Ginsenoside Characterization in Skin Cells

HaCaT (human keratinocytes) (Cell Line Service, Eppelheim, Germany) and human dermal fibroblasts (CCD986sk, ATCC, Manassas, VA, USA) were incubated in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C and humidified 5% CO₂ atmosphere.
Ginsenoside C-K, F1, F2, G17, G75, PPD, PPT, Rg2, Rg3, Rh1, and Rh2 (>95% pure) were prepared using enzymatic methods as previously reported [17 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link),47 (link)]. Ginsenosides Rb1, Rd, Re, and Rg1 were directly purified from protopanaxadiol (Hongjiu Biotech Co., Ltd., Dalian, China) type or protopanaxatriol (Da Nature Biological Engineering Co., Ltd., Fusong, China) type ginsenoside mixtures using a silica column (168 × 71 mm id, Biotage, Uppsala, Sweden), an ODS column (157 × 39 mm id, Biotage, Uppsala, Sweden), and recycling preparative HPLC (>95% purity). Ginsenosides were dissolved in dimethyl sulfoxide (DMSO). Madecassoside (MA) was purchased from Changzhou United Chemical Co., Ltd. (Changzhou, China). RU486 (GR antagonist) and DEX (GR agonist) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Control siRNA and GR siRNA were purchased from Cell Signaling Technology (Danvers, MA, USA).