Simultaneous determination of flavonoids and phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides by high-performance liquid chromatography

Abstract

Introduction Mullein (Verbascum) flowers are highly valued herbal drugs used in the treatment of inflammation, asthma, spasmodic coughs and other respiratory tract diseases. Their phenolic constituents are considered to be responsible for the anti-inflammatory and antimicrobial activity of the herb. However, knowledge about the contents of phenolics in flowers is limited and no HPLC method for their analysis is available. Objective To develop and validate an RP-HPLC-UV method for the simultaneous determination of eight flavonoids and two phenylethanoids in the flowers of Verbascum densiflorum and V. phlomoides. Methodology HPLC separation was accomplished on a C(18) Lichrosphere 100 column (5 microm, 250 mm x 4.6 mm, i.d.) with an acetonitrile gradient elution using aqueous 0.5% (w/v) orthophosphoric acid solution containing 1% (v/v) tetrahydrofurane. Results All the calibration curves showed good linear correlation coefficients (r > 0.997) over the wide test ranges. The relative standard deviation of the method was less than 3.4% for intra- and inter-day assays, and the average recoveries were between 93.5 and 101.9%. High sensitivity was demonstrated with detection limits of 0.062-0.083 microg/mL for flavonoid aglycones, 0.156-0.336 microg/mL for flavonoid glycosides and 0.390-0.555 microg/mL for phenylethanoids. The flower samples of V. phlomoides were found to contain high levels of diosmin and tamarixetin 7-rutinoside (2.327-2.392% of dry weight), whereas verbascoside (0.688-0.742% of dry weight) and luteolin 7-glucoside (0.204-0.279% of dry weight) dominated in the V. densiflorum flower. Conclusion The HPLC method established is appropriate for the quality assurance and the differentiation of V. phlomoides and V. densiflorum samples.