ИСТИНА

Introduction For thousands of years, herbal medicines made from terrestrial plants were used by humans to prevent and treat diseases. Extraction is the crucial first step in the analysis of medical plants. Researches have shown that most of the therapeutic properties of D. deltoidea and T. terrestris are due to the presence of steroidal glycosides. The main objectives of this study were to develop a complete strategy of isolation and HPLC-MS determination of steroidal glycosides. Methods The HPLC separation was conducted on a C18 column. The separation was carried out in a gradient elution mode. Detection were performed by MS in positive ion (SIM mode). Ultrasound-assisted (UAE), refluxing (RE), low pressure refluxing (LPRE) and Soxhlet (SE) extraction techniques were developed for determination steroidal saponin content. The results were confirmed using the multiple successive extraction method and spiking with standards before extraction. Results Extraction parameters for UAE, RE, LPRE and SE methods were optimized by single-factor-experiment for isolation of steroidal glycosides from T. terrestris [1]. Extraction parameters for UAE method were optimized by Latin Square experimental design at 4 levels for 4 factors for isolation of steroidal glycosides from D. deltoidea plant material and cell culture [2]. Thermal decomposition of protodioscin was observed after long term high temperature extraction process. The accuracy for all studied extraction techniques was confirmed by spiking of the plant material with standards before extraction and HPLC-MS analysis and the analytical method was validated for linearity, limits of detection, limit of quantification, precision and accuracy. Also, a high degree of extraction was confirmed by multiple successive extraction method (MSEM) and RE methods. At the same time, it was shown that the UAE method is completely inappropriate for a similar task when working with plant material. This may be due to the lack of mechanical tissues in the cell culture, in contrast to plant material. Conclusions In the course of the work cycle, a study was made of the patterns and features of extraction, chromatographic separation followed by mass spectrometric determination of the steroidal glycosides from plant material and cell cultures. The impossibility of using the UAE extraction method for working with plant material was also shown. Acknowledgements This work was supported by Russian Science Foundation (Grant No. 17-13-01146) for Moscow State University. Novel Aspect The method for HPLC-MS determination of steroid glycosides was developed and validated. The optimization and comparison of extraction techniques were performed. References 1. Sarvin B., Stekolshchikova E., Rodin I., Stavrianidi A., Shpigun O., JARMAP, 8, 75-82 (2018). 2. Sarvin B., Fedorova E., Shpigun O., Titova M., Nikitin M., Kochkin D., Rodin I., Stavrianidi A., Journal of Chromatography B, 1080, 64-70 (2018).