**1. Introduction**

Sea cucumbers belonging to the class *Holothuroidea* of the *Echinodermata* phylum are marine invertebrates that produce a range of compounds that have the potential to be used in agriculture, and as pharmaceuticals, nutraceuticals and cosmeceuticals [1,2].

Saponins are the most important characteristic and abundant secondary metabolites in this species [3]. Sea cucumber saponins exert a wide range of medicinal and pharmacological properties. Saponins are also the main bioactive compounds in many plant drugs and folk medicines, especially in the Orient.

Although sea cucumber saponins share common saponin features, their aglycones, also called sapogenins or genins, are significantly different from those reported in the plant kingdom [1]. These amphipathic compounds generally possess a triterpene or steroid backbone or aglycone (hydrophilic, lipid-soluble) connected glycosidically to a saccharide moiety (hydrophilic, water-soluble) [3–5]. Saponins are also produced by other marine organisms including asteroids [6], which also belongs to the phylum *Echinodermata,* and sponges of the phylum *Porifera* [7]. Sea cucumbers saponins are usually triterpene glycosides (derived from lanostane) [3] while those from starfish are steroid glycosides [6]. The sugar moieties mainly consist of D-xylose (Xyl), D-quinovose (Qui), 3*-O-*methyl-D-glucose (MeGlc), 3*-O*-methyl-D-xylose (MeXyl) and D-glucose (Glc), and sometimes 3*-O*-methyl-D-quinovose, 3-*O*-methyl-D-glucuronic acid and 6*-O*-acetyl-D-glucose. In the oligosaccharide chain, the first monosaccharide unit is always a xylose, whereas 3-*O*-methylglucose and/or 3-*O*-methylxylose are always the terminal sugars. The presence of two quinovose residues in a carbohydrate moiety is unique for sea cucumber and starfish glycosides.

There are more than 700 triterpene glycosides in various species of sea cucumbers [3,5,8–17], which are classified into four main structural categories based on their aglycone moieties: three holostane types containing a (1) 3β-hydroxyholost-9(11)-ene aglycone skeleton; (2) a 3β-hydroxyholost-7-ene skeleton and (3) an aglycone moiety different to other two holostane type aglycones, and a nonholostane aglycone [5,12,17,18]. The majority of saponins belong to the holostane type group [3, 12,13,19]. Most sea cucumber saponins comprise of a lanostane-3β-ol type aglycone with a γ-18 (20)-lactone in the D-ring of tetracyclic triterpene (3β,20*S*-dihydroxy-5α-lanostano-18,20-lactone) [5], sometimes containing shortened side chains; the glycone contains up to six monosaccharide units covalently connected to C-3 of the aglycone.

In sea cucumbers, the sugar residue has only one branch [13], whereas plant saponins may contain one, two or three saccharide chains, with a few having an acyl group bound to the sugar moiety [20]. One of the most noteworthy characteristics of many of the saponins from marine organisms is the sulfation of aglycone or sugar moieties [1], and in sea cucumbers the sulfation of one or more of Xyl, Glc, MeGlc and Qui residues have been reported [3,11]. Most of them are mono-sulfated glycosides with few occurrences of di- and tri-sulfated glycosides [13,17]. Another structural feature that has been found only in this series of aglycones is the presence of an acetoxyl group at C-16 and/ or in the lateral side of the aglycone (C-22 or C-23 and/or C-25). The other structural feature is the presence of a 12α-hydroxy group in the aglycone unit of saponins in the genus *Holothuria*; however some contain two hydroxy groups at positions 12α and 17α of the holostanol skeleton. Several triterpene glycosides (such as Holothurinoside X, Fuscocinerosides B and Scabraside B) isolated from the sea cucumber *Holothuria lessoni* contain a carbonyl group in the lateral chain [3,12]. The majority of saponins from Aspidochirotida sea cucumbers contain the 9(11)-double bond in their aglycone moiety, but most of the glycosides isolated from *Holothuria* are Δ9,11-glycosides.

Recently, a series of unusual non-holostane triterpene glycoside have been reported from sea cucumbers, belonging to order *Dendrochirotida*, which have a shortened side chain, and no lactone function. So far, only nine non-holostane acetylated saponins including Kurilosides A and C, Psolusoside B, Frondoside C, Cucumariosides A2-7, A2-8, A8, A9 and Koreoside A have been reported from the class *Holothuroidea*.

Similarities in structure of saponin glycosides leads to difficulties in purification, and this vitiates the complete structure elucidation of these molecules (especially isomers). High Performance Centrifugal Partition Chromatography (HPCPC) is effective in separating polar compounds and was employed successfully in obtaining purified saponins in this study.

The viscera of an Australian sea cucumber *Holothuria lessoni* (golden sandfish) [21] was selected as a source of saponins because we hypothesized that the internal organs contain high levels of compounds as the viscera are expelled from the sea cucumber in order to repel other sea animals. In relation to internal organs, the saponin content of the cuvierian tubules of *Holothuria* were found to be higher than the body wall on a weight basis [22,23]. We have recently reported [3,12] new saponins within the viscera of *H. lessoni*, and in this paper we present five new acetylated saponins and two related new non-acetylated isomers identified from the viscera using mass spectrometry.

Nuclear magnetic resonance (NMR) spectroscopy can provide extensive structural information for saponins, however larger quantities of high-purity samples are generally needed. This is complicated with fractions if the NMR signals overlap, making their assignments more difficult. Moreover, the measurement of the absolute configuration of the sugar moieties of a saponin cannot be completely solved by NMR methods alone [24]. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) and electrospray ionization mass spectrometry (ESI-MS) techniques have become the preferred techniques for analyses of saponins. Mass spectrometry provides a highly sensitive platform for the analyses of saponin structures by generating product ions by the cleavage of the glycosidic bond.

Several studies reported that individual species have specific saponin congeners. However some congeners are common among different species. Even if the diversity is great, saponins from closely related species still retain the same molecular motif [11,25] and this property of saponins can be utilized for their taxonomic classification. Because of their internal and external roles the molecular structure of these compounds was most likely to be conserved within the species.

Mass spectrometry has a long history in the structure elucidation of saponins in both negative and positive ion modes. It has been extensively used to determine the molecular weight and the structure of the native aglycones as well as the glycosidic linkages in the oligosaccharide chain without degradation of the glycosides. Knowledge of the chemical structure of compounds is very important to determine the specific correlation between the structure and their molecular and biological mechanism(s) of actions. We expect that the results of this project will transform the value of the viscera of sea cucumbers into sources of high value products, important to human health and industry.
