Preparation and In vitro Antioxidant Activities of 6-Amino-6-Deoxychitosan and Its Sulfonated Derivatives Jianhong Yang,1 Qinyue Xie,1 Jianfeng Zhu,1 Chang Zou,2 Lingyun Chen,3 Yumin Du,2 Dinglong Li1 1

Department of Environmental Engineering, School of Environmental and Safety Engineering, Changzhou University, Changzhou 213164, Jiangsu, China 2

Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan 430079, Hubei, China 3

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5

Received 26 January 2015; revised 25 March 2015; accepted 1 April 2015 Published online 6 April 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22656

ABSTRACT: The 6-amino-6-deoxychitosan (NC) and their 2, 6-di-Nsulfonated derivatives were prepared via Nphthaloylation, tosylation, azidation, hydrazinolysis, reduction of azide groups and N-sulfonation, and their structures were systematically characterized by FT-IR, 2D HSQC NMR, XRD, gel permeation chromatography (GPC), and elemental analysis. The 6-amino-6deoxychitosan showed effect in three selected antioxidant essays, including reducing power, superoxide anion radical scavenging ability, and hydroxyl radical scavenging

showed weak scavenging activity in a special inverse concentration-dependent manner. However, the incorporation of N-sulfonated groups significantly improved the scavenging activity, and the more N-sulfonated groups, the higher the concentrations, the stronger the activity was. The results could be due to the different conformations of NC and its sulfonated derivatives in aqueous C 2015 Wiley Periodicals, Inc. Biopolymers 103: solution. V

539–549, 2015. Keywords: 6-amino-6-deoxychitosan; sulfonation; antioxidant; influence factor

effect. But the factors affecting each activity were different. The reducing power and the superoxide anion radical scavenging ability of NC were strong and closely related to the amino groups in the molecular chains. Both introducing N-sulfonated groups into NC and the concentra-

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers editorial office at [email protected].

tion reduction of NC and its sulfonated derivatives decreased these activities. For the superoxide anion radical, the molecular charge property was also a significant

INTRODUCTION

influence factor. For the hydroxyl radical, NC only

eactive oxygen species (ROS) in the forms of superoxide anion (•O22), hydroxyl radical (•OH), and hydrogen peroxide (H2O2), which are generated by normal metabolic process or from exogenous factors and agents, can cause damage to a wide range of essential biomolecules, such as DNA, RNA, and proteins. Such damages have been associated with cancer, rheumatoid arthritis, Alzheimer’s disease, atherosclerosis, and the degenerative processes of aging.1–3 Thus, antioxidants that can

Correspondence to: J. Yang; e-mail: [email protected] Contract grant sponsor: Key Laboratory of Fermentation Engineering (Ministry of Education) Open Foundation Contract grant number: 2010KFJJ04 Contract grant sponsor: Changzhou city science and technology support program (social development) Contract grant number: CE20125036 C 2015 Wiley Periodicals, Inc. V

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scavenge and prevent the generation of ROS are widely used to protect biological systems. Natural antioxidants such as carotenoids, phenolic, and nitrogen compounds are preferable to synthetic ones which may have undesirable side effects.4 Recently, polysaccharides have attracted increasing interest as natural antioxidants, especially those based on chitosan because of their special structure with high percentage of nitrogen (5–8%).5–13 Chitosan is a deacetylated derivative of chitin, which is the second most abundant natural biopolymer, obtained from the shells of Crustacea and the cell walls of some fungi.14 Chitosan and its derivatives were reported to scavenge 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, carbon-centered, and superoxide radicals.15–17 Generally, their antioxidant activities are considered to be related to the free amino groups and hydroxyl groups in the pyranose ring.9,16,18 Some studies have suggested that the free amino groups play a major role in free radical scavenging activity.15,18,19 However, chitosan’s derivatives such as quaternized carboxymethyl chitosan,20 chitosan-N22-hydroxypropyl trimethyl ammonium chloride,21 chitosan Schiff base derivatives13 and sulfanilamide chitosan22 showed strong antioxidant activity although they possessed less free amino groups than chitosan itself. In addition, many recent researches have focused on antioxidant activities of the sulfated polysaccharides such as fucoidan,23 sulfated derivatives of polysaccharide from Momordica charantia L.,24 sulfated lacquer polysaccharide,3 sulfated porphyran,25 and sulfated chitosan17,22,26–29. Nevertheless, previous reports about the antioxidant activities of sulfated chitosan are controversial. For example, Xing et al.17 reported more pronounced scavenging activities of superoxide and hydroxyl radicals by sulfated chitosan than chitosan itself. On the contrary, Huang et al.26 suggested that sulfated chitosan exhibit no scavenging activity against hydroxyl radicals, but increase its generation. So far, the antioxidant activities of sulfated chitosan in relation to its functional groups, especially sulfated and amino groups are still ambiguous. 6-Amino-6-deoxychitosan is a chitosan derivative which 6hydroxyl groups are substituted by amino groups in chitosan molecule. It has two amino groups in the partial sugar units and higher positive charge density in acid solution compared with chitosan. Satoh et al.30 first prepared 6-amino-6deoxychitosan from 6-deoxy-6-halo-N-phthaloylchitosan via 6-azidation. 6-Amino-6-deoxychitosan and its galactosylated derivatives have been investigated as a gene carrier in COS-1 Cells and HepG2 cells, respectively, which exhibited enhanced gene transfer efficiency.31,32 Recently, 6-amino-6-deoxychitosan was used as suitable supports for Pd nanosized particles, and the catalytic activity of the supported Pd catalysts was evaluated with Suzuki–Miyaura and Heck carbon–carbon crosscoupling reactions.33 Moreover, the antibacterial activity of

6-amino-6-deoxychitosan and its trimethylated and triethylated derivatives were also evaluated.34,35 However, there are no articles about the antioxidant activity of 6-amnio-6deoxychitosan and its sulfonated derivatives, yet. In this article, 6-amino-6-deoxychitosan was prepared, as well as its 2, 6-Nsulfonated derivatives. Their antioxidant activities were evaluated using in vitro assays, including reducing power and scavenging capacities for superoxide radicals and hydroxyl radicals. Some influence factors on the antioxidant activities were investigated and discussed.

EXPERIMENTAL Materials and Chemicals Chitosan from a crab shell was supplied by Zhejiang Aoxin Biotechnology Co. (Taizhou, China). The molecular weight was 561 kDa and degree of deacetylation was 88%. Trimethylamine–sulfur trioxide (Me3NSO3) complex was purchased from Alfa Aesar (Tianjin, China). Trichloroacetic acid was purchased from Tianjin Kermel Chemical Reagents Development Centre (Tianjin, China). Nitroblue tetrazolium (NBT) was from Sigma Chemicals Co. (USA). Riboflavin was from Tianjin Chemical Reagents Co. (Tianjin, China). Methionine was from Shanghai Chemical Reagent Co. (Shanghai, China). Vitamin C (VC) and salicylic acid were purchased from Shanghai Sinopharm Chemical Reagent Co. (Shanghai, China). Dialysis tubing (molecular weight cut-off ;3500) was purchased from Wuhan Huashun Biotech Co. (Wuhan, China). Sodium borohydride and sodium azide were chemically pure reagents. All other chemicals and reagents were analytical grade.

Preparation of 6-Amino-6-Deoxychitosan 6-Amino-6-deoxychitosan was prepared according to our previous method with a slight modification.35 Chitosan (15 g) was suspended in 300 mL of N,N-dimethylformamide (DMF) containing 5% (v/v) water and stirred overnight. Then, 35.4 g of phthalic anhydride was added for reaction of 8 h, respectively, in nitrogen at 120 C. The resulting mixture was cooled and poured into ice water. The precipitate was collected, washed, and dried to give phthaloylated chitosan. The phthaloylated chitosan (87.46 mmol sugar residues) was added into a mixture solution of 18 mL of triethylamine and 207 mL of pyridine, followed by addition of 100.98 g tosyl chloride. After 4 h, the mixture was poured into distilled water, and the precipitate was collected, washed, and dried to give a solid. A mixture of the solid (50 mmol) and 125 mL dry DMF was then treated with NaN3 (150 mmol) at 100 C for 12 h. After reaction, the solution was poured into 500 mL of ice Biopolymers

Antioxidant Activities of 6-Amino-6-Deoxychitosan

water. The precipitate was collected, washed, and dried to give phthaloylchitosan azide derivative. The phthaloylchitosan azide derivative (20 mmol) was suspended in 131 mL of distilled water. Then, 66 ml of hydrazine monohydrate was added. After reaction at 70 C for 10 h, the solution was treated based on the method in the literature35 to obtain 6-azide-6-deoxychitosan. The obtained product (10 mmol) was then suspended in 160 mL of DMSO for 60 min at 60 C, followed by addition of NaBH4 (8.08 g, 214 mmol). After reaction of 70 h, 50 mL of distilled water was added in the suspension. The solution was adjusted to pH 5.5 with hydrochloric acid, dialyzed against NaOH solution at pH 9.0 for 24 h, and then adjusted to pH 7.5 with hydrochloric acid. After dialyzing against distilled water for 48 h, the dialysate was concentrated and lyophilized to obtain 6-amino-6deoxychtosan (labeled as NC).

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The 2D HSQC (Heteronuclear Single Quantum Correlation) NMR spectra were recorded on a Bruker AVANCE III 500 MHz NMR spectrometer (Germany) at 23 C. The samples were dissolved in D2O. X-ray diffraction patterns were recorded by a Rigaku D/ max 2500 PC-ray diffractometer (Japan) with a Cu target and Ka radiation at 40 kV and 100 mA. The scanning scope of 2h was 5o270o. C, N, and H% of samples were measured by Elemental Analyzer-MOD 1106 (Carlo Erba Strumentazione). The content of NH31 group in 6-amino-6-deoxychitosan was determined by potentiometric titration of the chloride ion.37 Sulfur content was determined colorimetrically by Colovos’ method.38 A calibration curve was constructed with sodium sulfate as standard.

Reducing Power Assay 2,6-Di-N-Sulfonation of 6-Amino-6-Deoxychitosan 2,6-Di-N-sulfonation of NC was prepared according to the method of Holme and Perlin36 with modifications. 6-Amino6-deoxychitosan (0.25 g) was dissolved in 50 mL of distilled water, then Me3NSO3 complex and Na2CO3 were added. Various molar ratios of Me3NSO3 complex and Na2CO3 to sugar unit were used. The solution was stirred at 65 C for 12 h, then cooled down to room temperature. Subsequently, the solution was dialyzed against distilled water for 4 d. The dialysate was concentrated under reduced pressure below 45 C, and then precipitated with anhydrous ethanol. The precipitate was collected after drying over phosphorous pentoxide in vacuum.

The reducing power of all samples was determined based on the method of Yen and Duh.39 Sample solutions with different concentrations (1 mL) were mixed with 2.5 mL of 0.2 mol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) aqueous potassium ferricyanide. The mixture was incubated at 50 C for 20 min. Then, 2.5 mL of 10% (w/v) trichloroacetic acid was added, and the mixture was centrifuged at 1500g for 10 min. The supernatant (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% (w/v) ferric chloride (0.5 mL) and its absorbance was measured with a 752N UV-Vis spectrophotometer (Jinke, Shanghai) at 700 nm. A high absorbance was indicative of strong reducing power.

Characterization

Superoxide Anion Radical Scavenging Assay

Weight average molecular weight (Mw) and polydispersity (PD) of sample were measured by a gel permeation chromatography (GPC) on a TSP P100 pump (Thermo Finnigan, USA) equipped with a TSKgel G3000-pw column (TOSOH, Japan). The eluent was 0.2M HAc/0.1M NaAc buffer for 6amino-6-deoxy chitosan and 0.1M NaCl solution for its sulfonated derivatives. The flow rate was 1.0 mL/min, the temperature of the column was maintained at 30 C, and the sample concentration was 5 mg/mL. The eluate was monitored with a RI 150 refractive index detector (Thermo Finnigan). The standards used for calibration the column were TOSOH pullulan of defined Mw ranging from 2.7 to 112 kDa. All data provided by the GPC system were collected and analyzed using the Jiangshen Workstation software package. The Fourier transform infrared (FT-IR) spectra were recorded with KBr pellets on a Nicolet FT-IR 5700 spectrophotometer (Thermo, Madison, USA). Thirty-two scans at a resolution of 4 cm21 were averaged and referenced against air. Biopolymers

This assay was based on the capacity of the samples to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) in the riboflavin–light–NBT system.40 Each test solution (3 mL) contained 50 mmol/L sodium phosphate buffer (pH 7.8), 13 mmol/L methionine, 2 lmol/L riboflavin, 100 lmol/L EDTA, 75 lmol/L NBT, and 1 mL sample solution. The production of blue formazan was followed by monitoring the increase in absorbance at 560 nm after a 10min illumination from a fluorescent lamp. The entire reaction assembly was enclosed in a box lined with aluminum foil. Identical tubes containing the reaction mixture were kept in the dark and served as blanks. The sample capacity to scavenge superoxide radical was calculated using the following equation: Scavenging capacity ð%Þ5½ðA0 2A1 Þ=A0  3 100

where A0 was the absorbance of the control and A1 was the absorbance of samples.

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SCHEME 1 The preparation route of 6-amino-6-deoxychitosan and its sulfonated derivative.

Hydroxyl Radical Scavenging Assay The scavenging activity for hydroxyl radicals was measured according to the method of Smirnoff and Cumbes41 with slight modifications. Reaction mixture contained 1 mL of samples at various concentrations (0.1–1.0 mg/mL), 1 mL of 1.5 mM FeSO4, 0.5 mL distilled water, 0.5 mL of 2 mM salicylic acid– ethanol and 1 mL of 6 mM H2O2. The reaction was started by adding H2O2. After incubation at 37oC for 1 h, the absorbance of the mixture at 510 nm was measured. The hydroxyl radical scavenging activity was calculated according to the following equation: Scavenging capacity ð%Þ 5½12ðA1 2A2 Þ=A0  3 100

where A0 was the absorbance of the control (blank, without samples), A1 was the absorbance in the presence of the sample, and A2 was the absorbance without salicylic acid–ethanol.

Fe21 Chelating Assay The ferrous ion-chelating potential of samples was investigated according to the method of Decker and Welch42 with minor modifications. In brief, the reaction mixture, containing the samples with different concentrations (1 mL), FeCl2 (0.1 mL, 2 mmol/L), and ferrozine (0.4 mL, 5 mmol/L), was adjusted to a total volume of 5.0 mL with water, shaken well, and incubated

for 10 min at room temperature. The absorbance of the mixture was measured at 562 nm against a blank. The ability of the sample to chelate ferrous ion was calculated using the following equation: Chelating ability ð%Þ5½ðA0 2A1 Þ=A0  3 100

where A0 was the absorbance of the control (distilled water, instead of sample) and A1 was the absorbance of the sample.

RESULTS AND DISCUSSION Molecular Structure Characterizations of 6-Amino-6Deoxychitosan 6-Amino-6-deoxychitosan (NC) was prepared via N-phthaloylation, tosylation, azidation, hydrazinolysis, and reduction of azide groups (Scheme 1). The FT-IR spectra of chitosan and NC are shown in Figure 1. In the spectrum of chitosan, the absorption band at 1598 cm21 was attributed to the bending vibration of amino group. The band at 1649 cm21 was assigned to the amide I which overlapped with the band of O– H deformation vibration of H2O at ;1640 cm21. In the FT-IR spectrum of 6-amino-6-deoxychitosan, the absorption band of amino group shifted to 1590 cm21,35 and the intensity significantly enhanced, indicating increased content of amino group Biopolymers

Antioxidant Activities of 6-Amino-6-Deoxychitosan

FIGURE 1 FT-IR spectra of chitosan (A), 6-amino-6deoxychitosan (B, NC) and 2,6-di-N-sulfonated 6-amino-6deoxychitosan (C, NCS1; D, NCS3).

after modification. In addition, the C–O stretching band of chitosan at 1032 cm21 related to the primary hydroxyl group disappeared in the spectra of 6-amino-6-deoxychitosan suggesting a high amination of 6-OH. Furthermore, a new absorption band appeared at 1538 cm21 in the spectrum of 6-amino6-deoxychitosan. This characteristic band was also found in the IR spectrum of chitosan hydrochloride, thus the result indicated that 6-amino-6-deoxychitosans were partially protonated to form a cationic derivative. Two-dimensional HSQC NMR was also performed to elucidate the structures of NC. The result is shown in Figure 2. In the 13C NMR spectrum, the signals at 125.21 and 129.44 ppm were attributed to –CH@ in the aromatic ring, which correlated in the 1H NMR spectrum with the protons at 7.20–8.20 ppm. The signals at 139.29 and 142.60 ppm were attributed to the carbon atoms in the aromatic ring linked to the carboxyl groups. These indicated the removal of the N-phthaloyl groups was incomplete in the hydrazinolysis process. The peak at 20.63 ppm was attributed to –CH3 of the acetyl group, which correlated with the protons at 1.98 ppm (Data not shown) and 2.25 ppm. The peaks at 40.02 and 38.51 ppm were assigned to C-6 linked to amino group.35 The former correlated with the proton signals at 3.21 and 3.44 ppm assigned to –CH2NH2 and –CH2NH31 at C-6. These suggested the amino groups were successfully Biopolymers

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incorporated into the C-6 position of chitosan. The latter correlated with the proton signals at 2.61 and 2.70 ppm. The ratio of the integral values of the proton signals at 2.25 and 2.70 ppm was about 3.0:1.0. Because acetyl group migration easily occurred in the reaction process,43 it was deduced that partial acetyl groups were migrated from the amino groups at C-2 to the amino groups at C-6. Similarly, the proton signal at 2.61 ppm was assigned to –CH2NH– at C-6 linked to phthaloyl group. The signal at 55.46 ppm was attributed to C-2, which correlated with the protons at 3.16 ppm. The peak at 60.23 ppm was assigned to C-6 linked to OH group, which correlated with the protons at 3.66 and 3.86 ppm. These signals were weak, indicating most hydroxyl groups had been substituted with amino groups. The signals at 69.84, 71.19, 77.18, and 97.42 ppm were attributed to C-3, C-5, C-4, and C-1at sugar ring, which correlated the protons at 3.80, 3.67, and 3.91, 3.87, and 4.85 ppm, respectively. In addition, the high degree of amino substitution (DAS) was also supported by the elemental analysis result. Table I showed that the degree of substitution was 0.94 for NC. Its molar ratio of NH31 group to sugar residue was 0.68. The result from GPC analysis showed that severe chain scission occurred during the sample preparation process as the Mw decreased from 561 kDa (original chitosan) to 47.9 kDa for NC after modification (Table I).

Preparation of Sulfonated 6-Amino-6-Deoxy Chitosans and Their Structure Characterization Sulfonation of 6-amino-6-deoxy chitosans was conducted using Me3NSO3 complex as sulfonation reagent in aqueous solution. This reaction is highly selective and only amino groups are sulfonated. To make the sulfonated NC with

FIGURE 2 (NC).

HSQC NMR spectrum of 6-amino-6-deoxychitosan

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Table I Molecular Structure Parameters of 6-Amino-6-Deoxychitosan Elemental analysis Sample NC

Yield (%)

C (%)

N (%)

H (%)

Mw (kDa)

PDa

N-Phthaloyl groupb

NH31 groupc

DASd

87

42.76

12.66

8.82

47.9

1.88

0.195

0.68

0.94

a

Polydispersity. Molar rate of the group to sugar residue, based on 1H NMR. c olar ratio of the group to sugar residue. d Degree of substitution of amino group at C-6 position, based on the C/N ratio of elemental analysis. b

different degree of sulfonation, the different molar ratios of Me3NSO3 complex to sugar unit (6:1, 5:1, 3.5:1, 2:1) were used. The results of four 2,6-di-N-sulfonated 6-amino-6-deoxy chitosans (NCS1, NCS2, NCS3, and NCS4) are listed in Table II. It can be seen that as the molar ratio of Me3NSO3 complex to sugar unit increased, Degrees of sulfonation (DS) of modified NCs increased. Degrees of sulfonation ranged from 0.83 to 1.52. It was obvious that N-sulfonation was incomplete. These samples were also studied by GPC. According to the result, the 2,6-di-N-sulfonated 6-amino-6-deoxy chitosan samples exhibited Mw ranging from 20.3 to 47.6 kDa. The result indicates that further degradation occurred in the sulfonation process. In addition, the more sulfonating reagents used, the smaller the Mw of the final products. Figure 1 shows FT-IR spectrum of 2,6-di-N-sulfonated derivatives (NCS1 and NCS3) of 6-amino-6-deoxy chitosan. The characteristic strong absorptions at 1228, 1199, and 1037cm21 are assigned to asymmetrical and symmetrical S@O stretching vibration of NH-SO32. The band corresponding to amino groups at 1590 cm21 still remained, but the absorption intensity was decreased, indicating N-sulfonation was incomplete. In the HSQC spectrum of NCS1 (Figure 3), the signals at 101.49 and 73–80 ppm were attributed to the anomeric carbon, the C-3, C-4, and C-5 at sugar ring. Compared with the spectrum of NC, these signals had a downfield shift because of the N-sulfonation. The signals at 55.48 ppm were attributed to the C-2 linked to acetylamino, phthalimide, and amino groups, respectively. The signal at 59.74 ppm was attributed to the C-2

linked to NHSO3Na, and the shift of the C-2 N-sulfo signal was around 4.5 ppm compared with the C-2 N-acetyl signal.44 The signal correlated with the protons at 3.03 ppm. The peak at 43.22 ppm which corrected with the protons at 3.32 ppm was attributed to the C-6 linked to NHSO32, and the signal had a shift of 3.2 ppm compared with the signal of the C-6 linked to amino group. These shifts indicate that N-sulfation occurred at positions C-2 and/or C-6. In addition, it can be seen from the HSQC spectrum that the C-6 linked to OH group still appeared at about 60 ppm. The proton signal at 2.62 ppm assigned to the phthaloyl group also existed. However, compared with the HSQC spectrum of NC, the intensity of the proton signal at 2.62 ppm became weak. At the same time, the intensity of the peaks at 7.3–7.6 ppm assigned to the protons in the aromatic ring also became weak. These further indicated that a small amount of phthaloyl groups could be migrated from –CHNH2 groups at C-2 to –CH2NH2 groups at C-6 in the preparation process of NC, but partial phthaloyl groups were removed in the N-sulfonation process. It can also be observed from the HSQC spectrum that the proton signal at 2.70 ppm assigned to the –CH2– at C-6 and the proton signal at 2.25 ppm assigned to –CH3 of the acetyl groups disappeared completely, indicating that the acetyl groups linked to the amino groups at C-6 were completely removed in the Nsulfonation process. Furthermore, NC and its 2,6-di-N-sulfonated derivative (NCS1) were analyzed by X-ray diffractometer. The results are shown in Figure 4. The diffraction pattern of NC shows two

Table II Preparation Conditions and Molecular Structure Parameters of 2,6-di-N-Sulfonated 6-Amino-6-Deoxychitosan Sample

n(SR)/n(SC)/n(SU)a

T (oC)

t (h)

Yield (%)

S wt%

DSb

Mw (kDa)

PDc

NCS1 NCS2 NCS3 NCS4

6: 3.3:1 5: 3.3:1 3.5:2:1 2:2:1

65 65 65 65

18 18 18 18

94 93 87 85

13.87 12.52 11.70 9.48

1.52 1.27 1.14 0.83

20.3 43.2 44.7 47.6

1.51 1.72 1.75 1.96

a

n(SR)/n(SC)/n(SU) was molar ratio among sulfonating reagent (Me3NSO3), sodium carbonate and sugar unit. Degree of N-sulfonation. c Polydispersity. b

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waals forces in sulfonated 6-amino-6-deoxychitosans in comparison with 6-amino-6-deoxychitosan.

Reducing Power

FIGURE 3 HSQC NMR spectrum of 2,6-di-N-sulfonated 6amino-6-deoxychitosan (NCS1).

crystalline peaks at 11.8o and 23.9o with the height of 998 and 1626, respectively. Whereas, the pattern of NCS1 exhibits only one major crystalline peak a 21.4o with the height of 1291. Meanwhile, compared with NC which showed a narrow peak at 23.9o, NCS1 had a relatively broader peak at 21.4o. It has been revealed that the width of X-ray diffraction peak is related to the size of crystallite, and the broadened peak usually results from small crystallites.37 In addition, peaks with smaller angle, corresponding to larger spacing, suggest a decrease in crystalline perfection.45 These changes in X-ray diffraction pattern indicate that the crystallinity of NC decreased after introduction of sulfonated groups, and the NCS1 had lower degree of structure order. This was probably due to weakened intermolecular and intra-molecular hydrogen bonding and Van der

The presence of a reductant such as antioxidant substance causes the reduction of the Fe31/ferricyanide complex to the ferrous form and the activity can be monitored by measuring the formation of Prussian blue at 700 nm. As shown in Figure 5A, the reducing power value of NC was 0.377 at 1mg/ml and increased with increasing its concentration. However, the reducing power decreased dramatically when it was Nsulfonated. The reducing power values of NCS1, NCS2, NCS3 and NCS4 were 0.059, 0.068, 0.076, and 0.089 at 1 mg/mL, respectively. And their reducing power only increased slightly with the increase of the sample concentration. In general, the reducing power is associated with the donating-hydrogen abilities. Some substances with high donating-hydrogen ability such as vitamin C46 and gallic acid-grafted chitosan10 usually showed excellent reductive ability. In our experiments, it was found that the reducing power of vitamin C was 0.612, 1.145, and 2.063 at 0.1, 0.2, and 0.4 mg/mL, respectively. Although the reducing power of NC was far lower than that vitamin C, it could be closely related to free amino groups as well, which could easily donate the hydrogen atoms. For N-sulfonated NC, the introduction of the sulfonated groups led to the diminution of free amino groups, subsequently decreased the reducing power. This result agreed with previous report that Nsulfonated chitosan showed weaker reducing power than the original chitosan.29 As to the effect of sulfated group itself, it was actually observed that some natural sulfated polysaccharides extracted from algae such as Laminaria japonica, Porphyra haitanensis, Enteromorpha linza, and Bryopsis

FIGURE 4 X-ray diffraction patterns of 6-amino-6-deoxychitosan (NC) and its 2,6-di-N-sulfonated derivative (NCS1).

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FIGURE 5 Antioxidant activities of 6-amino-6-deoxychitosan (NC) and its 2,6-di-N-sulfonated derivatives (NCS1, NCS2, NCS3, and NCS4). Values were means 6 SD of three times. (A) Reducing power; (B) Scavenging activity of hydroxyl radicals; (C) Fe21 chelating ability; (D) Scavenging activity of superoxide radicals.

plumose23,25,47,48 and most synthetic sulfated polysaccharides 3,25,29 only showed low reducing power. Thus, it seemed that sulfate group itself did not contribute to the reducing power. However, surprisingly, several special synthetic sulfated polysaccharides showed strong activity. The reducing power of TSCTS (C3,6 sulfated chitosan) was 0.92 at 0.08 mg/mL and 1.068 at 0.1 mg/mL.29 We also found that the reducing power of a sulfated lacquer polysaccharide (LPS5) was 1.2 at 1 mg/ mL.3 Such high reducing power of TSCTS and LPS5 could result from the formation of reductone-like structure due to the side reactions during the sulfation, as described previously in our studies.49,50

Hydroxyl Radical Scavenging Ability Hydroxyl radicals are mainly responsible for the oxidative injury of biomolecule generated by reaction of Fe(II) complex with H2O2. Salicylic acid has the ability to absorb •OH to produce a purple-red material. Added hydroxyl radical scavengers

compete with salicylic acid, which makes the content of purple-red material decrease. The method was used to evaluate the hydroxyl radical scavenging ability of NC and its derivatives. As shown in Figure 5B, NC was found to show the slight ability to scavenge hydroxyl radicals at concentration between 0.1 and 1 mg/mL. Its values of scavenging hydroxyl radicals were in the scope of 4.18–14.76%. For the vitamin C, its values of the hydroxyl radical scavenging ability only were 5.64 and 11.02% at 0.1 and 0.2 mg/mL, respectively. However, its value of the hydroxyl radical scavenging ability increased swiftly to 90.3% at 0.4 mg/mL. Thus, the hydroxyl radical scavenging ability of NC was only slightly higher at 0.1 mg/mL than that of vitamin C. Moreover, for NC, it was surprising to find that the color of the resultant solution gradually became deep and its scavenging hydroxyl radical ability gradually decreased with the increase of NC’s concentration. This was different from the chitosan’s result. It has been considered that chitosan eliminates hydroxyl radicals by the action of NH2 group on the C-2 position of the chitosan. In general, chitosan with high degree Biopolymers

Antioxidant Activities of 6-Amino-6-Deoxychitosan

of deacetylation exhibits the high scavenging hydroxyl radical activity and this scavenging ability increased with the increase of its concentration.15 Je and Kim51 prepared a aminoethylchitin derivative, and also found free amino group in the – CH2CH2NH2 plays an important role in the free radical scavenging activity. Obviously, besides the NH2 group and the concentration, NC’s scavenging ability against hydroxyl radicals should also be affected by other factors. Furthermore, it was interesting to notice that the four sulfonated NC samples had the high ability to scavenge hydroxyl radicals at the concentrations between 0.1 and 1 mg/mL and their scavenging abilities gradually increased with increasing their concentrations (Figure 5B). Their scavenging hydroxyl radical values were in the scope of 16.2–45.7%. At 1.0 mg/mL, the values were 45.7, 38.4, 32.4, and 29.0% for NCS1, NCS2, NCS3, and NCS4, respectively. It was also observed that they only showed higher hydroxyl radical scavenging ability at low concentrations (0.1 and 0.2 mg/mL) than the vitamin C (Figure 5B). Furthermore, compared with NC, these sulfonated NCs possessed less NH2 groups in the pyranose ring, but showed higher ability of scavenging hydroxyl radicals. At the same time, it was also observed from Figure 5B that the sulfonated NCs’ abilities of scavenging hydroxyl radicals increased in the order of NCS1 > NCS2 > NCS3 > NCS4, which was consistent with the order of their degree of sulfonation. The less the NH2 groups in the pyranose ring were, the higher the ability on scavenging hydroxyl radicals of sulfonated NCs were. This indicated that the sulfonated groups could be a positive factor affecting scavenging activity. It had been demonstrated that some natural sulfated polysaccharides were good scavengers for hydroxyl radicals, and their activities decreased with the decrease of the content of sulfated groups.52,53 Some sulfated chitosan also possessed higher scavenging hydroxyl radical activities than chitosan.17,29 However, Huang et al.26 found hydroxyethyl chitosan sulfate exhibit no scavenging activity against hydroxyl radicals, but increase its generation. Furthermore, other chitosan’s derivatives such as quaternized carboxymethyl chitosan,20 chitosan-N-2-hydroxypropyl trimethyl ammonium chloride,21 chitosan Schiff base derivatives13 showed strong scavenging radical activity, too. Therefore, it was not likely that the scavenging activity against hydroxyl radicals only resulted from the sulfonated groups. Some researchers also reported that, for hydroxyl radical, the scavenging activity was not due to the direct scavenging but inhibition of hydroxyl radical generation by chelating ions such as Fe21 and Cu21.54 Hydroxyl radicals can be generated by the reaction of Fe21 and H2O2, so the generation of hydroxyl radicals could be reduced by chelating the Fe21. However, in our research, the sulfonated NCs did not almost show any ferrous ion chelating capacity within the scope of Biopolymers

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studied concentration (Figure 5C). Obviously, the chelating effect of sulfonated NCs on Fe21 ions may not be responsible for the scavenging of hydroxyl radicals. In addition, we believed that the conformation of polysaccharide molecule in solution could be an important factor affecting the scavenging of hydroxyl radicals. NC had higher crystallinity and degree of structure order in the solid state than the sulfonated NCs as supported by the X-ray diffraction data. Thus, it was deduced that NC easily formed the ordered structure in aqueous solution, too. With the increase of NC’s concentration, these molecular chains of NC were in close proximity, the intermolecular and intramolecular hydrogen bonding could become stronger, and more molecule chains could easily assemble to form local ordered structures. Some active sites such as NH2 and OH groups were shielded, which blocked hydroxyl radicals to react with them. Thus, NC’s scavenging activity against hydroxyl radicals decreased gradually with the increase of its concentration. However, for the sulfonated NCs, introduction of sulfonated groups decreased Van der waals force and partly destroyed the intermolecular and intramolecular hydrogen bonds of NC as mentioned previously (Figure 4). The steric hindrance caused by the sulfonated groups and the electrostatic repulsion of the molecular chains of sulfonated NCs with negative charges also caused the change of the NC’s chain conformation in aqueous solution. These made the molecule chains difficult to assemble and form the ordered structures. Just like in a solid state, sulfonated NCs also had lower degree of structure order in aqueous solution than NC. Thus, more active sites such as NH2 and OH groups were exposed to strengthen the scavenging activity of hydroxyl radicals.

Superoxide Anion Radical Scavenging Ability Photochemical reduction of flavins generates •O22 which reduces NBT, resulting in the formation of blue formazan. The decrease of absorbance at 560 nm with addition of antioxidant indicates the consumption of superoxide anion radical in the reaction mixture. The superoxide anion radical scavenging capacity of NC and its 2,6-di-N-sulfonated derivatives is shown in Figure 5D. NC showed strong scavenging capacity against superoxide radicals in the concentration scope of 0.02–0.2 mg/ mL and increased with increasing sample concentration. The scavenging capacity of NC was 81.9% at 0.2 mg/mL. However, when the sample concentration was more than 0.2 mg/mL, it was found that the superoxide anion radical scavenging capacity of NC drastically decreased from 81.9 to 238.9% with increasing sample concentration from 0.2 to 1.0 mg/mL (Figure 5D). This was because some precipitates were formed, and the solution became turbid to cause the increase of

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absorbance. Actually, it was observed that the solution was not blue but colorless. Obviously, under high sample concentration, the scavenging capacity of NC should also be strong. After the solution was centrifuged, the results showed the superoxide anion radical scavenging capacity of NC increased with increasing sample concentration. The scavenging capacity of NC was 99.3% at 1.0 mg/mL. For the vitamin C, its scavenging capacity for superoxide radical gradually increased from 34.9 to 51.1% with increasing its concentration from 0.1 to 1.0 mg/mL (Figure 5D). Compared with this result, NC had stronger scavenging activity for superoxide radical than vitamin C. It has been demonstrated that the scavenging activity of chitosan on superoxide radicals is strongly dependent upon the – NH2 group on the C-2 position.9,15 Thus, it was deduced that the scavenging activity against superoxide radicals for NC also resulted from the amino groups. Furthermore, like the scavenging of the hydroxyl radical, the chain conformation of NC in aqueous solution should also affect the activity of scavenging superoxide anion radical. However, the scavenging activity of NC did not decrease with increasing its concentration, which was different from the result of the scavenging hydroxyl radical of NC. This could be related to the charge properties of superoxide anion and NC. Huang et al.26 reported that for the carbon-centered radicals with positive charge, hydroxyethyl chitosan sulfate exhibited high scavenging percentage. This is because the carbon-centered radicals can be easily approached and easy to be scavenged by the negatively charged hydroxyethyl chitosan sulfate. Similarly, superoxide anion radical is negatively charged. It was attracted by the NC with partially protonated amino groups and easily approached NC. This strengthened NC’s scavenging activity on superoxide anion radical, so it showed very high scavenging activity and this activity increased with the increase of its concentration. Thus, for the scavenging of superoxide anion radical, the charge properties could be a more important influence factor than the chain conformation of NC in aqueous solution. As for 2,6-di-N-sulfonated NCs (NCS1, NCS2, NCS3, and NCS4), the precipitates were not observed and the solution was transparent in the experimental process. Their results also showed all sulfonated samples had scavenging capacity against superoxide anion radicals and the scavenging effect increased with increasing sample concentration from 0.1 to 1.0 mg/mL (Figure 5D). All sulfonated samples also showed higher scavenging capacity against superoxide anion radicals than Vitamin C, but they had lower scavenging capacity than NC. This was probably due to the fact that they possessed less unsubstituted amino groups than NC. Moreover, as degree of sulfonation significantly decreased from 1.52 to 0.83, the scavenging capacity only slightly increased. 2,6-di-N-sulfonated samples NCS1,

NCS2, NCS3, and NCS4 demonstrated scavenging capacity values of 79.2, 81.9, 84.8, and 86.5% at 1 mg/mL, respectively (Figure 5D). Thus, the superoxide anion radical scavenging activity of the sulfonated NCs seemed not to be explained only using the number of NH2 groups in the pyranose ring. It could be affected by molecular charge properties as well. For sulfonated NCs, they carried negative charges due to the incorporation of sulfonated groups, and repelled the superoxide anion radicals. This could be a factor caused lower scavenging activity of the sulfonated NCs than NC. Furthermore, as degree of sulfonation gradually decreased and unsubstituted amino groups increased, the scavenging activity of sulfonated NCs did not show markedly increased, which could be related to the electrostatic repulsion occurred between negative charges as well.

CONCLUSION This study demonstrated 6-amino-6-deoxy chitosan and its sulfonated derivatives had the antioxidant activities such as superoxide anion radical scavenging capacity, hydroxyl radical scavenging ability and reducing power. Compared with vitamin C, they had better superoxide anion radical scavenging ability in the concentration scope of the study and higher hydroxyl radical scavenging ability only at low concentrations. However, their reduce power was always lower than that of vitamin C. In addition, factors affecting these activities were complex. The antioxidant activities of NC and its derivatives were not only dependent upon their structure such as degree of sulfonation, NH2 group, charge property and chain conformation, but also affected by the type of the radicals. For the reducing power and the superoxide anion radical scavenging activity, amino groups played a major role. The positively charged NC was advantageous to scavenge the superoxide anion radical with negative charge. For the scavenging of hydroxyl radical, NC’s conformation in aqueous solution could be a key factor. More work still need to be done to further expound the relationships between polysaccharide’s structure and antioxidant activity.

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Reviewing Editor: C. Allen Bush