Ultrasound Effects on the Degradation Kinetics, Structure, and

The effects of ultrasound on the molecular weight, structure, and antioxidant potential of a fucoidan found in Isostichopus badionotus were investigat...
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Ultrasound Effects on the Degradation Kinetics, Structure, and Antioxidant Activity of Sea Cucumber Fucoidan Xin Guo,∥ Xingqian Ye,∥ Yujing Sun, Dan Wu, Nian Wu, Yaqin Hu, and Shiguo Chen* College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technology and Equipment, Zhejiang University, Hangzhou 310029, China S Supporting Information *

ABSTRACT: The effects of ultrasound on the molecular weight, structure, and antioxidant potential of a fucoidan found in Isostichopus badionotus were investigated. The results showed the molecular weight (Mw) of fucoidan decreased obviously after ultrasound treatment. Higher ultrasonic intensity, lower temperature, and lower fucoidan concentrations led to a more effective sonochemical effect. The kinetic model for fucoidan degradation fitted to 1/Mwt − 1/Mw0 = kt at the tested temperature. The optimized degradation conditions by response surface methodology (RSM) were temperature, 12 °C, and intensity, 508 W/cm2. Structural analysis by FTIR and NMR indicated the fucoidan kept the linear tetrasaccharide repeating units as the original polysaccharides after the ultrasound treatment, with only slight destruction of the middle nonsulfated fucose units. Antioxidant activity assay showed the antioxidant activity was slightly improved by the ultrasound treatment. The results suggested that ultrasound treatment is an effective approach to decrease the Mw of fucoidan with only minor structural destruction. KEYWORDS: sea cucumber, fucoidan, ultrasound, degradation

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INTRODUCTION Fucoidan, also called sulfated fucan, is a special type of polysaccharide that consists of a substantial percentage of Lfucose and sulfate ester groups.1 Marine algae-derived fucoidan is widely considered to have great potential to be developed into functional foods.2 However, algae-derived fucoidan is a kind of heteropolysaccharide with a complex monosaccharide composition and structural features that would cause problems in quality control and in investigating the structure−activity relationships. Recently, fucoidan from marine animals has attracted more attention because of its simple composition and well-repeated structures,3 two features that greatly facilitate the understanding of its structure−function relationship and quality control in its production. The sea cucumber is a tonic food in China and some other Asian countries. Fucoidan from the sea cucumber is a linear polysaccharide that consists of regular di-, tri-, or tetrasaccharide repeating units, with well-defined glycosidic linkages and distinctive sulfation patterns.2 It has been reported to have various biological benefits, such as promotion of neural stem/ progenitor cell proliferation,4 protection against ethanolinduced gastric ulcer,5 inhibition of osteoclastogenesis,6 and anticoagulant and antithrombotic activities.7 Recently, we have isolated a novel fucoidan with wellrepeated tetrasaccharide units from the sea cucumber Isostichopus badionotus. It showed great potential in being developed into an antithrombic drug because of its excellent antithrombotic activity and lower risk of bleeding.7 However, the large molecular size of the fucoidan has limited its application. The molecular weight (Mw) and structure of polysaccharides are reported to be related to their biological activities. Specific Mw of polysaccharides is required for many special applications, particularly in medical treatments and cosmetics. Polysaccharides with lower Mw have certain © 2014 American Chemical Society

advantages over the ones with higher Mw due to their improved diffusion into biological tissues8 and bloodstreams. Several methods have been applied to degrade polysaccharides, such as chemical degradation, enzymatic hydrolysis, and physical depolymerization.9 Chemical degradations such as free radical degradation and acid hydrolysis rely on high temperature and hydrogen peroxide or acids. These methods not only are time-consuming but also produce unavoidable chemical wastes and lead to the severe destruction of structures. In addition, further purification is necessary due to the additives used to initiate reactions and the formation of side products. Enzymatic methods are in general more preferable than chemical reactions because of their gentle reaction conditions and their controllable product distribution.10 However, the high cost of different enzymes has limited their application on an industrial scale. Moreover, no commercial enzyme is available for the degradation of sulfated fucan. Ultrasonic treatment is an effective, energy-saving, and environmentally friendly way to prepare and process polymer particles, and it is particularly effective for breaking up aggregates and reducing particle size and Mw. Partial and controlled degradation of natural polysaccharides such as chitosan, starch, agarose, carrageenans, pectin, and other sulfated polysaccharides by ultrasound has been widely studied in the sonochemistry area.11−13 Ultrasound is a kind of mechanical wave having a frequency of >20 Hz. High-intensity low-frequency ultrasonication produces acoustic cavitation, which generates hot spots of short lifetimes with intense local heating of ∼5000 °C, Received: Revised: Accepted: Published: 1088

October 23, 2013 January 16, 2014 January 17, 2014 January 17, 2014 dx.doi.org/10.1021/jf404717y | J. Agric. Food Chem. 2014, 62, 1088−1095

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pressures of ∼1000 atm, and heating and cooling rates above 1010 K/s.14 The acoustic cavitations could generate a mechanical effect from shear force caused by rapid collapsing of the cavitation bubbles and free radicals from dissociation of the water. These two effects would degrade the huge polysaccharides into small particals.14−16 However, most of the research about ultrasonic degradation of sulfated polysaccharides has focused on heterostructural polysaccharides from sea algae, which induced difficulties in investigating the structural changes such as chain linkage and sulfation pattern during the degradation reaction, thus cause difficulty in eliciting the degradation mechanism of the ultrasonic degradation. To our best knowledge, there have been only a few reports on ultrasonic degradation of marine sulfated polysaccharides with well-repeated units, and investigations on the structural and bioactivity changes during the ultrasonication of the polysaccharides are even fewer. In the present study, we applied ultrasound to control degradation of the previously reported fucoidan from the sea cucumber, which was a linear sulfated polysaccharide with well-repeating tetrasaccharide units.7 Changes in the structures and antioxidant activities of the degraded products were also examined to investigate the degradation mechanism.

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M wt − M w0 = kt

(0)

Ln(M wt /M w0) = kt

(1)

1/M wt − 1/M w0 = kt

(2)

k is the rate constant (mol/g min) of Mw degradation during sonolysis; t is the sonolysis time (min); Mwt and Mw0 are the weight-average molar mass at time t and at time 0 (Da), respectively. The model with the best correlation coefficient (R2) was selected to predict the ultrasonic degradation. Optimization by Response Surface Methodology (RSM). The three-level−two-factor central composite design (CCD) was employed to optimize the ultrasonic degradation conditions. Temperature (X) and ultrasonic intensity (Y) were studied at three levels (−1, 0, +1). The complete experimental plan with respect to each value in actual and coded forms is shown in the Supporting Information (Supplementary Table 1). A set of 13 experiments was carried out. The response variable was fitted into a quadratic model as follows: Z = a + Bx + cY + dX2 + eY2 + f XY, where Z is the response calculated by the model, X represents temperature (°C), Y represents ultrasonic intensity (W/cm2), a is the intercept, b and c are linear coefficients, d and e are squared coefficients,and f is the interaction coefficient. The coefficients of the full regression model and their statistical significance were determined and evaluated using Minitab software. Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA). Determination of the Average Molecular Weight and Molecular Weight Distribution. The average molecular weight (average Mw) and polydispersity index were determined by GPC according to the method described by Houben18 with some modifications. The average Mw determination was carried out on a Waters 1525 HPLC system (Waters, Milford, MA, USA) with a TSKGEL 4000 column (Tosoh Bioscience, Tokyo, Japan). Twenty microliters of the sample solution was injected and eluted with 0.2 M NaCl at 40 °C for 25 min at a flow rate of 0.5 mL/min. The eluent was monitored with a Waters 2414 refractive index detector. The results were analyzed by the GPC software. The standard dextran (Sigma) with different molecular weights was used to obtain the calibration curves each time. Determination of Monosaccharide Composition. Monosaccharide compositions were determined using the PMP-HPLC method as described previously.19 In brief, 2 mg of fucoidan was hydrolyzed with 2 M trifluoroacetic acid at 110 °C for 8 h. The hydrolyzed samples were dissolved into 0.3 M NaOH (450 μL) and 0.5 M methanol solution of PMP (450 μL) at 70 °C for 30 min. Lactose was added as an internal standard to each sample before derivatization. The mixture was neutralized with 450 μL of HCl (0.3 M) after cooling to room temperature and extracted with 1 mL of chloroform three times. HPLC analyses were performed on an Agilent ZORBAX Eclipse XDB-C18 column (Agilent, Santa Clara, CA, USA; 5 μm, 4.6 mm × 150 mm) at 25 °C with UV detection at 250 nm. The mobile phase was aqueous 0.05 M KH2PO4 (pH 6.9) with 15% (solvent A) and 40% (solvent B) acetonitrile, respectively. A gradient of B (8−19%) within a period of 25 min was applied. Determination of Sulfate Content. Sulfate content was determined by ion chromatography.20 Two milligrams of fucoidan fraction was hydrolyzed with 2 M TFA at 110 °C under nitrogen for 8 h. The hydrolysate was dried under vacuum before dissolving in water prior to ion chromatography. The chromatographic system used was a Compact IC 761 (Metrohm) equipped with a separation column (Shodex IC SI-90E, 250 mm × 4.6 mm) and a suppressor system. The eluent for the ion chromatographic determination was 1 mM Na2CO3 + 4 mM NaHCO3 with 2.5% acetone at the flow rate of 1.2 mL min−1. The standard curve was obtained according to the peak area and known concentration of the standard sulfate. The sulfate contents of the samples were determined from the conductivity signal after the injection of 20 μL of sample. FTIR and NMR. Fucoidan samples (0.5 mg) were mixed with KBr (50 mg) and pressed into KBr pellets. FTIR spectra were collected by a Nicolet 5700 (Thermo Fisher Scientific, USA) at the absorbance

MATERIALS AND METHODS

Materials and Chemicals. Standard monosaccharides, dextran, and Trolox were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1-Phenyl-3-methyl-5-pyrazolone (PMP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), fluorescein (FL) sodium salt, azodiisobutyramidine dihydrochloride (AAPH), sodium chloride, and HPLC-grade potassium bromide were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A TSK-G4000 PWxl column (30 cm × 7.8 mm, i.d. 13 μm, 500A, optimum separation range of polysaccharides is 1−700 kDa) was acquired from TOSOH Biosep (Tokyo, Japan). A Compact IC 761 (Metrohm, Herisau, Switzerland) was equipped with a separation column (Shodex IC SI-90E, 250 mm × 4.6 mm). Fucoidan was extracted from I. badionotus after papain digestion as we previously described.7 Ultrasonic Treatment. Ultrasound process was administered with an ultrasonic processor (Scientz-IID, Ningbo Scientz Biotechnology Co., Ningbo, Zhejiang, China) equipped with a 10 mm horn microtip. The ultrasonic processor had a maximum power of 950 W and operated at the frequency of 21−25 kHz. The sample was dissolved in 20 mL of nanopure water in a glass tube (diameter = 3 cm, height = 10 cm), which was immersed into a water bath (DC-1006, Safe Corp., Ningbo, Zhejiang, China) to maintain a stable temperature. The ultrasonic probe was submerged into the solution 1 cm from the top surface of the suspension. The effects of the following ultrasonic parameters were investigated: duration (0−220 min), temperature (5, 15, 25, 35, and 45 °C), sample concentration (0.1, 1, 5, and 10 mg/mL), and input power level (5, 15, 25, 35, and 45% of the total input power). The general ultrasound conditions of all treatments were as follows: extraction temperature at 5 °C, input power level of 35%, extraction time of 20 min, and pulse mode (2 s on and 2 s off). The intensity of the ultrasonic power that dissipated from the microtip with a radius of 10 mm was calculated according to the formula I = P/(πr2), where I is the ultrasound intensity, P is the input power, and r is the radius of the ultrasound probe.17 The input power levels were set at 5, 15, 25, 35, and 45% of the total input power, which corresponded to ultrasonic powers of 61, 182, 303, 424, and 545 W/ cm2, respectively. Degradation Kinetics Models. The kinetic models of degradation under ultrasound with different temperatures were obtained by graphical analysis. A trial-and-error procedure was applied to find the best fit reaction order. The zero-order, first-order, second-order models are as follows: 1089

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Figure 1. Effects of ultrasonic duration on the average Mw and polydispersity values of fucoidan. Ultrasound treatment conditions: temperature, 5 °C; ultrasonic intensity, 424 W/cm2; concentration, 1 mg/mL.

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mode in the frequency range of 4000−400 cm−1, with a resolution of 4 cm−1. For NMR analysis, fucoidan fractions (50 mg) were coevaporated with 500 μL of D2O (99.8%) twice via lyophilization before final dissolution in 500 μL of high-quality D2O (99.96%) containing 0.1 μL of acetone. Both 13C NMR and 1H NMR spectra were obtained by a Bruker AVIII 600 M (Switzerland) at room temperature. The observed 1H and 13C chemical shifts were corrected by internal acetone standard (2.23 and 33.1 ppm, respectively). The number of scans (ns) in each experiment was dependent on the sample concentration. DPPH Radical Scavenging Activity Assay. Three milliliters of sample solution (1 mg/mL), 0.6 mL of DPPH solution (0.4 mM prepared in ethanol), and 6 mL of deionized water were mixed in test tube. The absorbance was determined at 515 nm after 30 min at room temperature. All samples were run in triplicate. A standard curve was prepared using different concentrations of Trolox. The DPPH scavenging effect was calculated as

RESULTS AND DISCUSSION

Effects of Different Ultrasonic Factors on the Molecular Properties of Fucoidan. Duration. The duration-dependent effects of ultrasound on changes in average Mw of the degraded fucoidan fractions are shown in Figure 1. No foam was formatted during the ultrasonic process. The average Mw decreased rapidly in the initial 30 min from 338 to 182 kDa and then gradually tended to a constant value of 91 kDa after 220 min. The polydispersity values, which indicate Mw distribution, increased quickly from 1.193 to 1.461 during the first 80 min of treatments and dropped gradually to 1.328 by the end of 220 min. It has been reported that fracture of the polysaccharide molecule by ultrasound was mainly caused by shear force produced from the rapid collapse of cavitation bubbles.22,23 Large polymers with longer chains are preferentially broken by shear force, whereas small molecules with shorter chains are not sensitive to it.11 Thus, the average Mw of the sample decreased obviously in the first 30 min, and then the rate slowed as most of the fucoidans fractured into small particles. The increase of PDI values in the first 80 min was because both native and fractured fucoidan molecules were present in the solution, which led to the solution changing from a homogeneous to an inhomogeneous state. After further ultrasonic treatment, more native fucoidans were fractured and shifted to the lower molecular weight region, thus leading to a narrow Mw distribution. Ultrasonic Intensity. Ultrasonic intensity is one of the most important factors affecting the distinct stages of acoustic cavitations, namely, nucleation, bubble growth, and collapse. The effect of ultrasound intensity on the average Mw of degraded fucoidan fractions is shown in Figure 2a. The average Mw decreased from 338 to 166, 142, 130, 124, and 122 kDa under ultrasonic intensities of 61, 182, 303, 424, and 545 W/ cm2, respectively, in 100 min. A significant increase in degradation efficiency was observed with the increase of ultrasonic intensity, indicating the importance of ultrasonic intensity on fucoidan sonolysis (P < 0.05).

scavenging effect (%) = [A 0 − (A − A b)/A 0] × 100% where A0 is the absorbance of DPPH without sample, A is the absorbance of sample and DPPH, and Ab is the absorbance of sample without DPPH. The inhibition percentage of absorbance was plotted versus the amount of fucoidan sample to obtain a regression line. The Trolox equivalent antioxidant activity (TEAC) was calculated by the ratio between the slopes of the sample and Trolox. Oxygen Radical Absorbance Capacity (ORAC) Assay. ORAC analyses were carried out in a Fluoroskan Ascent microplate reader (Thermo Electron Corp., Vantaa, Finland) using 96-well plates. The ORAC value was determined as previously described.21 The reaction was carried out in 75 mM phosphate buffer (pH 7.4). Twenty-five microliters of FL (504 nmol/L) and 25 μL of sample solution placed in the microplate were preincubated for 15 min at 37 °C, and then 150 μL of AAPH solution (17.07 mmol/L) was rapidly added. The fluorescence was recorded every 2 min for 180 min, with an excitation wavelength of 485 nm and an emission filter of 538 nm. The final results were calculated using the differences in areas under the FL decay curves between the blank and the sample. The results were expressed as TEAC values. Statistical Analysis. Each treatment was repeated three times. All data were expressed as the mean ± SD and statistically evaluated with SPSS/16.00 software. Duncan’s multiple-range tests were used to evaluate differences among groups. 1090

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Figure 3. Degradation kinetics curves of fucoidan under ultrasonic treatment in different temperatures obtained by plotting (1/Mwt − 1/ Mw0) of each sample versus time of ultrasonication (t).

Table 2. Reaction Rate Constants k and Half-Life Periods (t1/2) of Fucoidan under Ultrasonic Treatment

Figure 2. Effects of ultrasound factors on the average Mw of fucoidan: (a) ultrasonic intensity (ultrasonic intensity, 61, 182, 303, 424, or 545 W/cm2; temperature, 5 °C; concentration, 1 mg/mL; and duration, 20 min); (b) temperature (temperature, 5, 15, 25, 35, or 45 °C; amplitude, 35%; concentration, 1 mg/mL; and duration, 20 min); (c) solution concentration (concentration, 0.1, 1, 5, or 10 mg/mL; amplitude, 35%; temperature, 5 °C; and duration, 20 min).

zero order (Mt/M0) vs time

first order (ln Mt/M0) vs time

second order (1/Mt − 1/M0) vs time

5 15 25 35 45

0.521 0.668 0.734 0.760 0.877

0.758 0.846 0.860 0.869 0.954

0.913 0.955 0.945 0.943 0.991

k (mol/g min) × 10−8

t1/2 (min)

5 15 25 35 45

5.400 4.299 3.932 3.372 3.104

54.83 66.84 75.30 87.80 95.39

Figure 4. FTIR spectra of native fucoidan and ultrasonic degraded fucoidan fractions. Fuc-A, Fuc-B, Fuc-C, and Fuc-D were assigned to degraded fucoidan fractions prepared by ultrasonication for 30, 60, 120, and 200 min, respectively.

Table 1. Correlation Coefficients (R2) from Plots of Zero-, First-, and Second-Order Reactions temperature (°C)

temperature (°C)

In a certain range, higher ultrasonic intensity could increase the energy of cavitation, lower the threshold of cavitation, and enhance the quantity of the cavitation bubbles.24 This can explain the sharp decrease of average Mw from 166 to 124 kDa as the ultrasonic intensity increased from 61 to 424 W/cm2. However, as the ultrasonic intensity continued to rise, more cavitation bubbles were produced around the acoustic source, which dampened the efficiency of energy transmission into the reaction medium and hampered the propagation of the ultrasound wave.8,25 Thus, no obvious change of average Mw was observed when the ultrasonic intensity increased from 424 to 545 W/cm2. 1091

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Figure 6. Antioxidant activities of native fucoidan and degraded fucoidian fractions: (a) DPPH radical scavenging activity; (b) oxygen radical absorbance capacity. The results were expressed by TEAC (Trolox equivalent antioxidant activity) values. Fuc-A, Fuc-B, Fuc-C, and Fuc-D were assigned to degraded fucoidan fractions prepared by ultrasonication for 30, 60, 120, and 200 min, respectively. An asterisk (∗) indicates significant differences (p < 0.05).

concentration rose from 0.1 to 10 mg/mL. This trend could be explained by more diluted concentration leading to less intense entanglement between the polysaccharide chains in solution. Thus, the random coiled structure of the molecule extended more freely and became more vulnerable to the attack of shear force.27 Moreover, the velocity gradients around the collapsing bubbles are higher in more diluted solutions, which would also benefit the ultrasonic degradation.28 Degradation Kinetics of Fucoidan under Ultrasound Treatment. To further predict the degradation efficiency of ultrasonic treatment on fucoidan from the sea cucumber, the correlation coefficients (R2) among (Mwt − Mw0), (lnMwt/Mw0), (1/Mwt − 1/Mw0), and time at different temperatures are summarized in Table 1. The degradation kinetics model of fucoidan under ultrasonic treatments was best fitted to a second-order kinetic equation: 1/Mwt − 1/Mw0 = kt. Similar results can be found in ultrasound degradation on pectin,13 dextran,29 agarose, and carrageenans.12 The kinetic curves under different temperatures are presented in Figure 3 by plotting (1/Mwt − 1/Mw0) of each sample versus time of ultrasonication (t). The reaction rate constants (k) and half-life periods (t1/2) were calculated

Figure 5. NMR spectra of ultrasonic degraded fucoidan fractions: (a) 1 H NMR; (b) 13C NMR. Fuc-A, Fuc-B, Fuc-Cm and Fuc-D were assigned to degraded fucoidan fractions prepared by ultrasonication for 30, 60, 120, and 200 min, respectively.

Temperature. Figure 2b shows the effect of temperature on ultrasonic degradation efficiency. The result indicated higher temperature would lead to a lower degradation efficiency. The average Mw of the sample treated under 5, 15, 25, 35, and 45 °C was decreased from the initial 338 kDa to 130, 144, 155, 167, and 168 kDa after 100 min of treatment, respectively. This trend was due to the increasing reaction temperature allowing cavitation to be achieved with lower acoustic intensity, which was a direct consequence of the rising vapor pressure associated with the heated liquid.26 Initial Solution Concentration. The effect of concentration on degradation efficiency was investigated as shown in Figure 2c. The average Mw values of fucoidan after the ultrasonic processes were 131, 138, 151, and 160 kDa as the 1092

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Figure 7. Possible mechanism of ultrasonic action on the fucoidan: (1) mechanical effect broke the hydrogen bonds among the fucoidan molecules; (2) free radicals produced by ultrasonic treatment acted on the nonsulfated fucose in the backbone.

was performed under optimum conditions, and the average Mw of the degradation product was tested to be 208 kDa, which was quite close to the predicted value. Structure Analysis of Degraded Fucoidan Fractions. The structure of the fucoidan from the sea cucumber I. badionotus has been identified as a simple linear backbone with well-repeated units of tetrasaccharides (Figure 5). These features facilitated our investigation on the structural changes of the sample during ultrasonication treatments and the possible mechanism of degradation. Low Mw fucoidan fractions were prepared for further analyses of structure and antioxidant activity. The fractions treated under the optimum ultrasonic conditions for 30, 60, 120, and 200 min were named Fuc-A, Fuc-B, Fuc-C, and Fuc-D, respectively. Monosaccharide Composition and Sulfate Content. Monosaccharide composition analysis showed that fucose was the only monosaccharide detected in all fucoidan fractions. The sulfate contents of the native fucoidan and the degraded fucoidan fractions (Fuc-A, Fuc-B, Fuc-C, Fuc-D) were 31.22, 32.08, 33.42, 34.11, and 34.74%, respectively. The sulfate content was slightly increased with prolonged ultrasonic duration. This trend indicated that, on the one hand, the polysaccharide chain might have suffered minor destruction; on the other hand, the sulfate group, which was critical to the bioactivity, was not severely damaged throughout the ultrasound process. FTIR Spectra. The structures of the ultrasonic-treated fucoidan fractions were investigated by FTIR spectra, and the results are shown in Figure 4. The characteristic absorption peaks of the samples are assigned as follows:30,31 major absorption bands around 3430 cm−1 were attributed to OH stretching; the signal at 2932 cm−1 was due to CH stretching of CH2 groups; an absorption at 1650 arose from stretching vibration of CO, CN; the feature at 1555 cm−1 was due to the bending vibration of −NH; an absorption at 1461 cm−1 was the stretching vibration of CO from carboxyl group and the bending vibration of −OH; the signal at 1240 cm−1 was caused

according to the second-order reaction model (Table 2). The k value was much higher at lower temperatures. As the temperature increased from 5 to 45 °C, the k value dropped from 5.4 × 10−8 to 3.104 × 10−8 mol/g min while t1/2 increased from 54.83 to 95.39 min. The retarded depolymerization effect was attributed to the loss of cavitation energy, because the energy escaped more easily from the cavitation bubbles at a higher temperature. As a consequence, the degradation efficiency dropped continuously as the temperature increased from 5 to 45 °C. Optimization by Response Surface Methodology (RSM). On the basis of single-factor analysis, the relationship between response (average Mw) and variables (temperature and ultrasonic intensity) was further optimized by RSM. The results at each design point are shown in Supplementary Table 1 in the Supporting Information. The regression equation in terms of the coded value of the variables was calculated as Z = 243.12 + 2.954X − 17.785Y + 1.164X2 − 3.532Y2 − 5.135XY, which represents the average Mw of the degradation products (Z) as a function of temperature (X) and ultrasonic intensity (Y). The determination coefficient R2 of 0.9411 for average Mw of the degradation products suggested that the fitted model could explain 94.11% of the total variation, indicating that the regression model was reliable in predicting the average Mw changes of fucoidan during ultrasonic process by the model. ANOVA was conducted to assess the significance and adequacy of the model. As shown in Supplementary Table 2, the ANOVA of the quadratic regression model showed the model F value was 22.38, and a low value of “probability >F” (P < 0.001) demonstrated that the model was highly significant. XY is a significant model term (P < 0.05); the intercept and Y are highly significant model terms (P < 0.01). The “lack of fit F value” of 0.335 implied the lack of fit is significant. On the basis of the regression equation, the optimum temperature and ultrasonic intensity were calculated to be 12 °C and 508 W/cm2, respectively. The minimum average Mw of fucoidan was predicted to be 207 kDa under the optimized condition. To confirm the prediction, ultrasound degradation 1093

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by SO stretching; bands around 1044 cm−1 were assigned to C−O stretching vibrations (COH, COC); absorption at 839 cm−1 was attributed to COS bending vibration; and the feature at 578 cm−1 was assigned to the asymmetric and symmetric OSO deformation of sulfates. The similarity of the IR spectra between degraded and native focuidan indicated the main repeated units of the fucoidan chain were not destroyed by ultrasonication. Moreover, there was no obvious changing in the characteristic peaks at 1240 and 839 cm−1 caused by SO stretching and COS bending vibration,7 indicating that there was no obvious desulfation during the ultrasonic treatment. NMR Spectroscopy. The detailed structure, especially the chain linkage and sulfation pattern change, was further investigated by 1D NMR, and the results are shown in Figure 5. The assignment of the proton signals was based on our previous research, which gave in detail assignments of the signals based on mass spectra and 2D NMR.7 In the 1H NMR spectra (Figure 5a), all four samples showed very similar spectra: anomeric signals at 5.22 ppm (J = 3.92, A-α), 5.26 ppm (J = 3.60, B-α), 5.20 ppm (J = 3.81, C-α), and 5.00 ppm (J = 3.22, D-α) all appeared, and they were attributed to the anomeric proton of fucose residues A, B, C, and D, respectively, in Figure 5. The signals among 3.5−4.5 ppm were attributed to the cross-ring protons; those at 1.23 ppm were attributed to the methyl of the fucose units. No signal was obviously changed during the ultrasonic prosess, indicating that the unique repeated units of tetrasaccharides of the fucoidan remained. However, there were some minor changes after ultrasonic treatment. The strength of the anomeric signals at 5.00 ppm (unit D, the nonsulfated fucose in the tetrasaccharide repeat) of the ultrasonic-treated samples decreased slightly as the time increased. The proportions of D-1 at 5.00 ppm to the total anomeric proton (A1 to D-1), in Fuc-A, Fuc-B, Fuc-C, and Fuc-D, are 0.315, 0.306, 0.298, and 0.278, respectively. The slight decrease in proton of unit D indicated the nonsulfated fucose might be liable to destruction during ultrasonic treatment, which was a coincidence with the slight increase in the sulfate content. As the mechanical effect of the ultrasonic was usually acted on the hydrogen bond of the polysaccharides molecules, the destruction of unit D may have caused the free radicals produced during the ultrasonic treatment. A similar trend was identified during the acid hydrolysis of this fucodian in our previous research;7 the acid was also more likely to act on the middle nonsulfated fucose (unit D). 13 C NMR (Figure 5b) showed similar spectra for the four fractions. Although limited by the low resolution of the signals in the experimental conditions, we can still observe there were no obvious differences in the nonsulfated fucose anomeric carbon signals. Thus, we further confirmed the main backbone structure was not changed. Antioxidant Activities Change. The antioxidant activities of the degraded fucoidan fractions were evaluated by DPPH and ORAC assays, and the data are shown in Figure 6. Both of the experiments indicated that the antioxidant activity of the degraded sample increased rapidly during the first period of the ultrasonication and then showed a decreasing trend as the ultrasonication continued. The increase in the antioxidant activity in the first stage may due to the following reasons: First, lower Mw polysaccharides with a mild effect of intramolecular hydrogen bonds would have more free hydroxyl and amino groups.32 Second, lower Mw polysaccharides have higher

contents of reducing sugars at the same mass concentration level.33 However, in the second stage, the reduction of the average Mw became slower, and the destruction of the polysaccharide structure was more severe as more free radicals were produced, which resulted in the reduction of the antioxidant activity. Thus, it is important to control the ultrasonic time to control the molecular size as well as to improve its antioxidant activity. Mechanism of Ultrasonic Degradation of Fucoidan. The ultrasonic process is a mild, effective, and environmentally friendly method to degrade polysaccharides. It is widely acknowledged that the ultrasonic wave could yield the shear force and H• and HO• radicals.15 The current opinion on the mechanism in the ultrasonic degradation of polysaccharides, for example, the sulfated polysaccharide from a red algae, may be explained more by mechanical and less by radical effects.34 On the basis of the results of the structure analysis and the antioxidant activity, we speculated the possible ultrasonic mechanism acting on the fucoidan may be a combination a mechanical and free radical degradation (Figure 7). During the initial stage, the average Mw of fucodian decreased sharply and no obvious structure change was observed. Meanwhile, the antioxidant activities of the degraded fractions were significantly improved. Thus, we speculated that the degradation was mainly due to the mechanical effect on dissociated aggregate polymer rather than the chemical reaction. The active groups for the antioxidant activity were not damaged. Therefore, the broken hydrogen bonds among the fucoidan molecules was the main reason causing the average Mw to decrease during this stage. As the ultrasonication continues, the Mw could hardly decrease anymore because the polysaccharide chains have been broken to be a single chain and become harder and harder to fracture. At the same time, the decreasing antioxidant activity is an indication of the destruction of some chemical groups. Thus, it is difficult for the mechanical effect to act on the chain, and the degradation might be due to the free radicals that attacked the nonsulfated fucose in the backbone. However, the specific degradation bonds should be further confirmed by other analysis tools. Our research was based on a homogeneous fucoidan from the sea cucumber I. badionotus with well-defined tetrasaccharide repeating units, which showed excellent anticoagulant, antithrombotic, and antioxidant activities. Applying the ultrasonic process to obtain low molecular fuodian will not only improve its oral absorption and utilization rate but also help to understand the mechanism of ultrasonication on marine polysaccharides and further optimize the processing condition. The low Mw fucoidans prepared deserve further development as a marine antithrombotic drug.

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X.G. and X.Y. contributed equally to this work. dx.doi.org/10.1021/jf404717y | J. Agric. Food Chem. 2014, 62, 1088−1095

Journal of Agricultural and Food Chemistry

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Notes

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The authors declare no competing financial interest.

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dx.doi.org/10.1021/jf404717y | J. Agric. Food Chem. 2014, 62, 1088−1095