Separation, Purification Of Alfalfa Saponins And Their Structure Elucidation

Alfalfa(Medicago sativa L.)is a major legume forage crop.It has been served as a folk medicine used in the treatment of cough,indigestion,gravel,ect.in China.The development research on alfalfa has been mainly focused on forage and hay processing,and alfalfa is a protencial medicinal resources.Firstly,in this paper,a colorimetric method was established for determination of total triterpene saponins in Medicago sativa L.,Medicago lupulina L.and Medicago polymorpha L..The method was convenient,accurate and repeadable.The saponin content of different parts of three species,eg.roots,stems and leaves was also examined and compared.We also examined the saponin content of Medicago polymorpha L.at different growth period,this work has a certain significance on the reaserch on germplasm resource and standard control of Medicago sativa L..We also optimized the parameters in the purification of the triterpene saponins from alfalfa with D101 macroporous resin.Column packed with the D101 resin was also used to perform dynamic adsorption and desorption tests to optimize the separation process.The optimum parameters for the adsorption were:triterpene saponins in solution:2.2 mg/mL,processing volume 1 BV,flow rate:1.5BV/h,pH 4. For desorption:25%ethanol,4BV;then 60%ethanol(pH=12),4 BV as eluent,flow rate:2BV/h.After one run of adsorption and desorption,the contents of triterpene saponins were increased from 0.57%to 36.68%,the recovery yield was 73.5%.The chemical constituents of the alfalfa saponin were sdutied.After separation on D101 macroporous resin,RP-C18 column and preparative HPLC were used to isolate 2 triterpene saponins from crude alfalfa saponins.The structures of two compounds were identified by spectral methods:IR,MS,¹H-NMR,¹³C-NMR and HSQC spectrum.The structures of five compounds were identified as soyasaponin Bb and azukisaponin V,with azukisaponin V isolated first from Medicago sativa L..