Distribution of sapogenins in morphological Medicago sativa L. parts: Comparison of various extraction techniques.

Saponins in plant extracts were indirectly determined by estimation of the content of sapogenins. The first step of determination is extraction with high efficiency. One conventional extraction technique (maceration) and two modern ones (accelerated solvent extraction and supercritical fluid extraction) were compared. Methanol and ethanol were used as solvents or co-solvents. The results were supported by statistical analysis. Saponins were extracted from leaves, roots, and sprouts of Medicago sativa. Acid hydrolysis, purification, and determination by high-performance liquid chromatography with evaporative light scattering detector were used. The content of sapogenins was the highest in the roots. Smaller amounts of sapogenins were found in sprouts and the smallest ones in leaves. The main ingredient was medicagenic acid with mean concentration of 621.8 µg/g in roots, 456.7 µg/g in sprouts, and 471.3 µg/g in leaf extract. The highest content of sapogenins in extract was obtained after maceration with methanol; however, this method is nonselective in relation to biologically active compounds. Due to the possibility of using the obtained extracts with sapogenins in the cosmetic or pharmaceutical industry, the selection of extraction techniques and solvents is a very important aspect. Additionally, the chosen technique should be considered eco-friendly and consistent with the assumptions of "green chemistry."

Isolation and determination of saponin hydrolysis products from Medicago sativa using supercritical fluid extraction, solid-phase extraction and liquid chromatography with evaporative light scattering detection.

Saponins are widespread secondary metabolites with various beneficial properties: fungicidal, antibacterial, antiviral, and anticancer. Alfalfa saponin molecules contain mainly: medicagenic acid, hederagenin, bayogenin, and soyasapogenol B. Structural diversity of saponins makes their determination in Medicago sativa extracts very difficult. The most popular determination technique is high-performance liquid chromatography applied with evaporative light scattering detection. Qualitative and quantitative analysis of sapogenins from Medicago sativa by high-performance liquid chromatography with evaporative light scattering detection required hydrolysis and purification of extracts obtained by supercritical fluid extraction. Hydrolysis of saponins with concentrated hydrochloric acid provided high concentration of medicagenic acid. In the purification process, satisfactory results were obtained for solid-phase extraction using octadecyl. Recoveries were from 71 to 99% with a standard deviation from 2 to 8. Hydrolysis with concentrated hydrochloric acid was the only method that allowed identification of all four analyzed sapogenins. Moreover, it is characterized by a short time of preparation, simplicity of execution, a small amount of the sample and solvents. The hydrolysis and purification methods coupled with high-performance liquid chromatography and evaporative light scattering detection can be successfully used for qualitative and quantitative analysis of the main saponins present in Medicago sativa plant extracts obtained by supercritical fluid extraction.