KR102459434B1 - Composition for improvment, prevention or treatment in muscular atrophy comprising extracts of Potentilla chinensis or Indigo - Google Patents
Composition for improvment, prevention or treatment in muscular atrophy comprising extracts of Potentilla chinensis or Indigo Download PDFInfo
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- KR102459434B1 KR102459434B1 KR1020200093966A KR20200093966A KR102459434B1 KR 102459434 B1 KR102459434 B1 KR 102459434B1 KR 1020200093966 A KR1020200093966 A KR 1020200093966A KR 20200093966 A KR20200093966 A KR 20200093966A KR 102459434 B1 KR102459434 B1 KR 102459434B1
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- extract
- wireungchae
- muscle atrophy
- muscle
- indigo plant
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Abstract
본 발명은 근위축 개선, 예방 또는 치료용 조성물에 관한 것으로, 더욱 상세하게는 위릉채 추출물 또는 쪽 추출물을 함유하는 근위축 개선, 예방 또는 치료용 조성물에 관한 것이다. 본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 유비퀴틴 리가아제인 Atrogin1 및 MuRF1의 과발현을 억제하고, 근위축조절인자 Forkhead box(FOX) 0의 활성을 억제함으로써 근위축 개선에 효능을 발휘한다. 또한, 본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 근육조직 생성 억제에 관여하는 마이오스타틴(myostatin)을 억제하고 마이오스타틴의 억제제인 폴리스타틴(follistatin)의 증가를 통해 근육 강화를 시키고 근관(myotube)의 크기를 증가시킬 수 있으며, 히스톤탈아세틸화 효소(Histone deacetylases, HDACs)의 활성을 억제할 수 있다. 또한, 본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 기존에 근위축 개선과 근육 강화에 효능이 있다고 알려진 수베로일아닐리드 하이드록사믹산(suberoylanilide hydroxamic acid, SAHA, HDAC 억제제), 우르솔산(ursolic acid), 모노 크레아틴(creatine monohydrate)보다 더 뛰어난 효과를 발휘할 수 있다.The present invention relates to a composition for improving, preventing, or treating muscle atrophy, and more particularly, to a composition for improving, preventing or treating muscle atrophy containing an extract of Wireungchae or indigo plant. The composition containing the Wireungchae extract or the indigo plant extract of the present invention as an active ingredient is effective in improving muscle atrophy by inhibiting the overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibiting the activity of the muscle atrophy regulator Forkhead box (FOX) 0 to perform In addition, the composition containing the Wireungchae extract or the indigo plant extract of the present invention as an active ingredient suppresses myostatin, which is involved in the inhibition of muscle tissue production, and increases follistatin, an inhibitor of myostatin, through It can strengthen muscle, increase the size of myotube, and inhibit the activity of histone deacetylases (HDACs). In addition, the composition containing the extract of Wireungchae or indigo plant of the present invention as an active ingredient is suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), which is known to be effective in improving muscle atrophy and strengthening muscles, It may be more effective than ursolic acid or creatine monohydrate.
Description
본 발명은 근위축 개선, 예방 또는 치료용 조성물에 관한 것으로, 더욱 상세하게는 위릉채 추출물 또는 쪽 추출물을 함유하는 근위축 개선, 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for improving, preventing, or treating muscle atrophy, and more particularly, to a composition for improving, preventing or treating muscle atrophy containing an extract of Wireungchae or indigo plant.
근위축(muscle atrophy)은 근세포의 크기가 저하되어, 근육의 근원섬유 숫자가 감소하여 골격근 질량이 줄어드는 것을 말한다. 골격근은 체중의 40%를 차지하며, 골격근의 단백분해가 증가되고 단백합성이 감소하여 근위축이 초래된다. 또한, 근위축에 의한 근육량과 근력이 급격하게 감소하는 현상을 근감소증이라고 한다. 근위축은 활동저하(inactivity), 탈신경(denervation), 노화 및 스테로이드 남용으로 인해 유발된다.Muscle atrophy refers to a decrease in skeletal muscle mass due to a decrease in the size of muscle cells and a decrease in the number of myofibrils in the muscle. Skeletal muscle accounts for 40% of body weight, and proteolysis of skeletal muscle increases and protein synthesis decreases, resulting in muscle atrophy. In addition, a phenomenon in which muscle mass and muscle strength are rapidly reduced due to muscle atrophy is called sarcopenia. Muscle atrophy is caused by inactivity, denervation, aging, and steroid abuse.
스테로이드호르몬은 부신피질에서 분비되며 글루코코르티코이드(glucocorticoid)는 면역반응의 조절, 포도당, 지방, 항염증 반응의 조절 등에 관여하지만, 장기간 투여하면 근질량이 감소되는 근위축과 당뇨병 등의 부작용이 유발된다. 스테로이드 투여 후 속근섬유(fast-twitch muscle fiber)로 이루어진 type Ⅱ 근육은 선택적으로 영향을 받아서 스테로이드 치료를 받은 환자들은 근지구력의 변화보다도 최대 근력을 발생시키는 능력을 소실하게 된다.Steroid hormones are secreted from the adrenal cortex, and glucocorticoids are involved in the regulation of immune responses, glucose, fat, and anti-inflammatory responses. After steroid administration, type II muscle composed of fast-twitch muscle fibers is selectively affected, and patients who have received steroid treatment lose their ability to generate maximum muscle strength rather than change in muscle endurance.
노화나 스테로이드호르몬에 의한 근위축과 근감소증을 계속 방치할 경우 근육량이 최대 60%까지 줄고 근육의 기능도 현저히 잃을 수 있으며, 근육량이 줄어들면 기초대사량 자체가 줄어들기 때문에 혈중 포도당이 상승하게 되면서 당뇨 발병률이 증가하게 된다. 또한, 골절과 낙상, 우울증, 비만, 장애, 나아가 사망으로까지 이어질 수 있다. 따라서 근위축을 개선, 예방 또는 치료를 위한 연구와, 이에 대한 해결방안이 필요한 실정이다.If muscle atrophy and sarcopenia caused by aging or steroid hormones are left unattended, muscle mass may decrease by up to 60% and muscle function may be significantly lost. the incidence rate will increase. It can also lead to fractures and falls, depression, obesity, disability, and even death. Therefore, there is a need for a study for improving, preventing or treating muscular atrophy and a solution for this.
본 발명에서는 위릉채 추출물 또는 쪽 추출물을 이용하여 근위축을 개선할 수 있는 조성물을 개발하여 제공하고자 한다.In the present invention, it is intended to develop and provide a composition capable of improving muscle atrophy by using the Wireungchae extract or the indigo plant extract.
본 발명은 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 식품 조성물을 제공한다.The present invention provides a food composition for improving muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient.
또한, 본 발명은 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for improving muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient.
또한, 본 발명은 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy, characterized in that it contains the Wireungchae extract as an active ingredient.
또한, 본 발명은 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient.
또한, 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 동물 의약품 조성물을 제공한다.In addition, it provides an veterinary pharmaceutical composition for the prevention or treatment of muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient.
또한, 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 동물 의약품 조성물을 제공한다.In addition, there is provided an veterinary pharmaceutical composition for the prevention or treatment of muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient.
또한, 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 사료 조성물을 제공한다.In addition, there is provided a feed composition for improving muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient.
또한, 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 사료 조성물을 제공한다.In addition, there is provided a feed composition for improving muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient.
본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 유비퀴틴 리가아제인 Atrogin1 및 MuRF1의 과발현을 억제하고, 근위축조절인자 Forkhead box(FOX) 0의 활성을 억제함으로써 근위축 개선에 효능을 발휘한다.The composition containing the Wireungchae extract or indigo plant extract of the present invention as an active ingredient is effective in improving muscle atrophy by inhibiting overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibiting the activity of the muscle atrophy regulator Forkhead box (FOX) 0 to perform
또한, 본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 근육조직 생성 억제에 관여하는 마이오스타틴(myostatin)을 억제하고 마이오스타틴의 억제제인 폴리스타틴(follistatin)의 증가를 통해 근육 강화를 시키고 근관(myotube)의 크기를 증가시킬 수 있으며, 히스톤탈아세틸화 효소(Histone deacetylases, HDACs)의 활성을 억제할 수 있다.In addition, the composition containing the Wireungchae extract or the indigo plant extract of the present invention as an active ingredient suppresses myostatin, which is involved in the inhibition of muscle tissue production, and increases follistatin, an inhibitor of myostatin, through It can strengthen muscle, increase the size of myotube, and inhibit the activity of histone deacetylases (HDACs).
또한, 본 발명의 위릉채 추출물 또는 쪽 추출물을 유효성분으로 함유하는 조성물은 기존에 근위축 개선과 근육 강화에 효능이 있다고 알려진 수베로일아닐리드 하이드록사믹산(suberoylanilide hydroxamic acid, SAHA, HDAC 억제제), 우르솔산(ursolic acid), 모노 크레아틴(creatine monohydrate)보다 더 뛰어난 효과를 발휘할 수 있다.In addition, the composition containing the extract of Wireungchae or indigo plant of the present invention as an active ingredient is suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), which is known to be effective in improving muscle atrophy and strengthening muscles, It may be more effective than ursolic acid or creatine monohydrate.
도 1은 글루코코르티코이드(glucocorticoide)의 근위축 전사인자 FOX0 및 유비퀴틴 리가아제(MuRF1, Atrogin1)와의 작용에 따른 근위축 발생 기전에 대해 나타낸 그림이다.
도 2는 본 발명의 위릉채 추출물 또는 쪽 추출물이 분화시킨 근육세포에 덱사메타손을 처리한 후 근위축 인자(FOXO3A, FOXO1,MuRF1, atrogin-1)의 mRNA 발현에 미치는 영향을 확인한 결과 그래프이다.
도 3은 본 발명의 위릉채 추출물이 분화시킨 근육세포에 덱사메타손을 처리한 후 근육 생성 관련 인자(마이오스타틴(myostatin), 폴리스타틴(follistatin))의 mRNA 발현에 미치는 영향을 확인한 결과 그래프이다.
도 4는 본 발명의 위릉채 추출물 또는 쪽 추출물이 분화시킨 근육세포에 덱사메타손을 처리한 후 근위축 인자(FOXO3A, FOXO1, MuRF1, atrogin-1)의 단백질 발현에 미치는 영향을 확인한 결과 그래프이다.
도 5는 본 발명의 위릉채 추출물 또는 쪽 추출물이 분화시킨 근육세포에서 근위축 인자(FOXO3A, FOXO1, MuRF1, atrogin-1)의 mRNA 발현 및 단백질 발현에 미치는 영향을 확인한 결과 그래프이다.
도 6은 본 발명의 위릉채 추출물이 분화시킨 근육세포에 덱사메타손을 처리한 후 근관의 직경에 미치는 영향을 확인한 현미경 관찰 그림이다.
도 7은 본 발명의 위릉채 추출물 또는 쪽 추출물이 분화시킨 근육세포에서 FOXO3a, MuRF1과 Atrogin1의 프로모터에 글루코코르티코이드 수용체(Glucocorticoid receptor, GR), FOXO3a와 RNA polymerase Ⅱ(Pol Ⅱ)의 결합력을 조사한 결과 그래프이다.
도 8은 히스톤탈아세틸화효소(Histone deacetylases, HDACs)의 활성기작을 나타낸 모식도이다.
도 9는 본 발명의 위릉채 추출물 또는 쪽 추출물이 분화시킨 근육세포에서 HDAC(Histone deacetylases, 히스톤탈아세틸화효소) 6의 활성에 미치는 영향을 조사한 결과 그래프이다.1 is a diagram showing the mechanism of muscle atrophy according to the action of glucocorticoids with the muscular atrophy transcription factor FOX0 and ubiquitin ligase (MuRF1, Atrogin1).
Figure 2 is a graph showing the results confirming the effect of the Wireungchae extract or the indigo plant extract of the present invention on the mRNA expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) after treatment with dexamethasone in differentiated muscle cells.
3 is a graph showing the results of confirming the effect of the Wireungchae extract of the present invention on the mRNA expression of muscle production-related factors (myostatin, follistatin) after treatment with dexamethasone in differentiated muscle cells.
4 is a graph showing the results confirming the effect of the Wireungchae extract or the indigo plant extract of the present invention on the protein expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) after treatment with dexamethasone in differentiated muscle cells.
5 is a graph showing the results of confirming the effect of the Wireungchae extract or the indigo plant extract of the present invention on the mRNA expression and protein expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) in differentiated muscle cells.
Figure 6 is a microscopic observation confirming the effect of the Wireungchae extract of the present invention on the diameter of the root canal after dexamethasone treatment on differentiated muscle cells.
Figure 7 is the result of investigating the binding force of FOXO3a, MuRF1 and Atrogin1 promoters, glucocorticoid receptor (GR), FOXO3a and RNA polymerase Ⅱ (Pol Ⅱ) in muscle cells differentiated by Wireungchae extract or indigo plant extract of the present invention; It is a graph.
8 is a schematic diagram showing the activation mechanism of histone deacetylases (HDACs).
9 is a graph showing the results of investigating the effect of Wireungchae extract or indigo plant extract of the present invention on the activity of HDAC (Histone deacetylases, histone deacetylase) 6 in differentiated muscle cells.
근위축(muscle atrophy)은 근세포의 크기가 저하되어, 근육의 근원섬유 숫자가 감소하여 골격근 질량이 줄어드는 것을 말하며, 이를 유발하는 원인은 금식, 활동저하(inactivity), 탈신경(denervation), 노화 및 스테로이드 남용이 있다. 스테로이드 호르몬은 부신피질에서 분비되며 글루코코르티코이드(glucocorticoid)는 면역반응 조절, 포도당, 지방, 함염증 반응 조절 등에 관여하지만, 장기간 투여하면 근위축, 당뇨병 등의 부작용이 유발된다.Muscle atrophy refers to a decrease in skeletal muscle mass due to a decrease in the number of myofibrils due to a decrease in the size of muscle cells, and the causes of this are fasting, inactivity, denervation, aging and There is steroid abuse. Steroid hormones are secreted from the adrenal cortex, and glucocorticoids are involved in immune response regulation, glucose, fat, and anti-inflammatory responses, but long-term administration causes side effects such as muscle atrophy and diabetes.
글루티코르티코이드 수용체(GR)가 활성화되면 FOX0(Forknead box 0)가 근위축 전사인자로 작용하며, MuRF1과 Atrogin1은 근세포에 특이적으로 발현하는 유비퀴틴 리가아제(ubiquitin ligase)로 작용하게 된다. 이들의 발현이 증가되면 근육 단백질들이 유비퀴틴화(ubiquitination)되고 프로테아좀(proteasome)이 의존적으로 분해되어 근손실이 유발된다. 관련 기작에 대해서는 도 1에 나타냈다. When the glutcorticoid receptor (GR) is activated, FOX0 (Forknead box 0) acts as a muscle atrophy transcription factor, and MuRF1 and Atrogin1 act as ubiquitin ligases specifically expressed in muscle cells. When their expression is increased, muscle proteins are ubiquitinated and the proteasome is degraded in a dependent manner to induce muscle loss. A related mechanism is shown in FIG. 1 .
노화나 스테로이드호르몬에 의한 근위축과 근감소증을 계속 방치할 경우 근육량이 최대 60%까지 줄고 근육의 기능도 현저히 잃을 수 있으며, 골절과 낙상, 우울증, 비만, 장애, 나아가 사망으로까지 이어질 수 있기에 근위축을 개선, 예방 또는 치료를 위한 연구와, 이에 대한 해결방안이 필요하다. 기존에는 근위축 개선과 근육 강화를 위해 수베로일아닐리드 하이드록사믹산(suberoylanilide hydroxamic acid, SAHA, HDAC 억제제), 우르솔산(ursolic acid), 모노 크레아틴(creatine monohydrate)을 사용해왔다. 본 발명에서는 기존에 시판되는 물질들보다 근위축 개선 효능에 훨씬 뛰어난 효능을 발휘할 수 있는 물질을 개발하여 제공하고자 한다.If muscle atrophy and sarcopenia caused by aging or steroid hormones are left unattended, muscle mass can be reduced by up to 60% and muscle function can be significantly lost. Research to improve, prevent, or treat atrophy and a solution to this are needed. Previously, suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), ursolic acid, and creatine monohydrate have been used to improve muscle atrophy and strengthen muscles. In the present invention, it is intended to develop and provide a substance capable of exhibiting much superior efficacy in improving muscle atrophy than conventional commercially available substances.
본 발명은 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 식품 조성물을 제공한다. 또한, 본 발명은 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 식품 조성물을 제공한다. 또한, 본 발명은 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 약학 조성물을 제공한다. 또한, 본 발명은 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 약학 조성물을 제공한다. 또한, 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 동물 의약품 조성물을 제공한다. 또한, 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 예방 또는 치료용 동물 의약품 조성물을 제공한다. 또한, 위릉채 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 사료 조성물을 제공한다. 또한, 쪽 추출물을 유효성분으로 함유하는 것을 특징으로 하는 근위축 개선용 사료 조성물을 제공한다.The present invention provides a food composition for improving muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient. In addition, the present invention provides a food composition for improving muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient. In addition, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy, characterized in that it contains the Wireungchae extract as an active ingredient. In addition, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient. In addition, it provides an veterinary pharmaceutical composition for the prevention or treatment of muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient. In addition, there is provided an veterinary pharmaceutical composition for the prevention or treatment of muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient. In addition, there is provided a feed composition for improving muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient. In addition, there is provided a feed composition for improving muscle atrophy, characterized in that it contains an extract of indigo plant as an active ingredient.
위릉채는 장미과에 속하는 다년생 초본식물인 딱지꽃(Potentilla chinensis)의 뿌리나 뿌리가 달린 전초를 말한다. 이는 해독작용을 하고, 출혈성 질환, 이질, 각혈, 염증, 종기 등에 효과가 있고 근육이나 관절의 통증을 줄여 줄 수 있어 약재로 주로 사용한다. 봄에서 가을철 사이에 채취하여 햇볕에 말렸다가 달이거나 산제로 하여 복용한다.Wireungchae is a perennial herbaceous plant belonging to the Rosaceae family, Potentilla chinensis . It has a detoxifying effect, is effective for bleeding disorders, dysentery, keratostasis, inflammation, and boils, and is mainly used as a medicine because it can reduce pain in muscles and joints. It is collected between spring and autumn, dried in the sun, and taken as a decoction or powder.
쪽(Indigo)은 쌍떡잎식물 마디풀목 마디풀과에 속하는 한해살이풀로 과거에는 염료 자원으로 재배하였다. 쪽의 잎은 인디고라는 색소를 지니고 있어 남색의 염료로 사용한다. 이는 해열, 해독, 소종의 효능이 있어 감모, 황달, 이질, 토혈 등의 증상과 각종 염증에 약재로 주로 사용한다.Indigo is an annual plant belonging to the dicotyledon family, and was cultivated as a dye resource in the past. The leaves of the indigo plant have a pigment called indigo, which is used as a dye for indigo. It has antipyretic, detoxifying, and small-species effects, so it is mainly used as a medicine for symptoms such as loss of hair, jaundice, dysentery, and hematemesis and various inflammations.
본 발명에 있어서, 상기 위릉채 추출물 또는 쪽 추출물은, 물 또는 유기용매를 가하여 추출하는 용매추출법; 또는 초고압(초임계) 추출법;을 이용하여 수득하는 것일 수 있다. 이때, 상기 유기용매는, 일 예로, 탄소수 1-4의 무수 또는 함수 저급 알코올, 프로필렌글리콜, 부틸렌글리콜, 글리세린, 아세톤, 에틸 아세테이트, 클로로포름, 부틸 아세테이트, 디에틸에테르, 디클로로메탄, 헥산 및 이들의 혼합물로 구성된 군으로부터 선택되는 추출용매를 이용할 수 있다. In the present invention, the Wireungchae extract or the indigo plant extract is a solvent extraction method in which water or an organic solvent is added to extract; Or it may be obtained by using an ultra-high pressure (supercritical) extraction method. In this case, the organic solvent is, for example, anhydrous or hydrous lower alcohol having 1-4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl ether, dichloromethane, hexane and these An extraction solvent selected from the group consisting of a mixture of
이때, 바람직하게는 위릉채 추출물은 위릉채 전초 중량을 기준으로 10배(부피)의 70% 에탄올을 가하여 추출하는 용매 추출법을 사용하였으며, 쪽 추출물은 쪽의 전초 중량을 기준으로 10배(부피)의 70% 에탄올을 가하여 추출하는 용매 추출법을 사용하였다. 더 바람직하게는 용매 추출법을 사용하는 과정에서 각각 50∼70℃에서 22~26시간 침지시킨 후 실온에서 추출액을 수득하고, 3시간 정도 초음파를 가하여 추출하였다. 이때, 상기 추출 시간 및 추출 온도의 범위를 초과하거나 미만하는 경우, 추출물의 수율과 활성이 저하될 수 있다.At this time, preferably, for the Wireungchae extract, a solvent extraction method was used in which 10 times (volume) 70% ethanol was added based on the weight of the indigo plant, and the indigo plant extract was 10 times (volume) based on the weight of the indigo plant. A solvent extraction method in which 70% ethanol was added to extract was used. More preferably, in the process of using the solvent extraction method, the extract was obtained by immersion at 50 to 70° C. for 22 to 26 hours, respectively, at room temperature, followed by ultrasonication for about 3 hours. At this time, when the extraction time and the extraction temperature range are exceeded or less than the range, the yield and activity of the extract may be reduced.
한편, 하기 실험에 의하면, 본 발명의 위릉채 추출물 또는 쪽 추출물은 유비퀴틴 리가아제인 atrogin1 및 MuRF1의 과발현을 억제하고, 근위축조절인자 FOX0의 활성을 억제하였다. 또한, 본 발명의 위릉채 추출물 또는 쪽 추출물은 근육조직 생성 억제에 관여하는 마이오스타틴(myostatin)을 억제하고 마이오스타틴의 억제제인 폴리스타틴(follistatin)의 증가시켰고, 근관(myotube)의 크기를 증가시켰다. 또한, 본 발명의 위릉채 추출물 또는 쪽 추출물은 FOXO3a의 프로모터에 글루코코르티코이드 수용체와 RNA polymerase Ⅱ의 결합력을 감소시키고, MuRF1과 Atrogin1의 프로모터에 FOXO3a와 RNA polymerase Ⅱ의 결합력을 감소시켰으며, 히스톤탈아세틸화효소(Histone deacetylase)의 활성을 감소시켰다. 이를 통해 본 발명의 위릉채 추출물 또는 쪽 추출물은 근위축 개선 또는 근육 생성 억제 개선에 효능이 있는 물질임을 확인할 수 있다.On the other hand, according to the following experiment, the Wireungchae extract or indigo plant extract of the present invention inhibited the overexpression of atrogin1 and MuRF1, which are ubiquitin ligases, and inhibited the activity of the muscle atrophy regulator FOX0. In addition, the Wireungchae extract or indigo plant extract of the present invention inhibited myostatin involved in the inhibition of muscle tissue production, increased the myostatin inhibitor, follistatin, and increased the size of myotube. increased. In addition, the Wireungchae extract or indigo plant extract of the present invention reduced the binding affinity of the glucocorticoid receptor and RNA polymerase Ⅱ to the promoter of FOXO3a, and decreased the binding ability of FOXO3a and RNA polymerase Ⅱ to the promoter of MuRF1 and Atrogin1, and histone deacetylation. It reduced the activity of histone deacetylase. Through this, it can be confirmed that the Wireungchae extract or indigo plant extract of the present invention is a substance effective in improving muscle atrophy or inhibiting muscle production.
한편, 본 발명의 식품 조성물은 일 예로 육류, 곡류, 카페인 음료, 일반음료, 초콜렛, 빵류, 스넥류, 과자류, 피자, 젤리, 면류, 껌류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나일 수 있으며, 반드시 이에 한정되는 것은 아니다.On the other hand, the food composition of the present invention is, for example, meat, grains, caffeinated beverages, general drinks, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, vitamin complexes and other health It may be any one selected from supplements, but is not necessarily limited thereto.
한편, 본 발명의 약학 조성물은 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 선택되는 하나 이상을 사용할 수 있다. 또한, 치료 및 예방제가 약제인 경우 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등이 추가적으로 포함될 수 있다.On the other hand, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient. Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and at least one selected from among them may be used. In addition, when the therapeutic and prophylactic agent is a drug, a filler, an anti-aggregant, a lubricant, a wetting agent, a fragrance, an emulsifier, or a preservative may be additionally included.
한편, 본 발명의 약학 조성물 및 동물 의약품 조성물의 제형은 사용 방법에 따라 바람직한 형태로 제조될 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LOTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPESIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다.On the other hand, the pharmaceutical composition and the formulation of the veterinary pharmaceutical composition of the present invention may be prepared in a desired form according to the method of use, and in particular, it is known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is better to formulate by adopting a known method. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, syrups ( SYRUPS), OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), suspensions (SUSPESIONS), preparations (DECOCTIONS), infusions (INFUSIONS), eye drops (OPHTHALMIC SOLUTIONS), tablets (TABLETS), suppositories (SUPPOSITIORIES), injections (INJECTIONS), alcohol tablets (SPIRITS), CATAPLS ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pastas (PASTES), pills (PILLS), it may be any one selected from soft or hard gelatin capsules.
한편, 본 발명의 약학조성물 및 동물 의약품 조성물에 있어서, 투여량은 투여방법, 복용자의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분인 위릉채 추출물 또는 쪽 추출물을 기준으로 하였을 때 1일 0.000001 내지 100 mg/kg (체중)으로 1회 이상 경구 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.On the other hand, in the pharmaceutical composition and veterinary pharmaceutical composition of the present invention, the dosage is preferably determined in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, based on the active ingredient Wireungchae extract or indigo plant extract, it is possible to orally administer 0.000001 to 100 mg/kg (body weight) at least once per day. However, the above dosage is only an example for illustration, and may be changed by the condition of the user and the prescription of the doctor.
한편, 본 발명에서 '유효성분으로 함유하는'의 의미는 본 발명에서 요구하는 근위축 개선, 예방 또는 치료 효능이 본 발명의 성분인 위릉채 추출물 또는 쪽 추출물로부터 발생함을 의미하고, 그 외에 다른 성분으로 보조성분으로 포함할 수 있음을 의미한다.On the other hand, in the present invention, the meaning of 'contained as an active ingredient' means that the muscle atrophy improvement, prevention or treatment effect required in the present invention is generated from the Wireungchae extract or indigo plant extract, which is a component of the present invention, and other It means that it can be included as an auxiliary component as a component.
한편, 본 발명의 사료 조성물은 상기 위릉채 추출물 또는 쪽 추출물 외에 다른 성분들을 포함할 수도 있다. 예를 들어, 옥수수, 대두박, 팜박, 소맥(밀가루) 등과 비타민, 미네랄 등을 포함하여 조성될 수 있으며, 그 외에 기능성 물질로 동물의 성장을 촉진시키는 성장 촉진 물질 또는 동물의 면역력을 증가시키는 증강 물질을 더 포함할 수도 있다.Meanwhile, the feed composition of the present invention may include other ingredients in addition to the Wireungchae extract or indigo plant extract. For example, it may be composed of corn, soybean meal, palm meal, wheat (wheat), and vitamins, minerals, etc. may further include.
이하, 본 발명의 내용에 대해 하기 실시예 또는 실험예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the content of the present invention will be described in more detail through the following Examples or Experimental Examples. However, the scope of the present invention is not limited only to the following examples and experimental examples, and includes modifications of technical ideas equivalent thereto.
[실시예 1: 위릉채 추출물 제조][Example 1: Preparation of Wireungchae extract]
위릉채 추출물은 위릉채 전초 중량을 기준으로 10배(부피)의 추출용매(70% 에탄올)를 가하고 50∼70℃에서 22~26시간 침지시킨 후 실온에서 추출액을 수득하고, 3시간 정도 초음파를 가하여 추출하였다. 이 과정은 2~3회 반복할 수 있다. 이후, 추출액을 여과한 후 감압농축 및 건조시켜 70% 에탄올 추출물을 얻었다.For the Wireungchae extract, 10 times (volume) of the extraction solvent (70% ethanol) was added based on the weight of the Wireungchae whole plant, immersed at 50-70°C for 22-26 hours, and the extract was obtained at room temperature, followed by ultrasonication for about 3 hours. was added and extracted. This process can be repeated 2-3 times. Thereafter, the extract was filtered, concentrated under reduced pressure and dried to obtain a 70% ethanol extract.
[실시예 2: 쪽 추출물 제조][Example 2: Preparation of indigo plant extract]
쪽 추출물은 쪽의 전초 중량을 기준으로 10배(부피)의 추출용매(70% 에탄올)를 가하고 50∼70℃에서 22~26시간 침지시킨 후 실온에서 추출액을 수득하고, 3시간 정도 초음파를 가하여 추출하였다. 이 과정은 2~3회 반복할 수 있다. 이후, 추출액을 여과한 후 감압농축 및 건조시켜 70% 에탄올 추출물을 얻었다.For the indigo extract, 10 times (volume) of the extraction solvent (70% ethanol) is added based on the weight of the indigo plant, immersed at 50-70° C. for 22-26 hours, and the extract is obtained at room temperature, followed by ultrasonication for about 3 hours. extracted. This process can be repeated 2-3 times. Thereafter, the extract was filtered, concentrated under reduced pressure and dried to obtain a 70% ethanol extract.
[실험예 1: 위릉채 추출물 또는 쪽 추출물의 근위축(muscle atrophy) 개선 효능 평가][Experimental Example 1: Efficacy of improving muscle atrophy of Wireungchae extract or indigo plant extract]
본 실험예에서는 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하였을 때, 근위축이 개선되는지를 확인하기 위해 하기와 같은 실험을 수행하였다.In this experimental example, when the Wireungchae extract (Example 1) or the indigo plant extract (Example 2) was treated, the following experiment was performed to confirm whether muscle atrophy was improved.
1) 세포 배양 및 근관 세포로의 분화유도1) Cell culture and induction of differentiation into myotube cells
L6 쥐 근아세포(myoblast)는 ATCC(American Type Culture Collection, Manassa, VA, USA)에서 분양 받았으며, DMEM(Dulbecco's Modified Eagle's medium)에 10% FBS 및 0.5% 항생제-항진균제(antibiotic-antimycotic)가 첨가된 배지로 37℃, 5% CO2 항온반응기에서 배양하였다. 근아세포를 근관세포(myotube)로 분화시키기 위하여, 100% 세포 배양(confluent) 상태의 세포를 대상으로 2% 말 혈청이 포함된 DMEM 분화배지를 매 2일마다 교체하였다. 5-7일 후 근관(myotube)이 형성된 것을 현미경으로 관찰하고, 근위축 유도를 위하여 덱사메타손(Dexamethasone, DEXA, 20 nmol/L)을 처리하여 분화억제를 유도하였다. L6 mouse myoblasts were purchased from ATCC (American Type Culture Collection, Manassa, VA, USA), and 10% FBS and 0.5% antibiotic-antimycotic were added to DMEM (Dulbecco's Modified Eagle's medium). As a medium, 37° C., 5% CO 2 Incubated in an incubator. In order to differentiate myoblasts into myotubes, the DMEM differentiation medium containing 2% horse serum was replaced every 2 days for cells in 100% confluent state. After 5-7 days, the formation of myotubes was observed under a microscope, and differentiation inhibition was induced by treatment with dexamethasone (DEXA, 20 nmol/L) to induce muscle atrophy.
2) 분화시킨 근육세포에 덱사메타손을 처리한 후, 근위축 인자 및 근육생성 관련 인자의 mRNA 발현량 평가2) After treating the differentiated muscle cells with dexamethasone, mRNA expression levels of muscle atrophy factors and myogenesis-related factors were evaluated.
분화시킨 근육세포(L6)에 덱사메타손을 처리하여 근위축이 유발된 상기 세포에 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하여 근위축 인자인 FOX03A, FOX01, MuRF-1, atrogin-1 및 근육생성 관련 인자 마이오스타틴(myostatin)과 폴리스타틴(follistatin)의 유전자 발현 양상을 관찰하였다. 비교를 위해 근위축과 근육강화에 개선이 있다고 알려져 있고, 시판되는 약물인 수베로일아닐리드 하이드록사믹산(suberoylanilide hydroxamic acid, SAHA, HDAC 억제제), 우르솔산(ursolic acid), 모노 크레아틴(creatine monohydrate)을 실험에 사용하였다.Differentiated muscle cells (L6) were treated with dexamethasone to induce muscle atrophy, and the cells were treated with Wireungchae extract (Example 1) or indigo plant extract (Example 2) to FOX03A, FOX01, MuRF-1 as muscle atrophy factors. , the gene expression patterns of atrogin-1 and myostatin and follistatin, which are related to myogenesis, were observed. For comparison, suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), ursolic acid, and creatine monohydrate, which are commercially available drugs known to improve muscle atrophy and muscle strength, was used in the experiment.
LG 6 쥐 근아세포를 6-well 플레이트에 접종한 후, 2% 말 혈청 첨가 DMEM 배지로 분화시킨 후, 덱사메타손(Dexamethasone, DEXA, 20 nmol/L)과 각 추출물을 8시간 처리하였다. 세포를 회수하여 TRIzol reagent(Invitrogen, Carlsbad, CA)로 추출한 RNA를 분광광도계를 이용하여 정량하고, 3 ㎍ RNA를 RevertAidTM first strand cDNA synthesis kit(Fermentas)를 이용하여 cDNA를 합성하였다. qRT-PCR은 ABI Prism 7500 sequence etection system (Applied Biosystems; Foster City, CA)을 이용하여, 4 μL의 cDNA, 특이적인 프라이머와 Sybergreen master mix(Takara)를 이용하여 실시하였다. 반응결과는 2-ΔΔCt 값을 계산하여 발현양을 상대비교하였다.
사용된 유전자는 atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1, 마이오스타틴(myostatin)과 폴리스타틴(follistatin)이다. FOX(Forkhead box)0은 글루티코이드 수용체(GR)가 활성화되면 근위축 전사인자로 작용하며, atrogin-1과 MuRF(muscle RING-finger protein)1은 근세포에 특이적으로 발현하는 유비퀴틴 리가아제(ubiquitin ligase)로 작용한다. 이들의 발현이 증가되면 근육 단백질들이 유비퀴틴화(ubiquitination)되고 프로테아좀(proteosome)이 의존적으로 분해되어 근손실이 유발되는 것이다. 상기 타겟 mRNA의 발현량은 GAPDH로 보정(normalization) 한 후 비(ratio)로 나타내었다. The genes used were atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1, myostatin and follistatin. FOX (Forkhead box) 0 acts as a transcription factor for muscle atrophy when gluticoid receptor (GR) is activated, and atrogin-1 and MuRF (muscle ring-finger protein) 1 are ubiquitin ligases ( It acts as a ubiquitin ligase). When their expression is increased, muscle proteins are ubiquitinated and the proteasome is degraded in a dependent manner to induce muscle loss. The expression level of the target mRNA was expressed as a ratio after normalization with GAPDH.
위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)의 근위축 인자(FOXO3A, FOXO1,MuRF1, atrogin-1)의 mRNA 발현에 미치는 영향을 확인한 결과, 도 2와 같이 위릉채 추출물과 쪽 추출물은 FOXO3A, FOXO1,MuRF1, atrogin-1의 mRNA 발현을 농도 의존적으로 감소시켰으며, 이는 수베로일아닐리드 하이드록사믹산(suberoylanilide hydroxamic acid, SAHA), 우르솔산(ursolic acid), 모노 크레아틴(creatine monohydrate)보다 효과적으로 나타나는 것을 확인할 수 있었다. As a result of confirming the effect on the mRNA expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) of the Wireungchae extract (Example 1) or the indigo plant extract (Example 2), as shown in FIG. 2, the Wireungchae extract and indigo plant The extract reduced the mRNA expression of FOXO3A, FOXO1, MuRF1, and atrogin-1 in a concentration-dependent manner, which was suberoylanilide hydroxamic acid (SAHA), ursolic acid, and creatine monohydrate. ) was found to be more effective.
또한, 위릉채 추출물이 근육 생성을 억제하는 마이오스타틴(myostatin, growth differentiation factor 8)과, 마이오스타틴을 억제하고 근육세포를 증가시키는 폴리스타틴(follistatin)의 mRNA 발현에 미치는 영향을 확인하였다. 그 결과, 도 3과 같이 위릉채 추출물은 미오스타틴의 발현을 감소시켰고, 폴리스타틴의 발현을 증가시키는 것을 확인할 수 있었다. 이를 통해 위릉채 추출물 또는 쪽 추출물은 근위축 개선과 근육생성 억제 개선에 효능이 있는 물질임을 확인할 수 있었다.In addition, the effect of the Wireungchae extract on the mRNA expression of myostatin (growth differentiation factor 8), which inhibits muscle formation, and follistatin, which inhibits myostatin and increases muscle cells, was confirmed. As a result, as shown in FIG. 3 , it was confirmed that the Wireungchae extract decreased the expression of myostatin and increased the expression of follistatin. Through this, it was confirmed that the Wireungchae extract or indigo plant extract was effective in improving muscle atrophy and inhibiting muscle production.
3) 분화시킨 근육세포에 덱사메타손을 처리한 후, 근위축 인자의 단백질 발현량 평가3) After treating the differentiated muscle cells with dexamethasone, the protein expression level of the muscle atrophy factor was evaluated.
분화시킨 근육세포(L6)에 덱사메타손을 처리하여 근위축이 유발된 상기 세포에 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하여 근위축 인자인 FOX03A, FOX01, MuRF-1, atrogin-1의 단백질 발현 양상을 관찰하였다. Differentiated muscle cells (L6) were treated with dexamethasone to induce muscle atrophy, and the cells were treated with Wireungchae extract (Example 1) or indigo plant extract (Example 2) to FOX03A, FOX01, MuRF-1 as muscle atrophy factors. , The protein expression pattern of atrogin-1 was observed.
분화시킨 L6 세포에 덱사메타손(Dexamethasone, DEXA, 20 nmol/L)과 각 추출물을 2시간 처리하고, 세포를 용해완충액으로 균질화하였다. 그 후, 얼음 위에서 10분간 방치한 후 12,000rpm에서 원심 분리하여 상층액을 회수하였다. 회수된 상층액 속의 단백질량은 BCA 방법으로 정량하고 SDS-PAGE로 전기영동을 한 후에 니트로셀룰로오스막(nitrocellulose membrane)으로 옮겨 목적 단백질 항체로 면역 블롯(immunoblot)을 시행하였다. Horse-radish peroxidase가 결합된 이차항체를 두 시간 반응시키고 ECL로 필름에 인화하였다. 사용된 항체는 atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1과 β-actin이며 타겟 단백질의 발현량은 β-actin으로 보정(normalization) 한 후 비(ratio)로 나타내었다. Differentiated L6 cells were treated with dexamethasone (DEXA, 20 nmol/L) and each extract for 2 hours, and the cells were homogenized with a lysis buffer. Thereafter, the supernatant was recovered by centrifugation at 12,000 rpm after standing on ice for 10 minutes. The amount of protein in the recovered supernatant was quantified by BCA method, electrophoresed by SDS-PAGE, and then transferred to a nitrocellulose membrane and immunoblot was performed with the target protein antibody. Horse-radish peroxidase-conjugated secondary antibody was reacted for two hours and then printed on the film with ECL. The antibodies used were atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1 and β-actin, and the expression levels of the target proteins were expressed as ratios after normalization with β-actin.
위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)의 근위축인자(FOXO3A, FOXO1,MuRF1, atrogin-1)의 단백질 발현에 미치는 영향을 확인한 결과, 도 4와 같이 위릉채 추출물과 쪽 추출물은 FOXO3A, FOXO1, atrogin-1, MuRF1의 단백질 발현을 농도 의존적으로 감소시키는 것을 확인할 수 있었다. 이를 통해 상기 근위축 인자의 mRNA 발현 실험결과와 같이 위릉채 추출물 또는 쪽 추출물은 근위축 개선에 효능이 있는 물질임을 확인할 수 있었다.As a result of confirming the effect of Wireungchae extract (Example 1) or indigo plant extract (Example 2) on protein expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1), as shown in FIG. 4, Wireungchae extract and indigo plant It was confirmed that the extract reduced the protein expression of FOXO3A, FOXO1, atrogin-1, and MuRF1 in a concentration-dependent manner. Through this, it was confirmed that the Wireungchae extract or the indigo plant extract was effective in improving muscle atrophy as shown in the mRNA expression test result of the muscle atrophy factor.
4) 분화시킨 근육세포에서 근위축 인자의 mRNA 발현량 및 단백질 발현량 평가4) Evaluation of mRNA expression level and protein expression level of muscle atrophy factor in differentiated muscle cells
분화시킨 근육세포(L6)에 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하여 근위축 인자인 FOX03A, FOX01, MuRF-1, atrogin-1의 mRNA 발현 및 단백질 발현 양상을 관찰하였다. 실험방법은 덱사메타손을 처리하지 않는 것을 제외하고는 상기의 mRNA 발현 실험과 단백질 발현 실험과 동일하게 수행하였다.Differentiated muscle cells (L6) were treated with Wireungchae extract (Example 1) or indigo plant extract (Example 2) to examine the mRNA expression and protein expression patterns of muscle atrophy factors FOX03A, FOX01, MuRF-1, and atrogin-1. observed. The experimental method was performed in the same manner as the mRNA expression experiment and the protein expression experiment, except that dexamethasone was not treated.
위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)의 근위축인자(FOXO3A, FOXO1,MuRF1, atrogin-1)의 mRNA 발현 및 단백질 발현에 미치는 영향을 확인한 결과, 도 5의 A) 내지 C)와 같이 위릉채 추출물과 쪽 추출물은 덱사메타손을 처리하지 않고, 분화시킨 근육세포 자체에서 FOXO3A, MuRF1, atrogin-1의 mRNA 발현을 농도 의존적으로 감소시키는 것을 확인할 수 있었다. 또한, 도 5의 D) 내지 F)와 같이 위릉채 추출물과 쪽 추출물은 덱사메타손을 처리하지 않고, 분화시킨 근육세포 자체에서 FOXO3A, FOXO1의 단백질 발현을 농도 의존적으로 감소시키는 것을 확인할 수 있었다. 이를 통해 위릉채 추출물 또는 쪽 추출물은 분화시킨 근육세포 자체에서도 근위축 개선에 효능이 있는 물질임을 확인할 수 있었다.As a result of confirming the effect on mRNA expression and protein expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) of Wireungchae extract (Example 1) or indigo plant extract (Example 2), FIG. 5A) to As shown in C), it was confirmed that Wireungchae extract and indigo plant extract reduced the mRNA expression of FOXO3A, MuRF1, and atrogin-1 in a concentration-dependent manner in differentiated muscle cells without dexamethasone treatment. In addition, as shown in D) to F) of FIG. 5 , it was confirmed that the Wireungchae extract and the indigo plant extract reduced the protein expression of FOXO3A and FOXO1 in a concentration-dependent manner in the differentiated muscle cells themselves without dexamethasone treatment. Through this, it could be confirmed that the Wireungchae extract or the indigo plant extract was effective in improving muscle atrophy even in differentiated muscle cells themselves.
5) 근관의 직경 관찰5) Observation of the diameter of the root canal
분화시킨 근육세포(L6)에 덱사메타손을 처리하여 근위축이 유발된 상기 세포에 위릉채 추출물(실시예 1)을 처리하여 200배 현미경으로 근관의 직경을 관찰하였다. 각 그룹당 무작위로 직경을 측정하여 관찰하였다. 비교를 위해 시판되는 약물인 크레아틴을 사용하였다.Differentiated muscle cells (L6) were treated with dexamethasone to induce muscle atrophy, and the Wireungchae extract (Example 1) was treated, and the diameter of the root canal was observed under a 200-fold microscope. The diameters were measured and observed at random for each group. For comparison, a commercially available drug, creatine, was used.
그 결과, 도 6과 같이 분화시킨 근육세포에 덱사메타손을 처리한 경우, 근위축이 유발되어 근관(Myotube)의 크기가 감소되었다. 하지만, 위릉채 추출물은 처리함에 따라 근관의 크기 감소가 억제되는 것을 확인할 수 있었다. 이를 통해 위릉채 추출물은 근위축 개선에 효능이 있는 물질임을 확인할 수 있었다.As a result, when the differentiated muscle cells were treated with dexamethasone as shown in FIG. 6, muscle atrophy was induced and the size of the myotube was reduced. However, it was confirmed that the reduction in the size of the root canal was suppressed as the Wireungchae extract was treated. Through this, it was confirmed that the Wireungchae extract was effective in improving muscle atrophy.
6) 염색질면역침강(chromatin immunoprecipitation, ChIP)에 의한 근위축인자 글루코코르티코이드 수용체(Glucocorticoid receptor, GR), FOXO3a와 RNA polymerase Ⅱ의 결합력 조사6) Investigation of binding affinity between glucocorticoid receptor (GR), FOXO3a and RNA polymerase Ⅱ by chromatin immunoprecipitation (ChIP)
분화시킨 근육세포(L6)에 염색질 면역침강 분석(Chromatin immunoprecipitation assay, ChIP assay)에 의한 근위축 인자들의 결합력을 조사하였다. 분화시킨 근육세포(L6)에 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리한 세포를 1% 포르말린에 넣고 4℃에서 1시간 동안 충분히 고정시킨 후 단백분해 효소 길항제가 포함된 lysis buffer를 넣고 nucleosome이 200~1,000 bp 크기로 잘리도록 소니케이션(sonication)하였다. 결합력을 보는 항체를 1.0 ㎍을 첨가하여 16시간 반응시켰다. Protein G agarose를 첨가하여 반응 후 원심분리하여 상층액을 제거하고 씻어주었다. Elution buffer를 첨가하여 반응시킨 후 14,000 rpm에서 원심분리하여 얻은 상층액에 proteinase K 1 ㎕를 첨가하여 45℃에서 1~2시간 반응시켰다. DNA 정제 콜럼(column)을 이용해 DNA를 분리 후 qRT-PCR로 근위축 인자의 결합력을 분석하였다.The binding ability of muscle atrophy factors was investigated in differentiated muscle cells (L6) by chromatin immunoprecipitation assay (ChIP assay). Cells treated with Wireungchae extract (Example 1) or indigo plant extract (Example 2) in differentiated muscle cells (L6) were placed in 1% formalin and sufficiently fixed at 4° C. for 1 hour, including a proteolytic enzyme antagonist The lysis buffer was added and sonication was performed so that the nucleosome was cut to a size of 200 to 1,000 bp. 1.0 μg of the antibody showing binding force was added and reacted for 16 hours. After the reaction by adding Protein G agarose, the supernatant was removed and washed by centrifugation. Elution buffer was added to react, and 1 μl of proteinase K was added to the supernatant obtained by centrifugation at 14,000 rpm, followed by reaction at 45° C. for 1-2 hours. After DNA was isolated using a DNA purification column, the binding force of the muscle atrophy factor was analyzed by qRT-PCR.
분화시킨 근육세포(L6)에 위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하여 농도 누적적으로 처리하였을 때, FOXO3a, MuRF1과 Atrogin1의 프로모터에 글루코코르티코이드 수용체(Glucocorticoid receptor, GR), FOXO3a와 RNA polymerase Ⅱ(Pol Ⅱ)의 결합력을 염색질 면역침강 분석으로 조사하였다. When the differentiated muscle cells (L6) were treated with the Wireungchae extract (Example 1) or the indigo plant extract (Example 2) to accumulate concentrations, FOXO3a, MuRF1 and Atrogin1 promoters glucocorticoid receptor (Glucocorticoid receptor, GR), FOXO3a and RNA polymerase II (Pol II) were investigated by chromatin immunoprecipitation assay.
그 결과, 도 7의 a)와 같이 위릉채 추출물 또는 쪽 추출물은 FOXO3a의 프로모터에 글루코코르티코이드 수용체(Glucocorticoid receptor, GR)와 RNA polymerase Ⅱ(Pol Ⅱ)의 결합력을 감소시키는 것으로 나타났다. 또한, 도 7의 b) 및 c)와 같이 MuRF1와 Atrogin1의 프로모터에 FOXO3a와 RNA polymerase Ⅱ(Pol Ⅱ)의 결합력을 감소시키는 것으로 나타났다.As a result, as shown in FIG. 7 a), it was found that the Wireungchae extract or the indigo plant extract reduced the binding force of the glucocorticoid receptor (GR) and RNA polymerase II (Pol II) to the promoter of FOXO3a. In addition, as shown in b) and c) of FIG. 7 , it was shown to decrease the binding force of FOXO3a and RNA polymerase II (Pol II) to the promoters of MuRF1 and Atrogin1.
7) 히스톤탈아세틸화 효소(Histone deacetylases, HDACs) 6의 효소활성 조사.7) Investigation of enzymatic activity of histone deacetylases (HDACs) 6 .
후성유전학의 조절자인 HDAC(Histone deacetylases, 히스톤탈아세틸화 효소)은 히스톤의 아세틸화를 억제하여 세포증식 억제인자의 발현을 저해하여, 세포증식을 촉진시키고, 세포의 종양화 및 분화를 조절한다. 그리하여, HDACs 병리학적 활성 및 조절감소가 근위축(muscular dystrophy) 등 여러 질병에 관여한다. 즉, HDACs는 탈신경, 근육영양장애, 불사용으로 인한 근육 위축 및 근기능 장애에 영향을 미치고, 골격근에서의 부작용을 조절하고 있다. 특히, 근위축 모델에서 HDACs의 과발현을 보여주고, HDACs 억제제는 근위축 인자를 감소하였다. Class I HDACs는 골격근 대사에 관여하여, HDAC1/2가 결핍 된 쥐에서 근육 변성, 미토콘드리아 기능 장애, autophagy의 억제, 산화 환원대사를 특징으로 하는 근육 병증을 나타낸다.HDAC (Histone deacetylases, histone deacetylases), a modulator of epigenetics, inhibits the acetylation of histones and inhibits the expression of cell growth inhibitory factors, thereby promoting cell proliferation and regulating tumorigenesis and differentiation of cells. Thus, the pathological activity and decreased regulation of HDACs are involved in various diseases such as muscular dystrophy. That is, HDACs affect denervation, muscular dystrophy, and muscle atrophy and muscle dysfunction due to disuse, and control side effects in skeletal muscle. In particular, the overexpression of HDACs was shown in the muscle atrophy model, and the HDACs inhibitor decreased the muscle atrophy factor. Class I HDACs are involved in skeletal muscle metabolism, and in mice deficient in HDAC1/2, myopathy is characterized by muscle degeneration, mitochondrial dysfunction, inhibition of autophagy, and redox metabolism.
HDAC6는 HDACs 클래스 IIb 패밀리 멤버이고, 세포질 내에 위치한 유일한 탈아틸세틸화효소(cytoplasmic deacetylase)이다. 히스톤을 직접 촉매하지 않고, 알파-튜뷸린(α-tubulin) 및 열쇼크 단백질(HSP90)을 기질로 하여 세포의 수송, 부착과 운동을 조절한다, 따라서, 유전적으로 관련된 생리기능에 미치는 영향이 훨씬 적어 부작용이 줄어들고, 탈아세틸화시킨다. HDAC6의 효소 활성 억제는 근위축인자를 억제하고 근보호기능을 가진다. 도 8에 HDAC의 활성기작을 나타내었다.HDAC6 is a member of the HDACs class IIb family and is the only cytoplasmic deacetylase located in the cytoplasm. It does not directly catalyze histones, but uses α-tubulin and heat shock protein (HSP90) as substrates to regulate cell transport, adhesion and movement. Less side effects and deacetylation. Inhibition of enzymatic activity of HDAC6 inhibits muscle atrophy and has a myoprotective function. 8 shows the activation mechanism of HDAC.
위릉채 추출물(실시예 1) 또는 쪽 추출물(실시예 2)을 처리하여 농도 누적적으로 처리하여 히스톤아세틸화효소(Histone deacetylases, HDACs) 6의 효소활성을 조사하고자 하였다. 실험 방법은 HDACs가 아세틸 리신(K) 아미노산으로부터 아세틸기(O=C-CH3)를 제거하는 정도를 측정하였다. 즉, 아세틸레이트 라이신(Lys-Ac) 사이드 체인을 포함하는 HDAC 형광 기질을 정제된 HDAC 효소와 함께 배양하였다. 즉, HDAC에 의해 아세틸 그룹이 라이신(K)에서 제거된 경우, 이 용액은 형광성을 허용하는 염료를 방출한다. 그 형광 수치를 측정하여 HDAC 활성을 분석하였다. 비교를 위해 HDAC억제제인 트리코스타틴(Tricostatin) A를 사용하였다. It was intended to investigate the enzymatic activity of histone deacetylases (HDACs) 6 by treating the Wireungchae extract (Example 1) or the indigo plant extract (Example 2) and accumulating concentrations. The experimental method was to measure the extent to which HDACs remove the acetyl group (O=C-CH3) from the acetyl lysine (K) amino acid. That is, the HDAC fluorescent substrate containing the acetylate lysine (Lys-Ac) side chain was incubated with the purified HDAC enzyme. That is, when an acetyl group is removed from lysine (K) by HDAC, the solution releases a dye that allows fluorescence. HDAC activity was analyzed by measuring the fluorescence level. For comparison, an HDAC inhibitor, Tricostatin A, was used.
그 결과, 도 9와 같이 위릉채 추출물 또는 쪽 추출물은 농도의존적으로 히스톤아세틸화효소(Histone deacetylases, HDACs) 6의 효소활성을 억제하는 것으로 나타났다.As a result, as shown in Figure 9, Wireungchae extract or indigo plant extract was shown to inhibit the enzymatic activity of histone deacetylases (HDACs) 6 in a concentration-dependent manner.
Claims (8)
A food composition for improving muscle atrophy, characterized in that it contains the Wireungchae extract as an active ingredient to inhibit the overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibit the activity of the muscle atrophy regulator Forkhead box (FOX) O.
A pharmaceutical composition for the prevention or treatment of muscular atrophy, characterized in that it suppresses the overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibits the activity of the muscle atrophy regulator Forkhead box (FOX) O by containing the Wireungchae extract as an active ingredient.
Veterinary pharmaceutical composition for the prevention or treatment of muscular atrophy, characterized in that it contains an extract of Wireungchae as an active ingredient to inhibit the overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibit the activity of the muscle atrophy regulator Forkhead box (FOX) O .
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| 방면호 외 7인. 딱지꽃(Potentilla chinensis)의 지상부로부터 Triterpenoid 화합물의 분리. 한국작물학회 학술대회논문집. 2006년, pp. 600-601 1부.* |
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