*2.5. The Structure of Aglycones*

This analysis revealed the presence of at least seven different isomers with diverse aglycone and sugar components for the ion at *m/z* 1477.7. The glycosides differ in their aglycone structures or sugar moieties. The mass of the cationized aglycone and the deacetylated aglycone in Lessonioside C and E are 556 Da [M − sugar residue + Na]+ and 496 [M − AcOH − sugar residue + Na]+ , respectively, which are consistent with the mass of the aglycone reported by Elyakov and co-workers [37]. Their MS analyses showed *m/z* 556 [M]+, 541 [M − CH3] <sup>+</sup> and 496 [M − CH3COO]<sup>+</sup> for an aglycone moiety. Further, Rothberg and associates noted an aglycone with the same molecular weight having an acetoxy group at the C-23 from *Stichopus chloronotus* [38]. Analysis of the MS data for these isomers and comparison with those published for related saponin aglycones [3,12,31,39] shows that the aglycone part of Lessoniosides are a holostane skeleton featuring hydroxy groups at C-12 and C17. With other isomers, the mass of the cationized aglycone and the deacetylated aglycone was found to be 530 Da and 493 Da, respectively. Other prominent high mass ions *m/z* 1417 [M − AcOH + Na]<sup>+</sup> and 1241 [M − AcOH − MeGlc + Na]<sup>+</sup> or 1241 [1301 − AcOH + Na]<sup>+</sup> provide additional support for the acetate and lactone functions.

The aglycone structure of Lessoniosides C/E appears to be similar to that of Fuscocineroside A reported from the sea cucumber *Holothuria fuscocinerea* [32]. The lateral C-20 side chain of Lessoniosides C and E was found to be similar to Fuscocineroside A [32], and Arguside D [33]. However, they differed from Fuscocineroside A and Arguside D by the addition of a keto at C-16, a C-23 double bond and a 17-OH.

Three of these compounds, Lessoniosides A, B and D have identical holostane aglycones containing an 18(20)-lactone with a 9(11)-double bond and acetoxy group at C-16 and differ from each other in their sugar component. On the other hand, they differ from Lessoniosides C/E in the presence of an acetoxy group at the C-16 (*vs.* keto group in C/E) and absence of a C-22 keto group, a C-23 double bond and a C-25 acetoxy at the lateral chain.

These glycosides have holotoxinogenin, a genin containing 9(11)-double bond and 16-oxidized group in the aglycone. They possess two hydroxy groups at 12α and 17α positions that are characteristic for Aspidochirotid sea cucumber (the family *Holothuriidae*). This aglycone is common for glycosides from many different sea cucumbers [11].

Therefore, elucidation of the aglycone component of the saponins was performed by comparison with published data. Because the new identified compounds clearly have aglycone structures similar to that of other previously characterized compounds we can be confident that it is possible to elucidate the structure of these compounds based on MS analysis alone. However, NMR analysis will be required to confirm the structure of the aglycones and also to ascertain the stereochemistry and linkages of the sugar moieties. Whereas, in the case of a novel compound without any similar previously characterized components, detailed chemical analysis including the application of NMR would be required.

#### *2.6. Acetylated Saponins*

So far more than 700 triterpene glycosidic saponins have been reported from sea cucumber species of which more than 130 are acetylated saponin. The majority of these are of the holostane type. The known acetylated triterpene glycosides (saponins), isolated from sea cucumbers of the class *Holothuroidea*, possess an acetyl group (acetoxy) in their aglycone residues. In the *Holothuriidae* family, the acetoxy group is either located at C-16 of the aglycone core moiety such as in Arguside F [40] and Nobiliside C [31], or at the C-22, C23 or C-25 of the lateral chain, ie at C-25 of the Pervicosides A and D [40,41]. However, Cucumarioside A1-2 is the only example of a triterpene glycoside containing an acetate group at C-6 (6-OAc) of the terminal glucose unit (sugar residue) 6-*O*-acetylglucose [42].

The majority of the acetylated compounds, such as Fuscocineroside A from the sea cucumber *H. fuscocinerea,* contain a sulfate group in their structures [32]. However, the presence of a sulfate group was not observed in these new acetylated saponins from *H. lessoni*.

Acetylated saponins are mainly reported in the family *Cucucmarridae*. However, the presence of acetylated saponins for the genus *Holothuria* is only reported for *H. lessoni* (this work), *H. pervicax*, *H. forskalii*, *H. nobilis*, *H. hilla*, *H. fuscocinerea*, *H. (Microthele) axiloga* and *H. pervicax* [17,31,32,40,41,43,44]. The majority of reported acetylated saponins possess only one acetoxy group in their structure, whereas saponins containing two *O*-acetic groups in their aglycone moieties have also been reported [16].

The presence of 12α and 17α–hydroxy, which are characteristic for glycosides from holothurians belonging to the family *Holothuriidae* (order *Aspidochirotida*), in glycosides of Dendrochirotids confirms parallel and relatively independent character of evolution of glycosides.

Observations from numerous studies confirm that the biological activity of saponins is influenced both by the aglycone and the sugar moiety. In other words there is a close relationship between the chemical structure of saponins and their biological activities. It has been reported that the presence of acetyl groups usually increases cytotoxic potency [45]. Therefore Lessoniosides seem to be potential candidates for anti-cancer drugs development.

### **3. Experimental Section**

#### *3.1. Sea Cucumber Sample*

Twenty sea cucumber samples of *H. lessoni* [21], commonly known as Golden sandfish were collected off Lizard Island (latitude; 14◦41 29.46 S, longitude; 145◦26 23.33 E), Queensland, Australia on September 2010 [3,12]. The viscera (all internal organs) were separated from the body wall and kept separately in zip-lock plastic bags which were snap-frozen, then transferred to the laboratory and kept at −20 ◦C until use.
