A new digalactosyl diacylglycerol from a cultured marine dinoflagellate Heterocapsa circularisquama

Article abstract of DOI:10.1021/np0201265

A new digalactosyl diacylglycerol (1) has been isolated from the cultured marine dinoflagellate Heterocapsa circularisquama together with known monogalactosyl diacylglycerols 2 and 3, and their structures were elucidated by spectroscopic methods. The digalactosyl diacylglycerol 1 showed cytolytic activity toward heart cells of oysters.

Full text of DOI:10.1021/np0201265

1494
J . Nat. Prod. 2002, 65, 1494-1496
A New Diga la ctosyl Dia cylglycer ol fr om a Cu ltu r ed Ma r in e Din ofla gella te
Heter oca psa cir cu la r isqu a m a
Yoshikazu Hiraga,*,† Ken Kaku,† Daisuke Omoda,† Kinuko Sugihara,† Hiroshi Hosoya,‡ and Mizuki Hino‡
Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima
739-8526, J apan, and Department of Biological Science, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, J apan
Received March 20, 2002
A new digalactosyl diacylglycerol (1) has been isolated from the cultured marine dinoflagellate Heterocapsa
circularisquama together with known monogalactosyl diacylglycerols 2 and 3, and their structures were
elucidated by spectroscopic methods. The digalactosyl diacylglycerol 1 showed cytolytic activity toward
heart cells of oysters.
Unicellular algae comprise an important group of phy-
toplankton and form the foundation of many marine food
chains. However, harmful phytoplankton blooms have
caused serious social and environmental problems and
heavy damage to fisheries throughout the world.1 In recent
years, a novel dinoflagellate, Heterocapsa circularisquama,
has caused mass mortalities of bivalves, e.g., oysters, pearl
oysters, and mussels, in several inner bays of J apan.2
A
large number of studies have been reported on the toxic
effects of this phytoplankton to the bivalves.3,4 Recently,
hemolytic activity has been found in the ethanol extracts
of H. circularisquama cells.3j,k
In our search for the toxic substances in H. circularis-
quama, we have found that the EtOAc-soluble fraction of
a 1-BuOH extract of the cultured H. circularisquama
showed cytolytic activity toward heart cells of oysters.
Bioassay-guided purification of the EtOAc-soluble fraction
resulted in the isolation of a new digalactosyl diacylglycerol
(1) together with two known monogalactosyl diacylglycer-
ols, 2 and 3. Previously, it has been reported that gly-
coglycerolipids derived from microalgae can be sources of
biologically active substances including toxins.5 Although
a large number of studies have been made on the identi-
fication of galactolipids in some marine dinoflagellates,6
little is known about the galactolipids in H. circularis-
quama. In this paper, we report on the structure of 1, which
has been deduced from its spectroscopic data, and on its
biological activity.
25
The digalactosyl diacylglycerol 1, [R]D +54° (c 0.85,
The marine dinoflagellate H. circularisquama was cul-
tured in seawater media (SW II)7 in 10 L glass bottles at
22 °C. The cultivation was carried out applying a 16:8 h
light to dark cycle using cool-white fluorescent lights for 2
weeks. The cells harvested by centrifugation (800 rpm, 5
min) from 90 L of cultured media were extracted with
1-BuOH, and the 1-BuOH extract (9.0 g) was partitioned
between EtOAc and water. The EtOAc-soluble fraction (485
mg) was subjected to chromatography on silica gel using
2-100% methanol in chloroform as eluent to afford two
bioactive fractions containing a mixture of glycolipids.
Further separation by reversed-phase silica gel (ODS)
column chromatography and HPLC (ODS) yielded diga-
lactosyl diacylglycerol 1 from a latter fraction and mono-
galactosyl diacylglycerols 2 and 3 from a former fraction.
MeOH), was determined to have the molecular formula
51H78O15 by positive mode high-resolution fast atom
C
bombardment (HRFAB) mass spectrometry (m/z 953.5220,
[M + Na]+, ∆ -1.8 mmu). Analysis of the 1H and 13C NMR,
1H-1H COSY, and HMQC spectra of 1 allowed the assign-
ment of all of the 13C NMR signals and the 1H NMR signals
for the sugar and the glycerol moieties as shown in Table
1. The anomeric H-1′′′ proton of 1 showed HMBC cross-
peaks with C-3 and C-2′′′, and the anomeric H-1′′′′ proton
with C-6′′′ and C-2′′′′. The H and 13C NMR spectroscopic
1
findings were consistent with 1 bearing a 1,2-di-O-acyl-3-
O-[R-D-galactopyranosyl-(1′′′′f6′′′)-O-â-D-galactopyranosyl]-
sn-glycerol moiety. The 13C NMR spectrum of 1 showed two
carbonyl carbon signals arising from the acyl groups at δC
173.0 and 174.6, and their location in the molecule was
confirmed by long-range coupling detected by HMBC. Thus,
the carbonyl carbons at δC 174.6 (C-1′′) and 173.0 (C-1′)
were correlated with the proton signals of H-1 and H-2 of
the glycerol part, respectively. Moreover, C-1′′ was cor-
* To whom correspondence should be addressed. Tel: +81-824-24-7434.
Fax: +81-824-24-0747. E-mail: yhiraga@sci.hiroshima-u.ac.jp.
† Department of Chemistry.
‡ Department of Biological Science.
10.1021/np0201265 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/15/2002

Notes
J ournal of Natural Products, 2002, Vol. 65, No. 10 1495
Ta ble 1. 13C and 1H NMR Assignments for the Glycerol and
Sugar Moieties of 1
2-O-6,9,12,15-octadecatetraenoyl-3-O-[R-D-galactopyran-
osyl-(1′′′′f6′′′)-O-â-D-galactopyranosyl]-sn-glycerol.
Compounds 2 and 3 were identified on the basis of their
spectroscopical data as (2S)-1,2-di-O-3,6,9,12,15-octadeca-
pentaenoyl-3-O-â-D-galactopyranosyl-sn-glycerol, which was
previously isolated from the marine dinoflagellate Scrippsiel-
la trochoidea,6d and (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-
2-O-6,9,12,15-octadecatetraenoyl-3-O-â-D-galactopyranosyl-
sn-glycerol (heterosigma-glycolipid III), which was obtained
from the marine dinoflagellate Heterosigma akashiwo,6c
respectively.
Compound 1 exhibited cytolytic activity toward the heart
cells of oysters at a concentration of 0.5 µg/mL or greater.
The activities of 2 and 3 were the same as that of 1 under
the same concentrations. Thus, elucidation of the physi-
ological functions of the galactosyl diacylglycerols 1-3 in
H. circularisquama and their possible connection to the
mass mortalities of bivalves would be an interesting subject
for future investigation.
13Ca
64.4
1H b(mult, J in Hz)
1
4.30 (dd, 6.6, 11.9)
4.48 (dd, 3.0, 11.9)
5.30 (m)
3.78 (dd, 3.7, 10.8)
3.98 (dd, 5.3, 10.8)
4.28 (d, 7.1)
3.56 (dd, 7.1, 9.6)
3.52 (dd, 3.2, 9.6)
3.92 (br d, 3.2)
3.78 (m)
3.71 (dd, 6.4, 9.9)
3.94 (overlapped)c
4.91 (d, 3.7)
2
3
71.7
68.7
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
105.3
72.4
74.7
70.0
74.6
67.8
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
100.7
70.2
71.5
71.1
72.6
62.8
3.83 (dd, 3.7, 10.1)
3.78 (dd, 3.7, 10.1)
3.94 (br d, 3.7)
3.89 (m)
3.74 (dd, 5.5, 11.5)
3.75 (dd, 6.4, 11.5)
Exp er im en ta l Section
a
b
125 MHz; CD3OD (δ 49.0). 500 MHz; CD3OD (δ 3.35).
c Signal pattern is unclear due to overlap.
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with a J ASCO DIP-370 polarimeter. IR spectra
were recorded on a J ASCO FT/IR-7300 Fourier transform
infrared spectrometer. 1H and 13C NMR spectra were measured
and recorded on a J EOL ECP-500 spectrometer in CD3OD. The
resonances of CD3OD at δH 3.35 ppm and δC 49.0 ppm were
used as internal standards for NMR spectra. FABMS were
recorded on a J EOL J MS-SX102A spectrometer. ESIMS were
recorded on a Hitachi M-8000 mass spectrometer. GC-MS
were measured and recorded on a Shimadzu QP-5050A mass
spectrometer. HPLC was performed using a system composed
of a J ASCO PU-980 pump and a J ASCO UV-970 or a J ASCO
RI-1530 detector.
Cu ltiva tion . The dinoflagellate Heterocapsa circularis-
quama was obtained from Hiroshima Fisheries Experimental
Station. Cultures of H. circularisquama were grown in 10 L
glass bottles filled with seawater medium (SW II)7 at 22 °C
for 2 weeks with air bubbling, applying a 16:8 h light:dark
cycle provided by cool-white fluorescent lights.
related with proton signals at δH 2.40 (t, J ) 7.3 Hz, H-2′′)
and 1.67 (tt, J ) 7.3 and 7.8 Hz, H-3′′), and C-1′ was
correlated with proton signals at δH 3.18 (d, J ) 5.0 Hz,
H-2′) and 5.59 (m, H-3′ and H-4′). The connectivities
1
observed in the H-1H COSY and HMBC measurements
of the 1H and 13C signals of these ester chains were similar
to those observed for compound 3, thus indicating a 3,6,9,-
12,15-octadecapentaenoate side chain and a 6,9,12,15-
octadecatetraenoate being attached to 1-OH and 2-OH of
the glycerol moiety, respectively.
To confirm the structure of the fatty acids, compound 1
was treated with NaOMe-MeOH according to a reported
method,6c yielding a mixture of fatty acid methyl esters and
digalactosyl glycerol 4. The fatty acid composition was
determined by GC and GC-MS analyses of the above
methyl esters and found to be a 1:1 mixture of methyl
3,6,9,12,15-octadecapentaenoate (5) and methyl 6,9,12,15-
octadecatetraenoate (6) by comparison with samples ob-
tained by treatment of 3.6c
Extr a ction a n d Isola tion . The cultured cells were har-
vested by centrifugation (800 rpm, 5 min). The cells harvested
from the culture were extracted with 1-BuOH (1 L × 3) to give
a 1-BuOH extract (9.0 g). The 1-BuOH extract was partitioned
between EtOAc and water. The EtOAc-soluble fraction (485
mg) was subjected to silica gel column chromatography, eluting
with stepwise gradients of CHCl3-MeOH (98:2, 95:5, 90:10,
80:20) and finally with MeOH alone, to give five fractions
(I-V). Fraction III was further separated by an ODS silica
gel column eluting with MeOH-H2O (9:1) to give 1 in impure
form. Final purification was carried out by repetitive separa-
tion on an analytical HPLC column [Inertsil ODS-3 (GL
Science, 4.6 mm i.d. × 250 mm), flow rate 2.0 mL/min; solvent
MeOH-H2O (95:5); detection UV (215 nm)] to yield 1 (8 mg)
in pure form. Using the same method as above, the pure forms
of 2 (6 mg) and 3 (9 mg) were obtained from fraction II.
25
Diga la ctosyl d ia cylglycer ol (1): [R]D +54° (c 0.85,
MeOH); IR (Nujol) νmax 3338 (OH), 1736 cm-1 (CdO); 1H NMR
(500 MHz, CD3OD) δ 5.59 (2H, m, H-3′, H-4′), 5.39 (16H, m,
H-6′, H-7′, H-9′, H-10′, H-12′, H-13′, H-15′, H-16′, H-6′′, H-7′′,
H-9′′, H-10′′, H-12′′, H-13′′, H-15′′, H-16′′), 3.18 (2H, d, J )
5.0 Hz, H-2′), 2.89 (14H, m, H-5′, H-8′, H-11′, H-14′, H-8′′,
H-11′′, H-14′′), 2.40 (2H, t, J ) 7.3 Hz, H-2′′), 2.15 (2H, m,
H-5′′), 2.13 (4H, m, H-17′, H-17′′), 1.67 (2H, tt, J ) 7.3, 7.8
Hz, H-3′′), 1.45 (2H, quint, J ) 7.8 Hz, H-4′′), 1.02 (3H, t, J )
7.6 Hz), 1.01 (3H, t, J ) 7.6 Hz) (H-18′, H-18′′); 13C NMR (125
MHz, CD3OD) δ 174.6 (C-1′′), 173.0 (C-1′), 132.8, 132.8 (C-
16′, C-16′′), 132.5 (C-4′), 130.7 (C-6′′), 129.7, 129.5, 129.5, 129.3,
129.3, 129.3, 129.1, 129.0, 129.0, 128.9, 128.5 (C-6′, C-7′, C-9′,
C-10′, C-12′, C-13′, C-7′′, C-9′′, C-10′′, C-12′′, C-13′′), 128.2 (×2,
C-15′, C-15′′), 122.4 (C-3′), 35.0 (C-2′′), 33.6 (C-2′), 30.1
(C-4′′), 27.9 (C-5′′), 26.7, 26.6 (×2), 26.6(×2), 26.5 (×2) (C-5′,
Moreover, the glyceryl digalactoside 4, [R]25 +86° (c
D
0.33, H2O), was shown to be identical with (2R)-3-O-[R-D-
galactopyranosyl-(1′′′′f6′′′)-O-â-D-galactopyranosyl]-sn-glyc-
erol on the basis of a comparison with the optical rotation
and NMR data of previously reported data for this
compound.6c,8 Consequently, the chemical structure of 1
was determined as (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-

1496 J ournal of Natural Products, 2002, Vol. 65, No. 10
Notes
C-8′, C-11′, C-14′, C-8′′, C-11′′, C-14′′), 25.6 (C-3′′), 21.5 (×2,
C-17′, C-17′′), 14.7 (×2, C-18′, C-18′′); ESIMS (negative mode)
m/z 929 [M - H]-; HRFABMS (positive mode) m/z 953.5220
[M + Na]+ (calcd for C51H78O15Na, 953.5238); 13C NMR and
1H NMR data for sugars and glycerol moieties, see Table 1.
Meth a n olysis of 1. A solution of 1 (6 mg) in methanol (1.0
mL) was treated with 2.8% NaOMe-MeOH (1.0 mL), and the
mixture was stirred at room temperature for 30 min. The
reaction mixture was neutralized with AG 50W-X8 (BIO-RAD,
H+ form). Evaporation of the solvent under reduced pressure
yielded fatty acid methyl esters and digalactosyl glycerol 4.
The fatty acid methyl ester mixture was identified by GC and
GC-MS [OV-1, HiCap-CPB1, Shimadzu, 0.32 mm × 50 m;
column temperature, 100-300 °C, 3 °C/min] to be a 1:1
mixture of methyl 3,6,9,12,15-octadecapentaenoate (5) and
methyl 6,9,12,15-octadecatetraenoate (6). The mixture was
subjected to HPLC [Inertsil ODS-3, GL Science, MeOH-H2O
(93:7)] to furnish 4 (2 mg), 5 (2 mg), and 6 (2 mg). 1D and 2D
NMR spectroscopic data and MS data for (2R)-3-O-[R-D-
galactopyranosyl-(1′′′′f6′′′)-O-â-D-galactopyranosyl]-sn-glyc-
erol (4), methyl 3,6,9,12,15-octadecapentaenoate (5), and
methyl 6,9,12,15-octadecatetraenoate (6) were identical with
published data.6c,8
Bioa ssa y. Hearts obtained from oysters were homogenized
on 5 µm nylon mesh in Hank’s balanced salt solution (HBSS)
at room temperature.9 Filtration through 5 µm nylon mesh
gave a homogenate, which was treated with pronase for 10
min at 37 °C to afford small aggregates and single cells. After
another filtration through 5 µm nylon mesh and centrifugation
at 2000 rpm for 2 min at 4 °C, a homogeneous suspension of
the harvested cells in HBSS was obtained for assay of the
cytolytic activity. After incubation with a DMSO solution of
the galactosyl diacylglycerol for 30 min at room temperature,
the cells were stained with hematoxylin-eosin Y or fluorescein
diacetate-propidium iodide. Imaging was performed with an
Olympus CK40 microscope with a CK40-RFL fluorescence
attachment.
and 13024256) from the J apan Society for the Promotion of
Science and by the Regional Science Promoter Program of
Hiroshima Prefecture.
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, COSY, HMQC, and
HMBC spectra of 1. 1H and 13C NMR and MS data of 2-6. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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NP0201265