505www.kosac.or.kr

RESEARCH ARTICLE

.

Melanin

. ,

melanin

, ,

, , , , ,

(Ikeda et al., 2003). ,

melanin

, melanocyte

melanosome (Kim et al., 2002;

, 2003).

(dendrite) (keratinocyte)

,

(Han et al., 2012).

Melanin key enzyme tyrosinase

melanosome tyrosine

dopa, indole-5,6-quinone

melanin polymer

(Hearing & Ekel, 1976; Nappi & Vass, 1993).

melanin tyrosinase

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

In Vivo Zebrafish 1, 2, 3* 1 , 2, 3

Screening of Medicinal Herbs Against Melanin Biosynthesis Inhibition In Vivo Zebrafish ModelSeung Hwa Baek1, Jong Ho Park2, Jin Hee Kim3* 1Korea Department of Food Science & Biotechnology, Kyungpook National University 2Dainpia Inc., 3Department of Herbal Skin Care & Cosmetology, Daegu Haany University

Abstract The purpose of this study was to investigate whitening agent from a natural substance using in vivo model zebrafish. Tyrosinase is a key enzyme in melanogensis which was used for screening of whitening agent with tyrosinase inhibition assay and melanocyte cell melanin biosynthesis inhibition assay. However, pigmentation mechanisms were very a complicated procedure in human dermis. In this study, we used zebrafish embryo as an animal model for confirmed phenotype-based screening of melanin synthesis with natural sources. Zebrafish has most of same organs and tissues that can be seen in human. Especially, pigments were located in the epidermal layer on the surface of organism. In order to examine the whitening effects involved in pigment cell signaling, we treated zebrafish embryos with 10 kinds of methanol extracts of medicinal herbs. Among those medicinal herbs tested, 50 g/mL of MeOH extract of Forsythia suspensa was decreased the pigmentation on zebrafish larva. After solvent partitioning with MeOH extract of F. suspense, chloroform and ethyl acetate fractions showed the depigmentation effect on zebrafish. In contrast the tyrosinase inhibition effect of F. suspense has not shown. Those results suggest that different approaches to obtain depigmentation process, and confirm the new protocols to evaluate mleanogenesis using in vivo zebrafish model.

Keywords: Zebrafish, whitening agent, mleanogenesis, Forsythia suspense

Kor. J. Aesthet. Cosmetol.,Vol. 11 No. 3, 505-511, June 2013

*Corresponding author: Jin Hee Kim, Department of Herbal Skin Care, Daegu Haany University, 290 Yugok-dong, Gyeongsan, Gyeongbuk 712-715, Republic of KoreaTel.: +82 53 819 1588, Fax: +82 53 819 1572E-mail: jinheekim@dhu.ac.kr

Received February 18, 2013; Revised June 6, 2013;Accepted June 13, 2013; Published June 30, 2013

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Kor. J. Aesthet. Cosmetol.,Vol. 11 No. 3, 505-511, June 2013

2(dopachrome tautomerase)

(Brown, 2001; Kameyama et al., 1993).

estrogen, testosterone, glucocorticoid, prostaglandin

(Aroca et al., 1993; Busca & Ballotti, 2000),

cyclooxygenase-2 (COX-2) (Kim et al., 2012), interferon

(IFN) (Aoki & Moro, 2005), cyclic AMP (Jung et al., 2011)

melanocyte stimulation hormone (MSH) (Duval et al.,

2012), Vitamin D3 (Birlea et al., 2008), hitamine (Yoshida

et al., 2000) melanin

.

, ,

3

( , 2013).

Melanin type

. melanin key enzyme tyrosinase

type,

tyrosinase

melanin type

(Mishima et al., 1988).

:(i) tyrosinase,

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

protein-2 (TRP-2), and/or peroxidase

; (ii) keratinocytes melanosomes

; (iii) melanosome keratinocytes

(Li & Hill, 1997). melanin mechanism

tyrosinase

melanin (Briganti et al.,

2003). in vitro tyrosinase

melanin

.

tyrosinase

in vivo zebrafish

. Zebrafish (Danio rerio)

3

. 24

,

(Shin & Fishman, 2002; , 2006).

, , BHT

(dibutylated hydroxytoluene)

,

(Burke,

2007; Choi & Berson, 2006; Oricha, 2010).

(Baek et al., 2006; , 2012).

screening tyrosinase

in vivo zebrafish melanin

10

(Forsythia suspensa)

. (Oleaceae)

.

saponin, flavonoid, alkaloid, lignans

(Guo et al., 2007), (Piao et al., 2009), (

, 2005), (, 2012), (Qu et al.,

2012), (, 2006), (Li et al.,

2012) .

in vivo zebrafish

, zebrafish

.

.

1.

(Lonicera japonica Thunberg),

(Aucklandia lappa Decne), (Forsythia suspensa

Vahl), (Fallopia multiflora), (Plantaginis

Semen), (Liriopis Tuber), (Asiasari Radix),

(Polygoni Multiflori Radix), (Gentianae Scabrae

Radix), (Myristicae Semen) 10

5 3

(Whatman No. 2, Tokyo, Japan)

(Rotay Evaporator, R-200,

BUCHI, Switaerland) .

(Hexane),

(Cholorform), (Ethyl acetate),

(Butanol) .

tyrosinase (T3824), tyrosine (T3754),

sea salt (S9883) Sigma Chemical Co. (St. Louis, Mo,

USA) .

2. Tyrosinase

96 well plate (SPL, Korea) 0.1 M phosphate buffer

(pH 6.5) 150 mL, 1.5 mM L-tyrosine solution 38 mL

507www.kosac.or.kr

Zebrafish

2100 unit/mL mushroom tyrosinase (pH 6.5) 10 mL

, sample 3 mL 25 1

, microplate reader (Bio-Rad 3550) optical

density 470 nm . Tyrosinase

(%) , IC50

50% sample .

A :

B :

C :

D :

3. Zebrafish

zebrafish 14:10 28.5

,

(Kimmel et al., 1995; Westerfield, 1993). zebrafish

20 L 3~5 brine shrimp (San

Francisco BayBrand, Inc., Newark, CA, USA) 3

. 28.5

.

4. Zebrafish melanin

zebrafish

1~2 .

zebrafish embryo medium 24

, .

(chorion)

, 24

,

2 . 48

(Figure 1).

5.

3 (mean)

(standard deviation, SD) ,

Students t-test .

.

1. tyrosinase

tyrosinase Table 1

. tyrosinase 20%

.

tyrosinase

Figure 1. Scheme of melanin biosynthesis inhibition assay using zebrafish.

Table 1. Inhibitory activities of medicinal herbs on the mushroom tyrosinase

Scientic Name Park of use Tyrosinase activity (%)

Lonicera japonica Thunberg Vine 110.001**

Aucklandia lappa Decne Root 150.014**

Forsythia suspensa Vahl Fruit 80.008**

Fallopia multiora Rhizome and Root 40.006*

Plantaginis Semen Fruit 70.004**

Liriopis Tuber Root 190.031*

Asiasari Radix Rhizome and Root 140.007**

Polygoni Multiori Radix Tuberous root 180.005**

Gentianae Scabrae Radix Root 220.006**

Myristicae Semen Fruit 200.008**

Data are meansS.D of 3 experiments.*p.001 signicantly dierent as compared to control.

(%) =(D-C)-(B-A)

100D-C

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Kor. J. Aesthet. Cosmetol.,Vol. 11 No. 3, 505-511, June 2013

.

tyrosinase

hydroquinone (Li et al., 2005) 4-hydroxyanisole

(Di Nuzzo & Masotti, 2010)

, (Stenius et al.,

1993),

(Zhou et al., 2009) .

tyrosinase

TRP-1, 2

, tyrosinase

.

2. Zebrafish melanin

10 zebrafish embryo

48 Figure

2 (Forsythia suspensa)

. plate 11 zebrafish embryo

, 9

(data not shown). (yolk)

. (Liriopis Tuber),

(Asiasari Radix), (Polygoni Multiflori Radix)

.

, Figure 3

. Zebrafish

100~200 .

,

(Pasco & Leopold, 2012), (Rigo-Watermeier et al.,

2012), (Finn et al., 2012), zebrafish (Stanley et al.,

2009) . zebrafish

(Choi et al., 2007; O

Reilly-Pol & Johnson, 2008). zebrafish

(Sisman, 2011) (Huang X,

2011) .

in vivo

tyrosinase , zebrafish

.

tyrosinase

.

Figure 2. Effects of medicinal herbs on the pigmentation of zebrafish. Decline of pigment cells is shown (white arrowhead). The following morphologic malformations are evident; the swelling of yolk (red arrowhead). Photographed under the stereomicroscope MZ10 (Leica Microsystems, Ernst-Leitz-Strasse, Germany).

Figure 3. Effects of Forsythia suspensa solvent partition on the pigmentation of zebrafish. Decline of pigment cells is shown (white arrowhead). The following morphologic malformations are evident; the swelling of yolk (red arrowhead). Photographed under the stereomicroscope MZ10 (Leica Microsystems, Ernst-Leitz-Strasse, Germany).

509www.kosac.or.kr

Zebrafish

, (

, 2005). in vitro in

vivo .

.

tyrosinase

in vivo zebrafish

in vivo

. 10

tyrosianse ,

zebrafish

50 mg/mL zebrafish

.

zebrafish

.

tyrosinase

in vitro zebrafish in vivo

in vitro

. zebrafish

,

.

zebrafish

, in vivo

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