Down-regulation of tyrosinase, TRP-1, TRP-2 and MITF expressions by citrus press-cakes in murine B16 F10 melanoma

Abstract

Objective: To investigate the suitability of citrus-press cakes, by-products of the juice industry as a source for the whitening agents for cosmetic industry.

Methods: Ethylacetate extracts of citrus-press cakes (CCE) were examined for their anti-melanogenic potentials in terms of the inhibition of melanin production and mechanisim of melanogenesis by using Western Blot analysis with tyrosinese, tyrosinase-related protein-1 (TRP-1), TRP2, and microphthalmia-associated transcription factor (MITF) proteins. To apply the topical agents, citrus-press cakes was investigated the safety in human skin cell line. Finally flavonoid analysis of CCE was also determined by HPLC analysis.

Results: Results indicated that CCE were shown to down-regulate melanin content in a dose-dependent pattern. The CCE inhibited tyrosinase, TRP-2, and MITF expressions in a dose-dependent manner. To test the applicability of CCE to human skin, we used MTT assay to assess the cytotoxic effects of CCE on human keratinocyte HaCaT cells. The CCE exhibited low cytotoxicity at 50 µg/mL. Characterization of the citrus-press cakes for flavonoid contents using HPLC showed varied quantity of rutin, narirutin, and hesperidin.

Conclusions: Considering the anti-melanogenic activity and human safety, CCE is considered as a potential anti-melanogenic agent and may be effective for topical application for treating hyperpigmentation disorders.

Keywords: Citrus-press cakes; MITF; Melanin; Melanogenesis; TRP-1; TRP-2; Tyrosinase.

Figure 1.. Cell viability of the citrus-press cakes on the B16F10 cells and human keratinocyte HaCaT cells.

A: B16F10 cells; B: human keratinocyte HaCaT cells. MTT assay was performed after incubation of the B16F10 cells treated with varying concentrations (12.5, 25.0, and 50 µg/mL) of the EtOAc fraction of citrus-press cakes for 24 h at 37 °C in a 5% CO₂ atmosphere. The absorbance was measured at 570 nm with a spectrophotometer (Power Wave; Bio-tek, Winooski, VT). Values are the mean±SEM of triplicate experiments. *: P<0.05; **: P<0.01.

Figure 2.. Melanin content assay of the citrus-press cakes on the B16F10 cells.

B16F10 cells (2.0×10⁴ µg/mL) were pre-incubated for 18 h, and the melanin content was assayed after incubation of the B16F10 cells treated with α-MSH (100 nmol/L), melasolv (40 µmol/L), and the CCE (12.5, 25.0, and 50.0 µg/mL) for 72 h at 37 °C in a 5% CO₂ atmosphere. The absorbance was measured at 405 nm by an ELISA.

Figure 3.. Inhibitory effect on tyrosinase activity of citrus-press cakes in B16F10 cells.

B16F10 cells (2.0×10⁴ µg/mL) were pre-incubated for 18 h and tyrosinase activity was performed after incubation of B16F10 cells treated with α-MSH (100 nmol/L), melasolv (40 µmol/L) and EtOAc fraction of citrus-press cakes (12.5, 25.0, and 50.0 µg/mL) for 72 h at 37 °C in a 5% CO₂ atmosphere. Absorbance was measured at 405 nm with a ELISA. Values are mean±SEM of triplicate experiments. *: P<0.05; **: P<0.01.

Figure 4.. Inhibitory effect of the citrus-press cakes on the protein level related to melanogenic factors in the B16F10 cells.

B16F10 cells (1.0 × 105 cells/mL) were pre-incubated for 18 h and were stimulated with α-MSH (100 nmol/l) in the presence of melasolv (40 µmol/L) and the EtOAc fraction of citrus-press cakes (12.5, 25.0, and 50.0 µg/mL) for 24 h. The protein level was determined by immunoblotting.

Figure 5.. HPLC fingerprinting analysis of the CCE.

The wavelength of flavonoid absorbance is 280 nm. The injection volume was 10 µL. Ethanol stock solution containing (1) rutin, (2) narirutin, (3) naringin, (4) hesperidin, (5) neohesperidin, (6) neohesperidin dihydrochalcone (NHDC), (7) naringenin, (8) hesperetin, (9) aprgrnin, (10) nobiletin, (11) tangeretin, (12) auraptene was diluted with ethanol to obtain a series concentration of working solutions, and was subjected to HPLC analysis.