KR20200120884A - A Pharmaceutical Composition For Blocking AGEs Production And Promoting AGEs Decomposition Comprising Extracts Of Juglans Mandshurica Maxim., Its Fractions, Or A Physiologically Active Compound Derived Therefrom As Active Ingredient - Google Patents
A Pharmaceutical Composition For Blocking AGEs Production And Promoting AGEs Decomposition Comprising Extracts Of Juglans Mandshurica Maxim., Its Fractions, Or A Physiologically Active Compound Derived Therefrom As Active Ingredient Download PDFInfo
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Abstract
Description
본 발명은 가래나무 추출물, 이의 분획물 또는 그 유래 생리활성물질을 유효성분으로 함유하는 최종당화산물 생성 억제 및 분해 효과를 가지는 약학 조성물에 관한 것이다. 구체적으로, 본 발명은 가래나무 추출물, 이의 분획물 또는 그 유래 생리활성물질을 포함하는, 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition having an effect of inhibiting and decomposing a final saccharified product containing a sputum extract, a fraction thereof, or a physiologically active substance derived therefrom as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including sputum extract, fractions thereof, or physiologically active substances derived therefrom. will be.
당뇨병은 유전적 또는 환경적 요인에 의해서 인슐린의 분비량이 부족하거나 정상적인 기능이 이루어지지 않아 혈중 포도당의 농도가 높아져, 소변으로 포도당을 배출하는 만성질환이다. 당뇨병에 의해 체내 혈당이 높아진 상태가 장기간 지속됨에 따라, 당화 산물이 망막, 신장, 신경 또는 전신의 크고 작은 혈관들을 침범하면서 만성 합병증이 발병하게 된다. 당뇨병은 그 자체보다 당뇨 합병증이 더 위험하기 때문에, 오늘날 당뇨병 치료에 있어서 가장 큰 목표는 당뇨 합병증의 유발이나 진행을 억제하는 데에 있다. 대표적인 당뇨 합병증으로는 망막변증, 신증, 신경증 등이 있다. 당뇨병은, 상기 당뇨 합병증을 포함하여, 현재 우리나라 사망원인 중 네 번째로 높은 것으로 알려져 있을 만큼 현대에 접어들면서 일반적으로 발생하는 심각한 만성 질병이다.Diabetes is a chronic disease in which the amount of insulin secretion is insufficient due to genetic or environmental factors, or the concentration of glucose in the blood is high because the normal function is not performed, and glucose is discharged into the urine. As the state of elevated blood sugar in the body continues for a long time due to diabetes, the glycated product invades large and small blood vessels in the retina, kidneys, nerves, or whole body, leading to chronic complications. Because diabetes is more risky for diabetic complications than itself, the primary goal of diabetes treatment today is to suppress the initiation or progression of diabetic complications. Typical diabetes complications include retinopathy, nephropathy, and neurosis. Diabetes, including the complications of diabetes, is a serious chronic disease that generally occurs in the modern era as it is known to be the fourth highest cause of death in Korea.
이러한 당뇨 합병증을 유발하는 기전으로는 크게 단백질의 비효소적 당화반응(non-enzymatic glycation of protein)과 폴리올 경로(polyol pathway)의 기작 변화에 의한 삼투압 스트레스 및 자유라디칼에 의한 산화적 스트레스(oxidative stress) 등으로 설명되고 있다.The mechanisms that induce these diabetes complications are largely osmotic stress due to non-enzymatic glycation of protein and changes in the mechanism of the polyol pathway, and oxidative stress due to free radicals. ), etc.
단백질의 당화는 단백질과 당이 반응해서 먼저 쉬프 베이스(schiff base)를 형성하고, 반응이 더 진행되면 아마도리 생성물(amadori product)이 생성되며 계속해서 응축, 탈수 과정을 거치면 최종당화산물(advanced glycation end product, AGEs)을 형성한다. 이 반응은 반응 시작 단계에서 에너지 공급 없이 거의 자연발생적으로 일어나므로 식품이나 우리의 신체 내에서 일어나며, 일정단계 이후 비가역적인 특징을 가진다. 따라서 최종당화산물은 일단 생성되면 혈당이 정상으로 회복되어도 분해되지 않고, 단백질 생존 기간 동안 조직에 축적되어 조직의 구조와 기능을 비정상적으로 변화시킨다. 이처럼 비효소적 단백질 당화반응에 의하여 기저막, 혈장 알부민, 수정체 단백질, 피브린, 콜라겐 등의 단백질에서 당화가 일어나며, 생성된 최종당화산물(AGEs)은 조직의 구조와 기능을 비정상적으로 변화시켜 만성 당뇨 합병증을 유발시킨다. 이와 같이 폴리올 경로, 비효소적 당화 반응 및 산화적 스트레스 작용 기전들이 서로 연관되어 당뇨 합병증을 유발하므로, 당뇨 합병증의 발병을 지연시키거나 예방 또는 치료하기 위해서는 최종당화산물의 생성 억제 또는 분해 효과를 가지는 것이 중요하다.In the glycosylation of proteins, proteins and sugars react to form a schiff base first, and as the reaction proceeds further, amadori product is generated.Continuing through condensation and dehydration, the final glycosylation product (advanced glycation) is performed. end product, AGEs). Since this reaction occurs almost spontaneously without supplying energy at the beginning of the reaction, it occurs in food or in our body, and has an irreversible characteristic after a certain stage. Therefore, once the final glycated product is produced, it is not degraded even if blood sugar is restored to normal, but accumulates in the tissue during the protein survival period, causing abnormal changes in the structure and function of the tissue. In this way, by the non-enzymatic protein saccharification reaction, saccharification occurs in proteins such as basement membrane, plasma albumin, lens protein, fibrin, and collagen, and the resulting final glycated products (AGEs) abnormally change the structure and function of tissues, resulting in complications of chronic diabetes. Causes. In this way, the polyol pathway, the non-enzymatic saccharification reaction and the oxidative stress mechanisms are related to each other to cause diabetic complications, so in order to delay the onset of, prevent or treat the onset of diabetic complications, It is important.
최종당화산물은 당뇨 합병증 외에도 비알콜성 지방간염(non-alcoholic steatohepatitis, NASH) 질환과 관련이 있다고 최근 밝혀졌다. 비알콜성 지방간염의 발병 기전은 완전히 밝혀지지는 않았지만, 적어도 인슐린 저항성과 밀접한 관련이 있는 것으로 널리 받아들여지고 있다. 유전적 요인과 더불어 식이 및 운동 등 생활습관과 관련된 환경적 요인의 복합 작용으로 인슐린 저항이 증가하면, 간에서 과도한 유리 지방산(free fatty acid, FAA)이 생성된다. 유리 지방산은 간세포 안에서 독성이 없는 중성 지방(triglyceride, TG)으로 전환되어 일차적으로 단순 지방증의 상태가 된다(비특허문헌 1 및 2). 다양한 산화적 스트레스가 추가되면서 지방 과산화와 염증 사이토카인의 과생성으로 간세포 손상 및 염증반응이 일어나 비알콜성 지방간염으로 발전한다는 것으로 알려져 있다. 흥미롭게도 비알콜성 지방간염은 또 다른 인슐린 저항성 관련 질환인 제2형 당뇨병을 흔히 동반하는데, 이러한 사실은 당뇨 합병증의 주요 원인 물질로 알려진 최종당화산물과 비알콜성 지방간염의 연관성을 제안한다. 특히 글리세라알데하이드(glyceraldehyde, GA) 유래 최종당화산물(GA-AGEs)은 체내에서 생성되는 최종당화산물 중 가장 독성이 높아, TAGEs(toxic AGEs)라고 불리는데, 최근 TAGEs가 비알콜성 지방간염 환자의 혈청 및 간 조직에서 고농도로 존재한다고 밝혀졌다. 글리세라알데하이드뿐만 아니라 글리옥살 (glyoxal, GO), 메틸글리옥살 (methylglyoxal, MGO)과 같은 디카보닐 (dicarbonyl) 화합물로 유도된 최종당화산물의 경우도 비알콜성 지방간염을 촉진할 것이다.The final glycated product was recently found to be associated with non-alcoholic steatohepatitis (NASH) disease in addition to complications of diabetes. Although the pathogenesis of nonalcoholic steatohepatitis has not been fully elucidated, it is widely accepted that at least it is closely related to insulin resistance. When insulin resistance increases due to a combination of genetic factors and environmental factors related to lifestyle, such as diet and exercise, excess free fatty acid (FAA) is produced in the liver. Free fatty acids are converted into non-toxic triglycerides (TG) in hepatocytes, and are primarily in a state of simple steatosis (
최종당화산물은 또한 노인이나 노화된 동물의 뇌에서 증가되는 것으로 보고되어 있으며, 신경세포의 사멸을 촉진하여 알츠하이머병 등의 퇴행성 뇌질환에 영향을 미칠 수 있을 것이라고 알려져 있다(비특허문헌 3 및 4).The final glycated product is also reported to be increased in the brain of the elderly or aged animals, and it is known that it can affect degenerative brain diseases such as Alzheimer's disease by promoting the death of nerve cells (Non-Patent Documents 3 and 4 ).
당뇨 합병증, 비알콜성 지방간염 등과 관련된 최종당화산물 저해제들이 합성되고 있지만, 장기간 투여 시 독성을 나타낸다. 예를 들어, 합성 의약품인 아미노구아니딘(aminoguanidine)은 친핵성 히드라진(hydrazine)으로 축합반응의 산물과 결합하여 최종당화산물의 생성을 억제하여 당뇨 합병증으로 진전되는 것을 방지한다. 이는 당뇨 합병증의 예방 및 치료에 가장 유망한 의약품으로 제 3상 임상시험까지 진행되었으나, 장기간 투여 시 독성이 유발되는 문제점이 있어 더욱 안전한 약제의 개발이 필요한 실정이다. 나아가 비알콜성 지방간염은 간이식 외에는 뚜렷한 치료 방법이 없으므로 이러한 문제점을 해결할 수 있는 보다 안전한 새로운 치료법에 대한 개발이 요구되고 있다.Final glycated product inhibitors related to diabetes complications and non-alcoholic steatohepatitis have been synthesized, but show toxicity when administered for a long time. For example, aminoguanidine, a synthetic drug, is a nucleophilic hydrazine that binds to the product of the condensation reaction and inhibits the production of the final glycated product, thereby preventing the development of diabetes complications. This is the most promising drug for the prevention and treatment of diabetic complications, and has been conducted up to phase 3 clinical trials. However, there is a problem that toxicity is induced when administered for a long period of time, so the development of a safer drug is required. Furthermore, since there is no clear treatment method for non-alcoholic steatohepatitis other than liver transplantation, there is a need to develop a safer new treatment that can solve this problem.
가래나무 (Juglans mandshurica Maxim.)는 가래나무과 (Juglandaceae) 또는 호두나무과로 분류되며 나무 또는 관목 형태로 존재하며 아메리카, 유라시아 또는 남아시아에 원산지로 두고 있다. 이는 중국 원산의 호두나무와 비슷하며 한국 중부 이북 또는 중국 북동부 등지에서 주로 서식하며 약용식물, 한방 약재로 사용되며 추피라고 불리는 것은 한방에서 봄, 가을 사이에 가래나무의 수피를 채취하여 말린 것을 칭하며 수렴, 해열 및 눈을 맑게 하는 등의 효능이 있으며 장염, 설사, 맥립종 및 눈이 충혈 되고 붓는 통증 등에 처방하며 피부질환, 암, 위염 등의 치료에도 이용되고 가래나무의 열매 (가래; 추자)는 9~10월에 익은 것을 약용이나 식용으로 이용하며 열매의 씨는 먹거나 약재로 쓰인다. Juglans mandshurica Maxim. is classified as Juglandaceae or walnut family, and exists in the form of a tree or shrub, and is native to the Americas, Eurasia or South Asia. It is similar to a walnut tree native to China. It is mainly inhabited in the northeastern part of Korea or northeastern China. It is used as a medicinal plant and herbal medicinal material. The one called Chupi refers to collecting and drying the bark of a sputum tree between spring and autumn in oriental medicine. , Fever and clearing eyes. It is prescribed for enteritis, diarrhea, alleles, redness of the eyes, and swelling pain. It is also used in the treatment of skin diseases, cancer, gastritis, etc., and the fruit of the sputum tree (sputum; chuja) is 9 What is ripe in October is used for medicinal or edible use, and the seeds of the fruit are eaten or used as medicinal materials.
중국 및 북한에서는 가래나무 껍질과 열매를 암 치료제로 이용하며 만성장염, 이질, 간염, 간경화증, 요통, 신경통 및 각종 피부병에 사용되고 있다. 민간요법으로는 위염 (십이지장궤양; 열매를 짓찧어 즙을 내어 먹음), 무좀 (가래나무 가지를 물로 달여 식힌 후 발을 담금), 급성 장염 (가래나무 잎 및 가지를 채취 후 잘라 물로 달여 공복에 복용) 등에 이용되고 있다.In China and North Korea, sputum bark and fruit are used as cancer treatment and are used for chronic enteritis, dysentery, hepatitis, cirrhosis, low back pain, neuralgia and various skin diseases. Home remedies include gastritis (duodenal ulcer; crushed fruit and eaten with juice), athlete's foot (decoction of sputum branches with water and soaking feet), acute enteritis (severe leaves and branches are cut off with water and decoated with water. Taking), etc.
가래나무의 효능으로 보고된 문헌 중에는 대한민국등록특허 제10-1292837호(특허문헌 1)에서는 가래나무 잎의 추출물로부터 활성 화합물을 분리하고, 이의 피부 주름 개선 용도를 개시하고 있다. 이 외에도, 가래나무 수피의 추출물로부터 분리된 활성 화합물의 HIV-1 reverse transcriptase 및 Rnase 활성 억제 효과에 대하여 공지된 바 있다(비특허문헌 5). 가래나무 추출물의 항비만 효과와 관련하여, 가래나무의 물 추출물이 췌장 지방분해 효소의 활성 조절을 통해 항-비만 효과를 나타낼 수 있음이 공지되어 있다(비특허문헌 6). 하지만 가래나무의 최종당화산물의 생성 억제 또는 분해 효과, 특히, 가래나무 추출물뿐만 아니라 이로부터 분리되는 신규 화합물이 다양한 생리 활성, 특히 항-당뇨 합병증, 항-비알콜성 지방간염, 퇴행성 뇌질환에 활성을 나타낼 수 있을 것이라는 점에 대해서는 보고된 바 없다.Among the documents reported as the efficacy of sputum, Republic of Korea Patent No. 10-1292837 (Patent Document 1) discloses the use of separating the active compound from the extract of sputum leaves and improving skin wrinkles thereof. In addition, it has been known about the inhibitory effect of HIV-1 reverse transcriptase and Rnase activity of the active compound isolated from the extract of Sputum bark (Non-Patent Document 5). Regarding the anti-obesity effect of the sputum tree extract, it is known that the water extract of sputum tree can exhibit an anti-obesity effect through the regulation of the activity of pancreatic lipolytic enzymes (Non-Patent Document 6). However, the effect of inhibiting or decomposing the production of final glycated products of sputum sputum, in particular, sputum extract as well as novel compounds isolated therefrom have various physiological activities, especially anti-diabetic complications, anti-alcoholic steatohepatitis, degenerative brain diseases It has not been reported that it could be active.
본 발명의 목적은 최종당화산물의 생성을 억제하거나, 또는 생성된 최종당화산물을 분해함으로써, 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환을 예방 또는 치료하기 위한 약학 조성물 또는 상기 질환들을 개선 또는 예방하기 위한 건강기능식품 조성물을 제공하는 것이다.An object of the present invention is a pharmaceutical composition for preventing or treating diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases by inhibiting the production of final glycated products or decomposing the resulting final glycated products Or to provide a health functional food composition for improving or preventing the above diseases.
상기 목적을 달성하기 위하여, 본 발명은 가래나무 추출물, 이의 분획물, 또는 상기 분획물로부터 분리된 화합물을 포함하는 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention is for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including a sputum extract, a fraction thereof, or a compound isolated from the fraction Provides a pharmaceutical composition.
또한, 본 발명은 가래나무 추출물, 이의 분획물, 또는 상기 분획물로부터 분리된 화합물을 포함하는 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 개선 또는 예방용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract, a fraction thereof, or a compound isolated from the fraction. to provide.
일 실시 태양에서, 상기 화합물은 하기 화학식 1 내지 10 중 어느 하나로 표시되는 것일 수 있다.In one embodiment, the compound may be represented by any one of the following
본 발명에 따른 가래나무 추출물, 이의 분획물, 또는 상기 분획물로부터 분리된 화합물은 최종당화산물(AGEs)의 생성을 억제하고, 생성된 최종당화산물을 분해하는 효과를 가지므로, 최종당화산물(AGEs)에 의해 유발되는 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증 또는 퇴행성 뇌질환을 개선, 예방 또는 치료하는데 유용하게 사용될 수 있다.Phalaenopsis extract according to the present invention, a fraction thereof, or a compound isolated from the fraction inhibits the production of final saccharified products (AGEs) and has the effect of decomposing the final saccharified products, thus final saccharified products (AGEs) Diabetic complications caused by, non-alcoholic steatohepatitis, inflammatory disease, fibrosis or degenerative brain disease can be usefully used in improving, preventing or treating.
도 1 및 2는 가래나무 추출물과 이의 분획물의 최종당화산물(AGEs) 생성 억제 효과를 나타낸 것이다.
도 3 및 4는 가래나무 추출물, 이의 분획물, 및 분획물로부터 분리된 화합물의 최종당화산물(AGEs) 분해 효과를 나타낸 것이다.
도 5는 가래나무 추출물, 이의 분획물의 NO 농도를 대식세포에서 측정한 결과를 나타내는 그래프이다.
도 6a 및 6b는 가래나무 추출물, 이의 분획물의 세포 생존율을 대식세포에서 측정한 결과를 나타내는 그래프이다.
도 7a 및 7b는 IL-6 및 TNF-α생성량을 나타내는 그래프이다.
도 8은 가래나무 추출물, 이의 분획물의 NO 농도를 미세교세포에서 측정한 결과를 나타내는 그래프이다.
도 9a 및 9b는 가래나무 추출물, 이의 분획물의 혈관내피세포 보호 효능을 나타내는 그래프이다.
도 10은 가래나무 추출물, 이의 분획물의 α-SMA 발현 정도를 나타내는 그림 및 그래프이다.
도 11는 가래나무 추출물, 이의 분획물의 BDNF 발현 정도를 나타내는 그림 및 그래프이다.
도 12a 및 12b는 가래나무 추출물, 이의 분획물의 신경모세포 보호 효능을 나타내는 그래프이다.Figures 1 and 2 show the effect of inhibiting the production of final saccharified products (AGEs) of the sputum extract and its fractions.
3 and 4 show the effect of decomposing sputum extract, its fractions, and the final saccharified products (AGEs) of the compounds isolated from the fractions.
Figure 5 is a graph showing the result of measuring the NO concentration of the sputum extract, a fraction thereof in macrophages.
6A and 6B are graphs showing the results of measuring cell viability of sputum extract and fractions thereof in macrophages.
7A and 7B are graphs showing the amount of IL-6 and TNF-α production.
Figure 8 is a graph showing the result of measuring the NO concentration of the sputum extract, a fraction thereof in microglia.
9A and 9B are graphs showing the vascular endothelial cell protective efficacy of a sputum extract and a fraction thereof.
Figure 10 is a picture and graph showing the expression degree of α-SMA of a sputum extract, a fraction thereof.
11 is a diagram and a graph showing the expression level of BDNF of a sputum extract, a fraction thereof.
12a and 12b are graphs showing the neuroblastic protective efficacy of a sputum extract, a fraction thereof.
일 실시 태양에서, 본 발명은 가래나무 추출물 또는 이의 분획물을 포함하는 당뇨 합병증, 비알콜성 지방간염(non-alcholic steatohepatitis, NASH), 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다.In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcholic steatohepatitis (NASH), inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract or a fraction thereof Provides.
본원에서 가래나무는 수피, 뿌리, 잎, 꽃 또는 열매를 모두 사용할 수 있으며, 구체적으로 열매인 것이 보다 바람직하다. 또한, 상기 가래나무는 재배한 것 또는 시판되는 것 등 제한 없이 모두 사용할 수 있다.In the present application, the bark, root, leaf, flower, or fruit can be used as the sputum tree, and it is more preferable that it is a fruit. In addition, the sputum tree can be used without limitation, such as cultivated or commercially available ones.
상기 추출물은 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합물을 용매로 하여 추출하는 것이 바람직하며, 상기 저급 알코올은 바람직하게 에탄올, 메탄올 또는 부탄올일 수 있으나, 이에 제한되지 않는다.The extract is preferably extracted using water, a C1 to C4 lower alcohol or a mixture thereof as a solvent, and the lower alcohol may preferably be ethanol, methanol or butanol, but is not limited thereto.
상기 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조될 수 있으나, 이에 한정되지 않는다:The extract may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:
1) 가래나무에 추출용매를 가하여 추출하는 단계;1) extracting by adding an extraction solvent to the sputum tree;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하는 단계.3) A step of drying after concentrating the filtered extract of step 2) under reduced pressure.
상기 방법에 있어서, 가래나무 추출물의 추출 방법으로는 여과법, 열수 추출, 침지 추출, 환류냉각 추출 및 초음파추출 등 당업계의 통상적인 방법을 이용할 수 있다.In the above method, conventional methods in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used as the extraction method of the sputum extract.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용할 수 있으나, 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조일 수 있으나, 이에 한정하지 않는다.In the above method, the vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.
상기 분획물은 가래나무 추출물에 추가적으로 용매를 가하여 제조한 것일 수 있다. 상기 용매는 물, 헥산, 에틸 아세테이트, 또는 클로로포름일 수 있으나, 이에 제한되지 않는다.The fraction may be prepared by adding a solvent to the sputum extract. The solvent may be water, hexane, ethyl acetate, or chloroform, but is not limited thereto.
또 다른 실시 태양에서, 본 발명은 하기 화학식 1 내지 10 중 어느 하나의 화합물, 또는 이의 약학적으로 허용가능한 염을 포함하는 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다. In another embodiment, the present invention relates to diabetes complications, nonalcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including any one of the following
상기 화합물은 가래나무 추출물 또는 이의 분획물로부터 분리된 것일 수 있다.The compound may be isolated from a sputum extract or a fraction thereof.
본 발명의 당뇨 합병증은 당뇨성 망막증, 당뇨성 백내장, 당뇨성 신증, 당뇨성 신경병증, 당뇨성 심장병, 당뇨성 골다공증 또는 당뇨성 아테롬성 동맥경화일 수 있다.The diabetic complication of the present invention may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic osteoporosis or diabetic atherosclerosis.
본 발명의 염증성 질환은 포도막염, 결막염, 류마티즘 관절염, 골관절염, 강직성 척추염, 감염성 관절염, 크론병, 궤양성 대장염, 자가면역성 간염, 사구체 신염, 뇌수막염, 복막염, 골수염, 봉소염, 췌장염, 아토피 피부염, 천식, 외상 유발 쇼크, 알레르기성 비염, 이하선염, 급성 기관지염, 만성 기관지염, 건초염, 혈색소증 및 염증성 장질환일 수 있다.Inflammatory diseases of the present invention include uveitis, conjunctivitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, infectious arthritis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, glomerulonephritis, meningitis, peritonitis, osteomyelitis, cellulitis, pancreatitis, atopic dermatitis, asthma, Traumatic shock, allergic rhinitis, mumps, acute bronchitis, chronic bronchitis, tendonitis, hemoglobinosis and inflammatory bowel disease.
본 발명의 섬유증은 특발성 폐 섬유증, 낭포성 섬유증, 약물 유발된 섬유증, 환경 유발된 섬유증, 림프관평활근종증, 방사선 유발된 섬유증, 신장 섬유증, 간 섬유증, 피부 섬유증, 피부 경화증, 전신 경화증, 또는 골수섬유증일 수 있다.Fibrosis of the present invention is idiopathic pulmonary fibrosis, cystic fibrosis, drug-induced fibrosis, environment-induced fibrosis, lymphangioleiomyomatosis, radiation-induced fibrosis, renal fibrosis, liver fibrosis, skin fibrosis, skin sclerosis, systemic sclerosis, or myelofibrosis. Can be
본 발명의 퇴행성 뇌질환은 알츠하이머병 또는 뇌졸중일 수 있다.The degenerative brain disease of the present invention may be Alzheimer's disease or stroke.
본 발명의 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환은 최종당화산물(advanced glycation end product, AGEs)에 의해 유발되는 것일 수 있다. Diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases of the present invention may be caused by advanced glycation end products (AGEs).
하기 실시예를 통해 확인된 바와 같이, 본 발명의 가래나무 추출물, 이의 분획물, 또는 상기 화학식 1 내지 10의 화합물은 최종당화산물의 생성을 억제 또는 생성된 최종당화산물을 분해하므로, 본 발명의 가래나무 추출물, 이의 분획물, 또는 상기 화학식 1 내지 10의 화합물은 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 치료 또는 예방에 효과적이다.As confirmed through the following examples, the sputum tree extract of the present invention, its fraction, or the compounds of
본 발명은 또한 가래나무 추출물, 이의 분획물, 또는 상기 화학식 1 내지 10의 화합물을 포함하는 당뇨 합병증, 비알콜성 지방간염, 염증성 질환, 섬유증, 또는 퇴행성 뇌질환의 개선 또는 예방용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for the improvement or prevention of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract, a fraction thereof, or the compound of
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 상세하게 설명하기로 한다. 그러나 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다.Hereinafter, examples and the like will be described in detail to aid in understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples.
[실시예 1] 가래나무 추출물, 이의 분획물 및 활성화합물의 분리[Example 1] Separation of Phalaenopsis extract, its fraction, and active compound
가래나무는 2014년 대한민국 서울의 경동 약재시장에서 구입한 가래나무 열매를 사용하였다. 가래나무 과일(10kg)을 4시간 초음파 처리한 뒤, 메탄올(5L, 3회)로 추출하여 추출물(200g)을 얻은 후 현탁시켜 현탁액(JM1)을 얻었다. 상기 현탁액(JM1)을 물에 넣고 연속적으로 클로로포름 및 에틸아세테이트로 분배한 후, 용매를 제거하여, 클로로포름 분획물(JM1, 17.5 g), 에틸아세테이트 분획물(JM2, 45.7 g) 및 물 분획물(JM3, 78.6 g)을 얻었다. 클로로포름 분획물을 실리카 겔 컬럼에 로딩하고 헥산-아세톤(20:1→ 1:1, /v)의 구배(gradient)로 용출시켜 총 5개의 분획물을 수득하고, 이를 각각 JM1A(3.4 g), JM1B(4.1 g), JM1C(2.2 g), JM1D(1.2 g) 및 JM1E(2.5 g)으로 명명하였다.As for the sputum tree, the fruit of the sputum tree was purchased from the Gyeong-dong medicinal market in Seoul, Korea in 2014. After sonicating the sputum fruit (10 kg) for 4 hours, it was extracted with methanol (5L, 3 times) to obtain an extract (200g) and then suspended to obtain a suspension (JM1). The suspension (JM1) was added to water and continuously distributed with chloroform and ethyl acetate, and then the solvent was removed, and the chloroform fraction (JM1, 17.5 g), the ethyl acetate fraction (JM2, 45.7 g) and the water fraction (JM3, 78.6 g) were added. g) was obtained. The chloroform fraction was loaded on a silica gel column and eluted with a gradient of hexane-acetone (20:1 → 1:1, /v) to obtain a total of 5 fractions, which were JM1A (3.4 g) and JM1B ( 4.1 g), JM1C (2.2 g), JM1D (1.2 g) and JM1E (2.5 g).
상기 JM1C 분획물을 다시 실리카 겔 컬럼에 로딩하고, 클로로포름:메탄올을 5:1(v/v)로 혼합한 이동상 용매를 사용하여 5개의 소분획물을 용출하고, 이를 각각 JM1C1(0.4 g), JM1C2(0.3 g), JM1C3(0.8 g), JM1C4(0.8 g) 및 JM1C5(0.3 g)으로 명명하였다. 그런 다음, 다시 상기 JM1C2 소분획물을, J'sphere ODS H-80 컬럼(250 ㎜ X 20 ㎜)을 장착한 HPLC에 로딩하고, 22% MeCN 수용액을 3 ㎖/분의 유속으로 흘려주어 최종적으로 화합물 8B3 (4.3 mg)을 수득하였다. 또한, 상기 JM1C3 소분획물을 위와 동일한 조건으로 HPLC에 로딩하여 화합물 8C10 (6.3 mg), 8C11 (3.7 mg), 8D5 (3.5 mg), 8D4 (5.2 mg)를 수득하였다. 동일 조건 하에서 JM1C4 분획물로부터는 화합물 8C13 (2.7 mg), 8C14 (7.3 mg)을 수득하였다. 또한, 상기 JM1C5 소분획물을, J'sphere ODS H-80 컬럼(250 ㎜ x 20 ㎜)을 장착한 HPLC에 로딩하고, 15% MeCN 수용액을 3 ㎖/분의 유속으로 흘려주어 최종적으로 화합물 8E4 (5.7 mg)을 수득하였다.The JM1C fraction was again loaded on a silica gel column, and five small fractions were eluted using a mobile phase solvent mixed with chloroform:methanol at 5:1 (v/v), and these were JM1C1 (0.4 g) and JM1C2 ( 0.3 g), JM1C3 (0.8 g), JM1C4 (0.8 g) and JM1C5 (0.3 g). Then, the JM1C2 small fraction was again loaded on an HPLC equipped with a J'sphere ODS H-80 column (250 ㎜ X 20 ㎜), and a 22% MeCN aqueous solution was flowed at a flow rate of 3 mL/min. 8B3 (4.3 mg) was obtained. In addition, the JM1C3 small fraction was loaded on HPLC under the same conditions as above to obtain compounds 8C10 (6.3 mg), 8C11 (3.7 mg), 8D5 (3.5 mg), and 8D4 (5.2 mg). Compound 8C13 (2.7 mg) and 8C14 (7.3 mg) were obtained from the JM1C4 fraction under the same conditions. In addition, the JM1C5 small fraction was loaded on an HPLC equipped with a J'sphere ODS H-80 column (250 mm x 20 mm), and a 15% MeCN aqueous solution was flowed at a flow rate of 3 ml/min to finally compound 8E4 ( 5.7 mg) was obtained.
상기 JM2 분획물을 실리카 겔 컬럼에 로딩하여, 클로로포름:메탄올 (20:1 → 1:1, /v)의 구배(gradient)로 용출시켜 JM2A (7.0 g), JM2B (6.3 g), JM2C (5.8 g), JM2D (4.3 g), JM2E (8.2 g)의 5개의 소분획물을 얻었다. JM2C 분획물을 YMC RP-18 column에 로딩하여 아세톤:물 (1:1, v/v)으로 용출하여 JM2C1 (1.6 g)과 JM2C2 (2.3 g)의 2개의 소분획물을 확보하였다. JM2C1 분획물을 15% MeCN 수용액으로 J'sphere ODS H-80 컬럼(250 ㎜ x 20 ㎜)에서 용출하여 화합물 9C2 (2.3 mg)과 9B (3.3 g)를 수득하였다. 분리된 화합물들의 순도는 모두 95% 이상이었다.The JM2 fraction was loaded on a silica gel column, eluted with a gradient of chloroform:methanol (20:1 → 1:1, /v), JM2A (7.0 g), JM2B (6.3 g), JM2C (5.8 g) ), JM2D (4.3 g), JM2E (8.2 g) were obtained in five small fractions. The JM2C fraction was loaded on a YMC RP-18 column and eluted with acetone:water (1:1, v/v) to obtain two small fractions of JM2C1 (1.6 g) and JM2C2 (2.3 g). The JM2C1 fraction was eluted with a 15% MeCN aqueous solution on a J'sphere ODS H-80 column (250 ㎜ x 20 ㎜) to obtain compounds 9C2 (2.3 mg) and 9B (3.3 g). The purity of the isolated compounds was more than 95%.
[실시예 2] 최종당화산물 (AGEs)의 생성 억제 효과 확인[Example 2] Confirmation of the effect of inhibiting the production of final saccharified products (AGEs)
본 실시예에서는 가래나무 추출물, 이의 분획물 및 상기 분획물로부터 분리된 화합물의 최종당화산물(AGEs)의 생성 억제 효과를 확인하고자 하였다.In this example, the effect of inhibiting the formation of final saccharified products (AGEs) of the sputum extract, its fraction, and the compound isolated from the fraction was examined.
최종당화산물(AGEs) 시약은 5 mg/ml 소 혈청 알부민(BSA), 세균 증식을 막기 위한 0.025% 아지드화 나트륨, 및 메틸글리옥살(methylglyoxal, MGO) 또는 글리옥살(glyoxal, GO)을 넣어 제조하였다. 메틸글리옥살로부터 유도된 최종당화산물을 MGO-AGEs, 글리옥살로부터 유도된 최종당화산물을 GO-AGEs로 지칭하였다.The final glycosylated product (AGEs) reagent is 5 mg/ml bovine serum albumin (BSA), 0.025% sodium azide to prevent bacterial growth, and methylglyoxal (MGO) or glyoxal (GO). Was prepared. The final glycated products derived from methylglyoxal were referred to as MGO-AGEs, and the final glycated products derived from glyoxal were referred to as GO-AGEs.
각각 1, 5, 10 mg/ml의 가래나무 추출물 및 이의 분획물을 최종당화산물(AGEs) 반응 시약과 37℃에서 7일 동안 배양하였다. 배양 후, 반응 생성물의 형광 강도를 VICTORTMX3 멀티라벨 플레이트 리더기를 이용하여 여기 파장과 방출 파장을 각각 355 nm와 460 nm에서 측정하였다.Each of 1, 5, and 10 mg/ml of sputum extract and its fractions were incubated with a final saccharified product (AGEs) reaction reagent for 7 days at 37°C. After incubation, the fluorescence intensity of the reaction product was measured at 355 nm and 460 nm, respectively, using a VICTOR TM X3 multilabel plate reader.
최종당화산물의 억제제인 아미노구아니딘(aminoguanidine, AG)을 양성대조군으로 사용하였다.Aminoguanidine (AG), an inhibitor of the final glycated product, was used as a positive control.
(클로로포름 층)Sputum fraction
(Chloroform layer)
(클로로포름 층)Sputum fraction
(Chloroform layer)
그 결과, 본 발명에 따른 가래나무 추출물, 이의 분획물을 처리한 경우, 알부민과 메틸글리옥살 또는 알부민과 글리옥살과 반응하여 생성된 최종당화산물과 비교하여 최종당화산물의 생성이 억제된 것을 확인하였다(표 1 및 표 2). As a result, it was confirmed that the production of the final glycation product was suppressed compared with the final glycation product produced by reaction with albumin and methylglyoxal or albumin and glyoxal when treating the sputum tree extract and its fractions according to the present invention. (Table 1 and Table 2).
도 1 및 2에서 나타낸 바와 같이, 가래나무 메탄올 추출물(Total)과 클로로포름 분획물(CHCl3)을 처리한 경우, 최종당화산물(AGEs) 생성 억제 효과를 확인하였다.As shown in Figs. 1 and 2, when the methanol extract (Total) and the chloroform fraction (CHCl 3 ) were treated, the effect of inhibiting the production of final saccharified products (AGEs) was confirmed.
[실시예 3] 최종당화산물(AGEs)의 분해 효과 확인[Example 3] Confirmation of the decomposition effect of final saccharified products (AGEs)
본 실시예에서는 가래나무 추출물, 이의 분획물, 또는 상기 분획물로부터 분리된 화합물의 최종당화산물(AGEs) 분해 효과를 확인하였다.In this example, the effect of decomposing sputum extract, its fraction, or the compound isolated from the fraction was confirmed.
메틸글리옥살(MGO) 또는 글리옥살(GO)을 소 혈청 알부민(BSA)과 혼합하여 최종당화산물(AGEs)을 생성한 후, 1 mg/ml 최종당화산물에 가래나무 추출물, 이의 분획물을 각각 0.1, 0.5, 1.0 mg/ml의 농도로, 상기 분획물로부터 분리된 화합물을 각각 0.1, 0.4 mM의 농도로 24시간 동안 처리하였다. 반응 후, TNBSA(2,4,6-trinitrobenzene sulfonic acid), 4% 소듐바이카보네이트(sodium bicarbonate), 10% 소듐 도데실 설페이트(sodium dedecyl sulfate) 및 1N 염산용액을 포함하는 시약을 첨가하였다. 마이크로플레이트 리더기를 이용하여 최종당화산물의 분해물인 유리 아민(free amine)의 양을 335 nm에서 측정하여, 최종당화산물의 분해 정도를 확인하였다.Methylglyoxal (MGO) or glyoxal (GO) was mixed with bovine serum albumin (BSA) to produce final glycosylated products (AGEs), and then sputum extract and fractions thereof were each 0.1 mg/ml in the final glycation product. , At concentrations of 0.5 and 1.0 mg/ml, the compounds isolated from the fractions were treated at concentrations of 0.1 and 0.4 mM, respectively, for 24 hours. After the reaction, a reagent including 2,4,6-trinitrobenzene sulfonic acid (TNBSA), 4% sodium bicarbonate, 10% sodium dedecyl sulfate and 1N hydrochloric acid solution was added. Using a microplate reader, the amount of free amine, which is a decomposition product of the final saccharified product, was measured at 335 nm, and the degree of decomposition of the final saccharified product was confirmed.
최종당화산물의 억제제인 아미노구아니딘(AG)을 양성대조군으로 사용하였다.Aminoguanidine (AG), an inhibitor of the final glycated product, was used as a positive control.
(클로로포름 층)Sputum fraction
(Chloroform layer)
(클로로포름 층)Sputum fraction
(Chloroform layer)
도 3에 나타낸 바와 같이, 가래나무 메탄올 추출물(Total), 클로로포름 분획물(CHCl3), 및 분획물로부터 분리된 화합물(JM-8C13)에서, 음성 대조군(MGO-AGEs)과 비교할 때, 더 많은 유리 아민량이 검출되는 것을 확인하였다. As shown in Figure 3, in the methanol extract (Total), chloroform fraction (CHCl 3 ), and the compound isolated from the fraction (JM-8C13), as compared to the negative control (MGO-AGEs), more free amines It was confirmed that the amount was detected.
또한 도 4에 나타낸 바와 같이, 가래나무 메탄올 추출물(Total) 1.0 mg/ml를 처리한 경우, 클로로포름 분획물(CHCl3) 0.5 mg/ml 및 1.0 mg/ml을 처리한 경우, 및 분리물(JM-8C13) 0.4 mM을 처리한 경우에서, 음성 대조군(GO-AGEs)과 비교할 때, 더 많은 유리 아민량이 검출되는 것을 확인하였다.In addition, as shown in Figure 4, when treated with the methanol extract (Total) 1.0 mg/ml, the chloroform fraction (CHCl 3 ) 0.5 mg/ml and 1.0 mg/ml, and the isolate (JM- 8C13) In the case of treatment with 0.4 mM, it was confirmed that a higher amount of free amine was detected as compared to the negative control (GO-AGEs).
따라서, 가래나무 추출물, 분획물 및 분획물로부터 분리된 화합물은 모두 우수한 최종당화산물 분해 효과를 갖는 것을 알 수 있다(표 3 및 표 4). Therefore, it can be seen that the extract, the fraction and the compound isolated from the fraction have excellent decomposition effects of the final saccharified product (Tables 3 and 4).
[실시예 4] 대식세포에서 NO 생성 억제 효과 확인[Example 4] Confirmation of NO production inhibitory effect in macrophages
본 실시예에서는 본 발명의 가래나무 추출물, 이의 분획물 및 또는 상기 분획물로부터 분리된 화합물의 NO 생성 억제 효과 여부를 확인하기 위해 세포에 가래나무 추출물, 이의 분획물, 또는 상기 분획물로부터 분리된 화합물을 처리한 후 NO 생성량을 확인하였다. NO는 반응성이 크고 매우 불안정하므로 NO 농도를 직접 측정하는 대신 간접적으로 nitrite의 농도를 측정하였다.In this example, in order to determine whether the sputum tree extract of the present invention, its fraction, and the compound isolated from the fraction have an inhibitory effect on NO generation, cells are treated with a sputum tree extract, a fraction thereof, or a compound isolated from the fraction. After that, the amount of NO generation was confirmed. Since NO is highly reactive and very unstable, the concentration of nitrite was measured indirectly instead of directly measuring the NO concentration.
대식세포인 Raw 264.7 세포를 96 웰 플레이트에 각각 5ⅹ104 로 시딩하여 37℃, 5% O2 인큐베이터에서 24 시간 배양한 후, 가래나무 추출물, 이의 분획물(10, 50 μg/ml), 상기 분획물로부터 분리한 화합물(10, 20 μg/ml) 및 양성 대조군 L-NMMA (20 μM)를 30분 동안 전처리한 후 100 ng/ml의 LPS으로 24시간 동안 자극하였다. 24 시간 후, 상등액을 50 μl씩 취해 96 웰 플레이트에 옮겨준 후, Griess reagent (50 μl, % sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water)를 각각 1:1 로 섞어준 후에 50μl 상층액이 들어있는 플레이트에 처리하였다. 반응 후, microplate reader의 파장의 범위는 540 nm로 측정을 하였으며, NO 측정을 위하여 조정배지(conditioned medium)을 사용하였다. 이 때, 양성 대조군으로 사용한 L-NMMA는 NO 생성 억제제이다.Raw 264.7 cells, which are macrophages, were seeded in a 96-well plate with 5x10 4 each and incubated in an incubator at 37° C., 5% O 2 for 24 hours, and then sputum extract, a fraction thereof (10, 50 μg/ml), from the fraction The isolated compound (10, 20 μg/ml) and positive control L-NMMA (20 μM) were pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, 50 μl of the supernatant was taken and transferred to a 96-well plate, and then Griess reagent (50 μl,% sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water) was added 1:1. After mixing with, it was treated on a plate containing 50 μl supernatant. After the reaction, the wavelength range of the microplate reader was measured to be 540 nm, and a conditioned medium was used for NO measurement. At this time, L-NMMA used as a positive control is an inhibitor of NO production.
그 결과, 도 5에 나타낸 바와 같이, 가래나무 메탄올 추출물(Total), 물 분획물(H2O), EtOAc 분획물, CHCl3 분획물을 각각 50 μg/ml로 처리한 경우 NO 생성이 억제되는 것을 확인하였다. As a result, as shown in Figure 5, it was confirmed that NO generation was suppressed when the methanol extract (Total), water fraction (H 2 O), EtOAc fraction, and CHCl 3 fraction were treated with 50 μg/ml, respectively. .
[실시예 5] 대식세포에서 세포 독성 발현 여부 확인[Example 5] Confirmation of expression of cytotoxicity in macrophages
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물의 세포 독성 발현 여부를 확인하기 위해 세포에 본 발명의 가래나무 추출물 및 이의 분획물을 처리한 후 세포 생존율(cell viability)을 확인하였다.In this example, in order to determine whether the sputum sputum extract and its fractions of the present invention express cytotoxicity, cells were treated with the sputum sputum extract and its fractions of the present invention, and then cell viability was confirmed.
대식세포인 Raw 264.7 세포를 96 웰 플레이트에 각각 5ⅹ104 로 시딩하여 37℃, 5% O2 인큐베이터에서 24 시간 배양한 후, 가래나무 추출물, 이의 분획물(10, 50μg/ml) 및 양성 대조군 L-NMMA (20 μM)를 30분 동안 전처리한 후 100 ng/ml의 LPS으로 24시간 동안 자극하였다. 24 시간 배양한 후, 0.5 mg/ml MTT 용액 (3-[4,5-dimethylthiazol-2-yl]-2,5- bromide)을 1시간 동안 처리하였다. 이 후, DMSO 100 μl를 이용하여 세포를 용해시킨 뒤 570 nm 파장 값을 microplate reader기로 측정하여 세포 독성 여부를 평가하였다. 이 때, 양성 대조군으로 사용한 L-NMMA는 NO 생성 억제제이다.Raw 264.7 cells, which are macrophages, were seeded in a 96-well plate at 5x10 4 and cultured in a 37°C, 5% O 2 incubator for 24 hours, and then sputum extract, its fraction (10, 50 μg/ml) and positive control L- NMMA (20 μM) was pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After incubation for 24 hours, 0.5 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5- bromide) was treated for 1 hour. Thereafter, cells were lysed using 100 μl of DMSO, and then the 570 nm wavelength value was measured with a microplate reader to evaluate the cytotoxicity. At this time, L-NMMA used as a positive control is an inhibitor of NO production.
그 결과, 도 6a 및 6b에 나타낸 바와 같이, 가래나무 메탄올 추출물(Total), H2O 분획물, Hexane 분획물, EtOAc 분획물 및 CHCl3 분획물을 각각 50 μg/ml로 처리한 경우 세포 독성 없이 안전하게 사용할 수 있는 것을 확인하였다.As a result, as shown in Figs. 6a and 6b, when the methanol extract (Total), H2O fraction, Hexane fraction, EtOAc fraction, and CHCl 3 fraction are treated with 50 μg/ml, respectively, it can be safely used without cytotoxicity. Confirmed.
[실시예 6] 대식세포에서 염증 관련 인자 생성 억제 효과 확인[Example 6] Confirmation of the effect of inhibiting the production of inflammation-related factors in macrophages
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물이 염증 관련 인자인 IL-6 및 TNF-α의 생성을 억제하는지 확인하기 위해 세포에 본 발명의 가래나무 추출물 및 이의 분획물을 처리한 후 IL-6 및 TNF-α생성량을 확인하였다. In this example, in order to confirm whether the sputum extract of the present invention and its fractions inhibit the production of IL-6 and TNF-α, which are inflammation-related factors, cells were treated with the sputum sputum extract of the present invention and a fraction thereof, 6 and the amount of TNF-α production was confirmed.
대식세포인 Raw 264.7 세포를 24 웰 플레이트에 각각 3ⅹ105 로 시딩하여 37℃, 5% O2 인큐베이터에서 24 시간 배양한 후, 가래나무 추출물, 이의 분획물(50 μg/ml) 및 양성 대조군 L-NMMA (20 μM)를 30분 동안 전처리한 후 100 ng/ml의 LPS으로 24시간 동안 자극하였다. 24 시간 후, 배양된 세포의 배양액을 사용하여 IL-6 및 TNF-α 생성량을 ELISA 키트 (R&D system, USA)를 사용하여 제조사의 프로토콜의 지시에 따라 실험을 수행하였다. 이 때, 양성 대조군으로 사용한 L-NMMA는 NO 생성 억제제이다.Raw 264.7 cells, which are macrophages, were seeded with 3x10 5 in a 24-well plate and cultured in a 37°C, 5% O 2 incubator for 24 hours, followed by sputum extract, its fraction (50 μg/ml) and positive control L-NMMA (20 μM) was pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, the production amount of IL-6 and TNF-α was tested according to the manufacturer's protocol using an ELISA kit (R&D system, USA) using the culture medium of the cultured cells. At this time, L-NMMA used as a positive control is an inhibitor of NO production.
그 결과, 도 7a 및 7b에 나타낸 바와 같이, 가래나무 추출물 및 분획물이 IL-6 생성을 억제하였고, EtOAc 분획물 및 CHCl3 분획물이 TNF-α 생성을 억제하는 것을 확인하였다.As a result, as shown in Figs. 7a and 7b, it was confirmed that the sputum extract and the fraction inhibited IL-6 production, and the EtOAc fraction and the CHCl 3 fraction inhibited TNF-α production.
[실시예 7] 미세교세포에서 NO 생성 억제 효과 확인[Example 7] Confirmation of NO production inhibitory effect in microglia
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물의 NO 생성 억제 효과 여부를 확인하기 위해 세포에 본 발명의 가래나무 추출물 및 이의 분획물을 처리한 후 NO 생성량을 확인하였다. NO는 반응성이 크고 매우 불안정하므로 NO 농도를 직접 측정하는 대신 간접적으로 nitrite의 농도를 측정하였다.In this example, in order to check whether the sputum tree extract and its fractions of the present invention have the effect of inhibiting NO generation, cells were treated with the sputum tree extract and its fractions of the present invention, and then the amount of NO production was confirmed. Since NO is highly reactive and very unstable, the concentration of nitrite was measured indirectly instead of directly measuring the NO concentration.
미세교세포인 BV-2 세포를 96 웰 플레이트에 각각 4ⅹ104 로 시딩하여 37℃, 5% O2 인큐베이터에서 24 시간 배양한 후, 가래나무 추출물, 이의 분획물 (10, 50, 100 μg/ml) 및 양성 대조군 L-NMMA (20 μM)를 1시간 동안 전처리한 후 100 ng/ml의 LPS으로 24시간 동안 자극하였다. 24 시간 후, 상등액을 50 μl씩 취해 96 웰 플레이트에 옮겨준 후, Griess reagent (50μl, % sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water)를 각각 1:1 로 섞어준 후에 50 μl 상층액이 들어있는 플레이트에 처리하였다. 반응 후, microplate reader의 파장의 범위는 540 nm로 측정을 하였으며, NO 측정을 위하여 조정배지(conditioned medium)을 사용하였다. 이 때, 양성 대조군으로 사용한 L-NMMA는 NO 생성 억제제이다.BV-2 cells, which are microglial cells, were seeded with 4x10 4 in a 96-well plate, respectively, and cultured in a 37°C, 5% O 2 incubator for 24 hours, and then a sputum extract, a fraction thereof (10, 50, 100 μg/ml) and Positive control L-NMMA (20 μM) was pretreated for 1 hour and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, 50 μl of the supernatant was taken and transferred to a 96-well plate, and then Griess reagent (50 μl,% sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water) was added to each of 1:1. After mixing, the plate was treated with 50 μl supernatant. After the reaction, the wavelength range of the microplate reader was measured to be 540 nm, and a conditioned medium was used for NO measurement. At this time, L-NMMA used as a positive control is an inhibitor of NO production.
그 결과, 도 8에 나타낸 바와 같이, 가래나무 추출물의 분획물이 NO 생성을 억제하는 것을 확인하였다. 또한 농도가 증가할수록 더욱 우수한 효과를 나타내는 것을 확인하였다.As a result, as shown in Figure 8, it was confirmed that the fraction of the sputum extract inhibits NO generation. In addition, it was confirmed that more excellent effects were exhibited as the concentration increased.
[실시예 8] 혈관내피세포 보호 효능 확인[Example 8] Confirmation of vascular endothelial cell protection efficacy
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물의 메틸글리옥살(methylglyoxal; MGO) 유도 독성에 대한 혈관내피세포 보호 효능 여부를 확인하였다. 이를 위해 세포에 본 발명의 가래나무 추출물 및 이의 분획물을 처리한 후 세포 생존율을 확인하였다. 메틸글리옥살(MGO)는 최종당화산물이다.In this example, it was confirmed whether the effect of protecting vascular endothelial cells against methylglyoxal (MGO)-induced toxicity of the sputum tree extract and its fractions of the present invention. To this end, cells were treated with the sputum extract of the present invention and a fraction thereof, and then cell viability was confirmed. Methylglyoxal (MGO) is the final glycation product.
혈관내피세포 HUVECs를 96 웰 플레이트(12ⅹ103 cells/well)에 시딩하여 37℃, 5 % O2 배양기에서 24시간 동안 배양한 후, EBM-2 배양액으로 샘플을 만들고 1시간 전처리를 한 후, 메틸글리옥살(MGO)을 500 μM 처리하였다. 24 시간 배양한 후, 0.1 mg/ml MTT 용액(3-[4,5-dimethylthiazol-2-yl]-2,5- bromide)을 2시간 30분 동안 처리하였다. 이후, DMSO 100 μl를 이용하여 세포를 용해시킨 뒤 570 nm 파장 값의 ELISA plate reader기로 측정하여 세포독성 여부를 평가하였다.After seeding vascular endothelial cell HUVECs in a 96-well plate (12x10 3 cells/well) and incubating for 24 hours in a 37°C, 5% O 2 incubator, samples were made with EBM-2 culture solution and pretreated for 1 hour, followed by methylation. Glyoxal (MGO) was treated with 500 μM. After incubation for 24 hours, 0.1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-bromide) was treated for 2 hours and 30 minutes. Thereafter, cells were lysed using 100 μl of DMSO and then measured with an ELISA plate reader having a wavelength of 570 nm to evaluate whether cytotoxicity was present.
그 결과, 도 9a 및 9b에 나타낸 바와 같이, 가래나무 추출물 및 이의 분획물 중 EtOAc 분획물에서 메틸글리옥살 (MGO)에 대한 세포 보호 효능 나타내었으며, 세포 독성 없이 안전하게 사용할 수 있음을 확인하였다.As a result, as shown in Figs. 9a and 9b, it was confirmed that the sputum extract and the EtOAc fraction of the fractions thereof showed cell protective efficacy against methylglyoxal (MGO) and can be safely used without cytotoxicity.
[실시예 9] 항섬유증 활성 확인[Example 9] Antifibrotic activity confirmation
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물이 섬유화 지표인 α-SMA(α-Smooth muscle actin)의 발현 정도에 미치는 영향을 측정하여 항섬유증 활성을 확인하였다. 상기 α-SMA의 증가는 섬유화를 의미한다.In this example, antifibrotic activity was confirmed by measuring the effect of the sputum extract of the present invention and its fractions on the expression level of α-SMA (α-Smooth muscle actin), which is an index of fibrosis. The increase in α-SMA means fibrosis.
LX2 세포 (Human hepatic stellate cells)를 60 mm dish에 각각 1ⅹ106 로 시딩하여 37℃, 5 % O2 배양기에서 24시간 동안 배양한 후, 가래나무 추출물, 이의 분획물을 50μg/ml의 농도로 전처리하여 1시간 동안 배양하고, TGF-β를 5 ng/ml의 농도로 처리하여 24 시간 동안 배양하였다. 배양한 세포를 모은 후, 원심분리기 (5,000 rpm, 5 min, 4℃)로 세포 pellet을 spin down 시킨 후 lysis buffer를 첨가, 1일 동안 용해시킨 후 원심분리기 (12,000 rpm, 25 min, 4℃)하여 세포막 성분 등을 제거하였다. SDS-PAGE로 변성 분리하여, 이를 PVDF membrane에 transfer하였다. 그 후, 1차 항체를 하룻밤 동안 반응시킨 후, 2차 항체를 결합시키고 ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA)을 이용해서 α-SMA의 발현을 측정하였다.LX2 cells (Human hepatic stellate cells) were seeded with 1x10 6 each in a 60 mm dish and incubated in an incubator at 37°C and 5% O 2 for 24 hours, and then sputum extract and fractions thereof were pretreated at a concentration of 50 μg/ml. After incubation for 1 hour, TGF-β was treated at a concentration of 5 ng/ml and cultured for 24 hours. After collecting the cultured cells, spin down the cell pellet with a centrifuge (5,000 rpm, 5 min, 4℃), add lysis buffer, dissolve for 1 day, and then centrifuge (12,000 rpm, 25 min, 4℃) Thus, cell membrane components and the like were removed. After denatured separation by SDS-PAGE, it was transferred to a PVDF membrane. Then, after reacting with the primary antibody overnight, the secondary antibody was bound and the expression of α-SMA was measured using a ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA).
그 결과, 도 10에 나타낸 바와 같이, 가래나무 추출물, 분획물은 모두 우수한 항섬유증 효과를 갖는 것을 알 수 있다.As a result, as shown in Figure 10, it can be seen that both the extract and the fraction have excellent anti-fibrosis effect.
또한, H2O 분획물, CHCl3 분획물에서 α-SMA의 양이 현저하게 감소한 것을 확인하였다.In addition, it was confirmed that the amount of α-SMA significantly decreased in the H 2 O fraction and the CHCl 3 fraction.
[실시예 10] BDNF 발현 정도 확인[Example 10] BDNF expression level confirmation
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물이 뇌 유래 신경 영양 인자인 BDNF((Brain-Derived Neurotrophic Factor)의 발현 정도에 미치는 영향을 측정하여 시냅스의 가소성 및 신경세포 활성을 확인하였다.In this example, the effect of the sputum extract of the present invention and a fraction thereof on the expression level of brain-derived neurotrophic factor (BDNF) was measured to confirm synaptic plasticity and neuronal activity.
BDNF는 신경기능을 조절하는 인자로 중추신경계에서 신경세포의 생존, 분화 및 분자기전에 필수적이며 다양한 세포 간 신호전달 경로에 관여한다.BDNF is a factor that regulates nerve function and is essential for the survival, differentiation and molecular mechanisms of neurons in the central nervous system, and is involved in various intercellular signaling pathways.
신경 모세포주인 SH-SY5Y 세포를 60 mm dish에 각각 1ⅹ106 로 시딩하여 37℃, 5% O2 배양기에서 24시간 동안 배양한 후, 가래나무 추출물, 이의 분획물을 50μg/ml의 농도로 24 시간 동안 배양하였다. 배양한 세포를 모은 후, 원심분리기 (5,000 rpm, 5 min, 4℃)로 세포 pellet을 spin down 시킨 후 lysis buffer를 첨가, 1일 동안 용해시킨 후 원심분리기 (12,000 rpm, 25 min, 4℃)하여 세포막 성분 등을 제거하였다. SDS-PAGE로 변성 분리하여, 이를 PVDF membrane에 transfer하였다. 그 후, 1차 항체를 하룻밤 동안 반응시킨 후, 2차 항체를 결합시키고 ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA)을 이용해서 BDNF의 발현을 측정하였다.The neuroblastoma cell line SH-SY5Y cells were seeded with 1×10 6 each in a 60 mm dish and incubated for 24 hours in a 37° C., 5% O 2 incubator, and then a sputum extract, a fraction thereof, at a concentration of 50 μg/ml for 24 hours. Cultured. After collecting the cultured cells, spin down the cell pellet with a centrifuge (5,000 rpm, 5 min, 4℃), add lysis buffer, dissolve for 1 day, and then centrifuge (12,000 rpm, 25 min, 4℃) Thus, cell membrane components and the like were removed. After denatured separation by SDS-PAGE, it was transferred to a PVDF membrane. Then, after reacting with the primary antibody overnight, the secondary antibody was bound and the expression of BDNF was measured using a ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA).
그 결과, 도 11에 나타낸 바와 같이, H2O 분획물, EtOAC 분획물 및 CHCl3 분획물에서 BDNF의 양이 증가하는 것을 확인하였다.As a result, as shown in FIG. 11, it was confirmed that the amount of BDNF increased in the H 2 O fraction, the EtOAC fraction, and the CHCl 3 fraction.
[실시예 11] 신경모세포에서 세포 독성 발현 여부[Example 11] Whether or not cytotoxicity was expressed in neuroblastic cells
본 실시예에서는 본 발명의 가래나무 추출물 및 이의 분획물의 메틸글리옥살(methylglyoxal; MGO) 유도 독성에 대한 신경 모세포주 보호 효능 여부를 확인하였다. 이를 위해 세포에 본 발명의 가래나무 추출물, 이의 분획물을 처리한 후 세포 생존율을 확인하였다.In this example, it was confirmed whether the effect of protecting a neuroblastic cell line against methylglyoxal (MGO)-induced toxicity of the sputum sputum extract and its fractions of the present invention. To this end, the cells were treated with the sputum extract of the present invention, a fraction thereof, and then the cell viability was confirmed.
신경 모세포주인 SH-SY5Y를 96 웰 플레이트(4ⅹ104 cells/well)에 시딩하여 37℃, 5 % O2 배양기에서 24시간 동안 배양한 후, DMEM 배양액 (serum-free)으로 샘플을 만들고 1시간 전처리를 한 후, 메틸글리옥살(MGO)을 500 μM 처리하였다. 24 시간 배양한 후, 0.1 mg/ml MTT 용액(3-[4,5-dimethylthiazol-2-yl]-2,5- bromide)을 2시간 30분 동안 처리하였다. 이후, DMSO 100 ul를 이용하여 세포를 용해시킨 뒤 570 nm 파장 값의 ELISA plate reader기로 측정하여 세포독성 여부를 평가하였다.The neuroblastic cell line SH-SY5Y was seeded in a 96-well plate (4x10 4 cells/well) and incubated for 24 hours in a 37°C, 5% O 2 incubator, and then a sample was prepared with DMEM culture solution (serum-free) and pretreated for 1 hour. After performing, methylglyoxal (MGO) was treated with 500 μM. After incubation for 24 hours, 0.1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-bromide) was treated for 2 hours and 30 minutes. Thereafter, cells were lysed using 100 ul of DMSO and then measured with an ELISA plate reader having a wavelength of 570 nm to evaluate whether or not cytotoxicity.
그 결과, 도 12a 및 12b에 나타낸 바와 같이, 가래나무 추출물에서 메틸글리옥살 (MGO)에 대한 세포 보호 효능 보였으며, 세포 독성이 없이 안전하게 사용할 수 있음을 확인하였다.As a result, as shown in Figs. 12a and 12b, it was confirmed that the cell protective effect against methylglyoxal (MGO) was shown in the sputum extract, and can be safely used without cytotoxicity.
Claims (15)
A pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including sputum extract or fractions.
A pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or brain diseases, including a compound of any one of the following Formulas 1 to 10, or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of claim 2, wherein the compound of any one of Formulas 1 to 10 is isolated from a sputum extract or fraction.
The pharmaceutical composition according to claim 1 or 3, wherein the extract is extracted using water, C1 to C4 lower alcohol, or a mixture thereof as a solvent.
The pharmaceutical composition according to claim 1 or 3, wherein the fraction is fractionated by adding a solvent to the sputum extract.
The pharmaceutical composition of claim 5, wherein the solvent is selected from the group consisting of water, hexane, ethyl acetate, and chloroform.
The pharmaceutical according to claim 1 or 2, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic osteoporosis, or diabetic atherosclerosis. Composition.
The pharmaceutical composition according to claim 1 or 2, wherein the degenerative brain disease is Alzheimer's disease or stroke.
The method of claim 1 or 2, wherein the inflammatory disease is uveitis, conjunctivitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, infectious arthritis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, glomerulonephritis, meningitis, peritonitis, osteomyelitis, cellulitis , Pancreatitis, atopic dermatitis, asthma, allergic rhinitis, mumps, acute bronchitis, chronic bronchitis, systemic lupus erythematosus, tendonitis, hemoglobinosis and inflammatory bowel disease.
The method of claim 1 or 2, wherein the fibrosis is idiopathic pulmonary fibrosis, cystic fibrosis, drug-induced fibrosis, environmental-induced fibrosis, lymphangioleiomyomatosis, radiation-induced fibrosis, renal fibrosis, liver fibrosis, skin fibrosis, skin. A pharmaceutical composition, characterized in that it is sclerosis, systemic sclerosis, or myelofibrosis.
The pharmaceutical composition according to claim 1 or 2, wherein the diabetic complication, non-alcoholic steatohepatitis, inflammatory disease, fibrosis or degenerative brain disease is caused by the final glycated product.
The pharmaceutical composition according to claim 1 or 2, wherein the production of the final glycation product is inhibited or the resulting final glycation product is decomposed.
The pharmaceutical composition according to claim 1 or 2, which does not exhibit cytotoxicity.
A health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including sputum extract or fractions thereof.
A health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including a compound of any one of the following Formulas 1 to 10, or a pharmaceutically acceptable salt thereof.
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WO2023128452A1 (en) * | 2021-12-27 | 2023-07-06 | 엠테라파마 주식회사 | Composition for treating inflammatory bowel disease, comprising combined extract of isatidis folium and juglans mandshurica |
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WO2022045802A1 (en) * | 2020-08-26 | 2022-03-03 | 주식회사 뉴롤메드 | Composition for preventing and treating stroke comprising extract of juglans mandshurica fruit |
WO2023128452A1 (en) * | 2021-12-27 | 2023-07-06 | 엠테라파마 주식회사 | Composition for treating inflammatory bowel disease, comprising combined extract of isatidis folium and juglans mandshurica |
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