KR20110103102A - Composition for the prevention and treatment of glaucoma containing Cucumber extract, fractions thereof or compounds isolated therefrom as an active ingredient - Google Patents
Composition for the prevention and treatment of glaucoma containing Cucumber extract, fractions thereof or compounds isolated therefrom as an active ingredient Download PDFInfo
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- KR20110103102A KR20110103102A KR1020100022255A KR20100022255A KR20110103102A KR 20110103102 A KR20110103102 A KR 20110103102A KR 1020100022255 A KR1020100022255 A KR 1020100022255A KR 20100022255 A KR20100022255 A KR 20100022255A KR 20110103102 A KR20110103102 A KR 20110103102A
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Abstract
본 발명은 오죽 추출물, 이의 분획물 또는 이로부터 분리된 하기 [화학식 1]로 기재되는 화합물을 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물에 관한 것으로, 구체적으로 오죽을 유기용매로 추출하여 제조되는 추출물, 이의 분획물 및 상기 분획물에서 분리한 화합물인 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin 6-C-(6''-O-trans-caffeoylglucoside)]을 포함하는 녹내장 예방 및 치료용 조성물에 관한 것이다. 본 발명의 조성물은 녹내장의 원인인 망막 신경 세포의 퇴화를 보호해 주는 작용을 나타냄으로써, 녹내장의 예방 또는 치료용 조성물로 유용하게 사용될 수 있다:
[화학식 1]
[Formula 1]
Description
본 발명은 오죽(Phyllostachys nigra MUNRO) 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물, 또는 건강식품에 관한 것이다.
The present invention is Phyllostachys nigra MUNRO) relates to a composition for preventing and treating glaucoma, or health food containing the extract, a fraction thereof or a compound separated therefrom as an active ingredient.
세포자살(apoptosis)이란, 세포괴사(necrosis)와는 구별되는 세포 내의 자발적이고 계획된 세포사(programmed cell death)이고, 세포자살을 유발하는 다양한 기전들이 제시되어 왔다. 대표적인 유발인자로는 산화 스트레스(oxidative stress), 질소산화물 합성효소(nitric oxide synthase), 미토콘드리아(mitochondria)를 매개로 하는 케스페이즈 의존적(caspase-dependent) 또는 비의존적(caspase-independent) 기전 등이 있다. 특히 많은 안질환들이 특정 세포의 세포자살로 인해 발생하는 것으로 알려져 있으며, 그 대표적인 안질환으로는 연령 관련 황반 변성(age related macular degeneration) 및 녹내장(glaucoma)이 있다. 이러한 안질환의 억제 및 치료를 위하여 세포자살을 억제하거나 그 진행을 늦출 수 있는 약제에 대한 연구가 지속적으로 진행되고 있는 실정이다.
Apoptosis is spontaneous and programmed cell death in cells that is distinct from necrosis, and various mechanisms for causing apoptosis have been suggested. Representative triggers include oxidative stress, nitric oxide synthase, and caspase-dependent or caspase-independent mechanisms mediated by mitochondria. . In particular, many eye diseases are known to occur due to the apoptosis of specific cells, and representative eye diseases include age related macular degeneration and glaucoma. In order to suppress and treat such eye diseases, studies on drugs that can suppress or slow the progression of apoptosis have been continuously conducted.
눈의 각막, 수정체(렌즈) 및 유리체 등은 혈관이 없는 부위이기 때문에, 여기에 영양분을 공급하고 노폐물을 운반하는 방수라고 하는 물이 존재하고 있다. 그런데 방수는 정상으로 나오는데 반해, 빠져나가는 부분이 망가지게 되면, 방수는 계속 나오기 때문에 눈의 압력은 올라가게 된다. 이렇듯 눈의 압력이 높아지게 되면, 많은 혈액을 공급받아야 하는 눈 속에 혈액이 들어오지 못하게 되며, 약한 부위인 시신경에 지속적인 압력이 가하게 되고, 지속적인 압력을 받은 시신경은 치명적인 손상을 입게 되어 시력 및 시야의 결손을 초래하게 된다. 녹내장(glaucoma)은 이러한 안압의 상승으로 인해 시신경이 눌리거나 혈액 공급에 장애가 생겨 시신경의 기능에 이상을 초래하게 되는 질환으로, 말기에는 시력을 상실하게 되는 심각한 질환이다. 이러한 녹내장은 40세 이상의 성인 가운데 0.5 내지 2%의 빈도로 일어나게 되는데, 안압의 정상값이 15 내지 20 mmHg인데 반해, 녹내장이 진행되면 안압이 상승하면서, 동공 안쪽이 녹색으로 보이게 된다.Since the cornea, lens (lens), and vitreous body of the eye are areas without blood vessels, there exists water called waterproofing which supplies nutrients and carries waste products. However, the waterproofing comes out normal, but if the exiting part is broken, the waterproofing continues, so the pressure of the eye rises. As the pressure of the eye increases, blood does not enter the eye where a large amount of blood is supplied, and continuous pressure is applied to the optic nerve, which is a weak area, and the optic nerve under constant pressure causes fatal damage, resulting in loss of vision and vision. Will result. Glaucoma (glaucoma) is a disease that causes the optic nerve is depressed due to the increase in the intraocular pressure or impaired blood supply causing abnormalities in the function of the optic nerve. Such glaucoma occurs at a frequency of 0.5 to 2% among adults over 40 years old, whereas the normal value of the intraocular pressure is 15 to 20 mmHg, as the intraocular pressure increases as glaucoma progresses, the inside of the pupil appears green.
현재 녹내장에 대한 치료는 녹내장의 가장 중요한 위험요소인 안압 상승을 약물이나 수술적인 방법을 이용하여 막는 것이 대부분으로, 녹내장은 완치될 수 없고 평생 약물, 레이져 치료, 수술 등의 방법으로 안압을 조절하여 시신경의 장애를 최소화하는 방법밖에 없다.Currently, the treatment of glaucoma is to prevent intraocular pressure elevation, which is the most important risk factor of glaucoma, by using drugs or surgical methods. Glaucoma cannot be cured, and the intraocular pressure is controlled by drugs, laser treatment, and surgery. The only way to minimize optic nerve disorders is.
또한, 녹내장은 망막 신경 세포(망막 신경 cell)의 세포자살로 인한 시신경의 손상을 통해서 일어나게 되는데, 이러한 시신경의 손상은 특징적인 녹내장성 시야 손상을 동반한다. 녹내장에서 나타나는 망막 신경 세포의 세포사멸은 세포자살(apoptosis)로 알려져 있으며, 안압을 상승시킨 동물 모델뿐만 아니라 녹내장 환자의 사후 부검을 통해서도 녹내장 환자에서 망막 신경 세포의 세포자살이 관찰되었다(Quigley HA et al., Invest Ophthalmol Vis Sci., 36, 774-786, 1995; Garcia-Valenzuela E et al., Exp Eye Res., 61, 33-44, 1995). 세포 내에 세포사멸을 유도하는 기전과 억제하는 기전의 균형에 의하여 세포의 운명이 결정되는데, 녹내장에 있어서는 글루타메이트(glutamate), 사이토크롬 C(cytochrome c), 종양 괴사 인자-알파(TNF-α) 및 Bad(BCL2-antagonist of cell death) 단백질의 증가 또는 발현이 망막 신경 세포를 세포자살에 이르게 하는 것으로 알려져 있으며, 세포 내의 산화 스트레스(oxidative stress)도 녹내장의 중요한 원인으로 알려져 있다. 녹내장에서는 망막 신경 세포가 산화 스트레스를 받아 직접적으로 시신경을 손상에 이르게 할 뿐만 아니라, 방수 유출로를 구성하고 있는 섬유주 세포(trabecular meshwork cell)가 산화스트레스를 받음으로써, 그 결과 섬유주 세포의 기능저하가 일어나게 되고, 궁극에는 세포자살이 일어나게 되어 안압이 상승하게 된다(Sacca' SC et al, Arch Ophthalmol., 123, 458-463, 2005; Zhou L et al., J Cell Physiol., 180, 182-189, 1999). 섬유주 세포의 기능저하는 세포가 고유하게 가지고 있는 방수를 흡수하는 음세포작용(pinocytosis)에 영향을 주게 되어 안압의 상승을 유발하는데 특히 섬유주 세포의 기능저하가 안압상승의 주원인으로 알려져 있는 원발성 폐쇄 각 녹내장(primary open angle glaucoma)환자들의 섬유주 세포가 산화 스트레스에 더 취약한 것으로 알려져 있다(Izzotti A et al., Am J Med., 114, 638-646, 2003). 상기 환자의 방수를 분석한 결과, 총항산화역량(total antioxidant potential)이 유의하게 정상인에 비하여 낮은 것으로 나타났고(Ferreira SM at al., Am J Ophthalmol. 137, 62-69, 2004), 혈장에 산화스트레스를 조절하는 글루타치온-S- 전이효소(Glutachione-S-Transferase)가 증가되어 있음이 관찰되었다(Yang J et al., Invest Ophthalmol Vis Sci., 42, 1273-1276, 2001). 또한 최근에는 원발성 개방 각 녹내장 환자들에 있어서 나타나는 섬유주 세포의 손상이 항산화제에 의하여 억제됨이 알려져 있다(Yuan He et al., Invest Ophthalmol Vis Sci., 49, 1447-1458, 2008). 이처럼 망막 신경 세포와 섬유주 세포의 세포 자살을 억제하고 상기 세포의 산화 스트레스를 억제하는 것이 녹내장 치료의 새로운 접근방법으로 대두되고 있으나, 아직까지 이에 대한 연구는 많이 진행되어 있지 않다.
In addition, glaucoma occurs through damage to the optic nerve caused by apoptosis of retinal nerve cells (retinal nerve cells), which is accompanied by characteristic glaucoma visual field damage. Apoptosis of retinal neurons in glaucoma is known as apoptosis, and apoptosis of retinal nerve cells was observed in glaucoma patients through postmortem autopsy of glaucoma patients as well as in animals with elevated intraocular pressure (Quigley HA et al. al., Invest Ophthalmol Vis Sci., 36, 774-786, 1995; Garcia-Valenzuela E et al., Exp Eye Res., 61, 33-44, 1995). The fate of cells is determined by the balance between the mechanisms that induce apoptosis and the mechanisms that inhibit it. In glaucoma, glutamate, cytochrome c, tumor necrosis factor-alpha (TNF-α) and Increased or expressed Bad (BCL2-antagonist of cell death) protein is known to lead to retinal neuronal cell apoptosis, and oxidative stress in the cell is also known as an important cause of glaucoma. In glaucoma, retinal nerve cells are not only subjected to oxidative stress, which directly damages the optic nerve, but also trabecular meshwork cells, which constitute a waterproof outflow passage, are subjected to oxidative stress. And ultimately, apoptosis results in elevated intraocular pressure (Sacca 'SC et al, Arch Ophthalmol., 123, 458-463, 2005; Zhou L et al., J Cell Physiol., 180, 182-189). , 1999). The deterioration of the trabecular cells affects the pinocytosis, which absorbs the inherent waterproofness of the cells, leading to an increase in intraocular pressure. It is known that trabecular cells of patients with primary open angle glaucoma are more susceptible to oxidative stress (Izzotti A et al., Am J Med., 114, 638-646, 2003). Analysis of the waterproofness of the patients showed that total antioxidant potential was significantly lower than normal (Ferreira SM at al., Am J Ophthalmol. 137, 62-69, 2004). Increased glutathione-S-transferase that regulates stress has been observed (Yang J et al., Invest Ophthalmol Vis Sci., 42, 1273-1276, 2001). In recent years, it has been known that the damage of the trabecular cells in primary open-angle glaucoma patients is inhibited by antioxidants (Yuan He et al., Invest Ophthalmol Vis Sci., 49, 1447-1458, 2008). Inhibiting apoptosis of retinal nerve cells and trabecular cells and suppressing oxidative stress of the cells has emerged as a new approach for the treatment of glaucoma, but much research has not been done.
한편, 오죽(Phyllostachys nigra MUNRO)은 속명으로 자죽, 수죽자, 약죽이라 불리며, 화본과의 상록목본성 식물로서 높이 10 cm 내외이고, 지름 5-8 cm이다. 처음에는 백분으로 덮여 있지만 줄기가 흑색으로 된다. 6-7월에 꽃이 피고 꽃은 연한 황색이며, 죽순은 4-5월에 나오고 연한 적갈색이다. 10월에 열매가 성숙되며 영과이다. 용도는 식용, 관상용, 공업용, 약용으로 쓰이고, 어린순을 식용하며 관상수 및 세공용재, 건축용, 죽기용재로 줄기를 쓰고 한방과 민간에서 죽순을 구토, 소염, 경간, 주독, 유산, 익기, 객토혈 급창, 보약, 파상풍, 발한, 진통, 중풍 등에 약재로 쓴다. 본초 강목에 따르면 고죽엽(오죽 잎)은 성질이 서늘하며 맛이 쓰고 독이 없다. 잠 못 자는 것을 낫게 하며 소갈을 멈추고 술독을 풀며 번열을 없애고 땀을 낸다. 중풍으로 말을 못하는 것도 낫게 한다고 되어 있다.Meanwhile, Phyllostachys nigra MUNRO) is a genus name of succulent, bamboo, and yak-juk. It is an evergreen herbaceous plant with a flower, about 10 cm in height, and 5-8 cm in diameter. At first it is covered with white powder, but the stem is black. Flowers blossom in June-July, flowers are pale yellow, bamboo shoots come in April-May, light reddish brown. In October, the fruit matures and is spirit and fruit. Used for edible, ornamental, industrial, medicinal, edible young shoots, ornamental stems and ornamental materials, construction, and dying materials, stems vomiting in Chinese medicine and civilization, anti-inflammatory, span, poisoning, miscarriage, ripening, effusion blood It is used as medicine for medicine, tetanus, sweating, labor, paralysis. According to the herbaceous tree, the bamboo leaves are cool, tasteless and nontoxic. It helps to sleep better, stops to get rid of alcohol, dissolves heat and removes sweat. It is said that it is better to speak with a stroke.
'오죽차'는 간해독을 도와 피로해소에 좋고 고혈압에도 탁월한 효과가 있는 것으로 전해지고 있어서, 오죽엽차 및 이의 제조방법이 개발되어 보고되었다(대한민국 특허 제2001-0011491호). 또한 오죽의 추출물은 LDL 산화억제효과를 보이고, NF-B의 활성을 저해하며, ICAM, VCAM, MCP-1의 발현을 저해함으로써 동맥경화의 유발 위험을 감소시켜 항동맥경화제로서 사용될 수 있다고 보고되었고, 오죽 추출물을 0.25 내지 5.00 /플레이트 농도로 살모넬라 티피뮤리움 TA 100(Salmonella typhimurium TA 100)에 처리하였을 때 독성을 전혀 나타내지 않아, 대나무 추출물을 이용한 동물실험이 가능할 것으로 보고 되었다(대한민국 특허 2003-0014155). 그러나 망막 신경 세포를 보호함으로써 녹내장 질환을 예방 또는 치료할 수 있는 오죽 추출물의 효능에 관한 연구는 전무한 실정이다.
'Ojuk tea' is said to help liver detoxification and relieve fatigue and have an excellent effect on high blood pressure, and thus, Oju tea and its manufacturing method have been developed and reported (Korean Patent No. 2001-0011491). In addition, the extracts of Ojuk showed an LDL oxidation inhibitory effect, inhibited NF-B activity, and inhibited the expression of ICAM, VCAM, and MCP-1, thereby reducing the risk of atherosclerosis. , When the bamboo shoots were treated with Salmonella typhimurium
이에, 본 발명자들은 천연물을 대상으로 망막 신경 세포 보호제를 검색하던 중, 오죽 추출물, 이의 분획물 및 이로부터 분리한 화합물이 산화스트레스를 유도한 망막 신경 세포를 보호하여 세포사멸을 저해하고, 아폽토시스의 저해 효과를 나타내어, 산화스트레스를 유도한 망막 신경 세포의 퇴화로 인한 녹내장의 예방 및 치료제로 사용될 수 있음을 밝힘으로써 본 발명을 완성하였다.
Accordingly, the present inventors, while searching for a retinal neuronal cell protective agent in natural products, Ojuk extract, fractions thereof and compounds isolated therefrom protect the retinal neurons that induced oxidative stress to inhibit apoptosis, inhibition of apoptosis The present invention has been completed by revealing that it can be used as a prophylactic and therapeutic agent for glaucoma due to oxidative stress-induced degeneration of retinal nerve cells.
본 발명의 목적은 산화스트레스에 의한 망막 신경 세포의 퇴화를 저해하는 오죽(Phyllostachys nigra MUNRO) 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 녹내장의 예방 및 치료용 조성물, 또는 개선용 건강식품을 제공하는 것이다.
An object of the present invention is to prevent the degeneration of retinal nerve cells by oxidative stress ( Phyllostachys nigra MUNRO) It is to provide a composition for the prevention and treatment of glaucoma, or a health food for improvement, containing an extract, a fraction thereof or a compound separated therefrom as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 오죽(Phyllostachys nigra MUNRO) 추출물 또는 이의 분획물을 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물을 제공한다.In order to achieve the above object, the present invention is Phyllostachys nigra MUNRO) It provides a composition for the prevention and treatment of glaucoma containing an extract or a fraction thereof as an active ingredient.
또한, 본 발명은 하기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물을 제공한다:In addition, the present invention is the compound Lutein 6-C- (6 '' -O -trans-cafeoyl glucoside) represented by the following [Formula 1] [Luteolin6-C- (6 '' -O -trans- caffeoylglucoside)] is provided as a composition for the prevention and treatment of glaucoma:
또한, 본 발명은 오죽 추출물 또는 이의 분획물을 유효성분으로 함유하는 녹내장 예방 및 개선용 건강식품을 제공한다.In addition, the present invention provides a health food for preventing and improving glaucoma, containing the porridge extract or a fraction thereof as an active ingredient.
또한, 본 발명은 하기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 유효성분으로 함유하는 녹내장 예방 및 개선용 건강식품을 제공한다:In addition, the present invention is the compound Lutein 6-C- (6 '' -O -trans-cafeoyl glucoside) represented by the following [Formula 1] [Luteolin6-C- (6 '' -O -trans- caffeoylglucoside)] provides a health food for the prevention and improvement of glaucoma containing as an active ingredient:
[화학식 1][Formula 1]
본 발명의 오죽(Phyllostachys nigra MUNRO) 추출물, 이의 분획물 또는 이로부터 분리한 화합물은 산화스트레스를 유도한 망막 신경 세포의 퇴화를 보호하는 효과를 나타내고, 천연물 유래의 안전한 물질이므로, 녹내장 예방 및 치료용 조성물, 또는 개선용 건강식품의 유효성분으로 유용하게 이용될 수 있다.
Phyllostachys of the Invention nigra MUNRO) extracts, fractions thereof or compounds isolated therefrom have the effect of protecting the degeneration of retinal nerve cells that induced oxidative stress and are safe substances derived from natural products, thus preventing glaucoma from preventing and treating compositions, or improving health foods. It can be usefully used as an active ingredient of.
도 1 및 도 2는 본 발명에서 분리한 화합물의 구조를 나타낸 그림이다.
도 3은 망막 신경 세포 RGC-5에 오죽 분획물을 전처리하고, 산화스트레스를 유도한 다음, MTT 방법을 수행하여, 세포의 생존율을 비교하여 그래프로 나타낸 그림이다.
도 4는 망막 신경 세포 RGC-5에 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 전처리하고, 산화스트레스를 유도한 다음, MTT 방법을 수행하여, 세포의 생존율을 비교하여 그래프로 나타낸 그림이다.
도 5는 망막 신경 세포 RGC-5에 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 전처리하고, 산화스트레스를 유도한 다음, Hoechst 33342 및 프로피디움 요오드화물을 이용한 이중 염색 후, 이를 형광 현미경을 통해 확인한 사진을 나타낸 그림이다.
도 6은 망막 신경 세포 RGC-5에 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 전처리하고, 산화 라디칼에 의한 세포의 글루타티온 함량의 변화를 그래프로 나타낸 그림이다.
도 7은 망막 신경 세포(RGC-5)에서 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]을 전처리하고, 산화스트레스에 의해 변화되는 아폽토틱(apoptotic) 단백질의 발현양을 면역 블랏으로 확인한 결과를 나타낸 그림이다.1 and 2 is a diagram showing the structure of the compound isolated in the present invention.
Figure 3 is a graph showing the comparison of the survival rate of cells by pretreatment of the Oju fractions in retinal nerve cells RGC-5, induction of oxidative stress, MTT method.
4 is a retinal neuronal cell RGC-5 compound Lu Theo Lin 6-C- (6 '' - O - trans-cafe five days inclusive nose side) [Luteolin6-C- (6 'on' - O -trans-caffeoylglucoside)] After pretreatment and induction of oxidative stress, MTT method was performed to compare the survival rate of cells.
5 is a retinal neuronal cell RGC-5 compound Lu Theo Lin 6-C- (6 '' - O - trans-cafe five days inclusive nose side) [Luteolin6-C- (6 'on' - O -trans-caffeoylglucoside)] After pretreatment, induction of oxidative stress, and double staining with Hoechst 33342 and propidium iodide, this is a picture showing the picture confirmed by fluorescence microscopy.
The
7 is a retinal neuronal cell (RGC-5) in the base compound Theo Lin 6-C- (6 '' - O - trans-cafe five days inclusive nose side) [Luteolin6-C- (6 '' - O -trans-caffeoylglucoside Figure 2 shows the results of pretreatment and immunoblotting of the expression level of apoptotic protein changed by oxidative stress.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 오죽(Phyllostachys nigra MUNRO) 추출물 또는 이의 분획물을 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물을 제공한다.The present invention is Phyllostachys nigra MUNRO) It provides a composition for the prevention and treatment of glaucoma containing an extract or a fraction thereof as an active ingredient.
본 발명의 오죽 추출물은 하기와 같은 단계로 제조되는 것이 바람직하나 이에 한정되지 않는다;The porridge extract of the present invention is preferably prepared in the following steps, but is not limited thereto;
1) 건조한 오죽(Phyllostachys nigra MUNRO)을 추출용매를 가하여 추출하는 단계;1) dry porridge ( Phyllostachys nigra MUNRO) extract by adding an extraction solvent;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축하여 추출물을 제조하는 단계; 및3) preparing the extract by concentrating the filtered extract of step 2) under reduced pressure; And
4) 단계 3)의 추출물을 추가적으로 유기용매로 추출하여 분획물을 제조하는 단계.4) extracting the extract of step 3) with an additional organic solvent to prepare a fraction.
상기 방법에 있어서, 오죽은 재배한 것, 채취한 것 또는 시판되는 것 등이 제한 없이 사용될 수 있다. 상기 오죽은 식물 전체를 이용할 수 있고, 잎, 줄기 또는 뿌리를 이용하는 것이 바람직하나 이에 한정되는 것은 아니다.In the above method, cultivated, harvested or commercially available may be used without limitation. The porridge may use the entire plant, preferably using a leaf, stem or root is not limited thereto.
상기 추출 용매는 물, 알코올 또는 이들의 혼합물, 바람직하게는 C1 내지 C4의 저급 알코올 또는 이들의 혼합 용매로부터 선택된 용매를 사용하는 것이 바람직하며, 메탄올 또는 에탄올 수용액으로 추출하는 것이 더욱 바람직하나, 이에 한정되는 것은 아니다. 상기 추출 용매의 양은 오죽 건조 중량의 1 내지 20 배로 함이 바람직하고, 10 배로 하는 것이 더 바람직하나, 이에 한정되는 것은 아니다. 상기 추출 방법은 열수추출, 침지 추출, 환류 냉각 추출 및 초음파 추출 등의 추출 방법을 사용할 수 있으며, 바람직하게는 초음파 추출방법으로 1회 내지 5회 추출될 수 있다. 추출시 온도는 10℃ 내지 100℃ 인 것이 바람직하며 상온인 것이 더욱 바람직하나 이에 한정되지 않는다. 상기 추출 시간은 1일 내지 7일인 것이 바람직하고, 3일 내지 7일인 것이 더욱 바람직하나 이에 한정되지 않는다. The extraction solvent is preferably a solvent selected from water, alcohols or mixtures thereof, preferably C 1 to C 4 lower alcohols or mixed solvents thereof, and more preferably extracted with aqueous methanol or ethanol solution, It is not limited to this. The amount of the extraction solvent is preferably 1 to 20 times the dry weight of porridge, more preferably 10 times, but is not limited thereto. The extraction method may be an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction, it may be preferably extracted one to five times by the ultrasonic extraction method. When the extraction temperature is preferably 10 ℃ to 100 ℃ and more preferably room temperature, but is not limited thereto. The extraction time is preferably 1 day to 7 days, more preferably 3 to 7 days, but is not limited thereto.
상기 추출물을 제조하는 방법은 초임계추출, 아임계추출, 고온추출, 고압추출 또는 초음파추출법 등의 추출장치를 이용한 방법 또는 XAD 및 HP-20을 포함한 흡착 수지를 이용하는 방법 등 당업계의 통상적인 추출방법을 사용할 수 있으며, 가온하며 환류 추출 또는 상온에서 추출하는 것이 바람직하나, 이에 한정하는 것은 아니다. 상기 추출 회수는 1 내지 5회인 것이 바람직하며, 3회 반복 추출하는 것이 더욱 바람직하나 이에 한정되는 것은 아니다. The method of preparing the extract is conventional extraction in the art, such as a method using an extraction device such as supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction or ultrasonic extraction method or using an adsorption resin containing XAD and HP-20. The method may be used, but is preferably warmed and extracted at reflux or at room temperature, but is not limited thereto. The extraction number is preferably 1 to 5 times, more preferably 3 times repeated extraction is not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조, 상온건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다. In the above method, the reduced pressure concentration in step 3) is preferably a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying, room temperature drying or lyophilization.
상기 방법에 있어서, 단계 4)의 유기용매는 노르말-헥산, 염화 메틸렌, 에틸아세테이트 또는 노르말-부탄올인 것이 바람직하나 이에 한정하지 않는다. 상기 분획물은 오죽 추출물을 물에 현탁시킨 후 노르말-헥산, 염화 메틸렌, 에틸아세테이트, 노르말-부탄올 및 물로 순차적으로 계통 분획하여 수득한 노르말-헥산 분획물, 염화 메틸렌 분획물, 에틸아세테이트 분획물, 노르말-부탄올 분획물 또는 물 분획물 중 어느 하나인 것이 바람직하며, 에틸아세테이트 분획물임이 더욱 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 오죽 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the organic solvent of step 4) is preferably, but not limited to, normal-hexane, methylene chloride, ethyl acetate or normal-butanol. The fraction is obtained by suspending the Ojugi extract in water and then systematically fractionating the mixture into normal-hexane, methylene chloride, ethyl acetate, normal-butanol and water, and then extracting normal-hexane, methylene chloride, ethyl acetate and normal-butanol. Or it is preferably any one of the water fraction, it is more preferred that the ethyl acetate fraction, but is not limited thereto. The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times from the porridge extract, and preferably concentrated under reduced pressure after the fraction, but is not limited thereto.
본 발명의 구체적인 실시예에서, 건조된 오죽을 적당한 크기로 분쇄하여 추출용기에 넣고 에탄올 수용액을 가하여 실온에서 방치한 후, 거름종이로 여과한 휘, 여과액을 농출 및 동결건조하여 추출물을 얻었고, 상기 오죽 추출물을 분별깔때기를 이용하여, 노르말-헥산, 염화 메틸렌, 에틸아세테이트, 노르말-부탄올 및 물 분획물로 순차적으로 계통분석하여 오죽 추출물 및 이의 분획물을 제조하였다.In a specific embodiment of the present invention, the dried porridge was ground to an appropriate size, put in an extraction container and left at room temperature by adding an ethanol aqueous solution, the filtrate filtered with manure paper, the filtrate was concentrated and lyophilized to obtain an extract, The Ojuk extract was subjected to systematic analysis using a separatory funnel with normal-hexane, methylene chloride, ethyl acetate, normal-butanol and water fractions in order to prepare the Ojuk extract and its fractions.
상기 제조된 오죽 분획물의 망막 신경 세포(RGC-5) 보호 효과를 측정하기 위하여, 세포 배양 후, 오죽 분획물을 농도별로 전처리한 다음, 300 μM 과산화수소(hydrogen peroxide) 또는 0.5mM 1-부티오닌 설폭시민[1-buthionine-(S,R)-sulfoximine; BSO) 및 10mM L-글루탐산(L-glutamatmic acid; glutamate)을 37℃에서 24시간 동안 처리 후, MTT 방법을 수행하여 세포의 독성을 측정하였고, 그 결과, 망막 신경 세포 RGC-5에서 과산화수소 또는 BSO+글루탐산(glutamate)에 의해 산화스트레스가 유도된 경우, 상기 오죽의 노르말-헥산 분획물, 염화 메틸렌 분획물, 에틸 아세테이트 분획물 및 노르말-부탄올 분획물을 전처리한 경우에 과산화수소 또는 BSO+글루탐산만 처리한 경우에 비해, 더 높은 세포 생존율을 나타내었다(도 3 참조).In order to measure the protective effect of retinal nerve cells (RGC-5) of the prepared porridge fraction, after the cell culture, the porridge fraction was pretreated by concentration, and then 300 μM hydrogen peroxide or 0.5 mM 1-butionine sulfoxide Citizen [1-buthionine- (S, R) -sulfoximine; BSO) and 10 mM L-glutamatmic acid (gluamate) for 24 hours at 37 ℃, MTT method was performed to determine the toxicity of the cells, as a result, hydrogen peroxide or BSO + in retinal neurons RGC-5 When oxidative stress is induced by glutamate, the normal-hexane fraction, the methylene chloride fraction, the ethyl acetate fraction, and the normal-butanol fraction of the porridge are more pretreated than only hydrogen peroxide or BSO + glutamic acid. High cell viability was shown (see FIG. 3).
따라서, 오죽 분획물은 망막 신경 세포의 산화스트레스에 있어서, 세포 보호 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the Oju fraction exhibits a cytoprotective effect on the oxidative stress of retinal nerve cells.
또한, 본 발명은 하기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 유효성분으로 함유하는 녹내장 예방 및 치료용 조성물을 제공한다:In addition, the present invention is the compound Lutein 6-C- (6 '' -O -trans-cafeoyl glucoside) represented by the following [Formula 1] [Luteolin6-C- (6 '' -O -trans- caffeoylglucoside)] is provided as a composition for the prevention and treatment of glaucoma:
[화학식 1][Formula 1]
상기 [화학식 1]로 기재되는 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)] 화합물은 상기 오죽 추출물의 에틸아세테이트 분획물에 대한 실리카겔 크로마토그래피 또는 고성능액체크로마토그라피(HPLC) 수행에 의해 분리되는 것이 바람직하나, 이에 한정되지 않는다.The
상기 실리카겔 크로마토그래피는 크기 배제 크로마토그래피용 컬럼을 사용하여 수행될 수 있으며, 바람직하게는 세파덱스 LH-20을 충진한 컬럼을 사용할 수 있으나. 이에 한정되지 않는다.The silica gel chromatography may be performed using a column for size exclusion chromatography, preferably a column packed with Sephadex LH-20. It is not limited to this.
본 발명의 구체적인 실시예에서, 상기 제조된 오죽 추출물의 에틸아세테이트 분획물을 CH2Cl2-MeOH 용매로 실리카겔 컬럼 크로마토그래피를 수행하여 8개의 화합물을 분리하였고, 분리된 8개의 화합물의 분자량을 측정하고, NMR 및 IR의 기기분석을 통하여 구조를 결정하였으며(표 1 및 2 참조), 그 결과 6종의 플라보노이드계열의 화합물과 2종의 페닐프로판노이드(phenylpropanoids) 계열의 화합물을 제조하였다. 구조동정된 각 화합물의 구조는 (1)이소오리엔틴(Isoorientin), (2)오리엔틴(Orientin), (3)비텍신(Vitexin), (4)시스-쿠마르산(cis-coumaric acid), (5)p-쿠마르산(p-coumaric acid), (6)루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin 6-C-(6''-O-trans-caffeoylglucoside)],(7) 트리세틴 3',4',5'-트리메틸 에스터 7-O-b-D-글루코파이라노이드(tricetin 3',4',5'-trimethyl ether 7-O-b-D-glucopyranoside) 및 (8)트리신(Tricin)으로 확인되었다(도 1 및 2 참조). In a specific embodiment of the present invention, the ethyl acetate fraction of the prepared Ojuk extract was subjected to silica gel column chromatography with CH 2 Cl 2 -MeOH solvent to separate eight compounds, and the molecular weight of the separated eight compounds was measured. The structure was determined by instrumental analysis of NMR and IR (see Tables 1 and 2). As a result, six kinds of flavonoid compounds and two kinds of phenylpropanoids were prepared. The structure of each compound identified is: (1) isorientin, (2) orientin, (3) bitine, (4) cis-coumaric acid, (5) p-coumaric acid, (6) luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) [Luteolin 6-C- (6 ''- O -trans-caffeoylglucoside)], (7) tricetin 3 ', 4', 5'-trimethyl ester 7- O -bD-glucopyranoid (tricetin 3 ', 4', 5'-trimethyl ether 7- O -bD-glucopyranoside) and (8) Tricin (see Figures 1 and 2).
상기 분리된 8개의 화합물 중 상기 [화학식 1]로 기재되는 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물의 망막 신경 세포(RGC-5) 보호 효과를 측정하기 위하여, 배양된 망막 신경 세포에 상기 화합물을 농도별로 전처리 한 후, 과산화수소 또는 BSO+글루탐산을 처리한 다음, MTT 방법을 통하여, 세포독성을 측정하였고, 그 결과, 망막 신경 세포 RGC-5에서 과산화수소 또는 BSO+글루탐산(glutamate)에 의해 산화스트레스가 유도된 경우, 상기 화합물을 전처리한 경우에 과산화수소 또는 BSO+글루탐산만 처리한 경우에 비해, 더 높은 세포 생존율을 나타내었다(도 4 참조).The protective effect of the retinal nerve cells (RGC-5) of the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound described in the above [Formula 1] In order to measure, after pretreatment of the compound by concentration to the cultured retinal neurons, and then treated with hydrogen peroxide or BSO + glutamic acid, cytotoxicity was measured by MTT method, as a result, hydrogen peroxide in retinal neurons RGC-5 Alternatively, when oxidative stress was induced by BSO + glutamate, the compound showed higher cell viability when pretreated with hydrogen peroxide or only BSO + glutamic acid (see FIG. 4).
따라서, 상기 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)은 망막 신경 세포의 산화스트레스에 있어서, 세포 보호 효과를 나타냄을 확인하였다.Therefore, it was confirmed that the compound luteolin 6-C- (6 '' -O -trans-cafeoylclocoside) exhibits a cytoprotective effect in oxidative stress of retinal neurons.
또한, 상기 [화학식 1]로 기재되는 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물이 망막 신경 세포(RGC-5)의 세포사멸을 저해하는지 확인하기 위하여, 살아있거나 죽은 세포, 즉 전체 세포를 파란색으로 염색하는 Hoechst 33342와 죽은 세포를 빨간색으로 염색하는 프로피디움 요오드화물(propidium iodide, PI)을 이용하여 이중으로 염색한 후 세포분석을 수행하였고, 그 결과, 망막 신경 세포 RGC-5에 과산화수소 또는 BSO+글루탐산(glutamate)처리에 의해 유도된 산화스트레스에 의한 세포 사멸에서, 상기 화합물을 전처리한 경우, 과산화수소 또는 BSO+글루탐산(glutamate)만 처리한 경우와 비교하였을 때, 세포사멸을 나타내는 프로피디움 요오드화물에 의한 형광 적색 표지가 감소함을 확인하였다(도 5 참조).In addition, confirming that the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound described in [Formula 1] inhibits apoptosis of retinal nerve cells (RGC-5). For this purpose, cell analysis was performed after double staining using Hoechst 33342, which stains live or dead cells (ie, whole cells in blue), and propidium iodide (PI), which stains dead cells in red. As a result, the cell death by oxidative stress induced by hydrogen peroxide or BSO + glutamate treatment in the retinal nerve cell RGC-5 was compared with the case of pretreatment of the compound with only hydrogen peroxide or BSO + glutamate treatment. At this time, it was confirmed that the fluorescent red label by propidium iodide showing apoptosis was reduced (see FIG. 5).
따라서, 상기 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)은, 산화스트레스에 의한 망막 신경 세포의 세포사멸을 저해하는 효과를 나타냄을 확인하였다.Therefore, it was confirmed that the compound luteolin 6-C- (6 '' -O -trans-cafeoylclocoside) exhibits an effect of inhibiting apoptosis of retinal nerve cells by oxidative stress.
또한, 상기 [화학식 1]로 기재되는 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물의 망막 신경 세포(RGC-5)에서 글루타티온(Glutathione; GSH) 회복 효과를 확인하기 위하여, 전체 글루타티온(GSH) 함량을 Rahman et al(2006)의 방법을 변형하여 측정하였고, 그 결과, 망막 신경 세포 RGC-5에서 H2O2 라디칼, OH-. 라디칼 및 O2 -. 라디칼에 의해 유도된 산화 라디칼에 의한 글루타티온 함량의 감소에서, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, H2O2 라디칼, OH-. 라디칼 및 O2 -. 라디칼만 처리한 경우와 비교하였을 때, RGC-5의 전체 글루타티온 함량의 감소를 유의적으로 회복시킴을 확인하였다(도 6 참조).In addition, glutathione (GSH) recovery in retinal nerve cells (RGC-5) of the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound described in the above [Formula 1] In order to confirm the effect, the total glutathione (GSH) content was measured by modifying the method of Rahman et al (2006), and as a result, H 2 O 2 radical, OH- . Radicals and O 2- . H 2 O 2 radical, OH when pretreatment of compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) in the reduction of glutathione content by oxidative radicals induced by radicals -. Radicals and O 2- . When compared with the case of only treatment with radicals, it was confirmed that the significant recovery of the reduction of the total glutathione content of RGC-5 (see Fig. 6).
따라서, 상기 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)은, 산화 라디칼에 의한 망막 신경 세포의 글루타티온의 감소를 회복하는 효과를 나타냄을 확인하였다.Therefore, it was confirmed that the compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) exhibits an effect of restoring a decrease in glutathione of retinal nerve cells by oxidative radicals.
또한, 상기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)의 망막 신경 세포(RGC-5)에서 아폽토시스(Apoptosis) 저해 효과를 측정하기 위하여, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)의 처리에 의한 망막 신경 세포의 아톱토틱 단백질의 발현 양을 면역블랏(immunoblot)을 통하여 비교하였고, 그 결과, 망막 신경 세포 RGC-5에서 BSO+글루탐산(glutamate)에 의한 아폽토시스에 의해 발현양이 증가되는 잘려진 PARP, AIF 및 잘려진 카스페아제-3 단백질의 발현양이 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, BSO+글루탐산(glutamate)만 처리하여 증가된 단백질의 발현양을 농도의존적으로 감소시켰다(도 9).In addition, the effect of inhibiting apoptosis in retinal nerve cells (RGC-5) of the compound Lutein 6-C- (6 '' -O -trans-cafeoylglucoside) described in the above [Formula 1] To determine, the expression levels of atopic proteins in retinal neurons by treatment with compound Luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) were compared via immunoblot. As a result, the expression levels of the truncated PARP, AIF and the truncated caspease-3 protein increased in the retinal neuronal cell RGC-5 by apoptosis by BSO + glutamate. When (6 '' -O -trans-cafeoylglucoside) was pretreated, only BSO + glutamate was used to decrease the expression level of the increased protein (FIG. 9).
따라서, 상기 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)은, 망막 신경 세포의 아폽토시스를 저해하는 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the compound luteolin 6-C- (6 '' -O -trans-cafeoylclocoside) exhibits an effect of inhibiting apoptosis of retinal neurons.
상기 본 발명의 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 포함하는 조성물은, 조성물 총 중량에 대하여 상기 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 0.1 내지 50 중량%로 포함하는 것이 바람직하나 이에 한정되지 않는다.The composition comprising the Ojuk extract of the present invention, a fraction thereof or a compound separated therefrom, preferably comprises 0.1 to 50% by weight of the Ojuk extract, a fraction thereof or a compound separated therefrom based on the total weight of the composition It is not limited to this.
본 발명의 조성물은 약제의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.
본 발명에 따른 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태, 체중, 연령, 성별, 식이, 배설율, 질환의 중증도, 약물형태, 투여시간, 투여방법, 투여경로 및 투여기간 등에 따라 그 범위가 다양하다. 1일 투여량은 본 발명에 따른 추출물, 분획물 또는 화합물을 동결건조하였을 때의 양으로 0.0001㎎/㎏ 내지 500㎎/㎏, 바람직하게는 0.001㎎/㎏ 내지 100㎎/㎏ 이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.
The compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the patient's condition, weight, age, sex, The range varies depending on the diet, the rate of excretion, the severity of the disease, the form of the drug, the time of administration, the method of administration, the route of administration and the duration of administration. The daily dose is from 0.0001 mg / kg to 500 mg / kg, preferably from 0.001 mg / kg to 100 mg / kg in an amount when the extract, fraction or compound according to the invention is lyophilized, and daily if necessary. Administration may be from one to several times.
또한, 본 발명은 오죽 추출물 또는 이의 분획물을 유효성분으로 함유하는 녹내장 예방 및 개선용 건강식품을 제공한다.In addition, the present invention provides a health food for preventing and improving glaucoma, containing the porridge extract or a fraction thereof as an active ingredient.
아울러, 본 발명은 상기 [화학식 1]의 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin6-C-(6''-O-trans-caffeoylglucoside)]를 유효성분으로 함유하는 녹내장 예방 및 개선용 건강식품을 제공한다.
In addition, the present invention is the
본 발명의 오죽 추출물, 이의 분획물 및 이로부터 분리한 화합물이 산화스트레스를 유도한 망막 신경 세포를 보호하여 세포사멸을 저해하고, 아폽토시스의 저해 효과를 나타내어, 망막 신경 세포의 퇴화로 인한 녹내장 질환의 예방 및 개선용 건강식품으로 사용될 수 있다.
Ojuk extract of the present invention, fractions thereof and compounds isolated therefrom protect oxidative stress-induced retinal neurons, inhibit apoptosis, and inhibit apoptosis, thereby preventing glaucoma disease due to degeneration of retinal neurons. And it can be used as a health food for improvement.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 드링크제, 육류, 소세지, 빵, 비스켓, 떡, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강 식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of foods to which the above-mentioned substances may be added include dairy products including drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, Beverages, alcoholic beverages, vitamin complexes, and the like, and include all healthy foods in the conventional sense.
본 발명의 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강기능식품 중의 상기 추출물의 양은 전체 식품 중량의 0.1 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The porridge extract, fractions thereof or compounds separated therefrom of the present invention may be added to foods as is or used with other foods or food ingredients, and may be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the extract in the dietary supplement may be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 오죽 추출물, 이의 분획물 또는 이로부터 분리한 화합물은 100 중량부 당 0.1 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.
In addition to the above, the Ojuk extract of the present invention, its fractions or compounds separated therefrom are various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.) , Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the porridge extract of the present invention, a fraction thereof or a compound separated therefrom may contain flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but it is generally selected from the range of 0.1 to about 20 parts by weight per 100 parts by weight of the prickly extract, fractions thereof or compounds isolated therefrom.
이하, 본 발명을 실시예, 실험예 및 제조예에 의해 상세히 설명한다. Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.
단 하기 실시예, 실험예 및 제조예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 실험예 및 제조예에 의해 한정되지 않는다.
However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Experimental Examples, and Preparation Examples.
<실시예 1> 오죽 추출물의 제조Example 1 Preparation of Cucumber Extract
건조된 오죽(강릉시 오죽농원)을 적당한 크기로 분쇄하여 추출용기에 오죽 2.0 kg과 50 - 95%의 에탄올 수용액을 총 2.0 ℓ를 가하여 실온에서 7일간 방치한 후 거름종이로 여과하여 추출물을 얻었다. 추출과정은 5회 반복하였고, 이후 용매를 감압 농축하여 255.0 g의 에탄올 추출물을 수득하였다.
The dried Ojuk (Gangneung Ojuk Farm) was ground to an appropriate size, 2.0 kg of Ojuk and 50-95% ethanol aqueous solution were added to the extraction vessel, and the mixture was left at room temperature for 7 days and filtered through a filter paper to obtain an extract. The extraction process was repeated five times, after which the solvent was concentrated under reduced pressure to obtain 255.0 g of ethanol extract.
<실시예 2> 오죽 추출물로부터 분획물의 제조Example 2 Preparation of Fractions from Cucumber Extracts
<실시예 1>의 상기 오죽 추출물을 분별깔때기를 이용하여 노르말-헥산(106.0 g), 염화 메틸렌(11.0 g), 에틸 아세테이트(5.0 g), 노르말-부탄올(60.0 g) 및 물(72.6 g) 분획물로 순차적으로 계통분획하였다.
Using the separatory funnel, the extract of Example 1 was treated with a normal funnel, normal-hexane (106.0 g), methylene chloride (11.0 g), ethyl acetate (5.0 g), normal-butanol (60.0 g), and water (72.6 g). The fractions were systematically fractionated.
<< 실시예Example 3> 오죽 3> Oju 추출물로부터From extract 화합물의 제조 Preparation of compounds
상기 <실시예 2>의 에틸 아세테이트 분획물을 CH2Cl2-MeOH 용매로 실리카겔 컬럼 크로마토그래피를 수행하여 8개의 화합물을 분리하였다. 분리된 8개의 화합물의 분자량을 측정하고, NMR 및 IR의 기기분석을 통하여 구조를 결정하였고, NMR 결과는 8개의 화합물을 DMSO-d6에 녹인 후, 크라이오프로브(cryoprobe)를 이용하여 분광계로(Varian Unity Inova 500) 500과 25 MHz에서의 1H-와 13C-NMR 결과를 하기 [표 1] 및 [표 2]에 기록하였다. The ethyl acetate fraction of <Example 2> was subjected to silica gel column chromatography with a CH 2 Cl 2 -MeOH solvent to separate eight compounds. The molecular weight of the separated eight compounds was measured, and the structure was determined by instrumental analysis of NMR and IR. The NMR results were obtained by dissolving eight compounds in DMSO-
분석 결과, 6종이 플라보노이드계열의 화합물이었으며, 2종의 화합물은 페닐프로판노이드(phenylpropanoids) 계열의 화합물이었다. 구조동정 된 각 화합물의 구조는 (1)이소오리엔틴(Isoorientin), (2)오리엔틴(Orientin), (3)비텍신(Vitexin), (4)시스-쿠마르산(cis-coumaric acid), (5)p-쿠마르산(p-coumaric acid), (6)루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin 6-C-(6''-O-trans-caffeoylglucoside)],(7) 트리세틴 3',4',5'-트리메틸 에스터 7-O-b-D-글루코파이라노이드(tricetin 3',4',5'-trimethyl ether 7-O-b-D-glucopyranoside) 및 (8)트리신(Tricin) 이었다(도 1 및 도 2).
As a result, six compounds were flavonoid compounds, and two compounds were phenylpropanoids. The structure of each compound identified is: (1) isorientin, (2) orientin, (3) bitine, (4) cis-coumaric acid, (5) p-coumaric acid, (6) luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) [Luteolin 6-C- (6 ''- O -trans-caffeoylglucoside)], (7) tricetin 3 ', 4', 5'-trimethyl ester 7- O -bD-glucopyranoid (tricetin 3 ', 4', 5'-trimethyl ether 7- O -bD-glucopyranoside) and (8) Tricin (FIGS. 1 and 2).
<< 실험예Experimental Example 1> 오죽 1> Oju 분획물의Fraction 망막 신경 세포( Retinal nerve cells ( RGCRGC -5) 보호 효과 측정-5) protective effect measurement
망막 신경 세포인 RGC-5 세포(Alcon Research Ltd., 로부터 무상 공급받아 사용하였음)는 10% 우태아혈청(FBS)과 100 U/ml 페니실린/스트렙토마이신(penicillin/streptomycin)이 포함된 Dulbecco's modified Eagle's medium(DMEM, Hyclone, USA) 배지를 사용하여 T-75 조직 배양 플라스크(tissue culture flask)에 37℃에서 5% CO2, 95% 공기 및 포화 습도하의 항온기에서 배양하였다. 2일 내지 3일 후 포화 상태로 자란 세포는 트립신(trypsin-EDTA) 용액으로 플라스크로부터 떼어낸 후 계대 배양하였다. 세포를 96 웰 배양 접시에 분주한 후, 24시간 동안 배양기에 넣어 세포를 부착시킨 후, 배지를 제거하고 나서 혈청 단백질에 의한 항산화 효과를 배제하기 위하여 1%의 저농도 우태아혈청이 포함된 DMEM 배지로 교환하였다. 상기 <실시예 2>에서 분리한 각각의 분획물을 그 최종농도가 50, 10, 1 uM이 되게 1시간 동안 전처리한 후, 300 μM 과산화수소(hydrogen peroxide) 또는 0.5mM 1-부티오닌 설폭시민[1-buthionine-(S,R)-sulfoximine; BSO) 및 10mM L-글루탐산(L-glutamatmic acid; glutamate)을 37℃에서 24시간 동안 처리 후, MTT 방법을 수행하여 세포독성을 측정하였다. Retinal neurons RGC-5 cells (available from Alcon Research Ltd., gratuitously used) were Dulbecco's modified Eagle's containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin / streptomycin. medium and cultured in (DMEM, Hyclone, USA) using a medium T-75 tissue culture flasks at 37 ℃ to (tissue culture flask) 5% CO 2, 95% air and saturated humidity under thermostat. After 2 to 3 days, cells grown in saturation were detached from the flask with trypsin-EDTA solution and passaged. After dispensing the cells into a 96 well culture dish, the cells were placed in the incubator for 24 hours to attach the cells, and then, after removing the medium, DMEM medium containing 1% low concentration fetal calf serum to exclude the antioxidant effect by serum proteins. Exchanged with Each fraction separated in Example 2 was pretreated for 1 hour to have a final concentration of 50, 10, 1 uM, and then 300 μM hydrogen peroxide or 0.5 mM 1-butionine sulfoximine [ 1-buthionine- (S, R) -sulfoximine; BSO) and 10 mM L-glutamatic acid (L-glutamatmic acid; glutamate) were treated for 24 hours at 37 ℃, followed by MTT method to measure the cytotoxicity.
MTT(3-[4, 5-imethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, Sigma) 방법은 세포 생존과 세포독성의 선별 검사로 흔히 이용되고 있는 발색 검사의 일종으로, 세포의 미토콘드리아 탈수소효소(mitochondrial dehydrogenase)에 의해 테트라졸리움 염(tetrazolium salt)인 MTT가 불용성 포마잔(formazan crystal)으로 변화되는 것을 DMSO를 처리하여 용해한 후, 변화된 정도를 발색반응으로 확인하여, 세포 독성의 정도를 측정하는 방법이다. 구체적으로, MTT 방법을 수행하기 위하여, 상기 RGC-5 세포의 배지를 제거하고 각 웰당 최종농도가 0.5 mg/ml인 MTT를 첨가 후 1시간 동안 반응시켰다. 이후, 각 웰당 디메틸술폭시드(dimethylsulfoxide)를 100 ul씩 처리하고 15분간 흔든 후, 시너지 HT 마이크로플레이트 분석기[Synergy HT Multi-microplate reader(Bio-Tek instruments, Winooski, VT, U.S.A)]로 570 nm에서 흡광도를 측정하였다.MTT (3- [4,5-imethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide, Sigma) method is a colorimetric test commonly used for screening cell viability and cytotoxicity. Cellular mitochondrial dehydrogenation After dissolving the tetrazolium salt MTT, which is an enzyme (mitochondrial dehydrogenase), into an insoluble formazan crystal, by dissolving it in DMSO, the degree of change was confirmed by the color reaction, and the degree of cytotoxicity was measured. That's how. Specifically, to perform the MTT method, the medium of the RGC-5 cells was removed and reacted for 1 hour after the addition of MTT having a final concentration of 0.5 mg / ml per well. Subsequently, 100 ul of dimethylsulfoxide was treated per well and shaken for 15 minutes, followed by a Synergy HT Multi-microplate reader (Bio-Tek instruments, Winooski, VT, USA) at 570 nm. Absorbance was measured.
그 결과, 망막 신경 세포 RGC-5에서 과산화수소 처리에 의한 산화스트레스가 유도된 경우, 오죽의 노르말-헥산 분획물, 염화 메틸렌 분획물, 에틸 아세테이트 분획물 및 노르말-부탄올 분획물을 전처리한 경우, 과산화수소만 처리한 경우에 비해, 더 높은 세포 생존율을 나타내었다(도 3의 위쪽 그림).As a result, when oxidative stress induced by hydrogen peroxide treatment was induced in retinal nerve cell RGC-5, when normal-hexane fraction, methylene chloride fraction, ethyl acetate fraction, and normal-butanol fraction of Oju were treated with hydrogen peroxide only. In comparison, it showed higher cell viability (top figure in FIG. 3).
또한, 망막 신경 세포 RGC-5에서 BSO+글루탐산(glutamate) 처리에 의한 산화스트레스가 유도된 경우, 오죽의 노르말-헥산 분획물, 염화 메틸렌 분획물, 에틸 아세테이트 분획물 및 노르말-부탄올 분획물을 전처리한 경우, BSO+글루탐산(glutamate)만 처리한 경우에 비해, 더 높은 세포 생존율을 나타내었다(도 3의 아래쪽 그림).In addition, when oxidative stress induced by BSO + glutamate treatment was induced in retinal nerve cells RGC-5, BSO + glutamic acid when normal-hexane fraction, methylene chloride fraction, ethyl acetate fraction and normal-butanol fraction of Oju was pretreated. The cell viability was higher than when treated with (glutamate) alone (bottom figure of Figure 3).
따라서, 오죽 분획물, 특히 에틸아세테이트 분획물은, 망막 신경 세포의 산화스트레스에 있어서, 세포 보호 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the porridge fraction, especially the ethyl acetate fraction, exhibited a cytoprotective effect in the oxidative stress of retinal nerve cells.
<< 실험예Experimental Example 2> 2> 루테오린Luteolin 6-C-(6''- 6-C- (6 ''- OO -트랜스--Trans- 카페오일클루코사이드Cafe Oil Clocoside )의 망막 신경 세포(Retinal nerve cells () RGCRGC -5) 보호 효과 측정-5) protective effect measurement
망막 신경 세포를 96 웰 배양 접시에 분주한 후, 24시간 동안 배양기에 넣어 세포를 부착시킨 후, 배지를 제거하고 나서 혈청 단백질에 의한 항산화 효과를 배제하기 위하여 1%의 저농도 우태아혈청이 포함된 DMEM 배지로 교환하였다. 상기 <실시예 3>에서 분리한 상기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)[Luteolin 6-C-(6''-O-trans-caffeoylglucoside)]을 그 최종 농도가 50, 10, 1 uM이 되게 각각 1시간 동안 전처리한 후, 300 μM 과산화수소(hydrogen peroxide) 또는 0.5mM 1-부티오닌 설폭시민[1-buthionine-(S,R)-sulfoximine; BSO) 및 10mM L-글루탐산(L-glutamatmic acid; glutamate)을 37℃에서 24시간 동안 처리 후, MTT 방법을 수행하여 세포독성을 측정하였다.The retinal nerve cells were dispensed into a 96 well culture dish, placed in the incubator for 24 hours to attach the cells, and then the medium was removed to contain 1% low concentration fetal calf serum to exclude the antioxidant effect by serum proteins. Exchange with DMEM medium. The compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) described in the above [Formula 1] isolated in <Example 3> [Luteolin 6-C- (6 '' -O- trans-caffeoylglucoside)] was pretreated for 1 h each to its final concentrations of 50, 10 and 1 uM, followed by 300 μM hydrogen peroxide or 0.5 mM 1-butionine sulfoximine [1-buthionine -(S, R) -sulfoximine; BSO) and 10 mM L-glutamatic acid (L-glutamatmic acid; glutamate) were treated for 24 hours at 37 ℃, followed by MTT method to measure the cytotoxicity.
그 결과, 망막 신경 세포 RGC-5에서 과산화수소 처리에 의한 산화스트레스가 유도된 경우, 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, 과산화수소만 처리한 경우에 비해, 농도의존적으로 더 높은 세포 생존율을 나타내었다(도 4의 위쪽 그림).As a result, when peroxidation induced by hydrogen peroxide treatment was induced in retinal nerve cell RGC-5, and pretreated with luteolin 6-C- (6 '' -O -trans-cafeoylglucoside), only hydrogen peroxide Compared to the treatment, concentration-dependently higher cell viability was shown (upper figure in Figure 4).
또한, 망막 신경 세포 RGC-5에서 BSO+글루탐산(glutamate)에 의한 산화스트레스가 유도된 경우에도, 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, BSO+글루탐산(glutamate)만 처리한 경우에 비해, 농도의존적으로 더 높은 세포 생존율을 나타내었다(도 4의 아래쪽 그림).In addition, even when oxidative stress induced by BSO + glutamate was induced in retinal nerve cell RGC-5, luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) was pretreated. Compared to the case where only BSO + glutamate was treated, the cell viability was higher depending on the concentration (Fig. 4, bottom).
따라서, 상기 [화학식 1]로 기재되는 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물은, 망막 신경 세포의 산화스트레스에 있어서, 세포 보호 효과를 나타냄을 확인하였다.
Therefore, the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound described in [Formula 1] exhibits a cell protective effect in oxidative stress of retinal nerve cells. Confirmed.
<< 실험예Experimental Example 3> 3> 루테오린Luteolin 6-C-(6''- 6-C- (6 ''- OO -트랜스--Trans- 카페오일클루코사이드Cafe Oil Clocoside )의 망막 신경 세포(Retinal nerve cells () RGCRGC -5)의 세포사멸 저해 효과 측정-5) measurement of apoptosis inhibitory effect
상기 [화학식 1]로 기재되는 화합물인 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)가 망막 신경 세포(RGC-5)의 세포사멸을 저해하는지 확인하기 위하여, 살아있거나 죽은 세포, 즉 전체 세포를 파란색으로 염색하는 Hoechst 33342와 죽은 세포를 빨간색으로 염색하는 프로피디움 요오드화물(propidium iodide, PI)을 이용하여 이중으로 염색한 후 세포분석을 수행하였다. .To determine whether lutein 6-C- (6 '' -O -trans-cafeoylglucoside), a compound described in
구체적으로, 세포를 96 웰 배양 접시에 분주하여 부착시킨 후 배지를 1% 우태아혈청(FBS)으로 교환하고 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)을 그 최종농도가 50, 10, 1 uM이 되게 1시간 동안 전처리하였다. 이후, 300 μM 과산화수소(hydrogen peroxide) 또는 0.5mM 1-부티오닌 설폭시민[1-buthionine-(S,R)-sulfoximine; BSO) 및 10mM L-글루탐산(L-glutamatmic acid; glutamate)을 37℃에서 24시간 동안 처리 후, 세포를 차가운 D-PBS로 세척하고, 최종농도가 8 uM의 Hoechst 33342와 1.5 uM의 PI를 세포에 첨가하여 15분간 37℃에서 염색시킨 후, 형광 현미경(fluorescence microscope)을 사용하여 가시화하였다(도 5).Specifically, the cells were dispensed into 96 well culture dishes and attached to the medium, followed by exchange of medium with 1% fetal bovine serum (FBS), and the compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside ) Was pretreated for 1 hour to a final concentration of 50, 10, 1 uM. Then, 300 μM hydrogen peroxide or 0.5 mM 1-butionine sulfoximine [1-buthionine- (S, R) -sulfoximine; BSO) and 10 mM L-glutamatmic acid (gluamate) at 37 ° C. for 24 hours, then the cells were washed with cold D-PBS and the final concentrations were 8 uM Hoechst 33342 and 1.5 uM PI. After dyeing at 37 ° C. for 15 minutes, it was visualized using a fluorescence microscope (FIG. 5).
그 결과, 망막 신경 세포 RGC-5에서 과산화수소 또는 BSO+글루탐산(glutamate)처리에 의해 유도된 산화스트레스에 의한 세포 사멸에서, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, 과산화수소 또는 BSO+글루탐산(glutamate)만 처리한 경우와 비교하였을 때, 세포사멸을 나타내는 프로피디움 요오드화물에 의한 형광 적색 표지가 감소함을 확인하였다(도 5).As a result, in the cell death by oxidative stress induced by hydrogen peroxide or BSO + glutamate treatment in retinal nerve cells RGC-5, the compound luteolin 6-C- (6 '' -O -trans-cafeoylglu Coside) was pretreated, compared with the case of only hydrogen peroxide or BSO + glutamate (glutamate), it was confirmed that the fluorescent red label by the propidium iodide showing apoptosis is reduced (Fig. 5).
따라서, 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물은, 산화스트레스에 의한 망막 신경 세포의 세포사멸을 저해하는 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound exhibits an effect of inhibiting apoptosis of retinal nerve cells by oxidative stress.
<< 실험예Experimental Example 4> 4> 루테오린Luteolin 6-C-(6''- 6-C- (6 ''- OO -트랜스--Trans- 카페오일클루코사이드Cafe Oil Clocoside )의 망막 신경 세포(Retinal nerve cells () RGCRGC -5)에서 글루타티온(-5) to glutathione ( GlutathioneGlutathione ) 회복 효과 측정) Recovery effect measurement
상기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)의 망막 신경 세포(RGC-5)에서 글루타티온(Glutathione; GSH) 회복 효과를 확인하기 위하여, 전체 글루타티온(GSH) 함량을 Rahman et al(2006)의 방법을 변형하여 측정하였다. Glutathione (GSH) recovery effect in the retinal nerve cells (RGC-5) of the compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) described in [Formula 1] To confirm, total glutathione (GSH) content was measured by modifying the method of Rahman et al (2006).
구체적으로, 세포를 96 웰 배양 접시에 분주한 후, 24시간 동안 배양기에 넣어 세포를 부착시키고, 배지를 1% 우태아혈청(FBS)로 교환한 후, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 그 최종농도가 50, 10, 1 uM이 되게 1시간 동안 전처리하였다. 이후, 배지를 제거하고 1mM 과산화수소(H2O2 라디칼), 1mM 과산화수소 및 100 uM 철(II) 퍼클로레이트 염화물(iron(II) perchlorate hexahydrate; OH-. 라디칼), 1mM 과산화칼륨(KO2; O2 -. 라디칼)을 1시간 동안 세포에 노출시켜 산화적 손상에 의한 총 글루타티온의 감소를 측정한 후, 전체 글루타티온(GSH) 함량은 표준곡선에 의해서 산출하였고, nmol/ mg 단백질로 계산하여 대조군의 GSH 함량에 대한 시료를 처리한 GSH 함량의 비로 나타내었다. Specifically, after dispensing the cells in a 96 well culture dish, put the cells in the incubator for 24 hours to attach the cells, exchange the medium with 1% fetal bovine serum (FBS), and then the compound luteolin 6-C- (6 ' -O -trans-cafeoylglucoside) was pretreated for 1 hour to a final concentration of 50, 10, 1 uM. The medium was then removed and 1 mM hydrogen peroxide (H 2 O 2 radical), 1 mM hydrogen peroxide and 100 uM iron (II) perchlorate hexahydrate (OH- . Radical), 1 mM potassium peroxide (KO 2 ; O 2) - radical), a 1 is exposed to the cells for a time and then measuring the decrease in the total glutathione by oxidative damage, it was calculated total glutathione (GSH) content by a standard curve, the calculated as nmol / mg protein control group GSH It is expressed as the ratio of the treated GSH content to the content.
그 결과, 망막 신경 세포 RGC-5에서 H2O2 라디칼, OH-. 라디칼 및 O2 -. 라디칼에 의해 유도된 산화 라디칼에 의한 글루타티온 함량의 감소에서, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, H2O2 라디칼, OH-. 라디칼 및 O2 -. 라디칼만 처리한 경우와 비교하였을 때, RGC-5의 전체 글루타티온 함량의 감소를 유의적으로 회복시킴을 확인하였다(도 6).As a result, H 2 O 2 radical, OH − in retinal neurons RGC-5 . Radicals and O 2- . H 2 O 2 radical, OH when pretreatment of compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) in the reduction of glutathione content by oxidative radicals induced by radicals -. Radicals and O 2- . Compared with the case of only radical treatment, it was confirmed that the reduction of the total glutathione content of RGC-5 significantly recovered (FIG. 6).
따라서, 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물은, 산화 라디칼에 의한 망막 신경 세포의 글루타티온의 감소를 회복하는 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound exhibits an effect of restoring a reduction in glutathione of retinal nerve cells by oxidative radicals.
<< 실험예Experimental Example 5> 5> 루테오린Luteolin 6-C-(6''- 6-C- (6 ''- OO -트랜스--Trans- 카페오일클루코사이드Cafe Oil Clocoside )의 망막 신경 세포(Retinal nerve cells () RGCRGC -5)에서 At -5) 아폽토시스Apoptosis (( ApoptosisApoptosis ) 저해 효과 측정) Inhibition effect measurement
상기 [화학식 1]로 기재되는 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)의 망막 신경 세포(RGC-5)에서 아폽토시스(Apoptosis) 저해 효과를 측정하기 위하여, 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)의 처리에 의한 망막 신경 세포의 아톱토틱 단백질의 발현 양을 비교하였다.To measure the effect of inhibiting apoptosis in retinal nerve cells (RGC-5) of the compound luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) described above [Formula 1] In order to compare the expression levels of atopic proteins in retinal neurons by treatment with compound Luteolin 6-C- (6 '' -O -trans-cafeoylglucoside).
구체적으로, DMEM/10% FBS 용액을 RGC-5 세포 배양액과 혼합하여 세포의 수를 1 X 105 세포/ml로 조제한 후에 60 mm 배양접시에 5 ml씩 처리하여 5% CO2에서 37℃의 조건으로 배양하였다. 세포가 안정화된 후에 화합물 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)을 그 최종농도가 50, 10, 1 uM이 되게 1시간 동안 전처리한 후, 0.5mM 1-부티오닌 설폭시민[1-buthionine-(S,R)-sulfoximine; BSO) 및 10mM L-글루탐산(L-glutamatmic acid; glutamate)를 18시간 동안 처리하였다. 차가운 D-PBS(1x, pH 7.4) 완충 용액으로 3회 세척한 후 세포를 수득하였고, 수득된 세포를 세포 용해 완충 용액(cell lysis buffer[1M 트리스 pH 7.4, 2M 염화나트륨, 1M EDTA, 10% NP40, 프로테이즈 저해제 칵테일(Sigma, St. Louis, MO, U.S.A.)])을 가하여 10분 동안 얼음에 박아놓은 다음 소니케이션을 수행하여 세포를 파쇄하고, 4℃, 10,000 xg에서 30분 동안 원심분리하여 단백질을 추출하였다. 단백질의 농도는 Bio-Rad 단백질 분석방법을 사용하여 정량하였고, 4x 샘플 완충 용액(sample buffer[200mM Tris-HCl (pH 6.8), 40% 글리세롤, 8% SDS, 400mM DTT, 0.4% 브로모페놀 블루])과 1 : 4의 비율로 섞은 후, 5분 동안 끓여 10% 또는 12% 폴리아크릴아마이드 겔을 사용하여 SDS-폴리아크릴아마이드 전기영동을 수행하였다. 전기영동 후, 폴리아크릴아마이드 겔 상의 단백질을 폴리비닐리덴 디플루오라이드(PVDF) 멤브레인(Hybond-P; Amersham Biosciences, GE Healthcare, U.K)에 이동시키고 폰슈 S(Ponseau S, Sigma, St. Louis, USA)로 염색하여 동량의 단백질들이 전기영동 되었는지, 단백질의 이동이 잘 되었는지 확인하였다. 상기 멤브레인을 PBST 완충 용액(8g/L NaCl, 0.2g/L KCl, 1.44g/L NaH2PO4, 0.1% Tween-20)으로 세척하고, 5% 탈지 분유가 포함된 PBST 완충 용액으로 1시간 30분 동안 실온에서 블랏킹한 뒤, 1 : 500 희석 배수의 토끼 항-PARP 항체(catalog no.9542; Cell Signaling), 항-AIF 항체(catalog no. 4642; Cell Signaling) 및 항-잘려진 카스페아제-3 항체(catalog no. 9661; Cell Signaling)로 1시간 동안 실온에서 반응시킨 후 4℃에서 하룻밤 동안 반응시켰다. 일차항체 처리 후, 상기 멤브레인을 15분씩 3회 세척 후, 1 : 3,000 희석 배수의 이차항체(Santa Cruz Biotechnology, CA)로 2시간 동안 반응시키고, 발색을 유도하여 시각화하였고, 발현되는 단백질의 양은 Las-4000 이미지 판독기(image reader) 및 Multi Gauge 3.1 소프트웨어(Fuji Photo Film, Japan)을 사용하여 측정하였다.Specifically, DMEM / 10% FBS solution was mixed with RGC-5 cell culture to prepare the number of cells at 1
그 결과, 망막 신경 세포 RGC-5에서 BSO+글루탐산(glutamate)에 의한 아폽토시스에 의해 발현양이 증가되는 잘려진 PARP, AIF 및 잘려진 카스페아제-3 단백질의 발현양이 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드)를 전처리한 경우, BSO+글루탐산(glutamate)만 처리하여 증가된 단백질의 발현양을 농도의존적으로 감소시켰다(도 9).As a result, the expression levels of the truncated PARP, AIF and the truncated caspease-3 protein whose expression was increased by apoptosis by BSO + glutamate in the retinal nerve cell RGC-5 were expressed in luteolin 6-C- (6 When pre-treated with ' -O -trans-cafeoylglucoside), only BSO + glutamate was treated to decrease the expression level of the increased protein (FIG. 9).
따라서, 루테오린 6-C-(6''-O-트랜스-카페오일클루코사이드) 화합물은, 망막 신경 세포의 아폽토시스를 저해하는 효과를 나타냄을 확인하였다.
Therefore, it was confirmed that the luteolin 6-C- (6 '' -O -trans-cafeoylglucoside) compound exhibits an effect of inhibiting apoptosis of retinal neurons.
하기에 본 발명의 조성물을 위한 제조예를 예시한다.
The preparation examples for the compositions of the present invention are illustrated below.
<< 제조예Manufacturing example 1> 약학적 제제의 제조 1> Preparation of Pharmaceutical Formulations
1-1. 산제의 제조1-1. Manufacture of powder
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 2 gOju 2 g extract, fraction thereof or compound of
유당 1 g1 g lactose
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.
The above components were mixed and packed in airtight bags to prepare powders.
1-2. 정제의 제조1-2. Manufacture of tablets
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 100 ㎎
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.
After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
1-3. 캡슐제의 제조1-3. Preparation of Capsules
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 100 ㎎
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
1-4. 주사제의 제조1-4. Injection preparation
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 100 ㎎
만니톨 180 ㎎Mannitol 180 mg
Na2HPO4ㆍ2H2O 26 ㎎Na 2 HPO 4 2H 2 O 26 mg
증류수 2974 ㎎Distilled water 2974 mg
통상적인 주사제의 제조방법에 따라, 상기 성분들을 제시된 함량으로 함유시켜 주사제를 제조하였다.
According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.
<< 제조예Manufacturing example 2> 건강기능 식품의 제조 2> Manufacture of dietary supplements
2-1. 음료의 제조2-1. Manufacturing of beverages
꿀 522 ㎎522 mg of honey
치옥토산아미 5 ㎎
니코틴산아미드 10 ㎎
염산리보플라빈나트륨 3 ㎎Riboflavin Sodium Hydrochloride 3 mg
염산피리독신 2 ㎎Pyridoxine hydrochloride 2 mg
이노시톨 30 ㎎Inositol 30 mg
오르트산 50 ㎎Orthoic acid 50 mg
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 0.48-1.28 ㎎Oju Extract, fractions thereof, or compound of
물 200 ㎖200 ml of water
상기 조성 및 함량으로 하여 통상적인 방법을 사용하여 음료를 제조하였다.
A beverage was prepared using the above-mentioned composition and content by a conventional method.
2-2. 츄잉껌의 제조2-2. Preparation of Chewing Gum
껌베이스 20%
설탕 76.36-76.76%Sugar 76.36-76.76%
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 0.24-0.64%Oju Extracts, fractions thereof or compounds of
후르츠향 1%1% of fruit flavor
물 2%Water 2%
상기 조성 및 함량으로 하여 통상적인 방법을 사용하여 츄잉껌을 제조하였다.
Chewing gum was prepared using the above-mentioned composition and content by a conventional method.
2-3. 캔디의 제조2-3. Manufacture of candy
설탕 50-60%50-60% sugar
물엿 39.26-49.66%Starch syrup 39.26-49.66%
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 0.24-0.64%Oju Extracts, fractions thereof or compounds of
오렌지향 0.1%Orange flavor 0.1%
상기 조성 및 함량으로 하여 통상적인 방법을 사용하여 캔디를 제조하였다.
The composition and the content of the candy were prepared using a conventional method.
2-4. 밀가루 식품의 제조2-4. Manufacture of flour food products
본 발명에 따른 오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 0.5 내지 5 중량부를 밀가루 100 중량부에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.
Porridge according to the present invention 0.5 to 5 parts by weight of an extract, a fraction thereof, or the compound of
2-5. 유제품(dairy products)의 제조2-5. Manufacture of dairy products
본 발명에 따른 오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 5 내지 10 중량부를 우유 100 중량부에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.
Porridge according to the
2-6. 선식의 제조2-6. Manufacture of wire
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입고 60 메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted.
검은콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Black soybeans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.
상기에서 제조한 곡물류 및 종실류와 본 발명에 따른 오죽 추출물, 이의 분획물 또는 화학식 1의 화합물을 다음과 같은 비율로 배합하여 제조하였다.Cereals and seeds produced in the above and the porridge according to the present invention An extract, a fraction thereof or a compound of
현미 30%30% brown rice
율무 15%15% rate
보리 20%
들깨 7% Perilla 7%
검정콩 7% Black Bean 7%
검은깨 7%Black Sesame 7%
오죽 추출물, 이의 분획물 또는 화학식 1의 화합물 3%Oju Extracts, fractions thereof or compounds of
영지 0.5%Ganoderma 0.5%
지황 0.5%
Foxglove 0.5%
상기에서 살펴본 바와 같이, 본 발명 조성물인 오죽 추출물, 이의 분획물 및 이로부터 분리한 화합물은 산화스트레스를 유도한 망막 신경 세포의 퇴화를 보호하는 효과가 있고, 천연물 유래로 독성이 거의 없기 때문에, 망막 신경 세포의 퇴화로 인해 일어나는 녹내장의 예방 및 치료용 조성물의 유효성분으로 유용하게 이용될 수 있다.As described above, Ojuk extract, a fraction thereof, and a compound separated from the composition of the present invention have an effect of protecting the regression of oxidative stress-induced retinal nerve cells, and because of little toxicity due to natural products, retinal nerves It can be usefully used as an active ingredient of the composition for the prevention and treatment of glaucoma caused by the degeneration of cells.
Claims (11)
Phyllostachys nigra MUNRO) Glaucoma preventive and therapeutic composition containing an extract or a fraction thereof as an active ingredient.
The composition of claim 1, wherein the extract is extracted with water, C 1 -C 4 alcohols, or a mixed solvent thereof.
The composition of claim 2, wherein the C 1 to C 4 alcohol is methanol or ethanol.
The method of claim 1, wherein the fraction is a normal-hexane, methylene chloride, ethyl acetate, normal-butanol, normal-hexane fraction obtained by sequentially fractionating the extract of water, methylene chloride fraction, ethyl acetate fraction, normal-butanol fraction or Composition characterized in that the water fraction.
[화학식 1]
To Formula 1 the base Theo Lin 6-C- (6 '' - O - trans-cafe five days inclusive nose side) [Luteolin 6-C- (6 ' compound described in' - O -trans-caffeoylglucoside)] Composition for the prevention and treatment of glaucoma containing as an active ingredient:
[Formula 1]
6. The composition for preventing and treating glaucoma according to claim 5, wherein the compound is isolated from the bamboo shoot extract.
Health food for the prevention and improvement of glaucoma, containing the Ojuk extract or fractions thereof as an active ingredient.
The method of claim 7, wherein the extract is health food, characterized in that the extract of porridge with water, C 1 ~ C 4 alcohol or a mixed solvent thereof.
The method of claim 7, wherein the fraction is normal-hexane, methylene chloride, ethyl acetate, normal-butanol, normal-hexane fraction obtained by sequentially fractionating the water, methylene chloride fraction, ethyl acetate fraction, normal-butanol fraction or Health food, characterized in that the water fraction.
[화학식 1]
To Formula 1 the base Theo Lin 6-C- (6 '' - O - trans-cafe five days inclusive nose side) [Luteolin 6-C- (6 ' compound described in' - O -trans-caffeoylglucoside)] Health food for the prevention and improvement of glaucoma containing as an active ingredient:
[Formula 1]
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101422668B1 (en) * | 2011-12-21 | 2014-07-28 | 한국과학기술연구원 | Method for extracting bambusoideae's leaves using subcritical solvents |
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