J Korean Soc Food Sci Nutr 한국식품영양과학회지 42(5), 792~799(2013) http://dx.doi.org/10.3746/jkfn.2013.42.5.792 천연갈변저해제로서황금추출물의효소적갈변저해효과 박미지 1 장민선 1 정문철 2 김건희 1 1 덕성여자대학교건강기능신소재학과 2 한국식품연구원 Scutellaria baicalensis Extracts as Natural Inhibitors of Food Browning Miji Park 1, Min-Sun Chang 1, Moon-Cheol Jeong 2, and Gun-Hee Kim 1 1 Dept. of Health Functional Materials, Duksung Women's University, Seoul 132-714, Korea 2 Korea Food Research Institute, Gyeonggi 463-746, Korea Abstract This study was designed to develop natural browning inhibitors. The anti-browning effects of distilled water (SBD) and 80% ethanol extracts (SBE) of Scutellaria baicalensis Georgi in apple slices were investigated by L and ΔE values. Both SBD and SBE were effective in reducing the browning of apple slices and were successively fractionated into chloroform (CHCl 3), ethyl acetate (EtOAc), and water (H 2O) fractions. These extracts were measured for total phenolic content, flavonoid content, anti-oxidative activity (through free radical scavenging activity and the FRAP assay), ferrous ion chelation, and the inhibition of PPO (polyphenol oxidase) activity. Overall, fractions of SBE were better than fractions of SBD in all measurements. The highest total phenolic and flavonoid content were measured in the EtOAc and CHCl 3 fractions of SBE. EtOAc and CHCl 3 fractions also exhibited the highest anti-oxidative activities (in DPPH and ABTS free radical scavenging and the FRAP assay). Unusually, the highest ferrous ion chelating capacity was found in the H 2O fraction of SBD, but the other fractions showed more than triple the ascorbic acid already in use. Also, CHCl 3 fractions showed a stronger inhibition of PPO activity than ascorbic acid. All of these results suggest that EtOAc and CHCl 3 fractions from Scutellaria baicalensis can be used as natural anti-browning agents. Key words: Scutellaria baicalensis, antioxidative, antibrowning, PPO inhibition 서론황금 (Scutellaria(S.) baicalensis Georgi) 은다년생초본식물로꿀풀과 (Labiatae) 에속하며약용식물로전국에서재배되고있다. 예로부터뿌리를건조하여급성황달, 임신질환, 종기치료등에약재로사용되어왔다 (1). 황금추출물이나황금에서유래된단일물질의약리작용으로항산화, 항균, 항바이러스작용, 간기능보호, 항염, 항경련, 접촉성과민반응억제, 항고지혈및항불안효과등이보고되고있다 (2). 또한이러한효과들과관련하여간세포의지질과산화나암세포의증식을억제하였으나정상세포에는영향을끼치지않아부작용이없는치료효과를지니고있다고보고되고있다 (3,4). 이러한작용을하는주요활성물질은 baicalin, baicalein, wogonin, wogonoside 및 ganhuangenin으로이들모두 flavonoid 화합물이다. 이 flavonoid 화합물은 phenol기를함유하고있는구조로항산화활성을지닌다 (2). 최근경제발전에따른 1인및 2인가족의증가는단체급식의급증, 패스트푸드의소비량증가와함께편리성과간편성 을갖춘최소가공처리된신선편이제품의수요를증가시켰다 (5). 그러나신선편이제품은박피, 절단등과같은제조공정중의조직손상으로인하여저장이나유통기간중의연화나미생물번식, 갈변, 악취등의품질저하가문제시되고있다 (6,7). 특히갈변은소비자들이구매시바로확인할수있는품질지표로상품성을좌우한다 (8). 갈변은농산물이조직손상으로인해세포내에존재하는 catechin, tyrosine과같은 polyphenol 화합물이 polyphenol oxidase(ppo) 의작용으로산화되어 theaflavin, melanin 등의색소를형성하며일어나는현상이다 (3,9). 이러한갈변을저해하기위해 Vit. C, 환원제, 산미제, 킬레이팅약품등의화학물질들이전통적으로사용되어왔으나, 최근건강지향적식품에대한소비자들의관심증가로화학물질처리대신천연물질에대한요구가증가하고있는추세이다 (6,10,11). 이에따라생약재, 차, 과일및채소류등천연물의갈변저해능이보고되고는있으나실제로농산물에적용한경우는미비한실정이다 (12,13). 황금역시 tyrosinase 억제활성을본연구는있으나식품에적용하여갈변저해능을살펴본연구 Corresponding author. E-mail: ghkim@duksung.ac.kr Phone: 82-2-901-8496, Fax: 82-2-901-8474
황금의효소적갈변저해효과 793 는없는실정이다. 따라서본연구에서는황금추출물의사과슬라이스에대한갈변저해효과를살펴본후, 이추출물을다양한용매로분획하여분획물의항산화효과와 PPO 저해활성을조사하여황금의천연갈변억제제로서의가능성을검토하였다. 재료및방법재료본실험에사용한황금 (S. baicalensis Georgi) 은서울경동시장에서건조상태로구입하여사용하였다. 갈변억제대상물로사용한사과는 Fuji 품종으로경북영천에서 2012 년수확품을구입후외관이건전한것을선별하여실험에사용하였다. 추출물제조황금추출물은열수및 ethanol(etoh) 추출방법을적용하였다. 건조하여분쇄한황금 200 g에증류수및 80% EtOH 1,500 ml를넣고 60 o C의진탕수조에서 6시간 3회반복추출하였다. 각추출물은감압농축한후동결건조를거쳐분말형태로제조하여사과의갈변저해제로사용하였다. 또한각추출물을극성이다른 3가지용매로순차분획, 추출하여 chloroform(chcl 3), ethyl acetate(etoac) 와잔류 H 2O 분획물을각각얻었다. 이를농축한분말형태의시료를항산화및항갈변측정의시료로사용하였다. 갈변저해처리및저장사과를세라믹칼을이용하여 10 mm 두께로절단한후분말형태의추출물 0.1% 용액에 1분간침지처리한후종이타월로흐르는물기를제거하여상온에서 48시간동안보관하며외관의변화를관찰하였다. 사과슬라이스의표면색측정표면색은표준백판 (L=87.40, a=-0.49, b=1.96) 으로보정된 chromameter(cr-400, Minolta Co., Osaka, Japan) 를사용하여시료절단면의중심부위를 3반복으로 Hunter L, a, b값을측정하였고, 각처리구간색도의차이는색차 (color difference, ΔE) 를이용하여분석하였으며계산식은다음과같다 (14). ΔE=(ΔL 2 +Δa 2 +Δb 2 ) 1/2 총페놀함량측정 Folin-Ciocalteu법을이용하여측정하였으며 (15), 0.1% 시료 0.1 ml에 2 N Folin-Ciocalteu 용액 0.5 ml를첨가하여 3분간반응시킨후 20% Na 2CO 3 1 ml를첨가하여 1시간동안침전반응을시켰다. 반응용액을 3,000 rpm에서 3분간원심분리하여그상층액을취하여 microplate reader(m2, Molecular Device, Sunnyvale, CA, USA) 를이용하여 750 nm에서흡광도값을측정하였다. 총페놀함량은 gallic acid 를이용하여작성한표준곡선으로부터구하였다. 총플라보노이드함량측정 0.1% 시료 10 μl에 EtOH 60 μl와 10% aluminium chloride hexahydrate 4 μl, 1 M potassium acetate 4 μl, 증류수 122 μl를첨가하여실온에서 30분동안반응시킨후, microplate reader(m2, Molecular Device) 를이용하여 415 nm에서흡광도값을측정하였다. 총플라보노이드함량은 quercetin을이용하여작성한표준곡선으로부터구하였다 (16). DPPH radical 소거능측정 0.1% 농도의시료 50 μl를시험관에가하고 0.3 mm 1,1- diphenyl-2-picrylhydrazyl(dpph) 용액 150 μl를혼합하여 37 o C에서 30분간반응시킨후, 515 nm에서 microplate reader(m2, Molecular Device) 를이용하여측정하고, 아래의식으로부터 DPPH radical 소거활성을계산하였다 (17). 추출물대신양성대조군으로 ascorbic acid와 trolox를넣어같은방법으로실험을수행하여기존의갈변저해제및항산화제의효과와비교하였다. DPPH radical scavenging activity(%)=[1-(a/b)] 100 A, sample의흡광도 ; B, blank solution의흡광도 ABTS radical 소거능측정 ABTS radical의소거활성은 7.4 mm 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid)(abts) 와 2.6 mm potassium persulphate를하루동안암소에방치하여 ABTS 양이온을형성시킨후이용액을 735 nm에서흡광도값이 1.4±0.05가나오도록 buffer로희석하여사용하였다. 흡광도를맞춘 ABTS 용액 190 μl에 0.1% 농도의시료 10 μl를가하여 10분간반응시키고, 735 nm에서흡광도를측정하여아래의식으로부터 ABTS 라디칼소거활성을계산하였다 (18). 추출물대신양성대조군으로 ascorbic acid와 trolox를넣어같은방법으로실험을수행하여기존의갈변저해제및항산화제의효과와비교하였다. ABTS radical scavenging activity(%)=[1-(a/b)] 100 A, sample의흡광도 ; B, blank solution의흡광도 FRAP assay Ferric reducing antioxidant power(frap) assay에사용된시약은 0.3 M sodium acetate buffer(ph 3.6) 와 40 mm HCl로용해시킨 10 mm 2,4,6-tripyridyl-S-triazine(TPTZ) solution, 그리고 20 mm FeCl 3 solution을사용하였다. 미리제조된 sodium acetate buffer, TPTZ solution 및 FeCl 3 solution을 10:1:1(v/v/v) 의비율로혼합하여 37 o C에서 10~15 분간반응시켜 FRAP reagent를준비하였다. FRAP reagent 150 μl를 0.1% 시료 50 μl에혼합하여실온에서 20분간방치한후 593 nm에서흡광도를측정하였다. 이때 FRAP은 FeSO 4 를이용하여작성한표준곡선으로부터 Fe 2+ 의함량을구하였다 (19). 추출물대신양성대조군으로 ascorbic acid와
794 박미지 장민선 정문철 김건희 resorcinol을넣어같은방법으로실험을수행하여기존의갈변저해제의효과와비교하였다. 철이온 (Fe 2+ ) 에대한 chelate 효과측정 0.1% 시료 50 μl에 2 mm FeCl 2 5 μl를가한후, 실온에서 5분간방치한다음증류수 130 μl를가하였다. 이용액에 5 mm ferrozine 15 μl를가한후다시 10분간반응시킨후에 562 nm에서 microplate reader(m2, Molecular Device) 를이용하여측정하고, 아래의식으로부터철이온 (Fe 2+ ) 에대한킬레이트효과를 % 로나타내었다 (20). 추출물대신양성대조군으로 ascorbic acid와 ethylenediaminetetraacetic acid (EDTA) 를넣어같은방법으로실험을수행하여기존의갈변저해제및항산화제의효과와비교하였다. Chelating activity effect(%)=[1-(a/b)] 100 A, sample의흡광도 ; B, blank solution의흡광도 Polyphenol oxidase 활성저해력조사갈변효소인 PPO 저해활성은 50 mm phosphate buffer(ph 6.5) 1.7 ml와 PPO(500 units/mg) 0.2 ml를혼합한후 0.1% 의저해제를 0.1 ml를첨가하여 25 o C로조절된항온수조에서 15분간방치하고기질로서 4 mm catechin 용액 1 ml를각각첨가한후, microplate reader(m2, Molecular Device) 를이용하여 420 nm에서 5분간의변화를측정하였다. 효소의활성능은흡광도의변화를관찰한후 curve의직선부위로부터계산하였으며효소저해활성은흡광도감소량 % 로나타내었다 (21). 추출물대신양성대조군으로 ascorbic acid를넣어같은방법으로실험을수행하여기존의갈변저해제의효과와비교하였다. Inhibition of PPO activity(%)=[1-(a/b)] 100 A, sample의흡광도 ; B, blank solution의흡광도 IC 50 value 측정시료별 PPO 저해활성의상대적비교를위하여효소활성 의 50% 를저해하는시료의농도를환산하여 IC 50 값으로하였다. 통계처리통계처리는 SPSS Win program(version 19.0, SPSS Inc., Chicago, IL, USA) 을이용하여 ANOVA 처리를하였으며 least significance difference(lsd) test(p<0.05) 로유의성을검증하였다. 결과및고찰사과슬라이스의표면색사과슬라이스를황금추출물에침지하여상온에서 48시간동안의외관변화를관찰한결과, 황금추출물무처리군보다황금추출물처리군에서갈변이다소저해되는것이관찰되었다 (Fig. 1). 48시간후의표면색에있어서무처리군과 SBD, SBE의 L값이각각 64.83, 66.28, 66.89로측정되어무처리군의 L값이가장낮아육안으로판단한황금추출물의갈변저해를수치적으로확인할수있었다 (Table 1). 48시간후의 L값만을보면 SBD보다 SBE의값이높았으나 0시간의초기 L값을고려하여 L값의감소정도로비교하면 SBD가 SBE보다갈변저해효과가우수하다할수있다. 그러나 SBE 와 SBD의 L값의유의적차가존재하지않으므로 L값만으로추출용매에따른황금추출물의실제적인갈변저해효과를판단하기에는다소무리가있었다. 사과표면의색변화는 L, a, b값을모두고려한수치인 ΔE값으로나타낼수있으며값이클수록색변화가많이일어난것으로판단할수있다. ΔE값의경우무처리군의값이 4.77, SBD가 2.86, SBE가 3.34로무처리군에비해황금추출물처리군들의색변화가적게일어난것을알수있었으며, SBD의갈변저해효과가가장뛰어났다. Weller 등 (22) 은 L값의변화는 PPO의활성증가와관련이있으며 PPO는조직이노화되거나저장시 Fig. 1. Changes in the appearances of apple slices treated in 0.1% distilled water and ethanol extracts from S. baicalensis after 48 hours at room temperature. SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Table 1. Changes in the Hunter L, a, b and ΔE value of apple slices treated in 0.1% distilled water and EtOH extracts from S. baicalensis after 48 hours at room temperature Treatment 1) 0 hr 48 hr L a b L a b Untreated SBD SBE 68.86±1.07 a2) 67.71±0.21 a 69.82±2.20 a -2.75±0.28 a -2.19±0.35 b -2.47±0.13 ab 16.38±1.23 b 15.36±1.47 b 19.34±0.52 a 64.83±1.01 a 66.28±0.52 a 66.89±4.00 a -1.54±0.03 ab -2.15±0.07 b -1.38±0.71 a 18.62±1.44 a 17.84±0.57 b 20.50±0.63 ab ΔE 4.77±1.44 b 2.86±0.30 a 3.34±1.38 ab 1) SBD, distilled water extract of S. baicalentsis; SBE, 80% EtOH extract of S. baicalensis. 2) Each value represented mean±sd of triplicate analyses. Values within each column followed by different letters are significantly different (p<0.05).
황금의효소적갈변저해효과 795 스트레스를받았을때용해성이매우커지고활성화된다고하였다. PPO는산소와반응하여산화가촉진되는데, 갈변저해처리시일반적으로추출물에침지하는방법으로진행되므로과육이코팅된다. 이로인해산소가차단되어 PPO의활성을저해시키게되며, 황금이지닌항산화능으로인해 PPO의저해활성또한증가된것이라판단된다. 따라서다양한용매로분획한황금분획물의항산화능과 PPO 저해능을측정하여천연항갈변제로서의특성을조사하였다. 총페놀및플라보노이드함량페놀성화합물은다양한구조로식물체내에존재하고있으며, 플라보노이드는페놀성화합물중에서자연적으로생성되는가장큰그룹중하나이다. 이들은분자내에 phenolic hydroxyl기를지니고있어단백질또는효소단백질, Fe 2+, Cu 2+ 등의 2가금속이온과의결합력, 기타거대분자들과결합하는성질과높은항산화효과를가지는것으로알려져있다 (23,24). 황금추출물 100 g의페놀함량을측정한결과는 Fig. 2와같다. 전반적으로황금의열수추출분획물보다 EtOH 추출분획물의총페놀함량이더높았으며, 분획용매별로각각 EtOAc 분획물 (53.95, 70.80 g GAE/100 g)> CHCl 3 분획물 (53.30, 65.82 g GAE/100 g)> H 2O 분획물 (24.25, 23.22 g GAE/100 g) 의순으로 EtOAc 분획물의페놀함량이가장높았다. Hyun 등 (25) 의연구에따르면구절초의용매분획물별페놀함량은 EtOAc> CHCl 3> H 2O 분획물순으로높게조사되어본연구와같은양상을나타냈다. Cai 등 (26) 은황금의열수및 MeOH 추출물 100 g의페놀함량은각각 2.57, 8.17 g이었다고보고하여열수추출물보다 MeOH 혹은 EtOH 추출물이많은페놀을함유한다는점에서본연구와유사한경향을보였다. 황금추출물의플라보노이드함량을측정한결과는 Fig. 3과같다. 황금의열수및 EtOH 추출의 CHCl 3 분획물 (11.43, 14.83 g QE/100 g)> EtOAc 분획물 (6.13, 10.30 g QE/100 g)> H 2O 분획물 (1.63, 4.13 g QE/100 g) 의순으로 CHCl 3 Total flavonoid contents (g QE/100 g).. 20 15 10 5 0 b d SBD f a c SBE Fig. 3. Total flavonoid contents of 0.1% fractions which extracted from S. baicalensis. SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean±sd of triplicate analyses. Values that are followed by different letters are significantly different (p<0.05). 분획물에서플라보노이드함량이가장높았다. 총페놀함량과는다른경향을나타내었지만 EtOH 추출의분획에서높은함량을나타냈다는점에서유사하였다. DPPH radical 소거능 DPPH는짙은자주색을나타내며질소중심의안정한 free radical로서 515~520 nm에서최대흡수치를나타낸다. 황함유아미노산, ascorbic acid, 페놀성화합물등의항산화물질로부터전자나수소를제공받아 DPPH-H로환원되면서노란색으로탈색되며흡광도가감소된다 (27). 황금추출물 0.1% 의 DPPH radical 소거능을조사한결과는 Fig. 4와같다. 황금열수추출의용매분획물에대하여 EtOAc 분획물이 86.26% 로가장높았고, EtOH 추출의용매분획물에대하여 CHCl 3 분획물이 90.92% 로가장높았다. 양성대조군으로사용한 0.1% 의 ascorbic acid(96.23%) 와 trolox(96.21%) 의라디칼소거능과비교하였을때전반적으로황금열수추출의분획물은 73.45~89.64%, 황금 EtOH 추출의분획물은 73.58~94.49% 의값을나타내어 (p<0.05) 대체적으로 EtOH e Total phenolic content (g GAE/100 g).. 80 60 40 20 0 c c d SBD SBE Fig. 2. Total phenolic contents of 0.1% fractions which extracted from S. baicalensis. SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean±sd of triplicate analyses. Values that are followed by different letters are significantly different (p<0.05). b a d Fig. 4. DPPH radical scavenging activities of 0.1% fractions which extracted from S. baicalensis. AA, ascorbic acid; SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean±sd of triplicate analyses. Values that are followed by different letters are significantly different (p<0.05).
796 박미지 장민선 정문철 김건희 추출의분획물이열수추출의분획물보다라디칼소거능이우수한것으로조사되었다. 또한가장높은활성을나타낸 EtOH 추출의 CHCl 3 분획물의경우 ascorbic acid와 trolox 의값과유사한소거능을보였다. 이는총페놀및플라보노이드함량이높으면항산화능도우수하다는 Kim 등 (28) 의연구와도유사한경향을나타내었다. Park과 Kim(29) 이보고한황금추출물의 DPPH radical 소거능의경우열수추출물이 EtOH 추출물보다우수한활성을보여본연구와다소차이가있었으나, 이는시료의추출방법에따른차이로사료된다. Kim 등 (30) 이보고한황금 MeOH 추출의 EtOAc 분획층의소거능은양성대조군으로쓰인 BHT와비슷하였으며, 이는본연구결과와도유사하였다. ABTS radical 소거능 ABTS radical 소거능의경우 ABTS와 potassium persulfate가반응하여 ABTS cation radical이생성되면청록색을띠게되는데이때항산화물질과반응하여 ABTS cation radical이소거되면서청록색이탈색된다. 따라서높은탈색율을보일수록우수한항산화활성을기대할수있다 (27). 황금추출물 0.1% 의 ABTS radical 소거능을조사한결과는 Fig. 5와같다. 황금열수및 EtOH 추출모두 CHCl 3 와 EtOAc 분획물에서우수한활성을보였으나, H 2O 분획물에서는현저히낮은활성을보였다. 양성대조군으로사용한 0.1% 의 ascorbic acid(96.23%) 와 trolox(97.67%) 의라디칼소거능과비교하였을때황금열수추출의 CHCl 3 과 EtOAc 분획물의경우각각 87.30%, 87.17%, EtOH 추출의 CHCl 3 과 EtOAc 분획물의경우각각 96.34%, 99.07% 에해당하는수치를나타내어 EtOH 추출의분획물이 ascorbic acid 및 trolox의값과유사한소거능을보였다. 대체적으로 EtOH 분획물이열수분획물보다라디칼소거능이우수하였으며, 총페놀함량과유사한경향을나타내었다. Cai 등 (26) 은황금의 MeOH 추출물이열수추출물보다 ABTS radical 소거능이더높다고보고하였는데이는본연구의 EtOH 추출의용매분획물 이더높은소거능을보인것과같은경향을보였다. FRAP assay FRAP assay는환원력을측정하는방법으로 ferric tripyridyltriazine(fe 3+ -TPTZ) 복합체가시료의항산화성분에의해 ferrous tripyridyltriazine(fe 2+ -TPTZ) 으로환원되면서 593 nm에서의흡수치가증가한다 (31). 0.1% 의황금추출물의 FRAP assay에의한항산화활성을측정한결과는 Fig. 6과같으며, 분획물간의패턴은 DPPH radical 소거능 (Fig. 2) 과유사하였다. 황금열수추출의 EtOAc 분획물의항산화능이 260.04 mm Fe 2+ /ml로양성대조군으로사용한 ascorbic acid(280.03 mm Fe 2+ /ml) 의 92.86% 에해당하는수치를나타내며가장우수한활성을보였다. 열수및 EtOH 추출의다른분획물들또한 ascorbic acid의 77.20~88.42% 에이르는값을나타내어대체적으로우수한활성을보였다. 철이온 (Fe 2+ ) 에대한 chelating 효과 Ferrozine은 Fe 2+ 와복합체를형성하여붉은색을띠게되는데이때시료중의 chelating 효과를가진물질이 Fe 2+ - ferrozine 복합체의형성을방해하여발색을저해시킨다 (32). Metal chelating agent는제2의항산화제로불리며구연산, 주석산등의유기산및폴리페놀성분은 Fe 2+, Cu 2+ 와같은산화촉진제를 chelating하여 free radical의형성을억제하는효과를나타낸다고보고되고있다 (33). 본연구에서 PPO와직접연관된 Cu 2+ 에대한 chelating 효과를측정하진않았지만, 동일한전자가 (2+) 를지닌 Fe 2+ 에대한 chelate 효과를측정함으로써 Cu 2+ 에대한 chelating 효과를유추하였다. Chelating 효과가우수하다면 PPO 자체의활성을저하시켜갈변저해에효과가있다고할수있다. 황금추출물 0.1% 의 Fe 2+ 에대한 chelating 효과를측정한결과는 Fig. 7과같다. 황금열수및 EtOH 추출의 H2O 분획물이각각 71.02, 53.90% 로높은활성을나타내었고, CHCl 3 과 EtOAc 분획물의경우 25% 내외의활성을나타내었다. 이는현재 Fig. 5. ABTS radical scavenging activities of 0.1% fractions which extracted from S. baicalensis. AA, ascorbic acid; SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean±sd of triplicate analyses. Values that are followed by different letters are significantly different (p<0.05). Fig. 6. Ferric reducing antioxidant power of 0.1% ractions which extracted from S. baicalensis. AA, ascorbic acid; RES, resorcinol; SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean ±SD of triplicate analyses. Values that are followed by different letters are not significantly different (p<0.05).
황금의효소적갈변저해효과 797 Table 2. PPO inhibition activities of each fractions which extracted from S. baicalensis Distilled water Fractions Inhibition 1) 2) IC 50 Ascorbic acid 18.94±3.49 b3) 0.14±0.00 a CHCl 3 EtOAc H 2O 11.52±1.72 cd 13.17±4.34 cd 9.96±2.81 d 0.43±0.07 c 0.44±0.06 c 0.79±0.02 e 80% EtOH Inhibition IC 50 26.29±0.98 a 15.08±3.11 bc 10.93±5.93 d 0.35±0.02 b 0.56±0.07 d 0.77±0.05 e 1) PPO inhibition activities of 0.1% each fractions, % (w/v). 2) IC 50: the half maximal inhibitory concentration, % (w/v). 3) Each value represented mean±sd of triplicate analyses. Values within each column followed by different letters are significantly different (p<0.05). Fig. 7. Metal chelating activities of 0.1% fractions which extracted from S. baicalensis. AA, ascorbic acid; EDTA, ethylenediaminetetraacetic acid; SBD, distilled water extract of S. baicalensis; SBE, 80% EtOH extract of S. baicalensis. Each value represented mean±sd of triplicate analyses. Values that are followed by different letters are significantly different (p<0.05). 신선편이농산물의항갈변제로주로알려져있는 ascorbic acid의 chelating 효과가 8.12% 로측정된것과비교하여약 3배이상의효과로높은 chelating 효과를보였다. Polyphenol oxidase 활성저해력 PPO의반응은 monophenol이 diphenol이되는 hydroxylation과 diphenol이 quinone이되는산화과정에서작용하는것으로이 quinone이중합반응을거쳐갈색이나흑색을띄는 melanin이형성되면서갈변이일어난다 (34). 항산화제혹은항갈변제들은이두과정에서다양한매카니즘으로작용을하지만모두페놀의산화를저해시켜이루어지는반응으로본연구에서는 tyrosinase를효소로, catechin을기질로하였다 (35). 황금추출물 0.1% 의 PPO 활성저해력을조사한결과는 Table 2와같다. 황금열수추출의용매분획물은 EtOAc(13.17%)> CHCl 3(11.52%)> H 2O(9.96%) 의순으로, EtOH 추출의용매분획물은 CHCl 3(26.29%)> EtOAc(15.08 %)> H 2O(10.93%) 의순으로나타났다. 대체적으로 EtOH 추출의분획물이열수추출의분획물보다우수한활성을보였고, 열수추출에서는 EtOAc 분획물, EtOH 추출에서는 CHCl 3 분획물이가장뛰어난활성을나타내었다. 이는총페놀및플라보노이드함량과유사한경향을보여플라보노이드계열의페놀물질인황금의주요활성물질들이 PPO의활성을 저해한것이라추정된다. IC 50 의경우황금의열수와 EtOH 추출모두 H 2O 분획물 (0.79, 0.77%)> EtOAc 분획물 (0.44, 0.56%)> CHCl 3 분획물 (0.43, 0.35%) 의순으로높게나타나 CHCl 3 분획물이 PPO 활성저해력을 50% 에이르게하는농도가가장낮았다. 가장우수한활성을보인에탄올추출의 CHCl 3 분획물과현재갈변저해제로사용되고있는 ascorbic acid와비교하였을때, CHCl 3 분획물이 ascorbic acid 보다높은값을보여 ascorbic acid가더우수하였다. 반면두물질 0.1% 의 PPO 활성저해력은 CHCl 3 분획물이 ascorbic acid보다우수한것으로나타나, 갈변저해제를 0.1% 내외의저농도로사용한다면황금추출물이 ascorbic acid보다더효과적이라추정된다. 황금의다양한추출물의항산화및 tyrosinase 저해활성에대한연구는다수진행되어있지만 (2-4,30,36), 이추출물을농산물에적용하여갈변저해정도를실제적으로살펴본연구는거의없는실정이다. 따라서앞으로황금의다양한추출물에대한실제적인갈변저해정도를평가하는연구가추가적으로필요하다고사료된다. 요약천연갈변저해소재발굴을위해황금을열수와 EtOH로추출한후, 각각의추출물에사과슬라이스를침지하여외관의변화를관찰한결과황금의열수와에탄올추출물이사과의갈변억제에효과적이었다. 이에열수와 EtOH 추출물을각각상법에따라분획하여 CHCl 3, EtOAc, H 2O 분획물로총 6가지분획물을얻었다. 이분획물에대한총페놀과플라보노이드함량및라디칼소거능과환원력측정, 금속이온 chelating 등을통하여항산화효과와 PPO(polyphenol oxidase) 저해활성을측정하였다. 대체적으로 EtOH 추출의분획물이열수추출의분획물보다페놀과플라보노이드함량및항산화활성이뛰어났다. 총페놀의경우 EtOAc 분획물에서, 플라보노이드의경우 CHCl 3 분획물에서가장높은함량을나타내었다. DPPH와 ABTS radical 소거능, FRAP assay 결과 EtOAc 및 CHCl 3 분획물에서높은항산화능을보여총페놀및플라보노이드함량과유사한경향을나타내었다. 금속이온 chelating 활성은특이적으로 H 2O 분획물에서높은활성이나타났으나다른분획물들또한 ascorbic
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