Cellular Antioxidant Activity and Whitening Effects of Dendropanax morbifera Leaf Extracts

황칠나무 잎 추출물의 세포 항산화 활성과 미백활성 측정

박수아¹, 박준², 박찬일², 지영종², 황윤찬², 김용현², 전소하¹, 이혜미¹, 하지훈¹, 김경진¹, 박수남¹*

1서울과학기술대학교정밀화학과나노바이오화장품연구실

,

2서울과학고등학교

Received: November 1, 2013 / Revised: December 7, 2013 / Accepted: December 9, 2013

서 론

최근기능성화장품산업에서연구가가장활발하게진행 되고있는분야는피부노화방지와미백효능이라할수있 다

^.

자외선을꼽을수있다

[24, 25].

종

(reactive oxygen species, ROS)

^{. ROS}

superoxide anioin radical (O

), hydroxyl

radical (

^OH)

hydrogen peroxide (H

O

), singlet oxygen (

O

)

peroxyl radical (ROO

), alkoxyl radical (RO

⁾

[3, 16, 17]. ROS

있는인지질을산화시켜세포를파괴할뿐만아니라

^{, MMPs}

의발현을촉진시켜콜라젠및엘라스틴섬유등을분해시 킨다

^.

^.

^ROS

^DNA

^.

^ROS

Cellular Antioxidant Activity and Whitening Effects of Dendropanax morbifera Leaf Extracts. Park, Su Ah

, Jun Park

, Chan Il Park

, Young Jong Jie

, Yun Chan Hwang

, Yong Hyun Kim

, So Ha Jeon

, Hye Mi Lee

, Ji Hoon Ha

, Kyeong Jin Kim

, and Soo Nam Park

*.

Department of Fine Chemistry, Nanobiocosmetic laboratory, and Cosmetic R&D Center, Seoul National University of Science and Technology, Seoul 139-743, Korea,

Seoul Science High School, Seoul 110-521, Korea

In this study, we investigated the antioxidant activities on HaCaT and the whitening effects on B16F1 melanoma cells of Den- dropanax morbifera leaf extract. In an antioxidative activity assay using HaCaT cells, the ethyl acetate (50 μg/ml) and aglycone fractions (25 μg/ml) of the D. morbifera leaf extract didn't exhibit any characteristics of cytotoxicity. When HaCaT cells were exposed to a single large dose (800 mJ/cm

) of UVB, the extracts protected the cells against UVB radiation. When HaCaT cells were treated with 10 mM H

O

and 4 μM rose bengal, the ethyl acetate (6.25~50 μg/ml) and aglycone (6.25~25 μg/ml) frac- tions protected the cells against oxidative damage in a concentration dependent manner. When the whitening effects of D. mor- bifera leaf extract were tested in melanoma B16/F1 cells treated with the a-melanocyte stimulating hormone ( α-MSH), the extracts inhibited α-MSH-stimulated intra/extracellular melanogenesis in a concentration dependent manner. The inhibitory effects of the ethyl acetate and aglycone fractions of D. morbifera leaf extract were 21% and 44% at 25 μg/ml, respectively.

Both are more effective than arbutin (15% at 25 μg/ml) which is known as a whitening agent. These results indicate that frac- tions of the D. morbifera leaf can function as cell protectants and natural antioxidants in biological systems, particularly skins exposed to UV radiation by quenching and/or scavenging

O

and other ROS, and protecting cells against ROS. In addition, fractions of the D. morbifera leaf can be applied to new whitening cosmetics because of their inhibitory effects on α-MSH stim- ulated melanogenesis in B16F1 melanoma cells.

Keywords: Dendropanax morbifera, antioxidant activity, HaCaT, B16F1 melanoma, α-MSH, melanogenesis

*Corresponding author

Tel: +82-2-970-6451, Fax: +82-2-972-9585 E-mail: [email protected]

© 2013, The Korean Society for Microbiology and Biotechnology

있다

[1, 10, 26].

^{, ROS}

^.

^ROS

[13-15, 22].

멜라닌은사람의피부색을결정하는주요인자중하나로 서동식물계에존재하는생체고분자물질이며피부의기저

층에존재하는멜라닌생성세포

(melanocyte)

되고수지상돌기를통해각질형성세포

(keratinocyte)

송되어

melanin cap

각화과정에의해피부바깥층으로이동하여떨어져나간다

[2, 19].

tyrosinase, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2)

^,

tyrosinase

^.

,

^tyrosine

3,4-dihydroxy-phenylalanine (DOPA), DOPA quinone

^eumelanin

계열의

pheomelanin

^.

이동되어자외선등에의한피부의노화나일광각화증을억 제하여피부를보호하는긍정적인기능을가지고있는반면 에

^,

^,

^,

[6, 9, 20, 21, 23].

따라서피부노화를지연또는억제시키기위해서자외선 을차단하거나피부에서의

^ROS

^ROS

^ROS

[8, 12, 18].

황칠나무

(Dendropanax morbifera)

^,

^,

^,

^,

^,

[11].

sesquiterpene

^-selinene

, capnellane-8-one

이함유되어있다고알려져있다

^[7].

황칠나무추출물의항산화활성및항암활성등의생리 활성은일부제한적으로보고되고있으며

^,

^.

^,

1,1-diphenyl-picrylhydraxyl

(DPPH)

^[5],

흡한실정으로

^,

H

O

^O

^.

의에틸아세테이트분획과아글리콘분획으로멜라닌생합 성저해활성측정은아직연구가되지않았다

^.

(orientin, vitexin, rutin)

^,

^melanoma

^.

재료 및 방법

기기 및 시약

증감제로 사용된

rose-bengal, dihydroxyphenylalanine (DOPA),

^(arbutin)

Sigma (USA), H

O

^Dae Jung Chemical & Metals (Korea)

^.

^,

^(EtOAc),

^.

^CL-1000 Ultraviolet Crosslinker

^UVP

^(USA)

. UV-visible spectrophotometer

Varian (Australia)

Cary 50,

(D. morbifera)

2012

⁶

^.

황칠나무 잎의 추출 및 용매 분획

실험에사용한황칠나무잎은

^{Fig. 1}

출

^/

^.

^{100 g}

^50%

^{1 L}

^.

Fig. 1. Scheme for preparation of the fractions obtained from

D. morbifera leaf.

용하였다

^.

^50%

^(n-hexane)

^,

^,

³

^,

^.

에틸아세테이트 분획

(Ethyle acetate fraction of D.

morbifera leaf, DM EA)

(aglycone)

^:

반응을이용해서당을제거시킨후얻은아글리콘

^(aglycone

fraction of D. morbifera leaf, DM Agly)

^.

^{5% H}

SO

- 50%

^{, 4 h}

^,

각시킨다

^.

5% KOH-MeOH

적정한다

^.

^,

^,

^.

세포 보호 효과 측정

세포 배양: 사람각질형성세포주인

^HaCaT

^Dr.

Fusenig (German Cancer Research Center, DKFZ)

^.

(Dulbecco's modified Eagle's medium, DMEM)

10% fetal bovine serum (PAA, Austria), 1% penicillin-streptomycin (PAA, Austria)

^{, 37}

^{C, 5% CO}

^.

세포 독성 평가:

^HaCaT

96 well plate

^{24 h}

³⁷

^{C, 5% CO}

^,

황칠나무잎추출물을처리한다

^{. 24 h}

^PBS

세척하고

^MTT

⁽² μg/ml)

^{3 h}

^,

^formazan

^DMSO

^{570 nm}

^.

아무것도처리를하지않은비조사군을대조군으로하여

100%

^.

UVB 조사:

96 well plate

¹

¹⁰

cells/well

^HaCaT

^{24 h}

^.

UVB Crosslinker

UVB 800 mJ/cm

^,

^.

H2O2 처리:

96 well plate

¹

¹⁰

cells/well

킨

^HaCaT

물이함유된배지를넣고

^{30 min}

^{, 10 mM H}

O

함유한배지를처리하여

^{30 min}

^.

Rose bengal 처리:

96 well plate

¹

¹⁰

cells/well

로부착시킨

^HaCaT

무잎추출물이함유된배지를넣고

^{30 min}

^{, 4} μM rose bengal

^{15 min}

^.

세포 생존율 측정: 세포생존율은

3-(4,5-dimethythiazol- 2-yl)-2,5-di-phenytetrazolium bromide (MTT, Sigma, USA) assay

^.

mitochondria dehydro-

genase

^MTT

진청색의비수용성

^formazan

성된

^formazan

^.

HaCaT

96 well plate

^{24 h}

³⁷

^{C, 5%}

CO

^,

출물을 처리한 세포에

^{800 mJ/cm}

UVB, 10 mM H

O

, 4 μM rose bengal

^.

^{, 24 h}

^PBS

^MTT

⁽² μg/ml)

^{3 h}

^,

^formazan

^DMSO

^{570 nm}

^.

아무것도처리를하지않은비조사군을음성대조군으로하

여

^100%

^.

시료를처리하지않은조사군을양성대조군으로하였으며

^,

^.

멜라닌 생합성 저해능 측정

세포배양:

^B16F1

^melanoma

^.

^{10% fetal} bovine serum (PAA, Austria), 1% penicillin-streptomycine (PAA, Austria)

Dulbecco's modified Eagle's medium (DMEM)

³⁷

^{C, 5% CO}

^.

세포 독성 평가: 세포독성평가는

3-(4,5-dimethythiazol- 2-yl)-2,5-diphenytetrazolium bromide (MTT, Sigma, USA) assay

. B16 melanoma

⁹⁶ well plate

^{24 h}

³⁷

^{C, 5% CO}

^,

^.

^{72 h}

^MTT

(2 μg/

ml)

^{3 h}

^formazan

^DMSO

^ELISA

^{570 nm}

^.

세포의 형태학적 관찰:

B16 melanoma

^{6 well} plate

¹

¹⁰

cells/well

³⁷

^{C, 5% CO}

^{. 24 h}

-melanocyte stimulating hormone (

-MSH, Sigma, USA, 200 nM)

도별로처리하여

^{72 h}

^.

의변화와멜라닌배출정도를위상차도립현미경을이용하여 관찰하였다

^.

멜라닌 함량 측정: 멜라닌함량은

^Hosoi

^[4]

^{(%) =}

¹⁰⁰

대조군의흡광도

변형하여사용하였다

. B16 melanoma

6 well plate

¹

¹⁰

cells/well

³⁷

^{C, 5% CO}

^{. 24 h}

^PBS

¹

phenol red

^DMEM

^.

-melanocyte stimulating hormone (

^-MSH, Sigma, USA, 200 nM)

리하여

^{72 h}

^.

배양한후에각세포배양상층액

100 μl

^96-well

ELISA reader

^{405 nm}

^.

멜라닌함량은남은세포배양상층액을제거하고

^{1% triton}

X-100

백질함량을측정하고

^{, pellet}

^{10% DMSO}

1 N NaOH

¹⁰⁰ μl

96-well plate

^ELISA

^{405 nm}

^.

,

DC protein assay kit (Bio

Rad, USA)

^.

통계처리

모든실험은

³

^5%

Student's t-test

^.

결과 및 고찰

황칠나무 잎 추출물의 세포 독성 평가

황칠나무잎추출물의세포독성에미치는농도를조사하 고

^,

^{MTT assay}

^.

포독성을측정한결과

^{, 50} μg/ml

이

^80%

^.

, 50 μg/ml

^29%

^.

콘분획의농도를

^80%

농도로각각

^{50, 25} μg/ml

^{(Fig. 2).}

자외선 조사에 따른 황칠나무 잎 추출물의 세포 보호 효과 UVB 조사선량에 따른 세포 생존율:

UVB Crosslinker

를 이용하여

^HaCaT

^UVB

(600~900 mJ/cm

)

. 600 mJ/cm

^UVB

^78%

, 700 mJ/cm

65%, 800 mJ/cm

^50%,

^{900 mJ/cm}

^39%

^.

보호효과를측정하기위하여자외선의광량을

^{800 mJ/cm}

로설정하였다

^{(Fig. 3A).}

UVB로 유도된 세포 손상에 대한 황칠나무 잎 추출물 의 보호 작용:

^HaCaT

^{800 mJ/cm}

^UVB

^{. 24 h}

동안세포를배양하고세포의생존율을

^{MTT assay}

하였다

. 800 mJ/cm

^UVB

UVB

^44%

타내었다

^.

^HaCaT

^{12.5, 25}

50 μg/

ml

^{54, 57}

^59%

^.

^,

^{45, 53}

^22%

^,

⁵⁰ μg/ml

^UVB

나타났다

^.

50 μg/ml

콘분획자체의세포에대한독성을반영한결과로보여진 다

^{(Fig. 2).}

^,

물을처리하면

^{, UVB}

계를차단시켜서세포보호활성을나타냄을알수있었다

(Fig. 3B).

H2O2 처리에 따른 황칠나무 잎 추출물의 세포 보호 효과 H2O2 농도별 처리에 따른 HaCaT 세포의 생존율:자 외선에의해발생되어산화적손상을유발하는활성산소인

H

O

^HaCaT

(1, 5, 10, 15,

20 mM)

^.

1 mM

^H

O

^H

O

에비해

^89%

^{. 5 mM}

Fig. 2. HaCaT cell viability of the fractions of D. morbifera leaf extracts by MTT assay.

DM EA; ethyle acetate fraction of D. morbifera leaf, DM agly; agly-

cone fraction of D. morbifera leaf.

우세포생존율은

^75%

^{, 10 mM}

50%, 15 mM

^35%

^{20 mM}

^16%

(Fig. 4A).

^H

O

^H

O

^50%

^{10 mM}

^.

H2O2로 유도된 세포 손상에 대한 황칠나무 잎 추출물 의 보호 작용: 농도별황칠나무잎추출물을처리한후에

HaCaT

^{10 mM H}

O

^, 30 min

^PBS

^{. 24 h}

^{MTT assay}

^{. 10 mM H}

O

^H

O

^50%

^{. H}

O

^HaCaT

6.25, 12.5, 25

50 μg/ml

53, 53, 54

55%

^,

^H

O

^.

61, 63, 66

^40%

²⁵ μg/ml

것으로나타났다

^.

50 μg/ml

소만조사한경우보다세포생존율이더낮게나타났다

^.

는

50 μg/ml

의세포에대한독성을반영한결과로보여진다

^{(Fig. 2).}

과적으로황칠나무잎추출물이과산화수소로유도된세포

손상에대한세포보호활성을나타냄을알수있었다

^(Fig.

4B).

1O2으로 유도된 HaCaT 세포의 손상에 대한 세포보호 효과 Rose bengal 농도별 처리에 따른 HaCaT 세포의 생

존율:

^HaCaT

^,

성산소인¹

^O

rose bengal

(1, 2, 3, 4, 5 μM)

^{. 1} μM

^rose bengal

rose bengal

에비해

^89%

^{. 2} μM

우세포생존율은

^75%

^{, 3} μM

^{59%, 4} μM

50%, 5 μM

^37%

(Fig. 5A).

^O

rose bengal

^50%

Fig. 3. (A) Viability of HaCaT cells exposed to different irradi-

ation intensities of UVB irradiation. (B) Effect of the fractions of D. morbifera leaf extracts on UVB-induced cell damage in HaCaT cell system.

HaCaT cell were exposed to UVB 800 mJ/cm

and stained with MTT to show survival cells compared to non-irradiatied group (DM EA; ethyle acetate fraction of D. morbifera leaf, DM agly; aglycone fraction of D. morbifera leaf).

Fig. 4. (A) Cell viability on H

O

-induced cell damage in HaCaT cell system. (B) Cellular protective effect of the frac- tions of D. morbifera leaf extracts on H

O

-induced cell dam- age in HaCaT cell system.

HaCaT cells were treated with 10 mM H

O

and stained with MTT

to show survival cells compared to non-treated group (DM EA; eth-

yle acetate fraction of D. morbifera leaf, DM agly; aglycone fraction

of D. morbifera leaf).

율을나타낸

4 μM

^.

1O2으로 유도된 세포 손상에 대한 황칠나무 잎 추출 물의 보호 작용:

^HaCaT

⁴ μM

rose bengal

^{15 min}

^PBS

^{. 24 h}

동안세포를배양하고세포의생존율을

^{MTT assay}

하였다

^{. 4} μM rose bengal

rose bengal

^41%

^. Rose bengal

^HaCaT

6.25, 12.5, 25

및

⁵⁰ μg/ml

을처리한경우세포생존율은각각

54, 63, 72

^91%

, 50 μg/ml

^,

^control

^.

^,

75, 80, 84

^54%

^.

50 μg/ml

세포에대한독성을반영한결과로보여진다

^{(Fig. 2).}

황칠나무잎추출물이세포독성을갖지않는농도에서¹

^O

으로유도된라디칼연쇄반응의개시단계를차단시키고

^,

(Fig. 5B).

황칠나무 잎 추출물의 멜라닌 생성 저해활성 측정

황칠나무 잎 추출물의 B16F1 melanoma 세포에 대한 독성 평가:황칠나무잎추출물의멜라닌생성저해능측정 을위해황칠나무잎추출물과대조군으로사용된알부틴의 세포독성을알아보기위하여

^{MTT assay}

^.

, 50 μg/ml

도이하의세포생존율이

^80%

^.

경우

, 50 μg/ml

^56%

^.

100 μg/ml

^90%

^{(Fig. 6).}

분획의농도를

^80%

Fig. 5. Cell viability on

O

-induced cell damage in HaCaT cell system. (B) Cellular protective effect of the fractions of D.

morbifera leaf extracts on

O

-induced cell damage in HaCaT cell system.

HaCaT cell was treated with 4 μM rose bengal and stained with MTT to show cell viability compared to non-treated group (DM EA;

ethyle acetate fraction of D. morbifera leaf, DM agly; aglycone frac- tion of D. morbifera leaf (*p < 0.05).

Fig. 6. B16F1 melanoma cell viability of the fractions of D.

morbifera leaf extracts and arbutin as standard by MTT assay.

DM EA; ethyle acetate fraction of D. morbifera leaf, DM agly; agly-

cone fraction of D. morbifera leaf.

도로각각

^{50, 25} μg/ml

^,

¹⁰⁰ μg/ml

^.

B16F1 melanoma 세포의 형태학적 관찰: 황칠나무잎 추출물의멜라닌생성저해활성을측정하기위해서

^{, B16F1}

melanoma

고

⁷²

^.

^control

^-MSH

^,

^.

(6.25~50 μg/ml)

(3.13~25 μg/ml)

^,

^-MSH

(Fig. 7).

멜라닌 생성 저해활성 측정: 황칠나무잎추출물의미 백효과를알아보기위해α

^-MSH

B16 melanoma

^.

^.

^-MSH

(+ 66% of control)

6.25, 12.5, 25

50 μg/ml

13, 16, 21

^34%

^{, 50} μg/ml

처리군인

^control

^,

밖으로배출된멜라닌함량또한세포내멜라닌함량과비

슷한 경향성을보였다

(Fig. 8A).

^-

MSH

(+ 105% of control)

^3.13, 6.25, 12.5

²⁵ μg/ml

1, 14, 24

^44%

^{, 25} μg/ml

^control

^.

과비슷한경향을보였다

(Fig. 8B).

틴은α

^-MSH

^42%

증가하였고

^,

^-MSH

^12.5, 25, 50

¹⁰⁰ μg/ml

14, 15, 18

^20%

^.

^,

슷한경향성을보였다

(Fig. 8C).

틴은모두농도의존적으로멜라닌생합성저해활성을나타

내었다

^{. 25} μg/ml

이가장우수한멜라닌생합성저해활성

^(44%)

Fig. 7. Effect of D. morbifera leaf extracts on cellular morphology of cultured B16F1 melanoma cells under inverted phase con- trast microscope using a digital camera.

B16F1 melanoma cells were treated with the fractions of D. morbifera leaf extracts in the presence of 200 nM α-MSH for 72 h (DM EA;

ethyle acetate fraction of D. morbifera leaf, DM agly; aglycone fraction of D. morbifera leaf).