An in vitro comparison of the antioxidant activities of chitosan and green synthesized gold nanoparticles

Carbohydrate Polymers 211 (2019) 161–172

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

An in vitro comparison of the antioxidant activities of chitosan and green synthesized gold nanoparticles

T

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Shuai Pua,b, Jin Lia,b, , Lijun Sunb, Lian Zhongc, Qimin Maa,b a

Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao, 266100, China College of Environmental Science and Engineering, Ocean University of China, Qingdao, 266100, China c College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao, 266100, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Chitosan Gold nanoparticles Molecular weight Degree of deacetylation Antioxidant activity

Gold nanoparticles (AuNPs) were synthesized using chitosan with different degree of deacetylation (DD) and molecular weight (MW) as reducing agent and stabilizer. The synthesized AuNPs were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and Xray diffractometer (XRD). The OH%, ABTS%+ and DPPH scavenging activity and ferric reducing antioxidant power (FRAP) of chitosan, synthesized AuNPs and Au colloids were determined through in vitro assays. The antioxidant activity of chitosan decreased with the decreasing of chitosan DD and concentration, while increased with the decreasing of chitosan MW. Low molecular weight chitosan (LMWC, 47.8 kDa) showed the highest antioxidant activity at concentration of 0.3% (w/v). The antioxidant activities of AuNPs were dependent on their size, shape and amount. Spherical AuNPs showed the higher antioxidant activity than irregular or polygonal ones. The antioxidant activities of Au colloids were dependent on the DD, MW and concentration of chitosan. Au colloids synthesized by 0.3% (w/v) LMWC (47.8 kDa) showed the highest antioxidant activity. Compared with chitosan and Au colloids, the improved antioxidant activity of Au colloids may be due to the synergistic effect of AuNPs and chitosan remained in the colloids.

1. Introduction Appropriate oxidation is essential to many organisms for the production of energy to fuel biological processes. However, excessive oxidative stress, a result of imbalance between the antioxidant defense system and the uncontrolled production of free radicals, is involved in the onset of many severe diseases, such as Alzheimer’s and Parkinson’s diseases, ischemic injury, arthritis, myocardial infarction, atherosclerosis and cancer (Halliwell & Gutteridge, 1990). Antioxidants, which are scavengers of free radicals, can exert health-promoting and disease-preventing in humans and animals. However, some of synthetic antioxidants are suspected of being responsible for liver damage and carcinogenesis (Yuan, Zhang, Fan, & Yang, 2008; Zhou & Zheng, 1991). Consequently, the replacement of harmful antioxidants by biocompatible and nontoxic natural ones as preventive and therapeutic agents is of great interest to the important application in fields of pharmaceutical, cosmetic, and food because the risk of the formation of harmful byproducts is much smaller during their use than in the case of some synthetic antioxidants (Kirschweng, Tátraaljai, Földes, & Pukánszky, 2017).

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Gold nanoparticles (AuNPs), reported as a health adjuvant, have attracted tremendous interests in pharmacological and biomedical applications as well as relevant in therapy and imaging, due to unique tunable optical (Zhang, Qian, Wang, Wang, & He, 2013), distinct electronic properties (Zhang et al., 2014), excellent biocompatibility (Khan, Vishakante, & Siddaramaiah, 2013), ease of surface modification (Singh, Jagannathan, Khandelwal, Abraha, & Poddar, 2013) and excellent catalytic activities (Hutchings & Edwards, 2012). Among these possible applications, as one of the most effective heterogeneous oxidative catalyst (Stratakis & Garcia, 2012), there is considerable interest in using AuNPs for both in vitro and in vivo antioxidant application (Desai, Sangaokar, & Pawar, 2018; Lopez-Chaves et al., 2018). Generally, AuNPs could be efficiently prepared using a lot of different physical and chemical methods, such as chemical reduction (Ji et al., 2007), electrochemical reduction (Palanisamy et al., 2017), photochemical reduction (Uwada, Wang, Liu, & Masuhara, 2017), sonochemical reduction (Takahashi, Yamamoto, Todoriki, & Jin, 2018) and heat evaporation (Bonyár et al., 2018), most of these methods have utilized toxic and hazardous chemicals, have high costs of production, or are subject to difficulty in purification of the end product (Patra,

Corresponding author at: Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China, Qingdao, 266100, China. E-mail address: [email protected] (J. Li).

https://doi.org/10.1016/j.carbpol.2019.02.007 Received 26 September 2018; Received in revised form 6 December 2018; Accepted 1 February 2019 Available online 04 February 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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synthesized AuNPs and Au colloids. The identification of the relationship between AuNPs and Au colloids synthesis condition and the antioxidant activity of resultant AuNPs and Au colloids is significant for pursuing their practical application.

Kwon, & Baek, 2016). Hence, AuNPs synthesized by physical and chemical methods become unsuitable for biomedical application (Desai et al., 2018). In recent years, green synthesis of AuNPs has attracted a great deal of attention over the physical and chemical synthesis as it is highly eco-environmentally and possesses distinct advantages such as better control over the size and shape of AuNPs and enhanced stability. Polysaccharides, owning advantages of availability, multi-functionality and relatively low cost, are a good choice for reducing agent and/or stabilizer in the green synthesis of AuNPs (Pandey, Goswami, & Nanda, 2013; Pradeepa et al., 2016). Using polysaccharides as reducing agent and stabilizer, the synthesized AuNPs coated with polysaccharides, result in core-shell structures. Core-shell structures are challenging due to their multi-functionality originated from combination of different properties generated by core and shell (Nonkumwong et al., 2016). Among all of polysaccharides, chitosan has gained the highest popularity because it can participate in both reduction and stabilization processes in the synthesis of AuNPs (Bodnar, Hartmann, & Borbely, 2005; Tiwari, Mishra, Mishra, Arotiba, & Mamba, 2011). Chitosan, obtained by alkaline deacetylation of chitin, has excellent biocompatible, biodegradable, low toxicity and immune stimulating properties, and excellent antioxidant activity (Anraku et al., 2012; Kim & Thomas, 2007). Chitosan has attracted much attention to develop as natural antioxidant due to the plenty of eOH and eNH2 groups as Hatom donation (Feng, Du, Li, Hu, & Kennedy, 2008; Gue, Xing, Liu, Zhong, & Li, 2008). Although the eOH and eNH2 groups of chitosan exhibit many unique properties, such as antioxidant and reducing power, chitosan has its inherent drawback of non-reactivity and insolubility in water, aqueous bases and organic solvents because of strong inter- and intra-molecular hydrogen bonds (Kubota, Tastumoto, Sano, & Toya, 2000; Li, Du, & Liang, 2006), which is a vastest limitation to use chitosan in aqueous solution at neutral pH in a wide range of applications particularly biological media (Pasanphan, Rattanawongwiboon, Choofong, Güven, & Katti, 2015). However, the synthesized AuNPs coated with chitosan not only are in requirement of biological purpose due to biodegradability, sustainability and biocompatibility contributed to the coating chitosan (Esumi, Takei, & Yoshimura, 2003; Leiva et al., 2015; Regiel-Futyra et al., 2015) but also are stable in aqueous medium and more suitable in biomedical and bioanalytical areas (Silva, Bezerra, & Farias, 2012). As well known, the presence of eNH2, eCH2OH and eCHO groups in chitosan, which associated with chitosan degree of deacetylation (DD) and molecular weight (MW), not only affect its antioxidant activity (Feng et al., 2008; Gue et al., 2008), but also its reducing and stabilizing characteristics which influence the size and shape of synthesized AuNPs (Esumi et al., 2003; Sun et al., 2017). Synthesis of AuNPs of desired size and shape is very important in the field of scientific research because small changes in the size or shape of AuNPs can have great effect on a variety of physical properties of the material (Bhumkar, Joshi, Sastry, & Pokharka, 2007; Chithrani, Ghazani, & Chan, 2006; Duy et al., 2013). Therefore, the effect of the DD and MW on the shape and size of synthesized AuNPs and the antioxidant activity of resultant AuNPs required further study. In the present study, AuNPs and Au colloids were synthesized using different concentration chitosan with different DD and MW. The synthesized AuNPs were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffractometer (XRD). The antioxidant activities of synthesized AuNPs and Au colloids were evaluated through in vitro assays, including hydroxyl free radical (OH%), 2,2′-azino-bia 2,2′-azino-bis (3-ethylben-zothiazoline-6-sulphonic acid) (ABTS%+), 1′1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), in comparison with which of chitosan with different DD and MW used as reducing agent and stabilizer. The purpose of this paper is to investigate the effect of AuNPs synthesis conditions, including concentration and nature of reducing agent and stabilizer, on shape and size of resultant AuNPs and the antioxidant activity of

2. Experimental 2.1. Materials Chitosan (CS) with a weight-average molecular weight (Mw) of 490.2 kDa and a degree of deacetylation (DD) of 95% was supplied from Shandong Aokang Biotechnology Co. (Shandong, China). Neutral protease was purchased from Shanghai Solarbio Bioscience &Technology Co., Ltd. (China). HAuCl4 was obtained from Aldrich Chemical Co. and used without further purification. 1′1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Macklin Biochemical Co., Ltd. (China) and 2,2′-azino-bis (3-ethylben-zothiazoline-6-sulphonic acid) (ABTS) was from Aladdln Industrial Corporation (Shanghai, China). Salicylic acid, ferric chloride (FeCl3), potassium ferricyanide (K3Fe(CN)6), potassium persulfate (K2S2O8) and trichloroacetic acid (TCA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All other chemicals were of analytical grade and were used without further purification. Deionized water was used in all experiments.

2.2. Preparation of N-acetylated and different MW chitosans N-acetylated chitosans with different DD were prepared under homogeneous condition. Chitosan CS (2 g) was dissolved in aqueous 2.8% (v/v) acetic acid (50 mL). The mixture of acetic anhydride, which molar ratio to CS was 2:5, and anhydrous ethanol (50 mL) was added gently into the chitosan solution with magnetic stirring. After stirring at ambient temperature for 1 h, the reaction mixture was adjusted to pH 9 with 5 M aqueous KOH solution to precipitate water-insoluble Nacetylated chitosan. The precipitates were washed with distilled water until neutralization and then rinsed with anhydrous ethanol. The precipitates were collected after drying over phosphorus pentoxide in vacuum to get NA1. As the molar ratio of acetic anhydride to CS changed as 3:5, water soluble N-acetylated chitosan was obtained. The reaction mixture was dialyzed against distilled water for 3 days after adjusted to pH 9 with 5 M aqueous KOH solution. The solution was concentrated to about one-twentieth of the original volume with a rotary evaporator under reduced pressure at 55 °C and precipitated by adding anhydrous ethanol, then dried over phosphorus pentoxide in vacuum to get NA2. Chitosans with different MW were obtained by enzyme hydrolysis (Li, Du, & Liang, 2007). Neutral protease, a kind of nonspecific enzyme, can hydrolyze chitosan efficiently and obtain LMWC easily by controlling the mass ratio of enzyme to substrate and reaction time. The molecular weight of chitosan decreased with the increasing of the mass ratio of enzyme to substrate and reaction time. Chitosan CS (4 g) was dissolved in 100 mL of 1% (v/v) acetic acid and then adjusted to pH 5.4 using 1 M KOH. The solution in the reaction vessel was placed in a thermostatic water bath at 50 °C, and 2 mL neutral protease solution with different concentration was added to initial reaction. The mass ratio of neutral protease to CS was 1.25%, 2.5% and 5% (w/w), respectively. After hydrolysis for 1, 2 and 3 h, the mixture was taken out and boiled for 10 min to remove the enzyme. Then the hydrolysates were adjusted to pH 9 with 5 M KOH solution to precipitate the LMWC and coded as DC1, DC2 and DC3, respectively. The molecular parameters of N-acetylated chitosan and low molecular weight chitosan were summarized in Table 1. As shown in Table 1, CS and its N-acetylation derivatives have similar MW, while DD decreased from CS to NA2. Chitosan CS and its hydrolysates have similar DD, while MW decreased from CS to DC3.

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RM: molar ratio of acetic anhydride to chitosan; E/S: mass ratio of neutral protease to chitosan CS.

Transmission electron microscopy (TEM) was executed by H-7650 instrument (Hitachi High-Technologies Corporation) at an accelerating voltage of 100 KeV. The TEM samples were obtained by dropping small amount of Au colloidal solution onto carbon-coated copper grid and evaporating the water at room temperature. Fourier transform infrared (FT-IR) spectra were recorded with KBr pellets on a Tensor 27 spectrophotometer (Bruker Analytical Instruments Pvt. Ltd., Germany). Thirty-two scans at a resolution of 4 cm−1 were averaged and referenced against air. X-ray diffraction patterns (XRD) of synthesized AuNPs were measured by a Bruker D8 Advance instrument (Bruker Analytical Instruments Pvt. Ltd., Germany) and used a CuKα target at 40 kV and 40 mA at 20 °C. The relative intensity was recorded in the scattering range (2θ) of 30–80°.

2.3. Green synthesis of AuNPs

2.5. Evaluation of antioxidant activity

Chitosans were dissolved in 1% (v/v) acetic acid to obtain homogeneous solution with concentration of 0.01%, 0.1% and 0.3% (w/v). HAuCl4 solution (1.0 mM, 1 mL) was added to the chitosan samples solution (3 mL), reacted in a thermostatic magnetic stirring apparatus at 70 °C for 120 min. All glassware was thoroughly cleared with freshly prepared solution (HNO3:HCl = 1:3, v/v) and rinsed extensively with deionized water. Au colloids synthesized by CS, NA1, NA2, DC1, DC2 and DC3 were coded as CS-G, NA1-G, NA2-G, DC1-G, DC2-G and DC3G, respectively. The synthesized AuNPs were collected by ultracentrifugation (Himac CS120GHXL, Hitachi-Koki, Tokyo, Japan) at 10,000 rmp for 10 min at 4 ± 2 °C. Particle pellets were re-dissolved in deionized water and similarly centrifuged twice to remove the excess amount of potentially unreduced gold ions and unbound chitosan, if any. The resultant AuNPs were re-dissolved in deionized water, and used for further characterization. The synthesized AuNPs by CS, NA1, NA2, DC1, DC2 and DC3 were coded as CSG, NA1G, NA2G, DC1G, DC2G and DC3G, respectively.

2.5.1. Hydroxyl radical scavenging assay The hydroxyl radical (OH%) scavenging activity of chitosan with different molecular parameters, synthesized AuNPs and Au colloids were determined according to the method described by Fan et al. (2008) with few modifications. OH% was generated using the Fenton reaction system. Briefly, chitosan solutions, synthesized AuNPs or Au colloids (3 mL) were mixed with ethanol solution of salicylic acid (9 mM, 1 mL), FeSO4 (9 mM, 1 mL) and H2O2 (9 mM, 1 mL). The mixtures were incubated for 1 h and diluted to the volume of 10 mL with distilled water, then followed by centrifugation at 5000 rpm for 4 min. All determinations were performed in triplicate. The absorbance of supernatant was measured at 510 nm against distilled water as control. The OH% scavenging activity was calculated by the following formula:

Table 1 Preparation conditions of N-acetylated chitosans and low molecular weight chitosans and their physicochemical characteristics. Sample

CS NA1 NA2 DC1 DC2 DC3

RM

– 2:5 3:5 – – –

Hydrolysis condition E/S (%, w/w)

Time (h)

– – – 1.25 2.5 5.0

– – – 1 2 3

DD (%)

Mw (kDa)

Mw/Mn

95 63 53 93 93 94

490.2 531.5 568.9 285.9 81.3 47.8

6.56 6.98 7.12 4.23 3.12 2.33

OH·

2.5.2. ABTS radical scavenging assay The ABTS%+ scavenging activity of chitosan, synthesized AuNPs and Au colloids were evaluated using the method described by Duy et al. (2013) with slight modification. Briefly, 2 mL of 7.4 mM ABTS solution was mixed with K2S2O8 solution (2.6 mM, 2 mL) to generate free radical cation ABTS%+. The mixture was kept in the dark for 16 h at 30 °C to obtain a stable oxidative. ABTS%+ solution was diluted with sodium phosphate buffer (0.1 M, pH 7.4) to obtain absorbance of about 0.7 at 734 nm. For studying antioxidant activity of chitosan, synthesized AuNPs and Au colloids, 1.2 mL of samples at different concentrations were added into 2 mL ABTS%+ solution and reacted for 1 h in a dark condition, while distilled water was added as control. All determinations were performed in triplicate. The absorbance was measured over time at 734 nm. Efficiency of free radicals capture was calculated as follows:

The DD of chitosan was determined by elemental analysis (EA) (Xu, McCarthy, Gross, & Kaplan, 1996) which was performed using a Vario EL cube elemental analyzer (Elementar Analysensysteme GmbH, Germany), and the DD can be calculated as follow:

⎛ WC − 5.14⎞/1.72⎤ × 100% ⎥ ⎝ WN ⎠ ⎦

⎜

A 0 − A1 − A2 × 100 A0

where A0 is the absorbance of control, A1 is the absorbance of samples and A2 is the absorbance of sample blank, respectively.

2.4. Characterization

DD= ⎡1 − ⎢ ⎣

scavenging activity (%) =

⎟

where WC/WN is the ratio (w/w) of carbon to nitrogen. Weight-average molecular weight (Mw) and molecular weight distribution (Mw/Mn) of chitosan samples were measured by an Agilent 1260 size exclusion chromatography (SEC) equipped with TSK G5000PWXL (7.5 mm × 300 mm) combined with TSK G3000-PWXL (7.8 mm × 300 mm) columns and a refractive index detector. The eluent was 0.2 mol/L CH3COOH/0.1 mol/L CH3COONa aqueous solutions. Eluent and chitosan sample solutions were filtered through 0.45 μm Millipore filters. The flow rate was maintained at 1.0 mL/min. The column temperature was maintained at 30 °C. Dextran standards were used for a calibration curve. All data provided by the SEC system were collected and analyzed using JiangShen Workstation software package (Dalian, China). And the Mw was calculated by the following equation:

ABTS·+

scavenging activity

(%) =

A 0 − A1 − A2 × 100 A0

where A0 is the absorbance of control, A1 is the absorbance of samples and A2 is the absorbance of sample blank, respectively. 2.5.3. DPPH radical scavenging assay The DPPH radical scavenging activity of chitosan, synthesized AuNPs and Au colloids were measured based on the method reported by Muthuvel, Adavallan, Balamurugan, and Krishnakumar (2014) with minor modifications. Briefly, 2 mL of chitosan solution, synthesized AuNPs or Au colloids were mixed with ethyl alcohol-DPPH solution (0.1 M, 2 mL). After incubating in the dark at 25 °C for 30 min under shaking condition, the absorbance was measured at 517 nm. The distilled water was used as positive control and all determinations were

1ogMW = −0.4322Ve + 10.4203 UV–vis spectra of Au colloids were recorded by using a spectrophotometer (TU-1810 UV–vis, Purkinje General Instrument Co., Ltd. Beijing, China), which has a spectral range of 400–1100 nm. All spectra were measured in a 1 cm path length quartz cuvette and deionized water was used as a reference. 163

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used as reducing agent and stabilizer, respectively. It’s maybe due to the decrease of the amount of the protonation of eNH2 groups as well as the reducing of polyelectrolyte charge density with the decrease of chitosan DD, the larger size of AuNPs were more easily formed. The intensity of SPR, which is directly proportional to the density of AuNPs in solution, gradually increased with the decrease of chitosan DD. For four kinds of chitosans with different MW, i.e., CS, DC1, DC2 and DC3, the peak position of SPR slightly shifted towards lower wavelength. The average particle size of AuNPs was measured to be 10.01 ± 4.37, 11.03 ± 5.06 and 11.74 ± 5.49 nm, respectively, as DC1, DC2 and DC3 used according to Fig. 1(a4)–(a6). The results indicated that MW has little influence on the shape and size of synthesized AuNPs and spherical AuNPs with similar particle size were obtained as low concentration (0.01%, w/v) chitosans used as reducing agent and stabilizer, which are in accordance with the results in the previous literatures (Manivasagan, Bharathiraja, Bui, Lim, & Oh, 2016; Sun et al., 2019). As chitosan concentration increased to 0.1% (w/v), the UV–vis spectra of synthesized Au colloids using chitosan with different DD and MW as reducing agent and stabilizer were quite different as shown in Fig. 1(B). The UV–vis spectrum of Au colloids synthesized using CS showed a single broad adsorption band at 551 nm, which indicated that the AuNPs size distribution became wider and the shape changed to irregular morphology (Potara, Maniu, & Astilean, 2009). As confirmed in Fig. 1(b1), the shape of synthesized AuNPs changed to near-spherical or irregular AuNPs. As NA1 used as reducing agent and stabilizer, the UV–vis spectrum of synthesized Au colloids showed three adsorption bands at 540, 615 and 934 nm, which is attributed to spherical, anisotropic or non-spherical AuNPs, and the in-plane dipole resonance of the Au triangular nanoplates (Kan, Zhu, & Wang, 2006), respectively. As NA2 used as reducing agent and stabilizer, the UV–vis spectrum of synthesized Au colloids showed the two nearly overlapped absorption bands at 540 and 580 nm and another in-plane dipole resonance absorption band at 1019 nm. The in-plane dipole resonance absorption is sensitive to the edge length of the triangular Au nanoplates. It shifted to longer wavelength indicated that the edge lengths of synthesized triangular nanoplates increased with decreasing chitosan DD, which confirmed by Fig. 1(b2) and (b3). For three LMWC, i.e., DC1, DC2 and DC3, a broad absorption band attributed to the overlap of the transverse peak of spherical AuNPs at about 545 nm with longitudinal peak around 650 nm, respectively, due to the anisotropic or non-spherical structures of AuNPs were obtained as confirmed by Fig. 1(b4)–(b6). Fig. 1(C) shows the UV–vis spectra of Au colloids synthesized using 0.3% (w/v) chitosan as reducing agent and stabilizer. A broad SPR appeared at 544 as CS used as reducing agent and stabilizer, which indicated that the AuNPs size distribution became wider and nearspherical or irregular morphology AuNPs obtained (Potara et al., 2009). The maximum absorption wavelength of SPR blue shifted from 551 to 544 nm when CS concentration increased from 0.1% (w/v) to 0.3% (w/ v) may be due to the entanglement or aggregation of CS molecular chains caused by the strong inter-molecules hydrogen bonds forming at the higher concentration. The entangled CS molecules prevent the growth of AuNPs by covering fully on their surface and result in the slight decrement of particle size. As NA1 used as reducing agent and stabilizer, a single and wide SPR at 551 nm appeared, which indicated that AuNPs with wide size distribution and irregular morphology were obtained (Potara et al., 2009). As NA2 used as reducing agent and stabilizer, the UV–vis spectrum showed two absorption bands at 534 and 679 nm, respectively, indicated that larger special AuNPs and hexagonal AuNPs obtained (Hong, Shuford, & Park, 2011). It could be seen clearly from Fig. 1(c1)–(c3) that near-spherical or anisotropy AuNPs such as pentagonal, hexagonal and some other irregular shapes appeared. And the lower chitosan DD, the growth of anisotropy AuNPs was promoted. As LMWC (DC1, DC2 and DC3) used as reducing agent and stabilizer, the maximum absorption wavelength appeared around 540 nm and slightly decreased with the decrease of chitosan MW,

performed in triplicate. The DPPH radical scavenging activity was expressed in percentage using the following formula:

DPPH radical scavenging activity (%) =

A 0 − A1 − A2 × 100 A0

where A0 is the absorbance of control, A1 is the absorbance of samples and A2 is the absorbance of sample blank, respectively. 2.5.4. Ferric reducing antioxidant power (FRAP) assay The FRAP was determined through a method of Wanvimol Pasanphan with slight modification (Pasanphan et al., 2015; Zhang, Geng, Jiang, Li, & Huang, 2012). In this assay, the presence of antioxidants brought about reduction of Fe3+ to Fe2+ by donating an electron. In brief, 2 mL different concentration of chitosan, synthetized AuNPs and Au colloids were added to phosphate buffer (2.5 mL, pH 6.6) and K3Fe(CN)6 solution (0.1% (w/v), 2.5 mL). The mixtures were kept for 20 min at 50 °C, then TCA (10% (w/v), 2.5 mL) was added to the mixtures, followed by centrifugation at 5000 rpm for 10 min. 2.5 mL of the supernatant was mixed with distilled water (2.5 mL) and FeCl3 (0.1% (w/v), 0.5 mL). After reacting for 30 min at room temperature, the absorbance was measure at 700 nm. The higher the absorbance indicated the stronger reducing power. The distilled water was used as positive control. All determinations were performed in triplicate. 2.6. Statistical analysis All experiments of antioxidant activity were conducted in triplicate and expressed as mean ± standard deviation (SD). Statistical analysis was performed using analysis of variance (ANOVA). Statistically significant differences between groups were defined as p < 0.05. 3. Results and discussion 3.1. Influence of chitosan DD and MW on the synthesis of AuNPs DD and MW are two important characteristics of chitosan. The changes of chitosan DD and MW not only decide the amount of eNH2, eCH2OH and eCHO groups in chitosan, but also more or less effect the hydrogen bonds, electrostatic interaction and steric interaction, which have been proved to be associated with the reducing and stabilizing activity of chitosan in the synthesis of AuNPs (Sun et al., 2017). In order to investigate the effect chitosan DD and MW on the properties of the synthesized AuNPs, three chitosan with DD from 53% to 95% (with similar Mw around 500 kDa) and four chitosan with Mw from 490.2 to 47.8 kDa (with similar DD around 94%), described in Table 1, were used to synthesize AuNPs. Fig. 1 shows the UV–vis spectra and TEM images of Au colloids synthesized with the chitosan with different molecular parameters and at different concentration. The peak position, spectral bandwidth, and intensity of the surface plasmon resonance (SPR) are attributed to AuNPs size, shape and productivity (Jain, Lee, El-Sayed, & El-Sayed, 2006; Lee & El-Sayed, 2006; Mizutani et al., 2015). It is observed that SPR of Au colloids vary with varying chitosan concentration and molecular parameters, which are responsible for the size and shape change of synthesized AuNPs (Cai, Gao, Hong, & Sun, 2008). As shown in Fig. 1(A), a narrow and symmetric SPR appeared as 0.01% (w/v) chitosan used as reducing agent and stabilizer regardless of its DD and MW, indicated that the synthesized AuNPs were individual spherical and had a uniform size distribution (Njoki et al., 2007). For three kinds of different DD chitosans, i.e., CS, NA1 and NA2, the maximum absorption wavelength of Au colloids red shifted from 525 to 528 and 539 nm as chitosan DD decreased, which indicated that the average particle size of AuNPs certainly increased with the decrease of chitosan DD. These results are supported by TEM observations as shown in Fig. 1(a1)–(a3). The average particle size of AuNPs was 7.84 ± 2.53, 8.36 ± 2.66 and 29.33 ± 9.56 nm as CS, NA1 and NA2 164

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Fig. 1. UV–vis spectra of Au colloids synthesized using different molecular parameters chitosans at different concentrations. (A) 0.01% (w/v), (B) 0.1% (w/v) and (C) 0.3% (w/v). TEM image of Au colloids synthesized by different molecular parameters chitosans at (a) 0.01% (w/v), (b) 0.1% (w/v) and (c) 0.3% (w/v). (1) CS, (2) NA1, (3) NA2, (4) DC1, (5) DC2, (6) DC3.

groups, indicating the association of the hydrogen bonds between them. The CS spectrum shows three characteristic peaks at 1652.1, 1601.3 and 1318.9 cm−1 arising from the stretching vibration of eC]O of Nacetyl group in amide I, bending of free eNH2, and stretching vibration of CeN and bending vibration of NeH in amide III, respectively (Pereda, Aranguren, & Marcovich, 2008). The FT-IR spectra of LMWC, i.e., DC1, DC2 and DC3, are similar with that of CS, but the stretching vibration of eNH and eOH groups shift to higher frequency as the molecular weight of chitosan decrease, verifying an increase in the ordered structure (Focher, Naggi, Toci, Cosani, & Terbojevich, 1992) and the disruption of the hydrogen bonds (Chen & Du, 2002; Krishnan et al., 2016). For N-acetylated chitosan, i.e., NA1 and NA2, the adsorption bands at ˜1640 and 1562 cm−1 are considered as the contribution of amide I and amide II band, respectively, indicating the DD

indicated the effect of chitosan MW on the size and shape of synthesized AuNPs was very limited in the investigated chitosan MW range. As shown in Fig. 1(c4)–(c6), anisotropic AuNPs disappeared and small spherical occurred.

3.2. FT-IR analysis To confirm the specific interaction of chitosan functional groups with the AuNPs surface, FT-IR spectra of chitosan and AuNPs synthesized by 0.1% (w/v) chitosan with different molecular parameters were collected. Fig. 2(a) shows the FT-IR spectra of chitosan with different molecular parameters. The spectrum of CS shows a broad absorption peak spread over the spectral range of 3000–3670 cm−1, which could be interpreted as the overlapped stretching vibrations of eOH and eNH 165

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Fig. 2. FT-IR spectra of (a) chitosans with different molecular parameters and (b) the synthesized gold nanoparticles.

Fig. 3. XRD pattern of the synthesized gold nanoparticles using 0.1% (w/v) chitosan with different molecular parameters as reducing agent and stabilizer.

For AuNPs synthesized with NA1 and NA2, the shift of peak at ˜1640 cm−1 (stretching vibration of eC]O of N-acetyl group in amide I) to higher frequency side ˜1646 cm−1 indicated the coupling of AuNPs with eC]O group of chitosan. The shifting of this peak occurred due to attachment of heavy gold atom with O and N atoms of chitosan that caused an increase in band length with ultimate red frequency shift (Shah, Hussain, & Murtaza, 2018; Sherif, Khalil, Hegazi, Khalil, & Moharram, 2017). A clearly visible band appears at ˜1552 cm−1 attributed to amide II, which shifted from ˜1562 cm−1 to lower frequency side indicating the attachment of nitrogen atoms with AuNPs (Brugnerotto et al., 2001; Wei & Qian, 2008). These results indicated that a chemical bond between the gold atom and nitro and oxygen atom of eNH2, eOH and eC]O groups in chitosan has been formed. By this way, the chitosan molecules are strongly adsorbed on the surface of AuNPs, preventing the AuNPs from agglomeration. The FT-IR results confirm the chitosan as reducing agent and stabilizer plays an important role in the formation and growth of the AuNPs (Cheng, Hung, Chen, Liu, & Young, 2014).

of chitosan decrease (Kong, 2012). As shown in Fig. 2(b), the FT-IR spectrum of the synthesized AuNPs using CS as reducing agent and stabilizer shows significant changes in the 1800–1300 cm−1 region. The eNH vibration band at 1601.3 cm−1 in CS spectrum shifts to 1595.1 cm−1 accompanied by a gradual decrease in intensity and a new band appears at 1532.1 cm−1, which suggests the attachment of gold to nitrogen atoms of eNH2 (Brugnerotto et al., 2001; Wei & Qian, 2008). In addition to this, the absorption band at 1425.6 cm−1 in CS attributed to eOH groups bending vibration disappeared in the AuNPs, indicating that eOH groups participates in the chelation (Corma, Concepción, Domínguez, Forné, & Sabater, 2007). The broad absorption band at 3445.6 cm−1 in CS shift to lower frequency, also indicating that eNH2 or/and eOH were involved in the reduce and stabilization of AuNPs (CárdenasTriviňo & Cruzat-Contreras, 2018). AuNPs synthesized by DC1 shows the similar FT-IR spectrum with which synthesized by CS. The spectra of AuNPs synthesized by DC2 and DC3, compared with AuNPs synthesized by CS, the absorption band at 1601.3 cm−1 disappeared and the amide I at 1652.7 cm−1 shifted to lower frequency and appeared at 1642.9 and 1637.1 cm−1, respectively. The results indicate that the LMWC (DC2 and DC3) molecules coordinated with AuNPs through not only the eOH and eNH2, but the oxygen atom of the eC]O group in chitosan structure (Eisa, Abdelnaby, Mostafa, & Elzayat, 2018). LMWC has weaker inter- and intra-molecular hydrogen bonds and the molecular chain is stiff and extended, which resulted the reductive groups in the chitosan chains could have more chance to interact with Au(III).

3.3. XRD analysis The crystalline structures of synthesized AuNPs are confirmed from XRD analysis. The XRD pattern (Fig. 3) of AuNPs synthesized with CS (0.1%, w/v) as reducing agent and stabilizer show the characteristic diffraction peaks at 38.27°, 44.50°, 64.64° and 77.59° corresponding to (111), (200), (220) and (311) Bragg’s reflection are in good agreement with the face-centered-cubic (FCC) structure of AuNPs (JCPDS file no: 166

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O3eO5 and N2eO6. Higher molecular weight chitosan (HMWC) has compact structures due to the stronger hydrogen bonds, which weakens the activity of the hydroxyl and amino groups. In contrast, LMWC has a less-compact structure, so the effect of the intramolecular hydrogen bonds is weak and the more free eOH, eNH2 and eNH3+ can react with OH% radicals (Sun, Zhou, Xie, & Mao, 2007; Xing et al., 2005). For six chitosan samples, in the investigated concentration range, the OH% scavenging activity increased with the increase of chitosan concentration regardless of its DD and MW, which may be due to the increase of chitosan concentration resulted in the increase of hydroxyl and amine groups. AuNPs exist primarily in two oxidation states, i.e., Au(I) and Au(III), which might be considered as the primary cause behind the antioxidant potential of the d-block element (Vilas, Philip, & Mathew, 2016). So based on reaction conditions, AuNPs can accept or donate electrons in order to form stable compounds and to hence can scavenge free radicals (Ramamurthy et al., 2013). The OH% scavenging activities of synthesized AuNPs are shown in Fig. 4(b). In the case of the three investigated chitosan concentration, the AuNPs synthesized by 0.1% (w/v) chitosan showed the lowest OH% scavenging activity regardless its DD and MW. As shown in Fig. 1(b), irregular or polygonal AuNPs, such as triangular, rectangular and pentagonal, were synthesized using 0.1% (w/v) chitosan with different DD and MW as reducing agent and stabilizer. For the antioxidant activity, literature data available on the interaction of free radicals with AuNPs suggested the adsorption of free radicals on the AuNPs surface (Ionita, Spafiu, & Ghica, 2008) and the possibility of exchange interaction between the unpaired electron from the free radicals and the conduction-band electrons of AuNPs (Zhang, Berg, Levanon, Fessenden, & Meisel, 2003). The efficiency of the interaction of the AuNPs with the free radicals depends on the size, specific surface, and concentration of the particles (Mukherjee, Nethi, & Patra, 2017). It is widely accepted that high surface area AuNPs plays a vital role in scavenging the free radicals because spherical AuNPs in shape with larger surface area which can easily accept electrons from free radical. The results shown in Fig. 4(b) also proved there are some relationship between the OH% scavenging activity and the shape of synthesized AuNPs. Spherical AuNPs showed the higher OH% scavenging activity than irregular or polygonal ones, which may be attributed to the fact that the spherical AuNPs have the higher specific surface area (Yakimovich et al., 2008). Compared with the OH% scavenging activity of AuNPs synthesized by 0.01% and 0.3% (w/v) chitosan, except which synthesized by NA2, AuNPs synethsized by 0.3% (w/v) chitosan showed the higher scavenging activity. Because AuNPs synthesized by 0.01% and 0.3% (w/v) chitosan (CS, NA1, DC1, DC2 and DC3) are all spherical or near-spherical, the difference of the OH% scavenging activity may be due to the amount of AuNPs. In the case of NA2 used as reducing agent and stabilizer, AuNPs synthesized by 0.01% (w/v) NA2 showed the higher OH% scavenging activity than which synthesized by 0.3% (w/v) NA2, which may be also due to the shape of synthesized

04-0784) (Kongor et al., 2018). Apart from the Bragg peaks, the presence of additional peaks signifies the crystallization of reducing agent and stabilizer on the surface of the AuNPs (Jadhav et al., 2018). The characteristic diffraction peaks of AuNPs synthesized with different molecular parameters chitosan show no significant shift. It is noticed that the peak corresponding to the (111) plane is more intense than that of other planes suggesting the predominant growth of AuNPs along (111) directions (Kumar, Smitaa, Debut, & Cumbal, 2018). Generally, the breadth of a specific phase of material is directly proportional to the mean crystallite size of that material. The broader peaks indicating the crystallite size is small (Debnath et al., 2016). The results indicate that the crystallite size of synthesized AuNPs increases with the decreasing of chitosan DD and MW, which is in agreement with the observed TEM results as shown in Fig. 1(b). 3.4. Antioxidant activity It is known that the activity of scavenging free radical is of great importance, for the deleterious role of free radicals in biological systems as well as in food (Bursal & Köksal, 2011). Scavenging activity of OH%, ABTS, DPPH and FRAP were selected to evaluate the antioxidant activity of synthesized Au colloids and AuNPs compared with chitosan. 3.4.1. OH% scavenging activity OH% radicals are one of the most reactive oxygen species and can result in cell membrane disintegration and membrane protein damage (Korycka-Dahl & Richardson, 1978). OH% radical scavengers could serve as better alternatives for the protection of these tissue damages. The OH% scavenging activity of chitosan, AuNPs and Au colloids are shown in Fig. 4. Xie, Xu, and Liu (2001) reported that the OH% scavenging activity of chitosan may be derived from some or all of the following: (a) the eOH in the chitosan unit can react with OH% by the typical Habstraction reaction; (b) OH% can react with the residual free eNH2 to form stable macromolecule radicals; (c) the eNH2 can form eNH3+ by absorbing hydrion from the solution, then reacting with OH% through addition reaction. As shown in Fig. 4(a), the OH% scavenging activity of chitosan decreased with the decrease of chitosan DD. Chitosan CS which has the highest DD exhibited the highest OH% radicals scavenging activity. The results also demonstrated the important role of chitosan eNH2 and eNH3+ in the OH% scavenging activity, because the higher chitosan DD, the more active eNH2 and eNH3+ revealed at the C-2 position and the higher OH% scavenging activity obtained. Chitosan CS and its hydrolysates DC1 and DC2 showed similar OH% scavenging activity in each investigated concentration. DC3, which has the lowest MW, showed the highest OH% radicals scavenging activity (20.3 ± 1.9%) at concentration 0.3% (w/v) (n = 3, p < 0.01). The difference OH% scavenging activity of chitosan with different MW may be caused by intramolecular hydrogen bonds (Kim & Thomas, 2007; Tomida et al., 2009). Chitosan has many hydrogen bonds between

Fig. 4. Hydroxyl scavenging activity of (a) chitosan, (b) synthesized AuNPs and (c) Au colloids. Values were obtained from three independent experiments. * p < 0.05, **p < 0.01, ***p < 0.001 when compared to CS in (a) and CSG in (b), respectively. #p < 0.05, ##p < 0.01, ###p < 0.001 when compared to (a). 167

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In additional, it was reported that the eOH in chitosan also played an important role in ABTS%+ scavenging activity (Shang et al., 2018). The hydrogen bonds formed between inter- or intra-molecules of the chitosan could inhibit the reaction of ABTS%+ with the active eOH (Huang et al., 2015). The ABTS radical cation might be bleached by H-atom or the electron transfer mechanism from the functional eOH of chitosan (Li, Wang, Wang, & Xiong, 2016). LMWC showed the improved ABTS%+ scavenging ability may be due to the less-compact structure and the weakened inter- and intra-molecular hydrogen bonds. Fig. 5(b) showed the ABTS%+ scavenging activity of AuNPs synthesized by chitosan with different DD and MW at different concentration. It is obvious that anisotropic AuNPs synthesized by 0.1% (w/v) chitosan, regardless of its DD and MW, exhibited the lowest ABTS%+ scavenging activity which was similar with OH% scavenging activity. AuNPs synthesized by 0.01% (w/v) NA2 and LMWC (i.e. DC1, DC2 and DC3) showed the highest ABTS%+ scavenging activity in the investigated chitosan concentration, which may be due to the spherical AuNPs with the average particle size around 10 nm showed the higher specific surface area to react with ABTS%+ radicals. The results also indicated that the size and shape of synthesized AuNPs played more important role than its amount on the ABTS%+ scavenging activity. In the case of AuNPs synthesized by CS and NA1, AuNPs synthesized by 0.3% (w/v) CS and NA1 showed the higher ABTS%+ scavenging activity compared with which synthesized by 0.01% (w/v) CS and NA1. Because spherical AuNPs were obtained as 0.01% and 0.3% (w/v) CS and NA1 used as reducing agent and stabilizer, the difference of ABTS%+ scavenging activity may be due to the amount of AuNPs. Although the exact chemistry behind the scavenging activity of AuNPs against ABTS%+ still needs further investigation (Dauthal & Mukhopadhyay, 2018), AuNPs interact and scavenge ABTS%+ may be due to ambient electrostatic field and large surface area as described in pervious literatures (Das, Nath, Phukon, Kalita, & Dolui, 2013; Niraimathi, Sudha, Lavanya, & Brindha, 2013; Shanmugasundarama, Radhakrishnan, Gopikrishnana, Pazhanimurugana, & Balagurunathana, 2013). The ABTS%+ scavenging activity of Au colloids was shown in Fig. 5(c). The ABTS%+ scavenging activity increased with the increasing concentration of chitosan used as reducing agent and stabilizer. Au colloids synthesized by 0.3% (w/v) DC3 showed the highest ABTS%+ scavenging activity, which was reached 77.3 ± 1.9%. In the case of chitosan DD and MW, Au colloids synthesized by higher DD and lower MW chitosan exhibited the higher ABTS%+ scavenging activity, which showed the similar trends with the OH% scavenging activity as shown in Fig. 4(c). It is also observed that the scavenging activity higher in ABTS%+ assay as compared to OH% assay, which might be possible due to the difference in sensitivity of ABTS%+ and OH%. For Au colloids synthesized by CS and its N-acetylated derivatives (i.e. NA1 and NA2), the ABTS%+ scavenging activity were improved compared with chitosan at all three investigated chitosan concentration (n = 3, p < 0.05). For Au colloids synthesized by LMWC, compared with LMWC, the ABTS%+ scavenging activity of Au colloids synthesized

AuNPs. The OH% scavenging activity of Au colloids was enhanced compared with that of original chitosan and AuNPs as shown in Fig. 4(c). As 0.3% (w/v) DC3 used as reducing agent and stabilizer, synthesized Au colloids showed the highest OH% radicals scavenging activity as 64.3 ± 2.8%. OH% scavenging activity of synthesized Au colloids increased with the increasing of chitosan concentration regardless of its DD and MW, which showed the similar varying tendency of OH% scavenging activity as chitosan. It is well reported that antioxidants in the presence of AuNPs show enhanced scavenging as compare to the antioxidant alone (Nie et al., 2007; Razzaq et al., 2016). The OH% scavenging activity of Au colloids compared to chitosan significantly increased (n = 3, p < 0.05), which may be due to the synergistic effect of chitosan remained in the colloids and the AuNPs coated by chitosan (Yakimovich et al., 2008). Colloidal forms of AuNPs are particularly efficient antioxidant may be due to the high ratio of electrons remaining at the surface, and thus available to scavenging free radicals (Roucoux, Schulz, & Patin, 2002). 3.4.2. ABTS%+ scavenging activity Free radicals in the body were mainly divided into two categories, physiological free radical and non-physiological radical. ABTS belongs to non-physiological free radical, was produced by chemical pollution, tobacco smoke, pesticide residues, which could fast trigger the chain reaction of free radicals and seriously damage the body’s biological components, such as protein, DNA and lipids. Total antioxidant capacity was evaluated according to the ABTS assay. The free radical cation ABTS%+ generated from the oxidation of ABTS by potassium persulfate is an efficacious tool for determining the antioxidant activity of the chain breaking and hydrogen-donating antioxidants (Chen, Yue, Jiang, Liu, & Xia, 2018; Leong & Shui, 2002). Fig. 5 shows the ABTS%+ scavenging activity of chitosan, AuNPs and Au colloids. As shown in Fig. 5(a), ABTS%+ scavenging activity of chitosan increased with the increase of chitosan concentration regardless of its DD or MW. DC3, which with the lowest MW, showed the highest ABTS%+ scavenging activity as 75.1 ± 2.0%. At lower chitosan concentration (0.01% (w/ v)), the effect of chitosan DD and MW on the ABTS%+ scavenging activity were very limited. At higher chitosan concentration (0.1% and 0.3% (w/v)), ABTS%+ scavenging activity decreased significantly with the decreasing of chitosan DD but improved with the decreasing of chitosan MW (n = 3, p < 0.05). The ABTS%+ scavenging activity of chitosan may be explained by various mechanisms. One of them is chitosan eliminates ABTS%+ by the action of the nitrogen in the eNH2 (García et al., 2015). Xie et al. (2001) also reported that scavenging activity is related with the fact that the ABTS%+ can react with the H+ from the eNH3+ of chitosan. In general, these results are agree with these obtained in the present study, where the chitosan with the higher DD showed the higher ABTS%+ scavenging activity, and demonstrated the eNH2 and eNH3+ existed in chitosan played the important role in the ABTS%+ scavenging activity.

Fig. 5. ABTS%+ scavenging activity of (a) chitosan, (b) synthesized AuNPs and (c) Au colloids. Values were obtained from three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001 when compared to CS in (a) and CSG in (b), respectively. #p < 0.05, ##p < 0.01, ###p < 0.001 when compared to (a). 168

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chitosan concentration used as reducing agent and stabilizer. The DPPH scavenging activity of Au colloids especially synthesized by 0.1% (w/v) chitosan improved compared with AuNPs, which may due to the synergistic effects of AuNPs and chitosan remained in the colloids. Compared with chitosan, Au colloids synthesized by CS and its Nacetylated derivatives (NA1 and NA2) showed the significant improvement of DPPH scavenging activity at all three investigated chitosan concentration (n = 3, p < 0.05). DPPH scavenging efficiency of Au colloids synthesized by LMWC, especially by DC2 and DC3, improved very limited compared with chitosan. The results may be pointed out that the synergistic effects between AuNPs and higher MW chitosan played a more important role on the DPPH scavenging activity of Au colloids.

by 0.01% (w/v) chitosan improved significantly (n = 3, p < 0.01), which may be due to the synergistic effect of existed AuNPs and chitosan in colloids that not participate in reducing and stabilizing of AuNPs. However, the ABTS%+ scavenging activity of Au colloids synthesized by higher concentration chitosan (0.1% and 0.3% (w/v)) improved very limited, which may be due to the synthesized AuNPs are covered fully with chitosan molecules as the higher concentration of chitosan used as reducing agent and stabilizer (Esumi et al., 2003). 3.4.3. DPPH scavenging activity DPPH is a stable free radical and has the ability to accept electrons or hydrogen radical to become a stable diamagnetic molecule. DPPH is a stable nitrogen-centered and lipophilic-free radical that has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants (Nakkala, Mata, & Sadras, 2016; Patra & Beak, 2015). The scavenging mechanism of DPPH is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen donating antioxidant due to the formation of the nonradical form DPPH-H by the reaction (Wan, Xu, Sun, & Li, 2013). In the study, DPPH was used to determine the proton-scavenging activity of the chitosan and synthesized AuNPs and Au colloids. The DPPH scavenging activity of six different molecular parameters chitosan are shown Fig. 6(a). It’s obvious that chitosan with different molecular parameters exhibited similar dose dependent and reached the highest scavenging activity at 0.3% (w/v). The DPPH scavenging activity increased with the increasing of chitosan DD and decreasing of chitosan MW, which is similar with the % OH and ABTS%+ scavenging activity and indicated that the active eOH and eNH2 in the chitosan chains play important role in the DPPH scavenging. Chitosan CS has compact structure in solution, whose intraand inter-molecular hydrogen bonds are stronger than that of LMWC. The strong effect of hydrogen bonds weakens the activities of the hydroxyl and amino (Li, Xu, Chen, & Wan, 2014). The DPPH scavenging activities of AuNPs are shown in Fig. 6(b). AuNPs synthesized by 0.1% (w/v) chitosan, regardless of its DD and MW, exhibited the lowest DPPH scavenging activity. The anisotropic or polygonal AuNPs with larger sizes have lower specific surface area and harder to react with DPPH radicals. AuNPs synthesized by 0.3% (w/v) chitosan showed the highest DPPH scavenging activity, which may be due to the higher amount of synthesized AuNPs. As reported by Razzaq et al. (2016), DPPH could adsorb on AuNPs via N-12 atom owing to its greater charge density and non-planar nature of the phenyl groups. The presence of unpaired electron on the nitrogen atom of DPPH might result in the surface adsorption of DPPH on AuNPs since gold has higher affinity for the nitrogen as compared to oxygen. And then the adsorbed DPPH radicals on the AuNPs are capable for abstracting the radical hydrogen atoms from the hydrated surface of AuNPs to convert to DPPH-H molecules. Fig. 6(c) shows the DPPH scavenging activity of Au colloids synthesized by chitosan with different DD, MW and concentration. The DPPH scavenging activity of Au colloids increased with the increase of

3.4.4. Antioxidant capacity A direct correlation between the antioxidant and reducing capacity has been reported (Benzie & Strain, 1996). The capacity of antioxidants to donate electrons to free radicals by reducing ferric iron (Fe3+) to ferrous iron (Fe2+) were assessed using FRAP assays (Shimada, Fujikawa, Yahara, & Nakamura, 1992). Fig. 7 shows the FRAP of chitosan, AuNPs and Au colloids. As shown in Fig. 7(a), the FRAP of CS and LMWC increased with the increase of chitosan concentration. However, for NA1 and NA2, the effect of chitosan concentration on the FRAP was very limited. In the case of chitosan DD and MW, at the same chitosan concentration, chitosan with higher DD or lower MW showed the higher FRAP. The highest absorbance was 0.202 ± 0.003 for DC3 at 0.3% (w/ v). The FRAP of synthesized AuNPs are shown in Fig. 7(b). For CS and its N-acetylated derivatives (NA1 and NA2), there was little significant difference between the FRAP of chitosan and synthesized AuNPs. For AuNPs synthesized by LMWC, irregular or polygonal AuNPs obtained as 0.1% (w/v) LMWC showed the lowest FRAP. It also demonstrated that shape of AuNPs have more or less impact on FRAP and spherical AuNPs showed the greater FRAP than the other shapes. The FRAP of Au colloids was shown in Fig. 7(c). For synthesized Au colloids using 0.01% and 0.1% (w/v) CS, NA1 and NA2 as reducing agent and stabilizer, the effect of chitosan concentration and DD on the FRAP of synthesized Au colloids are very limited. As 0.3% (w/v) CS, NA1 and NA2 used as reducing agent and stabilizer, the FRAP of synthesized Au colloids decreased with the decreasing of chitosan DD. And there was no significant difference between the FRAP of chitosan and synthesized Au colloids. The FRAP of Au colloids synthesized by LMWC increased with the increase of chitosan concentration and the decrease of chitosan MW. Au colloids synthesized by 0.3% (w/v) DC3 exhibited the highest FRAP as 0.266 ± 0.007. 4. Conclusion Green synthesis of AuNPs is an environment-friendly and economically viable method. In the present study, AuNPs were synthesized in a

Fig. 6. DPPH scavenging activity of (a) chitosan, (b) synthesized AuNPs and (c) Au colloids. Values were obtained from three independent experiments. *p < 0.05, ** p < 0.01, ***p < 0.001 when compared to CS in (a) and CSG in (b), respectively. #p < 0.05, ##p < 0.01, ###p < 0.001 when compared to (a). 169

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Fig. 7. Ferric reducing antioxidant power (FRAP) of (a) chitosan, (b) synthesized AuNPs and (c) Au colloids. Values were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to CS in (a) and CSG in (b), respectively. #p < 0.05, ##p < 0.01, ###p < 0.001 when compared to (a).

green and convenient method in chitosan solution. The UV–vis spectra and TEM images revealed that the size, shape and productivity of synthesized AuNPs depended on the chitosan DD, MW and concentration. At the concentration of 0.1% (w/v), irregular or polygonal AuNPs obtained as all investigated chitosan samples used as reducing agent and stabilizer. The antioxidant activity of chitosan evaluated through four kinds of different in vitro assays increased with the increasing of chitosan concentration, chitosan DD and the decreasing of chitosan MW. The antioxidant activities of AuNPs were dependent on its size, shape and amount. Irregular or polygonal AuNPs, obtained using 0.1% (w/v) chitosan as reducing agent and stabilizer, showed the lowest antioxidant activity. Au colloids showed the more or less improved antioxidant activity compared with chitosan and synthesized AuNPs, which may due to synergistic effects of AuNPs and chitosan remained in the colloids that not participated in reducing or stabilizing AuNPs. Chitosan, synthesized AuNPs and Au colloids all showed different extent antioxidant activity in OH%, ABTS%+, DPPH and FRAP assays. Due to the compatibility of chitosan and unique characteristics of AuNPs, the Au colloids synthesized by green chemistry can be potentially applied in biomedicines, cosmetics and in other fields as well.

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