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Extraction, characterization and antioxidant activity of polysaccharide

from Pouteria campechiana seed

Jin-Shuang Ma, Hong Liu, Chang-Ri Han, Si-Jie Zeng, Xiao-Jun Xu,
Deng-Jun Lu, Hong-Ju He

PII: S0144-8617(19)31076-8
DOI: https://doi.org/10.1016/j.carbpol.2019.115409
Reference: CARP 115409

To appear in: Carbohydrate Polymers

Received Date: 24 June 2019

Revised Date: 21 September 2019
Accepted Date: 29 September 2019

Please cite this article as: Ma J-Shuang, Liu H, Han C-Ri, Zeng S-Jie, Xu X-Jun, Lu D-Jun, He
H-Ju, Extraction, characterization and antioxidant activity of polysaccharide from Pouteria
campechiana seed, Carbohydrate Polymers (2019),
doi: https://doi.org/10.1016/j.carbpol.2019.115409

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Extraction, characterization and antioxidant activity of polysaccharide from Pouteria campechiana seeds 1

Jin-Shuang Ma a, Hong Liu a, *, Chang-Ri Han b,*, Si-Jie Zeng c, Xiao-Jun Xu a, Deng-Jun Lu c, Hong-Ju He d

a Key Laboratory of the Ministry of Education of Tropical Medicine, Hainan Normal University, Haikou 570203, China
b
Key Laboratory of Medicinal and Edible Plants Resources of Hainan Province, Hainan Vocational University of
Science and Technology, Haikou 571125, China
c
College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
d
School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China

* Corresponding Author. Tel.:13976086314.

E-mail address: lhyd123@126.com(H. Liu), hchr116@126.com (C-R. Han)

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Highlights

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 Optimization extraction of polysaccharide from Pouteria campechiana seeds by RSM.

 PCSPa-1 is a high molecular weight polysaccharide with a glucose structure.


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The anti-DPPH radicals activity of the PCSPa-1 sample was comparable to that of Vc.
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ABSTRACT
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In this study, the seed polysaccharides (PCSP) was ultrasonic-assisted extracted from Pouteria campechiana and
optimized by response surface method (RSM). After separation and purification by DEAE-52 cellulose column and
Sephadex G-75 glucan gel column, the pure polysaccharide component of PCSPa-1 was obtained, and its structure
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and antioxidant activity were analyzed. The results showed that the optimal parameters of PCSP with maximum yields
(15.94%) were ultrasonic temperature of 79 °C, ultrasonic time of 69 min, and liquid to material ratio of 41 mL/g. The
molecular weight of PCSPa-1 was 67900 Da. PCSPa-1 consisted of glucose and mannose with a molar ratio of
86.65:4.62, and the glycosidic bond mainly included →4)-α-D-Glc(1→ and →6)-α-D-Glc(1→. Scanning electron
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microscopy showed that PCSPa-1 was a strip structure with a smooth surface. In addition, PCSPa-1 had strong
scavenging capacity to 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis- (3-ethylbenzthiaz oline-6-sulphonic
acid) (ABTS), hydroxyl radical, and superoxide radical. Polysaccharides of Pouteria campechiana seeds could be
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exploited as a natural antioxidant.

Keywords: Pouteria campechiana seeds; Polysaccharides; Structural characterization; Antioxidant

1. Introduction

Pouteria campechiana belongs to the family Sapotaceae, that is a perennial tropical fruit tree and widely

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distributes in South America and subtropical regions (Silva, Simeoni & Silveira, 2009). The yellow pulp is very
popular due to its sweet and tastes like egg yellow (Ma, Yang, Basile & Kennelly, 2004), has been used as antioxidant,
cosmetic and hepatoprotective with their richness in carotene (Kubola, Siriamornpun, & Meeso, 2011; Aseervatham et
al., 2014). Seeds are a major by-product of Pouteria campechiana consumption process and have not been fully utilized.
According to Morton and Elsayed (Morton, 1987; Elsayed, EI-Tanbouly, Moustafa, Abdou & EL Awdan, 2016),
Pouteria campechiana seeds are traditional medicine for inflammatory, pain, and ulcers. Elsayed separated
protocatechuic acid, quercetin, myricetin, myricetin-3-O-β-galactoside, gallicacid, and myricetin-3-O-α-L-rhamnoside
in the seeds from Pouteria campechiana (Andrade, Martins, & Sarzi, 2002; Padmathilake, Bandara, Qader, & Kumar,
2017). At present, the research on Pouteria campechiana seeds mainly focused on seed germination rate, endophytic
fungi, and biological activity. There are few reports on the extraction of polysaccharides from Pouteria campechiana
seeds.
Polysaccharides are a kind of macromolecular compounds widely existing in plants, animals and microorganisms,
and have biological activities including antioxidant, antiviral, antitumor, and anticoagulant (Chen et al., 2011; Chai &
Zhao, 2016). Bioactive polysaccharide has broad market, its discovery and evaluation has been a hot research topic.

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There are many methods for extracting polysaccharides, such as microwave, ultrasonic, enzymatic, and water
extraction (Ahmad, Alkharfy, Wani & Raish, 2015). Ultrasonic assisted method has been widely used in extraction of

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plant polysaccharides in recent years due to its advantages of simplicity, rapidity, and high extraction yield (Zhao,
Zhang, Li & Dong, 2015). In general, extraction yields of polysaccharides are affected by various factors including
ultrasonic temperature, ultrasonic time, ultrasonic power, and solvent extraction. Response surface methodology (RSM)

interactions on response values (Mkadmini et al., 2016).

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is an effective mathematical statistical technique for optimizing the influence of independent variables and their

In this study, RSM was used to optimize parameters of ultrasonic extraction of seed polysaccharides from
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Pouteria campechiana (PCSP), and it was further separated, purified, characterized by spectroscopy and chemical
techniques. In addition, the antioxidant activity of it also was evaluated. Provide a theoretical basis for in-depth
research and comprehensive utilization of Pouteria campechiana seeds.
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2. Materials and methods

2.1 Materials
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Pouteria campechiana from Vietnam was purchased from fruit wholesale market (Haikou, China) in 2017, and
identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University. Dried seeds were milled to
pass through 60 mesh sieve, and placed in a desiccator. Glucose (Glc), galactose (Gal), mannose (Man), arabinose
(Ara), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), and xylose (Xyl), ribose (Rib), glucosamine
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(GlcN), fucose (Fuc), guluronic acid (GulA), mannuronic acid (ManA), galactosamine (GalN), 1-phenyl-3-methyl-5-
pyrazolonde (PMP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulphonic acid)
(ABTS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dialysis bag (3500 Da) was
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purchased from Union Carbide Co. (Viskase, USA). All the salts were analytical grade and purchased from Aladdin
Biotechnology Co., Ltd. (Shanghai, China). Ethanol and other solvents were analytical grade and purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2 Extraction

Seed powders of Pouteria campechiana were refluxed (soxhlet extraction) at 85 ℃ for 8 hours to remove fat with
petroleum ether, followed with soaking in 95% ethanol for 48 hours to remove impurities ( Thirumavalavan, Lai, Lin &
Lee, 2010). After, they were filtered, dried, and extracted by ultrasonic method with a certain volume of distilled water. 5.0
g of the sample was placed in a conical flask and extracted three times according to a certain ultrasonic time, ultrasonic

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power and ultrasonic temperature.The extracts were mixed, centrifuged at 4000 g for 15 min and concentrated by rotary
evaporators. Four volumes of 95% ethanol solution were added and kept for 12 h at 4 ℃. The precipitate was collected by
filtration. Seed crude polysaccharides of Pouteria campechiana were obtained by lyophilizing. The content of
polysaccharide was determined by phenol-sulfuric acid method (Dubios, Gilles, Hamilion, Rebers, & Smith, 1956). The
yield of polysaccharide was calculated by the following equation：

Crude polysaccharide weight (g)

yield (%)  100% (1)
Raw material weight (g)

2.3 Single factor experimental design

Effects of ultrasonic power (100-600 W), ultrasonic time (40-90 min), ultrasonic temperature (50-100 °C), liquid to
material ratio (10-60 mL/g ) on the yield of polysaccharide were investigated.

2.4 Response surface experimental design

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Based on the single factor experiment results, the ultrasonic time, ultrasonic temperature, liquid to material ratio were
selected as independent variables, and the yields of PCSP were investigated as the response. The three-factor and three-level

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experiment using Box-Behnken center combination design principle are listed in Table 1. The experimental data were fitted
to the following quadratic polynomial model equation:

Y  β0  
3

i 1
βi X i  
3

i 1
βii X i2 
3

 β
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i 1 j i 1
ij X i X j
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Where Y is the predicted response, β0, βi, βii,and βij represent the regression coefficients of constant, linear, quadratic,
and interaction, respectively. Xi and Xj are independent variables.
Table 1 Factor and levels table
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Independent variables
Levels TemperatureX1 Time Liquid to material
(℃) X2 (min) ratio X3 (mL/g)
-1
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70 60 30
0 80 70 40
1 90 80 50
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2.5 Purification of crude polysaccharide

PCSP was deproteinized with trichloroacetic acid (15%). The protein-removed PCSP (200 mg) was dissolved in
distilled water, eluted with distilled water and NaCl (0.1, 0.2, 0.3, 0.4, 0.5 M) solution at the rate of 1 mL/min by
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DEAE-52 column (2.6 × 50 cm). 5 mL of eluent was collected each tube, and the content of polysaccharide was monitored
by the phenol-sulfuric acid method. The fraction with the highest polysaccharide content was collected and merged, then
concentrated, dialyzed (3500 Da), lyophilized. After that , fractions continued to be eluted with distilled water at 0.5 mL/min
by Sephadex G-75 (26 mm × 80 cm) column, 2 mL was collected each tube and monitored again by the phenol-sulfuric acid
method. The homogeneous fractions were collected, and lyophilized to generate purified seed polysaccharide (PCSPa-1)
(Wang & Luo, 2007).

2.6 Characterization of PCSPa-1

2.6.1 Homogeneity and molecular weight

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 The molecular weight and homogeneity of PCSPa-1 were determined by high-performance gel permeation
chromatography (HPLC, Waters 1515, USA) equipped with Ultrahydrogel 250 PKGD column and Waters 2414
refractive index detector. The column was eluted with sodium nitrate aqueous solution (0.1M) at a flow rate of 0.5
mL/min. The molecular weight of PCSPa-1 was calculated by the curve calibrated with T-series Dextran standards
(Mw 330,176, 82.500, 44, 25.3, 20.6, 12.6, 7.13, 4.29 and 1.4 kDa) .

2.6.2 Monosaccharide composition analysis

The monosaccharide composition of PCSPa-1 was analyzed by high performance liquid chromatography (HPLC)
( Dai et al., 2010). 20 mg sample and 5 mL trifluoroacetic acid (TFA, 2 M) were added to the clamp-mouth bottle to be
hydrolyzed in the oil bath at 120 °C for 2 h under the nitrogen atmosphere. Vacuum-dried monosaccharide sample
hydrolyzate was redissolved in pure water (5 mL) after TFA was removed with methanol. 400 μL of polysaccharide
hydrolyzate, 400 μL of PMP methanol solution (0.5 M) and NaOH (0.3 M) were reacted at 70 °C for 2 h, followed with 400
μL of HCl (0.3 M) for neutralization at room temperature. The mixture was dissolved in 1 mL chloroform and filtered
through a 0.45 μm microporous membrane for HPLC (Agilent 1100, USA) analysis. The standards monosaccharide mixed

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solution (400 μL, 0.4 mg/mL) was analyzed by HPLC as described methyl method. Chromatographic conditions: Column:
C18 (250 mm × 4.6 mm, 5 μm). Mobile phase A: 100 mM sodium phosphate buffer (pH = 6.7). Mobile phase B: acetonitrile.

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Detection wavelength: 250 nm. Column temperature 30 °C. Flow rate: 1 mL/min. Injection volume: 5 μL.

2.6.3 FT-IR spectrometric analysis

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The infrared spectrum of PCSPa-1 was determined using a FT-IR (Nicolet 6700, Thermo Scientific, USA). The dried
PCSPa-1 was ground with KBr at a ratio of 1:100 and pressed into pellets for FT-IR measurement in the frequency range of
500-4000 cm−1, then scanned 32 times with a resolution of 4 cm−1.
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2.6.4 Scanning electron microscopy

Samples were fixed on a copper platform with conductive adhesive and then sprayed with gold powder. The scanning
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electron microscope (SEM) images of PCSPa-1 at different magnification were observed by field emission scanning electron
microscopy (ISM-7100F, JEOL, Japan).

2.6.5 NMR spectrometric analysis

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The dried PCSPa-1 (25 mg) was dissolved in D2O and freeze-dried three times for hydrazine exchange, and then
samples were completely dissolved in 0.5 mL of D 2O before NMR analysis. 1H NMR and 13C NMR were recorded on a
Bruker AV-400 MHz spectrometer (Switzerland) at 25 °C (Pereira et al., 2005).
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2.7 Antioxidant activity

2.7.1 DPPH radical scavenging assay

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The DPPH radical scavenging capacity of PCSPa-1 was determined according to the method of Brand-williams,
Cuvelier & Berset (1995), with a slight modification. Briefly, 2 mL sample solution with different concentrations (0.2, 04,
0.6, 0.8, 1.0 mg/mL) was mixed with 2 mL of DPPH ethanol solution (1 mM) . The reaction was protected from light for 30
min at room temperature, and then the absorbance was measured at 517 nm. Ascorbic acid (Vc) was used as control. The
scavenging rate was calculated using the following equation:

1 -  A1  A2 
Scavenging rate (%)   100% (2)
A0

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where A0 is the absorbance of distilled water instead of sample, A1 is the absorbance of the sample, A2 is the absorbance of
distilled water instead of pyrogallol.

2.7.2 Superoxide radical scavenging assay

Superoxide radical scavenging experiments were performed by the method previously described by Wang & Liao
(2010), with a slight modification. 0.05 M Tris-HCI buffer solution (pH 8.2), 35 mM pyrogallol solution, and 8 mM HCl
solution were prepared for use. 0.1 mL sample solution with different concentrations (0.2, 04, 0.6, 0.8, 1.0 mg/mL) was
mixed with 4.5 mL of Tris-HCl buffer solution. After reacting for 8 min in a water bath at room temperature, 0.4 mL of
pyrogallol was added. The reaction was stopped by adding 0.1 mL of HCl after 5 min. The absorbance was measured at 325
nm. Vc was used as control. The scavenging rate was calculated with the following equation:

1 -  A1  A2 
Scavenging rate (%)   100% (3)
A0

where A0 is the absorbance of distilled water instead of sample, A1 is the absorbance of the sample, A2 is the absorbance of

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distilled water instead of pyrogallol.

2.7.3 ABTS radical scavenging assay

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The ABTS radical scavenging of PCSPa-1 was assessed with the method of Re, Pellegrini, Proteggente, Pannala,
Yang, & Evans (1999), with a slight modification. The ABTS solution was adjusted with absolute ethanol to make
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0.70±0.20 of the absorbance (734 nm). Reaction mixture contained 0.2 mL sample solution with different concentrations
(0.2, 04, 0.6, 0.8, 1.0 mg/mL) and 3.8 mL ABTS solution. After reacting for 6 min at room temperature, the absorbance was
determined at 734 nm. Vc was used as control. The scavenging rate was calculated with the following equation:
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1 -  A1  A2 
Scavenging rate (%)   100% (4)
A0
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where A0 is the absorbance of absolute ethanol instead of sample, A1 is the absorbance of the sample, A2 is the absorbance
of absolute ethanol instead of ABTS .

2.7.4 Hydroxyl radical scavenging assay

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Assessment of the scavenging ability of PCSPa-1 on hydroxyl radicals was performed according to the method described
by Smirnoff & Cumbes (1989), with a slight modification. 1 mL sample solution with different concentrations (0.2, 04, 0.6,
0.8, 1.0 mg/mL) was mixed with FeSO4 (6 mM, 1 mL), salicylic acid (6 mM, 1 mL) and H 2O2 (6 mM, 1 mL). The reaction
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was carried out in a water bath (37 °C) for 1 h. The absorbance was measured at 520 nm. Vc was used as control. The
scavenging rate was calculated with the following equation:

1 -  A1  A2 
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Scavenging rate (%)   100% (5)

A0

where A0 is the absorbance of absolute ethanol instead of sample, A1 is the absorbance of the sample, A2 is the absorbance
of absolute ethanol instead of salicylic acid.

2.8 Statistical analysis

All data were expressed as mean ± standard deviation (SD). The experiment was repeated three times. ANOVA was
used to analyze RSM results, and the difference was considered significant at P < 0.05.

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 3. Results and discussion

3.1 Effect of single factor on the yield of PCSP

As shown in Fig. 1A, the yield of PCSP gradually increased with the increase of ultrasonic time from 40 to 70 min and
reached the peak value when the extraction time was 70 min, the yield of PCSP started to decrease with increasing of the
ultrasonic time. This study indicated that the 70 min was enough to obtain the yield of polysaccharides. Sharifian-Nejad &
Shekarchizadeh (2019) pointed out that the yield of Agaricus blazei polysaccharide maintained a descending dynamic
equilibrium after reaching the optimal extraction time. The results may be explained that ultrasound would rupture the cell
wall of sample and accelerate release of polysaccharide in a short time. However, the destroyed structure of polysaccharide
resulted in the reduce of yield at a longer time with ultrasonic exaction (Rara, Li, Xu & Tang, 2017).
Temperature is an important factor in the extraction of polysaccharides. As shown in Fig. 1B, the yield of PCSP
increased with the increase of extraction temperature from 50 to 80℃ and reached a maximum at 80℃, then slowly decreased
with increasing temperature. This change was mainly due to an increase in temperature to accelerate molecular motion, but
when the temperature was too high to hydrology part of polysaccharide results to lower yields. Therefore, the optimum

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ultrasonic temperatures for PCSP was 80 °C, respectively (Samavati & Manoochehrizade, 2013).
As shown in Fig. 1C, the maximum yield of PCSP was obtained when the liquid to material ratio was 40 mL/g. The

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yield of polysaccharide decreased with the increase of liquid to material ratio after reached a maximum. The reason explained
that the diffusion rate of polysaccharide decreased slowly with the increase of solvent amount leading to a decrease of
polysaccharide yield. Therefore, 40 mL/g was selected as extraction conditions for PCSP (Szydłowska-Czerniak, Dianoczki,
Recseg, Karlovits & Szłyk, 2008). -p
As shown in Fig. 1D, the yield of PCSP increased with the increase of ultrasonic power, and reached a maximum at
400W. After the point of 400W, the yields changed slowly. Therefore, the optimum ultrasonic power of PCSP was 400W.
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24 20
(A)
(B)
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20
16

16
Yield(%)
Yield(%)

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12
12

8
8
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4 4
30 40 40 50 50 60 60 70 70 80 80 90 90 100 100 110
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Temperature(C)
Time(min)

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 24

20

16

Yield(%)
12

0
100 200 300 400 500 600
Power(W)

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20

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16
Yeild(%)

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4
0 10 20 30 40 50 60 70
liquid to material ratio(mL/g)

(C) (D)
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Fig. 1. Effect of different time(A), temperature(B), liquid to material ratio(C), power(D) on extraction yield of PCSP.

3.2 Optimization of extraction process of polysaccharides by RSM

According to the results of single factor experiments, the effects of ultrasonic time, ultrasonic temperature, and liquid
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to material ratio on polysaccharide yield were more significant. Therefore, these three factors were chosen as independent
variables. Box-Benkhen experimental design and data were listed in Table 2. The quadratic multiple regression model
equations for PCSP was obtained by linear fitting of the data：
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2 2 2
Yseed=15.44-0.21X1-0.28X2+0.18X3+0.19X1X2-0.14X1X3-0.18X2X3-1.27X1 -1.28X2 -0.85X3

Table 2 Box-Benkhen design with independent variables and response values

Run X1 X2 X3 Extraction yield(%)

1 1 0 -1 18.34
2 0 -1 -1 17.6
3 -1 0 -1 19.1
4 0 1 1 17.97

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 5 1 0 1 18.37
6 1 -1 0 18.15
7 0 -1 -1 17.5
8 0 0 0 19.56
9 -1 0 1 18.25
10 1 1 0 18.1
11 0 0 0 18.45
12 -1 -1 0 18.2
13 0 0 0 19.8
14 0 0 0 19.91
15 -1 1 0 19.32
16 0 0 0 19.75
17 0 0 0 20.00
The variance results of the regression model of PCSP was shown in Table 3. The significance of the model was
evaluated by F-value and p-value. The larger F-value and smaller p-value meant more significant of model. As shown in

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Table 3, F-value of PCSP model was 278.34, and p-value was <0.0001, indicating that the model was extremely significant.
The degree of fit of model was evaluated by the coefficient of determination (R2) and the coefficient of adjustment after
adjustment (R2adj). PCSP models’ R2 and R2adj was 0.9972 and 0.9936, respectively, indicating that model was well, and

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correlation between experimental and predicted values were very high. In addition, C.V.% of the PCSP model was 0.65%,
indicating the experimental values were accuracy and reliability. In summary, the regression model of PCSP was good (Ghavi,
2015; Guo, Zou & Sun, 2010; Fan, Hu, Fu, & Zhang, 2015).
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Response surface plots and contour plots clearly shown the interactions between various factors on the response values,
and the steeper response surface and more elliptical contour map meant more significant of interaction (Yu & Chao, 2013).
As shown in Fig. 2, when one of the factors was at 0 level, the yield of PCSP increased first and then decreased with the
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increase of other two factors. The curves of X1, X2, and X3 were steep, indicating that they had a great impact on extraction
process, which was consistent with results in the ANOVA of PCSP regression model. According to the contour plots,
interactions between X1X2, X1X3 and X2X3, have a significant effect on the yield of PCSP (Cheng et al., 2017).
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By using software Design-Expert, the optimal extraction conditions for PCSP were ultrasonic temperature 79 °C,
ultrasonic time 69 min, liquid-material ratio 41 mL/g, and the predicted value of 15.48. PCSP was extracted according to
optimal process parameters, and the actual extraction rate was 15.94%, which was close to the predicted value, indicating
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that the model was reliable.

Table 3 Analysis the variance of PCSP model

Seed extraction yield (%)

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Variable
DF Sum of squares F-value P-value
Model 9 20.25 278.34 <0.0001**
X1 1 0.36 44.68 0.0003**
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X2 1 0.62 76.19 <0.00018**

X3 1 0.25 31.17 0.0008**
X 1X 2 1 0.14 17.39 0.0042*
X 1X 3 1 0.076 9.35 0.01848*
X 2X 3 1 0.13 16.48 0.00488*
X 12 1 6.83 844.86 < 0.0001**
X 22 1 6.89 851.51 < 0.0001**
X 32 1 3.07 379.56 < 0.0001**

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 Residual error 7 0.057
Lack of fit 3 0.02 0.71 0.5964
Pure error 4 0.037
Cor Total 16 20.31
Adeq Precision 41.397
C.V.(%) 0.65 R2=0.9972 R2adj=0.9936

Note: *Significant at 0.05 level; **Significant at 0.001 level.

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(A) (B)
(C)
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Fig. 2. Response surface plots (A, B and C) showing the effects of ultrasonic time (X1), ultrasonic temperature (X2), liquid
to material ratio (X3) on the yield of PCSP.

3.3 Purification of PCSP

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PCSP was separated by DEAE-52 cellulose column to obtain main components PCSPa (Fig. 3A), which was further
separated by Sephadex G-75. The purified polysaccharides were obtained and named PCSPa-1 (Fig. 3B) .
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3.4 Characterization of PCSPa-1

3.4.1 Molecular weight and monosaccharide composition analysis

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The HPGPC chromatograms of PCSPa-1 (Fig. 4A) appeared as a single symmetrical elution peak, indicating the
relatively uniform distribution of their molecular weight. The average molecular weights of approximately of PCSPa-1
was 6.79× 104 Da. This value was lower than those reported for Descurainia sophia seed gum (2.1 × 106 Da) ( Sherahi
et al., 2017), Sage seed gum (4.0 × 105 Da) ( Razavi, Cui, Guo & Ding, 2013), vinal gum (1.43 × 10 6 Da) (Busch,
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Kolender, Santagapita & Buera, 2015), and Prosopis juliflora gum (1.73 × 106 Da) (Azero & Andrade, 2006), but also
a high molecular weight polymer. In addition, polysaccharides with high molecular weight have thickening or gelling
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properties, so it can be used a stabilizing in protein foams. (Martinez, Baeza, Millan & Pilosof, 2005). The
monosaccharide composition of polysaccharide was measured by HPLC, and the results were shown in Fig. 4B and
C. PCSPa-1 was mainly composed of glucose and mannose in a molar ratio of 86.65:4.62. These data clearly
demonstrated that PCSPa-1 was a heterogeneous polysaccharide with glucose as the dominant constituent. The
glucose/mannose ratio was the key property of heteropolysaccharides, which mainly determined their physicochemical
properties (Sherahi et al., 2017). PCSPa-1 was more soluble in water. The results of monosaccharide determination of
PCSPa-1 were consistent with the nuclear magnetic resonance spectrum (NMR).

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 3.0 3.0
PCSPa(H2O)
PCSPa-1
2.5 2.5

2.0 2.0
Absorbance

Absorbance
1.5 1.5

1.0 PCSPb 1.0

(0.1mol/LNaCl) PCSPe
(0.4mol/LNaCl)
PCSPd
0.5 PCSPc 0.5
(0.3mol/LNaCl)
(0.2mol/LNaCl)
0.0 0.0
0 20 40 60 80 100 0 10 20 30 40
Number of tube Number of tube

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(A) (B)
Fig. 3. DEAE-52 column chromatography of PCSP (A). Sephadex G-75 column chromatography of PCSPa-1 (B).

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3.4.2 FT-IR spectrum analysis

PCSPa-1 was characterized by FT-IR as shown in Fig. 4D. The broad band at 3403 cm-1 was attributed to O-H stretching
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vibration in the polysaccharide or moisture of sample, and the band at 2924cm -1 was attributed to C-H stretching vibration
of methylene or methyl groups in the polysaccharide. The band at 1630cm-1 was due to C=O stretching vibration or the H-
O-H vibration (Silverstein, Bassler &Morrill, 1992; Kaèuráková et al, 2000).. The bands at 1153 cm-1, 1081 cm-1, and 1023
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cm-1 were attributed to the stretching vibration of pyran ring (Dong et al., 2016). The band in the range of 1000-1200 cm -1
was also assigned to the bending or stretching vibration of C-O groups. In addition, the band at 848cm-1 was α-glycosidic
bands, indicating that both PCSPa-1 contained an α-glucopyranose ring (Percival, 1932).
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3.4.3 SEM analysis

SEM image of PCSPa-1 (water soluble white powder) was shown in Fig. 4E. The morphology of PCSPa-1 was irregular
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strip shape under low magnification mirror, and the surface was smooth and flat under high power microscope. The
intermolecular crosslinks between PCSPa-1 were very tight, which was also characteristic of most plant polysaccharides. It
is reported earlier that particle size and specific surface area influence their intrinsic viscosity and molecular mass. The
dissolution rate of polysaccharide powders generally increases with reduction in particle size. It can be shown from the
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figure that the particles of the PCSPa-1 were smaller (Malsawmtluangi et al, 2004).

3.4.4 NMR spectrum analysis

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1H
NMR of PCSPa-1 was shown in Fig. 4F. PCSPa-1 had two anomeric proton signal peaks at 5.32, 5.08 ppm.
According to references, 5.32 ppm was →)4-α-D-Glc(1→ anomeric proton signal peak; 5.08 was→6)-α-D-Glc(1→
anomeric proton signal peak. 13C NMR of PCSPa-1 was shown in Fig 4G. The 90-101 ppm range was α- anomeric
carbon region, and 170-185 ppm range was carboxylic acid region. 99.57 ppm was →)4-α-D-Glc(1→ anomeric carbon
signal peak; 97.88 ppm was →6)-α-D-Glc(1→ anomeric carbon signal peak (Wang et al., 2018; Xu et al., 2018; Li et
al., 2018).
According to the results of 1H NMR and 13C NMR of PCSPa-1, it was speculated that PCSPa-1 contained →4)-
α-D-Glc(1→ and →6)-α-D-Glc(1→ glycosidic bond. At the same time, there was no characteristic signal peak in the
range of 170~185 ppm, indicating that PCSPa-1 were no uronic acid.

10
 Table 4 Chemical shifts of resonances in the 1H and 13C spectra of PCSPa-1

Glycosidic bond C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6

→)4-α-D-Glc(1→ 99.58/5.32 69.16/3.85 72.40/3.64 76.74/3.66 71.22/3.89 60.45/3.76
→6)-α-D-Glc(1→ 97.88/5.08 70.69/3.68 73.35/3.79 71.67/3.56 71.54/3.95 66.85/3.74

30 320

25 PMP

20 240
Electrovoltage(mV)

15

mAU
10 160
6
5
5 3 4 13
1112
910 14

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0 80 12 7
8
-5

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-10 0
0 4 8 12 16 20 24 28 32
0 5 10 15 20 25 30
Retention time(min) Time(min)

800
(A) -p
100
(B)
re
9 848.82
600 90 1411.90
Transmittance(%)

2924.76 1630.82
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PMP
mAU

400 80
1153.05
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200 70
3403.86 1023.61
3
0 60
0 5 10 15 20 25 30
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4000 3500 3000 2500 2000 1500 1000 500

-1
Time(min) Wavenumber(cm )

(C) (D)
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（E）

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（F） （G）
Fig. 4. HPGPC chromatogram of PCSPa-1(A). HPLC chromatogram of standard monosaccharide (B) and PCSPa-1(C)
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(1:guluronic acid; 2:mannuronic acid; 3:mannose; 4:ribose; 5:rhamnose; 6:glucosamine; 7:glucuronic acid; 8:galacturonic
acid; 9:Glucose; 10:galactosamine;11:galactose; 12:xylose; 13:arabinose; 14:fucose). FT-IR spectrum of PCSPa-1(D). SEM
of PCSPa-1(E). 1H NMR spectrum of PCSPa-1(F) .13C NMR spectrum of PCSPa-1(G).

3.5 Antioxidant activity

In this study, DPPH, ABTS, hydroxyl, and superoxide radical scavenging activities of PCSPa-1 and Vc were
investigated to achieve comprehensive and accurate estimation of antioxidant activity, and the results are shown in Fig. 5.
DPPH was a very stable free radical whose ethanol solution has a strong absorption peak at 517 nm and appeared in purple.
With the strengthening of antioxidants, the purple color would gradually become lighter, which could be used to judge the

12
ability of antioxidants (Omp & Tejk, 2009). As shown in Fig. 5A, the IC50 values of the scavenging ability of PCSPa-1 and
Vc to DPPH radicals were 10.31 and 1.48 μg/mL, respectively. In the ABTS radicals assay, antioxidants can provide
electrons or hydrogen atoms to change the color of ABTS, and the antioxidant capacity of sample was calculated by
measuring the absorbance at 734 nm ( Lee & Yoon, 2018). As shown in Fig. 5B, the IC50 values of the scavenging ability of
PCSPa-1 and Vc to ABTS radicals were 29.9, and 3.14 μg/mL, respectively. Hydroxyl and superoxide were reactive oxygen
species produced during the metabolism of organisms, and the excess would break balance in the organism and promote
spread of various diseases. Therefore, it was very important to eliminate hydroxyl and superoxide radical (Wang et al., 2017).
As shown in Fig. 5C and D, the IC50 values of the scavenging ability of PCSPa-1 and Vc to hydroxyl radicals were 98.72,
and 8.92 ug/mL, respectively. The IC50 values of the scavenging ability of PCSPa-1 and Vc to superoxide radicals reached
358.22, and 7.74 μg/mL, respectively. Based on the results of the antioxidant evaluation, the scavenging ability of PCSPa-1
and Vc to DPPH, ABTS, hydroxyl, and superoxide radicals increased with the increasing sample concentration, the
scavenging activity of Vc was the best, and PCSPa-1 was slightly inferior.
Polysaccharides are one of the important antioxidants. In this experiment, the PCSPa-1 was assayed by various
methods such as DPPH, ABTS, superoxide and hydroxyl radical scavenging effect which was compared with Vc.

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Surprisingly, the IC50 of PCSPa-1 scavenging DPPH radical was lower than Vc, but more than the data reported for other
natural polymers like Prunus cerasoides D. Don gum (980 μg/mL), Hsian-tsao leaf gum (510 μg/mL) Prunus amygdalus

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gum(6600 μg/mL) and Albizia stipulate Boiv. gum (891 μg/mL) (Malsawmtluangi et al., 2014; Lai, Chou & Chao,2001;
Bouaziz et al., 2014; Thanzami et al., 2015), indicating PCSPa-1 was better antioxidant. In addition, the study also found
that the antioxidant capacity of sample depended on the concentration, and with the concentration increased, the
antioxidant capacity also increased. The observed results were consistent with those of other previous research (Malsaw-
mtluangi et al., 2014; Pu et al., 2016 ).
-p
re
100
DPPH radical scavenging ability(%)

ABTS radical scavenging ability(%)

100
lP

80
80

60 60
PCSPa-1
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Vc
40
40
PCSPa-1
20 Vc
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20
0
0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0
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Concentration(mg/mL) Concentration(mg/mL)

(A) (B)

13
 100

Superoxide radical scavenging ability(%)

100
Hydroxyl radical scavenging ability(%)

80 80

PCSPa-1
60 60 Vc

40 40
PCSPa-1
Vc
20 20

0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0
Concentration(mg/mL) Concentration(mg/mL)

(C) (D)

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Fig. 5. Antioxidant activity of PCSPa-1 and Vc. DPPH radical scavenging activity(A). ABTS radical scavenging
activity(B). Hydroxyl radical scavenging activity(C). Superoxide radical scavenging activity (D).

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4. Conclusion

In this study, the optimization of ultrasonic-assisted extraction of seed polysaccharides from Pouteria
campechiana (PCSP) was conducted by RSM. The optimal conditions for seed polysaccharide were temperature 79 ℃,
time 69 min, liquid to material ratio 41 mL/g, and polysaccharide yield was 15.94%. The main fractions PCSPa-1 was
isolated from crude PCSP with an average molecular weight of 67.9 kDa. Structural characterization showed that
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PCSPa-1 was mainly composed of glucose (more than 85%), and contained →4)-α-D-Glc(1→ and →6)-α-D-
Glc(1→glycosidic bond. PCSPa-1 was a strip structure with a smooth surface. In addition, PCSPa-1 had strong
antioxidant capacity. Therefore, the new technology can be used to develop and process the seeds of Pouteria
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campechiana to promote the comprehensive utilization of Pouteria campechiana resources.

Acknowledgments

This study was financially supported by Student's Innovation Training Program of Hainan Normal University
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(No. Hsyx2017-13).
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