J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(7), 972~979(2014) http://dx.doi.org/10.3746/jkfn.2014.43.7.972 쇠무릎잎과줄기추출물의항산화활성연구 서수정 김남우 대구한의대학교한약자원학과 Antioxidant Activities of Extracts from Leaves and Stems of Achyranthes japonica Soo Jung Seo and Nam Woo Kim Dept. of Herbal Biotechnology, Daegu Haany University ABSTRACT This study was conducted to investigate the physiological characteristics of water and ethanol extracts from leaves and stems of Achyranthes japonica. The highest contents of total polyphenol and flavonoid compounds were 58.27 and 42.22 mg/g in water extract from leaves, respectively. The protein content was the highest at 16.42 mg/g in water extract from leaves. Ethanol extract from stems showed the highest content of reducing sugars at 11.35 mg/g. In the measurement of electron donating ability (EDA), ethanol extract from stems showed the highest EDA at 93.41% at a concentration of 1.0 mg/ml. Superoxide dismutase-like activity of ethanol extract from leaves was the highest at 8.13% at a concentration of 1.0 mg/ml. In the analysis of nitrate scavenging activity, water extract from leaves showed the highest activity at 94.90% at ph 1.2, and the activity increased as concentration increased and ph decreased. In the measurement of xanthine oxidase inhibition, ethanol extract from stems showed the highest inhibitory activity at 66.67% at a concentration of 1.0 mg/ml. Especially, nitrate scavenging activities of water extract from leaves were the highest under all ph conditions. These results verify that extracts from leaves of A. japonica have strong antioxidant activity and can be used as an effective antioxidant source for nutraceutical foods, medicines, and cosmetic stuffs. Key words: Achyranthes japonica, antioxidant, electron donating ability, SOD-like activity, xanthine oxidase inhibition 서 현대의학및생활수준의향상으로인간의평균수명이늘어나고고령화에따른당뇨, 고혈압, 고지혈증과같은만성성인질환의증가로치료보다는질병의발생원인및예방에대한관심이높아지고있다 (1). 활성산소종 (reactive oxidative species, ROS) 은 peroxisome, xanthine oxidase (XO), NADPH oxidase 및 cyclooxygenase(cox) 등세포내에존재하는효소들에의해생성되는 O - 2, H 2O 2 등의 free radical을말하며세포내산화손상을일으켜만성성인질환의주요요인으로알려져있다 (2,3). 이에생물체는체내에서 superoxide dismutase, catalase, glutathione reductase 등의항산화효소와 vitamin C, vitamin E 등과같은천연항산화제가 free radicals을제거하지만과도하게생성된 ROS를제거하기에는부족하다 (4,5). 따라서약용및식용식물로부터인체에무해하고항산화력이우수한천연항산화제를개발하고자하는연구가활발히진행되고있 Received 3 March 2014; Accepted 26 May 2014 Corresponding author: Nam Woo Kim, Dept. of Herbal Biotechnology, Daegu Haany University, Gyeongsan, Gyeongbuk 712-882, Korea E-mail: tree@dhu.ac.kr, Phone: +82-53-819-1438 론 다 (6). 쇠무릎 (Achyranthes japonica NAKAI) 은비름과 (Amaranthaceae) 에속하는다년생초본으로우리나라각지의들판이나길가에서자생하고줄기의마디가두드러져소의무릎처럼보인다고하여쇠무릎이라한다. 쇠무릎의어린순은나물로먹기도하며, 한방에서는쇠무릎의건조된뿌리를우슬 ( 牛膝 ) 이라는한약재로사용하는데진통, 혈압강하, 류머티즘및관절통, 이뇨및간장해등의질병치료에사용한다 (7,8). 쇠무릎에관한연구로 Lee 등 (9) 은울릉도산쇠무릎의잎과씨에서 polyphenol과 flavonoids 함량및생리활성을측정하였으며뿌리에서 oleanolic계및 glycoside계 saponin(10,11) 을동정하였다. 그리고 Ogawa 등 (12) 은 caffeic acid를포함한약 7종의유효물질을분리하였고, Son 등 (13) 은해충방제에이용될수있는 inokosterone, ecdysterone, ponasterone A 등을분리동정한바있다. 그리고한방생약재로사용되는쇠무릎뿌리는 cytochrome p450 약물대사효소에대한억제작용 (14), 면역조절 (15), 항돌연변이 (16), 급성및아급성염증에대한항염증효과 (17) 를나타낸다고하였으며, 치아및치주질환완화 (18), 직장암저해 (19), cathepsin B 저해 (9), 간세포보호효과
쇠무릎잎과줄기추출물의항산화활성연구 973 (20), 류머티즘관절염완화 (21) 등다양한약리학적연구가이루어져있다. 지금까지한방생약재로사용되고있는쇠무릎뿌리인우슬에대한국내연구보고는다수있었으나지상부의부위별항산화연구는보고되지않았다. 이에본연구에서는약용뿐만아니라건강기능식품의첨가제로서응용가능한쇠무릎잎과줄기를대상으로항산화효과를탐색하고자하였다. 이에쇠무릎잎과줄기를물과에탄올을용매로추출하여각추출물에함유된단백질함량, 환원당및폴리페놀과플라보노이드화합물의함량을측정하고전자공여능, SOD 유사활성능, 아질산염소거능및 xanthine oxidase 저해활성을측정하여쇠무릎잎과줄기추출물에대한생리활성효과에대해알아보고자한다. 재료및방법실험재료본실험의재료인쇠무릎 (Achyranthes japonica) 은 2010년 10월에경북경산시일대에서동정후채집하였으며대구한의대학교한약자원학과실험실로운반하여잎과마디줄기로분리하여흐르는물에수회세척후열풍건조기 (DR-0160, Hankwang, Siheung, Korea) 를이용하여 30 o C의조건으로수분함량 12±1% 내외로건조하여본실험을위한시료로사용하였다. 추출물제조건조하여잘게세절된쇠무릎의잎과줄기는플라스크에무게당약 10배에해당하는증류수와 70% 에탄올을넣고수직으로환류냉각관을부착시킨환류추출기 (Changshin, Seoul, Korea) 를이용하여 80 o C와 60 o C의수욕상에서 3시간환류추출하였고이과정을 3회반복하였다. 각추출물은 filter paper(whatman No 2, Advantec, Tokyo, Japan) 로여과한다음 rotatory vacuum evaporator(400 series, Eyela, Tokyo, Japan) 로감압농축한후에동결건조 (Ilsin, Busan, Korea) 하여분말로제조한다음잎물추출물 (; A. japonica leaves water extract), 잎에탄올추출물 (; A. japonica leaves ethanol extract) 그리고줄기물추출물 (; A. japonica stems water extract), 줄기에탄올추출물 (; A. japonica stems ethanol extract) 로명명하였다. 분말화된시료를일정농도로 3차증류수에희석하거나 80% 에탄올에희석하여쇠무릎잎과줄기추출물의항산화활성을측정하기위한시료액으로사용하였다. 단백질함량쇠무릎잎과줄기의단백질함량은 Lowry 등 (22) 의방법에따라시료 0.2 ml를시험관에취하고혼합시약 (A : B= 50:1, A: 2% Na 2CO 3 in 0.1 N NaOH, B: 1% C 4H 4KNaO 6 in 0.5% CuSO 4 5H 2O) 을 1 ml 첨가하여상온에서 10분간 반응시켰다. 여기에 0.1 ml Folin-Ciocalteu's phenol reagent를첨가한후다시실온에서 30분간반응시켰다. 반응시료는 UV/VIS spectrophotometer(mecasys Optizen POP, Daejeon, Korea) 를사용하여 750 nm에서흡광도를측정하였으며, bovine serum albumin(sigma-aldrich Co., St. Louis, MO, USA) 으로표준곡선을작성하여각추출물의단백질함량을나타내었다. 환원당 (reducing sugar) 함량환원당함량은 Somogyi-Nelson(23) 방법에따라시료액 1 ml에혼합시약 (A : B=25:1, A: d 3H 2O 1 L in anhydrous Na 2HPO 4 25 g, C 4H 4O 6KNa 4H 2O 25 g, Na 2HCO 3 20 g, anhydrous Na 2SO 4 200 g, B: d 3H 2O 200 ml in CuSO 4 5H 2O 30 g, concentrate H 2SO 4 4 drop) 을 0.5 ml 첨가하여 20분간가열하였다. 가열한시료를냉각시킨후 C액 (total 500 ml store at 37 o C/day-(NH 4) 6Mo 7O 24 4H 2O 25 g in d 3H 2O 450 ml including, concentrate H 2SO 4 21 ml+na 2HSO 4 7H 2O 3 g in d 3H 2O 25 ml) 을 1 ml 첨가하여실온에반응시킨다음 spectrophotometer 를사용하여 520 nm에서흡광도를측정하고 glucose (Sigma-Aldrich Co.) 의표준곡선을기준으로쇠무릎잎과줄기추출물의환원당함량을산출하였다. 총폴리페놀화합물 (total polyphenol compound) 함량총폴리페놀화합물함량은쇠무릎잎과줄기추출물을 1 mg/ml의농도로증류수에희석하여 Folin-Denis법 (24) 으로측정하였다. 추출물 0.2 ml에증류수 1.8 ml를가한후 Folin-Ciocalteu's phenol reagent 0.2 ml를첨가, 혼합하여 3분간실온에서반응시켰다. 여기에 Na 2CO 3 포화용액 0.4 ml를가하여혼합하고증류수를 1.4 ml 넣고실온에서 1시간반응시킨후 UV/VIS spectrophotometer를이용하여 725 nm에서흡광도를측정하였다. 총폴리페놀화합물은 tannic acid를이용하여최종농도가 0, 31.25, 62.5, 125, 250 μg/ml가되도록취하고위와같은방법으로 725 nm에서흡광도를측정한후표준곡선으로부터쇠무릎잎과줄기각추출물의폴리페놀화합물함량을구하였다. 총플라보노이드화합물 (total flavonoid compound) 함량총플라보노이드함량은 Nieva Moreno 등 (25) 의방법을변형하여각농도별추출액 0.1 ml에 10% aluminum nitrate 0.1 ml와 1 M potassium acetate 0.1 ml 그리고 80% ethanol 4.7 ml를가하여 25 o C에서 40분간반응시킨후 415 nm에서흡광도를측정하였다. 총플라보노이드정량은 quercetin(sigma-aldrich Co.) 을이용하여최종농도가 0, 31.25, 62.5, 125, 250 μg/ml 가되도록취하고위와동일한방법으로측정한표준곡선으로부터플라보노이드화합물의함량을구하였다.
974 서수정 김남우 전자공여능 (EDA; electron donating ability) 측정전자공여능측정은 Blois(26) 의방법에따라각추출물의 DPPH(2,2-diphenyl-1-picrylhydrazyl) 에대한전자공여효과로써각시료의환원력을측정하였다. 일정농도의시료 2 ml에 0.2 mm DPPH 용액 (dissolved in 99% ethanol) 을 1 ml 넣고 vortex mixing 후 37 o C에서 30분간반응시켰다. 이반응액을 UV/VIS spectrophotometer를사용하여 517 nm에서흡광도를측정한다음시료첨가구와무첨가구사이의흡광도의차이를백분율 (%) 로나타내었다. Superoxide dismutase(sod) 유사활성능측정쇠무릎잎과줄기의 SOD 유사활성능측정은 Marklund 와 Marklund(27) 의방법에따라유해환원산소종을과산화수소 (H 2O 2) 로전환시키는반응을촉매하는 pyrogallol의산화된양을측정하여 SOD 유사활성을평가하였다. 일정농도의시료 0.2 ml에 ph 8.5로보정한 tris-hcl buffer [50 mm tris(hydroxymethyl)aminomethane+10 mm EDTA, ph 8.5] 2.6 ml와 7.2 mm pyrogallol 0.2 ml를첨가하여 25 o C에서 10분간반응후 1 N HCl 0.1 ml를가하여반응을정지시켰다. 반응액중산화된 pyrogallol의양은 UV/VIS spectrophotometer를사용하여 420 nm에서흡광도를측정하였다. SOD 유사활성은시료용액의첨가구와무첨가구사이의흡광도의차이를백분율 (%) 로나타내었다. 아질산염소거능 (nitrite-scavenging ability) 측정아질산염 (NaNO 2) 소거작용은 Kato 등 (28) 의방법에따라측정하였다. 1 mm의아질산염용액 1 ml에추출시료 1 ml를첨가하고 0.1 N HCl과 0.2 M citrate buffer 를완충용액으로하여반응용액의 ph를각각 1.2, 3.0, 6.0으로보정하여반응용액의부피를 10 ml로하였다. 이용액을 37 o C에서 1시간동안반응시킨후 1 ml씩취하여 2% acetic acid 5 ml와 Griess 시약 (A : B=1:1, A: 1% sulfanilic acid in 30% acetic acid, B: 1% naphthylamine in 30% acetic acid) 을 0.4 ml 첨가하여혼합후실온에서 15분간반응시켰다. 반응시킨시료를 UV/VIS spectrophotometer 를이용하여 520 nm에서흡광도를측정하여잔존하는아질산량을백분율 (%) 로나타내었다. 대조구는 Griess 시약대신증류수 0.4 ml를가하여상기와동일한방법으로실시하였다. 쇠무릎잎과줄기추출시료의첨가구와무첨가구사이의흡광도의차이를백분율 (%) 로나타내었다. Xanthine oxidase 저해활성 Xanthine oxidase 저해활성은 Stirpe와 Corte(29) 의방법에따라일정농도로희석한쇠무릎추출물 1 ml에 0.1 M potassium phosphate buffer(ph 7.5) 0.6 ml와 xanthine(1 mm) 을녹인기질액 0.2 ml를첨가하였다. 여기에 xanthine oxidase(0.2 U/mL) 0.1 ml를가하여 37 o C에서 15분간반응시킨후 1 N HCl 1 ml를가하여반응을정지시 킨다음반응액에생성된 uric acid를 292 nm에서흡광도를측정하였다. 쇠무릎잎과줄기추출물에대한 xanthine oxidase 저해활성은시료의첨가구와무첨가구의흡광도감소율을백분율 (%) 로나타내었다. 통계처리모든실험은독립적으로 3회이상반복실시하였고추출물의생리활성에대한결과는평균 (mean)± 표준편차 (standard deviation; SD) 로표시하였다. 실험군간의유의성을검정하기위하여 SPSS 19.0 for windows program(package for Social Science, SPSS Inc., Chicago, IL, USA) 을이용하여 ANOVA test를실시하고유의성이있는경우 P< 0.05 수준에서 Duncan's multiple range test를실시하였다. Table 1. Contents of the protein and reducing sugar from leaves and stems of A. japonica extracts Extracts 1) Extraction yield (%) 2.95 10.33 1.63 1.47 결과및고찰 추출수율쇠무릎잎과줄기를물과 70% 에탄올을용매로추출하여동결건조한각추출물의수율은 Table 1에나타내었다. 잎은에탄올을용매로추출한 에서 10.33% 로가장높은수율을나타내었으며, 줄기는물을용매로추출한 에서 1.63% 의추출수율을나타내어쇠무릎잎이줄기보다추출수율이더높았다. 잎은물보다에탄올을용매로추출시약 4.5배더많은고형분이추출되었으며, 줄기는물 (1.63%) 을용매로추출시에탄올 (1.47%) 보다약간높은수율을나타내었다. 단백질함량쇠무릎잎과줄기의단백질함량은 16.42 mg/g> 12.95 mg/g> 4.18 mg/g> 3.93 mg/g의순으로줄기보다잎의단백질함량이약 4배더많았으며, Reducing sugar Protein (mg/g) (mg/g) 16.42±0.14 A2) 1.70±0.03 C 12.95±0.85 B 3.68±0.03 B 3.93±0.03 C 3.12±0.04 B 4.18±0.01 C 11.35±0.22 A 1) : A. japonica leaves water extract using reflex extractor for 3 hours at 80 o C condition, : A. japonica leaves 80% ethanol extract using reflex extractor for 3 hours at 60 o C condition, : A. japonica stems water extract using reflex extractor for 3 hours at 80 o C condition, : A. japonica stems 80% ethanol extract using reflex extractor for 3 hours at 60 o C condition. 2) All values are mean±sd of triplicate determinations. Values with different capital letters within the respective extracts are significantly different at P<0.05 by Duncan's multiple range test.
쇠무릎잎과줄기추출물의항산화활성연구 975 잎은물을용매로그리고줄기는에탄올을용매로추출시단백질함량이높았다 (Table 1). 본실험결과는약용식물인우산나물의지상부에서 2.10 mg/g의단백질을함유하였다는결과 (30) 와비교하면쇠무릎은우산나물보다줄기는약 1.9배, 잎은 7.8배이상많은단백질을함유하는것으로분석되었다. 환원당함량환원당은당분자중에유리알데히드 (-CHO) 또는케톤 (-CO-) 기를갖고환원성을나타내는당 (sugar) 으로환원기는분자내히드록시기와결합하여헤미아세탈을만들어고리상구조를취하여알칼리용액을환원시키는성질이있는것으로알려져있다 (31). 쇠무릎잎과줄기추출물에함유된환원당함량을측정한결과, 줄기의에탄올추출물인 에서 11.35 mg/g으로가장많은환원당을함유하였으며, 는 3.68 mg/g으로물보다에탄올을용매로추출시더많은환원당을함유된것으로분석되었다 (Table 1). 이러한결과는약용식물인야생왕고들빼기잎의환원당함량이 4.75 mg/g이라는결과 (32) 와비교하면쇠무릎잎은야생왕고들빼기보다낮은환원당을함유하였다. 이상의결과 amylase 등의효소활성을증가시키고 (33) 식품의품질향상에큰영향을미치는것으로알려진환원당함량이높은쇠무릎줄기는식품으로서활용가능성이있을것으로사료된다. 총폴리페놀화합물함량페놀성물질은식물계에널리분포되어있는 2차대사산물로 phenolic hydroxyl기를가지기때문에단백질및기타거대분자들과결합하는성질을가지며, 활성산소의소거및산화적스트레스를막는역할을함으로써노화방지, 암및심장질환등을예방하거나지연시키는효과를나타내어오늘날식품, 의약품, 화장품등많은분야에서활용되고있다 (34-36). 쇠무릎잎과줄기에함유된총폴리페놀화합물함량을측정한결과쇠무릎잎은물을용매로추출한 가 58.27 mg/g으로가장많은폴리페놀화합물을함유하였고, 는 42.22 mg/g, 쇠무릎줄기는 16.13 mg/g, 3.91 mg/g으로잎이줄기보다약 3.6 8배많은폴리페놀화합물을함유하였다 (Table 2). 본실험의결과를초본류에속하는청나래고사리의폴리페놀함량이 25.08 mg/g이라는결과 (37) 와비교하면쇠무릎잎에함유된폴리페놀함량은청나래고사리보다약 2.7배높았다. 또한일부국내식물성식품에함유된폴리페놀화합물의함량을측정한 Lee와 Lee(38) 의보고에서상치 1.07 mg/g, 쑥 1.11 mg/g이라는결과와비교하여도쇠무릎잎과줄기의물과에탄올추출물에함유된폴리페놀함량이매우높은것으로분석되었다. 이에쇠무릎잎은총폴리페놀화합물의함량이식용및약용으로이용되고있는쑥이나상치보다더많이함유하고있으므로이를이용한기능성제품의활용가능성이높은것 Table 2. Contents of total polyphenol and flavonoid compounds from leaves and stems of A. japonica extracts (mg/g) Extracts 1) Total polyphenol compounds Total flavonoid compounds 58.27±0.75 A2) 42.22±0.64 A 42.22±0.64 B 21.85±0.19 C 16.13±0.42 C 3.91±0.22 D 27.40±0.54 B 4.52±0.75 D 1) The abbreviations of introductory remarks are the same as in Table 1. 2) All values are mean±sd of triplicate determinations. Values with different capital letters within the respective extracts are significantly different at P<0.05 by Duncan's multiple range test. 으로판단된다. 총플라보노이드화합물함량 플라보노이드는 C 6-C 3-C 6 의기본탄소골격을가지는페놀계화합물의총칭으로 (39), flavonol은 superoxide anion, 산소원자, 지질과산화라디칼등을제거하는항산화활성과금속이온과결합하는성질이있다고보고되었다 (40). 인체내에서 LDL의산화와세포독성을저해한다고알려져있는 quercetin을표준물질로하여플라보노이드화합물함량을측정한결과 와 에서각각 42.22 mg/g과 27.40 mg/g으로쇠무릎잎추출물이줄기보다약 1.5배많은플라보노이드를함유하였다. Choi 등 (41) 은전초를약용하는메꽃잎의물과에탄올추출물의플라보노이드분석결과각각 35.73 mg/g과 40.68 mg/g을함유한다고보고하여쇠무릎잎의물추출물은메꽃보다약 1.2배많이함유된것으로나타났다. 또한쇠무릎줄기물추출물은약용식물및기능성식품원료로많이사용되는 Jeong 등 (42) 이보고한당귀 11.06 mg/g, 상백피 10.30 mg/g 그리고홍삼 13.20 mg/g이라는결과와비교하면쇠무릎줄기의플라보노이드화합물이당귀, 상백피, 홍삼보다약 2 2.6배많이함유된것으로나타났다. 전자공여능측정안정된 free radical을갖는화합물인 DPPH(1,1-diphenyl-2-picrylhydrazyl) 를이용한전자공여측정법은항산화활성을갖는물질이 DPPH의 radical을소거시켜보라색이탈색되는특성을이용하여전자공여능을측정하는방법으로쇠무릎잎과줄기추출물의전자공여능을측정하였다 (Fig. 1). 1.0 mg/ml의농도에서 가 93.41% 로가장우수한전자공여능을나타내었으며, 와 에서도 90% 이상의활성을나타내었다. 0.1 mg/ml의농도에서는폴리페놀및플라보노이드화합물함량이가장많은쇠무릎잎의 가 63% 로가장우수한전자공여능을나타내었으며, 쇠무릎줄기는 가 51.49% 의활성을나타내었다. 그러나
976 서수정 김남우 Electron donating ability (%). 100 80 60 40 20 Ab Ab Aa Ba Bb Ca Bb Ba Ca Bc Dc Cc Ad Bc Cd Dd SOD-like activity (%). 10 8 6 4 2 Ac Ac Bc Cc Ab Bb Cb Db Aa Ba Ca Da 0 0.1 0.3 0.5 1.0 Concentration (mg/ml) Fig. 1. Electron donating abilities of water and ethanol extracts from leaves and stems of A. japonica. Bars with different capital letters within the respective extracts and small letters within the respective concentrations are significantly different at P< 0.05 by Duncan's multiple range test. The abbreviations of introductory remarks are the same as in Table 1. 농도가증가함에따라 가 보다높은전자공여활성을나타내어쇠무릎줄기에항산화활성이우수한유용물질이함유된것으로사료된다. 이러한결과를식용및약용으로이용하고있는머위추출물 (43) 의항산화효과측정결과에탄올추출물의잎과줄기에서각각 20.2% 와 34.9% 라는보고와비교하면쇠무릎의잎과줄기는머위보다약 3.5배이상의높은전자공여능을나타내었다. 또한 Park 등 (44) 이국내산생약추출물을 0.3 mg/ml의농도에서전자공여능이각각작약 86.6%, 목단 80.4%, 오미자 85.7% 라는나타낸결과와비교하면쇠무릎잎과줄기는생약추출물과비슷하거나약간낮은활성을나타내었다. Kang 등 (45) 은전자공여능이 phenolic acids 와플라보노이드및기타페놀성물질에대한항산화작용의지표이며환원력이클수록전자공여능이높다고보고하였고, Ryu(46) 는플라보노이드가유리기의형성을억제하며 -OH기가많은플라보노이드는 LDL(low density lipoprotein) 에대한항산화활성이높다고보고하여본실험의 와 의전자공여능이높은결과와도일치하였다. 이상의결과쇠무릎잎과줄기는약용식물로이용하고있는머위, 작약, 오미자보다우수한전자공여활성을나타내어이를이용하여항산화기능성식품이나첨가제로이용할수있을것으로사료된다. SOD 유사활성능 SOD는 superoxide를과산화수소로전환시키는반응을촉매하는효소로생체가상해를입는것으로부터보호기능을나타내는것으로알려져있다 (47). 이에 pyrogallol에대한자동산화반응을이용하여쇠무릎잎과줄기의 SOD 유사활성능을측정한결과 1.0 mg/ml의농도에서 와 가각각 8.13% 와 7.03% 의활성을내어쇠무릎잎이줄기보다약간더높은활성을나타내었다 (Fig. 2). 본실험결과를뿌리를약용하는맥문동잎의메탄올추출 0 0.3 0.5 1.0 Concentration (mg/ml) Fig. 2. Superoxide dismutase (SOD) like activities (%) of water and ethanol extracts from leaves and stems of A. japonica. Bars with different capital letters within the respective extracts and small letters within the respective concentrations are significantly different at P<0.05 by Duncan's multiple range test. The abbreviations of introductory remarks are the same as in Table 1. 물에서 2.07% 의활성을나타낸다는 Seo와 Kim의 (48) 의결과와비교하면쇠무릎잎은약 3.5배이상, 줄기는약 2배이상높은 SOD 유사활성을나타내었다. SOD 유사활성은페놀성화합물과 flavonoids 화합물함량이많을수록 SOD 유사활성또한증가하는것으로알려져있으나 (49), 본실험에서는잎의물추출물인 가폴리페놀및플라보노이드화합물함량이높았으나 SOD 유사활성능은낮게나타났다. 이는모든페놀성화합물이동일한항산화력을가지지않으므로쇠무릎의페놀성화합물의함량이높아도 SOD 유사활성을나타내는페놀성물질의함량이낮은것으로판단된다. 이에 superoxide의반응성을억제하여활성산소상태로부터생체를보호하는것으로알려져있는 SOD 유사활성능에대한추가적인연구가필요할것으로판단된다. 아질산염소거능니트로사민의생성을억제하기위해위장내의조건과유사한 ph 1.2와 3.0 그리고 6.0의조건에서쇠무릎잎과줄기의아질산염소거효과를측정한결과 는 ph 1.2, 1.0 mg/ml의농도에서 94.90%, 는 89.6% 로가장높은아질산염소거능을나타내었다 (Table 3). 쇠무릎줄기추출물인 와 는각각 15.43% 와 19.84% 로쇠무릎잎이줄기보다약 4.5 6배높았으며, 특히 는 0.3 mg/ml의낮은농도에서 61.03% 의우수한소거활성을나타내었다. ph 3.0의조건에서는 1.0 mg/ml의농도에서 와 가 45.20% 와 42.93% 로가장높았고, ph 6.0의조건에서는모든추출물이 10% 이하의낮은활성을나타내었다. 모든추출물은 ph가낮을수록그리고농도가증가함에따라아질산염소거활성도증가하였다 (P<0.05). 이러한결과를, 약용및식용식물로많이이용되고있는쑥추출물이 ph 1.2의 1.0 mg/ml의농도에서 40%, 에탄올추출물은 41% 의아질산염소거효과를나타낸다는 Park과
쇠무릎잎과줄기추출물의항산화활성연구 977 Table 3. Nitrite scavenging abilities (%) of water and ethanol extracts from leaves and stems from A. japonica (%) Extracts 1) (mg/ml) 0.3 0.5 1.0 28.93±0.40 Bc2) 55.09±1.44 Bb 94.90±0.40 Aa ph 1.2 61.06±0.92 Ac 86.40±2.11 Ab 89.60±0.80 Ba 6.54±0.02 Dc 8.76±0.01 Db 15.43±0.02 Da 10.98±0.48 Cc 12.52±0.69 Cb 19.84±0.51 Ca ph 3.0 ph 6.0 13.89±0.80 Ac 13.33±0.26 Ac 1.13±0.37 Cc 2.28±0.81 Bc 3.43±0.57 c nd 3) 1.20±0.55 b nd 22.42±0.45 Bb 24.44±0.67 Ab 7.23±1.79 Cb 4.20±0.72 Db 5.87±0.50 Ab 2.98±0.46 Bb 1.57±0.49 Cb 1.20±0.13 Ca 45.20±0.84 Aa 42.93±0.70 Ba 10.48±1.84 Ca 7.68±0.13 Da 8.80±0.20 Aa 7.61±0.36 Aa 3.14±0.37 Ba 1.30±0.76 Ca 1) The abbreviations of introductory remarks are the same as in Table 1. 2) All values are mean±sd of triplicate determinations. Value with different capital letters within the same column and small letters within the same row are significantly different at P<0.05 by Duncan's multiple range test. 3) Not detected. Kim(50) 의결과를비교하면쇠무릎잎은쑥보다약 2배이상우수한소거율을보였으며, Na 등 (51) 에의한두충의물과에탄올추출물에서각각 33.11% 와 34.18% 라는결과와비교하면쇠무릎의잎은두충보다약 2.5배높은아질산염소거율을나타내었다. Mirvish(52) 는폴리페놀화합물과플라보노이드는식품의조리과정중생성되는발암성니트로사민의생성을억제하는데효과가있고, 페놀화합물이다량으로함유된시료일수록산성조건에서아질산염소거효과가우수하며반응용액의 ph가높을수록효과는감소되는것으로보고하였는데, 본결과도폴리페놀과플라보노이드함량이가장높았던쇠무릎잎추출물이아질산염소거능이가장우수하여 Mirvish(52) 의보고와도일치하였다. 따라서쇠무릎잎은산성영역에서니트로사민의직접적인생성인자인아질산염을효과적으로분해할가능성이높다고판단되며, 이러한쇠무릎잎은아질산염소거를위한식품이나기능성제품의첨가소재로서활용될수있을것으로사료된다. Xanthine oxidase 저해활성생체내에서 purine 대사에관여하여통풍과신장질환의원인이되는 xanthine oxidase(xo) 를 0.1 1.0 mg/ml의농도에서쇠무릎추출물에대한저해활성을측정한결과는 Fig. 3에나타내었다. 1.0 mg/ml의농도에서 가 66.67% 로가장높은 XO 저해효과를나타내었으며 와 도각각 31.17% 와 32.06% 의활성을나타내었다. 쇠무릎잎은줄기보다물추출물은약 2배, 에탄올추출물은약 2.3배우수한저해율을보였으며농도가증가함에따라 XO 저해능도유의적으로증가하였다 (P<0.05). 이러한결과는 Kim 등 (53) 이연구한약용식물로이용하고있는차조기잎의물과에탄올추출물에서각각 41.2% 와 46.9% 의 XO 저해효과를나타내었다는보고와비교하면쇠무릎잎에탄올추출물은차조기잎보다낮았으나물추출 Xanthine oxidase inhibition (%). 100 80 60 40 20 0 Ad Bd Cd Dd Ac Bc Cc Dc Ab Bb Cb Db Aa Ba Ba 0.1 0.3 0.5 1.0 Concentration (mg/ml) Fig. 3. Xanthine oxidase inhibition of water and ethanol extracts from leaves and stems of A. japonica. Bars with different capital letters within the respective extracts and small letters within the respective concentrations are significantly different at P<0.05 by Duncan's multiple range test. The abbreviations of introductory remarks are the same as in Table 1. 물은차조기잎보다약 1.6배높은저해율을나타내었다. 또한현재식용으로많이이용되는고구마줄기에탄올추출물 (54) 의 XO 저해능이 0.25 mg/ml의농도에서 5.05% 라는결과와 0.3 mg/ml의농도에서 6.9% 의 XO 저해활성을나타낸쇠무릎줄기를비교하면쇠무릎줄기가고구마줄기보다약간높은저해능을나타내었다. 이에예로부터어린순을나물로식용하기도한쇠무릎잎은통풍질환에대한예방효과가있음을알수있으며이를이용한기능성식품및제품에효과적으로응용할수있을것으로기대된다. 요 한방에서건조된뿌리를 우슬 이라는약재로이용되는쇠무릎의잎과줄기를물과에탄올을용매로추출하여유용성분과항산화활성을측정하였다. 폴리페놀과플라보노이드화합물의총함량을측정한결과쇠무릎잎의물추출물인 약 Da
978 서수정 김남우 에서각각 58.27 mg/g과 42.22 mg/g으로가장많은함량을나타내었고, 단백질함량에서도 에서 16.42 mg/g으로가장많이함유된것으로분석되었다. 환원당함량은줄기의에탄올추출물인 에서 11.35 mg/g으로가장많았으며전자공여능은 1.0 mg/ml의농도에서 가 93.41% 로가장우수한전자공여효과를보였다. SOD 유사활성능은 1.0 mg/ml의농도에서모든추출물이 10% 미만의낮은활성을나타내었다. 아질산염소거능은 ph 1.2의조건의 1.0 mg/ml의농도에서 가 94.90% 의가장높은아질산염소거능을나타내었으며, ph 3.0에서도 가 45.20% 로가장높은소거활성을나타내었다. 는 1.0 mg/ml 66.67% 로가장높은 XO 저해효과를나타내었으며, 와 도각각 31.17% 와 32.06% 의 XO 저해율을나타내었다. 이상의결과쇠무릎잎은다량의폴리페놀및플라보노이드화합물을함유하였으며전자공여능과 SOD 유사활성능, 아질산염소거효과도줄기보다우수하며식용및약용식물로이용되는여러식물보다우수하여쇠무릎잎을식용뿐만아니라기능성식품의첨가제나천연항산화제로서활용될수있을것이라판단된다. 감사의글 본연구는 2012년대구한의대학교기린연구비지원에의한것임. REFERENCES 1. Kim MS, Yun SH, Na HS, Park HJ, Choi GG, Ynag SI, Lee JH. 2013. Chemical compositions and functional characteristics of Korean and imported pomegranate (Punica granatum L.). Korean J Food Preserv 20: 342-347. 2. Delanty N, Dichter MA. 1998. Oxidative injury in the nervous system. Acta Neurol Scand 98: 145-153. 3. Brüne B, Zhou J, von Knethen A. 2003. Nitric oxide, oxidative stress, and apoptosis. 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