OBJECTIVE: To explore the inhibitory effects of Nardostachys Jatamansi DC. volatile oil (NJVO) on psychological factors substance P (SP)/cortisol (CORT)-induced hyperpigmentation. METHODS: The model of psychologically-induced hyperpigmentation of B16F10 cells was created using SP (10 nmol/L) + CORT (10 µmol/L) for 72 h. The levels of melanin content, tyrosinase (TYR) activity using NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation methods were assessed, respectively. The effect of NJVO on SP/CORT-induced normal human skin tissue pigmentation was detected by Masson staining. Protein expressions of tyrosinase-related protein 1 (TRP-1), tyrosinase-relative protein 2 (DCT), and microphthalmia-associated transcription factor were determined using Western blot. The melanosome number, maturation, and melanosomal structure changes were detected through transmission electron microscopy and immunofluorescence experiments. In vivo, zebrafish pigment content was evaluated in SP/CORT-induced zebrafish hyperpigmentation model. RESULTS: NJVO significantly reduced the melanin content (P<0.01) and inhibited tyrosinase activity (P<0.01), the pigmentation of the normal skin tissue in the NJVO group was significantly lower than that in the SP/CORT group (P<0.05). And NJVO considerably downregulated expressions of melanogenesis-related proteins (TYR, TRP-1, DCT) in cells (P<0.01). In addition, the number of melanosomes was decreased and the dentrites formation of B16F10 cells was inhibited after NJVO treatment (P<0.01). In vivo, NJVO significantly reduced the pigment content in the zebrafish body (P<0.01). CONCLUSION NJVO effectively reversed SP/CORT-induced hyperpigmentation by suppressing the activity and expression of TYR and TRPs and inhibiting melanosome maturation in mouse B16F10 melanoma cells.
Animals ; Hyperpigmentation/psychology* ; Zebrafish ; Oils, Volatile/therapeutic use* ; Melanins/metabolism* ; Humans ; Monophenol Monooxygenase/metabolism* ; Mice ; Nardostachys/chemistry* ; Substance P ; Hydrocortisone ; Skin Pigmentation/drug effects* ; Cell Line, Tumor ; Melanosomes/ultrastructure* ; Microphthalmia-Associated Transcription Factor/metabolism* ; Melanoma, Experimental ; Oxidoreductases/metabolism* ; Intramolecular Oxidoreductases/metabolism*

Nigella sativa L. seeds have been traditionally utilized in Chinese folk medicine for centuries to treat vitiligo. This study revealed that the ethanolic extract of Nigella sativa L. (HZC) enhances melanogenesis and mitigates oxidative stress-induced cellular senescence and dysfunction in melanocytes. In accordance with established protocols, the ethanol fraction from Nigella sativa L. seeds was extracted, concentrated, and lyophilized to evaluate its herbal effects via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, tyrosinase activity evaluation, measurement of cellular melanin contents, scratch assays, senescence-associated β-galactosidase (SA-β-gal) staining, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis for expression profiling of experimentally relevant proteins. The results indicated that HZC significantly enhanced tyrosinase activity and melanin content while notably increasing the protein expression levels of Tyr, Mitf, and gp100 in B16F10 cells. Furthermore, HZC effectively mitigated oxidative stress-induced cellular senescence, improved melanocyte condition, and rectified various functional impairments associated with melanocyte dysfunction. These findings suggest that HZC increases melanin synthesis in melanocytes through the activation of the MAPK, PKA, and Wnt signaling pathways. In addition, HZC attenuates oxidative damage induced by H₂O₂ therapy by activating the nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway and enhancing the activity of downstream antioxidant enzymes, thus preventing premature senescence and dysfunction in melanocytes.
Oxidative Stress/drug effects* ; Melanocytes/cytology* ; Cellular Senescence/drug effects* ; Nigella sativa/chemistry* ; Plant Extracts/pharmacology* ; Seeds/chemistry* ; Mice ; Animals ; Melanins/metabolism* ; Monophenol Monooxygenase/metabolism* ; Humans

Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1β (IL-1β) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.
Animal Fur ; Animals ; Color ; Female ; Melanins ; Melanocytes ; enzymology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Monophenol Monooxygenase ; metabolism ; Pigmentation ; Skin ; physiopathology ; Stress, Physiological

Zinc oxide quantum dots (ZnO QDs) were synthesized by gel-sol method and employed as the transdermal aloesin (Alo) carriers. ZnO QDs were surface-functionalized with amino using aminopropyltriethoxysilane (APTES). Alo was covalently bonded on the surface of ZnO QDs via N,N'-carbonyldiimidazole to obtain Alo NPs, which were characterized by transmission electron microscope (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and thermal gravimetric analyzer (TGA). TEM images showed that ZnO QDs were analogously sphere and monodisperse with a reasonably narrow size distribution, of which was around 4 nm. The size of Alo NPs increased to around 8 nm due to the surface modification. The intense bands at around 3 400 cm and 1 200 cm in the FTIR spectrum of Alo NPs from the vibration of -OH indicated the linkage of Alo on the surface of ZnO QDs. The results of TGA analysis showed that the mass ratio of ZnO QDs and Alo were 39.27% and 35.14%, respectively. The penetration of Alo NPs was much higher than raw Alo according to the passive penetration experiments with Franz-type diffusion cells instrument using full-thickness cavy skin, which manifested the improvement of the penetration for Alo delivered by ZnO QDs. The pH-controlled drug release behavior was investigated. At pH 7.4, only a small amount of Alo (1.45% ± 0.21%) had been released after 2 h. In contrast, as incubation at pH 5.0 of which pH was similar to endosomal environment, Alo was released very fast (87.63% ± 0.46% in 2 h) from Alo NPs, confirming that Alo NPs could response to the pH and realize the intracellular drug release. The inhibitory effect of Alo NPs on tyrosinase was in a dose dependent manner. When the concentration of Alo NPs was 12.5 μg/mL, the inhibition rate was up to 40.32% ± 1.57%. All the results show that the Alo NPs hold a great potential in transdermal tyrosinase inhibition.
Administration, Cutaneous ; Animals ; Chromones ; administration & dosage ; Drug Delivery Systems ; Glucosides ; administration & dosage ; Guinea Pigs ; Monophenol Monooxygenase ; metabolism ; Nanoparticles ; Quantum Dots ; Zinc Oxide

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.
Alkaloids ; chemistry ; pharmacology ; Animals ; Benzodioxoles ; chemistry ; pharmacology ; Benzyl Compounds ; chemistry ; pharmacology ; Cholecalciferol ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Humans ; Kaempferols ; chemistry ; pharmacology ; Melanins ; genetics ; metabolism ; Monophenol Monooxygenase ; genetics ; metabolism ; Pigmentation ; drug effects ; Piperidines ; chemistry ; pharmacology ; Polyunsaturated Alkamides ; chemistry ; pharmacology ; Purines ; chemistry ; pharmacology ; Scopoletin ; chemistry ; pharmacology ; Vitiligo ; drug therapy ; enzymology ; metabolism ; Zebrafish

OBJECTIVETo confirm the inhibitory effect of Chinese herbs of acid taste on melanin synthesis.

METHODSActive ingredients of 22 kinds Chinese herbs of acid tastes were extracted by alkali extraction and acid precipitation, alcohol extraction, and water extraction, respectively, which was then dispensed into 25.00, 12.50, and 6.25 g/L suspension. Their effects on activities of tyrosinase were detected using mushroom-tyrosinase-DOPA speed oxidation. Their inhibition rates on activities of tyrosinase were respectively compared with inhibition rates of 1.0, 0.5, and 0.1 mmol/L arbutin.

RESULTSThe 22 kinds Chinese herbs of acid taste included Cornus Officinalis, Crataegus pinnatifida, dark plum fruit, Schisandra Chinensis, Chaenomeles sinensis Koehne, Reynoutria japonica Houtt, Achyranthes Bidentata, Sanguisorba officinalis L., Semen Ziziphi Spinosae, Herba Ecliptae, blueberry, immature bitter orange, submature bitter orange, Prunus mume Var, Hovenia acerba Lindl., Fructus Mori, Pomegranate Rind, white paeony root, Rosa laevigata Michx., Portulaca oleracea L, Terminalia chebula Retz, Rhus chinensis Mill. Their alkaline extractions showed inhibition to activities of tyrosinase to different degrees except Herba Ecliptae. Of them, the highest inhibition rate (88.49% ± 9.98%) was got by dark plum fruit at 25 g/L, while the lowest inhibition rate (11.22% ± 3.36%) was got by immature bitter orange at 6.25 g/L. Their alcohol extractions showed inhibition to activities of tyrosinase to different degrees except Herba Ecliptae. Of them, the highest inhibition rate (75.92% ± 5.57%) was got by Hovenia acerba Lindl. at 25 g/L, while the lowest inhibition rate (9.60% ± 1.15%) was got by submature bitter orange at 6.25 g/L. Their water extractions all had inhibition on activities of tyrosinase. Of them, the highest inhibition rate (54.23% ± 3.56%) was got by Fructus Mori at 25 g/L, while the lowest inhibition rate (10.25% ± 1.83%) was got by Semen Ziziphi Spinosae at 6.25 g/L. Compared with 1 mmol/L arbutin water solution, alkaline extractions of dark plum fruit, Schisandra Chinensis, Rhus chinensis Mill., Rosa laevigata Michx., blueberry, Chaenomeles sinensis Koehne, Portulaca oleracea L, Fructus Mori, Achyranthes Bidentata, Pomegranate Rind; alcohol extractions of dark plum fruit, Rhus chinensis Mill., Pomegranate Rind, Hovenia acerba Lindl., Crataegus pinnatifida, Achyranthes Bidentata; water extractions of Chaenomeles sinensis Koehne, blueberry, and Fructus Mori at 25 g/L got obviously higher inhibition rates (P < 0.05, P < 0.01). Compared with 0.5 mmol/L arbutin water solution, alcohol extraction of Chaenomeles sinensis Koehne and alcohol extraction of dark plum fruit at 12.5 g/L got obviously higher inhibition rates (P < 0.05, P < 0.01).

CONCLUSIONChinese herbs of acid taste could inhibit melanin synthesis, and its mechanism was related to inhibiting activities of tyrosinase.

Drugs, Chinese Herbal ; pharmacology ; Humans ; Melanins ; metabolism ; Monophenol Monooxygenase ; metabolism ; Oxidation-Reduction ; Schisandra ; Taste

OBJECTIVETo study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome.

METHODSEukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays.

RESULTSExpression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus.

CONCLUSIONThis study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

Amino Acid Sequence ; Animals ; Cell Line, Tumor ; Genetic Predisposition to Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luciferases ; genetics ; metabolism ; Mice ; Microphthalmia-Associated Transcription Factor ; genetics ; metabolism ; Microscopy, Confocal ; Monophenol Monooxygenase ; genetics ; metabolism ; Mutation ; NIH 3T3 Cells ; Nuclear Localization Signals ; genetics ; Transcriptional Activation ; Transfection ; Waardenburg Syndrome ; diagnosis ; genetics ; metabolism

Tea contains polyphenols and is one of the most popular beverages consumed worldwide. Because most tyrosinase inhibitors that regulate melanogenesis are phenol/catechol derivatives, this study investigated the inhibitory effects of Camellia sinensis water extracts (CSWEs), including black tea, green tea, and white tea extracts, on melanogenesis using immortalized melanocytes. CSWEs inhibited melanin accumulation and melanin synthesis along with tyrosinase activity in a concentration-dependent manner. These inhibitory effects were superior to those of arbutin, a well-known depigmenting agent. The anti-melanogenic activity of black (fermented) tea was higher than that of a predominant tea catecholamine, epigallocatechin gallate. CSWEs, especially black tea extract, decreased tyrosinase protein levels in a concentration-dependent manner. These results suggest that the anti-melanogenic effect of CSWEs is mediated by a decrease in both tyrosinase activity and protein expression, and may be augmented by fermentation. Thus, CSWEs could be useful skin-whitening agents in the cosmetic industry.
Animals ; Catechin/analogs & derivatives/metabolism ; Cell Line ; Melanins/*metabolism ; Melanocytes/enzymology/*metabolism ; Mice ; Monophenol Monooxygenase/*metabolism ; Plant Extracts/*pharmacology ; Plant Leaves/chemistry ; Tea/*chemistry

The anti-melanogenesis effect of glyceollins was examined by melanin synthesis, tyrosinase activity assay in zebrafish embryos and in B16F10 melanoma cells. When developing zebrafish embryos were treated with glyceollins, pigmentation of the embryos, melanin synthesis and tyrosinase activity were all decreased compared with control zebrafish embryos. In situ expression of a pigment cell-specific gene, Sox10, was dramatically decreased by glyceollin treatment in the neural tubes of the trunk region of the embryos. Stem cell factor (SCF)/c-kit signaling pathways as well as expression of microphthalmia-associated transcription factor (MITF) were determined by western blot analysis. Glyceollins inhibited melanin synthesis, as well as the expression and activity of tyrosinase induced by SCF, in a dose-dependent manner in B16F10 melanoma cells. Pretreatment of B16F10 cells with glyceollins dose-dependently inhibited SCF-induced c-kit and Akt phosphorylation. Glyceollins significantly impaired the expression and activity of MITF. An additional inhibitory function of glyceollins was to effectively downregulate intracellular cyclic AMP levels stimulated by SCF in B16F10 cells. Glyceollins have a depigmentation/whitening activity in vitro and in vivo, and that this effect may be due to the inhibition of SCF-induced c-kit and tyrosinase activity through the blockade of downstream signaling pathway.
Animals ; Embryo, Nonmammalian/drug effects ; Melanins/*biosynthesis ; Melanoma, Experimental/metabolism/pathology ; Mice ; Monophenol Monooxygenase/metabolism ; Phosphorylation/drug effects ; Pigmentation/drug effects ; Proto-Oncogene Proteins c-kit/*metabolism ; Pterocarpans/chemistry/*pharmacology ; SOXE Transcription Factors/metabolism ; Sesquiterpenes/chemistry/*pharmacology ; Signal Transduction/*drug effects ; Soybeans/*chemistry ; Stem Cell Factor/*pharmacology ; Zebrafish/embryology/metabolism

Tyrosinase is a key enzyme related to skin pigmentation disorders of elderly people, while self-aggregation of the amyloid-beta peptide, Abeta42, has been considered as a key event in the pathogenesis of Alzheimer's disease (AD). The present study was undertaken to investigate the inhibitory effects of 20 samples from Rhodiola species on tyrosinase and Abeta42 aggregation, and to isolate their corresponding bioactive components. The results demonstrated that the oligomeric proanthocyanidins (OPCs) commonly found in Rhodiola species were the major bioactive components corresponding to their anti-tyrosinase and anti-Abeta42 aggregation bioactivities. Salidroside, a representative compound of Rhodiola plants, proved not to be active in the present studies.
Amyloid beta-Peptides ; antagonists & inhibitors ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Glucosides ; isolation & purification ; pharmacology ; Monophenol Monooxygenase ; antagonists & inhibitors ; metabolism ; Peptide Fragments ; antagonists & inhibitors ; metabolism ; Phenols ; isolation & purification ; pharmacology ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Proanthocyanidins ; isolation & purification ; pharmacology ; Rhodiola ; chemistry