Bioactive Phytochemicals from Salix pseudolasiogyne Twigs: Anti-Adipogenic Effect of 2'- O-Acetylsalicortin in 3T3-L1 Cells

Abstract

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin (1) and 2'-O-acetylsalicortin (2). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin (1) in twigs of S. pseudolasiogyne. Here, we found that the isolated compounds, oregonin (1) and 2'-O-acetylsalicortin (2), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'-O-acetylsalicortin (2), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'-O-acetylsalicortin (2)-treated 3T3-L1 cells. Taken together, these results indicate that 2'-O-acetylsalicortin (2), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Keywords: 2′-O-acetylsalicortin; Salix pseudolasiogyne; adipocyte differentiation; adipogenesis; obesity.

Figure 1

Schematic representation of oregonin (1) and 2′-O-acetylsalicortin (2) isolation.

Figure 2

Chemical structure of oregonin (1) and 2′-O-acetylsalicortin (2).

Figure 3

Inhibitory effect of oregonin and 2′-O-acetylsalicortin on intracellular lipid contents. The 3T3-L1 preadipocytes were induced to differentiate and then treated with oregonin. (A) After differentiation, Veh or oregonin (10 or 50 µM)-treated cells were stained with Oil Red. (B) Lipid contents were quantified by spectrophotometry (n = 3 per group). (C) Veh or 2′-O-acetylsalicortin (10 or 50 µM)-treated cells were stained with Oil Red O solution. (D) Intracellular lipid accumulation was analyzed by spectrophotometry (n = 3 per group). The values indicate the mean ± SEM. Mean values followed by different letters are significantly different (p ≤ 0.05). The size of each scale bar in photomicrographs is 100 µm. Veh, vehicle treatment (negative control).

Figure 4

Inhibitory effect of 2′-O-acetylsalicortin on adipogenesis. (A) 3T3-L1 cells treated with various concentrations of 2′-O-acetylsalicortin during the adipocyte differentiation were stained with Oil Red O (upper) and analyzed by microscopy (bottom). (B) Quantification of intracellular lipid contents with spectrophotometry (n = 3 per group). (C) Expressions of PPARγ, FABP4, FASN, C/EBPα, and C/EBPβ genes were examined using qRT-PCR (n = 3 per group). (D) Protein levels of C/EBPβ, FASN, FABP4, C/EBPα, and PPARγ were analyzed by immunoblotting. (E) Cytotoxic effect of 2′-O-acetylsalicortin (n = 3 per group). The values represent the mean ± SEM. The size of each scale bar in photomicrographs is 100 µm. Mean values followed by different letters are significantly different (p ≤ 0.05). N.S., not significant. Veh, vehicle treatment (negative control).

Figure 5

The model on the action of the 2′-O-acetylsalicortin in 3T3-L1 differentiation. 2′-O-acetylsalicortin represses adipocyte differentiation through the downregulation of mRNA and proteins of lipogenic enzymes and adipogenic factors.