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pissn 1229-1153 / eissn 2465-9223 J. Food Hyg. Saf. Vol. 32, No. 5, pp. 371~380 (2017) https://doi.org/10.13103/jfhs.2017.32.5.371 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 유색농산물중안토시아닌과폴리페놀함량비교연구 정일형 * 오문석 전종섭 김한택 홍세라 박광희 윤미혜 경기도보건환경연구원보건연구기획팀 A Comparative Study on Anthocyanin and Polyphenol Contents in Colored Agricultural Products Il-Hyung Jeong*, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon Public Health Research Planning Team, Gyeonggi-do Institute of Health and Environment, Suwon, Korea (Received July 10, 2017/Revised August 3, 2017/Accepted September 21, 2017) ABSTRACT - In this study, 17 kinds of polyphenols and 5 kinds of anthocyanins were analyzed to compare the contents of polyphenols and anthocyanins in 76 colored agricultural products. A total of 17 polyphenols were analyzed simultaneously by 9 phenolic acids (gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, t-ferulic acid, t-cinnamic acid) and 8 flavonoids ((+)catechin, syringic aldehyde, rutin, epicatechin gallate, naringin, luteolin, naringenin, kaempferol) and 5 anthocyanins (delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside) were simultaneously analyzed. The total content of 17 polyphenols was determined as seoritae 255.1 ± 7.5 µg/g, seomoktae 275.8 ± 5.3 µg/g, black rice 78.5 ± 4.6 µg/g, black sesame 75.8 ± 3.2 µg/g, blueberry 143.3 ± 5.5 µg/g, aronia 195.2 ± 4.9 µg/g and blackcurrent 131.6 ± 3.2 µg/g, the highest content was found in the order of seomoktae > seoritae > aronia > blueberry > blackcurrant > black rice > black sesame. The total content of 5 anthocyanins was determined as seoritae 82.4 ± 17.2 µg/g, seomoktae 95.2 ± 6.1 µg/g, black rice 74.1 ± 9.7 µg/g, black sesame were not detected, blueberry 110.8 ± 1.9 µg/g, aronia 218.9 ± 6.1 µg/g and blackcurrent 209.7 ± 4.0 µg/g, the highest content was found in the order of aronia > blackcurrant > blueberry > seomoktae > seoritae > black rice. These results indicated that seomoktae and aronia possessed the high level of functional components and further study will be needed to develop high value-added foods based on the colored agricultural products. Key words : Colored Agricultural Products, Polyphenols, Anthocyanins 건강 100세시대를맞이하여컬러푸드 (color food) 가인기를끌고있으며, 조화로운식생활과건강한삶을유지하는데큰도움을주는건강식품으로노화를예방하는검정색식품, 면역력과항암효과를높이는주황색식품, 혈관과위장을깨끗하게하는초록색식품, 콜레스테롤을낮추는하얀색식품, 심장병예방과독소를제거하는보라색식품, 피부가좋아지는노란색식품, 항암효과와혈관을튼튼하게하는빨간색식품등이있다 1). 컬러푸드의빨강, 노랑, 초록, 보라, 검정, 흰색등의색깔은식품의파이토케미컬 (phytochemical) 이라는성분에의한것이며, 식 *Correspondence to: Il-Hyung Jeong, Public Health Research Planning Team Gyeonggi-do Institute of Health and Environment 95, Pajangcheon-ro, Jangan-gu, Suwon-si, Gyeonggi-do 16025, Korea Tel: 82-31-250-2571, Fax: 82-31-250-2606 E-mail: jih1010@gg.go.kr 품의색뿐만이아니라식품고유의독특한맛과향을부여하며, 항산화작용이나면역기능증가등을통해건강에이로운역할을하는자연발생의생리활성을가지는물질이다 2-3). 파이토케미컬은 carotenoids, phenolics, alkaloid, nitrogen-containing compounds, organosulfur compounds로분류된다 4). 그중에서도폴리페놀은녹색식물의광합성작용에의해생성된당의일부가변화한 2차대사산물로벤젠고리 (C 6 H 6 ) 의수소중하나가수산기 (OH ) 로치환된물질을페놀이라하고 2개이상갖고있는물질을폴리페놀또는다가페놀이라고한다 5). 폴리페놀은산화방지와종양세포억제, 자유라디칼소거역할을하며 6), 혈소판응집억제, 심혈관질환예방, 항암효과, 알츠하이머병에의한뇌손상예방역할을한다 7). 또한안토시아닌은식물계에널리분포되어있는페놀화합물중하나로열매, 꽃, 과실, 줄기, 잎, 뿌리등식물체각부위에폭넓게분포되어있 371

372 Il-Hyung Jeong, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon 는적색, 자색, 청색등의색을나타내는수용성 flavonoid 계색소이다 8). 안토시아니딘과당이결합된배당체를안토시아닌이라고하며, 자연계에다양한색깔로존재하고있으며, 항산화및항염활성뿐만아니라특정암세포에대하여세포증식억제, 세포주기정지, 세포사멸유도등의메커니즘을통해강력한항암효과를나타내는것으로알려져있다 9). 이처럼파이토케미컬에관한연구는성인병예방과관련된항암효과 10), 심혈관질환예방 11), 면역증진 12), 노화지연 13) 등건강증진효과와관련하여작용기작이나구조활성관계를중심으로연구가수행되어왔으며, 최근에는 ESI (electrospray ionization) 를기반으로질량분석기술을적극활용하여분석하고있으나 14-15), 대부분의연구가정성적특성평가에국한되어있으며 16-17), 여러개별성분을동시에분석할수있는정량분석의기본이되는분석조건에관한연구는아직미흡한실정이다. 따라서, 본연구에서는인터넷쇼핑몰에서유통되는유색농산물 76건을대상으로폴리페놀 17종및안토시아닌 5종을동일한전처리방법으로동시분석하여개별성분함량을비교조사하여이를이용한다양한기능성식품개발의기초자료를제공하고자하였다. Materials and Methods 실험재료본실험에서는인터넷쇼핑몰에서유통되는콩류 ( 서리태 10건, 서목태 10건 ) 20건, 곡류 ( 흑미 ) 20건, 유지종실류 ( 흑임자 ) 10건, 장과류 ( 블루베리 14건, 아로니아 7건, 블랙커런트 5건 ) 26건으로총 76건을구입하여시료로사용하였다. 표준물질및시약폴리페놀표준물질로는 gallic acid, protocatechuic acid, chlorogenic acid, (+)catechin, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, t-ferulic acid, t-cinnamic acid, syringic aldehyde, naringin, naringenin, kaempferol 등은 Sigma-Aldrich (St. Louis, MO, USA) 사의제품을사용하였고, rutin, epicatechin gallate, luteolin 등은 Wako Pure Chemical Industries (Osaka, Japan) 사의제품을사용하였다. 안토시아닌표준물질로는 delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-galactoside, cyanidin-3- glucoside, cyanidin-3-arabinoside 등은 Sigma-Aldrich (St. Louis, MO, USA) 사의제품을사용하였다. methanol과 acetonitrile의경우 Burdick & Jackson (Muskegon, MI, USA) 사의제품이며, trifluoroacetic acid와 acetic acid는 Sigma-Aldrich (St. Louis, MO, USA) 사의제품을사용하였고, potassium ferrocyanide trihydrate 및 zinc acetate dihydrate는 Daejung Chemicals & Metals (Siheung, Republic of Korea) 사의제품을사용하였다. water는 Thermo Scientific Barnstead NANO Pure Diamond (Reverse Osmosis, Model D126611/D11911, Dubuque, Iowa, USA) 로제조한 3차 water (> 18.2 MΩ cm resistivity) 를사용하였다. 장비 HPLC 분석은 Agilent사의 1100 series (Agilent Technologies, Boeblingen, Germany) 를사용하였고, HPLC-MS/MS 는 SPLC (Shiseido, Tokyo, Japan) 와 TSQ Quantum Ultra (Thermo Scientific, San Jose, CA, USA) 로구성되었으며, 원심분리기 (Hanil Scientific, Combi-514R, Gimpo, Republic of Korea) 와질소농축기 (Biotage, TurboVap-LV, Uppsala, Sweden) 를사용하였다. 실험방법 시료전처리 시료약 5g에 1% acetic acid in 50% methanol 50 ml 를첨가한후상온상태에서 24시간추출한후, Whatman No. 1 여과지 (Toyo Ltd., Tokyo, Japan) 로여과하여시료추출액으로사용하였다. 시료의단백질등간섭물질제거를위해다른연구에서사용된전처리법을변형하여추출액에카레스 (carrez) 침전제제1액 (15% 페로시안화칼륨용액 ) 1 ml를가해흔들어섞은다음다시제2액 (30% 초산아연용액 ) 1 ml를가해흔들어섞고, 원심분리 (3500 rpm, 10분, 4 o C) 하여상층액 2mL을취하였다 18-19). Sep-pak C18 cartrdge (Waters, Milford, MA, USA) 를 methanol 5 ml, H 2 O 5 ml의순으로흘려주어활성화시킨다음, 상층액 2 ml을 loading 후, 5 ml 1% acetic acid in 50% methanol 로 2회용출한후, 45 o C에서 N 2 가스로농축한다음, 50% methanol 2 ml로용해하고 Membrane syringe filter (PTFE 25 mm, 0.45 μm, Advantec, Toyo Ltd., Tokyo, Japan) 로여과하여시험용액으로사용하였다. 표준용액조제 각표준물질의표준원액 (stock standard) 은약 100 μg/ml 의농도가되도록 methanol에녹여조제하여냉장보관하여사용하였다. 혼합표준용액 (working standard mix) 는표준원액 (stock standard) 을 50% methanol로적정한농도로희석하여사용하였다. 폴리페놀정량 분석대상폴리페놀은 gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, t-ferulic acid, t-cinnamic acid 등 9종의 phenolic acid와 (+)catechin, syringic aldehyde, rutin, epicatechin gallate, naringin, luteolin, naringenin, kaempferol

A Comparative Study on Anthocyanin and Polyphenol Contents 373 Table 1. Conditions of polyphenol analysis by HPLC Parameter Condition Instrument Agilent 1100 series Column Column temp. Wavelength Mobile phase 등 8종의 flavonoid로총 17종의폴리페놀을서등 20) 의방법을변형하여동시분석하였고, HPLC 분석조건은 Table 1과같다. 안토시아닌정량 Zorbax Eclipse plus C18 (4.6 250 mm, 5 µm) 30 o C VWD Detector 280 nm A : 0.1% acetic acid B : 0.1% acetic acid in acetonitrile Time (min) A B 0.0 95 5 5.0 90 10 20.0 87 13 Gradient table 25.0 85 15 35.0 75 25 40.0 70 30 50.0 65 35 60.0 95 5 Flow rate 1.0 ml/min Injection volumn 10 µl Table 2. Conditions of anthocyanin analysis by HPLC Parameter Condition Instrument Agilent 1100 series Column Column temp. Wavelength Mobile phase YMC-Pack ODS-AM C18 (4.6 250 mm, 5 µm) 30 o C VWD Detector 530 nm A : 1% TFA in H 2 O B : acetonitrile Time (min) A B 0.0 89 11 Gradient table 25.0 85 15 30.0 85 15 40.0 89 11 Flow rate 1.0 ml/min Injection volumn 10 µl 분석대상안토시아닌은 delphinidin-3-glucoside, delphinidin- 3-rutinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside 등 5종을정 21) 의방법을변형하여동시분석하였고, HPLC 분석조건은 Table 2와같다. Table 3. Operation conditions of HPLC-MS/MS Parameter Condition Instrument SPLC/TSQ Quantum ultra HPLC-MS/MS 확인폴리페놀 17종및안토시아닌 5종의확인시험은 selected reaction monitoring (SRM) mode를이용하였고, 이온화는 electrospray ionization (ESI) 방식으로하였으며, 각성분의표준용액 (1 μg/ml) 을개별적으로질량분석기에직접주입하여 ESI Positive & Negative mode에서의이온화조건을검토하였고, 각성분의이온화된 parent ion과 product ion으로의이행조건을검토하여 product ion에대한최적의 collision energy를선정하였으며, 검액은정량시험용으로조제된시료를 50% methanol로 10배희석하여사용하였다. HPLC-MS/MS 분석조건은 Table 3에나타내었고, 다른연구에서사용된시험방법을응용하였다 22-23). 시험법의유효성검토 직선성 Column Column temp. Detector Mobile phase Thermo Hypersil gold C18 (2.0 150 mm, 3 µm) 40 o C MS/MS A : 0.1% acetic acid B : 0.1% acetic acid in acetonitrile Time (min) A B 0.0 95 5 2.0 90 10 Gradient table 10.0 50 50 15.0 50 50 18.0 90 10 20.0 95 5 Flow rate 0.3 ml/min Injection volume 5 µl Ionization source ESI + or ESI Spray voltage Capillary temp. Vaporizer temp. Sheath gas Aux gas Collision gas 4000V 3000V 330 o C 300 o C 40.0 psi 20.0 psi 1.5 mtorr Results and Discussion 직선성을평가하기위하여 5단계로희석한혼합표준용액을 HPLC로 5회반복측정하여검량선을구해 1차회

374 Il-Hyung Jeong, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon Table 4. Regression, R 2, LOD, LOQ and linear range for the studied compounds Compounds Regression equation R 2 LOD (µg/ml) LOQ (µg/ml) Linear range (µg/ml) Gallic acid y = 28.293x 3.9376 0.9997 0.0920 0.2789 0.6~10.0 Protocatechuic acid y = 15.676x + 0.9054 0.9999 0.0604 0.1831 0.6~10.0 Chlorogenic acid y = 16.787x 1.1093 0.9992 0.0831 0.2518 0.6~10.0 (+) Catechin y = 13.763x + 0.1070 0.9998 0.0762 0.2311 0.6~10.0 Vanillic acid y = 16.702x + 0.6885 0.9999 0.0223 0.0675 0.6~10.0 Caffeic acid y = 32.967x 0.7672 0.9998 0.0870 0.2636 0.6~10.0 Syringic acid y = 29.677x + 2.3212 0.9999 0.0766 0.2321 0.6~10.0 p-coumaric acid y = 32.828x 2.7234 0.9992 0.0472 0.1431 0.6~10.0 Syringic aldehyde y = 20.183x 0.8285 0.9998 0.0106 0.0322 0.6~10.0 t-ferulic acid y = 30.863x 2.8988 0.9995 0.1030 0.3124 0.6~10.0 Rutin y = 15.321x 2.5780 0.9991 0.0382 0.1158 0.6~10.0 Epicatechin gallate y = 18.067x 0.0287 0.9999 0.0367 0.1112 0.6~10.0 Naringin y = 17.943x + 0.5086 0.9999 0.0142 0.0432 0.6~10.0 Luteolin y = 17.820x 0.4748 0.9999 0.0246 0.0746 0.6~10.0 t-cinnamic acid y = 37.987x 1.2039 0.9998 0.0552 0.1673 0.6~10.0 Naringenin y = 32.487x 0.3756 0.9999 0.0371 0.1125 0.6~10.0 Kaempferol y = 15.284x + 0.1123 0.9999 0.0476 0.1445 0.6~10.0 Delphinidin-3-glucoside y = 20.669x 1.4986 0.9996 0.0975 0.2955 0.3~5.0 Delphinidin-3-rutinoside y = 18.428x 1.7369 0.9998 0.0450 0.1366 0.3~5.0 Cyanidin-3-galactoside y = 16.248x 0.0252 0.9994 0.0739 0.2240 0.3~5.0 Cyanidin-3-glucoside y = 30.222x + 1.5264 0.9997 0.0836 0.2534 0.3~5.0 Cyanidin-3-arabinoside y = 29.010x 0.9881 0.9998 0.0911 0.2761 0.3~5.0 귀방정식 (y = Sx + b) 으로결정계수 (determination coefficient, R 2 ) 를구하고직선성 (Linearity) 을검토하였다. 그결과폴리페놀 17종의결정계수 (R 2 ) 값은 0.9991~0.9999, 안토시아닌 5종의결정계수 (R 2 ) 값은 0.9994~0.9998로 1과거의유사한값이므로검량선범위내에있는각각의농도에대하여직선적인측정값을나타냄을알수있었고, Table 4에나타내었다. 정확성과정밀성 서리태, 흑미, 흑임자, 블루베리를유형별대표식품으로선정하여폴리페놀및안토시아닌의혼합표준용액을첨가한후혼합표준용액을첨가하지않은시료와동시에전처리한후혼합표준용액첨가시료와비첨가시료의폴리페놀및안토시아닌함량을 HPLC로구한후그차를회수율로이용하였고, 이과정을 3회반복하여정밀성을확인하였다. 그결과폴리페놀 17종의회수율은서리태 84.5~ 105.2%, 흑미 83.7~98.6%, 흑임자 85.1~102.5%, 블루베리 88.5~106.1% 사이에서확인되었고, 안토시아닌 5종의회수율은서리태 93.2~102.4%, 흑미 91.6~99.5%, 흑임자 90.8~97.3%, 블루베리 94.9~101.3% 이었다. 이는 AOAC (Association of official analytical chemists) 에서제시하는 회수율적정범위 75~120% 를만족한다 24). 또한상대표준편차 (Relative Standard Deviation) 는폴리페놀 17종은서리태 1.2~4.1%, 흑미 1.4~3.9%, 흑임자 1.3~4.5%, 블루베리 1.5~3.6% 사이에서확인되었고, 안토시아닌 5종은서리태 1.4~3.3%, 흑미 1.6~3.5%, 흑임자 1.5~3.9%, 블루베리 1.3~ 3.4% 로나타나 AOAC에서제시하는 8% 미만으로서 24), 정밀성이양호한시험법임을확인할수있었고, Table 5에나타내었다. 검출한계및정량한계 검출한계 (Limit of Detection, LOD) 와정량한계 (Limit of Quantitation, LOQ) 는직선성시험에서구한 1차회귀방정식으로검량선의기울기 (S) 를구하고반응의표준편차 (σ) 를이용하여검출한계는신호대잡음비 (S/N비) 의 3.3배, 정량한계는 10배의값으로 ICH (International Council for Harmonisation) 25) 에서제시한아래의식으로계산하였다. LOD = 3.3 σ/s LOQ = 10 σ/s σ = the standard deviation of the response S = the slope of the calibration curve

A Comparative Study on Anthocyanin and Polyphenol Contents 375 Table 5. Recovery of the studied compounds in colored agricultural products Compounds Concentration (µg/ml) Recovery Seoritae Black rice Black sesame Blueberry RSD 1) Recovery RSD Recovery RSD Recovery Gallic acid 5.1 98.5 1.2 92.1 1.5 94.6 1.8 98.0 1.5 Protocatechuic acid 5.1 97.7 1.7 95.4 1.4 102.5 1.3 96.4 1.6 Chlorogenic acid 4.7 91.6 2.8 90.5 2.5 95.8 2.1 106.1 2.6 (+) Catechin 4.6 92.4 3.2 89.6 2.6 91.3 3.4 96.5 3.1 Vanillic acid 5.1 89.3 3.0 96.2 2.6 98.7 2.8 92.5 2.2 Caffeic acid 5.1 88.2 2.9 89.7 2.4 97.9 2.2 91.1 2.6 Syringic acid 5.0 95.8 2.5 98.6 1.9 96.3 2.6 102.4 2.8 p-coumaric acid 5.2 93.6 3.3 90.5 3.9 92.5 3.2 95.8 3.5 Syringic aldehyde 5.2 89.2 2.6 92.8 2.2 87.4 3.0 88.5 2.8 t-ferulic acid 5.1 97.5 1.6 91.1 2.7 95.2 2.2 98.8 1.8 Rutin 5.0 88.9 3.4 89.2 2.5 98.1 2.4 94.7 2.1 Epicatechin gallate 5.3 93.8 3.5 83.7 3.0 94.9 3.6 96.3 3.3 Naringin 5.3 101.3 1.9 95.2 2.3 99.7 2.0 98.1 2.3 Luteolin 5.1 95.4 2.4 86.5 2.7 89.2 3.2 92.3 2.5 t-cinnamic acid 5.0 88.2 4.1 90.3 3.4 85.1 4.5 91.1 3.6 Naringenin 5.1 105.2 2.5 95.5 2.2 97.6 2.8 98.4 1.8 Kaempferol 5.1 84.5 2.6 86.1 2.8 88.7 2.0 90.2 2.5 Delphinidin-3-glucoside 5.1 98.2 1.7 94.7 2.0 98.6 2.1 98.9 1.3 Delphinidin-3-rutinoside 4.9 93.2 3.3 91.6 3.5 92.5 3.9 96.7 3.4 Cyanidin-3-galactoside 5.2 94.6 2.5 95.2 1.7 90.8 2.3 94.9 2.1 Cyanidin-3-glucoside 5.1 102.4 1.4 99.5 1.6 97.3 1.5 101.3 1.4 Cyanidin-3-arabinoside 5.0 96.1 1.9 97.2 1.6 93.9 2.2 99.5 1.8 1) RSD : Relative Standard Deviation RSD 그결과폴리페놀 17종의검출한계 (LOD) 는 0.0106~ 0.0920 μg/ml, 정량한계 (LOQ) 는 0.0322~0.3124 μg/ml이었고, 안토시아닌 5종의검출한계 (LOD) 는 0.0450~0.0975 μg/ml, 정량한계 (LOQ) 는 0.1366~0.2955 μg/ml로각성분별분석농도보다낮은정량한계를나타냄으로서정량이가능한수준을보여주었으며, Table 4에나타냈다. 폴리페놀의정량결과 폴리페놀 17종에대해정량분석한결과는 Table 6과같이나타났으며, 표준물질 17종에대한크로마토그램은 Fig. 1와같다. 서리태는 6종의폴리페놀이검출되었고, 이들중 p- coumaric acid 함량이 105.0 ± 18.3 μg/g으로가장높았으며, protocatechuic acid 95.1 ± 16.9 μg/g, naringenin 20.0 ± 9.6 μg/g, gallic acid 17.3 ± 3.4 μg/g, syringic acid 9.0 ± 2.3 μg/g, epicatechin gallate 8.7 ± 1.5 μg/g 순으로나타났고, 서목태는 7종의폴리페놀이검출되었고, 이들중 protocatechuic acid 함량이 93.8 ± 12.3 μg/g으로가장높았으며, (+)catechin 76.3 ± 16.2 μg/g, p-coumaric acid 42.8 ± 11.6 μg/g, naringenin 25.2 ± 9.4 μg/g, gallic acid 14.7 ± 2.7 μg/ g, epicatechin gallate 11.8 ± 2.3 μg/g, syringic acid 11.2 ± 4.8 μg/g 순으로검출되었다. 흑미는 protocatechuic acid 42.9 ± 13.9 μg/g, syringic acid 23.3 ± 8.8 μg/g, vanillic acid 12.3 ± 4.8 μg/g 순으로 3종의폴리페놀이검출되었다. 흑임자는 protocatechuic acid 31.8 ± 8.3 μg/g, chlorogenic acid 18.5 ± 4.7 μg/g, naringin 9.3 ± 1.6 μg/g, t-cinnamic acid 8.3 ±1.3μg/g, gallic acid 7.9 ± 0.9 μg/g 순으로 5종의폴리페놀이검출되었다. 블루베리는 6종의폴리페놀이검출되었고, 이들중 chlorogenic acid 함량이 62.2 ± 15.6 μg/g으로가장높았으며, epicatechin gallate 40.8 ± 11.6 μg/g, syringic acid 16.7 ± 6.3 μg/g, naringin 9.5 ± 3.6 μg/g, (+)catechin 8.0 ± 2.5 μg/g, gallic acid 6.1 ± 1.9 μg/g 순으로나타났으며, 아로니아는 protocatechuic acid 87.0 ± 14.8 μg/g, chlorogenic acid 75.2 ± 12.5 μg/g, (+)catechin 21.1 ± 7.5 μg/g, syringic acid 11.9 ± 3.9 μg/g 순으로 4종의폴리페놀이검출되었다. 블랙커런트는 protocatechuic acid 55.3 ±

376 Il-Hyung Jeong, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon Table 6. Contents of 17 polyphenol compounds in colored agricultural products Concentration, Mean ± S.D. 1) (µg/g) Compounds Seoritae Seomoktae Black rice (n = 20) Black sesame Blueberry (n = 14) Aronia (n = 7) Blackcurrent (n = 5) Gallic acid 17.3 ± 3.4 14.7 ± 2.7 N.D. 07.9 ± 0.9 06.1 ± 1.9 N.D. N.D. Protocatechuic acid 95.1 ± 16.9 93.8 ± 12.3 42.9 ± 13.9 31.8 ± 8.3 N.D. 87.0 ± 14.8 55.3 ± 13.3 Chlorogenic acid N.D. 2) N.D. N.D. 18.5 ± 4.7 62.2 ± 15.6 75.2 ± 12.5 N.D. (+) Catechin N.D. 76.3 ± 16.2 N.D. N.D. 08.0 ± 2.5 21.1 ± 7.5 N.D. Vanillic acid N.D. N.D. 12.3 ± 4.8 N.D. N.D. N.D. 17.4 ± 5.5 Caffeic acid N.D. N.D. N.D. N.D. N.D. N.D. N.D. Syringic acid 09.0 ± 2.3 11.2 ± 4.8 23.3 ± 8.8 N.D. 16.7 ± 6.3 11.9 ± 3.9 N.D. p-coumaric acid 105.0 ± 18.3 42.8 ± 11.6 N.D. N.D. N.D. N.D. N.D. Syringic aldehyde N.D. N.D. N.D. N.D. N.D. N.D. N.D. t-ferulic acid N.D. N.D. N.D. N.D. N.D. N.D. N.D. Rutin N.D. N.D. N.D. N.D. N.D. N.D. 27.5 ± 8.7 Epicatechin gallate 08.7 ± 1.5 11.8 ± 2.3 N.D. N.D. 40.8 ± 11.6 N.D. 31.4 ± 8.8 Naringin N.D. N.D. N.D. 09.3 ± 1.6 09.5 ± 3.6 N.D. N.D. Luteolin N.D. N.D. N.D. N.D. N.D. N.D. N.D. t-cinnamic acid N.D. N.D. N.D. 08.3 ± 1.3 N.D. N.D. N.D. Naringenin 20.0 ± 9.6 25.2 ± 9.4 N.D. N.D. N.D. N.D. N.D. Kaempferol N.D. N.D. N.D. N.D. N.D. N.D. N.D. Total 255.1 ± 7.5 275.8 ± 5.3 78.5 ± 4.6 75.8 ± 3.2 143.3 ± 5.5 195.2 ± 4.9 131.6 ± 3.2 1) S.D.: Standard Deviation 2) N.D.: Not Detected Fig. 1. Chromatogram of 17 polyphenol standards by HPLC. 13.3 μg/g, epicatechin gallate 31.4 ± 8.8 μg/g, rutin 27.5 ± 8.7 μg/g, vanillic acid 17.4 ± 5.5 μg/g 순으로 4종의폴리페놀이검출되었고, 분석한 17종의폴리페놀성분중서리태 6종, 서목태 7종, 흑미 3종, 흑임자 5종, 블루베리 6 종, 아로니아 4종, 블랙커런트 4종의개별성분을정량하였다. 서리태, 서목태, 흑임자, 블루베리, 아로니아, 블랙커런트는 phenolic acid, flavonoid 계열을함유하고있었지만, 흑미는 phenolic acid 계열만을함유하고있었다. 페놀성물질의정량방법으로 Folin-Ciocalteu 시약을이용하는 Folin-Denis 방법 26) 이실험자들에게있어서가장널리이용되고있다. 이방법은간단하면서도비교적감도가높 고, 페놀성물질들의중합도에관계없이정량성을나타낸다는점에서유용성을가지고있다 27). 하지만 Folin-Ciocalteu 시약과반응할수있는다양한종류의화합물들로인해시료중의페놀성물질정량에있어서간섭요인들이발생되어 28-29), 대략적인폴리페놀의함량을측정하는방법으로이용되고있다 30). 폴리페놀 17종의총함량은서리태 255.1 ± 7.5μg/g, 서목태 275.8 ± 5.3 μg/g, 흑미 78.5 ± 4.6 μg/g, 흑임자 75.8 ± 3.2 μg/g, 블루베리 143.3 ± 5.5 μg/g, 아로니아 195.2 ± 4.9 μg/g, 블랙커런트 131.6 ± 3.2 μg/g로나타나서목태 > 서리태 > 아로니아 > 블루베리 > 블랙커런트 > 흑미 > 흑임자순으로높은함량을나타내었다. 페놀성화합물은식물계에널리분포되어있는물질로다양한구조와분자량을가지며페놀성화합물의 phenolic hydroxyl기가단백질과같은거대분자와의결합을통해항산화, 항암및항균등의생리기능을가지는것으로알려져있다 31). 안토시아닌의정량결과 안토시아닌 5종에대해정량분석한결과 Table 7과같이나타났으며, 표준물질 5종에대한크로마토그램은 Fig. 2와같다. 서리태는 cyanidin-3-glucoside 63.9 ± 15.3 μg/g, delphinidin-

A Comparative Study on Anthocyanin and Polyphenol Contents 377 Table 7. Contents of 5 anthocyanin compounds in colored agricultural products Concentration, Mean ± S.D. 1) (µg/g) Compounds Seoritae Seomoktae Black rice (n = 20) Black sesame Blueberry (n = 14) Aronia (n = 7) Blackcurrent (n = 5) Delphinidin-3-glucoside 12.8 ± 6.1 18.6 ± 7.4 N.D. N.D. 24.2 ± 5.7 N.D. 039.5 ± 6.3 Delphinidin-3-rutinoside N.D. 2) N.D. 02.0 ± 1.2 N.D. N.D. N.D. 158.3 ± 11.2 Cyanidin-3-galactoside 01.0 ± 0.4 01.2 ± 0.5 06.1 ± 1.6 N.D. 28.7 ± 9.5 169.6 ± 15.9 N.D. Cyanidin-3-glucoside 63.9 ± 15.3 69.5 ± 14.3 61.9 ± 20.6 N.D. 31.4 ± 9.2 006.5 ± 3.8 011.9 ± 3.4 Cyanidin-3-arabinoside 04.7 ± 2.0 05.9 ± 3.0 04.1 ± 1.0 N.D. 26.5 ± 6.3 042.8 ± 10.7 N.D. Total 82.4 ± 17.2 95.2 ± 6.1 74.1 ± 9.7 N.D. 110.8 ± 1.9 218.9 ± 6.1 209.7 ± 4.0 1) S.D.: Standard Deviation 2) N.D.: Not Detected 등을함유하고있다고보고하였다. 안토시아닌 5종의총함량은서리태 82.4 ± 17.2 μg/g, 서목태 95.2 ± 6.1 μg/g, 흑미 74.1 ± 9.7 μg/g, 흑임자는불검출, 블루베리 110.8 ± 1.9 μg/g, 아로니아 218.9 ± 6.1 μg/g, 블랙커런트 209.7 ± 4.0 μg/ g로나타나아로니아 > 블랙커런트 > 블루베리 > 서목태 > 서리태 > 흑미순으로높은함량을나타내었다. Fig. 2. Chromatogram of 5 anthocyanin standards by HPLC. 3-glucoside 12.8 ± 6.1 μg/g, cyanidin-3-arabinoside 4.7 ± 2.0 μg/g, cyanidin-3-galactoside 1.0 ± 0.4 μg/g 순으로나타났으며, 서목태는 cyanidin-3-glucoside 69.5 ± 14.3 μg/g, delphinidin-3-glucoside 18.6 ± 7.4 μg/g, cyanidin-3-arabinoside 5.9 ± 3.0μg/g, cyanidin-3-galactoside 1.2 ± 0.5 μg/g 순으로나타났다. 흑미는 cyanidin-3-glucoside 61.9 ± 20.6 μg/g, cyanidin-3-galactoside 6.1 ± 1.6 μg/g, cyanidin-3-arabinoside 4.1 ± 1.0μg/g, delphinidin-3-rutinoside 2.0 ± 1.2 μg/g 순으로나타났으며, 흑임자는안토시아닌 5종이불검출되었다. 블루베리는 cyanidin-3-glucoside 31.4 ± 9.2 μg/g, cyanidin- 3-galactoside 28.7 ± 9.5 μg/g, cyanidin-3-arabinoside 26.5 ± 6.3 μg/g, delphinidin-3-glucoside 24.2 ± 5.7 μg/g 순으로나타났고, 아로니아는 cyanidin-3-galactoside 169.6 ± 15.9 μg/ g, cyanidin-3-arabinoside 42.8 ± 10.7 μg/g, cyanidin-3-glucoside 6.5 ± 3.8 μg/g 순으로나타났으며, 블랙커런트는 delphinidin-3-rutinoside 158.3 ± 11.2 μg/g, delphinidin-3-glucoside 39.5 ± 6.3 μg/g, cyanidin-3-glucoside 11.9 ± 3.4 μg/g 순으로검출되었고, 5종의안토시아닌성분중서리태 4 종, 서목태 4종, 흑미 4종, 흑임자 10종, 블루베리 4종, 아로니아 3종, 블랙커런트 3종의개별성분을정량하였다. 5 종의안토시아닌성분은흑임자만함유하지않았고, 서리태, 서목태, 흑미, 블루베리, 아로니아, 블랙커런트는폴리페놀성분과함께함유하고있었다. 방등 32) 의연구에서도블루베리는안토시아닌, 플라보노이드, 각종페놀화합물 HPLC-MS/MS의확인결과분석의신뢰도를향상시키고자 HPLC-MS/MS를이용하여확인시험을실시하였다. 분석대상의성분명, 분자량, parent 이온분자량, product 이온분자량, 충돌에너지 (CE, collision energy), polarity 등질량분석조건은 Table 8에나타내었다. 각성분의 product ion은 1μg/mL 표준용액을개별적으로질량분석기에직접주입하여, collision energy (CE) 를가하여 parent ion이 product ion으로깨어지도록하고 collison energy 값을미세하게조절하여정량이온과정성이온별로가장좋은 collision energy 값을결정하였고, 이동상과함께표준용액을주입하면서 MS parameter 의조건을최적화하였다. 폴리페놀 17종에대한 SRM chromatogram은 Fig. 3과같다. 그중 gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, t-ferulic acid, t-cinnamic acid, (+)catechin, rutin, epicatechin gallate, naringin, luteolin, naringenin, kaempferol이 ESI mode에서높은감도를나타내었고, ESI mode에서는 syringic aldehyde 가높은감도를나타내었다. 다른논문에서는페놀화합물분석시 ESI 방식의경우 mode보다 mode에서감도가더좋다고보고되어있다 33-34). 본연구에서는표준용액의검출조건을결정할때, mode를사용하여비교해본결과대체로 mode에서더높은감도를보였으나, syringic aldehyde만 ESI mode에서더높은감도를보였다. 안토시아닌 5 종에대한 SRM chromatogram은 Fig. 4에나타냈고 delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-

378 Il-Hyung Jeong, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon Table 8. SRM parameters of the studied compounds by HPLC- MS/MS Compounds MW 1) Parent ion Product ion CE 2) (V) Polarity Gallic acid 170 169 125 17 79 25 Protocatechuic acid 154 153 109 17 81 18 Chlorogenic acid 354 353 191 24 85 46 (+) Catechin 290 289 245 16 203 20 Fig. 3. SRM chromatogram of 17 polyphenol standards. Vanillic acid 168 167 152 17 108 20 Caffeic acid 180 179 135 19 134 28 Syringic acid 198 197 123 26 95 33 p-coumaric acid 164 163 119 17 93 37 Syringic aldehyde 182 183 155 7 77 23 t-ferulic acid 194 193 134 19 178 16 Rutin 610 609 300 38 271 58 Epicatechin gallate 442 441 289 19 125 43 Naringin 580 579 271 34 151 44 Luteolin 286 285 133 39 151 27 t-cinnamic acid 148 147 62 15 103 17 Naringenin 272 271 Kaempferol 286 285 119 32 151 21 93 40 117 47 Fig. 4. SRM chromatogram of 5 anthocyanin standards. Delphinidin-3- glucoside Delphinidin-3- rutinoside Cyanidin-3- galactoside Cyanidin-3- glucoside Cyanidin-3- arabinoside 1) MW : Molecular Weight 2) CE : Collision Energy 465 465 611 611 449 449 449 449 419 419 303 24 229 52 303 29 465 18 287 24 137 54 287 25 137 50 287 22 137 50 3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside 등은 ESI mode에서높은감도를나타내었다. 김등 35) 의연구에서도안토시아닌정성및정량 MS 분석시 electrospray ionization (ESI) source를이용하여 ionization mode로진행하였다. HPLC-MS/MS로 ESI 와 mode에서의이온화조건은폴리페놀 17종중 16종은 ESI mode, 1종은 ESI mode, 안토시아닌 5종은 ESI mode에서높은감도를확인할수있었다. 본실험결과폴리페놀은서목태, 안토시아닌은아로니아가가장높은함량을나타내었으며향후유색농산물을활용한기능식품개발이필요한것으로판 단된다.

A Comparative Study on Anthocyanin and Polyphenol Contents 379 국문요약 본연구에서는인터넷쇼핑몰에서유통되는컬러푸드중블랙푸드 76건을대상으로폴리페놀 17종및안토시아닌 5종을동일한전처리방법으로 HPLC, HPLC-MS/MS 분석하여정량및정성시험을실시하였다. 분석대상폴리페놀은 gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, t-ferulic acid, t-cinnamic acid 등 9종의 phenolic acid와 (+)catechin, syringic aldehyde, rutin, epicatechin gallate, naringin, luteolin, naringenin, kaempferol 등 8종의 flavonoid 로총 17종의폴리페놀을동시분석하였고, 안토시아닌은 delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin- 3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside 등 5종을동시분석하였다. 폴리페놀 17종의총함량은서 리태 255.1 ± 7.5 μg/g, 서목태 275.8 ± 5.3 μg/g, 흑미 78.5 ± 4.6 μg/g, 흑임자 75.8 ± 3.2 μg/g, 블루베리 143.3 ± 5.5 μg/ g, 아로니아 195.2 ± 4.9 μg/g, 블랙커런트 131.6 ± 3.2 μg/g 로나타나서목태 > 서리태 > 아로니아 > 블루베리 > 블랙커런트 > 흑미 > 흑임자순으로높은함량을나타냈다. 안토시아닌 5종의총함량은서리태 82.4 ± 17.2 μg/g, 서목태 95.2 ± 6.1 μg/g, 흑미 74.1 ± 9.7 μg/g, 흑임자불검출, 블루베리 110.8 ± 1.9 μg/g, 아로니아 218.9 ± 6.1 μg/g, 블랙커런트 209.7 ± 4.0 μg/g로나타나아로니아 > 블랙커런트 > 블루베리 > 서목태 > 서리태 > 흑미순으로높은함량을나타났다. 본실험결과폴리페놀은서목태, 안토시아닌은아로니아가가장높은함량을나타내었으며향후유색농산물을활용한기능식품개발이필요한것으로판단된다. References 1. Lee J.J.: Colored foods and diabetes. J. Korean Diabetes, 12, 219-224 (2011). 2. Kim D.O., Jeong S.W. and Lee C.Y.: Antioxidant capacity of phenolic phytochemicals from various cultivars of plum. Food Chemistry, 81, 321-326 (2003). 3. Oliver Chen, C.Y. and Blumberg, J.: Phytochemical composition of nuts. Asia Pac. J. Clin. Nutr., 17, 329-332 (2008). 4. Tsao, R.: Chemistry and biochemistry of dietary polyphenols. Nutrients, 2(12), 1231-1246 (2010). 5. Cieslik, E., Greda, A., Adamus, W.: Contents of polyphenols in fruit and vegetables. Food Chemistry, 94, 135-142 (2006). 6. Schijlen, E.G., de Vos, R.C.H., van Tune, A.J., Bovy, A.G.: Modification of flavonnoid biosysthesis in crop plants. Phytochemistry, 65, 2631-2648 (2004). 7. Havsteen, B.: Flavonoids a class of natural products of high pharmacological potency. Biochem. Pharmaco., 32, 1141-1148 (2003). 8. Kim Y.H., Yun H.T., Park K.Y. and Kim, S.D.: Extraction and separation of anthocyanins in black soybean. Korean J. Crop Sci., 39(1), 35-38 (1997). 9. Kong J.M., Chia L.S., Goh N.K., Chia T.F. and Brouillard, R.: Analysis and biological activities of anthocyanins. Phytochemistry, 64, 923-933 (2003). 10. D Incalci, M., Steward, W.P., Gesoher, A.J.: Use of cancer chemopreventive phytochemicals as antineoplastic agents. Lancet Oncol, 6, 899-904 (2005). 11. Steinberg, F.M., Bearden, M.M., Keen, C.L.: Cocoa and chocolate flavonoids: implications for cardiovascular health. Am J. Diet. Assoc., 103, 215-223 (2003). 12. Sforcin, J.M. Orsi, R.O., Bankova, V.: Effect of propolis, some isolated compounds and its source plant on antibody production. J. Ehthopharmacol, 98, 301-305 (2005). 13. Blaylock, R.L.: Neurodegeneration and aging of the central nerous system : prevention and treatment by phytochemicals and metabolic nutrients. Integ. Med., 1, 117-133 (1999). 14. Zhang M.W., Guo B.J., Zhang R.F., Chi J.W., Wei Z.C., Xu Z.H., Zhang Y. and Tang X.J.: Separation, purification and identification of antioxidant compositions in black rice. Agricultural Sciences in China, 5(6), 431-440 (2006). 15. Zhang M.W., Zhang R.F., Zhang F.X. and Liu R.H.: Phenolic profiles and antioxidant activity of black rice bran of different commercially available varieties. Journal of Agricultural and Food Chemistry, 58, 7580-7587 (2010). 16. Choung M.G., Han W.Y., Kang S.T., Baek I.Y., Shin D.C., Kim S.D., Kim S.C., Moon H.P. and Kang K.H.: Structural analysis of anthocyanins in black soybean. Korean Soybean Digest, 19(2), 68-77 (2002). 17. Kim S.L., Kim H.B., Choi H.Y., Park N.K., Son J.R., Yun H.T. and Kim S.J.: Variation of anthocyanins and isoflavones between yellow-cotyledon and green-cotyledon seeds of black soybean. Food Sci. Biotechnol., 14(6), 778-782 (2005). 18. Fujioka, K. and Shibamoto, T.: Chlorogenic acid and caffeine contents in various commercial brewed coffees. Food Chem., 106(1), 217-221 (2008). 19. Ceirwyn, S.J.: Analytical chemistry of foods. Blackie Academic and Professional, London, 153 (1995). 20. Seo M.C., Ko J.Y., Song S.B., Lee J.S., Kang J.R., Kwak D.Y., Oh B.G., Yoon Y.N., Nam M.H., Jeong H.S., Woo K.S.: Antioxidant compounds and activities of foxtail millet, proso millet and sorghum with different pulverizing methods. J. Korean Soc. Food Sci. Nutr., 40(6), 790-797 (2011). 21. Choung M.G.: Optimal HPLC Condition for Simultaneous Determination of Anthocyanins in Black Soybean Seed Coats. Korean J. Crop Sci., 53(4), 359-368 (2008). 22. Kim H.W., Kim J.B., Chu S.M., Kim S.Y., Kim S.N., Cho Y.S., Cho S.M., Baek H.J., Kim J.H., Park H.J., Lee D.J., Hiba A. Ali and Derek, S.: Analysis of Anthocyanin Composition and Content Contained from Grains of the Korean Purple Rice Varieties by Liquid Chromatography with Diode Array Detection and Electrospray Ionization/Mass Spectrometry (LC-DAD-ESI/MS). Korean J. Intl. Agri., 22(3), 267-272 (2010). 23. Shin S.O., Shin S.H., Lim S.G., Lee J.H., Kang N.S., Suh D.Y., Park K.Y., Ha T.J.: Isolation and Identification of New

380 Il-Hyung Jeong, Moon-Seog Oh, Jong-Sup Jeon, Han-Taek Kim, Se-Ra Hong, Kwang-Hee Park, and Mi-Hye Yoon Anthocyanins from the Black Seed Coat of Soybean (Glycine max L.) by HPLC-DAD-ESI/MS Analysis. Korean Soybean Digest, 24(1), 23-33 (2007). 24. AOAC. Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals (2002). 25. The International Council for Harmonisation (ICH): Validation of analytical procedures, text and methodology Q2(R1), 1-17 (2005). 26. Folin, O. and Denis, W.: On phosphotungstic-phosphomolybdic compounds as color reagents. J. Biol. Chem., 12, 239-249 (1912). 27. Prior, R., Wu, X., Schaich, K.: Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem., 53, 4290-4302 (2005). 28. Box, J.D.: Investigation of the Folin-Ciocalteu phenol reagent for the determination of polyphenolic substances in natural waters. Water Res., 17, 511-525 (1983). 29. Ikawa, M., Schafer, T., Dollard, C., Sasner, J.: Utilization of Folin-Ciocalteu reagent for the detection of certain nitrogen compounds. J. Agric. Food Chem., 51, 1811-1815 (2003). 30. Bradley, W.B., Oliver Chen, C.Y., Diane, L.M., Jeffrey, B.B.: Tree nut phytochemicals: composition, antioxidant, capacity, bioactivity, impact factors. A systemic review of almonds, brazils, cashews, hazelnuts, macadamia, pecans, pine nuts, pistachios and walnuts. Nutrition Research Reviews, 24, 244-275 (2011). 31. Rice-Evans, C.A., Miller, N.J., Paganga, G.: Antioxidant properties of phenolic compounds. Trends in Plant Sci., 2, 152-159 (1997). 32. Bang I.S., Yu C.Y., Lim J.D.: Effects of temperature and UV irradiation on stability of anthocyanin-polyphenol copigment complex in mulberry fruits. Korean Journal of Medicinal Crop Science, 18(3), 191-200 (2010). 33. Nováková, L1., Vildová, A., Mateus, J.P., Gonçalves, T. and Solich, P.: Development and application of UHPLC-MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts. Talanta, 82(4), 1271-1280 (2010). 34. Gruz, Jirí, Novák, Ondrej and Strnad Miroslav.: Rapid analysis of phenolic acids in beverages by UPLC-MS/MS. Food chemistry, 111(3), 789-794 (2008). 35. Kim H.W., Kim J.B., Chu S.M., Kim S.Y., Kim S.N., Cho Y.S., Cho S.M., Baek H.J., Kim J.H., Park H.J., Lee D.J., Ali, H.A. and Stewart, D.: Analysis of anthocyanin composition and content contained from grains of the korean purple rice varieties by liquid chromatography with diode array detection and electrospray ionization/mass spectrometry. Korean J. Intl. Agri., 22(3), 267-272 (2010). ê ê