International Journal of Biological Macromolecules 117 (2018) 1314–1325

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International Journal of Biological Macromolecules

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

Optimization extraction, characterization and anticancer activities of

polysaccharides from mango pomace
Huigang Hu a,1, Qiaoli Zhao a,⁎,1, Zhencai Pang a, Jianghui Xie c, Lijing Lin b, Quansheng Yao a
a
Ministry of Agriculture Key Laboratory of Tropical Fruit Tree Biology, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, China
b
Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, China
c
Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, 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 was used to optimize the extraction conditions for ultrasonic-assisted extraction of
Received 9 March 2018 polysaccharides from mango pomace. The Optimum extraction conditions consisted of extraction temperature of
Received in revised form 22 May 2018 74 °C, ultrasonic power of 170 W, extraction time of 100 min, and raw material-to-water ratio of 1:40 g/mL. Under
Accepted 30 May 2018
these conditions, the extraction yield was 3.71 ± 0.07%. Three novel polysaccharide fractions, MG-1, MG-2 and
Available online 31 May 2018
MG-3 were purified from the crude polysaccharides by using DEAE-52 cellulose and Sephadex G-100 column
Keywords:
chromatography. The molecular weight and monosaccharide composition of polysaccharide fractions (MPFs)
Mango pomace were analyzed by high performance liquid gel permeation chromatography (HPGPC) and HPLC analysis, respec-
Polysaccharides tively. The characterizations of MPFs were conducted with FT-IR, 1H NMR and SEM. Furthermore, the anticancer
Ultrasonic-assisted extraction activities of the polysaccharide fractions were also investigated in vitro. Results showed that MG-1, MG-2
Characterization and MG-3 exhibited significant anticancer activities against HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and
Anticancer activity BGC-823 cells in a dose-dependent manner. MPFs were also showed to promote apoptosis as seen in the nuclear
Response surface methodology morphological examination study using calcein acetyl methoxy methyl easter (calcein-AM) and propidium iodide
(PI) staining. This research could serve as a theoretical reference for the efficient utilization of MPFs in biomedical
and functional food.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction Polysaccharides, a group of biological macromolecules composed of

Mango (Mangifera indica L.), known as “the king of tropical fruit”, is
an important fruit crops in tropical and subtropical regions, native to
North India and the Malay peninsula. It is a favorite by consumers
worldwide because of the delicate flesh and rich nutrition [1–3]. The
mango planting area in tropical and subtropical regions of China is
8.5 × 103 ha, with a total output of 9.64 × 104 tons. However, the ined-
ible mango pomace accounts for 9%–16% of fresh fruits. Pomace is a by-
product generated during mango processing, it is usually discarded
as agricultural waste, which aggravates environmental pollution [4].
Mango pomace, which are rich in bioactive compounds, such as dietary
fiber, pectin, phenolic compounds, polysaccharides and flavonoids.
Studies have shown that mango pomace can promote alvine peristalsis,
anti-lipid peroxidation and lower cholesterol levels. Therefore, studying
the deep processing and utilization of mango pomace residue has im-
portant practical and economic significance.
⁎ Corresponding author.
E-mail address: zql2819557995@sina.com (Q. Zhao).
1
These authors contributed equally to this paper.
many monosaccharides linked together through glycosidic bonds [5],
widely exist in plants, animals, microorganisms and algal cell walls.
Moreover, polysaccharides from plants have attracted increasing
attention due to their many biological activities, such as antioxidant
[6, 7], antitumor [8], antidiabetic [9], anticancer [10], antiviral and
immunomodulating [11]. The conventional extraction method of poly-
saccharides from plant tissues is hot water extraction, the method
usually requires high extraction temperature and long extraction time,
but with low extraction yield and high energy consumption [12, 13].
Compared with the traditional methods, the ultrasonic-assisted extrac-
tion has been widely used to obtain active components from different
plant materials because of its lower energy consumption, shorter ex-
traction time, lower extraction temperature, and less solvent require-
ment [14, 15]. However, till date there is no investigation reported on
the optimization of ultrasonic-assisted extraction of polysaccharides
from mango pomace (MPP).
Response surface methodology (RSM) is an efficient statistical
technique for optimizing complicated processes such as extraction of
polysaccharides and it's advantageous for reducing the number of
experimental trials needed to evaluate multiple parameters and their
interactions [16]. Moreover, it is less laborious and time consuming

https://doi.org/10.1016/j.ijbiomac.2018.05.225
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1315

than other methods. Box-Behnken Design (BBD) is the most commonly centrifugation (4000 r/min, 10 min), washed two to three times with
used design in RSM duo to fewer runs. pure ethanol, acetone and ether. After lyophilization, the crude mango
In recent years, researches on the MPP had gained increasing atten- polysaccharides were obtained. The extraction yield of polysaccharides
tion over the past few years due to its unique properties. Recently, was determined by the phenol-sulfuric acid method using glucose as
studies on different extraction methods and experimental materials the standard reference, and the extraction yield was calculated by the
for mango polysaccharide had been reported. Maran et al. [4] obtained following formula:
polysaccharides from mango peels using microwave-assisted extraction
and optimized extraction conditions through single-factor tests and m1
Extraction yieldð%Þ ¼  100 ð1Þ
RSM. Chen et al. [1] obtained two purified polysaccharides (MSP I and m
MSP II) from mango seeds using hot water extraction and ethanol
where m1 is the content of crude polysaccharides (g) and m is the
precipitation. In their research, the extraction conditions, monosaccha-
weight of dried mango pomace powder (g).
ride composition and antioxidant activities of the polysaccharides
were investigated. Their results showed that all purified polysaccharide
2.3. Optimization of extraction conditions by RSM
fractions exhibit potential antioxidant activities.
Despite these numerous studies, it was obvious that researches on
Five single factor experiments were performed to determine
MPP in the last ten years was scattered, little was known about the
the preliminary range of the extraction factors, including extraction
ultrasonic-assisted extraction process, monosaccharide composition,
temperature (A), ultrasonic power (B), extraction time (C), raw
molecular weight, structural characteristics, and bioactive properties
material-to-water ratio (D) and extraction times (E). To simplify the
of MPP. Therefore, in this study, we extracted crude polysaccharides
experimental process, the number of extraction was set to 2 times,
by ultrasonic-assisted extraction method and optimized the extraction
and a four-factor-three-level BBD design was used to determine the op-
conditions using RSM, isolation and purification of MPP, determined
timal extraction conditions of ultrasonic-assisted extraction method.
their preliminary structural characteristics, and the anticancer activities
The extraction yield of MPP was selected as the response value, the
in vitro were also evaluated.
whole design consisting of 29 experimental points including 24 factorial
points and 5 axial points, and the level of encoding test factors are
2. Materials and methods
shown in Table 1. The quadratic model for predicting the optimal
point was expressed according to the Formula (2):
2.1. Materials and chemicals
X
4 X
4 3 X
X 4
Freshly harvested mango fruits were purchased from Ruihua Y ¼ β0 þ βi X i þ βii X 2i þ βij X i X j ð2Þ
Agriculture Company (Sichuan, China). Fruits without damage and in- i¼1 i¼1 i¼1 j¼iþ1
sect pest were selected. The pomace was collected and washed with
tap water, dried at 55 °C for 36 h, smashed in a blender, and then sieved where Y is the predicted response (experimental values) and β0 is an in-
at 40 mesh. tercept; βi, βj and βij are the regression coefficients for linear, quadratic
DEAE-52 cellulose, Sephadex G-100 gel and monosaccharide stan- and interaction terms, respectively; Xi and Xj are the independent
dards, including mannose, rhamnose, glucuronic acid, galacturonic
acid, glucose, galactose, xylose, arabinose and fucose, were purchased Table 1
from Solarbio Bioscience & Technology Co., Ltd. (Shanghai, China). Experimental design and results for response surface analysis.
1-Phenyl-3-methyl-5-pyrazolone (PMP) and dextrans T-series stan-
Run Factors Extraction
dards were purchased from Sigma Co., Ltd. (St. Louis, MO, USA). yield (%)
Trifluoroacetic acid (TFA), anhydrous ethanol, sulfuric acid, phenol, ace- Extraction Ultrasonic Extraction Raw
temperature power time material-to-water
tone and ethyl ether were obtained from Fuyu Fine Chemical Co., Ltd. (°C) (W) (min) ratio (g/mL)
(Tianjin, China). All reagents were domestic analytical pure, except for
1 −1 −1 0 0 2.56
acetonitrile, which was of HPLC grade.
2 1 −1 0 0 3.57
3 −1 1 0 0 2.99
2.2. Extraction of MPP 4 1 1 0 0 3.69
5 0 0 −1 −1 3.27
The mango pomace powder was extracted with 80% ethanol for 6 0 0 1 −1 3.09
7 0 0 −1 1 3.43
24 h by soaking in a beaker to remove some alcohol-soluble substances, 8 0 0 1 1 3.52
such as monosaccharide, oligosaccharide, lipids, colors and polyphenols 9 −1 0 0 −1 2.79
[17]. After filtration, the residues were collected, dried at 55 °C, and 10 1 0 0 −1 3.41
then stored in a desiccator at room temperature until use. The dried 11 −1 0 0 1 2.87
12 1 0 0 1 3.52
pretreated samples were subjected to ultrasonic-assisted extraction in
13 0 −1 −1 0 3.13
a PS-30ALD ultrasonic cleaning bath (Cleaning Electric Appliance Co., 14 0 1 −1 0 3.58
Ltd. Shenzhen). 1.0 g of pretreated sample was extracted in single- 15 0 −1 1 0 3.25
factor experiments under the following conditions: extraction tempera- 16 0 1 1 0 3.54
ture of 40–80 °C, ultrasonic power of 120–184 W, extraction time of 17 −1 0 −1 0 2.66
18 1 0 −1 0 3.43
20–120 min, and raw material-to-water ratio of 1:10–1:50 g/mL. Before 19 −1 0 1 0 2.72
extraction, the extraction temperature and ultrasonic power were 20 1 0 1 0 3.40
preset. When the set temperature was reached, the sample was placed 21 0 −1 0 −1 3.06
in an ultrasonic cleaning bath, after which time was recorded. After ex- 22 0 1 0 −1 3.12
23 0 −1 0 1 3.17
traction, the supernatant was separated by centrifugation at 6000 rpm
24 0 1 0 1 3.13
for 10 min, and the insoluble residue was treated again as mentioned 25 0 0 0 0 3.71
above. The collected supernatant was incorporated and concentrated 26 0 0 0 0 3.80
to 1/5 of original volume using a rotatory evaporator at 50 °C under re- 27 0 0 0 0 3.83
duced pressure and precipitated with 95% ethanol (24 h at 4 °C) to a 28 0 0 0 0 3.91
29 0 0 0 0 3.75
final concentration of 80% (v/v). The precipitates were obtained by
1316 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325
variables (i ≠ j). XiXj and X2i are the interaction and quadratic terms,
respectively.
2.4. Purification of crude polysaccharides
The freeze-dried samples were solubilized in distilled water,
deproteinization and decolorization with trichloroacetate-n-butanol solu-
tion and activated carbon, respectively. After dialysis (8000–14,000 Da)
for 48 h in distilled water, 95% ethanol precipitation and lyophiliza-
tion, the resulting precipitate was dissolved in ultrapure water and
purified sequentially by a DEAE-52 cellulose column (2.6 × 40 cm)
and Sephadex G-100 chromatography (1.3 × 60 cm) as previously
described [18]. The sample solution (10 mL, 50 mg/mL) was loaded
onto a DEAE-52 column and stepwise eluted with distilled water,
0.1, 0.2, 0.3, 0.4 and 0.5 mol/L NaCl solutions at a flow rate of
1.0 mL/min. The elution fraction (10 mL/tube) was collected and
determined by the phenol–sulfuric acid method at 490 nm using glu-
cose as the standard [19]. The main polysaccharide fractions (named
MG-1, MG-2 and MG-3) were pooled and concentrated at 50 °C by
rotary evaporator. Then, dialyzed, lyophilized and further purified
through a Sephadex G-100 column by distilled water at a flow rate
of 0.5 mL/min, and the resulting eluent (4 mL/tube) was pooled and
detected. The fractions with the largest peak profile were concentrated,
dialyzed and then lyophilized by the same methods as the above
mentioned. The refined polysaccharides were kept in dryer for further
analysis [20].
2.5. Physical and chemical properties analysis
2.5.1. I2-KI reaction
The 2 mL of different MPFs were added into the miniature centrifuge
tubes, then added 1.2 mL iodine solution, observed the color change.
Distilled water as negative control and 1 mg/mL starch solution as
positive contrast. If the reaction is negative, indicating there's no starch
in the MPFs [21].
2.5.2. Molish reaction
The 1 mL of different MPFs solution (1 mg/mL) was taken in the
10 mL tubes, then added to 1–3 drops, 2% α-naphthol ethanol solution.
Fig. 1. Effects of different extraction parameters: (a) extraction temperature, (b) ultrasonic powder, (c) extraction time, (d) water-to-raw material ratio, and (e) extraction times on the
yield of MPP (%).
After shaking, the sulfuric acid (500 μL) was added slowly along the wall
of the test tubes. To observe the color change between the two liquid
surfaces, distilled water as negative control and 0.5% starch solution as
positive contrast.
2.5.3. Chemical composition analysis
The total carbohydrate content was determined by the phenol-
sulfuric acid method. The protein content was measured by the
Coomassie brilliant blue G-250 method, using bovine serum albumin
for the standard curve. Uronic acid content was determined by photom-
etry with m-hydroxybiphenyl colorimetric method, using galacturonic
acid as the standard [22].
2.6. Molecular weight determination
The molecular weight of the MPFs were determined by high-
performance gel permeation chromatography (HPGPC) on an LC-
10A system equipped with a Ultrahydrogel 1000 column (30 cm ×
7.8 mm × 10 μm, Agilent technologies, CA, USA) and a refractive index
detector. The column was eluted with ultrapure water at a flow rate
of 1.0 mL/min. The refined samples (2 mg/mL) were dissolved in dis-
tilled water and filtered with a 0.22 μm membrane, and then 10 μL
was injected in each run. The calibration curve of log molecular weight
versus retention time was conducted using a series of dextrans (T-4.3,
T-12.6, T-73.8, T-110, T-289, T-496 and T-670) as standards. Then, the
molecular weight of the MPFs were determined in accordance with
the above standard curve [23]. The equation of the standard curve was
as follows:
LgðMw Þ ¼ −0:584T þ 10:262 ð3Þ
where Mw represents the molecular weight and T represents the reten-
tion time.
2.7. Determination of the monosaccharide composition
The monosaccharide composition of MPFs was determined by PMP-
pre-column derivatization. That is, the MPFs samples (10 mg) were
hydrolyzed with trifluoroacetic acid (2 mol/L, 2 mL) in a sealed flask
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1317

Table 2 at 110 °C for 4 h in an oven. Subsequently, the acid was completely re-
ANOVA of the regression equation and coefficients. moved by flash evaporation in a water bath temperature at 45 °C, and
Source Sum of df Mean square F-value p-value Significance the residue was co-distilled three times using methanol. The hydrolyzed
squares samples and the monosaccharide standards were labeled with PMP by
Model 3.51 14 3.51 10.96 b0.0001 ⁎⁎ adding 2 mL of 0.6 mol/L NaOH and 2 mL of 0.5 mol/L PMP methanol so-
A 1.64 1 1.64 71.42 b0.0001 ⁎⁎ lution. The mixture was reacted at 70 °C for 90 min. Then, the reaction
B 0.14 1 0.14 6.25 0.0255 ⁎
solution was neutralized with 2 mL of 0.3 mol/L HCl and extracted
C 3.33 × 10−5 1 3.33 × 10−5 1.46 × 10−3 0.9701
with chloroform three times. Later, the aqueous phase was filtered
D 0.067 1 0.067 2.95 0.1080
AB 0.024 1 0.024 1.05 0.3231 with a 0.22 μm nylon membrane. The PMP-labeled products were ana-
AC 2.02 × 10−3 1 2.02 × 10−3 0.088 0.7705 lyzed using a LC-2010A HPLC system equipped with an ultraviolet detec-
AD 2.25 × 10−4 1 2.25 × 10−4 9.83 × 10−3 0.9224 tor. The chromatographic conditions were as follows: column, ZORBAX
BC 6.40 × 10−3 1 6.40 × 10−3 0.28 0.6053 SB-C18 (4.6 × 250 mm, Agilent, USA); column temperature, 30 °C; mobile
BD 2.50 × 10−3 1 2.50 × 10−3 0.11 0.7460
CD 0.018 1 0.018 0.80 0.3874
phase, a mixture of phosphate buffer solution (0.1 mol/L, pH 6.7) and
A2 1.05 1 1.05 45.99 b0.0001 ⁎⁎ acetonitrile in a ratio of 87:13 (v/v); injection volume, 20 μL; flow rate,
B2 0.42 1 0.42 18.48 0.0007 ⁎⁎ 1.0 mL/min; and detector wavelength, 250 nm. The monosaccharide
C2 0.33 1 0.33 14.55 0.0019 ⁎⁎
standards included mannose, rhamnose, glucuronic acid, galacturonic
D2 0.61 1 0.61 26.64 0.0001 ⁎⁎
acid, glucose, galactose, xylose, arabinose and fucose.
Residual 0.32 14 0.023
Lack of fit 0.30 10 0.030 5.03 0.0666
Pure error 0.024 4 5.90 × 10−3
2.8. Fourier transform infrared spectroscopy (FT-IR) analysis
Cor Tatal 3.83 28
R2 0.9146
R2adj 0.8327 The dried MPFs (2 mg) were mixed with spectroscopic-grade potas-
C.V.% 4.58 sium bromide (KBr) powder, ground and pressed into a 1 mm pellet,
⁎ Indicates significant differences (p b 0.05). then analyzed using FT-IR spectroscopy (Tensor 27/37, Brandon,
⁎⁎ Indicates extremely significant differences (p b 0.01). Germany) at a frequency range of 4000–400 cm−1.

Fig. 2. 3D-response surface plots showing the effects of extraction temperature, ultrasonic power, extraction time and water-to-raw material ratio on the extraction yield of MPP and their
mutual effects.
1318 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325
2.9. 1H nuclear magnetic resonance (NMR) spectroscopy analysis
The freeze-dried MPFs (20 mg) were dissolved in 500 μL of D2O. 1H
NMR spectra were analyzed with a Bruker nuclear magnetic resonance
spectrometer at an operating frequency of 500 MHz.
2.10. Morphological analysis
The surface morphological traits and microstructures of the MPFs
were observed by scanning electron microscopy (S-3700 N, Hitachi,
Japan). Dried samples of the MPFs were glued on a specimen holder
by coating with a thin layer of gold under reduced pressure and then ob-
served at an accelerating potential of 5 kV under a high vacuum condi-
tion. Their surface morphology of 1.0 k and 5.0 k magnification were
compared [24–26].
2.11. Anticancer activities
The anticancer activities of MPFs were assayed using the CCK-8 kit
in vitro. HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and BGC-823
cells were cultured with dulbecco's modified eagle medium (DMEM)
Fig. 3. 2D-contour polts showing the effect of extraction temperature (A), ultrasonic powder (B), extraction time (C) and water-to-raw material ratio (D) on the MPP extraction yield.
containing 15% fetal bovine serum and 1% penicillin/streptomycin at
37 °C in a humidified incubator containing 5% CO2. The cell suspension
(100 μL/well) was inoculated in a 96-well plate at a density of 5
× 104/mL and allowed to adhere and spread for 24 h. Then, the cells
were treated with the MG-1, MG-2 and MG-3 solution at different con-
centrations (25–1000 μg/mL). After culturing for 48 h, CCK-8 reagents
(10 μL) were added to each well and cultured for 4 h. The absorbance
value at 450 nm was detected by the ELISA Reader. The inhibition rate
was calculated as follows:
Inhibition rate ð%Þ ¼ ð1−A1 =A0 Þ  100 ð4Þ
where A1 and A0 represents the absorbance of the test sample and con-
trol, respectively.
2.12. Apoptosis analysis
The effects of MG-1, MG-2 and MG-3 in inducing apoptosis in
HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and BGC-823 cells were
confirmed using fluorescent staining methodology with calcein-AM
and propidium iodide (PI). Cells were cultured on a 96-well plate and
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1319

Table 3 effect on the extraction yield of MPP when the extraction temperature
Molecular weight and monosaccharide compositions of MG-1, MG-2 and MG-3. varied from 40 to 80 °C, and the highest yield could be obtained at
Index MG-1 MG-2 MG-3 80 °C. The effect of temperature could be explained by the higher solubil-
Molecular weight (kDa) 17.8 32.6 2790
ity of the polysaccharides in the solvent and the higher diffusivities of the
polysaccharide at higher temperature [27–29]. Thus, 80 °C was selected
Monosaccharide components (mg/g) as the optimum extraction temperature. Ultrasonic power is another key
Mannose 8.66 27.13 34.46
Rhamnose 17.94 17.91 21.27
factor that affects the extraction yield of MPP. As shown in Fig. 1b, the ex-
Glucuronic acid 20.49 2.43 0.81 traction yield of MPP gradually increased with increasing of ultrasonic
Galacturonic acid 10.62 195.47 176.32 power. An increase in ultrasonic power strengthened the cavitation ef-
Glucose 4.23 4.83 5.07 fect and vibration and promoted cell wall fragmentation, which benefit-
Galactose 539.18 60.87 33.77
ted the dissolution and diffusion of the polysaccharides. Hence, 184 W
Xylose 1.00 1.47 2.69
Ababinose 230.83 80.72 52.58 was chosen as the best ultrasonic power. As shown in Fig. 1c, the extrac-
Fucose 2.04 2.20 2.33 tion yield of MPP increased significantly with prolonged extraction time
from 20 to 100 min, and then the extraction yield decreased slightly
when the extraction time exceeded 100 min. Excessive extraction time
incubated. After attachment, the cells were treated with fresh culture with high temperature and ultrasonic environment could lead to the in-
containing MPFs at different concentrations (50, 250 and 1000 μg/mL). stability and ultimately affected the polysaccharides yield. Therefore, the
After 72 h of incubation, the supernatant was washed with phosphate extraction time of 100 min was adopted. The effects of raw material-to-
buffer solution (PBS) for 2 times and digested by trypsin. Thorough water ratio on the extraction yield of MPP were also investigated.
removal of residual enzyme activity, suspension cells were stained As shown in Fig. 1d, the extraction yield of MPP first increased rapidly
with calcein-AM/PI (100 μL) for 15 min at 37 °C. Images were captured then decreased with increasing of raw material-to-water ratio, where
by an fluorescence microscope. an optimum value was 1:40 g/mL. An increase in raw material-to-
water ratio possibly improved the diffusion rate of the solvent into
2.13. Statistical analysis cells and enhanced the dissolution of MPP, but too much solvent could
cause further loss of polysaccharides. Therefore, the best raw material-
All data were expressed as means ± standard deviation. Statistical to-water ratio of 1:40 g/mL was chosen in the present work. Moreover,
analysis was performed by multiple-range test using SPSS 18.0 (SPSS, Fig. 1e shows that the extraction yield of MPP increased with prolonged
Inc., Chicago, IL). Difference at p b 0.05 was considered statistically extraction times. After two cycles of extraction, the polysaccharide yield
significant. showed no significant increase when the extraction times were further
prolonged. To simplify the experimental operation, 2 times was selected
3. Results and discussion as the optimum extraction times in the BBD experiments.

3.1. Effects of extraction parameters on the extraction yield of MPP 3.2. Optimization of MPP extraction conditions

The effects of the five extraction parameters (extraction tempera- On the basis of the single-factor experiments, a RSM experiment was
ture, ultrasonic power, extraction time, raw material-to-water ratio used to optimize the extraction conditions of MPP. Four factors with
and extraction times) on the MPP extraction yield are shown in Fig. 1. three variation levels were chosen for optimization, the Box–Behnken
As shown in Fig. 1a, the extraction temperature displayed a positive experimental design and the corresponding results are shown in

Fig. 4. Elution curve of crude polysaccharide extracted from the mango pomace by a DEAE-52 cellulose anion-exchange column (a). Purification of polysaccharide fractions of MG-1 (b),
MG-2 (c) and MG-3 (d) by a Sephadex G-100 column.
1320 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325

Table 1. By using quadratic regression analysis on the experimental Table 4

data, a multinomial regression equation was obtained as follows: Physical and chemical properties of MPFs.

Samples I2-KI Molish Carbohydrate Protein Uronic acid

Y ¼ −85:33 þ 0:70A þ 0:41B þ 0:48C þ 0:21D−4:84  10−4 AB−2:25 reaction reaction (%) (%) (%)
 10−4 AC þ 7:50  10−5 AD−2:50  10−4 BC−1:56  10−4 BD MG-1 Negative Positive 94.07 0.026 39.15
þ 6:75  10−4 CD−4:03  10−3 A2 −9:98  10−4 B2 −2:27 MG-2 Negative Positive 92.13 0.100 22.42
MG-3 Negative Positive 89.25 0.051 28.36
 10−3 C 2 −3:07  10−3 D2

where Y represent the extraction yield of MPP and A, B, C and D repre-

sent the extraction temperature, ultrasonic power, extraction time and
raw material-to-water ratio, respectively.
The analysis of variance (ANOVA) results for the two element re-
gression equation models and significance test analysis are shown in
Table 2. The model of high F-value and the low p-value (b0.0001) indi-
cated that the regression model was highly significant. The p-value of
lack of fit exceeded 0.05, indicated that the regression equation can
be used to replace the real point to analyze the experimental results.
Furthermore, the determination coefficient (R2) was 0.9146, which in-
dicated that the model fitting degree was good and the polysaccharide
yield can be accurately predicted. Additionally, the p-value of linear co-
efficients (A and B) and quadratic term coefficients (A2, B2, C2 and D2)
were lower than 0.05, indicating the significant effects on the extrac-
tion yield of MPP, while the other term coefficients were not significant
(p N 0.05). The effect of four factors on the extraction yield of MPP was
arranged as extraction temperature N ultrasonic power N raw material-
to-water ratio N extraction time.
We plotted 3D response surface and 2D contour plots to predict the
relationships between the independent variables and the response
variable (Figs. 2 and 3). Different shapes of the contour plots indicated
different interactions between the variables. A circular contour plot
implied that the interactions between the corresponding variables
were negligible, whereas an elliptical contour plot meant otherwise
[30, 31]. The extraction yield of MPP was researched when two factors
remained in the experimental range while the other factors were
fixed at zero level. For extraction temperature and ultrasonic power
(Figs. 2a and 3a), the contour plot was dense along the extraction tem-
perature, which indicated that the influence of extraction temperature
on the extraction yield of MPP was stronger than that of ultrasonic
power. In addition, the contour plot was not elliptical, indicating
that the interaction between the two factors was not significant. A sim-
ilar trend was found between extraction temperature and extraction
time (Figs. 2b and 3b), extraction temperature and raw material-to-
water ratio (Figs. 2c and 3c), ultrasonic power and extraction time

Fig. 5. HPLC profile of MPFs (a: standard monosaccharides, peaks from left to right:
Man, Rha, GluA, GalA, Glu, Xyl, Ara and Fuc;b: Monosaccharide composition of MG-1;
c: Monosaccharide composition of MG-2; d: Monosaccharide composition of MG-3). Fig. 6. FT-IR spectra of MG-1, MG-2 and MG-3.
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1321

(Figs. 2d and 3d), ultrasonic power and raw material-to-water ratio were high-molecular-weight biological molecules. Wiater et al. ob-
(Figs. 2e and 3e), and extraction time and raw material-to-water ratio tained a water-soluble polysaccharide from mango fruits with the
(Figs. 2f and 3f). Using the Design Expert 8.0.5 software to solve the re- molecular weight of 760 kDa, it was different from our findings, the
gression equation, the optimal extraction conditions were obtained as possible reason is that the different source of mango, extraction and
follows: extraction temperature of 74 °C, ultrasonic power of 170 W, ex- purification methods, etc.
traction time of 100 min, and raw material-to-water ratio of 1:41 g/mL.
Under these conditions, the predicted extraction yield of MPP was
3.89%. To verify the reliability of the model equation, the extraction pro-
cess was repeated by three times under optimal conditions (extraction
temperature of 74 °C, ultrasonic power of 170 W, extraction time of
100 min, and raw material-to-water ratio of 1:40 g/mL). The actual
extraction yield of MPP was 3.71 ± 0.07%, which was not significantly
different from the predicated value. The results show that the model is
effective for the prediction of MPP extraction yield.

3.3. Isolation and purification of MPP

The crude polysaccharides were loaded onto an anion-exchange

chromatography column of DEAE-52 cellulose to afford a neutral and
an acid polysaccharide. On the basis of the elution curve (Fig. 4a), four
independent elution peaks were obtained, and the main polysaccharide
fractions (MG-1, MG-2 and MG-3) were collected, concentrated, dia-
lyzed and further purified by Sephadex G−100 gel filtration chromatog-
raphy. The recovery rates of MG-1, MG-2 and MG-3 were 20.17%,
15.03% and 30.91%, respectively. As shown in Fig. 4b–4d, each fraction
generated a single elution peak, then collected, concentrated and lyoph-
ilized one by one for further analysis.

3.4. Physicochemical characteristics of MPFs

The physicochemical properties of the MPFs are summarized in

Table 4. The results show that MG-1, MG-2 and MG-3 contains no
starch. The molish reaction indicated that MG-1, MG-2 and MG-3 had
the general properties of polysaccharides. In addition, no absorption at
260 nm was detected, which shown that these MPFs were absence of
nucleic acid. The contents on carbohydrate and uronic acid of MG-1,
MG-2 and MG-3 were 94.07% and 39.15%, 92.13% and 22.42%, 89.25%
and 28.36%, respectively. In addition, MG-1, MG-2 and MG-3 contained
0.026%, 0.100% and 0.051% protein.
The monosaccharide compositions of MG-1, MG-2 and MG-3 were
determined by comparing the retention time with that of standards,
and the results are presented in Table 3 and Fig. 5. Nine monosaccha-
rides were detected in MG-1, MG-2 and MG-3, including Man, Rha,
GluA, GalA, Glu, Gal, Xyl, Ara and Fuc, in molar ratio of 3.5:7.2:
1.0:16.4:449.3:1.8:230.8:15.8:8.2, 2.7:15.4:1.0:11.1:34.5:1.4:56.2:1.3:
102.8 and 6.7:6.7:4.3:31.0:44.9:3.4:84.4:1.0:217.8, respectively. In
terms of peak area, Gal and Ara were the major monosaccharide of
MG-1, while GalA was the main monosaccharide of MG-2 and MG-3,
respectively. These results indicated that the monosaccharide composi-
tions of MG-1, MG-2 and MG-3 are extremely complicated. In our study,
polysaccharide fractions MG-2 and MG-3, especially MG-2, were mainly
comprised of GalA, and GluA was also existed among the three polysac-
charide fractions, especially MG-1. However, they were not detected by
Wiater et al. [32]. The result was also in agreement with Chen et al. [33].
In their research, two polysaccharide fractions (MSP I and MSP II) were
obtained from mango seeds, the monosaccharides occurred in MSP I
were determined to be Ara, Rha, Man, Gal and Glu. Besides these mono-
saccharides, fructose was detected as the major monosaccharide in
MSP II. The differences between these reports and our findings may be
caused by the different materials of mango, and different extraction
and purification methods.
The molecular weight of MG-1, MG-2 and MG-3 was determined
by HPGPC. From the calibration curve of the retention times of
dextran T-series standards, we estimated the molecular weight of
MG-1, MG-2 and MG-3 to be 1.78 × 104, 3.26 × 104 and 2.79 × 106 Da,
respectively. The results further indicated that all purified fractions Fig. 7. 1H NMR spectroscopy of MG-1 (a), MG-2 (b) and MG-3 (c).
1322 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325
FT-IR spectroscopy is used for the qualitative measurement of or-
ganic functional groups [34]. As shown in Fig. 6, the FT-IR spectra of
MG-1, MG-2 and MG-3 displayed a strong absorption at around
3423 cm−1, which was associated with O−H stretching vibration
[35]. A weak absorption peak at around 2973 cm−1 was related
to the stretching vibration of C−H [36], while the absorbance at
around 1414 cm−1 corresponded to the C−H bending vibration
[37], which was the main functional group of the polysaccharides.
Meanwhile, the absorption band at around 1630 cm−1 was attrib-
uted to the presence of C_O asymmetrical stretching vibration.
The absorbance at 1200–850 cm−1, referred to as the “finger print”
region, is usually used to identify the major chemical groups in poly-
saccharides [38, 39]. The absorption peak at around 1075 cm−1 indi-
cated the presence of a pyranoid ring in MG-1, MG-2 and MG-3. The
characteristic band at around 895 cm−1 demonstrated the presence
of α-glycosides in MG-1, MG-2 and MG-3 [40], and the absorbance
at 769 cm−1 was the characteristic absorption of D-pyranose ring
grape symmetric stretching vibration [41]. The peak at 643 cm−1
represented C\\C stretching vibration [42]. Based on the above re-
sults, it can be concluded that MG-1, MG-2 and MG-3 were pyranose
sugars.
The structural feature of MPFs were further analyzed by the NMR
spectra. The spectra of 1H NMR mainly solves the configuration of gluco-
side bonds. The effective proton signal in the glucoside structure of
polysaccharide was mainly concentrated in the region of 4.0–5.5 ppm,
but the overlap and interference of the proton signal in the solvent
Fig. 8. Scanning electron micrographs of MPFs (a: MG-1; b: MG-2; c: MG-3).
caused great difficulty in the resolution of the spectrogram in the actual
analysis. Therefore, the characteristic signals of the first carbon are usu-
ally judged and analyzed in the 4.8–5.5 ppm region. In general, 5.0 ppm
is the critical value to distinguish the proton signal of the configuration
of pyranose. If the first proton displacement of the samples exceeds
5.0 ppm, then it is an α-glucoside; if the displacement is b5.0 ppm,
then it is a β-glycoside. The 1H NMR spectra of MG-1, MG-2 and MG-3
are shown in Fig. 7. The results demonstrated that all of the fractions
contained heteropolysaccharides. One main single peak at 4.694 ppm
is due to D2O. 1H single peaks appeared in the anomeric region of
4.95–5.17 ppm. This results indicated that the glucosyl residues of
MG-1, MG-2 and MG-3 are α-glycosidically linked, which agree with
the FT-IR results.
3.5. SEM analysis
The scanning electron micrographs of MG-1, MG-2 and MG-3 at
magnifications of 1000× and 5000× are shown in Fig. 8. The surface
morphologies of the different MPFs exhibited significant differences in
shape and size. MG-1 had a fragmented surface with many irregular
particles adhered together (Fig. 8a), whereas MG-2 possessed a rough
surface and was composed of large and irregular particles (Fig. 8b). As
shown in Fig. 8c, MG-3 had a lamelliform morphology with many
small particles adhered together and exhibited smooth and incompact
structures. In addition, the irregular particles in MG-1 were smaller
than those in MG-2 and MG-3 under high magnification (5000×).
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1323

3.6. Anticancer activity analysis
The anticancer activities of polysaccharides were initially reported
by Nauts et al. in 1946. They found that certain polysaccharides could
stimulate complete remittance in patients with cancer [43]. Numerous
studies have reported that the anticancer activities of polysaccharides
could be influenced by molecular weight, monosaccharide composition,
degree of branching, form and solubility [18]. In addition, studies
showed that polysaccharides could suppress tumor growth via the fol-
lowing common mechanisms: (1) excessive levels of reactive oxygen

Fig. 9. Anticancer activities of MG-1 (a), MG-2 (b) and MG-3 (c) against HepG2 cells, MCF-7cells, A549 cells, Hela cells, A2780 cells, HCT-116 cells and BGC-823 cells. Cell viability was
determined by CCK-8 assay.
1324 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325

species were correlated well with the generation and malign transfor-
mation of cancer cells. If polysaccharides could enhance the level of an-
tioxidants and decrease the level of reactive oxygen species in cancer
cells, they might inhibit the cancer cell growth [18, 44]; (2) directly in-
hibits the proliferation of cancer cell in vitro, such as polysaccharides
from Poria cocos; (3) the induction of tumor cell apoptosis; (4) the inhi-
bition of tumor metastasis [16]; (5) the stimulation of the cell-mediated
immune response. For example, immunostimulatory activities were
found in the polysaccharides from Polyporus albicans, which suggested
that immunostimulatory effects might be the main mechanism for the

Fig. 10. Apoptosis study of HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and BGC-823 cells by the treatment of MPFs. Green color: live cells; Red color: dead cells. a: MG-1; b: MG-2; c: MG-3.
 H. Hu et al. / International Journal of Biological Macromolecules 117 (2018) 1314–1325 1325

anti-tumor activities of polysaccharides [22]. (6) the inhibition of tu- HeLa, A2780, HCT-116 and BGC-823 cells in vitro. Thus, these MPFs
morigenesis through the usage of oral active preparations; and (7) im- can be developed as potential agents to prevent and treat cancer and
mune potentiation function in combination with chemotherapy. other diseases.
To date, only few studies have evaluated the anticaner activity
of polysaccharides from mango pomace. In the present study, the Acknowledgements
anticaner activities of MG-1, MG-2 and MG-3 against HepG2, MCF-7,
A549, HeLa, A2780, HCT-116 and BGC-823 cells were investigated. As This work was financially supported by the scientific research
shown in Fig. 9, MG-1, MG-2 and MG-3 exhibited significant inhibitory from the Public Welfare industry (Agriculture) (201503142-13 and
effects on these cells in a dose-dependent manner within the concentra- 201503142-14).
tion range of 25–1000 μg/mL. This trend of the MPFs were similar to that
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