Syringin: A Phenylpropanoid Glycoside Compound in Cirsium brevicaule A. GRAY Root Modulates Adipogenesis

Abstract

Cirsium brevicaule A. GRAY is a wild perennial herb, and its roots (CbR) have traditionally been used as both food and medicine on the Japanese islands of Okinawa and Amami. The present study evaluated the antiadipogenic effect of CbR using mouse embryonic fibroblast cell line 3T3-L1 from JCRB cell bank. Dried CbR powder was serially extracted with solvents of various polarities, and these crude extracts were tested for antiadipogenic activity. Treatment with the methanol extract of CbR showed a significant suppression of lipid accumulation in 3T3-L1 cells. Methanol extract of CbR was then fractionated and subjected to further activity analyses. The phenylpropanoid glycosidic molecule syringin was identified as an active compound. Syringin dose dependently suppressed lipid accumulation of 3T3-L1 cells without cytotoxicity, and significantly reduced the expressions of peroxisome proliferator-activated receptor gamma, the master regulator of adipogenesis, and other differentiation markers. It was demonstrated that syringin effectively enhanced the phosphorylation of the AMP-activated protein kinase and acetyl-CoA carboxylase. These results indicate that syringin attenuates adipocyte differentiation, adipogenesis, and promotes lipid metabolism; thus, syringin may potentially serve as a therapeutic candidate for treatment of obesity.

Keywords: 3T3-L1 cells; Cirsium brevicaule A. GRAY; anti-adipogenesis; syringin.

Figure 1

Effects of extracts and fractions from Cirsium brevicaule A GRAY root (CbR) on 3T3-L1 cells. (A) Flowchart for the isolation of active anti-obesity compounds from Cirsium brevicaule A GRAY root (CbR). (B) Inhibitory effects of the CbR extract on cellular lipid accumulation. (C) Inhibitory effects of the fractions derived from the methanol extract on cellular lipid accumulation. 3T3-L1 cells were treated with 500 μg/mL of crude extracts or 100 μg/mL of individual fractions of the crude extracts with 0.5% DMSO as the vehicle. The results are presented as means ± SEM of three independent experiments (n = 3). The asterisk (*) indicates a significant difference between control and treatment groups by Dunnett’s test. ** p < 0.01, and *** p < 0.001 vs. control (Cntl) and preadipocytes (pre).

Figure 2

Isolation of active components from CbR and chemical structure identification. (A,B) Chromatograms of Fr-1 from crude methanol extract and purified syringin, respectively. (C) Chemical structure of syringin revealed by NMR analyses.

Figure 3

Effect of syringin on intercellular lipid accumulation in 3T3-L1 cells. (A) Time course of treatment with syringin during the cellular differentiation of 3T3-L1 adipocytes. (B) Inhibitory effects of syringin on lipid accumulation in 3T3-L1 adipocytes. The results are presented as means ± SEM of three independent experiments (n = 3). The asterisk (*) indicates a significant difference between control and treatment groups by the Student’s t-test. *** p < 0.001 vs. control (Cntl).

Figure 4

Effect of syringin on adipogenic differentiation through the regulation of adipogenic factors in 3T3-L1 cells. (A) Cytotoxic effect of syringin on 3T3-L1 cells. (B,C) Cellular lipid accumulation by Oil Red O staining (image 20×magnification and scale bar = 100 µm). (D–F) mRNA levels of (D) lipogenesis-, (E) lipolysis-, and (F) thermogenesis-related genes. Experiments were performed in triplicate. The results are presented as means ± SEM of three independent experiments (n = 3). The asterisk (*) indicates a significant difference between control and treatment groups by Dunnett’s test and Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (Cntl) and preadipocytes (pre).

Figure 5

Effect of syringin on the expression of adipogenesis-related genes and proteins. (A) mRNA levels of adipogenesis-related genes. (B) Representative Western blot of adipogenic protein factors. The results are presented as means ± SEM of three independent experiments (n = 3). The asterisk (*) indicates a significant difference between control and treatment groups tested by Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (Cntl).

Figure 6

Effects of syringin on AMPK, ACC, and Akt phosphorylation during the differentiation of 3T3-L1 adipocytes. (A–C) Ratios of relative phosphorylation to total protein levels of (A) p-AMPK/AMPK, (B) p-ACC/ACC, and (C) p-Akt/Akt. The results are presented as means ± SEM of three independent experiments (n = 3). The asterisk (*) indicates a significant difference between control and treatment groups by Student’s t-test. * p < 0.05 vs. control (Cntl).