A novel low-molecular-mass pumpkin polysaccharide: Structural characterization, antioxidant activity, and hypoglycemic potential

Article abstract of DOI:10.1016/j.carbpol.2020.117090

The novel natural low-molecular-mass polysaccharide (SLWPP-3) from pumpkin (Cucurbia moschata) was separated from the waste supernatant after macromolecular polysaccharide production and purified using a DEAE cellulose-52 column and gel-filtration chromatography. Chemical and instrumental studies revealed that SLWPP-3 with a molecular mass of 3.5 kDa was composed of rhamnose, glucose, arabinose, galactose and uronic acid with a weight ratio of 1: 1: 4: 6: 15, and primarily contained →3,6)-β-D-Galp-(1→, →4)-α-GalpA-(1→(OMe), →4)-α-GalpA-(1→, →2,4)-α-D-Rhap-(1→, →3)-β-D-Galp-(1→, →4)-α-D-Glcp, and →4)-β-D-Galp residues in the backbone. The branch chain passes were connected to the main chain through the O-4 atom of glucose and O-3 atom of arabinose. Physiologically, the ability of SLWPP-3 to inhibit carbohydrate-digesting enzymes and DPPH and ABTS radicals, as well as protect pancreatic β cells from oxidative damage by decreasing MDA levels and increasing SOD activities, was confirmed. The findings elucidated the structural types of pumpkin polysaccharides and revealed a potential adjuvant natural product with hypoglycemic effects.

Full text of DOI:10.1016/j.carbpol.2020.117090

Carbohydrate Polymers 251 (2021) 117090
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
A novel low-molecular-mass pumpkin polysaccharide: Structural
characterization, antioxidant activity, and hypoglycemic potential
,
,
,
,
,
,b,
Fei Lia b, Yunlu Weia b, Li Lianga b, Linlin Huanga b, Guoyong Yua b, Quanhong Lia
*
a College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, PR China
b National Engineering Research Center for Fruits and Vegetables Processing, Beijing, 100083, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The novel natural low-molecular-mass polysaccharide (SLWPP-3) from pumpkin (Cucurbia moschata) was
separated from the waste supernatant after macromolecular polysaccharide production and purified using a
DEAE cellulose-52 column and gel-filtration chromatography. Chemical and instrumental studies revealed that
SLWPP-3 with a molecular mass of 3.5 kDa was composed of rhamnose, glucose, arabinose, galactose and uronic
Low-molecular-mass polysaccharide
Cucurbia moschata
Structure
Hypoglycemic potential
Antioxidant activity
acid with a weight ratio of 1: 1: 4: 6: 15, and primarily contained →3,6)-β-D-Galp-(1→, →4)-
α
-GalpA-(1→(OMe),
→4)- -GalpA-(1→, →2,4)- -D-Rhap-(1→, →3)-β-D-Galp-(1→, →4)-α-D-Glcp, and →4)-β-D-Galp residues in the
α
α
backbone. The branch chain passes were connected to the main chain through the O-4 atom of glucose and O-3
atom of arabinose. Physiologically, the ability of SLWPP-3 to inhibit carbohydrate-digesting enzymes and DPPH
and ABTS radicals, as well as protect pancreatic β cells from oxidative damage by decreasing MDA levels and
increasing SOD activities, was confirmed. The findings elucidated the structural types of pumpkin poly-
saccharides and revealed a potential adjuvant natural product with hypoglycemic effects.
1. Introduction
recent decades. Zhao et al. (2017) isolated an acidic polysaccharide from
pumpkin (Cucurbita moschata) with a molecular mass of 22.6 kDa that
Pumpkin (Cucurbia moschata) is an annual herbaceous plant that
belongs to the genus Cucurbita and family Cucurbitaceae and is
considered a nutritional food with health protective properties without
side effects. Pumpkin is rich in carbohydrates (approximately 60 % of its
dry weight) and polysaccharides are a major bioactive component (Fu,
Shi, & Li, 2006; Maran, Mekala, & Manikandan, 2013). Reportedly, a
fundamental understanding of properties including molecular mass,
extraction method, monosaccharide composition, and type of glycosidic
linkage is pivotal in studying the biologically active mechanisms in
pumpkin (Li, Wu, Lv, & Zhao, 2013, 2019). Notably, the molecular mass
distribution affects polysaccharide properties, such as solubility and
viscosity. Unlike high- or medium-molecular-mass polysaccharides,
orally administered low-molecular-mass polysaccharides can be absor-
bed through the intestine into the blood stream, and have a systemic
biological effect (Du et al., 2011; Kurozumi, Kiyose, Noguchi, & Sato,
2019).
was composed of rhamnose, galacturonic acid, galactose, and arabinose
in a molar ratio of 7.4: 25: 28: 2.6. Song, Li, Hu, Ni, and Li (2011) iso-
lated a water-soluble heteropolysaccharide (LGPP2-1) with a molecular
mass of 10.1 kDa from Lady Godiva pumpkins (Cucurbita pepo Lady
Godiva), which mainly contained 1,6-disubstitued-α-galactopyranosyl
(33.33 %). However, low-molecular-mass pumpkin polysaccharides (<
1 × 104 Da) have rarely been investigated. In our previous study, a
certain amount of carbohydrates were found to remain in the waste
supernatant fraction after macromolecular polysaccharide precipitation,
which were hypothesized to be carbohydrates with lower molecular
mass (Liang, Liu, Yu, Song, & Li, 2019). Generally, the supernatant is
disposed of, and its potential for recycling is underutilized. Thus, it is of
our emphasis to extract, characterize a low-molecular-mass pumpkin
polysaccharide (SLWPP-3) in the waste supernatant fraction from the
macromolecular crude polysaccharide production.
Water-soluble pumpkin polysaccharides have attracted significant
attention due to their multiple pharmacological activities, such as hy-
poglycemic and antioxidant effects. For example, pumpkin
The isolation and characterization of macromolecular poly-
saccharides derived from pumpkin have been extensively investigated in
* Corresponding author at: College of Food Science and Nutritional Engineering, China Agricultural University, Qinghuadong Road No. 17, Haidian District,
Beijing, 100083, China.
E-mail addresses: pdlifei@163.com (F. Li), wei_yunlu@163.com (Y. Wei), gcfll@126.com (L. Liang), 454473661@qq.com (L. Huang), yuguoyong66@163.com
(G. Yu), quanhong_li@hotmail.com (Q. Li).
https://doi.org/10.1016/j.carbpol.2020.117090
Received 16 May 2020; Received in revised form 20 August 2020; Accepted 8 September 2020
Available online 15 September 2020
0144-8617/Published by Elsevier Ltd.

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
polysaccharides were found to exhibit good scavenging effects on su-
peroxide anions and hydroxyl radicals (Chen & Huang, 2018). The
antioxidant activity was reflected in its ability to increase GSH-Px and
SOD activities and reduce MDA levels in vivo (Chen, Long, Huang, &
Huang, 2020). The pumpkin (Cucurbita moschata) polysaccharide
(PPs-e) had the capacity to protect islet cell viability and lower the blood
glucose in allonxan-induced diabetic rats (Wang et al., 2017). Pumpkin
polysaccharides (ATPS-PPs) exerted significant protective effects
against STZ-induced islet β cells (Chen, Khan, Cheong, & Liu, 2019). To
the best of our knowledge, the antioxidant and hypoglycemic activities
of polysaccharides are strongly associated with structural characteris-
tics, among which the polysaccharide molecular mass plays a crucial
part (Xu et al., 2014). However, studies on the antioxidant activities and
hypoglycemic effects of the low-molecular-mass polysaccharides are
limited. In the present study, waste biomass from the supernatant frac-
tion after macromolecular pumpkin polysaccharide production was used
as the source of low-molecular-mass polysaccharides. Herein, the
structural properties and protective abilities of SLWPP-3 from pumpkin
against STZ-induced oxidative pancreatic β-cell redox imbalance, the
inhibitory activities of carbohydrate-hydrolyzing enzymes, and antiox-
idant activities in vitro were investigated. Our results are expected to
provide practical applications of the low-molecular-mass pumpkin
polysaccharide as a novel nutrient or food additive.
(CPP) was obtained.
LWPP was applied to a DEAE Cellouse-52 column (2.5 × 10 cm) and
then stepwise elution with distilled water (0.1, 0.3, 0.5, and 1.0 M NaCl)
at a flow rate of 1.0 mL/min was conducted to produce four fractions
(LWPP-1, LWPP-2, LWPP-3, and LWPP-4). Then, the main fraction
(LWPP-3) was filtered using 300 Da molecular mass membranes to
desalt. Then, LWPP-3 was further loaded into a Toyopearl HW-40 F gel-
filtration column (1.6 × 80 cm) with distilled water at a flow rate of 0.5
mL/min to obtain a purified pumpkin polysaccharide (SLWPP-3).
2.4. Characterization of SLWPP-3
2.4.1. Analysis of physicochemical characterization
Total carbohydrate content was analyzed using the phenol-sulfuric
acid method with D-glucose as the standard (Dubois, Gilles, Hamilton,
Rebers, & Smith, 1956). Uronic acid content was determined using the
m-hydroxybiphenyl colorimetric procedure with D-glucuronic acid as
the reference (Blumenkrantz & Asboe-Hansen, 1973). Protein content
was measured using the Coomassie Brilliant Blue G-250 method (Brad-
ford, 1976). Acetyl content was determined by hydroxylamine-ferric
trichloride method described by Notari and Munson (1969). Acetyl
content (%A) was obtained as the following equation:
[(V0 ꢀ Vn) × N × 43]
A(%) =
× 100
(1)
2. Materials and methods
M
2.1. Plant material
where V0 is the volume of hydrochloric acid consumed for the sample in
liters, Vn represents the volume of hydrochloric acid consumed for the
blank in liters, N stands for the normality of the hydrochloric acid, 43 is
the molecular weight of acetyl group and M is the mass of the sample in
grams.
Fresh mature pumpkins (Cucurbita moschata) were purchased at a
local commercial supermarket (Beijing, China) and selected for their
uniformity in shape, weight, and color. The crude samples were cleaned
thoroughly with tap water, peeled, deseeded, and cut into small pieces
(0.2 cm × 0.6 cm × 2.5 cm).
2.4.2. Homogeneity and molecular mass analysis
The homogeneity and molecular mass of SLWPP-3 were calculated
using high-performance gel- permeation chromatography (HPGPC) with
a Shimadzu LC-10A HPLC system equipped with a BRT102 column (7.8
2.2. Reagents
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethyl-
benzothiazoline-6-sulphonic acid) (ABTS), monosaccharide standards, a
mm × 30.0 cm) and a refractive index detector (RID). Sample (20 μL)
solution (5 mg/mL) was injected in each run, with ultrapure water as the
mobile phase at a flow rate of 0.8 mL/min (40 ℃). The molecular mass
of SLWPP-3 was analyzed by comparison with the retention times based
on the standard curve of a series of molecular mass standards (0.18,
0.342, 0.504, 0.828, 1.152, and 4.44 kDa).
series of molecular mass standards, α-amylase and α-glucosidase were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). DEAE cellulose-
52, Steptozozin (STZ) and 4‑nitrophenyl‑β‑D‑glucopyranoside (pNPG)
were obtained from Yuanye Biotechnology Inc. (Shanghai, China).
Roswell Park Memorial Institute (RPMI) 1640 and fetal calf serum (FBS)
were purchased from Gibco Life Technologies (Grand Island, NY, USA).
The MDA assay kit was obtained from Nanjing Jiancheng Bioengi-
neering Institute (Nanjing, China). Cell Counting Kit-8 (CCK-8), the SOD
assay kit was acquired from Beijing Solarbio Science & Technology Co.,
Ltd. (Beijing, China). Toyopearl HW-40 F resin was purchased from
TOSOH Co., Ltd. (Tokyo, Japan). The RINm5F cell line (rat pancreatic
carcinoma) was obtained from the American Type Culture Collection
(ATCC). All other reagents used were from Sinopharm Chemical Reagent
Beijing Co., Ltd. (Beijing, China) and were of analytical grade.
2.4.3. Monosaccharide composition determination
The monosaccharide composition of SLWPP-3 was evaluated using
GC–MS (Huang et al., 2014) with some modifications. SLWPP-3 (2 mg)
was hydrolyzed with 1 mL of 2 mol/L trifluoroacetic acid (TFA) at 120
ꢀ for 90 min, and the samples were reduced with NaBH4 and acetylated
using acetic anhydride. The alditol acetates were analyzed using GC–MS
(Shimadzu QP2010plus, Shimadzu Co., Kyoto, Japan) equipped with a
QP-2010 capillary column (30 m × 0.25 mm × 0.25 um). The temper-
ature program was as follows: increasing from 120 ◦C to 250 ◦C at 3
◦C/min and holding at 250 ◦C for 5 min. The temperatures of both the
injector and the detector were set at 250 ◦C. The flow rate was 1.0
mL/min and the carrier gas was nitrogen.
2.3. Extraction and purification of SLWPP-3
The samples were first crushed using a beater, the pumpkin purees
were then mixed with distilled water at a ratio of 1/4 (w/v) and placed
in a 90 ℃ water bath for 4 h with constant stirring. After centrifugation,
the supernatants were concentrated to a quarter of the original volume
by evaporation. Next, proteins in the extract were removed using Sevag
reagent (Sevag, Lackman, & Smolens, 1938). Ethanol (90 %) was added
to the solution and subsequently precipitated at 4 ℃ for 12 h. The su-
pernatants were obtained by centrifugation at 1000 rpm for 15 min,
dialyzed (membrane cut-off of 300 Da), and lyophilized. Then, the
low-molecular-mass crude polysaccharide (LWPP) was collected.
Furthermore, the macromolecular crude polysaccharide precipitate
2.4.4. Fourier transform infrared (FT-IR) spectrometric analysis
The infrared spectrum of SLWPP-3 was determined using a FT-IR
spectrophotometer (Tianjin Port East Science and Technology Devel-
opment Co., Ltd., Tianjin, China) at a wavelength range of 4000ꢀ 400
cmꢀ 1. Briefly, the samples (10 mg) and KBr (200 mg) were precisely
weighed and pressed into pellets, and the data collected with the FT-IR
650.
2.4.5. Methylation analysis
Prior to methylation, SLWPP-3 was reduced three times with CMC-
2

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
NaBD4 (Zhang, Liu, & Lin, 2014). The reduced sample was methylated
using the method of Needs and Selvendran (1993). Briefly, the sample
was dissolved in NaOH/DMSO and methylated with 2 mL CH3I.
Exhaustive methylation was determined based on the disappearance of
hydroxyl absorption (3200–3700 cmꢀ 1) on infrared spectroscopy. The
methylated sample was dissolved in 3 mL formic acid at 100 ◦C for 3 h
and then hydrolyzed with 3 mL trifluoroacetic acid (2 M) at 121 ◦C for 2
h. The hydrolysate was evaporated to dryness and the residue reduced
with NaBH4, neutralized with acetic acid, then acetylated by pyridine
and acetic anhydride, which was qualitatively and quantitatively
analyzed using a GC–MS system (Shimadzu GCMS-QP 2010) equipped
with a QP-2010 capillary column (30 m × 0.25 mm × 0.25 um). The
temperature program was as follows: initial column temperature was
120 ◦C for 2 min, followed by a programmed increase from 120 ◦C to 250
◦C at 3 ◦C/min (held for 5 min).
2.7.2. Establishment of a pancreatic β cell damaged model
The concentration of rat pancreatic β cells was seeded in 96-well
microplates at a density of 5 × 104 cells/well. Different STZ concen-
trations (0, 1, 3, 4, 6, 7, and 9 mmol/L) were added to the cells for 2 h.
The cell viability was determined based on the CCK-8 assay.
2.7.3. Cell viability analysis
RINm5F cells (100
μ
L) were diluted to 5 × 104 cells /well in the 96-
well plate at 37 ◦C for 12 h and divided into four groups: Group A
included the normal cells; Group B consisted of cells treated with STZ
and was the diabetic control group; Group C consisted of cells pre-
treated with the low, middle, or high dose of SLWPP-3 (200, 500, or
900 μg/mL, respectively) followed by treatment with STZ (5 mmol/L);
Group D included cells pre-incubated with different concentrations of
CCP (200, 500, or 900 μg/mL) followed by treatment with STZ (5 mmol/
L). Cell viability was estimated using the CCK-8 assay following the
2.4.6. NMR analysis
manufacturer’s instructions.
SLWPP-3 (20 mg) was dissolved into 0.5 mL of D2O with constant
stirring for 2 h at room temperature, and then freeze-dried. This
deuterium-exchanged procedure was repeated three times. After that,
the polysaccharides were re-dissolved in 0.5 mL D2O, and then trans-
ferred into 5 mm NMR tube for testing. High-resolution 1H and 13C NMR
spectra were documented at 600 and 150 MHz, respectively, with the
operating temperature of 25 ℃ using a Bruker AV-600 spectrometer
2.7.4. Determination of SOD activities and MDA levels
SLWPP-3 and macromolecular crude polysaccharide were investi-
gated for protective effects against oxidative stress in cells. RINm5F cells
(100
μ
L) were diluted to 3 × 105 cells /well. The experimental groups
were the same as described in Section 3. The SOD content of cells was
determined using the xanthine oxidase method with a commercial kit.
The intracellular lipid peroxidation products were measured using thi-
obarbituric acid (TBA) and tested with a commercially available kit.
(Brucker, Rheinstetten, Germany) and a 5 mm inverse geometry 1H/13
C
probe. 2D spectra (COSY, HSQC and HMBC) were recorded according to
the standard Bruker pulse sequences (Zhao et al., 2015). The chemical
shifts were provided in ppm, using the internal acetone-D6 signal (δ
2.8. Statistical analysis
H
=2.09 ppm) for 1H and the internal acetone-D6 signal (δC =30.89 ppm)
for 13C as references. Analysis of data was carried out using MestRe Nova
5.3.0 (Mestrelab Research S.L.) software.
The data were expressed as means ± standard deviation (SD) and
statistical analyses were performed using ANOVA with SPSS 21.0 soft-
ware. A p-value < 0.05 was considered statistically significant.
2.5. Analysis of antioxidant properties
3. Results and discussion
The DPPH and ABTS radical scavenging capacities were assessed
according to the methods of Gao et al. (2015) and Jeddou et al. (2016),
respectively. Samples with different concentrations (0.2–1.0 mg/mL)
were prepared and analyzed. Vc (0.2–1.0 mg/mL) was used as the
positive control. The percentage of DPPH and ABTS radical scavenging
activities was calculated as follows:
3.1. Characterization of SLWPP-3
3.1.1. Physicochemical compositions of SLWPP-3
The isolation and purification flow diagram is shown in Fig. 1A. After
purification on the DEAE-cellouse-52 column (2.6 × 10 cm), LWPP was
separated into four fractions (Fig. 1B). Then, LWPP-3 with a yield of 48.6
%, the most abundant component of LWPP, was further purified with the
Toyopearl HW-40 F gel-filtration column (1.8 × 80 cm) to obtain the
pure subfraction SLWPP-3 (Fig. 1C) with a yield of 88.9 % (Table 1). The
total neutral sugar content of SLWPP-3 was approximately 37.8 % based
on the phenol-sulfuric acid method (Table 1) and the absence of proteins
was determined based on the Bradford’s method. The UV–vis spectra
demonstrated that SLWPP-3 had no absorption peaks at 260 and 280 nm
(Fig. 1F), which further indicated the absence of proteins and nucleic
acids. The percentage of the acetyl content (10.2 %) in SLWPP-3 was
determined based on the hydroxylamine-ferric trichloride method. The
high percentage of uronic acid (53.8 %) in SLWPP-3 indicated that it
could be a member of the pectin family.
Scavenging rate (%) = (A0-A1)/A0 × 100
(2)
where A0 is the absorbance of the mixture without sample, and A1 is the
absorbance of the sample.
2.6. Analysis of
α-glucosidase and α-amylase
The hypoglycemic potential of SLWPP-3 was estimated by deter-
mining its inhibitory activities of α-glucosidase and α-amylase using the
methods of Cardullo et al. (2019) and Nampoothiri et al. (2011),
respectively. Acarbose was used as the positive control. Serial dilutions
of SLWPP-3 (0.2-2.0 mg/mL) were texted in the two experiments. The
percentage of inhibition was calculated using the following equation:
3.1.2. Homogeneity and molecular mass
Inhibitory activity (%) = (A0-A1)/A0 × 100
(3)
The homogeneity was confirmed by elution using the high-
performance gel permeation chromatography (HPGPC) to obtain a sin-
gle symmetrical peak at 7.01 min (Fig. 1D). As shown in Fig. 1E, the
calibration equation obtained using different molecular mass carbohy-
drates was as follows: log M = ꢀ 0.320x + 5.797 with a correlation co-
efficient of 0.9915 (M: the molecular mass of SLWPP-3; x: the retention
time); the molecular mass of SLWPP-3 was estimated to be 3.5 kDa.
where A0 is the absorbance of the mixture without sample, and A1 is the
absorbance of the sample.
2.7. Protective effects against STZ-induced toxicity
2.7.1. Cell culture
RINm5F pancreatic β cells were cultured in complete medium
(RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS),
3.1.3. Monosaccharide composition
100 U/mL penicillin, 100
μ
g/mL streptomycin), and incubated in a
Based on GC–MS analysis, SLWPP-3 contained rhamnose, glucose,
arabinose, galactose and uronic acid with a weight ratio of 1: 1: 4: 6: 15
humidified 5 % CO2 incubator at 37 ◦C.
3

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
Fig. 1. The procedure of SLWPP-3 extraction and purification (A); Elution profile of LWPP-3 on DEAE-cellulose-52 column (B); Elution profile of SLWPP-3 on
Toyopearl HW-40 F gel column (C); HPGPC chromatography of SLWPP-3 (D); Calibration curve of standards with different molecular mass (E); UV–vis spectrum of
SLWPP-3 (F).
(Table 1). The SLWPP-3 monosaccharide composition was quantita-
tively similar to peptic polysaccharide (PPc) obtained from pumpkin
(Cucurbita moschata) via a low-temperature alkali treatment and a
combination of gradual alcohol precipitation and ion-exchange with a
higher amount of galactose. However, the PPc molecular mass has been
estimated to be 20.3 kDa (Zhao et al., 2017) and may be associated with
the method of polysaccharide extraction and purification, which
significantly affects the structural properties and bioactivities, including
the molecular mass of polysaccharides (Chaves, Iacomini, & Cordeiro,
2019).
3.1.4. FT-IR analysis
The IR spectrum of SLWPP-3 is shown in Fig. 2. A broad and intense
peak at 3397 cmꢀ 1 was derived from the stretching vibration of O H,
–
4

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
→4)-GalpA-(1→, →3,6)-Galp-(1→, →3)-Galp-(→, and Galp-(1→,
respectively, in which →4)-GalpA-(1→ might be the main linkage of
SLWPP-3.
Table 1
Yield and chemical composition of SLWPP-3.
Parameters
SLWPP-3
Yield/%
48.6 ± 1.5a
88.9 ± 4.5b
37.8 ± 1.2
3.1.6. NMR spectrum analysis
The 1H, 13C, COSY, HSQC, and HMBC NMR spectra of SLWPP-3 at
600-MHz were investigated. As shown in Fig. 3A and B, most SLWPP-3
proton and carbon signals were obtained from the regions ranging from
δ 3.0–5.50 ppm and δ 60ꢀ 110 ppm, respectively, which were the
emblematic chemical shifts of polysaccharide (Cai, Zou, Liang, & Luan,
2018). In addition, six anomeric proton and carbon signals at δ
5.01/101.78 ppm, 4.55/97.58 ppm, 5.07/101.08 ppm, 5.16/110.71
ppm, 4.47/104.85 ppm, 5.01/108.79 ppm, 4.95/98.31 ppm, 5.24/93.56
ppm, 4.88/100.74 ppm, and 4.47/104.85 ppm were determined in the
1H and 13C NMR and HSQC spectra in Fig. 3A–C and denoted as A, B, C,
D, E, F, G, H, I, and J in SLWPP-3, respectively, indicating that SLWPP-3
Total neutral sugars/%
Monosaccharides Weight ratio
Rhamnose
1
Glucose
1
Arabinose
4
Galactose
6
Uronic acid
15
a
Relative to LWPP.
b
Relative to LWPP-3.
which was the typical peak of carbohydrate. In addition, the charac-
teristic absorption at 2933 cmꢀ 1 was associated with the bending vi-
bration of C–H of the methyl group in the sugar ring (Wang et al., 2019).
The shape peaks at 1623 cmꢀ 1 and 1749 cmꢀ 1 were attributed to the
carboxylate ion (COOꢀ ) stretching band and the methyl-esterified
carbonyl groups (COOꢀ CH3), respectively, confirming the presence of
uronic acid (Zuzana, Zdenka, & Anna, 2013). According to Singthong,
Cui, Ningsanond, and Douglasgoff (2004), the degree of esterification
(DE) can be evaluated based on its linear correlation with the value of
contained both α- and β-configurations (Zhu et al., 2019), which was in
accordance with the result of FT-IR spectroscopy.(Wang, Li et al., 2017)
The carbon signals at δ 54.25 ppm in the 13C NMR spectrum and the
proton signal at δ 3.72 ppm in the 1H NMR spectrum revealed the
presence of a methoxyl ester group in SLWPP-3. The δ 3.72/172.12 ppm
signals in the HMBC spectrum further proved the presence of the
methoxyl ester group. The carbon signals at δ 174.56 ppm and δ 21.67
ppm in the 13C NMR spectrum and the proton signal at δ 1.99 ppm in the
1H NMR spectrum revealed the presence of the acetyl group in SLWPP-3
(Wang et al., 2020; Wang, Li et al., 2017). The δ 1.99/174.56 ppm and δ
3.72/174.56 ppm signals in the HMBC spectrum, and the peaks at δ
1.99/21.6 ppm and δ 3.71/69.35 (BH2/BC2) ppm in the HSQC spectrum
revealed that the acetyl group was linked to a carbon with a chemical
shift of δ 69.35 ppm, which was C-2 chemical shift of residue C. The level
of O-acetylation could be calculated as 11.8 % using the methyl and
anomeric proton peak area ratios in the 1H-NMR spectrum (Fig. 3A),
which were from the acetyl group and the monosaccharide residues,
respectively (Yuan et al., 2020). Based on the monosaccharide compo-
sition and methylation analysis, the downfield signals at δ 170ꢀ 176 ppm
in the 13C NMR spectrum (Fig. 3B) proved the presence of the carboxyl
carbon of GalpA. As shown in the HSQC spectrum (Fig. 3C), the
anomeric signals that appeared at δ 5.01/101.78 ppm and 4.55/97.58
ppm in the HSQC spectrum were attributed to the H1/C1 of residue A
and residue C, respectively. The other proton chemical shifts of A and C
were obtained from the COSY spectrum (Fig. 3D), and the corresponding
13C signals were identified based on the HSQC spectrum (Fig. 3C). As
shown in Fig. 3E, the residue A showed intra-residue contacts of H-5
protons (δ 5.08 ppm) with the carboxyl carbon at δ 172.12 ppm (C-6)
correlating further with protons of the methoxyl group (δ 3.72 ppm); the
residue C units showed intra-residue contacts of H-5 protons (δ 4.83
ppm) with the carboxyl carbon at δ 175.51 ppm (C-6); Thus, the signals
A
1749/(A1749 + A1623) (here A refers to peak area). In the present study,
the DE value of SLWPP-3 was calculated to be 37.9 %. The ratio of -OMe
and -Ac could also be calculated as 4:1 by the degree of esterification
(37.9 %): the acetyl content (10.2 %). The absorption peak at around
1241 cmꢀ 1 also confirmed the appearance of C–H. In the range of
1200ꢀ 1000 cmꢀ 1, banks at 1149 cmꢀ 1, 1095 cmꢀ 1, and 1012 cmꢀ 1
–
–
were assigned to the C OH side groups and COC glycosidic bond
vibrations, indicating the presence of pyranose (Lu et al., 2019). In
addition, the weak bands near 895 cmꢀ 1 and 831 cmꢀ 1 demonstrated
that glucosyl residues could be present in both β- and α-configurations.
Furthermore, all peaks of SLWPP-3 were in accordance with the result of
pectin FT-IR spectroscopy (Fig. 2). These results further verified that
SLWPP-3 was a polysaccharide and may be the pectin with a low degree
of esterification.
3.1.5. Methylation analysis
The glycosidic linkage and molar ratios of sugar residues determi-
nation from SLWPP-3 was performed by methylation analysis (Table 2).
As summarized in Table 2, the PMAA derivatives of SLWPP-3 were
measured to be 2,3,5-Me3-Araf, 2,3-Me2-Araf, 3-Me1-Rhap, 2,3,6-Me3-
Galp, 2,4-Me2-Galp, 2,4,6-Me3-Galp and 2,3,4,6-Me4-Galp with the
molar of 4.3: 12.8: 4.61: 52.87: 4.96: 14.3: 5.76. As shown in 3.1.1, the
total uronic acid content was 53.8 % by weight, which therefore were
the dominate fraction in SLWPP-3. In addition, the content of galactose
increased after uronic acid reduction, indicating the presence of →4)-
GalpA-(1→ (Ru et al., 2019). This result suggested that SLWPP-3 mainly
contained seven linkages: Araf-(1→, →5)-Araf-(1→, →2,4)-Rhap-(1→,
of the C-6 of unesterified and esterified α-D-GalpA units were assigned at
δ 175.51 ppm and δ 172.12 ppm, respectively. The complete 1H and 13
C
chemical shifts for residues A and C are shown in Table 3, and were
consistent with the literature (Lin, Ji, Wang, Yin, & Peng, 2019), while
the downfield resonances of the C-4 of residue A and residue C at δ
79.94/80.05 ppm confirmed that residues
A
and
C
were
→4)-α-GalpA-(1→ (OMe) and →4)-α-2-O-ace-GalpA-(1→, respectively.
The β-configuration of residue B was confirmed based on the
anomeric chemical shifts at δ 4.55/97.58 ppm. The other proton
chemical shifts of H-2 (δ 3.45 ppm), H-3 (δ 3.71 ppm), H-4 (δ 4.16 ppm),
H-5 (δ 3.93 ppm), H-6a (δ 3.67 ppm), and H-6b (δ 3.78 ppm) were
successfully obtained from the COSY spectrum (Fig. 3D). Accordingly,
the 13C assignments of residue B were obtained from the HSQC spectrum
(Fig. 3C) and are shown in Table 3. Comparison of proton and carbon
resonances with the literature data (An et al., 2019) and the download of
the C-4 (δ 82.29 ppm) allowed assigning residue B to →4)-β-D-Galp.
The
α-configuration of residue D was confirmed based on the
anomeric chemical shifts at δ 5.16/110.71 ppm. The other proton signals
of residue D (from H-2 to H-6a) were δ 4.14 ppm, 3.87 ppm, 4.09 ppm,
Fig. 2. IR spectrum of SLWPP-3.
5

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
Table 2
Methylation analysis data for SLWPP-3.
Retention time
Methylated sugars
Mass fragments (m/z)
Molar ratio
Type of linkages
9.739
2,3,5-Me3-Araf
2,3-Me2-Araf
43,71,87,101,117,129,145,161
4.3
Araf-(1→
15.005
17.692
19.035
20.926
21.457
29.669
43,71,87,99,101,117,129,161,189
43,71,87,101,117,129,145,161,205
43,87,101,117,129,143,159,189
43,87,99,101,117,129,161,173,233
43,87,99,101,113,117,129,131,161,173,233
43,87,117,129,159,189,233
12.8
5.56
4.61
14.3
52.87
4.96
→5)-Araf-(1→
Galp-(1→
2,3,4,6-Me4-Galp
3-Me1-Rhap
→2,4)-Rhap-(1→
→3)-Galp-(1→
→4)-GalpAa /Galp-(1→
→3,6)-Galp-(1→
2,4,6-Me3-Galp
2,3,6-Me3-Galp
2,4-Me2-Galp
a
Dominate percentage in the mixture of 4-GalpA and 4-Galp.
3.76 ppm, and 3.65 ppm, respectively, based on the adjacent correlation
in the COSY spectrum (Fig. 3D), and the corresponding carbon signals
obtained using the HSQC spectrum (Fig. 3C) were δ 82.69 ppm, 77.90
ppm, 85.34 ppm, and 62.60 ppm for C-2, C-3, C-4, and C-5, respectively.
In addition, the complete 1H and 13C chemical shifts of residue D were
consistent with the literature (Li et al., 2020), confirming that residue D
A, O-1 of residue E was linked to C-3 of residue J, and O-4 of residue B
was linked to C-1 of residue C, respectively. Hence, a hypothetical
structure for SLWPP-3 was proposed as Fig. 3F, considering the
comprehensive results of monosaccharide compositions, methylation
analysis, and NMR analyses.
was α-L-Araf-(1→.
3.2. Antioxidant activities of SLWPP-3 in vitro
The anomeric proton and carbon shifts at δ 4.47/104.85 ppm in the
HSQC spectroscopy were attributed to H-1 and C-1, indicating that
residue E was a β-configuration residue. Based on the COSY spectrum
(Fig. 3D), the chemical shifts of H-2 to H-6a/b were determined, and the
corresponding carbon signals was obtained from the HSQC spectrum
(Fig. 3C). The complete 1H and 13C chemical shifts for residues E and J
were shown in Table 3. Based on the literature (Li et al., 2020), the
proosed unit of residues E and J were →3,6)-β-D-Galp-(1→ and
→3)-β-D-Galp-(1→, respectively.
In this study, the DPPH and ABTS radical scavenging capacities of
SLWPP-3, macromolecular crude polysaccharide and a positive control
(Vc) were investigated and the results are shown in Fig. 4.
All tested samples exhibited a concentration-dependent relationship
within the tested dosage range (0.2–1.0 mg/mL), and SLWPP-3
(17.5–43.7 %) displayed significantly stronger inhibitory effects than
the macromolecular crude polysaccharide (1.1–26.7 %), which was
significantly lower than the positive control acarbose (77.27–94.30 %)
as shown in Fig. 4. Furthermore, Wang, Liu et al. (2017) isolated a
polysaccharide with an average molecular mass of 21,100 Da from
Cucurbita moschata seeds with an inhibitory rate of 26.3 % at 1.0 mg/mL,
which was weaker than SLWPP-3 (43.7 %) at the same mass. As shown
in Fig. 4B, when the tested dosages were between 0.2 mg/mL and 1.0
mg/mL, the scavenging activities of all tested samples were
dose-dependent, and the SLWPP-3 scavenging rate (13.2–26.5 %)
increased more rapidly than did that of the macromolecular crude
polysaccharide (4–11.6 %), but showed a dramatically flat trend
compared with the Vc (66.4–92.8 %). The results of the two assays
revealed that SLWPP-3 exhibited better antioxidant potential compared
with the macromolecular polysaccharide at the same mass, which may
be associated with the SLWPP-3 molecular mass, including more
reducing terminal ends per unit mass for its low molecular mass (Yuan
et al., 2020).
The anomeric proton and carbon signals at δ 5.01/108.79 ppm were
described as H-1 and C-1, inferring residue F was the α-configuration
residue. All the chemical shifts from H-2 to H-6a were obtained from the
COSY spectrum (Fig. 3D), including H-2 (δ 3.93 ppm), H-3 (δ 3.83 ppm),
H-4 (δ 4.04 ppm), H-5 (δ 4.22 ppm), and H-6a (δ 4.29 ppm). Further-
more, the matching 13C chemical shifts were C-2 (δ 80.38 ppm), C-3 (δ
76.41 ppm), C-4 (δ 82.29 ppm), and C-5 (δ 66.93 ppm) in the HSQC
spectrum (Fig. 3C). The complete 1H and 13C chemical shifts for residue
F were consistent with the literature (Qin et al., 2019) and the downfield
shift in the C-5 of residue F at δ 80.14 ppm indicated that residue F was
→5)-
The cross peak at δ 1.19/16.79 ppm in the HSQC spectrum (Fig. 3C)
corresponds to H6/C6 (methyl group) of →2,4)- -L-Rhap-(1→. The cross
peaks at δ 5.24/93.56, 4.88/100.74 ppm (Fig. 3C) assigned as to H1/C1
of →4)- -D-Glcp, and -D-Galp-(1→, respectively. The complete proton
α-L-Araf-(1→.
α
α
α
chemical shifts of G, H, and I were obtained from the COSY (Fig. 3D)
spectrum, and the corresponding 13C signals were identified in the HSQC
spectrum (Fig. 3C). Their all signals were assigned by comparison with
the literature (Qin et al., 2019; Wu, Luo, Yao, & Yu, 2020). The NMR
data were summarized in Table 3.
3.3. Results of
α-glucosidase and α-amylase inhibition activities
The major strategy for decreasing postprandial hyperglycemia was
the inhibition of carbohydrate-hydrolyzing enzymes such as α-glucosi-
The glycosidic linkage sequence determined among the SLWPP-3
residues based on the correlation peaks obtained in the HMBC spec-
trum (Fig. 3E). As observed, the correlation peak between residue F (H-
5) and residue D (C-1) at δ 4.22/110.71 ppm indicated the presence of
dase and a-amylase (Zhang, Zhao, Shang, Guo, & Chen, 2020). There-
fore, the inhibitory activities of SLWPP-3 and macromolecular crude
polysaccharide against α-glucosidase and α-amylase were evaluated in
vitro to determine their hypoglycemic properties. As shown in Fig. 5A,
all tested samples displayed inhibitory activity against -glucosidase in a
α
-L-Araf-(1→5)-α-L-Araf-(1→. Similarly, the cross-peaks at δ 5.01/80.38
α
ppm (FH-1/EC-3), 4.47/79.94 ppm (EH-1/AC-4; JH-1/AC-4), δ 5.01/
80.05 ppm (AH-1/CC-4), δ 5.07/69.17 ppm (CH-1/EC-6), δ 4.95/69.17
ppm (GH-1/EC-6), δ 5.01/66.93 ppm (FH-1/FC-5), δ 4.88/66.93 ppm
(IH-1/FC-5), δ 5.01/77.99 ppm (FH-1/GC-4), δ 5.07/79.94 ppm (CH-1/
AC-4), δ 101.08/4.16 ppm (CC-1/BH-4), δ 4.47/82.29 ppm, (EH-1/JC-
3), and δ 101.08/3.48 ppm (CC-1/HH-4) showed O-5 of residue E was
linked to C-1 of residue D, O-1 of residue F was linked to C-3 of residue E,
O-1 of residue E was linked to C-4 of residue A, O-1 of residue J was
linked to C-4 of residue A, O-1 of residue A was linked to C-4 of residue
C, O-1 of residue C was linked to C-6 of residue E, O-1 of residue G was
linked to C-6 of residue E, O-1 of residue F was linked to C-5 of residue F,
O-1 of residue I was linked to C-5 of residue F, O-1 of residue F was
linked to C-4 of residue G, O-1 of residue C was linked to C-4 of residue
dose-dependent manner; however, the positive control, acarbose,
showed significantly better inhibitory ability than SLWPP-3 and the
macromolecular crude polysaccharide. The inhibitory capacities of
SLWPP-3 and the macromolecular crude polysaccharide were 17.8 %
and 11.7 %, respectively, at a concentration of 2.0 mg/mL, which was
higher than SCP, a polysaccharide isolated from sweet corncobs that
exhibited inhibitory rates of 10.5 % at 6 mg/mL (Ma, Wang, Gao, &
Zhang, 2016)). The results showed that the inhibitory effects on
α
-glucosidase were in the decreasing order of SLWPP-3 > macromo-
lecular crude polysaccharide > SCP.
The α-amylase inhibitory rates of SLWPP-3, macromolecular crude
polysaccharide, and the positive control (acarbose) in the 0.2–2.0 mg/
mL range are shown in Fig. 5B. The inhibitory effects of the tested
6

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
Fig. 3. 1H spectrum (A); 13C spectrum (B); HSQC spectrum (C); COSY spectrum (D); HMBC spectrum (E); Predicted repeating unit of SLWPP-3 (F).
samples against
α
-amylase improved as the analyzed dosage increased,
effect of several parameters, such as M and monosaccharide composi-
tion. Thus, the high content of galactose, arabinose, and galacturonic
acid, and low M content in SLWPP-3 could enhance the binding of
and the strongest inhibition (19.3 % for macromolecular crude poly-
saccharide, 30.4 % for SLWPP-3, and 94.9 % for acarbose) was at the
highest tested concentration (2 mg/mL). The polysaccharide F3 from
polysaccharides to
α-glucosidase and α-amylase (Deng et al., 2020;
Chaenomeles speciosa seeds (Deng et al., 2020), which showed
α-amylase
Zhang & Li, 2015).
inhibition of 20.2 % at 2 mg/mL concentration, exhibited better inhib-
itory capacities than the macromolecular crude polysaccharide, but was
evidently weaker than SLWPP-3. Based on these results, SLWPP-3
exerted a better in vitro effect on hypoglycemia, and the functional ac-
tivities of polysaccharides were associated with the comprehensive
3.4. Protective effects against STZ-induced toxicity
3.4.1. Effects of STZ on the viability of pancreatic β cells
STZ is toxin the most commonly used as diabetes model. The
7

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
Table 3
1H and 13C NMR chemical shifts (ppm) of SLWPP-3 in D2O.
Chemical shift δ H/C (ppm)
Glycosyl residues
H1/C1
5.01
H2/C2
3.68
H3/C3
3.93
H4/C4
4.18
H5/C5
4.83
H6a/C6
H6b
3.78
COOCH3
3.72
O2CCH3
→4)-
α-GalpA-(1→(OMe)
H
A
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
101.78
4.55
69.76
3.45
69.76
3.71
79.94
4.16
72.69
3.93
172.12
3.67
54.25
→4)-β-D-Galp
B
97.58
5.07
71.44
3.71
72.38
3.93
82.29
4.34
66.77
5.08
62.41
→4)-
α-GalpA-(1→
1.99
C
101.08
5.16
69.35
4.14
69.45
3.87
80.05
4.09
71.91
3.76
175.51
3.65
21.67
α
-L-Araf-(1→
D
110.71
4.47
82.69
3.57
77.90
3.65
85.34
4.04
62.60
3.87
→3,6)-β-D-Galp-(1→
3.7
3.8
3.8
E
104.85
5.01
71.70
3.93
80.38
3.83
69.76
4.04
74.28
4.22
69.17
4.29
→5)-
α-L-Araf-(1→
F
108.79
4.95
80.38
3.66
76.41
4.04
82.29
3.65
66.93
3.14
→2,4)-
α-L-Rhap-(1→
1.21
G
98.31
5.24
80.38
3.71
66.75
3.55
77.99
3.48
62.25
3.70
16.79
3.93
→4)-
α-D-Glcp
H
93.56
4.88
72.38
3.68
71.44
3.46
74.28
3.87
70.4
66.93
3.93
α
-D-Galp-(1→
4.09
I
100.74
4.47
67.11
3.55
72.38
3.65
76.4
74.28
3.83
62.41
3.7
→3)-β-D-Galp-(1→
4.09
J
104.85
71.72
82.29
69.26
73.29
62.41
Fig. 4. DPPH radical scavenging activity (A) and scavenging ABTS radical activity (B).
Fig. 5. Inhibition rates of SLWPP-3 on α-glucosidase activity (A) and α-amylase (B).
mechanism of STZ-induced β-cell toxicity relies on the generation of
both ROS and RNS, causing pancreatic cell dysfunction and death
(Al-Nahdi, John, & Raza, 2018). To measure STZ toxicity, RINm5F cells
were treated with various concentrations of STZ (0, 1, 3, 4, 5, 7, or 9
mM) for 24 h. Cell viability was assessed using the CCK-8 kit (Fig. 6). The
number of viable cells gradually decreased with increasing STZ con-
centration (p < 0.01). Treatment with 5.0 mM STZ induced cell death of
approximately 50 % compared with no STZ treatment, and this
concentration was used to investigate the efficacy of SLWPP-3 in the
following experiments.
3.4.2. Effects of SLWPP-3 on the viability of pancreatic β cells
Cell treated with SLWPP-3 and macromolecular crude poly-
saccharide (200, 500, or 900
μg/mL) exhibited that there were no
toxicity and had obvious protective effect on STZ-induced pancreatic
RINm5F cells (Fig. 7A). The survival of STZ-induced RINm5F cells
8

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
p < 0.05), and SLWPP-3 and macromolecular crude polysaccharide
showed superior protective ability. Among the groups, pre-treatment
with SLWPP-3 or macromolecular crude polysaccharide (500
μg/mL
or 900 μg/mL, respectively) resulted in a significant decline in contents
of MDA compared with the STZ control group (2.09, 1.92, 1.74, and
1.71; p < 0.01 and p < 0.05, respectively). Consequently, SLWPP-3 was
superior to macromolecular crude polysaccharide in promoting SOD
activities, which was in agreement with the in vitro antioxidant results.
4. Conclusion
Compared with previous studies, this paper focused on the elaborate
structure and biological activities of a novel natural low-molecular-mass
acidic polysaccharide from pumpkin in the waste supernatant fraction
after macromolecular polysaccharide precipitation. The hetero-
polysaccharide SLWPP-3 mainly consisted of galacturonic acid, galac-
tose, arabinose, rhamnose, and glucose. SLWPP-3 had a molecular mass
of 3.5 kDa and its backbone is likely composed of →4)-
(OMe), →4)- -GalpA-(1→, →3)-β-D-Galp-(1→, →2,4)-
→3,6)- β-D-Galp-(1→, →4)-
α-GalpA-(1→
Fig. 6. Effects of different concentrations of STZ on the viability of pancreatic
cells. n = 5. ##p < 0.01 vs no STZ treatment.
α
α-L-Rhap-(1→,
α
-D-Glcp, and →4)-β-D-Galp residues in the
backbone. The branch chain passes were connected to the main chain
through the O-4 atom of glucose and O-3 atom of arabinose. The
decreased to 41 % compared with normal control, whereas cells
increased significantly on co-treatment with SLWPP-3 and macromo-
lecular crude polysaccharide in tested doses (p < 0.01, p < 0.05; Fig. 7B).
branches were present as →5)-α-L-Araf-(1→, α-L-Araf-(1→, and α-D-Galp-
(1→ stretched from the O-3 position of→3,6)-β-D-Galp-(1→ and O-4
position of →2,4)- -L-Rhap-(1→. Acetyl groups were detected to be
connected to→4)- -D-GalpA-(1→ but not→4)- -D-GalpA -(1→(OMe). In
In particular, when at the concentration of 900
μg/mL, SLWPP-3/
α
macromolecular crude pumpkin polysaccharide along with STZ treat-
ment increased significantly cell viability to 51 %/46.6 %, respectively
(p < 0.01). Our present study displayed that protective effect by treating
with SLWPP-3 was relatively better.
α
α
addition, SLWPP-3 exhibited antioxidant activities in a dose-dependent
manner. Noticeable hypoglycemic activities were observed when the
inhibitory rates of two carbohydrate digesting enzymes were measured
and the protective effect in RINm5F pancreatic cells was evaluated. The
results of this study elucidate the structural types of pumpkin poly-
saccharides and illuminated that SLWPP-3 might be used as a potential
food supplement and in the pharmaceutical industry. However, the
complete structure-activity relationship remains unclear and future in-
depth studies should be conducted to clarify this association.
3.4.3. SLWPP-3 cellular antioxidant activity
STZ-induced diabetes leads to increased MDA levels and decreased
SOD activities (Subash-Babu, Alshatwi, & Ignacimuthu, 2014). The ef-
fects of SLWPP-3 on SOD activities and MDA levels in STZ-treated
RINm5F pancreatic β cells are shown in Fig. 8A and B. Compared with
cells in the normal group, cells in the STZ group exhibited an apparent
decrease in SOD activities (9.53 vs 14.04; p < 0.01). Conversely, MDA
levels were significantly higher in the STZ group compared with the
normal group (2.54 vs 1.57; p < 0.01). SOD values increased and MDA
values decreased with increased doses of SLWPP-3 and macromolecular
crude polysaccharide (Fig. 8A and B). After the cells were treated with
CRediT authorship contribution statement
Fei Li: Conceptualization, Methodology, Formal analysis, Writing -
original draft, Writing - review & editing. Yunlu Wei: Data curation,
Writing - original draft. Li Liang: Writing - review & editing. Linlin
Huang: Supervision, Resources. Guoyong Yu: Investigation, Valida-
900
μg/mL of either SLWPP-3 or macromolecular crude polysaccharide,
the SOD activities significantly increased (13.71 and 12.33; p < 0.01 and
tion.
Quanhong
Li:
Supervision,
Funding
acquisition,
Fig. 7. Effects of SLWPP-3 on the viability of STZ-treated pancreatic cells. n = 5; #p < 0.05 vs normal control group, ##p < 0.01 vs normal control group, *p < 0.01 vs
STZ group, **p < 0.01 vs STZ group, a p < 0.05 vs CPP group at the same concentration. CPP: macromolecular crude polysaccharide.
9

F. Li et al.
Carbohydrate Polymers 251 (2021) 117090
Fig. 8. Effects of SLWPP-3 on SOD activities (A) and MDA levels (B) in STZ-treated pancreatic cells. n = 5; ##p < 0.01 vs normal control group, **p < 0.01 vs STZ
group, *p < 0.05 vs STZ group, bp < 0.01 vs CPP group at the same concentration. CPP: macromolecular crude polysaccharide.
pulp and their prebiotic activities. International Journal of Food Science & Technology,
Conceptualization, Writing - review & editing.
46(5), 982–987.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chemistry, 28
Declaration of Competing Interest
(3), 350–356.
Fu, C. L., Shi, H., & Li, Q. H. (2006). A review on pharmacological activities and
The authors declare no conflict of interest.
utilization technologies of pumpkin. Plant Foods for Human Nutrition, 61(2), 73–80.
Gao, J., Zhang, T., Jin, Z. Y., Xu, X. M., Wang, J. H., & Zha, X. Q. (2015). Structural
characterisation, physicochemical properties and antioxidant activity of
polysaccharide from Lilium lancifolium Thunb. Food Chemistry, 169, 430–438.
Huang, F., Zhang, R. F., Yi, Y., Tang, X. J., Zhang, M. W., Su, D. X., et al. (2014).
Comparison of physicochemical properties and immunomodulatory activity of
polysaccharides from fresh and dried litchi pulp. Molecules, 19(4), 3909–3925.
Jeddou, K. B., Chaari, F., Maktouf, S., Nouri-Ellouz, O., Helbert, C. B., & Ghorbel, R. E.
(2016). Structural, functional, and antioxidant properties of water-soluble
polysaccharides from potatoes peels. Food Chemistry, 205, 97–105.
Acknowledgement
This project was supported by the opening fund of Beijing Advanced
Innovation Center for Food Nutrition and Human Health.
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