Effects of radix polygoni multiflori components on tyrosinase activity and melanogenesis

Radix Polygoni multiflori is a herb used effectively to prevent graying and treat skin depigmentation diseases in traditional Chinese medicine but its active ingredients have not been discovered yet. In this investigation, we tested six compounds isolated from Radix Polygoni multiflori, to discover the active component on melanogenesis. Three experiments were performed in the present investigation: mushroom tyrosinase activity, melanin content B16 cell proliferation assay. Among all the six components tested, THSG showed the most potent effects on tyrosinase activation and melanogenesis; it was shown to be a potent tyrosinase activator and a melanogenesis stimulator in this study. On the other hand, we found that gallic acid significantly inhibited tyrosinase and, in addition, anthraquinones were cytotoxic to melanoma cells. They were both harmful to melanogenesis. Therefore, we propose that THSG acts as the active ingredient of Radix Polygoni multiflori on melanogenesis.     

Effects of radix polygoni multiflori components on tyrosinase activity and melanogenesis

Radix Polygoni multiflori is a herb used effectively to prevent graying and treat skin depigmentation diseases in traditional Chinese medicine but its active ingredients have not been discovered yet. In this investigation, we tested six compounds isolated from Radix Polygoni multiflori, to discover the active component on melanogenesis. Three experiments were performed in the present investigation: mushroom tyrosinase activity, melanin content B16 cell proliferation assay. Among all the six components tested, THSG showed the most potent effects on tyrosinase activation and melanogenesis; it was shown to be a potent tyrosinase activator and a melanogenesis stimulator in this study. On the other hand, we found that gallic acid significantly inhibited tyrosinase and, in addition, anthraquinones were cytotoxic to melanoma cells. They were both harmful to melanogenesis. Therefore, we propose that THSG acts as the active ingredient of Radix Polygoni multiflori on melanogenesis.     

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Inhibitory Effects of Melanin Monomers,Dihydroxyindole-2-Carboxylic Acid (DHICA) and Dihydroxyindole (DHI) on Mammalian Tyrosinase,With a Special Reference to the Role of DHICA/DHI Ratio in Melanogenesis

DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase-controlled, melanogenic reaction, namely conversion of L-tyrosine to DOPAchrome (2-carboxy-2,3-dihydroindole-5,6-quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase-rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L-tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase—tyrosine-hydroxylation and DOPA-oxidation—more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase-catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase-catalyzed early phase of melanogenesis.     

Characterization of the Melanogenic System in Vibrio cholerae,ATCC 14035

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine. Other phenolic chemicals related to L-tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami-nases that transforms L-tyrosine into p-hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.     

Melanosomal Proteins – Role in Melanin Polymerization

Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific.     

Induction of melanogenesis suppression: cellular pharmacology and mode of differential action

Recent elucidation of regulatory mechanisms of eu- and pheomelanogenesis has led us towards an exciting new era of melanogenesis control. I will chiefly address our progress on inhibitory control of melanogenesis from the macromolecular level to human skin colour. In the past, the exploration and search for skin depigmenting agents has been focussed on and initiated from substances which can suppress isolated tyrosinase in vitro. Now, as I have classified below, many new melanogenic inhibitors have been discovered which, in spite of their non-suppressive effect on isolated naked tyrosinase, suppress melanin formation in the living pigment cell in vitro as well as in the natural world. I will also discuss a recently found unique disorder: unilateral suppression of mixed melanogenesis.     

Purification of Ribonuclease L from Aspergillus sp. by Affinity Chromatography

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3′-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of melanogenesis by aloesin: a competitive inhibitor of tyrosinase

Aloesin, [2-acetonyl-8-beta-d-glucopyranosyl-7-hydroxy-5-methylchromone], a compound isolated from the Aloe plant, is shown in these studies to modulate melanogenesis via competitive inhibition of tyrosinase. Aloesin inhibits purified tyrosinase enzyme and specifically inhibits melanin production in vitro. Enzyme kinetics studies using normal human melanocyte cell lysates and cell-based melanin production demonstrated that aloesin is a competitive inhibitor of tyrosinase from mushroom, human and murine sources. Tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase from normal human melanocyte cell lysates were inhibited by aloesin in a dose dependent manner. In a percutaneous absorption study a finite dose of aloesin penetrated the skin slowly and was recovered primarily in the surface wash. Aloesin shows promise as a pigmentation-altering agent for cosmetic or therapeutic applications.     

Synthesis and activity of Xenopus laevis oocyte tyrosinase

This study investigates the mechanisms that control pigment synthesis in Xenopus laevis oocytes. Although we find the molecular weight of oocyte tyrosinase to be similar to that of amphibian skin, we were unable to increase its activity by proteases or detergents, as has been reported for skin tyrosinase. On the other hand, by measuring the activity of polysomal-bound enzyme, we were able to correlate increased tyrosinase activity with increased levels of enzyme synthesis. We therefore suggest that in oocytes, the activity of tyrosinase is primarily dependent on its synthesis, whereas in skin, the rate-limiting step is the post-translational activation of the enzyme. We speculate on these differences in relation to the functional role of melanin in skin and oocytes.     

Quantitative assay of melanin in melanoma cells in culture and in tumors

A simple method has been developed for the quantitative fluorimetric determination of melanin. In these studies, we have used synthetic melanin prepared from l-dopa by mushroom tyrosinase and natural melanin present in B16 mouse melanoma cells grown in culture or as a solid tumor. In the assay procedure, the melanin is oxidatively degraded into soluble products by heating in an alkaline, dilute hydrogen peroxide solution under standard conditions. The resulting solution has a characteristic fluorescence which is quantitatively related to the melanin initially present. The assay is very sensitive and can be applied directly to tissues or cell cultures, without prior isolation of the melanin.     

Melanosomal proteins--role in melanin polymerization

Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific.     

Novel screening method for potential skin-whitening compounds by a luciferase reporter assay

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

链霉菌酪氨酸酶基因的克隆与表达

本文综述了近年来对链霉菌酪氨酸酶基因的研究。由酪氨酸酶合成黑色素是链霉菌属各个种的共同特性,并受到酪氨酸酶基因的控制。链霉菌几个种的酪氨酸酶基因已克隆到,其产黑色素的特性使之作为重要的标记基因得以广泛应用,同时,该基因在链霉菌的不同种及不同属的微生物中得到了高水平的表达。链霉菌酪氨酸酶基因表达调控的分子机制也得到较深入的研究。     

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.