Abstract:

A RP-HPLC method was established for the simultaneous determination of puerarin, daidzin and daidzein in roots, stems and leaves of Pueraria lobata (Wild) Ohwi. The procedure was based on extraction of test portions with 70% ethanol solution in an ultrasonic field for 20 min at 50 ℃ followed by direct HPLC analysis of the extracts. The chromatographic conditions were as follows: using a Hypersil ODS-2 column (4.6 mm × 250 mm, 5 μm) and a mobile phase composed of methanol and H2O, gradient elution [0－4 min 25:75 (methanol/water volume ratio), 4－7 min 25:75 → 32:78, 7－9min 32:68, 9－11 min 32:68 → 50:50, 11－24 min 50:50, 24－30 min 50:50 → 25:75], column temperature 35 ℃, detection wavelength 250 nm, and flow rate of the mobile phase 0.8 ml/min. A good linear relationship between peak area and concentration was found to puerarin, daidzin and daidzein in the ranges of 11.87－73.91 (r = 0.9998), 1.00－6.48 (r = 0.9997) and 0.28－1.53 μg/ml (r = 0.9994), respectively. The LODs were 0.43, 0.24 and 0.18μg/ml, respectively. Mean recoveries were 99.78%, 102.61% and 105.57% with the RSDs of 1.6%, 2.8% and 3.4% (n = 5), respectively. The precision, stability and reproducibility of the method all were good. The contents of puerain, daidzin, and daidzein in roots of Pueraria lobata (wild) Ohwi determined by this method were 3.98%, 0.36% and 0.039% respectively, higher than 3.28%, 0.26% and 0.028% of those analyzed according to the method stipulated in the Chinese Pharmacopoeia 2005 respectively, suggesting high efficiency of ultrasonic-assisted extraction of the three analytes. The contents of puerain, daidzin, and daidzein in stems of Pueraria lobata (wild) Ohwi were 0.004%, 0.0032% and 0.0007% respectively, while those in leaves were 0.0016%, 0.005% and 0.0012%, respectively. This method is accurate, fast and simple and therefore can be used for the simultaneous determination of puerain, daidzin, and daidzein in roots, stems and leaves of Pueraria lobata (wild) Ohwi. Our results demonstrate that the contents of puerain, daidzin, and daidzein in stems and leaves of Pueraria lobata (wild) Ohwi growned in Maoshan area of Jiangsu province are obviously lower than those in the roots.

Key words: Pueraria lobata (wild) Ohwi, puerarin, daidzin, daidzein, HPLC