Determination of Seven Biflavones of Selaginella Doederleinii by High Performance Liquid Chromatography

Abstract

Selaginella doederleinii Hieron (S. doederleinii) is widely used as an antitumor and anti-inflammatory agent in Asia, and is famous for containing biflavonoids, which have been reported as the bio-active constituents possessing anticancer and anti-oxidant effects. However, a simple and systematic quality control method for determining biflavonoids in S. doederleinii is lacking. In this study, seven biflavones, including amentoflavone, robustaflavone, 2″, 3″-dihydro-3′, 3″′-biapigenin; 3′, 3″′-binaringenin, delicaflavone, heveaflavone, and 7, 4′, 7″, 4″′-tetra-O-methyl-amentoflavone, were obtained from S. doederleinii. These seven compounds that are present in high concentration were used as detection markers for development of a quality control method by HPLC with a UV-visible detector. The chromatographic separation was accomplished on a C₁₈ column with a mobile phase composed of acetonitrile–water (0.5% acetic acid) in 52 min. With the optimized conditions, the seven biflavones showed good regression relationships (R² > 0.9989) within the test range and the recovery was in the range of 90.1–102.5%. The LODs of the seven analytes were between 0.2 and 1.17 µg/mL. The method was also validated for precision and accuracy and was successfully applied to the determination of these compounds in six batches of S. doederleinii from different areas. In conclusion, the present study provides a systematic and useful method for quality evaluation of S. doederleinii.

Keywords:

• Biflavonoids
• HPLC
• Preparative-HPLC
• Quality control
• Selaginella doederleinii Hieron

Acknowledgments

The authors gratefully acknowledge the financial support of the Foundation of National High Technology and Development of China (863 projects: 2012AA022604), the National Nature Science Foundation (No. 21275028 and 81202987), the Fujian Provincial Natural Science Foundation (2012J01131), the International Corporation Program of Science and Technology Department of Fujian Province (2009I0009), and Fujian Medical University Program (01601086).

Notes

^a y: peak area of analyte; x: concerntration of analyte (µg/mL).

^b Limits of detection were evaluated at signal-to-noise ratios (S/N) beyond 3.

The RSDs were calculated by the results of six determinations for each analyte except for stablility examination.

^a The RSDs for stability were calculated from the determination results for each analyte in the same sample solution after storing for 0, 2, 4, 8, 10, 12 and 24 hrs.