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1 | INTRODUCTION
Selenium (Se) is a necessar y microelement which is important for
the development and maintenance of organisms. Se has been found
to exhibit various biological effects, such as immunomodulatory,
antioxidant, antitumor, and hypoglycemic and hypolipidemic ef-
fects (Hartikainen, 2005). Se is a key component of Se- dependent
enzymes, such as glutathione peroxidase (GSH- Px) and thioredoxin
reductase (TrxR). Se deficiency could cause many diseases in hu-
mans, such as immune dysfunction, cancer, and hypothyroidism
(Rzymski et al., 2017). As a major source of Se, selenium supple-
ment is imperative for humans, especially in Se deficiency regions.
It shall be noted that organic Se shows a higher bioavailability, as
it can be readily absorbed in human digestive tracts and also has
higher threshold for the toxicity compared with inorganic Se. In ad-
dition, biotransformation and chemical synthesis of Se- proteins or
Received: 27 October 20 21
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Revised: 12 January 2022
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Accepted: 16 January 2022
DOI: 10.1002 /fsn3.2764
ORIGINAL RESEARCH
Immunostimulatory and antioxidant activities of the selenized
polysaccharide from edible Grifola frondosa
Qian Li1 | Linfei Zhu1 | Xingpu Qi2 | Ting Zhou1 | Yonglian Li1 | Mingjie Cai1 |
Yuting Yan3 | Jian- Ya Qian1 | Daxin Peng4
This is an op en access ar ticle under the terms of the Creative Commons Attribution License, whic h permits u se, distribution and reproduct ion in any medium,
provide d the original work is properly cited.
© 2022 The Authors. Food Science & Nutrition published by Wiley Pe riodicals LLC.
1College of Veterinary Medicine,
Yangzhou University, Yangzhou, China
2School of Food Science a nd Engineering,
Yangzhou University, Yangzhou, China
3College of Food Science a nd Technology,
Jiangsu Agri- animal Husbandry Vocational
College, Taizhou, China
4School of Agricultural Equipment
Engineering, Jiangsu University, Zhenjiang,
China
Correspondence
Jian- Ya Qian, School of Food Science
and Engin eering, Yang zhou Univer sity,
Huayang X ilu 196, Yangzhou, Jiangsu
225127, China.
Email: jyqian@yzu.edu.cn
Daxin Peng, College of Vete rinary
Medicine, Yangzhou University, Wenhui
Donglu 4 8, Yangzhou, Jiangsu 225009,
China.
Email: pengdx@yzu.edu.cn
Funding information
Underg raduate Science & Technolog y
Innovation Foundation of Yangzhou
University, Grant/Award Number:
X20190917 and X20200897; Research
Foundat ion of Jiangsu A gri- anim al
Husbandry Vocational College, Grant/
Award Number: NSF2021ZR07
Abstract
Grifola frondosa polysaccharide (GFP2) was extracted and purified by anion- exchange
chromatography. A selenized G. frondosa polysaccharide, SeGFP2, was modified in se-
lenylation by nitric acid– sodium selenite (HNO3- Na2SeO3) method. Structural features
were investigated, and the lymphocyte proliferation and antioxidant activities were
compared taking GFP2 as control. SeGFP2 with a molecular weight of 2.12 × 104 Da
was composed of mannose, glucose, and galactose with a ratio of 3.5:11.8:1.0. A typ-
ical absorption of selenium ester was observed in SeGFP2 molecule. SeGFP2 was
proposed as a branched polysaccharide, which consisted of 1,3- D- Glcp, 1,6- D- Glcp,
1 , 4 , 6 - D - G a l p, and 1,3,6- D- Manp. SeGFP2 showed a linear filamentous structure with
some branches. SeGFP2 could significantly promote T- or B- lymphocyte proliferation
and the enhancement was higher than GFP2. The in vitro antioxidant activities of
SeGFP2 were more potent than GFP2. These present data suggested that selenyla-
tion could significantly improve the lymphocyte proliferation and in vitro antioxidant
activities of GFP2.
KEYWORDS
antioxidant activity, Grifola frondosa, lymphocyte proliferation, polysaccharide, selenylation
modification
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Se- polysaccharides have been widely used to prepare organic Se
compounds, and attracted tremendous attention of researchers and
consumers recently (Hou et al., 2016; Zhang, Gao, et al., 2021).
Se- polysaccharides could exert the efficacy of both polysac-
charide and Se, and the biological activity is usually higher than
that of polysaccharide or Se (Zhang, Gao, et al., 2021). Generally,
Se- polysaccharides from biotransformation method mainly exist
in plants, mushrooms, and microorganisms. The quality of the Se-
polysaccharides is influenced by both the area and season. The Se
content and selenium translation rate of the Se- polysaccharides
from biotransformation method are relatively lower than chemical
selenylation, even in a high selenium area or liquid medium (Zhang,
Lu, et al., 2016). Recently, it has been reported that the chemical
selenylation of Se- polysaccharides involved nitric acid– sodium sele-
nite (HNO3- Na2SeO3), glacial acetic acid– selenous acid (CH3COOH-
H2SeO3), glacial acetic acid– sodium selenite (CH3COOH- Na2SeO3),
and selenium oxychloride (SeCl2O) method (Gao et al., 2016).
Grifola frondosa, an edible mushroom assigned to the
Polyporaceae family, has been found to have diverse medicinal val-
ues. Due to the existence of bioactive polysaccharides, G. frondosa
has become increasingly popular and widely cultivated in China
(Klaus et al., 2015). These polysaccharides have been reported to
possess potential biological effects, including immunostimulatory,
antioxidant, antitumor, antidiabetic, and antihypertensive activities
(Li et al., 2017; Meng et al., 2017). Bioactive polysaccharides ex-
tracted from the fruit bodies or mycelia of G. frondosa have attracted
the most attention due to their diverse structure and potentially sig-
nificant pharmacological activities.
In this study, the G. frondosa polysaccharides were extracted
and purified by anion- exchange chromatography, and modified in
selenylation by HNO3- Na2SeO3 method for the first time. The lym-
phocyte proliferation and antioxidant activities of selenized G. fron-
dosa polysaccharides (SeGFP2) were evaluated taking G . frondosa
polysaccharides as control. The structural features of SeGFP2 were
explored by Fourier transform- infrared (FT- IR) spectrometry, mono-
saccharide components analysis, methylation, gas chromatography–
mass spec trometry (GC- MS), high- performance size exclusion
chromatography– multiangle laser light scattering– refractive index
detector (HPSEC- MALLS- RI), Congo red spectrophotometric analysis,
circular dichroism (CD), and atomic force microscope (AFM). Overall,
this information would be helpful for the development of novel func-
tional foods or drugs using the Se- polysaccharide as an ingredient.
2 | MATERIALS AND METHODS
2.1 | Materials
The fruit bodies of G. fro ndosa (Bac te ri al nu mb er : Qing gr ay 151) wer e
harvest ed in Qing yu an, Zhejia ng Pr ovi nce of Chi na, and dr ied at roo m
temperature. Identity of G. frondosa was confirmed by Professor
Changwen Ye (Edible Fungus Research Center, Zhejiang, China). 3-
(4,5- Dimethylthiazol- 2- yl)- 2,5- diphenyltetrazolium bromide (MTT),
cyclophosphamide (CTX), concanavalin A (ConA), lipopolysaccha-
ride (LPS), and dimethyl sulfoxide (DMSO) were obtained from Sigma
Chemical Co. All solvents/chemicals used were of analytical grade.
2.2 | Animals
Balb/c strain mice (6 ~ 8 weeks old, 20 ± 2 g) were provided by the
Comparative Medicine Center of Yangzhou University, China (the
license number SCXK [SU] 2012– 0004). The animals were acclima-
tized for 1 week before the experiment. During the experiment, the
mice were housed under controlled environmental conditions of
temperature (25 ± 2°C) with a normal day/night cycle and humidity
(55 ~ 60%), and maintained on a basal diet and water ad libitum. All
animal experiments were performed in accordance with the Code of
Ethics of the World Medical A ssociation and approved by the Ethics
Committee of Yangzhou University.
2.3 | Extraction and purification of G. frondosa
polysaccharide
The crude polysaccharides were extracted from the fruiting bod-
ies of G. frondosa using a method reported before (Li et al., 2018).
Briefly, dry G. frondosa (40 g) was extracted with 120 0 ml double-
distilled water at 100°C for 3 h, and the extraction process was re-
peated for three times. After centrifugation, the supernatants were
combined and concentrated using a rotar y evaporator. Then, four
volumes of ethyl alcohol (EtOH) were added and the mixture was
stored at 4°C overnight to precipitate polysaccharides.
The crude polysaccharides were purified by trichloroacetic acid
method and column chromatography of DEAE- 52 cellulose. The
precipitates were redissolved in water and placed in an ice bath, fol-
lowed by a slow addition of 15% trichloroacetic acid until the pH
reached 2.0 ~ 3.0. After remaining for 4 h, the supernatants were
collected, centrifuged, and adjusted pH to 7.0 with 1 M NaOH.
The solution was extensively dialyzed for 72 h (MWCO 3500 Da),
and lyophilized to obtain the crude polysaccharides (GFP). GFP
was redissolved and subjected to a DEAE- 52 cellulose column
(1.6 cm × 50 cm), followed by a stepwise elution using an increasing
concentration of NaCl (0, 0.05, 0.10, 0.15, and 0.20 M) at a flow rate
of 1.0 ml/min. Frac tions were collected and the sugar profile was
monitored using the phenol- sulfuric acid method. Fractions with the
highest yield (0.05 M NaCl elution) were combined, concentrated,
and lyophilized, generating the purified polysaccharide (GFP2).
2.4 | Selenylation modification of GFP2
Grifola frondosa polysaccharide was selenylated using the HNO3-
Na2SeO3 method with minor modifications (Wang et al., 2016).
Briefly, the purified GFP2 (30 mg) was dissolved in 0.7% HNO3 and
stirred at room temperature for 10 h. Na2SeO3 of 24 mg and BaCl2 of
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LI et aL.
40 mg wer e added and re ac ted at 70°C for 6 h. Af ter the reacti on, the
mixture was cooled to room temperature and the pH was adjusted to
7.0 ~ 8.0. Na2SO4 of 40 mg was ad ded to rem ove the Ba2+. The super-
natant was collected and dialyzed (MWCO 3000 Da) using distilled
water until the reaction solution became colorless when detected by
ascorbic acid method (Zhang, Zhang, et al., 2021). The resulting solu-
tion was concentrated, precipitated with EtOH, and freeze dried to
obtain selenized G. frondosa polysaccharides (SeGFP2).
The selenium analysis was performed by atomic fluorescence
spectrophotometr y (AFS- 9950, Haiguang Analytical Co.) as re-
por ted by Li et al. (2017) and G ao et al. (2 016). Briefly, the concen-
trations of Se standard solution were set at 0, 2, 4, 8, 16, 32 μg/L .
The instrument automatically diluted, detected the fluorescence
intensity, and then drew the standard cur ve. The regression
equation was Y (fluorescence intensity) = 53.43X (Se concen-
tration, μg / L ) - 1 2 . 8 6 ( R2 = .9998). The GFP2 was digested with
HClO4- HNO3 (1:4) mixed solution for 12 h at 4°C, then heated at
180 ~ 190°C until it became clear accompanied with white smoke,
concentrated to 1 ~ 2 ml. Six M HCl was added, heated, and the
solution was concentrated to 1 ~ 2 ml, and then cooled and di-
luted into 10 ml with 6 M HCl. After detection, its Se content was
calculated according to the regression equation. The Se yield was
obtained by AFS over the weight of lyophilized extracts. All mea-
surements were performed in triplicate.
2.5 | FT- IR spectroscopy
The F T- IR spectrum (4000– 500 cm- 1) was obtained using a NEXUS
670 FT- IR spectrophotometer. Two milligram of SeGFP2 was com-
pletely mixed with 200 mg of KBr and pressed into flakes. Single-
beam spectra were collected against that of the background
reference and converted to the absorbance.
2.6 | Monosaccharide composition analysis
For GC analysis, SeGFP2 (5 mg) was hydrolyzed with 3 M H2SO4 at
110°C for 8 h. After totally removing the excess H2SO4, the resultant
monosaccharides were converted into alditol acetates as described
before (Li et al., 2018), and then analyzed by GC.
2.7 | Methylation and GC- MS analysis
The glycosidic linkage analysis of SeGFP2 was carried out using
the methylation method (Wang et al., 2017). Specifically, SeGFP2
(5.0 mg) was dissolved in anhydrous DMSO with a nitrogen inlet.
Dried NaOH (100 mg) was added and the mixture was stirred for 1 h.
CH3I of 1.0 ml was added and the mix ture was incubated in darkness
for 4 h. Th e react ion mix tu re was ex tracted with chl oro form, then the
organic phase was washed with distilled water and dried under vac-
uum. After being methylated several times, the methylated SeGFP2
was confirmed by FT- IR. The methylated SeGFP2 was hydrolyzed
with 85% formic acid at 100°C for 6 h and 2 M trifluoroacetic acid
(TFA) at 100°C for 6 h, and then reduced with NaBH4 and neutral-
ized with acetic acid. The sample was acetylated by a procedure as
mentioned in Section 2.6. Subsequently, the partially O- methylated
alditol acetates (PMAAs) were detected by a GC- MS (6890N/5975B
GC- MS, Agilent Co.) and the methylated SeGFP2 linkages were ob-
tained by the retention time and fragmentation pattern.
2.8 | HPSEC- MALLS- RI analysis
A high- performance size exclusion chromatograph (HPSEC) coupled
with a multiangle laser light scattering (MALLS) photometer (DAWN
HELEOS 8, Wyat t Technology Co.) and a refractive index (RI) de-
tector (2414 HPLC, Agilent) was used. Injection volume was 100 μl.
Samples with a concentration of 5.0 mg/ml were filtered through
0.22- μm syringe filters before injection. A TSK G- 6000PWXL col-
umn linked with a TSK G- 400 0PWXL column (30 cm × 7.8 mm i.d.,
TOSOH) was used at 633 nm and 35°C. The eluents were 0.15 M
NaNO3 with 0.05 M NaH2PO4 at 0.5 ml/min. Astra version 6.1.1
wyatt software (Wyatt Technology Co.) was used for the data ac-
quisition and analysis.
2.9 | Colorimetric determination with Congo red
The helix coil transition or random coils conformation of SeGFP2
was determined by Congo red test. Usually, SeGFP2 (5 mg) was dis-
solved in water and then mixed with 80 μM Congo red dye. One M
NaOH was dropwise added into the mixture to achieve 0 ~ 0.5 M
final concentrations, and the absorbance (A) was recorded on an
ultraviolet- visible spectrophotometer (UV- 2450, Shimadzu Co.). The
optical rotation of mixture alkaline solution without polysaccharides
was used as the reference.
2.10 | Microscopic analysis
The atomic force microscopy (AFM) was employed to observe the
molecular morphology of SeGFP2. SeGFP2 (10.0 µg/ml) was dis-
persed in water and filtered through a 0.45 μm syringe filter, and
10.0 µl of sample solution was deposited onto the freshly cleaved
mica and then air dried for 4 ~ 8 h at room temperature. The sample
was examined in the tapping mode with a Multimode 8 (Br uker) in air.
The Nanoscope software (Build R3Sr6.104281, Bruker Corporation)
was performed for image manipulation.
2.11 | Splenocyte proliferation assay
Lymphocyte proliferation was assessed by an MTT- based colorimet-
ric assay. Balb/c mice (6 ~ 8 weeks old, 20 ± 2 g) were sacrificed via
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cervical dislocation. Spleens were aseptically removed and placed in
cold RPMI- 1640 medium under aseptic conditions, then gently ho-
mogenized, passed through a 40 μm nylon cell strainer to generate
single- cell suspensions. After removal of er ythrocytes from the cell
mixture, the cells were washed twice and suspended in RPMI 1640
medium supplemented with 10% fetal bovine serum, adjusted to a
final density of 5 × 106 cells/ml. Aliquots of 100 μl of splenocy tes
(5 × 106 cells/ml) were placed in a 96- well plate with or without
ConA (10 μg/ml) or LPS (20 μg/ml). Samples of different concentra-
tions (0, 25, 50, or 100 μg/ml) were added to each well and the plate
was incubated at 37°C in a humidified 5% CO2 incubator for 72 h.
Twenty microliters of MTT (5 mg/ml) was added per well and in-
cubated for 4 h, followed by the addition of DMSO (150 μl/well).
The absorbance at 570 nm was measured using a microplate reader
(BioTek Synergy H4).
2.12 | Antioxidant activity analysis
The in vitro antioxidant ac tivities of SeGFP2 and GFP2 were
evaluated using the free radical scavenging activities and ferrous
ion- chelating abilities. The DPPH radical scavenging activity was
measured according to the method described by Li et al. (2017).
Briefly, 2 ml of aqueous aliquots (0, 200, 400, 600, 800, 1000, 1500,
and 200 0 μg/ml) was mixed with 2.0 ml DPPH solution (0.2 mM in
EtOH). The mixture was vor texed intensely, and then allowed to set-
tle for 30 min under dark condition. The DPPH radical scavenging ef-
fect was evaluated according to the following equation: Scavenging
rate (%) = [( AB- AS)/AB] ×100%, where AB and AS separately repre-
sent the absorbance of blank and test sample. Ascorbic acid was
used as a positive control to validate the assay.
The ABTS+ radical scavenging activity was measured follow-
ing a modified scheme based on Jeddou et al. (2016). The ABTS+
solution was produced by reacting 1.82 mM ABTS+ with 1.07 mM
potassium persulfate. Then, the mixture was left to settle for 24 h
under dark condition. The ABTS+ solution was diluted with 0.15 M
sodium phosphate- buffered saline (pH 7.4) to an initial absorbance
of 0.70 ± 0.02 (734 nm). Prior to the assay, 0.2 ml of sample solu-
tions was added to 3.8 ml of diluted ABTS+ radical solution. The
absorbance was measured at 734 nm after a 6- min incubation. The
ABTS+ radical scavenging activity was calculated using the following
equation: Scavenging rate (%) = [(AB- A S)/AB] ×100%, where AS and AB
separately represent the absorbance values of ABTS+ solution with
and without test sample. Ascorbic acid was used as a positive control.
The ferrous ion- chelating ability was performed following the
modified method described by Yuan et al. (2020). Briefly, the sam-
ple in different concentrations (0, 200, 400, 600, 80 0, 1000, 1500,
and 200 0 μg/ml) was mixed with 3.7 ml of distilled water, and then
reacted with 0.1 ml FeSO4 (2.0 mM). After 0.2 ml of 5.0 mM fer-
rozine was added, the solution was mixed, and allowed to remain
for 10 min at room temperature. The absorbance was determined at
562 nm. The chelating activity on ferrous ions was calculated using
the following equation: Chelating abilit y (%) = [(AB- AS) /AB] ×100% ,
where AB and AS separately represent the absorbance of blank and
test sample. EDTA was co- assayed as a positive control.
2.13 | Statistical analysis
Data analysis was performed with SPSS software (version rel. 18.0,
SPSS Inc.). Differences were considered statistically significant at
p < .05.
3 | RESULTS AND DISCUSSION
3.1 | Extraction, purification, and general analysis
of SeGFP2
A homogeneous polysaccharide fraction was purified by DEAE- 52
column and named GFP2 (Li et al., 2018). The UV spectrum of GFP2
exhibited a decreasing absorbance similar to that of most polysac-
charides, a negative response to the Bradford test and no absorption
peaks at 260 or 280 nm, indicating the absence of nucleic acids and
proteins.
The Se content of SeGFP2 was detected to be 445.39 μg/g.
Organic Se fortification of this mushroom source could help to allevi-
ate Se deficiency in the population of China, in addition, making more
Se- fortified food choices available. Similar Se content was also found
in the selenized polysaccharide (SeASP6) from Artemisia sphaeroceph-
ala after the HNO3- Na2SeO3 synthesis method (Wang et al., 2012).
The alditol acetates of acid hydrolyzed SeGFP2 were analyzed by
GC; in conclusion, it contained mannose, glucose, and galactose in a
ratio of 3.5:11.8:1.0 (Figure 1). SeGFP2 was a heteropolysaccharide,
in which D- glucose was the dominant constituent. A higher propor-
tion of glucose was indicated in SeGFP2 compared with the previous
reported study on G. frondosa polysaccharide, which consisted of glu-
cose (64.4%), galactose (25.7%), and mannose (9.9%) (Xu et al., 2010).
3.2 | FT- IR spectra
The characteristic absorptions of SeGFP2 and GFP2 were per-
formed using FT- IR spectra (4000 ~ 500 cm- 1 ) (Figure 2). In the
spectrum of GFP2, there were four characteristic absorption
peaks at 3387.6 cm- 1, 2925.2 cm- 1, 1652.0 cm- 1, and 1371.3 cm- 1,
respectively, being the stretching vibration absorption peaks
of O- H (350 0 ~ 3300 cm−1 ), C- H (300 0 ~ 2800 cm−1 ), C = O
(1700 ~ 1500 cm−1), and C- O (1400 ~ 1000 cm−1). The absorption
band in the region of 1000 ~ 1200 cm- 1 was dominated by ring vibra-
tion overlapped with the C- O- C glycosidic band vibration and C- OH
stretching vibration of pyranose in polysaccharides. The lack of car-
bonyl bands around 1750 cm- 1 indicated the absence of uronic acids
in GFP2 and SeGFP2 (Liu et al., 2013).
While most peaks were also shown in the spectrum of
SeGFP2, two new absorption peaks appeared at 667.9 cm- 1 and
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LI et aL.
1024.2 cm- 1, respectively, assigned to the Se- O- C stretching vibra-
tion (ν, 700 ~ 600 cm
- 1) and the O- Se- O stretching vibration (νas,
1040 ~ 1010 cm- 1) (Hou et al., 2016). This signified that Se had been
combined to the polysaccharide molecule. The absorption band at
864.0 cm- 1 was ascribed to β- type glycosidic linkages (Zhang, Zhou,
et al., 2016). Previous studies indicated that active polysaccharides
in those mushrooms appeared to have potential immunoregulatory
activity, primarily due to the polysaccharides with β- glucan struc-
tures (Xu et al., 2012).
3.3 | Glycosidic linkages
As summarized in Table 1 and Figure 3, the results showed the pres-
ences of five types of linkages: 2,3,4,6- Me4- Glcp, 2,4,6- Me3- Glcp,
2,3,4- Me3- Glcp, 2,3- Me2- Galp, and 2,4- Me2- Manp in a mole ratio
of 5.3:2.4:4.2:1:3.7. It suggested that SeGFP2 might be proposed
as a branched polysaccharide consisting of 1,3- linked- D- Glcp,
1 , 6 - l i n k e d - D - G l c p, 1 , 4 , 6 - l i n k e d - D - G a l p, a n d 1 , 3 , 6 - l i n k e d - D - M a n p
units. The mole ratio was nearly in agreement with the monosaccha-
ride composition. The degree of branching (DB) was 60.3% based on
the calculation method reported by Chen et al. (2015).
3.4 | Molecular weight and chain conformation
High- performance size exclusion chromatography– multiangle laser
light scat tering– refractive index detector was used as an efficient
method to determine the molecular conformation and related pa-
rameters of the polysaccharide in dilute polymer solution. As shown
in Figure 4, a single symmetrical peak was observed in the HPSEC
chromatogram, and indicated that SeGFP2 was a homogeneous
polysaccharide with the weight- average Mw of 2.12 × 104 Da. The
polydispersity index Mw/Mn was 1.068, suggesting a polydisperse
polymer in SeGFP2. The radius of gyration (Rg) is known as the
distance between the mass center and the segment. The value of
Z- average Rz was determined to be 13.5 nm. For a given polymer
solution, the gradient value (ν) may provide additional insights into
macromolecule conformation and architecture. Usually, the ν values
of 0.33, 0.50 ~ 0.60, and 1.0 separately exhibit the sphere, random
coil, and rigid rod of the polymer (Zhao et al., 2014). The ν value of
SeGFP2 was 0.40, which suggested that SeGFP2 molecules in an
aqueous solution might be in a state between spheres and random
coils.
Congo red test was performed to detect the triple helix or ran-
dom coils structure of polysaccharide chains in an aqueous alka-
line solution. Figure 5 shows the change of maximum absorbance
(λmax) of SeGFP2- Congo red complex at a NaOH concentration
(0 ~ 0.5 M). Obviously, the addition of SeGFP2 to the Congo red
solution did not cause any notable changes in λmax from 480 nm to
520 nm compared with that of Congo red alone, suggesting that
SeGFP2 chains existed as random coils instead of helical struc-
ture. Furthermore, our results are consistent with the other bio-
active polysaccharides, which also exhibited as random coils (Lavi
et al., 2006).
3.5 | Molecular morphology
Generally, AFM is useful for observing the surface and topography
of each sample (Kong et al., 2015). AFM images of SeGFP2 were
provided in Figure 6. SeGFP2 appeared as worm- like chains and
the molecular chains branched and entangled with each other at a
concentration of 10.0 μg/ml. The height of all observed chains was
around 0.3 ~ 8.1 nm, which is consistent with the thickness of mul-
tiple molecular chains (Liu et al., 2013). The molecular aggregation
was ascribed to the - OH groups of SeGFP2, which provided inter/
intra- molecular interactions with each other or water molecule
(Kong et al., 2015).
FIGURE 1 Gas chromatograms of the monosaccharide
compositions of SeGFP2
15 20 25
0
20000
40000
60000
23.05
22.48
22.16
Retention time (min)
Vu
FIGURE 2 FT- IR spectra of SeGFP2 and GFP2
4000 3500 3000 2500 2000 1500 1000 500
2359.1
1041.9
Wavenumber (cm
-1
)
)%(ecnattimsnarT
SeGFP2
GFP2
1371.3
1652.0
2360.7
2925.2
3387.6
578.8 578.7
761.3
1154.5
1081.3
864.0
1024.2
1652.0
2928.6
3387.4
1368.0
931.3
667.9
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3.6 | Lymphocyte proliferation
Many fungal polysaccharides can activate T lymphocyte and B
lymphocyte to show their effects on the immune system (Liu
et al., 2017). Lymphocyte proliferation is the most direct indicator
of immunoactivation. Usually, lymphocytes induced by ConA or LPS
are, respectively, used to evaluate T- or B- lymphocyte activity (Liu
et al., 2017). As shown in Figure 7a, the lymphocyte proliferation
rate in SeGFP2 or GFP2 group was significantly (p < .05) higher than
that of the control group. SeGFP2 groups at 25, 50, and 100 μg/ml
were significantly (p < .01) higher than corresponding ConA control
group (Fig ure 7b). Synergistic ef fect was obser ved between poly sac-
charide and LPS, especially at the medium and high concentrations
(Figure 7c). The T- lymphocyte proliferation effects of two polysac-
charides combined with ConA were presented in a concentration-
dependent manner. With the synergistic effect of SeGFP2 and LPS,
B- lymphocyte proliferation exhibited a dose- dependent trend. The
results confirmed that two polysaccharides at suitable concentra-
tions could significantly induce the lymphocyte proliferation, syner-
gistically with ConA or LPS. SeGFP2 treatment as an adjuvant could
significantly promote T- or B- lymphocyte proliferation combining
with ConA or LPS, and the enhancement was higher than GFP2.
Immunostimulation itself is regarded as one of the important
strategies to improve the host defense mechanism in humans as well
as cancer patients. Various experiments proved that polysaccha-
rides from mushrooms could enhance the host immune system by
stimulating T cells, B cells, natural killer cells, and macrophage cells
(Liu et al., 2017; Xu et al., 2012). Grifola frondosa polysaccharides
were reported to show immunostimulatory activities, such as the
improvement of RAW264.7 cells proliferation and the macrophage-
activating capability (Meng et al., 2017). In fact, immunostimulatory
activities of polysaccharides depend on the structural information
such as monosaccharide constituent, glycosidic linkage, molecular
weight, and function groups. It was reported that β- glucans from
mushrooms, especially β- 1,3- and β- 1,6- linkages, were important
for increasing cell immune activity (Liu et al., 2017; Xu et al., 2012).
Sun et al. (2012) reported that a relatively low molecular weight of
the polysaccharide was desired for it s immunostimulatory activity.
The current study firstly demonstrated that SeGFP2, with a Mw of
2.12 × 104 Da, consisted of 1,3- linked- D- Glcp, 1 , 6 - l i n k e d - D - G l c p,
TABLE 1 GC- MS data for methylation analysis of SeGFP2
Methylated sugar tR (min)bLinkage pattern MS (m/z) Molar ratio
2,3,4,6- Me4- Glcpa6.32 T- Glcp43,45,71,87,101,113,117,129,145,161, 205 5.3
2,4,6- Me3- Glcp7. 68 1,3- linked- Glcp43,58,71,87,101,117,129,161,233 2.4
2,3,4- Me3- Glcp8.02 1 , 6 - l i n k e d - G l c p43 ,87,99,101,117,129,161,189,233 4.2
2,3- Me2- Galp9.9 0 1 , 4 , 6 - l i n k e d - G a l p43, 85,99,101,117,127,142,161,261 1
2,4- Me2- Manp10.48 1 , 3 , 6 - l i n k e d - M a n p43,71 ,87,101,117,129,189,233 3.7
a1,5- di- O- a c e t y l - 2 , 3 , 4 , 6 - t e t r a - O- methyl- gluctiol.
btR, Relative retention time.
FIGURE 3 The total ion chromatograms from methylation
analysis of SeGFP2
6810 12 14 16
0
100000
200000
300000
400000
500000
600000
700000
1,6-glu
)V(ecnadnubA
Time (min)
T-glu
1,4,6-gal
1,3,6-man
1,3-glu
FIGURE 4 Light scattering signals of SEC chromatogram of
SeGFP2
0204
06
0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
40.27
DeIRIU
t/min
|
1295
LI et aL.
1 , 4 , 6 - l i n k e d - D - G a l p, a n d 1 , 3 , 6 - l i n k e d - D - M a n p units. Our results are
in agreement with these discussions.
Se- polysaccharides were immune response regulators as re-
ported in several studies (Gao et al., 2016). Haibo et al. (2016)
reported that the selenylation modification of Chuanminshen viola-
ceum polysaccharides (sCVPS) was obtained using HNO3- Na2SeO3
method. The selenylation of CVPS could significantly increase the
immunoregulatory activity both in vitro and in vivo, thus represent-
ing a powerful adjuvant for vaccine design. It was elucidated that
Se alone could improve the abnormal levels of cytokines and oxi-
dative damages in chicken spleen, thus ameliorating the injury in-
duced by heat stress. The combination of Se and polysaccharides
induced a higher immune function (Zhang, Gao, et al., 2021). Thus,
these structural features may be responsible for the higher lym-
phocy te proliferation activity of SeGFP2. Further studies should be
made to elucidate the immunostimulatory activity and its possible
mechanism.
3.7 | Antioxidant activity
As illustrated in Figure 8a, the DPPH radical scavenging abili-
ties of SeGFP2 and GFP2 were both concentration dependent
(200 ~ 2000 μg/ml). The DPPH radical scavenging ability ranged
from 25.18% to 48.24% for GFP2, while from 27.31% to 62.53% for
SeGFP2. At the concentration of 200 0 μg/ml, the inhibition percent-
age of SeGFP2 was 62.53% ± 1.85%, which was higher than that of
GFP2 (48. 24% ± 1.66%).
FIGURE 5 Absorption spectra of Congo red (control) and Congo
red with SeGFP2 at various concentrations of NaOH
0.00.1 0.20.3 0.40.5 0.6
470
480
490
500
510
520
530
Congo red
SeGFP2 + Congo red
NaOH (M)
λ
max
)mn(
FIGURE 6 AFM images of SeGFP2 obtained under tapping mode using a Multimode 8 instrument (Bruker)
1296
|
LI e t aL.
The ABTS+ radical has been widely used to measure the tot al
antioxidant activity of single compounds or complex mixtures
(Jeddou et al., 2016). As shown in Figure 8b, the effect of ABTS+ rad-
ical scavenging was presented in a concentration- dependent manner
(200 ~ 2000 μg/ml). The ABTS+ radical scavenging rate of SeGFP2 was
61.01% ± 2% at 2000 μg/ml, nearly 1.40- fold higher than that of GFP2
(43.67% ± 1.97%). It sugges te d that SeGFP2 sh owe d a not iceab le abil it y
on the scavenging of ABTS+ radicals, especially at a high concentration.
FIGURE 7 Evaluation of in vitro
antioxidant activity of SeGFP2. (a) DPPH
radical scavenging ac tivity, (b) ABTS+
radical scavenging ac tivity, and (c)
chelating effect on ferrous ions
0 500 1000 1500 2000
20
40
60
80
100
Vitamin C
GFP2
SeGFP2
Concentration (µg/mL)
)%(ytilibagnignevacsHPPD
(a)
0 500 1000 1500 2000
0
20
40
60
80
100
Vitamin C
GFP2
SeGFP2
Concentration (
µ
g/mL)
)%(ytilibagninevacs-lacidareerfSTBA
(b)
0 500 1000 1500 2000
0
20
40
60
80
100
EDTA
GFP2
SeGFP2
Concentration (
µ
g/mL)
)%(ytilibagnitalehC
(c)
FIGURE 8 Effect of SeGFP2
and GFP2 with or without ConA or
LPS on splenocyte proliferation. (a)
Polysaccharides, values are given as
means ± SD; *p < .05, **p < .01 vs.
negative control; (b) Polysaccharides
+ConA, values are given as means ±
SD; *p < .05, **p < .01 vs. negative
control; #p < .05, ##p < .01 vs. ConA; (c)
Polysaccharides +LPS, values are given
as means ± SD; *p < .05, **p < .01 vs.
negative control; #p < .05, ##p < .01 vs.
LPS
0.0
0.2
0.4
0.6
0.8
**
**
**
**
**
*
A
570nm
A
570nm
A
570nm
100
50
25
Control
SeGFP2
GFP2
0.0
0.5
1.0
1.5
**,##
**,##
**,##
**
**,##
**
**
ConA 100
50
25
Control
SeGFP2 + ConA
GFP2 + ConA
0.0
0.2
0.4
0.6
0.8
1.0
**,##
**,##
**,##
**
**
**
SeGFP2 + LPS
**
LPS 100
50
25
Control
GFP2 + LPS
(a) (b)
(c)
|
1297
LI et aL.
The chelating agents, which form bonds with metals, are ef-
fective as secondary antioxidants for the redox potential reduc-
tion, thus stabilizing the oxidized form of the metal ions (Yuan
et al., 2020). Both SeGFP2 and GFP2 showed antioxidant activi-
ties, and the Fe2+- chelating ability was, respectively, 62.83% and
45.9% at a dose of 200 0 μg/ml, lower than that of EDTA (Figure 8c).
Basically, the chelating ability of SeGFP2 was a little superior to
that of GFP2 under other six concentrations. As described by
Yuan et al. (2020), the chelating ability of polysaccharides on Fe2+
might affect the other radical scavenging activities to protect the
organism against oxidative damage. Since Fe2+ is the most effec-
tive pro- oxidant in food system, the high Fe2+- chelating abilities of
polysaccharides from G. frondosa fruit bodies would be somewhat
beneficial in the antioxidation.
It was reported that the antioxidant ability of polysaccharides
was due to their hydrogen- donating effects. The element Se in
SeGFP2 could activate the hydrogen atom of the anomeric carbon
(Turło et al., 2010; Zhang, Lu, et al., 2016). The higher irritation abil-
ity of the group led the hydrogen atom- donating abilit y stronger.
This suggested that selenylation modification could enhance the in
vitro antioxidant activity. In fact, a relatively low molecular weight
of polysaccharides was highly desired for the antioxidant ability
(Zhao et al., 2014). SeGFP2 with a MW of 2.12 × 104 Da exhibited a
stronger antioxidant ability than Se- GFP- 22 (4.13 × 106 Da), which
was repor ted in our previous study (Li et al., 2017). The antioxidant
ability of the polysaccharides also strongly depended on the type
of sugar monomers, the linkage pattern of the backbone, and the
degree of branching.
4 | CONCLUSION
In this study, G. frondosa polysaccharides were extracted and
purified by anion- exchange chromatography, and modified in
selenylation by HNO3- Na2SeO3 method for the first time. The
lymphocyte proliferation and antioxidant activities of SeGFP2
were also evaluated taking GFP2 as control. A typical absorp-
tion of selenium ester in SeGFP2 molecule was observed in FT-
IR. SeGFP2 was composed of mannose, glucose, and galactose
in a ratio of 3.5:11.8:1.0. SeGFP2 exhibited as a branched poly-
saccharide consisted of 1,3- D- Glcp, 1 , 6 - D - G l c p, 1 , 4 , 6 - D - G a l p,
a n d 1 , 3 , 6 - D - M a n p. The current study firstly demonstrated that
SeGFP2 (Mw 2.12 × 104 Da) possessed synergistic stimulation ef-
fects for T- or B- lymphocyte proliferation combining with ConA
or LPS. The lymphocyte proliferation in SeGFP2 group was higher
than in the GFP2 group. The in vitro antioxidant activities of
SeGFP2 were more potent than GFP2. In summary, selenylation
modification could enhance the antioxidant and immunostimula-
tory activities of GFP2, which might be related to its moderate
molecular weight, Se content, monosaccharide constituent, glyco-
sidic linkage, and function groups. Overall, SeGFP2 could be con-
sidered as a potential immunomodulator y agent with antioxidant
activity or a dietary Se- supplement. Further investigations of the
detailed mechanisms underlying the immunostimulatory activity
of SeGFP2 are still necessary.
ACKNOWLEDGEMENTS
This work was supported by the Undergraduate Science &
Technology Innovation Foundation of Yangzhou University
(X20190917, X20200897) and Research Foundation of Jiangsu Agri-
animal Husbandry Vocational College (NSF2021ZR07). We also ap-
preciate Dr. Samuel Jerry Cobbine for language polishing.
CONFLICT OF INTEREST
There are no conflicts to declare.
ETHICAL APPROVAL
Animal experiments were performed in accordance with the code of
ethics of the World Medical Association and approved by the Ethics
Committee of Yangzhou University.
DATA AVAIL ABILI TY STATEMENT
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Jian- Ya Qian https://orcid.org/0000-0003-4460-4634
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How to cite this article: Li, Q., Zhu, L., Qi, X., Zhou, T., Li, Y.,
Cai, M., Yan, Y., Qian, J., & Peng, D. (2022). Immunostimulatory
and antioxidant activities of the selenized polysaccharide from
edible Grifola frondosa. Food Science & Nutrition, 10, 1289–
1298. https://doi.org/10.1002/fsn3.2764
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