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Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties
Accepted Manuscript Title: Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties Author: Y. Wu D. Hui N.A.M. Eskin S.W. Cui PII: DOI: Reference:
S0141-8130(16)30500-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.088 BIOMAC 6149
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
22-2-2016 19-4-2016 25-5-2016
Please cite this article as: Y.Wu, D.Hui, N.A.M.Eskin, S.W.Cui, Watersoluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.088 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
1. It is the first time that antioxidant property of water-soluble yellow mucilage (WSM) was evaluated. 2. Water-soluble yellow mustard mucilage (WSM) demonstrated the strongest antioxidant activity compared to the other two commercial polysaccharides, xanthan gum and citrus pectin. 3. No relationship was found between antioxidant property and the physicochemical properties (uronic acid content, molecular weight, and apparent viscosity) of polysaccharides from different sources. 4. The study indicated a great potential of using WSM as a novel ingredient in food industries due to its superior antioxidant activities compared to citrus pectin and xanthan gum.
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Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties
Abstract The antioxidant properties of the water-soluble yellow mustard (Sinapis alba L.) mucilage (WSM) were compared with citrus pectin and xanthan gum using in vitro methods. The antioxidants ascorbic acid and butylated hydroxyanisole (BHA) were used as controls. The antioxidant activity, DPPH free radical scavenging ability, and reducing power on Fe were measured. Molecular weight (MW), uronic acid content, and viscosity for the three polysaccharides were obtained to investigate the relationships between the physicochemical properties and antioxidant activities of the three different polysaccharides. The results showed that the overall antioxidant activity of polysaccharides was lower than that for ascorbic acid and BHA. Of the three polysaccharides, WSM exhibited the strongest antioxidant properties, followed by citrus pectin and xanthan gum. Statistical analysis showed that the MW and uronic acid content had significant effects on antioxidant activity (P<0.05). MW, uronic acid and apparent viscosity had significant effects on reducing power on Fe (P<0.05). Concentration also significantly affected DPPH free radical scavenging effect and reducing power on Fe (P<0.05). The study indicated a great potential of using WSM as a novel ingredient in food industries due to its superior antioxidant activities compared to citrus pectin and xanthan gum.
Key words: Antioxidant activity, Water-soluble yellow mustard mucilage, Uronic acid content,
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Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties
Y. Wu1*, D. Hui2, N.A. M. Eskin 3, S. W. Cui 4
1. Department of Agricultural and Environmental Sciences, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN, 37209-1561, USA 2. Department of Biological Sciences, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN, 37209-1561, USA 3. Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada 4. Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada * Corresponding author. Tel: 615-963-6006; Fax: 615-963-5436. E-mail address: [email protected]
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1. Introduction Antioxidant can delay or prevent the oxidation of cellular oxidizable substances. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytolune (BHT), were commonly used for industrial processing to reduce damage to the human body and prolong the storage stability of food. However, synthetic antioxidants are now only used in nonedible products because of increasing health and safety concerns [1]. Naturally occurring antioxidants that have no adverse effects are drawing more and more attention. Polysaccharides have traditionally been widely used as food stabilizers, thickeners and emulsifiers [2]. In addition to their physicochemical functionalities in foods, polysaccharides have also demonstrated significant physiological effects as dietary fiber [3]. In recent years, many studies have also investigated the health and functional benefits of polysaccharides as antioxidants. A variety of polysaccharides from marine plants, microbes and high plants have been reported to demonstrate antioxidant properties [4-6]. Yellow mustard mucilage has been systematically studied in previous years including chemical composition, linkage and structural characteristics, as well as physicochemical properties [7-12]. These studies revealed that water-soluble yellow mustard mucilage (WSM) was mainly composed of pectic polysaccharides and a small portion of β-1,4-linked glucose with occasional side groups [11]. Yellow mustard mucilage also exhibits superior emulsification properties [9,12]. Eskin, Raju and Bird [13] reported that WSM has a potent anti-cancer effect in animal models for colon cancer. In the present study, antioxidant activities of WSM were investigated using in vitro methods by measuring the antioxidant activity in a lipid model, radical scavenging ability and reducing power on Fe. Pectin and xanthan gum were used as polysaccharide comparisons. In order to better understand the effects of physicochemical
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properties on the antioxidant activities of polysaccharides, molecular weight (MW), uronic acid contents, and apparent viscosity of the three polysaccharides were evaluated. BHA and ascorbic acid were used as positive antioxidant controls. The findings from this study may lead to a broader application of WSM in foods.
2. Materials and method
2.1. Preparation of polysaccharide solution All chemicals and reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Citrus pectin was with 90% purity, and the xanthan gum is with 90-94% purity. Yellow mustard bran was purchased from Minn-Dak Growers, Ltd. (Grand Forks, ND, USA). WSM was prepared according to the procedure described by Cui et al. [7] with slight modifications. The bran was immersed in the 70ºC distilled-water for 2 h at the bran to water ratio of 1:20, filtered using cheesecloth, and followed by centrifugation at 8000 g for 15 min. The supernatant was precipitated at 70% ethanol for 2 h and centrifuged at 8000g for 15 min. The precipitant was dried in fume hood and then grounded into powder for further study. The dried WSM powder contains 84% pectic polysaccharides and 16% cellulosic polysaccharides.
2.2. Antioxidant activity (AOA) Antioxidant activity was determined by the conjugated diene method [14]. Linolic acid (99%) was emulsified with the aid of an equal amount of Tween 20 in 0.1 M potassium phosphate buffer (pH 7.0). Polysaccharide solutions (0.1 ml, 0.1 – 2 mg/ml) were mixed with 2
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ml of 10 mM linoleic acid emulsion and placed in the dark at 37 ºC to accelerate oxidation. After incubation for 20 h, 0.2 ml of emulsion was solubilized in 2 ml absolute methanol, then 6 ml of 60% methanol in water were added. The absorbance of the mixture was measured at 234 nm against a blank (60% methanol in water) using a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The antioxidant activity was calculated as: AOA (%) = [(A234 of control – A234 of sample)/A234 of control] 100% The control consisted of water and the reagent solution without the polysaccharides. The AOA% value of 100 indicates the strongest antioxidant activity. Ascorbic acid and BHA were used as references.
2.3. DPPH free radical scavenging ability The radical scavenging ability of the polysaccharides was tested according to the method described by Shimada et al. [15] and Paiva-Martins and Gordon [16] with modifications. The 2 ml aqueous polysaccharide solutions (0.1-2 mg/ml) were added to 1 ml of methanolic DPPH (0.25 mM). The mixture was allowed to stand in the dark for 30 min followed by centrifugation. The supernatant was transferred to cuvettes. The absorbance was measured at 517 nm using the GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A lower absorbance of the reaction mixture indicated higher free radical scavenging activity. BHA and ascorbic acid were both used as positive controls. The capacity to scavenge the DPPH radical was calculated using the following equation: Scavenging effect (%) = [A0 – (A –Ab)/A0] 100%
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Where A0 is the absorbance of DPPH solution without sample; A is the absorbance of the test sample mixed with DPPH solution and Ab is the absorbance of the sample without DPPH solution.
2.4. Reducing power assay The reducing power was determined according to the method described by Oyaizu [17] with slight modifications. The 1 ml polysaccharide solutions (0.1 - 2 mg/ml) were mixed with 1 ml of 0.1 M sodium phosphate buffer (pH 7.0) and 1 ml of 1% potassium phosphate ferricyanide, and the mixture incubated at 50 ºC for 20 min. After 1ml of 10% trichloroacetic acid was added, the mixture was added with 2 ml of deionized water and 0.2 ml of 0.1% ferric chloride. The absorbance was measured at 700 nm against a blank using the GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A higher absorbance indicates a higher reducing power.
2.5.Molecular weight determination Molecular weight was measured by a Size Exclusion Chromatography (SEC) equipped with a triple detector: a low angle laser light scattering detector (LALS), a differential viscometer (DP) and a refractive index detector (RI) (Viscotek 305 TDA, Malvern Instruments Ltd., Westborough, MA, USA). The chromatographic system comprised of two Viscotek A6000M columns (Malvern Instruments, Westborough, MA, USA) in series. The columns and detectors were maintained at 40 ºC. The eluent was 0.1 M NaNO3 containing 0.02% (w/w) NaN3 at a flow rate of 0.6 ml/min.
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2.6.
Total uronic acid analysis Total uronic acid was determined by the m-hydroxydiphenyl method [18]. Briefly 0.2 ml
of 1mg/ml polysaccharide sample was mixed with 1.2 ml of concentrated sulfuric acid/0.0125 M tetraborate in an ice bath. The mixture was shaken in a Vortex mixer and the tubes heated in a water bath at 100°C for 5 min. After cooling in a water-ice bath, 20 l of the 0.15% (w/v) mhydroxydiphenyl reagent was added. The tubes were shaken and, within 5 min, absorbance measurements made at 520 nm using a spectrophotometer. A blank sample was run without addition of the reagent, which was placed by 20 l of 0.5% NaOH. The absorbance of the blank sample was subtracted from the total absorbance. Galacturonic acid was used as the standard.
2.7.
Viscosity measurement Viscosity of polysaccharide solutions was measured on a strain-controlled ARES G2
rheometer (TA Instruments, New Castle, DE, USA) using a cone-and-plate geometry (4/50 mm) with shear rate from 0.01 – 100 1/s at 25 C.
2.8.
Statistical analysis Statistical computations were performed using the GLM procedure of the Statistical
Analysis System (SAS Release 9.3, SAS Institute Inc., Cary, NC, USA). The data for the antioxidant activity, reducing power, and radical scavenging ability of pectin, xanthan gum and WSM at various concentrations were examined using a two-way ANOVA. Least square means of each of the property factors were calculated using the option of LSMEANS and statistical differences among the 3 types of antioxidants were identified at p < 0.05 using the option of PDIFF.
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3. Results and discussion 3.1. Antioxidant activity BHA showed the highest antioxidant activity, followed by ascorbic acid (Fig. 1). The antioxidant activities of the three polysaccharides were much lower than BHA and ascorbic acid in the concentration range examined. The three polysaccharides exhibited significant antioxidant activities (p< 0.05). However the effect of concentration on antioxidant activities varied, in which concentration of xanthan gum had significant effect on antioxidant activity (p< 0.05), while concentrations of WSM and citrus pectin showed no significant effect on antioxidant activity (p> 0.05). According to the FDA requirement, the limitation for BHA ranged from 11000 ppm depending on products. Polysaccharides are usually fall under the category of Generally Recognized as Safe (GRAS) and can be used at a much higher concentration (CFRCode of Federal Regulations Title 21).
Ascorbic Acid
BHA
Pectin
WSM
Xanthan
Antioxidant Activity (AA)%
80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
Concentration (mg/ml)
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Fig. 1. Antioxidant activity of WSM, xanthan gum, citrus pectin, BHA and asco rbic acid
Kishk and Al-sayed [19] investigated the antioxidant activities of some polysaccharides in emulsions. Their results showed xanthan gum had antioxidant activity, which is not concentration-dependent. Similarly, Trommer et al. [20] examined a variety of polysaccharides as potential antioxidative compounds against UV irradiation using a similar emulsion model. Their results showed that pectin and xanthan gum both exhibited antioxidant activity. In the concentration range from 0.4 mg/ml to 4 mg/ml, the antioxidant effect of xanthan showed no obvious concentration dependency. While in the case of pectin, their results varied based on the milling time used for pectin. The non-milled pectin did not show concentration dependency within the concentration range of 0.4-4.0 mg/ml. Gutterridge et al. [21] and Sipos et al. [22] proposed that the mechanism of the lipid protecting effects of polysaccharides seemed to be the chelation of transition metal ions. Sun et al. [23] examined the antioxidant activities of microalgae polysaccharides with different molecular weights. They proposed that inhibition of lipid peroxidation by the polysaccharides might be due to their ability to directly capture ROS generated by the lipid peroxidation chain by blocking or slowing down the process of lipid oxidation.
3.2. DPPH free radical scavenging ability BHA exhibited the strongest scavenging ability, followed by ascorbic acid and the three polysaccharides (Fig. 2). Among the three polysaccharides, WSM showed a higher reducing power compared to the other two. Xanthan gum showed the least scavenging effect. All materials, however, exhibited significant concentration-dependency (p <0.05).
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Radical scavenging is one of the various mechanisms for antioxidant activity. The oxidants scavenging free radicals by donating hydrogen atom to them before they attack molecules or electron followed by proton transfer to give a stable compound and antioxidantderived radical. The efficiency of scavenging radicals by antioxidants depends on the physical factors such as the mobility of antioxidant as well as its chemical reactivity [24]. DPPH is a stable radical that shows maximum absorption at 517 nm in methanol. When DPPH is mixed with an antioxidant, the radical would be scavenged and absorbance at 517 nm is reduced. The steric accessibility of DPPH radical is a major determinant of the reaction, since small molecules that have better access to the radical sites have relatively higher antioxidative capacity [25]. On the other hand, many large antioxidant compounds may react slowly in this assay [26]. The mobility of polysaccharide molecules varies with the structure, conformation and solubility of the polymer. Shimada et al. [15] attributed the potent scavenger properties of polysaccharides to their proton-donating ability. Chattopadhyay et al. [27] proposed that the polysaccharides with potent scavenger capacity might contain a higher amount of reduction, which could react with radicals to stabilize and terminate radical chain reaction. They suggested that the different bioactivities might be linked to their different molecular structures. Recently, Zhang et al. [28] measured DPPH radical scavenging effect of pectic polysaccharides and found that the scavening ability was concentration-dependent, which is similar to our results. Chen et al. [29] confirmed the concentraton-dependency of pectic polysaccharides extracted from the dry root of Astragalus membranaceus, a traditional Chinese herbal medcine. Xiong et al. [30] studied different xanthan gum oligosaccharide fractions and found that all fractions exhibited concentration dependency on DPPH scavenging proerty. The results from the current study are in agreenment with previous research [29-30].
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Ascorbic Acid
BHA
Pectin
WSM
Xanthan
DPPH Scavenging Property %
95 85 75 65 55 45 35 0.0
0.5
1.0
1.5
2.0
mg/mL
Fig. 2. DPPH redical scavenging activity of WSM, xanthan gum, citrus pectin, BHA and ascorbic acid
3.3. Reducing power The ferric reducing ability method treats the antioxidants in the sample as reductants in a redox linked colorimetric reaction, and the value reflects the reducing power of the antioxidant [31]. The individual antioxidant can cause the reduction of the Fe3+/ferricyanide complex to the ferrous form. The level of Fe2+ can then be monitored by measuring the formation of Prussian blue at 700 nm [32]. In this study, BHA showed the strongest reducing power, followed by ascorbic acid (Fig. 3). Compared to BHA and ascorbic acid, the polysaccharides had much weaker reducing power. However, the reducing power of all the three polysaccharides showed significant concentration dependency (p <0.05). Among them, WSM demonstrated the strongest capacity compared to citrus pectin and xanthan gum. This result is in agreement with the results on antioxidant activity (AOA) and the free radical scavenging capacity from previous studies
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[20,28]. The reducing properties are associated with the presence of the reductions, which had been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom [32]. Reductions are also reported to react with certain precursors of peroxide, thus preventing peroxide formation [33]. From the literature, polysaccharides have been reported to exhibit different levels of reducing power. In this study, WSM seemed to have a significantly higher level of reducing power than that of either xanthan gum or citrus pectin (p <0.05).
Ascorbic Acid
BHA
Pectin
WSM
Xanthan
3.5 3.0 2.5 2.0 1.5 1.0
Absorbance (700 nm)
0.5 0.3
0.2
0.1
0.0 0.0
0.5
1.0
1.5
2.0
Concentration (mg/ml) Fig. 3. Reducing power of WSM, xanthan gum and citrus pectin, BHA and ascorbic acid
3.4.
Relationships between physicochemical properties and antioxidant activities
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Diplok [34] summarized several mechanisms to explain the antioxidant activities of polysaccharides, including reducing power, prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, and prevention of continued hydrogen abstraction and radical scavenging. Some studies tried to relate the structure and molecular weight of polysaccharides to their antioxidant abilities [5,23,27], e.g. the factors like the content of uronic acid [35], sulphate group [33,36], and molecular weight [31,33]. In the current study, antioxidant activities of the three polysaccharides were compared in an attempt to relate the antioxidant activities to their physicochemical properties including uronic acid, MW and apparent viscosity. Large variations of MW, uronic acid content, and apparent viscosity at shear rate 1 s-1 were found among the three polysaccharides (Table 1).
Table 1. Uronic acid content, molecular weight and apparent viscosity of xanthan gum, WSM and pectin (Mean ± Standard Deviation) (WSM: water soluble yellow mustard mucilage) Viscosity (Pa.s) Polysaccharide
Molecular Weight
Type
(Dalton)
Uronic Acid %
at Shear Rate 1.0 (1/s)
Xanthan
2.92 ± 0.08 X10^6
21.76 ± 0.20
0.329 ± 0.037
WSM
4.38 ± 0.32X10^6
24.34 ± 1.91
0.062 ± 0.005
Pectin
1.45 ± 0.10X10^6
71.66 ± 2.24
0.011 ± 0.001
3.4.1. Effect of molecular weight
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Molecular weight was significantly different among the three polysaccharides (Table 1). WSM had the largest MW among the three (Fig. 4). Two peaks, representing two fractions, A and B were observed. The two peaks were not completely separated and fraction B is overlapping with fraction A. This is in agreement with previous finding that WSM is composed of two polysaccharide fractions, with 84% of pectic polysaccharide and 16% of cellulosic polysaccharides [7,10]. The MW of WSM was calculated by combining fractions A and B. It was reported that the MW of polysaccharides was an important parameter influencing antioxidant activities [37-38]. Chaouch et al. [38] depolymerized pectin extracted from a plant, Opuntia ficus indica (OFI), and found that the depolymerized pectin fractions exhibited higher antioxidant activities. Tomida et al. [4] studied the antioxidant properties of five polysaccharides for extended-release matrix tablets. They found that low MW chitosan and alginate demonstrated higher antioxidant ability than high MW controls. Wang et al. [33] extracted the polysaccharides from Potentilla anserine L. using two different methods: hot water extraction and microwave extraction. The lower MW polysaccharides extracted through microwave demonstrated stronger antioxidant activities. Sun et al. [23] examined the antioxidant activities of microalgae polysaccharides with different MWs. They showed that low MW fraction had exhibited a higher antioxidant effect. In the current study, xanthan gum possessed the highest MW, and the lowest antioxidant activities. Pectin possessed the lowest MW but didn’t show the strongest antioxidant activities. Therefore, MW is not the sole factor determining the degree of antioxidant activity.
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Xanthan Pectin
WSM-A
WSM-B
Fig. 4. Molecular weight of WSM, xanthan gum and citrus pectin (WSM-A: cellulosic polysaccharide fraction; WSM-B: pectic polysaccharide fraction).
3.4.2. Effect of uronic acid content All the three polysaccharides in the present study contain uronic acids in their primary structure. Pectin is composed of homogalacturonan and rhamnogalacturonan, where homogalacturonan is (1,4)-linked -D- galactopyranosyluronic acid (GalpA), and rhamnogalacturonan is constituted by an alternating sequence of (1,4)- linked -D-GalpA and (1,2)-linked -L-rhamnopyranose (Rhap) residues [39]. Xanthan gum consists of a -(1,4)-Dglucopyranosyl backbone substituted on every second unit with a charged trisaccharide sidechain with a glucuronic acid residue, and the terminal mannose may be substituted by a pyruvic acid residue [40]. WSM is majorly composed of two fractions with 84% of pectic polysaccharides and 16% of cellulosic polysaccharides [7-8,10-11]. Some researchers have investigated the effect of uronic acid on the antioxidant properties of polysaccharides. Popov et al. [41] extracted different pectin fractions from plums, and found that the fraction (PD-1) with 16
higher uronic acid content, up to 77.2%, exhibited higher antioxidant activity compared to the fraction (PD-E), which had a lower uronic acid content (57.5%). In another study, two xanthan oligosaccharides were obtained by degradation of soluble xanthan gum, and found that their antioxidant activities were different due to different levels of pyruvate acid and reducing sugar [30]. Citrus pectin has the highest uronic acid content (71.66%), followed by WSM (24.34%) and Xanthan gum (21.76%) (Table 1). WSM exhibited the highest antioxidant activities among the three even though its uronic acid content was much lower than that of pectin. Therefore, for polysaccharides from the same origin, such as degraded fractions from the same parent polysaccharides, antioxidant activities could be related to their uronic acid contents, considering that the major structural and chemical compositions remained the same [30,41]. In this study, the uronic acid content had significant effect on the antioxidant activity and DPPH free radical scavenging effect (p <0.05).
3.4.3. Effect of viscosity There is very limited information available on the relationship between viscosity and antioxidant activity of polysaccharides. Huang et al. [42] studied antioxidant activities of different polysaccharide fractions extracted from a medicinal fungus, Cordyceps sinensis. They proposed that the large size and high viscosity of polysaccharide might restrict the molecule mobility and accessibility to the radicals in solution. The result of this study showed that at the concentration of 0.2%, xanthan gum was the most viscous solution, followed by WSM and pectin (Fig. 5). Obviously the order of viscosity is not in agreement with the order of antioxidant activities (Figs. 2 and 3), where WSM exhibited the highest antioxidant activities. Although pectin possessed the highest uronic content, and the lowest viscosity and MW, its antioxidant
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activities were lower than that of WSM. Therefore, no trend was evident between antioxidant properties and its physicochemical properties including uronic acid content, MW and apparent viscosity among the three polysaccharides.
0.2% WSM
10
0.2% Xanthan
0.2% Pectin
Viscosity (Pa.s)
1
0.1
0.01
0.001 0.1
1
10
100
Shear rate (1/s)
Fig. 5. Viscosity of 0.2% (w/v) WSM, xanthan gum and citrus pectin. 4.
Conclusion In the current study, WSM, citrus pectin, and xanthan gum all exhibited antioxidant
properties with significant concentration-dependency (p <0.5). WSM demonstrated the strongest antioxidant activity compared to the other two commercial polysaccharides, xanthan gum and citrus pectin. Statistical analysis revealed that MW and uronic acid content had significant effects on antioxidant activity and DPPH free radical scavenging ability. All the three factors, MW, uronic acid content, and apparent viscosity, had significant effects on reducing power. In food applications, polysaccharides are used at much higher concentrations. Consequently their antioxidant activities may be greater than the tested range in the current study due to their 18
concentration-dependency. In addition to antioxidant ability, WSM is a good stabilizer in emulsions [12]. Therefore, further study should be conducted to investigate the possible applications of WSM in food for food quality, product shelf life, and replacement of the synthetic antioxidants in food products.
Acknowledgement The authors would like to thank U54 Cancer Partnership Grant (Grant No.: 5U54CA163066-02) for funding this research project.
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References
[1] L. Fan, N.A.M. Eskin, The use of antioxidants in the preservation of edible oils. In Handbook of Antioxidants for Food Preservation, Shahidi, F., Eds; Woodhead Publishing, ISBN: 978-178242-089-7 (2015) 373-388.
[2] P. A. Williams, G. O. Phillips, (2009) Gum arabic. In Handbook of hydrocolloids, Phillips, G. O., Williams, P. A. Eds; Cambridge: Woodhead Publishing Ltd. (2009) 252-273.
[3] S. W. Cui, K.T. Roberts, Dietary fiber: Fulfilling the promise of added-value formulations. In Modern Biopolymer Science, Kasapis, S., Norton, I., Ubbink, J. Eds, Academic Press (2009) 399-448.
[4] H. Tomida, T. Yasufuku, T. Fujii, Y. Kondo, T. Kai, M. Anraku, Carbohydr. Res. 345 (2010) 82-86.
[5] Y.-X. Sun, J.-C. Liu, J. F. Kennedy, Carbohydr. Polym. 82 (2010) 209-214.
[6] Y.-H. Tseng, J.-H. Yang, J.-L. Mau, Food Chem. 107 (2008) 732-738.
[7] W. Cui, N.A.M. Eskin, C.G. Biliaderis, Food Chem. 46 (1993) 169-176.
[8] W. Cui, N.A.M. Eskin, C.G. Biliaderis, Carbohydr. Polym. 27 (1995) 117-122.
20
[9] W. Cui, N. M.A. Eskin, C.G. Biliaderis, K. Marat, Carbohydr. Res. 292 (1996) 173-183.
[10] Y. Wu, W. Cui, N.A.M. Eskin, H.D. Goff, Food Hydrocoll. 23 (2009) 1535-1541.
[11] Y. Wu, W. Cui, N.A.M. Eskin, H.D. Goff, J. Nikiforuk, Carbohydr. Polym. 84 (2011) 6975.
[12] Y. Wu, N.A.M. Eskin, W. Cui, B. Pokharel, Food Hydrocoll. 47 (2015) 191-196.
[13] N.A.M. Eskin, J. Raju, R.P. Bird, Phytomed. 14 (2007) 479-485.
[14] H. Lingnert, K. Vallentin, C. E. Eriksson, Measurement of antioxidative effect in model system. J. Food Process. Preserv. 3 (1979) 87–103.
[15] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura, Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40 (1992) 945– 948.
[16] F. Paiva-Martins, M.H. Gordon, Isolation and characterization of the antioxidant component 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate from olive (Olea europaea) leaves. J. Agri. Food Chem., 49 (2001) 4214-4219.
21
[17] M. Oyaizu, Studies on product of browning reaction prepared from glucose amine. Jpn. J. Nutr. 44 (1986) 307–315.
[18] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489.
[19] Y.F.M. Kishk, H.M.A. Al-Sayed, LWT - Food Sci. Technol. 40 (2007) 270-277.
[20] H. Trommer, R.H.H. Neubert, Int. J. Pharm. 298 (2005) 153-163.
[21] J.M. Gutteridge, R. Richmond, B. Halliwell, Biochem. J. 184 (1979) 469–472.
[22] P. Sipos, O. Berkesi, E. Tombácz, T.G. St. Pierre, J. Webb, J. Inorg. Biochem. 95 (2003) 55–63.
[23] L. Sun, L. Wang, J. Li, H. Liu, Food Chem. 160 (2014) 1-7.
[24] E. Niki, Free Radic. Biol. Med., 49 (2010) 503-515.
[25] R. Huang, E. Mendis, S-K. Kim, Int. J. Biol. Macromol. 36 (2005) 120-127.
[26] L.M. Magalhães, M.A. Segundo, S. Reis, J.L.F.C. Lima, Anal. Chim. Acta 613 (2008) 1-19.
22
[27] N. Chattopadhyay, T. Ghosh, S. Sinha, K. Chattopadhyay, P. Karmakar, B. Ray, Polysaccharides from Turbinaria conoides: Structural features and antioxidant capacity. Food Chem. 118 (2010) 823-829.
[28] L. Zhang, W. Zhang, Q. Wang, D. Wang, D. Dong, H. Mu, X-S. Ye, J. Duan, Purification, antioxidant and immunological activities of polysaccharides from Actinidia Chinensis roots. Int. J. Biol. Macromol. 72 (2015) 975-983.
[29] R.Z. Chen, L. Tan, C.-G. Jin, J. Lu, L. Tian, Q.-Q. Chang, K. Wang, Extraction, isolation, characterization and antioxidant activity of polysaccharides from Astragalus membranaceus. Ind. Crops Prod. 77 (2015) 434-443.
[30] X. Xiong, M. Li, J. Xie, B. Xue, T. Sun, Preparation and antioxidant activity of xanthan oligosaccharides derivatives with similar substituting degrees. Food Chem. 164 (2014) 7-11.
[31] R. Chen, Z. Liu, J. Zhao, R. Chen, F. Meng, M. Zhang, W. Ge, Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu. Food Chem. 127 (2011) 434-440.
[32] H. Qi, Q. Zhang, T. Zhao, R. Hu, K. Zhang, Z. Li, In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorg. Med. Chem. Lett. 16 (2006) 2441-2445.
23
[33] J. Wang, J. Zhang, B. Zhao, X. Wang, Y. Wu, J. Yao A comparison study on microwaveassisted extraction of Potentilla anserina L. polysaccharides with conventional method: Molecule weight and antioxidant activities evaluation. Carbohydr. Polym. 80 (2010) 84-93.
[34] A.T. Diplock, Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease? Free Radical Res., 26 (1997) 565-583.
[35] C. Xue, G. Yu, T. Hirata, J. Terao, H. Lin, Antioxidative activities of several marine polysaccharides evaluated in a phospha- tidylcholine–liposomal suspension and organic solvents. Biosci., Biotechnol., Biochem. 62 (1998) 206–209.
[36] P. Rupérez, O. Ahrazem, J.A. Leal, Potential Antioxidant Capacity of Sulfated Polysaccharides from the Edible Marine Brown Seaweed Fucus vesiculosus. J. Agric. Food Chem. 50 (2002) 840–845.
[37] D. Fleita, M. El-Sayed, D. Rifaat, Evaluation of the antioxidant activity of enzymaticallyhydrolyzed sulfated polysaccharides extracted from red algae; Pterocladia capillacea. LWT Food Sci. Technol. 63 (2015) 1236-1244.
[38] M.A. Chaouch, J. Hafsa, C. Rihouey, D. Le Cerf, H. Majdoub, Int. J. Biol. Macromol. 79 (2015) 779-786.
[39] J.-P. Vincken, H.A. Schols, R.J.F.J. Oomen, M.C. McCann, P. Ulvskov, A.G.J. Voragen,
24
R.G.F. Visser, If homogalacturonan were a side chain of rhamnogalacturonan I. implications for cell wall architecture1. Plant Physiol. 132 (2003) 1781-1789.
[40] M. Rinaudo, M. Milas, F. Lambert, M. Vincendon, Proton and carbon-13 NMR investigation of xanthan gum. Macromolecules 16 (1983) 816–819.
[41] S.V. Popov, R.G. Ovodova, V.V. Golovchenko, D.S. Khramova, P.A. Markov, V.V. Smirnov, A.S. Shashkov, Y.S. Ovodov, Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory and antioxidant activities. Food Chem. 143 (2014) 106-113.
[42] Q.-L., Huang, K.-C., Siu, W.-Q., Wang, Y.-C. Cheung, J.-Y. Wu, Fractionation, characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus. Process Biochem. 48 (2013) 380-386.
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S0141-8130(16)30500-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.088 BIOMAC 6149
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
22-2-2016 19-4-2016 25-5-2016
Please cite this article as: Y.Wu, D.Hui, N.A.M.Eskin, S.W.Cui, Watersoluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.088 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
1. It is the first time that antioxidant property of water-soluble yellow mucilage (WSM) was evaluated. 2. Water-soluble yellow mustard mucilage (WSM) demonstrated the strongest antioxidant activity compared to the other two commercial polysaccharides, xanthan gum and citrus pectin. 3. No relationship was found between antioxidant property and the physicochemical properties (uronic acid content, molecular weight, and apparent viscosity) of polysaccharides from different sources. 4. The study indicated a great potential of using WSM as a novel ingredient in food industries due to its superior antioxidant activities compared to citrus pectin and xanthan gum.
1
Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties
Abstract The antioxidant properties of the water-soluble yellow mustard (Sinapis alba L.) mucilage (WSM) were compared with citrus pectin and xanthan gum using in vitro methods. The antioxidants ascorbic acid and butylated hydroxyanisole (BHA) were used as controls. The antioxidant activity, DPPH free radical scavenging ability, and reducing power on Fe were measured. Molecular weight (MW), uronic acid content, and viscosity for the three polysaccharides were obtained to investigate the relationships between the physicochemical properties and antioxidant activities of the three different polysaccharides. The results showed that the overall antioxidant activity of polysaccharides was lower than that for ascorbic acid and BHA. Of the three polysaccharides, WSM exhibited the strongest antioxidant properties, followed by citrus pectin and xanthan gum. Statistical analysis showed that the MW and uronic acid content had significant effects on antioxidant activity (P<0.05). MW, uronic acid and apparent viscosity had significant effects on reducing power on Fe (P<0.05). Concentration also significantly affected DPPH free radical scavenging effect and reducing power on Fe (P<0.05). The study indicated a great potential of using WSM as a novel ingredient in food industries due to its superior antioxidant activities compared to citrus pectin and xanthan gum.
Key words: Antioxidant activity, Water-soluble yellow mustard mucilage, Uronic acid content,
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Water-soluble yellow mustard mucilage: A novel ingredient with potent antioxidant properties
Y. Wu1*, D. Hui2, N.A. M. Eskin 3, S. W. Cui 4
1. Department of Agricultural and Environmental Sciences, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN, 37209-1561, USA 2. Department of Biological Sciences, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN, 37209-1561, USA 3. Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada 4. Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada * Corresponding author. Tel: 615-963-6006; Fax: 615-963-5436. E-mail address: [email protected]
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1. Introduction Antioxidant can delay or prevent the oxidation of cellular oxidizable substances. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytolune (BHT), were commonly used for industrial processing to reduce damage to the human body and prolong the storage stability of food. However, synthetic antioxidants are now only used in nonedible products because of increasing health and safety concerns [1]. Naturally occurring antioxidants that have no adverse effects are drawing more and more attention. Polysaccharides have traditionally been widely used as food stabilizers, thickeners and emulsifiers [2]. In addition to their physicochemical functionalities in foods, polysaccharides have also demonstrated significant physiological effects as dietary fiber [3]. In recent years, many studies have also investigated the health and functional benefits of polysaccharides as antioxidants. A variety of polysaccharides from marine plants, microbes and high plants have been reported to demonstrate antioxidant properties [4-6]. Yellow mustard mucilage has been systematically studied in previous years including chemical composition, linkage and structural characteristics, as well as physicochemical properties [7-12]. These studies revealed that water-soluble yellow mustard mucilage (WSM) was mainly composed of pectic polysaccharides and a small portion of β-1,4-linked glucose with occasional side groups [11]. Yellow mustard mucilage also exhibits superior emulsification properties [9,12]. Eskin, Raju and Bird [13] reported that WSM has a potent anti-cancer effect in animal models for colon cancer. In the present study, antioxidant activities of WSM were investigated using in vitro methods by measuring the antioxidant activity in a lipid model, radical scavenging ability and reducing power on Fe. Pectin and xanthan gum were used as polysaccharide comparisons. In order to better understand the effects of physicochemical
4
properties on the antioxidant activities of polysaccharides, molecular weight (MW), uronic acid contents, and apparent viscosity of the three polysaccharides were evaluated. BHA and ascorbic acid were used as positive antioxidant controls. The findings from this study may lead to a broader application of WSM in foods.
2. Materials and method
2.1. Preparation of polysaccharide solution All chemicals and reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Citrus pectin was with 90% purity, and the xanthan gum is with 90-94% purity. Yellow mustard bran was purchased from Minn-Dak Growers, Ltd. (Grand Forks, ND, USA). WSM was prepared according to the procedure described by Cui et al. [7] with slight modifications. The bran was immersed in the 70ºC distilled-water for 2 h at the bran to water ratio of 1:20, filtered using cheesecloth, and followed by centrifugation at 8000 g for 15 min. The supernatant was precipitated at 70% ethanol for 2 h and centrifuged at 8000g for 15 min. The precipitant was dried in fume hood and then grounded into powder for further study. The dried WSM powder contains 84% pectic polysaccharides and 16% cellulosic polysaccharides.
2.2. Antioxidant activity (AOA) Antioxidant activity was determined by the conjugated diene method [14]. Linolic acid (99%) was emulsified with the aid of an equal amount of Tween 20 in 0.1 M potassium phosphate buffer (pH 7.0). Polysaccharide solutions (0.1 ml, 0.1 – 2 mg/ml) were mixed with 2
5
ml of 10 mM linoleic acid emulsion and placed in the dark at 37 ºC to accelerate oxidation. After incubation for 20 h, 0.2 ml of emulsion was solubilized in 2 ml absolute methanol, then 6 ml of 60% methanol in water were added. The absorbance of the mixture was measured at 234 nm against a blank (60% methanol in water) using a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The antioxidant activity was calculated as: AOA (%) = [(A234 of control – A234 of sample)/A234 of control] 100% The control consisted of water and the reagent solution without the polysaccharides. The AOA% value of 100 indicates the strongest antioxidant activity. Ascorbic acid and BHA were used as references.
2.3. DPPH free radical scavenging ability The radical scavenging ability of the polysaccharides was tested according to the method described by Shimada et al. [15] and Paiva-Martins and Gordon [16] with modifications. The 2 ml aqueous polysaccharide solutions (0.1-2 mg/ml) were added to 1 ml of methanolic DPPH (0.25 mM). The mixture was allowed to stand in the dark for 30 min followed by centrifugation. The supernatant was transferred to cuvettes. The absorbance was measured at 517 nm using the GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A lower absorbance of the reaction mixture indicated higher free radical scavenging activity. BHA and ascorbic acid were both used as positive controls. The capacity to scavenge the DPPH radical was calculated using the following equation: Scavenging effect (%) = [A0 – (A –Ab)/A0] 100%
6
Where A0 is the absorbance of DPPH solution without sample; A is the absorbance of the test sample mixed with DPPH solution and Ab is the absorbance of the sample without DPPH solution.
2.4. Reducing power assay The reducing power was determined according to the method described by Oyaizu [17] with slight modifications. The 1 ml polysaccharide solutions (0.1 - 2 mg/ml) were mixed with 1 ml of 0.1 M sodium phosphate buffer (pH 7.0) and 1 ml of 1% potassium phosphate ferricyanide, and the mixture incubated at 50 ºC for 20 min. After 1ml of 10% trichloroacetic acid was added, the mixture was added with 2 ml of deionized water and 0.2 ml of 0.1% ferric chloride. The absorbance was measured at 700 nm against a blank using the GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A higher absorbance indicates a higher reducing power.
2.5.Molecular weight determination Molecular weight was measured by a Size Exclusion Chromatography (SEC) equipped with a triple detector: a low angle laser light scattering detector (LALS), a differential viscometer (DP) and a refractive index detector (RI) (Viscotek 305 TDA, Malvern Instruments Ltd., Westborough, MA, USA). The chromatographic system comprised of two Viscotek A6000M columns (Malvern Instruments, Westborough, MA, USA) in series. The columns and detectors were maintained at 40 ºC. The eluent was 0.1 M NaNO3 containing 0.02% (w/w) NaN3 at a flow rate of 0.6 ml/min.
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2.6.
Total uronic acid analysis Total uronic acid was determined by the m-hydroxydiphenyl method [18]. Briefly 0.2 ml
of 1mg/ml polysaccharide sample was mixed with 1.2 ml of concentrated sulfuric acid/0.0125 M tetraborate in an ice bath. The mixture was shaken in a Vortex mixer and the tubes heated in a water bath at 100°C for 5 min. After cooling in a water-ice bath, 20 l of the 0.15% (w/v) mhydroxydiphenyl reagent was added. The tubes were shaken and, within 5 min, absorbance measurements made at 520 nm using a spectrophotometer. A blank sample was run without addition of the reagent, which was placed by 20 l of 0.5% NaOH. The absorbance of the blank sample was subtracted from the total absorbance. Galacturonic acid was used as the standard.
2.7.
Viscosity measurement Viscosity of polysaccharide solutions was measured on a strain-controlled ARES G2
rheometer (TA Instruments, New Castle, DE, USA) using a cone-and-plate geometry (4/50 mm) with shear rate from 0.01 – 100 1/s at 25 C.
2.8.
Statistical analysis Statistical computations were performed using the GLM procedure of the Statistical
Analysis System (SAS Release 9.3, SAS Institute Inc., Cary, NC, USA). The data for the antioxidant activity, reducing power, and radical scavenging ability of pectin, xanthan gum and WSM at various concentrations were examined using a two-way ANOVA. Least square means of each of the property factors were calculated using the option of LSMEANS and statistical differences among the 3 types of antioxidants were identified at p < 0.05 using the option of PDIFF.
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3. Results and discussion 3.1. Antioxidant activity BHA showed the highest antioxidant activity, followed by ascorbic acid (Fig. 1). The antioxidant activities of the three polysaccharides were much lower than BHA and ascorbic acid in the concentration range examined. The three polysaccharides exhibited significant antioxidant activities (p< 0.05). However the effect of concentration on antioxidant activities varied, in which concentration of xanthan gum had significant effect on antioxidant activity (p< 0.05), while concentrations of WSM and citrus pectin showed no significant effect on antioxidant activity (p> 0.05). According to the FDA requirement, the limitation for BHA ranged from 11000 ppm depending on products. Polysaccharides are usually fall under the category of Generally Recognized as Safe (GRAS) and can be used at a much higher concentration (CFRCode of Federal Regulations Title 21).
Ascorbic Acid
BHA
Pectin
WSM
Xanthan
Antioxidant Activity (AA)%
80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
Concentration (mg/ml)
9
Fig. 1. Antioxidant activity of WSM, xanthan gum, citrus pectin, BHA and asco rbic acid
Kishk and Al-sayed [19] investigated the antioxidant activities of some polysaccharides in emulsions. Their results showed xanthan gum had antioxidant activity, which is not concentration-dependent. Similarly, Trommer et al. [20] examined a variety of polysaccharides as potential antioxidative compounds against UV irradiation using a similar emulsion model. Their results showed that pectin and xanthan gum both exhibited antioxidant activity. In the concentration range from 0.4 mg/ml to 4 mg/ml, the antioxidant effect of xanthan showed no obvious concentration dependency. While in the case of pectin, their results varied based on the milling time used for pectin. The non-milled pectin did not show concentration dependency within the concentration range of 0.4-4.0 mg/ml. Gutterridge et al. [21] and Sipos et al. [22] proposed that the mechanism of the lipid protecting effects of polysaccharides seemed to be the chelation of transition metal ions. Sun et al. [23] examined the antioxidant activities of microalgae polysaccharides with different molecular weights. They proposed that inhibition of lipid peroxidation by the polysaccharides might be due to their ability to directly capture ROS generated by the lipid peroxidation chain by blocking or slowing down the process of lipid oxidation.
3.2. DPPH free radical scavenging ability BHA exhibited the strongest scavenging ability, followed by ascorbic acid and the three polysaccharides (Fig. 2). Among the three polysaccharides, WSM showed a higher reducing power compared to the other two. Xanthan gum showed the least scavenging effect. All materials, however, exhibited significant concentration-dependency (p <0.05).
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Radical scavenging is one of the various mechanisms for antioxidant activity. The oxidants scavenging free radicals by donating hydrogen atom to them before they attack molecules or electron followed by proton transfer to give a stable compound and antioxidantderived radical. The efficiency of scavenging radicals by antioxidants depends on the physical factors such as the mobility of antioxidant as well as its chemical reactivity [24]. DPPH is a stable radical that shows maximum absorption at 517 nm in methanol. When DPPH is mixed with an antioxidant, the radical would be scavenged and absorbance at 517 nm is reduced. The steric accessibility of DPPH radical is a major determinant of the reaction, since small molecules that have better access to the radical sites have relatively higher antioxidative capacity [25]. On the other hand, many large antioxidant compounds may react slowly in this assay [26]. The mobility of polysaccharide molecules varies with the structure, conformation and solubility of the polymer. Shimada et al. [15] attributed the potent scavenger properties of polysaccharides to their proton-donating ability. Chattopadhyay et al. [27] proposed that the polysaccharides with potent scavenger capacity might contain a higher amount of reduction, which could react with radicals to stabilize and terminate radical chain reaction. They suggested that the different bioactivities might be linked to their different molecular structures. Recently, Zhang et al. [28] measured DPPH radical scavenging effect of pectic polysaccharides and found that the scavening ability was concentration-dependent, which is similar to our results. Chen et al. [29] confirmed the concentraton-dependency of pectic polysaccharides extracted from the dry root of Astragalus membranaceus, a traditional Chinese herbal medcine. Xiong et al. [30] studied different xanthan gum oligosaccharide fractions and found that all fractions exhibited concentration dependency on DPPH scavenging proerty. The results from the current study are in agreenment with previous research [29-30].
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Ascorbic Acid
BHA
Pectin
WSM
Xanthan
DPPH Scavenging Property %
95 85 75 65 55 45 35 0.0
0.5
1.0
1.5
2.0
mg/mL
Fig. 2. DPPH redical scavenging activity of WSM, xanthan gum, citrus pectin, BHA and ascorbic acid
3.3. Reducing power The ferric reducing ability method treats the antioxidants in the sample as reductants in a redox linked colorimetric reaction, and the value reflects the reducing power of the antioxidant [31]. The individual antioxidant can cause the reduction of the Fe3+/ferricyanide complex to the ferrous form. The level of Fe2+ can then be monitored by measuring the formation of Prussian blue at 700 nm [32]. In this study, BHA showed the strongest reducing power, followed by ascorbic acid (Fig. 3). Compared to BHA and ascorbic acid, the polysaccharides had much weaker reducing power. However, the reducing power of all the three polysaccharides showed significant concentration dependency (p <0.05). Among them, WSM demonstrated the strongest capacity compared to citrus pectin and xanthan gum. This result is in agreement with the results on antioxidant activity (AOA) and the free radical scavenging capacity from previous studies
12
[20,28]. The reducing properties are associated with the presence of the reductions, which had been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom [32]. Reductions are also reported to react with certain precursors of peroxide, thus preventing peroxide formation [33]. From the literature, polysaccharides have been reported to exhibit different levels of reducing power. In this study, WSM seemed to have a significantly higher level of reducing power than that of either xanthan gum or citrus pectin (p <0.05).
Ascorbic Acid
BHA
Pectin
WSM
Xanthan
3.5 3.0 2.5 2.0 1.5 1.0
Absorbance (700 nm)
0.5 0.3
0.2
0.1
0.0 0.0
0.5
1.0
1.5
2.0
Concentration (mg/ml) Fig. 3. Reducing power of WSM, xanthan gum and citrus pectin, BHA and ascorbic acid
3.4.
Relationships between physicochemical properties and antioxidant activities
13
Diplok [34] summarized several mechanisms to explain the antioxidant activities of polysaccharides, including reducing power, prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, and prevention of continued hydrogen abstraction and radical scavenging. Some studies tried to relate the structure and molecular weight of polysaccharides to their antioxidant abilities [5,23,27], e.g. the factors like the content of uronic acid [35], sulphate group [33,36], and molecular weight [31,33]. In the current study, antioxidant activities of the three polysaccharides were compared in an attempt to relate the antioxidant activities to their physicochemical properties including uronic acid, MW and apparent viscosity. Large variations of MW, uronic acid content, and apparent viscosity at shear rate 1 s-1 were found among the three polysaccharides (Table 1).
Table 1. Uronic acid content, molecular weight and apparent viscosity of xanthan gum, WSM and pectin (Mean ± Standard Deviation) (WSM: water soluble yellow mustard mucilage) Viscosity (Pa.s) Polysaccharide
Molecular Weight
Type
(Dalton)
Uronic Acid %
at Shear Rate 1.0 (1/s)
Xanthan
2.92 ± 0.08 X10^6
21.76 ± 0.20
0.329 ± 0.037
WSM
4.38 ± 0.32X10^6
24.34 ± 1.91
0.062 ± 0.005
Pectin
1.45 ± 0.10X10^6
71.66 ± 2.24
0.011 ± 0.001
3.4.1. Effect of molecular weight
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Molecular weight was significantly different among the three polysaccharides (Table 1). WSM had the largest MW among the three (Fig. 4). Two peaks, representing two fractions, A and B were observed. The two peaks were not completely separated and fraction B is overlapping with fraction A. This is in agreement with previous finding that WSM is composed of two polysaccharide fractions, with 84% of pectic polysaccharide and 16% of cellulosic polysaccharides [7,10]. The MW of WSM was calculated by combining fractions A and B. It was reported that the MW of polysaccharides was an important parameter influencing antioxidant activities [37-38]. Chaouch et al. [38] depolymerized pectin extracted from a plant, Opuntia ficus indica (OFI), and found that the depolymerized pectin fractions exhibited higher antioxidant activities. Tomida et al. [4] studied the antioxidant properties of five polysaccharides for extended-release matrix tablets. They found that low MW chitosan and alginate demonstrated higher antioxidant ability than high MW controls. Wang et al. [33] extracted the polysaccharides from Potentilla anserine L. using two different methods: hot water extraction and microwave extraction. The lower MW polysaccharides extracted through microwave demonstrated stronger antioxidant activities. Sun et al. [23] examined the antioxidant activities of microalgae polysaccharides with different MWs. They showed that low MW fraction had exhibited a higher antioxidant effect. In the current study, xanthan gum possessed the highest MW, and the lowest antioxidant activities. Pectin possessed the lowest MW but didn’t show the strongest antioxidant activities. Therefore, MW is not the sole factor determining the degree of antioxidant activity.
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Xanthan Pectin
WSM-A
WSM-B
Fig. 4. Molecular weight of WSM, xanthan gum and citrus pectin (WSM-A: cellulosic polysaccharide fraction; WSM-B: pectic polysaccharide fraction).
3.4.2. Effect of uronic acid content All the three polysaccharides in the present study contain uronic acids in their primary structure. Pectin is composed of homogalacturonan and rhamnogalacturonan, where homogalacturonan is (1,4)-linked -D- galactopyranosyluronic acid (GalpA), and rhamnogalacturonan is constituted by an alternating sequence of (1,4)- linked -D-GalpA and (1,2)-linked -L-rhamnopyranose (Rhap) residues [39]. Xanthan gum consists of a -(1,4)-Dglucopyranosyl backbone substituted on every second unit with a charged trisaccharide sidechain with a glucuronic acid residue, and the terminal mannose may be substituted by a pyruvic acid residue [40]. WSM is majorly composed of two fractions with 84% of pectic polysaccharides and 16% of cellulosic polysaccharides [7-8,10-11]. Some researchers have investigated the effect of uronic acid on the antioxidant properties of polysaccharides. Popov et al. [41] extracted different pectin fractions from plums, and found that the fraction (PD-1) with 16
higher uronic acid content, up to 77.2%, exhibited higher antioxidant activity compared to the fraction (PD-E), which had a lower uronic acid content (57.5%). In another study, two xanthan oligosaccharides were obtained by degradation of soluble xanthan gum, and found that their antioxidant activities were different due to different levels of pyruvate acid and reducing sugar [30]. Citrus pectin has the highest uronic acid content (71.66%), followed by WSM (24.34%) and Xanthan gum (21.76%) (Table 1). WSM exhibited the highest antioxidant activities among the three even though its uronic acid content was much lower than that of pectin. Therefore, for polysaccharides from the same origin, such as degraded fractions from the same parent polysaccharides, antioxidant activities could be related to their uronic acid contents, considering that the major structural and chemical compositions remained the same [30,41]. In this study, the uronic acid content had significant effect on the antioxidant activity and DPPH free radical scavenging effect (p <0.05).
3.4.3. Effect of viscosity There is very limited information available on the relationship between viscosity and antioxidant activity of polysaccharides. Huang et al. [42] studied antioxidant activities of different polysaccharide fractions extracted from a medicinal fungus, Cordyceps sinensis. They proposed that the large size and high viscosity of polysaccharide might restrict the molecule mobility and accessibility to the radicals in solution. The result of this study showed that at the concentration of 0.2%, xanthan gum was the most viscous solution, followed by WSM and pectin (Fig. 5). Obviously the order of viscosity is not in agreement with the order of antioxidant activities (Figs. 2 and 3), where WSM exhibited the highest antioxidant activities. Although pectin possessed the highest uronic content, and the lowest viscosity and MW, its antioxidant
17
activities were lower than that of WSM. Therefore, no trend was evident between antioxidant properties and its physicochemical properties including uronic acid content, MW and apparent viscosity among the three polysaccharides.
0.2% WSM
10
0.2% Xanthan
0.2% Pectin
Viscosity (Pa.s)
1
0.1
0.01
0.001 0.1
1
10
100
Shear rate (1/s)
Fig. 5. Viscosity of 0.2% (w/v) WSM, xanthan gum and citrus pectin. 4.
Conclusion In the current study, WSM, citrus pectin, and xanthan gum all exhibited antioxidant
properties with significant concentration-dependency (p <0.5). WSM demonstrated the strongest antioxidant activity compared to the other two commercial polysaccharides, xanthan gum and citrus pectin. Statistical analysis revealed that MW and uronic acid content had significant effects on antioxidant activity and DPPH free radical scavenging ability. All the three factors, MW, uronic acid content, and apparent viscosity, had significant effects on reducing power. In food applications, polysaccharides are used at much higher concentrations. Consequently their antioxidant activities may be greater than the tested range in the current study due to their 18
concentration-dependency. In addition to antioxidant ability, WSM is a good stabilizer in emulsions [12]. Therefore, further study should be conducted to investigate the possible applications of WSM in food for food quality, product shelf life, and replacement of the synthetic antioxidants in food products.
Acknowledgement The authors would like to thank U54 Cancer Partnership Grant (Grant No.: 5U54CA163066-02) for funding this research project.
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References
[1] L. Fan, N.A.M. Eskin, The use of antioxidants in the preservation of edible oils. In Handbook of Antioxidants for Food Preservation, Shahidi, F., Eds; Woodhead Publishing, ISBN: 978-178242-089-7 (2015) 373-388.
[2] P. A. Williams, G. O. Phillips, (2009) Gum arabic. In Handbook of hydrocolloids, Phillips, G. O., Williams, P. A. Eds; Cambridge: Woodhead Publishing Ltd. (2009) 252-273.
[3] S. W. Cui, K.T. Roberts, Dietary fiber: Fulfilling the promise of added-value formulations. In Modern Biopolymer Science, Kasapis, S., Norton, I., Ubbink, J. Eds, Academic Press (2009) 399-448.
[4] H. Tomida, T. Yasufuku, T. Fujii, Y. Kondo, T. Kai, M. Anraku, Carbohydr. Res. 345 (2010) 82-86.
[5] Y.-X. Sun, J.-C. Liu, J. F. Kennedy, Carbohydr. Polym. 82 (2010) 209-214.
[6] Y.-H. Tseng, J.-H. Yang, J.-L. Mau, Food Chem. 107 (2008) 732-738.
[7] W. Cui, N.A.M. Eskin, C.G. Biliaderis, Food Chem. 46 (1993) 169-176.
[8] W. Cui, N.A.M. Eskin, C.G. Biliaderis, Carbohydr. Polym. 27 (1995) 117-122.
20
[9] W. Cui, N. M.A. Eskin, C.G. Biliaderis, K. Marat, Carbohydr. Res. 292 (1996) 173-183.
[10] Y. Wu, W. Cui, N.A.M. Eskin, H.D. Goff, Food Hydrocoll. 23 (2009) 1535-1541.
[11] Y. Wu, W. Cui, N.A.M. Eskin, H.D. Goff, J. Nikiforuk, Carbohydr. Polym. 84 (2011) 6975.
[12] Y. Wu, N.A.M. Eskin, W. Cui, B. Pokharel, Food Hydrocoll. 47 (2015) 191-196.
[13] N.A.M. Eskin, J. Raju, R.P. Bird, Phytomed. 14 (2007) 479-485.
[14] H. Lingnert, K. Vallentin, C. E. Eriksson, Measurement of antioxidative effect in model system. J. Food Process. Preserv. 3 (1979) 87–103.
[15] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura, Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40 (1992) 945– 948.
[16] F. Paiva-Martins, M.H. Gordon, Isolation and characterization of the antioxidant component 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate from olive (Olea europaea) leaves. J. Agri. Food Chem., 49 (2001) 4214-4219.
21
[17] M. Oyaizu, Studies on product of browning reaction prepared from glucose amine. Jpn. J. Nutr. 44 (1986) 307–315.
[18] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489.
[19] Y.F.M. Kishk, H.M.A. Al-Sayed, LWT - Food Sci. Technol. 40 (2007) 270-277.
[20] H. Trommer, R.H.H. Neubert, Int. J. Pharm. 298 (2005) 153-163.
[21] J.M. Gutteridge, R. Richmond, B. Halliwell, Biochem. J. 184 (1979) 469–472.
[22] P. Sipos, O. Berkesi, E. Tombácz, T.G. St. Pierre, J. Webb, J. Inorg. Biochem. 95 (2003) 55–63.
[23] L. Sun, L. Wang, J. Li, H. Liu, Food Chem. 160 (2014) 1-7.
[24] E. Niki, Free Radic. Biol. Med., 49 (2010) 503-515.
[25] R. Huang, E. Mendis, S-K. Kim, Int. J. Biol. Macromol. 36 (2005) 120-127.
[26] L.M. Magalhães, M.A. Segundo, S. Reis, J.L.F.C. Lima, Anal. Chim. Acta 613 (2008) 1-19.
22
[27] N. Chattopadhyay, T. Ghosh, S. Sinha, K. Chattopadhyay, P. Karmakar, B. Ray, Polysaccharides from Turbinaria conoides: Structural features and antioxidant capacity. Food Chem. 118 (2010) 823-829.
[28] L. Zhang, W. Zhang, Q. Wang, D. Wang, D. Dong, H. Mu, X-S. Ye, J. Duan, Purification, antioxidant and immunological activities of polysaccharides from Actinidia Chinensis roots. Int. J. Biol. Macromol. 72 (2015) 975-983.
[29] R.Z. Chen, L. Tan, C.-G. Jin, J. Lu, L. Tian, Q.-Q. Chang, K. Wang, Extraction, isolation, characterization and antioxidant activity of polysaccharides from Astragalus membranaceus. Ind. Crops Prod. 77 (2015) 434-443.
[30] X. Xiong, M. Li, J. Xie, B. Xue, T. Sun, Preparation and antioxidant activity of xanthan oligosaccharides derivatives with similar substituting degrees. Food Chem. 164 (2014) 7-11.
[31] R. Chen, Z. Liu, J. Zhao, R. Chen, F. Meng, M. Zhang, W. Ge, Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu. Food Chem. 127 (2011) 434-440.
[32] H. Qi, Q. Zhang, T. Zhao, R. Hu, K. Zhang, Z. Li, In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorg. Med. Chem. Lett. 16 (2006) 2441-2445.
23
[33] J. Wang, J. Zhang, B. Zhao, X. Wang, Y. Wu, J. Yao A comparison study on microwaveassisted extraction of Potentilla anserina L. polysaccharides with conventional method: Molecule weight and antioxidant activities evaluation. Carbohydr. Polym. 80 (2010) 84-93.
[34] A.T. Diplock, Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease? Free Radical Res., 26 (1997) 565-583.
[35] C. Xue, G. Yu, T. Hirata, J. Terao, H. Lin, Antioxidative activities of several marine polysaccharides evaluated in a phospha- tidylcholine–liposomal suspension and organic solvents. Biosci., Biotechnol., Biochem. 62 (1998) 206–209.
[36] P. Rupérez, O. Ahrazem, J.A. Leal, Potential Antioxidant Capacity of Sulfated Polysaccharides from the Edible Marine Brown Seaweed Fucus vesiculosus. J. Agric. Food Chem. 50 (2002) 840–845.
[37] D. Fleita, M. El-Sayed, D. Rifaat, Evaluation of the antioxidant activity of enzymaticallyhydrolyzed sulfated polysaccharides extracted from red algae; Pterocladia capillacea. LWT Food Sci. Technol. 63 (2015) 1236-1244.
[38] M.A. Chaouch, J. Hafsa, C. Rihouey, D. Le Cerf, H. Majdoub, Int. J. Biol. Macromol. 79 (2015) 779-786.
[39] J.-P. Vincken, H.A. Schols, R.J.F.J. Oomen, M.C. McCann, P. Ulvskov, A.G.J. Voragen,
24
R.G.F. Visser, If homogalacturonan were a side chain of rhamnogalacturonan I. implications for cell wall architecture1. Plant Physiol. 132 (2003) 1781-1789.
[40] M. Rinaudo, M. Milas, F. Lambert, M. Vincendon, Proton and carbon-13 NMR investigation of xanthan gum. Macromolecules 16 (1983) 816–819.
[41] S.V. Popov, R.G. Ovodova, V.V. Golovchenko, D.S. Khramova, P.A. Markov, V.V. Smirnov, A.S. Shashkov, Y.S. Ovodov, Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory and antioxidant activities. Food Chem. 143 (2014) 106-113.
[42] Q.-L., Huang, K.-C., Siu, W.-Q., Wang, Y.-C. Cheung, J.-Y. Wu, Fractionation, characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus. Process Biochem. 48 (2013) 380-386.
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