Medical, Pharma, Engineering, Science, Technology and Business

^{1}College of Life and Environment Science, Hangzhou Normal University, Hangzhou 310036, People's Republic of China

^{2}Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, People's Republic of China

^{3}Guangxi Key Laboratory of Electrochemical and Magneto-chemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guilin, Guangxi 541004, People's Republic of China

- Corresponding Author:
- Xia Li

Guangxi Key Laboratory of Electrochemical and

Magneto-chemical, Functional Materials, College of Chemistry and

Bioengineering Guilin University of Technology, Guilin

Guangxi 541004, People's Republic of China

**Tel:**+8618107735083

**Fax:**+865896839

**E-mail:**[email protected]

**Received date: **November 22, 2016; **Accepted date:** December 12, 2016; **Published date:** December 15, 2016

**Citation: **Xiao-ri Z, Li-li X, Feng X, Xiao-lin L, Chun-na Y, et al. (2016) Optimization of Polysaccharide Extraction from Polygonatum odoratum by Response Surface
Methodology and Evaluation of its Antitumor Activity. J Bioprocess Biotech 6:292.
doi:10.4172/2155-9821.1000292

**Copyright:** © 2016 Barathikannan K, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.

**Visit for more related articles at** Journal of Bioprocessing & Biotechniques

This work established a microwave-assisted procedure for polysaccharide extraction from Polygonatum odoratum. Response surface methodology was used to optimize microwave-assisted extraction parameters (extraction temperature, water-to-raw material ratio, microwave power, and extraction time) by implementing a three-level, fourvariable Box–Behnken experimental design in a single-factor investigation. Three-dimensional response surfaces were plotted by Design-Expert, and result indicated the absence of interaction effects of extraction temperature and microwave power. A polysaccharide yield of approximately 17.49% was obtained under the following optimized conditions: temperature of 57°C, microwave power of 300 W, extraction time of 10 min, and water-to-raw material ratio of 23:1. The results of preliminary in vitro antitumor activity tests showed that polysaccharides derived from *P. odoratum* could inhibit growth of A549 cells in a dose-dependent manner. Moreover, 4-400 μg/mL of these polysaccharides exerted no significant cytotoxicity to Panc-1 cells.

Microwave-assisted extraction; Polysaccharides;
*Polygonatum odoratum*; Response surface methodology

Polysaccharides are essentially polymeric carbohydrates consisting of monosaccharides linked by glycosidic bonds. Depending on their structure, polysaccharides display functional properties distinct from those of their constituent monosaccharides; as a result, they play a wide array of important roles in nature [1]. Plant-derived polysaccharides demonstrate a variety of biological properties, including anti-oxidative [2-4], anti-viral [5], and anti-complementary activities [6,7]. Over the years, polysaccharides have become important raw materials for health foods, drugs, and cosmetics.

Rhizoma Polygonati Odorati (Fragrant Solomonseal Rhizome, Yu
Zhu) is the dried rhizome of *Polygonatum odoratum* (Mill.), which has
been used for centuries in traditional herbal medicine as an important
source of medicine and as base for a valuable nourishing tonic. The
Ministry of Health of the People’s Republic of China has defined *P. odoratum* as an affinal drug and a diet material. Traditionally, *P.
odoratum* is described to possess the capability to nourish the yin and
to promote fluid production to quench thirst. This medicinal plant
is applied to treat diabetes, palpitations, lung illnesses, and upset
stomachs. Currently, *P. odoratum* is usually used as a functional food
given that long-term consumption of this plant does not damage the
stomach [8-10]. *P. odoratum* contains various chemical components,
such as polysaccharides, flavonoids, steroidal saponins, and alkaloids.
Studies have shown that *P. odoratum* polysaccharides (POP)
demonstrate immunological, anti-tumor, and anti-aging activities [11].
However, studies on POP extraction remain low.

Microwave assists in solvent extraction of bioactive compounds from herbs [12-15]. Microwave extraction has many advantages, including reduced consumption of organic solvent and shorter extraction time. Thus, microwave extraction is more effective than traditional extraction methods.

When many factors and interactions affect desired response, response surface methodology (RSM) is an effective tool for optimizing the process, which was originally described by Box and Wilson. RSM is a collection of statistical and mathematical techniques used to determine the effects of several variables and to optimize various processes [16]. RSM has been successfully applied to optimize conditions in food and pharmaceutical research [16,17]. The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple variables and their interactions. Therefore, it is less laborious and time consuming than other approaches required optimizing a process. Usually, it applies an experimental design such as Box–Behnken (BBD), central composite (CCD) and Doehlert designs (DDD) to fit a secondorder polynomial by a least squares technique. An equation is used to describe how the test variables affect the response and determines the interrelationship among the variables.

This study investigated significant variables (ratio of water to raw
material, extraction temperature, extraction time, microwave power)
and further optimize the extraction process of polysaccharides from
*P. odoratum* using RSM, while employing a three-level, four-variable
BBD. Inhibitory effect of POP extraction on A549 and Panc-1 cell lines
was investigated.

**Materials and reagents**

A549 lung adenocarcinoma cells and Panc-1 human pancreatic carcinoma cells were obtained from the Department of Research Center for Biomedicine and Health, Hangzhou Normal University (Hangzhou).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI 1640 and FBS were obtained from Gibco. MAS-I Microwave Synthesis System was obtained from Shanghai Sineo Microwave Chemistry Technology Co., Ltd (Shanghai).

**Preparation of POP**

*P. odoratum* was dried at 40°C for 48 h and then pulverized in a
blender to obtain a fine powder (particle diameter is <500 μm). The
pulverized *P. odoratum* was soaked in 95% ethanol twice for defatting
and removal of colored materials, oligosaccharides, and small
molecules.

Each pretreated dried sample (5.0 g) was extracted with water under specified extraction temperature, water-to-raw material ratio, microwave power, and extraction time. The water extraction solutions were centrifuged at 3000 rpm for 15 min, and then the supernatant was separated from insoluble residue with four-layer filter cloth, which were precipitated by the addition of ethanol to a final concentration of 80% (v/v) and incubated overnight. The precipitates collected through centrifugation (3000 rpm for 15 min) were washed three times with dehydrated alcohol and ethyl ether and then dried under reduced pressure to obtain crude polysaccharides.

**Determination of polysaccharides yield**

The polysaccharide content was determined through the phenol– sulfuric acid method by using D-glucose as standard [18]. The crude polysaccharides were accurately weighed and dissolved in distilled water in a 100 mL volumetric flask. Afterwards, the sample solution was carefully transferred into a 10 mL cuvette. Phenol (5%, 1 mL) was added into the cuvette and then shaken. Sulfuric acid (7.0 mL) was subsequently added into the mixture, shaken, incubated at 40°C in a water bath for 30 min, and then allowed to stand in an ice bath for 5 min. Absorbance values were recorded by a UV spectrophotometer at 490 nm. The wash solution served as blank control and was measured in a similar manner. The regression equation between microgram value of glucose and OD value were obtained as: A=6.0667C+0.1078 (A: 490 nm OD, C: μg/mL), r=0.9972; linearity range: 10-40 μg/mL.

**Experimental design**

After the range of extraction variables was preliminarily determined
through a single-factor test, a three-level BBD was performed using
four independent variables (X_{1}, extraction temperature; X_{2}, water-toraw
material ratio; X_{3}, microwave power; and X_{4}, extraction time). For
statistical calculation, the variables were coded according to

(1)

where χ_{i} is a coded value of a variable; X_{i} is the actual value of a variable;
X_{0} is the actual value of X_{i} at the center point; and △X_{i} is the step change
value. **Table 1** shows the range of independent variables and their levels. The independent variables and their ranges were determined in
our preliminary experiments. Three experiments for each condition
were performed and the mean values were considered as observed
responses. The complete design consisted of 27 experimental points,
and the experiments were performed in a random order (**Table 2**).

Independent variables | Factor level | ||
---|---|---|---|

-1 | 0 | 1 | |

X_{1}: Extraction temperature (ºC) |
40 | 50 | |

60 | |||

X_{2}: Ratio of water to raw material |
15 | 20 | 25 |

X_{3}: Microwave power (W) |
200 | 300 | 400 |

X_{4}: Extraction time (min) |
5 | 10 | 15 |

**Table 1:** Independent variables and their levels used in the response surface
design.

Data from the BBD were analyzed by multiple regressions to fit the following quadratic polynomial model.

(2)

Y represents the response function. β_{0} is an intercept. β_{i}, β_{ii}, and
β_{ij} are the coefficients of the linear, quadratic, and interactive terms,
respectively. χ_{i} and χ_{j} represent the coded independent variables,
respectively. The fitted polynomial equation was expressed as surface
and contour plots to visualize the relationship between the response and
experimental levels of each factor and to deduce the optimum conditions
[19]. The regression coefficients of individual linear, quadratic, and
interaction terms were determined through analysis of variance. These
regression coefficients were used in statistical calculation to generate
dimensional and contour maps from the regression models. Designexpert
version 8.0.4 (Trial Version, State-Ease Inc., Minneapolis, MN,
USA) was used to analyze the experimental data. P-values of less than
0.05 indicated statistical significance.

**Cytotoxicity assay in vitro**

POP was dissolved in distilled water at a concentration of 10 g/L and then filtered through a 0.22 μm filter and stored at 4°C. The solution was further diluted to different concentrations (800 μg/mL, 80 μg/mL, 8 μg/mL) with RPMI 1640 medium in tissue plates separately.

Cell proliferation was analyzed through colorimetric MTT assay
as described previously [20]. Briefly, cells were seeded in a 96-well
microplate (1 × 10^{4} cells/well, in a volume of 100 μL), treated with or
without 5-Fu (1 μg/mL), DDP (10 μg/mL) and incubated in RPMI 1640
medium containing 10% FBS with 100 μL sample polysaccharides at a
concentration of 400 μg/mL, 40 μg/mL, 4 μg/mL. The experiments were
performed in triplicate. The cells were subsequently incubated at 37°C
under a humidified 5% CO_{2} atmosphere.

MTT (5 mg/mL, 20 μL) was added after 72 h. After the plates were incubated at 37°C for 4 h, the supernatant was aspirated and 150 μL of DMSO was added into each well. Absorbance was measured at 570 nm by a 96-well microplate reader (Bio-Rad, USA). The inhibition rate of cell growth was calculated as follows:

(mean value for control group−mean value for treated group)/ control group × 100%.

**Effect of extraction temperature on extraction yield of
polysaccharides**

Different extraction temperature was set at 30, 40, 50, 60 and 70°C,
respectively, to investigate the influence of extraction temperature
on the yield of POP. The other reaction conditions were as follows:
water-to-raw material ratio of 20:1, microwave power of 200 W, and
extraction time of 10 min. **Figure 1a** indicates that the maximum yield
was obtained when the extraction temperature was increased from
40°C to 50°C. However, when the extraction temperature was further
increased, the yield declined linearly. Therefore, the optimal extraction
temperature adopted in this work is 50°C.

**Effect of water to raw material ratio on extraction yield of
polysaccharides**

**Figure 1b** shows the effect of different water-to-raw material ratios
(5:1, 10:1, 15:1, 20:1, 25:1, and 30:1) on POP yield. When the extraction
temperature (50°C), microwave power (200 W), and extraction time (10
min) were set at level 0, the yield suddenly increased to the critical value
of 22.68% under 20:1 ratio. This result indicated that when the ratio is
higher than 20:1, the polysaccharide yield decreases possibly because
polysaccharides can be excessively dissolved in low-concentration
solvent, resulting in considerable yield loss during product collection.
Therefore, the water-to-raw material ratio of 20:1 was adopted in the
following work.

**Effect of microwave power on the extraction yield of
polysaccharides**

**Figure 1c** shows the effect of microwave power on POP yields.
When the extraction temperature, water-to-raw material ratio, and
extraction time were set at 50°C, 20:1, and 10 min, respectively, the
yield obviously increased under microwave power of 100-600 W. The
polysaccharide yield was 16.74% when the samples were extracted at
300 W. However, the yield decreased significantly when the microwave
power further increased, indicating that 200 W is sufficient for
polysaccharide extraction in the present work.

**Effect of extraction time on extraction yield of polysaccharides**

Extraction time influences the extraction efficiency and selectivity
of a fluid. A longer extraction time increases polysaccharide yields
[21,22]. This study investigated the effect of extraction time (5, 10,
15, 20, 25, and 30 min) on polysaccharide yield when the extraction
temperature is 50°C, water-to-raw material ratio is 20:1, and microwave
power is 200 W. **Figure 1d** shows that the maximum yield was obtained
when the extraction time was 10 min; beyond this period, the yield
decreased significantly; more over a descending dynamic equilibrium
was observed with extended extraction time. Therefore, the extraction
time used in this work is 5-15 min.

**Optimization of the extraction parameters of polysaccharides**

**Statistical analysis and the model fitting:** RSM optimization is
more advantageous than the traditional single-parameter optimization
because the former saves time, space, and raw materials [23]. A total of
29 runs were performed to optimize the four individual parameters in
BBD. **Table 2** shows the experimental conditions and the polysaccharide
yields according to the factorial design. By applying multiple regression
analysis on the experimental data, the response variables and the test
variables were related by the following second-order polynomial
equation:

Run | X_{1} |
X_{2} |
X_{3} |
X_{4} |
Yield (%) |
---|---|---|---|---|---|

1 | 1 | 0 | 0 | -1 | 13.98 |

2 | 0 | -1 | 1 | 0 | 9.26 |

3 | -1 | 0 | -1 | 0 | 6.99 |

4 | -1 | -1 | 0 | 0 | 11.07 |

5 | 0 | 0 | 1 | -1 | 9.24 |

6 | 0 | 0 | 0 | 0 | 16.74 |

7 | 1 | 0 | 1 | 0 | 10.58 |

8 | 0 | -1 | -1 | 0 | 2.37 |

9 | -1 | 0 | 1 | 0 | 7.07 |

10 | -1 | 0 | 0 | 1 | 10.27 |

11 | 1 | -1 | 0 | 0 | 1.12 |

12 | 0 | 0 | -1 | 1 | 6.02 |

13 | 1 | 0 | -1 | 0 | 9.68 |

14 | 0 | 0 | 0 | 0 | 16.74 |

15 | 0 | -1 | 0 | -1 | 3.37 |

16 | 0 | 0 | 1 | 1 | 10.83 |

17 | 0 | 0 | -1 | -1 | 10.22 |

18 | 1 | 0 | 0 | 1 | 8.80 |

19 | 1 | 1 | 0 | 0 | 17.23 |

20 | 0 | 0 | 0 | 0 | 16.74 |

21 | 0 | 1 | 1 | 0 | 8.84 |

22 | 0 | 0 | 0 | 0 | 16.74 |

23 | 0 | 1 | 0 | 1 | 13.75 |

24 | 0 | 1 | 0 | -1 | 6.56 |

25 | 0 | 1 | -1 | 0 | 9.54 |

26 | -1 | 1 | 0 | 0 | 1.16 |

27 | 0 | 0 | 0 | 0 | 16.74 |

28 | 0 | -1 | 0 | 1 | 4.06 |

29 | -1 | 0 | 0 | -1 | 5.03 |

**Table 2: **Box–Behnken experimental design and results for extraction yield.

Y=16.78+1.62 × X_{1}+1.61 × X_{2}+0.92 ×X_{3}−0.097 × X_{4}+6.59 ×X_{1}×
X_{2}+0.20 ×X_{1}×X_{3}−2.52 ×X_{1}×X_{4}−1.90 ×X_{2}× X_{3}+3.25 × v × X_{4}+1.45 ×
X_{3}×X_{4}−4.03 ×X_{1}×X_{1}−5.01 ×X_{2}×X_{2}−4.28 ×X_{3}×X_{3}−3.28 ×X_{4}× X_{4} (3)

**Table 3** shows the ANOVA results for the experimental BBD data.
The determination coefficient (R^{2}=0.9910) indicated that less than 1.0%
of the total variations were not explained by the model. Moreover, the
adjusted determination coefficient (AdjR^{2}=0.9437) confirmed that
the model was highly significant. Low values of coefficient of variance
(6.81) clearly indicated that the model was reproducible and reliable
[24]. A Pred R-Squared of 0.9457 was reasonably consistent with an
Adj R-Squared of 0.9820. Adequate Precision was used to measure the
signal-to-noise ratio. A ratio greater than 4 is desirable, and a ratio of
34.650 indicated adequate signal. This model can be used to navigate
the design space.

Source | SS | DF | MS | F value | P value Prob.>F |
---|---|---|---|---|---|

Model | 671.27 | 14 | 47.95 | 110.32 | < 0.0001 |

X_{1} |
32.42 | 1 | 32.42 | 74.60 | < 0.0001 |

X_{2} |
29.68 | 1 | 29.68 | 68.29 | < 0.0001 |

X_{3} |
10.08 | 1 | 10.08 | 23.20 | 0.0003 |

X_{4} |
0.11 | 1 | 0.11 | 0.25 | 0.6276ns |

X_{1}X_{2} |
190.08 | 1 | 190.08 | 437.34 | < 0.0001 |

X_{1}X_{3} |
0.17 | 1 | 0.17 | 0.39 | 0.5440ns |

X_{1}X_{4} |
27.67 | 1 | 27.67 | 63.67 | < 0.0001 |

X_{2}X_{3} |
14.40 | 1 | 14.40 | 33.14 | < 0.0001 |

X_{2}X_{4} |
36.67 | 1 | 36.67 | 84.38 | < 0.0001 |

X_{3}X_{4} |
8.38 | 1 | 8.38 | 19.28 | 0.0006 |

X_{12} |
103.48 | 1 | 103.48 | 238.08 | < 0.0001 |

X_{22} |
162.60 | 1 | 162.60 | 374.12 | < 0.0001 |

X_{32} |
116.86 | 1 | 116.86 | 268.86 | < 0.0001 |

X_{42} |
69.88 | 1 | 69.88 | 160.79 | < 0.0001 |

Residual | 6.08 | 14 | 0.43 | ||

Lack of Fit | 6.08 | 10 | 0.61 | ||

Pure Error | 0.000 | 4 | 0.000 | ||

Cor Total | 677.35 | 28 | |||

SD |
0.66 | R^{2} |
0.9910 | ||

Mean |
9.68 | Adj R^{2} |
0.9820 | ||

CV (%) |
6.81 | Pred R^{2} |
0.9457 | ||

PRESS |
36.79 | Adequate Precision |
34.650 |

**Table 3: **Analysis variance of experimental of the BBD.

The P value was used as a tool to determine the significance of each
coefficient; moreover, it indicated the strength of interaction between
independent variables. The smaller the P value, the more significant the
corresponding coefficient would be [21]. **Table 3** shows that the linear
coefficients (X_{1}, X_{2}, and X_{3}), quadratic term coefficients (X_{1}^{2}, X_{2}^{2}, X_{3}^{2}, and X_{4}^{2}), and cross product coefficients (X_{1}X_{2}, X_{1}X_{4}, X_{2}X_{3}, X_{2}X_{4}, and
X_{3}X_{4}) were significant, and their P-values were very low (P<0.05). The
other term coefficients were not significant (P>0.05). The full model
filled Eq. (3) was made into three-dimensional and contour plots to
predict the relationships between the independent and dependent
variables.

**Optimization of polysaccharide extraction conditions: **Response
surfaces were plotted using Design-Expert (version 8.0) software to
study the effect of parameters on polysaccharide yield, as well as the
interactions of these parameters. 3-D response surface plots and 2-D
contour plots (**Figures 2** and **3**) were very useful to assess the interaction
effects of the factors on the responses. These types of plots show the
effects of two factors on the response at a time. In all of the plots, the
two other factors were kept at level 0. In the two plots, the maximum
predicted value indicated by the surface was confined in the smallest
ellipse in the contour diagram. Elliptical contours are obtained when a
perfect interaction occurs between independent variables.

**Figures 2a** and **3a** show the effect of water-to-raw material ratio,
extraction temperature, and their reciprocal interaction on extraction
yield when extraction time and microwave power were fixed at level
0. A strong interaction between these factors were observed. The
polysaccharide yield decreased at the designed extraction temperature
range (40°C-60°C), and a quadratic effect on the response yield was
observed when the water-to-raw material ratio increased from 15 to
25. The maximum extraction yield could be achieved at a water-to-raw
material ratio and extraction temperature of approximately 20 and
50°C, respectively.

**Figures 2b** and **3b** show the 3-D response surface plot and contour
plot under varying extraction temperatures and microwave powers at a
fixed water-to-raw material ratio (level 0) and extraction time (level 0).
The plots indicate that the polysaccharide yield increased at 40°C-52°C and then decreased slightly at 52°C-60°C. The polysaccharide extraction
yield increased evidently with increased microwave power from 200 W
to 311 W; the polysaccharide yield decreased beyond 311 W.

The POP yield was affected by different extraction temperatures and
extraction times (**Figures 2c** and **3c**) when the water-to-raw material
ratio and microwave power were fixed at level 0. The maximum
polysaccharide yield was obtained when the extraction temperature
and extraction time.

**Figures 2d** and **3d** show the 3-D response surface plot and the
contour plot under varying water-to-raw material ratios and microwave
power values at a fixed extraction temperature (level 0) and extraction
time (level 0). During polysaccharide extraction, the water-to-raw
material ratio and microwave power demonstrated quadratic effects on
the extraction yields. The optimal range of microwave power was 250-
350 W and that of water-to-raw material ratio was 18-22.5.

**Figures 2e** and **3e** show the plots based on the independent variables
water-to-raw material ratio and extraction time when the extraction
temperature and microwave power were kept at level 0. The maximum
extraction yield could be achieved when the extraction time and waterto-
raw material ratio were 10 min and 20, respectively.

**Figures 2f** and **3f** show the plots under varying microwave power
and extraction time values under a fixed extraction temperature (level 0)
and water-to-raw material ratio (level 0). The extraction yield increased
with increased microwave power from 200 W to 325 W; beyond 325 W,
the yield decreased with increasing extraction time. The influence of
extraction time was similar to that of the microwave power, and 10 min
was the critical extraction time. Extraction time and microwave power
both exerted a positive impact on POP yields.

Based on **Figures 2** and **3** and on the results of the above singleparameter
study, the optimal extraction conditions for POP extraction
are as follows: a temperature of 57.21°C, a water-to-raw material
ratio of 23.23, a microwave power of 298.51 W, and an extraction
time of 10.12 min. Among the four extraction parameters studied,
extraction temperature was the most significant factor affecting the
POP extraction yield successively followed by water-to-raw material
ratio, microwave power, and extraction time based on the significance
of regression coefficients of the quadratic polynomial model (**Table 3**)
and on gradient of slope in the 3-D response surface plot (**Figure 2**).

**Verification of the predictive model:** The suitability of the
models in predicting optimum response values was tested under the
following conditions: extraction temperature of 57°C, water-to-raw
material ratio of 23:1, microwave power of 300 W, and extraction
time of 10 min. This set of conditions was determined to be optimum
through RSM optimization; moreover, this set of condition was used
to experimentally validate and predict response values by using the
model. A mean value of 17.49% ± 0.64% (n=3) was obtained from real
experiments. The results indicated that the RSM approach effectively
optimized the conditions for POP extraction, suggesting that the
regression model was accurate and adequate for polysaccharide
extraction (**Table 4**).

Extraction temperature (ºC) | Ratio of water to raw material | microwave power (W) | Extraction time (min) | Yield (%) | |
---|---|---|---|---|---|

Optimum condition |
57.21 | 23.23 | 298.51 | 10.12 | 17.87 (Predicted) |

Modified conditions |
57 | 23 | 300 | 10 | 17.49 (actual) |

**Table 4: **Predicted and experimental values of the responses at optimum conditions.

**Anti-tumor activity in vitro: **The inhibition effects of different
POP concentrations (4, 40, 400 μg/mL) were tested in two human
tumor cell lines for 72 h and measured using the MTT method

**Figure 4: **Effect of POP on the Panc-1 cells *in vitro*. Panc-1 was stimulated with
POP at the concentrations of 400, 40, 4 μg/mL for 72 h. After the incubation, cell
proliferation was measure by the MTT assay. DDP, positive, 10 μg/mL. Values
were expressed as mean ± SD. *P<0.05 vs control, **P<0.01 vs control.

Although 40 and 400 μg/mL of the polysaccharides exerted a certain degree of inhibitory effect on Panc-1, their effects were not statistically significant [25,26].

Growth of A549 cells was inhibited by POP in a dose-dependent
manner compared with the control, suggesting that POP (400 μg/mL)
are significantly cytotoxic to A549 cells in vitro (P<0.01) (**Figure 5**).

**Figure 5: **Effect of POP on the A549 cells in vitro. A549 was stimulated with
POP at the concentrations of 400, 40, 4 μg/mL for 72 h. After incubation, cell
proliferation was measured by the MTT assay. 5-Fu, positive, 1 μg/mL. Values
were expressed as mean ± SD. *P<0.05 vs control, **P<0.01 vs control.

The extraction conditions significantly influenced the POP yields. The contour and surface plots in RSM effectively estimated the effect of four independent variables (extraction temperature, water-to-raw material ratio, microwave power, and extraction time). The optimum set of the independent variables was achieved graphically in order to obtain the desired levels of crude polysaccharide. The optimal experimental yield of 17.49% was obtained when the conditions for POP extraction were as follows: extraction temperature of 57°C, waterto- raw material ratio of 23, microwave power of 300 W, and extraction time of 10 min. Under these optimized conditions, the experimental extraction yield is close to the predicted yield.

This research was financially supported by the National Natural Science Foundation of China (Nos. 31060193 and 31200269), the Natural Science Foundation of Guangxi Province (2014GXNSFBA118134), the Guangxi Key Laboratory of Electrochemical and Magneto-chemical Functional Materials (EMFM20162205).

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