As the less polar ginsenosides can be easily absorbed into blood vessels and act as the pharmacological agents with potential as drug candidates, the mass production or isolation of the less polar ginsenosides is of much interest in the ginseng industry [5]. Recent improvements in chromatographic techniques have led to the analysis and

isolation of the stereoisomers of minor ginsenosides in ginseng preparations [11]. The structure–activity relationships between the diverse ginsenosides isolated by these improved techniques has been studied in both cancer cells and noncancer cells [12]. In this study, we isolated 21 minor ginsenosides from a processed ginseng preparation selleckchem and unequivocally determined their structures by one-dimensional and two-dimensional NMR spectroscopy and compared these results with previously published data. The NMR data obtained for these minor ginsenosides will be useful in studying the structure–activity relationships between structural modifications such as the number of sugar groups, the sugar linkage at C-6, the number of hydroxyl groups, and the stereoisomers of 20(S) and 20(R), as well as in the identification of stereoisomers of ginsenosides. Column chromatography (CC) was carried out using Kiesgel 60 silica

gel (40–60 μm, 230–400 mesh, Merck, USA), YMC-GEL ODS-A (5–150 μm, YMC), and Sephadex LH-20 (25–100μM, Pharmacia, NJ, USA) columns. Thin-layer chromatography was selleck chemical carried out

using Kiesgel 60 F254 coated normal silica gel and RP-18 F254 coated reversed-phase (RP) silica gel columns. The 1H-NMR and 13C-NMR, 1H-1H COSY, HSQC, and HMBC spectra were recorded on a Bruker AMX 500 or 600 spectrometer in pyridine-d5. The solvent signals were used as internal standards. The high-performance liquid chromatography (HPLC) system consisted of a G-321 pump (Gilson, USA), a G-151 UV detector (Gilson), and a YMC-Pack Pro C18 column (250 mm × 10 mm i.d.; 5 μm); and all chromatograms were monitored at 210 nm. HPLC-grade solvents (Fisher Scientific, USA) were used in the MeOH–H2O or MeCN–H2O system. The processed ginseng preparation was gifted from Greencrosshs (Sungnam, Korea). these It was prepared using patented technology and a previously reported method [13]. Briefly, the harvested ginseng was repeatedly extracted with ethanol, followed by reaction with an enzyme containing ginsenoside-β-glucosidase. After acid hydrolysis of the residue, the reactant was purified with HP-20 resin followed by washing out with distilled water and, finally, 95% ethanol. Powders of the processed ginseng extract (GE) (90 g) were each subjected to normal silica CC (20 × 5 cm column) with a gradient elution of solvents (CHCl3:MeOH = 10:1, 7:1, 5:1, 3:1, 0:1; all 1-L volumes) and 24 sub-fractions (GE1–24) were obtained. 20(S/R)-AcetylRh2 (5, 6) (20 mg, Rt = 14.1 min) were obtained from the GE-5 (2.