Isolation, purification, characterization and antioxidant activity of polysaccharides from the stem barks of Acanthopanax leucorrhizus

Article abstract of DOI:10.1016/j.carbpol.2018.05.028

A novel water-soluble polysaccharide (named ALP-1) was successfully isolated from the stem barks of Acanthopanax leucorrhizus by hot-water extraction, and further purified by Cellulose DEAE-52 and Sephadex G-100 chromatography. The structure of ALP-1 was characterized by HPLC, HPGPC, partial acid hydrolysis, periodate oxidation, Smith degradation, methylation, together with UV, IR and NMR spectral analysis. The antioxidant activities also were evaluated in vitro. Structural analysis revealed that ALP-1 was a homogeneous galactan with the average molecular weight of 169 kDa, composed of galactose, glucose, mannose and arabinose in a molar ratio of 6.1:2.1:1.1:1.0, owning a backbone structure of 1,6-linked α-D-Galp residues with some branches of α-D-Manp-(1 → 3)-α-L-Araf residues at O-3 and α-D-Galp residues at O-4 of 1,6-linked α-D-Galp. Antioxidant assay showed that ALP-1 exhibited strong DPPH[rad] and HO[rad] scavenging activities, as well as ferric-reducing antioxidant power. These results provide a scientific basis for the further use of polysaccharides from A. leucorrhizus.

Full text of DOI:10.1016/j.carbpol.2018.05.028

CarbohydratePolymers196(2018)359–367
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Isolation, purification, characterization and antioxidant activity of
polysaccharides from the stem barks of Acanthopanax leucorrhizus
Hao-Bin Hua,⁎, Hai-Peng Liangb, Hai-Ming Lia, Run-Nan Yuanc, Jiao Sunc, La-La Zhanga,
Ming-Hu Hana, Yun Wua
a
College of Chemistry & Chemical Engineering, Longdong University, Qingyang 745000, PR China
Department of Oncology, Qingyang First People’s Hospital, Qingyang 745000, PR China
College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, PR China
b
c
A R T I C L E I N F O
A B S T R A C T
A novel water-soluble polysaccharide (named ALP-1) was successfully isolated from the stem barks of
Keywords:
Acanthopanax leucorrhizus
Polysaccharide
Isolation
Purification
Characterization
Antioxidant activity
Acanthopanax leucorrhizus by hot-water extraction, and further purified by Cellulose DEAE-52 and Sephadex G-
100 chromatography. The structure of ALP-1 was characterized by HPLC, HPGPC, partial acid hydrolysis, per-
iodate oxidation, Smith degradation, methylation, together with UV, IR and NMR spectral analysis. The anti-
oxidant activities also were evaluated in vitro. Structural analysis revealed that ALP-1 was a homogeneous ga-
lactan with the average molecular weight of 169 kDa, composed of galactose, glucose, mannose and arabinose in
a molar ratio of 6.1:2.1:1.1:1.0, owning a backbone structure of 1,6-linked α-D-Galp residues with some branches
of α-D-Manp-(1 → 3)-α-L-Araf residues at O-3 and α-D-Galp residues at O-4 of 1,6-linked α-D-Galp. Antioxidant
assay showed that ALP-1 exhibited strong DPPH% and HO% scavenging activities, as well as ferric-reducing
antioxidant power. These results provide a scientific basis for the further use of polysaccharides from A. leu-
corrhizus.
1. Introduction
treatment of rheumatoid arthritis and diabetes mellitus, and are po-
pularly used as a health supplement in China and Korea (Yoo, Lee, Jin,
Polysaccharides, present in almost all organisms, have many bio-
logical functions, such as energy storage, structure support, antigenic
determinant, and so on. Because the separation, purification, compo-
sition determination, and structural analysis of polysaccharides have
made remarkable progress, as well as because the biological functions
of polysaccharides are further understood, many bioactive poly-
saccharides have been widely studying and applying to the biochemical
and medical industry (Li, Li, You, Fu, & Liu, 2017). Recently, plant-
derived polysaccharides have been recognized as safe, highly stable and
effective natural antioxidants in pharmaceutical and food fields due to
their relatively nontoxicity and insignificant side-effects (Xu, Yao, Sun,
& Wu, 2009). Hence, the exploitation and utilization of plant-derived
polysaccharides as potential effective natural antioxidants could be
significant and worth to study further.
& Kim, 2008). Acanthopanax leucorrhizus Harms is deciduous shrub of
the genus Acanthopanax and an endemic medicinal plant growing
abundantly in Gansu province of China. Its root and stem bark possess
multiple biological functions such as antibacterial, antioxidant, anti-
tumour and so on, especially its stem barks have been used for a long
time to treat rheumatism, numbness, contracture, quadribblelegia,
hemiplegia, traumatic injury, edema, and itchy skin (Zhao et al., 1987).
Numerous studies show that Acanthopanax species are rich in poly-
saccharide, and that many polysaccharides have been isolated from A.
senticosus (Chen et al., 2011; Fu et al., 2012; Han et al., 2003; Li & Zhou,
2007), A. obovatus (Wang, Mao, Ito, & Shimura, 1991; Wang, Tsumura,
Ma, Shimura, & Ito, 1993), A. giraldii (Lu, Su, & Li, 2002; Wang,
Tsumura, Shimura, & Ito, 1992), A. sessiliflorus (Lee et al., 2003), A.
brachypus (Hu, Liang, & Wu, 2015), A. koreanum (Kang et al., 2015) and
A. sciadophylloides (Lee et al., 2015). However, to the best of our
knowledge, only few chemical constituents, such as volatile compo-
nents (Hu, Zheng, & Hu, 2012), sesquiterpenoids (Hu, Zhang, & Wu,
2014) and stilbenoids (Hu et al., 2018), have been isolated from A.
leucorrhizus. As part of an ongoing investigation of the chemical
The genus Acanthopanax, which belongs to the family Araliaceae
and comprises some 37 species (excluding varieties) in the world, are
widespread in Korea, Japan, China, and the far-eastern regions of
Russia (Ni & Liu, 2006; Phuong et al., 2006). Their roots and stem barks
have traditionally been used as tonic and sedative as well as for the
⁎
Corresponding author.
E-mail address: hhb-88@126.com (H.-B. Hu).
https://doi.org/10.1016/j.carbpol.2018.05.028
Received 18 February 2018; Received in revised form 7 May 2018; Accepted 9 May 2018
0144-8617/©2018ElsevierLtd.Allrightsreserved.

H.-B. Hu et al.
CarbohydratePolymers196(2018)359–367
constituents of this plant, a water-soluble polysaccharide (ALP-1) were
isolated from the root bark of A. leucorrhizus for the first time. Herein,
we report on the isolation, purification, structural characterization and
antioxidant activities of ALP-1.
on Sephadex G-100 gel-filtration column (1.6 cm × 80 cm) using ul-
trapure water as eluent at a flow rate of 15.0 mL/h. The main fractions
obtained was collected, concentrated, dialyzed against ultrapure water,
and finally lyophilized to obtain white fluffy pure polysaccharide
(4.5 g) named ALP-1 for next analyses.
2. Materials and methods
2.3. Physicochemical characterizations and structural analysis of ALP-1
2.1. Plant materials
2.3.1. Physicochemical characterizations of ALP-1
The stem barks of A. leucorrhizus was collected in September 2015
from Wushan County (Gansu Province of China), and identified by Prof.
Xiao-Qiang Guo (College of Life Science of Longdong University,
People’s Republic of China). A voucher specimen (No. 20150920012)
The physical characteristics were analyzed by color and texture
observation (Ge, Duan, Fang, Zhang, & Wang, 2009). The total carbo-
hydrate content of ALP was determined by the phenol-sulfuric acid
colorimetric method using D-glucose as the standard, and expressed as
glucose equivalents (Huang, Tan, Tan, & Peng, 2011). The content of
uronic acid was measured by photometry with m-hydroxydiphenyl at
525 nm using D-galacturonic acid as the standard (Gao et al., 2015). The
protein content was determined by the Bardford’s method (Bradford,
1976) using ovalbumin as a standard. ALP-1 was identified for the so-
lubility in water, ethanol, chloroform and acetone according to the
British pharmacopoeia (BP) specification (Kannan, Manivannan,
Balasubramaniam, & Kumar, 2010). Optical rotation of ALP-1 was
confirmed with a CDP-001 digital polarimeter according to the reported
method (Yi et al., 2015). The decolorization of polysaccharide was
measured using UV-spectrophotometry (Shimadzu UV-2401 spectro-
meter, Japan) at 420 nm (Xie, Shen, Nie, Li, & Xie, 2011). Gel per-
meation and anion exchange chromatography were monitored by as-
saying the total sugar content. D- or L-Configurations of sugars were
determined using the Gerwig method (Zhang, Liu, & Lin, 2014). The
nature of ALP-1 was verified through the following methods: Fehling’s
test, α-naphthol reaction, iodination reaction and FeCl3 reaction (Chen
& Kan, 2018).
was deposited in the Herbarium of College of Life Science
&
Technology, Longdong University, People's Republic of China. The stem
barks were washed and dried in the shade. Then, the dried plants were
crushed into powder (40 mesh) by a disintegrator.
Cellulose DEAE-52 and Sephadex G-100 were purchased from
Hengxin Chemical Reagent Co., (Shanghai, China). All standardes in-
cluding D-glucose (Glc), D-galactose (Gal), L-rhamnose (Rha), L-arabi-
nose (Ara), D-xylose (Xyl), D-mannose (Man), D-galacturonic acid
(GalA), erythritol, glycol and glycerol, as well as dextran series with
varying molecular weights of 5, 12, 25, 50, 80, 150, 270 and 410 kDa
were purchased from Shanghai Yuanju Bioscience Technology Limited
Company (China). Trifluoroacetic acid (TFA) and 1-phenyl-3-methyl-5-
pyrazolone (PMP) were purchased from Sinopharm Chemical Reagent
(Shanghai, China). 1-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimide
metho-p-toluenesulfonate (CMC), sodium borodeuteride (NaBD4), 1,1-
diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid (Vc), and ovalbumin
were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA).
Other chemicals and reagents used in this study were of analytical
grade from Xi’an Chemical Co. (Xi’an, China), and water was ultrapure
water.
2.3.2. Determination of homogeneity and relative molecular weight
The homogeneity and molecular weight of ALP-1 were identified by
high-performance gel- permeation chromatography (HPGPC) on an
Agilent 1100 HPLC system equipped with a TSK-GEL G4000PWXL
column (7.8 mm × 300 mm) and an Agilent RID-10A refractive index
detector. The determination procedure was carried out according to the
literature method (Cai, Xie, Chen, & Zhang, 2013). A sample solution
(15 μL) was injected for each run, and it was eluted with 0.1 M Na2SO4
solution at 40 °C and a flow rate of 0.6 mL/min. Data was analyzed by
Agilent GPC soft-ware. According to the peak shape of the HPGPC
chromatogram, the homogeneity of ALP-1 can be judged. The mole-
cular weight was estimated by reference to the calibration curve es-
tablished using the dextrans T-series of known molecular weights (5,
12, 25, 50, 80, 150, 270 and 410 kDa).
2.2. Extraction,isolation and purification of ALP-1
The extraction procedure of ALP was performed using the literature
method (Chen, Xie, Nie, Li, & Wang, 2008) with some modifications.
Briefly, the air-dried powder of A. leucorrhizus (300 g) was extracted
with 90% ethanol (2 L) for 24 h at room temperature to remove the
interfering components, including pigment, monosaccharide, dis-
accharide, oligosaccharide, volatile compounds and polyphenols. The
residues were filtrated and dried with a vacuum freeze dryer, and then
extracted three time (each time for 5.0 h) with ultrapure water (30:1,
water to material ratio, mL/g) at 80 °C. The combined aqueous extract
was concentrated to 25% of the original volume by a rotary evaporator
at 60 °C and then centrifuged at 4000g for 10 min. Whereafter, the su-
pernatant was collected and precipitated by adding five times of volume
of 95% (v/v) ethanol at 4 °C for 24 h. After centrifuging, the separated
precipitate was dissolved in appropriate volume of ultrapure water,
then deproteinated by the Sevag method (Yang, Huang, Wang, Cao, &
Sun, 2008). Briefly, the polysaccharide solution and the Sevag reagent
(CHCl3:n-C4H9OH = 4:1,v/v) were mixed (3:1, v/v) and shaken vigor-
ously for 30 min at room temperature and centrifuged at 3000g for
10 min. Then, the supernatant was collected and dialyzed for 72 h
against 100 vol of ultrapure water (cut-off Mw 8000 Da) to further re-
move the small molecular compounds (e.g., disaccharides or poly-
phenols). Finally, the dialyzate was lyophilized in vacuum freeze dryer
to obtain the crude polysaccharide (ALP, 22.7 g).
2.3.3. Monosaccharide composition analysis of ALP-1
The composition analysis of polysaccharide is an important step to
control the quality and got basic information about the polysaccharide.
The monosaccharide composition of ALP-1 was determined by the PMP-
labeling procedure (Li, Pan, Xia, Zhang, & Wu, 2014) with high per-
formance liquid chromatography (HPLC). Briefly, ALP-1 (5 mg) was
hydrolyzed with 2 M TFA (3 mL) at 120 °C for 3 h in a sealed glass tube.
Excess acid was removed by evaporating at reduced pressure with the
addition of methanol for five times after completing the hydrolysis. Dry
hydrolysis product was labeled with PMP through the addition of 0.6 M
NaOH (2 mL) and 0.5 M PMP-CH3OH solution (1 mL). The mixture was
reacted at 70 °C for 2 h. Later, the reaction product was neutralized with
0.3 M HCl (1 mL) and extracted with 20 mL chloroform three times. The
aqueous phase was finally filtered through 0.45 μm nylon membrane
for HPLC analysis. The resulting solution (20 μL) was injected into a RP-
C18 column (4.6 mm i.d. × 250 mm). The sample was eluted with a
mixture of acetonitrile and 0.1 M phosphate buffer (20:80, pH = 6.8) at
a flow rate of 1.0 mL/min and the injection volume was 20 μL. Standard
monosaccharides were determined by the same procedure. The sugar
ALP was redissolved in ultrapure water and purified by Cellulose
DEAE-52 column (2.6 cm × 40 cm) equilibrated with ultrapure water.
The polysaccharides were fractionated eluting stepwise with ultrapure
water, and followed by gradient NaCl solution (0–1.0 M) at a flow rate
of 1.0 mL/min. Each fraction (5 mL/tube) was monitored at 490 nm by
the phenol-sulphuric acid method (Cuesta, Suarez, Bessio, Ferreira, &
Massaldi, 2003). The eluted solution was collected and further purified
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CarbohydratePolymers196(2018)359–367
compositions of ALP-1 were identified by comparing retention times
with those of standard sugars (D-glucose, D-mannose, D-galactose, D-
galacturonic acid, L-rhamnose, D-xylose and L-arabinose), and the con-
tent of each sugar was calculated using the corresponding peak areas
and response factors.
follows: initial temperature of 140 °C was held for 3 min and then in-
creased to 250 °C by 3 °C/min and held for 10 min. The injector tem-
perature was 250 °C and helium was used as the carrier gas with a
constant flow rate of 1.0 mL/min. Erythritol, glycol, glycerol, manno-
pyranose, glucopyranose, galactopyranose and arabinofuranose were
used as standards.
2.3.4. Partial acid hydrolysis
2.3.7. UV and IR spectra analysis
Based on the fact that glycosidic linkages of the side chain were
more labile to acid than those of the main chain (Wu, Ai, Wu, & Cui,
2013), the main chain and the side chain of ALP-1 were determined by
partial acid hydrolysis. ALP-1 was hydrolyzed with 0.2 M TFA at 100 °C
for 1 h, then the hydrolyzate was evaporated to dryness. The resulting
residue was dialyzed with ultrapure water. After the retentate was
further purified by Sephadex G-100 gel-filtration chromatography, a
pure subfraction ALP-1P was isolated, which represented the partially
acid-degraded product of ALP-1. To determine the composition of
monosaccharides in ALP-1P, it underwent HPLC analysis and determi-
nation of relative molecular weight as described in Sections 2.3.2 and
2.3.3, respectively.
The ultraviolet spectra of ALP-1 (100 μg/mL) was recorded using a
Shimadzu UV-2401 spectro- photometer (Japan) in the wavelength
range of 200–700 nm. The organic functional groups of polysaccharide
were identified by Fourier Transform Infrared (FT-IR) method.
Polysaccharide (2 mg) was dried at 35–45 °C in vacuum over P2O5 for
48 h prior to grinding with spectroscopic grade KBr powder (100 mg)
and then pressed into a 1 mm pellet for FT-IR measurement using a
Shimadzu 8400S FT-IR infrared spectrometer (Japan). The experiment
conditions were 400–4000 cm−1 frequency, 8 cm−1 resolution, and 32
scans. The spectrum was analyzed using Nicolet Omnic 8.0 software
(Thermo Nicolet Corp.).
2.3.8. NMR analysis
2.3.5. Methylation analysis
The structural information of ALP-1 can be further acquired by NMR
spectroscopy (Li, Fan, & Ding, 2011). Briefly, the dried ALP-1 (30 mg)
was dissolved in 0.5 mL of 99.9% D2O in a NMR tube (5 mm diameter)
and kept at room temperature for 3 h. All one-dimensional (1D) and
two-dimensional (2D) NMR spectra were recorded with a Bruker AMX-
500 NMR spectrometer (Switzerland) with tetramethylsilane (TMS) as
internal standard and D2O as the solvents at 25 °C. The chemical shifts
were expressed in ppm, and the HOD signal fixed at 4.79 ppm.
Methylation analysis was accomplished according to the reported
method (Needs & Selvendran, 1993). ALP-1 was methylated three times
with CMC-NaBD4 (Zhang et al., 2014). The methylated products were
extracted by chloroform to obtain a corresponding neutral sugar (ALP-
1R), and examined by infrared (IR) spectroscopy. Completion of me-
thylation was examined through the disappearance of the O-H band
(3200–3700 cm−1) in the IR spectrum. The methylated product was
hydrolyzed with formic acid and 2 M TFA. The excess acid was eva-
porated by co-distillation with distilled water. Then the hydrolyzates
were reduced with NaBH4 and acetylated with acetic anhydride:pyr-
idine (1:1, V/V) and the partially methylated alditol acetates were
analyzed by GC–MS on an Agilent GC 7890N/MSD 5973N equipped
2.4. In vitro antioxidant activities analysis of ALP-1
2.4.1. DPPH% scavenging activity
DPPH% scavenging activity of ALP-1 was assessed according to the
reported method with minor modification (Li et al., 2014). In brief,
4 mL ALP-1 samples of various concentrations (0.25, 0.5, 1.0, 2.0,
4.0 mg/mL) were mixed with 1 mL 0.2 mM DPPH%-C2H5OH solution,
respectively. The mixture was shaken vigorously and incubated at room
temperature for 30 min in dark, and the reduction of DPPH% was
with
a HP-5 capillary column (30 m × 0.32 mm, film thinkness
0.25 μm). The degree of branching (DB) could be calculated with the
following equation (Wu et al., 2014):
DB = (NT + NB)/(NT + NB + NL)
(1)
Where NT, NB and NL are the total contents of the terminal residues,
branched residues, and backbone residues, respectively.
measured in the absorbance at 517 nm using
a UV–vis spectro-
photometer (T70, UV/VIS spectrometer, PG Instruments Ltd., China).
Ethanol was used as a negative control, while Vc was used as a positive
control at the same concentration. All tests were performed in triplicate.
DPPH% scavenging activity was calculated as follows:
2.3.6. Periodate oxidation and Smith degradation
Periodate oxidation is widely used in carbohydrate chemistry to
estimate patterns of substitution, frequency, and degree of branching.
Smith degradation is a useful extension of periodate oxidation, it can
selectively degrade a polysaccharide to either a polysaccharide with a
smaller repeat or an oligosaccharide, from which structural information
of the polysaccharide can be deduced (Zhang, Liu, & Lin, 2013).
ALP-1 (20 mg) was oxidized with 25 mL of 0.04 M NaIO4 and kept in
the dark place at 4 °C. Its absorption was monitored on a UV-2401
spectrophotometer at 223 nm every 4 h. Complete oxidation of ALP-1
occurred when the absorbance became stable. Glycol was then added to
decompose the excess HIO4 and end the oxidation reaction.
Consumption of NaIO4 was measured by spectrophotometer, and the
production of formic acid was determined by titrating the solution with
0.005 M NaOH. The periodate product solution was dialyzed against
ultrapure water for 48 h. The dialysate was reduced with 50 mg NaBH4
for 12 h at 25 °C, neutralized with 0.1 M CH3COOH to remove excess
NaBH4, and then dialyzed against ultrapure water. The retentate was
lyophilized to give polyalcohol. The polyalcohol was then hydrolyzed
with 1.0 M TFA at 100 °C for 3 h, and the acid was removed by co-
distillation with methanol under a vacuum. Finally, the product was
acetylated and analyzed by GC–MS on an Agilent GC7890N/MSD
5973N equipped with a HP-5 capillary column (30 m × 0.32 mm, film
thinkness 0.25 μm), and the oven temperature was programmed as
DPPH% scavenging activity (%) = [1 − (A1 − A0)/A2] × 100%
(2)
Where A0 is the absorbance of sample only (sample without DPPH%
solution), A1 is the absorbance of sample with DPPH% solution, A2 is the
absorbance of control (DPPH% solution without sample).
2.4.2. HO% scavenging activity
HO% scavenging activity of ALP-1 was determined as the reported
method (Sun, Wang, Shi, & Ma, 2009) with slight modifications. Briefly,
the reaction mixture contained 1 mL of polysaccharide at various con-
centrations (0.25, 0.5, 1.0, 2.0, 4.0 mg/mL), respectively. The reaction
was started by adding 1 mL of 2.5 mM phenanthroline, 1 mL of (2.5 mM
FeSO4) and 0.5 mL of 20 mM H2O2 in 1 mL of phosphate-buffer (20 mM,
pH = 7.4) for 60 min at 37 °C, then the absorbance of samples at
536 nm was measured, using Vc as a positive control at the same con-
centration. Percent inhibition of HO% was calculated according to the
following formula:
Inhibition rate (%) = [(A2 − A1)/(A0 − A1)] × 100%
(3)
Where A0 was the absorbance of the blank group where samples and
H2O2 were replaced with ultrapure water. A1 was the absorbance of the
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CarbohydratePolymers196(2018)359–367
control group where samples were replaced with ultrapure water. A2
was the absorbance of samples.
2.4.3. Reducing power
The reducing power of ALP-1 was measured according to the pre-
viously described method (Lin, Wang, Chang, Inbaraj, & Chen, 2009)
with slight modifications. Briefly, 1 mL of ALP-1 at various concentra-
tions (0.25, 0.5, 1.0, 2.0, 4.0 mg/mL) were mixed with 1.0 mL of K3[Fe
(CN)6] solution (1.0%, w/v) and 1.0 mL of phosphate-buffer (0.2 M,
pH = 6.6). After thorough mixing, each solution was heated in a water
bath at 50 °C for 20 min, and 1.0 mL of TFA (10.0%, w/v) was added.
Then supernatant (1 mL) was collected after centrifugation at 3000g for
10 min. The absorbance at 700 nm was measured in the reaction mix-
ture 10 min after 1 mL of deionized water and 1 mL of FeCl3 (1.0%, w/
v) were added at the room temperature. The reducing powers of sam-
ples were expressed using sample absorbances at 700 nm against a
blank (ultrapure water instead of the sample), as Vc was used as a
positive control at the same concentration.
Fig. 2. HPGPC profile of ALP-1.
difficultly dissolved in organic solvent (e.g. ethanol, chloroform and
acetone), with [α]2D0 = +164.6° (c = 0.5 mg/mL, H2O). This positive
value of optical rotation (+164.6°) suggested the dominating presence
of α-form glycosidic linkages in ALP-1 (Jahanbina, Abbasianb, &
Ahangb, 2017). The results obtained from the Fehling’s, α-naphthol,
iodination and FeCl3 reaction indicated that ALP-1 consisted of non-
reducibility polysaccharides but without starches and polyphenols. In
conclusion, the purification methods used in this study seemed to be
effective.
2.5. Statistical analysis
Results were presented as means
SD of three determinations.
Statistical analysis were performed using one-way analysis of variance
(ANOVA). Difference were considered statistically significant at
P < 0.05. All computations were done by employing the statistical
software (SPSS18.0, IL, USA).
3. Result and discussion
3.3. Homogeneity and molecular weight of ALP-1
3.1. Isolation and purification of polysaccharide
A calibration curve of standard dextrans was plotted as the loga-
rithm of relative molecular weight (MW, Da) versus the retention time (t,
min), the regression equation was log MW = 9.7197 − 0.3142 t with a
high correlation of R2 = 0.9988. As shown in Fig. 2. ALP-1 showed only
one single and symmetrical broad peak with elution time of about
14.3 min in the HPGPC chromatogram, indicating that it was a homo-
geneous polysaccharide. Based on the calibration with standard dex-
trans, the average molecular weight of ALP-1 was estimated to be about
169 kDa.
After hot-water extraction, ethanol precipitation, deproteinized by
Sevag method, dialyzed with water, and lyophilized in freeze-dry ap-
paratus, the crude ALP was isolated from the stem barks of A. leucor-
rhizus with a yield of 7.57% (dry weight), which was first separated
through anion-exchange chromatography of DEAE-52. As shown in
Fig. 1, the main fraction was obtained from the NaCl elution. To obtain
high-purity polysaccharide for structural elucidation and activity eva-
luation, the crude ALP was further purified by a Sephadex G-100 gel-
filtration column to obtain the pure polysaccharide (ALP-1, yield
1.50%).
3.4. Structural characterization of ALP-1
3.2. Physicochemical properties of ALP-1
3.4.1. Monosaccharide composition analysis
According to the phenol-sulfuric acid and m-hydroxydiphenyl
method, the total carbohydrate content existed in ALP-1 was 95.8%,
ALP-1 was classified as a neutral polysaccharide due to no uronic acid.
As shown in Fig. 3, the sugar composition analysis indicated that ALP-1
was a hetero-polysaccharide and consisted of Gal, Glc, Man and Ara in
the molar ratio of 6.1:2.1:1.1:1.0. The presence of galactose as the main
monosaccharide indicated that galactose could be the main backbone of
ALP-1.
The conventional physicochemical properties of ALP-1 were a white
powder, odorless, loose, freely soluble in water and DMSO, while
3.4.2. Partial acid hydrolysis
The partial hydrolysis was carried out to determinate the structure
of the main chain. After ALP-1 was partially hydrolyzed with 0.2 M
TFA, the hydrolyzate was dialyzed. The nondialysate was further pur-
ified on Sephadex G-100 chromatography to give a pure subfraction
ALP-1P. The composition analysis showed that ALP-1P is composed of
galactose, suggesting that the backbone of ALP-1 may be composed of
galactose. The absence of glucose, mannose and arabinofuranose im-
plied that three glycosyls may be located at the side chain.
3.4.3. Methylation analysis
ALP-1 was fully methylated and converted into the corresponding
alditol acetate derivatives for further analysis. Base on the data
Fig. 1. Elution curve of ALP on Cellulose DEAE-52 column.
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CarbohydratePolymers196(2018)359–367
Fig. 3. HPLC chromatograms of PMP derivatives of 7 standard monosaccharides (A) and component monosaccharides of ALP-1 (B).
consumed and 0.3 mol formic acid was produced per sugar residue. The
production of formic acid indicates that some of the residues may exist
in the pyran-hexose 1-linked or 1,6-linked form (about 60%). The
consumption of periodate was two times more than the amount of the
production of formic acid, indicating that many linkages may exist in
the pyran 1,2-; 1,4-; 1,2,6-; 1,4,6-linked forms; or furan 1-linked forms.
In addition, the ratio of the amount of monosaccharide to periodate was
about 1:1.4 for ALP-1. It prove that there may be 1,3-; 1,2,3-; 1,3,4-;
1,3,6-linked form, which cannot be oxidated by periodate (Jian, Chang,
Ablise, Li, & He, 2014).
Table 1
Glycosyl linkages analysis of ALP-1R.
Methylated sugars
Linkages type
Molar ratio
2,3,4-Me3-Gal
2,3-Me2-Gal
2,4-Me2-Gal
2,5-Me2-Ara
2,3,4,6-Me4-Glc
2,3,4,6-Me4-Man
→6)-Gal-(1→
→4,6)-Gal-(1→
→3,6)-Gal-(1→
→3)-Ara-(1→
Glc-(1→
3.1
2.0
1.0
1.0
2.1
1.1
Man-(1→
After the periodate-oxidized products of ALP-1 were reduced, hy-
drolyzed, and analyzed by GC–MS, a total of 5 compounds were de-
tected—galactose, arabinofuranose, erythritol and glycerin—in a molar
ratio of 1.1:1.0:2.1:6.1. The presence of galactose and arabinofuranose
indicated that some of galactose residues were 1,3-; 1,2,3-; 1,2,4-;
1,3,4-; 1,3,6- or 1,2,3,4-linkage, while arabinofuranose residues were
1,3-lingkage, which could not be oxidized. The degradation process
completely removed glucose and mannose residues, and produced a
certain amount of glycol and glycerol, indicating that these 2 glycoside
residues may exist in the 1- or 1,6-linked forms, which could be oxi-
dized. This inference also agrees with results from the methylation
analysis.
obtained from Table 1, the following conclusions can be drawn: (1)
ALP-1 contained six types of linkages, including 1,6-linked, 1,3,6-linked
and 1,4,6-linked Galp, 1,3-linked Araf, 1-linked Manp and 1-linked Glcp
residues in the molar ratio of 3.1:2.0:1.0:1.0:2.1: 1.1; (2) the Gal, Glc
and Man existed in pyranose form, and that the Ara in furanose form
(Jahanbina et al., 2017); (3) the galactose residues were present as 1,6-
linked, 1,3,6-linked and 1,4,6-linked Galp residues; (4) the glucose and
mannose residues were respectively present as 1-linked Glcp and 1-
linked Manp residues; (5) the arabinofuranose residues were present as
1,3-linked Araf residues. The number of these residues mentioned
above except 1,3-linked Araf, 1-linked Glcp and Manp residues account
for 59.2% of total methylated sugars, suggesting that the backbone of
ALP-1 may be composed of 1,6-linked Galp residues, with branches
attached to the O-3 or O-4 of 1,6-linked Galp, which agrees with the
results obtained from the partial acid hydrolysis analysis.
In ALP-1, all Glcp residues were found to be present as terminal
residues, and they were equal in number to the number of branched
1,4,6-linked Galp residues, implying that all Glcp residues are probably
attached to the O-4 position of the 1,6-linked Glcp residues. In addition,
the number of 1,3,6-linked Galp residues was approximately equal to
1,3-linked Araf and terminal Manp residues, suggesting that the Manp
residues are likely attached to the O-3 of the 1-linked Araf or 1,6-linked
Galp residues. Likewise, the DB value of ALP-1 was calculated to be
0.41, also implying a small-branched polysaccharide.
3.4.5. UV and FT-IR spectra analysis
ALP-1 had negative response to the Bradford test and had no any
characteristic absorption at 280 and 260 nm in the UV spectrum, de-
monstrating the absence of protein and nucleic acid. Furthermore, the
maximum absorbance peak around 199 nm suggested the presence of
polysaccharide (Chen & Kan, 2018).
As shown in Fig. 4. The FT-IR spectrum of ALP-1 displayed the ty-
pical characteristic peaks of polysaccharides, such as peaks at 3428,
2932, 1460, 1382, 1108 and 1036 cm−1 (Zou et al., 2017). The broad
stretching intense characteristic peak between 3428 cm−1, represented
the OeH stretching vibration of polysaccharide and water involved in
hydrogen banding. The weak absorption peaks at around 2932 and
1382 cm−1 was respectively attributed to the CeH stretching and
bending vibrations. The relatively strong absorption peak at 1651 cm−1
was due to associated water. The peaks at around 1460 and 1375 cm−1
were also assigned to the deformation vibration of CeH in the sugar
ring. Further, the peaks at 1108 and 1036 cm−1 range confirmed the
presence of CeOeC and CeOeH stretching vibrations, which was of
pyranose ring. Finally, the strong absorption peaks of 1075, 1029 and
1027 cm−1 indicated that ALP-1 was rich in galactose, the absorptions
at 812 cm−1 were typical for D-mannose as well as 918 and 757 cm−1
for D-glucose in the pyranose form (Cai et al., 2013), the peak at
843 cm−1 was characteristic of α-configuration of the sugar units
(Barker, Bourne, Stacey, & Whiffen, 1954), which was consistent with
its high positive value of optical rotation ([α]2D0 = +164.6° (c = 0.5 mg/
mL, H2O)).
Results obtained from the partial acid hydrolysis analysis suggested
that the backbone of ALP-1 may be mainly composed of galactose. This
information, along with information about the molar ratios of methy-
lated sugar, allowed us to predict the composition of side chains of ALP-
1. They are as follows: (1) a terminal Manp residue is likely attached to
the O-3 of a 1-linked Araf residue, and the latter is likely attached to the
O-3 of a 1,6-linked Galp residues in the backbone; (2) two terminal Glcp
residues are likely attached to the O-4 of two different 1,6-linked Galp
residues in the backbone.
3.4.4. Periodate oxidation and Smith degradation
The glycosidic linkage locations of ALP-1 were preliminarily de-
termined by Smith degradation and GC analysis (Qiao et al., 2010).
Results from periodate oxidation showed that 0.7 mol of periodate was
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H.-B. Hu et al.
CarbohydratePolymers196(2018)359–367
Fig. 4. FT-IR spectra of ALP-1.
Table 2
The chemical shift for related glycosyl residues of ALP-1.
Glucosyl residue
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 →6)-α-D-Galp-(1→
B →4,6)-α-D-GalP-(1→
C →3,6)-α-D-Galp-(1→
D α-D-GlcP-(1→
5.15/100.4
5.16/100.5
5.17/100.6
5.30/99.8
5.01/102.2
5.21/109.3
3.74/70.0
3.76/70.3
4.02/71.4
3.54/72.3
3.99/71.1
4.35/81.0
3.78/69.7
3.90/72.8
3.99/77.9
3.84/73.6
3.86/71.8
3.96/85.6
3.91/70.4
4.17/78.3
4.09/70.6
3.60/76.8
3.69/68.1
4.21/83.2
4.03/70.9
3.93/71.0
4.05/70.7
3.67/70.1
3.72/73.5
3.85, 3.76/63.5
3.76, 3.50/69.6
3.81, 3.58/69.3
3.79, 3.56/69.5
3.71, 3.64/61.2
3.88, 3.62/62.1
E α-D-Manp-(1→
F →3)-α-L-Araf-(1→
Fig. 5. The backbone structure of ALP-1.
Fig. 6. Structure of ALP-1.
3.4.6. NMR spectroscopy analysis
and 63.5 ppm, were attributed to the C-2 to C-5 of 1,3-linked α-L-Araf,
respectively. The carbon signals for terminal α-D-Glcp appeared at 72.3
(C-2), 73.6 (C-3), 76.8 (C-4), 70.1 (C-5), 61.2 (C-6) ppm, respectively.
The other carbon signals for terminal α-D-Manp appeared at 71.1 (C- 2),
71.8 (C-3), 68.1 (C-4), 73.5 (C-5), 62.1 (C-6) ppm, respectively. Ex-
cepting the anomeric carbon and arabinofuranose C-ring signals in all
glycosyl residues, the lowfield signals at 77.9 and 78.3 ppm, corre-
sponding to C-3 of 1,3,6-linked α-D-Galp and C-4 of 1,4,6-linked α-D-
Galp, arose because of the glycosidation effect (Bhanja et al., 2012).
Similarily, the lowfield signals (69.6, 69.3, 69.5 and 85.6 ppm) dis-
played the glycosidation position at C-6 of α-D-Galp and C-3 of α-L-Araf
residues, respectively.
The heteronuclear multiple bond correlation (HMBC) experiment
confirmed the sequence of glycoside residues in ALP-1. In the HMBC
spectrum of ALP-1, the cross-peaks between H-6 (α-D-Galp)/C-1 (α-D-
Galp), H-1 (α-D-Galp)/C-6 (α-D-Galp) were observed. According to the
afore-mentioned results, the backbone structure of ALP-1 was entirely
composed of galactose (Fig. 5), which was in agreement with the results
of methylation analysis.
Signals of ALP-1 in the NMR spectra were assigned as completely as
possible, based on the monosaccharide analysis, linkage analysis and
chemical shifts reported in the literature, the main chemical shifts of
ALP-1 were found, and they are presented in Table 2.
The 1H and 13C NMR spectra of ALP-1 were crowded in a narrow
region ranging from 3.5 to 5.5 ppm (1H NMR) and 60–110 ppm (13
C
NMR) which were the typical of polysaccharides, and the anomeric
proton signals at δ 5.15–5.17, 5.30, 5.01, 5.21 ppm and the anomeric
carbon signals at δ 100.4–100.6, 99.8, 102.2, 109.3 revealed that ALP-1
might contain four kinds of monosaccharide residues. The low-field
position of the anomeric protons suggested that the residues were α-
anomeric configuration because the signal at > 5.0 and < 5.0 ppm
were assigned as the anomeric protons of the α-anomeric configuration
and β-anomeric configuration according to the literature (Li et al.,
2014; Huang et al., 2016), which was in agreement with the presence of
FT-IR bands at 843 cm−1 and relatively high positive value of optical
rotation. Combined with 1H-1H COSY, HMBC spectra and document
data, the anomeric proton and carbon signals at δ 5.15/100.4 ppm,
5.16/100.5 ppm, 5.17/100.6 ppm, 5.30/99.8 ppm, 5.01/102.2 ppm,
and 5.21/109.3 ppm were assigned to the H1/C1 of 1,6-linked α-D-Galp,
1,4,6-linked α-D-Galp, 1,3,6-linked α-D-Galp, 1-linked α-D-Glcp, 1-linked
α-D-Manp and 1,3-linked α-L-Araf residues, respectively. The signals at
70.0, 69.7, 70.4, 70.9 and 69.6 ppm corresponded to C-2 through C-6 of
1,6-linked α-D-Galp, respectively. While the signals at 81.0, 85.6, 83.2
In addition, some of the Galp (∼50%) residues in the backbone
were branched. Their side chains consisted of [1] two terminal α-D-glcp
residues were respectively attached to the O-4 of two different 1,4,6-
linked α-D-Galp in the backbone; [2] a terminal α-D-Manp residue at-
tached to the O-3 of a 1,3-linked α-L-Araf residue, and the latter was
attached to the O-3 of 1,3,6-linked α-D-Galp in the backbone. Above
364

H.-B. Hu et al.
CarbohydratePolymers196(2018)359–367
Fig. 7. In vitro antioxidant activity of ALP-1. (a) Scavenging activity of ALP-1 on DPPH%, (b) Scavenging activity of ALP-1 on HO%, (c) Reducing power of ALP-1.
conclusion was also confirmed by the HMBC correlations of H-1 (D)/C-
4 (B), H-4 (B)/C-1 (D), H-1 (F)/C-3 (C), H-3 (C)/C-1 (F), H-1 (E)/C-3
(F), and H-3 (F)/C-1 (E).
Based on the methylation analysis, periodate oxidation and Smith
degradation, and the partial hydrolysis experiment, together with re-
sults from the UV, IR and NMR data studies, the predicted primary
structure of repeat unit in ALP-1 was confirmed as shown in Fig. 6.
damage to adjacent biomolecules (Chen et al., 2016). The scavenging
activities of ALP-1 and Vc against HO%, as given in Fig. 7B, revealed
ALP-1 had concentration-dependent manner and expressed noticeable
HO% scavenging ability, but it was weaker than that of Vc. Compara-
tively, the maximum scavenging effect of ALP-1 was 65.8
2.2% at
4.0 mg/mL which reached 70.45% that of Vc (93.4
3.2%), sug-
gesting that ALP-1 can be considered as a good scavenger of HO%, and
would be beneficial to a certain extent to protect against oxidative
damage.
3.5. Antioxidant activities of ALP-1
3.5.1. Ability of ALP-1to scavenge DPPH%
3.5.3. Reducing power of ALP-1
DPPH% is a very stable organic radical with nitrogen as its center,
which has strong absorption at 517 nm in ethanol. It was widely used to
evaluate the free-radical scavenging ability of antioxidants (Jin, Yang,
Tong, Jin, & Xu, 2016). Fig. 7A depicts the scavenging ability of ALP-1
on DPPH% compared with Vc as a positive standard. ALP-1 exhibited a
moderate antioxidant activity in dose-dependent manner within the
tested concentration range, while Vc displayed a stronger ability to
scavenge DPPH%. Specifically, ALP-1 was able to scavenge more DPPH%
as its concentration increased within the range of 0.25–4 mg/mL, but
no further increase in activity were detected above 4.0 mg/mL. At the
concentration of 4.0 mg/mL, the scavenging rates of ALP-1 and Vc on
The antioxidant activity of a compound directly relates to its re-
ducing power, this signifies that the stronger of reducing power, the
higher antioxidant activity is (Dou et al., 2015), and that the absor-
bance values can directly indicate the reducing power, a higher ab-
sorbance indicates a higher reducing power (Gao et al., 2015). As
shown in Fig. 7C, the reducing power of ALP-1 increased in a con-
centration-dependant manner in the concentration ranges of
0.25–4.0 mg/mL, although the effect was inferior to Vc at the same
doses, as Vc was a well recognized reducing agent. At a concentration of
4.0 mg/mL, the absorbance of ALP-1 was 0.482
0.075, which was
only of 52.7% that shown by Vc (0.915 0.091), indicating that ALP-
DPPH% were 46.1
1.8% and 95.15
3.1%, respectively. At the
1 has a moderate reducing power. The above results show that the re-
ducing power of ALP-1 correlates well its antioxidant activity, sug-
gesting that its reducing power is a significant indicator of its potential
antioxidant activity.
It is well known that the antioxidant activity of polysaccharide
largely depends on its structure, the influencing factors might be mo-
lecular weight, degree of branching, linkage, monosaccharide compo-
sition and conformation. Thus, the antioxidant activity of ALP-1 is not a
function of a single factor but rather a combination of several factors
(Wang et al., 2012).
same concentration (4.0 g/mL), the acid polysaccharide SP1, which was
isolated from Sisal in previous study, exhibited a greater ability than
ALP-1 to scavenge DPPH% (∼53%) (Zhang et al., 2013), implying that
carboxyl groups may play an important role in scavenging radicals,
possibly because carboxyl groups donate hydrogen more readily than
hydroxyl groups.
3.5.2. Ability of ALP-1 to scavenge HO%
HO% is dangerous to the organisms through inducing oxidative
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CarbohydratePolymers196(2018)359–367
4. Conclusions
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A new water-soluble polysaccharide, named ALP-1, was isolated
and purified from the stem barks of A. leucorrhizus using hot-water
extraction, ethanol precipitation, Cellulose DEAE-52 and Sephadex G-
100 chromatography, and its structure was elucidated by physico-
chemical and spectral analysis. Structural analysis revealed that ALP-1
was composed of Gal, Glc, Man and Ara in the molar ratio of
6.1:2.1:1.1:1.0, with the molecular weight of 169 kDa, and the back-
bone chain was composed of 1,6-linked α-D-Galp. Moreover, the in vitro
antioxidant activities as evaluated by their reducing power and their
scavenging abilities toward DPPH% and HO% demonstrated that ALP-1
had effective antioxidant activities. Based on these findings, we may
rationally assume that ALP-1 can be explored as novel nutriments, food
additives, or antioxidant supplements. However, the molecular me-
chanism of the antioxidant activity of ALP-1 needs to be further in-
vestigated.
Acknowledgements
This research was supported by the National Natural Science
Foundation of China (Grant No. 21462026) and the Key Constructive
Disciplines of Longdong University (Grant No. LDXYKCD 201704).
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