Countercurrent chromatography separation of saponins by skeleton type from Ampelozizyphus amazonicus for off-line ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry analysis and characterisation.

Ampelozizyphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria, is a climbing shrub, native to the Amazonian region, with jujubogenin glycoside saponins as main compounds. The crude extract of this plant is too complex for any kind of structural identification, and HPLC separation was not sufficient to resolve this issue. Therefore, the aim of this work was to obtain saponin enriched fractions from the bark ethanol extract by countercurrent chromatography (CCC) for further isolation and identification/characterisation of the major saponins by HPLC and MS. The butanol extract was fractionated by CCC with hexane - ethyl acetate - butanol - ethanol - water (1:6:1:1:6; v/v) solvent system yielding 4 group fractions. The collected fractions were analysed by UHPLC-HRMS (ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry) and MSn. Group 1 presented mainly oleane type saponins, and group 3 showed mainly jujubogenin glycosides, keto-dammarane type triterpene saponins and saponins with C31 skeleton. Thus, CCC separated saponins from the butanol-rich extract by skeleton type. A further purification of group 3 by CCC (ethyl acetate - ethanol - water (1:0.2:1; v/v)) and HPLC-RI was performed in order to obtain these unusual aglycones in pure form.

Spray-dried extract from the Amazonian adaptogenic plant Ampelozizyphus amazonicus Ducke (Saracura-mirá): Chemical composition and immunomodulatory properties.

Ampelozizyphus amazonicus Ducke is a medicinal plant used in the Amazon region to prepare a drink with tonic, immunomodulatory and adaptogenic properties. Due to the growing interest in dietary supplements with these properties and, to provide a new functional ingredient, barks from A. amazonicus were extracted. The extract was spray dried without drying adjuvants, resulting in a powder (SARF), which was characterized by its physico-chemical properties and proximate, mineral and saponin contents. The SARF saponins were characterized by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry (HPLC-HRMSn) analysis. The SARF particles tended to have a spherical shape and a unimodal size distribution. The particles also had good rehydration characteristics and high saponin content (33%). The effect of SARF on antibody production was investigated, and we found that SARF increased the basal levels of anti-ovalbumin, anti-LPS and anti-dextran IgM antibodies, and the anti-dextran IgG antibodies in unimmunized mice. No increase in antibody titers was observed after SARF treatment in immunized mice. These results suggest that SARF could be an interesting new functional ingredient for food applications or pharmaceutical products.