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    Bee pollen peptides as potent tyrosinase inhibitors with anti-melanogenesis effects in murine b16f10 melanoma cells and zebrafish embryos
    (2024-12-01)
    Sangtanoo, Papassara
    ;
    Srimongkol, Piroonporn
    ;
    Saisavoey, Tanatorn
    ;
    Puthong, Songchan
    ;
    Buakeaw, Anumart
    One important functional food ingredient today, valued for its health properties and ability to prevent disease, is bee pollen, which comprises a combination of nectar, pollen from plants, and the secretions of bees. In this research, the tyrosinase (TYR) inhibiting abilities of the peptides derived from bee pollen protein hydrolysates are investigated. Various proteases were utilized to generate these peptides, followed by testing at different concentrations. Tyrosinase inhibition activity was detected in all cases, while the hydrolysate drawn from 5.0% w/v neutrase exhibited the best IC<inf>50</inf> value and was thus investigated further via ultrafiltration to separate the active fractions. The highest potential for tyrosinase inhibition was recorded for the fractions below 0.65 kDa. Subsequent purification steps via SEC and RP-HPLC led to the identification of the VDGYPAAGY (named VY-9) peptide via LC-Q-TOF-MS/MS in fraction F<inf>1–2</inf>, known for its non-toxic and hydrophobic characteristics albeit poor water solubility. The synthesized VY-9 peptide demonstrated competitive inhibition, with IC<inf>50</inf> values of 0.55 ± 0.03 µM for mono-phenolase and 2.54 ± 0.06 µM for di-phenolase activities, as confirmed by molecular docking analysis revealing dominant hydrogen bond interactions with TYR. Effective concentrations of 0.2–1.6 µM of VY-9 showed negligible cytotoxicity in B16F10 cells. Melanin synthesis suppression was examined via qRT-PCR, and western blot in MITF, TYR, TRP-1, and TRP-2. Cell death in zebrafish embryos was evaluated in vivo using a toxicity assay which revealed no significant influence from VY-9, while anti-melanogenic effects were observed when the concentration was 4 µM, suggesting bee pollen-derived peptides’ potential in cosmetic and pharmaceutical depigmentation applications.
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    Synechococcus marine microalgae peptide: Melanogenesis inhibition in cellular and zebrafish models
    (2024-08-01)
    Srimongkol, Piroonporn
    ;
    Sangtanoo, Papassara
    ;
    Saisavoey, Tanatorn
    ;
    Puthong, Songchan
    ;
    Buakeaw, Anumart
    In this study, the AILQSYSAGKTK (named AK-12) peptide, also known as AK-12, from the Synechococcus marine microalgae cell extract is examined to determine the mechanism by which tyrosinase inhibition takes place. According to the docking simulation, it is expected that the peptides bind and interact at the tyrosinase active site, with the potential to support the inhibition of tyrosinase. For testing, the required peptides were first synthesized, before the results revealed tyrosinase inhibitory activity for which the respective IC<inf>50</inf> values for mono- and di-phenolase activities were 489.71 ± 0.01 μM, and 765.57 ± 0.01 μM. The characteristics of the different competitive types were shown in a Lineweaver-Burk plot. For the treatment of B16F10 cells, peptide concentrations of 50–400 μM were selected, confirming the absence of cytotoxicity. When the peptide was introduced, both tyrosinase activity and the production of melanin were significantly inhibited. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was then used to assess the suppression of melanin synthesis in MITF (microphthalmia-associated transcription factor, TYR (tyrosinase), TRP-1 (tyrosinase-related protein-1) and TRP-2 (tyrosinase-related protein-2). An in vivo toxicity assay was also carried out to evaluate zebrafish embryo cell death, revealing that AK-12 did not significantly affect cell death, while strong anti-melanogenic activity was observed for the concentration of 50 μM. From these findings it could be concluded that the peptide in question may offer potential in future hyperpigmentation treatments.
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    Zebrafish prss59.1 is involved in chorion development
    (2024-04-01)
    Rezanujjaman, Md
    ;
    Pachoensuk, Theeranukul
    ;
    Forhad Hossain, Md
    ;
    Maisum Sarwar Jyoti, Md
    ;
    Rubel Rana, Md
    The prss59.1 gene was identified as one of 11 genes that were highly upregulated during the induction of ovulation in zebrafish by using an in vivo ovulation assay. Previously, we conducted biochemical characterization of Prss59.1 and revealed it to be a trypsin-like proteolytic enzyme. In this study, we established a prss59.1 gene knockout strain using the CRISPR/Cas9 system. Phenotypic analysis of prss59.1 knockout fish showed that prss59.1 is associated with chorion elevation, a prominent event in egg activation during fertilization. The chorions of heterozygous and homozygous prss59.1 mutant zebrafish were smaller than those of the wild type. The results suggested that Prss59.1 is necessary for chorion expansion. The homozygous prss59.1 mutant strain, with a small chorion, showed an extremely low survival rate. Fiber-supported knob-like structures (KS) on the chorion showed an abnormal structure in prss59.1 mutants. Prss59.1 was detected in the KS on the chorion. The pores on the chorion were smaller in the prss59.1 mutants than in the wild type. Transmission electron microscopy (TEM) observations of the cross sections of the chorions showed abnormalities in the chorion structure in prss59.1 mutants. These results demonstrated that Prss59.1 is involved in chorion elevation and in proper formation of the chorion, which is necessary for embryo development.
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    Pax2a is expressed in oocytes and is responsible for early development and oogenesis in zebrafish
    (2020-12-10)
    Pachoensuk, Theeranukul
    ;
    Fukuyo, Taketo
    ;
    Wanlada, Klangnurak
    ;
    Rezanujjaman, Md
    ;
    Rahaman, Md Mostafizur
    Eleven genes, including pax2a, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. The purpose of this study was to investigate the role of the pax2a gene in the ovulation-inducing process. F2 pax2a homozygous mutant zebrafish possessing a deletion of 6 nucleotides were established in this study. However, the deletion included the start codon (ATG) of the pax2a gene, and the Pax2a protein was still detected, which indicated that the deletion caused a shift in the start codon to the next ATG, resulting in a 12-amino acid deletion. F2 pax2a homozygous mutant zebrafish showed ovulation. However, the embryos showed an abnormal oval shape at the epiboly stage that resulted in yolk and tail formation abnormalities and heart edema. The surviving F3 homozygous mutants did not develop ovaries. Pax2a was detected in oocytes and eggs but not after the Prim-22 stage. It is suggested that pax2a is expressed as a maternal gene in oocytes and is necessary for oogenesis and early development.