Mass spectrometry study of plant steroid glycosides and their interactions with biomolecules

A number of plant steroid glycosides (SGs) and genins nave been investigated by Cf plasma desorption mass spectrometry (Cf TOF-PDMS). PDMS was also applied for comparative studies of the interactions of SGs and their genins with basic amino acids, nucleosides and AMP. SGs can interact with these biomolecules to form heteroclusters of the types [SG + biomolecule + #7 and [SG + biomolecule + K]*, the affinity of SGs for biomolecules depends on the structures of the SG carbohydrate chain and the SG aglycone, and the biomolecule nature.

Steroid glycosides (SGs) are glycosylated secondary metabolites that commonly occur in the higher plants: more than 500 species belonging to nearly 80 dif ferent plant families can synthesise saponins [1][2][3].The SGs basic structure is a hydrophobic steroid ring (sapogenin or aglycone) of spirostan or furostan series attached to a hydrophilic chain of deferent sugar moieties.A sugar may be linked to the aglycone at one or two glycosylation sites, giving either monodesmosidic or bidesmosidic SGs, respectively [1][2][3].SGs represent one of the most promising classes of pharmacological substances used to treat more than 100 diseases [1][2][3].Most biological activities of SGs are ascribed to their interaction with biomembranes and separate biomolecules, e. g. with cholesterol and proteins [1][2][3].However, the mechanism underlying the SGs biochemical activity is not fully understood and requires further investigations.
SGs and genins of spirostan and furostan series have been investigated by 252 Cf plasma desorption mass spectrometry ( 252 Cf TOF-PDMS).The experi mental part connected with the procedure of extrac tion, purification and sample preparation of SGs and genins for PDMS investigation as well as principal schemes of PDMS method are described in [4][5][6].Firstly we recorded the mass spectra for well-charac terized aglycones and SGs.They were studied with the IR and NMR methods before the PDMS inves tigation, also complete acid and enzymatic hydrolysis and methylation were performed.[7][8][9].These in vestigations and detailed analysis of mass spectra permitted to make a conclusion about the main features of the PDMS spectra of SGs measured and served as a basis for mass spectrometry studies of genin and SG samples whose molecular mass and structure had been determined initially, but required further verification and confirmation, and to create a mass spectral database.The database allows 1) dis tinguishing between SGs and genins in the pure samples and crude plant extracts, 2) determination of molecular mass of SGs and genins, as well as 3) determination of aglycone (genin) and carbohydrate chain composition and structure of SGs.

PDMS data on steroid glycoside-amino acid interaction. Instrumental error in the m/z range 500 to J500 и is ±2 u. The dash indicates that there is no steroid glycoside-amino acid interaction
•Weak interaction; **average interaction; ***strong interaction.
of SG with certain biomolecule show that SGs can form heteroclusters with amino acids, nucleosides or AMP.The mass spectra indicate also that only SGs with carbohydrate chain containing more than one sugar can form heteroclusters of the types [SG +-+ biomolecule + Na Ґ, [SG + biomolecule + К f and [SG + biomolecule + H f. The intensity of peaks of heterocluster ions depends on structure of aglycone and amount of sugars moiety in carbohydrate chaine of SG.
For instance, the most striking results of analyses of SG-amino acid mixtures listed in Table, de monstrate that SGs with a larger carbohydrate com ponent (NT) and extra hydroxyl group (PD) in the aglycone have a greater affinity to amino acids, favoring polar negatively-charged at physiological pH and polar uncharged amino acids.
The same results we obtained in the case of model mixtures of SGs (NM, NB and PD) and genins (neotigogenin and gitogenin) with nucleosides and AMP [6].The most intense peaks of these hete roclusters in comparison with molecular ions were observed in case of mixtures SGs with Urd.The mass spectra of model mixtures of neotigogenin, gitogenin or NM with nucleosides and AMP show only peaks of individual mixture components.These results can be used for the explanation of some aspects of SG pharmacological activity and its ability to interact with the native biopolymers (pro teins, DNA or RNA) or with their components (amino acids, nucleosides or nucleotides).For example, the complexes formed by SGs with glycoplastic (Ala, Gly, Glu, His, Pro, Ser, Thr) and ketoplastic (Leu, Phe, Tyr) amino acids may be the cause of the SG effect on gluconeogenesis and coenzyme A synthesis in a cell, respectively [10].