Evaluating the Application Potential of a Recombinant Ganoderma Protein as Bioactive Ingredients in Cosmetics

Abstract

The aim of this study was to evaluate the application potential of a recombinant fungal immunomodulatory protein from Ganoderma lucidum (rFIP-glu). First, a recombinant plasmid pPIC9K::FIP-glu-His was transferred into Pichia pastoris for the production of protein. The protein was then to assess its free radical scavenging abilities and the effect on the viability of both human immortalized keratinocytes (HaCaT cells) and mouse B16-F10 melanoma cells (B16 cells) in vitro, followed by the effect on the melanin synthesis of B16 cells. The results of SDS-PAGE and western blot showed that rFIP-glu was successfully expressed. Furtherly, a bioactivity assay in vitro indicated that the scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals reached 84.5% at 6.0 mg/mL (p ≤ 0.0001) of rFIP-glu, showing strong antioxidant activity. Subsequently, a safety evaluation demonstrated that rFIP-glu promoted the proliferation of HaCaT cells, with the cell viability reaching 124.3% at 48 μg/mL (p ≤ 0.01), regarding the cell viability of B16 cells after exposure to rFIP-glu (48 μg/mL) significantly inhibited, to 80.7% (p ≤ 0.01). Besides, rFIP-glu inhibited the melanin synthesis of B16 cells in a dose-dependent manner from 100-1000 μg/mL, and rFIP-glu at 500 μg/mL (p ≤ 0.01) exhibited the highest intracellular melanin amount reduction of 16.8%. Furthermore, a mechanism analysis showed that rFIP-glu inhibited tyrosinase (TYR) activity by up-regulating the expression of the microphthalmia-associated transcription factor (MITF) and down-regulating the gene expression of TYR and tyrosinase-related protein-1 (TYRP-1), thus inhibiting melanin synthesis. The data implied that rFIP-glu had significant antioxidant activity and whitening potency. It should be used as raw materials for cosmeceutical applications.

Keywords: antioxidant activity; melanin; recombinant FIP-glu (rFIP-glu); tyrosinase; whitening potency.

Figure 1

Preparation of rFIP-glu in Pichia pastoris. (A) Construction of the recombinant plasmid pPIC9K::FIP-glu-His. (B) Identification of yeast transformants by PCR analysis. Lane M, DNA Ladder; Lane 1–3, rFIP-glu yeast transformant DNA; Lane 0, negative control. (C) SDS-PAGE analysis. Lane M, molecular weight marker; Lane 1, rFIP-glu. (D) Western blot analysis.

Figure 2

Safety evaluation. The effects of rFIP-glu on the cell viability of (A) HaCaT cells and (B) B16 cells. Vc (40 μg/mL) was used as the positive control. Data are expressed as means ± SD (n = 3). NS, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01 versus the control group.

Figure 3

rFIP-glu inducing the apoptosis of B16 cells. (A) Hoechst 33258 staining of B16 cells under a fluorescence microscope (400×). (B) B16 cells stained by Annexin/PI and analyzed by flow cytometry.

Figure 4

The effect of rFIP-glu on the scavenging rate of DPPH radicals. Vc (0.5 mg/mL) was used as the positive control. Data are expressed as means ± SD (n = 3). p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 versus the control group.

Figure 5

Effect of rFIP-glu on the melanin content of B16 cells. Arbutin (100 μg/mL) was used as the positive control. Data are expressed as means ± SD (n = 3). *, p ≤ 0.05; **, p ≤ 0.01 versus the control group.

Figure 6

The effects of rFIP-glu on tyrosinase activity and mRNA expression of related genes in B16 cells. Tyrosinase activity (A); mRNA expression of TYR (B), MITF (C), TYRP-1 (D), and TYRP-2 (E). Data are expressed as means ± SD (n = 3). NS, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, and p ≤ 0.0001 versus the control group.