Identification of HSP70-inducing activity in Arnica montana extract and purification and characterization of HSP70-inducers

Background: The expression of heat shock proteins (HSPs), particularly HSP70, is receiving considerable attention in the field of cosmetics, particularly given our recent report that ultraviolet-induced melanin production, skin damage and wrinkle formation were all suppressed in transgenic mice expressing HSP70. Objective: In the present study, we searched for HSP70-inducers from a library of herbal extracts that have already been approved as quasi-pharmaceutical products in Japan. We selected an ethanol extract of Arnica montana (. A. montana), based on its high HSP70-inducing activity and low cytotoxicity. Methods: Cell viability was determined by MTT method and expression of HPS70 was monitored by immunoblotting analysis. Results: From the extract, we purified and identified eight sesquiterpene lactones (AM1-8) as HSP70-inducers, among which AM-2 (helenalin 2-methylbutyrate) was selected due to its good HSP70-inducing properties and low cytotoxicity. Treatment of cultured mouse melanoma cells with AM-2 or A. montana extract up-regulated the expression of HSP70 in a dose-dependent manner. This treatment also activated heat shock factor-1, a transcription factor for hsp genes. Furthermore, pre-treatment of cells with AM-2 or A. montana extract decreased melanin production and expression and activity of tyrosinase. Conclusion: These results suggest that AM-2 and A. montana extract could be beneficial for use in hypopigmenting cosmetics as a consequence of their stimulatory effects on HSP70 expression.