KR102617562B1 - Composition for the prevention or treatment of sarcopenia comprising Udo peanut sprout extract as an active ingredient - Google Patents
Composition for the prevention or treatment of sarcopenia comprising Udo peanut sprout extract as an active ingredient Download PDFInfo
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Abstract
본 발명의 목적은 우도 땅콩 추출물을 유효성분으로 포함하는 근육 질환의 예방 또는 치료용 조성물을 제공하는 것으로서, 우도 땅콩 추출물은 지질 축적과 고지방/고자당 식이 유발된 근육 약화를 치료하는 것으로서, 우도 땅콩 추출물은 근육 약화된 마우스 모델에 투여시, 약력과 매달리는 능력을 향상시켰으며, 근육 위축 인자인 MuRF-1(Muscle RING-finger protein-1) 및 근육 위축 F-box(MAFbx/Atrogin-1)의 발현을 억제를 확인하였다. 또한, 골격근 세포(C2C12)에서 미토콘드리아 생합성(biogenesis) 및 기능 활성 마커인 페르옥시좀 증식인자 활성화 수용체 감마 공동활성제 1-알파(peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1-α) 발현을 증가시켜, 골격근 위축을 개선 시키며 미토콘드리아 기능을 활성화 시키는 것을 확인하여, 이와 관련된 사업에 유용하게 사용될 수 있다.The purpose of the present invention is to provide a composition for preventing or treating muscle diseases containing Udo peanut extract as an active ingredient, wherein the Udo peanut extract treats muscle weakness caused by lipid accumulation and high-fat/high-sucrose diet. When administered to a muscle-weakened mouse model, the extract improved strength and hanging ability, and inhibited the muscle atrophy factor MuRF-1 (Muscle RING-finger protein-1) and muscle atrophy F-box (MAFbx/Atrogin-1). Inhibition of expression was confirmed. In addition, it increases the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), a marker of mitochondrial biogenesis and functional activity in skeletal muscle cells (C2C12). It has been confirmed that it improves skeletal muscle atrophy and activates mitochondrial function, so it can be usefully used in related projects.
Description
본 발명의 목적은 우도 땅콩 새싹 추출물을 유효성분으로 함유하는 근감소증의 개선, 예방 또는 치료용 조성물을 제공하는 것이다.The purpose of the present invention is to provide a composition for improving, preventing or treating sarcopenia containing Udo peanut sprout extract as an active ingredient.
근육을 형성하는 근육세포는 근필라멘트로 구성되어 있는데, 이는 세포의 크기를 변하도록 하여 수축을 유발한다. 근육 조직이 수축할 때 장력이 발생하게 되는데, 이를 근력이라고 한다. 근육은 이러한 근력을 통하여 개체의 이동, 자세 유지, 체액분비 등을 담당하는 신체기관이다.The muscle cells that form muscles are composed of myofilaments, which change the size of the cells and cause contraction. When muscle tissue contracts, tension is generated, which is called muscle strength. Muscles are body organs that are responsible for moving an object, maintaining posture, and secreting body fluids through muscle strength.
근 위축증(muscle atrophy) 또는 근 감소증(sarcopenia)은 호르몬 불균형, 심한 부상, 패혈증, 암 및 노화로 인한 여러 가지 이화 상태(catabolic condition)에서 발생하는 근육세포 및 근조직의 크기 및 질량적 손실을 의미한다. 근 위축증 또는 근 감소증은 근육의 손실을 유발하여 근육 약화 및 피로감을 유발하고 합병증을 악화시킨다. 근 위축증 또는 근 감소증은 크레아틴 키나아제(Creatine kinase: CK), 근전도검사, 근육생검, 분자생물학적 유전자검사, 세포유전학적 검사 등을 통해 진단이 이루어진다. 현재 근 위축증 또는 근 감소증을 치료하기 위한 방법으로, 물리치료가 일반적으로 수행되며, 합병증, 기형 및 기능 장애 예방을 위한 작업치료, 호흡치료, 지지요법(supportive therapy)등이 병행되고 있으나, 이러한 치료는 대증요법으로써의 한계를 갖는다는 점에서, 근위축에 대한 치료제 개발이 절실한 상황이다.Muscle atrophy or sarcopenia is a loss of size and mass of muscle cells and muscle tissue that occurs in a variety of catabolic conditions due to hormonal imbalance, severe injury, sepsis, cancer and aging. . Muscular dystrophy or sarcopenia causes muscle loss, causing muscle weakness and fatigue, and worsening complications. Muscular dystrophy or sarcopenia is diagnosed through creatine kinase (CK), electromyography, muscle biopsy, molecular biological genetic testing, and cytogenetic testing. Currently, physical therapy is commonly performed as a method to treat muscular dystrophy or sarcopenia, and occupational therapy, respiratory therapy, and supportive therapy are also used to prevent complications, deformities, and functional disorders. Since it has limitations as a symptomatic treatment, there is an urgent need to develop a treatment for muscle atrophy.
근 위축증 또는 근 감소증 발병의 또 다른 원인으로 보고된 것은 산화물질 증가에 의한 근세포 손상, 스트레스 단백질 발현 감소로 인한 근단백질 생성 및 재생 감소, 프로테아좀-유비퀴틴 활성화에 따른 근단백질 분해 촉진 등이 알려져 있다. 또한, 항염증제로 잘 알려진 합성 글루코코르티코이드인 덱사메타손(DEX)의 투여에 의하여 근육량과 관련된 단백질의 분해가 촉진된다는 사실이 많은 연구에서 보고되었다.Other reported causes of muscular dystrophy or sarcopenia include muscle cell damage due to increased oxidative substances, decreased muscle protein production and regeneration due to decreased expression of stress proteins, and accelerated muscle protein degradation due to proteasome-ubiquitin activation. there is. In addition, many studies have reported that the administration of dexamethasone (DEX), a synthetic glucocorticoid well known as an anti-inflammatory agent, promotes the breakdown of proteins related to muscle mass.
근감소증의 예방, 개선 또는 치료를 위한 하나의 방법으로 운동이 있는데, 이는 단기적으로 골격근의 단백질 합성 능력을 증가시키며 노인들의 근육의 힘이나 운동성을 증가시킨다고 보고되고 있으나 장기적 치료방법으로 부적절하다. 근감소증의 예방, 개선 또는 치료를 위한 다른 방법으로 테스토스테론(Testosterone) 또는 아나볼릭 스테로이드(anabolic steroid)과 같은 약물을 투여하는 것인데, 이는 여성에게는 남성화를 유도하며 남성에게는 전립선 증상(prostate symptoms)을 유도하는 등의 부작용을 나타낸다. 이에 근감소증 예방, 개선 또는 치료를 위한 신규한 방법에 대한 연구가 필요한 실정이다.Exercise is one method to prevent, improve, or treat sarcopenia. It is reported to increase the protein synthesis ability of skeletal muscles in the short term and increase muscle strength or mobility in elderly people, but it is inappropriate as a long-term treatment method. Another method to prevent, improve, or treat sarcopenia is to administer drugs such as testosterone or anabolic steroids, which induce masculinization in women and prostate symptoms in men. It shows side effects such as: Accordingly, research into new methods to prevent, improve, or treat sarcopenia is needed.
덱사메타손은 암과 같은 여러 질병을 치료하는데 사용되지만, 유비퀴틴-프로테아좀 시스템을 통해 골격근에서 단백질 합성 속도를 감소시키고, 단백질 분해 속도를 증가시켜 근 위축증 또는 근 감소증을 유발한다. 2개의 근육 유비퀴틴 리가아제(ubiquitin ligase)는 Atrogin-1 및 MuRF1과 관련이 있는데, 덱사메타손은 Atrogin-1을 통해 MyoD(myogenic differentiation antigen)를 저하시키고, 이와 같은 기전을 통하여 근육량이 감소하게 된다.Dexamethasone is used to treat several diseases such as cancer, but it reduces the rate of protein synthesis and increases the rate of protein degradation in skeletal muscle through the ubiquitin-proteasome system, causing muscular dystrophy or sarcopenia. Two muscle ubiquitin ligases are related to Atrogin-1 and MuRF1. Dexamethasone reduces MyoD (myogenic differentiation antigen) through Atrogin-1, and this mechanism leads to a decrease in muscle mass.
이에, 본 발명자들은 땅콩 새싹 추출물의 생리활성에 대한 연구를 진행하던 중, 우도 땅콩 새싹 추출물이 근감소증을 억제할 수 있음을 확인함에 따라 본 발명을 완성하였다.Accordingly, while conducting research on the physiological activity of peanut sprout extract, the present inventors completed the present invention by confirming that Udo peanut sprout extract can suppress sarcopenia.
본 발명의 목적은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육 재생용 조성물을 제공하는 것이다.The purpose of the present invention is to provide a composition for muscle regeneration containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
또한, 본 발명의 목적은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육 손상 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle damage diseases containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
또한, 본 발명의 목적은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 운동 능력 개선용 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a composition for improving athletic performance containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
아울러, 본 발명의 목적은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육량 증대용 조성물을 제공하는 것이다.In addition, an object of the present invention is to provide a composition for increasing muscle mass containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육 재생용 조성물을 제공한다.The present invention provides a composition for muscle regeneration containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
또한, 본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육 손상 질환 예방 또는 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing or treating muscle damage diseases containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
또한, 본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 운동 능력 개선용 조성물을 제공한다.In addition, the present invention provides a composition for improving athletic performance containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
아울러, 본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육량 증대용 조성물을 제공한다.In addition, the present invention provides a composition for increasing muscle mass containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
본 발명의 우도 땅콩 추출물은 지질 축적과 고지방/고자당 식이 유발된 근육 약화를 치료하는 것으로서, 우도 땅콩 추출물은 근육 약화된 마우스 모델에 투여시, 약력과 매달리는 능력을 향상시켰으며, 근육 위축 인자인 MuRF-1(Muscle RING-finger protein-1) 및 근육 위축 F-box(MAFbx/Atrogin-1)의 발현을 억제를 확인하였다. 또한, 골격근 세포(C2C12)에서 미토콘드리아 생합성(biogenesis) 및 기능 활성 마커인 페르옥시좀 증식인자 활성화 수용체 감마 공동활성제 1-알파(peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1-α) 발현을 증가시켜, 골격근 위축을 개선 시키며 미토콘드리아 기능을 활성화 시키는 것을 확인하여, 이와 관련된 사업에 유용하게 사용될 수 있다.The Udo peanut extract of the present invention treats muscle weakness caused by lipid accumulation and a high-fat/high-sucrose diet. When administered to a muscle-weakened mouse model, the Udo peanut extract improved strength and hanging ability, and was a factor in muscle atrophy. The expression of MuRF-1 (Muscle RING-finger protein-1) and muscle atrophy F-box (MAFbx/Atrogin-1) was confirmed to be suppressed. In addition, it increases the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), a marker of mitochondrial biogenesis and functional activity in skeletal muscle cells (C2C12). It has been confirmed that it improves skeletal muscle atrophy and activates mitochondrial function, so it can be usefully used in related projects.
도 1은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군을 각 6일동안 복강내 주사 후, 체중 변화를 나타낸 도이다.
(control군:식염수 식이한 마우스 모델, Dex군:덱사메타손(dexamethasone) 만 주사한 마우스 모델, Dex+HFHS군:덱사메타손(dexamethasone)주사 및 HFHS:고자당/고지방(high-fat/high-sucrose diet)를 식이한 마우스 모델, Dex+HFHS+PSE군:덱사메타손(dexamethasone)주사 및 HFHS:고자당/고지방(high-fat/high-sucrose diet)식이 및 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE)을 식이한 마우스 모델)
도 2는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 포도당하부검사(Glucose tolerance test)를 나타낸 도이다.
도 3은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 공복 혈당 수치를 나타낸 도이다.
도 4는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 혈장 중성지방 함량을 나타낸 도이다.
도 5는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 혈장 총 콜레스테롤을 나타낸 도이다.
도 6은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 체중대비 앞다리 악력을 나타낸 도이다.
도 7은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 뒷다리, 앞다리의 악력정도를 나타낸 도이다.
도 8은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 몸무게 대비 악력 유지를 나타낸 도이다.
도 9는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 대퇴사두근을 헤마톡실린과 에오신(H&E)염색을 나타낸 도이다.
도 10은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 MIPAR 프로그램을 이용하여 대퇴사두근의 근섬유 크기를 나타낸 도이다.
도 11은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 비복근에서 근육 위축 인자인 MuRF-1, Atrogin-1, Myogenin의 mRNA 발현량을 나타낸 도이다.
도 12는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 비복근에서 MuRF-1, Atrogin-1 및 PGC1-α의 단백질 발현를 나타낸 도이다.
(대조군:GAPDH)
도 13은 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 염증성 마커인 TNF-α, IL-6 및 IL-1β의 RNA 발현량을 나타낸 도이다.
도 14는 각각의 Control군, Dex군, Dex+HFHS군, Dex+HFHS+PSE군들의 대퇴사두근에서 MuRF-1, Atrogin-1 및 Myogenin의 mRNA 발현량을 나타낸 도이다.
도 15는 골격근 세포(C2C12)에 각각의 Control, Dex+HFHS 및 Dex+HFHS+PSE 처리 시, PGC1-α의 단백질 및 mRNA 발현을 나타낸 도이다.
도 16은 골격근 세포(C2C12)에 우도 땅콩 추출물(PSE)을 처리 시, 세포 생존율을 나타낸 도이다.
도 17은 골격근 세포(C2C12)에 각각의 Control, Dex+HFHS 및 Dex+HFHS+PSE 처리 시, MuRF-1, Atrogin-1 및 PGC1-α의 mRNA 발현를 나타낸 도이다. Figure 1 is a diagram showing body weight changes after intraperitoneal injection for each of the Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group for 6 days.
(Control group: saline-fed mouse model, Dex group: mouse model injected only with dexamethasone, Dex+HFHS group: dexamethasone injection, and HFHS: high-fat/high-sucrose diet) Mouse model fed a diet, Dex+HFHS+PSE group: dexamethasone injection and HFHS: high-fat/high-sucrose diet and Udo peanut sprout extract (PSE) diet. one mouse model)
Figure 2 is a diagram showing the glucose tolerance test of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 3 is a diagram showing fasting blood sugar levels in each of the Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 4 is a diagram showing the plasma triglyceride content of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 5 is a diagram showing the total plasma cholesterol of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 6 is a diagram showing the front leg grip strength compared to body weight of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 7 is a diagram showing the grip strength of the hind limbs and front limbs of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 8 is a diagram showing the maintenance of grip strength compared to body weight in each of the Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 9 is a diagram showing hematoxylin and eosin (H&E) staining of the quadriceps muscles of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 10 is a diagram showing the muscle fiber size of the quadriceps femoris muscle using the MIPAR program for each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 11 is a diagram showing the mRNA expression levels of muscle atrophy factors MuRF-1, Atrogin-1, and Myogenin in the gastrocnemius muscle of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 12 is a diagram showing protein expression of MuRF-1, Atrogin-1, and PGC1-α in the gastrocnemius muscle of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
(Control: GAPDH)
Figure 13 is a diagram showing the RNA expression levels of inflammatory markers TNF-α, IL-6, and IL-1β in each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 14 is a diagram showing the mRNA expression levels of MuRF-1, Atrogin-1, and Myogenin in the quadriceps muscles of each Control group, Dex group, Dex+HFHS group, and Dex+HFHS+PSE group.
Figure 15 is a diagram showing protein and mRNA expression of PGC1-α when skeletal muscle cells (C2C12) were treated with Control, Dex+HFHS, and Dex+HFHS+PSE, respectively.
Figure 16 is a diagram showing cell survival rate when skeletal muscle cells (C2C12) are treated with Udo peanut extract (PSE).
Figure 17 is a diagram showing the mRNA expression of MuRF-1, Atrogin-1, and PGC1-α when skeletal muscle cells (C2C12) were treated with Control, Dex+HFHS, and Dex+HFHS+PSE, respectively.
이하, 첨부된 도면을 참조하여 본 발명의 구현 예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현 예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다, 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in detail as an example of implementation of the present invention with reference to the attached drawings. However, the following implementation examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited by this, and the present invention is capable of various modifications and applications within the description of the claims described later and the scope of equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어 (terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 사료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and feeds are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.
본 발명에 따른 추출물은 당업계에 공지된 추출 및 분리방법을 사용하여 천연으로부터 추출 및 분리하여 수득한 것을 사용할 수 있으며, 본 발명에서 정의된 "추출물"은 적절한 용매를 이용하여 창출으로부터 추출한 것이며, 예를 들어, 조추출물, 극성용매 가용 추출물 또는 비극성용매 가용 추출물을 모두 포함한다. 상기 창출으로부터 추출물을 추출하기 위한 적절한 용매로는 약학적으로 허용되는 유기용매라면 어느 것을 사용해도 무방하며, 물 또는 유기용매를 사용할 수 있으며, 이에 제한되지는 않으나, 예를 들어, 정제수, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol) 등을 포함하는 탄소수 1 내지 4의 알코올, 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다.The extract according to the present invention can be obtained by extraction and separation from nature using extraction and separation methods known in the art, and "extract" as defined in the present invention is extracted from natural sources using an appropriate solvent. For example, it includes crude extract, polar solvent soluble extract, or non-polar solvent soluble extract. An appropriate solvent for extracting the extract from the above can be any pharmaceutically acceptable organic solvent, and water or an organic solvent can be used, but is not limited thereto, for example, purified water, methanol ( Alcohols with 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform ( Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane can be used individually or in combination.
추출 방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법 중 어느 하나를 선택하여 사용할 수 있다. 또한, 목적하는 추출물은 추가로 통상의 분획 공정을 수행할 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다.As an extraction method, any one of the following methods can be selected and used: hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.
본 발명에서 사용되는 용어, "예방"이란, 본 발명에 따른 근육손상 질환의 예방 또는 치료용 약학 조성물을 개체에 투여하여 근육손상 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미할 수 있다.As used in the present invention, the term "prevention" may mean any act of suppressing or delaying the onset of a muscle-damaging disease by administering the pharmaceutical composition for preventing or treating muscle-damaging disease according to the present invention to an individual.
본 발명에서 사용되는 용어, "치료"란, 본 발명의 상기 조성물을 근육손상 질환 발병의 의심 개체에 투여하여 근육손상 질환의 증세가 호전되도록 하거나 이롭게 되도록 하 는 모든 행위를 의미할 수 있다.As used in the present invention, the term "treatment" may mean any act of administering the composition of the present invention to a subject suspected of developing a muscle-damaging disease to improve or benefit the symptoms of the muscle-damaging disease.
본 발명에서 사용되는 용어, "개선"은 치료되는 상태와 관련된 파라미터, 예 를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미할 수 있다.As used herein, the term “improvement” may mean any action that reduces at least the degree of a parameter related to the condition being treated, such as a symptom.
본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육 재생용 조성물을 제공한다.The present invention provides a composition for muscle regeneration containing peanut ( Arachis hypogaea L. ) sprout extract as an active ingredient.
일 실시예에 있어서, 상기 추출물은 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합용매로 이루어진 군에서 선택된 어느 하나의 용매로 추출된 것이나, 이에제한되지 않는다.In one embodiment, the extract is extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, or a mixed solvent thereof, but is not limited thereto.
일 실시예에 있어서, 상기 조성물은 대사 매개 변수를 유지시는 것이나, 이에 제한되지 않는다.In one embodiment, the composition maintains metabolic parameters, but is not limited thereto.
상기 대사는 체중, 포도당 내성 시험, 공복 혈당 수치, 혈장의 중성지방 함량, 또는 총 콜레스테롤인 것일 수 있다.The metabolism may be body weight, glucose tolerance test, fasting blood sugar level, plasma triglyceride content, or total cholesterol.
일 실시예에 있어서, 상기 조성물은 근 섬유를 증가시키는 것이나, 이에 제한되지 않는다.In one embodiment, the composition increases muscle fibers, but is not limited thereto.
상기 근 섬유는 대퇴사두근, 상완근, 상완이두근, 전완근, 복직근, 대퇴이두근, 박근, 반건양근, 반막양근, 비복근, 외측광근, 내측광근, 지골근, 장두, 내측근등으로 이루어진 군에서 1이상인 것일 수 있다.The muscle fibers include the quadriceps femoris, brachii muscle, biceps brachii muscle, forearm muscle, rectus abdominis muscle, biceps femoris muscle, gracilis muscle, semitendinosus muscle, semimembranosus muscle, gastrocnemius muscle, vastus lateralis muscle, vastus medialis muscle, phalanx muscle, long head, medial muscle, etc. It may be 1 or more.
일 실시예에 있어서, 상기 조성물은 근육 신생 및 분화에 관여하는 인자의 발현을 증진시키는 것이나, 이에 제한되지 않는다.In one embodiment, the composition promotes the expression of factors involved in muscle growth and differentiation, but is not limited thereto.
상기 인자는 MuRF-1(Muscle RING-finger protein-1), 아트로진-1(Atrogin-1) 및 미오게닌(Myogenin)으로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.The factor may be one or more types selected from the group consisting of MuRF-1 (Muscle RING-finger protein-1), Atrogin-1, and Myogenin.
일 실시예에 있어서, 상기 조성물은 미토콘드리아 생합성(biogenesis) 및 기능 활성 관여하는 인자의 발현을 증진시키는 것이나, 이에 제한되지 않는다.In one embodiment, the composition promotes the expression of factors involved in mitochondrial biogenesis and functional activity, but is not limited thereto.
상기 인자는 PGC1-α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha)인 것일 수 있다.The factor may be PGC1-α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha).
일 실시예에 있어서, 상기 조성물은 염증성 인자인 TNF-α, IL-6 및 IL-1β 감소시키는 것이나, 이에 제한되지 않는다.In one embodiment, the composition reduces inflammatory factors such as TNF-α, IL-6, and IL-1β, but is not limited thereto.
일 실시예에 있어서, 상기 조성물은 근육 재생으로 인한 악력 및 악력 유지를 증가시키는 것이나, 이에 제한되지 않는다.In one embodiment, the composition increases grip strength and grip strength maintenance due to muscle regeneration, but is not limited thereto.
본 발명은 땅콩 새싹 추출물을 유효성분으로 포함하는 근육 손상 질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating muscle damage diseases containing peanut sprout extract as an active ingredient.
본 발명의 약학 조성물에는 유효성분 이외에 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 면역성을 증가시킬 수 있다. The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase immunity.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 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 injection solutions according to conventional methods. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.
상기 약학 조성물은 다양한 경구 또는 비경구 투여 형태로 제형화될 수 있다.The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경질, 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/ 또는 폴리에틸렌 글리콜)를 추가로 포함할 수 있다. 또한, 상기 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유할 수 있다. 상기 제형은 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다.Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.
또한, 비경구 투여용 제형의 대표적인 것은 주사용 제제이며, 주사용 제제의 용매로서 물, 링거액, 등장성 생리식염수 또는 현탁액을 들 수 있다. 상기 주사용 제제의 멸균 고정 오일은 용매 또는 현탁 매질로서 사용할 수 있으며 모노-, 디-글리세라이드를 포함하여 어떠한 무자극성 고정오일도 이러한 목적으로 사용될 수 있다.In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.
또한, 상기 주사용 제제는 올레산과 같은 지방산을 사용할 수 있다.Additionally, the injectable preparation may use fatty acids such as oleic acid.
상기 상기 질환은 근육 좌상(muscle strain), 근육 파열(muscle rupture), 근육 열상(muscle tearing), 타박상(contusion), 염좌(distortion), 회전근개 증후근(roator cuff syndrome) 및 근육염(myositis)으로 이루어진 군으로부터 선택된 하나 이상의 근육 손상 질환인 것일 수 있으나, 이에 제한되지 않는다.The disease consists of muscle strain, muscle rupture, muscle tearing, contusion, sprain, rotator cuff syndrome, and myositis. It may be, but is not limited to, one or more muscle damage diseases selected from the group.
본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 운동 능력 개선용 조성물을 제공한다.The present invention provides a composition for improving athletic performance containing peanut ( Arachis hypogaea L.) sprout extract as an active ingredient.
본 발명은 땅콩(Arachis hypogaea L.) 새싹 추출물을 유효성분으로 포함하는 근육량 증대용 조성물을 제공한다.The present invention provides a composition for increasing muscle mass containing peanut ( Arachis hypogaea L.) sprout extract as an active ingredient.
일 실시예에서, 상기 정서행동장애는 주의력 결핍장애, 과잉행동장애, 대인관계장애, 우울증 장애, 반사회성 장애, 불안증 장애, ADHD(Attention Deficit Hyperactivity Disorder) 장애, 품행장애, 자폐증, 강박 장애, 조현병, 뚜렛 증후군 등에서 이루어진 군에서 하나 이상 선택된 것이나, 이에 제한되지 않는다.In one embodiment, the emotional behavior disorder includes attention deficit disorder, hyperactivity disorder, interpersonal relationship disorder, depression disorder, antisocial disorder, anxiety disorder, ADHD (Attention Deficit Hyperactivity Disorder) disorder, conduct disorder, autism, obsessive-compulsive disorder, and schizophrenia. , Tourette syndrome, etc., but is not limited thereto.
본 발명의 식품 조성물은 유효성분인 추출물을 함유하는 것 외에 통상의 Aa식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition to containing the extract as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical Aa food composition.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 본 발명의 식품 조성물은 상기 약학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.
또한 상기 식품 조성물은 유효성분인 추출물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the extract as an active ingredient, the food composition contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.
본 발명의 기능성 식품 조성물은, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공될 수 있다. 본 발명에서 '건강기능성 식품 조성물'이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. 본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 '식품 첨가물 공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다. 예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다. 캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다. 환 형태의 건강기능식품은 본 발명의 유효성분과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다. 과립 형태의 건강기능식품은 본 발명의 유효성분의 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high-quality pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 '%'는 별도의 언급이 없는 경우, 고체/고체는 (w/w) %, 고체/액체는 (w/v) %, 그리고 액체/액체는 (v/v) %이다.Throughout this specification, '%' used to indicate the concentration of a specific substance means (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/liquid is (v/v) %.
이하 하기 실시예를 통해 본 발명을 보다 상세히 설명한다. 그러나 하기 실 시예는 본 발명의 기술적 사상의 내용과 범위를 쉽게 설명하기 위한 예시일 뿐, 이 에 의해 본 발명의 기술적 범위가 한정되거나 변경되는 것은 아니다. 또한 이러한 예시에 기초하여 본 발명의 기술적 사상의 범위 안에서 다양한 변형과 변경이 가능 함은 당업자에 의해 용이하게 결정될 수 있다.Hereinafter, the present invention will be described in more detail through the following examples. However, the following examples are merely examples to easily explain the content and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed thereby. Additionally, based on these examples, it can be easily determined by those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention.
실시예 1. 실험 준비 Example 1. Experimental preparation
실시예 1-1. 실험용 화학물질 및 시료 준비Example 1-1. Laboratory chemicals and sample preparation
모든 세포 배양 접시와 플라스크는 달리 명시되지 않는 한 SPL(서울, 한국)에서 구입하였다. Dulbecco's Modified Eagle 's Medium (DMEM), 태아 소 혈청 (FBS), 말 혈청 (HS) 및 페니실린 / 스트렙토 마이신 (P/S)은 Gibco (Grand Island, NY, USA)에서 구입하였다. 다른 모든 화학 물질 및 시약들도 달리 명시되지 않는 한 Sigma Chemical Co.(St. Louis, MO, USA)에서 구입하였다. 9일 동안 발아된 우도 땅콩 새싹(Peanut sprouts, PS)은 WooYoung E&T(제주, 대한민국)에서 구매하였고, 건조 후 동결건조된 우도 땅콩 새싹(PS)을 분말화하여 -80℃ 냉동고에 보관하였다. 건조된 우도 땅콩 새싹(PS) 분말 10g을 100mL의 초순수 제조장치인Milli-Q로 수득한 100ml의 물과 함께 가압 열수 추출 방법을 사용하여 혼합하였다. 추출된 샘플을 3000rpm에서 3분 동안 원심분리하고 Whatman® 필터 페이퍼를 사용하여 여과하였다. 분말 추출물을 얻기 위해 여과액을 동결건조하였다. 마지막으로, 얻어진 샘플을 세포 및 동물 실험을 위해 각각 100 mg/mL 농도로 디메틸 설폭사이드(DMSO, Sigma, St. Louis, MO, USA)에 용해시켰다. All cell culture dishes and flasks were purchased from SPL (Seoul, Korea) unless otherwise specified. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), horse serum (HS) and penicillin/streptomycin (P/S) were purchased from Gibco (Grand Island, NY, USA). All other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified. Peanut sprouts (PS) germinated for 9 days were purchased from WooYoung E&T (Jeju, Korea), and after drying, the freeze-dried PS was powdered and stored in a -80°C freezer. 10 g of dried Udo peanut sprout (PS) powder was mixed with 100 ml of water obtained with Milli-Q, a 100 mL ultrapure water production device, using a pressurized hot water extraction method. The extracted sample was centrifuged at 3000 rpm for 3 minutes and filtered using Whatman® filter paper. The filtrate was freeze-dried to obtain a powder extract. Finally, the obtained samples were dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) at a concentration of 100 mg/mL for cell and animal experiments, respectively.
본 발명자들은 동물 실험에 사용하기 전에 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE) 스톡을 인산완충식염수(PBS)로 신선하게 희석하였다.The present inventors freshly diluted the Udo peanut sprout extract (PSE) stock with phosphate-buffered saline (PBS) before using it in animal experiments.
실시예 1-2. 동물과 식단Example 1-2. animals and diet
6주령 수컷 C57BL/6 마우스는 ORIENT BIO Animal Center(한국 성남시)에서 구입하여 식이와 물을 자유롭게 섭취하면서 12시간 암/광 주기를 유지하였다. 실험 전에 마우스는 경구 위관 영양법으로 1주일 동안 적응기를 가졌으며 모든 동물 실험 및 실험 프로토콜은 제주대학교 동물관리위원회(IACUC)의 승인을 받았으며(2021-0026), 기관 지침에 따라 수행되었다. 8주령 수컷 C57BL/6 마우스에게 10주 동안 식염수 또는 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE) (10mg/kg 체중(BW))와 함께 저지방 식이(fed low fat diet, LF) 또는 고지방/고당 식이(high-fat/high-sucrose diet, HFHS)를 먹였다. 본 발명의 덱사메타손(Dexamethasone, Dex, 10mg/kg BW)은 6일 동안 복강내 주사에 하여 쥐의 근육 위축을 악화시키기 위해 처리되었다: (ⅰ) 대조군, (ⅱ) Dex (ⅲ) Dex+HFHS 및 (ⅳ) Dex+HFHS+PSE를 마우스 동물모델에 투여하여 실험 하였다. 실험 종료 후 C57BL/6 마우스를 12시간 절식시킨 후 이산화탄소 마취로 희생시켰다. 희생 시킨 마우스로 부터 혈액, 간, 부고환 지방, 대퇴사두근 및 비복근을 수집하였다.Six-week-old male C57BL/6 mice were purchased from ORIENT BIO Animal Center (Seongnam-si, Korea) and maintained on a 12-hour dark/light cycle with free access to food and water. Before the experiment, mice underwent an adaptation period for 1 week by oral gavage, and all animal experiments and experimental protocols were approved by the Jeju National University Animal Care and Use Committee (IACUC) (2021-0026) and performed in accordance with institutional guidelines. 8-week-old male C57BL/6 mice were fed a low-fat diet (LF) or a high-fat/high-sugar diet with saline or Peanut sprout extracts (PSE) (10 mg/kg body weight (BW)) for 10 weeks. (high-fat/high-sucrose diet, HFHS). Dexamethasone (Dex, 10 mg/kg BW) of the present invention was treated to worsen muscle atrophy in rats by intraperitoneal injection for 6 days: (i) control group, (ii) Dex (iii) Dex+HFHS and (iv) Dex+HFHS+PSE was administered to a mouse animal model and tested. After completion of the experiment, C57BL/6 mice were fasted for 12 hours and then sacrificed by carbon dioxide anesthesia. Blood, liver, epididymal fat, quadriceps femoris, and gastrocnemius muscles were collected from sacrificed mice.
저지방 식단(LF, 11% kcal 지방) 및 고지방식(HF, 60% kcal 지방) 준비는 AIN-93G 식단으로 표 1로 수정되었다. 고자당수(High sucrose water, HS)는 115°C에서 15분 동안 20% 자당수를 고압멸균하여 제조하였다. 체중과 음식 섭취량은 매주 측정하였다.Low-fat diet (LF, 11% kcal fat) and high-fat diet (HF, 60% kcal fat) preparations were modified from Table 1 for the AIN-93G diet. High sucrose water (HS) was prepared by autoclaving 20% sucrose water at 115°C for 15 minutes. Body weight and food intake were measured weekly.
실시예 1-3. 포도당부하검사(Glucose tolerance test,Example 1-3. Glucose tolerance test, GTT) 분석GTT) Analysis
실험 동물은 실험 9주차에 포도당부하검사(GTT)를 하였다. 마우스를 12시간 절식시킨 후 10% D-glucose 용액(1g/kg BW)을 복강내 주사하였다. 혈당 수치를 측정하기 위해 혈당 측정기(Contour plus, BAYER)를 0, 15, 30, 60 및 120분에 사용하였다.The experimental animals underwent a glucose tolerance test (GTT) in the 9th week of the experiment. Mice were fasted for 12 hours and then intraperitoneally injected with 10% D-glucose solution (1g/kg BW). A blood glucose meter (Contour plus, BAYER) was used to measure blood glucose levels at 0, 15, 30, 60, and 120 minutes.
실시예 1-4. 혈장 생화학(Plasma biochemistry)Example 1-4. Plasma biochemistry
실험이 완료된 후 혈청 생화학을 결정하기 위해 희생된 마우스의 심장 천자에서 혈액을 수집하였다. 채혈한 혈액에 헤파린을 넣고 10,000rpm에서 10분간 원심분리하여 혈장을 얻었고, 혈장 중성지방(TG, mg/dL) 및 총 콜레스테롤(TC, mg/dL)의 농도는 분광광도계를 효소 분석 키트(아산제약, 서울, 한국)를 사용하여 각각OD 500, 550nm에서 흡광도에 따라 제조 프로토콜에 따라 측정하였다. After completion of the experiment, blood was collected from cardiac puncture of sacrificed mice to determine serum biochemistry. Heparin was added to the collected blood and centrifuged at 10,000 rpm for 10 minutes to obtain plasma. The concentrations of plasma triglycerides (TG, mg/dL) and total cholesterol (TC, mg/dL) were measured using a spectrophotometer with an enzyme analysis kit (Asan). Pharmaceutical Co., Seoul, Korea) was used to measure absorbance at OD 500 and 550 nm, respectively, according to the manufacturing protocol.
실시예 1-5.Examples 1-5. 행잉 테스트 및 악력 테스트(Hanging test and grip strength test)Hanging test and grip strength test
실험 10주차에 Grip Strength Meter(Ugo-Basile)로 쥐의 악력을 측정하였다. 앞다리의 악력은 3회 측정하여 가장 큰 값을 사용하였으며 앞다리와 뒷다리를 합하여 측정하였을 때도 동일하게 적용하였다.In the 10th week of the experiment, the rats' grip strength was measured using a Grip Strength Meter (Ugo-Basile). The grip strength of the forelimbs was measured three times and the highest value was used, and the same was applied when measuring the forelimbs and hindlimbs combined.
행잉 테스트(Hanging test)는 마우스가 스스로 철망을 잡고 있게 한 후 철망을 뒤집어 놓았을 때 마우스의 철망에 매달려 있는 지속 시간을 측정하였다. 이 실험은 각 마우스는 최소 3번 이상 측정하였으며 가장 큰 값을 사용하였다.In the hanging test, the mouse was allowed to hold onto a wire mesh by itself and then the length of time the mouse hung on the wire mesh was measured when the wire mesh was turned over. In this experiment, each mouse was measured at least three times and the largest value was used.
실시예 1-6.Example 1-6. 헤마톡실린 및 에오신(H&E) 염색 및 지방세포, 지방증 및 근섬유 크기 정량화Hematoxylin and eosin (H&E) staining and quantification of adipocyte, steatosis and muscle fiber size.
부검시 부고환 지방, 간 및 대퇴사 두근을 마우스에서 분리하고 10 % 포르말린 완충액으로 플래시 고정하였다. 파라핀 내장된 조직을 5-7 μm 섹션으로 절단하고 헤마톡실린 및 에오신(H&E)으로 염색하였다. 브라이트 필드 이미지(Bright-field image)는 10x, 20x 배율에서 Invitrogen 현미경(Invitrogen™ EVOS™ FL Digital Inverted Fluorescence Microscope, Invitrogen, CA, USA)으로 캡처하였다.At necropsy, epididymal fat, liver, and quadriceps muscle were isolated from mice and flash fixed in 10% formalin buffer. Paraffin-embedded tissue was cut into 5-7 μm sections and stained with hematoxylin and eosin (H&E). Bright-field images were captured with an Invitrogen microscope (Invitrogen™ EVOS™ FL Digital Inverted Fluorescence Microscope, Invitrogen, CA, USA) at 10x and 20x magnification.
부고환 지방 조직의 H&E 염색은 지방 세포 크기를 측정하기 위해 정량화되었다. 지방세포 크기의 정량적 분석을 위해 NIH(National Institutes of Health)의 Image J 및 Adiposoft를 사용하였다. MIPAR 소프트웨어(MIPARTM, Worthington, OH, USA)는 H&E 염색 간 조직의 지방증 정량화 및 H&E 염색 대퇴사두근의 근육 섬유 정량화 하는데 사용되었다.H&E staining of epididymal adipose tissue was quantified to determine adipocyte size. For quantitative analysis of adipocyte size, Image J and Adiposoft from the National Institutes of Health (NIH) were used. MIPAR software (MIPARTM, Worthington, OH, USA) was used to quantify steatosis in H&E-stained liver tissue and to quantify muscle fibers in H&E-stained quadriceps muscle.
실시예 1-7. 세포배양Example 1-7. cell culture
골격근세포인, C2C12 근아세포(myoblast cell)(American Type Culture Collection(ATCC), Manassas, VA, USA)를 1% P/S가 포함된 DMEM에서 10% FBS를 함유하는 기본 배지에서 배양하였다. C2C12 근아세포가 80-90% 융합에 도달한 후(0일째라고 함), 성장 배지를 1% P/S를 갖는 DMEM에서 2% HS를 함유하는 분화 배지로 교체하여 세포를 근관으로 분화시켰다. 이후, 격일로 배지를 교체하면서 세포를 최대 4-6일 동안 유지하였다.Skeletal muscle cells, C2C12 myoblast cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in basal medium containing 10% FBS in DMEM containing 1% P/S. After C2C12 myoblasts reached 80-90% confluence (termed day 0), the growth medium was replaced with differentiation medium containing 2% HS in DMEM with 1% P/S to differentiate the cells into myotubes. Thereafter, the cells were maintained for up to 4-6 days while changing the medium every other day.
실시예 1-8. 세포 생존율 분석Examples 1-8. Cell viability analysis
골격근 세포(C2C12)에 대한 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE)의 세포독성 효과를 알아보기 위해 EZ-cytox Cell Viability assay kit(Daeil Lab Service Co Ltd, Seoul, Korea)를 사용하여 측정하였고 제조사의 프로토콜을 따랐다.To determine the cytotoxic effect of Udo peanut sprout extracts (PSE) on skeletal muscle cells (C2C12), it was measured using the EZ-cytox Cell Viability assay kit (Daeil Lab Service Co Ltd, Seoul, Korea), and the manufacturer The protocol was followed.
골격근 세포(C2C12)는 성장 배지에서 대략 20,000개 세포/웰의 파종 밀도로 96-웰 플레이트에서 배양되었다. 세포를 DMSO 또는 다른 농도의 우도 땅콩 새삭 추출물(PSE)과 함께 24시간 동안 인큐베이션하였다. 그 후, 세포를 10% EZ-cytox 용액을 함유한 새로운 배지로 교체하고 37℃에서 인큐베이션하였다. 플레이트는 30분 에서 4시간 동안 마이크로플레이트 리더를 사용하여 OD 450nm에서 측정하였다.Skeletal muscle cells (C2C12) were cultured in 96-well plates at a seeding density of approximately 20,000 cells/well in growth medium. Cells were incubated with DMSO or different concentrations of Udo peanut sprout extract (PSE) for 24 hours. Afterwards, the cells were replaced with fresh medium containing 10% EZ-cytox solution and incubated at 37°C. Plates were measured at OD 450 nm using a microplate reader for 30 minutes to 4 hours.
실시예 1-9. 실시간 중합효소연쇄반응(실시간 PCR)에 의한 mRNA 분석Example 1-9. mRNA analysis by real-time polymerase chain reaction (real-time PCR)
근육과 세포의 total RNA는 Trizol 시약(Invitrogen)으로 추출하였다.Total RNA from muscles and cells was extracted with Trizol reagent (Invitrogen).
mRNA는 잠재적인 게놈 DNA 오염을 제거하기 위해 DNase(Mediatech)를 처리하였다. RNA 농도는 cDNA 합성 전에 Nanodrop(Nano-200 Micro-Spectrophotometer, Hangzhou City, China)으로 측정하였다. cDNA는 iScript cDNA 합성 키트(Biorad, Hercules, CA, USA)로 합성되었다. 유전자 발현은 실시간 qPCR(CFX96™ Real-Time PCR Detection System, Bio-Rad, CA, USA)에 의해 결정되었다. 히포크산틴포스포리보실전달효소(hypoxanthine-guanine phosphoribosyltransferase, HPRT) 및/또는 리보솜 단백질 가로줄기 서브유닛(ribosomal protein lateral stem subunit P0) (RPLP0/36B4)는 상대적인 유전자 발현을 표준화하는데 사용되었다.The mRNA was treated with DNase (Mediatech) to remove potential genomic DNA contamination. RNA concentration was measured with Nanodrop (Nano-200 Micro-Spectrophotometer, Hangzhou City, China) before cDNA synthesis. cDNA was synthesized with the iScript cDNA synthesis kit (Biorad, Hercules, CA, USA). Gene expression was determined by real-time qPCR (CFX96™ Real-Time PCR Detection System, Bio-Rad, CA, USA). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) and/or ribosomal protein lateral stem subunit P0 (RPLP0/36B4) were used to normalize relative gene expression.
qPCR을 위한 유전자 특이적 프라이머(표 2)는 Cosmo Genetech(서울, 한국)에서 구입하였다.Gene-specific primers for qPCR (Table 2) were purchased from Cosmo Genetech (Seoul, Korea).
실시예 1-10. 단백질 분리 및 웨스턴 블로팅Examples 1-10. Protein isolation and Western blotting
조직 샘플을 수확하고 프로테아제 및 포스파타제 억제제 홉합제가 포함된 RIPA(ice-col radioimmunoprecipitation) 분석 용해 완충액에서 균질화기로 균질화하였다. C2C12 세포를 프로테아제 및 포스파타제 억제제를 함유하는 얼음처럼 차가운 RIPA 분석 용해 완충액으로 긁어냈다. 그 후, 10,000 rpm, 4℃에서 10분간 원심분리하여 상층액을 모은 후, 8-10% SDS-PAGE를 사용하여 10-15 μg의 단백질을 분리하고 단백질을 Tris-buffered saline/Tween 20이 있는 PVDF(polyvinylidene difluoride) 멤브레인(Thermo Fisher Scientific, MA, USA)으로 옮기고 실온에서 1시간 동안 빛을 차단하였다. 막(membrane)을 Tris-buffered saline with tween 20(TBST) 용액으로 여러 번 세척하고, 근육 위축 F-box(MAFbx/Atrogin-1), 근육 특이적 링 핑거 단백질 근육 특이적 링 핑거 단백질 (Muscle specific RING finger protein, MuRF1) (Santa Cruz biotechnology, CA, USA), 글리세르알데히드-3-인산 수소 이탈 효소(glyceraldehyde-3-phosphate dehydrogenase GAPDH), β-엑틴(β-Actin)(Cell signaling Technology, Danvers, MA, USA), 산화적 인산화(oxidative phosphorylation, OXPHOS)(Abcam, Cambridge, MA, USA) 및 페르옥시좀 증식인자 활성화 수용체 감마 공동활성제 1-알파(peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1-α) (Novus biologicals)에 대한 1차 항체로 4℃에서 밤새 반응시켰다. 다음달, 막(membrane)을 1X TBST로 여러 번 세척하고 2차 항체, 염소 항토끼(Cell signaling Technology, Danvers, MA, USA) 또는 염소 항마우스 IgG-HRP(Santa Cruz biotechnology, CA, USA)와 함께 1시간 동안 인큐베이션하였다. 막을 세척하고 ECL(Enhanced chemiluminescence) 시약(PerkinElmer, Waltham, MA, USA)으로 반응시켰다. 밴드를 확인하기 위해 ChemiDoc MP(Bio-Rad, CA, USA)를 사용하였고, Image Lab(Bio-Rad, CA, USA) 또는 Image J(NIH, MD, USA)를 이용하여 발현 정도를 계산하였다.Tissue samples were harvested and homogenized with a homogenizer in ice-col radioimmunoprecipitation (RIPA) assay lysis buffer containing protease and phosphatase inhibitor combinations. C2C12 cells were scraped into ice-cold RIPA assay lysis buffer containing protease and phosphatase inhibitors. Afterwards, the supernatant was collected by centrifugation at 10,000 rpm at 4°C for 10 minutes, and then 10-15 μg of protein was separated using 8-10% SDS-PAGE, and the protein was separated in Tris-buffered saline/Tween 20. It was transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, MA, USA) and blocked from light for 1 hour at room temperature. The membrane was washed several times with Tris-buffered saline with tween 20 (TBST) solution, and incubated with muscle atrophy F-box (MAFbx/Atrogin-1), muscle-specific ring finger protein (Muscle specific ring finger protein). RING finger protein, MuRF1) (Santa Cruz biotechnology, CA, USA), glyceraldehyde-3-phosphate dehydrogenase GAPDH, β-Actin (Cell signaling Technology, Danvers) , MA, USA), oxidative phosphorylation (OXPHOS) (Abcam, Cambridge, MA, USA) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, The reaction was performed overnight at 4°C with a primary antibody against PGC1-α (Novus biologicals). The next month, the membrane was washed several times with 1X TBST and incubated with secondary antibodies, goat anti-rabbit (Cell signaling Technology, Danvers, MA, USA) or goat anti-mouse IgG-HRP (Santa Cruz biotechnology, CA, USA). Incubated for 1 hour. The membrane was washed and reacted with enhanced chemiluminescence (ECL) reagent (PerkinElmer, Waltham, MA, USA). ChemiDoc MP (Bio-Rad, CA, USA) was used to identify the band, and the expression level was calculated using Image Lab (Bio-Rad, CA, USA) or Image J (NIH, MD, USA).
실시예 1-11. 통계 분석Example 1-11. statistical analysis
실험 결과는 평균 ± 평균의 표준 오차(SEM)로 표시되었다. Bonferroni의 다중 비교 테스트 또는 학생 t-테스트와 함께 ANOVA(일방향 분산 분석)를 사용하여 통계 계산을 수행하였다. p-값 <0.05는 통계적으로 유의한 것으로 간주되었다. 모든 분석은 GraphPad Prism 8.02.263(La Jolla, California., USA)으로 수행하였다.Experimental results were expressed as mean ± standard error of the mean (SEM). Statistical calculations were performed using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test or Student's t-test. A p-value <0.05 was considered statistically significant. All analyzes were performed with GraphPad Prism 8.02.263 (La Jolla, California., USA).
실시예 2. 본 발명의 우도 땅콩 새싹 추출물(PSE)의 대사 매개변수에 영향 확인Example 2. Confirmation of the effect of Udo peanut sprout extract (PSE) of the present invention on metabolic parameters
우도 땅콩 새싹 추출물(PSE)이 대사(metabolic)에 영향을 미치는지 알아 보기 위해, 8주령 수컷 C57BL/6 마우스에 1)식염수(control) 투여 군, 2)저지방 식이(LF)군, 3)덱사메타손(Dex)만 주사한 군, 4)고지방/고당(HFHS) 식이+덱사메타손(Dex)군, 5)고지방/고당(HFHS)식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군을 두고, 각 10주 동안 식이하여, 대사의 변화를 알아 보았다. 모든 Dex 투여군은 지난 6일 동안 복강 내 주사로 투여하였다.To determine whether Udo peanut sprout extract (PSE) affects metabolism, 8-week-old male C57BL/6 mice were administered 1) saline (control) group, 2) low-fat diet (LF) group, 3) dexamethasone ( A group injected only with Dex), 4) a high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group, and 5) a high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group. After dieting for 10 weeks, changes in metabolism were observed. All Dex treatment groups were administered by intraperitoneal injection over the past 6 days.
표 3에 나와있듯이, Control 그룹은 HFHS+Dex 그룹보다 더 높은 음식 섭취량과 kcal 섭취를 나타냈다. 또한, 10주 동안 그룹 간에 마우스의 체중과 체중 증가에는 유의한 차이가 없음을 확인하였고(도 1), 포도당 부하 테스트(도 2), 공복 혈당(도 3), 부고환 지방 중량, 간 중량 및 근육 중량에는 유의한 차이가 없음을 확인하였다(표 3). 중성지방과 총 콜레스테롤 수치는 고지방/고당(HFHS) 식이+덱사메타손(Dex)군보다 고지방/고당(HFHS) 식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군에서 감소하는 것을 확인하였다(도 4,도 5). As shown in Table 3, the Control group showed higher food intake and kcal intake than the HFHS+Dex group. In addition, it was confirmed that there was no significant difference in body weight and body weight gain of mice between groups for 10 weeks (Figure 1), glucose tolerance test (Figure 2), fasting blood sugar (Figure 3), epididymal fat weight, liver weight, and muscle It was confirmed that there was no significant difference in weight (Table 3). Neutral fat and total cholesterol levels were confirmed to decrease in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group than in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group (Figure 4, Fig. 5).
실시예 3. 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE)의 운동 능력을 개선 확인Example 3. Confirmation of improvement in exercise capacity of Udo peanut sprout extracts (PSE)
우도 땅콩 새싹 추출물(PSE)이 운동능력을 개선에 영향을 미치는지 알아 보기 위해, 8주령 수컷 C57BL/6 마우스에 1)식염수(control) 투여 군, 2)저지방 식이(LF)군, 3)덱사메타손(Dex)만 주사한 군, 4)고지방/고당(HFHS) 식이+덱사메타손(Dex)군, 5)고지방/고당(HFHS)식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군을 두고, 각 10주 동안 식이하여, 마우스 모델들의 악력시험과 매달리기 시험으로 평가 하였다.To determine whether Udo peanut sprout extract (PSE) has an effect on improving exercise capacity, 8-week-old male C57BL/6 mice were administered 1) saline solution (control) group, 2) low-fat diet (LF) group, 3) dexamethasone ( A group injected only with Dex), 4) a high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group, and 5) a high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group. The diet was administered for 10 weeks and evaluated using grip strength tests and hanging tests in mouse models.
그 결과, 덱사메타손(Dex)만 주사한군과 비교하여, 고지방/고당(HFHS) 식이+덱사메타손(Dex)군에서 앞다리 뒷다리에 악력 강도가 유의적으로 약해졌음을 확인하였다(도 6,도 7). 또한, 매달리기 테스트(hanging test) 또한, 고지방/고당(HFHS) 식이+덱사메타손(Dex)군 낮아졌으나, 고지방/고당(HFHS) 식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)을 군에서 앞다리 뒷다리의 그립 강도가 유의적으로 높아졌음을 확인하였으며, 매달리기 테스트(hanging test) 능력 즉 악력 유지 향상됨을 확인 하였다(도 8).As a result, compared to the group injected only with dexamethasone (Dex), it was confirmed that the grip strength of the front and hind limbs was significantly weakened in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group (Figures 6 and 7). In addition, the hanging test was also lowered in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group, but the forelimb was lower in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + peanut sprout extract (PSE) group. It was confirmed that the grip strength of the hind legs was significantly increased, and the hanging test ability, that is, the maintenance of grip strength, was confirmed to be improved (FIG. 8).
따라서, 각각의 마우스 모델을 희생시켜 대퇴사두근을 헤마톡실린과 에오신(H&E)으로 염색한 뒤(도 9), MIPAR 프로그램을 이용하여 대퇴사두근의 근섬유 크기를 측정한 결과 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE)을 같이 처리한 군에서 근 섬유가 회복된 것을 확인 하였다(도 10).Therefore, each mouse model was sacrificed, the quadriceps femoris was stained with hematoxylin and eosin (H&E) (Figure 9), and the muscle fiber size of the quadriceps femoris was measured using the MIPAR program. As a result, Udo peanut sprout extract (Peanut sprout) was measured. It was confirmed that muscle fibers were recovered in the group treated with extracts (PSE) (FIG. 10).
실시예 4. 우도 땅콩 새싹 추출물(Peanut sprout extracts, PSE)의 골격근 위축 개선 효과Example 4. Effect of Udo peanut sprout extract (PSE) on improving skeletal muscle atrophy
우도 땅콩 새싹 추출물(PSE)이 골격근 위축을 개선시키는지 알아 보기 위하여, 8주령 수컷 C57BL/6 마우스에 1)식염수(control) 투여 군, 2)저지방 식이(LF)군, 3)덱사메타손(Dex)만 주사한 군, 4)고지방/고당(HFHS) 식이+덱사메타손(Dex)군, 5)고지방/고당(HFHS)식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군을 두고, 각 10주 동안 식이하여, 마우스 모델들의 근육 위축인자, 미토콘드리아 생성 마커 및 염증 마커를 확인 하였다.To determine whether Udo peanut sprout extract (PSE) improves skeletal muscle atrophy, 8-week-old male C57BL/6 mice were administered 1) saline (control) group, 2) low-fat diet (LF) group, and 3) dexamethasone (Dex). group, 4) high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group, and 5) high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group, each for 10 weeks. During the diet, muscle atrophy factors, mitochondrial production markers, and inflammatory markers in the mouse models were confirmed.
그 결과, 고지방/고당(HFHS) 식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군 에서, 종아리 뒤쪽의 두갈래로 갈라진 근육인 비복근(gastrocnemius)의 근육 위측 인자인 MuRF1 및 Atrogin1 단백질 및 mRNA 발현 수준이 덱사메타손(Dex) 군 및 고지방/고당(HFHS) 식이+덱사메타손(Dex) 군에 비해 억제된 것을 확인하였으며(도 11,도 12), 염증 마커인 TNF-α, IL-6 및 IL-1β의 mRNA 발현은 고지방/고당(HFHS) 식이+덱사메타손(Dex)군 보다 고지방/고당(HFHS) 식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군에서 억제된 것을 확인 하였다(도 13).As a result, in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group, MuRF1 and Atrogin1 proteins and mRNA, which are muscle upper factors of the gastrocnemius, a bifurcated muscle on the back of the calf, It was confirmed that the expression level was suppressed compared to the dexamethasone (Dex) group and the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) group (Figures 11 and 12), and the inflammatory markers TNF-α, IL-6, and IL- It was confirmed that the mRNA expression of 1β was suppressed in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + Udo peanut sprout extract (PSE) group than in the high-fat / high-sugar (HFHS) diet + dexamethasone (Dex) group (Figure 13) .
또한, 대퇴사두근에서 위축 유전자인 MuRF-1(Muscle RING-finger protein-1), 아트로진-1(Atrogin-1), 미오게닌(Myogenin)의 mRNA 발현 수준은 고지방/고당(HFHS) 식이+덱사메타손(Dex)군 보다 고지방/고당(HFHS) 식이+덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE)군에서 감소하는 되었으며(도 14), PGC1-α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha) 발현을 증가시키는 것을 확인하였다(도 15).In addition, the mRNA expression levels of atrophy genes MuRF-1 (Muscle RING-finger protein-1), Atrogin-1, and Myogenin in the quadriceps muscle were significantly affected by the high-fat/high-sugar (HFHS) diet. It was decreased in the high-fat/high-sugar (HFHS) diet + dexamethasone (Dex) + peanut sprout extract (PSE) group compared to the + dexamethasone (Dex) group (Figure 14), and PGC1-α (peroxisome proliferator-activated receptor gamma coactivator 1- alpha) expression was confirmed to be increased (Figure 15).
또한, 우도 땅콩 새싹 추출물(PSE)을 농도별로 골격근 세포(C2C12)에 처리시, 우도 땅콩 새싹 추출물 200 ug/ml에 농도에서 세포 독성 나타났으나, 그 외 농도에서는 세포 생존률에 영향이 없음을 확인하였다(도 16). 또한, 덱사메타손(Dex) 단독 처리 군과, 덱사메타손(Dex)+우도 땅콩 새싹 추출물(PSE) 처리군과 비교하여 근육 위축 유전자인 MuRF-1, Atrogin-1 발현은 감소하였다. 또한, 미토콘드리아 생합성 및 기능 활성 유전자인 PGC1-α의 mRNA 발현을 증가시킨 것을 확인하였다(도 17).In addition, when Udo peanut sprout extract (PSE) was treated with skeletal muscle cells (C2C12) at different concentrations, cytotoxicity was observed at a concentration of 200 ug/ml, but other concentrations showed no effect on cell viability. (Figure 16). In addition, compared to the group treated with dexamethasone (Dex) alone and the group treated with dexamethasone (Dex) + Udo peanut sprout extract (PSE), the expression of muscle atrophy genes MuRF-1 and Atrogin-1 was decreased. In addition, it was confirmed that the mRNA expression of PGC1-α, a mitochondrial biogenesis and functional activity gene, was increased (FIG. 17).
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본 질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관 점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으 로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (16)
상기 조성물은 체중, 포도당 내성 시험, 공복 혈당 수치, 혈장의 중성지방 함량 및 총 콜레스테롤로 이루어진 군으로부터 선택된 하나 이상의 대사 매개 변수를 유지시키는 것인, 조성물.According to clause 1,
The composition maintains one or more metabolic parameters selected from the group consisting of body weight, glucose tolerance test, fasting blood sugar level, plasma triglyceride content and total cholesterol.
상기 조성물은 근 섬유를 증가시키는 것인, 조성물.According to clause 1,
The composition of claim 1, wherein the composition increases muscle fibers.
상기 근 섬유는 대퇴사두근, 상완근, 상완이두근, 전완근, 복직근, 대퇴이두근, 박근, 반건양근, 반막양근, 비복근, 외측광근, 내측광근, 지골근, 장두, 내측근등으로 이루어진 군에서 1이상인 것인, 조성물.According to clause 5,
The muscle fibers include the quadriceps femoris, brachii muscle, biceps brachii muscle, forearm muscle, rectus abdominis muscle, biceps femoris muscle, gracilis muscle, semitendinosus muscle, semimembranosus muscle, gastrocnemius muscle, vastus lateralis muscle, vastus medialis muscle, phalanx muscle, long head, medial muscle, etc. A composition having at least 1.
상기 조성물은 MuRF-1(Muscle RING-finger protein-1), 아트로진-1(Atrogin-1) 및 미오게닌(Myogenin)으로 이루어진 군에서 선택된 1종 이상의 근육 신생 및 분화에 관여하는 인자의 발현을 증진시키는 것인, 조성물.According to clause 1,
The composition contains one or more factors involved in muscle growth and differentiation selected from the group consisting of MuRF-1 (Muscle RING-finger protein-1), Atrogin-1, and Myogenin. A composition that enhances expression.
상기 조성물은 미토콘드리아 생합성(biogenesis) 및 기능 활성에 관여하는 인자인 PGC1-α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha)의 발현을 증진시키는 것인, 조성물.According to clause 1,
The composition promotes the expression of PGC1-α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), a factor involved in mitochondrial biogenesis and functional activity.
상기 조성물은 염증성 인자인 TNF-α, IL-6 및 IL-1β 감소시키는 것인, 조성물.According to clause 1,
The composition reduces inflammatory factors TNF-α, IL-6, and IL-1β.
상기 조성물은 근육 재생으로 인한 악력 및 악력 유지를 증가시키는 것인, 조성물.According to clause 1,
The composition increases grip strength and grip strength maintenance due to muscle regeneration.
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