Development of a high-performance liquid chromatography procedure to identify known and detect novel C-13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W.

Abstract

As an aid for structure elucidation of new steviol glycosides, reversed-phase C18 high-performance liquid chromatography method was developed with several previously characterized diterpene glycosides, to identify known and detect novel aglycone-C13 oligosaccharide moieties and indirectly identify C-19 interlinkages. Elution order of several diterpene glycosides and their aglycone-C13 oligosaccharide substituted with different sugar arrangements were also summarized. Comparison of the retention time of a product obtained after alkaline hydrolysis with the aglycone-C-13 portions of known compounds reported herein allowed us to deduce the exact positions of the sugars in the C-13 oligosaccharide portion. The elution position of several steviol glycosides with an ent-kaurene skeleton was helpful to describe an identification key. Two previously uncharacterized diterpene glycosides together with two known compounds were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13-[(2-O-β-d-xylopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) ester (rebaudioside V), whereas the other was determined to be 13-[(2-O-β-d-xylopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-α-l-rhamnopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl) ester (rebaudioside W). Previously reported compounds were isolated in gram quantities and identified as rebaudioside J and rebaudioside H. In addition, a C-19 sugar-free derivative was also prepared from rebaudioside H to afford rebaudioside H1 . Chemical structures were partially determined by the high-performance liquid chromatography method and unambiguously characterized by using one-dimensional and two-dimensional nuclear magnetic resonance experiments.