Isolation of Soyasaponins I e VI from soybeans by semipreparatives HPLC and characterization by mass spectrometry.

INTRODUCTION 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 lower cholesterol level by inhibiting its absorption, be anti-carcinogenic and antihepatotoxic, and show anti-replicative properties against HIV. Lentils, one of the richest and cheapest sources of vegetable proteins, mainly contain soyasaponin I and soyasaponin VI. Because of the difficulties to obtain authentic, high purity soyasaponin standards, especially in the case of thermolabile soyasaponin VI, we started a project aiming at isolating pure soyasaponins I and VI from soybeans, to be used for quantitative analysis of soyasaponin content in various Italian cultivars of lentils. MATERIALS AND METHODS Extraction and purification of saponins were performed as follows: dried and finely ground soybeans were extracted with ethanol, then the alcoholic solution was filtered and concentrated. 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, containing 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. RESULTS AND DISCUSSION With the above described method, we have been able to obtain pure soyasaponins with high purity (soyasaponin I: 80%; soyasaponin VI: 91%), which was assessed using HPLC-DAD at 206 and 292 nm.