Anti-Adipogenic Activity of Secondary Metabolites Isolated from Smilax sieboldii Miq. on 3T3-L1 Adipocytes

Abstract

Smilax sieboldii, a climbing tree belonging to Smilacaceae, has been used in traditional oriental medicine for treating arthritis, tumors, leprosy, psoriasis, and lumbago. To evaluate the anti-obesity effects of S. sieboldii (Smilacaceae), we screened methylene chloride (CH₂Cl₂), ethyl acetate (EtOAc), aqueous-saturated n-butanol, and ethanol (EtOH) extracts of the whole plant at various concentrations to inhibit adipogenesis in adipocytes. The 3T3-L1 cell line with Oil red O staining with the help of fluorometry was used as an indicator of anti-obesity activity. Bioactivity-guided fractionation of the EtOH extract and subsequent phytochemical investigation of the active CH₂Cl₂- and EtOAc-soluble fractions resulted in the isolation of 19 secondary metabolites (1-19), including a new α-hydroxy acid derivative (16) and two new lanostane-type triterpenoids (17 and 18). The structures of these compounds were characterized using various spectroscopic methods. All the isolated compounds were screened for adipogenesis inhibition at a concentration of 100 μM. Of these, compounds 1, 2, 4-9, 15, and 19 significantly reduced fat accumulation in 3T3-L1 adipocytes, especially compounds 4, 7, 9, and 19, showing 37.05 ± 0.95, 8.60 ± 0.41 15.82 ± 1.23, and 17.73 ± 1.28% lipid content, respectively, at a concentration of 100 μM. These findings provide experimental evidence that isolates from S. sieboldii extracts exert beneficial effects regarding the regulation of adipocyte differentiation.

Keywords: Smilacaceae; Smilax sieboldii; adipogenesis; gallotannin; lanostane triterpenoid; phenylpropanoid glyceride.

Figure 1

Anti-adipogenic effects of S. sieboldii ethanol (EtOH) extract and three solvent fractions. 3T3-L1 cells were treated with varied concentrations of crude EtOH extract and solvent fraction from start of adipocyte differentiation (designated day 0) for eight days. (A) The representative Oil Red O staining pictures of differentiated adipocytes after eight days. (B) Quantitative analysis of Oil Red O staining levels. Values are represented as means ± SD of 3 experiments. Differences among groups were determined by one-way ANOVA followed by Tukey’s post-hoc test; the different superscript letters (a–d) are significantly different from each group (p < 0.05). MDI: 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin.

Figure 2

Chemical structures of compounds 1–19.

Figure 3

Key ¹H-¹H COSY (bold blue lines) and HMBC (red arrows) correlations of 16.

Figure 4

Key ¹H-¹H COSY (blue bold lines), HMBC (red arrows), and ROESY (blue arrow) correlations of 17.

Figure 5

Key ¹H-¹H COSY (blue bold lines), HMBC (red arrows), and ROESY (blue arrow) correlations of 18.

Figure 6

Anti-adipogenic activity of isolated constituents from whole plant of S. sieboldii CH₂Cl₂- and EtOAc-soluble extracts. UND: undifferentiated, one-way analysis of variance (ANOVA) and Student’s t-test were used to determine the significance of the results. Values of * p < 0.05 and ** p < 0.01 were considered statistically significant (compared with MDI-treated cells). MDI: 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin.

Figure 7

Dosage-dependent research of active compounds, 2-O-caffeoylglycerol (4), acertannin (7), maplexin D (9), and trans-ρ-ethyl coumarate (19) on lipid accumulation in adipocytes. (A) The representative Oil Red O staining pictures of differentiated adipocytes on day 8. (B) Quantitative analysis of Oil Red O staining contents. Differences among groups were determined by one-way ANOVA followed by Tukey’s post-hoc test; the different superscript letters (a–e) are significantly different from each group (p < 0.05). MDI: 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin.

Figure 8

The isolation scheme of compounds 1–19.