Anti-Obesity Activity of Euptelea Pleiosperma Ethanol Extract

Euptelea pleiosperma 에탄올 추출물의 항비만 활성

박정애¹, 진경숙¹, 권현주^1,2, 김병우^1,2*

1동의대학교블루바이오소재개발및실용화지원센터

2동의대학교생명응용학과

Received: September 1, 2015 / Revised: November 5, 2015 / Accepted: December 1, 2015

서 론

체내에지방조직이과다하게축적된상태를의미하는비 만은장시간에걸쳐에너지소비량에비해영양소를과다섭

취할경우발생하는에너지불균형이그원인이다

^{[8, 10].}

너지불균형을일으키는주요요인으로는과도한식이섭취

^,

^,

^,

^,

인 소인 등이 있으며

^adipokine

adipogenesis

의크기증가

(hypertrophy)

(hyperplasia)

^{[2, 3].}

세계보건기구

(World Health Organization, WHO)

¹⁹⁸⁰

²⁰¹⁴

¹⁸

¹⁹

^(39%)

체중이며이중

⁶

^(13%)

^[20].

우리나라또한서구식식습관과생활방식이증가하면서전

체인구의

^25%

과체중과비만율은빠른속도로증가하고있어

²⁰

^36%

^.

비만은심미적인관점에서뿐만아니라고혈압

^,

^,

^,

^,

^,

^·

^.

^,

^,

^,

[4, 6, 16].

시판되고있는대표적인비만치료제로는

sibutramine

orlistat

^orlistat

되는지방분해효소인

^lipase

중약

^30%

다

^[13].

래

^lipase

^{[12, 21].}

또한비만은지방전구세포의지방세포로의분화및지방

Anti-Obesity Activity of Euptelea Pleiosperma Ethanol Extract

Jung Ae Park

, Kyong-Suk Jin

, Hyun Ju Kwon

, and Byung Woo Kim

*

1

Blue-Bio Industry Regional Innovation Center,

Department of Life Science and Biotechnology, College of Natural Science and Human Ecology, Dong-Eui University, Busan 47340, Republic of Korea

Previously, Euptelea pleiosperma was identified as one of the useful sources containing anti-oxidative and anti-inflammatory activities for the first time in our research group. In this study, anti-obesity effect of E. pleiosperma ethanol extract (EPEE) was evaluated by using a pancreatic lipase enzyme inhibition assay and a cell culture model system. EPEE suppressed effectively pancreatic lipase enzyme activity dose dependently. Furthermore, EPEE significantly suppressed adipocyte differentiation, lipid accumulation, triglyceride contents, and triggered lipolysis activity on 3T3-L1 preadipocytes in a dose-dependent manner with- out cytotoxicity. Anti-adipogenic effect of EPEE was modulated by cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/

enhancer binding proteins α (C/EBPα), C/EBPβ and peroxisome proliferator-activated receptor γ (PPARγ) gene and protein expressions. Taken together, these results provide the important new insight that E. pleiosperma possesses anti-obesity activi- ties such as pancreatic lipase inhibition, anti-adipogenic, and lipolysis effects. It might be utilized as promising sources in the fields of nutraceuticals. The identification of active compounds that confer anti-obesity activity of EPEE might be needed.

Keywords: Euptelea pleiosperma, anti-obesity activity, lipase inhibition activity, anti-adipogenic and lipolysis effects

*Corresponding author

Tel: +82-51-890-2900, Fax: +82-505-182-6951 E-mail: [email protected]

© 2015, The Korean Society for Microbiology and Biotechnology

생성과정에의하여지방세포의세포내중성지방

(triglyceride, TG)

는것은비만억제의효과적인방법중하나이다

^{[1, 5].}

지방세포의분화는세포형태및유전자발현의다양한변 화가동반되는복합적인과정이다

^.

mesenchymal

precursor

^,

분화과정동안형태및생화학적변화를통해체내지방을 축적하면서크기가증가되는데이러한과정에는지방조직세 포에특이적으로발현되는유전자들의조절부위에작용하는 전사인자들이관여한다

^{[2, 19].}

대표적인세포실험계인

^3T3-L1

^insulin, dexamethasone(DEX), 3-isobutyl-1-methylxanthine(IBMX)

cytidine-cytidine-adenosine-adenosine- thymidine(CCAAT)/enhancer binding protein

^C/

EBP

^{, C/EBP}

용또는단독으로

peroxisome proliferator-activated receptor

^(PPAR

⁾

^C/EBP

^{. C/EPB}

PPAR

양한

adipogenic

^,

[15, 18].

한편전세계적으로비만치료제의개발을위한많은연 구가수행되고있으나시판되고있는비만치료제들의부작 용과내성등의문제가발생하면서사용기준이강화되고있 어우수한효능과함께안전성을보유한성분의개발이필

요하다

^{[12, 13].}

작용이없는항비만활성보유소재를발굴하기위한많은

노력이집중되고있다

^[21].

Euptelea pleiosperma

Eupteleaceae

^.

^,

^,

^,

^.

^,

^.

를통해

E. pleiosperma

효과에대해서처음으로밝힌바있다

^[11].

서는천연에서유래한생리활성보유신소재개발의일환으 로

E. pleiosperma 95%

^(EPEE)

^,

^,

^,

^.

재료 및 방법

E. pleiosperma 에탄올 추출물의 준비

본연구에서사용한

E. pleiosperma 95%

(EPEE)

^,

구입

⁽

FBM123-004)

^.

95%

⁴⁵

^C

³

수행하고추출이끝난시료를

^filter

앤후감압농축

(N-1000SW, EYELA, Japan)

(FDU2100, EYELA, Japan)

⁴

^C

^.

EPEE의 췌장 lipase 효소 활성 저해능 분석

EPEE

EPEE

^lipase

측정하였다

^.

1.5 ml tube

0.25 M Tris(pH 7.7), 250 mM CaCl

, 5 mM 4-nitrophenyl dodecanoate(PNPD)

³⁷

^C

⁵

^, 0.25 M Tris(pH 7.7)

^lipase

³⁷

^C

10

20% sodium dodecyl sulfate(SDS)

^.

⁴

C, 15,000 rpm

²⁰

^,

96-well tissue culture plate

multi-plate reader

412 nm

^{, 10}

흡광도로부터

⁰

^control

^.

³

^.

세포 배양 및 시료 처리

EPEE

cell model system

3T3-L1

American Type Culture Collection (ATCC, VA, USA)

10% fetal bovine serum(FBS)

penicillin/streptomycin

^DMEM

^{. 0.5}

^{M IBMX, 1}

^{M DEX, 10}

^g/ml

^insulin(

^MDI)

adipogenesis

EPEE

^,

지방세포분화관련인자의유전자및단백질발현조절등 의항비만활성을분석하였다

^[17].

세포 독성 유무 분석

항비만활성분석수행전시료가세포생존율에미치는영 향을확인함과동시에세포독성을유발하지않는시료의처 리농도를결정하기위해

water soluble tetrazolium(WST) assay

^{. 1}

¹⁰

^cell

24-well tissue culture

plate

²⁴

로처리하여

⁷²

^.

^WST

약이든배지로교체하여한시간동안반응시킨후

^multi-

plate reader

^{450 nm}

^.

정값은

³

^.

Oil Red O staining을 통한 지방세포 분화 및 TG 생성 저해능 분석

3T3-L1

^MDI

를 처리하여 유도하였다

. 12-well tissue culture plate

well

²

¹⁰

²

^{10% FBS}

^{. 2}

^MDI

2

4

insulin

^.

²

²⁰⁰

^,

방세포 분화 억제능 및

^TG

^{Oil Red O}

staining

^.

완료된세포를

¹

phosphate buffered saline(PBS)

10% formalin

Oil Red O staining solution

³⁰

^.

^100%

isopropanol

multi-plate reader

^{500 nm}

^.

EPEE

의 발현에 미치는 영향을 알아보기 위해

6-well tissue

culture plate

³

¹⁰

^RNA

리하여각유전자의발현을

^RT-PCR

^.

료 처리가 완료된 배양 세포의

^{total RNA}

^TRIzol

(Invitrogen, CA, USA)

NanoVue plus spectrophotometer(GE healthcare, WI, USA)

SuperScript

First-Strand Synthesis System (Invitrogen)

^cDNA

^PCR

^.

internal control

housekeeping

gene

glyceraldehydes-3-phosphate dehydrogenase (GAPDH)

Table 1

^.

Western blot hybridization을 통한 지방세포 분화 관련 단백질 발현 조절능 분석

상기의

^RT-PCR

한후시료처리가끝난세포에서단백질을분리하여지방생 성관련단백질의발현변화를

Western blot hybridization

^.

^cell lysate

Bradford assay

⁵⁰

^g

10% SDS-polyacrylamide gel electrophoresis

nitrocellulose membrane

^blotting

hybridization

^.

^C/EBP

^C/EBP

Cell Signaling Technology(Beverly, MA, USA)

^{, PPAR}

^actin

horse radish peroxidase

anti-goat, anti-rabbit, anti-mouse

Santa Cruz Biotechnology Inc.(Santa Cruz, CA, USA)

. Membrane

chemiluminescence detection system(FluoChem

FC2, AlphaInnotech, USA)

^.

지방세포내 중성지방 제거량 측정(lipolysis assay)

EPEE

^(lipolysis

activity)

. Confluence

3T3-L1

²

^MDI

DMEM

²

¹⁰

^g/ml

^DMEM

⁴

^.

화가완료된

3T3-L1 cell

48

^glycerol

glycerol-3-phosphateoxidase(GPO)-TRINDER kit(Sigma, St. Louis, MO, USA)

^{540 nm}

Table 1. Primer sequences used for RT-PCR.

Gene name Sequence

C/EBPα Sense

Antisense

5'-GTG TGC ACG TCT ATG CTA AAC CA-3' 5'-GCC GTT AGT GAA GAG TCT CAG TTT G-3'

C/EBPβ Sense

Antisense

5'-GTT TCG GGA GTT GAT GCA ATC-3' 5'-AAC AAC CCC GCA GGA ACA T-3'

PPARγ Sense

Antisense

5'-CGC TGA TGC ACT GCC TAT GA-3' 5'-TGC GAG TGG TCT TCC ATC AC-3'

GAPDH Sense

Antisense

5'-GGG AGT CAA CGG ATT TGG TCG TAT-3'

5'-AGC CTT CTC CAT GGT GGT GAA GAC-3'

정하였다

^.

³

^.

모든실험결과는최소

³

^(mean)

(standard deviation, SD)

^,

^.

SPSS 20.0 software

unpaired Student’s t-test

p

^0.05

( p < 0.05)

.

결과 및 고찰

EPEE의 췌장 lipase 효소 활성 억제능 분석

지방분해효소인

^lipase

^TG

지방산과글리세롤로가수분해하는효소이다

^.

치료를 위한 다양한 해법 중 천연에서 찾은 췌장

^lipase

inhibitor

^lipase

^[7].

^EPEE

lipase

위해농도별처리에따른

^lipase

^.

그결과

0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 1 mg/ml

^(100%)

63.5, 46.3, 38.9, 33.9, 32.0, 26.4, 12.8%

^lipase

^50%

(inhibitory concentration, IC

)

0.089 mg/ml

^{(Fig. 1).}

^EPEE

가췌장

^lipase

키는 것을 확인하였으며 이러한 결과는

^EPEE

^lipase

inhibitor

하였다

^.

EPEE가 3T3-L1 preadipocyte의 세포 생존율에 미치는 영향

세포실험계에서 지방생성억제능의평가를위해먼저

EPEE

3T3-L1 preadipocyte

^{WST assay}

^.

¹

¹⁰⁰

^g/

ml

^(Fig.

2A).

EPEE가 MDI로 유도한 3T3-L1 preadipocyte의 adipogenesis에 미치는 영향

EPEE

^MDI

분화를유도한

3T3-L1 preadipocyte

adipogenesis

^.

^{Fig. 2B}

^2C

억제되는것으로나타났고특히

⁵⁰

^g/ml

에서강한억제활성을보여

Oil Red O staining

^.

^TG

⁵⁰

^g/ml

^80.3%

^.

EPEE가 adipogenesis 관련 유전자 및 단백질 발현에 미 치는 영향

지방전구세포가지방세포로분화되는

adipogenesis

^,

^,

^{. MDI}

c-fos, c-jun, c-myc

^C/EBP

의발현또한유도된다

^{. C/EBP}

^MDI

등의분화인자에노출되었을때분화초기에가장먼저작 용하는전사 인자로서

^MDI

^C/EBP

^DEX

^{, C/}

EBP

^IBMX

다

^.

^C/EBP

^PPAR

^C/EBP

^,

PPAR

^C/EBP

특이적유전자의발현

(adipocyte specific gene expression)

을유도하여지방세포분화및지방형성을완성한다

^{[1, 9,}

14].

자들의발현조절유무는소재가보유한지방생성억제능 및그작용기전을판단하는주요지표중하나이다

^.

본연구에서는

^EPEE

^EPEE

adipogenesis

^C/EBP

^{, C/EBP}

^,

^PPAR

^.

Fig. 1. Lipase enzyme inhibition activity of EPEE. The effects

of EPEE on pancreatic lipase activity. Values are represented as

the mean ± SD (n = 3) *p < 0.01 vs vehicle control (0).

Fig. 3

^MDI

⁵⁰

^g/ml

은발현을보였다

^.

^EPEE

방세포분화억제능이

adipogenesis

^.

EPEE의 중성지방 제거능(lipolysis activity)

EPEE

Fig. 2. Effect of EPEE on 3T3-L1 cell proliferation (A), morphological change and lipid accumulation (B), and TG contents (C).

(A) Cells were treated with the indicated concentrations of EPEE for 72 h and viability was determined by WST assay. Data are expressed as the mean ± SD of triplicate experiments. (B) Differentiation of confluent 3T3-L1 preadipocytes was initiated with MDI treatment and maintained in DMEM containing 5% FBS in presence and absence of EPEE. After day 8, cells were fixed and stained with Oil red O.

The morphological change and lipid droplet accumulation were visualized using by inverted microscopy ( ×200). (C) TG contents were determined by Oil red O staining after treatment of EPEE. TG contents were measured at 500 nm by multi-plate reader. Data are expressed as the mean ± SD of triplicate experiments. *, # Significantly different from the undifferentiated cell control (Con) and untreated cell control (0), respectively (p < 0.05).

Fig. 3. Effect of EPEE on adipogenesis related gene and protein expressions. (A) Modulation of adipogenic transcription factors by

EPEE was evaluated by RT-PCR. GAPDH was used as an internal control. (B) Modulation of adipogenesis related protein expressions

by EPEE was evaluated by Western blot analysis. Actin was used as an internal control. The data are representative of three independent

experiments.

방의제거에도효과적인지를판단하기위해지방세포 내 중성지방제거능을

lipolysis activity assay

였다

^{. MDI}

^EPEE

과세포내축적되어있던중성지방의분해를통해배지로 방출된글리세롤의 양이농도의존적으로증가되는것으로 나타났다

(Fig. 4A).

Oil Red O staining

을수행하고현미경 관찰

^{(Fig. 4B)}

TG

^{(Fig. 4C)}

세롤양의증가와비례적으로세포내에남아있는

^TG

이감소되는것으로나타나

^EPEE

되어있는중성지방을농도의존적으로제거하는것을확인 하였다

^.

이상의결과를통해

^EPEE

^lipase

능

^,

^,

^.

E.

pleiosperma

며추후계속적인연구를통해활성물질의규명이필요할 것으로판단된다

^.

요 약

선행연구에서

Euptelea pleiosperma

.

E. pleiosperma

^(EPEE)

^.

^EPEE

^lipase

, 3T3-L1 preadipocyte

에서지방세포분화

^,

^{, TG}

없이농도의존적으로억제하였으며지방세포내중성지방을 유의적으로분해시키는것으로나타났다

^.

^EPEE

지방세포 분화 억제능은 핵심 작용 인자인

^C/EBP

^{, C/}

EBP

^,

^PPAR

^.

E. pleiosperma

^lipase

^,

^,

^.

Fig. 4. Stimulatory effect of EPEE on glycerol release in MDI-induced 3T3-L1 adipocytes (A), morphological change and lipid

accumulation (B), and TG contents (C). (A) Amount of released glycerol in culture media was measured after EPEE treatment. Glycerol

contents were measured 540 nm by multi-plate reader. (B) Differentiation of confluent 3T3-L1 preadipocytes was initiated with MDI treat-

ment and maintained in DMEM containing 5% FBS. After day 8, MDI-induced adipocytes were treated with EPEE for 48 h. Cells were

fixed and stained with Oil red O. The morphological change and lipid droplet accumulation was visualized using by inverted microscopy

( ×200). (C) TG contents were determined by Oil red O staining after treatment of EPEE. TG contents were measured at 500 nm by multi-

plate reader. Data are expressed as the mean ± SD of triplicate experiments. *, # Significantly different from the undifferentiated cell control

(Con) and untreated cell control (0), respectively (p < 0.05).

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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