Young Wan Ha

Simultaneous quantification of 14 ginsenosides in Panax ginseng C.A. Meyer (Korean red ginseng) by HPLC-ELSD and its application to quality control.

A new method of high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of 14 major ginsenosides, which are the marker compounds of Panax ginseng C.A. Meyer (Korean red ginseng). Various types of ginseng samples were extracted, and the amounts of the 14 ginsenosides (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, and Rh2) were determined by reverse-phase HPLC-ELSD using digoxin as an internal standard. The mobile phase consisted of a programmed gradient of aqueous acetonitrile. Calibration curves for each ginsenoside were determined for the quantification. The method was validated for linearity, precision, accuracy, limit of detection, and limit of quantification. This quantification method was applied to several finished ginseng products including white ginseng, red ginseng powder, and red ginseng concentrate. The amounts of the 14 ginsenosides in the various ginseng samples could be analyzed simultaneously. This validated HPLC method is expected to provide a new basis for the quality assessment of ginseng products.

Preparative isolation of six major saponins from Platycodi Radix by high-speed counter-current chromatography.

INTRODUCTION Platycosides, the primary constituents of Platycodi Radix, are known to have numerous and varied biological activities, exerting anti-inflammation, anti-allergy, anti-tumour, anti-obesity and anti-hyperlipidemia effects. However, effective methods for isolating and purifying platycosides from Platycodi Radix are not currently available. OBJECTIVE To develop an efficient method for the preparative separation of six platycosides from Platycodi Radix by high-speed counter-current chromatography (HSCCC) coupled with an evaporative light scattering detection (ELSD) system. METHODOLOGY Preparative separation was performed by water extraction using reversed-phase C(18) column chromatography on an HSCCC-ELSD system. A two-phase solvent system comprised hexane-n-butanol-water (1:40:20, v/v) and (1:10:5, v/v) was employed. Two other key parameters, revolution speed of the separation column and flow-rate of the mobile phase, were also investigated for optimum HSCCC performance. Each peak fraction obtained from separation of the platycosides was collected according to the ELSD elution profile and determined by HPLC. RESULTS Using the described method, six platycosides, all with purities of over 94%, could be isolated from 300 mg of the platycoside-enriched fraction. Their structures were characterized by electrospray ionisation mass spectrometry (ESI-MS), (1)H-NMR and (13)C-NMR. CONCLUSION Six of the main bioactive platycosides in Platycodi Radix could be isolated and purified systematically by HSCCC.

Isolation and tandem mass fragmentations of an anti-inflammatory compound from Aralia elata

One-step isolation of a saponin from Aralia elata was undertaken using high-speed countercurrent chromatography coupled with evaporative light scattering detection. A triterpenoid saponin, elatoside F, was purified with 96.8% purity using a two-phase-system comprising chloroform-methanol-water-isopropanol. The yield was 35.0 mg from 348.2 mg of the enriched saponin fraction. In vitro anti-inflammatory study demonstrated that elatoside F inhibited lipopolysaccharide-induced nitric oxide production, as well as nuclear factor κB activation, in a dose-dependent manner. Two types of mass ionization technique were compared on elatoside F to investigate characteristic fragmentation patterns. MALDI-TOF tandem mass spectrometric fragmentation patterns of sodiated ions provided structural information on glycosidic cleavages and on extensive cross-ring cleavages. Electrospray ionization multiple-stage tandem mass fragmentation of both sodiated and lithiated ions could provide information on glycosidic cleavages. All observed tandem mass fragmentation spectra provided valuable elatoside F structural information when unknown samples from crude extracts are under screening by mass spectrometry.

Enzymatic transformation of platycosides and one-step separation of platycodin D by high-speed countercurrent chromatography.

Platycosides, the saponins found in the roots of Platycodon grandiflorum (Platycodi Radix), are typically composed of oleanane triterpenes with two side chains. In platycosides, platycodin D, a glucose unit at C-3, is a major component, which has several pharmacological activities. Because of the high demand for this compound, we attempted to enzymatically convert platycodin D(3) and platycoside E, having two and three glucose units at C-3, respectively, into platycodin D. In this study, we tested the ability of several glycosidases to transform platycosides, or more specifically, the ability to transform platycoside E and platycodin D(3) into platycodin D. To obtain pure platycodin D on a preparative scale, high-speed countercurrent chromatography with a solvent system of ethyl acetate/n-butanol/water (1.2:1:2, v/v/v) was used for the separation of the enzymatically transformed product. Approximately 39.4 mg of platycodin D (99.8% purity) was obtained from 200 mg of the product in a one-step separation. The results strongly support the advantage of enzymatic transformation of the platycosides for the efficient enrichment of platycodin D in the complicated extract of the medicinal plant.

Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria.

Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MS(n)) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC-MS(n) analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na](+) ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.