Identification of Sixteen New Galactocerebrosides from the Starfish Protoreaster nodosus

Abstract

Cerebrosides, mainly glucocerebrosides and galactocerebrosides, are the simplest glycosphingolipids (GLSs) and known to be the biosynthetic precursors of most other GLSs. 1) While glucocerebrosides have been isolated from various starfishes, occurrence of galactocerebrosides seems to be limited to three species: Stellaster equestris, 2) Culcita novaeguineae, 3) and Oreaster reticulatus. 4) In our continuing research on GLSs from echinoderms, a galactocerebroside molecular species (PNC-1) was purified from the starfish Protoreaster nodosus (kobuhitode in Japanese) collected in Okinawa. Reversed-phase HPLC separation of PNC-1 afforded sixteen new galactocerebrosides (3-7, 9-17, 19, 21), along with five known galactocerebrosides (1, 2, 8, 18, 20). Further, we developed one-pot GC-MS analysis for cerebrosides which consist of phytosphingosinetype long chain base (LCB). Generally, the trimethylsilyl (TMS) derivatives of LCB are used for GC-MS analysis and give a useful structural information, 5) however, the preparation of phytosphingosine-type LCB from the parent cerebroside is in a quite low yield and not quantitative, because the glycosidic linkage between sugar and LCB is hard to cleavage against methanolysis. One-pot reaction following mathanolysis and periodate oxidation, and subsequent GC-MS analysis gave efficient structural information. In this paper, the isolation and structure elucidation of galactocerebroside molecular species (PNC-1) and constituent cerebrosides (1-21) of PNC-1 from the starfish Protoreaster nodosus are described.Isolation and Structure of PNC-1 The lipid fraction, obtained from CHCl 3 /MeOH extract of pyloric caeca, which was dissected from the fresh material of the starfish P. nodosus, was subjected to repeated silica gel, reversed-phase and Sephadex LH-20 column chromatography to give a less polar GLS, named PNC-1, showing a single spot on silica gel TLC plate.The positive ion FAB-MS spectrum of PNC-1 exhibited a series of homologous pseudomolecular ion peaks [MϩNa] )] in the total correlation spectroscopy (TOCSY) spectrum. Thus, a structural fragment from H 2 -1 to H 2 -6 was established, suggesting the LCB of ceramide was phytosphingosine-type. Analysis of COSY and TOCSY spectra revealed another structural fragment (H-2Ј to H 2 -4Ј) in the fatty acid part of ceramide. H-2Ј (d H 4.52) signal showed a heteronuclear multiple-bond correlation spectroscopy (HMBC) cross peak with the amide carbon signal [d C 175.7 (C-1Ј)], indicating the presence of a-hydroxy fatty acid. Further, H-2 (d H 5.18) signal in LCB showed a cross peak with amide carbon (C-1Ј), and the ceramide moiety was depicted as shown in Fig. 1.Identification of the compositions of fatty acid and LCB of ceremide moiety was conducted by GC-MS analysis following methanolysis and periodate oxidation of PNC-1 (see Experimental). The mixture of fatty acid methyl esters (FAMEs) obtained from methanolysis and a mixture of long chain aldehydes generated by periodate oxidation were simultaneously subjected to GC-MS, which revealed ...

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

“…IL-12 plays an important role in the generation of Th1 responses to human pathogens [23,24]. Although the induction of IL-12 by intracellular organisms is necessary for a protective host Th1 response, the overexpression of Th1 cytokines and IL-12 may contribute to the development and perpetuation of chronic inflammatory and autoimmune diseases.…”

“…However, studies on the biological activities of P. nodosus are limited. In previous studies, the main constituents of P. nodosus were found to be polyhydroxylated steroids [22], several of which exhibited moderate cytotoxicity, steroidal glycoside sulfates [23], galactocerebrosides [24], and gangliosides [25]. …”

Isolation and Structure Elucidation of GM4-Type Gangliosides from the Okinawan Starfish Protoreaster nodosus

“…Another fragment in FA, from C-2 to C-4, was also connected by extensive analysis of COSY and TOCSY spectra. The stereochemistry of the ceramide moiety was presumed to be identical to that of PNC-1 (Figure 3a), which was isolated from the same material [7]. …”

“…nodosus [7]. In a continuing investigation into GLS constituents of this animal, we isolated three new ganglioside molecular species and describe their structures in the present article.…”

Glycolipids from Marine Invertebrates

“…A mixture of related galactocerebrosides, namely molecular species CNC-2, was isolated from the starfish Culcita novaeguineae collected near Okinawa, Japan (Inagaki, Nakata, and Higuchi, 2006). Recently, 16 new b-galactopyranosides with saturated ceramides and 2-hydroxy FAs were isolated from a chloroform-methanol extract of pyloric ceaca dissected from the starfish Protoreaster nodosus as three series of homologous GSLs possessing normal, iso, and anteiso sphingosine chains (Pan et al, 2010). Among these, only four GSLs were isolated as pure compounds (see Table 5.3), the others being mixtures of homologs.…”