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Phenolic antioxidants-functionalized quaternized chitosan: Synthesis and antioxidant properties
International Journal of Biological Macromolecules 53 (2013) 77–81
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Phenolic antioxidants-functionalized quaternized chitosan: Synthesis and antioxidant properties Jianming Ren a,b , Qing Li a , Fang Dong a , Yan Feng a,b , Zhanyong Guo a,∗ a b
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Graduate School of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 25 September 2012 Received in revised form 30 October 2012 Accepted 8 November 2012 Available online 16 November 2012 Keywords: Quaternized chitosan Antioxidant Antioxidant–polymer conjugates
a b s t r a c t In this work, two kinds of phenolic antioxidants-functionalized quaternized chitosan were synthesized in order to develop aqueous soluble antioxidant–polymer conjugates. Quaternized chitosan conjugated with gallic acid or caffeic acid was carried out and the antioxidant properties of the products (namely gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan) against hydroxyl-radical, superoxideradical and DPPH-radical were evaluated in vitro, respectively. The scavenging activities of the obtained gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan exhibit a remarkable improvement over those of either chitosan or quaternized chitosan. And the scavenging effect indices of the products were all higher than 90% at a concentration of 1000 g/mL. Because gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan are convenient to prepare and possess improved potential activities, these materials may represent an attractive new platform for utilizations of chitosan. © 2012 Published by Elsevier B.V.
1. Introduction Reactive oxygen species (ROS), including superoxide anion (O2 •), hydroxyl radicals (•OH) and hydrogen peroxide (H2 O2 ), can cause pathological changes like cancer disease, diabetes in biological systems and lead to harmful alterations in foods and pharmaceutical industries [1]. As strong antioxidants and excellent free radical scavengers, most phenolic compounds could effectively prevent or slower those deleterious reactions [2,3]. Among phenolic compounds, gallic and caffeic acids have attracted much attention as promising antioxidant agents applicable to play a significant role in health-promoting benefits [4,5]. Unlike currently widely used synthetic antioxidant such as butylated hydroxyanisole and butylated hydroxytoluene, which have potential health hazards and toxicity, gallic and caffeic acids are both obtained from nature, and have been recognized as safety biological active molecules. However, sometimes it is lastly recommendable to directly employ gallic and caffeic acids in foods and pharmaceutical as antioxidant agents because they may result in undesirable reactions and change the chemical compositions of them. In recent years many reports proposed that appropriate macromolecular systems, namely antioxidant–polymer conjugates, could combine the merits of both components, which could retain the excellent biological activities of antioxidant molecules and show a higher stability and a slower degradation rate than the antioxidant molecules
∗ Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000. E-mail address: [email protected] (Z. Guo). 0141-8130/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2012.11.011
[6–8]. To synthesize antioxidant–polymer conjugates basing gallic and caffeic acids, chitosan is an ideal candidate for the polymer matrix. Chitosan, a kind of renewable, abundant natural polysaccharide, consists primarily of 2-amino-2-deoxy-glucopyranose units linked by -(1-4) linkage [9]. It has already been well studied and sufficiently reported that chitosan is non-toxic, biodegradable, and biocompatible, which help it to earn much scientific research attentions [10]. Meanwhile, known as a bioactive macromolecule, chitosan exhibits many bioactivities such as antitumor property, antifungal and antimicrobial activity and so on [11,12]. What is more, apart from these interesting properties, chitosan possesses antioxidant property too, which further makes this natural, renewable, bioactive polymeric material is suitable to be the polymer part of the “antioxidant–polymer conjugates”[13]. Our previous work has found that the antioxidant activity of chitosan is related to the forms of nitrogen atoms in chitosan molecules and chitosan quaternizied by methyl groups (N,N,N-trimethylchitosan) could obviously improve the antioxidant activity of chitosan [1,14]. In addition, better than chitosan, N,N,N-trimethylchitosan (TMC) is soluble in neutral and alkaline aqueous solutions, which can enlarge the applications of chitosan as a food preservative or bioactive matrix. Therefore, it is reasonable to propose that the coupling of TMC with gallic or caffeic acids is promisingly to give a stable, effective and water-soluble antioxidant–polymer conjugate. Given these characteristics of chitosan, TMC, gallic acid and caffeic acid, this paper concentrated on the development of two kinds of novel gallic acid-quaternized chitosan (G-TMC) and caffeic acid-quaternized chitosan (C-TMC) aqueous soluble antioxidant
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J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81
conjugates. The antioxidant activity of the synthesized conjugates was evaluated in vitro and compared to that of chitosan and TMC.
where Ablank 520 nm was the absorbance of the blank (distilled water instead of the samples) and Acontrol 520 nm was the absorbance of the control (distilled water instead of the H2 O2 ).
2. Experimental 2.1. Materials and methods Chitosan was purchased from Qingdao Baicheng Biochemical Corp. (China). Its degree of deacetylation was 97%, and the viscosityaverage molecular weight was 7.0 × 104 . Gallic acid and caffeic acid were purchased from the Sigma–Aldrich Chemical Co. (United States). The other reagents were all analytical grades and were used without further purification. The IR spectra were measured on a Jasco-4100 FT-TR spectrometer with KBr disks. The UV spectra were recorded on a Puxi-Tu1810 UV spectrometer and the elemental analyses (C, H, N) were performed on a Carlo-Erba 1106 elemental analyzer.
Scavenging effect (%) = 1 −
Asample 560 nm
Acontrol 560 nm
× 100
Acontrol 560 nm was the absorbance of the control (distilled water instead of the samples).
2.2. Synthesis 2.2.1. Preparation of TMC TMC was synthesized as follows: 3 g chitosan was dispersed in 100 mL of water, and formaldehyde (3 equiv. based on glucosamine units of chitosan) was added, and the mixture was stirred at room temperature. After 2 h, 10% NaBH4 (1.5-fold excess relative to mole mass of formaldehyde) was added, and the solution reacted for 2 h. The mixture was precipitated in acetone and filtered. The Nmethylchitosan was obtained after drying at 60 ◦ C in vacuum for 12 h. N-methylchitosan (1 g) was dispersed in 50 mL of N-methyl2-pyrrolidone (NMP) for 12 h at room temperature. Then, 0.5 mL NaOH (1 M), 1 g NaI and 4 mL CH3 I were added. The resulting mixture was heated to 60 ◦ C and stirred for 20 h. The product was obtained by precipitation with excess acetone, and the TMC was obtained by drying at 60 ◦ C in vacuum for 12 h. 2.2.2. Synthesis of antioxidant conjugates Gallic acid and caffeic acid were firstly transformed to reactive chloride form using thionyl chloride, in order to conveniently combine them with TMC. Gallic acid or caffeic acid (10 mmol) and thionyl chloride (100 mmol) were added to a dry flask with anhydrous THF (200 mL) under nitrogen. After the reaction was refluxed for 2 h, the excess thionyl chloride and THF were removed under reduced pressure and TMC (3 mmol) dispersed in NMP (150 mL) was added in. Then, the resulting mixture was stirred for 24 h at 80 ◦ C and the product was obtained by precipitation with excess acetone. The product was washed by ethanol and acetone, Soxhlet extracted with ethanol for 24 h and freeze dried. 2.3. The investigation of the antioxidant ability 2.3.1. Hydroxyl-radical scavenging ability assay The test of the hydroxyl-radical scavenging ability was carried out according to Liu’s methods [1]. The reaction mixture, a total volume 4.5 mL, containing the samples of the synthesized antioxidant conjugates, TMC and chitosan, was incubated with EDTA–Fe2+ (220 M), safranine O (0.23 M), H2 O2 (60 M) in potassium phosphate buffer (150 mM, pH 7.4) for 30 min at 37 ◦ C. The absorbance of the mixture was measured at 520 nm. Three replicates for each sample concentration were tested. Hydroxyl radicals bleached the safranine O, so increased absorbance of the reaction mixture indicated decreased hydroxyl radicals scavenging ability and the scavenging effect of the product was computed using the follow equation: Scavenging effect (%) =
2.3.2. Superoxide-radical scavenging ability assay The superoxide radical scavenging ability was assessed following the model of Xing [15]. Involving testing samples, phenazine mothosulfate (30 M), nicotinamide adenine dinucleotide reduced (338 M), and nitro blue tetrazolium (72 M) in phosphate buffer (0.1 M, pH 7.4), the reaction mixture was incubated at 25 ◦ C for 5 min and 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:
Asample 520 nm − Ablank 520 nm Acontrol 520 nm − Ablank 520 nm
× 100
2.3.3. DPPH-radical scavenging ability assay The DPPH• scavenging properties of the products were quantified by the following method: testing samples and 2 mL ethanol solution of DPPH (180 mol/L) was incubated for 30 min at 25 ◦ C in a water bath. Then, the absorbance of the remained DPPH 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 (%) =
Acontrol 517 nm − Asample 517 nm Acontrol 517 nm
× 100
Acontrol 560 nm was the absorbance of the control (distilled water instead of the samples). 2.4. Statistical analysis All data are expressed as means ± SD. Data were analyzed by an analysis of variance (P < 0.05) and the means were separated by Duncan’s multiple range test. The results were processed by the computer programs: Excel and Statistica software SPSS. 3. Results and discussion 3.1. Chemical syntheses and characterization Two kinds of water-soluble antioxidant–polymer conjugates containing the antioxidative groups of gallic acid or caffeic acid were constructed. The TMC was chosen as polymer part of the “antioxidant–polymer conjugates”, which is by virtue of its various unique qualities including stability, water-solubility, biocompatibility, antioxygenic property and so on. The conjugates of TMC and gallic or caffeic acids were synthesized following the route outlined in Scheme 1 (Scheme 1). The synthesized products were characterized by FT-IR (Fig. 1), UV (Fig. 2) and elemental analyses (Table 1). In Fig. 1, the FT-IR spectra present the comparison of transmission spectra data of the synthesized products with that of original chitosan. Chitosan was typically characterized by absorption regions at about 896, 1087 and 1600 cm−1 , which belongs to pyranose ring, glucoside and amine groups, respectively [16]. When chitosan was transformed to TMC, new peaks appeared at about 1660 cm−1 , which were assigned to the quaternary ammonium salt. There were peaks at about 1415–1430 cm−1 , which were ascribed to the characteristic absorb of N-CH3 [1]. After gallic or caffeic acids were combined with TMC through ester bond, strong new
J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81
79
Fig. 3. Hydroxyl radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
Fig. 1. FT-IR spectra of chitosan (1), TMC (2), G-TMC (3) and C-TMC (4).
Table 1 The elemental analyses results and the substitution degree of chitosan, TMC, G-TMC and C-TMC. Compounds
Chitosan TMC G-TMC C-TMC
Elemental analyses (%)
Substitution degreea
C
N
H
40.85 41.63 45.80 49.85
7.50 5.87 4.37 4.01
7.03 7.21 6.19 6.52
0.75 0.72
a Substitution degree refers to the C-6-O substitution degree of gallic acid in GTMC and caffeic acid in C-TMC.
peaks were observed at 1739 and 1720 cm−1 respectively, which correspond to C O vibrations [6]. Meanwhile, the aromatic C C vibrations were found at 748 and 1511 cm−1 in the FT-IR spectra of gallic acid functionalized TMC and at 744 and 3006 cm−1 in that of caffeic acid–TMC conjugate [6] (Fig. 1). Fig. 2 gives the UV spectra of each conjugate (0.5 mg/mL) with same concentration of TMC as baseline to remove the probable interference of TMC. As in Fig. 2, the UV spectra of both products show evident absorptions peaks in the aromatic region, which are attributed to the presence of gallic acid or caffeic acid respectively (Fig. 2). Results aforementioned results demonstrated that the aimed phenolic antioxidants-functionalized TMC were obtained. Finally, the results of elemental analyses are listed in Table 1 and the substitution degree of the products could be computed according to the percentage of N from the elemental analyses (Table 1). 3.2. Antioxidant activities Under normal condition, direct addition of Fe2+ to a reaction mixture containing phosphate buffer generates hydroxyl radicals which are harmful to body through reacting with such biological molecule as: amino acids or DNA. Fig. 3 reveals the •OH scavenging ability of the synthesized conjugates, TMC, and chitosan at various concentrations. According to the graph we could conclude the results as follows: firstly, all the scavenging effects of samples have a positive correlation with tested concentrations. Secondly, TMC has a better antioxidant activity than that of chitosan, which has
Fig. 2. UV spectra of water, G-TMC and C-TMC.
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O HOH2C
O
HOH2C O NaI NaOH n
R O
CH3I
OH NH2
C
SOCl2
O n
OH2C
O
O n
RCOOH OH N
OH N OH
R=
OH
or
OH
OH OH
Scheme 1. Synthesis pathway of G-TMC and C-TMC.
been reported and discussed in our previous work. Thirdly, of all the tested samples, the conjugate of gallic acid-TMC (G-TMC) and caffeic acid-TMC (C-TMC) exhibit the best scavenging ability against •OH IC50 value of G-TMC and C-TMC was 0.13 and 0.14 mg/mL respectively. IC50 , a good parameter to evaluate the scavenging activity, means the conjugates concentration to reduce the radical by 50%. Finally, significant scavenging effect (72.08–83.74%) of OH radicals was evident at tested concentrations of conjugates, which suggests the potential of the products to be developed as hydroxyl-radical scavenging reagents of food industry. It is reasonable to propose that the enhanced scavenging capability may mainly benefit from the phenolic compounds, specifically gallic and
caffeic acids, grafted on TMC since these phenolic compounds are effective antioxidant agents (Fig. 3). The scavenging properties of the chitosan, TMC and conjugates against superoxide-radical and DPPH radical are shown in Figs. 4 and 5, respectively. Similarly, G-TMC and C-TMC possess marked improved antioxidant activity than those of chitosan and TMC. It should be for the same reason that the increased antioxidant activity is primarily from the grafted phenolic compounds, specifically gallic and caffeic acids in this study (Figs. 4 and 5). 4. Conclusions In summary, in this paper, two kinds of antioxidant–polymer conjugates which are based on quaternized chitosan and natural products namely gallic acid and caffeic acid were synthesized via a convenient reaction, and their antioxidant activities against sorts of radicals was evaluated in vitro, respectively. For the synthesis of antioxidant–polymer conjugates, we have had gallic acid and caffeic acid transformed to reactive chloride form using thionyl chloride firstly, and then the obtained chloride could combine with TMC in an elegant way. The antioxidant assays in vitro models suggested that when grafted to quaternized chitosan, gallic acid or caffeic acid could enhance the antioxidant activities of TMC effectively. The antioxidant data suggested that to develop appropriate macromolecular antioxidant–polymer conjugates, the phenolic compounds associate with TMC is a promising way. However, comprehensive studies need to be carried out to ascertain the safety of the conjugates in experimental animal models and field experiments.
Fig. 4. Superoxide-radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
Acknowledgement This work was supported by the 12th Five-Year Science and Technology Plan for Agriculture (2012BAD32B09). References
Fig. 5. DPPH-radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
[1] J. Liu, H. Sun, F. Dong, Q. Xue, G. Wang, S. Qin, Z. Guo, Carbohydrate Polymers 78 (2009) 439–443. [2] R. Zadernowski, M. Naczk, J. Nesterowicz, Journal of Agricultural and Food Chemistry 53 (2005) 2118–2124. [3] M.P. Kahkonen, A.I. Hopia, H.J. Vuorela, J.-P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, Journal of Agricultural and Food Chemistry 47 (1999) 3954–3962. [4] Z. Lu, G. Nie, P.S. Belton, H. Tang, B. Zhao, Neurochemistry International 48 (2006) 263–274. [5] I. Gulcin, Toxicology 217 (2006) 213–220. [6] D. Arrua, M.C. Strumia, M.A. Nazareno, Journal of Agricultural and Food Chemistry 58 (2009) 9228–9234. [7] M. Curcio, F. Puoci, F. Iemma, O.I. Parisi, G. Cirillo, U.G. Spizzirri, N. Picci, Journal of Agricultural and Food Chemistry 57 (2009) 5933–5938. [8] W. Pasanphan, G.R. Buettner, S. Chirachanchai, Journal of Applied Polymer Science 109 (2008) 38–46.
J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81 [9] N. Bordenave, S. Grelier, V. Coma, Biomacromolecules 9 (2008) 2377–2382. [10] V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, International Journal of Pharmaceutics 274 (2004) 1–33. [11] C. Porporatto, I.D. Bianco, C.M. Riera, S.G. Correa, Biochemical and Biophysical Research Communications 304 (2003) 266–272. [12] Z. Zhong, R. Chen, R. Xing, X. Chen, S. Liu, Z. Guo, X. Ji, L. Wang, P. Li, Carbohydrate Research 342 (2007) 2390–2395.
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[13] R. Xing, S. Liu, H. Yu, Q. Zhang, Z. Li, P. Li, Carbohydrate Research 339 (2004) 2515–2519. [14] Z. Guo, R. Xing, S. Liu, Z. Zhong, P. Li, Carbohydrate Polymers 73 (2008) 173–177. [15] R.E. Xing, S. Liu, Z.Y. Guo, H.H. Yu, C.P. Li, X. Ji, J.H. Feng, P.C. Li, Bioorganic & Medicinal Chemistry 14 (2006) 1706–1709. [16] Z.Y. Guo, H.Y. Liu, X.L. Chen, X. Ji, P.C. Li, Bioorganic & Medicinal Chemistry Letters 16 (2006) 6348–6350.
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Phenolic antioxidants-functionalized quaternized chitosan: Synthesis and antioxidant properties Jianming Ren a,b , Qing Li a , Fang Dong a , Yan Feng a,b , Zhanyong Guo a,∗ a b
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Graduate School of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 25 September 2012 Received in revised form 30 October 2012 Accepted 8 November 2012 Available online 16 November 2012 Keywords: Quaternized chitosan Antioxidant Antioxidant–polymer conjugates
a b s t r a c t In this work, two kinds of phenolic antioxidants-functionalized quaternized chitosan were synthesized in order to develop aqueous soluble antioxidant–polymer conjugates. Quaternized chitosan conjugated with gallic acid or caffeic acid was carried out and the antioxidant properties of the products (namely gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan) against hydroxyl-radical, superoxideradical and DPPH-radical were evaluated in vitro, respectively. The scavenging activities of the obtained gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan exhibit a remarkable improvement over those of either chitosan or quaternized chitosan. And the scavenging effect indices of the products were all higher than 90% at a concentration of 1000 g/mL. Because gallic acid-quaternized chitosan and caffeic acid-quaternized chitosan are convenient to prepare and possess improved potential activities, these materials may represent an attractive new platform for utilizations of chitosan. © 2012 Published by Elsevier B.V.
1. Introduction Reactive oxygen species (ROS), including superoxide anion (O2 •), hydroxyl radicals (•OH) and hydrogen peroxide (H2 O2 ), can cause pathological changes like cancer disease, diabetes in biological systems and lead to harmful alterations in foods and pharmaceutical industries [1]. As strong antioxidants and excellent free radical scavengers, most phenolic compounds could effectively prevent or slower those deleterious reactions [2,3]. Among phenolic compounds, gallic and caffeic acids have attracted much attention as promising antioxidant agents applicable to play a significant role in health-promoting benefits [4,5]. Unlike currently widely used synthetic antioxidant such as butylated hydroxyanisole and butylated hydroxytoluene, which have potential health hazards and toxicity, gallic and caffeic acids are both obtained from nature, and have been recognized as safety biological active molecules. However, sometimes it is lastly recommendable to directly employ gallic and caffeic acids in foods and pharmaceutical as antioxidant agents because they may result in undesirable reactions and change the chemical compositions of them. In recent years many reports proposed that appropriate macromolecular systems, namely antioxidant–polymer conjugates, could combine the merits of both components, which could retain the excellent biological activities of antioxidant molecules and show a higher stability and a slower degradation rate than the antioxidant molecules
∗ Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000. E-mail address: [email protected] (Z. Guo). 0141-8130/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijbiomac.2012.11.011
[6–8]. To synthesize antioxidant–polymer conjugates basing gallic and caffeic acids, chitosan is an ideal candidate for the polymer matrix. Chitosan, a kind of renewable, abundant natural polysaccharide, consists primarily of 2-amino-2-deoxy-glucopyranose units linked by -(1-4) linkage [9]. It has already been well studied and sufficiently reported that chitosan is non-toxic, biodegradable, and biocompatible, which help it to earn much scientific research attentions [10]. Meanwhile, known as a bioactive macromolecule, chitosan exhibits many bioactivities such as antitumor property, antifungal and antimicrobial activity and so on [11,12]. What is more, apart from these interesting properties, chitosan possesses antioxidant property too, which further makes this natural, renewable, bioactive polymeric material is suitable to be the polymer part of the “antioxidant–polymer conjugates”[13]. Our previous work has found that the antioxidant activity of chitosan is related to the forms of nitrogen atoms in chitosan molecules and chitosan quaternizied by methyl groups (N,N,N-trimethylchitosan) could obviously improve the antioxidant activity of chitosan [1,14]. In addition, better than chitosan, N,N,N-trimethylchitosan (TMC) is soluble in neutral and alkaline aqueous solutions, which can enlarge the applications of chitosan as a food preservative or bioactive matrix. Therefore, it is reasonable to propose that the coupling of TMC with gallic or caffeic acids is promisingly to give a stable, effective and water-soluble antioxidant–polymer conjugate. Given these characteristics of chitosan, TMC, gallic acid and caffeic acid, this paper concentrated on the development of two kinds of novel gallic acid-quaternized chitosan (G-TMC) and caffeic acid-quaternized chitosan (C-TMC) aqueous soluble antioxidant
78
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conjugates. The antioxidant activity of the synthesized conjugates was evaluated in vitro and compared to that of chitosan and TMC.
where Ablank 520 nm was the absorbance of the blank (distilled water instead of the samples) and Acontrol 520 nm was the absorbance of the control (distilled water instead of the H2 O2 ).
2. Experimental 2.1. Materials and methods Chitosan was purchased from Qingdao Baicheng Biochemical Corp. (China). Its degree of deacetylation was 97%, and the viscosityaverage molecular weight was 7.0 × 104 . Gallic acid and caffeic acid were purchased from the Sigma–Aldrich Chemical Co. (United States). The other reagents were all analytical grades and were used without further purification. The IR spectra were measured on a Jasco-4100 FT-TR spectrometer with KBr disks. The UV spectra were recorded on a Puxi-Tu1810 UV spectrometer and the elemental analyses (C, H, N) were performed on a Carlo-Erba 1106 elemental analyzer.
Scavenging effect (%) = 1 −
Asample 560 nm
Acontrol 560 nm
× 100
Acontrol 560 nm was the absorbance of the control (distilled water instead of the samples).
2.2. Synthesis 2.2.1. Preparation of TMC TMC was synthesized as follows: 3 g chitosan was dispersed in 100 mL of water, and formaldehyde (3 equiv. based on glucosamine units of chitosan) was added, and the mixture was stirred at room temperature. After 2 h, 10% NaBH4 (1.5-fold excess relative to mole mass of formaldehyde) was added, and the solution reacted for 2 h. The mixture was precipitated in acetone and filtered. The Nmethylchitosan was obtained after drying at 60 ◦ C in vacuum for 12 h. N-methylchitosan (1 g) was dispersed in 50 mL of N-methyl2-pyrrolidone (NMP) for 12 h at room temperature. Then, 0.5 mL NaOH (1 M), 1 g NaI and 4 mL CH3 I were added. The resulting mixture was heated to 60 ◦ C and stirred for 20 h. The product was obtained by precipitation with excess acetone, and the TMC was obtained by drying at 60 ◦ C in vacuum for 12 h. 2.2.2. Synthesis of antioxidant conjugates Gallic acid and caffeic acid were firstly transformed to reactive chloride form using thionyl chloride, in order to conveniently combine them with TMC. Gallic acid or caffeic acid (10 mmol) and thionyl chloride (100 mmol) were added to a dry flask with anhydrous THF (200 mL) under nitrogen. After the reaction was refluxed for 2 h, the excess thionyl chloride and THF were removed under reduced pressure and TMC (3 mmol) dispersed in NMP (150 mL) was added in. Then, the resulting mixture was stirred for 24 h at 80 ◦ C and the product was obtained by precipitation with excess acetone. The product was washed by ethanol and acetone, Soxhlet extracted with ethanol for 24 h and freeze dried. 2.3. The investigation of the antioxidant ability 2.3.1. Hydroxyl-radical scavenging ability assay The test of the hydroxyl-radical scavenging ability was carried out according to Liu’s methods [1]. The reaction mixture, a total volume 4.5 mL, containing the samples of the synthesized antioxidant conjugates, TMC and chitosan, was incubated with EDTA–Fe2+ (220 M), safranine O (0.23 M), H2 O2 (60 M) in potassium phosphate buffer (150 mM, pH 7.4) for 30 min at 37 ◦ C. The absorbance of the mixture was measured at 520 nm. Three replicates for each sample concentration were tested. Hydroxyl radicals bleached the safranine O, so increased absorbance of the reaction mixture indicated decreased hydroxyl radicals scavenging ability and the scavenging effect of the product was computed using the follow equation: Scavenging effect (%) =
2.3.2. Superoxide-radical scavenging ability assay The superoxide radical scavenging ability was assessed following the model of Xing [15]. Involving testing samples, phenazine mothosulfate (30 M), nicotinamide adenine dinucleotide reduced (338 M), and nitro blue tetrazolium (72 M) in phosphate buffer (0.1 M, pH 7.4), the reaction mixture was incubated at 25 ◦ C for 5 min and 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:
Asample 520 nm − Ablank 520 nm Acontrol 520 nm − Ablank 520 nm
× 100
2.3.3. DPPH-radical scavenging ability assay The DPPH• scavenging properties of the products were quantified by the following method: testing samples and 2 mL ethanol solution of DPPH (180 mol/L) was incubated for 30 min at 25 ◦ C in a water bath. Then, the absorbance of the remained DPPH 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 (%) =
Acontrol 517 nm − Asample 517 nm Acontrol 517 nm
× 100
Acontrol 560 nm was the absorbance of the control (distilled water instead of the samples). 2.4. Statistical analysis All data are expressed as means ± SD. Data were analyzed by an analysis of variance (P < 0.05) and the means were separated by Duncan’s multiple range test. The results were processed by the computer programs: Excel and Statistica software SPSS. 3. Results and discussion 3.1. Chemical syntheses and characterization Two kinds of water-soluble antioxidant–polymer conjugates containing the antioxidative groups of gallic acid or caffeic acid were constructed. The TMC was chosen as polymer part of the “antioxidant–polymer conjugates”, which is by virtue of its various unique qualities including stability, water-solubility, biocompatibility, antioxygenic property and so on. The conjugates of TMC and gallic or caffeic acids were synthesized following the route outlined in Scheme 1 (Scheme 1). The synthesized products were characterized by FT-IR (Fig. 1), UV (Fig. 2) and elemental analyses (Table 1). In Fig. 1, the FT-IR spectra present the comparison of transmission spectra data of the synthesized products with that of original chitosan. Chitosan was typically characterized by absorption regions at about 896, 1087 and 1600 cm−1 , which belongs to pyranose ring, glucoside and amine groups, respectively [16]. When chitosan was transformed to TMC, new peaks appeared at about 1660 cm−1 , which were assigned to the quaternary ammonium salt. There were peaks at about 1415–1430 cm−1 , which were ascribed to the characteristic absorb of N-CH3 [1]. After gallic or caffeic acids were combined with TMC through ester bond, strong new
J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81
79
Fig. 3. Hydroxyl radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
Fig. 1. FT-IR spectra of chitosan (1), TMC (2), G-TMC (3) and C-TMC (4).
Table 1 The elemental analyses results and the substitution degree of chitosan, TMC, G-TMC and C-TMC. Compounds
Chitosan TMC G-TMC C-TMC
Elemental analyses (%)
Substitution degreea
C
N
H
40.85 41.63 45.80 49.85
7.50 5.87 4.37 4.01
7.03 7.21 6.19 6.52
0.75 0.72
a Substitution degree refers to the C-6-O substitution degree of gallic acid in GTMC and caffeic acid in C-TMC.
peaks were observed at 1739 and 1720 cm−1 respectively, which correspond to C O vibrations [6]. Meanwhile, the aromatic C C vibrations were found at 748 and 1511 cm−1 in the FT-IR spectra of gallic acid functionalized TMC and at 744 and 3006 cm−1 in that of caffeic acid–TMC conjugate [6] (Fig. 1). Fig. 2 gives the UV spectra of each conjugate (0.5 mg/mL) with same concentration of TMC as baseline to remove the probable interference of TMC. As in Fig. 2, the UV spectra of both products show evident absorptions peaks in the aromatic region, which are attributed to the presence of gallic acid or caffeic acid respectively (Fig. 2). Results aforementioned results demonstrated that the aimed phenolic antioxidants-functionalized TMC were obtained. Finally, the results of elemental analyses are listed in Table 1 and the substitution degree of the products could be computed according to the percentage of N from the elemental analyses (Table 1). 3.2. Antioxidant activities Under normal condition, direct addition of Fe2+ to a reaction mixture containing phosphate buffer generates hydroxyl radicals which are harmful to body through reacting with such biological molecule as: amino acids or DNA. Fig. 3 reveals the •OH scavenging ability of the synthesized conjugates, TMC, and chitosan at various concentrations. According to the graph we could conclude the results as follows: firstly, all the scavenging effects of samples have a positive correlation with tested concentrations. Secondly, TMC has a better antioxidant activity than that of chitosan, which has
Fig. 2. UV spectra of water, G-TMC and C-TMC.
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J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81
O HOH2C
O
HOH2C O NaI NaOH n
R O
CH3I
OH NH2
C
SOCl2
O n
OH2C
O
O n
RCOOH OH N
OH N OH
R=
OH
or
OH
OH OH
Scheme 1. Synthesis pathway of G-TMC and C-TMC.
been reported and discussed in our previous work. Thirdly, of all the tested samples, the conjugate of gallic acid-TMC (G-TMC) and caffeic acid-TMC (C-TMC) exhibit the best scavenging ability against •OH IC50 value of G-TMC and C-TMC was 0.13 and 0.14 mg/mL respectively. IC50 , a good parameter to evaluate the scavenging activity, means the conjugates concentration to reduce the radical by 50%. Finally, significant scavenging effect (72.08–83.74%) of OH radicals was evident at tested concentrations of conjugates, which suggests the potential of the products to be developed as hydroxyl-radical scavenging reagents of food industry. It is reasonable to propose that the enhanced scavenging capability may mainly benefit from the phenolic compounds, specifically gallic and
caffeic acids, grafted on TMC since these phenolic compounds are effective antioxidant agents (Fig. 3). The scavenging properties of the chitosan, TMC and conjugates against superoxide-radical and DPPH radical are shown in Figs. 4 and 5, respectively. Similarly, G-TMC and C-TMC possess marked improved antioxidant activity than those of chitosan and TMC. It should be for the same reason that the increased antioxidant activity is primarily from the grafted phenolic compounds, specifically gallic and caffeic acids in this study (Figs. 4 and 5). 4. Conclusions In summary, in this paper, two kinds of antioxidant–polymer conjugates which are based on quaternized chitosan and natural products namely gallic acid and caffeic acid were synthesized via a convenient reaction, and their antioxidant activities against sorts of radicals was evaluated in vitro, respectively. For the synthesis of antioxidant–polymer conjugates, we have had gallic acid and caffeic acid transformed to reactive chloride form using thionyl chloride firstly, and then the obtained chloride could combine with TMC in an elegant way. The antioxidant assays in vitro models suggested that when grafted to quaternized chitosan, gallic acid or caffeic acid could enhance the antioxidant activities of TMC effectively. The antioxidant data suggested that to develop appropriate macromolecular antioxidant–polymer conjugates, the phenolic compounds associate with TMC is a promising way. However, comprehensive studies need to be carried out to ascertain the safety of the conjugates in experimental animal models and field experiments.
Fig. 4. Superoxide-radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
Acknowledgement This work was supported by the 12th Five-Year Science and Technology Plan for Agriculture (2012BAD32B09). References
Fig. 5. DPPH-radical scavenging ability of chitosan, TMC G-TMC and C-TMC.
[1] J. Liu, H. Sun, F. Dong, Q. Xue, G. Wang, S. Qin, Z. Guo, Carbohydrate Polymers 78 (2009) 439–443. [2] R. Zadernowski, M. Naczk, J. Nesterowicz, Journal of Agricultural and Food Chemistry 53 (2005) 2118–2124. [3] M.P. Kahkonen, A.I. Hopia, H.J. Vuorela, J.-P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, Journal of Agricultural and Food Chemistry 47 (1999) 3954–3962. [4] Z. Lu, G. Nie, P.S. Belton, H. Tang, B. Zhao, Neurochemistry International 48 (2006) 263–274. [5] I. Gulcin, Toxicology 217 (2006) 213–220. [6] D. Arrua, M.C. Strumia, M.A. Nazareno, Journal of Agricultural and Food Chemistry 58 (2009) 9228–9234. [7] M. Curcio, F. Puoci, F. Iemma, O.I. Parisi, G. Cirillo, U.G. Spizzirri, N. Picci, Journal of Agricultural and Food Chemistry 57 (2009) 5933–5938. [8] W. Pasanphan, G.R. Buettner, S. Chirachanchai, Journal of Applied Polymer Science 109 (2008) 38–46.
J. Ren et al. / International Journal of Biological Macromolecules 53 (2013) 77–81 [9] N. Bordenave, S. Grelier, V. Coma, Biomacromolecules 9 (2008) 2377–2382. [10] V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, International Journal of Pharmaceutics 274 (2004) 1–33. [11] C. Porporatto, I.D. Bianco, C.M. Riera, S.G. Correa, Biochemical and Biophysical Research Communications 304 (2003) 266–272. [12] Z. Zhong, R. Chen, R. Xing, X. Chen, S. Liu, Z. Guo, X. Ji, L. Wang, P. Li, Carbohydrate Research 342 (2007) 2390–2395.
81
[13] R. Xing, S. Liu, H. Yu, Q. Zhang, Z. Li, P. Li, Carbohydrate Research 339 (2004) 2515–2519. [14] Z. Guo, R. Xing, S. Liu, Z. Zhong, P. Li, Carbohydrate Polymers 73 (2008) 173–177. [15] R.E. Xing, S. Liu, Z.Y. Guo, H.H. Yu, C.P. Li, X. Ji, J.H. Feng, P.C. Li, Bioorganic & Medicinal Chemistry 14 (2006) 1706–1709. [16] Z.Y. Guo, H.Y. Liu, X.L. Chen, X. Ji, P.C. Li, Bioorganic & Medicinal Chemistry Letters 16 (2006) 6348–6350.