Structure of an entangled heteropolysaccharide from Pholidota chinensis Lindl and its antioxidant and anti-cancer properties

Article abstract of DOI:10.1016/j.ijbiomac.2018.02.051

A major polysaccharide PCP-I was isolated and purified from Pholidota chinensis Lindl. The physicochemical and structural properties of PCP-I were studied using high-performance size-exclusion chromatography (HPSEC), gas chromatography (GC), Fourier transform infrared spectroscopy (FTIR), periodate oxidation-smith degradation, methylation-GC–MS analysis, nuclear magnetic resonance (NMR) spectroscopy and transmission electron microscopy (TEM) analysis. PCP-I was homogeneous with molecular weight (Mw) of 249 kDa and composed of xylose and fucose at a molar ratio of 2.45:1. The repeating structural units of PCP-I were →3)-α-D-Xylp-(1→ and →4)-α-L-Fucp-(1→ the terminal fractions were T-D-GalAp, and TEM further revealed that PCP-I was the entangled microstructure which was composed of four non-branched single chains. Compared with Vitamin C (Vc) and 5 fluorine urine (5-Fu), PCP-I showed scavenging effects of superoxide (EC₅₀ = 1.09 mg/mL) and hydroxyl (EC₅₀ = 0.11 mg/mL) radicals equivalent to Vc, and PCP-I (IC₅₀ = 69.54 μg/mL) also exhibited good anti-proliferation capability for human colon cancer cell line caco-2.

Full text of DOI:10.1016/j.ijbiomac.2018.02.051

International Journal of Biological Macromolecules 112 (2018) 921–928
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: https://www.journals.elsevier.com/ijbiomac
Structure of an entangled heteropolysaccharide from Pholidota chinensis
Lindl and its antioxidant and anti-cancer properties
a
a
b
Dianhui Luo a, , Zhaojing Wang , Zhiming Li , Xiao-qiang Yu
⁎
a
Department of Bioengineering and Biotechnology, Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, Xiamen, Fujian 361021, People's Republic of China
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A major polysaccharide PCP-I was isolated and purified from Pholidota chinensis Lindl. The physicochemical and
structural properties of PCP-I were studied using high-performance size-exclusion chromatography (HPSEC), gas
chromatography (GC), Fourier transform infrared spectroscopy (FTIR), periodate oxidation-smith degradation,
methylation-GC–MS analysis, nuclear magnetic resonance (NMR) spectroscopy and transmission electron mi-
croscopy (TEM) analysis. PCP-I was homogeneous with molecular weight (Mw) of 249 kDa and composed of xy-
lose and fucose at a molar ratio of 2.45:1. The repeating structural units of PCP-I were →3)-α-D-Xylp-(1→ and
→4)-α-L-Fucp-(1→, the terminal fractions were T-D-GalAp, and TEM further revealed that PCP-I was the
entangled microstructure which was composed of four non-branched single chains. Compared with Vitamin C
(Vc) and 5 fluorine urine (5-Fu), PCP-I showed scavenging effects of superoxide (EC50 = 1.09 mg/mL) and hy-
droxyl (EC50 = 0.11 mg/mL) radicals equivalent to Vc, and PCP-I (IC50 = 69.54 μg/mL) also exhibited good
anti-proliferation capability for human colon cancer cell line caco-2.
Received 29 November 2017
Received in revised form 5 February 2018
Accepted 9 February 2018
Available online 10 February 2018
Keywords:
Pholidota chinensis Lindl
Polysaccharides
Structural analysis
Antioxidant
Anti-cancer
© 2018 Elsevier B.V. All rights reserved.
1. Introduction
including NMR and TEM, and its antioxidant and anti-cancer activities
were investigated in order to study structure-function relationships.
Pholidota chinensis Lindl, a well-known functional food and a folk
medicine, has been used for a long time in southeast of China for allevi-
ating headache and coughing [1]. P. chinensis Lindl comprises various
classes of compounds, including stilbenoids, terpenoid, phytosterols, al-
kaloids, phenolic compounds and carbohydrates [1–3]. It has been re-
ported that extracts of P. chinensis Lindl have antioxidant, analgesic,
anti-inflammatory and antitumor activities [1,3–5]. Polysaccharides
from P. chinensis Lindl have attracted increasing attention in research
due to their potent biological and pharmacological functions [6]. How-
ever, previous studies mostly focused on the extraction and purification
of P. chinensis Lindl polysaccharides [7,8]. Although structural properties
and antioxidant activities of P. chinensis Lindl polysaccharides from dif-
ferent place of production have been reviewed by Yang (2016) [6], the
fine chemical and microcosmic structure of polysaccharides purified
from pseudobulbs of P. chinensis Lindl have not been investigated, and
the structure-function relationship has yet been established.
Our results will benefit the applications of P. chinensis Lindl in medical
and food industries.
2. Materials and methods
2.1. Materials and chemicals
Pholidota chinensis Lindl was purchased from
a local store
(Shaoguan, Guangdong Province, China) and dried at room tempera-
ture, and the pseudobulbs of P. chinensis Lindl were separated for the
study. The material was identified by Professor J. F. Liu, and a voucher
specimen (pc-20150609-001) has been deposited in Fan-hua experi-
mental building B403, Huaqiao University, Fujian, China. Monosaccha-
ride samples, dextran standard samples, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), phenazine methosulfate
(PMS) and dimethyl sulfoxide (DMSO) were obtained from Sigma
Chemical Co. (St. Louis, MO). Deuterium Oxide (D2O, Cambridge Isotope
Laboratories, Inc., Andover, MA, USA), DMEM medium and fetal bovine
serum (FBS, Hyclone, Logan, UT) and Penicillin-Streptomycin (MP Bio-
medicals, CA, USA) were purchased from a local agent (Taijing Co., Xia-
men). DEAE Sepharose CL-6B was obtained from Pharmacia, Co, now
available from GE-Healthcare. Ascorbic acid (vitamin C, Vc), hydrogen
peroxide (H2O2), Nitro Blue Tetrazolium (NBT), Nicotinamide Adenine
Dinucleotide Hydrogen (NADH) and ferrous sulfate (FeSO4) were
Fine structural features will help understand the relationship be-
tween chemical structures and biological activities of P. chinensis Lindl
polysaccharides. Therefore, in the present study, a polysaccharide
(PCP-I) was isolated and purified from pseudobulbs of P. chinensis
Lindl, its chemical structure was established using modern techniques
⁎
Corresponding author.
E-mail address: luodhwzj@hqu.edu.cn (D. Luo).
https://doi.org/10.1016/j.ijbiomac.2018.02.051
0141-8130/© 2018 Elsevier B.V. All rights reserved.

922
D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
obtained from Shanghai Macklin Chemical Reagent Company, China.
Human colon cancer cell line caco-2 was obtained from Shanghai Insti-
tutes for Biological Sciences, Chinese Academy of Sciences. All reagents
were analytical grades.
solution to stop the reaction. The remaining solution was dialyzed
against tap water and distilled water, each for 24 h. The dialyzed solu-
tion was concentrated, reduced with 80 mg NaBH4 at room temperature
for 24 h, neutralized to pH 7.0 with 50% acetic acid, dialyzed again as de-
scribed above, and concentrated to a volume of 10 mL. One-third of this
solution was freeze-dried and analyzed with GC (S1), and the remaining
solution was hydrolyzed by adding the same volume of 1 M sulfuric
acid, heating at 25 °C for 40 h, neutralizing to pH 7.0 with barium car-
bonate and filtering. The filtrate was dialyzed in distilled water for
24 h, the dialysis solution outside the dialysis tube was freeze-died for
GC analysis (S2), and the solution inside the dialysis tube was used for
GC analysis (S3) after adding 4 volumes of ethanol into the tube [14].
2.2. Extraction, isolation and purification of polysaccharide
The dry P. chinensis Lindl (100 g) was extracted with 3 L of distilled
water at 95 °C for 3 h. The extracts were concentrated, 4 volumes of eth-
anol were used to precipitate crude products, and the free proteins were
removed by Sevag reagent (trichloromethane:N-butyl alcohol = 4:1) to
obtain 14.85 g dry crude polysaccharides (PC) [9]. The PC (500 mg for
each experiment) was dissolved in 10 mL of distilled water and applied
to a column of DEAE-Sepharose CL-6B (4.6 cm × 40 cm), followed by
elution with distilled water at a flow rate of 50 mL/h (12 min/tube)
and gradient elution from 0 to 1 M NaCl aqueous solution at the same
flow rate. The solution (12 mL/tube) was collected using a fraction col-
lector and assayed by the phenol sulfuric acid method. The carbohy-
drate fraction corresponding to a major peak was collected, dialyzed
(MWCO 3500) against distilled water for 48 h, and dried in a freeze
drier to obtain a purified polysaccharide PCP-I.
2.6. Partial hydrolysis with acid
The PCP-I (120 mg) was hydrolyzed with 0.05 M CF3COOH at 95 °C
for 16 h and centrifuged. The sediment (P1) was dried and used for
GC analysis, the supernatant was dialyzed with distilled water for
48 h, and the solution outside the tube was dried and used for GC anal-
ysis. 4 volumes of ethanol were added into the solution in the tube, and
the precipitate (P2) and the supernatant (P3) were freeze-dried, respec-
tively, for GC analysis [15].
2.3. Homogeneity, molecular weight (MW), and monosaccharide
composition
Total sugar content was determined using the phenol sulfuric acid
method [10]. Total uronic acid was determined using the sulfuric acid
carbazole method (Bitter, & Muir, 1962) [11].
2.7. Methylation and GC–MS analysis
The PCP-I (30 mg) was dissolved in 9 mL dimethyl sulfoxide
(DMSO), and dry sodium hydroxide powder (360 mg) was also dis-
solved in DMSO (9 mL) by constant stirring for 24 h. The two DMSO so-
lutions were combined under constant stirring, and then 5.4 mL methyl
iodide was added to the DMSO solution for 7 min followed by addition
of 6 mL distilled water. The final solution was dialyzed against tap water
and distilled water, each for 24 h, and concentrated to 10 mL, and meth-
ylated polysaccharides were extracted with 10 mL trichloromethane
under constant oscillation for 30 min. The trichloromethane solution
(1 mL) was examined by FTIR spectrum, and no absorption peak of hy-
droxyl groups indicated the complete methylation. The remaining
trichloromethane solution was dried, and the desiccated methylated
polysaccharides were hydrolyzed by adding formic acid and
trifluoroacetic acid (TFA), reduced with NaBH4, and acetylated using
acetic anhydride–pyridine (1:1). Preparation of carboxyl-reduced poly-
saccharide was performed using the literature method [16]. The
resulting methylated alditol acetates were subjected to Gas chromatog-
raphy mass spectrometry (GC–MS) analysis. GC–MS was performed on
a HP7890A-5975C instrument (Agilent Technologies, Palo Alto, CA,
USA) with an HP-5 ms (30 m × 0.25 mm × 0.25 μm) column at the tem-
peratures programmed from 160 (held for 1 min) to 250 °C (held for
2 min) at 8 °C/min [17,18]. The injector and detector temperatures
were set at 250 °C, the ion source temperature was 230 °C, and helium
was used as the carrier gas.
The homogeneity and molecular weight (MW) of PCP-I were deter-
mined using high-performance size-exclusion chromatography
(HPSEC) (1100 system, Agilent Technologies, Palo Alto, CA, USA)
equipped with a gel-filtration chromatographic column of Shodex
Sugar KS-804 (Showa Denko K.K, Japan) and a refractive index detector
(RID). PCP-I water solution (0.5 mg/mL) was passed through a 0.22-μm
filter prior to injection into the column, and the column was eluted
using distilled water at 50 °C with a flow rate of 1 mL/min. MW was cal-
culated using a calibration curve of various dextran standards (Dextran
Blue, T10, T40, T70, T500 and Glucose) [12].
Gas chromatography (GC) was used for the identification and quan-
tification of monosaccharide composition of PCP-I. The polysaccharide
PCP-I (30 mg) was hydrolyzed with 1 M H2SO4 and kept at 100 °C for
8 h and neutralized to pH 7.0 with barium carbonate, then filtered, con-
centrated and finally freeze-dried to obtain hydrolysis products. Manni-
tol (3 mg) was added as the internal standard, and derivatization of the
products was then carried out using the trimethylsilylation reagent [13].
The derivatization products were injected into a gas chromatography
system (6890 system, Agilent Technologies, Palo Alto, CA, USA) fitted
with an HP-5 column (30 m × 0.25 mm × 0.25 μm) and a flame-ioniza-
tion detector (FID). Monosaccharide composition was identified by
comparison with the retention times of standard monosaccharide sam-
ples, and the molar ratio was calculated according to the peak areas.
2.4. Fourier transform infrared spectroscopy (FTIR) analysis
2.8. Nuclear magnetic resonance (NMR) spectroscopy
FTIR spectrum of PCP-I was determined using a Fourier Transform
Infrared Spectrometer (Nicolet iS10, Thermo Fisher Scientific, Waltham,
MA, USA) with a DTGS detector. The PCP-I (3 mg) was ground with KBr
powder (100 mg) and pressed into pellets for FTIR measurement at a
frequency range of 4000–400 cm−1 with 32 scans.
For NMR measurements, 30 mg PCP-I was exchanged with deute-
rium by lyophilizing with D2O for three times and then dissolved in
0.7 mL D2O (99.96%). Acetone was used as the internal standard. 1H
and 13C NMR spectra were recorded at 500 MHz and 125 MHz on an
AVANCE-500 NMR spectrometer (Bruker Inc., Rheinstetten, Germany)
at 25 °C. 2D NMR experiments were performed using standard Bruker
software, and the 2D NMR spectra included 1H/1H homonuclear correla-
2.5. Periodate oxidation-smith degradation
1
PCP-I (25 mg) was dissolved into 15 mmol/L NaIO4 solution and kept
in the dark, and an aliquot (0.1 mL) of the solution was diluted to 25 mL
with distilled water and used to read a value at 223 nm for 6 h intervals.
When the value at 223 nm was stable, glycol (2 mL) was added into the
tion spectroscopy (1H\\ H COSY), total correlation spectroscopy
(TOCSY), heteronuclear single quantum coherence (HSQC), nuclear
overhauser enhancement spectroscopy (NOESY), and heteronuclear
multiple-bond coherence (HMBC) [19–21].

D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
923
2.9. Microscopic analysis
570 nm [25]. The inhibition rate was calculated based on the following
equation:
PCP-I aqueous solution (1 mg/mL) was prepared and added into an
equal volume of SDS solution (1 mg/mL) at 80 °C for 2 h, diluted to a
final concentration of 5 μg/mL with distilled H2O and heated at 80 °C
again for 2 h. After that, a droplet of solution was deposited on a carbon
film specimen (200 mesh, Beijing Zhongjingkeyi Technology, Beijing,
China) and dried at an ambient temperature and humidity, and ob-
served by transmission electron microscopy (H-7650, Hitachi High-
Technologies Corporation, Tokyo, Japan) with an accelerating voltage
of 100 kV to visualize PCP-I molecular morphology [22–24].
Â
À
ÁÃ
Inhibition rate ð%Þ ¼ 1− A570nm;sample=A570nm;control ꢀ 100%
2.11. Statistical analysis
Three independent experiments were conducted, and data were
expressed as the mean standard deviation (SD). The significance of
differences between means was evaluated by one-way analysis of vari-
ance (ANOVA), and statistical significance was assigned at P b 0.05.
2.10. Bioactivities assay
3. Results and discussion
Superoxide, hydroxyl and DPPH radicals scavenging activities were
measured using the previously described method [16,25,26]. Samples
and vitamin C (Vc) were prepared at the final concentrations (0, 0.02,
0.08, 0.16, 0.33, 0.66, 1.3 and 2 mg/mL) for superoxide and hydroxyl
radicals scavenging experiments. For the DPPH radical scavenging ex-
periment, the final concentrations were 0, 0.8, 1.6, 3.3, 6.6 and
13 mg/mL. The percentage of the radical scavenging activities was cal-
culated according to the following equation, A0 was the absorbance of
the blank control, and A1 was the absorbance of the sample.
3.1. Extraction, purification and molecular characterization of PCP-I
Crude polysaccharides (PC) were obtained from P. chinensis Lindl
using hot-water extraction with a yield of 14.85% (Dry to Dry weight
ratio, D/D). After fractionation by the DEAE-Sepharose CL-6B, crude PC
gave two fractions (Fig. 1a), PCP-I was a major fraction from NaCl aque-
ous solution (50–60 tube) with a yield of 52.4% (D/D), and PCP-II was
another fraction (40–49 tube) with a yield of 1.9% (D/D), which was
not analyzed because of the low yield. The elution profile of PCP-I in
HPSEC with a Sugar KS-804 column was given in Fig. 1b, which showed
that PCP-I was homogeneous with one singular narrow peak in RID (Fig.
1b). PCP-I was composed of 95.72 0.71% (w/w) of total carbohydrates
and 4.49 0.15% (w/w) of uronic acid.
Using HPSEC coupled with RID, a calibration curve (y = −4 × + 6.3)
of standard dextrans was obtained, and the average molecular weight
(Mw) of PCP-I was calculated to be 249 kDa. The infrared spectrum of
PCP-I displayed characteristic polysaccharide peaks at approximately
3400 cm−1 for hydroxyl groups, 2900 cm−1 for C\\H stretching band,
1665 cm−1 for C_O bond, 1400 to 1200 cm−1 for C\\H variable angle
vibration bond, and 1100 cm−1 for C\\O bond (Fig. 1c). After hydrolysis
of PCP-I by 1 M H2SO4, GC chromatograms of hydrolyzed PCP-I and stan-
dard monosaccharide samples were showed in Fig. 1d and Fig. 1e,
Scavenging activity ð%Þ ¼ ð1−A0=A1Þ ꢀ 100%
The cytotoxicity of samples (PC, PCP-I and 5-Fu) against Caco-2 can-
cer cell line was determined using MTT assay. Caco-2 cells were cultured
in DMEM medium (high glucose) containing 10% FBS and 100 units/mL
penicillin-streptomycin with 5% CO2 at 37 °C to ~80% confluency, and
seeded in 96-well culture dishes at 100 μL/well. The Caco-2 cells were
cultured for 20 h, DMEM medium was discarded, and 100 μL DMED me-
dium with samples (25, 50, 250, 500 and 2500 μg/mL) were added to
each well of the 96-well culture dishes and the cells were cultured for
48 h. MTT (5 mg/mL) was added (20 μL/well) to cells and incubated
for 4 h, the media were removed, DMSO was added (150 μL/well), and
the cytotoxicity was determined by measuring the absorbance at
Fig. 1. (a) Sugar curve of PCP-I from NaCl elution of DEAE Sepharose CL-6B column chromatogram, (b) HPSEC chromatogram of PCP-I with a RID, (c) IR spectra of PCP-I and methylated
PCP-I, and (d) GC chromatograms of hydrolyzed PCP-I and standard samples (e). In the GC chromatogram of standards, the internal standard mannitol was peak 12, arabinose was peaks 1,
2 and 4, fucose was peaks 2,3 and 5, xylose was peaks 5 and 6, mannose was peaks 7 and 11, galactose was peaks 8, 9 and 11, glucose was peaks 10 and 13, and galacturonic acid was peaks
14 and 15.

924
D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
Table 1
and (1 → 3)-glycosidic linkage, respectively, which were in consistent
with the results of periodate oxidation and smith degradation.
The carboxyl-reduced polysaccharide was methylated, and the
methylated sugars were identified by GC–MS analysis, which showed
the presence of 1,4,5-O-Ac3–2,3-O-Me2–6-deoxy-L-galactiol, 1,3,5-O-
Ac3–2,4-O-Me2-D-xylitol and a new trace peak 1,5-O-Ac2–2,3,4,6-O-
Me4-D-galactiol. It could be confirmed that 1,5-O-Ac2–2,3,4,6-O-Me4-
D-galactiol was the carboxyl-group reduced product of galacturonic
acid, and the terminal fractions of PCP-I were T-D-GalAp.
The branched fractions of PCP-I were not identified in the methyla-
tion-GC–MS analysis, probably because their contents were the least
among the residues for detection or PCP-I was a non-branched polysac-
charide. PCP-I was further confirmed to be a non-branched polysaccha-
ride by the NMR and TEM results.
GC results from fractions of smith degradation and partial acid hydrolysis of PCP-I.
Fractions
Sugar components and molar ratios
Fucose Xylose Glycerol Erythritol
Partial acid
hydrolysis
Precipitation (P1)
Precipitation in the
sack (P2)
Supernatant in the
sack (P3)
Full acid hydrolysis
(S1)
Out of sack (S2)
Supernatant in the
sack (S3)
−
−
+
+
3.09
1
Smith
degradation
−
+
−
+
−
−
−
+
−
−
−
−
−: Undetectable; +: Detectable.
3.5. NMR spectroscopy
respectively. Calculating the ratio of retention times (each peak/the in-
ternal standard mannitol) and referring to the ratios of standards (Fig.
1e), we concluded that PCP-I was composed of xylose and fucose. The
molar ratio was 2.45:1 by calculating a ratio of the corresponding peak
areas.
NMR experiments were performed to identify sugar residues of PCP-
I. 1D NMR included 1H (Fig. 2a), 13C (Fig. 2b) and DEPT135 (Fig. 2c)
spectra. The 1H NMR spectrum showed two signals in anomeric regions
of δ 4.6 and δ 5.29 ppm, and the anomeric carbon signals appeared at δ
102 and δ 98.3 ppm in the 13C NMR and DEPT135 spectra. One signal at
high field (δ 1.27 ppm) was assigned to the proton of methyl (–CH3)
group, which is likely derived from fucose (C-6). The two sugar moieties
were designated as A and B according to their increasing chemical shifts
(Fig. 2, Table 3).
3.2. Periodate oxidation-smith degradation
The polysaccharide PCP-I showed HIO4 uptake while it was oxidized.
The periodate-oxidized products were hydrolyzed in 1 M H2SO4, and
the hydrolysate was examined by GC (Table 1). Xylose and erythritol
were identified in the fraction of full acid hydrolysis. The presence of xy-
lose indicated that its linkages, namely (1 → 3)-glycosidic linkages,
could not be oxidized. Fucose was absent, suggesting that fucose was
oxidized. In addition, erythritol was identified, further suggesting that
linkages of fucose in PCP-I were (1 → 4)-glycosidic linkages or (1 →
4,6)-glycosidic linkages. The conclusions were further confirmed by
the NMR results.
Because 1D NMR did not provide enough information, 2D NMR ex-
1
periments were also performed, including 1H\\ H COSY (Fig. 2d),
TOCSY (Fig. 2e), HSQC (Fig. 2f), NOESY (Fig. 2g) and HMBC (Fig. 2h).
In the 1H NMR spectrum of PCP-I, the chemical shift at δ 4.6 ppm was
assigned to residue A. C-1 chemical shift of the residue A was obtained
1
by HSQC at δ 102 ppm. Through 1H\\ H COSY spectroscopy, the H-2,
H-3, H-4 and H-5 chemical shifts of the residue A were determined at
δ 3.28, δ 3.43, δ 3.55 and δ 3.29 ppm, respectively. By the TOCSY spec-
trum, the H-3, H-4 and H-5 chemical shifts of the residue A were con-
firmed. The corresponding 13C chemical shifts of residue A could be
obtained through the data in the HSQC spectrum, and 1H and 13C chem-
ical shifts of residue A were listed in Table 3. According to 13C chemical
shifts, the residue A was identified as →3)-α-D-Xylp-(1→, and this as-
signment was consistent with the result reported in the literature
[27], and the results were in accordance with the results of monosaccha-
ride composition and GC–MS analysis.
3.3. Partial hydrolysis with acid
After partial acid hydrolysis of PCP-I, the fractions were subjected to
GC analysis (Table 1). Both hydrolysis precipitation and precipitation in
the sack contained xylose, indicating that xylose was the major compo-
nent of backbone structure in PCP-I. Due to high percentage of fucose in
the supernatant of the sack (Fuc:Xyl = 3.09:1) and by comparison with
the previous analysis of monosaccharide composition (Fuc:Xyl =
1:2.45) of PCP-I, it was concluded that fucose was mainly existed in
the position of edges in PCP-I.
The anomeric chemical shift for residue B was at δ 5.29 ppm, and the
anomeric carbon signal was observed at δ 98.3 ppm in the HSQC spec-
trum. 1H assignments (H-2, H-3, H-4 and H-5) of residue B were ob-
1
tained through the 1H\\ H COSY and TOCSY spectra. The carbon
signals of residue B were identified from the HSQC spectrum. The
cross peak in HSQC with the chemical shift of δ 1.27 ppm and δ
19.9 ppm confirmed that the –CH3 group was derived from residue B.
1H and 13C chemical shifts of residue B were listed in Table 3. According
to the chemical shifts, it was confirmed that residue B was →4)-α-L-
Fucp-(1→, and this assignment was consistent with the results of
Periodate oxidation-Smith degradation and GC–MS analysis.
NOESY correlates nuclei through space and reveals the glycosyl link-
ages between sugar residues, and intra- and inter-residue connectivities
are also obtained in NOESY spectrum (Fig. 3g) [28,29]. By the NOESY ex-
periment, the sequence of glycosyl residues in PCP-I was determined.
3.4. Methylation–GC–MS analysis of PCP-I
Comparing the FTIR spectrum of PCP-I with that of methylated PCP-I
(Fig. 1c), methylated PCP-I lacked an absorption peak at approximately
3400 cm−1 for hydroxyl group, indicating that PCP-I was completely
methylated. GC–MS analysis showed the presence of two components,
namely 1,4,5-O-Ac3–2,3-O– Me2–6-deoxy-L-galactiol and 1,3,5-O-
Ac3–2,4-O-Me2-D-xylitol (Table 2). The results of methylation-GC–MS
analysis showed that fucose and xylose were (1 → 4)-glycosidic linkage
Table 2
GC–MS chromatogram of methylated PCP-I products.
Methylation product
Molar ratio
Mass fragment (m/z)
Linkage type
1,4,5-O-Ac3-2,3-O-Me2-6-deoxy-L-galactiol
1,3,5-O-Ac3-2,4-O-Me2-D-xylitol
1
2.16
43,87,101,117,129,189
43,117
1,4-L-Fucp
1,3-D- Xylp

D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
925
1
Fig. 2. 1H NMR (a), 13C NMR (b), DEPT135 (c), 1H\\ H COSY (d), TOCSY (e), HSQC (f), NOESY (g) and HMBC (h) spectra of PCP-I polysaccharide. AH1 and AC1 represent the anomeric protons
of residue A. B (1,2) represents the correlation or the NOE contact between H-1 and H-2 of residue B, etc. In the NOESY, B1-A3 represents the NOE contact between H-1 of residue B and H-3
of residue A. A (H1-C1) means the correlation between H-1 and C-1 of residue A, etc.
Residue B had an NOE contact from H-1 to H-3 of residue A, in addition
to intra-residue NOE contacts with H-2, H-3, H-4 and H-5. Long-range
observed between the H-1 (δ 5.29) of B and the C-3 (δ 76.3) of A. The
above result indicated that residue B was linked to the 3-position of res-
idue A, and the above NOE result suggested the following sequence
1
13C\\ H correlations were obtained from HMBC. Cross-peaks were

926
D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
Table 3
its fully extended state, which was in agreement with the results of
methylation-GC–MS and NMR analyses, thus PCP-I was a non-branched
polysaccharide. In addition, PCP-I molecules tended to form entangled
chains under the experimental conditions, which were composed of
four signal chains as shown in Fig. 3. It has been reported that entangled
chains and helix conformations were beneficial for improving antioxi-
dant and anti-cancer activities of the polysaccharides [30], and PCP-I
showed strong inhibitory effect on the growth of colon cancer cell
Caco-2.
1H and 13C chemical shifts of the PCP-I recorded in D2O at 25 °C.
Label
Glycosyl residues
Chemical shifts (δ)
1
2
3
4
5
6
A
B
→3)-α-D-Xylp-(1→
→4)-α-L-Fucp-(1→
H
C
H
C
4.6
3.28
73.1
3.55
69.4
3.43
76.3
3.72
71.9
3.55
71.4
3.19
82.4
3.29
–
4.11
72.4
102.0
5.29
98.3
1.27
19.9
–: Not obtained.
3.7. Antioxidant and anti-proliferative activities of PCP-I
between residues A and B.
To test antioxidant activity of PCP-I, scavenging assays for superox-
ide, hydroxyl and DPPH radicals were performed, EC50 values were cal-
culated, and Vc was used as the positive control. As shown in Fig. 4, the
scavenging effects of PCP-I on the radicals increased with increasing
PCP-I concentrations. The EC50 values of PCP-I for superoxide, hydroxyl
and DPPH radicals were 1.09, 0.11 and 2.22 mg/mL, respectively, and
EC50 values of Vc for the three radicals were 1.667 mg/mL,
0.505 mg/mL and 2.72 μg/mL, respectively. The results of the EC50 values
showed that PCP-I had scavenging effects on superoxide and hydroxyl
radicals, which was equivalent to Vc according to their EC50 values (P
N 0.05).
The cytotoxicity induced by PCP-I and PC was investigated in human
colon cancer cells caco-2 using the MTT assay, and 5-Fu was used as the
positive control. As shown in Fig. 4d, PCP-I could inhibit proliferation of
cancer cells caco-2 at 25–2500 μg/ml concentration range, and the anti-
proliferation capability increased with the increasing PCP-I concentra-
tions. The IC50 values of PCP-I, PC and 5-Fu for caco-2 cells were 69.54,
677.95 and 37.95 μg/mL, respectively, indicating that PCP-I exhibited
good anti-proliferation capability for caco-2 cells. Comparing with the
chemical 5-Fu (IC50 = 37.95 μg/mL), PCP-I (IC50 = 69.54 μg/mL) from
the natural food can also effectively inhibit cancer cell growth, which
is not as good as 5-Fu (P b 0.05).
Compared with the polysaccharide previously found from P.
chinensis Lindl [6], PCP-I shows the different structural characteristics
and more noticeable antioxidant activity. The bioactivities of polysac-
charides might be influenced by molecule weight, glycosidic linkages
and chain conformation, the low MW exhibited stronger antioxidant ac-
tivity [31], and 1 → 3 glycosidic linkage showed a better promoting ef-
fect on anti-cancer activity [32]. Notably, chain conformation might
play an important role on the bioactivity of polysaccharides [30], and
entangled chain conformations may be related to good antioxidant
and anti-cancer activities [22,27].
→4Þ−α−L−Fucp−ð1→3Þ−α−D−Xylp−ð1→
Based on the result of monosaccharide composition of PCP-I (Fuc:
Xyl = 1:2.45), residue A (→3)-α-D-Xylp-(1→) corresponded to three
times of residue B (→4)-α-L-Fucp-(1→). The results of partial acid hy-
drolysis of PCP-I showed that the percentage of residue B obviously in-
creased in the supernatant of the sack (Fuc:Xyl = 3.09:1) compared to
the monosaccharide composition of PCP-I, indicating that residues A
and B are not alternating copolymerization (ABAB).
The terminal galacturonic acid of PCP-I was not identified in GC and
NMR, probably because the content was the least among the residues
for detection.
According to the results of the monosaccharide composition, partial
acid hydrolysis and NMR analysis, it was concluded that the residues A
were first connected to each other, and residues B was linked to them
later. The repeating units present in polysaccharide PCP-I was deter-
mined to be the following:
→½4Þ−α−L−Fucp−ð1ꢁn→½3Þ−α−D−Xylp−ð1ꢁ3n
→
The terminal fractions of PCP-I were T-D-GalAp according to the re-
sults of GC–MS.
3.6. Molecular morphology of PCP-I
To determine fine structure of macromolecules, TEM was performed
to illustrate the morphological information [27]. Sodium dodecyl sulfate
(SDS) was added as a surfactant to disperse PCP-I polysaccharides [24],
and then the microstructure of PCP-I was observed. A portion of PCP-I
molecule with extended conformations was shown in Fig. 3. Non-
branched conformation was observed from the TEM image of PCP-I in
Fig. 3. TEM image of PCP-I in sodium dodecyl sulfate (SDS) solution (left) and the local amplification of the left image (right).

D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
927
100
80
60
40
20
0
100
(b)
(a)
80
60
40
20
V
PCP-I
PC
c
V
PCP-I
PC
c
0
0.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
0.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
Concentration (mg/ml)
Concentration (mg/ml)
120
100
80
60
40
20
0
(c)
(d)
5-FU
PCP-I
PC
100
80
60
40
20
0
V
PCP-I
PC
c
0
2
4
6
8
10
12
14
25
50
250
500
2500
Concentration (mg/ml)
Final concentration(ug/mL)
Fig. 4. Bioactivities of PCP-I: (a) superoxide radical scavenging activity, (b) hydroxyl radical scavenging activity, (c) DPPH radical scavenging activity, and (d) inhibitory effects on the growth of colon
cancer cell Caco-2. PC and PCP-I were crude polysaccharides and the purified polysaccharide from P. chinensis Lindl, respectively. Each data point represents the mean SD (n = 3).
4. Conclusion
References
[1] S.W. Wen, The research progress of Shi-xian-tao, Mod. Chin. Med. 8 (6) (2006)
35–36.
According to our results, we concluded that the crude polysaccha-
ride PC from P. chinensis Lindl contained major polysaccharide PCP-I,
which was mainly purified using DEAE Sepharose CL-6B column chro-
matography with a yield of 52.4%. Using HPSEC coupled with a Sugar
KS-804 column and RID, PCP-I was purified to homogeneous and Mw
of PCP-I was 249 kDa. FTIR of PCP-I displayed the characteristic polysac-
charide peaks. PCP-I consisted of xylose and fucose with a molar ratio
2.45:1 by GC analysis. The results of periodate oxidation-smith degrada-
tion, partial hydrolysis with acid, methylation-GC–MS and NMR indi-
cated that →3)-α-D-Xylp-(1→ and →4)-α-L-Fucp-(1→ is the major
component of the main-chain structure of PCP-I, the terminal fractions
[2] B. Wu, H.B. Qu, Y.Y. Cheng, Cytotoxicity of new stilbenoids from Pholidota chinensis
and their spin-labeled derivatives, Chem. Biodivers. 5 (2008) 1803–1810.
[3] J. Wang, L.Y. Wang, S. Kitanaka, Stilbene and dihydrophenanthrene derivatives from
Pholidota chinensis and their nitric oxide inhibitory and radical-scavenging activities,
J. Nat. Med. 61 (2007) 381–386.
[4] Y.Y. Li, L.Y. Xu, R. Hu, M. Yao, X.Y. Zeng, The analgesic activity of different extracts
from Pholidota yunnanensis Rolfe, Guid. China Med. 10 (8) (2010) 51–52.
[5] J. Wang, N.L. Wang, X.S. Yao, R.I.S. Kitanaka, Inhibitory activity of Chinese herbal
medicines toward histamine release from mast cells and nitricoxide production by
macrophage-like cell line, RAW 264.7, J. Nat. Med. 60 (2006) 73–77.
[6] H.H. Yang, Y.J. Wu, C.J. Gan, T.L. Yue, Y.H. Yuan, Characterization and antioxidant ac-
tivity of a novel polysaccharide from Pholidota chinensis Lindl, Carbohydr. Polym.
138 (15) (2016) 327–334.
[7] H.H. Yang, H.M. Weng, Y.G. Guan, Y. Lin, C.J. Gan, Technology for extracting polysac-
charides of Pholidota chinensis Lindl, Food Sci. 2 (2009) 130–133.
[8] B. Li, S.X. Li, Y.X. Liu, Q. Zou, P.A. Liu, Study on extraction technology and content de-
termination of polysaccharides in Pholidota cantonensis, Hunan, J. Tradit. Chin. Med.
31 (7) (2015) 161–163.
of PCP-I were T-D-GalAp, and PCP-I was
a non-branched
heteropolysaccharide. TEM analysis revealed the entangled microstruc-
ture of PCP-I that was composed of four non-branched single chains.
Comparing the antioxidant activities of PCP-I with Vc, PCP-I showed
equal scavenging effects on superoxide and hydroxyl radicals, and the
EC50 values of PCP-I for superoxide and hydroxyl radicals were 1.09
and 0.11 mg/mL, respectively. The cytotoxicity experiment against
human colon cancer cells caco-2 showed that PCP-I and 5-Fu displayed
the IC50 values of 69.54 and 37.95 μg/mL, respectively.
[9] A.M. Staub, Removal of proteins: Sevag method, methods, Carbohydr. Chem. 5
(1965) 5–6.
[10] M. Dubois, K.A. Gillis, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for
determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356.
[11] T. Bitter, H.M. Muir, A modified uronic acid carbazole reaction, Anal. Biochem. 4
(1962) 330–334.
[12] D.H. Luo, B.S. Fang, Structural identification of ginseng polysaccharides and testing
of their antioxidant activities, Carbohydr. Polym. 72 (2008) 376–381.
[13] Z.J. Wang, D.H. Luo, Antioxidant activities of different fractions of polysaccharide pu-
rified from Gynostemma pentaphyllum Makino, Carbohydr. Polym. 68 (2007) 54–58.
[14] Z.J. Wang, Y.W. Zeng, D.H. Luo, Structure elucidation of a non-branched and
entangled heteropolysaccharide from Tremella sanguinea Peng and its antioxidant
activity, Carbohydr. Polym. 152 (2016) 33–40.
Acknowledgements
This work was supported by Promotion Program for Young and Mid-
dle-aged Teacher in Science and Technology Research of Huaqiao Uni-
versity (ZQN-PY316) and the National Nature Science Foundation of
China (31201314).
[15] D.H. Luo, X.M. Yuan, Y.W. Zeng, K.Y. Nie, Z.M. Li, Z.J. Wang, Structure elucidation of a
major fucopyranose-rich heteropolysaccharide (STP-II) from Sargassum thunbergii,
Carbohydr. Polym. 143 (2016) 1–8.

928
D. Luo et al. / International Journal of Biological Macromolecules 112 (2018) 921–928
[16] A.K. Ojha, D. Maiti, K. Chandra, S. Mondal, D.D.S.K. Roy, K. Ghosh, S.S. Islam, Struc-
tural assignment of a heteropolysaccharide isolated from the gum of Cochlospermum
religiosum (Katira gum), Carbohydr. Res. 343 (2008) 1222–1231.
[17] D.H. Luo, Identification of structure and antioxidant activity of a fraction of polysac-
charide purified from Dioscorea nipponica Makino, Carbohydr. Polym. 71 (2008)
544–549.
[18] D.L. Komura, A.C. Ruthes, E.R. Carbonero, P.A.J. Gorin, M. Iacomini, Water-soluble
polysaccharides from Pleurotus ostreatus var. florida mycelial biomass, Int. J. Biol.
Macromol. 70 (2014) 354–359.
[25] X.M. Yuan, Y.W. Zeng, K.Y. Nie, D.H. Luo, Z.J. Wang, Extraction optimization, charac-
terization and bioactivities of a major polysaccharide from Sargassum thunbergii,
PLoS One 10 (12) (2015).
[26] Z.Y. Wang, Y.Y. Zhao, T.T. Su, J. Zhang, F. Wang, characterization and antioxidant ac-
tivity in vitro of polysaccharide purified from Rana chensinensis, Carbohydr. Polym.
126 (2015) 17–22.
[27] L. Chen, J. Liu, Y. Zhang, Y. Niu, B. Dai, L. Yu, A novel alkaline hemicellulosic
heteroxylan isolated from alfalfa (Medicago sativa L.) stem and its thermal and
anti-inflammatory properties, J. Agric. Food Chem. 63 (2015) 2970–2978.
[28] D. Rout, S. Mondal, I. Chakraborty, S.S. Islam, The structure of a polysaccharide from
fraction-II of an edible mushroom, Pleurotus florida, Carbohydr. Res. 341 (2006)
995–1002.
[19] J. Liu, X.Y. Wen, J. Kan, C.H. Jin, Structural characterization of two water-soluble
polysaccharides from black soybean (Glycine max (L.) Merr.), J. Agric. Food Chem.
63 (2015) 225–234.
[20] M. Pramanik, S. Mondal, I. Chakraborty, D. Rout, S.S. Islam, Structural investigation of
polysaccharide (Fr. II) isolation from the aqueous extract of an edible mushroom,
Pleurotus sajor-caj, Carbohydr. Res. 340 (2005) 629–636.
[21] B. Li, X.J. Wei, J.L. Sun, S.Y. Xu, Structural investigation of a fucoidan containing a fu-
cose-free core from the brown seaweed, Hizikia fusiforme, Carbohydr. Res. 341
(2006) 1135–1146.
[22] L. Chen, J. Liu, Y. Zhang, B. Dai, Y. An, L. Yu, Structural, thermal, and anti-inflamma-
tory properties of a novel pectic polysaccharide from alfalfa (Medicago sativa L.)
stem, J. Agric. Food Chem. 63 (2015) 3219–3228.
[23] L. Chen, P.C.K. Cheung, Mushroom dietary fiber from the fruiting body of pleurotus
tuber-regium: fractionation and structural elucidation of nondigestible cell wall
components, J. Agric. Food Chem. 62 (2014) 2891–2899.
[29] E.R. Suarez, J.A. Kralovec, M.D. Noseda, H.S. Ewart, C.J. Barrow, M.D. Lumsden, T.B.
Grindley, Isolation, characterization and structural determination of a unique type
of arabinogalactan from an immunostimulatory extract of Chlorlla pyrenoidosa,
Carbohydr. Res. 340 (2005) 1489–1498.
[30] K. Wang, W. Li, X. Rui, T. Li, X. Chen, M. Jiang, M. Dong, Chemical modification, char-
acterization and bioactivity of a released exopolysaccharide (r-EPS1) from Lactoba-
cillus plantarum 70810, Glycoconj. J. 32 (2015) 17–27.
[31] Z.M. Li, K.Y. Nie, Z.J. Wang, D.H. Luo, Quantitative structure activity relationship
models for the antioxidant activity of polysaccharides, PLoS One 11 (9) (2016).
[32] Y.S. Jing, J.H. Zhu, T. Liu, S.X. Bi, X.J. Hu, Z.Y. Chen, L.Y. Song, W.J. Lv, R.M. Yu, Struc-
tural characterization and biological activities of a novel polysaccharide from cul-
tured Cordyceps militaris and its sulfated derivative, J. Agric. Food Chem. 63
(2015) 3464–3471.
[24] L. Chen, B.B. Zhang, J.L. Chen, P.C.K. Cheung, Cell wall structure of mushroom sclero-
tium (Pleurotus tuber-regium): part 2. Fine structure of a novel alkali-soluble hyper-
branched cell wall polysaccharide, Food Hydrocoll. 38 (2014) 48–55.