Using mass spectrometry techniques for the elucidation of structural features of immunostimulatory polysaccharides from medicinal plantsaqueous extracts
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Polysaccharides are well known for their immunostimulatory properties, which are closely related to
structural fèatures, such as molecular weight, branching, acetylation degree and acetylation pattem,
and presence of speciflc structures [1, 2]. TTie electrospray ionization-mass spectrometry (ESI-MS)
and electrospray ionization tandem mass spectrometry (ESI-MS/MS) have shown to be powerfül
techniques that can provide detailed information regarding the structural features of polysaccharides
and help to clarify its relation to some ofthe observed immunostimulatory properties [3-6]. TTie present
work desenhes the use of ESI-MS and ESI-MS/MS in the confimation and elucidation of structural
features of immunostimulatory polysaccharides present in lhe aqueous extracts of Fraxinus
angustifolia dned leaws and Ptemapaitum tridentatum dried inflorescences.
The polysaccharides present in the aqueous extracts of F. angustifolia dried leaves and P. fridentafum
dried inflorescences were isolated and fractionated using dialysis, ethanol precipitation, and anionexchange
chromatography. Sugar and linkage analysis suggested that the aqueous extracts of F.
angustifolia were mostly composed of pectic polysaccharides, while those from P. tridentatum
contained a mixture of pectic polysacchahdes, galactomannans, and also xyloglucans. Selected
fractions isolated from lhe aqueous extracts of F. angustifolia and P. tridentatum evidenced an in vitro
macrophage immunostimulatory activity, expressed through the production of nitric oxide (NO).
Enzymatic hydrolysis of the isolated polysaccharides, fbllowed by size exclusion chromatography of
the resulting oligosaccharides (OS), and ESI-MS and ESI-MS/MS characterization allowed to confirm
the presence of pectic polysaccharides, possibly with xylogalaturonan domains, in the aqueous
extracts of F. angustifolia. For the aqueous extracts of P. fridentatum it was possible to detect the
presence of galactomannans acetylated at 0-2. Moreover, it was possible to identify acetylated OS
that presented an acetylation pattem with much more acetyl groups than those observed for
galactomannans from coffee infusions [3]. The saponification ofthe fraction isolated from the aqueous
extracts of P. tridentatum dried inflorescences, which contained the acetylated galactomannans,
caused a decrease in the NO macrophage production, confirming lhe galactomannans acetylation as
an important structural feature for lhe expression of the in vitm macrophage immunostimulatory
activity previously registered.
