ИСТИНА

The steroidal saponins of Tribulus terrestris L. (Zygophyllaceae) are considered to be responsible for biological activity of products and preparations based on this plant. The activity depends on the concentration and the composition of active saponins. Furostanol-type saponin – protodioscin has been considered as the most dominant component of T. terrestris while its spirostanol-type less polar analogue – dioscin is also found in this plant. Therefore, several extraction techniques were applied in order to achieve comprehensive extraction of protodioscin and dioscin from T. terrestris leaves. Ultrasound-assisted (UAE), refluxing (RE), low pressure refluxing (LPRE) and Soxhlet (SE) extraction techniques were developed for isolation of steroidal saponin from T. terrestris leaves. Extraction parameters for UAE (solvent base: methanol, ethanol, isopropanol and acetonitrile; the ratio of solvent to soil; isopropanol and acetonitrile concentration; extraction time), RE (solvent base: isopropanol and acetonitrile; the ratio of solvent to soil; isopropanol concentration) and SE (extraction time) were optimized by single factor experiment. In the course of optimization RE and SE methods, thermal instability of protodioscin was detected, which leads to thermal degradation at a long extraction time. Extraction time and temperature were optimized via two factor experiment for most promising RE and LPRE techniques. Acclaim RSLC C18 column was used for HPLC separation of the extracts followed by ESI-MS/MS detection of these compounds in single ion monitoring mode. Two characteristic ions with m/z 415 and 397 corresponding to diosgenin and dehydrated diosgenin protonated molecules were monitored during chromatographic run. The optimal conditions were chosen for UAE in isopropanol and acetonitrile based solvents. Then UAE in water-organic solvent was compared to RE, LPRE and SE showing the equal maximum of extraction yield in optimized extraction conditions and allowing to achieve approximately 1.2 times better extraction yields. Also the ability of complete isolation of protodioscin and dioscin at the first extraction step was demonstrated in multi-step experiment for RE and LPRE methods. Good recovery values were achieved for all four extraction techniques in optimized conditions but thermal decomposition of protodioscin was observed after 90 min of RE, therefore 60 min extraction time (at 92°C, using isopropanol 50% solution in water as a solvent) is recommended in case of using RE instead of LPRE.