Anti-atopic Activity of Sargassum micracanthum Ethanol Extracts

잔가시 모자반 에탄올 추출물의 항아토피 효과

정다현¹, 김꽃봉우리², 김민지², 강보경¹, 박시우¹, 박원민¹, 김보람¹, 박홍민¹, 임무혁³, 안동현¹*

1부경대학교식품공학과

/

2부경대학교수산과학연구소

3대구식품의약품안전청유해물질분석과

Received: January 6, 2014 / Revised: March 10, 2014 / Accepted: March 11, 2014

서 론

아토피피부염은알레르기성질환중대표적인난치성질 환으로지속적인소양증을일으키는피부표층의염증을뜻 한다

^[5].

^,

^,

^,

^,

^,

개인및가족력과밀접한관련을맺고있다

^{[2, 24].}

종류에따라다양한위험인자가존재하나

^,

^,

^,

^,

[3, 5, 6, 31].

^Th2

위험인자로피부염환자의혈청내면역글로불린

^E(IgE)

생성량을높이는데영향을미친다

^[7].

보면

^,

^Th1

Th2 cytokines

^,

Th2 cytokine

IL-4,5,9,13

^B cell

^IgE

^[1].

^IgE

(mast cell)

^FcRI

^(cross- linked),

^,

^FcRI

specific IgE

^.

^,

^,

^{[21, 24].}

^,

고있다

^{[11, 20].}

Anti-atopic Activity of Sargassum micracanthum Ethanol Extracts

Da-Hyun Jeong

, Koth-Bong-Woo-Ri Kim

, Min-Ji Kim

, Bo-Kyeong Kang

, Si-Woo Bark

, Won-Min Pak

, Bo-Ram Kim

, Hong-Min Park

, Moo-Hyeog Im

, and Dong-Hyun Ahn

*

1

Department of Food Science & Technology/Institute of Food Science, Pukyong National University, Busan 608-737, Republic of Korea

2

Institute of Fisheries Sciences/Pukyong National University, Busan 619-911, Republic of Korea

3

Division of Hazardous Substances Analysis, Daegu Food & Drug Administration, Daegu 704-940, Republic of Korea Atopic dermatitis (AD) is a common chronic inflammatory disease preceding the development of allergic disorders. The aim of this study was to evaluate the effect of Sargassum micracanthum ethanol extract (SMEE) on AD. AD was induced by spread- ing 2,4-dinitrochlorobenzen (DNCB) on the back sides of BALB/c mice. The efficacy of SMEE was tested by observing the skin clinical severity score, proliferations of Raw 264.7 cells and the secretion of cytokines and IgE. The secretion of IL-4, and IgE was significantly decreased by SMEE in a dose dependent manner, while IFN- γ was increased. In addition, SMEE alleviated the AD symptoms better when compared to the positive controls. In conclusion, these results suggest that SMEE has an inhib- itory effect on AD, and may serve as a useful biomaterial for the development of cosmeceuticals.

Keywords: Sargassum micracanthum, atopic dermatitis, IgE, skin severity score

*Corresponding author

Tel: +82-51-629-5831, Fax: +82-51-629-5824 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

대부분외용제중심의증상완화제들이며

^,

⁽

⁾

[11, 20, 27, 34].

지구생물중약

^80%

^[15]

^,

^,

동아시아지역에서우수한먹거리로이용되어왔다

^[13].

중식용으로쓰이는해조류는일반적으로열량및영양성분 은낮은편이지만다양한생리활성물질들을함유하고있는 것으로보고되고있다

^.

^,

(agar, alginic acid, fucoidan, laminaran, starch)

^.

^,

^,

^,

[14, 16, 19, 22, 23].

^,

키는역할을하는것으로보고되어있다

^[4].

잔가시모자반

(Sargassum micracanthum)

^.

^,

^[18]

^{[8, 12]}

^.

DNCB(2,4-Dinitrochlorobenzene)

^cytokine

^,

IgE

탄올추출물의아토피억제효과를밝히고기능성소재로의 이용가능성을제시하고자연구하였다

^.

재료 및 방법

실험재료

잔가시모자반

(Sargassum micracanthum)

²⁰¹¹

^,

^,

²⁰

^C

^.

잔가시 모자반 추출물 제조

잔가시모자반분말에

¹⁰

^,

(H-0820, Dongwon Science Co., Busan, Korea)

²⁴

^.

(UNION 32R, Hanil Co., Incheon, Korea)

^{3,000 rpm}

¹⁰

^,

²

^.

³⁷

^C

감압농축기

(RE200, Yamoto Co., Tokyo, Japan)

^.

실험동물

생후

⁴

^BALB/c

^.

(Orient Co., Seongnam, Korea)

²⁰

²

^C,

⁵⁰

^{10%, 12}

⁶

.

아토피 피부염 유발 및 시료처리

Ahn

^[2]

하였다

^.

⁶

^BALB/c

^,

²⁴

^{. 24}

^,

^(3:1)

1% (w/v) DNCB(Sigma Chemical Co., St. Louis, MO, USA) 200

^l

³

^.

0.3% (w/v) DNCB 200

^l

^.

^{(30 mg/}

ml)

0.3% (w/v) DNCB

¹²

^{, 200}

^l

²

^.

비장 세포 분리 및 배양

Mishell

^[25]

^.

(NC: Negative control, PC: Positive control, and SMEE)

⁵

^BALB/c

diethyl ether

^,

^.

^RPMI

(RPMI Medium 1640, GIBCO, NY, USA)

^, tissue grinder

^.

1,800 rpm, 4

C

⁵

^{, RBC (Red} blood cells, Tris-buffered ammonium chloride; 0.87% (w/v) NH

Cl, pH 7.2) lysis buffer

¹⁰

^.

, 10% (w/v) FBS (Fetal bovine serum)- RPMI

²

¹⁰

^cells/ml

³⁷

^{C, CO}

incubator (MCO-15 AC, Sanyo, Osaka, Japan)

⁷²

^.

비장 세포 배양액의 cytokine 분비량 측정

비장세포로부터생성되는

^IFN-

IL-4 cytokine

ELISA-kit (Mouse ELISA set, BD Bioscience, San

Diego, USA)

. Micro-plate

^capture

antibody

anti-mouse IFN-

^IL-4

⁴

^C

^coating

^.

PBST (Phosphate bufferd

saline-Tween 20, 0.01 M PBS, pH 7.2, 0.05% (w/v) Tween 20)

, 10% (w/v) FBS

¹

^blocking

^{. 1}

^PBST

^,

상층액을분주하고실온에서

²

^PBST

로다시세척을하고희석한

biotinylated anti-mouse IFN-

^, IL-4 detection antibody

streptoavidin-horseradish peroxidase conjugated

¹

^.

^,

^PBST

, OPD (o-phenylenediamine, Sigma Chemical Co., USA)

^H

O

^citrate buffer (pH 5.0)

³⁰

^. 2 N H

SO

, micro-plate reader (Model 550, Bio-rad, Richmond, USA)

^{490 nm}

^.

비장세포의 증식능 측정

비장세포현탁액을

²

¹⁰

^cells/ml

96 well-plate

^{, 37}

^{C, CO}

incubator

⁷²

^.

, 5 mg/ml MTT (thiazol blue tetrazolium bromide, Sigma Chemical Co., USA)

²

formazan crystal

^.

2,000 rpm, 4

C

¹⁰

^,

^DMSO (dimethyl sulfoxide, Sigma Chemical Co., USA)

micro-plate reader (Model 550, Bio-rad, Richmond, CA, USA)

^{540 nm}

^.

^.

Proliferation Index (%) =

(Sample

^/Control

^{) × 100}

실험종료일에마우스를

Diethyl ether

동맥으로부터

Disposable syringe (Sungshim medical Co., LTD, Bucheon, Korea)

^{1.0 ml}

.

10,000 rpm, 4

C

5

^.

²⁰

^C

total IgE

^{ELISA kit}

^.

ELISA micro- plate

anti-mouse IgE

⁴

^C

coating

^.

^PBST

, 2% (w/v) BSA (Bovine serum albumin, Sigma Chemical Co., USA)

¹

^blocking

^{. 1}

^PBST

^,

²

^PBST

biotinylated anti-mouse IgE detection antibody

streptoavidin- horseradish peroxidase conjugated

1

^.

^,

^PBST

^, OPD

^H

O

citrate buffer (pH 5.0)

³⁰

^{. 2 N H}

SO

, micro-plate reader

^{490 nm}

^.

Skin clinical severity score

시험물질도포후

^{0, 7, 14}

^.

⁵

^.

(erythema),

(pruritus & dry skin),

(edema & excoriation),

^(erosion)

(lichemification)

^(0),

^(1),

^(2),

⁽³⁾

⁰

¹⁵

^[33].

통계 처리

모든실험에대한통계처리는

SAS program (Statistical analytical system V8.2, SAS Institute Inc., Cary, NC,

USA)

one way ANOVA

시하였으며

^,

^Duncan

p < 0.05

^.

결과 및 고찰

비장 세포 배양액의 cytokine 분비량 측정

IFN-

^Th1

^cytokine

macrophage

natural killer cell

시키며

^{, Th2}

^IL-4

으로써알레르기에대한보호효과를갖는다

^[26].

^IFN-

^(Fig.

1)

negative control (1583.23

4.14 pg/ml)

^(509.12

12.42 pg/ml)

^{, SMEE}

^1273.02

12.42 pg/ml

세포의

^IFN-

^Th1

성화시켜세포성면역을증진시킬것으로사료된다

^.

토피피부염은정상인에비해

^IL-4

^IFN-

는생산이줄어들어그현상으로

^IgE

려져있는데

[17], IL-4 cytokine

^(Fig.

2), Th2 cytokine

^IL-4

. Negative control

^2.38

^{3.63 pg/ml}

positive control

^81.89

^{5.44 pg/ml}

^PC

NC

^SMEE

^28.03

^{0.00 pg/ml}

^PC

^.

^Th1

^Th2

^cytokine

^.

비장세포의 증식능 측정

DNCB

²

^SMEE

를도포한후

^,

^{MTT assay}

실시하였다

^.

(Fig. 3), positive control

^DNCB

^{, SMEE}

negative control

한 수준까지 감소함을 보였다

^.

^SMEE

DNCB

^,

SMEE

^.

혈청 내 total IgE 함량 측정

아토피 피부염 환자의 급성 질환에서

Th1 cytokine

IFN-

^,

^IgE

^Th2

^.

^Th2 cytokine

^{, IgE}

^[30].

^,

^80~85%

^IgE

^,

^IgE

고알려져있다

^[9].

^DNCB

을유발하고

³

^SMEE

^{, BALB/c}

^IgE

^ELISA

^{(Fig. 4).}

그 결과

, negative control

^IgE

29.86

^{0.14 pg/ml}

positive control

IgE

^330.57

^{0.29 pg/ml}

^.

SMEE

^160.31

^{0.58 pg/ml}

^IgE

를나타내어

^SMEE

^IgE

의적으로억제된것을확인하였다

^.

Jeong [10]

Fig. 1. Effects of Sargassum micracanthum ethanol extract

on IFN- γ production in splenocytes from atopic dermatitis- like skin lesions mice. Negative control (NC, n = 5): untreated, Positive control (PC, n = 5): DNCB and vehicle, SMEE (n = 5):

DNCB and Sargassum micracanthum ethanol extract,

Means with different superscripts are significantly different (p < 0.05).

Fig. 2. Effects of Sargassum micracanthum ethanol extract on IL-4 production in splenocytes from atopic dermatitis-like skin lesions mice. Negative control (NC, n = 5): untreated, Positive control (PC, n = 5): DNCB and vehicle, SMEE (n = 5):

DNCB and Sargassum micracanthum ethanol extract,

Means with different superscripts are significantly different (p < 0.05).

Fig. 3. Effects of Sargassum micracanthum ethanol extract on the proliferation in splenocytes from atopic dermatitis-like skin lesions mice. Negative control (NC, n = 5): untreated, Positive control (PC, n = 5): DNCB and vehicle, SMEE (n = 5):

DNCB and Sargassum micracanthum ethanol extract,

Means

with different superscripts are significantly different (p < 0.05).

하며

^,

^[32],

^[35]

^IgE

^.

Skin clinical severity score

본연구에서는아토피성피부염에서일반적으로사용되는 임상적육안평가법

^[33]

(erythma),

(pruritus & dry skin),

(edema &

excoriation),

(erosion),

(lichenification)

⁵

^. DNCB

BALB/c mice

⁵

^{. SMEE}

negative control

^DNCB

positive control

^DNCB

에

^SMEE

^.

SMEE

¹

^{, PC}

^,

^,

^,

^SMEE

¹

²

(Fig.

5).

severity score

(Fig. 6). Positive control

적인차이는없었으나

²

^SMEE

하였을경우에는

positive control

^score

Fig. 4. Effects of Sargassum micracanthum ethanol extract on

total lgE production in sera from atopic dermatitis-like skin lesions mice. Negative control (NC, n = 5): untreated, Positive control (PC, n = 5): DNCB and vehicle, SMEE (n = 5): DNCB and Sargassum micracanthum ethanol extract.

Means with different superscripts are significantly different (p < 0.05).

Fig. 5. Effects of Sargassum micracanthum ethanol extract on clinical skin features of DNCB-applied BALB/c mice. Negative

control (NC, n = 5): untreated, Positive control (PC, n = 5): DNCB and vehicle, SMEE (n = 5): DNCB and Sargassum micracanthum

ethanol extract.

저히감소한것을확인할수있었다

^.

^[2],

^[28],

^[29]

등을 이용한 항아토피 연구 결과와 유사하였다

^.

SMEE

^.

요 약

본연구는

^DNCB

BALB/c mice

^SMEE

²

SMEE

^.

^IFN-

IL-4 cytokine

^,

^,

^IgE

^,

skin clinical severity score

^.

^SMEE

^IL- 4 cytokine

^IgE

^IFN-

^cytokine

^.

^{, SMEE}

negative control

^,

^SMEE

^.

skin clinical severity score

, SMEE

2

^,

^score

positive control

^.

^SMEE

Th1 cytokine

Th2 cytokine

^IgE

^.

Acknowledgments

This research was supported by Basic Science Research Pro- gram through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A1028677).

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