KR20150138478A - Pharmaceutical composition comprising extract of Geranium krameri FR. Et SAV for Prevention or Treatment of bone diseases - Google Patents
Pharmaceutical composition comprising extract of Geranium krameri FR. Et SAV for Prevention or Treatment of bone diseases Download PDFInfo
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- KR20150138478A KR20150138478A KR1020140065022A KR20140065022A KR20150138478A KR 20150138478 A KR20150138478 A KR 20150138478A KR 1020140065022 A KR1020140065022 A KR 1020140065022A KR 20140065022 A KR20140065022 A KR 20140065022A KR 20150138478 A KR20150138478 A KR 20150138478A
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Abstract
Description
본 발명은 선이질풀의 추출물을 유효성분으로 함유하는 골질환 예방 및 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing and treating osteoporosis, which contains an extract of Lepidoptera as an active ingredient.
또한 본 발명은 선이질풀의 추출물을 유효성분으로 함유하는 골질환 예방 및 개선용 건강기능식품에 관한 것이다. The present invention also relates to a health functional food for prevention and improvement of osteoporosis, which contains an extract of Lepidoptera as an active ingredient.
뼈(bone)는 인체의 연조직과 체중을 지탱해주고 내부기관을 둘러싸서 내부 장기를 외부의 충격으로부터 보호한다. 또한 근육이나 장기를 구조적으로 지탱할 뿐만 아니라 체내의 칼슘이나 다른 필수 무기질 즉 인이나 마그네슘과 같은 물질을 저장하는 인체의 중요한 부분 중 하나이다. 따라서 성장이 끝난 성인의 뼈는 멈추지 않고 죽는 날까지 오래된 뼈는 제거하고 새로운 뼈로 대체하는 생성과 흡수 과정을 매우 역동적, 지속적으로 반복 재생하면서 균형을 유지하게 된다. 이를 뼈의 재형성(bone remodeling)이라고 한다. 이러한 뼈의 재형성은 성장과 스트레스에 의해서 일어나는 뼈의 미세한 손상을 회복시키고 적절히 뼈의 기능을 유지하는데 필수적이다.The bone supports the body's soft tissues and body weight and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the body that not only supports the muscles or organs but also stores calcium or other essential minerals, such as phosphorus or magnesium, in the body. Thus, adult bones do not stop growing until the day they die, and the old bones are removed and replaced with new bones. This is called bone remodeling. Such bone reshaping is essential to restore fine bone damage caused by growth and stress and to maintain bone function properly.
뼈의 재형성에는 크게 두 종류의 세포가 관여하는 것으로 알려져 있다. 두 세포 중 하나는 뼈를 생성하는 조골세포(osteoblast)이고, 다른 하나는 뼈를 파괴하는 파골세포(osteoclast)이다. 조골세포는 RANKL(receptor activator of NF-κB ligand)과 이것의 유도 수용체(decoy receptor)인 OPG(osteoprotegerin)을 생성한다. RANKL이 파골 전구세포(osteoclast progenitor cells) 표면에 있는 수용체인 RANK(receptor activator of NF-κB)에 결합하면 파골 전구세포가 파골세포로 분화되어 골 흡수(resorption)가 일어난다. 자세하게는 RANKL 자극에 의한 세포 내 칼슘 신호 전달로 파골 전구세포에서 파골세포의 분화를 조절하는 핵심 전사인자로 알려져 있는 NFAT(nuclear factor of activated T cells)의 발현이 유도되고, 그 후 세포 외 신호조절인산화효소(extracellular signal-regulated kinase, ERK), NF-κB, p38 MAP kinase 등의 활성화가 NFAT의 자가증폭(autoamplification)을 유발하여 활성이 지속되는 것이다. 따라서 오래된 뼈의 흡수 또는 파괴는 상기와 같은 기작을 통해 파골세포에 의해 이루어지며, 이는 뼈에 구멍을 내어 적은 양의 칼슘이 혈류로 방출되어 신체기능을 유지하는데 사용된다(William J. et al., NATURE.,423, pp.337342, 2033).It is known that two kinds of cells are involved in bone remodeling. One of the cells is osteoblast that produces bone, and the other is osteoclast that destroys bone. Osteoblasts produce RANKL (receptor activator of NF-κB ligand) and its induction receptor (decoy receptor), OPG (osteoprotegerin). When RANKL binds to receptor RANK (receptor activator of NF-κB) on the surface of osteoclast progenitor cells, osteoclast precursor cells are differentiated into osteoclasts and bone resorption occurs. In detail, the expression of NFAT (nuclear factor of activated T cells), which is known as a key transcription factor controlling osteoclast differentiation in osteoclast precursor cells, is induced by intracellular calcium signaling by RANKL stimulation, Activation of extracellular signal-regulated kinase (ERK), NF-κB, p38 MAP kinase, etc., induces autoamplification of NFAT and is thus active. Thus, the absorption or destruction of old bone is carried out by the osteoclast through the above mechanism, which is used to keep the body function by piercing the bone and releasing a small amount of calcium into the bloodstream (William J. et al. , NATURE., 423, pp. 3737342, 2033).
한편 조골세포는 교원질로 구멍을 채우고 칼슘과 인의 침적물(hydroxyapatite)을 덮어서 단단한 새로운 뼈를 만들어 골격을 재건하게 된다. 성인의 경우 매년 골격의 약 10-30%가 이런 과정을 통하여 재형성되며, 파골 속도와 조골 속도가 동일해야만 전과 같은 골밀도를 유지할 수 있다. 정상 성인에서는 골 흡수 양과 골 형성 양 사이에는 항상 균형이 유지되고있다. 이와 같이 중요한 뼈에 균형이 깨질 경우 많은 질병이 야기될 수 있는데, 이러한 질환을 골 질환이라 한다.On the other hand, the osteoblasts fill holes with collagen and cover the calcium and phosphorus deposits (hydroxyapatite) to form new hard bones and rebuild the skeleton. In adults, about 10-30% of the bone is reformed every year through this process, and bone density can be maintained as long as the osteopenic rate and osteolysis rate are the same. In normal adults, there is always a balance between the amount of bone resorption and the amount of bone formation. If such an important bone is broken, many diseases can be caused. This disease is called bone disease.
골 질환 중 고령사회에서 큰 문제로 대두되고 있는 골다공증(osteoporosis)은 골의 화학적 조성에는 큰 변화 없이 단위 용적내의 골량이 감소하여 경미한 충격에도 쉽게 골절을 일으킬 수 있는 질환이다. 노인 특히 폐경 후 여성들의 경우 에스트로겐의 분비부족으로 인하여 가장 그 발생빈도가 높게 나타난다고 알려져 있다. 현재 에스트로겐이나 천연에 존재하면서 약한 에스트로겐 활성을 나타내는 식물 유래의 피토에스트로겐이 폐경 후 골다공증 예방에 매우 유용하다는 연구가 보고되었다. 그러나 이러한 방법은 폐경성 골다공증 이외의 원인에는 제한적으로 사용된다는 한계점을 가진다. 이 밖에 칼슘염, 우골분 등의 칼슘 보강제, 비타민 D 대사산물 요법 등이 비 폐경성 골다공증 치료에 이용되고 있으나, 이들은 용해성이나 흡수성, 미각적 측면에서 문제점을 가지고있다. 또한 약물 투여에는 비스포스포네이트(bisphosphonates)나 칼시토닌 제제 등이 사용되며 골다공증의 치료에 유효하다고 알려져 있으나, 이러한 약물은 전문의약품이고, 식도에 협착될 수 있는 등의 부작용이 있어 복용에 매우 주의를 요하며, 이명, 두통, 식욕 부진 등의 부작용도 나타낼 수 있다고 보고된 바 있다.
Osteoporosis, a major problem in the elderly, is a disease in which the amount of bone in a unit volume decreases without significant changes in the bone composition and can easily cause fractures even in the case of a minor impact. It is known that in the elderly, especially postmenopausal women, the most frequent occurrence is due to the lack of estrogen secretion. It has been reported that plant-derived phytoestrogens, which are present in estrogen or in nature and exhibit weak estrogenic activity, are very useful for the prevention of postmenopausal osteoporosis. However, these methods have limitations in that they are limited to causes other than menopausal osteoporosis. In addition, calcium supplementation such as calcium salt, bovine bone meal, and vitamin D metabolite therapy are used in the treatment of non-menopausal osteoporosis, but they have problems in terms of solubility, absorption, and taste. In addition, bisphosphonates and calcitonin preparations are known to be effective for the treatment of osteoporosis. However, these drugs are special medicines, and due to side effects such as constriction in the esophagus, It has been reported that side effects such as tinnitus, headache and anorexia can also be reported.
선이질풀(Geranium krameri FR. Et SA)은 60-80 cm 정도로 자라는 여러해살이풀로 꽃은 7-8월에 분홍 혹은 빨간 꽃이 핀다. 잎은 지름 7-10 cm로 양면에 짧은 갈라진 털이나 돌기(복모)가 있다. 그러나 선이질풀이 골 대사와 관련한 질환 치료에 효과를 가진다는 내용에 대해서는 전혀 보고된 바가 없다.
Geranium krameri FR. Et SA is a perennial plant that grows about 60-80 cm long. Flowers are pink or red in July-August. Leaves are 7-10 cm in diameter and have short split hairs or bumps on both sides. However, there has been no report on the fact that L. japonica has an effect on the treatment of diseases related to bone metabolism.
이에 본 발명자들은 골 질환 예방 및 치료제를 개발하기 위하여 연구하던 중, 선이질풀 추출물이 ERK 및 p38 MAPK(Mitogen-activated protein kinase)의 활성화를 억제하여 파골세포의 핵심 분화 조절 전사인자인 NFATc1과 c-FOS의 발현을 감소시킴으로써 파골세포 분화를 억제하고, 이에 따른 골 흡수 기능도 억제하며, 또한 LPS에 의해 골 손실이 유도된 동물모델에서 골손실을 억제하고 파골세포의 수를 유의하게 감소시키는 것을 확인함으로써 상기 선이질풀 추출물을 골 질환 예방 및 치료용 조성물의 유효성분으로 유용하게 사용될 수 있음을 밝힘으로써 본 발명을 완성하였다.Accordingly, the present inventors have conducted studies to develop a preventive and therapeutic agent for bone diseases, and found that the extract of L. japonica inhibited the activation of ERK and p38 MAPK (mitogen-activated protein kinase) It has been shown that inhibition of osteoclast differentiation by inhibiting the expression of FOS, thereby inhibiting bone resorption, and inhibiting bone loss and significantly reducing the number of osteoclasts in an animal model in which bone loss is induced by LPS The present invention has been accomplished on the basis of these facts that the above extracts can be effectively used as an active ingredient of a composition for preventing and treating bone diseases.
본 발명의 목적은 선이질풀(Geranium krameri FR. Et SA) 추출물을 유효성분으로 함유하는 골 질환 예방 및 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for preventing and treating bone diseases containing an extract of Geranium krameri FR. Et SA as an active ingredient.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명은 선이질풀(Geranium krameri FR. Et SA) 추출물을 유효성분으로 함유하는 골 질환 예방 및 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of bone diseases containing an extract of Geranium krameri FR. Et SA as an active ingredient.
또한, 본 발명은 선이질풀 추출물을 유효성분으로 함유하는 골 질환 예방 및 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for prevention and improvement of bone diseases containing an extract of L. japonica as an active ingredient.
본 발명의 선이질풀 추출물은 ERK 및 p38 MAPK의 활성화를 억제하여 NFATc1과 c-FOS의 발현을 감소시킴으로써 파골세포 분화를 억제하고, 이에 따른 골 흡수 기능도 억제하며, 또한 LPS에 의해 골 손실이 유도된 동물모델에서 골손실을 억제하고 파골세포의 수를 유의하게 감소시키므로 상기 선이질풀 추출물을 골 질환 예방 및 치료용 조성물, 골 질환 예방 및 개선용 건강기능식품의 유효성분으로 유용하게 사용될 수 있다. The present invention provides a method of inhibiting osteoclast differentiation by inhibiting the activation of ERK and p38 MAPK, thereby decreasing the expression of NFATc1 and c-FOS, inhibiting osteoclast differentiation, Inhibits osteoporosis and significantly reduces the number of osteoclasts in the animal model. Therefore, the above extract can be effectively used as an effective ingredient of a composition for preventing and treating bone diseases and a health functional food for preventing and improving bone diseases.
도 1은 RANKL 처리 후, 본 발명의 선이질풀 추출물 처리 유무에 따른 분화된 파골세포의 수를 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물.
도 2는 본 발명의 선이질풀 추출물 처리에 따른 세포 생존률을 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물.
도 3은 본 발명의 선이질풀 추출물 처리에 따른 파골세포 특이적 마커 유전자(칼시토닌리셉터, 카셉신 K)의 발현량을 RT-PCR을 이용하여 조사한 것을 나타낸 도이고,
CTR: Calcitonin receptor CTK: Cathepsin K GKF4: 선이질풀추출물
R+GKF4: RANKL+선이질풀추출물.
도 4는 본 발명의 선이질풀 추출물 처리에 따른 상아질 절편 상에서 골 흡수로 형성된 피트(pit)를 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물 R+GKF4: RANKL+선이질풀추출물.
도 5는 본 발명의 선이질풀 추출물 처리에 따른 NFATc1 단백질의 발현량을 웨스턴 블롯 방법으로 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물 R+GKF4: RANKL+선이질풀추출물.
도 6은 본 발명의 선이질풀 추출물 처리에 따른 c-FOS 단백질의 발현량을 웨스턴 블롯 방법으로 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물 R+GKF4: RANKL+선이질풀추출물.
도 7은 본 발명의 선이질풀 추출물 처리에 따른 ERK 단백질의 인산화 정도를 웨스턴 블롯으로 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물 R+GKF4: RANKL+선이질풀추출물.
도 8은 본 발명의 선이질풀 추출물 처리에 따른 p38 단백질의 인산화 정도를 웨스턴 블롯으로 조사한 것을 나타낸 도이고,
GKF4: 선이질풀추출물 R+GKF4: RANKL+선이질풀추출물.
도 9는 본 발명의 선이질풀 추출물을 마우스 두개관(頭蓋冠) 모델에 처리시, 골손실의 억제 정도를 나타낸 사진이고,
LPS: Lipopolysaccharide GKF4: 선이질풀추출물.
도 10는 본 발명의 선이질풀 추출물을 마우스 두개관(頭蓋冠) 모델에 처리시, 파골세포 특이적인 TRAP+로 염색된 면적을 조사한 것을 나타낸 도이다.
LPS: Lipopolysaccharide GKF4: 선이질풀추출물.FIG. 1 is a view showing the number of differentiated osteoclasts according to the presence or absence of the treatment of L. japonica extract of the present invention after RANKL treatment,
GKF4: Lentinus edodes extract.
FIG. 2 is a graph showing the cell viability according to the treatment with the extract of L. japonica according to the present invention.
GKF4: Lentinus edodes extract.
FIG. 3 is a graph showing the expression amount of an osteoclast-specific marker gene (calcitonin receptor, cassex K) according to the present invention of the present invention by RT-PCR,
CTR: Calcitonin receptor CTK: Cathepsin K GKF4: Ganoderma lucidum extract
R + GKF4: RANKL + line extract.
FIG. 4 is a view showing irradiation of a pit formed by bone resorption on dentin slices according to the present invention,
GKF4: Ginkgo biloba extract R + GKF4: RANKL + Ginkgo biloba extract.
FIG. 5 is a graph showing the amount of expression of NFATc1 protein by the Western blot method according to the present invention,
GKF4: Ginkgo biloba extract R + GKF4: RANKL + Ginkgo biloba extract.
FIG. 6 is a graph showing the expression level of c-FOS protein according to the Western blotting method according to the present invention,
GKF4: Ginkgo biloba extract R + GKF4: RANKL + Ginkgo biloba extract.
FIG. 7 is a graph showing Western blotting of the degree of phosphorylation of ERK protein according to the present invention,
GKF4: Ginkgo biloba extract R + GKF4: RANKL + Ginkgo biloba extract.
FIG. 8 is a graph showing Western blotting of the degree of phosphorylation of p38 protein according to the present invention,
GKF4: Ginkgo biloba extract R + GKF4: RANKL + Ginkgo biloba extract.
FIG. 9 is a photograph showing the degree of inhibition of bone loss when treated with a mouse double skull model of the extract of L. japonica according to the present invention,
LPS: Lipopolysaccharide GKF4: Lentinus edodes extract.
FIG. 10 is a diagram showing an area of osteoclast-specific TRAP + stained when treating the mouse skeletal model of the present invention with a line extract. FIG.
LPS: Lipopolysaccharide GKF4: Lentinus edodes extract.
본 발명에서 사용되는 용어를 설명한다.
Terms used in the present invention will be described.
본 발명에서 사용되는 용어 "예방"은 본 발명의 조성물의 투여로 관련질환을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다.The term "prophylactic " as used herein refers to any act that inhibits the progression of a disease or slows progression by administration of the composition of the present invention.
본 발명에서 사용되는 용어 "치료" 또는 "개선"은 본 발명의 조성물의 투여로 관련질환의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term " treatment "or" improvement "refers to any action by which administration of a composition of the invention improves or alleviates the symptoms of a related disorder.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에 소정의 본 발명의 조성물을 제공하는 것을 의미한다.
The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.
이하, 본 발명을 상세하게 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 선이질풀(Geranium krameri FR. Et SA) 추출물을 유효성분으로 함유하는 골 질환(bone disease) 예방 및 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing and treating bone disease containing an extract of Geranium krameri FR. Et SA as an active ingredient.
상기 선이질풀 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다.It is preferable, but not limited, that the above-mentioned extract of L. japonica is produced by a manufacturing method comprising the following steps.
1) 선이질풀에 추출용매를 가하여 추출하는 단계;1) extracting the extract by adding an extraction solvent to the extract;
2) 단계 1)의 추출물을 식힌 후 여과하는 단계; 및2) cooling the extract of step 1) and filtering; And
3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하는 단계.3) Concentrating the filtered extract of step 2) under reduced pressure and drying.
상기 방법에 있어서, 단계 1)의 추출용매는 물, C1 내지 C2 저급 알코올 또는 이들의 혼합물을 용매로 하여 추출하는 것이 바람직하며, 상기 저급 알코올은 에탄올 또는 메탄올인 것이 바람직하나, 이에 한정되는 것은 아니다. 상기 추출용매는 건조된 선이질풀의 0.1 내지 10배로 하는 것이 바람직하고, 0.3 내지 5배 첨가하는 것이 바람직하나, 이에 한정되지 않는다. 추출온도는 20 내지 70℃인 것이 바람직하나, 이에 한정되지 않는다. 또한, 추출시간은 12 내지 48시간인 것일 수 있으나, 이에 한정되지 않는다.In the above method, the extraction solvent in step 1) is preferably extracted with water, C1 to C2 lower alcohol or a mixture thereof, and the lower alcohol is preferably ethanol or methanol, but is not limited thereto . The extraction solvent is preferably 0.1 to 10 times, more preferably 0.3 to 5 times as much as the dried line extract, but is not limited thereto. The extraction temperature is preferably 20 to 70 DEG C, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것일 수 있으나 이에 한정되지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것일 수 있으나 이에 한정되지 않는다.In this method, the vacuum concentration of step 3) may be by using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling drying, spray drying or lyophilization.
상기 선이질풀 추출물은 파골세포 분화 억제 효능을 갖는 것일 수 있으나 이에 한정되지 않는다.The above extract may be one having an osteoclast differentiation inhibitory effect, but is not limited thereto.
상기 선이질풀 추출물은 RANKL(Receptor activator of NK-κB ligand)에 의한 파골세포 분화를 억제하는 것일 수 있으나 이에 한정되지 않는다.The above extract may inhibit osteoclast differentiation by RANKL (Receptor activator of NK-κB ligand), but is not limited thereto.
상기 선이질풀 추출물은 골손실을 억제하는 것일 수 있으나 이에 한정되지 않는다.The line extract may inhibit bone loss but is not limited thereto.
상기 골 질환은 골다공증, 파세트 골질환, 구루병, 골연화증, 신부전 환자의 신성골이영양증, 류머티스성 골질환, 퇴행성 골질환, 뼈전이성 병소(bone metastatic lesion), 원발성으로 뼈에 생성된 종양, 치주질환, 염증성 치조골 흡수질환 및 염증성 뼈 흡수질환으로 이루어지는 군으로부터 선택되는 어느 하나일 수 있으나 이에 한정되지 않는다.
The above-mentioned bone disease may be selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia, renal osteodystrophy, rheumatoid bone disease, degenerative bone disease, bone metastatic lesion, primary bone- , Inflammatory alveolar bone disease, and inflammatory bone resorption disease. However, the present invention is not limited thereto.
본 발명의 구체적인 실험예에서, 본 발명자들은 선이질풀 추출물이 파골세포의 분화를 억제하는지 조사하였다. RANKL을 마우스 골수세포에 처리하여, 파골세포로 분화를 유도하여 조사한 결과, 선이질풀 추출물을 처리한 경우 분화된 파골세포의 수를 감소시켰으며, 이에 상응하는 결과로써 파골세포 마커 유전자인 칼시토닌 리셉터(Calcitonin receptor: CTR) 및 카셉신 K(Cathepsin K: CATK) 발현량의 감소를 RT-PCR을 통해 확인할 수 있었다(도 1 및 도 3 참조). 또한, 이런 파골세포 분화를 억제하는 선이질풀 추출물의 효과는 세포독성에 기인하는 것이 아님을 MTT assay를 통해 확인하였다(도 2 참조).In a specific experimental example of the present invention, the present inventors investigated whether or not the extract of L. japonica inhibited osteoclast differentiation. RANKL was treated with mouse bone marrow cells to induce differentiation into osteoclasts. As a result, the number of differentiated osteoclasts decreased when treated with the extract of L. japonica, and as a result, calcitonin receptor Calcitonin receptor (CTR) and cathepsin K (CATK) expression were decreased by RT-PCR (see FIGS. 1 and 3). In addition, it was confirmed through MTT assay that the effect of the extract of L. japonica extract inhibiting osteoclast differentiation was not due to cytotoxicity (see FIG. 2).
파골세포의 분화를 억제하는 선이질풀 추출물이 실제 파골세포에 의한 골 흡수를 억제하는지를 알아보기 위하여 분화된 파골세포가 상아질을 파괴하여 생성되는 피트(pit) 형성 정도를 조사하였다. 그 결과, 선이질풀 추출물이 상아질 절편상의 피트 형성을 감소시킨다는 것을 알 수 있었다(도 4 참조). 이 결과는 선이질풀 추출물이 파골세포로의 분화를 억제하여 그 수를 감소시킴고 그로 인해 실제 골손실을 감소시킨다는 것을 제시한다. In order to investigate whether osteoporosis extract inhibiting osteoclast differentiation inhibits bone resorption by osteoclasts, the degree of formation of pits formed by the destruction of dentin by differentiated osteoclasts was investigated. As a result, it was found that the extract of L. japonica reduced pit formation on the dentin slice (see Fig. 4). These results suggest that the extract of L. japonica inhibits osteoclast differentiation and reduces the number of osteoclasts, thereby reducing the actual bone loss.
선이질풀 추출물이 파골세포의 분화를 억제하는 분자기전을 규명하기 위하여, 파골세포 분화와 관련된 전사인자, 상위 조절 단백질들에 대해 웨스턴 블롯을 수행하였다. 상기의 RANKL에 의한 분화유도실험에서, 선이질풀 추출물은 파골세포로의 분화에 중요한 전사인자인 NFATc1 및 c-FOS 단백질의 발현을 추출물을 처리하지 않은 대조군에 비해 유의성 있게 저해하였다(도 5 및 도 6 참조). 또한 NFATc1 및 c-FOS 단백질의 상위 조절자로 알려져 있는 ERK 및 p38 kinase의 인산화 정도를 살펴본 결과 선이질풀 추출물이 ERK 및 p38 kinase의 인산화를 통한 활성화 역시 마찬가지로 억제함을 확인할 수 있었다(도 7 및 도 8 참조). To elucidate the molecular mechanism of the inhibition of osteoclast differentiation by Ganoderma lucidum extract, western blotting was performed on the transcription factors and upstream regulatory proteins associated with osteoclast differentiation. In the above-mentioned RANKL-induced differentiation induction, the L. japonicum extract significantly inhibited the expression of NFATc1 and c-FOS protein, which are important transcription factors for osteoclast differentiation, as compared with the control without extract treatment (FIGS. 5 and 6 6). In addition, the degree of phosphorylation of ERK and p38 kinase, which are known to be the predominant regulators of NFATc1 and c-FOS proteins, was found to inhibit the activation of phosphorylation of ERK and p38 kinase as well (Fig. 7 and Fig. 8 Reference).
선이질풀 추출물이 실제 in vivo에서 파골세포로의 분화 및 골손실을 억제하는지를 마우스의 두개관 모델을 이용하여 조사하였다. 선이질풀 추출물을 처리한 경우, 염색분석을 통해 리포폴리사카라이드(LPS)에 의해 유도되는 TRAP+ 파골세포의 수가 줄어든 것을 알 수 있었고, 골손실 또한 줄어듬을 확인할 수 있었다(도 9 및 도 10 참조).Two different models of the mouse were used to investigate whether the extracts of Ganoderma lucidum inhibit osteoclast differentiation and bone loss in vivo. In the case of treatment with the extract of L. japonica, it was confirmed that the number of TRAP + osteoclasts induced by lipopolysaccharide (LPS) was reduced by the staining analysis, and the bone loss was also reduced (see Figs. 9 and 10) .
따라서, 본 발명의 선이질풀 추출물은 ERK 및 p38 kinase의 인산화를 억제, 그 하위조절자인 NFATc1 및 c-FOS 단백질의 발현량을 감소시킴으로써 파골세포로의 분화 및 그에 따른 골손실을 억제하므로 골질환 예방 또는 치료를 위한 약학적 조성물의 유효성분으로 유용하게 사용될 수 있다.
Therefore, the extract of the present invention inhibits the phosphorylation of ERK and p38 kinase and reduces the expression level of NFATc1 and c-FOS protein, which are subordinate regulators thereof, to inhibit osteoclast differentiation and subsequent bone loss, Or as an active ingredient of a pharmaceutical composition for treatment.
상기 본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.
상기 본 발명의 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여시 피부 외용 또는 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식을 선택하는 것이 바람직하며, 이에 한정되는 것은 아니다.The composition of the present invention can be administered orally or parenterally, and it is preferable to select an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, But is not limited thereto.
상기 본 발명의 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 엑기스제, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, aerosols and the like, oral preparations, suppositories, Can be used. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 생지황, 복령, 인삼 및 꿀의 혼합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate (calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 상기 조성물은 1일 0.0001 내지 1 g/㎏으로, 바람직하게는 0.001 내지 200 ㎎/㎏으로 투여하는 것이 바람직하나 이에 한정되지 않는다. 상기 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.
The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg per day, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
또한, 본 발명은 선이질풀 추출물을 유효성분으로 함유하는 골 질환 예방 및 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for prevention and improvement of bone diseases containing an extract of L. japonica as an active ingredient.
"건강기능식품"이란 일상 식사에서 결핍되기 쉬운 영양소나 인체에 유용한 기능을 가진 원료나 성분(기능성원료)을 사용하여 제조한 것으로, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선하는 식품을 의미한다."Health functional food" is produced by using raw materials and ingredients (functional raw materials) that have functions useful for nutrients and human body which are likely to be deficient in daily eating, and maintain healthy state through maintenance of normal function of human body or activation of physiological function Means improving food.
상기 선이질풀 추출물은 파골세포 분화 억제 효능을 갖는 것일 수 있으나 이에 한정되지 않는다.The above extract may be one having an osteoclast differentiation inhibitory effect, but is not limited thereto.
상기 골 질환은 골다공증, 파세트 골질환, 구루병, 골연화증, 신부전 환자의 신성골이영양증, 류머티스성 골질환, 퇴행성 골질환, 뼈전이성 병소(bone metastatic lesion), 원발성으로 뼈에 생성된 종양, 치주질환, 염증성 치조골 흡수질환 및 염증성 뼈 흡수질환으로 이루어지는 군으로부터 선택되는 어느 하나일 수 있으나, 이에 한정되지 않는다.The above-mentioned bone disease may be selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia, renal osteodystrophy, rheumatoid bone disease, degenerative bone disease, bone metastatic lesion, primary bone- , Inflammatory alveolar bone disease, and inflammatory bone resorption disease. However, the present invention is not limited thereto.
본 발명의 선이질풀 추출물은 ERK 및 p38 kinase의 인산화를 억제, 그 하위조절자인 NFATc1 및 c-FOS 단백질의 발현량을 감소시킴으로써 파골세포로의 분화 및 그에 따른 골손실을 억제하므로, 골 질환 예방 및 개선용 건강기능식품의 유효성분으로 유용하게 사용될 수 있다.
The present invention provides a method for preventing osteoporosis and osteoporosis by inhibiting phosphorylation of ERK and p38 kinase and decreasing the expression level of NFATc1 and c-FOS protein, which are subordinate regulators thereof, Can be usefully used as an effective ingredient of health functional foods for improvement.
이하, 본 발명을 실시예, 실험예 및 제조예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.
단 하기의 실시예, 실험예 및 제조예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예, 실험예 및 제조예에 한정되는 것은 아니다.
EXAMPLES The following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples, Experimental Examples and Production Examples.
<실시예 1> 선이질풀 메탄올 추출물의 제조Example 1 Preparation of Methanol Extract of Lycopodium L.
<1-1> 선이질풀 메탄올 추출물 제조≪ 1-1 > Preparation of Methanol Extract of Lycopene
본 발명에 사용된 선이질풀(Geranium krameri FR. Et SAV.)은 2008년 7월 수원시 서둔동 소재 작물과학원 약용작물시험포장에서 증식한 것을 채취한 후 종의 동정은 농촌진흥청 약용작물과 소속 식물분류전문가에게 확인받았다 (Voucher Number : MPS000259). 세척 및 건조한 선이질풀 뿌리는 세척 및 건조하고, 잘게 분쇄하여 가속용매추출장치 (accelerated solvent extractor, ASE)를 이용해 50°C에서 메탄올로 추출하였고 추출액을 여과한 후에 rotary evaporator (JP-SD1000, Eyela, Tokyo Rikikatai, Japan)로 감압농축하여 용매를 제거하였다. 선이질풀 뿌리 분말 207g으로부터 얻어진 추출물은 118g이었다.
The growth of Geranium krameri FR. Et SAV. In the medicinal crop test package of the Crop Science Institute, Seodun city, Suwon, Korea was investigated. The identification of the species was carried out by the RDA, (Voucher Number: MPS000259). The washed and dried lines were washed, dried, finely pulverized and extracted with methanol using an accelerated solvent extractor (ASE) at 50 ° C. The extracts were filtered and then washed with a rotary evaporator (JP-SD1000, Eyela, Tokyo Rikikatai, Japan) under reduced pressure to remove the solvent. 118 g of the extract obtained from 207 g of the root louse root powder was 118 g.
<1-2> 선이질풀 100 % 에탄올 추출물 제조<1-2> Preparation of 100% Ethanol Extract of Ganoderma Lucidum
추출용매로 100 % 에탄올을 사용한 것을 제외하고, 상기 실시예 <1-1>의 추출방법과 동일하게 추출하였다.
Extraction was carried out in the same manner as in Example <1-1> except that 100% ethanol was used as the extraction solvent.
<실험예><Experimental Example>
실험결과는 평균+표준편차로 표기하였고, Student's t-test로 분석하여 p값이 0.05미만일 때 통계적으로 유의하다고 판단하였다.
The test results were expressed as mean + standard deviation, and analyzed by Student's t-test to determine statistical significance when the p-value was less than 0.05.
<실험예 1> 선이질풀 추출물의 파골세포 분화 억제 효과(in vitro)<Experimental Example 1> Inhibitory effect on the osteoclast differentiation of the extracts of Ganoderma lucidum
<실험예 1-1> 선이질풀 추출물 처리에 따른 파골세포 분화 억제효과≪ Experimental Example 1-1 > Inhibitory effect on osteoclast differentiation according to the treatment with the extract of Ganoderma lucidum L.
본 발명의 유효성분인 선이질풀 추출물이 파골세포 분화에 미치는 영향을 조사하기 위하여, 마우스 골수세포를 파골세포로 분화시키고 그 수를 측정하였다. In order to investigate the effect of the extract of the present invention on the osteoclast differentiation, the bone marrow cells were differentiated into osteoclasts and the number thereof was measured.
골수세포를 얻기 위해 ICR 마우스(6-9주, 수컷)를 경추탈골한 후 70％ 에탄올로 소독하였다. 경골 부분의 피부를 절개하여 부착 근육을 떼어내고, 경골 중심부를 절단 , 슬개골을 탈골시킨 후 경골을 적출하였다. 경골 뼈 양끝을 조금 잘라 한 쪽 끝에 25 게이지의 주사바늘을 꽂고 a-MEM을 흘려보내 골수세포를 수거하였다. 원심 분리한 후 a-MEM에 현탁하고 2배의 Gey's solution을 가해 적혈구를 제거하고, 다시 원심 분리한 후 10 % FBS가 함유된 a-MEM으로 재현탁하여 배양하였다. 초기 배양된 골수세포를 대식세포증식자극인자(Macrophage colony stimulating fator, 이하 ‘M-CSF’라고 한다) 10ng/㎖로 하룻밤 배양한 후 부유세포를 M-CSF 30ng/㎖로 3일간 추가배양하여 골수 대식세포(Bone marrow Macrophage, 이하 ‘BMM’이라 한다)를 얻을 수 있었다. 생성된 BMM을 회수, well당 1×105개의 세포를 분주한 후 RANKL(receptor activator of NK-κB ligand) 100ng/㎖ 과 M-CSF 30ng/㎖ 존재하에서 4일간 배양하였다. 배양이 끝난 세포는 10％ 포르말린으로 10분간 고정한 후 에탄올-아세톤(1:1)로 1분간 재고정하여 TRAP(tartrate-resistant acid phosphatase)에 대해 Naphthol AS-MX phosphate(Sigma-Aldrich, St. Louis, MO) 0.1mg/ml, Fast Red violate LB salt(Sigma-Aldrich, St. Louis, MO) 0.6mg/ml, pH 5.2 acetate buffer 50mM이 포함된 TRAP-staining solution을 사용하여 상온에서 10분간 염색을 수행하였고,, 현미경하에서 관찰하여 3개 이상의 핵을 가진 TRAP+ 세포를 다핵 파골세포로 판정하였다.To obtain bone marrow cells, ICR mice (6-9 weeks, male) were cervical dislocated and disinfected with 70% ethanol. The skin of the tibia was cut to remove the attached muscles, and the tibia was excised after cutting the central portion of the tibia and dislocating the patella. Both ends of the tibial bone were cut a little, and a 25-gauge needle was inserted at one end and a-MEM was shed to collect the bone marrow cells. After centrifugation, the cells were suspended in a-MEM, 2 times of Gey's solution was added thereto to remove red blood cells, and the cells were centrifuged again and re-suspended in a-MEM containing 10% FBS. The cultured bone marrow cells were cultured overnight with 10 ng / ml of macrophage colony stimulating factor (M-CSF), followed by additional culturing of the floating cells with 30 ng / ml of M-CSF for 3 days. Bone marrow macrophages (BMMs) were obtained. The resulting BMM was recovered, and 1 × 10 5 cells were distributed per well. The cells were then cultured for 4 days in the presence of 100 ng / ml of RANKL (receptor activator of NK-κB ligand) and 30 ng / ml of M-CSF. The cultured cells were fixed with 10% formalin for 10 minutes and then resuspended in ethanol-acetone (1: 1) for 1 minute. Naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, Staining solution for 10 minutes at room temperature using a TRAP-staining solution containing 0.1 mg / ml of Fast Red violet LB salt (Sigma-Aldrich, St. Louis, MO) 0.6 mg / ml and pH 5.2
그 결과, 선이질풀 추출물은 농도 의존적으로 파골세포 분화를 억제하였으며, 10㎍/㎖ 농도에서 최대 억제 효과를 나타내는 것을 알 수 있었다(도 1 참조).
As a result, it was found that the extract of Ganoderma lucidum inhibited osteoclast differentiation in a concentration-dependent manner, and exhibited the maximum inhibitory effect at a concentration of 10 μg / ml (see FIG. 1).
<실험예 1-2> 선이질풀 추출물의 세포독성 시험≪ Experimental Example 1-2 > Cytotoxicity test
선이질풀 추출물의 파골세포 분화 억제 효과가 세포 독성에 기인하지 않음을 확인하기 위하여 MTT분석을 수행하였다.MTT assay was performed to confirm that osteoclast differentiation inhibition effect of Ganoderma lucidum extract was not due to cytotoxicity.
1×104 cells/well 농도로 96 웰플레이트에 분주한 BMM 세포에 시료를 처리하고 24 시간 동안 배양하였다. 이후, 배양액을 버리고 PBS로 세척한 후, MTT 용액(0.5mg/㎖ )을 well당 100㎕ 첨가하여 호일로 싼 상태에서 5시간 동안 배양하였다. 이후 solubilization buffer( 10％ SDS in 0.01M 염산)를 well당 100㎕ 첨가하고 다시 호일로 싼 채로 16시간 내지 17시간 경과 후, 570㎚ 에서 흡광도를 측정하였다.BMM cells were seeded in a 96-well plate at a concentration of 1 x 10 4 cells / well and cultured for 24 hours. Then, the culture medium was discarded, and the cells were washed with PBS. Then, 100 μl of MTT solution (0.5 mg / ml) was added thereto, and the cells were incubated in a foil for 5 hours. Then, 100 μl of a solubilization buffer (10% SDS in 0.01 M hydrochloric acid) was added per well, and the absorbance was measured at 570 nm after lapse of 16 to 17 hours while remaining in a foil.
그 결과, 대조군과 비교하여 선이질풀 추출물의 처리군에서 세포 생존률이 거의 차이를 보이지 않는 것을 통해, 파골세포 분화 억제 효과가 세포 독성에 기인한 것이 아님을 확인할 수 있었다(도 2 참조).
As a result, it was confirmed that the effect of inhibiting osteoclast differentiation was not due to cytotoxicity, as compared with the control group, in which the cell survival rate was hardly different in the treatment group of L. japonica extract (see Fig. 2).
<실험예 1-3> 선이질풀 추출물이 파골세포 특이적 마커 유전자의 발현에 미치는 영향≪ Experimental Example 1-3 > Effect of the extract of L. japonica on expression of osteoclast-specific marker gene
상기 <실험예 1-1>에서 보듯이 선이질풀 추출물은 파골세포의 분화를 억제하는데, 역전사-중합효소연쇄반응법(RT-PCR)을 이용하여, 파골세포 특이적 마커 유전자의 실제 발현 정도를 조사하였다.As shown in the above <Experimental Example 1-1>, the expression of the osteoclast-specific marker gene was expressed using the reverse transcription-polymerase chain reaction (RT-PCR) to inhibit osteoclast differentiation. Respectively.
Total RNA 추출은 Easy-blue(Intron, Biochemistry.INC.)를 이용하였다. 1㎍의 Total RNA를 oligoT primer, 10mM dNTP, 1 unit RNase inhibitor 및 4 unit scrip reverse transcriptase(Fermentas, Life science)로 42℃에서 60분 반응 시키고, 70℃에서 10분 처리함으로써 cDNA를 얻었다. PCR(polymerase chain reaction)의 조건과 사용한 프라이머(5’→3’)의 서열은 다음과 같다(표 1 참조).Total RNA was extracted with Easy-blue (Intron, Biochemistry.INC.). 1 μg of total RNA was reacted with oligoT primer, 10 mM dNTP, 1 unit RNase inhibitor and 4 unit scrip reverse transcriptase (Fermentas, Life science) at 42 ° C for 60 minutes and treated at 70 ° C for 10 minutes to obtain cDNA. The sequence of the polymerase chain reaction (PCR) and the used primer (5 '→ 3') are as follows (see Table 1).
Cassexin K
그 결과, RANKL에 의해 파골세포로 분화된 세포는 마커 유전자로 알려진 칼시토닌 리셉터(CTR)와 카셉신 K(CTK)의 mRNA 발현이 모두 증가되는 것을 알 수 있었다. 그러나 선이질풀 추출물을 첨가한 첨가군에서는 RANKL에 의해 유도된 이들 유전자의 발현이 모두 유의하게 억제되는 것을 확인할 수 있었다(도 3 참조). 이는 상기 실험예에서 살펴 본 선이질풀의 추출물이 파골세포의 분화를 억제하는 결과를 분자수준에서 그 마커 유전자의 발현을 통해 확인한 것으로 상호 일치하는 결과이다.
As a result, it was found that the cells differentiated into osteoclasts by RANKL showed both mRNA expression of calcitonin receptor (CTR) and caffecein K (CTK), which are known as marker genes. However, it was confirmed that the expression of these genes induced by RANKL was significantly inhibited in the addition group supplemented with the extract of L. japonica (see Fig. 3). This result is in agreement with the results of inhibiting the differentiation of osteoclast by the extract of L. japonica obtained in the above Experimental Example by confirming the expression of the marker gene at the molecular level.
<실험예 1-4> 선이질풀 추출물이 골 흡수능에 미치는 효과 ≪ Experimental Example 1-4 > Effect of extracts of Bombyx mori L. on bone resorption ability
상기 실험예들에서 살펴본 바와 같이 선이질풀 추출물은 파골세포로의 분화를 억제하는데, 실제 파골세포에 의한 골 흡수를 억제하는지 조사하였다. BMM세포를 M-CSF와 RANKL 존재 하에서 상아질 절편위에서 배양하면, BMM 세포가 파골세포로 분화됨에 따라 그 부분의 상아질을 파괴하여 피트(pit)라 불리는 골 흡수 자국이 생기는데, 피트 형성 정도를 분석함으로써 골 흡수능을 조사할 수 있다. As shown in the above Experimental Examples, it was examined whether or not the extract of L. japonica suppresses differentiation into osteoclasts and inhibits bone resorption by osteoclasts. When BMM cells are cultured on a dentin slice in the presence of M-CSF and RANKL, BMM cells are differentiated into osteoclasts, thereby destroying the dentin of the BMM cells, resulting in a bone resorption phenomenon called a pit. Bone resorption ability can be investigated.
구체적으로 BMM 세포를 상아 절편 상에서 RANKL 50ng/㎕, M-CSF 30ng/㎕ 존재하에서 6일간 배양하고, 상아 절편 표면의 세포를 닦아낸 후 톨루이딘블루(toluidin blue, J.T.Baker, UK, 1㎍/㎕)로 염색하였다. 상아 절편 상에서 골 흡수로 형성된 피트(pit)을 현미경 하에서 관찰하였다. Specifically, BMM cells were cultured for 6 days in the presence of 50 ng / R of RANKL and 30 ng / ㎕ of RANKL on the ivory slice, and the cells on the surface of the ivory slice were wiped off. Then, toluidine blue (JT Baker, UK, 1 / / ). Pits formed by bone resorption on the ivory section were observed under a microscope.
그 결과, 선이질풀 추출물을 처리한 처리군에서 상아질 절편상의 피트 형성이 유의하게 감소하는 것을 알 수 있었다. 결국, 선이질풀 추출물이 파골세포 분화를 억제하며 이에 따른 골 흡수 기능도 억제하는 것을 알 수 있었다(도 4 참조).
As a result, it was found that the pit formation on dentin slices was significantly reduced in the treatment group treated with the extract of L. japonica extract. As a result, it was found that the extract of Ganoderma lucidum inhibits osteoclast differentiation and thus inhibits bone resorption (see FIG. 4).
<실험예 2> 선이질풀 추출물의 파골세포 분화 억제 기전 규명(in vitro)EXPERIMENTAL EXAMPLE 2 Inhibition of Osteoclast Differentiation by the Extracts of Ganoderma lucidum
선이질풀 추출물이 RANKL에 의한 파골세골 분화와 이에 따른 골 흡수 기능을 억제하는 작용기전을 규명하고자 웨스턴 블롯을 수행하였다.Western blot analysis was performed to investigate the mechanism of RANKL - induced osteoclast differentiation and bone resorption inhibition by Ganoderma lucidum extract.
BMM 세포에 RANKL을 처리하고, 37℃, 5% CO2에서 24시간 배양 후, 세포를 수거, lysis buffer에 용해시킨 후 원심분리하여 상층액을 수득, 전체 단백질을 얻었다. 20μg의 단백질을 SDS-PAGE로 변성 분리하고, 이를 80v에서 105분 처리하여 PVDF 멤브레인에 이전시켰다. 이를 5% 탈지유(skim milk)가 함유된 PBST 용액 (0.05% Tween-20 in PBS)으로 블로킹하고, 1차 항체로서 항-NFAT (Santacruz, CA, USA), p-ERK (Cell Signaling, Danvers, MA, USA), ERK (Cell Signaling, Danvers, MA, USA), p-p38 (Cell Signaling, Danvers, MA, USA), p38 (Cell Signaling, Danvers, MA, USA), c-fos (Cell Signaling, Danvers, MA, USA), β-actin (Sigma Chemical Co., St. Louis, USA) 항체와 각각 반응시켰다. PBST로 5회 세정하고 호스라디시 페록시다제 (hoseradish peroxidase: HRP)가 결합된 2차 항체와 반응시킨 후 ECL Advance (Amersham Pharmacia Biotech, NY, USA)로 발색시켜 분석하였다.BMM cells were treated with RANKL and cultured at 37 ° C and 5% CO 2 for 24 hours. The cells were harvested, lysed in lysis buffer, centrifuged to obtain supernatant, and total protein was obtained. Twenty micrograms of protein was denatured by SDS-PAGE and transferred to PVDF membrane by treatment at 80 v for 105 min. (Santa Cruz, CA, USA), p-ERK (Cell Signaling, Danvers, USA) as the primary antibody and blocked with PBST solution (0.05% Tween-20 in PBS) containing 5% skim milk. MA, USA), ERK (Cell Signaling, Danvers, MA, USA), p-p38 (Cell Signaling, Danvers, MA, USA) Danvers, MA, USA) and β-actin (Sigma Chemical Co., St. Louis, USA). The cells were washed 5 times with PBST and reacted with secondary antibody conjugated with hoseradish peroxidase (HRP) and analyzed by ECL Advance (Amersham Pharmacia Biotech, NY, USA).
그 결과, BMM 세포를 RANKL로 24시간 처리하였을 때 기존에 보고된 바와 같이 NFAT 및 c-FOS 단백질 발현이 증가하였으나, 선이질풀 추출물 처리는 그 발현을 현저하게 감소시키는 것으로 나타났다. 이를 통해 선이질풀 추출물은 파골세포 분화에 중요한 전사인자인 NFATc1 및 c-FOS 단백질의 발현 억제를 통하여 파골세포 분화를 억제하는것을 알 수 있었다(도 5 및 6 참조). 또한, BMM 세포를 RANKL로 처리하였을 때 인산화된 ERK와 p38의 양이 증가하지만, 선이질풀 추출물은 ERK와 p38의 인산화를 억제함을 알 수 있었다(도 7 및 도 8 참조).As a result, when BMM cells were treated with RANKL for 24 hours, expression of NFAT and c-FOS protein was increased as reported previously, but treatment with L. japonica extract significantly decreased the expression thereof. These results indicate that the extract of L. japonica inhibits osteoclast differentiation by inhibiting the expression of NFATc1 and c-FOS protein, which are important transcription factors for osteoclast differentiation (see FIGS. 5 and 6). In addition, when BMM cells were treated with RANKL, the amounts of phosphorylated ERK and p38 were increased, but it was found that the extract of L. japonica inhibited the phosphorylation of ERK and p38 (see FIGS. 7 and 8).
<실험예 3> 선이질풀 추출물이 파골세포분화 및 골손실에 미치는 영향(in vivo)<Experimental Example 3> Effect of the extract of Ganoderma lucidum on osteoclast differentiation and bone loss (in vivo)
선이질풀 추출물이 in vitro에서 파골세포 분화 억제 효과를 보였으므로 마우스 두개관 모델을 이용해 선이질풀 추출물의 in vivo에서 파골세포 분화 및 골손실에 미치는 영향을 조사하였다.In vitro inhibition of osteoclast differentiation was observed in vitro. Therefore, we investigated the effects of Ganoderma lucidum extract on osteoclast differentiation and osteoporosis using a mouse two - view model.
ICR 마우스 (11-12주, 수컷)에 골손실을 유도하기 위해 리포폴리사카라이드 (0.5mg)를 실험 시작 1일 뒤 두개관(頭蓋冠)에 피하 주사하였다. 실험군에는 선이질풀 추출물(12.5mg/kg)을 DMSO에 용해시키고 콘 오일(corn oil)과 섞어 준비한 것(콘 오일:DMSO=9:1)을, 대조군에는 동량의 PBS를 실험 시작일부터 5일 뒤까지 매일 총 6회 복강내 주사하였다. 6일 뒤 마우스에서 두개관(頭蓋冠)을 채취한 후 4% 파라포름알데하이드(paraformaldehyde)에 고정하여 TRAP에 대해 Naphthol AS-MX phosphate(Sigma-Aldrich, St. Louis, MO) 0.1mg/ml, Fast Red violate LB salt(Sigma-Aldrich, St. Louis, MO) 0.6mg/ml, pH 5.2 acetate buffer 50mM이 포함된 TRAP-staining solution을 사용하여 상온에서 염색을 수행하였다. Lipopolysaccharide (0.5 mg) was subcutaneously injected into the two openings (calvaria) 1 day after the start of the experiment to induce bone loss in ICR mice (11-12 weeks, male). In the experimental group, the same amount of PBS was dissolved in DMSO and mixed with corn oil (cone oil: DMSO = 9: 1) and the same amount of PBS was added 5 days after the start of the experiment And 6 times daily. After 6 days, the two calves were harvested and fixed in 4% paraformaldehyde, and then 0.1 mg / ml of Naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO) Staining was performed at room temperature using a TRAP-staining solution containing 0.6 mg / ml of Fast Red violet LB salt (Sigma-Aldrich, St. Louis, MO) and 50 mM acetate buffer pH 5.2.
그 결과 리포폴리사카라이드를 마우스 두개관(頭蓋冠)에 피하 주사한 경우 TRAP+인 파골세포수가 증가하며 골손실을 일으킨 반면, 선이질풀 추출물의 투여 군에서는 리포폴리사카라이드에 의한 파골세포의 수를 유의하게 감소시키고 골손실도 감소시키는 것으로 나타났다(도 9 및 도 10 참조). 이는 선이질풀 추출물이 in vivo에서도 효과적으로 파골세포 분화 및 골손실을 억제하여, 골질환 예방의 예방 또는 치료용 조성물로 사용될 수 있음을 보여 준다.
As a result, when the lipopolysaccharide was subcutaneously injected into the mouse duoden (calvaria), the number of osteoclasts increased and the bone loss was caused by TRAP +, whereas the number of osteoclasts by lipopolysaccharide And decreased bone loss (see Figs. 9 and 10). This shows that the extract of L. japonica effectively inhibits osteoclast differentiation and bone loss in vivo, and can be used as a composition for preventing or treating bone disease prevention.
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| KR101899666B1 (en) | 2016-12-07 | 2018-09-17 | 건국대학교 글로컬산학협력단 | Composition for the prevention and treatment of bone diseases containing ginseng extract |
| KR102151643B1 (en) | 2019-03-20 | 2020-09-03 | 충북대학교 산학협력단 | Composition comprising extract of Lentinula edodes for prevention or treatment of bone diseases |
| KR102448913B1 (en) | 2019-05-23 | 2022-09-30 | 충북대학교 산학협력단 | Composition comprising extract of Abeliophyllum distichum for prevention or treatment of bone diseases |
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| KR20090056171A (en) * | 2007-11-30 | 2009-06-03 | 주식회사한국야쿠르트 | Anti-inflammatory composition containing geranium nepalense extract as an effective factor |
| KR20130001924A (en) * | 2011-06-28 | 2013-01-07 | 한림대학교 산학협력단 | Composition comprising extract from geranium thunbergii for preventing or treating various diabetic complications |
| KR101249930B1 (en) * | 2010-08-23 | 2013-04-03 | 윤유석 | Compositions comprising extracts of Sorbus commixta and Geranium nepalense for inhibiting osteoclastogenesis and stimulating chondrogenesis |
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| KR20090056171A (en) * | 2007-11-30 | 2009-06-03 | 주식회사한국야쿠르트 | Anti-inflammatory composition containing geranium nepalense extract as an effective factor |
| KR101249930B1 (en) * | 2010-08-23 | 2013-04-03 | 윤유석 | Compositions comprising extracts of Sorbus commixta and Geranium nepalense for inhibiting osteoclastogenesis and stimulating chondrogenesis |
| KR20130001924A (en) * | 2011-06-28 | 2013-01-07 | 한림대학교 산학협력단 | Composition comprising extract from geranium thunbergii for preventing or treating various diabetic complications |
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| 한국산학기술학회논문지, 2010* * |
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