HPLC-MS-Analysis of flavonoid-C-glycosides in the Mongolian medicinal plant Dianthus versicolor

Dianthus versicolor is used in Traditional Mongolian Medicine against liver diseases together with other Mongolian plants. Until now chrysoeriol-C-glycosides were found in the plant [1]. Our first investigations of an aqueous extract (OWE) by TLC indicated the occurrence of further flavonoids. For preparation of the OWE the plant material was powdered and extracted with water (pH 2, trifluoroacetic acid) at 40°C for 1h by shaking gently. To purify the extract and concentrate the flavonoids Solid Phase Extraction (SPE) was used as appropriate method. This fraction increased the bile flow in the perfused isolated rat liver [2]. For further investigations an analytical HPLC-method had to be developed to provide a separation technique which may be coupled to MS. HPLC-analyses were performed on a Shimadzu liquid chromatograph (LC-20 AD) using RP-18e, Luna Phenyl-hexyl and Aquasil C 18 as stationary phases. Aquasil C 18 showed the best separation of the seven main flavonoids. HPLC-ESI-MS experiments were carried out with a Perkin Elmer liquid chromatograph (Series 200) coupled to a PE Sciex API 150 EX single quadrupole instrument. According to retention time, mass- and UV-spectra, one flavonoid was supposed to be apigenin-6,7- O -diglucoside (saponarin). To confirm this hypothesis a small amount of this compound was isolated by semipreparative HPLC and compared to pure saponarin by TLC and UV-experiments using shift reagents (AlCl 3 /HCl and NaOCH 3 ). The isolated substance showed the same R f -value and fluorescence colour after detection with natural compound reagent A at UV 366nm. The reaction to UV-shift reagents was the same as the pure substance, therefore the compound was identified as apigenin-6,7- O -diglucoside. References: [1] Boguslavskaya, L.I. et al. (1983) Khim. Prir. Soedin. 6: 783–4; [2] Glasl S., Tsendayush D. et al. (2007) Planta Med. 73: 59–66