홈네트워크 시장 활성화 저해 요인과 주요 갈등2006-3-16 19 초 점 정보통신정책 제18권 5호 통권389호 홈네트워크 시장 활성화 저해 요인과
In Vivo Zebrafish 모델을 이용한 멜라닌 저해 생약재...
Transcript of In Vivo Zebrafish 모델을 이용한 멜라닌 저해 생약재...
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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: [email protected]
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
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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).
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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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.
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(No. 2011-
0009728).
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