International Journal of Biological Macromolecules 117 (2018) 473–482

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Structural characterization and antioxidant activities of a novel

polysaccharide fraction from the fruiting bodies of
Craterellus cornucopioides
Wei-Wei Yang a, Li-Ming Wang a, Li-Li Gong a, Yong-Ming Lu a,b, Wen-Juan Pan a, Ya Wang a,
Wen-Na Zhang a,⁎, Yan Chen a,b,⁎⁎
a
School of Life Sciences, Anhui University, Hefei 230601, Anhui, PR China
b
Key Laboratory of Modern Biomanufacturing of Anhui Province, Hefei 230601, Anhui, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A new polysaccharide fraction (CCPP-1) was obtained from Craterellus cornucopioides. CCPP-1 had an average
Received 19 April 2018 molecular weight of 9.2 × 105 Da, which was mainly composed of mannose, glucose, xylose, arabinose, fructose
Received in revised form 16 May 2018 in molar ratio of 0.7:0.05:0.18:1:0.05. Results of structural characterization revealed that the dominant linkage
Accepted 28 May 2018
types of CCPP-1 were →3, 6)-Manp(1→, T-Araf, →4, 6)-Manp (1→, →5)-Araf (1→ and →3)-Araf (1→. Interesting,
Available online 29 May 2018
in vitro antioxidant activities assays showed that CCPP-1 possessed strong scavenging abilities on DPPH and ABTS
Keywords:
radicals. The oxidative hemolysis induced by AAPH in mice erythrocytes was effectively reversed by incubation
Craterellus cornucopioides polysaccharide with CCPP-1. CCPP-1 significantly prevented AAPH-induced intracellular reactive oxygen species (ROS) genera-
Structural characterization tion. Moreover, CCPP-1 could significantly restore AAPH-induced increase of intracellular antioxidant enzyme
Antioxidant activities glutathione peroxidase (GPx) and catalase (CAT) activities to normal level, as well as inhibit intracellular
malondialdehyde (MDA) formation. Therefore, CCPP-1 could protect against AAPH-induced oxidative-stress in
erythrocytes, which would be explored as naturally potential antioxidant agent applied in food and cosmetic
fields.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction antioxidant defense systems. When an imbalance appears between

The medicinal properties of mushrooms have been exploited for
centuries in health maintenance and prevention of diseases, particularly
in Asian countries. Fungal polysaccharides, as one of functional compo-
nents existed in edible and medicinal mushrooms, have received more
attention due to the potential biological functions, such as, anti-aging,
antibacterial, antioxidant activities and immunomodulatory effects
[1–4]. Nowadays, it was demonstrated that oxidant damage played an
important role during the process of various chronic human diseases.
As we all know, excessive reactive oxygen species (ROS) and other
free radicals (FR) are indeed involved in the occurrence of oxidative
damage including peroxidation of membrane lipids and disturbance of
DNA integrity, which can lead to structural and functional disorders,
aging, inflammatory and neurodegenerative diseases [5]. Under normal
circumstances, ROS and FR production are counteracted by the
⁎ Corresponding author.
⁎⁎ Correspondence to: Y. Chen, Key Laboratory of Modern Biomanufacturing of Anhui
Province, Hefei 230601, Anhui, PR China.
E-mail addresses: vinale87@163.com, (W.-N. Zhang), chenyan91030@yahoo.com
(Y. Chen).
the generation and the elimination of ROS under pathological condi-
tions, oxidative damage occurs. Therefore, free radical-mediated oxida-
tive stress and antioxidant are studied by more and more researchers.
However, synthetic antioxidants frequently-used were restrict due to
their potential hazards to health. Thus, the focus has recently moved
to identify more benign natural antioxidants from edible or medicinal
fungi.
Erythrocytes, potentially powerful promoters of oxidative processes,
are extremely susceptible to oxidative damage because of the high poly-
unsaturated fatty acid content of their membranes and their high cellu-
lar oxygen hemoglobin concentrations [6]. In cell models, oxidative
stress can be induced by many reagents including 2, 2-azobis (2-
amidinopropane) dihydrochloride (AAPH). Thus, an AAPH-induced
erythrocyte hemolysis assay can visually reveal antioxidant activities
by measuring ROS and malondialdehyde (MDA) levels, as well as activ-
ities of intracellular antioxidant enzyme activities (glutathione peroxi-
dase, GPx; and catalase, CAT) [7].
Craterellus cornucopioides, belonged to the family of Cantharellaceae,
is a highly nutritious edible fungus distributed worldwidely [8]. Previ-
ous studies were focus on the content of phytonutrient including
ergosterol, sesquiterpenoids, total phenolic and flavonoid [9–11].

https://doi.org/10.1016/j.ijbiomac.2018.05.212
0141-8130/© 2018 Elsevier B.V. All rights reserved.
474 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482
O'Callaghan reported that the ethanolic extract of C. cornucopioides pos-
sessed certain anti-inflammatory effects by decreasing NO and IL-6 in
LPS-stimulated mouse macrophage cells [12]. Moreover, the ethanolic
and aqueous crude extract of C. cornucopioides exhibited moderate anti-
oxidant activities [13]. The sesquiterpenoids isolated from cultures of
C. cornucopioides possessed strong cytotoxic activities [14]. As we all
know, the structure-activity relationships of polysaccharides have
been extensively investigated, and many factors have been shown
to beneficial for exerting on the antioxidant activities including the
moderate molecular weight, higher branch degree, versatile linkage
types and complex conformation [15–18]. However, up to now, no in-
formation is available regarding the chemical characteristics and related
antioxidant activities of polysaccharides from the fruiting bodies of
C. cornucopioides.
Therefore, the aim of the study was to purify the polysaccharide frac-
tions from C. cornucopioides and determine the structure characteristics
by Fourier transform infrared (FT-IR), monosaccharide analysis and nu-
clear magnetic resonance (NMR). Furthermore, the antioxidant activi-
ties of polysaccharide were evaluated in vitro, including its DPPH and
ABTS free radical scavenging abilities and protective effect against 2,
2′-azo-bis (2-amidinopropane) hydrochloride (AAPH)-induced oxida-
tive stress in mice erythrocytes.
2. Materials and methods
2.1. Materials and reagents
Dried fruiting bodies of C. cornucopioides were obtained
from Yunnan Province, China. DEAE 52-Cellulose column and
Sepharose CL-4B were purchased from Pharmacia Biotech. A series
of different molecular weight dextrans, standard monosaccharides,
1,1-Diphenyl-1-picrylhydrazyl (DPPH), trifluoroaceticacid (TFA),
2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonate) (ABTS), 2,2-
azobis (2-amidinopropane) dihydrochloride (AAPH) and ascorbic
acid were purchased from the Sigma Chemical Co (St. Louis, MO,
USA). An MDA kit was purchased from Nanjing Jiancheng Institute
of Biotechnology (Jiangsu, China). Kits for the determination of cellu-
lar GPx, CAT and ROS were purchased from Beyotime Institute of Bio-
technology (Shanghai, China). Male Kunming mice were obtained
from Anhui Medical University. All other chemicals and reagents
were analytical grade.
2.2. Extraction and purification of the polysaccharide
The crude polysaccharides were extracted following the method of
previous study with some modifications [19]. The dried powder of
C. cornucopioides was refluxed with ethanol at 80 °C for 4 h to remove
the pigment and lipid [20]. Then, the ethanol-extracted residue was ex-
tracted with hot water (90 °C) for 3 times, each time for 2 h. The super-
natant was combined, concentrated, and precipitated with four
volumes of ethanol for 12 h at 4 °C. Precipitates were dissolved in dis-
tilled water, and then Sevag solution (chloroform:n-butyl alcohol, 4:1)
was added. The mixed solution was shaken vigorously for 2 h at room
temperature and centrifuged at 5000 ×g for 10 min. The procedure
was repeated until the protein was completely removed. The water
layers were collected and dialyzed with 14,000 Da for 72 h, After that,
the dialysis liquid was collected and lyophilized to obtain a yellowish
crude polysaccharide (CCPP). CCPP (10 mg/mL) were dissolved in dis-
tilled water and then centrifuged at 5000 ×g for 10 min. 10 mL of super-
natant was applied to DEAE 52-cellulose column (2.5 × 30 cm) and
eluted successively with distilled water and 0.1–0.5 M NaCl solutions
at a flow rate of 1 mL/min. The elution fractions (5 mL/tube) were col-
lected automatically and detected based on phenol sulfuric acid
method. The eluates obtained from 0.3 M NaCl was subjected to further
purification by Sepharose CL-4B (2 × 100 cm) chromatography and
eluted with distilled water at a flow rate of 0.5 mL/min. The eluate
(5 mL/tube) was collected and detected as described above. Finally,
the main polysaccharide fraction was concentrated, lyophilized and
named CCPP-1.
2.3. Structural characteristics of CCPP-1
2.3.1. Components analysis
Total carbohydrate content of CCPP-1 was determined by the
phenol sulfuric acid method with D-glucose as the standard [21]. Protein
contents were quantified according to Bradford assay with (bovine
serum albumin) BSA as the standard [22].
2.3.2. Determination of the average molecular weight
The purity and molecular weight of CCPP-1 were determined by
ELSD-HPLC. Based on our previously established method [23], an
Ultrahydrogel™ 1000 colum (300 × 7.8 mm, Tosoh Corp, Tokyo,
Japan) column was eluted with distilled water at 35 °C with flow rate
of 1.0 mL/min. The molecular weight of CCPP-1 was calculated accord-
ing to the calibrated curve established by standard dextrans T-series
(T-10, T-40, T-70, T-500 and T-1000).
2.3.3. UV–vis spectroscopic analysis
CCPP-1 was dissolved in distilled water, and then UV–vis absorption
spectra was recorded in wavelength range of 200–650 nm using a TU-
1901 UV–vis spectrophotometer (Beijing Purkinje General Analysis In-
strument Co., Ltd., Beijing, China).
2.3.4. Scanning electron microscope (SEM) analysis
CCPP-1 was coated with a thin layer of gold under reduced pressure
and then examined with a field emission scanning electron microscope
(Hitachi S-4800, Japan) at an accelerating voltage of 3.0 kV. The image
magnifications were set as 500×, 1000× and 2000×.
2.3.5. Fourier-transform infrared spectra (FT-IR) analysis
IR spectroscopic of the polysaccharide was recorded using a Fourier
transform infrared spectrophotometer (FT-IR). 1 mg of CCPP-1 was
mixed with 150 mg of dried KBr powder and then pressed into 1 mm
pellet for FT-IR determination in the frequency range of
4000–400 cm−1 [24].
2.3.6. Monosaccharide composition analysis
10 mg of CCPP-1 was hydrolyzed in 5 mL of 2 mol/L trifluoroacetic
acid (TFA), followed by complexing with 1-phenyl-3-methyl-5-
pyrazolone (PMP) (0.5 mol/L) [25]. The resulting products were
then analyzed by HPLC equipped with a ZORBAX SB-C18 column
(150 × 4.6 mm, particle size 5 μm, Agilent Technologies, CA, USA)
and a UV detection at 245 nm. Elution was carried out at a flow
rate of 1.0 mL/min at 30 °C. The mobile phase A consisted of acetoni-
trile and the mobile phase B was 200 mmol/L ammonium acetate
using a gradient elution of 86–80–74% B by a linear decrease from 0
to 20–30 min. The injection volume was 10 μL.
2.3.7. Methylation and GC–MS analysis
Methylation analysis was determined according to previous method
with some modifications [25]. The vacuum-dried polysaccharide CCPP-
1 (20 mg) was methylated with methyl iodide three times to methylate
completely. The completeness of methylation was confirmed by the dis-
appearance of the hydroxyl absorption in IR spectrum. Then methylated
products were hydrolyzed with TFA for 6 h at 100 °C, followed by reduc-
tion with NaBH4 and acetylation with acetic anhydride. The resulting
mixture of methylatedalditol acetates were analyzed by gas
chromatography–mass spectrometry. The degree of branching (DB)
could be calculated with the following formula [26]:
.
DB ¼ ðNBþNTÞ ð1Þ
ðNBþNTþNLÞ
 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482
where NT, NB and NL are the total numbers of the terminal residues,
branched residues, and linear residues, respectively.
2.3.8. NMR spectroscopy analysis
Briefly, CCPP-1 (50 mg) was dissolved with 1.0 mL D2O in NMR tube
and then the 13C NMR, 1H NMR, HSQC, HMBC and 1H\\1H COSY spectra
were recorded on a NMR apparatus (AVANCE IIIHD 600, Bruker, USA) at
298 K.
2.4. Antioxidant activity assay
2.4.1. Assay of DPPH free radical scavenging activity
The DPPH free radical scavenging activity was measured referring to
reported method with some modifications [27]. Briefly, 0.13 mmol/L so-
lution of DPPH in an hydrous methanol (2 mL) was added in 2 mL of
sample solution at various concentrations (50–400 μg/mL). The mixture
was shaken vigorously and kept at room temperature for 30 min in
dark, the absorbance of the mixture was measured at 517 nm using
the spectrophotometer (UV-1750, Shimadzu). Ascorbic acid (Vc) was
used as positive control. The scavenging activity was calculated as fol-
lows:
 . 
Scavenging activity ð%Þ ¼ 1−ðA2 −A1 Þ  100%
A0
where A0 is the absorbance value of DPPH-methanol solution (2 mL)
plus methanol (2 mL). A1 is the absorbance value of the mixture of
methanol (2 mL) plus sample (2 mL) with different concentrations. A2
is the absorbance value of DPPH-methanol solution (2 mL) plus sample
(2 mL) with different concentrations.
2.4.2. Total antioxidant capacity
The ABTS assay was carried out according to previous method with
slight modifications [28]. ABTS radical solution was produced by mixing
7 mmol/L ABTS• + solution with 2.5 mmol/L potassium persulfate, and
the mixture was incubated in the dark at room temperature for 14 h. At
the moment, the ABTS• + solution was diluted with PBS (PH 7.0) to an
absorbance of 0.70 ± 0.02 at 734 nm. The polysaccharide samples were
dissolved in PBS to form sample solutions with final concentrations of
50, 100, 200, 300, 400, 500 μg/mL, respectively. Then 10 μL of polysac-
charide samples were mixed with 200 μL of the diluted ABTS• + solution
and the absorbance value was measured at 734 nm. Ascorbic acid was
Fig. 1. Elution curve of CCPP on DEAE 52-Cellulose column (A), Elution curve of CCPP-1 on Sepharose CL-4B column (B).
475
used as positive control. The ABTS• + scavenging effect was calculated
by the following formula:
 . 
Scavenging activity ð%Þ ¼ 1−ðA2 −A1 Þ  100% ð3Þ
A0
where A0 is the absorbance value of the ABTS•+ solution with 10 μL of
PBS. A2 is the absorbance value of the ABTS•+ solution in presence of
samples with different concentrations. A1 is the absorbance value of
the samples solution in the absence of ABTS•+.
2.4.3. Preparation of erythrocyte suspension
The intracellular antioxidant activity of CCPP-1 was measured as the
inhibition of erythrocyte hemolysis based on the previous procedure
with some modifications [7]. Briefly, the blood was obtained from
healthy Male Kunming mice. Erythrocytes were isolated from the
plasma by centrifugation (1500 g, 5 min), then washed three times
with PBS buffer (PH 7.4) and finally re-suspended using the same buffer
to an hematocrit level of 10% and stored at 4 °C. 0.2 mL of CCPP-1 with
various concentrations (100–600 μg/mL, in PBS 7.4) or PBS was added
to 10% suspension of erythrocyte. The reaction mixture was incubated
at 37 °C for 20 min with gentle shaking, after which 0.4 mL of
100 mmol/L AAPH was added, and the incubation continued at the
ð2Þ same temperature for another 2 h. Finally, the reaction solution was di-
luted with 8 mL of PBS buffer and centrifuged at 1200 g for 10 min at 4
°C. The absorbance (A) of the supernatant was measured at 540 nm.
Similarly to achieve complete hemolysis, 8 mL of distilled water was
added to the mixture, which was then centrifuged at 1200 g for
10 min at 4 °C,and the absorbance (B) of the supernatant was deter-
mined at 540 nm. The percentage of hemolysis inhibition was calculated
using the following formula:
 . 
%hemolysis inhibition ¼ 1−A  100% ð4Þ
B
where A refers to the absorbance A and B refers to absorbance B as
mentioned above.
2.4.4. Determination of ROS generation
A ROS determination kit with DCFH-DA as a fluorescent probe was
used to determine the relative levels of intracellular ROS. Treated eryth-
rocytes were harvested by centrifugation at 1200g for 10 min at 4 °C,
washed twice with PBS buffer, and then suspended in PBS buffer. The
476 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482

Fig. 2. HPLC-ELSD of CCPP-1 (A), UV spectra of CCPP-1 (B), Chromatograms of monosaccharide compositions of CCPP-1 (C), Chromatograms of eight monosaccharide standards (D) (The
asterisk indicates solvent peak. 1-mannose, 2-galactose, 3-rhamnose, 4-glucuronic acid, 5-glucose, 6-xylose, 7-arabinos, and 8-fructose), Scanning electron micrographs of CCPP-1 (E), FT-
IR spectrum of CCPP-1 (F).

cell suspension was incubated with DCFH-DA to a final concentration of

10 μM at 37 °C for 20 min. DCFH-DA is converted to the fluorescent com-
pound 2′, 7′-dichlorofluorescin (DCF) within cells. The fluorescence in- Table 1
tensity of cells was measured by BioTek microplate reader (ex/em, 488/ GC–MS analysis of methylated CCPP-1.
525 nm) to monitor the generation of ROS within cells. Methylated Molar Mass fragments (m/z) Linkage
sugar ratios indicateda
2.4.5. Determination of MDA content, enzymes activities of CAT and GPx 2,3,5-Me3-Araf 8.23 43,59,71,87,101,117,129,161 T-
Erythrocytes were collected and washed as described above. A mi- 3,5-Me2-Araf 2.46 43,71,87,101,129,147,161,189 1,5
croscale MDA kit was used to monitor MDA content. The cellular GPx 2,3-Me2-Araf 1.08 43,71,87,101,117,129,161,189 1,2
Kit, and CAT assay Kit were used to measure the activities of GPx and 2,5-Me2-Araf 2.08 43,71,87,99,101,117,129,143,161,233 1,3
Total 13.85
CAT according to the manufacturer's instructions.
2, 7.98 43,71,87,99,101,117,129,161,189,233 1,3,6
4-Me2-Manp
2.4.6. Eryptosis analysis 2,3-Me2-Manp 2.50 43,85,99,117,161,201,261 1,4,6
Treated cells were collected by centrifugation at 300g at 4 °C for Total 10.48
2,3-Me2-Xylp 1.09 43,57,71,87,189 1,4
5 min. The cells were washed twice with pre-cooled PBS and centri-
3,4-Me2-Xylp 1.07 43,59,101,117,129,161,189 1,2
fuged at 300g at 4 °C for 5 min each time, which were then ended in Total 2.16
100 μL of binding buffer (Annexin V-FITC kit; Becton Dickinson, NJ, 2,6-Me2-Glcp 1.00 43,87,88,101,117,129,161 1,3,4
USA containing 5 μL of Annexin V-fluorescein isothiocyanate FITC). 2,4-Me2-Frup 1.02 43,58,71,85,101,117,129,161,189,233 1,3,6
After incubation for 10 min at room temperature in the dark, samples a
The sugar type and linkage were confirmed by both the literature and mass spectrum
were analyzed on Fluorescence microscopy. analysis.
 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482 477

Fig. 3. NMR spectrum of CCPP-1: HSQC spectrum (A1 means the correlation between C1 and H1 of residue A) (A and B), 1H-1H COSY spectrum (A (1, 2) means the correlation between H1
and H2 of residue A) (C).
478 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482

2.5. Statistical analysis
Each experiment was performed in triplicate and all data were pre-
sented as means ±standard deviation (SD). Statistical analysis was
evaluated using one way analysis of variance (ANOVA), where p b
0.05 was assumed to be statistically significant. The statistical analyses
were performed using the Statistical Package for the Social Sciences
(SPSS) software.
3. Results
3.1. Characterization of polysaccharide
3.1.1. Components analysis and molecular weight determination
The crude polysaccharide was isolated from C. cornucopioides
through hot-water extraction, ethanol precipitation, dialysis and lyoph-
ilization. It was fractionated usting DEAE-52 cellulose eluting with dis-
tilled water and 0.1–0.5 M NaCl to obtain three major fractions
(Fig. 1A). The eluates obtained from 0.3 M NaCl were further purified
by Sepharose CL-4B column. The polysaccharide fraction CCPP-1 was
obtained with the yield of 10.9% (Fig. 1B). The total sugar contents of
CCPP-1 were determined to be 99.15%. As shown in Fig. 2 A–B, CCPP-1
exhibited a single and symmetrical sharp peak on the HPLC chromato-
gram and had no peak at 260 nm or 280 nm in the UV spectrum, indicat-
ing the absence of protein and nucleic acid, which showed that CCPP-1
was a homogeneous polysaccharide. The molecular weight of CCPP-1
was calculated to be 9.2 × 105 Da according to the calibration curve of
standard.
ratios consistent with the overall monosaccharide composition. The
high proportion of T-linked Araf residue suggested that CCPP-1 was sig-
nificantly branched and that side chains were mainly terminated by Araf
residues. In addition, the DB was calculated to be 0.73, suggesting a
highly-branched structure.
3.1.6. NMR spectroscopy of CCPP-1
NMR is an effective method to analyze the structural characteristics
of polysaccharides [33]. Complex groups of peaks were exhibited in 1H
NMR spectrum of CCPP-1. The anomeric proton signals appeared in
the range of δ 5.30–4.45 ppm, suggesting CCPP-1 possessed α-(δ
N 5.0 ppm) and β-(δ b 5.0 ppm) configuration [16]. The corresponding
carbon signals were also found in the anomeric carbon region of δ
96.1–107.8 ppm in 13C NMR spectrum. In HSQC spectra, five main
pairs of 13C and 1H signals of anomeric CH at δ 98.5/4.98, δ 96.3/5.21, δ
103.4/4.53, δ 104.3/4.45, δ 102.9/5.12 were labeled as A, B, C, D and E
(Fig. 3A). By a combination analysis of these results in HSQC and H-H
COSY spectra (Fig. 3B and C) and previous reports, the chemical shifts
of protons and carbons of these five main residues could be determined
as shown in Table 2 [25,26,34,35]. On the basis of monosaccharide com-
position, methylation analysis and NMR spectroscopy, the sugar resi-
dues could be assigned as A β-L-Araf(1→, B → 3, 6)-α-D-Manp(1→, C
→ 5)-β-L-Araf(1→, D → 3)-β-L-Araf (1→ and E → 4, 6)-α-D-Manp(1→,
respectively. However, it remains difficult to identify the accurate struc-
ture of the polysaccharide due to its high molecular weight.
3.2. In vitro antioxidant activity assay

3.1.2. SEM analysis of CCPP-1 3.2.1. DPPH radical scavenging activity

The SEM images of the CCPP-1 were presented in Fig. 2E with the
magnifying power of 500×, 1000× and 2000×. CCPP-1 had an irregular,
large lamellar shape and a smooth surface at 2000 fold magnification,
which illustrated the CCPP-1 had amorphous structure. Additionally,
the SEM analysis showed that the molecules of CCPP-1 were not closely
arranged, indicating that the interaction forces between the polysaccha-
ride were not strong.
3.1.3. FT-IR spectrum
FT-IR spectroscopy has been widely used to characterize polysaccha-
rides since it provides useful functional groups information on the
chemical structure of polymer [29]. As shown in Fig. 2F, the intense
broad peak at 3396 cm−1 and a weak absorption peak at 2923 cm−1
were characteristic of O\\H and C\\H stretching vibration, respectively
[30]. The peaks at 1643 cm−1 and 1420 cm−1 were represented the
asymmetric and symmetric stretching vibrations of carboxyl group or
carboxylate, indicating that CCPP-1 was acidic polysaccharidem [31].
Furthermore, the strong peak at 1043 cm−1 was ascribed to the
stretching vibrations of pyranose ring. The absorption at 896 cm−1
and 670 cm−1 suggested the linkage of β-glycosides and α-glycosides,
respectively [32].
DPPH radical is a stable free radical which can accept an electron or
hydrogen radical to become a stable diamagnetic molecule [36]. There-
fore, scavenging ability on DPPH radical is used to determine the antiox-
idant activity of a substance. As shown in Fig. 4A, the results indicated
that CCPP-1 exhibited obvious scavenging activity on DPPH radical in
a concentration-dependent manner. Especially in the concentration of
400 μg/mL, scavenging activity of CCPP-1 was 81.2%, which was close
to that of Vc (86.3%). Therefore, it's clear that CCPP-1 might act as elec-
tron or hydrogen donator to scavenging DPPH radical.
3.2.2. ABTS radicals scavenging activity
ABTS assay was extensively used in evaluating the total antioxidant
power. Specific absorbance at 734 nm wavelength can be used as an
index which reflecting the antioxidant activity of polysaccharide
[37,38]. As shown in Fig. 4B, CCPP-1 showed obvious scavenging activity
against ABTS radical in a concentration-dependent manner. Moreover,
CCPP-1 exhibited significant radical scavenging activity (99.4%), which
was very closed to that of Vc at the concentration of 500 μg/mL. These
results indicated that CCPP-1 had strong scavenging power for ABTS
radical and could be explored as novel potential antioxidant.

3.1.4. Monosaccharide composition analysis

Table 2
As illustrated in Fig. 2 C-D, monosaccharide composition analysis re- 1
H and 13C NMR chemical shifts of CCPP-1 recorded in D2O.
vealed that CCPP-1 was mainly composed of mannose, glucose, xylose,
Sugar residue Chemical shifts (ppm)
arabinose, fructose in molar ratio of 0.7: 0.05: 0.18: 1: 0.05. Therefore,
mannose and arabinose were the two major monosaccharides of C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
CCPP-1, which was reported from C. cornucopioides for the first time. A: T-β-L-Araf 98.5 72.2 70.4 71.4 61.3
4.98 3.57 3.30 3.49 3.90/3.74
3.1.5. Methylation analysis B: →3,6)-α-D-Manp (1→ 96.3 75.9 73.5 69.9 72.8 65.9
5.21 3.50 3.71 4.18 3.22 3.97/3.74
The linkage types between monosaccharide residues in CCPP-1 were
C:→5)-β-L-Araf (1→ 103.4 70.4 70.5 70.6 65.7
determined by methylation analysis. The signals were identified by 4.53 3.51 4.07 3.91 3.29
their mass fragment patterns, and peak areas were integrated to deter- D: →3)-β-L-Araf (1→ 104.3 74.2 76.2 75.7 62.9
mine the relative molar ratio above. The results in Table 1 demonstrated 4.45 3.27 3.45 3.63 3.65
that the dominant linkage types of CCPP-1 were →3, 6)-Manp(1→, T- E: →4,6)-α-D-Manp (1→ 102.9 70.2 70.5 76.3 72.1 67.5
5.12 3.62 3.42 4.13 3.84 3.72
Araf, →4, 6)-Manp (1→, →5)-Araf (1→, →3)-Araf (1→ with their molar
 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482 479

Fig. 4. Scavenging effect of CCPP-1 and Vc on DPPH radical (A), Scavenging effect of CCPP-1 and Vc on ABTS radical (B).

3.3. Intracellular antioxidant activities analysis
3.3.1. Inhibition of mice erythrocyte hemolysis by CCPP-1
The antioxidant ability of CCPP-1 was further measured by erythro-
cyte hemolysis assay. AAPH, a well-known radical initiator, could be
decomposed at 37 °C to generate an alkyl radical. In the presence of ox-
ygen, these alkyl radicals will be converted to peroxyl radicals that can
cause the chain oxidation of lipid and protein, disturbing the membrane
organization and leading to eventually hemolysis [39]. Therefore, the in-
hibition rate of hemolysis is an indirect way to measure the antioxidant
activity of CCPP-1. As shown in Fig. 5A, CCPP-1 significantly inhibited
AAPH-induced hemolysis in dose-dependently manner. At a dose of
600 μg/mL, the inhibition rate of CCPP-1 on erythrocyte hemolysis was
72.9%. The results clearly showed that CCPP-1 could efficiently protect
normal erythrocytes against AAPH-induced hemolysis in vitro.
eventually cause oxidative damage. ROS have also been regarded as me-
diators of apoptosis. To confirm whether CCPP-1 could reduce AAPH-
induced oxidative stress in erythrocytes, intracellular ROS was detected
by using a fluorescein-labeled dye DCFH-DA, which can enter the cyto-
plasm through the plasma membrane, where it is hydrolyzed by non-
specific esterases into non-fluorescence DCFH. As shown in Fig. 5B,
compared with the normal group, a significant increase in ROS produc-
tion was noted in erythrocytes after AAPH treatment. However, those
groups of CCPP-1 pretreatment exposed to AAPH exhibited reduced
ROS generation in dose-dependent manner as compared to AAPH-
induced erythrocytes. Furthermore, no significant differences were
found in erythrocytes treated with CCPP-1 without AAPH addition com-
pared with the control. These results suggested that CCPP-1 protected
erythrocytes from AAPH-induced hemolysis by inhibiting ROS
production.

3.3.2. CCPP-1 inhibits AAPH-induced ROS generation in erythrocytes 3.3.3. CCPP-1 prevents AAPH-induced MDA accumulation and modulates
ROS, as a biomarker of oxidative stress, plays an important role in intracellular antioxidant enzyme (CAT and GPx) activities
cell signaling. Excessive intracellular ROS in cells may invade the lipid, AAPH-induced excess ROS generation can damage cell structure,
protein and DNA of cell membranes, inhibit their normal function, and leading to DNA damage, lipid peroxidation, and protein degradation.

Fig. 5. Protective effects of CCPP-1 on AAPH-induced erythrocyte hemolysis (A), Inhibitory activity of CCPP-1 on AAPH-induced ROS overexpression in human erythrocytes (B).
Erythrocytes were pretreated with different concentrations of CCPP-1 for 20 min prior to AAPH (100 mM) treatment for 2 h; the normal group was treated with PBS, the damage
group was treated with 100 mM AAPH only. *p b 0.05 and **p b 0.01 compared to damage group.
480 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482

Fig. 6. Change in MDA content (A) and enzyme activity of CAT (B) and GPx (C) in erythrocytes. Erythrocytes were pretreated with different concentrations (100–600 μg/mL) of CCPP-1 for
20 min prior to AAPH (100 mM) treatment for 2 h; the normal group was treated with PBS, the damage group was treated with 100 mM AAPH only. *p b 0.05 and **p b 0.01 compared to
damage group.

MDA, as one of the end products of lipid peroxidation, could alter the
structure and function of cell membrane, cause cell metabolism dys-
function and eventually lead to apoptosis [40]. As shown in Fig. 6A,
the MDA level in erythrocytes was significantly increased from 0.12 to
0.95 nmol/mg protein after treatment with 100 mM AAPH for 2 h, indi-
cating treatment of AAPH caused the occurrence of lipid peroxidation in
erythrocytes. However, when the cells were incubated with different
concentrations of CCPP-1(100, 200, 400, 600 μg/mL), the levels of
MDA in erythrocytes were decreased, especially at 600 μg/mL, where
the MDA concentration declined almost to that of the normal group.
For cells were incubated with CCPP-1 without AAPH supplementation,
the MDA level was comparable to that of the normal group, illustrating
that CCPP-1 itself didn't induce MDA formation. These results indicated
that CCPP-1 could alleviate the damage of AAPH on erythrocyte
membranes.
The CAT and GPx are the major radical scavenging antioxidant en-
zymes that constitute the intracellular defense system which can
coordinate the elimination of free radicals through a series of linkage re-
actions. It is crucial for maintaining a steady state of superoxide radicals
and hydrogen peroxide [7]. The cellular antioxidant system depends on
protection by CAT converts H2O2 to H2O and GPx catalyzes the reaction
of hydroperoxides with glutathione (GSH). As shown in Fig. 6B and C,
significant increase in the activities of CAT and GPx were observed
after 2 h treatment with 100 mM AAPH, indicating that the enzymatic
antioxidant defense systems in erythrocytes were activated by AAPH.
However, pretreatment with CCPP-1 provided effective control of
erythrocyte antioxidant defense systems as indicated by the lower
CAT and GPx. Especially at the concentration of 600 μg/mL, the activities
CAT and GPx were restored to normal levels. Thus, CCPP-1 could effec-
tively attenuated AAPH-induced oxidative stress in mice erythrocytes.
3.3.4. CCPP-1 inhibited AAPH-induced eryptosis
Erythrocytes lack mitochondria and nuclei but may undergo
eryptosis, an apoptosis-like suicidal cell death characterized by cell

Fig. 7. Indentification of the cell death by Annexin-V-PI straining. (A) normal group, (B) 100 mM AAPH treated only, (C)100 mM AAPH+100 μg/mL CCPP-1, (D) 100 mM AAPH+200 μg/mL
CCPP-1, (E) 100 mM AAPH+400 μg/mL CCPP-1; (F) 100 mM AAPH+600 μg/mL CCPP-1.
 W.-W. Yang et al. / International Journal of Biological Macromolecules 117 (2018) 473–482
shrinkage and cell membrane scrambling with phosphatidylserine (PS)
exposure at the cell surface. PS is distributed only on the inside of cell
membrane lipid bilayer. Due to action of oxidative stress, PS is trans-
formed from the inner leaflet to the outer leaflet. PS is externalized dur-
ing eryptosis and this eternalization can be measured using Annexin-V
binging [41]. Annexin-V is a Ca2+-dependent phospholipid-binding
protein, which is highly compatible with PS. As exhibited in Fig. 7B,
erythrocytes treated with AAPH for 2 h exhibited drastically increase
in fluorescence intensity compared with normal group, which indicat-
ing that AAPH-induce oxidative stress could trigger eryptosis at a cer-
tain extent. However, it was showed that the fluorescence intensity
decreased in a dose-dependent manner after treatment with CCPP-1
(100, 200, 400 and 600 μg/mL) in erythrocytes. Especially, at the con-
centration of 600 μg/mL, the fluorescence intensity returned to the
same as normal group. These results suggested that CCPP-1 could pro-
tect erythrocytes against oxidative stress.
4. Conclusion
In summary, a water-soluble polysaccharide (CCPP-1) was firstly
purified from the fruiting bodies of C. cornucopioides and its primary
chemical structure was characterized. Monosaccharide composition
analysis indicated that CCPP-1 was a heteropolysaccharide and mainly
consisted of mannose, glucose, xylose, arabinose, fructose in molar
ratio of 0.7:0.05:0.18:1:0.05. The main linkage types of CCPP-1 were
proven to →3, 6)-α-D-Manp(1→, T-β-L-Araf, →3)-β-L-Araf(1→, →5)-β-
L-Araf(1→, →4, 6)-α-D-Manp (1→. The in vitro antioxidant activities re-
vealed that CCPP-1 had strong radical scavenging activity on DPPH and
ABTS radicals. Besides, CCPP-1 could efficiently attenuate AAPH-
induced oxidative stress through inhibiting ROS generation and MDA
formation and regulating intracellular antioxidant enzyme activity
(GPx and CAT). Furthermore, the PS exposure assay showed that
CCPP-1 inhibited eryptosis in a certain extent. These results demon-
strated that CCPP-1 exhibited excellent antioxidant activities which
might be related to its hyper branch structure and complex linkage
types. Therefore, this study provides a theoretical basis for the further
research on the potential applications of CCPP-1 in functional food
industry.
Acknowledgements
This work was supported by the Key Project of Science and Technol-
ogy of Anhui (1501031099), the Provincial Program of Natural Science
of Anhui Higher Education (KJ2016A790) and Natural Science Founda-
tion of Anhui Province (1808085QC95). We express thanks to the test-
ing center of Hefei University of Technology for NMR measurements
and Anhui Medical University for providing mice.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2018.05.212.
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