6-Oxofurostane and (iso)spirostane type of saponins in Smilax sieboldii: UHPLC-QToF-MS/MS and GNPS-molecular networking approach for the rapid dereplication and biodistribution of specialized metabolites

Authors: AVULA, BHARATHI - University Of Mississippi; BAE, JI-YEONG - Jeju National University; AHN, JONGMIN - Korea Research Institue Of Bioscience & Biotechnology; WANG, YAN-HONG - University Of Mississippi; Wang, Mei; KWON, YONGSOO KWON - Kangwon National University; CHITTIBOYINA, AMAR - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2023
Publication Date: 7/14/2023
Citation: Avula, B., Bae, J., Ahn, J., Wang, Y., Wang, M., Kwon, Y., Chittiboyina, A.G., Khan, I.A. 2023. 6-Oxofurostane and (iso)spirostane type of saponins in Smilax sieboldii: UHPLC-QToF-MS/MS and GNPS-molecular networking approach for the rapid dereplication and biodistribution of specialized metabolites. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms241411487.
DOI: https://doi.org/10.3390/ijms241411487

Interpretive Summary: Smilax sieboldii Miq (Smilacaceae) is a deciduous climbing shrub, a key member of 350 species, widely distributed in Korea, China, and Japan. This plant’s young, tender leaves are used as edible vegetables, and the underground root/rhizomes have been used to treat various ailments in Korea and other traditional practices. Recently untargeted metabolomic approaches such as UHPLC-HRMS/MS proved promising to generate a holistic view of sample chemical composition, aka chemical fingerprints, without focusing on any specific chemical class. In this regard, an UHPLC-QToF-MS technique has been applied to the characterization of steroidal saponins from S. sieboldii with the advantage of avoiding the time-consuming derivatization steps and tedious purification of compounds from the crude extracts. Moreover, implementing an LC-MS/MS-based molecular networking approach has been proven to be a powerful method for rapidly dereplicating phytochemicals, specifically minor constituents in complex mixtures.

Technical Abstract: Identifying novel phytochemical secondary metabolites following classical pharmacognostic investigations is tedious and often involves repetitive chromatographic efforts. During the past decade, UHPLC-QToF-MS/MS, in combination with Global Natural Product Social Molecular Networking (GNPS), was successfully demonstrated for the rapid dereplication of novel natural products in complex mixtures. As a logical application of such innovative tools in botanical re-search, more than 40 unique, 3-oxy-, 3, 6-dioxy-, and 3, 6, 27-trioxy-steroidal saponins were iden-tified in aerial parts and rhizomes of botanically verified Smilax sieboldii, native to temperate re-gions of East Asia, mainly Korea, Japan, and China. The MS/MS diagnostic fragmentation patterns of aglycones, diosgenin, sarsasapogenin/tigogenin, and laxogenin were instrumental in estab-lishing unique nodes representing six groups of nineteen unknown steroidal saponins in S. sieboldii. The mass spectral dissociation pattern for these saponin glycosides allowed the unam-biguous establishment of the oligosaccharides appended to the C-3 position of (iso)spirostanes, including laxogenin and one glucose unit at the C-27 position of furostane-type metabolites. Moreover, mass fragmentation analysis resulted in the identification of 6-hydroxy sapogenins, which are believed to be key precursors in the biogenesis of characteristic smilaxins and sieboldins, along with other metabolites found in S. sieboldii. These analytes’ relative biodistri-bution and characteristic molecular networking profiles were generated by analyzing the leaf, stem, and rhizome of S. sieboldii. Deducing such profiles is anticipated to aid the overall product integrity of botanical dietary supplements while avoiding tedious pharmacognostic investiga-tions and helping identify exogenous components within the finished products.