KR100306913B1 - Safflower seed extract having bone fracture healing efficacy - Google Patents
Safflower seed extract having bone fracture healing efficacy Download PDFInfo
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- KR100306913B1 KR100306913B1 KR1019980057209A KR19980057209A KR100306913B1 KR 100306913 B1 KR100306913 B1 KR 100306913B1 KR 1019980057209 A KR1019980057209 A KR 1019980057209A KR 19980057209 A KR19980057209 A KR 19980057209A KR 100306913 B1 KR100306913 B1 KR 100306913B1
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- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/286—Carthamus (distaff thistle)
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A23V2200/00—Function of food ingredients
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- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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Abstract
Description
본 발명은 골절치유 효능이 있는 천연추출물에 관한 것으로 더욱 상세하게는 본 발명은 홍화씨로부터 분리한 골절치유 효능이 있는 홍화씨추출물 및 그 제조방법에 관한 것이다.The present invention relates to a natural extract having a fracture healing effect, and more particularly, the present invention relates to a safflower seed extract having a fracture healing effect separated from safflower seed and a method of manufacturing the same.
뼈는 독특한 생체조직으로서 보통 힘에 의해 쉽게 부서지지 않을 만큼 단단할 뿐 아니라 근육수축에 의해 움질일 수 있을 만큼 가벼운 것이 특징이다(krane et al., 1989). 이러한 특징은 뼈의 두가지 주요 형태와 관련되는데 그 중 하나는 석회화된 콜라겐이 빽빽이 층을 이루는 치밀골로서 견고한 성질을 가지며 관상골의 주요 구성성분이 된다. 다른 하나는 스폰지 형태로 되어 있는 해면골로 뼈에 강도와 탄력성을 주고 축골격의 주요부분을 구성한다. 치밀골에 결함이 생기거나 또는 치밀골이 빈약하면 장골골절이 생기는 반면 해면골에 결함이 생기거나 또는 해면골이 빈약하면 척추골절이 생긴다. 장골골절은 정상적인 해면골의 보강이 부족했을 때에도 일어날 수 있다(Ekhare et al., 1998).Bone is a unique biological tissue that is not only hard to break easily by force, but also light enough to be gripped by muscle contraction (krane et al., 1989). This feature is associated with two major forms of bone, one of which is densely packed with calcified collagen, which has a solid nature and is a major component of the coronal bone. The other is sponge sponge, which gives strength and elasticity to the bone and constitutes the main part of the axial skeleton. If the dense bone is defective or if the dense bone is poor, the iliac bone fracture occurs, while if the spongy bone is defective or the spongy bone is poor, the vertebral fracture occurs. Iliac fractures can occur even when there is a lack of normal cavernous bone reinforcement (Ekhare et al., 1998).
뼈 무게의 2/3는 무기질이 차지하고 나머지는 물과 콜라겐이 차지하고 있으며 무기질 중 특히 칼슘과 인이 풍부하고 소량의 중탄산염, 시트르산염, 마그네슘, 칼슘 및 나트륨 등이 존재한다( 박 등., 1992). 한편 유기물질의 95%는 제 1형 아교섬유(collagen)와 무형질(ground substance)로 구성되어 있고, 무형질로는 단백질과 연결되어 있는 프로테오글라이칸(proteoglycan)이 있으며 비아교섬유로는 특수한 당단백질인 시알로프로테인(sialoprotein)과 오스테오칼신(Osteocalcin)이 존재한다(Banks, 19860). 기질내 이들 콜라겐과 무형질들은 무기질화(mineralization)또는 석회화(Calcification)되어 인체의 지지기능을 수행하게 된다. 뼈는 일생을 통해 계속적으로 용출(골흡수)되고 생성(골형성)된다. 이러한 중요한 과정은 각기 다른 기능을 가지는 세가지 골세포에 의해 결정되는데 조골세포(osteoblast)는 파골세포(osteoclast)에 의해 미리 용출된 뼈의 표면에 새로운 뼈를 생성하는 역할을 한다. 조골세포는 뼈의 결합조직에 있는 간엽세포(mesenchymal cells)로부터 생긴 뼈 표면의 세포군으로부터 유래되며 조골세포는 뼈의 기질성분(주로 콜라겐)의 합성에 적극적으로 관여하고, 조골세포 매개에 의한 칼슘과 인의 수송이 콜라겐의 무기질화에 관여하며 뼈 생성에 결정적인 역할을 하는 것으로 알려져 있다. 뼈가 생성되는 동안 조골세포는 생성된 골기질(bone matrix)에 점점 둘러쌓이게 되고, 그 후 조골세포는 기능과 형태가 변하여 골세포가 된다. 이때 단백질 합성이 현저히 감소하고, 세포는 골조적에 있는 소강(lacunae)에 도달하여 골단위(osteon)내에 다른 골세포와 연결된다(Ekhare et al., 1998).Two-thirds of the bone's weight is taken up by minerals and the rest by water and collagen, especially minerals rich in calcium and phosphorus, with small amounts of bicarbonate, citrate, magnesium, calcium and sodium (Pak et al., 1992). . On the other hand, 95% of the organic material is composed of type 1 collagen and ground substance, and the intangible is proteoglycan, which is linked to protein. Glycoproteins, sialoprotein and osteocalcin, are present (Banks, 19860). These collagen and intangibles in the matrix are mineralized or calcined to perform the supporting functions of the human body. Bone is continuously eluted (bone absorbed) and produced (bone formed) throughout life. This important process is determined by three osteoblasts with different functions. Osteoblasts are responsible for producing new bone on the surface of the bone previously eluted by osteoclasts. Osteoblasts are derived from a group of cells on the surface of the bone that are derived from mesenchymal cells in the connective tissue of bone. Osteoblasts are actively involved in the synthesis of bone matrix components (mainly collagen). Phosphorus transport is involved in mineralization of collagen and is known to play a decisive role in bone formation. While bone is produced, osteoblasts are gradually enclosed in the bone matrix that is produced, and then the osteoblasts change in function and shape to become bone cells. At this time, protein synthesis is significantly reduced, and the cells reach the lacunae in the bone structure and connect with other bone cells in the bone unit (osteon) (Ekhare et al., 1998).
뼈대사에서 석회화된 뼈성분이 다시 파괴 또는 분해되는 것을 골흡수(bone resorption)라 하며 이에 관여하는 파골세포의 정확한 역할은 아직 뚜렷하게 발혀져 있지 않으나 뼈표면에 존재하면서 골흡수를 담당하며 뼈흡수 과정에서 형성된 세포의 찌꺼기를 제거하는 역할을 하는 것으로 알려져 있다(Rose et al., 1995). 파골세포는 다핵의 거대세포이며 뼈용출을 담당한다. 이 세포는 순환하는 단핵 대식세포로부터 유도되어 뼈주위에서 융합에 의해 성숙한 파골세포로 분화한다. 이 세포는 모든 효소성분을 포함하고 있어서 기질을 용해하여 칼슘과 인산을 방출하며 방출된 무기질은 파골세포를 통해 세포외액을 거쳐 결국 혈액으로 수송된다. 세포외액내의 무기질 항상성을 유지하기 위해 파골세포의 뼈용출과 조골세포에 의한 뼈표면으로부터 세포외액으로의 무기질 이동이 일어난다(Ekhare et al., 1998).Bone resorption is the destruction or disintegration of calcified bone components in bone metabolism. The exact role of osteoclasts involved in this is not yet clearly revealed, but it is present on the bone surface and is responsible for bone resorption. It is known to play a role in removing the debris of cells formed in (Rose et al., 1995). Osteoclasts are multinucleated giant cells and are responsible for bone elution. These cells are derived from circulating mononuclear macrophages and differentiate into mature osteoclasts by fusion around the bone. These cells contain all the enzymes that dissolve the substrate to release calcium and phosphoric acid, and the released minerals are transported through the osteoclasts through the extracellular fluid to the blood. To maintain mineral homeostasis in extracellular fluid, osteolysis of osteoclasts and mineral migration from bone surface by osteoblasts occurs to extracellular fluid (Ekhare et al., 1998).
뼈구조의 두가지 형태는 망상(woven)과 층판(lamellar)으로 치밀골이나 해면골에서 현미경으로 관찰할 수 있다. 망상골은 태생 뼈의 정상 구성요소이며, 성인의 뼈에서는 질환시에 발견된다. 층판골이 더 강하고 더 천천히 형성되며 출생 후 점진적으로 망상골을 대체한다. 콜라겐 섬유가 비평행(nonparallelism) 배열을 이루고 있는 망상골은 기질 단위면적당 골세포가 많고 콜라겐 근원섬유에 무기질이 적게 함유되어 있는 것이 특징이다. 반면 층판골은 콜라겐 섬유가 평행 배열을 이루며 기질 단위면적당 골세포가 적고 콜라겐 근원섬유내에 무기질층을 가지고 있다. 치밀성 층판골은 치밀골(골단위)의 하버스계(harversian system)를 구성하는 혈관통로를 둘러싼 동일한 중심을 갖는 층(concentric layers)에 존재한다. 대조적으로 해면성 층판골은 긴 다발과 얇은 판으로 층을 이루고 있다(Ekhare et al., 1998). 골대사에서 골흡수와 골형성(bone formation)이 짝지워져 일어날 때 이를 골의 재구성(bone ramodeling) 또는 골대사회전(bone turnover)이라한다(Banks, 1986, Shils et al., 1988). 골구성(modeling) 이라는 용어는 육안으로 볼 수 있는 골격의 형성과정을 의미하며, 따라서 골구성은 성인기(18 ~ 20세)에 종료된다. 재형성(remodeling)은 뼈의 구조적 완전 상태를 유지하기 위해 뼈표면에서 일어난다. 대사성 골질환에서는 뼈생성과 뼈용출 사이의 불균형으로 인해 뼈의 구조물이 감소되고 결국 골기능이 손상된다. 정상적인 상태에서는 성장이 멈춘 후에 골재형성이 지속적으로 일어나도 골격량이 실증가(net increase of bone mass)나 손실은 거의 없다. 이러한 결과는 뼈용출과 뼈생성이 밀접한 균형을 이루고 있으며 조골세포와 파골세포의 활성 조정에 의한 것이다. 즉 뼈의 일정량이 파골세포에 의해 용출되면(해면골의 표면과 치밀골에서 실질적인 결손이 생김) 조골세포에 의해 그 결손이 복구되어 콜라겐의 무기화로 진행된다(osteoid). 따라서 실 골절량을 변화시키는 주요인은 뼈용출과 뼈생성을 좌우하는 인자들이다(Ekhare et al., 1998).The two types of bone structures are woven and lamellar, which can be observed microscopically in dense or cavernous bone. Reticular bone is a normal component of native bone and is found in diseased bones in adults. Lamellar bones are stronger and formed more slowly and gradually replace reticular bones after birth. The reticular bone, in which collagen fibers form a nonparallelism arrangement, is characterized by a large amount of bone cells per unit area of the matrix and low mineral content in the collagen myofibrils. Laminar bone, on the other hand, has collagen fibers arranged in parallel, less bone cells per substrate area, and an inorganic layer in the collagen myofibril. Dense lamellar bone is present in concentric layers around the vascular pathways that make up the harversian system of dense bone. In contrast, spongy lamellar bones are layered with long bundles and laminae (Ekhare et al., 1998). When bone resorption and bone formation occur in the bone metabolism, this is called bone ramodeling or bone turnover (Banks, 1986, Shils et al., 1988). The term modeling refers to the process of the formation of the skeleton visible to the naked eye, and thus the bone formation ends in adulthood (18-20 years old). Remodeling occurs at the bone surface to maintain the structural integrity of the bone. In metabolic bone diseases, the imbalance between bone formation and bone elution reduces bone structure and ultimately impairs bone function. Under normal conditions, there is little net increase of bone mass or loss even when aggregate formation continues after growth stops. These results are in close balance with bone dissolution and bone formation and are due to the adjustment of osteoblast and osteoclast activity. In other words, when a certain amount of bone is eluted by osteoclasts (substantial defects occur at the surface of the sponges and dense bones), the defects are repaired by osteoblasts and proceed to mineralization of collagen (osteoid). Therefore, the main factors that change the amount of actual fractures are factors that influence bone dissolution and bone formation (Ekhare et al., 1998).
뼈질환 중 골절이 일어나면 혈관의 손상으로 부분적 출혈과 혈병이 형성되며 골절된 인접부위에 있는 뼈기질은 파괴되고 뼈세포도 역시 죽게된다. 치유과정 동안 혈병, 손상받은 뼈세포 및 뼈기질은 큰 포식세포에 의해 제거되며 골절부위 주위에 있는 골막(Perilsteum)과 골내막(endosteum)의 뼈선조세포(osteoprogenitor cell)는 활발하게 증식하여 골절 주변 부위에 세포성 조직(cellular tissue)을 형성하고 이들은 골절된 부위로 들어간다(박 등., 1992, Dellmann, 1993). 골절된 부위의 결합조직에서는 작은 연골 조각으로부터 연골내골화과정(endochondral ossification)이 일어나거나 막성뼈 발생과정(intramembranous ossification)을 통해서 미성숙뼈(immature bone)가 형성된다(Banks 1986, Ross et al., 1995, Dellmann, 1993, Hammersen, 1985, Bacha et al., 1990 강 등., 1996). 따라서 골절부위에서는 연골조직, 막성뼈 발생과정 그리고 연골내골화 과정이 동시에 관찰된다. 치유과정은 이와 같은 방식에 의해 진행되며 불규칙하게 형성된 미성숙뼈의 지주(trabecular)가 골절된 뼈의 양 끝을 일시적으로 결합시켜 가골(bony callus)를 형성한다. 골절부위에 형성된 가골의 일차뼈조직(woven bone)은 치유가 진행됨에 따라 점차적으로 흡수되어 층판뼈(lamellar bone)로 대체되면서 원래이 뼈구조로 복구된다(Ross et al., 1995, 강종구 외., 1996).Fractures of bone disease cause partial bleeding and blood clots due to damage to blood vessels, destruction of the bone matrix in the fractured adjacent area, and death of bone cells. During the healing process, blood clots, damaged bone cells and bone matrix are removed by large phagocytes. Perilsteum and osteoprogenitor cells around the fracture area actively proliferate and grow near the fracture. Form cellular tissue at the site and enter the fractured site (Pak et al., 1992, Dellmann, 1993). In connective tissue at the fracture site, endochondral ossification occurs from small pieces of cartilage or immature bone is formed through intramembranous ossification (Banks 1986, Ross et al., 1995, Dellmann, 1993, Hammersen, 1985, Bacha et al., 1990 River et al., 1996). Therefore, cartilage tissue, membranous bone development process and intrachondral ossification process are observed at the fracture site. The healing process proceeds in this way, with irregularly shaped trabecular trabeculars temporarily joining both ends of the fractured bone to form bony callus. The primary bone tissue (bone bone) formed at the fracture site is gradually absorbed and replaced with lamellar bone as healing progresses (Ross et al., 1995, Jong-gu Kang et al., 1996).
골절의 치유과정에서는 뼈형성에 관련된 여러 요인들이 영향을 미치는 것으로 추측되나 이와 관련된 골대사의 지표변화에 대해서는 아직 보고된 바가 없다. 뼈의 생성과정 또는 석회화 기전에는 막내골화 연골내골화가 있으며 골모세포와 파골세포의 작용과 함께 무기염류가 침착하여 딱딱한 골로 된다(Ross et al., 1995). 이때, 막내골화는 결합조직에서 바로 뼈가 되고 연골내골화는 연골에서 전환된다. 혈장에 존재하는 전체 알카라인 포스파타아제( total-ALP) 가운데 그 중 뼈의 석회학에 관여하는 것은 뼈-특이적 알카라인 포스파타아제(bone-specific alkaline phosphatase:bone-ALP)이다. 알카라인 포스파타아제(Alkaline phosphatase)의 아이소폼(isoform) 중의 하나인 bone-ALP는 조골세포 표면에서 발견되는 당단백질의 일종이며(Price, 1993), 이 효소의 기능은 골격 무기질화(Skeletal mineralization0에 관여한다고 보고되었다(Price 1993, Epstein, 1989, Price, 1995).It is speculated that various factors related to bone formation influence the fracture healing process, but there have been no reports of changes in the index of bone metabolism. In the bone formation process or calcification mechanism, there is endothelium osteochondral cartilage, and mineral salts are deposited with osteoblasts and osteoclasts to become hard bones (Ross et al., 1995). At this time, the endometrial ossification becomes bone in the connective tissue and the endochondralization is converted in the cartilage. Of the total alkaline phosphatase (total-ALP) present in the plasma is involved in bone calcification is bone-specific alkaline phosphatase (bone-ALP). Bone-ALP, an isoform of alkaline phosphatase, is a type of glycoprotein found on the surface of osteoblasts (Price, 1993), and its function is involved in skeletal mineralization. (Price 1993, Epstein, 1989, Price, 1995).
뼈표면에 존재하면서 골흡수를 담당하는 파골세포는 여러 호르몬의 작용을 받게 된다. 뼈에 있는 칼슘은 두 가지 기전에 의해 유리되는데, 첫 번째 기전은 하이드록시아파타이트 크리스탈(hydroxyapatite crystals)로부터 간질액으로의 단순한 칼슘이온 전이로서 빠르게 일어난다. 칼슘을 유리시키는 두 번째 기전은 부갑상선 호르몬(Parathroid hormone, PTH)이 뼈기질의 흡수를 촉진시키는 파골세포의 수를 증가 및 활성화시켜 칼슘을 유리시킨다. PTH와 반대로 칼시티닌(calcitinin)은 갑상선에서 합성되어 뼈기질 흡수를 방해한다. 골형성 지표 중의 하나인 오스테오칼신(osteocalcin)은 골형성시 조골세포의 활성을 간접적으로 반영하는데(Lee et al., 1990), 골흡수 증가로 인해 골밀도가 감소할 때 이차적으로 조골세포의 활동이 증가되며 오스테오칼신(osteocalcin)의 합성이 증가된다(박 등, 1992). 뼈에 특이적으로 작용하는 효소인 bone-ALP도 뼈의 석회화를 촉진하는 중요한 골형성 지표로서, 타 연구자들에 의하면(송 등., 1994)이 효소의 활성도는 혈중 오스테오칼신(osteocalcin) 농도와 독립적으로 작용할 수도 있다.Osteoclasts, which are present on the bone surface and are responsible for bone resorption, are subject to several hormones. Calcium in the bone is released by two mechanisms, the first of which rapidly occurs as simple calcium ion transitions from hydroxyapatite crystals to interstitial fluid. The second mechanism that releases calcium is that parathyroid hormone (PTH) releases calcium by increasing and activating the number of osteoclasts that promote the absorption of bone matrix. In contrast to PTH, calcitinin is synthesized in the thyroid gland and interferes with bone matrix absorption. Osteocalcin, one of the indicators of bone formation, indirectly reflects the activity of osteoblasts during bone formation (Lee et al., 1990). Secondary osteoblast activity increases when bone density decreases due to increased bone resorption. And the synthesis of osteocalcin is increased (Pak et al., 1992). Bone-ALP, an enzyme that acts specifically on bone, is also an important bone formation marker that promotes bone calcification. According to other researchers (Song et al., 1994), the activity of this enzyme is independent of blood osteocalcin concentration. It can also work.
종래 골조직을 얻어야만 판정이 가능하였던 골형성과 흡수의 정도를 최근에는 단순히 혈액 또는 소변에서 검체를 추출하여 간단한 반응에 의해 알 수 있는 새로운 방법들이 개발되었다. 이들 검사법의 일부는 조골세포와 파골세포들의 세포내 효소이면서 동시에 혈액으로 분비되는 효소들의 혈중농도를 측정하는 것이고 일부는 골형성과정에서 부분적으로 혈액으로 유리되는 새로 형성된 골의 구성성분이거나 또는 골흡수 과정 중에서 분비되는 골실질의 분절들을 측정하는 검사들이다. 최근에는 골흡수 정도를 더욱 정확하게 측정할 수 있는 생화학지표들이 개발됨에 따라 골질환의 치료와 관찰은 물론 약제선정에도 많은 도움이 된다(임., 1994).Recently, new methods have been developed to determine the degree of bone formation and absorption, which could be determined only by obtaining bone tissue, by simply extracting a sample from blood or urine. Some of these tests measure the blood levels of enzymes that are secreted into the blood and at the same time intracellular enzymes of osteoblasts and osteoclasts, and some are components of newly formed bone that are partially released into the blood during bone formation or bone resorption. These tests measure the segments of the bone parenchyma secreted during the process. In recent years, as biochemical indicators have been developed to more accurately measure the degree of bone resorption, it is very helpful in the treatment and observation of bone diseases as well as in the selection of drugs (I., 1994).
뼈에 영향을 미치는 요소들은 여러 가지 복합적 관계를 이루고 있는데, PTH와 칼시토닌(Calcitonin) 이외에 운동, 에스트로겐, 혈액내 칼슘과 인의 농도비율 등을 들 수 있다(kee, 1993). 뼈의 재형성시 필요한 영양인자로는 칼슘, 비타민 D, 비타민 A, 단백질 등이 관여하며 파라티로이드 호르몬(PTH) 및 칼시토닌 등의 호르몬이 영향을 미치는 것으로 알려져 있다(Shils et al., 1988). 한편 국내산 약초 중 홍화(또는 잇꽃, Carthamus tinctorius L.)의 작용기작은 전혀 밝혀진 바 없으니 그 씨앗성분이 금이 가거나 다친 뼈를 빠르고 튼튼하게 회복시키는 등 뼈질환에 뛰어난 효과가 있다고 민간에 알려지기 시작하였다(동의보감, 1989). 뿐만 아니라 보건복지부에서 98년 상반기에 홍화씨를 가공식품화하는 것을 허용하여 앞으로 국내 재배량도 급격히증가될 것으로 보인다. 이에 본 발명자들은 1997년 홍화의 기능성 성분을 탐색하는 연구의 일환으로 홍화씨 분말식의 급여가 늑골이 골절된 흰쥐의 골절치유과정에서 골대사에 미치는 영향을 조사하여 우리홍화인영농조합에 보고한 바 있으며 그 연구결과는 1997년에 국내 특허출원 후 1998년에 한국식품영양과학회지와 한국영양학회지에 게재되었다. 이 연구결과에서는 홍화씨 분말을 10% 수준으로 식이에 혼합하여 급여했을 때 골절치유기간이 단축되었고(전 등., 1998, 김 등., 1998) 그 과정에서 골형성이 활발히 촉진됨을 확인하였다.Factors affecting bone have a number of complex relationships, including PTH and calcitonin, as well as exercise, estrogen, and the concentration of calcium and phosphorus in the blood (kee, 1993). Nutrient factors necessary for bone remodeling include calcium, vitamin D, vitamin A, and protein, and hormones such as parathyroid hormone (PTH) and calcitonin are known to affect (Shils et al., 1988). . On the other hand, the mechanism of action of safflower (or safflower, Carthamus tinctorius L.) among the domestic herbs has not been revealed at all, so the seeds have started to be known to the public as having an excellent effect on bone diseases such as quick and strong recovery of cracked or injured bones. (Agreement, 1989). In addition, the Ministry of Health and Welfare will allow processed safflower seeds to be processed in the first half of 1998, and domestic cultivation is expected to increase rapidly. Therefore, the inventors investigated the effect of safflower seed powder on bone metabolism in the fracture healing process of rib fractured rats as part of a study to explore the functional components of safflower in 1997. The results of the research were published in the Korean Journal of Food and Nutrition Science and the Korean Journal of Nutrition in 1998, after a domestic patent application in 1997. The results showed that when the safflower seed powder was fed to the diet at 10% level, the period of fracture healing was shortened (Jeon et al., 1998, Kim et al., 1998) and bone formation was actively promoted in the process.
따라서 본 발명의 목적은 홍화씨로부터 골절치유효능이 있는 홍화씨추출물을 분리하여 제공함에 있다. 본 발명의 다른 목적은 상기 홍화씨로부터 분리한 골절치유효능이 있는 홍화씨추출물의 골강화 및 골절치유촉진을 위한 건강식품으로써의 용도를 제공함에 있다.Therefore, an object of the present invention is to provide a safflower seed extract having a fracture healing effect from safflower seed. Another object of the present invention is to provide a use as a health food for promoting bone reinforcement and fracture healing of safflower seed extract having effective fracture healing from the safflower seed.
본 발명의 상기 목적은 5% 홍화씨 분말, 홍화씨 열수추출물 및 에탄올추출물을 흰쥐에게 급여하여 사육한 후 골절을 유도하고 상기 5% 홍화씨 분말, 홍화씨 열수추출물 및 에탄올추출물을 계속 실험식이로 급여한 후 늑골을 채취하여 골조직을 형태학적으로 분석하고 골조직의 표본을 제작한 후 현미경으로 관찰하고 파골세포수 분석 및 혈액을 생화학적으로 분석하므로써 달성하였다.The object of the present invention is to feed the 5% safflower seed powder, safflower seed hot water extract and ethanol extracts to rats to induce fractures, and then continue feeding the 5% safflower seed powder, safflower seed hot water extract and ethanol extracts to the experimental diet ribs The results were obtained by morphological analysis of bone tissues, specimens of bone tissues, microscopic observation, osteoclast count, and biochemical analysis of blood.
이하 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.
도 1은 늑골골절 10일 및 20일 경과 후 분리된 늑골의 외형적인 변화를 나타낸 사진도이다.1 is a photograph showing the external appearance of the ribs separated after 10 days and 20 days after the rib fracture.
도 2는 늑골 골절 20일 경과 후 모의실험(Sham-operation)군의 조직적인 뼈구조를 나타낸 사진도이다.Figure 2 is a photograph showing the bone structure of the Sham-operation group 20 days after the rib fracture.
도 3은 늑골골절 10일 경과 후 조직적인 뼈구조의 모양을 나타낸 사진도이다.Figure 3 is a photograph showing the shape of the bone structure after 10 days of rib fractures.
도 4은 늑골골절 20일 경과 후 조직적인 뼈구조의 모양을 나타낸 사진도이다.Figure 4 is a photograph showing the shape of the bone structure after 20 days of rib fractures.
본 발명은 홍화씨 열수추출물과 에탄올추출물을 각각 제조하는 단계; 흰쥐를 5% 홍화씨 혼합식이로 사육한 후 골절유도 수술을 실시하고 골절 후 실험식이를 계속 급여한 후 늑골을 채취하고 골조직의 형태학적 분석을 실시하는 단계; 골절상을 유도하고 실험식이를 급여한 흰쥐의 체중중가량을 골절 후 10일째 및 20일째에 각각 측정하는 단계: 골절유도 후 10일째와 20일째에 사육한 각 실험동물군의 늑골을 절취하고 탈회시킨 후 연속절편을 제작하여 광학현미경으로 파골세포수를 분석하는 단계 및; 실험동물의 복부 대정맥으로부터 혈액을 채취하고 분광광도법으로 혈장을 생화학적으로 분석하는 단계로 구성된다.The present invention comprises the steps of preparing safflower seed hot water extract and ethanol extract, respectively; Breeding rats with a 5% safflower seed diet followed by fracture-induced surgery, and continuing feeding of the experimental diet after fracture; collecting ribs and performing morphological analysis of bone tissue; Measuring the weight of the rats that induced fracture and fed the experimental diet on the 10th and 20th day after the fracture, respectively: cutting and demineralizing the ribs of each experimental animal group raised on the 10th and 20th day after the fracture induction Analyzing the osteoclast number by an optical microscope by preparing a serial section; Blood is collected from the abdominal vena cava of the experimental animal and biochemical analysis of the plasma by spectrophotometry.
이하 본 발명의 구체적인 방법을 실시예를 들어 단계별로 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.
실시예 1: 증류수 및 에탄올로 홍화씨추출물 제조Example 1: Preparation of safflower seed extract with distilled water and ethanol
본 발명에 사용된 토종 홍화씨는 경북 의성군 소재 우리 홍화인 영농조합으로부터 제공된 시료를 사용하였다. 수확된 홍화씨는 정선, 선발하여 세척하고 180℃에서 15 ~ 20분간 로스팅(roasting)한 후 20메쉬 이하로 분쇄한 것을 사용하였으며, 본 실시예에서 분석된 홍화씨 분말의 일반성분 조성은 수분 5.2%, 회분 2.9%, 조단백질 18.4%, 조지방 22.9%, 당질 25.2%, 섬유소 25.4%로 확인되었다. 열수추출물은 볶음 홍화씨 분말 600g에 증류수 5배량(3000mL)을 가하여 90℃에서 6시간 동안 1회 추출한 후 와트만 No. 2 여과지를 이용하여 감압여과하고, 남은 잔류물에 다시 증류수 3000mL를 가하여 반복추출한 후 와트만 N0.2 여과지로 감압여과한 추출액을 40℃에서 다시 감압농축하여 용매를 제거하였다. 이러한 절차에 의해 얻어진 추출물은 -50℃에서 공업용 동결건조기를 사용하여 동결건조하여 분말화하였으며 동결건조된 열수추출물의 수율은 10.1%로 계산되었다. 에탄올추출물의 제조시에는 볶음 홍화씨 분말 600g에 80% 에탄올 5배량(3000mL)을 가하여 85℃에서 6시간동안 1회추출한 후 와트만 No. 2 여과지로 감압여과하고 남은 잔류물에 다시 80% 에탄올 3000mL을 가하여 반복추출한 후 와트만 No. 2 여과지로 감압여과한 추출액을 40℃에서 다시 감압농축하여 용매를 제거하였다. 이러한 절차에 의해 얻어진 추출물은 -50℃에서 동결건조하여 분말화하였으며 동결건조된 에탄올추출물의 수율은 4%로 계산되었다.The native safflower seed used in the present invention used a sample provided from Woori safflower farming cooperative in Uiseong-gun, Gyeongbuk. The harvested safflower seeds were selected, selected, washed, roasted at 180 ° C. for 15-20 minutes, and then pulverized to 20 mesh or less. The general composition of safflower seed powder analyzed in the present embodiment was 5.2% water, Ash 2.9%, crude protein 18.4%, crude fat 22.9%, sugar 25.2%, and cellulose 25.4%. Hot water extract was added to 600g of stir-fried safflower seed powder and 5 times (3000mL) of distilled water was extracted once at 90 ° C for 6 hours, and then Whatman No. 2 filtered under reduced pressure using a filter paper, and the remaining residue was added again 3000mL of distilled water and extracted repeatedly, and the extract was filtered under reduced pressure with Whatman N0.2 filter paper and concentrated under reduced pressure again to remove the solvent. The extract obtained by this procedure was lyophilized and powdered using an industrial lyophilizer at -50 ° C, and the yield of lyophilized hot water extract was calculated to be 10.1%. In the preparation of ethanol extract, 5 times (3000 mL) of 80% ethanol was added to 600 g of roasted safflower seed powder, and extracted once at 85 ° C. for 6 hours. 2 After filtration under reduced pressure with a filter paper, 3000 mL of 80% ethanol was added again to the residue, followed by repeated extraction. 2 The extract was filtered under reduced pressure with a filter paper and concentrated under reduced pressure again at 40 ° C. to remove the solvent. The extract obtained by this procedure was lyophilized at −50 ° C. to powder and the yield of lyophilized ethanol extract was calculated to be 4%.
실시예 2: 홍화씨추출물의 골절치유효능조사Example 2 Fracture Efficacy Study of Safflower Seed Extract
제 1단계: 실험식이 및 골절상 유도Phase 1: Experimental Diet and Fracture Induction
체중이 약 300 ~ 320g인 스프라그-도레이(Sprague-Dawley) 10주령 수컷 쥐 60마리를 한국화학연구소로부터 구입하여 7일간 고형사료로 적응시킨 후 표 1과 같이 2개의 대조군(골절후 10일째와 골절후 20일째 그룹)으로 나누고 각 그룹내에서 무작위로 대조군, 홍화씨 분말군(SS-powder), 홍화씨 에탄올군(SS-EtoH), 홍화씨물군(SS-H2O)으로 다시 나누었다. 늑골골절 유도수술을 실시하기 전 표 2의 식이조성을 가진 정상식이(AIN-76 Semipurified diet)와 5% 홍화씨 혼합식이로 각각 10일간 사육시켰다. 실험군의 식이조성은 홍화씨 분말의 일반성분 조성을 참고로 하여 두 군 식이의 영양소 밀도가 동일하도록 만들었다. 홍화씨 분획은 용매추출 전 홍화씨 분말상태로 계산하여 사료의 5% 수준이 되도록 첨가하였으며 신선한 상태로 공급하였다. 실험식이 11일째에는 수술방법에 의한 골절상을 유도하였는데 그 부위는 소형동물에서 골절상이 빈번히 일어나는 늑골(Statter, 1995) 선택하였다. 마취제인 Ketamine-HCl(유한양행)을 체중 1kg 당 75mg의 용량으로 근육주사한 후 가슴피부의 일부를 최소면적으로 개복하여 우측 제 9번 늑골을 수술용 가위로 자르고 골절부위를 육안으로 확인한 후 수술사로 봉합하였다. 모의실험(Sham-operation) 군은 골절을 가하지 않고 가슴부위의 개복수술만 실시하여 봉합함으로써 실험종료 후 골절그룹과 대조하였다. 골절 후 실험식이를 급여한 후 사육기간이 진행되는 동안 골절 후 10일과 20일째 되는 날 각각 희생하고 늑골을 채취하였다. 단 모의-실험(Shem-operation) 군은 대조군과 동일한 식이를 급여하여 마지막 30일째에 모두 희생하였다. 실험동물은 각기 스테인레스 케이지에서 한마씩 분리사육하였다. 사육실의 온도는 약 25℃, 습도는 약 60%를 유지하였으며 광주기는 매일 12시간이 되도록 조절하고 식이 섭취량은 매일 1회, 체중은 1주일에 한번씩 일정시간에 측정하였다. 채취한 늑골에 형성된 가골은 육안으로 확인하였고 가골부위는 골조직의 형태학적 분석에 사용되었다.60 Sprague-Dawley 10-week-old male rats weighing about 300-320 g were purchased from the Korea Research Institute of Chemical Technology and adapted to solid feed for 7 days, followed by two control groups (10 days after fracture). Group 20 days after the fracture) and randomly divided into the control group, safflower seed powder group (SS-powder), safflower ethanol group (SS-EtoH), safflower seed group (SS-H 2 O). Before the rib fracture induction procedure, the dietary diet (AIN-76 Semipurified diet) and the 5% safflower seed diet were mixed for 10 days. The dietary composition of the experimental group was based on the general composition of safflower seed powder so that the nutrient density of the two groups of diet was the same. The safflower seed fraction was calculated to be safflower seed powder before solvent extraction and added to 5% of the feed and fed fresh. On the 11th day of the experiment, fractures were induced by surgical methods, and the site was selected ribs (Statter, 1995). Inject an anesthetic, Ketamine-HCl, to a dose of 75 mg per kg of body weight, open the part of the chest skin with a minimum area, cut the right side rib with surgical scissors, and visually check the fractured area. It was closed with yarn. The Sham-operation group contrasted with the fracture group after the end of the experiment by suturing the chest by open surgery without fracture. After feeding the experimental diet after fracture, the ribs were collected and sacrificed on the 10th and 20th day after the fracture during the breeding period. The Shem-operation group fed the same diet as the control group and sacrificed at the last 30 days. Experimental animals were separated and bred one by one in the stainless cage. The temperature of the feeding room was maintained at about 25 ℃, humidity was about 60%, photoperiod was adjusted to 12 hours every day, dietary intake was measured once a day, weight was measured once a week. The bones formed on the ribs were visually identified and the bones were used for the morphological analysis of bone tissue.
제 2단계: 수술후 체중증가량 및 장기중량 측정Second step: measuring weight gain and organ weight after surgery
실험기간에 따라 나누어진 각 군을 대상으로 골절상 유도 후 10일 및 20일간의 식이섭취량 및 체중증가량을 표 3에 나타냈다. 수술 후 섭취한 평균 1일 섭취량은 모든 군에서 비슷한 수준이었으며 치유가 진행되는 동안의 체중증가량은 수술 10일 후 대조군을 제외하고는 수술에 의한 약간의 체중감소가 있었으나 수술 20일 후에는 모든 군에서 체중이 증가되어 군간 식이섭취량과 체중증가에 대한 유의적인 차이는 없는 것으로 나타났다. 표 4에 나타낸 바와 같이 간, 심장 및 신장 등의 장기중량은 각 식이군간 유의적 차이가 없었으며 SS-EtOH군의 간무게가 약간 증가된 경향을 보였다.Table 3 shows the dietary intake and body weight gain of 10 days and 20 days after the fracture induction in each group divided according to the experimental period. The average daily intake after surgery was similar in all groups, and the weight gain during healing was slightly reduced by surgery except for the control group after 10 days, but in all groups after 20 days There was no significant difference in dietary intake and weight gain between groups. As shown in Table 4, the organ weights of liver, heart and kidney were not significantly different among the dietary groups, and the liver weight of the SS-EtOH group tended to increase slightly.
제 3단계: 골조직표본의 제작, 현미경관찰 및 파골세포수 분석Step 3: Preparation, Microscopic Observation, and Analysis of Osteoblasts
골절유도 후 10일째와 20일째에는 사육된 각 군의 실험동물로부터 골절부위인 우측 9번 늑골을 절취 즉시 보우린액에 침지하여 24시간 이상 고정하였으며, 5 ~ 7% 질산용액에서 3일간 매일 질산액을 교환하면서 탈회를 실시하였다. 탈회완료 후 5% 소듐 설페이트 수용액에 24시간 침지시키고 에탄올 계열로 탈수시킨 후 통상적인 방법으로 파라핀 포매를 실시하여 5 ~ 10μm의 연속절편을 제작하고, 헤마톡실린-에오신(Hematoxylin-eosin) 염색과 Masson's trichrome 염색을 행하여 광학현미경으로 조직을 관찰하였다.(Statter, 1985, Culling, 1974). 파골세포수는 조직 1mm2당 10군데를 카운팅하여 평균과 표준편차를 구하였다. 실험결과, 도 1에 골절치유과정 중 분리된 늑골의 외형적인 변화를 나타냈다. 골절후 10일째에서는 군간 가골두께에 큰 차이가 없었으며 골절 후 20일째에서는 SS-EtOH군과 SS-H2O군이 다른 군에 비해 가골의 크기가 많이 축소되어 있었다. 늑골골절 치유에 있어서 가골이 형성되는 동안 뼈선조세포(osteoprogenitor cell)로부터 분화된 조골세포는 골절부위로부터 같은 거리상의 위치에 있는 뼈표면에 축적되면서 골절부위를 향해 새로운 뼈의 재형성을 진행시키는데 본 실시예에서는 골절손상부위에서는 섬유아세포(fibrablast)들과 모세혈관조직의 증식이 일어나고 골절부위에 새로이 형성된 느슨한 결합조직(granulation tissue)은 점차로 두꺼워지는 것이 보였으며, 섬유아세포들과 골절부변 세포들은 세포성 조직을 형성하였다. 이 실험에서도 골절된 늑골의 치유가 계속 진행되는 동안 가골내 생성된 콜라겐과 초자연골은 보니 칼루스(bony callus)로 교체되었는데 이 과정에서는 이미 알려진 것처럼 막성골화와 연골내골화 과정(Banks, 1986, Ross et al., 1995, rkd 등., 1996)에 의해 원래의 가골에 형성된 초자연골이 파골세포에 의해 파괴되고 좀더 단단한 뼈인 immature bone 또는 지주골(trabecular bone)로 교체되는 과정이 관찰되었다. 골절치유가 더욱 구체적으로 진행되는 동안 보니 칼루스의 이러한 연골지주골들이 연골파골세포(chondroclast)에 의해 파괴된 후 다시 석회화되어 치밀골(compact bone)인 층판뼈(lamellar bone)로 교체되는 골재구성(Banks, 1986) 과정이 진행된 것으로 나타났다. 이러한 골절 치유속도는 흰쥐의 늑골인 경우 본 실험결과에서 30일 이상이 걸리는 것으로 나타났고 사람의 경우 개인의 영양상태나 골절형태 등에 따라 다소 다르나 건강한 사람의 경우 6 - 12주가 걸리는 것으로 알려졌다(강 등., 1996). 본 실험에서는 홍화씨 분말의 급여가 이러한 골절치유과정에 호전적인 영향을 미쳐 치유에 걸리는 시간을 단축하는 것으로 나타났다. 골절된 조직의 치유진행에 따라 관찰된 홍화씨군들과 대조군의 조직형태를 구체적으로 비교하면 도 2에 나타낸 바와 같이 모의-실험군(Sham-operation)군의 늑골조직은 정상군과 동일한 조직소견이 인정되었는데 즉 하버시안 커넬(Harversian canal)을 중심으로 층판뼈(lamella bone)가 배열되어 있는 골단위(osteon)로 구성된 치밀뼈(compact bone)들이 골수강을 중심으로 존재하며 이들 치밀뼈 이외 지주골(trabecular bone), 초자연골(hyaline cartilage), 결합조직(connective tissue) 등은 거의 관찰되지 않았다. 골절후 10일째 골절조직은 표 5와 도 3에 나타낸 바와 같이 골절 선을 중심으로 결합조직, 초자연골 및 지주골로 구성된 기골의 형성이 관찰되었으며 특히 대조군에서는 가골내에서 다량의 결합조직이 여전히 남아 있는 것으로 관찰된 반면 SS-powder, SS-EtoH 및 SS-H2O군에서는 중등도의 결합조직이 관찰되었다. 또한 초자연골은 모든 홍화씨 실험군에서 비교적 풍부하게 관찰되었으나 대조군에서는 증등도로 관찰되어 다량의 초자연골이 관찰된 홍화씨 실험군과 차이를 나타냈다. 한편 골절치유 회복기부터 나타나는 지주골은 대조군, SS-powder 군 및 SS-EtOH 군에서 소량 관찰되어 별 다른 차이를 나타내지 않았으나 SS-H2O군에서는 증등도로 관찰되어 나머지군들에 비해 차이를 나타냈다. 골절 10일째 파골세포의 수적변화는 표 6과 같이 대조군에서는 3.80±0.84로 관찰되었으나, SS-powder군과 SS-H2O군에서는 각각 6.80±0.84와 5.60±0.55로 관찰되어 대조군에 비해 유의적으로(p<0.01) 증가되었다. 그러나 SS-EtOH 군에서는 3.60±0.89로 관찰되어 대조군과 거의 유사한 것으로 관찰되었다.On the 10th and 20th day after the fracture induction, the right rib, which is the fracture site, from the experimental animals of each group was cut and immediately immersed in Bowin solution for at least 24 hours, and nitric acid daily in 5-7% nitric acid solution for 3 days. The deliming was carried out while exchanging the liquid. After completion of deliming, it was immersed in 5% sodium sulfate aqueous solution for 24 hours, dehydrated with ethanol series, and paraffin embedding was carried out in a conventional manner to prepare 5-10 μm continuous sections, and hematoxylin-eosin staining and Masson's trichrome staining was performed to observe the tissue under an optical microscope (Statter, 1985, Culling, 1974). The number of osteoclasts was counted at 10 sites per 1 mm 2 of tissue, and the mean and standard deviation were calculated. As a result, Figure 1 shows the external appearance of the ribs separated during fracture healing. On the 10th day after the fracture, there was no significant difference in the bone thickness between the groups. On the 20th day after the fracture, the bone size of the SS-EtOH and SS-H 2 O groups were significantly reduced compared to the other groups. In the treatment of rib fractures, osteoblasts differentiated from osteoprogenitor cells accumulate on the bone surface at the same distance from the fracture site and progress to new bone remodeling toward the fracture site. In the embodiment, fibroblasts and capillary tissues proliferated in the fractured area, and newly formed loose granulation tissues were gradually thickened in the fractured areas. Sex tissue was formed. In this experiment, while the healing of fractured ribs continued, the collagen and supernatural bones produced in the bone were replaced with bony callus, which, as is known, is known as membrane and osteochondral processes (Banks, 1986). Ross et al., 1995, rkd et al., 1996) observed that supernatural bones formed in the original bones were destroyed by osteoclasts and replaced with immature bones or trabecular bones, which are harder bones. During bone healing, more specifically, these cartilage support bones of Callus are destroyed by chondroclasts and then calcified and replaced with lamellar bones, which are compact bones. , 1986). These fracture healing rates were found to take more than 30 days in the ribs of rats, and in humans, depending on the nutritional status and fracture type of individuals, it was known that it took 6-12 weeks in healthy people (river, etc.). ., 1996). The present study showed that the supplementation of safflower seed powder had a positive effect on the fracture healing process and shortened the healing time. When comparing the tissue types of the safflower seed group and the control group observed according to the progress of healing of the fractured tissue, as shown in FIG. 2, the rib tissue of the sham-operation group was recognized as the same as the normal group. In other words, compact bones composed of osteon, in which lamellar bones are arranged around the Harversian canal, are located around the bone marrow cavity. Trabecular bone, hyaline cartilage, and connective tissue were hardly observed. As shown in Table 5 and FIG. 3, the fractured tissue was formed on the fracture line at the 10th day after the fracture, and the formation of the airbone consisting of connective tissue, supernatural bone, and striatal bone was observed. On the other hand, moderate connective tissue was observed in SS-powder, SS-EtoH and SS-H 2 O groups. In addition, supernatural bones were relatively abundantly observed in all safflower seed groups, but in the control group, the supernatural bones were observed with abundance. On the other hand, a small amount of striatal bone from the fracture healing phase was observed in the control group, SS-powder group and SS-EtOH group, but there was no difference. However, SS-H 2 O group showed a slight increase compared to the other groups. On the 10th day of fracture, the number of osteoclasts was 3.80 ± 0.84 in the control group, as shown in Table 6, but 6.80 ± 0.84 and 5.60 ± 0.55 in the SS-powder and SS-H 2 O groups, respectively. Increased by (p <0.01). However, in the SS-EtOH group, it was observed as 3.60 ± 0.89, which is almost similar to the control group.
골절후 20일째 골절조직을 보면 표 5와 도 4에 나타낸 바와 같이 모든 실험군에서 골절 후 10일째와 유사하게 결합조직, 초자연골 및 지주골로 구성된 가골이 풍부하게 관찰되었으나 골절후 10일째에 비해 비교적 결합조직과 초자연골량은 감소한 반면 지주골의 양은 증가되었다. 대조군에서는 결합조직이 소량 관찰되어 골절 후 10일째에 비하여 현저히 감소되었으나 초자연골과 지주골은 중등도로 관찰되어 골절 후 10일째와 유사한 수준으로 나타났다. 한편, SS-powder군과 SS-H2O군의 결합조직량은 극소량만이 관찰되어 대조군에 비하여 현저히 작은 양이었으나 SS-EtOH군에서는 대조군과 유사한 양이 관찰되었다. 또한 초자연골의 양은 SS-powder군과 SS-EtOH군에서 소량 관찰되었으며, SS-H2O군에서는 극소량 관찰되어 대조군에 비하여 현저한 감소를 볼 수 있었다. 골절치유 후반기에 나타나는 지주골량은 대조군에 비해 SS-powder, SS-EtOH 및 SS-H2O군에서 다량 관찰되어 홍화씨 분말, 홍화씨 에탄올추출물 및 홍화씨 열수추출물이 늑골골절 치유기간을 단축시킨 것으로 나타났다. 또 골절후 20일째 파골세포의 수적변화는 표 6에서와 같이 대조군에서는 3.60±1.14로 관찰되었으나 SS-powder군과 SS-H2O군에서는 각각 7.20±0.84와 6.20±1.30으로 관찰되어 대조군에 비해 유의적으로(p<0.01) 증가되었으며 골절 후 10일째에 비해서도 다소 증가되었다. 그러나 골절 후 20일째 SS-EtOH군에서는 4.00±0.71로 관찰되어 대조군 및 골절 후 10일째에 비해 증가된 경향을 보였으나 유의성은 없는 것으로 나타났다.As shown in Table 5 and FIG. 4, the fractured tissues were found to have abundant bones composed of connective tissue, supernatural bones and striatal bones, as shown in Table 5 and FIG. Tissue and supernatural bone mass decreased, while the amount of superficial bone increased. A small amount of connective tissue was observed in the control group, which was significantly decreased compared to 10 days after the fracture, but the supernatural bone and the holding bone were moderately observed, similar to the 10 days after the fracture. On the other hand, only a small amount of connective tissue was observed in the SS-powder group and the SS-H 2 O group, which was significantly smaller than that of the control group. In addition, the amount of supernatural bone was observed in the SS-powder and SS-EtOH groups, and only a small amount was observed in the SS-H 2 O group. Skeletal bone mass in the second stage of fracture healing was observed in SS-powder, SS-EtOH, and SS-H 2 O groups compared to the control group, indicating that safflower seed, safflower ethanol extract and safflower hot water extract shortened the healing period of rib fracture. The number of osteoclasts on the 20th day after fracture was 3.60 ± 1.14 in the control group as shown in Table 6, but 7.20 ± 0.84 and 6.20 ± 1.30 in the SS-powder and SS-H 2 O groups, respectively. There was a significant increase (p <0.01) and a slight increase compared to 10 days after fracture. However, in the SS-EtOH group at 20 days after fracture, it was 4.00 ± 0.71, which was increased compared to the control group and 10 days after fracture.
제 4단계: 혈액채취 및 생화학적 분석Stage 4: Blood Collection and Biochemical Analysis
동물희생 전 12시간동안 공복시킨 후 ketamin-HCl(유한양행)로 마취시키고 복부 대정맥으로부터 채혈하였다. 채취한 혈액은 3,000rpm에서 20분간 원심분리하여 혈장을 분리하고 분석시까지 -60℃에 보관하였다. 혈장 칼슘과 인, GOT 및 GPT활성도는 Asanset의 칼슘과 무기인 측정용 키트, 그리고 GOT 및 GPT 측정용 키트에 의해 전체-ALP 활성도는 Assnset의 ALP 측정용 키트를 사용하여 분광광도법으로 측정하였다. 혈장 단백질 농도는 브래드포드 방법을 이용하여 정량하였다. 혈장 PTH 수준과 칼시토닌(calcitonin) 및 뇨 중 데옥시피리디놀린(deoxypyridinoline;DPD) 수준은 각각 면역분석법을 적용한 INTACT PTH(Nichols institute diagnostics사 제품)를, 칼시토닌 수준은 Calcitonin Double Antibody kit(Diagnostic products corgoration 사 제품)을 이용하였으며 동위원소 활성은 Gammal Counter로 측정하였다. Bone-ALP 활성도는 B-ALP ALKPASE-BTM kit(METRA BIOSYSTEM사 제품)를 이용하여 측정하였고 또한 혈장 오스테오칼신(osteocalcin) 농도 측정에는 Human Osteocalcin 키트(Nichols institute diagnostic 사 제품)가 사용되었다. 실험결과는 하기 1, 2, 3, 4 에 정리하였다.The animals were fasted for 12 hours prior to animal sacrifice, and then anesthetized with ketamin-HCl (final ambulance) and collected from the abdominal vena cava. The collected blood was centrifuged at 3,000 rpm for 20 minutes to separate plasma and stored at -60 ° C until analysis. Plasma calcium and phosphorus, GOT and GPT activity was measured by Asanset's calcium and inorganic phosphorus measurement kit, and GOT and GPT measurement kit, total-ALP activity was measured spectrophotometrically using Assnset's ALP measurement kit. Plasma protein concentrations were quantified using the Bradford method. Plasma PTH levels, calcitonin, and urinary deoxypyridinoline (DPD) levels were measured using the INTACT PTH (Nichols institute diagnostics) applied immunoassay, and calcitonin levels were calcitonin double antibody kit (Diagnostic products corgoration). Company) and isotope activity was measured by a Gammal Counter. Bone-ALP activity was measured using the B-ALP ALKPASE-BTM kit (manufactured by METRA BIOSYSTEM) and the Human Osteocalcin kit (manufactured by Nichols institute diagnostic) was used to measure plasma osteocalcin concentration. The experimental results are summarized in the following 1, 2, 3, 4.
1. 혈중 PTH와 칼시토닌 및 뇨중 DPDBlood PTH and Calcitonin and Urine DPD
혈중 칼슘농도가 정사치보다 감소되었을 때 골흡수를 촉진시켜 혈중으로 칼슘을 동원하는 PTH와 PTH의 작용과 상반되는 기능을 가진 칼시토닌의 혈중농도를 표 7에 나타냈다. PTH 수준은 골절유도 후 10일째에서는 SS-H2O군만이 다른 세군에 비해 유의적으로 높았고 골절 20일째에는 SS-powder군과 SS-H2O군이 유의적으로 높게 나타나 홍화씨 열수추출물 투여군인 SS-H2O군이 유의적으로 높은 수준을 보였다. 기간별 비교에서는 SS-powder군과 SS-EtOH군이 골절 후 20일째 유의적으로 높게 나타나 표 6에서 SS-powder군과 SS-H2O군의 파골세포수가 20일째 대조군에 비해 높게 나타난 결과와 일치하였다. 혈중 칼시토닌 수준 비교에서는 골절 후 10일째와 20일째의 각 식이군내 차이가 없었고 기간별 차이도 보이지 않았다. 즉 골형성을 도모하는 지표 중 칼시토닌은 홍화씨에 의한 골절치유과정에 크게 관여하지 않았음을 나타냈다. 골흡수 지표 중의 하나인 뇨중 DPD 농도는 골절 후 10일째 SS-EtOH군과 SS-H2O군이 유의적으로 높았으며 대조군과 SS-powder군은 10일째보다 20일째에 각각 유의적으로 DPD 수준이 증가되었다. 또한 골절 후 20일째 뇨중 DPD 배설수준은 거의 비슷하게 관찰되었다. 골흡수를 촉진하는 PTH 수준과 골흡수지표인 뇨중 DPD 농도간에는 SS-H2O 1군, SS-powder 2군, SS-EtOH 2군에서 비례경향을 볼 수 있었다. 특히 열수추출물을 급여한 SS-H2O군에서는 표 6의 파골세포수 변화결과와 표 7이 PTH 및 DPD 수준에서 나타난 바와 같이 골절 후 10일째 및 20일째까지 골흡수가 지속적으로 일어났음을 알 수 있다. 일반적으로 뼈의 재구성(bone remodelling)과 복구(repair)과정은 파골세포와 조골세포(osteoblast)의 협동작용에 의해 이루어지는데 파골세포의 작용은 홍화씨 특히 열수추출물의 투여에 의해 촉진됨을 알 수 있었다.Table 7 shows the blood concentrations of calcitonin, which has a function opposite to the action of PTH and PTH that promote calcium resorption by stimulating bone resorption when blood calcium concentration is lower than normal. PTH levels in the day 10 after the induction fracture SS-H 2 O gunman the day 20 significantly higher fracture than the other three groups, the SS-powder group and the SS-H 2 O group appeared significantly higher safflower hot water extract group SS-H 2 O group showed significantly higher levels. In comparison, the SS-powder and SS-EtOH groups were significantly higher on the 20th day after the fracture, and the number of osteoclasts in the SS-powder and SS-H 2 O groups was higher than the control group on the 20th day. It was. There was no difference in blood calcitonin levels in each diet group at 10 and 20 days after fracture and there was no difference in period. In other words, calcitonin was not significantly involved in the healing process of safflower seed. Urinary DPD concentration, one of the indexes of bone resorption, was significantly higher in the SS-EtOH and SS-H 2 O groups at 10 days after fracture, and the DPD level was significantly higher in the control and SS-powder groups at 20 days than 10 days, respectively. This has been increased. In addition, the levels of urinary DPD excretion were observed at 20 days after fracture. Proportional tendency was observed between SS-H 2 O 1 group, SS-powder 2 group, and SS-EtOH 2 group between PTH level promoting bone resorption and DPD concentration. In particular, the SS-H 2 O group fed hot water extract showed that the bone resorption occurred continuously until 10 and 20 days after fracture, as shown by the osteoclast counts in Table 6 and Table 7 in the PTH and DPD levels. Can be. In general, the bone remodeling and repair process is performed by the coordination of osteoclasts and osteoblasts. The osteoclasts are promoted by the administration of safflower seed, especially hot water extract.
2. 혈중 오스테오칼신 농도, 특정-뼈 알카라인 포스파타아제(bone-ALP) 및 전체 알카라인 포스파타아제(total-ALP) 활성도2. Blood osteocalcin concentration, specific-bone alkaline phosphatase (bone-ALP) and total alkaline phosphatase (total-ALP) activity
골형성 지표 중의 하나인 오스테오칼신(osteocalcin) 농도와 뼈의 석회화과정을 직접적으로 도모하는 뼈-ALP 활성도 및 간에서 혈중으로 분비된 전체-ALP 활성도를 표 8에 나타냈다. 혈중 오스테칼신(osteocalcin) 농도비교에서 골절 후 10일 째 대조군이 SS-powder 및 SS-EtOH군에 비해 유의적으로 높았고 골절 후 20일째에서도 대조군이 SS-H2O군에 비해 높게 나타났다. 그리고 기간별 비교에서는 SS-EtOH 군만이 다른 군에 비해 20일째에 유의적으로 높은 수준을 나타냈다. 뼈-ALP 활성도는 골절 후 10일째의 군내비교에서 유의적 차이는 없었으나 SS-powder군과 SS-H2O군이 높은 경향이었다. 골절후 20일째는 SS-EtOH군이 대조군과 SS-powder군에 비해 유의적으로 높윤 수준이였으며 SS-H2O군도 높은 경향을 보였다. 전체-ALP 활성도는 골절 후 기간별과 동일기간내 식이군간 비교에서 유의적인 차이가 전혀 없었다.Osteocalcin concentration, one of the bone formation indices, bone-ALP activity directly promoting the calcification process of bone, and total-ALP activity released into the blood in the liver are shown in Table 8. In the comparison of serum osteocalcin concentration, the control group was significantly higher than the SS-powder and SS-EtOH groups at 10 days after fracture, and the control group was higher than the SS-H 2 O group at 20 days after fracture. In comparison, the SS-EtOH group showed a significantly higher level at 20 days than the other groups. Bone-ALP activity was not significantly different in group comparison at 10 days after fracture, but SS-powder and SS-H 2 O groups tended to be higher. On the 20th day after the fracture, the SS-EtOH group showed a significantly higher level than the control group and the SS-powder group, and the SS-H 2 O group showed a higher tendency. Total-ALP activity was not significantly different between the groups after the fracture and during the same period.
3. 혈중 단백질, Ca, P 농도 및 Ca/P 비율3. Blood Protein, Ca, P Concentration and Ca / P Ratio
혈중 단백질 Ca 및 P농도의 Ca/P 비율은 표 9와 같다. 혈중 단백질농도 비교에서 기간별 비교 및 골절 후 20일째 식이군내 비교에서는 유의적 차이는 없었고 골절 후 10일째에는 그 이유는 알 수 없으나 SS-EtOH군이 대조군과 SS-powder군에 비해 유의적으로 낮게 나타났다. 골절 후 20일 째에는 홍화씨 투여군들이 대조군에 비해 혈장 단백질 농도가 낮은 경향으로 나타났다. 혈중 Ca 농도는 골절 후 10일째 군내 및 기간별 비교에서 유의 차이가 없었으나 골절 후 10일째 및 20일째의 홍화씨 실험군(SS-powder 2군, SS-EtOH 2군 및 SS-H2O군)이 대조군(대조군 2군)에 비해 높은 경향을 보였다. 한편 골절후 20일째에는 SS-EtOH군의 혈장 Ca 농도가 대조군에 비해 유의적으로 높게 나타났다. 혈중 P 농도 비교에서는 골절 후 10일째의 SS-EtOH군과 SS-H2O군이, 골절후 20일째에서는 SS-powder군, SS-EtOH군 및 SS-H2O군이 대조군보다 각각 유의적으로 높게 나타났다. 기간별로는 SS-EtOH군의 20일째 P 농도가 10일째 보다 유의적으로 낮았다. 한편 혈중 칼슘과 인의 비율을 보면 골절후 10일째에는 SS-EtOH 군이 대조군에 비해 유의적으로 낮았고 골절 후 20일 째에는 SS-powder군과 SS-H2O군이 대조군에 비해 유의적으로 낮은 비율로 나타나 골절 후 10일째와 20일째 모두 대조군이 홍화씨 실험군에 비해 높은 것으로 나타났다. 또한 기간별 비교에서는 SS-EtOH군만이 20일째에 유의적으로 높게 나타났다.Ca / P ratios of blood protein Ca and P concentrations are shown in Table 9. There was no significant difference in the comparison of blood protein concentrations in the period and in the diet group 20 days after the fracture. At 10 days after the fracture, the reason was unknown, but the SS-EtOH group was significantly lower than the control group and the SS-powder group. . At 20 days after the fracture, safflower seed group showed lower plasma protein concentration than the control group. Serum Ca concentration was not significantly different between the groups at 10 days after fracture and at the time of fracture, but the safflower group (SS-powder 2 group, SS-EtOH 2 group and SS-H 2 O group) at 10 and 20 days after fracture were It showed a higher tendency compared to (control group 2). On the other hand, 20 days after the fracture, the plasma Ca concentration of the SS-EtOH group was significantly higher than that of the control group. In the comparison of blood P concentration, the SS-EtOH and SS-H 2 O groups at the 10th day after the fracture and the SS-powder, SS-EtOH and SS-H 2 O groups at the 20th day after the fracture were significantly more significant than the control group. Appeared high. By period, the P concentration of the SS-EtOH group was significantly lower than that of the 10th day. On the other hand, the ratio of calcium and phosphorus in blood was significantly lower in the SS-EtOH group than the control group at 10 days after fracture, and significantly lower in the SS-powder and SS-H 2 O groups at 20 days after fracture. The control group was higher than the Safflower group in both 10 and 20 days after fracture. In comparison, the SS-EtOH group was significantly higher at 20 days.
4. 혈중 GOT와 GPT 활성도4. Blood GOT and GPT Activity
혈중 GOT 활성도와 GPT 활성도는 표 10에 나타낸 바와 같으며 혈중 GOT 활성도는 골절 후 10일 째 대조군이 나머지 세군에 비해 유의적으로 낮았으며 골절 후 20일째에서는 SS-EtOH군과 SS-H2O이 대조군과 SS-powder군에 비해 유의적으로 낮게 나타났다. 또한 기간별 비교에서는 대조군이 골절 후 10일째에 비해 20일째 유의적으로 높게 나타났고, SS-EtOH과 SS-H2O군은 20일째에 유의적으로 낮아졌다. GPT 활성도 비교에서는, 골절 후 10일째에는 대조군이 유의적으로 낮았고 골절 후 20일째에서는 SS-EtOH군과 SS-H2O군이 대조군과 SS-powder군에 비해 유의적으로 낮았다. 기간별로는 대조군과 SS-powder군이 10일째에 비해 20일째에 모두 유의적으로 낮게 나타났다. 간기능진단의 대표적인 지표인 이들 GOT와 GPT의 활성도는 대조군과 동일한 식이를 섭취한 sham군과 비교시 모두 정상적인 범위에 있었으므로 홍화씨의 독성은 전혀 없는 것으로 관찰되었다.Serum GOT activity and GPT activity are shown in Table 10. Serum GOT activity was significantly lower at 10 days after fracture compared to the other three groups, and SS-EtOH and SS-H 2 O were significantly decreased at 20 days after fracture. It was significantly lower than the control and SS-powder groups. In comparison, the control group was significantly higher on the 20th day than the 10th day after the fracture, and the SS-EtOH and SS-H 2 O groups were significantly lower on the 20th day. In comparison of GPT activity, the control group was significantly lower at 10 days after fracture and the SS-EtOH and SS-H 2 O groups were significantly lower than the control group and SS-powder group at 20 days after fracture. By period, both control and SS-powder groups were significantly lower at 20 days than at 10 days. The activity of these GOT and GPT, which is a representative indicator of liver function diagnosis, was in the normal range compared with the sham group fed the same diet as the control group, so that there was no toxicity of safflower seed.
이상 상기 실시예와 실험예를 통하여 설명한 바와 같이 본 발명은 식이의 5% 수준으로 급여된 토종 홍화씨 분말, 에탄올추출 분말 및 열수추출 분말은 흰쥐의 골절된 늑골의 치유속도를 빠르게 하여 복구에 걸리는 시간을 단축시켰고 특히 홍화씨 열수추출 분말을 급여한 SS-H2O군에서는 PTH, DPD, bone-ALP 및 파골세포수의 변화로 보아 골절 회복과정에서 뼈의 재구성을 지속적으로 활발히 진행시키는 뛰어난 효과가 있으므로 골절치유, 골다공증 예방 및 골강화을 위한 건강식품산업상 매우 유용한 발명인 것이다.As described above through the above Examples and Experimental Examples of the present invention, the safflower seed powder, ethanol extract powder, and hot water extract powder fed to the 5% level of the diet are fast recovery time of fractured ribs in rats In particular, SS-H 2 O group fed with safflower seed hot water extract powder showed excellent effect of continuously reconstructing bone during fracture recovery because of changes in PTH, DPD, bone-ALP and osteoclast count. It is a very useful invention in the health food industry for fracture healing, osteoporosis prevention and bone strengthening.
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| KR20030086158A (en) * | 2002-05-03 | 2003-11-07 | (주)메디엔스 | Composition for accerating growth |
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