International Journal of Biological Macromolecules 175 (2021) 473–480

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Characterization of an antioxidant pectic polysaccharide from

Platycodon grandiflorus
Yuan-Feng Zou a,b,1, Mengsi Chen a,1, Yu-Ping Fu a,c, Zhong-Kai Zhu a, Yan-Yun Zhang a, Berit Smestad Paulsen c,
Frode Rise d, Yu-Long Chen e,⁎, Yong-Zhi Yang e, Ren-Yong Jia b, Li-Xia Li a,b, Xu Song a,b, Hua-Qiao Tang a,b,
Bin Feng g, Cheng Lv a, Gang Ye a, Ding-Tao Wu h, Zhong-Qiong Yin a,b, Chao Huang b,f,⁎⁎
a
Natural Medicine Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, PR China
b
Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, PR China
c
Department of Pharmacy, Section Pharmaceutical Chemistry, Area Pharmacognosy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo, Norway
d
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo, Norway
e
Sichuan Academy of Forestry, Ecological Restoration and Conservation on Forest and Wetland Key Laboratory of Sichuan Province, Chengdu, Sichuan 610081, China
f
Laboratory of Experimental Animal Disease Model, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, PR China
g
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, PR China
h
Institute of Food Processing and Safety, College of Food Science, Sichuan Agricultural University, Yaan 625014, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Platycodonis Radix is widely used as homology of medicine and food in China; polysaccharides are thought to be
Received 9 November 2020 one of its functional constituents. In this study, a pectic polysaccharide, PGP-I-I, was obtained from the root of the
Received in revised form 3 February 2021 traditional medicine plant Platycodon grandiflorus through ion exchange chromatography and gel filtration. This
Accepted 4 February 2021
was characterized being mainly composed of 1,5-α-L-arabinan and both arabinogalactan type I (AG-I) and II
Available online 8 February 2021
chains linked to rhamnogalacturonan I (RG-I) backbone linked to longer galacturonan chains. In vitro bioactivity
Keywords:
study showed that PGP-I-I could restore the intestinal cellular antioxidant defense under the condition of hydro-
Platycodon grandiflorus gen peroxide (H2O2) treatment through promoting the expressions of cellular antioxidant genes and protect
Pectic polysaccharide against oxidative damages.
Antioxidant property © 2021 Published by Elsevier B.V.

1. Introduction asthma, hepatoprotective and hypoglycemic functions [3,4]. However,

most of the bioactivity studies are based on crude polysaccharides
Platycodonis Radix, the rhizomes of Platycodon grandiflorus (Jacq.) A. from P. grandiflorus [5–7], and/or some purified polysaccharide fractions
DC, distributed in China, North Korea, Japan and Mongolia, is widely without structure elucidation [8–13]. Besides, some well characterized
used for the treatment of cough, excessive phlegm, sore throat, and neutral polysaccharide fractions [14–18] and one homogalacturonan
displays anti-tumor, immunoregulatory, and anti-oxidant effects as from P. grandiflorus were reported [19].
medicine. Also, it is prepared as food like vegetables, fruits, tea, drinks Polysaccharides were demonstrated to exhibit amounts of bioactiv-
and cakes [1]. These effects were contributed by its various chemical ities, such as prebiotics and immunomodulating activity [8,17],
constituents, like triterpenoid saponins, flavonoids, phenolic acids, antiangiogenic activity [15,19] and so on. Among these biological activ-
polyacetylene and polysaccharides [2,3], and also high levels of starch, ities of polysaccharides mentioned in the literatures, antioxidant activ-
proteins, vitamins, and amino acids. The polysaccharide fraction from ity is one of the most frequently mentioned activities [6,9–12,20].
this plant was among those components reported in recent years for Although there are several reports about antioxidant activity of polysac-
the antioxidation, immunomodulation, anticancer, anti-fatigue, anti- charides from P. grandiflorus, they are crude polysaccharides and/or pu-
rified polysaccharide lack of detailed structure information. Therefore, it
is obviously necessary to elucidate the structural information of poly-
⁎ Corresponding author. saccharides with antioxidative activity from Platycodonis Radix, espe-
⁎⁎ Correspondence to: C. Huang, Key Laboratory of Animal Disease and Human Health of cially for the acidic fraction.
Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, The imbalance between the production and clearance of cellular re-
Chengdu 611130, PR China.
E-mail addresses: ww9987@163.com (Y.-L. Chen), huangchao@sicau.edu.cn
active oxygen species will result in oxidative stress that causes oxidative
(C. Huang). damages to DNA, proteins, and lipid membranes [21]. These events
1
Both authors contributed equally to this work. could further trigger cell apoptosis, autophagy or necrosis and lead the

https://doi.org/10.1016/j.ijbiomac.2021.02.041
0141-8130/© 2021 Published by Elsevier B.V.
Y.-F. Zou, M. Chen, Y.-P. Fu et al. International Journal of Biological Macromolecules 175 (2021) 473–480

overexpression of oncogenes, generation of mutagen compounds or in- Sepharose 6 FF after filtered through 0.45 μm filter. It was eluted with
duction of inflammatory reactions [22]. Oxidative stress and its related 10 mM NaCl at 1.0 mL/min. The purified fraction, PGP-I-I, was collected
responses are commonly occurred in intestinal diseases, like colorectal based on its elution profile, dialyzed and lyophilized as mentioned
cancers, colitis, and intestinal bowel disease (IBD) [23,24]. Therefore, above.
compounds that enhance intestinal antioxidant defense will be of
good prospects. Polysaccharide, as one of the multifunctional macromo- 2.3. Molecular weight, monosaccharide composition and glycosidic linkages
lecular carbohydrate polymers that is indigestible to reach intestine, is elucidation
benefit for intestinal function through the moderation of intestinal im-
mune response, gut microbes and antioxidant defense [25]. It has now The homogeneity and molecular weight of this purified polysaccha-
been identified several bioactive polysaccharides, including pectin, ride was determined by high performance size exclusion chromatogra-
with huge antioxidant function through scavenging ROS or regulating phy coupled with multi angle laser light scattering and refractive index
antioxidant response [26–35], especially on intestinal tissues, such as detector (Wyatt Technology Co., Santa Barbara, CA, USA) according to
polysaccharide from Hericium erinaceus [29], Ligusticum chuanxiong reported method [37]. PGP-I-I was separated using Shodex OHpak SB-
[31], Reishi Mushroom/Ganoderma lucidum [33] and Lycium barbarum 806M HQ (300 mm × 8.0 mm, i.d.) column at 30 °C, and data were ac-
[34]. So far, only one homogalacturonan from P. grandiflorus was re- quired and analyzed using the Astra software (version 7.1.3, Wyatt
ported exhibited anti-angiogenesis activity [19], the functions of pectic Technology Co., Santa Barbara, CA, USA). The Mw was calculated by
polysaccharides from P. grandiflorus in intestine have not been well de- the Zimm method of static light scattering based on the basic light scat-
fined. Considering that P. grandiflorus has been applied in the treatment tering equation is as follows [38].
of obesity, which is closely related with intestinal function [1], and total
"
#
polysaccharide and a selenium polysaccharide from P. grandifloras Kc 1 16πr 2 < S2 > z 2 θ
showed anti-oxidative functions in rat pheochromocytoma PC12 cells ¼ 1þ Sin þ 2A2 c þ . . .
Rθ Mw 3λ2 2
[6], but the structure elucidation of the polysaccharide mentioned
above was not performed further enough with only monosaccharide
composition and molecular weight, it is of interest to further elucidate where K is an optical constant equal to [4π2n2(dn/dc)2]/(NAλ4); c, the
the structure of the active pectic polysaccharides of P. grandiflorus and polysaccharide concentration in g/mL; Rθ, the Rayleigh ratio; Mw, weight
its functional mechanisms in intestine. average molecular mass; <S2>z, radius of gyration; λ, the wavelength; n,
The neutral polysaccharide from P. grandiflorus was identified as an the refractive index of the solvent (0.9% NaCl aqueous solution); dn/dc,
inulin-type fructan with prebiotic and immunomodulation effects in the refractive index increment of polysaccharides in 0.9% NaCl aqueous
our previous research [17]. In the present study, we mainly focus on solution; NA, the Avogadro's number; A2, the second virial coefficient.
the structure elucidation of acidic polysaccharide fraction from The specific refractive index increments (dn/dc) of PGP-I-I in 0.9% NaCl
P. grandiflorus and study its anti-oxidative effects in intestinal porcine aqueous solution was set as 0.147 mL/g. Then the content of water-
epithelial cells (IPEC-J2) with an in vitro oxidative stress model. soluble polysaccharides was calculated based on the refractive index
difference with dn/dc value according to the following equation [37,39],
2. Materials and methods  
α V i −V i,baseline
Ci ¼
2.1. Materials dn=dc

The root of Platycodon grandiflorus was bought from Hehuachi where Ci is the concentration of polymers; α is the RID calibration
Market of Chinese Traditional Medicine, and identified as Platycodon constant (in RI units per volt), which is determined as 3.4756 × 10−5
grandiflorus (Jacq.) A. DC. by Dr. Yuan-Feng Zou, College of Veterinary RIU/pixel; Vi and Vi,baseline are the RID voltages of sample and baseline,
Medicine, Sichuan Agricultural University. The voucher specimen No. respectively; dn/dc was set as 0.147 mL/g. The Astra software (version
20181110 was deposited at College of Veterinary Medicine, Sichuan 6.0.2, Wyatt Technology Co., Santa Barbara, CA, USA) was utilized for
Agricultural University. It was then dried and pulverized as the methods data acquisition and analysis.
mentioned before [17]. The monosaccharide composition was determined according to the
methods of [40–42]. It was determined by gas chromatography of the
2.2. Extraction and purification of polysaccharide fraction trimethylsilylated (TMS) derivatives of the methyl-glycosides obtained
after methanolysis of the polymer with 3 M hydrochloric acid in MeOH
The polysaccharide from P. grandiflorus (PGP) was obtained from for 20 h at 80 °C. It was analyzed by capillary gas chromatography on a
70 g powdered roots based on our previous research [17]. Briefly, the Focus GC (Thermo Scientific, Milan, Italy) and the internal standard was
dried materials were extracted with boiling water (30 mL/g) three mannitol.
times, 2 h each, after defat by refluxing petroleum. And PGP was ob- The glycosidic linkages were characterized by methylation studies.
tained after ethanol-precipitation and freeze-drying. 300 mg of PGP dis- The free uronic acids were first reduced with NaBD4 to their corre-
solved in 15 mL distilled water was applied to an anion exchange sponding neutral sugars, and then methylation, hydrolysis, reduction
column (GE Healthcare Bio-Sciences, Uppsala, Sweden) packed with and acetylation were carried out [43]. The derivatives were analyzed
DEAE-Sepharose Fast Flow (Beijing Rui Da Heng Hui Science Technology by the GCMS-QP2010 (Shimadzu, Kyoto, Japan). The identity of the
Development Co., Ltd., Beijing, China) after filtered through a 0.45-μm compound and relative amount of each peak of the linkage types were
filter membrane. The acidic fraction was eluted with a linear NaCl determined based on the interpretation of the mass spectra, retention
(0–1.5 mol/L) at 2 mL/min after eluting the neutral fractions with dis- times and the monosaccharide composition, respectively [42,44].
tilled water. The carbohydrate elution profiles were monitored using
the phenol-sulfuric acid assay [36], and the relevant tubes were pooled 2.4. NMR spectroscopy
together, concentrated and dialyzed (cut-off 3500 Da) to remove NaCl.
1
The lyophilized fraction without salt was the acidic fraction of PGP, H NMR, 13C NMR, and HSQC spectra of PGP-I-I were observed on an
named PGP-I. 800 NMR-spectrometer (800.03 MHz) after deuterium exchanged three
Then, 50 mg of PGP-I was dissolved in 10 mM NaCl elution buffer, times by freeze-drying in D2O. The 1H NMR spectra with solvent sup-
and subjected to gel filtration column (2.5 cm × 100 cm, Beijing Rui pression, 13C NMR and HSQC spectra with shaped decoupling pulses
Da Heng Hui Science Technology Development Co. Ltd) packing with and solvent suppression of the HOD line were recorded in D2O solution

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on a Bruker AVIIIHD 800 instrument (Bruker, Fällanden, Switzerland) at Table 1

60 °C. The primer sequences for qRT-PCR.

Gene NCBI no. Primer sequence

2.5. Precipitation with the Yariv β-glucosyl reagent Nrf2 XM_021075133.1 F: CACCACCTCAGGGTAATA
R: GCGGCTTGAATGTTTGTC
The Yariv β-glucosyl reagent is an identity indicator through a col- NQO1 NM_001159613.1 F: GATCATACTGGCCCACTCCG
ored radial precipitation formed with polymers containing R: GAGCAGTCTCGGCAGGATAC
CAT NM_214301.2 F: TGTGAACTGTCCCTTCCGTG
arabinogalactan type II (AG-II) structure [45], which is a typical linkage
R: CGTCTGTTCGGGAGCACTAA
unit in pectin type polysaccharide [44,46]. Samples of 2, 4 and 6 μL PGP- SOD1 NM_001190422.1 F: ACCTGGGCAATGTGACTG
I-I (5 mg/mL) and arabic gum (1 mg/mL, positive control) in water were R: TCCAGCATTTCCCGTCT
tested. GSH-Px (GPx) NM_214201.1 F: CGGACCACCTGTTGAAAGCTC
R: TCCGCCAGTTCTTGTTGTCCA
ZO-1 XM_021098896.1 F: TTGATAGTGGCGTTGACA
2.6. Cell culture R: CCTCATCTTCATCATCTTCTAC
β-Actin XM_003124280 F: GATGAGATTGGCATGGCTTT
The intestinal porcine epithelial cells (IPEC-J2) purchased from R: CACCTTCACCGTTCCAGTTT
Shanghai Institute of Biological Sciences, Chinese Academy of Sciences,
was cultured in DMEM/F-12 medium (Beijing Solarbio Science & Tech-
nology Co., Ltd.), containing 10% fetal bovine serum (FBS, Thermo Fisher
2.10. Statistical analysis
Scientific (China) Co., Ltd) and 1% penicillin-streptomycin (100 U/mL,
Beijing Solarbio Science & Technology Co., Ltd.). They were plated in
All data were represented as the mean and standard error of the
96-well cell plates (5 × 103 cells per well) in a cell incubator (5% CO2,
mean (SEM). One-way ANOVA and post hoc tests (LSD) were per-
37 °C), co-incubated with 0.2 mmol/mL H2O2 for 24 h for an oxidative
formed for all statistical significance analysis by IBM SPSS statistics soft-
stress damage. After that, PGP-I-I (with final concentration of 20, 10
ware (Version 24.0, USA), and displayed using GraphPad Prism software
and 5 μg/mL) treatment was performed for 24 h and the cells were sub-
(GraphPad Software, Inc., La Jolla, CA, USA).
jected into following assays.

3. Results and discussion

2.7. Cell viability assay

3.1. The acid polysaccharide extracted from Platycodon grandiflorus

Cell viability was determined by CCK-8 kit (Dojindo, CK04-11,
Minato-ku, Tokyo, Japan). All cells in 96-well plates were co-incubated
Totally 33 g PGP was obtained from P. grandiflorus with the yield of
with 10 μL of CCK-8 for 1 h, and then the absorbance of each well was
47.1% (w/w), being higher than those yields obtained under different
measured at 450 nm using a microplate reader (Thermo Fisher Scien-
extraction or precipitation methods in other studies, those ranged
tific, Inc.), and calculated according to the formula as follows:
from 6.7% to 42.15 (6.7% [15], 31.30% [11], and 42.51% [16]), while no
yield information from many studies. The ethanol-precipitation was
cell viability rate ð%Þ ¼ ðAtest −Ablank Þ=ðAcontrol −Ablank Þ  100% ð1Þ conducted with 3 folds ethanol in our research up to the final concentra-
tion of 75%, which was different with most of methods above. In addi-
tion, the original growth areas of these other P. grandiflorus materials
The Ablank means the absorbance of wells with medium and CCK-8 were different, including Anhui [15,16] and Inner Mongolia Province
but without cells; Acontrol means the absorbance of wells with cell and [11], and also from ours, being the Sichuan Province. The origin of mate-
CCK-8 solution; Atest means the absorbance of wells treated with PGP- rial and the extraction conditions are main factors for the variations in
I-I. yields.
After isolated by DEAE-Sepharose chromatography from PGP, an
2.8. Determination of antioxidant enzymes activity and malondialdehyde acidic fraction, PGP-I, was obtained (Fig. 1A). Further purification was
levels performed by gel filtration giving one fraction, PGP-I-I (Fig. 1B). The
chromatogram of size exclusion chromatography of PGP-I-I was
IPEC-J2 cells were cultured in 6-well plate. The oxidative stress dam- shown in Fig. 1C and D. Four peaks were obtained in Fig. 1C, and the
age model and the supplement of PGP-I-I were carried out as mentioned peaks around 22.0 min and 24.0 are assigned to NaCl solvent as
in Section 2.6. All wells were washed with PBS for three times, and the shown by Fig. 1D. The small peak around 16.5 min cannot be integrated
cells were collected and disrupted by cell ultrasonic breaker (Shanghai by automatic integration, which means the content of peak around
Huxi Industry Co., Ltd). After centrifuged at 12,000 rpm for 3 min, the 17.0 min is less than 5%. Those indicated that the peak 1 marked in
supernatants were obtained for the enzyme's activities determination Fig. 1C is the main peak of the PGP-I-I, which contribute more than
using Biochemical Detection Kits (Nanjing Jiancheng Bioengineering In- 95% of the polysaccharide fraction. The molecular weight of PGP-I-I
stitute, Nanjing, P.R China), including the total antioxidant capacity (T- was estimated to be 2.79 × 104 Da according to the calculation method
AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), mentioned in the method section (Fig. 1C), and was higher than most of
catalase (CAT) and malondialdehyde (MDA). both acidic and neutral polysaccharides from P. grandiflorus published
by Xu et al. (9200 Da) [15], Liu et al. [16] (4677, 10,233, and
2.9. Real-time PCR 23,442 Da), Xu et al. (8900 Da) [19], and lower than results from
Zheng et al. with the Mw of polysaccharides ranged from 1.1 × 104 to
The total RNAs of cells were isolated by Trizol reagent (Invitrogen; 2.67 × 104 Da [6].
Thermo Fisher Scientific, Inc.), and reversed transcribed into cDNA
using a PrimeScript RT Master Mix kit (Takara Biotechnology, Co., Ltd., 3.2. Monosaccharide composition and glycosidic linkage determination
Dalian, China). All the real-time PCR tests were performed using SYBR
Premix Ex Taq. II kit (Takara Biotechnology, Co., Ltd., Dalian, China). After methanolysis, monosaccharide determination was performed
All gene expressions were calculated as relative fold levels compared on PGP-I-I by GC. The polysaccharide consists of galacturonic acid
with β-actin (normalizer control), with primers showed in Table 1. (GalA), arabinose (Ara), galactose (Gal), rhamnose (Rha), glucose

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Y.-F. Zou, M. Chen, Y.-P. Fu et al. International Journal of Biological Macromolecules 175 (2021) 473–480

1.0 A a
1.0 DEAE ion chromatography showed no uronic acid; even though they
were eluted by NaCl solution. Our research demonstrated that the
0.8 0.8
uronic acid GalA was present in this acid fraction of PGP-I-I, in quite

NaCl(mol/L)
0.6 0.6
high amounts. This disparity could be due to the different elution
A490

method, as most of them were isolated under the fixed concentration

0.4 0.4 of NaCl solution, and quite lower than ours, such as those eluted with
salt concentrations ranged from 0.1 to 0.4 mol/L NaCl [15,16,19]. As
0.2 0.2 can be seen in Fig. 1A, the concentration of NaCl was around 0.5 mol/L
when the polysaccharide started to be eluted in our experiment. More-
0.0 0.0 over, the other monosaccharides types and ratio in PGP-I-I were incon-
0 20 40 60 80 100 120
sistent between the different studies, which could be due to the
Tubes (10mL/tube)
different material sources or purification procedures. The method
used for determination of the monosaccharide compositions could
also differ.
0.6 B b
After determination of the monosaccharide composition, the link-
ages between the units were determined by methylation analysis. It
was showed in Table 2, which PGP-I-I contained typical glycosidic link-
0.4
ages present in pectic polysaccharides. First, there was a backbone
A490

consisting mainly of homogalacturonan (HG) based on the high amount

0.2
of 1 → 4 linked GalpA. One homogalacturonan was purified from
P. grandiflorus and identified only contains 1 → 4 linked GalpA
[15,16,19]. The amount of Rhap linkage types (1 → 2, 4 and 1 → 2 linked)
0.0
typical of those belonging to the hairy region area shows that appr. 5% of
20 30 40 50 60 70 80 the main backbone of the polymer has branches attached [44]. This
Tubes (5mL/tube) hairy region, also called rhamnogalacturonan I (RG-I) will on position
4 of the Rha units as side chains consisting of different structures con-
taining Gal and Ara as the main components [46]. Chains consisting
mainly of 1,4-Gal with e.g. arabinose terminally are typical for the
chains normally termed arabinogalactan I (AG-I), while the Gal units
being 1,6-, 1,3- and 1,3,6-linked are typical for arabinogalactan type II
chains (AG-II) [46–48]. AG-I makes out approximately 9.1 mol% of the
polymer. The terminal Rhap could also be part of this domain [49]. The
polymer formed a red precipitate with the Yariv reagent (not shown)
which strongly indicate the presence of AG-II type polymer in PGP-I-I,
which also was shown as mentioned above based on the Gal linkages
present [50]. The AG-II constitute a minor part of the chains attached
to the RG-I region as it constitutes around 2.9 mol% of the total polysac-
charide, also being verified by the light-colored precipitate and the low
amount of 1, 3-linked Galp (0.9 mol%), which is important for binding
the Yariv reagent [48,50]. Additionally, with the monosaccharide units
Araf, are more than 30 mol% of the polymer. The backbone linkage is
1, 5-linked Araf with a high amount of branch points on position 3
(13.9 mol%) [51], closing to the amounts of terminal Ara units. This
arabinan part can be linked either directly to position 4 of Rha in the
RG-I region, or to Gal of the AG-I and/or AG-II chain [46]. These glyco-
sidic linkages identified in our studies were similar with only one previ-
ous study of a neutral polysaccharide from P. grandiflorus. That one was
found to constitute mainly 1,4-linked Galp and 1,5-linked Araf, followed

Table 2
The glycosidic linkage (mol %) present in the polysaccharide PGP-I-I from
Platycodon grandiflorus.

Monosaccharide Linkage type mol%

Ara Tf 12.9
Fig. 1. The purification elution profiles determined by phenol–sulfuric acid assay (A490 is
1➔ 5f 5.6
the absorbance at 490 nm). A. DEAE anion exchange chromatography elution file of
1➔ 3, 5f 13.9
PGP; a. PGP-I. B. gel filtration elution file of PGP-I; b, PGP-I-I. C. Chromatogram of high-
Rha Tp 1.3
performance size exclusion chromatography of PGP-I-I. Peak 1 is the peak of sample,
1➔ 2p 4.0
peak 2 is the peak of NaCl solvent. The blue line is the signal of RI, the green line is the
1➔ 3p 0.6
signal of UV and the red line is the signal of LS. D. Chromatogram of solvent NaCl. As
1➔ 2, 4p 2.4
seen from D, the peaks at 22.0 min and 24.0 min are assigned to NaCl solvent.
Gal Tp 2.6
1➔ 4p 9.1
1➔ 3p 0.9
1➔ 6p 0.7
(Glc) and fucose (Fuc), with a molar ratio at 43.2:32.4:14.6:8.3:0.7:0.1. 1➔ 3, 6p 1.3
As mentioned before, many studies on acidic polysaccharides from GalA Tp 1.1
1➔ 4p 42.1
P. grandiflorus have indicated that their polysaccharides isolated by

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Fig. 2. HSQC NMR spectrum of polysaccharide fraction PGP-I-I from Platycodon grandifloru.

with smaller amounts of terminal Araf, 1,3,5-Araf and 1,6-, 1,3,6-linked [44]. Only one signal at δ 177.8 ppm in the downfield region of 13C NMR
Galp, but with absence of GalpA [15]. The purification methods, espe- revealed carboxyl groups of non-methyl esterified α-D-GalA. This indi-
cially the elution system, were different from ours, and that could be cated that the GalA in the polysaccharide fraction PGP-I-I is not
the major factor of this difference. esterified.
The cross peak at 4.62/107.0 ppm (B1 in Fig. 2) corresponds to 1,
3.3. Structural features of PGP-I-I from NMR 4-β-Galp, and the H-2, H-3, H-4 signals at 3.69, 3.76, 4.16 ppm also indi-
cated the presence of fragments of 1, 4-β-Galp according to previous as-
The pectin polysaccharide fraction of P. grandiflorus was character- signments [52,53], which proposed the presence of β-(1,4)-galactan,
ized by assignment of signals in 1H and 13C NMR spectra using two- and manifested the AG-I side chain in PGP-I-I [44].
dimensional homonuclear HSQC, showed in Table 3 and Fig. 2. All The content of α-L-Araf residues was found in 1H, 13C NMR and HSQC
these assignments were determined by comparison with previous re- spectra by signals as in Table 3 and Fig. 2, and those cross peaks are δ
ports [44,52–55]. Briefly speaking, the result of 1H, 13C (Supplementary 5.25/111.8 (C1), δ 5.15/109.9 (D1), δ 5.80/110.3 (E1), and δ 5.11/110.2
Fig. 1) and 2D NMR spectra revealed the typical signals belonging to α- (F1) belonging to terminal α-L-Araf, 1,5–α-L-Araf, and 1,3,5–α-L-
1,4-GalpA, β-1,4-linked Galp and 1,3,5-linked Araf, demonstrating a Araf, respectively, as confirmed the presence of highly branched 1,5-α-
pectin polysaccharide with highly branched 1,5-α-L-arabinan and 1,4- L-arabinan (Table 2) [52,53]. Residue C and D are all assigned to terminal
β-D-galactan presented in PGP-I-I. However, signals from 1,3,6-β-D- linked Araf but with different chemical shifts, which may due to linked
Galp in the AG-II domain, cannot be observed due to their minor portion to different monosaccharides thus giving different assignments.
as shown in Table 2. The cross peaks at δ 1.25/19.35 ppm and δ 1.30/ As mentioned in introduction part, only one homogalacturonan was
19.50 ppm in the HSQC spectrum corresponds to H-6/C-6 of α-Rhap. isolated from P. grandiflorus and well characterized (P. grandiflorus)
The signals for other residues of Rhap cannot be assigned due to low [15,16,19]. Some similar side chains containing of 1,4-β-Galp, 1,6-β-
concentration. Galp, 1,3,6-β-Galp, and 1,3,5–α-L-Araf from the roots of P. grandiflorus
The signals in HSQC of PGP-I-I confirmed non-methyl esterified GalA were displayed by Xu et al. [15], but they were not characterized linked
residues from the HG regions, assigned with signals of H-1 at 5.11 ppm to RG moiety of a pectic backbone.

Table 3
1
H and 13C chemical shifts (δ in ppm) of polysaccharide fraction PGP-I-I from Platycodon grandiflorus.

Residues H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6

A →4)-α-GalpA-(1→ 5.11/101.8 3.75/71.4 3.98/71.7 4.39/80.0 4.75/74.3 177.8

B →4)-β-Galp-(1→ 4.62/107.0 3.69/74.6 3.76/76.0 4.16/80.3 3.71/77.3 3.80/63.6
C α-Araf-(1→ 5.25/111.8 4.22/84.1 3.96/79.4 4.04/86.7 3.82
3.73/64.0
D α-Araf-(1→ 5.15/109.9 4.22/84.5 3.96/79.4 4.04/86.1 3.83/63.9
E →5)-α-Araf-(1→ 5.08/110.3 4.14/83.9 4.03/79.5 4.21/85.0 3.79
3.80/69.2
F →3,5)-α-Araf-(1→ 5.11/110.2 4.28/82.0 4.09/85.1 4.30/84.2 3.88
3.94/69.2

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3.4. PGP-I-I promote the antioxidant defense of intestinal porcine epithelial cellular antioxidant defense may be resulted from increased expres-
cells (IPEC-J2) sions of these critical antioxidant genes as shown in Fig. 3G. These
genes contain specific nucleotide sequences in their promoters, defined
The improved antioxidant activity by bioactive polysaccharides is as the antioxidant-responsive element (ARE), and could contribute to
one of the mechanisms to exert their modulatory functions on gut mu- the protection of cells against oxidative stress [59]. Nuclear factor-
cosal immunity and reducing inflammation [25,32]. Hydrogen peroxide erythroid-2-related factor 2 (Nrf2) as a transcriptional factor, plays a
(H2O2), as both intracellular redox signaling and diffusible messenger in crucial role in the detoxification and elimination of reactive oxidants
the extracellular space, is now used frequently on oxidative damage and electrophilic agents through interacting with ARE and promoting
model of epithelial cells [56–58]. As shown in Fig. 3A, the cell viability the expressions of antioxidant genes [60]. In our study, the expression
of IPEC-J2 cells was dramatically decreased (p < 0.001) after H2O2 treat- of Nrf2 was reduced under H2O2 treatment and restored by PGP-I-I sup-
ment, probably due to the direct injures on lipid, proteins or DNA; but plementation (Fig. 3G), which suggests that the oxidative defense effect
those cells were then positively restored by PGP-I-I at all three dosages of PGP-I-I could be displayed via Nrf2 activation. Moreover, the junc-
(p < 0.001), which showed a possible protection effect of PGP-I-I. Corre- tional complexes are crucial determinants of the physical intestinal
spondingly, cellular total antioxidant capacity (T-AOC) and typical anti- barrier of epithelial cells, such as tight junctions. It consists of trans-
oxidant enzymes' activities, such as CAT, SOD, and GSH-Px, were membrane proteins like zonula occludens (ZO)-1, ZO-2, and regulatory
suppressed by H2O2, that resulted in enhanced MDA accumulation, proteins, which provide strong adhesive bonds to maintain the integrity
while all of them were significantly improved by PGP-I-I treatment of the epithelium [61]. The intestinal integrity is sensitive to oxidative
with a slight dosage-dependent manner in IPEC-J2 cells (Fig. 3B, C, D, stress, and we found that the tight junction can be destroyed by H2O2
E, F) similar to previous research on PC12 cells [6,10]. The enhanced and decrease ZO-1 expression [62–64]. Again, this suppression could

Fig. 3. The antioxidant defense effect of PGP-I-I on IPEC-J2 cells. Cells were treated with 0.2 mmol/L H2O2 for 24 h and then supplemented with PGP-I with the final concentration of 20, 10
and 5 μg/mL for further 24 h. A) Quantification shows that PGP-I-I could protect IPEC-J2 cells against H2O2-induced cell viability reduction; N = 12. B), C), D) and E) show that PGP-I-I could
promote T-AOC, CAT, SOD and GSH-Px release of damaged IPEC-J2 cells induced by H2O2; N = 8. F) Quantification shows that PGP-I-I could suppress MDA release of damaged IPEC-J2 cells
induced by H2O2; N = 8. G) qRT-PCR shows the mRNA levels of Nrf2, NQO1, ZO-1, and antioxidative enzymes (CAT and GPx) were upregulated by PGP-I-I on IPEC-J2 cells; N = 4. Error bars
indicate SEM. Differences are statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001 compared with model group, and #p < 0.05, ##p < 0.01, ###p < 0.001 compared with
normal group.

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Y.-F. Zou, M. Chen, Y.-P. Fu et al. International Journal of Biological Macromolecules 175 (2021) 473–480

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