KR101802696B1 - Pharmaceutical composition for preventing or treating obesity comprising extract of pueraria lobata leaf - Google Patents
Pharmaceutical composition for preventing or treating obesity comprising extract of pueraria lobata leaf Download PDFInfo
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Abstract
본 발명은 칡잎 추출물을 유효성분으로 함유하는 조성물에 관한 것으로, 본 발명의 칡잎 추출물은 천연추출물로 지방전구체세포의 분화과정에서 지방합성에 주요한 역할을 하는 ACC(acetly-CoA carboxylase) 유전자의 발현을 저해시키고 지방분화 및 지방합성에 관여하는 GPDH(glycerol-3-phosphate dehydrogenase)의 활성을 감소시켜, 지질축적을 저해하는 것이 확인됨에 따라, 본 발명의 칡잎 추출물을 유효성분으로 함유하는 조성물은 비만을 예방하거나 치료할 수 있는 약학조성물 또는 건강식품으로 제공될 수 있다.The present invention relates to a composition containing an extract of sesame leaf as an active ingredient, wherein the sesame leaf extract of the present invention is a natural extract, which expresses an acetyl-CoA carboxylase (ACC) gene, which plays a major role in lipogenesis in the differentiation of lipoprotein somatic cells (Glycerol-3-phosphate dehydrogenase), which is involved in lipid differentiation and lipid synthesis, is inhibited and lipid accumulation is inhibited. Therefore, the composition containing the safflower extract of the present invention as an active ingredient is useful for the prevention of obesity Or as a pharmaceutical composition or a health food that can prevent or treat the disease.
Description
본 발명은 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 치료용 약학조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating obesity containing an arrowroot leaf extract as an active ingredient.
비만이란 열량의 섭취와 소비의 불균형으로 발생하는 대사성 질환이며 과잉된 열량으로 인해 지방조직이 비정상적으로 증가된 상태를 말한다. 남자는 체지방이 체중의 25%, 여자는 체중의 30% 이상일 때 비만으로 보며, 임상적으로는 BMI(Body Mass Index: 체질량지수)가 24.0 내지 30.0은 과체중으로 정의하고 3.0 이상인 경우를 비만으로 정의한다.Obesity is a metabolic disease caused by an imbalance between intake and consumption of calories, and refers to a condition in which adipose tissue is abnormally increased due to excess calories. Men are considered to be obese when their body fat is 25% of their body weight and women are over 30% of their body weight. Clinically, BMI (Body Mass Index) of 24.0 to 30.0 is defined as overweight and 3.0 or more is defined as obesity. do.
비만이 발생하여 그 상태가 지속되면 여러 질환의 원인으로 작용하는데, 그러한 질환으로서 고혈압, 혈중 콜레스테롤 상승, 당뇨병, 신장 질환, 뇌졸증, 동맥경화증, 지방간, 관절염, 암, 수면 무호흡증, 당뇨병 등을 들 수 있다.If obesity occurs and the condition persists, it acts as a cause of various diseases. Such diseases include high blood pressure, elevated blood cholesterol, diabetes, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes. have.
비만의 원인으로는 고지방·고열량의 식생활, 바쁜 사회적 환경에 따른 운동 부족, 내분비 이상 등 환경적 요인과 유전적 요인을 들 수 있는데, 이 중 비만의 50 내지 70% 정도가 환경적 요인에 의한 것으로 알려져 있고, 나머지가 유전적 요인에 의한 것으로 알려져 있다.The causes of obesity include environmental and genetic factors such as high fat and high calorie diet, lack of exercise due to busy social environment, and endocrine abnormalities, of which 50 to 70% of obesity is attributed to environmental factors. It is known, and the rest is known to be due to genetic factors.
비만 치료제는 일반적으로 3가지 범주 즉 식욕 억제제, 체내 에너지 대사 촉진제 및 소화 흡수 억제제로 구분된다. 식욕을 억제하는 약리 기전을 이용하는 대표적인 비만 치료제로는 리덕틸(Reductil™, 애보트사, 미국)을 들 수 있고, 체내 에너지를 촉진하는 약리 기전을 이용하는 대표적인 비만 치료제로서는 엑소리제(Exorise™,아코파마사, 프랑스)를 들 수 있으며, 지방의 소화 흡수를 억제하는 약리기전을 이용하는 대표적인 비만 치료제로서는 제니칼(Xenical™, 로슈제약회사, 스위스)을 들 수 있다.Obesity treatments are generally divided into three categories: appetite suppressants, stimulators of energy metabolism in the body, and inhibitors of digestive absorption. A typical obesity treatment that uses a pharmacological mechanism to suppress appetite is Reductil™ (Abbott, USA), and Exorise™ (Aco Pharma) is a representative obesity treatment that uses a pharmacological mechanism that promotes energy in the body. , France), and Xenical™ (Roche Pharmaceuticals, Switzerland) is a representative obesity treatment that uses a pharmacological mechanism that inhibits the digestion and absorption of fat.
칡은 다년생 식물로 오래전부터 구황작물로 식용되었고 자양강장제 등 건강식품으로 이용되기도 하였다. 한방에서는 뿌리를 갈근이라하여 항염증, 항산화, 혈압강하, 항궤양, 간보호를 위한 약재로 사용하고 있으며, 현재까지는 에스트로겐 활성이 가장 높은 칡뿌리를 대상으로 한 지질 축적 저해 연구가 보고된 바 있으나, 칡잎의 비만 예방 또는 치료 효능에 대한 연구는 전무한 실정이다. Arrowroot is a perennial plant and has been edible as an oral crop for a long time, and has been used as a health food such as a nutrient tonic. In oriental medicine, the root is called prickly pear, and it is used as a medicinal for anti-inflammatory, antioxidant, blood pressure lowering, anti-ulcer, and liver protection. Until now, studies on the inhibition of lipid accumulation in arrowroot roots with the highest estrogen activity have been reported. , There is no research on the effectiveness of arrowhead leaves for preventing or treating obesity.
본 발명은 천연물인 칡잎 추출물을 유효성분으로 함유하는 조성물을 제공하여 지방전구세포의 분화를 저해함으로써, 지질합성 및 지질축적 억제시켜 비만을 예방하거나 치료할 수 있는 항비만용 조성물을 제공하고자 한다. The present invention is to provide a composition for preventing or treating obesity by inhibiting the differentiation of adipocytes by inhibiting the differentiation of adipocytes by providing a composition containing a natural product of arrowroot leaf extract as an active ingredient.
본 발명은 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating obesity containing an arrowroot leaf extract as an active ingredient.
또한, 본 발명은 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 개선용 건강식품을 제공한다.In addition, the present invention provides a health food for preventing or improving obesity containing an arrowroot leaf extract as an active ingredient.
본 발명은 칡잎 추출물을 유효성분으로 함유하며, 상기 칡잎 추출물은 생체 외(in vitro)에서 지방전구세포의 지방세포로의 분화를 억제하는 것을 특징으로 하는 지방세포분화 억제용 시약조성물을 제공한다.The present invention contains an arrowroot leaf extract as an active ingredient, and the arrowhead leaf extract provides a reagent composition for inhibiting adipocyte differentiation, characterized in that it inhibits differentiation of adipocytes into adipocytes in vitro.
또한, 본 발명은 인간을 제외한 개체로부터 분리된 지방전구세포에 칡잎 추출물을 처리하는 단계를 포함하는, 생체 외(in vitro) 지방세포 분화 억제방법을 제공한다.In addition, the present invention provides a method for inhibiting differentiation of adipocytes in vitro, comprising the step of treating an arrowroot leaf extract on adipocytes isolated from individuals other than humans.
본 발명에 따르면, 천연물인 칡잎 추출물은 지방전구세포의 분화과정에서 지방합성에 주요한 역할을 하는 ACC(acetly-CoA carboxylase) 유전자의 발현을 저해시키고 지방분화 및 지방합성에 관여하는 GPDH(glycerol-3-phosphate dehydrogenase)의 활성을 감소시켜, 지질축적을 저해하는 것을 확인하였다.According to the present invention, the arrowroot leaf extract, a natural product, inhibits the expression of the ACC (acetly-CoA carboxylase) gene, which plays a major role in adipogenesis in the differentiation process of adipocytes, and GPDH (glycerol-3), which is involved in adipogenesis and adipogenesis. -phosphate dehydrogenase) activity was confirmed to inhibit lipid accumulation.
따라서, 본 발명의 칡잎 추출물을 유효성분으로 함유하는 조성물은 비만을 예방하거나 치료할 수 있는 약학조성물 또는 건강식품으로 제공될 수 있다.Accordingly, the composition containing the arrowroot leaf extract of the present invention as an active ingredient may be provided as a pharmaceutical composition or health food capable of preventing or treating obesity.
도 1은 칡잎 에탄올 추출물의 세포 독성을 확인한 결과로, 3T3-L1 지방전구세포에 0 내지 800 μg/mL 칡잎 에탄올 추출물을 48시간 처리하고 세포생존도를 확인한 MTT 분석 결과이다.
도 2는 분화가 유도된 3T3-L1 지방전구세포에 25 내지 100 μg/mL 칡잎 에탄올 추출물을 8일간 처리하고 분화되는 동안 축적된 지질을 확인한 오일 레드 O 염색 결과로, a는 분화되지 않은 정상 3T3-L1 지방전구세포의 염색 결과이며, b는 지방세포로 분화가 유도된 3T3-L1 지방전구세포군에서 축적된 지질의 염색 결과이며, c는 분화가 유도된 3T3-L1 지방전구세포에 25 μg/mL 칡잎 에탄올 추출물이 처리된 세포군의 염색 결과이며, d는 분화기 유도된 3T3-L1 지방전구세포에 50 μg/mL 칡잎 에탄올 추출물이 처리된 세포군의 염색 결과이며, e는 분화가 유도된 3T3-L1 지방전구세포에 100 μg/mL 칡잎 에탄올 추출물이 처리된 세포군의 염색 결과이다.
도 3은 분화가 유도된 3T3-L1 지방전구세포에 25, 50 및 100 μg/mL 칡잎 에탄올 추출물을 8일간 처리하고 분화되는 동안 축적된 지질의 함량을 확인한 결과로, 분화된 대조군과 비교하여 ***p<0.001이며, N은 분화되지 않은 정상대조군이며, C는 분화된 대조군 세포이다.
도 4는 분화가 유도된 3T3-L1 지방전구세포에 25, 50 및 100 μg/mL 칡잎 에탄올 추출물을 8일간 처리하고 분화되는 동안 GPDH(glycerol-3-phosphate dehydrogenase) 활성을 확인한 결과로, 칡잎 에탄올 추출물이 처리되지 않은 세포분화 대조군과 비교하여 **p<0.01이며, N은 분화되지 않은 정상대조군이며, C는 세포분화 대조군이다.
도 5는 3T3-L1 지방전구세포에 50 및 100 μg/mL 칡잎 에탄올 추출물을 8일간 처리하고 분화가 유도된 3T3-L1세포에서 ACC(acetly-CoA carboxylase) mRNA 발현 수준을 확인한 RT-PCR 결과로, A는 겔에서 ACC mRNA 밴드를 확인한 결과이며, B는 ACC mRNA 발현을 정량한 결과이다.Figure 1 is a result of confirming the cytotoxicity of the arrowroot leaf ethanol extract, 3T3-L1 fat progenitor cells treated with 0 to 800 μg / mL arrowroot ethanol extract for 48 hours and confirmed the cell viability MTT analysis results.
Figure 2 is a result of oil red O staining confirming the lipids accumulated during differentiation after treatment with 25 to 100 μg/mL ethanol extract of arrowroot leaves on 3T3-L1 adipocytes induced differentiation for 8 days, a is a normal non-differentiated 3T3 -L1 is the result of staining of adipocytes, b is the result of staining of lipids accumulated in the 3T3-L1 adipocytes group in which differentiation into adipocytes is induced, and c is the result of staining of 3T3-L1 adipocytes in which differentiation is induced, 25 μg/ It is the staining result of the cell group treated with mL arrowroot leaf ethanol extract, d is the staining result of the cell group treated with 50 μg/mL arrowroot leaf ethanol extract in 3T3-L1 adipocytes induced differentiation, and e is the 3T3- induced differentiation. It is the staining result of the cell group treated with ethanol extract of 100 μg/mL arrowroot leaf to L1 adipocytes.
Figure 3 is a result of confirming the content of lipids accumulated during differentiation after treatment with 25, 50, and 100 μg/mL arrowroot leaf ethanol extract for 8 days on 3T3-L1 adipocytes from which differentiation was induced, compared with the differentiated control group * **p<0.001, N is an undifferentiated normal control, and C is a differentiated control cell.
FIG. 4 is a result of treating 3T3-L1 adipocytes in which differentiation is induced with ethanol extracts of 25, 50 and 100 μg/mL arrowroot leaves for 8 days and confirming the activity of glycerol-3-phosphate dehydrogenase (GPDH) during differentiation, arrowhead ethanol Compared to the untreated cell differentiation control with the extract, **p<0.01, N is the non-differentiated normal control, and C is the cell differentiation control.
5 is a RT-PCR result confirming the expression level of ACC (acetly-CoA carboxylase) mRNA in 3T3-L1 cells treated with 50 and 100 μg/mL arrowroot leaf ethanol extract for 8 days on 3T3-L1 adipocytes. , A is the result of confirming the ACC mRNA band in the gel, and B is the result of quantifying ACC mRNA expression.
본 발명은 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention can provide a pharmaceutical composition for preventing or treating obesity containing an arrowroot leaf extract as an active ingredient.
보다 상세하게는 상기 칡잎 추출물은 물, C 1 내지 4 알콜 및 이의 혼합 용매에 의해 추출된 것일 수 있으며, 보다 바람직하게는 에탄올 추출물일 수 있으나, 이에 한정되는 것은 아니다.More specifically, the arrowroot leaf extract may be extracted with water, C 1 to 4 alcohol, and a mixed solvent thereof, and more preferably, an ethanol extract, but is not limited thereto.
본 발명의 칡잎 추출물은 지방전구세포의 GPDH(glycerol-3-phosphate dehydrogenase) 활성 및 ACC(acetly-CoA carboxylase) 발현을 억제하여 지방세포로의 분화 및 지질합성을 억제할 수 있다.The arrowroot leaf extract of the present invention can inhibit differentiation into adipocytes and lipid synthesis by inhibiting the activity of glycerol-3-phosphate dehydrogenase (GPDH) and the expression of acetly-CoA carboxylase (ACC) in adipocytes.
보다 상세하게는 GPDH(glycerol-3-phosphate dehydrogenase)는 글리세롤과 지방산의 에스테르 결합을 촉매하는 효소로 GPDH의 활성은 세포내에서 중성지방 합성정도를 증가시키기 때문에 지방세포 분화에 대한 표지로 알려져 있다.In more detail, GPDH (glycerol-3-phosphate dehydrogenase) is an enzyme that catalyzes the ester bond between glycerol and fatty acids, and the activity of GPDH increases the degree of triglyceride synthesis in cells, so it is known as a marker for adipocyte differentiation.
본 발명의 일실시예에 따르면, 도 4와 같이 분화가 유도된 3T3-L1 지방전구세포에 25 및 50 μg/㎖농도의 칡잎 에탄올 추출물이 처리된 경우, 칡잎 에탄올 추출물이 처리되지 않은 분화세포보다 지방분화 및 지방합성에 관여하는 GPDH의 활성이 각각 16.8% 및 17.9% 감소된 것을 확인하였다.According to an embodiment of the present invention, as shown in FIG. 4, when the 3T3-L1 adipocytes in which differentiation is induced were treated with ethanol extract of arrowroot leaves at concentrations of 25 and 50 μg/ml, the ethanol extract of arrowroot leaves was not treated with the differentiated cells. It was confirmed that the activities of GPDH involved in lipodifferentiation and liposynthesis were reduced by 16.8% and 17.9%, respectively.
또한, ACC(acetly-CoA carboxylase)는 FAS의 기질인 malonyl-CoA를 형성하고 지방산 합성 속도를 조절하며 지방산 합성의 제 1반응 단계를 촉매하는 장쇄지방산 합성을 촉진하는 유전자로, 본 발명의 다른 일실시예에 따르면, 도 5와 같이 분화가 유도된 3T3-L1 지방전구세포에 25 및 50 μg/㎖농도의 칡잎 에탄올 추출물이 처리된 경우, 칡잎 에탄올 추출물이 처리되지 않은 분화세포보다 ACC 유전 발현이 각각 34.6 및 33.3% 감소된 것을 확인할 수 있었다. In addition, ACC (acetly-CoA carboxylase) is a gene that promotes the synthesis of long-chain fatty acids that forms malonyl-CoA, which is a substrate of FAS, regulates the rate of fatty acid synthesis, and catalyzes the first reaction step of fatty acid synthesis. According to an example, when differentiation-induced 3T3-L1 adipocytes are treated with ethanol extract at concentrations of 25 and 50 μg/ml as shown in FIG. 5, ACC genetic expression is more pronounced than differentiated cells not treated with the arrowroot ethanol extract. It could be confirmed that it was reduced by 34.6 and 33.3%, respectively.
본 발명의 또 다른 일실시예에 따르면, 지질성분에 특이적으로 민감하게 반응하여 붉은색을 나타냄으로써 지질 축적 정도를 확인할 수 있는 오일 레드 O 염색 결과, 도 2와 같이 분화가 유도된 3T3-L1 지방세포의 지방 방울이 칡잎 에탄올 추출물의 농도의존적으로 감소되는 것을 확인하였다.According to another embodiment of the present invention, as a result of oil red O staining that can confirm the degree of lipid accumulation by displaying red color by reacting specifically to lipid components, 3T3-L1 in which differentiation is induced as shown in FIG. 2 It was confirmed that the fat droplets of adipocytes decreased in a concentration-dependent manner of the ethanol extract of arrowroot leaves.
상기 결과로부터 칡잎 에탄올 추출물은 지방전구체세포의 분화과정에서 지방합성에 주요한 역할을 하는 ACC(acetly-CoA carboxylase) 유전자의 발현을 저해시키고 지방분화 및 지방합성에 관여하는 GPDH(glycerol-3-phosphate dehydrogenase)의 활성을 감소시켜, 지질축적을 저해하는 것이 확인되었다.From the above results, the ethanol extract of arrowroot leaves inhibits the expression of the ACC (acetly-CoA carboxylase) gene, which plays a major role in adipogenesis in the differentiation process of adipocytes, and glycerol-3-phosphate dehydrogenase (GPDH), which is involved in the differentiation of fat and fat synthesis. ) By reducing the activity, it was confirmed to inhibit lipid accumulation.
상기 결과들로부터 본 발명은 칡잎 추출물을 유효성분으로 함유하며, 상기 칡잎 추출물은 생체 외(in vitro)에서 지방전구세포의 지방세포로의 분화를 억제하는 것을 특징으로 하는 지방세포분화 억제용 시약조성물로 제공될 수 있다.From the above results, the present invention contains an arrowroot leaf extract as an active ingredient, and the arrowhead leaf extract is a reagent composition for inhibiting adipocyte differentiation, characterized in that it inhibits differentiation of adipocytes into adipocytes in vitro. Can be provided.
또한, 본 발명은 인간을 제외한 개체로부터 분리된 지방전구세포에 칡잎 추출물을 처리하는 단계를 포함하는, 생체 외(in vitro) 지방세포 분화 억제방법을 제공할 수 있다.In addition, the present invention can provide a method for inhibiting differentiation of adipocytes in vitro, comprising the step of treating an arrowroot leaf extract on adipocytes isolated from individuals other than humans.
본 발명의 약학조성물은 약학조성물 총 100 중량부에 대하여, 칡잎 추출물은 0.01 내지 90 중량부로 포함될 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may be included in an amount of 0.01 to 90 parts by weight based on 100 parts by weight of the total pharmaceutical composition, and the arrowroot leaf extract is not limited thereto.
본 발명의 한 구체예에서, 상기 칡잎 추출물을 유효성분으로 함유하는 자외선에 의한 세포손상 예방 또는 치료용 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition for preventing or treating cell damage caused by ultraviolet rays containing the arrowroot leaf extract as an active ingredient is an injection, granule, powder, tablet, pill, capsule, suppository, gel according to a conventional method. , Suspension, emulsion, drop or liquid may be used in any one of the selected formulation.
본 발명의 다른 구체예에서, 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 치료용 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition for preventing or treating obesity containing the arrowroot leaf extract as an active ingredient is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, which is commonly used in the manufacture of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants.
구체적으로 담체, 부형제 및 희석제는 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 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base material for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to an embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal It can be administered to the subject in a conventional manner via the route.
상기 칡잎 추출물의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.A preferred dosage of the arrowroot leaf extract may vary depending on the condition and weight of the subject, the type and degree of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several doses, whereby the scope of the present invention is not limited.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the'subject' may be a mammal including a human, but is not limited to these examples.
또한, 본 발명은 칡잎 추출물을 유효성분으로 함유하는 비만 예방 또는 개선용 건강식품을 제공할 수 있다.In addition, the present invention can provide a health food for preventing or improving obesity containing the arrowroot leaf extract as an active ingredient.
상기 건강식품은 상기 칡잎 추출물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다.유효성분의 혼합양은 그의 사용 목적 예를들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food may be used together with other foods or food additives in addition to the arrowroot leaf extract, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient is suitably used according to the purpose of use, such as prevention, health or therapeutic treatment. Can be determined.
상기 건강식품에 함유된 화합물의 유효용량은 상기 치료제의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range, and is effective. It is clear that the ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제등을 들 수 있다.There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.
<참고예> 시약 및 기구<Reference Example> Reagents and instruments
탄닌산(Tannic acid), folin-ciocalteu's 페놀 시약, 디(에틸렌글리콜) [di(ethylene glycol)] 시약, DPPH(1,1-diphenyl-2-picryl hydrazyl), 인슐린, 3-이소부틸-1-메틸잔틴[3-isobutyl-1-methylxanthine], 덱사메타손[dexamethasone], 오일 레드 O[Oil red O]는 Sigma 사(USA)의 제품을 사용하였고, DMEM(Dulbecco's modified Eagle's medium), 태아소혈청(fetal bovine serum; FBS), 페니실린/스트렙토마이신(P/S)은 Lonza 사(USA)의 제품을 구입하여 사용하였으며, 그 외 일반시약,들은 특급품을 사용하였다. 시료추출은 회전진공농축기(R-205, B-490, Buchi, Japan)를 사용하였다. 세포주 관찰은 도립현미경 (CKX41, Olympus, Japan)을 사용하였고, 세포주 배양은 CO2 배양기(MCO-15AC, Sanyo Electric, Japan)를 사용하였다.Tannic acid, folin-ciocalteu's phenol reagent, di(ethylene glycol) [di(ethylene glycol)] reagent, DPPH(1,1-diphenyl-2-picryl hydrazyl), insulin, 3-isobutyl-1-methyl Xanthine [3-isobutyl-1-methylxanthine], dexamethasone, and oil red O were manufactured by Sigma (USA), and DMEM (Dulbecco's modified Eagle's medium), fetal bovine serum (fetal bovine) were used. Serum; FBS) and penicillin/streptomycin (P/S) were purchased and used by Lonza (USA), and other general reagents were used with special products. The sample was extracted using a rotary vacuum concentrator (R-205, B-490, Buchi, Japan). The cell line was observed using an inverted microscope (CKX41, Olympus, Japan), and the cell line was cultured using a CO 2 incubator (MCO-15AC, Sanyo Electric, Japan).
<실시예 1> 칡잎 에탄올 추출물<Example 1> Arrowroot leaf ethanol extract
경상북도 칠곡에서 채취하여 음건시킨 칡잎 300g에 80% 에탄올 3L를 첨가하여 24시간 침적, 3회 반복 추출하여 여과지로 여과한 후 농축하고 동결건조시켜 분말상태의 시료를 제조하였다. 수율(yield)은 1.2%였다.A powdery sample was prepared by adding 3L of 80% ethanol to 300g of arrowroot leaves collected in Chilgok, Gyeongsangbuk-do and adding 3L of 80% ethanol to immersion for 24 hours, extracting 3 times repeatedly, filtering through filter paper, concentrating and freeze-drying. The yield was 1.2%.
<실시예 2> 칡잎 에탄올 추출물의 세포독성 확인<Example 2> Confirmation of cytotoxicity of ethanol extract of arrowroot leaves
1. 세포주 및 배양1. Cell line and culture
3T3-L1 지방전구세포를 계명대학교 의과대학 분자의학실에서 분양받아 계대 배양하여 사용하였다. 세포 배양 플레이트에 부착시키고 10% 우아혈청(bovine calf serum; BCS)와 1% 페니실린/스트렙토마이신(P/S)을 첨가한 DMEM 배지를 사용하여 37℃, 5% CO2 조건의 배양기에서 배양하였다. 3T3-L1 adipocytes were pre-sold in Molecular Medicine, Keimyung University College of Medicine, and were subcultured. Attached to the cell culture plate and cultured in an incubator at 37° C. and 5% CO 2 using DMEM medium to which 10% bovine calf serum (BCS) and 1% penicillin/streptomycin (P/S) were added. .
2. MTT 분석2. MTT analysis
3T3-L1 지방전구세포를 10% BCS와 1% 페니실린/스트렙토마이신(P/S)이 포함된 DMEM 배지에 풀어 37℃, 5% CO2 배양기에서 48시간 동안 안정화시킨 후, MTT 분석에 사용하였다. 안정화된 3T3-L1 지방전구세포를 96-웰 플레이트에 적정세포수(1×104 cells/well)로 200 ㎕씩 분주하고 37℃, 5% CO2 배양기에서 24시간 배양한 다음, PBS로 1회 씻은 다음 동결건조시킨 칡잎 에탄올추출물을 DMEM 배지에 25-800μg/㎖ 농도별로 희석하여 200㎕씩 넣은 후 37℃, 5% CO2 배양기에서 48시간 배양하였다. 3T3-L1 adipocytes were dissolved in DMEM medium containing 10% BCS and 1% penicillin/streptomycin (P/S) and stabilized in a 37° C., 5% CO 2 incubator for 48 hours, and then used for MTT analysis. . Dispense 200 µl of stabilized 3T3-L1 adipocytes into a 96-well plate at an appropriate number of cells (1×10 4 cells/well) and incubate for 24 hours in a 37°C, 5% CO 2 incubator, and then 1 with PBS. After washing twice, the freeze-dried ethanol extract of arrowroot leaves was diluted in DMEM medium by concentration of 25-800 μg/ml, added 200 µl each, and cultured in a 37°C, 5% CO 2 incubator for 48 hours.
그 후, PBS로 1회 씻고 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]가 0.5 mg/mL 함유된 DMEM 배지를 200 ㎕씩 넣은 후 37℃, 5% CO2 배양기에서 3시간 배양하였다. 배지를 버리고 DMSO를 200 ㎕씩 넣고 플레이트 교반기에서 15분간 세포를 녹여낸 다음 ELISA reader로 540 nm 파장에서 흡광도를 측정하였다. Then, wash once with PBS and add 200 µl of DMEM medium containing 0.5 mg/mL of MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] at 37°C, Incubated for 3 hours in a 5% CO 2 incubator. Discard the medium, add 200 µl of DMSO each, and dissolve the cells for 15 minutes in a plate stirrer, and then measure the absorbance at a wavelength of 540 nm with an ELISA reader.
그 결과, 도 1과 같이 100 μg/㎖ 농도의 칡잎 에탄올 추출물이 처리된 경우, 3T3-L1 지방전구세포가 89.3%의 세포생장률을 보인 반면, 200 μg/㎖ 농도의 칡잎 에탄올 추출물에서 68.8%로 나타나 세포독성을 보임으로써 최대허용농도는 100μg/㎖로 확인되었다. As a result, as shown in FIG. 1, when the ethanol extract of arrowhead leaves at a concentration of 100 μg/ml was treated, 3T3-L1 adipocytes showed a cell growth rate of 89.3%, whereas in the ethanol extract of arrowhead leaves at a concentration of 200 μg/ml, it was 68.8%. It appeared and showed cytotoxicity, and the maximum permissible concentration was confirmed to be 100 μg/ml.
상기 결과로부터 하기 모든 실험에서는 100 μg/㎖ 이하의 농도를 사용하였다. From the above results, in all the experiments below, a concentration of 100 μg/ml or less was used.
<실시예 3> 칡잎 에탄올 추출물의 지질 합성 억제 효과 확인<Example 3> Confirmation of lipid synthesis inhibitory effect of ethanol extract of arrowroot leaves
1. 세포 분화 및 시료 처리1. Cell differentiation and sample processing
3T3-L1 지방전구세포를 분화유도하기 위하여 세포를 60 mm 플레이트에 1×106 세포로 분주하고 2일 후 배지를 교환하여 4일째에 세포가 100% 밀집되게 배양하였다. 10% 태아소혈청(FBS)과 MDI 용액(0.5mM IBMX, 1μM 덱사메타손, 10/mL 인슐린)을 포함한 DMEM 배지를 2일 처리하였다. 이때 칡잎 에탄올 추출물이 지방세포분화에 미치는 영향을 관찰하기 위해 칡잎 에탄올 추출물을 25, 50 및 100/mL 농도로 함께 처리하였다. 분화 유도 2일 후 배지를 각 농도별 칡잎 에탄올 추출물, 10% FBS 및 10/mL 인슐린을 포함한 DMEM으로 3일간 처리하였고, 이후부터는 칡잎 에탄올 추출물과 함께 2일에 한 번씩 10% FBS와 1% 페니실린/스트렙토마이신(P/S)이 포함된 DMEM으로 교환하여 총 8일간 세포를 분화시켰다.In order to induce differentiation of 3T3-L1 adipocytes, the cells were aliquoted into 1×10 6 cells on a 60 mm plate, and the medium was exchanged 2 days later, and the cells were cultured to 100% densely on the 4th day. DMEM medium containing 10% fetal bovine serum (FBS) and MDI solution (0.5 mM IBMX, 1 μM dexamethasone, 10/mL insulin) was treated for 2 days. At this time, in order to observe the effect of the arrowroot leaf ethanol extract on adipocyte differentiation, the arrowroot leaf ethanol extract was treated with 25, 50 and 100/mL concentrations. After 2 days of induction of differentiation, the medium was treated with arrowhead ethanol extract at each concentration, DMEM containing 10% FBS and 10/mL insulin for 3 days, and thereafter, 10% FBS and 1% penicillin once every 2 days with arrowhead ethanol extract. / The cells were differentiated for a total of 8 days by exchange with DMEM containing streptomycin (P/S).
2. 오일 레드 O(Oil red O) 염색 및 지질 함량 확인2. Oil red O staining and lipid content check
지질성분에 특이적으로 민감하게 반응하여 붉은색을 나타냄으로써 지질 축적 정도를 확인할 수 있는 특수염색법인 오일 레드 O 염색을 이용하여 칡잎 에탄올 추출물의 지질 합성 억제 효과를 확인하였다.Oil red O staining, a special dyeing method that can confirm the degree of lipid accumulation by reacting specifically to lipid components and showing red color, was used to confirm the lipid synthesis inhibitory effect of the ethanol extract of arrowroot leaves.
상기 방법으로 분화시킨 3T3-L1 지방전구세포의 분화 8일째에 배지를 제거하고 PBS로 세포를 세척한 뒤 실온에서 10% 포름알데하이드(formaldehyde)로 60분 동안 고정하였다. 고정액을 제거하고 60% 이소프로판올(isopropanol)을 이용하여 세척한 후, 세포내 생성된 지질방울(lipid droplet)과 특이적으로 반응하는 오일 레드 O(Oil red O) 용액을 이용하여 실온에서 염색하였다. On the 8th day of differentiation of 3T3-L1 adipocytes differentiated by the above method, the medium was removed, the cells were washed with PBS, and then fixed with 10% formaldehyde at room temperature for 60 minutes. After removing the fixative and washing with 60% isopropanol, it was stained at room temperature using an oil red O solution that specifically reacts with lipid droplets generated in the cell.
그 결과, 도 2와 같이 분화가 유도된 3T3-L1 지방전구세포에 8일간 칡잎 에탄올 추출물을 처리하고 도립현미경을 사용하여 200 배율에서 관찰한 결과, 도 2a와 같이 미분화된 3T3-L1 지방전구세포에서는 붉은색이 거의 확인되지 않은 반면, 도 2b의 8 일간 분화시킨 대조군 지방세포에서는 많은 양의 붉은색이 확인됨에 따라, 많은 양의 지질이 축적된 것을 확인하였다. 또한, 대조군 지방세포와 비교하여 칡잎 에탄올 추출물 25 μg/㎖(도 2c), 50 μg/㎖(도 2d) 및 100 μg/㎖(도 2e)가 처리된 세포군에서는 농도 의존적으로 붉은색이 적게 관찰됨에 따라, 지질 축적이 억제된 것을 확인하였다. As a result, as a result of treatment with ethanol extract of arrowroot leaves for 8 days on 3T3-L1 adipocytes induced differentiation as shown in FIG. 2 and observation at 200 magnification using an inverted microscope, undifferentiated 3T3-L1 adipocytes as shown in FIG. 2A In contrast, red color was hardly observed in FIG. 2B, whereas a large amount of red color was observed in the control adipocytes differentiated for 8 days in FIG. 2B, indicating that a large amount of lipid was accumulated. In addition, compared to the control adipocytes, in the cell group treated with the ethanol extract of
또한, 염색된 세포는 현미경 관찰한 후 100% 이소프로판올을 이용하여 오일 레드 0 염색을 용해하여 추출한 뒤 490 nm에서 흡광도를 측정하여 지방세포 내에 있는 지질함량을 수치로 나타내어 대조군의 흡광도 값에 대한 백분율로 나타내었다.In addition, the stained cells were observed under a microscope, extracted by dissolving
그 결과, 도 3과 같이 8일간 분화시킨 3T3-L1 지방세포인 대조군(C)에 비해 칡잎 에탄올 추출물 25, 50 및 100 μg/㎖가 처리된 세포군에서는 농도의존적으로 각각 60.0%, 71.1% 및 73.3% 유의하게(p<0.001) 지질 함량이 낮게 나타났다.As a result, compared to the control group (C), which is 3T3-L1 adipocytes differentiated for 8 days as shown in FIG. 3, in the cell groups treated with arrowroot
상기 결과로부터, 칡잎 에탄올 추출물은 지질축적 저해 효능이 있음이 확인되었다.From the above results, it was confirmed that the ethanol extract of arrowroot leaves has an inhibitory effect on lipid accumulation.
3. 글리세롤-3-포스페이트 디하이드로게나아제(GPDH) 활성 확인3. Glycerol-3-phosphate dehydrogenase (GPDH) activity confirmation
지방세포에만 특이적으로 활성이 증가되는 GPDH 활성을 확인하여 칡잎 에탄올 추출물 처리에 따른 지방세포 분화 여부 및 중성지방의 축적 정도를 확인하였다.By confirming the GPDH activity, which increases the activity specifically only in adipocytes, the differentiation of adipocytes and the degree of accumulation of triglycerides according to the ethanol extract treatment of arrowroot leaves were confirmed.
GPDH 활성 측정은 GPDH activity kit(Takara Bio Inc., Japan)을 사용하였다. 8일 동안 분화 배지와 칡잎 에탄올 추출물을 함께 처리한 지방세포의 배양액을 제거하고, 효소 추출 버퍼(enzyme extraction buffer)를 넣어 세포를 수집하고 2-멜캡토에탄올(mercaptoethanol)이 포함된 희석버퍼로 세포를 희석하였다. GPDH activity was measured using a GPDH activity kit (Takara Bio Inc., Japan). Remove the culture medium of adipocytes treated with the differentiation medium and ethanol extract of arrowroot leaves for 8 days, collect the cells by adding an enzyme extraction buffer, and use a dilution buffer containing 2-melcaptoethanol. Was diluted.
기질을 넣은 플레이트에 희석한 세포를 넣어 혼합한 후, 분광광도계(spectrophotometer)에서 340 nm, 10분간 매 1분마다 흡광도의 변화를 측정하였다. GPDH 활성도는 1분 동안 기질 1 μM을 사용하기 위한 효소의 양을 계산하고, 계산된 값을 mg 단백질/ml로 환산하였다. 상층액의 단백질 함량 측정은 Bio-red protein assay kit(BioRad, CA, USA)로 정량하였다. After mixing the diluted cells in the plate containing the substrate, the change in absorbance was measured every 1 minute at 340 nm in a spectrophotometer for 10 minutes. GPDH activity was calculated by calculating the amount of enzyme to use 1 μM of the substrate for 1 minute, and the calculated value was converted to mg protein/ml. The protein content of the supernatant was measured using a Bio-red protein assay kit (BioRad, CA, USA).
그 결과, 도 4와 같이 대조군(C)과 비교하여 칡잎 에탄올 추출물 25 및 50 μg/㎖이 처리된 세포군은 GPDH 활성을 각각 16.8 및 17.9% 유의하게(p<0.01) 저해시키는 것이 확인되었다.As a result, it was confirmed that, compared to the control group (C), as shown in FIG. 4, the cell group treated with the ethanol extract of arrowroot leaves 25 and 50 μg/ml significantly inhibited the GPDH activity by 16.8 and 17.9%, respectively (p<0.01).
상기 결과로부터, 칡잎 에탄올 추출물은 효과적으로 지방세포 분화 및 지방합성을 저해하는 효능을 갖고 있음이 확인되었다. From the above results, it was confirmed that the ethanol extract of arrowroot leaves effectively inhibits adipocyte differentiation and adipogenesis.
4. ACC(아세틸-CoA 카르복실라아제) 유전자 발현 확인4. ACC (acetyl-CoA carboxylase) gene expression confirmation
FAS의 기질인 malonyl-CoA를 형성하고 지방산 합성 속도를 조절하며 지방산 합성의 제 1반응 단계를 촉매하는 장쇄지방산 합성을 촉진하는 아세틸-CoA 카르복실라아제(ACC) 유전자 발현에 칡잎 에탄올 추출물이 미치는 영향을 역전사 중합효소 연쇄반응(RT-PCR)으로 확인하였다.The effect of ethanol extract of arrowroot leaf on the expression of acetyl-CoA carboxylase (ACC) gene, which forms the substrate of FAS, malonyl-CoA, regulates the rate of fatty acid synthesis, and promotes the synthesis of long-chain fatty acids that catalyze the first reaction step in fatty acid synthesis. The effect was confirmed by reverse transcription polymerase chain reaction (RT-PCR).
상기 방법으로 분화 및 칡잎 에탄올 추출물이 처리된 3T3-L1 지방세포에 0.8 ㎖ Trizol-Reagent(Life Technologies, CA, USA)를 처리하여 총 RNA를 분리하였다. 분리한 RNA에 160 ㎕ 클로로포름(chloroform)을 넣고 혼합한 후 원심분리하는 과정을 2번 반복하여 상층액을 분리하였다. 400 ㎕ 이소프로필 알콜(isopropyl alcohol)을 이용하여 RNA를 침전시킨 후 70% 에탄올로 세척하고 자연 건조시킨 후 DEPC-water에서 RNA를 녹여 -80℃에 저장하였다.Total RNA was isolated by treatment with 0.8 ml of Trizol-Reagent (Life Technologies, CA, USA) on 3T3-L1 adipocytes differentiated and treated with ethanol extract of arrowroot leaves by the above method. 160 µl of chloroform was added to the separated RNA, mixed, and centrifuged twice to separate the supernatant. After precipitating RNA using 400 µl isopropyl alcohol, washing with 70% ethanol and air drying, the RNA was dissolved in DEPC-water and stored at -80°C.
상기 방법으로 대조군 및 실험군에서 각각 분리된 전체 RNA 5 μg을 M-MLV RT 5×버퍼 8 ㎕, 10 mM dNTPs의 3 ㎕, 10,000U RNase 억제제 0.45 ㎕, 50,000U M-MLV 역전사효소(Promega, USA) 0.3 ㎕ 및 20 pmol/ oligo dT(Bioneer, Korea) 3.75 ㎕가 포함된 용액 40 ㎕을 이용해 역전사하여 cDNA를 제조하였다. 5 μg of total RNA isolated from the control group and the experimental group by the above method, respectively, was mixed with 8 μl of M-MLV RT 5×buffer, 3 μl of 10 mM dNTPs, 0.45 μl of 10,000U RNase inhibitor, and 50,000U M-MLV reverse transcriptase (Promega, USA). ) CDNA was prepared by reverse transcription using 40 µl of a solution containing 0.3 µl and 20 pmol/oligo dT (Bioneer, Korea) 3.75 µl.
상기 방법으로 제조된 단일 가닥 cDNA를 5×green Go Taq flexi buffer 4 ㎕, 10 mM dNTPs 0.4 ㎕, 500U Taq polymerase 0.1 ㎕, 25 mM MgCl2(Promega, Madison, USA) 1.2 ㎕ 및 ACC, FAS, GAPDH의 특이적인 정방향 및 역방향 프라이머를 각각 20 pmol/L 0.4 ㎕를 사용하여 PCR로 증폭하였다. PCR에 사용한 프라이머 염기서열과 예상크기는 표 1과 같다. Single-stranded cDNA prepared by the above method was 5×green Go Taq flexi buffer 4 μl, 10 mM dNTPs 0.4 μl, 500U Taq polymerase 0.1 μl, 25 mM MgCl 2 (Promega, Madison, USA) 1.2 μl and ACC, FAS, GAPDH The specific forward and reverse primers of were amplified by PCR using 0.4 μl of 20 pmol/L, respectively. The primer sequence and expected size used for PCR are shown in Table 1.
내부표준물질로 GAPDH(50, 28 cycle), 실험물질로 ACC(94 30초, 56 1분, 72 1분, 30 cycle)를 사용하였으며, PCR 산물을 1.2% 아가로스 겔에 전기영동 시킨 후 분석하였다.GAPDH (50, 28 cycles) as an internal standard and ACC (94 30 seconds, 56 1 minute, 72 1 minute, 30 cycles) were used as the test material, and the PCR product was subjected to electrophoresis on a 1.2% agarose gel for analysis. I did.
ACC
351
GAPDH
248
그 결과, 도 5와 같이 칡잎 에탄올 추출물이 처리되지 않은 대조군 3T3-L1 지방세포와 비교하여 25 및 50 μg/㎖ 칡잎 에탄올 추출물이 처리된 세포군에서 ACC 유전자 발현이 각각 34.6 및 33.3% 감소된 것을 확인할 수 있었다. As a result, it was confirmed that ACC gene expression was reduced by 34.6 and 33.3%, respectively, in the cell groups treated with 25 and 50 μg/ml arrowroot ethanol extract compared to the control 3T3-L1 adipocytes not treated with the arrowroot leaf ethanol extract as shown in FIG. 5. Could.
상기 결과로부터, 칡잎 에탄올 추출물은 지방합성 초기단계에서 발현이 유도되는 ACC 유전자 발현을 감소시켜 3T3-L1 지방전구세포에서 지방합성을 저해하는 것을 확인할 수 있었다.From the above results, it was confirmed that the ethanol extract of arrowroot leaves reduced the expression of ACC genes induced in the early stage of adipogenesis, thereby inhibiting adipogenesis in 3T3-L1 adipocytes.
한편, 본 발명에 따른 칡잎 추출물은 목적에 따라 여러 형태로 제제화가 가능하다. 하기에서는 본 발명에 따른 칡잎 추출물을 활성성분으로 함유시킨 몇몇 제제화 방법을 예시한 것으로 본 발명이 이에 한정되는 것은 아니다.On the other hand, the arrowroot leaf extract according to the present invention can be formulated in various forms depending on the purpose. Hereinafter, some formulation methods containing the arrowroot leaf extract according to the present invention as an active ingredient are exemplified, and the present invention is not limited thereto.
<제제예 1> 약학조성물의 처방예<Formulation Example 1> Formulation example of pharmaceutical composition
<제제예 1-1> 주사제의 제조<Formulation Example 1-1> Preparation of injection
칡잎 에탄올 추출물 10 mg, 소디움 메타비설파이트 3.0 mg, 메틸파라벤 0.8 mg, 프로필파라벤 0.1 mg 및 주사용 멸균증류수 적량을 혼합하고 통상의 방법으로 최종 부피가 2 ㎖이 되도록 제조한 후, 2 ㎖ 용량의 앰플에 충전하고 멸균하여 주사제를 제조하였다.Arrowhead leaves ethanol extract 10 mg, sodium metabisulfite 3.0 mg, methylparaben 0.8 mg, propylparaben 0.1 mg, and an appropriate amount of sterile distilled water for injection were mixed and prepared so that the final volume became 2 ml by a conventional method, and then 2 ml dose Filled into ampoules and sterilized to prepare injections.
<제제예 2-1> 정제의 제조<Formulation Example 2-1> Preparation of tablets
칡잎 에탄올 추출물 10 mg, 유당 100 mg, 전분 100 mg 및 스테아린산 마그네슘 적량을 혼합하고 통상의 정제 제조방법에 따라 타정하여 정제를 제조하였다.Tablets were prepared by mixing 10 mg of arrowroot leaf ethanol extract, 100 mg of lactose, 100 mg of starch, and an appropriate amount of magnesium stearate, followed by tableting according to a conventional tablet preparation method.
<제제예 3-1> 캡슐제의 제조<Formulation Example 3-1> Preparation of capsules
칡잎 에탄올 추출물 10 mg, 유당 50 ㎎, 전분 50 ㎎, 탈크 2 ㎎ 및 스테아린산 마그네슘 적량을 혼합하고 통상의 캡슐제 제조방법에 따라 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.10 mg of arrowroot leaf ethanol extract, 50 mg of lactose, 50 mg of starch, 2 mg of talc, and an appropriate amount of magnesium stearate were mixed and filled into gelatin capsules according to a conventional capsule preparation method to prepare a capsule.
<제제예 2> 건강식품의 제조<Formulation Example 2> Preparation of health food
칡 잎 에탄올 추출물 0.5 ㎎, 비타민 혼합물 적량(비타민 A 아세테이트 70 ㎍, 비타민 E 1.0 ㎎, 비타민 B 1 0.13 ㎎, 비타민 B 2 0.15 ㎎, 비타민 B 6 0.5 ㎎, 비타민 B 12 0.2 ㎍, 비타민 C 10 ㎎, 비오틴 10 ㎍, 니코틴산아미드 1.7 ㎎, 엽산 50 ㎍, 판토텐산 칼슘 0.5 ㎎) 및 무기질 혼합물 적량(황산제1철 1.75 ㎎, 산화아연 0.82㎎, 탄산마그네슘 25.3 ㎎, 제1인산칼륨 15 ㎎, 제2인산칼슘 55 ㎎, 구연산칼륨 90㎎, 탄산칼슘 100 ㎎, 염화마그네슘 24.8 ㎎)을 혼합한 다음 과립을 제조하고 통상의 방법에 따라 건강식품을 제조하였다.Arrowroot leaf ethanol extract 0.5 mg, vitamin mixture appropriate amount (vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B 1 0.13 mg, vitamin B 2 0.15 mg, vitamin B 6 0.5 mg, vitamin B 12 0.2 μg, vitamin C 10 mg) , Biotin 10 µg, nicotinic acid amide 1.7 mg,
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION KEIMYUNG UNIVERSITY <120> Pharmaceutical composition for preventing or treating obesity comprising extract of pueraria lobata leaf <130> ADP-2015-0088 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ACC forward <400> 1 tgaccgtggg cacaaagtt 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACC reverse <400> 2 aggaggaacc gcatttatcg a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward <400> 3 gtatgactcc actcacggca aa 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse <400> 4 ggtctcgctc ctggaagatg 20 <110> INDUSTRY ACADEMIC COOPERATION FOUNDATION KEIMYUNG UNIVERSITY <120> Pharmaceutical composition for preventing or treating obesity comprising extract of pueraria lobata leaf <130> ADP-2015-0088 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ACC forward <400> 1 tgaccgtggg cacaaagtt 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACC reverse <400> 2 aggaggaacc gcatttatcg a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward <400> 3 gtatgactcc actcacggca aa 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse <400> 4 ggtctcgctc ctggaagatg 20
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Cited By (3)
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| KR20210093481A (en) | 2020-01-20 | 2021-07-28 | (주) 뉴젠바이오 | Composition for improving, treating or preventing obesity comprising glutinous rice fermented by lactic bacteria as an active ingredient |
| KR20220121432A (en) | 2021-02-25 | 2022-09-01 | 이주성 | Noole Including Leaf of Powder Arrowroot and the Method of Making the Same |
| KR20220121943A (en) | 2021-02-25 | 2022-09-02 | 이주성 | Bread and Snack Composition including Leaf of Powder Arrowroot and the Method of Making the Same |
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| KR100645385B1 (en) | 2005-10-05 | 2006-11-23 | 주식회사 안지오랩 | Composition for anti-obesity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210093481A (en) | 2020-01-20 | 2021-07-28 | (주) 뉴젠바이오 | Composition for improving, treating or preventing obesity comprising glutinous rice fermented by lactic bacteria as an active ingredient |
| KR20220121432A (en) | 2021-02-25 | 2022-09-01 | 이주성 | Noole Including Leaf of Powder Arrowroot and the Method of Making the Same |
| KR20220121943A (en) | 2021-02-25 | 2022-09-02 | 이주성 | Bread and Snack Composition including Leaf of Powder Arrowroot and the Method of Making the Same |
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