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1 치자 (Gardenia jasminoides Ellis fructus) 껍질용매별추출물의지질과산화저해및질소산화물소거능 진동혁 오다영 정헌식 이영근 성종환 김한수 부산대학교식품공학과 (2017 년 5 월 2 일접수 : 2017 년 6 월 18 일수정 : 2017 년 6 월 21 일채택 ) Evaluation of Lipid Peroxidation Inhibition and Nitrogen Oxide Scavenging Activity from Peel of Gardenia jasminoides Ellis Fructus Extracted by Various Solvents Dong-Hyeok Jin Da-Young Oh Hun-Sik Chung Young-Guen Lee Jong-Hwan Seong Han-Soo Kim Department of Food Science and Technology, Pusan National University, Miryang 50463, Korea (Received May 2, 2017; Revised June 18, 2017; Accepted June 21, 2017) 요약 : 치자껍질의 70% methanol, ethyl acetate (EA) 및 distilled water (DW) 의용매별추출물의 total phenol 함량및질소산화물소거능, 환원력, β-carotene 탈색을이용한항산화력및지질과산화저해능측정을통하여치자의기능성식품소재로서의가치를검토한결과, anthocyanin 함량은 3.519±0.635 mg/100 g DW 로나타났으며, 용매별추출수율은 DW (39.87%), 70% methanol (36.26%), EA (2.88%) 로관찰되었다. 추출용매별항산화활성은농도 (0.2, 0.4, 0.6 mg/ml) 가증가할수록유의적으로증가하였으며 control 로사용된 ascorbic acid, BHA, trolox 보다는낮은활성이확인되었다. 치자껍질의 total phenol 함량은 EA, 70% methanol, DW 추출물순으로 EA 추출물에서 26.59±0.20 CAE (caffeic acid equivalents) mg/g 으로가장높았으며, Nitric oxide (NO) radical 소거능에서는 70% methanol (70.32~76.15%), DW (52.66~59.31%), EA (34.65~46.98%) 추출물순으로나타났다. Nitrite (NO 2 ) 소거능은 70% methanol (34.57~39.33%), DW (32.53~38.47%), EA (27.59~32.62%) 순으로관찰되었다. β-carotene 탈색저해능은 DW (41.55~50.97%), 70% methanol (23.37~44.80%), EA (13.37~25.24%) 순으로동정되었다. Reducing power (optical density) 는 70% methanol (0.044~0.127), DW (0.033~0.099), EA (0.026~0.097) 순으로확인되었다. 지질과산화저해능은껍질추출물중 70% methanol (56.51~76.21%), EA (54.59~63.34%), DW (47.92~61.11%) 순으로관찰되었다 (p<0.05). 이에, 치자껍질은기능성식품및천연항산화제로서의가치가매우높을것으로판단된다. 주제어 : 치자껍질, 추출용매, 항산화활성, 안토시아닌, 총페놀 Corresponding author ( kimhs777@pusan.ac.kr)

2 2 진동혁 오다영 정헌식 이영근 성종환 김한수 Abstract : The aim of this study was to investigate the bioactivity and antioxidant activity of peel from Gardenia jasminoides Ellis fructus (GJE). We were separated into GJE peel. After that, we determined anthocyanin. GJE peel were extracted by 70% methanol, distilled water (DW) and ethyl acetate (EA) three solvents. To investigate by the solvent extract of total phenol content and value as a functional food ingredient of GJE peel through nitrogen oxide scavenging activity, antioxidant activity, reducing power and lipid peroxidation inhibition were performed. Solvent extract bioactivity of increasing concentrations (0.2, 0.4, 0.6 mg/ml) were significantly increased (p<0.05). GJE peel extracts showed lower activity than positive control (ascorbic acid, BHA, trolox). The total phenol contents of GJE peel extracts were highest in EA extract. However, the order of total phenol content of the solvent in the GJE peel and the results of analysis of various physiological activities were inconsistent. Considering the extraction yield and various physiological activities, it is expected to be effective when extracted from 70% methanol and DW extract. The results suggest that GJE peel is highly expected to be useful as a functional foods and natural antioxidant. Keywords : Peel of Gardenia jasminoides Ellis fructus, Solvent extracts, Antioxidant activity, Anthocyanin, Total phenol 1. 서론 식품의다양성과계절의존성감소로인한가격안정화의결과, 식품소비와공급량증가등많은변화를가져왔다 [1]. 식품은단순한영양원이아닌기능성을고려하게되어건강의중요성과인간수명연장에따라건강기능식품에대한관심과소비가증가하는추세이다 [2]. 산화질소합성효소에의해생성되는 reactive nitrogen species (RNS) 는 nitric oxide (NO ), nitrite (NO 2 -), peroxynitrite (ONOO-) 를형성하여이러한 free radical을가진물질은불안정하고홀수전자를가지므로반응성이매우높은것으로알려져있다 [3,4]. Reactive oxygen species (ROS) 와 RNS는그안정성을확보하기위해필요한전자를포착하여다른화합물과신속히반응하기때문에지질과산화및연쇄반응을개시하여여러산화생성물들을생성하여암을유발하고노화와관련하여생리적기능장애를일으킨다고알려져있다 [5]. 식물체의경우 2차대사산물의방어체계, 효소와같은보호기전이발달되어있어서식물체에서항산화물질을탐색, 연구하는것은중요한의미를지닌다고한다 [6]. 치자는꼭두서니과 (Rubiaceae) 에속하는치자나무 (Gardenia jasminoides Ellis) 의열매로국내를비롯한중국, 일본, 대만등의기온이따뜻한지 역에자생하고있다. 치자의주요생리활성물질은수용성 carotenoids 인 crocin 으로알려져있으며, crocin 과그대사산물인 crocetin 이동물실험에서췌장의 lipase 활성을억제하여지질흡수를감소시키는것으로보고되고있어고지방식이로인한고지혈증 (hyperlipidemia) 등을개선할수있다는것을시사하였다 [7]. 따라서, 본연구는치자껍질의 anthocyanin 함량을측정하고 70% methanol, distilled water (DW) 및 ethyl acetate (EA) 의용매별로추출하여 total phenol 함량을측정한뒤질소산화물소거능 (nitric oxide scavenging activity, nitrite scavenging activity), 항산화능 (antioxidant activity by β-carotene bleaching assay), 환원력 (reducing power), 지질과산화저해 (lipid peroxidation inhibition) 를측정하여추출용매에따른생리활성을비교하여생활습관병예방과건강기능식품개발의목적으로이들의소비효율성을높이고이에대한기초자료를제시함으로써고부가가치의천연항산화제의이용가능성을확인하고자하였다. 2. 재료및방법 2.1. 실험재료본실험에사용된시료인치자 (Gardenia

3 치자 (Gardenia jasminoides Ellis fructus) 껍질용매별추출물의지질과산화저해및질소산화물소거능 3 jasminoides Ellis) 는경상남도남해군에서 2014 년 11 월에수확하여반건조상태로되어있는시료를남해소재약재상에서 2015 년 1 월에구입하였다. 구입한치자의껍질을분리한뒤자연건조시켜분쇄기 (HMF-3250S, Han-Il Co., Seoul, Korea) 에분쇄하여분말로만든다음 deep freezer (DF-8514, Il-Shin BioBase Co., Daegu, Korea) 에 80 로저장하여실험에사용하였다 시료의추출시료의추출은동결저장된치자껍질분말 100 g 씩취하여 70% methanol, ethyl acetate (EA) 용매를각 10 배가하여 (1:10, w/v) 24 시간씩 2 회추출한뒤여과 (filter paper, Advantec, No.2, Tokyo, Japan) 하였고, distilled water (DW) 추출물은 70 에서 2 시간씩 2 회추출하여여과 (filter paper, Advantec, No.1, Tokyo, Japan) 하였다. 각추출물은 rotary vacuum evaporator (Hei-VAP Advantage, Heidolph Co., Germany) 를이용하여 40 에서감압농축하여용매를제거한후, 실험에사용하였으며, 시료의수율은추출전시료중량에대한추출후건조중량백분율 (%) 로나타내었다 [8] Anthocyanin 함량측정치자껍질의 anthocyanin 함량은 phdifferential method 를사용하여측정하였다 [9]. 건조된치자껍질분말 0.2 g 에 methanol-1m HCl (85:15, v/v) 15.0 ml 를가하여 30 분동안혼합한뒤여과 (filter paper, Advantec, No.2, Tokyo, Japan) 하여분석시료로사용하였다. 시료 1.0 ml 에 M potassium chloride buffer (ph 1.0) 3.0 ml 를가하고또다른시험관에시료 1.0 ml 와 0.4 M sodium acetate buffer (ph 4.5) 3.0 ml 를가한후암실에 20 분간방치하여 UV/Vis spectrophotometer (SP-200, Analytik Jena Co., Jena, Germany) 를사용하여 510 nm 및 700 nm 에서흡광도를측정하였다. 치자껍질의 anthocyanin 함량은 cyanidin-3- glucoside 의몰흡광계수 (ℇ=26,900 M -1 cm -1 ) 를이용하여 mg/100 g DW (dry weight) 로계산하여나타하였다 Total phenol 함량측정 Total phenol 함량은 Folin-Denis 방법을변형 하여실험하였다 [10]. 시료추출액 1.0 ml 에증류수 4.0 ml 을넣고, Folin-Denis reagent (Sigma-Aldrich, St. Louis, USA) 0.5 ml 를가한후, 잘섞어 3 분간실온에방치한뒤 10% sodium carbonate solution 0.5 ml 을첨가하여실온에 1 시간방치한후 750 nm 에서흡광도를측정하였다. 표준물질로는 caffeic acid (Sigma- Aldrich, St. Louis, USA) 를사용하여시료 1 g 당 mg CAE (mg of caffeic acid equivalents) 로나타내었다 Nitric oxide (NO) radical scavenging acticity 측정 Nitric oxide (NO) radical 소거능은 Rao (1997) 의방법을변형하여측정하였다 [11]. 용매별추출물각농도의시료 2.0 ml 에 10 mm sodium nitroferricyanide (III) dihydrate 및 0.2 M PBS (phosphate buffered saline, ph 7.4) 3.0 ml 를넣어잘섞은후 25 water bath 에서 150 분간반응시켰다. 반응액 0.5 ml 와 1% sulfanilamine 및 2% H 3 PO ml 을섞어 10 분간방치한뒤 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride 1.0 ml 를가하여 25 water bath 에서 30 분간방치하여 540 nm 에서흡광도를측정하였다. Positive control 은 trolox (6-hydroxy-2,5,7,8-tetramethylchroman -2-carboxylic acid, Sigma-Aldrich, St. Louis, USA) 를사용하였으며, 치자껍질의 NO 소거능은백분율 (%) 로계산하여표시하였다 Nitrite (NO 2 ) scavenging activity 측정 Nitrite 소거능은 Lim 등 [12] 의방법을변형하여측정하였다. 시료 2.0 ml 와 1 mm sodium nitrite 1.0 ml 를혼합하여 0.2 M citrate buffer (ph 2.5) 7.0 ml 를첨가한후, 37 로 1 시간동안반응시켰다. 반응액 1.0 ml 에 2% acetic acid 3.0 ml 를넣은후, Griess reagent (1% sulfanilic acid in 30% acetic acid:1% 1-naphthylamine 및 30% acetic acid, 1:1, v/v) 0.4 ml 와 15 분간반응시켜 520 nm 에서흡광도를측정하였다. Positive control 로 BHA 를사용하였으며백분율 (%) 로나타내었다 β-carotene bleaching 을이용한 antioxidant activity 측정 β-carotene 탈색을이용한치자껍질의항산화

4 4 진동혁 오다영 정헌식 이영근 성종환 김한수 활성은 Kato 등 [13] 의방법을변형하여측정하였다. Chloroform 10.0 ml에 β-carotene 1 mg을녹인용액 1.0 ml를 round-bottomed flask에넣은후 linoleic acid 20 mg과 Tween mg을가하여충분히혼합하였다. 남아있는 chloroform을 40 에서 rotary vacuum evaporator로제거한후잔여물을증류수 100 ml를넣고유화시킨 emulsion을실험직전에조제하여사용하였다. β-carotene-linoleic acid emulsion 4.0 ml와농도별시료추출물 0.4 ml 를시험관에넣고섞은뒤 470 nm에서흡광도를측정한후 (t=0 min), 50 의 water bath에서 2시간동안반응시켜 470 nm에서흡광도를재측정하였다 (t= 120 min). Positive control로 BHA를사용하였으며항산화활성은아래식에의해계산하였다. Antioxidant activity (%)= A s = the absorbance in the presence of sample extract. A b = the absorbance of the blank Reducing power 측정치자껍질의용매별추출물에따른 reducing power 의측정은각시료추출용액 1.0 ml 에 0.2 M sodium phosphate buffer (ph 6.6) 1.5 ml 와 1% potassium ferricyanide 1.0 ml 를넣고 50 의 water bath 에서 20 분간반응시켰다. 반응시킨혼합액에 10% trichloroacetic acid 1.5 ml 를가하여섞은후 3,000 rpm 에 10 분간원심분리하여분리된상등액 1.0 ml 에증류수 3.0 ml 와 0.1% ferric chloride solution 0.2 ml 를넣어잘혼합시켜, 10 분후 700 nm 에서흡광도를측정하였다 [14]. 또한 EC 50 은 positive control 인 BHA 를통하여계산하였다 Lipid peroxidation inhibition activity 측정치자껍질의각용매별추출물에서의지질과산화저해활성은 Siriwardhana 등 [15] 의방법을변형하여측정하였다. 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml 로희석한각용매별시료 1.0 ml 를취하여 2.5% linoleic acid emulsion 및 ethanol 2.0 ml 와 phosphate buffer (ph 7.0) 10 ml 를가한후혼합액이 20 ml 가되도록증류수로정 용하였다. 빛을차단한 40 water bath 에 24 시간동안혼합액을반응시킨후, 반응액 0.1 ml 에 30% ammonium thiocyanate 0.1 ml 와 ethanol 3.7 ml, 0.02 M ferrous chloride 및 3.5% HCl 0.1 ml 를가하여, 3 분간혼합하여산화정도에따른흡광도차이를 500 nm 에서측정하였다. positive control 로 BHA 를사용하였으며백분율 (%) 로계산하여지질과산화저해능을나타내었다 통계처리실험데이터는 3 회반복측정하였으며, mean±sd (n=3) 으로표시하였다. 실험결과값간의유의적인차이는 one-way ANOVA 로분석한뒤 p<0.05 수준에서 Duncan`s multiple range test 에의한각농도간의유의성을검정하였다. 시료농도별결과값에대한 IC 50 과 EC 50 은선형회귀분석을통하여구하였다. 통계처리에대한프로그램은 IBM SPSS statistic ver. 22 를사용하였다. 3. 결과및고찰 3.1. 수율치자껍질의 70% methanol, distilled water (DW) 및 ethyl acetate (EA) 에서의추출수율은 Table 1 에나타내었다. 각용매별추출수율은 70% methanol 에서 36.26%, DW 39.87%, EA 2.88% 로 DW 에서추출수율이가장높게나타났다 Anthocyanin 함량치자껍질의 anthocyanin 함량은 Table 1 과같이 3.519±0.635 mg/100 g DW (dry weight) 로관찰되었다. Anthocyanin 은적색과자색등식물체의수용성천연색소로식물이씨앗및열매를분산시키는데중요한역할을하며, 자외선조사에의한손상을보호하고벌과나비같은꽃가루매개자를끌어들이는역할을한다고알려져있다 [16]. 또한식물에서유래한 anthocyanin 은인체내의다양한생리활성중시각과뇌기능의향상, 비만과당뇨의관리, 심혈관계질환예방, 종양, 노화방지, 항암, 항염, DNA 손상방지등의효과가있는것으로알려져있고, 이와관련된연구가진행되고있다 [17]. 본실험에서치자

5 치자 (Gardenia jasminoides Ellis fructus) 껍질용매별추출물의지질과산화저해및질소산화물소거능 5 Table 1. Contents of anthocyanin, total phenol, IC 50 and EC 50 values in the bioactivity evaluation assays of peel from Gardenia jasminoides Ellis fructus Assays 1) Values Anthocyanin content (mg/100 g DW 2) ) 3.519±0.635 EA DW 70% methanol Extraction yields (%) Total phenol content (mg CAE 3) /g) 26.59±0.20 c4) 18.61±0.08 a 24.13±0.27 b NO (IC 50, mg/ml) 0.687±0.012 c 0.135±0.007 b 0.006±0.000 a NO 2 (IC 50, mg/ml) 1.966±0.077 c 1.377±0.014 a 1.518±0.097 b BC (IC 50, mg/ml) 1.425±0.019 c 0.547±0.002 a 0.698±0.003 b RP (EC 50, mg/ml) 1.717±0.065 c 1.798±0.040 b 1.407±0.059 a LPI (IC 50, mg/ml) 0.265±0.006 c 0.113±0.016 b 0.087±0.005 a 1) Nitric oxide radical scavenging activity (NO), nitrite scavenging activity (NO 2 ), antioxidant activity by β-carotene bleaching assay (BC), reducing power (RP), lipid peroxidation inhibition activity (LPI). 2) DW: dry weight. 3) CAE: caffeic acid equivalents. 4) The values are means±sd (n=3). Values with the different letters in the same row are significantly different (p<0.05) by Duncan`s multiple range tests. 껍질은 anthocyanin 성분을함유하고있는것으로확인되어그에따른여러생리활성효과가기대된다 Total phenol 함량치자껍질의용매별추출물에서 total phenol 함량은 Table 1 에나타내었으며, 껍질추출물중 EA 추출물 26.59±0.20 mg CAE/g. 70% methanol 추출물 24.13±0.27 mg CAE/g mg, DW 추출물 18.61±0.08 mg CAE/g 순으로 DW 추출물에서유의적인차이를보이며낮은값이관찰되었다 (p<0.05). Phenolic 화합물은항암, 항돌연변이, 항산화능뿐아니라, 단백질과같은고분자물질과결합하기도하여종양증식의개시, 촉진및진행을억제하며, 골수성백혈병세포를사멸시키는역할을하는등인체내에서다양한생리활성을가진다고보고되어있다 [18]. Cai et al.(2004) 은항암에관련된 112 가지중국약용작물의항산화활성실험중중국산치자열매의주요 phenolic 화합물은주로 phenolic acids (chlorogenic acid), flavones (gardenins) 로구성되어있으며 total phenol 함량은 methanol 추출물에서 10.0 mg/g, 물추출물에서 10.8 mg/g 으로보고하였다 [19]. 본실험결과는이보다높은수치의 phenol 함량이확인되었으며, 품종요인과재배환경조건에따라이와같은차이가나는것으로판단된다 Nitric oxide (NO) radical scavenging activity 치자껍질의용매별추출물과 positive control 인 trolox 의 nitric oxide (NO) radical 소거능을각농도별로비교한결과는 Fig. 1 과같으며, IC 50 값은 Table 1 에표시하였다. 각용매별추출물 0.2, 0.4, 0.6 mg/ml 의농도에서측정한결과, 농도가증가함에따라 NO radical 소거능또한유의적으로증가하는것으로나타났다 (p<0.05). 70% methanol 추출물 70.32±0.09%, 73.44±0.31%, 76.15±0.09%, IC ± mg/ml, DW 추출물에서 52.66±0.23%, 55.53±0.09%, 59.31±0.27%, IC ± mg/ml, EA 추출물 34.65±0.39%, 41.81±0.41%, 46.98±0.35%, IC ± mg/ml 순으로추출물중에서는 EA 용매추출물에서가장낮은활성과높은 IC 50 결과치가관찰되었다. Positive control 인 trolox 는각 83.37±0.23%, 86.34±0.15%, 89.36±0.09% 로확인되었으며, trolox 는산화적스트레스손상을감소시키는생리생화학적용도로사용되는항산화제이며, 항산화성분측정시항산화능력에

6 6 진동혁 오다영 정헌식 이영근 성종환 김한수 대한기준으로도사용된다고알려져있다 [20]. 질소산화물중 nitric oxide radical (NO) 는신경전달, 종양세포의면역및염증반응과정에관련이있다고보고되어있다 [21]. NO 합성효소 (EC ) 는 L-arginine 을 citrulline 과 NO radical 로변환시키며, 생성된 NO radical 은신경세포와내피세포, 간세포등에서여러 iso-form 으로전환되어독성을나타내는것으로알려져있다 [22]. 서는 EA 용매추출물에서유의적으로낮은활성과높은 IC 50 결과치가관찰되었다 (p<0.05). 70% methanol 추출물에서의 nitrite 소거활성은각농도에서 DW 추출물보다높았으나 IC 50 은 DW 추출물보다유의적으로약한것으로관찰되었다 (p<0.05). 이는농도간의소거활성증가폭이 DW 추출물보다낮기때문인것으로사료된다. Positive control 인 ascorbic acid 는각농도별로 49.07±0.21%, 68.51±0.08%, 84.01±0.21% 로확인되었다. 아질산염은 nitric oxide synthase 의활성및산화질소 radical 생성에대한바이오마커로서임상화학에서자주사용되고있다 [23]. Fig. 1. Nitric oxide (NO) radical scavenging activity of various solvent extracts from peel of Gardenia jasminoides fructus. 1) The values are means±standard deviation (n=3). Bars with the different letters are significantly different (p<0.05) by Duncan s multiple range tests. 2) EA: ethyl acetate. 3) DW: distilled water. 4) Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman -2-carboxylic acid Nitrite (NO 2 ) scavenging activity 치자껍질의각용매별추출물과 positive control 인 ascorbic acid 의 nitrite (NO 2 ) 소거활성을농도별로비교한결과를 Fig. 2 에나타내었으며, IC 50 값을구하여 Table 1 에표시하였다. 치자껍질의각용매별추출물을측정한결과, 농도가증가함에따라 NO 2 소거활성이유의적으로증가하였다 (p<0.05). 70% methanol 추출물 34.57±0.21%, 36.20±0.08%, 39.33±0.21%, IC ±0.097 mg/ml, DW 추출물 32.53±0.21%, 35.48±0.08%, 38.47±0.21%, IC ±0.014 mg/ml, EA 추출물 27.59±0.16%, 30.72±0.16%, 32.62±0.31%, IC ±0.077 mg/ml 순으로추출물중에 Fig. 2. Nitrite (NO 2 ) scavenging activity of various solvent extracts from peel of Gardenia jasminoides fructus. 1) The values are means±standard deviation (n=3). Bars with the different letters are significantly different (p<0.05) by Duncan s multiple range tests. 2) EA: ethyl acetate. 3) DW: distilled water. 4) AA: ascorbic acid β-carotene bleaching 을이용한 antioxidant activity 치자껍질의각용매별추출물과 control 인 BHA 에서의각농도별 β-carotene 탈색을이용한항산화활성의결과는 Fig. 3 에나타내었으며, IC 50 은 Table 1 과같다. 치자껍질의용매별추출물의각농도에서측정한결과, 농도가증가함에따라 β-carotene 탈색저해능이유의적으로증가되는것으로관찰되었다 (p<0.05). DW 추출물 41.55±0.20%, 47.10±0.12%, 50.97± 0.34%, IC ±0.002 mg/ml, 70% methanol 추출물에서 23.37±0.09%, 33.86±

7 치자 (Gardenia jasminoides Ellis fructus) 껍질용매별추출물의지질과산화저해및질소산화물소거능 %, 44.80±0.11%, IC ±0.003 mg/ml, EA 추출물은 13.37±0.04%, 20.16±0.04%, 25.24±0.04%, IC ±0.019 mg/ml 로추출물중 EA 용매추출물에서가장낮은탈색저해능과높은 IC 50 결과치가관찰되었다. β -carotene 은일정온도이상에서공기중에방치하게되면산화가진행되어탈색진행되며, flavonoid 나 phenolic 화합물과같은항산화효과가있는물질과함께존재할경우탈색의진행을어느정도억제하게되므로이러한 β-carotene 의탈색정도를이용한항산화능력분석방법이널리이용되고있다 [24] ±0.001, 0.099±0.001, EC ±0.040 mg/ml, EA 추출물 0.026±0.001, 0.070± 0.001, 0.097±0.001, EC ±0.065 mg/ml 순으로 DW 추출물과 EA 추출물 0.6 mg/ml 농도에서의환원력과그에따른 EC 50 에서는유의적인차이가없는것으로관찰되었다 (p<0.05). Senevirathne 등 [25] 은 nitric oxide 소거능의결과와환원력실험의결과는신뢰구간 95% 의유의적인관계가있는것으로보고하였다. 또한 total phenol, flavonoid 함량이높아짐에따라항산화활성이증가한다고알려져있다 [26], 반면본실험에서의 total phenol 함량은 EA 추출물에서가장높게나타났으나질소산화물소거능및환원력은 EA 추출물보다 70% methanol 추출물과 DW 추출물에서우세하게나타났다. 이러한결과는치자의주요생리활성성분중수용성 carotenoid 인 crocin 과본실험에서측정한치자껍질분말의 anthocyanin 및그밖의 flavonoid 성분이 70% methanol 및 DW 용매에추출되어영향을준것으로사료된다. Fig. 3. Antioxidant activity by β-carotene bleaching assay of various solvent extracts from peel of Gardenia jasminoides fructus. 1) The values are means±standard deviation (n=3). Bars with the different letters are significantly different (p<0.05) by Duncan s multiple range tests. 2) EA: ethyl acetate. 3) DW: distilled water. 4) BHA: butylated hydroxyanisole Reducing power 각용매별추출물과 positive control 인 BHA 의환원력을각농도별로비교한결과를 Fig. 4 에나타내었고, EC 50 값을구하여 Table 1 에표시하였다. 치자껍질의각용매별추출물 0.2, 0.4, 0.6 mg/ml 의농도에서환원력을측정한결과, 농도가증가함에따라유의적으로흡광도가증가하였다 (p<0.05). 껍질용매별추출물의환원력은 70% methanol 추출물 0.044±0.001, 0.096±0.001, 0.127±0.001, EC ±0.059 mg/ml, DW 추출물에서 0.033±0.001, Fig. 4. Reducing power of various solvent extracts from peel of Gardenia jasminoides fructus. 1) The values are means±standard deviation (n=3). Bars with the different letters are significantly different (p<0.05) by Duncan s multiple range tests. 2) EA: ethyl acetate. 3) DW: distilled water. 4) BHA: butylated hydroxyanisole Lipid peroxidation inhibition activity 치자껍질의각용매별추출물과 positive control 인 BHA 의지질과산화저해능을각농도별로비교한결과는 Fig. 5 와같으며, IC 50 값은

8 8 진동혁 오다영 정헌식 이영근 성종환 김한수 Table 1 에나타내었다. 70% methanol 추출물에서각농도별로 56.51±0.32%, 63.51±0.16%, 76.21±0.31%, IC ±0.005 mg/ml 으로확인되었으며, EA 추출물 54.59±0.28%, 56.89±0.16%, 63.34±0.28%, IC ± mg/ml, DW 추출물 47.92±0.37%, 54.37±0.26%, 61.11±0.16%, IC ± mg/ml 순으로추출물중 DW 추출물에서유의적으로낮은저해능과높은 IC 50 이관찰되었다 (p<0.05). 지질과산화는자동산화과정 (autoxidation) 중초기반응 (RH R +H ), 연쇄반응 (R +O2 RO2 ), 종결반응 (R +R RR, R +ROO ROOR, ROO +ROO ROOR+O2) 을통해활성이강한 free radical 들이서로중합하여중간생성물인 aldehyde 류, ketone 류, 산등의 carbonyl 화합물을형성하는것으로알려져있으며 [27], 유지의점도증가와체내흡수를어렵게하고필수지방산함량의감소가일어나영양적가치를감소시키는것으로보고되고있다 [28]. 또한 Esterbauer (1993) 는산화된지질을실험동물에게경구투여하였을때죽상동맥경화증위험증가, 간세포, 림프구및여러유전적독성을야기하여산화된지질섭취의위험성을시사하였다 [29]. 본실험결과, 치자껍질의추출물모두에서높은지질과산화저해능 Fig. 5. Lipid peroxidation inhibition activity of various solvent extracts from peel of Gardenia jasminoides fructus. 1) The values are means±standard deviation (n=3). Bars with the different letters are significantly different (p<0.05) by Duncan s multiple range tests. 2) EA: ethyl acetate. 3) DW: distilled water. 4) BHA: butylated hydroxyanisole. 이확인되어지질성분에서의천연산화방지제로서의효과가기대된다. 4. 결론 치자껍질의 anthocyanin 함량을알아보고 70% methanol, ethyl acetate (EA) 및 distilled water (DW) 의 3 가지용매를사용한추출물의용매별 total phenol 함량및질소산화물소거능, 환원력, β-carotene 탈색을이용한항산화력및지질과산화저해능측정을통하여치자의기능성식품소재로서의가치를검토하기위하여본실험을수행하였다. 치자껍질의 anthocyanin 함량을측정한결과 3.519±0.635 mg/100 g DW 로나타났으며, 치자껍질의용매별추출수율은 DW (39.87%), 70% methanol (36.26%), EA (2.88%) 로관찰되었다. 추출용매별항산화활성은농도 (0.2, 0.4, 0.6 mg/ml) 가증가할수록유의적으로증가하였으며 control 로사용된 ascorbic acid, BHA, trolox 보다는낮은활성이확인되었다. 치자껍질의 total phenol 함량은 EA, 70% methanol, DW 추출물순으로 EA 추출물에서 26.59±0.20 CAE mg/g 으로가장높았으며, Nitric oxide (NO) radical 소거능에서는 70% methanol (70.32~76.15%), DW (52.66~59.31%), EA (34.65~46.98%) 추출물순으로나타났다. Nitrite (NO 2 ) 소거능은 70% methanol (34.57~ 39.33%), DW (32.53~38.47%), EA (27.59~ 32.62%) 순으로관찰되었다. β-carotene 탈색저해능은 DW (41.55~50.97%), 70% methanol (23.37~44.80%), EA (13.37~25.24%) 순으로동정되었다. Reducing power (optical density) 는 70% methanol (0.044~0.127), DW (0.033~ 0.099), EA (0.026~0.097) 순으로확인되었다. 지질과산화저해능은껍질추출물중 70% methanol (56.51~76.21%), EA (54.59~63.34%), DW (47.92~61.11%) 순으로관찰되었다 (p<0.05). 이상의결과, 치자껍질의용매별 total phenol 함량순과질소산화물소거능, 환원력, 항산화능, 지질과산화저해능분석에서의결과는일치하지않고지질과산화저해능을제외한모든분석에서 EA 추출물이가장낮은활성을보였다. β-carotene 탈색저해능분석에서는다른분석에서와는다르게 70% methanol 추출물

9 치자 (Gardenia jasminoides Ellis fructus) 껍질용매별추출물의지질과산화저해및질소산화물소거능 9 이 DW 추출물보다낮은결과가관찰되었다. 이는치자껍질의수용성 carotenoid 인 crocin 이 70% methanol 과 DW 용매에추출되어영향을준것으로추정된다. 본실험결과치자의껍질은질소산화물소거능, 항산화능, 지질과산화저해능이우수하여본실험에서측정한 anthocyanin 및 phenolic 성분뿐만아니라여러유익한생리활성물질을가지고있는것으로생각되며, 추출수율과여러생리활성을고려하였을때 70% methanol 과 DW 추출물에서추출하였을때효과가좋을것으로생각되며, 그에따른기능성식품및천연항산화제로서의가치가매우높을것으로사료된다. References 1. J. Kearney, Food consumption trends and drivers, Philosoph. Transact. Royal Soc. B: Biol. Sci., Vol.365, No.1554 pp (2010). 2. K. Menrad, Market and marketing of functional food in Europe, J. Food Eng., Vol.56, No.2 pp (2003). 3. B. Halliwell, J. M. Gutteridge and C. E. Cross, Free radicals, antioxidants, and human disease: where are we now?, J. Lab. Clinic. Med., Vol.119, No.6 pp (1992). 4. N. Hogg, V. M. Darley-Usmar, M. T. Wilson and S. Moncada, Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochemical Journal, Vol.281, No.2 pp (1992). 5. J. S. Hwang, B. H. Lee, X. An, H. R. Jeong, Y. E. Kim, I. Lee, H. Lee and D. O. Kim, Total phenolics, total flavonoids, and antioxidant capacity in the leaves, bulbs, and roots of Allium hookeri, Korean J. Food Sci. Technol., Vol.47, No.2 pp (2015). 6. H. O. Edeoga, D. E. Okwu and B. O. Mbaebie, Phytochemical constituents of some Nigerian medicinal plants, African J. Biotechnol., Vol.4, No.7 pp (2005). 7. I. A. Lee, J. H. Lee, N. I. Baek and D. H. Kim, Antihyperlipidemic effect of crocin isolated from the fructus of Gardenia jasminoides and its metabolite crocetin, Biol. Pharm. Bull., Vol.28, No.11 pp (2005). 8 D. H. Jin, H. S. Kim, J. H. Seong and H. S. Chung, Comparison of total phenol, flavonoid contents, and antioxidant activities of Orostachys japonicus A. Berger extracts, J. Environ. Sci. Int., Vol.25, No.5 pp (2016). 9. T. Fuleki and F. J. Francis, Quantitative methods for anthocyanins, J. Food Sci., Vol.33, No.3 pp (1968). 10. T. Sun and C. T. Ho, Antioxidant activities of buckwheat extracts, Food Chem., Vol.90, No.4 pp (2005). 11. M. N. A. Rao, Nitric oxide scavenging by curcuminoids, J. Pharm. Pharmacol., Vol.49, No.1 pp (1997). 12. J. A. Lim, Y. S. Na and S. H. Baek, Antioxidative activity and nitrite scavenging ability of ethanol extract from Phyllostachys bambusoides, Korean J. Food Sci. Technol., Vol.36, No.2 pp (2004). 13. S. Kato, H. Aoshima, Y. Saitoh and N. Miwa, Highly hydroxylated or γ -cyclodextrin-bicapped water-soluble derivative of fullerene: The antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay, Bioorg. Med. Chem. Lett., Vol.19, No.18 pp (2009). 14. M. Singhal, A. Paul and H. P. Singh, Synthesis and reducing power assay of methyl semicarbazone derivatives, J. Saudi Chem. Soc., Vol.18, No.2 pp (2014). 15. N. Siriwardhana, K. W. Lee, Y. J. Jeon, S. H. Kim and J. W. Haw, Antioxidant activity of Hizikia fusiformis on reactive oxygen species scavenging and lipid peroxidation inhibition, Food Sci

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