| Summary: | The present study investigated potential bioactive natural products from the EtOH extract of <i>Salix chaenomeloides</i> twigs using column chromatography, leading to the isolation of six compounds (<b>1</b>–<b>6</b>), which were characterized as two proanthocyanidins, procyanidin B<sub>2</sub> (<b>1</b>) and procyanidin B<sub>1</sub> (<b>2</b>), and four phenolic compounds, 4-hydroxybenzoic acid <i>β</i>-D-glucosyl ester (<b>3</b>), di-<i>O</i>-methylcrenatin (<b>4</b>), <i>p</i>-coumaric acid glucoside (<b>5</b>), and syringin (<b>6</b>) by the comparison of their NMR spectra with the reported data and high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS) analysis. We investigated the potential of six compounds (<b>1</b>–<b>6</b>) to inhibit adipogenesis in 3T3-L1 preadipocytes, which showed that the compounds (<b>1</b>–<b>6</b>) significantly reduced lipid accumulation in 3T3-L1 adipocytes without affecting cell proliferation. Notably, compound <b>1</b> demonstrated a remarkable 60% and 90% reduction in lipid levels with 50 and 100 µM treatments, respectively. Oil Red O staining results indicated that compound <b>1</b> significantly inhibits the formation of lipid droplets, comparable to the effect of T863, an inhibitor of triglyceride used as a positive control, in adipocytes. Compound <b>1</b> had no effect on the regulators PPARγ, C/EBPα, and SREBF1 of adipocyte differentiation in 3T3-L1 preadipocytes, but compound <b>1</b> activated the fatty acid oxidation regulator, PPARα, compared to the lipogenic-induced control. It also suppressed fatty acid synthesis by downregulating the expression of fatty acid synthase (FAS). Finally, compound <b>1</b> induced the mRNA and protein levels of CPT1A, an initial marker of mitochondrial fatty acid oxidation in 3T3-L1. This finding substantiates the anti-lipogenic and lipolytic effects of procyanidin B<sub>2</sub> (<b>1</b>) in 3T3-L1 preadipocytes, emphasizing its pivotal role in modulating obesity-related markers.
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