Carboxymethylated degraded polysaccharides from Enteromorpha prolifera: Preparation and in vitro antioxidant activity

Food Chemistry 215 (2017) 76–83

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Carboxymethylated degraded polysaccharides from Enteromorpha prolifera: Preparation and in vitro antioxidant activity Mei-Jia Shi a, Xiaoyi Wei b, Jie Xu a, Bing-Jie Chen a, De-Yin Zhao a, Shuai Cui a, Tao Zhou a,⇑ a b

School of Food Science and Biotechnology, Zhejiang Gongshang University, 18 Xuezheng Street, Xiasha, Hangzhou, Zhejiang 310018, PR China College of Tourism & Food, Shanghai Business School, Shanghai 200235, PR China

a r t i c l e

i n f o

Article history: Received 14 April 2016 Received in revised form 22 June 2016 Accepted 28 July 2016 Available online 29 July 2016 Keywords: Carboxymethylation Enteromorpha prolifera Polysaccharides Antioxidant activity

a b s t r a c t In order to improve the bioactivities of the polysaccharide from Enteromorpha prolifera (PE), crude PE (Mw 1400 kDa) was degraded to low molecular weight polysaccharide (44 kDa) in the presence of hydrogen peroxide/ascorbic acid, followed by carboxymethylation. The reaction conditions for carboxymethylation of degraded polysaccharide (DPE) were optimized by Response Surface Methodology. The carboxymethyled degraded polysaccharide (CDPE) obtained under optimized conditions, with a degree of carboxymethylation of 0.849, was characterized by FT-IR and 13C NMR. The molecular weight of CDPE was measured to be 53.7 kDa. CDPE was evaluated for its antioxidant activity by determining the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals, and by determining the ferric reducing power. The antioxidant activity of CDPE was found to be greatly improved in comparison with degraded polysaccharide (DPE) and crude polysaccharide from Enteromorpha prolifera (PE). Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Enteromorpha prolifera, one of the most common fouling green algae, is distributed worldwide from the intertidal to the upper subtidal zones. It possesses high nutrient value and therapeutical properties (Lin, Shen, Wang, & Yan, 2008; Zhao et al., 2011). Several polysaccharides from this species have been extracted and their bioactivities, including blood lipid reduction (Teng, Qian, & Zhou, 2013), immunity (Zhang, Wang, Zhao, Yu, & Qi, 2013), and antiinflammatory (Jiao et al., 2009) have been reported. However, the biological activities of polysaccharide isolated from E. prolifera (PE) are of insufficient potential for a range of purposes in the food and pharmaceutical industries. Attempts have been made to enhance the activities of PE, and these are mainly focused on degradation and chemical modification of polysaccharide. Degradation of polysaccharide in principle can lead to the exposure of more active moieties. Indeed, it has been reported that the antioxidant activity of polysaccharides from Enteromorpha species can be enhanced by hydrolysis (Li et al., 2013; Zhang, Wang, Mo, & Qi, 2013). Introduction of functional groups on to polysaccharides by chemical modification has also been demonstrated to be an

⇑ Corresponding author. E-mail address: [email protected] (T. Zhou). http://dx.doi.org/10.1016/j.foodchem.2016.07.151 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

efficient protocol for the improvement of some bioactivities (Du et al., 2014). For example, it was reported that sulfation, acetylation and phosphorylation of polysaccharide from Enteromorpha species markedly increased the antioxidant activity (Wang, Zhang, Yao, Zhao, & Qi, 2013a, 2013b; Zhang et al., 2011). Selenylation of Enteromorpha prolifera also improves antibacterial activity (Lv, Gao, Shan, & Lin, 2014). In continuation of interests in enhancing the biological activities of polysaccharides (Xu et al., 2015, 2016), the effects of the carboxymethylation of degraded polysaccharide isolated from Enteromorpha prolifera on its antioxidant activity are herein reported.

2. Materials and methods 2.1. Materials and reagents E. prolifera was harvested in February 2013 in Zhoushan, Zhejiang, China. The raw sample was rinsed carefully with fresh water and air-dried. The dried seaweed was milled with a blender, sieved (0.125 mm) and stored at 20 °C before use. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and ascorbic acid were purchased from Aladdin Chemical Reagents Co. (Shanghai, China). Riboflavin, DL-methionine and nitrotetrazolium blue chloride were purchased from Sinopharm Chemical Reagents Co. (Hongkong,

M.-J. Shi et al. / Food Chemistry 215 (2017) 76–83

China). Dextran standards were purchased from Sigma Co. (USA). The solvents and other chemicals used in this work were of an analytically pure reagent grade. 2.2. Preparation of the degraded polysaccharides The extraction of crude polysaccharide from E. prolifera was carried out according to a previous report (Xu et al., 2015). Degradation of crude PE was achieved in the presence of hydrogen peroxide and ascorbic acid according to Zhang’s method, with slight modification (Zhang et al., 2013). Briefly, a solution of crude PE (1 g) in distilled water (100 ml) was heated to 30 °C, hydrogen peroxide and ascorbic acid (molar ratio 1:1) were then added (final concentration of the both: 0, 3, 6, 9, 12, 15 mmol/l). After the resulting mixture was stirred for 2 h, the reactant was concentrated under reduced pressure, followed by the precipitation with 4 folds volume of 95% ethanol, and then allowed to stand at 4 °C overnight. After centrifugation, the obtained precipitate was redissolved in distilled water. The resulting solution was dialyzed (MW cut off 3500) against distilled water for 48 h, then lyophilized, yielding the degraded polysaccharide from E. prolifera (DPE). The total antioxidant activity of DPE was assayed by FRAP method using a test kit (Luo, Li, & Kong, 2012). DPE possessing the best total antioxidant activity was used for carboxymethylation. 2.3. Preparation of carboxymethylated derivative of DPE (CDPE) Carboxymethylation of DPE was performed based on literature methods with modifications (Wang, Zhang, & Zhao, 2015; Yang et al., 2011). A solution of DPE (0.3 g) in DMSO (12.5 ml) and NaOH (20%, 5 ml) was stirred at 40 °C for 3 h, then chloroacetic acid solution in DMSO (12.5 ml) and NaOH (20%, 5 ml) was added (the final concentration of chloroacetic acid was 1.0, 1.5, 2.0, 2.5, 3.0 mol/l). The resulting solution was heated to a definite temperature (45, 50, 55, 60, 65 °C) for a definite time (2, 3, 4, 5, 6 h). The reaction mixture was cooled to room temperature and adjusted to pH 7 with 0.5 M HCl, diluted with water, and dialyzed (MW cut off 3500) in distilled water for 48 h, then lyophilised, yielding carboxymethylated extract (CDPE). The effect of chloroacetic acid concentration, reaction time and temperature on the degree of carboxymethylation (DS) was investigated. Monofactor tests indicated that the optimal chloroacetic acid concentration, reaction time and temperature were 2 mol/l, 4 h and 55 °C, respectively (detailed data shown in Supplementary Table 1 Central-composite experimental design of the independent variables along with the observed values for the degree of carboxymethylation. Experimental Temperature Time Concentration Degree of code (°C) (h) (mol/l) carboxymethylation (DS) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

50 60 55 55 60 55 55 55 60 55 50 55 55 50 55 50 60

4 5 5 4 3 3 5 4 4 3 4 4 4 5 4 3 4

2.5 2 2.5 2 2 2.5 1.5 2 2.5 1.5 1.5 2 2 2 2 2 1.5

0.744 0.802 0.785 0.851 0.691 0.689 0.618 0.870 0.737 0.629 0.625 0.855 0.866 0.736 0.827 0.773 0.657

77

materials). On the basis of the results of monofactor tests, the reaction conditions for carboxymethylation of DEP were further optimized by employing response surface methodology (RSM). A Box-Behnken design (BBD) was used to survey effects of independent variables (reaction temperature (A), reaction time (B) and chloroacetic acid concentration (C)) at three levels on the dependent variable (DS). Based on the results of preliminary experiments, a total of 17 randomised experiments, including 12 factorial and 5 zero point tests, were designed (Table 1). 2.4. Characterization of polysaccharide 2.4.1. Analysis of chemical compositions The total sugar contents of PE and DPE were analyzed with the phenol-sulfuric acid method, using glucose as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Uronic acid was estimated using a modified sulfuric acid-carbazole method with D-glucuronic acid as the standard (Bitter & Muir, 1962). Coomassie brilliant blue reaction was used to determine protein concentration (Bradford, 1976). Sulfate content was determined according to Kawai’s method (Kawai, Seno, & Anno, 1969). 2.4.2. Determination of the monosaccharide composition The monosaccharide composition was determined using Gas Chromatography–Mass Spectrometry (GC–MS). The samples were hydrolyzed by trifluoroacetic acid to monosaccharides. Then, the monosaccharides were derivatised to acetylated aldononitriles. Xylose, arabinose, glucose, galactose, mannose and rhamnose standards were also derivatised. Acetyl inositol was used as the internal standard. 10 mg samples were hydrolyzed with 2 M trifluoroacetic acid (4 ml) at 110 °C for 6 h in a sealed tube. The solution was mixed with 3 ml of methanol, and transferred to a glass tube. The solution was evaporated with nitrogen at 40 °C five times. Hydroxylamine hydrochloride (10 mg) and anhydrous pyridine (0.5 ml) were added to the tube. The sealed tube was immersed in a water bath at 90 °C for 30 min. Then, 0.5 ml of acetic anhydride was added. The mixture was kept at 90 °C for 30 min. The residue was dissolved in 1.0 ml chloroform, then inositol hexacetate was added. The resulting solution was analyzed using GC–MS. GC–MS was performed on a gas chromatography/mass spectrometer (Trace GC Ultra DSQ II, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a TR-5MS capillary column (30 m
0.25 mm
0.25 lm). The carrier gas was high purity nitrogen. The temperature program was set as follows: the initial temperature of the column was 80 °C and held for 3 min, then increased to 200 °C at 15 °C/min, and held for 1 min at 200 °C, and then increased to 250 °C at 10 °C/min, and held for 5 min at 250 °C. The flow rate was 1.0 ml/min. The injection temperature was 250 °C. The EI ion source of the mass spectrometer was set at 250 °C. Operational conditions for the mass spectrometer were 70 eV. EI source with a mass range between 33 and 500 Da. The split ratio was 10:1. 2.4.3. Molecular weight analysis Molecular weight determination of polysaccharides was measured using high-performance gel permeation chromatography (HP-GPC) which was undertaken on a Waters 1000 HPLC system, with a Grace 3300 Evaporative light Scattering Detector, ELSD. Samples (10.0 mg) were dissolved in distilled water (10.0 ml), passed through a 0.45 lm filter and applied to a gel-filtration chromatographic column of UltrahydrogelTM Linear (300 mm
7.8 mm. Waters., USA). Deionized water was used as the flow phase at a flow rate of 0.5 ml/min. The temperature of the column was maintained at 30 °C and the injection volume was 10 ll. Preliminary calibration of the column was carried out using Dextran standards with different molecular weights.

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2.4.4. Determination of degree of carboxymethyl substitution (DS) DS was determined using a colorimetric method (Eyler, Klug, & Diephuis, 1947; Yuen, Choi, Phillips, & Ma, 2009). In brief, CDPE solution (0.4 mg/ml) was prepared by dissolving it in 0.25 M NaOH. To a CDPE solution (1 ml) in a 25 ml test tube, with a stopper, chromotropic acid (5 ml, 0.1%) was added, followed by the addition of concentrated H2SO4 (1 ml). The resulting solution was heated with a boiling water bath for 0.5 h. After cooling to room temperature, ammonium acetate (18 ml, 30%) was added to a total volume of 25 ml. The absorbance of the solution was measured at 570 nm. The weight of glycolic acid was determined from a calibration curve by plotting the corrected absorbance at 570 nm against the amount of glycolic acid (mg) (Yuen et al., 2009). DS was calculated according to the following formula:

DS ¼

162B 76  80B

where B is the weight of glycolic acid (mg/mg of sample), 76 is the molecular weight of glycolic acid, 162 is the average molecular weight of anhydro-monosaccharide unit, and 80 is the net increase in weight of each unit of sodium carboxymethylate group substituted. 2.4.5. Fourier transform infrared spectra (FT-IR) analysis FT-IR spectra were determined on Nicolet 380 infrared spectrometer with KBr pallet. 2.4.6. 13C NMR 13 C NMR data were recorded on Avance 500 spectrometer, with an operating frequency of 125 MHz, using D2O as a solvent. 2.5. Assays for antioxidant activity 2.5.1. DPPH radical-scavenging activity assay The effect of scavenging DPPH radicals was measured according to a reported method, with minor modification (You, Zhao, Regenstein, & Ren, 2011). DPPH radical solution (0.1 mM) was prepared by dissolving in methanol. A polysaccharide solution (2 ml; 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) in distilled water was added to 2 ml of 0.1 mM DPPH solution. The resulting solution was shaken rigorously and incubated in the dark at room temperature for 1 h, and the absorbance of the solution was determined at 517 nm. Ascorbic acid was used as a positive control. The control was made in the same manner, except methanol replaced DPPHmethanol solution. Distilled water replaced sample solution for the blank. The DPPH radical scavenging activity of the sample was calculated as:

DPPH scavenging rate ð%Þ ¼ ½1  ðAsample  Acontrol Þ=Ablank 
100 2.5.2. Hydroxyl radical (HO) scavenging activity assay The hydroxyl radical scavenging activity was measured by Deng’s method, with modification (Deng et al., 2012). The polysaccharide solutions with different concentrations (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) were prepared by dissolving them in distilled water. A polysaccharide solution (2 ml) was mixed with a H2O2 solution (0.7 ml, 3%), FeSO4 solution (1 ml, 1.5 mM) and salicylic acid solution (0.3 ml, 20 mM in ethanol). The resulting mixture was shaken well and incubated at 37 °C for 30 min. The hydroxyl radical was detected by monitoring absorbance at 517 nm. Distilled water replaced salicylic acid-ethanol for the control, while distilled water replaced the sample solution for the blank. Ascorbic acid was used as a positive control. The hydroxyl radical scavenging activity was calculated as:

HO scavenging rate ð%Þ ¼ ½1  ðAsample  Acontrol Þ=Ablank 
100%

2.5.3. Superoxide anion radical (O 2 ) scavenging activity assay The effect of scavenging superoxide anion radicals was determined according to Prasad’s method, with minor modification (Prasad et al., 2009). The polysaccharide solutions with different concentrations (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) were prepared by dissolving them in distilled water. To a polysaccharide solution (1.5 ml) was added to a methionine solution (0.9 ml, 26 mM) in PBS buffer (pH 7.8, 0.1 M), nitroblue tetrazolium (0.3 ml, 0.75 mM) and riboflavin solution (0.3 ml, 20 lM) containing 1.0 lM EDTA. The reaction solution was exposed to a fluorescent lamp under the condition of light 3500 Lx for 30 min and the absorbance was measured at 560 nm. Distilled water replaced reaction liquid (riboflavin, methionine and nitroblue tetrazolium) for the control, while distilled water replaced sample solution for the blank. Ascorbic acid was used as a positive control. The superoxide anion radical scavenging activity of the sample was calculated as:

O 2 scavenging rate ð%Þ ¼ ½1  ðAsample  Acontrol Þ=Ablank 
100% 2.5.4. Determination of total antioxidant activity Ferric reducing ability of plasma (FRAP) method (Luo et al., 2012) was employed to measure the total antioxidant capacity of polysaccharide samples by a test kit. Stock solutions included detective buffer, 2,4,6-tripyridyl-s-triazine (TPTZ) solution, TPTZ dilution, 0.5 ml 10 mM FeSO4 solution and 0.1 ml 10 mM Trolox (an analogue of vitamin E) solution. A working solution was prepared freshly by mixing TPTZ dilution, detective buffer and TPTZ solution in a ratio of 10:1:1 (v/v), respectively. The working solution was warmed to 37 °C before use. Polysaccharide sample (5 ll, 1–5 mg/ml) was mixed with 180 ll of FRAP working solution and incubated for 5 min at 37 °C. The absorbance of the reaction mixture was then recorded at 593 nm. The standard curve was prepared using FeSO4 ranging from 0.15 to 1.50 mM. The absorbance of 1 mM ferrous salt at 593 nm is defined as 1 mM equivalent antioxidant capacity. The activity was expressed by FeSO4 values, which were calculated using standard curves. Meanwhile, vitamin E analogue (Trolox) standard curve was also prepared to calculate the Trolox equivalent antioxidant capacity of polysaccharide samples. 2.6. Statistical analysis All experiments were performed in triplicate. The data were statistically analyzed using analysis of variance test (SPSS Statistics 19 software). Significant differences between the treatments were examined by Duncan’s new multiple range test (DMRT). P < 0.05 was considered as statistically significant difference. 3. Results and discussion 3.1. Degradation of polysaccharide from E. prolifera PE was degraded in the presence of different concentrations of hydrogen peroxide and ascorbic acid. As shown in Fig. 1, the total antioxidant activity of the hydrolysate increased significantly (p < 0.05) with increasing the concentration of H2O2 and ascorbic acid until 9 mM, thereafter the activity decreased slightly. This is because a further increase of H2O2 and ascorbic acid concentration results in the decrease of molecular weight of degraded polysaccharide. Molecular weight of polysaccharide is an important factor which affects its activity (Wang et al., 2011). Degraded polysaccharide from E. prolifera (DPE), obtained in the presence of 9 mM of H2O2 and ascorbic acid, was found to possess the strongest total antioxidant activity (equivalent to 1.07 mM of FeSO4, or 0.54 mM

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Total antioxidant activity (mMFeSO4)

1.2

1.0

0.8

0.6

0.4

0.2 0

5

10

15

Concentration (mM) Fig. 1. Total antioxidant activity of DPE obtained at different concentrations of H2O2 and ascorbic acid. The concentration of DPE was 10 mg/ml.

of Trolox at 10 mg/ml). This was used for further carboxymethylation. The yield of DPE under these conditions was 67.5%. 3.2. Preparation of CDPE The carboxymethylation conditions, including reaction temperature, time and concentration of chloroacetic acid were optimized by Response Surface Methodology, using a Box-Behnken design to obtain the maximum degree of carboxymethylation. Optimization of the reaction conditions was described below. 3.2.1. Fitting the mathematical model The designed experiments were carried out for different combinations of the physical parameters (Table 1), and the empirical relationship between response variable and the test variables by a second-order polynomial equation was obtained as follows:

Y ¼ 0:78 þ 6:250
104 A þ 0:017B þ 0:045C þ 0:032AB  8:750
103 AC þ 0:023BC  0:039A2  0:048B2  0:098C2 where Y is DS; A, B, C are reaction temperature, time and concentration of chloroacetic acid, respectively.

A high F-value and a low P-value indicated the quadratic polynomial model was significant. As the results of ANOVA for the quadratic model shown in Table 2, the F-value of this model was 64.87, while the corresponding P-value was less than 0.0001, which implied the model was extremely significant. Nonsignificant lack-of-fit was desirable. The F-value and P-value of lack-of-fit in the regression model were 0.38 and 0.7379, respectively, implying that the lack-of-fit was not significant relative to the pure error and confirmed the validity of the model. The determined coefficient of model (R2 = 0.9885) indicates that the mode is applicable. The adjusted determination coefficient (R2adj = 0. 9729) was also high, showing the high degree of correlation between the experimental and predicted values. In addition, the R2adj was close to the R2 value, which exhibited the large enough sample scale. At the same time, a low value 1.93% of the coefficient of the variation (C.V.) indicated a high degree of precision and good reliability of the experimental values. As the results illustrated, factors with significant effects on degree of carboxymethylation (P < 0.05) were linear terms of B, C and interaction terms AB, BC and quadratic terms of A2, B2, C2, while linear term A and interaction term AC were insignificant (P > 0.05). The results of the study demonstrated that concentration of chloroacetic acid was the most significant single parameter which influenced the degree of carboxymethylation, followed by reaction time and temperature.

3.2.2. Analysis of the response surface As shown in Fig. 2a, at the fixed centre values for concentration of chloroacetic acid, DS increased significantly with the increase of reaction time at a fixed temperature until 4 h, after which the increasing rate slowed down and then DS decreased. When the reaction time was fixed, DS increased with increasing reaction temperature up to 55 °C. Fig. 2b is the response surface plot showing the effect of the concentration of chloroacetic acid and temperature on the response at the fixed centre values for reaction time. DS increased with increasing the concentration of chloroacetic acid and temperature reaction temperature up to 2.1 mol/l and 55.23 °C, respectively. Fig. 2c shows the effect of reaction time and concentration of chloroacetic acid on DS with the reaction temperature at the central level. When reaction time was fixed, DS increased appreciably with increasing concentration of chloroacetic acid until 2.1 mol/l, after which DS increased slightly. When the concentration of chloroacetic acid was fixed, DS increased with the increase of times at the beginning and then decreased slowly.

Table 2 Analysis of variance (ANOVA) for optimization of carboxymethylation conditions. Source

SS

DF

MS

Model 0.12 9 1.40
102 A 1.01
105 1 1.01
105 B 3.16
103 1 3.16
103 C 2.30
102 1 2.30
102 AB 5.48
103 1 5.48
103 AC 3.80
104 1 3.80
104 BC 2.86
103 1 2.86
103 A2 9.07
103 1 9.07
103 B2 1.40
102 1 1.40
102 C2 5.70
102 1 5.70
102 Residual error 1.46
103 7 2.09
104 Lack of fit 3.23
104 3 1.08
104 Pure error 1.14
103 4 2.85
104 Total 0.12 16 R2 = 0.9882 Adj R2 = 0.9729; Pred R2 = 0.9437; Adeq Precision = 21.082; C.V.% = 1.93

F-value

P-value

Significance

64.87 0.048 15.13 108.61 26.22 1.82 13.7 43.4 65.27 274.31

<0.0001 0.832 0.006 <0.0001 0.0014 0.2193 0.0076 0.0003 <0.0001 <0.0001

**

0.38

0.7746

* ** *

* * ** **

Note: DF, degree of freedom; SS, sum of squares; MS, mean squares; A, B and C are Reaction time (h), reaction temperature (°C) and concentration of chloroacetic acid (mol/l), respectively. ** Means the extremely significant difference (p < 0.0001). * Means that the difference is significant (p < 0.05).

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From the RSM tests, the optimal reaction time was found to be 4.10 h, at a temperature of 55.23 °C and with a concentration of chloroacetic acid 2.11 mol/l. Using this model under these conditions, DS was predicted to be 0.854. Considering the actual operation and the mathematical model, the reaction time, temperature and concentration of chloroacetic acid were modified to 4 h, 55 °C and 2.1 mol/l, respectively. Under these optimal conditions, DS was determined to be 0.849, indicating an excellent fit with the mathematical model. 3.3. Characterization of polysaccharides The contents of total sugar, uronic acid, protein and sulfate of DPE were not much different to those of PE (Table 3). The monosaccharide compositions of both PE and DPE were analyzed by gas chromatograph-mass spectrometry (GC–MS), and shown to be largely composed of rhamnose (Rha), glucose (Glu), xylose (Xyl), galactose (Gal) and mannose (Man) by matching with monosaccharide standards (Table 3). Gel permeation chromatography provides some information on the distribution of the polysaccharides. According to the standard curve obtained from Dextran standards with different molecular weights (log Mw = 0.3463Rt + 12.4796, R2 = 0.9991) and retention time (Rt) of polysaccharide peak, the weight-average molecular weight (Mw) of DPE was calculated to be 44 kDa, which was greatly reduced when compared with that of PE (1400 kDa). The Mw of CDPE obtained under optimized conditions was determined to be 53.7 kDa. FT-IR spectra of CDPE, DPE and PE are presented in Fig. 3. The signals at about 1050 cm1 and 1250 cm1 are attributed to

Fig. 2. Three-dimensional response-surface plots and contour plots of carboxymethylation conditions on degree of substitution (DS): (a) interaction between reaction time and temperature; (b) interaction between chloroacetic acid concentration and reaction temperature; (c) interaction between reaction time and chloroacetic acid concentration.

Fig. 3. Fourier transform infrared spectra of polysaccharides. Upper: CDPE; middle: DPE; down: PE.

Table 3 The chemical composition of polysaccharide from E. prolifera (PE) and degraded polysaccharide (DPE). Sample

Total sugar (%)

Uronic acid (%)

Protein (%)

Sulfate (%)

PE DPE

49.2 51.9

15.7 15.6

0.8 0.7

12.3 13.9

Monosaccharide composition (%) Rha

Glu

Xyl

Gal

Man

67.8 56.9

18.6 31.8

7. 7 6.4

4.0 2.5

1.4 2.5

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C-O-C symmetrical stretching vibrations and asymmetrical stretching vibrations, respectively. The absorption at 840 cm1 confirms the existence of b-glucopyranosidic linkages (Yuen et al., 2009). The broad band at about 3430 cm1 in the spectra of PE and DPE is assigned to the stretching vibration of OH groups. This band tends to shift to a higher wave number (3456 cm1) in the spectra of CDPE. The spectra of DPE and PE are very similar, indicating that functional groups on the polysaccharide chain were less affected during the degradation. The presence of carboxyl group in CDPE was confirmed by two new strong absorption bands in the 1607 and 1421 cm1, which were assigned to asymmetrical and symmetrical COO stretching vibrations, respectively (Kaur, Ahuja, Kumar, & Dilbaghi, 2012; Yang et al., 2011). In the 13C NMR spectrum of CDPE (Fig. S4), the appearance of signal at 177.62 ppm, was assigned to the carbonyl group, and provided further evidence of carboxymethyl substitution. The signal at 70.95 ppm was assigned to the methylene carbon atoms of the carboxymethyl substituents (Ren, Sun, & Peng, 2008). 3.4. Antioxidant activity analysis The activity of the polysaccharide is influenced by a range of factors, such as molecular weight, chain structure and chain conformation (Falch, Espevik, Ryan, & Stokke, 2000; Wang et al., 2011). In this study, crude PE was found to possess a relatively low activity. The antioxidant activity of DPE was significantly

45

PE DPE CDPE

3.4.1. Scavenging effects on DPPH radicals The DPPH free radical is stable and widely used to evaluate the free radical scavenging ability (Yuan et al., 2005). DPPH is reduced when receiving a hydrogen from a donor substance (such as an antioxidant), generating yellow coloured diphenyl-picryl-hydrazine and is accompanied by a decrease in absorption at 517 nm. The DPPH free radical scavenging effects of PE, DPE and CDPE were monitored (Fig. 4a). For all the samples, the effects of the scavenging DPPH occurred in a concentration-dependent manner ranging from 1 to 5 mg/ml. The antioxidant capacity of CDPE was significant higher than those of DPE and PE (p < 0.05). For instance, at 4 mg/ml the removal of DPPH by CDPE reached 36.2%, while DPE and PE were 19.8% and 11.3%, respectively. The IC50 values of CDPE, DPE and PE for scavenging DPPH were calculated to be 5.7, 8.3 and 14.5 mg/ml. Although the DPPH scavenging capacity of DPE was greatly improved by carboxymethylation, it was still considered low compared to vitamin C (IC50 0.03 mg/ml). Yang et al. have evaluated the antioxidant activity of carboxymethylated polysaccharides from A. auricular (Yang et al., 2011), and the carboxymethylated derivative showed DPPH-radical removal of 63.0% at a concentration of 1.6 mg/ml, while the data for non-modified polysaccharide was 46.4% at the same concentration.

a

70

b

35

Scavenging ability on ·O2- (%)

Scavenging ability on DPPH (%)

a 40

improved when compared to PE, probably due to the exposure of more active moieties. Carboxymethylation of DPE further enhanced the antioxidant activity.

30 25 20 15 10 5

60

PE DPE CDPE

b

50 40 30 20 10 0

0 1

2

3

4

1

5

2

Scavenging ability on ·OH (%)

c

c

PE DPE CDPE

PE DPE CDPE

1.2

d Total antioxidant ability (mM FeSO4)

100

3

4

5

4

5

Concentration (mg/mL)

Concentration (mg/mL)

80

60

40

20

d

1.0

0.8

0.6

0.4

0.2

0.0

0 1

2

3

Concentration (mg/mL)

4

5

1

2

3

Concentration (mg/mL)

Fig. 4. Antioxidant assays of CDPE, DPE and PE. (a) DPPH radical scavenging activity; (b) superoxide anion radical scavenging activity; (c) hydroxyl radical scavenging activity; (d) total antioxidant ability.

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3.4.2. Scavenging effects on superoxide anion radical Although the superoxide anion radical is a relatively weak oxidant, it indirectly initiates lipid peroxidation and can form stronger reactive oxidative species, such as hydroxyl radicals. The inhibitory effects of CDPE, DPE and PE are shown in Fig. 4b. The scavenging effects of all samples increased with increasing concentration ranging from 1 to 5 mg/ml. CDPE was found to scavenge the superoxide anion radical most effectively among the three samples, and PE the weakest. The scavenging rates of CDPE, DPE and PE at 4 mg/ml were determined to be 61.1, 49.7 and 28.7%, respectively. The IC50 values of CDPE, DPE and PE for scavenging superoxide anion radical were calculated to be 3.0, 4.9 and 5.9 mg/ml, indicating that the activity of these samples were lower than ascorbic acid (IC50 0.44 mg/ml). Wang et al. reported that carboxymethyled polysaccharides from Tremella fuciformis showed superoxide radical scavenging activities (60.8%) at 0.388 mg/ml, while the unmodified polysaccharide was only 19.6% at the same concentration (Wang et al., 2015). 3.4.3. Scavenging effects of hydroxyl radicals The hydroxyl radical possesses extremely high reactivity and can induce severe damage to functioning biomolecules in living cells, in principle this can be inhibited by antioxidants (RolletLabelle et al., 1998). Avoiding the influence of the hydroxyl radical is important for antioxidant defense in cell and food systems. In this study, the Fenton-type reaction was used to generate hydroxyl radicals, and the hydroxyl radical scavenging activity of the polysaccharide was determined using salicylic acid as a molecular probe. With increasing concentration, the scavenging activity of CDPE increased slightly ranging from 1 to 5 mg/ml, whereas the activity of DPE exhibited a marked concentration-dependent manner (Fig. 4c). Carboxymethylation of DPE greatly enhanced the hydroxyl radical scavenging activity (P < 0.05). At the concentration of 2 mg/ml, the inhibition rates of CDPE, DPE and PE on hydroxyl radical were determined to be 89.4, 44.4 and 17.9%, respectively. The IC50 values of CDPE, DPE and PE for scavenging hydroxyl radical were calculated to be 0.70, 2.5 and 6.2 mg/ml, which were higher than that of ascorbic acid (IC50 0.40 mg/ml). It has been reported that carboxymethylated polysaccharides from Tremella fuciformis also exhibit a strong ability to quench hydroxyl radicals (60.2%) at a concentration of 1.64 mg/ml, whereas the native polysaccharide was poorly soluble in water and showed weak scavenging activity (Rollet-Labelle et al., 1998). The great improvement of hydroxyl radical scavenging capacity could be attributed to the higher metal-binding ability of carboxymethyl group (Ueda, Saito, Shimazu, & Ozawa, 1996), as the hydroxyl radical-scavenging activity may be associated with the chelation of metal ions, such as Fe2+ and Cu2+, thus inhibiting their reaction with hydrogen peroxide (Li et al., 2013; Qi et al., 2005). 3.4.4. Total antioxidant activity The FRAP method was used to measure the total antioxidant capacity of polysaccharide samples, and was based on the measurement of a TPTZ–Fe(II) complex produced by the reduction of the TPTZ–Fe(III) complex by polysaccharides. The corresponding FeSO4 values were calculated using standard curves and regression equations. A higher FeSO4 value indicates a higher ferric reducing power. As shown in Fig. 4d, the total antioxidant capacities of CDPE, DPE and PE are concentration-dependent. It was found that the activity of CDPE was much higher than those of DPE and PE (p < 0.05). At 5 mg/ml, the total antioxidant activity of CDPE was determined to be equivalent to 1.16 mM FeSO4, while those of DPE and PE were 0.62 mM FeSO4, and 0.42 mM FeSO4, respectively.

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