Optimization of Synthesis of Seleno-Sargassum fusiforme ( Harv . ) Setch . Polysaccharide by Response Surface Methodology , Its Characterization , and Antioxidant Activity

The response surfacemethodology was employed to optimize the synthesis conditions of seleno-Sargassum fusiforme (Harv.) Setch. polysaccharide. Three independent variables (reaction time, reaction temperature, and ratio of Na 2 SeO 3 to SFPSI) were tested. Furthermore, the characterization and antioxidant activity of Se-SFPSI in vivo were investigated. The result showed that the actual experimental Se content of Se-SFPSI was 3.352mg/g at the optimum reaction conditions of reaction time 8 h, reaction temperature 71C, and ratio ofNa 2 SeO 3 to SFPSIB 1.0 g/g. A series of experiments showed that the characterization of Se-SFPSIBwas significantly different from that of SFPSIB. Additionally, antioxidant activity assay indicated that the Se-SFPSIB could increase catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity of mice bearing tumor S 180 in blood, heart, and liver while decreasing malondialdehyde (MDA) levels. It can be concluded that selenylation is a feasible approach to obtain selenopolysaccharide which was utilized as highly biological medicine or functional food.


Introduction
Sargassum fusiforme (Harv.)Setch., which is cultivated in the sea of China and eastern Asia, is a traditional edible and medicinal plant.It is used for treating some diseases, such as diabetes, high cholesterol, and thyroid disease [1].Moreover, research shows that Sargassum fusiforme (Harv.)Setch.polysaccharide (SFPS) as an active ingredient [2] has biological activity in vivo [3] and in vitro [4].
Meanwhile, selenium is a necessary micronutrient for human beings and animals [5], but it is usually poisonous in inorganics [6].As an organic selenium compound, selenopolysaccharide maintains the basic configuration of the polysaccharide and physiological functions of selenium.Moreover, seleno-polysaccharide can improve selenium bioavailability and reduce the toxicity [7] and side effects of inorganic selenium [8].
Due to the insufficiency of natural seleno-polysaccharide in nature, artificial method is the best way to increase the yield of seleno-polysaccharide.Compared with biotransformation, chemical synthesis is the most effective approach to harvest seleno-polysaccharide.It is also well known that the structure of polysaccharide is intimately related to its functions.The microstructure of seleno-polysaccharide may be changed by chemical modification.
Up to now, little information is available on the optimal conditions for seleno-seleno-Sargassum fusiforme (Harv.)Setch.polysaccharide (Se-SFPS) synthesis.Meanwhile, no experiments were employed to explore its characterization and antioxidant activity.Therefore, this paper optimized synthesis of seleno-Sargassum fusiforme (Harv.)Setch.polysaccharide by response surface methodology (RSM) [9,10] and investigated its characterization and antioxidant activity.

Purification and Isolation of SFPS.
The crude SFPS was extracted from the dried body of Sargassum fusiforme (Harv.)Setch.which was purchased from Wenzhou City, China.
Protein of crude polysaccharide solution was discarded by 0.1% (g/v) papain and Sevag, and pigment was treated with H 2 O 2 .Then, SFPSI was obtained from SFPS solution that was precipitated by dehydrated alcohol to a final concentration of 20% (v/v).After that, SFPSI was isolated by DEAE-52 column chromatogram with different NaCl solutions (0.0, 0.05, 0.1, 0.2, 0.3, 0.5, and 0.8 mol/L), respectively.The component eluted by 0.05 mol/L NaCl had the maximum proportion, named SFPSIB.SFPSI was used to optimize the synthesis, and Se-SFPSIB was used to detect the characterization and antioxidant activity.

Selenylation.
Different ratios of Na 2 SeO 3 and SFPSI were dissolved by HNO 3 (100 mL, 0.05%) in Erlenmeyer flasks.Then, the mixed solution was reacted in the designed reaction time, reaction temperature, and water bath shaking rate.After the reaction, Na 2 CO 3 was added to the reacted solution to adjust pH to 5-6.The solution was centrifuged to remove the insoluble residue (3000 ×g for 5 min, at 20 ∘ C) and then dialyzed with dialysis membrane (Molecular Weight Cutoff: 8000 ∼ 14000, Solarbio Science & Technology Co., Ltd., Beijing, China) in running water.Ascorbic acid was used to detect whether free SeO 3 2− was dialyzed out from the solution.When ascorbic acid did not redden the solution, the Se-SFPSI solution was collected and freeze-dried [10].

Optimization of Synthesis of Se-SFPSI.
According to the results of monofactor experiments for Se-SFPSI, a RSM was used to optimize the reaction conditions for Se-SFPSI.A multivariate study based on Box-Behnken design (BBD) was chosen to evaluate effects of reaction parameters.Three independent variables in a BBD ( 1 , reaction time;  2 , reaction temperature;  3 , ratio of Na 2 SeO 3 to SFPSI) at three levels were performed.Table 1 showed the range of independent variables and their levels.Se content as response value was calculated according to the linear equation ( = 0.0197 + 0.0343,  2 = 0.9998), which was detected by toluene-1,2phenylenediamine method and measured at 334 nm by 752 type UV-Vis spectrophotometer (Shanghai Spectrum Instruments Co., Ltd., Shanghai, China).4) Se-SFPSIB groups: mice of low-, medium-, and high-dose groups were treated with Se-SFPSIB at the dosage of 50, 100, and 150 mg/(kg⋅d), respectively.After 24 h of inoculation, the mice bearing tumor S 180 were treated in peritoneal injection (i.p.) for 10 days.Then, CAT, MDA, SOD, and GSH-Px of mice bearing tumor S 180 in blood, heart, and liver were detected by antioxidant assay kits, obtained from Nanjing Jiancheng Bioengineering Institute, China.

Statistical Analysis.
The experimental data was analyzed by SPSS 16.0 software (SPSS Inc., Chicago, USA).Statistical comparison within groups was carried out by one-way ANOVA.

Results and Discussion
3.1.Single Factor Assays.Figure 1 showed the effects of single factors on Se content in Se-SFPSI.As shown in Figure 1(a), Se content in Se-SFPSI reached a maximum value at 8 h, and Se content decreased after 8 h. Figure 1(b) showed that Se content in Se-SFPSI reached a maximum value at 70 ∘ C, and Se content decreased when the reaction temperature was 80 ∘ C. The reason for these results was that more and more monosaccharide and oligosaccharide were hydrolyzed from SFPSI by acidic water for the longer time and at the higher temperature.When the reacted solution was dialyzed with dialysis membrane in running water, the lower molecular weight monosaccharide and oligosaccharide were dialyzed out from solution.Se was introduced into monosaccharide, and oligosaccharide also was removed from dialysis membrane.Herein, 6-8 h and 60-80 ∘ C were considered to be optimal reaction time and reaction temperature in the RSM. Figure 1(c) indicated that Se content in Se-SFPSI significantly increased at the ratio of Na 2 SeO 3 to SFPSI 0.8 g/g, and then the curve kept a mild slope.Therefore, the ratio of Na 2 SeO 3 to SFPSI range of 0.6-1.0g/g was adopted for the reaction.Figure 1(d) showed that Se content in Se-SFPSI obviously increased with the increase of water bath shaking rate, but there was no significant increase after 40 r/min.This meant that 40 r/min was sufficient for the reaction.So, 40 r/min was determined as the reaction water bath shaking rate in next optimization experiments.

Experimental Results of Optimization by RSM.
The results of RSM analysis of the variation of Se content in Se-SFPSI with reaction time ( 1 ), reaction temperature ( 2 ), and ratio of Na 2 SeO 3 to SFPSI ( 3 ) are shown in Table 2.As shown in Table 2, Se content in Se-SFPSI ranged from 2.117 to 3.259 mg/g.By applying multiple regression analysis to the experimental data, the relationship between response variables and the test variables was obtained from the following secondorder polynomial equation: (1)

Model Fitting and Statistical Significance Analysis.
As shown in Table 3, Se content in Se-SFPSI was significantly affected by linear parameters ( 1 ,  2 ,  3 ), quadratic parameters ( 1 2 ,  2 2 ), and the interactive parameters ( 1  2 ,  2  3 ) ( < 0.05).However, the interactive parameter ( 1  3 ) and quadratic parameters ( 3 2 ) were not significant ( > 0.05).Meanwhile, ANOVA of quadratic regression model demonstrated that the variables were adequately fitted to the regression equation ( < 0.01) and adequate with satisfactory determination coefficients ( 2 = 0.9906, Adjusted  2 = 0.9785).This meant that the model could be used to analyze and predict selenylation process results.In addition, a low value 0.0162 of coefficient of the variation (C.V.) illustrated a high degree of precision and a good deal of reliability of the experimental values.

Optimization of Selenylation Conditions. Figure 2(a)
showed the interaction between reaction time and reaction temperature on the Se content in Se-SFPSI ( = 0.0003 < 0.05).When the reaction time and reaction temperature increased, the Se content increased before the medium level and then decreased.As shown in Figure 2(b), when the reaction time ranged from 7 h to 9 h and the ratio of Na 2 SeO 3 to SFPSI varied between 0.6 g/g and 0.8 g/g, the Se content in Se-SFPSI increased gradually.However, the interactive effect between reaction time and the ratio of Na 2 SeO 3 to SFPSI was not significant ( = 0.0659 > 0.05).As shown in Figure 2(c)  and Table 3, the interaction of reaction temperature and ratio of Na 2 SeO 3 to SFPSIB had a much higher effect on the selenylation ( = 0.0048 < 0.01).
According to the results of the analysis of regression model, the optimal reaction conditions for Se-SFPSI were at reaction time of 8.09 h, reaction temperature of 71.13 ∘ C, ratio of Na 2 SeO 3 to SFPSI of 1.0 g/g, and the predicted Se content in Se-SFPSI was 3.288 mg/g.

Verification Experiment.
For the actual operating convenience, the verification experiment was carried out under the optimum conditions: reaction time 8 h, reaction temperature 71 ∘ C, and ratio of Na 2 SeO 3 to SFPSI 1.0 g/g.The experimental value was (3.352 ± 0.04) mg/g, an average of triplicate experiments.The result confirmed the validity of the model, and the actual value was close to the predicted value.

Particle Size and Zeta Potential Distribution of Se-SFPSIB.
As shown in Figure 3, the particle size of Se-SFPSIB (95.07 nm) was significantly smaller than that of SFPSIB (177.40 nm).The reason was that SFPSIB was hydrolyzed into many smaller pieces by acidic water in selenylation.Figure 4 showed that the zeta potential of SFPSIB (0.489 mV) was higher than that of Se-SFPSIB (−0.194 mV).This meant that Se-SFPSIB was prone to clump and agglomerate.This also implied that stability of Se-SFPSIB was lower than that of SFPSIB in solution.
3.6.2.Differential Scanning Calorimetry Analysis.Figure 5 showed that the DSC spectrum shapes of Se-SFPSIB and SFPSIB were similar, and the major peaks of them were endothermic peak.However, the major endothermic peak temperature of Se-SFPSIB (176.12 ∘ C) was significantly lower than that of SFPSI (180.50 ∘ C).This phenomenon could be explained by chemical bonds and molecular orbital theory [10,11].The studies confirmed that the 6  -OH in polysaccharide could be substituted by HSeO 3 − in selenylation, as shown in Figure 6 [12,13].When HSeO 3 − was introduced into SFPSIB and substituted 6  -OH, p orbital of oxygen and Se=O bond formed the p- conjugated system in which the electron cloud of C-O transferred to Se=O.This caused C-O bond energy in Se-SFPSIB to be lower than that of SFPSIB, and C-O bond in Se-SFPSIB could be broken easily.Therefore, the lower C-O bond energy in Se-SFPSIB caused HSeO 3 − group to be easily removed from Se-SFPSIB in the lower temperature.Further, the removal of HSeO 3 − accelerated Se-SFPSIB decomposition.This confirmed that stability of Se-SFPSIB was lower than that of SFPSIB.3.6.3.X-Ray Diffraction Analysis.X-ray diffraction curves showed that Se-SFPSIB and SFPSIB were amorphous (Figure 7).This indicated that Se introduction had little effect on amorphous configuration of polysaccharide.This also explained that the regularity of polysaccharide molecules was poor and the configuration of polysaccharide was complex.

Shape in SEM.
It could be seen from Figure 8 that the shape of Se-SFPSIB was spherical and cylindrical, but the shape of SFPSIB presented long strip and rod.The figures demonstrated that the three-dimensional image of Se-SFPSIB was different from SFPSIB.This suggested that Se introduction could change polysaccharide morphology.

Antioxidant Activity Assay.
One of the major reasons for tumorigenesis is lipid peroxidation in which oxygen free radicals, MDA, and other peroxides are produced to damage body.
As one of the key enzymes in antioxidant system, CAT could promote H 2 O 2 decomposition and reduce H 2 O 2 damage to cells [14,15].It could be seen from Figure 9(a) that Se-SFPSIB could significantly improve the CAT activity in blood, heart, and liver ( < 0.01,  < 0.05, compared with negative group).Meanwhile, in comparison with the negative control, low Se-SFPSIB significantly improved CAT activity in blood, and mid-Se-SFPSIB group significantly improved CAT activity in liver.In addition, there were significant differences between Se-SFPSIB groups and SFPSIB group in heart ( < 0.01,  < 0.05).
MDA is a main product in lipid peroxidation.It reflects the degree of the body damage [16][17][18].According to  SOD is an important active enzyme to scavenge oxygen free radical, prevent cells from attacking, and repair damaged cells [19][20][21].As shown in Figure 9(c), there were significant differences between Se-SFPSIB groups and negative group ( < 0.01,  < 0.05).Meanwhile, mid-Se-SFPSIB significantly increased SOD compared with SFPSIB ( < 0.01).The result demonstrated that SFPSIB could enhance more SOD activity than SFPSIB.
Se is well known as an essential trace element and an essential cofactor for GSH-Px, which is involved in scavenging free radicals and maintaining the redox balance [22][23][24].Figure 9(d) showed that Se-SFPSIB significantly increased the GSH-Px activity in a dose-dependent manner (compared with the negative group,  < 0.01).Further, compared with SFPSIB group, Se-SFPSIB groups extremely significantly enhance GSH-Px activity in liver ( < 0.01).Especially, high-Se-SFPSIB group significantly enhances GSH-Px activity more than SFPSIB group in heart ( < 0.01).
In short, Se-SFPSIB could enhance CAT of mice bearing tumor S 180 to decompose H 2 O 2 and improve SOD to decrease MDA level.Meanwhile, GSH-Px activity was extremely significantly enhanced through Se supplements from Se-SFPSIB.The antioxidant results confirmed that antitumor ability of Se-SFPSIB was achieved by enhancing the antioxidant system.

Conclusion
In this paper, the selenylation conditions for Se-SFPSI were optimized by RSM.The confirmatory experimental optimum conditions were reaction time of 8 h, reaction temperature of 71 ∘ C, and the ratio of Na 2 SeO 3 to SFPSI of 1.0 g/g.The actual experimental Se content of (3.352 ± 0.04) mg/g closely matched the predicted value.
Furthermore, particle size, zeta potential, the thermal stability, and shape of Se-SFPSIB were significantly different from those of SFPSIB.Besides decreasing MDA, Se-SFPSIB also could enhance CAT, SOD, and GSH-Px activity.It demonstrated that Se-SFPSIB obtained from selenylation could significantly enhance antioxidant system and selenylation was a feasible approach to seek high biological functional seleno-polysaccharide.

Figure 1 :
Figure 1: Effect of reaction time, temperature, ratio of Na 2 SeO 3 to SFPSI, and water bath shaking rate on Se content.(a) Reaction temperature 70 ∘ C, ratio of Na 2 SeO 3 to SFPSI 0.8 g/g, and water bath shaking rate 40 r/min; (b) reaction time 8 h, ratio of Na 2 SeO 3 to SFPSI 0.8 g/g, and water bath shaking rate 40 r/min; (c) reaction time 8 h, reaction temperature 70 ∘ C, and water bath shaking rate 40 r/min; (d) reaction time 8 h, reaction temperature 70 ∘ C, and ratio of Na 2 SeO 3 to SFPSI 0.8 g/g.

Table 1 :
Coding schedule of response surface methodology.

Table 2 :
Box-Behnken design of three variables and the experimental results.

Table 3 :
Analysis of variance of regression model.