Preparation, characterization and antioxidant activity of phenolic acids grafted carboxymethyl chitosan

International Journal of Biological Macromolecules 62 (2013) 85–93

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Preparation, characterization and antioxidant activity of phenolic acids grafted carboxymethyl chitosan Jun Liu, Jian-feng Lu, Juan Kan, Ying-qing Tang, Chang-hai Jin ∗ College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China

a r t i c l e

i n f o

Article history: Received 19 July 2013 Received in revised form 17 August 2013 Accepted 22 August 2013 Available online 29 August 2013 Keywords: Antioxidant Carboxymethyl chitosan Graft copolymer Phenolic acid

a b s t r a c t In this study, three phenolic acids including gallic acid (GA), caffeic acid (CA) and ferulic acid (FA) were grafted onto N,O-carboxymethyl chitosan (NOCC) by a free radical mediated grafting method. The grafted copolymers obtained were all water-soluble samples. UV–vis absorption peaks of the grafted copolymers shifted toward longer wavelengths. FT-IR spectroscopy of the grafted copolymers exhibited additional phenolic characteristics of the aromatic ring C C stretching within 1450–1650 cm−1 . NMR spectroscopy of the grafted copolymers showed new peaks at 6.2–7.6 ppm assigned to the phenyl protons of phenolic acids. These results all confirmed the successful grafting of three phenolic acids to NOCC. The conjugation probably occurred at amine of NOCC and carboxyl groups of phenolic acids. The grafted copolymers exhibited decreased crystallinity as compared to NOCC and chitosan. Moreover, antioxidant activity in vitro assays showed that the antioxidant property decreased in the order of GA-g-NOCC > CA-g-NOCC > FAg-NOCC > NOCC > chitosan. Our results suggested the potential of phenolic acids grafted NOCC for the development of effective antioxidant agents. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Reactive oxygen species (ROS) and oxygen-derived free radicals may contribute to a variety of pathological effects (e.g. DNA damages, carcinogenesis and cellular degeneration) and induce many diseases including aging, cancer, atherosclerosis, diabetes and rheumatoid arthritis [1,2]. Although most organisms possess antioxidant defense and repair systems to protect them against oxidative damages, these systems are insufficient to prevent damages entirely. Therefore, it is essential to develop effective antioxidants to protect human body from free radicals and retard the progress of many chronic diseases. Recently, a number of natural polysaccharides and their derivatives have been demonstrated to possess potent antioxidant activity and potential applications as antioxidants [3–5]. Chitosan is a cationic linear polysaccharide consisting of ␤-1,4linked glucosamine with various N-acetyl glucosamine residues. As a natural renewable resource, chitosan exhibits unique properties such as biocompatibility, biodegradability, non-toxicity and non-antigenicity. And it has prospective applications in many fields including biomedicine, waste water treatment, functional membranes, cosmetics and food industries [6]. However, chitosan is only soluble in some dilute acid solutions due to its inter- and

∗ Corresponding author. Tel.: +86 514 87978009; fax: +86 514 87313372. E-mail address: [email protected] (C.-h. Jin). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.08.040

intra-molecular hydrogen bonds network, which greatly limits its applications. Thus, there has been a growing interest in the chemical modification of chitosan in order to improve its solubility and widen its applications [7,8]. Among the various modified products, carboxymethyl chitosan is a water soluble derivative of chitosan with several enhanced biological properties, such as antimicrobial, antioxidant and apoptosis inhibitory activities. And it has been considered as a promising candidate for different biomedical applications, including wound healing, tissue engineering, drug delivery, gene therapy and bioimaging [9]. Recently, synthesis of antioxidant–chitosan conjugation by grafting of antioxidant molecules onto chitosan chains has received increased attention. Phenolic acids, such as gallic acid (GA), caffeic acid (CA) and ferulic acid (FA) are all natural antioxidant molecules extractable from plants [10]. It has been reported that these antioxidant molecules can be grafted onto chitosan by the carbodiimide mediated coupling reaction, laccase catalyzed polymerization and free radical mediated reaction with enhanced antioxidant activity [11–16]. However, little attention has been paid to the grafting phenolic acids onto carboxymethyl chitosan [17]. In order to improve the solubility and antioxidant activity of carboxymethyl chitosan, three phenolic acids including GA, CA and FA were grafted onto N,O-carboxymethyl chitosan (NOCC) by a free radical mediated reaction in this study for the first time. The synthesized phenolic acids grafted NOCC was characterized by UV–vis, Fourier-transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction (XRD) to confirm

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the conjugation. The antioxidant activities in vitro of the grafted copolymers were also determined. The resulting data provide novel insight into the modification of NOCC and widen its applications as antioxidant agents. 2. Materials and methods 2.1. Materials and reagents Chitosan was purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). The degree of deacetylation and average molecular weight of chitosan were determined as 71% and 2.5 × 105 Da, respectively. Folin–Ciocalteau reagent, GA, CA, FA, pyrogallol, 2,2diphenyl-1-picrylhydrazyl (DPPH) were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade. 2.2. Synthesis of N,O-carboxymethylchitosan (NOCC) NOCC was synthesized by the method of Chen with some modifications [18]. Chitosan powder (10 g) was suspended in 100 ml of isopropyl alcohol and the resulting slurry was stirred in a 500 ml flask at room temperature. Then, 25 ml of 10 M NaOH solution was added to the stirred slurry over a period of 25 min. The alkaline slurry was stirred for additional 30 min. Subsequently, monochloroacetic acid (60 g) was added in five equal portions at 5 min intervals. The resulting mixture was heated at 60 ◦ C for 3 h, and then dialyzed against distilled water with a 14,000 Da molecular weight cut-off membrane for 72 h. Finally, the dialyzate was lyophilized to afford NOCC. 2.3. Preparation of phenolic acids grafted NOCC The preparation of NOCC grafted copolymers were performed by using ascorbic acid (Vc) and hydrogen peroxide (H2 O2 ) redox pair under inert atmosphere. Briefly, 0.5 g of NOCC was dissolved in 50 ml of 0.5% acetic acid solution (v/v) in a 0.5 L three-necked round bottom flask. Then, 0.1 g of Vc and 0.8 g of phenolic acid (GA or CA or FA) were added into the reactor. A slow stream of oxygen free nitrogen gas was passed through the reactor for 30 min with stirring. Afterwards, 2 ml of 5 M H2 O2 solution was added to initiate the reaction. The reaction was carried out under a continuous flow of oxygen free nitrogen gas for 12 h. Subsequently, the reaction mixture was dialyzed against distilled water with a 14,000 Da molecular weight cut-off membrane for 72 h to remove unreacted phenolic acid. Finally, the dialyzate was lyophilized to afford phenolic acid grafted NOCC, named GA-g-NOCC, CA-g-NOCC and FA-g-NOCC, respectively. 2.4. Determination of grafting ratio of phenolic acids grafted NOCC The grafting ratio of NOCC grafted copolymers was measured by the Folin–Ciocalteu method with slight modification [19]. Briefly, 10 mg of phenolic acid grafted NOCC was dissolved in 10 ml of distilled water. Afterwards, an aliquot of 1 ml of sample solution was mixed with 1 ml of Folin–Ciocalteu reagent (10 times dilution) and allowed to react at 30 ◦ C for 5 min in the dark. Then, 5 ml of saturated Na2 CO3 solution was added and the mixture was allowed to stand for 2 h before the absorbance of the reaction mixture was read at 760 nm. GA, CA and FA were used to calculate the standard curves, respectively. The grafting ratios of GA-g-NOCC, CA-g-NOCC and FA-g-NOCC were expressed as mg of GA equivalents per g (mg GAE/g), mg of CA equivalents per g (mg CAE/g) and mg of FA equivalents per g (mg FAE/g), respectively.

2.5. Characterization of NOCC and phenolic acids grafted NOCC Structural characterization of NOCC and phenolic acids grafted NOCC was determined by UV–vis, FT-IR, NMR and XRD spectra. The UV–vis spectra were determined by a Lambda 35 spectrophotometer (PerkinElmer Ltd., USA) by scanning from 200 to 500 nm. FT-IR spectra were recorded on a continuous scan Varian 670 FT-IR spectrometer (Varian Inc., USA) in the frequency range of 4000–400 cm−1 . Proton nuclear magnetic resonance (1 H NMR) spectra were recorded at 25 ◦ C for samples dissolved in CD3 COOD/D2 O (1%, v/v) using an AVANCE-600 spectrometer (Bruker Inc., Germany). Powder XRD spectra were acquired from 2 = 5–80◦ on a Bruker AXS D8 Advance X-ray diffractometer using Ni-filtered Cu K␣ radiation. 2.6. Assay of antioxidant activity in vitro 2.6.1. Assay of superoxide radical scavenging activity The superoxide radical scavenging activity was performed by the method of Jing and Zhao with some modifications [20]. Reaction was carried out in a mixture containing 4.5 ml of 50 mM Tris–HCl buffer (pH 8.2), 0.4 ml of 25 mM pyrogallol solution and 1 ml of sample (0.05–1 mg/ml) by incubating at 25 ◦ C for 5 min. Then, 1 ml of 8 mM HCl solution was dripped into the mixture promptly to terminate the reaction. The absorbance of the mixture was measured at 420 nm. Vc was used as the positive control. The superoxide radical scavenging activity was calculated by the following formula:



Scavenging activity (%) = 1 −

A1 − A2 A0



× 100

(1)

where A0 is the absorbance of the control (Tris–HCl buffer instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (Tris–HCl buffer instead of pyrogallol solution). The IC50 value represented the concentration of the compounds that caused 50% inhibition of superoxide radical formation. 2.6.2. Assay of hydroxyl radical scavenging activity The hydroxyl radical scavenging activity was determined according to the method of Zhong with some modifications [21]. The mixture containing 1 ml of sample (0.05–1 mg/ml), 1 ml of 9 mM FeSO4 and 1 ml of 0.3% H2 O2 in salicylic acid–ethanol solution was shaken vigorously and incubated at 37 ◦ C for 30 min. Then, the absorbance of the reaction mixture was determined at 510 nm. Vc was used as the positive control. The hydroxyl radical scavenging activity was calculated by the following formula:



Scavenging activity (%) = 1 −

A1 − A2 A0



× 100

(2)

where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (salicylic acid–ethanol solution instead of FeSO4 and H2 O2 solutions). The IC50 value represented the concentration of the compounds that caused 50% inhibition of hydroxyl radical formation. 2.6.3. Assay of H2 O2 scavenging activity The H2 O2 scavenging activity was determined according to the method of Ruch with some modifications [22]. The mixture containing 1 ml of sample (0.05–1 mg/ml), 2.4 ml of 0.1 M phosphate buffer (pH 7.4) and 0.6 ml of 40 mM H2 O2 solution was shaken vigorously and incubated at room temperature for 10 min. Then, the absorbance of the reaction mixture was determined at 230 nm. Vc

J. Liu et al. / International Journal of Biological Macromolecules 62 (2013) 85–93

87

(A) OH

OH

OH O

O O

O O

O OH

OH

OH NH2

NHCOCH3

NH2

n

Chitosan Chloroacetic acid

OH

NaOH/IPA

OCH2COO-

OH O

O O

O O

O OH

OH

OH

NH2

NHCH2COO-

NHCOCH3

NOCC

n

HO OCH2COO-

OH

OH

O

O O

O O

O OH

OH

OH NHCOCH3

NH

NHCH2COO-

n

NOCC macro radicals GA/CA/FA OH

OCH2COO-

OH O

O O

O O

O OH

OH

OH NHCOCH3

NHR

NHCH2COO-

n

GA-g-NOCC/CA-g-NOCC/FA-g-NOCC OH O

R=

OH

or

OH

C

OCH3

or

O

O

OH

OH

OH

(B) HO

H2O2

O

HO

OH

HO O

O

HO

O

O

HO

-H+

O

HO

pK = -0.86 HO

OH

Ascorbic acid (AscH2)

O

OH

AscH

O

O

Asc

Fig. 1. The proposed mechanisms for the synthesis of phenolic acids grafted NOCC. (A) Synthesis of NOCC and conjugation of phenolic acids onto NOCC by free radical mediated graft copolymerization. (B) Procedure for the generation of hydroxyl radical.

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was used as the positive control. The H2 O2 scavenging activity was calculated as follows: A1 − A2 A0



× 100

1.6

(3)

where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (phosphate buffer instead of H2 O2 solution). The IC50 value represented the concentration of the compounds that caused 50% inhibition of H2 O2 .

Absorbance



Scavenging activity (%) = 1 −

2.0

1.2

0.8 287

0.4

2.6.4. Assay of DPPH radical scavenging activity The DPPH radical scavenging activity was assayed according to the method of Shimada with some modifications [23]. Briefly, 0.2 ml of DPPH solution (0.4 mM DPPH in methanol) was mixed with 1.0 ml of sample (0.05–1 mg/ml) and 1.8 ml of water. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then, the absorbance of the mixture was measured at 517 nm. Vitamin C (Vc) was used as the positive control. The DPPH radical scavenging activity was calculated by the following formula: A1 − A2 A0



× 100

(4)

where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (water instead of DPPH). The IC50 value represented the concentration of the compounds that caused 50% inhibition of DPPH radical formation. 2.6.5. Assay of lipid peroxidation inhibition effect The lipid peroxidation inhibition effect was determined by thiobarbituric acid reactive substances (TBARS) assay using mouse liver homogenate as the lipid rich media with some modification [24]. Briefly, 1 ml of sample (0.05–1 mg/ml) was mixed with 1 ml of 1% liver homogenate (each 100 ml homogenate solution contains 1 g mouse liver), then 0.05 ml of 0.5 mM FeCl2 and 0.5 mM H2 O2 were added to initiate lipid peroxidation. After incubation at 37 ◦ C for 1 h, 1.5 ml of 20% trichloroacetic acid (w/v) and 1.5 ml of 0.8% thiobarbituric acid solution (w/v) were added to quench the reaction. The resulting mixture was heated at 100 ◦ C for 15 min, and then centrifuged at 5000 rpm for 30 min. The absorbance of the upper layer was measured at 532 nm. Vc was used as the positive control. The inhibition effect on lipid peroxidation was calculated as follows:



Inhibition effect (%) = 1 −

A1 − A2 A0



× 100

2.6.6. Assay of reducing power The reducing power was determined according to the method of Oyaizu with some modifications [25]. Reaction was carried out in a mixture containing 2.5 ml of sample (0.05–1 mg/ml), 2.5 ml of 0.1 M sodium phosphate buffer (pH 6.6) and 2.5 ml of K3 Fe(CN)6 (1%, w/v) by incubating at 50 ◦ C for 20 min. After addition of 2.5 ml trichloroacetic acid (10%, w/v), the mixture was centrifuged at 5000 × g for 10 min. The upper layer (5 ml) was mixed with 0.5 ml of fresh FeCl3 (0.1%, w/v), and the absorbance at 700 nm was measured against a blank. Vc was used as the positive control. The IC50 value represented the concentration of the compounds that provided 0.5 of absorbance.

318

FA-g-NOCC GA-g-NOCC

chitosan

200

300

400

500

Wavelength (nm)

Fig. 2. UV–vis spectra of chitosan, NOCC and phenolic acids grafted NOCC.

2.7. Statistical analysis Data were expressed as mean ± standard deviation (SD) of triplicates. The Duncan test and one-way analysis of variance (ANOVA) were used for multiple comparisons by the SPSS 13.0 software package. Difference was considered to be statistically significant if p < 0.05. 3. Results and discussion 3.1. Preparation of phenolic acids grafted NOCC In this study, three phenolic acids grafted NOCC were synthesized by using Vc and H2 O2 redox pair under inert air. The possible mechanism for the conjugation of phenolic acids with NOCC was proposed in Fig. 1. Firstly, NOCC was synthesized in isopropyl alcohol/NaOH/monochloroacetic acid system (Fig. 1A). Then, Vc reacted with H2 O2 to generate hydroxyl radical (HO• ) which subsequently initiated polymerization (Fig. 1B). The formed HO• could abstract hydrogen atom from NOCC molecules with consequent formation of NOCC macro radicals. The phenolic acids which were in close vicinity of the reaction site become acceptor of chitosan macro radicals, and thus phenolic acids grafted NOCC formed (Fig. 1A) [14,26]. The obtained NOCC grafted copolymers were all watersoluble samples. The grafting ratios of grafted copolymers were

(5)

where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (water instead of FeCl2 and H2 O2 ). The IC50 value represented the concentration of the compounds that caused 50% inhibition of lipid peroxidation.

CA-g-NOCC 285

NOCC 0

(A)

1595 1650

(B) Transmittance (%)



Scavenging activity (%) = 1 −

320

273

(C)

1320

1411 1601

(D)

1411 1601

(E)

1411 1601 1411 1601

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 3. FT-IR spectra of chitosan (A), NOCC (B), GA-g-NOCC (C), CA-g-NOCC (D) and FA-g-NOCC (E).

J. Liu et al. / International Journal of Biological Macromolecules 62 (2013) 85–93

Fig. 4.

1

H spectrum of chitosan (A), NOCC (B), GA-g-NOCC (C), CA-g-NOCC (D) and FA-g-NOCC (E).

89

90

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calculated by determining of phenolic contents in the grafted products. The grafting ratios of GA-g-NOCC, CA-g-NOCC and FA-g-NOCC were respectively determined as 45.8 mg CAE/g, 38.5 mg CAE/g and 36.7 mg FAE/g. The grafting ratios were much higher than chitosan grafted copolymers synthesized by adopting Vc and H2 O2 system under atmospheric air [14]. It was probably due to that oxygen existing in the reaction mixture served as a polymerization inhibitor. The graft polymerization catalyst was inhibited in the presence of oxygen as a radical capture under air atmosphere. Thus, our results suggested that Vc and H2 O2 redox pair under inert air could act as an effective radical initiator system for the syntheses of NOCC grafted copolymers. 3.2. Characterization of phenolic acid grafted NOCC 3.2.1. UV–vis spectra The UV–vis spectra of chitosan, NOCC and NOCC grafted copolymers were shown in Fig. 2. Chitosan and NOCC both showed no absorption peak ranging from 240 to 500 nm. GA exhibited two characteristic absorption bands at 212 nm and 262 nm, respectively, which should be assigned to the ␲-system of the benzene ring. Similarly, CA exhibited one band at 286 and another at 311 nm, whilst FA exhibited one at 285 and another at 312 nm (data not shown). When three phenolic acids were introduced into NOCC molecular chain as a graft, the UV–vis absorption peaks shifted toward longer wavelengths. These red shifts can be attributed to the less amount of energy required for the n–␲* and ␲–␲* transition due to the covalent linkage of phenolic acids with NOCC [13]. These results indicated that phenolic acids had been successfully grafted onto NOCC. This was consistent with Yu, who also observed the red shift in GA-g-NOCC synthesized by the carbodiimide mediated coupling reaction [17].

Fig. 5. XRD spectra of chitosan (A), NOCC (B), GA-g-NOCC (C), CA-g-NOCC (D) and FA-g-NOCC (E).

GA-g-NOCC showed a new peak at 6.9 ppm belonging to the phenyl protons of GA [15,17]. CA-g-NOCC and FA-g-NOCC exhibited new peaks at 6.2–7.6 ppm assigned to the methine protons of CA and FA, respectively [12,16]. Theses results further confirmed the successful grafting of phenolic acids onto NOCC.

3.2.2. FT-IR spectra The FT-IR spectra of chitosan, NOCC and NOCC grafted copolymers were shown in Fig. 3. For chitosan, the band at 1595 cm−1 was assigned to the N H bending of the primary amine. The bands at 1650, 1550 and 1320 cm−1 were attributed to the C O stretching (amide I), N H bending (amide II) and C N stretching (amide III) of the residual N-acetyl groups, respectively (Fig. 3A). This also indicated that chitosan was not fully deacetylated. In the FT-IR spectra of NOCC, two new bands at 1601 and 1411 cm−1 could be assigned to the asymmetry and symmetry stretch vibration of COO− , respectively (Fig. 3B). The result also suggested that the carboxymethyl substitution occurred at the C-6 position of chitosan [18,27]. As compared with NOCC, its grafted copolymers exhibited additional phenolic characteristics of the aromatic ring C C stretching within 1450–1650 cm−1 , causing the bands around 1601 and 1411 cm−1 becoming broader (Fig. 3C–E). This indicated that phenolic acids had been successfully grafted onto NOCC backbones. However, no additional band appeared at 1730 cm−1 in the grafted products, indicating no ester bond formed between hydroxyl groups of NOCC and carboxyl groups of phenolic acids [15]. Therefore, the conjugation of phenolic acids on NOCC probably occurred at C-2 position via the formation of amide linkage (as shown in Fig. 1).

3.2.4. Crystallographic structures The crystallographic structures of chitosan, NOCC and NOCC grafted copolymers were determined by XRD. As shown in Fig. 5, the diffraction pattern of original chitosan showed a characteristic peak at 2 = 20◦ , corresponding to its crystal form II [28]. For NOCC, the peak at 2 = 20◦ became broader and weaker. These indicated that chitosan and NOCC appeared to be semicrystalline and amorphous, respectively. However, GA-g-NOCC, CA-g-NOCC and FA-g-NOCC exhibited broader and weaker peaks at 2 = 23.1◦ , 23.6◦ and 22.4◦ , respectively, confirming the conjugation of phenolic acids with NOCC. The XRD results also revealed that the introduction of phenolic acids onto NOCC caused a remarkable decrease in crystallinity of the NOCC grafted copolymers. This suggested that inter- and intra-molecular hydrogen bonds of original NOCC had been greatly decreased after grafting, causing the increase in the solubility of grafted products.

3.2.3. 1 H NMR spectrum Further characterization of chitosan, NOCC and NOCC grafted copolymers was performed by using 1 H NMR. As shown in Fig. 4, Chitosan exhibited a single peak at 2.9 ppm (H-2), multiple peaks at 3.3–3.7 ppm (H-3 to H-6) and a small single peak at 4.4 ppm (H-1). The single peak at 1.8 ppm represents three protons of Nacetyl glucosamine units. In the spectrum of NOCC, the signals were considerably broader and less well-resolved than unmodified chitosan. The chemical shifts at around 4.4 ppm were attributed to the protons of CH2 COO− groups. As compared to NOCC,

3.3.1. Scavenging activity on superoxide radical Superoxide radical is considered as the primary ROS. It can interact with other molecules to generate secondary ROS, including hydroxyl radical, H2 O2 and singlet oxygen. Consequently, the formation of superoxide radical could induce oxidative damage in lipids, proteins and DNA [3]. The superoxide radical scavenging activities of chitosan, NOCC and its grafted copolymers were shown in Fig. 6A. The scavenging activities of all samples were correlated well with the increase of concentrations. This indicated that chitosan, NOCC and its grafted copolymers all had scavenging

3.3. Antioxidant activity in vitro of CA-g-chitosan and FA-g-chitosan

J. Liu et al. / International Journal of Biological Macromolecules 62 (2013) 85–93

(C)

100

100

80

80

Scavenging activity on H2O2 (%)

Scavenging activity on superoxide radical (%)

(A)

60 40 20

60 40 20

0

0 0

0.2

0.4

0.6

0.8

1

0.2

0

0.4

Concentration (mg/ml)

0.6

0.8

1

0.8

1

Concentration (mg/ml)

(D)

100 80

100

Scavenging activity on DPPH radical (%)

Scavenging activity on hydroxyl radical (%)

(B)

91

60 40 20

80 60 40 20

0

0 0

0.2

0.4

0.6

0.8

1

0.2

0

Concentration (mg/ml)

(E)

0.4

0.6

Concentration (mg/ml)

Inhibition effect on lipid peroxidation (%)

100 80 60 40 20 0 0

0.2

0.4

0.6

0.8

1

0.8

1

Concentration (mg/ml)

Absorbance at 700 nm

(F)

1.8 1.5 1.2 0.9 0.6 0.3 0 0

0.2

0.4

0.6

Concentration (mg/ml) Fig. 6. The superoxide radical (A), hydroxyl radical (B), H2 O2 (C) and DPPH radical scavenging activities (D), as well as lipid peroxidation inhibition effect (E) and reducing power (F) of chitosan (--), NOCC (--), FA-g-NOCC (--), CA-g-NOCC (-䊉-), GA-g-NOCC (--) and Vc (--). Data are presented as means ± SD of triplicates.

activity on superoxide radical. At the concentration of 1 mg/ml, the scavenging activities for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-g-NOCC were 35.18%, 42.51%, 55.36%, 62.21% and 73.85%, respectively. However, the scavenging activity of Vc was much higher than NOCC and its grafted copolymers, with the scavenging activity of 97.91% at 1 mg/ml. Statistical analysis showed that the

scavenging activity decreased in the order of Vc > GA-g-NOCC > CAg-NOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). Accordingly, the IC50 values for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC, GA-gNOCC and Vc were 2.12, 1.22, 0.85, 0.61, 0.32 and 0.05 mg/ml, respectively. Notably, the differences in scavenging activity of the three phenolic acids grafted NOCC may be due to their different

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chemical structures. Gallic acid is a trihydroxybenzoic acid, while caffeic acid is a dihydroxycinnamic acid and ferulic acids is a hydroxycinnamic acid. Consequently, more hydroxyl groups in the aromatic ring probably caused increased scavenging activity on superoxide radical. Thus, the scavenging activity decreased in the order of GA-g-NOCC > CA-g-NOCC > FA-g-NOCC. 3.3.2. Scavenging activity on hydroxyl radical Hydroxyl radical can attack and damage almost every biomacromolecule in living cells. When hydroxyl radical is generated close to membranes, it can attack the fatty acid side chains of the membrane phospholipids [19]. As shown in Fig. 6B, the scavenging activities of all samples increased with the increase of concentrations. At the concentration of 1 mg/ml, the scavenging activities for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-g-NOCC were 29.13%, 35.90%, 47.12%, 61.54% and 72.76%, respectively. In addition, Vc showed higher hydroxyl radical scavenging activity than NOCC and its grafted copolymers. Statistical analysis showed that the scavenging activity decreased in the order of Vc > GA-gNOCC > CA-g-NOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). The IC50 values for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC, GA-gNOCC and Vc were 5.07, 2.62, 1.46, 0.65, 0.36 and 0.09 mg/ml, respectively. 3.3.3. Scavenging activity on H2 O2 H2 O2 plays an important role as the radical-forming intermediate in the production of ROS molecules. It can react with Fe2+ or superoxide anion radical in the cell to form hydroxyl radical [29]. As depicted in Fig. 6C, chitosan, NOCC and its grafted copolymers all exerted concentration-dependent H2 O2 scavenging activities. At the concentration of 1 mg/ml, the H2 O2 scavenging activities for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-gNOCC were 5.44%, 44.83%, 58.62%, 62.07% and 75.86%, respectively. However, Vc exhibited higher H2 O2 scavenging activity than NOCC and its grafted copolymers. Statistical analysis showed that the scavenging activity decreased in the order of Vc > GA-g-NOCC > CAg-NOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). The IC50 values for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC, GA-g-NOCC and Vc were 432.92, 1.55, 0.71, 0.50, 0.26 and 0.07 mg/ml, respectively. 3.3.4. DPPH scavenging activity In the DPPH assay, antioxidants are able to reduce the stable DPPH radical (purple) to the non-radical form DPPH-H (yellow). As shown in Fig. 6D, the scavenging activities on DPPH radicals of chitosan, NOCC and its grafted copolymers increased with the increase of concentrations for all the samples. At a concentration of 1 mg/ml, the DPPH scavenging activities for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-g-NOCC were 26.11%, 51.85%, 59.26%, 64.81% and 74.54%, respectively. The scavenging activities of NOCC and its grafted copolymers were lower than Vc. Statistical analysis showed that the scavenging activity decreased in the order of Vc > GA-gNOCC > CA-g-NOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). The IC50 values for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC, GA-gNOCC and Vc were 29.60, 1.03, 0.59, 0.36, 0.23 and 0.03 mg/ml, respectively. 3.3.5. Lipid peroxidation inhibition effect Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acids in the cell membranes that generates a number of degradation products [30]. The lipid peroxidation inhibition effects of all samples increased with the increase of concentrations as shown in Fig. 6E. At the concentration of 1 mg/ml, the inhibition effects for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-g-NOCC were 35.88%, 50.54%, 65.88%, 71.30% and 80.32%, respectively. As the positive control, Vc showed higher lipid peroxidation inhibition effect than chitosan and its derivatives. Statistical analysis showed that

the inhibition effect decreased in the order of Vc > GA-g-NOCC > CAg-NOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). The IC50 values for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC, GA-g-NOCC and Vc were 3.59, 0.95, 0.40, 0.29, 0.19 and 0.08 mg/ml, respectively. 3.3.6. Reducing power The reducing capacity of a compound is a significant indicator of its potential antioxidant activity. As shown in Fig. 6F, the reducing power of all samples increased with the increase of concentrations. At the concentration of 1 mg/l, the reducing power for chitosan, NOCC, FA-g-NOCC, CA-g-NOCC and GA-g-NOCC were 0.11, 0.27, 0.44, 0.49 and 0.65, respectively. Vc showed reducing power of 1.6 at 0.5 mg/ml. Statistical analysis showed that the reducing power decreased in the order of Vc > GA-g-NOCC > CA-gNOCC > FA-g-NOCC > NOCC > chitosan (p < 0.05). The IC50 values for chitosan, CA-g-chitosan, FA-g-chitosan and Vc were 1358.57, 6.02, 1.57, 1.07, 0.56 and 0.04 mg/ml, respectively. All the antioxidant activity in vitro assays above suggested that antioxidant activity of chitosan and NOCC were greatly enhanced by graft copolymerization with phenolic acids. The enhanced bioactivities were probably due to the introduction of chemically modified phenolic acids groups onto NOCC that increased the hydrogen or electron donating ability of NOCC. 4. Conclusion The preparation, characterization and antioxidant activity of phenolic acids grafted NOCC were investigated in the present study. Results suggested that the phenolic acids could be easily grafted onto NOCC by using Vc and H2 O2 redox pair system under inert atmosphere. In addition, the conjugation probably occurred at amine of NOCC and carboxyl groups of phenolic acids with formation of amide linkages. Statistical analysis also indicated that antioxidant activity in vitro of NOCC was greatly enhanced by grafting with phenolic acids. Our data provided a novel approach for the modification of NOCC and indicated phenolic acids grafted NOCC could be explored as promising antioxidant agents. Acknowledgements This work was partly supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31101216) and the Natural Science Foundation of the Education Committee of Jiangsu Province (No. 11KJB550006), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors thank the testing center of Yangzhou University for NMR measurements. References [1] H.E. Seifried, D.E. Anderson, E.I. Fisher, J.A. Milner, J. Nutr. Biochem. 18 (2007) 567–579. [2] M. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur, J. Telser, Int. J. Biochem. Cell Biol. 39 (2007) 44–84. [3] J. Liu, J. Luo, H. Ye, X. Zeng, Food Chem. Toxicol. 50 (2012) 767–772. [4] M.L. Jin, Z.Q. Lu, M. Huang, Y.M. Wang, Y.Z. Wang, Int. J. Biol. Macromol. 48 (2011) 607–612. [5] P. Shao, M. Chen, Y. Pei, P. Sun, Int. J. Biol. Macromol. 59 (2013) 295–300. [6] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632. [7] N.M. Alves, J.F. Mano, Int. J. Biol. Macromol. 43 (2008) 401–414. [8] V.K. Mourya, N.N. Inamdar, React. Funct. Polym. 68 (2008) 1013–1051. [9] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Carbohydr. Polym. 91 (2013) 452–466. [10] R.J. Robbins, J. Agric. Food Chem. 51 (2003) 2866–2887. [11] A. Aljawish, I. Chevalot, B. Piffaut, C. Rondeau-Mouro, M. Girardin, J. Jasniewski, Carbohydr. Polym. 87 (2012) 537–544. [12] A.O. Aytekin, S. Morimura, K. Kida, J. Biosci. Bioeng. 111 (2011) 212–216. [13] M. Bozic, S. Gorgieva, V. Kokol, Carbohydr. Polym. 87 (2012) 2388–2398. [14] M. Curcio, F. Puoci, F. Iemma, O.I. Parisi, G. Cirillo, U.G. Spizzirri, J. Agric. Food Chem. 57 (2009) 5933–5938.

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