Monogalactosyldiacylglycerols, potent nitric oxide inhibitors from the marine microalga Tetraselmis chui

Article abstract of DOI:10.1080/14786419.2012.717285

Methanolic extracts of some marine and freshwater microalgae were tested for their nitric oxide (NO) inhibitory activity on lipopolysaccharide-induced NO production in RAW264.7 macrophage cells. Among the tested extracts, Tetraselmis chui extract showed the strongest NO inhibitory activity, thus selected for further study. NO inhibitory activity guided isolation led to identification of two monogalactosyldiacylglycerols (MGDGs) (2S)-1-O-(6Z,9Z,12Z,15Z- octadecatetranoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-O-β-D- galactopyranosylglycerol (1) and (2S)-1-O-(9Z,12Z,15Z-octadecatrinoyl)-2-O-(4Z, 7Z,10Z,13Z-hexadecatetranoyl)-3-O-β-D-galactopyranosylglycerol (2) from the MeOH extract of T. chui. The stereo-chemistry of 1 was elucidated by classical degradation method. MGDGs 1 and 2 showed strong NO inhibitory activity compared to N^G-methyl-L-arginine acetate salt, a well known NO inhibitor used as a positive control. Isolated MGDGs suppressed NO production through down-regulation of inducible NO synthase protein. A structure activity relationship study suggested that the polyunsaturated fatty acids of the MGDGs are responsible for NO inhibition. Moreover, increasing unsaturation on the fatty acid side chains enhanced the NO inhibitory potency of the MGDGs.

Full text of DOI:10.1080/14786419.2012.717285

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Monogalactosyldiacylglycerols, potent
nitric oxide inhibitors from the marine
microalga Tetraselmis chui
a
a
a
Arjun H. Banskota , Pamela Gallant , Roumiana Stefanova ,
a
a
Ronald Melanson & Stephen J.B. O’Leary
a
Aquatic and Crop Resource Development, National Research
Council Canada, 1411 Oxford Street, Halifax, Nova Scotia B3H
3Z1, Canada
Version of record first published: 14 Sep 2012.
To cite this article: Arjun H. Banskota, Pamela Gallant, Roumiana Stefanova, Ronald Melanson &
Stephen J.B. O’Leary (2012): Monogalactosyldiacylglycerols, potent nitric oxide inhibitors from the
marine microalga Tetraselmis chui , Natural Product Research: Formerly Natural Product Letters,
DOI:10.1080/14786419.2012.717285
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Natural Product Research
012, 1–7, iFirst
2
Monogalactosyldiacylglycerols, potent nitric oxide inhibitors from the
marine microalga Tetraselmis chui
Arjun H. Banskota*, Pamela Gallant, Roumiana Stefanova, Ronald Melanson and
Stephen J.B. O’Leary
Aquatic and Crop Resource Development, National Research Council Canada, 1411 Oxford Street,
Halifax, Nova Scotia B3H 3Z1, Canada
(
Received 3 November 2011; final version received 1 July 2012)
Methanolic extracts of some marine and freshwater microalgae were tested for
their nitric oxide (NO) inhibitory activity on lipopolysaccharide-induced NO
production in RAW264.7 macrophage cells. Among the tested extracts,
Tetraselmis chui extract showed the strongest NO inhibitory activity,
thus selected for further study. NO inhibitory activity guided isolation led
to identification of two monogalactosyldiacylglycerols (MGDGs) (2S)-1-O-
(
6Z,9Z,12Z,15Z-octadecatetranoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-
O-ꢀ-D-galactopyranosylglycerol (1) and (2S)-1-O-(9Z,12Z,15Z-octadecatrinoyl)-
-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-O-ꢀ-D-galactopyranosylglycerol (2)
2
from the MeOH extract of T. chui. The stereo-chemistry of 1 was elucidated by
classical degradation method. MGDGs 1 and 2 showed strong NO inhibitory
activity compared to N -methyl-L-arginine acetate salt, a well known NO
G
inhibitor used as a positive control. Isolated MGDGs suppressed NO production
through down-regulation of inducible NO synthase protein. A structure activity
relationship study suggested that the polyunsaturated fatty acids of the MGDGs
are responsible for NO inhibition. Moreover, increasing unsaturation on the fatty
acid side chains enhanced the NO inhibitory potency of the MGDGs.
Keywords: monogalactosyldiacylglycerol; nitric oxide; Tetraselmis chui; iNOS;
microalgae; galactolipid; glycolipid
1
. Introduction
Natural products are leading sources of novel molecules that have been used in the
pharmaceutical and nutraceutical industries since their inception. The majority of the
natural products currently on the market as therapeutic agents or as health supplements
are derived from terrestrial organisms including plants, animals and micro-organisms,
even though the oceans cover more than 70% of the Earth’s surface. The oceans contain
the majority of the biodiversity of the planet and are an enormous resource for new
biologically active compounds. Omega-3 fatty acids derived from fish oil provide an
example of valuable natural products from the ocean with several known health benefits
(
Simopoulos, 1991) and are widely used in the functional food and nutraceutical
industries. Microalgae, which are at the bottom of the marine food chain, are
considered a primary source of diverse bioactive natural products (Skulberg, 2000).
*
Corresponding author. Email: arjun.banskota@nrc-cnrc.gc.ca
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Her Majesty the Queen in Right of Canada
http://dx.doi.org/10.1080/14786419.2012.717285
http://www.tandfonline.com

2
A.H. Banskota et al.
Considerable interest has been focused on these unicellular photosynthetic organisms due to
their potential capacity to produce biofuels and as a source of a wide range of commercially
interesting byproducts including proteins, fats, sugars, carotenoids and many others
(
Spolaore, Joannis-Cassan, Duran, & Isambert, 2006). Currently, a small number of
microalgal species are intensively cultivated to support commercial shellfish aquaculture.
These include members of the genus Tetraselmis isolated for their favourable nutritional
qualities, such as high levels of the essential fatty acid 20:5 n À 3 (Wikfors et al., 1996).
Nitric oxide (NO) is an important signalling molecule that acts in many tissues to
regulate a diverse range of physiological processes. It is a weak radical produced from L-
arginine via the enzyme NO synthase (NOS). NO acts as a host defence mechanism by
damaging pathogenic DNA and as a regulatory molecule in homoeostatic activities (Kuo
&
(
Schroeder, 1995). However, excessive production of NO catalysed by inducible NOS
iNOS) is pathogenic to host tissues (Aktan, 2004). Thus, effective inhibition of NO
accumulation represents a beneficial therapeutic strategy for the treatment of NO-
mediated disorders.
National Research Council (NRC) Canada recently launched a national programme to
develop novel microalgae cultivation technologies with the long-term aim of converting
CO emissions from large point source emitters into renewable fuels and other value-added
2
products through the scaled production of microalgal biomass. Through this programme,
a diverse collection of algal strains have been cultivated and tested for potential biological
applications. In this report, we describe the isolation and identification of two
monogalactosyldiacylglycerols (MGDGs) from a marine microalga Tetraselmis chui,
and their NO inhibitory activity in lipopolysaccharide (LPS)-induced NO production in
RAW264.7 cells.
2
. Results and discussion
Marine and freshwater microalgae, namely Botryococcus braunii, Chlorella sorokiniana,
Chlorella vulgaris, Isochrysis galbana, Nannochloropsis granulata, Neochloris oleoabundans,
Pavlova lutheri, Pavlova pinguis, Phaeodactylum tricornutum, Porphyridium aerugineum,
Scenedesmus dimorphus and T. chui, acquired from various culture collections were
cultivated independently in f/2 medium under tightly controlled conditions in a 500 L
internally illuminated photobioreactor (Craigie, Armstrong, Staples, & Bauder, 2003).
Harvested algal biomass was freeze dried and extracted with MeOH. The MeOH extracts
were subsequently tested for NO inhibitory effects. MeOH extract of T. chui showed
strong and dose-dependent NO inhibition, reducing 47.7% of NO production in LPS-
À1
induced RAW264.7 macrophage cells at 50 mg mL concentration (Figure 1 and Table
S1), and was selected for further NO inhibitory activity guided isolation. The extract was
then partitioned into hexane, chloroform and aqueous fractions by liquid–liquid
extraction. The hexane and chloroform fractions showed significant NO inhibitory
activity while the aqueous fraction had very weak NO inhibitory activity (Figure 1a). The
1
H-NMR spectra of both hexane and chloroform fractions had identical signals
corresponding to polyunsaturated fatty acids (PUFAs) and sugar in addition to
chlorophyll signals, indicating that both fractions contained similar compounds respon-
sible for NO inhibition. The hexane fraction was sub-fractionated into three sub-fractions
by preparative TLC. The sub-fraction 2 containing MGDGs showed relatively stronger
NO inhibition among the three sub-fractions (Figure 1b), which led to isolation of two
MGDGs i.e. 1 and 2 by semi-preparative HPLC (Figure 2).
(2S)-1-O-(6Z,9Z,12Z,15Z-octadecatetranoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-
-O-ꢀ-D-galactopyranosylglycerol (1) was isolated as a colourless film with the molecular
3

Natural Product Research
3
(
a) 60.0
*
*
50 ug/mL
2
5 ug/mL
**
5
4
3
2
1
0.0
0.0
0.0
0.0
0.0
**
10 ug/mL
**
*
*
*
**
0
.0
MeOH Extract
70.0
Hexane Fraction Chloroform Fraction Aqueous Fraction
(
b)
*
5
2
1
0 ug/mL
5 ug/mL
0 ug/mL
*
60.0
50.0
40.0
30.0
20.0
10.0
*
*
*
0.0
Sub-fraction 1
Sub-fraction 2
Sub-fraction 3
Figure 1. NO inhibitory effects of T. chui extract, fractions and sub-fractions: (a) MeOH extract and
the hexane, chloroform and aqueous fractions and (b) sub-fractions 1, 2 and 3 obtained after
preparative TLC of hexane fraction. Significantly different from the LPS-treated control: *p 5 0.05
and **p 5 0.01.
1 13
formula C H O . The H- and C-NMR spectra of 1 (Table S2) displayed signals
4
3
66 10
corresponding to 8 unsaturated double bonds, 6 oxygenated methine groups, 17 methylene
groups including 3 oxygenated methylene, 2 primary methyl groups and two 2 carbonyl
groups suggesting that 1 was composed of a sugar, a glycerol and two PUFAs. In depth,
spectral analyses including 2D-NMR led to the identification of a terminal ꢀ-
D-galactopyranose and a glycerol moiety. This was further confirmed by alkaline
hydrolysis, which gave 2 R-1-O-ꢀ-galactopyranosylglycerol (Oshima, Yamada,
Matsunaga, Moriya, & Ohizumi, 1994), and two fatty acid methyl esters. The fatty acids
were identified as 6,9,12,15-octadecatetranoic acid and 4,7,10,13-hexadecatetranoic acid
attach to glycerol at C-1 and C-2, respectively, via ester bond by spectral analysis (Figures S1
and S2) and further confirmed by GC/mass spectrometry (MS) analysis of the methyl esters
obtained after alkaline hydrolysis. Since, allylic carbon signals of Z- and E-isomers are
observed at ꢁ 27–28 and 32–33, respectively (Andrianasolo et al., 2008; Jung, Lee, & Kang,
C
1
996), the 26.5 and 26.4 ppm shifts of all bis-allylic methylene carbons in 1 suggested that all

4
A.H. Banskota et al.
OH
6
'
HO
HO
O
1'
O
3
O
OH
2
4"
O 1"
16"
1
O
1"'
18"'
6
"'
O
OH
HO
HO
1
2
O
OH
O
O
O
O
O
Figure 2. Structure of MGDGs (1 and 2) isolated from T. chui.
double bonds have a cis-geometry (Z). Accordingly, stereo-structure of 1 was elucidated as
2S)-1-O-(6Z,9Z,12Z,15Z-octadecatetranoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-
(
O-ꢀ-D-galactopyranosylglycerol. Most recently, Xu, Chen, Yan, Chen, and Zhou (2010)
identified a similar MGDG from a marine diatom Stephanodiscus sp. using UPLC coupled
with electrospray ionisation-quadrupole-time of flight MS, though that report neither
described the position of the double bonds on the fatty acid chains nor their geometry. The
stereo-chemistry of the glycerol moiety at C-2 chiral centre was also unsolved. It is
worthwhile to mention here that this study describes the first time 1 was isolated and stereo-
structure was well established by spectral analyses and classical chemical degradation
method.
(2S)-1-O-(9Z,12Z,15Z-octadecatrinoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-O-
-D-galactopyranosylglycerol (2) was also isolated as a colourless solid with the molecular
ꢀ
formula C H O . The H- and C-NMR signals of 2 were identical to that of 1 except
1
13
4
3
68 10
the absence of one double bond. The fatty acid at C-1 position of 2 was determined
as 9,12,15-octadecatrinoyl acid instead of 6,9,12,15-octadecatetranoic acid as in 1.
Furthermore, spectral data of 2 were identical to the previously reported data (Greca,
Monaco, Previtera, & Parrilli, 1989). To the best of our knowledge this is the first report of
2
from T. chui.
Both monogalactosylglycerols 1 and 2 isolated from MeOH extract of T. chui were
tested for their NO inhibitory activity and demonstrated a potent NO inhibitory effect as
G
compared to N -methyl-L-arginine acetate salt (shortly L-NMMA), a well-known NO
inhibitor used as a positive control in this study (Figure 3). These results strongly
suggested that MGDGs are primarily responsible for the NO inhibitory activity of T. chui
MeOH extract. This is also supported by the fact that 1 and 2 are the major components of
the chloroform fraction as determined by HPLC analysis (Figure S4). Galactolipids and
pigments including carotenoids present in sub-fractions 1 and 3 and their possible
synergistic effect may also contribute to the NO inhibition of MeOH extract as these
fractions also possessed NO inhibitory activity (Murakami et al., 2000). MGDG 1, having
an additional double bond on the PUFA side chain attached at C-1 of the glycerol moiety

Natural Product Research
5
100.0
1
*
2
3
*
*
80.0
60.0
40.0
20.0
L-NMMA
*
*
*
*
*
*
*
*
*
*
*
*
*
0.0
100
80
60
40
20
Concentration uM
Figure 3. NO inhibitory effects of 1–3 and L-NMMA. Significantly different from the LPS-treated
control: *p 5 0.05 and **p 5 0.01.
possessed better NO inhibitory activity than 2, suggesting that increasing unsaturation in
the PUFA side chain may enhance the NO inhibitory activity of the glycolipid. To
examine the structure activity relationship, we further tested 2 R-1-O-ꢀ-D-galactopyr-
anosylglycerol
obtained
after
NaOMe
mediated
hydrolysis.
2 R-1-O-ꢀ-
Galactopyranosylglycerol showed no NO inhibition even at 100 mM concentration
suggesting that the PUFAs are responsible for NO inhibition. This was further supported
by the fact that the glycolipid with saturated fatty acids (3) (Figure S3) possessed very
weak NO inhibitory activity as compared to 1 and 2.
O’Donnell et al. (1999) described that unsaturated fatty acids react with the reactive
nitrogen species derived from NO to yield nitrated oxidation products. In order to
determine whether or not the isolated MGDGs simply inhibit NO production by reacting
with NO radicals, we further tested the effect of both MGDGs on iNOSs enzyme
regulation in RAW264.7 cells. Stimulation of macrophages by LPS increases iNOS protein
levels resulting in a high output of NO, one of the most important elements of
inflammatory reaction. Both MGDGs down regulated the iNOS protein levels (Figure 4)
in LPS-stimulated RAW264.7 macrophage cells suggesting MGDGs 1 and 2 inhibited the
NO production through the down-regulation of iNOS accumulation.
In a recent study, Jo et al. (2010) reported the anti-inflammatory effects of an 80%
MeOH extract of Tetraselmis suecica. They concluded that metabolites present in the
hexane and ethyl acetate fractions of the 80% MeOH extract are responsible for the
inhibition of NO production in LPS-stimulated RAW 264.7 macrophage cells. Based on
our finding in T. chui and almost identical extraction and fractionation techniques
employed in both studies, it is possible that MGDGs are responsible for the anti-
inflammatory activity of T. suecica as well. Similarly, Bruno et al. (2005) and Murakami,
Nakamura, Koshimizu, and Ohigashi (1995) reported that MGDGs isolated from a blue–
green alga and Citrus hystrix, respectively, showed grater anti-inflammatory activity than
indomethacin in in vivo experimental models. These finding further suggest that MGDGs 1
and 2 may have the potential to be new anti-inflammatory agents and further study is
warranted.

6
A.H. Banskota et al.
1
+
2
+
L
-NMMA
+
100 uM
LPS 1 ug/mL
-
+
iNOS(130 kDa)
3
2
2
1
1
0
5
0
5
0
5
0
LPS (-) LPS (+)
LPS + 1
LPS + 2 LPS + L-NMMA
Figure 4. Inhibitory effect of MGDGs (1 and 2) and L-NMMA on iNOS expression and the
À1
production of NO. Raw 264.7 cells were treated with LPS (1 mg mL ) for 24 h with or without tested
compounds. Protein levels of iNOS were determined by western blot analysis while NO production
was determined by absorbance spectrometry in the presence of the Griess reagent.
3
. Conclusions
This study demonstrated that the MGDGs isolated from T. chui inhibit NO production by
reducing iNOS accumulation in LPS-stimulated RAW 264.7 macrophage cells. The
structure activity relationship study suggested that the PUFA side chains are essential for
NO inhibition of the MGDGs.
Supplementary material
Experimental details are available online, alongside Tables S1–S3 and Figures S1–S4.
Acknowledgements
The authors are thankful to Dr S. Ewart for his support. This is NRC publication no. 54077.
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