Isolation of Soyasaponins I and VI from soybean by semipreparatives HPLC and characterization by mass spectrometry

Saponins are triterpenoid or steroid glycosides naturally occurring in plants. Soyasaponins from soy and other legumes are the primary dietary sources of food saponins. Soyasaponins have been demonstrated to possess multiple health-promoting properties, such as lowering of cholesterol level by inhibiting its absorption, being anti-carcinogenic and antihepatotoxic, and showing anti-replicative properties against HIV [1]. According to their molecular structures, soyasaponins can be divided into two groups, i.e. A and B. Soyasaponins belonging to group A are bidesmosidic saponins having two glycosylation sites on their aglycone moiety (soyasapogenol A), while group B soyasaponins are monodesmosidic saponins having one glycosylation site on two different aglycone moieties (soyasapogenol B and E). Lentils, one of the richest and cheapest sources of vegetable proteins, mainly contain soyasaponin I, also named soyasaponin Bb, and soyasaponin VI, otherwise named g, both belonging to the soyasaponins B group [2]. Because of the difficulties to obtain authentic, high purity soyasaponin standards [3], especially evident for soyasaponin VI that is thermolabile, we started a project aiming at isolating pure soyasaponins I and VI from soybeans, followed by quantitative analysis of soyasaponin content in various Italian cultivars of lentils. Extraction and purification of saponins were performed as follows: dried and finely ground soybeans were extracted with 70% aqueous ethanol at room temperature for 3 h, then the alcoholic solution was filtered and concentrated at temperature < 30°C by a rotary evaporator. The crude extract was purified on a C18 flash-chromatographic column using acetonitrile-water as eluting mixture (gradient); the collected fractions, containing both soyasaponins, were injected in a semipreparative HPLC system equipped with a C18 column using a mixture of methanol/water (83/17), containing 0.25 % of acetic acid, as mobile phase. Each saponin was detected at its specific wavelength: 206 nm for soyasaponin I, and 292 nm for soyasaponin VI. After HPLC analysis, the pure soyasaponins I and VI were collected and their structures identified using the HPLC-ESI-IT (Ion Trap), performing MSn experiments and studying their typical fragmentations. The purified soysaponins were used as pure standards for the quantitation of saponins in various cultivars of lentils. References [1] L. Gu, G. Tao, W. Gu, R.L. Prior Journal of Agricultural and Food Chemistry, 2002, 50, 6951-6959. [2] R.G. Ruiz, K.R. Price, A.E. Arthur, M.E. Rose, M.J.C. Rhodes, R.G. Fenwick Journal of Agricultural and Food Chemistry, 1996, 44, 1526-1530. [3] J. Hu, S.O. Lee, S. Hendrich, P.A. Murphy Journal of Agricultural and Food Chemistry, 2002, 50, 2587-2594.