KR101880515B1 - Composition for preventing, improving or treating ischemic disease comprising extract of Tetragonia tetragonoides as effective component - Google Patents
Composition for preventing, improving or treating ischemic disease comprising extract of Tetragonia tetragonoides as effective component Download PDFInfo
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- KR101880515B1 KR101880515B1 KR1020170060010A KR20170060010A KR101880515B1 KR 101880515 B1 KR101880515 B1 KR 101880515B1 KR 1020170060010 A KR1020170060010 A KR 1020170060010A KR 20170060010 A KR20170060010 A KR 20170060010A KR 101880515 B1 KR101880515 B1 KR 101880515B1
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Abstract
Description
본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, ameliorating or treating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.
허혈(ischemia)은 신체기관, 조직 또는 부위로의 혈류 공급 감소 상태를 말하며, 궁극적으로 비가역적인 손상인 세포 및 조직의 괴사(necrosis)로 이어지게 된다. 따라서, 혈액순환에 있어 특히 말초혈액순환은 모세혈관의 형성과 확장이 매우 중요하다 할 수 있다. 즉, 혈관에서의 혈류 증가는 반드시 혈관의 형성과 확장을 필요로 하는 기작이다. Ischemia refers to a state of reduced blood supply to organs, tissues or regions and ultimately leads to necrosis of cells and tissues, irreversible damage. Therefore, peripheral blood circulation, especially in blood circulation, can be considered to be very important for formation and expansion of capillary blood vessels. That is, the increase of blood flow in a blood vessel necessarily requires formation and expansion of blood vessels.
또한, 혈관신생(angiogenesis)은 기존의 혈관에서 새로운 혈관이 만들어지는 일련의 과정으로, 혈관을 구성하고 있는 혈관내피세포의 이동(migraition)과 세포간 장벽인 세포외기질(ECM)를 통과하는 침윤(invasion), 증식(proliferation), 혈관으로의 분화(tube 생성)의 과정을 통하여 진행된다. 생리적 상태의 혈관신생은 태아의 발생, 여성의 월경 및 국소의 산소부족에 따라 한시적으로 일어날 수 있으며, 허혈성 질환이나 골절 등에서는 혈류의 공급이 원활하지 못하여 치료성 혈관신생이 활용되고 있다. In addition, angiogenesis is a series of processes in which new blood vessels are formed in the existing blood vessels. In the blood vessels, migration of vascular endothelial cells and invasion through the extracellular matrix (ECM) invasion, proliferation, and differentiation into blood vessels (tube generation). The angiogenesis of the physiological state can occur for a short time according to the development of the fetus, female menstruation and local oxygen deficiency. Therapeutic angiogenesis is utilized because ischemic diseases or fractures do not supply blood flow smoothly.
한편, 번행초는 바닷가 모래땅에 자라는 여러해살이풀로, 줄기는 누워 자라거나 비스듬히 서며 길이는 40~50cm이고, 잎은 어긋나며 두껍고 잎자루는 2cm 정도이다. 잎몸은 난상 삼각형 또는 난형으로 길이 4~6cm, 폭 3~5cm이다. 꽃은 4월 초순부터 11월 하순에 잎겨드랑이에 1~2개씩 달리며, 꽃받침통은 4~5갈래로 얕게 갈라지는데 겉은 녹색이고 안쪽은 노란색이다. 꽃잎은 없고 열매는 견과로 윗부분에 가시 같은 돌기가 있다. 어린잎은 나물로 먹고, 전초를 약재로 쓴다. 우리나라 남부지방과 울릉도, 제주도에 자생하고, 일본, 중국을 비롯하여 남아시아와 호주, 북미와 남미에 분포한다. 번행초는 해열, 해독, 소종의 효능이 있고, 위장염과 위궤양, 위암, 자궁암, 피부의 땀구멍이나 기름구멍으로 화농균이 침입하여 생기는 부스럼 등을 치료하는 데 쓰인다. 전초에는 풍부한 철, 칼슘, 비타민 A와 각종 비타민 B가 함유되어 있다. On the other hand, Seonjangcho is a perennial plant that grows in the sandy seaside of the sea. The stem grows or stands diagonally. Its length is 40 ~ 50cm. Leaves are alternate and thick and petiole is about 2cm. Blade is egg-shaped triangle or oval, 4 ~ 6cm long, 3 ~ 5cm wide. The flowers hang 1-2 on the leaf axil from the beginning of April to the end of November, and the calyx tubers are shallowly divided into 4 to 5 branches, the surface is green and the inside is yellow. There are no petals, the fruit is nuts, and there are thorn like projections on the upper part. The young leaves eat herbs and use the outposts as medicines. It is native to southern Korea, Ulleungdo and Jeju Island, and is distributed in Japan, China, South Asia, Australia, North America and South America. It is used for the treatment of gastroenteritis, gastric ulcer, stomach cancer, uterine cancer, swelling of the skin, swelling caused by infestation by the oil hole, and so on. The outpost contains abundant iron, calcium, vitamin A and various vitamin B.
본 발명의 번행초 추출물의 의약 용도에 관한 종래기술로는 한국등록특허 제1108885호에 항당뇨 및 혈중 콜레스테롤 저해활성을 갖는 번행초 추출물에 관한 기술 및 한국등록특허 제0483183호에 세포사멸 억제 활성을 갖는 번행초 추출물 및 이를 포함하는 약학적 조성물이 알려져 있으나, 본 발명의 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방, 개선 또는 치료용 조성물에 대해서는 알려진 바 없다.As a conventional technology for the use of the medicinal herb extract of the present invention, Korean Patent No. 1108885 discloses a technique relating to an antidiabetic agent having antidiabetic and blood cholesterol inhibitory activity, and Korean Patent No. 0483183, Extracts and pharmaceutical compositions containing them are known, but a composition for preventing, ameliorating or treating ischemic diseases containing the extract of the present invention as an active ingredient is not known.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방, 개선 또는 치료용 조성물에 관한 것으로, 상기 유효성분인 번행초 추출물이 혈관내피세포의 이동, 생성 및 형성을 유도하여 혈류량을 증진시킬 수 있다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention provides a composition for preventing, ameliorating or treating ischemic diseases comprising the extract of Opadonthus edulis as an active ingredient. The present invention relates to a composition for preventing, improving or treating ischemic diseases, , Production and formation of blood, thereby enhancing blood flow volume, thereby completing the present invention.
상기 목적을 달성하기 위하여, 본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating an ischemic disease comprising an extract of Opium japonica as an active ingredient.
또한, 본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for preventing or ameliorating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.
본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방, 개선 또는 치료용 조성물에 관한 것으로, 본 발명의 번행초 추출물은 혈관내피세포의 이동, 생성 및 형성을 유도하여 혈류량을 효과적으로 증진하므로, 허혈성 질환의 예방, 개선 또는 치료용으로 의약품 또는 건강기능식품에 활용할 수 있다. The present invention relates to a composition for preventing, ameliorating or treating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient. Since the first-time extract of the present invention induces the migration, formation and formation of vascular endothelial cells, It can be used for medicines or health functional foods for prevention, improvement or treatment of diseases.
도 1은 본 발명의 하지 혈행장애 개선에 대한 레이저 도플러 영상 분석 결과를 나타낸 것이다.
도 2는 본 발명의 번행초 추출물의 농도별 처리에 따른 혈관내피세포의 증식율을 확인한 결과이다.FIG. 1 shows a result of a laser Doppler image analysis of the improvement of the bleeding disorder of the present invention.
FIG. 2 shows the results of confirming the proliferation rate of vascular endothelial cells according to the concentration of the first-time extract of the present invention.
본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.
상기 허혈성 질환은 하지혈관 허혈성 질환, 사지말단부 허혈성 질환, 허혈성 뇌혈관 질환, 허혈성 심장질환 및 상처나 화상 등에 의한 세포 괴사에 의한 허혈 중에서 선택된 하나 이상인 것이 바람직하며, 상기 하지혈관 허혈성 질환 또는 사지 말단부 허혈성 질환은 갱년기에 의한 것이 특징이지만 이에 한정하는 것은 아니다. The ischemic diseases are preferably at least one selected from the group consisting of lower limb ischemic diseases, limb ischemic diseases, ischemic cerebrovascular diseases, ischemic heart diseases, and ischemia due to necrosis caused by scars and burns, and the above ischemic ischemic diseases or limb ischemic diseases The disease is characterized by menopausal age, but is not limited thereto.
갱년기에 의한 허혈은 손 또는 발 저림, 시림 등 혈액 순환이 잘되지 않는 경우를 포함할 수 있다.Ischemia due to menopausal period may include cases in which blood circulation is not good, such as hand or foot numbness, palpitation.
상기 번행초 추출물은 하기의 단계를 포함하는 방법에 의해 제조할 수 있으나, 이에 한정하지 않는다:The progeny extract can be prepared by a method including, but not limited to, the following steps:
(1) 번행초에 추출용매를 가하여 추출하는 단계;(1) extracting by adding an extraction solvent in advance;
(2) 단계 (1)의 추출물을 여과하는 단계; 및 (2) filtering the extract of step (1); And
(3) 단계 (2)의 여과한 추출물을 농축하고 건조하여 추출물을 제조하는 단계. (3) concentrating the filtered extract of step (2) and drying to produce an extract.
상기 단계 (1)에서 추출용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물 중에서 선택하는 것이 바람직하며, 더 바람직하게는 에탄올이고, 더욱더 바람직하게는 70%(v/v) 에탄올이지만 이에 한정하지 않는다.In step (1), the extraction solvent is preferably selected from water, a C 1 -C 4 lower alcohol or a mixture thereof, more preferably ethanol, even more preferably 70% (v / v) ethanol But is not limited thereto.
상기 제조방법에 있어서, 추출방법은 여과법, 열수 추출, 침지 추출, 환류 냉각 추출 및 초음파 추출 등의 당 업계에 공지된 모든 통상적인 방법을 이용할 수 있다. 상기 추출용매는 건조된 번행초 중량의 1~20배 첨가하여 추출하는 것이 바람직하며, 더 바람직하게는 5~15배 첨가하는 것이고, 더욱더 바람직하게는 10배 첨가하는 것이다. 추출온도는 4~50℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10~100시간인 것이 바람직하나, 이에 한정하지 않는다. 상기 방법에 있어서, 단계 (3)의 농축은 진공 회전 농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무 건조 또는 동결건조하는 것이 바람직하며, 더 바람직하게는 동결건조이나 이에 한정하지 않는다.In the above production method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably added by 1 to 20 times the dry weight of the first step, more preferably 5 to 15 times, and further preferably 10 times by weight. The extraction temperature is preferably 4 to 50 ° C, but is not limited thereto. The extraction time is preferably 10 to 100 hours, but is not limited thereto. In the above method, the concentration of the step (3) is preferably, but not limited to, using a vacuum rotary condenser or a vacuum rotary evaporator. In addition, drying is preferably carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, more preferably freeze drying or the like.
상기 번행초 추출물은 혈관내피세포의 이동 및 증식을 유도하고, 혈관내피세포의 형성을 촉진하여 혈류량을 증가시키는 것을 특징으로 한다.The progeny extract of the present invention is characterized by inducing migration and proliferation of vascular endothelial cells and promoting formation of vascular endothelial cells to increase blood flow.
상기 번행초 추출물 이외에 추가로 약제학적으로 허용가능한 담체, 부형제 또는 희석제를 더 포함할 수 있다. In addition to the herbal extract, the composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent.
본 발명의 약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여 시 피부 외용 또는 복강 내, 직장, 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사 방식을 선택하는 것이 바람직하다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to use an intravenous or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebral injection method during parenteral administration.
본 발명의 약학 조성물은 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 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. 경구 투여를 위한 액상 제제로는 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. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성 용제 및 현탁 용제로는 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 61, 카카오지, 라우린지, 글리세로 젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention can be prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient. 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 may also be 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 sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 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 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. 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 side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도에 따라 그 범위가 다양하게 사용할 수 있다.The dosage of the composition of the present invention may be varied depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
또한, 본 발명은 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention also relates to a health functional food composition for preventing or ameliorating an ischemic disease comprising an effective amount of a first-time extract as an active ingredient.
상기 허혈성 질환은 하지혈관 허혈성 질환, 사지말단부 허혈성 질환, 허혈성 뇌혈관 질환 및 허혈성 심장질환 중에서 선택된 하나 이상인 것이 바람직하지만 이에 한정하지 않는다.Preferably, the ischemic disease is at least one selected from the group consisting of lower limb ischemic diseases, limb ischemic diseases, ischemic cerebrovascular diseases and ischemic heart diseases, but is not limited thereto.
상기 번행초 추출물을 유효성분으로 포함하는 허혈성 질환의 예방 또는 개선용 건강기능식품 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나로 제조하거나, 식품의 성분으로 첨가하여 제조될 수 있으며, 통상적인 방법에 따라 적절하게 제조될 수 있다. The health functional food composition for preventing or ameliorating an ischemic disease comprising the extract of the present invention as an active ingredient may be prepared from any one selected from powder, granule, ring, tablet, capsule, candy, syrup and beverage, And can be suitably prepared according to a conventional method.
본 발명의 번행초 추출물을 첨가할 수 있는 식품의 일례로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 중에서 선택된 어느 하나의 형태일 수 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.Examples of the food to which the extract of the present invention can be added include a dairy product including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, A tea, a drink, an alcoholic beverage, and a vitamin complex, all of which include health functional foods in a conventional sense.
상기 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 및 천연 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알킨산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. The health functional foods include various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alkynic acid and its salts, , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination.
본 발명의 건강기능식품 조성물은 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.The health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.
실시예Example 1. One. 번행초Early spring 추출물의 제조 Preparation of extract
본 발명에 사용된 번행초는 대한민국 제주도에서 채취 또는 유통품을 구입하여 사용하였으며, 건조한 번행초 시료양에 대하여 10배의 70%(v/v) 에탄올을 첨가하고, 상온에서 48시간 동안 침출한 후, 번행초 추출액을 수득하였다. 상기 수득한 번행초 추출액을 0.4㎛ 필터로 여과한 후, 진공회전농축기로 농축하여 동결건조하였다. In the present invention, the fresh shoots used in the present invention were collected or used in Jeju Island, South Korea. The dried seedlings were added with 10
실시예Example 2. 2. 번행초Early spring 추출물의 투여에 따른 혈류량 변화 분석 Analysis of blood flow changes by administration of extracts
암컷 C57BL/6 마우스 6주령을 수령하여 일주일의 순화기간을 거친 후 7주령에 난소절제술을 실시하였다. 8주령에 좌측 하지(left hind-limb) 두 구역의 혈관(동맥, 정맥)을 결찰 후, 분리하여 허혈모델을 완성하였다. Female C57BL / 6 mice were fed 6 weeks of age, followed by a week of fertilization, and then ovariectomized at 7 weeks of age. At the age of 8 weeks, left hind-limb blood vessels (arteries, veins) in two areas were ligated and separated to complete the ischemic model.
본 실험은 한국한의학연구원 실험동물 윤리위원회 심의(16-018)를 거쳤으며, 실험동물 사육 및 유지는 한국한의학연구원 실험동물연구센터의 동물 관리 및 사용 규정에 입각하여 온도 23±1℃ 습도 50±10% 내외, 12시간 명암주기로 일정하게 유지된 사육실에서 IVC(individually ventilated cage)에 사육하였다.This experiment was conducted by Korea Institute of Oriental Medicine (16-018), and the breeding and maintenance of experimental animals were conducted at 23 ± 1 ℃ and 50 ± 2 ℃, respectively, based on the animal care and use regulations of Korea Institute of Oriental Medicine. 10%, and 12 hours of light cycle, and maintained in an IVC (individually ventilated cage).
양성대조구 I(심바스타틴; SV), 양성대조구 II(에스트로겐, E2) 및 번행초 추출물 처치로는 AIN-76A의 사료에 각각의 농도로 시료를 포함시켜 자유식이 하도록 하였다.For the treatment of benignant control I (simvastatin; SV), positive control II (estrogen, E 2 ), and prosodic extract, samples were included in the diets of AIN-76A for freezing.
하지 허혈 모델 확립 28일 동안 레이저 도플러 영상장치를 이용하여 좌측 하지의 혈류(blood flow)를 측정하여, 모의수술 대조구인 우측 하지와 비교하여 허혈/비허혈 비율(ischemic/non-ischemic ratio)으로 혈류량(blood flow rate)을 구하였다. Blood flow of the left lower limb was measured using a laser Doppler imaging device for 28 days and compared with that of the right lower limb as a simulated surgical control, an ischemic / non-ischemic ratio (blood flow rate).
그 결과, 하지 허혈 직 후 혈류량(blood flow rate)은 하지 허혈 유발 전(-7일)에 비하여 모든 군에서 유의적으로 감소하였다. As a result, the blood flow rate after lower limb ischemia was significantly decreased in all groups compared with before ischemia induction (-7 days).
난소절제(OVX) 후 하지허혈(HLI)을 시행한 그룹인 음성대조구(vehicle; OH)에 비하여 양성대조구 I과 고농도 번행초 추출물(450mg/kg/day) 투여군에서 혈류량(blood flow rate)에 대한 유의적 효과가 관찰되었다. 이후 하지 허혈 유발 14일 및 28일 후에는 두 양성대조구(I, II) 및 번행초 추출물 투여군 모두에서 음성대조구(vehicle)에 비해 유의적인 효과가 확인되었다(표 1 및 도 1). In contrast to the control group (OH), which was treated with ovariectomy (OVX) after ischemia (HLI), there was a significant difference in the blood flow rate between the control group I and the high- The effect was observed. After 14 days and 28 days after induction of ischemic injury, significant effects were observed in both positive control (I, II) and nonsteroidal extract administration groups (Table 1 and Fig. 1) compared to negative control vehicle.
group
(OH+SV; 20mg/kg/day)Positive control I
(OH + SV; 20 mg / kg / day)
(OH+E2; 100㎍/kg/day)Positive control II
(OH + E 2 ; 100 μg / kg / day)
(OH+TTG; 150mg/kg/day)
(OH + TTG; 150 mg / kg / day)
(OH+TTG; 450mg/kg/day)
(OH + TTG; 450 mg / kg / day)
*p<0.05 vs. 수술 전, a p<0.05 vs. Vehicle, b p<0.05 vs. Positive I, c P<0.05 vs. Positive II. * p < Before surgery, a p <0.05 vs. Vehicle, b p < Positive I, c P < Positive II.
실시예Example 3. 하지 말초혈관 형성 확인 3. Identification of non-distal peripheral angiogenesis
혈관내피세포의 생체 표지자로, 사용되는 CD31(cluster of differentiation 31)을 이용하여 하지허혈이 유발된 좌측하지의 대퇴부(thigh) 및 장딴지 근육(calf muscle)에서 면역조직화학 염색을 실시하였다. 해당 조직은 파라핀 절편으로 5μm의 두께로 슬라이스 하고, 자일렌을 이용하여 탈파라핀하였다. 고농도에서 저농도의 에탄올을 이용 최종적으로 수돗물에서 함수시킨 후 사용하였다. 사용한 항체로는 Abcam사의 Anti-CD31(rabbit polyclonal to CD31)을 이용하였으며, 최종 염색된 positive cell(brown color)을 기준으로, 말초혈관(capillary)의 수를 세어 단위 면적(mm2)의 개수로 환산하였다. Immunohistochemical staining was performed on left lower limb thigh and calf muscle induced by lower limb ischemia using CD31 (cluster of differentiation 31), which is a biomarker of vascular endothelial cells. The tissues were sliced to a thickness of 5 mu m with paraffin sections, and deparaffinized with xylene. Low concentration of ethanol was used at high concentration and finally water was used in tap water. Antibody used is a number of Abcam Inc. Anti-CD31 (rabbit polyclonal to CD31 ) a was used, based on the final staining positive cell (brown color), peripheral blood vessel (capillary) counted per unit area (mm 2) the number of Respectively.
그 결과, 표 2에 개시한 바와 같이 번행초 추출물을 처리한 경우, 말초혈관 형성이 통계적으로 유의미하게 증가한 것을 확인하였다.As a result, it was confirmed that peripheral blood vessel formation was statistically significantly increased in the case of the treatment with the progeny extract as shown in Table 2. [
(OH)Negative control
(OH)
(OH+SV)Positive control I
(OH + SV)
(OH+E2)Positive control II
(OH +
(OH+TTG150)150 seconds
(OH + TTG150)
(OH+TTG450)450 seconds
(OH + TTG450)
aa
p<0.05 vs. Vehicle, p < Vehicle,
bb
p<0.05 vs. Positive I, p < Positive I,
cc
P<0.05 vs. Positive II, P < Positive II,
dd
P<0.05 vs. P <
TTG
실시예Example 4. 4. 번행초Early spring 추출물의 처리에 따른 혈관내피세포의 증식률 확인 Identification of vascular endothelial cell proliferation by treatment of extract
혈관내피세포를 96웰 플레이트에 4×103cell/well로 부착시킨 후, 37℃, 5% CO2 배양기에서 24시간 배양하였다. 번행초 추출물을 1, 5, 10, 50, 100㎍/㎖로 처리하여 24시간 동안 방치한 후, 배양액에 EZ-cytox(Biomax. Korea) 10㎕씩을 첨가하여 4시간 후, 450nm에서 흡광도를 측정하고, 그 측정값을 대조군과 비교하여 세포의 증식율을 계산하였다.The vascular endothelial cells were attached to a 96-well plate at 4 × 10 3 cells / well and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours. After incubation for 24 hours, 10 μl of EZ-cytox (Biomax. Korea) was added to the culture solution. After 4 hours, the absorbance was measured at 450 nm , And the measured value was compared with the control group to calculate the proliferation rate of the cells.
그 결과, 하기 표 3 및 도 2에 개시한 바와 같이 세포 증식율은 번행초 추출물(TTG)를 처리하지 않은 세포에 비해 5, 50, 100㎍/㎖의 번행초 추출물을 처리한 경우 통계적으로 유의미하게 농도의존적으로 증가하였다. As a result, as shown in the following Table 3 and FIG. 2, the cell proliferation rate was statistically significant in the case of treatment with 5, 50, and 100 / / ㎖ of the pre-treatment extracts compared with the cells not treated with the TTG Respectively.
(㎍/㎖)TTG
(占 퐂 / ml)
(%)Growth rate
(%)
aa p<0.001 p < 0.001 vsvs . . nonnon -- treatedtreated cellcell
실시예Example 5. 5. 번행초Early spring 추출물이 혈관내피세포의 이동에 미치는 영향 분석 Effect of Extracts on Vascular Endothelial Cell Migration
트랜스웰(transwell)의 윗쪽 웰(upper well)에 혈관내피세포(HUVAC) 3×103 세포와 혈청이 포함되지 않은(serum free) EGM2 배양액에 번행초 추출물을 각각 10 및 50㎍/㎖씩 첨가하여 50ng/㎖의 VEGF 처리군, 1nM의 E2 처리군, 10μM의 심바스타틴 처리군과 함께 6시간 동안 이동시켰다. 이동한 혈관내피세포는 H&E 염색법으로 염색하여 본 발명의 번행초 추출물이 혈관내피세포 이동에 미치는 영향을 대조군과 이동율을 비교하였다. To the upper well of the transwell, 3 × 10 3 HUVAC cells and serum-free EGM2 medium were added with 10 and 50 μg / ml, respectively, 50 ng / ml VEGF treated group, 1 nM E2 treated group, and 10 μM simvastatin treated group for 6 hours. The migrated vascular endothelial cells were stained with H & E staining method, and the effect of the progeny extract of the present invention on the vascular endothelial cell migration was compared with the control group and the migration rate.
그 결과, 표 4에 개시한 바와 같이, 번행초 추출물을 각각 10 및 50㎍/㎖씩 첨가한 경우, 아무것도 처리하지 않은 대조군(Normal)에 비해 이동 속도가 통계적으로 유의미하게 증가한 것을 확인하였다.As a result, as shown in Table 4, it was confirmed that the addition of 10 and 50 μg / ml of Puerariae Radix extract significantly increased the migration speed statistically compared to the control group without any treatment (Normal).
(50ng/㎖)VEGF
(50 ng / ml)
(1nM)E 2
(1 nM)
(10μM)Simvastatin
(10 [mu] M)
(10㎍/㎖)Early spring
(10 [mu] g / ml)
(50㎍/㎖)Early spring
(50 mu g / ml)
aa p<0.001 vs. Normal, p < Normal, bb p<0.001 vs. p < 심바스타틴Simvastatin
실시예Example 6. 6. 번행초Early spring 추출물이 혈관내피세포의 분화에 미치는 영향 분석 Effect of Extract on the Differentiation of Vascular Endothelial Cells
혈관내피세포를 1% fetal bovine serum이 포함된 EGM2 배지로 세포외 기질(extracellular matrix; ECM)인 Matrigel 코팅된 24웰 플레이트에 3×105이 되도록 분주하여 12시간 동안 37℃에서 혈관내피세포의 튜브(tube) 형성을 유도하였다. 12시간 후, Calsein AM으로 염색하고, 현미경으로 관찰 및 촬영하여 Image J 프로그램의 안지오제네시스(Angiogenesis) 분석기를 이용해 튜브의 브랜칭 간격(branching interval)을 측정하여 대조군과 비교하였다. The vascular endothelial cells were divided into 3 × 10 5 Matrigel-coated 24-well plates with extracellular matrix (ECM) in EGM2 medium containing 1% fetal bovine serum. Tube formation was induced. Twelve hours later, the cells were stained with Calsein AM, observed and photographed with a microscope and the tube branching interval was measured using the Image J program's Angiogenesis Analyzer and compared with the control group.
그 결과, 표 5에 개시한 바와 같이, 번행초 추출물을 각각 10 및 50㎍/㎖씩 첨가한 경우, 아무것도 처리하지 않은 대조군(Normal)에 비해 튜브 형성 속도가 통계적으로 유의미하게 증가한 것을 확인하였다.As a result, as shown in Table 5, it was confirmed that the tube formation rate was statistically significantly increased when 10 and 50 μg / ml of the first extracts were added, respectively, as compared with the control (Normal) without any treatment.
(1pM)Bin blastin
(1pM)
(50ng/㎖)VEGF
(50 ng / ml)
(1nM)E 2
(1 nM)
(10μM)Simvastatin
(10 [mu] M)
(10㎍/㎖)Early spring
(10 [mu] g / ml)
(50㎍/㎖)Early spring
(50 mu g / ml)
aa p<0.05,p < 0.05, b b p<0.01 p < 0.01 c c p<0.001 vs. Normal, p < Normal, dd p<0.05, p < 0.05, e e p<0.001 vs. p < 심바스타틴Simvastatin
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KR20030077690A (en) * | 2002-03-26 | 2003-10-04 | 김철호 | Extracts of Herba Tetragoniae inhibiting apoptosis and pharmaceutical preparation including thereof |
KR20160050747A (en) * | 2014-10-30 | 2016-05-11 | 농업회사법인 주식회사 비케이수 | Cosmetic composition containing new zealand spinach extract |
KR20160071155A (en) * | 2014-12-11 | 2016-06-21 | 단국대학교 천안캠퍼스 산학협력단 | Composition for antiinflammatory and inflammatory neurodegenerative diseases comprising tetragonia tetragonoides extract |
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KR20030077690A (en) * | 2002-03-26 | 2003-10-04 | 김철호 | Extracts of Herba Tetragoniae inhibiting apoptosis and pharmaceutical preparation including thereof |
KR20160050747A (en) * | 2014-10-30 | 2016-05-11 | 농업회사법인 주식회사 비케이수 | Cosmetic composition containing new zealand spinach extract |
KR20160071155A (en) * | 2014-12-11 | 2016-06-21 | 단국대학교 천안캠퍼스 산학협력단 | Composition for antiinflammatory and inflammatory neurodegenerative diseases comprising tetragonia tetragonoides extract |
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