ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2018; 16(1): R E S E A R C H

Size: px

Start display at page:

Download "ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2018; 16(1): R E S E A R C H"

지수 매
5 years ago
Views:

1 ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2018; 16(1): R E S E A R C H A R T I C L E Open Access Cnidium officinale Makino Extracts Inhibit α-msh-induced Melanogenesis in B16F10 Mouse Melanoma Cells Hwa Jun Cha Department of Beauty Care and Cosmetics, Osan University, Osan-si, Gyeonggi-do, Korea Corresponding author: Hwa Jun Cha, Department of Beauty Care and Cosmetics, Osan University, 45 Cheonghak-ro, Osan-si, Gyeonggido 18119, Korea Tel.: Fax: hjcha@osan.ac.kr Received October 30, 2017 Revised November 13, 2017 Accepted November 22, 2017 Published March 30, 2018 Abstract Purpose: Skin color is determined by melanin distributed in skin, hair, eye, heart, and brain. The melanin protects skin against ultraviolet (UV) and scavenges cellular reactive oxygen species (ROS). However, the abnormal accumulation of melanin induces hyperpigmentation diseases such as melisma, freckle etc.. Cnidium officinale Makino commonly used to natural medicine in East Asia such as Korea, Japan and China, is called Chun-kung in Korea. In present study, we identified the antimelanogenic effect of Cnidium officinale Makino in B16F10 cells. Methods: Cnidium officinale Makino extracts were prepared by leaching in 70% ethanol. Comparing with control and Cnidium officinale Makino extracts treated B16F10, we identified melanin contents measured by optical density at 450 nm, tyrosinase activity, tyrosinase mrna and protein expression, MITF activity using MITF. Results: As shown results, we demonstrated that Cnidium officinale Makino extracts down-regulated melanin synthesis using down-regulating activity and expression of tyrosinase which is key enzyme to produce melanin. In addition, we revealed expression of tyrosinase is regulated by MITF through repressing MITF transcriptional activity. Conclusion: Cnidium officinale Makino extracts has potential to repress melanogenesis and decreased of hyperpigmetation and to use as a cosmetic ingredient. Keywords: Cnidium officinale Makino, Melanogenesis, B16F10, Tyrosinase, MITF Introduction Melanin은대부분동물의피부, 모발, 눈, 내이, 뼈, 심장, 뇌등에분포되어있는물질로피부색을결정한다 (Alaluf et al., 2002; D Mello et al., 2016). 이러한 melanin은자외선으로부터피부를보호하고, 활성산소를제거하는역할을하는필수적인생체물질이다 (Brożyna et al., 2016; Herrling et al., 2008). 하지만비정상적인 melanin 축적은기미, 주근깨, 모반, 검버섯과같은색소이상증을일으킨다 (Slominski et al., 2004). 때문에 melanogenesis 조절을통해서색소이상증을조절할필요가있다. Melanogenesis는기저층에있는 melanocyte에서일어나며주로 tyrosinase에의해서조절된다 (Hearing, 2011). Tyrosinase (EC ) 은 polyphenol oxidase (PPO) 로알려져있으며 coppercontaining monooxygenase enzyme으로 melanin 합성에매우중요하다 (Parveen et al., 2010). 이러한 enzyme은 fungi, plant, animal 등에다양하게분포하고있으며 (van Gelder et al., 1997), L-tyrosine으로부터 melanin이합성되는초기과정인 3,4-dihydroxyphenylalanine (L-DOPA), dopaquinone이합성되는과정에서중요하게작용한다 (Hearing, 2011; Slominski et al., 2005; Slominski et al., 2012). 이러한 tyrosinase는 melanocyte-stimulating hormone (α-msh), adrenocorticotropic hormone (ACTH) 등과같은호르몬에의한신호전달기전에의해서 microphthalmia-associated transcription factor (MITF) 의전사활성을높이고, 이를통해 tyrosinase를발현시키게된다 (Brenner & Hearing, 2008; Costin & Hearing, 2007; Ha & You, 2016; Slominski et al., 2004). 때문에 melanogenesis의조절은관련신호전달기전을조절함으로써가능하다. Cnidium officinale Makino은산형과다년생식물로한국에서천궁으로불린다 (Choi et al., 2002). 이러한 Cnidium Copyright c Korea Institute of Dermatological Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Cnidium officinale Makino extracts inhibit melanogenesis officinale Makino은아시아를중심으로오랜기간동안진통제, 항염제, 생리불순치료등의목적으로전통의약제로사용하고있다 (Hong et al., 2017). 또한 Cnidium officinale Makino의주요성분인 phthalide 유도체들은진정제, 항빈혈제, 항진균제, 이완제, 항보체활성과같은다양한효능을가지고있고, Cnidium officinale Makino 추출물역시혈액순환개선및염증성질환억제의효능이있는것으로알려져있다 (Jang, 2017; Tahara et al., 1999; Wang et al., 1989). 특히, Higashi (1996) 의연구에서는 Cnidium officinale Makino가이완작용에효과적이고, 울혈과염증을억제하여염증성질환으로인한가려움증을완화할수있다고알려져있다. 또한간암모델에서 Cnidium officinale Makino 추출물이항암과항전이활성을가진다는것이입증되었다 (Haranaka et al., 1985; Hong et al., 2017; Onishi et al., 1998). 하지만아직까지도 Cnidium officinale Makino 가피부에존재하는세포들에대한연구는미비한편이다. 때문에본논문에서는 Cnidium officinale Makino 추출물이 melanocyte에서 melanogenesis에미치는영향을밝히고자한다. Methods 1. B16F10 mouse melanoma 세포배양 B16F10 mouse melanoma 세포는한국세포주은행 (Korean Cell Line Bank, Korea) 에서구매하였으며, Dulbecco s modified Eagle s medium (DMEM; Gibco, Thermo Fisher Scientific, USA) 에 10% fetal bovine serum (FBS; Sigma-Aldrich, USA) 와 10% penicillin (Gibco, 100 units/ml), 10% streptomycin (Gibco, 100 μ g/ml) 이포함된배지에배양하였다. 2. Cnidium officinale Makino 추출물제조 Cnidium officinale Makino을 1차세척후 60 건조기 (ON-50; Deahan Science, Korea) 에서완전히건조를한다. 건조된 Cnidium officinale Makino을분쇄기 (SMX- 5800LM; Shinil, Korea) 에넣고분말화하고, Cnidium officinale Makino 분말에 70% ethanol를첨가하여, 60 에서 30 min동안추출한다. 추출하는동안 20 khz이상의초음파 (Ultrasonic cleaner 8891; Cole-Parmer, USA) 를주어서추출효율을높였다. 추출후남은고형분을여과지 (Whatman No.2; GE Healthcare Life Sciences, USA) 로분리하고, 여과액을감압농축기 (EYELA N-3010; Tokyo Rikakikai, Japan) 와동결건조기 (LP 10-30; Ilshin, Korea) 로건조하였다. 건조된 Cnidium officinale Makino 추출물은다시농도에맞춰 dimethyl sulfoxide (DMSO; Biopure, Canada) 로녹였다. 3. 세포독성측정세포독성을측정하기위해서 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 를진행하였다. 96 well plate에 개의 B16F10 mouse melanoma 세포를접종하고, 48 hr 후추출물을 μ g/ml로처리하였다. 추출물이처리된세포를 48 hr 배양후 0.5 mg/ml MTT (Sigma-Aldrich) 로처리하고 37 에 1 hr 배양하였다. 1 hr 후배지를제거하고생성된 MTT formazan을 DMSO로녹여 595 nm에서 microplate reader (SpectraMax i3x; Molecular Devices, USA) 를이용하여흡광도를측정하였다. 측정된흡광도를비교하여세포독성을표현하였다. 4. Melanin 생성량측정 B16F10 mouse melanoma 세포에서의 melanin 함량측정은세포내의 melanin을추출하여흡광도를측정하는방법을사용하였다. 60-mm cell culture dish에 개의 B16F10 mouse melanoma 세포를접종하고 48 hr 배양하였다. 배양후세포에 melanin 생성유도물질인 α-msh와 Cnidium officinale Makino 추출물을처리하고 48 hr 배양하였다. 배양이끝난후세포를수확하여 PBS로 1회세척하고세포수를개수하였다. 각각의샘플당동일량의세포로분주하고 1 N sodium hydroxide (NaOH; Sigma-Aldrich) 100 μl를첨가하여 95 에서 15 min 세포를용해하였다. 그후 450 nm 파장에서 microplate reader를이용하여흡광도를측정하여 melanin 함량을확인하였다. 5. Tyrosinase 활성측정 Tyrosinase 활성측정은 0.1 M sodium phosphate buffer (ph 7.4) 에 2 mm L-Dopa (Sigma-Aldrich) 와 100 unit mushroom tyrosinase (Sigma-Aldrich) 또는 cell lysate 넣고반응하여측정하였다. Tyrosinase의직접적인저해효과를보기위해서 mushroom tyrosinase를이용하였고, 간접적인저해효과를보기위해서 cell lysate를이용하였다. Cell lysate는추출물을처리한세포를 1% Triton X-100 (Biopure), 150 mm sodium chloride (Biopure), 50 mm HEPES (ph 7.5, Biopure), 5 mm EDTA (Biopure) 가첨가된 lysis buffer를이용하여세포를용해하였다. 반응은 37 에서 30 min 반응하였으며, 반응을통해서합성된 melanin 은 450 nm 파장에서 microplate reader를이용하여흡광도 123

3 를측정하여확인하였다. Cell lysate를이용하는경우측정값의보정을위해서 Bradford protein assay (Thermo Fisher Scientific) 로전체단백질량측정후차이값을보정해주었다. 6. Tyrosinase mrna 발현량측정 Tyrosinase mrna의발현을확인하기위해서 total RNA는 TRIzol TM (Invitrogen, Thermo Fisher Scientific) 을이용하여추출하였다. 추출된 total RNA를 MaestroNano Pro Micro- Volume spectrophotometer (Maestrogen Inc., USA) 를이용하여정량하고, 동일량을 Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen) 로 cdna를합성하였다. 합성된 cdna를이용하여 tyrosinase의발현량을알아보는 quantitative real time polymerase chain reaction (qrt-pcr) 은 StepOnePlus TM Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) 에서진행하였다. PCR 반응액은 HOT FIREPol EvaGreen qpcr Mix Plus (ROX) (Solis BioDyne, Estonia), 0.5 pmoles/μ L forward primer, 0.5 pmoles/μl reverse primer, cdna 를혼합하여제조하였고, tyrosinase와보정을위해서사용된 β-actin의 primer 정보는 Table 1과같다. qrt-pcr 결과는 Ct값을측정하여 2 -ΔΔCT 의계산방법을이용하여상대정량하였고, 모든값은 β-actin을이용하여샘플간의차이를보정하였다. (M-19; Santa Cruz, USA) 와 β-actin (Santa Cruz) 를각각의 target 단백질에붙이고, secondary antibody를추가로붙여준후 Clarity TM Western ECL Substrate (Bio-Rad, USA) 를도포하여단백질이발생되게하고, 이를 Fusion FX7 imaging system (Vilber Lourmat, France) 에서촬영하여단백질량을확인하였으며확인된 tyrosinase 단백질량은 β-actin을이용하여보정하였다. 8. MITF 전사활성측정 MITF의전사활성을측정하기위해서 MITF의 binding site가들어있는 luciferase assay plasmid인 pgl3 (Invitrogen) 를사용하였다. Report plasmid인 pgl3- MITF와 normalization plasmid인 pcmv-β-gal을 Lipofectamine 2000 (Invitrogen) 을이용하여형질전환하였고, 24 hr의안정화시간을거친후 Cnidium officinale Makino 추출물로처리하였다. 처리후수확된세포는 1 luciferase lysis buffer (Promega, USA) 를이용하여 cell lysate를얻고, luciferase 기질인 Luciferase Reporter 1000 Assay System (Promega) 에넣어주어발광을유도하고, 발광정도는 Veritas Microplate Luminometer (Veritas, USA) 로측정하여 MITF의전사활성을확인하였다. 또한 O-nitrophenyl-β-D-galactopyranoside assays (β -Gal Assay Kit, Invitrogen) 를이용하여실험에서나올수있는오차값을보정하였다. 7. Tyrosinase protein 발현측정 Tyrosinase protein의발현을확인하기위해서 western blot을사용하여발현을분석하였다. 수확된세포를 1% SDS lysis buffer (Promega, USA) 로 95 에서 20 min 용해시킨후 5X sample buffer [10% SDS, 1M Tris-HCl (ph 6.8), 50% glycerol, 25% β-mercaptoethanol, 1% bromophenol blue] (Sigma-Aldrich) 에용해하고 Bradford protein assay kit로전체단백질량을확인하여동량의단백질을 5X sample buffer에섞어 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) 전기영동하여크기별로분리하였다. 이후단백질을 polyvinylidene difluoride (PVDF) membrane (Millipore, USA) 으로옮겨 tyrosinase 9. 통계처리본실험의모든실험은 3회반복하여진행하였고, 조건간의차이에대한유의성을입증하는방법으로 Student s t test을이용하였다. Student s t test 결과 p<0.05의값을유의성있다고판단하고, 유의적인값에는별표 ( * ) 를하였다. Results and Discussion 1. B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물의세포독성 B16F10 mouse melanoma 세포에 Cnidium officinale Table 1. Sequence of primers Primers Tyrosinase forward Tyrosinase reverse β-actin forward β-actin reverse Sequence 5 CAAGTACAGGGATCGGCCAAC3 5 GGTGCATTGGCTTCTGGGTAA3 5 CCCTGTATGCCTCTGGTC3 5 GTCTTTACGGATGTCAACG

4 Cnidium officinale Makino extracts inhibit melanogenesis Figure 1. Cytotoxicity of extracts of Cnidium officinale Makino in B16F10 mouse melanoma cells. Effects of Cnidium officinale Makino extracts on the cell viability. There is no significant difference between control and Cnidium officinale Makino extracts treated cells. Cytotoxicity was exhibited as a percentage of control at the indicated concentrations. Values are M±S.D. from triplicate experiments. M±S.D., mean±standard deviation. Makino 추출물에의한세포독성을확인하기위해서, Cnidium officinale Makino 추출물을 μg/ml 로 B16F10 mouse melanoma 세포에 24 h 처리후세포생존율을측정하였다. 200 μg/ml 농도까지세포의성장억제가일어나지않는것을확인하였고, 이에따라 Cnidium officinale Makino 추출물의독성이없는농도로확인된 200 μg/ml 이하에서 melanin 합성저해실험을진행하였다 (Figure 1). 2. B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물에의한 melanin 함량변화 Cnidium officinale Makino 추출물에의한 melanin 함량변화를확인하기위해서 200 ng/ml α-msh을처리하고동시에 Cnidium officinale Makino 추출물을 0, 100, 200 μg/ml 농도로처리하였다. 48 h 배양한후세포를파쇄하여 melanin 함량을측정하였다. α-msh 처리군에서는 melanin 함량이 % 까지증가였고, Cnidium officinale Makino 추출물 100, 200 μg/ml로처리시다시 %, % 까지감소하였다 (Figure 2). Melanin 합성은 melanosome에서 tyrosinase 에의해서생성되기때문에이후실험에서 tyrosinase 활성을측정하였다 (Pillaiyar et al., 2017). 3. B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물에의한 tyrosinase 활성변화 Cnidium officinale Makino 추출물에의한 melanin 생성감소가 tyrosinase에의해서일어나는지확인하기위해서 200 ng/ml α-msh을처리하고동시에 Cnidium officinale Makino 추출물을 0, 100, 200 μg/ml 농도로처리한후세 Figure 2. Effects of Cnidium officinale Makino extracts on melanin contents in B16F10 mouse melanoma cells. Cnidium officinale Makino extracts decreased melanin contents in B16F10 mouse melanoma cells. Melanin contents were exhibited as a percentage of control at the indicated concentrations. Values are M±S.D. from triplicate experiments. # p<0.05 compared with non-treated cells; * p<0.05 compared with α-msh-treated cells; COR ext., Cnidium officinale Makino extracts; α-msh, melanocyte-stimulating hormone; M±S.D., mean±standard deviation. 포내 tyrosinase 활성을확인하였다. Figure 3A에서와같이세포내 tyrosinase 활성이농도의존적으로감소됨을확인하였다. 하지만 Cnidium officinale Makino 추출물이 mushroom tyrosinase에의한 melanin 생성은억제하지못함을확인하였다 (Figure 3B). 때문에 Cnidium officinale Makino 추출물은간접적으로 tyrosinase 발현또는활성조절을통해서 melanin합성을저해함을확인하였다. 4. B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물에의한 tyrosinase 발현변화 Cnidium officinale Makino 추출물에의해 tyrosinase 발현이조절되는지알아보기위해 mrna와단백질발현량을확인하였다. Tyrosinase의단백질량을확인결과 Cnidium officinale Makino 추출물에의해서발현이의존적으로감소됨을확인하였다. 이러한 tyrosinase 발현감소가 mrna 억제에의해서일어나는지확인하기위해서 Cnidium officinale Makino 추출물을처리하고 tyrosinase의 mrna 발현량을확인하였다. Figure 4B에서와같이 tyrosinase mrna는 Cnidium officinale Makino 추출물에의해서감소됨을확인할수있었다. 5. B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물에의한 MITF 전사활성변화 α-msh에의한 melanin 합성에서 tyrosinase의발현은 MITF에의해서발현이조절된다 (D'Mello et al., 2016; 125

5 Figure 3. Effects of Cnidium officinale Makino extracts on tyrosinase activity. Cnidium officinale Makino extracts decreased tyroainase activity in B16F10 mouse melanoma cells. (A) Cellular tyrosianse activity, (B) mushroom tyrosinase activity. Melanin contents were exhibited as a percentage of control at the indicated concentrations. Values are M±S.D. from triplicate experiments. # p<0.05 compared with non-treated cells; * p<0.05 compared with α-msh-treated cells; COR ext., Cnidium officinale Makino extracts; α-msh, melanocyte-stimulating hormone; M±S.D., mean±standard deviation. Figure 4. Effects of Cnidium officinale Makino extracts on tyrosinase expression. Cnidium officinale Makino extracts decreased tyrosinase expression in B16F10 mouse melanoma cells. (A) Protein level of tyrosinase, (B) mrna level of tyrosinase. mrna level of tyrosinase were exhibited as a percentage of control at the indicated concentrations. Values are M±S.D. from triplicate experiments. # p<0.05 compared with non-treated cells; * p<0.05 compared with α-msh-treated cells; COR ext., Cnidium officinale Makino extracts; α-msh, melanocyte-stimulating hormone; M±S.D., mean±standard deviation. Hsiao & Fisher, 2014). 따라서 MITF의전사활성이 Cnidium officinale Makino 추출물에의해서조절되는지확인하였다. Figure 5에보는것과같이 MITF의전사활성은 α -MSH에서증가되었다가, Cnidium officinale Makino 추출물에의해서감소함을확인할수있었다. 때문에 Cnidium officinale Makino 추출물은 MITF의전사활성을감소시킴으로써 tyrosinase의발현및활성을억제하고, 이를통해서 melanin합성이저해됨을확인할수있었다. Conclusion 본연구에서는 B16F10 mouse melanoma 세포주실험모델활용을통해 Cnidium officinale Makino 추출물이피부 미백원료로서의효능이있음을 in vitro 에서알아보고, 화장품소재로서 Cnidium officinale Makino 추출물의효용성을확인하고자하였다. 첫째, α-msh가처리된 B16F10 mouse melanoma 세포에서 Cnidium officinale Makino 추출물이 melanin 합성을감소시킴을확인하였다. 둘째, Cnidium officinale Makino 추출물이 melanin 합성에가장중요한 tyrosinase 발현및활성을억제하는지분석한결과 α-msh 에의해서증가된 tyrosinase 발현및활성이 Cnidium officinale Makino 추출물에의해서감소함을확인할수있었다. 또한이러한 tyrosinase의발현감소는 MITF의전사활성변화에의해서일어남을확인할수있었다. 이러한내용을종합하여보았을때 Cnidium officinale Makino 추출물은피부의색소침착을저해하는미백기능성화장품소재로서가능성이있을것으로판단된다

6 Cnidium officinale Makino extracts inhibit melanogenesis Figure 5. Effects of Cnidium officinale Makino extracts on transcriptional activity of MITF. Cnidium officinale Makino extracts decreased MITF transcriptional activity in B16F10 mouse melanoma cells. Transcriptional activity of MITF was exhibited as a percentage of control at the indicated concentrations. Values are M±S. D. from triplicate experiments. # p<0.05 compared with nontreated cells; * p<0.05 compared with α-msh-treated cells; MITF, microphthalmia-associated transcription factor; COR ext., Cnidium officinale Makino extracts; α-msh, melanocytestimulating hormone; M±S.D., mean±standard deviation. Acknowledgements 이논문은 2017 년도오산대학교교내학술비지원에의해 서수행됨. References Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A. The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell & Melanoma Research, 15: , Brenner M, Hearing VJ. The protective role of melanin against UV damage in human skin. Photochemistry and Photobiology, 84: , Brożyna AA, Jóźwicki W, Roszkowski K, Filipiak J, Slominski AT. Melanin content in melanoma metastases affects the outcome of radiotherapy. Oncotarget, 7: , Choi HS, Kim MSL, Sawamura M. Constituents of the essential oil of Cnidium officinale Makino, a Korean medicinal plant. Flavour and Fragrance Journal, 17: 49-53, Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. The FASEB Journal, 21: , D'Mello SA, Finlay GJ, Baguley BC, Askarian-Amiri ME. Signaling pathways in melanogenesis. International Journal of Molecular Sciences, 17: E1144, Ha MJ, You SH. Bioactive characteristics of extracts of Opuntia humifusa fruit as functional cosmetic ingredients. Asian Journal of Beauty and Cosmetology, 14: , Haranaka K, Satomi N, Sakurai A, Haranaka R, Okada N, Kobayashi M. Antitumor activities and tumor necrosis factor producibility of traditional Chinese medicines and crude drugs. Cancer Immunology, Immunotheraphy, 20: 1-5, Hearing VJ. Determination of melanin synthetic pathways. Journal of Investigative Dermatology, 131: 8-11, Herrling T, Jung K, Fuchs J. The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 69: , Higashi K. The therapeutic effect of Unsei-in on facial redness (inflammatory congestion) in atopic dermatitis. The Japanese Journal of Oriental Medicine, 46: , Hong H, An JC, de La Cruz JF, Hwang SG. Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells. Experimental and Therapeutic Medicine, 14: , Hsiao JJ, Fisher DE. The roles of microphthalmia-associated transcription factor and pigmentation in melanoma. Archives of Biochemisty and Biophysics, 563: 28-34, Jang YA. Efficacy of a cosmetic material from complex extracts of Vaccinium spp., Phellinus linteus, Castanea crenata, and Cimicifuga heracleifolia. Asian Journal of Beauty and Cosmetology, 15: , Onishi Y, Yamaura T, Tauchi K, Sakamoto T, Tsukada K, Nunome S, Komatsu Y, Saiki I. Expression of the antimetastatic effect induced by Juzen-taiho-to is based on the content of Shimotsu-to constituents. Biological and Pharmaceutical Bulletin, 21: , Parveen I, Threadgill MD, Moorby JM, Winters A. Oxidative phenols in forage crops containing polyphenol oxidase 127

7 enzymes. Journal of Agricultural and Food Chemistry, 58: , Pillaiyar T, Manickam M, Jung SH. Recent development of signaling pathways inhibitors of melanogenesis. Cellular Signalling, 40: , Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiological Reviews, 84: , Slominski A, Wortsman J, Plonka PM, Schallreuter KU, Paus R, Tobin DJ. Hair follicle pigmentation. Journal of Investigative Dermatology, 124: 13-21, Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiological Reviews, 84: , Slominski A, Zmijewski MA, Pawelek J. L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions. Pigment Cell & Melanoma Research, 25: 14-27, Tahara E, Satoh T, Toriizuka K, Nagai H, Nunome S, Shimada Y, Itoh T, Terasawa K, Saiki I. Effect of Shimotsu-to (a Kampo medicine, Si-Wu-Tang) and its constituents on triphasic skin reaction in passively sensitized mice. Journal of Ethnopharmacology, 68: , van Gelder CW, Flurkey WH, Wichers HJ. Sequence and structural features of plant and fungal tyrosinases. Phytochemistry, 45: , Wang JD, Narui T, Kurata H, Takeuchi K, Hashimoto T, Okuyama T. Hematological studies on naturally occurring substances. II. effects of animal crude drugs on blood coagulation and fibrinolysis systems. Chemical and Pharmaceutical Bulletin, 37: ,

8 Cnidium officinale Makino extracts inhibit melanogenesis 국문초록 차화준오산대학교뷰티케어코스메틱과, 경기도오산시, 한국 목적 : 피부의색을결정하는것은피부, 모발, 눈, 심장, 뇌등에널리퍼져있는 melanin 이라는색소이다. Melanin은자외선을차단하고, 활성산소를제거하여피부를보호하는역할을한다. 그러나비이상적인 melanin의축적은과색소침착증이라는질환을일으키고, 대표적인예로는기미, 주근깨등이있다. Cnidium officinale Makino 는한국, 일본, 중국등의동아시아에서많이사용되는생약성분으로한국에서는천궁으로불린다. 본연구에서는 Cnidium officinale Makino 추출물의 melanin 생성억제효과를 B16F10 mouse melanoma 세포에서확인하고자한다. 방법 : Cnidium officinale Makino를 70% ethanol 에침지하여유효성분을추출하고, 이를 B16F10에처리하여대조군에비해서 melanin생성량이감소하는지를확인한다. 또한 tyrosinase 활성, tyrosinase mrna 와단백질발현, MITF의전사활성을확인하여 Cnidium officinale Makino 추출물의효능을평가한다. 결과 : Cnidium officinale Makino 추출물은 melanin합성을감소시키는것으로확인되었고, 이러한 melanin의감소는 melanin을합성하는효소인 tyrosinase 의 mrna 와단백질의발현감소로인한활성감소로인해일어나는것을확인하였다. 또한 tyrosinase 전사인자인 MITF의전사활성을확인한결과 Cnidium officinale Makino 추출물에의해서감소함을확인하였다. 결론 : 본연구결과를통해서 Cnidium officinale Makino 추출물의 melanin 합성저해능력을확인하였고, 과색소침착증을감소시킬수있는미백원료로가능성을보여주었다. 핵심어 : Cnidium officinale Makino, Melanogenesis, B16F10, Tyrosinase, MITF 이논문은 2017 년도오산대학교교내학술비지원에의해서수행됨. 참고문헌 장영아. 블루베리, 상황, 율피, 승마복합추출물의화장품소재로서의효능. 아시안뷰티화장품학술지, 15: , 하민정, 유선희. 천년초열매추출물의기능성화장품소재로서의생리활성특성. 아시안뷰티화장품학술지, 14: ,

9 中文摘要 Cnidium officinale Makino 提取物对 α-msh 诱导黑色素形成的 B16F10 mouse melanoma 细胞的抑制作用車和埈乌山大学美容化妆品学科, 京畿道乌山市, 韩国目的 : 皮肤颜色由分布在皮肤, 头发, 眼睛, 心脏和大脑中的黑色素决定黑色素保护皮肤免受紫外线并清除活性氧然而, 黑色素的异常蓄积导致色素沉着性疾病如色素沉着过度症, 祛斑等 Cnidium officinale Makino 在韩国日本和中国等东亚地区常用于天然药物, 在韩国被称为 Chun-kung 本研究探索 Cnidium officinale Makino 在 B16F10 细胞中的抗黑素生成作用方法 : Cnidium officinale Makino 提取物通过浸提在 70% 乙醇中制备在 Cnidium officinale Makino 提取物处理的 B16F10 中, 鉴定黑色素生成量与对照群相比此外, 还评价酪氨酸酶活性酪氨酸酶 mrna 和蛋白质表达以及 MITF 活性来确认 Cnidium officinale Makino 的效能结果 : 通过以上研究, 确认了 Cnidium officinale Makino 提取物降低黑色素合成, 这些黑色素合成量的减少由酪氨酸酶 mrna 和蛋白质表达的减少而形成的此外, 还确认了 Cnidium officinale Makino 提取物减少酪氨酸酶转录因子 microphthalmia-associated transcription factor (MITF) 的转录水平结论 : Cnidium officinale Makino 提取物具有抑制黑色素生成和色素沉着过度症, 作为美白化妆品原料充分具有可行性关键词 : Cnidium officinale Makino, 黑色素生成,B16F10, 酪氨酸酶,MITF 130

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,