Antioxidative, and Inhibitory Activities on Melanogenesis of Vitex negundo L. Leaf Extract

목형 잎 추출물의 항산화 활성과 멜라닌 생합성에 대한 저해활성

김아름, 박수아, 하지훈, 박수남*

서울과학기술대학교정밀화학과

,

Received : November 14, 2012 / Revised : January 14, 2013 / Accepted : January 15, 2013

서 론

의학의진보와사회

^,

^,

¹⁰⁰

^.

^.

^,

(anti-aging)

^,

^.

다

^[36].

하고

^,

^.

자외선이며

^,

UVC(200~280 nm), UVB(280~320 nm), UVA(320~400 nm)

^{. UVC}

^,

^UVB

와

^UVA

^{. UVB}

백질및

^DNA

^UVB

때문에주로표피의피부손상을야기하고

^{, UVA}

^.

⁽

^,

⁾

(reactive oxygen species, ROS)

^{[15, 16].}

^,

type I, type II

^{, type I}

^UVB

^{type II}

^UVA

^ROS

[8, 9]. ROS

⁽

^O

)

^,

⁽

^O

),

^(H

O

)

^(O

),

⁽

^OH)

Antioxidative, and Inhibitory Activities on Melanogenesis of Vitex negundo L. Leaf Extract. Kim, A Reum, Su Ah Park, Ji Hoon Ha, and Soo Nam Park*. Department of Fine Chemistry and Cosmetic R&D Center, Seoul National University of Sci- ence and Technology, Seoul 139-743, Korea

The aim of this study was to evaluate various aspects of Vitex negundo L. leaf extract, such as the antioxidative activity, tyrosi- nase inhibitory effects, and inhibitory activities on α-MSH induced melanogenesis, and active component analysis. The DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenging activities (FSC

) of the ethyl acetate fraction and aglycone fraction of V. negundo L.

leaf extract were 14.51 µg/ml and 13.96 µg/ml, respectively. A luminol-dependent chemiluminescence assay revealed that the reactive oxygen species (ROS) scavenging activity (OSC

) of the aglycone fraction of V. negundo L. leaf extract on ROS gener- ated in an Fe

-EDTA/H

O

system was the most prominent at 0.22 µg/ml. The protective effects of the extracts fractions of V.

negundo L. leaf against the rose-bengal sensitized photohemolysis of human erythrocytes were increased in a concentration dependent manner (1~50 µg/ml). In particular, there were greater protective effects of the aglycone fraction on the cellular mem- brane than that of the fat-soluble antioxidant (+)- α-tocopherol. The inhibitory effects (IC

) on mushroom tyrosinase were the highest for the ethyl acetate fraction (IC

= 48.58 µg/ml). The inhibitory effect on α-MSH induced melanogenesis in B16 mela- noma cells was 41.80% at 50 µg/ml of ethyl acetate fraction. Active component analyses by TLC, HPLC and LC/ESI-MS revealed luteolin and isoorientin. These results indicate that V. negundo L. leaf extract can be used as an antioxidant for ROS scavenging. Particularly, the luteolin and isoorientin of the ethyl acetate fraction may be applicable to new whitening cosmetics because of its inhibitory effect on mushroom tyrosinase and α-MSH induced melanogenesis in B16 melanoma cells.

Keywords: Vitex negundo, antioxidant activity, tyrosinase, melanogenesis

*Corresponding author

Tel: +82-2-970-6451, Fax: +82-2-972-9585

E-mail: [email protected]

같은산소중심의라디칼

^,

^ROS

^(ROO

),

^(RO

),

^(ROOH)

^.

^ROS

^,

^{, DNA}

^,

^{[1, 37].}

^ROS

superoxide dismutase (SOD), catalase, glutathione peroxidase

^vitamin E, vitamin C, glutathione

[19, 27, 32].

멜라닌은사람의피부색을결정하는주요인자중하나로 서동식물계에존재하는생체고분자물질로피부의기저층에

존재하는멜라닌생성세포

(melanocyte)

수지상돌기를통해각질형성세포

(keratinocyte)

melanin cap

에의해피부바깥층으로이동하게된다

^{[2, 30].}

(melanosome)

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

(eumelanin)

(pheomelanin)

^{[12, 20].}

[4, 10, 21].

(free radical scavenger)

[22, 23, 25, 26, 28].

butylhydroxytoluene (BHT), butylhydroxyanisole (BHA)

[13, 17],

연항산화제를개발하는노력이이루어지고있다

^.

목형

(Vitex negundo L. var. cannabifolia (Sieb. & Zucc.)

Hand-Mazz.)

식물이며주로동아시아에널리분포되어있으며바이텍스

(vitex negundo)

양

^,

^[34].

^,

negundoside, luteolin,

^- tocopherol,

-tocoquinone

^,

^{[18, 35].}

하지만국내에서생체내에서생성되는

^ROS

^,

⁽

^O

)

^,

^.

따라서

^,

^,

free radical

tyrosinase

^.

재료 및 방법

기기 및 시약

자유 라디칼 소거활성에 사용한

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

rose-bengal, luminol,

-ascorbic acid, (+)-

-tocopherol (1,000 IU vitamin E/g)

Sigma (USA)

^{. FeCl}

6H

O

Junsei (Japan)

^{, H}

O

^.

^Na

HPO

12H

O, NaH

PO

2H

O, NaCl,

trizma base, HCl,

^(EtOH),

^(MeOH),

^(EtOAc)

^.

Sonication

^(Korea)

Powersonic 410

^, UV-visible spectrophotometer

Varian (Australia)

^Cary 50,

Spectronic 20D

Milton Roy Co. (USA)

^,

Berthold (Germany)

^6- channel LB9505 LT

, pH meter

Istek (Korea)

^,

thin layer chromatography (TLC)

aluminum sheet silica gel 60 F254 (0.2 mm)

Merck (USA)

^HPLC

Shimadzu (Japan)

, LC/ESI-MS (Applied Biosystem, USA)

^.

(Vitex negundo L. var. cannabifolia (Sieb. &

Zucc.) Hand-Mazz.)

²⁰¹¹

⁹

^.

목형 잎의 분획 추출

건조된목형잎

^{10 g}

^50%

^{1 L}

^{1 h}

sonication (60 Hz)

³

^{Fig. 1}

과같은방법으로분액을하였다

^{. 50%}

압농축후

^n-

^.

^H

SO

5% KOH-MeOH

^,

^.

목형 잎 추출물의 항산화 효과 측정

DPPH법을 이용한 자유 라디칼 소거 활성: 목형잎추 출물과비교표준물질인

^(+)-

-tocopherol

거활성을

^DPPH

^[33].

자유라디칼인

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

^.

0.2 mM DPPH

^{1 ml}

^{1 ml}

^{1 ml}

^{10 min}

^{517 nm}

^.

^(control)

^,

(experiment)

으로하여다음식으로

^DPPH

며

^DPPH

^50%

^(free radical scavenging activity, FSC

,

^g/ml)

^.

Luminol 발광법을 이용한 Fe³⁺-EDTA/H2O2 계에 있 어서 활성산소 소거 활성(총항산화능):

^Fe

^+-EDTA/H

O

^{ROS (O}

, OH

^H

O

)

^,

hydroxyl radical (OH)

.

^ROS

^{[3, 31].}

^luminol

^ROS

타나는빛을화학발광기로검출하여측정하였다

^.

학발광측정용튜브에증류수

^{1.78 ml}

목형추출물과

2.5 mM EDTA 40

^l

5 mM FeCl

6H

O 10

^l

35 mM luminol 80

^l

cell holder

^{. 5 min}

^{150 mM H}

O

40

^l

^{25 min}

^.

^(control)

^(blank)

^H

O

^FeCl

6H

O

^.

6-channel LB9505 LT

^,

^50%

(reactive oxygen species scavenging activity, OSC

,

^g/ml)

^.

Photohemolysis법을 이용한 세포보호 효과 측정 적혈구 현탁액 제조: 적혈구는건강한성인남녀로부터 얻었다

^.

^{, 3,000}

rpm

^{5 min}

하였다

^.

0.9% saline phosphate buffer (pH 7.4, Na

HPO

12H

O 9.6 mM, NaH

PO

2H

O 1.6

mM)

였다

^{. 3}

^,

⁴

^C

^,

^{12 h}

^.

^.

^{700 nm}

^O.D.

^0.6

^1.5

¹⁰

^cells/ml

^.

목형 잎 추출물의 광용혈 억제 효과: 적혈구현탁액

^3.5 ml

^{(No. 9820)}

^,

⁵⁰

^l

^{30 min}

^pre- incubation

^.

rose-bengal (13

^{M) 0.5 ml}

(Whatman laboratory sealing film, UK)

^{15 min}

^[24].

광용혈에 필요한 광조사는 내부를 검게 칠한

^{50 cm}

20 cm

^{25 cm}

^{20 W}

^,

^{5 cm}

평행이되도록 배열되게하였다

^.

^(post-

incubation)

^{15 min}

^{700 nm}

(transmittance, %)

^.

^.

²⁰

^C

^.

post-incubation

^50%

Inhibition (%) 1 (AExperiment–ABlank) AControl

( ) ---

– ×100

=

Fig. 1. Scheme for preparation of compounds from V.

negundo leaf.

혈되는시간인τ50을구하여비교하였다

^.

대조군

^(control)

^{30 min}

± 1 min

^. Rose-bengal

^rose-

bengal

120 min

^.

용혈보호효과는아래와같이나타내었다

^.

멜라닌 생성 저해 측정

Mushroom tyrosinase 저해활성 측정[14]: 타이로시네 이즈는멜라닌생성과정중L

-tyrosine

3,4-dihydroxy-

- phenylananine (DOPA)

DOPA quinone

^.

^.

-

(0.3 mg/ml) 1.0 ml, potassium phosphate buffer (0.1 M, pH 6.8) 1.85 ml,

^{0.05 ml}

³⁷

^C

^{10 min}

^,

^,

DOPA chrome

^{475 nm}

^.

^{0.1 ml}

^50%

(inhibition concentration, IC

,

^g/ml)

^.

세포배양:

^B16F1

^melanoma

^.

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

Dulbecco's modified Eagle's medium (DMEM)

³⁷

^{C, 5% CO}

^.

passage number

¹⁵

^.

세포 생존율 측정:세포생존율은

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

assay

효소작용에의해노란색의수용성기질인

MTT tetrazolium

MTT formazan

[6]. B16 melanoma

^96- well plate

^{24 h}

³⁷

^{C, 5% CO}

^,

^arbutin

^.

^{24 h}

^MTT

⁽²

^g/ml)

^{3 h}

^formazan

^DMSO

^ELISA

^{570 nm}

^.

세포 내 멜라닌 함량 측정: 멜라닌함량은

^Hosei

^[11]

. B16 melanoma

^6- well plate

¹⁰

¹⁰

cells/well

³⁷

^{C, 5% CO}

조건으로항온배양하였다

^{. 24 h}

^PBS

¹

phenol red

DMEM

^.

-melanocyte stimulating hormone (

-MSH, Sigma, USA, 100 nM)

^{. 72 h}

1% triton X-100

^{, pellet}

^{10% DMSO}

^{1 N} NaOH

¹⁰⁰

^l

96-well plate

ELISA

^{405 nm}

^.

^,

DC protein assay kit (Bio Rad, USA)

^.

목형 잎 추출물의 성분분석

TLC 및 HPLC를 이용한 목형 잎 추출물 성분분석:

목형잎추출물의에틸아세테이트분획을

^100%

syringe filter (Milopore 0.45

^m)

^TLC

HPLC

^{. TLC}

아세테이트 분획의전개용매는

ethyl acetate : formic acid : chloroform : water = 8 : 1 : 1 : 1 (v/v)

^R

^{. HPLC}

^column

^Shim-pack VP-ODS (L : 250 mm, LD : 4.6 mm)

^detector

^SPD- M20A Diode array detector

^{. HPLC}

2% acetic acid

^(A

⁾

0.5% acetic acid

acetonitile

^(B

⁾

^B

0~5 min

20%, 5~35 min

20~40%, 35~75 min

40~80%, 75~95 min

^80~100%

^HPLC

^.

통계처리

모든실험은

³

^5%

Student's t-test

^.

결과 및 고찰

목형 잎 추출물의 항산화 활성

DPPH법을 이용한 자유 라디칼 소거활성:생체막에서는 활성산소종또는지질라디칼에의해개시되는지질과산화 반응은자동산화과정을경유하여연쇄반응이일어난다

^{. (+)-}

^-

Tocopherol,

칼에수소주개로작용하여연쇄반응을종결시킴으로써세포 막을보호한다

^.

free radical

DPPH

^DPPH

^.

Control τ50

---

=

Fig. 2. Free radical scavenging activities of extract/fractions of V. negundo leaf extracts and (+)- α-tocopherol.

Fig. 3. Reactive oxygen spesies scavenging activities of V.

negundo leaf extracts and

-ascorbic acid in Fe

-EDTA/

H

O

system by luminol-dependent chemiluminescence assay.

Table 1. Cellular Protective Effects of Extract/Fractions from V. negundo Leaf and Reference Compounds on the Rose- bengal Sensitized Photohemolysis of Human Erythrocytes.

τ

(Half time of hemolysis

)

Concentration, µg/ml 5 10 25 50

V. negundo leaf extract (50% EtOH extract) 36.8 (± 2.2) 52.0 (± 1.4) 67.8 (± 1.0) 91.7 (± 1.5) V. negundo leaf extract (EtOAc fraction) 38.5 (± 3.3) 49.5 (± 0.4) 58.5 (± 4.8) 66.8 (± 1.0) V. negundo leaf extract (Aglycone fraction) 56.8 (± 1.0) 71.5 (± 2.2) 103.7 (± 1.2) 120.3 (± 2.7)

(+)- α-Tocopherol - 38.0 (± 1.8) - 74.33 (± 6.35)

1)

Control, τ

= 30 ± 1.0 min

Fig. 4. Effects of aglycone fraction from V. negundo leaf extract on the rose-bengal sensitized photohemolysis of human erythrocytes.

Irradiation time: 15 min, pre-incubation time: 30 min, rose-bengal:

13 µM, ◆−◆: control, ▲−▲: 5 µg/ml, ■−■: 10 µg/ml, ×−×:

25 µg/ml.

목형잎추출물과비교물질인

^(+)-

-tocopherol

^(FSC

)

^{Fig. 2}

^.

목형 잎 추출물의

^50%

^FSC

50.53

^g/ml,

14.51

g/ml, 13.96

^g/ml

^.

^(+)-

^- tocopherol (FSC

, 8.98

^g/ml)

^.

루미놀 발광법을 이용한 Fe³⁺-EDTA/H2O2 계에 있 어서 활성산소 소거활성(총항산화능):루미놀은

^ROS

^(420~

450 nm)

^.

용한

^Fe

^-EDTA/H

O

^ROS

^{Fig. 3}

.

목형잎추출물의활성산소소거활성

⁽

^{, OSC}

)

^50%

^2.81

^g/ml,

^1.95

^g/ml,

^0.22

^g/ml

^.

-ascorbic acid

0.41 ± 0.14

^g/ml

-ascorbic acid

^.

1O2으로 유도된 사람 적혈구의 파괴에 대한 세포보호 효과 활성산소에의해야기되는세포손상에대한목형잎추출 물의세포보호효과를적혈구광용혈법으로측정하였고각 분획의농도에따른세포보호효과를

^{Table 1}

^.

^50%

⁽

)

^{(Fig. 4).}

^50%

5, 10, 25,

50

^g/ml

36.8, 52.0, 67.8, 91.7 min,

이트분획은동일한농도범위에서

38.5, 49.5, 58.5, 66.8 min

^.

56.8, 71.5, 103.7, 120.3 min

^(+)-

-tocopherol (10

^g/ml

38 min, 50

^g/ml

74 min)

^.

목형 잎 추출물의 멜라닌 생성 저해활성 측정

Mushroom tyrosinase 저해 활성: 타이로시네이즈는

멜라닌생성에있어서핵심효소로알려져있으며

^tyrosine

로부터시작되는 멜라닌생합성과정에서

3,4-dihydroxy-

- phenylananine (DOPA)

DOPA quinone

과정과

dihydroxyindole (DHI)

eumelanin

^.

즈저해활성을통해목형잎추출물의미백활성을측정하였다

^.

목형잎추출물중

^50%

즈저해활성

^(IC

)

^323.5

^g/ml

^,

^48.58

^g/ml,

97.57

^g/ml

^.

^(IC

= 226.88

^g/ml)

^{(Fig. 5).}

세포 생존률 측정: 목형잎추출물의멜라닌생성저해 능측정을위해목형잎추출물과대조군으로사용된알부 틴의세포독성을알아보기위하여

^{MTT assay}

^.

3 mM

(1~3 mM)

86.72 (± 6.70)%, 89.94 (± 4.29 )%, 97.42 (± 11.17)%

(Fig. 6A).

^50%

²⁵

^g/ml

92.10 (± 6.23)%

^80%

^.

^,

⁵⁰

^g/ml

²⁵

^g/ml

86.02(± 0.99)%, 97.84 (± 5.35)%

^80%

^{(Fig. 6B,} C, D).

세포 내 멜라닌 함량 측정:목형잎추출물의세포수준

Fig. 5. The inhibitory effect of extract/fractions obtained from

V. negundo leaf and reference compound on tyrosinase.

Fig. 6. Cell viability effect of arbutin and extract/fractions from V. negundo leaf by MTT assay.

A; Cell were treated with various doses of arbutin (1~5 mM). B, C, D; Cell were treated with various doses of extract/fractions

(12.5~100 µg/ml). (*p < 0.05, **p < 0.01)

에서 미백 효과를 알아보기 위해 α

^-MSH

^B16

melanoma

을측정하였다

^.

-MSH 100 nM

^(control)

16.19 (± 2.44)%

^.

^,

^-MSH

^18.56%

^,

^-MSH

^{3 mM}

58.38%, 2.5 mM

37.23%, 2 mM

^6.21%

생성을저해하였지만

^{1 mM}

나지않았다

(Fig. 7A).

^50%

^-MSH

^(+15.61%)

²⁵

^g/ml

28.14%, 12.5

^g/ml

14.03%, 6.25

^g/ml

8.74%, 3.125

^g/ml

^8.39%

(Fig. 7B),

^-MSH

^(+17.56%)

⁵⁰

^g/ml

^41.80%, 25

^g/ml

27.30%, 12.5

^g/ml

^18.70%, 6.25

^g/ml

^11.61%

(Fig. 7C).

^-MSH

^(+13.03%)

²⁵

^g/ml

^27%, 12.5

^g/ml

^{9%, 6.25}

^g/ml

^4.85%, 3.125

^g/ml

^0.90%

합성을저해하였다

(Fig. 7D).

mushroom tyrosinase

^{, mushroom}

tyrosinase

(IC

= 48.58

^g/ml)

⁵⁰

^g/ml

^41.80%

mushroom tyrosinase

^.

^{2 mM}

^54.45

^g/ml

^6.21%

^.

^,

^.

TLC 및 HPLC를 이용한 목형 잎 추출물의 luteolin 및 isoorientin 분석:목형잎추출물에는

^luteolin

luteolin

isoorientin (luteolin-6-C-glucoside)

^.

^luteolin

[5], isoorientin

^.

^luteolin

isoorientin

^.

Fig. 7. Melanin contents of arbutin and extract/fractions from V. negundo leaf.

A; Cell were treated with various doses of arbutin (1~3 mM, arbutin 2 mM = 54.45 µg/ml). B, C, D; Cell were treated with various

doses of extract/ethyl acetate fraction (6.25~50 µg/ml) and aglycone fraction (3.125~25 µg/ml). (*p < 0.05)