Purification, structural characterization and bioactivity evaluation of a novel proteoglycan produced by Corbicula fluminea

Article abstract of DOI:10.1016/j.carbpol.2017.08.063

A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea, which is a food source of freshwater bivalve mollusk. CFPS-11 had an average molecular weight of 807.7 kDa and consisted of D-glucose and D-glucosamine in a molar ratio of 12.2:1.0. The protein moiety (～5%) of CFPS-11 was covalently bonded to the polysaccharide chain in O-linkage type through both serine and thereonine residues. The polysaccharide chain of CFPS-11 was composed of (1 → 4)-α-D-glucopyranosyl and (1 → 3,6)-α-D-glucopyranosyl residues, which branched at O-6. The branch chain consisted of (1 →)-α-D-glucopyranosyl and (1 →)-α-D-N-acetylglucosamine residues. CFPS-11 exhibited significant antioxidant activity in a dose-dependent manner and remarkable inhibition activities against α-amylase and α-glucosidase by in vitro assays. These findings indicated that the CFPS-11 from C. fluminea has the potential for development as a health food ingredient.

Full text of DOI:10.1016/j.carbpol.2017.08.063

Accepted Manuscript
Title: Purification, structural characterization and bioactivity
evaluation of a novel proteoglycan produced by Corbicula
fluminea
Authors: Jing-Kun Yan, Yao-Yao Wang, Wen-Yi Qiu, Li-Xia
Wu, Zhi-Chao Ding, Wu-Dan Cai
PII:
S0144-8617(17)30940-2
DOI:
Reference:
http://dx.doi.org/10.1016/j.carbpol.2017.08.063
CARP 12670
To appear in:
Received date:
Revised date:
Accepted date:
13-6-2017
5-8-2017
12-8-2017
Please cite this article as: Yan, Jing-Kun., Wang, Yao-Yao., Qiu, Wen-Yi., Wu,
Li-Xia., Ding, Zhi-Chao., & Cai, Wu-Dan., Purification, structural characterization
and bioactivity evaluation of a novel proteoglycan produced by Corbicula
fluminea.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.08.063
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Submitted to: Carbohydrate Polymers
Original Research MS)
(
Purification, structural characterization and bioactivity evaluation of a novel
proteoglycan produced by Corbicula fluminea

Jing-Kun Yan , Yao-Yao Wang, Wen-Yi Qiu, Li-Xia Wu, Zhi-Chao Ding, Wu-Dan Cai
School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
Highlights


A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea
The structural feature of CFPS-11 was elucidated by physical and chemical analyses
CFPS-11 exhibited significant antioxidant activity in a dose-dependent manner
CFPS-11 showed remarkable inhibitory activities against α-amylase and
α-glucosidase



CFPS-11 could be explored as a potent food additive or nutritional supplement
Abstract
A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea, which is
a food source of freshwater bivalve mollusk. CFPS-11 had an average molecular weight of
07.7 kDa and consisted of D-glucose and D-glucosamine in a molar ratio of 12.2:1.0. The
8
protein moiety (~5%) of CFPS-11 was covalently bonded to the polysaccharide chain in
O-linkage type through both serine and thereonine residues. The polysaccharide chain of
CFPS-11 was composed of (1→4)-α-D-glucopyranosyl and (1→3,6)-α-D-glucopyranosyl
residues, which branched at O-6. The branch chain consisted of (1→)-α-D-glucopyranosyl

Corresponded authors: JK Yan, Tel: +08615952819661, E-mail: jkyan_27@163.com,
jkyan27@ ujs.edu.cn
1

and (1→)-α-D-N-acetylglucosamine residues. CFPS-11 exhibited significant antioxidant
activity in a dose-dependent manner and remarkable inhibition activities against α-amylase
and α-glucosidase by in vitro assays. These findings indicated that the CFPS-11 from C.
fluminea has the potential for development as a health food ingredient.
Keywords: Corbicula fluminea; Proteoglycan; Purification; Structural characterization;
Antioxidant activity
1
. Introduction
Nowadays, natural polysaccharides (PS) have been recognized as an important class of
biomacromolecules because of their useful properties. A major area of application of natural
PS is in the food industry as food additives or nutraceutical supplements because of their
distinct functional properties and numerous health benefits (Giavasis, 2014; Lovegrove et al.,
2
017). Proteoglycans are a class of protein- or peptide-PS complexes (PSPs) with the
protein/peptide chains being covalently bound to the PS side-chains in certain positions, and
the total carbohydrate content is significantly higher than that of protein (Murray et al., 2012;
Schwartz, 2009). Proteoglycans can have more prominent biological functions and
therapeutic properties, such as immunoregulation, antitumor, antioxidant, anticoagulant,
hpyerglyceric, and hypolipidemic, than PS, thereby endowing potential applications in
functional foods and pharmaceuticals (Fu et al., 2007; Pan et al., 2013; Schwartz, 2009). Over
the last few decades, a large number of proteoglycans with various structures and properties
have been isolated and purified from various natural resources, including animals, plants, and
microorganisms. Some of these proteoglycans include the acidic PSP from pumpkin (Fu et al.,
2
007), a glycosaminoglycan-like PS from abalone Haliotis discus hannai Ino (Li et al., 2011),
an arabinogalactan protein from Lycium ruthenicum (Peng et al., 2012), a
poly-N-acetylhexosamine from Cordyceps sinensis (Chen et al., 2013), a proteoglycan from
Ganoderma lucidum fruiting bodies (Pan et al., 2014), and a PSP from Herba Epimedii (Chi
et al., 2017). There is a continued interest in further research and development of novel
proteoglycans with remarkable bioactivities and nutritional values from natural resources.
2

Asian clam (Corbicula fluminea) belongs to a class of freshwater bivalve mollusk and is
widely distributed in China and some other Asian countries. It is consumed as a favorite food
because of good taste and nutrition and is also rich in functional ingredients beneficial to
health. C. fluminea, is also used in traditional Chinese medicine for a broad spectrum of
therapeutic effects, such as improved appetite and eyesight, dieresis, liver disease and
measles treatment, fever abatement, cough relief, reduced sputum, and antialcoholism (Lin et
al., 2012; Zhu, Lin, Wu & Lin, 2004). Modern pharmacological studies have demonstrated
that PS and PSPs of C. fluminea exhibit antitumor and antioxidant activities (Liao et al., 2013,
2
015, 2016; Zhu et al., 2004). For example, Liao et al. (2016) reported a novel protein-bound
PS, named CFPS-1 isolated from C. fluminea could effectively and inhibit breast cancer
MCF-7 and MDA-MB-23 cells. Recently, our research group attained a proteoglycan (CFPS)
from C. fluminea by three-phase partitioning (TPP) technique and the partially purified CFPS
shows notable radical scavenging and antioxidant activities in vitro (Yan, Wang, Qiu & Shao,
2
017). The biological activities of PS and PSPs depend mainly on chemical composition and
structural characteristics, such as monosaccharide composition, molecular weight (MW),
glycosidic bond, and chain conformation (Zhang, Cui, Cheung, & Wang, 2007). However, to
the best of our knowledge, no or little attention has been devoted to investigate the
purification, structural elucidation, and bioactivity evaluation of the CFPS isolated from C.
fluminea by using TPP approach.
The present work was on the purification and bioactivity assessment of a novel
proteoglycan, named CFPS-11 isolated from C. fluminea through column chromatographies.
CFPS-11 was structurally characterized by the combination of physical and chemical
methods. Additionally, the antioxidant and α-amylase, and α-glucosidase inhibition activities
of CFPS-11 were evaluated by in vitro assays.
2
. Materials and methods
2
.1. Materials and chemicals
The CFPS was obtained from C. fluminea by the three-phase partitioning (TPP) method
which was devloped in our previsous study (Yan et al., 2017). In brief, the TPP was
3

performed with 20% (w/v) mass fraction of (NH
4
)
2
SO , 9.8 mL of t-butanol at 35.3°C,
4
extraction time of 30 min, and pH 6.0. The CFPS recovered from the lower aqueous phase
was then dialyzed, concentrated, and lyophilized to yield the partially purified CFPS.
Acarbose, 1-phenyl-3-methyl-5-pyrazolone (PMP), porcine pancreatic α-amylase,
α-glucosidase, p-nitrophenyl-α-D-glucopyranoside (PNPG), 1,1-diphenyl-2-picrylhydrazyl
(DPPH), hydrogen peroxide (H
2
2
O ), and trifluoroacetic acid (TFA) were purchased from
Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). DEAE-Sepharose Fast Flow and
SuperdexTM 200pg were obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden).
All other chemicals and solvents were of laboratory grade and used without further
purification.
2
.2. Isolation and purification of CFPS-11
CFPS (1.0 g) was completely dissolved in 20 mL of deionized water, centrifuged at
5000 rpm for 30 min, and loaded onto a pre-equilibrated DEAE Sepharose Fast Flow
1
chromatography column (2.0×40 cm). Afterward, CFPS was eluted successively with
deionized water and NaCl aqueous solution (0.1 and 0.2 M) at a flow rate of 2.0 mL/min.
Each fraction (8.0 mL) was collected and separately detected by phenol-sulfuric acid method
at 490 nm (Dubois et al., 1956) and spectrophotometer method at 280 nm. The main fraction
was combined, concentrated, dialyzed (MWCO: 8-12 kDa), and lyophilized to obtain two PS
fractions, namely, CFPS-1 and CFPS-2 (Fig. 1a). CFPS-1 was further applied to a
SuperdexTM 200 pg gel filtration chromatography column (1.5×60 cm) by using deionized
water as elute at a flow rate of 2.0 mL/min. The purified PS named CFPS-11 was obtained
(Fig. 1b) and subsequently used for structural characterization and bioactivity evaluation in
this work.
2
.3. General physicochemical properties analysis
The total carbohydrate content, and protein content of CFPS-11 were determined by
phenol-sulfuric acid method using D-glucose as a standard, and Bradford method using
bovine serum albumin (BSA) as a standard, respectively (Dubois et al., 1956; Bradford,
4

1
976). Optical rotation was determined for the CFPS-11 dissolved in deionized water on a
Perkin-Elmer 341 digital polarimeter at 589 nm and 20 °C. UV-visible spectrum of CFPS-11
1.0 mg/mL) was recorded in a Varian Cary 100 spectrophotometer (Varian Co., USA) from
(
1
90 nm to 400 nm at 25 °C.
2
.4. Homogeneity and MW determination
The homogeneity and M
w
of CFPS-11 was determined by high-performance gel
permeation chromatography (HPGPC). HPGPC was performed on a Waters 1515 isocratic
pump and a Waters 2414 refractive index detector equipped with two ultrahydrogel columns
(250 and 2000, 7.8 mm × 300 mm, Waters Corp., Milford, MA, USA) at 50 °C and eluted
with deionized water at flow rate of 0.6 mL/min. CFPS-11 (2.0 mg/mL) was dissolved in
deionized water and filtered through 0.45 μm membranes (Millipore, USA) before injection.
Dextran MW standards ranging from 5.2 kDa to 1482 kDa (Sigma-Aldrich Chemical Co., St.
Louis, MO, USA) were used for calibration. Data were collected and analyzed by the online
Breeze software package (Waters Corp., Milford, MA, USA).
2
.5. Monosaccharide composition and amino acid analysis
Monosaccharide composition was analyzed by the PMP-HPLC method according to our
previous report (Chen et al., 2013). Briefly, CFPS-11 was treated through a series of
procedures, including hydrolysis, derivation, incubation, neutralization, and chloroform
extraction. The resultant aqueous layer was analyzed by HPLC on an Agilent 1100 instrument
with an Agilent ZORBAX Eclipse XDB-C18 column (5 μm, 4.6×150 mm) at 25 °C with UV
detection at 250 nm. The mobile phase was potassium phosphate-buffered saline (PBS, 0.05
M, pH 6.9) containing 15% acetonitrile (solvent A) and 40% acetonitrile (solvent B) at a flow
rate of 1.0 mL/min. D-arabinose (D-Ara), D-glucose (D-Glc), D-glucosamine (D-GlcN),
D-galactose (D-Gal), D-galacturonic acid (D-GalA), D-mannose (D-Man), L-rhamnose
(
L-Rha) and D-xylose (D-Xyl) were used as monosaccharide standards for the identification
and quantification of the corresponding peaks.
For amino acid composition, CFPS-11 (2.0 mg) was completely hydrolyzed with 1.0 mL
5

of HCl (6 M) at 110 °C for 24 h in a sealed tube. The hydrolysate was dried by evaporation
under reduced pressure and redissolved in 100 μL of citrate buffer. Amino acid composition
was recorded on an automated amino acid analyzer (HITACHI L-8800, Amino Acid Analyzer,
Japan).
2
.6. Methylation analysis
Freeze-dried CFPS-11 (10 mg) was methylated three times according to the reported
method with some modifications (Needs & Sevendran, 1993). The permethylated CFPS-11
was successively subjected to depolymerization by 90% formic acid at 100 °C for 6 h,
hydrolysis by TFA (2 M) at 100 °C for 4 h, reduction with NaBH , and acetylation with acetic
4
anhydride. The resulting methylated alditol acetate was dissolved in chloroform and analyzed
by GC-MS on an Agilent 6890 instrument with a HP-1 MS column. The column was
controlled at 50 °C for 2 min, increased to 170 °C at 30 °C/min and to 250 °C at 5 °C/min,
and held for 15 min.
2
.7. FT-IR and NMR spectroscopic analyses
The FT-IR spectrum of the CFPS-11 was performed with a Nexus 670 FT-IR
−
1
spectrometer (Thermo Nicolet Co., USA) in the wavenumber range of 500–4000 cm with
KBr pellets and referenced against air. For NMR analysis, 30 mg of freeze-dried CFPS-11
1
13
was dissolved in 0.5 mL of D
2
O. The H and C NMR spectra were obtained on a Bruker
AVANCEIII 600 MHz spectrometer (Bruker, Rheinstetten, Germany) at 25 °C. All chemical
1
13
shifts were expressed in reference to DDS (δ H 0.00 ppm, δ C 0.00 ppm) as an external
standard.
2
.8. β-Elimination reaction
CFPS-11 was subject to β-elimination reaction to analyze the linkage types between
protein and PS according to the reported method with minor modifications (Chen et al., 2008).
Briefly, CFPS-11 (5.0 μg/mL) was dissolved in 0.2 mol/L NaOH containing 1.0 mol/L
NaBH
4
and incubated at 25 °C for 2 h. Afterward, the resultant solution was measured by a
6

Varian Cary 100 spectrophotometer (Varian Co., USA) in the wavelength range of 210–270
nm. The CFPS-11 aqueous solution (5.0 μg/mL) without alkaline treatment was used as a
control.
2
.9. In vitro antioxidant activity
The in vitro antioxidant activities of the CFPS-11 were evaluated through DPPH and
hydroxyl free radicals scavenging abilities. The CFPS-11 was previously dissolved in
deionized water at different concentrations (0.5-5.0 mg/mL). Vitamin C (V ) was used as a
c
positive antioxidant reference. Details of the operation conditions and methods were
described as previously reported (Wang, Chen, Luo & Yan, 2016).
2
2
.10. α-Amylase and α-glucosidase inhibitory activity of CFPS-11
.10.1. α-Amylase inhibitory activity
The α-amylase inhibitory activity of CFPS-11 was determined through the reported
methods with slight modifications (Lordan et al., 2013). In this test, PBS (100 mM, pH 6.9)
was used as solvent. CFPS-11 was previously dissolved in PBS at various concentrations
(0.5–5.0 mg/mL). First, 500 μL of porcine pancreatic α-amylase (1 U/mL) was added in the
CFPS-11 solution and incubated at 37 °C for 10 min. Subsequently, 500 μL of potato starch
solution (1%, w/w) was mixed and continued to incubate for 10 min at 37 °C. The reaction
was terminated by addition of 1.0 mL of dinitrosalicylic acid reagent and boiling at 100 °C
for 5 min. The mixture was diluted by 10 mL of deionized water. Absorbance was measured
at 520 nm by a UV spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., China).
The α-amylase inhibitory activity (%) was estimated by the following equation: Inhibitory
activity (%) = (1 – A
1
/A
0
) × 100%, where A
1
and A are the absorbance values of the solution
0
with sample and without sample, respectively. Acarbose was used as a positive reference, and
deionized water was used as the blank control.
2
.10.2. α-Glucosidase inhibitory activity
The α-glucosidase inhibitory activity of CFPS-11 was investigated according to previous
7

reports with minor modifications (Lordan et al., 2013). PBS (100 mM, pH 6.9) was used as
solvent in this assay. CFPS-11 was completely dissolved in PBS at different concentrations
(0.5–5.0 mg/mL). Approximately 100 μL of the sample solution was mixed with 10 μL of
α-glucosidase solution (5.7 U/mL) and incubated at 37 °C for 10 min. Subsequently, 200 μL
of PNPG (6 mM) was added in the reaction mixture and kept warm at 37 °C for 20 min.
Afterward, 1.0 mL of Na
2
CO solution (1.0 M) was used for cessation reaction. Absorbance
3
was measured at 400 nm by a UV spectrophotometer (Beijing Ruili Analytical Instrument Co.,
Ltd., China). The α-glucosidase inhibitory activity (%) was calculated according to the
following formula: Inhibitory activity (%) = (1 – A
1
/A
0
) × 100%, where A
1
and A are the
0
absorbance values of the solution with sample and without sample, respectively. Acarbose
was used as a positive reference, and deionized water was used as the blank control.
2
.11. Statistical analysis
All experiments are conducted in three replicates and the mean ±standard deviation (SD)
is used in the analysis. The statistical analysis was performed by Student’s t-test and analysis
of variance (ANOVA) using OriginPro Software Version 8.0 (OriginLab Corp., MA, USA).
P<0.05 indicated statistically significant differences.
3
. Results and discussion
3
.1. Isolation, purification, and physicochemical properties of CFPS-11
The CFPS attained by the TPP method was furhter fractionate by ion exchange
chromatography on a DEAE Sepharose Fast Flow column. As shown in Fig. 1a, the CFPS
was separated into two fractions (CFPS-1 and CFPS-2), which were eluted with deionized
water and NaCl aqueous solution (0.1 M), respectively. Two peaks showed simultaneous
absorbances at 490 and 280 nm. The yields of CFPS-1 and CFPS-2 were 32.30% and 19.05%,
respectively, and the fraction CFPS-1 was considered as the main component. Given its high
yield and convenient preparation, CFPS-1 was subsequently subjected to further purification
by using gel filtration chromatography on a SuperdexTM 200 pg column (Fig. 1b). The
purified CFPS-11 was collected, concentrated, dialyzed, and lyophilized for further structural
8

characterization and bioactivety evaluation. The preliminary results obtained from column
chromatographies demonstrated that the CFPS-11 was a proteoglycan.
The purified CFPS-11 appeared as white fluffy material after lyophilization. The
carbohydrate and protein contents were 95.4%±1.5% and 4.5%±0.2%, respectively (Table 1).
Moreover, two characteristic bands were observed in the UV visible spectrum of the CFPS-11
(
data not shown) at approximately 205 and 280 nm; these bands were attributed to
carbohydrates and proteins, respectively. These results suggested that the purified CFPS-11
was a proteoglycan. In this work, HPGPC was used to determine the homogeneity and M of
w
CFPS-11, and the obtained results are shown in Fig. 1c and Table 1. CFPS-11 showed a
single and symmetrical sharp peak, which indicated that CFPS-11 was a homogeneous
proteoglycan. According to the calibration equation derived by linear regression of the
calibration curve with standard dextrans, the M
The M of CFPS-11 obtained in this study was considerably higher than that described by
Liao et al. (2013, 2016); the difference may be due to the variations in raw materials and
w
of CFPS-11 was around 807.7 kDa (Table 1).
w
2
0
separation protocols. In addition, the optical rotation of CFPS-11 was [α]
O) (Table 1), which indicated that CFPS-11 may mainly contain α-type glycosidic linkages
in its structure.
The HPLC-PMP derivatization method was applied to analyze the monosaccharide
D
= +32.3° (c 0.2,
H
2
composition of the CFPS-11, and the results are summarized in Table 1. Comparison with the
monosaccharide standards concluded that CFPS-11 was composed of D-Glc and D-GlcN in a
molar ratio of 12.2:1.0. Liao et al. (2013) found that a papain-released PS (CFPS-2) isolated
from C. fluminea was composed mainly of glucose and fucoseat a molar ratio of 0.68:0.86
and also minor contents of galactose, glucosamine, and galactosamine. Later, Liao et al.
(2016) reported a novel protein-bound PS (CFPS-1) isolated from C. fluminea which was
composed mainly of mannose and glucose in a molar ratio of 3.1: 12.7. In comparison,
CFPS-11 exhibited a distinct monosaccharide composition. Additionally, CFPS-11 was also
different from proteoglycans obtained from other mollusks, such as Anodonta anodonta
(Volpi & Maccari, 2005) and H. discus hannai Ino (Li et al., 2011).
9

As illustrated in Table 2, analysis of amino acids in CFPS-11 indicated that the protein
composition of CFPS-11 consisted of 17 kinds of amino acids, among which aspartic acid
(
1.38 mg/g), threonine (1.20 mg/g), serine (1.21 mg/g), glutamic acid (1.64 mg/g), leucine
1.40 mg/g), lysine (5.48 mg/g), and methionine (1.83 mg/g) were the main components. The
(
relatively high contents of threonine (1.20 mg/g) and serine (1.21 mg/g) revealed the possible
existence of O-glycosidic linkage between PS and protein in CFPS-11 (Chen et al., 2008).
Similar phenomena were also observed in previous studies (Li et al., 2011; Pan et al., 2014,
2
015; Peng et al., 2012). However, He et al. (2013) found that a novel protein-bound PS
(HS002-II) purified from the submerged cultures of Hirsutella sinensis Liu, Guo, Yu, and
Zeng consisted of 57.9% PS and 42.1% protein with N-type carbohydrate–protein linkage.
Liao et al. (2016) detected 15 amino acids in the protein-bound PS CFPS-1, and glutamic
acid, serine, and threonine were the main components. In addition, the existence of aspartic
acid (1.38 mg/g) and glutamic acid (1.64 mg/g) in CFPS-11 indicated that the CFPS-11 may
be an acidic proteoglycan. Linkages between PS chain and protein moiety in the proteoglycan
strucutures are divided into two types according to their stability against alkali, that is,
O-glycosidic linkages and N-glycosidic linkages (Matsunaga et al., 1998). In the present
study, Fig. 1d shows that β-elimination of CFPS-11 occurred after alkaline treatment due to
the distinct absorbance at around 240 nm. Such observation implied that the protein and PS
were covalently linked by an O-glycosidic linkages in CFPS-11. This result was in a good
agreement with the results of amino acid analysis.
3
.2. Structural characteristics of CFPS-11
The FT-IR spectrum of CFPS-11 is shown in Fig. 2a. A strong and wide absorption peak
−
1
at approximately 3386 cm corresponded to the stretching vibrations of O–H and N–H. A
−
1
weak absorption peak at about 2928 cm was attributed to the stretching vibration of C–H.
−
1
The characteristic absorption peak at around 1647 cm was ascribed to the bending vibration
−
1
of amide or amino group (–NH
2
). The absorption peak at about 1543 cm was due to the
secondary –CONH– group of proteins (Liao et al., 2016; Zhang, 1999), which indicated the
−
1
presence of protein in CFPS-11. The absorption bands between 934 and 1153 cm were due
1
0

to the stretching vibrations of pyranose rings (Liao et al., 2013). The three stretching peaks at
−
1
about 1153, 1059, and 1025 cm further implied the presence of pyranoside in CFPS-11. The
peak at around 839 cm−1 indicated the presence of α-glycosidic bonds in the PS fraction
(Zhang, 1999). This finding obtained from FT-IR spectroscopy indicated that the CFPS-11,
which contained α-glycosidic bonds, was a proteoglycan. Such result was in good agreement
with the results presented in Fig. 1 and Table 1.
The specific interglycosidic linkages between the monosaccharide residues of PS are
commonly elucidated by methylation analysis. In this work, CFPS-11 was methylated,
hydrolyzed, converted to alditol acetates, and analyzed by GC-MS. Table 3 presents the
identification and proportions of the methylated alditol acetates of CFPS-11 according to
relative literature and the standard data from the Complex Carbohydrate Research Center
(
CCRC, University of Georgia, USA) Spectral Database for PMAA (Sassaki et al., 2005;
Wang, He & Huang, 2007). Results showed that CFPS-11 was mainly composed of
,3,4,6-Me -Glcp, 2,3,4,6-Me -GlcNAc, 2,3,6-Me -Glcp, and 2,4-Me -Glcp in a molar ratio
of 1.96:1.00:7.12:2.86, which indicated the presence of Glcp(1→, GlcNAc(1→,
4)Glcp(1→, and →3,6)Glcp(1→ residues in the main chain of CFPS-11. This result also
2
4
4
3
2
→
suggested that the →4)Glcp(1→ residue might be regarded as the main consecutive repeating
unit of CFPS-11 due to its high proportion, and the →3,6)Glcp(1→ residue might be the
branching linkage in the CFPS-11 structure. Furthermore, the Glcp(1→ and GlcNAc(1→
might be considered as terminal residues attached to the O-6 position of →3,6)Glcp(1→
residue. This result showed a good correlation between terminal and branched residues.
Moreover, the methylation analysis result well agreed with that of monosaccharide
composition (Table 1).
NMR spectroscopy was used for further structural characterizations of the CFPS-11,
including monosaccharide composition, anomeric configuration, and the types and sequences
of the glycosidic linkages according to the available data reported in literature (Agrawal,
1
1
992; Yu & Yang, 1999; Zhang, 1999). As shown in the H NMR spectrum (Fig. 2b), four
anomeric proton signals at 5.29, 5.32, 5.14, and 4.89 ppm were observed; these signals
11

indicated that the repeating unit of CFPS-11 was composed of four residues, namely,
α-D-Glcp(1→, →4)-α-D-Glcp(1→, →3,6)-α-D-Glcp(1→, and α-D-GlcNAc(1→ residues,
which were designated as A, B, C, and D residues, respectively; these results were well
consistent with the methylation analysis results. The chemical shifts from δ 3.0 ppm to δ 4.2
ppm were assigned to the H-2 to H-6 protons. In addition, the resonance peaks observed in
the range of δ0.5–2.8 ppm were assigned to amino acid signals (Banfi & Patiny, 2008),
among which chemical shift at δ 1.97 ppm was ascribed to the N-acetyl group of GlcNAc
(Agrawal, 1992). This result was also in agreement with that from monosaccharide
composition analysis. As shown in the 13C NMR spectrum of CFPS-11 (Fig. 2c), four
anomeric carbon signals at δ 99.40, 99.09, 95.79, and 91.98 ppm were observed and
attributed to the C-1 of the →4)-α-D-Glcp(1→, α-D-Glcp(1→, →3,6)-α-D-Glcp(1→, and
α-D-GlcNAc(1→ residues in the CFPS-11 structure, respectively. Therefore, the strong
anomeric carbon signal at δ 99.40 ppm was ascribed to the C-1 in (1→4)-α-D-Glcp, and the
signals at 71.18, 73.24, 76.59, 72.43, and 60.26 ppm were assigned to C-2, C-3, C-4, C-5, and
C-6 of the (1→4)-α-D-Glcp residues, respectively. Moreover, the anomeric signal observed at
δ 95.79 ppm was attributed to (1→3,6)-α-D-Glcp. Other carbon signals from C-2 to C-6
1
3
within 60–78 ppm in the C NMR spectrum were assigned according to the reference data
Zhang, 1999). Resonances at δ 91.98, 54.61, 72.30, 71.82, 72.20, and 60.97 ppm were
(
assigned to C-1, C-2, C-3, C-5, and C-6 of α-D-GlcNAc(1→ residues, respectively. In
particular, a weak resonance at δ 176.82 ppm corresponded to the –CONH group of protein,
further indicating that CFPS-11 was a proteoglycan; this result was consistent with those
UV-vis results and FT-IR spectra. The presence of carbon signal at 21.42 ppm belonged to the
N-acetylated methyl group (Agrawal, 1992; Chen al., 2013; Pan et al., 2015). The entire
1
13
assignment of the H and C chemical shifts of CFPS-11 were achieved on the basis of the
1
13
H and C NMR spectra, and they are summarized in Table 4.
In summary, on the basis of the results of acid hydrolysis, methylation analysis, HPLC,
GC-MS, FT-IR, and NMR measurements, the CFPS-11 isolated from C. fluminea was
concluded as a novel water-soluble proteoglycan comprised of D-Glc and D-GlcN in a molar
1
2

ratio of 12.2:1.0 with a M of 807.7 kDa. The protein moiety was linked covalently to the PS
w
chain of CFPS-11 in O-linkage type through both serine and threonine residues. The CFPS-11
might possess a backbone of (1→4)-α-D-Glcp (55.02%) and (1→3,6)-α-D-Glcp (22.10%)
residues, which branched at O-6. The branch consisted of (1→)-α-D-Glcp (15.15%) and
(1→)-α-D-GlcNAc (7.73%) residues. Hence, on the basis of these data, the possible chemical
structure of repeating unit of the CFPS-11 was predicted and displayed in Fig. 2d. The
structural features of CFPS-11 were different from those of protein-bound PS (CFPS-1)
reported by Liao et al. (2016). The CFPS-1 possessed a backbone of 1,6-linked and
1
,4,6-linked-α-D-Glcp, which was terminated with a 1-linked-α-D-Manp residue at the O-4
position of 1,4,6-linked-α-D-Glcp in a molar ratio of 3:1:1. Regarding the other mollusks,
Volpi & Maccari (2005) reported that glycosaminoglycans from the body of the large
freshwater mollusk bivalve A. anodonta are composed of chondroitin sulfate (~38%),
nonsulfated chondroitin (~21%), and heparin (41%). Li et al. (2011) also found a novel
glycosaminoglycan-like sulfated PS (AAP) from the pleopods of H. discus hannai Ino, and
the AAP containing a chondroitin-like bone is composed of galactosamine, glucuronic acid,
fucose, and galactose with a ratio of 2.14:2.37:2.94:1; the sulfate content is 15.5%.
3
.3. Antioxidant activity of CFPS-11
DPPH·, as a stable nitrogen atom-centered free radical, has been frequently used to
evaluate the free radical scavenging capacity of antioxidants, such as plant extracts and
natural and synthesized compounds in food science (Li et al., 2008). The scavenging ability
of CFPS-11 on DPPH free radicals compared with vitamin C (V ) as a positive reference is
c
illustrated in Fig. 3a. Evidently, CFPS-11 showed an increased DPPH radical scavenging
ability in a dose-dependent manner in the concentration range of 0.5–5.0 mg/mL. At 5.0
mg/mL CFPS-11, the scavenging ability on DPPH radical was 61.28%, which was
considerably weaker than that of V
c
(91.62%, 0.5 mg/mL). The EC50 value of CFPS-11 was
3
.80 mg/mL, which was smaller than that of a papain-released PS (CFPS-2) (4.30 mg/mL)
from C. fluminea (Liao et al., 2013). Moreover, the scavenging effect of CFPS-11 on DPPH
radical was stronger than that of CFPS (58.91%, 5.0 mg/mL) obtained by TPP approach in
1
3

our previous study (Yan et al., 2017). This result indicated that CFPS-11 exhibited stronger
DPPH radical scavenging capacity than those of CFPS and CFPS-2; this difference may be
attributed to its hydrogen donation power to the free radicals, thereby effectively scavenging
the DPPH radicals. As reported by Li et al. (2013), an antioxidant glycoprotein (Fraction
AIV-2) of 27.2 kDa was purified from the ethanol-soluble protein hydrolysate of Mustelus
griseus muscle. The DPPH radical scavenging activity of fraction AIV-2 was up to about 97%
and was even higher than that of V at 5.0 mg/mL. Additionally, Xia et al. (2011) isolated a
c
water-soluble protein-bound PS (POPPS-a) from the fruiting bodies of Pleurotus ostreatus,
which exhibited potent radical scavenging activity in the three in vitro assays.
As the most active reactive oxygen, hydroxyl radical can induce severe damage to
adjacent biomolecules in the body, which results in a variety of diseases, such as aging,
cancer, and Alzheimer disease (Rollet-Labelle et al., 1998). Hence, scavenging on hydroxyl
free radicals by antioxidants may be protective against tissue injury. As shown in Fig. 3b, the
effects of CFPS-11 on hydroxyl radical scavenging ability were similar to those of DPPH
radical scavenging ability at the tested concentration range (0.5–5.0 mg/mL). At 5.0 mg/mL,
the scavenging ability of CFPS-11 on hydroxyl radical was 60.33%, which was slightly
stronger than that of CFPS (47.10%, 5.0 mg/mL) reported by our research group (Yan et al.,
2
017); by contrast, this value was far lower than that of V
value of CFPS-11 was 3.94 mg/mL. Zhang et al. (2013) reported that a mannoglucan sulfate
SF-2), which was isolated and purified from starfish, displayed significant antioxidant
c
(96.67%, 0.5 mg/mL). The EC50
(
activity in vitro. The hydroxyl radical scavenging activity of SF-2 was up to 100% at the
concentration of 10 mg/mL. Two fucosylated chondroitin sulfate (ACP and HOP) from sea
cucumber Acaudina molpadioidea and Holothuria nobilis, showed 30% and 24.6%
scavenging effects on hydroxyl radicals , respectively at 0.5 mg/mL (Zou, Pan, Dong, He, &
Wang, 2016). The scavenging capacity on hydroxyl free radicals may be related with several
factors, including monosaccharide composition, amino acid composition, MW, and chemical
structure of proteoglycans. This finding suggested that CFPS-11 would be beneficial to a
certain extent to protect against oxidative damage.
1
4

3
.4. Inhibitory effects of CFPS-11 on α-Amylase and α-glucosidase
Nowadays, the hypoglycemic activity in vitro of bioactive compounds is commonly
evaluated through inhibition assays of α-amylase and α-glycosidase activity (Lordan et al.,
013; Wu, Shi, Wang & Wang, 2016). The inhibition of α-amylase and α-glycosidase can
2
evidently delay the conversion of glucose to blood sugar and reduce the postprandial content
of blood glucose. Consequently, such inhibition can be regarded as one of the most effective
approach for treating type 2 diabetes (Tundis, Loizzo & Menichini, 2010; Wu et al., 2016).
Therefore, in the present work, the inhibitory effects of CFPS-11 on α-amylase and
α-glycosidase activities were evaluated. As shown in Fig. 3c, the inhibitory effect of CFPS-11
on α-amylase dose-dependently increased with the increasing concentration of CFPS-11 at
0
.5–5.0 mg/mL. At 5.0 mg/mL, the inhibitory ability of CFPS-11 on α-amylase was 51.18%,
which was higher than that of the protein-PS PSP-I from pumpkin (Cucurbita moschata)
seeds (Wang et al., 2017), but lower than that of the positive control acarbose (70.87%). Our
result indicated that the CFPS-11 showed evident α-amylase inhibitory activity with an EC50
value of 4.86 mg/mL in vitro. Furthermore, the CFPS-11 also presented a
concentration-dependent inhibitory effect on α-glycosidase activity in the determined
concentration range (0.5–5.0 mg/mL) (Fig. 3d). The inhibitory effect of CFPS-11 on
α-glycosidase was 65.74% at 5.0 mg/mL, which was less than that of the positive reference
acarbose (92.78%). Result suggested that CFPS-11 exhibited excellent α-glycosidase
inhibitory ability with an EC50 value of 3.92 mg/mL in vitro. Zhang et al. (2016) reported that
water-soluble PS (GP90 and P90) extracted from the fruit of Psidium guajava, exhibited
excellent α-glycosidase inhibition activity with an EC50 of 2.27 μg/mL and 0.18 mg/mL,
respectively. The inhibitory activities of GP90 and P90 were 1379- and 17-times higher,
respectively than the acarbose positive control (EC50 3.13 mg/mL). Notably, CFPS-11
showed the stronger inhibitory ability against α-glycosidase than that of α-amylase under the
same concentrations. This difference might be due to the different mechanisms of action
between α-amylase and α-glycosidase, and a moderated inhibition of α-amylase and a
stronger α-glycosidase inhibition should be preferred (Cho, Han & You, 2011). Similarly,
Kim et al (2014) found that fucoidan extracted from Ascophyllum nodosum inhibited both
1
5

α-amylase (IC50 0.12-4.64 mg/mL) and α-glycosidase (IC50 0.013-0.047 mg/mL), but the
quantities required for α-amylase inhibition were much higher than those required for
α-glycosidase. In contrast, Li et al. (2017) reported that Sargassum pallidum PSs exhibited
weaker inhibitory effects on α-glycosidase than α-amylase. The α-amylase and α-glycosidase
inhibitory activities may be attributed to the structural characterizations of the CFPS-11, such
as carbohydrate and protein contents, monosaccharide composition, amino acid composition,
MW, functional group, chemical structure, chain conformation, and extraction and isolation
methods. Therefore, these findings suggested that the CFPS-11 isolated from C. fluminea
could be useful as a potential component of new mediation as a food additive or as a food
supplement for an enzyme-targeted treatment of type 2 diabetes. Further studies on
hypoglycemic activity in vivo and molecular mechanism for type 2 diabetes are currently
underway in our laboratory.
4
. Conclusions
A novel proteoglycan (CFPS-11) was isolated from C. fluminea, and its structural
characterization and bioactivity were evaluated. Results showed that the CFPS-11 was
composed of D-Glc and D-GlcN in a molar ratio of 12.2:1.0 with a M of 807.7 kDa. The
w
protein moiety was linked covalently to the PS chain of CFPS-11 in O-linkage type through
both serine and thereonine residues. The CFPS-11 structure consisted of a (1→4)-α-D-Glcp
backbone, and the (1→3)-α-D-Glcp residues formed branches at the O-6 position with
1
-linked-α-D-Glcp and 1-linked-α-D-GlcNAc terminal residues. The CFPS-11 possessed
remarkable antioxidant activity and α-amylase and α-glycosidase inhibitory activities in vitro.
Thus, these results suggested that the CFPS-11 isolated from C. flumenia can be futher
explored as a potential functional food or nutritional supplement for improving human health.
Acknowledgements
This work was supported financially by the National Natural Science Foundation of
China (31671812), the Natural Science Foundation of Jiangsu Province (BK20140542),
Jiangsu Overseas Research & Training Program for University Prominent Young &
Middle-aged Teachers and Presidents, and the Priority Academic Program Development
1
6

(PAPD) of Jiangsu Higher Education Institutions.
1
7

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2
2

(a)
(b)
2.0
1.6
1.2
0.8
0.4
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.5
0.8
0.6
0.20
0.15
0.10
A
A
A
A
4
90
490
280
0.4
0.3
0.2
0.1
0.0
CFPS-1
CFPS-2
280
0
.4
0.2
0
0
.05
.00
0
.0
-0.1
0
20
40
60
80
100
120
0
10
20
30
40
50
60
Tube number
Tube number
(c)
(d)
0
0
0
0
0
0
0
.7
.6
.5
.4
.3
.2
.1
1
.0
.8
.6
.4
.2
.0
CFPS-11
CFPS-11 treated with NaOH
0
0
0
0
0
0
10
20
30
40
50
60
2
10
220
230
240
250
260
270
Elution time (min)
Wavelength (nm)
Fig. 1. (a) DEAE Sepharose Fast Flow chromatographic profile of CFPS eluted with
deionized water and NaCl solution (0.1 and 0.2 M). (b) SuperdexTM 200 pg chromatographic
profile of CFPS-1 eluted with deionized water. (c) High-performance gel permeation
chromatography chromatogram of CFPS-11. (d) UV spectrum of CFPS-11 before and after
β-elimination reaction.
2
3

(a)
(d)
8
39
8
6
4
2
0
9
34
0
0
0
0
2928
1543
1
647
1153
059
1
1025
3
386
3
500 3000 2500 2000 1500 1000
500
-1
Wavenumber (cm )
(b)
(c)
1
Fig 3. (a) Fourier transform infrared spectroscopy spectrum of CFPS-11. (b) H nuclear
1
3
magnetic resonance (NMR) spectrum of CFPS-11. (c) C NMR spectrum of CFPS-11. (d)
Possible chemical structure of repeating unit of CFPS-11.
2
4

(a)
(b)
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
CFPS-11
Vc
CFPS-11
Vc
0
1
2
3
4
5
0
1
2
3
4
5
Concentration (mg/mL)
Concentration (mg/mL)
(c)
(d)
8
0
0
0
0
0
100
8
6
4
2
0
0
0
0
0
6
4
2
CFPS-11
Acarbose
CFPS-11
Acarbose
0
1
2
3
4
5
0
1
2
3
4
5
Concemtration (mg/mL)
Concentration (mg/mL)
Fig 3. (a) Scavenging ability of CFPS-11 on DPPH radicals. (b) Scavenging ability of
CFPS-11 on hydroxyl radicals. (c) Inhibitory effect of CFPS-11 on α-amylase activity. (d)
Inhibition effect of CFPS-11 on α-glycosidase activity. Each value is expressed as mean ±
standard error (n = 3).
2
5

Table 1. Preliminary physicochemical properties and structural characteristics of CFPS-11
isolated from Corbicula fluminea
2
0
Sample
Carbohydrate Protein
[α]
D
M
w
Sugar composition (molar ratios) (mol %)
(
%)
(%)
(º)
(kDa)
807.7
GlcN
1.0
Glc
CFPS-11
95.4±1.5
4.5±0.2
32.3
12.2
2
6

Table 2. Amino acid composition of the purified CFPS-11 from C. fluminea
Amino acids
Aspartic acid
Threonine
Serine
Concentration (mg/g)
Amino acids
Leucine
Concentration (mg/g)
1.38
1.20
1.21
1.64
0.81
0.83
0.04
0.74
0.89
1.40
0.88
0.38
5.48
1.83
1.04
1.01
n.d.a
0.76
Tyrosine
Phenylalanine
Lysine
Glutamic acid
Glycine
Methionine
Histidine
Arginine
Alanine
Cysteine
Valine
Tryptophan
Proline
Isoleucine
a
Not determined.
2
7

Table 3. Gas chromatography-mass spectrometer results of the methylated products of
CFPS-11
Methylation product RT (min) a Mass fragments (m/z)
Linkage type
Glcp(1→
Molar ratio
1.96
2
2
2
,3,4,6-Me
4
4
-Glcp
1.00
43,59,71,87,101,117,129,145,161,205
159,161,203,205
,3,4,6-Me
-GlcNAc 1.02
GlcNAc(1→
→4)Glcp(1→
1.00
3
,3,6-Me -Glcp
1.08
1.15
43,87,101,113, 117,129,131,233
43,87,99, 129,161,189
7.12
2
,4-Me -Glcp
2
→3,6)Glcp(1→ 2.86
a
Retention time (RT) of sugars relative to that of 2,3,4,6-Me -Glcp
4
2
8

1
13
Table 4. H and C NMR chemical shifts of CFPS-11 recorded in D O at 25 °C
2
Sugar residue
Chemical shifts (ppm)
C-1/H-1 C-2/H-2
99.09/5.29 74.30/3.19 76.93/3.52 70.74/3.42 76.29/3.44 64.47/3.73
→4)-α-D-Glcp(1→ 99.40/5.32 71.18/3.69 73.24/3.75 76.59/3.77 72.43/3.89 60.26/3.59
C-3/H-3
C-4/H-4
C-5/H-5
C-6/H-6
MeCONH
A
B
α-D-Glcp(1→
C →3,6)-α-D-Glcp(1→ 95.79/5.14 70.58/3.53 76.76/3.70 72.74/3.58 75.83/3.99 69.46/3.67
α-D-GlcNAc(1→
91.98/4.89 54.61/3.84 72.30/3.73 71.82/3.50 72.20/3.94 60.97/3.81 176.82,21.42
1.97
D
/
2
9