Purification and Characterisation of κ-Carrageenan Oligosaccharides Prepared by κ-Carrageenase from Thalassospira sp. Fjfst-332

Article abstract of DOI:10.1016/j.carbpol.2017.10.043

κ-Carrageenan oligosaccharides (KCOs) are promising agents for treating inflammatory diseases. However, the lack of purification and structural elucidation of KCOs has limited structure-function evaluation. In this study, using a system coupling medium pressure liquid chromatography (MPLC) with an evaporative light scattering detector (ELSD), four types of KCOs were separated. The total yield of the four KCO powders was ～5.02% after purification (KCOs/κ-carrageenan, w/w). Their structural identities were characterised by ESI-MS, CID-MS/MS and NMR, as κ-neocarrabiose (α-DA-1,3-G4Srα/β), κ-neocarratetraose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β), κ-neocarrahexaose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β) and heterozygous κ/ι-neocarrahexaose (α-DA/DA2S-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β). KCOs showed no cytotoxicity in RAW264.7 macrophages, and the anti-inflammatory activity was closely correlated with the degree of polymerisation and the number of sulfated groups. κ/ι-Neocarrahexaose exhibited the highest inhibition of ROS (Reactive Oxygen Species) production in LPS-induced RAW264.7 macrophages. The MPLC-ELSD system provides a platform for large-scale fabrication of purified KCOs and affords a route to these compounds that may regulate immune defense.

Full text of DOI:10.1016/j.carbpol.2017.10.043

CarbohydratePolymers180(2018)314–327
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Research Paper
Purification and Characterisation of κ-Carrageenan Oligosaccharides
Prepared by κ-Carrageenase from Thalassospira sp. Fjfst-332
Juanjuan Guoa,b,c, Zhichang Zhenga,b,c, Xu Lua,b,c, Shaoxiao Zenga,b,c, Chi Chend,
⁎
Longtao Zhanga,b,c, Baodong Zhenga,b,c,
a
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
China-Ireland International Cooperation Centre for Food Material Science and Structure Design, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002,
b
China
c
Fujian Provincial Key Laboratory of Quality Science and Processing Technology in Special Starch, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002,
China
d
Department of Food Science and Nutrition, University of Minnesota,St. Paul, MN, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
κ-Carrageenan oligosaccharides
MPLC-ELSD
Anti-inflammatory
ROS
Structure-function relationship
κ-Carrageenan oligosaccharides (KCOs) are promising agents for treating inflammatory diseases. However, the
lack of purification and structural elucidation of KCOs has limited structure-function evaluation. In this study,
using a system coupling medium pressure liquid chromatography (MPLC) with an evaporative light scattering
detector (ELSD), four types of KCOs were separated. The total yield of the four KCO powders was ∼5.02% after
purification (KCOs/κ-carrageenan, w/w). Their structural identities were characterised by ESI-MS, CID-MS/MS
and NMR, as κ-neocarrabiose (α-DA-1,3-G4Srα/β), κ-neocarratetraose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β),
κ-neocarrahexaose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β) and heterozygous κ/ι-neo-
carrahexaose (α-DA/DA2S-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β). KCOs showed no cytotoxi-
city in RAW264.7 macrophages, and the anti-inflammatory activity was closely correlated with the degree of
polymerisation and the number of sulfated groups. κ/ι-Neocarrahexaose exhibited the highest inhibition of ROS
(Reactive Oxygen Species) production in LPS-induced RAW264.7 macrophages. The MPLC-ELSD system pro-
vides a platform for large-scale fabrication of purified KCOs and affords a route to these compounds that may
regulate immune defense.
1. Introduction
secretion of cytokines including NO, interleukin-1β (IL-1β), interleukin-
6 (IL-6), tumor necrosis factor-α (TNF-α) and reactive oxygen species
Carrageenan oligosaccharides (COs), the degraded carbohydrates of
carrageenans, are composed of an alternating backbone of β-1,3-D-ga-
lactose (G unit) and α-1,4-D-galactose (D unit). Based on the position
and number of sulfate ester units (S) and the presence of 3,6-anhy-
drogro-bridges (A) on D units, COs are classified into 13 types de-
nominated by Greek letter prefixes, including κ-(DA-G4S), ι-(DA2S-
G4S) and λ-(D2S6S-G2S) (Necas and Bartosikova, 2013). The functional
activities of COs are closely related to the degree of polymerisation
(Dp), as well as the number and position of sulfate units. However,
elucidating the relationship between biological activity and structure
remains challenging because large-scale fabrication the purification has
not been achieved.
(ROS) (Kofler, Nickel, & Weis, 2005). However, excessive expression of
macrophages results in tissue damage and cell death. Therefore, mod-
ulation of macrophage activation is an effective method for preventing
inflammatory responses (Alvarez-Suarez et al., 2017). COs are potential
pharmaceutical agents possessing multiple bioactivities including an-
tiviral, anti-tumour, antioxidant and immunoregulatory activities
(Wang et al., 2011; Yuan et al., 2005). In particular, the im-
munomodulation and antitumor activities of mixed κ-carrageenan oli-
gosaccharides (KCOs) have been investigated in S180-bearing mice, and
they exert their antitumor effects by promoting the immune system
(Yuan, Song, Li, Li, & Dai, 2006). The immunomodulatory function of
mixed KCOs has also been studied in lipopolysaccharide (LPS)-activated
microglial cells, and their biological function is closely correlated with
structure, especially the sulfate groups (Yao, Xu, & Wu, 2014). How-
ever, the immunomodulatory functions of KCOs with varying Dp are
Inflammation is regarded as a major risk factor for the pathogenesis
of cancer and various other chronic diseases. Immune cells such as
macrophages regulate inflammation and host defences through
⁎
Corresponding author at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
E-mail address: zbdfst@yeah.net (B. Zheng).
http://dx.doi.org/10.1016/j.carbpol.2017.10.043
Received 14 August 2017; Received in revised form 15 September 2017; Accepted 11 October 2017
Availableonline13October2017
0144-8617/©2017PublishedbyElsevierLtd.

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 1. Schematic diagram of KCOs enzymatic hydrolysis preparation
and purification.
Fig. 2. TLC analysis of KCOs. (A) The components prepared through
diverse enzymatic time. (B) I-V represented the components that se-
parated by MPLC-ELSD system, a was the standards of κ-carrabiose
alditols, b is the standards of κ-carratetraose alditols, c is the standards
of κ-carrahexaose alditols.
not well understood, due to low yield and difficulties with purification.
Thus, establishing a platform for the large-scale fabrication and pur-
ification of COs is necessary.
provides an ideal detection system for carbohydrates (Young and
Scientific, 2003). MPLC systems coupled with ELSD have been used for
separating lotus seed oligosaccharides but not COs (Lu et al., 2017),
which have a high polarity due to the sulfate groups, and varying Dp,
making the preparation CO monomers challenging. Gel filtration has
been used to purify COs using a Bio-Gel P-2 column (Sun et al., 2014), a
Superdex 30 pg column (Niu et al., 2015), a TSK gel GMPWxl column
(Zhang et al., 2010) and a Q-Sepharose Fast Flow column (Yang et al.,
2011). Compared with other columns, the Superdex 30 pg column
provides advantages such as high resolution, a rigid matrix, and good
retention properties (Caram-Lelham, Sundelöf, & Andersson, 1995).
However, when a Superdex 30 pg column is applied in HPLC or HPGPC,
the low loading amount remains a limiting factor preventing large-scale
purification and fabrication.
Column chromatographic systems consisting of a pump, detector
and chromatographic column are commonly used for the separation
and purification of natural products. Recently, high-pressure anion-
exchange chromatography (HPAEC) (Jouanneau, Boulenguer,
Mazoyer, & Helbert, 2010b), high-performance liquid chromatography-
evaporative light scattering detector (HPLC-ELSD) (Niu, Zhang,
Chen, & Yan, 2015), AKTA-fast protein liquid chromatography (FPLC)
(Zhang et al., 2010) and high-performance gel permeation chromato-
graphy (HPGPC) (Duan, Yu, Liu, Tian, & Mou, 2016) have been used to
purify COs. However, these technologies have drawbacks including a
low loading amount, and can be complex to operate. Therefore,
medium-pressure liquid chromatography (MPLC),
a
type of flash
In the present study, we used MPLC coupled with ELSD and a
HiLoad Superdex 30 prep grade column to separate KCOs from the
Thalassospira sp. Fjfst-332 strain (Fig. 1). Compared with traditional
methods, the approach removed the need for a pre-treatment operation
such as desalting and depigmentation, which simplified the processing
steps. Furthermore, the method provides an ideal automated, high-re-
solution, rapid separation compatible with a high loading amount. ESI-
MS (Electrospray Ionization Mass Spectrometry), CID-MS/MS (Collision
Induced Dissociation-Tandem Mass Spectrometry) and NMR (Nuclear
Magnetic Resonance) were used to characterise the structural sequence,
chromatography, has been developed to separate carbohydrates. This
high-resolution method can be easily automated with an autosampler
for peak collection (Weber, Hamburger, Schafroth, & Potterat, 2011).
Furthermore, compared with the aforementioned technologies, the
loading amount can be relatively high, ranging from milligrams to
hectograms, facilitating large-scale separation of naturally occurring
compounds (Challal et al., 2015). However, since carbohydrates do not
adsorb ultraviolet (UV) light, a phenol-sulfuric acid approach is usually
used for assessment, while evaporative light scattering detection (ELSD)
315

J. Guo et al.
CarbohydratePolymers180(2018)314–327
and structure-function relationships between KCO monomers and anti-
inflammatory activity were preliminarily established. The effective
fabrication of KCOs will lay the foundation for studying their structure-
function relationships in more detail.
seaweed Chondrus ocellatus (collection at the Fujian Agriculture and
Forestry University, China) (Guo et al., 2016).
2.2. Enzymatic preparation of KCOs
2. Materials and methods
Crude κ-carrageenases (pH = 7.34
0.05) (Guo et al., 2016) were
added to 0.5% (w/v) κ-carrageenan solution for preparation of KCOs
and incubated at 45 °C for 1 h, 4 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, and
72 h. Reactions were stopped by boiling at 100 °C for 5 min, then
cooled to room temperature. Products were centrifuged (10,528 × g,
10 min) to remove undigested fragments, and the supernatant was
2.1. Bacteria
The κ-carrageenan-hydrolysing bacterial strain Thalassospira sp.
Fjfst-332 was previously isolated from dried fragments of the red
Fig. 3. The chromatogram of κ-carrageenan oligosacchrides with (A) various flow rates (sample concentration is 0.6 g/mL, the loading amount is 1 mL, the mobile phase is 0.1 M
NH4HCO3); (B) various sample concentrations (flow rates is 4 mL/min, the loading amount is 1.0 mL, the mobile phase is 0.1 M NH4HCO3); (C) various loading amounts (flow rates is
4 mL/min, the sample concentration is 1.0 g/ mL, the mobile phase is 0.1 M NH4HCO3). The recovery rate of different components were calculated with dry powders, Rate% =
[component weight (g)/total weight (g)] × 100%. The total yield was calculated with the formula of Yield % = [Total weight of oligosaccharides (g)/Substrate of κ-carrageenan
(g)] × 100%. The component V was salty ions, which was analyzed with TLC (Fig. 2B).
316

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 3. (continued)
collected and filtered by tangential flow ultrafiltration using 10 kDa
and 3 kDa membranes (Pellicon XL, Millipore Inc., Massachusetts,
USA). Low molecular weight components ( < 3 kDa) were collected.
Fractions containing oligosaccharides were centrifuged and lyophilised
for further analysis.
column was equilibrated with 0.1 M NH4HCO3, and samples were in-
jected for purification. The effects of flow rate (1 mL/min, 2 mL/min,
3 mL/min and 4 mL/min), loading amount (0.5 mL, 1 mL, 2 mL and
3 mL) and sample concentration (0.3 g/mL, 0.6 g/mL, 1 g/mL and
1.2 g/mL) on the separation were investigated and optimal parameters
determined.
2.3. Separation and purification of KCOs
2.4. Resolution factor evaluation
KCOs were separated and purified by MPLC using a modified
method from that described previously (Lu et al., 2017). Data collection
and system operation were controlled using Interchim Software version
5.0 (Interchim Inc., Kennedy, France). A medium pressure HiLoad™
Superdex™ 30 prep grade (26 × 600 mm, max pressure: 5 bar, max
loading amount: 13 mL, adaptive flow rate: ∼0.9 − 4.4 mL/min) was
used for preparation with 0.1 M NH4HCO3 as eluent. Parameters for
Flash ELSD were as follows: purging nitrogen pressure: 3.4 bar, drift
tube temperature: 100 °C. KCO powders were dissolved in 0.1 M
NH4HCO3 and filtered through a 0.22-μm microporous membrane. The
The resolution factor (Rs) is an indicator of the separation efficiency
of chromatography (Urio and Masini, 2015) calculated using the fol-
lowing formula:
V − V
2
1
Rs =
.
(W + W )/2
1
2
where V is the appearance time of the peak, and W is the retention time
of the peak.
317

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 3. (continued)
2.5. TLC analysis of KCOs
were as follows: Drying gas: N2, flow rate: 5 L/min, temperature:
300 °C, nebulizer pressure: 45 psi, electrospray capillary voltage:
3500 V, temperature and flow rate of sheath gas: 300 °C and 11L/min,
respectively. Negative ionisation mode was used and the mass scan
range was ∼50 − 2500 amu. CID MS/MS was carried out with helium
(He) as the collision gas, and the collision energy was set at 70 V.
The KCOs were analyzed by the TLC (Thin Layer Chromatography).
The lyophilized samples were redissolved in deionized water (1 mg/
mL) and loaded on a 10 cm × 20 cm TLC plate. The plate was devel-
oped with a solvent mixture containing n-butane:ethanol:water (3:2:2,
v/v/v). The plate was stained by spraying a mixture of vitriol:ethanol
(3:17, v/v; with 0.2% resorcinol, w/v) and heating at 150 °C for 5 min
until the presence of visible bands.
2.7. NMR analysis
All NMR data were recorded on a Bruker Avance III HD 400 MHz
NMR spectrometer (Bruker Company, Fällanden, Switzerland) at 25 °C.
Purified samples were exchanged twice in D2O (99.7%) and redissolved
in D2O at 10 mg/mL. 1H-NMR spectra of KCOs were recorded using a
sweep width of 8223 Hz and 16 scans (Fidres of 0.125 Hz). 13C-NMR
spectra were recorded at 100 MHz with a sweep width of 24038 Hz and
1024 scans (Fidres of 0.367 Hz). 1H-1H COSY spectra were obtained
with a sweep width of 1014 Hz in both dimensions for KCO-1 (Fidres of
0.495 Hz), 925 Hz for KCO-2 (Fidres of 0.452 Hz), 927 Hz for KCO-3
2.6. ESI-MS and CID MS/MS analysis
Mass spectra of KCOs were analysed using a MS system (Agilent
7890A + 5975 B MS, California, USA) equipped with an electrospray
ionisation source. Data acquisition and processing were performed
using MassHunter Workstation software version B.04.00 (Agilent
Technologies Inc., California, USA). Analytes (1.0 mg/mL) were in-
jected into the mass spectrometer via a 5 μL loop. ESI-MS parameters
318

J. Guo et al.
CarbohydratePolymers180(2018)314–327
(Fidres of 0.452 Hz), and 2645 Hz for KCO-4 (Fidres of 1.292 Hz).
1H-13C HMQC experiments were recorded with a sweep width of
1114 Hz in both dimensions for KCO-1 (Fidres of 1.088 Hz), 793 Hz for
KCO-2 (Fidres of 0.775 Hz), 2645 Hz for KCO-3 (Fidres of 2.583 Hz),
and 2906 Hz for KCO-4 (Fidres of 2.906 Hz). 1H-13C HMBC spectra of
KCO-1 were recorded with a sweep width of 1014 Hz (Fidres of
0.495 Hz), 926 Hz for KCO-2 and KCO-3 (Fidres of 0.280 Hz), and
2645 Hz for KCO-4 (Fidres of 0.646 Hz).
2.8. Cell culture and treatment
The murine RAW264.7 macrophage cell line was purchased from
American Type Culture Collection (ATCC, Rockville, MD, USA).
RAW264.7 cells were cultured in 90% dulbecco's modified eagle
medium (DMEM) (12800-082 GIBCO, Carlsbad, California, USA) sup-
plemented with 10% fetal calf serum (FBS) (FSS500, ExCell Biology,
Carlsbad, California, USA), 100 U/mL of penicillin, 100 mg/mL of
Fig. 4. (A–D) ESI-MS and CID MS/MS spectrometry analysis of KCOs that were separated through MPLC-ELSD system. The fragment ions are annotated as [DA-G4S]n, where DA is 3,6-
anhydrogro-α-1,4linked-D-galactose, G4S is β-1,3-D-galactose with 4-O-sulfated groups, and n is the number of subunits. (E) The scheme of structural sequence. The non-reducing
terminal fragment ions are named as A-, B- or C- type ions, and reducing terminal are designed as X-, Y- or Z- type ions.
319

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 4. (continued)
320

J. Guo et al.
CarbohydratePolymers180(2018)314–327
488 nm and an emission wavelength of 530 nm.
Table 1
1H NMR data assignments for KCO-1, KCO-2, KCO-3 and KCO-4.
2.11. Statistical analysis
Residue
Chemical shift (ppm)
All experiments were performed in triplicate. Data were analysed
using the DPS statistical analysis system software (V9.50, Ruifeng
Information Company, Hangzhou, China) and Origin Pro (V8.5,
OriginLab, Wellesley Hills, Washington, USA). Data are presented as
H1
H2
H3
H4
H5
H6
KCO-1
α-DA(1→
→3)-G4Srα
→3)-G4Srβ
5.06
5.3
4.63
4.07
3.91
3.56
4.33
3.97
3.95
4.45
4.87
4.81
4.39
3.75
3.75
4.02/4.13
3.70/3.76
3.70/3.76
means
standard deviation (SD).
KCO-2
α-DA(1→
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
3. Results and discussion
5.06
4.63
5.04
5.27
4.63
4.11
3.56
4.06
3.87
3.56
4.38
3.95
4.31
3.97
3.95
4.46
4.81
4.59
4.87
4.81
4.39
3.79
4.39
3.75
3.79
4.02/4.18
3.70/3.76
4.02/4.18
3.71/3.76
3.70/3.76
3.1. Optimisation of parameters for MPLC separation of KCOs
A gel filtration column (HiLoad™ Superdex™ 30 prep grade) that is
capable of high resolution separation of the oligosaccharides and pep-
tides with molecular weight up to 10 kDa, was used in this study
(Knutsen et al., 2001). Its performance was evaluated using resolution
factor (Rs) (Ó’Fágáin et al., 2011). Before separation, the products of
various digested time were observed and monitored on TLC plates
(Fig. 2A). The components of various digested time were consistent, and
the oligosaccharides rang from disaccharide to hexasaccharide. There-
fore, the crude KCOs were prepared through 48 h degradation in the
next work.
KCO-3
α-DA(1→
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
5.06
4.63
5.04
5.28
4.63
4.1
4.31
3.95
4.31
4.12
3.95
4.44
4.82
4.59
4.85
4.82
4.37
3.77
4.61
3.75
3.77
4.02/4.21
3.70/3.77
4.02/4.18
3.71/3.76
3.70/3.77
3.56
4.06
3.87
3.56
KCO-4
α-DA(1→
α-DA2S(1→
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
5.04
5.25
4.61
5.07
5.28
4.61
4.07
4.65
3.6
4.11
3.86
3.55
4.3
4.44
4.64
4.82
4.56
4.87
4.82
4.37
4.61
3.78
4.63
3.78
3.78
4.01/4.18
4.01/4.18
3.74/3.78
4.01/4.18
3.74/3.78
3.74/3.78
4.82
3.99
4.64
4.11
3.98
3.1.1. Effect of flow rate on separation efficiency
The flow rate of mobile phase will affect the resolution and effi-
ciency. The effects of flow rate on the separation of κ-carrageenan
oligosaccharides were determined by comparing the chromatographic
performance at the flow rate ranging from 1–4 ml/min. A faster flow
rate significantly decreased the retention time of KCOs, but did not
decrease the chromatographic resolution of KCO peaks (Fig. 3A). When
the flow rate was 1 mL/min, the separation time was 5.5 h, and re-
sulting in overlap of peak IV. At a flow rate of 4 mL/min, the separation
time of KCOs was significantly shortened to less than 1.5 h. Compared
with the study that reported a elution time of 3 − 6 h (Knutsen et al.,
2001), the MPLC system performed faster separation time. On the other
hand, the Rs value (Rs1,2 = 1.78, Rs2,3 = 1.82, Rs3,4 = 1.62) and total
yield of KCOs (1.6%) at 4 mL/min were relatively higher, synthetically,
4 mL/min was the better flow rate for KCO separation.
*DA is 3,6-anhydrogro-α-1,4linked-D-galactose; DA’ is internal 3,6-anhydrogro-α-D-ga-
lactose; G4S is β-1,3-D-galactose-4-sulfate; DA2S is 3,6-anhydrogro-α-1,4linked-D-ga-
lactose-2-sulfate.
streptomycin, and maintained in an XD-101 incubator (SANYO,
Yamaguchi Prefecture, Japan) with 5% CO2 at 37 °C. Cells were pre-
incubated with purified KCOs (resuspended in DMEM to 10 μg/mL,
25 μg/mL and 50 μg/mL) and DXM (50 μg/mL, positive group) for 4 h,
exposed to 10 μg/mL LPS for 24 h, or treated with DMEM only (control
group) or LPS only (negative control) for 24 h.
2.9. Cell viability assay
RAW264.7 cells were seeded in 96-well plates at a density of
2 × 105 cells/mL and cultured overnight, then treated with KCO-1,
KCO-2, KCO-3 or KCO-4 for 24 h. Next, 20 μL of MTT (5 mg/mL in PBS,
0793 Amresco, California, USA) was added to each well and incubated
for another 4 h. The medium was removed carefully and 150 μL of
DMSO (Shanghai Yijiu Company, China) was added. Plates were shaken
for 10 min, and the optical density at 490 nm was measured with an
ELISA microplate reader (BioTek, ELx800, Norcross, Georgia, USA).
Cell viability was determined relative to untreated cells in the control
group.
3.1.2. Effect of sample concentration and loading amount on separation
efficiency
When the sample concentration was 1 g/mL, the cusp of peak III
was pointed and narrow, the Rs value (Rs1,2 = 2.44, Rs2,3 = 2,
Rs3,4 = 1.73) were higher than under other conditions, that indicating
better resolution (Fig. 3B). Furthermore, the total yield of KCOs (5.1%)
was the highest. Regarding loading amount, overloading can result in
severe superimposition and wider peaks, resulting in a lower yield.
When the loading amount was 1 mL, the Rs value was the best as
Rs1,2 = 2.63 Rs2,3 = 2 and Rs3,4 = 1.92 (Fig. 3C). Even through the
total yield of KCOs increased with the loading amount increased, the
resolution (Rs value) decreased significantly and the peak III was flat-
tened. Therefore, the loading amount of 1 mL is the most suitable
parameter. In summary, the optimal parameters for MPLC separation of
KCOs were a loading amount of 1 mL, a sample concentration of 1 g/
mL, and a flow rate of 4 mL/min.
2.10. Measurement of intracellular ROS
Intracellular ROS levels were measured using 2′-7′-dichloro-
fluorescin
diacetate
(DCFH-DA)
as
previously
reported
(Eruslanov & Kusmartsev, 2010). After treatment, cells were washed
with PBS, centrifuged (421 × g, 5 min), resuspended at a density of
1 × 106 cells/mL, and incubated with 10 μM DCFH-DA (Sigma Aldrich,
St. Louis, MO, USA) at 37 °C for 20 min in the dark. After washing with
PBS three times to remove free DCFH-DA, the fluorescence intensity
was captured using a FACS Calibur flow cytometer (Becton-Dickinson,
Franklin Lakes, New Jersey, USA) at an excitation wavelength of
3.2. ESI-MS Analysis of KCOs
After separation and purification, TLC was performed as a pre-
liminarily analysis of the molecular weight distribution of the five
321

J. Guo et al.
CarbohydratePolymers180(2018)314–327
components (Fig. 2B). Component V did not stain, indicating that it was
not a carbohydrate, and possibly corresponded to ions NaCl, NaNO3,
MgSO4·7H2O, K2HPO4, or CaCl2 that were added to produce κ-carra-
geenase in the fermentation medium (Guo et al., 2016). The other four
components were stained successfully, were of relatively high purity,
and were designated KCO-1, KCO-2, KCO-3 and KCO-4, based on their
structural sequences.
Thorough separation and detailed structural investigation of oligo-
monosaccharides are required before detailed structure and functional
relationship studies. MS analysis of KCOs was conducted at negative ESI
mode due to the presence of sulfate anion groups (Ekeberg,
Knutsen, & Sletmoen, 2001). In the mass spectrum of KCO-1 (Fig. 4A), a
solitary peak at m/z 403.1 was detected, which was assigned to κ-
carrabiose [(DA-G4S)]−, which has one sulfate group. The absence of
other signals indicated high purity. The MS image of KCO-2 (Fig. 4B)
revealed peaks at m/z 394.1 and m/z 806.2, corresponding to mole-
cular anions [(DA-G4S)2]2− and [(DA-G4S)2]− that were assigned to κ-
carratetraose with two sulfate units, and the doubly-charged [(DA-
G4S)2]2− anion. Peaks at m/z 391.1 and m/z 1209.1 were re-
presentative of κ-carrahexaose [(DA-G4S)3]3− and [(DA-G4S)3]− car-
rying three sulfate groups (Fig. 4C). The peak at m/z 547.2 corresponds
to desulfated doubly-charged κ-carrahexaose [(DA-G4S)2(DA-G)]2−
.
We speculated that desulfation due to the high cone voltage was re-
sponsible, rather than the presence of β-type COs (Anastyuk et al.,
2011; Sun et al., 2014). Peaks at m/z 587.1 and 1192.2 were assigned
to [(DA-G4S)3]2− and [(DA-G4S)3] −, which have lost a single OH−
group. Fig. 4D shows the mass spectrum of component KCO-4, with
peaks at m/z 313.1, 627.1 and 1272.1 corresponding to quadruple-,
doubly- and singly-charged molecular anions ι-carrahexaose [(An-
G4S)2(An2S-G4S)]4−
,
[(An-G4S)2(An2S-G4S)]2−-2OH− and [(An-
G4S)2(An2S-G4S)]−-2OH−, which all have four sulfate groups. Over-
sulfation was attributed to a common impurity of ι-carrageenan in
commercial κ-carrageenan (Sun et al., 2014). The peak at m/z 391.1
[(An-G4S)3]3− was also found in the mass spectrum, thus, component
KCO-4 might be the heterozygous κ/ι-carrahexaose. Furthermore, there
was no other mixed peak in the four MS spectra, indicating the high
purity of all four components. Characteristic ions of KCOs in mass
spectra are presented in supplementary material Table S1.
3.3. Analysis of CID-MS/MS spectra of KCOs
Detailed structural information on KCOs was obtained from CID
Fig. 5. (A) 1H-13C-HMQC spectra of KCO-1, KCO-2, KCO-3 and KCO-4. (B) 1H-1H-COSY spectra of KCO-1, KCO-2, KCO-3 and KCO-4. (C) 1H-13C-HMBC spectra of KCO-1, KCO-2, KCO-3
and KCO-4. The cross-peaks were labeled.
322

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 5. (continued)
MS/MS. In our study, there were no precursor ions added for deriva-
tisation, therefore, desulfation reactions occurred readily and the re-
sults were detected in the CID MS/MS spectra (Aguilan, Dayrit, Zhang,
Ninonuevo, & Lebrilla, 2006). Meanwhile, the method did not reduce
oligosaccharides using NaBH4 to generate oligosaccharide alditols, and
it was therefore not able to immediately differentiate between reducing
or non-reducing fragment ions with identical masses arising from gly-
cosidic cleavages (Sun et al., 2014; Yu et al., 2006).
were assigned (Table S2). The G4S groups arising from non-reducing
termini readily facilitate desulfation of B- and C-type ions (Yang et al.,
2011). However, there were no desulfated B- or C-type fragment ions
observed, indicating that oligosaccharides were assigned to κ-neo-
carratetraose with G4S at the reducing terminus, consistent with the
structural order displayed in Fig. 4E, and indicating that KCO-1 was κ-
neocarrabiose.
Based on enzyme specificity, component KCO-3 was κ-noecarra-
hexaose that carries G4S as the reducing terminal. When choosing
singly charged ions m/z 1209.2 as parent ions, peaks at m/z 997.4,
1014.8, 997.4, 1077.1, 1095.2, 1112.2, 1175.0 and m/z 1192.2 were
observed and sequences assigned (Table S2). These fragment structures
In the CID MS/MS profiles of κ-carrabiose using m/z 403.1 as the
parent ion, the fragment peaks m/z 113.1, 198.9 and 259.1 were re-
latively less abundant, since desulfation (HSO4−, m/z 97) would dis-
sipate energy. The peak at m/z 113.1 was assigned to the G4S group
following loss of HSO4 and CH2OH− [G4S-HSO4−-CH2OH−]. The
were consistent with κ-noecarrahexaose losing sulfate groups or OH−
.
−
peak at m/z 198.9 was the cross-ring cleavage fragment (G4S) corre-
The doubly charged ion m/z 587.1 was also chosen as a parent ion due
to the lower abundance of singly-charged ions. Peaks at m/z 97.0,
241.0, 385.1, 457.1, 529.0 and m/z 547.2 were assigned (Table S2). In
the obtained MS/MS profiles of κ/ι-neocarrahexaose (Fig. 4D), the
glycosidic cleavage fragment ions were detected at m/z 914.8, 1015.0,
1095.1 and 1175.0, but m/z 505 signals were not detected, indicating
that there were no [G4S-DA2S] fragment ions present. Thus, the com-
ponent was designated as κ/ι-neocarrahexaose with DA2S groups at the
non-reducing terminus, and the structural order is displayed in Fig. 4E.
sponding to 1,3A- or 1,3Z0-type. Additionally, we did not observe other
1
signals in the second-order spectrum, further indicating that KCO-1 was
κ-carrabiose.
Compared with doubly- or triply-charged molecular ions as pre-
cursors for CID MS/MS analysis, singly-charged ions can yield more
extensive structural information. MS/MS spectra of κ-carratetraose are
shown in Fig. 4B based on singly-charged m/z 806.2 as parent ions.
Fragment ions at m/z 97.0, 258.9, 385.2, 529.1, 565.1 and m/z 709.2
323

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 5. (continued)
3.4. Analysis of the structural sequence of KCOs
HMQC spectra made it possible to ascribe the correlations of H5 and H6
protons (Prechoux & Helbert, 2014). From the 13C-NMR spectrum of
KCO-2, the signal of the anomeric carbon at 101.96 ppm was found to
correspond to 1,3-β-G4S and the sulfated group substituted at position
4. Moreover, the non-reducing end of α-DA-C1 (94.21 ppm) indicated
that KCO-2 was κ-neocarratetraose with double repeating DA-G4S units
(α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β). The long-rang 1H-13C corre-
lations assigned in HMBC confirmed that G4S-C1 correlated with DA-
H4 through the 1,4-glycosidic linkage (Fig. 5C) (Zhang et al., 2010).
Based on the observation of the anomeric region of 1H-13C HMQC
spectra, four spin systems were found and the glycosyls were assigned
as pyranoses. Furthermore, the lack of a signal at 66.4 ppm (corre-
sponding to the G4S-C4 unit without a sulfate group) suggested that
desulfation did not occur during TF-332-κ-carrageenase enzymatic hy-
drolysis, which was also verified by ESI-MS analysis (Yuan & Song,
2005) (Table 2).
The end-products of the action of TF-332-κ-carrageenase were κ-
neocarrabiose, κ-neocarratetraose, κ-neocarrahexaose and κ/ι-neo-
carrahexaose, as confirmed by preparation and complete 1H and 13C
NMR spectra assignment (Table 1; 1H and 13C NMR spectra are shown
in Fig. S1). In the 1H NMR spectrum of KCO-1, the peak at 5.06 ppm is
the anomeric proton of 3,6-anhydrogro-α-D-galactose units, and re-
sonant signals at 5.3 ppm (G4Sre-α-H1) and 4.63 ppm (G4Sre-β-H1)
were observed. In the 13C-NMR spectrum, the anomeric carbon of G4S
with α and β configurations located at the reducing end were attributed
to peaks at 91.98 ppm and 96.17 ppm, which were shifted in the high-
field. In addition, the signal from G4S-C3 was shifted in the low-field,
indicating a glycosidic linkage located at position 3, which was con-
firmed by 1H-13C HMQC (Fig. 5A). Therefore, component KCO-1 was
designated as κ-neocarrabiose with a G4S unit at the reducing end, and
the structural formula is α-DA-1,3-G4Srα/β. The 1H-NMR spectrum of
KCO-2 was similar to that of KCO-1, except the signal at 5.04 ppm,
which was attributed to the α-anomeric proton of the internal 3,6-an-
hydrogro-α-D-galactose (α-DA’-H1). The ring proton of G4S was as-
NMR spectra of KCO-3 revealed similar characteristic peaks to those
of KCO-2, resulting from the close proximity of reducing and non-re-
ducing ends. In particular, the response value of 1,4-DA and 1,3-G4S
units were stronger than those observed for κ-neocarrabiose and κ-
neocarratetraose. Thus, component KCO-3 was designated as κ-
signed using 1H-1H COSY analyses from H1 to H4 (Fig. 5B). The 1H-13
C
324

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Table 2
sulfate groups on DA-2 may eliminate the cytotoxicity and benefit cell
growth, as observed with ι-carrageenan (G4S-DA2S) (Liang, Mao,
Peng, & Tang, 2014). In the present work, the heterozygous κ/ι-carra-
hexaoses appeared to have a synergistic effect for both G4S and DA2S,
since DA2S diminished the cytotoxicity of κ-carrahexaoses, resulting in
nontoxic compounds. Therefore, the degree of substitution and the
position of the sulfate groups on KCOs affected cytotoxicity, and an
acceptable concentration for anti-inflammatory assays was less than
75 μg/mL.
13C NMR data assignments for KCO-1, KCO-2, KCO-3 and KCO-4.
Residue
Chemical shift (ppm)
C1
C2
C3
C4
C5
C6
KCO-1
α-DA(1→
→3)-G4Srα
→3)-G4Srβ
94.22
91.98
96.17
69.2
66.53
70.2
80.5
77.92
77.98
69.67
74.58
73.54
76.99
74.19
74.19
68.65
61.05
61.05
KCO-2
α-DA(1→
94.21
101.96
94.27
91.97
96.3
69.71
69.2
69.71
66.86
70.2
80.45
78
78.76
77.93
78
69.58
73.39
77.9
74.84
73.43
76.96
74.36
76.96
74.51
74.16
69.13
60.84
69.2
61.13
61.03
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
3.6. Effect of purified KCOs on ROS levels in LPS-induced RAW 264.7
macrophages
ROS are key signalling molecules in the progression of inflammatory
disease, since they react and consequently damage cellular components
and induce stress signalling pathways (Mittal, Siddiqui, Tran,
Reddy, & Malik, 2014). The elevation of ROS production in in-
flammatory cells such as macrophages can cause tissue damage during
inflammatory responses (Venkatesan, Park, Choi, Lee, & Kim, 2017;
Zuo, Zhou, Pannell, Ziegler, & Best, 2015). In the present work, LPS
increased the production of ROS in RAW 264.7 macrophages, which
would damage mitochondrial membrane integrity and activate in-
flammatory signalling pathways. Pre-treatment with four purified KCOs
significantly decreased ROS levels in cells (Fig. 6B). Furthermore, κ-
neocarrahexaose displayed higher activity than κ-neocarrabiose and κ-
neocarratetraose, but κ/ι-carrahexaose performed the best of the four.
Inhibition of ROS production by κ/ι-carrahexaose on LPS-induced RAW
264.7 macrophages approached that of DXM (positive) controls at a
concentration of 50 μg/mL. These results suggest the degree of poly-
merisation, as well as the number of sulfate units of κ-neocarra-oligo-
saccharides, affects their anti-inflammatory activity. KCOs prepared
from H2O2 depolymerisation also suppress ROS production (Sun et al.,
2015). The lower ROS levels in RAW264.7 macrophages pre-treated
with KCOs might reduce the activities of MAPKs and NF-κB and en-
hance the mitochondrial membrane potential. Sulfated chitosan oligo-
saccharides reportedly inhibit LPS-induced iNOS in RAW 264.7 mac-
rophages via JNK (Kim et al., 2014). In addition, agar oligosaccharides
suppress pro-inflammatory mediator release by inducing heme oxyge-
nase 1 (Enoki et al., 2010), and diverse sulfated marine oligosacchar-
ides affect different signalling pathways depending on their structure.
However, the signalling pathways through which KCOs affect LPS-in-
duced RAW 264.7 macrophages remain unclear, and will be in-
vestigated in our future work.
KCO-3
α-DA(1→
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
94.24
101.98
94.24
91.9
69.26
68.94
69.71
66.86
68.94
80.41
77.74
80.45
77.93
77.74
69.54
73.34
77.9
74.84
73.34
76.9
68.83
60.82
69.2
61.13
60.82
74.37
76.96
74.51
74.37
96.3
KCO-4
α-DA(1→
α-DA2S(1→
→3)-β-G4S(1→
→4)α-DA’(1→
→3)-G4Srα
→3)-G4Srβ
94.28
91.63
101.92
94.34
91.97
96.16
68.99
78.31
68.73
80.43
77.42
77.58
79.02
74.5
69.54
69.39
73.31
77.76
71.73
73.31
76.94
76.48
74.33
76.24
74.33
74.33
69.16
69.16
60.78
69.16
61.03
61.03
66.86
68.64
77.58
*DA is 3,6-anhydrogro-α-1,4linked-D-galactose; DA’ is internal 3,6-anhydrogro-α-D-ga-
lactose; G4S is β-1,3-D-galactose-4-sulfate; DA2S is 3,6-anhydrogro-α-1,4linked-D-ga-
lactose-2-sulfate.
neocarrahexaose with the structural sequence α-DA-1,3-β-G4S-1,4-α-
DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β. Spectra of KCO-4 were similar to
those of κ-neocarrahexaose, yet the downfield signals observed at 5.25
and 91.96 ppm were attributed to sulfated DA at position 2 (α-DA2S)
(Jouanneau, Boulenguer, Mazoyer, & Helbert, 2010a). Therefore, the
constituent of KCO-4 was deemed to be heterozygous κ/ι-neocarra-
hexaose with DA and DA2S as non-reducing ends, and the structural
sequence is α-DA/DA2S-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-
G4Srα/β. The basic structures of κ-neocarrabiose, κ-neocarratetraose
and κ-neocarrahexaose are similar to those of other KCOs produced by
enzymatic hydrolysis, but different from those produced by acid hy-
drolysis of KCOs (Sun et al., 2015). In addition, heterozygous κ/ι-car-
rahexaoses are novel products from TF-332-κ-carrageenase that have
not been reported previously.
3.5. Cytotoxicity of the four purified KCOs on RAW264.7 macrophages
4. Conclusions
Previous work showed that KCOs exhibit immunomodulation ac-
tivity (Xu, Yao, Wu, Wang, & Zhang, 2012;Yao et al., 2014). However,
most research was conducted with mixed oligosaccharides. The cell
viability and potential cytotoxicity of purified κ-neo-oligosaccharides
(KCO-1, KCO-2, KCO-3 and KCO-4) on RAW264.7 macrophages were
evaluated (Fig. 6A). There was no evidence of cytotoxicity when the
four samples were used within a concentration of 75 μg/mL in MTT
assays. Cell viability was unaffected by the purified KCOs, while the
inhibition rate was increased at a concentration of 300 μg/mL. These
results were similar to those obtained with sulfated κ-carrageenan and
human umbilical vein endothelial cells (HUVEC), in which there was no
inhibition of cell growth when the concentration was lower than
100 μg/mL, and the viability was reduced to 50% when the con-
centration was higher than 600 μg/mL. Furthermore, substitution of
In the present study, four types of purified κ-carrageenan oligo-
saccharides were separated using MPLC-ELSD, and the total yield was
approximately 5.02%. The technique allows for large-scale separation,
and yielded κ-neocarrabiose, κ-neocarratetraose, κ-neocarrahexaose
and heterozygous κ/ι-neocarrahexaose, the structural sequences of
which were confirmed using ESI-MS, CID-MS/MS and NMR analyses. Of
the four, κ/ι-neocarrahexaose has highest degree of polymerisation and
the largest number of sulfated groups, and exhibited the highest in-
hibition of ROS production in LPS-induced RAW 264.7 macrophages.
Thus, the anti-inflammatory activity of κ-carrageenan oligosaccharides
is significantly affected by the degree of polymerisation and the number
of sulfated groups. Additional inflammatory cytokines and their effects
on signalling pathways will be studied in future in vivo and in vitro
work.
325

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Fig. 6. (A) Cytotoxicities of four purified KCOs in
RAW 264.7 macrophages determined by MTT assay.
▭ KCO-1,
KCO-2,
KCO-3, ■ KCO-4. (B)
Effect of four KCOs on ROS production in LPS-in-
duced RAW 264.7 cells.
326

J. Guo et al.
CarbohydratePolymers180(2018)314–327
Lu, X., Zheng, Z., Miao, S., Li, H., Guo, Z., Zhang, Y., Zheng, Y., Zheng, B., & Xiao, J.
(2017). Separation of Oligosaccharides from Lotus Seeds via Medium-pressure Liquid
Chromatography Coupled with ELSD and DAD. Scientific Reports, 7, 44174.
Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P., & Malik, A. B. (2014). Reactive oxygen
species in inflammation and tissue injury. Antioxidants Redox Signaling, 20,
1126–1167.
Acknowledgments
We gratefully acknowledge the financial support from the National
Marine Public Welfare Industry Special Scientific Research Projects
(No. 201505033) and the Science and Technology Major Project of
Fujian Province (No. 2015NZ0001-1).
Necas, J., & Bartosikova, L. (2013). Carrageenan: a review. Veterinami Medicina, 58,
187–205.
Niu, T. T., Zhang, D. S., Chen, H. M., & Yan, X. J. (2015). Modulation of the binding of
basic fibroblast growth factor and heparanase activity by purified lambda-carra-
geenan oligosaccharides. Carbohydrate Polymers, 125, 76–84.
Appendix A. Supplementary data
Ó’Fágáin, C., Cummins, P. M., & O’Connor, B. F. (2011). Gel-Filtration Chromatography.
Methods in Molecular Biology, 36, 1.
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.carbpol.2017.10.043.
Prechoux, A., & Helbert, W. (2014). Preparation and detailed NMR analyses of a series of
oligo-alpha-carrageenans. Carbohydrate Polymers, 101, 864–870.
Sun, Y., Liu, Y., Jiang, K., Wang, C., Wang, Z., & Huang, L. (2014). Electrospray ionization
mass spectrometric analysis of kappa-carrageenan oligosaccharides obtained by de-
gradation with kappa-carrageenase from Pedobacter hainanensis. Jouranl of
Agricultural and Food Chemistry, 62, 2398–2405.
Sun, Y., Yang, B., Wu, Y., Liu, Y., Gu, X., Zhang, H., Wang, C., Cao, H., Huang, L., & Wang,
Z. (2015). Structural characterization and antioxidant activities of kappa-carra-
geenan oligosaccharides degraded by different methods. Food Chemistry, 178,
311–318.
Urio, R. P., & Masini, J. C. (2015). Evaluation of Monolithic and Core-Shell Columns for
Separation of Triazine Herbicides by Reversed Phase High Performance Liquid
Chromatography. Journal of the Brazilian Chemical Society, 26, 2331–2338.
Venkatesan, T., Park, E. J., Choi, Y. W., Lee, J., & Kim, Y. K. (2017). Anti-inflammatory
activity of Ternstroemia gymnanthera stem bark extracts in bacterial lipopoly-
saccharide-stimulated RAW264.7 murine macrophage cells. Pharmaceutical Biology,
55, 837–846.
Wang, W., Zhang, P., Hao, C., Zhang, X. E., Cui, Z. Q., & Guan, H. S. (2011). In vitro
inhibitory effect of carrageenan oligosaccharide on influenza A H1N1 virus. Antiviral
Research, 92, 237–246.
Weber, P., Hamburger, M., Schafroth, N., & Potterat, O. (2011). Flash chromatography on
cartridges for the separation of plant extracts: rules for the selection of chromato-
graphic conditions and comparison with medium pressure liquid chromatography.
Fitoterapia, 82, 155–161.
Xu, L., Yao, Z., Wu, H., Wang, F., & Zhang, S. (2012). The immune regulation of kappa-
carrageenan oligosaccharide and its desulfated derivatives on LPS-activated micro-
glial cells. Neurochemistry International, 61, 689–696.
Yang, B., Yu, G., Zhao, X., Ren, W., Jiao, G., Fang, L., Wang, Y., Du, G., Tiller, C.,
Girouard, G., Barrow, C. J., Ewart, H. S., & Zhang, J. (2011). Structural character-
isation and bioactivities of hybrid carrageenan-like sulphated galactan from red alga
Furcellaria lumbricalis. Food chemistry, 124, 50–57.
Yao, Z. A., Xu, L., & Wu, H. G. (2014). Immunomodulatory function of kappa-carrageenan
oligosaccharides acting on LPS-activated microglial cells. Neurochemical Research, 39,
333–343.
References
Aguilan, J. T., Dayrit, F. M., Zhang, J., Ninonuevo, M. R., & Lebrilla, C. B. (2006).
Structural analysis of kappa-carrageenan sulfated oligosaccharides by positive mode
nano-ESI-FTICR-MS and MS/MS by SORI-CID. Journal of the American Society for Mass
Spectrom, 17, 96–103.
Alvarez-Suarez, J. M., Carrillo-Perdomo, E., Aller, A., Giampieri, F., Gasparrini, M.,
Gonzalez-Perez, L., Beltran-Ayala, P., & Battino, M. (2017). Anti-inflammatory effect
of Capuli cherry against LPS-induced cytotoxic damage in RAW 264.7 macrophages.
Food and Chemical Toxicology, 102, 46–52.
Anastyuk, S. D., Barabanova, A. O., Correc, G., Nazarenko, E. L., Davydova, V. N.,
Helbert, W., Dmitrenok, P. S., & Yermak, I. M. (2011). Analysis of structural het-
erogeneity of kappa/beta-carrageenan oligosaccharides from Tichocarpus crinitus by
negative-ion ESI and tandem MALDI mass spectrometry. Carbohydrate Polymers, 86,
546–554.
Caram-Lelham, N., Sundelöf, L. O., & Andersson, T. (1995). Preparative separation of
oligosaccharides from K −carrageenan, sodium hyaluronate: and dextran by
SuperdexTM 30 prep. grade. Carbohydrate Research, 273, 71–76.
Challal, S., Queiroz, E. F., Debrus, B., Kloeti, W., Guillarme, D., Gupta, M. P., &
Wolfender, J.-L. (2015). Rational and Efficient Preparative Isolation of Natural
Products by MPLC-UV-ELSD based on HPLC to MPLC Gradient Transfer. Planta
Medica, 81, 1636–1643.
Duan, F., Yu, Y., Liu, Z., Tian, L., & Mou, H. (2016). An effective method for the pre-
paration of carrageenan oligosaccharides directly from Eucheuma cottonii using
cellulase and recombinant κ-carrageenase. Algal Research, 15, 93–99.
Ekeberg, D., Knutsen, S. H., & Sletmoen, M. (2001). Negative-ion electrospray ionisation-
mass spectrometry (ESI-MS) as a tool for analysing structural heterogeneity in kappa-
carrageenan oligosaccharides. Carbohydrate Research, 334, 49–59.
Enoki, T., Okuda, S., Kudo, Y., Takashima, F., Sagawa, H., & Kato, I. (2010).
Oligosaccharides from agar inhibit pro-inflammatory mediator release by inducing
heme oxygenase 1. Bioscience Biotechnology Biochemistry, 74, 766–770.
Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and
flow-cytometry. Methods in Molecular Biology, 594, 57–72.
Guo, J., Zhang, L., Lu, X., Zeng, S., Zhang, Y., Xu, H., & Zheng, B. (2016). Medium
Optimization and Fermentation Kinetics for kappa-Carrageenase Production by
Thalassospira sp. Fjfst -332. Molecules, 21.
Jouanneau, D., Boulenguer, P., Mazoyer, J., & Helbert, W. (2010a). Complete assignment
of (1)H and (13)C NMR spectra of standard neo-iota-carrabiose oligosaccharides.
Carbohydrate Research, 345, 547–551.
Jouanneau, D., Boulenguer, P., Mazoyer, J., & Helbert, W. (2010b). Enzymatic de-
gradation of hybrid iota-/nu-carrageenan by Alteromonas fortis iota-carrageenase.
Carbohydrate Research, 345, 934–940.
Kim, J. H., Kim, Y. S., Hwang, J. W., Han, Y. K., Lee, J. S., Kim, S. K., Jeon, Y. J., Moon, S.
H., Jeon, B. T., Bahk, Y. Y., & Park, P. J. (2014). Sulfated chitosan oligosaccharides
suppress LPS-induced NO production via JNK and NF-kappaB inactivation. Molecules,
19, 18232–18247.
Knutsen, S. H., Sletmoen, M., Kristensen, T., Barbeyron, T., Kloareg, B., & Potin, P. (2001).
A rapid method for the separation and analysis of carrageenan oligosaccharides re-
leased by iota- and kappa −carrageenase. Carbohydrate Research, 331, 101–106.
Kofler, S., Nickel, T., & Weis, M. (2005). Role of cytokines in cardiovascular diseases: a
focus on endothelial responses to inflammation. Clinical Science, 108, 205–213.
Liang, W., Mao, X., Peng, X., & Tang, S. (2014). Effects of sulfate group in red seaweed
polysaccharides on anticoagulant activity and cytotoxicity. Carbohydrate Polymers,
101, 776–785.
Young, C. S., & Scientific, S. (2003). Success with Evaporative Light-Scattering Detection.
LC-GC Europe, 16, 132.
Yu, G., Zhao, X., Yang, B., Ren, S., Guan, H., Zhang, Y., Lawson, A. M., & Chai, W. (2006).
Sequence determination of sulfated carrageenan-derived oligosaccharides by high-
sensitivity negative-ion electrospray tandem mass spectrometry. Analytical Chemistry,
78, 8499–8505.
Yuan, H., & Song, J. (2005). Preparation: structural characterization and in vitro anti-
tumor activity of kappa-carrageenan oligosaccharide fraction from Kappaphycus
striatum. Journal of Applied Phycology, 17, 7–13.
Yuan, H., Song, J., Li, X., Li, N., & Dai, J. (2006). Immunomodulation and antitumor
activity of kappa-carrageenan oligosaccharides. Cancer Letters, 243, 228–234.
Yuan, H., Zhang, W., Li, X., Lu, X., Li, N., Gao, X., & Song, J. (2005). Preparation and in
vitro antioxidant activity of kappa-carrageenan oligosaccharides and their over-
sulfated, acetylated, and phosphorylated derivatives. Carbohydrate Research, 340,
685–692.
Zhang, W., Liu, M., Hansen, P. E., Yu, G. L., Yang, B., & Zhao, X. (2010). SEQUENCE AND
STRUCTURAL ANALYSIS OF kappa-CARRAGEENAN-DERIVED OLIGOSACCHARIDES
BY TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE1. Journal of Phycology,
46, 831–838.
Zuo, L., Zhou, T., Pannell, B. K., Ziegler, A. C., & Best, T. M. (2015). Biological and
physiological role of reactive oxygen species–the good, the bad and the ugly. Acta
Physiologica, 214, 329–348.
327