Structural characterization of an active polysaccharide of longan and evaluation of immunological activity

Article abstract of DOI:10.1016/j.carbpol.2019.03.007

An active polysaccharide (LPD2) was isolated from longan pulp by comparing the effects of polysaccharides on the phagocytosis of macrophages. LPD2 was composed of arabinose, mannose, glucose, and galactose in a molar ratio of 0.25:0.49:1:0.5 with average molecular weight of 9.64 × 10⁶ Da. The main linkages of the sugar residues of LPD2 were (1→4)-β-Glc and (1→6)-β-Man. LPD2 significantly enhanced the lymphocytes proliferation, phagocytosis and NO and IL-6 secretion by macrophage. The anti-TLR2 and anti-TLR4 mAbs markedly suppressed LPD2-mediated NO and IL-6 production. Furthermore, anti-TLR4 or anti-TLR2 plus anti-TLR4 treatment significantly decreased LPD2-induced increase of MyD88, IRAK4, TRAF6 and INOS mRNA expression. Moreover, western blotting analysis showed that LPD2 enhanced the expression of target proteins in MyD88/IRAK4-TRAF6- INOS pathways. These results suggested that LPD2 induced macrophage activation partly via the TLR2- and TLR4-mediated MyD88/IRAK4-TRAF6 signaling pathways. Knowing the structural features and activities of active polysaccharide of longan gives the insights into longan polysaccharide application as an immunomodulatory agent.

Full text of DOI:10.1016/j.carbpol.2019.03.007

CarbohydratePolymers213(2019)247–256
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structural characterization of an active polysaccharide of longan and
evaluation of immunological activity
Yu Ronga,1, Ruili Yangb,1, Yuzhe Yanga, Yazhou Wena, Sixin Liua, Congfa Lia, Zhuoyan Hub,
Xiangrong Chengc,⁎, Wu Lia,⁎
a Engineering Research Center of Utilization of Tropical Polysaccharide Resources, Ministry of Education, College of Food Science and Technology, Hainan University,
Haikou 570228, China
b College of Food Science, South China Agricultural University, Guangzhou 510642, China
c The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
An active polysaccharide (LPD2) was isolated from longan pulp by comparing the effects of polysaccharides on
the phagocytosis of macrophages. LPD2 was composed of arabinose, mannose, glucose, and galactose in a molar
ratio of 0.25:0.49:1:0.5 with average molecular weight of 9.64 × 106 Da. The main linkages of the sugar residues
of LPD2 were (1→4)-β-Glc and (1→6)-β-Man. LPD2 significantly enhanced the lymphocytes proliferation,
phagocytosis and NO and IL-6 secretion by macrophage. The anti-TLR2 and anti-TLR4 mAbs markedly sup-
pressed LPD2-mediated NO and IL-6 production. Furthermore, anti-TLR4 or anti-TLR2 plus anti-TLR4 treatment
significantly decreased LPD2-induced increase of MyD88, IRAK4, TRAF6 and INOS mRNA expression. Moreover,
western blotting analysis showed that LPD2 enhanced the expression of target proteins in MyD88/IRAK4-TRAF6-
INOS pathways. These results suggested that LPD2 induced macrophage activation partly via the TLR2- and
TLR4-mediated MyD88/IRAK4-TRAF6 signaling pathways. Knowing the structural features and activities of
active polysaccharide of longan gives the insights into longan polysaccharide application as an im-
munomodulatory agent.
Longan polysaccharides
Structural features
Immunomodulatory activities
Signaling pathways
1. Introduction
the function of T cells, B cells, macrophages cells, lymphocytes, den-
dritic cells and two-way regulations of immunologic function (Xie et al.,
Polysaccharides are macromolecules composed of monosaccharide
units joined by glycosidic bond, which play an important role in living
organism and widely involved in cell immune defense, cell adhesion,
cell differentiation and proliferation (Xie et al., 2016). In recent years,
polysaccharides from natural sources have attracted much attention in
the field of pharmacology and biochemistry due to their significant
biological activities, such as anti-diabetic, prebiotic, antitumor, anti-
viral, and immunomodulation activities (Ferreira, Passos, Madureira,
Vilanova, & Coimbra, 2015; Martins et al., 2017). Several poly-
saccharides have been used as immunotherapeutic agents for the
treatment of cancer and clinically applied in combination with che-
motherapy (Bisen, Baghel, Sanodiya, Thakur, & Prasad, 2010; Xie et al.,
2016). Astragalus polysaccharide, ginseng polysaccharide, anoderma lu-
cidum polysaccharide, lentinan, coriolus versicolor polysaccharide and
pachman are applied in clinical medicine (Yu, Shen, Song, & Xie, 2018).
Astragalus polysaccharide have been demonstrated that could improve
2016; Yu et al., 2018). The Morus alba polysaccharides had been re-
ported to enhance humoral immunity and nonspecific immunity by
increasing NK cell activity, promoting B and T cells proliferation (He
et al., 2017). The Ganoderma lucidum polysaccharide could facilitate
dendritic cell maturation thus inducing proliferation of T cells in the
course of cancer immunotherapy (Zhang et al., 2019). Meanwhile,
polysaccharide could improve gut immune function by regulating in-
testinal microbiota as well as the gut metabolites (Liu et al., 2019;
Zhang et al., 2017).
Although diverse biological activities of polysaccharides from nat-
ural sources have been constantly reported, the structures of most of the
bioactive polysaccharides are still vaguely characterized due to their
heavier molecular weights and complex compositions, which limit their
application.The biological activities of macromolecules are closely re-
lated to their structures (Li, Wu, Lv, & Zhao, 2013). Compared to pro-
teins, the studies of structure–bioactivity relationships of
⁎ Corresponding authors.
E-mail addresses: chengxr@jiangnan.edu.cn (X. Cheng), leewuu@163.com (W. Li).
1 These authors contributed equally to this article.
https://doi.org/10.1016/j.carbpol.2019.03.007
Received 3 October 2018; Received in revised form 1 March 2019; Accepted 3 March 2019
Availableonline04March2019
0144-8617/©2019ElsevierLtd.Allrightsreserved.

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
polysaccharides are limited. Previous reports showed that the chemical
structure, such as molecular weight, functional groups, branching, and
conformation were some of the structural features identified as con-
tributing to the polysaccharides bioactivities. However, no corre-
sponding definite rule has been proposed between the structures and
biological activities (Ferreira et al., 2015). It is necessary to clarify the
structure of individual polysaccharide from different sources, in order
to uncover and explore structure-bioactivity relationship of poly-
saccharides (Huang et al., 2016).
were obtained via freeze-drying by a freeze dryer (Martin Christ, Os-
terode, Germany) after dialysis. Crude polysaccharides were purified
with weak anion exchanger (DEAE- Fast Flow), which was eluted with a
step-wise gradient of 0, 0.05, and 0.5 mol/L NaCl aqueous solution.
Three segments were collected according to absorbance detected by
phenol-sulfuric acid method and further purified by liquid chromato-
graphy system (Shimadzu, Kyoto, Japan) with TSKgel-G4000PWXL
(7.8 cm I.D × 30 cm) and TSKgel-G3000PWXL (7.8 cm I.D × 30 cm)
(TOSOH, Shanghai, China) gel columns connected in series, eluted with
water at a flow rate of 0.5 mL/min. The column temperature was kept
at 60 °C. Different peaks were collected according to the retention time.
Longan (Dimocarpus longan Lour.) is a subtropical fruit, which has
long been used as a traditional medicine for treating insomnia, palpi-
tation, and forgetfulness in Asian countries (Park et al., 2010). Recent
medical studies have demonstrated that longan polysaccharides pos-
sessed a variety of bioactivities, including antioxidant (Jiang et al.,
2013), antitumor (Yi et al., 2013), and immunoregulatory activity
(Jiang et al., 2014). Several recent studies have shown that longan
polysaccharides could stimulate splenic lymphocyte proliferation and
macrophage phagocytosis (Jiang et al., 2014; Yi et al., 2012), increase
interferon-gamma (Meng et al., 2014), tumor necrosis factor-α and
interleukin-6 secretion (Jiang et al., 2014), and the structural char-
acteristics of longan polysaccharides were analyzed as well (Meng et al.,
2014; Yi et al., 2012). There are differences in the structural char-
acteristics of longan polysaccharides in these reports, which suggested
that there might be multiple polysaccharides contributing to im-
munoregulatory activity of longan. The structure and im-
munoregulatory activity analysis of individual longan polysaccharide is
necessary for revealing their biological activities and structure-bioac-
tivity relationship. In this study, the active polysaccharide was obtained
from longan pulp by comparing the effects of these purified poly-
saccharides on the phagocytosis of macrophages. Its structure proper-
ties were characterized using gas chromatography (GC–MS), multi-
angle laser light scattering (SEC-LLS), Fourier transform infrared (FT-
IR), and nuclear magnetic resonance (NMR) analysis. Moreover, its
immunomodulatory activities were evaluated.
2.3. Evaluation of the structure characteristics of LPD2
2.3.1. SEC-LLS analysis
Molecular parameters of the sample were measured by multi-angle
laser light scattering detection (Wyatt, Santa Barbara, California, USA)
equipped with Ultrahydrogel 2000 (Waters, Shanghai, China) and
Multi-angle laser photometer and refractive index detector. The data
was analyzed by ASTRA5.3.4.20 software.
2.3.2. GC analysis
Monosaccharide composition was analyzed by the previous method
(Jiang et al., 2013). Briefly, the 5 mg sample was hydrolyzed by 5 mL of
2 mol/L trifluoroacetic acid at 121 °C for 2 h, dried under vacuum. Then
500 μL pyridine, 200 μL hexamethyldisilazane and 100 μL trimethyl-
chlorosilane were added and kept at 30 °C for 10 min. The derivative
products were analyzed by gas chromatography system (Agilent, Santa
Clara, CA, USA) equipped with rtx-50 column (30 m × 0.25 mm ×
0.25 mm) and a flame ionization detector. The GC temperature pro-
gramme was as follow: the original temperature was 180 °C held for
3 min, increased to 210 °C by 0.3 °C/min, then increased to 215 °C by
6 °C/min and finally reached 240 °C and held for 30 min. The carrier gas
was nitrogen and the flow rate was 0.88 mL/min. The pressure was
110 kPa and the split ratio was 19:1.
2. Materials and methods
2.3.3. Methylation analysis
2.1. Materials
Methylation analysis was carried out according to a previously
method (Needs & Selvendran, 1993) with some modifications. Briefly,
5 mg samples were dissolved in 5.0 mL anhydrous dimethylsulfoxide
(DMSO) before 200 mg NaOH was added. After the mixture was stirred
for 4 h, 1.5 mL methyl iodide was added. The mixture was kept in dark
for 2 h before 4.0 mL of distilled water was added. The product was
then extracted with 3 mL chloroform and then dried at reduced pressure
by a rotary evaporator. The residues were hydrolyzed with 10 mL tri-
fluoroacetic acid (2 mol/L) at 100 °C for 8 h and then the hydrolysates
were dissolved into 4 ml of 1% (w/w) NaOH. 10 mg NaBH4 was added
to reduce hemiacetal bond and 100 μL of glacial acetic acid was added
after the reduction. The sample was dried under reduced pressure, and
then acetylated with 1.5 ml acetic anhydride and 1.5 ml pyridine at
100 °C for 2 h. The acetylated derivatives were extracted with 4 mL
chloroform and analyzed by GCeMS (Agilent 7890B-5977, Agilent,
Santa Clara, CA, USA). The operation condition for GC analysis was as
follows: the column (Agilent HP-5) temperature was increased from
120 °C to 180 °C at 10 °C/min, then increased to 280 °C at 10 °C/min
and held for 5 min at 280 °C Injection temperature was 170 °C. Helium
was used as the carrier gas and maintained at a flow rate of 1 mL/min.
Fresh longan (cv. Chuliang) fruits were harvested in 2014 in
Guangzhou, China. The mouse RAW264.7 cells were donated by
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou,
China. RPMI-1640 medium, DMEM medium and fetal bovine serum
were purchased from Gibco Life Technologies (Carlsbad, California,
USA). LPS, polymyxin B were purchased from Sigma-Aldrich (Shanghai,
China). Anti-mouse TLR2 (clone 6C2), TLR4 (clone MTS510) mAbs and
Isotype-control Abs were purchased from Thermo Fisher Scientific
(Waltham, MA, USA). Primary antibodies to MyD88 (ab 219413),
TRAF6 (ab 33915), inducible NO synthase (INOS, ab 3523), beat-Actin
(ab 8277) were purchased from Abcam (Cambridge, MA, USA), and
IRAK4 (4363S), Phospho-IRAK4 (D6D7) from Cell Signaling
Technology (Danvers, MA, USA). DEAE- Fast Flow was purchased from
GE Healthcare (New York, USA).
2.2. Preparation of polysaccharides
Fresh longan were peeled, stoned and dried at 65 °C via hot air
drying. The dried longan pulp were crushed by a high-speed disin-
tegrator for 5 min and extracted with distilled water (1:20, g/mL) at
80 °C for 2 h with continuously stirring. The filtrate was concentrated
into one- twentieth volume under vacuum circumstance at 55 °C by a
rotary evaporator (Shensheng Technology Ltd, Shanghai, China) and
then decolored and discarded protein by D301R macro-porous weak
base anion exchange resin according to a previous method (Yi et al.,
2012). After the removal of pigments and proteins, the filtrate was
concentrated and dialyzed (8000 ˜14,000 Da). Crude polysaccharides
2.3.4. FT-IR analysis
The sample were ground with potassium bromide and pressed into
slice. Fourier transform infrared spectrum of the sample was detected
by Fourier transform infrared spectrum spectrometer (Bruker,
Rheinstetten, German) at the wavelength range of 400–4000 cm−1
.
2.3.5. NMR analysis
1H and 13C spectra and HSQC spectrum of the sample were recorded
248

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
at 30 °C with AV-500 MHz spectrometer (Bruker, Rheinstetten,
German). Tetramethoxysilane was used as internal standard substance.
0.1 μg/mL of anti-TLR2, anti-TLR4, anti-TLR2 plus anti-TLR4 anti-
bodies or Isotype-control antibodies, for 30 min, and then 100 μg/mL
LPD2 was added for 24 h. The production of NO and IL-6 was examined
as described above.
2.4. Evaluation of the immunoregulatory activities of LPD2
2.4.1. Macrophage phagocytosis
2.4.5. RT-PCR analysis
Phagocytosis was examined as described previously (Hoppstädter
et al., 2019; Shiratsuchi, Watanabe, Takeuchi, Akira, & Nakanishi,
2004). Briefly, RAW 264.7 cells were plated into a 24-well plate at
2 × 105 cells/mL in DMEM. After incubated for 2 h (37 °C, 5% CO2), the
non-adherent cells were discarded. LPD2 (6.25 μg/mL～50 μg/mL;
control group: 0 μg/mL) were pre-incubated with or without polymyxin
B (PMB, 1000 units/mL) for 1 h at 4 °C and were then used to stimulate
the macrophages for 12 h. Subsequently, a suspension of FITC-labeled
latex beads (Polybead Microparticles; Polysciences, Warrington, PA,
USA) was added at a 30:1 bead/cell ratio in the cell cultures. After
incubation for 1.5 h, the cell suspensions were removed and the wells
were washed five times with pre-warmed PBS to remove remaining
fluorospheres. The intracellular fluorescence intensity was measured
with a Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA).
The formula of the phagocytic index of the cells was calculated as
follow: Phagocytic index (%) = (Fluorescence values of sample group -
Fluorescence values of control group)/ Fluorescence values of control
group.
After RAW 264.7 cells were pre-treated with or without 0.1 μg/mL
of anti-TLR4 or anti-TLR2 plus anti-TLR4 mAbs, the cells were in-
cubated with LPD2 (50 μg/mL and 100 μg/mL) for 4 h. The total RNA
was extracted from cells using TRIzol Reagent and transcribed into
cDNA using a Reverse Transcription System kit (TAKARA, DaLian,
China), according to the manufacturer's instructions. Real-time PCR
were performed with the incorporation of SYBR green using the real-
time PCR System 7500 (Applied Biosystems,Waltham, MA). PCRs were
carried out for 40 cycles using the following conditions: 95 °C for 10 s,
60 °C for 30 s. The mean Ct of the gene of interest was calculated from
triplicate measurements and normalized with the mean Ct of a control
gene, β-actin. The sequences of the primers used are listed in Table S1
(Supplement materials).
2.4.6. Western blot analysis
After incubated with 100 μg/mL LPD2 for 4 h, RAW 264.7 Cells
incubated in lysis buffer (Beyotime Institute of Biotechnology, Beijing,
China) on ice for 20 min. The cell lysates were centrifuged at
12,000×g, 4 °C for 10 min. The concentration of proteins was de-
termined with the BCA protein assay kit (Beyotime Institute of
Biotechnology, Beijing, China). The proteins were loaded and separated
by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE) and blotted onto PVDF membranes. Subsequently, the mem-
brane was blocked in TBST buffer (5.0% skim milk, 20 mM Tris-HCl,
137 mM NaCl, 0.1% Tween-20) for 1 h with shaking at room tem-
perature. Then the membranes were incubated with the primary anti-
bodies (diluted according to the manufacturer’s instructions) at 4 °C
overnight. After washing three times with TBST, the membranes were
incubated with the appropriate secondary antibodies for 1 h at room
temperature. After washing three with TBST, the antibody-specific
protein was visualized by enhanced chemiluminescence detection
system (Bio-Rad, Hercules, CA, USA).
2.4.2. Nitric oxide (NO) and IL-6 assay
The cells were plated into a 96-well plate at 2 × 105 cells/well and
exposed to the polysaccharide sample (25 μg/mL～100 μg/mL) for 24 h.
Equal volume of medium or LPS (10 μg/mL) was added as the control
group and positive group, respectively. The cell liquid supernatant was
collected by centrifugation at 5000 g for 10 min. The levels of NO and
IL-6 were measured using a NO ELISA kit (Beyotime institute of bio-
technology, Shanghai, China) and IL-6 ELISA kit (R&D systems,
Minneapolis, MN, USA), respectively.
2.4.3. Splenic lymphocyte proliferation assay
Mice (Kunming mouse) were purchased from Guangdong Medical
Laboratory Animal Center, China. All mice were bred and housed at
Institute of laboratory animal science, South China Agricultural
University, Guangzhong, China in specific pathogen-free conditions and
all experiments were performed according to protocols approved by the
Institutional Animal Care and Use committee of South China
Agricultural University (20160311). Mice anaesthetized and sacrificed
by cervical dislocation and their spleens were harvested and put into
aseptic PBS buffer solution. A single cell suspension was obtained by
mincing the spleens and passing them through a 200-mesh cell strainer.
Red blood cells were depleted with Tris–NH4Cl lysis (0.144 M NH4Cl,
0.017 M Tris–HCl). The remaining cells were washed twice and re-
suspended in RPMI 1640 medium supplemented with 10% fetal bovine
serum. the splenic lymphocytes were plated into a 96-well plate at
1 × 106 cells /mL in 0.1 mL of RPMI-1640 medium. The cells were
stimulated by a series of concentrations of the polysaccharide sample
(12.5 μg/mL～100 μg/mL) and incubated for 68 h at 37 °C with 5%
CO2. Lymphocytes were cultured for another 4 h after adding 0.02 mL
of 5 mg/mL MTT solution and then 100 μL of acidified isopropyl alcohol
were added. The plate was kept at room temperature for 2 h. The ab-
sorbance at 570 nm was detected with a microplate reader (SpectraMax
190, Molecular Devices, Sunnyvale, CA, USA). Equal of medium was
added as the control group. The proliferation index of splenic lym-
phocytes was calculated according to the formula as follow:
Proliferation index (%) = (A570 sample group - A570 control group)/
A570 control group ×100%.
2.5. Statistical analysis
Data were expressed as the mean
standard deviation and ana-
lyzed by SPSS V.13 (SPSS Inc., Chicago, IL, USA). One-way analysis of
variance (ANOVA) and Duncan’s New Multiple-range test were assessed
statistical differences between groups. Differences at p < 0.05 were
considered statistically significant.
3. Results
3.1. Purification of longan polysaccharides
Four longan polysaccharides were obtained via liquid chromato-
graphic and non-protein polysaccharides LPD2 showed higher stimu-
latory effect on macrophage phagocytosis among purified longan
polysaccharides (Unpublished). The homogeneity of LPD2 was eval-
uated by high performance liquid chromatography and SEC-LLS chro-
matograms. The peak shape of LPD2 was a single symmetrical peak
(Fig. 1A), similar to that in SEC-LLS chromatograms (Fig. 1B), which
indicated that the molecular weight of LPD2 was homogeneous.
3.2. Structural properties of LPD2
3.2.1. Molecular parameters
2.4.4. Antibody inhibition experiments
To determine whether TLR2 or TLR4 could be involved in the LPD2-
induced macrophage activation, RAW264.7 cells were pre-treated with
Molecular parameters of LPD2 were detected, including poly-
dispersity index, molar mass moments, and rms radius moments. The
value of Mw/Mn and Mz/Mn represented the polydispersity index of
249

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
Fig. 1. HPLC (A) and SEC-LLS (B) analysis of LPD2 purified from longan pulp.
2012). The carbonyl group bands at 1632.57 cm−1. The absorption
peaks from 1200 cm-1 to 1000 cm−1 were assigned to CeOeC glyco-
sidic bond vibrations and ring vibrations overlapped with stretching
vibrations of the group CeOeH (Huang et al., 2016). The absorption
peaks at 928.83 cm-1 was attributed to the vibrations of D-pyran ring
and the peak at 842.64 cm-1 is a typical characteristic of α-D-gluco-
pyranose ring (Yang & Zhang, 2009).
Table 1
Molecular characteristics of LPD2.
Molecular characteristics
Polydispersity
Parameter
Detection results
Mw/Mn
Mz/Mn
Mn
Mp
Mw
Mz
Rn
Rw
Rz
1.14
1.54
Molar mass moments (g/mol)
8.44 × 106
7.44 × 106
9.64 × 106
1.30 × 107
56.10
Rms radius moments (nm)
3.2.3. Monosaccharide composition
55.30
54.00
Monosaccharide composition was identified via comparing the re-
tention time of monosaccharide of LPD2 with that of standard mono-
saccharide and calculating its peak area. As shown in Table S2
(Supplement materials), LPD2 was composed of arabinose, mannose,
glucose, and galactose in a molar ratio of 0.25 : 0.49 : 1 : 0.5. Glucose
had the highest proportion and the proportion of mannose was almost
equal to galactose’s.
molecular weight of LPD2. As shown in Table 1, the Mw/Mn and Mz/
Mn of LPD2 was 1.14 and 1.54, respectively, which suggested that the
molar mass distribution of LPD2 was narrow, consistent with chroma-
tography (Fig. 1A, B). The Mw, Mn, Mp, and Mz values of LPD2 were
9.64 × 106 (g/mol), 8.44 × 106 (g/mol), 7.44 × 106 (g/mol), and
1.30 × 107(g/mol), and the Rn, Rw and Rz were 56.10 nm, 55.30 nm
and 54.00 nm, respectively.
3.2.4. Glycosidic linkage
The glycosidic linkage of LPD2 was determined by methylation
method and analyzed by GC/MS. As shown in Table 2, the →6)-Glc-
(1→, →4)-Glc-(1→, →6)-Gal-(1→, →3)-Gal-(1→, →6)-Man-(1→, →5)-
Ara-(1→, and Ara-(1→ were identified in LPD2. Moreover, the result
indicated that the major linking types of LPD2 were →4)-Glc-(1→
(41.29%) and →6)-Man-(1→ (21.89%) with a high molar percentage.
3.2.2. FT-IR spectrum characterization
The infrared spectrum of LPD2 was shown in Fig. S1 (Supplement
materials). The intense and broad brand at 3368.63 cm−1 was attrib-
uted to stretching vibration of hydroxyl group, the alkyl group bands at
2937.21 cm−1 and 1452.39 cm−1 (Wu, Zhu, Zhang, Yang, & Zhou,
250

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
3.2.5. NMR spectrum characterization
Table 2
1H-NMR, 13C-NMR, and HSQC spectra of LPD2 were shown in
Fig. 2A–C, respectively. The 1H-NMR signal at 1.92 ppm was assigned to
the protons of the acetyl groups (Muljana, Picchioni, Heeres, & Janssen,
2010). The 13C-NMR spectrum of LPD2 showed C-1 signals ranging
from 106.4 ppm to107.1 ppm. It had no signal at low field, which de-
monstrated LPD2 did not contain uronic acid (Liu, Chen, Chen, Huang,
The relative molar percentage of glycosidic linkages in LPD2.
Composition
glucose
Glycosidic linkages
relative molar percentage
→6)-Glc-(1→
→4)-Glc-(1→
→6)-Gal-(1→
Gal-(1→
3.37
41.29
12.91
9.35
galactose
&
Cheung, 2016; Liu, Zhu et al., 2016). The sharp signals at
mannose
arabinose
→6)-Man-(1→
→5)-Ara-(1→
Ara-(1→
21.89
8.39
2.80
106.4–107.1 ppm indicated β configuration of the glucosidic linkages
and the signals at 107.1, 78.1/4.10, 79.1/4.19, 72.5/3.71, 66.3/3.90,
3.58 implied the presence of (1→4)-β-glycosidic linkages (Yang &
Zhang, 2009). Meanwhile, the HSQC spectrum (Fig. 2C) showed a weak
signal at the range of 4.98–5.40 ppm, and suggested that LPD2 con-
tained a few α-glucosidic linkages, consistent with FT-IR data (Fig. S1,
Supplement materials).
The signals appearing at 4.47/105.4, 5.10/109.7, 5.20/109.0 and at
5.25/111.5 ppm in the HSQC spectrum (Fig. 2C) were deduced to ap-
pear from (1→6)-β-linked mannose residues with the aid of linkage
data from IR and literature of similar polysaccharides (Li et al., 2015;
Ližičárová, Matulová, Machová, & Capek, 2007). These results sug-
gested that LP-D had the backbone that mainly composed (1→4)-β-Glc
and (1→6)-β-Man.
3.3. Immunoregulatory activities of LPD2
3.3.1. Effects on macrophage phagocytosis
The stimulatory effects of LPD2 on mouse macrophage phagocytosis
were shown in Fig. 3A. LPD2 enhanced the cells phagocytosis in a dose-
dependent manner at 6.25–50 μg/mL (p < 0.05). Meanwhile, the ef-
fects of LPD2 on macrophage phagocytosis were not depressed by
treatment with PMB (p > 0.05). Treatment with 50 μg/mL LPD2 in-
creased the phagocytosis to 205.1
9.3% of that of control, which
suggested that the significant activity of the LPD2 to enhance the
macrophage phagocytosis.
3.3.2. Effects on NO and IL-6 production of macrophage
LPD2 showed significant stimulatory effect on the production of NO
(Fig. 3B) and IL-6 (Fig. 3C), with a dose-dependent manner (p < 0.05).
There were no significant changes in the number of RAW 264.7 cells
(MTT analysis), however, NO and IL-6 productions increased 8.15 fold
(100 μg/mL LPD2) and 5.08 fold (200 μg/mL LPD2), compared with the
control, respectively.
3.3.3. Effects on splenic lymphocyte proliferation
As shown in Fig. 3D, a significant proliferation was observed when
splenic lymphocytes were treated with LPD2 in the dose range of 12.5～
100 μg/mL. The splenic lymphocytes increased to 250
1.9% of the
control value when treatment with 50 μg/mL LPD2, which showed ef-
fectively stimulatory effect of LPD2 on the splenic lymphocyte pro-
liferation.
3.3.4. Effects of anti-TLR2 and anti-TLR4 antibodies on LPD2-induced NO
and IL-6 secretion
As shown in Fig. 4A and B, anti-TLR2 and/or anti-TLR4 treatment
significantly decreased the levels of NO and IL-6 in comparison to the
group treated with LPD2 only (p < 0.05). The levels of NO and IL-6 in
the group treated with anti-TLR2 plus anti-TLR4 were 31.77% and
42.18% of the group treated with isotype-control antibodies (isotype
group), respectively. Moreover, the isotypic antibodies treatment did
not significantly change the levels of NO and IL-6 in comparison to the
group treated with LPD2 only (no Ab group). NO and IL-6 productions
of ctrl+αR2+αR4 group did not have significant differences compared
with the control group (ctrl group). These results suggested that TLR2
and TLR4 could be involved in the LPD2-induced macrophage activa-
tion.
Fig. 2. The NMR spectra analysis of LPD2.
1HNMR (A), 13C NMR (B) and HSQC (C) spectra of LPD2.
RAW 264.7 cells were pre−treated with 0.1 μg/mL anti−TLR2
251

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
Fig. 3. Effects of LPD2 on macrophages and the proliferation of splenic lymphocyte.
(A) Effects of different concentration of LPD2 on macrophages phagocytosis. (B) and (C) were the effects of LPD2 on NO and IL-6 secretion of RAW 264.7 cells,
respectively. The group without LPD2 was used as control group, and LPS was used as the positive group. (D) Effects of LPD2 on the proliferation of splenic
lymphocyte. The proliferation index was measured by the MTT method. All data was given as means standard deviation (n=6). Values with different superscript
letters above the bar are significantly different (p < 0.05).
(aR2 group), anti−TLR4 (aR4 group), anti−TLR2 plus anti−TLR4
(aR2 + aR4 group, 0.1 μg/mL anti−TLR2 and 0.1 μg/mL anti−TLR4
antibodies) or isotype−control antibodies (isotype group) separately
for 30 min before adding LPD2 (100 μg/mL). The cells were
pre−incubated with the medium (do not add antibodies) for 30 min
and then LPD2 (100 μg/mL), equal volume of medium or LPS (10 μg/
mL) was added as the LPD2 group (no Ab group), control group (ctrl
group) and positive group (LPS group), respectively. After pre−treated
Fig. 4. Effects of anti-TLR2 and anti-TLR4 antibodies on LPD2-induced NO and IL-6 secretion.
252

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
Fig. 5. Effect of LPD2 on the mRNA and protein expression of MyD88, IRAK4, TRAF6 and INOS of macrophage.
(A) Effect of LPD2 and anti-TLR4 or anti-TLR2 plus anti-TLR4 on the mRNA expression of MyD88, IRAK4, TRAF6 and INOS of RAW 264.7 cells. mRNA expression
levels detected by RT-PCR were relative to that of the reference gene, β-actin. The cells were incubated with LPD2 (50 μg/mL, 100 μg/mL) for 8.0 h (n = 4). *
p < 0.05 compared with the control group; # p < 0.05 compared with the 100 μg/mL LPD2 group. (B), (C) Effect of LPD2 on the protein expression of MyD88, IRAK4
(t-IRAK4), phosphorylation IRAK4 (p- IRAK4), TRAF6 and INOS of RAW 264.7. The cells were incubated with LPD2 (100 μg/mL) for 4.0 h (n = 3). * p < 0.05
compared with the control group.
with 0.1 μg/mL anti−TLR2 plus 0.1 μg/mL anti−TLR4 for 30 min, the
cells were added the equal volume of medium as the antibodies control
(ctrl + aR2 + aR4 group). After 24 h, NO (A) and IL−6 (B) in the
culture supernatants were measured by ELISA. The values with dif-
ferent superscript letters above the bar are significantly different
(p < 0.05, n = 3).
after treatment by 100 μg/mL LPD2 were 2.71 0.2, 3.36 0.35,
2.07 0.08 and 3.96 0.49 times more than the control group, re-
spectively. Moreover, anti-TLR4 or anti-TLR2 plus anti-TLR4 treatment
significantly decreased the expression of these genes. The expression
levels of MyD88, IRAK4, TRAF6 and INOS after treatment by anti-TLR2
plus anti-TLR4 were reduced to 1.07 0.13 (p > 0.05), 0.97 0.13
(p > 0.05), 1.19 0.12 (p < 0.05) and 1.27 0.10 (p < 0.05)
times of the control group, respectively. These results suggested that
LPD2 induced macrophage activation partly through TLR2- and TLR4-
mediated MyD88/IRAK4-TRAF6 pathways. While, the expression of
TRAF6 and INOS was not completely suppressed by anti-TLR2 plus anti-
TLR4, implying that MyD88-independent pathway or other receptors
3.3.5. Effects of LPD2 on the mRNA and protein expression of MyD88,
IRAK4, TRAF6 and INOS
As shown in Fig. 5A, LPD2 up-regulated MyD88, IRAK4, TRAF6 and
INOS mRNA expression of macrophage (p < 0.05), in a dose-depen-
dent manner. The expression levels of MyD88, IRAK4, TRAF6 and INOS
253

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
may be involved in LPD2 induced macrophage activation.
To further confirm the involvement of MyD88 pathways in the
LPD2-induced macrophage activation, we examined the proteins levels
using western blot analysis. The results (Fig. 5B, C) indicated that LPD2
could enhance (p < 0.05) the expression of MyD88, TRAF6, INOS, and
phosphorylation level of IRAK4 (p-IRAK4), which were consistent with
the mRNA expression results.
immunostimulatory activity as well (Meng et al., 2014; Yi et al., 2015).
The results of the studies showed that LPD2, which exhibited stronger
immunostimulatory activity compared with longan polysaccharides
composed of (1→6)-α-D-Glc (Meng et al., 2014; Yi et al., 2015), is
mainly composed (1→4)-β-Glc containing mannose, galactose and
arabinose. The principal immunostimulatory polysaccharide from
Acetobacter xylinum (Li et al., 2004) and Rhizobium (Zhao, Chen, Ren,
Han, & Cheng, 2010) have been found to be mainly composed of (1→
4)-β-Glc. The mannose, arabinose, and galactose are identified as the
monosaccharides related to immunoregulatory activity (Kralovec et al.,
2007; Lo, Jiang, Chao, & Chang, 2007; Zhao et al., 2010). These specific
monosaccharides may serve as recognition sites of cell receptors and
facilitate the immune response (Ferreira et al., 2015), potentially re-
sulting in relatively high immunomodulatory activities of LPD2. In this
study, LPD2 mainly composed (1→4)-β-Glc containing mannose, ga-
lactose and arabinose, higher molecular weight, and with acetyl ex-
hibited stronger effects on macrophage activation and lymphocyte
proliferation.
The immunoregulatory activities of polysaccharides largely depend
on the modulation of the cell membrane receptor and the subsequent
intracellular signaling pathway. Toll-like receptors (TLRs) are a class of
proteins that play a key role in the recognition of invading pathogens
and activation of cytokine production by macrophages (Martinon,
Chen, Lee, & Glimcher, 2010). Recently, several reports have demon-
strated that the polysaccharides activated macrophages via TLRs (Liu,
Chen et al., 2016; Liu, Zhu et al., 2016; Zhang, Wang, Lai, & Wu, 2016).
Liu et al. (Liu, Su et al., 2018; Liu, Zhang et al., 2018) found that As-
tragalus polysaccharide protects against coxsackievirus B3-induced
myocardial damage through TLR-4/NF-κB p65 signal pathway. Mean-
while, Liu et al. (Liu, Su et al., 2018; Liu, Zhang et al., 2018) reported
that Astragalus polysaccharide could attenuate the immune stress in-
duced by OTA via activation of the AMPK/SIRT-1 signaling pathway.
Deng et al. (Deng, Fu, Shang, Chen, & Xu, 2018) found that TLR-4/NF-
κB p65 and Dectin1 receptor involving in the activation effect of Dic-
tyophora indusiate polysaccharides. In this study, the anti-TLR2 plus
anti-TLR4 mAbs significantly suppressed the LPD2-mediated produc-
tion of NO and IL-6. These results suggested that TLR2 and TLR4 were
involved in LPD2-mediated activation of macrophages. The stimulation
of TLRs could lead to the activation of several transcription factors,
including NF-κB. In this study, anti-TLR4 or anti-TLR2 plus anti-TLR4
treatment significantly decreased LPD2-induced increase of the mRNA
expression of MyD88, IRAK4, and TRAF6, which played an important
role in the activation of NF-κB by TRLs-induced MyD88-dependent
pathway. At the same time, our results suggested that there might be
other receptors or MyD88-independent pathway involved in LPD2-in-
duced macrophage activation. Taken together, LPD2 induced macro-
phage activation partly via the TLR2- and TLR4-mediated MyD88/
IRAK4-TRAF6 signaling pathway (Fig. 6).
4. Discussion
Polysaccharides from many natural sources have been reported
modulating immune function through interacting direct or indirectly
with the immune system (Ferreira et al., 2015). Though some poly-
saccharides separated, purified and identified from longan have been
proven to enhance immune function (Meng et al., 2014; Yi et al., 2015;
Zhu et al., 2013), the main active polysaccharides and structural
characteristics are still unclear. In the present study, we found the main
active polysaccharide of longan (LPD2) and its structure was char-
acterized. Furthermore, its immunoregulatory activities were in-
vestigated. LPD2 mainly consisted of (1→4)-β-Glc and (1→6)-β-Man
showed strong activation of the macrophage and lymphocyte.
Macrophages have crucial and distinct roles in immunity (Ginhoux
& Jung, 2014). Activation of macrophages plays a central role in the
immunomodulatory activity of polysaccharides (Huang, Nie, & Xie,
2017; Xie et al., 2016). In this study, LPD2 was found to strongly en-
hance the macrophage phagocytosis and the production of IL-6 and NO
indicating that LPD2 effectively induced the functional activation of
macrophages. Meanwhile, LPD2 showed strong ability to stimulate the
proliferation of splenic lymphocytes, which are key participants in the
innate and adaptive immunity. These results indicated that LPD2 has a
significant effect on the activation of immunity. In addition, compared
with the longan polysaccharides mainly composed of (1→6)-Glc (Yi
et al., 2015) and other previous reports (Jiang et al., 2014; Meng et al.,
2014; Yi et al., 2011, 2012), LPD2 showed stronger effect on activation
of macrophages phagocytosis, as well as on proliferation of splenic
lymphocytes than that of the longan polysaccharides of previous reports
(Meng et al., 2014; Yi et al., 2011, 2012).
Previous reports suggest that the structural features of poly-
saccharides have important bearing on their biological activities
(Ferreira et al., 2015). Polysaccharides with higher molecular weight
fractions usually exhibit considerable biological activity as high mole-
cular weight may be resulting in more repetitive structures, which
could cross-link receptors or other membrane targets (Schepetkin et al.,
2008). The immunomodulatory activity of polysaccharides isolated
from Opuntia polyacantha (Xie, Schepetkin IASiemsen, Kirpotina, Wiley,
& Quinn, 2008), Alchornea cordifolia (Kouakou et al., 2013), Artemisia
tripartite (Xie et al., 2008), and Tanacetum vulgare (Gang Xie,
Schepetkin, & Quinn, 2007) were positively correlated with their mo-
lecular weight. Consistent with the previous observations, the present
study showed that LPD2 has higher molecular weight and also exhibited
stronger immunostimulatory activity compared with longan poly-
saccharides of previous reports (Jiang et al., 2014; Meng et al., 2014; Yi
et al., 2011, 2012, 2015).
In addition, the presence of functional groups (such as acetyl, sul-
fate) can influence the bioactivity of polysaccharides by affecting the
charge, solubility, and binding capacity of receptor. The acetyl groups
played a key role for the maintenance of high immunoregulatory ac-
tivity of polysaccharides of coffee (Simões et al., 2010), Tremella aur-
antialba (Du et al., 2014), fucus evanescens (KhilâChenko et al., 2011).
The acetyl groups can promote the formation of hydrophobic pockets in
polysaccharides and trigger different immunostimulatory responses
(Ferreira et al., 2015), which may be a reason for the stronger im-
munostimulatory activity of LPD2.
5. Conclusion
In summary, an active polysaccharide of longan was obtained and
its structure was characterized. LPD2 was composed of arabinose,
mannose, glucose, and galactose in a ratio of 0.25 : 0.49 : 1 : 0.5 with
average molecular weight of 9.64 × 106Da. The main linkages of the
sugar residues were (1→4)-β-Glc and (1→6)-β-Man. Immunological
activity assay indicated that LPD2 strongly enhanced the lymphocytes
proliferation, phagocytosis and the production of NO and IL-6 of mac-
rophages. The immunostimulating properties of LPD2 on macrophages
were through a receptor-mediated mechanism, which involves TLR2
and TLR4 and resultant activation of MyD88/IRAK4-TRAF6 signaling
pathways.
Zhu et al. (Zhu et al., 2013) reported that a (1→6)-α-D-Glucan from
longan has anticancer activity. While two longan polysaccharides
mainly composed of (1→6)-α-D-Glc have been reported
Conflict of interest
The authors declare no competing financial interest.
254

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
Jiang, G., Wen, L., Chen, F., Wu, F., Lin, S., Yang, B., et al. (2013). Structural char-
acteristics and antioxidant activities of polysaccharides from longan seed.
Carbohydrate Polymers, 92(1), 758–764.
Jiang, J., Meng, F. Y., He, Z., Ning, Y. L., Li, X. H., Song, H., et al. (2014). Sulfated
modification of longan polysaccharide and its immunomodulatory and antitumor
activity in vitro. International Journal of Biological Macromolecules, 67, 323–329.
KhilâChenko, S., Zaporozhets, T., Shevchenko, N., Zvyagintseva, T., Vogel, U., Seeberger,
P., et al. (2011). Immunostimulatory Activity of Fucoidan from the Brown Alga Fucus
evanescens: Role of Sulfates and Acetates. Journal of Carbohydrate Chemistry, 30(4–6),
291–305.
Kouakou, K., Schepetkin, I. A., Yapi, A., Kirpotina, L. N., Jutila, M. A., & Quinn, M. T.
(2013). Immunomodulatory activity of polysaccharides isolated from Alchornea cor-
difolia. Journal of Ethnopharmacology, 146(1), 232.
Kralovec, J. A., Metera, K. L., Kumar, J. R., Watson, L. V., Girouard, G. S., Guan, Y., et al.
(2007). Immunostimulatory principles fromChlorella pyrenoidosa–part 1: isolation
and biological assessment in vitro. Phytomedicine, 14(1), 57–64.
Li, F., Cui, S. H., Zha, X. Q., Bansal, V., Jiang, Y. L., Asghar, M. N., et al. (2015). Structure
and bioactivity of a polysaccharide extracted from protocorm-like bodies of
Dendrobium huoshanense. International Journal of Biological Macromolecules, 72,
664–672.
Li, S. P., Wu, D. T., Lv, G. P., & Zhao, J. (2013). Carbohydrates analysis in herbal gly-
comics. Trac-Trends in Analytical Chemistry, 52, 155–169.
Li, W., Yajima, T., Saito, K., Nishimura, H., Fushimi, T., Ohshima, Y., et al. (2004).
Immunostimulating properties of intragastrically administered Acetobacter-derived
soluble branched (1,4)-beta-D-glucans decrease murine susceptibility to Listeria
monocytogenes. Infection and Immunity, 72(12), 7005.
Liu, L., Li, M., Yu, M., Shen, M., Wang, Q., Yu, Y., et al. (2019). Natural polysaccharides
exhibit anti-tumor activity by targeting gut microbiota. International Journal of
Biological Macromolecules, 121, 743–751.
Liu, C., Chen, J., Chen, L., Huang, X., & Cheung, P. C. (2016). Immunomodulatory
Activity of Polysaccharide-Protein Complex from the Mushroom Sclerotia ofPolyporus
rhinocerus in Murine Macrophages. Journal of Agricultural and Food Chemistry, 64(16),
3206–3214.
Liu, D., Su, J., Lin, J., Qian, G., Chen, X., Song, S., et al. (2018). Activation of AMPK-
dependent SIRT-1 by astragalus polysaccharide protects against ochratoxin A-in-
duced immune stress in vitro and in vivo. International Journal of Biological
Macromolecules, 120, 683–692.
Liu, T., Zhang, M., Niu, H., Liu, J., Ruilian, M., Wang, Y., et al. (2018). Astragalus
polysaccharide from Astragalus Melittin ameliorates inflammation via suppressing
the activation of TLR-4/NF-κB p65 signal pathway and protects mice from CVB3-
induced virus myocarditis. International Journal of Biological Macromolecules, 126,
179–186.
Fig. 6. Possible molecular mechanism of LPD2 activating macrophages.
Acknowledgements
Liu, X. C., Zhu, Z. Y., Tang, Y. L., Wang, M. F., Wang, Z., Liu, A. J., et al. (2016). Structural
properties of polysaccharides from cultivated fruit bodies and mycelium of Cordyceps
militaris. Carbohydrate Polymers, 142, 63–72.
The study was financially supported by the National Natural Science
Foundation of China (Grant No. 31360380), the Earmarked Fund for
China Agriculture Research System (Grant No. CARS-33) and the
Natural Science Foundation of Jiangsu Province, China (Grant No.
BK20140147).
Ližičárová, I., Matulová, M., Machová, E., & Capek, P. (2007). Cell wall mannan of human
pathogen Candida dubliniensis. Carbohydrate Polymers, 68(1), 191–195.
Lo, T. C., Jiang, Y. H., Chao, A. L., & Chang, C. A. (2007). Use of statistical methods to find
the polysaccharide structural characteristics and the relationships between mono-
saccharide composition ratio and macrophage stimulatory activity of regionally dif-
ferent strains of Lentinula edodes. Analytica Chimica Acta, 584(1), 50–56.
Martinon, F., Chen, X., Lee, A. H., & Glimcher, L. H. (2010). TLR activation of the tran-
scription factor XBP1 regulates innate immune responses in macrophages. Nature
Immunology, 11(5), 411–418.
Martins, V. M. R., Simoes, J., Ferreira, I., Cruz, M. T., Domingues, M. R., & Coimbra, M. A.
(2017). In vitro macrophage nitric oxide production by Pterospartum tridentatum(L.)
Willk. inflorescence polysaccharides. Carbohydrate Polymers, 157, 176–184.
Meng, F. Y., Ning, Y. L., Qi, J., He, Z., Jie, J., Lin, J. J., et al. (2014). Structure and
antitumor and immunomodulatory activities of a water-soluble polysaccharide from
Dimocarpus longan pulp. International Journal of Molecular Sciences, 15(3), 5140.
Muljana, H., Picchioni, F., Heeres, H. J., & Janssen, L. P. B. M. (2010). Green starch
conversions: Studies on starch acetylation in densified CO2. Carbohydrate Polymers,
82(3), 653–662.
Needs, P. W., & Selvendran, R. R. (1993). Avoiding oxidative degradation during sodium
hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide.
Carbohydrate Research, 245(1), 1–10.
Park, S. J., Park, D. H., Kim, D. H., Lee, S., Yoon, B. H., Jung, W. Y., et al. (2010). The
memory-enhancing effects of Euphoria longan fruit extract in mice. Journal of
Ethnopharmacology, 128(1), 160–165.
Schepetkin, I. A., Xie, G., Kirpotina, L. N., Klein, R. A., Jutila, M. A., & Quinn, M. T.
(2008). Macrophage immunomodulatory activity of polysaccharides isolated from
Opuntia polyacantha. International Immunopharmacology, 8(10), 1455.
Shiratsuchi, A., Watanabe, I., Takeuchi, O., Akira, S., & Nakanishi, Y. (2004). Inhibitory
effect of Toll-like receptor 4 on fusion between phagosomes and endosomes/lyso-
somes in macrophages. The Journal of Immunology, 172(4), 2039–2047.
Simões, J., Madureira, P., Nunes, F. M., Domingues, M. D. R., Vilanova, M., & Coimbra, M.
A. (2010). Immunostimulatory properties of coffee mannans. Molecular Nutrition &
Food Research, 53(8), 1036–1043.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2019.03.007.
References
Bisen, P. S., Baghel, R. K., Sanodiya, B. S., Thakur, G. S., & Prasad, G. B. (2010). Lentinus
edodes: a macrofungus with pharmacological activities. Critical Reviews in Food
Science and Nutrition, 17(22), 2419–2430.
Deng, C., Fu, H., Shang, J., Chen, J., & Xu, X. (2018). Dectin-1 mediates the im-
munoenhancement effect of the polysaccharide from Dictyophora indusiata.
International Journal of Biological Macromolecules, 109, 369–374.
Du, X. J., Zhang, J. S., Lv, Z. W., Ye, L. B., Yang, Y., & Tang, Q. J. (2014). Chemical
modification of an acidic polysaccharide (TAPA1) from Tremella aurantialba and
potential biological activities. Food Chemistry, 143(2), 336–340.
Ferreira, S. S., Passos, C. P., Madureira, P., Vilanova, M., & Coimbra, M. A. (2015).
Structure function relationships of immunostimulatory polysaccharides: A review.
Carbohydrate Polymers, 132, 378–396.
Ginhoux, F., & Jung, S. (2014). Monocytes and macrophages: Developmental pathways
and tissue homeostasis. Nature Reviews Immunology, 14(6), 392–404.
He, X., Fang, J., Ruan, Y., Wang, X., Sun, Y., Wu, N., et al. (2017). Structures, bioactivities
and future prospective of polysaccharides from Morus alba (white mulberry): A re-
view. Food Chemistry, 245, 899–910.
Hoppstädter, J., Diesel, B., Linnenberger, R., Hachenthal, N., Flamini, S., Minet, M., et al.
(2019). Amplified host defense by Toll-like receptor-mediated downregulation of the
glucocorticoid-induced leucine zipper (GILZ) in macrophages. Frontiers in
Immunology, 9, 3111.
Huang, F., Zhang, R., Liu, Y., Xiao, J., Su, D., Yi, Y., et al. (2016). Characterization and
mesenteric lymph node cells-mediated immunomodulatory activity of litchi pulp
polysaccharide fractions. Carbohydrate Polymers, 152, 496–503.
Huang, X., Nie, S., & Xie, M. (2017). Interaction between gut Immunity and poly-
saccharides. Critical Reviews in Food Science and Nutrition, 57(14), 2943–2955.
Wu, W., Zhu, Y., Zhang, L., Yang, R., & Zhou, Y. (2012). Extraction, preliminary structural
characterization, and antioxidant activities of polysaccharides from Salvia miltiorrhiza
Bunge. Carbohydrate Polymers, 87(2), 1348–1353.
Xie, G., Schepetkin, I. A., & Quinn, M. T. (2007). Immunomodulatory activity of acidic
polysaccharides isolated from Tanacetum vulgare L. International
Immunopharmacology, 7(13), 1639–1650.
Xie, G., Schepetkin IASiemsen, D. W., Kirpotina, L. N., Wiley, J. A., & Quinn, M. T. (2008).
Fractionation and characterization of biologically-active polysaccharides from
255

Y. Rong, et al.
CarbohydratePolymers213(2019)247–256
Yu, Y., Shen, M., Song, Q., & Xie, J. (2018). Biological activities and pharmaceutical
applications of polysaccharide from natural resources: A review. Carbohydrate
Polymers, 183, 91–101.
Zhang, J., Yang, G., Wen, Y., Liu, S., Li, C., Yang, R., et al. (2017). Intestinal microbiota
are involved in the immunomodulatory activities of longan polysaccharide. Molecular
Nutrition & Food Research, 61(11), 1700466.
Zhang, M., Wang, G., Lai, F., & Wu, H. (2016). Structural characterization and im-
munomodulatory activity of a novel polysaccharide from lepidium meyenii. Journal of
Agricultural and Food Chemistry, 64(9), 1921–1931.
Zhang, S., Pang, G., Chen, C., Qin, J., Yu, H., Liu, Y., et al. (2019). Effective cancer
immunotherapy by Ganoderma lucidum polysaccharide-gold nanocomposites through
dendritic cell activation and memory T cell response. Carbohydrate Polymers, 205,
192–202.
Zhao, L., Chen, Y., Ren, S., Han, Y., & Cheng, H. (2010). Studies on the chemical structure
and antitumor activity of an exopolysaccharide from Rhizobium sp. N613.
Carbohydrate Research, 345(5), 637.
Artemisia tripartita. Phytochemistry, 69(6), 1359.
Xie, J. H., Jin, M. L., Morris, G. A., Zha, X. Q., Chen, H. Q., Yi, Y., et al. (2016). Advances
on bioactive polysaccharides from medicinal plants. Critical Reviews in Food Science
and Nutrition, 56, S60–S84.
Yang, L. Q., & Zhang, L. M. (2009). Chemical structural and chain conformational
characterization of some bioactive polysaccharides isolated from natural sources.
Carbohydrate Polymers, 40(33), 349–361.
Yi, Y., Huang, F., Zhang, M. W., Zhang, R. F., Deng, Y. Y., Wei, Z. C., et al. (2013).
Solution properties and in vitro anti-tumor activities of polysaccharides from longan
pulp. Molecules, 18(9), 11601–11613.
Yi, Y., Liao, S. T., Zhang, M. W., Shi, J., Zhang, R. F., Deng, Y. Y., et al. (2011).
Physicochemical characteristics and immunomodulatory activities of three poly-
saccharide-protein complexes of longan pulp. Molecules, 16(7), 6148–6164.
Yi, Y., Wang, H., Zhang, R., Min, T., Huang, F., Liu, L., et al. (2015). Characterization of
polysaccharide from longan pulp as the macrophage stimulator. RSC Advances,
5(118), 97163–97170.
Yi, Y., Zhang, M. W., Liao, S. T., Zhang, R. F., Deng, Y. Y., Wei, Z. C., et al. (2012).
Structural features and immunomodulatory activities of polysaccharides of longan
pulp. Carbohydrate Polymers, 87(1), 636–643.
Zhu, Q., Jiang, Y., Lin, S., Wen, L., Wu, D., Zhao, M., et al. (2013). Structural identifi-
cation of (1→6)-α-d-glucan, a key responsible for the health benefits of longan, and
evaluation of anticancer activity. Biomacromolecules, 14(6), 1999–2003.
256