The Anti-Obesity Effect of Smilax china Extract

토복령 추출물의 항비만 활성

박정애¹, 진경숙¹, 권현주^1,2, 김병우^1,2*

1동의대학교블루바이오소재개발및실용화지원센터

2동의대학교생명응용학과

Received: July 22, 2014 / Revised: September 19, 2014 / Accepted: September 20, 2014

서 론

비만은신체조직에지방이과도하게축적되는것으로지 난수세기동안그유병율이꾸준히증가해왔다

^.

^,

^,

동성감소등이있다

[2, 15].

제가되나

^,

^adipokine

^,

[1, 6, 13, 26],

^,

^,

^,

[7, 9, 10, 24].

^,

^,

^,

이요법은비만의예방과치료에있어가장근본적이며중요 한방법이다

^[16].

한편대표적인비만치료제인

^orlistat

장에서분비되는지방분해효소인

^lipase

여섭취한지방중약

^30%

감소에도움을주는것으로알려져있으며이에

^orlistat

같은

^lipase

^[12].

또한비만은지방전구세포의분화및지방생성과정에의 하여지방세포의세포내중성지방

(triglyceride, TG)

만억제의효과적인치료방법으로알려져있다

^[4].

포의분화는세포형태및유전자발현의다양한변화가동

반되는복합적인과정을통하여일어난다

^{. 3T3-L1}

세포는지방세포로분화될때

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

^C/EBP

^{, C/EBP}

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

The Anti-Obesity Effect of Smilax china 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 614-714, Republic of Korea

In this study, the anti-obesity activity of Smilax china methanol extract (SCME) was evaluated using a pancreatic lipase enzyme inhibition assay, and a cell culture model system. Results indicated that, SCME effectively inhibited pancreatic lipase enzyme activity in a dose-dependent manner. Furthermore, SCME significantly suppressed insulin, dexamethasone, 3-isobutyl-1-meth- ylxanthine-induced adipocyte differentiation, lipid accumulation, and triglyceride contents on 3T3-L1 preadipocytes, in a dose- dependent manner. The anti-adipogenic effect was modulated by cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/

enhancer binding proteins (C/EBP) α, C/EBPβ, and the peroxisome proliferator-activated receptor γ gene and protein expres- sions. Moreover, SCME triggered lipolysis effects dose-dependently on adipocyte. Taken together, these results provide an important new insight into SCME, indicating that it possesses anti-obesity activity through pancreatic lipase inhibition, anti-adi- pogenic and lipolysis effects. SCME may therefore be utilized as a promising source in the field of nutraceuticals. The identifi- cation of active compounds that confer the anti-obesity activities of SCME may be a logical next step.

Keywords: Smilax china methanol extract, anti-obesity activity, lipase inhibition activity, anti-adipogenic and lipolysis effects

*Corresponding author

Tel: +82-51-890-2900, Fax: +82-51-890-2914 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

몬의 자극을 받아 초기 분화를 시작하며

peroxisome proliferator activated receptor

^(PPAR

^{), C/EBP}

(transcription factor)

서세포 내중성지방축적에 관여하는다양한

adipogenic

gene

[26]. C/EPB

^PPAR

adipogenic

^,

서발현양이현저하게증가되는양상을나타낸다

^{[22, 26].}

그러므로이러한지방세포분화유도인자의발현조절은지 방생성기전을조절할수있는중요한방법중하나이다

^.

한편전세계적으로비만치료제의개발을위한다양한연 구가수행되고있는가운데현재시판되고있는비만치료 제들의부작용들이보고되면서사용기준이강화되는등의 논란이일고있어우수한효능과함께안전성이높은물질 의개발이요구되고있다

^.

[11, 16, 22].

토복령

⁽ Smilax china)

목으로한국

^,

^,

^.

^,

^.

^5-6

^,

9-10

^.

민간에서토복령은해독

^,

^,

^,

되고있다

^[23].

은중독을푸는데효과적이며

^,

^,

^,

^,

^,

^,

^,

^,

^,

^{[25, 27].}

^[20],

^[15]

^,

^,

^,

있으나

^{[21, 29]}

된바없다

^.

이에본연구에서는다양한생리활성을보유하여한방에 서약재로사용되고있는토복령의항비만효과및그기전 을알아보고기능성소재로서의활용가능성을확인해보고 자하였다

^.

재료 및 방법

토복령 추출물의 제조

실험에사용한토복령은부산광역시소재㈜대한생약제품 에서구입하여사용하였다

^.

^{10 g}

⁵

methanol (MeOH)

75

C

³

³

^.

(N-1000S-W, EYELA, Japan)

(FDU2100, EYELA, Japan)

후중량법으로수율을계산하였다

^.

토복령 추출물의 pancreatic lipase 효소 활성 저해능 분석

Lipase

^TG

^glycerol

^fatty acid

pancreatic

lipase

하기에매우유용한시험법이다

^[8].

lipase

.

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

토복령의항비만활성을세포실험계에서분석하기위해항 비만활성분석에사용되는대표적인

cell model system

3T3-L1 preadipocyte

American Type Culture Collection (ATCC, VA, USA)

10% fetal bovine serum (FBS)

penicillin/streptomycin

Dulbecco's Modified Eagle Medium (DMEM)

^. 0.5

M 3-isobutyl-1-methylxanthine (IBMX), 1

^M dexamethasone (DEX), 10

^g/ml

^{insulin (}

^MDI)

처리하여

adipogenesis

비만활성을분석하였다

^[25].

세포 독성 유무 분석

항비만활성분석수행전시료가세포생존율에미치는영 향을확인함과동시에세포독성을유발하지않는시료의처 리농도를결정하기위해

water soluble tetrazolium (WST) assay

^{. 1}

¹⁰

^cell

24-well tissue culture

plate

²⁴

로처리하여

⁷²

^.

^WST

시약이든배지로교체하여한시간동안반응시킨후

^multi-

plate reader

^{450 nm}

^.

³

발하지않는농도범위에서이후실험을수행하였다

^.

3T3-L1

^MDI

를 처리하여 유도하였다

. 12-well tissue culture plate

well

²

¹⁰

²

^{10% FBS}

^{. 2}

^MDI

2

4

insulin

^.

²

²⁰⁰

^,

^TG

^{Oil Red} O staining

^.

¹

phosphate buffered saline (PBS)

씻어준 다음

10% formalin

^{Oil Red O}

staining solution

³⁰

^.

100% isopropanol

multi-plate reader

^{500 nm}

^.

Reverse transcription-polymerase chain reaction (RT-PCR)을 통한 지방세포 분화 관련 유전자 발현 조절능 분석

토복령이지방세포분화에중요한역할을담당하는유전

자의 발현에 미치는 영향을 알아보기 위해

^C/EBP

^,

C/EBP

^{, PPAR}

^RT-PCR

^. 6-well tissue culture plate

³

¹⁰

2

^{10% FBS}

^{. 2}

^MDI

²

²

^insulin

^.

^{total RNA}

TRIzol (Invitrogen, CA, USA)

NanoVue plus spectro- photometer (GE healthcare, WI, USA)

고

SuperScript

First-Strand Synthesis System (Invitrogen)

^cDNA

^PCR

^.

internal control

glyceraldehydes-3- phosphate dehydrogenase (GAPDH)

^{Table 1}

^.

상기의

RT-PCR

후시료처리가끝난세포에서단백질을분리하여지방생성 관련단백질의발현변화를

Western blot hybridization

^.

cell lysate

를 추출하여

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)

^.

지방세포 내 중성지방 제거량 측정(lipolysis assay)

토복령이보유한지방세포내중성지방제거능

^(lipolysis

activity)

. Confluent

^3T3-L1

²

^MDI

^DMEM

²

¹⁰

^g/ml

DMEM

⁴

다

^.

3T3-L1 cell

⁴⁸

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/EBP

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'

출된

^glycerol

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

540 nm

^.

³

^.

통계 분석

각 실험의 결과는 평균

^(mean)

^(standard

deviation, SD)

^,

unpaired Student’s t-test

p

0.05

( p <

0.05)

^.

결과 및 고찰

토복령 추출물의 제조 및 pancreatic lipase 활성 억제능 분석

먼저토복령

^{10 g}

^MeOH

^{0.69 g}

추출물을얻어추출수율은

^6.9%

^.

의

^lipase

물의농도별처리에따른

^lipase

^.

그결과

0.25, 0.5, 1.0, 1.5, 2.0, 2.5 mg/ml

46.1, 36.8, 24.8, 21.8, 21.0, 17.8%

^lipase

^lipase

^{(Fig. 1).}

lipase inhibitor

^.

시료의지방생성억제능의평가를위해먼저토복령추 출물이

3T3-L1 preadipocyte

을

^{WST assay}

^{. 50}

⁵⁰⁰

^g/ml

시료를농도별로처리한결과

¹⁰⁰

^g/ml

에큰변화를보이지않았고

^{, 150}

³⁰⁰

^g/ml

³⁰⁰

^g/ml

^83%

^{, 500}

^g/ml

^74%

(Fig. 2A).

^80%

³⁰⁰

^g/ml

^.

토복령 추출물이 MDI로 유도한 3T3-L1 preadipocyte의 adipogenesis에 미치는 영향

토복령추출물의항비만활성보유유무를알아보기위해

MDI

3T3-L1 preadipocyte

adipogenesis

^.

^Fig.

2B

^2C

Oil Red O staining

^.

TG

였으며

³⁰⁰

^g/ml

^43.3%

^.

토복령 추출물이 adipogenesis 관련 유전자 및 단백질 발 현에 미치는 영향

지방전구세포인

^3T3-L1

^.

(adipogenesis)

^PPAR

^C/EBPs

[11, 14, 27].

^.

본연구에서는토복령이보유한지방생성억제능의작용

기전을알아보기위하여토복령추출물이

adipogenesis

여하는 주요 핵심 조절자인

^C/EBP

^{, C/EBP}

^,

PPAR

다

^.

^{Fig. 3}

^MDI

^.

adipogenesis

현저해를통해나타나는것으로판단된다

^.

토복령이지방세포분화를억제할뿐만아니라생성된지 방의제거에도효과적인지를판단하기위해지방세포내중

Fig. 1. Lipase enzyme inhibition activity of Smilax china meth-

anol extract (SCME). The effect of SCME on pancreatic lipase

activity was evaluated. Values are represented as the mean ± SD

(n = 3) *p < 0.01 vs control.

성지방제거능을

lipolysis activity

^.

과

^{Fig. 4}

^MDI

토복령추출물을처리한결과세포내축적되어있던중성

지방의분해를통해배지로방출된

^glycerol

는것으로나타났다

^.

Oil Red O staining

^TG

^glycerol

^TG

Fig. 2. Effect of Smilax china methanol extract (SCME) on 3T3-L1 cell proliferation (A), morphological change and lipid accu- mulation (B), and TG contents (C). (A) Cells were treated with the indicated concentrations of SCME for 72 h and viability was deter- mined 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 SCME. 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 content was determined by Oil Red O staining after treatment of SCME. TG content was 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 Smilax china methanol extract (SCME) on adipogenesis related gene and protein expressions. (A) Modulation

of adipogenic transcription factors by SCME was evaluated by RT-PCR. GAPDH was used as an internal control. (B) Modulation of adi-

pogenesis related protein expressions by SCME was evaluated by Western blot analysis. Actin was used as an internal control. The data

are representative of three independent experiments.

에축적되어있는중성지방을제거하는것을확인하였다

^.

이러한결과를통해토복령추출물이

^lipase

제능

^,

^,

^.

^.

prosapogenin A, dioscin, gracillin, methyl proto-dioscin, methyl proto-gracillin, methyl proto-prosapogenin A

6

^steroid

^[17].

^dioscin

^[18],

^[28], PLA

^[3]

^dioscin

^lipase

[19].

분또한

^dioscin

정확히규명된점은없어이에대한추가적인연구가필요 할것으로판단된다

^.

요 약

본연구에서는토복령

( S. china)

(SCME)

pancreatic lipase

^.

^SCME

적으로

^lipase

^{, 3T3-}

L1 preadipocyte

^MDI

^,

지방축적

^{, TG}

^.

한토복령의지방세포분화억제능은핵심작용인자인

^C/

EBP

^{, C/EBP}

^,

^PPAR

^.

로 나타났다

^.

pancreatic

lipase

^,

^,

^.

Fig. 4. Stimulatory

effect of Smilax china methanol extract (SCME) on the 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 SCME

treatment. Glycerol contents were measured 540 nm by multi-plate reader. (B) Differentiation of confluent 3T3-L1 preadipocytes was ini-

tiated with MDI treatment and maintained in DMEM containing 5% FBS. After day 8, MDI-induced adipocytes were treated with SCME

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 SCME. TG content was measured

at 500 nm by multi-plate reader. Data are expressed as the mean ± SD of triplicate experiments. *,**Significantly different from the undif-

ferentiated 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 & Energy and Busan city.

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