Accepted Manuscript Title: Synthesis and Antioxidant Properties of, Gum Arabic-Stabilized Selenium Nanoparticles Author: Huiling Kong Jixin Yang Yifeng Zhang Yapeng Fang Katsuyoshi Nishinari Glyn O. Phillips PII: DOI: Reference:
S0141-8130(14)00012-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.01.011 BIOMAC 4091
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23-10-2013 24-12-2013 5-1-2014
Please cite this article as: H. Kong, J. Yang, Y. Zhang, Y. Fang,K. Nishinari, G.O. Phillips, Synthesis and Antioxidant Properties of, Gum Arabic-Stabilized Selenium Nanoparticles, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.01.011 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.
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Synthesis and Antioxidant Properties of
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Gum Arabic-Stabilized Selenium Nanoparticles
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Huiling Kong a, Jixin Yang b, Yifeng Zhang a, Yapeng Fang *, a, b, Katsuyoshi Nishinari a, Glyn O.
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Phillips a, c
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Department of Chemistry, Glyndwr University, Plas Coch, Mold Road, Wrexham, LL11 2AW, UK
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Engineering, Faculty of Light Industry, Hubei University of Technology, Wuhan 430068, China
Glyn O. Phillips Hydrocolloid Research Center, Glyndwr University, Plas Coch, Mold Road,
Wrexham, LL11 2AW, UK
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Glyn O. Phillips Hydrocolloid Research Centre at HUT, School of Food and Pharmaceutical
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TITLE RUNNING HEAD: Gum Arabic-Stabilized Selenium Nanoparticles *
To
whom
correspondence
should
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addressed:
Tel,
+86-(0)-2788015996;
Email,
[email protected] or
[email protected] 1
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ABSTRACT: Selenium nanoparticles (SeNPs) were prepared by using gum arabic (GA) as the
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stabilizer in a facile synthetic approach. The size, morphology, stability and antioxidant activity in vitro
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of the gum arabic-selenium nanocomposites (GA-SeNPs) were characterized by transmission electron
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microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR),
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atomic force microscopy (AFM) and ultraviolet/visible spectrophotometry (UV/Vis). SeNPs (particle
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size of ~34.9 nm) can be stabilized in gum arabic aqueous solutions for approximately 30 days. FTIR
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results show that SeNPs were combined to the hydroxyl groups of GA. In the present work, the alkali-
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hydrolyzed GA (AHGA) was also prepared and its efficiency in stabilizing SeNPs was compared with
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GA. It was concluded that the branched structure of GA was a significant factor for the functionality.
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The hydroxyl radical scavenging ability and DPPH scavenging ability of GA-SeNPs were higher than
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those of AHGA-SeNPs and could reach 85.3 ± 2.6%, 85.3 ± 1.9% at a concentration of 4 mg/ml,
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respectively.
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1. Introduction
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Selenium (Se) is a nutritional trace element with remarkable antioxidant characteristics that is of
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fundamental importance to human health [1-4]. It can inhibit many inflammatory cell mechanisms
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through antioxidant selenoenzymes as one selenium atom is absolutely required at the active site of all
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selenoenzymes in the form of the 21st amino-acid selenocystein [5, 6]. However, Se has a very narrow
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margin between the thresholds of functionality and toxicity. It was shown to suppress the growth of
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tumor cells in vivo and in vitro [7-10]. In particular, selenium nanoparticles (SeNPs) have excellent
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bioavailability, high biological activity and low toxicity [10]. For instance, consumption of 200 μg Se
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per day by cancer patients reduces mortality and depresses the incidence of many diseases including
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lung, colorectal and prostate cancers [11-13]. Nano-Se has a 7-fold lower acute toxicity than sodium
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selenite in mice (LD50 113 and 15 mg Se/kg body weight, respectively) [14]. Some studies
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demonstrated the antioxidant properties of hollow spherical selenium nanoparticles, which may have
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potential use as special anti-oxidative drugs [11, 15]. Moreover, aging cells accumulate oxidative
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damage [16-18]. It has been reported that SeNPs in the size range from 5 to 200 nm were efficient for 2
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free radical scavenging both in vivo and in vitro [19, 20]. Biologically synthesized selenium
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nanoparticles with diameter less than 100 nm have potential application as food additives with
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antioxidant properties [12, 21]. Gum arabic (GA) is one of the widely accepted ingredients in the food and pharmaceutical industry. It
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is a branched, neutral or slightly acidic complex polysaccharide existing as mixed calcium, magnesium,
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and potassium salts. The GA (Acacia Senegal species) has demonstrated high heterogeneity, which is
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made up of approximately 44% galactose, 13% rhamnose, 27% arabinose, and 16% glucuronic acid and
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4-O-methyl glucuronic acid [22-24]. It also contains 2-3% peptide moieties as an integral part of the
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structure. Three major fractions in GA were identified, including arabinogalactan protein complex
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(AGP), arabinogalactan (AG) and glycoprotein (GP) [25-27].
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Pure SeNPs do not represent a stable system in aqueous solutions, therefore stabilization and
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functionalization of them by suitable chemical reagents is essential towards their specific interaction
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with biological targets [28]. As is well known, biomacromolecules have been applied as templates for
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controlling inorganic crystal nucleation and growth [29-34]. Zhang et al. showed that a water-soluble
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hyperbranched polysaccharide (HBP) extracted from sclerotia of pleurotus tuber-regium functioned as a
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stabilizer and capping agent of SeNPs [31]. However, the extraction process of HBP was tedious and the
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yield was low. In recent years, the ability of GA to act as a biocompatible shell for nanostructures has
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triggered enormous interest in medical research [35]. GA, which is easily accessible and cheap, has
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many functional properties making it an ideal candidate as stabilizer and emulsifier in the food industry
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and beyond. In this work, a facile and green method to synthesize and stabilize selenium nanoparticles
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was developed by using gum arabic as a stabilizing agent. The SeNPs were prepared using GA and
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alkali-hydrolyzed GA (AHGA) to demonstrate their usefulness in stabilizing the nanostructures and
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clarify the effect of structural characteristics on the antioxidant ability of SeNPs in vitro. The size,
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morphology, bonding mechanism, stability and antioxidant action of GA-SeNPs were characterized by
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transmission electron microscope (TEM), atomic force microscope (AFM), Fourier transform infrared
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(FTIR), dynamic light scattering (DLS) and ultraviolet/visible spectroscopy (UV/Vis) etc. The
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stabilizing mechanism was found to be related to the branched structure of gum arabic and the
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interaction of hydroxyl groups with selenium nanoparticles. The anti-oxidant properties of the gum
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arabic-stabilized SeNPs were also studied, and found to be linked to the stability of the nanoparticles.
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2. Materials and methods
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2.1. Materials
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Gum arabic (GA) was provided by San-Ei Gen F.F.I. Inc. (Osaka, Japan) in spray dried form. The
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powder contains 5.56% moisture, as well as 8.76 ppm Fe and 1.43 ppm Cu. Selenium dioxide and
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ascorbic acid were purchased from Tianjin Chemical Reagent Institute (Tianjin, China) and Xilong
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Chemical Co. Ltd. (Puning, China), respectively. Iron (III) chloride hexahydrate, potassium
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ferricyanide, trichloroacetic acid (TCA), hydrogen peroxide, methanol and sodium borohydride (NaBH4)
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were purchased from Chinese Medicine Group Chemical Reagent Co., Ltd (Shanghai, China).
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Deoxyribose (DR) was purchased from Amresco and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was from
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Aladdin Industrial Corporation. 2-thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA)
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and protease (Type XIV from Streptomyces griseus) were purchased from Sigma–Aldrich (USA). All
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the chemicals were of analytical grade and used without further purification. Milli-Q water was used in
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all the experiments.
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2.2.
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Modification and characterization of GA
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Alkali-hydrolyzed GA (AHGA) was prepared according to the procedures reported in the literature
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[36]. Briefly, GA (2 g) was treated with 200 ml of 4 M NaBH4 and 2 M NaOH for 6 h at 100 ºC.
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Excessive NaBH4 was neutralized using 12.5 mL of 1 M acetic acid in 1062.5 mL of methanol in an ice
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bath. The resulting precipitate was washed three times with methanol, deproteinized, and dialyzed
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against water (Mw cutoff: 14 kDa) for 72 h to remove any free salts, followed by freeze drying.
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The GA samples before and after alkaline hydrolysis were characterized by gel permeation
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chromatography coupled with multi-angle laser light scattering (GPC-MALLS). The GPC-MALLS 4
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system consists of a Superose 6 10/300GL column (GE Healthcare, USA), a DAWN HELEOS
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multiangle light scattering detector (Wyatt Technology Corporation, USA) operated at 658 nm, an
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Optilab rEX refractometer (Wyatt Technology Corporation, USA), and a SPD-10Avp series UV
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detector (Shimadzu Technologies, Japan) carried out at 214 nm. 0.2 M aqueous NaCl solution filtered
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through a 0.2 µm Millipore filter was used as an eluent, delivered by a Waters 515 HPLC pump (Waters
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Corporation, USA) at a constant rate of 0.4 ml/min. A refractive index increment dn/dc of 0.141 ml/g
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was used for molecular parameter analysis of GA.
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2.3.
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Preparation of GA-SeNPs and AHGA-SeNPs
The same concentration of aqueous GA and AHGA solution (1 mg/mL, 11.25 ml) was added into a
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20 ml sealed bottle, respectively, and they were mixed with 150 μL of 0.6 M selenious acid (selenium
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dioxide dissolved in the water) and 2.85 ml of water under magnetic stirring for 6 h. 4.5 ml of 0.1 M
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aqueous ascorbic acid solution was added dropwise into the resulting mixture, which was then stirred
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for 0.5 h at room temperature.
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2.4.
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Size and morphology measurements of SeNPs
Transmission electron microscopy (TEM) of the diluted solutions of GA-SeNPs was measured on a
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JEOL JEM-2010 (HT) electron microscope at an accelerating voltage of 200 kV. The high-resolution
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transmission electron microscopy (HRTEM) image was acquired on a JEOL JEM 2010 FEF (UHR)
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microscope at 200 kV [34]. One drop of each sample solution was put onto copper grid and dried in air
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for 5 minutes for TEM observation. The average particle size of SeNPs was obtained from TEM
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measurements of three replications.
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The morphology and size distribution of the samples were examined using AFM (Agilent
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Technologies, USA) in a tapping mode. A 10 μL drop of either 10 μg/ml GA or GA-SeNPs aqueous
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solution was deposited onto freshly cleaved mica and dried by nitrogen at room temperature and 45-
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60% humidity. A scanning probe made of SiN4 with a cantilever length of 235 μm and a spring constant 5
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of 98 N/m was employed. Dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern) was used to monitor the change of
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particle size during storage. 1.0-1.5 ml of each sample was measured in a polystyrene cuvette at a fixed
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angle of 173° at 25ºC. Laser Doppler velocimety (LDV, Zetasizer Nano ZS, Malvern) was applied to
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obtain zeta potential values to check the bonding mechanism. Effect of pH was measured by pH-
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titration device (Zetasizer Nano ZS, Malvern). Both particle size, zeta potential and pHs of GA and GA-
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SeNPs were reported as the average values of triplicate measurements.
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2.5.
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4000-400 cm-1 using the KBr-disk preparation method.
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2.6.
The GA/AHGA-SeNPs aqueous solutions were recorded by photographs to compare the stability of SeNPs during storage of 60 days at 25 ± 1ºC.
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2.7.
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Antioxidant activity of Gum arabic (GA/AHGA)-SeNPs
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Stability of Gum Arabic (GA/AHGA)-SeNPs
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FTIR spectra of the samples were recorded on a Nicolet 170SX FTIR spectrometer in the range of
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Analysis of bonding between GA/AHGA and SeNPs
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Two different measurements of antioxidant behavior, hydroxyl radical scavenging activity and DPPH
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radical scavenging activity, were conducted. Each measurement was repeated by 3 times.
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2.7.1. Hydroxyl radical scavenging activity
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Deoxyribose assay was used to measure the hydroxyl radical scavenging activities of GA-SeNPs and
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AHGA-SeNPs [37-40]. The system consisted of Deoxyribose (16.8 mM), FeCl3 (300 mM), EDTA (1.2
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mM), H2O2 (16.8 mM), NaH2PO4/Na2HPO4 buffer (10 mM, pH=7.4) and ascorbic acid (0.6 mM)
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solutions. EDTA/FeCl3 solutions were prepared at a ratio of 1:1 (w/w). Gum arabic (GA/AHGA)-SeNPs
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aqueous solutions (0.1-4 mg/ml, 200 μL) was mixed with all the prepared solutions mentioned above,
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with ascorbic acid added last. The mixture was incubated at 37 ± 1ºC for 1 h. The TBA solution (1 ml, 6
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1% in 50 mM NaOH) and TCA solution (1 ml, 2.8% in water) were then added into the system at
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80±1ºC. After reaction for 20 min, absorbance was measured at 532 nm in a UV/Vis spectrometer.
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Hydroxyl radical scavenging activity (%) was calculated as: [1-(Aa-Ab) /Ac] × 100, where Aa was the
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absorbance of experimental group; Ab was the absorbance of sample control, which was Gum Arabic
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(GA/AHGA)-SeNPs with 1 ml ultrapure water, 1 ml of TBA, and 1 ml of TCA; and Ac was the
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absorbance of blank control without sample.
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2.7.2. DPPH radical scavenging activity
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Each GA-SeNPs aqueous solution (0.1-4 mg/ml, 1ml) was added into 2 ml of DPPH in methanolic
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solutions (0.09 mg/ml). The mixture was shaken by vortex mixer (Jiangsu Healthy Medical Supplies
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Co., Ltd, Jiangsu, China) and stored for 30 min in the dark. The absorbance of each solution was
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measured at 517 nm, spectrophotometrically [37, 41]. The scavenging ability was calculated in the same
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way as the deoxyribose test above.
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3. Results and discussion
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3.1.
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Synthesis of GA-SeNPs
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The reaction of selenious acid with ascorbic acid in water was formulated in Scheme 1 [42]. It could
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be clearly observed from its color change from yellow to orange-red which indicated an appearance of
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either amorphous or monoclinic selenium particles, since the color of trigonal selenium is black [20, 43]. OH
H2SeO3 + 2 161 162
OH
HO
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OH
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Scheme 1. Reaction of selenious acid with ascorbic acid in water at room temperature.
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Photographs of SeNPs aqueous solutions in the absence and presence of GA are shown in Figure
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1(a), both immediately and one week after their synthesis. The SeNPs solution in the presence of GA 7
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exhibited an orange-red colour due to the nano-size effect, and was much more stable, remaining
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transparent without any visible precipitation for approximately 30 days. However, for the control
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sample, brick-red particles were precipitated out 7 days after the preparation of SeNPs in the absence of
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GA. GA therefore played a vital role in improving their stability. The results suggest that GA with the
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large number of terminal hydroxyl groups had strong attraction to the surface of Se particles and thus
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stabilized them, where a similar mechanism was mentioned by Green et al. [44]. This can be further
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confirmed in Fig. 1(b). The size of SeNPs without GA was polydisperse after 7 days, while there is no
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obvious change of the size distribution of GA-SeNPs after the same period. As shown in Fig. 1(a),
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SeNPs without GA tend to aggregate and precipitate in the aqueous solutions and it was difficult to
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obtain the accurate size of SeNPs without GA after storage for 7 days, which was therefore not included
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in Fig. 1(b). The particle size of SeNPs without stabilizer was smaller than that of GA-SeNPs
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immediately after synthesis and the difference in diameter should be attributed to the GA. It indicates
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that GA, which was adsorbed on the surface of SeNPs, preventing their aggregation. Particle diameters
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of GA-SeNPs were determined by DLS to be 145, 161, 170, 158 and 156 nm, respectively, with the
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concentration of GA being 1, 5, 7, 13, and 19 mg/ml (Fig. 2(a)). In another word, when the GA
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concentration is over 1 mg/ml, the diameter of GA-SeNPs particles exceeded 150 nm. The SeNPs were
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stable for 30 days at 1 mg/ml GA. Selenious acid (Se (IV)) aqueous solutions at 0.1 M and 0.6 M
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resulted in smaller size (Fig. 2(b)). Moreover, 0.6 M selenious acid in the redox system resulted in a
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better stability during 60 days confirmed by DLS observation (data not shown). Thus 1mg/ml GA and
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0.6 M Se (IV) were considered as optimal preparation conditions and used in all subsequent experiments.
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3.2.
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Morphology and Size of GA-SeNPs
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Fig. 3 shows the TEM (a) and HRTEM (b) images of the SeNPs in the presence of GA and absence
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of GA ((c) and (d)). Fig. 3(a) clearly reveals that SeNPs were stabilized by 1 mg/ml GA showing
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monodisperse and homogeneous spherical structure, while the SeNPs aggregated to a large cluster in the
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absence of GA (Fig. 3(c) and (d)). The lattice pattern of the SeNPs can be clearly seen at room 8
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temperature (Fig. 3(b)), and the value was 0.15 nm. By counting more than 180 particles in several
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TEM images, the statistical results show that the mean particle size d = 34.9 nm with a standard
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deviation σ of 3.8 nm. The size is obviously smaller than that of the nanocomposites GA-SeNPs, where
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the hydrodynamic diameter contributed from the capping agent was measured in DLS. Peng et al.
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mentioned that the size of SeNPs played an important role in their biologic activity and, as expected, 5–
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200 nm SeNPs can directly scavenge free radicals in vitro. Torres et al. reported that chitosan CS-
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SeNPs remained stable with the size ranging from 120 to 150 nm, which supported their in vitro cell
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studies [21, 45-47]. Therefore, the GA-SeNPs (30-150 nm) were suitable to enhance the antioxidant
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capacity and cellular uptake. Nano elemental selenium (SeNPs) with the size range of 5~100 nm, can be
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synthesized in this work by reducing selenite in a clean environment containing GA, which can adhere
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to Se atoms and control the degree of their aggregation. In the present study, size-controlled SeNPs were
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prepared by adding GA to the redox system of selenious acid and ascorbic acid.
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To further confirm that the GA and SeNPs were packed closely to form spherical composites, AFM
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was used to analyze their morphology and size. Fig. 4(a) and (b) show the tapping mode AFM images of
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the GA and the GA-SeNPs aqueous solutions drop-cast on mica. The corresponding heights of arrow-
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marked particle in Fig. 4(a) and (b) are shown in Fig. 4(c) and (d), respectively. The height from top to
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bottom was used to determine the diameters of particles. The result indicates that the mean size of GA-
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SeNPs was about 138.5 nm, obviously larger than that of GA (65.2 nm). It was noted that the
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nanocomposites size from the AFM result was close to those obtained by DLS measurement (144.5 nm,
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Fig. 2(a)).
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3.3.
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Effect of pH on GA-SeNPs
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The highly branched structure of GA gives rise to compact molecules with a relatively small
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hydrodynamic volume (Rg is ~19.0 nm). The pHs of GA and as prepared GA-SeNPs aqueous solutions
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were 5.4 and 3.1, respectively. The effect of pH was examined to analyze the stability of SeNPs through
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observing the change of size and zeta potential (Fig. 5). The size of GA-SeNPs was larger than that of 9
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pure GA due to the contribution for the Se cores on to which GA was bound. The size of GA-SeNPs
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when treated with strong acid (pH 2-4) became smaller, which suggests that hydrogen ions made the
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interior structure of GA-SeNPs more compact. At pH > 4, the size of GA-SeNPs hardly changed, which
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is in line with the zeta potential results as shown in Fig. 5(b). The zeta potential of GA-SeNPs was
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nearly stable when pH > 4, whereas that of GA showed a sudden rise at pH ≥ 8. The results indicated
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that once SeNPs were formed in GA aqueous solution, the structure of GA-SeNPs was tight and stable.
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3.4.
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Stabilizing Mechanism of GA-SeNPs
To clarify the stabilizing mechanism of GA-SeNPs, GA and its alkali-hydrolyzed form AHGA with
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less branched structured are compared. The GPC-MALLS elution profiles of GA and AHGA samples
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obtained using light scattering (LS), refractive index (RI) and UV (at 214 nm) detectors are shown in
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Fig. 6. LS and RI are sensitive to molar mass and concentration, respectively, whereas UV absorbance is
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sensitive to the content of proteinaceous component. The UV elution profile of GA clearly consists of
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three peaks, arabinogalactan protein, AGP (~8.0 ml), arabinogalactan, AG (~13.0 ml) and glycoprotein,
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GP (~15.5 ml). After alkali-hydrolysis, the high molecular weight fraction AGP mostly disappeared
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with formation of some fragments even smaller than AG and GP (16.0-20.0 ml). The AG fraction, a
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protein-deficient component, however was not so much affected by akali-hydrolysis. The different
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behavior of AGP and AG responding to alkali-hydrolysis may be attributed to the difference in protein
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content in AGP and AG [36, 48, 49]. The calculated molecular parameters of GA and AHGA are
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included in Table 1. Alkaline hydrolysis reduced the molecular mass from ~ 8.38×105 to ~ 2.11×105.
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The AGP fraction in AHGA was close to zero (0.53%), with both AG and GP fractions slightly
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increased. The AGP in GA was supposed to take a wattle blossom-type branched structure with
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carbohydrate blocks attached to a common peptide chain [48]. The alkali-hydrolysis possibly broke the
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peptide chain, and degraded AGP into AG and GP. Thus the highly branched structure of GA was
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reduced after alkali-hydrolysis [36].
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Fig. 7 shows that GA-SeNPs remained stable for at least 30 days, while AHGA-SeNPs could only be
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stable for 15 days. The dispersion of SeNPs stabilized by AHGA was worse than that by GA as shown
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in Fig. 8. Statistical results obtained by counting more than 180 particles in several TEM images showed
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that the mean size of AHGA-SeNPs is d = 103.5 nm with standard deviation σ = 1.0 nm. This was larger
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than SeNPs covered by GA (34.9 nm shown in Fig. 3(a)). The longer stable time and smaller particle
244
size of GA-SeNPs indicate that highly branched structure of AGP contributed to the stability of SeNPs.
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3.5.
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Bonding mechanism of GA-SeNPs
To clarify the reason why the SeNPs and GA/AHGA can be bound, the interactions between them
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were studied by infrared spectroscopy. The FTIR spectra of GA/AHGA and GA/AHGA-SeNPs are
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shown in Fig. 9(a) and (b). The characteristic absorption peaks of hydroxyl group (OH) of GA-SeNPs
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and AHGA-SeNPs were at 3406 cm-1 and 3415 cm-1, respectively. Both of them shifted to lower
250
wavenumbers than that of pure GA/AHGA (3426 cm-1 and 3423 cm-1, respectively), and the shift of 20
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cm-1 for GA-SeNPs was larger than that of 8 cm-1 for AHGA-SeNPs. The shift of OH band indicates a
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strong bonding interaction between hydroxyl groups of GA and surface atoms of SeNPs, which plays an
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important role in stabilization of nanoparticles in this work [31], and the interaction is even more
254
intensive in GA-SeNPs than that in AHGA-SeNPs. The combination mode of GA-SeNPs was similar to
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what was reported for chitosan (CS)-SeNPs and hyperbranched polysaccharide (HBP)-SeNPs [44].
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3.6.
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Antioxidant action of GA/AHGA-SeNPs
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SeNPs have intriguing antioxidant activity, and this has been demonstrated in previous studies [16,
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21, 37, 48]. The method using deoxyribose was employed to measure the hydroxyl radical scavenging
259
activity of GA-SeNPs and DPPH assay was also used as previously reported by Jung et al [37, 39]. The
260
results are shown in Fig. 10(a). GA/AHGA-SeNPs exhibits the ability of scavenging free radicals in a
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dose-dependent manner at 0.1-4.0 mg/ml. DPPH scavenging ability was more sensitive to the
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concentration of SeNPs. In both cases of hydroxyl radical or DPPH, the scavenging ability of GA11
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SeNPs was higher than that of AHGA-SeNPs and the value can reach up to 85.3 ± 2.6% and 85.3 ±
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1.9% at 4.0 mg/ml (Fig. 10(a) and (b)), respectively. The AGP fraction seems again to play a crucial
265
role in the antioxidant action of GA-SeNPs. This can be related to their different stability. The inferior
266
stability of AHGA-SeNPs would lead to more aggregations of SeNPs, thus reducing the superficial area
267
of SeNPs that could react with the free radicals. Moreover, compared with GA, proteinaceous material
268
was mostly lost in AHGA. This could also possibly explain the decrease in antioxidant activity of
269
AHGA-SeNPs, as certain amino acids and their specific sequences are thought to scavenge free radicals
270
[50, 51]. Therefore, the highly branched structure of AGP and the presence of proteinaceous material
271
may contribute to the free radicals-scavenging ability of GA-SeNPs [38].
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4. Conclusions
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GA consists of abundant hydroxyl groups and some polypeptides. It has a highly branched structure
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and large specific surface, leading to the strong adsorption on some elements. SeNPs with mean particle
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size of approximately 34.9 nm were combined mainly through hydroxyl groups of GA. The results from
276
TEM, AFM and Nano ZS revealed that SeNPs were stabilized by the GA molecules to prevent
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aggregation of the grown SeNPs, as a consequence, SeNPs dispersed well in GA aqueous solution.
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Further, the branched structure of GA was analyzed to study the dominant factor of the stability and
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antioxidant ability. From our results, highly branched and polypeptide structure may both improve the
280
stability and antioxidant ability of SeNPs. The hydroxyl radical scavenging ability and DPPH
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scavenging ability of GA-SeNPs were higher, and both could reach 85.3 % at 4 mg/mL. This work
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provides the fundamental evidence where food grade macromolecules with high branched structure and
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abundant hydroxyl groups could be used to prepare and stabilize selenium nanoparticles. This represents
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a facile, economic and green route to the synthesis of functional selenium materials with potential
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impacts on the food industry and nutrition science areas.
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Glossary of Abbreviations
AFM, atomic force microscopy; AG, arabinogalactan; AGP, arabinogalactan protein; AHGA, alkali-
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hydrolyzed GA; DLS, dynamic light scattering; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; DR,
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deoxyribose; EDTA, ethylenediaminetetraacetic acid; FTIR, Fourier-transform infrared spectroscopy;
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GA, gum arabic; GP, glycoprotein; GPC, gel permeation chromatography; HBP, hyperbranched
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polysaccharide; HRTEM, high-resolution transmission electron microscopy; NaBH4, sodium
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borohydride; SeNPs, selenium nanoparticles; TBA, 2-Thiobarbituric acid; TCA, trichloroacetic acid;
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TEM, transmission electron microscopy; UV/Vis, ultraviolet/visible spectrophotometer.
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Acknowledgements
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The authors acknowledge the financial support from the National Natural Science Foundation of
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China (31322043, 31171751, 31101260), the Program for New Century Excellent Talents in University
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(NCET-12-0710), the Key Project of Chinese Ministry of Education (212117), the Key Project of
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Natural Science Foundation of Hubei Province (2012FFA004), the Team Project from the Hubei
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Provincial Department of Education (T201307) and the Project from the Ministry of Human Resources
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and Social Security of China.
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Table 1. Molecular parameters of GA and AHGA measured by GPC-MALLS.
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Peak 1:
Peak 2:
Peak 3:
fraction
AGP
AG
GP
Mw
Fraction
Mw
Fraction
Mw
Fraction
Mw
(g/mol)
(wt.%)
(g/mol)
(wt.%)
(g/mol)
(g/mol)
GA
8.38×105
15.52
1.76 ×106
72.57
3.94 ×105
ip t
Sample
Whole
AHGA
2.11×105
0.53
84.00
2.30×105
cr
11.91 15.47
1.44×105 2.13×104
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1.48×106
(wt.%)
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Figure captions
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Figure 1. Formation of selenium nanoparticles (SeNPs). (a) Photographs of SeNPs aqueous solutions in
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the presence and absence of gum arabic (GA) after one-week storage; and (b) the corresponding particle
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size distribution measured by dynamic light scattering (DLS). Day “0” = immediately after sample
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synthesis.
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Figure 2. Change in average particle size of GA-SeNPs with different concentration of GA at constant
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concentrations of Se (IV) 0.1M (a) and with different concentration of Se (IV) aqueous solutions at a
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constant concentration of GA 1mg/ml (b) for 7 days determined by DLS. Arrows here indicate the
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concentrations selected in the following experiments.
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Figure 3. Typical transmission electron microscope (TEM) images of SeNPs in the presence of GA
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aqueous solutions (a) and high resolution transmission electron microscope (HRTEM) image of the
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corresponding individual particle (b); TEM images of SeNPs in the absence of GA aqueous solutions in
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(c) and (d). All the samples were freshly prepared.
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Figure 4. 3D atomic force microscope (AFM) images of GA (a) and GA-SeNPs (b) (fresh samples); the
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bird’s eye view is shown in the bottom left corner. The corresponding heights of arrow-marked particles
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are shown in (c) and (d).
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Figure 5. pH effect on the average size (a), Zeta Potential (b) of GA and GA-SeNPs (fresh samples)
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measured by Zetasizer Nano ZS.
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Figure 6. Gel permeation chromatography (GPC) elution profiles from GA/AHGA obtained using LS,
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RI, UV (214 nm) detectors.
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Figure 7. Photographs of GA/AHGA-SeNPs during a 60 days period. Day “0” = immediately after
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sample synthesis, day “15”, “30”, “45”, “60”= standing time after synthesis.
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Figure 8. TEM result of SeNPs in AHGA aqueous solutions at (a) 0.2 μm and (b) 100 nm scale bars
398
(fresh samples).
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Figure 9. Fourier-transform infrared (FTIR) spectra of GA and GA-SeNPs (a), AHGA and AHGA-
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SeNPs (b).
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Figure 10. Antioxidant ability. Hydroxyl radical scavenging activity and 2,2-Diphenyl-1-
402
picrylhydrazyl (DPPH) radical scavenging ability of GA/AHGA-SeNPs at a variety of sample
403
concentrations.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Fig. 8.
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Fig. 9.
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Fig. 10.
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Graphic for table of contents
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