Louis, MO, USA). The following antibodies were used: poly (ADP-ribose) polymerase (PARP), Bid, DR5,
caspase-8, cleaved caspase-7, cleaved caspase-6, check details p53, β-actin (Cell signaling, Danvers, MA, USA); cytochrome C (BD Biosciences, San Jose, CA, USA); and Bcl-2, Bax, and DR4 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Fine Black ginseng (10 kg) was selected, dried, and powdered. Exactly 2 kg of powdered samples were refluxed two times with 10 L of 95% ethyl alcohol for 2 h in a water bath. The extracts were filtered through filter paper (Nylon membrane filters 7404-004; Whatman, Dassel, Germany) and concentrated by a vacuum evaporator (yield: 18.35%). FG-4592 supplier Ethyl alcohol extract (150 g) was dissolved in 1500 mL of water and extracted with 1500 mL of diethyl ether. The aqueous layer was extracted three times with 1500 mL of water-saturated n-butanol (n-BuOH). The n-BuOH fraction (84.50 g) was evaporated. The ginsenoside composition of the concentrate was analyzed by HPLC, as suggested by Ko and
colleagues  and . The total ginsenoside content and composition of each sample were analyzed three times. The 99% pure ginsenoside standards used in this experiment were purchased from Chromadex and the Ambo Institute. For the experiment, the Waters 1525 binary HPLC system (Waters, Milford, MA, USA) and the Eurospher Mirabegron 100-5 C 18 column (3 × 250 mm; Knauer, Berlin, Germany) were used. The mobile phase was a mixture of acetonitrile (HPLC grade) and distilled water (HPLC grade). The content of acetonitrile was sequentially
increased from 17% to 30% (35 min), from 30% to 40% (60 min), from 40% to 60% (100 min), from 60% to 80% (110 min), from 80% to 80% (120 min), from 80% to 100% (125 min), from 100% to 100% (135 min), and finally from 100% back to 17% (140 min, lasting for 5 min). The operating temperature was at room temperature and the flow rate was 0.8 mL/min. The elution profile on the chromatogram was obtained by using a UV/VIS detector at 203 nm (Waters 2487 dual λ absorbance detector; Waters) (Fig. 1A). The n-BuOH fraction (60 g) was chromatographed on a silica gel column (1 kg) with eluting solvents of CHCl3-MeOH-H2O (70:30:4) to obtain six subfractions (F1–F5). The F4 fraction (2.59 g) was further subjected to octadecylsilane (ODS) (C-18) column chromatography (500 g, 60% acetonitrile) to provide Rg5 (0.19 g) ( Fig. 1B). Ginsenoside Rg5: FAB–MS (negative); m/z: 465.48 [M-H]−, 603.6 [M-Glu]; 13C nuclear magnetic resonance (13C-NMR; pyridine-d6, 500 MHz ): δ 39.76 (C-1), 28.6 (C-2), 89.42 (C-3), 40.75 (C-4), 56.89 (C-5), 18.93 (C-6), 35.84 (C-7), 40.21 (C-8), 51.26 (C-9), 37.51 (C-10), 32.72 (C-11), 73.08 (C-12), 50.