Aminoglycoside 계열 항생제 tobramycin의 멜라닌 생성 증진 효과

文勝鉉 제주대학교 2019 국내석사

Since the first antibiotic, penicillin, the era of antibiotics has opened and many new antibiotics have been discovered and developed. However, in the 21st century, antibiotics discovered in the 20th century have been gradually reduced in usage or are no longer used due to antibiotic resistance, side effects, and the development of more effective antibiotics. In this study, we investigated the effects of streptomycin, kanamycin A, kanamycin B, tobramycin, fradiomycin, paromomycin, spertinomycin, hygromycin B, gentamicin, fosfomycin and lincomycin on melanogenesis in B16F10 melanoma cells. First, the effect of each antibiotic on the survival of B16F10 cells was confirmed by MTT assay. Subsequently, the groups of antibiotic alone treatment and the groups treated with antibiotics and α-MSH in the non-toxic concentration were divided to evaluate the effect on melanogenesis. Melanin production was decreased in kanamycin A and streptomycin, and melanin production was increased in tobramycin, fosfomycin and lincomycin. Melanin production was not changed in kanamycin B, fadomycin, paromomycin, spectinomycin, hygromycin B and gentamicin. Among them, melanin contents assay and tyrosinase activity assay were further performed with tobramycin, which increased melanin production remarkably. It was confirmed that tobramycin increased melanin production and increased the activity of tyrosinase, an enzyme important for melanin synthesis. We also confirmed that tobramycin phosphorylates p38, one of the MAPK proteins, by using western blotting and inhibitors, thereby increasing the expression of MITF protein. The increased expression of MITF increased the expressions of tyrosinase, TRP-1, TRP-2, an enzyme important for the synthesis of melanin, in a dose-dependent manner. We compared the effects of kanamycin A, kanamycin B with similar structure to tobramycin and 2-DOS, the central structure of these on the synthesis of melanin. As a result, the effect of tobramycin on the synthesis of melanin was presumed to be the structure of the aminosugar attached to the C-4 of 2-DOS, the central skeleton. Since melanocytes are mainly in the skin layer, the effect of tobramycin on keratinocytes was evaluated. At a concentration of 2 mg/mL, cell viability was over 90 %. In addition, the effect of RAW 264.7 cells on NO production, an inflammatory substance, was evaluated but not effective. Based on these results, we suggest that tobramycin may be helpful in the treatment of hypopigmentation.

Induction of Melanogenesis by Fosfomycin in B16F10 Cells Through the Upregulation of P-JNK and P-p38 Signaling Pathways

Sana Ullah 제주대학교 2020 국내석사

With the discovery of penicillin, the first antibiotic, the development of antibiotics has started and many new antibiotics have been discovered. In the earlier days, antibiotics were used to treat bacterial infections. However, in recent, antibiotics have been used for many other diseases. Gradually the old antibiotics were getting reduced in usage or are no longer used due to antibiotic resistance, side effects, and the development of more effective antibiotics. Previously tobramycin and some other antibiotics were used to target melanogenesis and skin coloration. In this study, we investigated the effects of, Fosfomycin disodium salt (FDS) on melanogenesis in B16F10 melanoma cells. First, we investigate the effect of FDS on the viability of B16F10 cells was confirmed with MTT assay. The antibiotic FDS showed no cytotoxicity in B16F10 cells along with α-MSH. We also confirmed the effect of FDS on Cellular tyrosinase and cellular melanin contents with and without α-MSH in B16F10 murine melanoma cells. The cellular tyrosinase (an enzyme important for melanin synthesis) and cellular melanin contents were increased by FDS with the increase in concentration compared with control. Furthermore, from the results of western blot, we also confirmed that FDS up regulates the phosphorylation of p38, JNK and ERK, one of the MAPK proteins, which increasing the expression of MITF protein. The increased expression of MITF increased the expressions of tyrosinase, TRP-1, TRP-2, an enzyme important for the synthesis of melanin, in a concentration dependent manner. These results are clear evidence of the melanogenesis-inducing effect of FDS in B16F10 murine melanoma cells.

제주 전통발효 식품 갈치속젓에서 분리한 Bacillus subtilis JNUCC의 유전체 분석과 항비만 및 미백 개선 효능 탐색

한지민 제주대학교 2020 국내석사

젓갈과 같은 발효 식품에서 분리된 미생물은 항균, 항산화, 항염증 효능이 있는 것 으로 알려져 기존의 화장품 및 의약품의 부작용을 줄일 수 있는 천연 원료 탐색에 있어서 중요성이 강대되고 있다. α-glucosidase는 탄수화물 소화를 촉진시키는 효소 로, α-glucosidase 저해제는 체내 탄수화물의 흡수 속도와, 탄수화물에 유도되는 저 밀도 지질 단백질 (Low density lipoprotein, LDL)의 생합성을 감소시켜 항비만제로 사용할 수 있다. tyrosinase는 피부의 색을 결정하는 색소인 멜라닌 생합성의 초기 반응에 작용하는 효소이며, tyrosinase 활성 억제제를 미백제로 사용할 수 있다. 이 번 연구는 제주도 지역 전통 발효 식품인 갈치속젓에서 분리한 Bacillus subtilis JNUCC 균주의 2차 대사산물 추출물로 항비만 및 미백 개선 원료로서의 가능성을 알아보고 균주의 유전체를 분석하여 미생물 유전자원의 확보를 하고자 하였다. 제주산 어린 갈치를 사용해 제조한 갈치속젓에서 17개의 균주를 분리하여 각 균주 의 배양액으로 항비만 및 미백 개선 효능을 탐색하고 결과를 종합적으로 판단하여 우수 균주를 선발하였다. 선발 균주는 16S rRNA 유전자 염기서열 database와 비교 하여 Bacillus subtilis와 가장 근연 종임을 확인하였고 균주 명은 JNUCC로 정하였 다. MiSeq 플랫폼 (Illumina Inc, USA)을 이용하여 균주의 전장 유전체를 분석하여 14개의 contig에 4,170,746 bp의 염기서열로 4,338개의 유전자를 가지고 있음을 확인 하였다. 이를 통해 G + C 함량은 43.6% 임을 알 수 있었다. Bacillus subtilis JNUCC 2차 대사산물 추출물을 서로 다른 농도로 각각 α -glucosidase 및 tyrosinase 억제 활동에 대해 평가하였으며, 각각 acarbose와 arbutin을 대조군으로 사용하였다. α-glucosidase 활성 저해 평가에서 균주의 2차 대사산물 추출물과 대조군의 반수 억제 농도(IC50)값은 각각 101.92 μg/mL, 100.94 μg/mL이었다. Tyrosinase 활성 저해 평가에서 균주의 2차 대사산물 추출물과 대조 군의 IC50값은 각각 669.52 μg/mL와 79.23 μg/mL이었다. 이번 연구는 Bacillus subtilis JNUCC 2차 대사산물 추출물이 acarbose와 흡사한 활성을 가졌으며 억제 활성이 농도에 의존한다는 것을 발견하였다. 또한 Bacillus subtilis JNUCC 2차 대 사산물 추출물의 tyrosinase 억제 활성은 대조군인 arbutin보다는 약한 활성을 가졌 으나 농도 의존적으로 증가함을 보였다. 따라서 갈치속젓에서 분리한 Bacillus subtilis JNUCC는 α-glucosidase 및 tyrosinase 억제제로서 잠재적인 기능성을 가졌 으며, 항비만제 및 미백 개선제로 화장품 및 의약품 산업에서 사용될 수 있음을 시 사한다.

마우스 B16F10 세포에서 Voglibose와 Miglitol의 Melanogenesis 억제 효과

김현미 제주대학교 대학원 2023 국내석사

In this study, the effects of Miglitol, Voglibose, and Validamycin A, which are used as α-glucosidase, on melanogenesis in B16F10 mouse melanoma cells were evaluated. In addition, it was confirmed that it can be used as a useful ingredient for external preparations and cosmetics for the treatment of hyperpigmentation due to the repurposing.Miglitol reduced the protein expression of the melanin-producing enzymes TRP-1, TRP-2, TYR and their transcription factor MITF. It was confirmed that Miglitol reduced melanin content through down-regulation of MITF in the Wnt/β-catenin, PKA, p38 MAPK, and MAPK/ERK signaling pathways. Voglibose also confirmed that TRP-1 and TRP-2 and their transcription factor, MITF, decreased their activity as a result of examining the protein expression levels. Regulation of Wnt/β-catenin, PKA/CREB, PI3K/Akt, and MAPKs signaling pathways reduces melanin synthesis through downregulation of MITF by phosphorylation and activation by voglibose. In addition, it was confirmed that melanin contents increased when the treated with an ERK-specific inhibitor (PD98059) and an Akt-specific inhibitor (LY294002). However, it seems that additional confirmation is needed by increasing the expression of Tyrosinase in B16F10 melanoma cells induced with α-MSH of the alpha-glucosidase inhibitors. Through human skin primary irritation test, Miglitol (125 μM and 250 μM) and Voglibose (50 μM and 100 μM) were tested for local use. Squalene was used as a negative control group, and the test substance was judged to be “non-irritating” in terms of its primary irritation potential to human skin.

마우스 B16F10 세포에서 쿠마린 유도체 Isoimperatorin과 Imperatorin의 멜라닌 생성 증진 효과

김태진 제주대학교 대학원 2023 국내석사

This study investigated the melanogenesis effect of isoimperatorin and imperatorin and the basic signaling pathway of imperatorin. The coumarin derivatives isoimperatorin and imperatorin were tested at concentrations of 6.25, 12.5 and 25 μM, which do not affect cell viability. Isoimperatorin and imperatorin increase melanin in the evaluation of melanin content and tyrosinase activity. Interestingly, there was no difference in melanin content and tyrosinase activity, despite the structural differences between isoimperatorin and imperatorin. Imperatorin increased melanin by regulating Tyrosinase, TRP-1 and TRP-2 enzymes, which play an important role in melanin activity. MITF is a protein factor that plays a major role in melenogenesis. Imperatorin increases melanin synthesis by finally regulating MITF through the ERK pathway of MAPKs, PKA/CREB pathway and AKT/GSK3β/β-catenin pathway in B16F10 cells. These pathways were further identified using specific inhibitors. In addition, it was confirmed that melanin production was induced in human epidermal melanocyte cells by treating imperetorin in HEMn-MP cells. Finally, the primary stimulation evaluation of the human skin was conducted to confirm whether the use of imperatorin had a stimulation or sensitization reaction to the skin. Through this test, it was confirmed that imperatorin was a hypoallergenic substance. In conclusion, the activity of coumarin derivatives on melanin production was evaluated through research. Both isoimperatorin and imperatorin showed increased melanin, and especially imperatorin can be used as a local treatment to treat hypochromic deposition.

추자도 멸치액젓의 Microbiom 분석 및 신종 미생물 특성 규명

변후돈 제주대학교 2020 국내박사

Fermentation is one of the most economical and classical methods for producing and preserving foods by humans. Besides preserving, fermentation also changes the organoleptic characteristics of foods through developing a wide diversity of flavors, aromas and textures. These fermented foods have been separately developed according to various fermentation conditions such as raw materials, combination of various microorganisms including seed bacteria, temperature, and pH. In particular, various microorganisms in fermented foods are the main factors that determine the taste and flavor of fermented foods, and the degree of fermentation can be determined depending on interactions among microbial communities. Therefore, microbiome analysis of fermented foods has recently emerged as a very important study field from a microbiological point of view in conjunction with the scientification of fermented foods. Therefore, with NGS technique, we want to find out the various microorganisms that exist in the fermented anchovy sauce of Chuja Island. Chuja Island became famous for anchovy, it is because when a spawning season of anchovy, many anchovies gather in Chuja island. In Chuja Island used anchovies to make salted anchovy sauce that can be used instead of soy sauce. The anchovy sauce for the Chuja Island is made by mixing salt and anchovies without adding any chemical seasoning and fermenting them for about three years. This MyeolchI-Aekjeot, traditional fermented anchovy sauce, has been consumed as an umami-tasting, seasoning ingredient. The umami of Myeolchi-Aekjeot is formed by exogenous enzymes of microorganisms, and despite the high concentration (20-30%) of salty environment, various microorganisms exist in fish sauces. Therefore, with NGS technique, we found out the various microorganisms that exist in the fermented anchovy sauce of Chuja Island. The fermented anchovy sauce, Myeolchi-Aekjeot, of Chuja island, which was used for the analysis of bacterial communities, was made by Chujado Federation of Fisheries Cooperatives in Jeju. It showed pH 6.1, 27% salinity, and 37.9 brix. To identify bacterial community, total DNA was extracted from fermented anchovy sauce and analyzed by an Illumina MiSeq platform. So total valid reads of 36,376 were obtained. Among total valid reads, reads of the species level was 19,661 reads (54%) and found 132 species. In results of analysis the bacterial community of fermented anchovy sauces of Chuja island, bacteria of phylum Firmicutes (82.5%) dominated at sample. And the Class Bacilli (76.4%), Order Bacillales (64.6%), Family Bacillaceae (64.3%) were the dominant in taxon, respectively. At the Genus level, the sample were dominated Lentibacillus (46.4%), Tetragenococcus (11.7%), and Pseudogracilibacillus (10.6%). Although there is no study on analyzed the bacterial community of fermented anchovy sauce in Jeju region, the results of the analysis of the bacterial community in 9 types of fermented anchovy sauce of Korea showed that unlike the results of this study, the bacteria of genus Tetragenococus and Halanaerobium in phylum Firmicutes were dominant in all samples. And study of analyzed the bacterial community of fermented anchovy salted fish (Myeolchi-Jeotgal) in jeju region, genus Tetragenococcus (90%) in phylum Firmicutes were dominant. According to the results of these studies, including this study, it can be confirmed that all phylum Firmicutes dominate. This is thought to be because a large number of bacteria belonging to this taxon form endospores and have the ability to exist in extreme environments. In addition, it appears to be the result because it contains a large number of the fermentation-related bacteria in this taxon. The difference in the results of these studies is thought to be due to the fermentation period. The fermentation period of fermented anchovy sauce of Chuja island analyzed in this study is 3 to 5 years, but the fermentation period of other fermented anchovy sauces and fermented anchovy salted fish is about 1 year, which is short. Through this, At the beginning of fermentation, bacteria that actively participate in fermentation, such as bacteria of genus Tetragenococus, appeared predominantly, and at the latter period of fermentation, genus Lentibacillus, which has a lategrowth characteristic, appears to be predominant. For this reason, it could be seen that the difference in dominant bacteria occurs over the fermentation period. In the future, if you conduct a bacterial community analysis by fermentation period of fermented anchovy sauce of Chuja island, we will see more accurate results. As a result of this analysis, it can be seen that fermented anchovy sauce of Chuja island is a well-fermented fish sauce. It is known that fish sauce such as fermented anchovy sauce, is rich in essential nutrients and tastes better with a longer fermentation period. It is thought that the presence of genus Lentibacillus in fish sauce can be used as an indicator of fermentation period. And in this study, strain JNUCC-1T was characterized and classified and this study proposed that strain JNUCC-1T isolated from fermented anchovy sauce in Chuja island is novel species via polyphasic taxonomy. Cells of strain JNUCC-1T are Gram-positive, aerobic, motile by means of polar flagella, and spore-forming rods (0.3-0.8 μm wide and 1.2-1.9 μm length). Spherical endospores form at the cell-terminal position. Colonies are circular with opaque of beige and below 1.0 mm in diameter. Growth occurs in 0-20% (w/v) NaCl (optimal in 5%) and pH 4.0-10.0 (optimal at pH 7.0) at 20-45 ℃ (optimal at 30 ℃). Positive results in tests for oxidase, catalase, nitrate reduction and hydrolysis of Tween 20 and 60. The major fatty acid (>20%) is anteiso-C15:0. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidyl-glycerol (PG), and one unidentified phospholipid (PL). The predominant respiratory isoprenoid quinone is menaquinone-7 (MK-7). Comparative analysis of 16S rRNA gene sequences showed that strain JNUCC-1T is most closely related to Lentibacillus alimentarius M2024T and Virgibacillus kekensis YIM-kkny16T. However, phylogenetic analysis showed that strain JNUCC-1T forms a phyletic lineage with members of the genus Lentibacillus. The topologies of phylogenetic trees built using the maximum-likelihood, maximum-parsimony, and neighbor-joining methods also supported these results, indicating that strain JNUCC-1T formed a stable clade. The genome size is 3,687,469 bp, and GC content of the genomic DNA is 43.3 %. Protein-coding sequences were predicted by Prodigal version 2.6.2, which showed that the genome contained 3,833 genes. In addition, 66 tRNA genes were identified by tRNAscan-SE version 1.3.1, and 17 rRNA genes were identified by Rfam version 12.0. As a result of analyzing the genes classified according to the COG functional categories, the genome of strain JNUCC-1T represented higher proportions in the categories of Replication, recombination and repair (7.7%), Amino acid transport and metabolism (7.7%), Inorganic ion transport and metabolism (5.6%), Carbohydrate transport and metabolism (5.3%), Energy production and conversion (5.2%), Transcription (5.1%), Cell wall/membrane/envelope biogenesis (4.81), Translation, ribosomal structure and biogenesis (4.6%). Analysis of phenotypic and genetic traits through COG functional categories gave the same results. The strain JNUCC-1T found the presence of motility and flagella, and through genomic analysis, it was confirmed that it contained 43 predicted genes related to motility or flagella. And the genome of strain JNUCC-1T contains 169 predicted genes associated with sporulation, such as spore germination protein GerD, GerM, and YaaH, and 33 predicted genes associated with antibiotic resistance such as pbp2A, ampC and multidrug resistance genes. The results of other biochemical tests, such as API system and hydrolysis of macromolecule test also related genes in COG functional categories of Amino acid transport and metabolism, Carbohydrate transport and metabolism, and Lipid transport and metabolism could confirm. This may be the basis for supporting the results of previous biochemical tests. Genes that could support the results of the chemotaxonomic test were identified. In genome of strain could confirm 9 predicted genes associated with phosphatidylglycerol and diphosphatidylglycerol such as umpA, lgt, and ltaS, and 14 predicted genes associated with menaquinone biosynthesis such as bioC, fadD, and ubiE, and 19 predicted genes associated with fatty acids biosynthesis such as fabG, fabH, and fabI. The genome of strain JNUCC-1T contains 19 predicted genes associated with osmotic stress regulation, such as choline glycine betaine transporter BetT2, osmoprotectant import permease protein OsmW. These genes could be key factors allowing strain JNUCC-1T to adapt to high-salt environments via osmotic regulation and adjustment of cytoplasmic pH, respectively. Metabolites produced by extreme halophile bacteria such as the strain JNUCC-1T have been utilized in various industries. Compatible solutes produced to overcome osmotic stress are utilized in the bio industry, enzymes that have activity at high concentrations of salinity are utilized in eco-friendly industries such as decomposition of pollutants and toxic substances, and enzymes such as esterase added in fermentation food used to improve the flavor of food. As a result of analyzing the genome of the strain, the strain is expected to have the potential ability to be used in various industries, and in the future, it will be able to derive the ability of the strain through additional studies such as metabolite regulation, efficacy evaluation, and genetic manipulation. The genome sequence of strain JNUCC-1T has been deposited in GenBank under accession no. WHOH00000000 (BioProject accession no. PRJNA577786 and BioSample accession no. SAMN13037569). The public version of the strain JNUCC-1T genome sequence deposited in GenBank was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP). In this study, Phylogenetic tree showed that strain JNUCC-1T forms a phyletic lineage with members of the genus Lentibacillus. The chemotaxonomic and phenotypic characterizations of strain JNUCC-1T showed typical features of members of the genus Lentibacillus, with MK-7 as the predominant respiratory isoprenoid quinone and major fatty acid including anteiso-C15:0. The strain JNUCC-1T is considered as a novel species of the genus Lentibacillus, for which the name Lentibacillus jejunsis sp. nov., (Type strain JNUCC-1T = KCTC 43912T) is proposed. This study analyzed the community of microorganisms present in fermented anchovy sauce of Chuja island using the Illumina Miseq platform to confirm that about species of 130 bacteria coexist. Among them, it was found that the bacteria in the genus Lentibacillus dominate, which means that fermentation is good for a long time, so that the essential nutrients are rich and the flavor is improved. In addition, the biodiversity was secured to respond to the implementation of the Nagoya Protocol by separating the new strains from the anchovy sauce and identifying their characteristics.

마우스 대식세포에서 사방오리 추출물의 항염증 효능 연구

임지연 제주대학교 대학원 2023 국내석사

It has been previously reported that Alnus firma have various physiological effects such as anti-bacterial, anti-cancer, anti-inflammatory, anti-obesity, anti-oxidative and anti-viral. However, the molecular mechanisms of A. firma on inflammatory has clearly not been identified. Therefore, this study mainly focused on anti-inflammatory effect of A. firma and its underlying molecular mechanism on LPS induced inflammation in RAW 264.7 cells. In the results, 60% EtOH extracts markedly reduced the production of nitric oxide (NO) increased by LPS in Raw 264.7 cells. To determine the effective fraction 60% EtOH extracts of A. firma were isolated using hexane, ethyl acetate, butanol, and water. Among them, hexane fraction showed the strongest anti-inflammatory effects. Hexane fraction reduced NO production and prostaglandin E2 (PGE2) release induced by LPS. Western blot analysis data indicates that the treatment of hexane fraction significantly reduced the expression of inducible nitric oxide synthase (iNOS) and phosphorylation of mitogen activated protein kinases (MAPKs; ERK1/2, JNK. and p38) increased by LPS. In addition total seven bioactive compounds were isolated from hexane fraction. Although they were cytotoxic at higher concentration all compounds significantly reduced NO production induced by LPS. In conclusion the results in this study showed that A. firma exhibits strong anti-inflammatory effect by inhibiting NF-κB, MAPKs, Akt, and iNOS signaling pathways. This suggests that A. firma can be a useful resource for relieving the inflammation. However, the structure and biological functions of the compounds from this study should be identified in the future.

비자 잎 열수추출물의 멜라닌 생성 저해 효과

고지혜 제주대학교 대학원 2019 국내석사

마우스 세포에서 D-(+)- cycloserine의 항염증 및 멜라닌 생성 억제효과 규명

강현규 제주대학교 2020 국내석사

Recently, additional therapeutic potentials of classical antibiotics are gaining considerable attention. The discovery of penicillin in the 1920s had a major i mpact on the history of human health. Penicillin has been used for the treatm ent for fatal microbial infections in humans and has led to the discovery of s everal new antibiotics. D-(+)-cycloserine (DCS) is an antibiotic isolated from Streptomyces orchidaceous and is used in conjunction with other drugs in the treatment of tuberculosis. To determine whether DCS has anti-inflammatory a nd anti-melanogenic effects, we investigated the ability of DCS in lipopolysac charide (LPS)-induced RAW 264.7 macrophages and α-melanocyte stimulating hormone (α-MSH)-induced B16F10 melanoma cells. DCS inhibited the product ion of nitric oxide (NO), prostaglandin E2 (PGE2) and the expression of proinf lammatory cytokines such as interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a concentration-dependent manner. However, it had no effect on the expres sion of TNF-α. Consistent with these findings, Western blot analysis demonst rated that DCS inhibited LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase type-2 (COX-2) expression via inhibition of phosphorylati on of inhibitory kappa B-α (IκB-α) through down-regulation of phosphorylate d Akt, ERK and p38. Furthermore, DCS markedly inhibited melanin synthesis and tyrosinase activity in concentration-dependent manner. Western blotting s howed that DCS treatment inhibited the expression of tyrosinase (TYR), tyros inase-related protein 1 (TRP-1) and tyrosine-related protein 2 (TRP-2) via in hibition of microphthalmia-associated transcription factor (MITF) expression. These results indicate that DCS may be used as potential drugs for the treat ment of inflammatory diseases and as melanogenesis inhibitor.

B16F10 마우스 세포에서 lincomycin의 멜라닌 합성 증진 규명

이민숙 제주대학교 대학원 2021 국내석사

Lincomycin hydrochloride monohydrate (L.H.M), isolated from Streptomyces lincolnensis, is a lincosamide-based antibiotic in the form of a hydrochlorid base. L.H.M was usually used to treat staphylococcus, streptococcus and Bacteroides fragilis infections. Although L.H.M has been utilized in clinical treatment for a long time, it has exhibited several side effects, leading to the introduction of more effective antibiotics, such as clindamycin. Therefore, we conducted an experiment focusing on new possibilities for clinical use of L.H.M. How L.H.M roles in skin melanin production has not been investigated. In this study we used the B16F10 melanoma cell to identify the melanin inducting properties of L.H.M. Melanin contents and tyrosinase activity in the cells were increased by L.H.M without any cytotoxicitiy. Western blot analysis indicated that the protein level of tyrosinase, tyrosinase-related-protenin-1 (TRP-1), and tyrosinase-related-protenin-2 (TRP-2) were increased after L.H.M treatment. In addition, L.H.M enhanced the microphthalmia-associated transmission factor (MITF) expression. Moreover, L.H.M increased phosphorylation of c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases in MAPK. Furthermore, the inhibition of tyrosinase activity by L.H.M was stimulated by treatment with SP600125 (a specific JNK inhibitor) and SB203580 (p38 inhibitor). We also found that L.H.M-induced melanogenesis was reversed by H89, which is a specific protein kinase an inhibitor. Finally, using MTT assay, the cell viability of L.H.M in HaCaT cell was confirmed. These results suggested that L.H.M may be used to treat or prevent hypopigmentation disorders or hair depigmentation.