170 Young-Mi Seo. Antioxidative Effect of Cornis fructus Extract ORIGINAL ARTICLE Korean J Clin Lab Sci. 2018;50(2):170-176 https://doi.org/10.15324/kjcls.2018.50.2.170 pissn 1738-3544 eissn 2288-1662 Antioxidative Effects of Cornis fructus Extract on Chromium Trioxide Toxicity Young-Mi Seo Department of Nursing, College of Medicine, Wonkwang Health Science University, Iksan, Korea Chromium Trioxide 의독성에대한산수유추출물의항산화효과 서영미 원광보건대학교간호학과 This study examined the antioxidative effects of Cornis fructus (C) extract on cultured C6 glioma cells. or this purpose, cytotoxicity analysis of chromium trioxide (CrO 3) was performed and the protective effects of the C extract on CrO 3 induced cytotoxicity was examined after the C6 glioma cells were cultured for 48 hours. The antioxidative effects, such as electron donating activity (EDA) and lactate dehydrogenase (LDH) activity were also analyzed. In this study, CrO 3 decreased the cell viability in a dose dependent manner. The XTT 50 value was determined to be 33 M after the cells were treated for 48 hours at a concentrations of 20 40 M CrO 3. The catalase (CAT) antioxidant increased significantly the cell viability that had been decreased by CrO 3 induced cytotoxicity. Regarding the protective effect of the C extract, the cell viability of the C extract was increased significantly compared to that of CrO 3 only. In addition, the C extract showed antioxidative effects, such as EDA and an inhibitory effect on the LDH activity. These findings suggest that the cytotoxicity of CrO 3 is correlated with oxidative stress, and the C extract effectively prevented CrO 3 induced cytotoxicity through the antioxidative effects. In conclusion, natural products, such as the C extract may be a useful therapeutic agent for the prevention or treatment of toxicity induced by heavy metals via oxidative stress. Key words: Antioxidative effect, Cytotoxicity, Natural products, Oxidative stress Corresponding author: Young-Mi Seo Department of Nursing, College of Medicine, Wonkwang Health Science University, 514 Iksan-daero, Iksan 54538, Korea Tel: 82-63-840-1314 ax: 82-63-840-1319 E-mail: dudn0408@wu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2018 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: May 15, 2018 Revised: May 27, 2018 Accepted: May 28, 2018 서론크롬을비롯한납, 수은과같은중금속류는인체에노출되면, 배출이어려울뿐만아니라체내에축적된후에는각기관의혈관을통해여러장기에손상을초래하게된다 [1]. 크롬은아말감을비롯한시멘트제조나도금, 내화벽돌및화학비료제조등여러산업공정에널리사용되어왔다 [2]. 그러나독성이강해인체가잦은노출에폭로될경우심각한질환을일으키는것으로잘 알려져있다 [1]. 특히, 뇌조직과같이혈액-뇌장벽 (bloodbrain barrier, BBB) 이잘형성된경우는크롬이나수은및카드뮴과같은중금속이직접뇌실질을통과하기어렵지만, 이들이분진이나흄 (fume) 에의해피부를통해체내로들어온경우, 혈액을통해허파를비롯한위, 신장및뇌조직등다양한장기에침투함으로서각종질환을유발한다는것은이미잘알려져있다 [3]. 특히, 6가크롬은 3가크롬과는달리생체막을쉽게통과할뿐만아니라독성이매우강하기때문에발암성과돌연변이
Korean J Clin Lab Sci. Vol. 50, No. 2, June 2018 171 성이높아취급시각별한주의가요구된다 [4]. 그러나, 현재구강또는호흡을통한크롬중독시환원제인 vitamin C의투여나기관지확장제와같은처치외에는특별한치료방법이없는실정이다 [4]. 최근, 크롬의독성이산화적손상 (oxidative stress) 과직접적인연관성이있다고제시되면서크롬중독에의한치료를항산화측면에서접근하려는시도가이루어지고있다 [5]. 그러나이에대한연구는초기단계일뿐만아니라연구가많이이루어지지않아매우미흡한상태이다 [2]. 특히, 산화적손상은세포내핵전사인자 (nuclear transcription factor) 인 N-kB (nuclear factor kb) 를활성화시켜세포고사인자 (apoptosis factor) 를활성화시키는동시에 [6], 세포막의 N-methyl-D aspartate (NMDA) 수용체를과활성시킨다 [7]. 또한막지질의과산화 [8] 및활성질소 (reactive nitrogen species, NOS) 와활성산소 (reactive oxygen species, ROS) 간의상호작용에의한 peroxynitrite 라는강력한독성물질을형성함으로서세포를퇴화내지는사멸에이르게한다 [9]. 최근, 각종식물에는항산화를비롯한항암, 항독, 항염등에유용한다양한생리활성성분들이많이함유되어있다고알려지고있다 [10]. 특히, 식물중산수유 (Cornis fructus, C) 는층층나무과 (Cornaceae) 에속하는낙엽관목으로우리나라전역에서자생하고있다. 산수유는 3 4월에잎보다황색꽃이먼저피는데 8 10 월경에는붉게열매를맺으며이의생약명으로는육조나석조라고도부른다 [11]. 산수유는 cornin을비롯한 loganin, linolic acid, saponin, gallic acid와같은성분들을함유하고있다. 특히, linoleic acid 나 gallic acid와같은성분은항산화에유효한생리활성성분으로잘알려져있다 [12]. 따라서, 산수유는오래전부터항균이나항바이러스를비롯한다뇨증이나월경과다및면역증강등의치료에사용되어왔다 [13]. 그러나아직까지산수유의항산화에대한연구는소수에불과하다 [11]. 근래, 세포배양법이발전되면서이를이용한병변의기전규명이나약제의안전성검사및신약에대한효능등을세포수준에서규명하려는연구가활발히이루어지고있다 [14]. 더욱이, 시험관내 (in vitro) 정량적분석방법이개발되면서정확하면서도빠르고간편하게분석할수있는계기가마련되었다 [15]. 본연구는중금속의일종인 CrO 3 의독성을산화적손상과관련하여조사함과동시에이의중독시천연소재로부터치료할수있는물질을항산화측면에서규명함으로서병변치료에유효한새로운물질의탐색을행하고자시행하였다. 재료및방법 1. 세포주본실험에사용한신경아교세포인 C6 glioma 세포주는 American Type Culture Collection (ATCC, Maryland, USA) 에서분양받아사용하였다. 2. 약제제조사용시약으로는 catalase (CAT) 를비롯한 chromium trioxide (CrO 3), ethyl alcohol, ethylenediaminetetraacetic acid (EDTA), trypsin, 1,1-diphenyl-2-picrylhydrazyl (DH), phosphate buffered saline (BS), dimethyl sulfoxide (DMSO) 및 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetra-zolium-5-carboxanilide (XTT) 는 Sigma-Alderich 사 (Sigma Chemical, Saint Louis, MO, USA.) 에서구입하였다. 또한, Hanker s balanced salt solution (HBSS) 과 fetal bovine serum (BS) 및 minimum essential medium (MEM) 은 Gibco 사 (Gibco Chemical, USA) 에서구입하였다. Lactate dehydrogenase (LDH) CytoTox detection kit는 Takara사 (Takara Biomedicals, Seoul, Korea) 에서구입하였다. CrO 3 의제조는 BS가들어있지않은 MEM을사용하여각각 10 um, 50 um 및 100 um의저장액을만들어냉암소에저장한후필요시희석하거나또는배양액에직접첨가하여사용하였다. XTT는 BS를이용하여 50 ug/ml의저장액을만든후냉암소에보관한다음필요한양을직접배양액에첨가하여사용하였다. 3. 세포배양 C6 glioma 세포의배양은 Kim 등 [16] 의방법에따라 trypsin 을이용한효소해리술로배양용기에부착된세포를분리하였다. 분리된세포들은튜브에모아원침시킨후 10% BS가함유된 MEM 배양액으로 1 10 5 cells/well이되도록 96-well 배양용기에분주하였다. 분주된세포들은 36 C, 5% CO 2 /95% air로조절된항온기내에서 72시간동안배양한후실험에사용하였다. 4. 산수유추출산수유는전초를채취하여깨끗이씻은후햇빛이들지않고서늘한곳에서건조시켜일정한크기로잘라냉암소에보관하여시료로사용하였다. 추출을위하여보관중인시료 250 g을이의 3배량의물과함께 1,000 ml의환저플라스크에넣고 24 시간동안가열하였다. 이를 3회반복적으로행한후추출액을모아 3,000 rpm에서 20분간원침하였다. 원침완료후여과하
172 Young-Mi Seo. Antioxidative Effect of Cornis fructus Extract 여진공농축기로감압농축한다음 24시간동안동결건조기에서건조하여 13 g의시료를얻었다. 이때수율은 5.2% 로나타났다. 5. 세포생존율조사세포생존율의분석은 Mosmann [17] 의방법에따라행하였다. 즉, 배양이완료된세포를 well당 1 10 5 /well의밀도로분주한후 72시간동안배양하였다. 배양이완료된세포에약제나시료를처리한다음, 전날제조한 XTT를각 well당 10 ul씩을넣고 37 C, 5% CO 2 /95% air로조절된항온기내에서 4시간동안배양하였다. 배양완료후 DMSO로처리하여 450 nm에서 ELISA reader (Spectra max 250, Molecular Devices, Sunnyvale, USA) 로흡광도를측정하여대조군과비교조사하였다. 6. CrO 3 에대한세포독성조사 CrO 3 의세포독성조사를위하여배양세포에 20 40 um의 CrO 3 가각각포함된배양액에서 48시간동안배양한후 450 nm 에서 ELISA reader 로흡광도를측정하여대조군과비교조사하였다. 7. CAT 의항산화능측정 CAT 의항산화능을조사하기위하여 H 2O 2 40 um를배양세포를처리하기 2시간전에 CAT 가 20 um과 40 um이포함된배양액에서세포를처리한후세포생존율을대조군과비교조사하였다. 8. CrO 3 에대한항산화제의영향 CrO 3 의독성에대한항산화제의일종인 CAT의영향을알아보기위하여 XTT 50 농도의 CrO 3 를배양세포에처리하기 2시간전에 CAT 가각각 20 um과 40 um이포함된배양액에서배양한후세포생존율을 CrO 3 만의처리와비교조사하였다. XTT 50 값은직선회귀식에의하여산출하였다. 9. CrO 3 에대한산수유추출물의영향 XTT 50 농도의 CrO 3 를배양세포에처리하기 2시간전에산수유추출물이각각 140 ug/ml과 160 ug/ml가들어있는배양액에서배양한후세포생존율을 CrO 3 로만처리한세포와비교조사하였다 10. 전자공여활성 (electron donating activity, EDA) 측정 EDA는항산화물질에대한라디칼소거활성을측정하는손 쉬운측정물이다. 항산화물질에대한항산화효과를측정하기위해 EDA에의한환원력을사용한다. EDA 측정은 Blois [18] 의방법에따라행하였다. 즉, 메탄올시료에 0.3 mm DH 메탄올용액 100 ul를첨가하여실온에서 30분간처리하였다. 처리후 ELISA reader로 517 nm에서흡광도를측정하였다. 전자공여능은시료첨가군과시료무첨가군간의차이를시료무첨가군에의한백분율로나타냈으며, 또한 CAT 의활성을양성대조군으로사용하였다. 11. Lactate dehydrogenase (LDH) 활성측정 LDH 측정을위하여약제나시료를처리한세포의배양액을모은후 1,500 rpm에서 15분동안원침시킨다음상등액 50 ul 를취하였다. 취한상등액에 LDH kit 반응액 50 um을넣고 30 분간반응시켰다. 반응완료후 ELISA reader 로 450 nm에서흡광도를측정하여대조군과비교조사하였다. LDH 활성은대조군에대한백분율로표시하였다. 12. 통계처리 실험의모든자료는 3회이상반복한결과를 mean±sd로나타냈으며, 실험결과는 SSS (Win Version 18.0, SSS, Chicago, USA) 를이용하여군간의차이를비교하기위하여 ANOVA 를시행하였다. 사후분석은 Tukey s HSD (honest significant difference) 로하였으며, 모든통계의유의수준은 -value가 0.05 미만의경우를유의한것으로채택하였다. 결과 1. CrO 3 의세포독성측정 CrO 3 에대한세포독성을측정하기위하여배양세포에 20 40 um의 CrO 3 가들어있는배양액으로각각처리한결과, 20 um Table 1. The cytotoxicity of chromium trioxide (CrO 3) in cultured C6 glioma cells Incubation of CrO 3 ( M) Control 0.21±0.02 60.51 <0.001 20 0.14±0.01 30 0.12±0.01 40 0.07±0.00 XTT 50 (CrO 3) 0.11±0.01 Abbreviation: XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetra-zolium-5-carboxanilide.
Korean J Clin Lab Sci. Vol. 50, No. 2, June 2018 173 처리에서는세포생존율이대조군인 100% (0.21±0.02) 에비하여 66.7% (0.14±0.01) 로나타났으며 30 um에서처리한경우 57.1% (0.12±0.01) 로나타났다. 또한, 40 um의경우세포생존율은 33.3% (0.07±0.00) 로나타났다. 이과정에서 XTT 50 값은 33 um에서나타났다 (<0.001) (Table 1). CrO 3 에대한세포독성에따른사후분석결과세포생존율이대조군, CrO 3 20 um, CrO 3 30 um, CrO 3 33 um, CrO 3 40 um 순으로높음을알수있었다. 다만 CrO 3 20 um, CrO 3 30 um, CrO 3 33 um은통계적으로유의한차이는아니었다. 2. CAT 의항산화능측정 CAT 의항산화능조사결과, H 2 O 2 40 um만의처리에서는세포생존율이대조군에비하여 45.0% (0.09±0.02) 로나타났다 (<0.001). 그러나, 세포생존율이 CAT 20 um의처리에서는 60.0% (0.12±0.00), CAT 40 um 처리에서는 90.0% (0.18± 0.02) 로나타났다 (<0.001) (Table 2). CAT 의항산화능에대한사후분석결과대조군과 CAT 40 um, CAT 20 um과 H 2 O 2 40 um 순으로세포생존율이높음을알수있었다. 3. CrO 3 의세포독성에대한항산화제의영향 CrO 3 의세포독성에대한항산화제의영향을알아본결과, XTT 50 농도인 CrO 3 만의처리에서 39.5% (0.68±0.10), CAT 20 um 처리에서 51.4% (0.89±0.07), CAT 40 um 처리에서 Table 2. The antioxidative activity of catalase (CAT) on the hydrogen peroxide (H 2O 2) in cultured C6 glioma cells 72.7% (1.25±0.05) 로통계적으로유의한차이를보였다 (< 0.001) (Table 3). CrO 3 의세포독성과항산화제의영향에따른사후검정결과대조군, CAT 40 um, CAT 20 um, XTT 50 (CrO 3 ) 순으로세포생존율이높음을알수있었다. 4. CrO 3 의세포독성에대한산수유추출물의영향 산수유추출물이 CrO 3 의세포독성에미치는영향을조사한결과 CrO 3 만의처리에서의세포생존율은대조군인 100% (1.83±0.03) 에비하여 41.5% (0.76±0.07) 로낮게나타났다. 반면, 140 ug/ml 산수유추출물처리에서의세포생존율은 52.5% (0.96±0.08), 산수유추출물 160 ug/ml의처리에서는 62.8% (1.15±0.05) 로나타났다 (<0.001) (Table 4). 산수유추출물이 CrO 3 의세포독성에미치는영향에대한사후검정결과대조군, 산수유 160 ug/ml, 산수유 140 ug/ml, XTT 50 (CrO 3 ) 순으로세포생존율이높은것을알수있었다. 5. EDA 측정 전자공여활성 (EDA) 의측정결과산수유추출물 140 ug/ml 농도에서는대조군에비하여활성이 80.4% (1.23±0.09) 로나타났으며, 산수유추출물 160 ug/ml 농도에서는 77.8% (1.19±0.09), CAT 20 um 처리에서는 21.6% (0.33±0.07) 로점차감소한것으로나타났다 (Table 5). 따라서, 산수유추출물 Table 4. The protective effect of Cornis fructus (C) extract on chromium trioxide (CrO 3)-induced cytotoxicity in cultured C6 glioma cells Concentrations of CAT ( M) Concentrations of C extract (ug/ml) Control 0.20±0.01 35.97 <0.001 40 H 2O 2 0.09±0.02 20 0.12±0.00 40 0.18±0.02 Abbreviation: See Table 1. Control 1.83±0.03 168.78 <0.001 XTT 50 (CrO 3) 0.76±0.07 140 0.96±0.08 160 1.15±0.05 Abbreviation: See Table 1. Table 3. The effect of catalase (CAT) on the cytotoxicity induced by chromium trioxide (CrO 3) in cultured C6 glioma cells Table 5. The electron donating activity (EDA) of Cornis fructus (C) extract Concentrations of CAT ( M) Concentrations of C extract (ug/ml) EDA (517 nm) Control 1.72±0.03 128.38 <0.001 XTT 50 (CrO 3) 0.68±0.10 20 0.89±0.07 40 1.25±0.05 Abbreviation: See Table 1. Control 1.53±0.06 130.58 <0.001 20 CAT 0.33±0.07 140 1.23±0.09 160 1.19±0.09 Abbreviation: CAT, catalase; See Table 1.
174 Young-Mi Seo. Antioxidative Effect of Cornis fructus Extract igure 1. Electron donating ability of C extract at concentrations of 140 ug/ml and 160 ug/ml, respectively. The data indicate the mean for 3 times on triplicate experiments. Electron donating activity of C extract were significantly different from negative control. CAT was used as positive control. igure 2. The LDH activity of C extract at concentrations of 140 ug/ml and 160 ug/ml, respectively. The data indicate the mean for 3 times on triplicate experiments. LDH activity of C extract were significantly different from negative control. CrO 3 was used as positive control. Table 6. The lactate dehydrogenase (LDH) activity of Cornis fructus (C) extract on chromium trioxide (CrO 3)-induced cytotoxicity in cultured C6 glioma cells Concentrations of C extract (ug/ml) LDH activity (450 nm) Control 1.76±0.04 35.56 <0.001 XTT 50 (CrO 3) 2.40±0.07 140 2.13±0.13 160 1.86±0.07 Abbreviation: See Table 1, 4. 140 ug/ml와 160 ug/ml의전자공여능은각각 19.6% 와 22.2%, CAT 20 um의전자공여능은 78.4% 로대조군에비하여통계적으로유의하였다 (<0.001) (igure 1). 전자공여능의사후검정결과 CAT 20 um, 산수유추출물 160 ug/ml와 140 ug/ml, 대조군순으로높은것으로나타났다. 6. LDH 활성측정 CrO 3 의세포독성에대한산수유추출물의 LDH 활성을조사한결과 CrO 3 만의처리에서는 LDH 활성이대조군인 100% (1.76±0.04) 에비하여 136.4% (2.40±0.07) 로매우높게나타났다. 이에비하여, 140 ug/ml 산수유추출물처리에서는 LDH 활성이 121.0% (2.13±0.13) 로나타나 CrO 3 만의처리에비하여매우감소한것을확인하였다 (<0.001) (Table 6). 또한, 160 ug/ml의산수유추출물처리에서는 105.7% (1.86±0.07) 로 CrO 3 만의처리에비하여유의한 LDH 활성감소를보였다 (< 0.001) (igure 2). CrO 3 의세포독성에대한산수유추출물의 LDH 활성에대한사후검정결과대조군과 160 ug/ml 산수유추출물, 140 ug/ml 산수유추출물, CrO 3 처리군순으로 LDH 활성감소가높은것으로나타났다. 고찰크롬을비롯한중금속의성분은인체에서소량으로존재하면서생명에필요한물질을구성하고있지만다량이인체내에축적되면심한중독을유발하여각종부작용을일으키고있다 [19]. 본연구에서는삼산화크롬인 CrO 3 의세포독성을조사하기위하여배양 C6 glioma 세포에 20 40 um의 CrO 3 를 48시간동안처리한결과처리농도에비례하여세포생존율이대조군에비해유의한감소를보였다. 이과정에서 XTT 50 값이 100 um 이하인 33 um로나타나 Borenfreund와 uerner [20] 에의한독성판정기준에의해고독성 (highly-toxic) 인것으로나타났다. 본연구결과는 CrO 3 가세포독성을가지고있음을말해주고있으며 Jung 등 [21] 에의한 CrO 3 의세포독성을보고한결과와일치하였다. 이같은현상은 CrO 3 가세포내핵산물질인 DNA (deoxyribonucleic acid) 의합성방해나또는단백질의합성을억제한결과도배제할수는없지만 [3], 그보다는 CrO 3 의산화적손상에의해세포가퇴화내지는고사한결과일가능성이클것으로생각된다. 따라서, 본연구에서는 CrO 3 의독성과산화적손상과의연관성을조사하였다. 항산화제의일종인 CAT 를배양세포에전처리한결과, 처리한 CAT 의처리농도에비례하여 CrO 3 에의해감소된세포생존율을유의하게증가시켰다. 본결과는 CrO 3 의독성이산화적손상과관련이있음을제시하고있으며, Jung 등 [21] 이 CrO 3 의독성을항산화제인 vitamin E가방어하였다는연구결과와도상호일치함을알수있었다. 이같은현상은 CrO 3 에의해생성된 hydroxyl radical 이나 superoxide anion radical과같은자유라디칼을 CAT 나
Korean J Clin Lab Sci. Vol. 50, No. 2, June 2018 175 vitamin E와같은항산화제가제거한결과인것으로생각된다 [4]. 한편, 산수유추출물이 CrO 3 의독성에미치는영향을알아보기위하여산수유추출물 140 ug/ml와 160 ug/ml를배양세포에각각전처리한결과, CrO 3 만의처리에비하여유의한세포생존율의증가를보였다. 본결과는산수유추출물이 CrO 3 에의한산화적손상을방어한결과임을말해주고있으며, 이는동시에산수유추출물의항산화능을제시하고있다. 이의증거하나로서 Lee 등 [22] 이산수유추출물이자유라디칼인과산화수소 (H 2 O 2 ) 의산화적손상을방어하였다는연구결과와도일치한다. 이같은현상은산수유추출물속에함유된 linolic acid를비롯한 gallic acid 및 saponin과같은항산화성분의상호작용의결과에기인한것으로생각된다 [12]. 따라서, 본연구에서는산수유추출물의항산화능을알아보기위하여전자공여활성 (EDA) 을비롯한 LDH 활성을조사하였다. 본연구의 EDA 측정을위해 140 ug/ml와 160 ug/ml의산수유시료를처리한결과대조군에비하여유의한전자공여능의증가를보였다. 이는산수유추출물이자유기제거능이있음을말해주고있으며이는곧산수유추출물의항산화능을증명해주고있다. 한편, 산수유추출물의 LDH 활성조사결과에서 140 ug/ml와 160 ug/ml의산수유추출물처리에서는 CrO 3 로만의처리에비하여유의한 LDH 활성저해를보였다. 위결과는산수유추출물의항산화능에의하여 CrO 3 의산화적손상으로야기되는막의지질과산화가방어된것으로서, 이는산수유추출물이과산화수소에의한산화적손상을방어하여 LDH 활성감소를나타냈다는연구보고와도일치하였으며 [22], EDA와함께산수유추출물의항산화능을증명하고있음을알수있다. LDH 활성분석은지질과산화반응 (reaction of lipid peroxidation) 의분석과함께막의손상정도를측정할수있는정량적분석방법의하나로알려져있다 [8]. 이상의결과로부터산수유추출물은산화적손상과관련이있는 CrO 3 의독성을항산화능에의하여효과적으로방어한다는것을확인하였다. 따라서, 산수유추출물과같은천연성분에대한항산화적생리활성분석은질병치료를위한약제의부작용염려는물론나아가서치료적효능이높은물질성분을이용해산화적손상으로야기되는질환치료적측면에서새로운물질개발을위한기초자료로서의활용도가높을것으로생각된다. 요약본연구목적은산수유추출물의항산화효과를배양 C6 glioma 세포를대상으로조사하였다. 이같은목적을위하여 C6 glioma 세포를 48시간동안배양한후 6가크롬의세포독성 및산수유추출물의방어효과를조사하였다. 이외에 EDA 및 LDH 활성과같은항산화효과를분석하였다. 본연구에서 CrO 3 는처리농도에비례하여세포생존율을감소시켰다. 또한이과정에서세포를 48시간동안 20 40 um로각각포함된배양액에서처리한결과 XTT 50 값은 33 um로나타났다. 항산화제인 CAT는 CrO 3 로유도된세포독성에의해감소된세포생존율을유의하게증가시켰다. 한편산수유추출물의보호효과에있어서, 산수유추출물은 CrO 3 만의처리군에비하여세포생존율을유의하게증가시켰다. 동시에산수유추출물은 EDA와 LDH 활성과같은항산화효과를보였다. 이와같은결과로부터 CrO 3 의독성에산화적손상이관련되어있는것으로나타났다. 또한산수유추출물은이의항산화효과에의하여 CrO 3 의세포독성을효과적으로방어하였다. 결론적으로, 산수유추출물과같은천연소재는산화적손상과관련된중금속에의해유발된세포독성을방어내지는치료하는데유용한치료적요소의하나로생각된다. Acknowledgements: This paper was supported by Wonkwang Health Science University in 2018. Conflict of interest: None REERENCES 1. attison DI, Davies MJ, Levina A, Dixon NE, Lay A. Chromium (VI) reduction by catecholamines results in DNA cleavage in vitro: relevance to chromium genotoxicity. Chem Res Toxicol. 2001;14:500-510. http://dx.doi.org/10.1021/tx000229s. 2. Tso TC, Lai HJ, Yang JL. Effects of mannitol or catalase on the generation of reactive oxygen species leading to DNA damaged by chromium (VI) reduction with ascorbate. Chem Res Toxicol. 1999;12:1002-1009. http://dx.doi.org/10.1021/tx9802264. 3. Zhang Q, Kluz T, Salnikow K, Costa M. Comparison of the cytotoxicity, cellular uptake, and DNA-protein induced by potassium chromate in lymphoblast cell line derived three different individuals. Biol Trace Elem Res. 2002;86:11-22. 4. Leonard S, Wang S, Zang L, Castranova V, Vallyathan V, Shi X. Role of molecular oxygen in the generation of hydroxyl and superoxide anion radicals during enzymatic Cr (VI)-induced carcinogenesis. J Environ athol Toxicol Oncol. 2000;19: 49-60. 5. Bagchi D, Bagchi M, Stohs SJ. Chromium (VI)-induced oxidative stress, apoptosis cell death and modulation of p53 tumor suppressor gene. Mol Cell Biochem. 2001;222:149-158. 6. Gracia-Lopez D, Cuevas MJ, Almar M, Lima E, De-az JA, Gonzalez-Gallego J. Effects of eccentric exercise on N-kB activation in blood mononuclear cells. Med Sci Sport Exerc. 2007; 39:653-664. http://dx.doi.org/10.1249/mss.0b013e31802f04f6. 7.Jung IJ. The effect of NMDA/glycin receptor antagonist,
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