KR102623896B1 - Composition for reducing body weight, body fat, or blood sugar including neohesperidin dihydrochalcone glycotransferase - Google Patents
Composition for reducing body weight, body fat, or blood sugar including neohesperidin dihydrochalcone glycotransferase Download PDFInfo
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- KR102623896B1 KR102623896B1 KR1020210048798A KR20210048798A KR102623896B1 KR 102623896 B1 KR102623896 B1 KR 102623896B1 KR 1020210048798 A KR1020210048798 A KR 1020210048798A KR 20210048798 A KR20210048798 A KR 20210048798A KR 102623896 B1 KR102623896 B1 KR 102623896B1
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
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Abstract
네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 체중, 체지방 및/또는 혈당 감소 효과가 있는 비만 예방, 개선 및/또는 치료용 약학적 및/또는 식품 조성물에 관한 것이다.It relates to a pharmaceutical and/or food composition for preventing, improving, and/or treating obesity, which contains neohesperidin dihydrochalcone glycotransferase as an active ingredient and has the effect of reducing body weight, body fat, and/or blood sugar.
Description
네오헤스페리딘 디하이드로칼콘 당전이체인 NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC)를 유효성분으로 포함하는 비만 예방, 개선 및/또는 치료용 조성물에 관한 것이다.Neohesperidin dihydrochalcone glycoside, NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β Composition for preventing, improving and/or treating obesity containing -D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC) as an active ingredient It's about.
세계 보건기구 (WHO)는 전세계적으로 1975년부터 2016년까지 비만이 약 3배 증가했다고 보고했다. 이러한 이유로 WHO는 비만에 관한 WHO 협의회에서 21 세기에 세계적 유행병으로 비만을 제안했다. 비만이 되는 것은 당뇨병, 심혈관 질환 및 암을 포함하는 대사 장애와 관련이 있다. 지방 생성 또는 지방 생성을 포함한 여러 지방 축적 관련 메커니즘이 있고, 반대로 β- 산화와 갈색 지방화는 지방 축적의 억제 메커니즘이다.The World Health Organization (WHO) reported that obesity worldwide increased approximately threefold from 1975 to 2016. For this reason, the WHO proposed obesity as a global pandemic in the 21st century at the WHO Consultation on Obesity. Becoming obese is associated with metabolic disorders, including diabetes, cardiovascular disease, and cancer. There are several fat accumulation-related mechanisms, including adipogenesis or adipogenesis, and conversely, β-oxidation and brown fat accumulation are inhibitory mechanisms of fat accumulation.
설탕 소비는 1960년대 중반 이후 급격히 증가했는데, 이는 설탕이 첨가된 음료가 섭취가 주요 원인 중 하나이다. WHO는 설탕 섭취량을 총 에너지 섭취량의 10% 미만으로 낮출 것을 강력히 권장했다. 이전 연구에 따르면 설탕 섭취가 증가하면 어린이와 성인의 비만 유병률이 높아질 수 있다. 설탕 소비를 줄이기 위한 해결책으로 대체 감미료가 제안됐다. 감미료는 bulk sweeteners와 intense sweeteners로 분류되고, 이는 칼로리 함량에 따라 나뉜다. bulk sweeteners는 칼로리를 생성하는 반면 intense sweeteners는 칼로리를 전혀 생성하지 않거나 최소한만 생성한다. 또한, intense sweeteners는 자당보다 상대적인 단맛이 더 높다. 따라서 설탕 대신 대체 감미료를 사용하여 비만이나 제2형 당뇨병을 예방할 수 있을 것으로 기대된다. 네오헤스페리딘 디하이드로칼콘 (neohesperidin dihydrochalcone, NHDC)은 intense sweeteners 중 하나이다. 이는 플라바논 (flavanone)이 모체인 네오헤스페리딘 (neohesperidin)에서 추출 및 가공된다.Sugar consumption has increased rapidly since the mid-1960s, with consumption of sugar-sweetened beverages being one of the main reasons. WHO strongly recommends reducing sugar intake to less than 10% of total energy intake. Previous research has shown that increased sugar intake may increase the prevalence of obesity in children and adults. Alternative sweeteners have been proposed as a solution to reduce sugar consumption. Sweeteners are classified into bulk sweeteners and intense sweeteners, which are divided according to calorie content. Bulk sweeteners produce calories, while intense sweeteners produce no or minimal calories. Additionally, intense sweeteners have a higher relative sweetness than sucrose. Therefore, it is expected that obesity and type 2 diabetes can be prevented by using alternative sweeteners instead of sugar. Neohesperidin dihydrochalcone (NHDC) is one of the intense sweeteners. It is extracted and processed from neohesperidin, the parent flavanone.
플라바논 글리코사이드 (glycoside)는 주로 오렌지 껍질에서 발견되며 디하이드로칼콘 형태는 수소화를 통해 합성된다. NHDC는 비터 오렌지 (bitter orange), citrus aurantium의 반천연 화합물로 높은 용해도와 안정성을 가지고 있다. NHDC의 상대적 단맛은 자당 용액보다 250 ~ 2000배 더 높다. 또한, NHDC는 다른 화합물의 쓴 맛을 없애는데 사용될 수 있다. 이 기능성 대체 감미료는 항산화 작용을 하는 것으로 보고됐다.Flavanone glycosides are mainly found in orange peels and the dihydrochalcone form is synthesized through hydrogenation. NHDC is a semi-natural compound from bitter orange and citrus aurantium and has high solubility and stability. The relative sweetness of NHDC is 250 to 2000 times higher than that of sucrose solution. Additionally, NHDC can be used to neutralize the bitter taste of other compounds. This functional alternative sweetener has been reported to have antioxidant properties.
글리코사이드는 당이 아닌 분자에 글리코사이드 결합으로 연결된 하나 이상의 당을 가진 화합물이다. 결합 위치에 따라 O-, C-, N- 및 S- 글리코사이드의 네 가지 유형이 있다. 글리코사이드는 항비만 또는 항당뇨 효과를 발휘하는 것으로 알려져있다. NHDC와 그 글리코사이드가 지방 조직과 그 메커니즘에 미치는 영향은 아직 확인되지 않았다. NHDC와 GNHDC 모두의 항비만 효과와 그 분자 메커니즘을 생체 내 및 시험관 내에서 조사했다.Glycosides are compounds that have one or more sugars linked to a non-sugar molecule by a glycosidic bond. Depending on the binding site, there are four types: O-, C-, N-, and S-glycosides. Glycosides are known to exert anti-obesity or anti-diabetic effects. The effects of NHDC and its glycosides on adipose tissue and its mechanisms have not yet been confirmed. The anti-obesity effects of both NHDC and GNHDC and their molecular mechanisms were investigated in vivo and in vitro.
상기와 같은 배경하에서, 본 발명자들은 NHDC 및 GNHDC의 항비만 효과에 대한 연구를 거듭하였다. 그 결과, NHDC 및 GNHDC의 항비만 효과를 관련 마커 확인을 통해 확인하여, 본 발명을 완성하였다.Against the above background, the present inventors conducted repeated studies on the anti-obesity effects of NHDC and GNHDC. As a result, the anti-obesity effect of NHDC and GNHDC was confirmed through confirmation of related markers, and the present invention was completed.
일 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 비만 예방 및/또는 치료용 약학적 조성물을 제공한다.One example provides a pharmaceutical composition for preventing and/or treating obesity, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 체중, 체지방 및/또는 혈당 감소용 약학적 조성물을 제공한다.Another example provides a pharmaceutical composition for reducing body weight, body fat, and/or blood sugar, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 비만 예방 및/또는 개선용 식품 조성물을 제공한다.Another example provides a food composition for preventing and/or improving obesity, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 체중, 체지방 및/또는 혈당 감소용 식품 조성물을 제공한다.Another example provides a food composition for reducing body weight, body fat, and/or blood sugar, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
본 명세서는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)의 항비만 효과를 관련 마커 확인을 통해 확인하여, 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)의 비만 예방, 비만 치료, 체중 감소, 체지방 감소 및/또는 혈당 감소 용도를 제공한다.This specification confirms the anti-obesity effect of neohesperidin dihydrochalcone glycotransferase (GNHDC) through identification of related markers, and determines the anti-obesity effect of neohesperidin dihydrochalcone glycotransferase (GNHDC) for obesity prevention, obesity treatment, weight loss, body fat reduction and/or Or it provides a use for reducing blood sugar levels.
이에, 일 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 Cd36, Lpl, Srebp1c, Fas, PPARγ 및 C/EBPα로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 감소 효과가 있는 조성물을 제공한다.Accordingly, one example includes neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of reducing the level of one or more genes selected from the group consisting of Cd36, Lpl, Srebp1c, Fas, PPARγ, and C/EBPα or their coding genes. Provides a composition containing
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 Acsl1, Cpt1α, Ucp1, Pgc1α, 및 Prdm16으로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 증가 효과가 있는 조성물을 제공한다.Another example provides a composition that contains neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of increasing the level of one or more genes selected from the group consisting of Acsl1, Cpt1α, Ucp1, Pgc1α, and Prdm16 or their coding genes. .
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 p-PI3K/PI3K, p-AKT/AKT 및 p-mTOR/mTOR로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 감소 효과가 있는 조성물을 제공한다.Another example includes neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and reduces the level of one or more genes selected from the group consisting of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR or their coding genes. An effective composition is provided.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 p-AMPK/AMPK 또는 이의 암호화 유전자 수준 증가 효과가 있는 조성물을 제공한다.Another example provides a composition that contains neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of increasing the level of p-AMPK/AMPK or its coding gene.
상기 조성물은 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는 비만 예방, 개선 및/또는 치료용 조성물일 수 있고, 이에 제한되는 것은 아니다.The composition may be a composition for preventing, improving and/or treating obesity containing neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient, but is not limited thereto.
상기 조성물은 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 체중, 체지방 및/또는 혈당 감소 효과가 있을 수 있고, 이에 제한되는 것은 아니다.The composition contains neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and may have the effect of reducing body weight, body fat, and/or blood sugar, but is not limited thereto.
상기 조성물은 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는 체중, 체지방 및/또는 혈당 감소용 조성물일 수 있고, 이에 제한되는 것은 아니다.The composition may be a composition for reducing body weight, body fat, and/or blood sugar containing neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient, but is not limited thereto.
상기 조성물은 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는 약학적 조성물 및/또는 식품 조성물 (예컨대, 건강기능식품)일 수 있고, 이에 제한되는 것은 아니다.The composition may be a pharmaceutical composition and/or a food composition (e.g., health functional food) containing neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient, but is not limited thereto.
이하, 본 출원을 보다 상세히 설명한다.Hereinafter, this application will be described in more detail.
네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)Neohesperidin dihydrochalcone glycotransferase (GNHDC)
본 명세서에서 "NHDC"는 “네오헤스페리딘 디하이드로칼콘” 또는 “(1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone)”을 의미하며, 하기 화학식 1의 화학식으로 나타낼 수 있다.In this specification, “NHDC” refers to “neohesperidin dihydrochalcone” or “(1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2 ,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone)”, and can be represented by the formula (1) below.
[화학식 1][Formula 1]
본 명세서에서 “GNHDC”는 “네오헤스페리딘 디하이드로칼콘 당전이체” 또는 “(1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone)”을 의미하며, 하기 화학식 2의 화학식으로 나타낼 수 있다.In this specification, “GNHDC” refers to “neohesperidin dihydrochalcone glycotransferase” or “(1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D -glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone)”, and can be expressed in the following formula (2) .
[화학식 2][Formula 2]
본 명세서는 GNHDC를 유효성분으로 포함하는 비만 예방, 비만 개선용, 비만 치료용, 체중 감소용, 체지방 감소용 및/또는 혈당 감소용 약학적 및/또는 식품 조성물을 제공한다.The present specification provides a pharmaceutical and/or food composition for preventing obesity, improving obesity, treating obesity, reducing body weight, reducing body fat, and/or reducing blood sugar, containing GNHDC as an active ingredient.
상기 조성물은 GNHDC를 0.001 내지 100,000μM, 0.01 내지 100,000μM, 0.1 내지 100,000μM, 1 내지 100,000μM, 5 내지 100,000μM, 10 내지 100,000μM, 15 내지 100,000μM, 20 내지 100,000μM, 25 내지 100,000μM, 30 내지 100,000μM, 35 내지 100,000μM, 40 내지 100,000μM, 45 내지 100,000μM, 0.001 내지 50,000μM, 0.01 내지 50,000μM, 0.1 내지 50,000μM, 1 내지 50,000μM, 5 내지 50,000μM, 10 내지 50,000μM, 15 내지 50,000μM, 20 내지 50,000μM, 25 내지 50,000μM, 30 내지 50,000μM, 35 내지 50,000μM, 40 내지 50,000μM, 45 내지 50,000μM, 0.001 내지 10,000μM, 0.01 내지 10,000μM, 0.1 내지 10,000μM, 1 내지 10,000μM, 5 내지 10,000μM, 10 내지 10,000μM, 15 내지 10,000μM, 20 내지 10,000μM, 25 내지 10,000μM, 30 내지 10,000μM, 35 내지 10,000μM, 40 내지 10,000μM, 45 내지 10,000μM, 0.001 내지 5,000μM, 0.01 내지 5,000μM, 0.1 내지 5,000μM, 1 내지 5,000μM, 5 내지 5,000μM, 10 내지 5,000μM, 15 내지 5,000μM, 20 내지 5,000μM, 25 내지 5,000μM, 30 내지 5,000μM, 35 내지 5,000μM, 40 내지 5,000μM, 45 내지 5,000μM, 0.001 내지 1,000μM, 0.01 내지 1,000μM, 0.1 내지 1,000μM, 1 내지 1,000μM, 5 내지 1,000μM, 10 내지 1,000μM, 15 내지 1,000μM, 20 내지 1,000μM, 25 내지 1,000μM, 30 내지 1,000μM, 35 내지 1,000μM, 40 내지 1,000μM, 45 내지 1,000μM, 0.001 내지 500μM, 0.01 내지 500μM, 0.1 내지 500μM, 1 내지 500μM, 5 내지 500μM, 10 내지 500μM, 15 내지 500μM, 20 내지 500μM, 25 내지 500μM, 30 내지 500μM, 35 내지 500μM, 40 내지 500μM, 45 내지 500μM, 0.001 내지 300μM, 0.01 내지 300μM, 0.1 내지 300μM, 1 내지 300μM, 5 내지 300μM, 10 내지 300μM, 15 내지 300μM, 20 내지 300μM, 25 내지 300μM, 30 내지 300μM, 35 내지 300μM, 40 내지 300μM, 45 내지 300μM, 0.001 내지 100μM, 0.01 내지 100μM, 0.1 내지 100μM, 1 내지 100μM, 5 내지 100μM, 10 내지 100μM, 15 내지 100μM, 20 내지 100μM, 25 내지 100μM, 30 내지 100μM, 35 내지 100μM, 40 내지 100μM, 45 내지 100μM, 0.001 내지 80μM, 0.01 내지 80μM, 0.1 내지 80μM, 1 내지 80μM, 5 내지 80μM, 10 내지 80μM, 15 내지 80μM, 20 내지 80μM, 25 내지 80μM, 30 내지 80μM, 35 내지 80μM, 40 내지 80μM, 45 내지 80μM, 0.001 내지 60μM, 0.01 내지 60μM, 0.1 내지 60μM, 1 내지 60μM, 5 내지 60μM, 10 내지 60μM, 15 내지 60μM, 20 내지 60μM, 25 내지 60μM, 30 내지 60μM, 35 내지 60μM, 40 내지 60μM, 45 내지 60μM, 0.001 내지 55μM, 0.01 내지 55μM, 0.1 내지 55μM, 1 내지 55μM, 5 내지 55μM, 10 내지 55μM, 15 내지 55μM, 20 내지 55μM, 25 내지 55μM, 30 내지 55μM, 35 내지 55μM, 40 내지 55μM 또는 45 내지 55μM의 농도로 포함할 수 있고, 이에 제한되는 것은 아니다.The composition contains GNHDC at 0.001 to 100,000 μM, 0.01 to 100,000 μM, 0.1 to 100,000 μM, 1 to 100,000 μM, 5 to 100,000 μM, 10 to 100,000 μM, 15 to 100,000 μM, 20 to 1 00,000 μM, 25 to 100,000 μM, 30 to 100,000 μM, 35 to 100,000 μM, 40 to 100,000 μM, 45 to 100,000 μM, 0.001 to 50,000 μM, 0.01 to 50,000 μM, 0.1 to 50,000 μM, 1 to 50,000 μM, 5 to 50,000 μM 50,000 μM, 10 to 50,000 μM, 15 to 50,000 μM, 20 to 50,000 μM, 25 to 50,000 μM, 30 to 50,000 μM, 35 to 50,000 μM, 40 to 50,000 μM, 45 to 50,000 μM, 0.001 to 10,000 μM, 0.01 to 10 ,000μM, 0.1 to 10,000μM, 1 to 10,000 μM, 5 to 10,000 μM, 10 to 10,000 μM, 15 to 10,000 μM, 20 to 10,000 μM, 25 to 10,000 μM, 30 to 10,000 μM, 35 to 10,000 μM, 40 to 10,000 μM, 45 to 10,000 μM, 0.001 to 5,000 μM, 0.01 to 5,000 μM, 0.1 to 5,000 μM, 1 to 5,000 μM, 5 to 5,000 μM, 10 to 5,000 μM, 15 to 5,000 μM, 20 to 5,000 μM, 25 to 5,000 μM, 30 to 5,000 μM; 35 to 5,000 μM, 40 to 5,000 μM, 45 to 5,000 μM, 0.001 to 1,000 μM, 0.01 to 1,000 μM, 0.1 to 1,000 μM, 1 to 1,000 μM, 5 to 1,000 μM, 10 to 1,000 μM, 15 to 1,000 μM; 20 to 1,000 μM, 25 to 1,000 μM, 30 to 1,000 μM, 35 to 1,000 μM, 40 to 1,000 μM, 45 to 1,000 μM, 0.001 to 500 μM, 0.01 to 500 μM, 0.1 to 500 μM, 1 to 500 μM μM, 5 to 500 μM; 10 to 500 μM, 15 to 500 μM, 20 to 500 μM, 25 to 500 μM, 30 to 500 μM, 35 to 500 μM, 40 to 500 μM, 45 to 500 μM, 0.001 to 300 μM, 0.01 to 300 μM, 0.1 to 300 μM μM, 1 to 300 μM, 5 to 300 μM 300 μM, 10 to 300 μM, 15 to 300 μM, 20 to 300 μM, 25 to 300 μM, 30 to 300 μM, 35 to 300 μM, 40 to 300 μM, 45 to 300 μM, 0.001 to 100 μM, 0.01 to 100 μM, 0 .1 to 100 μM, 1 to 100 μM, 5 to 100 μM, 10 to 100 μM, 15 to 100 μM, 20 to 100 μM, 25 to 100 μM, 30 to 100 μM, 35 to 100 μM, 40 to 100 μM, 45 to 100 μM, 0.001 to 80 μM, 0.01 to 80 μM, 0.1 to 80 μM, 1 to 80 μM 80 μM, 5 to 80 μM, 10 to 80 μM, 15 to 80 μM, 20 to 80 μM, 25 to 80 μM, 30 to 80 μM, 35 to 80 μM, 40 to 80 μM, 45 to 80 μM, 0.001 to 60 μM, 0.01 to 60 μM, 0.1 to 60 μM, 1 to 60 μM, 5 to 60 μM, 10 to 60 μM, 15 to 60 μM, 20 to 60 μM, 25 to 60 μM, 30 to 60 μM, 35 to 60 μM, 40 to 60 μM, 45 to 60 μM, 0.001 to 55 μM, 0.01 to 55 μM, 0.1 to It may be included at a concentration of 55 μM, 1 to 55 μM, 5 to 55 μM, 10 to 55 μM, 15 to 55 μM, 20 to 55 μM, 25 to 55 μM, 30 to 55 μM, 35 to 55 μM, 40 to 55 μM, or 45 to 55 μM. It is not limited.
의약 용도medicinal use
일 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.One example provides a pharmaceutical composition for preventing or treating obesity, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 체중, 체지방 또는 혈당 감소 효과가 있는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.Another example provides a pharmaceutical composition for preventing or treating obesity, which contains neohesperidin dihydrochalcone glycosyltransfer as an active ingredient and has an effect of reducing body weight, body fat, or blood sugar.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 체중, 체지방 또는 혈당 감소용 약학적 조성물을 제공한다.Another example provides a pharmaceutical composition for reducing body weight, body fat, or blood sugar, comprising neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 Cd36, Lpl, Srebp1c, Fas, PPARγ 및 C/EBPα로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 감소 효과가 있는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.Another example is a drug that contains neohesperidin dihydrochalcone glycosyltransferase as an active ingredient and has the effect of reducing the level of one or more genes selected from the group consisting of Cd36, Lpl, Srebp1c, Fas, PPARγ and C/EBPα or their coding genes, preventing obesity or A pharmaceutical composition for therapeutic use is provided.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 Acsl1, Cpt1α, Ucp1, Pgc1α, 및 Prdm16으로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 증가 효과가 있는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.Another example is a pharmaceutical for preventing or treating obesity, which contains neohesperidin dihydrochalcone glycosyltransfer as an active ingredient and has the effect of increasing the level of one or more genes selected from the group consisting of Acsl1, Cpt1α, Ucp1, Pgc1α, and Prdm16 or their encoding genes. A composition is provided.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 p-PI3K/PI3K, p-AKT/AKT 및 p-mTOR/mTOR로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 감소 효과가 있는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.Another example is a drug containing neohesperidin dihydrochalcone glycosyltransferase as an active ingredient and having the effect of reducing the level of one or more genes selected from the group consisting of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR or their coding genes. , and provides a pharmaceutical composition for preventing or treating obesity.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체를 유효성분으로 포함하고 p-AMPK/AMPK 및/또는 이의 암호화 유전자 수준 증가 효과가 있는, 비만 예방 또는 치료용 약학적 조성물을 제공한다.Another example provides a pharmaceutical composition for preventing or treating obesity, which contains neohesperidin dihydrochalcone glycosyltransfer as an active ingredient and has the effect of increasing the level of p-AMPK/AMPK and/or its encoding gene.
상기 단백질 또는 이의 암호화 유전자 수준 감소 또는 증가는 상기 약학적 조성물의 비투여군 (투여 전, 또는 비투여된 동종의 다른 대상)과 비교하여 판단할 수 있다The decrease or increase in the level of the protein or its encoding gene can be judged by comparing it with the non-administered group of the pharmaceutical composition (before administration or other subjects of the same species who were not administered).
상기 단백질 또는 이의 암호화 유전자 수준의 정량은 Quantitative real-time PCR, 면역조직화학검사법, 웨스턴 블랏, ELISA, mRNA 마이크로 어레이 등의 통상적인 정량 방법에 의하여 수행될 수 있고, 이에 제한되는 것은 아니다.Quantification of the protein or its coding gene level can be performed by conventional quantitative methods such as quantitative real-time PCR, immunohistochemistry, Western blot, ELISA, and mRNA microarray, but is not limited thereto.
일 예에서, 상기 비만 예방 또는 치료용 약학적 조성물은 Cd36, Lpl, Srebp1c, Fas, PPARγ, C/EBPα, p-PI3K/PI3K, p-AKT/AKT 및 p-mTOR/mTOR로 이루어지는 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준이 비만으로 판단되지 않은 개체보다 높은 대상에 투여하기 위한 것을 수 있다. 다른 예에서, 상기 비만 예방 또는 치료용 약학적 조성물은 Acsl1, Cpt1α, Ucp1, Pgc1α, Prdm16 및 p-AMPK/AMPK로 이루어지는 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준이 비만으로 판단되지 않은 개체보다 낮은 대상에 투여하기 위한 것일 수 있다. 이 때, 상기 비만으로 판단되지 않은 개체는 체질량지수 (BMI; 체중을 신장의 제곱으로 나눈 값; kg/m2)가 약 25kg/m2 이상인 개체를 의미할 수 있으나, 이에 제한되는 것은 아니다.In one example, the pharmaceutical composition for preventing or treating obesity is selected from the group consisting of Cd36, Lpl, Srebp1c, Fas, PPARγ, C/EBPα, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR. It may be administered to subjects whose levels of one or more genes or their encoding genes are higher than those of individuals not judged to be obese. In another example, the pharmaceutical composition for preventing or treating obesity has a level of at least one selected from the group consisting of Acsl1, Cpt1α, Ucp1, Pgc1α, Prdm16, and p-AMPK/AMPK or its encoding gene compared to an individual not judged to be obese. It may be intended for administration to low-level subjects. At this time, the individual not determined to be obese may mean an individual with a body mass index (BMI; weight divided by height squared; kg/m 2 ) of about 25 kg/m 2 or more, but is not limited thereto.
상기 네오헤스페리딘 디하이드로칼콘 당전이체는 앞서 설명한 바와 같다.The neohesperidin dihydrochalcone glycosyltransferase is as described above.
상기 혈당 감소는 혈액 내 포도당 농도의 감소를 의미한다.The decrease in blood sugar means a decrease in the concentration of glucose in the blood.
일 예에서, 비만은 체질량지수 (BMI; 체중을 신장의 제곱으로 나눈 값; kg/m2)가 약 25kg/m2 이상인 상태를 의미할 수 있으나, 이에 제한되는 것은 아니다.In one example, obesity may mean a body mass index (BMI; weight divided by height squared; kg/m 2 ) of about 25 kg/m 2 or more, but is not limited thereto.
본 명세서에서, “치료”는 질병 상태 또는 증상의 개선, 경감 또는 안정화, 부분적 또는 완전한 회복, 생존의 연장, 질환 범위의 감소, 질병 진행의 지연 또는 완화, 기타 다른 이로운 치료 결과 등을 모두 포함하는 의미로 사용된다. “예방”은 특정 질병을 갖지 않는 대상에게 작용하여 상기 특정 질병이 발병하지 않도록 하거나, 그 발병 시기를 늦추거나, 발병 빈도를 낮추는 모든 기작 및/또는 효과를 포함하는 의미로 사용된다.As used herein, “treatment” includes improvement, alleviation or stabilization of disease state or symptoms, partial or complete recovery, prolongation of survival, reduction of disease extent, delay or alleviation of disease progression, and other beneficial treatment results. It is used with meaning. “Prevention” is used to include all mechanisms and/or effects that act on subjects who do not have a specific disease to prevent the specific disease from developing, delay its onset, or reduce the frequency of its occurrence.
본 명세서에서 제공되는 약학적 조성물 내의 유효성분으로 사용된 네오헤스페리딘 디하이드로칼콘 당전이체의 함량은 사용 형태 및 목적, 사용 대상의 상태, 증상의 종류 및 경중 등에 의하여 적절하게 조절할 수 있으며, 고형분 (추출물에서 용매를 제거한 고형성분) 중량 기준으로 0.001 내지 99.9 중량%, 0.001 내지 90 중량%, 0.001 내지 75 중량%, 0.001 내지 50 중량%, 0.01 내지 99.9 중량%, 0.01 내지 90 중량%, 0.01 내지 75 중량%, 0.01 내지 50 중량%, 0.1 내지 99.9 중량%, 0.1 내지 90 중량%, 0.1 내지 75 중량%, 0.1 내지 50 중량%, 1 내지 99.9 중량%, 1 내지 90 중량%, 1 내지 75 중량%, 1 내지 50 중량%, 5 내지 99.9 중량%, 5 내지 90 중량%, 5 내지 75 중량%, 5 내지 50 중량%, 10 내지 99.9 중량%, 10 내지 90 중량%, 10 내지 75 중량%, 또는 10 내지 50 중량% 일 수 있으나, 이에 제한되지 않는다.The content of neohesperidin dihydrochalcone glycotransferase used as an active ingredient in the pharmaceutical composition provided herein can be appropriately adjusted depending on the form and purpose of use, the condition of the subject, the type and severity of symptoms, etc., and the solid content (extract solid component with solvent removed) 0.001 to 99.9% by weight, 0.001 to 90% by weight, 0.001 to 75% by weight, 0.001 to 50% by weight, 0.01 to 99.9% by weight, 0.01 to 90% by weight, 0.01 to 75% by weight %, 0.01 to 50% by weight, 0.1 to 99.9% by weight, 0.1 to 90% by weight, 0.1 to 75% by weight, 0.1 to 50% by weight, 1 to 99.9% by weight, 1 to 90% by weight, 1 to 75% by weight, 1 to 50% by weight, 5 to 99.9% by weight, 5 to 90% by weight, 5 to 75% by weight, 5 to 50% by weight, 10 to 99.9% by weight, 10 to 90% by weight, 10 to 75% by weight, or 10 It may be from 50% by weight, but is not limited thereto.
또한, 상기 네오헤스페리딘 디하이드로칼콘 당전이체의 함량은 상기 약학적 조성물을 투여하는 대상의 체중 기준으로 0.001 내지 400 mg/kg (body weight; BW), 1 내지 400 mg/kg, 50 내지 400 mg/kg, 100 내지 400 mg/kg, 0.001 내지 300 mg/kg, 1 내지 300 mg/kg, 50 내지 300 mg/kg, 100 내지 300 mg/kg, 0.001 내지 200 mg/kg, 1 내지 200 mg/kg, 50 내지 200 mg/kg, 100 내지 200 mg/kg, 0.001 내지 100 mg/kg, 1 내지 100 mg/kg 또는 50 내지 100 mg/kg일 수 있으나, 이에 제한되지 않는다.In addition, the content of the neohesperidin dihydrochalcone glycosyltransfer is 0.001 to 400 mg/kg (body weight; BW), 1 to 400 mg/kg, and 50 to 400 mg/kg based on the body weight of the subject administering the pharmaceutical composition. kg, 100 to 400 mg/kg, 0.001 to 300 mg/kg, 1 to 300 mg/kg, 50 to 300 mg/kg, 100 to 300 mg/kg, 0.001 to 200 mg/kg, 1 to 200 mg/kg , 50 to 200 mg/kg, 100 to 200 mg/kg, 0.001 to 100 mg/kg, 1 to 100 mg/kg, or 50 to 100 mg/kg, but is not limited thereto.
상기 약학적 조성물 또는 유효성분인 네오헤스페리딘 디하이드로칼콘 당전이체는 인간, 원숭이 등의 영장류, 마우스, 래트, 토끼 등의 설치류, 이외의 개, 고양이, 소, 돼지, 양, 말, 염소 등을 포함하는 포유류, 닭, 오리, 거위 등을 포함하는 조류, 뱀, 도마뱀, 거북, 악어 등을 포함하는 파충류, 양서류, 및 어류 등의 척추동물에서 선택된 투여 대상에 다양한 경로로 투여될 수 있다.The pharmaceutical composition or the active ingredient, neohesperidin dihydrochalcone glycotransferase, includes primates such as humans and monkeys, rodents such as mice, rats, and rabbits, as well as dogs, cats, cows, pigs, sheep, horses, and goats. It can be administered by various routes to vertebrates such as mammals, birds including chickens, ducks, and geese, reptiles including snakes, lizards, turtles, and crocodiles, amphibians, and fish.
상기 약학적 조성물의 투여 방식은 통상적으로 사용되는 모든 방식일 수 있으며, 예컨대, 경구 투여, 또는 정맥, 근육, 피하, 또는 복강 주사 등의 비경구 투여의 경로로 투여될 수 있다. 일 예에서, 상기 약학적 조성물은 경구 투여를 위한 것일 수 있으나, 이에 제한되는 것은 아니다.The method of administration of the pharmaceutical composition may be any commonly used method, for example, oral administration, or parenteral administration such as intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In one example, the pharmaceutical composition may be for oral administration, but is not limited thereto.
상기 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 또는 멸균 주사용액의 형태의 비경구 제형 등으로 제형화하여 사용될 수 있다.The pharmaceutical composition can be formulated and used in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., or parenteral dosage forms in the form of sterile injectable solutions, according to conventional methods. there is.
상기 약학적 조성물은 앞서 설명한 유효성분 이외에도, 일반적으로 투여방식과 표준 약제학적 관행 (Standard pharmaceutical practice)을 고려하여 선택된 약제학적 및/또는 생리학적으로 허용되는 담체, 부형제, 및 희석제 등으로 이루어진 군에서 선택된 1종 이상의 보조제와 혼합되어 투여될 수 있다. 예컨대, 상기 약제학적 및/또는 생리학적으로 허용되는 담체는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등으로 이루어진 군에서 선택된 1종 이상을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니며, 약제학 분야에서 통상적으로 사용되는 모든 담체일 수 있다.In addition to the active ingredients described above, the pharmaceutical composition generally consists of pharmaceutically and/or physiologically acceptable carriers, excipients, and diluents selected in consideration of administration method and standard pharmaceutical practice. It can be administered in combination with one or more selected adjuvants. For example, the pharmaceutically and/or physiologically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, It may contain one or more selected from the group consisting of cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. However, it is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.
상기 약제학적 및/또는 생리학적으로 허용되는 희석제 및/또는 부형제는 약학적 조성물의 적절한 제제화에 통상적으로 사용되는 모든 충진제, 증량제, 결합제, 습윤제, 붕해제, 윤할제, 계면활성제 등으로 이루어진 군에서 선택된 1종 이상일 수 있다.The pharmaceutically and/or physiologically acceptable diluents and/or excipients are from the group consisting of all fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, etc. commonly used in the appropriate formulation of pharmaceutical compositions. There may be one or more selected types.
예컨대, 정제, 환제, 산제, 과립제, 또는 캡슐제 등의 경구투여를 위한 고형 제제의 경우, 상기 부형제는 이러한 고형 제제의 제제화를 위한 적어도 1종 이상의 물질, 예컨대, 전분, 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose), 락토오스 (Lactose), 젤라틴 등으로 이루어진 군에서 선택된 1종 이상일 수 있다. 이외에도, 마그네슘 스티레이트 탈크 등과 같은 윤활제들도 사용될 수 있다. 또한, 현탁제, 내용액제, 유제, 시럽제 등의 경구투여를 위한 액상제제의 제제화의 경우, 통상적으로 사용되는 단순희석제인 물, 리퀴드 파라핀 등으로 이루어진 군에서 선택된 1종 이상이 사용될 수 있고, 이외에, 통상적으로 사용되는 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 및/또는 보존제 등이 포함될 수 있으나, 이에 제한되는 것은 아니다.For example, in the case of solid preparations for oral administration such as tablets, pills, powders, granules, or capsules, the excipient is at least one type of material for formulating such solid preparations, such as starch and calcium carbonate. It may be one or more types selected from the group consisting of , sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium styrate talc and the like may also be used. In addition, in the case of formulation of liquid preparations for oral administration such as suspensions, oral solutions, emulsions, syrups, etc., one or more types selected from the group consisting of water, liquid paraffin, etc., which are commonly used simple diluents, may be used. , various commonly used excipients, such as wetting agents, sweeteners, flavoring agents, and/or preservatives, may be included, but are not limited thereto.
예컨대, 상기 약학적 조성물은 전분 또는 락토오즈를 함유하는 정제 형태로, 또는 적절한 부형제를 함유하는 캡슐 형태로, 또는 맛을 내거나 색을 띄게 하는 화학 약품을 함유하는 엘릭시르 또는 현탁제 형태로 경구, 구강내 또는 혀밑 투여될 수 있다. 이러한 액체 제제는 현탁제 (예를 들면, 메틸셀룰로오즈, 위텝솔 (Witepsol)과 같은 반합성 글리세라이드 또는 행인유 (Apricot kernel oil)와 PEG-6 에스테르의 혼합물 또는 PEG-8과 카프릴릭/카프릭 글리세라이드의 혼합물과 같은 글리세라이드 혼합물)와 같은 약제학적으로 허용 가능한 첨가제와 함께 제형화 될 수 있으나, 이에 제한되는 것은 아니다.For example, the pharmaceutical composition may be administered orally, in the form of tablets containing starch or lactose, in the form of capsules containing appropriate excipients, or in the form of elixirs or suspensions containing flavoring or coloring chemicals. It may be administered intraorally or sublingually. These liquid preparations may contain suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as Witepsol or a mixture of Apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric It may be formulated with pharmaceutically acceptable additives such as a mixture of glycerides, but is not limited thereto.
식품 조성물food composition
일 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하는, 식품 조성물을 제공한다.One example provides a food composition containing neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 Cd36, Lpl, Srebp1c, Fas, PPARγ 및 C/EBPα로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 감소 효과가 있는, 식품 조성물을 제공한다.Another example includes neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of reducing the level of one or more genes selected from the group consisting of Cd36, Lpl, Srebp1c, Fas, PPARγ, and C/EBPα or their coding genes, A food composition is provided.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 Acsl1, Cpt1α, Ucp1, Pgc1α, 및 Prdm16으로 이루어진 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준 증가 효과가 있는, 식품 조성물을 제공한다.Another example is a food composition containing neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and having the effect of increasing the level of one or more genes selected from the group consisting of Acsl1, Cpt1α, Ucp1, Pgc1α, and Prdm16 or their coding genes. to provide.
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 p-PI3K/PI3K, p-AKT/AKT 및 p-mTOR/mTOR로 이루어진 군에서 선택된 1종 또는 이의 암호화 유전자 수준 감소 효과가 있는, 식품 조성물을 제공한다.Another example includes neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of reducing the level of one gene selected from the group consisting of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR or its encoding gene. Provides a food composition containing .
다른 예는 네오헤스페리딘 디하이드로칼콘 당전이체 (GNHDC)를 유효성분으로 포함하고 p-AMPK/AMPK 또는 이의 암호화 유전자 수준 증가 효과가 있는, 식품 조성물을 제공한다.Another example provides a food composition that includes neohesperidin dihydrochalcone glycotransferase (GNHDC) as an active ingredient and has the effect of increasing the level of p-AMPK/AMPK or its encoding gene.
상기 단백질 또는 이의 암호화 유전자 수준 감소 또는 증가는 상기 식품 조성물의 비투여군 (투여 전, 또는 비투여된 동종의 다른 대상)과 비교하여 판단할 수 있다The decrease or increase in the level of the protein or its encoding gene can be judged by comparing it with the non-administered group of the food composition (before administration or other non-administered subjects of the same species).
상기 단백질 또는 이의 암호화 유전자 수준의 정량은 Quantitative real-time PCR, 면역조직화학검사법, 웨스턴 블랏, ELISA, mRNA 마이크로 어레이 등의 통상적인 정량 방법에 의하여 수행될 수 있고, 이에 제한되는 것은 아니다.Quantification of the protein or its coding gene level can be performed by conventional quantitative methods such as quantitative real-time PCR, immunohistochemistry, Western blot, ELISA, and mRNA microarray, but is not limited thereto.
일 예에서, 상기 식품 조성물은 Cd36, Lpl, Srebp1c, Fas, PPARγ, C/EBPα, p-PI3K/PI3K, p-AKT/AKT 및 p-mTOR/mTOR로 이루어지는 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준이 비만으로 판단되지 않은 개체보다 높은 대상에 투여하기 위한 것을 수 있다. 다른 예에서, 상기 식품 조성물은 Acsl1, Cpt1α, Ucp1, Pgc1α, Prdm16 및 p-AMPK/AMPK로 이루어지는 군에서 선택된 1종 이상 또는 이의 암호화 유전자 수준이 비만으로 판단되지 않은 개체보다 낮은 대상에 투여하기 위한 것을 수 있다. 이 때, 상기 비만으로 판단되지 않은 개체는 체질량지수 (BMI; 체중을 신장의 제곱으로 나눈 값; kg/m2)가 약 25kg/m2 이상인 개체를 의미할 수 있으나, 이에 제한되는 것은 아니다.In one example, the food composition contains at least one selected from the group consisting of Cd36, Lpl, Srebp1c, Fas, PPARγ, C/EBPα, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, or encoding thereof. It may be intended for administration to subjects whose gene levels are higher than those not judged to be obese. In another example, the food composition is for administering to a subject whose level of one or more genes selected from the group consisting of Acsl1, Cpt1α, Ucp1, Pgc1α, Prdm16, and p-AMPK/AMPK or their encoding genes is lower than that of an individual not determined to be obese. You can do it. At this time, the individual not determined to be obese may mean an individual with a body mass index (BMI; weight divided by height squared; kg/m 2 ) of about 25 kg/m 2 or more, but is not limited thereto.
상기 식품 조성물은 비만 예방 및/또는 개선용 식품 조성물, 체중, 체지방 또는 혈당 감소용 식품 조성물일 수 있고, 이에 제한되는 것은 아니다.The food composition may be a food composition for preventing and/or improving obesity, or a food composition for reducing body weight, body fat, or blood sugar, but is not limited thereto.
본 명세서에서 제공되는 식품 조성물에서, 용어 "식품"은 영양소를 한 가지 이상 함유하고 있는 식용의 천연물 또는 가공품을 의미하며, 통상적인 의미로서, 각종 일반 식품, 건강기능식품, 음료, 식품 첨가제, 음료 첨가제 등으로 이루어진 군에서 선택된 하나 이상을 의미하기 위하여 사용될 수 있다. 용어 "식품 조성물"은 상기 식품을 제조하기 위한 재료의 조합을 의미할 수 있다. 일 예에서, 상기 식품은 건강기능식품을 의미할 수 있다.In the food composition provided herein, the term "food" refers to an edible natural product or processed product containing one or more nutrients, and in its usual sense, various general foods, health functional foods, beverages, food additives, and beverages. It may be used to mean one or more selected from the group consisting of additives, etc. The term “food composition” may refer to a combination of ingredients for producing the food. In one example, the food may refer to a health functional food.
상기 식품 조성물 내의 상기 네오헤스페리딘 디하이드로칼콘 당전이체의 함량은 식품사용 형태, 목적 등에 의하여 적절하게 조절할 수 있으며, 예컨대, 고형분 중량 기준으로 0.00001 내지 99.9 중량%, 0.0001 내지 99.9 중량%, 0.001 내지 99.9 중량%, 또는 0.001 내지 50 중량%일 수 있으나, 이에 제한되지 않는다.The content of the neohesperidin dihydrochalcone glycotransferase in the food composition can be appropriately adjusted depending on the type of food use, purpose, etc., for example, 0.00001 to 99.9% by weight, 0.0001 to 99.9% by weight, 0.001 to 99.9% by weight based on the weight of solid content. %, or 0.001 to 50% by weight, but is not limited thereto.
또한, 상기 네오헤스페리딘 디하이드로칼콘 당전이체의 함량은 상기 식품 조성물을 투여하는 대상의 체중 기준으로 0.001 내지 400 mg/kg (body weight; BW), 1 내지 400 mg/kg, 50 내지 400 mg/kg, 100 내지 400 mg/kg, 0.001 내지 300 mg/kg, 1 내지 300 mg/kg, 50 내지 300 mg/kg, 100 내지 300 mg/kg, 0.001 내지 200 mg/kg, 1 내지 200 mg/kg, 50 내지 200 mg/kg, 100 내지 200 mg/kg, 0.001 내지 100 mg/kg, 1 내지 100 mg/kg 또는 50 내지 100 mg/kg일 수 있으나, 이에 제한되지 않는다.In addition, the content of the neohesperidin dihydrochalcone glycosyltransfer is 0.001 to 400 mg/kg (body weight; BW), 1 to 400 mg/kg, 50 to 400 mg/kg, based on the body weight of the subject administering the food composition. , 100 to 400 mg/kg, 0.001 to 300 mg/kg, 1 to 300 mg/kg, 50 to 300 mg/kg, 100 to 300 mg/kg, 0.001 to 200 mg/kg, 1 to 200 mg/kg, It may be 50 to 200 mg/kg, 100 to 200 mg/kg, 0.001 to 100 mg/kg, 1 to 100 mg/kg, or 50 to 100 mg/kg, but is not limited thereto.
상기 식품 조성물의 투여 '대상'은 인간, 원숭이 등의 영장류, 마우스, 래트, 토끼 등의 설치류, 이외의 개, 고양이, 소, 돼지, 양, 말, 염소 등을 포함하는 포유류, 닭, 오리, 거위 등을 포함하는 조류, 뱀, 도마뱀, 거북, 악어 등을 포함하는 파충류, 양서류, 및 어류 등의 척추동물 중에서 선택된 개체, 상기 개체로부터 분리된 세포, 조직, 또는 세포 배양물 등일 수 있다.The 'subjects' of administration of the food composition include humans, primates such as monkeys, rodents such as mice, rats, and rabbits, mammals including dogs, cats, cows, pigs, sheep, horses, and goats, chickens, ducks, etc. It may be an individual selected from vertebrates such as birds including geese, reptiles including snakes, lizards, turtles, and crocodiles, amphibians, and fish, and cells, tissues, or cell cultures isolated from the individuals.
본 명세서에서 제공하는 네오헤스페리딘 디하이드로칼콘 당전이체인 NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC)를 유효성분으로 포함하는 조성물은 체중, 체지방 및/또는 혈당 감소 효과를 가져, 비만 예방, 치료 및/또는 개선 효과를 가진다.NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D), a neohesperidin dihydrochalcone glycotransfer provided herein A composition containing -glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC) as an active ingredient is used to measure body weight and body fat. and/or has a blood sugar reduction effect, thereby preventing, treating, and/or improving obesity.
도 1은 네오헤스페리딘 디하이드로칼콘 (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (NHDC) 및 네오헤스페리딘 디하이드로칼콘 당전이체인 NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC)의 구조를 보여주는 화학식이다.
도 2는 NHDC 및 GNHDC의 마우스의 공복 혈당에 대한 효과를 보여주는 그래프이다. Ctrl은 NHDC 및 GNHDC가 아닌 인산염 완충 식염수 (phosphate-buffered saline, PBS)를 먹인 대조군을 의미한다.
도 3은 NHDC 및 GNHDC가 마우스 피하 지방 세포에 미치는 효과를 보여주는 사진 및 그래프이다.
도 4는 NHDC 및 GNHDC가 마우스 피하 지방 조직에서 지방산 흡수, lipogenesis, adipogenesis, β-산화 및 갈색 지방화 관련 유전자에 미치는 효과를 quantitative real-time PCR로 분석한 결과를 보여주는 그래프이다.
도 5는 GNHDC가 3T3-L1 세포에서 트리글리세리드 축적에 미치는 효과를 oil red O staining으로 분석한 결과를 보여주는 사진 및 그래프이다.
도 6은 GNHDC가 3T3-L1 세포에서 세포 생존력에 미치는 효과를 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 분석으로 분석한 결과를 보여주는 그래프이다.
도 7 및 8은 GNHDC가 lipogenesis 및 adipogenesis에 미치는 효과를 quantitative real-time PCR로 분석한 결과를 보여주는 그래프이다.
도 9 내지 11은 GNHDC가 PI3K/AKT/mTOR 경로에 미치는 효과를 웨스턴 블랏으로 분석한 결과를 보여주는 그래프이다.Figure 1 shows neohesperidin dihydrochalcone (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3- [3-hydroxy-4-methoxyphenyl]-1-propanone) (NHDC) and NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α- L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) ( This is a chemical formula showing the structure of GNHDC).
Figure 2 is a graph showing the effect of NHDC and GNHDC on fasting blood sugar in mice. Ctrl refers to the control group fed with phosphate-buffered saline (PBS) rather than NHDC and GNHDC.
Figure 3 is a photograph and graph showing the effect of NHDC and GNHDC on mouse subcutaneous adipocytes.
Figure 4 is a graph showing the results of quantitative real-time PCR analysis of the effects of NHDC and GNHDC on genes related to fatty acid absorption, lipogenesis, adipogenesis, β-oxidation, and brown fat in mouse subcutaneous adipose tissue.
Figure 5 is a photograph and graph showing the results of oil red O staining analysis of the effect of GNHDC on triglyceride accumulation in 3T3-L1 cells.
Figure 6 is a graph showing the results of analyzing the effect of GNHDC on cell viability in 3T3-L1 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis.
Figures 7 and 8 are graphs showing the results of quantitative real-time PCR analysis of the effects of GNHDC on lipogenesis and adipogenesis.
Figures 9 to 11 are graphs showing the results of Western blot analysis of the effect of GNHDC on the PI3K/AKT/mTOR pathway.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.
실시예 1. 실험물질 및 실험동물 준비Example 1. Preparation of experimental materials and experimental animals
실시예 1-1. 실험물질 준비Example 1-1. Preparation of experimental materials
U.S. Horticultural Research Laboratory에서 네오헤스페리딘 디하이드로칼콘 ((1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone), NHDC) 및 네오헤스페리딘 디하이드로칼콘 당전이체인 NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone) (GNHDC)를 제공받았고, 상기 NHDC 및 GNHDC의 구조를 도 1에 나타내었다. 체외 연구를 위해 상기 NHDC를 0.2% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA)로, 상기 GNHDC를 0.1% 에탄올 (Merck, Darmstadt, Germany)로 30, 50 또는 100μM의 농도로 희석하였고, 별도로 체내 연구를 위해 상기 NHDC 및 GNHDC를 인산염 완충 식염수 (phosphate-buffered saline, PBS)에 용해시켰다.U.S. Neohesperidin dihydrochalcone ((1-(4-((2-O-[6-Deoxy-α-L-mannopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)- from the Horticultural Research Laboratory. 3-[3-hydroxy-4-methoxyphenyl]-1-propanone), NHDC) and NHDC-O-glycoside (1-(4-((2-O-[6-Deoxy- α-L-mannopyranosyl]-(4-O-α-D-glucopyranosyl]-β-D-glucopyranosyl)oxy)-2,6-dihydroxyphenyl)-3-[3-hydroxy-4-methoxyphenyl]-1-propanone ) (GNHDC) was provided, and the structures of NHDC and GNHDC are shown in Figure 1. For in vitro studies, NHDC was dissolved in 0.2% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). The GNHDC was diluted with 0.1% ethanol (Merck, Darmstadt, Germany) to a concentration of 30, 50, or 100 μM, and separately, the NHDC and GNHDC were dissolved in phosphate-buffered saline (PBS) for in vivo studies.
실시예 1-2. 실험동물 준비Example 1-2. Laboratory animal preparation
Central Lab Animal Inc. (서울, 한국)에서 5주령 수컷 C57BLKS/J db/db 마우스를 구매하고, 상기 마우스를 개별적으로 20 ~ 22℃의 온도, 45 ~ 55%의 습도에서 12시간은 밝은 곳, 12시간은 어두운 곳에 두는 사이클에 따라 준비하였다. 상기 마우스는 상기 조건으로 2주간의 적응 기간을 거친 후, 체중과 공복 혈당 수준을 기준으로 무작위로 다음과 같은 3개의 그룹 (그룹당 9마리의 마우스)으로 나누었다: PBS를 경구 투여한 마우스 (대조군); 상기 실시예 1-1에서 체내 연구를 위해 준비한 NHDC를 마우스 체중 (body weight; b.w.) 1kg당 100mg의 양으로 경구 투여한 마우스 (NHDC); 및 상기 실시예 1-1에서 체내 연구를 위해 준비한 GNHDC를 마우스 체중 1kg당 100mg의 양으로 경구 투여한 마우스 (GNHDC). 상기 마우스에 접종한 모든 시약은 4주동안 주5일 경구 위관 영양제로 제공되었다. 상기 마우스는 modified American Institute of Nutrition (AIN)-93G 식단 (Raonbio, Yongin, Korea)에 따라 급이하고 물을 자유롭게 섭취하도록 하였다. 실험 기간 동안 체중, 음식물 섭취량 및 수분 섭취량을 일주일에 두 번 기록하였다. 공복 혈당 수치는 꼬리 정맥에서 Accu-Check (Roche, Mannheim, Germany)를 사용하여 측정하였다. 상기 마우스를 희생한 후, 전혈을 13,000 rpm, 4℃ 온도에서 15분동안 원심분리 하였다. 피하 지방 조직을 PBS로 헹구고 액체 질소에서 냉동시켰다. 모든 샘플은 추가 분석까지 -80℃에서 보관하였다. 연구 절차 및 실험은 이화여자대학교 동물병원 동물 관리위원회 (IACUC 승인번호: 19-052)의 승인을 받았다.Central Lab Animal Inc. 5-week-old male C57BLKS/J db/db mice were purchased from (Seoul, Korea), and the mice were individually placed in a light place for 12 hours and a dark place for 12 hours at a temperature of 20 to 22°C and humidity of 45 to 55%. It was prepared according to two cycles. After a 2-week adaptation period under the above conditions, the mice were randomly divided into the following three groups (nine mice per group) based on body weight and fasting blood sugar level: Mice administered orally with PBS (control group) ; Mice (NHDC) to which the NHDC prepared for in vivo research in Example 1-1 was orally administered in an amount of 100 mg per 1 kg of mouse body weight (b.w.); and mice (GNHDC) to which GNHDC prepared for in vivo research in Example 1-1 was orally administered in an amount of 100 mg per 1 kg of mouse body weight. All reagents inoculated into the mice were provided by oral gavage 5 days a week for 4 weeks. The mice were fed according to a modified American Institute of Nutrition (AIN)-93G diet (Raonbio, Yongin, Korea) and allowed to freely consume water. Body weight, food intake, and water intake were recorded twice a week during the experiment. Fasting blood glucose levels were measured using Accu-Check (Roche, Mannheim, Germany) from the tail vein. After sacrificing the mouse, whole blood was centrifuged at 13,000 rpm and 4°C for 15 minutes. Subcutaneous adipose tissue was rinsed with PBS and frozen in liquid nitrogen. All samples were stored at -80°C until further analysis. Research procedures and experiments were approved by the Animal Care Committee of Ewha Womans University Animal Hospital (IACUC approval number: 19-052).
실시예 2. 각종 마우스 분석 방법Example 2. Various mouse analysis methods
실시예 2-1. 경구 포도당 내성 검사Example 2-1. oral glucose tolerance test
상기 실시예 1-2에서 2주간의 적응기간을 거친 PBS, NHDC 및 GNHDC를 각각 경구 투여한 3개의 마우스 그룹 각각의 마우스를 밤새 단식시키고, 마우스 체중 1kg당 1g의 포도당 용액을 경구로 제공하여 경구 포도당 내성 테스트 (oral glucose tolerance test, OGTT)를 수행하였다. 공복 혈당 수치는 꼬리 정맥에서 Accu-Check (Roche)를 사용하여 0, 30, 60, 90 및 120분에 모니터링 하였고, OGTT의 곡선 아래의 면적 (area under the curve, AUC)을 OGTT 곡선에서 계산하였다.In Example 1-2, mice in each of the three mouse groups each orally administered with PBS, NHDC, and GNHDC after a 2-week adaptation period were fasted overnight, and 1 g of glucose solution per 1 kg of mouse body weight was orally administered. An oral glucose tolerance test (OGTT) was performed. Fasting blood glucose levels were monitored at 0, 30, 60, 90, and 120 minutes using Accu-Check (Roche) in the tail vein, and the area under the curve (AUC) of OGTT was calculated from the OGTT curve. .
실시예 2-2. 마우스 내 생화학적 수치 분석Example 2-2. Analysis of biochemical levels in mice
전체 콜레스테롤 수치를 측정하기 위해, 상기 실시예 1-2에서 PBS로 헹군 피하 지방 조직을 이용하여 시판용 키트 (아산 제약, 서울, 한국)로 측정하였다.To measure total cholesterol level, subcutaneous adipose tissue rinsed with PBS in Example 1-2 was used and measured using a commercially available kit (Asan Pharmaceutical, Seoul, Korea).
상기 실시예 1-2에서 PBS로 헹군 피하 지방 조직을 이용하여 마우스 내 아디포넥틴 수치는 효소 결합 면역 흡착 분석 (enzyme-linked immunosorbent assay, ELISA) 키트 (Crystal Chem, Elk Grove Village, IL, USA)로 측정하였고, 비-에스테르화 지방산 (non-esterified fatty acid, NEFA) 수치를 효소 비색법 분석 (enzymatic colorimetric method assay) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)을 통해 측정하였고, 인슐린 수치도 ELISA 키트 (FUJIFILM Wako Pure Chemical Corporation)를 사용하여 측정하였다.In Example 1-2, adiponectin levels in mice were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Crystal Chem, Elk Grove Village, IL, USA) using subcutaneous adipose tissue rinsed with PBS. Non-esterified fatty acid (NEFA) levels were measured using an enzymatic colorimetric method assay (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and insulin levels were also measured using an ELISA kit (FUJIFILM Measured using Wako Pure Chemical Corporation).
실시예 2-3. 마우스 내 조직학적 분석Example 2-3. Histological analysis in mice
상기 실시예 1-2에서 PBS로 헹군 피하 지방 조직을 10% 포름 알데하이드로 고정하고 파라핀에 포매시킨 후, 자일렌으로 파라핀을 제거하고 재수화한 후 헤마톡실린 및 에오신 (Hematoxylin and eosin, H&E) 염색을 수행하였다. 염색된 부분을 현미경 (Nikon, 도쿄, 일본)으로 관찰하고 촬영한 후, 피하 지방 조직의 지방 세포 영역을 ImageJ (National Institutes of Health, Bethesda, Maryland, USA)를 사용하여 정량화하였다.The subcutaneous adipose tissue rinsed with PBS in Example 1-2 was fixed with 10% formaldehyde and embedded in paraffin, then deparaffinized with xylene, rehydrated, and then treated with hematoxylin and eosin (H&E). Staining was performed. After observing and photographing the stained sections under a microscope (Nikon, Tokyo, Japan), the adipocyte area of subcutaneous adipose tissue was quantified using ImageJ (National Institutes of Health, Bethesda, Maryland, USA).
실시예 2-4. Quantitative real-time PCRExample 2-4. Quantitative real-time PCR
Trizol 시약 (Invitrogen, Carlsbad, CA, USA)을 사용하여 샘플을 추출하고, RNA 농도와 품질을 Nanodrop (Thermo Scientific, Waltham, MA, USA)으로 확인하였고, cDNA를 RevertAid reverse transcriptase (Thermo Scientific)를 42℃에서 1시간 사용하고 72℃에서 3분동안 사용하여 준비하였다. 제조업체의 프로토콜에 따라 Rotor-Gene® Q (Qiagen, Hilden, Germany) 및 2X SYBR Green PCR master mix (Qiagen)를 사용하여 Quantitative real-time PCR을 수행하였다. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh)를 in vivo 및 in vitro 상 모두에서의 연구를 위한 대조군으로 사용하였다. 상기 PCR에서 사용된 Acsl1 (acyl-CoA synthetase long-chain family member 1), Cd36, C/EBPα (CCAAT/enhancer binding protein, alpha), Cpt1α (carnitine palmitoyltransferase 1a), Fas (fatty acid synthase), Lpl (lipoprotein lipase), Pgc1α (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha), PPARγ (peroxisome proliferator activated receptor gamma), Prdm16 (PR domain containing 16), Srebp1c (sterol regulatory element binding transcription factor 1), Ucp1 (uncoupling protein 1) 및 Gapdh (glyceraldehyde-3-phosphate dehydrogenase)의 프라이머 서열은 표 1에 나타내었다.Samples were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), RNA concentration and quality were checked with Nanodrop (Thermo Scientific, Waltham, MA, USA), and cDNA was purified using RevertAid reverse transcriptase (Thermo Scientific). It was prepared by using it at ℃ for 1 hour and at 72℃ for 3 minutes. Quantitative real-time PCR was performed using Rotor-Gene® Q (Qiagen, Hilden, Germany) and 2X SYBR Green PCR master mix (Qiagen) according to the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a control for both in vivo and in vitro studies. Acsl1 (acyl-CoA synthetase long-chain family member 1), Cd36, C/EBPα (CCAAT/enhancer binding protein, alpha), Cpt1α (carnitine palmitoyltransferase 1a), Fas (fatty acid synthase), Lpl ( lipoprotein lipase), Pgc1α (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha), PPARγ (peroxisome proliferator activated receptor gamma), Prdm16 (PR domain containing 16), Srebp1c (sterol regulatory element binding transcription factor 1), Ucp1 (uncoupling protein) 1) and Gapdh (glyceraldehyde-3-phosphate dehydrogenase) primer sequences are shown in Table 1.
실시예 2-5. 세포 배양Example 2-5. cell culture
뮤린 전지방 세포주인 3T3-L1을 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 구매하였고, 10% bovine calf serum (Gibco, Grand Island, NY, USA) 및 1% 페니실린/스트렙토마이신 (penicillin/streptomycin (P/S)) (Invitrogen)이 보충된 Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Gyeongsan, Korea)에서 37℃ 온도 및 5% CO2 환경에서 보관하였다. 상기 세포를 DMEM, 10% fetal bovine serum (FBS) (Gibco), 1% P/S, 500μM isobutylmethylxanthine (Sigma-Aldrich), 1μM dexamethasone (Sigma-Aldrich) 및 10μg/ml 인슐린 (Welgene)에서 2일동안 지방세포로 분화시켰다. 분화 후, 상기 지방세포를 10% FBS, 1% P/S 및 1μg/ml 인슐린에서 6일동안 배양하였다. 세포에 30, 50 또는 100μM 농도의 상기 실시예 1-1에서 체외 연구를 위해 준비한 NHDC 또는 GNHDC를 8일동안 처리하였다.3T3-L1, a murine pre-adipocyte cell line, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated with 10% bovine calf serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin. /streptomycin (P/S)) (Invitrogen) was stored in Dulbecco's modified Eagle's medium (DMEM) (Welgene, Gyeongsan, Korea) at 37°C and 5% CO 2 environment. The cells were grown in DMEM, 10% fetal bovine serum (FBS) (Gibco), 1% P/S, 500 μM isobutylmethylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/ml insulin (Welgene) for 2 days. Differentiated into adipocytes. After differentiation, the adipocytes were cultured in 10% FBS, 1% P/S, and 1 μg/ml insulin for 6 days. The cells were treated with NHDC or GNHDC prepared for the in vitro study in Example 1-1 at a concentration of 30, 50, or 100 μM for 8 days.
실시예 2-6. Oil red O stainingExample 2-6. Oil red O staining
트리글리세라이드 축적을 oil red O staining (Sigma-Aldrich)으로 측정하였다. 구체적으로, 상기 실시예 2-5에서 분화된 지방세포를 PBS로 헹구고 10% 포름알데히드로 고정하였다. 60% 이소프로필 알코올로 세척한 후 상기 고정한 지방세포를 oil red O staining 용액으로 염색하였다. 상기 oil red O staining 용액을 증류수로 씻어내고 슬라이드를 건조하였다. 상기 염색된 지방세포의 방울을 염색 용액이 100% 이소프로필 알코올에 용해되기 전에 Eclipse TS100 (Nikon)에 의해 촬영하였고, 정량화를 위해, 500nm에서 마이크로플레이트 리더 (Molecular Device, Sunnyvale, CA, USA)로 흡광도를 기록하였다.Triglyceride accumulation was measured by oil red O staining (Sigma-Aldrich). Specifically, the adipocytes differentiated in Examples 2-5 were rinsed with PBS and fixed with 10% formaldehyde. After washing with 60% isopropyl alcohol, the fixed adipocytes were stained with oil red O staining solution. The oil red O staining solution was washed off with distilled water and the slide was dried. Drops of the stained adipocytes were photographed by Eclipse TS100 (Nikon) before the staining solution was dissolved in 100% isopropyl alcohol, and for quantification, at 500 nm with a microplate reader (Molecular Device, Sunnyvale, CA, USA). Absorbance was recorded.
실시예 2-7. 세포 생존력 분석Example 2-7. Cell viability assay
세포 생존력을 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) 분석으로 측정하였다. 구체적으로, 3T3-L1 세포를 96-웰 플레이트에 뿌리고 50μM 농도의 상기 실시예 1-1에서 체외 연구를 위해 준비한 NHDC 또는 GNHDC로 8일동안 처리하였다. 상기 NHDC 또는 GNHDC를 처리한 세포에서 배양액을 제거하고 37℃, 5% CO2의 조건에서 500μg/ml MTT 용액이 함유된 배양액으로 3시간동안 처리하였다. 그 후, 560nm에서 마이크로플레이트 리더 (Molecular Device)를 사용하여 흡광도를 기록하였다.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) assay. Specifically, 3T3-L1 cells were seeded in a 96-well plate and treated with NHDC or GNHDC prepared for the in vitro study in Example 1-1 at a concentration of 50 μM for 8 days. The culture medium was removed from the NHDC or GNHDC-treated cells, and the cells were treated with a culture medium containing 500 μg/ml MTT solution at 37°C and 5% CO 2 for 3 hours. Afterwards, the absorbance was recorded using a microplate reader (Molecular Device) at 560 nm.
실시예 2-8. 웨스턴 블랏Example 2-8. western blot
분화된 지방세포의 단백질 농도를 Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA)를 사용하여 Bradford 단백질 분석으로 측정하였고, 상기 단백질 샘플을 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)를 사용하여 전기영동으로 변성 및 분리하였다. 그 후, 상기 단백질 샘플을 폴리비닐리덴 플로라이드 막 (polyvinylidene fluoride membranes) (Millipore, Billerica, MA, USA)으로 옮기고, Tween 20 (TBS-T)을 포함하는 Tris 완충 식염수에서 5% bovine serum albumin 또는 탈지유로 차단하였다. 상기 막은 4℃에서 상기 단백질에 대한 다음의 1차 항체와 함께 밤새 배양하였다: phosphoinositide 3 kinase (PI3K), phospho-phosphoinositide 3 kinase (p-PI3K), protein kinase B (AKT), phospho-protein kinase B (p-AKT), mammalian target of rapamycin (mTOR), phospho-mammalian target of rapamycin (p-mTOR), AMP-activated protein kinase (AMPK) 및 phospho-AMP-activated protein kinase (p-AMPK) (상기 항체 모두 Cell Signaling Technology, Danvers, MA, USA에서 입수) 및 β-actin (Abcam, Cambridge, UK). 상기 막을 TBS-T로 세척하고 2차 항체와 함께 1시간동안 배양하였다. 상기 검출된 단백질 밴드를 enhanced chemiluminescence (ECL) 시약 (Animal Genetics Inc., 수원, 한국)으로 검출하였다.The protein concentration of differentiated adipocytes was measured by Bradford protein analysis using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and the protein samples were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). ) were denatured and separated by electrophoresis. Afterwards, the protein samples were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and incubated with 5% bovine serum albumin or 5% bovine serum albumin in Tris-buffered saline containing Tween 20 (TBS-T). Blocked with skim milk. The membrane was incubated overnight at 4°C with the following primary antibodies against the proteins: phosphoinositide 3 kinase (PI3K), phospho-phosphoinositide 3 kinase (p-PI3K), protein kinase B (AKT), and phospho-protein kinase B. (p-AKT), mammalian target of rapamycin (mTOR), phospho-mammalian target of rapamycin (p-mTOR), AMP-activated protein kinase (AMPK) and phospho-AMP-activated protein kinase (p-AMPK) (above antibodies all from Cell Signaling Technology, Danvers, MA, USA) and β-actin (Abcam, Cambridge, UK). The membrane was washed with TBS-T and incubated with secondary antibody for 1 hour. The detected protein band was detected using enhanced chemiluminescence (ECL) reagent (Animal Genetics Inc., Suwon, Korea).
실시예 2-9. 통계분석Example 2-9. Statistical analysis
상기 실시예 2-1 내지 2-8의 분석결과를 평균 ± 평균의 표준오차 (standard error, SEM)로 표시하였고, Newman-Keuls’s 사후 테스트 또는 unpaired Student’s t-test를 사용하여 일원 분산 분석 (one-way ANOVA)으로 분석하였다. 상기 실험들은 적어도 세 번 수행되었다. P value가 0.05 미만인 경우 유의적인 결과로 간주하였다. GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) 소프트웨어는 데이터의 유의성을 평가하기 위해 사용되었다.The analysis results of Examples 2-1 to 2-8 were expressed as mean ± standard error of the mean (standard error, SEM), and one-way analysis of variance (one-way analysis of variance) was performed using Newman-Keuls's post hoc test or unpaired Student's t-test. way ANOVA). The experiments were performed at least three times. A P value of less than 0.05 was considered a significant result. GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) software was used to evaluate the significance of data.
실시예 3. NHDC 및 GNHDC가 마우스에 미치는 효과 분석 결과Example 3. Results of analysis of the effects of NHDC and GNHDC on mice
실시예 3-1. NHDC 및 GNHDC의 마우스 내 체중, 조직중량, 지질 프로필 및 사이토카인에 미치는 효과Example 3-1. Effects of NHDC and GNHDC on body weight, tissue weight, lipid profile and cytokines in mice
상기 실시예 1-2에서 4주동안 주5일 경구 위관 영양제로 PBS, NHDC 또는 GNHDC를 제공받은 3개의 마우스 그룹의 각 마우스 및 상기 실시예 1-2와 실질적으로 동일한 방법으로 준비한 상기 마우스의 피하 지방 조직에 대하여 하기 표 2의 측정 대상을 측정하여, 각 그룹별로 얻어진 결과의 평균값을 하기 표 2에 나타내었다.In Example 1-2, each mouse in the three groups of mice received PBS, NHDC, or GNHDC by oral gavage 5 days a week for 4 weeks, and the subcutaneous tissue of the mice prepared in substantially the same manner as in Example 1-2. The measurement targets shown in Table 2 below were measured for fat tissue, and the average values of the results obtained for each group are shown in Table 2 below.
구체적으로, 4주동안 시약을 제공한 각 그룹의 최종 체중을 측정하였고, 처음 (0주차) 체중 대비 최종 체중에서의 증가량 (최종 체중 - 처음 체중)를 통해 각 그룹별 증체량, 1일 섭취량, 1일 섭취 수분량을 측정하였고, 상기 마우스 피하 지방 조직의 무게를 측정하였고, 신장 지방, 내장 지방, 부고환 지방 및 피하 지방 무게를 합한 전체 지방 조직의 무게를 측정하였다.Specifically, the final body weight of each group provided with the reagent for 4 weeks was measured, and the increase in final body weight compared to the initial (week 0) body weight (final body weight - initial body weight) was used to determine the weight gain for each group, daily intake, 1 The daily water intake was measured, the weight of the subcutaneous adipose tissue of the mouse was measured, and the weight of the total adipose tissue including kidney fat, visceral fat, epididymal fat, and subcutaneous fat was measured.
전체 콜레스테롤 수치는 상기 실시예 2-2와 실질적으로 동일한 방법으로 상기 체중 시판용 키트를 사용하여 각 그룹별로 측정하였고, NEFA, 아디포넥틴 및 인슐린 수치는 상기 실시예 2-2와 실질적으로 동일한 방법으로 키트를 이용하여 각 그룹별로 측정하였고, 피하 지방 조직 수치는 상기 실시예 2-3과 실질적으로 동일한 방법으로 정량화하여 각 그룹별로 측정하였다. 상기 측정된 수치는 상기 실시예 2-9와 실질적으로 동일한 방법으로 분석결과를 표현 및 통계분석을 진행하였다.Total cholesterol levels were measured for each group using the weight commercially available kit in substantially the same manner as in Example 2-2, and NEFA, adiponectin, and insulin levels were measured using the kit in substantially the same manner as in Example 2-2. was measured for each group, and the subcutaneous fat tissue level was quantified and measured for each group in substantially the same manner as in Example 2-3. The measured values were expressed as analysis results and subjected to statistical analysis in substantially the same manner as in Example 2-9.
최종 체중은 NHDC를 먹인 그룹 (NHDC 그룹), GNHDC를 먹인 그룹 (GNHDC 그룹)에서 모두 감소하는 경향이 있었다. GNHDC 그룹의 체중 증가는 PBS를 먹인 그룹 (대조군 그룹)에 비해 29.1% (P<0.05) 유의하게 감소하였다.Final body weight tended to decrease in both the NHDC-fed group (NHDC group) and the GNHDC-fed group (GNHDC group). The body weight gain of the GNHDC group was significantly reduced by 29.1% (P<0.05) compared to the group fed PBS (control group).
전체 콜레스테롤은 GNHDC 그룹에서 대조군 그룹에 비해 8.3% (P<0.05) 유의하게 감소하였다. NEFA는 대조군 그룹에 비해 GNHDC 그룹에서 16.3% (P<0.05) 유의하게 감소하였다. 또한, GNHDC 그룹에서는 아디포넥틴 (adiponectin)이 증가하는 경향이 있었고, 인슐린은 NHDC 그룹에서 유의하게 증가하였고 (P<0.01), 대조군 그룹에 비해 GNHDC 그룹에서 약간 감소하였다. 인슐린은 대표적인 동화호르몬으로 지방 축적을 유도하기 때문에 인슐린 레벨의 감소는 항비만 효과에 도움이 될 수 있다. 상기 체중 감소, 지방 무게 감소, NEFA의 감소, 아디포넥틴의 증가 및 인슐린의 감소 결과를 통해 항비만 효과를 확인하였다.Total cholesterol was significantly reduced by 8.3% (P<0.05) in the GNHDC group compared to the control group. NEFA was significantly decreased by 16.3% (P<0.05) in the GNHDC group compared to the control group. Additionally, adiponectin tended to increase in the GNHDC group, and insulin significantly increased in the NHDC group (P<0.01) and slightly decreased in the GNHDC group compared to the control group. Insulin is a representative anabolic hormone that induces fat accumulation, so reducing insulin levels can help with anti-obesity effects. The anti-obesity effect was confirmed through the results of weight loss, fat weight loss, NEFA decrease, adiponectin increase, and insulin decrease.
실시예 3-2. NHDC 및 GNHDC가 마우스의 공복 혈당 수준 및 OGTT에 미치는 효과Example 3-2. Effects of NHDC and GNHDC on fasting blood glucose levels and OGTT in mice
마우스에서 고혈당증에 대한 NHDC 및 GNHDC의 효과를 평가하기 위해 상기 실시예 2-1과 실질적으로 동일한 방법으로 공복 혈당 수준을 모니터링 하였고, 그 결과를 도 2에 나타내었다. 2주간의 적응기간 후에 PBS를 먹인 그룹 (대조군)에 비해 공복 혈당 수치는 GNHDC를 먹인 그룹에서 35.5% (P<0.01) 유의하게 감소하였다.To evaluate the effect of NHDC and GNHDC on hyperglycemia in mice, fasting blood glucose levels were monitored in substantially the same manner as Example 2-1, and the results are shown in Figure 2. After a 2-week adaptation period, fasting blood sugar levels were significantly reduced by 35.5% (P<0.01) in the GNHDC-fed group compared to the PBS-fed group (control group).
실시예 3-3. NHDC 및 GNHDC가 마우스 피하 지방 세포 영역에 미치는 효과Example 3-3. Effects of NHDC and GNHDC on mouse subcutaneous adipocyte area
상기 실시예 2-3과 실질적으로 동일한 방법으로 상기 실시예 1-2에서 PBS로 헹군 피하 지방 조직에 대해 H&E 염색을 수행하고, 지방 세포 영역의 정량화를 통해 면적을 측정하여 지방 조직의 조직학적 변화를 관찰하여 NHDC 및 GNHDC를 먹인 마우스 그룹의 지방 조직에 미치는 효과를 평가하였고, 그 결과를 도 3에 나타내었다. 상기 지방 세포 영역의 크기를 정량화한 결과, PBS를 먹인 대조군 그룹에 비해 GNHDC를 먹인 그룹에서 39.2% (P<0.01) 감소하였다.H&E staining was performed on the subcutaneous adipose tissue rinsed with PBS in Example 1-2 in substantially the same manner as in Example 2-3, and the area was measured through quantification of the adipocyte area to determine histological changes in the adipose tissue. was observed to evaluate the effect on the adipose tissue of the mouse groups fed with NHDC and GNHDC, and the results are shown in Figure 3. As a result of quantifying the size of the fat cell area, it decreased by 39.2% (P<0.01) in the GNHDC-fed group compared to the PBS-fed control group.
실시예 3-4. NHDC 및 GNHDC로 인한 마우스 피하 지방 조직에서 지질 대사를 위한 유전자 발현 분석Example 3-4. Gene expression analysis for lipid metabolism in mouse subcutaneous adipose tissue due to NHDC and GNHDC
마우스 피하 지방 조직에서 NHDC 및 GNHDC가 지방산 흡수, lipogenesis, adipogenesis, β-산화 및 갈색 지방화 관련 유전자에 미치는 효과를 평가하기 위해 상기 실시예 1-2의 피하 지방 조직을 샘플로 하여 상기 실시예 2-4와 실질적으로 동일한 방법으로 quantitative real-time PCR을 수행하였고, 그 결과를 도 4에 나타내었다.In order to evaluate the effects of NHDC and GNHDC on genes related to fatty acid absorption, lipogenesis, adipogenesis, β-oxidation, and brown fat in mouse subcutaneous adipose tissue, the subcutaneous adipose tissue of Example 1-2 was used as a sample, and Example 2- Quantitative real-time PCR was performed in substantially the same manner as in 4, and the results are shown in Figure 4.
지방산 흡수에 대한 Cd36 및 Lpl의 mRNA 발현 수준을 분석하였고, Cd36 및 Lpl의 mRNA 발현은 PBS를 먹인 대조군 그룹에 비해 GNHDC를 먹인 그룹 (GNHDC 그룹)에서 각각 34.6%, 46.8% 하향 조절되었다 (모두 P<0.05). 지방 생성 관련 유전자에 대한 Srebp1c 및 Fas를 분석하였고, Srebp1c 및 Fas의 발현은 대조군 그룹에 비해 GNHDC 그룹에서 각각 41.0%, 49.7% 감소하였다 (P<0.05).The mRNA expression levels of Cd36 and Lpl in response to fatty acid absorption were analyzed, and the mRNA expression of Cd36 and Lpl was downregulated by 34.6% and 46.8%, respectively, in the GNHDC-fed group (GNHDC group) compared to the PBS-fed control group (all P <0.05). Srebp1c and Fas were analyzed for adipogenesis-related genes, and the expression of Srebp1c and Fas decreased by 41.0% and 49.7%, respectively, in the GNHDC group compared to the control group (P<0.05).
β-산화 관련 Acsl1 및 Cpt1α 유전자의 mRNA 발현 수준을 분석하였다. Acsl1의 발현은 대조군 그룹과 비교하여 GNHDC 그룹에서 약 1.8배 상향 조절되었다 (P<0.05). Cpt1α의 발현은 GNHDC 그룹에서 약 1.5배 상향 조절되었다 (P<0.05).The mRNA expression levels of β-oxidation-related Acsl1 and Cpt1α genes were analyzed. The expression of Acsl1 was upregulated approximately 1.8-fold in the GNHDC group compared to the control group (P<0.05). The expression of Cpt1α was upregulated approximately 1.5-fold in the GNHDC group (P<0.05).
갈색 지방화와 관련된 Ucp1, Pgc1α, 및 Prdm16 유전자의 mRNA 발현을 분석하였다. Ucp1의 발현은 대조군 그룹과 비교하여 GNHDC 그룹 (P<0.01)에서 상향 조절되었다. Pgc1α의 발현은 GNHDC 그룹에서 약 1.6배 상향 조절되었다 (P<0.01). Prdm16의 발현은 GNHDC 그룹에서 약 2.4배 상향 조절되었다 (P<0.01).The mRNA expression of Ucp1, Pgc1α, and Prdm16 genes related to brown localization was analyzed. The expression of Ucp1 was upregulated in the GNHDC group (P<0.01) compared to the control group. The expression of Pgc1α was upregulated approximately 1.6-fold in the GNHDC group (P<0.01). The expression of Prdm16 was upregulated approximately 2.4-fold in the GNHDC group (P<0.01).
실시예 3-5. GNHDC가 3T3-L1 세포의 지질 축적 및 세포 생존력에 미치는 효과 확인Example 3-5. Determination of the effects of GNHDC on lipid accumulation and cell viability of 3T3-L1 cells
GNHDC가 트리글리세리드 축적에 미치는 효과를 평가하기 위해 상기 실시예 2-5에서 준비한 체외 연구를 위해 준비한 GHDC를 처리한 지방세포를 이용하여 상기 실시예 2-6과 실질적으로 동일한 방법으로 oil red O staining을 수행하였고, 그 결과를 도 5에 나타내었다. 대조군으로는 GNHDC를 처리하지 않은 지방세포를 준비하였다. GNHDC를 처리한 그룹은 대조군 그룹에 비해 염색된 지방세포 방울의 양이 감소하였다. 대조군 그룹과 비교하여 50μM 농도의 GNHDC를 처리한 그룹에서는 16.0% (P<0.05), 100μM 농도의 GNHDC를 처리한 그룹에서는 20.0% (P<0.01) 감소하였다.To evaluate the effect of GNHDC on triglyceride accumulation, oil red O staining was performed in substantially the same manner as in Example 2-6 using the GHDC-treated adipocytes prepared for the in vitro study prepared in Example 2-5. This was performed, and the results are shown in Figure 5. As a control group, adipocytes not treated with GNHDC were prepared. The amount of stained adipocyte droplets in the GNHDC-treated group was reduced compared to the control group. Compared to the control group, it decreased by 16.0% (P<0.05) in the group treated with GNHDC at a concentration of 50μM and by 20.0% (P<0.01) in the group treated with GNHDC at a concentration of 100μM.
세포 생존력을 평가하기 위해 50μM GNHDC의 세포 독성을 상기 실시예 2-7과 실질적으로 동일한 방법으로 MTT 분석을 수행하였고, 그 결과를 도 6에 나타내었다. 두 감미료 처리에서 세포 생존력에는 유의한 차이가 없었다. 따라서, 50μM GNHDC를 사용하여 추가 시험관 내 실험을 수행하였다.To evaluate cell viability, MTT analysis was performed on the cytotoxicity of 50 μM GNHDC in substantially the same manner as Example 2-7, and the results are shown in Figure 6. There was no significant difference in cell viability between the two sweetener treatments. Therefore, additional in vitro experiments were performed using 50 μM GNHDC.
실시예 3-6. GNHDC가 3T3-L1 세포의 lipogenesis, adipogenesis에 미치는 효과 분석Example 3-6. Analysis of the effects of GNHDC on lipogenesis and adipogenesis in 3T3-L1 cells
GNHDC가 lipogenesis 및 adipogenesis에 미치는 효과를 평가하기 위해 상기 실시예 2-5에서 준비한 50μM 농도의 상기 실시예 1-1에서 체외 연구를 위해 준비한 GNHDC를 8일동안 처리한 지방세포를 샘플로 하여 상기 실시예 2-4와 실질적으로 동일한 방법으로 quantitative real-time PCR을 수행하였고, 그 결과를 도 7 및 8에 나타내었다. 대조군으로는 GNHDC를 처리하지 않은 지방세포를 준비하였다. Lipogenesis를 위한 Srebp1c 및 Fas (도 7)와 adipogenesis를 위한 PPARγ 및 C/EBPα의 mRNA 발현 (도 8)을 분석하였다. Srebp1c의 발현은 대조군 그룹에 비해 GNHDC 처리한 그룹 (GNHDC 그룹)에서 48.5% (P<0.01) 감소하였다. Fas 발현은 대조군 그룹에 비해 GNHDC 그룹에서 51.8% (P<0.01) 감소하였다. PPARγ의 mRNA 발현은 대조군 그룹에 비해 GNHDC 그룹에서 27.9% (P<0.01) 감소하였다. C/EBPα의 mRNA 발현은 대조군 그룹에 비해 GNHDC 그룹에서 44.2% (P<0.01) 감소하였다.In order to evaluate the effect of GNHDC on lipogenesis and adipogenesis, the above experiments were performed using adipocytes treated for 8 days with 50 μM concentration of GNHDC prepared in Example 2-5 for in vitro study in Example 1-1 as a sample. Quantitative real-time PCR was performed in substantially the same manner as Example 2-4, and the results are shown in Figures 7 and 8. As a control group, adipocytes not treated with GNHDC were prepared. The mRNA expression of Srebp1c and Fas for lipogenesis (Figure 7) and PPARγ and C/EBPα for adipogenesis (Figure 8) were analyzed. The expression of Srebp1c was decreased by 48.5% (P<0.01) in the GNHDC-treated group (GNHDC group) compared to the control group. Fas expression was decreased by 51.8% (P<0.01) in the GNHDC group compared to the control group. The mRNA expression of PPARγ decreased by 27.9% (P<0.01) in the GNHDC group compared to the control group. The mRNA expression of C/EBPα decreased by 44.2% (P<0.01) in the GNHDC group compared to the control group.
실시예 3-7. GNHDC의 3T3-L1 세포에서 PI3K/AKT/mTOR 경로 및 AMPK에 미치는 효과 분석Example 3-7. Analysis of effects on PI3K/AKT/mTOR pathway and AMPK in 3T3-L1 cells of GNHDC
GNHDC가 PI3K/AKT/mTOR 경로에 미치는 효과를 평가하기 위해 상기 실시예 2-5에서 준비한 50μM 농도의 상기 실시예 1-1에서 체외 연구를 위해 준비한 GNHDC를 8일동안 처리한 지방세포의 단백질 농도를 상기 실시예 2-8과 실질적으로 동일한 방법으로 웨스턴 블랏을 수행하여 측정하였고, 그 결과를 도 9 내지 11에 나타내었다. 대조군으로는 GNHDC를 처리하지 않은 지방세포를 준비하였다. PI3K/AKT/mTOR 경로의 활성화는 lipogenesis 및 adipogenesis를 통해 세포 증식과 지질 합성을 증가시킨다. p-PI3K/PI3K의 발현은 대조군 그룹에 비해 GNHDC를 처리한 그룹 (GNHDC 그룹)에서 19.3% (P<0.01) 감소하였다 (도 10). p-AKT/AKT의 발현은 대조군 그룹에 비해 GNHDC 그룹에서 46.1% (P<0.01) 감소하였다 (도 10). p-mTOR/mTOR 발현은 대조군 그룹에 비해 GNHDC 그룹에서 12.7% (P<0.01) 감소하였다 (도 11). AMPK는 상기 신호 전달 경로를 억제하고 mTOR 발현을 억제한다. p-AMPK/AMPK의 발현은 대조군 그룹에 비해 GNHDC 그룹에서 2.5배 (P<0.01) 증가하였다.Protein concentration of adipocytes treated for 8 days with GNHDC prepared for in vitro study in Example 1-1 at a concentration of 50 μM prepared in Example 2-5 to evaluate the effect of GNHDC on the PI3K/AKT/mTOR pathway was measured by Western blotting in substantially the same manner as Example 2-8, and the results are shown in Figures 9 to 11. As a control group, adipocytes not treated with GNHDC were prepared. Activation of the PI3K/AKT/mTOR pathway increases cell proliferation and lipid synthesis through lipogenesis and adipogenesis. The expression of p-PI3K/PI3K was decreased by 19.3% (P<0.01) in the GNHDC-treated group (GNHDC group) compared to the control group (FIG. 10). The expression of p-AKT/AKT was decreased by 46.1% (P<0.01) in the GNHDC group compared to the control group (FIG. 10). p-mTOR/mTOR expression was decreased by 12.7% (P<0.01) in the GNHDC group compared to the control group (Figure 11). AMPK inhibits the above signaling pathway and inhibits mTOR expression. The expression of p-AMPK/AMPK increased 2.5-fold (P<0.01) in the GNHDC group compared to the control group.
<110> Ewha University - Industry Collaboration Foundation Seoul National University R&DB Foundation <120> Composition for reducing body weight, body fat, or blood sugar including neohesperidin dihydrochalcone glycotransferase <130> DPP20210288KR <160> 24 <170> koPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acsl1 Forward <400> 1 tgccagagct gattgacatt c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acsl1 Reverse <400> 2 ggcataccag aaggtggtga g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd36 Forward <400> 3 gtgctctccc ttgattctgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd36 Reverse <400> 4 tgagaatgcc tccaaacaca 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C/EBPalpha Forward <400> 5 ccaagaagtc ggtggacaag a 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C/EBPalpha Reverse <400> 6 cggtcattgt cactggtcaa ct 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cpt1alpha Forward <400> 7 aacccagtgc cttaacgatg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cpt1alpha Reverse <400> 8 gaactggtgg ccaatgagat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fas Forward <400> 9 tgtgagtggt tcagaggcat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fas Reverse <400> 10 ttctgtagtg ccagcaagct 20 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Lpl Forward <400> 11 gagtttgacc gccttccg 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Lpl Reverse <400> 12 tcccgttacc gtccatcc 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1alpha Forward <400> 13 tcgagctgta cttttgtgga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1alpha Reverse <400> 14 tcatacttgc tcttggtgga 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma Forward <400> 15 gagcacttca caagaaatta cc 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma Reverse <400> 16 gaactccata gtggaagcct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 Forward <400> 17 agatgaacca ggcatccact 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 Reverse <400> 18 tctacgtcct ctggctttgc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Srebp1c Forward <400> 19 tagagcatat cccccaggtg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Srebp1c Reverse <400> 20 ggtacgggcc acaagaagta 20 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 Forward <400> 21 ccaagccagg atggtgaac 19 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 Reverse <400> 22 ccagcgggaa ggtgatgata 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Forward <400> 23 aactttggca ttgtggaagg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Reverse <400> 24 tgtgagggag atgctcagtg 20 <110> Ewha University - Industry Collaboration Foundation Seoul National University R&DB Foundation <120> Composition for reducing body weight, body fat, or blood sugar including neohesperidin dihydrochalcone glycotransferase <130> DPP20210288KR <160> 24 <170> koPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acsl1 Forward <400> 1 tgccagagct gattgacatt c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acsl1 Reverse <400> 2 ggcataccag aaggtggtga g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd36 Forward <400> 3 gtgctctccc ttgattctgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd36 Reverse <400> 4 tgagaatgcc tccaaacaca 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C/EBPalpha Forward <400> 5 ccaagaagtc ggtggacaag a 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C/EBPalpha Reverse <400> 6 cggtcattgt cactggtcaa ct 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cpt1alpha Forward <400> 7 aacccagtgc cttaacgatg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cpt1alpha Reverse <400> 8 gaactggtgg ccaatgagat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fas Forward <400> 9 tgtgagtggt tcagaggcat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fas Reverse <400> 10 ttctgtagtg ccagcaagct 20 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Lpl Forward <400> 11 gagtttgacc gccttccg 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Lpl Reverse <400> 12 tcccgttacc gtccatcc 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1alpha Forward <400> 13 tcgagctgta cttttgtgga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1alpha Reverse <400> 14 tcatacttgc tcttggtgga 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma Forward <400> 15 gagcacttca caagaaatta cc 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARgamma Reverse <400> 16 gaactccata gtggaagcct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 Forward <400> 17 agatgaacca ggcatccact 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 Reverse <400> 18 tctacgtcct ctggctttgc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Srebp1c Forward <400> 19 tagagcatat cccccaggtg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Srebp1c Reverse <400> 20 ggtacgggcc acaagaagta 20 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 Forward <400> 21 ccaagccagg atggtgaac 19 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 Reverse <400> 22 ccagcgggaa ggtgatgata 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Forward <400> 23 aactttggca ttgtggaagg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Reverse <400> 24 tgtgaggggag atgctcagtg 20
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