WAN Peng, YANG Xiaoman, CAI Bingna CHEN Hua SUN Huili CHEN Deke, and PAN Jianyu*

1)CAS Key Laboratory of Tropical Marine Bio-resources and Ecology,Guangdong Key Laboratory of Marine Materia Medica,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou510301,P.R. China

2)University of Chinese Academy of Sciences,Beijing100049,P.R. China

3)Ocean University of China,Qingdao266100,P.R. China

Ultrasonic Extraction of Polysaccharides from Laminaria japonica and Their Antioxidative and Glycosidase Inhibitory Activities

WAN Peng1),2), YANG Xiaoman3), CAI Bingna1), CHEN Hua1), SUN Huili1), CHEN Deke1),2), and PAN Jianyu1),*

1)CAS Key Laboratory of Tropical Marine Bio-resources and Ecology,Guangdong Key Laboratory of Marine Materia Medica,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou510301,P.R. China

2)University of Chinese Academy of Sciences,Beijing100049,P.R. China

3)Ocean University of China,Qingdao266100,P.R. China

In the present study, ultrasonic extraction technique (UET) is used to improve the yield of polysaccharides fromLaminaria japonica(LJPs). And their antioxidative as well as glycosidase inhibitory activities are investigated. Box-Behnken design (BBD) combined with response surface methodology (RSM) is applied to optimize ultrasonic extraction for polysaccharides. The optimized conditions are obtained as extraction time at 54 min, ultrasonic power at 1050 W, extraction temperature at 80℃ and ratio of material to solvent at 1：50 (g mL-1). Under these optimal ultrasonic extraction conditions, an actual experimental yield (5.75% ± 0.3%) is close to the predicted result (5.67%) with no significant difference (P＞ 0.05). Vitro antioxidative and glycosidase inhibitory activities tests indicate that the crude polysaccharides (LJP) and two major ethanol precipitated fractions (LJP1 and LJP2) are in a concentration-dependent manner. LJP2 (30%-60% ethanol precipitated polysaccharides) possesses the strongest α-glucosidase inhibitory activity and moderate scavenging activity against hydroxyl radicals (66.09% ± 2.19%, 3.0 mg mL-1). Also, the inhibitory activity againstα-glucosidase (59.08% ± 3.79%, 5.0 mg mL-1) is close to that of acarbose (63.99% ± 3.27%, 5.0 mg mL-1). LJP1 (30% ethanol precipitated polysaccharides) exhibits the strongest scavenging activity against hydroxyl radicals (99.80% ± 0.00%, 3.0 mg mL-1) and moderateα-glucosidase inhibitory activity (47.76% ± 1.92%, 5.0 mg mL-1). LJP shows the most remarkable DPPH scavenging activity (66.20% ± 0.11%, 5.0 mg mL-1) but weakestα-glucosidase inhibitory activity (37.77% ± 1.30%, 5.0 mg mL-1). However, all these LJPs exert weak inhibitory effects againstα-amylase. These results show that UET is an effective method for extracting bioactive polysaccharides from seaweed materials. LJP1 and LJP2 can be developed as a potential ingredient in hypoglycemic agents or functional food for the management of diabetes. This study provides scientific evidence and advances in the preparation technology and a hypoglycemic activities evaluation method for seaweed polysaccharides, especially glycosidase inhibition in combination with an antioxidative activity evaluation method.

polysaccharides;Laminaria japonica; ultrasonic extraction; antioxidative;α-glucosidase;α-amylase

1 Introduction

It is found that oxidative stress, causing by reactive oxygen species (ROS) of superoxide anion radicals, hydroxyl radicals, peroxyl radicals, and single oxygen, is a major factor in the pathogenesis of diabetes (Arricket al., 2011; El Zahraaet al., 2012; Orhanet al., 2009). In diabetes, free radicals are formed through glucose oxidation, glycation of proteins, and a series of oxidative degradations of glycated proteins. These are responsible for various cellular anomalies including DNA (deoxyribonucleic acid) damage, enzymes deactivation, membrane proteinsdamage, lipid peroxidation,β-cell dysfunction, impaired glucose tolerance (IGT) and insulin resistance (IR) (Qianet al., 2008; Wrightet al., 2006; Zhaoet al., 2011). Furthermore, chronic hyperglycaemia generates ROS, which in turn cause secondary complications in diabetes mellitus including kidney, eye, blood vessel, and nerve damage (Sanjay, 2012). Antioxidants provide aid to prevent the destruction ofβ-cells by inhibiting the peroxidation chain reaction and thus the following development of diabetes (Aslanet al., 2010). The therapeutic challenge of diabetes lies in the need for intensive glycaemic control and maintenance of glycaemia within a strict normal narrow range (Duet al., 2012). One of the therapeutic approaches to controlling postprandial blood glucose (PBG) is to retard the absorption of glucose via inhibition of glycosidase, such asα-glucosidase andα-amylase (Prathapanet al.,2012). Previous researches have demonstrated that glycosidase inhibitors exhibit anti-diabetic effects (Buet al., 2010; Liet al., 2012; Liuet al., 2012; Nwosuet al., 2011; Weiet al., 2012). Medications such as acarbose inhibitingα-glucosidase have been used clinically to treat diabetic patients. Therefore, screening for compounds with both antioxidative and glycosidase inhibitory activities have great significance for dietary therapy of diabetes.

Laminaria japonicais cultivated widely as the most important economic seaweed for edible-medicinal use in China (Wanget al., 2008). The rhizoid ofL. japonicais widely used in Traditional Chinese Medicine as a treatment for diabetes (Buet al., 2010). Moreover, the extracts ofL. japonicahave shown extensive biological activities, including antoxidative (Reddyet al., 1984), anticancer (Yamamoto and Maruyama, 1985), antiviral (Makarenkovaet al., 2009), anticoagulant (Wanget al., 2010), antimicrobial (Kimet al., 2013), anti-inflammatory (Khanet al., 2008), antiatherogenic (Zhaet al., 2012), and immunostimulatory (Leeet al., 2004) effects. These beneficial activities have been proved to be highly related toL. japonicapolysaccharides (LJPs) (Penget al., 2012). And it has been found that the extracts ofL. japonicacontain high polysaccharides content ranging from 36% to 61% of dry weight (Holdt and Kraan, 2011). However, only scarce reports have aimed to study the hypoglycemic effect of LJPs on diabetes.

To utilize the polysaccharides ofL.japonica, efficient extract methods are in great need and are significant and essential for research and industrialized application. At present, the conventional preparation ofL.japonicapolysaccharides mainly depends on water and acid extraction. Though these extraction techniques are simple and economical, some extraction conditions usually lead to degradations of the polysaccharides and reduction of bioactivities (Chenet al., 2012). As a green and large scale adaptable technique, ultrasonic extraction facilitates acoustic cavitation and diffusion through the cell walls (Caiet al., 2013). As a result, it accelerates polysaccharides releasing fromL. japonicaand reduces process time significantly compared with other conventional extraction methods.

To expand the exploration of the hypoglycemic potential ofL.japonicapolysaccharides, ultrasonic extraction, and subsequent antioxidative evaluation,α-glucosidase andα-amylase inhibitory activities assay were investigated in this paper.

2 Materials and Methods

2.1 Materials

L. japonica, the brown seaweed, was collected from Jinjiang, Fujian, China, in June 2013. The seaweed was washed with distilled water to remove epiphytes, dried at 75% humidity at room temperature for 3 days, and then in an oven dried for 6 hours at 50℃. Dried seaweed was ground into fine powder with a blender (1120×g, 3 min, 25℃), and screened through a 40-mesh sieve and stored at 4℃. Soluble starch was purchased from Sangong Biotech Co., Ltd. (Shanghai, China).α-glucosidase,p-nitrophenyl glycosides andα-amylase were purchased from Sigma-Aldrich Co. LLC. (St. Louis, USA). All the chemical regents were of analytical grade.

2.2 Ultrasonic Extraction

The seaweed powder sample (6.0 g) was extracted in an ultrasonic processor (BILON-2008, Beijing Bilon Lab Equipment Co. Ltd., Beijing, China) with distilled water, using selected material/solvent ratio, extraction time, temperature and ultrasonic power. The crude extraction was centrifuged (Anke TDL-5-A, Anting Scientific Instrument Factory, Shanghai, China) at 3640×gfor 15 min and the supernatant was concentrated with a rotary evaporator (EYELA N-1100, Tokyo Rikakikai Co. Ltd.) at 50℃ under vacuum. A portion of concentrated solution was mixed with anhydrous ethanol to the final ethanol concentration of 60%, kept overnight at 4℃ and the precipitates were collected by centrifugation for 20 min at 3640×g. The precipitates were lyophilized to obtain crude polysaccharide (LJP). Another portion of concentrated solution was mixed with anhydrous ethanol to the final ethanol concentration of 30% with the same treatment, and the precipitated fraction LJP1 was obtained. Then the supernatant of LJP1 was further precipitated in 60% ethanol in the same manner, and the precipitated fraction LJP2 was obtained. The two precipitated fractions of LJP1 and LJP2 were washed with anhydrous ethanol, acetone and ether in turn and the protein of them was removed by the Sevage method (Yang and Zhang, 2009).

2.3 Analytical Methods

The protein, uronic acid and sulfate of polysaccharide were assayed by folin phenol (Lowryet al., 1951), sulfuric acid-carbazole (Bitter and Muir, 1962), and barium sulfate turbidimetry (Daiet al., 2010), respectively. The yield of polysaccharide was determined by the phenol-sulphuric acid method (Duboiset al., 1956) and calculated as follows：

wherec(μg mL-1) is the concentration of polysaccharide,kis the conversion factor,w0(g),w1(g) andw2(g) are the weight of powder, precipitation polysaccharide, and polysaccharide for analysis, respectively.

2.4 Box-Behnken Design for Polysaccharides Extraction

Box-Behnken design was employed for optimization of ultrasonic extraction. The preliminary range of extraction variables was determined through a previous single-factor-test. As shown in Table 1, four independent variables used in this study are material/solvent ratio (X1), extraction time (X2), extraction temperature (X3), ultrasonic power (X4), with three levels for each variable which arecoded as +1, 0 and -1 for high, intermediate, and low values, respectively, while the dependent variable is the yield of polysaccharides. Then, 27 experimental points of the whole design were carried out in a randomized order to maximize the effects of unexpected variables in the observed responses. The variables are coded according to the equation：

wherexiis the (dimensionless) coded value of the variableXi,X0is the value ofxiat the centre point andδXis the step change. A second-order polynomial regression model established for the response surface analysis (RSM) is described with the following equation：

whereyis the predicted response, andβ0,βi, βii,βijrepresent the regression coefficients of intercept, linearity, square and interaction, respectively.xiandxjare the independent coded variables, whileεis the error. Analysis of the experimental design and calculation of predicted data were carried out using Design Expert Software (version 8.0) to estimate the response of the independent variables (Liet al., 2012).

Table 1 Coded and uncoded values of the experimental variables

2.5 Antioxidative Activities of Polysaccharidesin vitro

2.5.1 DPPH radical scavenging activity assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity of polysaccharides was then tested according to the method of Wanget al. (2010) with a slight modification. The mixture contained 2 mL polysaccharides solution and 2 mL freshly prepared DPPH solution (0.1 mmol L-1in ethanol). Ascorbic acid was used as the positive control.

2.5.2 ABTS radical scavenging activity assay

The ABTS radical scavenging activity was determined according to the method described by Heet al. (2014) with some modifications. The ABTS·+solution was prepared by mixing ABTS aqueous solution ( fi nal concentration 7.0 mmol L-1) with potassium persulphate solution ( fi nal concentration 2.45 mmol L-1). The mixture was kept in the dark at room temperature for 12-16 h before use. Prior to the assay, the ABTS·+solution was diluted with sodium phosphate buffer solution (0.2 mol L-1, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Then, 0.2 mL polysaccharides solution at different concentrations (0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg mL-1) was added to 4 mL diluted ABTS·+solution and mixed vigorously for 30 s. After reaction at room temperature for 6 min, the absorbance of the resultant solution was measured at 734 nm. Ascorbic acid was used as the positive control. The ABTS radical scavenging activity was calculated according to the following equation：

whereAcis the absorbance of the blank control (ABTS·+solution without test sample) andAsis the absorbance of the test sample.

2.5.3 Hydroxyl radical scavenging activity assay

The hydroxyl radical scavenging activity was measured by Fenton reaction (Wuet al., 2012) and ascorbic acid was used as the positive control.

2.5.4 Lipid peroxidation inhibitory activity assay

Measurement of the lipid peroxidation inhibition activity was based on the ferric thiocyanate method described by Gül?inet al. (2012) with a slight modification. The reaction solution was mixed vigorously, containing 0.5 mL samples at different concentrations (0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg mL-1), 2.0 mL sodium phosphate buffer solution (0.04 mol L-1, pH 7.0), 2.5 mL linoleic acid emulsion, and incubated at 37℃ in screw cap bottle. Prior to the reaction mixture, 5 mL linoleic acid emulsion system (15.5 μL linoleic acid, 17.5 mg tween-20, 0.04 mol L-1sodium phosphate buffer solution of pH 7.0) was prepared by mixing and homogenizing. A 100 μL of the reaction mixture was taken in 12 h and mixed with 4.7 mL ethanol (75%), 100 μL ammonium thiocyanate (30%), and 100 μL FeCl2(0.02 mol L-1in 3.5% HCl). Then, the absorbance at 500 nm was recorded after 3 min. The solution without sample was used as the blank. Ascorbic acid was used as the standard and the percentage of inhibition was calculated as follows：

whereAcis the absorbance of the blank control (reaction mixture without test sample) andAsis the absorbance of the test sample.

2.6 Determination ofα-Glucosidase Inhibitory Activity

Theα-glucosidase inhibitory activity was measuredaccording to the colorimetric method described by Hsuet al. (2013) with slight modification. Thep-nitrophenylα-D-glucopyranoside (pNPG) prepared with 0.1 mol L-1sodium phosphate buffer solution (pH 6.8) was used as substrate. The reaction mixture contained： 0.1 mol L-1sodium phosphate buffer solution (pH 6.8), 2 mL; sample at different concentrations, 0.5 mL; 1 mg mL-1reduced gluthatione, 50 μL; 0.56 U mL-1α-glucosidase, 20 μL. The mixed solution was incubated at 37℃ for 10 min and saturatedpNPG was added to initiate the enzymatic reaction for another 30 min. 10 mL 0.1 mol L-1Na2CO3solution was added to terminate the catalytic reaction. The absorbance of the mixture at 400 nm was determined. Acarbose was used as the positive control. The inhibitory rate of sample onα-glucosidase was calculated as follows：

whereAc,As,Abare the absorbance of 100% enzyme activity (only the solvent with the enzyme), test sample with the enzyme and test sample without the enzyme, respectively.

2.7 Determination of α-Amylase Inhibitory Activity

Inhibitory activity ofα-amylase was determined based on the method described by Saliuet al. (2012) with slight change. Briefly, 500 μL sample at different concentrations, 1000 μL 0.02 mol L-1sodium phosphate buffer solution (pH 6.9 with 0.006 mol L-1NaCl), and 100 μL Porcine pancreaticα-amylase (15 U mL-1, 0.02 mol L-1sodium phosphate buffer solution of pH 6.9) were incubated at 37℃ for 10 min. Then, 500 μL starch solution (1%, 0.02 mol L-1sodium phosphate buffer solution of pH 6.9) was added. The reaction mixture was incubated at 37℃ for 10 min and stopped with 2.0 mL dinitrosalicylic acid color reagent. Then, the mixture was incubated in boiling water for 5 min and cooled to room temperature. The absorbance was measured at 540 nm after 10 mL distilled water was added. Acarbose was used as the positive control. Theα-amylase inhibitory activity was expressed which the following formula：

whereAc+,Ac-,As,Abare defined as the absorbance of 100% enzyme activity (only the solvent with the enzyme), 0% enzyme activity (only the solvent without the enzyme), test sample with the enzyme and test sample without the enzyme, respectively.

2.8 Statistical Analysis

SPSS 16.0 software was used for statistical calculation. Analysis of variance (ANOVA) was adopted to analyses the statistical significance. All experiments were carried out in triplicate and the data was expressed as the mean and standard deviation.

3 Results and Discussion

3.1 Results of Single-Factor Experiments

3.1.1 Effect of Different Material/Solvent Ratios on the LJP Yield

Fig.1 Effect of (A) material/solvent ratio (750 W, 60℃, 45 min); (B) extraction time (750 W, 60℃, 1：40); (C) extraction temperature (45 min, 750 W, 1：40 ) and (D) ultrasonic power (45 min, 60℃, 1：40) on the LJP yield. Values are expressed as means with standard deviations.

The effect of different material/solvent ratios on the LJP yield was investigated in the rang from 1：20 to 1：70with three other factors fixed as follows： 750 W, 60℃and 45 min. The LJP yield increased from 1.27% to 3.35% when the material/solvent ratio was down to1：50. But the decline tendency of LJP yield was significant here after as the ratio continued to decrease (Fig.1A). Thus, a center point of 1：50 was selected for Box-Behnken design experiment.

3.1.2 Effect of Different Extraction Time on the LJP Yield

The effect of extraction time on the LJP yield was studied with the material/solvent ratio of 1：40 and the other conditions fixed at 750 W and 60℃. Results showed that the LJP yield increased from 2.22% to 3.06% rapidly within the first 45 min and then no significant increase was observed when the extraction time exceeded 45 min (Fig.1B). Meanwhile, significant differences among the extraction results with 30, 45 and 75 min (P＜ 0.05), 60 and 75 min (P＞ 0.05) were found. Thus, a centre point of 45 min was selected for Box-Behnken design experiment.

3.1.3 Effect of different extraction temperatures on the LJP yield

Effect of extraction temperature (40-90℃) on the LJP yield was investigated with the extraction time of 45 min and the other parameters fixed at 750 W and material/ solvent ratio 1：40. The LJP yield increased gradually from 3.2% to 4.41% as extraction temperature ascended from 40 to 80℃. However, the yield of polysaccharides decreased with further increase in temperature (Fig.1C). Moreover, significant differences among the extraction results of 40, 60 and 80℃ (P＜ 0.05), 80 and 90℃ (P＞ 0.05) were tested. Hence 60℃ was selected as a centre point.

3.1.4 Effect of different ultrasonic power on the LJP yield

The effect of ultrasonic power ranging from 450 to 1050 W on the LJP yield was investigated with other factors fixed at 60℃, 45 min and material/solvent ration of 1：40. As shown in Fig.1D, the LJP yield increased gradually when the ultrasonic power ranged from 450 to 900 W. After 900 W, the extraction yield achieved a plateau. Thus 900 W was chosen as a centre point.

3.2 Model Fitting and Statistical Analysis

The complete design matrix and the corresponding results of RSM experiments to determine the effects of the independent variables are given in Table 2. According to multiple regression analysis of the experimental data, the predicted model was obtained by the following second-order polynomial equation：

The analysis of variance (ANOVA) results demon-strates that the model is highly significant in view of a very high modelF-value (19.08) but a very lowP-value (P＜ 0.0001). In addition, both the determination coefficientR2(0.9570) and the adjusted correlation coefficientR2Adj(0.9069) indicate that the accuracy and general availability of the polynomial model are adequate. A very low value of coefficient of the variation (C.V., 7.68%) presents a very high accuracy and highly reliable experimental values. Meanwhile, the ‘Adeq. Precision’ of 15.72 (≥4) indicates an adequate signal and the model can be used to navigate the design space. The corresponding variables will be more significant with a largerF-value and a smallerP-value. In this case, the linear coefficients (x1,x2,x3,x4), the interaction term (x2x4) and quadratic term coefficients (x12,x22,x42) are significant, with smallP-values (P＜ 0.05). And the extraction temperature (x3) is the most significant factor affecting the LJP yield.

Table 2 Box-Behnken design matrix and response for the LJP yield

3.3 Analysis of Response Surfaces

Fig.2 Response surface and contours plots showing the effect of the material/solvent ratio, extraction time, extraction temperature and ultrasonic power on the LJP yield.

Based on the above regression equation, the three-dimensional (3D) response surfaces and two-dimensional (2D) contour plots were used to visualize the relationship between responses and experimental levels of each variable and the interactions between two tested variables with other variables fixed at level zero. The elliptical contour plot means that the interactions between the variables are significant while the circular contour plot means otherwise (Yanet al., 2011). As shown in Figs.2(A) and 2(a), when the extraction power and temperaturewere fixed at level zero, material/solvent ratio (x1) displayed a quadratic effect on the response of LJP yield. When material/solvent ratio was kept at a higher level, the LJP yield significantly increased initially with extraction time (x2) extension but with no further significant improvement thereafter. It can be seen that a maximum LJP yield (4.48%) was achieved when extraction time and material/solvent ratio were in the ranges of 48.9-52.1 min and 0.021-0.023 g mL-1, respectively. The same phenomenon was observed in Figs.2(B) and (b) with the maximum LJP yield (4.47%) when extraction power and material/solvent ratio were in the ranges of 950-980 W and 0.021-0.023 g mL-1, respectively. The attenuation of the LJP yield may relate to the decline of mass transfer rates with the decrease of material/solvent ratio (Wanget al., 2012) and degradation effects of polysaccharides by high ultrasonic wave and longer ultrasonic time (Zhaoet al., 2013; Zhaoet al., 2011). As shown in Figs.2(C) and (c), when the extraction temperature and material/solvent ratio were fixed at level zero, extraction time (x2) and ultrasonic power (x4) exhibited a quadratic effect on the LJP yield. The interaction between ultrasonic power and extraction time was significant with an elliptical contour in Fig.2(c). It is possible that higher extraction efficiency of LJP yield at higher ultrasonic power with a shorter time will increase the number of cavitation bubbles and enhance mass transfer. Meanwhile, most polysaccharides release from the disruption cell walls and broken cells at the early period of ultrasonic processing with a suitable ultrasonic power. However, part of LJP could be depolymerized with higher ultrasonic power and longer time (Claveret al., 2010). This is in good agreement with the ANOVA results and previous reports (Liet al., 2007, 2012).

3.4 Optimization of the Extraction Parameters and Validation of the Model

The optimum extraction conditions (X1= 1：50,X2= 54 min,X3= 80℃,X4= 1050 W) for the predicted maximum value of LJP yield (5.67%) were obtained by the regression equation and analysis of the response surface and contour plots.

The experimental value obtained from practical experiments is 5.75% ± 0.30%, which is not significantly different to the predicted value within 95% confidence interval and 0.25% of the percentage deviation. This demonstrates that the model is satisfactory and adequate for predicting ultrasonic extraction process in the yield of LJP. Furthermore, the LJP yield is higher and extraction time is much lower than that of acid and hot water extraction method, which have been reported by Vishchuket al. (2011), Luet al. (2013) and Penget al. (2012). The result demonstrates that ultrasonic extraction applied in the preparation of seaweed polysaccharides will be a green and environmentally friendly technique with short extraction time, but non-corrosive solvents.

3.5 Scavenging Activity of DPPH Radical

Fig.3 A) DPPH radical-scavenging activity; B) ABTS radical-scavenging activity; C) Hydroxyl radical-scavenging activity; D) Lipid peroxidation inhibition activity of LJPs. Values are expressed as means and standard deviations.

Table 3 IC50 values in antioxidative and glycosidase inhibitory properties of LJPs

The DPPH radical is a stable nitrogen-centered, lipophilic free radical that is widely used for evaluating the free-radical scavenging activities of antioxidants. The method is based on the reduction of DPPH radical solution at 517 nm in the presence of a hydrogen-donating antioxidant, thereby converting DPPH into the non-radical form, DPPH-H (Redouanet al., 2011). The scavenging ability of the samples on DPPH radical is shown in Fig.3A and compared with ascorbic acid. At a dose of 5 mg mL-1, the antioxidative activity of LJP and LJP1 were 66.20% ± 0.11% and 49.42% ± 2.12%, respectively, which was extremely higher than that of LJP2 but lower than that of ascorbic acid. The order of IC50value for LJPs was as follows： LJP ＜ LJP1 ＜ LJP2 (Table 3). The possible mechanism of LJPs acting as antioxidants may be attributed to their electron donation power to the free radicals. Meanwhile, the DPPH radical scavenging activities showed obvious correlation with the protein content of the LJPs, which was shown in Table 4. Many previous researches have considered that polysaccharide-protein conjugates reveal different antioxidative abilities depending on the protein content (Chaudhuriet al., 2007; Chenet al., 2008; Singh and Rajini, 2008). It is similar to the early report that the crude polysaccharides have better antioxidative effects than the protein-removed fractionated components (Chenet al., 2008; Jianget al., 2013).

3.6 Scavenging Activity of ABTS Radical

As showed in Fig.3B, the scavenging activities of LJPs on ABTS radical in a concentration-dependent manner were lower comparing with ascorbic acid. The scavenging activities of LJP, LJP1 and LJP2 were 26.40% ± 1.02%, 19.89% ± 0.23% and 24.32% ± 0.96%, respectively, at 5.0 mg mL-1. The order of IC50value for LJPs decreased in the following order： LJP1 ＞ LJP ＞ LJP2. These results indicate that LJPs have limited scavenging activity on ABTS radical. Obviously, the antioxidative activity of LJPs is owing to the electron transfer mechanism. One molecule of ABTS radical cation extracts an electron (or hydrogen atom) from the LJPs and regenerates the parent substrate, ABTS+(Osmanet al., 2006). Some previous studies have pointed to a relationship between the sulfate content and the antioxidative activity. Higher sulfate content in the polysaccharides exhibits stronger radical scavenging ability (Alveset al., 2012).To some extent, it is consistent with the sulfate content data in Table 4.

Table 4 Protein, uronic acid and sulfate content in antioxidative and glycosidase inhibitory properties of LJPs

3.7 Scavenging Activity of Hydroxyl Radical

Hydroxyl radical, the most harmful ROS, can easily cross cell membranes and react with most biomolecules causing tissue damage or cell death. Removing hydroxyl radical is important for protecting living system (Xieet al., 2012). The results of hydroxyl radical scavenging activities of the three fractions were given in Fig.3C. For LJPs, the effects of scavenging hydroxyl radical displayed a significant concentration-dependent increase pattern at tested concentrations. At the concentration of 3.0 mg mL-1, the scavenging activities of LJP, LJP1, LJP2 and ascorbic acid on the hydroxyl radical were 45.32% ± 2.71%, 99.83% ± 0.00%, 66.09% ± 2.19% and 100.00%, respectively. The scavenging activity of LJP1 was close to that of ascorbic acid at 3.0 mg mL-1. Among LJPs, LJP1 showed the strongest scavenging activities. The scavenging activities of LJPs increased in the order of LJP ＜ LJP2＜ LJP1 (Table 3). This trend agreed with the contents of uronic acids and sulphate groups (Table 4). For hydroxyl radical, there are two types of antioxidative mechanism：suppression against hydroxyl radical generation, and cleaning the hydroxyl radical generated. In the former, the antioxidative activity may ligate to the metal ions which react with H2O2to give the metal complexes. The metal complexes thus formed cannot further react with H2O2to give hydroxyl radicals (Wanget al., 2008; Zhanget al., 2011). Previous studies on polysaccharides have shown that the hydrogen-donating ability of uronic acids (Wanget al., 2010) and the sulphate groups of metal-chelating activity (Houet al., 2012) could facilitate scavenging activity against hydroxyl radicals. It indicats that LJPs might act as electron or hydrogen donator to scavenge hydroxyl radical.

3.8 Inhibitory Activity of Lipid Peroxidation

The end-products of lipid peroxidation, in terms of the thiobarbituric acid-reactive substances (TBARS), were measured in this study. The inhibitory activities of LJPs were shown in Fig.3D. The LJPs exhibited a dose-dependent behavior on lipid peroxidation inhibitory activity within the tested concentration. The inhibitory activities of LJP, LJP1 and LJP2 were 34.33% ± 1.45%, 26.60% ± 3.24% and 40.00% ± 0.11%, respectively, at 5.0 mg mL-1. These results are in accordance with ABTSscavenging activity determined above, which is possibly attributed to the sulfate content of the LJPs and its free radical scavenging potential to terminate chain reactions (Zhaoet al., 2011).

3.9 Inhibitory Activity ofα-amylase andα-glucosidase

Theα-glucosidase andα-amylase inhibitors are currently used for diabetic treatment as oral hypoglycemic agents for its high affinity toα-glucosidase andα-amylase (Liu, 2012). Theα-glucosidase inhibitors are mostly evaluated by determination ofα-glucosidase inhibitory activity usingpNPG as the reaction substrate (Xiaoet al., 2011). However, so far there is little information published about theα-amylase andα-glucosidase inhibitory activity of polysaccharides fromL. japonica.

Fig.4 (A)α-glucosidase and (B)α-amylase inhibition activity of LJPs. Values are expressed as means and standard deviations.

As shown in Fig.4B, LJPs exhibited weak inhibitory effects onα-amylase less than 10.0% at the concentration of 5.0 mg mL-1. However, it was observed from Table 3 that both the LJPs and acarbose showed concentration-dependent inhibitory activity onα-glucosidase with a decreased order of LJP ＜ LJP1 ＜ LJP2 ＜ acarbose. Furthermore, at the concentration of 5.0 mg/mL, the inhibitory activity of LJP2 (59.08% ± 3.79%）onα-glucosidase was close (P＞ 0.05 ) to that of acarbose (63.99% ± 3.27%), and higher than that of LJP1 (47.76% ± 1.92%) and LJP (37.77% ± 1.30%). The possible mechanism would be that the LJPs can inhibit degradations of disaccharides to monosaccharide and reduce the amount of glucose absorbed into the blood circulation. This result enhances the previous findings that plant phytochemicals are mildα-amylase but strongα-glucosidase inhibitors (Saliuet al., 2012). In the present study, all the samples contain sulphate groups, which may be the main factor in the inhibitory activity ofα-glucosidase, but it needs further studies to elucidate underlying mechanisms of this effect.

4 Conclusions

In this study, BBD combined with RSM is employed to optimize the ultrasonic extraction process of polysaccharides fromL. japonica.The actual experimental values identified under the optimal ultrasonic extraction conditions are closely correlated to the predicted ones. Compared with the previous studies on conventional extraction methods, ultrasonic processing is a green and environmentally friendly technique with short extraction time and non corrosive solvents, as well as higher polysaccharide yield. Antioxidative and glycosidase inhibitory activities testsin vitroindicate that LJP2 possesses the strongestα-glucosidase inhibitory activity and moderate scavenging activity against hydroxyl radicals. LJP1 shows the strongest scavenging activity against hydroxyl radicals and moderateα-glucosidase inhibitory activity. LJP exhibits the most remarkable DPPH scavenging activity but weakestα-glucosidase inhibitory activity. All these LJPs exert weak inhibitory effects againstα-amylase. The bioactivities of LJPs could be attributed to the different content of protein, uronic acid and sulfate, as well as the monosaccharide composition and molecular weight distribution of the polysaccharides, however, which needs further verification. Our results suggest that ultrasonic extraction technique is an effective method for extraction bioactive polysaccharides from seaweed materials. LJP1 and LJP2 have the potential to be an ingredient in hypoglycemic agents or functional food for the management of diabetes. Because of its glycosidase inhibition combined with antioxidative activities which probably depend on the protecting of the pancreaticβ-cell and inhibition of the digestive enzymes. Further studies need to verify the direct e ff ect of LJPs on diabetes in vivo and the underlying mechanisms.

Acknowledgements

This study is supported by the Project of National Key Technology Research and Development Program for the 12th Five-year Plan (No. 2012BAD33B10), Public Science and Technology Research Funds Projects of Ocean (No. 201305018-2), the Innovative Development of Marine Economy Regional Demonstration Projects (Nos. SZHY2012-B01-004, GD2013-B03-001), the National Science Foundation for Young Scientists of China (No. 31101271), the Natural Science Foundation of Guangdong Province (Nos. 2014A030310338, 2014A030310351), the Comprehensive Strategic Cooperation Programs between the Guangdong Province and Chinese Academy of Sciences (No.2011B090300057), and the Frontier Science Program for Young Scientists of South China Sea Institute of Oceanology, Chinese Academy of Science (No. SQ 201017).

Alves, M. G., C. F., Dore, C. M. P. G., Castro, A. J. G., Nascimento, M. S., Cruz, A. K. M., Soriano, E. M., Benevides, N. M. B., and Leite, E. L., 2012. Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red algaHypnea musciformis.Journal of Applied Phycology, 24 (5)： 1217-1227.

Arrick, D. M., Sun, H., Patel, K. P., and Mayhan, W. G., 2011. Chronic resveratrol treatment restores vascular responsiveness of cerebral arterioles in type 1 diabetic rats.American Journal of Physiology-Heart and Circulatory Physiology, 301 (3)： 696-703.

Aslan, M., Orhan, N., Orhan, D. D., and Ergun, F., 2010. Hypoglycemic activity and antioxidant potential of some medicinal plants traditionally used in Turkey for diabetes.Journal of Ethnopharmacology, 128 (2)： 384-389.

Bitter, T., and Muir, H. M., 1962. A modified uronic acid carbazole reaction.Analytical Biochemistry, 4 (4)： 330-334.

Bu, T., Liu, M., Zheng, L., Guo, Y., and Lin, X., 2010. α-glucosidase inhibition and the in vivo hypoglycemic effect of butyl-isobutyl-phthalate derived from theLaminaria japonica rhizoid.Phytotherapy Research, 24 (11)： 1588-1591.

Cai, B., Pan, J., Wan, P., Chen, D., Long, S., and Sun, H., 2014. Ultrasonic-assistant production of antioxidative polysaccharides fromCrassostrea hongkongensis.Preparative Biochemistry and Biotechnology, 44 (7)： 708-724.

Chaudhuri, S., Banerjee, A., Basu, K., Sengupta, B., and Sengupta, P. K., 2007. Interaction of flavonoids with red blood cell membrane lipids and proteins： Antioxidant and antihemolytic effects.International Journal of Biological Macromolecules, 41 (1)： 42-48.

Chen, H., Zhang, M., Qu, Z., and Xie, B., 2008. Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia sinensis).Food Chemistry, 106 (2)：559-563.

Chen, J., Tian, M. P., Deng, S. G., and Fang, X. B., 2012. Enzymatic extraction of polysaccharides fromLaminaria japonicaand their free radical scavenging activity and antimicrobial activity evaluation.Advanced Materials Research, 479-481： 605-610.

Chen, Y., Xie, M. Y., Nie, S. P., Li, C., and Wang, Y. X., 2008. Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies ofGanoderma atrum.Food Chemistry, 107 (1)： 231-241.

Claver, I. P., Zhang, H., Li, Q., Ke, Z., and Zhou, H., 2010. Optimization of ultrasonic extraction of polysaccharides from Chinese malted sorghum using response surface methodology.Pakistan Journal of Nutrition, 9 (4)： 336-342.

Dai, J., Wu, Y., Chen, S. W., Zhu, S., Yin, H. P., Wang, M., and Tang, J., 2010. Sugar compositional determination of polysaccharides from Dunaliella salina by modified RPHPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone.Carbohydrate Polymers, 82 (3)： 629-635.

Du, W. H., Peng, S. M., Liu, Z. H., Shi, L., Tan, L. F., and Zou, X. Q., 2012. Hypoglycemic effect of the water extract of Pu-erh Tea.Journal of Agricultural and Food Chemistry, 60 (40)： 10126-10132.

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T., and Smith, F., 1956. Colorimetric method for determination of sugars and related substances.Analytical Chemistry, 28 (3)：350-356.

El Zahraa, Z. E. A. F., Mahmoud, M. F., El Maraghy, N. N., and Ahmed, A. F., 2012. Effect ofCordyceps sinensisand taurine either alone or in combination on streptozotocin induced diabetes.Food and Chemical Toxicology, 50 (3-4)： 1159-1165.

Gül?in, ?., Elmasta?, M., and Aboul-Enein, H. Y., 2012. Antioxidant activity of clove oil - A powerful antioxidant source.Arabian Journal of Chemistry, 5 (4)： 489-499.

He, R., Ye, J., Zhao, Y., and Su, W., 2014. Partial characterization, antioxidant and antitumor activitiesof polysaccharides fromPhilomycusbilineatus.International Journal of Biological Macromolecules, 65： 573-580..

Holdt, S. L., and Kraan, S., 2011. Bioactive compounds in seaweed： Functional food applications and legislation.Journal of Applied Phycology, 23 (3)： 543-597.

Hou, Y., Wang, J., Jin, W., Zhang, H., and Zhang, Q., 2012. Degradation ofLaminaria japonicafucoidan by hydrogen peroxide and antioxidant activities of the degradation products of different molecular weights.Carbohydrate Polymers, 87 (1)： 153-159.

Hsu, W. K., Hsu, T. H., Lin, F. Y., Cheng, Y. K., and Yang, J. P. W., 2013. Separation, purification, andα-glucosidase inhibition of polysaccharides fromCoriolus versicolorLH1 mycelia.Carbohydrate Polymers, 92 (1)： 297-306.

Jiang, G., Wen, L., Chen, F., Wu, F., Lin, S., Yang, B., and Jiang, Y., 2013. Structural characteristics and antioxidant activities of polysaccharides from longan seed.Carbohydrate Polymers, 92 (1)： 758-764.

Khan, M. N., Choi, J. S., Lee, M. C., Kim, E., Nam, T. J., Fujii H., and Hong, Y. K., 2008. Anti-inflammatory activities of methanol extracts from various seaweed species.Journal of Environmental Biology, 29 (4)： 465-469

Kim, Y. H., Kim, J. H., Jin, H. J., and Lee, S. Y., 2013. Antimicrobial activity of ethanol extracts ofLaminaria japonicaagainst oral microorganisms.Anaerobe, 21： 34-38.

Lee, I. A., Popov, A. M., Sanina, N. M., Kostetsky, E. Y., Novikova, O. D., Reunov, A. V., Nagorskaya, V. P., and Shnyrov, V. L., 2004. Morphological and immunological characterization of immunostimulatory complexes based on glycoglycerolipids fromLaminaria japonica.Acta Biochimica Polonica, 51 (1)： 263-272.

Li, J. W., Ding, S. D., and Ding, X. L., 2007. Optimization of the ultrasonically assisted extraction of polysaccharides fromZizyphus jujubacv.jinsixiaozao.Journal of Food Engineering, 80 (1)： 176-183.

Li, X., Wang, Z., Wang, L., Walid, E., and Zhang, H., 2012. Ultrasonic-assisted extraction of polysaccharides fromHohenbuehelia serotinaby response surface methodology.International Journal of Biological Macromolecules, 51 (4)：523-530.

Li, Z. Q., Zuo, D. Y., Qie, X. D., Qi, H., Zhao, M. Q., and Wu, Y. L., 2012. Berberine acutely inhibits the digestion of maltose in the intestine.Journal of Ethnopharmacology, 142 (2)： 474-480.

Liu, G., 2012. Chemical compositions,α-glucosidase andα-amylase inhibitory activities of crude polysaccharides from the endodermis of shaddock (Citrus maxima).Archives of Biological Sciences, 64 (1)： 71-76.

Liu, G., Xu, S., and Chen, L., 2012. Chemical composition and bioactivities of a water-soluble polysaccharide from the endodermis of shaddock.International Journal of Biological Macromolecules, 51 (5)： 763-766.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., 1951. Protein measurement with the Folin phenol reagent.The Journal of Biological Chemistry, 193 (1)： 265-275.

Lu, J., You, L., Lin, Z., Zhao, M., and Cui, C., 2013. The antioxidant capacity of polysaccharide fromLaminaria japonicaby citric acid extraction.International Journal of Food Science & Technology, 48 (7)： 1352-1358.

Makarenkova, I., Deriabin, P., L'vov, D., Zviagintseva, T., and Besednova, N., 2009. Antiviral activity of sulfated polysaccharide from the brown algaeLaminaria japonicaagainst avian influenza A (H5N1) virus infection in the cultured cells.Voprosy Virusologii, 55 (1)： 41-45.

Nwosu, F., Morris, J., Lund, V. A., Stewart, D., Ross H. A., and McDougall, G. J., 2011. Anti-proliferative and potential antidiabetic effects of phenolic-rich extracts from edible marine algae.Food Chemistry, 126 (3)： 1006-1012.

Orhan, N., Aslan, M., Ho?ba?, S., and Deliorman, O. D., 2009. Antidiabetic effect and antioxidant potential ofRosa caninafruits.Pharmacognosy Magazine, 5 (20)： 309-315.

Osman, A., Wong, K., and Fernyhough, A., 2006. ABTS radicaldriven oxidation of polyphenols： Isolation and structural elucidation of covalent adducts.Biochemical and Biophysical Research Communications, 346 (1)： 321-329.

Peng, Z., Liu, M., Fang, Z., Wu, J., and Zhang, Q., 2012. Composition and cytotoxicity of a novel polysaccharide from brown alga (Laminaria japonica).Carbohydrate Polymers, 89 (4)： 1022-1026.

Peng, Z., Liu, M., Fang, Z., and Zhang, Q., 2012.In vitroantioxidant effects and cytotoxicity of polysaccharides extracted fromLaminaria japonica.International Journal of Biological Macromolecules, 50 (5)： 1254-1259.

Prathapan, A., Krishna, M. S., Nisha, V. M., Sundaresan, A., and Raghu, K. G., 2012. Polyphenol rich fruit pulp ofAegle marmelos(L.) Correa exhibits nutraceutical properties to down regulate diabetic complications - Anin vitrostudy.Food Research International, 48 (2)： 690-695.

Qian, Z. J., Jung, W. K., Byun, H. G., and Kim, S. K., 2008. Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster,Crassostrea gigasagainst free radical induced DNA damage.Bioresource Technology, 99 (9)： 3365-3371.

Reddy, B. S., Sharma, C., and Mathews, L., 1984. Effect of Japanese seaweed (Laminaria angustata) extracts on the mutagenicity of 7,12-dimethylbenz[a]anthracene, a breast carcinogen, and of 3,2’-dimethyl-4-aminobiphenyl, a colon and breast carcinogen.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 127 (2)： 113-118.

Redouan, E., Emmanuel, P., Michelle, P., Bernard, C., Josiane, C., and Cédric, D., 2011. Evaluation of antioxidant capacity of ulvan-like polymer obtained by regioselective oxidation of gellan exopolysaccharide.Food Chemistry, 127 (3)： 976-983.

Saliu, J. A., Ademiluyi, A. O., Akinyemi, A. J., and Oboh, G., 2012.In vitroantidiabetes and antihypertension properties of phenolic extracts from bitter leaf (Vernonia amygdalinaDel).Journal of Food Biochemistry, 36 (5)： 569-576.

Sanjay, K. K., Sagar, K. M., Dilipkumar, P., and Arijit, M., 2012. Isolation of β-sitosterol and evaluation of antidiabetic activity ofAristolochia indicain alloxan-induced diabetic mice with a reference toin-vitroantioxidant activity.Journal of Medicinal Plants Research, 6 (7)： 1219-1223.

Singh, N., and Rajini, P., 2008. Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage.Chemico-Biological Interactions, 173 (2)： 97-104.

Vishchuk, O. S., Ermakova, S. P., and Zvyagintseva, T. N., 2011. Sulfated polysaccharides from brown seaweedsSaccharina japonicaandUndaria pinnatifida： Isolation, structural characteristics, and antitumor activity.Carbohydrate Research, 346 (17)： 2769-2776.

Wang, J., Guo, H., Zhang, J., Wang, X., Zhao, B., Yao, J., and Wang, Y., 2010. Sulfated modification, characterization and structure-Antioxidant relationships ofArtemisia sphaero-cephalapolysaccharides.Carbohydrate Polymers, 81 (4)：897-905.

Wang, J., Zhang, Q., Zhang, Z., and Li, Z., 2008. Antioxidant activity of sulfated polysaccharide fractions extracted fromLaminaria japonica.International Journal of Biological Macromolecules, 42 (2)： 127-132.

Wang, J., Zhang, Q., Zhang, Z., Song, H., and Li, P., 2010. Potential antioxidant and anticoagulant capacity of low molecular weight fucoidan fractions extracted fromLaminaria japonica.International Journal of Biological Macromolecules, 46 (1)： 6-12.

Wang, X. P., Dong, S. X., and Chen, F., 2012. Ultrasonicassisted extraction of gardenia yellow fromFructus gardeniae.Advanced Materials Research, 396： 1075-1078.

Wei, X., Cai, X., Xiong, S., and Wang, Y., 2012. Hypoglycemic effect of oral crude tea flower polysaccharides on alloxan modeling Sprague-Dawley rats and the possible mechanism.CyTA-Journal of Food, 10 (4)： 325-332.

Wright, E., Scism-Bacon, J., and Glass, L., 2006. Oxidative stress in type 2 diabetes： The role of fasting and postprandial glycaemia.International Journal of Clinical Practice, 60 (3)：308-314.

Wu, W., Zhu, Y., Zhang, L., Yang, R., and Zhou, Y., 2012. Extraction, preliminary structural characterization, and antioxidant activities of polysaccharides fromSalvia miltiorrhizaBunge.Carbohydrate Polymers, 87 (2)： 1348- 1353.

Xiao, J., Huo, J., Jiang, H., and Yang, F., 2011. Chemical compositions and bioactivities of crude polysaccharides from tea leaves beyond their useful date.International Journal of Biological Macromolecules, 49 (5)： 1143-1151.

Xie, J. H., Shen, M. Y., Xie, M. Y., Nie, S. P., Chen, Y., Li, C., Huang, D., and Wang, Y., 2012. Ultrasonic-assisted extraction, antimicrobial and antioxidant activities ofCyclocarya paliurus(Batal.) Iljinskaja polysaccharides.Carbohydrate Polymers, 89 (1)： 177-184.

Yamamoto, I., and Maruyama, H., 1985. Effect of dietary seaweed preparations on 1, 2-dimethylhydrazine-induced intestinal carcinogenesis in rats.Cancer Letters, 26 (3)： 241-251.

Yan, Y. L., Yu, C. H., Chen, J., Li, X. X., Wang, W., and Li, S. Q., 2011. Ultrasonic-assisted extraction optimized by response surface methodology, chemical composition and antioxidant activity of polysaccharides fromTremella mesenterica.Carbohydrate Polymers, 83 (1)： 217-224.

Yang, L., and Zhang, L. M., 2009. Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources.Carbohydrate Polymers, 76 (3)： 349-361.

Ying, Z., Han, X., and Li, J., 2011. Ultrasound-assisted extraction of polysaccharides from mulberry leaves.Food Chemistry, 127 (3)： 1273-1279.

Zha, X. Q., Xiao, J. J., Zhang, H. N., Wang, J. H., Pan, L. H., Yang, X. F., and Luo, J. P., 2012. Polysaccharides inLaminaria japonica(LP)： Extraction, physicochemical properties and their hypolipidemic activities in diet-induced mouse model of atherosclerosis.Food Chemistry, 134 (1)：244-252.

Zhang, Z., Wang, X., Yu, S., and Zhao, M., 2011. Isolation and antioxidant activities of polysaccharides extracted from the shoots ofPhyllostachys edulis(Carr.).International Journal of Biological Macromolecules, 49 (4)： 454-457.

Zhao, Q., Kennedy, J. F., Wang, X., Yuan, X., Zhao, B., Peng, Y., and Huang, Y., 2011. Optimization of ultrasonic circulating extraction of polysaccharides fromAsparagus officinalisusing response surface methodology.International Journal of Biological Macromolecules, 49 (2)： 181-187.

Zhao, X., Li, B., Xue, C., and Sun, L., 2011. Effect of molecular weight on the antioxidant property of low molecular weight alginate fromLaminaria japonica.Journal of Applied Phycology, 24 (2)： 295-300.

Zhao, Z., Xu, X., Ye, Q., and Dong, L., 2013. Ultrasound extraction optimization ofAcanthopanax senticosuspolysaccharides and its antioxidant activity.International Journal of Biological Macromolecules, 59： 290-294.

(Edited by Ji Dechun)

(Received April 14, 2014; revised April 29, 2014; accepted May 20, 2014)

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-20-89023145 E-mail： jypan@scsio.ac.cn