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Anti-Oxidative and Anti-Inflammatory Effects of Malus huphensis, Ophiorrhiza cantonensis, and Psychotria rubra Ethanol Extracts

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Malus huphensis, Ophiorrhiza cantonensis, Psychotria rubra 에탄올 추출물의 항산화 및 항염증 활성

진경숙1, 권현주1,2, 김병우1,2*

1동의대학교블루바이오소재개발실용화지원센터

2동의대학교생명응용학과

Received: April 28, 2014 / Revised: August 6, 2014 / Accepted: August 12, 2014

서 론

활성산소종

(Reactive Oxygen Species, ROS)

호흡을 체내로유입된산소에의해생체대사과정에서끊임없이 발생하게되며산화적스트레스

(oxidative stress)

유발하 세포손상을일으킬아니라

,

염증유발인자를활성화 시킴으로써세포조직에염증을초래한다

[9, 24].

이러한 산화적스트레스는다양한질병의원인이되며

,

노화를일으

키는

·

간접적원인물질로작용한다

[14, 24].

그러므로세포 항상적정수준의항산화물질의보호를필요로하며

,

체에발생하는수많은질병에대응하기위해서는강한항산 화능을보유한생리활성소재의개발이매우중요하다

[17, 18, 26].

대표적인

cellular defensive phase 2 detoxifying anti- oxidant enzyme

으로알려진

heme oxygenase (HO)-1

도는산화적스트레스를방어하는중요한기전하나로

car-

cinogen

등의외부자극으로부터세포를보호하는

chemo-

prevention

에서중요한역할을한다

[5, 35].

특히천연유래 다양한

dietary phytochemical

nuclear factor E2- related factor 2 (Nrf2)

의해조절되는

phase 2 detoxify- ing antioxidant enzyme

발현증가를통해

chemopreven- Anti-Oxidative and Anti-Inflammatory Effects of Malus huphensis, Ophiorrhiza cantonensis, and Psychotria rubra Ethanol Extracts

Kyong-Suk Jin

1

, Hyun Ju Kwon

1,2

, and Byung Woo Kim

1,2

*

1

Blue-Bio Industry Regional Innovation Center,

2

Department of Life Science and Biotechnology, College of Natural Science, Dong-Eui University, Busan 614-714, Republic of Korea

This study was orchestrated with the purpose of uncovering new nutraceutical resources possessing biological activities in the plant kingdom. To fulfill our objective, we analyzed several Chinese plants and selected three possessing powerful anti-oxida- tive activities. The anti-oxidative and anti-inflammatory effects these three Chinese plants, Malus hupehensis, Ophiorrhiza can- tonensis, and Psychotria rubra ethanol extracts were then evaluated. First of all, they possessed potent scavenging activity against 1,1-diphenyl-2-picryl hydrazyl, similar with that of ascorbic acid, used as a positive control. Moreover, they inhibited lipo- polysaccharide (LPS)- and hydrogen peroxide-induced reactive oxygen species, in a dose-dependent manner, in RAW 264.7 cells. Also, they induced the expression of an anti-oxidative enzyme, heme oxygenase 1, and its upstream transcription factor, nuclear factor-E2-related factor 2. Furthermore, they suppressed LPS-induced nitric oxide (NO) formation, without cytotoxicity.

The inhibition of NO formation was the result of the down regulation of inducible NO synthase (iNOS). The suppression of NO and iNOS by the three extracts might be the result of modulation by the upstream transcription factors, nuclear factor κB and activator protein-1. Taken together, these results indicate that these three Chinese plants possess potent anti-oxidative and anti-inflammatory activities. Therefore, they might be utilized as promising materials in the field of nutraceuticals.

Keywords: Three Chinese plants, anti-oxidative activity, anti-inflammatory activity, upstream transcription factors

*Corresponding author

Tel: +82-51-890-2900, Fax: +82-51-890-2914 E-mail: bwkim@deu.ac.kr

© 2014, The Korean Society for Microbiology and Biotechnology

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tive function

나타낸다

[19].

이러한

chemoprevention

항산화활성을기초로하여

,

염증

,

심혈관계질환

,

노화등의예방치료기전과도상호작용하는것으로 려지고있어중요성이매우크다

[3, 15, 27, 33].

염증은외부자극에대한생체조직의방어기전하나이 지속적인염증반응의발생은조직의손상을일으켜

비롯한각종질병을유발한다

[4, 7].

생체염증반응은

대식세포

(macrophage)

에서 과량생산되는염증 매개인자

(inflammatory mediators)

부터유래되며

,

이러한염증 개인자로는

inducible nitric oxide synthase (iNOS)

의해 생산되는

nitric oxide (NO)

cyclooxygenase 2 (COX-2)

부터생산되는

prostaglandin E2 (PGE2)

등이알려져있다

.

이러한 염증 매개인자는

tumor necrosis factor

α

, inter-

leukin 1

β등과같은사이토카인의생산을유도하여염증

응을일으킨다

[15, 16].

염증반응의대표적인세포실험계

하나인

RAW 264.7 murine macrophage

염증유발 자인

lipopolysaccharide (LPS)

처리하면

iNOS

COX-2

발현유도에의해

NO

PGE2

등의염증매개인자가

생성되며이는사이토카인분비량증가를유도한다

.

이러 일련의반응은염증상위신호전달기전인

nuclear factor (NF)-

κ

B

activator protein (AP)-1

의해조절되는것으

알려져있다

[21, 28].

그러므로염증매개인자와상위

신호전달기전을효과적으로제어할있는물질의개발에 많은연구가집중되고있다

.

이에연구에서는천연에서유래한항산화항염증 리활성보유신소재개발의일환으로중국자생식물종의 생리활성을분석하여높은항산화능을보이는

3

종의 식물

,

Malus hupehensis, Ophiorrhiza cantonensis,

리고

Psychotria rubra

선별하였다

. M. hupehensis

미과

(Rosaceae)

속하는낙엽수로일본타이완을원산

지로하며

,

동아시아지역에널리분포한다

.

일반적인높이는

7 m

이고

,

개화시기는

4-5

월이며

, 10

월에씨를맺는다

.

매는식용가능하며

,

일반적으로는차의형태로복용한다

.

매가약용으로알려져있으나구체적인효능에대해서는 려진바가없다

. O. cantonensis

꼭두서니과

(Rubiaceae)

속하는아관목으로일반적으로

Rubiaceae

과의식물이 시아전역에널리분포하는데반해

O. cantonensis

중국에

분포하는것으로알려져있다

.

일반적인높이는

1-2 m

,

겨울과사이에개화하며

,

봄에서여름사이에씨를 는다

.

잎이식용가능한것으로알려져있으나구체적인효능 대해서는알려진바가없다

. P. rubra

또한

Rubiaceae

속하는하부층관목으로동아시아지역

,

특히중국

,

일본 이완에분포한다

.

일반적인높이는

0.5-3 m

이고

,

개화시기는

4-11

월이며

, 7-12

사이에열매를맺는다

. P. rubra

식용

약용에대해서는알려진바가없으며

,

특히상기소재 항산화항염증효과에대해서는전혀알려진없다

.

이에연구에서는

M. hupehensis, O. cantonensis,

그리고

P. rubra

95%

에탄올 추출물

(

이하

MHEE, OCEE,

PREE)

보유한항산화항염증활성을분석함으로써

소재의기능성자원으로서의활용가능성을확인해보고자 하였다

.

재료 및 방법

추출물의 제조

연구에서사용한

M. hupehensis, O. cantonensis,

리고

P. rubra

95%

에탄올추출물

(

이하

MHEE, OCEE,

PREE)

한국생명공학연구원

,

해외생물소재허브센터에서

구입

(

분양번호

FBM123-027, 077, 100)

하여사용하였으며

추출과정은 다음과같다

. MHEE

PREE

줄기부위

, OCEE

줄기를 포함한전초를 건조하여 분쇄하고

,

95%

에탄올을용매로하여

45

o

C

에서

3

일간추출을수행하였

.

추출이끝난시료를

filter

여과하여고형물을없애고 압농축

(N-1000SW, EYELA, Japan)

동결건조

(FDU2100, EYELA, Japan)

하여사용전까지

4

o

C

보관하였다

.

DPPH radical 소거능 측정을 통한 항산화능 분석

추출물의항산화능보유유무정도를알아보기 위해항산화능의주요지표로활용되고있는

1,1-diphenyl- 2-picryl hydrazyl (DPPH) radical

소거능분석을수행하였

[6]. DPPH

비교적 안정한

free radical

로써

, ascorbic acid, tocopherol, polyhydroxy

방향족화합물

,

방향족아민 등에의해환원되어짙은자색이탈색되며이러한원리 이용하여항산화활성을간단히측정할있는동시에 식물체의항산화활성과도연관성이매우높기때문에많이 이용되고있다

[13].

DPPH radical

소거능 측정을 위해 시료를 농도별

(0.512-12.8

μ

g/ml)

메탄올에녹여준비하고

96 well plate

메탄올에용해된

1.5 × 10

4

M DPPH 40

μ

l

시료

160

μ

l

분주한 혼합액을 실온에서

30

분간 반응시킨

,

multi-plate reader (Paradigm, Beckman, CA, USA)

용하여

520 nm

에서흡광도를측정하였다

.

시료를첨가하지

않은음성대조군과대비하여

free radical

소거정도를백분 율로 나타내고

, 50%

저해 농도

(Inhibitory Concentration,

IC

50

)

계산하였다

.

대표적인항산화제로

DPPH radical

거능측정양성대조군으로주로사용되는

ascorbic acid

함께비교분석하였으며측정값은

3

반복실험의평균 값으로나타내었다

.

(3)

RAW 264.7 murine macrophage의 배양

항산화 항염증 활성의 세포 실험 모델계로

murine

macrophage cell line

RAW 264.7

American Type Tissue Collection (ATCC

®

, TIB-71

TM

, Manassas, VA, USA)

로부터 구입하여

10% fetal bovine serum (FBS)

penicillin/streptomycin (Pen/Strep)

포함된

DMEM

배지 에서배양하였다

[23].

세포생존율 분석

활성분석수행시료가세포생존율에미치는영향을 인하고

,

세포독성을유발하지않는시료의처리농도를 정하기위해추출물에의한세포독성유발유무를

WST assay

통해 수행하였다

. 1.0

×

10

5

cell

24-well tissue culture plate

분주하여

24

시간동안부착시키고

,

농도별

시료처리

24

시간경과

WST

시약이배지로교체하여

시간동안반응시킨다음

multi-plate reader

이용하여

450 nm

에서흡광도를측정하였다

.

측정값은

3

반복실험

평균값으로나타내었으며독성을유발하지않는농도 위에서이후실험을수행하였다

.

Reactive oxygen species (ROS) 소거능 분석

ROS

과량생산핵산

,

단백질

,

지질등의생체 분자에산화적스트레스를유발하여다양한질병의원인이 되므로이러한

ROS

소거능은항산화력의중요한지표가 된다

[20]. Hydrogen peroxide (H

2

O

2

)

대표적인

ROS

하나로소재의항산화능을규명하기위한많은연구에서

ROS

유도제로사용되고있다

[29, 31, 34].

또한그람음성박테리 아의세포외벽구성인자인

lipopolysaccharide (LPS)

표적인염증유발인자로산화적스트레스또한유발하는 으로알려져있어항산화능항염증활성을규명하기

많은연구에서스트레스유도제로사용되고있다

[1, 22].

연구에서는소재가보유한항산화능을

H

2

O

2

LPS

유도한

ROS

생성에시료가미치는영향을통해분석하였

.

이를위해

RAW 264.7 cell

cell permeable fluorescent dye

50

μ

M

dichlorofluorescin diacetate (DCFH-DA)

2

시간동안처리한제거하고

500

μ

M

H

2

O

2혹은

1

μ

g/ml

LPS

농도시료와함께처리한시료에의한

ROS

생성억제능을

multiplate reader

이용한

fluorescence

측정을통해분석하였다

.

측정값은

3

반복실험의평균값 으로나타내었다

.

항산화 효소 HO-1 및 상위전사인자 Nrf2의 발현 조절능 분석 소재의항산화활성기전을알아보기위해대표적인

산화효소인

HO-1

상위전사인자인

Nrf2

시료처리에

의한단백질발현변화를

Western blot hybridization

으로

석하였다

. HO-1

일차항체는

Cell Signaling Technology (MA, USA)

로부터구입하였고

, Nrf2

그리고

Actin

일차항 체와

anti-goat, anti-rabbit

등의 이차항체는

Santa Cruz Biotechnology (CA, USA)

에서구입하여사용하였다

.

시료

처리가 끝난 배양 세포에서 단백질을추출하여

Bradford

assay

단백질농도를 결정한

50

μ

g

단백질을

10%

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

전기영동하고

nitrocellulose membrane

blotting

1:1,000

으로희석한대상단백질의일차항체

hybridization

하였다

. Membrane

수세

horse radish peroxidase (HRP)

부착된이차항체

(1:1,000)

시간 반응시키고

chemiluminescence detection system (Fluo- Chem

®

FC2, AlphaInnotech, USA)

이용하여단백질 현을분석하였다

.

실험의결과는

3

반복실험을통해유의 적인단백질발현변화를확인한대표적인데이터를 시하였다

.

NO 생성 억제능 분석

대표적인

free radical

하나인

NO

중요한세포신호 전달물질로서작용하나과잉생산산화적스트레스의유발 통해염증세포손상의원인이된다

[12].

이러한

NO

억제능의분석은

Park

[23]

방법을변형하여다음과 수행하였다

. RAW 264.7 cell

24-well tissue culture plate

well

1.0

×

10

5

cell

분주하여부착시킨

1

μ

g/

ml

LPS

처리하여

NO

생성을유도하고식물추출물에

의한

NO

생성저해능을

Griess reaction

통해분석하였으 측정값은

3

반복실험의평균값으로나타내었다

.

항염증 활성 기전 분석

소재가보유한

NO

생성억제능의기전을밝히기위해

NO

생성유전자인

iNOS

단백질발현을분석하였다

.

또한

추출물에의한

NO

생성

iNOS

발현저해능이

NF-

κ

B

AP-1

의해조절될가능성을알아보기위해

LPS

유도된

NF-

κ

B p65

inhibitory

κ

B

α

(I

κ

B

α

),

그리고

AP-1

subunit

c-Jun

인산화에추출물이미치는영향을 분석하였다

. Western blot hybridization

위한

iNOS, p- p65, p-I

κ

B

α

,

그리고

p-c-Jun

일차항체는

Cell Signaling Technology (MA, USA)

부터구입하여사용하였다

.

시료

처리가 끝난 배양 세포에서 단백질을추출하여

Bradford

assay

단백질농도를 결정한

50

μ

g

단백질을

10%

SDS-PAGE

전기영동하고

nitrocellulose membrane

blotting

대상단백질의항체와

hybridization

하였다

.

Membrane

수세

HRP

tagging

이차항체로시간 동안 반응시키고

chemiluminescence detection system

이용하여단백질발현을분석하였다

.

실험의결과는

3

(4)

실험을통해유의적인단백질발현변화를확인한 표적인데이터를제시하였다

.

통계 분석

실험의결과는평균

(mean)

± 표준편차

(standard deviation, SD)

나타내었고

,

데이터의 통계 분석은

SPSS 20.0 software

이용한

unpaired Student’s t-test

통해

p

0.05

미만

( p < 0.05)

경우유의성이있는것으로판단하 였다

.

결과 및 고찰

추출물의 항산화능 분석

3

소재의항산화능보유유무정도를알아보기

먼저항산화능의주요지표하나인

DPPH radical

거능을분석하였다

.

결과

Table 1

제시된바와 같이

MHEE, OCEE,

그리고

PREE

모두농도증가에따른강한

radical

소거능을 보여

12.8

μ

g/ml

시료 처리에 의해

DPPH radical

소거능이각각

97.32, 95.42, 95.01%

나타 양성대조군으로 사용한

ascrobic acid,

vitamin C

97.49%

유사한정도의활성을보여매우강한항산화능을

보유함을확인하였다

.

소재의

DPPH radical

대한

50%

소거농도를나타내는

IC

50값은

MHEE, OCEE, PREE

각각

1.10, 1.49, 1.38

μ

g/ml

으로나타났다

.

이에소재가 보유한항산화능의정도기전을세포수준에서확인하고 하였다

.

추출물의 ROS 소거능 분석

DPPH radical

소거능분석에의해소재가보유한

항산화능이확인됨에따라작용기전을자세히

알아보기위해먼저

RAW 264.7 cell

대표적인산화적

트레스유도인자인

H

2

O

2

LPS

각각처리하여소재의

ROS

소거능을분석하였다

.

결과

Fig. 1

Fig. 2

제시 바와같이

H

2

O

2

LPS

의해각각유도된

ROS

생성이 소재의농도별처리에의해효과적으로저해되는것으로

타나소재가

DPPH radical

뿐만아니라세포수준에서

H

2

O

2

LPS

의해유도된산화적스트레스또한효과적으 감소시킴을확인하였다

.

Table 1. DPPH radical scavenging activity of MHEE, OCEE, and PREE.

Reagent Concentration ( µg/ml)

Inhibition rate (%)

MHEE 10.512 38.10 ± 0.19

12.56 79.86 ± 0.37

*

12.8 97.32 ± 0.29

*

OCEE 10.512

12.56 12.8

33.58 ± 0.27 67.84 ± 0.46

*

95.42 ± 0.25

*

PREE 10.512

12.56 12.8

33.14 ± 0.23 73.13 ± 0.62

*

95.01 ± 0.10

*

Ascorbic acid

(Positive control)

10.512 30.10 ± 0.15 12.56

12.8

96.68 ± 0.18

*

97.49 ± 0.06

*

*Significantly different from the inhibition rate of each reagent’s first concentration, 0.512 µg/ml (p < 0.05).

Fig. 1. Effect of MHEE (A), OCEE (B), and PREE (C) on H

2

O

2

-

induced ROS scavenging activity in RAW 264.7 cells. Values

are represented as the mean ± SD (n = 6). *, #Significantly different

from the vehicle control [Con (−)] and H

2

O

2

-induced control [Con

(+)], respectively (p < 0.05).

(5)

각 추출물이 항산화 효소 HO-1 및 상위 전사인자 Nrf2의 발현에 미치는 영향

강한항산화능을보유한천연소재들이

Nrf2

의한항산 효소계의발현유도를통해활성을나타낸다는것이 연구를통해밝혀짐에따라소재가보유한항산화능 작용기작을알아보고자하였다

[11, 27].

이를위해

재가대표적인항산화효소인

HO-1

상위전사인자인

Nrf2

단백질발현에미치는영향을분석하였다

.

결과

Fig. 3

제시된바와같이

10-50

μ

g/ml

시료처리에의해

HO-1

상위전사인자인

Nrf2

단백질발현이증가되는

Fig. 2. Effect of MHEE (A), OCEE (B), and PREE (C) on LPS- induced ROS scavenging activity in RAW 264.7 cells. Values are represented as the mean ± SD (n = 6). *, #Significantly different from the vehicle control [Con ( −)] and LPS-induced control [Con (+)], respectively (p < 0.05).

Fig. 3. Modulation of an anti-oxidative enzyme, HO-1, and its

upstream transcription factor, Nrf2 protein expression in RAW

264.7 cells by MHEE (A), OCEE (B), and PREE (C). Actin was

used as an internal control. *Significantly different from the vehicle

control (0) (p < 0.05).

(6)

것으로나타나소재에의한

HO-1

발현유도가

Nrf2

발현증가에서기인할것으로판단되었다

[2, 5].

추출물의 세포생존율 및 항염증 활성 분석

소재가보유한항염증활성을알아보기추출물이

RAW 264.7

세포생존율에미치는영향을살펴보았다

.

Fig. 4

제시한바와같이추출물모두

100

μ

g/ml

지의처리에서심한세포독성을유발하지않는것을확인 하였으며이후실험의농도를

100

μ

g/ml

까지로결정하여 행하였다

.

항염증활성의확인을위해소재의

NO

생성억제능을 분석하였다

. LPS

자극을 유도한 대식세포주

RAW 264.7 cell

에서농도별시료의처리에따른

NO

생성양의

변화를분석한결과

Fig. 5

제시된바와같이시료

10-100

μ

g/ml

시료처리에의해농도의존적인

NO

저해능을보였으며이는

NO

생성단백질인

iNOS

저해에서기인하는것으로나타났다

(Fig. 6).

이러한결과

통해소재가항산화능뿐만아니라항염증활성또한 보유함을확인하였다

.

Fig. 4. Effect of MHEE (A), OCEE (B), and PREE (C) on the via- bility of RAW 264.7 cells. Values are represented as the mean ± SD (n = 3). *Significantly different from the LPS-induced control [Con (+)] (p < 0.05).

Fig. 5. Modulation of LPS-induced NO formation in RAW 264.7

cells by MHEE (A), OCEE (B), and PREE (C). Values are repre-

sented as the mean ± SD (n = 3). *, #Significantly different from the

vehicle control [Con, LPS ( −)] and LPS-induced control [Con, LPS

(+)], respectively (p < 0.05).

(7)

추출물의 항염증 활성 상위신호전달기전 분석

추출물이

iNOS

발현저해를통한

NO

생성억제능

보임에따라항염증활성의상위신호전달기전인

NF-

κ

B

AP-1

연관성을알아보기위해추출물의처리가

LPS

의해 유도된

NF-

κ

B p65

I

κ

B

α

,

그리고

AP-1

subunit

하나인

c-Jun

인산화에미치는영향을분석하였다

.

결과

Fig. 7

제시된바와같이

2

시간동안의

LPS

처리에 의해유도된전사인자의인산화가시료농도의증가에 유의적으로억제되는것으로나타났다

.

이러한결과는 소재가보유한항염증활성이

NF-

κ

B

AP-1

같은상위 신호전달인자들의일련의조절기작을통해이루어질가능 성을시사하였다

.

이러한결과를통해중국자생식물인

M. hupehensis, O.

cantonensis,

그리고

P. rubra

에탄올추출물이항산화능과 항염증활성을보유함을확인하였다

.

이러한결과는기능성 대한보고가거의없는상기의소재에대한생리활성을 음으로규명한것으로신규소재에대한새로운기능성데이 터를구축함과동시에향후생리활성보유기능성소재로서 활용을위한근거자료로사용될있을것으로판단된다

.

M. hupehensis

호북꽃사과나무로불리는데열매인꽃사

과는과당

,

포도당

,

주석산

,

비타민

C

등의함유하고

,

한방에 서는소화불량

,

식욕부진

,

위산분비조절위장관계관련 환에이용되며

,

민간에서는고기를부드럽게하는연육제로

,

과즙은숙취해소에사용된다

.

보고된바로는

M. hupehensis

잎의

polyphenol

flavonoid

doxorubicin

으로유도한 세포사멸에대한보호효과를나타내며

,

동물모델에서 기생충으로유도한간섬유증의억제효과를보유하는것으로 밝혀졌다

[30, 32].

규명된

M. hupehensis

활성성분으로는 잎으로부터 분리한

12

종의

flavonoid

3

종의

biflavonoid

glycoside

있으며중에는강한항산화능을보유하는것으

알려진

quercetin

포함하였다

[30].

또한

M. hupehensis

flavon

조성을분석한결과

,

사과나무껍질등에서주로

채취하는배당체인

phlorizin

1.6%

함유되어있는것으 나타났다

[8]. Phlorizin

포도당수용체인

SGLT-2

제제로작용하여당뇨병치료제개발의기초물질로활용되 있다

[25].

연구에서사용한

M. hupehensis

줄기부위 대해서는기능성활성성분에대한보고는전무하다

.

다만상기의연구결과에서보고된활성성분이줄기부위 에도함유되어있을가능성이있다고판단되며

,

대부 분이항산화와함염증활성을보유할것으로생각되어이에 대한분석이필요할것으로사료된다

. O. cantonensis

능성에대해보고된문헌은현재까지없으며

, P. rubra

능성또한에탄올추출물에서분리된신종

naphthoquinone

psychorubrin

항암활성에대한보고가유일하다

[10].

추후계속적인연구를통해상기소재가보유한항산

Fig. 6. Modulation of LPS-induced iNOS protein expression in

RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C). Actin

was used as an internal control. *, #Significantly different from the

vehicle control [Con ( −)] and LPS-induced control [Con (+)], respec-

tively (p < 0.05).

(8)

항염증활성의작용물질작용기작의세부적인 규명이필요할것으로판단된다

.

요 약

연구에서는식물자원으로부터생리활성을보유한새로 기능성소재를찾고자하였다

.

이를위해수종의중국 식물을 분석하여 강한 항산화능을 보유한

3

( Malus hupehensis, Ophiorrhiza cantonensis,

그리고

Psychotria

rubra)

선별하고

,

추출물의항산화능과항염증생리활

성을 분석하였다

.

먼저 추출물의 항산화능을

DPPH

radical

소거능을통해분석한결과모두양성대조군으로

용한

ascorbic acid

유사한정도의높은활성을보여 재가매우강한항산화능을보유함을확인하였다

.

또한

RAW

264.7

세포주에서

H

2

O

2

LPS

의해유도된

ROS

대한 추출물의소거능을분석한결과에서도농도의존적인 소거능을보였다

.

뿐만아니라대표적인항산화효소 하나로항산화능보유천연물에의해발현이유도되는

HO-

1

전사인자인

Nrf-2

단백질발현이추출물의

리에의해증가됨을보였다

.

한편소재가

LPS

의해 도된

NO

생성에미치는영향을분석한결과모두농도의존 적인

NO

생성저해능을보였으며이는

NO

생성단백질인

iNOS

발현저해에서기인함을확인하였다

.

이와같은

소재의

NO

생성

iNOS

발현억제효과는염증상위신호

전달계인

NF-

κ

B

AP-1

조절을통해일어날가능성을 보였다

.

이러한결과를통해중국자생식물

3

종의높은 산화능과항염증활성을처음으로확인하였으며향후기능 소재로서유용하게활용될있을것으로판단된다

.

Fig. 7. Modulation of upstream signaling pathways for the anti-inflammatory activity by MHEE (A), OCEE (B), and PREE (C) in

RAW 264.7 cells. Actin was used as an internal control. *, #Significantly different from the vehicle control [Con ( −)] and LPS-induced control

[Con (+)], respectively (p < 0.05).

(9)

Acknowledgments

This work was supported by Blue-Bio Industry Regional Innova- tion Center (RIC08-06-07) at Dong-Eui University as a RIC pro- gram under Ministry of Trade, Industry and Energy (MOTIE) and Busan city.

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수치

Table 1. DPPH radical scavenging activity of MHEE, OCEE, and PREE. Reagent Concentration ( µg/ml) Inhibition rate(%) MHEE 10.512 38.10 ± 0.19 12.56 79.86 ± 0.37 * 12.8 97.32 ± 0.29 * OCEE 10.512 12.56 12.8 33.58 ± 0.2767.84± 0.46 *95.42 ± 0.25 * PREE 10.51
Fig. 3. Modulation of an anti-oxidative enzyme, HO-1, and its upstream transcription factor, Nrf2 protein expression in RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C)
Fig. 5. Modulation of LPS-induced NO formation in RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C)

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