pissn 1229-1153 / eissn 2465-9223 J. Fd Hyg. Saf. Vl. 32, N. 5, pp. 389~395 (2017) https://di.rg/10.13103/jfhs.2017.32.5.389 Jurnal f Fd Hygiene and Safety Available nline at http://www.fdhygiene.r.kr 루이보스차 (Asphalthus linearis) 의추출방법에따른페놀릭류함량변화연구 박신희 * 도영숙 김윤성 김난영 이진희 김종화 윤미혜 경기도보건환경연구원안양농수산물검사소 Determinatin f Phenlic Cntents in Ribs (Asphalthus linearis) Tea Depending n the Steeping Temperature and Time Sin-Hee Park*, Yung-Suk D, Yun-Sung Kim, Nan-Yung Kim, Jin-Hee Lee, Jng-Hwa Kim, and Mi Hye Yn Anyang Agr-fishery Prducts Inspectin Center, Gyenggi prvince Institute f Health and Envirnment, Krea (Received May 31, 2017/Revised June 10, 2017/Accepted September 25, 2017) ABSTRACT - A simultaneus determinatin f 5 phenlic acids (gallic acid, chlrgenic acid, caffeic acid, p- cumaric acid, trans ferulic acid) and 9 flavnids (prcyanidin b1, aspalathin, rutin, vitexin, hyperside, isquercitrin, lutelin, quercetin, chryseril) in ribs tea has been carried ut by ultra-high perfrmance liquid chrmatgraphy cupled t tandem mass spectrmetry (UPLC-MS/MS). A validated analysis methd in this study was applied t ribs aqueus infusins. Ribs tea is an antixidant-rich tea which has anti-cancer, anti-aging, anti-inflammatry, anti-diabetic effect. Extractin yield f phenlics depends n steeping time and temperature f water. Tea infusins were prepared by placing 1 g f tea leaves r 1 tea bag in 100 ml f biled water, and then at 3, 6 and 30 minutes intervals the infused teas were taken t carry ut the analysis f phenlic cntents. Anther tea infusin was cnducted with cld water (25-30 C) fr 30 minuntes. As a result, the ttal amunt f phenlics was highest in ribs tea steeped with ht water fr 30 minutes, fllwed by 6 minutes, 3 minutes and cld water 30minutes and the result has statistical significance. Key wrds : Phenlics, Ribs Tea, Phenlic Acids, Flavnids, UPLC-MS/MS 페놀릭류는식물계에광범위하게분포되어있는대표적인 phytchemical이며지금까지 8,000종이상이발견되었다. 페놀릭류는적어도하나이상의 armatic ring에하나이상의 hydrxyl grup을가진다. 저분자량의 simple armatic ring 구조부터고분자량의 cmplex tannin이나 plyphenl 까지매우다양하게존재한다. 물질의구조에따라크 1,2) 게 flavnids와 nn-flavnids로구분되는데 nn-flavnid 에는 phenlic acids, phenlic alchls, stilbene과 lignan등이속한다. Flavnids는차, 과일, 야채, 향신료, 코코아, 1) 와인등과같이식물유래의식품에다양하게존재하며 flavnids는섭취시인체내에서항산화, 항미생물, 심장보호, 소화관보호, 항골다공증, 항염증, 항암작용을함으로써건강에매우유익하다 3). 루이보스는남아프리카의 Western Cape 지역에있는세 *Crrespndence t: Sin-hee Park, Fd and Drug Research Divisin, Gyenggi-D Institute f Health and Envirnment, Suwn 16205, Krea Tel: 82-31-250-2562, Fax: 82-31-250-2569 E-mail: psh75@gg.g.kr 더버그산인근지역의척박한환경에서서식하고있으며, 콩과에속하는관목으로키는 1.5-2 m정도로자라고밝은녹색의침엽수이면서작은노란색꽃이핀다 4). 수확후발효과정동안녹색에서붉은색으로변하게되는데이는구성페놀릭류들의산화로인한결과이며이때문에 red tea 혹은 red bush tea라고불리기도한다. 1999-2000년까지루이보스는연 4,500-6,000 metric tnne이생산되었으나생산량의 70-75% 가남아프리카현지에서소비되었다 5). 하지만 2015년현재루이보스티의연재배량은 12,000 metric tnne이넘으며로컬마켓뿐아니라세계적시장에서그수요가늘어나고있다. 루이보스티의가장큰특징은카 6) 페인이거의없으며, 녹차 (Camellia sinensis tea) 에비해매우적은정도의 tannin을함유하고있다는것이다 7). 카페인에민감한어린이나임산부에게도부작용이거의없으며 tannin의양이적으므로일반녹차보다오랜시간우려내어마셔도상대적으로떫은맛이매우적다. 루이보스티에는다른식물에서는아직까지발견되지않은유일한페놀릭류인 aspalathin과 aspalalinin이라는 flavnid 단량체가존재한다. 또한 quercetin, isquercetin, vitexin, 8,9) 389
390 Sin-Hee Park, Yung-Suk D, Yun-Sung Kim, Nan-Yung Kim, Jin-Hee Lee, Jng-Hwa Kim, and Mi Hye Yn chryseril, lutelin, hyperside, rutin, cumaric acid, ferulic acid등다양한페놀릭류가루이보스티에서검출되었다. 10) 차는우려내는물의온도와우려내는시간에따라그속에함유된유용성분의추출효율이달라진다. 루이보스 11) 티는녹차나홍차, 우롱차에비해세계시장에소개된지오래되지않았으나여러연구결과들을통해항산화, 항암, 항알러지등인체에유익한작용을많이하는것으로보고되고있다. 가장접하기쉽고대중적으로소비량이높 12-14) 은녹차의음용방법은보통끓인물을 80 C정도로식힌 후녹차에붓고약 3분간우려내어마시는것을권장하고있으나루이보스티는우려내는물의온도와시간에따른유용성분함량에대한연구결과가많지않다. 이번연구의목적은유익한페놀릭류를다량포함하고있는것으로알려진루이보스티를음용할때가장많은페놀릭류를추출할수있는물의온도와추출시간을밝히는것이다. Materials and Methds 시료본연구에사용한루이보스티는경기도내대형유통매장및백화점에서총 30건구매하였다. 구매한루이보스티의원산지는 30건중 29건은남아프리카, 1건은독일이었다. 표준용액및시약페놀릭류표준품인 gallic acid, prcyanidine B1, chlrgenic acid, caffeic acid, p-cumaric acid, aspalathin, vitexin, hyperside, trans-ferulic acid, isquercitrin, rutin, lutelin, quercetin, chryseril은 Extrasynthese (Genay, France) 제품을사용하였다. Hexane, methanl은 Wak (Tky, Japan) 에서잔류농약분석급을사용하였고 acetnitrile은 Merck (Darmstadt, Germany) GR급을사용하였다. 각물질의 stck standards 는 400~500 mg/l의농도로 methanl에녹여갈색바이알에담아밀봉한후사용전까지 20 C에서냉동보관하 였다. Wrking standard mix는 stck standard slutin을 methanl을이용하여원하는농도로단계희석한후사용하였다. (Waters, Milfrd, MA, USA) 을이용하였고수집된데이터는 Masslynx 4.1 sftware를이용하여가공하였다. 여러논문에서페놀릭류분석시 ESI 방식의경우 psitive mde보다 negative mde에서감도가더좋다고보고되어있다 15,16). 이번연구에서도표준품의검출조건을결정할때 psitive, negative mde를사용하여비교해본결과 negative mde에서더높은감도를보였으므로실제실험에서도 ESI negative mde를이용하여분석하였다. 나머지 TQD 조건은 Table 2와같다. 시료전처리방법시중유통중인루이보스티의포장상태에따라티백인경우는 1티백으로혹은건조된분말인경우 1g을정밀히취하여찻잔에넣고전기주전자를이용하여끓인물 100 ml를찻잔에부어차를우려내었다. 루이보스티속의페놀릭류추출을돕기위해 2분에 1번씩찻잔속의물을티스푼을이용하여부드럽게저어주었고찻잔에물을부은후 3분, 6분, 30분이지난시점에추출된액을 2mL씩 glass tube에취하고 40 C로세팅된워터배스에서압축공기를가 하여증발농축시켰다. 완전히증발된추출액에메탄올 2mL을가하고 vrtexing 하여잘녹인후 0.22 μm PTFE syringe filter로여과하여분석하였다. 뜨거운물을이용한추출과는별도로차가운물추출은상온 (20~25 C) 의물을 Table 1. UPLC mbile phase cnditins Flw rate Time A(%) B(%) Flw rate (ml/min) 0.0 100 0 1.0 100 0 10.0 70 30 15.0 0 100 0.3 18.0 0 100 18.1 100 0 20.0 100 0 A- 0.1% acetic acid B- 0.1% acetic acid in Acetnitrile Table 2. TQD cnditins 분석기기및 sftware 루이보스티시료의페놀릭류함량분석에는 Waters UPLC system (Waters, Milfrd, MA, USA) 과칼럼은 Cadenza CL-C18, 100 * 2 mm를이용하여이동상과유속을 Table 1 과같은조건으로분석하였다. 분석온도는 40 C, injectin vlume은 5μL를주입하였으며분석시간은 20분이소요되었다. 컬럼을통해분리된 cmpund들의함량을측정하기위해 mass spectrmetry 를사용하였는데 Waters Acquity TQD tandem quadruple mass spectrmeter system ESI mde Capillary vltage Extractr vltage Surce temp. Deslvatin temp. Cne gas flw (N 2 ) Deslvatin gas flw (N 2 ) Cllisin gas (He) Cllisin gas pressure Negative 3.0 kv 3.0 V 150 C 400 C 50 ml/min 750 ml/min 0.17 ml/min 3.7 10 mbar 3
Determinatin f Phenlic Cntents in Ribs (Asphalthus linearis) Tea Depending n the Steeping Temperature and Time 391 동일량가하고 30분간추출한후동일한방법으로전처리하여페놀릭류의함량을측정하였다. 분석물질의정성을위한 In rati 구하는방법 In rati (%) = 정성이온 peak area / 정량이온 peak area 100 분석방법의유효성검증방법시험법의유효성검증항목으로직선성, LOD, LOQ, linear range, 회수율 (%), 정밀성에대한실험을실시하였다. 페놀릭류의검출한계 (Limit f Detectin, LOD) 와정량한계 (Limit f Quantitatin, LOQ) 는 32, 65, 125, 250, 500, 1000, 2000 μg/l standard 용액을각 6회분석하여얻어진데이타를바탕으로 ICH (Internatinal Cnference n Harmnisatin f Technical Requirements fr Registratin f Pharmaceuticals fr Human Use) 가이드라인따라 18) 아래의방법으로계산하였다. LOD = 3.3 σ/s LOQ = 10 σ/s σ = the standard deviatin f the respnse S = the slpe f the calibratin curve 직선성을평가하기위하여 0.032 mg/l ~ 4.0 mg/l의 8 단계농도의표준품을조제하고분석하였는데, 그결과얻어진크로마토그램의 peak 면적과농도간의상관관계를회귀분석을통해결정하였다. 회수율시험은루이보스티제품하나를대표로선택하여농도 1mg/L의표준품혼합 액을가한후뜨거운물로 30분추출하여페놀릭류의함량을구하고표준품혼합액을가하지않은샘플의페놀릭류함량을따로구하여그차이를계산한후회수율로하였다. 정밀도를측정하기위하여 1mg/L의표준품 mix를하루동안 6회반복분석하여 interday precisin을구하였고, 연속하지않은 3일동안하루당 6회씩을분석하여 intraday precisin을구하였다. 통계처리페놀릭류의함량데이타결과는 3회평균 ± 표준편차로표현하였다. GraphPad Prism 5 (GraphPad Sftware, Inc. San Dieg, CA) 을사용하여 ANOVA분석과 Bnferrni s test 를통해유의성을검정하였다. p <0.05 일때유의한것으로판단하였다. Results and Discussin TQD (MS/MS) detectr 분석조건설정 UHPLC system을이용하여샘플을분리하기위한이동상으로 A (5% acetnitrile) 용액과 B (100% acetnitrile) 용액에각각 MS용 acetic acid를 0.1% 농도가되도록가하여이용하였다. TQD detectr는분석대상물질에따라그물질에대한민감도를최적화하기위해 cne vltage와 cllisin energy 값을각각정해주어야한다. 감도의최적화를위한방법은다음과같다. 먼저페놀릭류표준품을개별적으로 5mg/L의농도로조제하고각각의표준품용 Table 3. MS/MS parameters Cmpunds Retentin time (min) Cne Vltage Quantificatin transitin* Gallic acid 1.42-1.43 30 169 > 125(13) Cnfirmatin transitin* In rati (%) Prcyanidin B1 3.69 30 577 > 289(25) 577 > 407(18) 45.7 Chlrgenic acid 4.03 25 353 > 191(20) Caffeic acid 4.73-4.74 25 179 > 135(15) p-cumaric acid 6.06-6.09 25 163 > 119(15) 163 > 93(25) 5.0 Aspalathin 6.13-6.16 25 451 > 331(14) 451 > 361(20) 15.0 Rutin 6.36-6.46 50 609 > 300(35) 609 > 271(55) 43.9 Vitexin 6.46-6.53 40 431 > 312(25) 431 > 284(35) 2.2 Hyperside 6.63-6.73 40 434 > 300(25) trans-ferulic acid 6.7 25 193 > 134(25) 193 > 149(10) 24.8 Isquercitrin 6.64-6.74 45 463 > 300(25) 463 > 271(40) 49.4 Lutelin 9.71-9.72 45 285 > 133(35) 285 > 107(35) 12.1 Quercetin 9.71-9.74 35 301 > 151(22) 301 > 179(20) 31.6 Chryseril 11.41 40 299 > 284(20) 299 > 256(30) 23.3 * Cllisin energy (ev) is given in brackets.
392 Sin-Hee Park, Yung-Suk D, Yun-Sung Kim, Nan-Yung Kim, Jin-Hee Lee, Jng-Hwa Kim, and Mi Hye Yn Fig. 1. Chrmatgrams f 14 phenlic standards (1 mg/kg). 액을이동상 A와 B를비율 50 : 50, 유속은 0.1 ml/min 으로 direct infusin 방식으로주입한다. 표준용액주입시표준품에해당하는분자량 [M-H] 에서 parent in의 beam 이나오는것을확인한후 cne vltage를조절하여 parent in의감도가가장높게나오는 cne vltage를결정하는데 cne vltage가결정되면 cllisin energy를조금씩높여가면서 parent in을 fragmentatin시키고 daughter in이가장잘검출되는 cllisin energy값을찾는다. Daughter in중가장감도가높게잘검출되는것을정량이온으로정하고두번째로잘나오는이온을정성이온으로정하는데이와같은방법으로각 14개의페놀릭류 standard별로최적의 cne vltage값과정량정성이온별 cllisin energy 값을결정하였고 (Table 3) 14종페놀릭류의크로마토그램은 Fig. 1과같다. 이렇게최적화된조건으로샘플을분석할때샘플에서나온 peak이표준품과동일한물질인지확인하기위해 SANTE기준을적용하였다. SANTE 기준 17) 에의하면 MS/MS분석시샘플의크로마토그램상에나온피크가분석하려는물질인지확인하기위해먼저분석물질 peak의 retentin time을 standard와비교할때 ±0.1 min 이내의범위에속해야한다. 두번째로는질량분석기에의해수집된분자량데이터를이용하여확인하는데, 샘플을분석할때 standard와동일한 parent in이일차적으로검출되는지, 또한 parent in에 cllisin energy를가하여 fragmentatin시켜나온 daughter in의분자량과정성, 정량이온의비율인 in rati가 standard와비교하여 ± 30% (relative) 범위내에속하는지비교하여이를만족하면 standard와동일한물질로판단한다. 하지만우리가분석하려는 20개물질중 gallic acid, chlrgenic acid, caffeic acid는정성이온이검출되지않았다. 위의두가지기준에의해샘플에서검출된 peak이우리가분석하려는물질과동일한것으로확인되면 peak의 intensity를이용하여정량하였다. 분석방법의유효성검증결과시험법의유효성검증항목으로직선성, LOD, LOQ, linear range, 회수율 (%) 에대한실험을실시한결과 Table 4와같았다. LOQ는 0.001~0.101 mg/l로확인되었고 linear range는물질에따라최소 0.032 mg/l에서최대 4.0 mg/l로결정되었다. 직선성은평가결과는 R 2 값이 0.9817~0.9992로양호한결과를보였다. 회수율시험은 SANTE 기준에의하면 70-120% 이내이어야하며이번실험에서회수율은 asphalatin 94.9% ~ quercetin 112.2% 사이에서확인되었다. Precisin은반복실험을통하여값이일정하게나오는지를확인하기위한유효성검증인자인데 SANTE 기준에의하면 RSD (relative standard deviatin) 가 20% 이어야한다. Precisin 측정결과 interday precisin (RSD%) 는 chryseril 1.8 ~ rutin 9.2% 로나왔으며 intraday precisin (RSD%) 은 caffeic acid 1.8 ~ aspalathin 10.0% 로 SANTE 기준을만족하였다. 루이보스티우려내는물의온도및채취시간별페놀릭류함량루이보스티에서검출된 14종의페놀릭류중 rutin, aspalathin, vitexin, hyperside, isquercitrin, quercetin등이주로검출되었고, 그외에 lutelin, chryseril, trans-ferulic acid, p-cumaric acid, caffeic acid, chlrgenic acid, gallic
Determinatin f Phenlic Cntents in Ribs (Asphalthus linearis) Tea Depending n the Steeping Temperature and Time 393 Table 4. Regressin, R, LODs, LOQs, linear range and recvery fr studied cmpunds 2 Cmpunds Regressin equatin R 2 LOD (mg/l) LOQ (mg/l) Linear range (mg/l) Recvery (%) Gallic acid 35794.8 X + 103.901 0.9979 0.018 0.055 0.032-2.0 100.2 Prcyanidin B1 6694.14 X 13.754 0.9992 0.010 0.029 0.032-2.0 97.9 Chlrgenic acid 25860.8 X + 40.6025 0.9963 0.010 0.031 0.032-4.0 99.3 Caffeic acid 0.61815 X + 2059.13 0.9931 0.006 0.018 0.032-4.0 103.3 p-cumaric acid 61918.3 X + 2246.84 0.9933 0.006 0.018 0.032-2.0 105.7 Aspalathin 36985.5 X + 368.238 0.9986 0.003 0.009 0.032-4.0 94.9 Rutin 0.43515 X + 194.98 0.9938 0.000 0.001 0.032-4.0 108.3 Vitexin.115194 X + 2689.71 0.9934 0.001 0.003 0.032-4.0 107.1 Hyperside.110797 X + 1194.44 0.9976 0.000 0.001 0.032-2.0 102.1 trans-ferulic acid 47273.1 X + 2618.42 0.9817 0.002 0.006 0.032-2.0 104.4 Isquercitrin.160823 X + 3378.03 0.9939 0.004 0.013 0.032-2.0 105.9 Lutelin.144598 X + 2033.31 0.9933 0.001 0.002 0.032-2.0 104.2 Quercetin 69678.4 X + 751.953 0.9935 0.034 0.101 0.032-2.0 112.2 Chryseril.196271 X + 9826.48 0.9902 0.002 0.007 0.032-2.0 95.2 Table 5. Cmparing individual phenlic cntents f Ribs tea steeping with ht water at 3 min, 6 min, 30 min interval and cld water at 30 min. (unit : mg/100 ml) Ht water 3 min Ht water 6 min Ht water 30 min Cld water 30 min Gallic acid 12.0-83.4 06.7-94.3 05.8-102.7 16.3-66.0 Prcyanidin B1 03.0-119.9 05.8-126.9 03.9-148.6 03.2-140.2 Chlrgenic acid 09.1-716.6 09.4-824.9 10.2-814.7 07.6-823.3 Caffeic acid 02.3-82.0 04.0-89.3 03.7-89.7 09.3-60.4 p-cumaric acid 01.8-39.4 01.8-44.4 07.0-50.0 06.2-30.6 Asphalatin 02.4-3166.7 04.5-3444.3 10.3-2686.5 01.4-3193.9 Rutin 05.9-1343.6 07.9-1381.3 18.6-1430.1 35.4-179.2 Vitexin 01.2-252.3 02.4-253.4 08.0-236.4 14.7-207.5 Hyperside 00.4-321.9 01.1-334.5 03.1-326.7 03.4-973.1 trans-ferulic acid 01.6-211.4 02.8-222.0 05.0-220.6 02.0-169.0 Isquercitrin 37.3-301.4 41.2-366.7 02.1-349.3 21.2-206.8 Lutelin 00.2-143.7 00.4-151.1 01.0-141.0 01.6-46.2 Quercetin 10.7-345.6 11.3-331.0 18.8-310.5 11.2-107.9 Chryseril 01.3-71.3 02.0-69.5 06.4-68.1 01.3-20.8 ttal 2734.3 ± 6.7 2875.6 ± 15.9 a 2998.3 ± 8.2 a 1824.9 ± 6.9 a a: significantly different (p > 0.001) frm "Ht water 3 min" acid, prcyanidin B1등이미량검출되었다. 루이보스티를뜨거운물에 3분, 6분, 30분, 차가운물 30 분에우려낸결과 14종각페놀릭류의함량범위는 Table 5와같다. 최근연구결과에의하면 aspalathin은 green ribs에서 red ribs로발효되면서그양이감소하며결과적으로 1.2 mg/g ~ 49.9 mg/g이검출되었다고보고된바있다 19). 이번실험에이용된루이보스는전체가 red ribs로써 aspalathin 함량이 0.01~3.3 mg/g범위에서검출되었다 ( 뜨 거운물 30분기준 ). Aspalathin를비롯한다른페놀릭류의함량도검체별로크게차이가났는데이는루이보스차의제품별표시함량, 생산날짜및포장상태가다르기때문으로유추된다. 루이보스티는 aspalathin 이외에도 rutin (1.3-1.7 mg/g), isquercitrin (0.01-0.11 mg/g), lutelin (0.02-0.03 mg/g), chryseril (0.01-0.02 mg/g) 을함유하고있는것으로보고되었고 19), 이번실험에서는 rutin 0.02-1.4 mg/ g, isquercitirn 0.002-0.35 mg/g, lutelin 0.001-0.14 mg/g, chryseril은 0-0.06 mg/g을함유하고있음이확인되었다.
394 Sin-Hee Park, Yung-Suk D, Yun-Sung Kim, Nan-Yung Kim, Jin-Hee Lee, Jng-Hwa Kim, and Mi Hye Yn Fig. 2. The ttal quantities f phenlics represented as a histgram. (n = 3 experiment, '***' dentes significantly different frm H- 3min, p < 0.001). Hyperside를제외한 13종의페놀릭류성분들은뜨거운물로추출시추출시간이길어짐에따라총함량이증가하였다. 동일실험을 3회반복시험하여평균값을계산한결과 (Fig. 2) 루이보스티를우려내는온도와시간에따라페놀릭류의총합이유의성있게변하였다 (p < 0.001). 뜨거운물 3분추출을기준으로할때, 뜨거운물 6분추출은페놀릭류의총합이 5.2% 증가하였고, 뜨거운물 30분추출은 9.7% 증가하였으나차가운물 30분추출은오히려 33.3% 감소하였다. 대부분의페놀릭류는뜨거운물에서더많이추출되거나차가운물에서도비슷한수준으로추출되었으나특이하게 hyperside는뜨거운물 3분과비교했을때차가운물 30분에서추출양이약 3배가량증가하였다. Sara 등에의하면 green tea는추출시간과온도에따라 20) 항산화능력이민감하게변화하는데차가운물에서 2시간우려낸것에서가장높은항산화능력을보였고, black tea 는뜨거운물로짧은시간동안우려낸것에서가장높은항산화능을나타냈다. 이처럼 tea의종류에따라구성페놀릭류의종류와양이다르므로가장높은항산화능을나타내는추출온도와시간은다를수밖에없다. 루이보스티의페놀릭류추출효율은물의온도와시간두가지모두영향을받는것으로판단되며상온의물보다는끓는물로우려내는시간이길수록더많은페놀릭류가추출되었다. 총페놀의양과항산화능력은양의상관관계가있으므로 21,22) 페놀릭류의총합이가장많은뜨거운물로 30분추출할때항산화능력이가장높을것으로추측된다. 루이보스티를위의조건과같이추출하였을때실제항산화능력도유사하게증가하는지를확인하기위해서는추가적인항상화능측정실험이진행되어야할것으로보인다. Cnclusin 국내유통중인루이보스티 30 건에대한페놀릭류함량 을 triple-quadruple을이용하여동시분석하였다. 1. 루이보스티는 rutin, aspalathin, quercetin, isquercitirn, vitexin, hyperside 등의페놀릭류를함유하고있었다. 2. 루이보스티를우려낼때물의온도와추출시간에따라추출되는총페놀릭류의양이달라졌다. 물의온도가높을수록우려내는시간이길수록더많은양의총페놀릭류가추출되었다. 루이보스티는보통녹차나홍차를마실때우려내는시간 (3min) 보다는비교적길게우려내는것이그속에함유된여러항산화물질들을더많이섭취할수있다. 뜨겁게마실경우 3분보다는 6분이더많은유용성분을음용할수있고차갑게마시고싶은경우라도소량의뜨거운물로먼저 6분이상우려내서유용성분을충분히추출시킨후얼음등을가하여차갑게하여마시는방법을추천한다. 국문요약 루이보스티에서 5종의 phenlic acid (gallic acid, chlrgenic acid, caffeic acid, p-cumaric acid, trans-ferulic acid) 와 9종의 flavnid (prcyanidin b1, aspalathin, rutin, vitexin, hyperside, isquercitrin, lutelin, quercetin, chryseril) 를 UPLC-MSMS를이용하여동시분석하였다. 14종페놀릭류를동시분석하기위하여기기조건과유효성을검증하였고확립된분석방법을이용하여시중에유통중인루이보스티 30건을채취하여페놀릭류를분석하였다. 루이보스티 1g 혹은 1티백에뜨거운물 100 ml을가하여 3분, 6분, 30분이경과후그리고차가운물 (25-30 C) 에 30분우려낸루이보스티의페놀릭류함량을구하였다. 루이보스티에서전체실험대상페놀릭류중 rutin과 aspalathin이가장많이추출되어나왔으며각각물질의함량은제품별로달랐다. 페놀릭류성분의추출효율은 14종페놀릭류의총합기준으로뜨거운물 30분 >6분 >3분 > 차가운물 30분순으로높았다. References 1. Crzier, A., Jaganath, I. B. and Cliffrd, M. N.: Dietary phenlics: chemistry, bia-vailability and effects n health. Natural Prduct Reprts, 26, 1001-1043 (2009). 2. Tsa, R.: Chemistry and bichemistry f dietary plyphenls. Nutrients, 2(12), 1231-1246 (2010). 3. Daniel Del ri, Ana Rdrigues-Mates, Jeremy P. E. Spencer, Massimilian Tgnlini, Gina Brges, and Alan Crzier.: Dietary(Ply)phenlics in Human Health : Structures, biavaility, and Evidence f Prtective Effects Against Chrnic Diseases, Antixidants & Redx Signaling, 18(14), 1818-1892 (2013). 4. Van Wyk, B. E. and Gericke, M.: Peple s plants. A Guide t Useful Plants f Suthern Africa. Briza Publicatins, Pret-
Determinatin f Phenlic Cntents in Ribs (Asphalthus linearis) Tea Depending n the Steeping Temperature and Time 395 ria, Suth Africa, pp. 351, (2000). 5. Wilsn, N. L. W.: Cape natural tea prducts and the US market: ribs rebels ready t raid. Applied Ecn. Perspectives and Plicy, 27(1), 139-148 (2005). 6. Republic f suth africa, Agriculture, Frestry & Fisheris department: A prfile f the Suth Africa Ribs market value chain, pp. 1-10 (2015). 7. Mrtn, J. F.: Ribs tea, Aspalathus linearis, a caffeineless, lw-tannin beverage. Ecn. Btany, 37(2), 164-173 (1983). 8. Jubert, E., Gelderblm, W., Luw, A. and De Beer, D.: Suth African herbal teas: Aspalathus linearis, Cyclpia spp. and Athrixia phylicides - a review. J. f Ethn-pharmaclgy, 119(3), 376-412 (2008a). 9. Keppen, B. and Rux, D.: C-glycsylflavnids. The chemistry f aspalathin. Bichem. J., 99(3), 604-609 (1966). 10. Rabe, C., Steenkamp, J. A., Jubert, E., Burger, J. F. W. and Ferreira, D.: Phenlic metablites frm ribs tea (Aspalathus linearis). Phytchemistry, 35(6), 1559-1565 (1994). 11. Xingu Su, Jun Duan, Xuewu Duan and Feng Chen.: Plyphenlic prfile and antixidant activities f lng tea infusin under varius steeping cnditins. Ml. Sci., 8(12), 1196-1205 (2007). 12. Shulz H, Jubers E and Shutze W.: Quantificatin f quality parameters fr reliable evaluatin f green rivs (Asphalthus linearis). Eur. Fd Res. Technl. 216(6), 539-543 (2003). 13. Edenharder R, Sager JW, Glatt H, Muckel E and Platt KL.: Prtectin by beverages, fruits, vegetables, herbs, and flavnids against gentxicity f 2-acetylaminflurene and 2-amin-1-methyl-6-phenylimidaz[4,5-b]pyridine (PhlP) in metablically cmpetent V79 cells. Mutat. Res., 521, 57-72 (2002). 14. Hasseling PB and Jubert JR.: The effect f ribs tea n the type I allergic reactin. S. Afr. Med. J., 62, 1037-1038 (1982). 15. Nvákvá L1, Vildvá A, Mateus JP, Gnçalves T and Slich P.: Develpment and applicatin f UHPLC-MS/MS methd fr the determinatin f phenlic cmpunds in Chammile flwers and Chammile tea extracts, Talanta, 82(4), 1271-1280 (2010). 16. Gruz, Jirí, Nvák, Ondrej and Strnad Mirslav.: Rapid analysis f phenlic acids in beverages by UPLC-MS/MS, Fd Chem., 111(3), 789-794 (2008). 17. SANTE/EU: Methd Validatin and Quality Cntrl Prcedures fr Pesticide Residues Analysis in fd and feed. pp. 1-42 (2015). 18. ICH Harmnised Tripartite Guideline, Validatin f Analytical Prcedures : Test and Methdlgy Q2(R1). pp. 1-17 (2005). 19. Bramati L, Aquilan F and Pietta P.: Unfermented ribs tea : Quantitative charaterizatin f flavnids by HPLC-UV and determiantin f the ttal antixidant activity. J. Agri. Fd Chem., 51(25), 7472-7474 (2003). 20. Sara H.A. alipur, Junedah Sanusi and Kanthimathi M S.: Temperature and time f steeping affect the antixidant prperties f white, green, and black tea infusins. J. Fd Sci., 81(1), H246-254 (2016). 21. M. Amzad Hssain and Muhammad Dawd Shah.: A study n the ttal phenls cntent and antixidant activity f essential il and different slvent extracts f endemic plant Merremia brneensis. Aravian J. Chem., 8(1), 66-71 (2015). 22. Ali Ghasemzadeh, Hawa Z.E. Jaafar and Asmah Rahmat.: Antixidant activities, ttal phenlics and flavnids cntent in tw varieties f Malysia yung ginger (Zinziber fficinale Rsce). Mlecules. 15(6), 4324-4333 (2010). ê ê