Oxylipins from the microalgae Chlamydomonas debaryana and Nannochloropsis gaditana and their activity as TNF-α inhibitors

Article abstract of DOI:10.1016/j.phytochem.2014.03.011

The chemical study of the microalgae Chlamydomonas debaryana and Nannochloropsis gaditana has led to the isolation of oxylipins. The samples of C. debaryana have yielded the compounds (4Z,7Z,9E,11S,13Z)-11-hydroxyhexadeca-4, 7,9,13-tetraenoic acid (1), (4Z,7E,9E,13Z)-11-hydroxyhexadeca-4,7,9,13- tetraenoic acid (2), (4Z,6E,10Z,13Z)-8-hydroxyhexadeca-4,6,10,13-tetraenoic acid (3), (4Z,8E,10Z,13Z)-7-hydroxyhexadeca-4,8,10,13-tetraenoic acid (4), and (5E,7Z,10Z,13Z)-4-hydroxyhexadeca-5,7,10,13-tetraenoic acid (5), which are derived from the fatty acid 16:4Δ^4,7,10,13 together with the compound (5Z,9Z,11E,15Z)-13-hydroxyoctadeca-5,9,11,15-tetraenoic acid (7) derived from coniferonic acid (18:4Δ^5,9,12,15). In addition, the known polyunsaturated hydroxy acids 11-HHT (6), (5Z,9Z,11E)-13- hydroxyoctadeca-5,9,11-trienoic acid (8), (13S)-HOTE (9), (9E,11E,15Z)-13- hydroxyoctadeca-9,11,15-trienoic acid (10), 9-HOTE (11), 12-HOTE (12), 16-HOTE (13) and (13S)-HODE (14) have also been obtained. The chemical study of N. gaditana has led to the isolation of the hydroxy acid (15S)-HEPE (15) derived from EPA (20:5Δ^5,8,11,14,17). The structures of the isolated compounds were established by spectroscopic means. The optical activity displayed by oxylipins 1, 2, 6, 7, 9, 10, 14, and 15 suggests the occurrence of LOX-mediated pathways in C. debaryana and N. gaditana. In anti-inflammatory assays, all the tested compounds inhibited the TNF-α production in LPS-stimulated THP-1 macrophages. The most active oxylipin was the C-16 hydroxy acid 1, which at 25 μM caused a 60% decrease of the TNF-α level.

Full text of DOI:10.1016/j.phytochem.2014.03.011

Phytochemistry 102 (2014) 152–161
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Oxylipins from the microalgae Chlamydomonas debaryana
and Nannochloropsis gaditana and their activity as TNF-
a inhibitors
Carolina de los Reyes a, Javier Ávila-Román b, María J. Ortega a, Adelina de la Jara c,
Sofía García-Mauriño b, Virginia Motilva d, Eva Zubía a,
⇑
a Departamento de Química Orgánica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain
b Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla, Spain
c Instituto Tecnológico de Canarias, Playa de Pozo Izquierdo, 35119 Santa Lucía-Gran Canaria, Spain
d Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemical study of the microalgae Chlamydomonas debaryana and Nannochloropsis gaditana has led to
the isolation of oxylipins. The samples of C. debaryana have yielded the compounds (4Z,7Z,9E,11S,13Z)-
11-hydroxyhexadeca-4,7,9,13-tetraenoic acid (1), (4Z,7E,9E,13Z)-11-hydroxyhexadeca-4,7,9,13-tetrae-
noic acid (2), (4Z,6E,10Z,13Z)-8-hydroxyhexadeca-4,6,10,13-tetraenoic acid (3), (4Z,8E,10Z,13Z)-7-
hydroxyhexadeca-4,8,10,13-tetraenoic acid (4), and (5E,7Z,10Z,13Z)-4-hydroxyhexadeca-5,7,10,13-tetra-
enoic acid (5), which are derived from the fatty acid 16:4D4,7,10,13 together with the compound
(5Z,9Z,11E,15Z)-13-hydroxyoctadeca-5,9,11,15-tetraenoic acid (7) derived from coniferonic acid
Received 7 October 2013
Received in revised form 7 March 2014
Available online 1 April 2014
Keywords:
Chlamydomonas debaryana
Chlamydomonaceae
Nannochloropsis gaditana
Eustigmataceae
Oxylipins
Polyunsaturated hydroxy acids
Anti-inflammatory activity
(18:4D
5,9,12,15). In addition, the known polyunsaturated hydroxy acids 11-HHT (6), (5Z,9Z,11E)-13-
hydroxyoctadeca-5,9,11-trienoic acid (8), (13S)-HOTE (9), (9E,11E,15Z)-13-hydroxyoctadeca-9,11,15-tri-
enoic acid (10), 9-HOTE (11), 12-HOTE (12), 16-HOTE (13) and (13S)-HODE (14) have also been obtained.
The chemical study of N. gaditana has led to the isolation of the hydroxy acid (15S)-HEPE (15) derived
from EPA (20:5D
5,8,11,14,17). The structures of the isolated compounds were established by spectroscopic
TNF-a inhibition
means. The optical activity displayed by oxylipins 1, 2, 6, 7, 9, 10, 14, and 15 suggests the occurrence of
LOX-mediated pathways in C. debaryana and N. gaditana. In anti-inflammatory assays, all the tested com-
pounds inhibited the TNF-
was the C-16 hydroxy acid 1, which at 25
a
production in LPS-stimulated THP-1 macrophages. The most active oxylipin
M caused a 60% decrease of the TNF- level.
Ó 2014 Elsevier Ltd. All rights reserved.
l
a
Introduction
transformed by the action of different enzymes into an array of
metabolites, including hydroxy and oxo fatty acids, divinyl ethers,
volatile aldehydes, and jasmonates (Mosblech et al., 2009), which
play important roles as signal molecules and defensive compounds
(Blée, 2002; Howe and Schilmiller, 2002; Weber, 2002).
Oxylipins are a large and structurally diverse family of lipid
metabolites generated by the oxidation of polyunsaturated fatty
acids (PUFAs) (Mosblech et al., 2009). Oxylipins are widespread
in nature occurring in animals (Shimizu, 2009), plants, mosses, al-
gae, bacteria, and fungi (Andreou et al., 2009). In general, oxylipins
are bioactive metabolites involved in regulating developmental
processes and in environmental and pathological responses.
Most plant oxylipins are derived from linoleic acid (LA,
Red and brown macroalgae have also been source of a variety of
metabolites derived from C18 and C20 PUFAs such as arachidonic
acid (ARA, 20:4D5,8,11,14
)
and eicosapentaenoic acid (EPA,
20:5D
5,8,11,14,17) via LOX-mediated pathways. Although studies on
green seaweeds are much scarcer, oxylipin formation seems
mainly based on the oxidation of C18 PUFAs (Andreou et al.,
2009; Gerwick, 1994; Gerwick and Singh, 2002; Guschina and
Harwood, 2006).
18:2
(16:3
-dioxygenase
D
D
9,12),
7,10,13), whose enzymatic peroxidation is catalyzed by
-DOX) or, mainly, by lipoxygenase (LOX)
a-linolenic acid (ALA, 18:3D
9,12,15) or roughanic acid
a
(a
enzymes (Andreou and Feussner, 2009; Liavonchanka and Feussner,
2006). The hydroperoxides thus formed can subsequently be
With respect to microalgae, initial research on some diatom
species showed that the oxidation of PUFAs followed by chain
cleavage leads to an array of polyunsaturated aldehydes involved
in the chemical defense against predation (Cadwell, 2009; Fontana
et al., 2007; Pohnert, 2005). Later, a series of hydroxy acids,
⇑
Corresponding author. Tel.: +34 956016021; fax: +34 956016193.
E-mail address: eva.zubia@uca.es (E. Zubía).
http://dx.doi.org/10.1016/j.phytochem.2014.03.011
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
153
hydroxy-epoxy acids, and oxoacids derived from the LOX-medi-
ated oxidation of the PUFAs 16:3
6,9,12, 16:4 6,9,12,15, and EPA
food in aquaculture and are renown by their high content in EPA
(Forján et al., 2011). The study of the biomass of N. gaditana has
led to the isolation of the C20 hydroxy acid 15. In addition, the ma-
D
D
have also been identified in various species of diatoms (Cutignano
et al., 2011; Fontana et al., 2007; Lamari et al., 2013). Only a few
additional accounts of oxylipins from microalgae have been re-
ported and they are restricted to the identification of hydroxy acids
derived from LA and ALA in the chlorophycean Dunaliella acidophila
(Pollio et al., 1988) and in the cyanobacteria species Lygnbya
majuscula (Cardellina and Moore, 1980), Anabaena flos-aquae
f. flos-aquae (Murakami et al., 1992), Oscillatoria redekei HUB 051
(Mundt et al., 2003), and Nostoc spp. (Lang and Feussner, 2007;
Lang et al., 2008).
As a part of our project aimed to the study of bioactive com-
pounds from microalgae grown in outdoor mass cultures, we have
examined the biomass derived from cultures of the freshwater spe-
cies Chlamydomonas debaryana (Class Chlorophyceae) and of the
seawater species Nannochloropsis gaditana (Class Eustigmastophy-
ceae). The genus Chlamydomonas comprises a high variety of mic-
roalgal species, mostly from freshwaters and often abundant in
nutrient-rich environments (Klochkova et al., 2008). The biomass
samples obtained from cultures of C. debaryana have yielded an ar-
ray of monohydroxylated C16 and C18 acids (Fig. 1). The C16 deriv-
atives include the new compounds 1–5 and the known related acid
6. The C18 hydroxy acids include the new compound 7, together
with the known acids 8–14. On the other hand, microalgae of the
genus Nannochloropsis are mostly marine species that are used as
jor isolated compounds have been tested in TNF-
a inhibition as-
says to evaluate their anti-inflammatory properties.
Results and discussion
Compound 1 was obtained as an optically active oil ([a]D = +7.5
(c 0.08, MeOH)), whose molecular formula C16H24O3 was deter-
mined by HRMS. The 13C NMR spectrum exhibited a signal at dC
177.1 attributable to a carboxylic acid function, eight signals be-
tween 120 and 140 ppm due to four disubstituted double bonds,
and a signal at dC 73.2 assigned to a hydroxy-bearing methine
(Table 1). These functions accounted for the five unsaturation
degrees deduced from the molecular formula and indicated that
compound 1 was a monohydroxylated hexadecatetraenoic acid.
The positions of the double bonds and the hydroxy group were
defined from the COSY and HMBC spectra (Fig. 2). The location of a
double bond at C-13,C-14 was readily inferred from the COSY
coupling of the methyl group at dH 0.95 (t, J = 7.5 Hz, Me-16) with
themethyleneprotonsat dH 2.05(H2-15)whichinturnwerecoupled
with the olefinic proton at dH 5.46 (H-14). The olefinic carbons C-13
and C-14 showed HMBC correlations with two allylic methylene
protons at dH 2.31/2.26 (H2-12) that were coupled in the COSY
7
4
9
13
11
1 COOH
COOH
1 COOH
16
13
9
18
15
11
5
OH
OH
OH
OH
7
8
1
2
COOH
COOH
11
9
7
OH
9
4
8
COOH
COOH
13
10
6
13
OH
COOH
COOH
18
15
11
9
OH
3
10
16
10
8
7
OH
4
11
12
OH
18
15
13
12
4
5
COOH
COOH
9
9
OH
16
10
7
5
4
COOH
COOH
12
16
OH
18
14
OH
13
14
7
11
COOH
16
13
9
OH
OH
6
11
8
5
15
COOH
20
17
13
OH
15
Fig. 1. Structures of the hydroxy acids 1–14 isolated from Chlamydomonas debaryana and of the hydroxy acid 15 isolated from Nannochloropsis gaditana.

154
C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
Table 1
1H and 13C NMR data of compounds 1, 2, and 6 (CD3OD).
Position
1a
2b
6a
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
1
2
177.1
35.1
177.4
35.2
177.9
35.1
2.32 m
2.32 m
2.26 t (7.5)
3
23.9
2.38 m
23.9
2.35 m
26.0
1.60 m
4
5
6
7
8
9
10
11
12
129.5
129.8
26.9
130.7
129.3
126.3
137.3
73.2
5.40 m
5.40 m
2.97 brdd (7.3,5.8)
5.36 m
5.97 dd (11.1,10.8)
6.54 dddd (15.2,11.1,1.2,1.2)
5.67 dd (15.2,6.5)
4.12 dt (6.5,6.5)
2.31 m
130.1
129.1
31.2
133.4
131.3
131.6
135.0
73.3
5.43 m
5.43 m
29.8
30.4
28.5
132.8
129.4
126.6
136.8
73.3
1.36 m
1.41 m
2.85 dd (6.4,6.4)
5.65 dt (15.2,6.4)
6.04 dd (15.2,10.3)
6.16 dd (15.2,10.3)
5.56 dd (15.2,6.4)
4.05 dt (6.4,6.4)
2.29 m
2.20 dt (7.6,7.6)
5.40 dt (10.9,7.6)
5.96 dd (11.2,10.9)
6.49 dd (15.3,11.2)
5.63 dd (15.3,6.8)
4.10 dt (6.6,6.6)
2.30 m
36.2
36.2
36.3
2.26 ddd (14.5,7.4,7.4)
5.36 m
5.46 dtt (10.7,7.3,1.5)
2.05 qdd (7.5,7.5,1.3)
0.95 t (7.5)
2.24 m
5.33 m
5.45 m
2.04 qdd (7.8,7.3,1.5)
0.95 t (7.6)
2.24 m
13
14
15
16
125.5
134.6
21.7
125.6
134.5
21.7
125.5
134.6
21.7
5.35 dtt (10.6,7.3,1.5)
5.46 dtt (10.6,7.3,1.5)
2.05 qdd (7.5,7.5,0.9)
0.95 t (7.6)
14.5
14.5
14.5
a
1H at 600 MHz and 13C at 150 MHz.
1H at 500 MHz and 13C at 125 MHz.
b
Compound 2 ([a]D = +12.0 (c 0.10, MeOH)) possessed the molec-
11
1
7
4
COOH
16
14
13
9
ular formula C16H24O3 that indicated that it was an isomer of com-
pound 1. The 13C NMR spectrum, which was closely similar to that
of compound 1, indicated that compound 2 was also a C-16 acid
containing four disubstituted double bonds and a secondary hy-
droxy group. The analysis of the COSY and HMBC correlations
established that these functions were located at the same positions
than in compound 1 and therefore compound 2 had to be a stereo-
isomer of 1. The difference between compounds 1 and 2 was found
at the geometry of the double bond at C-7,C-8 upon observation in
2 of a coupling constant of 15.2 Hz between H-7 and H-8, that indi-
cated the trans relationship between these protons. This proposal
was also consistent with the upfield shift of the bis-allylic methy-
lene protons H2-6 at d 2.85 (d 2.97 in 1) and the downfield shift of
the C-6 resonance at dC 31.2 (dC 26.9 in 1). These data led to pro-
pose for compound 2 the structure (4Z,7E,9E,13Z)-11-hydroxy-
hexadeca-4,7,9,13-tetraenoic acid. The absolute configuration at
C-11 remains unassigned because the small amount of compound
available precluded the preparation of the MPA esters.
OH
1
Fig. 2. Key COSY (bold) and HMBC (arrow) correlations observed for compound 1.
spectrum with the oxymethine proton at dH 4.12 (H-11), thus defin-
ing the location of the hydroxy group at C-11. The sequence of COSY
couplings observed from the oxymethine proton along four olefinic
protons at dH 5.67 (H-10), 6.54 (H-9), 5.97 (H-8), and 5.36 (H-7) indi-
cated that the hydroxy-bearing methine at C-11 was adjacent to a
conjugated diene system at C-7,C-8 and C-9,C-10. The HMBC corre-
lation of the carboxylic carbon (C-1) with the allylic methylene pro-
tons at dH 2.38 (H2-3) which were also coupled in the COSY spectrum
with the olefinic protons signal at dH 5.40 (m, H-4 and H-5) defined
the location of the remaining double bond at C-4,C-5. The proton
coupling constants J7,8 = 10.8 Hz, J9,10 = 15.2 Hz, and J13,14 = 10.7 Hz
indicated the 7Z,9E,13Z configuration. The Z geometry of the double
bond at C-4,C-5 was supported by the chemical shifts of the allylic
methylenecarbonC-3at dC 23.9 and thebis-allylic methylene carbon
C-6 at dC 26.9 (Breitmaier and Voelter, 1989). The absolute configu-
ration at C-11 was determined by NMR analysis of the derivatives 1r
and 1s obtained by esterification of 1 with TMSCH2N2 and subse-
quent treatment of the methyl ester 1a with (R)- and (S)-
oxy- -phenylacetic acids (MPA), respectively. Positive chemical
shift differences (
H-9, and H-10 while negative values were obtained for H-12, H-13,
H-14, H-15, and Me-16 (Fig. 3), indicating an S configuration at
C-11 (Seco et al., 2004). All these data led to define for compound 1
the structure (4Z,7Z,9E,11S,13Z)-11-hydroxyhexadeca-4,7,9,13-tet-
raenoic acid.
The molecular formula C16H24O3 determined for compounds 3,
4, and 5 indicated that they also were isomers of compound 1.
The 13C NMR spectrum of each compound contained a signal
attributable to a carboxylic acid function, eight signals due to
olefinic carbons and one signal due to a secondary hydroxy group
(Table 2). Furthermore, similar to compound 1 the 1H NMR spectra
of 3–5 exhibited between 4.0 and 6.6 ppm a set of signals diagnostic
of a moiety featuring the hydroxy group adjacent to a E,Z conju-
gated diene. This assignment was confirmed by the COSY spectra
that showed, for each compound, a spin system consisting of the
oxymethine proton and four consecutive olefinic protons. In addi-
tion, the three compounds exhibited an HMBC correlation between
the terminal methyl group and an olefinic carbon, indicating the
a-meth-
a
D
d = dR ꢀ dS) were obtained for H-6, H-7, H-8,
presence of a double bond at the n-3 position (D
13). These data
suggested that compounds 3–5 were, as compound 1, hydroxy
acids derived from the oxidation of the fatty acid 16:4D4,7,10,13 at
different positions of the chain.
In compound 3 the conjugated diene was deduced to be located
at D4Z,6E and the hydroxy group at C-8. Key data were the HMBC
correlations of the methylene protons H2-3 (dH 2.47) with two ole-
finic carbons at dC 131.0 (C-4) and 130.1 (C-5), the correlation of
this latter carbon with the trans-olefinic proton at dH 5.67 (dd,
J = 15.2 and 6.4 Hz, H-7), and the COSY coupling of this proton with
the oxymethine proton at dH 4.13 (H-8). The presence of a double
-0.09
-0.12
-0.12
+0.11 +0.09 +0.09
+0.24 +0.15
OR
COOCH3
-0.07 -0.14 -0.22
H
1a R= H
1r R= MPA(R)
R= MPA(S)
1s
Fig. 3.
Dd (dR–dS) observed for the MPA esters 1r and 1s.

C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
155
Table 2
1H and 13C NMR data of compounds 3–5 (CD3OD).a
Position
3
4
5
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
177.7
35.5
24.5
131.0
130.1
126.3
137.4
73.1
178.5
35.9
24.5
131.4
127.3
36.3
179.1
32.3
33.9
2.33 t (7.3)
2.47 dt (7.3,7.3)
5.40 m
5.99 dd (10.8,10.8)
6.53 dddd (15.2,10.8,1.5,1.0)
5.67 dd (15.2,6.4)
4.13 dt (6.4,6.4)
2.33 m
5.40 m
5.43 m
2.79 dd (6.1,6.1)
5.28 m
5.37 m
2.30 t (6.9)
2.33 m
5.45 m
5.45 m
2.33 m
4.13 dt (6.9,6.4)
5.67 dd (15.2,6.4)
6.54 dddd (15.2,10.8,1.5,1.0)
5.97 dd (10.8,10.3)
5.35 m
2.93 dd (7.8,6.4)
5.30 m
5.40 m
2.33 t (7.6)
1.80 m
72.6
4.15 dt (6.8,6.8)
5.66 dd (15.2,6.6)
6.57 dddd (15.2,10.8,1.5,1.0)
5.99 dd (11.3,11.3)
5.36 m
2.97 dd (7.3,5.9)
5.36 m
5.36 m
2.82 dd (6.4,5.9)
5.30 m
5.38 m
2.08 qdd (7.8,7.8,1.5)
0.96 t (7.6)
137.3
126.5
129.3
130.8
26.9
128.5
129.8
26.4
73.1
137.2
126.4
129.1
131.0
26.8
127.8
133.1
21.5
36.3
126.3
131.2
26.6
128.2
132.8
21.5
128.1
132.9
21.5
2.07 m
0.96 t (7.3)
2.09 m
0.97 t (7.6)
14.6
14.6
14.6
a
1H at 500 MHz and 13C at 125 MHz.
bond at C-10,C-11 was proposed from the additional COSY cou-
pling of the oxymethine proton H-8 with an allylic methylene at
dH 2.33 (H2-9) that was correlated in the HMBC with the olefinic
carbons at dC 126.3 (C-10) and 131.2 (C-11). The 10Z,13Z geometry
was supported by the chemical shift of the bis-allylic methylene
carbon C-12 at dC 26.6 (Breitmaier and Voelter, 1989). These data
led to propose for compound 3 the structure (4Z,6E,10Z,13Z)-8-
hydroxyhexadeca-4,6,10,13-tetraenoic acid.
in the 13C NMR spectrum (Table 1). The analysis of the COSY and
HMBC correlations defined for compound 6 a structure identical
to that of compound 1 from the terminal methyl group up to
C-7. The remaining signals of the spectra of 6 were due to a sequence
of five methylenes that connected C-7 to the carboxylic group.
Therefore compound 6 was identified as (7Z,9E,13Z)-11-hydroxy-
hexadeca-7,9,13-trienoic acid (11-HHT). This compound has been
previously identified as an oxylipin formed in plants (Montillet
et al., 2004; Polkowska-Kowalczyk et al., 2008), used in bioactivity
studies (Prost et al., 2005), and prepared for MS analysis (Montillet
et al., 2004). However, to the best of our knowledge, this is the first
description of the NMR spectroscopic data of 11-HHT (6).
In compound 4 the methylene protons adjacent to the carboxy
group (H2-2, dH 2.30) exhibited an HMBC correlation with the ole-
finic carbon at dC 131.4 (C-4) indicating the presence of a double
bond at C-4,C-5. Following this, the HMBC correlation of the ole-
finic carbon C-5 (dC 127.3) with the oxymethine proton at dH
4.13 (H-7) defined the position of the hydroxy group at C-7. Conse-
quently, the accompanying conjugated diene system had to be at
Compound 7 was obtained as an optically active compound
([a]D = +7.1 (c 0.07, MeOH)) whose molecular formula C18H28O3
was determined by HRMS. The 13C NMR spectrum (Table 3) exhib-
ited signals due to a carboxylic acid function (dC 179.0), four disub-
stituted double bonds (dC 136.9, 134.6, 132.3, 130.8, 130.5, 129.6,
126.6, and 125.5), a hydroxy-bearing methine (dC 73.3/dH 4.11),
and a terminal methyl group (dC 14.5/dH 0.95), indicating that com-
pound 7 was a monohydroxylated octadecatetraenoic acid. The
location of a double bond at C-15,C-16 was readily inferred from
the COSY coupling of the methyl group at dH 0.95 (t, J = 7.5 Hz,
Me-18) with the methylene protons at dH 2.05 (H2-17) which in
turn were coupled with the olefinic proton at dH 5.46 (H-16)
(Fig. 4). The olefinic carbons C-15 and C-16 showed HMBC correla-
tions with two allylic methylene protons dH 2.30/2.23 (H2-14) that
showed COSY coupling with the oxymethine proton at dH 4.11 (H-13),
thus defining the location of the hydroxy group at C-13. The
sequence of COSY couplings observed from the oxymethine proton
at dH 4.11 (H-13) along four consecutive olefinic protons at dH 5.64
(H-12), 6.50 (H-11), 5.97 (H-10) and 5.42 (H-9), indicated the pres-
ence of two conjugated double bonds at C-9,C-10 and C-11,C-12.
The 9Z,11E,15Z geometry of the double bonds was assigned on
the basis of the proton coupling constants J9,10 = 10.5 Hz,
J11,12 = 15.3 Hz, and J15,16 = 11.1 Hz. The remaining double bond of
the molecule was located at C-5,C-6 from the HMBC correlation
of the olefinic carbon at dC 130.5 (C-5) with the methylene protons
at dH 1.64 which were identified as H2-3 from their HMBC correla-
tion with the carboxylic carbon (C-1). The Z geometry of the double
bond at C-5,C-6 was deduced from the chemical shifts of the allylic
methylene carbons C-4 and C-7 at dC 27.7 and 28.3, respectively
(Breitmaier and Voelter, 1989). The absolute configuration at
C-13 could not be investigated due to the paucity of compound
for further studies. All these data and the remaining COSY and
D8E,10Z The chemical shifts of the allylic methylene carbons C-3
.
(dC 24.5) and C-12 (dC 26.8) were consistent with the 4Z,13Z config-
uration. Therefore, the structure (4Z,8E,10Z,13Z)-7-hydroxyhexa-
deca-4,8,10,13-tetraenoic acid was defined for compound 4.
In compound 5 the hydroxy group was located at C-4 on the ba-
sis of the COSY correlation between the oxymethine proton at dH
4.15 (H-4) and the methylene protons at dH 1.80 (m, H2-3) which
in turn were correlated in the HMBC with the carboxylic carbon
(dC 179.1, C-1). Therefore, the two conjugated double bonds were
placed at
D
5E,7Z. The 1H NMR spectrum showed the signals of
two bis-allylic methylenes at dH 2.97 (H2-9) and 2.82 (H2-12).
These data indicated that the molecule featured three methy-
lene-interrupted double bonds. Therefore, after the double bond
at C-7,C-8 another two double bonds had to be located at C-10,C-11
and C-13,C-14. This proposal was confirmed upon observation of
the HMBC correlations of the methylene protons at dH 2.97 (H2-
9) with carbons at dC 129.3 (C-7), 130.8 (C-8), 128.5 (C-10) and
129.8 (C-11) and those of the protons at dH 2.82 (H2-12) with
C-10,C-11, and the olefinic carbons at dC 128.1 (C-13) and 132.9
(C-14), this latter one also exhibiting a correlation with the termi-
nal methyl group. The chemical shifts of the bis-allylic methylene
carbons C-9 (dC 26.9) and C-12 (dC 26.4) indicated the 10Z,13Z
geometry of the double bonds. All these data led to propose for
compound 5 the structure (5E,7Z,10Z,13Z)-4-hydroxyhexadeca-
5,7,10,13-tetraenoic acid.
Compound 6 was obtained as an optically active oil ([a]D = +6.7
(c 0.06, MeOH)) whose molecular formula C16H26O3 was obtained
by HRMS. The NMR spectra of 6 were similar to those of compound
1 although only six signals due to olefinic methines were observed

156
C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
Table 3
1H and 13C NMR data of compounds 7, 8, and 15 (CD3OD).
Position
7a
8b
15a
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
dc
dH, m (J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
179.0
35.3
26.4
178.9
35.4
26.5
177.6
34.4
26.0
2.25 t (7.5)
2.25 t (7.3)
1.64 tt (7.3,7.3)
2.08 dt (7.3,7.3)
5.40 m
5.40 m
2.12 dt (6.8,6.8)
2.24 m
2.29 t (7.3)
1.66 tt (7.3,7.3)
2.13 m
5.37 m
5.37 m
2.83 brdd (5.4,5.4)
5.37 m
5.37 m
2.96 m
5.37 m
1.64 tt (7.5,7.5)
2.09 dt (7.5,7.2)
5.40 m
5.40 m
2.12 dt (7.8,7.2)
2.23 m
5.42 dt (10.5,7.5)
5.97 dd (11.1,10.8)
6.50 dd (15.3,11.1)
5.64 dd (15.3,6.7)
4.11 dt (6.5,6.5)
2.30 m
27.7
27.7
27.6
130.5
130.8
28.3
130.5
130.8
28.3
130.1
129.8
26.5
128.7
129.6
27.0
130.7
129.3
126.4
137.3
28.8
28.8
132.3
129.6
126.6
136.9
73.3
132.2
129.7
126.5
137.5
73.4
5.42 m
5.98 dd (11.2,10.8)
6.49 dd (15.2,11.2)
5.62 dd (15.2,6.8)
4.07 dt (6.8,6.8)
1.52 m
5.97 dd (10.9,10.9)
6.55 dd (15.2,10.7)
5.67 dd (15.2,6.5)
36.2
38.4
2.23 m
1.46 m
15
16
125.5
134.6
5.35 m
26.3
33.0
1.38 m
1.33 m
1.33 m
73.2
36.3
4.12 dt (6.5,6.5)
5.46 qtt (11.1,7.2,1.4)
2.31 m
2.25 m
17
18
19
20
21.7
14.5
2.05 qd (7.5,7.5)
0.95 t (7.5)
23.7
14.4
1.33 m
0.90 t (6.9)
125.5
134.6
21.7
5.37 m
5.46 dtt (10.9,7.3,1.5)
2.05 qd (7.5,7.5)
0.95 t (7.6)
14.5
a
1H at 600 MHz and 13C at 150 MHz.
1H at 500 MHz and 13C at 125 MHz.
b
was established by HRMS. The analysis of the NMR data led to con-
clude that compound 15 was (5Z,8Z,11Z,13E,17Z)-15-hydroxyeico-
sa-5,8,11,13,17-pentaenoic acid (15-HEPE). This acid has been
cited in a variety of pharmacological studies (eg. Vang and Ziboh,
2005) and it has also been identified as an oxylipin of diatoms
(Cutignano et al., 2011; d’Ippolito et al., 2009). However, to the
best of our knowledge, this is the first description of the NMR data
of 15-HEPE (15). Further, the optical activity of any of the enantio-
mers of 15-HEPE has not been found reported in literature. There-
fore, the absolute configuration of 15 was determined by NMR
analysis of the diastereomeric esters 15r and 15s. Positive chemical
9
13
COOH
11
18
5
16
OH
7
Fig. 4. Key COSY (bold) and HMBC (arrow) correlations observed for compound 7.
HMBC correlations defined the structure (5Z,9Z,11E,15Z)-13-
hydroxyoctadeca-5,9,11,15-tetraenoic acid for compound 7.
The HRMS and NMR data of compound 8 led to define the struc-
ture (5Z,9Z,11E)-13-hydroxyoctadeca-5,9,11-trienoic acid. The
only previous account on this compound was found in a Russian-
written paper that deals with the enzyme-catalyzed oxidation of
shift differences (
D
d = dRꢀdS) were obtained for H-10, H-12, H-13
and H-14 while negative values were obtained for H-16, H-17, H-
18, H-19, and Me-20 (Fig. 5), in agreement with an S configuration
at C-15 (Seco et al., 2004).
pinolenic acid (18:3D
5,9,12) (Kuklev et al., 1993). The HRMS, NMR,
and optical rotation data of compound 9 allowed to identify this
compound as (9Z,11E,13S,15Z)-13-hydroxyoctadeca-9,11,15-trienoic
acid [(13S)-HOTE] (Yadav et al., 1992; Zheng et al., 2009). Com-
pound 10 was identified as (9E,11E,15Z)-13-hydroxyoctadeca-
9,11,15-trienoic acid, recently described by Zheng et al. (2009).
Although these authors proposed a 13R configuration for the com-
Compounds 1–14 isolated from C. debaryana and 15 from
N. gaditana are polyunsaturated hydroxy acids whose formation
can be explained through the oxidation either enzymatic or chem-
ical of the corresponding fatty acids (Table 4). The optical activity
exhibited by compounds 1, 2, 6, 7, 9, 10, 14, and 15 strongly indi-
cates that these metabolites are enzymatically formed, likely
through the action of LOXes. The first step in LOX-mediated lipid
peroxidation is a stereoselective hydrogen removal from a bis-
allylic methylene to yield an intermediate fatty acid radical, whose
reaction with oxygen can lead to hydroperoxide formation at [+2]
or [ꢀ2] position (Andreou and Feussner, 2009; Liavonchanka and
Feussner, 2006). It is well known that in plants LOX enzymes
mediate the specific insertion of molecular oxygen into either the
pound ([
ment was not supported by any data nor study of the absolute
configuration. Therefore, although compound 10 ([ D = +4.0,
a]D = +4.3, c 1.0, MeOH), it is worth noting that this assign-
a
]
c 0.1, MeOH) obtained from the microalga C. debaryana is identical
to the acid described by Zheng et al. (2009), in the absence of fur-
ther data we present the structure 10 with the absolute configura-
tion unassigned. Compounds 11, 12, and 13 were obtained as
optically inactive compounds that were identified as
(10E,12Z,15Z)-9-hydroxyoctadeca-10,12,15-trienoic acid (9-HOTE)
(McLean et al., 1996), (9Z,13E,15Z)-12-hydroxyoctadeca-9,13,15-
trienoic acid (12-HOTE) (Pagani et al., 2011), and (9Z,12Z,14E)-
16-hydroxyoctadeca-9,12,14-trienoic acid (16-HOTE) (Dong et al.,
2000), respectively. Compound 14 was identified, on the basis of
its optical rotation, MS, and NMR data as (9Z,11E,13S)-13-hydroxy-
octadeca-9,11-dienoic acid [(13S)-HODE] (Dong et al., 2000; Yadav
et al., 1992).
-0.15
-0.12
-0.10 +0.10
+0.09
+0.23
COOCH3
3
-0.07 -0.15
+0.15
-0.22 H
OR
15a R= H
15r R= MPA(R)
15s R= MPA(S)
Compound 15 was obtained as an optically active oil
([a
]D = +4.9 (c 0.11, MeOH)) whose molecular formula C20H30O3
Fig. 5. Dd (dR–dS) observed for the MPA esters 15r and 15s.

C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
157
Table 4
species of microalgae that have been subjected to the same isola-
tion procedures were devoid of this class of hydroxy acids.
The results herein described for C. debaryana, together with a
first report of the hydroxy acids (12R)-HOTE and (9S)-HOTE in
the green microalga D. acidophila (Pollio et al., 1988), represent
the only accounts of the isolation of oxylipins from green
microalgae.
During our research we have studied two batches of C. debaryana
cultured at different dates. Differences in the total content of oxy-
lipins per dry weight and in their relative amounts were observed
among the two biomass samples (See Experimental). Since these
polyenoic hydroxy acids proved to be rather unstable, chemical
differences among the two biomass samples could partly be due
to different extent of degradation of compounds during the purifi-
cation steps. Nonetheless, it is well known that the production of
oxylipins in plants, seaweeds, and microalgae is induced by a vari-
ety of biotic and abiotic factors (Blée, 2002; Cutignano et al., 2011;
Potin, 2008). Thus, it can also be considered that differences in the
profile of oxylipins among samples of C. debaryana can be due to
culture, harvesting, or environmental conditions, a topic that is
currently the focus of new studies in our laboratories.
Fatty acid composition (% of total fatty acids SD, n = 3) of the samples of C. debaryana
and N. gaditana.
Fatty acids
C. debaryana
N. gaditana
sample 0902-12
sample-1702-12
C14
2.46 0.01
11.45 0.02
1.88 0.00
0.33 0.00
1.13 0.01
0.43 0.00
1.78 0.00
21.00 0.01
1.46 0.00
0.96 0.00
3.70 0.00
6.33 0.00
3.25 0.01
40.70 0.02
3.14 0.00
0.00
2.71 0.09
11.44 0.02
1.80 0.00
0.38 0.00
1.22 0.00
0.45 0.00
2.14 0.01
20.61 0.09
1.45 0.01
0.78 0.00
3.55 0.00
6.02 0.01
3.90 0.01
40.50 0.07
3.07 0.02
0.00
7.11 0.26
25.02 0.63
0.00
32.97 1.06
0.00
0.00
0.00
0.00
0.18 0.01
4.07 0.04
0.00
2.72 0.10
0.00
0.00
0.00
2.88 0.15
25.05 1.83
C16
C16D7
C16D9
C16D7,10
C16D4,7,10
C16D7,10,13
C16D4,7,10,13
C18
C18D9
C18D11
C18D9,12
C18D5,9,12
C18D9,12,15
C18D5,9,12,15
C20D5,8,11,14
C20D5,8,11,14,17
0.00
0.00
The occurrence of oxylipin 15 (15S-HEPE) in the biomass sam-
ple of N. gaditana strongly suggests that this microalga features a
15-LOX and represents the first account of oxylipins from microal-
gae of the class Eustigmatophyceae. The oxidation of EPA
position 9 (9-LOX) or 13 (13-LOX) of the C18 fatty acids ALA and LA
to yield the corresponding 9- and 13-hydroperoxy derivatives, in
most cases with S configuration (Andreou et al., 2009). Analogous
pathways for fatty acid oxidation have been proposed to explain
the production of oxylipins in some green macrophytic algae
(Andreou et al., 2009; Tsai et al., 2008; Gerwick, 1994). Similar to
plants and green algae, oxylipins 9 (13S-HOTE) and 14 (13S-HODE)
could arise in the microalga C. debaryana through the 13S-LOX-
mediated oxidation of ALA and LA, respectively, to yield the corre-
sponding (13S)-hydroperoxides, whose subsequent reduction
would lead to the hydroxy fatty acids. In this context it is worth
noting that it has been sequenced a LOX from the related species
Clamydomonas reinhardtii, showing about 35% homology to plant
and moss LOXes. (Andreou et al., 2009). On the other hand, studies
(20:5D
5,8,11,14,17) by different LOXes has been previously proposed
to explain the biosynthesis of a variety of metabolites obtained
from red and brown algae (Gerwick, 1994; Guschina and Harwood,
2006). More recently, the 15-LOX mediated oxidation of EPA to
yield the 15-hydroperoxide and then (15S)-HEPE has also been de-
scribed in diatoms (d’Ippolito et al., 2009; Lamari et al., 2013).
From the pharmacological point of view, some monohydroxy-
lated compounds derived from the oxidation of PUFAs are known
to be ligands of peroxisome proliferator-activated receptors
(PPARs) (Itoh et al., 2008; Willson et al., 2000). These are nuclear
transcription factors that play a central role in regulating the stor-
age and catabolism of dietary fatty acids, being also involved in
pathological processes such as inflammation, atherosclerosis, and
cancer (Willson et al., 2000). In this regard, (13S)-HODE (14) has
been shown to induce apoptosis in colorectal cancer cells through
binding and down-regulating PPAR-d (Shureiqi et al., 2003). The
EPA-derived hydroxy acid (15S)-HEPE (15) together with (15S)-HETrE,
on plants containing high amounts of roughanic acid (16:3D
7,10,13),
the so-called ‘‘16:3 plants’’, have indicated that 9- and 13-LOXes
that oxidize C18 PUFAs also mediate the oxidation of the
16:3D7,10,13 acid to yield the 7- and 11-hydroperoxides, respec-
tively (Montillet et al., 2004; Polkowska-Kowalczyk et al., 2008).
Thus, in the microalga C. debaryana the 11-hydroxy acid 6 can be
proposed to arise from the action of a 13S-LOX on roughanic acid.
We have not found data regarding to oxylipins derived from the
tetraenoic acids 16:4D4,7,10,13 and 18:4D5,9,12,15 (coniferonic acid),
but it seems likely that these acids in C. debaryana have also expe-
rienced a 13-LOX mediated oxidation to yield the hydroxy acids 1
and 7, respectively. The hydroxy acids 2 and 10, which are geomet-
ric isomers of 1 and 9, respectively, could also derive from the oxi-
dation of the acid 16:4D4,7,10,13 and ALA with concomitant cis to
trans double bond isomerisation (Fukushige et al., 2005). The major
oxylipins isolated from C. debaryana were compounds 1 and 9, a re-
sult consistent with the prominence of the fatty acids 16:4D4,7,10,13
and ALA (ca. 20% and 40% of total fatty acids, respectively) in the
examined biomass (Table 4).
On the other hand, the optically inactive hydroxy acids 3–5, 8,
and 11–13 would arise from the ROS-mediated oxidation of the
corresponding C16 and C18 fatty acids. The coexistence of metab-
olites arisen from enzymatic and chemical oxidation of PUFAs has
often been recorded, since physiological and environmental condi-
tions may affect the intensity and nature of lipid peroxidation
(Berger et al., 2001; Montillet et al., 2004; Polkowska-Kowalczyk
et al., 2008).The possibility of compounds 3–5, 8, and 11–13 be
artefacts formed during the purification steps may also be consid-
ered. Nonetheless, other extracts obtained from this and other
the analogous derivative of dihomo-c-linolenic acid (20:3D
8,11,14),
have shown to inhibit the growth and the production of ARA-de-
rived metabolites in human prostatic cancer cells with a possible
mechanism consisting on binding to and activating PPAR-c (Vang
and Ziboh, 2005). 13-HODE (14) has also been described to play
a role in modulating cutaneous hyperproliferation and in the sup-
pression of the activity of the epidermal protein kinase C-b (Ziboh
et al., 2000). On the other hand, monohydroxy acids derived from
LA, ALA, and EPA have shown activity in assays aimed to detect
anti-inflammatory activity. Thus, 13-HOTE (9) has been described
to suppress the IL-1b induced expression of matrix metalloprotein-
ases (MMPs), enzymes that degrade the cartilage-specific extracel-
lular matrix (Schulze-Tanzil et al., 2002); compounds 16-HOTE
(13), 13-HODE (14), and the methyl ester of 9-HOTE (11) suppress
the PMA-induced inflammation on mouse ears (Dong et al., 2000),
and 15-HEPE (15) inhibits the production of the proinflammatory
leukotriene B4 in RBL-1 cells (Ziboh et al., 2000).
In the present study, the polyunsaturated hydroxy acids 1, 9, 11,
and 14, which are the major oxylipins obtained from C. debaryana,
together with 15 obtained from N. gaditana, were tested for their
activity as inhibitors of the tumour necrosis factor
a (TNF-a), a po-
tent proinflammatory cytokine mainly produced by monocytes and
macrophages in immunologic and inflammatory responses (Iqbal
et al., 2013). For compounds 6 and 13 there were not enough

158
C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
amounts of pure samples and an HPLC fraction composed by 6 and
13 (1:1) was evaluated. Assays were performed on the human THP-
1 macrophages using lipopolysaccharide (LPS) as the triggering
Experimental section
General experimental procedures
factor to stimulate the TNF-
toxic effects, the compounds were assayed at a maximum concen-
tration of 100 M which do not affect THP-1 cell viability in the
SRB assay. To test the effects of the oxylipins on the TNF- produc-
tion, THP-1 macrophages were pretreated with each oxylipin, then
stimulated with LPS and finally analyzed to quantify TNF- (Fig. 6).
During the incubation time of 24 h, control THP-1 macrophages
produced 16.77 ng/mL of TNF- After stimulation with LPS
(1 g/mL) the TNF- production increased about tenfold, up to
153.17 ng/mL. The treatment of cells with oxylipins 1, 9, 11, 14,
or 15 at 100 M strongly inhibited the TNF- production by 87%,
a production. In order to rule out cyto-
Optical rotations were measured on a Perkin-Elmer 241 polar-
imeter. IR spectra were recorded on a Perkin-Elmer FT-IR System
Spectrum BX spectrophotometer. UV/Vis analysis were performed
l
a
on a Helios
c
Unicam spectrophotometer. 1H and 13C NMR spectra
a
were recorded on Varian INOVA 600 or Agilent 500 spectrometers
using CDCl3 or CD3OD as solvents. Chemical shifts were referenced
using the corresponding solvent signals [dH 7.26 and dC 77.0 for
CDCl3, and dH 3.30 and dC 49.0 for CD3OD]. COSY, HSQC, and HMBC
experiments were performed using standard Varian pulse se-
quences. High resolution mass spectra (HRMS) were obtained on
a Waters SYNAPT-2G spectrometer. GC/MS analyses were per-
formed on a QUATTRO Micro GC instrument. Column chromatogra-
phy was carried out on Merck Silica gel 60 (70–230 mesh). HPLC
separations were performed on a LaChrom-Hitachi apparatus using
a.
l
a
l
a
86%, 85%, 98%, and 90%, respectively, upon comparison with LPS-
stimulated untreated THP-1 cells. The mixture of 6 and 13 was
rather less active and only 45% of inhibition was observed. It is
noticeable the activity exhibited by compound 14, which caused
an almost total inhibition of the TNF-
tration of 50 M compounds 1 and 11 again exhibited high activity,
causing 78% and 72% decrease of the TNF- level, respectively.
Compounds 9 and 14 were also significantly active at 50 M, inhib-
iting the production of TNF- by 54% and 52%, respectively. When
compounds were tested at 25 M, the new oxylipin 1 proved to be
the most potent TNF- inhibitor, causing 60% decrease of the TNF-
level upon comparison with LPS-stimulated untreated THP-1 cells.
At the same concentration of 25 M, compound 11 inhibited the
production of TNF- by 44% and compounds 9, 14, and 15 exhib-
a production. At the concen-
LiChrospher Si-60 (Merck, 250 ꢁ 10 mm, 10
(Phenomenex, 250 ꢁ 4.6 mm, m) columns (flow rates
mL/min and 1 mL/min, respectively, volume of injection 150
and monitored by a differential refractometer RI-71 or an UV
detector L-7400 (Merck) set at k = 254 nm. PMA, dexamethasone,
and LPS were purchased from Sigma.
lm) and Luna Si (2)
l
5
l
3
a
lL),
l
a
l
a
a
Biological material
l
The species used in the present study were C. debaryana (fresh-
water species, strain ITC-09; Phylum Chlorophyta, Class Chloro-
phyceae, Order Volvocales, Family Chlamydomonaceae) and
N. gaditana (seawater species, strain ITC-Nano-01; Phylum Hetero-
kontophyta, Class Eustigmatophyceae, Order Eustigmatales, Family
Eustigmataceae). Stocks of both strains are maintained at the Mic-
roalgae Collection of the Instituto Tecnológico de Canarias (Gran
Canaria, Spain). Maintenance of stocks, scale-up of inocula and out-
door production of biomass were performed using the minimal
medium (Sueoka, 1959) for C. debaryana and f/2 medium (Guillard,
1975) for N. gaditana. Scale-up of the inocula was performed in-
door using 10 L polycarbonate containers (Carboys, Nalgene) at
12:12 h light–dark cycle, 20 °C, and aeration with 3% CO2. Outdoor
production was done in batch mode. For C. debaryana cultures,
225 L of minimal medium were prepared using previously chlori-
nated freshwater. Similarly, 225 L of f/2 medium were prepared
for N. gaditana cultures, using previously chlorinated seawater.
Every batch was inoculated with 10% (v/v) of inoculum. Cultures
were performed during February 2012 using a 3 m2 conventional
paddle-wheel driven raceway pond with 15 cm culture depth,
placed in a greenhouse at Instituto Tecnológico de Canarias (Playa
de Pozo Izquierdo, Gran Canaria, Spain). Ponds were supplied with
pure CO2 to maintain the culture pH between 7 and 7.5. Cell
growth was daily monitored by optical density measurements at
750 nm using a UV/Vis spectrophotometer. Cultures were har-
vested in the late stationary phase, when maximal biomass was
achieved, using a Westfalia industrial centrifuge. After centrifuga-
tion, the microalgal biomass was immediately frozen at ꢀ20 °C un-
til lyophilized to dryness.
a
ited a weaker inhibitory effect, less than 25%.
In conclusion, we have found that the cultured biomass of the
green microalga C. debaryana contains an array of oxylipins, includ-
ing new monohydroxy acids derived from the highly unsaturated
16:4D4,7,10,13 and 18:4D5,9,12,15 fatty acids. The occurrence of opti-
cally active oxylipins suggests the existence of LOX-mediated path-
ways in C. debaryana that oxidatively transform C16 and C18
PUFAs. The obtention of the EPA-derived metabolite (15S)-HEPE
(15) from the biomass of N. gaditana indicates that this microalga
features a 15-LOX and represents the first account of oxylipins in
microalgae of the Eustigmatophyceae class. Bioactivity assays have
shown the significant activity of the tested hydroxy acids as TNF-
a
inhibitors, in particular of the new oxylipin 1, and evidence the po-
tential of microalgal biomass to contain, in addition to the well
known long chain PUFAs, other minor bioactive lipid metabolites.
Isolation of oxylipins
Microalgal dry biomass was extracted with acetone-MeOH (1:1,
75 mL/g dry wt.) at room temperature. After filtration, the solvent
was evaporated under reduced pressure (bath temp. 35 °C). Two
samples of lyophilized biomass of C. debaryana, ITC09-0902-12
(72 g) and ITC09-1702-12 (106 g), were extracted to yield 15.0 g
and 19.0 g of extract, respectively. Each extract was chromato-
Fig. 6. Effect of oxylipins 1, 9, 11, 14, 15, and 6 + 13 on LPS-induced TNF-
production by THP-1 macrophages. Cells were incubated with compounds (25, 50,
and 100 M) for 1 h and then stimulated for 24 h with LPS (1 g/mL). TNF- was
quantified in supernatants using ELISA assay. Dexamethasone (Dex) was used as
positive reference compound at concentration of 1 M. Data are means SEM from
six independent experiments. Statistical analysis was performed by ANOVA
followed by Tukey test (n = 6; +++p < 0.001 vs control; ⁄⁄⁄p < 0.001 vs LPS;
⁄⁄p < 0.01 vs LPS).
a
l
l
a
l
graphed on
a
silica gel column (25 ꢁ 7 cm and 30 ꢁ 7 cm,

C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
159
respectively) using solvents of increasing polarities starting with
(7Z,9E,13Z)-11-hydroxyhexadeca-7,9,13-trienoic acid (11-HHT) (6)
the combinations of hexanes-Et2O (9:1, 1:1, 3:7, v/v, 1.5 L each),
Et2O 100% (3.0 L), some combinations of CHCl3–MeOH (9:1 and
8:2, v/v, 1.5 L each), and finally MeOH 100% (1.5 L). The fractions
eluted with hexanes-Et2O (3:7, v/v), Et2O 100%, and CHCl3–MeOH
(9:1, 8:2, v/v) were dried and resuspended in MeOH–H2O (9:1,
v/v, 50 mg/mL), transferred onto RP-18 cartridges preconditioned
with MeOH-H2O (9:1, v/v, 1 mL) and eluted with 10 mL of the same
solvent. After evaporation of the solvent, each mixture was repeat-
edly separated by normal phase HPLC using CHCl3–MeOH
(99.8:0.2, v/v) or hexanes-propan-2-ol-AcOH (97:3:0.1, v/v/v) as
the mobile phase until pure compounds were obtained. The extract
of ITC09-0902-12 yielded dry wt. of compounds 1 (1.6 ꢁ 10ꢀ2% dry
Colorless oil; [a]
D = +6.7 (c 0.06, MeOH); 1H NMR (CD3OD,
600 MHz) see Table 1; 13C NMR (CD3OD, 150 MHz) see Table 1;
HRESIMS(ꢀ) m/z 265.1809 [MꢀH]ꢀ (calcd for C16H25O3, 265.1804).
(5Z,9Z,11E,15Z)-13-hydroxyoctadeca-5,9,11,15-tetraenoic acid (7)
Colorless oil; [a]D = +7.1 (c 0.07, MeOH); IR (film) mmax 3416,
1700, 1580, 1406; 1H NMR (CD3OD, 600 MHz) see Table 3; 13C
NMR (CD3OD, 150 MHz) see Table 3; HRESIMS(ꢀ) m/z 291.1956
[MꢀH]ꢀ (calcd for C18H27O3, 291.1960).
(5Z,9Z,11E)-13-hydroxyoctadeca-5,9,11-trienoic acid (8)
Colorless oil; 1H NMR (CD3OD, 500 MHz) see Table 3; 13C NMR
(CD3OD, 125 MHz) see Table 3; HRESIMS(ꢀ) m/z 293.2115 [MꢀH]ꢀ
(calcd for C18H29O3, 293.2117).
wt.),
3
(1.1 ꢁ 10ꢀ3
%
dry wt.),
4
(1.3 ꢁ 10ꢀ3
%
dry wt.),
5
%
(2.1 ꢁ 10ꢀ3% dry wt.), 9 (9.5 ꢁ 10ꢀ3% dry wt.), 11 (8.1 ꢁ 10ꢀ3
dry wt.), 12 (2.4 ꢁ 10ꢀ3% dry wt.), 13 (8.6 ꢁ 10ꢀ3% dry wt.), and
14 (4.2 ꢁ 10ꢀ4% dry wt.). The extract of ITC09-1702-12 yielded
compounds 1 (4.8 ꢁ 10ꢀ3% dry wt.), 2 (3.1 ꢁ 10ꢀ3% dry wt.), 6
(1.1 ꢁ 10ꢀ3% dry wt.), 7 (2.0 ꢁ 10ꢀ3% dry wt.), 8 (6.6 ꢁ 10ꢀ4% dry
(5Z,8Z,11Z,13E,15S,17Z)-15-hydroxyeicosa-5,8,11,13,17-pentaenoic
acid (15S-HEPE) (15)
(1.7 ꢁ 10ꢀ2
%
dry wt.), 10 (4.7 ꢁ 10ꢀ4
% dry wt.), 11
Colorless oil; [
a]D = +4.9 (c 0.11, MeOH); IR (film) mmax 3417,
wt.),
9
1700, 1595, 1432 cmꢀ1
;
1H NMR (CD3OD, 600 MHz) see Table 3;
(2.6 ꢁ 10ꢀ3% dry wt.), 12 (1.5 ꢁ 10ꢀ3% dry wt.), 13 (1.5 ꢁ 10ꢀ3
%
13C NMR (CD3OD, 150 MHz) see Table 3; HRESIMS(ꢀ) m/z
dry wt.), and 14 (3.5 ꢁ 10ꢀ3% dry wt.). Extraction of the lyophi-
lized biomass of N. gaditana (25 g) as described above yielded
7.0 g of extract, that was chromatographed on a silica gel col-
umn (28 ꢁ 3.5 cm) using solvents of increasing polarities starting
with the combinations of hexanes-Et2O (9:1, 1:1, 3:7, v/v,
0.5 L each), Et2O 100% (0.2 L), some combinations of CHCl3–
MeOH (9:1 and 8:2, v/v, 0.2 and 0.4 L, respectively), and finally
MeOH 100% (0.3 L). The fractions eluted with hexanes-Et2O
(3:7, v/v), Et2O 100%, and CHCl3–MeOH (9:1, 8:2, v/v) were
317.2123 [MꢀH]ꢀ (calcd for C20H29O3, 317.2117).
Synthesis of the MPA esters 1r, 1s, 15r, 15s
The acid (1 or 15) was dissolved in MeOH (1 mL) and treated
with a solution of TMSCH2N2 2.0 M in Et2O (100 lL). After stirring
the mixture at room temp. for 30 min, the solvent was evaporated.
Treatment of 1 (2.8 mg, 0.011 mmol) and 15 (7.1 mg, 0.022 mmol)
as described above yielded the methyl esters 1a and 15a, respec-
tively, with quantitative yield in both cases. Each methyl ester
was dissolved in CH2Cl2 (0.5 mL) and treated with CH2Cl2 solutions
(0.5 mL each) of N,N0-dicyclohexylcarbodiimide (tenfold excess
over the starting compound), N,N-dimethylaminopyridine (fivefold
excess) and the MPA acid (fivefold excess). The mixture was stirred
overnight at room temp. and then purified by preparative TLC
(hexanes-EtOAc 85:15, v/v) to obtain the MPA ester. Treatment
of 1a (1.6 mg, 5.8 ꢁ 10ꢀ3 mmol) and 15a (5.4 mg, 1.6 ꢁ 10ꢀ2
mmol) with (R)-MPA as described above yielded compounds 1r
and 15r, respectively. Treatment of 1a (1.2 mg, 4.3 ꢁ 10ꢀ3 mmol)
and 15a (1.7 mg, 5.1 ꢁ 10ꢀ3 mmol) with (S)-MPA as described
above yielded compounds 1s and 15s, respectively.
separated as described above to yield compound 15 (6.0 ꢁ 10ꢀ2
%
dry wt.).
Characterization of compounds
(4Z,7Z,9E,11S,13Z)-11-hydroxyhexadeca-4,7,9,13-tetraenoic acid (1)
Colorless oil; [ D = +7.5 (c 0.08, MeOH); IR (film) mmax 3452,
1711, 1591, 1411 cmꢀ1
a
]
;
1H NMR (CD3OD, 600 MHz) see Table 1;
13C NMR (CD3OD, 150 MHz) see Table 1; HRESIMS(ꢀ) m/z
263.1647 [MꢀH]ꢀ (calcd for C16H23O3, 263.1647).
Compound 1r: 1H NMR (CDCl3, 600 MHz) (selected data, assign-
ments aided by a COSY experiment) d 6.51 (1H, dd, J = 15.3 and
11.2 Hz, H-9), 5.94 (1H, dd, J = 11.4 and 11.2 Hz, H-8), 5.61 (1H,
dd, J = 15.3 and 7.3 Hz, H-10), 5.41 (1H, m, H-7), 5.38 (1H, m, H-
11), 5.34 (1H, m, H-14), 5.03 (1H, m, H-13), 2.91 (2H, br dd,
J = 6.2 and 5.9 Hz, H2-6), 2.32 (1H, m, H-12a), 2.26 (1H, m, H-
12b), 1.91 (2H, qd, J = 7.6 and 7.6 Hz, H2-15), 0.88 (3H, t,
J = 7.5 Hz, Me-16).
(4Z,7E,9E,13Z)-11-hydroxyhexadeca-4,7,9,13-tetraenoic acid (2)
Colorless oil; [a]
D = +12.0 (c 0.10, MeOH); 1H NMR (CD3OD,
500 MHz) see Table 1; 13C NMR (CD3OD, 125 MHz) see Table 1;
HRESIMS(ꢀ) m/z 263.1646 [MꢀH]ꢀ (calcd for C16H23O3,
263.1647).
(4Z,6E,10Z,13Z)-8-hydroxyhexadeca-4,6,10,13-tetraenoic acid (3)
Colorless oil; 1H NMR (CD3OD, 500 MHz) see Table 2; 13C NMR
(CD3OD, 125 MHz) see Table 2; HRESIMS(ꢀ) m/z 263.1644 [MꢀH]ꢀ
(calcd for C16H23O3, 263.1647).
Compound 1s: 1H NMR (CDCl3, 600 MHz) (selected data, assign-
ments aided by a COSY experiment) d 6.27 (1H, dd, J = 15.2 and
10.9 Hz, H-9), 5.85 (1H, dd, J = 11.2 and 10.9 Hz, H-8), 5.50 (1H,
dd, J = 15.2 and 6.5 Hz, H-10), 5.48 (1H, m, H-14), 5.40 (1H, m, H-
11), 5.32 (1H, m, H-7), 5.25 (1H, m, H-13), 2.76 (2H, br dd, J = 7.3
and 7.0 Hz, H2-6), 2.44 (1H, m, H-12a), 2.35 (1H, m, H-12b), 2.03
(2H, m, H2-15), 0.95 (3H, t, J = 7.6 Hz, Me-16).
(4Z,8E,10Z,13Z)-7-hydroxyhexadeca-4,8,10,13-tetraenoic acid (4)
Colorless oil; 1H NMR (CD3OD, 500 MHz) see Table 2; 13C NMR
(CD3OD, 125 MHz) see Table 2; HRESIMS(ꢀ) m/z 263.1649 [MꢀH]ꢀ
(calcd for C16H23O3, 263.1647).
Compound 15r: 1H NMR (CDCl3, 600 MHz) (selected data,
assignments aided by a COSY experiment) d 6.51 (1H, dd, J = 15.1
and 11.0 Hz, H-13), 5.94 (1H, dd, J = 11.0 and 10.7 Hz, H-12), 5.61
(1H, dd, J = 15.1 and 7.3 Hz, H-14), 5.34 (1H, m, H-15), 5.33 (1H,
m, H-18), 5.03 (1H, dddd, J = 14.2, 7.2, 3.2, and 1.6 Hz, H-17),
2.91 (2H, dd, J = 7.3 and 7.3 Hz, H2-10), 2.29 (1H, m, H-16a), 2.26
(1H, ddd, J = 14.5, 7.3, and 7.3 Hz, H-16b), 1.91 (2H, m, H2-19),
0.88 (3H, t, J = 7.4 Hz, Me-20).
(5E,7Z,10Z,13Z)-4-hydroxyhexadeca-5,7,10,13-tetraenoic acid (5)
Colorless oil; 1H NMR (CD3OD, 500 MHz) see Table 2; 13C NMR
(CD3OD, 125 MHz) see Table 2; HRESIMS(ꢀ) m/z 263.1649 [MꢀH]ꢀ
(calcd for C16H23O3, 263.1647).

160
C. de los Reyes et al. / Phytochemistry 102 (2014) 152–161
Compound 15s: 1H NMR (CDCl3, 600 MHz) (selected data,
appropriate amounts of fresh medium. At the highest concentra-
tion of oxylipin used in the assay the concentration of DMSO was
assignments aided by a COSY experiment) d 6.28 (1H, dddd,
J = 15.2, 11.1, 1.3, and 1.0 Hz, H-13), 5.85 (1H, dd, J = 11.1 and
10.8 Hz, H-12), 5.51 (1H, dd, J = 15.2 and 6.7 Hz, H-14), 5.48 (1H,
m, H-18), 5.40 (1H, m, H-15), 5.25 (1H, m, H-17), 2.76 (2H, m,
H2-10), 2.44 (1H, ddd, J = 14.6, 6.9, and 6.9 Hz, H-16a), 2.36 (1H,
ddd, J = 13.4, 6.6, and 6.6 Hz, H-16b), 2.03 (2H, qdd, J = 7.6, 7.6,
and 1.3 Hz, H2-19), 0.95 (3H, t J = 7.4 Hz, Me-20).
1%, v/v. At this dose DMSO did not affect cell viability or TNF-
duction. Positive controls were incubated with a dexamethasone
solution (500 L/well, final concentration of 1 M) that was pre-
a pro-
l
l
pared by dilution of a stock solution (20 mM in DMSO) with fresh
medium. Control groups were incubated with growth medium
(500
sponse was induced by addition of lipopolysaccharide (LPS, final
concentration of 1 g/mL). The stock LPS solution (5 mg/mL) was
prepared in DMSO and then diluted with fresh medium for a final
volume of 5 L/well. An unestimulated control containing DMSO
1%, v/v, but without LPS, was also assayed. After 24 h incubation,
supernatant fluids were collected and stored at ꢀ80 °C until TNF-
measurements. Commercial enzyme-linked immunosorbent assay
(ELISA) kits (Diaclone GEN-PROBE) were used to quantify TNF-
lL/well) containing DMSO 1%, v/v. The inflammatory re-
Fatty acid analysis
l
An aliquot (15–30 mg) of the acetone-MeOH extract of each
microalga was dissolved in 1 mL of MeOH–HCl (10:1, v/v). 100 lL
l
of a solution of heptadecanoic acid in hexanes (50 mg in 5 mL)
were added as internal standard and the mixture was refluxed
for 1 h. After cooling, the reaction mixture was extracted with hex-
anes (3 ꢁ 3 mL). The organic layers were collected, rinsed with
brine (3 mL) and dried over MgSO4. After filtration and evaporation
of the solvent under reduced pressure, the residue was purified on
a small silica gel column eluting with hexanes-Et2O (95:5, v/v). The
FAME mixture was dissolved in CH2Cl2 and subjected to CG/MS
analysis in a QUATTRO Micro GC (Waters) instrument fitted with
a
a
according to the manufacturer’s protocol. Samples were diluted
by 1:100 prior to reading the absorbance at 450 nm with a micro-
plate reader. To calculate the concentration of TNF-a, a standard
curve (r2 = 0.99) was constructed using serial dilutions of cytokine
standards (range 25–1600 pg/mL) provided with the kit.
an Supelcowax10 column (250
l
m ꢁ 30 m, 0.25
lm film) with
Acknowledgements
He as carrier gas (1 mL/min), operating at 70 eV. The column tem-
perature was elevated from 50 to 220 °C (4 °C/min) and held at
220 °C for 20 min. Fatty acids were identified by comparison of
retention time and mass spectral data with FAME standards and
a NIST library and, when necessary, by analysis of pyrrolidine
derivatives as described in literature (Kajikawa et al., 2006).
This research was supported by a grant from MINECO (Spain)
and FEDER (research project IPT-2011-1370-060000). We thank
P. Assunçao (ITC, Spain) for help with microalgal taxonomy.
Appendix A. Supplementary data
Anti-inflammatory assays
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
03.011.
Cell culture
The THP-1 human monocytic leukemia cell line was obtained
from the American Type Culture Collection (TIB-202, ATCC, USA)
and cultured in RPMI 1640 medium containing 10% heat-inacti-
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