Journal of Life Science 2015 Vol. 25. No. 11. 1311~1318 Changes in Phenolic Composition, Antioxidant and Antidiabetic Properties of Jeju Citrus sudachi as Influenced by Maturity Ji Eun Lee 1, Ji Hye Kim 1 and Min Young Kim 1,2 * 1 Toxicology Laboratory, Major in Biomaterials, College of Applied Life Science, SARI, Jeju National University, Jeju 690-756, Korea 2 Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju 690-756, Korea Received August 24, 2015 /Revised November 10, 2015 /Accepted November 17, 2015 The effects of fruit maturation on changes in the total phenolics, flavonoids, and carotenoids of methanolic extracts of Citrus sudachi, in addition to its antioxidant and antidiabetic activities, were determined. Generally, the concentration of these chemical constituents increased as C. sudachi reached maturity. C. sudachi contained high levels of total phenolics, flavonoids, and carotenoids at maturity, contributing 6339.5 mg of gallic acid equivalent per 100 g, 2364.2 mg of rutin equivalent per 100 g, and 678.7 mg/ml, respectively. The scavenging activities of 1,1-diphenyl-2-picryl hydrazyl (DPPH), hydrogen peroxide and nitric oxide radicals and the reducing power of mature fruits were significantly higher at all data points than those of immature fruits (p<0.05). In contrast, the ferrous ion chelating activity of mature and immature C. sudachi fruits was similar. The 50% effective concentrations (EC 50) of mature fruits were 4.1±0.10 mg/ml for scavenging DPPH radicals, 3.1±0.02 mg/ml for scavenging hydrogen peroxide, 3.9±0.01 mg/ml for scavenging nitric oxide, and 3.8±0.02 mg/ml for chelating ferrous ion. The antidiabetic activity of C. sudachi was studied in vitro using the α-glucosidase inhibitory method. The inhibitory activity of mature C. sudachi fruits on α-glucosidase was higher than that of immature fruits. These results suggest that the content of bioactive compounds and the antioxidant and antidiabetic activities of C. sudachi change during maturation. These findings can be further extended to exploit them for their possible application for the preservation of food products, as well as their use as health supplements and nutraceuticals. Key words : Antioxidant, α-glucosidase, Citrus sudachi, harvest date, polyphenol ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 DOI : http://dx.doi.org/10.5352/jls.2015.25.11.1311 - Note - 서 활성산소란 (reactive oxygen species, ROS) 세포의대사과 정중필연적으로생성되는반응성이매우큰산소종으로, 세 포에손상을가하고 DNA 변성, 지질산화, 단백질분해등을 유발한다 [2, 41]. 체내에는 superoxide dismutase (SOD), glutathione peroxidase, catalase 등의산화억제효소들이존재하 여발생된활성산소를소거하지만 [28] 불규칙적인식습관, 스 트레스, 환경오염등에노출된현대인의생활습관으로산화 억제효소들의균형이깨지게되고심질환, 당뇨, 신경질환 등과같은만성질환으로이어지게된다 [17]. 현재다양한항산 화제개발이활발하게이루어지고있지만여러합성항산화제 의부작용이보고되면서천연물소재를이용하여활성산소를 Authors contributed equally. *Corresponding author *Tel : +82-64-754-3349, Fax : +82-64-756-3351 *E-mail : jeffmkim@jejunu.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/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 억제하려는시도가활발히진행중이다 [13]. 2013년기준우리나라사망원인 5위인당뇨병은인슐린이제대로작용하지못해체내포도당의대사가정상적으로작동하지않아발생되는대사장애를말하며심혈관계질환, 망막증, 족부병변등의합병증을유발한다 [31]. 당뇨병성합병증의주요원인중하나는고혈당으로인한산화스트레스현상으로, 이는지속적인고혈당상태에서포도당의당화과정중생성된 ROS로인해지질과산화물생성이촉진되고단백질의당화과정이일어나합병증이발생하게된다 [39]. 따라서효과적인당뇨병치료를위해서는혈당유지와더불어 ROS의감소가함께이뤄져야한다. 무농약 유기농공법으로생산되는제주영귤 (Citrus sudaci Hort. ex Sirai) 은운향과감귤속후생감귤아속에속하는유자의근친종이며, 우리나라에서는제주도에서 80년대초일부농가가재배하기시작해일본명인스다치로불리다 1997년에 신선이살만한곳 이라는옛제주명칭인영주 ( 瀛州 ) 에서영 ( 瀛 ) 자를따서영귤이라명명되었다. 영귤의크기는직경 30~40 mm, 무게는 35 g 내외이며산이풍부해신맛이강하고독특한향을지닌향산성감귤의일종으로과피가녹색인미숙과상태에서가장향이좋아 8월말에서 9월말이주수확기이며, 생식용으로는적합하지않아주로향미제와조미료등가공식품으로이용되고있다 [33]. 영귤의성분과생리활성에대한국외연구는영귤에서발견된플라보노이드인 sudachi-
1312 생명과학회지 2015, Vol. 25. No. 11 tin [8, 11], 영귤의 limonoid [30], 영귤에서나온새로운 flavone glucoside인 sudachiin A의구조및합성 [9], 영귤에서분리된추출성분에서의점액산합성 [37], 감귤류의항돌연변이성 [7], 영귤과즙의칼슘에대한생체이용률향상 [32] 등의다양한연구보고가존재한다. 감귤대체작목장려정책지원에의해일시적으로 1990년대말에서 2000년대초에주로가공식품의산업화등응용연구중심으로진행되었으나, 성분및생리활성에관한연구는수확시기별성분특성 [12], 항균효과 [25], 가열온도에따른영귤과즙의성분변화 [23] 등으로그수가적어영귤에대한기초자료가상대적으로부족한실정이다. 따라서본연구에서제주지역에서재배 수확되고있는영귤을수확시기별로수거하고기능성성분및항산화, 항당뇨활성에관한기초연구자료를제시하여고부가가치천연식 의약품소재로서의이용가능성을조사하고자하였다. 재료및방법시료준비본연구에서사용한영귤은제주녹색농원 ( 제주특별자치도제주시봉개동소재 ) 에서 9월 ( 미숙과 ) 과 11월 ( 성숙과 ) 에수확한것을제공받아사용하였다. 영귤시료는흐르는물로 3~4 회깨끗이세척하고 0.5 cm로 slice하여 -70 에서 1차동결한후동결건조기 (PVTFD10A, 일신랩, Korea) 에서 3일동안건조하였다. 건조된시료는믹서로분말화한후 25 g을 500 ml의 100% methanol에 shaking incubator를이용하여 25 에서 150 rpm으로 72시간동안추출하였다. 추출된시료는원심분리후상층액을 0.45 μm syringe filter로여과하여실험에사용하였다. 좀더자세히예를들어기술하자면준비된영귤추출물원액의농도가 50 mg/ml 이므로 (5 g/100 ml의농도로추출하였으므로 ), 본실험결과중 DPPH radical scavenging activity의경우 0.5, 1, 2, 4, 8 mg/ml의시료를만들기위해영귤추출물원액을 8 mg/ml로희석한뒤 duplication하여실험을진행하였다. 영귤의기능성성분함량측정총폴리페놀함량측정은 Folin-Denis의방법 [3] 을변형하여측정하였다. 96 well plate에여과한추출물 30 μl 을놓고 95% ethanol 30 μl, 증류수 150 μl, 50% Folin-Ciocalteu reagent 15 μl 를첨가해실온에 5분간방치한다. 그후, 5% Na 2CO 3 30 μl 를넣어잘혼합하여암실에서 1시간동안반응시켜 microplate reader (15PA-0065, Dynex, USA) 로 725 nm에서흡광도를측정하였다. 함량은 gallic acid를표준물질로사용하여 mg gallic acid equivalent (GAE)/100 g으로나타내었다. 총플라보노이드함량은 Zhishen의방법 [45] 을변형하여측 정하였다. 96 well plate에여과한추출물 30 μl를놓고증류수 120 μl, 5% NaNO 2 9 μl를혼합해실온에 6분방치후 10% AlCl 3 을넣고다시 6분간방치한다. 그다음 1 M NaOH 60 μl 와증류수 72 μl를첨가해실온에 15분간반응시켰다. 함량은 rutin을표준물질로사용하여 mg rutin equivalent (RE)/ 100 g으로나타내었다. 총카로티노이드함량은 Rainha등의방법 [36] 을이용하여측정하였다. 추출직후 96 well plate에추출물 200 μl를넣고 470, 653, 666 nm에서흡광도를측정하여아래의식으로계산하였다. Total carotenoids (mg/l) = (1,000 A 470) - (2.860 Ca) - (129.2 Cb/245) Chlorophyll a (Ca) (mg/l) = (15.65 A 666) - (7.340 A 653) Chlorophyll b (Cb) (mg/l) = (27.05 A 653) - (11.21 A 666) A 470: absorption of 470 nm, A 653: absorption of 653 nm, A 666: absorption of 666 nm 2,2-diphenyl-2-picrylhydrazyl (DPPH) 라디칼소거능측정 DPPH 라디칼소거능은 Blois의방법 [1] 을변형하여측정하였다. 시료추출액 100 μl에 0.4 mm DPPH 용액 100 μl를혼합하여 10분동안암실에서반응시켜 517 nm에서흡광도를측정하였다. 소거능은시료첨가구와무첨가구간흡광도차이를계산한백분율과 DPPH radical을 50% 소거하는농도 (EC 50) 로나타내었다. Hydrogen peroxide 소거능측정 Hydrogen peroxide 소거능측정은 Kim의방법 [20] 을이용하였다. 시료추출용액 80 μl에 10 mm hydrogen peroxide 20 μl, 0.1 M phosphate buffer (ph 5.0) 을혼합하여 37 에서 5분간반응시킨후 1.25 mm 2,2'-azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) 30 μl, peroxidase (1 U/ml) 30 μl 를첨가해 37 에서 10분간반응시키고 405 nm에서흡광도를측정하여시료첨가구와무첨가구간흡광도차이의백분율과 hydrogen peroxide를 50% 소거하는농도 (EC 50) 로나타내었다. Nitric oxide소거능측정 Nitric oxide 소거능역시 Kim의방법 [20] 으로측정하였는데, 추출액 50 μl에 phosphate-buffered saline (PBS, ph 7.0) 에녹인 10 mm sodium nitroprusside 50 μl를첨가후 25 에서 3시간반응시킨다. 그다음 Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylene diamine dihydrochloride in 2.5% polyphosphoric acid) 100 μl를잘혼합해실온에 15분간방치하여 540 nm에서흡광도를측정하였다. 소거능은시료첨가구와무첨가구간흡광도차이의백분율과 nitric oxide를
Journal of Life Science 2015, Vol. 25. No. 11 1313 50% 소거하는농도 (EC 50) 로나타내었다. 금속결합력측정금속결합력은 Kim의방법 [19] 을이용하여측정하였다. 시료액 250 μl에 2 mm ferrous chloride 5 μl를섞은후 5 mm ferrozine solution 10 μl를놓은후실온에서 10분동안방치한다음 562 nm에서흡광도를측정하였다. 환원력측정환원력은 Oyaizu의방법 [34] 을변형하여측정하였다. 시료용액 400 μl와 1% potassium ferricyanide가포함된 200 mm sodium phosphate buffer (ph 6.6) 400 μl를 e-tube에혼합하여 50 에서 20분동안반응시킨다. 10% (w/v) trichloroacetic acid 400 μl를넣어반응을멈추게한후상층액 100 μl를 96 well plate에분주한후증류수 100 μl와 0.1% (w/v) ferric chloride 20 μl를넣어 700 nm에서흡광도를측정하였다. α-glucosidase 저해활성측정 2 U/ml α-glucosidase 50 μl에추출물또는 50 mm sodium phosphate buffer (ph 6.5) 10 μl를혼합하여실온에서 5분간반응시킨후 5 mm ρ-nitrophenol- α-glucopyranoside (PNPG) 50 μl 를첨가하여실온에 5분간방치하고 405 nm에서흡광도를측정하였다. 통계처리모든실험결과는 3회반복측정후평균값 ± 표준편차로나타내었고 SPSS program (18.0) 의 independent sample t-test 를실시하여 p<0.05 수준에서유의성을검정하였다. 결과및고찰영귤의기능성성분함량측정영귤미숙과와성숙과의총폴리페놀함량을측정한결과 Table 1과같이성숙과의폴리페놀함량이 6339.5 mg GAE/ 100 g로미성숙과의폴리페놀함량 5191.4 mg GAE/100 g 보다유의적으로높은것으로나타났다 (p<0.05). Lee 등은영귤착즙액의총폴리페놀함량을 28.4 mg GAE/100 ml라고 보고하여 [27] 본연구의함량이높게측정되었는데이는 Lee 의연구에서는껍질을제거해가식부만을착즙하고별도의추출과정이없었던반면본연구에서는과실전체를사용하고 100% methanol에서추출하여 methanol에용해된다양한폴리페놀성분이포함되었기때문이라사료된다. 일반적으로 Kim 등의보고와같이대부분의감귤류는수확시기가늦어질수록총폴리페놀함량이감소하는경향을보이나 [22] Yoo 등의연구에서유자 (Citrus junos SIEB ex TANAKA) 의경우에는성숙할수록과피와과육의폴리페놀함량이높아진다고보고하였는데 [43] 이는본연구의경향과일치한다. 영귤미숙과와성숙과의총플라보노이드함량은 Table 1과같이성숙과가 2833.3 mg RE/100 g으로미숙과의함량 2364.2 mg RE/100 g 보다높은것을확인할수있었다 (table 1). Hyon 등의연구에서진귤과온주밀감진피의플라보노이드함량은 50.7~63.9 mg/100 g로본연구보다낮게측정되었는데 [11] 이는추출농도및시간차이와본연구에서는과실전체를사용한반면 Hyon 등은과피만을사용한점에서차이가발생한것으로사료된다. Kim 등은제주재래종감귤착즙액의플라보노이드함량이대부분성숙할수록감소하는경향을보이나품종에따라함량이증가하는경우도있다고보고하여 [22] 본연구결과의경우도예외적으로성숙과의플라보노이드의함량이높아진것으로생각된다. 영귤미숙과와성숙과의총카로티노이드함량을측정한결과성숙과가 678.7 mg/ml로미숙과의함량 447.2 mg/ml보다유의적으로높았다. 이는성숙과정중에클로로필이분해되면서카로티노이드가급속히합성된것에기인하는것으로사료된다 [15]. DPPH 라디칼소거능 DPPH 소거법은항산화능측정에널리쓰이는방법으로 DPPH radical이항산화활성을가진물질과만나면전자를받아들이고환원되어진한보라색이노란색으로탈색되는데이변화정도를측정하여소거능을계산한다 [18]. 영귤추출물의 DPPH 라디칼소거능을측정한결과는 Fig. 1과같다. 미성숙과와성숙과의라디칼 50% 소거농도 (EC 50) 를비교한결과성숙과가 4.1 mg/ml로미성숙과의 6.1 mg/ml 보다낮아 DPPH 라디칼소거능이유의적으로높은것으로 Table 1. Total phenolic, flavonoid and carotenoids content of Citrus sudachi Harvesting time Immature Mature Content Total phenolic (mg GAE/100 g) 1) Total flavonoid (mg RE/100 g) 2) Total carotenoids (mg/ml) 5191.4±385.5 6339.5±244.5* 2364.2±46.6 2833.3±121.4* 447.2±1.5 678.7±0.6* 1) Total phenolic content, expressed as mg gallic acid equivalent (GAE) per 100 g of dried weight; 2) Total flavonoid content, expressed as mg rutin equivalent (RE) per 100 g of dried weight. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature (p<0.05).
1314 생명과학회지 2015, Vol. 25. No. 11 Fig. 1. DPPH radical scavenging activity and 50% effective concentration (EC 50) of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05). Fig. 2. Hydrogen peroxide scavenging activity and 50% effective concentration (EC 50) of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05). 나타났다 (p<0.05). 이는폴리페놀함량과자유라디칼소거능이상관관계를가지는경향으로미루어보아 [35] 성숙과의폴리페놀함량이높기때문에성숙과의 DPPH 라디칼소거능이미성숙과보다뛰어난것으로사료된다. Kim의연구에서도영귤과피의라디칼소거능이 8월보다 11월이높게측정되어본연구와경향과일치하였다 [21]. 수확시기별유자 (Citrus junos SIEB ex TANAKA) 추출물 [43] 과산지별유자의수확시기에따른라디칼소거능을측정한실험 [29] 에서도수확시기가늦을수록소거능이증가하였다는보고가있다. Hydrogen peroxide 소거능측정 Hydrogen peroxide는 DNA 및단백질을손상시키거나과산화지질의생성을촉진하는것으로알려져있으며그결과노화및성인병등을초래한다고보고되고있다 [11]. Hydrogen peroxide 소거능을측정한결과는 Fig. 2와같이농도의존적으로소거능이증가하는경향을보였으며성숙과의 EC 50 이 3.1 mg/ml로미숙과의 EC 50 3.5 mg/ml와비교하여유의적으로차이가있는것으로나타나성숙과의 hydrogen peroxide 소거능이더뛰어남을알수있었다 (p<0.05). Kim 등은제주감귤재래종감귤과피의수확시기별 hydrogen peroxide 소거활성을조사한결과대부분소거능이감소한다고하였으나지각과진귤은 9월수확분보다 11월수확분이더높은활성을나타내었다고보고하여본실험과유사한경향을보였다 [22]. Kim의연구에서 hydrogen peroxide 소거활성의 IC 50 이 0.72~1.03 mg/ml에비해 [20] 본연구의 EC 50 의수치가높은데이는감귤품종및재배환경의차이등에의한것으로판단된다. Nitric oxide소거능측정 Nitric oxide synthase (NOS) 에서생성되는 nitric oxide는 평활근이완을통한혈관등의이완, 혈소판응집억제, 신경전달, 면역조절, 세포독성등의중요한기능을한다 [42]. 그러나 nitric oxide가 superoxide (O - 2 ) 와반응하여 peroxynitrite (ONOO - ) 를생성하며세포독성을발생시키며과량의염증반응을일으키고면역체계에이상을일으키며종양을발생시킬수있다. Nitric oxide 소거능은 Fig. 3과같이농도가증가할수록영귤추출물의소거능이증가하였으며성숙과의 EC 50 값은 3.9 mg/ml로미숙과의 4.3 mg/ml 보다낮게나와 nitric oxide 소거능또한성숙과가높았으며 (p<0.05), Kim 등이보고한 IC 50 값 (0.66~3.08 mg/ml) 보다는높게측정되었다 [20]. Kim 등은대부분의감귤종이성숙할수록 nitric oxide 소거능이증가하는추세를보였으나변화폭은낮다고보고하였다 [24]. Hwang 등이감귤과피의 nirtic oxide 생성저해능을측정한 Fig. 3. Nitric oxide scavenging activity and 50% effective concentration (EC 50) of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05).
Journal of Life Science 2015, Vol. 25. No. 11 1315 결과감귤류 7종중영귤이두번째로활성이높은것으로보고하였고 [10] Yuasa 등은영귤에서발견되는 polymethoxyflavone인 sudachitin이 LPS로유도된 RAW264 세포주에서항염증효과를보였다고보고하였다 [44]. 이를종합하여볼때영귤이 nitric oxide 소거뿐만아니라생성또한효과적으로저해하므로 nitric oxide로유발된질병예방에효과적일것이라사료된다. 금속결합력측정 Fe, Cu 등과같은산화환원이용이한금속은지질산화과정에서촉매로작용하며 O - 2, H 2O 2, RO, ROO 등의 2차라디칼을생성시켜산화를촉진시킨다. 따라서이들금속을결합시키면간접적으로항산화활성을나타낼수있다 [4]. 영귤추출물의금속결합력측정결과는 Fig. 4와같다. 농도의존적으로결합력이증가함을보였으나 EC 50 값이 3.76 mg/ml로미숙과와성숙과간의유의적인차이는보이지않았다. 이는금속결합력과 ROS소거는작용기전이달라폴리페놀, 플라보노이드함량과의상관관계가 ROS 소거능보다낮기때문에 [5] 미성숙과와성숙과에서큰차이를보이지않은것으로판단된다. Kim은온주밀감의야생형및돌연변이형의금속결합력의 IC 50 을 1.35~7.59 mg/ml 로보고하였다 [19]. 영귤이금속결합활성을보이는것으로보아폴리페놀, 플라보노이드외의유용물질들이존재해금속이온을제거하는기능을하는것으로추측되며이에대한추가적인분석이필요하다고생각된다. 본실험에서실시한 in vitro 실험뿐만아니라 in vivo 실험에서노령흰쥐에게감귤전체, 감귤과피, 감귤과육를섭취하게한후지질과산화의척도가되는 Thiobarbituric acid reactive substances (TBARS) 값을측정한결과감귤전체와감귤과피에서대조군에비해유의적으로감소했다는보고가있다 [16]. 이와마찬가지로감귤류에속하는영귤도 in vivo 실 험을실시한다면지질과산화방지효과가있을것으로사료된다. 환원력측정환원력은 ferric ion/ferricyanide복합체를환원시켜 Perl s Prussian blue complex를 700 nm의흡광도로측정하여나온 OD 값으로항산화활성을나타낸다. 환원력이클수록녹색에가깝게발색되어높은흡광도값을가지는물질일수록항산화활성이뛰어나다 [18]. 본실험에서영귤미숙과와성숙과의환원력을측정한결과는 Fig. 5처럼 0.5~4 mg/ml에서미성숙과는 0.16~0.86, 성숙과는 0.21~1.19로성숙과의흡광도가미성숙과보다높게측정되었다 (p<0.05). 유자과피열수추출물의환원력은 500~10,000 μg/ml에서 0.03~0.97로본연구의흡광도가더높았다 [38]. Park 등은감귤 4종류의항산화효과를비교한결과총폴리페놀의함량과환원력의연관성이 0.946으로상관관계가높다고보고하여 [35] 본실험에서도총폴리페놀함량이많은성숙과의환원력이미숙과보다높게나타난것으로판단된다. α-gucosidase 저해활성측정 α-glucosidase는소장점막의미세융모막에서존재하는효소로다당류를단당류로분해하는효소이다. Acarbose와같은경구혈당강하제는 α-glucosidase저해를통해다당류의분해를방해해소장에서 glucose의흡수를지연시켜주어식후혈당의급격한상승을방지해주는데이들저해제는복부팽만감, 구토, 설사등다수의부작용이보고되어 [6] 천연소재를이용한저해제개발연구가활발하게이루어지고있다. 또한포도당대사과정에서발생하는 ROS에의해조직손상이나타나기때문에 ROS를소거하는항산화활성과 α-glucosidase 저해활성이동시에작용할때더욱효율적으로당뇨병을관리할수있다 [14]. Fig. 4. Ferrous ion chelating activity and 50% effective concentration (EC 50) of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05). Fig. 5. Reducing power of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05).
1316 생명과학회지 2015, Vol. 25. No. 11 생리활성이미성숙과보다뛰어나수확시기를놓쳐상품가치가떨어진성숙영귤도활용가능성이있다고사료된다. 본연구를통해영귤의항산화및항당뇨효과를확인하였으며수확시기가제한되어있다는한계를보완할수있어영귤의부가가치상승및활용을극대화할수있을것으로판단된다. 영귤이기능성식품및의약품소재로폭넓게사용되기위해서는항산화와항당뇨이외의다른생리활성에대한추가적인 in vitro 및 in vivo 실험이진행되어야할것으로사료된다. 감사의글 Fig. 6. α-glucosidase inhibitor activity of Citrus sudachi. Values are means ± SD of three separated experiments. Data were statistically analyzed by independent sample t-test. *Values are significantly different from immature fruit (p<0.05). 영귤추출물의 α-glucosidase 저해활성을확인한결과 Fig. 6와같이 25, 50, 100, 200 μg/ml에서미숙과와성숙과의저해활성이각각 5.5, 18.8, 25.7, 38.2% (EC 50 = 212.3±5.67 μg/ml) 와 14.2, 32.3, 41.9, 54.9% (EC 50 = 212.7±17.82 μg/ml) 로성숙과의저해도가유의적으로높은것을알수있었다 (p<0.05). Lee 등은주요감귤성분을대사성질환마우스에게주요감귤성분을경구투여한결과공복혈당이감소함을보였고 Akt의인산화가크게증가하여당뇨관련세포결손을억제시킬수있다고하였다 [26]. Tsutsumi 등은영귤과피에있는플라보노이드중하나인 sudachitin을고지방식이로비만이유발된 C57BL/6 J마우스와당뇨질환모델 db/db마우스에게 12주동안구강투여한결과 sudachitin이에너지대사를향상시켜이상지질혈증과대사증후군을개선시켜주고대사장애를막아미토콘드리아생합성을유도하여에너지소모를늘리고체중이늘어나는것을막아준다고보고하였다 [40]. 본실험과위보고들을종합하여볼때영귤의항당뇨효과가기대되며항산화효과를통해효율적인당뇨병의관리또한가능할것으로생각되어향후천연소재를이용한의약품및건강기능식품개발소재로활용될수있을것이라사료된다. 본연구에서는제주산영귤의수확시기별항산화및항당뇨효과를분석하였다. 영귤미성숙과와성숙과의총폴리페놀, 총플라보노이드및총카로티노이드함량을분석한결과성숙과의함량이각 6339.5 mg GAE/100 g, 2833.3 mg RE/100 g, 678.7 mg/ml으로미성숙과보다기능성물질의함량이높았다. 항산화활성을비교한결과 DPPH radical, hydrogen peroxide, nitric oxide 등의 ROS소거능과환원력에서성숙과의활성이유의적으로높았으나금속결합능에서는유의적인차이가없는것으로나타났다. 영귤미성숙과와성숙과의 α- glucosidase 저해활성을분석한결과성숙과의저해활성이유의적으로우수한것을알수있었다. 결론적으로성숙과의 이논문은교육부의지방대학특성화사업 (CK-I) 에의해이루어진것입니다. References 1. Blois, M. S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199-1200. 2. Dröge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47-95. 3. Folin, O. and Denis, W. 1912. On phosphotungstic-phosphomolybdic compounds as color reagents. J. Biol. Chem. 12, 239-243. 4. Fridovich, I. 1986. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247, 1-11. 5. Graf, E. and Eaton, J. W. 1990. Antioxidant functions of phytic acid. Free Radic. Biol. Med. 8, 61-69. 6. Hanefeld, M. 1998. The role of acarbose in the treatment of non insulin-dependent diabetes mellitus. J. Diabetes Complications 12, 228-237. 7. Higashimoto, M., Yamato, H., Kinouchi, T. and Ohnishi, Y. 1998. Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol. Mutat. Res. 415, 219-226. 8. Horie, T., Masumura, M. and Okumura, F. S. 1961. Sudachitin, a new flavone pigment of sudachi. Bull. Chem. Soc. Jpn. 34, 1547-1548. 9. Horie, T., Tsukayama, M. and Nakayama, M. 1982. The structure and synthesis of sudachiin A, a new flavone glucoside from citrus sudachi. Bull. Chem. Soc. Jpn. 55, 2928-2932. 10. Hwang, J. H., Park, K. Y., Oh, Y. S. and Lim, S. B. 2013. Phenolic compound content and antioxidant activity of citrus peels. J. Kor. Soc. Food Sci. Nutr. 42, 153-160. 11. Hyon, J. S., Kang, S. M., Senevirathne, M., Koh, W. J., Yang, T. S., Oh, M. C., Oh, C. K., Jeon, Y. J. and Kim, S. H. 2010. Antioxidative activities of extracts from dried citrus sunki and C. unshiu peels. J. Kor. Soc. Food Sci. Nutr. 39, 1-7. 12. Jeong, S. W., Lee, K. M., Jeong, J. W., Lee, Y. C., Lee, M. S. and Um, S. S. 1999. Physicochemical properties of korean citrus sudachi fruit by harvesting time and region. Kor. J. Food Sci. Technol. 31, 1503-1510. 13. Joo, S. Y. 2013. Antioxidant activities of medicinal plant
Journal of Life Science 2015, Vol. 25. No. 11 1317 extracts. J. Kor. Soc. Food Sci. Nutr. 42, 512-519. 14. Kang, J. R., Hwang, C. R., Sim, H. J., Kang, M. J., Kang, S. T. and Shin, J. H. 2015. Biological activities of yellow garlic extract. J. Kor. Soc. Food Sci. Nutr. 44, 983-992. 15. Kim, B. S., Kim, H. S., Koh, J. S., J. and Kang, Y. 1996. carotenoid, color value, uv spectrum, organic acid and free sugar contents of citrus varieties produced in Cheju. Kor. J. Post-Harvest Sci. Technol. Agri. Products 3, 23-32. 16. Kim, J. H. and Kim, M. S. 2003. Effect of different part of mandarin intake on antioxidative capacity in 15-month-old rats. Kor. J. Nutr. 36, 559-569. 17. Kim, M. J., Hong, C. O., Nam, M. H. and Lee, K. W. 2011. Antioxidant effects and physiological activities of pumpkin (Cucurbita moschata Duch.) extract from different aerial parts. Kor. J. Food Sci. Technol. 43, 195-199. 18. Kim, M. J. and Park, E. J. 2011. Feature analysis of different in vitro antioxidant capacity assays and their application to fruit and vegetable samples. J. Kor. Soc. Food Sci. Nutr. 40, 1053-1062. 19. Kim, M. Y. 2013. Free radical scavenging and ferrous ion chelating activities of citrus fruits derived from induced mutations with gamma irradiation. Life Sci. 10, 9s. 20. Kim, M. Y., Kim, I. J., Lee, H. Y., Lee, D. S., Im, S. J., Kim, J. H., Byun, J. H., Kim, J. Y., Lee, Y. J. and Jeong, S. R. 2012. Characterization of the antioxidant properties of citrus mutants induced by Gamma-rays. Life Sci. 9, 1495-1500. 21. Kim, Y. D. 2009. Study on the composition of flavonoids and biological activities from jeju citrus fruits. Jeju National University Graduate School. 22. Kim, Y. D., Ko, W. J., Koh, K. S., Jeon, Y. J. and Kim, S. H. 2009. Composition of flavonoids and antioxidative activity from juice of Jeju native citrus fruits during maturation. Kor. J. Nutr. 42, 278-290. 23. Kim, Y. D., Lee, Y. C., Oh, Y. J. and Kang, Y. J. 2001. Changes of components of citrus sudachi juice heated at various temperatures. Kor. J. Food Sci. Technol. 33, 238-244. 24. Kim, Y. D., Mahinda, S., Koh, K. S., Jeon, Y. J. and Kim, S. H. 2009. Reactive oxygen species scavenging activity of Jeju native citrus peel during maturation. J. Kor. Soc. Food Sci. Nutr. 38, 462-469. 25. Kim, Y. J., Kim, Y. D., Oh, S. W., Kang, Y. J. and Lee, Y. C. 1999. Antimicrobial activities of solvent extracts from citrus sudachi juice and peel. Kor. J. Food Sci. Technol. 31, 1613-1618. 26. Lee, Y. J., Choi, Y. H., Lee, S. Y., Park, K. J., Park, S. M., Hong, H, J. and Han, C. H. 2010. A study on the treat cause of diabetes due to dry citrus flavonoids. Kor. J. Hort. Sci. Technol. 105-106. 27. Lee, Y. J., Kim, D. B., Cho, J. H., Bailc, O. H. and Lee, O. H. 2013. Physicochemical characteristics and antioxidant activities of bioresource juices from Jeju. Kor. J. Food Sci. Technol. 45, 293-298. 28. Lee, Y. M., Bae, J. H., Jung, H. Y., Kim, J. H. and Park, D. S. 2011. Antioxidant activity in water and methanol extracts from korean edible wild plants. J. Kor. Soc. Food Sci. Nutr. 40, 29-36. 29. Moon, S. H., Assefa, A. D., Ko, E. Y. and Park, S. W. 2015. Comparison of flavonoid contents and antioxidant activity of yuzu (Citrus junos Sieb. ex Tanaka) based on harvest time. Kor. J. Hort. Sci. Technol. 33, 283-291. 30. Nakagawa, H., Duan, H. and Takaishi, Y. 2001. Limonoids from citrus sudachi. Chem. Pharm. Bull. (Tokyo) 49, 649-651. 31. Nam, S. M., Kang, I. J. and Shin, M. H. 2015. Anti-diabetic and anti-oxidative activities of extracts from crataegus pinnatifida. J. East Asian Soc. Dietary Life 25, 270-277. 32. Nii, Y., Fukuta, K., Sakai, K. and Yamamoto, S. 2004. Japanese citrus fruit (sudachi) juice is associated with increased bioavailability of calcium from whole small fish and suppressed bone resorption in rats. J. Nutr. Sci. Vitaminol. (Tokyo) 50, 177-183. 33. Oh, H. S. 2009. Cooking potentiality for japanese dishes using domestic citrus sudachi. Kor. J. Culin. Res. 15, 18-27. 34. Oyaizu, M. 1986. Studies on products of browning reaction--antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutri. 44, 307-315. 35. Park, G. H., Lee, S. H., Kim, H. Y., Jeong, H. S., Kim, E. Y., Yun, Y. W., Nam, S, Y. and Lee, B. J. 2011. Comparison in antioxidant effects of four citrus fruits. J. Fd Hyg. Saafety 26, 355-360. 36. Rainha, N., Lima, E., Baptista, J. and Rodrigues, C. 2011. Antioxidant properties, total phenolic, total carotenoid and chlorophyll content of anatomical parts of Hypericum foliosum. J. Med. Plan. Res. 5, 1930-1940. 37. Sengoku, T., Murata, Y., Mitamura, H., Takahashi, M. and Yoda, H. 2012. Synthesis of novel mucic acid 1,4-lactone methyl ester 3-O-ferulate related to an extractive component isolated from the peels of citrus sudachi. Tetrahed. Lett. 53, 435-437. 38. Shin, J. H., Lee, S. J., Seo, J. K., Chen, E. W. and Sung, N. J. 2008. Antioxidant activity of hot-water extract from yuza (Citrus junos SIEB ex TANAKA) peel. J. Life Sci. 18, 1745-1751. 39. Shin, K. N., Lee, H. S. and Kwon, C. S. 2011. Effects of nutrition education in type 2 diabetes mellitus on diabetes control and blood antioxidant status. J. Kor. Soc. Food Sci. Nutr. 40, 689-695. 40. Tsutsumi, R., Yoshida, T., Nii, Y., Okahisa, N., Iwata, S., Tsukayama, M., Hashimoto, R., Taniguchi, Y., Sakaue, H. and Hosaka, T. 2014. Sudachitin, a polymethoxylated flavone, improves glucose and lipid metabolism by increasing mitochondrial biogenesis in skeletal muscle. Nutr. Metab. 11, 1-14. 41. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M. and Telser, J. 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44-84. 42. Yim, C. Y. 2010. Nitric oxide and cancer. Kor. J. Med. 78, 430-436. 43. Yoo, K. M. and Hwang, I. K. 2004. In vitro effect of yuza (Citrus junos SIEB ex TANAKA) extracts on proliferation of human prostate cancer cells and antioxidant activity. Kor J. Food Sci. Technol. 36, 339-344.
1318 생명과학회지 2015, Vol. 25. No. 11 44. Yuasa, K., Tada, K., Harita, G., Fujimoto, T., Tsukayama, M. and Tsuji, A. 2012. Sudachitin, a polymethoxyflavone from citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells. Biosci. Biotechnol. Biochem. 76, 598-600. 45. Zhishen, J., Mengcheng, T. and Jianming, W. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555-559. 초록 : 수확시기별제주산영귤의항산화및항당뇨활성비교이지은 1 김지혜 1 김민영 1,2 * ( 1 제주대학교생명자원과학대학바이오소재전공독성학실험실, 2 제주대학교아열대농업생명과학연구소 ) 본연구는 9월과 11월에수확한영귤미성숙과와성숙과의항산화및항당뇨활성을비교하였다. 총폴리페놀, 플라보노이드및카로티노이드함량을분석한결과모두성숙과의함량이높았다. 항산화활성을 DPPH radical, hydrogen peroxide, nitric oxide 소거능, 금속결합능및환원력으로평가하고이를 EC 50 으로나타낸결과금속결합능을제외하고성숙과의활성이유의적으로높았다. α-glucosidase 저해활성을통해항당뇨활성을확인한결과성숙과가높은저해도를나타내었다. 이를통해출하시기를놓친영귤성숙과도건강기능식품및의약품소재로개발가능성이있다고판단되며이를활용하기위한추가적인연구가필요하다고사료된다.