HPLC determination of flavonoid glycosides in Mongolian Dianthus versicolor Fisch. (Caryophyllaceae) compared with quantification by UV spectrophotometry.

INTRODUCTION: Dianthus versicolor is used in traditional Mongolian medicine against liver impairment. Fractions enriched in flavone-di- and triglycosides were shown to enhance bile secretion. Therefore, reliable and accurate analytical methods are needed for the determination of these flavonoids in the crude drug and extracts thereof. OBJECTIVE: To provide a validated HPLC-DAD (diode array detector) method especially developed for the separation of polar flavonoids and to compare the data obtained with those evaluated by UV spectrophotometry. METHODOLOGY: Separations were carried out on an Aquasil® C18-column (4.6 mm × 250.0 mm, 5 µm) with a linear gradient of acetonitrile and water (adjusted to pH 2.8 with trifluoroacetic acid) as mobile phase. Rutoside was employed as internal standard with linear behavior in a concentration range of 0.007-3.5 mg/mL. Accuracy was determined by spiking the crude drug with saponarin resulting in recoveries between 92% and 102%. RESULTS: The method allows the quantification of highly polar flavonoid glycosides and the determination of their total content. For saponarin a linear response was evaluated within the range 0.007-3.5 mg/mL (R² > 0.9999). It was proven that threefold sonication represents a time-saving, effective and cheap method for the extraction of the polar flavonoid glycosides. The contents determined by HPLC were shown to be in agreement with those obtained employing UV spectrophotometry. CONCLUSION: The study has indicated that the newly developed HPLC method represents a powerful technique for the quality control of D. versicolor. Ultraviolet spectrophotometry may be used alternatively provided that the less polar flavonoids are removed by purification.

Flavonoid C- and O-glycosides from the Mongolian medicinal plant Dianthus versicolor Fisch.

Eighteen flavonoids were identified from an aqueous extract of the aerial parts of Dianthus versicolor, a plant used in traditional Mongolian medicine against liver diseases. The flavonoid C- and O-glycosides isoorientin-7-O-rutinoside, isoorientin-7-O-rhamnosyl-galactoside, isovitexin-7-O-rutinoside, isovitexin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-rutinoside, isoscoparin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-galactoside, and isoorientin-7-O-galactoside were isolated and structurally elucidated. Their structures were established on the basis of extensive spectroscopic techniques including LC-UV-DAD, LC-MS(n), LC-HRMS, 1D and 2D NMR spectroscopy, and by GC-MS analysis after hydrolysis. Flavonoids with such a high glycosylation pattern are rare within the genus Dianthus. Furthermore, isovitexin-7-O-glucoside (saponarin), isovitexin-2″-O-rhamnoside, apigenin-6-glucoside (isovitexin), luteolin-7-O-glucoside, apigenin-7-O-glucoside, as well as the aglycons luteolin, apigenin, chrysoeriol, diosmetin, and acacetin were identified by TLC and LC-DAD-MS(n) in comparison to reference substances or literature data. The NMR data of seven structures have not been reported in the literature to date.

Quantification of flavonoid glycosides in an aqueous extract from the traditional Mongolian medicinal plant Dianthus versicolor FISCH.

An HPLC-diode array detection (DAD) method was established in order to investigate dried aerial parts of Dianthus versicolor FISCH. (Caryophyllaceae), a plant used in traditional Mongolian medicine against liver impairment. Aqueous extracts were separated on an Aquasil(®) C(18) column with a linear gradient of acetonitrile (ACN) and water (adjusted to pH 2.8 with formic acid) as the mobile phase. LC-IT-MS facilitated the assignment of 26 flavonoids, among them a series of rare C-glycosylated as well as O-glycosylated derivatives, which are assumed to be the active principles. Quantification was performed and validated using isovitexin-7-O-glucoside (saponarin) as the external standard. The method showed good linear behaviour (r(2) ≥0.9999) over the investigated concentration range (0.007-3.5 mg/mL). The good precision of the method allowed the successful qualitative and quantitative analysis of flavonoid-glycosides in the aqueous extracts prepared from five different D. versicolor samples. Depending on the origin of the samples, the total flavonoid content was found to vary considerably from 0.41 to 3.30% in the aqueous extracts and from 0.07 to 0.57% in the crude drug. In addition, the relative composition of the various flavonoids was found to differ strongly. These results highlight the need for proper quality control for this herbal drug.

Extracts from the Mongolian traditional medicinal plants Dianthus versicolorFisch. and Lilium pumilum Delile stimulate bile flow in an isolated perfused rat liver model.

AIM OF THE STUDY: Dianthus versicolor (Caryophyllaceae) and Lilium pumilum (Liliaceae) are two medicinal plants used in traditional Mongolian medicine to treat hepatic and gastrointestinal disorders. In this study aqueous (AE) and methanolic (ME) extracts of Dianthus versicolor and Lilium pumilum were investigated for their influence on the bile flow. The aqueous extracts of both plants were tested in absence and presence of 10 µM taurocholic acid at three different concentrations (100, 250, and 500 mg/L). The aqueous extract of Dianthus versicolor was further purified in order to locate the active principles. Two resulting fractions, one enriched in flavonoids and the other in sugars, were investigated for their influence on the bile flow in absence of taurocholic acid at 10, 20, and 40 mg/L. The aqueous extracts of both plants were analysed qualitatively by LC-MS(n) and quantitatively by UV-spectrophotometry. MATERIALS AND METHODS: The bile flow experiments were performed in the isolated perfused rat liver. The compounds were identified by LC-DAD-MS(n) and TLC using references. The UV-spectrophotometric analysis was based on the monograph "Passiflorae herba" of the European Pharmacopoeia, and the total flavonoid contents were calculated and expressed as vitexin. RESULTS: AE and ME of both plants increased the bile flow dose-dependently (between 9% and 30%), and no hepatotoxic effect was seen even during longer perfusions. Stimulation of bile secretion was comparable in the presence and in the absence of taurocholic acid. The flavonoid fraction of Dianthus versicolor increased the bile flow by 18% (p<0.05) at 40 mg/L, which was comparable to the positive control cynarin. The phytochemical investigations of the Dianthus versicolor AE (total flavonoid content 1.78%) revealed the presence of the isovitexin derivative saponarin. In the AE of Lilium pumilum (total flavonoid content 1.04%) the flavonoids rutoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were detected. CONCLUSIONS: The results show that choleresis under extract application is due to a stimulation of the bile-salt-independent bile flow which might be caused by the osmotic power of the extracts (hydrocholeresis). The flavonoids seem to contribute to the bile-flow-stimulating effect of Dianthus versicolor. Both plants exhibit a considerable choleretic effect that contributes to their use in traditional Mongolian medicine against gastrointestinal disorders.