Hamid2019 Article MetabolomeProfilingOfVariousSe

Planta (2019) 249:1921–1947
https://doi.org/10.1007/s00425-019-03134-1
ORIGINAL ARTICLE
Metabolome profiling of various seaweed species discriminates

between brown, red, and green algae
Shahlizah Sahul Hamid1,2 · Masataka Wakayama1,2 · Kensuke Ichihara3 · Katsutoshi Sakurai4 · Yujin Ashino2 ·
Rie Kadowaki2 · Tomoyoshi Soga1,2 · Masaru Tomita1,2
Received: 28 January 2019 / Accepted: 7 March 2019 / Published online: 19 March 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Main Conclusion Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and
green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated.
Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed
groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not
been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two
green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-
dried seaweed samples were processed by methanol–water extraction with or without chloroform and analysed by capillary
electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration
profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not
have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only
sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas
fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some
characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles
generated in this study will be a resource and provide guidance for nutraceutical research studies because the information
about metabolites in seaweeds is still very limited compared to that of terrestrial plants.
Keywords Metabolites · Red algae · Brown algae · Green algae · Extraction methods
Abbreviations LC Liquid chromatography

CE Capillary electrophoresis NMR Nuclear magnetic resonance
MS Mass spectrometry GC Gas chromatography
PCA Principle component analysis
Electronic supplementary material The online version of this MeOH Methanol
article (https://doi.org/10.1007/s00425-019-03134-1) contains CHCl3 Chloroform
supplementary material, which is available to authorized users. H2O Water
MES 2-(N-morpholino)ethanesulfonic acid
* Masataka Wakayama
wakayama@ttck.keio.ac.jp CSA d-camphor-10-sulfonic acid
IS Internal standard
1
Institute for Advanced Biosciences, Keio University, 246‑2, ESI Electrospray ionisation
Kakuganji‑Mizukami, Tsuruoka, Yamagata 997‑0052, Japan TOF Time-of-flight
2
Systems Biology Program, Graduate School of Media SD Standard deviation
and Governance, Keio University, Fujisawa 252‑8520, Japan Asp Aspartate
3
Muroran Marine Station, Field Science Centre for Northern Glu Glutamate
Biosphere, Hokkaido University, 1‑133‑31, Funami‑cho, Asn Asparagine
Muroran, Hokkaido 051‑0013, Japan
Pro Proline
4
Yamagata Prefecture Fisheries Experiment Station, Kamo Gly Glycine
Ookuzure 594, Tsuruoka, Yamagata 997‑1204, Japan
13
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1922 Planta (2019) 249:1921–1947
Phe Phenylalanine lipids and its derivatives (Goulitquer et al. 2012; Kumari
Arg Arginine et al. 2013), targeted compounds synthesised as defence
Trp Tryptophan response, such as mycosporine-like amino acids and halo-
Ala Alanine genated compounds (Laturnus 1996; Hartmann et al. 2015),
Ser Serine or examined mono- and polysaccharide extracts of various
Gln Glutamine seaweeds (Percival 1979; Costa et al. 2010; Skriptsova et al.
DNA Deoxyribonucleic acid 2010; Wu et al. 2014; Hamed et al. 2015; Robin et al. 2017;
HPLC High-performance liquid chromatography Garcia-Vaquero et al. 2017). Those studies were often lim-
3PG 3-Phosphoglycerate ited to either a few algae species or certain treatment effects
2PG 2-Phosphoglycerate on several species. However, a comprehensive analysis
2AB 2-Aminobutyrate comparing the chemical profiles of various seaweeds is still
G6P Glucose-6-phosphate lacking.
kV Kilovolt Metabolomics is an emerging field that greatly contrib-
i.d. Inner diameter utes to the latest insights in systems biology as it captures
the immediate biological information generated by genomic
and transcriptomic activity. Most studies have been focused
Introduction on select metabolites involved in amino acid and/or lipid
metabolism (Wahbeh 1997; Hwang et al. 2013; Zhou et al.
Seaweeds or marine macroalgae are photosynthetic non- 2015; Parjikolaei et al. 2016; Astorga-España et al. 2016).
flowering plant-like organisms that are divided into three More than 50,000 metabolites of terrestrial plants have been
major groups based on their dominant pigmentation: brown deposited in the KNApSAck database (http://kanaya .naist. jp/
(Phaeophyceae, approximately 1755 species), red (Rhodo- KNApSAcK/) (Nakamura et al. 2014), whereas the infor-
phyta, approximately 6000 species), and green algae (Chlo- mation on seaweed metabolites is still limited; the seaweed
rophyta, approximately 1500 species) (West et al. 2016; metabolite database (SWMD; http://www.swmd.co.in/) only
Guiry and Guiry 2018). More than 3000 different com- contains 1109 metabolites mostly from the red algae Lau-
pounds have been identified in seaweeds, indicating a diver- rencia sp. (Davis and Vasanthi 2011).
sity that is linked to their polyphyletic origin and the differ- Several analytical platforms are being used in metabo-
ent marine environments in which they thrive (Leal et al. lomics (Lei et al. 2011; Heyman and Dubery 2016), includ-
2013; Belghit et al. 2017). Among the seaweeds, the brown ing a variety of NMR spectroscopy and mass spectrometry
algae are phylogenetically distant from the red and green (MS) methods, such as MS coupled with gas chromatogra-
algae; whereas the red and green algae originated through phy (GC), liquid chromatography (LC), or capillary elec-
the primary endosymbiosis of photosynthetic prokaryotes, trophoresis (CE) (Gupta and Abu-Ghannam 2011; Gu et al.
the evolution of brown algae included a secondary endosym- 2015; de Raad et al. 2016). Although NMR analysis gener-
biosis event (De Clerck et al. 2012; Groisillier et al. 2014). ates precise structural information, it requires larger sample
Therefore, many morphological and metabolic properties of volumes with higher concentrations for quantification than
brown algae differ greatly from those of red and green algae. MS-based methods. Using MS, small sample amounts are
For instance, brown algae have plastids with four membranes sufficient to obtain comprehensive metabolite analyses (Cai
and a specific cell wall composition associated with unique et al. 1995; Ramautar et al. 2009; Hirayama et al. 2014),
metabolic pathways (Cavalier-Smith 2000; Rousvoal et al. but sample preparation typically includes extraction steps.
2011). Presently, there is no comprehensive extraction and drying
Previous studies already indicated that the chemical com- technique to recover all compound classes with high yield
position of seaweeds varies with the species, habitat, salin- and reproducibility. Thus, to simultaneously compare several
ity, temperature, light intensity, and other environmental seaweed species, we had to identify an optimal extraction
conditions such as stress due to the threat from an invasive and drying method.
species or pathogen (Robledo and Freile Pelegrín 1997; La Previous studies on seaweed metabolomics or nutrients
Barre et al. 2004; Date et al. 2012; Tabarsa et al. 2012; Pei- used different extraction and drying methods (Chan et al.
nado et al. 2014; Endo et al. 2015; Rodrigues et al. 2015; 1997; Wong and Cheung 2001a; Michalak and Chojnacka
Collins et al. 2016; Palanisamy et al. 2018). However, those 2015). For example, to examine amino acids in seaweeds,
studies were mostly focused on select species, for example, some studies targeted only free amino acids (Norziah and
using the whole genome sequence data of Chondrus crispus Ching 2000; Omar et al. 2017), but others investigated
(red algae) and Ectocarpus siliculosus (brown algae) (Cock amino acids in acid-hydrolysed materials (Qasim 1991;
et al. 2010; Collén et al. 2013; Nishitsuji et al. 2016). Some Dawczynski et al. 2007; Astorga-España et al. 2016).
other studies investigated specific molecule classes, such as However, due to the differences in sample processing
13
Planta (2019) 249:1921–1947 1923
methods, it is difficult to compare the metabolite con- Materials and methods

centrations among the studies. We previously compared
various extraction and drying methods on edible blanched Sample collection
brown algae and observed some differences in metabolite
concentrations depending on the pre-treatment (Hamid Various seaweeds were collected at two different sites in
et al. 2018). Thus, to conduct a comparative metabolome the northern part of Japan, the Shonai coastal area in the
analysis of the three seaweed groups, we needed to develop Yamagata Prefecture (Sea of Japan) and Muroran in Hok-
an optimised extraction and drying procedure that would kaido (Pacific Ocean), between February 2017 and August
generate comparable results with samples recovered from 2017. To assess the differences in metabolite profiles based
different seaweed species. on seaweed taxonomy, we collected samples of four brown
In the present study, we conducted a comprehensive algae species, Sargassum micracanthum (Togemoku; Japa-
profiling of water-soluble metabolites, such as free amino nese name), Sphaerotrichia firma (Ishimozuku), Papenfuss-
acids, organic acids, and sugars, in seaweed species of red, iella kuromo (Kuromo), and Saccharina Japonica (Kombu),
brown, and green algae. To assess the effect of the extrac- samples of five red algae species, Pyropia pseudolinearis
tion conditions on the profiling results, different extrac- (Uppurui Nori), Gelidium elegans (Makusa), Neodilsea
tion methods, including methanol–water extraction with yendoana (Akaba), Dasya sessilis (Enashi Dajia), and Bot-
or without chloroform, were compared using CE–MS and ryocladia wrightii (Taoyagisou), and samples of two green
LC–MS. This study will provide guidance on seaweed spe- algae species, Ulva australis (Ana Aosa) and Chaetomor-
cies characterisation based on metabolite profiling using pha moniligera (Tamajuzumo) (Fig. 1, Table 1). After the
only a single pre-treatment procedure. sample collection, the seaweed material was washed using
filtered seawater and the excess water was removed using
Brown
algae
Sargassum micracanthum Sphaerotrichia firma Papenfussiella kuromo Saccharina japonica (Kombu)

(Togemoku) (Ishimozuku) (Kuromo)
Red
algae
Pyropia pseudolinearis Gelidium elegans Neodilsea yendoana Dasya sessilis Botryocladia wrightii
(Uppurui Nori) (Makusa) (Akaba) (Enashi Dajia) (Taoyagisou)
Green
algae
Ulva australis Chaetomorpha moniligera

(Ana Aosa) (Tamajuzumo)
Fig. 1 The photograph of analysed samples. Each of the species in all 3 seaweed groups; brown, red, and green algae used in this study. Details
of each seaweeds species is described in Table 1. Scale bars indicate 5 cm length of the original sample
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1924 Planta (2019) 249:1921–1947
Table 1 The sample information; the species name, collection date, location, and drying percentage after freeze-drying
Scientific Japanese Phylum Class Family Collection Collection location GPS location Drying
name name date percentage
(%)
Brown algae
Sargassum Togemoku Ochrophyta Phaeophyceae Sargassaceae 16-Feb-17 Wasada beach, 38°34′48.2″N 21.68
micracan- Shonai Coastal 139°33′25.2″E
thum area, Yamagata
Sphaerotrichia Ishimo- Ochrophyta Phaeophyceae Chordariaceae 12-Jun-17 Wasada beach, 38°34′48.2″N 13.06
firma zuku Shonai Coastal 139°33′25.2″E
area, Yamagata
Papenfussiella Kuromo Ochrophyta Phaeophyceae Chordariaceae 3-Jul-17 Wasada beach, 38°34′48.2″N 15.00
kuromo Shonai Coastal 139°33′25.2″E
area, Yamagata
Saccharina Kombu Ochrophyta Phaeophyceae Laminariaceae 25-Aug-17 Denshi beach, 42°18′50.2″N 20.30
japonica Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
Red algae
Pyropia pseu- Uppurui Rhodophyta Bangiophyceae Bangiaceae 16-Feb-17 Wasada beach, 38°34′48.2″N 19.66
dolinearis Nori Shonai Coastal 139°33′25.2″E
area, Yamagata
Gelidium Makusa Rhodophyta Florideophyceae Gelidiales 13-Jul-17 Kobato port, Sho- 38°41′30.8″N 39.35
elegans nai Coastal area, 139°38′37.0″E
Yamagata
Neodilsea Akaba Rhodophyta Florideophyceae Dumontiaceae 25-Aug-17 Denshi beach, 42°18′50.2″N 27.14
yendoana Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
Dasya sessilis Enashi Rhodophyta Florideophyceae Dasyaceae 25-Aug-17 Denshi beach, 42°18′50.2″N 12.11
Dajia Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
Botryocladia Taoyagisou Rhodophyta Florideophyceae Rhodymeni- 25-Aug-17 Denshi beach, 42°18′50.2″N 5.94
wrightii aceae Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
Green algae
Ulva australis Ana Aosa Chlorophyta Ulvophyceae Ulvaceae 25-Aug-17 Denshi beach, 42°18′50.2″N 20.54
Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
Chaetomorpha Tamaju- Chlorophyta Ulvophyceae Clad- 25-Aug-17 Denshi beach, 42°18′50.2″N 10.46
moniligera zumo ophoraceae Muroran Marine 140°58′05.1″E
Station, Hok-
kaido
paper towels. The fresh seaweed specimens were weighed Sample preparation by freeze‑drying
and divided into five samples replicated (n = 5). Each sample
was placed into a polyethylene-polypropylene non-woven To eliminate the effect of sample moisture content on
fabric bag (11.0 × 10.5 cm, Dashi bag, DAISO, Hiroshima, metabolite quantification, all samples were preserved using
Japan) and quenched in liquid nitrogen before storage in a a freeze-drying method (Hamid et al. 2018). Prior to sam-
− 80 °C freezer (MDF-U482-PJ, Panasonic Corp., Kadoma, ple loading into the freeze-dryer, the freeze trap (Freeze
Osaka, Japan). Trap VA-800R, Taitec Corp., Saitama, Japan) was set to
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Planta (2019) 249:1921–1947 1925
− 70 °C and < 20 Pa. Then, completely frozen samples at 4 °C in the MX-307 centrifuge (Tomy Seiko Co. Ltd.,
(− 80 °C, > 24 h) were placed into freeze-dryer vessels. Tokyo, Japan). After the centrifugation, the aqueous of the
The temperature and pressure were maintained at < − 55 °C top layer (~ 400 µL) were transferred into ultra-filtration
and < 50 Pa using a vacuum pump (GLD-137CC ULVAC, tubes (MW 5000 kDa; HMT, Inc., Tsuruoka, Japan) and
Taitec Corp., Saitama, Japan) for 7 days. Then, once the centrifuged at 9100×g and 4 °C for 3 h. Filtrate aliquots
freeze-dryer pressure was stabilised at < 0.5 Pa for more (30 µL) were collected and transferred into LC vials for
than 24 h, the samples were removed from the vessels and sugar analysis. Sample vials were stored in the freezer at
reweighed. The dried samples were stored in a − 80 °C − 80 °C before the LC–MS analysis. An aliquot of 100 µL of
freezer until further processing. each remaining filtrate was evaporated at 4 °C in a refriger-
ated spin dryer (CentriVap Concentrator, Labconco Corp.,
Chemicals and reagents Kansas City, MO, USA). The dried samples were stored at
− 80 °C until CE–MS analysis. The dried samples were dis-
Reagents of analytical or higher grade purity were used. solved in Milli-Q water mixed with 20 µL of 200 µM trimes-
The standard reagents were purchased and prepared for ate and 3-aminopyrrolidine (migration time marker). Then,
identification and quantification of each metabolite. To nor- two aliquots of 10 µL per sample were dispensed into two
malise the quantification, the following internal standard separate CE–MS vials for cationic and anionic metabolite
solutions (IS1) were prepared as 200 µM stock solutions analysis using CE–MS.
in methanol: 2-(N-morpholino) ethanesulfonic acid (MES)
(No. 349-01,623; Dojindo Laboratories, Kumamoto, Japan) Free sugar analysis by quadrupole liquid
and D-camphor-10-sulfonic acid (CSA) (No. 037-01032; chromatography–tandem mass spectrometry (LC/
Wako Pure Chemical Industries, Ltd., Osaka, Japan); cati- MS/MS)
onic standard, l-methionine sulfone (No. 502-76641; Wako
Pure Chemical Industries, Ltd., Osaka, Japan); and sugar The analysis was performed using an API 3000 Quadrupole
standard, 13C6-glucose (No. 404624; Sigma-Aldrich Corp., LC/MS/MS system (Sciex, Framingham, MA, USA) fitted
St. Louis, MO, USA). To have a standard for CE–MS migra- with an Agilent 1100 series column oven (Agilent Tech-
tion time corrections, Milli-Q water was used to prepare nologies, Santa Clara, CA, USA), LC binary pump, and an
200-µM stock solutions of trimesate (No. 206-03641; Wako autosampler. We used a hydrophilic interaction chromatog-
Pure Chemical Industries, Ltd., Osaka, Japan) and 3-amino- raphy (HILIC) amino column (Asahipak NH2P-4E; 4.6 mm
pyrrolidine (No. 404624; Sigma-Aldrich Corp., St. Louis, inner diameter × 250 mm length; 5 µm; Showa Denko K.K.,
MO, USA). Tokyo, Japan) for sample separation. At the start, the mobile
phase was set to 80% acetonitrile and 20% Milli-Q water
Sample extractions with a flow rate of 0.8 mL min−1. The acetonitrile gradi-
ent was changed to 70%, 60%, and 80% after a run time of
Aliquots of approximately 100 mg of each of the five freeze- 23 min, 35 min, and 40 min, respectively. The duration of
dried sample replicates were placed into 13-mL disruption each sample analysis was 40 min; the column oven tem-
tubes. The sample material was mechanically disrupted in perature was set to 30 °C. Only 0.3 µL aliquots per sample
a cell disruptor (Multi beads shocker, Yasui Kikai, Osaka, were injected into the system. Using the system in turbo
Japan) without solvent using a 1 cm height × 1 cm diam- spray mode, the pressure in the nebuliser, curtain, and colli-
eter metal cylinder at 1500 rpm for 120 s. Aliquots of the sion gas chamber, the ion spray voltage, and the ion source
powdered samples were recovered and weighed (~ 10 mg temperature were set at 15 psi, 11 psi, 8 psi, − 4500 V, and
each) before being processed using two extraction methods: 500 °C, respectively. The negative-ion and MRM modes
(A) methanol–water and (B) methanol–chloroform–water were selected for MS analysis.
extraction. For the methanol–water extraction method (A),
the samples were suspended in 500 µL of 200 µM IS1 in CE–MS analysis of free amino acids, organic acids,
methanol using a micro-mixer (Micromixer E36, Taitec and charged metabolites
Corp., Saitama, Japan) for 3 min and diluted with 500 µL
Milli-Q water (Wakayama et al. 2010). For the metha- All CE–MS analyses were performed using an Agilent
nol–chloroform–water extraction method (B), the samples CE–MS system consisting of an Agilent G6220A LC/
were mixed with 500 µL of 200 µM IS1 in methanol, fol- MSD TOF, an Agilent 1100 series isocratic HPLC pump, a
lowed with 500 µL chloroform and 200 µL Milli-Q water G1603A Agilent CE–MS adapter kit, and a G1607A Agilent
(Wakayama et al. 2015). Then, all extraction samples were CE-ESI–MS sprayer kit (Agilent Technologies, Santa Clara,
vortexed for 3 min and centrifuged at 120,000×g for 10 min CA, USA).
13

1926 Planta (2019) 249:1921–1947
Cationic metabolite analysis the fragmentor, skimmer, and OCT RFV voltages were set
to 100 V, 50 V, and 500 V, respectively. MS ionisation was
Cationic metabolites, i.e., amino acids and amines, were stabilised by maintaining the drying nitrogen gas flow rate at
analysed using an uncoated fused silica capillary (50 µm 10 L min−1 and the heater temperature at 300 °C. The nebu-
inner diameter × 100 cm length) filled with 1 M formic acid liser gas pressure was set to 7 psi (48.2 kPa). Each acquired
as an electrolyte (Soga and Heiger 2000; Hirayama et al. spectrum was automatically recalibrated with two refer-
2014). The sheath liquid (methanol/water, 50% v/v) contain- ence masses, [13C isotopic ion of deprotonated acetic acid
ing 0.1 μM hexakis (2,2-difluoroethoxy)phosphazene was dimer (2CH3COOH–H)]−, m/z 120.03834, and [hexakis(2,2-
delivered at 10 μL min−1 via an HPLC pump in isocratic difluoroethoxy)phosphazene + deprotonated acetic acid
mode to stabilise the ESI sprayer ionisation. The samples (CH3COOH-H)] −, m/z 680.03554. Exact mass data were
were injected into the system at 50 hPa for 5 s (3 nL). Dur- acquired at 1.5 cycles s−1 within the range of 50–1000 m/z.
ing the CE, the voltage was kept at +30 kV, the capillary
temperature thermostat was set at 20 °C, and the sample Statistical analysis
tray temperature was cooled using a cooler system (TRL
108H Circulation type handy, Thomas, Tokyo, Japan). Time- For the sugar analysis by LC/MS/MS, the Analyst v.
of-flight mass spectrometry (TOF–MS) was conducted in 1.4.2 software was used for peak annotation and quantita-
positive ion mode. The capillary, fragmentor, skimmer, and tion. To analyse the cationic and anionic metabolites by
OCT RFV voltages were set at 4000 V, 75 V, 50 V, and CE–TOF–MS, the Agilent Mass Hunter v. B06.00 software
500 V, respectively. The MS ionisation was stabilised by was used for data collection and Masterhands v. 2.17.3.17,
in−1,
maintaining the drying nitrogen gas flow rate at 10 L m an original software from Keio University (Sugimoto et al.
the heater temperature at 300 °C, and the nebuliser gas pres- 2012), was used for peak annotation and peak area integra-
sure at 7 psi (48.2 kPa). The exact mass data of spectrum tion. The data sets were merged using in-house created mac-
were acquired at 1.5 cycles s−1 in the range of 50–1000 m/z ros in Microsoft Excel v. 2013 (Microsoft Corp, Redmond,
and automatically recalibrated using the reference masses WA, USA). Multivalent statistical analysis was performed
of the sheath liquid ([2MeOH + H2O + H]+, m/z 66.06306) on all data using JMP v. 13.2.1 (SAS Corp., Cary, NC, USA)
and protonated hexakis(2,2-difluoroethoxy)phosphazene to evaluate the metabolite profiles of different seaweeds
([M + H]+, m/z 622.02896). (Figs. 2, 3, 4, 5, 6, 7, 8, S1–S23, Tables 1, 2, 3, S1). Tukey’s
honestly significant difference (HSD) analysis, the principal
Anionic metabolite analysis component analysis (PCA), and the hierarchical clustering
analysis (HCA) were performed based on both the fresh and
Anionic metabolites, i.e. organic carboxylic acids and sugar dry weight. Both PCA and HCA were used to summarise
phosphates, were analysed using a cationic polymer-coated the statistical metabolite profile variations among species
COSMO (+) capillary (50 µm i.d. × 105 cm length) (Nacalai of each major seaweed group and between the brown, red,
Tesque, Kyoto, Japan) (Soga et al. 2009) filled with 50 mM and green algae. The PCA data were visualised using a score
ammonium acetate (pH 8.5) as electrolyte. Prior to using a and loading plot. The score plot shows the sample separation
new capillary, the pre-treatment included a 10 min flush- distribution based on metabolite concentration variations in
ing step with an electrolyte solution, followed with 50 mM the data set, whereas the loading plot provides the informa-
acetic acid (pH 3.4), and again with the electrolyte solution on the spectral position of metabolites corresponding to
tion. The capillary temperature thermostat was set at 20 °C the score plot. HCA conducted by two-way clustering using
and the sample tray was cooled using cooler system. The the Ward’s method (Murtagh and Legendre 2014) presents
sheath liquid of 5 mM ammonium acetate in methanol/water the metabolite characteristics in terms of hierarchical or tree-
(50% v/v) containing 0.1 μM hexakis(2,2-difluoroethoxy) like structure and heatmap. The heatmap shows the relative
phosphazene was delivered to the electrospray interface at concentrations of each metabolite. It indicates tendencies in
10 μL min−1 with an Agilent 1100 series isocratic pump each species for all seaweed groups separated according to
fitted with a 1:100 splitter. The sheath liquid circulated the two extraction methods.
around the outside of the electrospray and CE capillary to
provide a stable electrical connection between the capillary
tip and the grounded electrospray needle. Before starting Result
each injection, the capillary was flushed with 50 mM acetic
acid (pH 3.4) for 2 min followed with an electrolyte solu- Dry weight
tion for 5 min. The samples were injected at 50 hPa for 30 s
(30 nL). After the injection, CE voltage was maintained at The dry weight percentage was different for each algae spe-
− 30 kV. The MS capillary voltage was set to − 3500 V, and cies (Table 1). The highest dry weight percentage of 39%
13
Planta (2019) 249:1921–1947 1927
Fig. 2 Principle component analysis (PCA) score plots of dry weight- ▸ a

based metabolites concentrations in all data set of this research. a
PC1–PC2 plot and b PC2–PC3 plots. Four brown algae are indicated
by blue colour symbols; Sargassum micracanthum (Togemoku), Sac-
charina japonica (Kombu), Sphaerotrichia firma (Ishimozuku), and
Papenfussiella kuromo (Kuromo), five red algae are indicated by red U. australis
colour symbols; Pyropia pseudolinearis (Uppurui Nori), Gelidium (Ana Aosa)
elegans (Makusa), Neodilsea yendoana (Akaba), Dasya sessilis
(Enashi Dajia), and Botryocladia wrightii (Taoyagisou), and two
PC2 (13.2 %)
green algae are indicated by green colour symbols; Ulva australis
(Ana Aosa) and Chaetomorpha moniligera (Tamajuzumo). Filled
and unfilled colour indicates the methanol–water extractions without
chloroform and with chloroform, respectively. The details of each
P. pseudolinearis
colour and markers were listed on the figure. The percentage of each
(Uppurui Nori)
axis indicated separation variance. Precise PCA (PC1–PC5) including
score and loading plots are shown in Fig. S1
was observed in G. elegans, whereas the lowest percentage

of approximately 5.9% was observed in B. wrightii. Both
species were red algae. The dry weight percentage range was PC1 (22.0 %)
larger in red algae (5.9–39%) than in green (13–21%) and b
brown algae (10–20%). The average dry weight percentage
in brown, red, and green algae were 20.84%, 17.51%, and
15.50%, respectively.
Overview of the metabolite profiles D. sessilis

(Enashi Dajia) U. australis
(Ana Aosa)
PC3 (10.5 %)
To evaluate general tendencies of metabolites in seaweeds

and to assess species-specific differences between their pro-
files as well as the effect of the extraction method on the
results, the entire data set was subjected to a multivalent
analysis (11 species, 2 extraction methods) (Figs. 2, 3, 4, 5,
6, 7, 8, S1–S23). The principle component analyses (PCA)
indicated that the seaweed species was the dominating fac-
tor for metabolite concentrations (Fig. 2a, S1: PC1–PC5).
The separation variance of PC1 (22%) and PC2 (13.2%)
were associated with the metabolite characteristics of P.
pseudolinearis (red algae) and U. australis (green algae), PC2 (13.2 %)
respectively (Fig. 2a: positive plot PC1–PC2, Fig. S1). A (A) (B)
MeOH-Water MeOH-CHCl3-
clear separation variance between the brown, red, and green Extraction Water Extraction
algae was identified in the PC2 and PC3 plots (Fig. 2b, S1: Method Method
PC2–PC3). Furthermore, the plots from PC1 to PC5 indi- Sargassum micracanthum (Togemoku)
Brown
algae
cated that the separation patterns were mostly species-spe- Saccharina japonica (Kombu)
cific and not linked to the extraction methods (Fig. 2, S1). Sphaerotrichia firma (Ishimozuku)
An HCA was performed on 110 individual samples Papenfussiella kuromo (Kuromo)

Pyropia pseudolinearis (Uppurui Nori)
derived from 11 seaweed species to simultaneously deter-
Gelidium elegans (Makusa)
mine the concentrations of 175 metabolites (Fig. 3, S8–S12).
algae
Red
Neodilsea yendoana (Akaba)

Importantly, the main factor in hierarchical clustering of Dasya sessilis (Enashi Dajia)
metabolites was the species (Fig. 3, see colour boxes). Botryo wrightii (Taoyagisou)
Interestingly, the species did not cluster into the three sea-
Green
algae
Ulva australis (Ana Aosa)

weed groups of algae; instead, they were clustered in several Chaetomorpha moniligera (Tamajuzumo)
clades (Fig. 3, S2) that depended on the taxonomic class or

family. For example, S. firma and P. kuromo of the brown
algae family Chordariaceae were clustered closely by metab-
olite profiling. Furthermore, if comparing metabolite profiles
13

1928 Planta (2019) 249:1921–1947
Ulva Dasya Saccharina Botryocladia Neodilsea Geldium Papenfussiella Sphaerotrichia Chaetomorpha Sargassum Pyropia
australis sessilis japonica wrightii yendoana elegans kuromo firma moniligera micracanthum pseudolinearis
(Ana Aosa) (EnashiDajia) (Kombu) (Taoyagisou) (Akaba) (Makusa) (Kuromo) (Ishimozuku) (Tamajuzumo) (Togemoku) (UppuruiNori)
Most of all the

algae
S. firma (Ishimozuku)
&
P. kuromo (Kuromo)
N. yendoana
(Akaba)
S. micracanthum
(Togemoku)
S. japonica
(Kombu)
C. moniligera
(Tamajuzumo)
B. wrightii
(Taoyagisou)
G. elegans
(Makusa)
D. sessilis
(Enashi Dajia)
U. australis
(Ana Aosa)
P. pseudolinearis
(Uppurui Nori)
P. pseudolinearis
(Uppurui Nori)
&
other algae
Low concentration High concentration A: MeOH-Water extraction method

B: MeOH-CHCl3-Water extraction method
13
Planta (2019) 249:1921–1947 1929
◂Fig. 3 Hierarchical clustering analysis (HCA) and heat map of methanol–chloroform–water extraction method, nine out
dry weight-based metabolite concentrations in all data set of this of 175 metabolites, i.e. nicotine, taurocyamine, Glu–Glu,
research. All quantified concentration were normalised to Z-scores
for each metabolite. HCA was performed by Ward’s method, and
orotate, 2, 3-DPG, galacturonate 1-phosphate, prostaglan-
two-way clusterisation was done on both metabolites concentration din F2alpha, IDP, and GDP, were not detected (Fig. S12).
and each algae species after 2 extraction methods. Alphabet A indi- The efficiency of the two extraction methods was com-
cates the methanol–water extraction method; alphabet B indicates the pared for several major metabolites, such as amino acids,
methanol–chloroform–water extraction method. In the heat map, blue
and red colours indicate low and high abundance of metabolites con-
TCA and glycolysis organic acids, and sugars, by plotting
centrations, respectively, as shown in the bottom of colour bar. Blue, each of the extraction methods on x–y axis plots (Fig. S13).
red, and green fonts indicate the seaweed species groups, brown, red, The graph shows that most metabolite concentrations were
and green algae, respectively. Colour framed boxes indicated the not affected by either extraction method. However, some
significant high concentration of each seaweed species; 2,3-DPG:
2,3-diphosphoglycerate, 2AB: 2-aminobutyrate, 3′AMP: 3′-ade-
metabolites, such as Ala and Glu, had 2–3 times higher
nylic acid, 3PG: 3-phosphoglycerate, ADMA: asymmetric dimethyl concentrations in samples processed by methanol–water
arginine, ADP: adenosine diphosphate, AICAR: 5-aminoimidazole- extraction as compared to the concentrations in the other
4-carboxamide ribonucleotide, AMP: adenosine monophosphate, samples (Fig. S13a), and peaks of 2, 3-DPG (Fig. S13b)
CMP: cytidine monophosphate, DOPA: 3,4-dihydroxyphenylalanine,
F6P: fructose-6-phosphate, G1P: glucose-1-phosphate, G6P: glucose-
were only detectable in methanol–water extraction sam-
6-phosphate, GABA: gamma-aminobutyric acid, GDP: guanosine ples. The PCA scores and loading plots of the individual
5′-phosphate, IDP: Inosine 5-diphosphate, IMP: inosine monophos- extraction methods patterns did not differ significantly.
phate, S7P: sedoheptulose 7-phosphate, SAH: S-adenosyl homocyst- Thus, although the extraction methods were different, the
eine, SAM+: S-adenosylmethionine, SDMA: symmetric dimethylar-
ginine, UDP: uridine diphosphate, UMP: uridine-monophosphate
metabolite profiles were mainly determined by the sea-
weed species (Fig. 5, S6–S7).
within each species, the two extraction methods were clus-

tered together (Fig. 3, S2). Metabolite characteristics of three groups
We also calculated the fresh weight metabolite concentra- of seaweeds
tions for evaluation by PCA and HCA (Fig. S14–S23). Simi-
lar to dry weight metabolite concentration data, the PCA The PCA of the entire data set indicated a clear separation
separation and HCA cluster patterns depended mainly on the variance between the three seaweed groups in the PC2 and
species and not on the extraction method. The fresh weight PC3 plots (Fig. 2b, S1). The PC2 positive plot was domi-
metabolite concentrations in the samples of some species, nated by the high concentrations of certain metabolites
such as B. wrightii, were relatively low for performing an identified in green algae, such as sucrose (Fig S1: PC2).
HCA (Fig. S20, S22), and the clustering tendencies were The positive plot of PC3 was dominated by high metabo-
difficult to interpret. Thus, we used only dry weight metabolite concentrations in red algae, whereas the negative PC3
lite concentrations for further evaluation of the metabolite plot was dominated by the brown algae. Significantly high
profiles of those samples. concentrations of the major metabolites proline betaine,
panose, p-hydroxyphenylacetate, and DOPA were detected
Effect of extraction methods in certain red algae species, such as D. sessilis (Fig S1:
positive plot PC3), whereas mannitol and arabitol were
Two extraction methods, the methanol–water and the dominant in all brown algae (Fig S1: negative plot PC3).
methanol–chloroform–water extraction methods, were
compared. An extraction method separation variance could Characteristic metabolites in brown algae
not be clearly identified using all metabolite concentration
data in PCA (Fig. 2, S1). Instead, by restricting the data The concentrations of mannitol (at least 3 times) and cysta-
to seaweed groups, PCA separation variances were iden- thionine (at least 5 times) were significantly higher in brown
tified by performing the PC1–PC5 multivalent analysis algae than in red or green algae (Tables 2, 3, S1). Some of
(Fig. 4, S3–S5). In brown algae, a separation difference the metabolites were only detected in certain species, e.g.
that aligned with the extraction methods was found in PC4 arabinose in S. micracanthum and S. japonica or 5-methyl-
with a 5.46% variance (Fig. S3: PC4, see colour rectan- cytosine in S. japonica. Significantly high concentrations
gular), in red algae, it was found in PC5 with a 3.36% of tyramine, succinate, urate, and AMP were detected in S.
variance (Fig. S4: PC5, see colour eclipse), and in green micracanthum samples and of carnitine in S. firma samples.
algae, the separation variance was 8.1% in PC2 (Fig. S5: Twenty-one of 175 metabolites, including sorbitol, malto-
PC2). Using the methanol–water extraction method, only triose, and panose, were not detected in any brown algae
one out of 175 metabolites, i.e. 1, 3-diaminopropane, species (Figs. 3, S8, Table S1).
was not detected (Fig. S11, Table S1), whereas using the
13

1930 Planta (2019) 249:1921–1947
Fig. 4 PCA score plots of dry weight-based metabolites concentra- ▸

a
tions in three seaweed groups. a 4 species of brown algae; S. micra- S. firma S. micracanthum
canthum (Togemoku), S. firma (Ishimozuku), P. kuromo (Kuromo), (Ishimozuku) (Togemoku)
and S. japonica (Kombu), b 5 species of red algae; P. pseudolinearis
(Uppurui Nori), G. elegans (Makusa), N. yendoana (Akaba), D.
sessilis (Enashi Dajia), and B. wrightii (Taoyagisou), and c 2 species
of green algae; U. australis (Ana Aosa) and C. moniligera (Tama-
juzumo). Filled and unfilled colour indicates the methanol–water
Brown algae
PC2 (26.8 %)
extractions without chloroform and with chloroform, respectively.
Different shapes indicate different species in the different seaweed
type (See Fig. 1 legend for species details). The horizontal axis value P. kuromo
indicates PC1 separation variance and the vertical axis value indicates (Kuromo)
PC2 separation variance. Precise PCA (PC1 to PC5) including score
and loading plots are shown in Fig. S2–S4
S. japonica
(Kombu)
The PCA separation variances and HCA clusters demon-
strated that the metabolites profiles of the four brown algae
species display significant differences (Fig. 4a, S3). The
PC1 (29.8 %)
PC1 plot illustrates the separation between the Chordari-
aceae family (S. firma and P. kuromo) (Fig. 4a: negative plot b
PC1, Fig. S3) and the Sargassaceae (S. micracanthum) and
Laminariaceae (S. japonica) family (Fig. 4a: positive plot D. sessilis
(Enashi Dajia)
PC1, Fig. S3) with a separation variance of 29.8%. S. firma
and P. kuromo from the same family were clustered because
they shared some metabolites with different concentrations, B. wrightii P.pseudolinearis
such as rhamnose, betaine, Trp, Phe, and Thr (Fig. 4a, S3).
PC2 (17.5 %)
(Taoyagisou)
Red algae
(Uppurui Nori)
The positive PC1 plot separated S. micracanthum and S.
japonica from the other two species.
N. yendoana G. elegans
Characteristic metabolites in red algae (Akaba) (Makusa)
In red algae, each species displayed a characteristic metab-

olite profile. The concentrations of some metabolites were
significantly higher in certain red algae species than in
brown or green algae, e.g. isoamylamine (except P. pseu-
dolinearis), citrulline (except G. elegans), and isethion- PC1 (36.6 %)
ate (except D. sessilis) (Tables 2, 3, S1). Among the 175
profiled metabolites, ten were not detected in red algae,
c
Water extraction
i.e. 5-methylcytosine, nicotine, Phe–Phe, orotate, urate,

MeOH-CHCl3-
method
galacturonate 1-phosphate, ribose, arabinose, xylose, and

rhamnose (Fig. 3, S9). Some metabolites were detected
only in certain species. For example, sorbitol, GDP, UDP,
Green algae
and IDP was detected only in P. pseudolinearis, glycolate

PC2 (8.1 %)
and UDP only in N. yendoana, panose and p-hydroxy- C. moniligera U. australis

phenylacetate only in D. sessilis, and 3′-AMP only in B. (Tamajuzumo) (Ana aosa)
wrightii (Fig. 3, S9, Table S1).
The PCA including the five red algae species, P. pseu-
MeOH-Water
dolinearis, G. elegans, N. yendoana, D. sessilis, and B.

extraction
method
wrightii, showed that the PC1-4 plots mainly illustrate

species-specific variations (Fig. 4b, S4), whereas the PC5
plot indicates the effect of the extraction methods (Fig.
S4: PC5). The clade of P. pseudolinearis was clustered
distantly (Figs. 2, 3; positive plot PC1; Fig. 4b, S4) from PC1 (64.6 %)
the other red algae species with a separation variance of
36.6% (Fig. 2: negative plot PC1; Fig. 4b, S4) in alignment
13
Planta (2019) 249:1921–1947 1931
a MeOH-Water extraction methods P. pseudolinearis by high concentrations of amino acids

(Trp, Asn, Arg, Gly–Gly, and Gly-Leu), CMP, and gamma-
U. australis butyrobetaine (Fig. S4: positive plot PC1). These metabo-
(Ana Aosa) lites were significantly concentrated in P. pseudolinearis
but not in the other red algae species. The PC2:PC4 plot
S. firma provides a clear separation of each red algae species (Fig.
C. moniligera P. kuromo (Ishimozuku)
(Tamajuzumo) (Kuromo) S4a: see circle in PC2:PC4).
PC2 (14.0 %)
S. micracanthum
B. wrightii
(Togemoku) Characteristic metabolites in green algae
(Taoyagisou)
N. yendoana The concentration of thymine (at least 2 times), fructose (at

(Akaba) P. pseudolinearis least 15 times), glucose (at least 2 times), sucrose (at least
(Uppurui Nori)
S. japonica 4 times), and inositol (at least 1.3 times) was significantly
G. elegans
(Kombu) D. sessilis (Makusa) higher in green algae than in brown or green algae (Tables 2,
(Enashi Dajia) 3, S1). Among the profiled metabolites, orotate and Phe–Phe
were specific for U. australis. High concentrations of several
metabolites, pipecolate, Met, guanine, uridine, threonate,
and cis-aconitate, were detected at high concentrations in U.
PC1 (23.4 %) australis but only one, betaine, in C. moniligera. There were
42 out of 175 profiled metabolites that were not detected in
b MeOH-CHCl3-Water extraction methods green algae (Fig. 3, S10, Table S2).
The PCA indicated a clear separation variance between
U. australis the two green algae species in relation to the two extraction
(Ana Aosa) methods (Fig. 4c, S5). The PC1 plot shows the separation
S. firma
(Ishimozuku)
of U. pertusa and C. monilligera with a separation variance
C. moniligera of 64.6% (Fig. 4c: PC1; S5). The positive and negative PC2
(Tamajuzumo) S. micracanthum plot shows the separation by extraction method using metha-
PC2 (13.9 %)
P. kuromo (Togemoku) nol–water with and without chloroform, respectively, at a

(Kuromo) separation variance of 8.1% (Fig. 4c: PC2; Fig. S5).
B. wrightii N. yendoana
(Taoyagisou) (Akaba)
Amino acid and sugar profiling in various seaweeds
P. pseudolinearis
S. japonica
(Kombu) G. elegans (Uppurui Nori)
(Makusa) Most of the amino acids were detected at different concen-
D. sessilis trations in all seaweeds with the exception of Cys (Fig. 6,
(Enashi Dajia) Tables 2, 3). However, cystine, which is a dimer of Cys, was
detected in most seaweeds except in S. micracanthum, S.
japonica, D. sessilis, and C. moniligera. There were other
amino acids that could not be detected in some algae, Met
PC1 (22.9 %) (P. kuromo, N. yendoana, and D. sessilis), Asn (B. wrightii),
Lys (D. sessilis), and Trp (N. yendoana, D. sessilis and B.
Fig. 5 PCA score plots of dry weight-based metabolites concentra- wrightii). Moreover, in all seaweeds samples, the Asp con-
tions in all data set after two extraction methods. a Methanol–water centration was higher than that of Asn (Tables 2, 3). High
extractions methods and b methanol–water with chloroform methods. concentrations of Ala were detected in all species, and Pro
Different shapes indicate different species of in the different seaweed
was detected at the highest concentration in D. sessilis. In
types (See Fig. 2 legends for species details). The horizontal axis
value indicates PC1 separation variance, and the vertical axis value green and red algae except in P. pseudolinearis, the concen-
indicates PC2 separation variance. Precise PCA (PC1 to PC5) includ- tration of Glu was higher than that of Gln. In brown algae,
ing score and loading plots are shown in Fig. S5–S6 except in S. japonica, Gln concentration was higher than
Glu. The variations in the amino acid concentrations were
mainly related to the individual species in this algae group
with a different taxonomic classification. P. pseudolinearis that was not significantly affected by the different extraction
belongs to the Bangiophyaceae, whereas the other red algae methods (Fig. 6, Tables 2, 3).
species are classified as Florideophyceae (Fig. 2: positive To determine the characteristic properties of major
plot PC1; Figs. 3, 4b, S4). Loading plot of PC1 indicates metabolite groups (i.e. amino acids, TCA and glycolysis
13

1932 Planta (2019) 249:1921–1947
Fig. 6 HCA and heat map of

dry weight based on amino
acids concentrations in all spe-
Pyropia pseudolinearis
cies and extraction methods. All Pyropia pseudolinearis
legends and abbreviations are Pyropia pseudolinearis
the same as in Fig. 3 Pyropia pseudolinearis
Sphaerotrichia firma
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Sargassum micracanthum (Togemoku) A
Sargassum micracanthum
Sargassum fulvellum (Togemoku) A
Sargassum fulvellum
micracanthum
Sargassum fulvellum (Togemoku) A
fulvellum
Sargassum fulvellum
Sargassum micracanthum (Togemoku) B
Sargassum fulvellum (Togemoku) B
fulvellum
fulvellum (Togemoku) B
Sargassum fulvellum (Togemoku) B
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Papentussiella kuromo
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Chaetomorpha monoligera
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
A: MeOH-Water extraction method
B: MeOH-CHCl 3-Water extraction method
Low concentration High concentration
13
Planta (2019) 249:1921–1947 1933
Fig. 7 HCA and heat map

of dry weight based on TCA
and glycolysis organic acids
concentrations in all species and
extraction methods. All legends
and abbreviations are the same Pyropia pseudolinearis
as in Fig. 3 Pyropia pseudolinearis
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
B: MeOH-CHCl 3-Water extraction method
13

1934 Planta (2019) 249:1921–1947
Fig. 8 HCA and heat map of

dry weight based on sugars
concentrations in all species and
extraction methods. All legends
and abbreviations are the same Pyropia pseudolinearis
as in Fig. 3 Pyropia pseudolinearis
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Gelidium elegans
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Botryocladia wrighi
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Neodilsea yendoana
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Botryocladia wrighi
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Dasya sessilis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Ulva australis
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
Saccharina japonica
B: MeOH-CHCl3-Water extraction method
13
Table 2 Amino acids, sugars, and major organic acids of 11 seaweeds after methanol–water extraction method
Methanol–water extraction methods
Metabolites Brown algae Red algae
Sargassum micracanthum Sphaerotrichia firma (Ishi- Papenfussiella kuromo (Kuromo) Saccharina japonica (Kombu) Pyropia pseudolinearis
(Togemoku) mozuku) (Uppurui Nori)
Amino acids
Planta (2019) 249:1921–1947
Gly 736.39 ± 36.75ef 546.45 ± 51.57fgh 285.24 ± 81.90ghi 185.57 ± 24.37hi 4278.75 ± 481.72a

Ala 14777.00 ± 746.18e 29971.00 ± 1754.79c 4247.00 ± 757.72g 2446.00 ± 303.52g 100818.00 ± 9381.19a
Ser 930.06 ± 27.61cdef 1101.54 ± 122.06cdef 318.94 ± 73.07f 616.10 ± 86.36def 6949.48 ± 1493.02a
Pro 1275.90 ± 187.83cdef 1686.30 ± 451.19c 384.10 ± 75.06defg 882.10 ± 109.63cdefg 1092.70 ± 153.36cdefg
Val 2807.73 ± 227.46a 1651.99 ± 178.00b 595.42 ± 190.45defg 199.01 ± 39.81fg 3009.32 ± 505.94a
Thr 1050.34 ± 30.37def 2379.17 ± 258.36b 1702.41 ± 329.50c 638.24 ± 61.62efghi 3044.47 ± 483.78a
Ile 663.02 ± 55.10bc 723.81 ± 51.56b 417.14 ± 119.55ef 54.22 ± 14.14g 1309.73 ± 250.03a
Leu 1180.95 ± 91.64c 1035.06 ± 112.64c 553.46 ± 176.12de 104.52 ± 26.74f 1926.48 ± 301.44ab
Asn 9982.58 ± 389.77a 2089.68 ± 391.19d 504.57 ± 194.98ef 269.73 ± 41.80ef 9712.72 ± 1185.88a
Asp 18619.30 ± 713.38a 2993.90 ± 755.02gh 1706.40 ± 392.09hijk 9043.70 ± 615.23d 11068.90 ± 1301.71c
Gln 21320.80 ± 735.62c 27317.50 ± 3873.72b 4699.10 ± 1335.76fg 793.40 ± 209.32ghi 43085.80 ± 4055.55a
Glu 5930.20 ± 245.67de 3756.70 ± 947.02ef 3726.20 ± 800.70ef 6154.90 ± 1244.38de 7901.90 ± 829.56d
Lys 306.15 ± 27.78fgh 995.82 ± 204.64b 609.26 ± 244.88de 91.82 ± 11.75ghi 791.82 ± 162.33bcd
Met 53.76 ± 25.10def 32.08 ± 3.89ef NDf 7.31 ± 3.35f 166.06 ± 29.54c
His 150.29 ± 13.38ef 291.01 ± 30.71c 52.09 ± 15.71ij 56.13 ± 6.44hij 481.48 ± 87.78a
Phe 345.82 ± 20.99hij 1301.04 ± 152.78bc 759.16 ± 185.89efg 269.93 ± 28.56ij 579.33 ± 76.25fghi
Arg 350.43 ± 39.57efg 848.58 ± 205.81cd 129.21 ± 35.14hi 89.60 ± 18.81i 1330.19 ± 217.92a
Tyr 300.36 ± 18.26c 784.52 ± 95.35a 300.26 ± 67.24c 114.41 ± 16.89efg 862.76 ± 104.08a
Trp 96.69 ± 7.78e 414.00 ± 52.04a 285.57 ± 39.84b 25.69 ± 5.46g 203.11 ± 34.88cd
Cystine NDc 3.52 ± 4.81c 0.48 ± 1.08c NDc 113.83 ± 24.06a
Sugars
Ribose 3993.82 ± 565.94a 1745.22 ± 726.43e 3143.74 ± 413.92bc 1437.75 ± 356.81ef NDg
Arabinose 1495.59 ± 211.76b NDd NDd 1224.12 ± 326.15c NDd
Xylose NDc NDc NDc 118.51 ± 29.40a NDc
Xylitol 292.67 ± 22.13c 213.63 ± 43.52c 887.67 ± 154.73a 608.08 ± 73.85b 71.36 ± 16.84d
Arabitol 202.65 ± 10.97ef 566.35 ± 101.81c 431.39 ± 92.28cd 1203.48 ± 142.29a 395.43 ± 55.68d
Rhamnose NDf 66.55 ± 6.67b 56.67 ± 24.29bc NDf NDf
13
1935

Table 2 (continued)
1936
13
Sargassum micracanthum Sphaerotrichia firma (Ishi- Papenfussiella kuromo (Kuromo) Saccharina japonica (Kombu) Pyropia pseudolinearis
(Togemoku) mozuku) (Uppurui Nori)
Fructose 316.00 ± 29.64d 73.00 ± 11.67d 158.00 ± 21.51d 249.00 ± 23.13d 150.00 ± 18.10d

Glucose 1040.50 ± 24.31cde 446.80 ± 146.29cde 1122.10 ± 229.07cde 146.00 ± 75.51e 2594.40 ± 357.92cde
Sorbitol NDb NDb NDb NDb 198.32 ± 15.08a
Mannitol 275498.00 ± 11393.90b 27678.00 ± 8818.40g 103188.00 ± 7287.40e 347984.00 ± 14768.70a 121.00 ± 22.80h
Inositol 172.20 ± 17.07fgh 352.27 ± 93.37cd 398.09 ± 43.56c 60.98 ± 9.45ij 230.39 ± 23.61ef
Sucrose 704.50 ± 124.81e 10.30 ± 5.51e 97.00 ± 34.98e 162.00 ± 30.53e 6.90 ± 5.70e
Maltotriose NDe NDe NDe NDe 715.37 ± 127.23a
Panose NDc NDc NDc NDc NDc
Major organic acids
Lactate 1328.97 ± 316.25c 1732.88 ± 253.18c 1972.92 ± 486.72c 1809.81 ± 469.20c 1998.67 ± 271.23c
Fumarate 183.45 ± 43.47bcd 67.03 ± 64.34de 275.45 ± 48.85b 108.32 ± 76.11cde 661.90 ± 240.38a
Succinate 1922.13 ± 226.50a 393.71 ± 60.66fghi 610.77 ± 152.01defg 425.78 ± 167.32fghi 775.83 ± 285.55cde
Malate 655.36 ± 120.39c 220.24 ± 96.69c 348.67 ± 54.74c 775.45 ± 136.28c 6049.82 ± 1885.67a
trans-Aconitate 302.96 ± 145.85b 51.15 ± 71.85c 699.13 ± 313.14a NDc NDc
cis-Aconitate 145.43 ± 29.14c 1541.24 ± 309.22b 464.26 ± 89.31c NDc 6.95 ± 15.54c
3PG 132.09 ± 59.72bc 65.03 ± 53.02cd 120.13 ± 24.95bcd 108.29 ± 26.07bcd 538.70 ± 208.58a
Isocitrate 258.48 ± 23.56b 90.67 ± 85.87cdef 145.59 ± 55.04cd NDf NDf
Citrate 1341.26 ± 128.58fg 755.75 ± 409.64fg 2540.63 ± 839.19ef 5300.88 ± 794.68bcd 8945.23 ± 2453.75a
G1P 112.90 ± 22.50cd 11.28 ± 25.22e 15.94 ± 35.65e 32.42 ± 49.87de 376.80 ± 128.56a
F6P 259.63 ± 31.02c 9.00 ± 20.13g 45.73 ± 63.38fg 138.86 ± 10.60ef 789.77 ± 113.92a
G6P 914.90 ± 84.86c 131.10 ± 61.59d 304.84 ± 78.51cd 474.64 ± 106.99cd 5585.82 ± 1198.53a
2,3-DPG NDb NDb 15.50 ± 34.67b NDb 189.12 ± 133.23a
6-Phosphogluconate NDe NDe NDe 157.36 ± 36.81c 850.28 ± 129.78a
S7P 649.95 ± 102.69cd NDg 184.90 ± 290.48fg 902.94 ± 298.72bc 1389.00 ± 158.53a
UDP-glucose 372.53 ± 19.64a 132.18 ± 52.29fg 162.46 ± 40.58efg 359.24 ± 58.86ab 193.36 ± 100.97def
Planta (2019) 249:1921–1947
Red algae Green algae
Metabolites Gelidium elegans (Makusa) Neodilsea yendoana Dasya sessilis (Enashi Dajia) Botryocladia wrightii Ulva australis (Ana Aosa) Chaetomorpha moniligera
(Akaba) (Taoyagisou) (Tamajuzumo)
Amino acids
Planta (2019) 249:1921–1947
Gly 1886.07 ± 263.85d 476.36 ± 204.87fghi 1029.74 ± 81.06e 674.16 ± 50.38ef 3339.20 ± 205.82b 412.57 ± 22.29fghi

Ala 2771.00 ± 656.15g 2423.00 ± 535.45g 16216.00 ± 1797.01de 1891.00 ± 134.09g 3176.00 ± 230.91g 1146.00 ± 61.30g
Ser 1835.74 ± 533.41c 336.12 ± 178.09f 277.26 ± 23.16f 536.97 ± 43.23ef 1520.16 ± 139.94cd 630.42 ± 30.92def
Pro 1354.30 ± 410.95cde 125.20 ± 20.11g 28052.50 ± 1644.27a 267.80 ± 65.87fg 401.60 ± 138.05defg 1488.30 ± 92.38c
Val 318.46 ± 176.83efg 320.11 ± 107.77efg 1631.52 ± 185.45b 1065.13 ± 208.76c 825.68 ± 121.55cd 146.20 ± 9.54g
Thr 1169.27 ± 317.08d 431.62 ± 82.55ghi 854.71 ± 76.99defgh 420.40 ± 35.50ghi 1113.48 ± 137.32de 208.83 ± 14.19i
Ile 64.05 ± 53.08g 127.59 ± 63.27g 590.89 ± 43.44bcde 34.55 ± 17.87g 459.00 ± 71.71def 57.91 ± 5.67g
Leu 29.90 ± 24.86f 71.19 ± 33.72f 890.72 ± 93.12cd 15.97 ± 5.43f 2130.03 ± 374.04a 223.08 ± 8.14ef
Asn 25.35 ± 8.85f 326.32 ± 35.86ef 917.51 ± 100.64e NDf 349.14 ± 55.10ef 126.25 ± 24.25f
Asp 8594.30 ± 926.78de 2184.50 ± 254.56ghi 1791.00 ± 107.65hij 1646.00 ± 167.74ijk 602.60 ± 47.73jk 8874.60 ± 552.88d
Gln 11803.30 ± 4156.67e 245.00 ± 21.92hi 1507.00 ± 70.99ghi 578.70 ± 79.32hi 739.50 ± 126.76ghi 83.10 ± 9.73i
Glu 38041.60 ± 6103.94a 1168.00 ± 849.53f 17649.20 ± 2151.44b 5713.00 ± 431.26de 1558.80 ± 110.65f 1151.80 ± 211.16f
Lys 602.07 ± 203.88de 64.42 ± 17.90hi NDi 74.43 ± 8.70ghi 867.49 ± 98.13bc 290.86 ± 15.56fgh
Met 103.84 ± 50.71cde NDf NDf 9.01 ± 2.20f 898.67 ± 114.92a 66.29 ± 7.58def
His 97.43 ± 20.22fghi 37.29 ± 5.39ij 126.58 ± 16.39efg 29.11 ± 2.10j 245.40 ± 23.20cd 32.00 ± 1.30j
Phe 1469.49 ± 282.03b 1360.87 ± 121.87bc 2816.21 ± 336.65a 413.80 ± 67.59ghij 930.56 ± 132.52def 115.15 ± 6.14j
Arg 365.55 ± 138.08ef 157.33 ± 49.42ghi 199.66 ± 22.88fghi 139.02 ± 18.88hi 1009.54 ± 102.30bc 370.67 ± 22.66ef
Tyr 169.85 ± 27.08cdef 1.61 ± 1.19g 237.00 ± 104.57cde 4.16 ± 0.71g 562.59 ± 87.93b 47.79 ± 5.12fg
Trp 29.62 ± 12.04g NDg NDg NDg 245.08 ± 33.48bc 39.51 ± 3.57fg
Cystine 16.11 ± 4.53c 5.20 ± 2.06c NDc 9.23 ± 1.30c 8.38 ± 1.86c ND
Sugars
Ribose NDg NDg NDg NDg 2924.37 ± 367.30bc 2063.63 ± 363.73de
Arabinose NDd NDd NDd NDd NDd NDd
Xylose NDc NDc NDc NDc NDc NDc
Xylitol 10.77 ± 1.85d 70.20 ± 14.13d 317.85 ± 33.80c 35.09 ± 10.46d 11.18 ± 3.44d 9.04 ± 2.89d
Arabitol 21.52 ± 1.19h 106.83 ± 32.95fgh 37.59 ± 9.35gh 156.40 ± 24.47fgh 53.26 ± 6.04gh 15.67 ± 2.12h
Rhamnose NDf NDf NDf NDf 33.78 ± 10.08de NDf
Fructose 552.00 ± 1143.43d 60.00 ± 14.85d 347.00 ± 125.71d 58.00 ± 16.01d 11336.00 ± 1830.66c 106271.00 ± 3962.32a
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1938
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Red algae Green algae
Metabolites Gelidium elegans (Makusa) Neodilsea yendoana Dasya sessilis (Enashi Dajia) Botryocladia wrightii Ulva australis (Ana Aosa) Chaetomorpha moniligera
(Akaba) (Taoyagisou) (Tamajuzumo)
Glucose 790.70 ± 1257.70cde 112.00 ± 32.31e 3977.00 ± 282.88c 120.90 ± 43.33e 11799.20 ± 1980.38b 43512.50 ± 3144.48a

Sorbitol NDb NDb NDb NDb NDb NDb
Mannitol 1520.00 ± 652.30h 7797.00 ± 1699.20h 2948.00 ± 647.50h 287.00 ± 37.70h 1533.00 ± 212.70h 655.00 ± 80.70h
Inositol 286.00 ± 16.90de 110.92 ± 23.99ghij 49.86 ± 12.48j 61.38 ± 10.77ij 841.79 ± 56.78a 634.77 ± 51.54b
Sucrose 81.40 ± 33.72e 13.70 ± 4.65e 69.30 ± 24.27e 53.60 ± 21.62e 11705.00 ± 1306.03a 4020.10 ± 700.63c
Maltotriose NDe NDe 377.58 ± 53.97bc NDe 269.58 ± 106.65cd 436.32 ± 48.24b
Panose NDc NDc 44.55 ± 15.13a NDc NDc NDc
Major organic acids
Lactate 1376.60 ± 320.81c 1784.51 ± 365.54c 9658.82 ± 1932.49a 4713.29 ± 901.28b 1502.59 ± 319.61c 1827.52 ± 372.93c
Fumarate NDe NDe 83.84 ± 83.43de NDe NDe 84.74 ± 80.92de
Succinate 150.24 ± 35.51i 876.46 ± 162.59cd 1069.33 ± 163.62c 313.54 ± 57.10ghi 425.97 ± 30.12fghi 604.87 ± 64.86defg
Malate 463.47 ± 109.54c 353.14 ± 60.88c 744.03 ± 101.39c 214.28 ± 28.97c 643.90 ± 83.62c 685.99 ± 30.34c
Trans-Acon- ND NDc 177.29 ± 89.75bc NDc 649.07 ± 74.10a NDc
itate
Cis-Aconitate 6.97 ± 15.58c NDc 28.23 ± 27.36c NDc 3570.73 ± 706.09a 241.55 ± 44.23c
3PG 124.47 ± 51.59bcd 101.44 ± 57.61bcd 43.87 ± 44.07cd 43.34 ± 41.10cd NDd NDd
Isocitrate 185.76 ± 22.28bc NDf 36.89 ± 51.62def NDf 888.40 ± 129.01a 10.57 ± 23.63ef
Citrate 4694.88 ± 674.32bcd 2184.00 ± 707.42ef 5174.99 ± 1335.36bcd 5538.30 ± 598.00bc 44.31 ± 22.87g 1765.01 ± 241.84efg
G1P 13.75 ± 30.74e 25.28 ± 56.54de 15.06 ± 33.67e 31.41 ± 43.23de 130.81 ± 35.29c 43.30 ± 41.37cde
F6P 43.51 ± 59.66fg 97.61 ± 55.28efg 133.18 ± 77.00ef NDg NDg 279.04 ± 42.96c
G6P 412.99 ± 47.87cd 260.88 ± 110.08cd 688.74 ± 70.75cd 176.73 ± 7.32d NDd 323.48 ± 28.14cd
2,3-DPG NDb NDb NDb NDb NDb NDb
6-Phospho- NDe 300.23 ± 116.23b NDe NDe NDe 107.90 ± 13.61cd
gluconate
S7P NDg NDg NDg NDg NDg 263.54 ± 367.58efg
UDP-glucose 119.16 ± 38.91fg 110.60 ± 29.93fg 77.01 ± 13.23gh NDh 80.10 ± 22.44gh 263.15 ± 41.93bcd
The values indicate as mean ± SD (nmol g−1) of the metabolite concentration of dry weight (n = 5). ND, not detected. Different superscript letters indicate the significant differences (p < 0.05)
between each type of species by Tukey HSD analysis. Statical analysis were compared between all data set (species and extraction methods (Tables 2, 3, S1)
Planta (2019) 249:1921–1947
Table 3 Amino acids, sugars, and major organic acids of 11 seaweeds after methanol–chloroform–water extraction method
Methanol–chloroform–water extraction methods
Sargassum micracanthum Sphaerotrichia firma (Ishimo- Papenfussiella kuromo Saccharina japonica (Kombu) Pyropia pseudolinearis
(Togemoku) zuku) (Kuromo) (Uppurui Nori)
Amino acids
Planta (2019) 249:1921–1947
Gly 649.19 ± 50.60efg 604.76 ± 81.26fg 271.30 ± 69.60ghi 153.04 ± 14.83i 4067.37 ± 226.47a

Ala 9563.00 ± 574.74f 20834.00 ± 1239.88d 2978.00 ± 412.96g 1673.00 ± 269.34g 36888.00 ± 2705.91b
Ser 824.73 ± 50.44def 1253.71 ± 126.52cde 309.13 ± 73.43f 529.13 ± 67.12ef 5971.16 ± 660.81b
Pro 1150.60 ± 147.18cdefg 1603.80 ± 425.46c 369.90 ± 56.46defg 797.00 ± 112.93cdefg 1011.70 ± 99.66cdefg
Val 2673.50 ± 235.13a 1641.00 ± 133.14b 609.99 ± 189.77cdef 175.70 ± 40.10fg 2639.62 ± 346.44a
Thr 971.82 ± 64.09def 2598.55 ± 174.90ab 1719.45 ± 325.96c 592.01 ± 63.34fghi 2902.00 ± 303.81a
Ile 533.05 ± 20.86cdef 632.13 ± 28.07bcd 379.89 ± 123.06f 45.27 ± 9.52g 1185.06 ± 146.76a
Leu 1205.99 ± 105.68c 1075.56 ± 93.48c 587.64 ± 195.62d 95.48 ± 22.51f 1724.71 ± 131.00b
Asn 6583.31 ± 402.54c 2221.94 ± 135.92d 404.43 ± 186.20ef 185.19 ± 61.59ef 7682.69 ± 553.53b
Asp 12758.80 ± 641.30b 3387.80 ± 219.34g 1496.10 ± 244.78ijk 6038.70 ± 679.84f 8837.60 ± 681.66d
Gln 15865.20 ± 601.33d 24715.90 ± 1847.37bc 4131.10 ± 1000.94fgh 631.90 ± 137.07hi 25520.10 ± 1648.95b
Glu 4004.40 ± 158.09ef 3706.50 ± 322.80ef 3059.50 ± 491.27ef 4211.50 ± 1101.22def 5679.40 ± 368.06de
Lys 253.21 ± 28.49fghi 1271.70 ± 123.86a 487.54 ± 172.68ef 81.11 ± 9.25ghi 711.14 ± 101.03cde
Met 21.05 ± 9.55f 11.71 ± 3.04f NDf 6.39 ± 0.87f 126.76 ± 11.88cd
His 117.67 ± 15.39fgh 361.64 ± 24.97b 52.47 ± 13.17ij 44.38 ± 3.69ij 445.57 ± 61.44a
Phe 323.59 ± 30.75hij 1131.48 ± 71.60bcd 690.27 ± 174.59fgh 222.45 ± 13.67ij 487.07 ± 47.22ghi
Arg 300.38 ± 41.51efgh 1199.24 ± 68.25ab 112.81 ± 22.98hi 91.33 ± 23.00i 1184.39 ± 73.09ab
Tyr 267.59 ± 9.57cd 816.59 ± 60.29a 304.65 ± 65.23c 111.23 ± 9.19efg 825.56 ± 50.68a
Trp 84.87 ± 5.81ef 378.07 ± 30.08a 279.75 ± 50.89b 23.59 ± 4.18g 170.30 ± 26.06d
Cystine 1.02 ± 0.60c 5.16 ± 0.91c 2.16 ± 0.25c NDc 89.95 ± 23.57b
Sugars
Ribose 3468.96 ± 451.91ab 1921.73 ± 159.20de 2551.00 ± 111.09cd 1056.72 ± 342.31f NDg
Arabinose 1642.00 ± 149.04b NDd NDd 2321.15 ± 235.85a NDd
Xylose 44.27 ± 9.97b NDc NDc 103.11 ± 10.81a NDc
Xylitol 274.75 ± 29.84c 249.77 ± 41.55c 770.65 ± 129.59a 544.33 ± 88.88b 58.69 ± 7.98d
Arabitol 180.39 ± 14.35fg 576.00 ± 46.90c 351.22 ± 50.42d 1034.95 ± 188.75b 341.02 ± 38.46de
Rhamnose NDf 86.95 ± 18.79a 48.71 ± 6.63cd 18.01 ± 2.74e NDf
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1940
13
Sargassum micracanthum Sphaerotrichia firma (Ishimo- Papenfussiella kuromo Saccharina japonica (Kombu) Pyropia pseudolinearis
(Togemoku) zuku) (Kuromo) (Uppurui Nori)
Fructose 311.00 ± 16.22d 68.00 ± 15.57d 175.00 ± 20.08d 253.00 ± 24.25d 123.00 ± 11.63d

Glucose 951.70 ± 91.98cde 480.00 ± 116.48cde 1093.60 ± 159.19cde 158.50 ± 69.33de 2228.70 ± 399.78cde
Sorbitol NDb NDb NDb NDb 206.28 ± 15.21a
Mannitol 189028.00 ± 8076.60d 27376.00 ± 6452.20g 84350.00 ± 5324.60f 225793.00 ± 9377.40c 101.00 ± 18.10h
Inositol 147.27 ± 22.80fghij 361.35 ± 31.69cd 433.84 ± 130.19c 44.84 ± 5.84j 161.92 ± 12.83fghi
Sucrose 479.60 ± 103.41e 10.90 ± 4.32e 75.20 ± 18.57e 127.60 ± 27.37e 5.10 ± 2.35e
Maltotriose NDe NDe NDe NDe 710.86 ± 107.34a
Panose NDc NDc NDc NDc NDc
Major organic acids
Lactate 1572.33 ± 128.19c 1287.87 ± 715.57c 772.68 ± 143.46c 1189.72 ± 203.22c 1051.14 ± 537.64c
Fumarate 111.26 ± 26.06cde 117.31 ± 14.33bcde 263.41 ± 55.73bc 173.17 ± 86.72bcd 509.73 ± 67.68a
Succinate 1435.31 ± 149.60b 524.14 ± 78.50efgh 622.41 ± 152.68defg 439.22 ± 240.47fghi 521.03 ± 62.75efgh
Malate 411.00 ± 69.17c 268.45 ± 31.80c 303.39 ± 35.14c 764.02 ± 208.14c 3885.64 ± 305.17b
trans-Aconitate 204.37 ± 33.66bc 119.30 ± 36.84bc 676.60 ± 271.33a NDc NDc
cis-Aconitate 66.84 ± 23.00c 1454.89 ± 136.61b 363.56 ± 63.75c 41.11 ± 10.72c 19.78 ± 3.28c
3PG 41.39 ± 9.24cd 44.95 ± 14.54cd 78.55 ± 8.88bcd 35.53 ± 38.74cd 195.94 ± 23.70b
Isocitrate 122.80 ± 10.98cd 89.77 ± 14.10cdef 112.81 ± 29.34cde NDf NDf
Citrate 805.46 ± 87.55fg 844.00 ± 182.66fg 2381.47 ± 574.59ef 3731.79 ± 774.12cde 6054.64 ± 561.76b
G1P 59.29 ± 7.14cde NDe 61.62 ± 7.94cde 62.77 ± 16.23cde 253.51 ± 32.28b
F6P 142.06 ± 13.86def NDg 71.89 ± 11.30efg 110.40 ± 28.52efg 581.48 ± 128.53b
G6P 509.49 ± 38.78cd 142.55 ± 29.60d 238.91 ± 32.84cd 357.70 ± 80.57cd 3953.17 ± 765.54b
2,3-DPG NDb NDb NDb NDb NDb
6-Phosphogluconate NDe NDe NDe NDe 317.27 ± 89.06b
S7P 348.59 ± 44.77def NDg 159.56 ± 54.48fg 572.34 ± 150.89de 1045.93 ± 205.50b
UDP-glucose 320.65 ± 34.29abc 148.24 ± 22.48fg 149.61 ± 30.96fg 297.97 ± 70.98abc 140.92 ± 35.82fg
Planta (2019) 249:1921–1947
Metabolites Red algae Green algae
Gelidium elegans (Makusa) Neodilsea yendoana Dasya sessilis (Enashi Botryocladia wrightii Ulva australis (Ana Aosa) Chaetomorpha moniligera
(Akaba) Dajia) (Taoyagisou) (Tamajuzumo)
Amino acids
Planta (2019) 249:1921–1947
Gly 1634.75 ± 200.07d 422.10 ± 165.07fghi 1028.04 ± 59.17e 618.43 ± 41.44fg 2810.42 ± 196.89c 444.18 ± 30.94fghi

Ala 1598.00 ± 426.63g 1575.00 ± 300.67g 11530.00 ± 1360.28ef 1323.00 ± 84.23g 1946.00 ± 101.17g 964.00 ± 84.82g
Ser 1410.88 ± 460.50cde 312.06 ± 163.86f 270.57 ± 26.45f 502.03 ± 57.51ef 1318.60 ± 139.41cde 677.43 ± 54.33def
Pro 1035.20 ± 195.79cdefg 104.10 ± 15.24g 22173.10 ± 1026.78b 230.70 ± 52.51fg 312.70 ± 89.63efg 1427.40 ± 138.32cd
Val 262.85 ± 150.79efg 282.17 ± 75.61efg 1664.29 ± 221.77b 1029.53 ± 146.37cd 673.15 ± 88.26cde 166.45 ± 28.30fg
Thr 1039.94 ± 345.48def 392.66 ± 46.15hi 889.15 ± 96.24defg 392.75 ± 29.04hi 963.69 ± 115.36def 220.44 ± 20.18i
Ile 49.56 ± 49.61g 100.65 ± 43.50g 548.25 ± 44.90bcdef 26.52 ± 7.63g 448.96 ± 53.76def 56.47 ± 8.30g
Leu 23.43 ± 26.71f 76.88 ± 33.54f 945.21 ± 130.36c 15.50 ± 2.89f 1721.81 ± 279.48b 239.97 ± 34.73ef
Asn 20.67 ± 10.31f 262.79 ± 21.78ef 890.03 ± 102.40e NDf 275.76 ± 36.54ef 101.17 ± 19.49f
Asp 5776.20 ± 640.58f 1688.70 ± 204.29hijk 1583.50 ± 176.75ijk 1300.70 ± 174.46ijk 455.40 ± 33.28k 7496.50 ± 675.27e
Gln 7313.50 ± 2291.48f 187.90 ± 21.06hi 1361.70 ± 142.78ghi 463.00 ± 61.34hi 565.90 ± 77.60hi 90.40 ± 35.43i
Glu 20319.70 ± 2402.72b 855.50 ± 617.49f 13806.80 ± 1424.25c 4301.90 ± 334.87def 1094.10 ± 51.51f 931.30 ± 136.02f
Lys 470.28 ± 146.85ef 53.94 ± 14.05hi 2.23 ± 0.82i 78.50 ± 7.71ghi 654.48 ± 57.11cde 331.03 ± 43.24fg
Met 74.93 ± 46.30def NDf NDf 30.11 ± 6.37ef 704.18 ± 67.59b 54.02 ± 8.17def
His 81.36 ± 19.04ghij 29.67 ± 2.31j 127.14 ± 16.16efg 28.11 ± 1.46j 184.66 ± 13.79de 33.13 ± 4.18j
Phe 1430.44 ± 298.19bc 1085.50 ± 128.29cde 2504.23 ± 314.48a 251.21 ± 43.80ij 691.27 ± 70.69fgh 97.96 ± 12.89j
Arg 286.01 ± 90.42efghi 141.66 ± 30.53hi 210.11 ± 30.08fghi 152.95 ± 21.50ghi 792.56 ± 64.51d 443.90 ± 56.75e
Tyr 147.64 ± 25.21def 1.58 ± 1.39g 272.92 ± 127.93cd 3.82 ± 1.36g 504.43 ± 53.12b 55.43 ± 6.00fg
Trp 23.67 ± 9.97g 4.97 ± 0.76g 1.02 ± 0.47g 1.02 ± 0.67g 205.31 ± 23.55cd 39.55 ± 4.42fg
Cystine 9.80 ± 3.14c 3.37 ± 2.49c NDc 5.81 ± 1.39c 6.22 ± 1.81c NDc
Sugars
Ribose NDg NDg NDg NDg 2544.94 ± 174.21cd 1819.01 ± 300.14e
Arabinose NDd NDd NDd NDd NDd NDd
Xylose NDc NDc NDc NDc NDc NDc
Xylitol 5.43 ± 0.84d 54.50 ± 5.69d 265.96 ± 37.38c 33.91 ± 10.79d 4.11 ± 0.95d 9.72 ± 3.23d
Arabitol 15.60 ± 1.77h 78.21 ± 19.55fgh 27.85 ± 3.46h 124.46 ± 14.93fgh 40.57 ± 4.45gh 14.68 ± 3.69h
Rhamnose NDf NDf NDf NDf 22.73 ± 5.18e NDf
Fructose 28.00 ± 5.18d 45.00 ± 7.42d 263.00 ± 48.19d 59.00 ± 12.27d 9000.00 ± 1521.81c 96088.00 ± 9764.49b
Glucose 192.20 ± 51.28de 92.30 ± 30.06e 3725.70 ± 183.70cd 118.50 ± 14.36e 9691.70 ± 1313.73b 43757.80 ± 5779.09a
Sorbitol NDb NDb NDb NDb NDb NDb
Mannitol 1177.00 ± 657.80h 6126.00 ± 1579.90h 2412.00 ± 495.80h 255.00 ± 19.40h 1057.00 ± 272.40h 674.00 ± 83.10h
13
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1942
13
Metabolites Red algae Green algae
Gelidium elegans (Makusa) Neodilsea yendoana Dasya sessilis (Enashi Botryocladia wrightii Ulva australis (Ana Aosa) Chaetomorpha moniligera
(Akaba) Dajia) (Taoyagisou) (Tamajuzumo)
Inositol 194.23 ± 16.38efg 86.85 ± 11.61hij 42.17 ± 7.82j 48.03 ± 14.05j 616.61 ± 64.11b 565.01 ± 63.39b

Sucrose 46.60 ± 16.60e 7.00 ± 2.17e 53.40 ± 13.40e 42.10 ± 12.22e 7863.80 ± 990.57b 3041.70 ± 515.42d
Maltotriose NDe NDe 265.27 ± 25.03cd NDe 164.00 ± 64.50d 276.24 ± 45.72cd
Panose NDc NDc 34.10 ± 4.43b NDc NDc NDc
Major organic acids
Lactate 850.93 ± 223.49c 773.87 ± 172.93c 8532.86 ± 1477.16a 3947.28 ± 605.71b 1024.36 ± 164.11c 1323.23 ± 190.35c
Fumarate 36.54 ± 11.32de 38.77 ± 10.20de 146.25 ± 25.98bcde 33.55 ± 11.35de 89.63 ± 37.48de 111.50 ± 24.54cde
Succinate 133.23 ± 29.02i 568.56 ± 112.64defgh 871.65 ± 107.09cd 270.23 ± 31.55hi 436.30 ± 101.86fghi 679.74 ± 45.23def
Malate 375.37 ± 105.98c 317.87 ± 60.77c 779.64 ± 128.14c 184.22 ± 23.50c 621.55 ± 142.07c 717.18 ± 62.85c
trans-Acon- NDc NDc 193.50 ± 106.02bc NDc 709.60 ± 155.34a NDc
itate
cis-Aconitate 26.31 ± 10.60c 19.94 ± 5.87c 36.01 ± 12.47c 12.59 ± 4.91c 3860.52 ± 852.51a 248.29 ± 11.93c
3PG 44.31 ± 18.41cd NDd 38.02 ± 39.26cd NDd NDd NDd
Isocitrate 105.37 ± 20.32cdef 54.96 ± 15.20def 70.76 ± 14.14def NDf 786.54 ± 117.83a 41.55 ± 11.27def
Citrate 3410.95 ± 677.47de 1778.86 ± 519.10efg 5028.01 ± 1522.49bcd 4586.35 ± 709.24bcd NDg 1798.41 ± 203.45efg
G1P NDe 66.57 ± 20.54cde 91.49 ± 14.24cde 56.96 ± 21.11cde 113.21 ± 5.52cd 63.07 ± 25.74cde
F6P 46.69 ± 8.42fg 102.91 ± 28.00efg 169.09 ± 35.26cde NDg NDg 257.56 ± 27.73cd
G6P 223.92 ± 24.61cd 221.22 ± 105.25cd 670.21 ± 97.88cd 122.00 ± 49.53d 38.03 ± 18.10d 307.83 ± 18.93cd
2,3-DPG NDb NDb NDb NDb NDb NDb
6-Phosphoglu- NDe 188.06 ± 48.05c NDe NDe NDe 11.94 ± 26.70de
conate
S7P 11.68 ± 26.11g 38.83 ± 86.82fg NDg NDg NDg 194.59 ± 95.75fg
UDP-glucose 77.37 ± 37.85gh 103.66 ± 23.42fg 92.44 ± 31.76gh NDh 79.04 ± 26.75gh 255.83 ± 63.54cde
The values indicate as mean ± SD (nmol g −1) of the metabolite concentration of dry weight (n = 5). ND, not detected. Different superscript letters indicate the significant differences (p < 0.05)
between each type of species by Tukey HSD analysis. Statical analysis were compared between all data set (species and extraction methods (Table 2, 3, S1)
Planta (2019) 249:1921–1947
Planta (2019) 249:1921–1947 1943
organic acids, and sugars) in seaweeds, each metabolite The formation of distinct clades linked to the algae spe-
group was subjected to an HCA (Figs. 6, 7, 8). Importantly, cies has been observed in related studies. A fatty acid pro-
sugar concentration profiling by HCA determined clear dif- filing study on various tropical marine algae indicated clear
ferences that aligned with the taxonomy of seaweeds, i.e. trends within the respective phyla in the PCA separation
the brown, red, and green algae (Fig. 8), unlike the HCA of variance and HCA (Kumari et al. 2013). Endosymbiotic
amino acids (Fig. 6) and TCA and glycolysis organic acids relationships mainly contributed to the fatty acid profiles
(Fig. 7). Mannitol was found at significantly high concentra- because red and green algae evolved by primary endosym-
tions in brown algae, whereas the concentrations of fructose, biosis of cyanobacteria, whereas brown algae by secondary
glucose, and sucrose were significantly high in green algae endosymbiosis of red algae (Ryall et al. 2003; Chan et al.
(Fig. 8, Tables 2, 3). Among species-specific metabolites 2012; Kumari et al. 2013), which may cause the differences
found in red algae, sorbitol was detected at high concentra- of the clade arrangements. Among all metabolite profiling
tions only in P. pseudolinearis and panose was detected only studies of various seaweeds including our work, several data
in D. sessilis. Arabinose was only detected only in the brown sets indicated clear clusters of the PCA (Fig. 2b) or HCA
algae S. micracanthum and S. japonica. Depending on the (Fig. 8), but some data sets do not (Figs. 2a, 3), depend-
seaweed group, some sugars were not detectable; ribose and ing on the selection of the metabolites and algae species.
rhamnose were not detected in red algae, whereas maltotri- Because the type of data affects the cluster patterns gener-
ose was not detected in brown algae (Fig. 8, Tables 2, 3). ated by PCA and HCA, we also discuss the metabolite types
(sugars and amino acids) selected for HCA.
Discussion Sugar profiling
Taxonomic differences of metabolite profiles Monosaccharides play important roles in food storage, mac-
romolecule synthesis, and energy transfer from phototropic
In our study, the PCA and HCA generated characteristic to heterotrophic pathways that are essential for living organ-
clusters from metabolite profiles that were typical for each isms (Ortiz-Tena et al. 2016). The sugar profiles (Fig. 8)
species, not the extraction method (Figs. 2, 3). By focus- formed a taxonomic tree representing the three seaweed
ing on the clades of the HCA clusters, several taxonomic groups, the brown, red, and green algae, which were not
characteristics were identified by metabolite profiling created using amino acids and major organic acids (Fig. 6)
(Fig. 3). Among the red algae, P. pseudolinearis was not involved in TCA and glycolysis (Fig. 7). The sugar charac-
clustered with its taxonomic group in the HCA (Fig. 3, S9) teristics mainly contributed to the formation of the seaweed
and the PCA score plot (Figs. 2, 4b, S1, S3). P. pseudolin- groups (Fig. 8). Mannitol is the main constituent in brown
earis belongs to class Bangiophyaceae, whereas the other algae, which was consistent with a other studies (Rousvoal
red algae species were from class Florideophyceae. Among et al. 2011; Groisillier et al. 2014; Belghit et al. 2017),
the four brown algae, S. firma and P. kuromo were nearly whereas fructose, sucrose, and glucose are characteristic in
clustered by metabolite profiling, matching their taxonomic green algae (Thompson 1996; Kim et al. 2016). The char-
classification in the same family, the Chordariaceae (Figs. 3, acteristic metabolites in green algae are similar to that in
4a, S3, S8). A previous study performed metabolic profil- land plants due to the evolutionary relationship; green plants
ing on various seaweeds from Norway, including 11 brown originated from green algae. Thus, the taxonomic clades
algae, 7 red algae, and 3 green algae (Belghit et al. 2017). were closely clustered (De Clerck et al. 2012). In red algae,
This study showed that the HCA of 391 metabolites were the sugar profiles were depended on the species. Sorbitol
clustered independently in the brown and red algae clade, was significantly characteristic in P. pseudolinearis and pan-
but there were two clades of green algae. Although two red ose in D. sessilis (Fig. 8). The red algae sugar profiles have a
algae classes, Bangiophyaceae and Florideophyceae, were similar tendency as the monosaccharides in a study on algae
included, the red algae species clustered under one clade. from the Eastern Mediterranean Sea, in which the results
In our HCA of the entire data set, the three seaweed groups showed that the red algae species had the largest variation in
were not clustered together (Fig. 3). PC1 was mainly affected monosaccharide diversity as compared to those in brown and
by the metabolite characteristics of P. pseudolinearis. How- green algae (Robin et al. 2017). This study suggested that
ever, the PCA score plot (PC2–PC3) suggested that charac- the characteristic profiles were closely linked to the species.
teristic metabolite profiles existed in three seaweed groups
(Fig. 2b, Fig. S1: PC2–PC3). The main contribution factors Amino acid profiling
of PCA separation in three groups were caused by the high
concentration of some metabolites, such as mannitol (brown Our analysis showed that most amino acids were found in
algae) and sucrose (green algae). all species; however, individual concentrations depended
13

1944 Planta (2019) 249:1921–1947
mainly on the species and varied between the algae groups profiles followed by different pre-treatment methods (Hamid
(Fig. 6). It is well established that both essential and non- et al. 2018).
essential amino acids are available at different concentra-
tions throughout the year depending on the season and Metabolite characteristics of each species
the environmental conditions (Wong and Cheung 2001b).
Among the amino acids, the Ala concentration was the high- The specimens analysed in this study were dried before fur-
est in all the algae species (Figs. 3, 6, Tables 2, 3). Unlike in ther processing to avoid bias in metabolite quantification
terrestrial or freshwater plants, many marine algae experi- due to the difference in moisture content (Gupta et al. 2014).
ence changing water levels due to the high and low tide that Our results showed that the dry weight percentage varied
generate an oxygen flux, which stimulates changes in the significantly between the species (5–47% (w/w) dry weight),
metabolism to increase the Ala concentration (Narsai et al. but it did not depend on the seaweed group (Table 1). These
2011). Glu and Asp, the acidic amino acids, are linked to results were consistent with our previous study on three
the umami taste of the seaweeds and play an important role edible brown algae, which indicated that species identity is
in the nitrogen transport system and macromolecule storage the main factor affecting the moisture content (Hamid et al.
(Bolton 2009; Fleurence 2010; Biancarosa et al. 2017). Stud- 2018). The moisture content also contributed to the diversity
ies conducted by other research groups showed that brown of the metabolite profiles of each algae species. In addition
algae have a higher level of Glu and Asp as compared to to the moisture content, previous research showed that the
those in red and green algae (Lourenço et al. 2002; Diniz metabolite diversity may also be related to the variation in
2011; Biancarosa et al. 2017). However, in our study, the the environmental conditions that affect the metabolite pro-
levels of total Glu and Asp (Figs. 3, 6, Tables 2, 3) were file of an organism, such as the season, sample collection
species-dependent and not based on the seaweed group. time, and location (Kim and Verpoorte 2010; Gupta et al.
In terms of nutritional aspect, nine essential amino acids 2013; Manns et al. 2014; Ernst et al. 2014; Hamid et al.
(His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) were neces- 2018).
sary for human life which cannot be produced by the human Seaweed studies such as Undaria pinnatifida (Zhou
body. Among the various edible seaweeds, P. pseudolinearis et al. 2015), Fucus spiralis (Paiva et al. 2018), and some
had higher concentrations of five essential amino acids (Thr, seaweeds from Mediterranean Sea coast, Egypt (El-Said
Ile, His, Leu, and Val) and eight non-essential amino acids and El-Sikaily 2013), showed that the chemical composi-
(cystine, Gly, Ser, Tyr, Arg, Ala, Gln, and Asn), while high tion such as total nitrogen, amino acid, and antioxidant
Met and Phe were contained in U. pertusa and G. elegans, properties of seaweed are differed due to seasonal vari-
respectively. In Japan, the dried Pyropia sp. were commonly ability, growth condition, location, plant parts, and envi-
consumed as Nori (Taboada et al. 2013). The high nutri- ronmental changes. In the case of amino acids, our data
tional value contained in these edible seaweeds is not only showed no seaweed group characteristics were observed.
able to promote good health, but also to develop into new Instead, the distinct seaweeds group characteristics were
functional food ingredient. found in sugar compounds, even though we collected at
different locations, seasons, and time. By focusing on the
Effect of the extraction methods on the metabolite environmental conditions, further metabolomics stud-
profiles ies will contribute to the knowledge about the effects
on metabolite profiles depending on the species and
To identify an optimised extraction method, sample process- environment.
ing was performed by extraction in methanol–water with or
without chloroform. We did not find significant differences
in the metabolic profiles of all the species that could be
linked to the extraction method (Figs. 2, 3, 4, 5). However, Conclusion
some effect of the extraction method on the metabolite con-
centrations was observed in the individual seaweed groups, This study assessed the water-soluble metabolite content of
the brown (Fig. S3: PC4), red (Fig. S4: PC5), and green seaweeds biomass to derive metabolite profiles from various
algae (Fig. S5: PC2), but the effect on the outcome was very seaweeds including brown, red, and green algae collected
limited. In summary, the study showed that species-specific from northern Japan; specimens were freeze-dried and pro-
differences had the most effect on the metabolite profiles cessed by studying the effect of different extraction methods
in various seaweeds (Figs. 2, 3, S1). Our previous study on the metabolite concentrations. Metabolite profiles were
generated a similar result, in which species characteristics evaluated by multivariate analysis; PCA and HCA identified
were the main contributor for differences of the metabolite individual metabolic characteristics within each species of
13
Planta (2019) 249:1921–1947 1945
different seaweed groups. Mannitol and cystathionine were Cai J, Henion J, Toxicology A, Drive W (1995) Capillary electropho-
characteristic in brown algae, whereas sorbitol was specific resis-mass spectrometry. J Chromatogr A 703:667–692. https://
doi.org/10.1016/0021-9673(94)01178-H
in P. pseudolinearis and panose in D. sessilis of red algae. Cavalier-Smith T (2000) Membrane heredity and early chloroplast evo-
Fructose, sucrose, glucose, and inositol were characteristic lution. Trends Plant Sci 5:174–182. https://doi.org/10.1007/978-
metabolites in green algae. Sugar profiles, but not amino 3-319-06947-0_19
acid profiles, were characteristically aligned with the tax- Chan JC-C, Cheung PC-K, Ang PO (1997) Comparative studies on
the effect of three drying methods on the nutritional composition
onomy tree representing the three seaweed groups. Brown of seaweed Sargassum hemiphyllum (Turn.) C. Ag. J Agric Food
and green algae could be characterised by the distinctive Chem 45:3056–3059. https://doi.org/10.1021/jf9701749
metabolites; however, in red algae, metabolite profiles of Chan CX, Zauner S, Wheeler G et al (2012) Analysis of porphyra
individual species should be evaluated. The comprehensive membrane transporters demonstrates gene transfer among pho-
tosynthetic eukaryotes and numerous sodium-coupled transport
results of the metabolite analysis in this study can be used systems. Plant Physiol 158:2001–2012. https://doi.org/10.1104/
to further explore the uses of seaweeds in foods and health- pp.112.193896
related fields, such as in nutraceutical research. Cock JM, Coelho SM, Brownlee C, Taylor AR (2010) The ectocarpus
genome sequence: insights into brown algal biology and the evo-
lutionary diversity of the eukaryotes. N Phytol 188:1–4. https://
Author contribution statement SSH and MW designed, doi.org/10.1111/j.1469-8137.2010.03454.x
sample collection, performed the experiments, and wrote Collén J, Porcel B, Carré W et al (2013) Genome structure and meta-
the manuscript. KI and KS collected and identified the sea- bolic features in the red seaweed Chondrus crispus shed light
weeds samples. SSH, MW, YA, RK analysed the data. MT, on evolution of the Archaeplastida. Proc Natl Acad Sci USA
110:5247–5252. https://doi.org/10.1073/pnas.1221259110
TS, KI, and KS provided their comments and contributed to Collins K, Fitzgerald G, Stanton C, Ross R (2016) Looking beyond
substantial revision of the manuscript. All authors read and the terrestrial: the potential of seaweed derived bioactives to
approved the manuscript. treat non-communicable diseases. Mar Drugs 14:60. https://doi.
org/10.3390/md14030060
Costa LS, Fidelis GP, Cordeiro SL et al (2010) Biological activities of
sulfated polysaccharides from tropical seaweeds. Biomed Phar-
Acknowledgements For samples collection and species identification, macother 64:21–28. https: //doi.org/10.1016/j.biopha .2009.03.005
the authors would like to thank all those who have contributed directly Date Y, Sakata K, Kikuchi J (2012) Chemical profiling of complex bio-
or indirectly in this research; all staffs of Muroran Marine Station, chemical mixtures from various seaweeds. Polym J 44:888–894.
Yamagata Prefecture Fisheries Experiment Station, and Mr. Yohichi https://doi.org/10.1038/pj.2012.105
Homma, fishermen from Wasada port (Tsuruoka city). This study was Davis GDJ, Vasanthi AHR (2011) Seaweed metabolite database
funded by the Yamagata prefectural government and Tsuruoka city. It (SWMD): a database of natural compounds from marine algae.
was also supported by a scholarship from the Ministry of Education, Bioinformation 5:361–364. https://doi.org/10.6026/9732063000
Culture, Sports, Science, and Technology (MEXT), Japan, for an inter- 5361
national student (Hamid, S.S). Dawczynski C, Schubert R, Jahreis G (2007) Amino acids, fatty
acids, and dietary fibre in edible seaweed products. Food Chem
Compliance with ethical standards 103:891–899. https://doi.org/10.1016/j.foodchem.2006.09.041
De Clerck O, Bogaert KA, Leliaert F (2012) Diversity and evolution
Conflict of interest The authors declare that they have no conflicts of of algae. In: Piganeau G (ed) Advances in botanical research,
interest. vol 64. Elsevier, pp 55–86
de Raad M, Fischer CR, Northen TR (2016) High-throughput plat-
forms for metabolomics. Curr Opin Chem Biol 30:7–13. https
://doi.org/10.1016/j.cbpa.2015.10.012
Diniz GS (2011) Gross chemical profile and calculation of nitrogen-
References to-protein conversion factors for five tropical seaweeds. Am J
Plant Sci 02:287–296. https://doi.org/10.4236/ajps.2011.23032
El-Said GF, El-Sikaily A (2013) Chemical composition of some
Astorga-España MS, Rodríguez-Galdón B, Rodríguez-Rodríguez EM, seaweed from Mediterranean Sea coast, Egypt. Environ
Díaz-Romero C (2016) Amino acid content in seaweeds from the Monit Assess 185:6089–6099. https://doi.org/10.1007/s1066
Magellan Straits (Chile). J Food Compos Anal 53:77–84. https:// 1-012-3009-y
doi.org/10.1016/j.jfca.2016.09.004 Endo H, Suehiro K, Kinoshita J, Agatsuma Y (2015) Combined
Belghit I, Rasinger JD, Heesch S et al (2017) In-depth metabolic profil- effects of temperature and nutrient enrichment on palatability
ing of marine macroalgae confirms strong biochemical differences of the brown alga Sargassum yezoense (Yamada) Yoshida & T.
between brown, red and green algae. Algal Res 26:240–249. https Konno. Am J Plant Sci 06:275–282. https://doi.org/10.4236/
://doi.org/10.1016/j.algal.2017.08.001 ajps.2015.62031
Biancarosa I, Espe M, Bruckner CG et al (2017) Amino acid composi- Ernst M, Silva DB, Silva RR et al (2014) Mass spectrometry in
tion, protein content, and nitrogen-to-protein conversion factors plant metabolomics strategies: from analytical platforms to data
of 21 seaweed species from Norwegian waters. J Appl Phycol acquisition and processing. Nat Prod Rep 31:784. https://doi.
29:1001–1009. https://doi.org/10.1007/s10811-016-0984-3 org/10.1039/c3np70086k
Bolton MD (2009) Primary metabolism and plant defense—fuel Fleurence J (2010) Seaweed proteins: biochemical, nutritional
for the fire. Mol Plant Microb Interact 22:487–497. https://doi. aspects and potential uses. Trends Food Sci Technol 10:25–28.
org/10.1094/MPMI-22-5-0487 https://doi.org/10.1088/1751-8113/44/8/085201
13

1946 Planta (2019) 249:1921–1947
Garcia-Vaquero M, Rajauria G, O’Doherty JV, Sweeney T (2017) Phytochem Rev 3:371–379. https : //doi.org/10.1007/s1110
Polysaccharides from macroalgae: recent advances, innova- 1-005-1459-3
tive technologies and challenges in extraction and purification. Laturnus F (1996) Volatile halocarbons released from Arctic mac-
Food Res Int 99:1011–1020. https: //doi.org/10.1016/j.foodr roalgae. Mar Chem 55:359–366. https://doi.org/10.1016/S0304
es.2016.11.016 -4203(97)89401-7
Goulitquer S, Potin P, Tonon T (2012) Mass spectrometry-based Leal MC, Munro MHG, Blunt JW et al (2013) Biogeography and bio-
metabolomics to elucidate functions in marine organisms and discovery hotspots of macroalgal marine natural products. Nat
ecosystems. Mar Drugs 10:849–880. https://doi.org/10.3390/ Prod Rep 30:1380–1390. https://doi.org/10.1039/c3np70057g
md10040849 Lei Z, Huhman DV, Sumner LW (2011) Mass spectrometry strate-
Groisillier A, Shao Z, Michel G et al (2014) Mannitol metabolism gies in metabolomics. J Biol Chem 286:25435–25442. https: //doi.
in brown algae involves a new phosphatase family. J Exp Bot org/10.1074/jbc.R111.238691
65:559–570. https://doi.org/10.1093/jxb/ert405 Lourenço SO, Barbarino E, De-Paula JC et al (2002) Amino acid com-
Gu H, Zhang P, Zhu J, Raftery D (2015) Globally optimized targeted position, protein content and calculation of nitrogen-to-protein
mass spectrometry: reliable metabolomics analysis with broad conversion factors for 19 tropical seaweeds. Phycol Res 50:233–
coverage. Anal Chem 87:12355–12362. https://doi.org/10.1021/ 241. https://doi.org/10.1046/j.1440-1835.2002.00278.x
acs.analchem.5b03812 Manns D, Deutschle AL, Saake B, Meyer AS (2014) Methodology
Guiry MD, Guiry GM (2018) Algaebase: listing the world’s algae. for quantitative determination of the carbohydrate composition
In: World-wide electron. Publ. Natl. Univ. Ireland, Galway, Irel. of brown seaweeds (Laminariaceae). RSC Adv 4:25736–25746.
http://www.algaebase.org/. Accessed 27 Sep 2018 https://doi.org/10.1039/C4RA03537B
Gupta S, Abu-Ghannam N (2011) Bioactive potential and possible Michalak I, Chojnacka K (2015) Algae as production systems of
health effects of edible brown seaweeds. Trends Food Sci Technol bioactive compounds. Eng Life Sci 15:160–176. https://doi.
22:315–326. https://doi.org/10.1016/j.tifs.2011.03.011 org/10.1002/elsc.201400191
Gupta V, Thakur RS, Reddy CRK, Jha B (2013) Central metabolic Murtagh F, Legendre P (2014) Ward’s hierarchical agglomerative clus-
processes of marine macrophytic algae revealed from NMR based tering method: Which algorithms implement ward’s criterion? J
metabolome analysis. RSC Adv 3:7037. https://doi.org/10.1039/ Classif 31:274–295. https://doi.org/10.1007/s00357-014-9161-z
c3ra23017a Nakamura Y, Mochamad Afendi F, Kawsar Parvin A et al (2014)
Gupta V, Thakur RS, Baghel RS et al (2014) Seaweed metabolomics. KNApSAcK metabolite activity database for retrieving the rela-
Advances in botanical research, vol Serial. Academic Press, Cam- tionships between metabolites and biological activities. Plant Cell
bridge, pp 31–52 Physiol 55:1–9. https://doi.org/10.1093/pcp/pct176
Hamed I, Özogul F, Özogul Y, Regenstein JM (2015) Marine Narsai R, Rocha M, Geigenberger P et al (2011) Comparative analysis
bioactive compounds and their health benefits: a review. between plant species of transcriptional and metabolic responses
Compr Rev Food Sci Food Saf 14:446–465. https : //doi. to hypoxia. N Phytol 190:472–487. https : //doi.org/10.111
org/10.1111/1541-4337.12136 1/j.1469-8137.2010.03589.x
Hamid SS, Wakayama M, Soga T, Tomita M (2018) Drying and extrac- Nishitsuji K, Arimoto A, Iwai K et al (2016) A draft genome of the
tion effects on three edible brown seaweeds for metabolomics. brown alga, Cladosiphon okamuranus, S-strain: a platform for
J Appl Phycol 30:3335–3350. https://doi.org/10.1007/s1081 future studies of ‘mozuku’ biology. DNA Res 23:561–570. https
1-018-1614-z ://doi.org/10.1093/dnares/dsw039
Hartmann A, Becker K, Karsten U et al (2015) Analysis of Norziah MH, Ching CY (2000) Nutritional composition of edible sea-
mycosporine-like amino acids in selected algae and cyanobacte- weed Gracilaria changgi. Food Chem 68:69–76
ria by hydrophilic interaction liquid chromatography and a novel Omar MMA, Elbashir AA, Schmitz OJ (2017) Capillary electropho-
MAA from the red alga Catenella repens. Mar Drugs 13:6291– resis method with UV-detection for analysis of free amino acids
6305. https://doi.org/10.3390/md13106291 concentrations in food. Food Chem 214:300–307. https://doi.
Heyman HM, Dubery IA (2016) The potential of mass spectrometry org/10.1016/j.foodchem.2016.07.060
imaging in plant metabolomics: a review. Phytochem Rev 15:297– Ortiz-Tena JG, Rühmann B, Schieder D, Sieber V (2016) Revealing
316. https://doi.org/10.1007/s11101-015-9416-2 the diversity of algal monosaccharides: fast carbohydrate finger-
Hirayama A, Wakayama M, Soga T (2014) Metabolome analysis printing of microalgae using crude biomass and showcasing sugar
based on capillary electrophoresis-mass spectrometry. TrAC distribution in Chlorella vulgaris by biomass fractionation. Algal
Trends Anal Chem 61:215–222. https : //doi.org/10.1016/j. Res 17:227–235. https://doi.org/10.1016/j.algal.2016.05.008
trac.2014.05.005 Paiva L, Lima E, Neto AI, Baptista J (2018) Seasonal variability of the
Hwang ES, Ki KN, Chung HY (2013) Proximate composition, amino biochemical composition and antioxidant properties of Fucus spi-
acid, mineral, and heavy metal content of dried laver. Prev Nutr ralis at two Azorean Islands. Mar Drugs. https://doi.org/10.3390/
Food Sci 18:139–144. https://doi.org/10.3746/pnf.2013.18.2.139 md16080248
Kim HK, Verpoorte R (2010) Sample preparation for plant metab- Palanisamy SK, Arumugam V, Rajendran S et al (2018) Chemical
olomics. Phytochem Anal 21:4–13. https: //doi.org/10.1002/ diversity and anti-proliferative activity of marine algae. Nat Prod
pca.1188 Res. https://doi.org/10.1080/14786419.2018.1488701
Kim DH, Lee SB, Kim SK et al (2016) Optimization and evaluation of Parjikolaei BR, Bruhn A, Eybye KL et al (2016) Valuable biomolecules
sugars and chemicals production from green macro-algae Enter- from nine north atlantic red macroalgae: amino acids, fatty acids,
omorpha intestinalis. Bioenergy Res 9:1155–1166. https://doi. carotenoids, minerals and metals. Nat Resour 07:157–183. https
org/10.1007/s12155-016-9759-6 ://doi.org/10.4236/nr.2016.74016
Kumari P, Bijo AJ, Mantri VA et al (2013) Fatty acid profiling of Peinado I, Girón J, Koutsidis G, Ames JM (2014) Chemical composi-
tropical marine macroalgae: an analysis from chemotaxonomic tion, antioxidant activity and sensory evaluation of five different
and nutritional perspectives. Phytochemistry 86:44–56. https:// species of brown edible seaweeds. Food Res Int 66:36–44. https
doi.org/10.1016/j.phytochem.2012.10.015 ://doi.org/10.1016/j.foodres.2014.08.035
La Barre SL, Weinberger F, Kervarec N, Potin P (2004) Monitoring
defensive responses in macroalgae—limitations and perspectives.
13
Planta (2019) 249:1921–1947 1947
Percival E (1979) The polysaccharides of green, red and brown sea- Taboada MC, Millán R, Miguez MI (2013) Nutritional value of the
weeds: their basic structure, biosynthesis and function. Br Phycol marine algae wakame (Undaria pinnatifida) and nori (Porphyra
J 14:103–117. https://doi.org/10.1080/00071617900650121 purpurea) as food supplements. J Appl Phycol 25:1271–1276.
Qasim R (1991) Amino acid composition of some common seaweeds. https://doi.org/10.1007/s10811-012-9951-9
Pak J Pharm Sci 4:49–54 Thompson GA (1996) Lipids and membrane function in green algae.
Ramautar R, Somsen GW, de Jong GJ (2009) CE–MS in metabolomics. Biochim Biophys Acta Lipids Lipid Metab 1302:17–45. https://
Electrophoresis 30:276–291. https://doi.org/10.1002/elps.20080 doi.org/10.1016/0005-2760(96)00045-8
0512 Wahbeh MI (1997) Amino acid and fatty acid profiles of four species
Robin A, Chavel P, Chemodanov A et al (2017) Diversity of mono- of macroalgae from Aqaba and their suitability for use in fish
saccharides in marine macroalgae from the Eastern Mediterra- diets. Aquaculture 159:101–109. https://doi.org/10.1016/S0044
nean Sea. Algal Res 28:118–127. https://doi.org/10.1016/j.algal -8486(97)00183-X
.2017.10.005 Wakayama M, Aoki N, Sasaki H, Ohsugi R (2010) Simultaneous
Robledo D, Freile Pelegrín Y (1997) Chemical and mineral composi- analysis of amino acids and carboxylic acids by capillary elec-
tion of six potentially edible seaweed species of Yucatán. Bot trophoresis–mass spectrometry using an acidic electrolyte and
Mar 40:301–306. https://doi.org/10.1515/botm.1997.40.1-6.301 uncoated fused-silica capillary. Anal Chem 82:9967–9976. https
Rodrigues D, Freitas AC, Pereira L et al (2015) Chemical composi- ://doi.org/10.1021/ac1019039
tion of red, brown and green macroalgae from Buarcos bay in Wakayama M, Hirayama A, Soga T (2015) Capillary electrophoresis-
central west coast of Portugal. Food Chem 183:197–207. https:// mass spectrometry. In: Bjerrum JT (ed) Metabonomics: methods
doi.org/10.1016/j.foodchem.2015.03.057 and protocols, methods in molecular biology. Springer, New York,
Rousvoal S, Groisillier A, Dittami SM et al (2011) Mannitol-1-phos- pp 113–122
phate dehydrogenase activity in Ectocarpus siliculosus, a key role West J, Calumpong HP, Martin G (2016) Seaweeds. In: Nations U
for mannitol synthesis in brown algae. Planta 233:261–273. https (ed) The first global integrated marine assessment. Cambridge
://doi.org/10.1007/s00425-010-1295-6 University Press, Cambridge, pp 223–228
Ryall K, Harper JT, Keeling PJ (2003) Plastid-derived type II fatty acid Wong K, Cheung PC (2001a) Nutritional evaluation of some subtropi-
biosynthetic enzymes in chromists. Gene 313:139–148. https:// cal red and green seaweeds part II. In vitro protein digestibil-
doi.org/10.1016/S0378-1119(03)00671-1 ity and amino acid profiles of protein concentrates. Food Chem
Skriptsova AV, Shevchenko NM, Zvyagintseva TN, Imbs TI (2010) 72:11–17. https://doi.org/10.1016/S0308-8146(00)00176-X
Monthly changes in the content and monosaccharide composi- Wong K, Cheung PC (2001b) Influence of drying treatment on three
tion of fucoidan from Undaria pinnatifida (Laminariales, Phaeo- Sargassum species 2. Protein extractability, in vitro protein digest-
phyta). J Appl Phycol 22:79–86. https://doi.org/10.1007/s1081 ibility and amino acid profile of protein concentrates. J Appl Phy-
1-009-9438-5 col 13:51–58
Soga T, Heiger DN (2000) Amino acid analysis by capillary electro- Wu X, Jiang W, Lu J et al (2014) Analysis of the monosaccharide
phoresis electrospray ionization mass spectrometry. Anal Chem composition of water-soluble polysaccharides from Sargassum
72:1236-1241. https://doi.org/10.1021/ac990976y fusiforme by high performance liquid chromatography/electro-
Soga T, Igarashi K, Ito C et al (2009) Metabolomic profiling of anionic spray ionisation mass spectrometry. Food Chem 145:976–983.
metabolites by capillary electrophoresis mass spectrometry. Anal https://doi.org/10.1016/j.foodchem.2013.09.019
Chem 81:6165–6174. https://doi.org/10.1021/ac900675k Zhou AY, Robertson J, Hamid N et al (2015) Changes in total nitrogen
Sugimoto M, Kawakami M, Robert M et al (2012) Bioinformatics tools and amino acid composition of New Zealand Undaria pinnatifida
for mass spectroscopy-based metabolomic data processing and with growth, location and plant parts. Food Chem 186:319–325.
analysis. Curr Bioinform 7:96–108. https: //doi.org/10.2174/15748 https://doi.org/10.1016/j.foodchem.2014.06.016
9312799304431
Tabarsa M, Rezaei M, Ramezanpour Z, Waaland JR (2012) Chemical Publisher’s Note Springer Nature remains neutral with regard to
compositions of the marine algae Gracilaria salicornia (Rhodo- jurisdictional claims in published maps and institutional affiliations.
phyta) and Ulva lactuca (Chlorophyta) as a potential food source.
J Sci Food Agric 92:2500–2506. https: //doi.org/10.1002/jsfa.5659
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Hamid2019 Article MetabolomeProfilingOfVariousSe

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Planta (2019) 249:1921–1947

Metabolome profiling of various seaweed species discriminates

Abbreviations LC Liquid chromatography

methods, it is difficult to compare the metabolite con- Materials and methods

Sargassum micracanthum Sphaerotrichia firma Papenfussiella kuromo Saccharina japonica (Kombu)

Ulva australis Chaetomorpha moniligera

Fig. 2 Principle component analysis (PCA) score plots of dry weight- ▸ a

was observed in G. elegans, whereas the lowest percentage

Overview of the metabolite profiles D. sessilis

To evaluate general tendencies of metabolites in seaweeds

An HCA was performed on 110 individual samples Papenfussiella kuromo (Kuromo)

Neodilsea yendoana (Akaba)

Ulva australis (Ana Aosa)

clades (Fig. 3, S2) that depended on the taxonomic class or

Most of all the

Low concentration High concentration A: MeOH-Water extraction method

within each species, the two extraction methods were clus-

Fig. 4 PCA score plots of dry weight-based metabolites concentra- ▸

In red algae, each species displayed a characteristic metab-

i.e. 5-methylcytosine, nicotine, Phe–Phe, orotate, urate,

galacturonate 1-phosphate, ribose, arabinose, xylose, and

and IDP was detected only in P. pseudolinearis, glycolate

and UDP only in N. yendoana, panose and p-hydroxy- C. moniligera U. australis

dolinearis, G. elegans, N. yendoana, D. sessilis, and B.

wrightii, showed that the PC1-4 plots mainly illustrate

a MeOH-Water extraction methods P. pseudolinearis by high concentrations of amino acids

N. yendoana The concentration of thymine (at least 2 times), fructose (at

P. kuromo (Togemoku) nol–water with and without chloroform, respectively, at a

Fig. 6 HCA and heat map of

Low concentration High concentration

Fig. 7 HCA and heat map

Fig. 8 HCA and heat map of

Low concentration High concentration

Gly 736.39 ± 36.75ef 546.45 ± 51.57fgh 285.24 ± 81.90ghi 185.57 ± 24.37hi 4278.75 ± 481.72a

Methanol–water extraction methods

Fructose 316.00 ± 29.64d 73.00 ± 11.67d 158.00 ± 21.51d 249.00 ± 23.13d 150.00 ± 18.10d

Gly 1886.07 ± 263.85d 476.36 ± 204.87fghi 1029.74 ± 81.06e 674.16 ± 50.38ef 3339.20 ± 205.82b 412.57 ± 22.29fghi

Methanol–water extraction methods

Glucose 790.70 ± 1257.70cde 112.00 ± 32.31e 3977.00 ± 282.88c 120.90 ± 43.33e 11799.20 ± 1980.38b 43512.50 ± 3144.48a

Gly 649.19 ± 50.60efg 604.76 ± 81.26fg 271.30 ± 69.60ghi 153.04 ± 14.83i 4067.37 ± 226.47a

Methanol–chloroform–water extraction methods

Fructose 311.00 ± 16.22d 68.00 ± 15.57d 175.00 ± 20.08d 253.00 ± 24.25d 123.00 ± 11.63d

Gly 1634.75 ± 200.07d 422.10 ± 165.07fghi 1028.04 ± 59.17e 618.43 ± 41.44fg 2810.42 ± 196.89c 444.18 ± 30.94fghi

Methanol–chloroform–water extraction methods

Inositol 194.23 ± 16.38efg 86.85 ± 11.61hij 42.17 ± 7.82j 48.03 ± 14.05j 616.61 ± 64.11b 565.01 ± 63.39b

Discussion Sugar profiling

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Abbreviations LC Liquid chromatography

Fig. 2 Principle component analysis (PCA) score plots of dry weight- ▸ a

Fig. 4 PCA score plots of dry weight-based metabolites concentra- ▸

Fig. 6 HCA and heat map of

Fig. 7 HCA and heat map

Fig. 8 HCA and heat map of