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Preparation of low-molecular-weight carboxymethyl chitosan and their superoxide anion scavenging activity
EUROPEAN POLYMER JOURNAL
European Polymer Journal 43 (2007) 652–656
www.elsevier.com/locate/europolj
Short communication
Preparation of low-molecular-weight carboxymethyl chitosan and their superoxide anion scavenging activity Tao Sun *, Dongxiang Zhou, Fang Mao, Yingna Zhu College of Food Science, Shanghai Fisheries University, Shanghai 200090, China Received 22 October 2006; received in revised form 11 November 2006; accepted 12 November 2006 Available online 26 December 2006
Abstract Low-molecular-weight carboxymethyl chitosans (CMCTSs) were prepared by oxidative degradation method involving hydrogen peroxide (H2O2) without or with microwave radiation. Viscosity determination and end group analysis were applied to measure molecular weights of CMCTSs. Effects of concentration of H2O2 and degradation time on molecular weights of CMCTSs were studied. The degradation process of CMCTSs will be accelerated with the aid of microwave radiation and degradation time may be reduced greatly. The superoxide anion scavenging activity of CMCTSs was evaluated by application of flow injection chemiluminescence technology. The 50% inhibition concentrations (IC50s) of CMCTSs A, B, and C (1130, 2430 and 4350 Da) were 10.36, 17.57, and 23.38 mg/mL, respectively. The above results showed that CMCTSs with lower molecular weight had better superoxide anion scavenging activity. 2006 Elsevier Ltd. All rights reserved. Keywords: Carboxymethyl chitosan; Oxidative degradation; Superoxide anion scavenging activity; Chemiluminescence technology
1. Introduction Chitosan, poly-b-(1 ! 4)-D-glucosamine, has received much attention for its interesting properties such as nontoxicity, biocompatibility, and biodegradability [1]. However, the applications of chitosan are restricted because it is essentially insoluble in water at neutral pH. Chemical modification and depolymerization were used to improve its aqueous solubility [2].
* Corresponding author. Tel.: +86 21 65710032; fax: +86 21 65684287. E-mail address: [email protected] (T. Sun).
The solubility of chitosan can be improved remarkably by introducing carboxymethyl groups to the chitosan molecules. Carboxymethyl chitosan (CMCTS) is an important derivative for its good water-solubility, low toxicity and special biological characteristics [3,4]. Some studies have focused on the relationship between the structure and properties of CMCTS and molecular weight was found to be one of the most important factors [5–8]. However, there is little study on degradation of CMCTS [9]. H2O2 is a strong oxidant agent and may be disassociated to hydroxyl radicals, which can attack the b ! (1,4)glycoside bond. It has been used in degradation of many polysaccharides because it is handy, easily
0014-3057/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2006.11.014
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
available and environmentally friendly [10]. Microwave radiation is a useful way to degrade polysaccharides [11]. In order to get low-molecular-weight CMCTS, initial CMCTS (7.5 · 106 Da) was oxidative degraded by H2O2 without and with microwave radiation. The superoxide anion scavenging activity of CMCTSs was evaluated by application of flow injection chemiluminescence technology. 2. Experimental 2.1. Materials Carboxymethyl chitosan (7.5 · 106 Da) was obtained from Shanghai Weina Science and Technology Co., Ltd. (Shanghai, China). Luminol (5-amino2,3-dihydro-1,4-phthalazinedione) was purchased from Sigma Chemical Co. (Shanghai, China). All other chemicals were of analytical grade and supplied by Shanghai Chemicals Co. (Shanghai, China). Double distilled water was used to prepare solutions for antioxidant test. 2.2. Preparation of low-molecular-weight carboxymethyl chitosan 2.0 g carboxymethyl chitosan was added into 50.0 mL deionized water, and then stirred at room temperature to obtain a homogenous solution. Moderate 30% H2O2 solution was added into CMCTS solution and kept for 5–28 h. Then the solution was concentrated to about 10 mL and was precipitated by adding ethanol. The precipitate was filtrated off, washed with ethanol several times, and then was dried under vacuum at 60 C for 48 h to get the degraded CMCTS. The preparation of low-molecular-weight CMCTS under microwave radiation was similar to the above process. The CMCTS solution was placed on the center of the turntable of an 800 W microwave oven (Galanz, Shunde, China) and irradiated at high fire for 4–14 min. The sample was taken outside to let it cool down to room temperature every one minute to avoid the degrading at too high temperatures and the cross-linking of CMCTS polymer chains. Results from degradation were presented as means of triplicates. The structural changes of carboxymethyl chitosan after degraded were confirmed by Fourier Transform Infrared (FTIR) spectra, which were taken with KBr pellets on an EQUNOX55 FT-IR-
653
Laman spectrophotometer with a revolution of 0.8 cm1 in the range of 400–4000 cm1. 2.3. Determination of molecular weight Molecular weights of degraded CMCTSs were measured by viscosity determination or the method of end group analysis [10]. The viscosities of chitosan samples were measured in a solvent of 0.10 mol/L NaCl at 25 C using an Ubbleohde capillary viscometer. And molecular weights were determined using the classic Mark–Houwink equation [g] = 7.92 · 105 M [12]. The end group analysis was processed similar to the published literature and here introduced briefly. 0.375 mg/mL potassium ferricyanide dissolved in 0.3 mol/L Na2CO3 was used as colorproducing reagent and 10.0 mg/mL D-glucosamine hydrochloride (GAH) solution as standard solution. Different concentrations of GAH solution was prepared by diluting with distilled water, and then reacted with potassium ferricyanide in a boiling water bath for 15 min. The absorbance of colorreducing reactions at 420 nm was recorded to obtain the standard curve of absorbance and concentration of GAH. Solutions of degraded CMCTS were prepared and processed the color-reducing reaction with potassium ferricyanide. Their molecular weights were calculated by the following equation: Mn = (W2/W1) · 215.5, where W1 and W2 are the weight percent of GAH and degraded CMCTS, respectively. 2.4. Evaluation of superoxide anion scavenging activity Superoxide anion scavenging activity of CMCTSs was evaluated by flow injection chemiluminescence on a chemical luminometer (IFFM-D, Xi’an, China). Superoxide anion was produced by a luminol-enhanced autoxidation of pyrogallol. The chemiluminescencent reaction was processed in a Na2CO3–NaHCO3 (pH 10.20, 0.05 mol/L) buffered solution at room temperature. The CMCTS samples were dissolved in Na2CO3– NaHCO3 buffered solution to prepare scavenger solutions at different concentrations. The scavenging activity of CMCTS samples against superoxide anion was evaluated according to their quenching effects on the chemiluminescence (CL) signal of the luminol-pyrogallol system. The inhibiting efficacy for superoxide anion O 2 was calculated as: IE(%) = (A0 Ai)/A0, where A0 and Ai represent
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
3. Results and discussion The structural changes of carboxymethyl chitosan after degradation were confirmed by FTIR. In the FTIR spectrum of initial CMCTS (7.5 · 106 Da), there are characteristic peaks assigned to the saccharide structure at 1031, 1068, 1113 and 1155 cm1. The peak at 1400 cm1 should be assigned to the symmetrical stretch vibration absorption of COO. The strong absorption at 1558 cm1 should be owed to the overlap of asymmetrical vibration of COO at 1560 cm1 and amide II band at 1550 cm1. The peak at 1650 cm1, amide I band, indicated that there are some residue acetylamide groups in CMCTS. As for degraded CMCTS (2430 Da), the absorption peak of extending vibration of ether bond at 1074 cm1 became stronger, and the peak of C–O of primary alcohol at 1030 cm1 and the peak of C–O of secondary alcohol were not so obvious. The fact should be related to the low-molecularweight and the decrease of intermolecular hydrogen bond. The peaks at 1402 and 1602 cm1 (overlap of 1560 and 1650 cm1) became stronger, which indicated that the relative content of COO group in degraded CMCTS increased. The fact could be related to the COO group produced from the oxidation of 1 ! 4 b-glucoside by H2O2 during the degradation of CMCTS. CMCTS was oxidative degraded without and with microwave radiation and the effects of concentration of H2O2 on degradation of CMCTS were evaluated with a fixed degradation time. Without microwave radiation, the molecular weights of degraded CMCTS decreased from 4350 Da to 1250 Da with the increase of concentration of H2O2 when the concentration of H2O2 changed from 0.9 to 1.3 mol/L (Fig. 1). At higher H2O2 concentration (1.3–2.3 mol/L), effect of H2O2 concentration on degradation was not so important, and the molecular weights of degraded CMCTS was about 1200–1100. Under microwave radiation, the molecular weights of degraded CMCTS samples
4500 Molecular weight (Mn)/Da
CL peak areas of the blank group and test group, respectively. The free radical produced in the system was proved to be superoxide anion tested by superoxide dismutase (SOD), catalase, and mannitol. Data of radical scavenging were expressed as mean ± standard error of the mean (n = 3) and independent student’s t-test was used to determine the level of significance (Originpro 6.1, p < 0.05).
4000 3500 3000 2500 2000 1500 1000 0.8
1.0
1.2 1.4 1.6 1.8 2.0 2.2 Concentration of H2O2 /(mol/L)
2.4
Fig. 1. Effect of concentration of H2O2 on degradation of CMCTS at 4 h.
7000 Molecular weight (Mn)/Da
654
6000 5000 4000 3000 2000 1000 0.0
0.2
0.4
0.6
0.8
1.0
Concentration of H2O2/(mol/L) Fig. 2. Effect of concentration of H2O2 on degradation of CMCTS at 8 min under microwave radiation.
decreased from 6230 Da to 1800 Da then about 2000 Da when the H2O2 concentration changed from 0.02 to 0.2 then 0.9 mol/L (Fig. 2). The results may be owed to the rapid decomposition rate of H2O2 at high concentrations or high temperature in the reaction system. Effects of degradation time on molecular weight of CMCTS without and with microwave radiation were shown in Figs. 3 and 4, respectively. Without microwave radiation, the molecular weights of CMCTS samples changed from 4350 Da at 4 h to 1620 Da at 16 h, then slightly decreased with the increasing of degradation time (Fig. 3). As shown in Fig. 4, molecular weights of CMCTSs decreased with the prolonging of degradation time but slightly changed after 8 min when the concentration of H2O2 was 0.20 mol/L. It meant that CMCTSs with
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
100 90
4000
80 Inhibiting efficacy/%
Molecular weight (Mn)/Da
4500
3500 3000 2500 2000 1500 0
655
1130 Da 2430 Da 4350 Da
70 60 50
10.35
17.57
23.28
40 30 20 10
5
10 15 20 Degradation time/h
25
30
Fig. 3. Effect of degradation time on degradation of CMCTS at the H2O2 concentration of 0.9 mol/L.
0 0
5 10 15 20 25 30 Concentration of CMCTS/(mg/mL)
35
Fig. 5. Superoxide anion scavenging effect of CMCTSs with different molecular weights.
Molecular weight (Mn)/Da
6000 5000 4000 3000 2000 1000 4
6
8 10 Reaction time/min
12
14
Fig. 4. Effect of degradation time on degradation of CMCTS at the H2O2 concentration of 0.9 mol/L under microwave radiation.
low-molecular-weights would be obtained at a long degradation time, but the effect of degradation time was not so important. When the degradation time changed from 8 min to 14 min, the molecular weight of CMCTS was about 2000 Da. Fig. 5 showed the superoxide anion inhibiting efficacy of CMCTS A, B, and C (1130, 2430 and 4350 Da, respectively) at different concentrations. Three CMCTSs showed scavenging ability against superoxide anion, but had different inhibiting efficacy at the same concentration. When the concentration of CMCTS A (1130 Da) was 10.35 mg/mL in the final solution, a superoxide anion inhibiting efficacy of approximately 50% was achieved, this means that the 50% inhibition concentration (IC50) was 10.35 mg/mL. As for CMCTS B and C (2430 and 4350 Da), the IC50s were 17.57 and 23.38 mg/mL, respectively. At the concentration of 25 mg/mL,
the maximal inhibiting efficacy of superoxide anion by CMCTS A, B and C were 93%, 71%, and 52.5%, respectively. The results showed that CMCTS with lower molecular weight would have relative stronger scavenging ability against superoxide anion. The antioxidant activity of chitosan and its derivatives has indicated that the active hydroxyl and amino groups in the polymer chains may take part in free radicals scavenging and contributed to the antioxidant activity. And the contents of active hydroxyl and amino groups in their polymer chains may affect the antioxidant activity of chitosan derivatives [13–16]. The initial CMCTS (7.5 · 106 Da) showed no scavenging activity against superoxide anion at the maximal concentration of 40 mg/mL in the antioxidant evaluation system. CMCTS with lower-molecular-weight would have relative stronger antioxidant activity. The above results showed that the antioxidant activity of was closely related to their molecular weight. Acknowledgements This project was supported by the President’s Special Foundation of Shanghai Fisheries University (SFU200305) and Shanghai Leading Academic Discipline (Project No. T1102). References [1] Kofuji K, Qian CJ, Nishimura M, Sugiyama I, Murata Y, Kawashima S. Eur Polym J 2005;41:2784–91. [2] Jia ZS, Shen DF. Carbohyd Polym 2002;49(4):393–6.
656
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
[3] Liu SQ, Qiu B, Chen LY, Peng H, Du YM. Rheumatol Int 2005;26:52–7. [4] Chen Q, Liu SQ, Du YM, Peng H, Sun LP. Eur J Pharm 2006;541(1–2):1–8. [5] Chen LY, Du YM, Wu HQ, Xiao L. J Appl Polym Sci 2002;83:1233–41. [6] Chen LY, Du YM, Zeng XQ. Carbohyd Res 2003; 338:333–40. [7] Chen XG, Wang Z, Liu WS, Park HJ. Biomaterials 2002; 23:4609–14. [8] Shi XW, Du YM, Yang JH, Zhang BZ, Sun LP. J Appl Polym Sci 2006;100:4689–96. [9] Huang L, Zhai ML, Peng J, Li JQ, Wei GS. Carbohyd Polym; 2006. doi:10.1016/j.carbpol.2006.05.023.
[10] Shao J, Yang YM, Zhong QQ. Polym Degrad Stabil 2003;82:395–8. [11] Ge HC, Luo DK. Carbohyd Res 2005;340:1351–6. [12] Nishimura SI, Nishi N, Nishimura ST, Azuma I. Carbohyd Res 1986;146:251–8. [13] Xie WM, Xu PX, Liu Q. Bioorg Med Chem Lett 2001; 11:1699–701. [14] Sun T, Xie WM, Xu PX. Carbohyd Polym 2004; 58(4):379–82. [15] Zhu XY, Wu JM, Jia ZS. Chinese Chem Lett 2004; 15(7):808–10. [16] Huang RH, Mendis E, Kim SK. Int J Biol Macromol 2005;36(1–2):120–7.
European Polymer Journal 43 (2007) 652–656
www.elsevier.com/locate/europolj
Short communication
Preparation of low-molecular-weight carboxymethyl chitosan and their superoxide anion scavenging activity Tao Sun *, Dongxiang Zhou, Fang Mao, Yingna Zhu College of Food Science, Shanghai Fisheries University, Shanghai 200090, China Received 22 October 2006; received in revised form 11 November 2006; accepted 12 November 2006 Available online 26 December 2006
Abstract Low-molecular-weight carboxymethyl chitosans (CMCTSs) were prepared by oxidative degradation method involving hydrogen peroxide (H2O2) without or with microwave radiation. Viscosity determination and end group analysis were applied to measure molecular weights of CMCTSs. Effects of concentration of H2O2 and degradation time on molecular weights of CMCTSs were studied. The degradation process of CMCTSs will be accelerated with the aid of microwave radiation and degradation time may be reduced greatly. The superoxide anion scavenging activity of CMCTSs was evaluated by application of flow injection chemiluminescence technology. The 50% inhibition concentrations (IC50s) of CMCTSs A, B, and C (1130, 2430 and 4350 Da) were 10.36, 17.57, and 23.38 mg/mL, respectively. The above results showed that CMCTSs with lower molecular weight had better superoxide anion scavenging activity. 2006 Elsevier Ltd. All rights reserved. Keywords: Carboxymethyl chitosan; Oxidative degradation; Superoxide anion scavenging activity; Chemiluminescence technology
1. Introduction Chitosan, poly-b-(1 ! 4)-D-glucosamine, has received much attention for its interesting properties such as nontoxicity, biocompatibility, and biodegradability [1]. However, the applications of chitosan are restricted because it is essentially insoluble in water at neutral pH. Chemical modification and depolymerization were used to improve its aqueous solubility [2].
* Corresponding author. Tel.: +86 21 65710032; fax: +86 21 65684287. E-mail address: [email protected] (T. Sun).
The solubility of chitosan can be improved remarkably by introducing carboxymethyl groups to the chitosan molecules. Carboxymethyl chitosan (CMCTS) is an important derivative for its good water-solubility, low toxicity and special biological characteristics [3,4]. Some studies have focused on the relationship between the structure and properties of CMCTS and molecular weight was found to be one of the most important factors [5–8]. However, there is little study on degradation of CMCTS [9]. H2O2 is a strong oxidant agent and may be disassociated to hydroxyl radicals, which can attack the b ! (1,4)glycoside bond. It has been used in degradation of many polysaccharides because it is handy, easily
0014-3057/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2006.11.014
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
available and environmentally friendly [10]. Microwave radiation is a useful way to degrade polysaccharides [11]. In order to get low-molecular-weight CMCTS, initial CMCTS (7.5 · 106 Da) was oxidative degraded by H2O2 without and with microwave radiation. The superoxide anion scavenging activity of CMCTSs was evaluated by application of flow injection chemiluminescence technology. 2. Experimental 2.1. Materials Carboxymethyl chitosan (7.5 · 106 Da) was obtained from Shanghai Weina Science and Technology Co., Ltd. (Shanghai, China). Luminol (5-amino2,3-dihydro-1,4-phthalazinedione) was purchased from Sigma Chemical Co. (Shanghai, China). All other chemicals were of analytical grade and supplied by Shanghai Chemicals Co. (Shanghai, China). Double distilled water was used to prepare solutions for antioxidant test. 2.2. Preparation of low-molecular-weight carboxymethyl chitosan 2.0 g carboxymethyl chitosan was added into 50.0 mL deionized water, and then stirred at room temperature to obtain a homogenous solution. Moderate 30% H2O2 solution was added into CMCTS solution and kept for 5–28 h. Then the solution was concentrated to about 10 mL and was precipitated by adding ethanol. The precipitate was filtrated off, washed with ethanol several times, and then was dried under vacuum at 60 C for 48 h to get the degraded CMCTS. The preparation of low-molecular-weight CMCTS under microwave radiation was similar to the above process. The CMCTS solution was placed on the center of the turntable of an 800 W microwave oven (Galanz, Shunde, China) and irradiated at high fire for 4–14 min. The sample was taken outside to let it cool down to room temperature every one minute to avoid the degrading at too high temperatures and the cross-linking of CMCTS polymer chains. Results from degradation were presented as means of triplicates. The structural changes of carboxymethyl chitosan after degraded were confirmed by Fourier Transform Infrared (FTIR) spectra, which were taken with KBr pellets on an EQUNOX55 FT-IR-
653
Laman spectrophotometer with a revolution of 0.8 cm1 in the range of 400–4000 cm1. 2.3. Determination of molecular weight Molecular weights of degraded CMCTSs were measured by viscosity determination or the method of end group analysis [10]. The viscosities of chitosan samples were measured in a solvent of 0.10 mol/L NaCl at 25 C using an Ubbleohde capillary viscometer. And molecular weights were determined using the classic Mark–Houwink equation [g] = 7.92 · 105 M [12]. The end group analysis was processed similar to the published literature and here introduced briefly. 0.375 mg/mL potassium ferricyanide dissolved in 0.3 mol/L Na2CO3 was used as colorproducing reagent and 10.0 mg/mL D-glucosamine hydrochloride (GAH) solution as standard solution. Different concentrations of GAH solution was prepared by diluting with distilled water, and then reacted with potassium ferricyanide in a boiling water bath for 15 min. The absorbance of colorreducing reactions at 420 nm was recorded to obtain the standard curve of absorbance and concentration of GAH. Solutions of degraded CMCTS were prepared and processed the color-reducing reaction with potassium ferricyanide. Their molecular weights were calculated by the following equation: Mn = (W2/W1) · 215.5, where W1 and W2 are the weight percent of GAH and degraded CMCTS, respectively. 2.4. Evaluation of superoxide anion scavenging activity Superoxide anion scavenging activity of CMCTSs was evaluated by flow injection chemiluminescence on a chemical luminometer (IFFM-D, Xi’an, China). Superoxide anion was produced by a luminol-enhanced autoxidation of pyrogallol. The chemiluminescencent reaction was processed in a Na2CO3–NaHCO3 (pH 10.20, 0.05 mol/L) buffered solution at room temperature. The CMCTS samples were dissolved in Na2CO3– NaHCO3 buffered solution to prepare scavenger solutions at different concentrations. The scavenging activity of CMCTS samples against superoxide anion was evaluated according to their quenching effects on the chemiluminescence (CL) signal of the luminol-pyrogallol system. The inhibiting efficacy for superoxide anion O 2 was calculated as: IE(%) = (A0 Ai)/A0, where A0 and Ai represent
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
3. Results and discussion The structural changes of carboxymethyl chitosan after degradation were confirmed by FTIR. In the FTIR spectrum of initial CMCTS (7.5 · 106 Da), there are characteristic peaks assigned to the saccharide structure at 1031, 1068, 1113 and 1155 cm1. The peak at 1400 cm1 should be assigned to the symmetrical stretch vibration absorption of COO. The strong absorption at 1558 cm1 should be owed to the overlap of asymmetrical vibration of COO at 1560 cm1 and amide II band at 1550 cm1. The peak at 1650 cm1, amide I band, indicated that there are some residue acetylamide groups in CMCTS. As for degraded CMCTS (2430 Da), the absorption peak of extending vibration of ether bond at 1074 cm1 became stronger, and the peak of C–O of primary alcohol at 1030 cm1 and the peak of C–O of secondary alcohol were not so obvious. The fact should be related to the low-molecularweight and the decrease of intermolecular hydrogen bond. The peaks at 1402 and 1602 cm1 (overlap of 1560 and 1650 cm1) became stronger, which indicated that the relative content of COO group in degraded CMCTS increased. The fact could be related to the COO group produced from the oxidation of 1 ! 4 b-glucoside by H2O2 during the degradation of CMCTS. CMCTS was oxidative degraded without and with microwave radiation and the effects of concentration of H2O2 on degradation of CMCTS were evaluated with a fixed degradation time. Without microwave radiation, the molecular weights of degraded CMCTS decreased from 4350 Da to 1250 Da with the increase of concentration of H2O2 when the concentration of H2O2 changed from 0.9 to 1.3 mol/L (Fig. 1). At higher H2O2 concentration (1.3–2.3 mol/L), effect of H2O2 concentration on degradation was not so important, and the molecular weights of degraded CMCTS was about 1200–1100. Under microwave radiation, the molecular weights of degraded CMCTS samples
4500 Molecular weight (Mn)/Da
CL peak areas of the blank group and test group, respectively. The free radical produced in the system was proved to be superoxide anion tested by superoxide dismutase (SOD), catalase, and mannitol. Data of radical scavenging were expressed as mean ± standard error of the mean (n = 3) and independent student’s t-test was used to determine the level of significance (Originpro 6.1, p < 0.05).
4000 3500 3000 2500 2000 1500 1000 0.8
1.0
1.2 1.4 1.6 1.8 2.0 2.2 Concentration of H2O2 /(mol/L)
2.4
Fig. 1. Effect of concentration of H2O2 on degradation of CMCTS at 4 h.
7000 Molecular weight (Mn)/Da
654
6000 5000 4000 3000 2000 1000 0.0
0.2
0.4
0.6
0.8
1.0
Concentration of H2O2/(mol/L) Fig. 2. Effect of concentration of H2O2 on degradation of CMCTS at 8 min under microwave radiation.
decreased from 6230 Da to 1800 Da then about 2000 Da when the H2O2 concentration changed from 0.02 to 0.2 then 0.9 mol/L (Fig. 2). The results may be owed to the rapid decomposition rate of H2O2 at high concentrations or high temperature in the reaction system. Effects of degradation time on molecular weight of CMCTS without and with microwave radiation were shown in Figs. 3 and 4, respectively. Without microwave radiation, the molecular weights of CMCTS samples changed from 4350 Da at 4 h to 1620 Da at 16 h, then slightly decreased with the increasing of degradation time (Fig. 3). As shown in Fig. 4, molecular weights of CMCTSs decreased with the prolonging of degradation time but slightly changed after 8 min when the concentration of H2O2 was 0.20 mol/L. It meant that CMCTSs with
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
100 90
4000
80 Inhibiting efficacy/%
Molecular weight (Mn)/Da
4500
3500 3000 2500 2000 1500 0
655
1130 Da 2430 Da 4350 Da
70 60 50
10.35
17.57
23.28
40 30 20 10
5
10 15 20 Degradation time/h
25
30
Fig. 3. Effect of degradation time on degradation of CMCTS at the H2O2 concentration of 0.9 mol/L.
0 0
5 10 15 20 25 30 Concentration of CMCTS/(mg/mL)
35
Fig. 5. Superoxide anion scavenging effect of CMCTSs with different molecular weights.
Molecular weight (Mn)/Da
6000 5000 4000 3000 2000 1000 4
6
8 10 Reaction time/min
12
14
Fig. 4. Effect of degradation time on degradation of CMCTS at the H2O2 concentration of 0.9 mol/L under microwave radiation.
low-molecular-weights would be obtained at a long degradation time, but the effect of degradation time was not so important. When the degradation time changed from 8 min to 14 min, the molecular weight of CMCTS was about 2000 Da. Fig. 5 showed the superoxide anion inhibiting efficacy of CMCTS A, B, and C (1130, 2430 and 4350 Da, respectively) at different concentrations. Three CMCTSs showed scavenging ability against superoxide anion, but had different inhibiting efficacy at the same concentration. When the concentration of CMCTS A (1130 Da) was 10.35 mg/mL in the final solution, a superoxide anion inhibiting efficacy of approximately 50% was achieved, this means that the 50% inhibition concentration (IC50) was 10.35 mg/mL. As for CMCTS B and C (2430 and 4350 Da), the IC50s were 17.57 and 23.38 mg/mL, respectively. At the concentration of 25 mg/mL,
the maximal inhibiting efficacy of superoxide anion by CMCTS A, B and C were 93%, 71%, and 52.5%, respectively. The results showed that CMCTS with lower molecular weight would have relative stronger scavenging ability against superoxide anion. The antioxidant activity of chitosan and its derivatives has indicated that the active hydroxyl and amino groups in the polymer chains may take part in free radicals scavenging and contributed to the antioxidant activity. And the contents of active hydroxyl and amino groups in their polymer chains may affect the antioxidant activity of chitosan derivatives [13–16]. The initial CMCTS (7.5 · 106 Da) showed no scavenging activity against superoxide anion at the maximal concentration of 40 mg/mL in the antioxidant evaluation system. CMCTS with lower-molecular-weight would have relative stronger antioxidant activity. The above results showed that the antioxidant activity of was closely related to their molecular weight. Acknowledgements This project was supported by the President’s Special Foundation of Shanghai Fisheries University (SFU200305) and Shanghai Leading Academic Discipline (Project No. T1102). References [1] Kofuji K, Qian CJ, Nishimura M, Sugiyama I, Murata Y, Kawashima S. Eur Polym J 2005;41:2784–91. [2] Jia ZS, Shen DF. Carbohyd Polym 2002;49(4):393–6.
656
T. Sun et al. / European Polymer Journal 43 (2007) 652–656
[3] Liu SQ, Qiu B, Chen LY, Peng H, Du YM. Rheumatol Int 2005;26:52–7. [4] Chen Q, Liu SQ, Du YM, Peng H, Sun LP. Eur J Pharm 2006;541(1–2):1–8. [5] Chen LY, Du YM, Wu HQ, Xiao L. J Appl Polym Sci 2002;83:1233–41. [6] Chen LY, Du YM, Zeng XQ. Carbohyd Res 2003; 338:333–40. [7] Chen XG, Wang Z, Liu WS, Park HJ. Biomaterials 2002; 23:4609–14. [8] Shi XW, Du YM, Yang JH, Zhang BZ, Sun LP. J Appl Polym Sci 2006;100:4689–96. [9] Huang L, Zhai ML, Peng J, Li JQ, Wei GS. Carbohyd Polym; 2006. doi:10.1016/j.carbpol.2006.05.023.
[10] Shao J, Yang YM, Zhong QQ. Polym Degrad Stabil 2003;82:395–8. [11] Ge HC, Luo DK. Carbohyd Res 2005;340:1351–6. [12] Nishimura SI, Nishi N, Nishimura ST, Azuma I. Carbohyd Res 1986;146:251–8. [13] Xie WM, Xu PX, Liu Q. Bioorg Med Chem Lett 2001; 11:1699–701. [14] Sun T, Xie WM, Xu PX. Carbohyd Polym 2004; 58(4):379–82. [15] Zhu XY, Wu JM, Jia ZS. Chinese Chem Lett 2004; 15(7):808–10. [16] Huang RH, Mendis E, Kim SK. Int J Biol Macromol 2005;36(1–2):120–7.