KR101797717B1 - A pharmaceutical composition comprising derhamnosylmasin or pharmaceutically acceptable salt thereof as an active ingredient for prevention and treatment of diabetes mellitus - Google Patents
A pharmaceutical composition comprising derhamnosylmasin or pharmaceutically acceptable salt thereof as an active ingredient for prevention and treatment of diabetes mellitus Download PDFInfo
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- KR101797717B1 KR101797717B1 KR1020160152876A KR20160152876A KR101797717B1 KR 101797717 B1 KR101797717 B1 KR 101797717B1 KR 1020160152876 A KR1020160152876 A KR 1020160152876A KR 20160152876 A KR20160152876 A KR 20160152876A KR 101797717 B1 KR101797717 B1 KR 101797717B1
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- diabetes
- pharmaceutical composition
- pparγ
- pharmaceutically acceptable
- active ingredient
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Abstract
본 발명은 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 치료용 약학적 조성물에 관한 것으로, 구체적으로 디람노실메이신을 근육세포에 처리하여 그 효과를 확인한 결과, 세포 내 포도당 흡수율을 증가시키고, PPARγ(peroxisome proliferator-activated receptor γ)활성을 증가시키며, 인슐린 신호전달 경로를 활성화 시킴을 확인함으로써, 디람노실메이신은 당뇨병의 예방 및 치료에 유용하게 사용될 수 있다.The present invention relates to a pharmaceutical composition for preventing and treating diabetes comprising derhamnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient, and specifically relates to a pharmaceutical composition for treating or preventing diabetic dyslipidemia, As a result of confirming that diramnosylmecin is useful for the prevention and treatment of diabetes by confirming that it increases intracellular glucose uptake rate, increases PPARγ (peroxisome proliferator-activated receptor γ) activity and activates insulin signal transduction pathway have.
Description
본 발명은 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing and treating diabetes comprising deramnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient.
최근 우리나라도 급속한 경제성장과 생활수준의 향상 및 서구화로 신체발달 측면에서는 매우 바람직한 현상을 보이는 반면, 성인들의 경우 과다한 고칼로리 음 식의 섭취, 운동부족 및 산업사회의 고도화에 따른 스트레스로인하여 질병양상도 점점 성인병 위주로 서구화되고 있다. 대표적인 성인병의 예로는 간질환, 고혈압, 당뇨병, 비만증, 고지혈증 등을 들 수 있다.In recent years, Korea has shown a very desirable phenomenon in terms of rapid economic growth, improvement of living standards, and westernization. On the other hand, adults suffer from stress due to excessive intake of high calorie foods, lack of exercise, It is increasingly becoming westernized mainly by adult diseases. Representative examples of adult diseases include liver disease, hypertension, diabetes, obesity, and hyperlipemia.
대표적인 성인병의 하나인 당뇨병은 비전염성 만성질환으로, 췌장의 β세포에서 분비되는 인슐린이 부족하거나 그 기능을 제대로 발휘하지 못하여 체내에서 포도당이 에너지원으로 이용되지 못하고 혈액 내에 고농도로 남아 있다가 소변으로 배설되는 질환이다. 당뇨병은 그 발병원인 및 증상의 치료방법에 따라 크게 인슐린 의존형 당뇨병(insulin dependent diabetes mellitus, IDDM)과 인슐린 비의존형 당뇨병(non-insulin dependent diabetes mellitus, NIDDM)으로 분류할 수 있다. 우리나라의 경우 당뇨병 환자의 95% 이상이 NIDDM 환자이다. 당뇨병은 그 자체가 큰 질환이라기 보다는 장기간 이 질환에 걸려 있음으로 해서 발병하는 합병증, 예를 들어 당뇨병성 신경병증(neuropathy), 망막병증(retinopathy), 백내장(cataract), 신장병(nephropathy) 등으로 인해 환자들이 정상적인 삶을 영위할 수 없을 뿐 아니라 치명적인 결과까지 초래할 수 있기 때문에 사회적으로 큰 문제가 되는 것이다.Diabetes mellitus is a non-communicable chronic disease that lacks insulin secreted from the pancreatic β-cells and does not function properly. Glucose can not be used as an energy source in the body and remains in high concentrations in the blood. It is an excreted disease. Diabetes mellitus can be categorized into insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM), depending on the cause and symptoms of the disease. In Korea, more than 95% of diabetic patients are NIDDM patients. Diabetes is not a large disease in itself, but rather a complication that arises from long-term illnesses such as diabetic neuropathy, retinopathy, cataract, and nephropathy. It is a societal problem because patients can not only lead a normal life but also cause catastrophic consequences.
현재 흔히 사용되는 당뇨병 치료제는 경구용 혈당강하제와 인슐린 주사제로 크게 구분된다. 일반적으로, 체내에서 인슐린 분비가 없는 인슐린 의존형 당뇨병 환자, 임신성 당뇨병 환자 및 경구용 혈당강하제로 혈당조절이 제대로 안 되는 인슐린 비의존형 당뇨병 환자에게는 인슐린 주사를, 식사용법과 운동요법을 병행함에도 불구하고 적절한 혈당조절이 되지 않는 인슐린 비의존형 당뇨병 환자는 경구용 혈당강하제를 복용하게 하는 것이 일반적이며, 경구용 혈당강하제는 설포닐우레아계 약물과 비구아니드계 약물 등으로 구분될 수 있다.Currently, commonly used diabetes drugs are broadly divided into oral hypoglycemic agents and insulin injections. In general, insulin-dependent diabetes mellitus patients who do not have insulin secretion in the body, gestational diabetes mellitus patients, and oral insulin-dependent diabetes mellitus patients whose insulin-dependent diabetes mellitus is poorly controlled due to oral hypoglycemia, Non-insulin dependent diabetes mellitus patients who do not control blood glucose levels are generally given oral hypoglycemic agents. Oral hypoglycemic agents can be classified into sulfonylurea drugs and acetaminophen drugs.
글리피자이드(glipizide), 글리클라자이드(gliclazide), 글리퀴돈(gliquidone), 글리벤클라마이드(glibenclamide) 및 클로르프로파마이드(chlorpropamide) 등의 설포닐우레아계 약물은 췌장에서 인슐린 분비를 촉진하는 작용을 나타내지만, 췌장에서 인슐린 분비가 전혀 없는 인슐린 의존형 당뇨병 환자에게는 사용할 수 없으며, 췌장에서 인슐린 분비 능력이 상대적으로 감소된 인슐린 비의존형 당뇨병 환자나 기형아 출산, 유산, 사산 등이 우려되므로 가임기 여성에게는 사용할 수 없다는 단점이 있다. 또한, 대부분의 설포닐우레아계 약물은 간에서 대사되어 신장으로 배설되므로 간기능 및 신장기능 장애 환자에게는 주의하여 사용하여야 한다.Sulfonylurea drugs such as glipizide, gliclazide, gliquidone, glibenclamide, and chlorpropamide show actions that promote insulin secretion in the pancreas However, it can not be used in patients with insulin-dependent diabetes without any insulin secretion in the pancreas. It can not be used in fertile women because of concerns about non-insulin dependent diabetes mellitus or aberrant birth, abortion, and stillbirth in which the insulin secretory ability is relatively decreased in the pancreas There are disadvantages. In addition, since most sulfonylurea drugs are metabolized in the liver and excreted in the kidney, they should be used with caution in patients with hepatic and renal dysfunction.
메트포르민(metformin) 등의 비구아니드계 약물은 작용기전이 분명하지 않지만 췌장에서 인슐린 분비를 증가시키는 작용은 없는 것으로 밝혀져 있으며, 설포닐우레아계 약물보다 혈당강하효과가 약한 반면에, 저혈당을 일으킬 가능성도 낮다. 그러나 소화기계 부작용의 발생빈도가 높아 치료초기에 오심, 구토, 설사, 발진 등 이 나타나며, 젖산혈증(lactic acidosis)을 유발하여 생명을 위협하는 치명적인 부작용을 일으키므로, 현재 미국에서는 실험용 약제로만 사용하고 있다.Metformin and other acetaminophen drugs have not been shown to have a mechanism of action, but they do not appear to increase insulin secretion in the pancreas. They have a lower blood glucose lowering effect than sulfonylurea drugs, low. However, the incidence of gastrointestinal adverse reactions is high, causing nausea, vomiting, diarrhea, and rash at the beginning of treatment, causing lactic acidosis and causing life-threatening side effects. have.
따라서, 현재 사용되고 있는 설포닐우레아계 또는 비구아니드계의 약물은 상기의 단점이나 부작용이 있으므로 국내외적으로 증가 추세에 있는 당뇨성 질환을 더 바람직하게 치료할 수 있는 보다 부작용이 적고 안전한 혈당 강하제를 개발할 필요가 있다.Therefore, there is a need to develop a safe hypoglycemic agent which can treat diabetes mellitus which is increasing in domestic and foreign countries with fewer side effects, because the currently used sulfonylurea or acetaminophen drugs have the disadvantages and side effects described above .
이와 같이, 현재 화학적으로 합성된 항당뇨제의 여러가지 심각한 부작용의 발생과 함께 약물 내성의 발생으로 인한 약물 효과가 떨어지고 있으므로, 새로운 항당뇨 활성을 갖는 신규 물질을 찾고자 하는 많은 연구가 진행중이다.Thus, many serious side effects of currently chemically synthesized anti-diabetic drugs occur and drug effects due to the occurrence of drug resistance are lowered. Therefore, a lot of research is underway to find new substances having novel anti-diabetic activity.
한편, 디람노실메이신의 당뇨병에 대한 효과와 관련한 선행기술로서, 대한민국 공개특허 10-2015-006115에는 메이신의 비만예방 효과에 관하여 개시하고 있고, 박형준 등은 센티페드그라스 추출물(centipedegrass, Eremochloa ophiuroides)의 항-지방형성 활성에 관하여 개시하고 있으나(BMC Complementary and Alternative Medicine 2012, 12:230), 상기 선행문헌을 포함하여 현재까지 디람노실메이신에 대한 연구내용은 식물 추출물로부터 디람노실메이신의 성분을 확인하는 것일 뿐, 이에 대한 직접적인 효과에 대해서는 알려진 바가 없다.As a prior art relating to the effect of diramnosylchemicin on diabetes, Korean Patent Laid-Open No. 10-2015-006115 discloses the effect of Mechin on the prevention of obesity, and Hyung Jun Park discloses a centipedegrass extract ( Eremochloa ophiuroides ) (BMC Complementary and Alternative Medicine 2012, 12: 230), the contents of the study on diramnosylmeycine to date, including the above-mentioned prior art, It is only to identify the ingredients, but the direct effect on it is unknown.
이에, 본 발명자들은 당뇨병에 대한 효과를 갖는 새로운 물질을 찾고자 노력하던 중, 디람노실메이신을 근육세포에 처리하여 그 효과를 확인한 결과, 세포 내 포도당 흡수율을 증가시키고, PPARγ(peroxisome proliferator-activated receptor γ)활성을 증가시키며, 인슐린 신호전달 경로를 활성화 시킴을 확인하여, 디람노실메이신이 당뇨병의 예방 및 치료에 유용하게 사용될 수 있음을 밝힘으로써, 본 발명을 완성하였다.Accordingly, the present inventors tried to find a new substance having an effect on diabetes. As a result of examining the effect of diramnosylmethacin on muscle cells, the present inventors have found that PPARγ (peroxisome proliferator-activated receptor γ ) Activity and activates the insulin signaling pathway, thus confirming that diramnosylchemycin can be effectively used for the prevention and treatment of diabetes. The present invention has been completed based on this finding.
본 발명의 목적은 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for prevention and treatment of diabetes comprising deramnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 치료용 약학적 조성물을 제공한다:In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating diabetes comprising as an active ingredient deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[화학식 1][Chemical Formula 1]
또한, 본 발명은 상기 화학식 1로 표시되는 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for preventing or ameliorating diabetes comprising deramnosylmasin represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 상기 화학식 1로 표시되는 디람노실메이신 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 혈당 강하용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for reducing blood glucose content comprising diramnosylmecine represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
아울러, 본 발명은 상기 화학식 1로 표시되는 디람노실메이신 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 혈당 강하용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for hypoglycemia comprising diramnosylmecin or its pharmaceutically acceptable salt represented by the above formula (1) as an active ingredient.
본 발명의 디람노실메이신(derhamnosylmaysin)은 근육세포로의 포도당 흡수율을 증가시키고, PPARγ(peroxisome proliferator-activated receptor γ) 활성을 증가시키며, 인슐린 신호전달 경로를 활성화 시킴을 확인함으로써, 디람노실메이신은 당뇨병의 예방 및 치료에 유용하게 사용될 수 있다.By confirming that the diramnosylmaysin of the present invention increases glucose uptake into muscle cells, increases PPARy (peroxisome proliferator-activated receptor gamma) activity and activates the insulin signaling pathway, And can be usefully used for prevention and treatment of diabetes.
도 1은 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질의 성분 분석을 나타낸 도이다.
도 2a는 디람노실메이신, 오리엔틴, 람노실오리엔틴, 메이신, 이레모네틴, 이소오리엔틴, 클로로젠산, 및 루테올린의 포도당 흡수율을 나타낸 도이다:
대조군(control): 아무것도 처리하지 않은 군.
도 2b는 센티페드그라스 추출물(50% CGE)의 농도에 따른 포도당 흡수율을 나타낸 도이다.
도 3a는 디람노실메이신의 인슐린 신호전달 분자의 발현 양상을 확인하기 위한 RT-PCR 실험 결과를 나타낸 도이고,
도 3b는 디람노실메이신의 인슐린 신호전달 부자의 인산화를 확인하기 위한웨스턴 블롯 실험결과를 나타낸 도이다:
IR: insulin receptor, IRS-1: insulin receptor substrate-1, GLUT4: glucose transporter 4, pIRS: phosphorylated insulin receptor substrate.
도 4a는 디람노실메이신, 오리엔틴, 람노실오리엔틴, 메이신, 이레모네틴, 이소오리엔틴, 클로로젠산, 및 루테올린의 PPARγ의 전사 활성 정도를 확인하기 위한발광효소분석(luciferase assay) 실험 결과를 나타낸 도이다:
control: 아무것도 처리하지 않은 군.
GW1929: PPARγ agonist.
도 4b는 디람노실메이신의 농도 증가에 따른 PPARγ의 전사 활성 정도를 확인하기 위한 발광효소분석(luciferase assay) 실험 결과를 나타낸 도이다.
도 4c는 센티페드그라스 추출물(50% CGE)의 농도에 따른 PPARγ의 전사 활성 정도를 확인하기 위한 발광효소분석(luciferase assay) 실험 결과를 나타낸 도이다.
도 5는 디람노실메이신 처리에 의한 시간에 따른 PPARγ와 RXRα의 결합의 변화를 웨스턴블롯(western blot)으로 나타낸 도이다.
도 6은 디람노실메이신도 PPARγ의 잘 알려진 부위에 결합하여 전사인자의 활성 증가를 확인하기 위한 분석키트((LanthaScreen TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit; Invitrogen, CA) 실험 결과를 나타낸 도이다.
도 7은 PPARγ의 활성을 조절하는데 필요한 물질과의 결합부위로 잘 알려진 부위와 디람노실메이신이 결합할 것이라고 예측되는 알려지지 않은 부위를 나타낸 도이다.
도 8은 PPARγ과 디람노실메이신의 결합부위를 나타낸 도이다.
도 9는 점 돌연변이를 발생시킨 PPARγ과 디람노실메이신의 결합여부를 확인하기 위한 발광효소분석(luciferase assay) 실험 결과를 나타낸 도이다.BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a compositional analysis of a single substance fraction extracted from a centipedegrass.
Figure 2a shows glucose uptake rates of diramnosylmecin, orientin, rhamnosylorientin, meicin, irremotin, isoorientin, chlorogenic acid, and luteolin;
Control: You have not done anything.
FIG. 2B is a graph showing the glucose uptake rate according to the concentration of the centipede glass extract (50% CGE).
FIG. 3A is a graph showing the results of RT-PCR experiments for confirming the expression pattern of insulin signaling molecules of diramnosylmecine,
FIG. 3B is a graph showing the results of western blot experiments for confirming phosphorylation of the insulin signal transduction of diramnosylmecine.
IR: insulin receptor, IRS-1: insulin receptor substrate-1, GLUT4: glucose transporter 4, pIRS: phosphorylated insulin receptor substrate.
4A is a luciferase assay for confirming the transcriptional activity of PPARγ of diramnosylmecin, orientin, rhamnosylorientin, meicin, irremotin, isoorientin, chlorogenic acid, and luteolin ) Shows experimental results:
control: You have not done anything.
GW1929: PPARgamma agonist.
FIG. 4B is a graph showing the results of luciferase assay for confirming the transcriptional activity of PPARγ according to an increase in the concentration of diramnosylmecine.
FIG. 4c is a graph showing the results of luciferase assay for confirming the degree of transcriptional activity of PPARγ according to the concentration of the centipede glass extract (50% CGE).
FIG. 5 is a western blot diagram showing changes in the binding of PPARγ and RXRα with time by diramnosylmecine treatment. FIG.
FIG. 6 is a graph showing an experiment result of an assay kit ((LanthaScreen TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit; Invitrogen, CA) for binding to well-known sites of diramnosylmethionine PPARγ .
FIG. 7 is a diagram showing a region well known as a binding site with a substance necessary for regulating the activity of PPARy and an unknown region predicted to bind diramnosylmethacin.
8 is a view showing the binding site of PPARγ and diramnosylmecine.
FIG. 9 is a graph showing the results of luciferase assay for confirming the binding of diramnosylmethacin to PPARγ causing point mutation. FIG.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기 화학식 1로 표시되는 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 치료용 약학적 조성물을 제공한다:The present invention provides a pharmaceutical composition for preventing and treating diabetes comprising as an active ingredient deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[화학식 1][Chemical Formula 1]
또한, 본 발명은 상기 화학식 1로 표시되는 디람노실메이신 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 혈당 강하용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for reducing blood glucose content comprising diramnosylmecine represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 조성물은 근육세포로의 포도당 흡수율을 증가시키고, PPARγ(peroxisome proliferator-activated receptor γ) 활성을 증가시키며, 인슐린 신호전달 경로를 활성화 시키는 것이 바람직하다.Preferably, the composition increases glucose uptake into muscle cells, increases PPARy (peroxisome proliferator-activated receptor gamma) activity, and activates the insulin signaling pathway.
또한, 상기 당뇨병은 제 1형 당뇨병(인슐린-의존형 당뇨병(insulin-dependent diabetes mellitus)) 또는 제 2형 당뇨병(인슐린-비의존형 당뇨병(insulin-independent diabetes mellitus))인 것이 바람직하다.It is also preferred that the diabetes is
인슐린 신호전달 분자들은 발현은 PPARγ(peroxisome proliferator-activated receptor γ)에 의하여 조절되는 것으로 이미 많은 선행연구들에 의하여 보고가 되어있다. PPAR(Peroxisome Proliferator Activated Receptor, 퍼옥시좀 증식인자 활성화 수용체)은 물질대사와 에너지 생산에 관여하고 있으며, 섭식 후는 PPARγ의 발현이 가장 높은 것으로 알려져 있다. PPARγ 활성화는 지방산을 지방세포 안으로 집어넣어 혈중 내의 농도를 낮추고 이 결과로 지방산이 근육에서의 이용이 감소되어 인슐린 저항성을 감소시킨다. 이러한 PPAR의 작용에 이상이 발생하면 당뇨병이나 고지혈증, 비만을 유발하게 된다.Expression of insulin signaling molecules is regulated by PPARγ (peroxisome proliferator-activated receptor γ) and has been reported by many previous studies. PPAR (Peroxisome Proliferator Activated Receptor, Peroxisome Proliferator Activated Receptor) is involved in metabolism and energy production and is known to have the highest expression of PPARγ after ingestion. Activation of PPARγ lowers the concentration in the blood by putting fatty acids into fat cells, which results in a decrease in insulin resistance due to reduced use of fatty acids in muscle. Such abnormalities of PPAR may cause diabetes, hyperlipemia and obesity.
PPAR는 레티노이드-X-수용체(RXRs, retinoid X receptor α)와 이질이합체를 형성하는데, 리간드가 결합하면 AF2 (activation function 2)에 구조적인 변화가 일어나면서 보조억제물질(corepressor)이 떨어져나가는 대신 보조활성물질(coactivator)이 결합하고 PPAR-RXR 이성체는 DNA 서열 중 PPRE (peroxisome proliferator-response element)에 결합함으로써 목표유전자의 전사를 촉진하거나 억제하는데, 이때 가장 많은 영향을 받는 분자들이 인슐린 신호전달 분자들이다. PPARs form heterodimers with retinoid X receptor α (RXRs), which, when coupled with ligands, cause a structural change in AF2 (activation function 2) The coactivator binds and the PPAR-RXR isoform promotes or inhibits the transcription of the target gene by binding to the peroxisome proliferator-response element (PPRE) in the DNA sequence, where the most affected molecules are insulin signaling molecules .
본 발명의 구체적인 실시예에서, 디람노실메이신을 근육세포에 처리하여 그 효과를 확인한 결과, 세포 내 포도당 흡수율을 증가시키고(도 2a 참조), PPARγ(peroxisome proliferator-activated receptor γ)활성을 증가시키며(도 4 참조), 인슐린 신호전달 경로를 활성화 시킴을 확인하였다(도 3 참조). In a specific example of the present invention, diramnosylmecin was treated on muscle cells to examine the effect thereof. As a result, it was found that it increased the intracellular glucose uptake rate (see FIG. 2A), increased PPARγ (peroxisome proliferator-activated receptor γ) 4) to activate the insulin signaling pathway (see FIG. 3).
또한 디람노실메이신을 처리하는 경우 PPARγ와 RXRα의 결합을 빠르게 유도하고(도 5 및 표 4 참조), 디람노실메이신은 PPARγ의 리간드 결합 부위가 아닌 다른 부위에 결합하는 것을 확인하였고(도 6 및 도 7 참조), 그 중 447번째 아미노산인 트레오닌(threonine)이 가장 중요한 역할을 하는 것을 확인하였다(도 8 및 도 9 참조).It was also confirmed that when diramnosylmethacin was treated, the binding of PPARγ and RXRα was rapidly induced (see FIGS. 5 and 4), and that diramnosylmecine binds to a site other than the ligand binding site of PPARγ 7), and it was confirmed that threonine, which is the 447th amino acid, plays the most important role (see FIGS. 8 and 9).
따라서, 디람노실메이신은 당뇨병의 예방 및 치료를 위한 약학적 조성물 또는 혈당 강하용 약학적 조성물로써 유용하게 사용될 수 있다.Therefore, diramnosylmecin can be usefully used as a pharmaceutical composition for prevention and treatment of diabetes or as a pharmaceutical composition for lowering blood glucose.
본 발명은 화학식 1로 표시되는 화합물뿐만 아니라, 이의 약학적으로 허용되는 염, 이의 프로드럭(prodrug), 이로부터 제조될 수 있는 가능한 용매화물, 수화물, 라세이체 또는 입체이성질체를 모두 포함한다.The present invention includes all of the compounds represented by formula (1) as well as pharmaceutically acceptable salts thereof, prodrugs thereof, and possible solvates, hydrates, racemates or stereoisomers thereof which can be prepared therefrom.
본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용되는 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요오드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸이도에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트,니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 숙시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, 하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.The present invention can be used in the form of a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면 화학식 1로 표시되는 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한, 동량의 화학식 1로 표시되는 화합물, 및 산 수용액 또는 알코올을 가열하고, 이어서 이 혼합물을 증발시켜서 건조하거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile To precipitate it. It is also possible to prepare the same by heating the compound represented by the general formula (1) and an acid aqueous solution or alcohol, followed by evaporating the mixture or drying the precipitated salt by suction filtration.
또한, 염기를 사용하여 약학적으로 허용가능 한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조 시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
본 발명의 약학적 조성물은 통상적으로 사용되는 부형제, 붕해제, 감미제, 활택제, 향미제 등을 추가로 포함할 수 있으며, 통상적인 방법에 의해 정제, 캅셀제, 산제, 과립제, 현탁제, 유제, 시럽제, 기타 액제로 제형화 될 수 있다.The pharmaceutical composition of the present invention may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, Syrups, and other liquid preparations.
본 발명의 약학적 조성물은 당뇨병 또는 당뇨병 합병증을 예방 또는 치료하기 위한 약제로서 사용될 수 있다.The pharmaceutical composition of the present invention can be used as a medicine for preventing or treating diabetes or diabetic complications.
상기 당뇨병 합병증은 신경병증(neuropathy), 신장병(nephropathy), 망막증(retinopathy), 거대혈관병증(macroangiopathy), 관상동맥질환(coronary artery disease), 골다공증(osteopenia)을 포함하고, 또한 본 발명의 약학적 조성물은 당내성 손상을 치료하기 위한 약제로서 사용될 수 있다.Such diabetic complications include neuropathy, nephropathy, retinopathy, macroangiopathy, coronary artery disease, osteopenia, and also the pharmacological activity of the present invention The compositions can be used as medicaments for treating intraspinal gingival damage.
구체적으로 본 발명의 약학적 조성물은 경구 투여용 제형, 예를 들면 정제, 트로치제(troches), 로젠제(lozenge), 수용성 또는 우성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제(elixirs)로 제제화된다. 정제 및 캡슐 등의 제형으로 제제하기 위해 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕해제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌 글리콜 왁스와 같은 윤활유가 함유된다. 캡슐제형의 경우는 상기에서 언급한 물질 이외에도 지방유와 같은 액체 담체를 함유한다.Specifically, the pharmaceutical compositions of the present invention may be formulated for oral administration, for example, tablets, troches, lozenges, aqueous or aqueous suspensions, prepared powders or granules, emulsions, hard or soft capsules, It is formulated into elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, Lubricating oil such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.
또한, 본 발명의 약학적 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여시 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식을 선택하는 것이 바람직하다. 비경구 투여용 제형으로 제제화하기 위해서는 본 발명의 혼합 추출물을 안정제 또는 완충제와 함께 물에서 혼합하여 현탁액으로 제조하고 이를 앰플 또는 바이알(vial)의 단위 투여형으로 제제하며, 본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection method for parenteral administration. In order to formulate the composition for parenteral administration, the mixed extract of the present invention is mixed with a stabilizer or a buffer in water to prepare a suspension, which is formulated into a unit dosage form of ampoule or vial, , Or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
본 발명에 따른 유효성분의 투여량은 체내에서 활성성분의 흡수도, 불활성화율 및 배성속도, 환자의 연령, 성별 및 상태, 치료할 질병의 중증 정도에 따라 적절히 선택된다.The dosage of the active ingredient according to the present invention is appropriately selected depending on the degree of absorption, inactivation rate and rate of absorption of the active ingredient in the body, age, sex and condition of the patient, and severity of the disease to be treated.
또한, 본 발명은 하기 화학식 1로 표시되는 디람노실메이신(derhamnosylmasin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 당뇨병 예방 및 개선용 건강기능식품을 제공한다:The present invention also provides a health functional food for prevention and improvement of diabetes comprising deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[화학식 1][Chemical Formula 1]
아울러, 본 발명은 상기 화학식 1로 표시되는 디람노실메이신 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 혈당 강하용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for hypoglycemia comprising diramnosylmecin or its pharmaceutically acceptable salt represented by the above formula (1) as an active ingredient.
상기 당뇨병은 제 1형 당뇨병 또는 제 2형 당뇨병인 것이 바람직하다.The diabetes is preferably
본 말명에서 디람노실메이신을 근육세포에 처리하여 그 효과를 확인한 결과, 세포 내 포도당 흡수율을 증가시키고, PPARγ(peroxisome proliferator-activated receptor γ)활성을 증가시키며, 인슐린 신호전달 경로를 활성화 시킴을 확인함으로써, 디람노실메이신은 당뇨병의 예방 및 개선, 또는 혈당 강하용 건강기능식품으로써 유용하게 사용될 수 있다.As a result of confirming the effect of treatment of diramnosylmethacin on muscle cells, it was confirmed that it increased intracellular glucose uptake rate, increased PPARγ (peroxisome proliferator-activated receptor γ) activity and activated the insulin signal transduction pathway , Diramnosylmecin can be usefully used as a health functional food for prevention and improvement of diabetes or blood sugar lowering.
본 명세서의 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 진정, 수면 및 항경련의 효과를 증진시키기 위한 보조제로 섭취가 가능하다.As used herein, the term " health functional food "refers to foods prepared and processed in the form of tablets, capsules, powders, granules, liquids, and circles by using raw materials and components having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there are no side effects that may occur when a medicine is taken for a long time by using a food as a raw material, and it is excellent in portability and the health functional food of the present invention is effective in improving the effect of sleep, It can be taken as an adjuvant for.
유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 화학식 1의 화합물은 원료 조성물 중 1 ~ 10 중량%, 바람직하게는 5 ~ 10중량%의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the compound of the formula (1) of the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight of the raw material composition in the production of food. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.
본 발명의 건강기능식품은 통상의 식품과 같이 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 of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient like ordinary foods. 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, 본 발명을 실시예 및 실험예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해서 한정되는 것은 아니다.However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
<실시예 1> 센티페드그라스의 유효성분 함량 확인≪ Example 1 > Determination of the content of active ingredients of sentiped grass
가을에 수확된 센티페드그라스(centipedegrass)를 80% 메탄올을 이용하여 일주일간 추출하였다. 센티페드그라스와 80% 메탄올의 비율은 1:20 (v/w)으로 하였으며, 추출이 끝나면 센티페드그라스 제거 및 필터 과정을 통하여 불순물(찌꺼기)를 제거하였다. 이후 농축기를 이용하여 메탄올을 제거하였고, 물 층 추출물을 회수하였다. 회수된 추출물은 헥산을 이용하여 지질 성분 및 엽록소 등을 제거하는 과정을 거치는데, 이때 헥산층이 녹색을 띄지 않을 때까지 진행하였다. 이후, 에틸아세테이트를 이용하여 유효성분들을 재차 추출하는 과정을 진행하였으며, 그런 다음 농축하여 분말 상태로 만들었고, 하기 과정의 실험들에서 사용될 수 있게 냉장 보관하였다. 분말 형태의 센티페드그라스 추출물은 성분 분석을 위하여 1 mg/ml 농도로 50%, 70%메탄올에 각각 녹인 후 하기 표 1의 조건의 HPLC 분석 방법으로 유효성분을 분석하였다. The centipedegrass harvested in autumn was extracted with 80% methanol for one week. The ratio of centipedegrass to 80% methanol was 1:20 (v / w). After the extraction, impurities (residue) were removed through centipede grasping and filtering. Methanol was then removed using a concentrator, and the water layer extract was recovered. The recovered extract was treated with hexane to remove lipid components and chlorophyll, and the hexane layer proceeded to a green state. Then, the active ingredients were extracted again by using ethyl acetate, and then concentrated to a powder state, which was refrigerated for use in the following experiments. The powdered Centipedgrass extract was dissolved in 50% and 70% methanol at a concentration of 1 mg / ml for analysis of the components, and the active ingredient was analyzed by HPLC analysis under the conditions shown in Table 1 below.
그 결과, 도 1에 나타난 바와 같이, 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질에 디람노실메이신(derhamnosylmaysin), 오리엔틴(orientin), 람노실오리엔틴(rhamnosylisoorientin), 메이신(maysin), 이소오리엔틴(isoorientin), 및 클로로젠산(cholorogenic acid), 이레모네틴(eremonetin)이 포함됨을 확인하였으며(도 1), 각각의 단일물질의 함량을 조사한 결과 총 1mg의 센티페드그라스 추출물(50% CGE)에는 디람노실메이신이 57㎍(약 5%) 포함되어 있는 것을 확인하였다(표 2). As a result, as shown in Fig. 1, a single substance extracted and fractionated from centipedegrass was treated with a solution of diramnosylmaysin, orientin, rhamnosylisoorientin, maysin, , Isoorientin, and cholorogenic acid and eremonetin (FIG. 1). As a result of the examination of the content of each single substance, a total of 1 mg of Sentifedgrass extract ( 50% CGE) contained 57 μg (about 5%) of diramnosylchemicin (Table 2).
각각 단일물질의 화학식은 하기의 표에 나타내었다. The chemical formulas of each single substance are shown in the following table.
<< 실시예Example 2> 2> 디람노실메이신의Diramnosylmecine 준비 Ready
디람노실메이신(derhamnosylmaysin)의 분리를 위하여, Toyopearl HW-40 gel 충진제를 이용하여 1차 column chromatography를 실시하였다. 먼저 직경 2.5 cm × 54 cm의 칼럼을 세운 뒤 gel을 충진시켰다. 그 후, 칼럼 부피 3배의 메탄올을 이용하여, 칼럼 충진제를 세척 및 활성화 시킨 후 칼럼 용리의 시작 용매인 20% 메탄올로 칼럼을 치환시키고 안정화시켰다. 분말 상태의 에틸아세테이트층을 소량의 20% 메탄올에 녹인 후, 충진된 gel에 흡착시킨 다음 H2O:MeOH, step wise gradient mode의 용매계를 이용하여 20%, 40%, 60%, 100% 메탄올로 칼럼을 용리시켰다. 그런 다음, 각각의 용리층은 감압 농축을 통해 용매를 제하여 버린 후 분말 상태로 확보하였다. For the separation of diramnosylmaysin, primary column chromatography was performed using Toyopearl HW-40 gel filler. First, a column having a diameter of 2.5 cm × 54 cm was set up and filled with gel. The column was then washed and activated with a column volume of methanol three times, then the column was replaced with 20% methanol, the starting solvent for column elution, and stabilized. The ethyl acetate layer was dissolved in a small amount of 20% methanol, adsorbed on packed gel, and then eluted with 20%, 40%, 60% and 100% of H 2 O: MeOH using a solvent system of stepwise gradient mode. The column was eluted with methanol. Then, each of the eluting layers was concentrated under reduced pressure to remove the solvent, and then secured in powder form.
다음으로, 디람노실메이신의 최종 분리를 위해 디람노실메이신의 함유가 추정되는 40% 메탄올 용리층을 이용하여, ODS AQ 120S 충진제를 이용한 2차 column chromatograpy를 수행하였다. 칼럼 사용, gel 충진 및 활성화 방식은 위의 방식과 동일하게 진행하였고, 분말로 준비된 40% 메탄올 용리층을 칼럼 용리의 시작 용매인 소량의 H2O에 녹인 뒤 충진제에 흡착시킨 다음 H2O:MeOH, step wise gradient mode의 용매계를 이용하여, 5%씩 메탄올의 함량을 증가시켰다. 각 용리층은 분획(fraction)기기를 이용하여 200 drops씩 시험관에 분획하였고, HPLC를 이용한 모니터링을 통해 순도가 높은 분획만을 수집하여 농축 후 물질을 확보하였다.Next, a second column chromatograpy using an ODS AQ 120S filler was performed using a 40% methanol elution layer containing diramnosylmecine, which was presumed to contain diramnosylmecicin. The column was eluted with a small amount of H 2 O, which was the starting solvent for column elution, and adsorbed on a packing material. Then, H 2 O: The methanol content was increased by 5% using a solvent system of MeOH and step wise gradient mode. Each elution layer was fractionated in a test tube by 200 drops using a fraction device, and only a fraction of high purity was collected through HPLC monitoring to obtain a substance after concentration.
분리 후 농축된 디람노실메이신 추정 물질의 순도 및 분자량을 HPLC-MS를 이용하여 확인하였고, 순도 확인 후 1H, 13C의 1D NMR을 측정하여 화합물의 구조를 동정 후 하기의 실험에 사용하였다.After the separation, the purity and molecular weight of the diramnosylmecine-enriched substance were confirmed by HPLC-MS. After confirming the purity, 1D NMR of 1H and 13C was measured and the structure of the compound was used for the following experiments.
<< 실험예Experimental Example 1-1> 1-1> 디람노실메이신의Diramnosylmecine 포도당 흡수 효과 확인 Confirm glucose absorption effect
디람노실메이신의 포도당 농도 조절 효과를 확인하기 위하여, C2C12 근육세포(American Type Culture Collect; ATCC, 미국)을 이용하여 하기의 방법으로 포도당 흡수(glucose uptake) 실험을 실시하였다.Glucose uptake experiments were carried out using C2C12 muscle cells (American Type Culture Collection, ATCC, USA) in the following manner to confirm the effect of controlling the glucose concentration of diramnosylchemicin.
구체적으로, 미분화 상태의 C2C12 세포를 6 well plate의 각 well에 1 × 105 개를 넣고 세포가 90%까지 자랄 수 있게 10% FBS(Fetal bovine serum)가 포함된 DMED 배지(Dulbecco’s modified Eagle’s medium; Gibco, Life technologies, 미국)를 이용하여 37℃로 항온 유지되는 5% CO2 incubator에서 24시간 동안 배양하였다. 그런 다음, 배양중인 배지를 버리고 근육세포로 분화를 도와주는 배지를 넣어 이틀 동안 근육세포로의 분화를 유도하였다. 상기 근육세포 분화를 도와주는 배지의 조성은 2% FBS가 들어있는 DMEM 배지이다. 이후 근육세포로 분화하는 동안 상기 <실시예 1 및 2>의 방법으로 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질(디람노실메이신(derhamnosylmaysin), 오리엔틴(orientin), 람노실오리엔틴(rhamnosylisoorientin), 메이신(maysin), 이레모네틴(eremonetin(luteoin-6-C-boivinopyranoside)), 이소오리엔틴(isoorientin), 클로로젠산(cholorogenic acid), 루테올린(luteolin))을 상기 세포에 처리하여 성숙한 근육세포가 될 때까지 4~6 일간 배양하였다. 그 후, 이틀마다 분화를 유도하는 배지를 바꿔주며, 상기 단일물질들을 10 μg/ml의 농도가 되도록 처리하였다. 성숙한 근육세포로 자라게 되면 포도당이 배제된 무혈청배지(serum free media)에서 12시간 동안 배양하였고, 이후 2-NBDG(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose)를 처리하여 세포 내 포도당이 유입되는 것을 형광 현미경 및 형광 분석기(Infinite M200 Tecan, 오스트리아)를 통해 관찰하였다. 2-NBDG는 포도당의 deoxy-form에 형광을 띠는 구조가 결합되어 있는 구조로, 세포 내로 포도당과 같이 들어가지만 분해되지 않고, 쌓여서 형광을 관찰할 수 있다. 육안으로 형광을 확인하고 사진으로 비교분석하고, 형광 분석기를 이용하여 세포 내 2-NBDG의 상대적인 양을 수치상으로 비교하였다.Specifically, C2C12 cells in an undifferentiated state were added to each well of a 6-well plate at 1 × 10 5 , and DME medium (Dulbecco's modified Eagle's medium containing 10% FBS (Fetal bovine serum) Gibco, Life technologies, USA) in a 5% CO 2 incubator maintained at 37 ° C for 24 hours. Then, the culture medium was discarded and a medium for assisting differentiation into muscle cells was added to induce differentiation into muscle cells for two days. The composition of the culture medium for supporting the differentiation of muscle cells is DMEM medium containing 2% FBS. Thereafter, a single substance (derhamnosylmaysin, orientin, rhamnosylorientin, or the like) extracted and fractionated in a centipedegrass by the method of Examples 1 and 2 during differentiation into muscle cells rhamnosylisoorientin, maysin, eremonetin (luteoin-6-C-boivinopyranoside), isoorientin, cholorogenic acid, luteolin) And cultured for 4-6 days until mature muscle cells were obtained. Thereafter, the medium for inducing differentiation was changed every two days, and the single substances were treated to a concentration of 10 μg / ml. When grown to mature muscle cells, the cells were cultured in serum-free media for 12 hours, and then 2-NBDG (2- (N- (7-Nitrobenz-2-oxa-1,3- diazol- 4-yl) Amino) -2-Deoxyglucose), and the intracellular glucose was observed through a fluorescence microscope and a fluorescence analyzer (Infinite M200 Tecan, Austria). 2-NBDG is a structure in which the deoxy-form of glucose is bound to a fluorescent structure. It enters the cell like glucose but does not decompose, so it can accumulate fluorescence. Fluorescence was confirmed by naked eyes and compared with photographs, and the relative amounts of 2-NBDG in the cells were numerically compared using a fluorescence analyzer.
그 결과, 도 2a에 나타난 바와 같이, 디람노실메이신의 포도당 흡수율이 다른 성분들보다 현저하게 높음을 확인하였다(도 2a).As a result, it was confirmed that the glucose absorption rate of diramnosylmecin was significantly higher than that of the other components as shown in FIG. 2A (FIG. 2A).
<< 실험예Experimental Example 1-2> 1-2> 센티페드그라스Sentiped Grass 추출물의 포도당 흡수 효과 확인 Identification of glucose absorption effect of extract
상기 <실시예 1>에서 분리한 분말 형태의 센티페드그라스 추출물을 50% 메탄올(50% CGE)에 녹인 후 <실험예 1-1>과 동일한 방법으로 6.25, 12.5, 25, 50, 100μg/ml 농도로 처리하여 포도당 흡수(glucose uptake) 실험을 실시하였다. 그 결과, 도 2b에서 나타난 바와 같이 센티페드그라스 추출물의 농도가 증가할수록 세포내 포도당 흡수율이 증가하는 것을 확인하였다(도 2b). 그러나 도 2a의 결과와 비교하면, 디람노실메이신의 단일물질을 처리하는 경우 유사한 농도에서 센티페드그라스 추출물을 처리했을 때보다 포도당 흡수율이 더 많이 증가하는 것을 확인할 수 있었다. In the same manner as in EXPERIMENTAL EXAMPLE 1-1, 6.25, 12.5, 25, 50, and 100 μg / mL of the centrifuged granular extract isolated in Example 1 were dissolved in 50% methanol (50% Glucose uptake experiments were performed. As a result, it was confirmed that the intracellular glucose uptake increased as the concentration of the centipedgras extract increased as shown in FIG. 2B (FIG. 2B). However, in comparison with the results in Fig. 2a, it was confirmed that the treatment of a single substance of diramnosylmecine increased the glucose uptake rate at a similar concentration as compared with the treatment with the centipedragus extract.
<< 실험예Experimental Example 2> 인슐린 신호전달 분자의 발현 조절 2> Regulation of insulin signaling molecule expression
IR (insulin receptor), IRS-1 (insulin receptor substrate-1), GLUT4 (glucose transporter 4)는 혈중에 혈당이 높을 때, 인슐린에 의해 활성화되고 포도당을 세포내로 들어오게 유도하는 핵심 인슐린 신호전달 물질이다. derhamnosylmaysin의 인슐린 신호전달 분자들의 발현 조절 효과를 확인하기 위하여 위 3물질의 mRNA 발현과 단백질의 활성을 비교하는 실험을 실시하였다. 구체적으로, 미분화 상태의 C2C12 세포를 <실험예 1-1>에서 분화하는 방법과 동일한 방법으로 세포를 넣고 배지에서 배양하였다. 분화를 시키는 시작시점부터 디람노실메이신을 10 μg/ml의 농도로 4일간 처리를 하였으며, 이후 처리구와 무처리구의 세포들을 모아서 total RNA와 단백질들을 분리하였다. Insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) are key insulin signaling molecules that are activated by insulin and induce glucose to enter the cells when blood glucose levels are high . In order to investigate the effect of derhamnosylmaysin on the expression of insulin signaling molecules, experiments were conducted to compare the mRNA expression and protein activity of the above three substances. Specifically, the cells were cultured in a culture medium in the same manner as in the method of differentiating C2C12 cells in undifferentiated state in <Experimental Example 1-1>. From the starting point of differentiation, diramnosylmecin was treated at a concentration of 10 μg / ml for 4 days, and then total RNA and proteins were isolated by collecting the cells from the treatment and control groups.
우선, total RNA를 분리하기 위해서 Trizol reagent(MRC, Cincinnati, OH, 미국)를 이용하였으며, 이후 RT-PCR실험법을 통하여 IR, IRS-1, GLUT4의 mRNA의 발현양을 비교하였다(도 3a). 상기 total RNA 분리는 제조사의 지시대로 수행하였다. 그리고 단백질의 분리를 위하여 RIPA lysis buffer를 이용하였으며, 용해된 단백질을 분석하기 위해서 SDS-PAGE를 통하여 단백질들을 나누었다.First, Trizol reagent (MRC, Cincinnati, OH, USA) was used to isolate total RNA, and then the expression levels of IR, IRS-1 and GLUT4 mRNA were compared through RT-PCR experiments (FIG. The total RNA isolation was performed according to the manufacturer's instructions. RIPA lysis buffer was used for the separation of proteins, and proteins were separated by SDS-PAGE to analyze the dissolved proteins.
그리고, 웨스턴블롯을 통하여 나누어진 단백질들 중에서 목적 단백질인 IRS-1와 Akt의 양과 활성정도를 확인하였다(도 3b). 활성화 정도는 각 단백질의 인산화 정도를 확인함으로써 비교할 수 있다. 웨스턴블롯은 당업계에 공지된 일반적인 방법으로 수행하였고, 여기에 사용된 항체는 모두 cell signaling technology(미국)에서 구입하여 사용하였다.Then, the amount and activity of the target proteins IRS-1 and Akt among the proteins separated through Western blotting were confirmed (FIG. 3B). The degree of activation can be compared by confirming the degree of phosphorylation of each protein. Western blotting was performed by a conventional method known in the art, and all of the antibodies used therein were purchased from cell signaling technology (USA).
그 결과, 도 3a에서 나타난 바와 같이, 인슐린에 의해서 조절되는 인슐린 신호전달 인자들의 발현정도가 디람노실메이신을 처리함에 따라 증가하는 것을 확인할 수 있다. 뿐만 아니라 도 3b에서 나타난 바와 같이, 발현이 증가된 인슐린 신호전달 인자인 IRS-1은 양이 늘어난 만큼 그의 활성(인산화)도 상대적으로 증가된 패턴으로 나타나며, 이러한 현상은 하위 신호전달물질인 Akt의 활성화(인산화)를 유발한다. 이것은 디람노실메이신이 혈당을 조절할 수 있는 물질이라는 것을 의미한다(도 3). As a result, as shown in FIG. 3A, it can be seen that the degree of expression of insulin signal transduction factors regulated by insulin increases with the treatment of diramnosylchemicin. In addition, as shown in FIG. 3B, IRS-1, which is an increased insulin signal transduction factor, exhibits a relatively increased pattern of its activity (phosphorylation) as the amount thereof is increased. Activation (phosphorylation). This means that diramnosylmeicin is a substance capable of regulating blood sugar (FIG. 3).
<< 실험예Experimental Example 4> 4> PPARγPPARγ 활성 조절 Active regulation
인슐린 신호전달 분자들은 발현은 PPARγ(peroxisome proliferator-activated receptor γ)에 의하여 조절되는 것으로 이미 많은 선행연구들에 의하여 보고가 되어있다. 본 발명자들은 인슐린 신호전달 분자의 발현을 증가시키는 디람노실메이신이 PPARγ의 전사 활성에도 미치는 영향을 확인하기 위하여 발광효소분석(luciferase assay)을 실시하였다. 발광효소분석은 특정 유전자의 전사 활성을 측정하는 방법으로 특정한 유전자에 발광효소유전자를 연결한 융합유전자를 배양세포에 도입하여 그 발광량으로부터 전사활성을 측정한다. Expression of insulin signaling molecules is regulated by PPARγ (peroxisome proliferator-activated receptor γ) and has been reported by many previous studies. The present inventors performed a luciferase assay to confirm the effect of diramnosylmeicin, which increases the expression of insulin signaling molecules, on the transcriptional activity of PPARγ. Luminescent enzyme analysis is a method for measuring the transcriptional activity of a specific gene. A fusion gene in which a luminescent enzyme gene is linked to a specific gene is introduced into cultured cells, and transcription activity is measured from the amount of luminescence.
구체적으로, 293T 세포(ATCC; American Type Culture Collection, 미국)에 PPARγ와 결합 파트너인 RXRα(retinoid X receptor α)가 충분히 존재할 수 있도록 각각을 발현시킬 수 있는 플라스미드를 형질전환하여 세포에서 과발현 시켰다. 그리고 이 전사인자들이 결합하여 전사활성을 나타낼 수 있게 결합부위가 존재하고, 그 결과로 발광효소가 발현되는 플라스미드 역시 함께 형질전화 하였다. 상기 형질전환은 Lipofectamine LTX(Thermofisher, 미국)을 사용하여, 제조사의 지시대로 수행하였다. 이후, 형질전환된 293T 세포에 <실험예 1-1>에서 사용한 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질들을 10 μg/ml의 농도로 24시간 처리하였고, 그런 다음 세포를 파쇄하여 발광효소분석(luciferase assay)을 실시하였다. 발광효소분석은 Luciferase Assay System(Promega, Madison, WI, 미국)을 이용하였으며, 제조사의 지시대로 실험을 수행하였다. 그리고, 디람노실메이신의 경우 0~20 μg/ml의 농도로 다양하게 실험을 진행하였다. Specifically, a plasmid capable of expressing PPARγ and a binding partner RXRα (retinoid X receptor α) sufficiently existed in 293T cells (ATCC, American Type Culture Collection, USA) was transformed and overexpressed in cells. Also, the binding sites exist so that these transcription factors can bind and express transcriptional activity, and as a result, plasmids expressing the luminescent enzyme are also transfected. The transformation was carried out using Lipofectamine LTX (Thermofisher, USA) according to the manufacturer's instructions. Subsequently, the transformed 293T cells were treated with the single substances extracted from centipedegrass used in <Experimental Example 1-1> at a concentration of 10 μg / ml for 24 hours, and then the cells were disrupted to produce luminescence enzyme Luciferase assay was performed. Luciferase Assay System (Promega, Madison, Wis., USA) was used for the luminescence analysis and the experiment was conducted as instructed by the manufacturer. In the case of diramnosylmecin, various experiments were conducted at a concentration of 0 to 20 μg / ml.
그 결과, 도 4에 나타난 바와 같이, 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질 중에서 디람노실메이신만이 PPARγ의 활성을 유의적으로 증가시키는 것을 확인하였으며(도 4a), 이러한 활성 증가 현상은 디람노실메이신의 농도에 비례하는 것을 확인하였다(도 4b).As a result, as shown in FIG. 4, it was confirmed that diramnosylmecin alone significantly increased the activity of PPARγ among the single substances extracted and fractionated in centipedegrass (FIG. 4A) (Fig. 4B). ≪ tb > < TABLE >
상기 <실험예 1-2>에서 사용된 센티페드그라스 추출물(50% CGE)을 처리하여 발광효소분석(luciferase assay)을 실시한 결과(도 4c), 디람노실메이신의 단일물질을 처리하는 경우 센티페드그라스 추출물(50% CGE)을 처리했을 때보다 유사한 농도에서 PPARγ의 활성이 더 많이 증가하는 것을 확인할 수 있었다. (Luciferase assay) (FIG. 4c) was performed by treating the centipede glass extract (50% CGE) used in Experimental Example 1-2, and when a single substance of diramnosylmecine was treated, It was confirmed that the activity of PPARγ was increased more at a similar concentration than when treated with a glass extract (50% CGE).
<< 실험예Experimental Example 5> 5> 디람노실메이신의Diramnosylmecine PPARγ와PPARγ and RXRα의Of RXRα 결합력에 미치는 영향 Effect on binding force
디람노실메이신의 PPARγ와 RXRα의 결합력에 미치는 영향을 확인하기 위하여 면역침강법을 수행하였다. 면역침강법은 두 분자들간의 결합력을 알아보는 실험으로 하나의 분자에 표지인자를 붙이고 나머지 분자와 결합반응을 시켜준 다음 표지인자를 인식하여 분리할 수 있는 물질을 처리하여 표지인자가 붙어있는 분자만을 선별해 낸다. 이때 표지인자가 있는 분자와 다른 분자가 결합을 하고 있다면 함께 분리되는 원리이다. 이러한 방법으로 얼마나 많은 분자들이 결합을 하는지, 혹은 얼마나 빨리 결합을 하는지 확인할 수 있다.Immunoprecipitation was performed to confirm the effect of diramnosylmecin on the binding strength of PPARγ and RXRα. Immunoprecipitation is an experiment in which the binding force between two molecules is examined. After attaching a marker to one molecule and binding reaction with the other molecules, the substance capable of recognizing and separating the marker is treated, . At this time, if the molecule having the marker and another molecule are binding, the principle is separated. This way you can see how many molecules are binding, or how fast they are binding.
구체적으로, 293T 세포(ATCC; American Type Culture Collection, 미국)에 PPARγ에 표지인자를 붙여서 발현시킬 수 있는 플라스미드를 형질전환하여 세포에서 과발현시키고 이것을 파쇄하여 샘플로 이용하였다. 상기 형질전환은 Lipofectamine LTX (Thermofisher, 미국)을 사용하여, 제조사의 지시대로 수행하였다. 그런 다음, 다른 293T 세포에 RXRα를 발현시키는 플라스미드를 형질전환하여 함께 반응할 수 있는 샘플을 준비하였다. 그 후, 준비된 두 분자샘플을 이용하여 면역침강 실험을 진행하였다. 상기 샘플에 디람노실메이신을 함께 처리하여 그 활성 유무를 판단하였다. PPARγ 단백질이 과발현된 샘플에 디람노실메이신을 전처리하여 활성이 더 확실히 나타나도록 준비하였다. 이후, RXRα 단백질이 과발현된 샘플과 디람노실메이신을 전처리한 PPARγ가 과발현된 샘플을 1:1의 비율로 혼합하여 반응을 유도하였다. 이때 반응 시간을 다르게 하여 시간별로 확인하였으며, 반응이 끝나면 바로 침강시켜 상층액을 제거하고 계면활성제가 첨가된 용출액을 통하여 반응이 더 진행되지 않도록 하였다. 이후 웨스턴블롯을 통하여 PPARγ와 RXRα의 결합을 확인하였다. 웨스턴블롯은 당업계에 공지된 일반적인 방법으로 수행하였고, 여기에 사용된 항체는 모두 cell signaling technology(미국)에서 구입하여 사용하였다.Specifically, a plasmid capable of expressing PPARγ by attaching a marker to PPARγ was transfected into 293T cells (ATCC; American Type Culture Collection, USA), overexpressed in cells, and disrupted and used as a sample. The transformation was carried out using Lipofectamine LTX (Thermofisher, USA) according to the manufacturer's instructions. Then, plasmids expressing RXRa were transfected into other 293T cells to prepare samples capable of reacting together. Subsequently, the immunoprecipitation experiment was carried out using the prepared two molecular samples. The sample was treated with diramnoxyline to determine its activity. A sample in which the PPARgamma protein was over-expressed was prepared so that the activity becomes more apparent by pretreatment of diramnosylchemicin. Thereafter, a sample in which RXRα protein was overexpressed and diramnosylmecin-pretreated PPARγ-overexpressed sample were mixed at a ratio of 1: 1 to induce the reaction. At this time, the reaction time was different, and the reaction time was checked. After completion of the reaction, the reaction solution was settled to remove the supernatant, and the reaction was not allowed to proceed through the eluent with the surfactant. Thereafter, binding of PPARy with RXR [alpha] was confirmed by Western blotting. Western blotting was performed by a conventional method known in the art, and all of the antibodies used therein were purchased from cell signaling technology (USA).
그 결과, 도 5에 나타난 바와 같이, 디람노실메이신은 PPARγ와 RXRα의 결합을 더 빠르게 유도함을 확인하였다. 5분간 두 단백질의 결합반응을 유도하였을 때는 크게 차이를 보이지 않지만, 10분 동안 결합반응을 시켰을 때는 디람노실메이신을 처리해 준 군과 처리하지 않은 군 간의 차이가 확연하게 나타났으며, 30분 동안 결합반응을 시킨 군에서는 이러한 차이가 더욱 선명하게 나타나는 것을 확인하였다(도 5).As a result, as shown in Fig. 5, it was confirmed that diramnosylmecin induced binding of PPARy with RXRa more rapidly. There was no significant difference in the binding of the two proteins for 5 minutes. When the binding reaction was performed for 10 minutes, the difference between the treated and untreated diramnosylchemic groups was remarkable, And the difference was more apparent in the group to which the reaction was performed (Fig. 5).
<< 실험예Experimental Example 6> 6> 디람노실메이신이Diramnosylmeysin PPARγ와PPARγ and RXRα의Of RXRα 결합에 미치는 영향을 확인하기 위한 표면 Surface to identify the effect on binding 플라스몬Plasmon 공명 실험 Resonance experiment
디람노실메이신이 PPARγ와 RXRα의 결합에 미치는 영향을 더욱 자세하게 살펴보기 위하여 표면 플라스몬 공명(Surface plasmon resonance) 실험을 수행하였다. 표면 플라스몬 공명 실험은 분자간의 결합이 이루어지는 정도를 실시간으로 확인, 두 분자간의 결합이 이루어지는 최적의 환경을 비교 분석하여 두 분자간의 결합 친화력을 수치로 나타낼 수 있다. Surface plasmon resonance experiments were conducted to examine in more detail the effect of diramnosylmethacin on binding of PPARγ and RXRα. In the surface plasmon resonance experiment, the degree of binding between molecules can be confirmed in real time, and the optimal affinity for binding between two molecules can be compared and analyzed to reveal the binding affinity between two molecules.
구체적으로, 표면 플라스몬 공명실험을 수행하기 위하여 고정상과 이동상을 지정한다. 고정상은 골드칩에 코딩되어 이동상이 일정속도로 흘러갈 때 이동상에 존재하는 표적분자와 결합하게 된다. 이때 이동상에 들어있는 분자의 농도가 결합 친화력을 나타내는데 중요한 역할을 한다. 고정상으로 PPARγ 단백질을 골드칩에 코팅하여 준비를 하였고, 이동상으로 RXRα 단백질을 PBS 버퍼에 녹여서 준비하였다. 이 실험에서 사용된 PPARγ와 RXRα 단백질은 CUSABIO에서 제조된 것을 구입하여 사용하였다. PPARγ 단백질이 코팅된 골드칩을 Reichert SR7500DC system에 장착하여 실험을 준비한다. 그리고 이동상인 RXRα 단백질을 흘려보내기 전에 비교군으로 사용될 로지글리타존이나 실험군으로 사용될 디람노실메이신을 전처리로 흘려준다. 5분간 전처리로 디람노실메이신이나 로지글리타존을 흘려준 후, RXRα 단백질이 포함된 이동상을 흘려주어 결합 친화력을 비교한다. 결합 친화력을 분석하기 위해서 Scrubber2 소프트웨어를 사용하였으며, 이 실험은 우정 BSC Inc. 에서 진행 되었다. Specifically, a stationary phase and a mobile phase are designated to perform surface plasmon resonance experiments. The immobilized phase is coded on the gold chip, and when the mobile phase flows at a constant rate, it binds to the target molecule present in the mobile phase. At this time, the concentration of molecules in the mobile phase plays an important role in showing the binding affinity. The PPARγ protein was coated on a gold chip in a stationary phase, and the RXRα protein was prepared as a mobile phase by dissolving the protein in PBS buffer. The PPARγ and RXRα proteins used in this experiment were purchased from CUSABIO. The gold chip coated with the PPAR gamma protein is mounted on the Reichert SR7500DC system to prepare the experiment. Before dripping the mobile phase of the RXRα protein, diramnosylcysine to be used as a comparator or to be used as an experimental group is flowed into the pretreatment. Diramnosylmethacin or roglitazone is flowed through the pretreatment for 5 minutes, and then the mobile phase containing the RXRα protein is poured to compare the binding affinities. Scrubber2 software was used to analyze the binding affinity. .
그 결과, 표 4에 나타난 바와 같이 디람노실메이신의 처리는 PPARγ와 RXRα의 결합을 약 3배 증가시키는 것으로 확인되었으며, 이것은 당뇨병 치료제로 이미 알려진 로지글리타존(rosiglitazone)가 보여주는 수치보다 높은 것으로 나타난다. As a result, as shown in Table 4, the treatment of diramnosylmecin was found to increase the binding of PPARγ and RXRα by about 3 times, which is higher than that of rosiglitazone, which is already known as a therapeutic agent for diabetes.
<실험예 7-1> PPARγ단백질에서 디람노실메이신의 결합부위 확인EXPERIMENTAL EXAMPLE 7-1 Confirmation of binding site of diramnosylmecine in PPARy protein
인슐린 신호전달 인자들의 발현을 조절하는 PPARγ의 활성은 이미 잘 알려져 있는 리간드 결합 부위에 어떠한 물질이 결합하여 그 활성을 높이게 된다. 이때 리간드 결합 부위에서도 473번째의 아미노산 서열인 타이로신 잔기가 깊게 관여를 한다고 보고가 되어있다. 따라서 디람노실메이신도 PPARγ의 잘 알려진 부위에 결합하여 단백질의 활성을 높이는지 확인하기 위하여, PPARγ의 리간드 결합 부위만이 존재하며 직접적으로 리간드 결합 부위에 결합하여야 특정한 활성을 나타나게 되는 분석키트((LanthaScreen TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit; Invitrogen, CA)를 이용한 실험을 수행하였다. The activity of PPARγ, which regulates the expression of insulin signaling factors, is enhanced by the binding of certain substances to well-known ligand binding sites. It is reported that the 473rd amino acid sequence tyrosine residue deeply participates in the ligand binding site. Therefore, in order to confirm whether diramnosylmethionine binds to a well-known site of PPARγ to increase the activity of the protein, an assay kit ((LanthaScreen) in which only a ligand binding site of PPARγ exists and a specific activity is required to bind directly to the ligand binding site TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit (Invitrogen, CA).
구체적으로, PPARγ의 리간드 결합 부위에 결합을 하는지 분석하기 위하여 <실시예 1>의 방법으로 센티페드그라스(centipedegrass)에서 추출 분획된 단일물질(디람노실메이신(derhamnosylmaysin), 오리엔틴(orientin), 람노실오리엔틴(rhamnosylisoorientin), 메이신(maysin), 이레모네틴(eremonetin(luteolin-6-C-boivinopyranoside)), 이소오리엔틴(isoorientin), 클로로젠산(cholorogenic acid), 루테올린(luteolin))을 메탄올에 녹여 준비한다. 이때 단일물질들의 농도는 최종 처리 농도가 10 μg/ml이 되게 한다. 그리고 PPARγ의 리간드 결합 부위에 결합하여 그 활성을 증가시키는 GW1929와 리간드 결합 부위에 결합하여 그 활성을 억제시키는 GW9662을 비교군으로 준비하여 함께 실험을 진행하였다. 실험 방법은 분석키트에서 제공하는 방법대로 실행하였다.Specifically, a single substance extracted from centipedegrass (derhamnosylmaysin, orientin, and the like) was analyzed by the method of Example 1 to analyze whether it binds to the ligand binding site of PPARγ. Isomers of rhamnosylisoorientin, maysin, eremonetin (luteolin-6-C-boivinopyranoside), isoorientin, cholorogenic acid, luteolin, ) Is dissolved in methanol and prepared. At this time, the concentration of the single substances makes the
그 결과, 도 6에서 나타나 바와 같이 디람노실메이신을 처리한 경우 PPARγ의 활성이 증가되지 않는 것으로 보아, 디람노실메이신은 잘 알려진 PPARγ의 리간드 결합 부위에 결합하지 않는 것으로 확인되었다. As a result, as shown in FIG. 6, when diramnosylmecin was treated, the activity of PPARγ was not increased, and it was confirmed that diramnosylmecin did not bind to the well-known ligand binding site of PPARγ.
<실험예 7-2> PPARγ단백질에서 디람노실메이신의 결합부위 확인<Experimental Example 7-2> Confirmation of diramnosylmecine binding site in PPARγ protein
디람노실메이신이 PPARγ의 활성을 증가시키는데 있어, 이미 알려진 부위에 결합하지 않는 것으로 확인되었으므로, 본 발명자들은 디람노실메이신이 PPARγ의 어느 부위에 결합하는지 컴퓨터 모델링을 통하여 검색하였다.Since diramnosylmethacin has been found not to bind to known sites in increasing the activity of PPARy, the present inventors have searched through computer modeling which part of diramnosylmeicin binds to PPARy.
구체적으로, 컴퓨터 모델링을 위하여 문헌으로 보고가 되어있는 PPARγ의 엑스레이 크리스탈 구조(PDB ID: 4JAZ)를 바탕으로 실험을 진행하였다. 컴퓨터 모델링을 위한 디람노실메이신과 PPARγ의 수치 계산은 GOLD Suite 5.2.2(The cambridge Crystallographic Data Center, 영국) 프로그램을 이용하였다. Discovery Studio (DS) 3.5 프로그램의 Clean Protein 프로토콜을 이용하여 디람노실메이신이 PPARγ에 결합 가능한 부위를 분석하였다. PPARγ의 아미노산 잔기와 디람노실메이신 분자간의 결합이 15Å 이내인 모든 결합가능한 부위를 일차적으로 선별하고, 이후 Minimization 프로토콜을 이용하여 가장 결합 가능성이 높은 두 부위를 선정하였다. Specifically, experiments were conducted based on the X-ray crystal structure (PDB ID: 4JAZ) of PPARγ reported in the literature for computer modeling. Numerical calculations of diramnosylcysine and PPARγ for computer modeling were performed using the GOLD Suite 5.2.2 (The Cambridge Crystallographic Data Center, UK) program. Using the Clean Protein protocol of the Discovery Studio (DS) 3.5 program, diramnosylmethacin was analyzed for binding sites to PPARγ. All binding sites within 15 Å of the amino acid residues of PPARγ and the diramnosylmecin molecule were first selected, and then the two sites most likely to bind were selected using the Minimization protocol.
그 결과, 도 7에서 나타난 바와 같이 디람노실메이신이 PPARγ과 결합할 것으로 예상되는 부위로 아직까지 알려지지 않은 다른 한 부위가 선정되었다. 알려지지 않은 새로운 부위에서 디람노실메이신이 결합하는데 관여할 것으로 추정되는 아미노산 서열들은 Tyr320, His323, Glu324, Arg443, Gln444, Thr447, Ile472잔기들로 나타난다. 그 중에서 결합력이 강력한 Glu324, Arg443, Thr447이 중요할 것으로 판단된다(도 8).As a result, as shown in Fig. 7, another site not yet known was selected as a site where diramnosylmeicin is expected to bind PPARy. The amino acid sequences that are believed to be involved in binding of diramnosylchemicin in unknown new sites are represented by Tyr320, His323, Glu324, Arg443, Gln444, Thr447, Ile472 residues. Among them, Glu324, Arg443 and Thr447, which have strong binding strength, are considered to be important (FIG. 8).
<실험예 7-3> PPARγ단백질에서 디람노실메이신의 결합부위 확인<Experimental Example 7-3> Confirmation of diramnosylmecine binding site in PPARγ protein
디람노실메이신과 PPARγ이 결합하는 것으로 예상되는 Tyr320, His323, Glu324, Arg443, Gln444, Thr 447, Ile472 아미노산 염기서열 중에서, 결합에 있어 중요한 역할을 할 것으로 예상되는 아미노산으로 Glu324, Arg443, Thr447을 선정하였다(도 8). Glu324, Arg443, and Thr447 were selected as the amino acids expected to play an important role in binding among the amino acid sequences of Tyr320, His323, Glu324, Arg443, Gln444, Thr 447, and Ile472, which are expected to bind PPARγ with diramnosylcysine (Fig. 8).
본 발명자들은 상기 아미노산의 점 돌연변이(point mutation)를 통하여 PPARγ의 활성을 조절하는데 디람노실메이신이 직접적으로 관여하며, 이러한 현상은 알려지지 않은 부위에 결합하여 이루어 질 것이라는 것을 luciferase activity를 통하여 확인하였다. The present inventors have confirmed through the luciferase activity that diramnosylmethacin is directly involved in regulating the activity of PPARγ through point mutation of the above amino acid and that this phenomenon will be linked to an unknown site.
구체적으로, PPARγ의 점 돌연변이를 발생시키기 위하여 각 부위의 DNA염기서열에서 하나의 염기서열을 교체하는 방법을 사용하였고, 이 실험을 위하여 QuikChange ⅡXL Site Direacted Mutagenesis Kit (Agilent, 미국)를 사용하였으며, 실험 방법은 제조사에서 제공하는 메뉴얼대로 진행하였다. 점 돌연변이를 발생시킨 PPARγ는 각각의 아미노산 서열이 Ala로 치환 되었다. 이렇게 점 돌연변이가 된 PPARγ를 발현시키는 플라스미드를 293T 세포에 형질전환 시켜서 luciferase assay를 진행하였다. 이때 실험 방법은 <실험예 4>에서 사용한 것과 동일하게 진행하였으며, 단지 PPARγ를 과발현 시키는 플라스미드만 점 돌연변이가 이루어진 것으로 대체하여 실험을 진행하였다. 이렇게 형질전환된 293T 세포에 디람노실메이신을 10 μg/ml와 20 μg/ml의 농도로 24시간 동안 처리하였고, 이후 세포들을 파쇄하여 luciferase assay에 사용하였다. Specifically, in order to generate a point mutation of PPARγ, a single base sequence was substituted for the DNA sequence of each site, and QuikChange IIXL Site Directed Mutagenesis Kit (Agilent, USA) was used for this experiment. The method was carried out according to the manual provided by the manufacturer. Each amino acid sequence of PPARγ that caused point mutation was replaced with Ala. The plasmid expressing the point mutated PPARγ was transformed into 293T cells and luciferase assay was carried out. At this time, the experimental procedure was the same as that used in Experimental Example 4, except that the plasmid only overexpressing PPARγ was replaced with a point mutation. The transformed 293T cells were treated with diramnosylchemicin at concentrations of 10 μg / ml and 20 μg / ml for 24 hours, and then cells were disrupted and used for luciferase assay.
그 결과, 도 9에 나타난 바와 같이 정상적인 아미노산 염기서열을 가지는 PPARγ은 디람노실메이신에 의하여 그 활성이 증가하는 것을 확인할 수 있다. 또한 443번째 아미노산인 글루타메이트(glutamate)를 알라닌(alanine)으로 치환한 경우 PPARγ의 활성이 더욱 증가되는 것을 확인하였다. As a result, as shown in FIG. 9, it can be confirmed that the activity of PPARγ having a normal amino acid sequence is increased by diramnosylmethacin. It was also confirmed that the activity of PPARγ was further increased when alanine (glutamate), which is the 443rd amino acid, was substituted with alanine.
하지만, 447번째 아미노산인 트레오닌(threonine)을 알라닌(alanine)으로 치환한 PPARγ는 디람노실메이신에 의한 PPARγ의 활성 증가가 나타나지 않는 것을 확인하였다. 이것은 PPARγ에 디람노실메이신이 결합할 수 없어 활성을 증가시키지 못하는 것으로 판단된다. However, PPARγ in which threonine, which is the 447th amino acid, was replaced with alanine showed no increase in the activity of PPARγ due to diramnosylmecin. This suggests that diramnosylmethacin can not bind to PPARγ and thus does not increase its activity.
이것으로 디람노실메이신은 인슐린 신호전달 인자들의 발현을 조절하는 전사인자의 활성을 조절함으로써 세포내 포도당을 유입하여 비정상적인 혈당을 조절하는데, 이때 전사인자의 이미 잘 알려진 부위에 결합을 하여지 않고, 알려지지 않는 부위에 결합하여 활성을 조절하고, 이때 447번째 아미노산인 트레오닌(threonine)이 중요한 역할을 하는 것으로 확인되었다.Thus, diramnosylchemicin modulates the activity of transcription factors that regulate the expression of insulin signaling factors, thereby regulating abnormal glucose levels by introducing intracellular glucose, which does not bind to already well known regions of the transcription factor, (Threonine), which is the 447th amino acid, plays an important role.
Claims (9)
[화학식 1]
A pharmaceutical composition for prevention and treatment of diabetes comprising derhamnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]
The pharmaceutical composition for preventing and treating diabetes according to claim 1, wherein the composition increases glucose uptake into muscle cells.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the composition increases PPARy (peroxisome proliferator-activated receptor gamma) activity.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the composition activates an insulin signaling pathway.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the diabetes is type 1 diabetes or type 2 diabetes.
[화학식 1]
A dietary supplement for preventing and improving diabetes comprising deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]
7. The dietary supplement according to claim 6, wherein the diabetes is type 1 diabetes or type 2 diabetes.
[화학식 1]
A pharmaceutical composition for lowering blood glucose comprising diramnosylmecine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]
[화학식 1]
A health functional food for hypoglycemia comprising diramnosylmecine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]
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