• 한국식품과학회지
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• 제52권4호
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• pp.369-376
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• 2020

본 연구에서는 HR02/04(8:2)-W에 대한 항산화 성분 및 생리활성을 평가하기 위하여, p-coumaric acid 함량, 총 플라보노이드 및 총 페놀 함량, DPPH, ABTS 라디칼 소거능, FRAP 활성, reducing power, ORAC value를 측정하였다. 또한, 피부 섬유아세포에서 hydrogen peroxide에 의하여 산화적 스트레스가 유도된 세포 모델에서의 HR02/04(8:2)-W의 산화 손상 보호효과를 측정하기 위하여 XTT assay 및 H2-DCFDA assay를 진행하였다. HPLC를 이용한 p-coumaric acid 함량 분석 결과, HR02/04(8:2)-W 내의 p-coumaric acid는 75.62±1.56 mg/100 g 함유되어 있는 것으로 나타났으며, 총 플라보노이드 함량 및 총 페놀 함량은 각각 21.57±0.84 mg RE/g, 21.25±1.31 mg GAE/g으로 측정되었다. DPPH 및 ABTS 라디칼 소거활성 시험에서 HR02/04(8:2)-W의 농도 유의적으로 소거활성이 증가하는 것을 확인하였으며, FRAP 활성 및 reducing power 측정에서도 우수한 항산화 활성을 보였다. ORAC assay 결과 control 보다 5배 이상 높은 수치를 보이며 효과적으로 라디칼 저해 활성을 보였다. 항산화 실험에서의 뛰어난 항산화 활성은 HR02/04(8:2)-W에 함유된 페놀류들에 의한 것으로 판단된다. 피부 섬유아세포를 이용한 실험에서, HR02/04(8:2)-W을 300 ㎍/mL의 농도까지 세포의 생존력에 영향을 미치지 않는 것으로 나타났으며, hydrogen peroxide 처리에 의하여 56%까지 감소되었던 세포 생존율을 최대 78%까지 증가시키며 효과적으로 세포를 보호했음을 확인하였다. H₂-DCFDA 염색을 통하여 세포 내의 ROS의 양을 측정하였을 때, hydrogen peroxide처리에 의하여 control군 대비 137%까지 증가하였던 ROS를 최대 89% 까지 감소시킴으로써 세포 내 활성산소를 효과적으로 억제하였다. 이상의 결과를 종합하여 볼 때, HR02/04(8:2)-W이 뛰어난 항산화 생리활성 기능을 가지고, 세포 내 활성산소를 효과적으로 제거할 수 있는 소재임이 확인되었으며, 천연물 유래 기능성 식품원료로서의 활용 가능성이 매우 넓을 것으로 판단된다.

• 한국식품위생안전성학회지
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• 제33권2호
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• pp.140-145
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• 2018

본 연구는 예부터 약용으로 사용되었거나, 현재 식품공전에 식품원료로 사용이 가능한 것으로 등록된 국내 산림지역에 자생하는 식물을 식품산업에 활용하고자 선행연구에서 우수한 항산화 활성을 보인 갈참나무 잎을 본 연구에서 사용하였다. 70% 에탄올을 이용하여 추출한 갈참나무 잎을 이용하여, hydrogen peroxide로 산화적 스트레스를 유도한 피부 섬유아세포에서의 세포 보호효과, 세포내 항산화 효과 및 항노화 효과를 측정하였다. 세포 독성을 평가한 결과 25, 50 및 $100{\mu}g/mL$의 갈참나무 잎 추출물을 처리하였을 때 모두 독성을 나타내지 않았으며, hydrogen peroxide로 산화적 스트레스를 유도한 상태에서는 세포를 보호하여 농도 유의적으로 세포생존율이 증가하였다. 특히, $100{\mu}g/mL$의 농도에서는 양성대조군으로 사용한 $50{\mu}M$ ascorbic acid 수준까지 세포 생존율이 증가하였다. 세포내 항산화 효과를 확인 하기위해 사용한 $H_2-DCFDA$ assay에서는 형광현미경과 형광흡광도 측정에서 모두 농도 유의적으로 세포내 ROS 저감 활성을 확인하였고 갈참나무 잎 추출물을 $100{\mu}g/mL$ 농도로 처리했을 때는 $50{\mu}M$ ascorbic acid와 비슷한 세포내 항산화 효과를 나타내었다. 또한 SA-${\beta}$-galactosidase assay를 이용한 갈참나무 잎 추출물의 피부 섬유아세포에 대한 항노화활성은 ROS 생성 억제 효과와 유사한 경향으로 갈참나무 잎 추출물의 농도 유의적으로 세포 노화 억제효과를 확인하였다. 이상의 결과를 종합하여 볼 때 갈참나무 잎 추출물이 hydrogen peroxide로 인한 산화적 스트레스 상태에서 세포 보호효과, 항산화 효과 및 항노화 효과가 관찰되어 기능성 식품원료로서의 활용도가 매우 넓을 것으로 판단된다.

• Restorative Dentistry and Endodontics
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• 제39권2호
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• pp.89-94
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• 2014

Objectives: The aim of this study was to evaluate the cytotoxicity, setting time and compressive strength of MTA and two novel tricalcium silicate-based endodontic materials, Bioaggregate (BA) and Biodentine (BD). Materials and Methods: Cytotoxicity was evaluated by using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide (XTT) assay. Measurements of 9 heavy metals (arsenic, cadmium, chromium, copper, iron, lead, manganese, nickel, and zinc) were performed by inductively coupled plasma-mass spectrometry (ICP-MS) of leachates obtained by soaking the materials in distilled water. Setting time and compressive strength tests were performed following ISO requirements. Results: BA had comparable cell viability to MTA, whereas the cell viability of BD was significantly lower than that of MTA. The ICP-MS analysis revealed that BD released significantly higher amount of 5 heavy metals (arsenic, copper, iron, manganese, and zinc) than MTA and BA. The setting time of BD was significantly shorter than that of MTA and BA, and the compressive strength of BA was significantly lower than that of MTA and BD. Conclusions: BA and BD were biocompatible, and they did not show any cytotoxic effects on human periodontal ligament fibroblasts. BA showed comparable cytotoxicity to MTA but inferior physical properties. BD had somewhat higher cytotoxicity but superior physical properties than MTA.

• 대한본초학회지
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• 제22권3호
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• pp.27-37
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• 2007

Objectives: Hepatocellular carcinoma is the most common primary malignant tumor of the liver worldwide. In-Jin-Ho-Tang(IJHT) has been used as a traditional Chinese herbal medicine since ancient time. and today it is widely applied as a medication for jaundice which is associated with inflammation in liver. In this study, I investigated whether methanol extract of IJHT induced HepG2 cancer cell death. Methods: Cytotoxic activity of IJHT on HepG2 cells was using XTT assay. Apoptosis induction by Ros A in HCT116 cells was verified by the induction of cleavage of poly ADP-ribose polymerase (PARP). and activation of caspase-3, -8 and -9. The release of cytochrome c from mitochondria to cytosol. the level of Bcl-2 and Bax and the expression of p53 and p21 were examined by western blotting analysis. Furthermore, MAPKs activation was analyzed by western blotting analysis. Results: IJHT induced apoptosis in HepG2 cells. And treatment of IJHT resulted in the release of cytochrome c into cytosol, decreased anti-apoptotic Bcl-2, and increased pri-apoptotic Bax expression. IJHT markedly inactivated extracellular signal-regulated kinase (ERK1/2), and activated p38 mitogen-activated protein (MAP) kinase. Sodium orthovanadate (SOV), a phosphatase inhibitor, to reverse IJHT-induced ERK1/2 inactivation and SB203580, a specific p38 MAP Kinase inhibitor efficiently blocked apoptosis of HepG2. Thus, IJHT induces apoptosis in HepG2 cells via MAP kinase modulation. Conclusion: These results indicated that IJHT has some potential for use as an anti-cancer agent.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.241-248
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• 2020

Luminespib (AUY922), a heat shock proteins 90 inhibitor, has anti-neoplastic and antitumor effects. However, it is not clear whether AUY922 affects events in vascular diseases. We investigated the effects of AUY922 on the platelet-derived growth factor (PDGF)-BB-stimulated proliferation and migration of vascular smooth muscle cells (VSMC). VSMC viability was detected using the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reagent. To detect the attenuating effects of AUY922 on PDGF-BB-induced VSMCs migration in vitro, we performed the Boyden chamber and scratch wound healing assays. To identify AUY922-mediated changes in the signaling pathway, the phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 was analyzed by immunoblotting. The inhibitory effects of AUY922 on migration and proliferation ex vivo were tested using an aortic ring assay. AUY922 was not cytotoxic at concentrations up to 5 nM. PDGF-BB-induced VSMC proliferation, migration, and sprout outgrowth were significantly decreased by AUY922 in a dose-dependent manner. AUY922 significantly reduced the PDGF-BB-stimulated phosphorylation of Akt and ERK1/2. Furthermore, PD98059 (a selective ERK1/2 inhibitor) and LY294002 (a selective Akt inhibitor) decreased VSMC migration and proliferation by inhibiting phosphorylation of Akt and ERK1/2. Greater attenuation of PDGF-BB-induced cell viability and migration was observed upon treatment with PD98059 or LY294002 in combination with AUY922. AUY922 showed anti-proliferation and anti-migration effects towards PDGF-BB-induced VSMCs by regulating the phosphorylation of ERK1/2 and Akt. Thus, AUY922 is a candidate for the treatment of atherosclerosis and restenosis.

• 대한한의학방제학회지
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• 제15권2호
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• pp.127-137
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• 2007

The purpose of this study was to investigate the effect of Yong-dam-sa-gan-tang (YST) on apoptosis in HepG2 cells, First of all. to study the cytotoxic effect of methanol extract of YST on HepG2 cells, the cells were treated with various concentrations of YST and then cell viability was determined by XTT reduction method and trypan blue exclusion assay. YST reduced proliferation of HepG2 cells in a dose-dependent manner. To confirm the induction of apoptosis, HepG2 cells were treated with various concentrations of YST. The cleavage of poly AD P-ribose polymerase (P ARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of caspase-3, procaspase-8 and procaspase-8 were examined by western blot analysis. YST decreased procaspase-3, procaspase-8 and procaspase-9 levels in a dose-dependent manner and induced the clevage of PARP. YST triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome c from mitochondria to cytosol. Furthermore, YST also downregulated the anti-apoptotic Bcl-2 and upregulated the pro-apoptotic-Bax. Therefore, this result suggest that YST induced HepG2 cell death through the mitochondrial pathway. Sustained activation of the Ras/Raf/MEK/ERK cascade in cells results in a cell cycle arrest and has been implicated in the differentiation of certain cell types, in many cases acting to promote differentiation. YST decreased the activation of Ras/Raf/MEK/ERK cascade in a dose-dependent manner. These results suggest that YST is potentially useful as a chemo-therapeutic agent in HepG2.

• 대한본초학회지
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• 제21권2호
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• pp.37-46
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• 2006

Objectives : The purpose of this study was to investigate the effect of Dangguibohyultang (DB) and its combination (DB-I; Astragali membraneus BUNGE : Angelica gigas NAKAI=5:1, DB-II; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:1, DB-III; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:5,) on apoptosis in human colorectal adenocarcinoma HCT116 cells. Methods : To study the cytotoxic effect of methanol extract of DB-I, DB-II and DB-III on HCT116 cells, the cell viability was determined by XTT reduction method and ttypan blue exclusion assay. To confirm the induction of apoptosis, the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of procaspase-3, -8 and -9 were examined by western blot analysis. Furthermore, DB-induced apoptosis was confirmed by DNA fragmentation. The release of cytochrome C from mitochondria to cytosol, the level of Bcl-2 and Bax, and the expressions of Raf/MEK/ERK were examined by western blot analysis. Results : DB-I and DB-II reduced proliferation of HCT116 cells in a dose-dependent manner. DB-I and DB-II decreased procaspase-3, -8, -9 levels in a dose-dependent manner and induced the clevage of PARP. DB-I and DB-II also triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome C from mitochondria to cytosol, decreasing of anti-apoptotic Bcl-2, and increasing of pro-apoptotic Bax. DB-I and DB-II decreased the activation of Ras/Raf/MEK/ERK cascade in a dose-dependent manner. Conclusion : These results suggest that DB-I and DB-II induce apoptosis via mitochondrial pathway in HCT116 cells. Furthermore, Raf/MEK/ERK cascade is involved in DB-induced apoptosis. These results suggest that DB is potentially useful as a chemotherapeutic agent in human liver cancer.

• 대한본초학회지
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• 제21권4호
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• pp.27-36
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• 2006

Objectives : This study was performed for the investigation of anticancer effects of methanol extract of Rheum Rhizoma (MeOH-RR) on a human liver cancer cell line (HepG2). Methods : To study the cytotoxic effect of MeOH-RR on HepG2 cells, the cell viability was determined by XTT reduction method and trypan blue exclusion assay. The cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of procaspase-3, -8 and -9 were examined by western blot analysis. Furthermore, MeOH-RR-induced apoptosis was confirmed by DNA fragmentation. The release of cytochrome c from mitochondria to cytosol, the level of Bcl-2 and Bax were examined by western blot analysis. Results : MeOH-RR reduced proliferation of HepG2 cells in a dose-dependent manner at 24 h and 48 h treatment. MeOH-RR induced the activation of caspase-3, -8, and -9 and the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3. Furthermore, treatment with MeOH-RR resulted in internucleosomal DNA fragmentation, evidenced by the formation of a DNA ladder on agarose gel, a hallmark of cells undergoing apoptosis. MeOH-RR downregulated Bcl-2, upregulated Bax, and increased the release of cytochrome c from the mitochondria into cytosol in a dose-dependent manner. Moreover, MeOH-RP increased caspase-3 activity. Conclusion : There results suggest that MeOH-RR induce apoptosis via mitochondrial pathway and caspase-3-dependent pathway in HepG2 cells. There results suggest that MeOH-RR is potentially useful as a chemotherapeutic agent in human liver cancer.

• 한국식품영양과학회지
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• 제41권1호
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• pp.7-13
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• 2012

본 연구에서는 산화적 스트레스에 의한 간 손상 개선 소재 개발을 위하여 꾸지뽕나무 각 부위별(잎, 줄기, 열매), 용매별(80% 에탄올, 10% 에탄올, 물) 추출물의 항산화 활성 및 간세포 보호효과를 측정하였다. 총 폴리페놀 함량과 플라보노이드 함량은 꾸지뽕 잎 80% 에탄올 추출물에서 가장 높게 나타났으며, 부위별로는 잎>줄기>열매 순이었고, 추출용매별로는 80% 에탄올>10% 에탄올>물 추출물 순으로 나타났다. DPPH 라디칼 소거능과 ABTS 라디칼 소거능 또한 잎80% 에탄올 추출물이 가장 높았으며, HepG2 세포에서 $H_2O_2$로 유도된 산화적 손상에 대해서는 꾸지뽕 잎 80% 에탄올 추출물만 유의적으로 높은 세포보호활성을 나타내었으며, HepG2/2E1 세포에서 알코올로 유도된 산화적 손상에 대한 각 부위별, 용매별 추출물의 간세포보호효과 또한 꾸지뽕잎 80% 에탄올 추출물이 가장 높게 나타났다. 부위별로는 잎>줄기>열매 순이었고, 추출 용매별로는 꾸지뽕 잎의 경우 80% 에탄올>10% 에탄올>물 순이었으며, 줄기와 열매의 경우는 용매별로 유의적인 차이가 나타나지 않았다. 이상의 결과로부터 꾸지뽕나무 잎 추출물은 우수한 항산화활성을 가질 뿐만 아니라 $H_2O_2$와 알코올로 유도된 간 손상으로부터 간세포 보호활성을 보임을 확인하였다. 이에 꾸지뽕나무 잎 추출물은 산화적 스트레스에 의한 간 손상으로부터 간세포 보호효과를 갖는 기능성 소재로 활용될 수 있을 것으로 사료된다.

• Biomolecules & Therapeutics
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• 제18권1호
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• pp.32-38
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• 2010

Activation of JNK has long been associated with the apoptotic response induced by various anti-cancer drugs including doxorubicin, vinblastine, and etoposide. In this study, we examined and compared patterns of apoptosis and JNK activation according to three different anti-cancer drugs (daunorubicin, vinblastine, and etoposide) and two different sources of HL60 cells (Jackson Laboratory and ATCC). HL60 cells from Jackson Laboratory (HL60/RPMI) were maintained in RPMI 1640 containing 5% fetal bovine serum and those from ATCC (HL60/IMDM) in IMDM containing 20% fetal bovine serum as to each manufacture's guideline. In general, HL60/RPMI cells were more sensitive to anti-cancer drugs compared to HL60/IMDM cells, demonstrated by the XTT and flow cytometric analyses. Apoptotic pathways after treatment with anti-cancer drugs seemed to be different between HL60/RPMI (daunorubicin and etoposide, caspase 3 dependent, but caspase 8 or 9 independent; vinblastine, caspase 3 independent) and HL60/IMDM (caspase 3 and caspase 9 dependent). The expression of apoptotic protein, BID, was consistent with caspase 3 activation. Immunoblotting of phospho-JNK and JNK kinase assay showed JNK activation by all three anti-cancer drugs in HL60/RPMI, while JNK activation was observed only in vinblastine-treated cells in HL60/IMDM. Our study results suggest that in vitro environmental conditions have a significant influence on JNK mediated apoptosis of HL60 cells by anti-cancer drugs and in vitro culture conditions are important factors in JNK or possibly other MAPK related studies.