Determination of Biflavonoids in Four Mexican Species of Selaginella by HPLC

The genus Selaginella comprises several species, some of which grow on the American continent. Among these species, S. nothohybrida Valdespino, S. lepidophylla Hook et Grey, S. pallescens (Presl) Spring, and S. reflexa Underw. are found in several states of the Mexican Republic. The aforementioned three species are used medicinally, typically to treat renal disorders. This paper describes the development of an HPLC-UV method for the determination of amentoflavone (1), robustaflavone (2), and (25)-2,3-dihydrorobustaflavone (3) as major components of the above-mentioned species of Selaginella. Components 1, 2, and 3 were quantitatively determined using an XBridge Waters C-18 5 mu m column, with a gradient system consisting of mixtures of acetonitrile and water with 0.4% acetic acid. The flow rate was 0.4 mL/min, with UV detection at 367 nm. LOD and LOQ values were in the range of 0.025 to 0.216 mu g/mL. Compounds 1, 2, and 3 showed good linearity in the 1.2 to 18 mu g/mL range; recovery was within 98.2 and 101.9% for all three cases. Compounds 1 and 2 were detected in all eight samples; their concentration ranged from 0.35 to 1.79 mg/g of plant. Thus, compounds 1 and 2 could be used as markers for S. nothohybrida, S. lepidophylla, S. pallescens, and S. reflexa. In addition, trehalose was detected in all samples as two peaks at 1.5 and 2.0 min. The HPLC method described here was shown to be reliable, reproducible, and accurate and can be used for QC of Selaginella medicinal materials.