Accepted Manuscript

Title: Chemical characterization, antioxidant,

immune-regulating and anticancer activities of a novel
bioactive polysaccharide from Chenopodium quinoa seeds

Authors: Yichen Hu, Jinming Zhang, Liang Zou, Chaomei Fu,

Peng Li, Gang Zhao

PII: S0141-8130(16)32295-4
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.019
Reference: BIOMAC 7182

To appear in: International Journal of Biological Macromolecules

Received date: 16-11-2016

Revised date: 2-2-2017
Accepted date: 3-3-2017

Please cite this article as: Yichen Hu, Jinming Zhang, Liang Zou, Chaomei
Fu, Peng Li, Gang Zhao, Chemical characterization, antioxidant, immune-
regulating and anticancer activities of a novel bioactive polysaccharide from
Chenopodium quinoa seeds, International Journal of Biological Macromolecules
http://dx.doi.org/10.1016/j.ijbiomac.2017.03.019

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<AT>Chemical characterization, antioxidant, immune-regulating and anticancer
activities of a novel bioactive polysaccharide from Chenopodium quinoa seeds
<AU>Yichen Hua†, Jinming Zhangb†, Liang Zoua, Chaomei Fub, Peng Lic*, Gang
Zhaoa*
<AFF>aSchool of pharmacy and bioengineering, Chengdu University, Chengdu
610106, China
<AFF>bSchool of Pharmacy, Chengdu University of Traditional Chinese Medicine,
Chengdu 611137, China
<AFF>cState Key Laboratory of Quality Research in Chinese Medicine, Institute of
Chinese Medical Sciences, University of Macau, Macao 999078, China
<AFF>†These authors equally contribute to this work
<PA>*Correspondence Dr. Peng Li, Institute of Chinese Medical Sciences,
University of Macau, Macao 999078, China. E-mail: pengli@umac.mo(Dr. Peng L)
Prof. Gang Zhao, School of pharmacy and bioengineering, Chengdu University,
Chengdu 610106, Sichuan Province, China. E-mail: prozhao1@126.com.

<ABS-HEAD>Abstract

<ABS-P>Chenopodium quinoa, a promising nutraceutical cereal, has attracted

increasing research interest, yet its polysaccharides remains to get few systematic
studies. In this study, we employed orthogonal experimental design to optimize the
ultrasound-assisted extraction process for highest yield of C. quinoa polysaccharides.
A novel C. quinoa polysaccharide (CQP) fraction with high content and low
molecular weight (8852 Da) was subsequently purified by column chromatography,
constituted by galacturonic acid and glucose monosaccharides. The purified CQP
exhibited significantly antioxidant effect against DPPH+ and ABTS+, with even higher
efficiency than some other reported polysaccharides. Moreover, CQP could promote
the RAW264.7 macrophage proliferation, while suppress the nitri oxide production on
inflammatory RAW264.7 macrophage in a dose- and time-dependent manner. In view
of the pathological correlation of free radical, inflammation and carcinogenesis, the
anticancer effect of CQP was further investigated on human liver cancer SMMC 7721
and breast cancer MCF-7 cells. Interestingly, CQP displayed cytotoxicity against
cancer cells, while none proliferation inhibition on normal cells. These results suggest
that the bioactive polysaccharide from C. quinoa provided the promising potential as a
natural antioxidant, immune-regulating and anticancer candidate for food and even
drug application.

<KWD>Keywords: Chenopodium quinoa seed; Polysaccharides; Biological

macromolecule

<H1>1. Introduction
Chenopodium quinoa (Chenopodium quinoa Willd.), an ancient pseudocereal
originated from the Andes of South America, attracts increasing interest worldwide
during the last two decades [1]. Due to its confirmed complete nutrition, C. quinoa
becomes an excellent example of ``functional food'', which could benefit to reduce the
risk of various diseases [2]. In the early 1970s, the National Academic Science (NAS)
considered C. quinoa as one of the promising and recommended plants to improve the
nutrition and quality of life of the population on ``developing'' countries [3]. The
higher nutritive value in C. quinoa seeds than other common cereals has been found,
containing a higher content of lysine-rich protein and balanced distribution of
essential amino acids. Additionally, there is also rich in lipids, fibers, vitamins and
minerals [4]. Most importantly, the flavonoids, phenolic acids and saponins in C.
quinoa contribute to its biological functions such as antimicrobial [5], antioxidant [6],
and anti-inflammatory [7, 8]. However, little attention has been paid to
polysaccharides from quinoa, let alone their bioactivity.
Currently, polysaccharides widely derived from plants, animals and microorganisms
become attractive sources for food and medicinal application, with unique biological
activities such as antioxidant, immunomodulation and antitumor [9-12]. Four
polysaccharide sub-factions were isolated and purified from quinoa by Ren et al.,
shown significant antioxidant and immunoregulatory activities [13]. However, to
better exploit and utilize polysaccharides in quinoa, its extraction procedure and
physicochemical property need further investigation.
In the present study, we primarily optimized the extraction procedure on the highest
extraction yield of polysaccharides by ultrasonic-assisted extraction technology,
taking advantage of orthogonal experiment design. Following the purification process,
a novel polysaccharide from quinoa was obtained, with an evident preliminary
structural feature. Subsequently, its antioxidant, immune-regulatory, and anticancer
effects in vitro have been evaluated.

<H1>2. Materials and Methods

<H2>2.1. Materials

C. quinoa seeds were collected from Jintang (Sichuan, China) in November,

2015, and identified by Prof. Gang Zhao. The voucher specimen has been deposited at

the herbarium of Chengdu University.

DEAE-52 and Sephadex G-100 were purchased from Solarbio Bioscience &
Technology Co., Ltd. (Beijing, China). The human normal liver (L02) cells, human
normal breast epithelial (MCF 10A) cells, human liver cancer (SMMC 7721) cells,
human breast cancer (MCF-7) cells and mice macrophage (RAW264.7) cells were
obtained from American Type Culture Collection (ATCC) and cultured in DMEM
containing 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin
solution at 37 °C in a humidified CO2 (5%) incubator. Monosaccharides standards
including mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose,
were obtained from Aladdin Co., Ltd. (Shanghai, China). 1-phenyl-3-methyl-5-
pyrazolone (PMP), Trifluoroacetic acid (TFA), 2,2-diphenyl-1-picrylhydrazyl
(DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). All reagents were of analytical grade,
except for acetonitrile for HPLC grade.

<H2>2.2. Extraction and purification of polysaccharides from C. quinoa

C. quinoa seeds were extracted with petroleum ether (boiling range: 60-90 °C) at 60
°C for three times to remove lipids and pigments, with 2 h per time. The residue was
then extracted with distilled water using ultrasonic-assisted extraction technology in
combination of orthogonal experiment design [14]. A three-factor, three-level
orthogonal test, with the extraction variables, namely, ultrasonic temperature (X1),
ultrasonic time (X2), and water to raw material ratios (X3), was applied to optimize the
extraction condition. Subsequently, the cooled extraction solution was centrifuged at
5000 × g for 20 min. The supernatant was then concentrated to one tenth of its volume
by a rotary evaporator at 50 °C and precipitated with 95% ethanol (1:4, v/v) at 4 °C
for 12 h. The resulting precipitate (crude polysaccharides) was collected by
centrifugation. The crude polysaccharides were put into blue cap bottles and treated
with 1/5th the volume of the sample of the Sevag reagent (butyl alcohol / chloroform =
1/4, v/v) to remove proteins [15]. This treatment was repeated until there were no
white denatured proteins in the interphase. The crude C. quinoa polysaccharides were
stored at -20 °C after freeze drying. The extraction yield was calculated by the weight
ratio of crude C. quinoa polysaccharides and dried C. quinoa seeds. The content of
total sugar in crude polysaccharides and the purified polysaccharide fraction was
determined by the phenol-sulfuric acid colorimetric method [16] using glucose as the
standard reference (y=11.597x+0.091, R2=0.9991).
The crude C. quinoa polysaccharides were dissolved in deionized water with the
concentration of 0.1mg/mL. The supernatant was collected by centrifugation, and
loaded onto a DEAE-52 cellulose column (3.0 × 50 cm). The column was then
stepwise eluted with 0, 0.1 and 0.3 M NaCl solution at a flow rate of 1.0 mL/min.
Fractions eluted with different concentrations of NaCl solution were collected,
dialyzed, and lyophilized. The polysaccharide fraction was further purified by gel-
filtration (2.0 × 100 cm) on Sephadex G-100 column using distilled water as the
eluant at a flow rate of 0.5 mL/min.

<H2>2.3. Characterization of purified polysaccharide fraction

In the study, infrared spectroscopy (IR), high performance gel permeation

chromatography (HPGPC), and scanning electron microscope (SEM) were utilized to
characterize the purified polysaccharide fraction from C. quinoa seeds (CQP),
according to the previously similar reports [17, 18].
The purified CQP fraction was analyzed with a Fourier transform infrared
spectrometer (FTIR, Thermo Fisher Scientific). The molecular weight of purified
polysaccharide fraction was determined by a Waters e2695 HPLC instrument
equipped with a refractive index detector (Malvern Instrument, UK). 100 μL of
polysaccharide solution (0.1 mg/mL) was injected and performed in a
chromatographic column (300 mm × 7.8 mm, Waters UltrahydrogelTM1000, Serial
number: WAT011535, USA). Milli-Q water was selected as mobile phase, and the
flow rate was set at 0.5 mL/min. GPC peaks were detected by RI detector. The
number average molecular weight (Mn), the weight average molecular weight (Mw),
and the polydispersity (Mw/Mn) were calculated. Polyethylene oxide/glycol was
chosen as standard materials. The morphological features of the polysaccharide
fraction were recorded by a Quanta 2000FEG SEM (FEI, Chicago, USA), following
the polysaccharide sample sputtered with gold.
The monosaccharide composition of purified CQP fraction was analyzed by the
HPLC method as previously reported [19]. Initially, the dried polysaccharide sample
(10 mg) was hydrolyzed with 4 mL of 2.0 M TFA at 110 °C for 4 h under nitrogen
atmosphere. After hydrolysis, excess TFA was removed by the addition of methanol
and then removed TFA residue by rotary evaporation at reduced pressure. The dried
hydrolyzed polysaccharide sample was dissolved in 2 mL of water for subsequent
PMP derivatization [20]. Briefly, the hydrolyzed polysaccharides or monosaccharide
standards were mixed with 0.3 M NaOH and 0.5 M PMP-methanol solution, with
sequential reaction for 90 min at 70 °C. When the reaction mixture was cooled, it was
neutralized with 0.3 M HCl. After that, the solution was extracted with chloroform for
3 repeated times, in which the chloroform layer was discarded. Conversely, the
aqueous layer was filtered through a 0.45 μm membrane and analyzed by HPLC
system equipped with a reverse phase C18 column (5μm, 250 × 4.6 mm) (Agilent
Zorbax, USA) at the maximum wavelength of 245 nm. The mobile phase consisted of
0.1 M phosphate buffer (pH 6.7) and acetonitrile (83:17, v/v) at a flow rate of 1
mL/min [19].

<H2>2.4. Antioxidant activities of purified CQP fraction in vitro

<H3>2.4.1. DPPH radical scavenging activity

Evaluation of the scavenging capability of purified CQP fraction on DPPH free

radicals was performed by the reported method [21] with a slight modification.
Briefly, 2 mL of DPPH ethanol solution (0.1 mM) was added into tubes that
containing 2 mL CQP in water (0.125~5 mg/mL), and co-incubated for 30 min at
room temperature in the dark. The absorbance was detected at 517 nm. Vitamin C
(Vc), with a favorable antioxidant efficiency in the previous report [22], was used as a
positive control at the same concentration. The ability to scavenge the DPPH radical
was calculated using the following equation [23]: scavenging rate (%) = [1 − (A1 − A2)
/ A0] × 100, where A0 was the absorbance of control (without samples), A1 was the
absorbance in the presence of the sample and DPPH radical dot, and A2 was the
absorbance of the sample blank (without DPPH radical dot).

<H3>2.4.2. ABTS+ radical scavenging activity

The ABTS+ radical scavenging activity of CQP was measured by the reported method
[24] with a slight modification. ABTS+ stock solution was prepared by the mixture of
ABTS (7 μM) and potassium persulfate (2.45 μM), and diluted to an absorbance of
0.7 (±0.02) at 734 nm. Briefly, 0.2 mL of CQP samples with different concentrations
were mixed with 4 mL ABTS+ diluent for 6 min at room temperature in the dark. The
absorbance was measured at 734 nm and Vc was used as a positive control.

<H2>2.5. Immunomodulation activities of purified polysaccharide fraction in vitro

<H3>2.5.1. Inhibition of Raw264.7 cells proliferation

To evaluate the proliferation inhibition activity, the cell viability of RAW264.7

macrophage cells treated with CQP was determined by MTT assay. In brief,
RAW264.7 cells were pre-incubated in 96-well plates at a density of 6×103 cells/well
for 12 h. The supernatant were replaced with a new culture medium in the absence or
presence of CQP at a series of concentrations (12.5, 25, 50, and 100 μg/mL), and then
cultured at 37 °C in a humidified incubator with 5% CO2 for 24 h and 48 h.
Lipopolysaccharide (LPS) was used as a positive control. MTT solution (5 mg/mL in
medium) was added into each well. After 4 h incubation, the supernatant was
removed and 100 μL of DMSO was added to dissolve formazan crystal [25]. The
absorbance was measured by the microplate reader at 570 nm. The inhibition of cell
growth was calculated by formula: inhibition (%) = (1-OD/OD0) × 100 %, where OD
and OD0 indicated the absorbance of treated cells and untreated cells, respectively.

<H3>2.5.2. Measurement of nitric oxide (NO) release

The levels of NO production in the Raw264.7 cells treated by CQP were monitored
by Griess reagent (1% sulphanilamide and 0.1% naphtylethylene diamine
dihydrochloride in 2% H3PO4) [26]. Briefly, Raw 264.7 cells (1×104 cells/well) were
plated in 96-wells plate and cultured with several concentrations (12.5, 25, 50, 100
and 200 μg/mL) of CQP or LPS (10 μg/mL) for 24 h and 48 h. Then, 50 μL of cell
culture medium was removed from individual wells and mixed with equal volumes of
Griess reagent at room temperature for 10 min. The optical density was measured
using an automated microplate reader at 550 nm. A standard curve using a standard
solution of NaNO2 in culture medium was employed to calculate the nitrite
concentration.

<H2>2.6. Anticancer activities of purified polysaccharide fraction in vitro

The human normal liver (L02) cells, human normal breast epithelial (MCF 10A) cells,
human liver cancer (SMMC 7721) cells, human breast cancer (MCF-7) cells were
employed to evaluate the anticancer effect of CQP fractions by MTT assay in vitro.
Cells were pre-plated at a density of 5×103 cells/well and treated by various
concentration of CQP fractions (12.5~200 μg/mL), and then cultured at 37 °C in a
humidified incubator with 5% CO2 for 24 h and 48 h. MTT solution (5 mg/mL in
medium) was added into each well. After 4 h incubation, the supernatant was
removed and 100 μL of DMSO was added to dissolve formazan crystal. The
absorbance was measured by the microplate reader at 570 nm. Cell viability rate was
calculated by the ratio of the absorbance of wells treated with samples and the
absorbance of wells untreated.

<H2>2.7. Statistical analysis

All data were presented as means ± standard deviation. All experiments were

repeated at least three times.

<H1>3. Results and discussion

<H2>3.1. Optimization for extraction of crude C. quinoa polysaccharides

Various factors potentially affect the ultrasonic-assisted extraction, the optimum

extraction conditions represent a critical step in the extraction method. Herein, we
initially integrated the results from the single factor pre-experiments, three key factors
influencing the polysaccharide yield were selected, i.e. ultrasonic temperature (°C),
ultrasonic time (min) and water to raw material ratio (mL/g). Subsequently, a three-
factor and three-level orthogonal experiment (Table 1) was designed according to the
L9 (33) table. The results were shown in Table 2.
According to the variance analysis result in Table 3, both ultrasonic temperature (X1)
and ultrasonic time (X2) exhibited significance on extraction yield of crude C. quinoa
polysaccharides. Additionally, although water to raw material ratio (X3) exerted no
significance, there was still difference on extraction yield among three levels. Thus,
the optimum conditions of CQP were (X1)3(X2)2(X3)3, namely, ultrasonic
temperature of 90 °C, ultrasonic time of 30 min, and water to raw material ratio 25
mL/g. The extraction yield of crude C. quinoa polysaccharides was 9.65%.

<H2>3.2. Purification and physicochemical properties

<H3>3.2.1. Isolation and purification of CQP fraction

After deproteinization procedure by Sevag method, the polysaccharide content in
crude C. quinoa polysaccharides was determined as 68.6%±2.64%, according to the
established standard reference. The crude C. quinoa polysaccharides was further
purified using an anion-exchange chromatography column of DEAE-52 cellulose
based on the ionic groups as previous method [27]. As shown in Fig.1A, two main
fraction were collected with stepwise concentrations of NaCl solutions. However, the
yield of fraction at 250~500 mL elution volume with 0.1M NaCl solution was
considerable low, which was less than 8%. Thus, we gave a further purification to the
first main fraction (CQP), obtained by water eluant, using a Sephadex G-100 gel
filtration column by its molecular distribution. A single symmetrical peak was
observed in Fig.1B, suggesting it was a homogeneous polysaccharide fraction.

<H3>3.2.2. Physicochemical properties of purified CQP

The structure of purified CQP fraction was primarily characterized by FT-IR (Fig.
2A). A broad and intense peak at 3391 cm-1 was derived from the characteristic
absorption of plentiful hydroxyl groups (-OH) in polysaccharides. The peaks at 2930
cm-1 and 1415 cm-1 were contributed to the stretching vibration and symmetrical
deformation vibration of –CH2 group [28]. The peaks at 1652 cm-1 and 1240 cm-1
indicated the presence of –COOH groups in polysaccharide chain [29]. Additionally,
the wide absorption bands between 1000 cm-1 and 1154 cm-1 were derived from the
stretching vibration of C-O-C groups [30]. The peaks around 847 cm-1 suggested the
glycosidic linkages in polysaccharides [31].
The molecular weight distribution of CQP fraction was determined by GPC. As
shown in Fig. 2B, the single peak with approximatively symmetrical shape suggested
that it was a homogeneous polysaccharide, with the average Mn and Mw of 4833 Da
and 8852 Da, respectively. Ren et al. [13] reported four homogeneous polysaccharides
from quinoa with Mw as 26, 37, 34, and 22 kDa, respectively. Thus, the homogeneous
polysaccharide we obtained herein was a novel polysaccharide which was different
from those in previously reports. As shown in Fig. 2C, the SEM image of CQP with
50K-fold magnification provided its surface morphology, existing in the state of
aggregation and platy structure.
According to the previous study [14], seven derivative monosaccharides by PMP were
analyzed using HPLC as shown in Fig. 2D-1. Similarly, CQP fraction was hydrolyzed
with TFA and then generated PMP derivatives. As shown in Fig.2D-2, galacturonic
acid (Gal A) and glucose (Glc) were detected with the proportion of 77.35% and
22.65%, respectively. Thus, the obtained CQP fraction was a kind of
heteropolysaccharide constituted by Gal A and Glc.

<H2>3.3. Antioxidant activities

Antioxidants could effectively avoid degenerative disease and ageing caused by

excessive free radicals [32]. Like other natural components such as flavones [33],
various polysaccharides have been demonstrated as the promising antioxidants that
have gained attention. Herein, the antioxidant activities of the highly purified CQP
fraction were evaluated by DPPH and ABTS radical scavenging assay.
DPPH is a very stable nitrogen center of free radicals with a deep purple color.
Antioxidants could reduce the absorbance of DPPH at 517 nm. The DPPH radical
scavenging effect of CQP fraction was determined, with Vc as a positive control. As
shown in Fig. 3A, CQP showed scavenging effect in a dose-dependent manner. The
IC50 value of CQP was 1.859 mg/mL. Although the DPPH radical scavenging ability
of CQP was weaker than Vc, it exhibited the close efficiency as the previously
reported polysaccharides [34, 35]. Additionally, ABTS assay is often a useful method
to measure the total antioxidant power of a potential antioxidant [36]. Concentration-
dependent radical scavenging activity was also shown in Fig. 3B. Its IC50 value for
ABTS radical scavenging activity was 1.108 mg/mL. Accordingly, antioxidant
activity of CQP exceeded that of some reported natural polysaccharides like those
derived from Rhizoma Panacis Majoris [37] and Chuanminshen violaceum [38].
Thus, the purified CQP fraction from quinoa may be potential antioxidants and
enhance its nutritive properties.

<H2>3.4. Immunomodulation activities

The effects of purified CQP at various concentrations on the viability of RAW264.7

macrophages were evaluated by MTT assay. As shown in Fig. 4A and Fig. 4B, LPS,
an acknowledged stimulant for immune system, displayed a potent enhanced
proliferation on RAW264.7 cells, as a positive control. Although the low
concentration of CQP did not exert significantly influence on the proliferation of
RAW264.7 cells, the remarkable growth of CQP at higher concentrations ranging
from 25~100 μg/mL was observed in a both dose- and time-dependent manner.
Particularly, both 50μg/mL and 100μg/mL of CQP could significantly rise the
RAW264.7 cell viability with 24 h and 48 h incubation, compared to control group
(p<0.05▲). Thus, CQP fraction could significantly enhance the immune capacity of
RAW264.7 macrophages.
Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory
responses and plays a pivotal role in immunity [39, 40]. Subsequently, the effect of
CQP on NO release in LPS-stimulated RAW264.7 macrophages was evaluated. As
shown in Fig. 4C and Fig. 4D, 0.1 μg/mL of LPS could significantly stimulate the NO
production. NO concentration in medium of LPS-unstimulated RAW264.7 cells was
much lower than that of LPS-stimulated condition(p<0.05▲), indicated the
inflammatory response caused by LPS. However, CQP fraction could inhibit NO
production of LPS-stimulated RAW264.7 cells in a concentration- and time-
dependent manner. Especially, in comparison with LPS-stimulated group, both 50
μg/mL and 100 μg/mL of CQP could significantly decrease NO production after 24 h
and 48 h incubation (p<0.05*). Inhibition rate of 100 μg/mL of CQP for 24 h and 48 h
treatment could reach to 34.3% and 52.7%, respectively. This result suggested that the
purified CQP exhibited immune-potentiation as well as anti-inflammatory effect.
<H2>3.5. Anticancer activities

Excessive free radicals can be hazardous to body, damage normal cells and induce
inflammation. These reactive radicals may injure cells and tissue directly via
oxidative degradation of essential cellular components as well as injure cells
indirectly by altering the protease/antiprotease balance [41]. Even, inflammation is
also a critical component of carcinogenesis [42]. Thus, the antioxidants and anti-
inflammatory agents would be effective therapeutic approaches to treat cancer.
Herein, the in vitro growth inhibition of CQP against SMMC 7721 and MCF-7 cells
were studies. Additionally, to demonstrate its biosafety, the normal cells (both L02
and MCF 10A) were employed. As shown in Fig. 5, CQP showed a significant
proliferation inhibition against cancer cells in a dose- and time-dependent manner.
The IC50 values of CQP in SMMC 7721 cells for 24 h and 48 h were 121.4 μg/mL
and 53.4 μg/mL. The IC50 values of CQP in MCF-7 cells for 24 h and 48 h were 83.48
μg/mL and 64.67 μg/mL. Nevertheless, no remarkable influence in normal cells was
observed, in which the inhibition rates of normal cell viability were less than 10%.
The antioxidant and anticancer activities of C. quinoa leaves extracts containing
phenolic compounds have been revealed [43]. Thus, the results indicated that
polysaccharides in C. quinoa also exhibited potent anticancer activity. Certainly, the
anticancer mechanism and the relationship among its antioxidant, anti-inflammatory
and anticancer activities need to be further investigated.

<H1>4. Conclusion

In present study, a novel polysaccharide with the molecular weight of 8852 Da,
constituted by galacturonic acid and glucose, were successfully isolated from C.
quinoa seeds based on orthogonal experimental design, and purified by cellulose
column and subsequent sephadex gel. This low molecular weight polysaccharide
exhibited significantly antioxidant, immune-regulatory and anticancer effects in vitro.
Most importantly, no significant inhibition on proliferation of normal cells was
observed, indicated that the obtained new polysaccharide fraction exerted high
biosafety. Nevertheless, further detailed studies are required to investigate the
involved cellular signaling processed and the potential relationship among
antioxidant, immune-regulatory and anticancer activities.

<H1>Conflict of interest

Authors declare no conflict of interest.

<ACK>Acknowledgements

The authors gratefully acknowledge the financial supports by Special Fund for Agro-
scientific Research in the Public Interest (201303069-08), Chengdu University
Research Fund (2080516032), Applied Basic Research Program of Science and
Technology Department, Sichuan Province (2014JY0016), and Earmarked Fund for
China Agriculture Research System (CARS-08-B-3). And we are also grateful for the
experimental guidance from Dr. Jie Hu and Xuejing Jia in University of Macau.

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</BIBL>

<Figure>Fig. 1 Elution profiles of crude C. quinoa polysaccharides fraction on

DEAE-52 chromatography column with gradient of NaCl solution (0, 0.1, and 0.3 M)

(A), and on Sephadex G-100 gel chromatography column with distilled water.

<Figure>Fig. 2 FT-IR spectrum (A), GPC analysis spectrum (B), SEM image (C) of

CQP; HPLC chromatograms of seven kind of standard monosaccharides (D-1) and

monosaccharide composition in purified CQP (D-2). Note: Man, mannose; Rha,

Rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glucose; Gal,

galactose; Ara, Arabinose.

<Figure>Fig. 3 Antioxidant activity of purified CQP by DPPH free radical

scavenging assay (A) and ABTS radical scavenging assay (B).

<Figure>Fig. 4 Immunomodulation activities of CQP fraction in vitro. Effect of CQP

on the viability of RAW264.7 cells at different concentrations for 24 h (A) and 48 h

(B) incubation; Effect of CQP on LPS-stimulated NO production in RAW264.7

macrophages for 24 h (C) and 48 h (D) incubation. ▲ indicated p<0.05 versus control

and LPS treated groups, * indicated p<0.05 versus LPS treated groups and LPS-

stimulated groups with CQP treatment.

<Figure>Fig. 5 In vitro anticancer effect of CQP against human liver cancer SMMC

7721 cells, human breast cancer MCF-7 cells, normal liver L02 cells and normal

breast epithelial MCF 10A by MTT assay after 24 h (A) and 48 h (B) treatment.

<Table>Table 1 Factors and levels for orthogonal test

Levels
Variable
1 2 3

X1 ultrasonic temperature (°C) 70 80 90

X2 ultrasonic time (min) 20 30 40

X3 water to raw material ratio (mL/g) 15 20 25

<Table>Table 2 Result analysis of L9 (3)3 test

No. X1 X2 X3 Yields (%)

1 1 1 1 4.25

2 1 2 2 5.38

3 1 3 3 5.81

4 2 1 2 4.96

5 2 2 3 7.38

6 2 3 1 6.98

7 3 1 3 7.84

8 3 2 1 9.46

9 3 3 2 9.23
 K1 15.44 17.05 20.69

K2 19.32 22.22 19.57

K3 26.53 22.02 21.03

R 3.696666667 1.723333 0.486666667

<Table>Table 3 Variance and significance analysis result

Sum of
Factors Mean of square F value P value
deviation square

X1 21.114 10.557 228.013 0.0043▲▲

X2 5.718 2.859 61.758 0.0159▲

X3 0.389 0.194 4.201 0.1922

Error 0.092 0.046

Note: significance ▲P<0.05; ▲▲P<0.01

TDENDOFDOCTD