KR102687978B1 - A composition for improving, preventing and treating of obesity comprising peanut shell extract - Google Patents
A composition for improving, preventing and treating of obesity comprising peanut shell extract Download PDFInfo
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- KR102687978B1 KR102687978B1 KR1020210135788A KR20210135788A KR102687978B1 KR 102687978 B1 KR102687978 B1 KR 102687978B1 KR 1020210135788 A KR1020210135788 A KR 1020210135788A KR 20210135788 A KR20210135788 A KR 20210135788A KR 102687978 B1 KR102687978 B1 KR 102687978B1
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- extract
- obesity
- peanut
- composition
- extraction
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/332—Promoters of weight control and weight loss
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/48—Ultrasonic treatment
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- Health & Medical Sciences (AREA)
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- Natural Medicines & Medicinal Plants (AREA)
- Engineering & Computer Science (AREA)
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Abstract
본 발명은 비만의 개선, 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 땅콩 겉껍질 추출물을 유효성분으로 포함함으로써, 비만에 의해 유발될 수 있는 질환을 개선, 예방 또는 치료할 수 있으므로, 식품 조성물, 나아가 건강기능식품 또는 약학 조성물로 활용될 수 있다.The present invention relates to a composition for improving, preventing or treating obesity, and more specifically, to a composition for improving, preventing or treating diseases that may be caused by obesity by containing peanut skin extract as an active ingredient. , Furthermore, it can be used as a health functional food or pharmaceutical composition.
Description
본 발명은 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 개선, 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for improving, preventing or treating obesity containing peanut skin extract as an active ingredient.
우리나라는 물론 세계적으로 경제발전 및 의료기술의 발달로 인하여 인간의 평균수명이 길어지고 삶의 질이 빠른 속도로 향상되고 있어 이에 부합하는 적극적인 웰빙시대의 대응전략이 시급히 요청되고 있다. 특히, 가속화되고 있는 노령화, 성인병의 발생에 따른 국민건강의 보호 및 정부와 개인의 의료비 절감의 차원에서도 효과적이고 안전한 의약품 및 건강기능식품의 개발이 필요하다.Due to economic development and medical technology development not only in Korea but also around the world, the average human lifespan is lengthening and the quality of life is improving at a rapid pace. Accordingly, an active response strategy for the era of well-being is urgently required. In particular, the development of effective and safe medicines and health functional foods is necessary to protect public health due to the accelerating aging and occurrence of adult diseases and to reduce medical costs for the government and individuals.
심장마비, 고혈압, 동맥경화, 뇌졸중 등의 심혈관질환은 세계적으로 전체 사망률의 30%를 차지하고 있다. 특히 비만, 당뇨병, 고혈압, 동맥경화증, 심장 질환, 뇌졸중 등은 각기 다른 질병으로 보이지만 체지방 증가로 인한 인슐린 저항성이라는 공통 병인을 갖는 하나의 증후군으로 이해되고 있다.Cardiovascular diseases such as heart attack, high blood pressure, arteriosclerosis, and stroke account for 30% of all deaths worldwide. In particular, obesity, diabetes, high blood pressure, arteriosclerosis, heart disease, and stroke appear to be different diseases, but they are understood as one syndrome with a common etiology of insulin resistance caused by increased body fat.
비만은 전 세계적으로 가장 흔한 영양장애 중의 하나로서 WHO 통계자료에 의하면 현재 2억 5천여만명이 비만 환자로 분류되며 20년 후에는 약 3억명이 비만으로 고통받을 것으로 예측된다.Obesity is one of the most common nutritional disorders worldwide. According to WHO statistics, approximately 250 million people are currently classified as obese patients, and it is predicted that approximately 300 million people will suffer from obesity in 20 years.
상기 비만은 과량의 에너지 섭취 또는 에너지 소비 저하로 인해 열량 대사가 불균형하여 체지방이 과도하게 축적된 상태를 지칭하며, 최근에는 현대인들의 서구적인 식생활 패턴과 운동 부족으로 인해 비만의 발병율이 급격히 증가하는 추세이다. 또한, 수치상의 개념으로는 BMI(body mass index, 체질량지수) 측정 시 25 이상일 경우를 비만으로 정의하며, BMI 측정법은 체중(kg)을 키의 제곱(㎡)으로 나눈 값을 통해 체지방의 양을 추정하는 비만측정법이다. The above-mentioned obesity refers to a state in which excessive accumulation of body fat occurs due to imbalance in calorie metabolism due to excessive energy intake or low energy consumption. Recently, the incidence of obesity has rapidly increased due to the Western eating patterns and lack of exercise of modern people. am. In addition, as a numerical concept, obesity is defined as a BMI (body mass index) of 25 or more when measuring, and the BMI measurement method calculates the amount of body fat by dividing body weight (kg) by the square of height (㎡). It is a method of measuring obesity.
비만치료제 중에서 식욕억제제에는 휴터민정, 펜터민 및 로카세린이라는 성분이 있고 부작용으로는 불안감, 현기증과 불면증 등이 있으며 지방분해효소 억제제에는 오르리스타트, 로카세린과 마진돌 등이 있고 부작용으로는 기름변, 복통과 변실금 등이 있는데, 상기 상업화된 약물들의 부작용이 보고됨에 따라 효과가 뛰어나면서 부작용이 적은 천연물 유래의 비만치료제가 요구되는 실정이다.Among obesity treatments, appetite suppressants include Hutermine tablets, phentermine, and lorcaserin, and side effects include anxiety, dizziness, and insomnia, while lipolytic enzyme inhibitors include orlistat, lorcaserin, and mazindol, and side effects include oily stool. , abdominal pain and fecal incontinence, etc., and as the side effects of the commercialized drugs are reported, there is a need for obesity treatments derived from natural products that are highly effective and have fewer side effects.
한편, 땅콩(Peanut, Arachis hypogaea)은 대표적인 유지작물로 콩과에 속하는 일년생의 초본식물이며 한국, 인도와 미국 등 세계 각국에서 재배되어 단백질, 불포화지방산, 비타민과 아미노산 등의 다양한 영양성분을 풍부하게 함유하고 있다. 땅콩 중량의 35~40%를 차지하는 땅콩 겉껍질은 소화율이 낮아 가축 사료로의 사용에 제한이 있어 대부분 폐기되는 실정으로 부산물 처리비용과 환경문제를 발생시키므로 이를 해결하기 위해 땅콩 겉껍질의 활용을 통한 고부가가치 소재 개발이 요구되고 있다.Meanwhile, peanut ( Arachis hypogaea ) is a representative oil crop and an annual herbaceous plant belonging to the legume family. It is cultivated in countries around the world, including Korea, India, and the United States, and is rich in various nutrients such as protein, unsaturated fatty acids, vitamins, and amino acids. It contains. Peanut shells, which account for 35-40% of the weight of peanuts, have a low digestibility and are therefore limited in their use as livestock feed, so most of them are discarded, causing by-product disposal costs and environmental problems. To solve this problem, use the peanut shells. The development of high value-added materials is required.
비만은 지방전구세포가 지방세포로 분화되는 정상적인 지방세포의 이상발달이 발생하는데 지방세포로의 과잉분화와 지방세포 내 지방의 축적은 지방세포의 수나 크기를 증가시키고 그에 따라 비만 증상이 더욱 심해지는 것으로 알려져 있다. 지방전구세포가 confluence 상태가 되면 세포 주기가 정지되어 세포의 증식이 중단되고 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone (DEX) 및 insulin 등의 호르몬에 의해 지방세포로 분화(adipogenisis)가 유도되고 비만은 조직 내 지방세포가 비대(hypertrophy)와 과형성(hyperplasia)으로 초래된다. 지방전구세포의 초기분화 과정은 DEX에 의해 cAMP의 농도가 증가되면서 sterol regulatory element binding proteins-1c (SREBP-1c) 발현이 유도된 후 IBMX에 의해 peroxisome proliferator activated receptor-γ (PPAR-γ)와 CCAAT enhancer binding protein-α (CEBP-α)가 상호 발현되어 분화를 촉진한다. 이들의 발현은 불포화지방산에서 포화지방산으로 합성될 때 포화 지방산을 단일 불포화지방산으로 전환시키는 fatty acid synthase (FAS) 및 stearoyl-CoA desaturase 1 (SCD1) 발현과 함께 지방세포로의 분화를 유도한다. Obesity is caused by abnormal development of normal adipocytes, in which preadipocytes differentiate into adipocytes. Hyper-differentiation into adipocytes and accumulation of fat within adipocytes increases the number and size of adipocytes, thereby making obesity symptoms more severe. It is known. When preadipocytes become confluent, the cell cycle stops, cell proliferation stops, and differentiation into adipocytes occurs by hormones such as 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone (DEX), and insulin (adipogenis). is induced, and obesity is caused by hypertrophy and hyperplasia of fat cells in tissues. The initial differentiation process of preadipocytes involves increasing the concentration of cAMP by DEX, inducing the expression of sterol regulatory element binding proteins-1c (SREBP-1c), and then peroxisome proliferator activated receptor-γ (PPAR-γ) and CCAAT by IBMX. Enhancer binding protein-α (CEBP-α) is co-expressed and promotes differentiation. Their expression induces differentiation into adipocytes along with the expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), which convert saturated fatty acids into monounsaturated fatty acids when unsaturated fatty acids are synthesized into saturated fatty acids.
따라서 지방전구세포 분화에 핵심적인 전사인자의 발현 조절을 통해 지방세포 분화를 억제하는 것은 비만을 예방하는 효과적인 전략이며, 이에 본 연구는 천연물로부터 독성 및 부작용이 없는 천연물질을 이용한 비만치료제가 요구되고 있다.Therefore, suppressing adipocyte differentiation by regulating the expression of transcription factors key to preadipocyte differentiation is an effective strategy to prevent obesity. Accordingly, this study calls for an obesity treatment using natural substances without toxicity and side effects. there is.
본 발명의 목적은 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 예방 또는 치료용 약학 조성물을 제공하는데 있다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating obesity containing peanut skin extract as an active ingredient.
또한, 본 발명의 다른 목적은 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 예방 또는 개선용 식품 조성물을 제공하는데 있다.In addition, another object of the present invention is to provide a food composition for preventing or improving obesity containing peanut skin extract as an active ingredient.
상기한 목적을 달성하기 위한 본 발명의 비만을 예방 또는 치료할 수 있는 약학 조성물은 땅콩 겉껍질 추출물을 유효성분으로 포함할 수 있다.The pharmaceutical composition capable of preventing or treating obesity of the present invention to achieve the above object may include peanut skin extract as an active ingredient.
상기 땅콩 겉껍질 추출물은 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로 추출된 것일 수 있다.The peanut skin extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
상기 혼합용매는 20 내지 99 부피%의 메탄올, 에탄올, 부탄올 또는 프로판올 수용액일 수 있다.The mixed solvent may be a 20 to 99% by volume aqueous solution of methanol, ethanol, butanol, or propanol.
상기 땅콩 겉껍질 추출물은 20 내지 50 kHz의 진동수 및 50 내지 700 W의 파워의 초음파기로 5 내지 60 분 동안 처리된 것일 수 있다.The peanut skin extract may be treated with an ultrasonicator with a frequency of 20 to 50 kHz and a power of 50 to 700 W for 5 to 60 minutes.
상기 초음파 처리 시 추출온도는 40 내지 100 ℃일 수 있다.The extraction temperature during the ultrasonic treatment may be 40 to 100 °C.
또한, 상기한 다른 목적을 달성하기 위한 본 발명의 비만을 예방 또는 개선할 수 있는 식품 조성물은 땅콩 겉껍질 추출물을 유효성분으로 포함할 수 있다.In addition, the food composition capable of preventing or improving obesity of the present invention to achieve the above-described other purposes may include peanut outer shell extract as an active ingredient.
본 발명의 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 개선, 예방 또는 치료용 조성물은 리파아제 저해 활성이 시판되는 리파아제 저해제의 저해율에 상당하는 높은 저해 효과를 나타내며, 알파 글루코시데이즈 저해 활성 역시 시판되는 알파 글루코시데이즈 저해제의 저해율보다 높은 저해 효과를 나타내므로 비만에 의해 유발될 수 있는 질환을 개선, 예방 또는 치료할 수 있으므로, 식품 조성물, 나아가 건강기능식품 또는 약학 조성물로 활용될 수 있다.The composition for improving, preventing or treating obesity containing the peanut skin extract of the present invention as an active ingredient exhibits a high inhibitory effect in lipase inhibitory activity equivalent to the inhibition rate of commercially available lipase inhibitors, and also has alpha glucosidase inhibitory activity. Since it exhibits a higher inhibition effect than that of alpha-glucosidase inhibitors, it can improve, prevent, or treat diseases that may be caused by obesity, and can be used as a food composition, and further as a health functional food or pharmaceutical composition.
또한, 본 발명의 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 개선, 예방 또는 치료용 조성물은 고지방 식이 동물에서 체중 감소효과가 있으므로 항비만 효과 있을 뿐만 아니라 항산화 효과도 우수하다.In addition, the composition for improving, preventing or treating obesity containing the peanut skin extract of the present invention as an active ingredient has a weight reduction effect in animals on a high-fat diet, so it not only has an anti-obesity effect but also has an excellent antioxidant effect.
도 1a는 에탄올 농도와 추출시간에 따른 추출물의 총 폴리페놀 활성 변화를 나타내는 반응표면 곡선이며, 도 1b는 에탄올 농도와 추출온도에 따른 추출물의 총 폴리페놀 활성 변화를 나타내는 반응표면 곡선이다.
도 2a는 에탄올 농도와 추출시간에 따른 추출물의 총 플라보노이드 활성 변화를 나타내는 반응표면 곡선이며, 도 2b는 에탄올 농도와 추출온도에 따른 추출물의 총 플라보노이드 활성 변화를 나타내는 반응표면 곡선이다.
도 3a는 에탄올 농도와 추출시간에 따른 추출물의 DPPH 라디칼 소거능 변화를 나타내는 반응표면 곡선이며, 도 3b는 에탄올 농도와 추출온도에 따른 추출물의 DPPH 라디칼 소거능 변화를 나타내는 반응표면 곡선이다.
도 4는 본 발명의 실시예에 따라 제조된 땅콩 겉껍질의 초음파 최적 추출조건을 찾기 위하여 상기 도 1, 도 2 및 도 3의 반응표면곡선을 겹치기기법으로 나타낸 그래프이다.
도 5는 본 발명의 실시예 18에 따라 제조된 땅콩 겉껍질 추출물로 처리 시 항비만 기작의 핵심 단백질인 SREBP-1c, PPAR-γ및 FAS의 발현을 나타내는 웨스턴 블롯이다.
도 6a 내지 도 6c는 상기 도 5에서 SREBP-1c, PPAR-γ및 FAS의 발현을 정량화한 그래프이다. Figure 1a is a response surface curve showing the change in total polyphenol activity of the extract according to ethanol concentration and extraction time, and Figure 1b is a response surface curve showing the change in total polyphenol activity of the extract according to ethanol concentration and extraction temperature.
Figure 2a is a response surface curve showing the change in total flavonoid activity of the extract according to ethanol concentration and extraction time, and Figure 2b is a response surface curve showing the change in total flavonoid activity of the extract according to ethanol concentration and extraction temperature.
Figure 3a is a response surface curve showing the change in DPPH radical scavenging ability of the extract according to ethanol concentration and extraction time, and Figure 3b is a response surface curve showing the change in DPPH radical scavenging ability of the extract according to ethanol concentration and extraction temperature.
Figure 4 is a graph showing the response surface curves of Figures 1, 2, and 3 using an overlapping method to find the optimal ultrasonic extraction conditions for peanut skins manufactured according to an embodiment of the present invention.
Figure 5 is a Western blot showing the expression of SREBP-1c, PPAR-γ, and FAS, which are key proteins of the anti-obesity mechanism, when treated with peanut skin extract prepared according to Example 18 of the present invention.
Figures 6A to 6C are graphs quantifying the expression of SREBP-1c, PPAR-γ, and FAS in Figure 5.
본 발명은 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 개선, 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for improving, preventing or treating obesity containing peanut skin extract as an active ingredient.
이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 비만을 개선, 예방 또는 치료할 수 있는 조성물은 땅콩 겉껍질 추출물을 유효성분으로 포함한다.The composition that can improve, prevent or treat obesity of the present invention includes peanut skin extract as an active ingredient.
상기 땅콩(Penut, Arachis hypogaea)에서 발생되는 부산물인 땅콩 겉껍질은 종실을 보호하는 것으로서, 땅콩 겉껍질에는 염증질환이나 암과 같은 여러 질병에 효과가 있는 루테올린과 함께 플라보노이드 계열 화합물로서 강력한 항산화 효능이 있는 에리오딕티올을 함유되어 있다. Peanut outer skin, a by-product produced from the peanut ( Arachis hypogaea ), protects the seed. Peanut outer skin contains luteolin, which is effective in various diseases such as inflammatory diseases and cancer, and a flavonoid compound with strong antioxidant effect. Contains eriodictyol.
본 발명은 폐기되는 땅콩 겉껍질을 이용하는 것으로서, 종실을 제거한 땅콩 겉껍질만을 이용한다.The present invention uses discarded peanut shells, and only the peanut shells from which the seeds have been removed are used.
상기 땅콩 겉껍질은 추출용매 하에서 초음파처리를 통해 추출되는 것으로서, 구체적으로 땅콩 겉껍질과 추출용매가 1 : 5 내지 25의 중량비, 바람직하게는 1 : 8 내지 15의 중량비로 혼합되어 20 내지 50 kHz, 바람직하게는 30 내지 40 kHz의 진동수 및 50 내지 700 W, 바람직하게는 150 내지 400 W 파워의 초음파기로 40 내지 100 ℃, 바람직하게는 50 내지 95 ℃하에서 5 내지 60분, 바람직하게는 15 내지 50분 동안 처리된다. The peanut outer shell is extracted through ultrasonic treatment under an extraction solvent. Specifically, the peanut outer skin and the extraction solvent are mixed at a weight ratio of 1:5 to 25, preferably 1:8 to 15, and extracted at 20 to 50 kHz. , preferably at a frequency of 30 to 40 kHz and a power of 50 to 700 W, preferably 150 to 400 W, at 40 to 100°C, preferably 50 to 95°C, for 5 to 60 minutes, preferably 15 to 15°C. Processed for 50 minutes.
땅콩 겉껍질을 추출 시 초음파 추출이 아니라 열수 추출인 경우에는 유효성분이 소량 추출될 뿐만 아니라 항비만 및 항산화 효과가 낮을 수 있다.When extracting peanut skins, if hot water extraction is used instead of ultrasonic extraction, not only a small amount of active ingredients may be extracted, but the anti-obesity and antioxidant effects may be low.
상기 땅콩 겉껍질과 추출용매의 중량비가 상기 범위를 벗어나는 경우에는 추출물에 땅콩 겉껍질의 유효성분이 적은 양으로 추출될 수 있다. If the weight ratio of the peanut skin and the extraction solvent is outside the above range, the active ingredient of the peanut skin may be extracted in a small amount in the extract.
또한, 초음파기의 진동수 및 파워가 상기 하한치 미만인 경우에는 땅콩 겉껍질의 유효성분이 적은 양으로 추출될 수 있으며, 상기 상한치 초과인 경우에는 유효물질의 열분해, 중합 또는 유효성분 외에 다른 물질도 다량으로 추출되어 효과가 저하될 수 있다. In addition, if the frequency and power of the ultrasonicator are below the above lower limit, the active ingredients of the peanut skin can be extracted in a small amount, and if it is above the above upper limit, a large amount of substances other than the active ingredients may be extracted due to thermal decomposition, polymerization, or other substances in addition to the effective ingredients. Effectiveness may be reduced.
또한, 추출온도 및 추출시간이 상기 하한치 미만인 경우에는 땅콩 겉껍질의 유효성분이 적은 양으로 추출될 수 있으며, 상기 상한치 초과인 경우에는 독성물질이 발생할 수 있다.In addition, if the extraction temperature and extraction time are less than the above lower limit, the active ingredients of the peanut skin may be extracted in a small amount, and if the extraction temperature and extraction time exceed the above upper limit, toxic substances may be generated.
상기 추출물을 추출하는 추출용매는 물, 탄소수 1 내지 4의 저급알코올, 에틸렌글리콜, 에틸에테르 또는 이들의 혼합용매이다. 상기 저급알코올로는 20 내지 99 부피%의 메탄올, 에탄올, 부탄올 또는 프로판올 수용액을 들 수 있으며, 바람직하게는 우수한 항비만 효과를 위하여 40 내지 99 부피%의 에탄올 수용액을 들 수 있다.The extraction solvent for extracting the extract is water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The lower alcohol may include a 20 to 99 vol% aqueous solution of methanol, ethanol, butanol or propanol, and preferably a 40 to 99 vol% aqueous ethanol solution for excellent anti-obesity effect.
본 발명의 땅콩 겉껍질물 추출물은 약학 조성물 외에 건강기능식품에도 사용될 수 있다.The peanut skin extract of the present invention can be used in health functional foods in addition to pharmaceutical compositions.
또한, 본 발명은 반응표면분석법을 이용하여 항산화용 땅콩 겉껍질 초음파 추출물을 제조하는 방법을 제공한다.Additionally, the present invention provides a method for producing an antioxidant peanut skin ultrasonic extract using response surface analysis.
본 발명의 반응표면분석법을 이용한 땅콩 겉껍질 초음파 추출물의 제조방법은 (A) 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매인 추출용매, 40 내지 100 ℃의 추출온도, 5 내지 60분의 추출시간의 추출조건 하에서 20 내지 50 kHz의 진동수 및 50 내지 700 W의 파워의 초음파기로 추출한 후 원심분리하여 수득한 상등액의 DPPH 라디칼 소거능에 대한 실험값을 획득하는 단계; (B) 상기 (A)단계의 실험값을 이용하여 2차 회귀식 모델을 도출하는 단계; (C) 상기 (B)단계에서 도출된 2차 회귀식 모델을 변량분석(ANOVA)하여 신뢰도를 입증하고 상기 추출조건에 대한 반응표면곡선을 수득하는 단계; (D) 상기 (A)단계에서 수득한 상등액의 총 폴리페놀 함량에 대한 실험값을 획득하는 단계; (E) 상기 (D)단계의 실험값을 이용하여 하기 2차 회귀식 모델을 도출하는 단계; (F) 상기 (E)단계에서 도출된 2차 회귀식 모델을 변량분석(ANOVA)하여 신뢰도를 입증하고 상기 추출조건에 대한 반응표면곡선을 수득하는 단계; (G) 상기 (A)단계에서 수득한 상등액의 총 플라보노이드 함량에 대한 실험값을 획득하는 단계; (H) 상기 (G)단계의 실험값을 이용하여 2차 회귀식 모델을 도출하는 단계; (I) 상기 (H)단계에서 도출된 2차 회귀식 모델을 변량분석(ANOVA)하여 신뢰도를 입증하고 상기 추출조건에 대한 반응표면곡선을 수득하는 단계; 및 (J) 상기 (C), (F) 및 (I)단계에서 수득된 반응표면곡선을 겹치기기법(superimposing)으로 DPPH 라디칼 소거능, 총 폴리페놀 함량 및 총 플라보노이드 함량에 대한 공통의 최적화 조건을 예측하는 단계;를 포함한다. 이렇게 예측된 조건을 이용하여 땅콩 겉껍질을 초음파 추출하면 DPPH 라디칼 소거능, 총 폴리페놀 함량 및 총 플라보노이드 함량에 대하여 우수한 효과를 보인다. The method for producing peanut skin ultrasonic extract using the response surface analysis method of the present invention is (A) an extraction solvent that is water, a lower alcohol with 1 to 4 carbon atoms, or a mixed solvent thereof, an extraction temperature of 40 to 100 ° C., and 5 to 60 minutes. Obtaining experimental values for the DPPH radical scavenging ability of the supernatant obtained by centrifugation after extraction with an ultrasonicator with a frequency of 20 to 50 kHz and a power of 50 to 700 W under extraction conditions of an extraction time of; (B) deriving a quadratic regression model using the experimental values of step (A); (C) performing analysis of variance (ANOVA) on the quadratic regression model derived in step (B) to prove reliability and obtain a response surface curve for the extraction conditions; (D) obtaining an experimental value for the total polyphenol content of the supernatant obtained in step (A); (E) deriving the following quadratic regression model using the experimental values of step (D); (F) performing analysis of variance (ANOVA) on the quadratic regression model derived in step (E) to prove reliability and obtain a response surface curve for the extraction conditions; (G) obtaining an experimental value for the total flavonoid content of the supernatant obtained in step (A); (H) deriving a quadratic regression model using the experimental values of step (G); (I) performing analysis of variance (ANOVA) on the quadratic regression model derived in step (H) to prove reliability and obtain a response surface curve for the extraction conditions; and (J) predicting common optimization conditions for DPPH radical scavenging ability, total polyphenol content, and total flavonoid content by superimposing the response surface curves obtained in steps (C), (F), and (I). It includes; Ultrasonic extraction of peanut skin using these predicted conditions shows excellent effects on DPPH radical scavenging ability, total polyphenol content, and total flavonoid content.
상기 DPPH 라디칼 소거능, 총 폴리페놀 함량 및 총 플라보노이드 함량에 대한 공통의 최적화 조건에 따른 추출조건은 추출 시간 35.8분, 추출 온도 82.7 ℃ 및 에탄올 농도 96.0 부피%이다.The extraction conditions according to the common optimization conditions for the DPPH radical scavenging ability, total polyphenol content, and total flavonoid content are extraction time of 35.8 minutes, extraction temperature of 82.7°C, and ethanol concentration of 96.0% by volume.
본 발명에서 사용되는 용어 ‘추출물’은 상기 용매를 이용하여 땅콩 겉껍질에 포함된 성분을 추출한 추출물, 이들로부터 분획한 분획물, 이들 추출물 또는 분획물을 추가적으로 농축한 농축물, 이를 정제 또는 분리한 정제물도 포함하고, 상기 추출물, 분획물, 농축물 또는 정제물을 건조한 건조물 또는 그를 분쇄한 분말을 포함하는 의미로 사용된다. The term 'extract' used in the present invention refers to extracts obtained by extracting components contained in the outer shell of peanuts using the above solvent, fractions separated from them, concentrates obtained by additionally concentrating these extracts or fractions, and purified products obtained by purifying or separating them. It is used to include the dried product of the extract, fraction, concentrate or purified product or the powder obtained by pulverizing the same.
상기 정제물의 제조를 위해 분자량 컷-오프 값을 갖는 한외 여과막을 통과시키거나, 또는 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 부가할 수 있다.For the preparation of the purified product, various additional methods are used, such as passage through an ultrafiltration membrane with a molecular weight cut-off value, or separation by various chromatographs (designed for separation according to size, charge, hydrophobicity or affinity). Purification methods can be added.
본 발명은 땅콩 겉껍질 추출물을 유효성분으로 포함하는 비만의 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for preventing or improving obesity containing peanut skin extract as an active ingredient.
상기 ‘식품 조성물’은 유효성분으로 땅콩 겉껍질 추출물 이외에, 식품 제조에 통상적으로 사용되는 식품의 기준 및 규격(‘식품공전’)에 기재된 식품으로 사용가능한 식품 원료, 식품첨가물 공전에 기재된 식품첨가물을 포함한다.The 'food composition' includes, in addition to peanut shell extract as an active ingredient, food raw materials usable as foods and food additives listed in the Food Additives Code as described in the Food Standards and Specifications ('Food Code of Standards') commonly used in food production. Includes.
특별히 한정할 필요는 없으나 예를 들어 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상기 탄수화물은 단당류, 예를 들어, 포도당, 과당 등; 이당류, 예를 들어 말토스, 설탕, 유당 등; 올리고당 또는 폴리사카라이드, 예를 들어 덱스트린, 물엿, 사이클로덱스트린 등; 당알코올, 예를 들어 자일리톨, 소르비톨, 에리트리톨 등을 사용할 수 있다. 상기 향미제는 천연 향미제[타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다.There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).
상기 땅콩 겉껍질 추출물을 유효성분으로 식품 조성물을 제조하는 경우에 땅콩 겉껍질 추출물은 비만 질환을 예방 또는 치료할 수 있는 함량이면 특별히 한정할 필요는 없으나, 예를 들어 0.1 내지 99 중량%, 0.5 내지 95 중량%, 1 내지 90 중량%, 2 내지 80 중량%, 3 내지 70 중량%, 4 내지 60 중량%, 5 내지 50 중량%로 포함될 수 있다.When manufacturing a food composition using the peanut skin extract as an active ingredient, there is no need to specifically limit the content of the peanut skin extract as long as it can prevent or treat obesity diseases, for example, 0.1 to 99% by weight, 0.5 to 95% by weight. It may be included in weight%, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, and 5 to 50% by weight.
상기 식품 조성물에서 유효성분인 땅콩 겉껍질 추출물은 섭취자의 상태, 체중, 질병의 유무나 정도 및 기간에 따라 다르지만, 통상의 기술자에 의해 적절하게 선택될 수 있다. 예들 들어 1일 투여량을 기준으로 1 내지 5,000 mg, 바람직하게는 5 내지 2,000 mg, 더욱 바람직하게는 10 내지 1,000 mg, 더더욱 바람직하게는 20 내지 800 mg, 가장 바람직하게는 50 내지 500 mg일 수 있고, 투여 횟수는 특별히 한정할 필요는 없으나 1일 3회 내지 1주일에 1회의 범위 내에서 통상의 기술자가 조절할 수 있다. 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있다.The peanut skin extract, which is an active ingredient in the food composition, varies depending on the condition, body weight, and presence, degree, and duration of the disease of the consumer, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.
상기 식품 조성물은 특별히 한정할 필요는 없으나 예를 들어 산제, 과립제, 정제, 캡슐제, 환제, 엑스제, 젤리 제형, 티백 제형 또는 음료 제형일 수 있다.The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.
또한 일반 식품에 비만의 예방 또는 개선의 기능성을 부여하기 위하여 상기 땅콩 겉껍질 추출물을 첨가할 수 있으며, 첨가가 가능한 식품은, 특별히 한정할 필요는 없으나 예를 들어 식품위생법 제7조에 따른 식품의 기준 및 규격(‘식품공전’)에 예시된 과자류, 빵 또는 떡류, 코코아가공품류 또는 초콜릿류, 식육 또는 알가공품, 어육가공품, 두부류 또는 묵류, 면류, 다류, 커피, 음료류, 특수용도식품, 장류, 조미식품, 드레싱류, 김치류, 젓갈류, 절임식품, 조림식품, 주류, 건포류, 기타 식품류 등에 첨가될 수 있다. 또한 축산물위생관리법 제4조에 따른 축산물의 가공기준 및 성분규격(‘축산물공전’)에 예시된 유가공품, 식육가공품 및 포장육, 알가공품에 첨가될 수 있다.In addition, the peanut shell extract can be added to general foods to provide functionality for preventing or improving obesity, and there is no need to specifically limit the foods that can be added, but for example, the food standards under Article 7 of the Food Sanitation Act and confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish products, tofu or jelly, noodles, tea, coffee, beverages, special-purpose foods, sauces, etc. as exemplified in the standards ('Food Code of Conduct'). It can be added to seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.
한편, 상기 땅콩 겉껍질 추출물을 유효성분으로 하는 식품 조성물은 단독으로 “비만의 예방 또는 개선용 건강기능식품”으로 이용될 수 있다. Meanwhile, the food composition containing the peanut skin extract as an active ingredient can be used alone as a “health functional food for preventing or improving obesity.”
상기 ‘건강기능식품’은 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 법적 기준에 따라 제조(가공을 포함)한 식품(건강기능식품에 관한 법률 제3조 제1호)을 말한다. 상기 ‘건강기능식품’은 국가마다 용어나 범위에 차이가 있을 수 있으나, 미국의 ‘식이 보충제(Dietary Supplement)’, 유럽의 ‘식품 보충제(Food Supplemnet)’, 일본의 ‘보건기능식품’ 또는 ‘특정보건용식품(Food for Special Health Use, FoSHU)’, 중국의 ‘보건식품’ 등에 해당할 수 있다.The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.
상기 식품 조성물 또는 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, 식품첨가물로서의 적합여부는 다른 규정이 없는 한 ‘식품첨가물공전’의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 따른다.The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.
또한 상기 건강기능식품에는 상기 땅콩 겉껍질 추출물과 함께 “비만의 예방 또는 개선용 건강기능식품”에 사용되는 ‘기능성 원료’로 고시된 원료 또는 개별인정된 원료로서, Lactobacillus gasseri BNR17, L-카르니틴타르트 레이트, 가르시니아캄보지아껍질추출물, 공액리놀렌산(유리지방산), 공액리놀렌산(트리글리세라이드), 그린마떼추출물, 그린커피빈추출물, 깻잎추출물, 녹차추출물, 대두배아추출물 등 복합물, 돌외잎주정추출분말, 락토페린(우유정제단백질), 레몬 밤 추출물 혼합분말, 마테열수추출물, 미역 등 복합추출물(잔티젠), 발효식초석류복합물, 보이차추출물, 서목태(쥐눈이콩) 펩타이드 복합물, 식물성유지 디글리세라이드, 와일드망고 종자추출물, 중쇄지방산(MCFA)함유 유지, 콜레우스포스콜리추출물, 키토산, 키토올리고당, 풋사과추출폴리페놀, 핑거루트추출분말, 히비스커스 복합추출물 등의 체지방감소와 관련된 건강기능식품 소재를 복합하여 비만의 예방 또는 개선용 건강기능식품을 제조할 수 있다.In addition, the above-mentioned health functional food includes raw materials notified as 'functional raw materials' or individually recognized raw materials used in "health functional food for preventing or improving obesity" along with the above-mentioned peanut shell extract, Lactobacillus gasseri BNR17, L-carnitine tart. Complexes such as late, garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green mate extract, green coffee bean extract, perilla leaf extract, green tea extract, soybean germ extract, ginseng leaf alcohol extract powder, lactoferrin ( Milk purified protein), lemon balm extract mixed powder, mate thermal water extract, seaweed and other complex extracts (xanthigen), fermented vinegar pomegranate complex, pu-erh tea extract, soybean peptide complex, vegetable oil diglyceride, wild mango seed Prevents obesity by combining health functional food ingredients related to body fat reduction, such as extracts, medium-chain fatty acid (MCFA)-containing fats and oils, Coleus forskohlii extract, chitosan, chitooligosaccharide, green apple extract polyphenol, finger root extract powder, and hibiscus complex extract. Alternatively, health functional foods for improvement can be manufactured.
또한 본 발명은 인간, 또는 인간을 제외한 동물에게 상기 조성물을 투여하는 비만의 치료방법을 제공한다.Additionally, the present invention provides a method of treating obesity by administering the composition to humans or non-human animals.
또한 본 발명은 비만의 예방 또는 치료용 의약, 또는 동물용 의약 제조를 위한 땅콩 겉껍질 추출물의 신규 용도를 제공한다.In addition, the present invention provides a new use of peanut skin extract for the manufacture of medicine for preventing or treating obesity, or medicine for animals.
상기 ‘약학 조성물’또는 ‘의약’은 유효성분으로 땅콩 겉껍질 추출물 이외에, 약학 조성물 등의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The ‘pharmaceutical composition’ or ‘medicine’ may further include appropriate carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions, etc., in addition to peanut skin extract as an active ingredient.
상기 ‘담체’는 세포 또는 조직 내로의 화합물의 부가를 용이하게 하는 화합물이다. 상기 ‘희석제’는 대상 화합물의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 화합물을 용해시키게 되는 물에서 희석되는 화합물이다. The ‘carrier’ is a compound that facilitates the addition of the compound into cells or tissues. The ‘diluent’ is a compound diluted in water that not only stabilizes the biologically active form of the target compound, but also dissolves the compound.
상기 담체, 부형제 및 희석제로는 특별히 한정할 필요는 없으나 예를 들어, 유당, 포도당, 설탕, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다.There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, 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.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약의 사용량은 환자 또는 치료대상 동물의 나이, 성별, 체중에 따라 달라질 수 있으며, 무엇보다도, 치료대상 개체의 상태, 치료 대상 질환의 특정한 카테고리 또는 종류, 투여 경로, 사용되는 치료제의 속성에 의존적일 것이다.The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, sex, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 체내에서 활성성분의 흡수도, 배설속도, 환자 또는 치료대상 동물의 연령 및 체중, 성별 및 상태, 치료할 질병의 중증정도 등에 따라 적절히 선택되나, 일반적으로 1일 0.1 내지 1,000 mg/kg, 바람직하게는 1 내지 500 mg/kg, 더욱 바람직하게는 5 내지 250 mg/kg, 가장 바람직하게는 10 내지 100 mg/kg으로 투여하는 것이 바람직하다. 이렇게 제형화 된 단위 투여형 제제는 필요에 따라 일정시간 간격으로 수회 투여할 수 있다.The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , it is generally preferred to administer 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 개별적으로 예방제 또는 치료제로서 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다.The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent, or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
상기 약학조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 트로키제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구 제형으로 제형화하여 사용될 수 있다. 제형화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical compositions, medicines, pharmaceutical compositions for animals, or medicines for animals can be used by formulating them into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods. there is. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 카보네이트, 설탕 또는 유당, 젤라틴 등을 섞어 조제될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .
상기 비만의 치료방법은 인간, 또는 인간을 제외한 동물, 특히 포유동물에게 상기 조성물을 투여 하는 것으로, 예를 들어 비만인 치료대상 개체에게 상기 조성물을 투여하는 것이다.The method for treating obesity is administering the composition to humans or non-human animals, especially mammals, for example, administering the composition to an obese subject to be treated.
상기 치료를 위한 투여량, 투여 방법 및 투여 횟수는 상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약의 투여량, 투여 방법 및 투여 횟수를 참고할 수 있다.The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.
실시예 1 내지 17. Examples 1 to 17.
국내산 피땅콩을 수세하여 종실을 제거한 후 얻은 땅콩 겉껍질을 60 ℃ 건조오븐에서 24 시간 건조하여 더 이상의 중량이 낮아지지 않는 것을 확인하고 건조한 시료를 분쇄기로 분쇄한 후 25 mesh로 여과하였다. 상기 분쇄된 땅콩 겉껍질과 농도가 상이한 에탄올 수용액을 1 : 10의 중량비로 혼합하여 초음파기에 투입 후 초음파(40 kHz, 200 W)를 이용하여 추출하였다. After washing domestic blood peanuts and removing the seeds, the obtained peanut outer shell was dried in a drying oven at 60°C for 24 hours to confirm that the weight did not decrease further. The dried sample was pulverized with a grinder and filtered through a 25 mesh. The pulverized peanut shells and ethanol aqueous solutions of different concentrations were mixed at a weight ratio of 1:10, placed in an ultrasonicator, and extracted using ultrasonic waves (40 kHz, 200 W).
이때 추출 공정의 추출시간, 추출온도 및 에탄올 농도를 다르게 하여 각각의 추출물을 수득하였다.At this time, each extract was obtained by varying the extraction time, extraction temperature, and ethanol concentration of the extraction process.
[항산화][Antioxidant]
<시험예 I><Test Example I>
시험예 1. DPPH 라디칼 소거능, 총 폴리페놀(TPC) 및 총 플라보노이드(TFC) 측정Test Example 1. Measurement of DPPH radical scavenging ability, total polyphenols (TPC) and total flavonoids (TFC)
1-1. DPPH 라디칼 소거능(%): DPPH(2,2-Diphenyl-1-picrylhydrazyl) free radical 소거활성을 측정하는 Lee등의 방법을 변형하여 사용하였다. 라디칼이 항산화 물질로 인하여 환원되면 고유의 보라색을 잃고 노란색으로 변색되는데 이를 흡광도로 계산하여 라디칼 소거능을 측정하였다. DPPH 시약을 MtOH로 희석하여 분광광도계를 이용해 517 nm에서 O.D 값을 1.0로 맞춰 DPPH 희석액을 준비한다. 준비한 시료 0.25 ml에 희석한 DPPH 시약 1.25 ml를 가하여 암실에서 20분간 정치반응 후 원심분리하여 517 nm에서 흡광도를 측정하고 대조군과 비교하여 아래와 같이 free radical 소거활성을 백분율로 표현하였다. 대조군으로는 ascorbic acid를 이용하였다.1-1. DPPH radical scavenging activity (%): A modified method of Lee et al. to measure DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging activity was used. When radicals are reduced by antioxidants, they lose their original purple color and change color to yellow. This was calculated as absorbance to measure the radical scavenging ability. Prepare a DPPH dilution solution by diluting the DPPH reagent with MtOH and setting the O.D. value to 1.0 at 517 nm using a spectrophotometer. 1.25 ml of diluted DPPH reagent was added to 0.25 ml of the prepared sample, allowed to stand in the dark for 20 minutes, centrifuged, and absorbance was measured at 517 nm. The free radical scavenging activity was expressed as a percentage compared to the control group as follows. Ascorbic acid was used as a control.
[수학식 1][Equation 1]
라디칼 소거활성(%)={1-(Abs(test)-Abs(color))/Abs(control)}×100Radical scavenging activity (%)={1-(Abs (test) -Abs (color) )/Abs (control) }×100
1-2. 총 폴리페놀: 총 폴리페놀(Total Polyphenol Content) 함량은 변형된 Folin-Ciocalteu’s 방법을 이용하여 측정하였다. Folin-Ciocalteu’s 페놀 용액을 시료에 첨가하여 폴리페놀 화합물에 의해 환원되어 청색으로 발색되는 원리에 근거하여 흡광도 변화로 시료의 총 폴리페놀 농도를 측정하였다. 각 시료 0.14 mL를 취하고 0.2 N F.C 페놀용액을 0.7 mL 가하여 8분간 실온에 반응시켰다. 0.56 mL의 7.5% Sodium carbonate 용액을 첨가하여 실온에서 1시간 반응시킨 후 10초간 vortexing하고 분광광도계로 756 nm에서 흡광도를 측정하였다. 표준물질로 Gallic acid를 사용하였고 총 폴리페놀 함량은 gallic acid를 농도별로 희석하여 검량선을 작성한 후 mg Gallic Acid Equivalent (GAE) / g Dried Material (DM)으로 표시하였다. 1-2. Total polyphenol: Total polyphenol content was measured using a modified Folin-Ciocalteu’s method. Folin-Ciocalteu's phenol solution was added to the sample, and the total polyphenol concentration of the sample was measured by the change in absorbance based on the principle that the polyphenol compound is reduced and turns blue. Take 0.14 mL of each sample, add 0.7 mL of 0.2 N F.C. phenol solution, and react at room temperature for 8 minutes. 0.56 mL of 7.5% sodium carbonate solution was added and reacted at room temperature for 1 hour, vortexed for 10 seconds, and absorbance was measured at 756 nm with a spectrophotometer. Gallic acid was used as a standard material, and the total polyphenol content was expressed as mg Gallic Acid Equivalent (GAE) / g Dried Material (DM) after creating a calibration curve by diluting gallic acid by concentration.
1-3. 총 플라보노이드: 총 플라보노이드(Total Flavonoid Content) 함량은 Zhishen 등의 방법을 변형하여 사용하였다. 플라보노이드에 알칼리를 작용시키면 황색으로 발색되는 원리에 근거하여 흡광도를 측정해 총 플라보노이드 농도를 측정하였다. 각 시료 0.5 mL에 DW 2.5 ml와 99% 에탄올 1.5 ml 가한 후 1M potassium acetate 0.1 ml와 10% aluminum chloride 0.1 ml를 가하여 vortexing 후 실온에서 30분간 방치하였다. 415 nm에서 흡광도를 측정하였으며 quercetin을 200, 100, 50, 25 ul/ml로 농도 별로 희석하여 검량선을 구하여 플라보노이드 함량을 mg Quercetin Equivalent (QE) / g Dried Material (DM)로 나타내었다. 1-3. Total flavonoid: Total flavonoid content was determined by modifying the method of Zhishen et al. The total flavonoid concentration was measured by measuring absorbance based on the principle that when alkali is added to flavonoids, they turn yellow. 2.5 ml of DW and 1.5 ml of 99% ethanol were added to 0.5 ml of each sample, then 0.1 ml of 1M potassium acetate and 0.1 ml of 10% aluminum chloride were added, vortexed, and left at room temperature for 30 minutes. Absorbance was measured at 415 nm, and quercetin was diluted by concentration to 200, 100, 50, and 25 ul/ml to obtain a calibration curve, and the flavonoid content was expressed as mg Quercetin Equivalent (QE) / g Dried Material (DM).
(min)Extraction time
(min)
(℃)Extraction temperature
(℃)
(mg GAE/mg DW)Total polyphenols
(mg GAE/mg DW)
(mg QE/mg DW)Total flavonoids
(mg QE/mg DW)
총 폴리페놀 함량에 미치는 조건 탐색Exploration of conditions affecting total polyphenol content
각각의 추출 조건에 따른 추출물의 활성 변화를 반응표면 곡선으로 나타낸 결과는 도 1과 같다. 도 1a는 에탄올 농도와 추출시간에 따른 추출물의 총 폴리페놀 활성 변화를 나타내는 반응표면 곡선이며, 도 1b는 에탄올 농도와 추출온도에 따른 추출물의 총 폴리페놀 활성 변화를 나타내는 반응표면 곡선이다.The results showing the change in activity of the extract according to each extraction condition as a response surface curve are shown in Figure 1. Figure 1a is a response surface curve showing the change in total polyphenol activity of the extract according to ethanol concentration and extraction time, and Figure 1b is a response surface curve showing the change in total polyphenol activity of the extract according to ethanol concentration and extraction temperature.
위 표 1 및 도 1에 도시된 바와 같이, 실험값을 기반하여 최적화 기법으로 예측된 땅콩 겉껍질 추출물의 총 폴리페놀의 최대 수율은 추출 시간 55.0 분, 추출 온도 94.0 ℃ 및 에탄올 농도 74.3%의 조건에서 10.2 mg GAE/g DM으로 확인되었다. As shown in Table 1 and Figure 1 above, the maximum yield of total polyphenols of peanut outer skin extract predicted by optimization technique based on experimental values was under the conditions of extraction time of 55.0 minutes, extraction temperature of 94.0 °C, and ethanol concentration of 74.3%. It was found to be 10.2 mg GAE/g DM.
총 플라보노이드 함량에 미치는 조건 탐색Exploration of conditions affecting total flavonoid content
각각의 추출 조건에 따른 추출물의 활성 변화를 반응표면 곡선으로 나타낸 결과는 도 2와 같다. 도 2a는 에탄올 농도와 추출시간에 따른 추출물의 총 플라보노이드 활성 변화를 나타내는 반응표면 곡선이며, 도 2b는 에탄올 농도와 추출온도에 따른 추출물의 총 플라보노이드 활성 변화를 나타내는 반응표면 곡선이다.The results showing the change in activity of the extract according to each extraction condition as a response surface curve are shown in Figure 2. Figure 2a is a response surface curve showing the change in total flavonoid activity of the extract according to ethanol concentration and extraction time, and Figure 2b is a response surface curve showing the change in total flavonoid activity of the extract according to ethanol concentration and extraction temperature.
플라보노이드는 다양한 생리적 기능을 갖는 페놀계 화합물의 총칭이며 유리 상태로 존재하거나 당류와 결합한 배당체의 형태로 존재하며 열수와 유기용매를 이용한 추출을 통해 생산되어 식품, 화장품 및 의약품 산업에서 널리 이용된다.Flavonoids are a general term for phenolic compounds with various physiological functions and exist in a free state or in the form of glycosides combined with saccharides. They are produced through extraction using hot water and organic solvents and are widely used in the food, cosmetics and pharmaceutical industries.
위 표 1 및 도 2에 도시된 바와 같이, 땅콩 겉껍질 추출물의 총 플라보노이드 활성에 최적 추출 조건을 최적화 기법으로 예측한 결과, 총 플라보노이드의 최대 수율은 추출 시간 49.0 분, 추출 온도 94.0 ℃ 및 에탄올 농도 77.1%의 조건에서 1.18 mg QE/g DM으로 확인되었다. As shown in Table 1 and Figure 2 above, as a result of predicting the optimal extraction conditions for the total flavonoid activity of the peanut outer skin extract using an optimization technique, the maximum yield of total flavonoids was extracted at an extraction time of 49.0 minutes, an extraction temperature of 94.0°C, and an ethanol concentration. It was confirmed to be 1.18 mg QE/g DM in 77.1% of conditions.
추출 온도가 총 플라보노이드 함량에 유의한 영향을 미치나 추출 시간이 미치는 영향이 크지 않다는 것을 재차 확인하였으며 추출 온도에 비해 에탄올 농도에 의존성이 높다는 것을 확인하였다. It was confirmed again that the extraction temperature had a significant effect on the total flavonoid content, but the effect of the extraction time was not significant, and it was confirmed that it was highly dependent on the ethanol concentration compared to the extraction temperature.
DPPH 라디칼 소거능에 미치는 조건 탐색Exploration of conditions affecting DPPH radical scavenging ability
각각의 추출 조건에 따른 추출물의 활성 변화를 반응표면 곡선으로 나타낸 결과는 도 3과 같다. 도 3a는 에탄올 농도와 추출시간에 따른 추출물의 DPPH 라디칼 소거능 변화를 나타내는 반응표면 곡선이며, 도 3b는 에탄올 농도와 추출온도에 따른 추출물의 DPPH 라디칼 소거능 변화를 나타내는 반응표면 곡선이다.The results showing the change in activity of the extract according to each extraction condition as a response surface curve are shown in Figure 3. Figure 3a is a response surface curve showing the change in DPPH radical scavenging ability of the extract according to ethanol concentration and extraction time, and Figure 3b is a response surface curve showing the change in DPPH radical scavenging ability of the extract according to ethanol concentration and extraction temperature.
위 표 1 및 도 3에 도시된 바와 같이, 땅콩 겉껍질 추출물의 총 플라보노이드 활성에 최적 추출 조건을 최적화 기법으로 예측한 결과, DPPH 라디칼 소거능의 최대값은 추출 시간 55.0 분, 추출 온도 81.2 ℃ 및 에탄올 농도 73.2%의 조건에서 89.4%로 확인되었다.As shown in Table 1 and Figure 3 above, as a result of predicting the optimal extraction conditions for the total flavonoid activity of the peanut skin extract using an optimization technique, the maximum value of DPPH radical scavenging ability was extracted at an extraction time of 55.0 minutes, an extraction temperature of 81.2°C, and ethanol. It was confirmed to be 89.4% under the condition of concentration of 73.2%.
최적조건 탐색Search for optimal conditions
땅콩 겉껍질 추출물에 있어 주요 3 조건인 DPPH 라디칼 소거능(RSA), 총 폴리페놀(TPC) 및 총 플라보노이드(TFC)의 개별 최적화 결과를 바탕으로 Design Expert를 이용하여 반응표면곡선을 겹치기기법(superimposing)으로 3개 변수의 공통 최적화 조건을 예측하여 도 6(X1: 추출시간, X2: 추출온도)에 나타내었다. Superimposing the response surface curve using Design Expert based on the individual optimization results of DPPH radical scavenging ability (RSA), total polyphenol (TPC), and total flavonoid (TFC), which are the three main conditions in peanut skin extract. The common optimization conditions of the three variables were predicted and shown in Figure 6 (X 1 : extraction time, X 2 : extraction temperature).
도 4에 도시된 바와 같이, 모든 종속 변수(TPC, TFC, RSA)의 최대화를 동시에 만족하는 최적 조건 중 경제적인 운전 조건을 위한 추출 온도가 최소화된 지점을 선택했을 때, 추출 시간은 35.8 분, 추출 온도는 82.7 ℃ 및 에탄올 농도는 96.0 부피%로 예측되었다. 이때, TPC, TFC 및 RSA는 각각 8.67 mg GAE/g DM, 0.84 mg QE/g DM 및 85.3%로 확인되었다. 통계학적 최적화를 통해 생리활성물질 추출의 최적 조건인 실시예 18은 추출 시간 49.0 분, 추출 온도 94.0 ℃ 및 에탄올 농도 77.1 부피%이며 이를 이용하여 향후 실험을 진행하였다 As shown in Figure 4, when the point where the extraction temperature for economical operating conditions is minimized is selected among the optimal conditions that simultaneously satisfy the maximization of all dependent variables (TPC, TFC, RSA), the extraction time is 35.8 minutes, The extraction temperature was predicted to be 82.7°C and the ethanol concentration was 96.0% by volume. At this time, TPC, TFC, and RSA were confirmed to be 8.67 mg GAE/g DM, 0.84 mg QE/g DM, and 85.3%, respectively. Example 18, which is the optimal condition for extraction of bioactive substances through statistical optimization, is extraction time of 49.0 minutes, extraction temperature of 94.0°C, and ethanol concentration of 77.1% by volume, and future experiments were conducted using these.
[항비만][Anti-obesity]
<시험예 Ⅱ><Test Example Ⅱ>
시험예 2. 리파아제 활성 억제능(LAI) 및 α-글루코시다아제 활성 억제능(GAI)Test Example 2. Lipase activity inhibition ability (LAI) and α-glucosidase activity inhibition ability (GAI) 측정measurement
실시예 1 내지 18에 따라 제조된 추출물을 이용하여 비만의 지표인 리파아제 활성 억제능(Lipase activity inhibition , LAI)와 α-글루코시다아제 활성 억제능(α-Glucosidase activity inhibition, GAI)를 측정하였다.Lipase activity inhibition (LAI) and α-Glucosidase activity inhibition (GAI), indicators of obesity, were measured using the extracts prepared according to Examples 1 to 18.
중심합성계획법(central composite design, CCD)을 이용하여 추출시간(X1), 추출온도(X2), 용매농도(X3)에 대하여, 5단계의 -1.68, -1, 0, 1 및 1.68로 코드화하여 17개 실험범위를 설계하고, 설계된 하기 조건과 앞선 최적화 조건(실시예 18)를 통해 도출된 조건에 따라 항비만 실험을 통해 실험값을 수득하였다.Using central composite design (CCD) , extraction time ( 17 experimental ranges were designed by coding, and experimental values were obtained through anti-obesity experiments according to the designed conditions below and the conditions derived through the previous optimization conditions (Example 18).
2-1. 리파아제 활성 억제능(%): 리파아제 효소 활성 억제능이 우수하면 장내에서 지방의 흡수를 억제할 수 있어 비만 예방에 효과가 있다. 돼지 췌장 리파아제로 lipase 활성 억제능을 측정하는 실험을 수행하였다. 시료를 0.1 mg/mL의 농도로 희석한 후에 0.167 mM의 p-nitrophenylpalmitate용액, 0.061 M의 Tris-HCl buffer (pH 8.5) 및 0.3 mg/mL의 리파아제와 함께 plate에 넣고 25 ℃에서 10 분간 반응시켰다. 반응 후, 405 nm에서 흡광도를 측정하였으며, 대조군(control)은 시료를 용매로 대체하여 실험을 수행하였다. 이후 리파아제 억제활성은 하기 수학식으로 계산하였다.2-1. Lipase activity inhibition ability (%): If the lipase enzyme activity inhibition ability is excellent, it can inhibit the absorption of fat in the intestines and is effective in preventing obesity. An experiment was performed to measure the ability to inhibit lipase activity with porcine pancreatic lipase. After diluting the sample to a concentration of 0.1 mg/mL, it was added to a plate with 0.167 mM p-nitrophenylpalmitate solution, 0.061 M Tris-HCl buffer (pH 8.5), and 0.3 mg/mL lipase and reacted at 25°C for 10 minutes. . After the reaction, the absorbance was measured at 405 nm, and the control experiment was performed by replacing the sample with a solvent. Thereafter, lipase inhibitory activity was calculated using the following equation.
[수학식 2][Equation 2]
리파아제 억제 활성(%) = 1-(시료의 흡광도/control의 흡광도) X 100Lipase inhibitory activity (%) = 1-(absorbance of sample/absorbance of control)
2-2. α-글루코시다아제 활성 억제능(%): 시료 50 ㎕에 0.5 U/ml 알파-글루코시데이즈 효소액 50 ㎕를 첨가하고 여기에 200 mM 인산나트륨 완충액(pH 6.8) 50 ㎕를 혼합하여 37 ℃에서 10 분간 예비 배양한 후, 인산나트륨 완충액(pH 6.8)을 100 ㎕ 가하여 37 ℃에서 10 분간 반응시켰다. 반응액에 100 mM Na2CO3 0.75 ml를 첨가하여 반응을 정지시키고 420 nm에서 흡광도를 측정하였으며 음성 대조구는 시료 대신 증류수를 사용하여 동일한 방법으로 진행하여 흡광도의 차이를 하기 수학식에 의해 백분율(%)로 산출하였다.2-2. α-Glucosidase activity inhibition ability (%): Add 50 ㎕ of 0.5 U/ml alpha-glucosidase enzyme solution to 50 ㎕ of sample, mix with 50 ㎕ of 200 mM sodium phosphate buffer (pH 6.8), pre-incubate at 37°C for 10 minutes, and then incubate in sodium phosphate buffer (pH 6.8). 6.8) was added in 100 ㎕ and reacted at 37°C for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na 2 CO 3 to the reaction solution, and the absorbance was measured at 420 nm. The negative control was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was expressed as a percentage ( %) was calculated.
[수학식 3][Equation 3]
알파-글루코시다아제 저해활성(%) = [1-(음성대조구 흡광도 ÷ 실험구 흡광도)] X 100Alpha-glucosidase inhibitory activity (%) = [1-(absorbance of negative control group ÷ absorbance of experimental group)]
(min)Extraction time
(min)
(℃)Extraction temperature
(℃)
위 표 2에 나타낸 바와 같이, 리파아제 활성 억제능에 대한 실험값은 실시예 18에 따라 제조된 추출물이 다른 군에 비하여 우수한 것을 확인하였고, α-글루코시다아제 활성 억제능은 실시예 15에 따라 제조된 추출물이 다른 군에 비하여 우수한 것을 확인하였다. 항비만에 효과를 가지는 두 가지 효소에 대한 억제 활성은 실시예 18이 다른 군에 비하여 우수한 것으로 나타났다. 특히, 리파아제 저해제로 시판되고 있는 orlistat는 85.74±1.35%의 저해율을 나타내므로 실시예 18에 따라 제조된 추출물이 orlistat 대비 유의적으로 높은 저해 활성을 나타내는 것을 확인하였다. 양성대조군에 상당하는 높은 억제효과를 나타내며 리파아제 저해 활성이 높은 땅콩 겉껍질 추출물로부터 항비만 활성을 기대할 수 있다.As shown in Table 2 above, the experimental values for the ability to inhibit lipase activity confirmed that the extract prepared according to Example 18 was superior to the other groups, and the ability to inhibit α-glucosidase activity was found to be excellent for the extract prepared according to Example 15. It was confirmed that it was superior to other groups. The inhibitory activity of Example 18 against two enzymes effective in anti-obesity was found to be superior to that of the other groups. In particular, orlistat, commercially available as a lipase inhibitor, showed an inhibition rate of 85.74 ± 1.35%, so it was confirmed that the extract prepared according to Example 18 showed significantly higher inhibitory activity than orlistat. Anti-obesity activity can be expected from peanut skin extract, which shows a high inhibitory effect equivalent to that of the positive control group and has high lipase inhibitory activity.
또한, 알파 글루코시데이즈 저해제로 알려진 acarbose는 86.65±0.24%의 저해율을 나타내므로 실시예 18에 따라 제조된 추출물이 acarbose 대비 유의적으로 높은 저해 활성을 보여 항비만 및 식후 혈당 조절제로서의 활용 가능성이 뛰어날 것으로 판단된다.In addition, acarbose, known as an alpha-glucosidase inhibitor, exhibits an inhibition rate of 86.65 ± 0.24%, so the extract prepared according to Example 18 showed significantly higher inhibitory activity than acarbose, showing excellent potential for use as an anti-obesity and postprandial blood sugar control agent. It is judged that
반면, 정제수를 사용한 실시예 13은 리파아제 활성 억제능 및 α-글루코시다아제 활성 억제능 모두 수치가 낮은 것을 확인하였다. On the other hand, in Example 13 using purified water, it was confirmed that both lipase activity inhibition ability and α-glucosidase activity inhibition ability were low.
리파아제 활성 억제능 및 α-글루코시다아제 활성 억제능이 모두 고르게 우수한 군은 실시예 7, 15와 18인 것을 확인되었으며 실시예 18에서 최대값을 가짐을 확인하였다.It was confirmed that Examples 7, 15, and 18 were the groups that were equally excellent in both lipase activity inhibition ability and α-glucosidase activity inhibition ability, and it was confirmed that Example 18 had the maximum value.
시험예 3. 항비만 유전자 발현 Test Example 3. Anti-obesity gene expression
분화가 완료된 지방전구세포를 인산완충용액으로 세척한 후 추출 키트를 사용하여 전체 RNA를 추출한 후 상기 추출된 전체 RNA를 diethyl pyrocarbonate (DEPC)로 희석하고 260 ㎚에서 흡광도를 측정하여 정량한 다음 RNA (1 μg)에 cDNA synthesis 완충용액과 cDNA synthsis enzyme mix를 혼합하여 역전사 반응하여 cDNA를 합성하였다. 항비만 핵심유전자인 SREBP-1c, PPAR-γ와 FAS 프라이머를 첨가해 RT-PCR을 수행하였다. 지방전구세포는 지방세포로 분화되면서 중성지방 축적에 관여하는 전사인자를 유도하며, 분화 초기과정에서 cAMP의 농도가 증가하게되면 SREMP-1c 발현이 유도됨에 따라 PPAR-γ와 CEBP-α가 상호 발현되고 중성지방 합성에 관여하는 효소인 FAS와 함께 지방세포의 분화를 유도하게 된다.After washing the differentiated preadipocytes with phosphate buffer solution, total RNA was extracted using an extraction kit. The extracted total RNA was diluted with diethyl pyrocarbonate (DEPC) and quantified by measuring absorbance at 260 nm. RNA ( cDNA was synthesized by reverse transcription reaction by mixing cDNA synthesis buffer solution and cDNA synthesis enzyme mix (1 μg). RT-PCR was performed by adding anti-obesity core genes SREBP-1c, PPAR-γ, and FAS primers. As preadipocytes differentiate into adipocytes, they induce transcription factors involved in neutral fat accumulation. When the concentration of cAMP increases in the early stage of differentiation, SREMP-1c expression is induced, and PPAR-γ and CEBP-α are mutually expressed. and induces the differentiation of adipocytes along with FAS, an enzyme involved in neutral fat synthesis.
도 5는 본 발명의 실시예 18에 따라 제조된 땅콩 겉껍질 추출물(1 mg/ml)로 처리 시 SREBP-1c, PPAR-γ및 FAS의 발현을 나타내는 웨스턴 블롯이다. 도 6a 내지 도 6c는 상기 도 5에서 SREBP-1c, PPAR-γ및 FAS의 발현을 정량화한 그래프이다. Figure 5 is a Western blot showing the expression of SREBP-1c, PPAR-γ, and FAS upon treatment with peanut skin extract (1 mg/ml) prepared according to Example 18 of the present invention. Figures 6A to 6C are graphs quantifying the expression of SREBP-1c, PPAR-γ, and FAS in Figure 5.
도 5 및 도 6에 도시된 바와 같이, 지방전구세포에서 지방세포로 분화된 세포를 대조군 1로 이용하였으며, 실시예 18에 따라 제조된 땅콩 겉껍질 추출물 처리군은 대조군 1 대비 SREBP-1c, PPAR-γ와 FAS의 유전자 발현이 유의적으로 감소하는 것을 확인하였다(p < 0.05). As shown in Figures 5 and 6, cells differentiated from preadipocytes into adipocytes were used as control group 1, and the group treated with the peanut skin extract prepared according to Example 18 had higher levels of SREBP-1c and PPAR compared to control group 1. It was confirmed that gene expression of -γ and FAS was significantly decreased ( p < 0.05).
이에 따라, 땅콩 겉껍질 추출물이 보유한 항비만 활성이 지방전구세포 분화과정에 관여하는 핵심 유전자 발현 저해 확인을 통해 지방전구세포와 지방축적 억제에 효능이 있음을 확인하였다. Accordingly, it was confirmed that the anti-obesity activity of peanut skin extract is effective in suppressing preadipocytes and fat accumulation through confirmation of inhibition of the expression of key genes involved in the preadipocyte differentiation process.
시험예 4. 동물실험Test Example 4. Animal testing
200~250 g의 sprague-dawley계 마우스(5주령, 숫컷)를 실험에 사용하였다. 아크릴 케이지(45 X 60 X 25 cm)에서 사육되었으며, 충분한 사료와 물이 공급되며 적절한 인공조도로 12시간의 낮, 밤을 조절하였다(am 8:00부터 낮). 그리고 일정한 온도(20~24℃) 및 습도(45~65%)를 유지시켜 주었다. 바뀐 환경에 적응하도록 일주일간 살펴보며 수면주기를 유지하고, 이상행동을 확인하였다. Sprague-Dawley mice (5 weeks old, male) weighing 200-250 g were used in the experiment. They were raised in acrylic cages (45 And constant temperature (20-24℃) and humidity (45-65%) were maintained. We monitored them for a week to help them adapt to the changed environment, maintained their sleep cycle, and checked for abnormal behavior.
동물의 프로토콜은 선문대학교의 기관 동물 관리 및 사용위원회(IACUC)에 의해 승인되었다. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Sunmoon University.
실험동물은 체중변화가 일정하고 건강한 동물만을 선별하여 임의 배치법에 의해 일반식이를 급여한 정상군, 고지방식이를 급여한 대조군 2, 고지방식이 및 실시예 4의 추출물을 급여한 군으로 나누었으며 실험군마다 8마리씩 사용하였다. The experimental animals were selected by selecting only healthy animals with consistent body weight changes and divided into a normal group fed a normal diet by a random placement method, a control group 2 fed a high-fat diet, and a group fed the high-fat diet and the extract of Example 4. Eight animals were used in each experimental group.
실시예 4의 추출물은 총 식이를 기준으로 5 중량%로 혼합하여 사용하였다.The extract of Example 4 was mixed and used at 5% by weight based on the total diet.
-3개 군의 마우스--3 groups of mice-
정상군: 일반식이 급여 8마리Normal group: 8 animals fed regular diet
대조군: 고지방식이 급여 8마리 Control group: 8 animals fed high-fat diet
실시예 18: 고지방식이 + 실시예 18의 추출물 급여 8마리Example 18: 8 animals fed high-fat diet + extract of Example 18
정상군, 대조군 및 실시예 18에 급여된 식이의 조성은 하기와 같다.The composition of the diet fed to the normal group, control group, and Example 18 is as follows.
4-1. 마우스의 무게 측정4-1. Measure the weight of the mouse
마우스의 초기 무게와 4주 후의 무게를 측정하였다.The initial weight of the mouse and the weight after 4 weeks were measured.
위 표 4에 나타낸 바와 같이, 정상군, 대조군 및 실시예 18군에 있어서 세 군 모두 초기 체중 조건 및 식이 섭취량이 유사하였으나, 4주 후 최종 체중에 있어 실시예 18군이 대조군에 비하여 체중감소 효과가 있는 것을 확인하였다. As shown in Table 4 above, in the normal group, control group, and Example 18 group, the initial body weight conditions and dietary intake were similar for all three groups, but the Example 18 group had a weight loss effect compared to the control group in terms of final body weight after 4 weeks. It was confirmed that there is.
즉, 실시예 18군의 체중 증감을 보면 정상군에 비해서는 증가하였으나 대조군 2에 비해서는 감소된 것을 확인하였다. That is, when looking at the weight gain or loss of the Example 18 group, it was confirmed that the weight increased compared to the normal group but decreased compared to the control group 2.
아래에 본 발명의 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Below, a formulation example of a composition containing the extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.
제제예 1: 산제의 제조Formulation Example 1: Preparation of powder
실시예 4의 추출물 분말 20 mg20 mg of extract powder of Example 4
유당 100 mg100 mg lactose
탈크 10 mgTalc 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled into an airtight bubble to prepare a powder.
제제예 2: 정제의 제조Formulation Example 2: Preparation of tablets
실시예 4의 추출물 분말 10 mg10 mg of extract powder of Example 4
옥수수전분 100 mgCorn starch 100 mg
유당 100 mg100 mg lactose
스테아린산 마그네슘 2 mgMagnesium stearate 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.
제제예 3: 캡슐제의 제조Formulation Example 3: Preparation of capsules
실시예 4의 추출물 분말 10 mg10 mg of extract powder of Example 4
결정성 셀룰로오스 3 mg3 mg crystalline cellulose
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.
제제예 4: 과립제의 제조Formulation Example 4: Preparation of granules
실시예 4의 추출물 분말 1,000 mg1,000 mg of extract powder of Example 4
비타민 혼합물 적량Vitamin mixture dosage
비타민 A 아세테이트 70 ㎍Vitamin A acetate 70 μg
비타민 E 1.0 mgVitamin E 1.0 mg
비타민 B1 0.13 mgVitamin B1 0.13 mg
비타민 B2 0.15 mgVitamin B2 0.15 mg
비타민 B6 0.5 mgVitamin B6 0.5 mg
비타민 B12 0.2 ㎍Vitamin B12 0.2 ㎍
비타민 C 10 mgVitamin C 10 mg
비오틴 10 ㎍Biotin 10 μg
니코틴산아미드 1.7 mgNicotinamide 1.7 mg
엽산 50 ㎍Folic acid 50 μg
판토텐산 칼슘 0.5 mgCalcium pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture appropriate amount
황산제1철 1.75 mgFerrous sulfate 1.75 mg
산화아연 0.82 mgZinc oxide 0.82 mg
탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg
제1인산칼륨 15 mgMonobasic Potassium Phosphate 15 mg
제2인산칼슘 55 mgDibasic calcium phosphate 55 mg
구연산칼륨 90 mgPotassium citrate 90 mg
탄산칼슘 100 mgCalcium carbonate 100 mg
염화마그네슘 24.8 mgMagnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 건강기능식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강기능식품 조성물 제조에 사용할 수 있다.The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a normal health functional food manufacturing method. Next, granules can be prepared and used to manufacture a health functional food composition according to a conventional method.
제제예 5: 음료 제형의 제조Formulation Example 5: Preparation of beverage formulation
실시예 4의 추출물 분말 1,000 mg1,000 mg of extract powder of Example 4
구연산 1,000 mg1,000 mg citric acid
올리고당 100 g100 g oligosaccharides
매실농축액 2 g2 g plum concentrate
타우린 1 g1 g taurine
정제수를 가하여 전체 900 mLAdd purified water to make a total of 900 mL.
통상의 음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1 시간 동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 L 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 기능성 음료 조성물 제조에 사용한다.After mixing the above ingredients according to a typical beverage production method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, stored in the refrigerator, and then used according to the present invention. It is used in the production of functional beverage compositions.
Claims (4)
상기 땅콩 겉껍질 초음파 추출물은 땅콩 겉껍질과, 77.1 부피%의 에탄올 수용액을 94 ℃ 하에서 40 kHz의 진동수 및 200 W의 파워의 초음파기로 49 분 동안 처리한 것을 특징으로 하는 비만의 예방 또는 치료용 약학 조성물.Contains peanut hull ultrasonic extract as an active ingredient,
The peanut skin ultrasonic extract is a pharmaceutical for preventing or treating obesity, characterized in that peanut skin and 77.1% by volume ethanol aqueous solution were treated with an ultrasonicator at a frequency of 40 kHz and a power of 200 W at 94° C. for 49 minutes. Composition.
상기 땅콩 겉껍질 초음파 추출물은 땅콩 겉껍질과, 77.1 부피%의 에탄올 수용액을 94 ℃ 하에서 40 kHz의 진동수 및 200 W의 파워의 초음파기로 49 분 동안 처리한 것을 특징으로 하는 비만의 예방 또는 개선용 식품 조성물. Contains peanut hull ultrasonic extract as an active ingredient,
The peanut skin ultrasonic extract is a food for preventing or improving obesity, characterized in that peanut skin and a 77.1% by volume ethanol aqueous solution were treated for 49 minutes with an ultrasonicator with a frequency of 40 kHz and a power of 200 W at 94 ° C. Composition.
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