Discovery of novel saponins from the viscera of the sea cucumber Holothuria lessoni

Abstract

Sea cucumbers, sometimes referred to as marine ginseng, produce numerous compounds with diverse functions and are potential sources of active ingredients for agricultural, nutraceutical, pharmaceutical and cosmeceutical products. We examined the viscera of an Australian sea cucumber Holothuria lessoni Massin et al. 2009, for novel bioactive compounds, with an emphasis on the triterpene glycosides, saponins. The viscera were extracted with 70% ethanol, and this extract was purified by a liquid-liquid partition process and column chromatography, followed by isobutanol extraction. The isobutanol saponin-enriched mixture was further purified by high performance centrifugal partition chromatography (HPCPC) with high purity and recovery. The resultant purified polar samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)/MS and electrospray ionization mass spectrometry (ESI-MS)/MS to identify saponins and characterize their molecular structures. As a result, at least 39 new saponins were identified in the viscera of H. lessoni with a high structural diversity, and another 36 reported triterpene glycosides, containing different aglycones and sugar moieties. Viscera samples have provided a higher diversity and yield of compounds than observed from the body wall. The high structural diversity and novelty of saponins from H. lessoni with potential functional activities presents a great opportunity to exploit their applications for industrial, agricultural and pharmaceutical use.

Figure 1

The thin-layer chromatography (TLC) pattern of a saponin mixture (A) and the high performance centrifugal partition chromatography (HCPCP) fractions (B) from the purified extracts of the viscera of the Holothuria lessoni sea cucumber using the lower phase of CHCl₃-MeOH-H₂O (7:13:8) system. The numbers under each lane indicate the fraction number of fractions in the fraction collector. Here, only the fractions 52 to 61 of one analysis (of 110 fractions) are shown as a representative.

Figure 2

The full-scan matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrum of HPCPC Fraction 55 in the (+) ion mode.

Figure 3

Schematic fragmentation patterns of the ion detected at m/z 1303.6; (A) Fraction 15; (B) Fraction 14 and (C) Fraction 12. Full and dotted arrows show the two main feasible fragmentation pathways. The predominant peak (A and B) at m/z 507 corresponds to the key sugar residue and aglycone moiety. The major abundant peak (C) at m/z 523 corresponds to both the key sugar residue and aglycone moiety. Abbreviations; G = Glc, MG = MeGlc, Q = Qui, X = Xyl.

Figure 4

The full-scan MALDI mass spectrum of the isobutanol-enriched saponin extract from the viscera of the H. lessoni. A mass range of 600 to 1500 Da is shown here. It is noted that this spectrum is unique for this species.

Figure 5

The schematic diagram of the proposed isomeric structures of ion at m/z 1303.6.

Figure 6

(+) ion mode ESI-MS/MS spectrum of saponins detected at m/z 1287.6. This spectrum shows the presence of two different aglycones, which led to the isomeric saponins. Full and dotted arrows illustrate the two main possible fragmentation pathways.

Figure 7

A schematic diagram of the proposed isomeric structures of ion at m/z 1287.6.

Figure 8

Positive tandem MALDI spectrum analysis of the precursor ion (saponin) detected at m/z 1243.5. The figure shows the collision-induced fragmentation of parent ions at m/z 1243.5. The consecutive losses of sulfate group, aglycone, xylose (Xyl), quinovose (Qui) and 3-O-methylglucose (MeGlc) residues affords product ions detected at m/z 1123, 639, 507, 361and 185, respectively.

Figure 9

The structure of identified saponins in the viscera of H. lessoni.

Figure 10

(+) ESI-MS spectrum of saponins extract from the viscera of H. lessoni.

Figure 11

(+) ion mode ESI-MS spectrum of saponins extract from Fraction 14.

Figure 12

(+) Ion mode ESI-MS/MS spectrum of saponin detected at 1243.5 (Holothurin A). Full and dotted arrows show the two main feasible fragmentation pathways. The structure of saponin was elucidated on the base of tandem mass spectrometry.