A strategy for screening antioxidants in Ginkgo biloba extract by comprehensive two-dimensional ultra high performance liquid chromatography.

Recently, screening of bioactive compounds by on-line ultra-high performance liquid chromatography (UHPLC) draws increasing attentions for the advantages of rapidity and intuition. Nevertheless, most on-line methods were limited to the shortcoming like low resolution and peak capacity, which could interfere the active ingredient identification. Comprehensive two-dimensional UHPLC (LC×LC) has revealed to be a powerful tool to separate complex mixtures. Herein, a strategy based on LC×LC analysis coupled with pre-column 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was proposed to screen the antioxidants from the extract of Ginkgo biloba (EGB). A total of 61 compounds were identified in EGB, and 25 of them showed appreciable radical scavenging capacity. This work may offer pharmacodynamics base for further research about EGB, also the strategy is likelihood to be applied in screening antioxidant in other herbal medicine.

Accurate analysis of ginkgolides and their hydrolyzed metabolites by analytical supercritical fluid chromatography hybrid tandem mass spectrometry.

Hydrolysis plays an important role in the metabolic transformations of lactones. Analysis of lactones and their hydrolyzed metabolites in biological samples is challenging, because unexpected hydrolysis or reversed dehydration may occur depending on the test environments. In this work, a supercritical fluid chromatography hybrid triple-quadrupole mass spectrometry (SFC-QQQ MS) method has been proposed for simultaneous analysis of ginkgolides and hydrolyzed metabolites. The SFC utilizes carbon dioxide as the mobile phase, avoiding hydrolysis of ginkgolides that always happens during reversed phase liquid chromatographic detection. Compared with normal phase liquid chromatography, the SFC provides good resolutions especially for ginkgolide with similar structures. This SFC-QQQ MS method was validated in linearity, precision, accuracy, and stability for ginkgolides and metabolites. Then this method was successfully applied to pharmacokinetic study of 3 ginkgolides and their 6 hydrolyzed metabolites after intravenous administration of total ginkgolide extract. Ginkgolides and metabolites showed different clear rates and excluded in 2-4h. The developed SFC-QQQ MS method allows accurate determination of ginkgolides and metabolites with high resolutions, and can be extended to analysis of other water-unstable compounds.

Pharmacokinetic studies of ginkgolide K in rat plasma and tissues after intravenous administration using ultra-high performance liquid chromatography-tandem mass spectrometry.

Ginkgolide K (GK), a derivative compound of ginkgolide B, has been recently isolated from the leaves of Ginkgo biloba. It is a powerful natural platelet activate factor (PAF) antagonist, and also has obvious protect effects for cerebral ischemia. However, no reports have been described for the pharmacokinetic study of GK. In this study, a simple, sensitive and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the determination of GK in rat plasma and tissues. Biological samples were pretreated by an efficient liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent ZORBAX SB-Aq column (4.6 mm × 50 mm, 1.8 µm) with a mobile phase of 0.5% aqueous formic acid (A)-menthol (B). Quantitation was carried out on a triple quadruple mass spectrometry using positive electrospray ionization in multiple reaction monitoring mode. Diazepam was used as internal standard (IS). The ion transitions monitored were set at m/z 407.10 → 389.20 and m/z 285.08 → 193.10 for GK and IS, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of GK after intravenous administration. The current results have indicated that pharmacokinetic parameters of GK vary in a dose-dependent manner with rapid elimination in 4h. The major distribution tissues of GK in rats were liver and kidney. This study would provide critical information to promote the future study of GK.

Rapid preparation of rare ginsenosides by acid transformation and their structure-activity relationships against cancer cells.

The anticancer activities of ginsenosides are widely reported. The structure-activity relationship of ginsenosides against cancer is not well elucidated because of the unavailability of these compounds. In this work, we developed a transformation method to rapidly produce rare dehydroxylated ginsenosides by acid treatment. The optimized temperature, time course, and concentration of formic acid were 120°C, 4 h and 0.01%, respectively. From 100 mg of Rh1, 8.3 mg of Rk3 and 18.7 mg of Rh4 can be produced by acid transformation. Similarly, from 100 mg of Rg3, 7.4 mg of Rk1 and 15.1 mg of Rg5 can be produced. From 100 mg of Rh2, 8.3 mg of Rk2 and 12.7 mg of Rh3 can be generated. Next, the structure-activity relationships of 23 ginsenosides were investigated by comparing their cytotoxic effects on six human cancer cells, including HCT-116, HepG2, MCF-7, Hela, PANC-1, and A549. The results showed that: (1) the cytotoxic effect of ginsenosides is inversely related to the sugar numbers; (2) sugar linkages rank as C-3 > C-6 > C-20; (3) the protopanaxadiol-type has higher activities; (4) having the double bond at the terminal C20-21 exhibits stronger activity than that at C20-22; and (5) 20(S)-ginsenosides show stronger effects than their 20(R)-stereoisomers.