Using various chromatographic methods, a polyhydroxylated sterol 5α-cholestane-3β,6β,7α,8β,15α,16β,26-heptol (1) and an asterosaponin sodium salt of 6α-[(O-β-D-fucopyranosyl-(l®2)-O-β-D-galactopyranosyl-(l®4)-O-[β-D-quinovopyranosyl-(l®2)]-O-β-D-xylopyranosyl-(l®3)-O-β-D-quinovopyranosyl)oxy]-5α-pregn-9(11)-ene-20-one (2), were isolated from the methanol extract of the starfish Culcita novaeguineae. Their structures were elucidated by 1D and 2D-NMR experiments and comparison of their NMR data with reported values. Compounds 1 was isolated from         C. novaeguineae for the first time

Cuong, Nguyen Xuan

Diep, Chau Ngoc

Hong Hanh, Tran Thi

Kiem, Phan Van

Minh, Chau Van

Nam, Nguyen Hoai

Ngoan, Bui Thi

Thanh, Nguyen Van

Thao, Nguyen Phuong

Thung, Do Cong

Vien, Le Thi

Vietnam Academy of Science and Technology: Journals Online

 23 
VIETNAM JOURNAL OF CHEMISTRY         VOL. 53(2e) 23-27                                               APRIL 2015 
DOI: 10.15625/0866-7144.2015-2e-006 
  
 
 
A POLYHYDROXYLATED STEROL AND A SAPONIN ISOLATED FROM 
THE STARFISH CULCITA NOVAEGUINEAE 
 
Bui Thi Ngoan
1
, Tran Thi Hong Hanh
1
, Le Thi Vien
1
, Chau Ngoc Diep
1
, Nguyen Phuong Thao
1
,  
Nguyen Van Thanh
1
, Nguyen Xuan Cuong
1
, Nguyen Hoai Nam
1
,  Do Cong Thung
2
,  
Phan Van Kiem
1
, Chau Van Minh
1*
 
 
1
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) 
2
Institute of Marine Environment and Resources, VAST  
Received 23 January 2015; Accepted for Publication 18 March 2015 
  
Abstract 
Using various chromatographic methods, a polyhydroxylated sterol 5α-cholestane-3β,6β,7α,8β,15α,16β,26-heptol 
(1) and an asterosaponin sodium salt of 6α-[(O-β-D-fucopyranosyl-(l2)-O-β-D-galactopyranosyl-(l4)-O-[β-D-
quinovopyranosyl-(l2)]-O-β-D-xylopyranosyl-(l3)-O-β-D-quinovopyranosyl)oxy]-5α-pregn-9(11)-ene-20-one (2), 
were isolated from the methanol extract of the starfish Culcita novaeguineae. Their structures were elucidated by 1D 
and 2D-NMR experiments and comparison of their NMR data with reported values. Compounds 1 was isolated from         
C. novaeguineae for the first time. 
Keywords. Culcita novaeguineae, Oreasteridae, starfish, polyhydroxylated sterol, asterosaponin.  
 
1. 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. 
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 on 
Vietnamese starfish, we address herein the isolation 
and structure identification of a polyhydroxylated 
sterol and an asterosaponin from the starfish Culcita 
novaeguineae. 
 
2. EXPERIMENTAL 
2.1. General experimental procedures 
The 
1
H-NMR (500 MHz) and 
13
C-NMR (125 
MHz) spectra were recorded on a Bruker AM500 
FT-NMR spectrometer, TMS was used as an internal 
standard. The electrospray ionization mass spectra 
(ESI-MS) were obtained on an Agilent 1260 series 
single quadrupole LC/MS system (Waldbronn, 
Germany). Medium pressure liquid chromatography 
(MPLC) was carried out on a Biotage - Isolera One 
system (SE-751 03 Uppsala, Sweden). Column 
chromatography (CC) was performed on silica gel 
(Kieselgel 60, 70–230 mesh and 230–400 mesh, 
Merck) and YMC RP-18 resins (30–50 μm, Fuji 
Silysia Chemical Ltd.). Thin layer chromatography 
(TLC) used pre-coated silica gel 60 F254 
(1.05554.0001, Merck) and RP-18 F254S plates 
(1.15685.0001, Merck). Compounds were visualized 
by spraying with aqueous 10 % H2SO4 and heating 
for 3–5 minutes.  
2.2. Marine materials 
The sample of the starfish C. novaeguineae 
Muller & Troschel, 1842 was collected at 
Quangninh, Vietnam, in October 2013, and 
identified by Prof. Do Cong Thung. A voucher 
specimen (DAB-DG-CN-01/2013) was deposited at 
the Institute of Marine Biochemistry and Institute of 
Marine Environment and Resources, VAST, 
Vietnam. 
2.3. Isolation 
The fresh body walls of C. novaeguineae (10 kg) 
were cut into small pieces and extracted in hot 
methanol (three times for 6h each) to afford a MeOH 
residue (125 g, A) after removal of the solvent under 
reduced pressure. This extract was partitioned 
between H2O and CH2Cl2 (31.0 L) to give CH2Cl2 
 24 
VJC, Vol. 53(2e), 2015                                                                                                      Chau Van Minh, et al. 
extract (C, 15.2 g) and water layer. The CH2Cl2 
extract (C, 15.2 g) was separated by silica gel MPLC 
using gradient elution of CH2Cl2MeOH 
(100:11:1, v/v) to obtain nine fractions, C1C9. 
Fraction C-8 (2 g) was separated into six 
subfractions, C-8.1C8.6, by YMC RP-18 MPLC 
using gradient elution of MeOHH2O (1:15/1, v/v). 
Further separation of subfraction C-8.4 (0.17 g) by 
silica gel CC eluting with EtOAcMeOHH2O 
(10:1:0.1, v/v), followed by YMC CC with 
MeOHH2O (1.5:1, v/v) to obtain compound 1 (5.4 
mg). The latter was passed through 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 W4 (6.5 g) was separated 
into five subfractions, W4AW4D, by silica gel 
MPLC using gradient elution of CH2Cl2MeOH 
(20:11:1, v/v). Subfraction W4D (1.5 g) was 
further separated on YMC RP-18 CC eluting with 
MeOHH2O (1:1, v/v), followed by silica gel CC 
using CH2Cl2MeOHH2O (3:1:0.15, v/v) as eluent 
furnished compound 2 (4.5 mg).  
5α-cholestane-3β,6β,7α,8β,15α,16β,26-heptol 
(1): White powder; 
1
H-NMR (500 MHz, DMSO-d6) 
and 
13
C-NMR (125 MHz, DMSO-d6) see table 1; 
ESI-MS: m/z 507 [M+Na]
+
 and 519 [M+Cl]

 
(C27H48O7, M = 484). 
 
 
Figure 1: Chemical structures of 1 and 2
Table 1: NMR data of 1 and reported compounds 
C 
a
δC 
b
δC δC
c,d δH
c,e
 mult. (J in Hz) COSY (H  H) HMBC (H  C) 
1 39.6 41.4 39.70 0.85 m/1.55 m 2  
2 31.5 31.7 30.80 1.32 m/1.60 m 1, 3  
3 72.3 72.5 69.99 3.40 m 2, 4  
4 32.3 35.8 35.09 1.35 m/1.65 m 3, 5  
5 44.5 42.8 40.64 1.40 m 4, 6  
6 68.9 74.1 75.53 3.53 br dd (3.0, 4.5) 5, 7  
7 76.5 78.4 72.02 3.67 t (3.0) 6 5, 9 
8 77.7 77.8 76.53 -   
9 51.2 51.4 49.39 1.09 m 11  
10 37.8 36.4 34.78 -   
11 19.3 18.3 17.98 1.42 m/1.72 m 9, 12  
12 43.2 43.2 41.53 1.10 m/1.82 m 11  
13 45.5 45.6 43.66 -   
14 59.6 59.5 57.86 1.27 m 15  
15 79.3 80.1 78.25 3.98 m 14, 16  
16 82.7 82.8 80.18 3.82 dt (1.5, 7.0) 15, 17 13 
17 61.4 61.6 59.61 1.08 m 16, 20  
18 16.6 16.8 16.19 1.03 s  12, 13, 14, 17 
19 13.9 16.4 15.33 1.00 s  1, 5, 9, 10 
20 30.6 30.6 28.74 1.77 m 17, 21, 22  
21 18.4 18.3 17.65 0.83 d (6.5) 20 17, 20, 22 
22 37.1 37.0 35.32 0.96m/1.35 m 20, 23  
23 24.9 24.8 23.24 1.12 m/1.37 m 22, 24  
24 35.0 35.0 33.48 0.94 m/1.35 m 23, 25  
25 37.1 37.1 35.49 1.45 m 24, 26, 27   
26 68.5 69.6 66.25 3.15 m/3.24 m 25 14, 25, 27 
27 17.4 17.2 17.00 0.81 d (6.5) 25 24, 25, 26 
8-OH - - - 4.07 s  7, 8, 14 
aC of 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol in CD3OD [6], 
bC of 5α-cholestane-3β,6β,7α,8β,15α,16β,26-heptol 
in CD3OD [7], 
c
recorded in DMSO-d6, 
d
125 MHz, 
e
500 MHz. 
 25 
VJC, Vol. 53(2e), 2015                                                                     A polyhydroxylated sterol and a saponin… 
Table 2: 
1
H-NMR (500 MHz) and 
13
C-NMR (125 MHz) data of 2 and reported compound 
C 
aδC δC
b δH
b
 mult. (J = Hz) C 
aδC δC
b δH
b
 mult. (J = Hz) 
1 35.9 35.33 1.28 m/1.63 m Xyl    
2 29.4  28.43 1.39 m/2.12 m 1 104.4 102.65 4.53 d (7.0) 
3 77.6  75.40 3.86 m 2 82.2 82.69 3.35
 c
 
4 30.7  29.57 1.08 m/2.37 m 3 75.5 74.11 3.57
 c
 
5 49.2  48.49 1.10 m 4 79.2 77.17 3.60
 c
 
6 80.2  78.06 3.47 m 5 64.5 63.16 3.30/3.95
 c
 
7 41.6  40.62 0.86 m/2.26 m Qui II    
8 35.5  34.97 2.03 m 1 105.1  104.75 4.43 d (7.5) 
9 146.0  145.78 - 2 75.4 74.99 3.07 dd (7.5, 9.0) 
10 38.3  37.92 - 3 76.9 75.60 3.14 t (9.0) 
11 116.0  115.57 5.31 d (4.5) 4 76.2 74.75 2.86 t (9.0) 
12 40.6  40.09 2.20 m/2.28 m 5 73.9 72.17 3.22
 c
 
13 42.5  41.99 - 6 18.5 17.40 1.18 d (6.0) 
14 53.7  52.97 1.37 m Gal    
15 23.1  22.38 1.58 m/2.03 m 1 102.4  100.58 4.41 d (7.5) 
16 25.6  25.01 1.20 m/1.73 m 2 83.4 81.86 3.48
 c
 
17 63.3  62.44 2.65 t (9.5) 3 75.0 72.61 3.33
 c
 
18 13.1  12.90 0.43 s 4 69.0 67.48 3.67 br s 
19 19.2  19.12 0.88 s 5 76.8 75.34 3.42
 c
 
20 208.3  208.75 - 6 62.0 60.37 3.50
 c
 
21 31.0  30.94 2.05 s Fuc    
Qui I    1 107.2  105.73 4.21 d (7.0) 
1 105.1 102.85 4.31 d (8.0) 2 71.9 71.09 3.40
 c
 
2 74.1 73.17 3.13
 c
 3 75.0 73.21 3.51
 c
 
3 90.2 87.87 3.28
 c
 4 72.6 70.88 3.27
 c
 
4 74.5 73.08 2.91 t (9.0) 5 71.9 70.59 3.53
 c
 
5 71.9 70.59 3.27
 c
 6 17.2 16.76 1.12 d (6.0) 
6 17.9 17.96 1.14 d (6.0)     
aC of sodium salt of 6α-[(O-β-D-fucopyranosyl-(l2)-O-β-D-galactopyranosyl-(l4)-O-[β-D-quinovopyranosyl-
(l2)]-O-β-D-xylopyranosyl-(l3)-O-β-D-quinovopyranosyl)oxy]-5α-pregn-9(11)-ene-20-one in pyridine-d5 [8], 
b
recorded in DMSO-d6, 
c
overlapped signals. 
Sodium salt of 6α-[(O-β-D-fucopyranosyl-
(l2)-O-β-D-galactopyranosyl-(l4)-O-[β-D-
quinovopyranosyl-(l2)]-O-β-D-xylopyranosyl-
(l3)-O-β-D-quinovopyranosyl)oxy]-5-α-pregn-
9(11)-ene-20-one (2): White powder; 
1
H-NMR (500 
MHz, DMSO-d6) and 
13
C-NMR (125 MHz, DMSO-
d6) see table 2; ESI-MS: m/z 1143 [MNa]

 
(C50H79NaO27S, M = 1166). 
3. RESULTS AND DISCUSSION 
Compound 1 was obtained as a white powder. 
The NMR features are typical for a 
polyhydroxylated sterol, one main constituent from 
starfish [1]. The 
1
H-NMR spectrum revealed signals 
of two tertiary methyl [H 1.03 (H-18) and 1.00     
(H-19), each 3H, s] and two secondary methyl 
groups [H 0.83 (H-21) and 0.81 (H-27), each 3H, d, 
J = 6.5 Hz]. In addition, five oxymethine groups [C 
69.99 (C-3), 75.65 (C-6), 72.02 (C-7), 78.25 (C-15), 
and 80.18 (C-16)/H 3.40 (1H, m, H-3), 3.53 (1H, br 
dd, J = 3.0, 4.5 Hz, H-6), 3.67 (1H, t, J = 3.0 Hz,    
H-7), 3.98 (1H, m, H-15), and 3.82 (1H, dt, J = 1.5, 
7.0 Hz, H-16)], one oxygenated quaternary carbon 
[C 76.53 (C-8)], and one oxymethylene group [C 
66.25 (C-26)/H 3.15 (1H, m, Ha-26) and 3.24 (1H, 
m, Hb-26)] were determined in the 
1
H- and 
13
C-NMR 
spectra of 1. All 
1
H-NMR data were assigned with 
 26 
VJC, Vol. 53(2e), 2015                                                                                                      Chau Van Minh, et al. 
the relevant 
13
C-NMR data by HSQC experiment 
and the results were shown in the table 1.  
Analysis of 
1
H-
1
H COSY correlations led to 
identify the connectivities of H-1/H-2/H-3/H-4/H-
5/H-6/H-7, H-9/H-11/H-12, H-15/H-16/H-17/H-
20/H-22, H-23/H-24/H-25/H-26, H-20/H-21, and H-
25/H-27. These data and the HMBC cross-peaks of 
H-18 (H 1.03) with C-12 (C 41.53), C-13 (C 
43.66), C-14 (C 57.86), and C-17 (C 59.61); H-19 
(H 1.00) with C-1 (C 39.70), C-5 (C 40.64), C-9 
(C 49.39), and C-10 (C 34.78); and those of OH-8 
(H 4.07) with C-7 (C 72.02), C-8 (C 76.53)/C-14 
(C 57.86), clearly confirmed the planar structure of 
1 (figure 2), which was also supported by 
comparison of the NMR data with the reported 
values [6, 7]. In the ROESY spectrum, the spatial 
proximities were observed between H-5 (H 1.40) 
and H-3 (H 3.40)/H-6 (H 3.53), H-6 (H 3.53) and 
OH-7 (H 5.45), and H-16 (H 3.82) and H-17 (H 
1.08) confirmed the α-orientation for H-3, H-6, OH-
7, and H-16. Moreover, the correlations of H-15 
(3.98) with H-7 (H 3.67) and H-18 (H 1.03), and 
those of OH-8 (H 4.07) with H-7 (H 3.67) and H-
19 (H 1.00) indicated β-orientation of H-7, OH-8, 
and H-15. Thus, compound 1 was elucidated as 5α-
cholestane-3β,6β,7α,8β,15α,16β,26-heptol. This is 
the first report of 1 from the starfish C. 
novaeguineae. However, based on 2D-NMR 
experiments, the reported 
13
C-NMR data at C-6 and 
C-7 [7] must be reversed as shown in the table 1. 
 
 
Figure 2: Key ROESY correlations of 1 
 The NMR spectral data of 2 indicated an 
asterosaponin [2, 3] containing five sugar units. All 
carbon signals were assigned with relevant protons 
by mean of an HSQC experiment (Table 2). 
Comparison of the 
13
C-NMR of 2 with published 
values and analysis of HMBC cross-peaks led to 
assignment of 2 as sodium salt of 6α-[(O-β-D-
fucopyranosyl-(l2)-O-β-D-galactopyranosyl-
(l4)-O-[β-D-quinovopyranosyl-(l2)]-O-β-D-
xylopyranosyl-(l3)-O-β-D-quinovopyranosyl) 
oxy]-5α-pregn-9(11)-ene-20-one in pyridine-d5 [8, 
9]. The HMBC correlation of H-1 (H 4.21) with 
C-2 (C 81.86), H-1 (H 4.41) with C-4 (C 
77.17), H-1 (H 4.43) with C-2 (C 82.69), and 
that of H-1 (H 4.53) with C-3 (C 87.87) 
confirmed positions of all inner linkages. Moreover, 
anomeric proton H-1 (H 4.31) had an HMBC cross-
peak with C-6 (C 78.06) confirming the attachment 
of the pentaglycoside chain at C-3 (figure 3). 
 
Acknowledgements. This work was financially 
supported by Vietnam Academy of Science and 
Technology (Codes: VAST.TÐ.ÐAB.03/13–15 and 
VAST.HTQT.Nga.08/2014-2015).  
 
 
Figure 3: Key HMBC correlations of 2 
 
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VJC, Vol. 53(2e), 2015                                                                     A polyhydroxylated sterol and a saponin… 
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Corresponding author: Chau Van Minh 
                       Institute of Marine Biochemistry, Vietnam Academy of Science and Technology 
                       18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 
                       E-mail: cvminh@vast.vn. 
 
 


A polyhydroxylated sterol and a saponin isolated from the starfish culcita novaeguineae

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A polyhydroxylated sterol and a saponin isolated from the starfish culcita novaeguineae

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