Studies On Separation And Preparation Of Soyasaponins With High-speed Counter Current Chromatography

Soyasaponin is one of the important components in soybean, which has important bioactivities and physiological activities, such as reducing plasma cholesterol and guarding against atherosclerosis. Since it has great economic potential in pharmaceutical and food industries, soyasaponin's development is a key area of deep processing for soybean industry. However, as a strong polar compound and a surfactant, soyasaponin is difficult to be separated from other components of soybean. Until now, no good methods can be applied to isolate and purify these components with high efficiency.High Speed Counter Current Chromatography (HSCCC) is a new separation method. It can be applied in a wide range of chemicals, analyze large sample sizes and keep the bio-activity of target components. As HSCCC has not been used in soyasaponin purification, this research aims to optimize the solvent systems and operating conditions for soyasaponin separation by HSCCC.The paper mainly contains two parts: 1) isolating soyasaponins from the crude extracts with HSCCC, 2) purifying single soyasaponins with HSCCC.In the first part, n-butanol-water(1:1, v/v) and ethyl acetate-methanol- n-butanol-water (4:0.8:1:6, v/v/v/v) solvent systems were selected following the previous HSCCC studies for other saponins. The two solvent systems were also verified by scanning the crude extracts with HPLC and determining soyasaponins'partition coefficient. Then the two solvent systems, with different acetic acid concentration and different sample loading sizes, are carried out to isolate soyasaponin.In the second part, chloroform-methanol-n-butanon-water (4:3:0.1:2) and ethyl acetate-n-butanon-water(1:6:7) solvent systems were employed after studying the previous HSCCC studies in other single saponins'seperation. The two solvent systems were tested and the flow rate of mobile phase was optimized.It was discovered that:1) Ethyl acetate-methanol-n-butanol-water (4:0.8:1:6, v/v/v/v) solvent system has low stationary phase retention rate, and fails to isolate soyasaponins appropriately.2) n-butanol-water(1:1, v/v) with 5% acetic acid is the optimized solvent system to completely separate soyasaponins from the crude extract.3) Sample sizes can affect the separation and the optimized sample size for 300mL HSCCC is 100mg.4) Three single soyasaponins, Ae, Ab and DDMP soyasaponin were successfully purified after two HSCCC steps.Based on these results, it was concluded that with its high efficiency and ability to keep DDMP soyasaponins'bio-activity, HSCCC is a very promising method for soyasaponin separation.