• Korean Chemical Engineering Research
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• v.54 no.6
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• pp.817-821
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• 2016

Levodopa or L-3,4-dihydroxyphenylalanine (L-DOPA) is the direct precursor of the neurotransmitter dopamine. L-DOPA is a well-known neuroprotective agent for the treatment of Parkinson's disease symptoms. L-DOPA was synthesized using the enzyme, tyrosinase, as a biocatalyst for the conversion of L-tyrosine to L-DOPA and an electrochemical method for reducing L-DOPAquinone, the product resulting from enzymatic synthesis, to L-DOPA. In this study, three electrode systems were used: A glassy carbon electrode (GCE) as working electrode, a platinum, and a Ag/AgCl electrode as auxiliary and reference electrodes, respectively. GCE has been modified using electropolymerization of pyrrole to facilitate the electron transfer process and immobilize tyrosinase. Optimum conditions for the electropolymerization modified electrode were a temperature of $30^{\circ}C$ and a pH of 7 producing L-DOPA concentration 0.315 mM. After 40 days, the relative activity of an enzyme for electropolymerization remained 38.6%, respectively.

• Microbiology and Biotechnology Letters
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• v.24 no.2
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• pp.222-226
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• 1996

The enzymatic synthesis of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal-5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96% by solubilizing and recrystallyzing the precipitates.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1446-1451
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• 2012

Levodopa or L-3,4-dihydroxyphenylalanine (L-DOPA) is the precursor of the neurotransmitter dopamine. L-DOPA is a famous treatment for Parkinson's disease symptoms. In this study, electroenzymatic synthesis of L-DOPA was performed in a three-electrode cell, comprising a Ag/AgCl reference electrode, a platinum wire auxiliary electrode, and a glassy carbon working electrode. L-DOPA had an oxidation peak at 376 mV and a reduction peak at -550 mV. The optimum conditions of pH, temperature, and amount of free tyrosinase enzyme were pH 7, $30^{\circ}C$, and 250 IU, respectively. The kinetic constant of the free tyrosinase enzyme was found for both cresolase and catacholase activity to be 0.25 and 0.4 mM, respectively. A cyclic voltammogram was used to investigate the electron transfer rate constant. The mean heterogeneous electron transfer rate ($k_e$) was $5.8{\times}10^{-4}$ cm/s. The results suggest that the electroenzymatic method could be an alternative way to produce L-DOPA without the use of a reducing agent such as ascorbic acid.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.98-102
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• 1996

A thermostable tyrosine phenol-lyase gene of a thermophilic Symbiobacterium species was cloned and overexpressed in Escherichia coli in order to produce the biocatalyst for the synthesis of 3, 4-dihy-droxyphenyl-L-alanine (L-DOPA). The substrates used for the synthetic reaction were pyrocatechol, so-dium pyruvate, and ammonium chloride. The enzyme was stable up to $60^{\circ}C$, and the optimal temperature for the synthesis of L-DOPA was $37^{\circ}C$ . The optimal pH of the reaction was about 8.3. Enzyme activity was highly dependent on the amount of ammonium chloride and the optimal concentration was estimated to be 0.6 M. In the case of pyrocatechol, an inactivation of enzyme activity was observed at con-centrations higher than 0.1 M. Enzyme activity was increased by the presence of ethanol. Under op-timized conditions, L-DOPA production was carried out adding pyrocatechol and sodium pyruvate to the reaction solution intermittently to avoid substrate depletion during the reaction. The concentration of L-DOPA reached 29.8 g/l after 6 h, but the concentration didn t increase further because of the formation of byproducts by a non-enzymatic reaction between L-DOPA and pyruvate.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.2 s.51
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• pp.153-159
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• 2005

In this study, we investigated the application of an extract from Ephedra sinica which has been composed of traditional Korean medicine as a whitening ingredient. The extract of Ephedra sinica which was obtained from the mixture of methanol and water (1:1) the inhibitory effect of tyrosinase. Then, Ephedra sinica was extracted by two different solvents. One was water and the other was methylene chloride. Only, the water extract of Ephedra sinica showed the inhibitory effort of tyrosinase; the anti-tyrosinase activity with $0.2\%$ of the water extract was $60.6\%$. But the extract of Ephedra sinica in methylene chloride fraction showed little inhibitory effect on tyrosinase. The inhibitory effect of the concentrated water extract of Ephedra sinica was tested on L-DOPA auto-oxidation and melanin synthesis in B-16 melanoma. In L-DOPA auto-oxidation, $0.5\%$ of the concentrated water extract showed $87\%$ of inhibition of L-DOPA auto-oxidation and the $0.75\%$ concentrated Ephedra sinica extract in wafer fraction inhibited $98.8\%$ of that. In melanin synthesis of B-16 melanoma, the concentrated water effect of Ephedra sinica inhibited $70.2\%$ or $79.9\%$ of inhibitory effect on that at the concentration of $0.05\%$ or $0.075\%$, respectively. For verifying the skin whitening effect of the concentrated water extract of Ephedra sinica in vivo, we performed the clinical test of that. The research showed the significant skin whitening effect of a cream containing $0.5\%$ Ephedra sinica extract and the statistical analysis showed a significant difference (p < 0.05) between sample (containing $0.5\%$ Ephedra sinica extract) and placebo after 10 weeks.

• Journal of Life Science
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• v.28 no.3
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• pp.331-338
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• 2018

Melanin is a polymer substance that plays an important role in the determination of hair growth and skin color in vivo. However, melanin, which is over-produced by reactive oxygen species, is known to cause stains, freckles, and hypercholesterolemia, which are associated with aging. Previous studies have shown that polyphosphate, one of the components of Rhynchosia Nulubilis, inhibits skin aging induced by ultraviolet rays. The aim of this study is to investigate the direct effect of Rhynchosia Nulubilis ethanolic extract (RNEE) on melanin synthesis. In this study, RNEE showed no antioxidative effects on scavenging activity of DPPH radical in addition to reducing power. The cytotoxicity of RNEE was increased in a dose-dependent manner in an MTT assay. In addition, RNEE increased tyrosinase activity and melanin synthesis in DOPA-oxidation experiments. RNEE did not promote the conversion L-DOPA into melanin in live cells, but melanin production was promoted in the RNEE-treated group after H2O2 pretreatment compared to the control group in which melanin production was reduced by treatment with H2O2. In addition, RNEE increased the expression level of tyrosinase related protein-2 (TRP-2) and increased the expression level of tyrosinase related protein-1 (TRP-1) at a concentration of $16{\mu}g/ml$. In particular, it was found that RNEE increased the expression level of SOD-3, by which superoxide anion is converted to hydrogen peroxide, higher than the control and ${\alpha}$-MSH used as a positive control at a concentration of more than $16{\mu}g/ml$. The results suggest that RNEE can induce melanogenesis related to black hair.

• Journal of Life Science
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• v.28 no.7
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• pp.802-810
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• 2018

Aging is accompanied by changes in the body, such as graying hair, wrinkles, and black spots composed of lipid peroxides and proteins. Melanin is a polymer substance produced by an oxidation polymerization reaction from tyrosine, and it determines the color of hair and skin. It has been reported that melanin is synthesized by melanocyte, and its excessive production by reactive oxygen species is associated with aging. The purpose of this study was to determine the direct effects of Musa paradisiaca peel ethanolic extract (MPEE) on antioxidative activity and melanin synthesis. It was observed that the antioxidant activity of MPEE was similar to that of vitamin C, a positive control, in both DPPH radical scavenging assay and reducing power assay. In order to examine cytotoxicity prior to cell experimentation, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed for B16F1 cells. MPEE was not cytotoxic at $32{\mu}g/ml$ or less. In addition, MPEE increased melanin synthesis in live cells in addition to tyrosinase activity and melanin synthesis in dihydroxyphenylalanine (DOPA)-oxidation assay in vitro. Moreover, MPEE increased melanin synthesis in cells aged by pretreatment with $H_2O_2$. The expression levels of tyrosinase-related protein (TRP)-1, TRP-2, and superoxide dismutase (SOD)-2 by western blot analysis were increased in the presence of MPEE. These results suggest that MPEE could promote the melanin synthesis as an antioxidative substance.

• Journal of Life Science
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• v.29 no.4
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• pp.461-469
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• 2019

The aim of this study was to investigate the effect of a mixture of ethanol extracts of Black oryzasativa, Sesamum indicum, Oryza sativa, Rhynchosia Nulubilis, and Polygoni multiflori radix (MIXEE) on melanogenesis to develop a natural product for black hair growth. An accumulation of hydrogen peroxide ($H_2O_2$) in hair follicles, which reduces melanin synthesis, is responsible for hair graying. In the present study, MIXEE showed scavenging activity against DPPH radicals and reducing power. In addition, it reduced the cellular $H_2O_2$ level, indicating that it could inhibit oxidation and promote melanin synthesis, which was decreased by $H_2O_2$. On the other hand, it did not affect tyrosinase activity in vitro but promoted the turnover of L-DOPA into melanin. MIXEE promoted melanin synthesis at the cellular level in B16F1 cells. Furthermore, MIXEE increased the expression levels of superoxide dismutase 2 (SOD2) and SOD3 in western blot analysis. In addition, MIXEE increased the expression levels of tyrosinase-related protein (TRP)-2, which promoted melanin synthesis from L-DOPA. The results suggested that MIXEE could promote melanogenesis. Therefore, MIXEE may have potential as a natural product for promotion of melanin production and reversal of gray hair to black hair.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.3
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• pp.528-532
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• 2011

This study was designed to investigate the collagen metabolism and tyrosinase activity of Meretrix extracts (ME). The effect of ME on type I procollagen production and collagenase activity in human normal fibroblasts HS68 after UVB (312 nm) irradiation was measured by ELISA method. The tyrosinase activity after treatment of ME was measured as well. Type I procollagen production was recovered by ME in UVB damaged HS68 cells. The increased collagenase activity after UVB damage was significantly recovered by ME. The tyrosinase activity and L-DOPA oxidation were significantly reduced as well. However, the effects on tyrosinase activity and L-DOPA oxidation were not powerful enough to be used as whitening agents. ME showed the anti-wrinkle effects and some whitening effects in vitro. These results suggest that ME may be a useful drug as an anti-wrinkle treatments.

• The Korean Journal of Mycology
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• v.38 no.2
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• pp.167-171
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• 2010

The present study aims to evaluate Cordyceps militaris water extract (CMWE) with a view to develop melanogenesis inhibitors. Inhibitory activities of CMWE against tyrosinase, L-DOPA(L-3,4-dihydroxyphenylalanine) oxidation, and melanin biosynthesis in B16 mouse melanoma cells were investigated. CMWE, at $5000\;{\mu}g/ml$, inhibited tyrosinase activity of 71% and DOPA oxidation of 40% as reacting with L-DOPA. Furthermore, B16 mouse melanoma cell survived over 50% from low to high dose on MTT assay, and CMWE markedly inhibited (> 50%) melanin synthesis at $5000\;{\mu}g/ml$. The inhibitory effect of CMWE on melanogenesis was attributed to enhancement of tyrosinase degradation. Key enzyme of melanin biosynthesis is tyrosinase which catalyses a beginning step from tyrosine to DOPA quinine and melanin formation step, respectively. These results indicated that CMWE may be a potential source of novel whitening agents for cosmetic or therapeutic application.