Anti-Adipogenic Effects of Salicortin from the Twigs of Weeping Willow (Salix pseudolasiogyne) in 3T3-L1 Cells

Abstract

Salix pseudolasiogyne (Salicaceae), the "weeping willow," has been used in traditional Korean medicine to treat pain and fever due to its high concentrations of salicylic acid and salicin. The present study investigated bioactive compounds from S. pseudolasiogyne twigs to discover bioactive natural products. Phytochemical investigation of the ethanol (EtOH) extract of S. pseudolasiogyne twigs followed by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of two salicin derivatives, salicortinol and salicortin, the structures of which were determined by interpretation of their NMR spectra and data from the LC/MS analysis. To the best of our knowledge, this is the first report of salicortinol isolated from S. pseudolasiogyne. The isolated compounds were evaluated for their anti-adipogenic effects in 3T3-L1 cells. Both salicortinol and salicortin were found to significantly inhibit adipocyte differentiation in 3T3-L1 cells. In particular, salicortin exhibited a strong inhibitory effect on lipid accumulation. Furthermore, salicortin inhibited the expression of lipogenic and adipogenic transcription factors, including FASN, FABP4, C/EBPα, C/EBPβ, and PPARγ, without inducing cytotoxicity. These results suggest that salicortin could be a potential therapeutic compound for the prevention or treatment of metabolic disorders such as obesity.

Keywords: 3T3-L1 cells; Salix pseudolasiogyne; adipocyte differentiation; lipid metabolism; salicortin.

Figure 1

Separation scheme leading to salicortinol and salicortin identification.

Figure 2

Chemical structure of salicortinol (1) and salicortin (2).

Figure 3

Inhibitory effect of salicortinol and salicortin on lipid accumulation in 3T3-L1 cells: (A) microscopy images of cells treated with different concentrations of salicortinol (as indicated) during adipocyte differentiation, followed by oil red O staining; (B) Quantification of intracellular lipid accumulation in salicortinol-treated 3T3-L1 cells; (C) microscopy images of cells treated with different concentrations of salicortin (as indicated) during adipocyte differentiation, followed by oil red O staining; and (D) Quantification of intracellular lipid accumulation quantification in salicortin-treated 3T3-L1 cells. B and D, n = 3 per group. The values represent the mean ± SEM. Scale bar = 100 µm. * p < 0.05, ** p < 0.01. Veh, vehicle (negative control).

Figure 4

Inhibitory effect of salicortin on adipogenesis: (A) quantification of intracellular lipid accumulation in salicortin-treated 3T3-L1 cells compared to that in control cells; (B) Oil red O staining images (top) and microscopy (bottom) images in 3T3-L1 cells after 7 days of differentiation in either the absence or presence of salicortin at different concentrations. Scale bar = 100 µm; (C) gene expression quantification of PPARγ, FASN, FABP4, C/EBPβ, and C/EBPα, performed by real time qPCR; (D) immunoblot images of PPARγ, FASN, FABP4, C/EBPβ, and C/EBPα; and (E) evaluation of salicortin cytotoxicity. (B,C,E), n = 3 per group. N.S., not significant; Veh, vehicle (negative control). The values represent the mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 5

Model of mechanism of salicortin’s action. Salicortin suppresses adipocyte differentiation by reducing the mRNA and protein levels of lipogenic and adipogenic factors.