Lotus (Nelumbo nuficera) flower essential oil increased melanogenesis in normal human melanocytes

Abstract

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.

Figure 1

Effects of lotus flower essential oil on melanogenesis in human melanocytes. Supplement-reduced melanocytes were untreated or treated with various concentrations of lotus flower oil or 10 µM forskolin (FK). After the melanocytes were incubated for 5 days, melanin content was determined and MTT assays were conducted (A). Supplement-reduced melanocytes were treated with 10 µg/ml of lotus flower essential oil for 0.5, 1, or 2 h, and for 1, 3, or 5 d. The expression of MITF-M, TRP-1, TRP-2, tyrosinase and GAPDH mRNA were assessed at the indicated times. The intensity of each band was quantitated by densitometry and normalized versus GAPDH. The error bars indicate S.E. (n = 4) (B). After treatment with lotus flower oil, tyrosinase and MITF-M protein expression were assessed at 1, 3, or 5 d. The intensity of each band was quantitated by densitometry and normalized versus β-actin. The data represent the means ± S.E. of three independent experiments (C).The error bars indicate S.E. (n = 5); ^*P < 0.01; ^**P < 0.001 (B).

Figure 2

Effect of the main ingredients of lotus flower essential oil on tyrosinase activity and expression. Supplement-reduced melanocytes were stimulated with 10 µg/ml of lotus flower oil for 5 days in the presence of linolic acid (LA) 12.5 µM, linolic acid methyl ester (mLA) 12.5, 25, 50 µM, palmitic acid (PA) 12.5, 25, 50 µM or palmitic acid methyl ester (mPA) 12.5, 25, 50 µM. Tyrosinase expression was assessed. β actin was used as a control (A). The intensity of each band was quantitated by densitometry and normalized versus β-actin. The data represent the means ± S.E. of three independent experiments (B). Tyrosinase activity, melanin content and cell viability (MTT) were measured as described in the Methods section. The error bars indicate S.E. (n = 4) (C); ^*P < 0.01; ^**P < 0.001.

Figure 3

Signaling pathways activated by lotus flower essential oil in human melanocytes. Supplement-reduced melanocytes were treated with 10 µg/ml of lotus flower essential oil (A) or 50 µM of palmitic acid methyl ester (mPA) (B) for 0.5, 1, or 2 h. The whole lysates were electrophoresed via SDS-PAGE and analyzed by immunoblotting with each antibody. The intensity of phosphorylation and total ERKs and CREB bands were quantitated by densitometry and the amounts of phosphorylated ERKs and CREB were normalized versus total ERKs and CREB. The data represent the means ± S.E. of four independent experiments. Supplement-reduced melanocytes were treated with 10 µg/ml of lotus flower essential oil (L) or 50 µM of palmitic acid methyl ester (mPA) in the absence or presence of 10 βM PD98059 (PD98059) for 2 days. The whole lysates were electrophoresed via SDS-PAGE and analyzed by immunoblotting with each antibody. Tyrosinase expression was normalized versus β-actin. The data represent the means ± S.E. of three independent experiments C: unstimulated melanocytes. ^*P < 0.01; ^**P < 0.001.