Structural Characterization and Immunostimulatory Activity of Polysaccharides from Brassica rapa L.

Article abstract of DOI:10.1021/acs.jafc.7b03902

Two neutral polysaccharides (BRNP-1, 6.9 kDa; BRNP-2, 4.8 kDa) were purified from the common edible plant Brassica rapa L. via the combined techniques of ion-exchange chromatography and high-performance gel permeation chromatography. Monosaccharide composition analysis showed that BRNP-1 and BRNP-2 were composed of glucosyl residues. Methylation and 1D- and 2D-NMR analyses revealed that both BRNP-1 and BRNP-2 contained a backbone chain that was composed of α-D-(1 → 4)-linked Glcp residues and side chains that were composed of terminally linked Glcp residues attached at the O-6 position of backbone-glycosyl residues. BRNP-1 and BRNP-2, however, differed in branch degree and molecular weight. Bioassay results showed that treatment with the higher dosage (400 μg/mL) of BRNP-1 and BRNP-2 stimulated the proliferation, NO release, and cytokine secretion (IL-6 and TNF-α) of RAW264.7 macrophages. These results suggested that BRNP-1 and BRNP-2 may enhance macrophage-mediated immune responses.

Full text of DOI:10.1021/acs.jafc.7b03902

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Article
Structural characterization and immunostimulatory
activity of polysaccharides from Brassica rapa L.
Zhuo-Er CHEN, Reziyamu WUFUER, Jin-Hu JI, Jin-Fang LI, Yu-
Feng CHENG, Caixia Dong, and Hailiqian TAOERDAHONG
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03902 • Publication Date (Web): 10 Oct 2017
Downloaded from http://pubs.acs.org on October 10, 2017
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Page 1 of 30
Journal of Agricultural and Food Chemistry
Structural characterization and immunostimulatory activity of polysaccharides from
Brassica rapa L.
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‖
1‖
2
3
1
Zhuo-Er CHEN , Reziyamu WUFUER , Jin-Hu JI , Jin-Fang LI , Yu-Feng CHENG ,
4*
1*
Cai-Xia DONG , Hailiqian TAOERDAHONG
1College of Pharmacy, Xinjiang Medical University,Urumqi, Xinjiang Uygur Autonomous
Region,China;
2Medical Engineering Technology Institute, Xinjiang Medical University, Urumqi,
Xinjiang Uygur Autonomous Region, China;
3The Experimental Teach Center, College of HouBo, Xinjiang Medical University,
Karamay, Xinjiang Uygur Autonomous Region, China;
4Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics
and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
Corresponding Authors
*
*
(C.-X.D.) Tel: +86-183-2247-3708; E-mail: dongcaixia@tmu.edu.cn
(H.T.) Tel: +86-139-9987-9870; E-mail: hailiqian2471@sina.com
Author Contributions
‖
Z.-R.C. and R.W. contributed equally.
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ABSTRACT
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Two neutral polysaccharides (BRNP-1 6.9 kD, BRNP-2 4.8 kD) were purified from
the common edible plant Brassica rapa L. via the combined techniques of ion-exchange
chromatography and high-performance gel permeation chromatography. Monosaccharide
composition analysis showed that BRNP-1 and BRNP-2 were composed of glucosyl
residues. Methylation and 1D- and 2D-NMR analyses revealed that both BRNP-1 and
BRNP-2 contained a backbone chain that was composed of α-D-(1→4)-linked Glcp
residues and side chains that were composed of terminally linked Glcp residues attached at
the O-6 position of backbone-glycosyl residues. BRNP-1 and BRNP-2, however, differed
in branch degree and molecular weight. Bioassay results showed that treatment with the
higher dosage (400 µg/mL) of BRNP-1 and BRNP-2 stimulated the proliferation, NO
release, and cytokine secretion (IL-6 and TNF-α) of RAW264.7 macrophages. These
results suggested that BRNP-1 and BRNP-2 may enhance macrophage-mediated immune
responses.
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KEYWORDS: Brassica rapa L., polysaccharides, structural characterization,
immunostimulatory activity
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. INTRODUCTION
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The innate immune system comprises macrophages, monocytes, granulocytes, and
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humoral elements . Macrophages, the first line of immune defense, perform a variety of
complex biological activities, including phagocytosis, surveillance, chemotaxis, and
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destruction of targeted organisms . These activities suggest that regulating macrophage
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activity is a potential strategy against disease .In recent years, many kinds of plant-derived
polysaccharides have been widely investigated by researchers because of their
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immunostimulatory activity on macrophages , such as water-soluble polysaccharide (SNP)
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from Sipunculus nudus L. , a Laminaria japonica polysaccharide , and Ginseng fruits
polysaccharides , as well as Astragalus polysaccharides 8-10, and polysaccharides from
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,11
Tinospora cordifolia .And studies demonstrated that polysaccharides may mainly exert
immunomodulatory effects on macrophage RAW264.7 via TLR4-mediated
MyD88-dependent signaling pathways8-11
.
Brassica rapaL., a common edible plant that belongs to the family of Cruciferae, is
widely distributed at high-altitude regions in Xinjiang Uygur Autonomous Region, China.
The root of B. rapa is a traditional Uyghur folk medicine for moistening lungs and
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relieving cough and asthma .Studies have shown that the pharmacological activities of B.
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rapa result from its nutritional benefits, including vitamins , glucosinolates , flavonoids ,
ferredoxin-sulfite reductase , chalcone glycosides , and polysaccharides18,19. Among
them, glucosinolates (GLSs) have attracted the more attention in view of their
chemopreventive activities against numerous chronic degener-ative diseases, together with
cancer, cardiovascular diseases, neurodegeneration and diabetes.14,18 Compared with GLSs,
however, polysaccharides as the main water-soluble components in B. rapa were few
investigated, especially in structural characterization18,19. It has been reported that only
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three polysaccharides with relatively low molecular weight and three polysaccharides
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with large molecular weight were isolated from B. rapa. However, there is no more
information reported on the structural characterization except the monosaccharide
composition. In addition, increasing evidence over recent years has demonstrated that
polysaccharides exhibit immunomodulatory activity. Experimental evidencefor the
immunodulatory activities of other plant-derived polysaccharides and the use of B. rapa as
traditional folk medicine imply that polysaccharides from B. rapamay also have
immunomodulatory activity. To the best of our knowledge, however, no other studies have
investigated polysaccharides from B. rapaand their immunological activity. In this paper,
we report the isolation, structural characterization, and in vitro immunostimulatory
activity of polysaccharides from B. rapa L.
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. MATERIALS AND METHODS
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2.1 Materials. B. raparoots were collected from a commercial market in Urumqi,
Xinjiang Uygur, Autonomous Region of China. Toyopearl DEAE 650 M was purchased
from Tosoh (Tokyo, Japan). PL aquagel-OH MIXED-H (7.5 mm × 300 mm, 8 μm) and
Sugar-pack TM (6.5 mm × 300 mm, 10 μm) columns were purchased from Agilent (USA)
and Waters Co., respectively. Sepharose 6B and Sephacryl S-300 HR were obtained from
GE Healthcare (Amersham Biosciences AB, Uppsala, Sweden). Other chemicals were
obtained from Sigma-Aldrich Co. LLC. Fetal bovine serum (FBS), trypsin, Dulbecco
Modified Eagle Medium (DMEM), phosphate buffered solution (PBS), streptomycin,
penicillin, and dimethyl sulfoxide (DMSO) were obtained from Hyclone Co. (UT, USA).
Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. (MO, USA). Cell
Counting Kit-8 (CCK-8 kit), and IL-6 and TNF-α ELISA kits were purchased from Wuhan
Boster Co. (Wuhan, China). Nitric oxide (NO) kit was supplied by Promega Co.
(Wisconsin, USA).
HPLC analyses were conducted on an Agilent 1200 system that was coupled with a
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refractive index detector (RID). GC–MS analysis was performed on an Agilent GC-MS
7890A-5975 instrument with helium as the carrier gas. Fourier transform infrared (FT–IR)
spectra were recorded on a Nicolet 380 FT–IR spectrophotometer.
2.2 Extraction, Isolation, and Purification of Polysaccharides. Extraction was
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performed in accordance with a previously described method . Dried B. rapa powder was
defatted by soaking in 95% ethanol (EtOH) at room temperature. After removing the
solvent, residues were extracted with hot water thrice for 2 h per extraction. The residues
were then filtered through defatted cotton and concentrated in vacuo. The supernatant was
precipitated with 80% EtOH. The precipitates were redissolved and dialyzed against
distilled water (cut off, 7000 Da). Crude B. rapa polysaccharides (BRP) were obtained
after freeze-drying.
BRP (3.0 g) was suspended in H O. The suspension was stirred at room temperature
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and then centrifuged at 4000 rpm for 15 min to remove insoluble portions (17.6%). The
supernatant was then subjected to an ion-exchange chromatography on a Toyopearl DEAE
650 M column (5.0 i.d. × 20 cm). The supernatant was successively eluted with distilled
0.8 Lof H O, 0.5 M, 1.0 M NaCl, 2.0 M NaCl, and 0.2 M NaOH to yield BRN (B. rapa L.
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neutral polysaccharide fraction) (11.2%), BRA1 (32.9%), BRA2(0.7%), BRA3 (0.1%),
and BRA4 (1.4%), respectively.Fractions of 15 mL were collected and monitored at 490
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nm via phenol–H
2
SO method .
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The water-eluted polysaccharide fraction BRN (500 mg) was loaded on a Sepharose
6B (5.0 i.d. × 90 cm) column and eluted with 0.1 M NaCl (1.2 L) to yield two major
fractions, BRN-1 and BRN-2. BRN-1 was further purified by gel filtration on Sephacryl
S-300 (2.2 i.d. × 90 cm) and eluted with 0.1 M NaCl (0.4 L) to yield BRNP-1 (240 mg).
BRN-2 was purified to yield BRNP-2 (250 mg). Fractions of 10 mL were collected and
monitored at 490 nm via phenol–H
2
SO
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method and at 280 nm via UV absorbance
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spectroscopy.
2.3 Estimation of Homogeneity and Apparent Molecular Weight.The apparent
molecular weights of BRNP-1 and BRNP-2 were estimated with high-performance gel
permeation chromatography (HPGPC) analysis on an Agilent 1200 system equipped with
an RID detector. Samples (5 mg/mL, 10 μL) were applied to a PL Aquagel-OH MIXED-H
column (7.5 mm × 300 mm, 8 μm) and eluted with 0.1M NaNO at 0.6 mL/min with the
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column temperature maintained at 35 C. Commercially available T-series dextrans (MW
2000, 670, 410, 270, 150, 80, 50, 12, 5, and 1 kD) were used as standard molecular
markers.
2.4 Colorimetric Analyses. Total carbohydrate and uronic acid contents were
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determined by phenol–sulfuric acid and m-hydroxybiphenyl methods with galactose as
the standard. Protein content was analyzed using a Bio-Rad protein assay kit with bovine
serum albumin (BSA) as the standard.
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2.5 Sugar Composition Analysis. The sugar components of BRNP-1 and BRNP-2
were directly hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 °C for 2 h in
accordance with the routine method of complete hydrolysis for neutral polysaccharides.
After the removal of TFA under nitrogen gas, the hydrolysates were converted into alditol
acetates for GC–MS analysis, which was conducted with a fused silica capillary column
(HP-5 MS, 30 m × 0.25 mm, 0.25 μm, Agilent, USA). Injection and detector temperatures
were maintained at 280 °C. The oven temperature was programmed to increase from 160
°C to 190 °C at 2 °C/min, then to 240 °C at 5 °C/min, and maintained at 240 °C for 5 min.
Helium was used as the carrier gas.
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2.6 Methylation Analysis. The methylation analyses of BRNP-1 and BRNP-2 were
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performed in accordance with a previously described method . After posttreatment, the
resultant products were hydrolyzed with 2 M TFA at 120 °C for 2 h, followed by
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reduction with NaBD
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and acetylation with acetic anhydride to yield partially methylated
alditol acetates. These acetates were analyzed with GC–MS using a HP-5 MS fused silica
capillary column (30 m × 0.25 mm, 0.25 μm, Agilent). The column temperature was set to
120 °C during injection, then increased by 4 °C/min to 280 °C, and maintained at 280 °C
for 5 min. Helium was used as the carrier gas. Mass spectra were interpreted to identify
the compounds that corresponded to each peak. The molar ratio of each residue was
calculated on the basis of peak areas.
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2.7 Infrared Spectral Analysis. The infrared spectra of the polysaccharide samples
were obtained with a FT–IR spectrophotometer. The purified polysaccharides were ground
with KBr powder and then pressed into a polymer film for FT–IR measurement in the
–1
frequency range of 4000–400 cm .
2.8 NMR Analysis. Approximately 20 mg BRNP-1 and BRNP-2 were dissolved in
0.6 mL D
then obtained.
2.9 Cell Line and Culture Medium. The macrophage RAW 264.7 cell line was
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O and then analyzed via NMR at 30 °C. The maps of 1D- and 2D-NMR were
purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai. The cell line was grown in DMEM medium that
contained 10% FBS and 100 U/mL of penicillin and 100 μg/mL of streptomycin.
2.10 Macrophage Viability Test by CCK-8 Assay. RAW 264.7 cells were seeded at
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a density of 1.0  10 cells/mL in 96-well plates and cultured for 24 h at 37 °C in a
humidified incubator with 5% CO
concentrations of 0, 1.6, 3.2, 8, 40, 80, 200, 400, 1000, and 2000 μg/mL. After 24 h of
incubation at 37 °C in 5% CO humidified atmosphere, 10 μL of CCK-8 solution was
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added to each well. After 1 h of incubation, absorbance was recorded at 450 nm using an
ELISA plate reader and then converted into macrophage viability ratio for comparison.
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2.11 Nitric Oxide Production and Cytokine Production after BRNP-1 and
BRNP-2 Treatment. After 24 h of incubation in the above conditions, RAW 264.7 cells
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(2×10 cells/mL) were treated with BRNP-1 and BRNP-2(0, 80, or 400 μg/mL) or LPS(1
μg/mL) in DMEM medium. The treated cells were then incubated for an additional 24 h.
The conditioned culture medium was collected to analyze nitric oxide (NO) and cytokine
release by RAW 264.7 cells. NO, TNF-α, and IL-6 contents were determined using
commercial kits in accordance with the manufacturer’s instructions. Cells that were treated
with 1 μg/mL LPS were used as the positive control. Cells that were cultured in DMEM
medium without polysaccharides and LPS were used as the normal control.
2.12 Statistical Analysis. All experiments were repeated thrice. Results were
expressed as the mean ± SD of triplicate analyses. Statistical significance was analyzed by
one-way ANOVA using SPSS 16.0 software. Comparisons with P values less than 0.05
were considered as statistically significant.
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. RESULTS AND DISCUSSION
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3.1 Structural Characterization of BRNP-1 and BRNP-2. The purified
polysaccharides BRNP-1 and BRNP-2 were colorless, highly water-soluble powders. The
two polysaccharides were eluted as a single and symmetrical sharp peak on the HPGPC
chromatogram. As shown in Fig.1, these results indicated that BRNP-1 and BRNP-2 were
homogeneous. The apparent molecular weight of these polysaccharides were estimated as
6.9 kD and 4.8 kD from a dextran standard curve. The total carbohydrate, uronic acid, and
protein contents of BRNP-1 and BRNP-2 were determined through colorimetric analysis.
Results showed that carbohydrates dominated the chemical composition of BRNP-1
(99.8%) and BRNP-2 (99.9%), whereas protein was present only in trace amounts
(BRNP-1: 0.11%; BRNP: 0.07%).Uronic acid was not detected, thus suggesting that the
two polysaccharides are neutral polysaccharides.
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The monosaccharide compositions of BRNP-1 and BRNP-2 were hydrolyzed and
acetylated intoalditol acetates for GC–MC analysis. As shown in Fig.2, only one peak
appeared at 21.1 min. Comparing the retention time of this peak with those of
authenticated standards revealed its identity as glucose (Glc). Therefore, BRNP-1 and
BRNP-2are composed of glucosyl residues.
The FT–IR spectra of BRNP-1 and BRNP-2 were similar (Fig. 3). The absorbance
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band at 3409.3 cm represented the stretching vibration of O–H bonds in constituent
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sugar residues. Bands at approximately 2929.4 cm were associated with the stretching
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vibration of C–H in sugar rings. The band at approximately 1649.1 cm indicated bound
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water . Absorbance bands at 1154.1 cm , 1078.0 cm , and 1025.7 cm indicated the
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bending vibrational modes of C–O stretching in pyranose . In addition, the absorption
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peak at 842.2 cm suggestedthat glucosyl residues are mainly present inα-configuration ,
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whereas that at approximately 928.4 cm isa characteristic of α-glucans . These wave
numbers indicated that the major glucosyl residues in these two polysaccharides are of
α-configuration and are α-glucans.
To identify the linkage type between glucosyl residues, BRNP-1 and BRNP-2 were
subjected to methylation analyses. Results are shown in Fig. 4 and are summarized in
Table 1.The total ionization chromatogram (TIC) showed that derivatized BRNP-1 and
BRNP-2 exhibited the same peak-signal pattern, which was composed of three peak
signals (1–3). These signals were identified based on the combination of fragment analysis
and
monosaccharide
composition
results.
Peak
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was
identified
as1,5-di-O-acetyl-(1-deuterio)-2,3,4,6-tetra-O-methyl glucitol as deduced from the major
primary ion fragments at m/z 161 and 162 that appeared in nearly equal amounts and the
diagnostic ion fragments at m/z 205 and 118.These ion fragments confirmed the presence
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1,4,5-tri-O-acetyl-(1-deuterio)-2,3,6-tri-O-methyl glucitol, as deduced from the major
primary ion fragments at m/z 233 and 118 and the diagnostic fragment at m/z 162. These
fragments indicated the presence of (1→4)-linked Glcp units. As deduced from the
diagnostic
pair
m/z
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and
261,
peak
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assigned
to
1,4,5,6-tetra-O-acetyl-(1-deuterio)-2,3-di-O-methyl glucitol, suggesting the presence of
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(1→4,6)-linked Glcp . Taken together, both BRNP-1 and BRNP-2 were predominantly
composed of (1→4)-linked Glcp residues, suggesting the presence of a (1→4)-linked
glucan backbone. In addition, (1→4,6)- and t-linked Glcp residues were also present in
small amounts, indicating that the glucan backbone, which is attached by t-linked Glcp
residues, branches at the O-6 position. As summarized in Table 1, BRNP-1 and BRNP-2
exhibited similar glycosyl linkage composition but different apparent molecular weight
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and branch degree (DB). DB was calculated according to the formula (DB) =
(NT+NB)/(NT+NB+NL)，where NT, NB, and NL are the total numbers of the terminally
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linked residues, branched residues, and linear residues, respectively. On the basis of this
formula, the DB values of BRNP-1 and BRNP-2 were calculated as 0.243 and 0.238,
respectively.
To further interpret the structures of BRNP-1 and BRNP-2, the two polysaccharides
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were analyzed via 1D- ( H- and C-NMR)and 2D-NMR (HSQC and HMBC). The
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obtained spectra were data from the literature. As shown in Fig.5A-a, the H-
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and C-NMR spectra of these two polysaccharides have similar signal patterns. In the
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low-field region, three anomeric hydrogen signals appeared at δ 4.5-5.5 ppm in H-NMR
spectra. These signals corresponded to three anomeric carbon signals at δ 90–110 ppm in
13C-NMR spectra. Taking the results of methylation analysis into consideration, the
presence of these anomeric signals suggested three glucopyranosyl residues in the
repeating units of BRNP-1 and BRNP-2. In the case of BRNP-1, signals at δ4.88 (98.63),
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5.30 (99.83), and 5.27 (100.02) ppm were assigned to the H1 (C1) of t-linked Glcp residue
(residue A), (1→4)-linked Glcp residue (residue B), and (1→4, 6)-linked Glcp residue
(residue C), respectively. Consistent with the results of IR analysis, the signals in the
lower field indicated that all glucosyl residues haveα-configuration. In high field, the
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major signals at δ 3.76, 3.87, 3.57, 3.55, and 3.77/3.76 in H-NMR spectrum were
assigned to H-2, H-3, H-4, H-5, and H-6a/H-6b of residue B in accordance with the
literature30,31.Corresponding carbons were easily determined from correlations in HSQC,
as summarized in Table 2. Other signals were assigned by the combination of 2D-NMR
and evidence from the literature30,31
.
The linkage sequence was deduced from HMBC spectra (Fig.5B-b). A strong
cross-peak signal at δ 3.57/99.83 ppm was assigned to the correlation between H-4 and
C-1 of inter-residue B, suggesting a(1→4)-α-linked glucan backbone. This deduction was
also supported by the weak cross-peak signal at δ 5.30/77.12 ppm; on the basis of data
from the literature, this signal was ascribed to the correlation between the H-1 and C-4 of
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inter-residue B . In addition, a very weak cross-peak signal at 4.88/69.44 ppm was
attributed to the correlation between the H-1(δ 4.88) of residue A and the C-6(δ 69.44) of
residue C, indicating that the H-1 of residue A is linked to the O-6 of residue C. In
accordance with the results of methylation analysis, deductions from the HMBC spectra
suggested that BRNP-1 contains a (1→4)-linked glucan backbone that branches at the O-6
position by t-linked Glcp residues.
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BRNP-2 showed similar H- and C-NMR and HMBC spectra (not shown here) as
BRNP-1, suggesting that BRNP-1 and BRNP-2 have similar structural characteristics.
Considering all of the information obtained from NMR spectra and methylation analyses,
both BRNP-1 and BRNP-2 contain a (1→4)-α-linked glucan backbone chain that branches
at the O-6 position of the backbone units with t-α-linked Glcp residues as side chains. The
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two polysaccharides, however, differ in apparent molecular weight and DB. The possible
structures of BRNP-1 and BRNP-2 are shown in Fig. 6.
3.2 Immunomodulatory Activity of BRNP-1 and BRNP-2. The cytotoxicity of
BRNP-1 and BRNP-2 against RAW 264.7 macrophages was evaluated by CCK-8 assay.
The viability of control wells (no sample) was considered to be 100%. The viability ratios
of macrophages that were activated by samples were determined at the indicated
concentrations. As shown in Fig.7A, BRNP-1 showed low cytotoxicity at the low
concentration of 1.6–80 μg/mL, exerted no cytotoxicity at concentrations higher than 80
μg/mL, and exhibited the best stimulatory activity on macrophages at 400 μg/mL.
BRNP-2 exhibited significant proliferation activity in a dose-dependent manner and
exerted no cytotoxic effects at concentrations of 1.6 to 2000 μg/mL. Thus, our results
revealed that higher concentrations of BRNP-1 and BRNP-2 stimulate macrophage
proliferation. Therefore, the concentrations of 80, 400 μg/mL of BRNP-1 and BRNP-2
were used as treatments in the following experiments.
NO is released from various mammalian cells, including vascular endothelial cells,
macrophages, mesangial cells, spleen lymphocytes, glial cells, and fibroblasts. As a
messenger molecule, NO exerts cytotoxic effects on bacteria, fungi, and tumor cells, and
32
is implicated in inflammatory responses in response to tissue injury . We determined NO
production from RAW 264.7 cells treated with BRNP-1 and BRNP-2 to evaluate their
activation effects on macrophages. As shown in Fig. 7B, treatment with higher dosage
(400 μg/mL) of BRNP-1 or BRNP-2 on RAW264.7 cells significantly enhanced the
release of NO in the culture medium compared with that in the normal control group,
especially for BRNP-1 which showed even a comparative level compared to LPS group.
As compared with the normal control group, however, treatment with the lower dosage (80
μg/mL) of BRNP-1 or BRNP-2 showed no significant effect on the release of NO. It is
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well known that NO is a major mediator of macrophages and essential for the resistance of
immune system to pathogens invasion. Glucans that are composed of 1,4-α-D-glucosidic
linkages have been reported to have a significant effect on NO production from
33
macrophages . In our study, both BRNP-1 and BRNP-2 stimulated the NO release of
RAW 264.7 cells at higher dosage (400 μg/mL). BRNP-1 showed more significant effects
on NO production than BRNP-2. Given the similar backbones and side chains of the two
polysaccharides, the different effects of BRNP-1 and BRNP-2 on NO production may be
closely related to molecular weight and DB.
We examined the ability of BRNP-1 and BRNP-2 to stimulate cytokine secretion in
macrophages. As illustrated in Fig. 7C, BRNP-1 or BRNP-2 exhibited a marked
enhancement in TNF-α secretion when treated RAW 264.7 cells at higher dosage 400
μg/mL compared with the control group. The TNF-α secretion was observed to increase to
4.5 folds of that in control group with a level of 81 pg/mL, and 6.1 folds with a level of
110 pg/mL. BRNP-2 showed a stronger effect on TNF-α secretion than BRNP-1 at the
higher dosage. Consistent with the effect of two polysaccharides on the NO release, the
lower dosage of BRNP-1 and BRNP-2 showed little even suppressed effect on the TNF-α
secretion. Unlike the case in TNF-α, both BRNP-1 and BRNP-2 exhibited a significant
effect on the secretion of IL-6 (Fig. 7D) compared with that both in control group and in
LPS group, but no difference were observed between the higher and lower dosage
treatments.
The activation of macrophages results in the production of a large amount of NO. NO
contributes to the killing of pathogens and mediates a variety of biological functions as an
34
intracellular messenger molecule . In addition, macrophage activation produces various
cytokines, including TNF-α and IL-6. In turn, TNF-α and IL-6 induce the proliferation of
35
other immune cells, including B-cells and T-cells . In the present study, we found that
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polysaccharides from B. rapa L. induced the activation of macrophages in vitro, as
manifested by NO production and cytokine (TNF-α and IL-6) secretion. Therefore, these
two polysaccharides may potentially enhance macrophage-mediated innate immune
response.
In summary, two neutral polysaccharides, BRNP-1 and BRNP-2, with apparent
molecular weights of 6.9 kD and 4.8 kD, respectively, were isolated from the medicinal
plant B. rapa L. via the combination of ion-exchange chromatography and
high-performance gel permeation chromatography. The structures of BRNP-1 and
BRNP-2 were characterized by chemical derivatization, HPGPC, GC–MS, and 1D- and
2D-NMR technologies. BRNP-1 contains a glucan backbone chain that is composed of
α-D-(1→4)-linked Glcp residues. Moreover, BRNP-1contains side chains that are
composed oft-linked-α-D-Glcp residues that are attached at the O-6 position of
α-D-(1→4)-linked Glcp at every eight backbone-glycosyl residues. Furthermore, the DB
of BRNP-1 is 0.243. BRNP-2 has the same backbone chain and side chains as BRNP-1
but differs in DB (0.238), which indicates that its side chains are attached at the O-6
position of α-D-(1→4)-linked Glcp at every nine backbone-glycosyl residues. Bioassay
results showed that both BRNP-1 and BRNP-2 stimulate macrophage proliferation and
significantly enhance NO production and cytokine (TNF-α and IL-6) secretion when
treated RAW 264.7 cells with higher dosage of 400 μg/mL. Therefore, BRNP-1 and
BRNP-2 may potentially enhance macrophage-mediated immune response. This study
provides preliminary information for further investigations on the antitumor effects of B.
rapa polysaccharides.
FUNDINGS
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This work was financially supported by the National Natural Science Foundation of China
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(No. 81460615), the Foundation of Key Laboratory of Xinjiang Uygur Autonomous
Region of China (XJDX0208-2012-1), and Tianjin Municipal Science and Technology
Commission (No. 15JCYBJC50700).
REFERENCES
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
(1) Chávez-Sánchez, L.; Espinosa-Luna, J. E.; Chávez-Rueda, K.; Legorreta-Haquet, M.
V.; Montoya-Díaz, E.; Blanco-Favela, F., Innate Immune System Cells in
Atherosclerosis. Arch. Med.Res. 2014, 45, 1-14.
(2) Sica, A.; Allavena, P.; Mantovani, A., Cancer related inflammation: the macrophage
connection. Cancer Lett. 2008, 267, 204-215.
(3) Grivennikov, S. I.; Greten, F. R.; Karin, M., Immunity, inflammation, and cancer. Cell.
2010, 140, 883-899.
(4) Schepetkin, I. A.; Quinn, M. T., Botanical polysaccharides: macrophage
immunomodulation and therapeutic potential. Int. Immunopharmacol. 2006, 6,
317-333.
(5) Zhang, C. X.; Dai, Z. R., Immunomodulatory activities on macrophage of a
polysaccharide from Sipunculus nudus L. Food Chem. Toxicol. 2011, 49, 2961-2967.
(6) Zha, X.-Q.; Lu, C.-Q.; Cui, S.-H.; Pan, L.-H.; Zhang, H.-L.; Wang, J.-H.; Luo, J.-P.,
Structural identification and immunostimulating activity of a Laminaria japonica
polysaccharide. Int. J. Biol. Macromol. 2015, 78, 429-438.
(7) Wang, Y.; Huang, M.; Sun, R.; Pan, L., Extraction, characterization of a Ginseng fruits
polysaccharide and its immune modulating activities in rats with Lewis lung
carcinoma. Carbohydr. Polym. 2015, 127, 215-221.
(8) Wei, W.;Xiao, H.; Bao, W.; Ma, D.; Leung, C.; Han, X.; Ko, C.; Lau, C.; Wong, C.;
Fung, K.; Leung, P.; Bian, Z.; Han, Q.TLR-4 may mediate signaling pathways of
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 30
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Astragalus polysaccharide RAP induced cytokine expression of RAW264.7 cells.J.
Ethnopharmacol. 2016, 179, 243-252.
(9) Zhou, L.; Wang, Z.; Yu, S.; Long, T.; Zhou, X.; Bao, Y., Astragalus polysaccharides
exerts immunomodulatory effects via TLR4-mediated MyD88-dependent signaling
pathway in vitro and in vivo., Sci. Rep. 2017, 7, 1-13.
(10)Zhang, W.; Ma, W.; Zhang, J.; Song, X.; Sun, W.; Fan, Y., The immunoregulatory
activities of Astragalus polysaccharide liposome on macrophages and dendritic cells.
Int. J. Biol. Macromol., 2017, in press.
(11)Kumar, G.P.; Rajan, M.G.R.; Kulkami,S. Activation of murine macrophages by
G1-4A, a polysaccharide from Tinospora cordifolia, in TLR4/MyD88 dependent
manner. Int. Immunopharmacol. 2017, 50, 168-177.
(12)Liu, Y.M. Brassica rapa L. In Uygur medicine tzu, edition 1.; Liu, Y.M., Eds.; Health
science and technology publishing house in Xinjiang, China; 1999; Vol. 9.; pp.
334-335.
(13)Dominguez-Perles, R.; Mena, P.; Garcia-Viguera, C.; Moreno, D. A., Brassica foods
as a dietary source of vitamin C: a review. Crit Rev Food Sci Nutr. 2014, 54,
1076-1091.
(14)Jeong, J.; Park, H.; Hyun, H.; Kim, J.; Kim, H.; Oh, H. I.; Hwang, H. S.; Kim, D. K.;
Kim, H. H., Effects of glucosinolates from Turnip (Brassica rapa L.) root on bone
formation by human osteoblast-like MG-63 cells and in normal young rats. Phytother
Res. 2015, 29, 902-909.
(15)Annalisa R., P. V., Laura I., Francesca I., Daniela H., HPLC-DAD/MS
characterization of flavonoids and hydroxycinnamic derivatives in Turnip Tops
(Brassica rapa L. Subsp. sylvestris L.). J.Agric. Food Chem. 2006, 54，1342-1346.
3
60
(16)Takahashi, S.; Yip, W. C.; Tamura, G., Purification and characterization of
1
6
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Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
ferredoxin-sulfite reductase from turnip (Brassica rapa) leaves and comparison of
properties with ferredoxin-sulfite reductase from turnip roots. Biosci. Biotechnol.
Biochem.1997, 61, 1486-1490.
(17)Hara, H.; Nakamura, Y.; Ninomiya, M.; Mochizuki, R.; Kamiya, T.; Aizenman, E.;
Koketsu, M.; Adachi, T., Inhibitory effects of chalcone glycosides isolated from
Brassica rapa L. 'hidabeni' and their synthetic derivatives on LPS-induced NO
production in microglia. Bioorg. Med. Chem. 2011, 19, 5559-5568.
(18)Xie, Y.; Jiang, S.; Su, D.; Pi, N.; Ma, C.; Gao, P., Composition analysis and
anti-hypoxia activity of polysaccharide from Brassica rapa L. Int. J. Biol. Macromol.
2010, 47, 528-533.
(19)Wang, W.; Wang, X.; Ye, H.; Hu, B.; Zhou, L.; Jabbar, S.; Zeng, X.; Shen, W.,
Optimization of extraction, characterization and antioxidant activity of
polysaccharides from Brassica rapa L. Int. J. Biol. Macromol. 2016, 82, 979-988.
(20)Bao-lin HOU, Q. W., HailiqianTaoerdahong., Research on polysaccharide extraction
and the content of Brassica rapaL. Lishizhen Medicine And Materia Medica Research
2009, 20，2759-2761.
3
3
3
3
3
3
3
3
3
77
78
79
80
81
82
83
84
85
(21)Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F., Colorimetric
method for determination of sugars and related substances. Anal. Chem., 1956, 28,
350-356.
(22)Blumenkrantz, N.; Asboe-Hansen, G., New method for quantitative determination of
uronic acids. Anal. Biochem. 1973, 54, 484-489.
(23)Ciucanu, I.; Kerek, F., A simple and rapid method for the permethylation of
carbohydrates. Carbohydr. Res. 1984, 131, 209-217.
(24) Parker, F.S., Application of I.R Spectroscopy in Biochemistry, In Biology and
Medicine, edition 1.; Parker, F.S., Eds.; Plenum Press, New York, 1971, Vol. 14.;
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 30
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
pp.100-104.
(25)Xu, F.; Liao, K.; Wu, Y.; Pan, Q.; Wu, L.; Jiao, H.; Guo, D.; Li, B.; Liu, B.,
Optimization, characterization, sulfation and antitumor activity of neutral
polysaccharides from the fruit of Borojoa sorbilis cuter. Carbohydr. Polym. 2016, 151,
364-372.
(26)Wu, Y.; Li, Y.; Liu, C.; Li, E.; Gao, Z.; Liu, C.; Gu, W.; Huang, Y.; Liu, J.; Wang, D.;
Hu, Y., Structural characterization of an acidic Epimedium polysaccharide and its
immune-enhancement activity. Carbohydr. Polym. 2016, 138, 134-142.
(27)Wang, Y.-Y.; Khoo, K.-H.; Chen, S.-T.; Lin,C.-C.; Wong,C.-H.; Lin, C.-H.,Studies
on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi)
polysaccharides: Functional and proteomic analyses of a fucose-containing
glycoprotein fraction responsible for the activities.Bioorg. Med.Chem. 2002, 10,
1057-1062.
(28)Carpita, N.C., Shea, E.M. Linkage structure of carbohydrates by gas
chromatography-mass spectroscopy (GC-MS) of partially methylated alditol acetates.
In Analysis of Carbohydrate by GLC and MS, edition 1.; Biermann, C. J., McGinnis,
G. D., Eds.; CRC Press, FL: Boca Raton, 1988; Vol. pp. 157-216.
(29)Tao, Y.; Zhang, L.; Fan, Y.; Wu,X., Chain conformation of water-insoluble
hyperbranched polysaccharide from fungus. Biomacromolecules. 2007, 8, 2321-2328.
(30)Xu, X.; Gu, Z.; Liu, S.; Gao, N.; He, X.; Xin, X., Purification and characterization of
a glucan from Bacillus Calmette Guerin and the antitumor activity of its sulfated
derivative. Carbohydr. Polym. 2015, 128, 138-146.
(31)Jiang, J.; Kong, F.; Li, N.; Zhang, D.; Yan, C.; Lv, H., Purification, structural
characterization and in vitro antioxidant activity of a novel polysaccharide from
Boshuzhi. Carbohydr. Polym. 2016, 147, 365-371.
1
8
ACS Paragon Plus Environment

Page 19 of 30
Journal of Agricultural and Food Chemistry
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11
(32)Huang, M.; Mei, X.; Zhang,S., Mechanism of nitric oxide production in macrophages
treated with medicinal mushroom extracts (Review). Int.J.Med.Mushrooms. 2011, 13,
1-6.
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
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20
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31
32
33
34
35
(33)Wu, D.-T.; Xie, J.; Wang, L.-Y.; Ju, Y.-J.; Lv, G.-P.; Leong, F.; Zhao, J.; Li, S.-P.,
Characterization of bioactive polysaccharides from Cordyceps militaris produced in
China using saccharide mapping. J.Funct. Foods. 2014, 9, 315-323.
(34)Baugh, J. A.; Bucala, R., Mechanisms for modulating TNF alpha in immune and
inflammatory disease. Curr. Opin. Drug Disc. 2001, 4, 635-650.
(35)Sobota, R. M.; Müller, P. J.; Khouri, C.; Ullrich, A.; Poli, V.; Noguchi, T.; Heinrich, P.
C.; Schaper, F., SHPS-1/SIRP1α contributes to interleukin-6 signalling. Cell. Signal.
2008, 20, 1385-1391.
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Figure captions
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Fig. 1. HPGPC chromatogram of polysaccharides BRNP-1 (A) and BRNP-2 (B). Samples
were applied to PL Aquagel-OH MIXED-H column (7.5mm×300mm, 8μm) and eluted
with 0.1M NaNO
3
at 0.6mL/min with column temperature maintained at 35 °C.
Commercially available T-series dextrans were used as standard molecular markers.
Fig. 2. GC-MS chromatograms of standard monosaccharides (A) and hydrolysates of
BRNP-1 (B) and BRNP-2 (C). Rha: rhamnose; Fuc: fucose; Ara: arabinose; Xyl: xylose;
Man: mannose; Glc: glucose; Gal: galactose.
-1
Fig. 3. FT-IR spectrum of BRNP-1 and BRNP-2 between 400 and 4000cm .
Fig. 4. GC-MS profile of partially methylated alditol acetates of BRNP-1. (A) TIC profile;
(B) MS fragments; (C) deduced residues. Peaks: 1, 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6-
tetra-O-methyl glucitol; 2, 1,3,4-tri-O-acetyl-(1-deuterio)-2,3,6-tri-O-methyl glucitol; 3,
1,4,5,6-tetra-O-acetyl-(1-deuterio)-2,3-di-O-methyl glucitol.
13
1
Fig. 5. C and H NMR spectra of BRNP-1 (A-a) and BRNP-2 (A-b) in D
2
O as recorded
on a Varian INOVA 600 NMR spectrometer at 30 °C. HSQC (B-a) and HMBC (B-b)
spectra of BRNP-1 in D
°C.
2
O as recorded on a Varian INOVA 600 NMR spectrometer at 30
Fig. 6. Predicted structure of the repeating units of BRNP-1 (A) and BRNP-2 (B).
Fig.7. The effect of BRNP-1 and BRNP-2 on the proliferation (A), NO production (B),
*
and TNF-α (C) and IL-6 (D) secretion of RAW264.7 macrophages. P ＜ 0.05, vs the
#
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control group; P＜0.05, vs the LPS positive control group.
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Table 1. The Deduced Glycosidic Linkage Type And Composition Of BRNP-1 And
BRNP-2 Determined By Methylation And GC-MS Analyses
mol%
BRNP-2
Deduced
residues
Sugar derivatives
BRNP-1
1
,5-di-O-acetyl-(1-deuterio)-2,3,4,6-tetr
a-O-methyl glucitol
1
7
4.9
5.7
15.3
76.2
8.5
t-Glcp
1
,4,5-tri-O-acetyl-(1-deuterio)-2,3,6-tri-
O-methyl glucitol
(1→4)-Glcp
(1→4,6)-Glcp
1
,4,5,6-tetra-O-acetyl-(1-deuterio)
2,3-di-O-methyl glucitol
9
.4
-
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Table 2. The H-, C-NMR Chemical Shifts of BRNP-1 Isolated From Brassica rapa
L.
Chemical shifts, δ (ppm)
Glycosyl residues
1
2
3
4
5
6
H
C
H
C
H
C
4.97
98.49
5.27
99.67
5.30
99.99
3.51
74.58
3.53
71.42
3.50
71.52
3.95
76.71
3.88
73.27
3.55
73.45
3.68
70.30
3.88
76.54
3.57
77.05
3.87
76.44
3.76
71.18
3.88
71.20
3.68
60.47
3.76
60.43
3.33
69.28
t-Glcp
(
1→4)-Glcp
(
1→4,6)-Glcp
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