RSC Advances

PAPER
Central metabolic processes of marine macrophytic
algae revealed from NMR based metabolome analysis3
Cite this: RSC Advances, 2013, 3,
7037

Vishal Gupta,a Rajendra Singh Thakur,*b C. R. K. Reddy*a and Bhavanath Jhaa

Metabolites, the regulatory components of cellular functioning, represent the biological phenomes.
Metabolome profile of select marine macrophytic algal species of Chlorophyta, Rhodophyta and
Phaeophyta were characterized for the first time using NMR spectroscopy. A facile and tractable method
suitable for metabolite profiling in marine macroalgal species was also developed and validated against
the one commonly employed for terrestrial plants. The identified metabolites and their fluxes enabled to
gain newer insights into the biochemical regulations adaptive to marine environment. Among the salient
metabolites identified, lignin precursors (acetates of guaiacyl and syringyl units) in Ulva lactuca support the

Received 23rd November 2012,

Accepted 27th February 2013

hypothesis of conserved evolution of lignification trait. Also, the metabolites of non-neuronal cholinergic
system and non-proteinaceous cysteine-sulfinic acid oxoforms identified in this study are the constituents
of redox sensing mechanism. Further, the fluxes of lactate and acetate are indicative for the regulatory
switching of oxidative respiration to fermentative glycolysis. The biological functions of identified

DOI: 10.1039/c3ra23017a

metabolites were then associated with the known metabolic pathways in a metabolic model summarizing

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the central metabolic processes of marine macroalgae.

Introduction
The metabolome analysis offers a snapshot of cells catalytic
and regulatory processes. Such analysis is often of more
biological relevance than other -omes as it reflects the
immediate biochemical consequences of genomic and transcriptomic activity.1 Metabolomics is therefore gaining prominence in the area of integrated systems biology and emerging
as an essential analytical tool in the post-genomic era.24
Metabolomics has made major contributions in trait development among agricultural crops, functional annotation of
unknown genes,5 correlating genotype with phenotype6,7 and
provide greater understanding of the stress tolerance mechanisms.810
The challenge with metabolomics, however, lies in addressing the vast complexity and chemical heterogeneity of
biological metabolites. As a result, many metabolomic studies
employ a range of spectroscopic techniques with Nuclear
Magnetic Resonance (NMR) spectroscopy and Mass spectrometry (GC-MS and LC-MS) being the most commonly
preferred.11 Each technique has its distinct advantage and
a

Discipline of Marine Biotechnology and Ecology, Gijubhai Badheka Marg,

Bhavnagar, Gujarat, India. E-mail: [email protected]
b
Analytical Discipline and Centralized Instrument Facility CSIR-Central Salt and
Marine Chemicals Research Institute, Bhavnagar, 364002, India.
E-mail: [email protected]; Fax: + 91 278 256 6970/256 7562;
Tel: +91 278 256 5801/256 3805 Extn 6140
3 Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ra23017a

This journal is The Royal Society of Chemistry 2013

disadvantage. NMR spectroscopy is less sensitive than MS, but

is generally robust and displays little inter-laboratory variance
provided sample preparation is standardized with respect to
the solvent type, pH and run-time temperature.12 It also allows
detection and quantification of metabolites in a complex
mixture without prior knowledge of sample composition even
under in vivo state.13 Additionally, NMR based analysis of
extracts is non-destructive as it circumvents the need of
sample derivatization.1417 Recently, NMR spectroscopy has
shown extensive utilization in plant metabolomics mainly for
discrimination of species, ecotypes, and mutants,1820 transgenic plants from their wild type siblings,2123 infected plants
from healthy ones24 and also led to understand the metabolic
transitions during disease.25 According to Fernie et al.,26
NMR represents the gold standard in structure identification. The usefulness of NMR spectroscopy to both species
management and monitoring of agricultural product quality27
led to compiled reference library of metabolites for terrestrial
plants.
For marine macroalgae, although, a large amount of
transcriptome data is being accumulated, information on
their metabolite compositions is elusive. Marine macroalgal
species are evolutionarily more ancient and metabolically
distinct from terrestrial plants. Earlier studies on chemical
profiling of macroalgal species were largely restricted to
targeted identification of selected class of compounds.28,29
Nevertheless, a few studies on untargeted metabolite profiling
using GC-MS has been carried out for brown alga Ectocarpus
siliculosus to get insights into its central metabolic processes30

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and to understand the short term responses against saline and
oxidative stress.31 Macroalgae play an important role in
sustaining the productivity of coastal ecosystems, and have a
variety of economic uses.32,33 Metabolite profile of macroalgal
species will be of interest to understand their adaptive
mechanisms against the concomitant acute stress induced
by marine environment and will also allow to identify bioactive
compounds of human health benefits.
In this study metabolite profiling of marine macrophytic
algal species was carried out using NMR spectroscopy, and
their functional relevance was discussed following various
metabolic pathways. One dimensional 1H NMR spectra,
together with two dimensional homo- and hetero-nuclear
spectra (TOCSY and HSQC), indispensable tools for metabolite
identification,34 were employed.

Results and discussion

This study is the first to characterize the metabolome profile of
marine macrophytic algae using NMR spectroscopy. NMR
spectral acquisitions were made directly from algal aqueous
extracts utilizing the pulse programmes available for metabolite analysis of biofluids (urine, serum, tissue extracts etc.).
Different biological systems require different extraction
strategies due to lack of a standard procedure. Metabolite
extraction using CH3OH-d4-KH2PO4 buffer in D2O (pH 6.0) is
one among the most suitable strategy for metabolite analysis
in terrestrial plants as it captures both intermediate polar and
polar metabolites.3537 The spectral information from algal
aqueous extracts were therefore compared with that of the
aforementioned solvent system.
Spectral information from both aqueous and solvent extract
was found similar and is annotated in Fig. 1. Similarity among
the spectra from both the extracts was reproduced from the
macroalgal samples collected during different months in a
year (Fig. 1 and 2). Sharp signals were observed for aqueous
extracts of all algal samples except for G. dura which was
affected with line broadening due to higher viscosity arising
from cell wall polysaccharide agar (Fig. 1 and 2). Nevertheless,
the metabolite information for G. dura was successfully
obtained after 2D analysis. Kaiser et al.38 while comparing
different extraction strategies revealed that the information
generated from deuterated water (D2O) extract was superior to
organic solvent extract. However, authors raised concern about
the stability of D2O extract. Interestingly, the spectral profile of
the aqueous extract remained unchanged after an incubation
of 12 h at 20 uC (ESI, Fig. S13). This could mainly be attributed
to the heat treatment employed during sonication that would
have inactivated the active soluble enzymes. Further, no
degradation of metabolite was observed as a result of the
heat treatment employed. The extraction strategy thereby
overcame the stability issue38 and also circumvented the use of
D2O for preparing the extract.
General practice of metabolome analysis by either GC-MS
or NMR spectroscopy involves water removal from samples by

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freeze drying.35 NMR spectra from freeze dried algal thalli on
comparison with that of the spectra of direct aqueous extract
in this study revealed loss of signals for acetate and
coniferaldehyde in U. lactuca (Fig. 3). Because of the loss of
metabolites, spectra for other freeze dried algal species were
not acquired. Identification of these metabolites provided
preliminary evidence for the existence of lignification trait and
fermentative metabolic regulations in marine macroalgal
species. Concomitantly, freeze drying is a time consuming
and energy intensive step. Another additional advantage of the
developed method is that the spectra were free from the
residual proton resonances of d4-methanol (d 3.3) which
otherwise mask the betaine signal at d 3.27.37 Betaine is a
primary osmoprotectant and requires to be identified unambiguously for water submerged plants. The developed method
thereby minimizes the sample preparation efforts and maximizes the throughput.
The aforementioned indication about the existence of
ligninification trait was confirmed by derivatization followed
by reductive cleavage (DFRC) method. DFRC method is
considered diagnostic for lignin as it specifically cleaves at bO-4 linkages and releases the acetylated mono-lignols.39
Lignin precursors identified in this study include acetates of
guaiacyl and syringyl units of coniferyl and sinapyl alcohol
(Fig. 4). These lignin precursors were reported from protoxylem and metaxylem vessels and fibres of angiosperm
respectively.39 Lignification trait is a key innovation in the
evolution of terrestrial plants from aquatic ancestors. Martone
et al.,39 although, discovered lignification in coralline red
algae Calliarthron cheilosporiodes, the trait was considered to
be non-existing in green algae.40 Red algae have diverged from
vascular plants more than 1 billion years ago thereby finding
of lignification trait in green alga has direct ancestral
eira et al.41 identified peroxidases capable
relevance.41 Espin
of oxidizing monolignols in Ulva rigida but unequivocal
evidence about the existence of lignin was not shown even
after employing the confirmatory thioacidolysis method.
Recently, Srensen et al.42 after thioacidolysis method confirmed the release of guaiacyl and syringyl monomers from
Coleochaete species (Charophyta) indicating the presence of
lignin or lignin-like polymers. The finding of acetates of
guaiacyl and syringyl units in U. lactuca (Chlorophyta), the
monophyletic lineage of Charophyta, supports the hypothesis
of evolutionary transition of this trait from aquatic to land
habitat (Fig. 5). The phenylpropanoid metabolism such as
biosynthesis of lignin in land plants is an adaptation of
secondary metabolism which evolved from algal ancestors.43
Therefore finding of the lignification trait in green algae
supports for the inter-relationship among plant cell wall
architecture with algal ancestors (Fig. 5).
All the identified metabolites and their respective 1H and
1
H13C correlation peaks are listed in Table 1 and are shown in
ESI, Fig. S2 and S3.3 The NMR spectra acquired for the samples
collected during the December month showed anomeric
signals of carbohydrates such as glucose (d 4.48 d), galactose
(d 4.0 t) and sucrose (d 5.4 d) in U. lactuca; glucose (d 4.2 d) and

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Fig. 1 1H NMR spectra of (1) Ulva lactuca, (2) Gracilaria dura and (3) Sargassum tenerrimum acquired during the month of April/May using (a) organic solvent extract
and (b) aqueous extract.

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Fig. 2 1H NMR spectra of (1) Ulva lactuca, (2) Gracilaria dura and (3) Sargassum tenerrimum acquired during month of December using (a) organic solvent extract and
(b) aqueous extract.

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Fig. 3 1H NMR spectra of Ulva lactuca (a) after freeze drying and (b) direct aqueous extract. Arrows indicated the peaks found missing after freeze drying.

sorbitol (d 3.8) in S. tenerrimum; and galactose (d 4.2 d) in G.

dura. In the amino acid region (d 0.8d 4.0), signals were
assigned to alanine, cysteine and glutamic acid in U. lactuca;
alanine, proline and threonine in G. dura; and alanine,
aspartate, glutamate and glutamine in S. tenerrimum. The
investigated amino acid profile corroborate with earlier reports
where macroalgae were shown to be rich in acidic amino acids
such as aspartic acid and glutamic acid.44 These amino acids
play a vital role in nitrogen transport and serve as storage
molecules.45
Seasonal variation in metabolic composition of all life
forms has been well described and the spectral information
for algal samples collected during different periods also
attributed for the same. The metabolites identified from
different algal samples collected during AprilMay months
include alanine, cysteine and glycine in U. lactuca; alanine and
glycine in G. dura; and alanine, glutamine and glycine in S.
tenerrimum. Further, 1D NMR spectra of aqueous extracts of
samples collected during AprilMay months were found to be
less sensitive, showing lower peak intensities, than those
collected during December. This sensitivity constraint made
the recording of HSQC spectra challenging and required
concentrated aqueous extracts (1 g fresh wt) even then signals
in the aliphatic region (d 0.94.0), corresponding mainly to
amino acids, were weak (ESI, Fig. S43). These findings attribute
for an arrest in metabolic activities during AprilMay months.
The data corroborate well with the result of earlier phenological studies from the Indian subcontinent indicating
OctoberFebruary as a period of active growth for macroalgal
species.46,47 The spectral variations among the samples
collected during different periods were also observed in
overlapped HSQC spectra (ESI, Fig. S53).
Interestingly, NMR spectra for S. tenerrimum recorded during
AprilMay months showed signature for O-acetylcholine along
with choline (ESI, Fig. S43). Acetylcholine is synthesized from
choline and regulates the membrane permeability for ions such

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as sodium, potassium and calcium for cellular homeostasis.48

Spectra showed higher prominence for choline than
O-acetylcholine during active growth period (December) while
trend reversed during unfavourable period (April-May) (ESI, Fig.
S43). The rise in concentration of acetylcholine with shown
metabolic arrest during AprilMay months provides an evidence
of existence of non-neuronal cholinergic system. Such system is
known since phylogenetically ancient palaeozoicum era (about
600 million years ago) in both pro- and eukaryotic cells49 but
poorly defined in marine macroalgae despite the long standing
knowledge about the existence of choline containing lipids.
These results provided newer insights into the metabolic level
regulations for this plant group.
1
H NMR spectra for different macroalgal species belonging
to the investigated genus Ulva, Gracilaria and Sargassum and
also for an out group species Odontothalia veravalensis showed
species specific spectral signatures (ESI, Fig. S63). This initial
attempt of showing significant differences in spectral information at inter-species and inter-genus level is a complement to
conventional taxonomic approach. Recently, NMR based
metabolomic analysis has been shown as a rapid and efficient
fingerprint tool for Curcuma species of different origin.50
Identification of acetate and lactate for all macroalgal
species indicates the existence of fermentative regulatory
switching. These compounds mainly accumulate during lower
oxygen availability to accomplish the increased energy
demands.51,52 However acetaldehyde, an intermediate of this
catalytic fermentative pathway, was not detected in this study
probably due to its high reactivity with hydroxyl radicals.
Accumulation of acetate mitigates the effect of lactate and
detoxifies the anaerobic fermentative intermediates. The
existence of both acetate and lactate indicates the presence
of PDH bypass which regenerates NAD+ and allows the
tricarboxylic acid (TCA) cycle to continue.53 Recently, Rocha
et al.54 proposed a metabolic model in terrestrial plants
dwelling in water logged hypoxic conditions explaining

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Fig. 4 Characterization of lignin monomers from Ulva lactuca using DFRC method. (a) GC retention time of the derivatized extract and mass fragmentation pattern of
(b) guaiacyl and (c) syringyl units released. Inset image represent the mass fragmentation of respective units determined by Martone et al. (2009).

accumulation of these metabolites along with alanine.

However, the authors recommended confirmation of such
pathway in other plant species. The increase in concentration

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of alanine and lactic acid was also summarized by Narsai

et al.55 as hypoxia induced metabolic change. Unlike terrestrial
or freshwater plants, marine macroalgae experience different

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Fig. 5 Evolutionary relationship of lignification trait of marine chlorophycean alga U. lactuca with terrestrial plants. Figure is modified from Martone et al. (2009)
where * represents the gap filled by this study.

water submergence level due to periodic oceanic tidal rhythms

that generate the oxygen flux. The experienced submergence
level by the studied seaweed genus is in the order of Sargassum
. Gracilaria . Ulva. Consequently, the relative peak intensities for acetate, lactate and alanine were estimated highest in
S. tenerrimum, followed by G. dura and U. lactuca (Fig. 1 and 2).
S. tenerrimum also possesses additional morphological and
anatomical developments such as parenchymal tissue and gas
vesicles to mitigate the consequences of water submergence.56,57 These results are indicative for a metabolic switching mechanism in marine macroalgal species that regulates
the concomitant acute stress of oxygen flux generated from
tidal rhythms. These insights pave the way for reverse genetic
approach to understand the adaptive regulatory and network
pathways in marine macroalgae.
The other salient metabolites identified include nonproteinogenic cysteine-oxoforms such as hypotaurine, isethionic acid and cysteinesulfinic acid in U. lactuca, G. dura and S.
tenerrimum, respectively. Recently, cysteine-oxoforms are sug-

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gested to have promising role in free radical detoxification.58

Chemically such oxoforms possess rapid transition rate for
different oxidation states and could quench free radicals
effectively. Identification of these compounds presented
another adaptation of macroalgal species under a habitat
prone to generate high amount of free radicals from photocatalysed reactions. Further, hypotaurine, the sulfinic acid
compound identified in U. lactuca, has health promoting
properties such as anti-hypertensive and hypocholesterolemic.59 Abundance of this compound in marine macroalgae
extracts strengthens their functional food applications.
Isethionic acid detected in G. dura is reported to disrupt the
acyl homoserine lactone (AHL) signalling. The other macroalgal species reported for isethionic acid production and
inhibition of bacterial quorum-sensing are Grateloupia turuturu60 and Ahnfeltiopsis flabelliformis.61
All the identified metabolites were then summarized in a
metabolic model according to their functional correlations
known from current biochemical pathways (Fig. 6). The

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Table 1 Metabolites identified using 1D and 2D NMR analysis from the aqueous extract of investigated marine macrophytic algal species

Chemical Shift d, ppm

Metabolites

Glucose
Galactose
Sucrose
Sorbitol
Lactate
Acetate
Alanine
Glutamic acid
Hypotaurine
Coniferaldehyde
a-Ketoglutaric acid
Isethionic acid
Proline
Aspartate
Glutamine
Creatinine
L-Cysteinesulfinic acid
Cysteine
Threonine
Ethanolamine
Choline
Betaine
Glycine
Triethanolamine
Creatine
Succinic acid
Acetylcholine

4.48 (d), 3.64, 3.75, 3.82 (m)

5.2 (d), 4.0 (t)
5.4 (d)
3.8 (m)
1.33 (d), 4.1 (m)
1.92 (s)
1.48 (d), 3.65 (q)
2.15 (m), 2.4 (m)
2.72 (t), 3.45 (t)
3.64 (d), 6.85 (bd), 8.5 (s)
2.37 (t), 3.02 (t)
3.15 (t), 3.95 (t)
2.03 (bm), 2.4 (bm)
2.6 (dd), 2.8 (dd), 3.88 (dd), 3.05 (d), 3.07 (d)
2.1 (q), 2.43 (m)
3.1 (s), 3.9 (s)
2.7 (bq), 4.1 (bm)
2.98 (m), 4.01 (dd)
1.38 (d), 4.1 (m)
3.37 (d), 3.9 (d)
3.19 (s), 3.5 (m)
3.19 (s), 3.9 (s)
3.55 (s)
3.88 (s), 3.4 (s)
2.89 (s), 3.84 (s)
2.42 (s)
2.17 (s), 3.23 (s), 3.8 (t)

metabolic model deciphers information on central metabolic

processes of marine macroalgae involved in maintaining the
cellular homeostasis under periodic environmental fluctuations.

Experimental
Sample collection
The macroalgal samples of Ulva lactuca (Chlorophyta), and
Gracilaria dura (Rhodophyta) were collected from Veraval (N
20u 54.879; E 70u 20.839) and Sargassum tenerrimum
(Phaeophyta) from Okha (N 22u 27.049; E 69u 03.58), Gujarat
coast of India. All sample collections were made from
intertidal area between 1011 a.m. from the respective sites
during different months of the years 2011 and 2012. The
collected algal samples were transported to the laboratory in
seawater under constant temperature of 20 uC. After bringing
to the laboratory, samples were rinsed several times in
autoclaved seawater to remove dirt and adhering particles
and thereafter, cleaned with a drawing brush to remove
epiphytes from thallus.
Extraction of algal metabolites
The metabolite extraction strategies employed in this study
includes: 1) aqueous extract and 2) the organic solvent
extract.3537 These extracts were prepared from three biological
replicates of each algal thalli of U. lactuca and G. dura and
from the leafy blades of S. tenerrimum. The solvent extracts

7044 | RSC Adv., 2013, 3, 70377047

HSQC (1H13C)

5.995; 3.578.5; 3.775.37; 3.874.7; 3.763.3

5.2103.4; 5.294; 3.660
5.494.8; 4.379.4; 3.7575.18; 3.8475.1; 3.8365
3.872.2; 3.773.5; 3.673.8; 3.8465.7
1.322.8; 4.1271.2
2.026.0
1.3518.8; 3.853.5
2.129.48; 2.436.22; 3.857.5
2.758.1; 3.436.4
3.656.4; 7.21124.2
2.9438.7; 2.4533.75
3.1555.5; 3.9559.6
2.132.2; 4.064.1; 3.449.1
2.738.8; 3.955.2
3.856.8; 2.4933.92; 2.1729.0
4.059.1; 2.7833.1
4.152.3
2.9828.4; 4.0159.0
1.3823; 4.167
3.8460.2, 3.444.01
4.059.0; 3.2154.89; 3.658.5
3.1956.0; 3.8967.1
3.544.1
3.8457.8; 3.3457.8
3.9255.7; 2.8939.0
2.3936.8
4.5560.1; 3.7266.6; 3.2556.5; 2.1823.2

were prepared following the protocol described by Kim et al.35

Briefly, algal thalli (200 mg fresh wt) were ground to powder in
liquid nitrogen and then added a mixture of CH3OHd4 : KH2PO4 buffer in D2O (pH 6.0) (1 : 1). For aqueous extract,
algal thalli were blotted on paper towel to remove excess saline
water and then thalli of 200 mg fresh wt were macerated using
mortar and pestle. While maceration about 200 mL of 50 mM
phosphate buffer adjusted to pH 6.0 was added. Both the
solvent and aqueous extracts were vortexed for one minute and
then sonicated for 30 min. For aqueous extract sonication was
carried out with heating at 55 uC, after which samples were
centrifuged at 10,000 rpm for 2 min. Both the aqueous and
organic solvent extract were collected and re-centrifuged
likewise to obtain a clear solution. An aliquot of this solution
was transferred directly to a 5 mm NMR tube to which a few
drops of D2O containing a reference standard (TSP) was added.
Stability of aqueous extract was examined by acquiring the 1H
NMR spectra after an incubation of 12 h at 20 uC. NMR spectra
were also acquired from the freeze dried algal samples.
NMR measurements and metabolite identification
NMR spectra were acquired on a Bruker Avance II 500 MHz
spectrometer, equipped with a 5 mm BBI probe. Samples were
spun at 20 Hz at temperature 20 uC. Each spectra consisted of
80 scans of 2 s acquisition time with a spectral width of 7000
Hz. Spectra were Fourier transformed using an exponential
line broadening value of 0.3 Hz. Spectra were then manually
phased, baseline corrected and calibrated to the internal
standard (TSP = 0.0 ppm). The solvent signal suppression was
applied at 4.8 ppm during the recycle delay of 1 s with a low

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Fig. 6 Metabolic model framed by mapping the functional relevance of the identified metabolites providing information onto the central metabolic processes of
marine macrophytic algae.

strength (bandwidth of 49 Hz) RF pulse. Ambiguities among

the 1D pattern recognition were resolved by mapping the
correlation among cross peaks of TOCSY (pre-saturation
version) and HSQC experiments. HSQC spectra were recorded
by echo-antiecho method using gradients where 64 scans for
256 t1 points were acquired with recycle delay of 1.5 s. The
total acquisition time was around 7 h. The broad residual
water signal in HSQC spectra was removed by correcting the
baseline with Gaussian function of width 0.3 ppm. TOCSY
spectra were acquired with 24 scans for 128 t1 points.
Metabolite identification was performed by comparing 1H
resonances and 2D correlation data against the reported
literature and available online databases (http://prime.psc.
riken.jp/, http://www.bmrb.wisc.edu/metabolomics/, http://
www.hmdb.ca/), and Chenomx software (evaluation version).
The spectral data hereby generated was examined for relative
intensity variations among the identified metabolites by
scaling the peak intensity which were then used as indicators
of the metabolic fluxes.

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Derivatization Followed by Reductive Cleavage (DFRC) method

Mono-lignols were identified from U. lactuca following the
DFRC procedure.62 In brief, algal sample (100 mg) were
suspended in acetyl bromide : acetic acid solution (1 : 4 by
volume) and stirred at room temperature for 16 h. After
incubation, solvent was completely removed by rotary evaporation. The residue was then dissolved in dioxane : acetic
acid : water (5 : 4 : 1 v/v/v) and zinc dust (50 mg) was added
while stirring and continued to stir for 30 min. The mixture
was transferred to a separatory funnel to which dichloromethane (10 mL) was added. The mixture was vigorously
mixed and the organic layer was separated and dried under
reduced pressure. The residue was then acetylated in the
solvent dichloromethane containing 1 : 1 ratio of pyridine :
acetic anhydride and incubated for 40 min thereafter solvents
were evaporated under reduced pressure. The acetylated
compounds were re-dissolved in dichloromethane and used
for GC-MS analysis.62

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Conclusion
Metabolome analysis aids in linking the metabolites level
mechanisms with other complementary -omics and also lead
to identify active compounds of human health benefits. The
untargeted metabolite information generated for the first time
for marine macrophytic algae provided newer insights into the
key aspects about their functionality and biochemical regulations. The identification of lignin precursors in green alga Ulva
lactuca supports the hypothesis of conserved evolution of
lignification traits in terrestrial plant from aquatic algal
ancestors. Also, biochemical regulatory mechanisms including
non-neuronal cholinergic system and sulfinic acid switch for
redox signalling were determined. Further, a facile and
tractable method particularly suitable for metabolite profiling
of marine macroalgal species was developed. The method
minimizes the duration of sample preparation efforts and
maximizes the throughput and also circumvents the use of
organic deuterated solvents. These advantages reflected the
potentials for utilization of this method as a rapid fingerprinting tool to complement the conventional taxonomic
approaches. Moreover, the generated snapshots of cellular
activities are a complement for other -omic approaches and
pave the way for further investigation.

Acknowledgements
We thank the Council of Scientific and Industrial Research
(CSIR), New Delhi for the financial support under network
project NWP018 and OLP 0007 for maintenance of sophisticated analytical instruments. Mr. Vishal thanks CSIR for
awarding Senior Research Fellowship. We sincerely thank Dr
Pia Winberg and Dr Lisa Kirkendale, Wollongong University,
Australia, and Prof. John H. Bothwell, Queens University,
Belfast for reviewing the manuscript. We also thank Mr.
Harshad Brahmbhatt for GC-MS analysis.

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