Flavonoids in Astragalus corniculatus

ARTIGO

Flavonoids in Astragalus corniculatus

Ilina Krasteva^*; Stefan Nikolov

Department of Pharmacognosy, Faculty of Pharmacy, Medical University, 2 Dunav St., 1000 Sofia, Bulgaria

ABSTRACT

Nine flavonoids were identified in aerial parts of Astragalus corniculatus Bieb. (Fabaceae) by liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection. Vitexin, orientin and eriodictyol-7-O-glucoside are obtained for the first time in genus Astragalus L, and isorhamnetin-3-O-glucoside in the species.

Keywords: Fabaceae; Astragalus corniculatus; flavonoids.

INTRODUCTION

Astragalus corniculatus Bieb. (Fabaceae) is a perennial herbaceous plant, distributed in Moldova, South Ukraine and Southeastern Romania¹. The species was recently found in Bulgarian flora². Our earlier investigations of the ethyl acetate extract of this species, containing flavonoids, resulted in a low acute oral toxicity and a remarkable antihypoxic activity, especially in a model of circulatory hypoxia³.

The aim of this study is to determine the flavonoids in the ethyl acetate extract of A. corniculatus using liquid chromatography coupled with ionspray mass spectrometry in tandem mode (LC/MS/MS).

EXPERIMENTAL

General procedures

LC analysis was performed on a Agilent 1100, Model G1312A O (Hewlet Packard, Palo Alto, CA, USA). An Aqua C18 125 A (150 x 3.0 mm i.d., 5 mL) (Phenomenex, Torrance, CA, USA) was used. Gradient elution was carried out with water - 0.1% formic acid and water acetonitrile - 0.1% formic acid at a constant flow rate of 400 µL min^-1. The MS and MS/MS data were obtained by using an API 365 tripe-quadroupole mass spectrometer (Perkin-Elmer Sciex, Concord, ON, Canada). All the analyses were performed using a Turbo Ionspray source. The operating parameters as follows: capillary voltage – 3500 V, nebolizer gas (N₂; 10 arbitrary units), curtain gas (N₂; 8 arbitrary units), draying gas (N₂; 7000 cm³/min^-1), collision gas (N₂; 5 arbitrary units), focusing potential – 240 V and entrance potential 10 V. The collision energy (CE) and declustering potential (DP) were optimized for each standard. Thin layer chromatography (TLC) was performed on silica gel plates (Kieselgel 60 F₂₅₄, Merck, Germany). The spots were visualized by spraying with 1% methanolic solution of diphenylboric acid aminoethyl ester (NST). Column chromatography (CC) was performed on cellulose (Watman, Germany), Polyamid S (Riedel-de Haën, Germany) and Sephadex LH-20 (Pharmacia, Sweden).

Plant material

Astragalus corniculatus herbs were collected in July 1999 near the town of Svishtov, Bulgaria and identified by Dr. D. Pavlova. A voucher specimen has been deposited in the Herbarium of the Faculty of Biology, Sofia University, Bulgaria (SO95265).

Extraction and obtained of purified flavonoid fractions

Air-dried plant material (800 g) was powdered and extracted with 50% EtOH under reflux. After the removal of ethanol in vacuo the aqueous residue was consecutively treated with CHCl₃ and EtOAc. The EtOAc extract was evaporated to dryness to give a solid residue (14 g), which was submitted to column chromatography on cellulose, using 0-95% EtOH linear gradient. Further purification of combined fractions (TLC analysis) was achieved by rechromatography over Polyamid and Sephadex LH-20. Four main purified flavonoid fractions were obtained and analysed by LC/MS/MS.

RESULTS AND DISCUSSION

Solvent partition and repeated column chromatography over cellulose, Polyamide S and Sephadex LH-20 of the ethyl acetate extract to was submitted to fractionation, and the flavonoid fractions were analysed by LC/MS/MS. Standard solutions of 12 flavonoids were studied in the negative ion mode using MS/MS product ion scans (multiple reactions monitoring (MRM). For flavonol and flavanon O-glycosides, the spectra present both the deportment molecule [M-H]^– of the glycosides and the ion corresponding to the deprotonated aglycone [A-H]^–. The latter ion is formed by loss of the sugar residue from the glycosides⁴. Fragmentation of aglycones provided characteristic ions for each family of flavonoids⁵. Isorhamnetin exhibits specific fragmentation with the loss of methyl radical, thus giving m/z 315 ® m/z 300⁶. Hyperoside and isoquercetin have the same molecular mass and were identified together. Different fragmentation patterns were observed in MS/MS experiments for flavone-C-glycosides. Losses of 120 and 90 u were observed, corresponding to cross-ring cleavages in the sugar unit^7,8 (Figure 1).

After MRM analysis of flavonoid fractions, nine flavonoids were identified as quercetin, quercetin-3-O-rutinoside (rutin), quercetin-3-O-galactoside (hyperoside), quercetin-3-O-glucoside (isoquercitrin), kaempferol, isorhamnerin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside and eriodictyol-7-O-glucoside (Table 1).

Apigenin-8-C-glucoside (vitexin) and luteolin-8-C-glucoside (orientin) were identified on the bases of the product ion spectrum and comparison with literature data⁹. Vitexin shows the ions at m/z 431 (deprotoned molecule), m/z 341 (loss of 90 u) and m/z 311 (loss of 120 u) as characteristic ions in the MS/MS mode (Table 2). The product ion spectra of the ion m/z 447 of orientin differ in relative abundance of the m/z 357 (loss of 90 u) and m/z 327 (loss of 120 u) ions (Table 2). Moreover, the ion spectra of the ion at m/z 429 in the spectrum of isoorientin, was not present in that of orientin and for isovitexin – the ion at m/z 353 wasn't absent in the spectrum of vitexin¹⁰.

CONCLUSIONS

LC/MS/MS analysis of ethyl acetate fractions from the aerial parts of Astragalus corniculatus led to the identification of nine flavonoids. Vitexin, orientin and eriodictyol-7-O-glucoside are identified for the first time in genus Astragalus L, and isorhamnetin-3-O-glucoside – in the species.

ACKNOWLEDGEMENTS

This work was partially supported by EU Socrates-Erasmus programme (Bilateral collaboration between Medical University, Sofia, Bulgaria and Fachhochschule Halle – Anhalt, Köthen, Germany).

Recebido em 10/11/06; aceito em 25/5/07; publicado na web em 19/12/07

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Publication Dates

• Publication in this collection
  22 Feb 2008
• Date of issue
  2008

History

• Accepted
  19 Dec 2007
• Reviewed
  25 May 2007
• Received
  10 Nov 2006