Silica column

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).

Minor ginsenosides including F1, F2, Rh1(S), Rh2(S), Rg3(S), and CK (>95% pure) were prepared using enzymatic methods previously reported^{23 (link)–28 (link)}. Briefly, PPT type (Daziran Co. Ltd.) or PPD type (Hongjiou Biotech Co. Ltd.) major ginsenosides mixtures were converted into minor ginsenosides using various recombinant β-glucosidases and the produced minor ginsenosides were purified using a silica column (168 × 71 mm id, Biotage, Sweden) and ODS column (157 × 39 mm id, Biotage, Sweden). They were then further purified by Recycling-Preparative HPLC (Japan Analytical Industry Co. Ltd.) with a JAIGEL-ODS-AP column (10 μm, 500 × 20 mm id, Japan Analytical Industry Co., Ltd.). Major ginsenosides including Rg1, Re, Rb1, and Rd were directly purified from PPT- or PPD-type major ginsenoside mixtures using a silica column, ODS column and Recycling-Preparative HPLC. The compounds were dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with the medium for the sample preparation.

NHKs were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dermal Cell Basal Medium (ATCC) supplemented with the Keratinocyte Growth kit (ATCC) at 37 °C with 5 % CO₂. Ginsenosides compound K (C-K), F1, F2, gypenoside XVII (G17), gypenoside LXXV (G75), protopanaxadiol (PPD), protopanaxatriol (PPT), Rb1, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, and Rh2 were prepared as previously described [39 (link),40 (link)]. PPD and PPT were purchased from Hongjiu Biotech Co. Ltd. (Dalian, China) and Da Nature Biological Engineering Co. Ltd. (Fusong, China), respectively. Ginsenoside C-K, F1, F2, G17, G75, Rg2, Rg3, Rh1, and Rh2 were transformed from PPD or PPT type ginsenosides using enzymatic bioconversion methods. Ginsenoside Rb1, Rb3, Rc, Rd, Re, and Rg1 were directly purified from PPD or PPT type ginsenosides using a silica column (Biotage, Uppsala, Sweden), an octadecyl silica (ODS) column (Biotage), and recycling preparative high-performance liquid chromatography (HPLC). The purity of each ginsenoside was greater than 95%. Ginsenosides were dissolved in dimethylsulfoxide (DMSO). DMSO, DEX, recombinant human TNF-α, Poly I:C, and bay 11-7082 were purchased from Sigma Aldrich (St. Louis, MO, USA). All kits were used as per the manufacturers’ instructions, unless otherwise specified.