Antioxidant and skin-whitening effects of aerial part of Euphorbia supina Raf. Extract

Abstract

Background: Euphorbia supina (ES) has been widely used in folk medicine owing to its antibacterial, hemostatic, and anti-inflammatory properties. The aim of this study was to evaluate the antioxidant and skin-whitening effects of a 70% ethanol extract of ES.

Methods: The aerial parts of ES plant were extracted with 70% ethanol. The viability of B16F10 cells was evaluated by MTT assay to determine the non-toxic doses for further experiments. The tyrosinase and cellular tyrosinase activities were then measured using an enzyme-substrate assay. In addition, the expression of whitening-related proteins was measured using western blot.

Results: The antioxidant activity of the ES samples increased in a dose-dependent manner, as confirmed by their radical scavenging activities in the 2,2-diphenyl-1-1-picrylhydrazyl and 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) assays. The ES extract significantly reduced tyrosinase activity and melanin content in a dose-dependent manner. Furthermore, it decreased α-melanocyte stimulating hormone (MSH)-induced protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF).

Conclusions: Our results indicate that the ES extract attenuated α-MSH-stimulated melanin synthesis by modulating tyrosinase and MITF expression. Therefore, the ES extract could be a promising therapeutic agent to treat hyperpigmentation and as an ingredient for skin-whitening cosmetics.

Keywords: DPPH; Euphorbia supina (ES); MITF; Melanogenesis; Tyrosinase; α-MSH.

Fig. 1

Cell viability of the ES extract on the B16F10 cells. B16F10 cells were treated with various concentration of ES extract (8, 40, 200 μg/ml) for 24 h and the cell viability was measured by MTT assay. The absorbance was measured at 540 nm on a VersaMax microplate reader. Values represent the mean ± S.D. of triplicate experiments

Fig. 2

Antioxidant activities of ES extract. a Scavenging effect of ES on DPPH radical (b) ABTS⁺ radical scavenging activity of the extract. The ES ethanol extract (ESEE) (8, 40, 200 μg/ml), ascorbic acid (20 μg/ml) were incubated with DPPH, ABTS⁺ solution, respectively. Results are represented as percentages of control, and the data represent mean ± S.D. for three separate experiments. Values are significantly different by comparison with control. *: p < 0.05, **: p < 0.01

Fig. 3

Inhibitory effect of ES extract on mushroom tyrosinase activity, B16F10 melanin content and intracellular tyrosinase activity. a Different concentrations of the ES extract (8, 40, 200 μg/ml) or arbutin (10 mM) was incubated with the same units of mushroom tyrosinase. Following incubation, the amount of dopachrome produced was determined at 490 nm. b and c B16F10 melanoma cells were stimulated with α-MSH (100 nM) for 48 h, and then the melanin content or intracellular tyrosinase activity were measured after treatment with various concentrations of the ES extract (8, 40,200 μg/ml) or arbutin (10 mM) for another 24 h. Results are represented as percentages of control, and data are presented as mean ± S.D. for three separate experiments. Values are significantly different by comparison with control. *: p < 0.05, **: p < 0.01 α-MSH treatment alone

Fig. 4

Effect of ES extract on melanogenesis-related proteins expression. a B16F10 cells were cultured with α-MSH (100 nM) for 24 h and then treated with various concentration of the ES extract (8, 40, 200 μg/ml) or arbutin (10 mM) for another 24 h. Then the content of cellular MITF, tyrosinase proteins were analyzed by western blotting assay. b Densitometry was normalized to the α-MSH group. Values are mean ± S.D. Data were analyzed by Student’s t-test. *: p < 0.05, **: p < 0.01 versus α-MSH treatment alone

Fig. 5

GC-MS Chromatograms of the methanol extract ES