Structural elucidation, antioxidant and immunomodulatory activities of a novel heteropolysaccharide from cultured Paecilomyces cicadae (Miquel.) Samson

Article abstract of DOI:10.1016/j.carbpol.2019.03.104

The fine structure and chain conformation of a heteropolysaccharide (PCIPS3) from mycelium of Paecilomyces cicadae were investigated via the analysis of HPLC, IR, methylation, NMR spectroscopy and multiangle light scattering. It was determined to be a 2.23 × 10⁴ g/mol heteropolysaccharide primarily composed of glucose, galactose and mannose in a molar ratio of 23.8:2.1:1.0. The PCIPS3 backbone consisted of 1,4-linked α-D-Glcp and 1,4-linked 6-O-Me-α-D-Glcp residues, which were occasionally interrupted by branched β-Galf residues through 1,6-linkage. Moreover, the α (0.60) from Mark–Houwink–Sakurada (MHS) equation suggested that PCIPS3 adopted a flexible chain conformation in 0.1 mol/L NaNO₃ at 25 °C. The worm-like chains model parameters for PCIPS3 were estimated as following: M_L = 437 nm^?1, q = 0.46 nm and 0.79 nm, which were further evidenced by AFM. Furthermore, PCIPS3 showed excellent scavenging capacities of 2,2-diphenyl-1-picrylhydrazyl radical, superoxide radical, hydroxyl radical, ORAC radical and moderate immunomodulatory activity.

Full text of DOI:10.1016/j.carbpol.2019.03.104

CarbohydratePolymers216(2019)270–281
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structural elucidation, antioxidant and immunomodulatory activities of a
novel heteropolysaccharide from cultured Paecilomyces cicadae (Miquel.)
Samson
Yanbin Wanga, Pengfei Heb, Liang Hea,⁎, Qingrong Huangd, Junwen Chenga, Weiqi Lic, Yu Liuc,
Chaoyang Weic
a Key Laboratory of State Forest Food Resources Utilization and Quality Control, Zhejiang Provincial Key Laboratory of Forest Food, Zhejiang Academy of Forestry,
Hangzhou 310023, China
b Department of Processing, Marine Fisheries Research Institute of Zhejiang, Zhoushan 316021, China
c Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
d Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The fine structure and chain conformation of a heteropolysaccharide (PCIPS3) from mycelium of Paecilomyces
cicadae were investigated via the analysis of HPLC, IR, methylation, NMR spectroscopy and multiangle light
scattering. It was determined to be a 2.23 × 104 g/mol heteropolysaccharide primarily composed of glucose,
galactose and mannose in a molar ratio of 23.8:2.1:1.0. The PCIPS3 backbone consisted of 1,4-linked α-D-Glcp
and 1,4-linked 6-O-Me-α-D-Glcp residues, which were occasionally interrupted by branched β-Galf residues
through 1,6-linkage. Moreover, the α (0.60) from Mark–Houwink–Sakurada (MHS) equation suggested that
PCIPS3 adopted a flexible chain conformation in 0.1 mol/L NaNO3 at 25 °C. The worm-like chains model
parameters for PCIPS3 were estimated as following: ML = 437 nm−1, q = 0.46 nm and 0.79 nm, which were
further evidenced by AFM. Furthermore, PCIPS3 showed excellent scavenging capacities of 2,2-diphenyl-1-pi-
crylhydrazyl radical, superoxide radical, hydroxyl radical, ORAC radical and moderate immunomodulatory
activity.
Paecilomyces cicadae
Heteropolysaccharide
Structural elucidation
Biological activity
AFM
1. Introduction
discovered from the artificial culture and mycelium including poly-
saccharides, cordycepic acid, ergosterol peroxide and effective nucleo-
Cordyceps cicadae Shing (called “Chan Hua” in Chinese), which
belongs to the Ascomycota, Hypocreales, Claviceptaceae, Cordeceps,
and parasites on the larvae of Cicada flammata Dist, is a valued Chinese
caterpillar fungus that has been extensively used as tonic food and
medicine for a long time (Sun et al., 2017). It has attracted considerable
attention due to its wide-range of nutritional and pharmacological
benefits on the immune, circulatory, cardiovascular, hematogenic and
respiratory systems (Ke & Lee, 2018; Weng, Chou, Lin, Wsai, & Kuo,
2002). However, the natural recalcitrance of Cordyceps slows down its
commercialization, including the specific host, the strictly conditioned
environment and artificial destruction.
sides (Chai et al., 2007; Shen et al., 2007; Xiao et al., 2004). For human
beings, these secondary metabolites can be developed to be biofunc-
tional agents with amazing potential in improving health and pre-
venting diseases. Among them, some hetero-polysaccharides derived
from P. cicadae have attracted great attention due to their intriguing
biological activities, including anti-tumor (Shen et al., 2007; Sun et al.,
2017), immunomodulation (Cheng et al., 2012; Xu et al., 2018), anti-
inflammation (Ke & Lee, 2018), anti-bacteria (Zhang et al., 2017) and
anti-oxidation (He, Wu, Cheng, Li, & Lu, 2010; Ren, He, Cheng, &
Chang, 2014).
Many studies have been reported that the biological activities of
polysaccharides are not only dependent on their chemical structures,
but also closely related to their unique architectures, especially the
advanced helical conformations (Yang & Zhang, 2009). Therefore, it is
interesting and essential to elucidate the role of chemical features and
Paecilomyces cicadae (Miquel) Samson is thought to be the ana-
morph stage of Cordyceps. Culture of this entomogenous fungi in sub-
merged fermentation has become a promising way to meet the needs of
human consumption. More and more secondary metabolites have been
⁎ Corresponding author.
E-mail address: kite006@126.com (L. He).
https://doi.org/10.1016/j.carbpol.2019.03.104
Received 25 December 2018; Received in revised form 29 March 2019; Accepted 30 March 2019
Availableonline07April2019
0144-8617/©2019ElsevierLtd.Allrightsreserved.

Y. Wang, et al.
CarbohydratePolymers216(2019)270–281
chain conformations of polysaccharides on their biological activities
(Hu et al., 2017; Ding, Qian, Goff, Wang, & Cui, 2018). In our previous
research (Cheng et al., 2012; Wei, Li, Shao, He, & Cheng, 2016), crude
polysaccharides obtained from the mycelium of P. cicadae has been
fractionated into three sub-fractions, namely PCIPS1, PCIPS2 and
PCIPS3. Further, the fine structure and chain conformation of hetero-
mannan (PCIPS2) has been characterized, which might shed light on its
immuno-stimulating activity on murine macrophage RAW264.7 pro-
liferative response. Unfortunately, other fractions still need definitive
evidence to develop this kind of fermented resources.
Therefore, in order to obtain a comprehensive knowledge of the
polysaccharides from the mycelium of P. cicadae, we further purified
the third fraction (PCIPS3) and characterized its chemical structure and
advanced architecture by monosaccharide composition, Fourier trans-
form infrared spectroscopy (FT-IR), methylation, 1D/2D nuclear mag-
netic resonance (NMR), size exclusion chromatography combined with
multiangle laser light scattering. Moreover, its antioxidant activities
were detected by different assays for better understanding the re-
lationship of the structure-function.
phenyl-3-methyl-5-pyrazolone (PMP) derivatization procedures with
some modification (Lv et al., 2009). Briefly, 3 mg of PCIPS3 was dis-
solved in 1 mL of 4 mol/L trifluoroacetic acid (TFA) in a sealed tube and
hydrolyzed for 6 h at 121 °C. The excess TFA was removed with 200 μL
methanol under a reduced pressure after the complete hydrolyzation.
Then the dried hydrolysate of PCIPS3 or 100 μL of standard mono-
saccharides (2 mmol/L) were mixed with 100 μL of 0.6 mol/L aqueous
NaOH and 100 μL of 0.5 mol/L PMP in methanol solution. The mixture
was subsequently neutralized with 0.5 mol/L hydrochloric acid after
cooling to room temperature. The resulting solution was successively
extracted with the same volume of water and chloroform for three
times. The aqueous layer was finally filtered through 0.45 μm pore
membrane for HPLC analysis.
Analysis of the PMP-labeled monosaccharides was operated on a
Dionex Ultimate3000 HPLC system (Sunnyvale, USA) equipped with a
UV 6999 detector (Sunnyvale, USA). The analytical column used was a
Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm, Agilent,
USA). The wavelength of UV detection used was 245 nm. The process
was carried out at a flow rate of 1.0 mL/min at 25 °C for 40 min, in
which the eluent consisted of the mobile phase A (acetonitrile) and the
mobile phase B (0.05 mol/L sodium phosphate (KH2PO4-NaOH, pH 6.9)
buffer) in a ratio of 17:83. The injection volume was 10 μL.
2. Materials and methods
2.1. Materials and reagents
2.4. FT-IR spectroscopy of PCIPS3
The strain of P. cicadae ZJ001 was screened and collected from
Zhejiang Academy of Forestry, Hangzhou city, Zhejiang Province,
China. And it was maintained on potato dextrose agar (PDA) slant
subcultured every 4 weeks. According to our previous report (Cheng
et al., 2012; Wei et al., 2016), the crude polysaccharide was extracted
from the mycelia of P. cicadae with distilled water at 80 °C and pre-
cipitated with 75% ethanol. The fractionation of PCIPS3 was collected
after DEAE-Sepharose Fast Flow chromatography.
2 mg of dried PCIPS3 was mixed with 200 mg spectroscopic-grade
potassium bromide (KBr) powder and pressed into 1 mm thick pellet for
FT-IR spectroscopy. FT-IR spectra were recorded on a Nexus IS10 FT-IR
spectrometer (Thermo Nicolet, USA) at the frequency range of
4000–400 cm−1 with resolution of 4 cm−1 and 64 numbers of scans.
Dextran standard (Mw = 41 400 Da) was obtained from American
Polymers Standards Co. (Mentor, OH, USA). The compounds 1,1-di-
phenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)
hydroxyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH),
nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium
(NBT), potassium ferricyanide and lipopolysaccharide (LPS) were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA). Deuterium oxide
(99.9%) was from J&K Scientific Ltd (Beijing, China). Ultrapure water
was produced by Milli-Q system (Millipore, Billerica, USA). All other
reagents were of analytical grade and purchased from local chemical
suppliers in China.
2.5. Methylation analysis of PCIPS3
The determination of the linkage types of PCIPS3 was performed by
methylation analysis according to the method of Ciucanu and Kerek
(1984) with some slight modification. Briefly, freeze-dried PCIPS3
(4 mg) was dissolved in 0.6 mL of anhydrous DMSO (dimethylsulfoxide)
and then methylated with adding 0.6 mL of cold CH3I dropwise and
0.6 mL of NaOH-DMSO solution. After 30 min of reaction under the
ultrasonic bath, the solution was extracted by 3 mL chloroform. The
organic phase was washed with 6 mL distilled water before being eva-
porated to dryness. The disappearance of the O–H band
(3200–3700 cm−1) in the IR spectrum was used to confirm complete
methylation. After successive hydrolysis with 3 mL of 90% aqueous
formic acid for 3 h and 2 mol/L trifluoroacetic acid (4 mL) for 6 h at
100 °C, the permethylated sample was converted into partially methy-
lated alditol acetates (PMAAs) and analyzed by gas chromato-
graphy–mass spectrometer (GC–MS). The methylation GC–MS program
was carried out on Agilent 7890A/5975C instrument with a HP-5MS
column (30 m × 0.25 mm × 0.5 mm) (Thermo Co., Austin, TX, USA)
equipped with flame-ionization detector. The temperature was in-
creased from 150 to 250 °C at a rate of 6 °C/min then maintained at
250 °C for 15 min. The injector and the detector temperatures were both
set at 250 °C.
2.2. General analysis of PCIPS3 from cultured P. cicadae
PCIPS3 (10 mg/mL) was initially loaded onto a Sephadex G-100 gel
column (1.6 cm × 80 cm) and then purified on a Sephadex G-75 gel
column (1.6 cm × 80 cm), which was both eluted by 0.05 mol/L PBS
containing 0.1 mol/L NaCl (pH 7.0) at a flow rate of 0.5 mL/min. The
eluate (3 mL/tube) was collected with a fraction collector, dialyzed
against distilled water for 48 h and finally freeze-dried to afford the
purified product.
The total sugar content was determined by phenol-sulphuric acid
method using glucose as standard (Dubois, Gilles, Hamilton, Rebers, &
Smith, 1956). The protein content was evaluated with the Bradford
method, using bovine serum albumin as the standard (Bradford, 1976).
The uronic acid content was determined by photometry with m-hy-
droxybiphenyl at 525 nm using D-galacturonic acid as the standard
(Blumenkrantz & Asboe-Hansen, 1973).
2.6. Partial hydrolysis with acid
Partial degradation by acid hydrolysis was subjected to elucidate
the structure of branches and backbone of PCIPS3. PCIPS3 was hy-
drolyzed with 0.2 mol/L TFA at 100 °C for 2 h, then the excess acid was
removed by evaporation. The resulting hydrolysate was dialyzed with
distilled water (molecular weight cutoff 3500 Da). The retentates (the
fractions in dialysis sack, PCIPS3-D1) and the dialysates (the fractions
outside dialysis sack, PCIPS3-D2) were both collected and lyophilized.
2.3. Monosaccharide composition analysis of PCIPS3
The monosaccharide composition of PCIPS3 was determined by
high-performance liquid chromatography (HPLC) according to 1-
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CarbohydratePolymers216(2019)270–281
2.7. NMR spectra
measured at 517 nm. Ascorbic acid as a standard was used to be com-
pared with the samples. Triplicate measurements were carried out for
each sample. The inhibition percentage of DPPH scavenging capability
was calculated as the following equation:
PCIPS3 and PCIPS3-D1 were dried for 48 h in a vacuum dryer with
P2O5 and exchanged with deuterium by lyophilizing for three times,
and finally dissolved in 0.5 mL D2O at room temperature before starting
the NMR experiments. Chemical shifts were referenced to internal 4,4-
dimethyl-4-silapentane-1-sulfonic acid (1H and 13C at 0.00 ppm). The
1H NMR, 13C NMR, 1H–1H correlation spectroscopy (COSY), Total
correlation spectroscopy (TOCSY), heteronuclear multiple quantum
coherence (HMQC), heteronuclear multiple bond correlation (HMBC)
and nuclear overhauser effect spectroscopy (NOESY) were recorded on
a Bruker AVANCE 600 MHz spectrometer (Bruker Group, Fäl-landen,
Switzerland) at 333.15 K with a Bruker 5 mm 1H–19F/15N–31P TXI
switchable broadband probe. All the data was processed and analyzed
using standard Bruker Topspin-NMR software (Smallcombe, Patt, &
Keifer, 1995).
Scavenging rate (%) = [1 − (A − Ab)/A0) × 100
(1)
where A0 was the absorbance of the mixture with sample replaced by
95% ethanol, A and Ab were the absorbances of DPPH solution with or
without the tested sample.
2.10.2. Superoxide radical scavenging activity
The superoxide radical scavenging activity of sample was in-
vestigated by a slightly modified method (Liu, Ooi, & Chang, 1997). In
this assay, PCIPS3 was dissolved in distilled water at 0, 0.1, 0.2, 0.4,
0.6, 0.8, 1.0 or 1.2 mg/mL. 1.0 mL of the sample solution was mixed
with 3.0 mL of Tris–HCl buffer (16 mmol/L, pH 8.0) containing 1.0 mL
of NADH (78 μmol/L) solution and 1.0 mL of NBT (50 μmol/L) solution.
The reaction system was triggered by adding 1.0 mL of PMS (10 μmol/L,
pH 8.0) solution and incubated at 25 °C for 5 min. Finally, the absor-
bance was measured at 560 nm against blank sample. Ascorbic acid was
used as the reference compound. All the measurements were carried out
in triplicate. The scavenging effect of the superoxide radical was de-
fined as:
2.8. Chain conformation of PCIPS3
The solubilized PCIPS3 chains were characterized by size-exclusion
chromatography
(SEC
column,
TSK
G3000PW
column,
7.8 mm × 300 mm) combined with multiangle-laser photometer
(DAWN HELEOS II, Wyatt Technology, USA), a degasser (GASTORR
TG-14, Gen Tech Scientific Inc., USA), a pump (S-1500, SSI, USA) and a
sampler (High-Pressure Injection system, Wyatt Technology, USA). A
differential viscometer (ViscoStar™ II, Wyatt Technology, USA) and a
refractive index detector (RID-10A, Shimazu Corporation, Japan) were
simultaneously connected as well. The eluent was 0.1 mol/L aqueous
NaNO3 (containing 0.02% NaN3) with a flow rate of 0.5 mL/min. The
PCIPS3 sample was prepared with 0.1 mol/L NaNO3 to be a con-
centration of 3 mg/mL and kept stirring for at least 24 h. Then all the
samples were filtered on a 0.22 μm pore membrane before the injection
in order to eliminate large aggregates. The refractive index increment
(dn/dc) value of PCIPS3 in 0.1 mol/L aqueous NaNO3 solution was set
to be 0.138 mL/g at 658 nm (Bednar & Hennessey, 1993). All the tests
were maintained at 25 °C. ASTRA software (Version 7.1.2, Wyatt
Technology) was used for data acquisition and further analysis
Scavenging effect (%) = (1 − As/A0) × 100
(2)
where As was the absorbance of sample and A0 was the blank control
solution without the sample.
2.10.3. Hydroxyl radical scavenging activity
The hydroxyl radical system generated by the Fenton reaction was
evaluated in vitro according to the method described by Zhong, Lin,
Wang, and Zhou (2012). Briefly, PCIPS3 of various concentrations (0,
0.1, 0.2, 0.4, 0.6, 0.8, 1.0 or 1.2 mg/mL) were mixed with 2.0 mL of
FeSO4 (6 mmol/L) solution and 2.0 mL H2O2 (6 mmol/L) solution. After
thoroughly shaken, the mixture was left standing at room temperature
for 10 min. Then the reaction was performed by adding 2.0 mL of sal-
icylic acid (6 mmol/L) and allowed to stay at room temperature for
another 10 min. The hydroxyl radical was detected by monitoring ab-
sorbance at 510 nm. Ascorbic acid was used as positive control. All
experiments were performed three times, and the capability of hydroxyl
radical scavenging activity was calculated using the following equation:
2.9. Atomic force microscopy of PCIPS3
The atomic force microscope used in this study was a ParkProbe XE-
70 system (Park Scientific Instruments, Suwon, Korea) equipped with a
Z-scanner. The experiment was performed in ambient air at room
temperature and a relative humidity of 30–35%. The image process was
obtained in non-contact mode using a classical silicon cantilever (Si3N4)
with a spring constant of 26 N/m and a resonance frequency of ap-
proximately 300 kHz. PCIPS3 was initially dissolved in Milli-Q water at
a concentration of 1 mg/mL with continuous stirred for 2 h and in-
cubated at 80 °C for 2 h. Then the solution was diluted to a con-
centration of 20 μg/mL and 10 ng/mL, successively. After filtered
through a 0.22 μm filter, 2.5 μL of sample solution was dropped onto
freshly cleaved mica substrate and dried in desiccator before imaging.
The scan area was set at 5 μm2, while the resolution was 512 × 512
points. The scanning linear velocity was 0.8 Hz. XEI data processing
software (Version 4.3.0, Park Systems Corporation, Korea) was used for
image manipulation.
Scavenging ability (%) = [1 − (As − Aj)/A0] × 100
(3)
where A0 was the absorbance control (water instead of sample solu-
tion), As was the absorbance in the presence of the sample, and Aj was
the absorbance of the blank reagent (water instead of H2O2).
2.10.4. Oxygen radical absorbance capacity (ORAC) assay
ORAC assay was performed according to a previously reported
protocol (You et al., 2013) with minor modification in a Synergy 2
Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). All of
fluorescein (FL), AAPH, Trolox (antioxidant standard) and sample were
prepared in 75 mmol/L phosphate buffer (pH 7.4). Briefly, the final
reaction mixture was 200 μL containing 40 μL of fluorescein (3.5 nmol/
L), 20 μL of sample solution (0.05 mg/mL) or 20 μL of Trolox (6.25,
12.5, 25, 40 and 50 μmol/L) and 140 μL of AAPH (12.8 mmol/L), where
the FL and sample or Trolox solution were shaken and pre-incubated at
37 °C for 15 min in 96-well polystyrene black microplates before rapidly
adding the AAPH solution. The fluorescence was read every 2 min for
98 min, where excitation and emission wavelengths were 485 and
538 nm, respectively. All determinations were carried out in triplicate.
ORAC value was expressed as Trolox equivalents (μmol Trolox/g).
2.10. Antioxidant activities in vitro
2.10.1. DPPH scavenging activity
The DPPH scavenging ability of sample was determined by the
method of Chen, Xie, Nie, Li, and Wang (2008) with some modifica-
tions. Briefly, fifty microliters of PCIPS3 solutions (0.1, 0.2, 0.4, 0.6,
0.8, 1.0 and 1.2 mg/mL) were mixed with 2.5 mL of DPPH solution
(0.2 mmol/L DPPH in 95% ethanol). Then, the mixtures were incubated
at room temperature in the dark for 30 min, and the discolorations were
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2.11. Immunomodulatory activity of PCIPS3
Table 1
Monosaccharide compositions analysis of PCIPS3, PCIPS3-D1 and PCIPS3-D2.
2.11.1. Cell viability
Fractions
Molar ratios
Glucose
A murine macrophage cell line, RAW264.7 cells were collected from
Shanghai Institute of Cell Biology (Shanghai, China) and maintained in
RPMI 1640 that was supplemented with 100 U/mL penicillin, 100 U/
mL streptomycin and 10% fetal bovine serum. Cells were grown at 37 °C
in a humidified 5% CO2 incubator. After incubation, cell viability was
determined by CCK-8 assay.
Galactose
Mannose
PCIPS3
23.8
20.0
3.3
2.1
1.0
n.d.
1.0
PCIPS3-D1a
PCIPS3-D2b
n.d.c
1.9
a
The retentates after dialysis of hydrolysate of PCIPS3.
The dialysates after dialysis of hydrolysate of PCIPS3.
Not detected.
b
c
2.11.2. Immunomodulatory activity
For drug treatment experiments, the cells were inoculated into 24-
well culture plates at 3 × 105 cells per well and incubated for 24 h. The
medium was then incubated with 6.25–100 μg/mL of PCIPS3 or 1 μg/
mL of lipopolysaccharide (LPS) and incubated for 24 h. 10 μg/mL of
Polymyxin B was also added to each well to exclude the effects of LPS,
which was used as a positive reference drug in the test. Cytokines TNF-
α, IL-1β, IL-6 and IL-10 in the culture supernatant were assayed with
the corresponding sandwich Enzyme-Linked ImmunoSorbent Assay
(ELISA) kits (eBioscience) according to manufacturer's instructions. All
treatments were performed in triplicate and repeated at least once, and
Bradford method. Additionally, no uronic acid content was determined
by photometry method at 525 nm.
The sugar analysis of PCIPS3 by the HPLC analysis based on pre-
column PMP derivatization was shown in Table 1. The results indicated
that PCIPS3 was a neutral heteropolysaccharide including glucose,
galactose and mannose in a molar ratio of 23.8: 2.1: 1.0. In term of
molar ratios, the predominant monosaccharide was glucose, which was
extremely different from the former biomacromolecules from both the
natural fruiting body of C. cicadae and the fermented mycelium derived
from P. cicadae. Ukai, Matsuura, Hara, Kiho, and Hirose (1982) found
that the C-3, isolated from the rod-like ascocarps of C. cicadae through
Sephadex G-100 column, was primarily composed of mannose and
galactose in a ratio of 4:3. Kiho, Miyamoto, Nagai, and Ukai (1988)
pointed out that two minor polymers contained mannose and galactose
in a molar ratio of 1: 0.85 for CI-P and 1:0.57 for CI-A. A neutral het-
eropolysaccharide named as PCIPS2 had also been reported in our
previous work to consist of mannose, rhamnose, 3-O-methyl-galactose,
glucose and galactose with a molar ratio of 47.9:3.1:6.4:0.9:0.8 (Wei
et al., 2016). It seems that the growth environment and fermentation
processes might contribute to the differences of monosaccharide com-
position of C. cicadae.
the results were expressed by their means
SD (standard deviation).
Statistical significance of the treatment effects was determined by
Duncan's multiple range t-test.
3. Results and discussion
3.1. Chemical properties of PCIPS3
According to our previous work (Wei et al., 2016), the third fraction
(PCIPS3) obtained from the mycelium of P. cicadae was separated on
Sephadex G-100 gel chromatography. As shown in Fig. 1A, the profile
yielded a major peak with a very small hump in the front, which could
be neglected. Then the major fraction was further purified to give one
symmetrical peak (Fig. 1B) through gel-filtration chromatography on
Sephadex G-75. It was indicated to contain 1.78% protein using the
3.2. FT-IR spectrum of PCIPS3
The FT-IR spectrum (Fig. 2) of PCIPS3 exhibited typical character-
istics of polysaccharide. The broad peaks around at 3411 cm−1 was
attributed to -OH stretching vibration, and the weak absorption at
2933 cm−1 represented C-H stretching vibration of a CH2 group. A
symmetrical absorption signal at 1633 cm−1 indicated the presence of
C]O groups or C = C groups vibration in the structure. The absence of
absorption peaks at 1730 cm−1 confirmed that there was no uronic
acids in PCIPS3 (Wang et al., 2015). The prominent bands between
1100 and 1000 cm−1 were assigned to the characteristic of CeOeC
glycosidic bond vibrations and ring vibrations overlapped with C-O-H
stretching vibrations of side group bounds. The characteristic
Fig. 1. The elution profile pf PCIPS3 from P. cicadae on Sephadex G-100 column
(A) and Sephadex G-75 column (B).
Fig. 2. IR spectrum of fraction PCIPS3 from P. cicadae.
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Table 2
Methylation analysis of PCIPS3 and PCIPS3-D1.
Methylated sugars
Linkagesa
Major mass fragment
Molar ratios
PCIPS3
PCIPS3-D1
2,3,4,6-Me4-Glc
3,4,6-Me3-Gal
2,4,6-Me3-Glc
2,3,6-Me3-Man
2,3,6-Me3-Glc
2,3,4-Me3-Glc
2,3-Me2-Gal
1-linked Glcp
43,71,87,102,113,118,129,145,162,205
43,71,88,101,129,145,161,190,205
43,71,87,101,118,129,161,174,234
59,71,87,102,118,129,143,162,173,203,233
71,87,101,118,129,142,162, 173,233
43,71,87,102,113,118,129,145,162,189,233
59,85,102,118,127,159,201, 261
1.35
0.38
0.14
0.48
10.93
0.27
1.00
n.d.b
n.d.
n.d.
n.d.
10.0
n.d.
n.d.
1,2-linked Galp
1,3-linked Glcp
1,4-linked Manp
1,4-linked Glcp
1,6-linked Glcp
1,5,6-linked Galf
a
Glcp: glucopyranose, Galp: galactopyranose, Manp: mannopyranose, Galf: galactofuranose.
Not detected.
b
absorption peaks at 892 and 860 cm−1 were due to the existence of α-
and β-configurations simultaneously (Zhang, Tian, Jiang, Miao, & Mu,
2014). In addition, a signal at 812 cm−1 suggested the presence of
mannose. These results were consistent with the outcomes of HPLC
analysis for PCIPS3.
of PCIPS3 beyond α-Glucopyranose.
3.5. NMR analysis of PCIPS3
In the 1H NMR spectrum (Fig. 3A), three resonance absorptions
were clearly observed at δ 5.40, 5.19 and 4.97 in the anomeric proton
regions (δ 4.40–5.90). δ 5.40 containing two overlapped anomeric
protons was evident from two cross resonance signals corresponding to
δ 5.40 (δ 5.40/3.64 and δ 5.40/3.57) in COSY spectrum. In addition,
several very weak anomeric proton peaks were found at around δ 5.23,
5.05, 4.66 and 4.64. The 13C NMR spectrum (Fig. 3B) exhibited four
major (δ 109.84, 102.64, 102.50 and 101.42) and several minor (δ
110.56, 107.18, 105.42, 103.38, 100.54 and 98.55) signals in the
anomeric carbon regions (δ 90–112). The proton signal at δ 3.23 had a
corresponding carbon signal at δ 56.85, suggesting the presence of O-
Me. Seven residues were finally identified by a series of 2D correlation
experiments (COSY, TOCSY, HSQC and HMBC) which allowed the as-
signment of most of the main signals of the residues in Fig. 3. To fa-
cilitate the determination of the residues, the seven anomeric carbons
were labeled as A-G from high to low intensity. Those proton and
carbon chemical shifts were assigned in detailed and summarized in
Table 3.
For residue A, B and D, the proton resonances for H-1, H-2, H-3, H-
4, H-5 and H-6 at the same system spin system were easily assigned
from the correlation peaks in the TOCSY along with 1H–1H COSY
spectra (Supplemental Figs. 2 and 3). All three residues belong to dif-
ferent types of glucose units according to upfield chemical shifts of H-2
(δ 3.64, 3.57, 3.59) compared to its normal position in reference. The
chemical shifts of anomeric proton at δ 5.40, 5.40 and 4.97, respec-
tively, and the small coupling constant values JH-1,H-2 (< 3 Hz) and JC-
3.3. Methylation analysis of PCIPS3
Methylation analysis was conducted for identification of the gly-
cosidic linkages involved in PCIPS3. The substitution patterns of the O-
acetyl groups on the PMAA reflected the linkage positions of the cor-
responding sugar residues. The PMAA prepared from PCIPS3 were
analyzed through GC-MS and identified by their retention time and
main characteristic ion fragments. Methylation analysis of PCIPS3 gave
PMAAs corresponding to terminal Glcp, 2-O-substituted Galp, 3-O-
substituted Glcp, 4-O-substituted Manp, 4-O-substituted Glcp, 6-O-sub-
stituted Glcp and 5,6-Di-O-substituted Galf (see Table 2). 4-O-Sub-
stituted Glcp accounted for around 84.4% of all residues, suggesting
that the backbone of PCIPS3 may be composed mainly of (1→4)-D-
glucopyranan. The presence of linkage of 5,6-linked Galf indicated that
PCIPS3 is a slightly branched polymer. This kind of structure was ex-
tremely different from that of PCIPS2, which had a backbone of 1,4-
linked Rhap residues and 1,6-linked Manp residues instead (Wei et al.,
2016). In addition, the presence of galactofuranose was evidenced by
the following NMR analysis.
3.4. Partial acid hydrolysis of PCIPS3
Partial degradation of polysaccharides controlled by acid hydrolysis
is on basis of the fact that glycosidic linkages in carbohydrates shows
different resistance to acid treatment (Wu, Ai, Wu, & Cui, 2013).
Generally, furanose is more easily hydrolyzed in dilute acid with a
faster rate than pyranose. The glycosidic linkages in the side chain are
more prone to be broken down than those in backbone. In this work,
PCIPS3-D1 (the retentates after dialysis) and PCIPS3-D2 (the dialysates
after dialysis) were obtained from partial acid hydrolysis of PCIPS3.
Therefore, PCIPS3-D1 should be primarily originated from the back-
bone of PCIPS3. The sugar composition analysis showed that PCIPS3-D1
was exclusively composed of glucose (Table 1), implying that the
backbone of PCIPS3 consisted mainly of glucopyranose. Glucose, ga-
lactose and mannose existed in PCIPS3-D2 with a ratio of 3.3:1.9:1.0
(Table 1), indicating they were presented in side chains or in furanose
form. PCIPS3-D1 was also subjected to methylation analysis, and the
results (Table 2) corroborated that (1→4)-D-glucopyranan might be
main components of the backbone of PCIPS3. 1H NMR experiments was
carried out to further determine these partial hydrolysis products. By
reference to proton chemical shifts of PCIPS3 (see Section 3.5), signals
in 1H NMR spectrum (Supplemental Fig. 1) of PCIPS3-D1 were ascribed
to (1→4)-α-D-Glcp, α-Glcp and (1→4)-α-6-O-Me-α-Glcp, suggesting that
6-O-Me-α-Glucopyranose was also presented in (1→4)-D-glucopyranan
(∽170 Hz) indicated that A, B and D were α-linked residues.
1,H-1
Compared with chemical shifts of reference analogous compounds, the
downfield shift of C-4 (δ 79.69) confirmed that residue A was →4)-α-D-
Glcp-(1→ and residue B was α-Glcp-(1→. Two obvious inter-residual
cross signals at δ 3.23/68.40 and δ 3.68/56.85 in HMBC spectrum
(Fig. 3d and e) suggested that O-Me group was linked to residue D. The
downshift resonance of C-6 at δ 68.80 and C-4 at δ 79.69 was further
proved that residue D was →4)-α-6-O-Me-α-Glcp-(1→.
Likewise, residue C was of Gal-type evident from the uncompleted
spin system from H-1 to H-6 and its small coupling constant between H-
4 and H-5 (Staaf, Urbina, Weintraub, & Widmalm, 1999), and of β-
configuration supported by the presence of the chemical shift of C-1 at δ
109.84. It was noteworthy that the clear chemical resonance of C-4 at δ
84.21 readily revealed the existence of galactofuranose unit in this re-
sidue. The chemical shifts from C-1 to C-6 for residue C corresponded
nearly to the documented reference values (Parra et al., 1994), and the
downfield shifts of C-5 (δ 78.34) and C-6 (δ 68.80) indicated that re-
sidue C should be designated as →5,6)-β-Galf-(1→.
The proton signals of H-1, 2 and 3 for residue E were assigned from
the cross peaks in TOCSY spectrum. Other proton positions were
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CarbohydratePolymers216(2019)270–281
Fig. 3. NMR spectra of PCIPS3 in D2O. (a) 1H NMR; (b) 13C NMR; (c) 1H–13C HSQC; (d and e) parts of HMBC correlation spectrum.
deduced with the combination of COSY and NOESY spectra
(Supplemental Fig. 4). The correlated carbon shifts could be obtained
by HSQC spectrum (Fig. 3C). With regards to the downfield shifts of H-2
(δ 4.13), C-4 (δ 79.27) and no carbon signal evident in the δ 76–82,
residue E was a 1,4-disubstituted mannopyranose. In comparison with
the carbon chemical signals of →4)-β-Manp-(1→ reported by Jansson,
Lindberg, Widmalm, and Leontein (1987), residue E was undoubtedly
designated as →4)-α-Manp-(1→.
In addition, another two anomeric proton signals could be found at
δ 4.66 and 4.64 after careful identification of 1H NMR spectrum. The
other protons in the same residues were partly assigned from COSY and
TOCSY spectra and corresponding carbon signals were partly provided
by HSQC spectrum due to the very weak resonances. Regarding to the
reference data previously reported, residue F and residue G were →6)-
β-Glcp-(1→ and →2)-β-Galp-(1→, respectively.
Both NOESY and HMBC experiments were carried out to determine
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Table 3
Chemical shifts assignments of 1H NMR and 13C NMR spectra of PCIPS3.
Residues
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6a,b/C-6
O-Me
A
5.40
102.50
5.40
102.64
5.19
109.84
4.97
101.42
5.05
105.42
4.66
98.55
4.64
107.13
3.64
74.39
3.57
74.34
4.15
84.21
3.59
74.34
4.13
–
3.97
76.19
3.70
75.60
4.11
79.24
4.03
76.26
4.08
72.81
3.55
78.47
3.79
74.02
3.66
79.69
3.43
72.18
4.13
84.21
3.66
79.69
3.85
79.27
3.52
3.85
74.04
3.75
75.74
3.96
78.44
3.91
73.85
3.77
75.88
3.68
77.38
3.73
3.85
→4)-α-Glcp-(1→
B
α-Glcp-(1→
C
→5,6)-β-Galf-(1→
D
→4)-6-O-Me-α-Glcp-(1→
E
→4)-α-Manp-(1→
F
→6)-β-Glcp-(1→
G
→2)-β-Galp-(1→
63.28
3.77, 3.86
63.90
3.68
68.80
3.68
68.80
3.68, 3.73
65.50
3.77, 4.00
68.85
–
3.23
56.85
3.28
76.60
3.68
79.66
72.18
3.97
72.84
77.36
–
Bold values represent glycosylation sites.
the inter/intra-linkage sequences among all the residues of PCIPS3. The
cross peak δ 5.40/3.66 in NOESY spectrum implied that residue A was
joined to O-4 of residue A or D. The strong inter-cross peaks at δ 5.40/
79.69 and δ 3.66/102.50 in HMBC spectrum also implied that the
backbone chain of the polymer mainly consists of (1→4)-D-glucopyr-
anan. The inter-residual NOE correlations at δ 4.97/3.68 arising from
H1 of residue D to H6 of residue C, indicated that residue D was linked
to O-6 of residue C. The intra-residual cross signals occurring at C H-6/
D C-1 (δ 3.68/101.42) and C C-6/D H-1 (δ 68.80/4.97) were attributed
to residue D linked to residue C at the position of O-6. The NOE cor-
relation at δ 5.40/3.85, δ 5.40/4.00, δ 5.40/3.68 suggested that O-4 of
residue E, O-6 of residue F and O-2 of residue G were terminated by
residue B.
Based on the monosaccharide composition analysis, the methylation
results and NMR spectra (COSY, TOCSY, HSQC and HMBC), the struc-
ture character of PCIPS3 was proposed as one kind of slightly branched
glucan as shown in Fig. 4. It had a main backbone consisted of 1,4-
linked α-D-Glcp and 1,4-linked 6-O-Me-α-D-Glcp residues, which were
occasionally interrupted by branched β-Galf residues through 1,6-
linkage. These β-Galf residues were attached via O-5 position by 1,4-
linked-α-Manp, 1,6-linked-β-Glcp and 1,2-linked-β-Galp residues,
which were terminated by α-Glcp residues. In this regard, PCIPS3 was
similar with NCSP-50 isolated from natural Cordyceps sinensis, the latter
was determined to have a main chain of (1→4)-linked a-D-Glcp (Wang
et al., 2017).
weight.
The relationship between intrinsic viscosity and molecular weight
can be obtained by the Mark–Houwink–Sakurada (MHS) equation
([η] = KMwα), where α is usually used to estimate the conformation of
PCIPS3. Generally, the exponent of α lies in the range of 0.5–0.8 in-
dicate a flexible chain in good solvent increases with a climb in chain
stiffness (Burchard, 1999). The architecture information about PCIPS3
in 0.1 mol/L NaNO3 solution was investigated by plotting log Mw vs log
[η] (Fig. 5B). After fitting the curve with linear regression, the Mar-
k–Houwink equation was established as [η] = 9.53 × 10−3Mw0.60 (mL/
g). The slope (α = 0.60) indicated that PCIPS3 existed as a flexible
chain in 0.1 mol/L NaNO3 aqueous solution at room temperature,
which was in concert with above conclusion. Further, the molecular
parameter of PCIPS3 was convinced according to the power-law func-
tion (Rh = KMwv) by plotting log Rh vs log Mw (Fig. 5C), and the ex-
ponent of v for PCIPS3 was found to be 0.53, which was in agreement
with random coil conformation.
The Bushin–Bohdanecký theory is another popular method to cal-
culate the parameters of the chain rigidity for flexible polymers by
explaining the relationship between [η] and Mw. Based on that theory,
Yamakawa and Fujii (1974) later developed a worm-like cylinder
model to estimate the related conformational parameters, the molar
mass per unit contour length (ML) and persistence length (q) for the
stiffness of polysaccharide chain. According to the Yamakawa–Fu-
jii–Yoshizaki (YFY) model, the (Mw2/[η])1/3 and Mw could be ex-
2
pressed as the following equation:
(Mw2 /[η])1/3 = 1.516 × 10−8A0 ML (g1/3 cm−1
)
3.6. Chain conformation of PCIPS3
+ 1.516 × 10−8B0 (2q/ML)−1/2(g1/3 cm−1)Mw2
(1)
The conformation property of PCIPS3 was investigated by using SEC
combined with MALLS, viscometer and refractive index, which has
been widely applied to analyze absolute molecular mass, molecular
parameters and molecular morphology. The SEC-MALLS-RI-VISC
chromatogram of PCIPS3, shown in Fig. 5A, reflected a single peak in
the elution profile with small amounts of negligible aggregates detected
by the laser light signal (LS), differential pressure (DP) signal and re-
fractive index signal (RI) simultaneously. The Mw and the hydro-
dynamic radius (Rh) of PCIPS3 in 0.1 mol/L NaNO3 were estimated to
be 2.23 × 104 g/mol and 2.4 nm respectively. The polydispersity (Mw/
Mn) of the biopolymer was 1.03, close to 1.0, indicating a homogeneous
heteropolysaccharide with relatively narrow distribution of molecular
log(dr2/A0) = 0.173 + 2.158 log dr (dr ≤ 0.1)
(2)
(3)
d = dr × 2q
where the value of A0 and B0 depend on q and d (the hydrodynamic
diameter of cylinder), and can be acquired according to the literature.
Fig. 5D showed the Bohdanecký plot of PCIPS3 in 0.1 mol/L NaNO3
solution, in which the intercept (45.56) and slope (3.47) could be ex-
tracted by fitting the curve with linear regression. Based on the A0 and
B0 value, the molecular parameters of ML, q and d for PCIPS3 were
assumed as 437 nm-1, 0.46 nm and 0.79 nm. These results further de-
scribed that PCIPS3 adopted as a flexible chain conformation in
0.1 mol/L aqueous NaNO3 solution, which was similar to a PCIPS2 with
ML = 580 nm−1, q = 2.3 nm, d = 0.8 nm (Wei et al., 2016).
3.7. AFM analysis of PCIPS3
AFM is an emerging technique which can overcome some limitation
of methods by allowing for direct imaging of individual macromolecule
Fig. 4. Proposed structure of PCIPS3.
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CarbohydratePolymers216(2019)270–281
Fig. 5. SEC-MALLS-RI-VISC chromatograms of PCIPS3 (A), logarithmic plot of Mw versus [η] for PCIPS3 (B), logarithmic plot of Mw versus Rh (C) for PCIPS3 and
Bohdanecký plot of (Mw2/[η])1/3 vs Mw
for PCIPS3 in 0.1 mol/L aqueous NaNO3 solution at 25 °C (D).
1/2
at nanoscale level. Fig. 6 showed the topography of AFM for PCIPS3,
which was deposited with doubly distilled water at the concentration of
20 μg/mL and 10 ng/mL. There were many small dotlike aggregates
with diameters of about 150–180 nm and cross-sectional heights of
approximately 0.8–1.0 nm in Fig. 6A. Those spherical-like aggregates
may be entangled chains of PCIPS3 molecules in such relative low
concentration solution. To achieve better continuous microfibrous and
non-twined structures, the whole set of PCIPS3 was diluted into 10 ng/
mL and imaged with same probe under lower-frequency scanning
conditions. Fig. 6B presented a series of representative AFM images
with relative discrete and flexible chains. Based on the polymer statis-
tical analysis of an average of 50 chains, PCIPS3 were approximately
DPPH radical at relative low amount of addition. Similar scavenging
ability has been reported on previous research. The purified exopoly-
saccharide (PEPS) from mycelial culture of P. cicadae (Miq.) Samson
presented a similar scavenging ability on DPPH radical with 64.1% at a
dose of 1.0 mg/mL (He et al., 2010). FPCPS obtained from the solid-
state fermentation of P. cicadae had strong DPPH radical inhibition from
dose of 0.1 to 0.6 mg/mL (Ren et al., 2014). Those data were fully
consistent with the findings in this study.
3.8.2. Superoxide radical-scavenging assay
Superoxide anion, as a reduced form of molecular oxygen, could be
initiated to evaluate one's antioxidant capacity due to its transformation
into more reactive species, such as hydroxyl radical, singlet oxygen and
hydrogen peroxide, which have been reported to induce oxidative da-
mage in lipids, proteins and DNA (Jayakumar, Thomas, & Geraldine,
2009). In this assay, superoxide anion radicals were generated by non-
enzymatic PMS-NADH system in the reduction of NBT. The scavenging
activity of PCIPS3 on superoxide anion radical was shown in Fig. 7B by
determination of absorbance at 560 nm. The comparison standard vi-
tamin C showed valuable high radical scavenging activity (89.2–93.5%)
in the doses from 1.0 to 1.2 mg/mL. More favorably, PCIPS3 exhibited
strong power as a notable scavenger in the tested concentration range.
The percentage inhibition of superoxide anion generation at 0.8 mg/mL
concentration of PCIPS3 was found as 76.4%, which could bear com-
parison with that of FPCPS from solid-state fermented P. cicadae and
PEPS from mycelial culture of P. cicadae (Ren et al., 2014; He, Wu et al.,
2010). The IC50 value of PCIPS3 for eliminating superoxide radicals was
about 0.46 mg/mL, which was slightly lower than that of W-CBP50
obtained from the fruiting bodies of cultured Cordyceps militaris (Chen,
Wu, & Huang, 2013). W-CBP50 was characterized to have similar
structure containing of α-glucose, α-mannose, α-galactose and α-ara-
binose with α-type glycosidic linkage.
0.75
0.15 nm in height and 70 nm in width. The result is consistent
with the former studied chain model parameters of ML = 437 nm−1
,
q = 0.46 nm, d = 0.79 nm. Further it confirmed that PCIPS3 exists as a
flexible conformation in 0.1 mol/L NaNO3 solution, which was slightly
smaller and shorted than the size of PCIPS2 (Wei et al., 2016).
3.8. Antioxidant activities in vitro
3.8.1. DPPH radical-scavenging assay
DPPH is a stable N-centered free radial, which can be easily reduced
by hydrogen-donating antioxidant with a rapid reduction of maximum
absorbance at 517 nm. Therefore, it is commonly used as an effective
and sensitive way to evaluate the antioxidant capacity of natural
compounds. Fig. 7A depicts the DPPH radical scavenging power of
PCIPS3. It was obvious that PCIPS3 exhibited significant scavenging
ability on DPPH in a concentration-dependent manner. And the IC50
value of PCIPS3 was 0.28 mg/mL. There was an increase in the
scavenging effect of PCIPS3 up to 0.8 mg/mL concentration (90.4%),
beyond which there was no significant increase even up to 1.2 mg/mL.
The results suggested that PCIPS3 had a strong scavenging effect on
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Y. Wang, et al.
CarbohydratePolymers216(2019)270–281
Fig. 6. Section analysis of AFM images of PCIPS3 by scan size of 4.0 nm × 4.0 nm at the concentration of 20 μg/mL (A) and 10 ng/mL (B).
3.8.3. Hydroxyl radical-scavenging assay
higher capacity than those of PPM and PPE reported by Jiang, Yuan,
Cai, Jiao, and Zhang (2015). PPM and PPE were both neutral hetero-
polysaccharides consisting of mannose, galactose, and glucose with
molecular ratios of 2.99:1.00:0.34 and 38.40:1.00:1.76. Therefore, our
data confirmed that PCIPS3 has potential antioxidant ability of
scavenging hydroxyl radical.
Hydroxyl radical is the most harmful and reactive oxygen species
and is mainly responsible for the oxidative injury and lipid peroxida-
tion. There are two types of reaction mechanism on scavenging hy-
droxyl radicals: one suppresses the generation of the hydroxyl radical,
and the other removes the hydroxyl radicals (Tseng, Yang, & Mau,
2008). In this assay, the hydroxyl radical is generated by using Fenton's
reaction of the iron (II) complex with H2O2 in the presence of salicylic
acid. The results of hydroxyl radical scavenging activities of PCIPS3 and
ascorbic acid at different concentrations were given in Fig. 7C. As il-
lustrated in the figure, the scavenging powers of PCIPS3 and Vc both
correlated well with increasing concentrations. Below the dose of
0.4 mg/mL, the inhibitory effects of Vc was markedly stronger than
PCIPS3, and after that, no longer obviously increased, whereas the in-
hibitory effect of PCIPS3 continued to increase and was comparable to
Vc at the addition concentration of 1.2 mg/mL. Within the tested con-
centration range, the scavenging ability of PCIPS3 was more pro-
nounced than that of bioactive components obtained from natural and
cultured Cordyceps sinensis (Wang et al., 2015). Although the scaven-
ging ability of the sample was not as remarkable as that of W-CBP50,
the IC50 value (0.32 mg/mL) of PCIPS3 was very close to those of FPCPS
and PEPS (Chen et al., 2013; Ren et al., 2014; He et al., 2010). In ad-
dition, the scavenging effect of this biomacromolecule demonstrated
3.8.4. Oxygen radical absorbing capacity (ORAC) assay
The ORAC assay was used to determine the antioxidant activity
capacity of PCIPS3 against peroxyl radicals, which are the most
common free radicals in vivo and are highly reactive and unstable. The
fluorescein decay curves induced by AAPH were shown in Fig. 7D,
suggesting the potent scavenging capacity for PCIPS3 (1623.44 μmol
Trolox/g), which was higher than those of polysaccharides from La-
minaria japonica (Lu, You, Lin, Zhao, & Cui, 2013) and Polyporus um-
bellatus sclerotia (He, Zhang, Zhang, Linhardt, & Sun, 2016). Normally,
the antioxidant ability of polysaccharide is supposed to relate to the
configuration of the sugar units and monosaccharide compositions.
Concerning to its flexible chain with small branches in PCIPS3, the
effect of antioxidant on peroxyl radicals scavenging was conceived to be
due to its hydrogen-donating ability (Cui et al., 2016). These data
suggested that PCIPS3 could be an effective electron donor capable of
reacting with free radicals to convert them into more stable products.
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Fig. 7. Antioxidant activities of PCIPS3 from P. cicadae by different methods. DPPH radical scavenging assay (A); Superoxide anion scavenging assay (B); hydroxyl
radical scavenging assay (C); ORAC assay (D).
3.9. Immunomodulatory activities of PCIPS3 in vitro
4. Conclusion
RAW264.7 cells are known to produce cytokines in response to the
addition of LPS, and this system is usually conducted to detect the
modulating activities of compounds on cytokine production (Meng
et al., 2018). For investigation of the viability of RAW 264.7 macro-
phages, there was no significant effect of PCIPS3 on the cell viability
within the range of 6.25–100 μg/mL after 24 h incubation in Fig. 8A,
which indicated that the polysaccharide had no toxicity against RAW
264.7 cells in vitro. Three typical pro-inflammatory cytokines including
TNF-α, IL-1β, IL-6 were adopted to explain the defense mechanism of
macrophages against pathogens. Fig. 8B–D showed the levels of three
cytokines, TNF-α, IL-1β and IL-6 in the culture medium of macrophage
RAW264.7 cells after treatment with PCIPS3 and LPS. Compared with
those in the control, all three cytokines were stimulated to higher levels
by PCIPS3 and LPS at suitable concentrations. With regard to TNF-α, it
was clear that PCIPS3 could increase the TNF-α production in a dose-
dependent manner with the highest level of 318.2 pg/mL. Moreover,
with respect to the other two cytokines, the secretions of IL-1β and IL-6
were significantly stimulated by PCIPS3 treatment (6.25–12.5 μg/mL,
p < 0.05; 25–100 μg/mL, p < 0.01). The results suggested that
PCIPS3 held a moderate potential in promoting the secretions of cyto-
kines in RAW 264.7 cells, which further evidenced the findings made by
Cheng et al., 2012. Similar phenomenon has been found in some other
glucans. YCP, composed of α-D-(1–4)-linked glucose residues, inter-
acted with TLR2 and TLR4 to induce B cells proliferation and activation
(Zhu et al., 2014). Another homogenous glucan, named NCSP-50, was
revealed to stimulate the proliferation of macrophages, promote nitric
oxide production and enhance cytokine secretion (Wang et al., 2017).
The structure of NCSP-50 closely had a backbone of α-D-(1–4)-linked
glucose with terminal glucose on O-6 position.
The purified third fractionation (PCIPS3) was analyzed by HPLC, IR,
methylation and 1D/2D NMR spectroscopy. The results revealed that
PCIPS3 consisted of glucose, galactose and mannose in the molar ratio
of 23.8: 2.1: 1.0. It had a main backbone consisted of 1,4-linked α-D-
Glcp and 1,4-linked 6-O-Me-α-D-Glcp residues, which were occasionally
interrupted by branched β-Galf residues through 1,6-linkage. These β-
Galf residues were attached via O-5 position by 1,4-linked-α-Manp, 1,6-
linked-β-Glcp and 1,2-linked-β-Galp residues, which were terminated
by α-Glcp residues. Moreover, the results of SEC-MALLS-RI-VISC
showed that the values of Mw, Rh and [η] for PCIPS3 were 2.23 × 104
g/mol, 2.4 nm and 9.53 × 10−3 mL/g, respectively. The α (0.60) from
the Mark–Houwink–Sakurada (MHS) equation suggested that PCIPS3
adopted a flexible chain conformation with a coil-like structure in
0.1 mol/L NaNO3 at 25 °C. The worm-like chains model parameters for
PCIPS3 were estimated as following: ML = 437 nm−1, q = 0.46 nm and
0.79 nm, which were further evidenced by AFM. Furthermore, PCIPS3
showed excellent scavenging capacities of DPPH radical
(IC50 = 0.28 mg/mL), superoxide radical (IC50 = 0.46 mg/mL), hy-
droxyl radical (IC50 = 0.32 mg/mL) and ORAC radical (1623.44 μmol
Trolox/g). In addition, it exhibited a moderate immunomodulatory
activity by enhancing the secretion of major inflammatory cytokines in
macrophages such as TNF-α, IL-1β, IL-6. These results suggested that
PCIPS3 could be developed as an immunomodulator for functional
foods and medicines.
Acknowledgements
We gratefully acknowledge the financial grants from the National
Natural Science Foundation of China (No. 31571795), the National
Natural Science Foundation of China (No. 31501815), the project of
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CarbohydratePolymers216(2019)270–281
Fig. 8. Effects of PCIPS3 on the RAW 264.7 macrophages. (A) The cell viability, (B) production of TNF-α, (C) production of IL-1β, and (D) production of IL-6.
Lipopolysaccharides (LPS, 1 μg/mL) were used as a positive control. Results are represented as mean
group, n = 5.
SD. *p < 0.05, **p < 0.01 compared with the control
Zhejiang Science and Technology (No. 2016F30012) and the project of
Zhejiang Science and Technology (No. 2016C02057-8). The authors
also thank Dr. Qiang Han from North Carolina State University,
Raleigh, NC, for proofreading the manuscript.
He, P., Zhang, A., Zhang, F., Linhardt, R. J., & Sun, P. (2016). Structure and bioactivity of
a polysaccharide containing uronic acid from Polyporus umbellatus sclerotia.
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Hu, T., Huang, Q. L., Wong, K., Yang, H., Gan, J. S., & Li, Y. R. (2017). A hyperbranched
β-D-glucan with compact coil conformation from Lignosus rhinocerotis sclerotia. Food
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.carbpol.2019.03.104.
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