Using various chromatographic experiments, four sterols as 5α,8α-epidioxy-24(S)-ethylcholest-6-en-3β-ol (1), (22E,24S)-5α,8α-epidioxy-24-ethyl-cholesta-6,22-dien-3β-ol (2), 7-oxocholesterol (3), and (22E)-3β-hydroxycholesta-5,22-dien-7-one (4) were isolated from the methanol extract of the blood cockle Anadara granosa. The structures of isolated compounds were elucidated by 1D and 2D-NMR experiments in comparison with reported data. This is the first report of these compounds from A. granosa

Cuong, Nguyen Xuan

Giang, Nguyen Hoang

Hong Hanh, Tran Thi

Minh, Chau Van

Thanh Huong, Phan Thi

Thanh, Nguyen Van

Thung, Do Cong

Vietnam Academy of Science and Technology: Journals Online

 Vietnam Journal of Chemistry, International Edition, 54(6): 786-789, 2016 
DOI: 10.15625/0866-7144.2016-00405 
786 
 
 
 
Steroids from the blood cockle Anadara granosa 
Phan Thi Thanh Huong
1
, Nguyen Hoang Giang
2
, Tran Thi Hong Hanh
1
, Do Cong Thung
3
, 
Nguyen Xuan Cuong
1
, Nguyen Van Thanh
1
, Chau Van Minh
1* 
 
1
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology 
2
Faculty of Chemistry, VNU University of Science 
3
Institute of Marine Environment and Resources, VAST 
Received 24 November 2016; Accepted for publication 19 December 2016 
  
Abstract 
Using various chromatographic experiments, four sterols as 5α,8α-epidioxy-24(S)-ethylcholest-6-en-3β-ol (1), 
(22E,24S)-5α,8α-epidioxy-24-ethyl-cholesta-6,22-dien-3β-ol (2), 7-oxocholesterol (3), and (22E)-3β-hydroxycholesta-
5,22-dien-7-one (4) were isolated from the methanol extract of the blood cockle Anadara granosa. The structures of 
isolated compounds were elucidated by 1D and 2D-NMR experiments in comparison with reported data. This is the first 
report of these compounds from A. granosa. 
Keywords. Anadara granosa, Arcidae, blood cockle, steroid.  
 
1. INTRODUCTION 
 
With more than 100,000 species, the phylum 
Mollusca is the second largest after the Arthropoda 
and, like the latter, the Mollusca includes as many 
marine species as terrestrial species. Of these, 
mollusk shells have been of considerable economic 
importance since antiquity [1]. 
The blood cockle Anadara granosa is one 
important sea product in Vietnam and some Asian 
countries. As a part of our investigations on 
chemical constituents of Vietnamese mollusks, we 
report herein the isolation and structure 
identification of four steroids from A. granosa. 
 
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 with TMS used as an internal 
standard. 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, Darmstadt, Germany), YMC*GEL (ODS-A, 
12 nm S-150 mm, YMC Co., Ltd., Japan), Sephadex 
LH-20 (Sigma-Aldrich, USA), and Diaion HP-20 
(Supelco, USA) resins. For thin layer 
chromatography (TLC) there 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 samples of blood cockle A. granosa were 
collected in Cat Ba, Hai Phong, Vietnam, in August 
2014 and identified by Professor Do Cong Thung. 
Voucher specimens (No. VAST-TM09) were 
deposited at the IMBC, VAST, Vietnam. 
2.3. Isolation 
The muscle of blood cockle Anadara granosa 
was cut into small pieces and dried at 50
o
C. The 
dried powder (1.5 kg) was extracted with MeOH in 
ultrasonic condition (3×1h). The obtained solutions 
were filtered, combined, and concentrated under 
reduced pressure to afford a MeOH residue (M, 150 
g). This was suspended in water and partitioned in 
turn with n-hexane, CH2Cl2, and EtOAc to give the   
n-hexane (H, 50g), CH2Cl2 (D, 20g), EtOAc (E, 5g) 
extracts, and water layer (W). 
Extract H (50 g) was crudely separated by silica 
gel MPLC with gradient concentrations of acetone in 
n-hexane (from 2 to 100 %) to yield five fractions, 
H1-H5. Fraction H2 (6 g) was separated by silica gel 
 VJC, 54(6) 2016                                                                                                                Chau Van Minh, et al. 
787 
CC eluting with n-hexane/acetone (7/1) to give three 
subfractions, H2a-H2c. Subfraction H2c (350 mg) 
was divided into two smaller fractions, H2c1 and 
H2c2, by YMC CC eluting with acetone/H2O (5/1). 
Fraction H2c1 (45 mg) was purified by Sephadex 
LH-20 CC eluting with MeOH/H2O (3/1) to give 
compounds 3 (5.0 mg) and 4 (4.0 mg). Compounds 
1 (4.5 mg) and 2 (25 mg) were purified from fraction 
H2c2 (150 mg) using silica gel CC with                   
n-hexane/EtOAc (4/1) as eluent.  
 
Figure 1: Chemical structures of compounds 1 4 
Table 1: 
1
H-NMR (500 MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3) data of 1 and 2 
C 
aδC 
1
 
bδC 
2
 
δC δH mult. (J = Hz) δC δH mult. (J = Hz) 
1 34.75 34.72 1.68 m/1.94 m 34.76 34.70 1.68 m/1.94 m 
2 30.16 30.15 1.52 m/1.83 m 30.19 30.13 1.53 m/1.83 m 
3 66.52 66.49 3.96 m 66.54 66.47 3.97 m 
4 37.00 36.97 1.90 m/2.10 m 37.01 36.94 1.89 m/2.10 m 
5 82.24 82.16 - 82.22 82.15 - 
6 135.46 135.41 6.24 d (8.5) 135.46 135.41 6.24 d (8.5) 
7 130.83 130.79 6.51 d (8.5) 130.83 130.76 6.50 d (8.5) 
8 79.54 79.47 - 79.49 79.42 - 
9 51.10 51.11 1.49 m 51.14 51.11 1.49 m 
10 37.00 36.97 - 37.01 36.98 - 
11 23.48 23.43 1.23 m/1.50 m 23.48 23.41 1.22 m/1.50 m 
12 39.49 39.45 1.23 m/1.97 m 39.42 39.36 1.23 m/1.96 m 
13 44.80 44.75 - 44.79 44.55 - 
14 51.63 51.61 1.55 m 51.76 51.70 1.57 m 
15 20.68 20.65 1.42 m/1.62 m 20.77 20.70 1.40 m/1.60 m 
16 28.32 28.26 1.38 m/1.92 m 29.05 28.88 1.36 m/1.78 m 
17 56.36 56.34 1.20 m 56.21 56.15 1.23 m 
18 12.69 12.63 0.80 s 12.51 12.86 0.82 s 
19 18.24 18.17 0.88 s 18.23 18.17 0.88 s 
20 36.01 35.73 1.36 m 40.09 39.95 2.04 m 
21 18.71 18.71 0.91 d (6.5) 21.16 21.12 1.01 d (6.5) 
22 34.75 33.73 0.99 m/1.36 m 137.72 137.60 5.15 dd (8.5, 15.0) 
23 26.11 26.41 1.02 m/1.30 m 129.97 129.97 5.04 dd (8.5, 15.0) 
24 45.86 46.05 0.92 m 51.29 51.18 1.52 m 
25 29.20 28.98 1.67 m 31.92 31.79 1.52 m 
26 19.89 19.59 0.83 d (6.5) 21.22 20.90 0.83 d (6.5) 
27 18.76 18.98 0.80 d (6.5) 19.00 18.94 0.78 d (6.5) 
28 23.11 23.03 1.13 m/1.32 m 25.42 25.35 1.18 m/1.42 m 
29 12.38 12.31 0.85 t (6.5) 12.92 12.44 0.82 t (6.5) 
a
C of 5α,8α-epidioxy-24(S)-ethylcholest-6-en-3β-ol [2], 
b
 of (22E,24S)-5α,8α-epidioxy-24-ethyl-cholesta-6,22-dien-
3β-ol [2]. 
 VJC, 54(6) 2016                                                                     Steroids from the blood cockle Anadara granosa. 
788 
Table 2: 
1
H-NMR (500 MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3) data of 3 and 4 
C 
aδC 
3 bδC 
4 
δC δH mult. (J = Hz) δC δH mult. (J = Hz) 
1 36.46 36.36 1.36 m/1.97 m 36.3 36.37 1.20 m/1.95 m 
2 31.29 31.22 1.63 m/1.97 m 31.2 31.20 1.62 m/1.94 m 
3 70.61 70.55 3.67 m 70.5 70.52 3.68 m 
4 41.90 41.83 2.42 m/2.52 m 41.8 41.83 1.82 m 
5 165.23 165.04 - 165.0 165.10 - 
6 126.27 126.15 5.69 s 126.1 126.44 568 d (1.5) 
7 202.40 202.28 - 202.2 202.20 - 
8 45.43 45.43 2.24 t (10.5) 45.4 45.40 2.23 m 
9 50.07 49.99 1.35 m 50.0 49.97 1.51 m 
10 38.38 38.29 - 38.3 38.29 - 
11 21.34 21.24 1.01 m 21.2 21.22 1.59 m 
12 38.82 38.73 1.17/2.04 38.6 38.60 1.17 m/2.00 m 
13 41.90 43.12 - 43.0 43.02 - 
14 50.07 49.96 1.53 m 51.2 50.06 1.34 m 
15 26.41 26.33 1.25 m/2.41 m 51.2 26.35 1.24 m/2.39 m 
16 28.60 28.55 1.09 m/1.95 m 28.8 28.75 1.28 m/1.78 m 
17 54.91 54.83 1.13 m 54.6 54.68 1.14 m 
18 12.07 11.98 0.68 s 12.2 12.20 0.69 (s) 
19 17.40 17.32 1.20 s 17.3 17.32 1.20 (s) 
20 35.80 35.72 1.40 m 39.9 39.89 2.05 m 
21 18.96 18.88 0.92 d (6.5) 21.0 21.06 1.01 d (6.5) 
22 36.28 36.20 1.01 m/1.25 m 137.9 137.88 5.25 m 
23 23.92 23.84 1.18 m/1.36 m 126.4 126.11 5.28 m 
24 39.58 39.49 1.17 m 41.9 41.95 2.39 m/2.50 m 
25 28.09 28.01 1.13 m 28.5 28.56 1.59 m 
26 22.64 22.56 0.86 d (6.5) 22.3 22.30 0.86 d (6.5) 
27 22.88 22.81 0.86 d (6.5) 22.3 22.30 0.86 d (6.5) 
a
C of 7-oxocholesterol [3], 
b
C of (22E)-3β-hydroxycholesta-5,22-dien-7-one [4]. 
5α,8α-Epidioxy-24(S)-ethylcholest-6-en-3β-ol 
(1): White powder; 
1
H-NMR (500 MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3), see table 1. 
(22E,24S)-5α,8α-Epidioxy-24-ethyl-cholesta-
6,22-dien-3β-ol (2): White powder; 1H-NMR (500 
MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3), see 
table 1. 
7-Oxocholesterol (3): White powder; 
1
H-NMR 
(500 MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3), 
see Table 2. 
(22E)-3β-Hydroxycholesta-5,22-dien-7-one 
(4): White powder; 
1
H-NMR (500 MHz, CDCl3) and 
13
C-NMR (125 MHz, CDCl3), see table 2.  
 
3. RESULTS AND DISCUSSION 
Compound 1 was obtained as a white powder. 
The NMR features indicated a steroid, one of main 
constituents of many marine organisms. The 
1
H-
NMR spectrum revealed typical signals of two tert-
methyl [ H 0.80 (H-18) and 0.88 (H-19), each 3H, s], 
three sec-methyl [ H 0.91 (H-21), 0.83 (H-26), and 
0.80 (H-27), each 3H, d, J = 6.5 Hz], and one pri-
methyl [ H 0.85 (3H, t, J = 6.5 Hz, H-29)] groups 
suggesting a ethylcholesterol-type steroid. The 
proton signals of an oxymethine group [ H 3.96 (1H, 
m, H-3)] and a disubstituted endocyclic double bond 
[ H 6.24 (H-6) and 6.51 (H-7), each 1H, d, J = 8.5 
Hz] were also observed. In addition, the 
13
C-NMR 
spectrum of 1 revealed 29 signals containing one 
oxymethine group [ C 66.49 (C-3)], two oxygenated 
quaternary carbons [ C 82.16 (C-5) and 79.47 (C-9)], 
and a 1,2-disubstituted double bond [ C 135.41 (d, 
C-6) and 130.79 (d, C-7)]. The signals of two 
oxygenated quaternary carbons were strongly shifted 
downfield suggesting  the presence of a peroxide 
bridge. The 
13
C-NMR data of 1 were totally identical 
(table 1) to those of 5α,8α-epidioxy-24(S)-
ethylcholest-6-en-3β-ol [2]. Moreover, the structure 
of 1 was further confirmed by HMBC experiment. 
The HMBC cross-peaks of H-4 with C-2, C-3, C-5 
and C-10; H-6 with C-8 and C-10; H-7 with C-5 and 
 VJC, 54(6) 2016                                                                                                                Chau Van Minh, et al. 
789 
C-9; H-18 with C-12, C-13, C-14, and C-17; and 
those of H-19 and C-1, C-5, C-9, and C-10 (figure 2) 
clearly confirmed the structure of 1 as 5α,8α-
epidioxy-24(S)-ethylcholest-6-en-3β-ol.  
 
 
Figure 2: Key HMBC (H  C) correlations of 1 
The 
1
H- and 
13
C-NMR spectroscopic data of 2 
were similar to those of 1, except for an additional 
presence of a trans disubstitued double bond [ C 
137.60 (C-22) and 129.97 (C-23)/ H 5.15 (H-22) and 
5.04 (H-23), each 1H, d, J = 8.5, 15.0 Hz]. Analysis 
of the structure of 1 indicated a unique position of 
the additional double bond at C-22/C-23 in 2, which 
was further confirmed by a good agreement of the 
13
C-NMR data of 2 (table 1) with those of 
(22E,24S)-5α,8α-epidioxy-24-ethyl-cholesta-6,22-
dien-3β-ol [2] and by 2D-NMR evidence. 
Compounds 3 and 4 were also isolated as white 
powders. Their NMR features indicated 3β-
hydroxycholesta-5-en-7-one steroid nucleus with 
signals of one oxymethine, one trisubstituted 
endocyclic double bond, and one α, -unsaturated 
ketone. Similar as in case of 1 and 2, compound 4 is 
different from 3 only with presence of a trans 
disubstitued double bond at C-22/C-23. Comparison 
of their 
13
C-NMR values with those reported (table 
2) and detailed analysis of HSQC and HMBC data 
confirmed  these two compounds as  7-oxo-
cholesterol (3) [3] and (22E)-3β-hydroxycholesta-
5,22-dien-7-one (4) [4]. To the best of our 
knowledge, this is the first report of compounds 1-4 
from blood cockle A. granosa. 
 
Acknowledgement. This work was financially 
supported by Vietnam Academy of Science and 
Technology, code VAST04.08/14-15.  
 
REFERENCES 
 
1. J.-M. Kornprobst. Mollusks. In Encyclopedia of 
Marine Natural Products, Second ed.; Wiley-VCH 
Verlag GmbH & Co. KGaA, 2014, p. 1333. 
2. A. Gauvin, J. Smadja, M. Aknin, R. Faure, E.-M. 
Gaydou. Isolation of bioactive 5α,8α-epidioxy sterols 
from the marine sponge Luffariella cf. variabilis, 
Canadian Journal of Chemistry, 78, 986-992 (2000). 
3. G. Notaro, V. Piccialli, D. Sica. New steroidal 
hydroxyketones and closely related diols from the 
marine sponge Cliona copiosa, Journal of Natural 
Products, 55, 1588-1594 (1992). 
4. M. Nasir, S. Saeidnia, A. Mashinchian-Moradi, A. R. 
Gohari. Sterols from the red algae, Gracilaria 
salicornia and Hypnea flagelliformis, from Persian 
Gulf, Pharmacognosy Magazine, 7, 97-100 (2011). 
 
Corresponding author: Chau Van Minh 
Institute of Marine Biochemistry 
Vietnam Academy of Science and Technology 
No 18, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 
E-mail: cvminh@vast.vn. 
 
 


Steroids from the blood cockle Anadara granosa

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Steroids from the blood cockle Anadara granosa

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