KR101652206B1 - Composition for stimulating bone or bone tissue comprising extracts of leaves of stauntonia hexaphylla - Google Patents
Composition for stimulating bone or bone tissue comprising extracts of leaves of stauntonia hexaphylla Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
본 발명은 조골세포분화 또는 연골세포 분화 촉진용 조성물에 관한 것으로서, 보다 구체적으로는 천연원료를 이용하여 독성 및 부작용 없이 안전하게 사용될 수 있는 멀꿀 잎 추출물을 함유하는 골 및 연골 조직 손상의 억제 및 치료를 위한 용도로 사용될 수 있는 골(조직) 생성 촉진용 조성물에 관한 것이다. 본 발명에서의 멀꿀 잎 추출물을 유효성분으로 포함하는 기능성 식품 조성물은 연골조직생성촉진 또는 예방하기 위한 기능성 건강식품으로 사용될 수 있다. The present invention relates to a composition for promoting osteoblast differentiation or chondrocyte differentiation promotion, and more particularly, to a composition for preventing and treating bone and cartilage tissue damage containing a mulberry leaf extract which can be safely used without toxicity and side effects using natural raw materials To a composition for promoting bone formation. The functional food composition containing the extract of alfalfa leaf as an active ingredient in the present invention can be used as a functional health food for promoting or preventing the formation of cartilage tissue.
Description
본 발명은 조골 또는 연골조직 생성 촉진용 기능성 식품 조성물에 관한 것으로서, 보다 구체적으로는 멀꿀 잎 조추출물을 함유하는 천연원료를 이용하여 독성과 부작용이 없이 안전하게 사용될 수 있는 조골 또는 연골 조직 손상 억제와 치료를 위한 용도로 사용될 수 있는 연골조직 생성 촉진용 기능성 식품 조성물에 관한 것이다.
The present invention relates to a functional food composition for promoting osteoarthritis or cartilage tissue formation, and more particularly, to a functional food composition for preventing osteoarthritis or cartilage tissue damage which can be safely used without toxicity and side effects by using a natural raw material containing mulberry leaf crude extract And to a functional food composition for promoting cartilage formation.
골 조직은 대체로 뼈의 표면이 튼튼한 치밀골질로 이루어지고, 중심부 또는 장골(長骨)의 양 끝은 골질이 그물눈같이 연합된 해면골질(海綿骨質)로 되어 있다. 대부분의 뼈는 처음에 결합조직 중의 연골로서 발생하여 이것이 나중에 골조직으로 바뀌는데, 일부 뼈는 결합조직 중에서 직접 만들어진다. Bone tissue usually consists of a hard bone with a hard surface, and both ends of the center or long bone are associated with a spongy bone quality like bone. Most bones initially develop as cartilage in connective tissue, which later translates into bone tissue, some of which are made directly from connective tissue.
골단(骨端)에서는 이웃뼈와 접하는 부분에 관절면이 있고, 그 표면은 초자연골(硝子軟骨)인 관절연골로 덮여 있다. 해면질 가운데의 해면소주(海綿小柱)는 일정한 배열로 되어 있는 것이 특징이다. 골 간부(骨幹部)의 넓은 수강(髓腔)은 해면소주로 된 소강(小腔)과 연속되어 있고 모두 골수로 채워져 있다. At the tip of the bone, there is a joint surface in contact with the neighboring bone, and its surface is covered with articular cartilage, which is supernatural bone (vitreous cartilage). The marine spoil (sponge small pillar) in the spongy is characterized by a constant arrangement. The large intestine of the caudal trunk is continuous with a small lumen of spongy soju and is filled with bone marrow.
조혈작용을 하는 골수는 혈관이 많이 분포되어 붉은색을 띠며, 적색골수라고 한다. 골질의 구조는 치밀질이나 해면질 모두 두께 5∼12㎛의 골판이 겹쳐 있는데, 치밀질에서는 동심원상으로 몇 층이 겹친 골층판(하버스 층판)이 여러 방향으로 배열되어 있고, 각 층판의 중심에는 하버스관이 있어 혈관이 통한다.The hematopoietic bone marrow is reddish and red marrow. The bone structure is composed of a corrugated layer with a thickness of 5 to 12 μm in both dense quality and spongy matter. In the dense layer, a plurality of concentric corrugated laminae (laminae) are arranged in various directions. There is ha bus pipe, and blood vessel runs.
골 세포는 골층판 사이에 배열되어 있으며, 불규칙한 별 모양으로 가는 원형질 돌기로 인접한 다른 골세포와 연결되어 있다. 뼈의 표면에는 질긴 결합조직성 골막이 있으며, 신경과 혈관이 분포되어 뼈의 보호와 영양을 맡고 있다. 골막이 결손되면 뼈의 생존, 신생 및 재생 등이 곤란하게 된다. 골질의 성분은 수분 20%, 세포를 포함한 유기질 35%, 무기질 45%인데, 뼈의 일정한 탄력성은 유기질이 있기 때문이다. 연령이 증가함에 따라 무기질(주로 인산칼슘)이 증가하여 뼈의 경도도 증가한다.The osteocytes are arranged between the stratum corneum and are connected to other adjacent osteoclasts by protruding protrusions with irregular star shape. There is a rigid connective tissue periosteum on the surface of the bones, and nerve and blood vessels are distributed to protect and nourish the bones. If the periosteum is lost, it is difficult to survive, start and regenerate the bone. The bone is composed of 20% moisture, 35% organic and 35% inorganic, because the elasticity of the bone is organic. As age increases, minerals (mainly calcium phosphate) increase and bone hardness increases.
한편 멀꿀은 학명 Stauntonia hexaphylla로서 식물계, 속씨식물문, 쌍떡잎식물강, 미나리아재비목에 속하는 식물이다. 주로 한국, 일본, 타이완, 중국 등지에 분포하고, 원줄기는 5m 정도 뻗어가고 잎은 어긋나며 5∼7개의 작은 잎으로 된 손바닥모양 겹잎(掌狀複葉)이다. 작은 잎은 두껍고 달걀모양 또는 타원형이며 가장자리가 밋밋하다. 잎자루는 길이 6∼8cm, 작은 잎자루는 3cm이다. 꽃은 5월에 피고 1가화(一家花)이며 황백색이고 총상꽃차례[總狀花序]에 달린다. Meanwhile, Stauntonia hexaphylla is a plant belonging to the plant family, herbaceous gates, dicotyledonous plant river and buttercups. It is mainly distributed in Korea, Japan, Taiwan and China. The main stem is 5 ~ 7m long, palm shaped compound leaf with 5 ~ 7 small leaves. Leaves are thick, ovate or oval, with flat edges. The petiole is 6 ~ 8cm long and the petiole is 3cm. The flower blooms in May and is a single flower. It is yellowish white and runs on a flower stem.
암꽃의 작은 꽃가지는 가을에 적갈색으로 되고 많은 피목(皮目)이 있어 거칠다. 열매는 장과(漿果)로 달걀모양 또는 타원형이고 길이 5~10cm이며 10월에 적갈색으로 익고 과육(果肉)은 으름보다 맛이 좋다. 종자는 달걀모양의 타원형으로 흑색이다. The small flower blooms of female flower become reddish brown in autumn and there are many shrubs. Fruits are ovate or elliptical in length, 5 ~ 10cm long, ripen in reddish brown in October, and flesh is tastier than sunflower. Seeds are oval oval and black.
본 발명은 우리나라에서 흔히 구할 수 있는 천연재료로서 멀꿀 잎 추출물을 이용하여 치주염 또는 골다공증 치료, 예방을 위한 기능성 식품 조성물을 제공할 목적으로 멀꿀 잎 열수추출물, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물을 추출하여 조골세포 ALP 활성 및 조골세포 분화 촉진실험 및 골 또는 연골조직의 생성 촉진 실험을 실시하였다. The present invention relates to a functional food composition for the treatment and prevention of periodontitis or osteoporosis using a mulberry leaf extract as a natural material which is commonly available in Korea. The extract of the mulberry leaf hot water extract and the ethyl acetate fraction of the mulberry leaf hot water extract are extracted The osteoblast ALP activity and osteoblast differentiation promotion experiment and bone or cartilage tissue promotion experiment were performed.
실험 결과 멀꿀 잎 조추출물이 연골조직 생성 촉진 및 예방과 관련한 효과가 있다는 것을 실험적으로 확인함으로서 멀꿀 잎 조추출물을 이용한 골생성 촉진용 기능성 식품 조성물 및 치료제를 제공하고자 한다.
As a result of experiment, it is experimentally confirmed that a mulberry leaf crude extract has an effect on promotion and prevention of cartilage tissue formation, thereby providing a functional food composition and a therapeutic agent for promoting osteogenesis using mulberry leaf crude extract.
본 발명은 천연물인 멀꿀 잎 유래 추출물을 유효성분으로 사용함으로써 장기간 복용하여도 부작용 없이 안전한 골 및 연골 조직 손상의 억제 및 치료를 위한 용도로 사용될 수 있는 연골 조직 생성 촉진용 멀꿀 잎 조추출물 함유 기능성 식품조성물을 제공하고자 한다.
The present invention relates to a functional food containing mulberry leaf extract for promoting cartilage tissue formation which can be used for the prevention and treatment of safe bone and cartilage tissue damage without side effects even if taken for a long time by using extract of mulberry leaf which is a natural product as an active ingredient Composition.
본 발명의 목적을 달성하기 위해, 멀꿀 잎 조추출물 또는 비극성가용 추출물을 유효성분으로 포함하는 연골 조직 손상의 억제, 치료를 위한 연골조직 생성 촉진용 기능성 식품조성물을 제공한다. In order to accomplish the object of the present invention, there is provided a functional food composition for promoting cartilage tissue formation for inhibiting and treating cartilage tissue damage comprising an extract of mulberry leaf or a nonpolar soluble extract as an active ingredient.
멀꿀 잎 조추출물은 물, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 또는 이들의 혼합용매 중 어느 하나에서 가용한 추출물로서 상기 비극성 용매는 헥산, 클로로포름, 디클로메탄 및 에틸아세테이트 중에서 선택되는 어느 하나일 수 있다. The mulberry leaf crude extract is an extract which is soluble in water, methanol, ethanol, propanol, isopropanol, butanol or a mixed solvent thereof. The nonpolar solvent may be any one selected from hexane, chloroform, dichloromethane and ethyl acetate have.
멀꿀 잎 열수추출물은 조골세포에서 생성되는 ALP 활성 및 조골세포 분화를 증가시키는 기능이 있고, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물은 조골세포에서 생성되는 ALP 활성 및 조골세포 분화를 증가시키는 기능을 갖는다. The hot - water extract of mulberry leaves has the function of increasing ALP activity and osteoblast differentiation produced in osteoblast. Ethyl acetate fraction of mulberry leaf hot - water extract has the function of increasing ALP activity and osteoblast differentiation produced in osteoblast.
또한, 멀꿀 잎 조추출물은 전체 기능성 식품조성물의 0.01 내지 99.9중량%로 포함되며, 기능성 식품조성물의 1일당 투여량은 상기 추출물이 10 내지 1000mg/kg 체중으로 제공되는 것을 특징으로 한다.
The mullen leaf crude extract contains 0.01 to 99.9% by weight of the total functional food composition, and the daily dose of the functional food composition is 10 to 1000 mg / kg body weight of the extract.
본 발명의 멀꿀 잎 열수추출물, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물의 조골세포 ALP 활성 및 조골세포 분화를 촉진시킴으로써 연골조직의 생성을 촉진하는 효과가 확인됨으로써 멀꿀 잎 조추출물을 유효성분으로 포함하는 기능성 식품 조성물은 연골 생성촉진을 위한 기능성 식품으로 사용될 수 있다.
As a result of promoting the osteoblast ALP activity and osteoblast differentiation of the ethyl acetate fraction of the mulberry leaf hot water extract of the present invention and the hot water extract of mulberry leaves, the effect of promoting the formation of cartilage tissue was confirmed, The food composition can be used as a functional food for promoting cartilage formation.
도 1은 멀꿀 잎 열수 추출물과 분획물의 제조 모식도이다
도 2는 멀꿀 잎 열수 추출물의 ALP활성에 미치는 영향을 나타낸 그래프(골아세포 분화를 통한 골형성 촉진 활성)이다.
도 3은 멀꿀 잎 열수 추출물 에틸아세테이트 분획물의 ALP활성에 미치는 영향을 나타낸 그래프이다.
도 4는 멀꿀 잎 열수추출물, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물의 ALP 활성 염색 결과를 나타낸 그래프이다.1 is a schematic diagram showing the preparation of hot water extract and fractions of mulberry leaf
Fig. 2 is a graph showing the effect of ALP activity on mulberry leaf hot-water extract (osteogenesis promoting activity through osteoblast differentiation).
FIG. 3 is a graph showing the effect of ethyl acetate fraction of hot water extract of mulberry leaves on ALP activity.
4 is a graph showing the results of ALP activity staining of the ethyl acetate fraction of hot water extract of mulberry leaves and hot water extract of mulberry leaves.
본 발명은 멀꿀 잎 조추출물 또는 비극성가용추출물을 유효성분으로 포함하는 골 및 연골 조직 손상의 억제 및 치료를 위한 골조직 생성 촉진용 기능성 식품조성물 및 이를 이용한 치주염 또는 골다공증 치료제를 제공한다.
The present invention provides a functional food composition for promoting bone tissue formation for inhibiting and treating bone and cartilage tissue damage comprising extracts of mulberry leaf or nonpolar soluble extract as an active ingredient, and a therapeutic agent for periodontitis or osteoporosis using the same.
1. One. 멀꿀Mellow 잎 leaf 열수추출물Hot water extract 제조 Produce
도 1은 멀꿀 잎 열수 추출물과 분획물의 제조를 위한 모식도를 나타낸다. 멀꿀 (Stauntonia hexaphylla) 잎 10kg을 증류수로 수세한 다음, 증류수 200L를 가하고, 전기약탕기로 110℃에서 4시간 동안 가열하여 추출하였다. 400 메쉬 여과포로 여과한 다음 감압회전농축기로 농축하였다. Figure 1 shows a schematic diagram for the preparation of hot water extracts and fractions of mulberry leaves. 10 kg of Stauntonia hexaphylla leaf was washed with distilled water, and then 200 L of distilled water was added thereto, followed by heating at 110 ° C. for 4 hours in an electric bath. Filtered through 400 mesh filter cloth, and then concentrated using a rotary evaporator under reduced pressure.
여과 후 남은 잔사에 다시 동량의 증류수를 사용하여 동일 과정으로 2번 더 추출, 여과 및 감압 농축한다. 농축된 열수추출물을 동결건조기(Freeze dryer)에서 동결건조 하였다. 이 과정을 통해 멀꿀 잎 열수추출물 1kg (10%)을 얻었다.
After the filtration, the same amount of distilled water is again used for the remaining residue, which is further extracted twice, filtered and concentrated under reduced pressure. The concentrated hot water extract was lyophilized in a freeze dryer. Through this process, 1 kg (10%) of hot water extract of mulberry leaves was obtained.
2. 2. 멀꿀Mellow 잎의 극성용매, Leaf polar solvent, 비극성용매Nonpolar solvent 가용 Available 분획물의Fraction 제조 Produce
도 1에서 도시된 바와 같이 제조된 멀꿀 잎 열수추출물을 유기 용매를 이용하여 다음과 같이 분획하였다.The hot water extract of mulberry leaves prepared as shown in Fig. 1 was fractionated as follows using an organic solvent.
2.1. 헥산 가용성 분획 분리2.1. Hexane-soluble fraction separation
멀꿀 잎 열수추출물로서 얻어진 멀꿀잎 추출물 250g을 5L의 증류수에 완전히 용해시킨 후에 분획여두에 넣고 헥산 5L를 첨가하여 헥산 불용성층(수층)과 헥산가용성층을 분리하였다. 다시 헥산 불용성층(수층)을 대상으로 동일한 공정을 3번 반복하여 헥산 불용성 분획 및 가용성 분획을 수집하였다. 250 g of mulberry leaf extract obtained as a hot water extract of mulberry leaves was completely dissolved in 5 L of distilled water, and then put into a separating funnel. 5 L of hexane was added to separate the hexane insoluble layer (water layer) and the hexane soluble layer. Again, the hexane insoluble fraction and the soluble fraction were collected by repeating the same process three times for the hexane insoluble layer (water layer).
2.2. 클로로포름 가용성 분획분리2.2. Chloroform-soluble fractionation
헥산불용성 분획(수층)에 클로로포름 5L를 가하여 섞은 후에 클로로포름가용성 분획 및 불용성 분획을 분리하였고, 클로로포름 불용성층(수층)을 대상으로 동일한 공정을 3번 반복하여 클로로포름 불용성 분획 및 가용성 분획을 수집하였다.The hexane insoluble fraction (aqueous layer) was mixed with 5 L of chloroform, and then the chloroform-soluble fraction and the insoluble fraction were separated. The chloroform-insoluble fraction and the soluble fraction were collected by repeating the same process three times for the chloroform insoluble layer (water layer).
2.3. 에틸아세테이트 가용성 분획분리2.3. Ethyl acetate soluble fraction separation
클로로포름 불용성 분획(수층)에 에틸아세테이트 5L를 가하여 섞은 후에 에틸아세테이트 가용성 분획 및 불용성 분획을 분리하였고, 에틸아세테이트 불용성층(수층)을 대상으로 동일한 공정을 3번 반복하여 에틸아세테이트 불용성 분획 및 가용성 분획를 수집하였다.After adding 5 L of ethyl acetate to the chloroform insoluble fraction (aqueous layer), the ethyl acetate soluble fraction and the insoluble fraction were separated. The ethyl acetate insoluble layer (water layer) was subjected to the same process three times to collect the ethyl acetate insoluble fraction and the soluble fraction Respectively.
2.4. 부탄올 가용성 분획분리2.4. Butanol soluble fraction fractionation
에틸아세테이트 불용성 분획(수층)에 부탄올 5L를 가하여 섞은 후에 부탄올 가용성 분획 및 불용성 분획을 분리하였고, 부탄올 불용성층을 대상으로 동일한 공정을 3번 반복하여 부탄올 불용성 분획 및 가용성 분획을 수집하였다.After adding 5 L of butanol to the ethyl acetate insoluble fraction (water layer), the butanol soluble fraction and the insoluble fraction were separated, and the butanol insoluble fraction was repeated three times to collect the butanol insoluble fraction and the soluble fraction.
2.5. 물층 분획분리2.5. Fractionation of water layer
멀꿀 잎 열수추출물 250g을 5L의 증류수에 완전히 용해시킨 후에 분획여두에 넣고 상기 헥산 가용성층, 클로로포름 가용성층, 에틸아세테이트 가용성층 그리고 부탄올 가용성층을 분획 분리 후 농축하여 남아있는 유기용매를 제거하고 물 분획을 수집하였다.The hexane-soluble layer, the chloroform-soluble layer, the ethyl acetate-soluble layer and the butanol-soluble layer were separated by fractionation and the remaining organic solvent was removed, and the water fraction Were collected.
이와 같은 멀꿀 잎 열수 추출 및 분획물 수득과정을 통해 제조된 멀꿀 잎 열수추출물 250g에서 헥산 가용성 분획, 클로로포름 가용성 분획, 에틸아세테이트 가용성 분획 및 부탄올 가용성 분획을 감압 농축한 후에 동결 건조함으로서 헥산분획 0.02g(0.015), 클로로포름 분획 0.67g(0.27%), 에틸아세테이트 분획 2.62g(1.05%), 부탄올 분획 68.75g(27.5%), 물 분획 150.14g(60.06%) 을 얻어 시료로 사용하였다.
The hexane-soluble fraction, the chloroform-soluble fraction, the ethyl acetate-soluble fraction and the butanol-soluble fraction were concentrated under reduced pressure and then lyophilized in 250 g of the hot water extract of mulberry leaves prepared from the hot water extraction and fractionation of mulberry leaves to obtain 0.02 g ), Chloroform fraction 0.67 g (0.27%), ethyl acetate fraction 2.62 g (1.05%), butanol fraction 68.75 g (27.5%) and water fraction 150.14 g (60.06%).
3. 3. 멀꿀Mellow 잎 leaf 열수Heat number 추출물에 의한 조골세포의 Extract-induced osteoblast APAP 및 And ALPALP 의 활성측정 Activity measurement
도 2는 멀꿀 잎 열수 추출물의 ALP활성에 미치는 영향을 나타낸 그래프(골아세포 분화를 통한 골형성 촉진 활성)이다.Fig. 2 is a graph showing the effect of ALP activity on mulberry leaf hot-water extract (osteogenesis promoting activity through osteoblast differentiation).
3.1 멀꿀 잎 열수 추출물에 의한 조골세포의 AP 활성측정 3.1 Measurement of AP activity of osteoblasts by hot water extract of mulberry leaves
조골세포는 ALP 활성을 나타내므로, 상기에서 수득된 멀꿀 잎 열수 추출물이 조골세포에서 ALP 활성에 미치는 영향을 측정하였다. 구체적으로, 조골모세포인 C2C12 cells을 48well에 5 x 10^4 cell/well이 되도록 분주하고 세포성장 배지인 10% FBS, 0.1% p/s을 함유한α-MEM essential medium에서 3일 동안 세포를 배양하였다. 3일 뒤에 조골세포 분화를 유도하기 위하여 1% horse serum, 0.1% p/s을 함유한 α-MEM essential medium으로 교환해 주었다. 그 후 10ng/ml의 BMP-2(bone morphogenic protein-2)를 처리하고, 시료를 농도(10, 50ug/ml)별로 동시에 처리한 후 4일간 배양하였다. 이어 세포를 0.01% Triton X를 포함한 AP assay buffer (kit)로 처리한 후, 1000x g에서 5분간 원심분리하여 ALP 활성검색에 필요한 시료를 수득 하였다. Since osteoblasts exhibit ALP activity, the effect of the hot water extract of mulberry leaves obtained above on the ALP activity in osteoblasts was measured. Specifically, C2C12 cells, which are osteoblasts, were plated at 48 × 10 4 cells / well in α-MEM essential medium containing 10% FBS and 0.1% p / s for 3 days. Lt; / RTI > After 3 days, α-MEM essential medium containing 1% horse serum and 0.1% p / s was exchanged to induce osteoblast differentiation. Then, 10ng / ml BMP-2 (bone morphogenic protein-2) was treated and the samples were treated simultaneously at a concentration of 10, 50 ug / ml and then cultured for 4 days. The cells were then treated with an AP assay buffer (kit) containing 0.01% Triton X and centrifuged at 1000 x g for 5 minutes to obtain samples for ALP activity detection.
ALP가 p-니트로페닐포스페이트 (p-nitrophenylphosphate)를 p-니트로페놀 (p-nitrophenol)과 포스페이트(phosphate)로 분해시키는 것을 이용하여 405 nm에서의 흡광도의 변화를 이용하여 ALP 활성을 측정하였다. 단백질의 농도는 BioRad 단백질 분석키트를 사용하였다. ALP 활성은 PNP uM/min/mg protein으로 나타냈다. ALP activity was measured using the change in absorbance at 405 nm using ALP decomposition of p-nitrophenylphosphate into p-nitrophenol and phosphate. Protein concentrations were determined using a BioRad protein assay kit. ALP activity was expressed as PNP uM / min / mg protein.
3.2. 멀꿀 잎 열수 추출물에 의한 조골세포의 ALP 활성측정 3.2. Measurement of ALP activity of osteoblasts by hot water extract of mulberry leaves
멀꿀 잎 열수 추출 및 분획물 수득과정을 통해 얻어진 멀꿀 잎 열수 추출물들을 각각 농도별로 즉, 열수추출물(10, 50ug/ml)을 조골모세포(C2C12 cell)에 처리하고, 4일간 배양한 다음 조골세포의 ALP 활성에 미치는 영향을 측정하고 그 결과를 도 2에 그래프로 나타내었다. 도 2에서 보는 바와 같이, BMP-2와 멀꿀 잎 열수추출물(10, 50ug/ml)을 동시에 처리한 실험군의 경우, BMP-2를 처리한 대조군에 비해 농도 의존적으로 ALP 활성이 증가하는 것으로 관찰되었다. (10, 50 ug / ml) was treated with osteoblast (C2C12 cell) and cultured for 4 days. The alumina extracts of alum And the results are shown in FIG. 2 as a graph. As shown in FIG. 2, in the case of the experimental group treated with BMP-2 and hot water extract of mulberry leaves (10, 50 ug / ml), the ALP activity was increased in a concentration-dependent manner compared to the control group treated with BMP-2 .
또한 멀꿀 잎 열수추출물(10, 50ug/ml)을 단독으로 처리한 경우, BMP-2를 처리하지 않은 정상군에 비해 농도 의존적으로 ALP 활성이 증가하는 것으로 관찰되었다. 이와 같은 결과는 ALP가 조골세포의 분화 및 활성 증가에 의해 방출되는 효소로서 이러한 ALP의 활성증가는 조골세포 활성 및 증가와 직접적으로 연관되어 있으므로, 멀꿀 잎 열수 추출물에 의한 ALP 활성의 증가는 직접적인 조골세포의 분화 및 활성증가에 의한 골형성 촉진효과를 증명하는 것이다.
In addition, ALP activity was increased in a concentration-dependent manner compared to the normal group not treated with BMP-2 when the extract of hot water of mulberry leaves (10, 50 ug / ml) alone was treated. These results indicate that ALP is an enzyme released by the differentiation and activity of osteoblast. Since the increase of ALP activity is directly related to the osteoblast activity and increase, the increase of ALP activity by alfalfa leaf hot- And demonstrate the effect of promoting osteogenesis by increasing cell differentiation and activity.
4. 4. 멀꿀Mellow 잎 leaf 열수Heat number 추출물의 에틸아세테이트 The ethyl acetate extract 분획물에In the fraction 의한 조골세포의 Osteoblast APAP 및 And ALPALP 의 활성측정 Activity measurement
도 3은 멀꿀 잎 열수 추출물 에틸아세테이트 분획물의 ALP활성에 미치는 영향을 나타낸 그래프이다. FIG. 3 is a graph showing the effect of ethyl acetate fraction of hot water extract of mulberry leaves on ALP activity.
4.1 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물에 의한 조골세포의 AP 활성측정 4.1 Measurement of AP activity of osteoblast by ethylacetate fraction of hot-water extract of mulberry leaves
조골세포는 ALP 활성을 나타내므로, 앞서 수득된 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물의 조골세포에서 ALP 활성에 미치는 영향을 측정하였다. 구체적으로, 조골모세포인 C2C12 cells을 48well에 5 x 10^4 cell/well이 되도록 분주하고 세포성장배지인 10% FBS, 0.1% p/s을 함유한α-MEM essential medium에서 3일 동안 세포를 배양하였다. Since osteoblasts exhibit ALP activity, the effect of ALP activity on the osteoblast activity of the ethyl acetate fraction of the hot-water extract of mulberry leaves obtained above was measured. Specifically, C2C12 cells, which are osteoblasts, were plated at 48 × 10 4 cells / well in α-MEM essential medium containing 10% FBS and 0.1% p / s for 3 days. Lt; / RTI >
3일 뒤에 조골세포 분화를 유도하기위하여 1% horse serum, 0.1% p/s을 함유한 α-MEM essential medium으로 교환해 주었다. 그 후 10ng/ml의 BMP-2(bone morphogenic protein-2)를 처리하고, 멀꿀 잎 열수 추출물의 부탄올 분획물의 HP-20 column 용출물 HP20-2를 농도(5, 10ug/ml)별로 동시에 처리한 후 4일간 배양하였다. 이어 세포를 0.01% Triton X를 포함한 AP assay buffer (kit)로 처리한 후, 1000 x g에서 5분간 원심분리하여 ALP 활성검색에 필요한 시료를 수득하였다. After 3 days, α-MEM essential medium containing 1% horse serum and 0.1% p / s was exchanged to induce osteoblast differentiation. Then, 10ng / ml of bone morphogenic protein-2 (BMP-2) was treated and the HP20 column effluent HP20-2 of the butanol fraction of mulberry leaf hot water extract was treated simultaneously (5, 10ug / ml) And then cultured for 4 days. The cells were then treated with an AP assay buffer (kit) containing 0.01% Triton X and centrifuged at 1000 x g for 5 minutes to obtain a sample required for ALP activity detection.
ALP가 p-니트로페닐포스페이트 (p-nitrophenylphosphate)를 p-니트로페놀 (p-nitrophenol)과 포스페이트(phosphate)로 분해시키는 것을 이용하여 405 nm에서의 흡광도의 변화를 이용하여 ALP 활성을 측정하였다. 단백질의 농도는 BioRad 단백질 분석키트를 사용하였다. ALP 활성은 PNP uM/min/mg protein으로 나타냈다. ALP activity was measured using the change in absorbance at 405 nm using ALP decomposition of p-nitrophenylphosphate into p-nitrophenol and phosphate. Protein concentrations were determined using a BioRad protein assay kit. ALP activity was expressed as PNP uM / min / mg protein.
4.2 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물에 의한 조골세포의 AP 활성측정 4.2 Measurement of AP activity of osteoblasts by ethyl acetate fraction of hot-water extract of mulberry leaves
멀꿀 잎 열수 추출물의 에틸아세테이트 분획물을 각각 농도별로 즉, 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물 (5, 10ug/ml)을 조골모세포(C2C12 cell)에 처리하고, 4일간 배양한 다음 조골세포의 ALP 활성에 미치는 영향을 측정하고 그 결과를 도 3에 그래프로 나타내었다. The ethyl acetate fraction of the hot water extract of mulberry leaves was treated with the ethyl acetate fraction (5, 10 ug / ml) of hot water extract of mulberry leaf for 2 days, and the osteoblast cell (C2C12 cell) And the results are shown in FIG. 3 as a graph.
도 3에서 보는 바와 같이, BMP-2와 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물(5, 10ug/ml)을 동시에 처리한 실험군의 경우, BMP-2를 처리한 대조군에 비해 농도 의존적으로 ALP 활성이 증가하는 것으로 관찰되었다. 또한 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물 (5, 10ug/ml)을 단독으로 처리한 경우, BMP-2를 처리하지 않은 정상군에 비해 농도 의존적으로 ALP 활성이 증가하는 것으로 관찰되었다.As shown in FIG. 3, in the experimental group treated with the ethyl acetate fraction (5, 10 ug / ml) of BMP-2 and hot water extract of mulberry leaves, the ALP activity was increased in a concentration-dependent manner compared with the control group treated with BMP-2 Respectively. In addition, when the ethyl acetate fraction (5, 10 ug / ml) of the hot water extract of mulberry leaves was treated alone, the ALP activity was increased in a concentration-dependent manner compared to the normal group without BMP-2 treatment.
ALP는 조골세포의 분화 및 활성 증가에 의해 방출되는 효소로 이러한 ALP의 활성증가는 조골세포 활성 및 증가와 직접적으로 연관되어 있으므로, 본 발명의 멀꿀 잎 열수 추출물의 에틸아세테이트에 의한 ALP 활성의 증가는 직접적인 조골세포의 분화 및 활성증가에 의한 골형성 촉진효과를 증명하는 것이다.Since ALP is an enzyme released by osteoblast differentiation and activity increase, the increase of ALP activity is directly related to the osteoblast activity and increase. Therefore, the increase of ALP activity by ethyl acetate in the hot water extract of honeycomb leaf of the present invention And demonstrate the effect of promoting osteogenesis by increasing the differentiation and activity of direct osteoblasts.
4.3. 멀꿀 잎 열수 추출물의 에틸아세테이트 분획물에 의한 조골세포의 AP activity 염색 4.3. AP activity staining of osteoblast by ethylacetate fraction of hot water extract of mulberry leaves
조골모세포인 C2C12 cells을 48well에 5 x 104 cell/well이 되도록 분주하고 세포성장배지인 10% FBS, 0.1% p/s을 함유한α-MEM essential medium에서 3일 동안 세포를 배양하였다. 3일 뒤에 조골세포 분화를 유도하기 위하여 1% horse serum, 0.1% p/s을 함유한 α-MEM essential medium으로 교환해 주었다. 그 후 10ng/ml의 BMP-2(bone morphogenic protein-2)를 처리하고, 시료를 농도별로 동시에 처리한 후 4일간 배양하였다. Cells were cultured for 3 days in α-MEM essential medium containing 10% FBS, 0.1% p / s, and cell growth medium, and C2C12 cells, osteoblasts, were added to 48 wells at 5 x 104 cells / well. After 3 days, α-MEM essential medium containing 1% horse serum and 0.1% p / s was exchanged to induce osteoblast differentiation. Then, 10ng / ml of BMP-2 (bone morphogenic protein-2) was treated and the samples were treated at the same concentration and cultured for 4 days.
본 발명에서는 조골세포의 분화 유도인자인 BMP-2 (10ng/ml)를 처리한 양성대조군, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물(5, 10ug/ml)을 단독 처리한 실험군, 아무것도 처리하지 않은 정상군으로 분류하여 각각 조골세포 분화(osteoblast differentiation) 정도를 NBT/BCIP 기질을 이용한 AP 활성자리 염색을 통해 측정하였다. In the present invention, the positive control group treated with BMP-2 (10 ng / ml), osteoblast differentiation inducer, the test group treated with the ethyl acetate fraction (5, 10 ug / ml) And the degree of osteoblast differentiation was measured by AP active site staining using NBT / BCIP substrate.
구체적으로 4일간 배양이 끝난 세포의 배양액을 제거하고 1xPBS로 3번 cell을 씻는다. 10% 포르말린 용액으로 실온에서 15분동안 cell을 고정시키고 1xPBS로 3번 cell을 씻어 잔여 포르말린을 제거한다. 1x alkaline phosphaste 용액으로 cell을 다시 씻어낸 다음 NBT/BCIP 기질용액으로 AP 활성자리를 염색하였다. Specifically, after removing the culture solution of the cultured cells for 4 days, wash the cell 3 times with 1xPBS. The cells are fixed with 10% formalin solution for 15 minutes at room temperature, and the cells are washed with 1 × PBS for 3 times to remove residual formalin. Cells were washed again with 1x alkaline phosphaste solution and stained AP active sites with NBT / BCIP substrate solution.
도 4는 멀꿀 잎 열수추출물, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물의 ALP 활성 염색 결과를 나타낸 그래프이다. 도 4에서 나타낸 바와 같이, 실험결과 BMP-2만을 처리한 양성대조군의 경우, BMP-2와 시료를 처리하지 않은 정상군에 비해 NBT/BCIP에 의한 세포 염색이 더 많이 되었음을 확인하였다. 4 is a graph showing the results of ALP activity staining of the ethyl acetate fraction of hot water extract of mulberry leaves and hot water extract of mulberry leaves. As shown in FIG. 4, in the positive control group treated with BMP-2 only, it was confirmed that NBT / BCIP induced more cell staining than BMP-2 and the untreated normal group.
멀꿀 잎 열수추출물의 에틸아세테이트 분획물(5, 10ug/ml)을 단독 처리한 실험군의 경우, BMP-2와 시료를 처리하지 않은 정상군에 비해 NBT/BCIP에 의한 세포 염색이 더 많이 되었음을 확인하였다.In the group treated with the ethyl acetate fraction (5, 10 ug / ml) of the hot water extract of mulberry leaves, the NBT / BCIP-induced cell staining was higher than that of the normal group not treated with BMP-2.
ALP는 조골세포의 분화 및 활성 증가에 의해 방출되는 효소로 이러한 ALP의 활성증가는 조골세포 활성 및 증가와 직접적으로 연관되어 있으므로, 본 발명의 멀꿀 잎 열수추출물의 에틸아세테이트 분획물에 의한 ALP 활성의 증가는 직접적인 조골세포의 분화 및 활성증가에 의한 골형성 촉진효과를 나타낸다고 할 수 있다.
Since ALP is an enzyme released by osteoblast differentiation and activity increase, the increase of the activity of ALP is directly related to the osteoblast activity and increase. Therefore, the increase of ALP activity by the ethyl acetate fraction of the hot water extract of mulberry leaf of the present invention Suggests the effect of promoting osteogenesis by direct osteoblast differentiation and increased activity.
5. 5. 멀꿀Mellow 잎 leaf 조추출물을Crude extract 유효성분으로 포함하는 연골 조직 생성 촉진용 기능성 식품조성물 및 연골조직생성 촉진용 예방 치료제 A functional food composition for promoting cartilage tissue formation and an agent for preventing cartilage formation
연골조직 생성 촉진용 기능성 식품 조성물을 이용한 골다공증 예방 또는 치료제는 상기 조성물이 0.01 내지 99.9중량%로 포함되도록 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸, 경피제, 좌제 또는 멸균 주사용 액으로 제형화하여 제조할 수 있다. The agent for preventing or treating osteoporosis using the functional food composition for promoting cartilage tissue formation may be in the form of a powder or granules, a tablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, a transdermal agent, And can be manufactured by formulating it into a use liquid.
멸균주사액의 경우, 상기 기능성 식품조성물이 0.01 내지 99.9중량%로 포함하도록 하고 정제수 또는 포도당을 99.9 내지 0.01 중량%를 혼합하여 제조 가능하다. 캡슐제의 경우, 상기 기능성 식품조성물을 동결건조하여 0.01 내지 99.9중량%로 포함하도록 하고, 비타민제, 칼슘제을 99.9 내지 0.01 중량%를 혼합하여 제조 가능하다. In the case of a sterile injectable solution, the functional food composition may be contained in an amount of 0.01 to 99.9% by weight and 99.9 to 0.01% by weight of purified water or glucose may be mixed. In the case of capsules, the functional food composition may be prepared by lyophilization to 0.01 to 99.9% by weight, and 99.9 to 0.01% by weight of a vitamin and calcium preparation.
상기 제조된 기능성 식품조성물의 1일당 투여량은 상기 추출물이 10 내지 1000mg/kg 체중 포함되는 함량으로 제공한다. The daily dose of the above-prepared functional food composition is such that the extract contains 10 to 1000 mg / kg body weight.
또한 상기 기능성 식품 조성물을 0.01 내지 99.9중량%로 포함하는 골다공증 개선, 예방용 기능성 건강식품으로도 제조 가능하다.
Also, the functional food composition can be manufactured as a functional health food for improving and preventing osteoporosis comprising 0.01 to 99.9% by weight of the functional food composition.
본 발명의 멀꿀 잎 열수추출물, 멀꿀 잎 열수추출물의 에틸아세테이트 분획물의 조골세포 ALP 활성 및 조골세포 분화를 촉진시킴으로써 연골조직의 생성을 촉진하는 효과가 확인됨으로서 멀꿀 잎 추출물을 유효성분으로 포함하는 기능성 식품 조성물은 치주염 또는 골다공증을 치료 또는 예방하기 위한 골다공증 치료제로 사용될 수 있고 제조 원료를 자연에 서식하는 식물로 대체함으로 제조생산단가 절감과 산업화를 통한 수입대체 및 수출효과를 기대할 수 있을 것이다.As a result of promoting the osteoblast ALP activity and osteoblast differentiation of the ethyl acetate fraction of the mulberry leaf hot water extract of the present invention and the hot water extract of mulberry leaves, the effect of promoting the formation of cartilage tissue was confirmed, and thus, a functional food containing mulberry leaf extract as an active ingredient The composition can be used as a therapeutic agent for osteoporosis to treat or prevent periodontal disease or osteoporosis, and by replacing the raw material with a plant inhabited by nature, it is possible to expect the effect of import substitution and export by reducing manufacturing production cost and industrialization.
Claims (8)
Functional food composition for promoting osteoarthritis or cartilage tissue formation containing mulberry leaf crude extract as an active ingredient
[3] The method according to claim 1, wherein the crude extract of Mulberry leaf is an extract soluble in water, methanol, ethanol, propanol, isopropanol, butanol or a mixed solvent thereof. Or a functional food composition for promoting cartilage tissue formation
[3] The method according to claim 2, wherein the mullen leaf crude extract extracted using the solvent is fractionated using a fraction solvent selected from the group consisting of hexane, chloroform, dichloromethane and ethyl acetate as a non- Functional food composition for promoting bone formation or cartilage tissue formation containing an extract
A functional food composition for promoting osteoarthritis or cartilage formation, comprising a crude extract of mulberry leaf, comprising 0.01 to 99.9% by weight of a composition according to any one of claims 1 to 3
[Claim 5] The method according to claim 4, wherein the daily dose of the mulberry leaf crude extract is in an amount of 10 to 1000 mg / kg per kg of body weight. Composition
The composition of claim 4, wherein the composition is formulated as a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, transdermal preparation, suppository or sterile injectable solution Functional food composition for promoting bone formation or cartilage tissue formation
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| KR100497945B1 (en) | 2001-06-26 | 2005-06-29 | 한국 한의학 연구원 | Extract of herb for promoting release of growth hormone |
| KR101167589B1 (en) | 2011-08-18 | 2012-07-27 | 재단법인 전라남도생물산업진흥재단 | Anti-inflammatory agent containing stauntonia hexaphylla fruit extract |
| KR101221617B1 (en) | 2012-04-16 | 2013-01-14 | 재단법인 전라남도생물산업진흥재단 | Anti-inflammatory agent containing stauntonia hexaphyllafruit leaf extract |
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| KR20130020095A (en) | 2011-08-18 | 2013-02-27 | 재단법인 전라남도생물산업진흥재단 | Hepatoprotective composition containing stauntonia hexaphylla extract |
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| KR100497945B1 (en) | 2001-06-26 | 2005-06-29 | 한국 한의학 연구원 | Extract of herb for promoting release of growth hormone |
| KR101167589B1 (en) | 2011-08-18 | 2012-07-27 | 재단법인 전라남도생물산업진흥재단 | Anti-inflammatory agent containing stauntonia hexaphylla fruit extract |
| KR101221617B1 (en) | 2012-04-16 | 2013-01-14 | 재단법인 전라남도생물산업진흥재단 | Anti-inflammatory agent containing stauntonia hexaphyllafruit leaf extract |
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