Industrial Crops & Products 159 (2021) 113079

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Industrial Crops & Products

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Extraction, structure characterization, carboxymethylation and antioxidant

activity of acidic polysaccharides from Craterellus cornucopioides
Yuntao Liu a, *, 1, Xiaoyu Duan a, 1, Mingyue Zhang a, Cheng Li a, Zhiqing Zhang a, Bin Hu a,
Aiping Liu a, Qin Li a, Hong Chen a, Zizhong Tang b, Wenjuan Wu c, Daiwen Chen d
a
College of Food Science, Sichuan Agricultural University, Yaan 625014, China
b
College of Life Science, Sichuan Agricultural University, Yaan 625014, China
c
College of Science, Sichuan Agricultural University, Yaan 625014, China
d
Key Laboratory of Animal Disease-Resistance Nutrition, Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China

A R T I C L E I N F O A B S T R A C T

Keywords: Herein, we isolated and purified polysaccharides from Craterellus cornucopioides. Chemical composition analysis
Craterellus cornucopioides revealed that both polysaccharides (CCPs-1 amd CCPs-2) were composed of different molar ratios of rhamnose,
Polysaccharides fucose, arabinose, xylose, mannose, glucose, and galactose. The structure of polysaccharides was determined
Structure characterization
using FT-IR, periodate oxidation, smith degradation, methylation analysis, and NMR, which demonstrated that
Carboxymethylation
Antioxidant activity
the two polysaccharides were primarily connected by mannose with (1→3)-linked. Notably, the configuration of
the two polysaccharides exhibited random coil with pyranoid polysaccharide containing α or β glycosidic bond.
Furthermore, to increase the antioxidant activity of polysaccharides (CCPs-1 amd CCPs-2), two carboxymethy­
lated polysaccharides were obtained as cmCCPs-1 and cmCCPs-2 with the degree of substitution values of 0.34
and 0.52, respectively. Structural analysis elucidated that nonselective carboxymethylation occurred in cmCCPs-
1 and cmCCPs-2, where C-2, C-4 or C-6 were partially substituted. Of note, cmCCPs-2 exhibited the most sig­
nificant antioxidant activity.

1. Introduction solubility and antioxidant abilities of polysaccharides (Xu et al., 2009;

Functional polysaccharides are naturally abundant materials that
possess essential pharmacological activities including immunoregula­
tory, anti-inflammatory, antioxidant, and antimicrobial. Therefore, they
are considered as one of the most promising new resources for the
development of drugs and nutraceuticals (Xu et al., 2009; Wang et al.,
2016a; Liu et al., 2020f). Specifically, previous works have demon­
strated that bioactivity of polysaccharides primarily depended on its
physicochemical property and molecular structure, such as water solu­
bility, chemical composition, main-chain, and branched-chain, etc.
(Ferreira et al., 2015; Liu et al., 2017a, 2017b). Chemical modifications
such as sulfated modification, acetylation, and selenylation (Liu et al.,
2017a, 2017b) are deemed to be an effective strategy to introduce the
functional groups, improve the waters solubility of those poorly
water-soluble fractions and increase the bioactivity of polysaccharides
(Xu et al., 2009; Wang et al., 2016a). Furthermore, it has been widely
accepted that carboxymethyl modification might enhance the water
Wang et al., 2016a; Liu et al., 2017a).
In the past few years, there has been a growing body of literature,
emphasizing separation, characterization, modification, and utilization
of different polysaccharides from various edible fungus (Xu et al., 2009;
Guo et al., 2019; Yang et al., 2011). For instance, polysaccharides
extracted from C. cornucopioides have been outlined to exhibit various
bioactive effects. In our previous study, we obtained C. cornucopioides
polysaccharides (CCPs) with a specific antioxidant activity (Liu et al.,
2020c). In addition, studies on the structure-activity relationship of
polysaccharides have shown that the bioactivities of most of the natural
polysaccharides were directly or indirectly affected by their structures
(Duan et al., 2020). However, the detailed structures and types of
polysaccharides from C. cornucopioides remains elusive, thus hindering
the investigation of their structure and activity relationships. Therefore,
it is necessary to further purify the CCPs to obtain high purity, and to
clarify its structural characteristics, so as to understand its
structure-activity relationship. Notably, compared with the positive

* Corresponding author at: College of Food Science, Sichuan Agricultural University, 46# Xinkang Road, Yaan, Sichuan 625014, China.
E-mail addresses: lyt_taotao@163.com, nutritionlab@sicau.edu.cn (Y. Liu).
1
The authors contributed equally to this article.

https://doi.org/10.1016/j.indcrop.2020.113079
Received 2 September 2020; Received in revised form 24 October 2020; Accepted 25 October 2020
Available online 4 November 2020
0926-6690/© 2020 Elsevier B.V. All rights reserved.
Y. Liu et al.
control, CCPs and their purified components depicted insufficient po­
tential to improve its biological properties and pharmacological appli­
cations. The structural modification of polysaccharides may change its
spatial configuration and biological activities. Besides, to the best of our
knowledge, there were no references to the antioxidant activity of car­
boxymethylated CCPs as well as the structural changes caused by car­
boxymethyl. In this context, we synthesized carboxymethylated
polysaccharides and then investigated their structure and antioxidant
activities compared with the native one. This was executed for further
understanding of the structure-function relationships of CCPs and ex­
pands the application of C. cornucopioides resources.
2. Materials and methods
2.1. Materials and chemicals
Craterellus cornucopioides was purchased and collected from Kunm­
ing, Yunnan, China, while DPPH (1, 1-diphenyl-2-picrylhydrazyl), BHT
(2, 6-di-tert-butyl-4-methylphenol), EDTA (ethylene diamine tetraacetic
acid), and standard monosaccharide samples were obtained from San­
gon Biotech (China). All other analytical reagent grade chemicals used
in this study were purchased from Sinopharm Chemical Reagent Co. Ltd,
China.
2.2. Isolation and purification of the polysaccharide
To obtain CCPs from crude polysaccharides of C. cornucopioides, they
were extracted according to the method we previously described (Liu
et al., 2020c), through hot water extraction, Sevag to remove protein
and ethanol for precipitation.
Here, CCPs were dissolved with ultra-pure water (100 mg/mL) and
then purified using a DEAE-52 column (3.5 cm × 20 cm). After loading
the CCPs solution, the column was eluted with twice the column volume
of ultra-pure water. Afterward, the column was also eluted with a
stepwise gradient of 0.1 M to 2.0 M NaCl at a flow rate of 1 mL/min. The
eluent was automatically collected (10 mL per tube), while the sugar
distribution was monitored using the phenol-sulfuric acid method.
Notably, the CCPs were divided into 2 peaks. The ratios based on crude
polysaccharide content were 32.36 % and 67.64 %. The obtained eluent
containing the carbohydrate was collected, concentrated, and lyophi­
lized. Sephadex G-200 chromatography column (2.6 cm × 80 cm) was
used to purify further the two fractions, which were then dissolved in
distilled water (30 mg/mL). Subsequently, the column was washed with
ultra-pure water at a flow rate of 0.3 mL/min. Finally, the eluent was
detected using the phenol-sulfuric acid method, then collected and
concentrated. The resultant solution was freeze-dried to obtain CCPs-1
and CCPs-2.
2.3. Characterization
2.3.1. Chemical composition analysis
The CCPss’ (CCPs-1 and CCPs-2) chemical compositions were
analyzed as previously described (Liu et al., 2020c). Afterward, the
meta-hydroxybiphenyl method was used to assess the content of uronic
acid, while the monosaccharide compositions of polysaccharides were
evaluated using gas chromatography (GC, Shimadzu GC-14A) equipped
with flame ionization detector (250 ◦ C) and PEG20 M silica capillary
column (30 m ×0.53 mm I.D.). Setting procedure is as follows: initial
column temperature was maintained at 150 ◦ C for 3 min, increased to
240 ◦ C at a rate of 3 ◦ C/min and maintained for 20 min.
2.3.2. Structure determination of C. cornucopioides polysaccharides
(CCPs)
2.3.2.1. Molecular weight (Mw). The molecular weight of CCPs-1 and
2
Industrial Crops & Products 159 (2021) 113079
CCPs-2 was measured using HPGPC with an Agilent 1100 HPLC system.
Setting procedure is as follows: the column was maintained at 30 ◦ C and
eluted with ultrapure water at a flow rate of 0.8 mL/min. According to a
technique described in our previous study (Liu et al., 2020c).
2.3.2.2. Fourier transform infrared (FT-IR) spectra. The infrared spec­
trum was acquired using the FT-IR spectrometer at room temperature
(25 ◦ C). In particular, the polysaccharides were blended with KBr
powder (1:100, w/w), and pressed into pellets for the collection of
infrared spectra in a range of 400–4000 cm− 1 at 4 cm− 1 resolution using
32 scans for three or more times.
2.3.2.3. Analysis of glycosidic bond connection mode. Periodate oxida­
tion and Smith degradation analysis were performed based on a previ­
ously established method (Liu et al., 2020g), whereas methylation
analysis was assessed according to a reported method with some mod­
ifications (Liu et al., 2020g).
Fifty mg polysaccharides were proton-exchanged in deuterium oxide
(D2O, 99.9 %) three times, lyophilized, and then redissolved in 1 mL of
D2O. Chemical shifts were expressed in ppm using tetramethylsilane
(TMS). Thereafter, 1H nuclear magnetic resonance (1H NMR), 13C nu­
clear magnetic resonance (13C NMR), 1H-1H correlated spectroscopy
(1H-1H COSY), and heteronuclear single-quantum coherence (13C-1H
HSQC) were recorded using an NMR apparatus (AVANCE II 400 M,
Bruker, Germany). The NMR test parameters were given in Table S1.
2.3.3. Conformation studies of C. cornucopioides polysaccharides (CCPs)
2.3.3.1. Solubility determination. The water-solubility test of poly­
saccharides from C. cornucopioides was performed based on a previously
reported method with slight modification (Sharifian-Nejad and Shek­
archizadeh, 2019). Specifically, polysaccharide samples (100 mg) were
placed into a centrifuge tube and weighed as W0. Then, distilled water
(10 mL) was added and mixed for a certain time (0, 5, 10, 15, 20, 25, 30,
40, 50, 60, 120, 180, 240, 300 min) at room temperature. After centri­
fugation (8000 rpm, 30 min), we removed supernatant and dried the
centrifuged tubes at 55 ◦ C for 30 min. Consequently, the centrifuged
tubes were weighed as W1. Lastly, the water-solubility of polysaccharide
samples was computed using the following formula:
Water-solubility (%) = (W0-W1)/ W0 × 100 %
2.3.3.2. Congo red analysis. Congo red staining was used to test the
interaction of polysaccharides by examining the visible absorption
maximum wavelength (range of 400–600 nm) according to a previous
method (Liu et al., 2017a).
2.3.3.3. The I2-KI analysis. To determine the degree of branching of
polysaccharides and their derivatives, the I2-KI experiment was used,
while dextran T10 was applied as a positive control to detect the
absorbance in the wavelength range of 300–700 nm after reacting with
I2-KI reagent (0.02 % I2 and 0.2 % KI, w/v) at 20 ◦ C for 15 min (Guo
et al., 2019).
2.4. The carboxymethylation of polysaccharides
2.4.1. The cmCCPs preparation
The carboxymethylation modification of polysaccharide from
C. cornucopioides (cmCCPs) was performed as previously described with
slight modification (Liu et al., 2017a). Briefly, 200 mg CCPs, 8 mL iso­
propyl alcohol, 4 mL 20 % (v/v) dropwise sodium hydroxide solution
and carboxymethyl agent (including 4 g chloroacetic acid, 2 mL 20 %
NaOH and 4 mL isopropanol) were added one at a time to form a mixed
solution and allowed to react at 60 ◦ C for 3 h. After the reaction was
Y. Liu et al. Industrial Crops & Products 159 (2021) 113079

complete, the solution was cooled and the pH adjusted to 7. Then, the Table 1
mixture was dialyzed (3 kDa) against ultra-pure water for 48 h and Molecular weight (Mw), uronic acid, and monosaccharides composition (%) of
precipitated with 95 % ethanol. After centrifugation (5000 rpm, CCPs-1 and CCPs-2.
10 min), the precipitates were lyophilized for 48 h to yield cmCCPs-1 Process CCPs-1 CCPs-2
and cmCCPs-2. Mw (Da) 1.38 × 105 2.73 × 105
Uronic acid content (%) 5.50 ± 0.03a 11.36 ± 0.25b
2.4.2. Characterization of cmCCPs Monosaccharides composition (%)
After the yields of cmCCPs-1 s were obtained, the DS (degree of Rhamnose 0.19 0.19
Fucose 8.28 6.05
substitution) was examined according to our previous study (Liu et al.,
Arabinose 0.33 0.40
2017a). Next, the structure of cmCCPs-1 and cmCCPs-2 was character­ Xylose 28.05 31.78
ized (FT-IR and NMR) as elucidated in section 2.3.2.2 and 2.3.2.3, while Mannose 42.75 43.76
their conformation (Water-solubility, the interaction of polysaccharides, Glucose 6.36 9.38
and branching of polysaccharides) was determined as outlined in section Galactose 14.05 8.44

2.3.3. CCPs, polysaccharides from C. cornucopioides; CCPs-1, eluted with ultra-pure

2.5. Determination of the antioxidant activity in vitro
Antioxidant activities (DPPH free radical scavenging activity,
reducing power, and metal chelating activity) were determined as pre­
viously described (Zhang et al., 2018).
2.6. Statistical analysis
All numerical variables are presented as the mean plus standard
deviation of the mean. The statistical significance was assessed using
Tukey’s, while its significance level was set at p < 0.05.
water obtained polysaccharides by DEAE-52, and purified further by Sephadex
G-200; CCPs-2, eluted with 0.1 M NaCl obtained polysaccharides by DEAE-52,
and purified further by Sephadex G-200.
However, the composition of the monosaccharides differed from those
of C. cornucopioides polysaccharides noted in other previous studies (Guo
et al., 2019; Yang et al., 2018), and the differences could be attributed to
the enhanced release of more C. cornucopioides polysaccharides due to
change in the extraction time or the concentration of acid, temperature
and time of hydrolysis (Shi et al., 2020).
3.3. Fourier transform infrared (FT-IR) analysis

3. Results and discussion The features of functional groups in CCPs-1 and CCPs-2 were
examined using FT-IR spectroscopy (Fig. 1). The FT-IR spectra of CCPs-1
3.1. Extraction and purification of CCPs and CCPs-2 exhibited absorption in bands at 3382, 2925, 1601, 922, and
893 cm− 1, which are typical bands for carbohydrates (range
Herein, CCPs were obtained using hot water extraction and ethanol 4000− 400 cm− 1). Moreover, the FT-IR spectra of CCPs (CCPs-1 and
sedimentation method resulting in a yield of 3.89 % based on the dry CCPs-2) were similar, thereby indicating that purification induced no
weight of raw material. After deproteinization and decolorization, the significant effects on the structure feature of CCPs (Liu et al., 2020c). In
DEAE-52 column and Sephadex G-200 chromatography column were
employed to fractionate and purify CCPs. As depicted in Figure S1, the
fractions obtained after the purification using the DEAE-52 cellulose
column and Sephadex G-200 chromatography column, yielded a single
and symmetrical peak named CCPs-1 and CCPs-2. The calculated rela­
tive weight average molecular weights of CCPs-1 and CCPs-2 were
1.38 × 105 and 2.73 × 105 Da, respectively.
Based on the previous reports, the DEAE 52-cellulose and Sepharose
CL-4B column was adopted to purify polysaccharide extracted from
CCPs, and the relative weight average molecular weight of CCPs ob­
tained was 9.2 × 105 Da (Yang et al., 2018). In another study by Guo
et al. (2019) used a DEAE-sepharose CL-6B column, and the relative
weight average molecular weight of the water-soluble polysaccharide
was 1.97 × 103 kDa. A major reason for this discrepancy in the molec­
ular weight of polysaccharides between these two studies and our
research might be the source or extraction method (Zhang et al., 2020).

3.2. Chemical components of CCPs-1 and CCPs-2

The contents of uronic acid and monosaccharides composition are

outlined in Table 1. These findings suggest that the two polysaccharides
are acidic, where CCPs-2 contained the highest amount of uronic acid
residues.
The GC results of monosaccharides composition analysis revealed
that CCPs-1 was mainly composed of rhamnose, fucose, arabinose,
xylose, mannose, glucose, and galactose in the ratio of 0.19 %, 8.28 %,
0.33 %, 28.05 %, 42.75 %, 6.36 %, and 14.05 %, respectively, whereas
CCPs-2 was primarily comprised of rhamnose, fucose, arabinose, xylose,
mannose, glucose, and galactose in the ratio of 0.19 %, 6.05 %, 0.40 %,
31.78 %, 43.76 %, 9.38 %, and 8.44 %, respectively. Notably, mannose
may be the backbone of the main chain of CCPs-1 and CCPs-2. This Fig. 1. Fourier-transforms infrared spectroscopy analysis of the CCPs-1,
finding was consistent with the previous research by Guo et al. (2019). cmCCPs-1 (A) and CCPs-2, cmCCPs-2 (B).

3
Y. Liu et al.
summary, CCPs (CCPs-1 and CCPs-2) possessed the characteristic ab­
sorption patterns of polysaccharides, which primarily composed of py­
ranose rings with different molecular vibrations and structures.
3.4. Periodate oxidation and Smith degradation analysis
Based on the calculation, the ratio of consumed NaIO4 and produced
HCOOH during oxidation was 7.95:1 of CCPs-1 (consumed NaIO4 of
0.735 mmol and the production of HCOOH was 0.092 mmol), which
indicated the existence of (1→)-linked or (1 → 6)-linked or little branch.
In addition, the consumption of periodate was much higher than the
release of HCOOH, which implied the presence of (1→2), (1→2,6),
(1→4), or (1→4,6) glycosidic bonds in CCPs-1. Besides, periodate was
not completely consumed, which also indicates that linkage types which
cannot be oxidized by periodate such as (1→3), (1→2,3), (1→3,4),
(1→3,6) and (1→2,4), might either exist or extensive in CCPs-1 (Guo
et al., 2019; Liu et al., 2020e).
After dialysis, reduction and acetylation reaction, GC–MS analysis
was conducted (Table S2). The presence of rhamnose, fucose, xylose,
mannose, glucose, and galactose implied that their partial residues
contained (1→3)-linked, (1→3,4)-linked, (1→2,4)-linked or (1→2,3,4)-
linked, which were not oxidized. Additionally, the mannose content was
88.37 %, which indicated that it is the primary main chain of CCPs-1.
Besides, a few glycerols were detected in the products, which reflected
that there were either (1→)-linked, (1→2)-linked or (1→6)-linked in
CCPs-1. However, there was no erythritol found in the products, thus
suggesting that (1→4)-linkage or (1→4,6)-linkages residues were ab­
sent. Of note, the amount of ethylene glycol produced was higher than
that of glycerol, and there was no erythritol, which might be the
oxidative degradation of xylose (five-carbon sugar). Therefore, there
were contain (1→2)-linked or (1→4)-linked xylose in CCPs-1. Hence, the
linkages of the backbone of the polysaccharide of CCPs-1 were inferred
to be (1→)-linked, (1 → 3)-linked, (1 → 6)-linked, (1→2)-linked, (1→4)-
linked or (1 → 3,6)-linked.
Similarly, the periodate oxidation findings and the changes in smith
degradation products of CCPs-2 were close to CCPs-1. In this context, the
linkages of the backbone of polysaccharide of CCPs-2 were also inferred
to be (1→)-linked, (1 → 3)-linked, (1 → 6)-linked, (1→2)-linked, (1→4)-
linked or (1 → 3,6)-linked.
Fig. 2. The NMR spectra of CCPs-1 and cmCCPs-1, (A) 1H NMR of CCPs-1; (B) 13C HMR of CCPs-1; (C) DEPT 135 of CCPs-1; (D) 1H NMR of cmCCPs-1; and (E) DEPT
135 of cmCCPs-1.
4
Industrial Crops & Products 159 (2021) 113079
3.5. Methylation analysis
Results of methylation analysis (Table S3) revealed that the largest
residue in the CCPs-1 structure was the glycosidic bond of mannose
which included Manp-(1→ (18.38 %) and →3,6) Manp-(1→ (29.56 %).
The second-largest residue in the structure was the glycosidic bond of
xylose containing 15.39 % of →3)-Xylp-(1→ and 10.21 % of Xylp-(1→.
These were followed by the glycosidic bond of galactose containing →3)-
Galp-(1→ (9.67 %), Galp-(1→ (3.82 %) and →2,4)-Galp-(1→ (1.24 %),
glycosidic bond of fucose including →3,4)-Fucp-(1→ (6.24 %), glyco­
sidic bond of glucose, consisting of Glcp-(1→ (2.57 %) and →6)-Glcp-
(1→ (2.61 %), and glycosidic bond of arabinose, →3)-Araf-(1→ (0.31
%). The inferences were consistent with the results of the periodate
oxidation and Smith degradation.
Similarly, the following 12 components →3,4)-Fucp-(1→, →3)-Araf-
(1→, →3)-Xylp-(1→, Xylp-(1→, →4)-Xylp-(1→, Manp-(1→, →3,6)-
Manp-(1→, Glcp-(1→, →6)-Glcp-(1→, →3)-Galp-(1→, Galp-(1→, →2,4)-
Galp-(1→ —were found at a molar ratio of 5.13: 0.34: 6.26: 8.15: 17.56:
19.86: 25.37: 2.79: 4.23: 7.84: 1.68: 0.79.
3.6. NMR spectroscopy analysis
The 1H, 13C and 13C-135 NMR spectra for CCPs-1 and CCPs-2 are
shown in Figs. 2 and 3 . The signals of protons derived from − CH3 (C-6)
of deoxy-sugar were generally seen in the region of 0.8–1.4 ppm (here is
fucose) (Li et al., 2020), and the signals for − CH3 protons of O-acetyl
groups (− OCOCH3) were in the region of 1.8–2.2 ppm (Liu et al.,
2020a). Generally, residues with anomeric protons chemical shifts
exceeding δH 4.95 ppm were in α-configuration, because chemical shift
below δH 4.95 ppm indicated the existence of β-configuration (Li et al.,
2015). In the 13C NMR spectrum, the resonances in the region of δC
95.0–110.0 ppm were assigned to the anomeric carbon atoms, and the
chemical shift range of the α-configuration is usually δC 90− 102 ppm
(Liu et al., 2020a). Thus, the residues in CCPs-1 and CCPs-2 contained
both α-configuration and β-configuration. The absence of 13C NMR
spectrum of signals in the region δ 83–88 reflected that all sugar residues
of CCPs-1 and CCPs-2 were in the pyranose form (Velichko et al., 2020),
which consistent with the results of FT-IR.
Since the 13C NMR signals of the two polysaccharide samples were
crowded and the content of some monosaccharide residues was low, the
Y. Liu et al.
Fig. 3. The NMR spectra of CCPs-2 and cmCCPs-2, (A) 1H NMR of CCPs-2; (B) 13C HMR of CCPs-2; (C) DEPT 135 of CCPs-2; (D) 1H NMR of cmCCPs-2; and (E) DEPT
135 of cmCCPs-2.
number of sugar residues cannot be determined from 13C NMR. There­
fore, it was necessary to determine the type of the primary glycosidic
bond and the specific position of the corresponding H and C atoms by
combining 2D NMR correlation spectra (Figure S2, S3, S4, S5).
Analysis of the 1H NMR spectrum (Fig. 2A) indicated that CCPs-1 had
eight main anomeric proton signals, indicating that CCPs-1 contained
many types of sugars. The eight major anomeric proton signals at 5.27,
5.20, 5.03, 4.98, 4.89, 4.83, 4.67 and 4.31 ppm, exhibited relative in­
tegrals of 1.0: 1.37: 4.89: 2.11: 2.62: 0.48: 0.45: 5.14. The 1H NMR spin
systems of the polysaccharide were assigned based on the 1H-1H COSY
spectrum (Figure S2) and the direct CH coupling was determined by the
1 13
H- C HSQC spectrum (Figure S3). Eight pairs of 1H signals of anomeric
CH attributed to eight different types of glycosidic bonds were assigned
to δ5.27/99.48, 5.20/99.71, 5.03/101.32, 4.98/101.55, 4.89/97.79,
4.83/98.26, 4.83/98.26, 4.65/102.91 and 4.31/103.48 based on their
chemical shifts in the 1H and 13C analysis and corresponding HSQC
spectra, labeled A1-H1. Comparing the chemical shift data of similarly
substituted sugar residues with the above monosaccharide composition,
periodate oxidation and smith degradation, methylation and infrared
spectrum results indicated that the nine types of glycosidic bonds
confirmed the existence of nine organic residues in CCPs-1. For example,
the signal at δC 99.48 ppm that correlated with the anomeric proton
signal at δH 5.27 was found in the HSQC spectrum for CCPs-1. Combined
with the COSY spectrum and published data, H-2 (3.57 ppm), H-3
(3.79 ppm), H-4 (3.97 ppm), H-5 (4.14 ppm) and H-6 (1.16 ppm) were
unambiguously assigned (Table 2). The values of signals for C-1 to C-6
were assigned based on the HSQC spectra. The field signals of C-2
(72.45 ppm), C-3 (66.53 ppm), C-4 (69.82 ppm), C-5 (66.44 ppm) and
C-6 (15.42 ppm) suggested that residue was →1)-α-L-Fuc- (Li et al.,
2020). Similarly, the other residues were assigned as follows: residual
B1: →2)-α-L-Xylp-(1→ (Shakhmatov et al., 2020), residual C1:
α-D-Manp-(1→ (Huo et al., 2020), residual D1: →3)-α-D-GalpA-(1→
(Kostalova and Hromadkova, 2019), residual E1: →3)-α-D-Xylp(1→
(Huo et al., 2020; Hu et al., 2020), residual F1: →3)-α-D-Manp-(1→
(Kokoulin et al., 2020), residual G1: →1)-β-D-Glcp-(6→ (Perepelov et al.,
2018; Liu et al., 2020g), residual H1: →6)-β-D-Manp-(1→ (Wang and
Guo, 2020; Liu et al., 2020b).
The 1H NMR spectrum of fraction CCPs-2 contained nine main peaks
in the anomeric region, which were labeled A2 to I2. The 13C NMR
spectrum of CCPs-2 had a signal at a low field ranging from δC
5
Industrial Crops & Products 159 (2021) 113079
160–180 ppm (Fig. 3B), which illustrated that CCPs-2 contained uronic
acid. This was consistent with the result of the determination of uronic
acid test in section 3.2 (Wang et al., 2016b). Similarly, these structural
residuals were determined individually as follow: residues A2:
→1)-α-L-Fuc- (5.27/99.52) (Li et al., 2020), residues B2:
→2)-α-L-Xylp-(1→ (5.20/99.58) (Shakhmatov et al., 2020), residues C2:
α-D-Manp-(1→ (5.03/101.35) (Huo et al., 2020), residues D2:
→3)-α-D-GalpA-(1→ (4.97/101.51) (Kostalova and Hromadkova, 2019),
residues E2: →3)-α-D-Xylp(1→ (4.89/97.82) (Huo et al., 2020; Hu et al.,
2020), residues F2: →3)-α-D-Manp-(1→ (4.82/98.39) (Kokoulin et al.,
2020), residues G2: →1)-β-D-Glcp-(6→ (4.66/102.87) (Perepelov et al.,
2018; Liu et al., 2020g), residues H2: →4)-β-D-Xylp-(1→ (4.42/102.66)
(Sigida et al., 2020), residues I2: →6)-β-D-Manp-(1→ (4.31/103.46)
(Wang and Guo, 2020; Liu et al., 2020b). The entire assignment of
chemical shifts is summarized in Table 2. Monosaccharide composition
analysis confirmed that CCPs-2 was mainly composed of fucose, xylose,
mannose, glucose and galactose, which is consistent with glycoside
residues.
3.7. Carboxymethylation modification
3.7.1. Characterization of cmCCPs
In the past, we have done much research to improve the activities of
polysaccharides (Liu et al., 2020c, 2020d). Afterward, the findings
indicated that carboxymethylation is an effective method to modify the
polysaccharides. This method of modifying polysaccharides included a
two-step etherification reaction. Herein, the calculated DS values for
CCPs-1 and CCPs-2 were 0.34 ± 0.04 and 0.52 ± 0.06, respectively,
which deemed that substitution occurred. Additionally, the yields of the
native polysaccharides for CCPs-1 and CCPs-2 were 71.85 ± 1.04 % and
80.45 ± 0.69 %, respectively (data not shown).
Fourier-transforms infrared (FT-IR) spectrum of cmCCPs-1 and
cmCCPs-2 (Fig. 1) revealed three new strong peaks at 1601 cm− 1,
1419 cm− 1, and 1313 cm− 1 after carboxymethylation modification.
Thereafter, these peaks were assigned to asymmetrical and symmetrical
COO- stretching vibrations (Liu et al., 2017a). Remarkably, these find­
ings sufficiently indicated that the native polysaccharide was carbox­
ymethylated successfully.
The 1H and 13C NMR spectrum (DEPT-135) of cmCCPs-1 and
cmCCPs-2 provided more detailed structural information (see Figs. 2
Y. Liu et al. Industrial Crops & Products 159 (2021) 113079

Table 2 activity value. Additionally, its solubility and dissolution kinetics are
1
H and 13C NMR chemical shift data (δ, ppm) for CCPs-1 and CCPs-2. thus pivotal specifications in a large number of technological application
Residue H/ 1.00 2.00 3.00 4.00 5.00 6.00 processes. Figure S6A illustrates the solubility of CCPs, CCPs-1, CCPs-2,
C cmCCPs-1, and cmCCPs-2 as a function of dissolution time. Notably,
CCPs-1 CCPs were steadily soluble in water and reached their maximum degree
→1)-α-L- 1
H 5.27 3.57 3.79 3.97 4.14 1.16 of solubility (around 95 %) after 5 h. In particular, its two fractions
A1
Fuc- 13
C 99.48 72.45 66.53 69.82 66.44 15.42 (CCPs-1 and CCPs-2) were almost completely dissolved in water after
1
→2)-α-L- H 5.20 3.69 3.88 4.06 40 min. The water solubility of cmCCPs-1 and cmCCPs-2 was enhanced
B1 13
Xylp-(1→ C 99.71 71.77 71.27
α-D- 1
H 5.03 3.81 4.12 4.35
compared with original polysaccharide, and this perhaps may be due to
C1 Manp- 13
the increase in hydrophilic carboxyl groups (Liu et al., 2017a).
C 101.32 78.73 75.19
(1→
1
→3)-α-D- H 4.98 4.00 4.14 4.31 3.7.3. Congo red test
D1 GalpA- 13
C 101.55 71.81 66.44 Congo red (CR) test is a useful, simple, and fast method that has been
(1→
→3)-α-D- 1
H 4.89 3.74 4.04 4.14 extensively applied to examine the helical conformation of poly­
E1 13 saccharides. Furthermore, the order-disorder transitions in a helical
Xylp(1→ C 97.79 77.40 70.09 66.44
→3)-α-D- 1
H 4.83 3.69 3.87 4.05 polymer are usually accompanied by changes in the visible absorption
F1 Manp- 13 spectra of the complexes formed with congo red (Liu et al., 2017a).
C 98.26 71.77 78.46 70.09
(1→
→1)-β-D- 1
H 4.65 3.44 3.78 3.97 4.14
Elsewhere, compared with the congo red (control group), the λmax of the
G1 13 complexes with a polysaccharide, which possessed a triple helix struc­
Glcp-(6→ C 102.91 71.70 69.49 69.82 66.44
→6)-β-D- 1
H 4.31 3.21 3.55 3.86 4.04 ture, showed a bathochromic shift and thereby forming a metastable
H1 Manp- 13
C 103.48 65.14 76.26 78.46 70.09 region in a certain concentration range. Here, the comparison results of
(1→
the congo red experiment of C. cornucopioides polysaccharides samples
CCPs-2
→1)-α-L- 1
H 5.27 3.56 are depicted in Figure S6B. The λmax of the CCPss and the cmCCPss,
A2 13 when mixed with congo red, gradually decreased and indicated irregular
Fuc- C 99.52

B2
→2)-α-L- 1
H 5.20 3.70 changes with the increase in NaOH concentration (0− 0.8 mol/L).
13
Xylp-(1→ C 99.58 Collectively, the above arguments indicated that CCPs-1, CCPs-2,
1
α-D- H 5.03 3.81 4.10
cmCCPs-1, and cmCCPs-2 exhibited no triple helix structure. Simulta­
C2 Manp- 13
C 101.35 66.55 68.62 neously, the descending behavior of the λmax for the CCPss-CR and
(1→
→3)-α-D- 1
H 4.97 3.71 4.00 cmCCPss-CR complex was elucidated in low NaOH concentration
D2 GalpA- 13
C 101.51 71.61 66.80 (0− 0.3 mol/L). These findings enumerated that CCPss posses a rigid
(1→
1 conformation, whereas their highly ordered conformation may convert
→3)-α-D- H 4.89 3.75
E2
Xylp(1→ 13
C 97.82 69.47 into a flexible conformation under alkaline conditions (Wang et al.,
→3)-α-D- 1
H 4.82 3.71 2016c).
F2 Manp- 13
C 98.39 71.61
(1→ 3.7.4. The I2-KI analysis
1
H 4.66 3.47
The result (Figure S6C) showed that the maximum absorption of
→1)-β-D-
G2 13
Glcp-(6→ C 102.87 71.67
→4)-β-D- 1
H 4.42 3.25 3.87 polysaccharides reacting with I2-KI reagent was 350 nm, whereas there
H2
Xylp-(1→ 13
C 102.66 73.36 65.10 was no absorption peak at around 565 nm for each sample. Thus, indi­
1
→6)-β-D- H 4.31 3.21 3.36 3.52 3.85 4.12 cating that there were many branches on the CCPss and cmCCPss mo­
I2 Manp- 13
C 103.46 73.36 69.43 69.33 65.10 68.62 lecular backbone. This finding corresponded with the study of Guo et al.
(1→
(2019).

and 3). The 5 major anomeric proton signals at 5.20, 5.06, 4.91, 4.73, 3.8. The antioxidant activity in vivo
and 4.64 ppm for cmCCPs-1, compared with those of CCPs-1, revealed
that it was difficult to identify the carbon signals in 13C NMR spectrum of The antioxidant activities of CCPss (CCPs-1 and CCPs-2) and its
cmCCPs-1, which severely overlapped with one another (Fig. 2E). This carboxymethylation fractions (cmCCPs-1 and cmCCPs-2) are depicted in
may due to the impact of the carboxymethyl group that may have Table 3. Herein, the polysaccharide of cmCCPs-2 exhibited the greatest
changed the chemical environment around every group thus weakening activity to scavenge DPPH radicals (EC50 = 136.0 μg/mL), while CCPs-1
the 13C NMR resonance signals (He et al., 2015). Compared with the possessed the lowest scavenging activity (EC50 = 233.2 μg/mL). These
spectrum of CCPs-1, the new negative peaks that appeared around δC findings implied that chemically modified polysaccharides have
71.26, 70.16, 69.59, 68.12 and 67.20 ppm were assigned to C-6 (the five
major C-6 signals at 66.43, 65.17, 61.00, 60.72 and 60.21 ppm for Table 3
CCPs-1), and this down-field shift may due to the carboxymethylation of Antioxidant properties of polysaccharides and its carboxymethylation fractions.
C-6 (Liu et al., 2017a). Furthermore, the enhancement in peak in­
Samples DPPH (EC50) Reducing power Metal chelating activity
tensities 69.42 ppm, and the new peak appeared at around 76.01, 75.24 (EC0.5) (EC50)
were possibly due to the substitution of − CH2COOH group at C-4 or C-2
CCPs-1 233.2 ± 14.4c 210.5 ± 13.4c 535.7 ± 45.7d
position. Based on the above information, we proposed that the sub­ CCPs-2 191.8 ± 19.5bc 190.1 ± 11.2c 480.6 ± 17.8c
stitutions occurred at C-6, C-4, or C-2 (Yang et al., 2011). Similarly, as cmCCPs-1 180.6 ± 16.4bc 191.5 ± 16.3bc 475.9 ± 24.1c
illustrated in Fig. 3D, E, it revealed that nonselective carbox­ cmCCPs-2 136.0 ± 9.9ab 148.9 ± 14.1ab 418.3 ± 25.9b
ymethylation has occurred for cmCCPs-2, that is, C-2, C-4 or C-6 were Positive 89.0 ± 13.9a 138.5 ± 14.8a 304.0 ± 22.6a
control
being partially substituted.
Each value is expressed as the mean ± standard deviation (n = 3). Means with
3.7.2. Solubility determination different letters within a column are significantly different (p < 0.05). Positive
Polysaccharides are commonly prepared in a powdery form and must control, BHT was used as a positive control for DPPH radical scavenging activity
be dissolved in an aqueous solution before being of any significant and reducing power activity, while EDTA was used as a positive control for metal
chelating activity.

6
Y. Liu et al.
favorable DPPH radical scavenging activity. However, the discrepancy
in scavenging activity between the two polysaccharides might be due to
the difference in the degree of substitution, molecular weight, uronic
acid content, and monosaccharide composition of polysaccharides.
Moreover, the activity of polysaccharide to provide hydrogen is an
important factor in antioxidant activity. In another study, it was
demonstrated that the carboxymethylation components of poly­
saccharides elucidated a stronger antioxidant effect than native poly­
saccharides, which was consistent with the results of our study (Liu
et al., 2017a). Their carboxyl groups behave like electrophiles to facil­
itate the intramolecular hydrogen abstraction.
Based on Table 3, the reducing power of the polysaccharides from
caps ranked as cmCCPs-2 > CCPs-2 > cmCCPs-1 > CCPs-1. In particular,
the reducing power of CCPs-2 was greater compared to that of the CCPs-
1, which may correlate with the structural properties of the
polysaccharides.
Similar to DPPH free radical scavenging activity and reducing power,
carboxymethylated polysaccharides exhibited higher metal chelating
activity. The findings are also similar to the reports of the antioxidant
activities of the carboxymethylated derivatives from blackcurrant fruits
which improved scavenging OH activity compared to native poly­
saccharides (Daun et al., 2020).
3.9. Correlation relationship analysis between structure and activity
Notably, it is well known that the antioxidant activity of poly­
saccharide largely depends on its structure (molecular weight, mono­
saccharide composition, amount of hydroxyl group, type of glycosidic
linkage, degree of branching, the changes of advanced stereotactic
structure, and chain conformation). However, in this case, it was noted
that the antioxidant activity of CCPss (CCPs-1 and CCPs-2) was not a
function of a single factor but rather a combination of several factors. In
this study, the polysaccharides that exhibited higher Mw possessed
stronger antioxidant properties, which agreed with our previous
research (Zhang et al., 2018). On the other hand, Gu et al. (2020) re­
ported that the polysaccharides from Sagittaria sagittifolia L. with lower
Mw possessed stronger antioxidant activity because the polysaccharide
with low Mw can present a higher content of reducing end and accept
more free radicals. Therefore, the inconsistency in findings between
these studies suggests that the polysaccharide Mw is not the primary
factor affecting its antioxidant activity. For instance, the effects of
glucose and mannose on the activity of antioxidants were significant
according to the result of Zhang et al. (2016). Also, galactose, mannose,
arabinose, rhamnose, xylose, etc. are important compositions of the
polysaccharide, which have antioxidant activity (An et al., 2020). Be­
sides, studies have outlined that high acidic monosaccharide contents
are favorable for the antioxidant activities of polysaccharides (Miao
et al., 2020), this is consistent with our research results. Polysaccharides
(CCPs-1 and CCPs-2) possessed better performances in antioxidant as­
says, acidic polysaccharides were more likely act as secondary antioxi­
dants because the carboxyl groups of the uronic acid of the
polysaccharides might play the role of hydrogen-donating and
electron-transfer agent (Zhang et al., 2013).
In addition, the DPPH radical scavenging activity and ferrous ion
reducing power of cmCCPs-2 were close to BHT, which indicated that
cmCCPs-2 possessed high antioxidant activity. Studies have shown that
the presence of polyelectron materials can enhance the free radical
scavenging activity of antioxidants, a major reason for this finding may
be attributed to the fact that the carboxymethylated samples were more
water-soluble and had a higher surface area, which facilitated their
contact with the radical (Liu et al., 2017a). According to the previous
study, some functional groups such as OH, COOH, PO3H2, and CO can
combine with Fe2+. Therefore, this perhaps may explain the high Fe2+
chelation activity of cmCCPs associated with these functional groups
(Cao et al., 2020).
Numerous previous studies have isolated different polysaccharides
7
Industrial Crops & Products 159 (2021) 113079
from C. cornucopioides and demonstrated that they have antioxidant
activities themselves through in vitro studies (Yang et al., 2018; Liu et al.,
2020d). However, the relationship between their antioxidant property
and the Nrf2 signaling pathway remains enigmatic. Hence, this neces­
sitates more research to explore the relationship between the structure
and activity of C. cornucopioides polysaccharides and their role in the
signaling pathway, particularly through in vivo experiments.
4. Conclusions
In summary, CCPs-1 (1.38 × 105 kDa) and CCPs-2 (2.73 × 105 kDa)
obtained from C. cornucopioides, are all pyranoid polysaccharides con­
taining α or β glycosidic bond formed by (1→3)-links with mainly
monosaccharides (mannose, xylose). Structural analysis revealed that
nonselective carboxymethylation occurred in cmCCPs-1 and cmCCPs-2.
Besides, the relationships between structure and activities of CCPss and
its derivatives were established based on antioxidant activities in vitro.
Results indicated that cmCCPss had more significant effects than CCPss
at the same dosage, showing that carboxymethylation modification is an
effective method for enhancing the antioxidant activities of natural
polysaccharides. Introduction of functional groups improved the water-
solubility and surface area of the polysaccharides, making them interact
with free radicals. Collectively, our results show that derivatization of
CCPss improves its bioactivities, hence makes it applicable in functional
food and pharmaceutical materials.
CRediT authorship contribution statement
Yuntao Liu: Validation, Conceptualization, Visualization, Writing -
review & editing, Funding acquisition, Supervision. Xiaoyu Duan:
Conceptualization, Data curation, Formal analysis, Methodology,
Writing - original draft. Mingyue Zhang: Methodology, Writing - orig­
inal draft. Cheng Li: Supervision. Zhiqing Zhang: Resources. Bin Hu:
Methodology. Aiping Liu: Visualization. Qin Li: Software. Hong Chen:
Project administration. Zizhong Tang: Validation. Wenjuan Wu:
Investigation. Daiwen Chen: Supervision.
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgments
This work was financially supported by the Sichuan Science and
Technology Program (2020JDRC0085); the Scientific Research Project
of Sichuan Cuisine Development Research Center (CC20Z09); the
Technology Innovation R&D Project of Chengdu City (2019-YFYF-
00045-SN).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2020.113079.
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