Discrimination of Regioisomeric and Stereoisomeric Saponins from Aesculus hippocastanum Seeds by Ion Mobility Mass Spectrometry

  • Emmanuel Colson
  • Corentin Decroo
  • Dale Cooper-Shepherd
  • Guillaume Caulier
  • Céline Henoumont
  • Sophie Laurent
  • Julien De Winter
  • Patrick Flammang
  • Martin Palmer
  • Jan Claereboudt
  • Pascal GerbauxEmail author
Research Article


Modern mass spectrometry methods provide a huge benefit to saponin structural characterization, especially when combined with collision-induced dissociation experiments to obtain a partial description of the saponin (ion) structure. However, the complete description of the structures of these ubiquitous secondary metabolites remain challenging, especially since isomeric saponins presenting small differences are often present in a single extract. As a typical example, the horse chestnut triterpene glycosides, the so-called escins, comprise isomeric saponins containing subtle differences such as cis-trans ethylenic configuration (stereoisomers) of a side chain or distinct positions of an acetyl group (regioisomers) on the aglycone. In the present paper, the coupling of liquid chromatography and ion mobility mass spectrometry has been used to distinguish regioisomeric and stereoisomeric saponins. Ion mobility arrival time distributions (ATDs) were recorded for the stereoisomeric and regioisomeric saponin ions demonstrating that isomeric saponins can be partially separated using ion mobility on a commercially available traveling wave ion mobility (TWIMS) mass spectrometer. Small differences in the ATD can only be monitored when the isomeric saponins are separated with liquid chromatography prior to the IM-MS analysis. However, gas phase separation between stereoisomeric and regioisomeric saponin ions can be successfully realized, without any LC separation, on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The main outcome of the present paper is that the structural analysis of regioisomeric and stereoisomeric natural compounds that represents a real challenge can take huge advantages of ion mobility experiments but only if increased ion mobility resolution is attainable.


Saponins Ion mobility Cyclic ion mobility TWIMS Stereoisomers Regioisomers Natural products Escin 



The MS laboratory acknowledges the “Fonds de la Recherche Scientifique (FRS-FNRS)” for its contribution to the acquisition of the Waters QToF Premier and the Waters SYNAPT G2-Si mass spectrometers. P.F. is Research Director of the FRS-FNRS. E.C. and C.D. are grateful to the F.R.I.A. for the financial support.

Supplementary material

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  1. 1.
    Lorent, J.H., Quetin-Leclercq, J., Mingeot-Leclercq, M.P.: The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Org. Biomol. Chem. 12, 8803–8822 (2014)CrossRefGoogle Scholar
  2. 2.
    Segal, R., Schlösser, E.: Role of glycosidases in the membranlytic, antifungal action of saponins. Arch. Microbiol. 104, 147–150 (1974)CrossRefGoogle Scholar
  3. 3.
    Podolak, I., Galanty, A., Sobolewska, D.: Saponins as cytotoxic agents : a review. Phytochem. Rev. 9, 425–474 (2010)CrossRefGoogle Scholar
  4. 4.
    Vincken, J.P., Heng, L., de Groot, A., Gruppen, H.: Saponins, classification and occurrence in the plant kingdom. Phytochemistry. 68, 275–297 (2007)CrossRefGoogle Scholar
  5. 5.
    Sparg, S.G., Light, M.E., Van Staden, J.: Biological activities and distribution of plant saponins. J. Ethnopharmacol. 94, 219–243 (2004)CrossRefGoogle Scholar
  6. 6.
    Madl, T., Sterk, H., Mittelbach, M., Rechberger, G.N.: Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa. J. Am. Soc. Mass Spectrom. 17, 795–806 (2006)CrossRefGoogle Scholar
  7. 7.
    Bahrami, Y., Zhang, W., Franco, M.M.: C.: Distribution of saponins in the Sea Cucumber Holothuria lessoni; the body wall versus the viscera, and their biological activities. Mar. Drugs. 16, 423 (2018)CrossRefGoogle Scholar
  8. 8.
    Yamanouchi, T.: On the poisonous substance contained in holothurians. Seto. Mar. Biol. Lab. 4, 183–203 (1995)Google Scholar
  9. 9.
    Caulier, G., Mezali, K., Soualili, D.L., Decroo, C., Demeyer, M., Eeckhaut, I., Gerbaux, P., Flammang, P.: Chemical characterization of saponins contained in the body wall and the Cuvierian tubules of the sea cucumber Holothuria (Platyperona) sanctori (Delle Chiaje, 1823). Biochem. Syst. Ecol. 68, 119–127 (2016)CrossRefGoogle Scholar
  10. 10.
    Kitagawa, I., Kobayashi, M.: On the structure of the major saponin from the starfish Acanthaster Planci. Tetrahedron Lett. 18, 859–862 (1977)CrossRefGoogle Scholar
  11. 11.
    Demeyer, M., De Winter, J., Caulier, G., Eeckhaut, I., Flammang, P., Gerbaux, P.: Molecular diversity and body distribution of saponins in the sea star Asterias rubens by mass spectrometry. Comp. Biochem. Physiol. B. 168, 1–11 (2014)CrossRefGoogle Scholar
  12. 12.
    Palagiano, E., Zollo, F., Minale, L., Iorizzi, M., Bryan, P., McClintock, J., Hopkins, T.: Isolation of 20 glycosides from the starfish Henricia downeyae, collected in the Gulf of Mexico. J. Nat. Prod. 59, 348–354 (1996)CrossRefGoogle Scholar
  13. 13.
    Maier, M.S.: Biological activities of sulfated glycosides from echinoderms. Stud. Nat. Prod. Chem. 35, 311–354 (2008)CrossRefGoogle Scholar
  14. 14.
    D’Auria, M.V., Minale, L., Riccio, R.: Polyoxygenated steroids of marine origin. Chem. Rev. 93, 1839–1895 (1993)CrossRefGoogle Scholar
  15. 15.
    Van Dyck, S., Gerbaux, P., Flammang, P.: Qualitative and quantitative saponin contents in five sea cucumbers from the Indian ocean. Mar. Drugs. 8, 173–189 (2010)CrossRefGoogle Scholar
  16. 16.
    Van Dyck, S., Gerbaux, P., Flammang, P.: Elucidation of molecular diversity and body distribution of saponins in the sea cucumber Holothuria forskali (Echinodermata) by mass spectrometry. Comp. Biochem. Physiol. B. 152, 124–134 (2009)CrossRefGoogle Scholar
  17. 17.
    Shvartsburg, A.A., Jarrold, M.F.: An exact hard-spheres scattering model for the mobilities of polyatomic polyatomic ions. Chem. Phys. Lett. 261, 86–91 (1996)CrossRefGoogle Scholar
  18. 18.
    Mesleh, M.F., Hunter, J.M., Shvartsburg, A.A., Schatz, G.C., Jarrold, M.F.: Structural information from ion mobility measurements: effects of the long-range potential. J. Phys. Chem. 100, 16082–16086 (1996)CrossRefGoogle Scholar
  19. 19.
    Decroo, C., Colson, E., Lemaur, V., Caulier, G., De Winter, J., Cabrera-Barjas, G., Gerbaux, P.: Ion mobility mass spectrometry of saponin ions. Rapid Commun. Mass Spectrom. 33(S2), 22–33 (2019)CrossRefGoogle Scholar
  20. 20.
    Decroo, C., Colson, E., Demeyer, M., Lemaur, V., Caulier, G., Eeckhaut, I., Gerbaux, P.: Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration. Anal. Bioanal. Chem. 409, 3115–3126 (2017)CrossRefGoogle Scholar
  21. 21.
    Patlolla, J.M.R., Rao, C.V.: Anti-inflammatory and anti-cancer properties of β-escin, a triterpene saponin. Curr. Pharmacol. Rep. 1, 170–178 (2015)CrossRefGoogle Scholar
  22. 22.
    Abudayeh, Z.H.M., Al Azzam, K.M., Naddaf, A., Karpiuk, U.V., Kislichenko, V.S.: Determination of four major saponins in skin and endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using high performance liquid chromatography with positive confirmation by thin layer chromatography. Adv. Pharm. Bull. 5, 587–591 (2015)CrossRefGoogle Scholar
  23. 23.
    Yoshikawa, M., Harada, E., Murakami, T., Matsuda, T., Wariishi, N., Yamahara, Y., Murakami, N., Kitagawa, I.: Escins-Ia, Ib, IIa, IIb, and IIIa, Bioactive triterpene oligoglycosides from the seeds of Aesculus hippocastanum L.: their inhibitory effects on ethanol absorption and hypoglycemic activity on glucose tolerance test. Chem. Pharm. Bull. 42, 1357–1359 (1994)CrossRefGoogle Scholar
  24. 24.
    Yoshikawa, M., Murakami, T., Yamahara, J., Matsuda, H.: Bioactive saponins and glycosides. XII. Horse chestnut. (2): Structures of escins IIIb, IV, V, and VI and isoescins Ia, Ib, and V, acylated polyhydroxyoleanene triterpene oligoglycosides, from the seeds of horse chestnut tree (Aesculus hippocastanum L., Hippocastanaceae). Chem. Pharm. Bull. 46, 1764–1769 (1998)CrossRefGoogle Scholar
  25. 25.
    Yoshikawa, M., Murakami, T., Yamahara, J., Matsuda, H.: Bioactive saponins and glycosides. III. Horse chestnut. (1): the structures, inhibitory effects on ethanol absorption, and hypoglycemic activity of escins Ia, Ib, IIa, IIb, and IIIa from the seeds of Aesculus hippocastanum L. Chem. Pharm. Bull. 44, 1754–1764 (1996)CrossRefGoogle Scholar
  26. 26.
    Matsuda, H., Li, Y., Murakami, T., Ninomiya, K., Yamahara, J., Yoshikawa, M.: Effects of escins Ia, Ib, IIa, and IIb from horse chestnut, the seeds of Aesculus hippocastanum L., on acute inflammation in animals. Biol. Pharm. Bull. 20, 1092–1095 (1997)CrossRefGoogle Scholar
  27. 27.
    Duez, Q., Chirot, F., Liénard, R., Josse, T., Choi, C.M., Coulembier, O., Dugourd, P., Cornil, J., Gerbaux, P., De Winter, J.: Polymers for traveling wave ion mobility spectrometry calibration. J. Am. Soc. Mass Spectrom. 28, 2483–2491 (2017)CrossRefGoogle Scholar
  28. 28.
    Giles, K.; Ujma, J.; Wildgoose, J.; Green, M.; Richardson, K.; Langridge, D.; Tomczyk, N. Design and performance of a second-generation cyclic ion mobility enabled Q-TOF. June 6 (Poster Presentation). In: Proceedings of the 65th Conference on Mass Spectrometry and Allied Topics, Indianapolis in June 4–8 (2017);Google Scholar
  29. 29.
    Giles, K., Ujma, J., Wildgoose, J., Pringle, S., Richardson, K., Langridge, D., Green, M.: A cyclic ion mobility-mass spectrometry system. Anal. Chem. 91, 8564–8573 (2019)CrossRefGoogle Scholar
  30. 30.
    Smith, D., Knapman, T., Campuzano, I., Malham, R., Berryman, J., Radford, S., Ashcroft, A.: Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur. J. Mass Spectrom. 15, 113 (2009)CrossRefGoogle Scholar
  31. 31.
    Dodds, J.N., May, J.C., McLean, J.A.: Correlating resolving power, resolution, and collision cross section: unifying cross-platform assessment of separation efficiency in ion mobility spectrometry. Anal. Chem. 89, 12176–12184 (2017)CrossRefGoogle Scholar
  32. 32.
    Giles, K., Williams, J.P., Campuzano, I.: Enhancements in travelling wave ion mobility resolution. Rapid Commun. Mass Spectrom. 25, 1559–1566 (2011)CrossRefGoogle Scholar
  33. 33.
    Rathore, D., Dodds, E.D.: Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes. J. Am. Soc. Mass Spectrom. 25, 1600–1609 (2014)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  • Emmanuel Colson
    • 1
    • 2
  • Corentin Decroo
    • 1
    • 2
  • Dale Cooper-Shepherd
    • 3
  • Guillaume Caulier
    • 2
  • Céline Henoumont
    • 5
  • Sophie Laurent
    • 5
  • Julien De Winter
    • 1
  • Patrick Flammang
    • 2
  • Martin Palmer
    • 3
  • Jan Claereboudt
    • 4
  • Pascal Gerbaux
    • 1
    Email author
  1. 1.Organic Synthesis and Mass Spectrometry Laboratory (S2MOs)University of MonsMonsBelgium
  2. 2.Biology of Marine Organisms and Biomimetics Unit (BOMB)University of MonsMonsBelgium
  3. 3.Waters CorporationWilmslowUK
  4. 4.Waters CorporationZellikBelgium
  5. 5.Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging LaboratoryUniversity of MonsMonsBelgium

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