Steroid glycosides from the starfish Pentaceraster gracilis

Abstract Using combined chromatographic separations, two new steroid glycosides namely pentacerosides A (1) and B (2), and four known compounds were isolated from the methanol extract of the starfish Pentaceraster gracilis. Their structures were determined on the basis of spectroscopic data (1H and 13C NMR, HSQC, HMBC, 1H-1H COSY, ROESY, and FT-ICR-MS) and by comparing obtained results to the literature values. Among the isolated compounds, only maculatoside (5) showed significant cytotoxic effect against Hep-G2 (IC50 = 16.75 ± 0.69 μM) and SK-Mel2 (IC50 = 19.44 ± 1.45 μM) cell lines and moderate effect on KB (IC50 = 36.53 ± 0.78 μM), LNCaP (IC50 = 39.75 ± 3.34 μM), and MCF7 (IC50 = 47.34 ± 7.01 μM) cell lines.


Introduction
Steroid glycosides are a class of widespread natural products having either terrestrial or marine origins. In living echinoderms, starfish is a richest biological source of steroid glycosides, as any studied species contains a wide diversity of steroid glycosides [1]. Pentaceraster starfish (phylum Echinodermata, class Asteroidea, order Valvatida, family Oreasteridae) are little investigated species with few steroid glycosides [2,3], steroids [4], glycolipids [4], and glycosphingolipids [5] isolated and identified to date.
In continuation of our ongoing investigations on steroid glycosides of Vietnamese starfish [6,7], the present paper deals with the isolation, structure elucidation, and cytotoxic evaluation of six steroid glycosides (Figure 1), including two new compounds namely pentacerosides A (1) and B (2), from the starfish Pentaceraster gracilis.
Pentaceroside A (1) was isolated as a white powder. Its molecular formula, C 37 H 64 O 14 , was determined by a quasi-molecular ion peak at m/z 755.4193 [M + Na] + on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The 1D and 2D NMR features of 1 were indicative for a polyhydroxysteroid glycoside, one main constituent of starfish [10]. These data were similar to those of nodososide (3) [8], except for differences in the data of sugar moieties. The 1 H and 13 C NMR spectra for the aglycon of 1 confirmed the presence of four oxymethine groups [δ C 68. This evidence and the heteronuclear multiple bond correlation (HMBC) cross-peaks of H 3 -19 (δ H 1.33) with C-1 (δ C 34.4), C-5 (δ C 76.4), C-9 (δ C 49.2), and C-10 (δ C 39.2); H-6 (δ H 3.61) with C-8 (δ C 77.4) and C-10 (δ C 39.2); and that of H-7 (δ H 2.11) with C-5 (δ C 76.4) confirmed positions of the four hydroxy groups at C-3, C-5, C-6, and C-8. Detailed analysis of other COSY and HMBC correlations ( Figure 2) clearly confirmed the planar structure of 1. The relative configurations for the aglycon of 1 were assigned to be identical to those of nodososide (3) [8] on the basis of a good agreement of their 1 H and 13 C NMR data as well as the coexistence in P. gracilis, which were also supported by ROESY experiment (Figure 3).

Biological material
The sample of the starfish P. gracilis (Lütken, 1871) was collected at Bac Van, Co To, Quangninh, Vietnam, in March 2014, and identified by Prof. Do Cong Thung. A voucher specimen (BV-SB1) was deposited at the Institute of Marine Biochemistry and Institute of Marine Environment and Resources, VAST, Vietnam.

Extraction and isolation
Dried body walls of the starfish P. glacilis (3.5 kg) were extracted three times with MeOH under ultrasonic condition to obtain 400 g residue after removal of MeOH in vacuum. This was suspended in water (2 L) and partioned three times with CH 2 Cl 2 (2 L each time) to obtain CH 2 Cl 2 residue (70 g) and water layer. The latter was passed through a Diaion HP-20 CC eluting with increasing concentration of MeOH in water (0, 25, 50, 75, and 100%) to obtain four fractions, W1-W4, after removal of the fraction eluted with water. Fraction