Synthesis, Characterization, and the Antioxidant Activity of Double Quaternized Chitosan Derivatives

With the specialty of improving the water solubility of chitosan, quaternary ammonium salts have broadened the application of this polysaccharide in food, medicine and pesticides. To identify the effect of quaternary ammonium salts’ quantity, single quaternized chitosan N-phenmethyl-N,N-dimethyl chitosan (PDCS), double quaternized chitosan N-(1-pyridylmethyl-2-ylmethyl)-N,N-dimethyl chitosan (MP2MDCS), N-(1-pyridylmethyl-3-ylmethyl)-N,N-dimethyl chitosan (MP3MDCS), and N-(1-pyridylmethyl-4-ylmethyl)-N,N-dimethyl chitosan (MP4MDCS) were designed and synthesized successfully through chemical modification of chitosan. Besides, three kinds of antioxidant activities, including hydroxyl radicals, superoxide radicals, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals were tested in vitro. As shown in this paper, the scavenging ability was ranking in order of MP3MDC > MP4MDCS > MP2MDCS > PDCS > chitosan at 1.6 mg/mL in all assays. All double quaternary ammonium salts were better than chitosan or the single quaternary ammonium salt. In addition, MP3MDCS could scavenge hydroxyl radicals totally at 1.6 mg/mL. MP2MDCS and MP4MDCS with more than 90% scavenging indices both had great scavenging ability on hydroxyl radicals or DPPH radicals. Furthermore, these data demonstrated that the increasing number of the positive charge would improve the antioxidant property of chitosan derivatives, and the N-pyridinium position would influence the scavenging radical ability.


Introduction
Free radicals, especially oxygen free radicals in one's body, may damage the chemical structure of the organized cell and cause symptoms, such as ruptures of the main chain of the nucleic acid and protein peptide bond, membrane lipid peroxidation, enzyme inactivation, and cell apoptosis in certain pathological conditions [1][2][3][4][5]. Fortunately, free radicals can be removed by the antioxidant, where the composite agent plays an important role in cleaning while protecting body cells from damaging [6]. Meanwhile, it was reported that some polysaccharides with free hydroxyl and amino group have antioxidant ability, and the order of scavenging hydroxyl radicals ability is chitosan > hyaluronan > starch [7]. Chitosan and chitosan derivatives, in consequence, have attracted numerous attentions as the natural antioxidants with inestimable potentials [8].
Chitosan, the natural cationic amino polysaccharide copolymer of glucosamine and N-acetylglucosamine, is usually obtained from the exoskeletons of the shellfish and the insects. As a natural renewable resource, chitosan has advantages in unique physicochemical characteristics

Structure of the Chitosan Derivative
The synthetic procedures of the quaternary ammonium salts chitosan are shown in Scheme 1. N-(2-pyridylmethy), N- (3-pyridylmethy), and N-(4-pyridylmethy) chitosan were synthesized based on the reaction between the primary amino group of chitosan and aldehyde group of pyridine carboxaldehyde following by the reduction with sodium borohydride. Then, the secondary amine and N-pyridine were attacked by iodomethane to obtain quaternary ammonium salts, respectively. 1068.37 cm −1 , and 898.67 cm −1 indicated the β glycosidic bond. After quaternized, a new peak appeared at about 1546.63 cm −1 for PDCS, which was assigned to the benzene ring, and the peak at about 1461.78 cm −1 was the characteristic absorption of N-CH3 [35]. The peaks of quaternary ammonium salts of MP2MDCS, MP3MDCS, and MP4MDCS appeared at 1515.78 cm −1 , 1515.78 cm −1 , and 1546.63 cm −1 , respectively. The absorption of N-CH3 was at about 1461.78 cm −1 , 1465.64 cm −1 , and 1469.49 cm −1 for MP2MDCS, MP3MDCS, and MP4MDCS, respectively. Moreover, double quaternized chitosan MP2MDCS, MP3MDCS, and MP4MDCS had new peaks at 779.10 cm −1 , 806.10 cm −1 , and 813.81 cm −1 , respectively, corresponding to the pyridine groups with different substitution position. Above results demonstrated preliminarily that quaternized chitosan derivatives were obtained. Figure 2 showed the 1 H-NMR spectra of PDCS, MP2MDCS, MP3MDCS, and MP4MDCS, respectively. It was known that all of the signals at 5.12 to 3.81 ppm were assigned to the protons of glucose skeleton of chitosan. It exhibited characteristic resonance of N-CH3 at about 3.35 ppm for C7 in the molecules of PDCS, MP2MDCS, MP3MDCS, and MP4MDCS, respectively. At the same time, the peaks at 4.42, 4.38, and 4.39 ppm should correspond to methyl protons grafted to pyridine for MP2MDCS, MP3MDCS, and MP4MDCS, respectively. And 8.0-9.3 ppm should correspond to the pyridine ring with different substitution position. The signal at 7.5 ppm was assigned to the benzene ring. The above mentioned results demonstrated further that PDCS, MP2MDCS, MP3MDCS and MP4MDCS were obtained successfully. 1068.37 cm −1 , and 898.67 cm −1 indicated the β glycosidic bond. After quaternized, a new peak appeared at about 1546.63 cm −1 for PDCS, which was assigned to the benzene ring, and the peak at about 1461.78 cm −1 was the characteristic absorption of N-CH 3 [35]. The peaks of quaternary ammonium salts of MP2MDCS, MP3MDCS, and MP4MDCS appeared at 1515.78 cm −1 , 1515.78 cm −1 , and 1546.63 cm −1 , respectively. The absorption of N-CH 3 was at about 1461.78 cm −1 , 1465.64 cm −1 , and 1469.49 cm −1 for MP2MDCS, MP3MDCS, and MP4MDCS, respectively. Moreover, double quaternized chitosan MP2MDCS, MP3MDCS, and MP4MDCS had new peaks at 779.10 cm −1 , 806.10 cm −1 , and 813.81 cm −1 , respectively, corresponding to the pyridine groups with different substitution position. Above results demonstrated preliminarily that quaternized chitosan derivatives were obtained. Figure 2 showed the 1 H-NMR spectra of PDCS, MP2MDCS, MP3MDCS, and MP4MDCS, respectively. It was known that all of the signals at 5.12 to 3.81 ppm were assigned to the protons of glucose skeleton of chitosan. It exhibited characteristic resonance of N-CH 3 at about 3.35 ppm for C7 in the molecules of PDCS, MP2MDCS, MP3MDCS, and MP4MDCS, respectively. At the same time, the peaks at 4.42, 4.38, and 4.39 ppm should correspond to methyl protons grafted to pyridine for MP2MDCS, MP3MDCS, and MP4MDCS, respectively. And 8.0-9.3 ppm should correspond to the pyridine ring with different substitution position. The signal at 7.5 ppm was assigned to the benzene ring. The above mentioned results demonstrated further that PDCS, MP2MDCS, MP3MDCS and MP4MDCS were obtained successfully.

Antioxidant Activity
Chitosan has poor solubility in neutral water due to the high polymerization degree. We used the water-soluble chitosan with low molecular weight in all antioxidant activity tests. All quaternized

Antioxidant Activity
Chitosan has poor solubility in neutral water due to the high polymerization degree. We used the water-soluble chitosan with low molecular weight in all antioxidant activity tests. All quaternized

Antioxidant Activity
Chitosan has poor solubility in neutral water due to the high polymerization degree. We used the water-soluble chitosan with low molecular weight in all antioxidant activity tests. All quaternized chitosan derivatives had good solubility in water, and were prepared as aqueous solutions at the concentration of 0.1 to 1.6 mg/mL. Figure 3 showed the superoxide radicals' scavenging ability of chitosan and all quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. According to the graph, we concluded the results as follows: Firstly, the superoxide radicals' scavenging ability of all samples enhanced with the increasing concentration. Secondly, scavenging indices were listed as follows at the concentration of 1.6 mg/mL: chitosan 40.75%, PDCS 43.04%, MP2MDCS 67.98%, MP3MDCS 82.53%, and MP4MDCS 76.80%. These data showed that MP2MDCS, MP3MDCS and MP4MDCS had better superoxide radicals' scavenging ability than chitosan and PDCS at 1.6 mg/mL. And all double quaternized chitosan derivatives had higher density of positive charges than chitosan and PDCS, which might conclude that the higher density of positive charges could contribute to the scavenging on the superoxide radicals' activity. Thirdly, in the three double quaternized chitosan derivatives, the scavenging properties of MP2MDCS, MP3MDCS, and MP4MDCS were similar at the lower concentration, but MP3MDCS gave much stronger scavenging ability at 1.6mg/mL, which might conclude that the different position of N-pyridinium might have some influence on the scavenging activity. chitosan derivatives had good solubility in water, and were prepared as aqueous solutions at the concentration of 0.1 to 1.6 mg/mL. Figure 3 showed the superoxide radicals' scavenging ability of chitosan and all quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. According to the graph, we concluded the results as follows: Firstly, the superoxide radicals' scavenging ability of all samples enhanced with the increasing concentration. Secondly, scavenging indices were listed as follows at the concentration of 1.6 mg/mL: chitosan 40.75%, PDCS 43.04%, MP2MDCS 67.98%, MP3MDCS 82.53%, and MP4MDCS 76.80%. These data showed that MP2MDCS, MP3MDCS and MP4MDCS had better superoxide radicals' scavenging ability than chitosan and PDCS at 1.6 mg/mL. And all double quaternized chitosan derivatives had higher density of positive charges than chitosan and PDCS, which might conclude that the higher density of positive charges could contribute to the scavenging on the superoxide radicals' activity. Thirdly, in the three double quaternized chitosan derivatives, the scavenging properties of MP2MDCS, MP3MDCS, and MP4MDCS were similar at the lower concentration, but MP3MDCS gave much stronger scavenging ability at 1.6mg/mL, which might conclude that the different position of N-pyridinium might have some influence on the scavenging activity.  Figure 4 showed the curve chart of the hydroxyl radicals' scavenging ability of chitosan and the synthesized quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. The results were similar to above results on the superoxide radicals' scavenging activity. Firstly, the scavenging indices enhanced with the increasing concentration. Secondly, the scavenging ability against hydroxyl radicals was in order of MP3MDCS > MP4MDCS > MP2MDCS > PDCS > chitosan at the 1.6 mg/mL. Thirdly, MP3MDCS could scavenge hydroxyl radicals totally at 1.6 mg/mL.   Figure 4 showed the curve chart of the hydroxyl radicals' scavenging ability of chitosan and the synthesized quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. The results were similar to above results on the superoxide radicals' scavenging activity. Firstly, the scavenging indices enhanced with the increasing concentration. Secondly, the scavenging ability against hydroxyl radicals was in order of MP3MDCS > MP4MDCS > MP2MDCS > PDCS > chitosan at the 1.6 mg/mL. Thirdly, MP3MDCS could scavenge hydroxyl radicals totally at 1.6 mg/mL. chitosan derivatives had good solubility in water, and were prepared as aqueous solutions at the concentration of 0.1 to 1.6 mg/mL. Figure 3 showed the superoxide radicals' scavenging ability of chitosan and all quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. According to the graph, we concluded the results as follows: Firstly, the superoxide radicals' scavenging ability of all samples enhanced with the increasing concentration. Secondly, scavenging indices were listed as follows at the concentration of 1.6 mg/mL: chitosan 40.75%, PDCS 43.04%, MP2MDCS 67.98%, MP3MDCS 82.53%, and MP4MDCS 76.80%. These data showed that MP2MDCS, MP3MDCS and MP4MDCS had better superoxide radicals' scavenging ability than chitosan and PDCS at 1.6 mg/mL. And all double quaternized chitosan derivatives had higher density of positive charges than chitosan and PDCS, which might conclude that the higher density of positive charges could contribute to the scavenging on the superoxide radicals' activity. Thirdly, in the three double quaternized chitosan derivatives, the scavenging properties of MP2MDCS, MP3MDCS, and MP4MDCS were similar at the lower concentration, but MP3MDCS gave much stronger scavenging ability at 1.6mg/mL, which might conclude that the different position of N-pyridinium might have some influence on the scavenging activity.  Figure 4 showed the curve chart of the hydroxyl radicals' scavenging ability of chitosan and the synthesized quaternized chitosan derivatives composed at 0.1 to 1.6 mg/mL. The results were similar to above results on the superoxide radicals' scavenging activity. Firstly, the scavenging indices enhanced with the increasing concentration. Secondly, the scavenging ability against hydroxyl radicals was in order of MP3MDCS > MP4MDCS > MP2MDCS > PDCS > chitosan at the 1.6 mg/mL. Thirdly, MP3MDCS could scavenge hydroxyl radicals totally at 1.6 mg/mL.  The scavenging abilities of chitosan, PDCS, MP2MDCS, MP3MDCS, and MP4MDCS against DPPH radicals were shown in Figure 5. The results were similar to those of scavenging superoxide radicals and hydroxyl radicals too. Firstly, the sample had a positive correlation with the increasing concentration. Secondly, the scavenging indices were listed as followed: Chitosan 16.93%, PDCS 62.60%, MP2MDCS 94.80%, MP3MDCS 97.80%, and MP4MDCS 95.08%. All double quaternary ammonium salts could improve the ability of scavenging DPPH radicals significantly.
Based on the results mentioned above, the scavenging ability of the products against superoxide radicals, hydroxyl radicals, and DPPH radicals were almost in order of MP3MDCS > MP4MDCS > MP2MDCS > PDCS > Chitosan at 1.6 mg/mL, which could conclude that the antioxidant ability might associate with the density of the positive charge, as the positive charge could attract the single electron of free radicals to damage the free radical chain reaction. All double quaternized chitosan derivatives with higher density positive charges than chitosan and PDCS would attract more single electron of free radicals, which could improve the antioxidant ability. Furthermore, different N-pyridinium positions could have different influences on the antioxidant activity. The delocalization of pyridine was remarkable at the 2-and 4-position, which was enhanced if the nitrogen was protonated. So the distribution electronic cloud of MP2MDCS and MP4MDCS were more uniform than MP3MDCS in pyridine ring, which could explain MP3MDCS had a better antioxidant ability than MP2MDCS and MP4MDCS [30,36,37]. Based on the above results, it will be reasonable to presume that the density of positive charges and the different N-pyridinium position can influence the antioxidant property of chitosan derivatives. Further comprehensive investigation to ascertain the antioxidant mechanism and the structure-activity relationship would be studied in the future. The scavenging abilities of chitosan, PDCS, MP2MDCS, MP3MDCS, and MP4MDCS against DPPH radicals were shown in Figure 5. The results were similar to those of scavenging superoxide radicals and hydroxyl radicals too. Firstly, the sample had a positive correlation with the increasing concentration. Secondly, the scavenging indices were listed as followed: Chitosan 16.93%, PDCS 62.60%, MP2MDCS 94.80%, MP3MDCS 97.80%, and MP4MDCS 95.08%. All double quaternary ammonium salts could improve the ability of scavenging DPPH radicals significantly.
Based on the results mentioned above, the scavenging ability of the products against superoxide radicals, hydroxyl radicals, and DPPH radicals were almost in order of MP3MDCS > MP4MDCS > MP2MDCS > PDCS > Chitosan at 1.6 mg/mL, which could conclude that the antioxidant ability might associate with the density of the positive charge, as the positive charge could attract the single electron of free radicals to damage the free radical chain reaction. All double quaternized chitosan derivatives with higher density positive charges than chitosan and PDCS would attract more single electron of free radicals, which could improve the antioxidant ability. Furthermore, different N-pyridinium positions could have different influences on the antioxidant activity. The delocalization of pyridine was remarkable at the 2-and 4-position, which was enhanced if the nitrogen was protonated. So the distribution electronic cloud of MP2MDCS and MP4MDCS were more uniform than MP3MDCS in pyridine ring, which could explain MP3MDCS had a better antioxidant ability than MP2MDCS and MP4MDCS [30,36,37]. Based on the above results, it will be reasonable to presume that the density of positive charges and the different N-pyridinium position can influence the antioxidant property of chitosan derivatives. Further comprehensive investigation to ascertain the antioxidant mechanism and the structure-activity relationship would be studied in the future.

Analytical Methods
FT-IR spectrometers were recorded on a Jasco-4100 ranging from 4000 cm −1 to 400 cm −1 (Japan, provided by JASCO Co., Ltd., Shanghai, China) with KBr disks. 1 H NMR was recorded on a Bruker AVIII 500 spectrometer (Fällanden, Switzerland, provided by Bruker Biospin CN/Bruker (Beijing, China) Tech. and Serv. Co., Ltd., Beijing, China), using D 2 O as solvents with tetramethylsilane (TMS) as internal standard. Chemical shift values were given in δ (ppm). The elemental analyses (C, H, and N) were performed on a Vario EL III (Elementar, Langenselbold, Germany). The Degree of Substitution (DS) of chitosan derivatives were calculated based on the percentages of carbon and nitrogen, which is acquired by following Fonseca et al.'s method [38]. The UV-vis absorbance of the tested mixture were measured with a T6 New Century UV spectrometer (China, provided by P General Co., Ltd., Beijing, China). The results are processed by computer programs Excel (Microsoft, Redmond, WA, DC, USA) and Origin 8 (OriginLab, Northampton, MA, USA) and reported as mean ± SD.

Synthesis of Single Quaternized Chitosan (PDCS)
Single quaternized chitosan PDCS was prepared according to an earlier method [34]. In brief, 1.61 g chitosan was dissolved into 50 mL 1% acetic acid aqua and 50 mL ethanol in flask at 25 • C, and 3.05 mL benzaldehyde were added with stirring at 25 • C. After 2 h, 1.8 g NaBH 4 was added and the reaction was carried out for 2 h. The solution was precipitated into acetone and the precipitants were filtered. Then, the N-substituted chitosan derivative was obtained after drying at 60 • C for 24 h. Then, 0.5 g N-substituted chitosan was dispersed into 30 mL N-methyl-2-pyrrolidone (NMP) for 12 h at 25 • C. To this mixture, 0.1 mL NaOH (1 M), 0.75 g NaI, and 2 mL CH 3 I were added, and the reaction was refluxed gently with stirring at 60 • C for 4 h. The solution was precipitated by excess acetone and the precipitations were filtered. The single quaternized chitosan derivative was obtained by drying at 60 • C for 24 h (Scheme 1), yield: 90.30%; DS: 78.32% (Table 1). Double quaternized chitosan MP2MDCS, MP3MDCS, and MP4MDCS were synthesized as follows: 1.61 g chitosan was dissolved into 50 mL 1% acetic acid aqua and 50 mL ethanol in flask at 25 • C, and 30 mmol 2-pyridinecarboxaldehyde (2.85 mL), 3-pyridinecarboxaldehyde (2.82 mL), and 4-pyridinecarboxaldehyde (2.86 mL)were added, respectively, with stirring at 25 • C. After 2 h, 1.8 g NaBH 4 was added and the reaction was carried out continuously for 2 h. The solution was precipitated into excess acetone and the precipitant were filtrated. Then, the N-methylpyridine chitosan derivatives were obtained after drying at 60 • C for 6 h. In addition, 0.5 g above synthesized N-methylpyridine chitosan was dispersed into 30 mL NMP for 12 h at 25 • C. The reaction was carried out at 60 • C for 4 h with reflux stirring after 0.2 mL NaOH solution (1 M), 1.5 g NaI and 4 mL CH 3 I were added. The solution was precipitated by excess acetone and the precipitations were filtered. The double quaternized chitosan derivatives were obtained by drying at 60 • C for 24 h (Scheme 1), MP2MDCS yield: 93.54%; DS: 88.0%; MP3MDCS yield: 94.62%; DS: 76.5%; MP4MDCS yield: 93.80%; DS: 77.0% (Table 1).

Hydroxyl Radicals' Scavenging Activity Assay
The reaction of Fe-EDTA complex with H 2 O 2 in phosphate buffer can generate ·OH, which is harmful to the body through reacting with biological molecule such as amino acid or DNA. The hydroxyl radical scavenging activity was measured according to Guo and Liu [5,34]. The reaction mixture, total volume 4.5 mL, containing the samples of chitosan or chitosan derivatives (10 mg/mL, 0.045, 0.09, 0.18, 0.36, and 0.72 mL), were incubated with EDTA-Fe 2+ (220 µM), potassium phosphate buffer (150 mM, pH 7.4), safranine T (0.23 µM) and H 2 O 2 (60 µM) for 30 min at 37 • C. The absorbance of the mixture was measured at 520 nm. Three replicates for each sample concentration were tested. The ·OH bleached the safranine T, so decreased absorbance of the reaction mixture indicated decreased ·OH scavenging ability, and the capability of scavenging ·OH was calculated using the follow equation: Scavenging effect (%) = (A sample 520nm − A blank 520nm )/(A control 520nm − A blank 520nm ) × 100, where A blank 520nm was the absorbance of the blank (distilled water instead of the samples), and A control 520nm was the absorbance of the control (distilled water instead of H 2 O 2 ).

Superoxide Radicals' Scavenging Ability Assay
The superoxide radical ability was assessed by the method of Nishikimi et al. [39]. Superoxide radicals can generate single oxygen or hydroxyl radicals which could cause the peroxidation of lipids [40], which would be deleterious to the body. Involving testing samples of chitosan or chitosan derivatives (5 mg/mL, 0.06, 0.12, 0.24, 0.48, and 0.96 mL), 30 µM phenazine methosulfate (PMS), 338 µM nicotinamide adenine dinucleotide reduced (NADH), and 72 µM nitro blue tetrazolium (NBT) in Tris-HCl buffer (16 mM, pH 8.0), the reaction mixture was incubated at 25 • C for 5 min. The absorbance was read at 560 nm against a blank. Three replicates for each sample concentration were tested and the capability of scavenging superoxide radical was calculated using the following equation: Scavenging effect (%) = [1 − (A sample 560nm − A control 560nm )/A blank 560nm ] × 100, where A control 560nm is the absorbance of the control (distilled water instead of NADH for each concentration) and A blank 560nm is the absorbance of the blank (distilled water instead of the samples).

DPPH Radicals' Scavenging Ability Assay
According to HU [35], the DPPH radical scavenging ability of chitosan, PDCS, MP2PDCS, MP3MDCS, and MP4MDCS were measured as followed: testing samples (10 mg/mL, 0.03, 0.06, 0.12, 0.24 and 0.48 mL) and 2 mL ethanol solution of DPPH (180 µmol/L) was incubated for 30 min at 25 • C. Then, the absorbance of the remained DPPH radical was measured at 517 nm against a blank. Three replicates for each sample concentration were tested and the scavenging effect was obtained according to the following equation: Scavenging effect (%) = [1 − (A sample 517nm − A control 517nm )/A blank 517nm ] × 100, where A control 517nm is the absorbance of the control (ethanol instead of DPPH for each concentration) and A blank 517nm is the absorbance of the blank (distilled water instead of the samples).

Conclusions
Via N-pyridylmethyl chitosan, a series of derivatives of chitosan with single or double quaternary ammonium salts were synthesized successfully. In addition, antioxidant activities of chitosan and quaternized chitosan derivatives against hydroxyl radicals, DPPH radicals, and superoxide radicals were tested in vitro. It was found that all quaternized chitosan derivatives had good water solubility and stronger antioxidant ability compared with chitosan, especially double quaternized chitosan derivatives that might be further developed into more effective antioxidant biomaterials. These data demonstrated that the higher positive charge density of quaternized chitosan derivatives might contribute to antioxidant activities. Furthermore, MP3MDCS was more effective than MP2MDCS and MP4MDCS in all assays especially at 1.6 mg/mL. It was reasonable to presume that the N-pyridinium position of double quaternized chitosan derivatives could influence the antioxidant property. Besides, it was reported that pyridinium derivatives were showed to be non-toxic for genen delivery in vitro among the quaternary ammonium chitosans [30], so our double quaternized chitosan derivatives might have lower toxicity, which needs to be studied further. Finally the mechanism of the antioxidant activity and the structure-activity relationship need to be further investigated in the future.