Simultaneous extraction and purification of polysaccharides from Gentiana scabra Bunge by microwave-assisted ethanol-salt aqueous two-phase system

An effective method of concurrent extraction and purification for polysaccharides from Gentiana scabra Bunge stems was developed by microwave-assisted aqueous two-phase extraction (MA-ATPE). Ethanol/sodium dihydrogen phosphate was employed to form aqueous two-phase system (ATPS) by evaluating the extraction properties of five combinations of ethanol/salt systems. The important factors, namely, the composition of ATPS, extraction temperature, solvent-to-material ratio, extraction time and the particle size were investigated by single factor experiment and response surface methodology (RSM) combined with Box-Behnken Design (BBD). The final extraction conditions were concluded as follows: ethanol concentration 21.73% (w/w) and NaH2PO4 concentration 23.27% (w/w) for the ATPS, extraction temperature 95 °C, extraction time 5.8 min, solvent-to-material ratio 21:1 (mL/g) and particle size 80 mesh. Under the optimum conditions, the extraction yield of polysaccharides were 15.97 ± 0.32% for the top phase, 0.58 ± 0.26% for the bottom phase and 16.55 ± 0.13% for total polysaccharides, and the purity of water-soluble polysaccharides was 78.19 ± 0.42%. Compared with smashing tissue extraction (STE), microwave assisted-extraction (MAE), ultrasonic-assisted extraction (UAE) and heat reflux extraction (HRE), microwave-assisted aqueous two-phase extraction not only improved the yield and purity of water-soluble polysaccharides, but also obtained a variety of polysaccharides. In addition, the chemical characterization of polysaccahrides from both the two phases were carried out by Fourier transforms infrared spectrum (FT-IR) and High performance liquid chromatography derivatization with 1-phenyl-3-methy-5-pyrazolone (HPLC-PMP). The extraction mechanism was also discussed by scanning electron microscope (SEM). Therefore, as an efficient and promising technique, microwave-assisted aqueous two-phase extraction may be a viable option for the simultaneous extraction and purification of polysaccharides from plants.