Asterosaponins and glycosylated polyhydroxysteroids from the starfish Culcita novaeguineae and their cytotoxic activities

Using combined chromatographic methods, two asterosaponins (compounds 1 and 2), including a new compound novaeguinoside E (compound 1), and six glycosylated polyhydroxysteroids (compounds 3–8) were isolated from a methanol extract of the starfish Culcita novaeguineae. Their structures were determined on the basis of spectroscopic data (1H and 13C NMR, HSQC, HMBC, 1H–1H COSY, ROESY, and HRESI-MS) and by comparison with the literature values. The new compound 1 represents the third example of asterosaponins containing the 5α-cholesta-9(1l)-en-3β,6α,20,22-tetraol aglycone. Among isolated compounds, 4–7 exhibited moderate to weak cytotoxic activities against five human cancer cell lines such as Hep-G2 (hepatoma), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma).


Introduction
Starfish are invertebrates belonging to the class Asteroidea, phylum Echinodermata. The secondary metabolites from starfish are characterized by a diversity of polar steroids, including polyhydroxylated steroids and steroid glycosides. There are two main structural groups of steroid glycosides from starfish, namely asterosaponins and glycosylated polyhydroxysteroids. These compounds have exhibited a variety of biological activities, such as cytotoxic, hemolytic, and anti-microbial effects [1 -5].
As a part of our ongoing investigations to catalog the chemical constituents and biological effects of Vietnamese starfish [6 -11], we studied to this concern the species C. novaeguineae. The present study addresses the isolation and structure elucidation of two asterosaponins, including a new compound novaeguinoside E (1), and six glycosylated polyhydroxysteroids ( Figure 1) from this starfish. Their in vitro cytotoxic activities against five human cancer cell lines were also evaluated using the sulforhodamine B (SRB) method.
Novaeguinoside E (1) was isolated as a white amorphous powder. Its molecular formula was determined as C 56 H 91 NaO 27 S by high-resolution electrospray ionization mass spectrometry (HRESI-MS) at m/z 1273.5257 [M þ Na] þ . The NMR features indicated an asterosaponin, one of the main constituents of starfish. The 1 H and 13 C NMR data (in DMSO-d 6 ) of compound 1 were similar to those of protoreasteroside [20], except for differences in the data of sugar moieties. Analysis of 1D and 2D NMR spectra confirmed that the aglycone of compound     Figure 2). The 13 C NMR chemical shift of C-21 at d C 19.9 (in DMSO-d 6 ) indicated the relative R* configuration at C-20 [20]. For determination of the stereochemistry at C-22, the 1 H NMR spectrum of compound 1 was recorded again in pyridine-d 5 . The 1 H NMR chemical shift of H-21 at d H 1.64 (pyridine-d 5 ) clearly indicated the relative S* configuration at C-22 [20].

General experimental procedures
Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo, Japan). High-resolution mass spectra were recorded on a MicroQ-TOF III mass spectrometer (Bruker Daltonics, Bremen, Germany). The 1 H NMR (500 MHz) and 13

Extraction and isolation
The fresh body walls of C. novaeguineae (10 kg) were cut into small pieces and extracted in hot methanol (

Cytotoxic assays
Cytotoxic evaluations were performed by following the protocols described previously [23,24].

Supplementary data
1D, 2D-NMR, and HR-ESI mass spectra for the new compound 1.