**4. Conclusions**

In recent years, there has been a great improvement in the number of MS applications. The tandem MS approach coupled with HPCPC separation revealed the structure of isomeric compounds containing different aglycones and/or sugar residues. Therefore, a creative and sensitive method has been developed for the structure elucidation of triterpene glycosides in sea cucumber and related products using HPCPC and MS. The result showed that this method is a rapid, accurate and reliable technique for the structure determination of triterpene glycosides in sea cucumber extracts.

This study proved the occurrence of both glycoside and cross-ring cleavages in the sugar moieties of sea cucumber saponins. The sequence of monosaccharide units and the presence of an acetoxy group, clearly reflected by the loss of 60 Da from the parent ions, were noted in five of the seven new saponins.

Tandem mass spectrometry data suggested that the most prominent ions generally stemmed from the losses of aglycones and/or the key diagnostic sugar moieties (*m/z* 493, 507, 511, 639, 643 and 625).

Our results also illustrate that some saponins are unique to the species, whilst others are common between multiple species. The MS analysis revealed that individual species possesses a unique saponin pattern in which some congeners are very specific to one species. This feature can be used for the taxonomic classification of sea cucumber species.

Characterization of some of these saponins were easier since their MS<sup>2</sup> spectra possessed the key diagnostic signal at *m/z* 493, corresponding to the oligosaccharide chain [MeGlc-Glc-Xyl + Na+] in addition to the vital peak at *m/z* 643, corresponding to the oligosaccharide moiety [MeGlc-Glc-Xyl-Xyl-H2O + Na+]. Lessoniosides C, D and E contain 3-*O*-methylxylose as a terminal monosaccharide unit, which is a rare structural feature in sea cucumber triterpene glycosides.

The ion at *m/z* 1477.7 was identified as the major component of the glycoside fraction 18, containing holostane aglycones with 9(11)-double bond and 18(20)-lactone, characteristic for most of the known sea cucumber glycosides. The structures of these glycosides are quite different from those reported in this species. These substances contain aglycones with an oxidized position at C-16, (acetate group or keto group). Lessoniosides have the aglycone unit like that in Holothurinoside Y with an acetoxyl instead of hydrogen at position 16, but another aglycone moiety with the saturated side chain and with an acetoxyl (a 16β-acetate group) instead of a ketone at position 16.

Our results to date highlight the abundance of new saponins in the viscera indicating the viscera as a major source of these compounds with diverse structures. This paper is the first not only to deduce the structure of several new acetylated isomeric saponins, (Lessoniosides A–G) but also to present the structural diversity of triterpene glycoside congeners in the viscera of *H. lessoni*.

These new saponins (Lessoniosides A–G) have the potential to be consumed with applications as dietary supplements, food preservatives (because of their emulsifying and foaming properties), food additives and development of high value products for various industrial applications and as anti-cancer agents.

Our findings demonstrate that the marine world, in particular sea cucumbers, have much to offer human society in the way of nutraceuticals, pharmaceuticals, agrochemicals, cosmeceuticals, and research biochemicals.

**Acknowledgments:** We would like to express our sincerest thanks to the Australian SeaFood CRC for financially supporting this project and the Iranian Ministry of Health and Medical Education for their scholarship to YB, Ben Leahy and Tasmanian SeaFoods for supplying the sea cucumber samples. The authors gratefully acknowledge the technical assistance provided by Daniel Jardine and Jason Young at Flinders Analytical Laboratory, Elham Kakaei and Associate Prof. Michael Perkins at Flinders.

**Author Contributions:** Y.B. and C.F. designed the experiments. Y.B. carried out the experiments with guidance from C.F. Y.B. worked on chemical structure elucidation and both authors contributed in writing the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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