Orthogonal test design for optimizing the extraction of total flavonoids from Flos pueraria

The conditions for extraction of the total flavonoids from Flos pueraria was optimized and the purification and separation process were conducted to identify the main constituents of the total flavonoids as well. The solvent for extraction and its concentration, the solid-to-liquid ratio, extraction duration, temperature and ultrasonic frequency were investigated through a single-factor experiment. An orthogonal design (L9 (34) was constructed to achieve the best extraction conditions. The crude extract was then purified sequentially by petroleum ether, ethanol and chloroform, n-butyl alcohol, and eluted gradually with mixed mobile phase of methanol-chloroform solution in the silica gel column system. The ingredients were further separated by color reaction, ultraviolet spectrophotometry, high performance liquid chromatography, infrared and mass spectral analysis. The optimum extraction condition for the total flavonoids from F. pueraria was as follows: extraction  by 50% (v/v) methanol solution, the solid-to-liquid ratio at 1:30, extraction duration 2.0 h, the temperature of 70°C, ultrasound 3 times and 30 min each time. Five isoflavones were separated and identified as irisolidone, genistein, daidzein, kakkalide and puerarin, respectively. Under these optimal conditions, the yield of total flavonoids in the extracts was up to 17.5%, while the output rate previously reported is about 8% in general. Our study provided means for further development and utilization of the bioactive components from F. pueraria. Key words: Flos pueraria, flavonoids, orthogonal design, purification, ultrasound-assisted extraction.