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Tyrosinase-based TLC Autography for anti-melanogenic drug screening.

ABSTRACT: 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??g, 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).

SUBMITTER: Hsu KD

PROVIDER: S-EPMC5762723 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Tyrosinase-based TLC Autography for anti-melanogenic drug screening.

Hsu Kai-Di KD Chan Yu-Hin YH Chen Hong-Jhang HJ Lin Shi-Ping SP Cheng Kuan-Chen KC

Scientific reports 20180110 1

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 a ...[more]

PMID: 29321636

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Project description:BACKGROUND:Aspergillus fumigatus, an opportunistic fungal pathogen is associated with a wide array of diseases. It produces 1, 8-dihydroxy naphthalene (DHN) melanin that imparts greenish grey color to conidia and is an important virulence factor. It masks various molecular patterns associated with A. fumigatus and protects the fungus from host immune system. Myristica fragrans, enriched with secondary metabolites has been traditionally used for the treatment of infectious and inflammatory diseases. The present study was aimed to explore the anti-melanogenic effect of M. fragrans extracts on A. fumigatus. METHODS:M. fragrans extracts (hexane, chloroform, methanol and ethanol) were prepared through polarity guided extraction. Phytochemical analysis was performed to detect the chemical constituents of the extracts. The minimum effective concentration (MEC) of the extracts against A. fumigatus melanin was determined by broth micro-dilution assay. Various virulence factors were assayed by spectrophotometric methods. Electron microscopic studies were performed to evaluate the effect of the hexane extract of M. fragrans on A. fumigatus cell surface morphology. The major active compounds of the extract were detected by gas chromatography-mass spectrometry (GC-MS). Docking was performed to study the interaction between the major identified compounds and the ketosynthase domain of polyketide synthase protein. RESULTS:The results indicated that the hexane extract of M. fragrans inhibited melanin production (76.09%), reduced ergosterol content (83.63%) and hydrophobicity of the cell (72.2%) at the MEC of 0.078?mg/mL. Altered conidial surface, disappearance of protrusions and absence of melanin layer on outer cell surface was observed in electron microscopy. Forty-two compounds were identified by GC-MS. The main constituents were identified as sabinene (12.2%), linoleic acid (11.7%), hexadecanoic acid (10.5%), safrole (8.1%) and elemicin (7.8%). Docking studies revealed that hexadecanoic acid, its derivative compound cis-9-hexadecenal and isoeugenol have lower binding energy forming proper hydrogen bond with ketosynthase domain of polyketide synthase protein. CONCLUSION:The study concludes that the extract of M. fragrans has potential antifungal properties that can be explored in combination with available antifungals. This combination approach may be helpful for large number of patients suffering with A. fumigatus infections.

Dataset Information

Tyrosinase-based TLC Autography for anti-melanogenic drug screening.

Publications

Tyrosinase-based TLC Autography for anti-melanogenic drug screening.

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