KR102670918B1 - Composition for preventing, ameliorating or treating muscular disease comprising Moutan Radicis Cortex extract as effective component - Google Patents
Composition for preventing, ameliorating or treating muscular disease comprising Moutan Radicis Cortex extract as effective component Download PDFInfo
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- KR102670918B1 KR102670918B1 KR1020210081465A KR20210081465A KR102670918B1 KR 102670918 B1 KR102670918 B1 KR 102670918B1 KR 1020210081465 A KR1020210081465 A KR 1020210081465A KR 20210081465 A KR20210081465 A KR 20210081465A KR 102670918 B1 KR102670918 B1 KR 102670918B1
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Abstract
본 발명은 목단피 추출물을 유효성분으로 포함하는 근육질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. 본 발명의 유효성분인 목단피 추출물은 세포 독성이 거의 없으며, 근관세포로의 분화를 유도 및 촉진할 수 있으며, 근세포 사멸 관련 유전자의 발현량을 억제시키는 효과가 있다. 또한 갱년기에 의해 감소된 근육의 단면적을 증가시키고 근력을 증가시키는 효과가 있는 것이다. 따라서 본 발명의 조성물은 근육성장 촉진 또는 갱년기에 의한 근 손실의 예방 또는 개선용 건강기능식품과 근육질환의 예방 또는 치료용 의약품으로 유용하게 사용할 수 있다.The present invention relates to a composition for the prevention, improvement or treatment of muscle disease containing extract from the bark of the plant as an active ingredient. The active ingredient of the present invention, the extract of woody stem bark, has little cytotoxicity, can induce and promote differentiation into myotube cells, and has the effect of suppressing the expression level of genes related to muscle cell death. It also has the effect of increasing the cross-sectional area of muscles reduced by menopause and increasing muscle strength. Therefore, the composition of the present invention can be usefully used as a health functional food for promoting muscle growth or preventing or improving muscle loss due to menopause, and as a medicine for preventing or treating muscle diseases.
Description
본 발명은 목단피 추출물을 유효성분으로 포함하는 근육질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for the prevention, improvement or treatment of muscle disease containing extract from the bark of the plant as an active ingredient.
우리 몸의 근육은 뼈에 붙어 뼈를 보호하고 체형을 바르게 유지시켜 주는 등의 여러 가지 기능을 한다. 또한, 근육은 칼슘 유입을 촉진시켜 골 밀도를 높여 주기도 한다. 그러나 신체는 노화하면서 구성성분의 변화로써 체지방과 체단백질의 재분포가 일어나며, 약 50세가 되면 근 세포 내 단백질의 합성속도가 분해속도보다 느려져 근육이 급격하게 퇴화를 시작하게 되며, 근육 감소 질환에 노출될 수 있다.The muscles in our body are attached to the bones and perform various functions, such as protecting the bones and maintaining the correct body shape. Additionally, muscles promote calcium influx and increase bone density. However, as the body ages, redistribution of body fat and body protein occurs due to changes in composition, and at about age 50, the rate of protein synthesis within muscle cells becomes slower than the rate of decomposition, causing muscles to begin to rapidly degenerate, leading to muscle loss diseases. may be exposed.
근육 감소 질환의 하나인 근육 감소증은 평소 자기 체질량의 약 13~24%가 감소한 상태를 말하는 것으로, 근육 감소증이 있으면 행동량이 현격하게 줄어 정신건강을 해칠 뿐만 아니라 생활의 만족도도 떨어지며, 용이한 일상 생활에서도 쉽게 부상을 입고 심각한 중상에 이르기도 한다. 근육 감소증의 원인 중 하나는 노화가 진행됨에 따라 일어나는 골격근의 양과 질의 점진적 감소 및 부적절한 식이 에너지 섭취에 따른 지방과 체지방성분을 포함하는 체중감소 등을 원인으로 꼽을 수 있다.Sarcopenia, one of the muscle loss diseases, refers to a state in which approximately 13 to 24% of one's body mass is reduced. If sarcopenia occurs, the amount of movement is significantly reduced, which not only harms mental health, but also reduces satisfaction with life and makes daily life easier. You can easily get injured and even get seriously injured. One of the causes of sarcopenia is the gradual decrease in the quantity and quality of skeletal muscle that occurs with aging and weight loss, including fat and body fat components, due to inadequate dietary energy intake.
전구세포(precursor cell)의 분열과 결정 과정을 통해 형성된 근모세포(myoblast)는 근세포(myocyte)로 분화하고, 인접한 세포들과 융합하여 근관세포(myotube)로 분화한다. 분화된 근관세포는 근원섬유(myofibril)를 형성하는 과정을 통해 근육이 형성된다. 근모세포의 일부는 줄기 위성세포(satellite cell)로서 정지 상태로 있다가, 근육조직이 외상 또는 근육위축병(muscular dystrophy)과 같은 질병에 노출되면, 위성세포로 알려진 근육 줄기세포들은 손상된 부위의 근육을 치유하고 재생시킨다. 근육위축병과 같은 질환에 있어서, 이들 세포들은 근육의 안정성과 치유에 있어 특히 중요하다.Myoblasts, formed through the division and determination of precursor cells, differentiate into myocytes and fuse with adjacent cells to differentiate into myotubes. Differentiated myotube cells form muscles through the process of forming myofibrils. Some of the myoblasts are called stem satellite cells, which remain in a quiescent state. When muscle tissue is exposed to trauma or a disease such as muscular dystrophy, muscle stem cells, known as satellite cells, are released into the muscle of the damaged area. Heals and regenerates. In diseases such as muscular dystrophy, these cells are particularly important for muscle stability and healing.
한편, 목단피(Moutan Radicis Cortex)는 '모란(Paeonia suffruticosa) 뿌리의 껍질'을 약재로 이르는 말로, 성분으로는 패오노사이드(paeonoside; paeonol glucoside), 패오노라이드(paeonolide; paeonol-rhamnoglucoside) 및 패오놀(paeonol)을 함유하며, 패오노사이드는 저장중에 분해되어 당과 패오놀을 생성한다. 또한, 패오니플로린(paeoniflorin), 옥시패오니플로린(oxypaeoniflorin), 벤조일페오니플로린 (benzoylpaeoniflorin), 패오노라이드 탄닌(paeonolide tannin), 프로시아니딘 B1 (procyanidin B1), 벤조일옥시패오니플로린(benzoyloxypaeoniflorin), 패오닌 (paeonin), 아스트라갈린(astragalin), 페라고닌(pelargonin) 등의 성분이 함유되어 있다. 이러한 성분은 근피뿐만 아니라 목심부에도 존재함이 조직화학적으로 확인되었다. 구어혈약으로서의 항염증 작용은 모노페르펜 글리코사이드(Monoterpene glycosides) 성분이 관여하는 것으로 추측되고 있다. 목단피는 성질이 차서 한방에서 소염성 구어혈약으로서 이용되며, 그 약효는 하복부 장기의 혈관계의 염증, 울혈에 의한 동통, 발열, 화농, 출혈등을 대상으로, 특히 부인과 영역에서 월경불순, 자궁 및 부속기의 염증, 울혈, 견인통에 대해서 소염, 진통, 진경의 효과가 있으며, 치질, 충수염에도 응용되고 있다.Meanwhile, Moutan Radicis Cortex refers to the bark of the root of the peony ( Paeonia suffruticosa ) as a medicine. Its ingredients include paeonoside (paeonol glucoside), paeonolide (paeonol-rhamnoglucoside), and It contains paeonol, and paeonoside decomposes during storage to produce sugar and paeonol. In addition , paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, paeonolide tannin, procyanidin B 1 , benzoyloxypaeoniflorin ), paeonin, astragalin, and pelargonin. It was confirmed histochemically that these components exist not only in the root skin but also in the neck core. It is presumed that the anti-inflammatory action of oral blood medicine is due to the component monoperpene glycosides. Because of its cold properties, the bark of the radish tree is used as an anti-inflammatory herbal medicine in oriental medicine. Its medicinal effects target inflammation of the vascular system of the lower abdominal organs, pain due to congestion, fever, purulence, bleeding, etc., especially in the field of gynecology, menstrual irregularities, uterine and It has anti-inflammatory, analgesic, and antispasmodic effects on inflammation, congestion, and traction pain in the appendages, and is also applied to hemorrhoids and appendicitis.
목단피 추출물 관련 기술로서, 한국등록특허 제1256273호에 목단피 추출물을 포함하는 간질환 예방 또는 치료용 조성물이 개시되어 있고, 한국등록특허 제1528190호에 목단피로부터 분리된 2,5-디하이드록시-4-메톡시아세토페논을 유효성분으로 함유하는 세포 노화 억제용 조성물이 개시되어 있으나, 아직까지 본 발명의 목단피 추출물을 유효성분으로 포함하는 근육질환의 예방, 개선 또는 치료용 조성물에 대해 개시된 바 없다.As a technology related to wood bark extract, Korean Patent No. 1256273 discloses a composition for preventing or treating liver disease containing wood bark extract, and Korean Patent No. 1528190 discloses 2,5-dihydroxy-4 isolated from wood bark extract. - A composition for inhibiting cellular aging containing methoxyacetophenone as an active ingredient has been disclosed, but a composition for preventing, improving, or treating muscle disease containing the extract of woody stem bark of the present invention as an active ingredient has not yet been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 목단피 추출물을 유효성분으로 포함하는 근육질환의 예방, 개선 또는 치료용 조성물을 제공하고, 본 발명의 유효성분인 목단피 추출물이 세포 독성이 거의 없고, 근관 세포로의 분화를 유도 및 촉진할 수 있으며, 근세포 사멸 관련 유전자의 발현량을 억제시키는 효과가 있을 뿐만 아니라, 갱년기에 의해 감소된 근육의 단면적을 증가시키고 근력을 증가시키는 효과가 있다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs. The present invention provides a composition for preventing, improving, or treating muscle diseases containing the extract of the bark of the plant as an active ingredient, and the extract of the bark of the plant, which is the active ingredient of the present invention, is not cytotoxic. There is little, and it can induce and promote differentiation into myotube cells, and has the effect of suppressing the expression level of genes related to muscle cell death, as well as increasing the cross-sectional area of muscle reduced by menopause and increasing muscle strength. By confirming this, the present invention was completed.
상기 목적을 달성하기 위하여, 본 발명은 목단피 추출물을 유효성분으로 함유하는 근육성장 촉진 또는 갱년기에 의한 근 손실의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In order to achieve the above object, the present invention provides a health functional food composition for promoting muscle growth or preventing or improving muscle loss due to menopause, which contains an extract of dandruff bark as an active ingredient.
또한, 본 발명은 목단피 추출물을 유효성분으로 함유하는 근육질환의 예방 또는 치료용 약학 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle disease containing the extract of the dandelion bark as an active ingredient.
본 발명은 목단피 추출물을 유효성분으로 포함하는 근육질환의 예방, 개선 또는 치료용 조성물에 관한 것으로, 본 발명의 유효성분인 목단피 추출물이 세포 독성이 거의 없고, 근관 세포로의 분화를 유도 및 촉진할 수 있으며, 근세포 사멸 관련 유전자의 발현량을 억제시키는 효과가 있을 뿐만 아니라, 갱년기에 의해 감소된 근육의 단면적을 증가시키고 근력을 증가시키는 효과가 있는 것이다.The present invention relates to a composition for the prevention, improvement or treatment of muscle diseases containing an extract of the bark of the plant as an active ingredient. The extract of the bark of the plant, which is an active ingredient of the present invention, has little cytotoxicity and can induce and promote differentiation into root canal cells. It not only has the effect of suppressing the expression level of genes related to muscle cell death, but also has the effect of increasing the cross-sectional area of muscle reduced by menopause and increasing muscle strength.
도 1은 본 발명의 목단피 추출물의 세포 생존율을 확인한 결과이다.
도 2는 본 발명의 목단피 추출물의 처리에 따른 근모세포의 분화정도를 나타낸 것이다. PS는 목단피 추출물을 의미하고, E2는 에스트라디올을 의미한다.
도 3은 본 발명의 목단피 추출물의 처리에 따른 근관세포의 직경을 확인한 결과이다. ###은 정상군 대비 TNFα 처리군의 근관세포 직경이 통계적으로 유의미하게 감소한 것을 의미하며, p<0.001이고, ***은 TNFα 처리군 대비 목단피 추출물 처리군 또는 양성대조군(에스트라디올, 10nM)의 근관세포 직경이 통계적으로 유의미하게 증가하였다는 것으로, p<0.001이다.
도 4는 근모세포(myoblast)에서 근관세포(myotube)로 분화시킨 근육 세포에 TNFα를 처리한 근육세포에서는 Cleaved PARP 단백질 및 Cleaved caspase 3의 발현이 증가되었으나, 본 발명의 목단피 추출물을 처리한 근육세포에서의 Cleaved PARP 단백질 및 Cleaved caspase 3 단백질의 발현이 감소한 것을 확인한 웨스턴 블랏 결과이다.
도 5는 갱년기 동물모델에 목단피 추출물을 경구투여한 후, 동물 모델의 뒷다리 근육 중에서 Tibialis anterior 근육만 분리하여 면역조직화학염색기법으로, 근육의 단면적에 대한 변화를 확인한 결과이다. ###은 Control군 대비 OVX군의 근육 단면적이 통계적으로 유의미하게 감소하였다는 것으로, p<0.001이고, ***은 OVX군 대비 본 발명의 목단피 추출물 투여군 또는 양성대조군(에스트라디올) 투여군의 근육 단면적이 통계적으로 유의미하게 증가하였다는 것으로 p<0.001이다. n.s.은 통계적으로 유의미한 차이가 없다는 것이다.
도 6은 갱년기 모델에 목단피 추출물을 경구투여한 후, 동물 모델의 행동분석 결과로, (A)는 로타로드 테스트 결과이고, (B)는 인버티드 그립 테스트 결과이다. ###은 Control군 대비 OVX군의 로타로드에 매달려 있는 시간 또는 인버티드 그립에서 버티는 시간이 통계적으로 유의미하게 감소하였다는 것으로, p<0.001이고, *, **은 OVX군 대비 본 발명의 목단피 추출물 투여군 또는 양성대조군(에스트라디올) 투여군의 로타로드에 매달려 있는 시간 또는 인버티드 그립에서 버티는 시간이 통계적으로 유의미하게 증가하였다는 것으로 *은 p<0.05이고, **은 p<0.01이다. n.s.은 통계적으로 유의미한 차이가 없다는 것이다.Figure 1 shows the results of confirming the cell viability of the Mokdan bark extract of the present invention.
Figure 2 shows the degree of differentiation of myoblasts according to treatment with the wood dandruff extract of the present invention. PS refers to wood dandelion bark extract, and E2 refers to estradiol.
Figure 3 shows the results of confirming the diameter of root tube cells according to treatment with the wood dandelion bark extract of the present invention. ### indicates a statistically significant decrease in the diameter of myotube cells in the TNFα-treated group compared to the normal group, p<0.001, and *** indicates a wood bark extract-treated group or positive control group (estradiol, 10nM) compared to the TNFα-treated group. The myotube cell diameter increased statistically significantly, p<0.001.
Figure 4 shows that the expression of Cleaved PARP protein and Cleaved caspase 3 was increased in muscle cells differentiated from myoblasts into myotubes and treated with TNFα, but in muscle cells treated with the wood bark extract of the present invention. This is a Western blot result confirming that the expression of Cleaved PARP protein and Cleaved caspase 3 protein was decreased.
Figure 5 shows the results of confirming changes in the cross-sectional area of the muscle using an immunohistochemical staining technique by isolating only the Tibialis anterior muscle from the hind limb muscles of the animal model after oral administration of the extract of the bark extract to a menopausal animal model. ### indicates a statistically significant decrease in the cross-sectional area of the muscle in the OVX group compared to the Control group, p<0.001, and *** indicates the muscle of the group administered with the bark extract of the present invention or the positive control group (estradiol) compared to the OVX group. The cross-sectional area increased statistically significantly, which is p<0.001. ns means there is no statistically significant difference.
Figure 6 shows the results of behavioral analysis of the animal model after oral administration of the extract from the bark of Mokdan to a menopausal model. (A) is the result of the rotarod test, and (B) is the result of the inverted grip test. ### indicates a statistically significant decrease in the time hanging on the rotarod or the time holding in the inverted grip in the OVX group compared to the Control group, p < 0.001, *, ** are the neck hem of the present invention compared to the OVX group There was a statistically significant increase in the time hanging on the rotarod or the time holding in the inverted grip in the extract-administered group or the positive control (estradiol)-administered group. * means p < 0.05, and ** means p < 0.01. ns means there is no statistically significant difference.
본 발명은 목단피 추출물을 유효성분으로 함유하는 근육성장 촉진 또는 갱년기에 의한 근 손실의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a health functional food composition for promoting muscle growth or preventing or improving muscle loss due to menopause, containing extract from the bark of the plant as an active ingredient.
상기 목단피 추출물은 하기의 단계를 포함하는 방법에 의해 제조할 수 있으나, 이에 한정하지 않는다:The wood bark extract can be prepared by a method comprising the following steps, but is not limited to this:
(1) 건조한 목단피에 추출용매를 가하여 추출하는 단계;(1) Extracting by adding an extraction solvent to dried wood bark;
(2) 단계 (1)의 추출물을 여과하는 단계; 및 (2) filtering the extract of step (1); and
(3) 단계 (2)의 여과한 추출물을 농축하고 건조하여 추출물을 제조하는 단계. (3) Concentrating and drying the filtered extract of step (2) to prepare an extract.
상기 단계 (1)에서 추출용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물 중에서 선택하는 것이 바람직하며, 더 바람직하게는 에탄올이며, 더욱더 바람직하게는 70%(v/v) 에탄올이지만 이에 한정하지 않는다. 상기 제조방법에 있어서, 추출방법은 여과법, 열수 추출, 침지 추출, 환류 냉각 추출 및 초음파 추출 등의 당 업계에 공지된 모든 통상적인 방법을 이용할 수 있다. 상기 추출용매는 목단피 중량의 1~20배 첨가하여 추출하는 것이 바람직하며, 더 바람직하게는 5~15배 첨가하는 것이고, 더욱더 바람직하게는 10배 첨가하는 것이다. 추출온도는 20~100℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 1~48시간인 것이 바람직하며, 1~24시간이 더욱 바람직하고, 3시간이 가장 바람직하나 이에 한정하지 않는다. 상기 방법에 있어서, 단계 (3)의 농축은 진공 회전 농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무 건조 또는 동결건조하는 것이 바람직하며, 더 바람직하게는 동결건조이나 이에 한정하지 않는다.In step (1), the extraction solvent is preferably selected from water, lower alcohols of C 1 to C 4 , or mixtures thereof, more preferably ethanol, and even more preferably 70% ( v/v ) ethanol. It is not limited to this. In the above manufacturing method, the extraction method can be any conventional method known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent is preferably added at 1 to 20 times the weight of the bark, more preferably at 5 to 15 times, and even more preferably at 10 times. The extraction temperature is preferably 20 to 100°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 48 hours, more preferably 1 to 24 hours, and most preferably 3 hours, but is not limited thereto. In the above method, the concentration in step (3) is preferably performed using a vacuum rotary concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and more preferably freeze drying, but is not limited to these.
본 발명에 따른 건강기능식품 조성물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강기능식품 중에 포함되는 상기 유효성분의 양은 전체 건강기능식품 중량의 0.1~90 중량부로 가할 수 있다. 하지만, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취하는 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The health functional food composition according to the present invention can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, the amount of the active ingredient contained in the health functional food can be added in the range of 0.1 to 90 parts by weight of the total weight of the health functional food. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in amounts above the above range. .
본 발명의 건강기능식품 조성물은 음료, 환, 정제(tablet), 캡슐제(capsule) 및 산제 중에서 선택된 어느 하나의 제형으로 제조될 수 있으며, 음료로 사용되는 경우, 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 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 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.The health functional food composition of the present invention can be manufactured in any one formulation selected from beverages, pills, tablets, capsules, and powders, and when used as a beverage, it contains the above essential ingredients in the indicated ratio. Other than containing the active ingredient, there are no particular restrictions on other ingredients, and like regular drinks, various flavoring agents or natural carbohydrates can be included as additional ingredients. 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. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.
본 발명의 건강기능식품 조성물은 상기 유효성분 이외에 추가로, 영양제, 비타민, 전해질, 풍미제, 착색제, 증진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 및 탄산음료에 사용되는 탄산화제 중에서 선택된 하나 이상을 더 첨가하여 함유할 수 있다. 이외에도 본 발명의 건강기능식품 조성물은 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 상기 과육은 독립적으로 또는 조합하여 사용할 수 있다. 상기 다양한 첨가제의 비율은 중요하진 않지만, 본 발명의 목단피 추출물 100 중량부 당 0.1~20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above active ingredients, the health functional food composition of the present invention contains nutrients, vitamins, electrolytes, flavors, colorants, enhancers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, and stabilizers. It may further contain one or more selected from topicals, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice and vegetable drinks. The pulp can be used independently or in combination. The ratio of the various additives is not critical, but is generally selected in the range of 0.1 to 20 parts by weight per 100 parts by weight of the wood bark extract of the present invention.
또한, 본 발명은 목단피 추출물을 유효성분으로 함유하는 근육질환의 예방 또는 치료용 약학 조성물에 관한 것이다.Additionally, the present invention relates to a pharmaceutical composition for the prevention or treatment of muscle disease containing the extract of dandruff bark as an active ingredient.
상기 근육질환은 긴장감퇴증(atony), 근위축증(muscular atrophy), 근이영양증(muscular dystrophy), 근육 퇴화, 근경직증, 근디스트로피, 근위축성 축삭경화증, 근무력증, 악액질(cachexia) 및 근육감소증(sarcopenia) 중에서 선택된 어느 하나인 것이 바람직하지만 이에 한정하는 것은 아니다. The above muscle diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscular rigidity, muscular dystrophy, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, and sarcopenia. It is preferable to select any one, but it is not limited to this.
본 발명의 약학 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 상기 조성물을 제제화할 경우에는 통상적으로 사용하는 담체, 부형제 또는 희석제를 사용하여 조제할 수 있으나 이에 한정하는 것은 아니다. 경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제 또는 좌제 등이 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제(base)로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, it can be prepared using commonly used carriers, excipients, or diluents, but is not limited thereto. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, or preservatives. You can. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, or suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
본 발명의 약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여시 피부 외용 또는 복강 내, 직장, 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사 방식을 선택하는 것이 바람직하지만 이에 제한하지 않는다. 본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select an injection method for external application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebrovascular injection, but is limited thereto. I never do that. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type of patient's disease, severity, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도에 따라 그 범위가 다양하며, 일일 투여량은 목단피 추출물의 양을 기준으로 0.01~1000㎎/㎏이고, 바람직하게는 30~500㎎/㎏이고, 더욱 바람직하게는 50~300㎎/㎏이며, 하루 1~6회 투여될 수 있으나, 투여 경로, 근육질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.The dosage range of the composition of the present invention varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, and the daily dosage is based on the amount of wood bark extract. It is 0.01 to 1000 mg/kg, preferably 30 to 500 mg/kg, and more preferably 50 to 300 mg/kg, and can be administered 1 to 6 times a day, depending on the route of administration, severity of muscle disease, Since it may increase or decrease depending on gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
실시예 1. 목단피 에탄올 추출물의 제조Example 1. Preparation of ethanol extract from Mokdan bark
500g의 목단피를 50℃에서 7일 동안 열풍 건조한 후, 10배의 70%(v/v) 에탄올을 첨가하여 3시간씩 2회에 걸쳐 환류 냉각 추출 및 여과하였다. 이후 상기 목단피 추출액을 45℃에서 감압농축하여 에탄올을 제거한 후, -80℃에서 7일 동안 동결건조하여 목단피 추출물을 획득하였다. 상기 목단피(Moutan Radicis Cortex)는 '모란(Paeonia suffruticosa) 뿌리의 껍질이다.500 g of wood bark was dried with hot air at 50°C for 7 days, then 10 times 70% ( v/v ) ethanol was added, refluxed, cooled, extracted and filtered twice for 3 hours each. Thereafter, the extract was concentrated under reduced pressure at 45°C to remove ethanol, and then freeze-dried at -80°C for 7 days to obtain a woody bark extract. The Moutan Radicis Cortex is the bark of the root of the peony ( Paeonia suffruticosa ).
실시예 2. 목단피 추출물의 처리에 따른 세포 생존율 분석Example 2. Analysis of cell viability according to treatment of wood dandelion bark extract
세포 생존율 분석을 위해, 1×104 cells/㎖의 C2C12 세포를 96웰 플레이트에 깔아주고, 24~72시간 동안 배양한 후 1, 10, 25, 50, 100 및 500㎍/㎖의 목단피 추출물을 각 웰에 처리하고 48시간 동안 배양하였다. 이후, 490nm에서 흡광도를 측정하여 세포 생존율을 분석하였으며, 3번 반복실험하여 평균±표준편차로 나타냈다. For cell viability analysis, 1 × 10 4 cells/ml of C2C12 cells were spread on a 96-well plate, cultured for 24 to 72 hours, and then 1, 10, 25, 50, 100, and 500 ㎍/㎖ of wood bark extract was added. Each well was treated and cultured for 48 hours. Afterwards, cell viability was analyzed by measuring absorbance at 490 nm, and the experiment was repeated three times and expressed as mean ± standard deviation.
그 결과 도 1에 개시한 바와 같이, 본 발명의 목단피 추출물은 세포 독성이 거의 없는 것으로 나타났다.As a result, as shown in Figure 1, it was shown that the extract of the dandelion bark of the present invention had almost no cytotoxicity.
실시예 3. 목단피 추출물의 처리에 따른 근모세포(myoblast)로부터 근관세포(myotube)로의 분화 효과 확인 Example 3. Confirmation of the differentiation effect from myoblasts to myotubes according to the treatment of wood bark extract
2×105 cells/㎖의 C2C12 세포를 6웰 플레이트에서 24시간 동안 배양한 후, 목단피 추출물을 처리하고 5일 동안 배양하였다. 이후, 목단피 추출물을 처리하고, 30분 후 근손실을 유도하는 10ng/㎖의 TNFα를 처리하면서 근관세포로의 분화를 3일 동안 유도하였다. 이후, 근육의 두께를 정량하였고, TNFα에 의한 근소실은 세포 사멸을 유도하므로 본 발명의 목단피 추출물이 이를 극복하는지를 확인하기 위하여 웨스턴 블랏으로 PARP(Poly(ADP-ribose)polymerase) 및 Caspase 3 단백질의 발현량을 확인하였다.2×10 5 cells/ml of C2C12 cells were cultured in a 6-well plate for 24 hours, then treated with woody bark extract and cultured for 5 days. Afterwards, the cells were treated with the extract of the bark of the plant, and after 30 minutes, they were treated with 10 ng/ml of TNFα, which induces muscle loss, and differentiation into myotube cells was induced for 3 days. Afterwards, the thickness of the muscle was quantified, and since muscle loss due to TNFα induces cell death, the expression of PARP (Poly(ADP-ribose)polymerase) and Caspase 3 protein was tested by Western blot to confirm whether the extract of the bark of the present invention overcomes this. The amount was confirmed.
그 결과, 본 발명의 목단피 추출물을 처리한 경우, 근모세포가 근관세포로 분화하는 것을 촉진하며(도 2), 근관세포의 두께가 TNFα만 처리한 군에 비해 두껍고(도 3), PARP 단백질의 발현량이 상대적으로 감소된(도 4) 것으로부터, 근육세포의 사멸을 억제한다는 것을 알 수 있었다.As a result, when treated with the wood bark extract of the present invention, differentiation of myoblasts into myotube cells is promoted (Figure 2), the thickness of myotube cells is thicker than the group treated only with TNFα (Figure 3), and the PARP protein Since the expression level was relatively reduced (Figure 4), it was found that it inhibits the death of muscle cells.
실시예 4. 갱년기 모델에서 목단피 추출물의 경구투여에 따른 뒷다리 근육의 변화 확인Example 4. Confirmation of changes in hind limb muscles following oral administration of Mokdan bark extract in a menopausal model
난소적출 후 5개월이 지난 마우스(C57BL/6, n=8)에 100 및 300mg/kg의 목단피 추출물을 2개월 동안 경구투여하고 마우스를 희생시킨 후, 마우스의 뒷다리 근육 중에서 Tibialis anterior 근육만 분리하여 면역조직화학(immunohistochemistry: IHC) 분석을 실시하였다. 이미지 분석은 이미지 J 분석프로그램을 이용하여 근육의 단면적(Cross sectional area)을 분석하였다. Mice (C57BL/6, n=8) 5 months after ovariectomy were orally administered 100 and 300 mg/kg of wood bark extract for 2 months. The mice were sacrificed, and only the Tibialis anterior muscle was isolated from the hind limb muscles of the mouse. Immunohistochemistry (IHC) analysis was performed. For image analysis, the cross sectional area of the muscle was analyzed using the Image J analysis program.
그 결과, 난소 절제에 의해 유도된 갱년기 군(OVX)은 정상대조군(Control)에 비해 근육의 단면적이 통계적으로 유의미하게 감소하였고, 300mg/kg의 목단피 추출물 투여군 및 에스트라디올 투여군(양성 대조군, 0.1mg/kg)은 OVX 군에 대비하여 통계적으로 유의미하게 근육의 단면적이 증가하였다(도 5).As a result, the menopausal group (OVX) induced by ovarian resection had a statistically significant decrease in the cross-sectional area of muscle compared to the normal control group (Control), and the group administered 300 mg/kg of bark extract and the group administered estradiol (positive control group, 0.1 mg /kg) showed a statistically significant increase in the cross-sectional area of the muscle compared to the OVX group (Figure 5).
실시예 5. 갱년기 모델에 목단피 추출물의 경구투여에 따른 행동분석Example 5. Behavioral analysis following oral administration of Mokdan bark extract to a menopausal model
(1) 로타로드 테스트(Rotarod Test)(1) Rotarod Test
난소를 절제하여 갱년기를 유도한 후, 2개월 동안 목단피 추출물을 경구 투여하거나, 1개월 동안 에스트라디올을 복강 내 주사한 마우스(C57BL/6, n=8)를 로타로드 머신의 봉에 올려놓고, 20rpm의 회전속도로 회전하여 마우스가 떨어질 때까지의 시간을 측정하였다. 상기 로타로드 테스는 3번 반복하여 평균 값을 실험값으로 채택하였고, 각 군의 평균과 표준편차를 구하였다.After inducing menopause by removing the ovaries, mice (C57BL/6, n = 8) that were orally administered extracts of the dandruff bark for 2 months or injected intraperitoneally with estradiol for 1 month were placed on the rod of the rotarod machine. It was rotated at a rotation speed of 20 rpm and the time until the mouse fell was measured. The rotarod test was repeated three times and the average value was taken as the experimental value, and the average and standard deviation for each group were calculated.
그 결과 도 6(A)에 개시한 바와 같이, 정상군(N) 대비 OVX군이 로타로드에 매달려 있는 시간이 통계적으로 유의미하게 감소하였고, 이에 대비하여 본 발명의 목단피 추출물 투여군과 양성 대조군인 에스트라디올 투여군이 로타로드에 매달려 있는 시간이 통계적으로 유의미하게 증가하였다.As a result, as shown in Figure 6 (A), the time the OVX group hung on the rotarod was statistically significantly reduced compared to the normal group (N), and in contrast, the group administered the wood bark extract of the present invention and the positive control group, Estura. The time the diol administration group spent hanging on the rotarod increased statistically significantly.
(2) Inverted grip test(2) Inverted grip test
그물망에 마우스를 올려 좋고, 그 그물망을 뒤집은 후 마우스가 얼마나 오래 버티는지를 확인하였다.I hovered the mouse over the net, flipped it over, and checked how long the mouse would last.
그 결과, 정상대조군(Control) 대비 OVX군이 그물망에서 버티는 시간이 통계적으로 유의미하게 감소하였고, 이에 대비하여 본 발명의 목단피 추출물 투여군과 양성 대조군인 에스트라디올 투여군이 그물망에서 버티는 시간이 통계적으로 유의미하게 증가하였다(도 6B).As a result, compared to the normal control group (Control), the time that the OVX group stayed in the net was statistically significantly reduced, and in contrast, the time that the group treated with the wood bark extract of the present invention and the estradiol-treated group, which was a positive control group, stayed in the net was statistically significantly reduced. increased (Figure 6B).
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