Estimation of Harvest Period and Cultivated Region of Commercial Green Tea by Pattern Recognition

(1)

패턴인식법에 의한 시판 녹차의 산지 및 채엽시기 추정

주홍매·김정숙·박경래·조정원·김영섭

*

^·김정우

**

^·유시용

*

^,#^·강종성^#

충남대학교 약학대학, *한국화학연구원, **배재대학교 생명공학과

(Received October 23, 2008; Revised December 20, 2008; Accepted January 20, 2009)

Estimation of Harvest Period and Cultivated Region of Commercial Green Tea by Pattern Recognition

Hongmei Zhu, Jung Sook Kim, Kyung Lae Park, Cheong Weon Cho, Young Sup Kim*, Jung Woo Kim**, Shi Yong Ryu*

^,#

and Jong Seong Kang

^#

College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea

*Korea Research Institute of Chemical Technology, Daejeon 305-606, Korea

**Dept. of Life Science and Technology, Pai Chai University, Daejeon 302-735, Korea

Abstarct

— Quantitative analysis of (+)-catechin (C), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and caffeine in commercial green tea was carried out by HPLC employing gradient elution of 0.1% acetic acid and acetonitrile on ODS column. The optimized HPLC method provided satisfactory lin- earity, accuracy and precision. The relationship between the concentration of the components and cultivated region of the commercial green tea was not significant, while the concentration of EGCG, ECG and caffeine decreased significantly in the later harvested green tea samples (p<0.01). Multivariate analysis of the components was performed in order to characterize and evaluate the cultivated region and harvest period-related variation. Hierarchical clustering and discriminant analysis were applied to classify the geographical and seasonal origins of the green tea samples. The classification accuracy of the cultivated region and harvest period by discriminant analysis was 95% and 91%, respectively, indicating that this methode could be reliable and convenient for the quality control of herbal products with different origin.

Keywords □

green tea, catechin, discriminant analysis, hierarchical clustering, harvest period

차나무

(Camellia sinensis)

^는 ^{동백나무과}

(Camelliaceae or

Theaceae)

에속하는다년생상록관목으로우리나라를비롯한아

시아를중심으로아프리카

,

러시아등아열대온대에걸쳐광범 위하게재배되고있다

.

^녹차는^차나무의^잎을^말려^가공한^것으

로국민소득의향상과건강기호음료의인식전환으로그소비가 급증하고있다

.

^녹차가^중요한^{기호음료로서}^발전해^온^가장^큰

이유는대중적인기호성을가지고있을뿐아니라복잡한생명 활동의조절기능을갖는기능성식품으로서중요성이새삼강조 되고있기때문이다

.

^최근^녹차를^음용하는^인구가^늘어남에^따

라녹차의성분과효능에대한연구가다양하게진행되고있다

.

^1-4)

차의주요성분은카페인성분

,

폴리페놀성분

(

카테킨

,

카테킨

산화물

,

^플라보놀

),

^단백질과^그^밖의^{질소화합물}

(

^아미노산

,

^아미

드

),

탄수화물성분

(

섬유소

,

전분

,

덱스트린

,

당

,

팩틴

),

각종식물 성색소

(

엽록소

,

카로틴

,

키산트필

,

안트키안

),

방향류

(

정유성분

),

유기산성분

(

^구연산^사과산

,

^호박산

),

^지방산^성분

(

^초산

,

^길초산

),

비타민성분

(

비타민

A, B1, B2, C,

니코틴산

),

무기성분

(

약

5~

6%

^의^망간

,

^요오드

)

^등이^있으며^특히^{폴리페놀류를}^많이^함유하

고있어강한항산화력을나타낸다

.

^녹차의^{폴리페놀류는}^카테킨

류로 알려진

(+)-catechin, (-)-epicatechin, (-)-epigallocatechin gallate, (+)-epigallocatechin, (-)-epicatechin gallate

^등이다

.

⁵⁾^이

런화학성분은차엽의채엽시기

,

^6-8)^품종

,

⁹⁾^차나무가^자라는^기

상및주위환경

,

¹⁰⁾제다법¹¹⁾등에따라차이가나는데채엽시기 가빠른차일수록총질소

,

^카페인^및^{아미노산등의}^함량이^많고

카테킨류는늦게딴차잎일수록함량이많은것으로알려져있 다

.

¹²⁾카페인은뿌리와종자에는함유되어있지않지만

,

차잎의 경우종자의발아시에생성되며

,

^{계절별로는}^봄철보다^여름철

#본논문에관한문의는저자에게로

(

^전화

) 042-821-5928 (

^팩스

) 042-823-6566

(E-mail) [email protected]

(2)

에함량이증가한다

.

¹³⁾^이런^차의^{주요성분들은}^여러^가지^생리

활성과약리효과를나타내는것으로보고되고있다

.

^카테킨은^항

산화작용

,

^14,15)항암작용

,

^1,16)해독작용

,

¹⁷⁾혈당저하작용

,

¹⁸⁾항균작 용

,

^{충치예방효과}

,

^{노화억제작용}

,

¹⁹⁾^진정작용²⁰⁾^등에^효과가^있는

것으로알려져있으며

,

카페인에의한중추신경흥분

,

강심

,

이 뇨

,

항천식및대사항진

,

²¹⁾

aflatoxin

생성억제등의효과가계속 적으로밝혀지고있다

.

^이러한^녹차의^기능이^{밝혀지면서}^성분

에대한분석방법도여러방면으로시도되고있다

.

카테킨류의 일반적인분석법으로는비색법이알려져있지만잘사용되지않 고대신자외선분광법

,

²²⁾^{모세관전기영동법}

,

^23,24)

HPLC

^법8,12,25-27)

등이많이사용되고있다

.

최근에는

LC-MS

를이용하여녹차에

서²⁸⁾뿐아니라녹차를음용한사람의혈액이나뇨중에서도카

테킨류를분석하는연구도진행되고있다

.

^29,30)

차나무는광범위한분포와오랜기간을통해품종내에서형태 적또는생리생태적으로특유한형질변이를일으키는것으로알 려져있다

.

^그러므로^녹차는^생산지의^{재배환경이나}^채엽시기

,

제다법등에따라맛

,

색

,

향이다르고

,

이에따라등급도달라질 수있다

.

^녹차의^{등급판정이나}^서로^다른^제다법에^대한^품질의

관리는그동안주로관능시험에의존하였으나근래에는기기나 각종센서³¹⁾를이용한패턴인식법을활용하려는시도가활발하 다

.

패턴인식법은녹차의미지^31,32)또는기지^33-35)의다성분을변 량으로하여녹차의품질이나차종을판정할수있는방법으로주 로주성분분석

,

판별분석

,

군집분석등의통계적기법을활용한 다

.

^국내에서^시판되는^녹차의^경우^{채엽시기에}^따라^첫물차

,

^두

물차

,

^세물차

,

^네물차^또는^우전

,

^세작

,

^중작

,

^대작^등으로^구분

하고있는데이것이차의등급및가격을결정짓는중요한요소 가되고있다

.

^본^{연구에서는}^시판되는^{녹차로부터}^칸테킨류^및

카페인의함량을분석하고

,

^성분^함량과^생산지^및^{채엽시기와}

의관계를패턴인식법으로검토하였다

.

실험재료 및 방법

시약및기기

수소및탄소핵자기공명스펙트럼은

Bruker(NJ, USA)

의

AM-

300

및

AMX-500

을이용하여측정하였고화학적이동치는사용

한용매의피크를기준으로 δ

(ppm)

^으로^{표시하였다}

. HPLC

^는

Futecs HPLC system(NS-3000i integrated HPLC System, AT-4000 Column Oven, Korea)

및

ProntoSIL Eurobond C18 (250×4.0 mm, 5

^µ

m)(Bischoff Chromatography, Germany)

^컬

럼을사용하였다

. HPLC

분석에사용한

(+)-catechin(C)

표준품 은

Aldrich(MI, USA)

사에서 구입하였고

,

나머지 표준품

(-)- epigallocatechin(EGC), caffeine, (-)-epicatechin(EC), (-)-epigallocatechin gallate(EGCG), (-)-epicatechin gallate(ECG)

는 직접

분리하여그순도가

99%

이상되는것을사용하였다

.

시약은국

내외 특급 및일급 시약을 사용하였고

,

^물은

3

^차 ^증류수를

0.22

^µ

m

^{막여과기로}^여과하여^{사용하였다}

.

표준품의분리

건조된녹차잎

(Camellia sinensis) 20 kg

을메탄올로상온에

서일주일간냉침한후감압농축하여메탄올추출물

7 kg

을얻

었다

.

^추출물을^헥산과^증류수로^현탁시킨^후^물층을^에틸아세

테이트와부탄올로추출농축하여에틸아세테이트엑스

622 g

과 부탄올엑스

1942 g

을얻었다

.

부탄올엑스

760 g

을메틸렌클로라 이드

/

^메탄올^{혼합용매를}^{용출용매로}^사용하여^기울기^용리^방식

으로실리카겔컬럼

(70~230 mesh, 3 kg, 15.0×100 cm)

크로마 토그래피를실시하여

Fr-1, 2, 3, 4, 5, 6

등

6

개의분획으로나 누었다

.

^각각의^분획을^실리카^겔^컬럼^{크로마토그래피}

, RP-18

컬럼크로마토그래피및세파덱스컬럼크로마토그래피를반복 적으로실시하여정제한결과카페인과

4

종의카테킨을분리하 였다

.

^이들^{화합물들의}^{물리화학적}^성상

,

¹

H-NMR,

¹³

C-NMR,

MS

및선광도를종합검토하고표품과비교하여본결과각각

caffeine,

³⁶⁾

EC,

^37,38)

EGC,

³⁹⁾

EGCG,

³⁹⁾

ECG

³⁹⁾^로^동정할^수^있

었다

.

Caffeine(1)의분리 −

Fr-1(106 g)

를메탄올에수일방치하여 흰색의 결정을얻었다

(24.7 g):

¹

H-NMR(300 MHz, DMSO-d

₆

)

δ

: 7.97(1H,s), 3.86(3H,s), 3.38(3H,s), 3.19(3H,s)

¹³

C-NMR (125 MHz, DMSO-d

6

)

δ

: 154.5, 151.0, 148.1, 142.8, 106.6, 33.1, 29.3, 27.4.

(-)-epicatechin(2)의분리 −

Fr-2(5 g)

^를

50%

^메탄올을^용출액

으로세파덱스

LH-20

컬럼

(2.0×80 cm)

크로마토그래피를실시 하여세번째분획

(Fr-23, 2.7 g)

^을^농축하고^다시

RP-18

^컬럼^크

로마토그래피를실시하여 (2)

1.4 g

^을^{분리하였다}

: [

^α

]

D

=-41.3 (C: 1.0, in MeOH)

¹

H-NMR(300 MHz, DMSO-d

6

)

δ

: 9.11 (1H, 5-OH), 8.90(1H, s, OH-7), 8.81(1H, s, OH-4'), 8.72(1H, s, OH-3'), 6.89(1H, br s, H-2'), 6.66(2H, br s, H-5',6'), 5.89 (1H, d, J=2.2 Hz, H-8), 5.72(2H, d, J=2.2 Hz, H-6), 4.74(1H, br s, H-2), 4.66(1H, d, OH-3), 4.01(1H, t, J=3 Hz, H-3), 2.57 (2H, m, H-4)

¹³

C-NMR(125 MHz, DMSO-d

6

)

δ

: 156.6(C-5), 156.3(C-7), 155.8(C-9), 144.5(×2)(C-3',4'), 130.7(C-1'), 118.1 (C-6'), 114.9(C-5'), 114.8(C-2'), 98.6(C-10), 95.1(C-6), 94.2(C- 8), 78.1(C-2), 65.0(C-3), 28.3(C-4).

(-)-epigallocatechin(3) 및 (-)-epigallocatechin gallate(4) 의분리 −

Fr-4(300 g)

^중

34 g

^을^{메틸렌클로라이드}

/

^메탄올^혼합

용매를용출용매로사용하여기울기용리방식으로실리카겔컬 럼

(70~230 mesh, 980 g, 5.0×100 cm)

^{크로마토그래피를}^실시

하여

6

^개의^분획을^얻고^세^번째^분획인

Fr-43(6.5 g)

^을^메탄올

/

물혼합용매를용출용매로사용하여기울기용리방식으로

MCI

겔컬럼

(1.5×60 cm)

크로마토그래피하여(3)

1.65 g,

(4)

1.9 g

(3)

을분리하였다

.

(-)-epigallocatechin(3)

[

^α

]

D

=-60.0(C : 1.0, in MeOH)

¹

H-NMR(300 MHz, DMSO-d

6

)

δ

: 9.11(1H, 5-OH), 8.90(1H, s, OH-7), 8.71(2H, s, OH-3',5'), 7.95(1H, s, OH-4'), 6.37(2H, s, H-2'.6'), 5.88(1H, d, J=1.8 Hz, H-8), 5.71(1H, d, J=1.8 Hz, H-6), 4.66(1H, s, H-2), 4.62(1H, d, OH-3), 4.12 (1H, m, H-3), 2.56(2H, m, H-4)

¹³

C-NMR(125 MHz, DMSO- d

6

)

^δ

: 156.6(C-9), 156.3(C-7), 155.8(C-5), 145.4(×2)(C-3',5'), 132.2(C-4'), 129.8(C-1'), 106.1(×2)(C-2'.6'), 98.6(C-10), 95.1 (C-6), 94.1(C-8), 78.2(C-2), 65.1(C-3), 28.3(C-4).

(-)-epigallocatechin gallate(4)

[

^α

]

D

=-179(C : 1.0, in MeOH)

¹

H- NMR(300 MHz, DMSO-d

6

)

δ

: 6.83(2H, s, H-2''.6''), 6.42(2H, s, H-2'.6'), 5.94(1H, d, J=2.0 Hz, H-8), 5.84(1H, d, J=2.0 Hz, H-6), 5.38(1H, m, H-3), 4.97(1H, s, H-2), 2.64~2.98(2H, m, H-4)

¹³

C-NMR(125 MHz, DMSO-d

6

)

δ

: 165.4(C-7''), 156.6 (×2), 155.8, 145.8(×2), 145.5(×2), 138.7, 132.5, 128.8, 119.4, 108.8, 105.6, 97.5(C-10), 95.6(C-6), 94.5(C-8), 76.6(C- 2), 68.2(C-3), 25.9(C-4).

(-)-epicatechin gallate(5)의 분리 −

Fr-5(152 g)

^중

10 g

^을

RP-18

^컬럼^{크로마토그래피를}^실시하여

5

^개의^분획을^얻고

,

^이

중

Fr-52(2.7 g)

와

Fr-54(400 mg)

을각각메틸렌클로라이드

/

메탄 올혼합용매를용출용매로사용하여기울기용리방식으로실리 카겔

(70~230 mesh)

^컬럼^{크로마토그래피를}^실시하여

compound 5(970 mg)

을분리하였다

: [

α

]

D

=-160.6(C : 1.0, in MeOH)

¹

H- NMR(300 MHz, DMSO-d

6

)

^δ

: 6.88(1H, dd, J=7.2,1.8 Hz, H- 2'), 6.83(2H, s, H-2'', 6''), 6.76(1H, dd, J=7.2, 1.8 Hz, H-6'), 6.76(1H, d, J=7.2 Hz, H-5'), 5.95(1H, d, J=2.1 Hz, H-8), 5.84 (1H, d, J=2.1 Hz, H-6), 5.36(1H, m, H-3), 5.03(1H, s, H-2), 2.65~2.98(2H, m, H-4)

¹³

C-NMR(125 MHz, DMSO-d

6

)

δ

: 165.3(C-7''), 156.6(×2)(C-5,7), 155.7(C-9), 145.5(×2)(C-3'',5''), 144.8(×2)(C-3',4'), 138.7(C-4''), 129.5(C-1'), 119.3(C-1''), 117.7 (C-6'), 115.2(C-5'), 114.3(C-2'), 108.7(2)(C-2'',6''), 97.3(C-10), 95.6(C-6), 94.4(C-8), 76.6(C-2), 68.2(C-3), 25.8(C-4).

카테킨류및카페인의추출효율

녹차로부터성분을추출시최적의온도및시간을설정하기 위하여추출시간을

3, 6, 9, 12

^분

,

^{추출온도를}

40, 60, 80, 100

^o

C

등으로변화시키면서추출하고

,

추출된카테킨류및카페인함 량을분석하였다

.

HPLC를이용한녹차중카테킨류및카페인의분석 실험에사용한녹차는

2004

^년

1

^월부터

5

^월^사이^시중에서^무

작위로구입하였으며제주산

9

^종

(S1-S9),

^보성산

14

^종

(S10-S23)

등총

23

종이었다

.

시료의확인은전문가의자문을거쳤고

,

표본 은충남대학교약학대학약품분석실에보관하였다

.

시판녹차

3 g

^을

80

^o

C

^의^물

150 ml

^에

6

^분간^가온^침출하여^시

료액을

0.2

^µ

m

^{막여과기로}^여과하여^분석에^{사용하였다}

.

^크로

마토그래피를위하여

ProntoSIL Eurobond C18

컬럼에이동상

A(0.1%

^초산

)

^와

B(

^{아세토니트릴}

)

^를^분당

1 ml

^의^속도로

0

^분

10%B, 12

분

15%B, 25

분

30%B

와같이기울기용리하였다

.

이때 시료주입량은

30

µ

l,

오븐온도는

35

^o

C,

검출파장은

UV 280 nm

였다

.

^{카테킨류와}^카페인^표준품을^물에^녹여

EGC 0.1~2 mg/

ml, C 0.01~0.1 mg/ml, EC 0.01~0.5 mg/ml, EGCG 0.1~2 mg/ml, ECG 0.1~1 mg/ml, caffeine 0.1~1 mg/ml

의농도로표 준액을조제하고이것을이용하여분석법의타당성을검증하였다

.

패턴인식법을위한다변량자료처리

시료의물추출액을각각

HPLC

^로^분석하고^그^{크로마토그램}

으로부터피크의면적를구하여검량선으로부터각성분의농도 를계산하였다

.

현재표준녹차가제공되고있지않으므로모든 시료의성분에대한중위수를표준녹차의성분함량으로하였다

.

다변량자료는인터넷으로제공되는

R

통계프로그램⁴⁰⁾으로처 리하였으며유의수준은

0.01

^로^하였다

.

유사도의평가 − 유클리드거리

(d),

^코사인

(C),

^면적비

(A),

^상관

계수

(r)

등의척도를이용하여표준녹차와각시료와의유사도를 평가하였다

.

유클리드거리

코사인

면적비

상관계수

계층적군집방법을이용한시료의군집분석 − 산지또는채엽 시기의정보를무시하고성분의차이만으로각각의시료가어떤 군집을형성하는지를관찰하였다

.

계층적방법은사전에군집수 를정하지않고단계적으로군집을형성하는방법으로

R

^프로그

램

cluster

^팩키지의

agnes(agglomerative nesting), hclust (hierarchical clustering), diana(divisive analysis clustering)

등 을사용하였다

.

d

ir

=

(

X

ik

-X

rk)² k=1

∑

m ^1/2

C

ir

= X

ik⋅

X

rk k=1

∑

m

X

ik2 k=1

∑

m

⎝ ⎠

⎜ ⎟

⎛ ⎞

X

rk2 k=1

∑

m

⎝ ⎠

⎜ ⎟

⎛ ⎞ ---

A

ir

= 2 min X

( ik,

X

rk) k=1

∑

m

X

ik

+X

rk ( ) k=1

∑

m

---

r

ir

=

(

X

ik

-X

i)(

X

rk

-X

r) k=1

∑

m

X

ik

-X

i

( )² (

X

rk

-X

r)² k=1

∑

m k=1

∑

m

---

(4)

판별분석에의한시료의채엽시기및산지평가 − 산지또는채 엽시기의정보와성분의차이를이용하여판별식을계산하고이 로부터산지또는채엽시기를추정하였다

.

판별분석은

R

프로그 램

MASS

^팩키지의

linear discriminant analysis(LDA)

^를^이용하

였다

.

실험결과 및 고찰

최적분석조건의설정

C, EC, EGCG, EGC, ECG

^등^카테킨류

5

^종^및^카페인에^대

한최적분석조건을설정하기위하여이동상의변화에따른분 리능을검토하였다

. 10%B

를이동상으로한등용매용리로

6

개의 성분을모두분리할수있었지만분리시간이

60

^분^이상으로^너

무길었고

, 20%B

를이동상으로한등용매용리로는분리가완전

하지못하였다

.

^이상의^조건을^고려하여^이동상

A

^와

B

^를

0

^분

10%B, 12

^분

15%B, 25

^분

30%B

^와^같이^기울기^{용리하였을}^경

우

6

개의성분을

25

분이내로완전히분리할수있었다

(Fig. 1).

최적추출시간및온도의설정

시료로부터카테킨류와카페인의최적추출조건을검토하였다

(Fig. 2).

^차는^물에^우려서^마시는^것을^감안하여^{추출용매는}^물

을이용하였다

. 80

^o

C

에서

3

분정도추출할경우

EGCG

를제외 한대상성분이대부분추출되었다

. EGCG

도

6

분이면거의추 출이되므로추출시간을

6

^분으로^하였다

.

^녹차^중의^{대상성분은}

온도가높을수록잘추출되었으며추출시간을

6

분으로하였을 경우

60

^o

C

에서

EGCG

를제외한대상성분이대부분추출되었 다

.

^온도를

80

^o

C

^로^하였을^경우

EGCG

^를^포함한^{대상성분이}^모

두

6

분이내에추출되므로추출조건을

80

^o

C, 6

분으로결정하였다

.

Fig. 1 −

HPLC chromatograms of 5 catechins and caffeine from (a) green tea extract and (b) mixed standards. HPLC: column; Prontosil

Eurovond C18, mobile phase; A 0.1% acetic acid in water, B acetonitrile, gradient elution; 0 min 10%B, 12 min 15%B, 25 min 30%B

for 1.0 ml/min, detection; UV 280 nm. Peaks: 1. EGC, 2. C, 3. caffeine, 4. EC, 5. EGCG, 6. ECG.

(5)

직선성,정확성,정밀성

각각의표준물질을증류수에녹인표준용액을

HPLC

로분석 하여각성분에해당하는피크의넓이로부터각성분의검량선 을작성하였다

.

^검량선의^{상관계수는}^모두

0.99

^이상으로^양호한

직선성을나타내었다

.

각성분에대한정확성과정밀성은저농 도

,

두개의중농도

,

고농도에해당하는품질관리시료로평가하 였으며일내정밀성은

5

^회의^측정

,

^{일간정밀성은}

5

^일간의^측정으

로하였다

. Table I

에서보는바와같이대상성분에대하여일내

정밀성은

9%

이하

,

일간정밀성은

10%

이하

,

정확성은

98%~

105%

^로^비교적^양호하여^본^방법은^녹차^중

EGC, C, caffeine, EC, EGCG, ECG

등

6

개성분의분석에타당함을알수있었다

.

녹차중카테킨류및카페인함량분석

시판녹차

23

종에대해서

HPLC

의방법으로분석하고카테킨

류및카페인의함량을조사하였다

.

^총

23

^개^{녹차시료에}^대한

EGC, C, EC, EGCG, ECG

^및

caffeine

^의^평균값은^각각

2.20, 0.022, 0.79, 5.03, 1.22

및

2.56

으로이미발표된자료와비슷하 였다

.

^보성산^녹차는^제주산^녹차에^비해^각^성분^함량이^약간

낮은것으로나타났으나통계적유의성은없었다

(p>0.01).

^보성

산녹차의경우함량이낮게나온이유는채엽시기가늦은시료 가절반섞여있었기때문으로풀이된다

.

^녹차의^품질^및^가격은

채엽시기에따라달라지는데첫물차인경우대체로

5

^월^초순이

기준이되고있다

.

시료를채엽시기에따라

5

월초순이전과중 순이후로나누어분석하였을경우

5

월중순이후에채엽된녹차 의

EGCG, ECG

^및

caffeine

^함량이^{초순이전에}^채엽된^녹차에

비해유의적으로감소하였다

(p<0.01).

이결과는

5

월차는

4

월차 에비해

EGCG, ECG

및

caffeine

의함량이감소한다는김등¹²⁾ 의발표와 유사하였다

.

^하지만 ^총^카테킨^함량

(EGC, C, EC, EGCG, ECG

성분의합

)

은채엽시기가늦을수록감소하여

(9.88%

→

6.91%, p<0.01)

^김^등¹²⁾^의^발표와는^다르게^나타났다

.

패턴인식법에의한다변량분석

시판녹차

23

^종에^대해서^조사된^카테킨류^및^카페인의^함량

을변수로하여다변량분석을실시하였다

.

^각^성분에^대한

23

^종

시료의중위수로부터표준녹차의성분함량을구하고이를이용 하여각시료의유사도를평가하였다

.

^유클리드^거리

,

^코사인

,

^면

적비

,

^상관계수^등의^유사성^척도는

23

^개의^시료가^{표준녹차와}

상당한동질성을가지고있는것을보여주었으며

,

산지나채엽

시기에따른특징은표현되지않았다

(Fig. 3).

^{성분함량을}^기초한

Table I −

Precision and accuracy for the analysis of 6 standards by HPLC method

Compound Concentration (mg/m l ) intraday interday Precision (%) Accuracy (%) EGC

0.100 8.68 9.98 105.1

0.500 1.46 9.88 102.4

1.000 1.46 6.87 099.8

2.000 2.11 1.95 099.9

C

0.010 0.94 5.56 103.4

0.025 3.57 7.74 102.2

0.050 1.91 3.30 099.9

0.100 1.84 3.68 099.5

Caffeine

0.100 0.94 6.51 105.4

0.250 3.57 2.68 102.1

0.500 1.91 1.55 100.3

1.000 1.84 4.19 101.2

EC

0.010 1.12 9.01 104.3

0.050 7.37 7.04 102.1

0.100 1.42 2.58 099.2

0.500 1.03 2.25 098.3

EGCG

0.100 2.89 4.87 100.7

0.500 4.26 7.85 101.2

1.000 1.38 5.41 099.1

2.000 1.52 2.90 099.5

ECG

0.100 4.71 9.01 101.4

0.250 8.62 7.04 098.2

0.500 2.13 2.58 099.5

1.000 1.70 2.58 097.8

Fig. 2 −

Extraction efficient of (a) time at 80

^o

C, and (b) temperature

for 6 min on the extraction of catechins and caffeine from

green tea samples.

(6)

계층적군집방법에서는

23

개의녹차시료가크게두개의군집으 로나누어졌다

.

^제

1

^군집은

S1-S9, S14, S16, S19, S21-S23

^등

15

개의시료로

,

^제

2

^군집은^나머지

8

^개의^시료로^{구성되었다}

.

^이^군

집분류에는주로성분

EGCG, ECG

및

caffeine

함량의영향이

컸으며

, Table II

^에서^보는^바와^같이^{산지보다는}^{채엽시기가}^중

요한인자로작용한것으로파악된다

(Fig. 4).

^제

1

^군집에는^다수

의

5

월초순이전채엽된시료와제주산이포함되는것으로나타

났다

. S2-S5

^에^대한^정확한^{채엽시기는}^알려져^있지^않지만^군

집분석에서제

1

군집에속하는것으로보아

5

월초순이전에채엽 된것일가능성이높은것으로판단된다

.

알려진산지또는채엽시기정보와성분함량을이용하여판

별분석으로부터산지또는채엽시기를추정하였다

. Fig. 5a

는채

엽시기가불확실한시료

S2-S5

^를^제외한^나머지^시료를^기준으

로채엽시기와성분함량으로판별식을작성하고이판별식을이 용하여각시료의채엽시기를추정한것이다

.

판별식의값

(Out)

이양수인것은채엽시기가

5

^월^{초순이전이고}

,

^음수인^것은

5

^월

중순이후이다

.

^이^판별식을^{이용하였을}^경우^{채엽시기가}

5

^월^초

순이전이었던시료

(

○

)

는모두양수로

,

채엽시기가

5

월중순이 후였던시료

(

^△

)

^는

S21

^을^{제외하고는}^모두^음수로^분류되어^약

95%

의정확성을보였다

. S21

의채엽시기는

6

월하순이나함수 의값은양수로나타나채엽시기가아주늦어질경우판별식에 또다른인자가영향을미치는것으로생각할수있다

.

^채엽시기

Fig. 3 −

(a) Cosine, (b) area ratio, (c) correlation and (d) distance between standard green tea and green tea samples for similarity measurements.

Table II −

Changes of catethins and caffeine contents in green tea from different cultivation region and harvest period

EGC C EC EGCG ECG Caffeine

All samples 2.20±0.77 0.022±0.010 0.79±0.27 5.03±1.38 1.22±0.46 2.56±0.45

Cultivated in Jeju 2.62±1.04 0.022±0.010 0.92±0.27 5.82±0.65 1.30±0.46 2.84±0.46

Bosung 1.93±0.36 0.021±0.011 0.71±0.23 4.52±1.50 1.17±0.47 2.39±0.36

Harvested

before May 10. 1.91±0.53 0.023±0.011 0.83±0.21 5.75±0.89 1.49±0.38 2.76±0.40

after May 10. 1.95±0.39 0.019±0.009 0.57±0.24 3.51±1.24 0.87±0.43 2.18±0.38

Values represent mean±standard deviation (n=3) in %.

*p<0.01 when compared with the data of green tea harvested after May 10.

(7)

가불확실하였던시료

(+)

는모두함수의값이양수로

5

월초순 이전에채엽된것으로추정되며이것은군집분석의결과와도일 치하였다

.

^녹차의

6

^개의^성분^중

EGCG

^가^{채엽시기를}^추정하는

판별함수의값에가장큰영향을미치고다음으로

C, EC, EGC

의순으로영향을미치는것으로나타났다

. Fig. 5b

^는^산지와^성

분함량으로판별식을작성하고이판별식을이용하여각시료의 산지를추정한것이다

.

판별식의값이양수인것은제주산

,

음수 인것은보성산이다

.

^이^판별식을^{이용하였을}^경우

S9

^를^제외한

제주산

(

○

)

은모두양수로

, S23

을제외한보성산

(

△

)

은모두음 수로분류되어약

91%

의정확성을보였다

.

무작위로추출된

S7, S18, S19

^를^동일한^{판별식으로}^{판별하였을}^경우^각각^제주산

(S7),

보성산

(S18, S19)

으로올바르게판별되었다

.

산지를추정하는판 별식에는채엽시기를추정한판별식과는달리녹차의

6

개성분 이고르게영향을미친것으로나타났다

.

^이것은

Table II

^에서^나

타난바와같이산지의차이에대해

6

개의성분모두가유의한 차이를보이지않았다는것과도일맥상통한다고생각된다

.

결 론

본연구에서는녹차의성분을분석하고성분함량과녹차의 산지및채엽시기와의관계를패턴인식법으로검토하였으며다 음과같은결론을얻었다

.

1.

^녹차의^{주요성분인}

C, EC, EGCG, EGC, ECG

^등^카테킨

류

5

종및카페인을

HPLC

로분석하였다

.

이때역상컬럼에서 용매

A(0.1%

^초산

)

^와

B(

^{아세토니트릴}

)

^를

0

^분

10%B, 12

^분

15%B, 25

^분

30%B

^로^기울기^{용리하였고}

,

^{검출파장은}

280 nm

였다

.

2.

산지차이에대해녹차의각성분은유의적인차이를보이

지않았으나

,

^{채엽시기에}^대하여

EGCG, ECG

^및

caffeine

^함량

은채엽시기가늦어짐에따라유의적으로감소하였다

(p<0.01) 3.

유사성척도로판단할경우

23

개의시료는성분상으로표 준녹차와상당한동질성을가지고있는것으로나타났다

.

^판별

식을이용하여채엽시기및산지를추정하였을경우채엽시기는 약

95%,

산지는약

91%

의정확성을보여본방법은녹차의산 지및채엽시기를어느정도추정할수있을것으로판단된다

.

Fig. 4 −

The hierarchical clustering analysis of 23 green tea extract, harvested in Jeju (

^●

) or Bosung (

^○

). The harvest period was before May 10. (

■

) or after May 10. (

□

), or not known (×).

Fig. 5 −

Results of linear discriminant analysis of the 23 green tea extract for the estimation of (a) harvest period and (b) cultivated region. Harvest period:

^○

before May 10,

^△

after May 10, + estimated. Cultivated region:

○

Jeju,

△

Bosung, + estimated.

(8)

감사의 말씀

본연구결과는보건복지부의한의학연구개발사업

(B070044)

의 지원에의하여수행되었으며이에감사드린다

.

^또한^기기^측정

에도움을주신기초과학지원연구원에감사드린다

.

참고문헌

1) Zhang, X., Zhang, H., Tighiouart, M., Lee, J. E., Shin, H. J., Khuri, F. R., Yang, C. S., Chen, Z. and Shin, D. M. : Synergistic inhibition of head and neck tumor growth by green tea (-)- epigallocatechin-3-gallate and EGFR tyrosine kinase inhibitor.

Int. J. Cancer

¹²³

, 1005 (2008).

2) Anderson, M. W., Goodin, C., Zhang, Y., Kim, S., Estensen, R. D., Wiedmann, T. S., Sekar, P., Buncher, C. R., Khoury, J. C., Garbow, J. R., You, M. and Tichelaar, J. W. : Effect of dietary green tea extract and aerosolized difluoromethylornithine during lung tumor progression in A/J strain mice. Carcino- genesis

²⁹

, 1594 (2008).

3) Jochmann, N., Lorenz, M., Krosigk, A., Martus, P., Boehm, V., Baumann, G., Stangl, K. and Stangl, V. : The efficacy of black tea in ameliorating endothelial function is equivalent to that of green tea. Br. J. Nutr.

99

, 863 (2008).

4) Cho, H. S., Kim, S., Lee, S. Y., Park, J. A., Kim, S. J. and Chun, H. S. : Protective effect of the green tea component, - theanine on environmental toxins-induced neuronal cell death.

Neurotoxicol.

29

, 656 (2008).

5) Graham, H. N. : Green tea composition, consumption, and polyphenol chemistry. Prev. Med.

²¹

, 334 (1992).

6) Wee, J. H., Moon, J. H. and Park, K. H. : Catechin content and composition of domestic tea leaves at different packing time.

Kor. J. Food Sci. Technol.

31

, 20 (1999).

7) Oh, M. J. and Hong, B. H. : Variation in chemical components of Korean green tea (Camellia sinensis L.) resulted from developing stages and processing recipe. Kor. J. Crop Sci.

⁴⁰

, 518 (1995).

8) Oh, M. J. and Hong, B. H. : Variation of pectin, catechins and caffeine contents in Korean green tea (Camellia sinensis L.) by harvesting time and processing recipe. Kor. J. Crop Sci.

40

, 775 (1995).

9) Nakagawa, M. and Buruya, G. M. : Differences in amino acid, tannin, total nitrogen contents in leaves of cultivated species of green tea. Bull. Japan Tea-Tech. Assoc.

⁴⁸

, 84 (1975).

10) Park, J. H. : Studies on chemical composition in Korean native tea plants. PhD thesis, Chonnam National Univ. Kwangju, Korea (1997).

11) Ko, Y. S. and Lee, I. S. : Quantitative analysis of free amino acids and free sugars in steamed and roasted green tea by

HPLC. J. Kor. Soc. Food Nutr.

14

, 301 (1985).

12) Kim, S. H., Han, D. S. and Park, J. D. : Changes of some chemical compounds of Korean (Posong) green tea according to harvest periods. Kor. J. Food Sci. Technol.

³⁶

, 542 (2004).

13) Kim, J. T. : Science and culture of tea, Borim Press. Seoul, Korea, 160 (1996).

14) Munoz-Munoz, J. L., Garcia-Molina, F., Molina-Alarcon, M., Tudela, J., Garcia-Canovas, F. and Rodriguez-Lopez, J. N. : Kinetic characterization of the enzymatic and chemical oxidation of the catechins in green tea. J. Agric. Food Chem.

56

, 9215 (2008).

15) Gao, D. F., Zhang, Y. J., Yang, C. R., Chen, K. K. and Jiang, H. J. : Phenolic antioxidants from green tea produced from Camellia taliensis. J. Agric. Food Chem.

56

, 7517 (2008).

16) Shimizu, M., Weinstein, I. B. and Moriwaki, H. : Targeting receptor tyrosine kinases and multiple downstream signaling pathways by green tea catechin EGCG. Cancer: Disease Prog.

Chemoprev. 203 (2007).

17) Lee, S. J., Kim, M. J. and Yoon, Y. H. : Effect of Korean green tea, oolong tea and black tea beverage of the removal of cadmium and antioxidative detoxification in cadmium administered rats. The 3rd International Symposium on Green Tea. 21 (1995).

18) Asai, H., Ogawa, K., Hara, Y. and Nakamura, K. : Effect of alumina-tea catechin complex on the blood sugar in spontaneous diabetic mice. Clin. Rep.

²¹

, 163 (1987).

19) Chung, H. Y. and Yokozawa, T. : Studies on antioxidative and antimutagenic mechanism of epicatechin 3-O-gallate isolated from green tea. The 3rd International Symposium of Green Tea. 65 (1995).

20) Adachi, N., Choi, Y. H., Suenaga, R., Tomonaga, S., Denbow, D. M. and Furuse, M. : Green tea component, (-)- epigallocatechin gallate, but not L-theanine, has sedative effects in chick under acute stress conditions. Cur. Topics Nutraceut. Res.

5

, 107 (2007).

21) Buchanan, R. L. and Fletcher, A. M. : Methylxanthine inhibition of aflatoxin production. J. Food Sci.

⁴³

, 654 (1978).

22) Yamauchi, Y., Nakamura, A., Kohno, I., Kitai, M., Hatanaka, K.

and Tanimoto, T. : Simple and rapid UV spectrophotometry of caffeine in tea coupled with sample pre-treatment using a cartridge column filled with polyvinylpolypyrrolidone (PVPP).

Chem. Pharm. Bull.

56

, 185 (2008).

23) Huang, Y., Chiang, C., Lin, Y., Liu, K., Hu, C., Bair, M. and Chang, H. : Capillary electrophoretic separation of biologically active amines and acids using nanoparticle-coated capillaries.

Electrophoresis

²⁹

, 1942 (2008).

24) Lee, B. L. and Ong, C. N. : Comparative analysis of tea

catechins and theaflavins by high-performance liquid chro-

matography and capillary electrophoresis. J. Chromatogr. A

(9)

881

, 439 (2000).

25) Woo, H. S., Choi, H. J., Han, H. S., Park, J. H., Son, J. H., An, B. J., Son, G. M. and Choi, C. : Isolation of polyphenol from green tea by HPLC and its physiological activities. Kor. J. Food Sci. Technol.

³⁵

, 1199 (2003).

26) Spacil, Z., Novakova, L. and Solich, P. : Analysis of phenolic compounds by HPLC and ultra performance liquid chromatography. Talanta

76

, 189 (2008).

27) Sultana, T., Stecher, G., Mayer, R., Trojer, L., Qureshi, M. N., Abel, G., Popp, M. and Bonn, G. K. : Quality assessment and quantitative analysis of flavonoids from tea samples of different origins by HPLC -DAD-ESI-MS. J. Agric. Food Chem.

56

, 3444 (2008).

28) Pongsuwan, W., Bamba, T., Harada, K., Yonetani, T., Kobayashi, A. and Fukusaki, E. : High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS). J.

Agric. Food Chem.

⁵⁶

, 10705 (2008).

29) Ito, H., Gonthier, M., Manach, C., Morand, C., Mennen, L., Remesy, C. and Scalbert, A. : Polyphenol levels in human urine after intake of six different polyphenol-rich beverages. Br. J.

Nutrition

⁹⁴

, 500 (2005).

30) Li, C., Meng, X., Winnik, B., Lee, M., Lu, H., Sheng, S., Buckley, B. and Yang, C. : Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry. Chem. Res. Toxicol.

¹⁴

, 702 (2001).

31) Chen, Q., Zhao, J. and Vittayapadung, S. : Identification of the green tea grade level using electronic tongue and pattern recognition. Food Res. Int.

⁴¹

, 500 (2008).

32) Chen, Q., Zhao, J., Zhang, H., Liu, M. and Fang, M. : Qualitative identification of tea by near infrared spectroscopy based on soft independent modelling of class analogy pattern recognition. J. Near Inf. Spectrosc. Copy

¹³

, 327 (2005).

33) Dou, J., Lee, V. S. Y., Tzen, J. T. C. and Lee, M. R. : Identification and comparison of phenolic compounds in the preparation of oolong tea manufactured by semifermentation and drying processes. J. Agric. Food Chem.

55

, 7462 (2007).

34) Alcazar, A., Ballesteros, O., Jurado, J. M., Pablos, F., Martin, M. J., Vilches, J. L. and Navalon, A. : Differentiation of Green, White, Black, Oolong, and Pu-erh teas according to their free amino acids content. J. Agric. Food Chem.

⁵⁵

, 5960 (2007).

35) Del Rio, D., Stewart, A. J., Mullen, W., Burns, J., Lean, M. E. J., Brighenti, F. and Crozier, A. : HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea. J. Agric. Food Chem.

⁵²

, 2807 (2004).

36) Talebpour, Z., Maesum, S., Jalali-Heravi, M. and Shamsipur, M. : Simultaneous determination of theophylline and caffeine by proton magnetic resonance spectroscopy using partial least squares regression techniques. Anal. Sci.

¹⁹

, 1079 (2003).

37) Kwon, Y. M., Lee, J. H. and Lee, M. W. : Phenolic compounds from barks of Ulmus macrocarpa and its antioxidative activities. Kor. J. Pharmacogn.

³³

, 404 (2002).

38) Lee, Y. A. and Lee, M. W. : Tannins from Rubus coreanum. Kor.

J. Pharmacogn.

26

, 27 (1995).

39) Cho, Y. J., An, B. J. and Choi, C. : Isolation and enzyme inhibition of tannins from Korean green tea. Kor. Biochem. J.

26

, 216 (1993).