확인하고자 지방전구세포를 지방세포로 분화시킨 후 게니핀과 게니포사이드를 처리하였다. 지방전구세포를 지방세포로 분화시키는 과정은 상기 실험예 2와 동일하였다. In this experiment, to confirm the inhibitory effect of genipin and geniposide on fat accumulation in mature adipocytes, the adipose precursor cells were differentiated into adipocytes and treated with genipin and geniposide. The process of differentiating the lipid precursor cells into adipocytes was the same as in Experimental Example 2 above.
3T3-L1 지방전구세포를 지방세포로 완전히 분화시킨 후 게니핀 (25, 50, 100 μM)과 게니포사이드 (100 μM)를 4일간 처리하였다. 4일 후 세포 내에 함유된 지방을 오일 레드 오 용액으로 염색하였다. 오일 레드 오 염색 과정은 상기 실험예 2와 동일하였다.3T3-L1 adipose precursor cells were completely differentiated into adipocytes and treated with genipin (25, 50, 100 μM) and geniposide (100 μM) for 4 days. After 4 days, the fat contained in the cells was stained with an oil red oozing solution. The procedure of dyeing oil red was the same as in Experimental Example 2 above.
실험결과, 게니핀의 농도 의존적으로 성숙한 지방세포 내 지방 축적량이 감소함을 확인할 수 있었다. 또한, 게니포사이드 100 μM 처리시 게니핀 25 μM 처리시와 비슷한 수준의 성숙한 지방세포 내 지방 축적량 감소 효과를 나타냄을 확인할 수 있었다 (도 5).
As a result, it was confirmed that fat accumulation in mature adipocytes was decreased in a concentration dependent manner of genipin. In addition, it was confirmed that treatment with 100 μM of geniposide showed a similar effect of reducing fat accumulation in mature adipocytes as in the case of treatment with 25 μM of genipin (FIG. 5).
[실시예 1: 비만 개선용 식품 조성물 제조][Example 1: Preparation of food composition for obesity improvement]
본 실시예에서는 하기와 같이 비만 개선용 식품 조성물을 제조하였다.In this Example, a food composition for obesity improvement was prepared as follows.
(1) 선식 제조(1) Manufacturing of wire
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60메쉬의 분말로 준비하였다. 검정콩, 검정깨 및 들깨 각각을 공지의 방법으로 쪄서 건조시킨 후 배전 및 분쇄하여 입도 60메쉬의 분말로 준비하였다. 이후, 현미 30중량%, 율무 15중량%, 보리 20중량%, 찹쌀 9중량%, 들깨 7중량%, 검정콩 8중량%, 검정깨 7중량%, 게니핀 3중량%, 영지 0.5중량% 및 지황 0.5중량%을 혼합하여 선식을 제조하였다.
Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then the mixture was prepared into powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds and perilla seeds were each steamed and dried by known methods, and then power distribution and pulverization were carried out to prepare powder having a particle size of 60 mesh. Thereafter, 30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley, 9% by weight of glutinous rice, 7% by weight of perilla seeds, 8% by weight of black soybeans, 7% by weight of black sesame seeds, 0.5 wt% were mixed to prepare an electric wire.
(2) 츄잉껌 제조(2) Production of chewing gum
껌 베이스 20중량%, 설탕 76.9중량%, 향료 1중량%, 물 2중량% 및 게니핀 0.1중량%를 배합하여 통상의 방법으로 츄잉껌을 제조하였다.
Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of a flavor, 2% by weight of water and 0.1% by weight of genipin.
(3) 캔디 제조(3) Candy manufacturing
설탕 60중량%, 물엿 39.8중량%, 향료 0.1중량% 및 게니핀 0.1중량%를 배합하여 통상의 방법으로 캔디를 제조하였다.
Sugar, 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of flavor and 0.1% by weight of genipin were mixed to prepare a candy by a conventional method.
(4) 비스킷 제조(4) Manufacturing of biscuits
박력 1급 25.59중량%, 중력 1급 22.22중량%, 정백당 4.80중량%, 식염 0.73중량%, 포도당 0.78중량%, 팜쇼트닝 11.78중량%, 암모니움 1.54중량%, 중조 0.17중량%, 중아황산나트륨 0.16중량%, 쌀가루 1.45중량%, 비타민 B1 0.0001중량%, 비타민 B2 0.0001중량%, 밀크향 0.04중량%, 물 20.6998중량%, 전지분유 1.16중량%, 대용분유 0.29중량%, 제일인산칼슘 0.03중량%, 살포염 0.29중량%, 분무유 7.27중량% 및 게니핀 1중량%를 배합하여 통상의 방법으로 비스킷을 제조하였다.
By weight of starch, 0.77% by weight of glucose, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonia, 0.17% by weight of sodium bicarbonate, 0.16% by weight of sodium bisulfite 1.45 wt% of rice flour, 0.0001 wt% of vitamin B 1, 0.0001 wt% of vitamin B 2 , 0.04 wt% of milk fractions, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% 0.29% by weight of spray salt, 7.27% by weight of spray oil and 1% by weight of genipin were mixed to prepare a biscuit by a conventional method.
(5) 건강음료 제조(5) health drink manufacturing
꿀 0.26중량%, 치옥토산아미드 0.0002중량%, 니코틴산아미드 0.0004중량%, 염산리보플라빈나트륨 0.0001중량%, 염산피리독신 0.0001중량%, 이노시톨 0.001중량%, 오르트산 0.002중량%, 물 98.7362중량% 및 게니핀 1중량%를 배합하여 통상의 방법으로 건강 음료를 제조하였다.
0.0001 wt.% Of niacinamide, 0.0001 wt.% Of sodium riboflavin, 0.0001 wt.% Of pyridoxine hydrochloride, 0.001 wt.% Of inositol, 0.002 wt.% Of orthoacetic acid, 98.7362 wt.% Of water, 1% by weight was added to prepare a health drink.
(6) 소시지 제조(6) Production of sausages
돈육 65.18중량%, 계육 25중량%, 전분 3.5중량%, 대두단백 1.7중량%, 식염 1.62중량%, 포도당 0.5중량%, 글리세린 1.5중량% 및 게니핀 1중량%를 배합하여 통상의 방법으로 소시지를 제조하였다.
Sausage was prepared by blending 65.18% by weight of pork, 25% by weight of chicken meat, 3.5% by weight of starch, 1.7% by weight of soybean protein, 1.62% by weight of salt, 0.5% by weight of glucose, 1.5% by weight of glycerin and 1% .
(7) 건강보조식품 제조(7) Health supplement manufacturing
스피루리나 55중량%, 구아검효소 분해물 10중량%, 비타민 B1 염산염 0.01중량%, 비타민 B6 염산염 0.01중량%, DL-메티오닌 0.23중량%, 스테아린산 마그네슘 0.7중량%, 유당 22.2중량%, 옥수수전분 1.85중량% 및 게니핀 11중량%를 배합하여 통상의 방법으로 정제형 건강보조식품을 제조하였다.
Spirulina 55% by weight, guar gum enzyme degradation product of 10 wt%, vitamin B 1 hydrochloride 0.01% by weight vitamin B 6 hydrochloride 0.01% by weight, 0.23% by weight of DL- methionine, 0.7 wt% magnesium stearate, 22.2 wt% lactose, corn starch 1.85 By weight, and 11% by weight of genipin, were mixed together to prepare a refillable health supplement.
(8) 주류 제조(8) Liquor production
게니핀 0.5중량%를 소주, 맥주, 양주 또는 과실주와 혼합하여 에멀전 상태로 만든 후, 고속믹서기로 9,000 rpm에서 혼합하여 게니핀이 함유된 주류를 제조하였다.
0.5% by weight of genipin was mixed with soju, beer, liquor or fruit wine into an emulsion state, and then mixed at 9,000 rpm with a high-speed mixer to prepare a mainstream containing genipin.
[실시예 2: 비만 예방 및 치료용 약제 조성물 제조][Example 2: Preparation of pharmaceutical composition for prevention and treatment of obesity]
본 실시예에서는 하기와 같이 비만 예방 및 치료용 의학 조성물을 제조하였다.In this Example, a medical composition for the prevention and treatment of obesity was prepared as follows.
(1) 산제 제조(1) Manufacture of powders
게니핀 2 g에 유당 1 g을 혼합하고, 기밀포에 충진하여 산제를 제조하였다.
2 g of Gunipin was mixed with 1 g of lactose, and filled in an airtight container to prepare a powder.
(2) 정제 제조(2) Preparation of tablets
게니핀 100 mg, 옥수수전분 100 mg, 유당 100 mg 및 스테아린산 마그네슘 2 mg을 혼합한 후, 통상의 정제 제조방법에 따라 타정하여 정제를 제조하였다.
100 mg of genipin, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed, and tablets were prepared by tableting according to a conventional tablet preparation method.
(3) 캡슐제 제조(3) Manufacture of capsules
게니핀 100 mg, 옥수수전분 100 mg, 유당 100 mg 및 스테아린산 마그네슘 2 mg을 혼합한 후, 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
100 mg of genipin, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and filled in a gelatin capsule to prepare a capsule.
(4) 주사제 제조(4) Injection preparation
게니핀 100 mg에 주사용 증류수 적량을 가하여 용해시키고, pH를 약 7.5로 조절한 다음 2 ㎖ 용량의 앰플에 충진 및 멸균시하여 주사제를 제조하였다.
100 mg of Gunipin was dissolved in an appropriate amount of distilled water for injection, pH was adjusted to about 7.5, and the solution was filled and sterilized in a 2 ml volume ampoule to prepare an injection.
[실시예 3: 비만 예방 및 치료용 동물 의약품 제조][Example 3: Preparation of animal drugs for prevention and treatment of obesity]
본 실시예에서는 하기와 같이 비만 예방 및 치료용 동물 의약품을 제조하였다.In this Example, animal drugs for the prevention and treatment of obesity were prepared as follows.
(1)정제 제조(1) Preparation of tablets
게니핀 100 mg에 단백질 혼합물 100 mg, 말분 100 mg을 혼합한 후 통상의 정제 제조방법에 따라 타정하여 정제를 제조하였다.
100 mg of genipin was mixed with 100 mg of the protein mixture and 100 mg of the ending powder, and tablets were prepared by tableting according to a conventional tablet preparation method.
(2)주사제 제조(2) Injection preparation
게니핀 100 mg에 주사용 증류수 적량을 가하여 용해시키고, pH를 약 7.5로 조절한 다음 2 ㎖ 용량의 앰플에 충진 및 멸균시하여 주사제를 제조하였다.
100 mg of Gunipin was dissolved in an appropriate amount of distilled water for injection, pH was adjusted to about 7.5, and the solution was filled and sterilized in a 2 ml volume ampoule to prepare an injection.
[실시예 4: 비만 개선용 동물 사료 첨가제 제조][Example 4: Preparation of animal feed additive for obesity improvement]
본 실시예에서는 게니핀과 닭기름을 1 : 9의 중량비로 혼합하여 비만 개선용 동물(개, 애완견) 사료 첨가제를 제조하였다.
In this Example, genipin and chicken oil were mixed at a weight ratio of 1: 9 to prepare feed additive for animals (dogs, pets) for obesity improvement.
[실시예 5: 비만 개선용 동물 사료 조성물 제조][Example 5: Preparation of animal feed composition for obesity improvement]
본 실시예에서는 하기와 같이 비만 개선용 동물(개, 애완견) 사료 조성물을 제조하였다.In this Example, an animal (dog, pet dog) feed composition for obesity improvement was prepared as follows.
옥수수 25.5 kg, 소맥 15.04 kg, 소맥분 8.15 kg, 미강 7.4 kg, 대두박 18 kg, 옥구르텐 1kg, 닭부산물 14 kg, 동물성유지 9 kg, 가공염 0.3 kg, 인산제삼칼슘 0.3 kg, 석회석 1 kg, 염화콜린 0.01 kg, 비타민 0.05 kg, 미네랄 0.05 kg, 소화효소제 0.1 kg 및 게니핀을 0.1 kg을 혼합하여 비만 개선용 동물(개, 애완견) 사료 조성물을 제조하였다.
Corn 25.5 kg, wheat 15.04 kg, wheat flour 8.15 kg, rice bran 7.4 kg, soybean meal 18 kg, oogurtene 1 kg, chicken by-product 14 kg, animal fat 9 kg, processed salt 0.3 kg, trisodium phosphate 0.3 kg, (Dog, pet dog) feed composition was prepared by mixing 0.01 kg of choline, 0.05 kg of vitamin, 0.05 kg of mineral, 0.1 kg of digestive enzyme, and 0.1 kg of genipin.
Claims (15)
상기 게니핀은 지방전구세포의 지방세포로의 분화 억제 효능 및 지방세포 내 지방축적 억제 효능을 갖는 것을 특징으로 하는 비만 개선용 식품 조성물.
[화학식 1]
A pharmaceutical composition comprising genipin having a structure represented by the following formula (1) as an active ingredient,
Wherein the genipin has an inhibitory effect on the differentiation of adipocyte precursor cells into adipocytes and an effect of inhibiting adipocyte accumulation in adipocytes.
[Chemical Formula 1]
상기 게니핀은,
비만 개선용 식품 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것을 특징으로 하는 비만 개선용 식품 조성물.
The method according to claim 1,
The above-
Wherein the composition is contained in an amount of 0.000001 to 50% by weight based on the total weight of the food composition for improving obesity.
상기 게니핀은,
농도가 10 μM ~ 1 mM인 것을 특징으로 하는 비만 개선용 식품 조성물.
The method according to claim 1,
The above-
And the concentration is 10 μM to 1 mM.
상기 게니핀은 지방전구세포의 지방세포로의 분화 억제 효능 및 지방세포 내 지방축적 억제 효능을 갖는 것을 특징으로 하는 비만 예방 또는 치료용 약학 조성물.
[화학식 1]
A pharmaceutical composition comprising genipin having a structure represented by the following formula (1) as an active ingredient,
Wherein the genipin has an inhibitory effect on the differentiation of adipocyte precursor cells into adipocytes and an effect to inhibit adipocyte accumulation in adipocytes.
[Chemical Formula 1]
상기 게니핀은,
비만 예방 또는 치료용 약학 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것을 특징으로 하는 비만 예방 또는 치료용 약학 조성물.
5. The method of claim 4,
The above-
A pharmaceutical composition for preventing or treating obesity, which comprises 0.000001 to 50% by weight based on the total weight of the pharmaceutical composition for preventing or treating obesity.
상기 게니핀은,
농도가 10 μM ~ 1 mM인 것을 특징으로 하는 비만 예방 또는 치료용 약학 조성물.
5. The method of claim 4,
The above-
Wherein the concentration is 10 [mu] M to 1 mM.
상기 게니핀은 지방전구세포의 지방세포로의 분화 억제 효능 및 지방세포 내 지방축적 억제 효능을 갖는 것을 특징으로 하는 비만 예방 또는 치료용 동물 의약품 조성물.
[화학식 1]
A pharmaceutical composition comprising genipin having a structure represented by the following formula (1) as an active ingredient,
Wherein the genipin has an inhibitory effect on the differentiation of adipocyte precursor cells into adipocytes and an effect to inhibit adipocyte accumulation in adipocytes.
[Chemical Formula 1]
상기 게니핀은,
비만 예방 또는 치료용 동물 의약품 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것을 특징으로 하는 비만 예방 또는 치료용 동물 의약품 조성물.
8. The method of claim 7,
The above-
Animal pharmaceutical composition for preventing or treating obesity, which comprises 0.000001 to 50% by weight based on the total weight of the composition for preventing or treating obesity.
상기 게니핀은,
농도가 10 μM ~ 1 mM인 것을 특징으로 하는 비만 예방 또는 치료용 동물 의약품 조성물.
8. The method of claim 7,
The above-
Wherein the concentration is 10 μM to 1 mM.
상기 게니핀은 지방전구세포의 지방세포로의 분화 억제 효능 및 지방세포 내 지방축적 억제 효능을 갖는 것을 특징으로 하는 비만 개선용 동물 사료 첨가제.
[화학식 1]
A pharmaceutical composition comprising genipin having a structure represented by the following formula (1) as an active ingredient,
Wherein said genipin has an inhibitory effect on the differentiation of adipocyte precursor cells into adipocytes and an effect of inhibiting adipocyte accumulation in adipocytes.
[Chemical Formula 1]
상기 게니핀은,
비만 개선용 동물 사료 첨가제 전체 중량에 대하여 0.000001~50중량% 함유되는 것을 특징으로 하는 비만 개선용 동물 사료 첨가제.
11. The method of claim 10,
The above-
Animal feed additive for obesity improvement comprising 0.000001 to 50% by weight based on the total weight of the animal feed additive for obesity improvement.
상기 게니핀은,
농도가 10 μM ~ 1 mM인 것을 특징으로 하는 비만 개선용 동물 사료 첨가제.
11. The method of claim 10,
The above-
Wherein the concentration is 10 μM to 1 mM.
상기 게니핀은 지방전구세포의 지방세포로의 분화 억제 효능 및 지방세포 내 지방축적 억제 효능을 갖는 것을 특징으로 하는 비만 개선용 동물 사료 조성물.
[화학식 1]
A pharmaceutical composition comprising genipin having a structure represented by the following formula (1) as an active ingredient,
Wherein the genipin has an inhibitory effect on the differentiation of adipocyte precursor cells into adipocytes and an effect of inhibiting adipocyte accumulation in adipocytes.
[Chemical Formula 1]
상기 게니핀은,
비만 개선용 동물 사료 조성물 전체 중량에 대하여 0.000001~50중량% 함유되는 것을 특징으로 하는 비만 개선용 동물 사료 조성물.
14. The method of claim 13,
The above-
Wherein the composition is contained in an amount of 0.000001 to 50% by weight based on the total weight of the animal feed composition for obesity improvement.
상기 게니핀은,
농도가 10 μM ~ 1 mM인 것을 특징으로 하는 비만 개선용 동물 사료 조성물.14. The method of claim 13,
The above-
And the concentration is 10 μM to 1 mM.
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