P synephrine

Indigo, Aliso B acetate, gentiopicroside, peimine, perillaldehyde, wedelolactone, bergapten, picroside I, berberine, cinnamaldehyde, ephedrine, p-synephrine, 6-shogao, and 6-gingerol were purchased from Sigma-Aldrich. Phellodendrine was purchased from Abcam. Baicalein, baicalin, wogonin, wogonoside, and Ephedra sinica were kindly provided by Y.L. Leu (Chang Gung University, Taiwan). Scutellaria baicalensis (precursor of Baicalein, baicalin, wogonin, wogonoside) and Ephedra sinica were supplied and authenticated by Department of Pharmacy Services, Chang Gung Memorial Hospital at Taoyuan, Taiwan. See Supplementary Information for more details on compound preparation.

The mouse preadipocyte cell line (3T3-L1) was obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) containing 10% bovine calf serum (BCS; Gaithersburg, MD, USA) and 1% penicillin/streptomycin antibiotics (P/S; Gaithersburg, MD, USA). To induce adipogenesis, 3T3-L1 preadipocytes (4 × 10⁴ cells/well) were grown in a 24-well plate for 2 days, and then the culture medium was replaced with the adipogenic differentiation medium containing 0.4 μg/mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS; Gaithersburg, MD, USA), 1-methyl-3-isobutylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO, USA), 1% P/S antibiotics, and 5 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 days, the culture medium was replaced with DMEM supplemented with 10% FBS, 5 µg/mL insulin, and 1% P/S antibiotics every 2 days. Finally, the culture medium was replaced with DMEM containing 10% FBS and 1% P/S antibiotics, which was changed every 2 days, as previously described [50 ]. Hispidulin (5, 10, 20, and 40 μM) and p-synephrine (5, 10, 20, and 40 μM) were added individually or in combination in the culture medium during adipogenic differentiation. Hispidulin (≥98%) and p-synephrine (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).