• Applied Biological Chemistry
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• v.22 no.4
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• pp.198-209
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• 1979

L-Tyrosine, 2-chloro-L-tyrosine, 2-bromo-L-tyrosine, and 2-iodo-L-tyrosine were synthesized by ${\beta}-tyrosinase$ obtained from cells of Escherichia intermedia A-21, through the reversal of the ${\alpha},{\beta}-elimination$ reaction, and their molecular structures were analyzed by element analysis, NMR spectroscopy, mass spectrometry and IR spectroscopy. Rates of synthesis and hydrolysis of halogenated tyrosines by ${\beta}-tyrosinase$, inhibition of the enzyme activity by halogenated phenols, and effects of addition of m-bromophenol on the synthesis of 2-bromotyrosine were determined. The results obtained were as follows: 1) In the synthesis of halogenated tyrosines, the yield of 2-chlorotyrosine from m-chlorophenol were approximately 15 per cent, that of 2-bromotyrosine from m-bromophenol 13.8 per cent, and that of 2-iodotyrosine from m-iodophenol 9.8 per cent. 2) Rate of synthesis of halogenated tyrosines by ${\beta}-tyrosinase$ was slower than that of tyrosine and the rates were decreased in the order of chlorine, bromine and iodine, that is, by increasing the atomic radius. Relative rate of 2-chlorotyrosine synthesis was determined to be 28.2, that of 2-bromotyrosine to be 8.13, and that of 2-iodotyrosine to be 0.98, respectively, against 100 of tyrosine. However 3-iodotyrosine was not synthesized by the enzyme. 3) The relative rate of 2-chlorotyrosine hydrolysis by ${\beta}-tyrosinase$ was 70.7, that of 2-bromotyrosine was 39.0, and that of 2-iodotyrosine was 12.6 against 100 of tyrosine, respectively. The rate of hydrolysis appeared to be decreased in the order of chlorine, bromine and iodine, that is, by increasing the atomic radius or by decreasing the electronegativity. But 3-iodotyrosine was not hydrolyzed by the enzyme. 4) The activity of ${\beta}-tyrosinase$ was inhibited by phenol markedly. Of the halogenated phenols, o-, or m-chlorophenol and o-bromophenol gave marked inhibition on the enzyme action, however inhibition by iodophenol was not strong. Plotting by Lineweaver-Burk method, a mixed-type inhibition by m-chlorophenol was observed and its Ki value was found to be $5.46{\times}10^{-4}M$. 5) During the synthesizing reaction of 2-bromotyrosine by the enzyme, sequential addition of substrate which was m-bromophenol with time intervals and in a small amount resulted in better yield of the product. 6) The halogenated tyrosines which were produced by ${\beta}-tyrosinase$ from pyruvate, ammonia and m-halogenated phenols were analysed to determine their molecular structures by element analysis, NMR spectroscopy, mass spectrometry, and IR spectroscopy. The result indicated that they were 2-chloro-L-tyrosine, 2-bromo-L-tyrosine, and 2-iodo-L-tyrosine, respectively.

• Journal of Photoscience
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• v.9 no.2
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• pp.209-212
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• 2002

Cutaneous hyperpigmentation is a well-known consequence of both acute and chronic X-irradiation, although the molecular mechanisms involved are not well understood. Recently, protein kinase C-$\beta$ (PKC-$\beta$) was shown to activate tyrosinase, a key and the rate-limiting enzyme in melanogenesis [1]. In this study, we have investigated its role in mediating ionizing radiation-induced pigmentation by exposing cultured human melanocytes to X-irradiation. Increased tyrosinase activity after the 4 Gys exposure was observed within 48 hrs and total melanin content doubled after 7 days. Interestingly, tyrosinase mRNA level was not affected by X-irradiation. However, there was a 2-3 fold increase in PKC-$\beta$ mRNA after 48 hours of irradiation, coinciding with the increase in tyrosinase activity. This induction was not due to non-specific heat generated during the irradiation because when melanocytes were incubated at 4$0^{\circ}C$, there was no induction of PKC-$\beta$ mRNA. Taken together, these data suggest that X-irradiation induces cutaneous hyperpigmentation, at least in part, by up-regulating the level of PKC-$\beta$.

• Biomolecules & Therapeutics
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• v.10 no.3
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• pp.152-158
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• 2002

The present study elucidated the effect of $\beta$-carbolines (harmaline and harmalol) on brain mitochondlial dysfunction caused by the tyrosinase-induced oxidation of dopamine. Harmaline, harmalol and antioxidant enzymes (SOD and catalase) attenuated the dopamine-induced alteration of membrane potential, cytochrome c release and thiol oxidation in mitochondria. In contrast, antioxidant enzymes failed to reverse mitochondrial dysfunction induced by dopmnine plus tyrosinase. $\beta$-Carbolines decreased the damaging effect of dopamine plus tyrosinase against mitochondria, except no effect of harmalol on thiol oxidation. Antioxidant enzymes decreased the melanin formation from dopamine in the reaction mixture containing mitochondria but did not reduce the formation of dopamine quinone caused by tyrosinase. Both harmalol and harmaline inhibited the formation of reactive quinone and melanin. Harmalol being more effective for quinone formation and vise versa. The results indicate that compared to MAO-induced dopamine oxidation, the toxic effect of dopamine in the presence of tyrosinase against mitochondria may be accomplished by the dopamine quinone and toxic substances other than reactive oxygen species. $\beta$-Carbolines may decrease the dopamine plus tyrosinase-induced brain mitochondrial dysfunction by inhibition of the formation of reactive quinone and the change in membrane permeability.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.37 no.3
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• pp.211-218
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• 2011

Effects of Kenpaullone, a specific inhibitor of GSK3${\beta}$, on melanin synthesis in B16 melanoma cells and human melanocytes were investigated. Kenpaullone showed a melanogenesis stimulation activity in a concentrationdependent manner in murine B16 melanoma cells and human melanocytes without any significant effects on cell proliferation. Tyrosinase activity was increased 48 h after treatment of B16 cells with Kenpaullone. The protein expression level of tyrosinase was dose-dependently enhanced after the treatment with Kenpaullone. At the same time, the expression level of tyrosinase mRNA was also increased after addition of Kenpaullone. The stimulatory effect of Kenpaullone mainly resulted from increased expression of tyrosinase. These findings suggest that the application of GSK3${\beta}$ inhibitors may be a potential therapeutic agent for the treatment of hypopigmentation disorder.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.1
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• pp.53-61
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• 2018

Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in ${\alpha}$-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) and reduced the level of ${\beta}-catenin$, suggesting that ethyl linoleate inhibits melanogenesis through $Akt/GSK3{\beta}/{\beta}-catenin$ signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.

• BMB Reports
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• v.43 no.12
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• pp.824-829
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• 2010

Melanin synthesis is regulated by melanocyte specific enzymes and related transcription factors. $\beta$-carboline alkaloids including harmaline and harmalol are widely distributed in the environment including several plant families and alcoholic beverages. Presently, melanin content and tyrosinase activity were increased in melanoma cells by harmaline and harmalol in concentration- and time-dependent manners. Increased protein levels of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 were also evident. In addition, immunofluorescence and Western blot analyses revealed harmaline and harmalol increased cAMP response element binding protein phosphorylation and microphthalmia-associated transcription factor expression. In addition to studying the signaling that leads to melanogenesis, roles of the p38 MAPK pathways by the harmaline and harmalol were investigated. Harmaline and harmalol induced time-dependent phosphorylation of p38 MAPK. Harmaline and harmalol stimulated melanin synthesis and tyrosinase activity, as well as expression of tyrosinase and TRP-1 and TRP-2 indicating that these harmaline and harmalol induce melanogenesis through p38 MAPK signaling.

• Korean Journal of Pharmacognosy
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• v.44 no.1
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• pp.1-5
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• 2013

Nelnumbo nucifera Gaerth. (Nymphaeaceae) has been used in a korean traditional medicine to treat fever, sunstroke and dizziness. The receptaculums of this plant were refluxed with MeOH, and then fractionated with organic solvents ($CH_2Cl_2$, EtOAc and n-BuOH) to screen whitening activity using tyrosinase inhibitory activity. EtOAc ($IC_{50}$, 45.23 ${\mu}g/ml$) fractions showed a good tyrosinase inhibitory activity. Column chromatographic separation of $CH_2Cl_2$ and EtOAc fractions of Receptaculum nelumbinis led to the isolation 3 compounds. Their chemical structures were characterized as ${\beta}$-sitosterol (1), quercetin 3-O-${\beta}$-D-galactopyranoside (2) and kaempferol 3-O-${\beta}$-D-glucopyranoside (3) by comparison NMR spectral data and with those in references, respectively. Isolated compounds 1 and 3 were firstly isolated from Receptaculums nelumbinis. Compounds 2 and 3 showed potent whitening activities.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.298.1-298.1
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• 2002

Tyrosinase (monophenol. 3.4-${\beta}$-dihydroxyphenylalanin oxygen oxidoreductase. EC 1.14.18.1 J. which plays a pivotal role in melanogenesis. It is single chain glycoprotein catalyzing the hydroxylation of tyrosine to ${\beta$\mid$$-3.4-dihydroxyphenylalanin (DOPA) and the oxidation of DOPA to DOPA quinone. To investigate whitening effect of chitosan oligosaccharide. we obtained chitosan oligosaccharide ［(glucosamine)2-6］ by NaNO2 oxidation and measured the effect of chitosan oligosaccharide on tyrosinase activity. Chitosan oligosaccharide dose-dependently inhibited tyrosinase (2 unit) activity and inhibited by 18.8% at dose of 100${\mu}$g/ml. Vitamin C. arbutin and kojic acid that are well known to be inhibitor of melanin production dose-dependently inhibited tyrosinase (2unit) activity. These results suggest that chitosan oligosaccharide may be used as inhibitor of melanin production in melanocyte. which will be further studied.

• YAKHAK HOEJI
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• v.42 no.4
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• pp.353-358
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• 1998

The MeOH extract of the bark of Paeonia moutan showed potent inhibitory effect on the mushroom tyrosinase activity in vitro. The activity-guided fractionation of t he MeOH extract resulted in the isolation of three active compounds. The chemical structures of these compounds were elucidated by chemical and spectroscopic evidence as catechin, 1,2,3,6-tetra-O-galloyl-${\beta}$-D-glucose and 1,2,3,4,6-penta-0-galloyl-${\beta}$-D-glucose, respectively. Among them, the inhibitory activity by 1,2,3,6-tetra-galloyl-${\beta}$-D-glucose on mushroom tyrosinase was more potent $(IC_{50}=3.5\;{\mu}M)$ than that of kojic acid $(IC_{50}=8.7\;{\mu}M)$ ,but catechin enhanced the mushroom tyrosinase activity 50% in the concentration of 34.5M.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.33-38
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• 2004

Melanin pigmentation in human skin is a major defense mechanism against ultraviolet light of the sun, but abnormal pigmentation such as freckles, liver spot could be a serious aesthetic problem. Nearly all studies are mainly concentrated on searching for the materials that have inhibitory activities on tyrosinase. In this work, to isolate phenolic compounds from Gastrodia elata, we purified the extract through solvent fractionation, column chromatography, and recrystallization. They were identified as 4-hydroxybenzyl alcohol 1, bis(4-hydroxyphenyl)methane 2, gastrodin (4-${\beta}$-D-glucopyranosyloxybenzyl alcohol) 3 on the base of spectroscopic evidences. In order to investigate their depigmentation effect, inhibitory activity against mushroom tyrosinase and inhibitory activity of melanin synthesis in B16 melanoma cells were evaluated in vitro. We have found that 4-hydroxybenzyl alcohol 1 and gastrodin (4- ${\beta}$-D-glucopyranosyloxybenzyl alcohol) 3 have no tyrosinase inhibitory activity, but inhibit the melanin synthesis in B16 melanoma cells. Tyrosinase inhibitory activities of bis(4-hydroxyphenyl)methane 2 (IC$\_$50/ = 400 $\mu\textrm{g}$/mL) and butanol fraction (IC$\_$50/ = 46 $\mu\textrm{g}$/mL) were lower/higher than that of arbutin (IC$\_$50/ = 114 $\mu\textrm{g}$/mL), but inhibitory activities of melanin synthesis in B16 melanoma cells were much higher than that of arbutin. Especially, tyrosinase inhibitory activities of isolated phenolic fraction (IC$\_$50/ = 2.37 $\mu\textrm{g}$/mL) from butanol fraction was very higher than that of arbutin (IC$\_$50/ = 114 $\mu\textrm{g}$/mL). Therefore, these results suggest that isolated phenolic compounds from Gastrodia elata have inhibitory activity against mushroom tyrosinase and inhibitory activity of melanin synthesis in 816 melanoma cells in vitro.