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1 ORIGINAL ARTICLE Korean J Clin Lab Sci. 2018;50(2): pissn eissn Korean J Clin Lab Sci. Vol. 50, No. 2, June DNA Breakage by Salvianolic acid B in the Presence of Cu (II) Pyeongjae Lee 1, Cheol Moon 2, Yoon Seon Choi 3, Hyun Kyu Son 4 1 School of Industrial Bio-pharmaceutical Science, Semyung University, Jecheon, Korea 2 Department of Clinical Laboratory Science, Semyung University, Jecheon, Korea 3 Department of Cosmetic Science, Semyung University, Jecheon, Korea 4 Department of Natural Medicine Resources, Semyung University, Jecheon, Korea 구리이온 (II) 이존재할때 Salvianolic acid B 에의한 DNA 절단 이평재 1, 문철 2, 최윤선 3, 손현규 4 1 세명대학교바이오제약산업학부, 2 세명대학교임상병리학과, 3 세명대학교화장품과학과, 4 세명대학교자연약재과학과 Salvianolic acid B, which is a compound in the Salvia miltiorrhiza, has diverse biological activities, In particular, the antioxidative effects were reported to be involved in the protection of hepatocytes, neurons, and various cell types. On the other hand, some phenolic compounds, such as ferulic acid, which is regarded as an antioxidant, plays a pro-oxidative role in the specific transitional metal environment, which could explain the anticancer effect. This study examined the pro-oxidative effects of salvianolic acid B in the presence of Cu 2+. Treatment with both salvianolic acid B and Cu 2+ induced the transition of supercoiled DNA to the open circular or linear form but not in the sole salvianolic acid B or Cu 2+ treatments. Salvianolic acid B reduced the Cu 2+ to Cu + using neocuproine, a Cu + specific chelator. In addition, catalase, an enzyme that breaks down the H 2O 2 to water and molecular oxygen, inhibited the DNA breakage. H 2O 2, a reactive oxygen species, has detrimental effects on biological molecules, particularly DNA. Overall, the reduction of Cu 2+ by salvianolic acid B could lead to the production of H 2O 2 followed by DNA breakage. These results suggest that the pro-oxidative effects could be the one of the anti-cancer mechanisms of salvianolic acid B, which remains to be explained. Key words: Antioxidant, Copper ion, Hydroxyl radical, Pro-oxidant, Salvianolic acid B Corresponding author: Pyeongjae Lee School of Industrial Bio-pharmaceutical Science, Semyung University, 65 Semyung-ro, Jecheon 27136, Korea Tel: Fax: pjlee1@semyung.ac.kr 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. Copyright 2018 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: February 9, 2018 Revised 1 st : March 21, 2018 Revised 2 nd : May 16, 2018 Revised 3 rd : May 21, 2018 Accepted: May 22, 2018 서론산소는세포에서 ATP 를생성하기위한호흡기능에필수적요소이다. 미토콘드리아에서전자전달계를거친전자는최종적으로산소가수용하는데전자전달계를이탈한전자는산소와반응하여 superoxide radical ( O 2 ), hydrogen peroxide (H 2O 2), hydroxyl radical ( OH) 등반응성이강한활성산소종 (Reactive Oxygen Species: ROS) 을생성하게된다. 때로는이와다른화학적반응을통해 ROS를생성하기도하는데이렇게 생성된활성산소종은세포를구성하는핵산, 단백질, 지질의구조를변화시키고이는세포의기능에심각한손상을줄수있다. 이를 oxidative stress (OS) 라부르며퇴행성질환및노화에있어주요한원인으로여겨지고있다. 생명체는 ROS를방어하는시스템을구축하여대응한다. Superoxide dismutase (SOD) 와 catalase 등의효소시스템은생성된 ROS를안전한 H2O로변환시키며체내 ROS를제거하는항산화물질을가지고있기도하다 [1-2]. ROS가세포에부정적영향을주기도하지만생명체는 ROS

2 206 Pyeongjae Lee, et al. DNA Breakage by Salvianolic acid B 를유용하게이용하기도한다. DNA, 단백질, 지질의변형을가져오는 ROS는외부로부터침입한병원체등을제거하기위한자기생체방어시스템으로서의역할을수행하기도하고세포간의신호전달물질로서의역할을하기도한다 [3]. OS로인한질병의발생과노화를예방혹은치료를위해그동안생명체의방어시스템을강화시키거나혹은 ROS를소거하는항산화효능을가지는소재에대해연구되어보고되어왔다. 하지만항산화효능이뛰어나다고알려진물질도 Cu 2+ 과같은산화형의전이금속이있는환경에서오히려 pro-oxidant 로서 ROS를만들어낼수있음 (Figure 1) 이알려져있고 [4-5] 이것이암세포를사멸시키는기전중에하나라고보고있기도하다 [6-8]. 이는물질이가지는다양한면을보여주고있는것으로때론항산화물질로서때론 pro-oxidant 로유용하게이용할수도있음을보여주고있다고생각한다. 단삼 (Salvia miltiorrhiza Bunge) 은중앙ㆍ서아메리카, 중앙ㆍ동아시아에분포하는꿀풀과에속하는다년생초본식물로서뿌리를약용으로사용한다. 단삼에포함되어있는주요성분은 diterpene 화합물에포함되는 tanshinone I, IIA, IIB 등과 phenolic 화합물에포함되는 danshensu, protocatechuic aldehyde, salvianolic acid B 등이알려져있다 [9]. 이전연구에따르면단삼에포함되어있는주요성분중 Salvianolic acid B는간보호, 혈관보호, 신경세포보호등의생리활성기능과함께 superoxide radical 과 hydroxyl radical 를소거하는항산화효과를가지고있다 [10-14]. 위에서설명했듯이 phenolic compounds 들이 pro-oxidant로작용하여 ROS 중특히 hydroxyl radical을생성한다고보고가있으며이를 phenolic compound의항암작용의하나의기전으로설명하기도한다. 본실험에서는단삼의주요한생리활성물질이며항산화효과가있다고알려진 salvianolic acid B를대상으로 pro-oxidant 로서의기능하는지확인하는실험을진행하였다. 재료및방법 1. 실험재료본실험에사용한 3-(2-Pyridyl)-5,6-diphenyl-1,2,4- triazine-4`,4``-disulfonic acid sodium salt (Ferrozine), Figure 1. Mechanism of hydroxyl radical formation mediated by Cu 2+. neocuproine, salvianolic acid B, Calf DNA는 Sigma Aldrich (St. Louis, MO, USA) 에서구입한제품을사용하였고, pbr322 (Plasmid) 는 New England Biolabs (NEB) 에서, FeSO 4 와 CuSO 4 는 Samchun Chemical 에서구입한제품을사용하였다. 2. DNA nicking assay pbr322가 hydroxyl radical에의해절단이일어나는현상에 salvianolic acid B가어떠한영향을주는지확인하기위해실행하였다. pbr322 (250 g/ml), FeSO 4 혹은 CuSO 4, salvianolic acid B를혼합한후 phosphate buffer (ph 7.4) 를이용하여총부피를 20 L로맞추었다. 실험에따라 catalase 혹은 EDTA 를넣어 20 L로맞추었다. Heating block을이용하여 37 C에서 60분간반응시킨후 TAE buffer (1X) 로만든 1% agarose gel을만들어전기영동시킨후 gel image analysis system을사용하여밴드의위치를확인하였다. 3. Calf DNA break assay Calf DNA가 hydroxyl radical 에의하여절단이일어나는현상에대하여 salvianolic acid B가어떠한영향을주는지확인하기위해실행하였다. Calf DNA (0.4 mg/ml in phosphate buffer), FeSO 4 (2 mm), salvianolic acid B (200 M, 10 M, 100 M, 200 M) 을혼합한후 H 2O를이용하여총부피를 40 L로맞추었다. Heating block을이용하여 37 C에서 60분간반응시킨후 TAE buffer (1X) 로만든 1% agarose gel을만들어전기영동시킨후 gel image analysis system을사용하여결과를확인하였다. 4. Neocuproine assay Salvianolic acid B에의해 Cu 2+ 가 Cu + 로변환되는지를확인하기위해 Cu + 과특이적으로결합하는 neocuproine 를이용하였다. CuSO 4 (100 M), neocuproine (400 M), salvianolic acid B (1, 5, 10, 100, 200 M) 를혼합한후 phosphate buffer (ph 7.4) 를이용하여 400 L로맞추었다. 실온에서 10분간반응시킨후 450 nm에서흡광도를측정하였다. 5. Iron 킬레이팅 assay Salvianolic acid B가 Fe 2+ 에대하여 chelating 효과가있는지확인하기위해실행하였다. FeSO 4 (200 M), ferrozine (200 M), salvianolic acid B (10 M, 100 M, 200 M) 를혼합한후 H 2O를이용하여총부피를 400 L로맞추었다. 실온에서 10분간반응시킨후 562 nm에서흡광도를측정하였다.

Korean J Clin Lab Sci. Vol. 50, No. 2, June 2018 207 6. 통계분석실험은독립적으로 3회반복하였다. 분석한실험결과값의통계처리는평균값 ± 표준편차로나타내었으며, 실험결과값의통계적유의성은대조군과각각의시료처리군의실험결과값으로부터 Student s t-test를통하여검증하였다. P-value< 0.

플라 스미드 DNA에절단이일어나면 supercoiled (form I) 에서 open circular form (form II) 이되었다가더절단이일어나게되면 linear form (form III) 이되어 agarose gel에서그위치가변한다. 따라서 DNA분해를일으키는물질을처리했을때겔에서의 DNA 위치를관찰하면 DNA 절단정도를확인할수있다.

3 Korean J Clin Lab Sci. Vol. 50, No. 2, June 통계분석실험은독립적으로 3회반복하였다. 분석한실험결과값의통계처리는평균값 ± 표준편차로나타내었으며, 실험결과값의통계적유의성은대조군과각각의시료처리군의실험결과값으로부터 Student s t-test를통하여검증하였다. P-value< 0.05 인것을통계적으로유의한것으로하였다. 결과 1. Fe 2+ 이온조건에서 salvianolic acid B의 DNA 절단억제효과플라스미드는 circular 형태로 supercoiled 되어있다. 플라 스미드 DNA에절단이일어나면 supercoiled (form I) 에서 open circular form (form II) 이되었다가더절단이일어나게되면 linear form (form III) 이되어 agarose gel에서그위치가변한다. 따라서 DNA분해를일으키는물질을처리했을때겔에서의 DNA 위치를관찰하면 DNA 절단정도를확인할수있다. 단삼이함유하고있는여러물질중에 salvianolic acid B는주요한생리활성효능을보이는물질로서항산화효과가보고된바있다 [14]. 본실험에서는환원형금속이온인 Fe 2+ 환경에서 salvianolic acid B의항산화효과를확인하였다. FeSO M 을플라스미드에처리했을때 DNA가절단되어 supercoiled 형태가사라지고 open circular 형태와일부 linear 형태를확인할수있었다. FeSO M을 salvianolic acid B와같이처리했을때처리농도를증가할수록 linear 형태가사라지고 supercoiled 형태가증가함을알수있다 (Figure 2A). DNA를플라스미드대신 Calf DNA로바꾸었을때 salvianolic acid B가 Fe 2+ 에의한 DNA 절단억제를좀더뚜렷이알수있다 (Figure 2B). 항산화효과를측정하는방법으로 Fe 2+ 킬레이팅능을측정하는데본실험에서 salvianolic acid B는 Fe 2+ 과 ferrozine의복합체형성을억제하지못하였다 (Figure 3). 이는 salvianolic acid B가 Fe 2+ 를킬레이팅능이뛰어나지않음을의미하며 Zhao 등 [14] 의내용과부합하는결과이다. 2. Cu 2+ 이온환경에서 salvianolic acid B 의 DNA 절단 Figure 2. Inhibitory effect of salvianolic acid B on nicking and breakage of plasmid (A) or calf DNA (B). Electrophoresis was performed on 1% agarose gel. Form I, supercoiled form: Form II, open circular form: Form III, linear form. CuSO 4 를단독으로 200 M 처리했을때, FeSO 4 처리와달리 supercoiled 형태의변화를관찰할수없었다. CuSO 4 를 200 M 로고정하고 salvianolic acid B의처리농도를증가할때 1 M 에서 open circular 형태를관찰할수있었으며 100 M에서는 linear 형태가보이고 200 M에서는 linear 형태위치에서부터 smearing 형태를관찰할수있었다 (Figure 4A). Salvianolic Figure 3. Complex of ferrozine and Fe 2+ has the absorbance on 562 nm. Salvianolic acid B had no effect on binding of ferrozine on Fe 2+. Values represent mean±sd of three independent measurements. ***P<0.001; significantly different from the mixture of ferrozine and Fe 2+ by Student s t-test. Figure 4. Induction of DNA breakage by salvianolic acid B in the presence of CuSO 4 (A and B). Electrophoresis was performed on 1% agarose gel. Form I, supercoiled form: Form II, open circular form: Form III, linear form.

CuSO 4 와 salvianolic acid B 단독처리에서는 DNA 절단을확인할수없었으나같이처리했을때는 DNA의절단을확인할수있었다. 이는 Cu 2+ 와 salvianolic acid B의반응으로 DNA 절단하는물질을생성했음을의미한다. 환원된형태의금속은용액내산소와의반응을통해 ROS을생산한다 (Figure 1).

4 208 Pyeongjae Lee, et al. DNA Breakage by Salvianolic acid B acid B 처리농도에비례하여 DNA의절단정도가심해짐을알수있다. Salvianolic acid B를 10 M로고정하고 CuSO 4 의처리농도를증가할경우 20 M에서 open circular 형태를관찰할수있었고 100 M에서는완전히 open circular 형태만을관찰가능하며 200 M에서는 100 M처리때와는달리더이상의 DNA 분해는관찰되지않았다 (Figure 4B). CuSO 4 와 salvianolic acid B 단독처리에서는 DNA 절단을확인할수없었으나같이처리했을때는 DNA의절단을확인할수있었다. 이는 Cu 2+ 와 salvianolic acid B의반응으로 DNA 절단하는물질을생성했음을의미한다. 환원된형태의금속은용액내산소와의반응을통해 ROS을생산한다 (Figure 1). 특히 hydroxyl radical 은가장반응성이큰 ROS로서 DNA 절단의주요한원인이된다 [15]. Cu 2+ 는산화형태로서 salvianolic acid B에의해 Cu 2+ 가환원되어 Cu + 가되고이는산소와의반응을통해 hydroxyl radical 생성되어 DNA를절단하였다고가정해볼수있다. 3. Cu 2+ 이온환경에서 salvianolic acid B에의한 DNA 절단기전이실험에서 DNA 절단의원인이 hydroxyl radical 임을확인하기위해중간단계생성물로여겨지는 H 2 O 2 를제거하는 catalase를처리하여 DNA 절단을관찰하였다. CuSO 4 와 salvianolic acid B의처리농도를각각 100 M과 10 M로고정하고 catalase 처리농도를증가할수록 open circular 형태가사라짐을관찰할수있다 (Figure 5A). 이는 catalase로인해 H 2 O 2 가제거되어이후 hydroxyl radical 의생성이억제되었음을추론해볼수있다. Cu 2+ 는 DNA의특정위치에결합하여 ROS를생성함으로서 DNA의절단이용이해지며 EDTA 는전이금속킬레이터로서 Cu 2+ 의 DNA 결합을억제한다 [16]. 본실험에서 CuSO 4 와 salvianolic acid B의처리농도를각각 100 M과 10 μm 로고정하고 EDTA 를처리하여 DNA 절단을관찰하였다. EDTA 처리농도를증가시킬수록 open circular 형태가줄어드는것을관찰할수있었다 (Figure 5B). 이는본실험에서 Cu 2+ 가 DNA에결합하고 salvianolic acid B와의상호작용을통해 H 2O 2 를생성하고최종적으로 hydroxyl radical을생성하였다고생각해볼수있다. 4. Cu + 의생성 Cu 2+ 는산화형으로 hydroxyl radical을생성하기위해환원형으로바뀌어야한다. Salvianolic acid B가 Cu 2+ 를 Cu + 로변환시키는지알아보기위해 Cu + 에만특이적으로결합하는킬레이터인 neocuproine 를넣어흡광도측정하였다. Cu + 와 neocuproine의결합은흡광도를증가시킨다. Salvianolic acid B를처리농도를증가할수록농도의존적으로흡광도증가하였다 (Figure 6). 이는 salvianolic acid B에의해 Cu 2+ 가 Cu + 로변환됨을의미한다. 위결과를종합하여보면 Cu 2+ 가 DNA에결합하고 salvianolic acid B에의해 Cu + 로환원되고환원된 Cu + 는산소와의반응을통해 H 2 O 2 를거쳐 hydroxyl radical 를생성하며이것이 DNA절단의원인이됨을추론해볼수있다. 고찰본실험에서환원형태의 Fe 2+ 는 DNA 절단을일으키지만 salvianolic acid B는플라스미드와 Calf DNA에서절단을억제하였고산화형태의 Cu 2+ 는단독으로 DNA 절단을일으키지않 Figure 5. Preventive effect of catalase (A) and EDTA (B) on DNA breakage in the presence of salvianolic acid B and CuSO4. Electrophoresis was performed on 1% agarose gel. Form I, supercoiled form: Form II, open circular form: Form III, linear form. Figure 6. Transition of Cu 2+ into Cu+ by salvianolic acid B. Neocuproine is Cu 2+ -specific chelator. Values represent mean±sd of three independent measurements. ***P<0.01, ***P<0.001; significantly different from the mixture of buffer and neocuproine without salvianolic acid B by Student s t-test.

5 Korean J Clin Lab Sci. Vol. 50, No. 2, June 으나 salvianolic acid B를첨가함으로서 DNA의절단을확인하였다. Catalase 및 EDTA 처리는플라스미드의절단을억제하였고 neocuproine 를처리했을때흡광도가증가함을확인하였다. 이를종합해보면 Cu 2+ 가 DNA와결합하고 salvianolic acid B와의산화환원반응을통해 Cu + 로변환된후산소와의일련의반응을통해 H 2 O 2 를거쳐 hydroxyl radical을생성한다고볼수있다. 즉산화형의금속이존재할때전자를부여하는능력이산화형의금속을환원시키면 hydroxyl radical의생성을늘리며이것이 DNA을절단한다고생각되어진다. 이는항산화능력이있다고여겨지는물질도주위금속이온의환경에따라 pro-oxidant 로서기능을수행함을의미한다. 항산화물질이 pro-oxidant 로서기능을수행한다는것이유용하게활용되어질수있는데특히항암효과를나타내는기전으로여겨진다. 정상세포와암세포의차이점으로구리이온의증가한다는보고 [17] 는구리이온이증가된암세포의경우 phenolic compounds 등이 pro-oxidant 로서세포사멸에기여할수있음을의미한다. 최근 salvianolic acid B의항암효과를보고한논문이발표되었다. Wu 등 [18] 은 salvianolic acid B가 acute lymphoblastic leukemia 세포에대해독성이있고 Guo 등 [19] 은약물저항성을갖는 colorectal 암세포에서 salvianolic acid B가 ROS를증가시켜 apoptosis 를유발한다고보고하였다. 즉 salvianolic acid B의 pro-oxidant 기능이암세포를사멸시키는데기여했다는것이다. 본연구에서는 salvianolic acid의 pro-oxidant 기능을시험관내실험으로확인한것일뿐암세포및동물실험에서확인한것은아니다. 따라서이런 salvianolic acid B의 DNA 절단효과가생체내에서나타날것이라할수없다. 다만이런결과는 salvianolic acid B의항암효과를설명하는하나의기전으로 ROS 생성을고려해볼수있다고생각한다. 앞으로다양한암세포에대한 salvianolic acid B의독성효과와그기전을설명하는데있어전이금속상태와 ROS 생성관계를면밀히살펴볼필요가있다고생각한다. 요약단삼의성분인 salvianolic acid B는다양한생리활성이알려져있다. 특히항산화효과는간세포, 신경세포를포함한다양한세포유형에서보호효과가있다고보고되었다. 하지만 ferulic acid와같이항산화제로여겨지는몇몇페놀성물질은특정전이금속이있으면산화작용을하며이것이항암효능을설명하기도한다. 본실험에서 salvianolic acid B가 Cu 2+ 환경에서산화작용을하는지알아보았다. salvianolic acid B와 Cu 2+ 를동 시처리하면 supercoilded 형태의 DNA가 open circular 혹은 linear 형태로바뀌었으나 salvianolic acid B 혹은 Cu 2+ 를단독처리했을때는그렇지않았다. Cu + 에만특정적인킬레이터 neocuproine 을이용하여 salvianolic acid B가 Cu 2+ 를 Cu + 로환원시킴을알았으며 H 2 O 2 를물과산소로분해하는 catalase 를처리하면 DNA 분해가일어나지않았다. 활성산소종중하나인 H 2O 2 는생체분자특히 DNA를공격하여정상기능을수행하지못하게한다. 정리하면 salvianolic acid B에의한 Cu 2+ 의환원은 H 2 O 2 를생성하며 H 2 O 2 는 DNA 분해를일으킨다. 이런결과는 salvianolic aicd B의항암효과가 salvianolic acid의 H 2O 2 생성때문일수있다는작은단서를줄수있으며이는좀더실험이이뤄져야한다. Acknowledgements: This paper was supported by the Semyung University Research Grant of Conflict of interest: None REFERENCES 1. Halliwell B. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radic Res. 1999;31: Deponte M. Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochim Biophys Acta. 2013;1830: Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, et al. Antioxidants and human diseases. Clin Chim Acta. 2014;436: Murakami K, Haneda M, Qiao S, Naruse M, Yoshino M. Prooxidant action of rosmarinic acid: transition metal-dependent generation of reactive oxygen species. Toxicol In Vitro. 2007;21: Yoshino M, Haneda M, Naruse M, Htay HH, Iwata S, Tsubouchi R, et al. Prooxidant action of gallic acid compoounds: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA. Toxicol In Vitro. 2002;16: Sarwar T, Zafaryab M, Husain MA, Ishqi HM, Rehman SU, Rizvi MM, et al. Redox cycling of endogenous copper by ferulic acid leads to cellular DNA breakage and consequent cell death: A putative cancer chemotherapy mechanism. Toxicol Appl Pharmacol. 2015;289: Quassinti L, Ortenzi F, Marcantoni E, Ricciutelli M, Lupidi G, Ortenzi C, Buonanno F, et al. DNA binding and oxidative DNA damage induced by climacostol-copper(ii) complexes: implications for anticancer properties. Chem Biol Interact. 2013;206: Bhat SH, Azmi AS, Hadi SM. Prooxidant DNA breakage induced by caffeic acid in human peripheral lymphocytes: involvement

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical