International Journal of Biological Macromolecules 94 (2017) 335–344

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Optimization of ultrasonic-assisted extraction of antioxidant

polysaccharides from the stem of Trapa quadrispinosa using response
surface methodology
Aun Raza a , Feng Li b,c , Xiuquan Xu a,∗ , Jian Tang a
a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, PR China
b
Affiliated Hospital of Jiangsu University, Zhenjiang, 212001, PR China
c
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, PR China

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

Article history: Response surface methodology (RSM) was used to optimize experimental conditions for ultrasonic-
Received 30 March 2016 assisted extraction (UAE) of antioxidant crude polysaccharides (CPS) from Trapa quadrispinosa stems.
Received in revised form 7 October 2016 Antioxidant capacity of polysaccharides was determined by Ferric-Reducing Antioxidant Capacity (FRAC)
Accepted 13 October 2016
assay with the method of 1, 10-phenanthroline. The maximum yield of polysaccharides (2.78 ± 0.16%)
Available online 14 October 2016
was obtained under optimal extraction conditions of extraction time, 41 min; ratio of water to material,
31.5 mL/g; and extraction temperature, 58 ◦ C. The maximum antioxidant capacity (19.02 ± 0.24 ␮mol
Keywords:
Fe2+ /g) was obtained under the optimal extraction conditions of extraction time, 38 min; ratio of water
Trapa quadrispinosa Roxb.
Antioxidant polysaccharides
to material, 32 mL/g; and extraction temperature, 56 ◦ C. These two values were agreed well with predicted
Ultrasonic-assisted extraction yield (2.75%) and antioxidant capacity (18.77 ␮mol Fe2+ /g). Antioxidant activities of CPS were investigated
Response surface methodology by various assays. The results showed that CPSUAE obtained by UAE showed higher 1,1-Diphenyl-2-
picrylhydrazxyl (DPPH), 2,2-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) radical scavenging
activities and stronger reducing power, total antioxidant capacity compared with CPSHWE obtained by
hot water extraction (HWE).The results indicated that UAE is an advisable method for extraction of
antioxidant polysaccharides from the stem of T. quadrispinosa and polysaccharides could be explored as
potential antioxidant to use in medicine or functional food.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction used to extract polysaccharides. Among these techniques, UAE is

a simple, rapid, inexpensive and efficient method alternative to
Polysaccharides are essential and important biological macro- conventional extraction method. It is most useful technique for
molecules in the plants. Polysaccharides have been described the extraction of polysaccharides because of low cost, low tem-
to possess a variety of therapeutic effects including anti- perature, and maximum extraction yield with stable biological
inflammatory, antioxidant, anti-tumor, anti-diabetic and neuro- properties [13,14]. In general, extraction of polysaccharides using
protective activities [1–5]. Most of these therapeutic effects are ultrasonic-assisted extraction from plant source is influenced by
mainly due to the antioxidant activity of polysaccharides. Several multiple parameters such as ultrasonic power, extraction temper-
polysaccharides with strong scavenging activities on DPPH and ature, extraction time and ratio of liquid to material and their
ABTS free radical, superoxide anion and hydroxyl radical have been effects may be either independent or interactive. Response sur-
used to treat cancer, diabetes and Alzheimer’s disease [6–8]. face methodology (RSM) has been described to be an effective
Normally, conventional extraction techniques such as hot water statistical technique for optimization of the polysaccharides extrac-
or boiling extraction and novel extraction methods including tion process when the independent variables have a combined
ultrasonic-assisted extraction (UAE), microwave-assisted extrac- effect on the desired response [15,16]. Furthermore, the yield of
tion (MAE), and pressurized water extraction (PWE) [9–12] are polysaccharides is considered to measure the advantages and dis-
advantages of extraction process. However, during these extraction
processes, polysaccharides can easily be lost along with decrease
of their antioxidant activity due to degradation and oxidation
∗ Corresponding author. because of long extraction time and high extraction temperature
E-mail address: xxq781026@ujs.edu.cn (X. Xu).

http://dx.doi.org/10.1016/j.ijbiomac.2016.10.033
0141-8130/© 2016 Elsevier B.V. All rights reserved.
336 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344

in water heating extraction [17–19]. Therefore, development of a Table 1

Coded and uncoded levels of independent variables used for Box-Behnken design
simple, quick and accurate method for determination of antioxi-
(BBD).
dant capacity of polysaccharides to evaluate the extraction process
is desirable. However, as far as we know, few reports described Independent variables Coded Levels
that the antioxidant method was applied to evaluate the extraction −1 0 1
process.
Ultrasonic time(X1 ) (min) 30 40 50
T. quadrispinosa Roxb., also known as Sijiaoling, an aquatic free- Liquid to material ratio(X2 ) (mL/g) 25 30 35
floating plant, is one of the most popular vegetables in China due Extraction temperature (X3 ) (◦ C) 50 60 70
to its medical functions and special taste [20]. The pericarps and
stems of T. quadrispinosa are commonly discarded as waste. Recent
studies have reported that the pericarps of T. quadrispinosa exhib- doubly distilled water for polysaccharides yield and antioxidant
ited excellent antioxidant and anti-cancer activities on behalf of its capacity determination. For comparison, in hot water extraction, a
excellent content of polyphenols [21,22]. However, the composi- 5.0 g sample was extracted using 150 mL of distilled water at 90 ◦ C
tions and biological activities of the stem of T. quadrispinosa had for 4 h.
rarely been examined to the best of our knowledge. The percentage polysaccharide yield from T. quadrispinosa stems
In our study, polysaccharides were detected in the stem of T. were determined by phenol-sulfuric method [23]. Glucose was cho-
quadrispinosa and exhibited good antioxidant activities. Thus, the sen as standard, and the polysaccharide yield (%) was calculated as
objective of present study was to explore the potential of ultrasonic follows:
technology in the extraction of crude polysaccharide (CPS) from content of crude polysaccharides (g)
polysaccharideyield (%) = × 100% (1)
weight ofsample powder (g)
the stem of T. quadrispinosa and to optimize the extraction con-
ditions using response surface methodology (RSM). A simple and The ferric-ion-based antioxidant capacity of polysaccharides
accurate ferric-ion-based total antioxidant capacity method with 1, was determined according to the method of 1,10-phenanthroline
10-phenanthroline was used to investigate the antioxidant capacity [24] with minor modification. Briefly, 0.2 mL of each polysaccha-
of CPS. The effects of ultrasonic power, extraction time, extraction ride solution mixed with 1 mL of 0.2% FeCl3 solution and 0.5 mL of
temperature, and liquid to material ratio on the extraction yield 0.5% 1,10-phenanthroline solution, followed by adjusting the final
and antioxidant capacity of CPS were optimized using Box-Behnken volume up to 10 mL with 50% methanol, mixing each adequately,
design (BBD). Furthermore the antioxidant properties of the CPS keeping in the dark for 30 min and measuring the absorbance at
were investigated by various assays in vitro. 510 nm. FeSO4 ·7H2 O was chosen as standard, and the antioxidant
capacity of polysaccharides was expressed as Fe2+ equivalent per
gram of weight material (␮mol Fe2+ /g).
2. Materials and methods
2.3. Experimental design
2.1. Plant material and chemical reagents
Based on the preliminary single factor results, RSM was used
The plant materials were collected from Weishan Lake,
to investigate and optimize the UAE conditions for CPS from the
Shandong province of China and authenticated as stems of T.
stem of T. quadrispinosa. Ultrasonic time (X1 , min), liquid to material
quadrispinosa .The air-dried and crushed (about 60 mesh) powder
ratio (X2 , mL/g), and ultrasonic temperature (X3 , ◦ C) were preferred
of T. quadrispinosa was degreased with petroleum ether (60–90 ◦ C)
for independent variables. The BBD with three variables and three
for 12 h in a Soxhlet apparatus and pretreated twice with 95%
levels was constructed. The yield and FRAC values of polysaccha-
ethanol for 6 h to remove some colored materials, monosaccha-
rides obtained from triplicates were designated as the response
ride, oligosaccharides and some small molecule materials [9].
dependent values. The variables and their levels, with both coded
Thereafter, the samples were obtained by filtration and dried at
and actual values are presented in Table 1. Based on experimen-
60 ◦ C until it attained the constant weight. D-glucose, phenol,
tal data from BBD, regression analysis was carried out, and the
Iron sulfate heptahydrate (FeSO4 ·7H2 O), Iron chloride hexahydrate
second-order polynomial model performed as follows:
(FeCl3 ·6H2 O), 1, 10-phenanthroline, DPPH and ABTS were pur-
chased from Sigma Chemicals Co. (St. Louis, MO, USA). All other 
3

3

2

3

chemical reagents used in experiment were of analytical grade and Y = A0 + Ai Xi + Aii Xi2 + Aij Xi Xj (2)
doubly distilled water was used throughout the experiment. i=1 i=1 i=1 j=i+1

Where Y is the predicted response; A0 is a constant, Ai is linear coef-

2.2. Ultrasonic-assisted extraction (UAE) ficient, Aii is quadratic coefficient and Aii is interaction coefficient.
Xi and Xj are independent variables.
The UAE was performed in an ultrasonic device (KQ-250DB,
Kunshan Ultrasonic Co. Ltd., China) to which the frequency was 2.4. Antioxidant activity of CPS
fixed as 40 kHz, electric power of 200W and equipped with a tem-
perature controller and a digital timer. Sample (1.0 g) was extracted The antioxidant activities of the two CPS named CPSUAE and
with distilled water under these various conditions: ultrasonic CPSHWE obtained by UAE and HWE respectively were evaluated by
power ranging from 120 to 200 W, ultrasonic time from 10 to DPPH radical scavenging, ABTS radical scavenging, reducing power
50 min, extraction temperature from 30 to 70 ◦ C and liquid to and total antioxidant capacity assay methods.
material ratio from 20 to 40 mL/g. After extraction, the mixtures
were centrifuged at 4000 rpm for 10 min to remove the insolu- 2.4.1. DPPH radical scavenging activity
ble materials. The supernatants were collected and concentrated The activity of the CPS on scavenging DPPH radical was mea-
to a certain volume under reduced pressure at 60 ◦ C, and then sured as reported previously [25] with slight modification. Briefly,
precipitated by the addition of absolute ethanol to a final concen- 1.0 mL of CPS water solution (5–50 ␮g mL−1 ) was thoroughly
tration of 80% (v/v) overnight at 4 ◦ C. The crude polysaccharides mixed with 2.0 mL of freshly prepared DPPH methanol solution
were obtained by centrifugation of precipitates at 5000 rpm for (0.1 mmol L−1 ). Then the mixture was incubated at room temper-
15 min. The obtained crude polysaccharides were re-dissolved in ature for 30 min in the dark. The absorbance of the solution was
 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344 337

3.0 20 3.0 20
Yield (%)
Yield (%)
FRAC (μM/g)

Polysaccharide yield (%)

Polysaccharide yield (%)

A FRAC (μM/g) B
2.5 2.5

FRAC (μM/g)
FRAC (μ M/g)
16 16
2.0 2.0

1.5 1.5
12 12

1.0 1.0
120 140 160 180 200 10 20 30 40 50
Ultrasonic power (W) Ultrasonic time (min)

3.0 20 3.0 20
Yield (%) Yield (%)

Polysaccharide yield (%)

Polysaccharide yield (%)

C FRAC (μM/g) D FRAC (μM/g)

2.5 2.5

FRAC (μ M/g)
FRAC (μ M/g )
16 16
2.0 2.0

1.5 1.5
12 12

1.0 1.0
20 25 30 35 40 30 40 50 60 70
Liquid to material ratio (mL/g) Extraction Temperature( OC)

Fig. 1. Effects of different extraction parameters on the yield and of antioxidant capacity of CPS: (A) ultrasonic power, (W); (B); ultrasonic time, (min) (C); liquid to material
ratio, (mL/g); (D) extraction temperature, (◦ C).

measured at 517 nm. The scavenging activity was calculated with at 700 nm against a blank after a 10 min reaction time. A higher
the following equation: absorbance indicated a higher reducing power.
A0 − (A1 − A2 )
Scavengingrate (%) = × 100 (3)
A0 2.4.4. Total antioxidant capacity assay
The total antioxidant capacity of CPS was evaluated by phos-
Where A0 was the absorbance of DPPH solution without any sam-
phomolybdenum method[28]. In a typical procedure, 1.0 mL of CPS
ple, A1 was the absorbance of a mixture solution of the sample and
(50–400 ␮g mL-1) solution were mixed with 3.0 mL of reagent solu-
DPPH, and A2 was the absorbance of the absorbance of the sample
tion (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM
solution without DPPH.
ammonium molybdate). The mixture was incubated at 90 ◦ C for
90 min. After cooling to room temperature, the absorbance of
2.4.2. ABTS radical scavenging activity the mixture was measured at 695 nm against a blank. A higher
The activity of the CPS on scavenging ABTS radical was per- absorbance indicated a higher total antioxidant capacity.
formed following a previously described method [26] with some Ascorbic acid was carried out as the positive control. And all the
modifications. The prepared ABTS+ solution was diluted to the experiments were carried out in triplicate.
absorbance of 0.70 ± 0.02 at 734 nm. An aliquot of 0.1 mL of
CPS water solution (5–50 ␮g mL−1 ) was mixed with 2.9 mL of
ABTS+ solution. After reacting for 6 min at room temperature, the 2.5. Fourier transform infrared (FT- IR) spectroscopy analysis
absorbance was immediately measured at 734 nm The ABTS radical
scavenging activity was calculated according to Eq. (3) with ABTS The FT-IR spectroscopic analysis of CPS from the stem of T.
solution instead of DPPH. quadrispinosa was measured by a spectrophotometer (Avater370,
Nicolet Co., USA). About 2 mg of CPS was ground with 100 mg
spectroscopic grade KBr powder and pressed into pellets for FT-IR
2.4.3. Reducing power assay
measurement in the range 4000–400 cm−1 at a resolution of 8 cm−1
The reducing power of CPS was determined through the trans-
[11].
formation of Fe3+ to Fe2+ according to a previously described
method [27] with a slight modification. An aliquot of 1.0 mL of
CPS (50–400 ␮g mL−1 ) solution were mixed with 2.5 mL phos- 2.6. Statistically analysis
phate buffer (pH 6.6, 0.2 mol L−1 ) and 2.5 mL of potassium
ferricyanide (1%, w/v). After incubation at 50 ◦ C for 20 min, 2.5 mL of Design-Expert version 8.0.5b, (Stat-Ease Inc., Minneapolis, MN,
trichloroacetic acid (10%, w/v) was added to the mixtures and cen- USA) was used to analyze the experimental data. The analysis of
trifuged at 4000 rpm for 10 min. Finally, 2.5 mL of the supernatant variance (ANOVA) was used to evaluate the significant terms in the
solution was mixed with 2.5 mL of distilled water and 0.5 mL of fer- models. The various statistical analysis parameters including lack-
ric chloride in water (1%, w/v), and the absorbance was measured of fit test, p value, F value, determination coefficient (R2 ), adjusted
338 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344

determination coefficient (Radj 2 ), coefficient of variation (C.V.%) Table 2

Box-Behnken design with independent variables and response values.
were preferred to evaluate the adequacy of the models.
Run X1 X2 X3 Response

Yield (%) FRAC (␮M/g)

3. Results and discussion
1 −1 −1 0 2.40 17.3
2 −1 1 0 2.40 17.6
3.1. Single-factor experimental analysis 3 −1 0 −1 2.10 17.7
4 −1 0 1 1.84 16.5
In this study, four key parameters influence on the UAE includ- 5 1 −1 0 2.43 16.4
ing ultrasonic power, ultrasonic time, liquid to material ratio and 6 1 1 0 2.47 17.4
7 1 0 −1 2.37 17.1
extraction temperature were picked out for investigation. The 8 1 0 1 1.74 13.8
effect of ultrasonic power on the yield and FRAC value of CPS 9 0 −1 −1 2.30 17.4
were studied from 120 to 200 W, with other fixed extraction con- 10 0 −1 1 2.04 15.6
ditions as follows: ultrasonic time, 40 min; liquid to material ratio, 11 0 1 −1 2.43 18.1
12 0 1 1 2.05 15.7
30 mL/g; and extraction temperature, 60 ◦ C. As shown in Fig. 1A,
13 0 0 0 2.68 18.2
the yield and FRAC value of CPS increased linearly and reached a 14 0 0 0 2.72 18.5
maximum level at 160 W, and after 160 W, the two values no longer 15 0 0 0 2.76 18.7
changed during extraction process. Therefore, 160 W was selected
as the optimal ultrasonic power according to the purpose of saving
energy. The effect of ultrasonic time on the yield and FRAC value of three parameters were preferred for optimizing the ultrasonic-
CPS was explored from 10 to 50 min with other extraction fixed as assisted extraction of antioxidant polysaccharides from the stem
follows: ultrasonic power, 160 W; liquid to material ratio, 30 mL/g; of T. quadrispinosa using RSM. The parameters and their levels are
and extraction temperature, 60 ◦ C. The effect of ultrasonic time on shown in Table 1.
the yield and FRAC value of CPS is presented in Fig. 1B. It can be
seen that, the yield and FRAC value of CPS increased quickly as the
3.2. Fitting the models
ultrasonic time ascended from 10 to 40 min and then, the yield
decreased along with significant decrease of FRAC value. These
The process variables and experimental data are presented in
results can be explained as the increasing extraction time led to
Table 2. The percentage yield and FRAC value of CPS from the
the increasing of material cell disruption and speeded up both the
stem of T. quadrispinosa ranged from 1.74 to 2.76% and from 13.8
release and diffusion of the CPS into water. On the other hand,
to 18.7 ␮mol Fe2+ /g respectively. The experiment 15 (Ultrasonic
excessively lengthening ultrasonic time will cause oxidization or
time 40 min, liquid to material ratio 30 mL/g, and extraction tem-
degradation of CPS; it will decrease the yield and FRAC value of CPS.
perature 60 ◦ C) represented the maximum yield and FRAC value,
Hence, 30–50 min was suitable ultrasonic time for the extraction of
while experiment 8(Ultrasonic time 50 min, liquid to material ratio
CPS from the stem of T. quadrispinosa. Fixing the ultrasonic power,
30 mL/g, and extraction temperature 70 ◦ C) produced the least yield
160 W; extraction time, 40 min; and extraction temperature, 60 ◦ C;
and FRAC value.
the influence of the ratio of liquid to material on the extraction yield
With analysis of variance (ANOVA), the second-order polyno-
and FRAC value of CPS is shown in Fig. 1C. The yield and of antiox-
mial models were applied to calculate the predicted response as
idant capacity of CPS increased quickly with the increased ratio of
follows:
liquid to material from 20 to 35 mL/g, while above 35 mL/g, the
yield and FRAC value of CPS decreased slowly. A larger ratio of liq- Y Yield = 2.72 + 0.034X 1 + 0.022X 2 − 0.19X 3 + 0.01X 1 X 2
uid to material indicates greater concentration difference between
the interior plant cells and the exterior solvent, and the diffusion − 0.093X 1 X 3 − 0.03X 2 X 3 − 0.24X 1 2
of polysaccharides occurred more quickly, which could lead to an − 0.051X 2 2 − 0.46X 3 2 (4)
enhancement of the extraction yield [29]. However, when the ratio
of liquid to material was increased markedly, the ultrasonic energy
attached to the unit volume would decrease, resulting in decrease
of extraction yield [24]. Therefore, the ratio of liquid to material was Y FRAC = 18.47 − 0.55X 1 + 0.26X 2 − 1.09X 3 + 0.18X 1 X 2
selected from 25 to 35 mL/g. The high extraction temperature could − 0.53X 1 X 3 − 0.15X 2 X 3 − 0.86X 1 2 − 0.43X 2 2 − 1.33X 3 2 (5)
increase the diffusion coefficient and solubility of polysaccharides,
therefore increase the polysaccharides yield. To study the effect
of extraction temperature on yield and FRAC value of CPS, extrac- The ANOVA for fitted quadratic polynomial model of extraction
tion process was carried out under different extraction temperature of polysaccharides and their antioxidant capacity are presented in
from 30 to 70 ◦ C, while other extraction conditions were as follows: Table 3. The statically significance of the model equations were
ultrasonic power, 160 W; liquid to material ratio, 30 mL/g; and evaluated by the F-test and p-value. According to the results, the
extraction time, 40 min. As shown in Fig. 1D, the extraction yield high F-values (56.05 for yield and 16.30 for FRAC value) and small
and FRAC value of CPS increased remarkably with the increased p-values (0.0002 for yield and 0.0034 for FRAC value, both p-values
temperature from 30 to 60 ◦ C, while above 60 ◦ C, the yield and <0.01) suggested that the regression models are more significant.
antioxidant capacity decreased significantly. The two extraction The goodness of fit of regression model was carried out by deter-
values increased with increasing the extraction temperature due to mination coefficient (R2 ) and adjusted determination coefficient
the increase of the polysaccharides solubility and decreased with (R2 adj ) [31]. The high values of R2 (yield and FRAC values 0.9902
higher extraction temperature due to the polysaccharides oxidiza- and 0.9670, respectively) and R2 adj (yield and FRAC values 0.9725
tion. [30]. Therefore, extraction temperature range of 50–70 ◦ C was and 0.9077, respectively) were reasonably close to 1, indicating a
considered to be optimal in the present experiment. high degree of correlation between the experimental and predicted
According to the single factor results, ultrasonic time, ratio values. Furthermore, the low values of coefficient variation (C.V.)
of liquid to material and ultrasonic temperature were more sig- (yield and FRAC values 2.19% and 2.31%, respectively) and the high
nificant on the yield and antioxidant capacity of CPS. So these values of Adeq. precision (yield and FRAC values 23.110 and 13.552
 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344 339

Table 3
Analysis of variance (ANOVA) of the quadratic model and lack of fit.

Variable Extraction yield (%) FRAC (␮M/g)

DF Sum of squares F-value P-value DF Sum of squares F value P value

Model 9 1.30 56.05 0.0002*** 9 22.72 16.30 0.0034**

X1 1 9.11 × 10−3 3.54 0.1187 1 2.42 15.63 0.0108*
X2 1 4.05 × 10−3 1.57 0.2652 1 0.55 3.56 0.1178
X3 1 0.29 113.64 0.0001*** 1 9.46 61.11 0.0005***
X1 X2 1 4.00 × 10−4 0.16 0.7097 1 0.12 0.79 0.4145
X1 X3 1 0.034 13.29 0.0148* 1 1.10 7.12 0.0444*
X2 X3 1 3.60 × 10−3 1.40 0.2902 1 0.09 0.58 0.4802
X1 2 1 0.22 85.19 0.0003*** 1 2.72 17.57 0.0086**
X2 2 1 9.70 × 10−3 3.77 0.1100 1 0.69 4.48 0.0879
X3 2 1 0.79 308.38 <0.0001*** 1 6.56 42.39 0.0013**
Residual error 5 0.013 5 0.77
Lack of fit 3 9.67 × 10−3 2.02 0.3486 3 0.65 3.41 0.2351
Pure error 2 3.20 × 10−3 2 0.13
Total 14 1.31 14 23.59
Adeq Precision 23.110 13.552
C.V. (%) 2.19 2.14
R2 0.9902 R2 adj 0.9725 R2 0.9670 R2 adj 0.9077

Notes: * Significant at 0.05 level; ** Significant at 0.01 level; *** Significant at 0.001 level.

Table 4
Comparison of experimental results obtained by the fitted model and the results obtained by the conventional method.

Extraction Methods Extraction conditions Response

◦
Time(min) L/M ratio(mL/g) Temperature( C) Yield (%) FRAC (␮mol Fe2+ /g)

UAE 41 31.5 58 2.78 ± 0.16 –

UAE 38 32 56 – 19.02 ± 0.24
HWE 240 30 90 2.32 ± 0.21 13.17 ± 0.19

respectively) indicated a very high degree of precision and a good ables exhibited significant negative quadratic effects in response
deal of reliability of the experimental values. of extraction yield (Eq. (4)). The decreased value of extraction yield
was mainly due to the thermal degradation of polysaccharides with
a long extraction time.
3.3. Response surface analysis of extraction yield of CPS

The F-value and P-value are used to check the significance of 3.4. Response surface analysis of FRAC value of CPS
each coefficient. The larger F-value and smaller P-value means the
more significant of coefficient. According to Table 3, it can be seen The effect of extraction time, liquid to material ratio and extrac-
that the yield of CPS was significantly influenced by one linear tion temperature on the antioxidant capacity of CPS are shown in
(X3 ) and two quadratic (X1 2 , X3 2 ) parameters (p < 0.01), and influ- Table 3. The FRAC value was significantly influenced by one linear
enced by one interaction effect of X1 X3 (0.01 < p < 0.05). However, (X3 ) and two quadratic (X1 2 , X3 2 ) parameters (p < 0.01) that were
the linear(X1 X2 ), quadratic (X2 2 ) and interaction effect of X1 X2 , similar to the yield value of CPS, while influenced by one linear (X1 )
X1 X3 had less influence on the yield of CPS due to higher p-value and one interaction effect of X1 X3 (p < 0.05).
(p > 0.05). The three-dimensional surface plots of FRAC value (Fig. 3) are
Moreover, three-dimensional surface plots about the relation- also similar to the extraction yield plots. It was observed that
ship between extraction parameters and extraction yield of CPS the FRAC value increased with increase in liquid to material ratio
were investigated by response surface methodology according to (Figs. 3A and C). This suggested that liquid to material ratio had a
Eq. (4). Fig. 2 showed the effect of ultrasonic time, liquid to material positive linear impact on the FRAC value of CPS. Furthermore, first
ratio and ultrasonic temperature and their mutual interaction on increasing and then decreasing trend of FRAC value was observed
the extraction yield of CPS. From Figs. 2A and C, when extraction with increase in extraction time and temperature (Fig. 3B). How-
time and temperature are fixed at level 0, the highest extraction ever, extraction time and temperature had negative quadratic
yield was observed with increase of liquid to material ratio. When impact on the FRAC value of CPS. The oxidization, degradation or
liquid to material ratio kept fixed (level 0), the extraction yield the change of physicochemical characteristics of CPS accompanied
increased at first with the increase of extraction time and temper- with further decrease of antioxidant capacity of CPS. This behav-
ature, and then decreased. Long extraction time or high extraction ior was similar to previously reported extraction of antioxidant
temperature could increase the diffusion and solubility rate of polysaccharides from Flammulina velutipes using different methods
polysaccharides from plant resulting in a highest extraction yield [15].
[32]. However, excessive extraction time and temperature could
also lead to the hydrolyzation or degradation of polysaccharides 3.5. Optimization and verification of predictive models
[10]. Therefore, the decrease of extraction yield with a long extrac-
tion time or a high extraction temperature could be explained by The objective of this study was to optimize the ultrasonic-
thermal degradation of polysaccharides. From Fig. 2B, we could assisted extraction conditions with maximum extraction yield and
observe the remarkable decrease of polysaccharides with higher antioxidant capacity of crude polysaccharides from the stem of
extraction temperature and longer extraction time. These two vari- Trapa quadrispinosa Roxb. The optimum extraction conditions for
340 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344

2.8 2.8

ld(%)
ld (%)
A B
2.6

Polysaccharide yie
Polysaccharide yie

2.6
2.4

2.4 2.2

2.0
2.2
1 .0 g) 1.8 1 .0
/ 0 o )
mL
0 -1 .
-1 . 0 .5 0 .5 C
-0 .
5 io( e(
0 .0
ra
t
Ex-t0
.5 0 .0 ur
Ext 0 .0 0 at
rac ial -0 . rac 0 . -0 . pe
r
tion 0.5 5 ter tion .5 5
em
tim - me tim 0 -1 . t
e (m 1.0 1 . 0 to e (m 1.0 0 on
in) id in) cti
qu t ra
Li Ex

2.8
ld (%)

C
2.6
Polysaccharide yie

2.4

2.2

2.0 1 .0
- 1 .0 0 .5 o
C
)
re (
5
Liq -0 . 0 .0
uid 0 .0 atu
- 0 .5 r
to m
0.5 pe
eter
ial 0 -1 . tem
1. 0
rat on
io (
mL acti
tr
/ g) Ex

Fig. 2. Surface plots of interactive effects of extraction time, liquid to material ratio and extraction temperature on the yield of CPS from the stem of T. quadrispinosa.

maximum extraction yield of polysaccharides were as follows: able for extraction antioxidant polysaccharides from the stem of
extraction time of 41.2 min, ratio of liquid to material of 31.5 mL/g, T. quadrispinosa.
and extraction temperature of 57.7 ◦ C.Under these optimal condi-
tions, the predicted highest yield of polysaccharides is 2.75%. For 3.6. Antioxidant activity of CPS
highest antioxidant capacity, optimal extraction conditions were
as follows: extraction time of 38.3 min, ratio of liquid to mate- 3.6.1. DPPH radical scavenging activity
rial of 31.7 mL/g and extraction temperature of 56.1 ◦ C.Under these DPPH a stable free radical can accept an electron or hydro-
optimal conditions, the predicted highest antioxidant capacity of gen radical to become a stable diamagnetic molecule and decrease
CPS is 18.77 ␮mol Fe2+ /g. Based on reliability conditions of exper- the absorbance at 517 nm, which has been widely used to inves-
imental process, the optimum extraction conditions for maximum tigate radical scavenging activity of natural polysaccharides [33].
extraction yield of polysaccharides were as follows: extraction The DPPH radical scavenging effects of CPSUAE , CPSHWE and ascor-
time, 41 min; ratio of water to material, 31.5 mL/g; and extrac- bic acid were investigated. As shown in Fig. 4A, the CPSUAE , CPSHWE
tion temperature, 58 ◦ C.Under these optimal conditions, the actual and positive control ascorbic acid exhibited obvious scavenging
maximum yield of polysaccharides is 2.78 ± 0.16%. And extrac- activity on DPPH radical with a concentration − dependent man-
tion time of 38 min, ratio of water to material of 32 mL/g and ner at a concentration range of 5–50 ␮g mL−1 . At a concentration
extraction temperature of 56 ◦ C.Under these optimal conditions, of 50 ␮g mL−1 , the DPPH radical scavenging activities of CPSUAE ,
the actual highest antioxidant capacity of CPS is19.02 ± 0.24 ␮mol CPSHWE and ascorbic acid were 87.4, 78.8 and 99.1% respectively.
Fe2+ equivalent/g. The experimental values obtained under the opti- At all tested concentration, CPSUAE displayed a higher scaveng-
mal extraction conditions with extraction yield of 2.78 ± 0.16% ing activity compared with CPSHWE although weaker than ascorbic
and antioxidant capacity of 19.02 ± 0.24 ␮mol Fe2+ /g were agreed acid. The result indicated that CPSUAE had a more powerful ability
well with the predicted values, that validated the suitability to transfer electron or hydrogen atom to DPPH than CPSHWE .
of the fitted response surface model. Moreover, comparing the
hot water extraction (HWE) with ultrasonic-assisted extraction 3.6.2. ABTS radical scavenging activity
(Table 4), UAE presented more extraction yield and better antiox- The ABTS assay is a decolorization assay applicable to both
idant capacity with increase of 16.5% and 30.7%, respectively. lipophilic and hydrophilic antioxidants at different pH levels [34].
These results indicated that ultrasonic-assisted method is suit- The results of ABTS radical scavenging activity of CPSUAE , CPSHWE
 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344 341

19 19

A B
18
M/g)

M/g)
18
Fe equivalent (μ

Fe equivalent (μ
17

16
17
2+

15

2+
16
1 .0
L/
g) 14 1 .0
(
o C)
-1 .
0
0 .5
i o(
m -1 .
0
0 .5 re
at tu
-0 .
5
lr Ext -
0 .5 ra
pe
0 .0 0 .0
Ext
rac 0 .0 ri a rac 0 .0
tion -0 .
5
m
et e t io n
tim .5
-0 .
5
n tem
tim 0.5
e (mi -1 . dt
o e (m 0 -1 . c tio
i
n) 1.0 0
qu
in) 1.0 0
tra
Li Ex

19

C
μM /g)

18
Fe equivalent (

17
2+

16
1 .0
)
(C
0
o
-1 . 0 .5
e
Liq .5 t ur
uid - 0 0 .0
ra
to m
eter
0 .0
mpe
ial 5
-0 .
5 te
rat 0. on
io (
mL -1 .
a cti
1.0 0
/g)
E xtr

Fig. 3. Response surface plots of interactive effects of extraction time, liquid to material ratio and extraction temperature on the FRAC value of CPS from the stem of T.
quadrispinosa.

and ascorbic acid were described in Fig. 4B. Similar to the results CPSHWE .in the concentration range of 50–400 ␮g mL−1 . Although
of DPPH method, all the samples showed remarkable effect on the reducing power of CPSUAE was lower than that of ascorbic acid, it
scavenging ABTS activities and the abilities increased with their still reached 0.559 at the concentration of 400 ␮g mL−1 . The reduc-
concentration. The scavenging effect of CPSUAE reached 90.4% at ing property was generally associated with the capacity of reacting
50 ␮g mL−1 which was close to that of ascorbic acid (99.6%) and with certain precursors of peroxide and preventing peroxide for-
higher than CPSHWE (84.7%) at the same concentration. The result mation [37]. Based on that theory, CPSUAE could have a stronger
indicated that CPSUAE had a stronger ABTS radical scavenging activ- ability to donate electrons and reduce peroxide than CPSHWE .
ity than CPSHWE .
3.6.4. Total antioxidant capacity assay
3.6.3. Reducing power assay The phosphomolybdenum method was based on the reduc-
In the reducing power assay, the antioxidants would result in the tion of Mo (VI) to Mo (V) and subsequent formation of a green
reduction of the Fe3+ /ferricyanide complex to its ferrous form (Fe2+ ) phosphate/Mo (V) complex at acid medium [28].The result of total
by donating an electron. Hence, the Fe2+ can then be monitored by antioxidant capacity of CPSUAE , CPSHWE and ascorbic acid were
measuring the formation of Perl’s Prussian blue at 700 nm [35]. The given in Fig. 4D. Similar to the results of reducing power, the total
reducing capacity of a compound have been served as a significant antioxidant capacity of CPSUAE was more pronounced than that
indicator of its potential antioxidant activity [36]. of CPSHWE . When concentration of samples reached 400 ␮g mL−1 ,
Fig. 4C showed the reducing power of CPSUAE , CPSHWE and ascor- CPSUAE and CPSHWE all exhibited obvious total antioxidant capac-
bic acid. The reducing power of CPSUAE was higher than that of ity. But compared with ascorbic acid (absorption of 2.12), CPSUAE ,
342 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344

100
100 B
A

Scavenging activity (%)

Scavenging activity (%)
80
80

60
60

40 40
CPS UAE
CPS UAE
CPS HWE
CPS HWE
20 20
ascorbic acid ascorbic acid

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Concentration(μ g/mL) Concentration (μg/mL)

1.6

Total antioxidant capacity(A695nm)

2.0 D
C
Reducing power (A 700nm )

1.2 CPS UAE

1.5 CPS UAE
CPS HWE CPS HWE
ascorbic acid
0.8 ascorbic acid
1.0

0.4 0.5

0.0 0.0
0 100 200 300 400 0 100 200 300 400
Concentration(μg/mL) Concentration (μg/mL)

Fig. 4. Antioxidant activities of CPS UAE obtained by UAE and CPSHWE obtained by HWE, (A) scavenging of DPPH radical; (B) scavenging of ABTS radical; (C) reducing power
and (D) total antioxidant capacity.

CPSHWE exhibited relatively lower activities of 0.67 and 0.51 respec-

tively.
In all assays, the antioxidant activities of CPSUAE were much
Transmittance(%)

higher than those of CPSHWE , which was agree with previous stud-
ies [38–41]. These studies revealed that the antioxidant activity
1238
of natural polysaccharides may be related to their composition, 1735
water solubility, molecular weight, molecular structure, monosac-
1420
charide component, structure and conformation, polarity and 2929 1369
intramolecular hydrogen bonds. The antioxidant activities of CPS
may be affected by the different of physicochemical characteris- 1157
tics between the CPSUAE and CPSHWE . CPS obtained by different
3380 1080
methods have a lot of differences, for example, polysaccha-
1022
ride content, protein content, molecular weight distribution and
3500 3000 2500 2000 1500 1000 500
monosaccharide compositions [41,42]. The antioxidant activities -1
of polysaccharides are affected by all of these differences. Several Wavenumber(cm )
researchers reported that CPS obtained from different methods
Fig. 5. FT-IR spectrum of CPS from the stem of T. quadrispinosa.
had different average molecular weight and distributions which
were the important factors on influence the antioxidant activ-
ity of polysaccharides [43–45].Therefore, the further purification,
characterization, and the more potential activities of the polysac-
charides from the stem of T. quadrispinosa should be further studied.

3.7. FT-IR analysis 2929 cm−1 was associated with stretching vibration of C H in the
sugar ring. The absorption bands at 1735 cm−1 represented the
FT-IR spectroscopy is a useful technique for the identification asymmetric stretching vibration of carbonyl double-bond (C O)
of characteristic organic groups in the polysaccharides. In order [46]. The bands at 1420, 1367 and 1238 cm−1 were assigned to
to confirm the identity of CPS, FT-IR was applied to analyze the C O stretching vibrations and O H deformation vibrations [47].
CPS. As shown in Fig. 5, the FTIR spectra of CPS showed a strong The strong absorption band between 1200 − 1000 cm−1 at 1157,
absorbance band at 3380 cm−1 . This represented the stretching 1080 and 1022 cm−1 were assigned to the stretching vibration of
vibration of O H in the constituent sugar residues. The band at C O C and C O H bonds [48,49].
 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344 343

4. Conclusions evaluation of its biological activities, Ultrason. Sonochem. 19 (6) (2012)

1160–1168.
[11] Y. Yuan, D. Macquarrie, Microwave assisted extraction of sulfated
UAE has been confirmed a simple, rapid, inexpensive and effi- polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant
cient method for the extraction of bioactive constituent from activity, Carbohydr. Polym. 129 (2015) 101–107.
medicinal plant. In this present study, UAE was used to extract [12] Y. Zhao, C. Sun, F. Shi, C.K. Firempong, J. Yu, X. Xu, W. Zhang, Preparation,
characterization, and pharmacokinetics study of capsaicin via
antioxidant polysaccharides from the stem of T. quadrispinosa. hydroxypropyl-beta-cyclodextrin encapsulation, Pharm. Biol. 54 (1) (2016)
Response surface methodology was successfully employed to opti- 130–138.
mize extraction conditions. At fixed ultrasonic power of 160 W, [13] S. Shen, D. Chen, X. Li, T. Li, M. Yuan, Y. Zhou, C. Ding, Optimization of
extraction process and antioxidant activity of polysaccharides from leaves of
the optimal extraction conditions were as follows: extraction time
Paris polyphylla, Carbohydr. Polym. 104 (2014) 80–86.
of 41 min, ratio of water to material of 31.5 mL/g and extraction [14] Q. You, X. Yin, C. Ji, Pulsed counter-current ultrasound-assisted extraction and
temperature of 58 ◦ C.Under these conditions, the highest polysac- characterization of polysaccharides from Boletus edulis, Carbohydr. Polym.
101 (2014) 379–385.
charides yield was 2.78%. And extraction time of 38 min, ratio
[15] W. Chen, W.P. Wang, H.S. Zhang, Q. Huang, Optimization of ultrasonic-assisted
of water to material of 32 mL/g and extraction temperature of extraction of water-soluble polysaccharides from Boletus edulis mycelia using
56 ◦ C for the maximum antioxidant capacity of 19.02 ␮mol Fe2+ /g. response surface methodology, Carbohydr. Polym. 87 (1) (2012) 614–619.
The antioxidant activities of the CPS were investigated by vari- [16] J.P. Maran, V. Mekala, S. Manikandan, Modeling and optimization of
ultrasound-assisted extraction of polysaccharide from Cucurbita moschata,
ous assays. The results showed that CPS obtained by UAE showed Carbohydr. Polym. 92 (2) (2013) 2018–2026.
higher DPPH, ABTS radical scavenging activity and stronger reduc- [17] W. Yongjiang, C. Zhong, M. Jianwei, F. Minger, W. Xueqian, Optimization of
ing power, total antioxidant capacity compared with CPS obtained ultrasonic-assisted extraction process of Poria cocos polysaccharides by
response surface methodology, Carbohydr. Polym. 77 (4) (2009) 713–717.
by HWE. The results showed that the application of ultrasound sig- [18] C. Chen, L.J. You, A.M. Abbasi, X. Fu, R.H. Liu, Optimization for ultrasound
nificantly decreased extraction time, and improved the extraction extraction of polysaccharides from mulberry fruits with antioxidant and
efficiency and the antioxidant activity of polysaccharides compared hyperglycemic activity in vitro, Carbohydr. Polym. 130 (2015) 122–132.
[19] C. Chen, Y. Shao, Y. Tao, H. Wen, Optimization of dynamic microwave-assisted
with the hot water method. Further study needs to purify the extraction of Armillaria polysaccharides using RSM, and their biological
polysaccharides and reveal the relationship between the structure activity, LWT-Food Sci. Technol. 64 (2) (2015) 1263–1269.
and the antioxidant activities. Hereby, we present a prospective [20] P.-Y. Chiang, J.-Y. Ciou, Effect of pulverization on the antioxidant activity of
water caltrop (Trapa taiwanensis Nakai) pericarps, LWT-Food Sci. Technol. 43
extraction method for isolation of polysaccharides from stem of T.
(2) (2010) 361–365.
quadrispinosa, and provide a theoretical basis for extraction of other [21] H. Yu, S. Shen, Phenolic composition, antioxidant, antimicrobial and
natural polysaccharides. This study also indicates that the polysac- antiproliferative activities of water caltrop pericarps extract, LWT-Food Sci.
Technol. 61 (1) (2015) 238–243.
charides from the stem of T. quadrispinosa could be explored as a
[22] Q.S. Lin, H.N. Yu, U.N. Das, S.R. Shen, Water caltrop pericarps extracts
good antioxidant agent with a potential application in food and attenuate H2O2-induced human umbilical vein endothelial cell injury, Adv.
medicine industry Mater. Res. Trans. Tech. Publ. (2012) 1757–1761.
[23] M. Dubois, K.A. Gilles, J.K. Hamilton, P. Rebers, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (3) (1956)
Acknowledgements 350–356.
[24] A. Szydłowska-Czerniak, C. Dianoczki, K. Recseg, G. Karlovits, E. Szłyk,
Determination of antioxidant capacities of vegetable oils by ferric-ion
The financial support for this investigation given by Chinese spectrophotometric methods, Talanta 76 (4) (2008) 899–905.
Government Scholarship (CSC No: 2014DFH792), Natural Science [25] Z. Wu, H. Li, D. Tu, Y. Yang, Y. Zhan, Extraction optimization, preliminary
characterization, and in vitro antioxidant activities of crude polysaccharides
Foundation of Jiangsu province, China (LZ13244) and Doctoral from finger citron, Ind. Crops Prod. 44 (2013) 145–151.
Innovation Project of Jiangsu province, China (CXLX13-686). [26] Q. Wang, Y. Sun, B. Yang, Z. Wang, Y. Liu, Q. Cao, X. Sun, H. Kuang,
Optimization of polysaccharides extraction from seeds of Pharbitis nil and its
anti-oxidant activity, Carbohydr. Polym. 102 (2014) 460–466.
References [27] B. Zhao, J. Zhang, X. Guo, J. Wang, Microwave-assisted extraction, chemical
characterization of polysaccharides from Lilium davidii var. unicolor Salisb
[1] C. Chen, R. Kasimu, X. Xie, Y. Zheng, W. Ding, Optimised extraction of and its antioxidant activities evaluation, Food Hydrocoll. 31 (2) (2013)
Erythronium sibiricum bulb polysaccharides and evaluation of their 346–356.
bioactivities, Int. J. Biol. Macromol. 82 (2016) 898–904. [28] I. Lassoued, L. Mora, R. Nasri, M. Jridi, F. Toldrá, M.C. Aristoy, A. Barkia, M.
[2] J.A. Batista, E.G. Dias, T.V. Brito, R.S. Prudêncio, R.O. Silva, R.A. Ribeiro, M.H.L. Nasri, Characterization and comparative assessment of antioxidant and ACE
Souza, R.C. de Paula, J.P. Feitosa, L.S. Chaves, Polysaccharide isolated from inhibitory activities of thornback ray gelatin hydrolysates, J. Funct. Foods 13
Agardhiella ramosissima: chemical structure and anti-inflammation activity, (2015) 225–238.
Carbohydr. Polym. 99 (2014) 59–67. [29] Z.Y. Zhao, Q. Zhang, Y.F. Li, L.L. Dong, S.L. Liu, Optimization of ultrasound
[3] X. Jia, C. Zhang, J. Qiu, L. Wang, J. Bao, K. Wang, Y. Zhang, M. Chen, J. Wan, H. extraction of Alisma orientalis polysaccharides by response surface
Su, Purification, structural characterization and anticancer activity of the methodology and their antioxidant activities, Carbohydr. Polym. 119 (2015)
novel polysaccharides from Rhynchosia minima root, Carbohydr. Polym. 132 101–109.
(2015) 67–71. [30] V. Samavati, A. Manoochehrizade, Dodonaea viscosa var. angustifolia leaf: new
[4] J. Gao, Y.L. Han, Z.Y. Jin, X.M. Xu, X.Q. Zha, H.Q. Chen, Y.Y. Yin, Protective effect source of polysaccharide and its anti-oxidant activity, Carbohydr. Polym. 98
of polysaccharides from Opuntia dillenii Haw. fruits on (1) (2013) 199–207.
streptozotocin-induced diabetic rats, Carbohydr. Polym. 124 (2015) 25–34. [31] P.P. Ghavi, The extraction process optimization of antioxidant
[5] Y.S. Ho, M.S. Yu, X.F. Yang, K.F. So, W.H. Yuen, R.C.C. Chang, Neuroprotective polysaccharides from Marshmallow (Althaea officinalis L.) roots, Int. J. Biol.
effects of polysaccharides from wolfberry, the fruits of Lycium barbarum, Macromol. 75 (2015) 51–57.
against homocysteine-induced toxicity in rat cortical neurons, J. Alzheimer’s [32] V. Samavati, Polysaccharide extraction from Abelmoschus esculentus:
Dis. 19 (3) (2010) 813–827. optimization by response surface methodology, Carbohydr. Polym. 95 (1)
[6] J. Wang, Y. Zhao, W. Li, Z. Wang, L. Shen, Optimization of polysaccharides (2013) 588–597.
extraction from Tricholoma mongolicum Imai and their antioxidant and [33] H. Wang, X.D. Gao, G.C. Zhou, L. Cai, W.B. Yao, In vitro and in vivo antioxidant
antiproliferative activities, Carbohydr. Polym. 131 (2015) 322–330. activity of aqueous extract from Choerospondias axillaris fruit, Food Chem. 106
[7] J. Fu, J. Yuan, Y. Tu, J. Fu, N. Zhang, B. Gao, G. Fu, Y. Zhang, A polysaccharide (3) (2008) 888–895.
from Acanthopanax senticosus improves the antioxidant status in [34] P. Shao, X. Chen, P. Sun, Chemical characterization, antioxidant and antitumor
alloxan-induced diabetic mice, Carbohydr. Polym. 88 (2) (2012) 517–521. activity of sulfated polysaccharide from Sargassum horneri, Carbohydr. Polym.
[8] M. Wozniak, T. Bell, Á. Dénes, R. Falshaw, R. Itzhaki, Anti-HSV1 activity of 105 (2014) 260–269.
brown algal polysaccharides and possible relevance to the treatment of [35] G. Zhang, M. Hu, L. He, P. Fu, L. Wang, J. Zhou, Optimization of
Alzheimer’s disease, Int. J. Biol. Macromol. 74 (2015) 530–540. microwave-assisted enzymatic extraction of polyphenols from waste peanut
[9] K. Feng, W. Chen, L. Sun, J. Liu, Y. Zhao, L. Li, Y. Wang, W. Zhang, Optimization shells and evaluation of its antioxidant and antibacterial activities in vitro,
extraction, preliminary characterization and antioxidant activity in vitro of Food Bioprod. Process. 91 (2) (2013) 158–168.
polysaccharides from Stachys sieboldii Miq. tubers, Carbohydr. Polym. 125 [36] Z. Zhang, G. Lv, H. Pan, L. Shi, L. Fan, Optimization of the microwave-assisted
(2015) 45–52. extraction process for polysaccharides in himematsutake (Agaricus blazei
[10] R. Chen, Y. Li, H. Dong, Z. Liu, S. Li, S. Yang, X. Li, Optimization of ultrasonic Murrill) and evaluation of their antioxidant activities, Food Sci. Technol. Res.
extraction process of polysaccharides from Ornithogalum Caudatum Ait and 17 (6) (2011) 461–470.
344 A. Raza et al. / International Journal of Biological Macromolecules 94 (2017) 335–344

[37] Y. Song, Y. Ni, X. Hu, Q. Li, Effect of phosphorylation on antioxidant activities water and enzymolysis from Grifola frondosa, Int. J. Biol. Macromol. 48 (5)
of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide, Int. J. Biol. (2011) 798–803.
Macromol. 81 (2015) 41–48. [44] X. Li, L. Wang, Effect of extraction method on structure and antioxidant
[38] Z.M. Wang, Y.C. Cheung, P.H. Leung, J.Y. Wu, Ultrasonic treatment for activity of Hohenbuehelia serotina polysaccharides, Int. J. Biol. Macromol. 83
improved solution properties of a high-molecular weight exopolysaccharide (2016) 270–276.
produced by a medicinal fungus, Bioresour. Technol. 101 (14) (2010) [45] Z.-y. Zhu, F. Dong, X. Liu, Q. Lv, F. Liu, L. Chen, T. Wang, Z. Wang, Y. Zhang,
5517–5522. Effects of extraction methods on the yield, chemical structure and anti-tumor
[39] B. Yang, M. Zhao, J. Shi, N. Yang, Y. Jiang, Effect of ultrasonic treatment on the activity of polysaccharides from Cordyceps gunnii mycelia, Carbohydr. Polym.
recovery and DPPH radical scavenging activity of polysaccharides from 140 (2016) 461–471.
longan fruit pericarp, Food Chem. 106 (2) (2008) 685–690. [46] Z. Ying, X. Han, J. Li, Ultrasound-assisted extraction of polysaccharides from
[40] K. Zhong, L. Tong, L. Liu, X. Zhou, X. Liu, Q. Zhang, S. Zhou, Immunoregulatory mulberry leaves, Food Chem. 127 (3) (2011) 1273–1279.
and antitumor activity of schizophyllan under ultrasonic treatment, Int. J. [47] J. Chen, T. Zhang, B. Jiang, W. Mu, M. Miao, Characterization and antioxidant
Biol. Macromol. 80 (2015) 302–308. activity of Ginkgo biloba exocarp polysaccharides, Carbohydr. Polym. 87 (1)
[41] Z. Zhang, G. Lv, W. He, L. Shi, H. Pan, L. Fan, Effects of extraction methods on (2012) 40–45.
the antioxidant activities of polysaccharides obtained from Flammulina [48] W.C. Zeng, Z. Zhang, H. Gao, L.R. Jia, W.Y. Chen, Characterization of
velutipes, Carbohydr. Polym. 98 (2) (2013) 1524–1531. antioxidant polysaccharides from Auricularia auricular using
[42] S. Jia, F. Li, Y. Liu, H. Ren, G. Gong, Y. Wang, S. Wu, Effects of extraction microwave-assisted extraction, Carbohydr. Polym. 89 (2) (2012) 694–700.
methods on the antioxidant activities of polysaccharides from Agaricus blazei [49] Q. Yuan, Y. Xie, W. Wang, Y. Yan, H. Ye, S. Jabbar, X. Zeng, Extraction
Murrill, Int. J. Biol. Macromol. 62 (2013) 66–69. optimization, characterization and antioxidant activity in vitro of
[43] Y. Fan, X. Wu, M. Zhang, T. Zhao, Y. Zhou, L. Han, L. Yang, Physical polysaccharides from mulberry (Morus alba L.) leaves, Carbohydr. Polym. 128
characteristics and antioxidant effect of polysaccharides extracted by boiling (2015) 52–62.