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1 Korean J. Plant Res. 31(2): (2018) Print ISSN Online ISSN Original Research Article 노종현, 심미옥, 정호경, 이무진, 장지훈, 정다은, 성태경, 안병관, 조현우 * 한약진흥재단 Anti-colorectal Cancer and Anti-oxidant Activities of Rubiae radix Ethanol Extract in vitro Jong Hyun Nho, Mi Ok Sim, Ho Kyung Jung, Mu Jin Lee, Ji Hun Jang, Da Eun Jung, Tae Kyoung Sung, Byeong Kwan An and Hyun Woo Cho* National Development Institute of Korean Medicine, Jangheung-gun 59338, Korea Abstract - Rubiae radix is root of Runia akane Nakai, it has been used to hemostasis and blood stasis in Korean and China. This study investigated that anti-oxidant and anti-colorectal cancer effect of ERA (ethanol extract of Rubiae radix) and WRA (water extract of Rubiae radix) using RAW (murine macrophage from blood) and HCT-116 cells (human colorectal cancer cell line). ERA contained polyphenol (45.77 ± 2.03 mg /g) and flavonoid (22.82 ± 1.33 mg /g). 500 μm H 2 O 2 -induced ROS generation was diminished by 500 μg / ml ERA treatment in RAW cells, but not WRA (125, 250, and 500 μg / ml ). Moreover, caspase-3 activity and DNA fragmentation increased by 500 μg / ml ERA treatment during apoptotic cell death in HCT-116. Results demonstrated that anti-cancer effect of ERA against human colorectal cancer cells is mediated apoptotic cell death and DNA fragmentation through caspase-3 activation. However, further study is required to what active ingredient of ERA are important for anti-oxidant and anti-colorectal cancer effect in vivo. Key words - Anti-oxidative effect, Colorectal cancer, Rubia akane Nakai, Rubiae radix 서언 우리나라에서대표적인만성질환으로알려져있는암은신체조직을구성하고있는세포가제한없이증식되어악성종양을형성하게되는질병으로서, 산업발달과식생활의변화또는유전적인요인에의해발병하게된다 (Lewis et al., 2005). 대장암은우리나라뿐만아니라서양에서도흔히발생하는악성종양중하나이며여성보다남성에서발생률이더높은것으로알려져있다 (Kim et al., 2013). 대장암을치료하는방법으로, 일반적인방법인수술이외에화학요법과방사선요법등이시행되고있으나이는면역기능저하, 탈모, 신장독성, 심장독성과같은여러부작용을야기할수있다고알려져있다 (Shariati et al., 2010; Andersen et al., 2006). * 교신저자 : thej01234@gmail.com Tel Both authors contributed equally to this work. 이러한이유로부작용이없고항암효과를가지고있는천연자원소재의탐색과개발이요구되며, 이를이용한기능성식품또는의약품을개발하는연구가활발하게진행되고있다 (Shin et al., 2004; Kim et al., 2012; Kwon et al., 2011). 산화스트레스는건강에쉽게영향을미칠뿐만아니라, 질병에쉽게관여한다고알려져가장많이연구되고있는분야중하나로알려져있고 (Lee et al., 2013), 악성종양의원인들중하나로알려져있는활성산소는정상세포에게영향을끼쳐암또는기능장애를유발하게된다고알려져있다 (Kim and Kim, 2018). 천초근 (Rubiae radix) 은꼭두서니 (Rubia akane Nakai) 의뿌리를건조한것으로, 우리나라에서예로부터어혈을없애고지혈작용을하는것으로알려져다양하게이용되었다 (Kim et al., 2012; Bae et al., 2005). 이와같은이유에의해최근항산화효능을가진천연자원소재를찾기위해여러분야에서연구가진행되고있다 (Woo et al., 2018; You and Moon, 2018; Sim et al., 2017; Jeong et al., 2017). c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 여러연구에따르면, 천초근추출물은 human promyelocytic leukemia( 전골수구백혈병 ) 세포의증식을세포자멸사 (apoptosis) 과정을통해억제시킨다고알려져있으며 (Choi et al., 2008), Bacillus cereus 와 Pseudomonas aeruginosa 같은식중독균의증식을억제한다고알려졌다 (Bae et al., 2005). 현재까지천초근에탄올추출물을이용하여, 대장암을억제할수있는효능에대한연구가이루어지지않았다. 따라서본연구는천초근물추출물과에탄올추출물을이용하여대장암세포에대한항암효능과항산화효능이있는지알아보고자하였다. 재료및방법실험재료와추출물의제조본연구에사용한천초근은꼭두서니과 (Rubiaceae) 에속하는 Rubia akane Nakai의뿌리로서 2015 년 5월 27일에전남완도 ( 위도 N, 경도 E) 에서채취하였으며, 목포대학교한약자원학과김휘교수님의식물학적동정을거쳤다. 실험에사용한시료의확증표본 (TKM-2097) 은한약진흥재단한약자원본부에보관하고있다. 천초근은멸균수로수세한뒤, 50 조건에서건조기를이용하여일주일간건조하였다. 건조된천초근은분쇄하여, 증류수또는에탄올을칭량한시료무게의 10배를넣고환류냉각추출방법을통해각각물추출은 100 조건에서, 에탄올추출은 70 온도에서 3시간, 3회반복하여추출하였다. 추출물은 filter paper (Thermo, Waltham, MA, USA) 를이용하여여과하고, 여과액을동결건조하여시료를 PBS 에녹인뒤사용하였다. 세포배양 RAW 그리고 HCT-116 세포주는한국세포주은행 (KCLB, Seoul, Korea) 에서분양받아사용하였다. Dulbecco s modified Eagel s medium, DMEM (Thermo, Waltham, MA, USA) 에 10% FBS (fetal bovine serum), 1% penicillin/streptomycin 을첨가하여사용하였고. 37, 5% CO 2 조건에서배양하고유지하였다. 세포생존율분석세포생존율은 CellTiter 96 AQueous One Solution Cell proliferation assay kit (Promega, Fitchburg, WI, USA) 를사용하였으며, 제조사의프로토콜에따라서측정하였다. HCT-116 세포를 96 well plate에 cells/ ml농도가되도록분주한 뒤 37, 5% 배양기에서 24시간배양한후천초근에탄올추출물을각각 31.3, 62.5, 125, 250, 그리고 500 μg / ml농도로 24시간동안처리하였으며, MTS 시약 20 μl를넣고 2시간동안배양한후 microplate reader Infinite 200 PRO (TECAN, Mannedorf, Switzerland) 를이용하여 490 nm에서흡광도를측정하였다. 세포생존율은정상대조군에대한생존율로표시하였고이에따라추출물들의암세포사멸효과를측정하였다. Tunel 분석 Tunel 분석은 DeadEnd Fluorometric TUNEL System (Promega, Fitchburg, Wisconsin, USA) 을사용하였으며, 제조사의프로토콜에따라서수행하였다. HCT-116 세포를 12 well plate에서 cover slip 위에 cells/ ml농도가되도록분주한뒤, 37, 5% 배양기에서 24시간배양했다. 배양된세포에천초근에탄올추출물 (ERA) 을 24시간동안처리하였다. 그후 10% neutralized buffered formalin (NBF) 로 4 에서 20분동안고정하였고, phosphate buffered saline (PBS) 로 2번씻어주었다. 다음 0.2% Triton X-100 in PBS 용액으로 5분간투과화한뒤 PBS 로 2번씻어주었다. 남은액체가없도록모두제거하고 Equilibration buffer 를 5분간처리하였으며 100 μl rtdt incubation buffer 를처리하고 37 에서 1시간동안배양하였다. 그후 2 SSC buffer 를처리하고, PBS 로 2번씻은뒤 Prolong Gold antifade mountant with DAPI (Thermo, Waltham, MA, USA) 로봉입하여 Epi-fluorescence microscope (Carl Zeiss, Oberkochen, Germany) 를이용해형광정도를관찰하였다. Caspase-3 활성도측정 Caspase-3 의활성도는 Caspase-3 colorimetric detection kit (Enzo, Farmingdale, NY, USA) 를이용해실험을진행했다. HCT-116 세포는 PBS로세척한뒤 Pierce protease and phosphatase inhibitor mini tablets (Thermo, Waltham, MA, USA) 가포함된 RIPA cell lysis buffer 2 (Enzo, Farmingdale, NY, USA) 로 1시간동안용해하였다. 세포용해액은원심분리기로 4, 13,000 g 조건에서 10분간원심분리하였고, 단백질농도를 bradford assay 를이용하여측정하였다. 총 20 μg의단백질을사용해제조사가제공한프로토콜에따라실험을진행했다. FACS 분석 FACS를이용한분석에는 annexin V-FITC apoptosis detection kit (Enzo, Farthingale, NY, USA) 와 DCFDA cellular ROS -103-

3 韓資植誌 KoreanJ. Plant Res. 31(2) : 102~108(2018) detection assay kit (abcam, Cambridge, England) 를사용했다. 세포에천초근에탄올추출물처리후유도되는 apoptosis 의비율을구하기위해제조사에서제공한 protocol에따라 Annexin V-FITC와 PI 시약으로염색시켰으며, 마찬가지로 H 2 O 2 처리에따른활성산소종을확인하기위해 DCFDA 시약으로염색하였다. 염색된세포는 CytoFLEX (Beckman clulter, Indianapolis, IN, USA) 를이용해분석하였다. 폴리페놀함량분석총페놀함량은 Folin-Ciocalteau 시약을이용하여 Folin 과 Denis (1912) 의방법을일부변형시켜측정하였다. 표준물질의검량선작성을위해 gallic acid를사용하였으며, 각각추출물에 Folin-Ciocalteu's phenol solution 을각각 160 μl를첨가하고, 3분간상온에서반응시킨뒤 10% Na 2 CO 3 용액을 160 μl를첨가하여 1시간동안반응시켰다. 다음실온에서 10,000 rpm, 10분동안원심분리하였으며상등액을 microplate reader Infinite 200 PRO (TECAN, Mannedorf, Switzerland) 를이용하여 750 nm에서흡광도를측정하였다. 플라보노이드함량분석각각추출물의플라보노이드함량은 96 well plate 에농도별시료 10 μl를넣고, 10% aluminum nitrate 와 1 M potassium acetate를각각 4 μl, methanol 82 μl를첨가하였다. 40 분간암소반응한뒤 microplate reader Infinite 200 PRO (TECAN, Mannedorf, Switzerland) 를이용해 415 nm에서흡광도를측정 하였다. 표준물질의검량선작성을위해 rutin을이용하였다 (Moreno et al., 2000). 통계처리통계처리는평균 ± 표준편차 (mean ± SD) 로나타냈다. 유의성을검정하기위해 SPSS (Statistical Package for Social Science Inc., Chicago, IL, USA) 통계프로그램을사용하였다. 일원변량분석 (one way ANOVA) 을실시하였으며, 유의성이있는경우 p < 0.05 수준에서, Duncan's Multiple Range Test (DMRT) 를실시하였다. 결과및고찰천초근의 ROS 생성억제효과및폴리페놀, 플라보노이드함량 WRA( 천초근물추출물 ) 와 ERA( 천초근에탄올추출물 ) 의폴리페놀 (Polyphenol) 및플라보노이드 (Flavonoid) 함량을알아보기위해실험을진행하였을때, WRA는폴리페놀이 ± 0.86 mg /g, 플라보노이드가 ± 1.30 mg /g이포함되어있는것으로나타났다. 반면 ERA는폴리페놀이 ± 2.03 mg /g, 플라보노이드가 ± 1.33 mg /g으로 WRA에비해각각의함량이조금더높은것으로확인되었다 (Table 1). 폴리페놀은식물에서흔히발견할수있는화합물로, 식물의대사에의해형성되며항염증, 항당뇨, 항비반등여러질병에 Fig. 1. Effect of extracts on H 2 O 2 -induced ROS generation in RAW cells. (A-B) WRA; water extract of Rubiae radix, ERA; ethanol extract of Rubiae radix. After pretreatment with iridin for various concentration (125, 250, and 500 μg / ml ), RAW cells were treated with 500 μm H 2 O 2 for 24 h. ROS was measured by flow cytometry. Representative images were taken from at least three independent experiments

Table 1. Polyphenol and flavonoid contents of Rubiae radix extracts Groups Polyphenol ( mg /g) Flavonoid ( mg /g) WRA z 14.50 ± 0.86 x 11.81 ± 1.30 x ERA y 45.77 ± 2.03 22.82 ± 1.

4 Table 1. Polyphenol and flavonoid contents of Rubiae radix extracts Groups Polyphenol ( mg /g) Flavonoid ( mg /g) WRA z ± 0.86 x ± 1.30 x ERA y ± ± 1.33 z WRA; water extract of Rubiae radix y ERA; ethanol extract of Rubiae radix x Each value in the table is represented as mean ± S.D. (n=9) 대한효능을가지고있음이밝혀져있다 (Pandey and Rizvi, 2009; Horakova, 2011). ERA의폴리페놀및플라보노이드함량은각각 ± 2.03 mg /g, ± 1.33 mg /g으로, Kwon et al. (2014) 에의해알려진모링가 (Moringa oleifera Lam.) 잎에탄올추출물 ( 폴리페놀 ; ± 1.53, 플라보노이드 ; ± 0.30) 과비교하여조금더높은것을확인할수있었으며, 모링가잎은 β-carotene, 플라보노이드및폴리페놀함량이높아항산화제품소재로매우널리이용되고있기때문에 (Kwon, 2014; Anwar, 2007), 천초근추출물또한추가실험을통해항산화제로이용될수있을것이라고사료된다. 다음으로각각추출물의 ROS (reactive oxygen species) 억제효과를확인하기위해실험을진행하였다. RAW 세포주에각각추출물을여러농도 (125, 250, and 500 μg / ml ) 로 30분동안전처리한뒤, 500 μm H 2 O 2 를 24시간동안처리하여 ROS 생성량을 flow cytometry 로분석했다. WRA는 H 2 O 2 에의해증가된 ROS를억제하지못했으며 (Fig. 1A), ERA는 H 2 O 2 (99.97%) 와비교하여 500 μg / ml농도에서 34.39% 까지 ROS 생성을억제하였다 (Fig. 1B). ROS 는세포내부대사에의해생성되며, 세포분화와유전자발현등에관여한다 (Rhee et al., 2006). 과도한 ROS생성은세포자멸사를유도할뿐만아니라, 염증세포의응집을유도하여염증반응을일으키는요인으로알려져있다 (Kang, 2013; Mittal et al., 2014). 이에따라 ERA는 H 2 O 2 에의해증가된 ROS 생성을억제하는것으로판단되며, 결과를종합하면 WRA 보다 ERA가항산화효능이높은것으로나타났다. 천초근에탄올추출물의 HCT-116 세포주사멸효과대장암세포사멸효과를 in vitro 수준에서확인하기위해대장암세포주인 HCT-116 세포를이용하여실험을진행하였다. WRA를 HCT-116 세포에여러농도 (31.3, 62.5, 125, 250, and 500 μg / ml ) 로 24시간동안처리하여세포생존율을확인한결과, 세포생존율이유의적으로감소하지않았다 (Fig. 2A). 하지만 EAR 를같은조건으로처리하였을때 31.3 μg / ml농도부터세포생존율이농도의존적으로감소하면서 500 μg / ml농도에선 HCT-116 세포의생존율이 ± 3.13% 까지감소하였다 (Fig. 2B). 이와같은조건에서 Kim (2015) 의보고에의하면, 개똥쑥 (Artemisia annua Linne) 에탄올추출물은 100 μg / ml농도에서대장암세포주인 HCT-116 세포에대해뛰어난사멸효과를나타낸다고보고하였다. Fig. 2. ERA (ethanol extract of Rubia akane Kakai) induced cell death in HCT-116 cells. (A-B) WRA; water extract of Rubiae radix, ERA; ethanol extract of Rubiae radix. HCT-116 cells were treated with various concentration (31.3, 62.5, 125, 250, and 500 μg / ml ) for 24 h. Cell viability was measured by MTS assay. Means values ± SD from triplicate separated experiments are shown. *Means with difference letters are significantly different at p < 0.05 by Duncan s multiple range test

韓資植誌 KoreanJ. Plant Res. 31(2) : 102~108(2018) Fig. 3. ERA induced caspase-3 activation and DNA fragmentation during apoptotic cell death in HCT-116 cells. ERA; ethanol extract of Rubiae radix.

5 韓資植誌 KoreanJ. Plant Res. 31(2) : 102~108(2018) Fig. 3. ERA induced caspase-3 activation and DNA fragmentation during apoptotic cell death in HCT-116 cells. ERA; ethanol extract of Rubiae radix. HCT-116 cells were treated with various concentration (125, 250, and 500 μg / ml ) for 24 h. (A) Annexin V and propidium iodide staining were analyzed by flow cytometry. (B) Caspase-3 activity was measured by capsase-3 activity assay. and (C) HCT-116 cells visualized by tunel assay. Representative images were taken from at least three independent experiments. Means values ± SD from triplicate separated experiments are shown. *Means with difference letters are significantly different at p < 0.05 by Duncan s multiple range test. 개똥쑥은항암효과가매우뛰어난것으로알려져있으며 (Singh, 2004), 이에비교하였을때천초근에탄올추출물의대장암세포사멸효과는개똥쑥에탄올추출물보다낮은것으로보아천초근에탄올추출물에존재하는유효성분을탐색하는추가적인실험이필요할것으로생각된다. 천초근에탄올추출물의 HCT-116 세포주에대한 caspase-3 활성화, DNA 단편화유도활성천초근에탄올추출물이 HCT-116 세포주에대해세포자멸사 (apoptosis) 또는세포괴사 (necrosis) 를일으키는지알아보기위해, HCT-116 세포주에 ERA를여러농도 (125, 250, and 500 μg / ml ) 로 24시간동안처리하고, Annexin Vand propidium iodide (PI) 염색을통해유세포분석기를이용하여세포사의비율을확인하였다. 그결과 500 μg / ml ERA를 24시간동안처리하였을때 apoptotic cell death (apoptosis, Q2-4 + cell death, Q2-2) 를 25.32% 까지증가시키는것으로확인했다 (Fig. 3A). 다음으로세포사에관여하는단백질인 caspase-3 의활성도를확인한결과, ERA를 500 μg / ml농도로처리하였을때정상대조군에비교하여 ± 3.65% 까지증가하였다 (Fig. 3B). 앞선결과와마찬가지로, DNA fragmentaion을확인하기위해 Tunel 염색을수행한결과 ERA를 500 μg / ml농도로처리하였을때 Tunel positive signal 이가장많이나타났다 (Fig. 3C). 결과를종합하면, ERA는대장암세포주인 HCT-116 세포에 500 μg / ml농도로처리될때 apoptotic cell death와 DNA 단편화및 caspase-3 활성화를나타낸다. DNA 단편화 (DNA fragmentation) 는 apoptosis 의여러특징중하나로알려져있으며, 이과정에는 caspase-3, cytochrome c, and Apaf-1 같은단백질들이관여한다고밝혀져있다 (Kitazumi and Tsukahara, 2010; Zhang and Xu, 2000). 밝혀진바와같이 ERA를처리하였을때 500 μg / ml농도에서 apoptosis 뿐만아니라 caspase-3 활성화와 DNA 단편화가유 -106-

6 도되는것으로확인되었으며, 이는 ERA가 HCT-116 세포주에서 apoptosis 를통해항암효과를나타내는것으로생각된다. 하지만이는다른연구논문들과비교하였을때농도대비효과가미미한것으로생각되며, 천초근에탄올추출물에대장암세포의성장을억제하는유효성분을찾을뿐만아니라 HCT-116 세포를제외한다른대장암세포주를이용해그효능을탐색하는추가적인실험이필요할것이라고사료된다. 적요본연구는꼭두서니의뿌리인천초근의물추출물과에탄올추출물을이용하여대장암세포에대한암세포성장억제및사멸효과가있는지알아보고자수행하였다. ERA( 천초근에탄올추출물 ) 은폴리페놀 (45.77 ± 2.03 mg /g) 과플라보노이드 (22.82 ± 1.33 mg /g) 를함유하고있었으며, H 2 O 2 에의해증가된 ROS (reactive oxygen species) 를억제하는효과를나타냈지만 WRA ( 천초근물추출물 ) 은효과가없었다. 또한 ERA는 500 μg / ml의농도로대장암세포주 (HCT-116) 에처리했을때세포사멸을유도할뿐만아니라 caspase-3 단백질활성화, DNA fragmentation 및 apoptotic cell death를일으키는것으로확인되었다. 이는 ERA가 HCT-116 세포주에대해 apoptosis( 세포자멸사 ) 를통해항암효과를나타내는것으로생각되지만다른연구결과들과비교하였을때농도대비효능이미미하다. 따라서천초근에탄올추출물에대장암세포의성장을억제하는유효성분을분석하여그효능을탐색하는추가실험이필요할것으로생각된다. 사사본연구는한의약의과학화, 한의약육성및산업진흥을통해국민의건강한삶과국가경제에기여할수있도록보건복지부한국토종자원의한약재기반구축사업지원에의해이루어진결과로이에감사드립니다. References Andersen, C., L. Adamsen, T. Moeller, J. Midtgaard, M. Quist, A. Tveteraas and M. Rorth The effect of a multidimensional exercise programme on symptoms and side-effects in cancer patients undergoing chemotherapy-the use of semi-structured diaries. Eur. J. Oncol. Nurs. 10: Anwar, F., S. Latif, M. Ashraf and A.H. Gilarni Moringa oleifera : a food plant with multiple medicinal uses. Phytother. Res. 21: Bae, J.H., H.J. Jang and J.I. Jung Antimicrobial effect of Rubia akane Nakai on food-borne pathogens. J. Korean Soc. Food Sci. Nutr. 34: Choi, H.S., J.M. Park, S.M. Ju, S.H. Kim, D.K. Kim, W.S. Kim and B.H. Jeon Influence of Rubiae radix extract on the mechanism of apoptosis in HL-60 cells. J. Physiol and Pathol. Korean Med. 22: Folin, O. and W. Denis On phosphotungstic-phosphomolybdic compounds as color reagents. J. Biol. Chem. 12: Horakova, L Flavonoids in prevention of diseases with respect to modulation of Ca-pump function. Interdiscip. Toxicol. 4: Jeong, J.H., W.H. Hwang, S.H. An, H.Y. Jeong, H.S. Lee, J.S. Baek, K.J. Choi, G.H. Lee, J.E. R, N.J. Chung, S.J. Lee and S.J. Yun Seed germination, plant growth and antioxidant capacity of Limonium tetragonum under different salt concentrations. Korean J. Plant Res. 30: Kitazumi, I. and M. Tsukahara Regulation of DNA fragmentation: the role of caspases and phosphorylation. FEBS J Kwon, H.N., W.S. Bang, J.Y. Kim, J.R. Park and J.R. Jeon Effect of Acer tegmentosum M. extracts on hepatocarcinoma cell. Korean J. Food Sci. Technol. 43: Kwon, H.N., W.S. Bang, J.Y. Kim, J.R. Park and J.R. Jeon Effect of Acer tegmentosum M. extracts on hepatocarcinoma cell. Korean J. Food Sci. Technol. 43: Kim, J.Y., E.J. Jung, Y.S. Won, J.H. Lee, D.Y. Shin and K.I. Seo. 2012a. Cultivated Orostachys japonicus induces apoptosis in human colon cancer cells. Korean J. Food Sci. Technol. 44: Kim, D.H., J.H. Park, S.H. Kim and S.H. Sung. 2012b. Case study: regression of a residual tumor and prolongation of overall survival with allergen-removed Rhus verniciflua stokes after chemoradiotherapy in locally advanced non-small cell lung cancer. Korean J. Intern. Med. 36: Kim, Y.Z., J.H. Kim, J.Y. Lee, S.E. Park, J.M. Choi and Y.G. Park symptoms and initial diagnostic test in colorectal cancer patients. Korean J. Fam. Pract. 3: Kwon, Y.R. and K.S. Youn Antioxidant activity and physiological properties of moringa (Moringa oleifera Lam.) leaves extracts with different solvents. Korean J. Food Preserv. 21: Kim, B.M., G.T. Kim, E.G. Lim, E.J. Kim, S.Y. Kim, S.H. Ha -107-

7 韓資植誌 KoreanJ. Plant Res. 31(2) : 102~108(2018) and Y.M. Kim Cell cycle arrest of extract from Artemisia annua Linne via Akt-mTOR signaling pathway in HCT116 colon cancer cells. Korean Soc. Biotechnol. Bioeng. J. 30: Kim, J.H. and E.J. Kim Evaluation of anti-oxidative, anti-thrombin, anti-invasive and pro-apoptotic activities of Paeonia japonica. Korean J. Plant Res. 31: Lee, S.E., J.H. Choi, J.H. Lee, H.J. Noh, G.S. Kim, J.K. Kim, H.Y. Chung and S.Y. Kim Screening of useful plants with anti-inflammatory and antioxidant activity. Korean J. Plant Res. 26: Lewis, J.K. and W. Addison Principles of cancer biology. San Francisco, USA. pp Moreno, M.I., M.I. Isla, A.R. Sampietro and M.A. Vattuone Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J. Ethnopharmacol. 71: Pandey, K.B. and S.I. rizvi Plant polyphenols as dietary antioxidants in human disease. Dxid. Med. Cell Longev. 2: Rhee, S.G Cell signaling. H 2 O 2, a necessary evil for cell signlaing. Science 312: Shin, Y.J., D.Y. Jung, H.K. Ha and S.W. Park Anticancer effect of Erythronium japonicum extract on ICR mouse and L1210 cells with alteration of antioxidant enzyme activities. Korean J. Food Sci. Technol. 36: Singh, N.P. and H.C. Lai Artermisinin induces apoptosis in human cancer cells. Anticancer Res. 24: Shariati, A., S. Haghighi, S. Fayyazi, H. Tabesh and M.M. Kalboland The effect of exercise on the severity of the fatigue in colorectal cancer patients who received chemotherapy in ahwaz. Iran J. Nurs. Midwifery Res. 15: Sim, M.O., H.J. Lee, J.H. Jang, H.E. Lee, H.K. Jung, T.M. Kim, J.H. No, J.K. Jung and H.W. Cho Anti-inflammatory and antioxidant effects of Spiraea prunifolia Sieb. et Zucc. var. simpliciflora Nakai in RAW cells. Korean J. Plant Res. 30: Woo, K.W., M.O. Sim, H. Bak, H.K. Jung, B.K. An, S.H. Ham, J.H. Park and H.W. Cho Protective effects of flavonoids from Boehmeria quelpaertense against H 2 O 2 -induced cytotoxicity in H9c2 cardiomyblast cells. Korean J. Plant Res. 31:1-9. Yu, J.M and H.I. Moon Antioxidants and acetyl-cholinesterase inhibitory activity of solvent fractions extracts from Dendropanax morbiferus. Korean J. Plant Res. 30: Zhang, J.H. and M. Xu DNA fragmentation in apoptosis. Cell Res. 10: (Received 22 September 2017 ; Revised 11 December 2017 ; Accepted 19 January 2018) -108-

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,