KR101514791B1 - A composition for preventing or treating blood vessel disorders, comprising extracts of Acanthopanax - Google Patents
A composition for preventing or treating blood vessel disorders, comprising extracts of Acanthopanax Download PDFInfo
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Abstract
본 발명은 오갈피(Acanthopanax) 추출물을 유효성분으로 포함하는 조성물에 관한 것으로, 구체적으로 오갈피 추출물을 포함하는 동맥경화를 비롯한 혈관질환의 예방 또는 치료용 약학적 조성물, 오갈피 추출물을 포함하는 동맥경화를 비롯한 혈관질환의 예방 또는 개선용 식품 조성물, 및 상기 조성물의 제조방법에 관한 것이다.The present invention relates to a composition comprising an Acanthopanax extract as an active ingredient, and more particularly to a pharmaceutical composition for prevention or treatment of vascular diseases including arteriosclerosis including an extract of Ogaki, an arteriosclerosis including an extract of Ogaki A food composition for preventing or ameliorating vascular diseases, and a process for producing the composition.
Description
본 발명은 오갈피(Acanthopanax) 추출물을 유효성분으로 포함하는 조성물에 관한 것으로, 구체적으로 오갈피 추출물을 포함하는 동맥경화를 비롯한 혈관질환의 예방 또는 치료용 약학적 조성물, 오갈피 추출물을 포함하는 동맥경화를 비롯한 혈관질환의 예방 또는 개선용 식품 조성물, 및 상기 조성물의 제조방법에 관한 것이다.The present invention relates to a composition comprising an Acanthopanax extract as an active ingredient, and more particularly to a pharmaceutical composition for prevention or treatment of vascular diseases including arteriosclerosis including an extract of Ogaki, an arteriosclerosis including an extract of Ogaki A food composition for preventing or ameliorating vascular diseases, and a process for producing the composition.
혈관질환은 혈관이 좁아져 발생하는 질환을 의미하는 것으로, 혈전 등의 혈액응고 덩어리 또는 지방의 축적으로 혈류가 방해를 받아 조직으로의 가스 및 영양 공급이 원활하게 이루어지지 못해 발생하는 질환들을 포함한다. 이외에도 혈관이 좁아지는 원인으로는 동맥의 염증 및 괴사와 갑작스럽게 조밀해지는 혈관연축 등이 있다. Vascular disease refers to a disease caused by narrowing of blood vessels and includes diseases in which blood flow is interrupted due to the accumulation of blood clots or fat such as thrombus and gas and nutrient supply to the tissue is not smoothly performed . Other causes of narrowing of blood vessels include inflammation and necrosis of the arteries and vasospasm suddenly becoming dense.
이러한 혈관질환 중 대표적인 예의 하나가 동맥경화이다. 동맥경화는 심장, 뇌, 창자와 같이 생명에 중요한 장기에 대한 혈액 공급이 심각하게 감소할 정도로 동맥이 좁아진 상태를 말한다. 뇌 혈관에 생기면 뇌졸중, 심장에 생기면 심근 경색증, 협심증이 생기고, 동맥이 완전히 막히면 막힌 동맥이 혈액을 공급하는 신체 각 부위가 죽는 괴사가 발생하는 치명적인 질병이다.One of the representative examples of such vascular diseases is arteriosclerosis. Atherosclerosis is a condition in which arteries become so narrow that blood supply to life-critical organs, such as the heart, brain, and gut, is severely reduced. It is a fatal disease that occurs in the cerebral blood vessels, in which stroke occurs, in the heart, myocardial infarction and angina, and when the artery is completely blocked, necrosis occurs in which each part of the body supplying blood supplies blood.
동맥경화는 여러 가지 원인에 의하여 야기될 수 있으며, 이는 혈관에서 흔히 발생하는 염증질환이다. 동맥경화의 초기 발달 단계는 염증반응의 전형적인 과정으로 진행한다. 염증의 진행과정을 보면, 염증반응이 시작되는 병소부위에서 화학주성 사이토카인(chemotactic cytokine)들이 분비되고, 단핵구 같은 혈구 세포가 병소 부위의 내피층과 부착이 되면서, 단핵구 혹은 백혈구 등이 내피층 안으로 이동(trans-endothelial migration)한다. 동맥경화반의 형성은 내막 안으로 들어간 단핵구가 대식세포(macrophage)와 포말세포(foam cell)로 변환된 후, 콜레스테롤 등의 축적으로 이루어진다. Atherosclerosis can be caused by a variety of causes, which are inflammatory diseases that are common in blood vessels. The early stages of atherosclerosis develop into a typical process of inflammatory response. In the process of inflammation, chemotactic cytokines are secreted from the lesion where the inflammatory reaction begins, and hematopoietic cells such as mononuclear cells adhere to the endothelial layer of the lesion, and monocytes or leukocytes enter into the endothelial layer Trans-endothelial migration. The formation of atherosclerotic plaque is caused by the accumulation of cholesterol after the mononuclear cells in the inner membrane are converted into macrophages and foam cells.
동맥경화 발생의 예방은 식이요법, 운동요법 및 약물요법 등이 알려져 있으나 동맥경화 발생을 예방하는 예방법에 대하여 더 많은 연구가 필요한 상황이며, 현재 동맥경화 발생을 억제할 수 있는 예방법 중의 하나는 천연물의 효능탐색을 통한 신약개발이다.
Although prevention of arteriosclerosis is known as diet therapy, exercise therapy and drug therapy, more research is needed to prevent prevention of arteriosclerosis. One of the preventive measures that can prevent the occurrence of arteriosclerosis is to use natural products Development of new drugs through efficacy search.
한편, 오가피라고도 불리는 오갈피(Acanthopanax)는 인삼, 산삼과 함께 오가과에 속하는 낙엽관목이다. 오갈피는 인삼과 유사한 점이 많아 ‘시베리아 인삼’이라고도 불리우고 있으며, 중풍, 고혈압, 당뇨병 등의 한약재로 사용되어 왔다. 오갈피 추출물은 한방에서 오랫동안 사용되어 왔던 약재 중의 하나로 다양한 연구가 최근까지 계속해서 수행되면서 약리학적 또는 생리학적으로 의미 있는 연구 결과들이 보고되었다.
한편 대표적인 혈관질환인 혈전 생성, 혈액 응고와 관련하여, 마늘의 유효성분인 알린(alliin)의 혈구세포의 응집 저해 효과가 알려진바 있으나 (K. M. Park et al., Arch Pharm Res Vol 35, No 12, 2153-2162, 2012), 흰털오갈피 추출물의 혈전 생성, 혈액 응고 저해 효과는 알려진바 없다.
On the other hand, senticosus (Acanthopanax), also known as Acanthopanax is a deciduous shrub belonging to ohgagwa with ginseng, wild ginseng. Ogallip is also called 'Siberian ginseng' because it has many similarities with ginseng and has been used as herbal medicine such as stroke, hypertension and diabetes. The extract of O. clark is one of the medicinal herbs that have been used for a long time in the oriental medicine. As a result of various studies until recently, pharmacological or physiologically meaningful research results have been reported.
In addition, alliin, an effective component of garlic, has been known to inhibit the aggregation of blood cells (KM Park et al., Arch Pharm Res Vol 35,
본 발명자들은 오갈피 추출물의 효능에 대하여 예의연구한 결과, 오갈피 추출물의 혈관 기능 개선 및 동맥경화 예방 효과를 세포생물학적 분석을 통하여 확인하여 본 발명을 완성하였다.As a result of intensive studies on the efficacy of the extract of O. clark, the present inventors confirmed the vascular function improvement and the arteriosclerosis-preventing effect of the O. clark extract through cell biological analysis and completed the present invention.
본 발명의 하나의 목적은 오갈피(Acanthopanax) 추출물을 유효성분으로 포함하는, 혈관질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. It is an object of the present invention to provide a pharmaceutical composition for preventing or treating vascular diseases, which comprises an extract of Acanthopanax as an active ingredient.
본 발명의 다른 목적은 오갈피 추출물을 유효성분으로 포함하는, 혈관질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다.It is another object of the present invention to provide a food composition for prevention or improvement of vascular diseases, which comprises an extract of Ogaki as an active ingredient.
본 발명의 또 다른 목적은 혈관질환 의심 개체에 상기 약학적 조성물을 투여하는 단계를 포함하는, 혈관질환의 치료방법을 제공하는 것이다.It is still another object of the present invention to provide a method for treating vascular diseases, which comprises administering the pharmaceutical composition to a subject suspected of having a vascular disease.
본 발명의 또 다른 목적은 상기 조성물의 제조방법을 제공하는 것이다. It is still another object of the present invention to provide a method for producing the composition.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 오갈피(Acanthopanax) 추출물을 유효성분으로 포함하는, 혈관질환, 구체적으로는 동맥경화의 예방 또는 치료용 약학적 조성물을 제공한다.
In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating vascular diseases, in particular atherosclerosis, comprising an Acanthopanax extract as an active ingredient.
본 발명에서 용어, "오갈피(Acanthopanax)"는 오갈피나무속(Eleutherococcus) 수종으로 두릅나무과(Araliaceae)에 속하는 나무를 의미하며, 섬오갈피, 가시오갈피, 흰털오갈피, 참오갈피 등이 있다. 오갈피는 잎, 줄기, 뿌리 및 열매로 나누어져 있으며, 특히 뿌리는 주근, 지근, 세근으로 나누어져 있다. 상기 오갈피는 간질환 치료 효능 및 면역력 증강 효능을 갖는 것으로 알려져 있으나, 혈관질환, 특히 동맥경화의 치료 효능은 알려진 바가 없으며, 본 발명자들에 의해 새롭게 규명되었다. 상기 오갈피는 자연에서 채취한 것을 사용하거나 상업적으로 판매되는 것을 구입하여 사용할 수 있으나, 이에 제한되지 않는다. The term "senticosus (Acanthopanax)" in the invention means a tree belonging to Araliaceae (Araliaceae) by senticosus hawthorn species (Eleutherococcus), and the like island senticosus, Acanthopanax senticosus, huinteol senticosus, indeed senticosus. It is divided into leaf, stem, root and fruit, and especially root is divided into main root, root and fine root. Although it is known that it has an efficacy for treating liver diseases and an immunity enhancing effect, the treatment effect of vascular diseases, especially arteriosclerosis, has not been known and it has been newly discovered by the present inventors. The above-mentioned ogalopsis may be collected from natural sources or commercially available, but the present invention is not limited thereto.
본 발명에서 용어, "오갈피(Acanthopanax) 추출물"은 오갈피를 추출하여 수득한 추출물을 의미하며, 바람직하게는 흰털오갈피(Acanthopanax divaricatus var . albeofructus)를 추출하여 수득한 추출물을 의미하며, 보다 바람직하게는, 흰털오갈피의 뿌리 및/또는 줄기를 추출하여 수득한 추출물을 의미한다. The term " Acanthopanax extract" in the present invention means an extract obtained by extracting an acaricus, preferably Acanthopanax divaricatus there is . refers to an extract obtained by extracting albeofructus , and more preferably extracts obtained by extracting roots and / or trunks of white mulberry.
상기 오갈피 추출물은 오갈피의 뿌리 및/또는 줄기, 바람직하게는 줄기와 뿌리의 혼합물을 이용하여 제조될 수 있으며, 줄기와 뿌리의 혼합비는 특별히 제한되지 않으나 4:1 내지 1:4의 혼합비로 이용될 수 있다. 추출 용매로서는 물을 이용할 수 있으며, 추출용매는 오갈피 시료 중량의 1배 내지 20배의 양으로 이용될 수 있으며, 바람직하게는 2배 내지 10배, 더욱 바람직하게는 7배의 양으로 이용될 수 있다.The extract may be prepared by using a mixture of roots and / or stems, preferably stems and roots of an aphid, and the mixing ratio of stems and roots is not particularly limited, but may be used in a mixing ratio of 4: 1 to 1: 4 . As the extraction solvent, water may be used, and the extraction solvent may be used in an amount of 1 to 20 times, preferably 2 to 10 times, more preferably 7 times, have.
추출은 25℃ 내지 120℃에서 1시간 내지 15시간 동안 냉침 추출, 열수 추출, 초음파 추출, 환류냉각 추출, 초고압 추출, 가열 추출법 또는 이들의 조합을 이용하여 이루어질 수 있으며, 바람직하게는 1시간 내지 5시간 동안의 냉침 추출 및 이어서 90℃ 내지 120℃에서 5시간 내지 9시간 동안의 열수추출에 의해 이루어질 수 있다. Extraction may be carried out at 25 ° C to 120 ° C for 1 hour to 15 hours using cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, ultrahigh pressure extraction, heat extraction or a combination thereof, preferably 1 hour to 5 Followed by cold extraction at a temperature of < RTI ID = 0.0 > 90 C < / RTI > to 120 C for 5 to 9 hours.
상기 추출과정을 거친 추출액은 여과, 농축 및 건조 과정을 순차적으로 거쳐 본 발명의 추출물로 제조될 수 있으나, 특별히 이에 제한되지 않고, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들의 조정제물 또는 정제물을 모두 포함한다. 또한, 상기 오갈피 추출물은 천연, 잡종, 변종 오갈피 식물의 다양한 기관으로부터 추출될 수 있으며, 본 발명의 일 실시예에서는 흰털오갈피의 뿌리 및 줄기의 혼합물로부터 물을 이용하여 추출하였다(실시예 1).The extract solution obtained through the extraction process may be prepared as an extract of the present invention through filtration, concentration, and drying process in sequence, but is not particularly limited thereto, and may be a dried product obtained by drying an extract, a diluent or concentrate of an extract, Of a controlled release or purified product. In addition, the extract can be extracted from various organs of natural, hybrid, and variegated oregano plants. In one embodiment of the present invention, water is used to extract from a mixture of roots and stem of a white gallccass (Example 1).
본 발명에서 용어, "혈관질환"은 혈관이 좁아져 발생하는 질환을 의미하는 것으로 예를 들어, 동맥경화성 질환, 뇌혈관질환, 말초혈관질환, 혈관염, 정맥류 등을 포함한다.The term "vascular disease" in the present invention means a disease caused by narrowing of blood vessels and includes, for example, atherosclerotic disease, cerebrovascular disease, peripheral vascular disease, vasculitis and varicose vein.
본 발명에서 용어 "동맥경화"는 심장, 뇌, 등과 같이 생명에 중요한 장기에 대한 혈액 공급이 심각하게 감소할 정도로 동맥이 좁아진 상태를 의미한다.The term "arteriosclerosis" in the present invention means a condition in which the arteries are narrowed to such an extent that blood supply to vital organs such as heart, brain, etc. is seriously reduced.
본 발명에서 용어, "예방"은 상기 조성물의 투여에 의해 동맥경화를 억제하거나 발병을 지연시키는 모든 행위를 의미하며, "치료"는 상기 조성물의 투여에 의해 동맥경화 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, the term "prevention" means any action that inhibits or slows the onset of atherosclerosis by administration of the composition, and "treatment" Means action.
본 발명의 오갈피 추출물을 포함하는 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형체 또는 희석제를 추가로 포함할 수 있다. 이때, 상기 조성물에 포함되는 오갈피 추출물의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 99.99중량%로, 바람직하게는 0.001 중량% 내지 50 중량%로 포함될 수 있다.The pharmaceutical composition comprising the extract of Orgaliae of the present invention may further comprise a suitable carrier, adduct or diluent conventionally used in the production of pharmaceutical compositions. At this time, the content of the extract of Ogalloko contained in the composition is not particularly limited, but it may be 0.0001 to 99.99% by weight, preferably 0.001 to 50% by weight based on the total weight of the composition.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 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 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories May have one formulation, and may be various forms of oral or parenteral administration. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The composition of the present invention may be administered in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 그러나 바람직한 효과를 위해서는 본 발명의 약학 조성물은 1일 0.001 내지 100㎍/㎏으로, 더욱 바람직하게는 0.01 내지 100㎍/㎏의 범위로 비경구 또는 경구 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면에서든 본 발명의 범위를 한정하는 것은 아니다. 상기 조성물은 쥐, 가축, 인간 등의 다양한 포유동물에 다양한 경로로 투여할 수 있으며, 투여의 방식은 당업계의 통상적인 방법이라면 제한없이 포함하며, 예를 들어, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다.The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably parenterally or orally administered in the range of 0.001 to 100 μg / kg per day, more preferably 0.01 to 100 μg / kg per day. The administration can be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The composition may be administered to various mammals such as rats, livestock, humans, and the like in a variety of routes. The mode of administration is not limited as long as it is a conventional method in the art. For example, oral, rectal or intravenous, Subcutaneous, intra-uterine dural or intracerebral injection.
본 발명의 일 실시예에서는 흰털오갈피의 뿌리 및 줄기를 물을 이용하여 냉침 및 열수추출함으로써 오갈피 추출물을 제조하였고, 오갈피 추출물을 염증유발인자인 리포폴리사카라이드(LPS)와 함께 단핵구세포에 처리한 결과, 리포폴리사카라이드에 의해 증가하였던 혈관부착이 감소함을 확인하였으며, 더 나아가 오갈피 추출물의 처리에 의해 단핵구 세포표면의 L-셀렉틴(selectin) 분자의 발현이 감소되고 이로 인해 단핵구와 혈관내피세포의 부착이 감소됨을 확인함으로써, 본 발명의 오갈피 추출물이 혈관 염증 발생을 억제함으로써 동맥경화를 비롯한 혈관질환의 발생의 예방 및 치료를 위해 유용하게 사용될 수 있음을 확인하였다(실험예 5 및 실험예 6).
In one embodiment of the present invention, the extracts of O. clark extract were prepared by cold-watering and hot-water extraction using water for roots and stems of P. falciparum and treated with monocyte cells with lipopolysaccharide (LPS) As a result, it was confirmed that the adhesion of the blood vessels increased by lipopolysaccharide was reduced. Furthermore, the expression of L-selectin molecules on the surface of mononuclear cells was decreased by the treatment with the extract of O. clark, It was confirmed that the extract of Ogallippi of the present invention inhibits the occurrence of vascular inflammation and thus can be usefully used for the prevention and treatment of the occurrence of vascular diseases including arteriosclerosis (Experimental Example 5 and Experimental Example 6 ).
또 다른 양태로서, 본 발명은 동맥경화를 비롯한 혈관질환 의심 개체에 상기 조성물을 투여하는 단계를 포함하는, 동맥경화를 비롯한 혈관질환의 치료 방법을 제공한다.In another aspect, the present invention provides a method for treating vascular diseases, including arteriosclerosis, comprising administering the composition to a subject suspected of having a vascular disease, including arteriosclerosis.
상기 조성물 및 동맥경화에 대해서는 상기에서 설명한 바와 같다.The above composition and atherosclerosis are as described above.
구체적으로, 본 발명의 치료 방법은 상기 약학적 조성물을 약학적 유효량으로 동맥경화를 비롯한 혈관질환 의심 개체 내에 투여하는 것을 포함한다. 상기 개체는 개, 소, 말, 토끼, 마우스, 랫트, 닭 또는 인간을 포함하는 포유류 전체를 의미하나, 상기 예에 의해 본 발명의 포유류가 한정되는 것은 아니다. 상기 약학적 조성물은 비 경구, 피하, 복강 내, 폐 내 및 비강 내로 투여될 수 있고, 국부적 치료를 위해, 필요하다면 병변 내 투여를 포함하는 적합한 방법에 의하여 투여될 수 있다. 본 발명의 상기 약학적 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.
Specifically, the method of treatment of the present invention comprises administering the pharmaceutical composition in a pharmaceutically effective amount into a suspect vascular disease including atherosclerosis. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples. The pharmaceutical compositions may be administered by non-oral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes and may be administered by a suitable method, including localized administration, if necessary, for localized treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.
또 다른 양태로서, 본 발명은 오갈피 추출물을 유효성분으로 포함하는, 동맥경화를 비롯한 혈관질환의 예방 또는 개선용 식품 조성물을 제공한다.In another aspect, the present invention provides a food composition for prevention or amelioration of a vascular disease, including arteriosclerosis, which comprises an extract of Oerca as an active ingredient.
상기 오갈피 추출물 및 동맥경화에 대해서는 상기에서 설명한 바와 같다. The abovementioned peel extract and atherosclerosis are as described above.
구체적으로, 본 발명의 추출물은 동맥경화를 비롯한 혈관질환의 예방 또는 개선을 목적으로 식품 조성물에 첨가할 수 있다. Specifically, the extract of the present invention can be added to a food composition for the purpose of preventing or improving vascular diseases including arteriosclerosis.
본 발명의 오갈피 추출물을 식품 첨가물로 사용할 경우, 상기 추출물 또는 이의 분획물을 그대로 첨가하거나 다른 식품 또는 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합량은 사용 목적에 따라 적합하게 결정할 수 있다. When the extract of the present invention is used as a food additive, the extract or its fractions can be directly added or used together with other foods or ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.
또한, 본 발명의 식품의 종류에는 특별한 제한은 없다. 상기 오갈피 추출물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있고, 통상적인 의미에서의 식품을 모두 포함할 수 있으며, 동물을 위한 사료로 이용되는 식품을 포함한다.There is no particular limitation on the kind of the food of the present invention. Examples of the foods to which the auger extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, drinks, tea, , An alcoholic beverage, and a vitamin complex, and can include all foods in a conventional sense, and includes foods used as feed for animals.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄 산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 또한, 상기 식품은 공지의 제조방법에 따라 정제, 과립, 분말, 캅셀, 액상의 용액 및 환 등의 제형으로도 제조될 수 있다. 본 발명에 의한 오갈피 추출물을 유효성분으로 포함하는 것 이외에는 다른 성분에는 특별한 제한이 없으며, 통상의 여러가지 향미제 또는 천연 탄수화물 등을 추가성분으로 포함할 수 있다.
In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in a carbonated drink, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. The food may also be prepared in the form of tablets, granules, powders, capsules, solutions in liquid form, and the like according to known production methods. There are no particular limitations on other ingredients other than those containing the extract of Ocgakii according to the present invention as an active ingredient, and it may contain various conventional flavors or natural carbohydrates as an additional ingredient.
또 다른 양태로서, 본 발명은 상기 오갈피 추출물; 및 이를 포함하는 조성물의 제조방법을 제공한다. In another embodiment, the present invention relates to a composition comprising the auger extract; And a method for producing a composition comprising the same.
상기 오갈피 및 오갈피 추출물에 대해서는 상기에서 설명한 바와 같다. The above-mentioned aphid and scallop extracts are as described above.
구체적으로, 상기 제조방법은 (a) 건조한 오갈피 시료 중량의 1 내지 20배(v/w)의 물을 가하여 25℃ 내지 120℃에서 1시간 내지 15 시간동안 추출하는 단계; 및 (b) 상기 (a) 단계에서 추출한 물을 여과, 농축 및 건조하는 단계를 포함한다.Specifically, the method comprises: (a) extracting 1 to 20 times (v / w) water by weight of the dry oat sample at 25 ° C to 120 ° C for 1 hour to 15 hours; And (b) filtering, concentrating and drying the water extracted in the step (a).
상기 (a) 단계는 추출 용매를 이용하여 오갈피로부터 추출하는 단계로서, 건조하여 절단한 오갈피의 줄기 및 뿌리의 혼합물을 물을 이용하여 추출하는 단계로서, 바람직하게는 25℃ 내지 120℃에서 1시간 내지 15시간 동안 냉침 추출, 열수 추출, 초음파 추출, 환류냉각 추출, 초고압 추출, 가열 추출법 또는 이의 조합을 이용하여 이루어질 수 있으며, 바람직하게는 1시간 내지 5시간 동안의 냉침 추출 및 이어서 100℃ 내지 120℃에서 5시간 내지 9시간 동안의 열수추출에 의해 이루어질 수 있다. 보다 바람직하게는, 3시간 동안의 냉침 추출 및 이어서 105℃에서 6 내지 7시간 동안의 열수추출에 의해 이루어질 수 있다.The step (a) is a step of extracting a mixture of stem and roots of an augaru which is dried and cut by using water, preferably at 25 to 120 ° C for 1 hour The extraction may be performed by cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, ultra-high pressure extraction, heat extraction or a combination thereof for 15 to 15 hours, preferably 1 to 5 hours of cold extraction followed by 100 to 120 RTI ID = 0.0 > 5 C < / RTI > to 9 hours. More preferably, this can be accomplished by 3-hour cold extraction followed by hot water extraction at 105 ° C for 6 to 7 hours.
상기 (b) 단계는 (a) 단계에서 추출한 물을 여과, 농축 및 건조하는 단계이다.The step (b) is a step of filtering, concentrating and drying the water extracted in the step (a).
상기 단계를 포함하는 제조방법을 통하여 본 발명의 오갈피 추출물; 및 이를 포함하는 조성물을 제조할 수 있다.The ocher extract of the present invention through the manufacturing method including the above step; And a composition containing the same.
본 발명의 오갈피 추출물은 오랫동안 천연약재로 사용되어온 천연물에서 유래되어 부작용이 없으면서도 동맥경화를 비롯한 혈관질환에 대한 예방 및 치료 효과를 가지므로, 오갈피 추출물을 포함하는 본 발명의 조성물은 동맥경화의 예방 또는 치료에 유용하게 사용될 수 있다.Since the extract of Ogalliae of the present invention is derived from a natural product which has been used for a long time as a natural medicinal material and has no side effects and has a preventive and therapeutic effect on vascular diseases including atherosclerosis, the composition of the present invention containing Ogalli extract, Or can be used therapeutically.
도 1은 오갈피 추출물의 농도에 따른 혈관 내피세포 성장에 대한 영향을 보여주는 그래프이다.
도 2는 오갈피 추출물의 농도에 따른 인산화된 eNOS의 발현양을 보여주는 젤 사진이다.
도 3은 오갈피 추출물의 처리에 따른 THP-1 세포의 응집 변화를 보여주는 사진이다.
도 4는 혈관 내피세포의 이동에 대한 오갈피 추출물의 영향을 보여주는 사진이다.
도 5는 오갈피 추출물의 처리에 따른 단핵구 세포의 혈관 내피세포 부착 억제 효과를 보여주는 사진이다.
도 6은 오갈피 추출물의 처리에 따른, 단핵구 세포의 부착 분자들의 RNA 생성 정도를 보여주는 젤 사진이다.
도 7은 오갈피 추출물의 처리에 따른, 단핵구 세포의 부착 분자들의 발현 정도를 보여주는 젤 사진이다.
도 8은 오갈피 추출물의 처리에 의해 THP-1 세포에서 L-셀렉틴의 발현이 증가함을 보여주는 사진이다.FIG. 1 is a graph showing the effect on the growth of vascular endothelial cells according to the concentration of the extract of Ogaki.
FIG. 2 is a photograph showing the amount of phosphorylated eNOS expressed by the concentration of the extract of Ocarpa.
FIG. 3 is a photograph showing the agglutination change of THP-1 cells according to the treatment with the extract of O. ancariae.
Fig. 4 is a photograph showing the effect of the extract of augari on the migration of vascular endothelial cells.
FIG. 5 is a photograph showing the effect of inhibiting the adhesion of monocyte cells to vascular endothelial cells according to the treatment of the extract of Ogaki.
FIG. 6 is a gel photograph showing the degree of RNA production of adhesion molecules of monocytic cells according to the treatment of aphthae extract.
FIG. 7 is a gel photograph showing the degree of expression of adhesion molecules of monocyte cells according to the treatment of aphthae extract. FIG.
FIG. 8 is a photograph showing that the expression of L-selectin in THP-1 cells is increased by treatment with an extract of O. anthracis.
이하 본 발명을 하기 예에 의해 상세히 설명한다. 다만, 하기 예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples.
실시예Example
1: 오갈피 추출물 제조 1: Manufacture of augaru extract
충남 천안에서 채취한 흰털오갈피의 줄기 및 뿌리를 세정한 후, 수분 함량이 15 ~ 17% 되도록 30 ℃ 미만에서 열풍건조하였다. 건조된 시료를 적절한 길이, 약 1 ~ 3 cm로 절단한 후, 줄기와 뿌리를 4:1의 중량비로 혼합하고, 상기 혼합물에 7배 (v/w)의 물을 가하여 3시간 동안 순환시키며 냉침시켰다. 냉침 후, 105℃에서 6-7시간 동안 열수추출 하였다. 추출이 완료된 후 필터로 여과하고 1차 및 2차 농축 및 동결건조하여 오갈피 추출물을 수득하여 하기 실험예의 시료로 사용하였다.
The stems and roots of white clover collected from Cheonan, Chungnam Province were washed and then hot-air dried at less than 30 ℃ to have a moisture content of 15 ~ 17%. The dried sample was cut to an appropriate length of about 1 to 3 cm, and the stem and root were mixed at a weight ratio of 4: 1. Seven times (v / w) water was added to the mixture and circulated for 3 hours. . After cooling, it was subjected to hot water extraction at 105 ° C for 6-7 hours. After the extraction was completed, the mixture was filtered with a filter, and subjected to primary and secondary condensation and lyophilization to obtain an Ocgaki extract, which was used as a sample in the following experimental example.
실시예Example
2: 혈관내피세포와 2: Vascular endothelial cells and
단핵구세포의Monocytic cell
배양 culture
혈관내피세포는 세포 외 기질에 부착하여 자라는 부착세포(adhesion cell)인 소 대동맥 내피세포(bovine aortic endothelial cell, BAEC)를 소의 대동맥에서 추출한 후 6~9차 계대(passage) 배양한 세포를 사용하였다. 배양방법은 20% 소태아혈청(fetal bovine serum, Wel GENE Inc., Korea)과 0.5% 항생제(streptomycin/penicillin)가 포함된 DMEM (glucose 1g/liter Wel GENE Inc.)을 배지로 이용하여 37℃, 5% CO2 조건에서 배양하였다.
The vascular endothelial cells were obtained from bovine aortic endothelial cells (bovine aortic endothelial cell), an adhesion cell that adheres to the extracellular matrix, and then passaged for 6 to 9 days . The cultures were incubated at 37 ° C with 20% fetal bovine serum (Wel GENE Inc., Korea) and DMEM (glucose 1 g / liter Wel GENE Inc.) containing 0.5% antibiotic (streptomycin / penicillin) , 5% CO 2 Lt; / RTI >
단핵구 세포인 THP-1은 액체 배지에 부유 상태로 성장하는 부유세포(suspension cell)로서, 배양배지는 RPMI1640(W디 GENE Inc., Korea)에 10% 소태아혈청(fetal bovine serum, Wel GENE Inc., Korea)과 0.5% 항생제(streptomycin/penicillin)가 첨가된 배지를 이용하여 세포를 배양하였다. 기타 배양 조건은 BAEC의 배양 조건과 동일하였다.
THP-1, a mononuclear cell, was suspended in a liquid medium. The culture medium was RPMI 1640 (WD GENE Inc., Korea) supplemented with 10% fetal bovine serum (Wel GENE Inc ., Korea) and 0.5% antibiotic (streptomycin / penicillin). Other culture conditions were the same as BAEC culture conditions.
실험예Experimental Example
1: 혈관 내피세포의 성장 및 사멸에 대한 오갈피 추출물의 영향 확인 1: Identification of the effect of augaru extract on the growth and death of vascular endothelial cells
혈관 기능을 평가할 때, 특정 약물이 내피층에 미치는 효과는 혈관의 내부 표면을 둘러싸고 있는 내피세포의 사멸 또는 성장에 영향을 미치지 않아야 한다. 따라서 오갈피 추출물이 혈관 내피세포의 성장과 사멸에 미치는 영향을 측정하여 이러한 영향을 미치지 않는 오갈피 추출물을 선별하여 실험에 사용하였다.
When assessing vascular function, the effect of a particular drug on the endothelial layer should not affect the death or growth of endothelial cells surrounding the inner surface of the vessel. Therefore, we examined the effects of the extract of O. clark on the growth and death of vascular endothelial cells, and used it for the experiment.
WST(High sensitive water soluble tetrazolium salt)가 살아있는 세포내의 미토콘드리아의 탈수소효소에 의해 환원되면서 색을 발하는 염료인 포르마잔(formazan)으로 전환되며, 따라서 측정된 염료의 양으로 살아있는 세포의 수를 측정할 수 있는 WST 분석을 이용하여 내피세포의 성장을 관찰 하였다.
The high sensitive water soluble tetrazolium salt (WST) is converted to formazan, a coloring dye, by the reduction of mitochondrial dehydrogenase in living cells, so that the number of living cells can be measured by the amount of dye measured Endothelial cell growth was observed using the WST assay.
먼저, 혈청기아 상태인 DMEM에서 BAEC을 16시간 동안 배양한 뒤, 양성대조군으로는 20% FBS를 처리하고 음성대조군은 아무것도 처리하지 않았다. 오갈피 추출물을 여러 가지 농도(0.01~200㎍/㎖)로 처리하고 시간별로 UV 스펙트럼을 이용하여 450nm에서 측정하였다.
First, BAEC was cultured in serum-starved DMEM for 16 hours, followed by 20% FBS as a positive control and no negative control. The ocher extracts were treated at various concentrations (0.01-200 μg / ml) and measured at 450 nm using UV spectra over time.
측정 결과, 도 1에 나타난 바와 같이, 오갈피 추출물의 농도와 시간 별로 세포성장과 사멸에 큰 변화가 없지만, 세포의 성장과 사멸에 영향이 가장 적은 농도인 200㎍/㎖을 선별하여 다음 실험을 진행하였다.
As shown in FIG. 1, 200 μg / ml, the least effect on cell growth and death, was selected by the concentration and time of the extract of O. clark extract Respectively.
실험예Experimental Example
2: 혈관 내피세포의 2: vascular endothelial cells
eNOSeNOS
활성 측정 Active measurement
내피세포의 기능에 중요한 물질인 활성 질소(Nitric Oxide(NO))는 혈관 확장, 혈소판에 의한 혈액응고 억제, 혈관형성, 동맥경화 발생 억제와 같은 다양한 기능을 하는 혈관 조절인자이다. 혈관 내피세포에 대한 오갈피 추출물의 영향을 확인하기 위하여, 오갈피 추출물을 내피세포에 처리하여 신호전달 경로 활성을 평가하였다.
Nitric oxide (NO), an important substance in the function of endothelial cells, is a vascular regulator of various functions such as vasodilation, inhibition of blood coagulation by platelets, inhibition of angiogenesis and arteriosclerosis. To determine the effect of the extract on the endothelial cells of the vascular endothelial cells, the activity of the signal transduction pathway was evaluated by treating the endothelial cell extract with the endothelial cells.
구체적으로, 내피세포를 35mm 배양접시에 분주한 후 24시간 동안 37℃, 5% CO2 조건으로 배양을 한 후, 혈청 기아 배지(DMEM에 0.5% FBS와 0.5% streptomycin/penicillin을 첨가한 배지)로 16시간 동안 배양한 후, 200㎍/㎖의 오갈피 추출물을 처리한 후 시간 별로 세포를 분해 완충액(50mM HEPES, 150mM NaCl, 0.1mM PMSF, 1.0mM Na Vanadate, 1.0% Triton X-100, 10% Glycerol, pH 7.4)으로 분해하였다. 세포 분해액의 단백질 함량을 측정하여 동일한 양의 분해액을 SDS-PAGE에 의해 단백질을 분리시켰다. 분리된 단백질을 PVDF(Polyvinylidene fluoride) 막으로 전단시킨 후 eNOS의 활성형태인 인산화된 eNOS의 항체를 처리하고 2차 항체를 처리한 후, 엑스선(X-ray) 필름으로 현상하여 개별적인 단백질을 검출하였다.
Specifically, the endothelial cells were placed in a 35 mm culture dish and incubated for 24 hours at 37 ° C in 5% CO 2 After culturing, cells were cultured in serum starvation medium (medium supplemented with 0.5% FBS and 0.5% streptomycin / penicillin in DMEM) for 16 hours, treated with 200 μg / And digested with digestion buffer (50 mM HEPES, 150 mM NaCl, 0.1 mM PMSF, 1.0 mM Na Vanadate, 1.0% Triton X-100, 10% Glycerol, pH 7.4). The protein content of the cell lysis solution was measured, and proteins were separated by SDS-PAGE in the same amount of the degraded solution. Separated proteins were sheared with PVDF (polyvinylidene fluoride) membrane, treated with the antibody of phosphorylated eNOS, the active form of eNOS, treated with secondary antibody, and developed with X-ray film to detect individual proteins .
그 결과, 도 2에 나타난 바와 같이, 오갈피 추출물을 처리하지 않은 대조군에 비하여 오갈피 추출물을 처리한 경우에 인산화된 eNOS의 단백질양이 증가하는 것을 확인할 수 있었다. 이러한 결과는 오갈피 추출물 처리에 의해, 혈관 내피세포에서 항동맥경화 등의 여러 중요한 기능을 하는 활성질소의 생성이 촉진됨을 의미한다. As a result, as shown in FIG. 2, it was confirmed that the amount of phosphorylated eNOS protein was increased in the case of treatment with the extract of O. clark compared with the control without O. clark extract. These results indicate that the treatment of the extract of O. augma stimulates the production of active nitrogen which plays various important functions such as atherosclerosis in vascular endothelial cells.
실험예Experimental Example
3: 3:
THPTHP
-1 세포의 응집 측정-1 cell aggregation measurement
오갈피 추출물에 의한 혈전형성을 평가하기 위해 혈구세포의 응집에 대해 오갈피 추출물이 미치는 영향을 관찰하였다.
In order to evaluate the thrombus formation by the extract of Ogalli, we examined the effect of Ogalli extract on the aggregation of hemocyte.
구체적으로, THP-1 세포를 72시간 동안 배양한 후, 혈청기아 배지(RPMI1640에 0.5% FBS와 0.5% streptomycin/penicillin을 첨가한 배지)에 1㎍/㎖의 리포폴리사카라이드를 처리하거나 오갈피 추출물 10㎍/㎖과 200㎍/㎖을 각각 처리한 후 24시간까지 시간 별로 진행되는 세포 응집 현상을 현미경으로 관찰하였다.
Specifically, THP-1 cells were cultured for 72 hours, and treated with 1 쨉 g / ml of lipopolysaccharide in serum starvation medium (RPMI1640 medium supplemented with 0.5% FBS and 0.5% streptomycin / penicillin) After 10 ㎍ / ㎖ and 200 ㎍ / ㎖ respectively, cell aggregation progressed by time to 24 hours was observed under a microscope.
그 결과, 도 3에 나타난 바와 같이, 리포폴리사카라이드를 처리하였을 때, THP-1 세포는 응고되는 현상(하얀색 화살표)을 보였으나, 오갈피 추출물을 처리하였을 때는 저농도 및 고농도 모두의 경우, THP-1세포의 응집현상이 일어나지 않았다. 이러한 결과는 오갈피 추출물이 혈전형성에 영향을 미치지 않는다는 것을 보여준다.
As a result, as shown in FIG. 3, when lipopolysaccharide was treated, THP-1 cells showed a phenomenon of coagulation (white arrow). However, when the extract was treated with olive oil extract, THP- 1 cell aggregation did not occur. These results demonstrate that the extract of Ocagi does not affect thrombus formation.
실험예Experimental Example
4: 혈관 내피세포의 이동 측정 4: Measurement of migration of vascular endothelial cells
혈관 내피세포의 이동은 혈관형성 과정에서 일어나는 한 단계로 혈관형성에 오갈피 추출물이 어떤 영향을 미치는지 쉽게 평가할 수 있는 방법이다. 따라서 세포 이동 정도를 측정하여 혈관형성에 미치는 오갈피 추출물의 효능을 간접적으로 확인하였다.
The migration of vascular endothelial cells is a step that takes place during the angiogenesis process, and it is a method to easily evaluate the influence of the augaru extract on angiogenesis. Therefore, the degree of cell migration was measured to indirectly confirm the efficacy of the extract of O. clark extract on angiogenesis.
구체적으로, in vitro 세포 이동 분석은 세포의 성장을 중지시킨 조건하에서 수행하였다. 세포 성장의 첫 번째 중지는 BAEC에 2mM thymidine을 18시간 처리 후 PBS(phosphate buffered saline)로 2회에 걸쳐 세척하고, 9시간 동안 배양한 후, 두 번째 성장 중지(2mM thymidine, 17시간 배양)를 수행하였다. 그리고 스크레이퍼 찰과기(scraper)로 창상선을 만든 후 오갈피 추출물 200㎍/㎖을 처리한 실험군과 아무것도 처리하지 않은 음성 대조군과 그리고 20% FBS를 처리한 양성 대조군에서 세포이동 정도를 측정하였다. 세포 이동 정도는 일정한 시야에서 이동한 세포수를 현미경 상에서 평가하였다.
Specifically, in vitro cell migration assays were performed under conditions in which cell growth was discontinued. For the first stop of cell growth, BAEM was treated with 2 mM thymidine for 18 hours, washed twice with PBS (phosphate buffered saline), incubated for 9 hours, and incubated for 2 hours with 2 mM thymidine for 17 hours Respectively. After sclerotial scraping, the cell migration was measured in the experimental group treated with 200 μg / ml of the extract of O. clark, the negative control group without any treatment, and the positive control group treated with 20% FBS. The degree of cell migration was evaluated microscopically by the number of cells migrated in a certain field of view.
그 결과, 도 4에 나타난 바와 같이, FBS를 처리한 양성 대조군에서는 내피세포의 이동이 잘 일어나고 있으나, 오갈피 추출물을 처리한 실험군은 아무것도 처리하지 않은 음성 대조군과 차이가 없었다. 따라서, 오갈피 추출물이 혈관형성에 영향을 미치지 않는다는 것을 알 수 있었다.
As a result, as shown in FIG. 4, the endothelial cell migration was well occurred in the positive control group treated with FBS, but the test group treated with the extract of O. clark did not differ from the negative control group treated with nothing. Thus, it was found that the extract of O. clark did not affect angiogenesis.
실험예Experimental Example
5: 혈관 내피세포와 단핵구 세포의 부착 측정 5: Measurement of adhesion of vascular endothelial cells and mononuclear cells
동맥경화 발병의 초기 단계는 단핵구 또는 대식세포가 혈관 내피세포에 부착되는 현상이 나타나므로, 단핵구의 내피층 부착 정도를 평가함으로써 동맥경화 발생 가능성을 평가할 수 있다. 따라서, 오갈피 추출물이 혈관 내피세포와 단핵구 세포 사이의 부착에 미치는 영향을 확인하였다.
The early stage of arteriosclerosis develops the adherence of monocytes or macrophages to vascular endothelial cells, so the possibility of arteriosclerosis can be evaluated by assessing the degree of endothelial adhesion of monocytes. Therefore, we examined the effect of the extract on the adhesion between vascular endothelial cells and mononuclear cells.
먼저, BAEC을 전면 성장시키고 THP-1은 배양후 15μM의 Calcein AM (Sigma)을 37℃, 5% CO2 조건에서 45분간 반응 시키고, PBS를 이용하여 5번 세척하였다. Calcein AM 으로 염색시킨 THP-1을 BAEC가 부착된 기판 위에 동량으로 첨가하여 1시간 동안 배양하였다. BAEC가 부착된 배양접시에서 내피세포와 부착되지 않은 THP-1세포는 PBS로 세척하여 제거하였다.First, BAEC was grown to the full, THP-1 was cultured and 15 μM of Calcein AM (Sigma) was added at 37 ° C., 5% CO 2 For 45 minutes, and washed five times with PBS. Calcein AM-stained THP-1 was added to the BAEC-loaded substrate in the same volume and incubated for 1 hour. In the BAEC-adhered culture dish, endothelial cells and unattached THP-1 cells were removed by washing with PBS.
THP-1 세포 안으로 들어간 Calcein AM은 세포내의 에스테라제(esterase)에 의해 가수 분해되어 형광을 나타낸다. 따라서 형광현미경을 이용하여 세포를 관찰하여 내피세포와 부착되어있는 THP-1 세포를 확인할 수 있었다.
Calcein AM, which enters into THP-1 cells, is hydrolyzed by intracellular esterases and fluoresces. Therefore, by observing the cells using fluorescence microscopy, it was possible to identify THP-1 cells adhering to endothelial cells.
확인 결과, 도 5에 나타난 바와 같이, 아무것도 처리하지 않은 대조군에 비하여 리포폴리사카라이드를 처리한 양성 대조군에서 THP-1세포의 혈관내피세포 부착이 의미 있게 상승하였다. 그러나 THP-1세포에 리포폴리사카라이드와 오갈피 추출물을 함께 처리한 경우에는, 리포폴리사카라이드에 의해 상승하였던 혈관부착이 감소하는 것을 관찰할 수 있었으며, 또한 오갈피 추출물을 농도별로 처리했을 경우 농도가 올라갈수록 세포 부착이 감소하는 것을 관찰할 수 있었다. 이러한 결과는 오갈피 추출물이 혈관에서 혈관 내피세포 부착 억제를 통해 동맥경화 발생 억제 효과를 가짐을 보여주는 것이다.
As a result, as shown in Fig. 5, the vascular endothelial cell adhesion of THP-1 cells was significantly increased in the positive control treated with lipopolysaccharide, compared with the control group treated with nothing. However, when THP-1 cells were treated with lipopolysaccharide and aripara extract, it was observed that the adhesion of blood vessels increased by lipopolysaccharide decreased, And the cell adhesion was decreased as it went up. These results demonstrate that the extract of O. clark extract has an inhibitory effect on vascular endothelial cell adhesion inhibition of arteriosclerosis.
실험예Experimental Example
6: 단핵구 세포의 세포부착분자 발현에 대한 오갈피 추출물의 영향 6: Effect of augaru extract on the expression of cell adhesion molecule in mononuclear cells
오갈피 추출물이 단핵구 세포와 혈관 내피세포 사이의 부착을 억제하는 기전을 확인하기 위하여, 오갈피 추출물의 처리시에 단핵구 세포에서 발현되는 세포부착분자들의 발현량을 하기와 같이 관찰하였다.In order to confirm the mechanism of inhibiting the adhesion between the extract of O. clark and the endothelial cells, the expression level of the cell adhesion molecules expressed in the mononuclear cells during the treatment of the O. clark extract was observed as follows.
단핵구 세포에서 발현하는 세포부착분자인 PAF 수용체, 인테그린(Integrin) αL, 인테그린 αM, 인테그린 β2, L-셀렉틴 다섯 가지 분자의 발현을 RNA와 단백질의 양으로 각각 확인하였다. Expression of five molecules of PAF receptor, integrin alpha L, integrin alpha M,
먼저, RNA양을 확인하기 위해서 다섯 가지의 부착분자 각각의 프라이머를 제작하여 RT-PCR을 수행하였다. 각 부착분자를 위한 프라이머 서열은 하기와 같다.
First, to confirm the amount of RNA, primers for each of five adhesion molecules were prepared and subjected to RT-PCR. The primer sequences for each attachment molecule are as follows.
PAF 수용체 PAF receptor
전방 프라이머 : ATCAACACCTACTGCTCTG (서열 번호 1)Front primer: ATCAACACCTACTGCTCTG (SEQ ID NO: 1)
역방 프라이머 : GCTGAACACGATGAAGATG (서열 번호 2)Reverse primer: GCTGAACACGATGAAGATG (SEQ ID NO: 2)
인테그린 αL Integrin αL
전방 프라이머 : CATCCACGACCACAACAT (서열 번호 3)Front primer: CATCCACGACCACAACAT (SEQ ID NO: 3)
역방 프라이머 : CTCCTGCCTGAAGACAAC (서열 번호 4)Reverse primer: CTCCTGCCTGAAGACAAC (SEQ ID NO: 4)
인테그린 αM Integrin αM
전방 프라이머 : AGAACAACATGCCCAGAACC (서열 번호 5)Forward primer: AGAACAACATGCCCAGAACC (SEQ ID NO: 5)
역방 프라이머 : GCGGTCCCATATGACAGTCT (서열 번호 6)Reverse primer: GCGGTCCCATATGACAGTCT (SEQ ID NO: 6)
인테그린 β2 Integrin β2
전방 프라이머 : CAAGCTGGCTGAAAACAAACA (서열 번호 7)Forward primer: CAAGCTGGCTGAAAACAAACA (SEQ ID NO: 7)
역방 프라이머 : ATTGCTGCAGAAGGAGTCGT (서열 번호 8)Reverse primer: ATTGCTGCAGAAGGAGTCGT (SEQ ID NO: 8)
L-셀렉틴L-selectin
전방 프라이머 : AAACCCATGAACTGGCAAAG (서열 번호 9)Forward primer: AAACCCATGAACTGGCAAAG (SEQ ID NO: 9)
역방 프라이머 : CGCAGTCCTCCTTGTTCTTC (서열 번호 10)
Reverse primer: CGCAGTCCTCCTTGTTCTTC (SEQ ID NO: 10)
먼저 THP-1 세포를 60mm 배양접시에 분주한 후 0, 0.5, 1, 2, 6 시간 동안 리포폴리사카라이드를 단독으로 처리하거나 리포폴리사카라이드와 오갈피 추출물을 함께 처리하였다. 시간이 다 되면 세포를 용해하여 RNA만 분리해 내었다. RNA를 정량하여 모두 2 ㎍의 같은 양을 cDNA로 역전사 시켰다. 이 cDNA를 이용하여 PCR을 수행하였고 결과는 1% 아가로즈 젤을 이용하여 확인하였다.
First, THP-1 cells were dispensed into a 60 mm culture dish and treated with lipopolysaccharide alone for 0, 0.5, 1, 2, or 6 hours, or treated with lipopolysaccharide and augarifolia extract together. At the end of the time, the cells were lysed to separate the RNA. RNA was quantitated and the same amount of all 2 μg was reverse transcribed with cDNA. PCR was carried out using this cDNA and the result was confirmed using 1% agarose gel.
확인 결과, 도 6에 나타난 바와 같이, 리포폴리사카라이드를 처리한 시간에 비례하여 부착분자의 발현이 증가하는 반면, 리포폴리사카라이드와 오갈피 추출물을 함께 처리한 경우에는 부착분자의 발현이 감소하는 분자가 인테그린 β2와 L-셀렉틴인 것을 확인할 수 있었다. 따라서 이 두 분자의 발현을 단백질 수준에서도 확인하기 위하여 웨스턴 블롯팅(western blotting)을 수행하였다.
As a result, as shown in Fig. 6, the expression of the adhesion molecule was increased in proportion to the time of treatment with the lipopolysaccharide, whereas when the lipopolysaccharide and the extract of Ocarki were treated together, the expression of the adhesion molecule decreased It was confirmed that the molecule was integrin [beta] 2 and L-selectin. Therefore, western blotting was performed to confirm the expression of these two molecules at the protein level.
먼저, THP-1 세포를 60mm 배양접시에 분주한 후 0, 4, 8, 12, 24 시간 동안 리포폴리사카라이드를 단독으로 처리한 것과 리포폴리사카라이드와 오갈피 추출물을 함께 처리한 것을 분해 완충액(150mM NaCl, 0.1mM PMSF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris-Hcl pH 8.0)으로 분해시켰다. 원심분리를 통해 상층액을 획득하여 BCA 분석을 통하여 단백질을 정량하고 동일한 양의 단백질을 SDS-PAGE를 이용하여 분리시켰다. 분리시킨 단백질을 PVDF막으로 이동시켜 고정하고, 분석하고자 하는 표적 단백질에 해당하는 1차 항체와 반응 시킨 후, 2차 항체를 반응시켜 화학발광 검출법을 통해 X-ray 필름으로 현상하여 세포 부착분자의 발현 변화를 관찰하였다. First, THP-1 cells were dispensed into a 60-mm culture dish, treated with lipopolysaccharide alone for 0, 4, 8, 12, and 24 hours, and treated with lipopolysaccharide and augarifolia extract in dissolution buffer 150 mM NaCl, 0.1 mM PMSF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0). The supernatant was obtained by centrifugation, and proteins were quantified by BCA analysis and the same amount of protein was separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane, fixed, reacted with a primary antibody corresponding to the target protein to be analyzed, and then reacted with a secondary antibody, and developed with X-ray film through chemiluminescence detection, Expression changes were observed.
그 결과, 도 7에서 확인할 수 있는 것과 같이, 인테그린 β2의 단백질 발현은 오갈피 추출물에 의해 감소되지 않았지만 L-셀렉틴의 경우에는 리포폴리사카라이드만 처리한 경우 시간이 지남에 따라 발현이 증가하였고 리포폴리사카라이드와 오갈피 추출물을 함께 처리한 경우 시간이 지남에 따라 발현이 억제되는 것을 관찰하였다. 이러한 결과는 RNA 수준에서 관찰한 실험과 동일한 결과로서, 본 발명의 오갈피 추출물이 단핵구 세포의 L-셀렉틴 발현을 억제시킴으로써 단핵구 세포와 혈관 내피세포의 부착을 억제하여 동맥경화를 예방할 수 있음을 잘 보여주는 것이다.
As a result, as can be seen from Fig. 7, protein expression of integrin [beta] 2 was not decreased by the extract of O. clark but in the case of L-selectin, expression was increased over time only with lipopolysaccharide, When the saccharide and acaricidal extracts were treated together, the expression was inhibited over time. These results are the same as those of the experiment observed at the RNA level. As a result, it is shown that the extract of Ogallipuccus of the present invention inhibits the expression of L-selectin in monocytic cells, thereby inhibiting the adhesion of monocytes and vascular endothelial cells to prevent arteriosclerosis will be.
실험예Experimental Example
7: 7:
THPTHP
-1 세포의 L--1 cells < / RTI >
셀렉틴Selectin
발현에 대한 오갈피 추출물의 영향 Influence of the extract of augarugi on the expression
THP-1 세포의 L-셀렉틴 발현에 대한 본 발명의 오갈피 추출물의 영향을 확인하기 위하여, THP-1 세포에서 발현하는 L-셀렉틴을 면역세포화학법을 이용하여 관찰하였다.
In order to confirm the effect of the extract of the present invention on the expression of L-selectin in THP-1 cells, L-selectin expressed in THP-1 cells was observed by immunocytochemistry.
60mm 배양접시에서 배양한 THP-1을 PBS를 이용하여 세척하고 2% 파라로름알데하이드를 이용하여 세포를 고정하였다. 후에 PBS를 이용하여 두 차례 더 세포를 세척한 다음 L-셀렉틴에 특이적으로 붙는 1차 항체를 1시간동안 4℃에서 반응시켰다. 그리고 형광을 띄는 2차 항체를 처리하고 4℃에서 1시간이 지난 후 형광현미경을 이용하여 세포를 관찰하였다.
The THP-1 cultured in a 60 mm culture dish was washed with PBS and fixed with 2% para-formaldehyde. After washing the cells twice with PBS, the primary antibody specific for L-selectin was reacted at 4 ° C for 1 hour. The cells were observed using a fluorescence microscope after 1 hour at 4 ° C after treatment with a fluorescent secondary antibody.
그 결과, 도 8에 나타난 바와 같이, 아무것도 처리하지 않은 대조군에 비해 리포폴리사카라이드를 처리했을 경우 THP-1세포 표면에 L-셀렉틴을 발현하고 있는 세포가 많아졌다. 또한 리포폴리사카라이드와 오갈피 추출물을 함께 처리했을 때 리포폴리사카라이드에 의해 야기된 L-셀렉틴의 과발현이 감소됨을 관찰할 수 있었다. 이는 총 단백질량을 측정한 웨스턴 블롯팅 에서도 확인했듯이 본 발명의 오갈피 추출물이 단핵구 세포의 L-셀렉틴 발현을 억제하여 동맥경화를 예방할 수 있음을 보여주는 것이다.
As a result, as shown in Fig. 8, when lipopolysaccharide was treated as compared with the control group in which nothing was treated, the number of cells expressing L-selectin on the surface of THP-1 cells was increased. In addition, it was observed that over-expression of L-selectin caused by lipopolysaccharide was reduced when lipopolysaccharide and Ocarpa extract were treated together. This was shown in the Western blotting method in which the total protein amount was measured, showing that the extract of the present invention can prevent arteriosclerosis by inhibiting the expression of L-selectin in monocyte cells.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
<110> Industry-Academic Cooperation Foundation, Dankook University <120> A composition for preventing or treating blood vessel disorders, comprising extracts of Acanthopanax <130> PA121157KR <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 atcaacacct actgctctg 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gctgaacacg atgaagatg 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 catccacgac cacaacat 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ctcctgcctg aagacaac 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 agaacaacat gcccagaacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gcggtcccat atgacagtct 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 caagctggct gaaaacaaac a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 attgctgcag aaggagtcgt 20 <110> Industry-Academic Cooperation Foundation, Dankook University <120> A composition for preventing or treating blood vessel disorders, comprising extracts of Acanthopanax <130> PA121157KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 atcaacacct actgctctg 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gctgaacacg atgaagatg 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 catccacgac cacaacat 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ctcctgcctg aagacaac 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 agaacaacat gcccagaacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gcggtcccat atgacagtct 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 caagctggct gaaaacaaac a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 attgctgcag aaggagtcgt 20
Claims (10)
The roots and stem samples of Acanthopanax divaricatus var. Albeofructus were frosted with water of 1 to 20 times (v / w) of the weight of the sample, and then subjected to hot water extraction at 100 to 120 ° C to obtain a hot water extract of white- , Or a pharmaceutical composition for preventing or treating blood vessel formation or blood coagulation inhibition, or vasculitis.
상기 조성물은 혈전형성 또는 혈액 응고 억제용인 것인, 약학적 조성물.
The method according to claim 1,
Wherein said composition is for thrombus formation or for inhibiting blood coagulation.
The roots and stem samples of Acanthopanax divaricatus var. Albeofructus were frosted with water of 1 to 20 times (v / w) of the weight of the sample, and then subjected to hot water extraction at 100 to 120 ° C to obtain a hot water extract of white- , Or a composition for prevention or improvement of blood vessel formation or blood coagulation inhibition or vasculitis.
(a) extracting 1 to 20 times (v / w) water of dry white sclerotium sample in water at 25 ° C to 120 ° C for 1 hour to 15 hours by adding water to a white sclerotium sample; And (b) filtering, concentrating and drying the water extracted in the step (a).
10. The method of claim 9, wherein the extraction in step (a) is performed by hot water extraction at 100 DEG C to 120 DEG C for 5 to 9 hours after cooling for 1-5 hours.
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