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Zooxanthellactone, a Novel γ-Lactone-type Oxylipine

from Dinoflagellates of Symbiodinium sp.: Structure,
Distribution, and Biological Activity
a a a b
Ken-ichi ONODERA , Takuya FUKATSU , Nozomi KAWAI , Yukio YOSHIOKA , Tetsuji
b a a
OKAMOTO , Hideshi NAKAMURA & Makoto OJIKA
a
Graduate School of Bioagricultural Sciences, Nagoya University
b
Graduate School of Biomedical Sciences, Hiroshima University
Published online: 22 May 2014.

To cite this article: Ken-ichi ONODERA, Takuya FUKATSU, Nozomi KAWAI, Yukio YOSHIOKA, Tetsuji OKAMOTO, Hideshi
NAKAMURA & Makoto OJIKA (2004) Zooxanthellactone, a Novel γ-Lactone-type Oxylipine from Dinoflagellates of
Symbiodinium sp.: Structure, Distribution, and Biological Activity, Bioscience, Biotechnology, and Biochemistry, 68:4,
848-852, DOI: 10.1271/bbb.68.848

To link to this article: http://dx.doi.org/10.1271/bbb.68.848

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 Biosci. Biotechnol. Biochem., 68 (4), 848–852, 2004
Zooxanthellactone, a Novel -Lactone-type Oxylipine from Dinoflagellates
of Symbiodinium sp.: Structure, Distribution, and Biological Activity
Ken-ichi O NODERA,1 Takuya FUKATSU,1 Nozomi K AWAI,1 Yukio Y OSHIOKA,2
Tetsuji O KAMOTO,2 Hideshi N AKAMURA,1; * and Makoto O JIKA1; y
1
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2
Graduate School of Biomedical Sciences, Hiroshima University,
1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
Received November 6, 2003; Accepted December 9, 2003
A novel fatty acid derivative named zooxanthellac-
tone (ZL) was isolated from several strains of symbiotic
microalgae, dinoflagellates of the genus Symbiodinium.
The metabolite is structurally related to docosahexae-
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noic acid (DHA) and seems to be biosynthesized by
oxidation and subsequent lactonization. The absolute
stereochemistry was determined from the specific
rotation of the perhydro derivative. The distribution
of ZL within several Symbiodinium isolates was quanti-
tatively analyzed by HPLC techniques and suggested a
relationship between the productivity of this metabolite
and the Symbiodinium phylogeny. The cytotoxicity of ZL
was evaluated by using human squamous cell carcinoma
cell lines in comparison with that of DHA and other
common fatty acids, suggesting that the long unsaturat-
ed chain was important rather than the -lactone
moiety.
Key words: zooxanthellactone; oxylipine; cytotoxic; di-
noflagellate; Symbiodinium
Zooxanthellae are unicellular microalgae that occur as
symbionts in many hundreds of common marine
invertebrates, e.g., sponges, corals, shellfishes, etc. Most
zooxanthellae have been placed in the dinoflagellate
genus Symbiodinium as one or several species that are
not easily distinguished.1) Although the dinoflagellates
have produced a number of toxic and complex secon-
dary metabolites, most of which might be accumulated
into marine food animals,2–4) the secondary metabolites
of the genus Symbiodinium have not been investigated
very well until recently. Nakamura et al. isolated novel
polyhydroxylated polyene metabolites named zooxan-
thellatoxins from the Y-6 strain of Symbiodinium sp. as
vasoconstrictive substances in 1993.5) In the course of
the search for novel secondary metabolites in several
Symbiodinium isolates, we have recently discovered a
large novel polyol metabolite named zooxanthellamide
y
To whom correspondence should be addressed. Tel/Fax: +81-52-789-4284; E-mail: ojika@agr.nagoya-u.ac.jp
* Deceased 9th November 2000.
Abbreviations: ZL, zooxanthellactone; ZL-H12 , dodecahydrozooxanthellactone
A from a butanol-soluble extract of the algal cells.6) On
the other hand, an examination of a cytotoxic non-polar
fraction yielded a unique oxylipine named zooxanthel-
lactone (ZL) which is structurally related to docosahex-
aenoic acid (DHA). We describe here the isolation,
structural elucidation, distribution, and cytotoxic activ-
ity of ZL.
Materials and Methods
General. HPLC analyses were performed by using
Jasco PU-980 pumps equipped with a Jasco MD-915
multi-wavelength detector operated by a Dell OptiPlex
Gxi computer. NMR spectra were recorded by a Bruker
AMX2-600 instrument (600 MHz) in a CDCl3 solution
(99.9% atom enriched). NMR chemical shifts were
referenced to the solvent peak of
H 7.26 (residual
CHCl3 ) or
C 77.0 (CDCl3 ). Optical rotation were
measured with a Jasco DIP-370 digital polarimeter. IR
spectra were recorded by a Jasco FT/IR-8300 instru-
ment, and UV spectra were recorded by a Jasco V-530
spectrophotometer. Mass spectra were recorded by an
Applied Biosystems Mariner mass spectrometer equip-
ped with an electrospray ion source in the positive mode.
High-resolution mass measurements were performed
with a 1 M mixture of angiotensin, bradykinin, and
neurotensin in 50% MeCN–1% AcOH as an internal
standard. DHA and linoleic acid were purchased from
Sigma Co., Ltd., and linolenic acid was from Aldrich
Co., Ltd.
Dinoflagellate materials. All isolates of the dinofla-
gellates used in this study were kindly provided by the
following researchers: Symbiodinium sp. Y-6 and Am-
phidinium sp. Y-5 from Dr. T. Yamasu at Ryukyu
University, Symbiodinium sp. SHIN-SADO and Amphi-
dinium carterae from Dr. K. Kogame at Hokkaido
University, and all other Symbiodinium isolates from Dr.
 Zooxanthellactone, a Novel Oxylipine from Dinoflagellates
T. Maruyama at the Japan Marine Science and Tech-
nology Center and Dr. M. Kawachi at the National
Institute for Environmental Studies. Culture and sub-
culture of these algae were carried out in an ES or f/2
medium as described later.
Production and isolation of ZL. A portion (20 ml) of a
100
ES medium (containing 70 mg of NaNO3 , 15 mg

of Na2 glycerophosphate 5.5H2 O, 1 mg of FeEDTA

3H2 O, 5 mg of Na2 EDTA 2H2 O, 100 mg of Tris, 1 mg

of H3 BO3 , 100 g of thiamine HCl, 1 g of biotin, 2 g

of vitamin B12 , 314 g of MnCl2 4H2 O, 32 g of
 
ZnCl2 2.5H2 O, and 8 g of CoCl2 6H2 O; adjusted to
pH 7.8 with 3 N HCl) was sterilized by filtration and
added to 2 liters of natural sea water in a 3-liters glass
bottle that had been autoclaved at 121  C for 20 min. To
this medium, a portion (50 ml) of a subculture of
Symbiodinium sp. (P083-2 isolate) was inoculated and
subjected to a static culture at 25  C under a 12 h light
(3000–5000 lux)-dark cycle. After 6 weeks, when the
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growth had reached a plateau, the culture (total of 156
liters) was filtered to collect the algal cells (138.7 g wet
wt.) which were stored at 80  C until needed.
The cells were homogenized in 70% EtOH (200 ml)
by an Ultra-Turrax T25 (Janke & Kunkel GmbH & Co.
KG IKA-Labortechnik, Germany) at 24,000 rpm for
2 min three times on an ice bath. After centrifuging the
homogenate at 3,000 rpm for 10 min at 4  C, the super-
natant was separated by decantation. This extraction
procedure with 70% EtOH was conducted three times.
The combined aqueous EtOH extracts (600 ml) were
concentrated under reduced pressure. The resulting
aqueous solution was extracted with EtOAc (200 ml)
three times and then with water-saturated BuOH
(150 ml) three times. The combined EtOAc extracts
were concentrated under reduced pressure to give a
brown oil (1.04 g). A portion (403 mg) of the oil was
chromatographed on silica gel (silica gel 60, Wako,
1.7 cm i.d.
25 cm), eluting with CH2 Cl2 :MeOH (99:1,
98:2, 96:4, 92:8, 84:16, and 68:32; 120 ml each) and
then MeOH (180 ml) to afford 21 fractions. A portion
(20.4 mg) of the 3rd fraction (22.9 mg) eluted with
CH2 Cl2 :MeOH (99:1) was chromatographed on ODS
(Cosmosil 75C18-OPN, Nacalai Tesque, 1.2 cm
i.d.
14 cm), eluting with MeOH:H2 O (85:15, 90:10,
95:5, and 100:0; 30 ml each), and then with
CH2 Cl2 :MeOH (1:1, 45 ml) to give 6 fractions. The
second fraction eluted with 90% MeOH was zooxan-
thellactone (ZL, 1.0 mg) as a colorless oil, ½D 27
þ64:6 (c 0.24, CHCl3 ); UV max (CH3 CN) nm (")
234 (28500); IR max (film) cm1 : 3013, 2960, 2935,
2875, 1775, 1655, 1455, 1175, 1010, 980, 950, 913, and
681; HRMS (ESI positive): found, m=z 327.2317
½M þ Hþ ; calcd. for C22 H31 O2 , 327.2319.
Hydrogenation of ZL. A mixture of ZL (10.3 mg,
0.0316 mmol) and 10%-Pd/C (2.0 mg) in EtOAc
(2.5 ml) was stirred in a hydrogen atmosphere at room
849
temperature for 15 h. The catalyst was removed by
filtration and washed with ether. The filtrate and
washings were combined and concentrated. The result-
ing residue was purified by preparative TLC (silica gel,
CH2 Cl2 ) to give dodecahydrozooxanthellactone (ZL-
H12 , 5.5 mg, 51%) as a white powder, ½D 27 þ21:4 (c
0.23, THF); IR max (KBr) cm1 : 2920, 2850, 1755,
1470, 1195; 1 H-NMR (600 MHz, CDCl3 )
: 4.48 (1H,
 quint, J ¼ 6:9 Hz, H-4), 2.55 (1H, m, H-2), 2.50 (1H, m,
H2), 2.31 (1H, m, H3), 1.85 (1H, m, H-3), 1.73 (1H, m,
H-5), 1.59 (1H, m, H-5), 1.44 (1H, m, H-6), 1.36 (1H, m,
H-6), 1.35–1.21 (24H, m, H-8 to H-19), 1.30 (2H, m, H-
7), 1.28 (2H, m, H-21), 1.24 (2H, m, H-20), 0.88 (3H, t,
J ¼ 7:0 Hz, H-22); 13 C-NMR (150 MHz, CDCl3 )
:
177.3 (s, C-1), 81.1 (d, C-4), 35.6 (t, C-5), 31.9 (t, C-20),
29.7–29.2 (t, C-8 to C-19), 29.3 (t, C-7), 28.9 (t, C-2),
28.0 (t, C-3), 25.2 (t, C-6), 22.7 (t, C-21), 14.1 (q, C-22).
HRMS (ESI, positive): found, m=z 339.3256 ½M þ Hþ ;
calcd. for C22 H43 O2 , 339.3258.
Quantitative analysis of ZL within 11 dinoflagellate
isolates. Cultivation of all the microalgae in the ES
medium was performed as already described. Another
medium, f/2, was also used for culturing the same
microalgae under the same conditions. A portion (0.2
liters) of a 10
f=2 medium (containing 147 mg of

NaNO3 , 12 mg of NaH2 PO4 2H2 O, 9 mg of Na2 EDTA 

2H2 O, 293 g of thiamine HCl, 1 g of biotin, 1 g of

vitamin B12 , 6 mg of FeCl3 6H2 O, 352 g of MnCl2 

4H2 O, 43 g of ZnSO4 7H2 O, 19 g of CoCl2 6H2 O, 

19 g of CuSO4 , 13 g of Na2 MoO4 2H2 O, and a 10-ml
portion of a soil extract prepared by autoclaving a
mixture of 150 g of natural soil and 1 liter of water for
1 h with subsequent filtration) was added to 1.8 liters of
sea water in a 3-liters glass bottle which had been
autoclaved. A portion (10 g) of each EtOAc-soluble
material in MeOH (1 l) was injected into an HPLC
instrument (Develosil ODS-HG-5 column, 4.6 mm
i.d.
25 cm, 50% to 100% MeCN in H2 O, 50 min linear
gradient, 1.0 ml/min flow rate, photodiode array UV
detection). ZL was eluted at a retention time of 36.4 min.
The amount of ZL in each EtOAc extract was quantified
by using a calibration curve and normalized to the
weight per 1 g of wet cells. The detection limit was
estimated as 4 g/g of wet cells.
Cell lines and cytotoxic assay. The A431 human
vulval-derived epidermoid carcinoma cell line7) and
Nakata oral squamous cell carcinoma cell line8) were
used in this study. These cell lines were maintained in a
humidified atmosphere of 5% CO2 /95% air at 37  C in
an RD medium (1:1 mixture of an RPMI1640 medium
and Dulbecco’s modified Eagle’s medium (DMEM;
Sigma Co., St. Louis, MO, USA) supplemented with 5%
calf serum (CS; Hyclone Laboratories, Logan, UT,
USA).
The cytotoxic effects of the compounds on the cell
growth of A431 and Nakata cells were studied under
 850 K. ONODERA et al.

serum-free defined culture conditions as reported pre- Table 1. NMR Data for Zooxanthellactone (ZL) in CDCl3
viously.9,10) The cells at the density of 1
104 cells/ml Position
C
H mult. (J in Hz)
were inoculated into an RD medium containing the five
factors of 10 g/ml of bovine insulin, 5 g/ml of 1 176.8 —
2 28.6 2.57, 2.53 m
human transferrin, 10 M 2-mercaptoethanol, 10 M 2- 3 28.9 2.42, 2.01 m
aminoethanol, and 10 nM sodium selenite11,12) (all from 4 80.6 5.00 dt (7.0, 7.0)
Sigma Chemical Co.) in type I collagen (Cell Matrix 5 130.0 5.68 dd (15.1, 7.0)
type I-A; Nitta Gelatin Co., Osaka, Japan)-coated 24- 6 128.0 6.62 dd (15.1, 10.8)
well culture plates (BD Bioscience, Franklin, NJ, USA). 7 127.1 6.01 dd (10.8, 10.8)
8 132.5 5.51 dt (10.8, 7.5)
The cells were allowed to attach and spread for 12 hours 9 26.1 2.97 t (7.5)
prior to being incubated with a sample at various 10 127.2 5.36 m
concentrations (0.002, 0.02, 0.2, 2, and 20 g/ml) and 11 128.9 5.40 m
further cultured at 37  C. On day 4, the cells were 12 25.6 2.85 m
harvested by 0.05% trypsin/0.01% EDTA, and their 13 127.8 5.36 m
14 128.6 5.37 m
numbers were counted with a model Zf Coulter counter 15 25.7 2.85 m
(Coulter Electronics, Hialeah, FL, USA). 16 127.8 5.38 m
17 128.4 5.38 m
Results and Discussion 18 25.5 2.81 t (6.5)
19 127.0 5.31 m
20 132.1 5.39 m
Production, isolation, and structural elucidation of 21 20.5 2.07 sextet (7.6)
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ZL 22 14.3 0.97 t (7.6)

The dinoflagellate, Symbiodinium sp. strain P083-2,
which had originally been isolated from the foraminifer,
Amphisorus hemprichii, was cultured in the ES medium
at 25  C for 6 weeks. The algal cells obtained from the
culture (156 liters) were extracted with aqueous EtOH,
and the extract was partitioned into EtOAc, BuOH and
aqueous fractions. The EtOAc fraction, which showed
cytotoxicity against the human tumor cell lines
(IC50 ¼ 0:13 and 0.16 g/ml against A431 and Nakata
cells, respectively), was successively subjected to silica
gel and ODS column chromatography to afford ZL
(1.0 mg) as a cytotoxic component.
ZL was obtained as a colorless oil and had the
molecular formula of C22 H30 O2 as determined by high-
resolution ESI-TOF MS data. The IR absorption bands
at 1775 and 1655 cm1 respectively indicated the
presence of a -lactone and a conjugated diene. The
presence of the conjugated diene was supported by the
UV absorption at 234 nm. The 1 H- and 13 C-NMR data
(Table 1) indicated the presence of an ester carbonyl
group (
C 176.8) and six disubstituted olefins, suggest-
ing that this substance was a highly unsaturated fatty
acid derivative. The COSY correlations revealed the
partial structures of C-2 to C-9 and C-18 to C-22. The -
lactone moiety (C-1 to C-4) was determined by the
HMBC correlation between C-1 and H-4. The remaining
parts, three disubstituted olefins (C-10, C-13, and C-16)
and two double allylic methylene groups (C-12 and C-
15), could be arranged one after the other, because these
olefins were detached from each other in the COSY data.
The double-bond geometry of the conjugated diene
system was determined to be 5E, 7Z based on the
olefinic proton-proton coupling constants. The Z geom-
etry of other double bonds at C-10, C-13, C-16, and C19
was determined from the 13 C chemical shifts (
C 25.5–
26.1) of the double-allylic positions at C-12, C-15, and
C-18.13) Based on these findings, we concluded the
13
Measured at 150 MHz and 600 MHz for C and 1 H, respectively.
structure of ZL to be that shown in Fig. 1.
We next tried to determine the absolute stereochem-
istry of ZL, ½D þ64:6 (c 0.24, CHCl3 ). Catalytic
hydrogenation of ZL gave the dodecahydro derivative,
½D þ21:4 (c 0.23, THF). The positive specific
rotation value indicated the R configuration of this
perhydro derivative based on the report that various 4-
alkyl -lactones with the R configuration unexception-
ally showed positive specific rotations: þ47:4 to þ28:3
for the 4-ethyl to 4-tridecyl groups in order.14) The
absolute stereochemistry of ZL was thus determined to
be of the S configuration.
Distribution of ZL within 11 dinoflagellate isolates
The presence of ZL in Symbiodinium sp. is quite
interesting from the following points of view: 1) This is
the first discovery as a secondary metabolite from nature
and is unknown even as a synthetic compound, although
a number of saturated 4-alkyl -lactones are known (for
example, see ref. 14). 2) No other congeners with a
different chain length and degree of unsaturation were
observed. 3) Polyunsaturated fatty acids in dinoflagel-
lates attract attention in respect of symbiosis,15) and
DHA is known as one of the most abundant fatty acids
in dinoflagellates.16,17) These points suggest that there
might be a specific metabolic pathway from DHA to ZL
in dinoflagellates, especially of Symbiodinium sp. To
demonstrate this, we examined the distribution of ZL
within nine Symbiodinium sp. and two different dino-
flagellates of the genus Amphidinium that are kept in our
laboratory. Two kinds of culture medium, ES and f/2,
were used for all the types of microalga. A total of 22
EtOAc-soluble fractions were obtained by the same
extraction procedure as that used for the Symbiodinium
 Zooxanthellactone, a Novel Oxylipine from Dinoflagellates 851

Fig. 1. Structure of Zooxanthellactone (ZL), Dodecahydrozooxanthellactone (ZL-H12 ), and DHA.

Table 2. Distribution of Zooxanthellactone (ZL) within 11 Dinoflagellate Isolates

Weight (mg/g of wet cells) Genotypeb

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Isolate
Hostb
or Species ES medium f/2 medium (clade)

Symbiodinium
HA3-5 —a — A1 free-living
P083-2 0.24 0.27 A1 foraminifer
PL-TS-1 0.11 0.77 A3 giant clam
PL-TM-1 — — A3 giant clam
PHQUTC2A — — A3 giant clam
JCUSG-1 0.28 0.39 A2 soft coral
JCUCS-1 — — B jelly fish
SHIN-SADO — — n.d. free-living
Y-6 1.25 0.94 n.d. flatworm
Amphidinium
Y-5 — — flatworm
A. carterae — — free-living
a
—: <4 g/g of cells (detection limit).
b
Refer to ref. 18 for the genotypes and hosts of Symbiodinium isolates.
n.d.: not determined.

P083-2 isolate and were directly analyzed by HPLC.
The results are summarized in Table 2. Four strains of
the nine Symbiodinium sp. contained more than 0.1 g/g
of ZL cells, whereas neither strain of Amphidinium sp.
did. Although there was no significant correlation
between the content of ZL and dinoflagellate strain
(genus, genotypes, and hosts), it should be noted that ZL
was not discovered by chance and could be frequently
detected in the genus Symbiodinium.
Cytotoxicity of ZL and its related compounds
ZL was obtained as a cytotoxic constituent from an
isolate of Symbiodinium sp. To obtain information on
the structure-activity relationship, we tested the cyto-
toxicity of this metabolite in comparison with that of the
perhydro derivative, DHA, and other common fatty
acids by using two human tumor cell lines, A431 and
Nakata cells.7,8) The results are summarized in Table 3;
the IC50 values for ZL, DHA, ZL-H12 , linoleic acid, and
linolenic acid were determined to be 0.23, 0.21, 7.1, 2.2,
and 2.2 M, respectively, against A431 cells, and 0.27,
0.24, 3.5, 1.7, and 2.3 M, respectively, against Nakata
cells. The low specific activity of ZL in comparison with
that of the crude EtOAc extract would have been due to
other cytotoxic metabolites in the EtOAc extract, one of
which was confirmed to be the carotenoid, peridinin.
DHA, which is a highly unsaturated long-chain (C22 )
fatty acid and a plausible biosynthetic precursor of ZL,
showed the same cytotoxicity as ZL did, whereas the
dodecahydro derivative of ZL was less toxic. Two
common unsaturated fatty acids (C18 ), linoleic acid and
linolenic acid, showed lower toxicity. These results
suggest that the polyunsaturated long chain was im-
portant for cytotoxicity, rather than the presence of the
-lactone moiety.
Acknowledgments
We are grateful to Dr. T. Yamasu of Ryukyu
University, Dr. K. Kogame of Hokkaido University,
Dr. T. Maruyama of the Japan Marine Science and
Technology Center, and Dr. M. Kawachi of the National
 852 K. ONODERA et al.
Table 3. Cytotoxicity of Zooxanthellactone (ZL) and Related Compounds against Two Human Cancer Cell Lines

Concentration (g/ml) IC50

0.002 0.02 0.2 2 20 (M)

A431 cells
ZL 104:1  1:1 96:6  3:7 15:6  1:8 — — 0.23
DHA 92:5  2:0 94:5  1:4 9:2  0:5 — — 0.21
ZL-H12 — 108:4  1:4 104:7  1:2 58:3  4:1 5:6  0:8 7.1
linoleic acid — 100:9  2:1 97:0  0:5 1:0  0:4 0:5  0:1 2.2
linolenic acid — 98:7  6:3 95:4  1:8 0:4  0:1 0:3  0:2 2.2
Nakata cells
ZL 102:5  1:8 99:5  0:5 21:2  0:4 — — 0.27
DHA 105:7  3:9 105:7  4:9 14:9  0:8 — — 0.24
ZL-H12 — 98:7  1:4 71:5  1:6 41:9  4:7 4:3  0:5 3.5
linoleic acid — 99:7  2:5 79:7  8:7 1:5  1:1 0:6  0:0 1.7
linolenic acid — 99:6  4:5 105:4  3:7 1:4  0:8 0:9  0:2 2.3

Each value (n ¼ 2) indicates the percentage of the numbers of cells relative to the control (without a compound) on day 4.

Institute for Environmental Studies for donating the Anim., 30A, 790–795 (1994).
algal isolates, and to Dr. M. Watanabe of the National 10) Michimukai, E., Kitamura, N., Zhang, Y., Wang, H.,
Institute for Basic Biology for supplying sea water. This Hiraishi, Y., Sumi, K., Hayashido, Y., Toratani, S.,
Downloaded by [122.54.65.35] at 21:30 04 March 2015

study was financially supported by grant-aid for explor-
atory research (14658186).
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