Article

Oxyresveratrol and trans-dihydromorin from the twigs of Cudrania tricuspidata as hypopigmenting agents against melanogenesis

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Abstract

The depigmenting effects of oxyresveratrol and trans-dihydromorin were investigated in b16F10 cell line and its synergetic melan-a cells, as well as in 3-dimensional skin models. But their inhibitory effects were different in two cell lines. In b16 cells, oxyresveratrol showed stronger depigmenting effect than trans-dihydromorin, with greater suppression on tyrosinase activity. Both of them induced post-transcriptional degradations of microphthalmia-associated transcription factor (MITF), leading to significant decreases of tyrosinase related protein 1 (TRP-1) and tyrosinase related protein 2 (TRP-2) production. In melan-a cells, trans-dihydromorin exhibited stronger inhibition on melanin synthesis. It also induced greater suppression on the levels of tyrosinase, TRP-1 and TRP-2 via MITF down-regulation at both transcription and translation level. Evaluations in artificial skin model also demonstrated the depigmenting effects of trans-dihydromorin. In conclusion, we found that trans-dihydromorin is an effective hypopigmenting agent in normal skin cells. Furthermore we demonstrated that hypopigmenting agents effective in melanoma system may not be effective on normal melanocytes, indicating that a non-tumor melanocyte system is more suitable for the screening of hypopigmenting agents.

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... The cells were treated with serial concentrations of RTPs for 72 h. After the indicated incubation time, cells were treated with 100 μL 10% Cell Counting Kit (CCK)-8 solution for 1.5 h (Dojindo China Co., Ltd., Shanghai, China) (Hu et al., 2015). The absorbance was measured at 490 nm using Victor X4 Multilabel Plate Reader (PerkinElmer). ...
... The cells were treated with α-MSH (1 µm) alone or with α-MSH plus different concentrations of testing agents for 72 h. T. Cellular tyrosinase activity was determined by measuring the oxidation of L-DOPA (Sigma-Aldrich) to dopachrome in cell lysates (Hu et al., 2015). The dopa oxidase activity of tyrosinase was measured using 3-methyl-2-benzothiazolinone hydrazone (Sigma-Aldrich) assay (Winder, 1994). ...
... Only Kuwanon O and Sanggenon T induced more increase of *L value, as well as stronger inhibition on melanin content and distribution than Kojic acid without significant cytotoxicity in 3-dimensional artificial skin model. Moreover, our previous depigmentation study on Oxyresveratrol, another recorsinol type phenolic compound which also identified from the root extract of M. australis, substantiated this hypothesis as well (Hu et al., 2015). ...
Article
Ethnopharmacological relevance: Morus australis, one of the major Morus species growing in East Asia, is rich in phenolic compounds. The extract of M. australis has been used as skin whitening components for a long period. The action mechanisms of its principal constituents are still unclear. This study aims to evaluate the skin lightening effects of phenolic compounds extracted from the root of M. australis in different melanocyte systems and artificial skin models. Materials and methods: The depigmenting effect of resorcinol type polyphenols (RTPs) from the root extract of M. australis was evaluated in murine b16 and melan-a cell lines using a combined sulforhodamine B assay. Tyrosinase activity and the expression of melanogenesis proteins were evaluated for the mechanism study. The artificial skin model is used as a replacement of the animal test. Results: Only Kuwanon O and Sanggenon T were found to have significant depigmenting effects in both murine b16 and melan-a cell lines. Their depigmenting mechanisms are slightly different in the two cell systems. In b16 cells, Kuwanon O and Sanggenon T, together with the other two RTPs, induced post-transcriptional degradations of MITF without suppressing its mRNA expression, leading to significant decreases of TRP-1 and TRP-2 production. While in melan-a cells, the levels of tyrosinase families were suppressed via MITF downregulation at both transcription and translation level by RTPs, with Kuwanon O inducing the greatest suppression. Further evaluations in artificial skin model demonstrated the outstanding depigmenting effects of Kuwanon O and Sanggenon T. Conclusions: Kuwanon O and Sanggenon T from M.australis root extract are two potential skin whitening ingredients. To screen resorcinol flavonone derivatives with an isoprenyl group in the Diels-Alder substituent might be an option for the search of potent hypopigmenting agents from plants.
... Mushroom tyrosinase [14,34,37,49,61,63,67,77,84,97,109,111,124,128,138,139,141,[148][149][150][151][152][153][154][155] Murine tyrosinase from cell lysates [128,149] Human tyrosinase from cell lysates [128] Cellular tyrosinase/melanogenesis [49,61,68,77,97,124,128,138,[152][153][154][156][157][158][159] Hypopigmentation in animals [49,77,97,159] Depigmentation in humans [160] Anti-browning effect [78] ORV inhibited mushroom tyrosinase in a non-competitive manner when L-dopa was employed as the substrate [139,148,150]. This was also confirmed by a detailed study using combined techniques of fluorescence spectroscopy and circular dichroism (CD) [49]. ...
... Numerous studies of ORV were conducted on cellular tyrosinase. ORV showed suppression of melanogenesis in murine melanoma B16/B16-F10 cells [49,61,68,128,153,154,[156][157][158] and human PIG1 melanocytes [152], all in a dose-dependent manner. ORV reduced tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) productions in B16-F10 cells via downregulation of microphthalmia-associated transcription factor (MITF). ...
... ORV reduced tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) productions in B16-F10 cells via downregulation of microphthalmia-associated transcription factor (MITF). [68]. In a study on the binding of ORV to tyrosinase using fluorescence and circular dichroism spectroscopic techniques, it was found that there was a static interaction between ORV and the enzyme through van der Waals forces and hydrogen bonding [153]. ...
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Oxyresveratrol has recently attracted much research attention due to its simple chemical structure and diverse therapeutic potentials. Previous reviews describe the chemistry and biological activities of this phytoalexin, but additional coverage and greater accessibility are still needed. The current review provides a more comprehensive summary, covering research from 1955 to the present year. Oxyresveratrol occurs in both gymnosperms and angiosperms. However, it has never been reported in plants in the subclass Sympetalae, and this point might be of both chemotaxonomic and biosynthetic importance. Oxyresveratrol can be easily obtained from plant materials by conventional methods, and several systems for both qualitative and quantitative analysis of oxyresveratrol contents in plant materials and plant products are available. Oxyresveratrol possesses diverse biological and pharmacological activities such as the inhibition of tyrosinase and melanogenesis, antioxidant and anti-inflammatory activities, and protective effects against neurological disorders and digestive ailments. However, the unfavorable pharmacokinetic properties of oxyresveratrol, including low water solubility and poor oral availability and stability, have posed challenges to its development as a useful therapeutic agent. Recently, several delivery systems have emerged, with promising outcomes that may improve chances for the clinical study of oxyresveratrol.
... The cytotoxicity of ORT, an antioxidant, is reportedly lower than that of DTIC (26). Moreover, ORT is not cytotoxic in normal cells, such as human keratinocyte and mouse melanocyte cells, at relatively high concentrations (27,28). The DPPH radical scavenging activity assay is widely utilized to measure the antioxidant potential of chemicals (29). ...
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Various therapies have been developed to target malignant melanoma, which is associated with a high mortality rate worldwide. Although dacarbazine (DTIC) is employed for treating melanoma, it is associated with several side effects. Hence, patients with melanoma are co-treated with additional drugs to mitigate the side effects of DTIC. In the present study, synergistic therapeutic effects of the DTIC/oxyresveratrol (ORT) combination were examined using the human malignant melanoma WM-266-4 cell line. Treatment with ORT and DTIC inhibited the proliferation of WM-266-4 cells. Compared with those in the ORT- and DTIC-treated groups, the proportion of cells arrested at the S phase, as well as apoptotic rates, were increased in the ORT and DTIC co-treatment group. In WM-266-4 cells, synergistic proliferation-inhibitory activities of the ORT/DTIC combination were assessed based on cell viability and migration, antioxidant capacity, cytokine production, cell cycle arrest, apoptotic rate and protein expression through WST-1 assay, wound healing assay, flow cytometry and western blotting. Furthermore, the expression levels of proteins, including NOTCH, involved in the pathogenesis of solid cancers, such as melanoma, were examined. Overall, the ORT/DTIC combination synergistically promoted cell cycle arrest at the S phase and the apoptosis of WM-266-4 cells. Thus, this combination treatment may serve as a novel therapeutic strategy for treating malignant melanoma.
... Tyrosinase (EC 1.14.18.1) is a metalloenzyme group of polyphenol oxidases that is found in various organisms and exhibits a specific function in melanogenesis [1]. Tyrosinase promotes the transformation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) (monophenolase activity) by hydroxylation, and then, l-DOPA is converted to DOPA-quinone (diphenolase activity) [2][3][4]. When l-tyrosine or l-DOPA are the substrates, the product of the reaction catalyzed by tyrosinase is dopaquinone, an intermediate in the melanin biosynthesis pathway [5]. ...
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A series of (E)-1-(furan-2-yl)prop-2-en-1-one derivatives (compounds 1–8) were synthesized and evaluated for their mushroom tyrosinase inhibitory activity. Among these series, compound 8 (2,4-dihydroxy group bearing benzylidene) showed potent tyrosinase inhibitory activity, with respective IC50 values of 0.0433 µM and 0.28 µM for the monophenolase and diphenolase as substrates in comparison to kojic acid as standard compound 19.97 µM and 33.47 µM. Moreover, the enzyme kinetics of compound 8 were determined to be of the mixed inhibition type and inhibition constant (Ki) values of 0.012 µM and 0.165 µM using the Lineweaver-Burk plot. Molecular docking results indicated that compound 8 can bind to the catalytic and allosteric sites 1 and 2 of tyrosinase to inhibit enzyme activity. The computational molecular dynamics analysis further revealed that compound 8 interacted with two residues in the tyrosinase active site pocket, such as ASN260 and MET280. In addition, compound 8 attenuated melanin synthesis and cellular tyrosinase activity, simulated by α-melanocyte-stimulating hormone and 1-methyl-3-isobutylxanthine. Compound 8 also decreased tyrosinase expressions in B16F10 cells. Based on in vitro and computational studies, we propose that compound 8 might be a worthy candidate for the development of an antipigmentation agent.
... Cellular tyrosinase activity was measured using a previously described method with some modifications. (Hu, Zheng, Zhang, Chen, & Wang, 2015;Kim et al., 2008) Cells cultured and harvested as described in the melanin content assay were rinsed with PBS and detached with 0.5% Trypsin (30 μl/well). Then, the cells were harvested by centrifugation at 20,000 g at 4°C for 5 min and lysed by adding 100 μl of 1% Triton X-100 and two freeze-thaw cycles. ...
Article
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The efficacy of oily components is often difficult to evaluate due to their incompatibility with most models. Here, we emulsified adlay bran oil (ABO), processed it to a nanoscale, and investigated its anti‐hyperpigmentation efficacy, assessed for its inhibitory effects against tyrosinase activity and melanin production, in an in vitro system (mouse melanoma B16F10 cells) and an in vivo system (zebrafish embryos). ABO induced dose‐dependent reductions in tyrosinase activity and melanin production in both the melanoma cells and zebrafish, without affecting viability. The efficacy of ABO was strongly influenced by emulsion particle size in the zebrafish but not in the cells. These results indicate that ABO has potential as a tyrosinase inhibitor and anti‐hyperpigmentation agent and that the emulsion system is an effective method for delivering the bioactive components of ABO to living systems that could be utilized for other oily components. The by‐product from adlay processing was used as the source of functional food ingredient. Nanoemulsion was developed for oral delivery of adlay bran oil and was effective to inhibit tyrosinase activity and melanin synthesis as shown in the in vitro B16F10 cells model and in vivo zebrafish were used in the study.
... 41 Recent in vitro study reported that low concentration of oxyresveratrol than 20 µM able to inhibit melanin synthesis potently on B16 cells whereas on melan-a cells, concentration used between 20 to 100 µM did not give significant difference in melanin content. 44 Based on above studies, oxyresveratrol has a potential to be an antimelanogenic agent. To the best of our knowledge, data of oxyresveratrol based on in vivo study is much lesser than in vitro studies and thus, the anti-melanogenic activity of oxyresveratrol should be further explored be ↓ melanin index, tyrosinase, TRP-1, MITF [38] it in vivo study or in vitro study to evaluate the effectiveness of oxyresveratrol in exerting its antimelanogenic activity in all aspects. ...
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Ultraviolet radiation (UVR) from sunlight is an environmental human carcinogen. Skin exposure to UVR would increase the oxidative stress, deoxyribonucleic acid (DNA) damage, melanogenesis and photocarcinogenesis. Therefore, development of photoprotective agent is necessary in order to reduce the cutaneous toxicity. The use natural active compounds like stilbenes and its derivatives have gained attention as photoprotection to skin due to its broad biological activities such as antioxidant, anti-inflammatory, anti melanogenesis and chemoprevention. This review article aims to analyse the existing literature on the photoprotective effect of stilbenes and its derivatives which include the resveratrol, pterostilbene, piceatannol and oxyresveratrol on in vitro and in vivo studies. This article describes the stilbenes and its derivatives protect and prevent UVR induced skin disorders via the reduction of oxidative stress, alleviation of DNA damage, inhibition of melanogenesis and anti photocarcinogenic effect.
... However, in vivo animal models often requires long experimental periods and extensive work, not to mention the considerable consumption of testing drugs used in animals for large-scale screening 5 . In view of the aforementioned disadvantages, in vitro assays such as cell-free mushroom tyrosinase activity assay, cell-based and MelanoDerm 3D tissue models 6,7 are preferable to in vivo assays during preliminary screening of tyrosinase inhibitors from mixtures of natural products in terms of cost and efficiency 5 . Although these in vitro techniques provide effective approaches to evaluate depigmenting effects of unknown samples, the development of compound-oriented screening method is urgently required for the identification of bioactive compound(s). ...
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Tyrosinase-based TLC (thin layer chromatography) was developed for screening of anti-melanogenic drugs. In particular, this technique enables researchers to identify melanogenic inhibitor(s) in tested mixtures with the naked eye. In comparison with traditional colorimetric screening assays for tyrosinase inhibitor(s), not only is tyrosinase-based TLC a more cost-effective option (nearly one-tenth the enzyme cost of colorimetric methods) but also is a more sensitive detection approach for kojic acid (KA), a standard anti-melanogenic drug. The detection limit of tyrosinase-based TLC and colorimetric tyrosinase assay for KA was 0.0125 and 1.25 μg, respectively, demonstrating that the former was 100-fold more sensitive than the latter to determine the tyrosinase inhibitory rate of KA. Furthermore, the results of this method have demonstrated excellent precision by Gage Repeatability and Reproducibility (Gage R&R), with the variation of total Gage R&R being 28.24%. To verify the applicability of tyrosinase-based TLC, this platform was employed to screen melanogenic inhibitor(s) from Ganoderma formosanum extracts and two of all fractions (GFE-EA F4, F5) obtained showed depigmenting activity. It is noteworthy that these two fractions also exerted anti-melanogenesis activity on zebrafish, therefore verifying the credibility of tyrosinase-based TLC. In sum, this technique provides new insight into the discovery of novel melanogenic inhibitor(s).
... Tyrosinase is a vital enzyme for melanin synthesis, and observing its intracellular activity is constantly the first step in studying melanin synthesis (Hu et al., 2015). As shown in Table 4. FPE effectively inhibited intracellular TYR activity of B16 cells in dose-dependent manner; this inhibition is similar to change in melanin content. ...
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Rape bee pollen possesses many nutritional and therapeutic properties because of its abundant nutrimental and bioactive components. In this study, free (FPE) and bound (BPE) phenolic extracts of rape bee pollen were obtained, phenolic and flavonoid contents were determined, and composition of phenolic acids was analyzed. In vitro antioxidant and anti-tyrosinase (TYR) activities of FPE and BPE were compared, and inhibitory melanogenesis of FPE was further evaluated. Results showed FPE and BPE contain total phenolic contents of 11.76 and 0.81 mg gallic acid equivalents/g dry weight (DW) and total flavonoid contents of 19.24 and 3.65 mg rutin equivalents/g DW, respectively. Phenolic profiling showed FPE and BPE fractions contained 12 and 9 phenolic acids, respectively. FPE contained the highest rutin content of 774.87 μg/g. FPE and BPE showed the high antioxidant properties in vitro and high inhibitory activities for mushroom TYR. Higher activities of FPE than those of BPE can be attributed to difference in their phenolic compositions. Inhibitory melanogenesis activities of FPE against B16 were further evaluated. Results showed suppressed intracellular TYR activity, reduced melanin content, and promoted glutathione synthesis (p < 0.05) in FPE-treated cells. FPE reduced mRNA expression of TYR, TYR-related protein (TRP)-1 and TRP-2, and significantly suppressed cyclic adenosine monophosphate (cAMP) levels through down-regulation of melanocortin 1 receptor gene expression (p < 0.05). FPE reduced mRNA expression of microphthalmia-associated transcription factor (MITF), significantly inhibiting intracellular melanin synthesis (p < 0.05). Hence, FPE regulates melanogenesis of B16 cells involved in cAMP/MITF/TYR pathway. These results revealed that FPE can be used as pharmaceutical agents and cosmetics to protect cells from abnormal melanogenesis.
... well-established model for melanogenic inhibitors discovery as previous studies suggested 2,23,24 . In our research, the melanin content of B16-F10 melanoma cells without drug treatment was assigned as 100%. ...
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In this study, the inhibitory effect of Ganoderma formosanum mycelium extracts on tyrosinase, the central regulatory enzyme being responsible for cutaneous pigmentation, was investigated in both cell-free and cellular enzymatic systems, as well as in phenotype-based zebrafish model. Bioassay-guided purification indicated that the ethyl acetate fraction of G. fromosanum mycelium ethanolic extract (GFE-EA) demonstrated the highest inhibition toward cell-free tyrosinase (IC 50 = 118.26 ± 13.34 ppm). The secreted and intracellular melanin of B16-F10 cells were reduced by GFE-EA through suppression of tyrosinase activity (IC 50 = 102.27 ± 9.49 ppm) and its protein expression. Moreover, GFE-EA decreased surface pigmentation level of zebrafish via down-regulation of tyrosinase activity. Most of all, there is no significant difference in morphology and mortality between control and GFE-EA treated groups. Not only does GFE-EA exhibit similar depigmenting efficacy to kojic acid with lower dosage (approximately one-seventh of dose), but show less toxicity to zebrafish. It is worth noting that GFE-EA is extracted from mycelium, which subverts the general concept that mycelium lacks certain bioactivities possessed by fruit bodies. Altogether, it would appear that GFE-EA has great potential for application in the cosmetics industry.
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The combined application of polyphenols and anticancer drugs may be of great help to current cancer treatment. In this paper, the individual and combined interactions of oxyresveratrol (OXY)/piceatannol (PIC) and mitoxantrone (MIT) with human serum albumin (HSA) in phosphate buffer of pH 7.4 were studied by multi-spectroscopic techniques and molecular docking. The quenching mechanism of HSA by OXY, PIC and MIT was determined by fluorescence quenching experiments and UV difference spectra. The binding location of the three ligands on HSA was identified by site marking fluorescence spectra, synchronous fluorescence spectra, and molecular docking. For the OXY/PIC/MIT + HSA binary system and the OXY/PIC + MIT + HSA ternary system, the thermodynamic parameters were obtained. The results indicated the competitive binding of OXY/PIC and MIT to HSA. The driving forces in the binding process were discussed. The circular dichroism spectroscopy and dynamic light scattering were employed to investigate the effects of OXY/PIC/MIT single drug and OXY/PIC + MIT combined drugs on the secondary structure and particle size of HSA. The effects of drug combination and HSA interaction on the in vitro cytotoxicity against HeLa cells were determined by MTT assay. The results would provide a theoretical basis for the combined application of polyphenols and traditional anticancer drugs.
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The aim of this study was to explore how the toxic trans‐crotonaldehyde (TCA) in mitochondria or aldehyde dehydrogenase (ALDH) at different pHs was intercepted by oxyresveratrol (Oxy‐Res) contributing to anticancer. Ultraviolet–visible (UV–vis) spectroscopy and Raman spectroscopy were employed. UV–vis spectra showed that the Oxy‐Res red shifted the peak of the toxic TCA from 316 nm to 325 nm, while the peaks of the Oxy‐Res shifted from 329 nm with 290 nm and 300 nm to 325 nm with 303 nm. In the mitochondria, the Oxy‐Res blue shifted the peaks of the toxic TCA from 325 nm with 303 nm to 321 nm with 301 nm. Raman spectra revealed that the Oxy‐Res caused shifting of the CHO of the toxic TCA from 1,689 cm−1 to 1,671 cm−1 with band decline. The CC of the toxic TCA at 1641 cm−1 was split into 1,639 cm−1 and 1,642 cm−1 with band decline. The bands of the Oxy‐Res at 1634 cm−1, 1,617 cm−1, and 1,595 cm−1 disappeared. In the mitochondria, the CC of the toxic TCA at 1641 cm−1 splitting disappeared. In ALDH, with the decrease of pH from 7.8 to 6.5, the CHO of the toxic TCA did not red shift from 1,689 cm−1 to 1,674 cm−1 up to pH 6.5. There was no change in the CC of the toxic TCA at 1640 cm−1 in ALDH at different pHs. The conclusion of the study was that the CHO of the toxic TCA was intercepted by the Oxy‐Res under the action of ALDH in the mitochondria, particularly at pH 7.8.
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Cudrania tricuspidata is a perennial plant of the family Moraceae with numerous medicinal and nutritional applications. It has been widely used in East Asia as an important traditional folk medicine for the treatment of many ailments such as eczema, mumps, tuberculosis, contusions, insomnia and acute arthritis. The whole plant of C. tricuspidata, including the roots, leaves, bark, stems and fruits, has been found to contain diverse phytochemicals, including xanthones, flavonoids, organic acids, and polysaccharides, with various bioactivities. In particular, xanthones and flavonoids, as the main active constituents, isolated from C. tricuspidata have been proven to possess notable anti-inflammatory, antioxidative, antitumor, hepatoprotective, neuroprotective and anti-obesity effects. This review summarizes the botany, traditional uses, phytochemistry and pharmacology of C. tricuspidata, and the limitations of studies on this species have also been discussed such that to serve as the basis for further research and development on this medicinal plant.
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The production of melanin pigment in mammals requires tyrosinase, an enzyme which hydroxylates the amino acid tyrosine to DOPA (3,4-dihydroxyphenylalanine), thus allowing the cascade of reactions necessary to synthesize that biopolymer. However, there are other regulatory steps that follow the action of tyrosinase and modulate the quantity and quality of the melanin produced. DOPAchrome tautomerase is one such melanogenic enzyme that isomerizes the pigmented intermediate DOPAchrome to DHICA (5,6-dihydroxyindole-2-carboxylic acid) rather than to DHI (5,6-dihydroxyindole), which would be generated spontaneously. This enzyme thus regulates a switch that controls the proportion of carboxylated subunits in the melanin biopolymer. Efforts to clone the gene for tyrosinase have resulted in the isolation of a family of tyrosinase related genes which have significant homology and encode proteins with similar predicted structural characteristics. Using specific antibodies generated against synthetic peptides encoded by unique areas of several of those proteins, we have immuno-affinity purified them and studied their melanogenic catalytic functions. We now report that TRP-2 (tyrosinase related protein-2), which maps to and is mutated at the slaty locus in mice, encodes a protein with DOPAchrome tautomerase activity.
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Tyrosinase is responsible for the molting process in insects, undesirable browning of fruits and vegetables, and coloring of skin, hair, and eyes in animals. To clarify the mechanism of the depigmenting property of hydroxystilbene compounds, inhibitory actions of oxyresveratrol and its analogs on tyrosinases from mushroom and murine melanoma B-16 have been elucidated in this study. Oxyresveratrol showed potent inhibitory effect with an IC(50) value of 1.2 microm on mushroom tyrosinase activity, which was 32-fold stronger inhibition than kojic acid, a depigmenting agent used as the cosmetic material with skin-whitening effect and the medical agent for hyperpigmentation disorders. Hydroxystilbene compounds of resveratrol, 3,5-dihydroxy-4'-methoxystilbene, and rhapontigenin also showed more than 50% inhibition at 100 microm on mushroom tyrosinase activity, but other methylated or glycosylated hydroxystilbenes of 3,4'-dimethoxy-5-hydroxystilbene, trimethylresveratrol, piceid, and rhaponticin did not inhibit significantly. None of the hydroxystilbene compounds except oxyresveratrol exhibited more than 50% inhibition at 100 microm on l-tyrosine oxidation by murine tyrosinase activity; oxyresveratrol showed an IC(50) value of 52.7 microm on the enzyme activity. The kinetics and mechanism for inhibition of mushroom tyrosinase exhibited the reversibility of oxyresveratrol as a noncompetitive inhibitor with l-tyrosine as the substrate. The interaction between oxyresveratrol and tyrosinase exhibited a high affinity reflected in a K(i) value of 3.2-4.2 x 10(-7) m. Oxyresveratrol did not affect the promoter activity of the tyrosinase gene in murine melanoma B-16 at 10 and 100 microm. Therefore, the depigmenting effect of oxyresveratrol works through reversible inhibition of tyrosinase activity rather than suppression of the expression and synthesis of the enzyme. The number and position of hydroxy substituents seem to play an important role in the inhibitory effects of hydroxystilbene compounds on tyrosinase activity.
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Tyrosinase (TYR), tyrosinase‐related protein‐1 (TYRP1/gp75) and dopachrome tautomerase (DCT/TYRP2) belong to a family of melanocyte‐specific gene products involved in melanin pigmentation. During melanocyte development expression of tyrosinase family genes is thought to be orchestrated in part by the binding of a shared basic helix–loop–helix transcription factor MITF to the M box, a regulatory element conserved among these genes. In transformed melanocytes, expression of tyrosinase and TYRPs is highly variable. Whereas TYR expression in melanoma cells is regulated by both transcriptional and post‐translational mechanisms, TYRP1/gp75 transcription is often completely extinguished during melanoma tumor progression. In this study, we investigated the mechanisms of selective repression of TYRP1 transcription. Interestingly, in early stage melanoma cells TYRP1 mRNA could be induced by inhibition of protein synthesis. Transient transfection experiments with a minimal TYRP1 promoter showed that the promoter activity correlates with expression of the endogenous TYRP1 gene. Nucleotide deletion analysis revealed novel regulatory sequences that attenuate the M box‐dependent MITF activity, but which are not involved in the repression of TYRP1. Gel mobility shift analysis showed that binding of the transcription factor MITF to the TYRP1 M box is selectively inhibited in TYRP1– cells. These data suggest that protein factors that modulate the activity of MITF in melanoma cells repress TYRP1 and presumably other MITF target genes.
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The twigs of Cudrania tricuspidata were found to show strong tyrosinase inhibitory activity, and further detailed component analysis resulted in the isolation of a new flavanol glucoside, (2S,3S)-2,3-trans-dihydromorin-7-O-β-D-glucoside (1), plus twenty-seven known compounds (2-28). Their structures were elucidated on the basis of ESI-MS and NMR spectral data. Among the isolated compounds, trans-dihydromorin (8), oxyresveratrol (9), and steppogenin (12) were found to exhibit significant tyrosinase inhibition activities. Moreover, the structure-activity relationship of these isolated compounds was also discussed.
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A major obstacle in the clinical management of malignant melanoma is its intrinsic resistance to chemotherapy and radiation therapy. Consequently, most patients with melanoma often do not respond to conventional anticancer therapy in a clinically significant manner. Recent advances in cancer research have provided new insights into the mechanisms of intrinsic resistance in melanomas. We have recently reported that the over-expression of tyrosinase-related protein 2 (TYRP2), an enzyme that is well characterized for its function in melanin synthesis, is associated specifically with resistance to DNA damaging drugs and radiation treatment. This review will summarize our findings as well as discuss the possible mechanisms by which TYRP2 over-expression contributes to intrinsic resistance in human malignant melanoma.
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To investigate the efficacy of traditional Chinese medicine (TCM) in the treatment of hyperpigmentation problems, extracts of herbs selected based on traditional Chinese medical literature were screened. Forty extracts were extracted from 10 selected herbs using hexane, dichloromethane, methanol and water. They were then screened using melan-a cells, an immortalized non-tumorigenic mouse melanocyte cell line. Sulphorhodamine B (SRB) assay and measurement of melanin production were performed to examine the effects of the extracts as well as some natural compounds from these herbs on melanogenesis in the melan-a cells. The hexane and dichloromethane extracts of Angelica sinensis exhibited strong hypopigmentary effects. Natural compounds occurring in this herb were also investigated. Among them 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol demonstrated positive effects in attenuating melanin synthesis in the cultured cells.
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Please cite this paper as: Antioxidative characteristics and inhibition of α-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Experimental Dermatology 2010; 19: 742–750. Abstract: The antioxidant activities of vanillin and vanillic acid isolated from Origanum vulgare are investigated. These compounds may serve as agents for antimelanogenesis. Vanillic acid is a stronger antioxidant than vanillin, in terms of free radical scavenging activity, reducing power and inhibition of lipid peroxidation. The inhibition of cellular reactive oxygen species (ROS) in H2O2-treated BNLCL2 cells by vanillic acid exceeds that of ascorbic acid (AA) or trolox. In B16F0 cells stimulated with α-melanocyte-stimulating hormone (α-MSH), vanillic acid reduced cellular tyrosinase activity, DOPA oxidase and melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins 2 (TRP-2) and TRP-1. Vanillin did not express inhibition of tyrosinase activity. These results supported that vanillic acid is a significantly stronger antioxidant than vanillin and exhibited stronger antimelanogenesis performance because of the structural presence of the carboxyl group.
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An immortal line of pigmented melanocytes, "melan-a", has been derived from normal epidermal melanoblasts from embryos of inbred C57BL mice. The conditions favouring proliferation of these cells largely resemble those for normal, non-established mouse melanoblasts and melanocytes, and include a low extracellular pH and the presence of a tumour promoter, tetradecanoyl phorbol acetate (TPA) or teleocidin. Melan-a cells have the diploid chromosome number and do not form tumours in syngeneic or nude mice. They are therefore the first known line of non-tumorigenic mouse melanocytes, although an aneuploid melanocyte line of untested tumorigenicity has been reported (Sato et al., 1985). Melan-a cells are syngeneic with the B16 melanoma and its sublines, and provide an excellent parallel non-tumorigenic line for studies of the cellular and molecular basis of melanoma malignancy.
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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.
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Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).
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Tyrosinase is the rate limiting enzyme critically associated with melanin synthesis. The melanosomes are specialized membrane-bound organelles within melanocytic cells in which melanin polymers are ultimately deposited. To determine whether tyrosinase correlates with the number of melanosomes, we examined the relationship between tyrosinase activity, tyrosinase mRNA levels, and the number of melanosomes in B16 murine melanoma cells, using melanogenesis regulatory agents. 12-O-Tetradecanoylphorbol-13-acetate (TPA) or linoleic acid decreased tyrosinase activity, while dibutyryl cyclic adenosine monophosphate (dbcAMP) or palmitic acid increased it. The tyrosinase mRNA levels were not always correlated with tyrosinase activity, i.e., TPA down-regulated, dbcAMP upregulated, while linoleic acid or palmitic acid did not alter the message levels, indicating that fatty acid regulation of melanogenesis was due to post-transcriptional events. The number of melanosomes changed when agents which modulate the tyrosinase gene expression were added, since TPA decreased, dbcAMP increased, and linoleic acid or palmitic acid did not alter their number. These results suggest that the number of melanosomes changed in relation to tyrosinase mRNA level but not to tyrosinase activity in response to melanogenesis regulatory agents.
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Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.
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A stopped spectrophotometric assay for the dopa oxidase activity of tyrosinase has been developed to enable large numbers of samples to be screened very rapidly. The assay measures the pink pigment formed by the reaction of Besthorn's hydrazone (3-methyl-2-benzothiazoninone hydrazone, or MBTH) with dopaquinone, the product of oxidation of L-dopa by tyrosinase. Addition of perchloric acid stops the reaction and precipitates protein, enabling turbid as well as non-turbid samples to be assayed. Stability of the pink product is enhanced in acid solution and the pigment has a sharp absorbance maximum at 505 nm such that it is easily measured spectrophotometrically. Using the stopped assay, tyrosinase is detectable only in mammalian cell lines expected to express the enzyme, and the specificity of the assay has also been confirmed using tyrosinase inhibitors. The stopped MBTH assay is approx. 15-times more sensitive than the widely used dopachrome assay and can reliably detect the formation of as little as 350 pmol of product.
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Variations in human pigmentation among different racial groups are due to differences in the production and deposition of melanin in the skin. Although melanin synthesis is known to be controlled by the rate-limiting enzyme tyrosinase, the role of this enzyme as the principal determinant of skin pigmentation is unclear. Results from studies with human melanocyte cultures derived from different racial skin types reveal an excellent correlation between the melanin content of melanocyte cultures and the in situ activity of tyrosinase. Melanocytes derived from black skin have up to 10 times more tyrosinase activity and produce up to 10 times more melanin than melanocytes derived from white skin. However, the higher level of tyrosinase activity in melanocytes derived from black skin is not due to a greater abundance of tyrosinase. Results from immunotitration experiments and Western immunoblots reveal that the number of tyrosinase molecules present in white-skin melanocytes may equal the number found in highly pigmented black skin types. Moreover, approximately equivalent levels of tyrosinase mRNA are present in white and black skin cell strains. In contrast, melanocytes derived from red-haired neonates with low tyrosinase activity contain low numbers of tyrosinase molecules and low levels of tyrosinase mRNA. These results show that tyrosinase activity and melanin production in most light-skinned people is controlled primarily by a post-translational regulation of pre-existing enzyme and not by regulating tyrosinase gene activity. In contrast, melanocytes from red-haired (type I) people have low levels of tyrosinase protein and mRNA, suggesting that transcriptional activity of the tyrosinase gene is suppressed.
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Varied effects of chemical or biological compounds on mammalian pigmentation have been reported by many groups, but to date, no standardized method has established necessary and/or optimal parameters for testing such agents. A standardized method has been developed to screen compounds with potential effects on pigmentation. The protocol comprises basic parameters to analyze melanogenic effects and allows for further characterization of candidate compounds, providing important insights into their mechanism of action. In this protocol (termed STOPR, for standardized testing of pigmentation regulators), compounds are initially screened using purified tyrosinase and are then tested on melanocytes in culture. After treatment of melanocytes with potentially bioactive compounds, cell proliferation and viability, total melanin accumulated, and melanogenic potential are measured. This protocol is an important first step in characterizing chemical regulation of effects on melanogenesis. When bioactive candidate compounds are identified, testing may proceed for pharmacological or otherwise commercial applications in coculture and/or organ culture models followed by in vivo testing. As an application of this method, results for compounds known to stimulate and/or inhibit melanogenesis (including arbutin, hydroquinone, kojic acid, melanocyte-stimulating hormone, and thymidine dimers) as well as some commercial skin whiteners are reported.
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During photoaging, the density of melanin chromatophores is heterogeneous in the epidermis. To define the patterns of pheomelanin-enriched melanotic hypermelanosis of the face in phototype II subjects and to assess the effect of depigmenting agents. Azelaic acid and glycolic acid were tested as well as a soy extract, reported to reduce pigmentation through interaction with the protease-activated receptor 2 (PAR-2) of keratinocytes. Evaluations were made by image analysis of high magnification pictures obtained by a video camera equipped with an internal ultraviolet-emitting unit (Visioscan((R))). Three patterns of subclinical facial hypermelanosis were recognized including the spotty perifollicular type, the accretive globular type and the elongated type of the sunny side of wrinkles. Azelaic acid and the soy extract led to significant skin lightening after a 3-week treatment. By contrast, glycolic acid showed an inconsistent effect. Sensitive fluorescence video recording combined with image analysis represents an advance in the noninvasive assessment of the mottled subclinical skin pigmentation. The depigmenting effect observed with the soy extract indicates that the inhibition of PAR-2 may be a novel way to approach certain pigmentary disorders of the skin.
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Treatment of human melanoma cells with the differentiation-inducing agent hexamethylene bisacetamide (HMBA) results in reciprocal changes in expression of melanocyte-specific genes tyrosinase-related proteins-1 and -2 (TYRP1 and TYRP2). In this study, we investigated the effects of HMBA on cultured neonatal human cutaneous melanocytes. Flow cytometric analysis showed that HMBA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent growth of melanocytes by reducing the population of cells entering the DNA synthesis phase of cell cycle. Melanocyte growth inhibition was accompanied by an increase in the number of cells exhibiting polydendritic morphology. This morphologic change was less pronounced when HMBA was added to melanocytes in the absence of TPA. Northern blot analyses of total cellular RNA showed that expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), TYRP1, Silver (SILV/Pmel17) gene was down-regulated by HMBA, while TYRP2 mRNA was up-regulated (> 10-fold). When the inducer was added to cells in the absence of TPA, there was > 50-fold increase in TYRP2 mRNA with a moderate increase in MITF, tyrosinase and SILV gene mRNAs and complete repression of TYRP1 gene. Studies using inhibitors for protein kinases involved in cell signaling pathways suggested that stress-activated kinase p38 and mitogen-activated protein kinase kinase MEK are involved in TPA-independent regulation of TYRP2 expression in melanocytes. These data show that treatment of proliferating melanocytes with the differentiation inducer HMBA results in a distinct change in morphology and up-regulation of TYRP2, while quiescent melanocytes respond by a dramatic increase in expression of TYRP2 without change in morphology. These results suggest an inverse relationship of TYRP2 gene regulatory mechanisms to melanocyte growth regulatory pathways.
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Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte-keratinocyte co-cultures, melanocyte-keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (alpha-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.
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Eight new isoprenylated xanthones, cudratricusxanthones A-H (1-8), were isolated from the roots of Cudrania tricuspidata, together with ten known compounds, cudraxanthones H (9) and M (10), xanthone V(1a) (11), toxyloxanthone C (12), macluraxanthone B (13), 1-hydroxy-3, 6, 7-trimethoxyxanthone (14), cycloartocarpesin (15), artocarpesin (16), cudraflavone B (17), and kaempferol (18). Their structures were characterized by spectroscopic methods. Xanthones 5, 7, 10, and 12 showed inhibitory effects on four kinds of human digestive apparatus tumor cell lines (HCT-116, SMMC-7721, SGC-7901, and BGC-823) with IC(50) values of 1.6-11.8 microg/mL. Xanthones 2, 4, and 11 displayed significant cytotoxicity against HCT-116, SMMC-7721, and SGC-7901 (IC(50)=1.3-9.8 microg/mL). Flavonoids 15-17 were almost inactive.
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Microphthalmia-associated transcription factor (MITF) acts as a master regulator of melanocyte development, function and survival by modulating various differentiation and cell-cycle progression genes. It has been demonstrated that MITF is an amplified oncogene in a fraction of human melanomas and that it also has an oncogenic role in human clear cell sarcoma. However, MITF also modulates the state of melanocyte differentiation. Several closely related transcription factors also function as translocated oncogenes in various human malignancies. These data place MITF between instructing melanocytes towards terminal differentiation and/or pigmentation and, alternatively, promoting malignant behavior. In this review, we survey the roles of MITF as a master lineage regulator in melanocyte development and its emerging activities in malignancy. Understanding the molecular function of MITF and its associated pathways will hopefully shed light on strategies for improving therapeutic approaches for these diseases.
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The catecholic xanthones and flavonoids 1-13 were isolated from the root bark of Cudrania tricuspidata. Compounds 1 and 3-8 exhibited significant antioxidant activity against low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance (TBARS) assay. Among them, prenylated flavonoids 10-12 showed an inhibitory effect on the NO production and iNOS expression in RAW264.7 cells. Also, compounds 1, 2, 5, 7, 9, and 11 preferentially inhibited hACAT-2 than hACAT-1, whereas compounds 3, 4, 6, and 8 showed a similar specificity against hACAT-1 and -2. However, flavonoids 10, 12, and 13 dominantly inhibited hACAT-2, not hACAT-1.
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An overview of agents causing hypopigmentation in human skin is presented. The review is organized to put forward groups of biological and chemical agents. Their mechanisms of action cover (i) tyrosinase inhibition, maturation and enhancement of its degradation; (ii) Mitf inhibition; (iii) downregulation of MC1R activity; (iv) interference with melanosome maturation and transfer; (v) melanocyte loss, desquamation and chemical peeling. Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as this enzyme catalyses the rate-limiting step of pigmentation. Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce effects in clinical trials. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their efficacy and safety. Successful treatments need the combination of two or more agents acting on different mechanisms to achieve a synergistic effect. In addition to tyrosinase inhibition, other parameters related to cytotoxicity, solubility, cutaneous absorption, penetration and stability of the agents should be considered. The screening test system is also very important as keratinocytes play an active role in modulating melanogenesis within melanocytes. Mammalian skin or at least keratinocytes/melanocytes co-cultures should be preferred rather than pure melanocyte cultures or soluble tyrosinase.
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Melanocytes are phenotypically prominent but histologically inconspicuous skin cells. They are responsible for the pigmentation of skin and hair, and thereby contribute to the appearance of skin and provide protection from damage by ultraviolet radiation. Pigmentation mutants in various species are highly informative about basic genetic and developmental pathways, and provide important clues to the processes of photoprotection, cancer predisposition and even human evolution. Skin is the most common site of cancer in humans. Continued understanding of melanocyte contributions to skin biology will hopefully provide new opportunities for the prevention and treatment of skin diseases.
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Antibrowning activities of Morus alba L. twig extracts, oxyresveratrol, and mulberroside A isolated from mulberry twig on cloudy apple juices and fresh-cut apple slices were evaluated by monitoring the change of a* value, total color difference (DeltaE), and visual observation. It was found, similar to 4-hexylresorcinol, that oxyresveratrol could effectively inhibit browning in cloudy apple juices at a concentration as low as 0.01% and that mulberry twig extract also showed remarkable antibrowning effects on cloudy apple juices. However, for fresh-cut apple slices, mulberry twig extract and oxyresveratrol needed to be used in combination at least with ascorbic acid to exhibit their antibrowning effects. Apple slice samples treated by dipping in a solution containing 0.001 M oxyresveratrol, 0.5 M isoascorbic acid, 0.05 M calcium chloride, and 0.025 M acetylcysteine did not undergo any substantial browning reaction for 28 days at 4 degrees C. However mulberroside A did not show antibrowning effects on cloudy apple juices although it is also a good mushroom tyrosinase inhibitor.
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We have proven that xanthones 1-8 isolated from the root of C. tricuspidata possess highly potent alphaalpha-glucosidase inhibition properties. Compound 1 was identified as a new isoprenylated tetrahydroxy xanthone, 1,3,6,7-tetrahydroxy-2-(3-methylbut-2-enyl)-8-(2-methylbut-3-en-2-yl)-9H-xanthen-9-one (1). These are the first natural xanthones documented to exhibit such inhibition. The IC(50) values of compounds 1-8 inhibiting alpha-glucosidase activity were determined to be up to 16.2microM. Mechanistic analysis showed the xanthones 1-8 exhibited full mixed inhibition.
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Skin colour typology depends on the amount and location of its chromophores. Among them, eumelanins derived from 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), and phaeomelanins are of utmost importance. These biomolecules result from the multi-step enzymatic and non-enzymatic conversion of tyrosine into melanins. Pigmentation disorders are multiple and depend on alterations in the density in active melanocytes, and on specific abnormalities of any of the complex melanogenesis mechanisms. This review presents some of the main skin-lightening agents with respect to their mechanisms of action and side-effects. Some of the novel compounds may lead to new perspectives in the fields of dermatology and cosmetology. The methods commonly used to assess efficacy of skin-lightening products rely on in vitro models including cell-free enzymatic assays, melanocyte cultures and reconstructed epidermis bioassays. Animal models have little relevance. By contrast, human testing with the support of instrumental evaluations is the most informative.
Hypopigmenting agents: An updated review on biological, chemical and clinical aspects
  • F Solano
  • S Briganti
  • M Picardo
  • G Ghanem
Solano, F., Briganti, S., Picardo, M., & Ghanem, G. (2006). Hypopigmenting agents: An updated review on biological, chemical and clinical aspects. Pigment Cell Research, 19(6), 550571.