Human tyrosinase 유전자의 대장균에서의 발현

박소영,공광훈,박희중 中央大學校 遺傳工學硏究所 1997 遺傳工學硏究論集 Vol.10 No.1

Tyrosinase는 활성부위에 한 쌍의 구리이온을 함유하고 있는 metalloprotein으로 멜라닌 생합성에 중요한 역할을 한다. Human tyrosinase의 메카니즘을 연구하기 위해 human tyrosinase 유전자에 대한 대장균에서의 대량발현계를 구축하였다. Plasmid pRHOHT2 (Shibahara et al, 1988. Tohoku J. Exp. Med. 156. 403)로부터 human tyrosinase cDNA sequence는 polymerase chain reaction에 의해 증폭되었으며, 발현벡터 pGEX-KG와 pTrcHis에 subcloning 되었다. 이렇게 작성된 plasmid pKG-Tyrosinase와 pHis-Tyrosinase를 대장균주 MV1190에 형질전환시켜, 유도제 isopropyl- β-D-thiogalactopyranoside로 human tyrosinase를 대량발현시켰다. 발현된 tyrosinase는 GSH affinity column과 immobilized metal affinity column으로 정제하였고, 정제된 재조합 tyrosinase는 SDS-gel 전기영동상에서 각각 84,000과 66,000 정도를 나타내었다. 또한 정제된 재조합 tyrosinase는 dopa oxidation 활성을 나타내었다. Tyrosinase is a bifunctional copper-containing metalloprotein and play a central role and of melanin biosynthesis. In the study, we constructed several E. coil-expression system of human tyrosinase using a cDNA encoding human tyrosinase for its mechanism study. Human tyrosinase cDNA sequence, plasmid pRHOHT2 (Shibahara et al. 1988, Tohoku J. Exp. Med. 156, 403), was amplified by the polymerase chain reaction and was subcloned with pGEX-KG and pTrcHis expression vector. These recombinant tyrosinases were produced by induction with isopropyl-β-D-thiogalactopyranoside. Overexpressed human tyrosinases were purified by GSH affinity column and immobilized metal affinity column. The molecular weights of the purified recombinant tyrosinases determined by SDS-polyacrylamide gel electrophoresis was about 84,000 (pKG-Tyrosinase) and 66,000 (pHis-Tyrosinase), respectively. These enzymes also showed dopa oxidation activity.

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Ullah, Sultan,Park, Chaeun,Ikram, Muhammad,Kang, Dongwan,Lee, Sanggwon,Yang, Jungho,Park, Yujin,Yoon, Sik,Chun, Pusoon,Moon, Hyung Ryong Academic Press 2019 Bioorganic chemistry Vol.87 No.-

<P><B>Abstract</B></P> <P>Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (<I>E</I>)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (<I>E</I>)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (<B>4</B>, <B>9</B>, <B>14</B>, and <B>19</B>) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (<B>9</B>) = cyclopentylamino (<B>14</B>) < cyclohexylamino (<B>19</B>) < <I>N</I>-methylpiperazino (<B>4</B>) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Twenty cinnamamide derivatives were synthesized as potential tyrosinase inhibitors. </LI> <LI> Four cinnmamides exhibited over 90% mushroom tyrosinase inhibitions than kojic acid. </LI> <LI> In B16F10 cells, four compounds strongly decreased tyrosinase activity and melanin. </LI> <LI> In docking study, four cinnamamides bind to tyrosinase strongerly than kojic acid. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Lactobacillus plantarum M23 균주를 이용한 Tyrosinase 저해 활성 발효유 생산의 최적화

임상동,김기성,Lim, Sang-Dong,Kim, Kee-Sung 한국축산식품학회 2012 한국축산식품학회지 Vol.32 No.5

본 연구는 tyrosinase 활성 저해효과가 있는 젖산균인 L plantarum M23 균주를 이용하여 멜라닌 형성을 억제하기 위한 최적 발효유 제조조건을 설정하는데 그 목적이 있다. 3수준 4인자 중심합성 계획법을 통해 실험설계하여 반응표면분석법을 사용하였다. 독립변수로는 yeast extract 농도(%, $X_1$), 포도첨가량(%, $X_2$), 배양온도($^{\circ}C$, $X_3$), 배양시간(h, $X_4$)을 설정하였고, 종속변수로는 pH(pH, $Y_1$), 종합적 기호도(score, $Y_2$)와 tyrosinase inhibitory activity(%, $Y_3$)를 설정하였으며, tyrosinase inhibitory activity가 가장 높고 pH는 4.4를 동시에 만족하는 최적화를 실시한 결과 yeast extract 농도는 0.13%, 포도 첨가량은 2.95%, 배양 온도는 $37.1^{\circ}C$, 배양 시간은 14.8 h일 때 예상되는 pH는 4.42, 기호도는 7.06, tyrosinase inhibitory activity은 86.65%이었으며, 실제 실험 결과 pH는 4.35, 기호도는 6.86, tyrosinase inhibitory activity은 84.05%로서 큰 차이를 나타내지 않았다. 이러한 결과를 토대로 melanin 생합성과정의 key enzyme인 tyrosinase을 활성억제하는 L. plantarum M23을 이용한 발효유는 피부의 미백 작용과 노화 억제 작용에 기여할 것으로 사료되었다. The melanin pigment in human skin is a major defense mechanism against ultraviolet light to the skin, but darken skin color. Tyrosinase is mainly responsible for melanin biosynthesis (melanogenesis) in animals and enzymatic browning (melanosis) in plants. The purpose of this study was to optimize the fermented milk process for the melanin formation inhibition by using Lactobacillus plantarum M23 with tyrosinase inhibitory activity. We used 4-factor-3-level central composite design combining with response surface methodology. Yeast extract concentration (%, $X_1$), addition of grape (%, $X_2$), incubation temperature ($^{\circ}C$, $X_3$) and incubation time (h, $X_4$) was used as an independent factor, on the other hand, pH (pH, $Y_1$), overall palatability (score, $Y_2$) and tyrosinase inhibitory activity (%, $Y_3$) was used as a dependant factor. Based on the optimization for the highest tyrosinase inhibitory activity with pH 4.4, the expected data of pH, palatability and tyrosinase inhibitory activity with 14.8 h incubation at $37.1^{\circ}C$ by the addition of 0.127% of yeast extract, 2.95% of grape was 4.42, 7.06 and 86.65%, but the real data was 4.35, 6.86 and 84.05%, respectively. Based on the previous results, fermented milk using Lactobacillus plantarum M23 with the tyrosinase inhibitory activity could contribute for the whitening and antiaging of human skin.

A potent tyrosinase inhibitor, (E)-3-(2,4-dihydroxyphenyl)-1-(thiophen-2-yl)prop-2- en-1-one, with anti-melanogenesis properties in á-MSH and IBMX-induced B16F10 melanoma cells

( Hee Jin Jung ),( Sang Gyun Noh ),( Dae Hyun Kim ),( Eun Jin Bang ),( Sugyeong Ha ),( A Kyoung Lee ),( Min Kyung Hyun ),( Byeong Moo Kim ),( Chang Seok Kim ),( Hae Young Chung ) 한국장기요양학회 2018 한국장기요양학회 추계학술대회자료집 Vol.2018 No.-

In this study, we designed and synthesized eight heterocyclic chalcone derivatives (1a - 1h) as tyrosinase inhibitors and evaluated their mushroom tyrosinase inhibitory activities. Of these 8 compounds, (E)-3-(2,4-dihydroxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1c) showed strong competitive inhibition activity against mushroom tyrosinase with IC50 values of 0.013 μM for tyrosine hydroxylase and 0.93 μM for dopa oxidase. As an underlying mechanism of 1c mediated anti-melanogenic effect, we investigated the inhibitory activity against melanin contents and cellular tyrosinase. In addition, we used a kinetics study and docking program to further evaluate the inhibitory mechanism of 1c toward tyrosinase. The enzyme kinetics and docking results supports that 1c highly interacts with tyrosinase residues in the tyrosinase active site and it can directly inhibit tyrosinase as competitive inhibitor. Moreover, we examined the inhibitory effect of 1c on tyrosinase expression through protein kinase A (PKA), cAMP response element-binding protein (CREB) and microphthalmia-associated transcription factor (MITF) signaling on á-melanocyte stimulating hormone (á-MSH) and IBMX-induced B16F10 melanoma cells. As the results, 1c decreased the expression levels of PKA, CREB, MITF, and tyrosinase pathways. Furthermore, 1c interfered with the phosphorylation of PKA and tyrosinase demonstrating potent anti-melanogenic effects on á-MSH and IBMX-induced B16F10 cells in cytosolic fractions. Overall, our results suggested that 1c might be considered potent candidates for use in the development of therapeutic agents for diseases associated with hyperpigment disorders.

방선균 F-97이 생산하는 Tyrosinase 저해제의 정제 및 특징

이동희,방병호,이문수,김진오 한국응용생명화학회 2008 Journal of Applied Biological Chemistry (J. Appl. Vol.51 No.3

An actinomycetes F-97 producing tyrosinase inhibitor was isolated from soil samples. Isolation and purification of tyrosinase inhibitor produced by F-97 was performed as follows: IRC-120 (NH4 + type) column chromatography, silica gel column chromatography, C18 column chromatography and Sephadex LH-20 column chromatography were used successively after the centrifuged supernatant was adjusted to pH 4.0. To identify the purity of the inhibitor, octadecylsilyl(ODS) HPLC was carried out with 5% methanol as a mobile phase. Finally, the purification yield of a tyrosinase inhibitor was 5.24%. The inhibitor was very soluble in water, methanol and ethanol but insoluble in acetone, butanol, ethylacetate and chloroform. The λmax value of this inhibitor in water was 194 nm under UV light. The biochemical test of the inhibitor was positive in Molish, Benedict, conc. H2SO4, and KMnO4 tests but negative in iodine, ninhydrin, Million, Sakaguchi, xanthoproteic and Emerson tests. The tyrosinase inhibitor was stable against heat treatment of 100oC for 50 minutes and pH 4~9. The IC50 value of this inhibitor was 19.2 μg/ml for mushroom tyrosinase. In 1,000 μg/ ml inhibitor concentration, inhibition zone was 27 mm for Streptomyces bikiniensis NRRL B-1049. The inhibition of F-97 against mushroom tyrosinase was competitive with tyrosine. An actinomycetes F-97 producing tyrosinase inhibitor was isolated from soil samples. Isolation and purification of tyrosinase inhibitor produced by F-97 was performed as follows: IRC-120 (NH4 + type) column chromatography, silica gel column chromatography, C18 column chromatography and Sephadex LH-20 column chromatography were used successively after the centrifuged supernatant was adjusted to pH 4.0. To identify the purity of the inhibitor, octadecylsilyl(ODS) HPLC was carried out with 5% methanol as a mobile phase. Finally, the purification yield of a tyrosinase inhibitor was 5.24%. The inhibitor was very soluble in water, methanol and ethanol but insoluble in acetone, butanol, ethylacetate and chloroform. The λmax value of this inhibitor in water was 194 nm under UV light. The biochemical test of the inhibitor was positive in Molish, Benedict, conc. H2SO4, and KMnO4 tests but negative in iodine, ninhydrin, Million, Sakaguchi, xanthoproteic and Emerson tests. The tyrosinase inhibitor was stable against heat treatment of 100oC for 50 minutes and pH 4~9. The IC50 value of this inhibitor was 19.2 μg/ml for mushroom tyrosinase. In 1,000 μg/ ml inhibitor concentration, inhibition zone was 27 mm for Streptomyces bikiniensis NRRL B-1049. The inhibition of F-97 against mushroom tyrosinase was competitive with tyrosine.

꽃송이버섯에서 추출한 β-glucan의 tyrosinase 활성과 멜라닌 합성 억제 효능

오철현(Chul Hyun Oh),구미정(Mi Jung Ku),이용환(Yong Hwan Lee) 한국생명과학회 2021 생명과학회지 Vol.31 No.11

천연물에서 피부 미백 효능을 가진 화합물을 개발하기 위한 노력이 많이 이루어지고 있다. 꽃송이 버섯(Sparassis crispa)은 β-glucan의 함량이 건조중량의 40% 이상으로서 항암과 면역증강 효과가 있는 것으로 입증되어 약용으로도 인정받고 있다. 이 연구에서는 S. crispa β-glucan의 멜라닌 생합성 및 tyrosinase 활성억제 효능을 확인함으로써 피부 미백제로서의 활용 가능성을 평가하고자 하였다. 외부 자극제인 α-melanocyte stimulating hormone (α-MSH)와 S. crispa β-glucan (10, 100, 1,000 μg/ml)을 B16F1 melanoma 세포에 투여하여 멜라닌 생성량과 tyrosinase 활성도를 측정하였다. Tyrosinase, tyrosinase related protein-1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF)의 발현정도에 대해서는 western blot으로 분석하였다. S. crispa β-glucan을 10, 100, 1,000 μg/ml의 농도로 투여하였을 때 α-MSH 만 투여군에 비하여 멜라닌 생성이 각각 13.9%, 18.7%, 39.5% 감소하였다. S. crispa β-glucan을 10, 100, 1,000 μg/ml의 농도로 투여하였을 때 β-glucan을 투여하지 않은 α-MSH 유도군에 비하여 tyrosinase 활성도는 각각 15.6%, 26.9%, 43.2% 억제되었다. 또한 tyrosinase와 TRP-1, TRP-2, MITF 단백 발현도 효과적으로 감소시켰다. 본 연구 결과에 따르면 S. crispa β-glucan은 MITF 발현을 억제함으로써 tyrosinase 발현을 감소시켜 멜라닌 생성을 억제시킨 것으로 판단된다. 따라서 S. crispa β-glucan은 미백제로서 유용하게 활용할 수 있을 것으로 생각한다. There are a lot of efforts to develop new compounds having skin whitening effect from natural products. Sparassis crispa is a medicinal mushroom containing more than 40% β-glucan, which exhibits anticancer and immunostimulating effects. The aim of this study was to assess the availability of β-glucan extracted from cauliflower mushroom S. crispa as a skin whitener through the evaluation of inhibitory effects of melanin synthesis and tyrosinase activity and their mechanisms. B16F1 cells were treated with S. crispa β-glucan (10, 100, and 1,000 μg/ml, respectively) and α-melanocyte stimulating hormone (α-MSH), simultaneously. Content of melanin synthesis and tyrosinase activity were determined. The expressions levels of tyrosinase, tyrosinase related protein-1 (TRP-1), TRP-2 and microphthalmia-associated transcription factor (MITF) were also measured by western blotting. Treatment with 10, 100 and 1,000 μg/ml S. crispa β-glucan and 200 nM α-MSH significantly decreased melanin synthesis by 13.9%, 18.7% and 39.5%, respectively, and tyrosinase activity by 15.6%, 26.9% and 43.2%, respectively, compared to the α-MSH alone group. In addition, S. crispa β-glucan inhibited expressions of tyrosinase, TRP-1, TRP-2 and MITF induced by α-MSH. These results indicated that S. crispa β-glucan inhibited MITF expression, thereby reducing tyrosinase expression and inhibiting melanin production in B16F1 melanoma cells. Therefore, S. crispa β-glucan might be available as a skin whitener.

Fucoidan의 멜라닌 합성과 tyrosinase 활성도 억제 효과

정숙희(Sook Hee Jung),구미정(Mi Jung Ku),문희정(Hee Jung Moon),유병철(Byeng Chul Yu),전만중(Man Joong Jeon),이용환(Yong Hwan Lee) 한국생명과학회 2009 생명과학회지 Vol.19 No.1

미역, 다시마 등 갈조류의 추출물로서 생리활성성분으로 각광 받고 있는 fucoidan이 멜라닌 합성에 미치는 영향을 알아봄으로써 미백제로서의 개발 가능성 여부를 확인하기 위하여 B16F10 melnoma 세포를 이용하여 멜라닌 합성과 tyrosinase 활성도에 대한 실험의 결과 B16F10 melnoma 세포의 멜라닌 생성과 tyrosinase 활성도는 fucoidan의 농도가 증가함에 따라 농도 의존적으로 억제되었다. B16F10 melnoma 세포에 α-MSH를 투여 후 멜라닌 생성과 tyrosinase의 활성도 역시 fucoidan의 농도가 증가할수록 멜라닌 생성과 tyrosinase활성도가 억제되는 경향을 보였다. 또한 DOPA 염색의 결과 fucoidan은 농도 의존적으로 tyrosinase 활성도를 억제하였다. 이상의 결과 해조 추출 다당류인 fucoidan은 B16F10 melanoma 세포의 멜라닌 합성과 tyrosinase 활성도 억제 작용을 나타내므로 미백 활성 물질로서의 가능성을 가지고 있는 것으로 생각된다. Melanogenesis is a physiological process that results in the synthesis of melanin pigments. Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be improved by treatment with depigmenting agents. Among the possible melanin-reducing compounds, tyrosinase inhibitors are most promising for preventing and treating pigmentation disorder and are used as skin-whitening agents in the cosmetic industry. In the present study, the effects of fucoidan on melanogenesis and tyrosinase activity of B16F10 melanoma cells were investigated. Melanin synthesis and tyrosinase activity in B16F10 melanoma cells were decreased in a dose-dependent manner by fucoidan. Melanin production and tyrosinase activity in B16F10 melanoma cells stimulated by α-melanocyte stimulating hormone (α-MSH) were inhibited by fucoidan with a dose-dependent manner compared to control. Fucoidan inhibited tyrosinase activity of B16F10 melanoma cells with a dose-dependent manner as assessed by 3,4-dihydroxyphenylalanine (DOPA) staining. In conclusion, these findings indicate that fucoidan, which inhibit melanin synthesis and tyrosinase activity, is an effective skin-whitening agent.

2-Phenyl-1,4-benzopyrone 유도체(Flavones)의 Tyrosinase 저해활성에 관한 3D-QSARs 분석과 분자도킹

성낙도,박준호 한국응용생명화학회 2010 Journal of Applied Biological Chemistry (J. Appl. Vol.53 No.4

To understand the inhibitory activity with changing hydroxyl substituents (R1-R9) of polyhydroxy substituted 2-phenyl-1,4-benzopyrone analogues (1-25) against tyrosinase (PDB ID: oxy-form; 1WX2), molecular docking and the three dimensional quantitative structure-activity relationships (3D-QSARs: Comparative molecular field analysis (CoMFA) & Comparative molecular similarity indices analysis (CoMSIA)) were studied quantitatively. The statistically best models were CoMFA 1 and CoMSIA 1 model from the results. The optimized CoMSIA 1 model with the sensitivity of the perturbation and the prediction produced (dq2'/dr2yy'=1.009 & q2=0.511) by a progressive scrambling analysis were not dependent on chance correlation. The inhibitory activities with optimized CoMSIA 1 model were dependent upon electrostatic factor (51.4%) of substrate molecules. Contour mapping the 3D-QSAR models to the active site of tyrosinase provides new insight into the interaction between tyrosinase as receptor and 2-phenyl-1,4-benzopyrone analogues as inhibitor. Therefore, the results will be able to apply to the optimization of a new potent tyrosinase inhibitors. 기질분자로서 polyhydroxy 치환된 2-phenyl-1,4-benzopyrone 유도체(Flavones)(1-25)들의 hydroxyl 치환기(R1-R9)가 변화함에 따른 Tyrosinase(PDB ID: oxy-form; 1WX2)에 대한 저해활성을 이해하기 위하여 분자도킹과 3차원적인 정량적 구조-활성관계(3D-QSARs: CoMFA 및 CoMSIA)가 연구되었다. 그 결과, 통계적으로 CoMFA 1 및 CoMSIA 1 모델이 가장 양호한 3D-QSARs 모델이었다. 또한, 순차 혼합화 분석결과로부터 CoMSIA 1 모델(dq2'/dr2yy'=1.009 및 q2=0.511)이 우연상관성에 저촉되지 않는 최적화 모델이었으며 최적화된 CoMSIA 1 모델의 tyrosinase에 대한 저해활성은 기질분자의 정전기장(51.4%)에 의존적이었다. Tyrosinase의 반응점에 대한 3D-QSAR 모델의 등고도는 수용체로서 tyrosinase과 저해제로서 2-phenyl-1,4-benzopyrone 기질분자 사이의 새로운 상호작용 관계를 이해하는 계기가 되었다. 그러므로 이 결과들은 새로운 잠재적인 tyrosinase 저해제의 최적화에 적용될 수 있을 것이다.

SK-MEL-2에서 상백피 추출물의 Tyrosinase 활성 억제 및 Melanin 생성 억제 효과

유민정 한국피부과학연구원 2011 대한피부미용학회지 Vol.9 No.4

This study was to investigate the ethanol extracts of Morus alba on tyrosinase activity and melanogenesis. In the present study , we examined the effects of Morus alba ethanol extract on mushroom tyrosinase activity in vitro, on SK-MEL-2 cell tyrosinase activity , melanin contents, expression of melanogenic enzyme proteins including tyrosinase, tyrosinase-related protein1(TRP-1) and and tyrosinase-related protein2(TRP-2). Mushroom tyrosinase activity of dose dependant and about 75% at concentration of 400 ㎍/㎖. Morus alba ethanol extracts reduced melanin contents of SK-MEL-2 melanoma cella in a dose dependant manner and decreased to about 83.2% at a cocentration of 100 ㎍/㎖ without cell cytotoxicity(below 100 ㎍/㎖) and intracellular tyrosinase activity about 54.2% at concentration of 100 ㎍/㎖. Morus alba ethanol extracts inhibited tyrosinase and TRP-2 expression at protein level. These results suggest that Morus alba ethanol extracts reduced melanin formation by the inhibitied of tyrosinase activity and expression in SK-MEL-2 melanoma cells. Therefor, we confirmation that Morus alba ethanol extracts could be used as a useful whitening agent.

멜라닌 세포에서 홍차 열수추출물의 멜라닌 합성 저해능과 작용기전

최소영,김영철,장병수,Choi, So-Young,Kim, Young-Chul,Chang, Byung-Soo 한국현미경학회 2011 Applied microscopy Vol.41 No.3

홍차 열수추출물의 멜라닌 합성 저해능과 작용기전을 알아보기 위해 in vitro 실험으로 항산화능을 확인한 후, melan-a 세포를 사용하여 멜라닌 합성 저해능, tyrosinase 활성 저해능 및 발현저해능 등에 대한 실험을 실시하여 다음과 같은 결과를 얻었다.(1) 항산화능 실험에서 홍차 열수추출물의 총 폴리페놀 및 플라보노이드 함량은 각각 102, 87 mg/g으로 양성대조군인 알부틴(25, 11 mg/g)보다 높았고, 전자공여능 역시 800 ${\mu}g$/mL에서 82%로 알부틴(48%)보다 높았다. (2) Melan-a 세포 실험에서 홍차 열수추출물은 수지상돌기의 발달과 멜라닌 생합성을 효과적으로 저해하였고, 멜라닌 합성 저해능과 tyrosinase 활성 저해능이 알부틴보다 유의하게 (p<0.001) 높았다. (3) Tyrosinase 단백질 발현 저해능 실험에서 알부틴은 발현에 유의한 영향을 미치지 않은 반면, 홍차 열수추출물은 발현을 유의하게 (p<0.001) 저해하였다. (4) 유전자 발현 저해능 실험에서 알부틴과 홍차 열수추출물 모두 tyrosinase, TRP-1과 TRP-2의 mRNA 발현에 영향을 미치지 않았다. 본 연구를 통하여, 홍차 열수추출물은 우수한 항산화능과 함께 멜라닌 합성 저해능을 확인하였으며, 이는 tyrosinase의 생합성을 저해시키기 보다는 tyrosinase 단백질 발현량을 줄이고 tyrosinase 활성을 감소시킴으로써 멜라닌 합성을 저해시키는 것으로 사료된다. To evaluate the whitening effect of Camellia sinensis water extract (CSWE), CSWE was treated to melan-a cells. Total polyphenol contents and flavonoid contents of CSWE were 102 mg/g and 87 mg/g, respectively. The electron-donating ability of CSWE revealed a dose-dependent response, showing the excellent ability of 82% at 800 ${\mu}g$/mL, and which was higher than the arbutin (48%). The CSWE significantly (p<0.001) suppressed the melanin synthesis and the development of melanocyte dendrites was inhibited in a dose-dependent manner. The CSWE significantly (p<0.001) inhibited both intra-cellular and cell-extracted tyrosinase activities. And inhibitory efficacies of CSWE on both melanin synthesis and tyrosinase activity were significantly (p<0.001) higher than the arbutin. The tyrosinase protein expression was not influenced by arbutin treatment. However, CSWE treatment significantly (p<0.001) reduced it. Both arbutin and CSWE treatment did not influence on mRNA expressions of tyrosinase, tyrosinase-related protein-1 and tyrosinase related protein-2.