KR20130049660A - Compositions and functional food for prevention or treatment of diabetic complications comprising using extract of hedera rhombea - Google Patents
Compositions and functional food for prevention or treatment of diabetic complications comprising using extract of hedera rhombea Download PDFInfo
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- KR20130049660A KR20130049660A KR1020110114806A KR20110114806A KR20130049660A KR 20130049660 A KR20130049660 A KR 20130049660A KR 1020110114806 A KR1020110114806 A KR 1020110114806A KR 20110114806 A KR20110114806 A KR 20110114806A KR 20130049660 A KR20130049660 A KR 20130049660A
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- ethyl acetate
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Abstract
Description
본 발명은 송악 추출물, 또는 이의 분획물을 유효성분으로 포함하는 당뇨합병증 예방 또는 치료를 위한 약학적 조성물 및 건강기능식품에 관한 것이다.
The present invention relates to a pharmaceutical composition and health functional food for preventing or treating diabetic complications comprising Songak extract, or a fraction thereof as an active ingredient.
당뇨병은 전 세계적으로 중요한 성인병 중의 하나로서, 최근 우리나라에서도 급속한 경제 성장과 더불어 당뇨병 유병률이 10%에 달하며, 현재 전 세계적으로 2억4천만명이 넘었으며, 2025년에는 전 세계적으로 3억8천만명으로 증가하고, 이중 60%가 아시아 지역에서 발병할 것이라고 2009년 미국의사협회(JAMA)에서 발표하였다. 특히, 당뇨병 발병시기가 중장년으로 당겨졌으며, 또한 수명이 연장됨으로서 합병증으로 진전되는 것을 피할 수 없는 상황이 되었다. 일반적으로 당뇨병에 걸린 후 10~20년이 지나면 체내 거의 모든 기관이 손상을 받아 당뇨성 망막병증(diabetic retinopathy), 당뇨성 백내장(diabetic cataract), 당뇨성 신증(diabetic nephropathy), 당뇨성 신경병증(diabetic neuropathy), 심장병, 암, 골다공증 등으로 나타난다. 만성 당뇨성 신증은 혈액 투석 치료 및 말기 신부전의 가장 중요한 원인이 되고 있으며, 당뇨성 백내장과 망막증은 실명을 초래하고 결국엔 죽음에 이르게 한다.
Diabetes is one of the world's most important adult diseases. Recently, with the rapid economic growth in Korea, the prevalence rate of diabetes reached 10%, and now it is over 240 million people worldwide, and 380 million people worldwide by 2025. Increasing, of which 60 percent will occur in Asia, according to the 2009 American Medical Association (JAMA). In particular, the onset of diabetes mellitus has been extended to the middle age, and the lifespan is prolonged, and the development of complications is inevitable. In general, after 10-20 years of diabetes, almost all organs in the body are damaged, resulting in diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. diabetic neuropathy), heart disease, cancer and osteoporosis. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end-stage renal failure, and diabetic cataract and retinopathy cause blindness and eventually death.
모든 연령대에서 당뇨병보다는 당뇨합병증 환자 증가율이 높아졌다. 특히 40?50대의 경우 말초순환장애 당뇨합병증 환자 증가율이 당뇨 환자보다 6.5배, 당뇨성 망막병증은 2.2배 높게 보고되고 있다. 말초순환장애 당뇨합병증의 진료비는 2006년 807억원에서 2010년 1530억원으로, 당뇨병성 망막증은 327억원에서 505억원으로 54.4% 증가하였다(중앙일보 2011년 8월 29일자). 이는 평균 수명이 늘어나면서 당뇨병 병력이 오래된 환자가 늘고 있기 때문이다. 따라서, 당뇨병 환자가 당뇨합병증으로 발병되지 않도록 사전에 조치를 취하는 것이 중요하게 고려되고 있다.
In all age groups, diabetic complications increased more than diabetics. In particular, patients in their 40s and 50s reported 6.5 times higher rates of diabetic complications than diabetic patients and 2.2 times higher rates of diabetic retinopathy. The cost of diabetic complications of peripheral circulation increased from 80.7 billion won in 2006 to 1530 billion won in 2010, and diabetic retinopathy increased 54.4% from 32 billion won to 50.5 billion won (Jongang Ilbo, August 29, 2011). This is due to an increase in life expectancy, with an increasing number of older diabetic patients. Therefore, it is important to take measures in advance so that diabetics do not develop diabetic complications.
미국의 경우 25세에서 74세 연령대의 실명의 원인이 당뇨병이며, 당뇨 발병 후 15년이나 20년이 지나면 60%가 실명으로 이어진다. 따라서, 당뇨 환자에게 당뇨합병증이 발병하는 기간이 5년이나 10년 정도만 지연되더라도, 환자와 그 가족의 삶의 질이 달라질 것이며, 국가재정에도 커다란 영향을 끼칠 것이다.
In the United States, diabetes is the cause of blindness in the 25- to 74-year-old age group, and 60% of blindness after 15 or 20 years of diabetes develops blindness. Therefore, even if the delay in the development of diabetic complications in diabetic patients is only about 5 or 10 years, the quality of life of the patient and his family will be different and will have a major impact on national finances.
이러한 당뇨합병증을 유발하는 대표적인 인자 중의 하나로 폴리올 경로(polyol pathway)로 설명되고 있다. 폴리올 경로란 (1) 알도스나 케토스로부터 알도스 환원효소(AR)작용에 의해 환원되어 솔비톨을 형성하는 단계 및 (2) 솔비톨이 솔비톨 탈수소효소에 의해 산화되어 과당을 생성하는 단계로 이루어지는 과정이다. 정상상태에서는 알도스 환원효소가 포도당에 대하여 친화력이 매우 낮지만, 고혈당 상태에 의하여 폴리올 경로의 첫 번째 효소인 알도스 환원효소(AR)가 과도하게 활성화되어, 이로 인해 과도한 고혈당이 솔비톨과 과당으로 전환되어 조직에 축적되어 삼투압의 균형이 깨져 합병증이 유발된다. 즉, 증가된 삼투압으로 인하여 수분이 인입되어 당뇨성 망막병증(diabetic retinopathy), 당뇨성 백내장(diabetic cataract), 당뇨성 신경병증(diabetic neuropathy) 등으로 진행된다(당뇨병학, 김응진 외, 대한 당뇨병학회, 고려의학, 483쪽; Soulis-Liparota, T., et al., 1995, Diabetologia, 38: 357-394).
One of the representative factors causing the diabetic complications has been described as the polyol pathway (polyol pathway). The polyol pathway is a process consisting of (1) reduction by aldose reductase (AR) from aldose or ketose to form sorbitol and (2) sorbitol is oxidized by sorbitol dehydrogenase to produce fructose. . At steady state, aldose reductase has a very low affinity for glucose, but the hyperglycemic state causes excessive activation of aldose reductase (AR), the first enzyme in the polyol pathway, resulting in excessive hyperglycemia to sorbitol and fructose. It is converted and accumulated in tissues, causing the osmotic pressure to be unbalanced, causing complications. In other words, due to the increased osmotic pressure water is introduced into the diabetic retinopathy, diabetic cataract, diabetic neuropathy (Diabetes, Kim Eung-jin et al., Korean Diabetes Association) , Korea Medicine, p. 483; Soulis-Liparota, T., et al., 1995, Diabetologia, 38: 357-394).
이에 본 발명자는, 이상과 같은 당뇨합병증을 예방 또는 치료하기 위한 천연 약재를 연구하던 중, 송악 추출물 또는 이의 분획물이 알도즈 환원효소의 효능을 억제하고 고혈당으로 인한 제브라피쉬의 눈혈관의 비후를 예방 또는 치료함으로써, 당뇨합병증의 예방 또는 치료 효능이 있음을 확인하고 본 발명을 완성하였다.
Accordingly, the present inventors, while studying natural medicines for preventing or treating diabetic complications as described above, Songak extract or fractions thereof inhibit the efficacy of aldose reductase and prevent the thickening of zebrafish eye blood vessels due to hyperglycemia Or by treating, it was confirmed that the prevention or treatment efficacy of diabetic complications and completed the present invention.
본 발명은 당뇨합병증을 예방 또는 치료할 수 있는 송악 추출물, 또는 이의 분획물을 유효성분으로 포함하는 약학적 조성물 및 건강기능식품을 제공하기 위한 것이다.
The present invention is to provide a pharmaceutical composition and health functional food comprising Songak extract, or a fraction thereof, as an active ingredient, which can prevent or treat diabetic complications.
상기 과제를 해결하기 위하여, 본 발명은 송악 추출물, 또는 이의 분획물을 유효성분으로 포함하는 당뇨합병증 예방 또는 치료용 약학적 조성물을 제공한다.
In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications comprising Songak extract, or a fraction thereof as an active ingredient.
본 발명에서 사용되는 용어 "송악"은, 학명은 Hedra rohombea이며 한국에서는 제주도 등의 남부지방과 따뜻한 난류대를 따라 인천 앞바다와 울릉도 등에 분포한다.
The term " songak " as used herein, the scientific name is Hedra It is rohombea and is distributed in the sea off the coast of Incheon and Ulleungdo along the warm turbulent zone and southern part of Jeju Island.
본 발명에서 사용되는 용어 "추출물"은, 송악으로부터 액체의 용매를 사용하여 추출된 특정 성분을 의미한다. 상기 용매로는 물, 메탄올, 에탄올, 부탄올, 에틸아세에이트 또는 이들의 혼합물을 사용할 수 있다. 유효성분을 효율적으로 추출하기 위하여 메탄올, 에탄올 또는 이들과 물의 혼합물로 추출하는 것이 바람직하다. 메탄올과 에탄올의 농도는 10 내지 90%가 바람직하나 이에 제한되는 것은 아니다.
As used herein, the term "extract" means a specific component extracted from the songak using a solvent of a liquid. As the solvent, water, methanol, ethanol, butanol, ethyl acetate or a mixture thereof may be used. In order to efficiently extract the active ingredient, it is preferable to extract with methanol, ethanol or a mixture of these and water. The concentration of methanol and ethanol is preferably 10 to 90%, but is not limited thereto.
본 발명의 송악 추출물은, 송악의 가지, 잎 또는 뿌리를 추출한 것을 사용할 수 있다. 추출방식으로는 상온추출, 열수 추출, 냉침 추출, 환류 냉각 추출, 초음파 추출 및 증기 추출로 이루어진 군으로부터 선택된 어느 하나를 사용할 수 있다.
Songak extract of the present invention can be used to extract the branches, leaves or roots of Songak. Extraction method may be any one selected from the group consisting of room temperature extraction, hot water extraction, cold needle extraction, reflux cooling extraction, ultrasonic extraction and steam extraction.
또한, 본 발명에서 상기 송악 추출물로부터 분획할 수 있으며, 송악 에탄올 추출물을 물에 현탁시킨 후 에틸아세테이트로 분획하여 얻은 에틸아세테이트 분획물인 것이 바람직하다. 또한, 송악 에탄올 추출물을 물에 현탁시킨 후 에틸아세테이트로 분획하여 얻은 물 분획물을, 부탄올로 분획하여 얻은 부탄올 분획물 또는 물 분획물인 것이 바람직하다.
In addition, the present invention may be fractionated from the Songak extract, it is preferable that the ethyl acetate fraction obtained by suspending Songak ethanol extract in water and fractionated with ethyl acetate. In addition, the water fraction obtained by suspending Songak ethanol extract in water and fractionating with ethyl acetate is preferably a butanol fraction or a water fraction obtained by fractionation with butanol.
또한, 송악 에탄올 추출물을 물에 현탁시킨 후 부탄올로 분획하여 얻은 부탄올 분획물 또는 물 분획물인 것이 바람직하다.
Also, it is preferable that the Sok ethanol extract is a butanol fraction or water fraction obtained by suspending it in water and then fractionating it with butanol.
본 발명에서 사용되는 용어 "당뇨합병증"은, 당뇨병이 장기간 지속되는 경우 유발되는 증상을 의미한다. 당뇨합병증은, 당뇨병의 발병 기준 및 판단 기준과 상이하며, 당뇨합병증 치료제는 당뇨병 치료제와는 별개로 사용되고 있다.
The term "diabetic complication" used in the present invention means a symptom caused when the diabetes persists for a long period of time. Diabetic complications are different from the criteria for the onset and judgment of diabetes, and diabetic complications are used separately from diabetic agents.
당뇨합병증 유병의 원인은 알도즈 환원효소(aldose reductase)의 비정상적인 활성과 가속화된 산화성 스트레스에 의해 유발되므로, 당뇨합병증의 치료제로서의 개발시 알도즈 환원효소의 활성과 항산화 효능을 측정하여 치료제의 효능을 판단할 수 있다. 또한, 당뇨합병증으로 당뇨성 망막병증, 당뇨성 백내장, 신증, 신경병증, 당뇨병성 심장병 등이 야기되므로, 직접적으로 망막병, 백내장, 신증 및 신경병증 등을 억제하는 것으로 직접적인 치료제의 효능을 나타낸다.
The cause of diabetes complication is caused by abnormal activity of aldose reductase and accelerated oxidative stress. Therefore, the activity of aldose reductase and the antioxidative activity of aldose reductase are measured in the development of diabetic complication, It can be judged. In addition, since diabetic complications cause diabetic retinopathy, diabetic cataracts, nephropathy, neuropathy, diabetic heart disease, and the like, direct inhibition of retinopathy, cataracts, nephropathy and neuropathy shows the efficacy of a direct therapeutic agent.
본원발명은 당뇨합병증 예방 또는 치료 효과가 있으며 특히 상기 당뇨합병증은 당뇨성 망막병증, 당뇨성 백내장, 신증, 신경병증 또는 당뇨병성 심장병이다.
The present invention has the effect of preventing or treating diabetic complications. In particular, the diabetic complications are diabetic retinopathy, diabetic cataract, nephropathy, neuropathy or diabetic heart disease.
본 발명에서는 송악 추출물, 또는 이의 분획물이 당뇨합병증 예방 또는 치료에 효과가 있음을 확인하였다. 본 발명의 일실시예에 따르면, 당뇨합병증 치료의 지표가 되는 알도즈 환원효소의 활성 억제효능을 확인함으로서, 당뇨합병증의 예방 또는 치료 효능이 있음을 확인하였다.
In the present invention, it was confirmed that Songak extract, or a fraction thereof, is effective in preventing or treating diabetic complications. According to one embodiment of the present invention, it has been confirmed that the inhibitory effect of aldose reductase, which is an index of diabetic complication treatment, has an effect of preventing or treating diabetic complications.
본 발명에서 사용되는 용어 "예방"은, 상기 송악 추출물, 또는 이의 분획물을 포함하는 조성물의 투여로 질환을 억제 또는 지연시키는 모든 행위를 의미한다. 또한, 본 발명에서 사용되는 용어 "치료"는, 상기 송악 추출물, 또는 이의 분획물을 포함하는 조성물의 투여로 질환의 증세가 호전되거나 완치되는 모든 행위를 의미한다.
As used herein, the term "prevention" refers to any action that inhibits or delays a disease by administration of a composition comprising the Songak extract, or a fraction thereof. In addition, the term "treatment" as used in the present invention means all the actions that improve or cure the symptoms of the disease by administration of the composition comprising the Songak extract, or a fraction thereof.
본 발명의 조성물은 투여를 위하여, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀롤로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.
The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하나, 이에 한정되는 것은 아니다. 이러한 고형제제는 상기 송악 추출물, 또는 이의 분획물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.
The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the Songak extract, or a fraction thereof. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 상기 송악 추출물, 또는 이의 분획물의 일일 투여량은 바람직하게는 1 mg/kg 내지 500 mg/kg이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.
The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dosage of the Songak extract, or fractions thereof is preferably 1 mg / kg to 500 mg / kg, and may be administered once to several times daily if necessary.
또한, 본 발명은 송악 추출물, 또는 이의 분획물을 유효성분으로 포함하는 당뇨합병증 예방 또는 개선용 건강기능식품을 제공한다.
The present invention also provides a health functional food for preventing or improving diabetic complications comprising the Songak extract, or a fraction thereof as an active ingredient.
상기 건강기능식품은 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, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품은 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알콜 음료 및 비타민 복합제 중 어느 하나의 형태일 수 있다.
The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. In addition, the health functional food is any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcoholic beverage and vitamin complex Can be.
또한 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합여부는 다른 규정이 없는 한 식품의약품안정청에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.
In addition, the health functional food may further include food additives, and the suitability as a "food additive" is related to the relevant items according to the General Regulations and General Test Act of the Food Additives Code approved by the Food and Drug Administration unless otherwise specified. Judging by the standards and standards.
또한 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합여부는 다른 규정이 없는 한 식품의약품안정청에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.
In addition, the health functional food may further include food additives, and the suitability as a "food additive" is related to the relevant items according to the General Regulations and General Test Act of the Food Additives Code approved by the Food and Drug Administration unless otherwise specified. Judging by the standards and standards.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀롤로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합 제제류들을 들 수 있다.
Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulosic, high- , A sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent, and the like.
이때, 건강기능식품을 제조하는 과정에서 음료를 포함한 식품에 첨가되는 본 발명에 따른 추출물은 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100 중량%에 1 내지 15 중량% 포함되도록 첨가하는 것이 바람직하다.
At this time, the extract according to the present invention is added to the food containing the beverage in the process of manufacturing a health functional food can be appropriately added or subtracted as needed, preferably 1 to 15% by weight in 100% by weight food It is preferable to add.
본 발명은 당뇨합병증의 예방 또는 치료에 효과적인 천연 추출물로서, 당뇨합병증 예방 또는 치료 조성물로 약학적으로 이용 가능할 뿐 아니라 건강기능식품으로서도 유용하게 이용될 수 있다.
The present invention is a natural extract effective for the prevention or treatment of diabetic complications, as well as pharmacologically available as a preventive or therapeutic composition for diabetic complications can be usefully used as a health functional food.
도 1은, 본 발명의 일실시예에 따른 송악 추출물의 계통분획 단계를 나타낸 것이다.
도 2는, 본 발명의 일실시예에 따른 송악 에탄올 추출물의 제브라피쉬에서의 혈관두께 변화를 나타낸 것이다. 도 2a는 형광이미지를 나타낸 것이고, 도 2b는 혈관두께의 변화를 그래프로 나타낸 것이다.
도 3은, 본 발명의 일실시예에 따른 송악 분획물의 제브라피쉬에서의 혈관두께 변화를 나타낸 것이다. 도 3a는 형광이미지를 나타낸 것이고, 도 3b는 혈관두께의 변화를 그래프로 나타낸 것이다 Control은 정상군을 의미하고 HG는 고혈당 처리군을 의미하며 +3E-1는 송악 에칠아세테이트 분획 1 ug/ml 처리군을 의미하며 +3E-5는 송악 에칠아세테이트 분획 5 ug/ml 처리군을 의미하며 +3B-1은 송악 부탄올 분획 1 ug/ml 처리군을 의미하며 +3B-5는 송악 부탄올 분획 5 ug/ml 처리군을 의미하며 +3W-1는 송악 물 분획 1 ug/ml 처리군을 의미하며 +3W-5은 송악 물 분획 5 ug/ml 처리군을 의미한다.Figure 1 shows the phylogenetic fraction of Songak extract according to an embodiment of the present invention.
Figure 2 shows the change in blood vessel thickness in zebra fish of Songak ethanol extract according to an embodiment of the present invention. Figure 2a is a fluorescence image, Figure 2b is a graph showing the change in blood vessel thickness.
Figure 3 shows the blood vessel thickness change in the zebrafish of the Songak fraction according to an embodiment of the present invention. Figure 3a is a fluorescence image, Figure 3b is a graph showing the change in blood vessel thickness Control means a normal group, HG means a hyperglycemic treatment group + 3E-1 is Songak
이하, 하기 제조예 및 실시예에 의하여 본 발명을 더욱 상세하게 설명하고자 한다. 단, 하기 제조예 및 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to the following Production Examples and Examples. However, the following Preparation Examples and Examples are for illustrating the present invention, but the scope of the present invention is not limited thereto.
실시예Example : 송악 가지의 추출물, 및 이의 : Extract of Songak eggplant, and its 분획물Fraction 제조 Produce
충청남도 태안 외연도에서 채집 및 동정한 송악의 가지, 잎, 뿌리 전부로 이하의 추출물 및 분획물을 제조하였다.
The following extracts and fractions were prepared from all the branches, leaves, and roots of Songak, which were collected and identified in Taeyeon, Taean, Chungcheongnam-do.
1) 추출단계1) Extraction step
음건·세절한 송악의 전초를 분쇄한 후 1.0L 80% 에탄올을 넣고, 추출용기에서 상온상태로 48 시간동안 3시간 반복 추출한 후, 40℃에서 감압 하에 농축시켜 에탄올 추출물을 제조하였다.
After crushing the dry and fine grain of starch, 1.0L 80% ethanol was added, and extracted repeatedly for 3 hours at room temperature in an extraction vessel for 3 hours, and then concentrated under reduced pressure at 40 ℃ to prepare an ethanol extract.
2) 농축, 건조단계2) concentration, drying
상기 에탄올 추출액을 여과한 후 감압 상태에서 농축하였다. 이때, 구성성분의 분해 및 가수분해를 방지할 수 있도록 농축시 온도를 40℃ 내지 45℃로 유지하였다.
The ethanol extract was filtered and concentrated under reduced pressure. At this time, the temperature during the concentration was kept at 40 캜 to 45 캜 so as to prevent decomposition and hydrolysis of the constituents.
3) 계통분획 단계3) System fractionation stage
도 1에 도시된 바와 같이, 송악의 분획물을 제조하였다. 먼저, 에탄올 추출물에 500㎖의 물과 에틸아세테이트 500㎖를 넣고, 분액깔때기를 이용하여 물층과 에틸아세테이트층을 분리하였다. 그 후 에틸아세테이트 분획물을 감압농축시켜 에틸아세테이트 분획물을 수득하였다.
As shown in FIG. 1, a fraction of Songak was prepared. First, 500 ml of water and 500 ml of ethyl acetate were added to the ethanol extract, and the water layer and the ethyl acetate layer were separated using a separatory funnel. Thereafter, the ethyl acetate fractions were concentrated under reduced pressure to obtain an ethyl acetate fraction.
또한, 상기 물층은 다시 n-부탄올 500 ㎖를 넣고, 분액깔때기를 이용하여 물층과 부탄올층을 분획하였다. 그 후 부탄올 분획물과 물분획물을 각각 감압농축시켜 n-부탄올 분획물과 물분획물을 각각 수득하였다.
In addition, 500 ml of n-butanol was added to the water layer, and the water layer and the butanol layer were partitioned using a separatory funnel. Thereafter, the butanol fraction and the water fraction were concentrated under reduced pressure, respectively, to obtain n-butanol fraction and the water fraction, respectively.
실험예Experimental Example 1: One: 알도즈Aldoz 환원효소( Reductase ( aldosealdose reductasereductase ) 활성 억제 효능 분석Activity Inhibition Effect Analysis
상기 실시예 1에서 제조한 송악 추출물과 이의 계통분획물들을 시험관내에서 알도즈 환원효소(aldose reductase) 활성 억제 효능을 분석 하기 위해서 하기와 같이 분석하였다.
Songak extract prepared in Example 1 and its lineage fractions were analyzed as follows to analyze the effect of inhibiting aldose reductase activity in vitro.
1) 실험방법1) Experimental method
듀프란(Dufrane)(1984) 방법으로 SD 랫트(Sprague-Dawley rat, 250-280g)의 안구로부터 천연상태의 알도즈 환원효소(aldose reductase)를 얻기 위하여, 135 mM Na, K-인산 완충제(Na, K-phosphate buffer) (pH 7.0)와 10 mM 2-머캡토에탄올(2-mercaptoethanol)을 적출한 렌즈와 함께 균질기(Homogenizer)와 초음파분쇄기(Sonicater)를 이용해 분쇄하였다. 14000 rpm에서 30분간 원심 분리한 다음 상층액을 0.2 um의 거름기에 여과 후 실험에 사용하였다.
In order to obtain natural aldose reductase from the eyes of Sprague-Dawley rats (250-280 g) by the method of Dufrane (1984), 135 mM Na, K-phosphate buffer (Na , K-phosphate buffer (pH 7.0) and 10 mM 2-mercaptoethanol were extracted using a homogenizer and a sonicator together with the extracted lens. After centrifugation at 14000 rpm for 30 minutes, the supernatant was filtered through a 0.2 um filter and used for the experiment.
위 과정은 모두 4℃에서 수행하였다. 효소(Enzyme)의 단백질원으로 BSA을 이용하여 라우리(lowry) 방법으로 정량하였다. 135 mM Na, K-인산 완충제(Na, K-phosphate buffer)(pH 7.0), 100 mM 황산 리튬(Lithium sulfate), 0.03 mM NADPH, 0.04 mM DL-글리세알데하이드(DL-glycealdehyde) 와 100 ug/ml 효소(enzyme)의 혼합물을 0.1% DMSO에 녹인 후 각 농도로 희석한 시료 50 ul에 첨가하여 전체 부피가 1 ml가 되도록 한 뒤 37℃에서 10분간 반응시켰다. 이때 BLK는 0.04 mM DL-글리세알데하이드(DL-glycealdehyde)를 첨가하지 않은 혼합물을, STD는 135 mM Na, K-인산 완충제(Na, K-phosphate buffer)(pH 7.0), 100 mM 황산 리튬(Lithium sulfate)에 50 ul NADP (0.2-5 uM)를 첨가한 것을 사용하였다.
All of the above process was carried out at 4 ℃. Enzyme protein source was quantified by a lowry method using BSA. 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM lithium sulfate, 0.03 mM NADPH, 0.04 mM DL-glyceraldehyde and 100 ug / ml The mixture of enzymes was dissolved in 0.1% DMSO and added to 50 ul of the sample diluted to each concentration to a total volume of 1 ml, followed by reaction at 37 ° C. for 10 minutes. At this time, BLK is a mixture without addition of 0.04 mM DL-glycerycealdehyde, STD is 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM lithium sulfate (Lithium). sulfate to 50 ul NADP (0.2-5 uM) was used.
시료(Sample) 0.3 ml의 0.5 N HCl을 첨가하여 반응을 종료시킨 뒤, 10 mM imidazole이 첨가된 6 M NaOH 1 ㎖을 가하여 60℃에서 10분간 반응시켜 NADP가 fluorescent product로 전환되는 정도를 측정하였다. 시료는 3회(triplicate) 수행하였다. 효능 정도는 Spectrofluorophotometric detector (Bio-TEK, Synergy HT, USA)를 이용하여 Ex. 360 nm, Em. 460 nm에서 측정하여, IC50 으로 나타내었다. After completion of the reaction by adding 0.3 ml of 0.5 N HCl, 1 ml of 6 M NaOH added with 10 mM imidazole was added and reacted at 60 ° C. for 10 minutes to measure the degree of NADP conversion into fluorescent product. . Samples were triplicated. Efficacy was measured by using a spectrofluorophotometric detector (Bio-TEK, Synergy HT, USA). 360 nm, Em. Measured at 460 nm, indicated as IC50.
송악 추출물은 1.0 ug/ml에서 25 ug/ml 농도 사이로 조제하였다.
Songak extract was prepared at a concentration of 1.0 ug / ml to 25 ug / ml.
2) 실험결과2) Experimental results
송악 추출물 및 이의 분획물들을 상기 실험방법에 따라 시험관 내에서 알도즈 환원효소 활성 억제 효능을 측정하였으며, 그 결과는 하기 표 1에 나타낸 바와 같다.
Songak extract and its fractions were measured in vitro aldose reductase activity inhibitory effect in accordance with the experimental method, the results are shown in Table 1 below.
사용농도(ug/ml)
Concentration (ug / ml)
Inhibition(%)
Inhibition (%)
IC 50(ug/ml)
IC 50 (ug / ml)
2.5
5.01.0
2.5
5.0
36.81±0.33
57.90±1.6217.02 ± 1.31
36.81 ± 0.33
57.90 ± 1.62
1
2.50.5
One
2.5
34.69±2.09
59.91±1.218.55 ± 0.39
34.69 ± 2.09
59.91 ± 1.2
2.5
5One
2.5
5
36.17±1.70
53.29±0.3416.79 ± 0.50
36.17 ± 1.70
53.29 ± 0.34
255
25
67.30±0.8625.00 ± 2.76
67.30 ± 0.86
상기 실험을 통해 송악 추출물 및 이의 분획물이 알도즈 환원효소의 활성을 억제한다는 사실을 확인하였다.
The experiment confirmed that Songak extract and its fractions inhibit the activity of aldose reductase.
실험예Experimental Example 2: 송악 추출물의 2: of Songak extract inin vivovivo 시스템에서 On the system 항당뇨Anti-diabetic 합병증 효능 실험 Complication efficacy experiment
제브라피쉬(Zebrafish)는 척추동물 중의 하나이며 실험기간이 상대적으로 짧으며, 쥐나 마우스 등의 설치류에 비해 가격이 비교적 낮은 장점으로 최신에 동물 실험으로 각광을 받고 있는 in vivo 시스템 모델이다(Disease models & Mechanisms, 3, 236-245(2010); Journal of Molecular Endocrinology (2007) 38, 433-440). 이하에서, 제브라피쉬를 이용하여 송악 추출물의 in vivo 시스템에서 항당뇨 합병증 효능을 측정하였다.
Zebrafish is one of the vertebrates, has a relatively short duration of experiments, and is relatively inexpensive compared to rodents such as mice and mice. Mechanisms, 3, 236-245 (2010); Journal of Molecular Endocrinology (2007) 38, 433-440). Hereinafter, the anti-diabetic complication effect was measured in the in vivo system of Songak extract using zebrafish.
1) 실험방법1) Experimental method
① 제브라피쉬 발생배 준비① Preparation of zebrafish breeding ship
혈관내피세포에 특이적으로 형광단백질(green fluorescence protein)이 발현하는 형질전환 제브라피쉬(Tg(kdr:EGFP)) 암수를 교배하여 발생배(embryo)를 획득하였다.
Embryos were obtained by crossing male and female transgenic zebrafish (Tg (kdr: EGFP)) expressing a fluorescent protein (green fluorescence protein) specifically in vascular endothelial cells.
② 고혈당 유도 및 약물처리② Hyperglycemia induction and drug treatment
수정 후 24시간이 지난 시점에서 형광을 발현하는 발생배를 선별하여 24 well plate에 5개체씩 분주한 후 30 mM glucose를 이용하여 고혈당을 유도하였다. 이때, 확인하고자 하는 약물(송악 추출물, 또는 이의 분획물)을 glucose 용액에 같이 희석하여 처리하였다. 약물 처리 후 3일째 새로운 용액으로 교체하였다.
After 24 hours after fertilization, the embryos expressing fluorescence were selected, divided into 5 wells in 24 well plates, and hyperglycemia was induced using 30 mM glucose. At this time, the drug (songak extract, or fractions thereof) to be identified was treated by diluting it together in a glucose solution. Three days after drug treatment, the new solution was replaced.
③ 유리체 혈관 변화 분석③ Analysis of vitreous blood vessel change
고혈당 조건에서 5일간 처리한 후, 4% 파라포름알데하이드(paraformaldehyde)를 이용하여 하루 동안 고정하였다. 고정된 개체에서 수정체를 분리하여 형광실체현미경 하에서 유리체혈관(hyaloid vasculature)의 변화를 분석하였다.
After treatment for 5 days in hyperglycemic conditions, it was fixed for 1 day using 4% paraformaldehyde (paraformaldehyde). Lenses were isolated from fixed subjects and analyzed for changes in hyoidoid vasculature under fluorescence stereomicroscopy.
2) 실험결과2) Experimental results
① 송악 에탄올 추출물의 효능① Efficacy of Songak Ethanol Extract
송악 에탄올 추출물을 처리한 결과를 도 2에 나타내었다. 도 2에 나타난 바와 같이, 30 mM 글루코오스(glucose) 용액에서 5일간 배양한 고혈당군(HG)의 유리체 혈관은 대조군(Control)에 비해 유의적으로 확장되었다. 글루코오스와 송악추출물을 동시에 처리한 실험군(+1 ug/ml, +5 ug/ml)에서는 고혈당에 의한 혈관 확장이 유의성 있게 억제되었다. 특히 송악 추출물 5 ug/ml 투여군은 정상군의 혈관과 거의 비슷한 건강상태로 예방 또는 치료되었음을 확인하였다.
The result of treating Songak ethanol extract is shown in FIG. As shown in FIG. 2, the vitreous vessels of the hyperglycemic group (HG) cultured in 30 mM glucose solution for 5 days were significantly expanded compared to the control group. Hyperglycemia was significantly inhibited in the experimental group (+1 ug / ml, +5 ug / ml) treated with glucose and pineal extract at the same time. In particular, 5 ug / ml administration of Songak extract was confirmed to be prevented or treated with a state of health almost similar to the blood vessels of the normal group.
② 송악 추출물의 분획물의 효능② Efficacy of fractions of Songak extract
송악 추출물로부터 계통분획한 에틸아세테이트, 부탄올 및 물 분획물들을 처리한 결과를 도 3에 나타내었다. 도 3에 나타난 바와 같이, 물 분획물을 제외한 에틸아세테이트와 부탄올 분획물(Control: 정상군, HG: 고혈당 처리군, +3E-1: 송악 에칠아세테이트 분획 1 ug/ml 처리군, +3E-5: 송악 에칠아세테이트 분획 5 ug/ml 처리군, +3B-1: 송악 부탄올 분획 1 ug/ml 처리군, +3B-5: 송악 부탄올 분획 5 ug/ml 처리군, +3W-1: 송악 물 분획 1 ug/ml 처리군, +3W-5: 송악 물 분획 5 ug/ml 처리군)이 고혈당으로 유도된 망막혈관의 확장을 유의성 있게 억제함을 입증하였다. 특히 에틸아세테이트 분획물 5 ug/ml 처리군은 거의 정상 수준으로까지 회복(예방)되었다.
The result of treating ethyl acetate, butanol and water fractions lineage fractions from the Songak extract is shown in FIG. As shown in FIG. 3, the ethyl acetate and butanol fractions except for the water fraction (Control: normal group, HG: hyperglycemic treatment group, + 3E-1: Songak ethyl acetate
Claims (9)
Pharmaceutical composition for the prevention or treatment of diabetic complications comprising the Songak extract, or fractions thereof as an active ingredient.
According to claim 1, wherein the Songak extract is a pharmaceutical composition, characterized in that extracted from the branches, leaves or roots of Songak.
The pharmaceutical composition of claim 1, wherein the Songak extract is extracted with water, methanol, ethanol, butanol, ethyl acetate, or a mixture thereof.
The pharmaceutical composition of claim 1, wherein the extraction is at room temperature extraction, hot water extraction, cold needle extraction, reflux cooling extraction, ultrasonic extraction or steam extraction.
The pharmaceutical composition according to claim 1, wherein the fraction is an ethyl acetate fraction obtained by suspending Songak ethanol extract in water and fractionating with ethyl acetate.
The pharmaceutical composition according to claim 1, wherein the fraction is a butanol fraction or a water fraction obtained by dividing Songak ethanol extract in water and fractionating with ethyl acetate, butanol fraction.
The pharmaceutical composition according to claim 1, wherein the fraction is a butanol fraction or a water fraction obtained by suspending Songak ethanol extract in water and fractionating it with butanol.
The pharmaceutical composition according to claim 1, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, nephropathy, neuropathy or diabetic heart disease.
Health functional food for preventing or improving diabetic complications comprising Songak extract, or fractions thereof as an active ingredient.
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