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

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.
Animals ; Oryzias/genetics* ; Gene Editing/methods* ; CRISPR-Cas Systems ; Codon ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Monophenol Monooxygenase/genetics* ; CRISPR-Associated Protein 9/genetics* ; RNA, Transfer/genetics* ; Staphylococcus aureus/genetics* ; PAX6 Transcription Factor/genetics*

Twelve flavonoids were isolated and purified from the ethyl acetate fraction of 95% ethanol extract of Dalbergia odorifera by heat reflux extraction, solvent extraction, recrystallization, normal phase silica gel, Sephadex LH-20, MCI gel and HPLC methods. The structures were identified with multiple spectroscopic methods, including 1 D-NMR, 2 D-NMR and MS. The compounds were identified as 6,7,8-trimethoxy-5,4'-dihydroxy isoflavone(1), medicarpin(2), 7,2'-dihydroxy-4'-methoxy-isoflavanol(3), biochanin A(4), prunetin(5), genistein(6), pratensein(7), 3-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-4H-chromen-4-one(8), tectorigenin(9), irisolidone(10), vestitol(11), and formononetin(12). Compound 1 was a new isoflavone, and compound 8 was isolated from D. odorifera for the first time. The results showed that compounds 1-3 had inhibitory effects on tyrosinase, with inhibition rates of 35.58%, 38.63% and 51.34% at the concentration of 1.0 mmol·L~(-1), respectively.
Dalbergia/chemistry* ; Ethanol ; Flavonoids/chemistry* ; Genistein ; Isoflavones/pharmacology* ; Monophenol Monooxygenase ; Plant Extracts/pharmacology* ; Silica Gel ; Solvents

OBJECTIVE: To identify the causative variants in 13 Chinese pedigrees affected with oculocutaneous albinism (OCA) so as to provide genetic counseling and prenatal diagnosis to them. METHODS: Thirteen unrelated pedigrees with clinically diagnosed OCA were collected and classified based on the manifestation of skin and eyes. With informed consent obtained from the participants, peripheral blood samples were collected from the probands and their family members for the extraction of genomic DNA. Candidate variants were screened by targeted capture and next generation sequencing, and the results were validated by Sanger sequencing. Prenatal diagnosis was provided to the families upon their subsequent pregnancies. RESULTS: Causative variants were detected in all probands, including 10 with compound heterozygotes or homozygotes for TYR gene variants and 3 with compound heterozygotes for OCA2 gene variants. Among these, two variants [TYR: c.650G>C (p.Arg217Pro) and OCA2: c.516-2A>T] were unreported previously. The pathogenicity of the novel TYR: c.650G>C (p.Arg217Pro) variant was verified through bioinformatic analysis and prediction of three dimensional structure of the protein. Prenatal diagnosis was provided to 6 fetuses with a high risk for OCA. Four fetuses were found to be carriers, one did not carry the variants of the proband, and one was affected with OCA. CONCLUSION Identification of the pathogenic variants in the 13 probands, including 2 novel ones, has expanded the mutational spectrum of OCA and enabled genetic counseling and prenatal diagnosis for the families.
Albinism, Oculocutaneous/genetics* ; China ; Female ; Genetic Testing ; Humans ; Membrane Transport Proteins/genetics* ; Monophenol Monooxygenase/genetics* ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis

Sophorae Flavescentis Radix,derived from the root of Sophora flavescens in the Leguminosae family,has been widely used in the medicine,agriculture,animal husbandry,and daily chemical industry. A pharmacophore model-based method for rapid discovery of tyrosinase inhibitors( TIs) from S. flavescens was established by molecular docking under Lipinski rules,and verified by enzyme assays. Briefly,the chemical constituent database of S. flavescens( CDSF) was established based on the previous papers. Theoptimal pharmacophore model( OPM) was constructed by DS 2019 on the basis of known active TIs. Eighty-three hits predominated by flavonoids having higher fitting scores with OPM than the positive control were screened out,and subjected to molecular docking based on the three-dimensional structure of tyrosinase crystal protein. The potential TIs such as kurarinone and nor-kurarinone were rapidly discovered from the compounds with higher docking scores than the positive control under the Lipinski rules. The results were verified by the in vitro enzyme assays. The inhibition activities of tyrosinase from non-medicinal parts of S. flavescens were also tested to explore the relationship between the inhibition activity and chemical compositions. This study is expected to provide data support for the comprehensive application and development of S. flavescens and also a new method for the rapid discovery of active substances or functional constituents in the complex systems.
Animals ; Flavonoids ; Molecular Docking Simulation ; Monophenol Monooxygenase ; Plant Extracts/pharmacology* ; Plant Roots ; Sophora

Melanin is an important factor affecting human skin color. This study defines its synthetic pathways and regulatory pathways have the practical significance for the treatment of diseases caused by pigmentation problems, such as chloasma. Besides, it also provides a theoretical basis for screening out melanin inhibitors and developing whitening and freckle products. At present, the melanin inhibitors used in whitening and freckle products mainly come from natural products and traditional Chinese medicine. This article first introduces the melanin biosynthesis pathway with tyrosinase as the core, defines the synthesis, transport and catalytic activity of tyrosinase as the three key factors affecting melanin synthesis, and then reviews two common types of melanin inhibitors, namely tyrosinase synthesis inhibitors and tyrosinase inhibitors derived from natural products and traditional Chinese medicine. This article provides guidance for the development of new melanin inhibitors, and puts forward the idea for combining and synergizing inhibitors according to different mechanisms, in order to develop new whitening formulas.
Biological Products ; Enzyme Inhibitors ; Humans ; Medicine, Chinese Traditional ; Melanins ; Monophenol Monooxygenase

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

PURPOSE: Barely sprout is a well-known oriental herbal medicine with a wide range of health benefits. Recent studies have provided scientific evidence of its therapeutic effects with expanded application. This study investigated anti-melanogenic effect of barley sprout water extract (BSE) in murine melanocyte B16F10. METHODS: Various concentrations (0, 50, 125, and 250 µg/mL) of BSE and arbutin (150 ppm) were applied to B16F10 stimulated with or without alpha-melanocyte stimulating hormone (100 nM) for 72 hours. The whitening potency of BSE was determined altered cellular melanin contents. Activity and expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were also assayed. RESULTS: Experimental results revealed that treatment with BSE reduced cellular melanin production by approximately 40% compared to the control. Molecular findings supported that suppressed activity and expression of tyrosinase and MITF proteins by BSE were associated with declined cellular melanogenesis. Furthermore, anti-melanogenic effect of BSE (250 µg/mL) was similar to that of arbutin, a commonly used whitening agent. Lastly, polyphenols including p-coumaric, ferulic, and vanillic acids were identified in BSE using HPLC analyses. They might be potential active ingredients showing such melanogenesis-reducing effect. CONCLUSION: BSE was evident to possess favorable anti-melanogenic potency in an in vitro model. As a natural food sourced material, BSE could be an effective depigmentation agent with potential application in pharmaceutical and cosmetic industries.
Arbutin ; Chromatography, High Pressure Liquid ; Herbal Medicine ; Hordeum ; In Vitro Techniques ; Insurance Benefits ; Melanins ; Melanocytes ; Melanoma ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Polyphenols ; Therapeutic Uses ; Vanillic Acid ; Water