Protective Effect of Gardenia jasminoides Against Carbon Tetrachloride-Induced Acute Hepatotoxicity

(1)

사염화탄소 유도 급성 간독성 모델에서 치자의 간 보호 효과

신전규·김효연*^·이선미*^,#

경희대학교 생명과학부, *성균관대학교 약학대학

(Received December 28, 2009; Revised January 5, 2010; Accepted January 5, 2010)

Protective Effect of Gardenia jasminoides Against Carbon Tetrachloride-Induced Acute Hepatotoxicity

Jun-Kyu Shin, Hyo-Yeon Kim* and Sun-Mee Lee*^,#

College of Life Science, Kyung Hee University, Yongin 446-701, Korea

*School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea

Abstract

— Gardenia jasminoides is one of the most widely used herbal preparations for the treatment of liver disorders.

This study evaluated the potential beneficial effect of G. jasminoides in a mouse model of carbon tetrachloride (CCl

4

)- induced liver injury. The mice were treated intraperitoneally with CCl

4

(10

^µ

l/kg). They received G. jasminoides (30, 100, 300 mg/kg) 48 h, 24 h and 2 h before and 6 h after administering CCl

4

. The serum activities of aminotransferase and the hepatic level of malondialdehyde were significantly higher 24 h after the CCl

4

treatment, while the concentration of reduced glutathione was lower. These changes were attenuated by G. jasminoides. CCl

4

increased the level of circulating tumor necrosis factor-

^α

(TNF-

^α

) markedly, which was reduced by G. jasminoides. The levels of hepatic inducible nitric oxide syn- thase (iNOS) and cyclooxygenase-2 (COX-2) protein expression were markedly higher after the CCl

4

treatment. G. jas- minoides diminished these alterations. CCl

4

increased the level of TNF-

^α

, iNOS and COX-2 mRNA expressions, and these increases were attenuated by G. jasminoides. These results suggest that G. jasminoides alleviates CCl

4

-induced liver injury, and this protection is likely due to the reduced oxidative stress and the downregulation of proinflammatory mediators.

Keywords □

carbon tetrachloride, Gardenia jasminoides, hepatotoxicity, inflammation

간질환의원인은다양한것으로알려져있으며병인학적으로 볼때바이러스에기인된간질환

,

약물

(

독성물질

)

중독에기인된 간질환및담도기능부전에의한간질환등으로분류할수있다

.

이러한간질환들은간세포파괴에의한해독능력저하와담즙분 비억제에의한독성물질배설장애

,

비타민

B

1

, B

2및

C

의흡수 억제및축적저하에의한전신권태감

,

^소양감

,

^식욕부진^및^피

로등의임상증상을나타낸다

.

¹⁾^현재^{국내외적으로}^사용되고^있

는간질환치료제로는합성약물인인터페론과

malotilate

가있 으나이들은간독성물질에대한보호작용만으로그의유효성을 인정받고있는수준이며

,

^부작용^또한^빈번히^나타난다

.

^또한^마

리아엉겅퀴의활성성분인

silymarin

과천연물유래합성간질환 치료제인

biphenyl dimethyl dicarboxylate

^의^개발로^천연물에^대

한관심이고조되고있으나

,

²⁾^대다수의^천연물의^경우^유효^용

량뿐아니라정확한약리효과에대한기초연구결과가미약하다

.

사염화탄소는 지방성 퇴행

(fatty degeneration),

³⁾ 섬유증

(fibrosis),

⁴⁾간세포사멸및간암등을유발시키며

,

⁵⁾독성물질이 간세포에미치는영향과그대사과정을규명하는데대표물질로 서주로사용되어오고있다

.

사염화탄소는

1

차적으로일산소첨 가효소계

(mixed function oxidase system)

^의^활성에^의해

CCl

3

라디칼로활성화되어세포막의지질과산화를일으켜간독성을 유발하며

,

⁶⁾

2

차적으로는간장대식세포인

Kupffer cell

을활성화 시켜염증매개인자를생성한다

.

⁷⁾

치자는꼭두서니과에 속한상록관목인치자나무

(Gardenia jasminoides)

의성숙한과실을건조한것이다

.

치자의효능은열 독을없애고황달을낫게하며소갈을멎게하며눈이붓고아 픈것

,

^문둥병

,

^창양

(

^瘡瘍

)

^을^낫게^하고^지충

(

^地蟲

)

^의^독을^없앤

다고알려져있다

.

치자의추출물은

hexobarbital

의수면효과를 현저히연장시키고

,

⁸⁾^생쥐의^운동성을^{감소시키며}^{체온강하와}^부

교감신경계활성화를통하여혈압강하작용도나타낸다

.

^치자의

활성성분인

geniposide

는α

-naphthylisothiocyanate

로유도한간

#본논문에관한문의는저자에게로

(

^전화

) 031-290-7712 (

^팩스

) 031-292-8800 (E-mail) [email protected]

종설

(2)

독성모델에서혈중

bilirubin, ALT

^및

AST

^등의^생화학적^수치

뿐만아니라병리학적조직병변을개선시켰으며

,

⁹⁾

genipin

^과

crocetin

도담즙분비를증가시켰다

.

¹⁰⁾또한

genipin

은위산의분 비를촉진하고항산화작용및

nitric oxide(NO)

^생성^저해를^통

해소염작용을나타낸다

.

¹¹⁾

따라서

,

저자등은본연구에서사염화탄소급성간독성모델 을사용하여치자추출물의간장보호작용을확인하고

,

^더^나아

가산화적손상과염증매개인자를중심으로작용기전도알아보 고자하였다

.

실험방법

실험동물

실험동물은대한바이오링크

(

주

)

로부터생후

8

주수컷

ICR

생 쥐를 공급받아 온도

23±1

^o

C,

상대습도

55±15%, 300~500 Lux

^및

12

^시간^간격으로^명암이^조절되는^{성균관대학교}^약학대

학동물사육실에서

7

일이상순화시킨후육안적증상을관찰 하여정상적인동물만을실험에사용하였으며

,

^{실험동물용}^고형

사료

(Dae-Han Biolink, Korea)

^및^물은^자유롭게^{섭취시켰다}

.

시료의추출및제조

실험에사용된치자는약초약업사

(

^주

)

^에서^구입하여^시료로^사

용하였다

.

조절

(

操切

)

한치자

500 g

을

70% EtOH 2.5 l

에가한 후실온에서

24

^시간^{냉침하였다}

.

^냉침^후

70% EtOH 2.5 l

^로

3

회반복하여환류추출하였고

,

^추출하여^얻은^총^추출액을^여과

후감압농축후동결건조하여치자시료

125 g(

수율

25%)

얻어 기밀용기에보존후실험에사용하였다

.

약물및사염화탄소투여

약물투여는치자

(30, 100

^및

300 mg/kg)

^를^{생리식염수에}^용해

시킨후

,

^{사염화탄소}^투여

48

^시간

, 24

^시간

, 2

^시간^전^및^투여^후

6

시간후에경구투여하였다

.

사염화탄소는올리브유에희석

(1 : 999, v/v)

^하여^복강투여^하였으며

,

^최종^용량은

10

^µ

l/kg

^이었

다

.

모든실험동물은사염화탄소투여후

24

시간에채혈한후간 을적출하여실험에사용하였다

.

혈청아미노산전이효소활성

혈청중

alanine

전이효소

(ALT)

와

aspartate

전이효소

(AST)

의 활성도는 각각

ALT

^및

AST assay kit(IVDLab Co., Ltd., Korea)

를이용하여

UV spectrophotometer(Shimadzu, Japan)

으 로흡광도를측정하였다

.

조직학적분석

간의좌엽부분을

10%

중성완충포르말린으로고정시킨후

파라핀에넣고

5

^µ

m

^의^관상^절편으로^{제작하였다}

. Xylene

^으로

파라핀을제거시키고

,

^알코올로^{친수화시킨}^후

hematoxylin

^과

eosin

으로염색하여광학현미경을통해조직병리학으로간조직

을관찰하였다

.

지질과산화물및glutathione함량

간장 내 지질 과산화 함량은

thiobarbituric acid reactive

substances

의 형광법으로 측정하였으며

,

표준물질로서는

malondialdehyde(1,1,3,3-tetraethoxypropane, Sigma, USA)

를 사용하였다

.

¹²⁾^총

glutathione

^은^간조직을

1% picric acid 5

^배^부

피에

glutathione reductase, yeast glutathione reductase, 5,5'- dithio-bis(2-nitrobenzoic acid),

및

NADPH

를가하여흡광도변 화량을이용하여측정하였고

,

¹³⁾^단백질^농도는

BCA

^TM

Protein assay kit(Pierce, USA)

를사용하여측정하였다

.

¹⁴⁾

혈중TNF-α농도

Tumor necrosis factor-

α

(TNF-

α

)

농도는

TNF-

α

ELISA assay kit(BD Biosciences, USA)

^를^사용하여^{정량하였다}

.

Western blot분석

정량한단백질을

SDS-PAGE

^로^분리한^후

Semi-Dry Trans- Blot Cell(Bio-Rad Laboratories, USA)

^를 ^이용하여

PVDF (Polyvinyllidene fluoride) membrane(Millipore, USA)

에전기영 동하고

membrane

^을

TBS/T(Tris-Buffered Saline/Tween-20)

^로

세척한후

5%(w/v) skim milk

^를^넣은

TBS/T

^로^상온에서

1

^시간

동안

blocking

하였다

. 1

차항체와

4

^o

C

에서

12

시간반응시킨후

, horseradish peroxidase-conjugated 2

^차항체에 ^반응시켜

ECL detection system(iNtRON Biotechnology Co., Ltd., Korea)

를 사용하여발색시켰다

.

각각의 밴드는

ImageQuant

^TM

TL (Amersham Biosciences/GE Healthcare, USA)

^{밀도측정법으로}

평가하였다

.

^사용한

1

^차항체는 ^다음과^같다

: inducible nitric oxide synthase(iNOS, 1 : 500

희석

, Transduction Laboratories, USA), cyclooxygenase-2(COX-2, 1 : 1000

^희석

, Cayman, USA)

및 β

-actin(1 : 5000

희석

, Sigma, USA).

역전사-중합효소연쇄반응(RT-PCR)

Total RNA 2

µ

g

을

Oligo(dT)

12-18

prime

와

SuperScript

^TM

ii RNase H

^-

Reverse Transcriptase(Invitrogen Tech-Line

^TM

, USA)

^를^이용하여^{역전사하였다}

. Primer

^의^종류^및^서열은

Table

I

에 표시한 바와 같다

. PCR

반응 조건은

GeneAmp 2700

thermocycler(Applied Biosystems, USA)

에서

94

^o

C, 5

분간변성

, 72

^o

C, 7

^분간^{연장하였으며}

,

^각각의

primer

^에^대한^증폭^주기의

조건은다음과같다

.: TNF-

α

, 28

주기

94

^o

C 30

초

, 65

^o

C 30

초

,

72

^o

C 60

초

; iNOS, 35

주기

94

^o

C 30

초

, 65

^o

C 30

초

, 72

^o

C 30

초

;

(3)

COX-2, 35

^주기

94

^o

C 30

^초

, 60

^o

C 30

^초

, 72

^o

C 30

^초

;

^β

-actin, 25

주기

94

^o

C 30

초

, 56

^o

C 30

초

, 72

^o

C 30

초

.

이후반응 생성물을

ethidium bromide

^로^염색된

1.5% agarose gel

^을^이용하여

100 V

에서전기영동하였고

,

^각

PCR

^산물은

SLB Mylmager(UVP Inc., USA)

와

ImageQuant

^TM

TL(Amersham Biosciences/GE Healthcare, USA)

^를^사용하여^{반정량적으로}^{분석하였다}

.

통계처리

모든실험결과는

one-way ANOVA

^를^{사용하였으며}

P<0.05

일때유의성있는차이가있는것으로판정하였다

.

실험 결과 및 고찰

혈중ALT및AST활성도

아미노산전이효소는세포질에존재하며

, ALT

^및

AST

^활성

증가는알코올

,

^유기용매^및^기타^독소에^의해^간장해가^발생할

때혈중으로유리되어총혈중농도가증가하므로독성지표로널 리이용되고있다

.

¹⁵⁾

ALT

^및

AST

^는^세포^내^위치에^따라^다른

동종효소로존재하는데

,

^특히

ALT

^는^간에^많이^존재하며

AST

는심장

,

간

,

골격근에많아그특이성이인정된다

.

혈중

ALT

및

AST

^활성도를^측정한^결과는

Table II

^와^같다

.

^{사염화탄소}^단독

투여군의혈중

ALT

수치는

2115.6±235.0 IU/l

으로대조군에비 해현저히증가하였으나치자

30, 100

및

300 mg/kg

투여군의 경우 각각

903.0±80.0 IU/l, 1012.7±76.5 IU/l

^및

1112.5±53.9 IU/l

로사염화탄소단독투여군에비해현저히감소하였다

.

혈중

AST

수치는

ALT

수치와유사하게사염화탄소단독투여시대

조군에비해현저히증가하였으나

,

^치자

30, 100

^및

300 mg/kg

투여군모두에서사염화탄소단독투여군에비해현저히감소하 였다

.

^따라서^간독성^지표인^혈중

ALT

^및

AST

^의^수치가^치자

30, 100, 300 mg/kg

^모두에서^대조군에^비해^현저하게^감소한^것

으로보아치자가간보호에탁월한효과가있음을알수있었 다

.

^이를^바탕으로^이후^조직학적^분석^및^작용기전^{연구에서는}

치자

30 mg/kg

^용량을^선택하여^실험을^{진행하였다}

.

조직학적분석

Fig. 1

^는^{사염화탄소}^유도^급성^간독성^모델에서^치자가^간의

조직학적변화에미치는영향을살펴본것이다

.

그림에서보는 바와같이간조직의염색결과

,

^{사염화탄소}^단독^투여군

(Fig. 1B)

에서는문맥주위염증

,

^간세포^괴사^및

Kupffer cell

^비대가^관

찰되었으나치자

30 mg/kg

투여군

(Fig. 1C)

에서이러한변화가 현저히완화되었다

.

^이는^치자의^혈중

ALT

^및

AST

^결과를^일

관성있게뒷받침하였으며

,

^이로서^치자가^{사염화탄소로}^유도된

급성간독성에있어보호효과가있음을확인할수있었다

.

지질과산화물및glutathione정량

지질과산화는활성산소가세포막의불포화지방산을공격함으 로써일어나며

,

^이로^인하여^세포막뿐^아니라^{세포막으로}^둘러

싸인소기관

,

즉미토콘드리아및소포체막등에도기능손상을 유발한다

.

최근연구에따르면사염화탄소그자체는독성이강 하지않으나간장내일산소첨가효소계에의해활성화되어산 화적손상과세포막인지질내불포화지방산을공격하여간세 포의기능과구조를파괴시킨다고한다

.

¹⁶⁾

Table III

에서와같이 Table I −

Characteristics of specific primers used for RT-PCR analysis

Gene (accession number) Primer sequences (5'-3') Product length (bp)

TNF-

^α

(M11731) Sense: AGCCCACGTCGTAGCAAACCACCAA

Antisense: AACACCCATTCCCTTCACAGAGCAAT 446

iNOS (NM_010927) Sense: AAGCTGCATGTGACATCGACCCGT

Antisense: GCATCTGGTAGCCAGCGTACCGG 598

COX-2 (NM_011198) Sense: ACTCACTCAGTTTGTTGAGTCATTC

Antisense: TTTGATTAGTACTGTAGGGTTAATG 582

β

-Actin (X03672) Sense: TGGAATCCTGTGGCATCCATGAAA

Antisense: TAAAACGCAGCTCAGTAACAGTCCG 348

Table II −

Effects of G. jasminoides on the serum aminotransferase activities in the CCl

4

-induced mice

Group Dose (mg/kg) ALT (IU/ l ) AST (IU/

^ℓ

)

Control 0041.6±3.4 0030.2±5.5

CCl

4

Vehicle 2115.6±235.0 1629.6±127.4

G. jasminoides 030 0903.0±80.0 0662.6±63.1

^,++

100 1012.7±76.5**

^,++

0861.1±85.4**

^,++

300 1112.5±53.9**

^,+

0979.5±115.0**

^,++

Silymarin 200 1365.0±176.5 0832.2±94.7

^,++

The results are presented as the mean±S.E.M. of 8 mice per group. ** Denotes significant differences ( P <0.01) versus control group;

+,++

denote significant differences ( P <0.05, P <0.01) versus CCl

4

group.

(4)

사염화탄소단독투여군의

MDA

^함량은^대조군의^약

5.1

^배로^현

저히증가되었으나

,

^치자

30 mg/kg

^{투여군에서}^{사염화탄소}^단독

투여군의

MDA

함량의약

65.0%

정도로현저히억제되었다

.

Glutathione

^은^세포의^산화적^손상에^대해

1

^차적^{방어역할을}^하

는중요한비단백성물질로서

,

세포가산화적손상을받으면

GSH

가

GSSG

로산화됨으로써활성산소를무독화시킨다

.

¹⁷⁾본실험

에서사염화탄소단독투여후간조직에남아있는

GSH

^의^양은

대조군의

82.9%

로감소하였으나

,

치자

30 mg/kg

투여는이러한 감소를억제하였다

.

이는치자의간보호작용의일부가사염화 탄소로인한세포막의파괴를유발하는지질과산화물의생성억 제와간장내

glutathione pool

을증가시킴으로써세포의산화적 손상에대한항산화방어기작을통해나타냄을알수있었다

.

혈중TNF-α농도및유전자발현

간은인체주요염증기관

(inflammatory organ)

으로다양한간 독소노출후염증반응은일련의병리학적변화에관여한다

.

^간

Fig. 1 −

Liver sections stained with hematoxylin and eosin. Mice were treated orally with vehicle or G. jasminoides (30 mg/

kg) 48 h, 24 h and 2 h before 6 h after CCl

4

injection. (A):

Control group, showing normal hepatic architecture; (B):

CCl

4

group, showing hepatocellular degeneration and necrosis with inflammatory infiltration; (C): G. jasminoides (30 mg/kg) + CCl

4

group, showing mild hepatocellular necrosis and inflammatory infiltration. Original magnification

×400.

Table III −

Effects of G. jasminoides on the lipid peroxidation and hepatic glutathione content in the CCl

4

-induced mice

Group Dose (mg/kg) MDA (nmol/mg protein) GSH (

^µ

mol/g liver)

Control 0.34±0.01**

^,++

7.76±0.31

CCl

4

Vehicle 1.76±0.14**

^,++

6.43±0.03**

G. jasminoides 30 1.16±0.06**

^,++

7.17±0.16*

^,++

The results are presented as the mean±S.E.M. of 8 mice per group. *

^,

** Denote significant differences ( P <0.05, P <0.01) versus control group;

⁺⁺

denotes significant differences ( P <0.01) versus CCl

4

group.

Fig. 2 −

Effect of G. jasminoides (30 mg/kg) on the serum TNF-

^α

level of the CCl

4

-treated mice. The values are reported as the means±S.E.M. of 8 mice per group. ** Denotes significant differences ( P <0.01) versus control group;

⁺⁺

denotes significant differences ( P <0.01) versus CCl

4

group.

(5)

장내대식세포인

Kupffer cell

은간세포괴사혹은다양한간독 소에반응하여염증매개인자를유리하며이는사염화탄소로유 도된간손상의악화에도관여된다

.

¹⁸⁾

TNF-

^α는^조직^손상시^대

식세포에서빠르게생성되는대표적인염증성사이토카인이다

.

사염화탄소노출후활성화된

Kupffer cell

^에서

TNF-

^α는

TNF-

α수용체와함께작용하여간조직괴사를일으킨다

.

¹⁹⁾^본^연구

에서사염화탄소투여후혈중

TNF-

α농도는

149.3±0.5 pg/ml

로대조군에비해현저히증가하였으나치자투여군에서

127.1±

1.3 pg/ml

^로^{감소하였고}

,

^이와^유사하게^유전자^발현량도^사염화

탄소단독투여군에서대조군에비해약

3.8

배로증가하였으나 치자투여는이러한증가를현저히억제하였다

(Figs. 2

^및

3).

iNOS및COX-2단백질과유전자발현

NO

^는^반응성이^매우^높은^산화제로

iNOS

^의^작용에^의해

L-

arginine

^으로부터^간장의^{실질세포와}^{비실질세포에서}^생성된다

.

정상시

NO

^는^세포^내에^낮은^농도로^존재하나^염증^반응^시

TNF-

α에의해서

iNOS

가유도되면장시간동안다량의

NO

를생성하

며

,

^생성된

NO

^는^혈관확장

,

^세포독성^및^조직손상^등과^같은^생

체유해작용을 나타낸다

.

²⁰⁾^또한

iNOS

^에^의해 ^생성된

NO

^는

superoxide anion

과반응하여좀더강력한산화물질인

peroxy- nitrite

^를^생성하며

,

^세포^내

oxidant-sensitive

^{전사인자인}

NF-

^κ

B

을활성화시켜다른염증매개체의생성을촉진하여염증반응 을 심화시킨다

.

²¹⁾ 다수의 염증 억제 약물들의 작용기전은

prostaglandin

^합성^억제이며

,

^이는

COX-2

^의^생성^및^활성저해

에의한것이다

. COX-2

^는^동물이나^인간의^염증^반응^부위에서

발현되는효소로

, COX-2

에의한

prostaglandin

의합성은염증반 응을매개한다

.

^{연구보고에}^따르면^염증^반응에^있어

COX-2

^외

Fig. 3 −

Effect of G. jasminoids (30 mg/kg) on the protein expression of iNOS and COX-2. Whole Hepatic proteins were extracted from mice 24 h after CCl

4

injection. iNOS and COX-2 were detected by western blotting as described in Materials and Methods and

^β

-actin was used as a loading control. The results are presented as the mean±S.E.M. of 8 mice per group. *

^,

** Denote significant differences ( P <

0.05, P <0.01) versus control group;

^+,++

denote significant differences ( P <0.05, P <0.01) versus CCl

4

group.

Fig. 4 −

Effect of G. jasminoides (30 mg/kg) on the mRNA expression of TNF-

^α

, iNOS and COX-2. The levels of TNF-

^α

, iNOS and COX-2 mRNA expression were measured by using RT- PCR as described in Materials and Methods. Also,

^β

-actin was used as a loading control. The result is presented as the mean±S.E.M. of 8 mice per group. *

^,

** Denote sig- nificant differences ( P <0.05, P <0.01) versus control group;

+,++

denote significant differences ( P <0.05, P <0.01) versus

CCl

4

group.

(6)

생성은사염화탄소로유도된간독성의이차적효과에도관여한 다고한다

.

²²⁾

Figs. 3

^및

4

^에서^보는^바와^같이

iNOS

^및

COX- 2

의단백질발현량은대조군에비해사염화탄소단독투여군에 서각각약

4.4

^배

,

^및

3.3

^배로^현저히^{증가하였으나}^치자^투여^군

에서이러한증가가현저히감소되었다

.

이와유사하게유전자

발현량에있어서도

iNOS

및

COX-2

의유전자발현량이사염화

탄소단독투여군에서각각약

3.8

^배^및

5.8

^배로^현저히^증가하

였으나치자투여군에서이러한증가가현저히감소되었다

.

이 는아마도치자가전사단계

(transcriptional level)

에서사염화탄 소로유도된염증매개인자인

iNOS

^및

COX-2

^의^생성을^조절하

여간보호작용을나타내는것으로여겨진다

.

결 론

치자의간보호작용과산화성스트레스및염증매개인자발현 억제에대한치자의역할을규명하기위하여사염화탄소를처치 한생쥐의간조직에서

ALT, AST,

지질과산화생성과염증매개 인자

TNF-

^α

, iNOS

^및

COX-2

^의^혈중^농도

,

^유전자^및^단백질

발현량을관찰하였다

.

혈중

ALT

및

AST

의수치는사염화탄소단독투여군에비해

치자투여군에서더낮은활성도를나타내었고

,

^{지질과산화량}

은치자투여군에서감소하였고이와는반대로

GSH

^는^치자^투

여군에서증가하였다

.

치자투여군에서염증매개인자인

TNF-

α 혈중농도및유전자발현량이사염화탄소단독투여군에비해 낮게나타났으며

, iNOS

^및

COX-2

^의^단백질^및^유전자^발현량

역시사염화탄소단독투여군에비해현저히감소하였다

.

이상의결과들을종합하여볼때치자는사염화탄소로유도된 간독성모델에서산화성스트레스와염증매개인자발현억제를 통하여간보호작용을나타내는것으로여겨진다

.

감사의 말씀

본과제

(

^결과물

)

^는교육과학기술부·지식경제부의출연금으로 수행한산학협력중심대학육성사업의연구결과입니다

.

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