KR20240058030A - Composition for inhibiting fat accumulation comprising Senna Tora sprouts and Fagopyrum Tataricum sprouts - Google Patents
Composition for inhibiting fat accumulation comprising Senna Tora sprouts and Fagopyrum Tataricum sprouts Download PDFInfo
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Abstract
본 발명은 결명 새싹 및 메밀 새싹을 포함하는, 지방 축적을 억제하기 위한 혼합조성물 및 이의 용도에 관한 것이다.
본 발명의 혼합조성물은 지방전구세포의 지방생성을 억제하고, 복부에 축적되는 복부 지방과 지방의 과도한 축적으로 증가되는 간의 중량을 현저하게 감소시킴으로써 체내에 지방 축적을 효과적으로 억제하는 효과가 있다. 또한 NASH 유도 실험동물에서 간 조직의 세포 손상을 억제하며, 다양한 염증 및 세포사멸 관련 인자들을 효율적으로 억제하는 것이 관찰되어 NASH 질환의 특이적인 병리기전에 유효하다. The present invention relates to a mixed composition for inhibiting fat accumulation, comprising chestnut sprouts and buckwheat sprouts, and its use.
The mixed composition of the present invention has the effect of effectively suppressing fat accumulation in the body by inhibiting lipogenesis in preadipocytes and significantly reducing abdominal fat accumulated in the abdomen and liver weight increased due to excessive accumulation of fat. In addition, it was observed to suppress cell damage in liver tissue in NASH-induced experimental animals and to efficiently suppress various inflammation- and apoptosis-related factors, making it effective in the specific pathogenesis of NASH disease.
Description
본 발명은 결명 새싹 및 메밀 새싹을 포함하는 지방 축적 억제용 조성물에 관한 것이다.The present invention relates to a composition for inhibiting fat accumulation containing stem sprouts and buckwheat sprouts.
지방간은 간 내 지방침착으로 인한 무게가 간 내 무게의 5% 이상으로 이루어진 경우로 정의된다. 서구화된 식습관 등으로 인해 비알코올성 지방간 질환(non-alcoholic fatty liver disease; NAFLD)이 증가되고 있다. 상기 비알코올성 지방간 질환은 만성 간질환으로 간 내 단순 지방증(simple steatosis)부터 지방간염(non-alcoholic steatohepatitis), 간경변에 이르는 일련의 과정을 모두 포함한다. 지방간은 전세계적으로 증가 추세에 있는 만성 질환으로 임상적인 증상이 두드러지지 않아 치료가 늦어지고 중등도의 지방간으로 발전하여 간경변으로 이행되는 심각한 질환이다. 중등도 지방간에서 염증과 간 조직 석회화가 진행될 경우 그 손상은 복구하기 어려운 상태에 진입하며 이후 간경변의 발병률이 급격하게 올라간다. Fatty liver is defined as a case where the weight due to fat deposition in the liver is more than 5% of the weight of the liver. Non-alcoholic fatty liver disease (NAFLD) is increasing due to westernized eating habits. The non-alcoholic fatty liver disease is a chronic liver disease and includes a series of processes ranging from simple steatosis in the liver to non-alcoholic steatohepatitis and cirrhosis. Fatty liver is a chronic disease that is on the rise worldwide. It is a serious disease that has no noticeable clinical symptoms, so treatment is delayed and it develops into moderate fatty liver disease and cirrhosis. If inflammation and liver tissue calcification progress in moderate fatty liver, the damage becomes difficult to repair, and the incidence of cirrhosis rises rapidly thereafter.
비알코올성 지방간질환은 간 내에 중성 지방이 과다하게 축적되어 있는 상태로 단순 지방증(simple steatosis)에서부터 비알코올성 지방간염(non-alcoholic steatohepatitis, NASH) 나아가 간경변증까지를 포함하는 광의의 개념이다(Cohen JC, Horton JD, Hobbs HH. Science 2011;332:1519-23).Non-alcoholic fatty liver disease is a condition in which excessive neutral fat is accumulated in the liver and is a broad concept that includes simple steatosis, non-alcoholic steatohepatitis (NASH), and even cirrhosis (Cohen JC, Horton JD, Hobbs HH. Science 2011;332:1519-23).
비알코올성 지방간질환의 가장 초기 단계는 단순 지방증이며 건강하고 마른 사람들 사이에서 간 내의 중성지방 함량이 상위 95% 이상에 해당되거나 간 세포 내에서 세포질 내의 중성지방 입자 비율이 5% 이상일 때로 정의한다. 단순 지방증은 대부분 저절로 호전되는 경과를 보이나 일부에서는 비알코올성 지방간염으로 진행하게 된다. The earliest stage of non-alcoholic fatty liver disease is simple steatosis, which is defined as when the neutral fat content in the liver is in the upper 95% or higher among healthy, lean people or when the proportion of neutral fat particles in the cytoplasm of liver cells is higher than 5%. Simple steatosis mostly improves spontaneously, but in some cases, it progresses to non-alcoholic steatohepatitis.
상기 비알코올성 지방간염은 단순 지방증과 달리 팽창성 변성(ballooning degeneration) 및 세포 사멸, 염증성 침윤 등의 병리학적 소견을 보이며, 비알코올성 지방간염이 심화되면, 콜라겐 축적 등의 섬유화 소견을 보이기도 한다. 비알코올성 지방간염(Non-alcoholic hepatitis, NASH)은 심각한 알코올 섭취 문제를 가지지 않은 인구에서 고지방 및 고칼로리 식이섭취 습관을 통해 중등도 지방간에서 비가역적인 간 손상 상태로 이행되는 중요한 중간 단계이다. 다만, 현재까지 비침습적인 방법으로 NASH를 진단할 수 없기에 지방간이 발생한 단계에서부터 식이 조절과 함께 장기간 안전하게 복용 가능한 NASH 예방 및 치료용 건강기능식품 또는 의약품의 개발이 필요한 상태이다. Unlike simple steatosis, non-alcoholic steatohepatitis shows pathological findings such as ballooning degeneration, cell death, and inflammatory infiltration, and when non-alcoholic steatohepatitis worsens, it may show fibrosis such as collagen accumulation. Non-alcoholic hepatitis (NASH) is an important intermediate stage in the transition from moderate fatty liver to irreversible liver damage through high-fat and high-calorie dietary habits in populations without serious alcohol consumption problems. However, since NASH cannot be diagnosed through non-invasive methods to date, there is a need to develop health functional foods or medicines for the prevention and treatment of NASH that can be safely taken for a long time along with dietary control from the stage of fatty liver development.
또한, 비만은 일반적으로 체내에 지방조직이 과다한 상태인 것을 의미하며, 음식물로 섭취한 에너지가 신체활동 등으로 소비한 에너지와 균형을 이루지 못하여 잉여의 에너지가 체지방으로 축적되는 현상이다. 오랜 기간에 걸쳐 에너지 소비량에 비해 영양소를 과다 섭취할 경우 에너지 불균형에 의해 비만이 유발된다. 유전적으로 특정 유전자의 돌연변이에 의해 식욕 조절 중추 기능에 문제가 있거나, 쿠싱증후군과 같은 내분비 질환, 식욕을 증가시키는 다양한 약제에 의해 발생하는 경우도 있으나, 일반적으로는 에너지 섭취량이 에너지 소비량보다 커서 발생한다. 일반적인 비만의 경우 유전적 영향 및 환경적 영향이 복합적으로 작용하여 발생한다. 특히 칼로리가 높은 식품이 풍부하고 신체 활동을 덜 해도 사는데 불편이 없는 현대의 생활환경이 비만의 폭발적 증가를 초래하고 있다. 따라서, 지방 축적을 억제할 수 있으면서, 인체에 안전성이 입증된 천연물질을 이용한 비만 치료제의 개발 역시 필요한 상태이다. Additionally, obesity generally refers to a state of excessive fatty tissue in the body, and is a phenomenon in which the energy consumed through food is not balanced with the energy consumed through physical activity, and the excess energy is accumulated as body fat. Obesity is caused by energy imbalance when nutrients are consumed excessively compared to energy consumption over a long period of time. It may be caused by a genetic problem in the central appetite control function due to a mutation in a specific gene, an endocrine disease such as Cushing's syndrome, or various medications that increase appetite. However, it is generally caused by energy intake being greater than energy expenditure. . In the case of general obesity, it is caused by a combination of genetic and environmental influences. In particular, the modern living environment, where high-calorie foods are abundant and people can live comfortably even with less physical activity, is causing an explosive increase in obesity. Therefore, there is a need to develop a treatment for obesity using natural substances that can suppress fat accumulation and have proven safety in the human body.
본 발명에서는 결명 새싹 및 메밀 새싹을 포함하는 혼합조성물이 지방전구세포의 지방생성을 억제하고, 복부에 축적되는 복부 지방과 지방의 과도한 축적으로 증가되는 간의 중량을 현저하게 감소시킴으로써 체내에 지방 축적을 효과적으로 억제하는 것을 확인하였다. 또한 NASH 유도 실험동물에서 간 조직의 세포 손상을 억제하며, 다양한 염증 및 세포사멸 관련 인자들을 효율적으로 억제하는 것이 관찰되어 NASH 질환의 특이적인 병리기전에 유효한 것을 확인하고 본 발명을 완성하였다. In the present invention, a mixed composition containing stem sprouts and buckwheat sprouts inhibits lipogenesis in preadipocytes and significantly reduces abdominal fat accumulated in the abdomen and liver weight increased due to excessive accumulation of fat, thereby preventing fat accumulation in the body. Effective inhibition was confirmed. In addition, it was observed to suppress cell damage in liver tissue in NASH-induced experimental animals and to efficiently suppress various inflammation- and apoptosis-related factors, confirming that it is effective in the specific pathological mechanism of NASH disease, and completing the present invention.
상기한 목적을 달성하기 위하여, 본 발명의 목적은 결명 새싹 및 메밀 새싹을 포함하는, 지방 축적을 억제하기 위한 혼합조성물을 제공하는 것이다. In order to achieve the above-described object, the object of the present invention is to provide a mixed composition for inhibiting fat accumulation, comprising stem sprouts and buckwheat sprouts.
본 발명의 다른 목적은 상기 혼합조성물을 유효성분으로 포함하는, 비만 또는 비알코올성 지방간 질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or non-alcoholic fatty liver disease, comprising the mixed composition as an active ingredient.
본 발명의 또 다른 목적은 상기 혼합조성물을 유효성분으로 포함하는, 비만 또는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a food composition for preventing or improving obesity or non-alcoholic fatty liver disease, comprising the mixed composition as an active ingredient.
이하 본 명세서에 대하여 더욱 상세히 설명한다.Hereinafter, this specification will be described in more detail.
본 발명에서 개시된 각각의 설명 및 실시형태는 각각에 대한 다른 설명 및 실시형태에도 적용될 수 있다. 즉, 본 발명에 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기에 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description set forth below.
본 명세서에서 사용되는 「포함하는」과 같은 표현은, 해당 표현이 포함되는 문구 또는 문장에서 특별히 다르게 언급되지 않는 한, 다른 실시예를 포함할 가능성을 내포하는 개방형 용어(open-ended terms)로 이해되어야 한다.Expressions such as “comprising” used in this specification are understood as open-ended terms that imply the possibility of including other embodiments, unless specifically stated otherwise in the phrase or sentence containing the expression. It has to be.
본 발명의 설명 및 청구범위에서 사용된 용어나 단어는, 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선을 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.Terms or words used in the description and claims of the present invention should not be construed as limited to their ordinary or dictionary meanings, and the inventor should appropriately use the concept of the term to explain his or her invention in the best way. Based on the principle of definability, it must be interpreted with meaning and concept consistent with the technical idea of the present invention.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 결명 새싹 및 메밀 새싹을 포함하는, 지방 축적을 억제하기 위한 혼합조성물을 제공한다.As one aspect for achieving the above object, the present invention provides a mixed composition for suppressing fat accumulation, including shoots of the shoot and buckwheat sprouts.
본 발명의 용어 "결명"은 학명은 Senna Tora으로 결명 새싹(sprout)은 결명의 어린 새싹의 추출물로, 결명의 종자가 발아하여 2일 내지 31일 동안 자연 생장하였거나 재배한 새싹 또는 어린 식물체일 수 있으며, 상기 새싹은 씨앗을 발아시켜 단기간에 재배한 것으로, 실내에서 종자를 발아시켜 재배한 것일 수 있고, 또는 일반적으로 판매되는 새싹인 것일 수 있다.The term "salmon" in the present invention refers to the scientific name Senna Tora. Sprout is an extract of young shoots of a shoot. The seed of the shoot is germinated and grows naturally for 2 to 31 days, or it can be a cultivated sprout or young plant. The sprouts may be those grown in a short period of time by germinating seeds, and may be grown by germinating seeds indoors, or may be sprouts that are generally sold.
상기 결명 새싹 또는 어린 식물체는 결명 새싹 또는 어린 식물체의 전체 또는 부분일 수 있다. 상기 부분은 전초 또는 지상부 또는 지하부일 수 있다. 상기 지상부는 줄기, 잎, 꽃 또는 이들의 조합일 수 있다. 상기 지하부는 뿌리일 수 있다. The shoot or young plant may be the whole or part of the shoot or young plant. The portion may be an outpost or an above-ground portion or an underground portion. The above-ground part may be a stem, leaf, flower, or a combination thereof. The underground part may be the root.
상기 식물체는 자연적으로 생장하였거나 또는 인위적으로 재배된 것일 수 있다. 본 발명의 결명 새싹은 실내에서 용이하게 재배할 수 있으며 별도의 영양분 공급 없이 수 주 이내의 단기간에 재배하여 사용할 수 있으므로 산업적 활용성이 크다는 장점을 가진다.The plant may have grown naturally or may have been artificially cultivated. The shoots of the present invention can be easily grown indoors and have the advantage of great industrial utility because they can be grown and used in a short period of time, within a few weeks, without additional nutrient supply.
본 발명의 용어 "메밀"은 학명이 Fagopyrum Tataricum으로 메밀 새싹(sprout)은 메밀의 어린 새싹의 추출물로, 메밀의 종자가 발아하여 2일 내지 31일 동안 자연 생장하였거나 재배한 새싹 또는 어린 식물체일 수 있으며, 상기 새싹은 씨앗을 발아시켜 단기간에 재배한 것으로, 실내에서 종자를 발아시켜 재배한 것일 수 있고, 또는 일반적으로 판매되는 새싹인 것일 수 있다.The term "buckwheat" in the present invention has the scientific name Fagopyrum Tataricum . Buckwheat sprouts are an extract of young sprouts of buckwheat. Buckwheat seeds are germinated and grown naturally for 2 to 31 days, or they can be cultivated sprouts or young plants. The sprouts may be those grown in a short period of time by germinating seeds, and may be grown by germinating seeds indoors, or may be sprouts that are generally sold.
상기 메밀 새싹 또는 어린 식물체는 메밀 새싹 또는 어린 식물체의 전체 또는 부분일 수 있다. 상기 부분은 전초 또는 지상부 또는 지하부일 수 있다. 상기 지상부는 줄기, 잎, 꽃 또는 이들의 조합일 수 있다. 상기 지하부는 뿌리일 수 있다. The buckwheat sprouts or young plants may be the whole or part of the buckwheat sprouts or young plants. The portion may be an outpost or an above-ground portion or an underground portion. The above-ground part may be a stem, leaf, flower, or a combination thereof. The underground part may be the root.
상기 식물체는 자연적으로 생장하였거나 또는 인위적으로 재배된 것일 수 있다. 본 발명의 메밀 새싹은 실내에서 용이하게 재배할 수 있으며 별도의 영양분 공급 없이 수 주 이내의 단기간에 재배하여 사용할 수 있으므로 산업적 활용성이 크다는 장점을 가진다.The plant may have grown naturally or may have been artificially cultivated. The buckwheat sprouts of the present invention can be easily grown indoors and have the advantage of great industrial utility because they can be grown and used in a short period of time, within a few weeks, without additional nutrient supply.
본 발명에서 상기 혼합조성물은 결명 새싹 추출물과 메밀 새싹 추출물이 1:0.5 내지 1:2의 중량비로 혼합되거나 또는 결명 새싹과 메밀 새싹이 1:0.5 내지 1:2의 중량비로 혼합된 혼합물의 추출물일 수 있다.In the present invention, the mixed composition is an extract of a mixture in which the shoot extract and buckwheat sprout extract are mixed at a weight ratio of 1:0.5 to 1:2, or the shoot shoot and buckwheat sprout are mixed at a weight ratio of 1:0.5 to 1:2. You can.
구체적으로, 상기 혼합조성물은 결명 새싹 추출물과 메밀 새싹 추출물이 1:1의 중량비로 혼합되거나 또는 결명 새싹과 메밀 새싹이 1:1의 중량비로 혼합된 혼합물의 추출물일 수 있다.Specifically, the mixed composition may be an extract of a mixture of shoot extract and buckwheat sprout extract mixed at a weight ratio of 1:1 or a mixture of shoot extract and buckwheat sprout mixed at a weight ratio of 1:1.
상기 결명 또는 메밀 새싹은 물, C1-C4의 알코올 또는 이들의 혼합용매로 환류 또는 초음파 추출한 것일 수 있다. 상기 물은 증류수(distilled water, DW)일 수 있고, 상기 알코올은 에탄올일 수 있으며, 구체적으로 30%(v/v) 내지 100%(v/v) 에탄올일 수 있다.The shoot or buckwheat sprouts may be refluxed or ultrasonically extracted with water, C 1 -C 4 alcohol, or a mixed solvent thereof. The water may be distilled water (DW), and the alcohol may be ethanol, specifically 30% (v/v) to 100% (v/v) ethanol.
상기 추출물은 결명 새싹, 메밀 새싹의 환류 추출물 또는 초음파 추출물일 수 있으며, 또는 결명 새싹과 메밀 새싹이 1:1의 중량비로 혼합된 혼합물의 환류 또는 초음파 추출물일 수 있다. The extract may be a reflux extract or ultrasonic extract of shoot shoots or buckwheat sprouts, or may be a reflux or ultrasonic extract of a mixture of shoot shoots and buckwheat sprouts in a weight ratio of 1:1.
구체적으로, 상기 결명 새싹의 환류 추출물은, 결명 새싹을 에탄올을 포함하는 제1수용액에 분산시켜 제1분산액을 형성하는 제1분산단계; 및 상기 제1분산액을 환류 냉각 추출을 하여 결명 새싹의 환류 냉각 추출물을 추출하는 제1추출단계; 로 추출될 수 있다. 상기 에탄올을 포함하는 제1수용액은, 에탄올이 전체 수용액 대비 30%(v/v) 내지 100%(v/v)일 수 있다. 상기 제1추출단계는, 1 내지 4시간 동안 환류 냉각 추출을 진행하여 결명 새싹 추출물을 얻을 수 있다. Specifically, the refluxed extract of the shoots of the shoots includes a first dispersion step of dispersing the shoots of the shoots in a first aqueous solution containing ethanol to form a first dispersion; And a first extraction step of extracting the reflux-cooled extract of the shoot shoot by subjecting the first dispersion to reflux-cooled extraction; It can be extracted as The first aqueous solution containing ethanol may contain 30% (v/v) to 100% (v/v) of ethanol based on the total aqueous solution. In the first extraction step, reflux cooling extraction may be performed for 1 to 4 hours to obtain a shoot extract.
상기 메밀 새싹의 환류 추출물은, 상기 메밀 새싹을 에탄올을 포함하는 제2수용액에 분산시켜 제2분산액을 형성하는 제2분산단계; 및 상기 제2분산액을 환류 냉각 추출을 하여 메밀 새싹의 환류 냉각 추출물을 추출하는 제2추출단계;로 추출될 수 있다. 상기 에탄올을 포함하는 제2수용액은, 에탄올이 전체 수용액 대비 30%(v/v) 내지 100%(v/v)일 수 있다. 상기 제2추출단계는, 1 내지 4시간 동안 환류 냉각 추출을 진행하여 메밀 새싹 추출물을 얻을 수 있다. The refluxed extract of the buckwheat sprouts includes a second dispersion step of dispersing the buckwheat sprouts in a second aqueous solution containing ethanol to form a second dispersion; And a second extraction step of extracting the reflux-cooled extract of buckwheat sprouts by subjecting the second dispersion to reflux-cooled extraction. The second aqueous solution containing ethanol may contain 30% (v/v) to 100% (v/v) of ethanol based on the total aqueous solution. In the second extraction step, buckwheat sprout extract can be obtained by performing reflux cooling extraction for 1 to 4 hours.
상기 혼합조성물은 상기와 같은 과정에 의해 얻은 결명 새싹의 환류 추출물과 메밀 새싹의 환류 추출물을 1:0.5 내지 1:2의 중량비로 혼합할 수 있다. 구체적으로 상기와 같은 분산단계 및 추출단계로 추출된 결명 새싹의 환류 냉각 추출물과 메밀 새싹의 환류 냉각 추출물을 1:0.5 내지 1:2의 중량비, 보다 구체적으로, 1:1의 중량비로 혼합하여 제조할 수 있다.The mixed composition may be a mixture of the refluxed extract of buckwheat sprouts obtained through the above process and the refluxed extract of buckwheat sprouts at a weight ratio of 1:0.5 to 1:2. Specifically, it is prepared by mixing the reflux-cooled extract of the shoots and the reflux-cooled extract of the buckwheat sprouts extracted through the dispersion and extraction steps as described above at a weight ratio of 1:0.5 to 1:2, more specifically, a weight ratio of 1:1. can do.
또한, 상기 결명 새싹의 초음파 추출물은, 결명 새싹에 물 또는 알코올을 가한 후 초음파 수조에서 초음파 추출하여 결명 새싹의 초음파 추출물을 제조할 수 있다. 상기 물은 증류수(distilled water, DW)일 수 있고, 상기 알코올은 에탄올일 수 있으며, 구체적으로 30%(v/v) 내지 100%(v/v) 에탄올일 수 있다.In addition, the ultrasonic extract of the shoots can be prepared by adding water or alcohol to the shoots and then performing ultrasonic extraction in an ultrasonic water bath. The water may be distilled water (DW), and the alcohol may be ethanol, specifically 30% (v/v) to 100% (v/v) ethanol.
또한, 상기 메밀 새싹의 초음파 추출물은, 메밀 새싹에 물 또는 알코올을 가한 후 초음파 수조에서 초음파 추출하여 메밀 새싹의 초음파 추출물을 제조할 수 있다. 상기 물은 증류수(distilled water, DW)일 수 있고, 상기 알코올은 에탄올일 수 있으며, 구체적으로 30%(v/v) 내지 100%(v/v) 에탄올일 수 있다.In addition, the ultrasonic extract of buckwheat sprouts can be prepared by adding water or alcohol to buckwheat sprouts and then performing ultrasonic extraction in an ultrasonic water bath. The water may be distilled water (DW), and the alcohol may be ethanol, specifically 30% (v/v) to 100% (v/v) ethanol.
이후, 상기 제조된 결명 새싹의 초음파 추출물과 메밀 새싹의 초음파 추출물을 1:0.5 내지 1:2의 중량비, 보다 구체적으로, 1:1의 중량비로 혼합하여 혼합조성물을 제조할 수 있다.Thereafter, a mixed composition can be prepared by mixing the prepared ultrasonic extract of the shoot and the ultrasonic extract of the buckwheat sprout at a weight ratio of 1:0.5 to 1:2, more specifically, 1:1.
또한, 상기 혼합조성물은 결명 새싹과 메밀 새싹이 1:0.5 내지 1:2의 중량비로 혼합된 혼합물의 추출물일 수 있다.Additionally, the mixed composition may be an extract of a mixture of milkweed sprouts and buckwheat sprouts mixed at a weight ratio of 1:0.5 to 1:2.
상기 혼합조성물은 상기 결명 새싹과 메밀 새싹을 1:0.5 내지 1:2의 중량비로 혼합하고, 물 또는 알코올을 가한 후 초음파 수조에서 초음파 추출하여 제조할 수 있다. 상기 물은 증류수(distilled water, DW)일 수 있고, 상기 알코올은 에탄올일 수 있으며, 구체적으로 30%(v/v) 내지 100%(v/v) 에탄올일 수 있다.The mixed composition can be prepared by mixing the shoots and buckwheat sprouts at a weight ratio of 1:0.5 to 1:2, adding water or alcohol, and then performing ultrasonic extraction in an ultrasonic water bath. The water may be distilled water (DW), and the alcohol may be ethanol, specifically 30% (v/v) to 100% (v/v) ethanol.
상기 결명 새싹의 환류 추출물은 Aurantio obtusin을 포함하며, 결명 새싹의 환류 추출물의 건조 중량 대비 0.02~0.07 중량%을 포함한다. The refluxed extract of the shoots of the shoots contains Aurantio obtusin, and contains 0.02 to 0.07% by weight based on the dry weight of the refluxed extracts of the shoots of the shoots.
상기 메밀 새싹의 환류 추출물은 Rutin을 포함하며, 메밀 새싹의 환류 추출물의 건조 중량 대비 3.0 내지 10 중량%을 포함한다. The refluxed extract of buckwheat sprouts contains Rutin, and contains 3.0 to 10% by weight based on the dry weight of the refluxed extract of buckwheat sprouts.
상기 결명 새싹 및 메밀 새싹을 포함하는 혼합조성물은 지방전구세포의 지방생성을 억제하고, 지방 축적을 억제하는 효과를 가진다. 복부에 축적되는 복부 지방과 지방의 과도한 축적으로 증가되는 간의 중량을 현저하게 감소시킴으로써 체내에 지방이 축적되는 것을 억제한다. 또한, 간 조직 내 지방 축적을 억제하는 효과가 있다.The mixed composition containing the chestnut sprouts and buckwheat sprouts has the effect of inhibiting lipogenesis in preadipocytes and inhibiting fat accumulation. It suppresses the accumulation of fat in the body by significantly reducing abdominal fat accumulated in the abdomen and the weight of the liver that increases due to excessive accumulation of fat. Additionally, it has the effect of suppressing fat accumulation in liver tissue.
다른 하나의 양태로, 본 발명은 상기 혼합조성물을 유효성분으로 포함하는, 비만 또는 비알코올성 지방간 질환의 예방 또는 치료용 약학 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for preventing or treating obesity or non-alcoholic fatty liver disease, comprising the mixed composition as an active ingredient.
상기 혼합조성물은 결명 새싹 추출물과 메밀 새싹 추출물이 1:0.5 내지 1:2의 중량비로 혼합되거나 또는 결명 새싹과 메밀 새싹이 1:0.5 내지 1:2의 중량비로 혼합된 혼합물의 추출물일 수 있다.The mixed composition may be an extract of a mixture of shoot extract and buckwheat sprout extract mixed at a weight ratio of 1:0.5 to 1:2, or a mixture of shoot shoot and buckwheat sprout extracted at a weight ratio of 1:0.5 to 1:2.
본 발명에서 용어 "결명 새싹", "결명 새싹 추출물", "메밀 새싹", "메밀 새싹 추출물", "혼합조성물"에 대한 설명은 전술한 바와 같다. In the present invention, the descriptions of the terms “barbecue sprout,” “barbecue sprout extract,” “buckwheat sprout,” “buckwheat sprout extract,” and “mixed composition” are as described above.
본 발명에서 상기 비만은 일반적으로 체내에 지방조직이 과다한 상태인 것을 의미하며, 음식물로 섭취한 에너지가 신체활동 등으로 소비한 에너지와 균형을 이루지 못하여 잉여의 에너지가 체지방으로 축적되는 현상이다.In the present invention, obesity generally refers to a state in which there is excessive adipose tissue in the body, and is a phenomenon in which the energy consumed through food is not balanced with the energy consumed through physical activities, and the excess energy is accumulated as body fat.
본 발명에서 상기 비알코올성 지방간 질환(Nonalcoholic Fatty Liver Disease, NAFLD)은 단순 지방증(simple steatosis), 또는 비알코올성 지방간염(non-alcoholic steatohepatitis, NASH)일 수 있다. In the present invention, the nonalcoholic fatty liver disease (NAFLD) may be simple steatosis or non-alcoholic steatohepatitis (NASH).
상기 단순 지방증은 지방성 간질환 중 간세포의 지방 침착만이 보이고, 간세포의 괴사·염증이나 섬유화를 수반하지 않는 증세를 나타낸다.The simple steatosis is a type of fatty liver disease in which only fat deposition in hepatocytes is visible and is not accompanied by necrosis, inflammation, or fibrosis of hepatocytes.
상기 비알코올성 지방간염은 단순 지방증이 심화된 질병의 유형으로서, 지방간에 의한 간세포의 염증이 유발된 상태를 의미한다. 이는 알코올성 간 질환과 조직학적으로 매우 유사하나, 알코올을 거의 또는 전혀 마시지 않는 음주력이 없는 사람들에서 발생한 경우는 NASH로 정의하며, 간에서의 비정상적 지방 축적 또는 침적(간 지방증), 간 염증(간염), 간 손상이 이를 특징화하고, 대사 기원의 다른 간 질환과 구별한다. 초기 단계에서 지방 축적만 관찰된 경우는 비알코올성 지방간, 또는 단순 지방증으로 진단되나, 이에 발전되어 간엽 염증, 간세포 손상을 동반하는 경우 NASH로 구분된다. The non-alcoholic steatohepatitis is a type of disease in which simple steatosis becomes more severe and refers to a state in which inflammation of hepatocytes is induced by fatty liver. It is histologically very similar to alcoholic liver disease, but when it occurs in non-drinking people who drink little or no alcohol, it is defined as NASH, which is characterized by abnormal accumulation or deposition of fat in the liver (hepatic steatosis) and liver inflammation (hepatitis). , liver damage characterizes it and distinguishes it from other liver diseases of metabolic origin. If only fat accumulation is observed in the early stages, it is diagnosed as non-alcoholic fatty liver disease or simple steatosis, but if it progresses and is accompanied by mesenchymal inflammation and liver cell damage, it is classified as NASH.
또한 본 발명에서 "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다. In addition, in the present invention, “active ingredient” refers to an ingredient that can exhibit the desired activity alone or in combination with a carrier that is not active on its own.
상기 약학 조성물은, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredients described above. The carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 상기 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 구체적으로, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하지만, 이에 제한되는 것은 아니다. 이러한 고형제제는 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제 등도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods. there is. Specifically, when formulating, it may be prepared using commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition to oral liquid and liquid paraffin, it can be prepared by adding various excipients, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.
본 발명의 상기 약학 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) according to the desired method, and the dosage is determined by the patient's condition and weight, and the severity of the disease. It varies depending on the degree, drug form, administration route and time, but can be appropriately selected by a person skilled in the art.
본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어, "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 예를 들면, 상기 결명 새싹 추출물 및 메밀 새싹 추출물은 유효성분으로 1일 0.001 내지 500 mg/kg으로, 구체적으로 0.1 내지 100 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한 번 또는 수회 나누어 투여할 수도 있다. 또한, 본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 결명 새싹 추출물 및 메밀 새싹 추출물을 0.001 내지 50 중량 %로 포함할 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and drug. activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the field of medicine. For example, the chestnut sprout extract and buckwheat sprout extract can be administered as active ingredients at a dose of 0.001 to 500 mg/kg per day, specifically 0.1 to 100 mg/kg, and the administration is once a day or It may be administered in several divided doses. Additionally, the pharmaceutical composition of the present invention may include 0.001 to 50% by weight of the Cassia shoot extract and buckwheat sprout extract based on the total weight of the composition.
또 다른 하나의 양태로, 본 발명은 상기 혼합조성물을 유효성분으로 포함하는, 비만 또는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물을 제공한다.In another aspect, the present invention provides a food composition for preventing or improving obesity or non-alcoholic fatty liver disease, comprising the mixed composition as an active ingredient.
상기 혼합조성물은 결명 새싹 추출물과 메밀 새싹 추출물이 1:0.5 내지 1:2의 중량비로 혼합되거나 또는 결명 새싹과 메밀 새싹이 1:0.5 내지 1:2의 중량비로 혼합된 혼합물의 추출물일 수 있다.The mixed composition may be an extract of a mixture of shoot extract and buckwheat sprout extract mixed at a weight ratio of 1:0.5 to 1:2, or a mixture of shoot shoot and buckwheat sprout extracted at a weight ratio of 1:0.5 to 1:2.
본 발명에서 용어 "결명 새싹", "결명 새싹 추출물", "메밀 새싹", "메밀 새싹 추출물", "혼합조성물", "비만", "비알코올성 지방간 질환"에 대한 설명은 전술한 바와 같다. In the present invention, the terms "Buckwheat sprout", "Buckwheat sprout extract", "Buckwheat sprout", "Buckwheat sprout extract", "Mixed composition", "Obesity", and "Non-alcoholic fatty liver disease" are as described above.
본 발명에서 상기 비알코올성 지방간 질환(Nonalcoholic Fatty Liver Disease, NAFLD)은 단순 지방증(simple steatosis), 또는 비알코올성 지방간염(non-alcoholic steatohepatitis, NASH)일 수 있다. In the present invention, the nonalcoholic fatty liver disease (NAFLD) may be simple steatosis or non-alcoholic steatohepatitis (NASH).
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함할 수 있으며, 본 발명의 결명 새싹 추출물 및 메밀 새싹 추출물을 첨가할 수 있는 식품의 종류에는 별다른 제한이 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있다.The food composition of the present invention may include the form of pills, powders, granules, steeps, tablets, capsules or liquids, and there are no particular restrictions on the types of foods to which the Cassia shoot extract and buckwheat sprout extract of the present invention can be added. does not exist. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes.
상기 식품 조성물에는 결명 새싹 추출물 및 메밀 새싹 추출물 이외에도 다른 성분을 추가할 수 있으며, 그 종류는 특별히 제한되지 않는다. 예를 들어, 통상의 식품과 같이 여러 가지 생약 추출물, 식품학적으로 허용되는 식품보조첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있으며, 이에 제한되지 않는다.Other ingredients may be added to the food composition in addition to the buckwheat sprout extract and the buckwheat sprout extract, and the types are not particularly limited. For example, like regular foods, it may contain various herbal extracts, foodologically acceptable food additives, or natural carbohydrates as additional ingredients, but is not limited thereto.
본 발명에서 용어, "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.In the present invention, the term "food supplement" refers to a component that can be added as an auxiliary food additive, and can be appropriately selected and used by a person skilled in the art as it is added to prepare each type of food. Examples of food supplements include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplements of the present invention are not limited to the above examples.
상기 천연 탄수화물의 예는 포도당, 과당 등의 단당류; 말토스, 수크로스 등의 이당류; 및 덱스트린, 시클로덱스트린 등의 다당류와, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 있으며, 상기한 것 이외의 향미제로서 천연 향미제(타우마틴 등), 스테비아 추출물(레바우디오시드 Ａ, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Flavoring agents other than those mentioned above include natural flavoring agents (such as thaumatin) and stevia extract (rebaudioside A, glycyrrhizin). hygin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be used advantageously.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에서 사용된 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The food composition of the present invention may include health functional foods. The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, functionality means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.
유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 유효성분은 원료 조성물 중 0.01 내지 50 중량 %, 바람직하게는 0.1 내지 10 중량 %의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food, the active ingredient of the present invention may be added in an amount of 0.01 to 50% by weight, preferably 0.1 to 10% by weight, of the raw material composition, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be used even below the above range.
본 발명은 결명 새싹 및 메밀 새싹을 포함하는, 지방 축적을 억제하기 위한 혼합조성물 및 이의 용도에 관한 것이다. The present invention relates to a mixed composition for inhibiting fat accumulation, comprising chestnut sprouts and buckwheat sprouts, and its use.
지방전구세포의 지방생성을 억제하고, 복부에 축적되는 복부 지방과 지방의 과도한 축적으로 증가되는 간의 중량을 현저하게 감소시킴으로써 체내에 지방 축적을 효과적으로 억제하는 효과가 있다. 또한 NASH 유도 실험동물에서 간 조직의 세포 손상을 억제하며, 다양한 염증 및 세포사멸 관련 인자들을 효율적으로 억제하는 것이 관찰되어 NASH 질환의 특이적인 병리기전에 유효하다.It has the effect of effectively suppressing fat accumulation in the body by suppressing fat production in preadipocytes and significantly reducing abdominal fat accumulated in the abdomen and liver weight increased due to excessive accumulation of fat. In addition, it was observed to suppress cell damage in liver tissue in NASH-induced experimental animals and to efficiently suppress various inflammation- and apoptosis-related factors, making it effective in the specific pathogenesis of NASH disease.
도 1은 실험동물들의 6개월 동안의 체중 측정 결과를 나타낸다.
도 2는 실험동물들의 복부지방 및 간 중량 측정 결과를 나타낸다.
도 3은 실험동물들의 혈장 내 지질 및 간 손상 지표 측정 결과를 나타낸다.
도 4는 간 조직내 NASH 질환 기전 관련 인자 단백질 발현량 측정 결과를 나타낸다.
도 5는 간 조직내 NASH 질환 기전 관련 인자 단백질 발현량을 정량적으로 나타낸 결과이다.
도 6은 조직 분석(Histological analysis)를 통한 간 조직에 지방이 축적되는 정도를 평가한 결과이다.
도 7은 3T3-L1 세포에서 결명 새싹과 메밀 새싹의 최적 용매 조건 및 혼합 조건에 따른 지방 축적 억제 효능을 나타낸 것이다.
도 8은 3T3-L1 세포에서 다양한 천연 식물, 또는 이들을 혼합한 혼합물을 DW를 이용하여 추출한 초음파 추출물의 지방 축적 억제 효능을 비교한 것이다.Figure 1 shows the results of measuring body weight of experimental animals for 6 months.
Figure 2 shows the results of measuring abdominal fat and liver weight of experimental animals.
Figure 3 shows the results of measuring lipids and liver damage indicators in the plasma of experimental animals.
Figure 4 shows the results of measuring protein expression levels of factors related to the NASH disease mechanism in liver tissue.
Figure 5 is a quantitative result showing the protein expression level of factors related to the NASH disease mechanism in liver tissue.
Figure 6 shows the results of evaluating the degree of fat accumulation in liver tissue through histological analysis.
Figure 7 shows the efficacy of suppressing fat accumulation in 3T3-L1 cells according to the optimal solvent conditions and mixing conditions of buckwheat sprouts and buckwheat sprouts.
Figure 8 compares the fat accumulation inhibition efficacy of ultrasonic extracts extracted from various natural plants or mixtures thereof using DW in 3T3-L1 cells.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 첨부한 도면을 참고로 하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.
[A. 결명 새싹과 메밀 새싹 환류 추출물의 제조 및 효능 분석][A. Preparation and efficacy analysis of refluxed extracts of sesame sprouts and buckwheat sprouts]
실시예 1. 결명 새싹의 환류 추출물의 제조Example 1. Preparation of reflux extract of Foliage shoots
1-1. 30%(v/v) 에탄올을 이용한 결명 새싹 환류 추출물 제조1-1. Preparation of refluxed fruit shoot extract using 30% (v/v) ethanol
결명(Senna Tora) 새싹을 건조시킨 다음, 5g을 저울에 달아 50mL의 30%(v/v) 에탄올로 3시간 동안 1회 환류 냉각 추출하였다. 상기 추출액을 거름종이로 여과하여 얻어진 여과액을 35℃에서 감압 농축하여 추출물 약 700mg을 얻었다.After drying Senna Tora sprouts, 5 g was weighed on a scale and extracted with 50 mL of 30% (v/v) ethanol once by refluxing for 3 hours. The extract was filtered through filter paper, and the filtrate was concentrated under reduced pressure at 35°C to obtain about 700 mg of extract.
1-2. 100%(v/v) 에탄올을 이용한 결명 새싹 환류 추출물 제조1-2. Preparation of refluxed fruit shoot extract using 100% (v/v) ethanol
100%(v/v) 에탄올을 사용하여 추출하는 것을 제외하고, 실시예 1-1과 동일하게 결명 새싹 환류 추출물을 제조하였다. A reflux extract of F. fern shoot was prepared in the same manner as in Example 1-1, except that extraction was performed using 100% (v/v) ethanol.
실시예 2. 메밀 새싹의 환류 추출물의 제조Example 2. Preparation of reflux extract of buckwheat sprouts
2-1. 30%(v/v) 에탄올을 이용한 메밀 새싹 환류 추출물 제조2-1. Preparation of buckwheat sprout reflux extract using 30% (v/v) ethanol
메밀(Fagopyrum Tataricum)새싹을 건조시킨 다음, 5g을 저울에 달아 50mL의 30%(v/v) 에탄올로 1시간 동안 1회 환류 냉각 추출하였다. 결명 새싹 추출물과 같은 방법으로 여과, 감압 농축하여 추출물 약 226mg을 얻었다.Buckwheat ( Fagopyrum Tataricum ) sprouts were dried, then 5 g was weighed on a scale and extracted with 50 mL of 30% (v/v) ethanol by refluxing once for 1 hour. About 226 mg of extract was obtained by filtering and concentrating under reduced pressure in the same manner as the shoot extract.
2-2. 50%(v/v) 에탄올을 이용한 메밀 새싹 환류 추출물 제조2-2. Preparation of buckwheat sprout reflux extract using 50% (v/v) ethanol
50%(v/v) 에탄올을 사용하여 추출하는 것을 제외하고, 실시예 2-1과 동일하게 메밀 새싹 환류 추출물을 제조하였다. Buckwheat sprout reflux extract was prepared in the same manner as Example 2-1, except that extraction was performed using 50% (v/v) ethanol.
2-3. 70%(v/v) 에탄올을 이용한 메밀 새싹 환류 추출물 제조2-3. Preparation of buckwheat sprout reflux extract using 70% (v/v) ethanol
70%(v/v) 에탄올을 사용하여 추출하는 것을 제외하고, 실시예 2-1과 동일하게 메밀 새싹 환류 추출물을 제조하였다. Buckwheat sprout reflux extract was prepared in the same manner as Example 2-1, except that extraction was performed using 70% (v/v) ethanol.
2-4. 100%(v/v) 에탄올을 이용한 메밀 새싹 환류 추출물 제조2-4. Preparation of buckwheat sprout reflux extract using 100% (v/v) ethanol
100%(v/v) 에탄올을 사용하여 추출하는 것을 제외하고, 실시예 2-1과 동일하게 메밀 새싹 환류 추출물을 제조하였다. Buckwheat sprout reflux extract was prepared in the same manner as Example 2-1, except that extraction was performed using 100% (v/v) ethanol.
실시예 3. 결명 새싹 추출물과 메밀 새싹 추출물의 혼합조성물 제조Example 3. Preparation of a mixed composition of Buckwheat sprout extract and buckwheat sprout extract
상기 실시예 1-1에서 제조된 결명 새싹 추출물 50mg을 저울에 달아 2mL의 30%(v/v) 에탄올을 첨가하여 "결명 새싹추출물 용해액"을 제조하고, 상기 실시예 2-1에서 제조된 메밀 새싹 추출물 50mg을 저울에 달아 2mL의 30%(v/v) 에탄올을 첨가하여 "메밀 새싹 추출물 용해액"을 제조한 뒤 결명 새싹 추출물의 용해액 1mL과 메밀 새싹 추출물의 용해액 1mL을 혼합하여 회전 감압 농축하여 30mg의 최종 조성물을 제조하였다.Weigh 50 mg of the Cassia shoot extract prepared in Example 1-1 on a scale and add 2 mL of 30% (v/v) ethanol to prepare a “Castle shoot extract solution” prepared in Example 2-1. Weigh 50 mg of buckwheat sprout extract on a scale and add 2 mL of 30% (v/v) ethanol to prepare “buckwheat sprout extract solution”, then mix 1 mL of buckwheat sprout extract solution with 1 mL of buckwheat sprout extract solution. 30 mg of the final composition was prepared by rotation and concentration under reduced pressure.
실험예 1. 결명 새싹 추출물의 지표성분 분석Experimental Example 1. Analysis of indicator components of Foliage sprout extract
결명 새싹 추출물의 지표성분인 Aurantio obtusin의 함량수준을 정량하기 위해, 10μL 루프를 포함하는 자동시료주입기가 장착된 Waters 2695 separation module(MA, USA) 시스템에서 분석을 수행하였다.To quantify the content level of Aurantio obtusin, an indicator component of the extract of the shoot extract, analysis was performed on a Waters 2695 separation module (MA, USA) system equipped with an autosampler containing a 10 μL loop.
크로마토그래피 분석은 5μm 직경의 입자로 채워진 C18 역상컬럼 (Cosmosil AR-2, 4.6mmХ250mm)에서 수행되었다. 용매 A(100% 아세트로니트릴)와 용매B(100% 물)의 두가지 이동상이 사용되었다. 구배 흐름은 1.0mL/min의 유속 및 30분의 구배시간에서 10:90에서 100:0(A:B 부피%)까지 사용되었다. 컬럼 온도는 30℃로 설정되었다. UV 검출파장은 280nm로 설정하였다. 그 구체적인 작동조건은 하기 표 1에 정리된 바와 같다. Chromatographic analysis was performed on a C18 reversed phase column (Cosmosil AR-2, 4.6mmХ250mm) packed with 5μm diameter particles. Two mobile phases were used: solvent A (100% acetronitrile) and solvent B (100% water). Gradient flow was used from 10:90 to 100:0 (A:B volume %) at a flow rate of 1.0 mL/min and a gradient time of 30 min. The column temperature was set at 30°C. The UV detection wavelength was set to 280 nm. The specific operating conditions are summarized in Table 1 below.
지표 성분 분석을 위한 시료는 실시예 1-1에서 제조된 30%(v/v) 에탄올을 이용하여 추출한 결명 새싹 추출물과 실시예 1-2에서 제조된 100%(v/v) 에탄올을 이용하여 추출한 결명 새싹 추출물의 지표성분(오란티오 옵투신, Aurantio obtusin) 함량을 분석하였다.Samples for indicator component analysis were the shoot extract extracted using 30% (v/v) ethanol prepared in Example 1-1 and 100% (v/v) ethanol prepared in Example 1-2. The content of indicator components (Aurantio obtusin) in the extracted shoot extract was analyzed.
결명 새싹 추출물의 Aurantio obtusin 식별은 Aurantio obtusin 표준물질 (Avention, Korea)과 비교한 머무름 시간 및 흡광 스펙트럼을 기반으로 하였다. Aurantio obtusin의 함량은 표준물질의 검량선에 의해 결정되었으며, 결명 새싹 추출물의 건조 중량에 대비하여 mg/100g의 단위로 계산하였으며, 시료의 지표성분 함량은 표 2와 같다. Identification of Aurantio obtusin from the shoot extract was based on retention time and absorbance spectrum compared with Aurantio obtusin standard (Avention, Korea). The content of Aurantio obtusin was determined by the calibration curve of the standard substance and calculated in units of mg/100g compared to the dry weight of the shoot extract. The content of the indicator component of the sample is shown in Table 2.
실험예 2. 메밀 새싹 추출물 내의 지표성분 분석Experimental Example 2. Analysis of indicator components in buckwheat sprout extract
메밀 새싹추출물의 지표성분인 Rutin 함량 수준을 정량하기 위해, 10μL 루프를 포함하는 자동시료주입기가 장착된 Waters 2695 separation module(MA, USA) 시스템에서 분석을 수행하였다.To quantify the level of rutin content, an indicator component of buckwheat sprout extract, analysis was performed on a Waters 2695 separation module (MA, USA) system equipped with an autosampler containing a 10 μL loop.
크로마토그래피 분석은 5μm 직경의 입자로 채워진 C18 역상컬럼 (Cosmosil AR-2, 4.6mmХ250mm)에서 수행되었다. 용매 A(100% 아세트로니트릴)와 용매B(100% 물)의 두가지 이동상이 사용되었다. 구배 흐름은 1.0mL/min의 유속 및 30분의 구배시간에서 10:90에서 100:0 (A:B 부피%)까지 사용되었다. 컬럼 온도는 30℃로 설정되었다. UV 검출파장은 350nm로 설정하였다. 그 구체적인 작동조건은 하기 표 3에 정리된 바와 같다.Chromatographic analysis was performed on a C18 reversed phase column (Cosmosil AR-2, 4.6mmХ250mm) packed with 5μm diameter particles. Two mobile phases were used: solvent A (100% acetronitrile) and solvent B (100% water). Gradient flow was used from 10:90 to 100:0 (A:B volume %) at a flow rate of 1.0 mL/min and a gradient time of 30 minutes. The column temperature was set at 30°C. The UV detection wavelength was set to 350 nm. The specific operating conditions are summarized in Table 3 below.
지표 성분 분석을 위한 시료는 실시예 2-1에서 제조된 30%(v/v) 에탄올을 이용하여 추출한 메밀 새싹 추출물, 실시예 2-2에서 제조된 50%(v/v) 에탄올을 이용하여 추출한 메밀 새싹 추출물, 실시예 2-3에서 제조된 70%(v/v) 에탄올을 이용하여 추출한 메밀 새싹 추출물, 실시예 2-4에서 제조된 100%(v/v) 에탄올을 이용하여 추출한 메밀 새싹 추출물로 지표성분(루틴, Rutin) 함량을 분석하였다.Samples for indicator component analysis were buckwheat sprout extract extracted using 30% (v/v) ethanol prepared in Example 2-1, and 50% (v/v) ethanol prepared in Example 2-2. Buckwheat sprout extract extracted, buckwheat sprout extract extracted using 70% (v/v) ethanol prepared in Example 2-3, buckwheat extracted using 100% (v/v) ethanol prepared in Example 2-4 The content of indicator components (rutin) was analyzed using sprout extract.
메밀 새싹 추출물의 Rutin 식별은 Rutin 표준물질(Tokyo Chemial, Japan)과 비교한 머무름 시간 및 흡광 스펙트럼을 기반으로 하였다. Rutin의 함량은 표준물질의 검량선에 의해 결정되었으며, 메밀 새싹 추출물의 건조 중량에 대비하여 mg/100g의 단위로 계산하였으며, 시료의 지표성분 함량은 표 4와 같다. Identification of Rutin in buckwheat sprout extract was based on retention time and absorption spectrum compared with Rutin standard (Tokyo Chemial, Japan). The content of rutin was determined by the calibration curve of the standard substance and was calculated in units of mg/100g compared to the dry weight of the buckwheat sprout extract, and the content of the indicator component of the sample is shown in Table 4.
실험예 3. 비알코올성 지방간염 유도 동물 모델 제조 및 시험물질 투여Experimental Example 3. Preparation of non-alcoholic steatohepatitis inducing animal model and administration of test substances
본 실험에 사용된 실험동물은 오리엔트바이오사에서 구입한 6주령의 C57BL/6 수컷 마우스로 실험군 당 10마리를 사용하였다. 총 6개월의 시험기간 동안 NASH 질환 유도군들은 FPC 특수사료(FPC diet: Fructose, Palmitate, Cholesterol & Trans-fat diet)와 과당 함유 음용수를 섭취하였으며, 대조군(Control)은 일반 사료와 일반 음용수를 섭취하였다. The experimental animals used in this experiment were 6-week-old C57BL/6 male mice purchased from Orient Bio, and 10 mice were used per experimental group. During the total 6-month test period, the NASH disease induction group consumed FPC special feed (FPC diet: Fructose, Palmitate, Cholesterol & Trans-fat diet) and fructose-containing drinking water, while the control group consumed regular feed and regular drinking water. did.
대조군(Control)은 시험물질 대신 식염수를 주 5 회 경구 투여하였다. NASH 질환유도군에서 지방간이 충분히 유도된 3개월차부터 6개월차까지 총 4개월의 기간 동안 시험물질을 주 5회 경구 투여했으며, 시험물질은 식염수에 녹여서 사용했다. 시험물질로는 실시예 3에서 제조한 메밀/결명 혼합추출물 소재(CNS01)를 50 mg/kg/10 ml 용량으로 투여하였다.The control group was orally administered saline solution 5 times a week instead of the test substance. In the NASH disease-induced group, the test substance was administered orally 5 times a week for a total of 4 months from the 3rd month to the 6th month when fatty liver was sufficiently induced, and the test substance was dissolved in saline solution and used. As a test substance, the buckwheat/columbine mixed extract material (CNS01) prepared in Example 3 was administered at a dose of 50 mg/kg/10 ml.
본 실험에서 사용된 NASH 유도 마우스 모델은 장기간(6개월)에 걸친 고지방/고열량 특수사료(FPC diet: Fructose, Palmitate, Cholesterol & Trans-fat diet) 및 과당 함유 음용수 섭취를 통해 중등도의 지방간 유도 끝에 염증과 간 석회화를 동반하는 비알코올성 지방간염(Non-alcoholic steatohepatitis, NASH)이 유도된 동물 모델이다. The NASH-induced mouse model used in this experiment suffered from inflammation after inducing moderate fatty liver disease through long-term (6 months) consumption of a high-fat/high-calorie special feed (FPC diet: Fructose, Palmitate, Cholesterol & Trans-fat diet) and fructose-containing drinking water. It is an animal model in which non-alcoholic steatohepatitis (NASH) is induced, accompanied by liver calcification.
실험예 4. 실험동물들의 체중 측정 결과Experimental Example 4. Weight measurement results of experimental animals
시험 시작일부터 매주 동물들의 체중을 측정하였고 그 결과는 도 1과 같다. 지방간 상태 유도 첫 2개월 동안 실험동물들의 체중은 꾸준히 증가하였으며 특히 FPC 특수사료와 과당 함유 음용수를 섭취한 실험동물들의 체중이 더 빠르게 증가하였다. 이후 시험물질 투여를 진행한 실험군 모두에서 FPC 특수사료와 과당 함유 음용수를 계속적으로 섭취하였음에도 불구하고 체중의 증가 추세는 감소하여 일반 사료를 섭취한 Control군과 차이가 없는 체중 상태를 유지하였다. 6개월이 지난 최종 시점에서 Control군 대비 NASH 유도군은 체중이 현저히 증가하였으나 메밀/결명 혼합추출물 소재(CNS01) 시험물질 투여군은 유의적인 체중 감소 효능을 확인할 수 있었다.The animals' weight was measured every week from the start of the test, and the results are shown in Figure 1. During the first two months of inducing fatty liver disease, the weight of the experimental animals steadily increased, and in particular, the weight of the experimental animals that consumed FPC special feed and fructose-containing drinking water increased more rapidly. Afterwards, despite the continuous consumption of FPC special feed and fructose-containing drinking water in all experimental groups administered test substances, the trend of weight gain decreased and the body weight was maintained with no difference from the control group that consumed regular feed. At the final time point of 6 months, compared to the control group, the NASH induction group significantly increased body weight, but the buckwheat/columbine mixed extract material (CNS01) test substance administration group showed significant weight loss efficacy.
실험예 5. 실험동물들의 복부지방 및 간 중량 측정 결과Experimental Example 5. Results of measuring abdominal fat and liver weight of experimental animals
시험이 종료된 후 실험동물을 희생하여 복부지방 및 간의 중량을 측정한 결과는 도 2와 같다. 체중의 증가 추세와 유사하게 복부지방 조직과 간의 무게(중량)도 NASH 유도군에서 현저하게 증가하였다. 메밀/결명 혼합추출물 소재(CNS01)를 투여한 군에서 복부지방은 Control군과 유사한 수준으로 감소되었으며, 간 중량도 Control군 보다는 증가된 수준이지만 NASH 유도군 대비 유의성 있게 감소되었다.After the test was completed, the experimental animals were sacrificed and the weight of abdominal fat and liver was measured, and the results are shown in Figure 2. Similar to the trend of increase in body weight, the weight of abdominal adipose tissue and liver also significantly increased in the NASH-induced group. In the group administered buckwheat/columbine mixed extract material (CNS01), abdominal fat was reduced to a similar level as the control group, and liver weight was also increased compared to the control group, but significantly decreased compared to the NASH induction group.
실험예 6. 혈중 콜레스테롤 및 간 손상지표 측정 결과Experimental Example 6. Blood cholesterol and liver damage index measurement results
시험이 종료된 실험동물로부터 채취한 혈장에서 총 콜레스테롤(Total cholesterol), 중성지방(Triglyceride), 간 손상 지표인 알라닌아미노전달효소 (alanine aminotransferase; ALT) 및 아스파르테이트아미노전달효소(Aspartate Aminotransferase; AST)를 전용 측정 키트를 이용하여 측정하였으며 그 결과는 도 3과 같다.Total cholesterol, triglyceride, alanine aminotransferase (ALT), an indicator of liver damage, and aspartate aminotransferase (AST) were collected from plasma collected from experimental animals after the test was completed. ) was measured using a dedicated measurement kit, and the results are shown in Figure 3.
혈장 내의 지질 성분 중 Triglyceride의 양은 NASH 유도군에서 큰 변화가 없 었으나, Total cholesterol의 양은 NASH 유도군에서 현저하게 증가하였다. 메밀/결명 혼합추출물 소재(CNS01) 투여군에서는 Total cholesterol이 감소하는 경향을 나타냈다. 혈장 내에서 측정할 수 있는 간 세포 손상의 대표적인 효소인 AST 및 ALT의 경우 NASH 유도군에서 모두 현저하게 증가하였다. 메밀/결명 혼합추출물 소재(CNS01) 투여군은 AST 및 ALT의 수치들이 현저하게 감소하여 NASH 유도에 따른 간 세포의 직접적인 손상을 억제하는 효능이 있음을 시사하였다.Among the lipid components in plasma, the amount of triglyceride did not change significantly in the NASH induction group, but the amount of total cholesterol significantly increased in the NASH induction group. Total cholesterol tended to decrease in the group administered buckwheat/columbine mixed extract material (CNS01). AST and ALT, representative enzymes of liver cell damage that can be measured in plasma, both significantly increased in the NASH-induced group. In the group administered buckwheat/columbine mixed extract material (CNS01), the levels of AST and ALT were significantly reduced, suggesting that it is effective in suppressing direct damage to liver cells caused by NASH induction.
실험예 7. 간 조직 단백질을 이용한 NASH 연관 인자 측정 결과 (Western blot)Experimental Example 7. Measurement results of NASH-related factors using liver tissue proteins (Western blot)
시험이 종료된 후 실험동물의 간 조직을 냉동 채취한 후 단백질을 추출하여 Western blot 실험법으로 NASH와 연관되어 있는 여러 가지 기전 관련 인자들의 발현량을 측정하였다. 그 결과는 도 4와 같으며, 도 5는 이를 정량적으로 수치화하여 나타낸 것이다. After the test was completed, the liver tissues of the experimental animals were frozen, the proteins were extracted, and the expression levels of various mechanism-related factors related to NASH were measured using Western blot testing. The results are as shown in Figure 4, and Figure 5 shows them quantitatively.
Fatty acid synthase(FASN)은 간 조직에서 지방산을 합성하는 복합 효소로 본 실험에서는 NASH 유도군에서 발현량이 현저히 증가하였다. 또한, 메밀/결명 혼합추출물 소재(CNS01) 투여군에서 발현량은 특별히 변화되지 않았다. Fatty acid synthase (FASN) is a complex enzyme that synthesizes fatty acids in liver tissue, and its expression level was significantly increased in the NASH-induced group in this experiment. In addition, the expression level was not particularly changed in the group administered buckwheat/columbine mixed extract material (CNS01).
지방산의 섭취, 저장과 함께 염증 및 인슐린 관련 혈당 항상성 조정에 관여하는 PPAR-gamma의 경우 NASH 유도군에서 현저히 증가하였고, CNS01 투여군에서 유의적으로 감소하였다. PPAR-gamma, which is involved in regulating inflammation and insulin-related blood sugar homeostasis along with intake and storage of fatty acids, was significantly increased in the NASH-induced group and significantly decreased in the CNS01 administered group.
또한, 염증 관련 매개인자인 iNOS, COX-2, TNF-alpha 등이 NASH 유도군에서 현저하게 증가하였으며 염증 및 세포사멸 기전에 관여하는 p-NF-kB, p-IkB-alpha 및 Cleaved-Caspase-3의 발현 역시 NASH 유도군에서는 모두 현저하게 증가하였다. CNS01 투여군은 이러한 인자들의 발현을 현저하게 감소시키는 것을 확인할 수 있었다. In addition, inflammation-related mediators such as iNOS, COX-2, and TNF-alpha were significantly increased in the NASH-induced group, and p-NF-kB, p-IkB-alpha, and Cleaved-Caspase-, which are involved in inflammation and cell death mechanisms, were significantly increased in the NASH-induced group. The expression of 3 also significantly increased in all NASH-induced groups. It was confirmed that the CNS01 administration group significantly reduced the expression of these factors.
한편, NASH 유도군에서는 간섬유화와 관련된 TGF-beta의 발현량도 높게 증가하였으나, CNS01은 이의 발현 감소에는 효과가 거의 없었다. Meanwhile, in the NASH-induced group, the expression level of TGF-beta, which is related to liver fibrosis, also increased significantly, but CNS01 had little effect on reducing its expression.
실험예 8. 간 조직에 지방 축적 정도 평가Experimental Example 8. Evaluation of the degree of fat accumulation in liver tissue
다음으로 간 조직의 지방 축적 정도를 측정하였다. 그 결과는 도 6과 같다. 간 조직의 지방 축적 정도를 측정하기 위하여 간 조직 절편을 Oil red O 염색과 H&E 염색을 하여 간 조직을 현미경으로 관찰하였다. 그 결과 NASH 유도군에서 지방 축적이 유의적으로 증가하였고, CNS01를 투여할 경우 간에 지방 축적이 유의적으로 감소되는 것이 관찰되었다(도 6).Next, the degree of fat accumulation in liver tissue was measured. The results are as shown in Figure 6. To measure the degree of fat accumulation in liver tissue, liver tissue sections were stained with Oil red O and H&E, and the liver tissue was observed under a microscope. As a result, fat accumulation significantly increased in the NASH-induced group, and when CNS01 was administered, fat accumulation in the liver was observed to be significantly reduced (Figure 6).
본 실험의 모든 결과를 종합해보면, 6개월간 유도되는 NASH 질환 유도 마우스 모델에서, 지방간이 유도되는 시점부터 4개월간 꾸준하게 메밀/결명 혼합추출물 소재(CNS01)를 경구 투여한 결과 체중 증가 양상이 정상 수준으로 감소되었다. Summarizing all the results of this experiment, in the NASH disease-induced mouse model induced for 6 months, the body weight gain pattern returned to a normal level as a result of oral administration of buckwheat/pollinated mixed extract material (CNS01) for 4 months consistently from the time fatty liver was induced. decreased.
또한, 복부에 축적되는 복부 지방과 지방의 과도한 축적으로 증가되는 간의 중량을 현저하게 감소시킴으로써 체내에 지방이 축적되는 것을 효과적으로 억제하였다. 이러한 효능은 지방산의 섭취, 저장과 함께 염증 및 인슐린 관련 혈당 항상성 조정에 관여하는 PPAR-gamma와 관련된 작용기전을 통해 발현될 수 있을 것으로 판단된다.In addition, it effectively suppressed fat accumulation in the body by significantly reducing abdominal fat accumulated in the abdomen and liver weight increased due to excessive accumulation of fat. It is believed that this effect may be expressed through an action mechanism related to PPAR-gamma, which is involved in regulating inflammation and insulin-related blood sugar homeostasis along with the intake and storage of fatty acids.
NASH 유도 실험동물에서 가장 큰 손상을 받는 간 조직의 세포 손상을 억제하는 효능에 대해서도 CNS01(결명 새싹 추출물과 메밀 새싹 추출물의 혼합조성물)은 우수한 결과를 나타냈다. 다양한 염증 및 세포사멸 관련 인자들을 효율적으로 억제하는 것이 관찰되어 NASH 질환의 특이적인 병리기전에 유효한 물질로 사용될 수 있을 것으로 판단된다.CNS01 (a mixed composition of bokwheat sprout extract and buckwheat sprout extract) showed excellent results in terms of its efficacy in suppressing cell damage in liver tissue, which is the most damaged in NASH-induced experimental animals. It was observed to efficiently inhibit various inflammation- and cell death-related factors, so it is believed that it can be used as an effective substance for the specific pathological mechanism of NASH disease.
[B. 결명과 메밀을 포함하는 초음파 추출물의 제조 및 지방생성 억제 효능 분석][B. Preparation of ultrasonic extracts containing grains and buckwheat and analysis of their effectiveness in inhibiting lipogenesis]
실시예 4. 결명과 메밀을 포함하는 초음파 추출물의 제조Example 4. Preparation of ultrasonic extract containing grains and buckwheat
4-1. 시서스 지상부의 추출물4-1. Extract of Cissus aerial parts
시서스 (Cissus Quadrangularis) 지상부 추출물은 Development Zone (Xianshanxi, China)에서 구입한 시료를 이용하여 사용하였다. Cissus Quadrangularis aerial extract was used using samples purchased from Development Zone (Xianshanxi, China).
4-2. DW를 이용한 녹차 잎의 초음파 추출물 제조4-2. Preparation of ultrasonic extract from green tea leaves using DW
건조된 녹차(Camellia Sinensis) 잎 5 g에 DW 50mL를 가하고, 40 kHz의 초음파 수조에서 35℃에 2시간 동안 초음파 추출하였다. 상기 추출액을 여과하여 얻어진 여과액을 35℃에서 감압 농축하여 추출물 시료를 얻었다.50 mL of DW was added to 5 g of dried green tea ( Camellia Sinensis ) leaves, and ultrasonic extraction was performed for 2 hours at 35°C in an ultrasonic bath at 40 kHz. The filtrate obtained by filtering the extract was concentrated under reduced pressure at 35°C to obtain an extract sample.
4-3. DW를 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-3. Preparation of ultrasonic extracts from a mixture of chestnut sprouts and buckwheat sprouts using DW.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 4-2와 동일하게 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물을 제조하였다. Cassia sprouts and buckwheat sprouts were prepared in the same manner as in Example 4-2, except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts at a weight ratio of 1:1. An ultrasonic extract of the mixture was prepared.
4-4. DW를 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-4. Preparation of ultrasonic extracts from a mixture of chestnut sprouts and buckwheat sprouts using DW.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 1:3의 중량비로 혼합하는 것을 제외하고 실시예 4-3과 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. A mixture of ultrasonic extracts of shoots and buckwheat sprouts was prepared in the same manner as in Example 4-3, except that dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts were mixed at a weight ratio of 1:3. .
4-5. DW를 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-5. Preparation of ultrasonic extracts from a mixture of chestnut sprouts and buckwheat sprouts using DW.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 3:1의 중량비로 혼합하는 것을 제외하고 실시예 4-3과 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. A mixture of ultrasonic extracts of shoots and buckwheat sprouts was prepared in the same manner as in Example 4-3, except that dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts were mixed at a weight ratio of 3:1. .
4-6. 30%(v/v) 에탄올을 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-6. Preparation of ultrasonic extract of a mixture of cassia sprouts and buckwheat sprouts using 30% (v/v) ethanol.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 1:1의 중량비로 혼합한 혼합물 5 g에 30%(v/v) 에탄올 50mL을 가하는 것을 제외하고 실시예 4-3과 동일하게 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물을 제조하였다. Example 4-3, except that 50 mL of 30% (v/v) ethanol was added to 5 g of a mixture of dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts at a weight ratio of 1:1. In the same manner, ultrasonic extracts of a mixture of cassia sprouts and buckwheat sprouts were prepared.
4-7. 30%(v/v) 에탄올을 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-7. Preparation of ultrasonic extract of a mixture of buckwheat sprouts and buckwheat sprouts with 30% (v/v) ethanol.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 1:3의 중량비로 혼합한 혼합물 5 g에 30%(v/v) 에탄올 50mL을 가하는 것을 제외하고 실시예 4-3과 동일하게 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물을 제조하였다. Example 4-3, except that 50 mL of 30% (v/v) ethanol was added to 5 g of a mixture of dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts at a weight ratio of 1:3. In the same manner, ultrasonic extracts of a mixture of cassia sprouts and buckwheat sprouts were prepared.
4-8. 30%(v/v) 에탄올을 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-8. Preparation of ultrasonic extract of a mixture of buckwheat sprouts and buckwheat sprouts with 30% (v/v) ethanol.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 3:1의 중량비로 혼합한 혼합물 5 g에 30%(v/v) 에탄올 50mL을 가하는 것을 제외하고 실시예 4-3과 동일하게 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물을 제조하였다. Example 4-3 except that 50 mL of 30% (v/v) ethanol was added to 5 g of a mixture of dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts at a weight ratio of 3:1. In the same manner, ultrasonic extracts of a mixture of cassia sprouts and buckwheat sprouts were prepared.
4-9. DW를 이용한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물 제조4-9. Preparation of a mixture of ultrasonic extracts of buckwheat sprouts and buckwheat sprouts using DW
건조된 결명(Senna Tora) 새싹 5 g에 DW 50mL를 가하여 실시예 4-2와 동일하게 결명 새싹의 초음파 추출물을 제조하였다. 또한, 건조된 메밀(Fagopyrum Tataricum) 새싹 5 g에 DW 50mL를 가하여 실시예 4-2와 동일하게 메밀 새싹의 초음파 추출물을 제조하였다.An ultrasonic extract of Senna Tora shoots was prepared in the same manner as in Example 4-2 by adding 50 mL of DW to 5 g of dried Senna Tora sprouts. In addition, 50 mL of DW was added to 5 g of dried buckwheat ( Fagopyrum Tataricum ) sprouts to prepare an ultrasonic extract of buckwheat sprouts in the same manner as in Example 4-2.
이후, 이들 초음파 추출물을 1:1의 중량비로 혼합하여 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. Afterwards, these ultrasonic extracts were mixed at a weight ratio of 1:1 to prepare a mixture of ultrasonic extracts of chestnut sprouts and buckwheat sprouts.
4-10. DW를 이용한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물 제조4-10. Preparation of a mixture of ultrasonic extracts of stem sprouts and buckwheat sprouts using DW
결명 새싹 추출물과 메밀 새싹 추출물을 1:3의 중량비로 혼합하는 것을 제외하고 실시예 4-9와 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. A mixture of ultrasonic extracts of chestnut shoots and buckwheat sprouts was prepared in the same manner as in Examples 4-9, except that the extracts of the shoots and buckwheat sprouts were mixed at a weight ratio of 1:3.
4-11. DW를 이용한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물 제조4-11. Preparation of a mixture of ultrasonic extracts of buckwheat sprouts and buckwheat sprouts using DW
결명 새싹 추출물과 메밀 새싹 추출물을 3:1의 중량비로 혼합하는 것을 제외하고 실시예 4-9와 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다.A mixture of ultrasonic extracts of chestnut shoots and buckwheat sprouts was prepared in the same manner as in Examples 4-9, except that the extracts of the shoots and buckwheat sprouts were mixed at a weight ratio of 3:1.
4-12. 30%(v/v) 에탄올을 이용한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물 제조4-12. Preparation of a mixture of ultrasonic extracts of stem sprouts and buckwheat sprouts using 30% (v/v) ethanol
건조된 결명(Senna Tora) 새싹 5 g에 30%(v/v) 에탄올을 가하여 실시예 4-2와 동일하게 결명 새싹의 초음파 추출물을 제조하였다. 또한, 건조된 메밀(Fagopyrum Tataricum) 새싹 5 g에 30%(v/v) 에탄올을 가하여 실시예 4-2와 동일하게 메밀 새싹의 초음파 추출물을 제조하였다.An ultrasonic extract of Senna Tora shoots was prepared in the same manner as in Example 4-2 by adding 30% (v/v) ethanol to 5 g of dried Senna Tora shoots. In addition, an ultrasonic extract of buckwheat sprouts was prepared in the same manner as in Example 4-2 by adding 30% (v/v) ethanol to 5 g of dried buckwheat ( Fagopyrum Tataricum ) sprouts.
이후, 이들 초음파 추출물을 1:1의 중량비로 혼합하여 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. Afterwards, these ultrasonic extracts were mixed at a weight ratio of 1:1 to prepare a mixture of ultrasonic extracts of chestnut sprouts and buckwheat sprouts.
4-13. 30%(v/v) 에탄올을 이용한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물 제조4-13. Preparation of a mixture of ultrasonic extracts of stem sprouts and buckwheat sprouts using 30% (v/v) ethanol
결명 새싹 추출물과 메밀 새싹 추출물을 1:3의 중량비로 혼합하는 것을 제외하고 실시예 4-12와 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. A mixture of ultrasonic extracts of chestnut shoots and buckwheat sprouts was prepared in the same manner as in Examples 4-12, except that the extracts of the shoots and buckwheat sprouts were mixed at a weight ratio of 1:3.
4-14. 30%(v/v) 에탄올을 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조4-14. Preparation of ultrasonic extract of a mixture of cassia sprouts and buckwheat sprouts using 30% (v/v) ethanol.
결명 새싹 추출물과 메밀 새싹 추출물을 3:1의 중량비로 혼합하는 것을 제외하고 실시예 4-12와 동일하게 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물을 제조하였다. A mixture of ultrasonic extracts of chestnut shoots and buckwheat sprouts was prepared in the same manner as in Examples 4-12, except that the extracts of the shoots and buckwheat sprouts were mixed at a weight ratio of 3:1.
실시예 4-1에서 실시예 4-14까지 제조한 식물 추출물의 혼합비율, 추출용매, 추출방법을 정리하면 표 5와 같다. Table 5 summarizes the mixing ratio, extraction solvent, and extraction method of the plant extracts prepared from Example 4-1 to Example 4-14.
(혼합추출)2h sonication
(mixed extraction)
(혼합추출)2h sonication
(mixed extraction)
(혼합추출)2h sonication
(mixed extraction)
(혼합추출)2h sonication
(mixed extraction)
(혼합추출)2h sonication
(mixed extraction)
(혼합추출)2h sonication
(mixed extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
(추출 후 혼합)2h sonication
(mixed after extraction)
실험예 9. 지방전구세포에서 결명새싹과 메밀새싹의 최적 용매 조건 및 혼합조건에 따른 지방 축적 억제 기능 검증Experimental Example 9. Verification of fat accumulation inhibition function according to optimal solvent conditions and mixing conditions of stem sprouts and buckwheat sprouts in preadipocytes.
3T3-L1 지방전구세포를 ATCC (American Type Culture Collection)에서 구입하여 사용하였다. 세포배양배지는 10% 소태아혈청(FBS), 1% 페니실린(100U/ml), 1% 스트렙토마이신(100U/ml), 1% HEPES(1M), 1% 소듐 피루베이트(100mM), 1% L-글루타민(200 mM), 1% NEAA(100X)를 포함하는 DMEM(Dulbecco's Modified Eagle's Medium)를 사용하였고 CO2 배양기에 37℃, 5% CO2 조건에서 배양하였다. 3T3-L1 preadipocytes were purchased from ATCC (American Type Culture Collection) and used. Cell culture medium contains 10% fetal bovine serum (FBS), 1% penicillin (100U/ml), 1% streptomycin (100U/ml), 1% HEPES (1M), 1% sodium pyruvate (100mM), 1% DMEM (Dulbecco's Modified Eagle's Medium) containing L-glutamine (200 mM) and 1% NEAA (100X) was used and cultured in a CO 2 incubator at 37°C under 5% CO 2 conditions.
먼저, 3T3-L1 세포를 24-well 플레이트에서 1x105 cells/well씩 접종(seeding)하여 3일간 배양하였다. 세포가 포스트-융합(post-confluent) 할 때 호르몬 칵테일인 0.25 μM 덱사메타손, 5 μg/mL 인슐린, 0.5mM IBMX가 포함된 배지에 시료를 50 μg/mL로 하여 1 mL 처리 하였다. 이틀 뒤에, 인슐린 5㎍/mL이 포함된 배지에 시료를 50 μg/mL로 만든 후 1 mL 로 교체하였고, 다시 이틀 뒤에 시료와 인슐린 5㎍/ml이 포함된 배지에 시료를 50 μg/mL로 만든 후 1 mL 배지를 추가하여 지방세포로 분화를 유도하였다. 지방세포의 분화가 끝난 후 PBS 용액으로 2회 세척한 뒤 PBS 용액을 완전히 suction 하였다. 이후 10% formalin을 이용하여 2시간 동안 실온에서 고정시킨 후 Oil red O solution 용액을 첨가하여 60분 동안 실온에서 지방구를 염색하였다. 염색 후 증류수로 5번 세척하여 잘 건조된 상태에서 100% isopropanol을 500 μL 첨가하여 Oil red O dye를 용출한 뒤 96 well plate에 100 μL씩 옮겼으며, Microplate Reader(Molecular Devices, SpectraMax M2)를 이용하여 560 nm 파장에서 optical density를 측정하였다. 지방 분화 억제능은 (%) = (시료 첨가구의 흡광도 / 무 첨가구의 흡광도) * 100으로 계산하였다.First, 3T3-L1 cells were seeded at 1x10 5 cells/well in a 24-well plate and cultured for 3 days. When the cells were post-confluent, 1 mL of the sample was treated at 50 μg/mL in medium containing the hormone cocktail of 0.25 μM dexamethasone, 5 μg/mL insulin, and 0.5mM IBMX. Two days later, the sample was adjusted to 50 μg/mL in a medium containing 5 μg/mL of insulin and then changed to 1 mL, and two days later, the sample was adjusted to 50 μg/mL in a medium containing the sample and 5 μg/mL of insulin. After making, 1 mL medium was added to induce differentiation into adipocytes. After differentiation of adipocytes was completed, the cells were washed twice with PBS solution and the PBS solution was completely suctioned. Afterwards, the fat spheres were fixed at room temperature for 2 hours using 10% formalin, and then Oil red O solution was added to stain the fat spheres at room temperature for 60 minutes. After staining, the dye was washed five times with distilled water and dried well. 500 μL of 100% isopropanol was added to elute the Oil red O dye, and then 100 μL each was transferred to a 96 well plate using a Microplate Reader (Molecular Devices, SpectraMax M2). The optical density was measured at a wavelength of 560 nm. The ability to inhibit adiposity was calculated as (%) = (absorbance of the sample added / absorbance of the sample not added) * 100.
도 7은 3T3-L1 세포에서 결명 새싹과 메밀 새싹의 최적 용매 조건 및 혼합 조건에 따른 지방 축적 억제 효능을 나타낸 것이다. 사용된 시료는 표 5에 나타낸 바와 같다. Figure 7 shows the efficacy of suppressing fat accumulation in 3T3-L1 cells according to the optimal solvent conditions and mixing conditions of buckwheat sprouts and buckwheat sprouts. The samples used are shown in Table 5.
그 결과, 도 7에서 확인되는 바와 같이, 결명 새싹과 메밀 새싹을 1:1의 중량비로 혼합하고, 증류수를 이용하여 추출한 시료를 사용한 경우 지방전구세포의 지방생성 억제 효과가 가장 높았으며, 지방생성 억제 효과가 있다고 알려진 녹차 잎이나 시서스의 지상부의 초음파 추출물 보다 지방생성 억제 효과가 높았다.As a result, as can be seen in Figure 7, the effect of inhibiting lipogenesis in preadipocytes was the highest when a sample was mixed with buckwheat sprouts and buckwheat sprouts at a weight ratio of 1:1 and extracted using distilled water, and the effect of suppressing lipogenesis in preadipocytes was highest. The effect of inhibiting lipogenesis was higher than that of ultrasonic extracts from green tea leaves or aerial parts of Cissus, which are known to have an inhibitory effect.
구체적으로, 결명 새싹과 메밀 새싹을 1:1의 중량비로 혼합한 혼합물에 DW(증류수)를 가하여 초음파 추출한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 시료(실시예 4-3)를 처리한 경우, 지방전구세포의 지방생성 억제 효과가 가장 높았다. 또한, 증류수를 이용한 결명 새싹의 초음파 추출물과 증류수를 이용한 메밀 새싹의 초음파 추출물을 1:1의 중량비로 혼합한 결명 새싹과 메밀 새싹의 초음파 추출물의 혼합물(실시예 4-9)에서 지방생성 억제 효과가 높았다. Specifically, when processing an ultrasonic extract sample (Example 4-3) of a mixture of shoots and buckwheat sprouts, which was ultrasonically extracted by adding DW (distilled water) to a mixture of shoots and buckwheat sprouts at a weight ratio of 1:1, the fat The effect of suppressing adipogenesis in progenitor cells was the highest. In addition, the effect of inhibiting lipogenesis in a mixture of the ultrasonic extract of the buckwheat sprout and the ultrasonic extract of the buckwheat sprout using distilled water and the ultrasonic extract of the buckwheat sprout using distilled water mixed at a weight ratio of 1:1 (Example 4-9) was high.
[C. 다양한 천연 식물의 초음파 추출물 제조 및 지방생성 억제 효능 비교][C. Production of ultrasonic extracts from various natural plants and comparison of lipogenesis inhibition efficacy]
실시예 5. 다양한 천연 식물의 초음파 추출물 제조Example 5. Preparation of ultrasonic extracts from various natural plants
5-1. 시서스 지상부의 추출물5-1. Extract of Cissus aerial parts
시서스 (Cissus Quadrangularis) 지상부 추출물은 Development Zone (Xianshanxi, China)에서 구입한 시료를 이용하여 사용하였다. Cissus Quadrangularis aerial extract was used using samples purchased from Development Zone (Xianshanxi, China).
5-2. DW를 이용한 녹차 잎의 초음파 추출물 제조5-2. Preparation of ultrasonic extract from green tea leaves using DW
건조된 녹차(Camellia Sinensis) 잎 5 g에 DW 50mL를 가하고, 40 kHz의 초음파 수조에서 35℃에 2시간 동안 초음파 추출하였다. 상기 추출액을 여과하여 얻어진 여과액을 35℃에서 감압 농축하여 추출물 시료를 얻었다.50 mL of DW was added to 5 g of dried green tea ( Camellia Sinensis ) leaves, and ultrasonic extraction was performed for 2 hours at 35°C in an ultrasonic bath at 40 kHz. The filtrate obtained by filtering the extract was concentrated under reduced pressure at 35°C to obtain an extract sample.
5-3. DW를 이용한 결명 새싹의 초음파 추출물 제조5-3. Preparation of ultrasonic extracts from stem shoots using DW
건조된 결명(Senna Tora) 새싹을 사용하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹의 초음파 추출물을 제조하였다. An ultrasonic extract of Senna Tora shoots was prepared in the same manner as in Example 5-2, except that dried Senna Tora buds were used.
5-4. DW를 이용한 메밀 새싹의 초음파 추출물 제조5-4. Preparation of ultrasonic extracts from buckwheat sprouts using DW
건조된 메밀(Fagopyrum Tataricum) 새싹을 사용하는 것을 제외하고 실시예 5-2와 동일하게 메밀 새싹의 초음파 추출물을 제조하였다. An ultrasonic extract of buckwheat sprouts was prepared in the same manner as Example 5-2, except that dried buckwheat ( Fagopyrum Tataricum ) sprouts were used.
5-5. DW를 이용한 루꼴라 지상부의 초음파 추출물 제조5-5. Preparation of ultrasonic extract from arugula aerial parts using DW
건조된 루꼴라 (Eruca Sativa) 지상부(잎, 가지 및 줄기)를 사용하는 것을 제외하고 실시예 5-2와 동일하게 루꼴라 지상부의 초음파 추출물을 제조하였다. An ultrasonic extract of the aerial part of Arugula ( Eruca Sativa ) was prepared in the same manner as in Example 5-2, except that dried aerial parts (leaves, branches, and stems) were used.
5-6. DW를 이용한 서양 민들레 지상부의 초음파 추출물 제조5-6. Preparation of ultrasonic extract from aerial parts of Western dandelion using DW
건조된 서양 민들레(Taraxacum Officinale) 지상부(잎, 가지 및 줄기)를 사용하는 것을 제외하고 실시예 5-2와 동일하게 서양 민들레 지상부의 초음파 추출물을 제조하였다. An ultrasonic extract of the aerial parts of Western dandelion ( Taraxacum Officinale ) was prepared in the same manner as in Example 5-2, except that dried aerial parts (leaves, branches, and stems) were used.
5-7. DW를 이용한 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물 제조5-7. Preparation of ultrasonic extracts from a mixture of cassia sprouts and buckwheat sprouts using DW.
건조된 결명(Senna Tora) 새싹과 건조된 메밀(Fagopyrum Tataricum) 새싹을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹과 메밀 새싹 혼합물의 초음파 추출물을 제조하였다. Cassia sprouts and buckwheat sprouts were prepared in the same manner as in Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora sprouts and dried buckwheat ( Fagopyrum Tataricum ) sprouts at a weight ratio of 1:1. An ultrasonic extract of the mixture was prepared.
5-8. DW를 이용한 결명 새싹과 루꼴라 지상부 혼합물의 초음파 추출물 제조5-8. Preparation of ultrasonic extract from a mixture of aerial shoots and arugula using DW
건조된 결명(Senna Tora) 새싹과 건조된 루꼴라 (Eruca Sativa) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹과 루꼴라 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2 except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora sprouts and dried arugula ( Eruca Sativa ) aerial parts (leaves, branches and stems) at a weight ratio of 1:1. In the same way, an ultrasonic extract of a mixture of aerial shoots and arugula was prepared.
5-9. DW를 이용한 결명 새싹과 서양 민들레 지상부 혼합물의 초음파 추출물 제조5-9. Preparation of ultrasonic extracts from a mixture of stem buds and aerial parts of Western dandelion using DW
건조된 결명(Senna Tora) 새싹과 건조된 서양 민들레(Taraxacum Officinale) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹과 서양 민들레 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora buds and dried Western dandelion ( Taraxacum Officinale ) aerial parts (leaves, branches, and stems) at a weight ratio of 1:1. In the same manner as above, an ultrasonic extract of a mixture of shoots and aerial parts of Western dandelion was prepared.
5-10. DW를 이용한 결명 새싹과 녹차 잎 혼합물의 초음파 추출물 제조5-10. Preparation of ultrasonic extracts from a mixture of stem buds and green tea leaves using DW.
건조된 결명(Senna Tora) 새싹과 건조된 녹차(Camellia Sinensis) 잎을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹과 녹차 잎 혼합물의 초음파 추출물을 제조하였다. Cassia shoots and green tea leaves were prepared in the same manner as in Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora buds and dried green tea ( Camellia Sinensis ) leaves at a weight ratio of 1:1. An ultrasonic extract of the mixture was prepared.
5-11. DW를 이용한 결명 새싹과 시서스 지상부 혼합물의 초음파 추출물 제조5-11. Preparation of ultrasonic extracts from a mixture of cassia shoots and Cissus aerial parts using DW.
건조된 결명(Senna Tora) 새싹과 건조된 시서스(Cissus Quadrangularis) 지상부(잎, 가지 및 줄기)를 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 결명 새싹과 시서스 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried Senna Tora sprouts and dried Cissus Quadrangularis aerial parts (leaves, branches and stems) at a weight ratio of 1:1. In the same manner as above, an ultrasonic extract of a mixture of cassia shoots and Cissus aerial parts was prepared.
5-12. DW를 이용한 메밀 새싹과 루꼴라 지상부 혼합물의 초음파 추출물 제조5-12. Preparation of ultrasonic extracts from a mixture of buckwheat sprouts and arugula aerial parts using DW.
건조된 메밀(Fagopyrum Tataricum) 새싹과 건조된 루꼴라(Eruca Sativa) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 메밀 새싹과 루꼴라 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2 except that 50 mL of DW was added to 5 g of a mixture of dried buckwheat ( Fagopyrum Tataricum ) sprouts and dried arugula ( Eruca Sativa ) aerial parts (leaves, branches and stems) at a weight ratio of 1:1. An ultrasonic extract of a mixture of buckwheat sprouts and arugula aerial parts was prepared in the same manner.
5-13. DW를 이용한 메밀 새싹과 서양 민들레 지상부 혼합물의 초음파 추출물 제조5-13. Preparation of ultrasonic extracts from a mixture of buckwheat sprouts and dandelion aerial parts using DW.
건조된 메밀(Fagopyrum Tataricum) 새싹과 건조된 서양 민들레(Taraxacum Officinale) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 메밀 새싹과 서양 민들레 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried buckwheat ( Fagopyrum Tataricum ) sprouts and dried Western dandelion ( Taraxacum Officinale ) aboveground parts (leaves, branches, and stems) at a weight ratio of 1:1. An ultrasonic extract of a mixture of buckwheat sprouts and aerial parts of Western dandelion was prepared in the same manner as above.
5-14. DW를 이용한 메밀 새싹과 녹차 잎 혼합물의 초음파 추출물 제조5-14. Ultrasonic extract preparation of buckwheat sprout and green tea leaf mixture using DW
건조된 메밀(Fagopyrum Tataricum) 새싹과 건조된 녹차(Camellia Sinensis) 잎을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 메밀 새싹과 녹차 잎 혼합물의 초음파 추출물을 제조하였다. Buckwheat sprouts and green tea leaves were prepared in the same manner as in Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried buckwheat ( Fagopyrum Tataricum ) sprouts and dried green tea ( Camellia Sinensis ) leaves at a weight ratio of 1:1. An ultrasonic extract of the mixture was prepared.
5-15. DW를 이용한 메밀 새싹과 시서스 지상부 혼합물의 초음파 추출물 제조5-15. Preparation of ultrasonic extracts from a mixture of buckwheat sprouts and Cissus aerial parts using DW.
건조된 메밀(Fagopyrum Tataricum) 새싹과 건조된 시서스(Cissus Quadrangularis) 지상부(잎, 가지 및 줄기)를 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 메밀 새싹과 시서스 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried buckwheat ( Fagopyrum Tataricum ) sprouts and dried Cissus ( Cissus Quadrangularis ) aerial parts (leaves, branches, and stems) at a weight ratio of 1:1. An ultrasonic extract of a mixture of buckwheat sprouts and Cissus aerial parts was prepared in the same manner as above.
5-16. DW를 이용한 녹차 잎과 루꼴라 혼합물의 초음파 추출물 제조5-16. Preparation of ultrasonic extract from green tea leaf and arugula mixture using DW
건조된 녹차(Camellia Sinensis) 잎과 건조된 루꼴라(Eruca Sativa) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 녹차 잎과 루꼴라 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2 except that 50 mL of DW was added to 5 g of a mixture of dried green tea ( Camellia Sinensis ) leaves and dried arugula ( Eruca Sativa ) aerial parts (leaves, branches and stems) at a weight ratio of 1:1. An ultrasonic extract of a mixture of green tea leaves and arugula aerial parts was prepared in the same manner.
5-17. DW를 이용한 녹차 잎과 서양 민들레 지상부 혼합물의 초음파 추출물 제조5-17. Preparation of ultrasonic extracts from a mixture of green tea leaves and dandelion aerial parts using DW.
건조된 녹차(Camellia Sinensis) 잎과 건조된 서양 민들레(Taraxacum Officinale) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 녹차 잎과 서양 민들레 지상부 혼합물의 초음파 추출물을 제조하였다. Example 5-2, except that 50 mL of DW was added to 5 g of a mixture of dried green tea ( Camellia Sinensis ) leaves and dried Western dandelion ( Taraxacum Officinale ) aerial parts (leaves, branches, and stems) at a weight ratio of 1:1. In the same manner as above, an ultrasonic extract of a mixture of green tea leaves and aerial parts of Western dandelion was prepared.
5-18. DW를 이용한 시서스 지상부와 루꼴라 혼합물의 초음파 추출물 제조5-18. Ultrasonic extract preparation of Cissus aerial parts and arugula mixture using DW
건조된 시서스(Cissus Quadrangularis) 지상부(잎, 가지 및 줄기)와 건조된 루꼴라(Eruca Sativa) 지상부(잎, 가지 및 줄기)를 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50 mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 시서스 지상부와 루꼴라 지상부 혼합물의 초음파 추출물을 제조하였다. Add 50 mL of DW to 5 g of a mixture of dried Cissus ( Cissus Quadrangularis ) aerial parts (leaves, branches, and stems) and dried Arugula ( Eruca Sativa ) aerial parts (leaves, branches, and stems) at a weight ratio of 1:1. An ultrasonic extract of a mixture of Cissus aerial parts and Arugula aerial parts was prepared in the same manner as in Example 5-2, except that.
5-19. DW를 이용한 시서스 지상부와 서양 민들레 지상부 혼합물의 초음파 추출물 제조5-19. Preparation of ultrasonic extracts from a mixture of Cissus aerial parts and dandelion aerial parts using DW
건조된 시서스(Cissus Quadrangularis) 지상부(잎, 가지 및 줄기)와 건조된 서양 민들레(Taraxacum Officinale) 지상부(잎, 가지 및 줄기)을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 시서스 지상부와 서양 민들레 지상부 혼합물의 초음파 추출물을 제조하였다.Add 50 mL of DW to 5 g of a mixture of dried Cissus ( Cissus Quadrangularis ) above-ground parts (leaves, branches, and stems) and dried Western dandelion ( Taraxacum Officinale ) at a weight ratio of 1:1. An ultrasonic extract of a mixture of Cissus aerial parts and Western dandelion aerial parts was prepared in the same manner as in Example 5-2, except that.
5-20. DW를 이용한 시서스 지상부와 녹차 잎 혼합물의 초음파 추출물 제조5-20. Ultrasonic extract preparation of Cissus aerial parts and green tea leaf mixture using DW
건조된 시서스(Cissus Quadrangularis) 지상부(잎, 가지 및 줄기)와 건조된 녹차(Camellia Sinensis) 잎을 1:1의 중량비로 혼합한 혼합물 5 g에 DW 50 mL를 가하는 것을 제외하고 실시예 5-2와 동일하게 시서스 지상부와 녹차 잎 혼합물의 초음파 추출물을 제조하였다. Example 5-, except that 50 mL of DW was added to 5 g of a mixture of dried Cissus ( Cissus Quadrangularis ) aerial parts (leaves, branches and stems) and dried green tea ( Camellia Sinensis ) leaves in a weight ratio of 1:1. In the same manner as in 2, an ultrasonic extract of a mixture of Cissus aerial parts and green tea leaves was prepared.
실시예 5-1에서 실시예 5-20까지 제조한 식물 추출물의 혼합비율, 추출용매, 추출방법을 정리하면 표 6과 같다. Table 6 summarizes the mixing ratio, extraction solvent, and extraction method of the plant extracts prepared from Example 5-1 to Example 5-20.
실험예 10. 지방전구세포에서 천연 식물, 또는 이들을 혼합한 혼합물을 DW를 이용하여 추출한 초음파 추출물의 지방 축적 억제 효능 비교Experimental Example 10. Comparison of the fat accumulation inhibition efficacy of ultrasonic extracts extracted from preadipocytes from natural plants or a mixture thereof using DW.
3T3-L1 지방전구세포를 ATCC (American Type Culture Collection)에서 구입하여 사용하였다. 세포배양배지는 10% 소태아혈청(FBS), 1% 페니실린(100U/ml), 1% 스트렙토마이신(100U/ml), 1% HEPES(1M), 1% 소듐 피루베이트(100mM), 1% L-글루타민(200 mM), 1% NEAA(100X)를 포함하는 DMEM(Dulbecco's Modified Eagle's Medium)를 사용하였고 CO2 배양기에 37℃, 5% CO2 조건에서 배양하였다. 3T3-L1 preadipocytes were purchased from ATCC (American Type Culture Collection) and used. Cell culture medium contains 10% fetal bovine serum (FBS), 1% penicillin (100U/ml), 1% streptomycin (100U/ml), 1% HEPES (1M), 1% sodium pyruvate (100mM), 1% DMEM (Dulbecco's Modified Eagle's Medium) containing L-glutamine (200 mM) and 1% NEAA (100X) was used and cultured in a CO 2 incubator at 37°C under 5% CO 2 conditions.
먼저, 3T3-L1 세포를 24-well 플레이트에서 1x105 cells/well씩 접종(seeding)하여 3일간 배양하였다. 세포가 포스트-융합(post-confluent) 할 때 호르몬 칵테일인 0.25 μM 덱사메타손, 5 μg/mL 인슐린, 0.5mM IBMX가 포함된 배지에 시료를 50 μg/mL로 하여 1 mL 처리하였다. 이틀 뒤에, 인슐린 5㎍/mL이 포함된 배지에 시료를 50 μg/mL로 만든 후 1 mL 로 교체하였고, 다시 이틀 뒤에 시료와 인슐린 5㎍/ml이 포함된 배지에 시료를 50 μg/mL로 만든 후 1 mL 배지를 추가하여 지방세포로 분화를 유도하였다. 지방세포의 분화가 끝난 후 PBS 용액으로 2회 세척한 뒤 PBS 용액을 완전히 suction 하였다. 이후 10% formalin을 이용하여 2시간 동안 실온에서 고정시킨 후 Oil red O solution 용액을 첨가하여 60분 동안 실온에서 지방구를 염색하였다. 염색 후 증류수로 5번 세척하여 잘 건조된 상태에서 100% isopropanol을 500 μL 첨가하여 Oil red O dye를 용출한 뒤 96 well plate에 100 μL씩 옮겼으며, Microplate Reader(Molecular Devices, SpectraMax M2)를 이용하여 560 nm 파장에서 optical density를 측정하였다. 지방 분화 억제능은 (%) = (시료 첨가구의 흡광도 / 무 첨가구의 흡광도) * 100으로 계산하였다.First, 3T3-L1 cells were seeded at 1x10 5 cells/well in a 24-well plate and cultured for 3 days. When the cells were post-confluent, 1 mL of the sample was treated at 50 μg/mL in medium containing the hormone cocktail of 0.25 μM dexamethasone, 5 μg/mL insulin, and 0.5mM IBMX. Two days later, the sample was adjusted to 50 μg/mL in a medium containing 5 μg/mL of insulin and then changed to 1 mL, and two days later, the sample was adjusted to 50 μg/mL in a medium containing the sample and 5 μg/mL of insulin. After making, 1 mL medium was added to induce differentiation into adipocytes. After differentiation of adipocytes was completed, the cells were washed twice with PBS solution and the PBS solution was completely suctioned. Afterwards, the fat spheres were fixed at room temperature for 2 hours using 10% formalin, and then Oil red O solution was added to stain the fat spheres at room temperature for 60 minutes. After staining, the dye was washed five times with distilled water and dried well. 500 μL of 100% isopropanol was added to elute the Oil red O dye, and then 100 μL each was transferred to a 96 well plate using a Microplate Reader (Molecular Devices, SpectraMax M2). The optical density was measured at a wavelength of 560 nm. The ability to inhibit adiposity was calculated as (%) = (absorbance of the sample added / absorbance of the sample not added) * 100.
도 8은 3T3-L1 세포에서 다양한 천연 식물, 또는 이들을 혼합한 혼합물을 DW를 이용하여 추출한 초음파 추출물의 지방 축적 억제 효능을 비교한 것이다. 사용된 시료는 표 6에 나타낸 바와 같다. Figure 8 compares the fat accumulation inhibition efficacy of ultrasonic extracts extracted from various natural plants or mixtures thereof using DW in 3T3-L1 cells. The samples used are as shown in Table 6.
그 결과, 도 8에서 확인되는 바와 같이, 결명 새싹 단독 추출물과 메밀 새싹 단독 추출물에 비해, 결명 새싹과 메밀 새싹을 1:1의 중량비로 혼합한 혼합물을 증류수를 이용하여 추출한 시료(실시예 5-7)를 사용한 경우 지방전구세포의 지방생성 억제 효과가 가장 높았다. 또한, 지방생성 억제 효과가 있다고 알려진 녹차 잎이나 시서스의 지상부, 서양민들레 지상부, 루꼴라 지상부의 단독 초음파 추출물이나, 결명 새싹, 메밀 새싹, 녹차 잎, 시서스의 지상부, 서양민들레 지상부, 루꼴라 지상부의 혼합물의 초음파 추출물의 시료보다 지방생성 억제 효과가 높았다.As a result, as seen in Figure 8, compared to the extract of only shoot shoots and buckwheat sprouts alone, the sample extracted using distilled water was a mixture of shoot shoots and buckwheat sprouts at a weight ratio of 1:1 (Example 5- When 7) was used, the effect of inhibiting lipogenesis in preadipocytes was the highest. In addition, the exclusive ultrasonic extract of green tea leaves, aerial parts of Cissus, aerial parts of dandelion, and aerial parts of arugula, which are known to have an effect of inhibiting lipogenesis, or aerial parts of aerial shoots, buckwheat sprouts, green tea leaves, aerial parts of Cissus, aerial parts of dandelion, and aerial parts of arugula. The effect of inhibiting lipogenesis was higher than that of the ultrasonic extract of the mixture.
이상에서 본 발명의 바람직한 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다.Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.
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