ORIGINAL ARTICLE Korean J Clin Lab Sci. 2017;49(3):271-278 https://doi.org/10.15324/kjcls.2017.49.3.271 pissn 1738-3544 eissn 2288-1662 Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 271 The Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum of Dementia Inducer Jai-Yun Jung 1, In-Ju Jung 2, Seung-Joo Jekal 3 1 Department of Anesthesiology and Pain Medicine, Sanbon Hospital, Wonkwang University College of Medicine, Gunpo, Korea 2 Department of Cosmetology, Dongshin University, Naju, Korea 3 Department of Clinical Laboratory Science, Wonkwang Health Science University, Iksan, Korea 치매유발제인알루미늄으로손상된배양 C6 Glioma 세포에대한금은화추출물의보호효과 정재윤 1, 정인주 2, 제갈승주 3 1 원광대학교의과대학산본병원마취통증의학과, 2 동신대학교뷰티미용학과, 3 원광보건대학교임상병리학과 This study was performed to evaluate the cytotoxicity of aluminum chloride (AlCl 3), and the protective effect of Lonicerae flos (LF) extract on AlCl 3-induced cytotoxicity in the cultured C6 glioma cells. Here, the cell viability and antioxidative effects, such as DPPH-radical scavenging activity, superoxide anion scavenging activity, and lipid peroxidation (LP), were assessed. AlCl 3 significantly decreased the cell viability in a dose-dependent manner; the XTT 50 value was measured at 128.8 M of AlCl 3. The cytotoxicity of AlCl 3 was determined as highly toxic the y Borenfreund and Puerner's toxic criteria. The butylated hydroxytoluene (BHT) and antioxidant both significantly increased the cell viability, which was damaged by AlCl 3-induced cytotoxicity in these cultures. In the protective effect of LF extract on AlCl 3-induced cytotoxicity, the LF extract significantly increased the superoxide anion scavenging activity and inhibitory activity of LP, as well as the DPPH-radical scavenging activity. From these results, it is suggested that the oxidative stress may have been involved in the cytotoxicity of AlCl 3, and LP extract effectively protected AlCl 3-induced cytotoxicity through the antioxidative effects. Conclusively, the natural resources, like LP extract, may be a putative therapeutic agent for the treatment of dementia induced by allergen like aluminum correlated with the oxidative stress. Key words: Aluminum, Dementia, Cytotoxicity, Antioxidative effect Corresponding author: Seung-Joo Jekal Department of Clinical Laboratory Science, Wonkwang Health Science University, 514 Iksan-daero, Iksan 54538, Korea Tel: 82-63-840-1215 Fax: 82-63-840-1219 E-mail: sjjei@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 2017 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: July 31, 2017 Revised 1 st : August 13, 2017 Revised 2 nd : August 23, 2017 Revised 3 rd : August 28, 2017 Accepted: August 28, 2017 서론치매 (dementia) 는혈관손상에의한혈관성치매 (vascular dementia, VD) 와연령증가에따른노인성치매인알쯔하이머병 (Alzheimer's disease, AD) 으로구분된다 [1]. 최근대부분선 진국에서는급속한노인인구의증가로인하여고령화시대로접어들면서 AD가사회적문제로관심의대상이되었다. AD의발병에는많은요인들이관여하고있는데예를들면, 염색체에의한유전적요인을비롯하여신경전달물질, 신경영양인자및유해중금속류등다양한인자들이작용하고있다 [2]. 처음으로
272 Jai-Yun Jung, et al. Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum 발견된알츠하이머병에서관련유전자는 21번염색체에존재하는 -amyloid precursor protein ( APP) 으로밝혀졌으며 [3], 이밖에도 apoprotein E (APOE) 나 presenilin (PS) 역시 AD의발병가능성을높여준다고알려져있다 [4]. 특히, PS의돌연변이와 -amyloid protein ( A) 의침적에의한노인반 (senile plaque) 형성과신경원섬유의변화 (neurofibrillary tangle, NFT) 에따른신경세포의파괴가 AD 발병을유발한다고알려져있다 [5]. 또하나의원인으로뇌조직의가교역할을하는미세아교세포 (microglia) 나별아교세포 (astrocyte) 가염증전구물질인 interleukin (IL)-1 나 tumor necrosis factor (TNF)- 와같은사이토카인을과도히생성한결과라고제시된바있다 [6]. 현재 AD의치료에주로아세틸콜린보충요법이많은효과를나타내고있으나그밖에도카테콜아민이나감마아미노부틸산과같은여러신경전달물질이증가할수있는약제개발도우선적으로요구되고있다 [1]. 최근, AD의요인의하나로자유라디칼설 (free radical theory) 이제시된바있다. 인체내의생체반응과정에서세포내사립체 (mitochondria) 의전자전달계에의해서약간의슈퍼옥사이드 (superoxide) 나히드록실라디칼 (hydroxylradical) 과같은활성산소 (reactive oxygen species, ROS) 가생성되지만이들은항산화계에속하는 superoxide dismutase (SOD) 나 catalase와같은항산화효소에의하여분해되어소실되어지는데만약그렇지않고뇌에과량이축적될경우뇌조직에손상을초래하여 AD발병을유발한다고한다 [7]. 따라서, AD를비롯한파킨슨병및근위축성측삭경화증 (ALS) 과같은난치성뇌질환의병인의하나로산화적손상 (oxidative stress) 이제시되고있으며, 이를근거로항산화측면에서뇌질환에대한치료적방법을찾기위한노력이시도되고있다 [8]. AD 유발물질로알려진알루미늄은중금속의일종으로대기나토양오염에의한먼지나음용수를통하여인체내에들어오게되는데, 특히이는혈관-뇌장벽 (blood-brain barrier, BBB) 을쉽게통과함으로서뇌조직에축적되어 AD와같은뇌병변을유발한다고알려져있다 [9]. 최근알루미늄을비롯한카드뮴이나수은과같은몇몇중금속들은이들의붕괴시자유라디칼을생성한다고알려짐으로써이들의독성이산화적손상과관련성이있음이제시되었다. 따라서, 이들의독성에대한방어를항산화측면에서접근하려는연구가이루어지고있다 [10]. 한약재를비롯한많은식물에는항산화를비롯한항염, 항독및항균에유효한각종생리활성물질이다량함유되어있다고밝혀지고있다. 예를들면, 카로티노이드를비롯한페놀화합물및이소플라빈과같은다양한성분들이각종식물의잎이나줄 기, 뿌리및꽃과같은부위에많이들어있다고알려져있다 [11]. 식물중금은화 (Lonicerae flos, LF) 는인동과 (Caprifoliaceae) 에속하는반상록수의활엽수인인동덩굴 (Lonicera japonica) 의건조꽃봉오리로서꽃은 6 7 월경에개화하는데이때꽃봉오리를채취하여그늘이나햇빛에말려건조후약제로사용한다 [4]. 인동덩굴은우리나라전국에분포하고있는데주로산비탈의덤불속에서식하고있으며, 맛은달고성은냉하여오래전부터관절염을비롯한종독, 임파선염및인후염의치료에사용하여왔었다 [12]. 금은화의성분으로는 saponin을비롯하여 luteolin, tannin, triterpenoid glycoside, loniceran과같은다양한물질들이함유되어있으며, 페놀화합물또는플라보노이드계통인 luteolin 이나 tannin 성분이나 saponin 같은성분들은항산화를비롯한항염이나항균작용이강하다고알려져있다 [11]. 금은화의효능은시험관내에서는물론생체에서도동물을대상으로피부염과같은질환에대해일부이루어지고있으나 [4], 치매에대한연구는매우드물다. 그러나시험관내에서의연구와함께동물을대상으로한실험에서도금은화가기억력과학습회복에효과가있다는것이제시되고있다 [13]. 근래에세포배양기법이빠르게발전되면서독성물질의독성정도를비롯하여신약제의효능분석및안전성검정과같은다양한분석도구로활용되고있다 [14]. 본연구는치매유발제인염화알루미늄 (AlCl 3 ) 의세포독성을산화적손상측면에서조사하였으며, 동시에, AlCl 3 에대한금은화추출물의영향을항산화측면에서조사하였다. 재료및방법 1. 세포주본실험에사용한대뇌신경세포인 C6 glioma 세포주는 American Type Culture Collection (ATCC) 에서분양받아사용하였다. 2. 약제제조본실험에사용한시약으로 aluminum chloride (AlCl 3 ) 를비롯한 trypsin, phosphate buffer (ph 7.5), xanthine, butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), dimethylsulfoxide (DMSO), linoleic acid, ammonium thiocyanate, ferrous chloride, methanol, hydrogen peroxide (H 2O 2) 및 XTT는 Sigma사 (St. Louis, MO, USA) 에서, 구입하였다. AlCl 3 의제조는 fetal bovine serum (FBS, Gibco, USA) 이
Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 273 없는 minimum essential medium (MEM, Gibco, USA) 을사용하여각각 10 M, 50 M, 150 M 및 300 M의저장액을만들어사용하였다. XTT (2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-caboxanilide, disodium salt) 는 phosphate buffered saline (PBS) 을이용하여 50 ug/ml의저장액을만든후냉암소에보관한다음필요한양을직접배양액에첨가하여사용하였다. 3. 금은화 (LF) 추출 전북익산시야산에서 5 6 월경인동덩굴로부터금은화를채취한후시에위치하고있는원광대학교부설생명자원과학연구소에서동정확인하였다. 추출은 Han 등 [15] 의방법에따라채취하여보관중인건조꽃봉오리 39.6 g을잘게파쇄한다음시료의 3배정도의물과함께 1,000 ml의환저플라스크에넣고 3시간동안가열하였다. 위의과정을 4회반복추출하여여과한다음 3,000 rpm에서 30분동안원침시켰다. 원침후진공농축기에서농축감압시킨다음 2.1 g의시료를얻었으며, 이때수율은 5.3% 로나타났다. 금은화의화학적성분분석은 Accela high-speed LC system (Thermo Scientific, MA, USA) 에의한표준물질 (macranthoidins A, B, dipsacoside, B, sweroside, luteolin-7-o-d-glucoside) 의 UHPLC-QQQ-MS 분석방법결과는 Table 1과같다 [16]. 4. 세포배양 C6 glioma 세포의배양은 Park 등 [17] 의방법에따라배양용기에부착된세포를 0.025% trypsin을사용하여분리하였다. 분리된세포들은원침후 10% FBS가함유된 MEM 배양액에넣고 1 10 5 cells/well이되도록조절한후 96-well 배양용기에분배하였다. 분배된세포들은 36 C, 5%CO 2 로조절된항온기내에서 72시간동안배양하였다. Table 1. Chemical constituents of Lonicerae flos Components Content (mg) Iridoids Sweroside 0.245 Flavonoids Luteolin-7-O-D-glucoside 0.173 Triterpenoids (Saponin) Macranthoidin B 1.06 Macranthoidin A 3.78 Dipsacoside B 40.96 5. 치매유발제 (AlCl 3) 처리배양중인 C6 glioma 세포에 AlCl 3 가 120 160 M 농도로각각포함된배양액에서세포를 48시간동안배양한후세포생존율을대조군과비교조사하였다. 6. BHT의항산화능측정 BHT의항산화능을조사하기위하여활성산소의일종인 H 2 O 2 15 M를배양세포를처리하기 2시간전에 BHT가 10 50 M 농도로각각포함된배양액에서세포를처리한다음세포생존율을대조군과비교조사하였다. 7. AlCl 3 에대한 BHT의영향 XTT 50 농도의 AlCl 3 를배양세포에처리하기 2시간전에 BHT가 30 M과 50 M로각각포함된배양액에서세포를배양한다음세포생존율에의하여대조군과비교조사하였다. 8. 금은화 (LF) 추출물처리 AlCl 3 에대한 LF 추출물의영향을조사하기위하여배양 C6 glioma 세포에 AlCl 3 XTT 50 농도를처리하기 2시간전에 80 g/ml와 100 g/ml의 LF 추출물이각각포함된배양액에서세포를배양한다음이의영향을세포생존율에의하여대조군과비교조사하였다. 9. 세포생존율분석세포생존율의분석은 Mosmann [18] 의방법에따라행하였다. 즉, 배양세포에약제나시료추출물을농도별로처리한다음실험당일제조한 XTT (50 g/ml) 를 well당 20 L씩넣고 36 C로조절된항온기에서 4시간동안배양하였다. 배양완료후 dimethylsulfoxide (DMSO) 를넣어실온에서정치한다음 ELISA reader (Spectra max 250, Molecular Devices, Sunnyvale, USA) 로 450 nm에서흡광도를측정하였다. XTT 50 값의산출은회귀직선식에의하여산정하였다. 10. DPPH- 라디칼소거능측정 DPPH-라디칼소거능 (DPPH-radical scavenging activity) 의측정은 Blois [19] 의방법에따라, 메탄올시료에 0.3 mm DPPH 메탄올용액 100 L를첨가하여실온에서 30분간처리하였다. 처리완료후 ELISA reader (Spectra max 250, Molecular Devices, Sunnyvale, USA) 로 517 nm에서흡광도를측정하였다. 라디칼소거능은시료첨가군과시료무첨가군간의차이를
274 Jai-Yun Jung, et al. Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum 시료무첨가군에의한백분율로나타냈다. 또한 BHT의활성을양성대조군으로사용하였다. 11. Superoxide anion-radical (SAR) 제거능측정 SAR 제거능측정은 nitroblue tetrazolium (NBT) 의환원방법에따라, 시료용액 0.1 ml와 0.4 ml potassium phosphate buffer (ph 7.5) 에 0.4 mm xanthine 과 NBT를가하여 37 C에서 20분동안반응시켰다. 반응후 1 N HCl I ml를가하여반응을정지시켜생성된 SAR 양을 ELISA reader (Spectra max 250, Molecular Devices, Sunnyvale, USA) 로 560 nm에서흡광도를측정하였다. 제거능의측정은시료첨가군과무첨가군의차이에의한백분율로표시하였다. 또한 BHT의활성을양성대조군으로사용하였다. 12. 지질과산화 (lipid peroxidation, LP) 측정 LP 측정은 Kikuzaki 와 Nakatani [20] 의방법에따라, 2.52% linoleic acid와 0.05 M PBS (ph7.0) 용액 12.1 ml에에탄올과시료 3.9 ml 혼합액을첨가한후 24시간동안 40 C에서배양하였다. 배양완료후에탄올과 30% ammonium thiocyanate 와 0.02 M ferrous chloride를 0.1 ml를넣은후실온에서 3분동안반응시켰다. 반응후 ELISA reader (Spectra max 250, Molecular Devices, Sunnyvale, USA) 로 500 nm에서흡광도를측정하였다. 또한 BHT의활성을양성대조군으로사용하였다. 13. 통계처리실험결과는 SPSS/WIN 18.0을이용하여군간의차이를비교하기위하여 ANOVA 를시행하였고사후분석은 Tukey's post-hoc test에의하였으며, 모든통계의유의수준은 p<.05 에서채택하였다. 결과 1. 염화알루미늄 (AlCl 3 ) 의독성측정 AlCl 3 의독성을조사하기위하여배양 C6 glioma 세포에 AlCl 3 120 160 M 농도각각을 48시간동안처리한결과, 처리농도에비례하여 AlCl 3 는세포생존율을대조군에비하여유의하게감소시킴으로서독성을나타냈다 (p<0.001). 이과정에서 XTT 50 값은 128.8 M의처리에서나타났다 (Table 2). 2. BHT의항산화능측정 BHT의항산화능을조사하기위하여자유라디칼인 15 M의 H 2 O 2 를배양세포에처리하기전에 BHT가각각 10 50 M로포함된배양액에서 2시간동안전처리하였다. 그결과, 10 M 의 H 2 O 2 만을처리한경우대조군에비하여세포생존율이 41.1% (0.097±0.021) 로나타난반면, 10 M과 30 M의 BHT의처리에서는각각 53.4% (0.126±0.016) 와 75.4% (0.178±0.013) 로나타났다. 또한, 50 M 농도의 BHT의처리에서는 83.0% (0.198±0.030) 나이는모두 H 2 O 2 의처리에비하여유의한세포생존율의증가를나타냈다 (p<0.001) (Table 3). 3. AlCl 3 에대한 BHT의영향 AlCl 3 의세포독성에대한항산화제인 BHT의영향을알아보기위하여 AlCl 3, XTT 50 농도를배양세포에처리하기전에 BHT 가각각 30 M과 50 M로포함된배양액에서 2시간동안전처리하였다. 그결과, XTT 50 농도의 AlCl 3 만의처리에서는세포생존율이대조군에비하여 42.9% (0.079±0.015) 로나타난것에비하여 30 M과 50 M의 BHT의처리에서는각각 63.6% (0.117±0.022) 와 79.3% (0.146±0.012) 로나타나이는 AlCl 3 의처리에비하여모두유의한세포생존율을나타냈다 (p<0.001) (Table 4). Table 2. The cytotoxicity of aluminum chloride (AlCl 3) on cultured C6 glioma cells by XTT assay Incubation XTT assay (450 nm) Concentration of AlCl 3 ( M) Mean±S.D. (% of control) Control 0.412±0.028 100 120 0.232±0.021 56.3*** 140 0.173±0.022 42.0*** 160 0.135±0.021 32.8*** Cultured C6 glioma cells were treated with 120, 140 and 160 um of AlCl 3 for 48 hours. The data indicate the mean±sd for triplicate experiments. Significantly different from the control. Table 3. The antioxidative effect of butylated hydroxytoluene (BHT) on the hydrogen peroxide (H 2O 2) in cultured C6 glioma cells Incubation XTT assay (450 nm) Concentration of BHT ( M) Mean±S.D. (% of control) Control 0.236±0.018 100 15 H 2O 2 0.097±0.021 41.1 10 0.126±0.016 53.4*** 30 0.178±0.013 75.4*** 50 0.198±0.030 83.0*** Cultured C6 glioma cells were pretreated with 10, 30 and 50 M of BHT for 2 hours. The data indicate the mean±sd for triplicate experiments. Significantly different from H 2O 2-treated group.
Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 275 4. AlCl 3 의세포독성에대한금은화 (LF) 추출물의영향 LF 추출물이 AlCl 3 의세포독성에미치는영향을조사하기위하여배양세포에 XTT 50 농도의 AlCl 3 를처리하기전에 80 g/ml 와 100 g/ml의 LF 추출물을각각처리한결과, AlCl 3 만의처리에서는세포생존율이대조군에비하여 36.2% (0.059±0.014) 로나타난데비하여 80 g/ml 추출물처리에서는 78.5% (0.128±0.018) 로나타났으며, 또한 100 g/ml 추출물처리에서는 88.3% (0.144±0.018) 로나타나이는모두 AlCl 3 만의처리에비하여유의한증가를보였다 (p<0.001) (Table 5). 5. DPPH- 라디칼소거능측정 DPPH-라디칼소거능을측정하기위하여 80 g/ml와 100 g/ml 농도의 LF 추출물시료를분석한결과 80 g/ml 농도처리에서는소거능이 32.6% 로나타났으며, 100 g/ml의처리에서는 47.5% 로나타나이는모두대조군에비하여유의한소거능의증가를나타냈다 (p<0.001). 특히, 100 g/ml LF 추출물의소거능은양성대조군으로사용한 50 M BHT 소거능인 79.8% 값의 60% 에근접한것으로나타났다 (Table 6). 6. Superoxide anion-radica (SAR) 제거능측정 LF 추출물에대한 SAR-소거능측정을위하여 80 g/ml와 100 g/ml의농도의추출물시료를각각분석한결과 80 g/ml 추출물의처리에서는제거능이 21.2% 로나타났으며, 100 g/ml의처리에서는 35.8% 로나타나이는모두대조군에비하여유의한제거능의증가를나타냈다 (p<0.001). 특히, 100 g/ml의 LF 추출물의제거능은양성대조군으로사용한 50 M BHT 활성인 75.2% 값의 40% 이상인것으로나타났다 (Table 7). 7. 지질과산화 (LP) 측정 AlCl 3 에대한 LF 추출물이 LP에미치는영향을조사하기위하여 AlCl 3 XTT 50 농도를배양세포에처리하기전에 80 g/ml 와 100 g/ml의 LF 추출물을처리한결과, LF 추출물 80 g/ml의처리에서는 LP가 81.4% 로나타났다. 또한, 100 g/ml의처리에서는 62.7% 로나타났다. 따라서 LF 추출물의 LP 억제능은추출물 80 g/ml와 100 g/ml의처리에서각각 Table 4. The effect of butylated hydroxytoluene (BHT) on the cytotoxicity induced by aluminum chloride (AlCl 3) in cultured C6 glioma cells Incubation XTT assay (450 nm) Concentration of BHT ( M) Mean±S.D. (% of control) Control 0.184±0.024 100 AlCl 3 (XTT 50) 0.079±0.015 42.9 30 0.117±0.022 63.6*** 50 0.146±0.012 79.3*** Cultured C6 glioma cells were pretreated with 30 and 50 M of BHT for 2 hours. The data indicate the mean±sd for triplicate experiments. Significantly different from AlCl 3-treated group. Table 6. The DPPH-radical scavenging activity of Lonicerae flos (LF) extract determined at a wavelength of 517 nm Concentration of LF extract ( g/ml) DPPH-radical scavenging activity (517 nm) % of control 50 M BHT 79.8**** 80 32.6*** 100 47.5*** The data indicate the mean±sd for triplicate experiments. Significantly different from the control. BHT was used as positive control. Table 5. The protective effect of Lonicerae flos (LF) extract on aluminum chloride (AlCl 3)-induced cytotoxicity in cultured C6 glioma cells Incubation XTT assay (450 nm) Concentration of LF ( g/ml) Mean±S.D. (% of control) Control 0.163±0.013 100 AlCl 3 (XTT 50) 0.059±0.014 36.2 80 0.128±0.018 78.5*** 100 0.144±0.018 88.3*** Cultured C6 glioma cells were pretreated with 80 and 100 g/ml of LF extract for 2 hours. The data indicate the mean±sd for triplicate experiments. Significantly different from the AlCl3-treated group. Table 7. The superoxide anion-radical (SAR) scavenging activity of Lonicerae flos (LF) extract determined at a wavelength of 560 nm Concentration of LF extract ( g/ml) SAR scavenging activity (560 nm) % of control 50 M BHT 75.2**** 80 21.2*** 100 35.8*** The data indicate the mean±sd for triplicate experiments. Significantly different from the control. BHT was used as positive control.
276 Jai-Yun Jung, et al. Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum Table 8. The lipid peroxidation (LP) of Lonicerae flos (LF) extract determined at wavelength 500 nm Concentration of LF extract ( g/ml) Lipid peroxidation (500 nm) % of control Control 100 50M BHT 73.3*** 80 18.6*** 100 37.3*** The data indicate the mean±sd for triplicate experiments. Significantly different from the control. BHT was used as positive control. 18.6% 와 37.3% 로나타남으로서이는모두대조군에비하여유의한 LP 억제능을보였다 (p<0.001). 특히 100 g/ml의 LF 추출물처리에서는양성대조군인 BHT의 LP 억제능값인 73.3% 의 50% 이상의억제능을보였다 (Table 8). 고찰 치매는기억력감퇴와인지기능의상실로인한학습및정상적인생활적영위가어려운뇌질환으로서이의원인물질중의하나로알루미늄이알려져있으며이는또한산화적손상과도밀접한관련이있다고제시된바있다 [2]. 본연구에서는치매유발제인염화알루미늄 (AlCl 3 ) 의세포독성을조사하기위하여대뇌신경교세포의일종인 C6 glioma 세포에 120 160 M의 AlCl 3 농도를각각처리한결과농도의존적으로세포생존율이유의하게감소되었으며이때 XTT 50 값이 128.8 M에서나타났다. 따라서, AlCl 3 는배양 C6 glioma 세포에세포독성을나타냈으며이는 Borenfreund 와 Puerner [21] 의독성판정기준에따라중간독성 (mid-toxic) 인것으로나타났다. 이들은약제의독성을 XTT 50 값이 100 M 이하인경우는고독성으로, 100 1,000 M 이면중간독성 (mid-toxic) 으로각각판정하였다. 본연구결과는 Verstraeten 등 [22] 이알루미늄의뇌독성을보고한연구결과나, 또한 Kim 등 [2] 이배양신경교종세포에대한 AlCl 3 의신경독성에대한연구보고와일치하였다. 이같이 AlCl 3 가배양 C6 glioma 세포에독성을보인것은 AlCl 3 가신경전달물질에손상을주어신경연접간원활한자극전달을방해함으로서세포손상을초래하였거나 [23], 또는세포내신경원섬유의타우단백질의인산화를초래하여세포손상에영향을주었을가능성도배제할수는없지만 [24], 그보다는 AlCl 3 가자유라디칼의생성을유발함으로서산화적손상으로인한세포독성을나타냈을가능성이클것으로생각된다 [2]. 따라서본연구에서는 AlCl 3 의세포독성과산화적손상간의상호관련성을조사하였다. 이를위하여먼저항산화제의일종인 BHT의라디칼제거능을조사하기위하여자유라디칼의일종인 20 M H 2 O 2 를배양세포에처리하기전에 10 50 M BHT 각각의농도를배양세포에전처리한결과 H 2 O 2 의처리에비하여모두유의한세포생존율의증가를나타냈다. 본실험의결과에서 BHT의처리가 H 2O 2 의처리에비하여세포생존율이유의하게증가된것은 BHT가자유라디칼을제거할수있는항산화능을가지고있음을증명하고있다. 본연구결과는 Kim과 Jekal [14] 이자유라디칼에대한 BHT의소거능을보고한연구결과와도일치함을알수있었다. 한편, AlCl 3 와산화적손상간의관련성을알아보기위하여 XTT 50 농도의 AlCl 3 를배양세포에처리하기전에 30과 50 M의 BHT를각각전처리한결과모두 AlCl 3 의처리에비하여유의한세포생존율의증가를보였다. 본실험결과는 BHT가 AlCl 3 의세포독성을방어한것을말해주고있으며이는다시말해 AlCl 3 의세포독성에산화적손상이관여하고있음을제시하고있다. 또한, 본실험결과는 Kim 등 [2] 이 AlCl 3 에대한항산화제인 vitamin E의독성방어효과에대한연구보고와서로상통함을알수있었다. AlCl 3 에대한금은화 (LF) 추출물의영향조사에있어서, XTT 50 농도의 AlCl 3 를배양세포에처리하기전에 80과 100 g/ml의추출물을각각전처리한결과 AlCl 3 의처리에비하여모두유의한세포생존율의증가를보였다. 본연구결과는 LF 추출물이 AlCl 3 의독성을방어한것으로서 Kim 등 [25] 이 LF 추출물이 AlCl 3 와같은중금속류인크롬의독성을방어하였다는연구보고와일치하였다. 이같은결과는 LF 추출물속에함유된 tannin이나 luteolin, saponin과같은성분들이 AlCl 3 의산화적손상을방어한결과라고생각되며, 그이유의하나로플라보노이드계통인 luteolin이나페놀화합물의일종인 tannin 및 saponin 등의성분들은강력한항산화성분들로알려져있기때문이다 [26]. 따라서본연구에서는 LF 추출물시료에대한항산화효과를알아보기위하여 DPPH-라디칼소거능을비롯하여 superoxide anion-radical (SAR) 제거능및지질과산화 (LP) 억제능을조사하였다. DPPH-라디칼소거능조사에서, 80과 100 g/ml 추출물에서대조군에비하여각각 32.6% 와 47.5% 로나타나모두유의한라디칼소거능을보였다. 본실험결과는 LF 추출물이자유라디칼소거능이있음을말해주고있으며, 이같은항산화효과는 LF 추출물성분중항산화능이강한플라보노이드또는페놀화합물의성분들에의한것으로생각된다 [4]. 페놀화합물은수산기 (-OH) 를분자구조에하나또는그이상을가지고있어타물
Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 277 질과의결합력이매우뛰어나기때문에항산화를비롯한항염, 항독과같은유효한생리활성을나타낸다고알려져있다 [27]. 한편, SAR 제거능조사에있어서는 80과 100 g/ml 추출물처리에있어서각각 21.2% 와 35.8% 로나타나이는모두대조군에비하여유의한 SAR 제거능을보였다. 본실험결과는 LF 추출물이자유라디칼의일종인 SAR을제거할수있는항산화능이있음을말해주고있다. 이같은항산화능은 LF 추출물성분인 saponin이나 secoiridoid glycoside 및페놀화합물성분들때문인것으로생각된다 [28]. 한편, 본연구의 LP 억제능에있어서 80과 100 g/ml 추출물처리에서대조군에비하여각각 18.6% 와 37.3% 로나타나이는모두대조군에비하여유의한 LP 억제능을보였다. 본연구결과는 Kim 등 [25] 이 LF 추출물에대한 LP 억제능과 lactate dehydrogenase (LDH) 활성저해를보고한연구결과와일치하였다. 지질과산화는 LDH 활성과함께산화적손상에의한지질과산화의막손상정도를측정하는정량적분석방법의하나로알려져있다 [29]. 이상의결과로부터 LF 추출물과같은천연성분은치매와같이산화적손상과관련된병변에대한치료적약물개발을위해활용가치가클것으로생각된다. 그러나천연성분에대한산화적손상에대한생리활성분석은생화학이나약리및분자적측면에서보다자세한연구가필요할것으로생각된다. 요약치매유발제인염화알루미늄 (aluminum chloride, AlCl 3) 의세포독성을대뇌교종세포인 C6 glioma 세포를대상으로산화적손상측면에서조사하였으며, 또한 AlCl 3 의세포독성에대한금은화 (Lonicerae flos, LF) 추출물의영향을세포생존율을비롯한 DPPH-라디칼소거능, superoxide anion-radical (SAR) 제거능및지질과산화 (lipid peroxidation, LP) 억제능과같은항산화측면에서분석하였다. 본실험에서배양 C6 glioma 세포에 120 160 M의 AlCl 3 가각각포함된배양액에서처리한결과처리농도에비례하여세포생존율이대조군에비하여유의한감소를보였으며, 이때 XTT 50 값은 128.8 M에서나타나중간독성 (mid-toxic) 인것으로나타났다. 또한, 항산화제인 BHT는 AlCl 3 의세포독성을유효하게방어하였다. 한편, AlCl 3 의세포독성에대한금은화 (LF) 추출물의영향에대한조사에있어서, LF 추출물은 AlCl 3 에의하여감소된세포생존율을유의하게증가시킴으로써세포독성을방어하였다. 이와동시에, LF 추출물은 DPPH-라디칼소거능을비롯하여 SAR 제거능및 LP 억제능을보임으로서항산화효과를나타냈다. 위의결과로부 터 AlCl 3 의세포독성에산화적손상이관여하고있으며, 또한 LF 추출물은 AlCl 3 의독성을항산화능에의하여효과적으로방어하였다. 따라서, LF 추출물과같은천연성분은알루미늄과같이산화적손상과관련된치매유발제의독성을방어함으로써차후치매와같은산화적손상관련의질환치료를위한항산화물질로서의개발적가치가있다고사료된다. Acknowledgements: None Funding: None Conflict of interest: None REFERENCES 1. Ellis JM. Cholinesterase inhibitors in the treatment of dementia. J Am Osteopath Assoc. 2005;105(13):145-158. 2. Kim SJ, Yu YW, Lee JK. Cytotoxicity and protective effect of Portulaca oleracea L. extract on cultured neuroglioma cells damaged by aluminum of dementia-induced agent. J Kor Soc Plants Environ. 2013;16(5):251-256. 3. Kourie JI. Mechanisms of amyloid beta protein-induced modification in ion transfort systems; implications for neurodegenerative disease. Cell Mol Neurobiol. 2001;21(3):173-213. 4. Kim SC, Lee JR, Choi KI, Park ST, Kwon YK, Byun SH. Effect of Lonicerae flos on contact hypersensitivity induced by repeat elicitation of DNCB. Kor J Herbol. 2006;21(1):9-15. 5. Bryan-Sisneros AA, Fraser SP, Suh YH, Djamgoz MB. Toxic effect of the beta-amyloid precursor protein C-terminus fragment and Na+/Ca 2+ gradients. Neuroreport. 2000;11(15):3357-3360. 6. Stephen LY, Loyd HB, June KA, Joyce MA, Michael DD, Paula BE, et al. Amyloid and amylin fibrils induce increase in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia. J Neurochem. 2000;74(3):1017-1025. 7. Kontush A. Amyloid-beta ; an antioxidant that becomes a pro-oxidant and critically contributes to Alzheimer s disease. Free Radic Bio Med. 2001;31(19):1120-1131. 8. Kim YJ. Etiology and pathogenesis of Parkinson s disease. J Kor Neurol Assoc. 2004;22(5):421-432. 9. Roger DG, Olivier F, Clank P, Courtosis M, Piriou A. Aluninum transfer as complex through blood-brain barrier. Biological Rrace Element Res. 1990;25(5):39-45. 10. Son YW, Choi YS. Protective of Chenopodium album var. exract by oxidative effect in cultured human skin fibroblasts damaged by cadmium. J Kor Soc People plants Environ. 2012;15(3): 155-161. 11. Li YL, Gan GP, Zhang HZ, Wu HZ, Li CL, Hung YP, et al. A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines. J Ethnopharmacol. 2007;113(1):115-124. 12. Choi CW, Jung HA, Kang SS, Choi JS. Antioxidant constituents and a new triterpenoid glycoside from Lonicerae flos. Arch
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