KR102114378B1 - Composition for preventing and treating bone-related diseases comprising Jeobgolgongjindan - Google Patents
Composition for preventing and treating bone-related diseases comprising Jeobgolgongjindan Download PDFInfo
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- KR102114378B1 KR102114378B1 KR1020180060912A KR20180060912A KR102114378B1 KR 102114378 B1 KR102114378 B1 KR 102114378B1 KR 1020180060912 A KR1020180060912 A KR 1020180060912A KR 20180060912 A KR20180060912 A KR 20180060912A KR 102114378 B1 KR102114378 B1 KR 102114378B1
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Abstract
본 발명은 골 재생 및 골 세포 분화를 위한 접골공진단을 포함하는 조성물에 관한 것으로, 구체적으로 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향을 혼합하여 제조된 접골공진단 추출물은 조골세포에서 골세포로의 분화를 유도하고, 골형성(osteogenesis) 관련 유전자 및 단백질의 발현을 증가시켜, 골형성 및 골세포 분화 촉진 효과를 나타냄으로써, 골 관련 질환의 예방 또는 치료에 유용하게 사용될 수 있다.The present invention relates to a composition comprising a osteoclast diagnosis for bone regeneration and bone cell differentiation. Specifically, the osteoclast diagnosis extract prepared by mixing antler, Angelica, cornus, safflower, grafted wood and acupuncture from osteoblasts to bone cells By inducing the differentiation of, and increasing the expression of genes and proteins related to osteogenesis, by showing the effect of promoting bone formation and bone cell differentiation, it can be usefully used for the prevention or treatment of bone-related diseases.
Description
본 발명은 골 재생 및 골 세포 분화를 위한 접골공진단을 포함하는 조성물에 관한 것으로, 구체적으로는 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물의 조골세포 및 골세포 분화 유도 및 골 형성 관련 유전자와 단백질 발현 촉진으로 인한 골 재생 및 골 세포 분화 촉진 효과에 관한 것이다.The present invention relates to a composition comprising a osteoclast for bone regeneration and bone cell differentiation, specifically, induction of osteoblast and bone cell differentiation induction and bone formation related genes of deer antler, Angelica, Cornus officinalis, safflower, grafted tree and aloe extract And the effect of promoting bone regeneration and bone cell differentiation by promoting protein expression.
국내에서의 뼈, 연골을 치료하기 위한 시장은 약 900억원의 큰 시장의 규모를 형성하고 있다. 이에, 많은 관련 제품과 이식재료에 대하여 다양한 연구와 개발이 진행되고 있다. 또한, 고령화가 되면서 노인환자들에게 골절이 빈번하게 일어나고 있고, 장년이나 청년에 비해 회복의 시기가 늦어짐으로써 그로 인한 합병증과 더불어 경제적 부담도 매우 크기 때문에, 기존의 골 재생을 위해 치료약을 복용하는 방법과 함께, 골절부위에 scaffold나 membrane에 encapsulation을 해서 전달하는 것을 병행함으로써 과도한 약물 복용으로 인한 부작용의 가능성을 줄이고 효과적인 전달을 통해 회복의 시기를 단축시키기 위한 목적으로 TGF-beta, BMP 등의 성장인자, 이를 이용한 전달방법 및 전달 재료에 대해서도 많은 연구가 진행되고 있다. The market for treating bone and cartilage in Korea is forming a large market of about 90 billion won. Accordingly, various researches and developments have been conducted on many related products and implant materials. In addition, as aging, fractures occur frequently in elderly patients, and the recovery period is slower than that of older adults or young adults, so the complications and economic burden are very high. In addition, the growth factors such as TGF-beta and BMP are aimed at reducing the possibility of side effects caused by excessive drug use and shortening the recovery period through effective delivery by concurrently delivering the scaffold or membrane to the fracture site by encapsulation. , A lot of research has been conducted on delivery methods and delivery materials using the same.
골 재생의 일반적인 회복과정을 살펴보면 골절이 된 후 빠르게 일주일 이내에 염증반응이 일어나게 된다. 손상된 혈관에서 염증성 삼출액이 분비되며 골절된 뼈 주의조직은 괴사가 일어나게 된다. 이때, 파골세포(osteoclasts)들은 괴사된 뼈 주변 조직을 제거한다. 그 다음 부드러운 캘러스 단계가 진행되어, 통증과 부종이 가라앉으면서 캘러스 층이 생기고, 섬유성 조직 및 연골의 생성과 더불어서 혈관이 자라나기 시작한다. 캘러스 내의 모세혈관의 성장은 혈류를 증가시키며 캘러스를 통하여 mesenchymal progenitor cell들이 확산되고 여러 세포들의 상호작용과 함께 재생을 하기 시작한다. 그리고 단단한 캘러스 단계로 넘어가게 되어, 연골 내 골화로 인해 연부조직 내 틈은 원래의 코엑스를 이어주는 woven 골로 대체된다. 그리고 리모델링이 일어나는데 이 과정은 몇 년 동안에 걸쳐 골절부위에서 일어나는 모든 과정과 더불어서 회복과정을 의미한다.Looking at the general recovery process of bone regeneration, an inflammatory reaction occurs quickly within a week after fracture. Inflammatory effusion is secreted from the damaged blood vessels and necrosis occurs in the fractured bone tissue. At this time, osteoclasts (osteoclasts) remove the tissue surrounding the necrotic bone. Then a soft callus stage proceeds, as the pain and swelling subsides, a callus layer is formed, and along with the production of fibrous tissue and cartilage, blood vessels begin to grow. The growth of capillaries in the callus increases blood flow, through which the mesenchymal progenitor cells spread and begin to regenerate with the interaction of several cells. And it goes to the hard callus stage, and due to the ossification in the cartilage, the gap in the soft tissue is replaced by the woven bone connecting the original COEX. And remodeling takes place, which means the recovery process, along with all the processes that occur at the fracture site over the years.
뼈는 mineralized connective tissue로서, 이들 tissue에 조골세포(osteoblasts), 골표면세포(bone lining cells), 골세포(osteocytes), 파골세포(osteoclasts) 등 다양한 세포들이 존재하여야만 만들어질 수 있다. 이러한 조골세포 및 골세포들은 뼈의 세포 외 기질(Extra Cellular Matrix, ECM)을 collagenous matrix로 만들고 나서 mineralization화 하기 위해 crystalline hydroxyapatite들을 골의 ECM에 축적한다. 뼈의 mineralized tissue ECM에 조골세포들은 다양한 상태로 존재하게 되며, apoptosis를 하거나 골표면세포, 골세포로 분화된다. 분화된 골세포들은 골 mineralized tissue ECM에서 항상성을 유지하기 위해 조골세포들과 파골세포들을 자극하는 기능을 한다. 그러므로 골세포로의 분화 유도는 골 재생을 위한 가장 효과적인 방법으로 이용된다. Bone is a mineralized connective tissue, which can be made only when various cells such as osteoblasts, bone lining cells, osteoblasts, and osteoclasts exist. These osteoblasts and bone cells make the extracellular matrix (ECM) of the bone into a collagenous matrix and then accumulate crystalline hydroxyapatite in the bone ECM to mineralize. Osteoblasts in the mineralized tissue ECM of the bone exist in a variety of states, apoptosis or differentiation into bone surface cells and bone cells. Differentiated bone cells function to stimulate osteoblasts and osteoclasts to maintain homeostasis in bone mineralized tissue ECM. Therefore, induction of differentiation into bone cells is used as the most effective method for bone regeneration.
한편, 골 세포로의 분화를 확인하기 위한 실험에서는 Saos-2 세포가 사용되고 있다. 골육종 세포(osteosarcoma cells)인 Saos-2 세포는 조골세포와 유사한(osteoblast-like) 성격을 가지고 있으며, 특히, osteogenic markers, ALP(alkaline phosphatase), OPN(osteopontin), COL1(Collagen type1), BMP(Bone morphogenetic protein) 등 골 분화와 형성에 중요한 유전자 및 단백질들의 발현이 나타나므로, 골세포 분화실험들에서 유전자들의 발현 양을 상대적으로 비교하기 위해서 많이 이용되고 있다.On the other hand, Saos-2 cells are used in experiments to confirm differentiation into bone cells. Saos-2 cells, which are osteosarcoma cells, have osteoblast-like characteristics, in particular, osteogenic markers, alkaline phosphatase (ALP), osteopontin (OPN), collagen type 1 (COL1), BMP ( Since the expression of genes and proteins important for bone differentiation and formation, such as bone morphogenetic protein), is widely used to compare the expression of genes in bone cell differentiation experiments.
이에, 본 발명자들은 골분화 유도 시기를 단축시킬 수 있는 새로운 골질환 치료제를 개발하기 위한 연구를 수행한 결과, 여러 한약재들을 조합한 추출물이 Saos-2 세포에서 조골세포에서 골세포로의 분화를 유도하고, 골형성 관련 유전자들 및 단백질의 발현을 증가시켜 골 분화를 촉진시킬 수 있음을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors conducted a study to develop a new therapeutic agent for bone disease that can shorten the induction period of bone differentiation, and extracts combining various herbal medicines induce differentiation from Saos-2 cells to osteoblasts to bone cells The present invention was completed by confirming that it is possible to promote bone differentiation by increasing the expression of genes and proteins related to bone formation.
본 발명자들은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향을 포함하는 접골공진단 추출물이 골분화를 유도하고 ALP, COL 및 OPN의 유전자 발현 및 BMP-2 단백질 발현을 증가시켜 골형성을 촉진하는 것을 확인함으로써 본 발명을 완성하였다.The present inventors suggest that the osteoclast diagnosis extract containing deer antler, Angelica, Cornus officinalis, safflower, grafted graft and aloe induces bone differentiation and promotes bone formation by increasing ALP, COL and OPN gene expression and BMP-2 protein expression. The present invention was completed by confirming.
따라서, 본 발명의 목적은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방, 개선 또는 치료용 약학적 조성물, 건강기능식품 조성물 및 건강식품 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition, a health functional food composition and a health food composition for the prevention, improvement or treatment of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, grafted tree and aloe extract as active ingredients Is to do.
상기 목적을 달성하기 위하여, In order to achieve the above object,
본 발명은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, phlegmon and aloe extract as active ingredients.
또한, 본 발명은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for the prevention or improvement of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, phlegmon and aloe extract as active ingredients.
아울러, 본 발명은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방 또는 개선용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for the prevention or improvement of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, grafted tree and aloe extract as an active ingredient.
본 발명에 따르면, 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향을 혼합하여 제조된 접골공진단 추출물은 조골세포에서 골세포로의 분화를 유도하고, 골형성(osteogenesis) 관련 ALP, OPN 및 COL1 유전자들의 발현 및 BMP-2 단백질 발현을 촉진하며, 골형성 및 골세포 분화를 촉진하는 효과를 나타냄으로써, 골 관련 질환의 예방 또는 치료에 유용하게 사용될 수 있다.According to the present invention, antler, Angelica, Cornus officinalis, safflower, a phalaenopsis co-producing extract prepared by mixing acupuncture and acupuncture induces differentiation from osteoblasts to osteoblasts, and ALP, OPN and COL1 genes related to osteogenesis It promotes the expression and BMP-2 protein expression, and by showing the effect of promoting bone formation and bone cell differentiation, it can be useful for the prevention or treatment of bone-related diseases.
도 1은 접골공진단 추출물 처리에 의한 조골세포-유사 세포주 Saos-2의 세포 생존율을 확인한 결과이다.
도 2는 접골공진단 추출물 처리에 의한 Saos-2의 세포 모양 변화를 확인한 결과이다.
도 3은 접골공진단 추출물 처리에 의한 Saos-2 세포에서의 골분화 관련 유전자(ALP, OPN 및 COL1)의 발현 변화를 확인한 결과이다; (a) ALP 유전자 Fold changes 측정 결과, (b) OPN 유전자 Fold changes 측정 결과 및 (c) COL1 유전자 Fold changes 측정 결과.
도 4는 접골공진단 추출물 또는 공진단 추출물 처리에 의한 Saos-2 세포에서의 골형성 관련 단백질 BMP-2의 발현을 확인한 결과이다.1 is a result confirming the cell viability of the osteoblast-like cell line Saos-2 by treatment with the osteoclast diagnosis extract.
Figure 2 is a result of confirming the change in cell shape of Saos-2 by treatment with the osteoclast diagnosis extract.
3 is a result of confirming the expression change of bone differentiation related genes (ALP, OPN and COL1) in Saos-2 cells by treatment with the osteoclast diagnosis extract; (a) ALP gene Fold changes measurement result, (b) OPN gene Fold changes measurement result and (c) COL1 gene Fold changes measurement result.
4 is a result of confirming the expression of BMP-2, a bone-forming protein, in Saos-2 cells by treatment with a grafting cavity extract or a resonating stage extract.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
골 관련 질환 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating bone-related diseases
본 발명은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, phlegmon and aloe extract as active ingredients.
본 발명의 상기 녹용(Cervi Parvum Cornu)은 사슴과 사슴속(Cervidae)에 속한 매화록(Cervus nippon Temminck), 마록(C. elaphus Linne) 또는 대록(C. canadensis Erxleben)의 숫사슴의 털이 밀생하고 아직 골질화되지 않았거나 약간 골질화 된 어린 뿔을 자른 다음 말린 것이다. 동의보감에서 '녹용은 오장을 강건하게 하고 음위(정력)을 보충해주며 허약체질 면역력 강화에 도움이 된다' 라고 전해오고 있다. 또한, 녹용은 조혈 작용과 혈행 개선 작용을 하며, 혈중 콜레스테롤을 낮춰 동맥경화증 예방과 심혈관 질환을 예방한다.The antler (Cervi Parvum Cornu) of the present invention is a deer and deer (Cervidae) belonging to the plum tree (Cervus nippon Temminck), Marok (C. elaphus Linne) or Daerok (C. canadensis Erxleben) male hair Young horns that have not yet been boned or slightly boned are cut and dried. In Donguibogam, it has been said, 'Anti-yellowing strengthens the intestine, replenishes the sound (energy), and helps strengthen the weakened body immunity.' In addition, antler acts to improve hematopoiesis and blood circulation, and lowers cholesterol in the blood to prevent arteriosclerosis and prevent cardiovascular disease.
본 발명의 상기 당귀(Angelicae gigantis Radix)는 미나리과(Umbelliferae)에 속하는 당귀(Angelica gigas N.)의 뿌리이다. 당귀는 동의치료에서 진정약, 강장약으로 빈혈증, 월경불순에 사용하며, 자궁에 대하여 쌍향성 조절작용이 있어 자궁의 근육수축을 강화시킬 수도 있고 이완시킬 수도 있다. 산후회복, 보혈, 양혈의 중요한 약재이며, 기타 부인병에 뛰어난 효과가 있다. 그리고 대장균, 디푸스균 등에 대하여 억제작용을 하는 것으로 알려져 있다.The Angelica (Angelicae gigantis Radix) of the present invention is the root of the Angelica gigas N. belonging to the family Apiaceae (Umbelliferae). Angelica is used as an sedative and tonic in the treatment of anemia, and is used for anemia and dysmenorrhea. It also has a bidirectional control effect on the uterus, which can strengthen or relax muscle contractions in the uterus. It is an important medicine for postpartum recovery, blood and sheep blood, and has excellent effects on other gynecological diseases. In addition, it is known to have an inhibitory effect on Escherichia coli and Dipus bacteria.
본 발명의 상기 산수유(Corni fructus)는 층층나무과에 속하는 산수유나무(Cornus officinalis)의 과육으로, 가을에 성숙한 붉은색 열매의 씨를 제거한 건조한 과육을 산수유라 하며, 예로부터 우리나라를 비롯하여 중국과 일본 등에서 중요한 한약재로 많이 사용되어 왔다. 산수유는 그 맛이 시고 성질은 따뜻하며, 다뇨증, 요통, 이명 및 폐결핵 등의 치료제로 사용되어 왔으며, 그 과실은 자양, 강장, 음위 및 이조에 약효가 있고, 간경화와 신경에 좋고, 이뇨작용, 혈압강하작용, 항암 및 항균 작용 등의 약리작용이 있다고 한방자료에 기록되어 있다. The Cornus fructus of the present invention is a flesh of the Cornus officinalis belonging to the Dogwoodaceae family, and the dried flesh with the seeds of mature red fruits removed in the fall is called Cornus officinalis, and is important in Korea, China and Japan since ancient times. It has been used a lot as an herbal medicine. Cornus officinalis tastes sour and warm, and has been used as a treatment for polyuria, low back pain, tinnitus and pulmonary tuberculosis, and its fruit has medicinal effects on nourishment, tonicity, sound, and irritation, good for cirrhosis and nerves, diuretic action It has been documented in herbal data that it has pharmacological effects such as lowering blood pressure, anti-cancer and antibacterial effects.
본 발명의 상기 홍화자(Carthami Semen)는 홍화(Carthamus tinctorius L.)의 씨로 잇꽃의 종자가 성숙한 여름철에 채취하고 볕에 건조하여 약제로 쓰는데, 성질이 따뜻하고 맛이 달다. 홍화자에 대한 한방의 기록은 홍화의 약능과 같은 이혈작용에 관하여 알려져 있으며, 최근에는 관상 동맥 확장 및 혈압을 낮추는 작용이 있으며 고콜레스테롤혈증을 향상시키고 혈전을 치료하는 효과가 있다고 보고된 바 있고, 미백 및 피지분비조절 효과가 있는 것으로도 알려져 있다.The safflower seed (Carthami Semen) of the present invention is a seed of safflower (Carthamus tinctorius L.). The seeds of safflower are collected in the mature summer season and dried in the sun to be used as a medicament. The properties are warm and sweet. Oriental medicine records for safflowerers are known for their hemorrhagic effects such as safflower's medicinal properties, and recently have been reported to have coronary artery expansion and blood pressure lowering effects and to improve hypercholesterolemia and to treat blood clots. And sebum secretion control effect.
본 발명의 상기 접골목(Sambuci Lignum)은 인동과 식물인 딱총나무(Sambucus williamsii) 또는 동속 근연식물의 줄기 및 가지를 말린 것이다. 한방과 민간에서는 신경통, 발한, 이뇨, 폐염, 수종, 해열, 신경염 등에 사용하는 약재로 알려져 있다. 접골목은 혈액순환을 좋게 하며 통증을 멎게 하는 효능이 있고, 두드러기 및 피부 가려움증에도 효과가 있는 것으로 보고된 바 있다.The grafted alley (Sambuci Lignum) of the present invention is a dried plant stem and branch of a common plant, such as the stalk (Sambucus williamsii). It is known as a medicine used for neuralgia, sweating, diuresis, pneumonia, hydrocephalus, fever, and neuritis in oriental medicine and folk. It has been reported that the allograft has an effect of improving blood circulation and relieving pain, and is also effective for hives and itching of the skin.
본 발명의 상기 침향(Aquilariae Resinatum Lignum)은 침향 나무(Aquilaria agallocha Roxburgh)에 천연적으로 분비된 수지가 침착하여 침향나무의 심재부위에 조직학적으로 단단한 덩어리 모양을 이루고 있는 부분을 말한다. 침향은 한국, 중국, 일본 등에서는 예로부터 피부 소양, 복통, 천식 등의 치료에 사용되어 왔으며 진정, 건위, 소화불량, 식욕부진 등에 전통약재로 사용되고 있다. 최근 연구에서 항알러지, 항균, 항산화 등의 효능이 보고되었다.The acupuncture (Aquilariae Resinatum Lignum) of the present invention refers to a portion that forms a histologically hard lump on the core of the acupuncture tree by depositing a resin naturally secreted in the acupuncture tree (Aquilaria agallocha Roxburgh). Acupuncture has been used in Korea, China, and Japan for the treatment of skin irritation, abdominal pain and asthma, and has been used as a traditional medicine for sedation, dry stomach, indigestion, and anorexia. In recent studies, the efficacy of anti-allergic, antibacterial, and antioxidant properties has been reported.
본 발명에서, 상기 추출물은 침향 1.0 중량부를 기준으로 녹용 6 내지 10 중량부, 당귀 6 내지 10 중량부, 산수유 6 내지 10 중량부, 홍화자 0.1 내지 4.0 중량부 및 접골목 0.1 내지 4.0 중량부를 혼합한 혼합물을 추출한 것일 수 있다. 바람직하게는, 침향 1.0 중량부를 기준으로 녹용 7 내지 9 중량부, 당귀 7 내지 9 중량부, 산수유 7 내지 9 중량부, 홍화자 1 내지 3 중량부 및 접골목 1 내지 3 중량부를 혼합한 혼합물을 추출한 것일 수 있다. 본 발명의 일실시예에서는, 침향 1 중량부를 기준으로 녹용 8 중량부, 당귀 8 중량부, 산수유 8 중량부, 홍화자 2 중량부, 접골목 2 중량부를 혼합하여 환 형태로 제조한 것을 '접골공진단(Jeobgolgongjindan)'이라 명명하였고, 상기 접골공진단을 추출하여 접골공진단 추출물을 제조하였다. 상기 접골공진단은 녹용, 당귀, 산수유 및 침향으로 제조되는 것으로 알려진 공진단에 홍화자 및 접골목을 더 혼합한 것을 의미하는 것일 수 있다.In the present invention, the extract is a mixture of 6 to 10 parts by weight of antler, 6 to 10 parts by weight of Angelica, 6 to 10 parts by weight of Cornus officinalis, 0.1 to 4.0 parts by weight of safflower and 0.1 to 4.0 parts by weight of grafted tree based on 1.0 parts by weight of aloe. The mixture may be extracted. Preferably, a mixture of 7 to 9 parts by weight of antler antler, 7 to 9 parts by weight of Angelica, 7 to 9 parts by weight of Cornus officinalis, 1 to 3 parts by weight of safflower and 1 to 3 parts by weight of grafted tree is extracted based on 1.0 parts by weight of acupuncture. May be In one embodiment of the present invention, based on 1 part by weight of aloe, 8 parts by weight of antler, 8 parts by weight of Angelica, 8 parts by weight of Cornus officinalis, 2 parts by weight of safflower, and 2 parts by weight of grafted wood are prepared in a ring form. (Jeobgolgongjindan) ', and the extract of the osteoclast was extracted to prepare an extract of the osteoclast. The grafting cavity diagnosis may mean that a safflower and a grafting tree are further mixed with a resonating stage known to be made of antler, Angelica, Cornus and aloe.
본 발명에서 사용되는 용어 "추출물(extract)"은 생약을 적절한 침출액으로 짜내고 침출액을 증발시켜 농축한 제제를 의미하는 것으로, 이에 제한되지는 않으나, 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 이들의 조정제물 또는 정제물일 수 있다. 상기 추출물은 통상의 기술분야에 공지된 일반적인 추출방법, 분리 및 정제방법을 이용하여 제조할 수 있다. 상기 추출방법으로는, 이에 제한되지는 않으나, 바람직하게 열탕 추출, 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등의 방법을 사용할 수 있다.As used in the present invention, the term "extract" refers to a formulation in which a crude drug is squeezed with an appropriate leach solution and concentrated by evaporating the leach solution, but is not limited thereto, but an extract obtained by an extraction treatment, a dilution or concentrate of the extract, It may be a dried product obtained by drying the extract, a crude product or a purified product thereof. The extract may be prepared using general extraction methods, separation and purification methods known in the art. The extraction method is not limited to this, but may preferably use methods such as hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction.
본 발명에서, 상기 추출용매는 물, 유기용매 또는 이들의 혼합용매 등을 사용할 수 있으며, 상기 유기용매는 탄소수 1 내지 4의 알코올이나, 에틸아세테이트 또는 아세톤 등의 극성용매, 헥산 또는 디크로로메탄의 비극성용매 또는 이들의 혼합용매를 사용할 수 있다. 또한, 바람직하게는 물, 탄소수 1 내지 탄소수 4의 알코올 또는 이들의 혼합용매를 사용할 수 있으며, 보다 바람직하게는 에탄올을 사용할 수 있다. 본 발명의 일실시예에서는 상기 용매로서 100% 에탄올을 이용하여 추출한 뒤 감압 농축한 추출물을 제조하였다.In the present invention, the extraction solvent may be water, an organic solvent or a mixed solvent thereof, and the organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dicroromethane. Non-polar solvents or mixed solvents of these may be used. In addition, preferably, water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof may be used, and more preferably, ethanol may be used. In one embodiment of the present invention, an extract using 100% ethanol as the solvent and then concentrated under reduced pressure was prepared.
본 발명에 따른 상기 추출물은 골형성(osteogenesis) 관련 유전자와 단백질들의 발현을 촉진시키는 것을 특징으로 할 수 있고, 조골세포(osteoblast) 및 골세포(osteocyte)로의 분화를 촉진시키는 것을 특징으로 할 수 있다. The extract according to the present invention may be characterized in that it promotes the expression of genes and proteins related to osteogenesis and may be characterized by promoting differentiation into osteoblasts and osteoblasts. .
본 발명의 일실시예에 있어서, 상기 골 질환의 예방 또는 치료는 골형성 관련 유전자와 단백질들의 발현 증가 및 조골세포에서 골세포로의 분화 촉진으로 인한 골 재생 및 골 분화 유도를 통해 달성될 수 있다.In one embodiment of the present invention, the prevention or treatment of the bone disease may be achieved through bone regeneration and bone differentiation induction due to increased expression of genes and proteins related to bone formation and differentiation from osteoblasts to bone cells. .
본 발명의 일 실시예에서는 접골공진단 추출물 처리에 의해 조골세포 유사 세포 Saos-2가 조골세포 또는 골세포로의 분화가 유도됨을 확인하였으며(실험예 2 및 도 2 참조), 골형성 관련 유전자 ALP, COL 및 OPN의 발현이 증가하는 것을 확인하였고(실험예 3 및 도 3 참조), 골형성 관련 단백질 BMP-2 발현이 증가하는 것을 확인함으로써(실험예 4 및 도 4 참조), 본 발명의 접골공진단 추출물이 골형성 관련 유전자와 단백질들의 발현을 촉진시키고, 골형성 및 골세포 분화를 촉진시킴으로써, 골 관련 질환을 예방 또는 치료할 수 있음을 확인하였다.In one embodiment of the present invention, it was confirmed that differentiation of osteoblast-like cells, Saos-2, into osteoblasts or osteoblasts is induced by treatment of the osteoblastic diagnostic extract (see Experimental Example 2 and FIG. 2), the bone formation-related gene ALP, It was confirmed that the expression of COL and OPN increased (see Experimental Examples 3 and 3), and by confirming that the expression of BMP-2 related to bone formation was increased (see Experimental Examples 4 and 4), the osteoclast diagnosis of the present invention It was confirmed that the extract can prevent or treat bone-related diseases by promoting the expression of genes and proteins related to bone formation and promoting bone formation and bone cell differentiation.
본 발명에서, 상기 골 관련 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손, 고관절 감소증, 골관절염, 퇴행성 관절염, 무릎관절증, 고관절증, 전신 홍반루푸스(SLE), 척추관절염, 류마티스성 다발성 근육통, 강직성 척추염, 라이터 증후군, 건선성 관절병증, 장병성 관절염, 류마티스 관절염, 신경병관절병증, 급성 류마티스 열, 통풍, 연골석회화증, 치조골 파괴에 의한 치주질환, 염증성 치조골 흡수질환, 염증성 뼈 흡수질환, 칼슘 수산화인회석 결정 침전 질환 및 라임 질환으로 구성된 군으로부터 선택되는 어느 하나일 수 있으며, 바람직하게는 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손, 고관절 감소증 등을 포함하는 대사성 골 질환 또는 정형외과적 골 질환일 수 있으며, 골다공증인 것이 바람직하다.In the present invention, the bone-related diseases are osteoporosis, osteogenic dysplasia, osteomalacia, osteopenia, fractures, bone defects, hip reduction, osteoarthritis, degenerative arthritis, knee joints, hip joints, systemic lupus erythematosus (SLE), spondylitis, rheumatism Sexual multiple myalgia, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, enteric arthritis, rheumatoid arthritis, neuropathic arthrosis, acute rheumatic fever, gout, chondrocalcification, periodontal disease caused by alveolar bone destruction, inflammatory alveolar absorption disease, inflammatory bone Absorption disease, calcium hydroxide apatite crystal precipitation disease and may be any one selected from the group consisting of Lyme disease, and preferably, osteoporosis, bone dysplasia, osteomalacia, osteopenia, fracture, bone defect, hip joint reduction, etc. It may be a bone disease or an orthopedic bone disease, and is preferably osteoporosis.
상기 "대사성 골 질환"은 파골세포의 과다한 생성 또는 활성으로 인해 나타나는 상태 또는 질병을 의미하는 것으로, 골량 저하 질환을 포함할 수 있다. 상기 골량 저하 질환이란 골밀도의 저하, 골조직의 연화 등의 증상을 수반하는 골량의 저하가 나타나는 상태 또는 질환을 의미한다.The "metabolic bone disease" refers to a condition or a disease that occurs due to excessive production or activity of osteoclasts, and may include a disease that decreases bone mass. The bone loss disease refers to a condition or disease in which a decrease in bone mass accompanied by symptoms such as a decrease in bone density and softening of bone tissue occurs.
상기 "치주질환"은 치조골 또는 상기 치조골을 치은에 지지 및 결합시키는 치조골 주변조직에 영향을 미치는 질환을 포괄적으로 의미하며, 더욱 구체적으로, 상기 치조골 또는 치조골 주변조직에 감염된 치주균에 의하여 분비되는 병독성 대사산물에 의하여 치아의 구조와 치은의 세포조직이 손상되고, 치은조직의 면역력을 약화시키는 병변이 발생되며, 치주균이 증식하여 병변 부위가 반복적으로 확장되는 질환을 의미할 수 있다.The "periodic disease" refers to a disease that affects the alveolar bone or the surrounding tissue of the alveolar bone that supports and binds the alveolar bone to the gingiva, and more specifically, the virulence secreted by the periodontal bacteria infected with the alveolar bone or the surrounding alveolar tissue By metabolites, the structure of the teeth and the tissues of the gingiva are damaged, lesions that weaken the immunity of the gingival tissue are generated, and the periodontal bacteria multiply and the lesion area may be repeatedly expanded.
본 발명에서 사용되는 용어 "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 골 관련 질환의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미하고, "치료"란 상기 약학적 조성물의 투여에 의해 골 관련 질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.The term "prevention" as used in the present invention means any action that inhibits or delays the development, spread and recurrence of bone-related diseases by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to the pharmaceutical composition. Refers to all acts of improving or beneficially altering the symptoms of a suspected and developing individual of bone-related diseases by administration. If the person having ordinary knowledge in the technical field to which the present invention belongs, can refer to the data presented by the Korean Medical Association and the like to know the exact criteria of the disease in which the composition of the present invention is effective, and to determine the degree of improvement, improvement and treatment. will be.
본 발명의 추출물은 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있으며, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다. The extract of the present invention may be administered in various dosage forms, oral and parenteral, during clinical administration, and when formulated, a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are usually used, can be used. Is manufactured.
상기 경구투여를 위한 고형 제제에는 정제, 환자, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 본 발명의 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스(sucrose), 락토오스(lactose) 또는 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 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, 보존제 등이 포함될 수 있다.The solid preparation for oral administration includes tablets, patients, powders, granules, capsules, troches, etc. These solid preparations include at least one excipient, for example, starch, calcium carbonate, in one or more extracts of the present invention. It is prepared by mixing sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate talc are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions or syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. are included. Can be.
상기 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 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, 글리세롤, 젤라틴 등이 사용될 수 있다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspension solutions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.
본 발명의 일실시예에 따르면, 본 발명의 추출물은 환 형태로 제조하여 경구 투여되는 것일 수 있다(실시예 1 참조).According to an embodiment of the present invention, the extract of the present invention may be prepared in a ring form and administered orally (see Example 1).
본 발명의 추출물의 인체에 대한 효과적인 투여량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 일반적으로 약 0.001-100 mg/kg/일이며, 바람직하게는 0.01-35 mg/kg/일이다. 몸무게가 70㎏인 성인 환자를 기준으로 할 때, 일반적으로 0.07-7000 mg/일이며, 바람직하게는 0.7-2500 ㎎/일이며, 의사 또는 약사의 판단에 따라 일정시간 간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다.The effective dose to the human body of the extract of the present invention may vary depending on the patient's age, weight, sex, dosage form, health status and disease level, and is generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, and once a day at regular time intervals according to the judgment of a doctor or pharmacist It can also be administered in multiple doses.
또한, 본 발명의 상기 약학적 조성물은 골 관련 질환의 예방 또는 치료를 위하여, 인공골, 골 고정재, 골 대체제, 골 수복재, 골 충진재 또는 골 이식재에 적용되기 용이한 형태로 제조될 수 있다. In addition, the pharmaceutical composition of the present invention may be prepared in a form that is easy to apply to artificial bones, bone fixatives, bone substitutes, bone repair materials, bone fillers, or bone grafts for the prevention or treatment of bone-related diseases.
예컨대, 상기 골 이식재나 골 대체체는 협의로는 골 조직 내의 공간을 충진하기 위하여 사용될 수도 있고, 광의로는 뼈나 치아의 일부를 대체하는데 사용될 수 있으므로, 상기 약학적 조성물은 압착, 압축, 가압접촉, 패킹, 압박, 굳힘 등의 방법을 사용하여 퍼티, 페이스트, 주형 가능한 스트립, 블록, 칩 등의 형태로 사용될 수 있고, 화학적 첨가물을 이용하여, 겔, 과립, 페이스트, 정제, 펠렛 등의 형태로 제형화하여 사용될 수 있으며, 분말 형태로도 사용될 수 있으나, 이에 한정되지는 않는다. For example, the bone graft material or the bone substitute may be used to fill a space in the bone tissue in a narrow sense, or in a broad sense, it may be used to replace a part of a bone or a tooth, so the pharmaceutical composition is compressed, compressed, or pressed in contact. It can be used in the form of putty, paste, moldable strip, block, chip, etc. using packing, pressing, hardening, etc., and using chemical additives, in the form of gels, granules, pastes, tablets, pellets, etc. It may be used in a formulation, may be used in powder form, but is not limited thereto.
또한, 본 발명의 약학적 조성물은 골 재생 또는 골 이식 보조제로 사용될 수 있다. 상기 골재생 또는 골이식 보조제는 본 발명의 추출물을 뼈의 세포 외 기질(Extra Cellular Matrix, ECM)을 재생할 수 있도록 하는 osteoblasts, osteocytes로의 조골세포와 골세포로의 분화를 유도하는 growth factors 대체제로 사용되는 것일 수 있고, scaffold나 membrane에 encapsulation하여 골절 부위에 부착하거나 implant 되어 전달체에 의해 전달되어 이식되는 형태로 사용되는 것일 수 있다.In addition, the pharmaceutical composition of the present invention can be used as a bone regeneration or bone graft adjuvant. The bone regeneration or bone graft supplements use the extract of the present invention as a replacement agent for growth factors that induce osteoblasts and osteoblast differentiation into osteoblasts and osteocytes that can regenerate the extracellular matrix (ECM) of bone. It may be, it may be used in the form of being attached to the fracture site by encapsulation to a scaffold or membrane, or implanted and delivered by a carrier.
건강기능성식품Health Functional Food 및 건강식품 조성물 And health food composition
본 발명은 녹용, 당귀, 산수유, 홍화자, 접골목 및 침향 추출물을 유효성분으로 포함하는 골 관련 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of bone-related diseases, including deer antler, Angelica, Cornus officinalis, safflower, phlegmon and aloe extract as active ingredients.
본 발명에서 사용되는 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 '기능성'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 통상의 기술분야에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 통상의 기술분야에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한, 상기 건강기능식품의 제형은 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 골 관련 질환 치료제의 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using ingredients or ingredients having useful functional properties for the human body. Here, the term 'functional' refers to obtaining a useful effect for health use, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the general technical field, and may be prepared by adding raw materials and components conventionally added in the general technical field. In addition, the formulation of the health functional food can be prepared without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material, unlike general drugs, and has excellent portability, and the present invention The health functional foods of can be consumed as supplements to enhance the effectiveness of treatments for bone-related diseases.
본 발명의 조성물을 건강기능식품 조성물 또는 건강식품 조성물로 사용하는 경우, 상기 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 상기 조성물은 유효성분 이외에 식품학적으로 허용 가능한 식품보조첨가제를 포함할 수 있으며, 유효성분의 혼합량은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the composition of the present invention is used as a health functional food composition or a health food composition, the extract may be added as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method. The composition may include a food additive that is food-acceptable in addition to the active ingredient, and the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).
본 발명에서 사용되는 용어 "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품 또는 건강식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.The term "food supplement additive" used in the present invention means a component that can be supplementally added to food, and is added to prepare a health functional food or health food of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food supplements include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like, but the types of food supplements of the present invention are not limited by the examples.
또한, 본 발명의 조성물이 사용될 수 있는 건강기능식품 또는 건강식품의 종류에는 제한이 없다. 아울러 본 발명의 추출물을 활성성분으로 포함하는 조성물은 당업자의 선택에 따라 건강기능식품에 함유될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림 류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다.In addition, there are no restrictions on the types of health functional foods or health foods to which the composition of the present invention can be used. In addition, the composition containing the extract of the present invention as an active ingredient may be prepared by mixing a known additive with other suitable auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gums, ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and There are vitamin complexes, etc., and can be prepared by adding the extract according to the present invention as a main component, adding juice, tea, jelly, and juice.
본 발명의 상기 추출물은 천연 식물을 원료로 하므로 약학적 조성물 또는 식품 조성물로 사용할 경우에도 일반적인 합성 화합물에 비하여 부작용이 덜할 수 있으므로, 안전하게 약학적 조성물, 건강기능식품 및 건강식품에 포함되어 유용하게 사용될 수 있다.Since the extract of the present invention uses natural plants as raw materials, even when used as a pharmaceutical composition or a food composition, side effects may be less than that of a general synthetic compound, so it is safely contained in pharmaceutical compositions, functional foods, and health foods to be usefully used. Can.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereto.
<< 제조예Manufacturing example 1> 1> 공진단Resonant stage 및 And 공진단Resonant stage 추출물의 제조 Preparation of extract
녹용, 당귀 및 산수유 각 16g, 침향 2g을 혼합하여 100mesh 이하의 크기로 분말화 한 뒤, 자동제환기를 이용하여 제환하여 공진단을 제조하였다.Deer antler, Angelica and Cornus officinalis, each mixed with 16g and 2g of aloe, powdered to a size of 100 mesh or less, and then exchanged using an automatic changer to prepare a resonant stage.
제조된 상기 공진단은 100% 에탄올을 용매로 이용하여 20 내지 25℃의 온도에서 일주일 동안 추출한 후, -70 내지 -90℃의 온도에서 24 내지 48시간동안 감압농축하여 공진단 추출물을 제조하였다.The prepared resonant stage was extracted by using 100% ethanol as a solvent at a temperature of 20 to 25 ° C for a week, and then concentrated under reduced pressure at a temperature of -70 to -90 ° C for 24 to 48 hours to prepare a resonant stage extract.
<< 실시예Example 1> 1> 접골공진단Osteopathic Diagnosis 및 And 접골공진단Osteopathic Diagnosis 추출물의 제조 Preparation of extract
녹용, 당귀 및 산수유 각 16g, 홍화자 및 접골목 각 4g 및 침향 2g을 혼합하여 100mesh 이하의 크기로 분말화한 뒤, 자동제환기를 이용하여 제환하여 접골공진단을 제조하였다.After antler, Angelica and Cornus officinalis each 16g, safflower and grafted tree each 4g and aloe 2g were mixed and powdered to a size of 100 mesh or less, and then exchanged using an automatic ventilator to prepare a grafted cavity diagnosis.
제조된 상기 접골공진단은 100% 에탄올을 용매로 이용하여 20 내지 25℃의 온도에서 일주일 동안 추출한 후, -70 내지 -90℃의 온도에서 48시간동안 감압농축하여 접골공진단 추출물을 제조하였다.The prepared osteoclast was extracted by using 100% ethanol as a solvent at a temperature of 20 to 25 ° C for a week, and then concentrated under reduced pressure at a temperature of -70 to -90 ° C for 48 hours to prepare a phylogenetic co-existence extract.
<< 실험예Experimental Example 1> 1> 접골공진단의Osteopathic diagnosis SaosSaos -2 세포에 대한 세포 독성 확인-2 Cytotoxicity check for cells
상기 실시예 1에서 제조한 접골공진단 추출물의 골세포에 대한 독성 및 골세포분화에 미치는 영향을 확인하기 위하여, 조골세포-유사(osteoblast-like) 세포인 Saos-2(한국세포주은행)를 100㎕씩 96-웰 플레이트에 1×104 세포/웰이 되도록 분주하고, 37℃의 5% CO2 인큐베이터(MCO-15AC, Sanyo, Osaka, Japan)로 24시간 배양하였다. 24시간 배양 후, 상기 실시예 1에서 제조한 접골공진단 추출물을 12.5, 25, 50, 100 및 200㎍/mL 농도로 100㎕씩 처리하고, 24시간 동안 인큐베이션 하였다. 인큐베이션한 세포에 EZ-Cytox 시약(DoGen)을 배지의 1/10인 10㎕ 처리한 다음, 클린밴치의 불을 끄고 호일을 세포 플레이트에 씌우고 2시간 동안 인큐베이션하였고, 그 후, 마이크로플레이트 리더기(Bio-rad Model 680) 450nm에서의 흡광도를 측정하여 세포 생존율을 확인하였다.In order to confirm the effect of the osteoclast diagnosis extract prepared in Example 1 on the toxicity to bone cells and the differentiation of osteoblasts, 100 μl of osteoblast-like cells, Saos-2 (Korea Cell Line Bank), The cells were dispensed to 1 × 10 4 cells / well in each 96-well plate, and cultured for 24 hours in a 37% 5% CO 2 incubator (MCO-15AC, Sanyo, Osaka, Japan). After incubation for 24 hours, the osteoblastic diagnostic extract prepared in Example 1 was treated with 100 μl at concentrations of 12.5, 25, 50, 100 and 200 μg / mL, and incubated for 24 hours. The incubated cells were treated with 10 μl of EZ-Cytox reagent (DoGen), which is 1/10 of the medium, and then the fire was turned off and the foil was put on a cell plate and incubated for 2 hours, after which the microplate reader (Bio -rad Model 680) The cell viability was confirmed by measuring absorbance at 450 nm.
그 결과, 접골공진단 추출물은 200㎍/mL 농도 이하의 범위에서 Saos-2 세포에 독성이 없는 것으로 나타나, 안전한 약물임을 확인하였고, Saos-2 세포 생존율을 유의하게 증가시키는 것으로 나타나, 접골공진단 추출물 처리에 의해 Saos-2 세포의 활성과 증식이 일어나는 것을 확인하였다(도 1).As a result, the osteoclast diagnosis extract was found to be non-toxic to Saos-2 cells in a range of 200 μg / mL or less, confirming that it is a safe drug, and it was shown to significantly increase the survival rate of Saos-2 cells, and treated with the osteoclast diagnosis extract By confirming that the activity and proliferation of Saos-2 cells occur (Fig. 1).
<< 실험예Experimental Example 2> 2> 접골공진단에Osteopathic diagnosis 의한 조골세포에서 골세포로의 분화 유도 효과 확인 Check the effect of induction of differentiation from osteoblasts to osteoblasts
상기 실시예 1에서 제조한 접골공진단 추출물이 조골세포-유사 세포인 Saos-2 세포들에서의 조골세포(osteoblast)에서 골세포로(osteocyte)의 분화에 영향을 미치는지 알아보기 위하여, Saos-2 세포들에 접골공진단 추출물을 처리한 후, 세포의 모양 변화를 확인하였다. 구체적으로, Saos-2 세포들에 접골공진단 추출물을 25, 50 및 100㎍/mL 농도로 처리한 후, 0, 1, 2 및 14일째의 세포의 변화되는 모양을 현미경(ZEISS, Axio, Observer)으로 관측하였다. 이때, 배지는 2~3일마다 갈아주었다.In order to determine whether the osteoblastic diagnostic extract prepared in Example 1 affects the differentiation of osteoblasts to osteoblasts in osteoblast-like cells, Saos-2 cells, Saos-2 cells After treating the grafting cavity diagnostic extract on the field, the shape change of the cells was confirmed. Specifically, after treating the osteoclast extract with Saos-2 cells at a concentration of 25, 50, and 100 µg / mL, the changing shape of the cells at 0, 1, 2, and 14 days under a microscope (ZEISS, Axio, Observer) Was observed. At this time, the medium was changed every 2-3 days.
그 결과, 도 2에 나타난 바와 같이, 접골공진단 추출물 처리 후 1일부터 Saos-2 세포의 모양이 조골세포(osteoblast)의 특징인 방추형(spindle shape)으로 서서히 변화가 일어나는 것으로 나타났고, 접골공진단 추출물 처리 14일 후에는 세포들이 골세포(osteocyte)의 특징인 별모양(stellate shape)으로 변화되는 것으로 나타났다. 따라서, 접골공진단 추출물 처리에 의해 Saos-2 세포가 조골세포에서 골세포로의 분화가 유도됨을 확인하였다.As a result, as shown in FIG. 2, it was found that the shape of Saos-2 cells gradually changed to a spindle shape, characteristic of osteoblasts, from
<실험예 3> 접골공진단에 의한 골형성 관련 유전자 발현 증가 효과 확인<Experimental Example 3> Confirmation of the effect of increasing gene expression related to bone formation by osteoclast diagnosis
접골공진단 추출물 처리에 따른 조골세포-유사 세포주 Saos-2 세포들에서의 ALP, COL 및 OPN의 유전자 발현 정도를 알아보았다. 상기 유전자들은 골형성(osteogenesis)을 촉진하는 것으로 알려진 유전자이다.The gene expression level of ALP, COL and OPN in osteoblast-like cell line Saos-2 cells according to the treatment of the osteoconjugation diagnosis extract was examined. The genes are genes known to promote osteogenesis.
먼저, Saos-2 세포들을 6-웰 플레이트에 2×105 세포/웰로 분주하고, 접골공진단 추출물을 50ug/mL 농도로 처리 한 다음, 7 및 14일 뒤에 원래 있던 배지를 모두 버리고 세포들에 트리졸(Trizol) 1mL를 넣고 lysis시켰다. 5분 동안 RT(37℃)에서 두고, 5분 동안 최대의 속도로 센트리퓨즈를 돌린 다음, 세포찌거기는 버리고 상층액은 새 튜브로 옮겨 상층액과 클로로포롬을 섞어 다시 센트리퓨즈를 돌려서 상층액을 분리하였다. 분리된 상층액과 70% 에탄올을 섞은 용액을 Qiagen kit의 column tube를 이용해서 prep을 실시하였다. Kit 내에 버퍼를 이용해서 워싱과 RNase free water를 넣고 purified 된 RNA를 회수하였고, 회수된 RNA의 1μg을 Prime Script Reverse Transcriptase with Prime ScriptTM 1st strand cDNA Synthesis Kit(Takara, Otsu, Shiga, Japan)을 이용해서 cDNA로 합성하였다. First, Saos-2 cells were dispensed into a 6-well plate at 2 × 10 5 cells / well, treated with the osteoclast extract at a concentration of 50 ug / mL, and after 7 and 14 days, the original medium was discarded and the cells were added to the cells. 1 ml of zol (Trizol) was added and lysis was performed. Leave at RT (37 ° C) for 5 minutes, spin the centrifuge at maximum speed for 5 minutes, discard the cell debris, transfer the supernatant to a new tube, mix the supernatant and chloroform, and turn the centrifuge again to turn the supernatant. Separated. The solution of the separated supernatant and 70% ethanol was prep using a column tube of Qiagen kit. Purified RNA was collected by washing and RNase free water using a buffer in the kit, and 1 μg of the recovered RNA was collected from Prime Script Reverse Transcriptase with
상기 합성된 cDNA 0.5 ㎍, 2.5 unit의 Taq 중합효소(TaKaRa TaqTM, Takara, Japan), 1.5 mM dNTP, 1×완충액(10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1 % Triton X-100), 유전자 각각의 정방향(forward) 및 역방향(reverse) 프라이머쌍 20 pmol씩을 섞고 증류수로 총 부피를 10 ㎕로 맞춘 다음 기계(TaKaRa Biotechnology, Seoul, Korea)를 이용하여 PCR 반응을 실시하였다. The synthesized cDNA 0.5 μg, 2.5 units of Taq polymerase (TaKaRa Taq TM , Takara, Japan), 1.5 mM dNTP, 1 × buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1% Triton X-100) , Mixing 20 pmol of forward and reverse primer pairs for each gene, adjusting the total volume to 10 μl with distilled water, and then conducting PCR reaction using a machine (TaKaRa Biotechnology, Seoul, Korea).
PCR 반응에 이용한 각 프라이머의 시퀸스(sequence)는 하기 표 1에 나타낸 바와 같다. PCR 증폭은 stage1은 95℃에서 30초 동안 한 사이클 실시하였고, stage 2는 95℃ 5초 및 60℃ 10초 변성 과정을 40 사이클을 실시하였으며, stage3은 95℃ 15초, 60℃ 1분 및 95℃ 15초 실시하는 조건으로 수행하였다.The sequence of each primer used in the PCR reaction is shown in Table 1 below. For PCR amplification,
PCR의 결과분석은 접골공진단 추출물로 인한 골형성관련 유전자들의 발현의 차이를 비교하였다. 접골공진단 추출물을 처리하지 않은 대조군과 접골공진단 추출물을 처리한 실험군에서 발현차이를 비교 분석하는 fold change를 이용하였다.Analysis of the results of PCR compared the differences in expression of genes related to bone formation due to the extract of the osteoclast. A fold change was used to compare and analyze the expression difference between the control group not treated with the osteoclast diagnostic extract and the experimental group treated with the osteoclast diagnostic extract.
그 결과, 도 3에 나타난 바와 같이, 접골공진단 추출물 처리 14일 후에 Saos-2 세포들에서 골분화에 중요한 ALP, OPN 및 COL1 유전자들의 발현이 모두 증가된 것으로 나타났다. As a result, as shown in FIG. 3, after 14 days of treatment with the osteoclast extract, the expression of ALP, OPN and COL1 genes important for bone differentiation in Saos-2 cells was all increased.
따라서, 본 발명에 따른 접골공진단은 골형성(osteogenesis) 관련 유전자들의 발현을 촉진하며, 골형성 및 골세포 분화를 촉진함을 확인할 수 있었다.Accordingly, it was confirmed that the osteoclast diagnosis according to the present invention promotes the expression of genes related to osteogenesis and promotes bone formation and bone cell differentiation.
<< 실험예Experimental Example 4> 4> 접골공진단에Osteopathic diagnosis 의한 by 골형성Bone formation 단백질 발현 증가 효과 확인 Confirm the effect of increasing protein expression
접골공진단 추출물 처리에 따른 조골세포-유사 세포주 Saos-2 세포들에서의 골형성 단백질 BMP2의 발현 정도를 알아보았다. 상기 단백질은 골형성(osteogenesis)을 촉진하는 것으로 알려진 단백질이다.The expression level of osteogenic protein BMP2 in the osteoblast-like cell line Saos-2 cells according to the treatment of the osteoconjugation extract was examined. The protein is a protein known to promote bone formation.
Saos-2 세포들을 2×105 세포/웰로 계산하여 2㎖의 배양배지와 함께 6웰에 분주한 후 24시간 배양하였다. 다음날 안정적으로 세포가 부착한 것을 확인한 다음, 상기 실시예 1의 접골공진단 추출물과 제조예 1의 공진단 추출물을 각각 100㎍/㎖농도로 처리한 후, 0, 7 및 14일 동안 배양하였다. 0, 7 및 14일 각 회수 시기마다 배지를 모두 버리고, DPBS로 워싱 한 뒤, scarper를 이용해서 세포들을 회수하였다. 세포들로부터 원하는 단백질들을 분리해 내기 위해 lysis 과정을 실시하였다. 먼저, lysis buffer(RIPA buffer 20mM Tris-Hcl(pH 7.5), 150mM NaCl, 1mM NaEDTA, 1mM EGTA, 1% NP-40, 1% sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na3VO4 및 1㎍/㎖ leupeptin(Cell signaling)을 100㎕씩 각각 처리하였다. vortex를 이용하여 10분 간격으로 30분간 3번씩 세포를 파쇄하였고, 이 때, 세포들로부터 분리되어서 나오는 단백질들의 분해를 막기 위해 얼음에 박아두고 진행하였다. 각 샘플들을 15,520×g에서 15분동안 원심분리를 하여 세포찌꺼기와 단백질들을 분리하였다. 그 다음, western blot으로 단백질들의 크기에 따라 분리한 후, 원하는 단백질을 확인하기 위해, BMP-2(sc-137087) 1차 항체(anti-body)를 붙여서 24시간 처리하고 나서 TBST Buffer로 워싱하였다. 워싱은 15분마다 두 번 TBST Buffer를 갈아주는데 워싱 과정마다 동일하게 진행하였으며, 2차 항체는 1시간 처리하고 나서 다시 TBST Buffer로 워싱하였다. 분리 회수된 단백질들을 RC DCTM Protein Assay Kit II(protein assay kit, Bio-rad, USA)를 이용하여 정량하였다.Saos-2 cells were counted at 2 × 10 5 cells / well and dispensed into 6 wells with 2 ml of culture medium and cultured for 24 hours. The next day, after confirming that the cells were stably attached, the osteoclast diagnosis extract of Example 1 and the resonant stage extract of Preparation Example 1 were respectively treated at a concentration of 100 μg / ml, and then cultured for 0, 7 and 14 days. After each recovery period of 0, 7 and 14, the medium was discarded, washed with DPBS, and cells were recovered using a scarper. A lysis process was performed to separate the desired proteins from the cells. First, lysis buffer (RIPA buffer 20 mM Tris-Hcl (pH 7.5), 150 mM NaCl, 1 mM NaEDTA, 1 mM EGTA, 1% NP-40, 1% sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 And 100 μl of 1 μg / ml leupeptin (Cell signaling) was treated respectively. Cells were crushed three times for 30 minutes at 10-minute intervals using vortex, and at this time, they were placed in ice to prevent decomposition of proteins from cells. Each sample was centrifuged at 15,520 × g for 15 minutes to separate cell debris and proteins. Then, after separating according to the size of proteins by western blot, in order to confirm the desired protein, BMP-2 (sc-137087) primary antibody (anti-body) was attached and treated for 24 hours, and then washed with TBST buffer. . Washing changes TBST buffer twice every 15 minutes. The washing process was performed in the same manner, and the secondary antibody was treated for 1 hour and then washed again with TBST buffer. The separated and recovered proteins were quantified using RC DC TM Protein Assay Kit II (protein assay kit, Bio-rad, USA).
그 결과, 도 4에 나타난 바와 같이, Saos-2 세포에서 공진단 추출물 및 접골공진단 추출물 처리 7일 후에는 BMP-2 단백질 발현 정도가 각각 2배 및 3배 증가된 것으로 나타났고, 14일 후에는 각각 7배 및 14배 증가된 것으로 나타나, 접골공진단 추출물이 공진단 추출물보다 BMP-2 단백질 발현을 더 촉진시킬 수 있음을 확인하였다.As a result, as shown in FIG. 4, the expression level of BMP-2 protein was increased by 2 and 3 times, respectively, after 7 days of treatment with the resonant end extract and the osteoconjugation extract in Saos-2 cells, and after 14 days, respectively It has been shown to be increased 7-fold and 14-fold, and it was confirmed that the osteoclast diagnosis extract can promote BMP-2 protein expression more than the resonance group extract.
따라서, 본 발명에 따른 접골공진단은 골형성(osteogenesis) 관련 단백질 BMP-2의 발현을 증가시키며, 일반 공진단보다 더 우수한 골형성 촉진 효과를 가짐을 확인할 수 있었다.Accordingly, it was confirmed that the osteoclast according to the present invention increases the expression of BMP-2, a protein related to osteogenesis, and has a better bone formation promoting effect than the general resonant stage.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.
Claims (11)
골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손, 고관절 감소증 및 치조골 파괴에 의한 치주질환 중 선택되는 골 관련 질환의 예방 또는 치료용 약학적 조성물.Deer antler, Angelica, Cornus officinalis, safflower, grafted alley and aloe extract as active ingredients,
A pharmaceutical composition for the prevention or treatment of bone-related diseases, selected from periodontal disease due to bone dysplasia, osteomalacia, osteopenia, fractures, bone defects, hip reduction and alveolar bone destruction.
상기 추출물은 침향 1.0 중량부를 기준으로 녹용 6 내지 10 중량부, 당귀 6 내지 10 중량부, 산수유 6 내지 10 중량부, 홍화자 0.1 내지 4.0 중량부 및 접골목 0.1 내지 4.0 중량부를 혼합한 혼합물을 추출하여 제조되는 것을 특징으로 하는 약학적 조성물.According to claim 1,
The extract extracts a mixture of 6 to 10 parts by weight of antler, 6 to 10 parts by weight of Angelica, 6 to 10 parts by weight of Cornus officinalis, 0.1 to 4.0 parts by weight of safflower and 0.1 to 4.0 parts by weight of grafted tree based on 1.0 parts by weight of aroma Pharmaceutical composition characterized in that it is prepared.
상기 추출물은 물, 탄소수 1 내지 탄소수 4의 알코올 또는 이들의 혼합용매로 추출한 것을 특징으로 하는 약학적 조성물.According to claim 1,
The extract is a pharmaceutical composition, characterized in that extracted with water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
상기 추출물은 에탄올로 추출한 것을 특징으로 하는 약학적 조성물.According to claim 3,
The extract is a pharmaceutical composition, characterized in that extracted with ethanol.
상기 추출물은 ALP(alkaline phosphatase), COL1(Collagen type1) 및 OPN(osteopontin) 중 1종 이상의 골형성(osteogenesis) 관련 유전자 또는 BMP2(Bone morphogenetic protein 2) 단백질의 발현을 촉진시키는 것을 특징으로 하는 약학적 조성물.According to claim 1,
The extract is a pharmaceutical characterized in that it promotes the expression of one or more bone-forming genes or BMP2 (Bone morphogenetic protein 2) proteins among ALP (alkaline phosphatase), COL1 (Collagen type 1) and OPN (osteopontin). Composition.
상기 추출물은 조골세포(osteoblast)에서 골세포(osteocyte)로의 분화를 촉진시키는 것을 특징으로 하는 약학적 조성물.According to claim 1,
The extract is a pharmaceutical composition characterized in that to promote the differentiation of osteoblasts (osteoblast) to bone cells (osteocytes).
상기 약학적 조성물은 인공골, 인공관절, 골 고정재, 골 대체제, 골 수복재, 골 충진재 및 골 이식재로 이루어진 군으로부터 선택되는 어느 하나 이상의 물질에 사용되는 것을 특징으로 하는 약학적 조성물.According to claim 1,
The pharmaceutical composition is used in any one or more materials selected from the group consisting of artificial bones, artificial joints, bone fixatives, bone substitutes, bone repair materials, bone fillers, and bone grafts.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101751398B1 (en) * | 2016-08-05 | 2017-06-27 | 영농조합법인 한꿀팜스원 | Composition comprising Angelica gigas Nakai, Cornus officinalis, Cervi Parvum Cornu, Red ginseng, Rehmanniae Radix Preparata, Aquilaria agallocha Roxburgh and Honey for anti-inflammation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101193540B1 (en) | 2009-09-23 | 2012-10-22 | 경희대학교 산학협력단 | Composition comprising the extract of Astragalus membranaceus BGE.,Cinnamomum cassia and Phellodendron amurensis for preventing and treating of osteoporesis and bone disease |
| KR20110053151A (en) * | 2009-11-13 | 2011-05-19 | 박의신 | Removal of knee pain and treatment of arthritis ointments |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101751398B1 (en) * | 2016-08-05 | 2017-06-27 | 영농조합법인 한꿀팜스원 | Composition comprising Angelica gigas Nakai, Cornus officinalis, Cervi Parvum Cornu, Red ginseng, Rehmanniae Radix Preparata, Aquilaria agallocha Roxburgh and Honey for anti-inflammation |
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