Polysaccharides from Pyracantha fortuneana and its biological activity

Journal Pre-proofs Polysaccharides from Pyracantha fortuneana and its biological activity Yi-Lan Yao, Chang Shu, Ge Feng, Qing Wang, You-Yu Yan, Yang Yi, HongXun Wang, Xi-Feng Zhang, Li-Mei Wang PII: DOI: Reference:

S0141-8130(19)32895-8 https://doi.org/10.1016/j.ijbiomac.2019.10.125 BIOMAC 13626

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

18 April 2019 9 October 2019 13 October 2019

Please cite this article as: Y-L. Yao, C. Shu, G. Feng, Q. Wang, Y-Y. Yan, Y. Yi, H-X. Wang, X-F. Zhang, L-M. Wang, Polysaccharides from Pyracantha fortuneana and its biological activity, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.125

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Polysaccharides from Pyracantha fortuneana and its biological activity Yi-Lan Yao1,# ,Chang Shu1,#,Ge Feng1, Qing Wang1, You-Yu Yan1, Yang Yi2, Hong-Xun Wang1, Xi-Feng Zhang1,3, Li-Mei Wang1,* 1.College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China; 2.College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China. 3.China.College of Veterinary medicine, Qingdao Agricultural University, Qingdao 266100, China.

# Yi-Lan Yao and Chang Shu are co-first authors *Correspondence: [email protected], Tel.: +86-27-8395-6793

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Abstract: This study used response surface methodology to determine the optimal conditions for extraction of polysaccharides from Pyracantha. fortuneana (PSPF), and studied the mechanism of PSPF-inducing apoptosis in human ovarian carcinoma Skov3 cells. Response surface methodology (RSM) were adopted to extract PSPF. The maximum value of polysaccharide yield was obtained under these optimal conditions. PSPF had good potential as an antioxidant. Exposure of cells to PSPF resulted in cytotoxicity through the induction of apoptosis, and the reactive oxygen species were increased, mitochondrial membrane potential decreased, DNA damage (detected as γ- H2AX and RAD51 foci) was observed in Skov3 cells. In addition, PSPF could induce apoptosis of cancer cells. Therefore, PSPF should be explored as novel potential antioxidants and an anti-tumor drug in a clinical setting. Keywords: P. fortuneana; Polysaccharides; Antioxidant activity; Apoptosis; Cancer

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1. Introduction Polysaccharides are important natural biopolymers occurring in almost all organisms, which play important roles in some physiological functions, such as immunity, regulating cell growth and senescence [1]. Polysaccharides have become a hot topic in many disciplines, as they can be widely used in medicine, health products, materials and functional foods. The study of the biological activity of polysaccharides can be traced back to the discovery of anti-tumor effects of polysaccharides in 1936. Later, more fungal and plant polysaccharides were found to have obvious antibacterial and anti-tumor activities. It has also been reported that polysaccharides isolated from plants have some strong antioxidant actions on free radicals and should be further explored as novel potential antioxidants [2]. In recent years, the anti-tumor activity of plant polysaccharides (especially those extracted from Chinese herbs) has attracted research interest. Plant and microbial polysaccharides affect a variety of tumor cells, mainly through inhibiting tumor growth, inducing apoptosis, enhancing immune function and synergizing chemotherapy drugs [3]. Inducing the apoptosis of tumor cells is considered useful in inhibiting tumor growth and enhancing tumor therapy as well as in the preventing cancer [4]. A variety of active polysaccharides have been used clinically as anti-tumor, or as tumor adjuvant therapeutic drugs, together with drugs for alleviating tumor chemotherapy side-effects. P. fortuneana (Maxim.) Li, a plant species of Maloideae, locally known as Huo-ji [5],which is widely distributed in southern and northwestern China and has been used as a traditional Chinese medicine for the treatment of indigestion [6]. Several previous studies have shown that P. fortuneana has great medicinal and edible development value [7-9]. At present, P. fortuneana 3

is widely used in the fields of medicine, health, and functional food.To further elucidate the biological activity of P. fortuneana, we extracted its water-soluble polysaccharides. Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models and evaluating the effects of factors on the response and searching for the optimum conditions [10-12]. The principal advantage of using RSM is that the number of experimental runs required to evaluate multiple factors and their interactions can be reduced, consequently, which can be less time-consuming in optimizing a process compared with other approaches. RSM has been used to optimize the polysaccharide extraction process variables and their interactions [13-15]and which is a good way to determine optimal conditions for extraction of PSPF. Many studies had been confirmed that polysaccharides from Pyracantha fortuneana (PSPF) [5,16-17] have biological activities; however, there are no reports on its extraction methods, antitumor activity, chemical composition and molecular weight. Therefore, this study aims to determine the extraction conditions of PSPF through RSM. Various established methods were used to study their antioxidant activity and their properties were characterized by Fourier transform infrared (FT-IR) spectra. In addition, Skov3 cells were employed as a model to study the anti-tumor activity of PSPF, which provides a theoretical basis for anti-tumor research and treatment. 2.Materials and Methods 2.1. Materials Pyracantha fortuneana fruits were purchased from Hubei Province of China. All other 4

chemicals and solvents were all of analytical grade. 2.2. PSPF preparation The P. fortuneana fruits were dried at 50℃, pulverized and passed through 40 mesh sieve, and then ground in a high-speed disintegrator (Model SF-2000,Chinese Traditional Medicine Machine Works, Tianjin, China) to obtain a fine powder. The powder was extracted twice with 75% ethanol at 85℃ to degrease and remove some colored materials as well as small molecule materials through reflux. A single-factor experiment was performed after the insoluble residue was dried in an oven at 60 ℃ to get a pretreated sample: every pretreated sample (1g) was extracted with water in a designed heating times (X1: 1-5 h), extraction temperature (X2: 5090 ℃) and the ratio of water-to-raw material (X3: 10-50mL/g). All the extraction solutions have been removed colored materials with Column adsorption chromatography and then concentrated by evaporation under reduced pressure to remove protein with Sevage method [18]. Three volumes of 95% ethanol were added for precipitation of polysaccharide at 4 ℃ overnight. The precipitates were redissolved for eliminating protein with Sevage method,which was repeated 3 times and the supernatant were collected by centrifugation (10,000 rpm for 10min) and freeze drying for 12 h. 2.3. Preparation of standard curve Glucose standard liquid (0.1mg/ml) 0.2, 0.4, 0.6, 0.8 and 1.0 mL was pipetted into tubes respectively and distilled water was added to make them up to 1.0 ml,then 1.4 ml of 5% liquid phenol together with 5.0 ml of concentrated sulfuric acid were added and the solution was mixed 5

by vortex, reacted in a boiling water bath for 30 min and absorbency determined at 490 nm. As a blank control, glucose standard liquid was substituted with 1.0 ml of distilled water and subsequently the glucose contents of the mixed solutions were determined with phenol-sulfuric acid method [19], and a glucose standard curve obtained with regression equation : y=8.4245x 0.0204 (R2=0.999), where y is absorbance at 490 nm and x is glucose concentration (mg/mL). The standard curve had a good linear relationship between 0.01-0.1mg/mL. 2.4. Determination of PSPF purity Certain concentrations of polysaccharide solution were prepared and their absorbance values were used to construct a standard curve. The yield of crude polysaccharide was determined as follows: Yield (%) = W 1 /W 0 ×100% W 1 = weight of extracted polysaccharide W 0 = weight of raw material. 2.5. Experimental design of RSM Basing on the experimental design and the results of single-factor experiments, three variables were applied to statistically optimize the extraction of PSPF. The three independent variables considered were heating time (X1, A), extraction temperature (X2 , B) and the ratio of water to raw material (X3 , C) ,as a result, the response variable was the yield of PSPF and the three levels for the three factors are shown in Table 1,the uncoded independent variables were coded according to the following transfer equation [20]:

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Xj=

X X i

0

X

i  1,2,3

Where Xj is the dimensionless value of an independent variable; Xi′ – X0 is the real value of an independent variable with X 0 at the center point and ΔX is the step change value[20]. In order to predict the optimal conditions, experimental data were analyzed using Design– Expert software (v.8.0.6.1 trial, Stat-Ease Inc., Minneapolis, USA) and the quadratic equation for the variables was given below:

Y=

3

3

i 1

i 1

2

A0 +  Ai X i +  Aii X i +  2

3

X X

i 1 j i 1

i

j

Where Y is the predicted response variables associated with each three - level combination,

A0 is a constant coefficient, and Ai , Aii and Aij are coefficients estimated by the model. The Xi and X are coded independent variables that represent the linear, quadratic and cross-product effects of the X1, X2 and X3 factors on the response, respectively [21-22]. The model evaluated the effects of each independent variable on the response. Analysis of the experimental design and calculation of predicted data were performed with Design-Expert software to estimate the response of the independent variables [23]. 2.6. Verification test In order to verify the accuracy of the regression equation and the model obtained by response surface analysis, five parallel experiments were conducted under the optimum conditions and the final conditions were determined.

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2.7. Infrared spectroscopy analysis Samples of PSPF were dried under vacuum at 50℃ to remove the free water in the sample subsequently to eliminate interference of water-molecule absorption peaks [24]. 200 mg potassium bromide (KBr) powder with spectroscopic -grade, was weighed and placed in an agate mortar, together with about 2 mg of PSPF immediately, which weight was 1% of KBr. Then the sample was milled to allow it to be fully blended and then pressed. Finally, the organic functional groups of the polysaccharide fraction were identified by FT-IR spectrometry (PerkinElmer, Spectrum 400, USA) with a scanning wavelength range of 4000-400 cm −1 . 2.8. Determination of molecular weight The PSFP molecular was measured with HPSEC-MALLS-RID on the basis of the dn/dc method according to previous reported methods [25]. The HPSEC-MALLS-RID was carried out in a HPSEC columns (TSKgel SuperMultipore PW-M, Tosoh, Japan) coupled with a MALLS detector (DAWN HELEOSII, Wyatt Technology, Santa Barbara, CA, USA), and refractive index detector (2414, Waters, USA) with the refractive index increment (dn/dc) of 0.135 mL/g. The MALLS instrument was equipped with a He-Ne laser (λ = 663.6 nm). The PSPF solution was filtered by a membrane with 0.45 μm pore size before injection and eluted with 0.1 M NaCl (0.5 M/min). The column temperature was kept at 40℃. Astra software (version 6.0.2, Wyatt Technology Co) was utilized for data acquisition and analysis. 2.9. SEM analysis Scanning electron microscope (Hitachi, Japan) was adopted to investigate the 8

morphological features of purified PSPF and the dried PSPF was installed on a metal stage and sputtered with gold for rendering the powder conductive. 2.10 Analysis of monosaccharide composition Pre-column derivatization high performance liquid chromatography (HPLC) was employed to analyze the monosaccharide species and the composition ratio of PSPF [26]. The PSPF (10 mg) was hydrolyzed to prepare monosaccharides under 2 mol/L trifluoroacetic acid (2.0 mL) at 70˚C for 100 min in a sealed ampule and the remaining trifluoroacetic acid was evicted by vacuum concentrator after hydrolysis for 6 h. Then, the dried sample was dissolved in 50 μL of .distilled water and analysis of monosaccharide compositions was performed by a HPLC 1260 with Extend-C18(4.6 mm × 250 mm, 5 µm) (Agilent, USA),along with following chromatography conditions : the mobile phase was composed of phosphate buffer (0.1 mol/L, pH 6.7) and phosphate - acetonitrile gradient elution and the flow-rate was 1.0 mL/min. Standard monosaccharides were subjected to the same conditions described above as a reference. According to the retention time of each chromatographic peak, the composition of monosaccharide

sample was determinedand the molar ratios of various monosaccharides were

calculated from the peak areas of the respective chromatographic peaks. 2.11. Antioxidant activity of PSPF 2.11.1. ABTS radical scavenging activity The capability to scavenge ABTS radical was investigated as described by Re et al [27] with some modifications. 0.5 mL ABTS solution with concentration of 15 mM was added to 2.5 9

mL of 15 mM potassium persulfate solution,then the ABTS+ cationic solution was formed at room temperature without light for more than 16 h, which was diluted with distilled water until absorbance at 734 nm was between 0.6800 and 0.7200, and then placed in the darkness. Polysaccharide solution at different concentration (1, 2, 4, 8 and 10 mg/mL), 50 μL was added to 200 μL of ABTS+ cationic solution and the absorbance of hydrogen peroxide at 734 nm was determined after incubation for 10 min at room temperature. The scavenging activity of ABTS radical was calculated as follows: scavenging effect (%) = [1- (A1 - A2)/A0] × 100%, where A1 is absorbance of sample, A2 is absorbance of the blank and A0 is absorbance of the control. 2.11.2. Metal ion scavenging activity The scavenging activity of metal ions was assayed according to Gülçin et al [28]. 200μLpolysaccharide solutions at different concentration (1, 2, 4, 8 and 10 mg/mL),was added to 10μL of FeCl2 solution, along with 20 μL of phenanthroline test solution and 0.27 mL of distilled water. Absorbance of hydrogen peroxide was determined at 562 nm. The scavenging activity on metal ions was calculated as follows: scavenging effect (%) = [1- (A1 - A2)/A0]×100%, where A1 is absorbance of sample, A2 is absorbance of the blank and A0 is absorbance of the control. 2.11.3. DPPH radical scavenging activity The antioxidant activity was also determined by DPPH radical scavenging activity measured as described by Ganie et al [29]. Solution containing 200μL of various concentration (1, 2, 4, 8 and 10 mg/mL) of each sample was added 200μL of DPPH solution at room temperature and the 10

absorbance was measured at 517 nm after 30 min incubation,then the scavenging activity on DPPH radical was calculated as follows: scavenging effect (%) = [1- (A1 - A2)/A0] × 100%, where A1 is absorbance of sample, A2 is absorbance of the blank and A0 is absorbance of the control. 2.11.4. Superoxide-radical scavenging activity The superoxide anion scavenging activities of polysaccharide samples with various concentrations were measured on the basis of a previously published method with some modifications[30]. 3 mL Tris-HCl buffer (pH 8.2) was kept in water bath at 25℃ for 20 min. Then 1 mL of sample solution and 0.3 mL of 25 mM 1,2,3-phentriol were added and incubated at 25°C for 5 min. Finally 1 mL of 10 mM HCl was added quickly to terminate the reaction and the absorbance of the mixture was determined at 420 nm. Scavenging activity of the plant extract on superoxide anion radical was expressed as the following: % inhibition = [(A0 ‐ A1)/A0] × 100 %, where A0 is absorbance of the control and A1 is absorbance in the presence of Podophyllum hexandrum extract (a known antioxidant). 2.12. Cell culture and cytotoxicity assay of PSPF Human ovarian carcinoma cells (Skov3) were cultured in DME/F12 medium(Hyclone, Logan, UT,USA) supplemented with fetal bovine serum (10%) and antibiotics (100 U/mL penicillin together with 100 µg/mL streptomycin) with a 5% CO2 atmosphere at 37°C in humidified incubator. The cells were used to evaluate the cytotoxicity of PSPF by MTT assay. The cytotoxicity of PSPF was calculated according to reported method [31]. The cell suspension 11

(100 μL/well) were seeded on a 96-well culture plate and incubated in humidified incubator for 24 h. Then the culture medium was replaced by

a fresh medium containing different

concentrations of PSPF(0, 25, 50, 100, 200μg/mL) when cells proliferated to 70-80% confluence. After treatment for 24 h, the suspension was replaced by 50 μL of MTT (1.0 mg/mL). The cells were incubated in humidified incubator for 4 h. Finally, 150 μL of DMSO was added to dissolve the formazan crystals for 1 h. Subsequently, absorbance at 630 nm and at 570 nm was determined. The experiment was repeated three times. 2.13. Measurement of ROS production Intracellular ROS levels were measured under the detection kit no S0033 (Beyotime, Jiangsu, China). Upon oxidation by ROS, the nonfluorescent DCFH-DA was converted to the highly fluorescent DCF [32]. The DCFH-DA probe was applied to detect the intracellular ROS levels. Cells were seeded in six-well plates for 24 h, then incubated with H2DCF-DA (10 µM) in darkness at 37°C for 30 min after the cells were treated with different concentrations of PSPF for 24 h. The DCF fluorescence intensity was measured by a fluorescence microscope with image analysis software. 2.14. Jc-1 assays Decreased ∆ψm is a hallmark of early apoptosis [33]. The JC-1 was detected by JC-1 Mitochondrial Membrane Potential Kit (Beyotime). Cells were cultured with different concentrations of PSPF for 24 h, then incubated in DMEM containing 10 µM JC-1 at 37℃ for 20 min before washing with JC-1 staining buffer and rapidly mounted for observation by a 12

fluorescent microscope. 2.15. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling analysis The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was employed to detect apoptotic cells in the PSPF-treated groups. The apoptotic cells were detected by TUNEL BrightRed Apoptosis Detection Kit (Vazyme). The cells were washed in PBS, fixed in 4% paraformaldehyde for 15 min, and incubated in a TUNEL reaction mixture at 37℃ for 1 h after culturing with different concentrations of PSPF for 24 h. Cells were visualized by a fluorescent microscope after mounted with Vectashield fluorescence. 2.16. Immunohistochemistry Cells were fixed in 4% paraformaldehyde, and blocked with 1% bovine serum albumin for 30 min after being hatched with different concentrations of PSPF for 24 h, then incubated with primary antibodies against γ-H2AX, Rad51 ,Bax or Caspase-3 at 4°C overnight. Then labeled Cy3-labeled Goat Anti-Rabbit IgG(H+L) or FITC-labeled Goat Anti-Mouse IgG(H+L) at a dilution of 1:100 at 37°C for 1h. After being washed three times with PBS, the samples were counterstained with Hochest and visualized under a confocal microscope. 2.17. Western blot analysis Cells were treated with 200 µg/mL PSPF for 12 h and harvested cell pellets were prepared for western blot analysis. The following primary antibodies were used: anti-γH2AX(Abcam ab26350, London, UK), anti-Rad51 (Abcam ab88572), anti-p53 (Sangon Biotech, D120082, Shanghai, China), anti-BAX (Cell Signaling Technology, #2772, Boston, USA), anti-Bcl2 13

(ImmunoWay YT0470, SuZhou JiangSu, China) and anti-β-action (Abcam ab8227). 2.18. Statistical analysis Data are presented as mean±SD for every duplicate in independent experiments which were performed at least three times. All the basic data were evaluated by one-way ANOVA (Scheffe’s F-test) followed by Tukey's test processed with Graph Pad Prism analysis software and the differences were regarded as significant at P<0.05.

3. Results and Discussion 3.1. Optimization of extraction parameters from polysaccharides 3.1.1. Optimization of yield of polysaccharide extraction In this experiment, Box-Behnken design (BBD) with three-levels, three-variables was applied to statistically optimize PSPF extraction. The design consisted of 17 experimental points carried out in random order (Table 2). Response surface regression analysis was performed and results were estimated as regression coefficients of a second-order polynomial model for yield of polysaccharides. Based on results in Table 2, a second-order polynomial model equation was fitted: Y=2.57475+0.073675A+0.10698B+0.2995C+5×10-5AB+3.75×10-3AC-2.5×10-4BC-1.38×10-3A28.8×10-4B2-0.1055C2. Table 3 showed the analysis of variance (ANOVA) of the BBD experimental results. The P-values were used to determine the significance and contribution of 14

each factor as well as the polynomial model equation [34]. An F-test was used to check statistical significance of the regression equation. The ANOVA was performed by Design–Expert software to determine whether or not the quadratic model was significant [30]. The larger the magnitude of the F-value and the smaller p-value, the higher is the significance of the corresponding coefficient [35-36]. The F-test showed a high model F-value (160.43) and a very low P-value (P < 0.0001; Table 3). These results showed that the regression model fitted the experimental data and the model can be used to analyze and predict the PSPF extraction process. Values of p < 0.0500” indicated model terms are significant, but p > 0.1 implied the model was not significant. In the case shown in Table 3, A,B, AC, A2, B2 and C2 are significant model terms. The results showed that the influence of various factors on the extraction of PSPF was not a simple linear relationship and the three factors were important factors in the extraction process. The fitted quadratic polynomial model of extraction of polysaccharides was determined by determination coefficient (R2; Table 4). The “Pred R-Squared” = 0.9788 was in reasonable agreement with the “Adj R-Squared” = 0.9890 (Table 4). The very low value of coefficient of the variation (CV%, 0.57%) clearly indicated a high degree of precision and reliability of the experimental values. The “R2”=0.9952 was used to measure the signal-to-noise ratio and showed that the model was significant, with a good fit and the experimental factors had a strong effect on the response value. The ratio of being greater than 4 along with the“Adeq. Precision” of 33.987 indicated that this model could be used to navigate the design space. In addition, the item F-measure was 0.32 (P = 0.8092>0.05) showed no significant relationship with pure error, and so the regression model could be used to guide the experiment. 15

Response surfaces were plotted using Design–Expert software to show effects of the three parameters and their interactions on extraction yield of PSPF. The response surface and the contour of the multiple regression equation are shown in Figure 1. Using the dynamic maps, we analyzed and evaluated the effect of the interactions between every two factors, then determined the best factor range. The best horizontal range was the top of the response surface or its vicinity. The response surface is a three-dimensional spatial surface diagram of response value and each experimental factor - a steeper curve of the surface indicates a greater effect on the experimental results. The shape of contour reflects the degree of interaction between the two factors; circles indicate no significant interaction of the two factors,by contrast, significantlywith elliptical shapes . The optimal relative variables are located at the coordinates of the central point which represents the highest level of each figure. The interaction effect of the ratio of water to raw material and extraction temperature resulted in circular contour plots, indicating non-significant interaction (Figure 1A and a). while significantly between the interaction of extraction time and ratio of water to raw material (Figure 1B and b), also, the interaction of extraction time and temperature was not (Figure 1C and c)and the extraction temperature and the ratio of water to raw material had the most significant effect on the yield of PSPF. The Box-Behnken experiment and the quadratic polynomial regression equation indicated the following optimal ultrasonic-assisted extraction conditions: the ratio of water to raw material, 36.3, extraction time of 2.1 h and extraction temperature as 61.5℃. 3.1.2. Verification of the mode

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The optimum extraction conditions (A = 2.09 h, B = 61.51℃ and C = 36.28 mL/g) for polysaccharide extraction were verified five times. The extraction yield of five experiments was 2.08, 2.10, 2.13, 1.98 and 2.11% respectively, while the predicted value was 2.083% from fitted equations in the above conditions. A mean of 2.08% for extraction yield of the polysaccharides was gained by five independent real experiments, which confirmed that the response model was adequate for optimization. All crude polysaccharides were purified several times and then the total carbohydrate content of 93.5% was determined by phenol-sulfuric acid method. 3.2. IR spectroscopy The FT-IR spectra of PSPF displayed a broad and intense peak around 3421.5cm-1 (Figure 2A), which was assigned to the stretching vibration of hydroxyl-group. Besides, It was shown that the moisture content in the sample. The weak peak at 2924.31cm-1 was attributed to C-H and C-H2 stretching vibration signal, the absorption peak near 1403.33 cm-1 also indicated the presence of C-H. The absorption peak at 1636 cm−1 was caused by the C=O symmetric stretching vibration of carboxyl group. In addition, FT-IR spectra of each polysaccharide fraction showed specific band maxima in the 1000–1200 cm−1 region, which was attributed to ring vibrations overlapping with stretching vibrations of C-OH side-groups and the C-O-C glycosidic-bond vibration [37-38]. The absorption peaks around 1100 cm−1(PSPF at 1105.47) suggested that the sugar rings of the two polysaccharide fractions were pyranose rings. Furthermore, absorptions at 619.07 and 540.05 cm−1 were due to C-H deformation-induced vibration [39]. 3.3. Molecular weight

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The molecular weight of PSPF was determined by HPSEC-MALLS-RID, which has advantages of simple operation, high sensitivity and high precision. The distribution curve of the laser light signal and the differential detection signal (dRI) of PSPF showed two different components corresponding to two different peaks at about 8.4-17.1 and 17.1-20.4 mins (Figure 2B). T The weight average molecular weight (Mw) of peak1 was 2.513×106 (±6.499%) g/mol and the number average molecular weight (Mn) was 3.279×105 (±22.677%)g/mol. And the weight average molecular weight (Mw) of peak2 was 4.974×104 (±45.544%) g/mol and the number average molecular weight (Mn) was 2.311×104 (±43.325%)g/mol. The mass fraction of peak1 and peak2 were 7.4% and 92.6% respectively. In addition, the polydispersity index (Mw / Mn) indicated the breadth of the molecular weight distribution of the polymer-with the larger the Mw / Mn, the wider distribution of molecular weight. The Mw / Mn of

peak1 and peak2

were 7.663 (±23.590%) and 2.152 (±62.859%) respectively, indicating that the molecular weight distribution of PSPF was relatively wide. Thus, polysaccharide needed further purification. 3.Scanning electron microscopy of PSPF Scanning electron microscopy (SEM) can be a useful tool to analyze the surface morphology of polysaccharide powder. The SEM of PSPF (Figure.3) revealed the defined shape of PSPF, which was shown aggregates of irregular shaps and dimensions. 3.4. Monosaccharide composition of PSPF Monosaccharides are the basis of polysaccharide chemical structure,which contribute to the biological activity of polysaccharides. Thus, it was very important to analyze the 18

monosaccharide composition of PSPF. The monosaccharide composition of LJP (Table 5) implied the dominance of sorbose(Sor), rhamnose (Rha), mannose (Man), glucose (Glu), galactose (Gal), xylose (Xyl) and arabinose (Ara) in PSPF. 3.5. Antioxidant activity of PSPF 3.5.1. DPPH radical scavenging activity The DPPH radical scavenging rate increased from 21.0% to nearly 93.5%when the PSPF concentration increased from 1.0 to 8.0 mg/mL (Figure 4A), which decreased yet to 90.51% with PSPF concentration of 10.0 mg/mL, In a word, PSPF should be explored as a potential antioxidant. 3.5.2. Metal ion scavenging activity Metal chelating activity is generally acknowledged as an antioxidant mechanism because it reduces the concentration of transition metals catalyzing lipid peroxidation [40]. When the concentration of PSPF was 10 mg/mL, its scavenging effect on metal ions increased to 67.13% (Figure 4B). The scavenging activity increased with risingsample concentration indicating concentration dependence. 3.5.3. ABTS radical scavenging activity The ABTS assay is often used to measure the total antioxidant power of a potential antioxidant and can also be adopted as an index of antioxidant activity [41]. The ABTS radical scavenging activity rose as the sample concentration increased (Figure 4C) with very obvious 19

changes within 1- 4 mg/mL. 3.5.4 Superoxide-radical scavenging activity The highly toxic superoxide anion radical is generated first of the reactive oxygen species(ROS) by numerous photochemical and biological reactions. The superoxide-radical scavenging activity of PSPF rose as sample concentration increased within 1- 4 mg/mL (Figure 4D). However, s decreased for sample concentration of 8.0 - 10.0 mg/mL. 3.6. Effect of PSPF on cell viability and cell morphology The Skov3 cells were significantly dose-dependent for cells exposed to different PSPF concentration (Figure 5). The cell viability clearly decreased during 24 h of exposure to 100–200 µg/mL, In addition, many experiments [42-44] have shown that plant polysaccharides can strongly induce apoptosis of tumor cells. The apoptotic status of cells can be determined by way of cell morphology and the control cells showed a distinct shape with elongated spindle morphology and a regular cell margin (Figure 5A). However, some cells become rounded up after treatment with 200µg/mL of PSPF. The viability of human ovarian cancer cells decreased significantly during the 24 h exposure to 100 and 200µg/mL PSPF (P<0.05; Figure 5B).

3.7. PSPF induce increased ROS level The effect of PSPF on cell viability indicated that PSPF increased ROS level in the treated Skov3 cells. Many studies have shown that ROS are closely related to apoptosis; increasing the ROS level leads to decrease

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in the level of the antioxidative system in cells

and so results in oxidative damage to cell components [45-46]. The fluorescent probe DCFH-DA is commonly used for detection of ROS. The DCFH-DA without fluorescence can freely cross the cell membrane and enter living cells and is then hydrolyzed to produce DCFH, which cannot easily pass through the cell membrane. In the presence of ROS, DCFH is oxidized to produce the fluorescent 2′,7′dichlorofluorescein (DCF) [47-48]. The fluorescence intensity of DCF was increased, indicating the higher ROS level, in the PSPF-treated compared with the control group (Figure 6A). The results demonstrated that the intercellular ROS in Skov3 cells was upregulated by PSPF. Additionally, fluorescence intensity of the PSPF-treated group increased in a dose-dependent manner (P < 0.01, Figure 6B). Together, the results suggested that PSPF enhanced the ROS production and was responsible for cell death.

3.8. Loss of mitochondrial membrane potential (∆ψm) and apoptosis induction In this study, PSPF induced an increase in ROS level and cell death usually along with ROS production, which is mainly derived from mitochondria. The ∆Ψm decline is an early manifestation of apoptosis. Once ∆Ψm is lost, cells enter an irreversible apoptosis process. The JC-1 is an ideal fluorescent probe widely used to detect ∆Ψm. When ∆Ψm is high, JC-1 accumulates in the mitochondrial to form a polymer that can generate red fluorescence. But when ∆Ψm is low, JC-1 cannot accumulate in the matrix and produces a green fluorescence [49-50]. The decrease in cell membrane potential can be easily detected by the transition from red to green fluorescence of JC-1. Green fluorescence was apparent in cells with the increase of PSPF concentration (Figure 7A). The cells exposed to PSPF exhibited significant (P < 0.01) mitochondrial damage in a dose-dependent manner (Figure 7B). We then investigated the level of apoptosis in PSPF-treated Skov3 cells using TUNEL assay, which is an effective method to 21

evaluate cell apoptosis by detecting DNA rupture in the early stage of apoptosis[51]. The Skov3 cells treated with PSPF showed higher levels of TUNEL-positive cells compared with the control group, and cells showing fluorescence in the high-concentration treatment group were higher in number and intensity- so apoptosis was more pronounced.

3.9. PSPF induces nuclear DNA breakage Apoptosis is always accompanied by DNA damage. One of the earliest reactions after DNA double-strand breaks is phosphorylation of the C-terminal serine residue of histone H2AX near the breakpoint to form γ-H2AX. Up to now, γ-H2AX has been the most important DNA damagesensing molecule and a growing number of studies have suggested it is a good protein marker for DNA damage [52-53]. The Rad51 protein plays a major role in the repair process after DNA damage, of which an appropriate level is necessary to repair spontaneous DNA damage [54]. We measured the distribution of γ-H2AX and Rad51 in the nucleus after exposure to PSPF for 24 h, The histone γ-H2AX was measured using immunofluorescent images of γ-H2AX phosphorylation (Figure 8A), which showed that green fluorescence representing γ-H2AX was enhanced significantly in treated samples compared with the controls and that the signals were stronger after treatment with 100 µg/mL and 200 µg/mL (P<0.01). In the control group, Skov3 cells had no γ-H2AX foci in the nuclei. We concluded that increasing the PSPF concentration led to an increased percentage of γ-H2AX-positive cells（Figure 8a). Moreover, DNA damage in such cells was confirmed by the nuclear positivity for RAD51 staining( Figure 8B and b ),which were consistent with our results showing that PSPF induced apoptosis and DNA damage in ovarian cancer cells in a dose-dependent manner. 22

3.10 PSPF induce apoptosis by activation of bax and Caspase-3 expression Apoptosis is an active and programmed death process in which cells are precisely regulated by multiple genes under certain physiological or pathological conditions [55]. Among the numerous apoptosis-regulating genes, the Bcl-2 protein and the caspase families are currently the most closely studied,, of which Bcl-2 and bax are the two most important regulatory genes currently known to function in apoptosis regulation. The Bax protein has a role in promoting apoptosis in cells and is activated to change the molecular structure once the apoptotic signal stimulus is received, and then destroys the integrity of the mitochondrial membrane and prevents the anti-apoptotic effect of Bcl-2 [56]. Caspase-3 is the most critical downstream apoptotic factor in the caspase cascade,whose activation depends to a large extent on the release of cyt-c. The Bax gene can release cyt-c and other substances through the mitochondrial pathway and activate Caspase-3, then the caspase cascade reaction was initiated [57-58].The cells treated with PSPF showed higher numbers of Bax and Caspase-3 positive cells compared to controls, and fluorescence was detectable only at a polysaccharide concentration of 200 µg/mL, but not at lower concentrations (Figure 9). The expression of Bax and Caspase-3 protein in Skov3 cells treated with sufficient PSPF concentration were increased in a dose-dependent manner. These results further demonstrate that PSPF induces apoptosis in ovarian cancer cells. 3.11. Activation of signaling molecules involved in apoptotic Next we examined the mechanism of cell death caused by PSPF using western blot analysis, through which expressions of apoptosis-related proteins such as p53, Bax, Bcl-2, RAD51 and

23

γH2AX were examined (Figure.10). Levels of Bax and p53 proteins were significantly upregulated but Bcl-2 was significantly downregulated in PSPF-treated Skov3 cells, also expressions of γ-H2AX and RAD51 were significantly upregulated compared with the control group, as was consistent with immunofluorescence data (Figure 9). 4. Conclusions The results were analyzed using a response surface model and under optimal conditions (heating time 2.09 h, extraction temperature 61.51°C and ratio of water to raw material of 36.28mL/g), and the yield of polysaccharides was 2.08%. Validation experiments were also performed to verify the accuracy of the mode – the predicted value was in good agreement with the experimental value, and the carbohydrate content was 93.5%. Additionally, antioxidation tests in vitro indicated that PSPF had scavenging activities on superoxide, DPPH and ABTS radical and metal ion. We also demonstrated that PSPF could induce significant cytotoxicity to Skov3 cells via induction of intracellular ROS, which directly affected a mechanical pathway of cell viability by apoptosis, caused mitochondrial disruption and consequent DNA damage. Thus, PSPF could be developed as a potential antioxidant ingredient for functional foods and pharmaceutical industries. It also has good prospects as a functional dietary additive for dietary fiber and an anti-tumor drug in clinical settings.

Author Contributions: Wang LM designed the study. Yao YL, Shu C, Feng G, Wang Q, Yan YY, Yi Y, Wang HX and Zhang XF collected data. All authors agreed the final version.

Funding: This research was funded by the Nature Science Foundation of China [21602166]. 24

Conflicts of Interest: The authors declare no conflict of interest.

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Figure legends 32

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Figure 1. Response surface plots showing effects of variables on the extraction yield of PSPF. A. Ratio of water to raw material (mL/g), B. extraction temperature (°C) and C. extraction time (h).

Figure 2. FT-IR spectrum and GPC chromatogram of PSPF. A. FT-IR spectrum of PSPF; B. GPC chromatogram of PSPF for molecular weight determination with HPSECMALLS-RID system.

Figure 3. Scanning election microscope (SEM) photographs of PSPF

Figure 4. Antioxidant activity of PSPF. A.Scavenging effects of PSPF on DPPH. Value are means±S.D(n=3).B. Scavenging effects of PSPF on superoxide anion radical. Value are means±S.D(n=3).C.Scavenging effects of PSPF on ABTS radical. Value are means±S.D(n=3).D.Scavenging effects of PSPF on hydroxyl radical. Value are means±S.D(n=3).

Figure 5. Effects of PSPF on cell viability and cell morphology. Skov3 cells were incubated with different PSPF concentrations for 24 h, and then cell viability was determined by MTT assay. A. Phase contrast microscopy showing the morphology of Skov3 cells after PSPF treatment for 24 h. Scale bars =100 µm. B. Results are expressed as the mean ± standard deviation of three independent experiments. A significant difference was observed between 33

control and treated cells.The viability of treated cells was compared to that of untreated cells using Student’s t-test (P<0.05).

Figure 6. Evaluation of ROS level in Skov3 cells after PSPF treatment. A. Intracellular ROS levels were measured with fluorescence imaging using the DCFH-DA probe in cells cultured in the presence of PSPF (0, 25, 50, 100, and 200 µg/mL) for 24 h. Scale bars = 100 µm. B. Average intensity of fluorescence in Skov3 cells. Results are expressed as the mean ± standard deviation of three independent experiments(Student’s t-test, P<0.05).

Figure 7. Loss of mitochondrial membrane potential (∆ψm) and apoptosis induction A. The∆ψm was evaluated using JC-1 in treated cells. Red fluorescence indicates JC-1 aggregates within mitochondria in healthy cells, whereas green fluorescence indicates JC-1 monomers in cytoplasm and loss of ∆ψm. Scale bars = 100 µm. B. Ratio of JC-1 monomers to JC-1 aggregates. C. Apoptosis was assessed in a Tunel assay; nuclei were counterstained with Hoechst and apoptotic Skov3 cells are labeled red while cell nuclei are labeled blue, scale bars = 100 µm. D.Average intensity of TUNEL fluorescence in Skov3 cells.

Figure 8. Nuclear DNA damage in Skov3 cells after PSPF treatment using immunocytofluorescence with γ-H2AX and Rad51 antibody. A. Nuclear γ-H2AX foci in cells exposed in vitro to PSPF for 24 h. Scale bars = 100 µm. a. Average intensity of γ34

H2AX fluorescence in Skov3., B. Nuclear rad51 foci in cells exposed in vitro to PSPF for 24 h. b. Average intensity of Rad51 fluorescence in Skov3. Scale bars =100 µm.

Figure 9. PSPF exposure increases apoptosis in cultured Skov3 cells in 24h. A. Baxstained Skov3. Scale bars = 100 µm. a. Average intensity of Bax fluorescence in Skov3. Scale bars = 100 µm. B. caspase-3-stained Skov3. b. Average intensity of Caspase-3 fluorescence in Skov3.

Figure 10. Assessment of Western blotting. Western blotting of Bax, Bcl2, RAD51, γH2AX and p53 expression in Skov3 cells. Cells were treated with PSPF at 200µg/mL for 24h.

35

Table 1 Levels and code of extraction variables used in Box-Behnken design Variable

Symbols

Coded levels

Uncoded

Coded

-1

0

Ration of water to raw material

X1

Exaction temperature(℃) Exaction time(h)

A

20

30

40

X2

B

50

60

70

X3

C

1

2

3

Table 2 Box-Behnken experimental design and the results for extraction yield of polysaccharides

36

Run

A

B

C

Yield (%)

1

0

-1

1

1.91

2

0

1

-1

1.97

3

0

0

0

2.12

4

0

0

0

2.11

5

1

-1

0

1.89

6

-1

0

1

1.83

7

1

0

-1

1.87

8

0

1

1

1.96

9

-1

1

0

1.90

1

10

1

1

0

1.94

11

0

0

0

2.12

12

-1

0

-1

1.92

13

1

0

1

1.93

14

0

-1

-1

1.91

15

0

0

0

2.14

16

0

0

0

2.14

17

-1

-1

0

1.88

Table 3 Analysis of variance of the experimental results of the BBD

37

Variables

Sum of squares

df

Mean square

F-value

p-value Prob.>F

Model

0.18

9

0.021

160.43

< 0.0001**

A

1.513E-03

1

1.513E-03

11.83

0.0108

B

4.513E-03

1

4.513E-03

35.29

0.0006**

C

2E-04

1

2E-04

1.56

0.2515

A×B

1E-04

1

1E-04

0.78

0.4058

A×C

5.625E-03

1

5.625E-03

43.99

0.0003**

B×C

2.5E-05

1

2.5E-05

0.2

0.6717

A2

0.08

1

0.08

627.15

< 0.0001**

B2

0.033

1

0.033

255.02

< 0.0001**

C2

0.043

1

0.043

332.62

< 0.0001**

Residual

8.95E-04

7

1.279E-04

-

-

Lack of fit

1.75E-05

3

5.833E-05

0.32

0.8092

Pure error

7.2E-04

4

1.8E-04

-

-

Table 4 Analysis of variance of the fitted quadratic polynomial model of extraction of PSPF Item

Std.dev

Mean

C.V.

Press

R2

R2Adj

R2Pred

Adep.precision

3.925E-003

0.9952

0.989

0.9788

33.987

% Value

38

0.011

1.97

0.57

Table 5. Monosaccharide compositions of PSPF Mol(%)

PSPF

39

Sor

Rha

Man

Glu

Gal

Xyl

Ara

9.3

19.5

37.6

10.5

6.6

4.8

11.7

Figure.1 A

a

B

b

a

c

c

C

b

A:Ration of water to raw material, B:Exaction temperature, C:Exaction time.

Figure.2

% Transmittance

A

Wave numbers(cm-1)

B

Relative Scale

0.5

0.0

-0.5

-1.0

0.0

10.0

20.0 time(min)

30.0

Figure.3

Superoxide anion radical scavenging rate(%)

ABTS radical scavenging rate(%)

Metal ion radical scavenging rate(%)

DPPH radical scavenging rate(%)

Figure.4 A

B

Concentration of PSPF(mg/mL)

C

Concentration of PSPF(mg/mL) Concentration of PSPF(mg/mL)

D

Concentration of PSPF(mg/mL)

Figure.5 0μg/mL

50μg/mL

200μg/mL

B

25μg/mL

100μg/mL

Cell viability (% of control)

A

120 100 80

*

*

100

200

60 40 20 0 0

25

50

Concentration of PSPF (μg/mL)

Figure.6

Average intensity of Dihydroethidium fluorescence

200 200

3

**

100

4

2

**

50

B

25

**

0

**

A

1 0 0

25

50 100 200

Concentration of PSPF(μg/mL)

200

Figure.7

a

Concentration of PSPF (μg/mL)

Merged

JC-1 monomer

Average intensity of Dihydroethidium fluorescence

JC-1 aggregate

200

B 50

100

1.2 0.8 0.4 0 0

25 50 100 200

Concentration of PSPF(μg/mL)

200 6 Apoptosis rate(%)

Hoechst Tunel

25

1.6

b

Concentration of PSPF (μg/mL) 0

2

5 4

** *

100

**

50

*

25

**

0

**

A

3 2

Merge

1 0 0

25 50 100 200

Concentration of PSPF(μg/mL)

Figure.8

Merge

γH2AX

Average intensity of Dihydroethidium fluorescence

2.5 2

** **

Hoechst

a ** **

Concentration of PSPF (μg/mL) 0 25 50 100 200

A

1.5 1 0.5 0 0

25 50 100 200

Concentration of PSPF(μg/mL)

b Average intensity of Dihydroethidium fluorescence

Hoechst Rad51 Merge

200

3 2.5 2 1.5 1 0.5 0

**

0

Concentration of PSPF (μg/mL) 25 50 100

**

B

0 25 50 100200 Concentration of PSPF(μg/mL)

Figure.9 25

50

100

200

Bax Merge

25

50

100

200

1 0.5 0 0

25 50 100 200

b 2.5 Average intensity of Dihydroethidium fluorescence

Hoechst Caspase3

1.5

2

**

0

Merge

2

Concentration of PSPF(μg/mL)

Concentration of PSPF (μg/mL)

B

a Average intensity of Dihydroethidium fluorescence

Hoechst

0

**

A

** **

Concentration of PSPF (μg/mL)

1.5 1 0.5 0 0

25 50 100 200

Concentration of PSPF(μg/mL)

Figure.10

Control 200μg/mL

β-action

42 kDa

Bax

21 kDa

Bcl2

27 kDa

RAD51

37 kDa

γH2AX

16 kDa

p53

44 kDa