KR102553040B1 - Composition of preventing or improving bone disease comprising Vitex rotundifolia extract - Google Patents
Composition of preventing or improving bone disease comprising Vitex rotundifolia extract Download PDFInfo
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- KR102553040B1 KR102553040B1 KR1020200179312A KR20200179312A KR102553040B1 KR 102553040 B1 KR102553040 B1 KR 102553040B1 KR 1020200179312 A KR1020200179312 A KR 1020200179312A KR 20200179312 A KR20200179312 A KR 20200179312A KR 102553040 B1 KR102553040 B1 KR 102553040B1
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Abstract
본 발명은 순비기나무 (Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물, 및 골질환 개선용 식품 조성물에 관한 것으로, 본 발명에 따른 순비기나무 추출물은 파골세포 분화 억제에 우수한 효과를 나타내는 바, 다양한 골질환 개선, 예방 또는 치료에 유용한 약제학적 조성물 또는 식품 조성물로 이용될 수 있다.The present invention relates to a pharmaceutical composition for preventing or treating bone disease comprising an extract of Vitex rotundifolia as an active ingredient, and a food composition for improving bone disease. Since it shows an excellent effect on inhibition, it can be used as a pharmaceutical composition or food composition useful for improving, preventing or treating various bone diseases.
Description
본 발명은 순비기나무 (Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물, 및 골질환 개선용 식품 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating bone disease, and a food composition for improving bone disease, comprising an extract of Vitex rotundifolia as an active ingredient.
골 (bone)의 대사는 조골세포 (osteoblast)와 파골세포 (osteoclast)의 활성에 의해 뼈가 파괴, 흡수되거나 새로운 뼈를 형성한다. 정상인은 조골세포와 파골세포의 상호작용에 의해 골 리모델링 (bone remodeling)이 일어난다. 하지만, 이러한 골 흡수와 골 형성의 항산성은 여성의 경우 폐경 이후 급격한 골 손실이 더 일어나게 되어 균형이 깨지기 쉬우며, 노화로 골 형성 기능이 퇴화되어 골 손실이 지속됨에 따라 골다공증이 일어날 수 있다.In the metabolism of bone, bone is destroyed, resorbed, or new bone is formed by the activity of osteoblast and osteoclast. In normal people, bone remodeling occurs through the interaction between osteoblasts and osteoclasts. However, the anti-acidity of bone resorption and bone formation is easily out of balance because rapid bone loss occurs after menopause in women, and osteoporosis may occur as bone loss continues due to deterioration of bone formation function due to aging.
골다공증은 초기에 골 흡수로 인하여 골 손실이 일어났는데, 골 형성이 불충분하여 일어나기 때문에 조골전구세포가 조골세포로 분화하는 능력에 의해서도 결정된다. 따라서, 조골세포의 분화를 촉진하거나 파골세포의 분화를 억제하여 골 흡수를 감소시키는 것이 필요하다.Osteoporosis initially causes bone loss due to bone resorption, and since it occurs due to insufficient bone formation, it is also determined by the ability of osteoblast progenitor cells to differentiate into osteoblasts. Therefore, it is necessary to reduce bone resorption by promoting the differentiation of osteoblasts or inhibiting the differentiation of osteoclasts.
현재 골다공증 치료제로써 사용되고 있는 제제는 에스트로겐, 라톡시펜, 알렌드로네이트 및 비스포스포네이트 등이 있으며, 이들은 골 흡수 억제제로써 골절 위험을 줄여 주기 위해 사용되고 있다. 이와 같은 종래의 치료제 이외에도 칼슘, 비타민 D, 칼시토닌, 바이오포스포네이트, 안드로겐 및 플라보노이드 등을 이용한 골다공증 예방, 개선 또는 치료제에 대한 연구도 활발히 진행되고 있다. 그러나 이러한 치료제들은 인체에 부작용이 있고 근본적인 골다공증의 치료제라고 하기에는 한계가 있다. 따라서, 기존 치료제의 단점을 보완하면서 부작용이 적은 천연 소재 유래의 골다공증 예방, 개선 또는 치료제의 개발이 절실히 요구되는 실정이다.Agents currently used as osteoporosis therapeutic agents include estrogen, latoxifene, alendronate and bisphosphonate, which are used as bone resorption inhibitors to reduce the risk of fracture. In addition to these conventional therapeutic agents, studies on preventing, improving or treating osteoporosis using calcium, vitamin D, calcitonin, biophosphonate, androgens, flavonoids, and the like are also being actively conducted. However, these treatments have side effects on the human body and have limitations in being a fundamental treatment for osteoporosis. Therefore, there is an urgent need to develop a preventive, ameliorative, or therapeutic agent for osteoporosis derived from natural materials with fewer side effects while supplementing the disadvantages of existing therapeutic agents.
순비기나무 (Vitex rotundifolia)는 행안가에 서식하면서 해풍을 맞으며 자라는 식물로, 염에 내성을 갖고 자라는 염생식물로 알려져 있다. 순비기나무 꽃은 자줏빛을 띄고 향이 우수하며, 그 열매는 두통, 안질 등의 질병 치료제로 민간요법에서 이용되고 있다. 순비기나무는 피부 미용, 항산화, 항염증 및 항균 등의 일부 생리활성에 관한 연구가 보고된 바 있지만, 순비기나무 잎 추출물의 골다공증 예방 또는 치료 효과를 대상으로 한 구체적인 연구는 알려진 바 없다.Vitex rotundifolia is a plant that grows in the sea breeze while inhabiting coastal areas, and is known as a salt plant that grows with tolerance to salt. The flowers of japonica japonica are purple and have an excellent fragrance, and the fruit is used in folk medicine as a treatment for diseases such as headache and eye disease. Although studies on some physiological activities such as skin beauty, antioxidant, anti-inflammatory and antibacterial have been reported, no specific study has been conducted on the preventive or therapeutic effect of osteoporosis of the leaf extract of Sunbikina.
따라서, 순비기나무 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물 및 골질환 예방 또는 개선용 식품 조성물의 개발이 시급히 요구되고 있다.Therefore, there is an urgent need for the development of a pharmaceutical composition for preventing or treating bone disease and a food composition for preventing or improving bone disease, which includes an extract of Astragalus sinensis as an active ingredient.
이에 본 발명자들은 골질환 개선, 예방 또는 치료용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과, 순비기나무 추출물이 파골세포 분화 억제에 효과를 나타낸 다는 것을 규명함으로써, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive research efforts to develop a composition for improving, preventing or treating bone disease. As a result, the present invention was completed by clarifying that the extract of Astragalus sinensis exhibits an effect on inhibiting osteoclast differentiation.
이에, 본 발명의 목적은 순비기나무 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases comprising an extract of Astragalus sinensis as an active ingredient.
본 발명의 다른 목적은 순비기나무 추출물을 유효성분으로 포함하는 골질환 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for improving bone disease comprising an extract of Astragalus sinensis as an active ingredient.
본 발명의 또 다른 목적은 순비기나무 추출물의 골질환 예방 또는 치료 유효량으로 이를 필요로 하는 대상에 투여하는 단계를 포함하는 골질환 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating bone disease comprising the step of administering to a subject in need of it in an effective amount for preventing or treating bone disease of the extract of Astragalus sinensis.
본 발명의 또 다른 목적은 순비기나무 추출물의 골질환 개선, 예방 또는 치료 용도에 관한 것이다.Another object of the present invention relates to the improvement, prevention or treatment of bone diseases of the extract of Astragalus sinensis.
본 발명은 순비기나무 (Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물, 및 골질환 개선용 식품 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating bone disease, and a food composition for improving bone disease, comprising an extract of Vitex rotundifolia as an active ingredient.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 순비기나무(Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물에 관한 것이다.One aspect of the present invention relates to a pharmaceutical composition for preventing or treating bone disease comprising an extract of Vitex rotundifolia as an active ingredient.
본 명세서상 용어 "순비기나무"는 마편초과의 (Verbenaceae)의 나무로 해안가에서 자주 볼 수 있는 모래에서도 자라는 나무이다. 또한, 염분이 있어서 자라는 염생 식물이다. 낙엽수이며 잎은 두꺼우며 난형, 도란형, 넓은 타원형으로 마주난다. 열매는 해열, 진정, 소염에 효능이 있어 감창, 두통, 관절염, 안질동통, 풍습비통, 이명 등에 치료제로 쓰인다. 잎과 가지에는 향기가 있어 향료로도 쓰인다.In the present specification, the term "sunbigi tree" is a tree of the verbenaceae family ( Verbenaceae ) and is a tree that grows even in the sand that can often be seen on the coast. It is also a halophyte that grows in salt. It is a deciduous tree and the leaves are thick and meet each other in an egg shape, an obovate shape, and a wide oval shape. The fruit is effective in antipyretic, sedative, and anti-inflammatory, so it is used as a remedy for sore throat, headache, arthritis, eye pain, customs, and tinnitus. The leaves and branches are fragrant and are also used as spices.
본 발명에 있어서 순비기나무는 잎 및 줄기로 이루어진 군에서 선택된 1종 이상인 것일 수 있으며, 예를 들어, 잎인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pure ginseng tree may be one or more selected from the group consisting of leaves and stems, for example, may be leaves, but is not limited thereto.
본 명세서상 용어 "추출물"은 용매 조추출물, 특정 용매 가용 추출물(용매 분획물) 및 용매 조추출물의 용매 분획물을 포함하며, 상기 순비기나무 추출물은 용액, 농축물 또는 분말 상태일 수 있다.The term "extract" in the present specification includes a crude solvent extract, a specific solvent-soluble extract (solvent fraction), and a solvent fraction of the crude solvent extract, and the extract of Astragalus sinensis may be in a solution, concentrate or powder state.
본 발명에 있어서 순비기나무 추출물은 물, 및 탄소수 1 내지 4개의 직쇄 또는 분지형 알코올로 이루어진 군에서 선택된 1종 이상의 용매로 추출하여 얻어진 추출물인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the extract of Astragalus sinensis may be an extract obtained by extraction with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, but is not limited thereto.
본 발명에 있어서 용매는 알코올 함량이 10 %(v/v) 이상 내지 100 %(v/v) 미만, 20 %(v/v) 이상 내지 100 %(v/v) 미만, 30 %(v/v) 이상 내지 100 %(v/v) 미만, 40 %(v/v) 이상 내지 100 %(v/v) 미만, 50 %(v/v) 이상 내지 100 %(v/v) 미만, 50 %(v/v) 이상 내지 95 %(v/v) 미만, 50 %(v/v) 이상 내지 92 %(v/v) 미만, 60 %(v/v) 이상 내지 100 %(v/v) 미만, 60 %(v/v) 이상 내지 95 %(v/v) 미만, 60 %(v/v) 이상 내지 92 %(v/v) 미만, 70 %(v/v) 이상 내지 100 %(v/v) 미만, 70 %(v/v) 이상 내지 95 %(v/v) 미만, 70 %(v/v) 이상 내지 92 %(v/v) 미만, 80 %(v/) 이상 내지 100 %(v/v) 미만, 80 %(v/v) 이상 내지 95 %(v/v) 미만, 80 %(v/v) 이상 내지 92 %(v/v) 미만, 예를 들어, 90 %(v/v)의 알코올 수용액인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent has an alcohol content of 10% (v/v) or more to less than 100% (v/v), 20% (v/v) or more to less than 100% (v/v), 30% (v/v) or more. v) greater than or equal to 100% (v/v), greater than or equal to 40% (v/v) and less than 100% (v/v), greater than or equal to 50% (v/v) to less than 100% (v/v), 50 % (v/v) or more to less than 95% (v/v), 50% (v/v) or more to less than 92% (v/v), 60% (v/v) or more to 100% (v/v) ) less than, 60% (v/v) or more to less than 95% (v/v), 60% (v/v) or more to less than 92% (v/v), 70% (v/v) or more to 100% Less than (v/v), 70% (v/v) or more to less than 95% (v/v), 70% (v/v) or more to less than 92% (v/v), 80% (v/) or more to less than 100% (v/v), from 80% (v/v) to less than 95% (v/v), from 80% (v/v) to less than 92% (v/v), for example, It may be a 90% (v/v) aqueous alcohol solution, but is not limited thereto.
본 발명에 있어서 용매는 무수 알코올인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent may be anhydrous alcohol, but is not limited thereto.
본 발명에 있어서 용매는 에탄올, 부탄올, 에틸에테르 및 아세트산에틸로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent may be at least one selected from the group consisting of ethanol, butanol, ethyl ether and ethyl acetate, but is not limited thereto.
본 발명에 있어서 골질환은 골다공증 (osteoporosis), 치주질환 (periodontal disease), 류마티스 관절염 (rheumatoid arthritis), 다발성 골수종 (multiple myeloma), 퇴행성 관절염 (degenerative arthritis), 염증성 치조골 흡수 질환 및 염증성 뼈 흡수 질환으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, bone diseases include osteoporosis, periodontal disease, rheumatoid arthritis, multiple myeloma, degenerative arthritis, inflammatory alveolar bone resorption disease and inflammatory bone resorption disease. It may be one or more selected from the group consisting of, but is not limited thereto.
본 명세서상 용어 "골다공증"은 뼈의 양이 감소하고 질적인 변화로 인해 뼈의 강도가 약해져서 골절이 일어날 가능성이 높은 상태를 의미한다. 뼈는 인체 내 여러 기관들을 보호하고 칼슘과 같은 체내에 필요한 물질들을 보관하는 저장소로서 기능하며, 뼈 조직에 존재하는 뼈를 분해하는 파골세포 (osteoclast) 및 뼈를 생성하는 조골세포 (osteoblast) 간의 균형에 이어 항상성이 유지된다. 골다공증의 경우에는 두 세포 활성이 균형이 깨지게 되면서 파골세포에 의한 지나친 뼈의 파괴가 일어나 발병 및 진행된다. 골다공증에서 주요한 분자로는 RANKL (receptoractivator of nuclear factor kappa-B ligand)이 있으며, RANKL이 이의 수용체에 결합하여 다양한 전사인자를 활성화시켜 파골세포의 분화를 촉진시킨다.As used herein, the term "osteoporosis" refers to a condition in which bone strength is weakened due to a decrease in bone quantity and qualitative changes, resulting in a high possibility of fracture. Bone protects various organs in the human body and functions as a reservoir for storing substances necessary for the body, such as calcium, and balances between osteoclasts that decompose bones existing in bone tissue and osteoblasts that create bones. Then, homeostasis is maintained. In the case of osteoporosis, as the balance between the two cell activities is broken, excessive destruction of bone by osteoclasts occurs, resulting in onset and progression. RANKL (receptoractivator of nuclear factor kappa-B ligand) is a major molecule in osteoporosis, and RANKL binds to its receptor to activate various transcription factors to promote osteoclast differentiation.
상기 RANKL은 대식세포에 처리할 경우 RANK (Receptor activator of nuclear factor kappa-Β에 결합과 함께 TRAP 양성 세포로 분화하게 되는데, 이러한 TRAP 양성 세포에 RANKL, TNF-α와 같은 염증인자로 자극하면 세포끼리 융합되어 다핵형 TRAP 양성 세포로 분화된다.When the RANKL is treated with macrophages, it binds to RANK (Receptor activator of nuclear factor kappa-Β) and differentiates into TRAP-positive cells. When these TRAP-positive cells are stimulated with inflammatory factors such as RANKL and TNF-α, the cells They fuse and differentiate into multinucleated TRAP-positive cells.
본 명세서상 용어 "치주질환"은 세균에 의해 야기되는 치아지지 조직의 염증상태를 말하며, 치은염 및 치주염으로 분리할 수 있다. 발병원인은 불량한 구강 위생상태로 인한 구강 세균이 치면 세균막을 형성하는 데 있다. 치면 세균막이란 침에 있는 끈끈한 물질을 접착제로 이용하여 세균이 치아 표면에 달라붙은 후 증식한 세균덩어리를 말한다. 치면 세균막은 그냥 방치해 두면, 염증이 생겨 가끔 잇몸에서 피가 나고, 구취가 나는 경우가 있으며 이러한 증상을 치은염이라고 한다. 치은염이 더 진행되면, 치아와 잇몸 사이의 벌어진 틈이 더 깊어져서 치주낭이 생기고, 여기에 치주질환을 일으키는 세균들이 번식하여 치주염이 발생된다. 치주염이 진행되면 칫솔질과 같은 약한 자극에도 잇몸에서 피가 나기도 하며 붓고, 종종 급성염증으로 변화되어 통증을 유발한다. 이러한 염증은 골을 만드는 기능은 저하시키고, 골을 흡수하는 작용이 높아져서 치조골이 점점 낮아지게 되어 치조골이 파괴되고 결국 치아를 상실하게 된다.As used herein, the term "periodontal disease" refers to an inflammatory state of tooth supporting tissue caused by bacteria, and can be separated into gingivitis and periodontitis. The cause of the disease is the formation of a bacterial film when oral bacteria are struck due to poor oral hygiene. Tooth surface bacterial film refers to a mass of bacteria that proliferates after bacteria adhere to the tooth surface using sticky substances in saliva as an adhesive. If left unattended, the bacterial film on the toothbrush becomes inflamed, sometimes leading to bleeding gums and bad breath, and these symptoms are called gingivitis. As gingivitis progresses further, the gap between the teeth and the gums deepens to form periodontal pockets, where bacteria causing periodontal disease proliferate to cause periodontitis. As periodontitis progresses, the gums bleed and swell even with weak stimuli such as brushing, and often cause pain due to acute inflammation. This inflammation reduces the function of making bone and increases the function of absorbing bone, so that the alveolar bone is gradually lowered, resulting in destruction of the alveolar bone and eventually loss of teeth.
본 명세서상 용어 "관절염"은 하나 이상의 관절 부위에 염증을 수반하는 관절 질환을 의미한다. 상기 관절염의 일반적인 형태는 관절을 보호하고 있는 연골의 점진적인 손상이나 퇴행성 변화로 인해 관절을 이루는 뼈와 인대에 손상이 일어나서 염증과 통증이 생기는, 골 관절염(osteoarthritis)이다.As used herein, the term "arthritis" refers to a joint disease involving inflammation in one or more joint areas. A common form of arthritis is osteoarthritis, in which inflammation and pain occur due to damage to bones and ligaments constituting the joint due to gradual damage or degenerative change of cartilage protecting the joint.
본 발명의 다른 양태는 순비기나무 추출물을 제조하는 방법에 관한 것이다.Another aspect of the present invention relates to a method for preparing an extract of Astragalus sinensis.
상기 순비기나무 추출물은 순비기나무의 용매 추출물을 포함하며, 상술한 바와 같다.The extract of japonica japonica includes a solvent extract of japonica japonica, and is as described above.
본 발명에 따른 순비기나무 추출물의 제조 과정을 보다 상세하게 설명하면 다음과 같다: 순비기나무의 중량에 대하여 약 10 내지 50배 중량의 추출용매로 상온 추출한다. 추출 후 여과하여 여과액을 모은다. 추출 온도는 특별한 제한은 없지만 예를 들어, 15 내지 110 ℃, 20 내지 100 ℃, 20 내지 90 ℃, 20 내지 70 ℃, 20 내지 50 ℃, 20 내지 30 ℃, 예를 들어, 25℃인 것이 좋다.A more detailed description of the manufacturing process of the extract of the present invention is as follows: Room temperature extraction is performed with an extraction solvent of about 10 to 50 times the weight of the extract of the genus japonica. After extraction, filter and collect the filtrate. The extraction temperature is not particularly limited, but is preferably 15 to 110 °C, 20 to 100 °C, 20 to 90 °C, 20 to 70 °C, 20 to 50 °C, 20 to 30 °C, for example, 25 °C. .
추출공정은 1회 또는 수회 반복할 수 있으며, 본 발명의 한 바람직한 예에서는 1차 추출 후 다시 재추출하는 방법을 채택할 수 있는데, 이는 추출물을 대량 생산하는 경우 효과적으로 여과를 한다 하더라도 자체의 수분 함량이 높기 때문에 손실이 발생하게 되어 1차 추출만으로는 추출효율이 떨어지므로 이를 방지하기 위함이다. 또한, 각 단계별 추출효율을 검증한 결과 2차 추출에 의해 전체 추출량의 80 내지 90% 정도가 추출되는 것으로 밝혀졌다.The extraction process may be repeated once or several times, and in one preferred example of the present invention, a method of re-extracting again after the first extraction may be adopted, which means that even if the extract is effectively filtered in the case of mass production, its water content Since this is high, loss occurs, and the extraction efficiency drops only with the first extraction, so this is to be prevented. In addition, as a result of verifying the extraction efficiency at each stage, it was found that about 80 to 90% of the total extraction amount was extracted by the secondary extraction.
본 발명의 일 예에서, 추출공정을 2회 반복하는 경우, 상기 얻어진 잔사에 다시 추출용매, 약 5 내지 15 부피배, 예를 들어, 8 내지 12 부피배로 환류 추출한다. 추출 후 여과하고 이전에 얻어진 여과액과 합쳐서 감압 농축을 하여 순비기나무 추출물을 제조한다. 이와 같이 2차에 걸친 추출 및 각각의 추출 후 얻어진 여과액을 혼합함으로써 추출 효율을 높일 수 있으나, 본 발명의 추출물이 추출 회수에 한정되는 것은 아니다.In one example of the present invention, when the extraction process is repeated twice, the extraction solvent is again extracted with about 5 to 15 times the volume, for example, 8 to 12 times the volume of the obtained residue under reflux. After extraction, it is filtered and combined with the previously obtained filtrate, and concentrated under reduced pressure to prepare an extract of Astragalus sinensis. Although the extraction efficiency can be increased by mixing the filtrate obtained after the second extraction and each extraction, the extract of the present invention is not limited to the number of extractions.
상기 순비기나무 추출물 제조 시에 사용되는 용매의 양이 너무 적으면 교반이 어렵게 되고 추출물의 용해도가 낮아져 추출효율이 떨어지게 되고, 지나치게 많은 경우는 다음의 정제단계에서 사용되는 용매의 사용량이 많아져 경제적이지 못하여 취급상 문제가 발생할 수 있으므로, 용매의 사용량은 상기 범위로 하는 것이 좋다.If the amount of the solvent used in the preparation of the extract of Sunbinata is too small, stirring becomes difficult and the solubility of the extract decreases, resulting in a decrease in extraction efficiency. Failure to do so may cause problems in handling, so the amount of solvent used is preferably within the above range.
이와 같이 얻어진 여과된 추출물은 의약품 원료로 사용하기에 적합하도록 잔존하는 저급 알코올의 함량을 조절하기 위하여 농축물 총량의 약 10 내지 30배, 예를 들어, 15 내지 25배, 예를 들어, 약 20 중량배의 물로 1 내지 5회, 예를 들어, 2 내지 3회 공비 농축하고 재차 동량의 물을 가하여 균질 하게 현탁시킨 후 동결 건조하여 분말상태의 순비기나무 추출물로 제조될 수 있다.The filtered extract obtained in this way is about 10 to 30 times, for example, 15 to 25 times, for example, about 20 times the total amount of the concentrate in order to adjust the content of the remaining lower alcohol to be suitable for use as a pharmaceutical raw material. After azeotropically concentrating 1 to 5 times, for example, 2 to 3 times with water by weight, adding the same amount of water again to homogeneously suspending, freeze-drying can be prepared as a powdery Astragalus japonica extract.
본 발명에 사용된 추출 방법은 통상적으로 사용되는 모든 방법일 수 있으며, 예컨대, 열수추출, 초음파 추출, 또는 환류 추출법일 수 있으나, 이에 한정되는 것은 아니다.The extraction method used in the present invention may be any method commonly used, and may be, for example, hot water extraction, ultrasonic extraction, or reflux extraction, but is not limited thereto.
본 발명의 약제학적 조성물은 순비기나무 추출물의 약제학적 유효량 및/또는 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물로 이용될 수 있다.The pharmaceutical composition of the present invention may be used as a pharmaceutical composition comprising a pharmaceutically effective amount of the extract of Astragalus sinensis and/or a pharmaceutically acceptable carrier.
본 명세서상의 용어 "약제학적 유효량"은 상술한 순비기나무 추출물의 효능 또는 활성을 달성하는데 충분한 양을 의미한다.The term "pharmaceutically effective amount" in the present specification means an amount sufficient to achieve the efficacy or activity of the above-described Astragalus sinensis extract.
본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
본 발명에 따른 약제학적 조성물은 인간을 포함하는 포유동물에 다양한 경로로 투여될 수 있다. 투여 방식은 통상적으로 사용되는 모든 방식일 수 있으며, 예컨대, 경구, 피부, 정맥, 근육, 피하 등의 경로로 투여될 수 있으며, 예를 들어, 경구로 투여될 수 있다.The pharmaceutical composition according to the present invention may be administered to mammals including humans by various routes. The administration method may be any method commonly used, and may be administered through, for example, oral, dermal, intravenous, intramuscular, subcutaneous, or the like routes, for example, orally.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
본 발명의 조성물은 상기 혼합 추출물 이외에 약제학적으로 적합하고 생리학적으로 허용되는 담체, 부형제 및 희석제 등의 보조제를 추가로 함유하는 것일 수 있다. The composition of the present invention may further contain adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients and diluents in addition to the mixed extract.
본 발명의 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, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 추출물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose) 또는 락토오스 (lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다.In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose ( It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구를 위한 제제로는 현탁제, 내용액제, 유제, 시럽제, 연고제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Oral preparations include suspensions, solutions for oral use, emulsions, syrups, ointments, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제, 경피제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌 글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, transdermal preparations, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
좌제의 제제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.As preparations for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 조성물을 인간에게 적용하는 구체예에 있어서, 본 발명의 생약 추출물 조성물은 단독으로 투여될 수 있으나, 일반적으로 투여방식과 표준 약제학적 관행 (standard phamaceutical practice)을 고려하여 선택된 약제학적 담체와 혼합되어 투여될 수 있다.In the specific embodiment of applying the composition of the present invention to humans, the herbal extract composition of the present invention may be administered alone, but generally with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice. They may be administered in combination.
예를 들면, 본 발명의 생약 추출물 함유 조성물은 전분 또는 락토오즈를 함유하는 정제 형태로, 또는 단독 또는 부형제를 함유하는 캡슐 형태로, 또는 맛을 내거나 색을 띄게 하는 화학 약품을 함유하는 엘릭시르 또는 현탁제 형태로 경구, 구강 내 또는 혀 밑 투여될 수 있다. 이러한 액체 제제는 현탁제 (예를 들면, 메틸셀룰로오즈, 위텝솔 (witepsol)과 같은 반합성 글리세라이드 또는 행인유 (apricot kernel oil)와 PEG-6 에스테르의 혼합물 또는 PEG-8과 카프릴릭/카프릭 글리세라이드의 혼합물과 같은 글리세라이드 혼합물)와 같은 약제학적으로 허용 가능한 첨가제와 함께 제형화 될 수 있다.For example, the herbal extract-containing composition of the present invention may be in the form of tablets containing starch or lactose, either alone or in the form of capsules containing excipients, or in the form of elixirs or suspensions containing flavoring or coloring chemicals. It can be administered orally, buccally or sublingually in all forms. Such liquid formulations may be prepared by suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as witepsol or mixtures of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric acid). It can be formulated with pharmaceutically acceptable excipients such as mixtures of glycerides).
본 발명의 추출물 함유 조성물의 투여 용량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 의사 또는 약사의 판단에 따라 일정 시간간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다.The dosage of the extract-containing composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, state of health and degree of disease, and may be administered once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. Split doses may also be administered.
본 발명에 있어서 약제학적 조성물은 순비기나무 추출물의 함량을 체중 60 kg 당 100 내지 1000 mg/day, 100 내지 900 mg/day, 100 내지 800 mg/day, 150 내지 1000 mg/day, 150 내지 900 mg/day, 150 내지 800 mg/day, 170 내지 1000 mg/day, 170 내지 900 mg/day, 170 내지 800 mg/day, 예를 들어, 180 mg/day 내지 720 mg/day 범위로 하여 투여되는 것일 수 있다.In the present invention, the pharmaceutical composition contains the content of the extract of chinensis 100 to 1000 mg / day, 100 to 900 mg / day, 100 to 800 mg / day, 150 to 1000 mg / day, 150 to 900 per 60 kg of body weight administered in the range of mg/day, 150 to 800 mg/day, 170 to 1000 mg/day, 170 to 900 mg/day, 170 to 800 mg/day, for example, 180 mg/day to 720 mg/day it could be
본 발명의 순비기나무 추출물 함유 조성물의 1일 투여량이 상기 투여 용량 미만이면 유의성 있는 효과를 얻을 수 없으며, 그 이상을 초과하는 경우 비경제적일 뿐만 아니라 상용량의 범위를 벗어나므로 바람직하지 않은 부작용이 나타날 우려가 발생할 수 있으므로, 상기 범위로 하는 것이 좋다.Significant effects cannot be obtained if the daily dose of the composition containing the extract of Astragalus sinensis of the present invention is less than the above dose, and if it exceeds more than that, it is not only uneconomical but also outside the range of the usual dose, so undesirable side effects may occur. Since concerns may arise, it is good to set it as the above range.
본 발명의 또 다른 양태는 순비기나무(Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 개선용 식품 조성물에 관한 것이다.Another aspect of the present invention relates to a food composition for improving bone disease comprising an extract of Vitex rotundifolia as an active ingredient.
본 발명에 있어서 순비기나무는 잎 및 줄기로 이루어진 군에서 선택된 1종 이상인 것일 수 있으며, 예를 들어, 잎인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pure ginseng tree may be one or more selected from the group consisting of leaves and stems, for example, may be leaves, but is not limited thereto.
본 발명에 있어서 순비기나무 추출물은 물, 및 탄소수 1 내지 4개의 직쇄 또는 분지형 알코올로 이루어진 군에서 선택된 1종 이상의 용매로 추출하여 얻어진 추출물인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the extract of Astragalus sinensis may be an extract obtained by extraction with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, but is not limited thereto.
본 발명에 있어서 용매는 알코올 함량이 10 %(v/v) 이상 내지 100 %(v/v) 미만, 20 %(v/v) 이상 내지 100 %(v/v) 미만, 30 %(v/v) 이상 내지 100 %(v/v) 미만, 40 %(v/v) 이상 내지 100 %(v/v) 미만, 50 %(v/v) 이상 내지 100 %(v/v) 미만, 50 %(v/v) 이상 내지 95 %(v/v) 미만, 50 %(v/v) 이상 내지 92 %(v/v) 미만, 60 %(v/v) 이상 내지 100 %(v/v) 미만, 60 %(v/v) 이상 내지 95 %(v/v) 미만, 60 %(v/v) 이상 내지 92 %(v/v) 미만, 70 %(v/v) 이상 내지 100 %(v/v) 미만, 70 %(v/v) 이상 내지 95 %(v/v) 미만, 70 %(v/v) 이상 내지 92 %(v/v) 미만, 80 %(v/) 이상 내지 100 %(v/v) 미만, 80 %(v/v) 이상 내지 95 %(v/v) 미만, 80 %(v/v) 이상 내지 92 %(v/v) 미만, 예를 들어, 90 %(v/v)의 알코올 수용액인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent has an alcohol content of 10% (v/v) or more to less than 100% (v/v), 20% (v/v) or more to less than 100% (v/v), 30% (v/v) or more. v) greater than or equal to 100% (v/v), greater than or equal to 40% (v/v) and less than 100% (v/v), greater than or equal to 50% (v/v) to less than 100% (v/v), 50 % (v/v) or more to less than 95% (v/v), 50% (v/v) or more to less than 92% (v/v), 60% (v/v) or more to 100% (v/v) ) less than, 60% (v/v) or more to less than 95% (v/v), 60% (v/v) or more to less than 92% (v/v), 70% (v/v) or more to 100% Less than (v/v), 70% (v/v) or more to less than 95% (v/v), 70% (v/v) or more to less than 92% (v/v), 80% (v/) or more to less than 100% (v/v), from 80% (v/v) to less than 95% (v/v), from 80% (v/v) to less than 92% (v/v), for example, It may be a 90% (v/v) aqueous alcohol solution, but is not limited thereto.
본 발명에 있어서 용매는 무수 알코올인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent may be anhydrous alcohol, but is not limited thereto.
본 발명에 있어서 용매는 에탄올, 부탄올, 에틸에테르 및 아세트산에틸로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the solvent may be at least one selected from the group consisting of ethanol, butanol, ethyl ether and ethyl acetate, but is not limited thereto.
본 발명에 있어서 골질환은 골다공증 (osteoporosis), 치주질환 (periodontal disease), 류마티스 관절염 (rheumatoid arthritis), 다발성 골수종 (multiple myeloma), 퇴행성 관절염 (degenerative arthritis), 염증성 치조골 흡수 질환 및 염증성 뼈 흡수 질환으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, bone diseases include osteoporosis, periodontal disease, rheumatoid arthritis, multiple myeloma, degenerative arthritis, inflammatory alveolar bone resorption disease and inflammatory bone resorption disease. It may be one or more selected from the group consisting of, but is not limited thereto.
본 발명의 식품 조성물을 식품 첨가물로 사용할 경우, 상기 식품조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 식품조성물은 원료에 대하여 15 중량% 이하, 예를 들어, 10 중량% 이하의 양으로 첨가될 수 있다.When the food composition of the present invention is used as a food additive, the food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. In general, when preparing food or beverage, the food composition of the present invention may be added in an amount of 15% by weight or less, for example, 10% by weight or less based on the raw material.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all foods in a conventional sense.
상기 음료는 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrates may be appropriately determined by a person skilled in the art.
상기 외에 본 발명의 식품조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives can also be appropriately selected by those skilled in the art.
상기 식품 조성물에 있어서, 상기 약제학적 조성물과 중복되는 내용은 본 명세서의 복잡성을 고려하여 생략한다.In the food composition, the contents overlapping with the pharmaceutical composition are omitted in consideration of the complexity of the present specification.
본 발명은 순비기나무 (Vitex rotundifolia) 추출물을 유효성분으로 포함하는 골질환 예방 또는 치료용 약제학적 조성물, 및 골질환 개선용 식품 조성물에 관한 것으로, 본 발명에 따른 순비기나무 추출물은 파골세포 분화 억제에 우수한 효과를 나타내는 바, 다양한 골질환 개선, 예방 또는 치료에 유용한 약제학적 조성물 또는 식품 조성물로 이용될 수 있다.The present invention relates to a pharmaceutical composition for preventing or treating bone disease comprising an extract of Vitex rotundifolia as an active ingredient, and a food composition for improving bone disease. Since it shows an excellent effect on inhibition, it can be used as a pharmaceutical composition or food composition useful for improving, preventing or treating various bone diseases.
도 1은 본 발명의 일 실시예에 따른 순비기나무 잎 추출물의 파골세포 억제 효과를 보여주는 사진 및 그래프이다.1 is a photograph and a graph showing the osteoclast inhibitory effect of the leaf extract of Astragalus sinensis according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1. 순비기나무 잎의 에탄올 추출물의 제조Example 1. Preparation of ethanol extracts from leaves
순비기나무 잎은 2020년 6월 전라남도 완도군 해안가에서 채취하였다. 구체적으로, 순비기나무 잎 100 g을 90 %(v/v) 에탄올 1L에 넣고, 호모겐나이저 (Homogenizer)로 분쇄하여 상온에서 24시간 동안 추출하였다. 그리고 No.2 정성 여과지 (8 um, Whatman사) 여과지로 여과하여 에탄올 추출액을 얻었다. 얻어진 잔사에 90 %(v/v) 에탄올 (0.5 L)를 가하여 상기와 동일한 방법으로 다시 추출하였다. 수득한 에탄올 추출액은 아스피레이터 (aspirator, CCA-1110, Eyela사, Tokyo, Japan)를 사용하여 45 ℃에서 감압 농축하여 순비기나무 잎 에탄올 추출물 (0.81 g)을 제조하였다.Sunbigi leaves were collected from the coast of Wando-gun, Jeollanam-do in June 2020. Specifically, 100 g of chinensis leaves were put in 1 L of 90% (v/v) ethanol, pulverized with a homogenizer, and extracted at room temperature for 24 hours. Then, the mixture was filtered with No.2 qualitative filter paper (8 um, Whatman) to obtain an ethanol extract. 90% (v/v) ethanol (0.5 L) was added to the obtained residue, and extraction was performed again in the same manner as above. The obtained ethanol extract was concentrated under reduced pressure at 45° C. using an aspirator (CCA-1110, Eyela, Tokyo, Japan) to prepare an ethanol extract (0.81 g) of the leaves of A.
실시예 2. 파골세포 분화 억제 효과Example 2. Osteoclast differentiation inhibitory effect
2-1. 마우스 골수 유래 대식세포 (bone marrow-derived macrophages, BMDMs) 분리 및 배양2-1. Isolation and culture of mouse bone marrow-derived macrophages (BMDMs)
8 내지 12주령의 C57BL/6 마우스의 대퇴골 (femur)에서 골수를 분리하였다. 그리고 얻어진 골수에 10 %(v/v) 소 태아 혈청 (Fetal bovine serum), 1 %(v/v) 페니실린 및 대식세포 콜로니 자극인자 (Macrophage-colony stimulating factor, M-CSF) 25 ng/ml를 포함한 MEM-알파 배지(alpha modification of eagle's minimum essential media, MEM alpha)에 도말하여 3일 동안 37 ℃의 5 %(v/v) CO2 인큐베이터에서 배양하여 골수 유래 대식세포 (Bone marrow-derived macrophage, BMDMs)를 얻었다.Bone marrow was isolated from the femurs of 8 to 12 week old C57BL/6 mice. Then, 10% (v/v) fetal bovine serum, 1% (v/v) penicillin, and 25 ng/ml of macrophage-colony stimulating factor (M-CSF) were added to the obtained bone marrow. Bone marrow - derived macrophages (Bone marrow-derived macrophages, BMDMs) were obtained.
2-2. 파골세포 분화 억제 검증2-2. Verification of inhibition of osteoclast differentiation
골수 유래 대식세포 (Bone marrow-derived macrophage, BMDMs)를 10% FBS, 1% 페니실린 (penicillin) 및 M-CSF (Macrophage-colony stimulating factor)가 첨가된 배지에 웰 (well) 당 2 x 105 세포 밀도로 접종하여 12-웰-플레이트 (12-well plate)에 24시간 동안 배양하였다. 상기와 동일한 조성의 배지로 교체한 후, 순비기나무 잎 에탄올 추출물 10, 12.5, 15 및 20 μg/ml을 2시간 동안 처리하였다. 그 다음, RANKL (Receptor activator of nuclear factor kappa-Βligand)을 처리하여 24시간 동안 반응시켰다.Bone marrow-derived macrophages (BMDMs) were 2 x 10 5 cells per well in a medium supplemented with 10% FBS, 1% penicillin and M-CSF (Macrophage-colony stimulating factor) Inoculated at a density and cultured for 24 hours in a 12-well plate. After replacing the medium with the same composition as above, 10, 12.5, 15, and 20 μg/ml of ethanol extracts of chinensis chinensis leaves were treated for 2 hours. Then, RANKL (Receptor activator of nuclear factor kappa-Βligand) was treated and reacted for 24 hours.
상기한 방법과 동일한 방법으로 4일간 파골 세포 분화를 유도하였다. 그리고 파골세포의 세포 화학적 표지 효소인 (Tartrate resistance acid phosphatase, TRAP)에 발색성 기질을 첨가하여 핵을 염색하였다. 그 다음, 핵이 3개 이상으로 다핵화된 세포를 이미지화 및 정량화 하여 하기 표 1 및 도 1에 나타내었다.Osteoclast differentiation was induced for 4 days in the same manner as described above. Nuclei were stained by adding a chromogenic substrate to tartrate resistance acid phosphatase (TRAP), a cytochemical marker of osteoclasts. Then, the multinucleated cells with three or more nuclei were imaged and quantified, and are shown in Table 1 and FIG. 1 below.
도 1에서 확인할 수 있듯이, 대식세포에 RANKL을 처리할 경우 파골 세포의 분화가 증가하는 것을 확인하였다. 반면, 순비기나무 잎 에탄올 추출물을 대식세포에 처리할 경우, RANKL로부터 유도된 파골세포는 추출물의 농도에 의존적으로 억제되는 것을 확인할 수 있었다. 이는 순비기나무 잎 에탄올 추출물이 파골세포 억제에 우수한 효과가 있음을 나타낸다.As can be seen in Figure 1, it was confirmed that the differentiation of osteoclasts increased when macrophages were treated with RANKL. On the other hand, it was confirmed that when macrophages were treated with the ethanol extract of the leaves of Astragalus sinensis, osteoclasts induced from RANKL were inhibited in a concentration-dependent manner. This indicates that the ethanol extract from the leaves of Astragalus sinensis has an excellent effect on inhibiting osteoclasts.
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