Development of analytical method for esculetin in medicinal plants using LC-MS/MS and esculetin-induced growth inhibition effect in vascular smooth muscle cells

윤은선 건국대학교 대학원 2012 국내박사

A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis method was developed to determine the contents of esculetin in medicinal plants. The analysis was performed using multiple reaction monitoring (MRM) in negative mode, and an XBridgeTM C18 column (2.1×100 mm, 3.5 m) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r2>0.9993). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.07 ng/ml respectively. The intra- and inter-day precisions were 1.5-6.8% and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini cortex showed the highest content of esculetin (761.5-5,475.0 mg/kg). In Mori folium and Artemisiae capillaris herba, 5.2-21.5 mg/kg and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive, and reproducible. The pharmacological effects of esculetin isolated from medicinal plants were investigated as part of the new medicines development. Esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes.

Esculetin의 A253 인간 두경부 편평세포 암종에서 세포 사멸 단백질 조절을 통한 항 종양 효과

박수빈 전북대학교 일반대학원 2021 국내석사

Esculetin is a natural lactone commonly derived from coumarin. In several human cancer types, this compound is applied as a useful chemotherapeutic agent. This compound is used for proliferation hindering and apoptosis induction of the cancer cells. In the present study, the esculetin anti-proliferative characteristic on head and neck squamous cancer cell line, A253, was investigated. In vitro and in vivo analysis were utilized for this evaluation. The apoptotic effects of esculetin were examined through MTT assay, TUNEL assay, apoptosis protein array, RT-PCR, and western blot analysis in both A253 cells and a xenograft model of head and neck cancer. Experiments have confirmed through MTT analysis that the proliferation of A253 cells treated with esculetin was significantly reduced, and TUNEL analysis has confirmed the increase in apoptosis positive cells. In addition, western blot analysis, protein array and RT-PCR confirmed that signal pathways to regulate apoptosis by esculetin were increased. In animal experiments using xenograft models, the anti-tumor effect was identified through apoptosis by esculetin. The results showed antiproliferative impact of esculetin on the growth of A253 human head and neck squamous cell carcinoma. The observed effect depended on dose and time of the treatment. Cell growth and induced apoptosis were significantly declined A253 treatment, regarding to the regulation of apoptosis regulatory protein. Overall, the observed results illustrated that esculetin could be a great candidate as an oral anticancer drug in the head and neck squamous cell carcinoma cases. Esculetin (6,7-dihydroxycoumarin)은 coumarin 유도체이다. 여러 유형의 인간 암세포에서 증식을 억제하고 세포 사멸을 유도하는 것으로 알려져 있으며 유망한 화학 요법 제로 간주된다. 본 연구의 목적은 in vitro 및 in vivo를 통해 두 경부 암 세포주 A253에 대한 esculetin의 항 증식 효과를 조사하는 것이다. 이 실험에서 MTT assay, TUNEL assay, apoptosis protein array, RT-PCR, western blot analysis, H&E staining 및 A253 세포를 이용한 인간 두경부 암종 이종 이식 모델에 대한 esculetin의 세포 사멸 효과를 측정했다. 실험 결과 esculetin을 처리한 A253 세포의 증식이 현저히 감소된 것을 MTT assay를 통해 확인했고, TUNEL assay 결과 apoptosis에 양성인 세포가 증가된 것을 확인할 수 있었다. 또한 western blot analysis, protein array 및 RT-PCR을 통해 esculetin에 의해 세포사멸을 조절하는 signal pathway가 늘어나는 것을 검증하였다. 이종 이식 모델을 이용한 동물실험에서도 esculetin에 의한 세포사멸을 통해 항 종양 효과가 확인되었다. 종합하면, 본 연구의 결과는 esculetin이 A253 인간 두경부 편평 세포 암종 (HNSCC) 세포의 성장에 용량 및 시간 의존적 방식으로 항 증식 효과를 보여주었다. A253 세포에 esculetin을 처리하면 세포 사멸 조절 단백질의 조절에 의해 성장이 크게 감소하고 세포 사멸이 유도되었다. 이는 esculetin이 A253 세포에서 세포 사멸 조절 단백질을 조절하여 세포 성장을 억제하고 세포 사멸을 유도하여 구강암에 대한 강력한 항암제 후보임을 나타낸다.

천연물 유래 esculetin과 적혈구 생성인자 erythropoietin의 역할과 효과 규명

박성석 충주대학교 산업대학원 2012 국내석사

본 연구에서는 천연물 한인진(韓茵蔯)이라 불리는 사철쑥에서 추출한 천연 유래 물질인 esculetin을 이용하여 대장암의 항암 효과 및 작용기전 을 확인 하였으며, 적혈구 형성 1차 조절 인자인 erythropoietin (EPO)이 방광암과 동맥 경화의 주요 병태 요인인 혈관 평활근세포 (VSMC)의 증 식에 미치는 영향을 in vitro 상의 실험을 통해 분자생물학적 기초 자료로 제시하고자 연구하였다. 연구방법 1. Esculetin의 대장암 작용기작 및 항암 효과 학인. esculetin을 농도별 처리하여 MTT assay와 [3H]Thymidine incorporation assay 를 이용하여 세포 성장억제 및 DNA 합성능력을 확인하였다. cell cycle analysis (FASC)를 이용하여 세포주기를 확인하였으며, cell cycle 조절인자인 cyclins/CDKs의 변화를 실험하였다. cell cycle negative 조절 단백질 p21WAF1, p27, p53의 증가를 확인하였으며, 세포 신호전달을 하는 MAP kinase (ERK, JNK/SAPK, P38) 의 인산화를 실험하였다. 2. Erythropoietin (EPO)의 방광암 증식 작용기작 및 효과 확인. 세포의 전이 및 이동성을 확인하기 위하여 wound healing migration assay를 실험 하였으며, 세포의 전이와 증식에 관련이 있다고 알려진 MMP-2와 MMP-9 효소를 zymography 실험으로 확인하였다. 증식과 전이에 중요하다고 알려진 전사인자인 NF-κB, SP-1, AP-1의 활성은 EMSA (Electrophoresis mobility shift assay)를 통해 확인 하였다. Immuno blot를 통해 신호전달계인 ERK, JNK/SAPK, p38 MAP kinase를 실험하여 신호전달 경로를 확인하였고, MAP kinase inhibitor 와 EPO receptor siRNA를 이용하여 결과에 대한 확인 실험을 하였다. 3. 혈관평활근세포(VSMC)에서의 EPO에 의한 세포 증식 및 작용기작 확인 Wound healing migration assay를 통해 세포 이동성을 확인하였으며, zymography를 이용하여 MMP-2, MMP-9 발현을 확인하였다. 핵내 전사인자( NF-κB, SP-1, AP-1 )는 electrophoresis mobility shift assay (EMSA) 법을 이용하여 확인 하였으며, MAP kinase ( ERK, JNK/SAPK, p38 MAP kinase )의 인산화 활성은 immunoblot 법으로 확인했다. 결과 1. 대장암 HCT116 세포의 MTT assay와 [3H]Thymidine incorporation 결과 세포 증식 능력과 DNA 합성 능력이 esculetin 농도 의존적으로 저해되었고, G1-cell cycle의 조절인자인 CDKs와 cyclins 복합체 형성을 억제 시키면서 증식을 저해 한다는 것을 알 수 있었으며, 또한 p27KIP의 활성화가 G1-cell cycle arrest와 ERK1/2의 인산화에 관련 되었다는 것을 확인 하였다. 2. 5637 세포에 EPO 처리 시 EPO receptor를 통해 ERK1/2, JNK/SAPK, p38 MAP kinase의 활성이 증가되었고, 또한 NF-κB, AP-1, SP-1의 증가 활성을 통하여 MMP-9 유전자를 발현 하였다. T24 세포는 EPO receptor를 통해 ERK1/2 신호가 전달되어 전사인자 NF-κB, SP-1를 활성화시켜 MMP-9 gene expression을 유도 하였며, 결과적으로 세포의 전이가 일어났다. 3. 혈관평활근세포 VSMC는 EPO 처리로 인해 MAP kinase (ERK1/2, p38) 인산화를 증가시켰으며, 이로 인해 NF-κB, AP-1의 활성이 생겨나고 최종적으로 MMP-9 gene expression을 유도 하어 세포 전이가 일어났다. This study examined the anticancer effect and action mechanism of esculetin, which is natural substance extracted from Artemisia iwayomogi Kitamura called Haninjin (韓茵蔯), against colon cancer and analyzed the effects of erythropoietin (EPO), primary regulator of erythropoiesis, on the multiplication of vascular smooth muscle cell(VSMC), the major pathological factor of bladder cancer and arteriosclerosis, through in vitro experiments in order to provide molecular data. 1. Esculetin, a phenolic compound, has inhibited the growth of colon tumors in animal study. However, the roles of signaling pathways and cell cycle regulation in esculetin-induced inhibition of cancer cell growth remain to be examined. The present study suggests a novel mechanism of the Ras/ERK1/2 pathway in esculetin-treated human colon cancer HCT116 cells. Treatment of cells with esculetin resulted in significant growth inhibition and G1 phase cell cycle arrest, which led to down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1 phase cell-cycle arrest was associated with up-regulation of p27KIP expression. In addition, ERK1/2 was activated by esculetin. Pretreatment of cells with the MEK1/2-specific inhibitor, PD98059, blocked p27KIP expression induced by esculetin. Blockage of the ERK1/2 function consistently prevented the inhibition of cell proliferation and decreased G1 phase cell cycle proteins. Furthermore, Ras activation was increased by esculetin treatment. Transient transfection of the dominant negative Ras (RasN17) mutant gene abolished both ERK1/2 activity and p27KIP expression induced by esculetin. Finally, overexpression of RasN17 suppressed the esculetin-induced reduction in cell proliferation and cell cycle proteins. In conclusion, these results indicate that the Ras/ERK1/2 pathway is mediated by p27KIP1 induction, leading to a reduction in cyclin /CDK complexes in esculetin-induced inhibition of colon cancer cell growth. Overall, these findings indicate that the molecular action of esculetin has therapeutic potential for the treatment of colon malignancies. 2. Analysis of the action mechanism and effect of erythropoietin (EPO) on the multiplication of bladder cancer cells. we conducted an experiment using wound healing migration assay in order to see cell metastasis and motility, and examined enzymes MMP-2 and MMP-9 known to be involved in cell metastasis and multiplication through zymography experiment. The activity of transcription factors NF-κB, SP-1 and AP-1 known to be important in multiplication and metastasis was tested through EMSA (electrophoresis mobility shift assay). In addition, signal transduction systems ERK, JNK/SAPK and p38 MAP kinase were tested through immuno blot, and the results were verified using MAP kinase inhibitor and EPO receptor siRNA. According to the results, when 5637 cell was treated with EPO, the activity of ERK1/2, JNK/SAPK and p38 MAP kinase increased through the EPO receptor, and MMP‐9 gene was expressed through the increased activity of NF-κB, AP-1 and SP-1. For T24 cell, ERK1/2 signal was transduced through the EPO receptor and activated transcription factors NF-κB and SP-1, and this induced the expression of MMP-9 gene and consequently cell metastasis occurred. 3. Analysis of cell proliferation and action mechanism induced by EPO in vascular smooth muscle cell (VSMC). cell motility was measured through wound healing migration assay, and the expression of MMP-2 and MMP-9 was examined through zymography. Intranuclear transcription factors (NF-κB, SP-1 and AP-1) were analyzed by electrophoresis mobility shift assay (EMSA), and the phosphorylation activity of MAP kinases (ERK, JNK/SAPK, and p38 MAP kinase) was tested by immunoblot. According to the results, due to treatment with EPO, VSMC increased the phosphorylation of MAP kinase (ERK1/2, p38), which in turn activated NF-κB and AP-1 and, ultimately, induced the expression of MMP-9 gene and cell metastasis. key word: Esculetin, colon cancer, erythropoietin (EPO), vascular smooth muscle cell (VSMC), bladder cancer cells, p27KIP, ERK1/2, MMP-2, MMP-9, NF-κB, SP-1, AP-1, EMSA (electrophoresis mobility shift assay), zymography, wound healing migration assay, MAP kinases (ERK, JNK/SAPK, and p38 MAP kinase).

Esculetin의 RAW 264.7 세포 및 제브라피쉬 파골세포 분화에 대한 영향

안은미 숙명여자대학교 교육대학원 2020 국내석사

Bone remodeling system is performed by balanced interaction between bone resorption of osteoclasts and bone formation of osteoblasts. Bone remodeling is important to maintain normal bone condition and bone homeostasis. The mechanism of metabolic bone diseases like osteoporosis is regulated by osteoclasts and osteoblasts. In this study, effects of esculetin on osteoclast differentiation and bone formation were investigated. Esculetin is a naturally occurring coumarin compound isolated from various plant species like Artemisia scoparia. Previous studies show that esculetin has pharmacological activities such as antioxidant, anti-inflammatory activities and also inhibits osteoclastogenesis. Effects of esculetin on inhibition of osteoclast differentiation were investigated in RAW 264.7 cells and zebrafish (Danio rerio). Esculetin inhibits mRNA expression of osteoclast-specific markers such as TRAP (TRACP, tartrate-resistant acid phosphatase), cathepsin K, MMP-9 (matrix metalloproteinase-9), and suppresses TRAP activity in RAW 264.7 cells. The whole-mount skeletal staining (Alizarin red staining) was performed in zebrafish larvae. As a result, the area of stained bone tissue is decreased in osteoporosis-induced group of prednisolone treatment. But the area of stained bone tissue is increased in esculetin-treated group, and it shows that bone formation is increased. Esculetin inhibits mRNA expression of osteoclast-related genes such as RANKL (receptor activator of nuclear factor-κB ligand), NFATc1 (nuclear factor of activated T cell, cytoplasmic 1), TRAP (TRACP, tartrate-resistant acid phosphatase) in zebrafish. Also, effects of esculetin on TGF-β1 (transforming growth factor-β1) gene were investigated. Previous studies show that TGF-β1 is one of the important regulator factors of bone remodeling. Esculetin suppresses mRNA expression of TGF-β1 in RAW 264.7 cells and zebrafish. These results show that esculetin inhibits osteoclastogenesis and also is involved in regulation of TGF-β signaling related to bone remodeling. 골 리모델링은 오래된 골 조직은 제거되고, 새로운 골 조직이 형성되면서 일생 동안 일어나는 지속적인 골 재형성 과정이다. 골 리모델링은 파골세포의 골 흡수 작용과 조골세포의 골 형성 작용의 균형적인 상호작용에 의해 이루어지며, 정상적인 골 상태와 골 항상성 유지에 중요하다. 골다공증과 같은 대사성 골 질환과 관련된 기전은 파골세포와 조골세포에 의해 조절된다. 본 연구에서는 esculetin이 파골세포 분화와 골 형성에 미치는 영향에 대해 살펴보았다. Esculetin은 쑥과 같은 다양한 식물에서 추출되는 coumarin 유도체로 항산화, 항염증 등과 같은 약리학적 활성을 가지고 있을 뿐만 아니라, 파골세포의 분화를 억제한다고 보고되어 있다. RAW 264.7 세포와 zebrafish (Danio rerio)를 사용해 esculetin의 파골세포 분화 억제에 대한 효과를 살펴보았다. Esculetin은 RAW 264.7 세포에서 파골세포 분화의 특이적 마커인 TRAP (TRACP, tartrate-resistant acid phosphatase), cathepsin K, MMP-9 (matrix metalloproteinase-9)의 mRNA 발현과 TRAP activity를 감소시켰다. Zebrafish larvae를 이용한 in vivo 시험에서 whole-mount skeletal staining (Alizarin red staining)을 실행한 결과, prednisolone을 처리하여 골다공증을 유도한 군에서는 골 염색 정도가 감소하였으나, prednisolone과 esculetin을 함께 처리한 군에서는 골 염색 정도가 다시 증가되어 골 형성을 유도하는 것을 확인하였다. Esculetin은 zebrafish에서 파골세포 관련 유전자인 RANKL (receptor activator of nuclear factor-κB ligand), NFATc1 (nuclear factor of activated T cell, cytoplasmic 1), TRAP (TRACP, tartrate-resistant acid phosphatase)의 mRNA 발현을 저해하는 것을 확인하였다. 또한, esculetin이 골 리모델링에 중요한 조절인자 중 하나로 보고된 TGF-β1 (transforming growth factor-β1)의 발현에 미치는 영향에 대해서도 살펴보았다. Esculetin은 RAW 264.7 세포와 zebrafish에서 TGF-β1의 mRNA 발현을 감소시켰다. 이를 통해 esculetin이 파골세포 형성을 억제하는 효과가 있을 뿐만 아니라, 골 리모델링과 관련된 TGF-β signaling의 조절에 관여한다는 것을 확인하였다.

Protective effect of esculetin against oxidative stress induced cell damage via scavenging reactive oxygen species : 산화적인 스트레스에 의해 유도된 세포손상에서 활성산소 제거를 통한 Esculetin의 세포보호 효과

Kim, So Hyung 제주대학교 대학원 2009 국내박사

Esculetin(6, 7-dihydroxycoumarin)은 phenol성의coumarin 유도체로서 Fraxius chinensis (물푸레나무)의 피층에 주로 함유되어 있다. 본 논문에서는 esculetin의 항산화 작용을 조사하여 H₂O₂에 의한 중국 햄스터 폐 섬유아 세포(V79-4) 손상에 대해 세포 보호 효과가 나타나는지를 연구 하였다. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, 그리고 세포 내 활성 산소종에 의한 라디칼 소거율을 측정하였더니 esculetin에 의해 DPPH radical 제거, hydroxyl radical 제거와 세포 내 활성 산소종 제거 능력을 보였다. 지방 과산화의 척도인 thiobarbituric acid 와 반응하는 물질을 측정한 결과 esculetin이 지방 과산화를 방지함을 알 수 있었다. Protein carbonyl ELISA kit 를 이용하여 단백질 과산화의 척도인 carbonyl formation을 측정한 결과 esculetin에 의해 단백질에서 carbonyl formation 도 방지됨을 확인하였다. 또한, western blot과 면역형광상을 통해 세포 DNA 손상을 측정한 결과 esculetin을 처리하였을 때 DNA손상을 억제함을 알 수 있었다. 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide 법으로 세포 생존능을 측정하였고 역시 esculetin이 H₂O₂에 노출된 세포의 생존능을 회복시켰다. 결론적으로 esculetin 이 그 항산화 작용을 통해 산화 스트레스를 효과적으로 줄일 수 있으므로 기능성 식품이나 화장품의 원료 개발에 이용될 수 있을 것으로 생각한다. Aim: To investigate the antioxidant properties of esculetin (6, 7-dihydroxycoumarin) against H₂O₂-induced Chinese hamster lung fibroblast (V79-4) damage. Methods: The radical scavenging activity was assessed by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, and intracellular reactive oxygen species (ROS). In addition, lipid peroxidation was assayed by the measure of related substances which react with thiobarbituric acid. The amount of carbonyl formation in protein was determined using a protein carbonyl ELISA kit. As well, cellular DNA damage was detected by western blot and immuno-fluorescence image. Cell viability was assessed by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide assay. Results: Esculetin exhibited DPPH radical scavenging, hydroxyl radical scavenging, and intracellular ROS scavenging activities. The radical scavenging activity of esculetin resulted in the protection of cells from lipid peroxidation, protein carbonyl, and DNA damage induced by H₂O₂. Therefore, esculetin recovered cell viability exposed to H₂O₂. Conclusion: Esculetin efficiently attenuated the oxidative stress induced cell damage via its antioxidant properties. As a result, esculetin may be useful in the development of functional food and raw materials of medicine.

Esculetin inhibited cell proliferation through the Ras/ERK1/2 pathway in human colon cancer cells : 대장암 억제 효능을 지닌 에스큘레틴의 작용 기전 연구

박성규 상지대학교 2012 국내박사

Esculetin, a phenolic compound, has inhibited the growth of colon tumors in animal study. However, the roles of signaling pathways and cell cycle regulation in esculetin-induced inhibition of cancer cell growth remain to be examined. The present study suggests a novel mechanism of the Ras/ERK1/2 pathway in esculetin-treated human colon cancer HCT116 cells. Treatment of cells with esculetin resulted in significant growth inhibition and G1 phase cell cycle arrest, which led to down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1 phase cell-cycle arrest was associated with up-regulation of p27KIP expression. In addition, ERK1/2 was activated by esculetin. Pretreatment of cells with the MEK1/2-specific inhibitor, PD98059, blocked p27KIP expression induced by esculetin. Blockage of the ERK1/2 function consistently prevented the inhibition of cell proliferation and decreased G1 phase cell cycle proteins. Furthermore, Ras activation was increased by esculetin treatment. Transient transfection of the dominant negative Ras(RasN17) mutant gene abolished both ERK1/2 activity and p27KIP expression induced by esculetin. Finally, overexpression of RasN17 suppressed the esculetin-induced reduction in cell proliferation and cell cycle proteins. In conclusion, these results indicate that the Ras/ERK1/2 pathway is mediated by p27KIP1 induction, leading to a reduction in cyclin /CDK complexes in esculetin-induced inhibition of colon cancer cell growth. Overall, these findings indicate that the molecular action of esculetin has therapeutic potential for the treatment of colon malignancies. 페놀화합물의 일종인 에스큘레틴은 동물실험에서 결장암 용종의 성장을 억제하였다. 그러나 암세포 성장에서 에스큘레틴 유발 억제 경로와 세포주기 조절신호의 역할은 아직 연구해야 할 부분이다. 현재 에스큘레틴으로 처리된 연구에서 인간 결장암 HCT116 세포에서 새로운 Ras/ERK1/2 경로 기작이다. 에스큘레틴으로 세포를 치료한 결과 상당한 억제효과와 cyclins의 하향조절과 cyclin 의존 키나제 (CDK) 발현을 이끄는 G1 단계 세포주기 체포효과를 보여주었다. G1 단계 세포주기 체포는 p27KIP 발현의 상향조절과 관련 있다. 또한, ERK1/는 에스큘레틴에 의해 활성화 되었다. MEK1/2의 특이적 억제제로 전 처리된 PD98059는 에스큘레틴에 의해 유도된 p27KIP 발현을 억제하였다. ERK1/의 기능장애가 지속적으로 세포 증식의 억제를 방해하고 G1 단계 세포주기 단백질을 감소시켰다. 또한, RAS 활성화는 에스큘레틴 처리에 의해 증가되었다. 우성-음성 RAS(RasN17) 돌연변이 유전자의 일시적 형질주입은 에스큘레틴에 의해 유도된 ERK1/2 활동과 p27KIP 발현을 모두 없애 버렸다. RasN17의 과도 발현은 세포증식 및 세포주기 단백질에 있어서 에스큘레틴 유발 감소를 억제하였다. 이러한 결과는 Ras/ERK1/2 경로가 결장 암 세포 성장을 에스큘레틴 유도 억제에서 cyclin/CDK 화합물의 감소로 이어지는 p27KIP1 유도에 의해 매개되고 있는 것으로 나타났으며, 전반적으로, 이러한 연구 결과는 에스큘레틴의 단순한 작용이 결장 악성종양에 대한 치료 가능성이 있다는 것을 보여준다.

Hepatoprotective Effects of Esculetin on Alcohol-induced Liver Injury in Human HepG2 cells and C57BL/6J Mice

이재민 충북대학교 2017 국내석사

Esculetin (6,7-dihydroxycoumarin), a coumarin derivative, is present in various natural plants such as Artemesia capillaris, Artemisia scoparia, Citrus limonia and Cichorium intybus. It has been reported to display anti-oxidant, anti-obesity and anti-inflammatory activities. However, the effect of esculetin in the ALD has not yet been investigated. This study was designed to decision the pharmacological actions of esculetin on the development of alcoholic liver disease, especially ROS accumulation and antioxidant defense system, in alcohol-induced HepG2 cells. In the present study, we investigated the effect of esculetin on oxidative stress in alcohol-induced HepG2 cells. HepG2 cells were treated with 3% alcohol containing with or without esculetin (10, 50, and 100 μM) and silymarin (100 μM) as a positive control. The results revealed that esculetin markedly inhibited oxidative stress in HepG2 cells. Esculetin also reduced the expression of ALD factors, including Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Glutathione depletion (GSH) and Malondialdehyde (MDA). Moreover, esculetin significantly increased phase II enzyme including NADPH Quinone Oxidoreductase (NQO-1), Heme Oxygenase 1 (HO-1) and ɤ-Glutamate-cystein ligase catalytic (GCLC). Also, esculetin significantly increased Nuclear factor erythroid 2-related factor (Nrf-2) translocation. We evaluated the liver injury protective effect of esculetin in alcohol-induced mice. C57BL/6J mice were fed a normal diet (ND) or alcohol-fed diet with or without esculetin (200mg/kg body weight) for 1 week. Liver weight of mice fed alcohol + esculetin was significantly increase compared to those in alcohol diet. Esculetin significantly lowered Glutathione depletion (GSH), Malondialdehyde (MDA), triglyceride and total cholesterol concentration in liver. Moreover, esculetin significantly lowered Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), triglyceride and total cholesterol concentration in serum. These results suggest that esculetin may attenuate the alcohol-induced oxidative stress in HepG2 hepatocytes through modulation of Nrf-2 signaling pathway, therefore preventing alcoholic liver disease.

Dimethyl-esculetin이 Carbon tetrachloride 負荷 家兎血淸 Transaminase活性度에 미치는 影響

이돈일 朝鮮大學校 大學院 1970 국내박사

Effect of Dimethyl-esculetin on the serum transaminarse activity of rabbit that was pretreated with Carbon tetrachloride In order to confirm whether or not Dimethyl-esculetin inhibits the increase of serum transaminase activity, this paper conductive. Ten rabbits mere equally divided into two groups: A and B. The A group was injected carbon tetrachloride of 0.5㎖/kg for seven days. The B group was pretreated with Carbon tetrachloride for seven days before was administered Dimethyl-esculetin. The result of the serum transaminase activity of each group were illustrated in the Fig Ⅰ& Ⅱ From the data, therefore, following conclusion might be drawn. 1. 30mg of Dimethy-esculetin was decreased serum-Glutamic Oxaloscetic transaminase activity of Rabbit which was pretreated with carbon tetrac chloride. 2. After was pretreated with carbon tetrachloride for seven days. the serum-Glutamic pyruvic transaminase activity was decreased by Dimethyl-esculetin. 3. Dimethyl-esculetin was, showed remarkable protective effect on the liver damage of Rabbit.

Dimethyl-esculetin의 血中 cholesterol에 대한 生化學的인 硏究

이돈일 朝鮮大學校 大學院 1970 국내박사

Biochemical Studios on the Dimethyl-esculetin of berum Total Cholesterol In order to confirm whether or not Dimethyl esculetin effect on the Change of serum total Cholesterol, this paper is conducted Ton rabbite were equally divided into two groups A and B The A group was administered Dimethyl-esculotin of 30 mg/kg for seven days before was tested serum total Cholesterol. The B group was injected Carbon tetra chlorido 0.1 ml/kg for seven days. The Chango of the serum total Cholesterol of each group is following Conclusion. The group of protreated with Dimothyl-esculotin was showed available protective effect on the liver damage of rabbit with injected Carbon totrachlorido

Molecular Mechanisms Underlying the Neuroprotective Effects of Esculetin on Oxidative Stress-induced Apoptosis in SH-SY5Y Cells

송선숙 서울大學校 大學院 2006 국내석사