Hydrolysis of Helleborus niger L. saponins for aglycone analysis

Saponins are secondary metabolites in plants holding a defensive role. They are chemically split up into triterpene and steroid saponins. Generally, saponin structures may attain a great diversity due to various possible substituent patterns. They possess a wide range of pharmacological properties including cytotoxic and antitumor activities. Especially steroid saponins of the diosgenyl group showed remarkable effects [1]. Macranthogenin, a sapogenin similar to diosgenin, is the only aglycone described for H. niger L. so far [2]. Therefore additional research effort is needed to get a more complete view of its sapogenin composition.

Due to the risk of artefact formation, saponin hydrolysis is the crucial step for aglycone analysis. Acid hydrolysis (1N HCl, 100 °C, 1h, GC-MS) of the whole H. niger L. saponin fraction resulted in a complex peak mixture. Further experiments on isolated macranthosid I showed the introduction of up to 3 artefact double bonds in the aglycone structure. In order to achieve unaltered aglycons, several systems for enzymatic hydrolysis were tested. While β-glucosidase was not able to yield the macranthogenin aglycone, further experiments revealed pectinases and a glucuronidase to be powerful cleaving enzymes working under mild conditions (40 °C, pH 4.8). After precipitation, the aglycone fraction was re-dissolved and analysed via GC-MS and LC-MS. In addition to macranthogenin, two further diosgenin-like sapogenins namely sarsasapogenin/smilagenin and sceptrumgenin were detected.

This study underlines that carefully chosen working conditions upon saponin hydrolysis are crucial to achieve an authentic aglycone profile in the course of phytochemical characterization of plant extracts.

Fig. 1

References:

[1] Man S., Gao W., Zhang Y., Huang L., Changxiao L. (2010) Fitoterapia 81: 703 – 714.

[2] Linde, H.F., Isaac O., Linde H.H., Zivanov D. (1971) Helv Chim Acta 54: 1703 – 1708.