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Antioxidative activities of water-soluble disaccharide chitosan derivatives
Food Research International 37 (2004) 883–889 www.elsevier.com/locate/foodres
Antioxidative activities of water-soluble disaccharide chitosan derivatives Hsia-Yin Lin, Cheng-Chun Chou
*
Graduate Institute of Food Science and Technology, National Taiwan University 59, Lane 144, Keelung Rd., Section 4, Taipei 106-17, Taiwan Received 1 February 2004; accepted 23 April 2004
Abstract Water-soluble chitosan derivatives, having various degrees of substitution with disaccharide (DS 20–30%, 40–50%, 60–70%), were prepared by reductive alkylation of a-chitosan with lactose, maltose or cellobiose. Antioxidative activities were determined in the present study, including radical scavenging effect for a,a-diphenyl-b-picryl-hydrazyl (DPPH) radicals, superoxide anion radicals, hydrogen peroxide and copper ion chelating ability of these chitosan derivatives. It was found that the tested chitosan derivatives exhibited multiple antioxidative activities that varied with the concentration, DS with disaccharides and the kind of disaccharide present in the derivative molecule. In general, the antioxidative activities increased as the concentration of these chitosan derivatives increased up to a certain extent. A stronger scavenging effects for superoxide anion radicals, DPPH radicals and H2 O2 was noted with the chitosan derivatives having lower DS with disaccharide than those with higher DS. Among the various antioxidative activities tested, the disaccharide chitosan derivatives were found to show the highest excellent hydrogen peroxide scavenging activity. At a concentration of 400 lg/ml, all the chitosan derivatives tested exhibited 60% or greater scavenging activity for hydrogen peroxide. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Disaccharide chitosan derivative; Antioxidative activity
1. Introduction Chitosan, the deacetylated derivative of chitin, is a polymer of glucosamine. It is also one of the nontoxic and biodegradable carbohydrate polymers. Numerous studies have demonstrated that chitosan and its derivatives have various biological activities such as antimicrobial activity (Kobayashi, Watanabe, Suzuki, & Suzuki, 1990; Tokoro et al., 1989), antitumor activity (Saiki, Murata, Nakajima, Tokura, & Azuma, 1990; Tsukada et al., 1990) and immune-enhancing effects (Maeda, Murakami, Ohta, & Tajima, 1992; Nishimura et al., 1985; Shibata, Foster, Metzger, & Myrvik, 1997). Furthermore, antioxidant activity of chitosan derivatives has also been observed by several investigators over the past few years. For example, Xie, Xu, and Liu *
Corresponding author. Tel.: +886-2-2363-0231x2717; fax: +886-22362-0849. E-mail address: [email protected] (C.-C. Chou). 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.04.007
(2001) reported that the hexanoyl chitin and an N-benzoylhexanoyl chitosan could trap peroxide radicals in organic solvent when the radical chain reaction had been initiated by 2,20 -azobis (2,4-dimethylvaleronitrile). In addition, Matsugo et al. (1998) obtained chitosan derivatives through the acylation of chitosan with acid anhydride, and observed that these derivatives inhibited thiobartituric acid reactive substrate formation in t-butylhydroperoxide and benzoyl peroxide induced lipid peroxidation. Furthermore, Xue, Yu, Hirata, Terao, and Lin (1998) indicated that the chitosan derivatives prepared by graft copolymerization of maleic acid sodium onto hydroxypropyl chitosan and carboxymethyl chitosan sodium had scavenging activities against hydroxyl radicals. Due to these unique properties, chitosan and its derivatives have been proposed for diverse applications in biomedical, food, agriculture, biotechnology and pharmaceutical fields (Felse & Panda, 1999; Shahidi, Arachchi, & Jeon, 1999). However, its insolubility at
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neutral or high pH often limits the application of chitosan and its derivatives (Sugimoto, Morimoto, Sashiwa, Saimoto, & Shigemasa, 1998). To overcome the limited solubility, we previously prepared water-soluble chitosan derivatives through the reductive N-alkylation of chitosan with different disaccharides (Yang, Chou, & Li, 2002). These disaccharide chitosan derivatives were demonstrated to exhibit antibacterial activity that might be more useful than that of native chitosan as an antimicrobial agent in a food product or a system with a pH at 7.0 or higher (Yang, Chou, & Li, 2004). In the present study, the antioxidant activity of these derivatives is further examined.
2. Materials and methods 2.1. Preparation of N-alkylated disaccharide chitosan derivatives In this study, antioxidative activity of N-alkylated disaccharide (lactose, manose and cellobiose) derivatives with different degrees of substitution (DS) was tested. The disaccharide chitosan derivatives were prepared according to the procedures described by Yang et al. (2002). Briefly, Chitosan (0.5 g) from shrimp shells with 99% N-deacetylation, as determined by colloid titration (T€ oei & Kohara, 1976), was first obtained from China League Biotechnology Associates. Ltd. Lytone Enterprise, Inc. (Taipei, Taiwan). Then, dissolved in a mixture (1:1, pH 5.5) of methanol and 1% aqueous acetic acid, chitosan was mixed with various amounts of disaccharides (0.5–1.5 g). After resting at room temperature for 1 h, it was supplemented with NaCNBH3 (1 g) and was kept at room temperature for a period of 4–48 h, generally until a white solidified mass was formed. The solidified mass was then collected and washed with methanol for four times followed by washing with diethyl ether, then air-dried and finally dried under vacuum. If no solidified mass formed, the mixture was dialyzed against distilled water, then freeze-dried. The residue was dissolved in distilled water, and centrifuged (8000 rpm, 15 min). The supernatant and precipitate were separated by decanting, and the supernatant was freeze-dried again. The methods in T€ oei and Kohara (1976), with minor modifications as described by Yang et al. (2002), were followed to determine the DS of the prepared chitosan disaccharide derivatives. 2.2. Measurement of free radical scavenging activity The free radical scavenging activity of chitosan disaccharide derivatives was measured by 1,1-diphenyl2-picrylhydrazyl radical (DPPH) (Nacalai Tesque,
Kyoto, Japan) using the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). Briefly, 400 lM DPPH solution in methanol was prepared and 3.0 ml of this solution was added to 1.0 ml test samples at different concentrations. After a 90 min incubation period at ambient temperature, the absorbance at 517 nm was measured. The inhibitory percentage of DPPH was calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100:
2.3. Measurement of superoxide anion radical scavenging activity The method described by Robak and Gryglewski (1988) was used to measure the ability to scavenge superoxide anion radicals. First the reagents were all prepared in 100 mM phosphate buffer (pH 7.4). The reaction mixture containing 50 ll of test sample, 50 ll of 300 lM nitrobluetetrazolium (Sigma, St. Louis, MO, USA), 50 ll of 936 lM NADH and 50 ll of 120 lM phenazine methosulfate (Sigma) was incubated at room temperature for 5 min. The absorbance at 560 nm was then measured by an automated microplate reader (OPTImaxTM Tunable microplate reader, Molecular Devices Corporation, CA, USA). The superoxide anion radical scavenging activity of the sample was then calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100: 2.4. Measurement of hydrogen peroxide scavenging activity Hydrogen peroxide scavenging activity was measured by the method of Pick and Mizel (1981) with minor modification. Briefly, 50 ll of sample was first mixed with 50 ll of 5 mM H2 O2 solution (Shimakyu, Osaka, Japan) and incubated at room temperature for 20 min. It was then supplemented with 100 ll of horseradish peroxidase-phenol red solution (HRPase 300 lg/ml and phenol red 4.5 mM in 100 mM phosphate buffer). HRPase was the product of Sigma. After another 10 min of incubation, the sample absorbance at 610 nm was monitored by an automated microplate reader. The scavenging effect was then calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100:
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2.5. Measure of copper chelating ability Cu2þ -binding activity was measured according to the method of Shimada et al. (1992) with minor modification. In this assay method, tetramethyl murexide (TMM, Sigma) was a chelating agent, forming a complex with free Cu2þ , except for the Cu2þ that was bound by test samples. The TMM–Cu2þ complex showed an absorption maximum at 485 nm. To start the assay procedure, a mixture containing 25 ll of 10 mM hexamine (Sigma), 25 ll of 30 mM potassium chloride and 50 ll of 3 mM copper sulfate was added to 100 ll of test sample. To begin the colorational reaction, 0.75 ll of 1 mM TMM was added. After incubation at room temperature for 5 min, the absorbance at 485 nm was detected by automated microplate reader. Cu2þ -binding activity was obtained by according to the following equation: Chelating ability; % ¼ ½1 absorbancesample =absorbancecontrol 100:
3. Results and discussion 3.1. Scavenging of DPPH radical One important mechanism of antioxidation involves the scavenging of hydrogen radicals. DPPH has a hydrogen free radical and shows a characteristic absorption at 517 nm (Brand-Williams, Cuvelier, & Berset, 1995). After encountering the proton-radical scavengers, the purple color of the DPPH solution fades rapidly (Yamaguchi, Takamura, Matoba, & Terao, 1998). In this study, DPPH was used to determine the protonscavenging activity of the various disaccharide chitosan derivatives. The dose–response curve for the various N-alkylated disaccharide chitosan derivatives with different DS and ascorbic acid, used as the positive scavenger, is shown in Fig. 1. The degree of substitution was found to affect the DPPH radical-scavenging activity of the prepared chitosan derivative. The chitosan derivatives having the same disaccharide and possessing a DS of 20–30% exhibited the highest radical-scavenging activity, followed by the derivatives with DS 40–50% and 60–70%. It was noted that, in general, the DPPH radical-scavenging effect increased as the concentration of the chitosan derivatives increased to a certain extent, and then leveled off even with further increase in the concentration. For example, at a concentration of 50–100 lg/ml, the lactose chitosan derivative with DS 20–30% showed 65–95% scavenging activity of DPPH radical. Further increasing the dosage did not significantly increase the DPPHscavenging effect.
Fig. 1. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid on DPPH radicals. Each value represents mean SD (n ¼ 3). Ascorbic acid, O; DS 20– 30%, d; DS 40–50%, N; DS 60–70%, j.
Table 1 summarizes the calculated half-inhibition concentrations (IC50 ), which is the efficient concentration required to decrease initial DPPH concentration by 50%, of the various chitosan derivatives and ascorbic acid. These IC50 values were obtained by interpolation from linear regression analysis of data shown in Fig. 1. As shown in Fig. 1 and Table 1, variation in the DPPH free radical-scavenging activity with an IC50 of 32.3– 290.6 lg/ml or more was noted with the disaccharide chitosan derivatives tested. Lactose and maltose chitosan derivatives with DS 20–30%, among all the samples tested, showed the highest DPPH radical-scavenging activity. The IC50 of these chitosan derivatives was about 2.5 times that of ascorbic acid. On the other hand, maltose and cellobiose chitosan derivatives with DS 60– 70%, having an IC50 of more than 800 lg/ml, exhibited the lowest DPPH radical-scavenging activity. 3.2. Scavenging of hydrogen peroxide Hydrogen peroxide can be generated in biological and food systems. Being a nonradical oxygen-containing reactive agent, it can form hydroxyl radical, the most
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Table 1 IC50 of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid in scavenging DPPH radicals Degree of substitution (%)
IC50 (lg/ml)a of disaccharide chitosan derivative Lactose
20–30 40–50 60–70 Ascorbic acid
b
A 35.91 13.80a B 109.28 2.21a C 290.64 10.10 14.49 0.16
Maltose
Cellobiose
A 32.34 1.20a B 113.39 32.18a >800
A 56.76 5.43b B 119.52 1.88a >800
a
The efficient concentration of antioxidant that decreases initial DPPH concentration by 50%. Each value is given as mean SD ðn ¼ 3Þ. Means within a column with the same capital letters are not significantly different ðp > 0:05Þ. Means within a row with the same lowercase letters are not significantly different ðp > 0:05Þ. b
highly reactive oxygen radical known, in the presence of transition metal ions and participate in free-radical reaction (Halliwell, Murcia, Chirico, & Aruoma, 1995). High levels of hydrogen peroxide can attack several cellular energy-producing systems; for example it inactivates the glycolytic enzyme glyceraldehydes3-phosphate dehydrogenase (Hyslop et al., 1988). Fig. 2 shows the dose–response effect of both the various chitosan derivatives and ascorbic acid on the H2 O2 -scavanging activity. It was found that the H2 O2 scavenging effect of the tested disaccharide chitosan derivatives increased as their concentration increased up to 500 lg/ml. At this dosage level, the chitosan derivatives, regardless of DS, showed a scavenging effect of 80% or more for H2 O2 DS was also found to influence the H2 O2 -scavenging effect of the chitosan derivatives, since increasing the DS resulted in the reduction of the scavenging activity for H2 O2 . Furthermore, it was also observed that with similar degree of DS, the chitosan derivatives with different kinds of disaccharide exhibited different extents of H2 O2 -scavenging activity. As shown in Table 2 the chitosan derivatives shows a IC50 ranging between 163.2 and 305.4 lg/ml, whereas the IC50 for ascorbic acid was found to be ca. 115.5 lg/ ml. This indicates that all the disaccharide chitosan derivatives tested have substantial H2 O2 -scavenging activity, although less than ascorbic acid. It was previously reported that oxidative destruction of chitosan occurred in presence of hydroxy radicals which were generated from H2 O2 (Kabal’Nova et al., 2001; Qin, Du, & Xiao, 2002). Therefore, the reduced amount of hydrogen after this reaction may account for the H2 O2 -scavenging effect of the chitosan derivatives observed in the present study.
Fig. 2. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid on hydrogen peroxide. Each value represents mean SD ðn ¼ 3Þ. Ascorbic acid, O; DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
3.3. Scavenging of superoxide anion radical Superoxides are radicals in which the unpaired electron is located on oxygen. Although they have limited chemical reactivity, superoxides are able to generate more dangerous species, such as highly reactive hydroxyl radicals and the protonated form of superoxide (Halliwell & Chirico, 1993).
Fig. 3 shows the dose–response curves of the chitosan derivatives on the scavenging effect for superoxide anion radicals. Contrary to the report of Matsugo et al. (1998), which indicated that the water-soluble chitosan derivatives prepared by acylation of chitosan did not show any scavenging activity toward superoxide, various extents of superoxide anion-scavenging effects were observed
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Table 2 IC50 of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid in scavenging hydrogen peroxide Degree of substitution (%)
IC50 ( lg/ml)a of disaccharide chitosan derivative Lactose
20–30 40–50 60–70 Ascorbic acid
b
A 230.59 12.24b AB 255.90 19.04ab B 284.31 13.79ab 115.53 0.48
Maltose
Cellobiose
A 248.77 7.50b A 248.44 0.72a B 305.38 13.08b
A 163.16 21.49a B 285.36 13.74b B 270.62 3.76a
a
The efficient concentration of antioxidant that decreases initial hydrogen peroxide concentration by 50%. Each value is given as mean SD ðn ¼ 3Þ. Means within a column with the same captical letters are not significantly different ðp > 0:05Þ. Means within a row with the same lowercase letters are not significantly different ðp > 0:05Þ. b
DS 20–30% showed the highest scavenging effect followed by those with DS 40–50%, and the chitosan derivatives DS 60–70% exhibited little or no scavenging effect for superoxide anion.Furthermore, no significant difference was noted among the IC50 of the various disaccharide chitosan derivative with DS 20–30% ðp > 0:05Þ (Table 2). Xue et al. (1998) indicated that the scavenging activities of chitosan derivatives against hydroxyl radical, the strongest reactive oxygen species, was derived from: (1) the hydroxyl groups in the polysaccharide unit that can react with hydroxyl radical by the typical H-abstraction reaction, (2) the residual free amino groups that can react with hydroxyl radical or (3) the NH2 groups can form NHþ 3 then reacting with hydroxyl radical through addition reaction. Our results again demonstrated the important role of NH2 and NHþ 3 in exhibiting the antioxidative activity of disaccharide chitosan derivatives. The more free amino groups present (low DS) in chitosan derivatives, the higher scavenging effect toward DPPH radicals (Fig. 1) and superoxide anion radicals (Fig. 3) was noted. Furthermore, ability to dissociate the strong hydrogen bond of chitosan derivatives varies with the kinds of disaccharide on the chitosan molecules and thus might lead to the different antioxidative activities observed with various disaccharide chitosan derivatives tested in the present study. 3.4. Copper ion chelating ability
Fig. 3. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution on superoxide anion radicals. Negative value showed that the samples do not have any scavenging effects. Each value represents mean SD ðn ¼ 3Þ. DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
with the water-soluble disaccharide chitosan derivatives tested in the present study. As shown in Fig. 3, in addition to concentration, DS was also found to affect the scavenging effect of the chitosan derivatives for superoxide anion radicals. Regardless of the kinds of disaccharide on the molecules, the chitosan derivatives with
Transition metal ions can initiate lipid peroxidation and start a chain reaction, which leads to the deterioration of flavor and taste in food (Gordon, 1990). It has also been proposed that the catalysis of metal ions might correlate to cancer and arthritis (Halliwell et al., 1995). In the present study, the chelating ability of the disaccharide chitosan derivatives with different DS toward copper ions was investigated. As shown in Fig. 4, all the tested chitosan derivatives exhibited Cu2þ ion-chelating ability, which increased as the concentration increased. While their chelating activities were less than that observed with EDTA. EDTA at a concentration of 1.0 mg/ml showed a
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80
chelating ability of chitosan is strongly affected by the degree of acetylation, with the fully acetylated chitosan showing very little chelating activity. On the other hand, Kurita, Sannan, and Iwakura (1979) reported that although the metal ion absorption capability of chitin is closely related to its amino acid content, other factors such as affinity for water and crystallinity also affected the ion-chelating activity.
60 40 20 Lactose chitosan derivative
Scavenging effect (%)
0 80
4. Conclusions
60 40 20 Maltose chitosan derivative
0 80 60 40 20 Cellobiose chitosan derivative
0
0
1
2 3 Concentration (mg/mL)
4
Fig. 4. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and EDTA on cupric ion. Each value represents mean SD ðn ¼ 3Þ. EDTA, O; DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
This study demonstrated that the water-soluble N-alkylated disaccharide chitosan derivative has various extents of antioxidant properties, including scavenging activities for DPPH radicals, hydrogen peroxide, superoxide anion radicals and Cu2þ ion-chelating activity. These antioxidative effects may vary with the concentration, DS and the kinds of disaccharide on the chitosan derivative molecule. In general, within a certain concentration range, the antioxidative activity increased with the concentration. Furthermore, it was noted that the derivatives with low DS (20–30%) exhibited a higher DPPH radicals- superoxide anion radicals- and H2 O2 scavenging activity than those with higher DS (60–70%). In light of the antioxidative activity observed in the present study and the water-soluble characteristics (Yang et al., 2002), it is suggested that these disaccharide chitosan derivatives may be used as a source of antioxidants, as a possible food supplement or in the pharmaceutical industry, especially in areas where the use of chitosan and its derivatives is limited due to their insolubility.
References Cu2þ -scavenging effect of ca. 60%. while a dosage of 4.0 mg/ml or more of the chitosan derivatives tested was required to show similar degree of scavenge effect on Cu2þ . Unlike the effects observed on the scavenging effect for DPPH (Fig. 1, Table 1) and H2 O2 (Fig. 2, Table 2), no regular trend concerning the effect of DS on the Cu2þ -scavenging activity of the disaccharide chitosan derivatives can be drawn (Fig. 4). Among the lactose chitosan derivatives tested, those with DS 40–50% showed the least Cuþ -scavenging effect (Fig. 4). Whereas, among the cellobiose chitosan derivatives tested the lowest scavenging effect was noted with samples having a DS of 60–70%. Factors affecting the ion-chelating ability of chitosan and chitin are rather complex. Inoue, Baba, Yoshizuka, Noguchi, and Yoshizaki (1988) suggested that chelating of Cu2þ by chitosan involves the binding of Cu2þ with the hydroxyl group on C6 and amino group on C2 of the chitosan molecule. Qin (1993) indicated that the ion-
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LebensmittelWissenschaft und-Technologie, 25, 25–30. Felse, P. A., & Panda, T. (1999). Studies on applications of chitin and its derivatives. Bioprocess Engineering, 20, 505–512. Gordon, M. H. (1990). The mechanism of antioxidant action in vitro. In B. J. F. Hudson (Ed.), Food antioxidants (pp. 1–18). London and New York: Elsevier Applied Science. Halliwell, B., & Chirico, S. (1993). Lipid peroxidation: Its mechanism, measurement, and significance. The American Journal of Clinical Nutrition, 57, 715S–725S. Halliwell, B., Murcia, M. A., Chirico, S., & Aruoma, O. I. (1995). Aruoma, free radicals and antioxidants in food and in vivo: What they do and how they work. Critical Reviews in Food Science and Nutrition, 35, 7–20. Hyslop, P. A., Hinshaw, D. B., Halsey, W. A., Jr., Schraufstater, I. U., Sauerheber, R. D., Spragg, R. G, Jackson, J. H., & Cochrane, C. G. (1988). Mechanisms of oxidant-mediated injury. The Journal of Biological Chemistry, 263, 1665–1675. Inoue, K., Baba, Y., Yoshizuka, K., Noguchi, H., & Yoshizaki, M. (1988). Selectivity series in the adsorption of metal ions on a resin prepared by crosslinking copper(II)-complexed chitosan. Chemistry Letters, 1281–1284.
H.-Y. Lin, C.-C. Chou / Food Research International 37 (2004) 883–889 Kabal’Nova, N. N., Murinov, K. Y., Mullagaliev, I. R., Krasnogorskaya, N. N., Shereshovets, V. V., Monakov, Y. B., & Zaikov, G. E. (2001). Oxidative destruction of chitosan under the effect of ozone and hydrogen peroxide. Journal of Applied Polymer Science, 81, 875–881. Kobayashi, M., Watanabe, T., Suzuki, S., & Suzuki, M. (1990). Effect of N-acetylchitogexaose against Candida albicans infection of tumor-bearing mice. Microbiology and Immunology, 34, 413–426. Kurita, K., Sannan, T., & Iwakura, Y. (1979). Studies on chitin. VI. Binding of metal cations. Journal of Applied Polymer Science, 2, 511–515. Maeda, M., Murakami, H., Ohta, H., & Tajima, M. (1992). Stimulation of IgM 15 production in human-human hybridoma HB4C5 cells by chitosan. Bioscience, Biotechnology, and Biochemistry, 56, 427–431. Matsugo, S., Mizuie, M., Matsugo, M., Ohwa, R., Kitano, K., & Konishi, T. (1998). Synthesis and antioxidant activity of watersoluble chitosan derivatives. Biochemistry and Molecular Biology International, 44, 939–948. Nishimura, K., Nishimura, S., Nishi, N., Numata, F., Tone, Y., Tokura, S., & Azuma, I. (1985). Adjuvant activity of chitin derivatives in mice and guinea-pigs. Vaccine, 3, 379–384. Pick, E., & Mizel, D. (1981). Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. Journal of Immunological Methods, 46, 211–226. Qin, Y. (1993). The chelating properties of chitosan fibers. Journal of Applied Polymer Science, 49, 727–731. Qin, C. Q., Du, Y. M., & Xiao, L. (2002). Effect of hydrogen peroxide treatment on the molecular weight and structure of chitosan. Polymer Degradation and Stability, 76, 211–218. Robak, J., & Gryglewski, R. J. (1988). Gryglewski, flavonoids are scavengers of superoxide anions. Biochemical Pharmacology, 17, 837–841. Saiki, I., Murata, J., Nakajima, M., Tokura, S., & Azuma, I. (1990). Inhibition by sulfated chitin derivatives of invasion through extracellular matrix and enzymatic degradation by metastatic melanoma cells. Cancer Research, 50, 3631–3637. Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food applications of chitin and chitosans. Trends in Food Science and Technology, 10, 37–51.
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Shibata, Y., Foster, L., Metzger, W., & Myrvik, Q. (1997). Alveloar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D -glucosamine, in mice. Infection and Immunity, 65, 1734–1741. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the anti-oxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945–948. Sugimoto, M., Morimoto, M., Sashiwa, H., Saimoto, H., & Shigemasa, Y. (1998). Preparation and characterization of watersoluble chitin and chitosan derivatives. Carbohydrate Polymer, 36, 49–59. T€ oei, K., & Kohara, T. (1976). A conductometric method for colloid titrations. Analytica Chimica Acta, 83, 59–65. Tokoro, A., Kobayashi, M., Tatewaki, N., Suzuki, K., Okawa, Y., Mikami, T., Suzuki, S., & Suzuki, M. (1989). Protective effect of Nacetylchitohexaose on Listeria monocytogenes infection in mice. Microbiology and Immunology, 33, 357–367. Tsukada, K., Matsumoto, T., Aizawa, K., Tokoro, A., Naruse, R., Suzuki, S., & Suzuki, M. (1990). Antimetastatic and growthinhibitory effects of N-acetylchitohexaose in mice bearing lewis lung carcinoma. Japanese Journal of Cancer Research, 81, 259– 265. Xie, W., Xu, P., & Liu, Q. (2001). Antioxidative activity of watersoluble chitosan derivatives. Bioorganic and Medicinal Chemistry Letters, 11, 1699–1701. Xue, C., Yu, G., Hirata, T., Terao, J., & Lin, H. (1998). Antioxidative activities of several marine polysaccharides evaluated in a phosphatidylcholine-liposomal suspension and organic solvents. Bioscience, Biotechnology, and Biochemistry, 62, 206–209. Yamaguchi, T., Takamura, H., Matoba, T., & Terao, J. (1998). HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry, 62, 1201–1204. Yang, T. C., Chou, C. C., & Li, C. F. (2002). Preparation, water solubility and rheological property of the N-alkylated mono or disaccharide chitosan derivatives. Food Research International, 35, 707–713. Yang, T. C., Chou, C. C., & Li, C. F. (2004). Antibacterial activity of Nalkylated disaccharide chitosan derivatives. International Journal of Food Microbiology (in press), doi: 10.1016/S0168-1605(03)00083-7.
Antioxidative activities of water-soluble disaccharide chitosan derivatives Hsia-Yin Lin, Cheng-Chun Chou
*
Graduate Institute of Food Science and Technology, National Taiwan University 59, Lane 144, Keelung Rd., Section 4, Taipei 106-17, Taiwan Received 1 February 2004; accepted 23 April 2004
Abstract Water-soluble chitosan derivatives, having various degrees of substitution with disaccharide (DS 20–30%, 40–50%, 60–70%), were prepared by reductive alkylation of a-chitosan with lactose, maltose or cellobiose. Antioxidative activities were determined in the present study, including radical scavenging effect for a,a-diphenyl-b-picryl-hydrazyl (DPPH) radicals, superoxide anion radicals, hydrogen peroxide and copper ion chelating ability of these chitosan derivatives. It was found that the tested chitosan derivatives exhibited multiple antioxidative activities that varied with the concentration, DS with disaccharides and the kind of disaccharide present in the derivative molecule. In general, the antioxidative activities increased as the concentration of these chitosan derivatives increased up to a certain extent. A stronger scavenging effects for superoxide anion radicals, DPPH radicals and H2 O2 was noted with the chitosan derivatives having lower DS with disaccharide than those with higher DS. Among the various antioxidative activities tested, the disaccharide chitosan derivatives were found to show the highest excellent hydrogen peroxide scavenging activity. At a concentration of 400 lg/ml, all the chitosan derivatives tested exhibited 60% or greater scavenging activity for hydrogen peroxide. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Disaccharide chitosan derivative; Antioxidative activity
1. Introduction Chitosan, the deacetylated derivative of chitin, is a polymer of glucosamine. It is also one of the nontoxic and biodegradable carbohydrate polymers. Numerous studies have demonstrated that chitosan and its derivatives have various biological activities such as antimicrobial activity (Kobayashi, Watanabe, Suzuki, & Suzuki, 1990; Tokoro et al., 1989), antitumor activity (Saiki, Murata, Nakajima, Tokura, & Azuma, 1990; Tsukada et al., 1990) and immune-enhancing effects (Maeda, Murakami, Ohta, & Tajima, 1992; Nishimura et al., 1985; Shibata, Foster, Metzger, & Myrvik, 1997). Furthermore, antioxidant activity of chitosan derivatives has also been observed by several investigators over the past few years. For example, Xie, Xu, and Liu *
Corresponding author. Tel.: +886-2-2363-0231x2717; fax: +886-22362-0849. E-mail address: [email protected] (C.-C. Chou). 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.04.007
(2001) reported that the hexanoyl chitin and an N-benzoylhexanoyl chitosan could trap peroxide radicals in organic solvent when the radical chain reaction had been initiated by 2,20 -azobis (2,4-dimethylvaleronitrile). In addition, Matsugo et al. (1998) obtained chitosan derivatives through the acylation of chitosan with acid anhydride, and observed that these derivatives inhibited thiobartituric acid reactive substrate formation in t-butylhydroperoxide and benzoyl peroxide induced lipid peroxidation. Furthermore, Xue, Yu, Hirata, Terao, and Lin (1998) indicated that the chitosan derivatives prepared by graft copolymerization of maleic acid sodium onto hydroxypropyl chitosan and carboxymethyl chitosan sodium had scavenging activities against hydroxyl radicals. Due to these unique properties, chitosan and its derivatives have been proposed for diverse applications in biomedical, food, agriculture, biotechnology and pharmaceutical fields (Felse & Panda, 1999; Shahidi, Arachchi, & Jeon, 1999). However, its insolubility at
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neutral or high pH often limits the application of chitosan and its derivatives (Sugimoto, Morimoto, Sashiwa, Saimoto, & Shigemasa, 1998). To overcome the limited solubility, we previously prepared water-soluble chitosan derivatives through the reductive N-alkylation of chitosan with different disaccharides (Yang, Chou, & Li, 2002). These disaccharide chitosan derivatives were demonstrated to exhibit antibacterial activity that might be more useful than that of native chitosan as an antimicrobial agent in a food product or a system with a pH at 7.0 or higher (Yang, Chou, & Li, 2004). In the present study, the antioxidant activity of these derivatives is further examined.
2. Materials and methods 2.1. Preparation of N-alkylated disaccharide chitosan derivatives In this study, antioxidative activity of N-alkylated disaccharide (lactose, manose and cellobiose) derivatives with different degrees of substitution (DS) was tested. The disaccharide chitosan derivatives were prepared according to the procedures described by Yang et al. (2002). Briefly, Chitosan (0.5 g) from shrimp shells with 99% N-deacetylation, as determined by colloid titration (T€ oei & Kohara, 1976), was first obtained from China League Biotechnology Associates. Ltd. Lytone Enterprise, Inc. (Taipei, Taiwan). Then, dissolved in a mixture (1:1, pH 5.5) of methanol and 1% aqueous acetic acid, chitosan was mixed with various amounts of disaccharides (0.5–1.5 g). After resting at room temperature for 1 h, it was supplemented with NaCNBH3 (1 g) and was kept at room temperature for a period of 4–48 h, generally until a white solidified mass was formed. The solidified mass was then collected and washed with methanol for four times followed by washing with diethyl ether, then air-dried and finally dried under vacuum. If no solidified mass formed, the mixture was dialyzed against distilled water, then freeze-dried. The residue was dissolved in distilled water, and centrifuged (8000 rpm, 15 min). The supernatant and precipitate were separated by decanting, and the supernatant was freeze-dried again. The methods in T€ oei and Kohara (1976), with minor modifications as described by Yang et al. (2002), were followed to determine the DS of the prepared chitosan disaccharide derivatives. 2.2. Measurement of free radical scavenging activity The free radical scavenging activity of chitosan disaccharide derivatives was measured by 1,1-diphenyl2-picrylhydrazyl radical (DPPH) (Nacalai Tesque,
Kyoto, Japan) using the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). Briefly, 400 lM DPPH solution in methanol was prepared and 3.0 ml of this solution was added to 1.0 ml test samples at different concentrations. After a 90 min incubation period at ambient temperature, the absorbance at 517 nm was measured. The inhibitory percentage of DPPH was calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100:
2.3. Measurement of superoxide anion radical scavenging activity The method described by Robak and Gryglewski (1988) was used to measure the ability to scavenge superoxide anion radicals. First the reagents were all prepared in 100 mM phosphate buffer (pH 7.4). The reaction mixture containing 50 ll of test sample, 50 ll of 300 lM nitrobluetetrazolium (Sigma, St. Louis, MO, USA), 50 ll of 936 lM NADH and 50 ll of 120 lM phenazine methosulfate (Sigma) was incubated at room temperature for 5 min. The absorbance at 560 nm was then measured by an automated microplate reader (OPTImaxTM Tunable microplate reader, Molecular Devices Corporation, CA, USA). The superoxide anion radical scavenging activity of the sample was then calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100: 2.4. Measurement of hydrogen peroxide scavenging activity Hydrogen peroxide scavenging activity was measured by the method of Pick and Mizel (1981) with minor modification. Briefly, 50 ll of sample was first mixed with 50 ll of 5 mM H2 O2 solution (Shimakyu, Osaka, Japan) and incubated at room temperature for 20 min. It was then supplemented with 100 ll of horseradish peroxidase-phenol red solution (HRPase 300 lg/ml and phenol red 4.5 mM in 100 mM phosphate buffer). HRPase was the product of Sigma. After another 10 min of incubation, the sample absorbance at 610 nm was monitored by an automated microplate reader. The scavenging effect was then calculated according to the following equation: Scavenging effect; % ¼ ½1 absorbancesample =absorbancecontrol 100:
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2.5. Measure of copper chelating ability Cu2þ -binding activity was measured according to the method of Shimada et al. (1992) with minor modification. In this assay method, tetramethyl murexide (TMM, Sigma) was a chelating agent, forming a complex with free Cu2þ , except for the Cu2þ that was bound by test samples. The TMM–Cu2þ complex showed an absorption maximum at 485 nm. To start the assay procedure, a mixture containing 25 ll of 10 mM hexamine (Sigma), 25 ll of 30 mM potassium chloride and 50 ll of 3 mM copper sulfate was added to 100 ll of test sample. To begin the colorational reaction, 0.75 ll of 1 mM TMM was added. After incubation at room temperature for 5 min, the absorbance at 485 nm was detected by automated microplate reader. Cu2þ -binding activity was obtained by according to the following equation: Chelating ability; % ¼ ½1 absorbancesample =absorbancecontrol 100:
3. Results and discussion 3.1. Scavenging of DPPH radical One important mechanism of antioxidation involves the scavenging of hydrogen radicals. DPPH has a hydrogen free radical and shows a characteristic absorption at 517 nm (Brand-Williams, Cuvelier, & Berset, 1995). After encountering the proton-radical scavengers, the purple color of the DPPH solution fades rapidly (Yamaguchi, Takamura, Matoba, & Terao, 1998). In this study, DPPH was used to determine the protonscavenging activity of the various disaccharide chitosan derivatives. The dose–response curve for the various N-alkylated disaccharide chitosan derivatives with different DS and ascorbic acid, used as the positive scavenger, is shown in Fig. 1. The degree of substitution was found to affect the DPPH radical-scavenging activity of the prepared chitosan derivative. The chitosan derivatives having the same disaccharide and possessing a DS of 20–30% exhibited the highest radical-scavenging activity, followed by the derivatives with DS 40–50% and 60–70%. It was noted that, in general, the DPPH radical-scavenging effect increased as the concentration of the chitosan derivatives increased to a certain extent, and then leveled off even with further increase in the concentration. For example, at a concentration of 50–100 lg/ml, the lactose chitosan derivative with DS 20–30% showed 65–95% scavenging activity of DPPH radical. Further increasing the dosage did not significantly increase the DPPHscavenging effect.
Fig. 1. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid on DPPH radicals. Each value represents mean SD (n ¼ 3). Ascorbic acid, O; DS 20– 30%, d; DS 40–50%, N; DS 60–70%, j.
Table 1 summarizes the calculated half-inhibition concentrations (IC50 ), which is the efficient concentration required to decrease initial DPPH concentration by 50%, of the various chitosan derivatives and ascorbic acid. These IC50 values were obtained by interpolation from linear regression analysis of data shown in Fig. 1. As shown in Fig. 1 and Table 1, variation in the DPPH free radical-scavenging activity with an IC50 of 32.3– 290.6 lg/ml or more was noted with the disaccharide chitosan derivatives tested. Lactose and maltose chitosan derivatives with DS 20–30%, among all the samples tested, showed the highest DPPH radical-scavenging activity. The IC50 of these chitosan derivatives was about 2.5 times that of ascorbic acid. On the other hand, maltose and cellobiose chitosan derivatives with DS 60– 70%, having an IC50 of more than 800 lg/ml, exhibited the lowest DPPH radical-scavenging activity. 3.2. Scavenging of hydrogen peroxide Hydrogen peroxide can be generated in biological and food systems. Being a nonradical oxygen-containing reactive agent, it can form hydroxyl radical, the most
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Table 1 IC50 of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid in scavenging DPPH radicals Degree of substitution (%)
IC50 (lg/ml)a of disaccharide chitosan derivative Lactose
20–30 40–50 60–70 Ascorbic acid
b
A 35.91 13.80a B 109.28 2.21a C 290.64 10.10 14.49 0.16
Maltose
Cellobiose
A 32.34 1.20a B 113.39 32.18a >800
A 56.76 5.43b B 119.52 1.88a >800
a
The efficient concentration of antioxidant that decreases initial DPPH concentration by 50%. Each value is given as mean SD ðn ¼ 3Þ. Means within a column with the same capital letters are not significantly different ðp > 0:05Þ. Means within a row with the same lowercase letters are not significantly different ðp > 0:05Þ. b
highly reactive oxygen radical known, in the presence of transition metal ions and participate in free-radical reaction (Halliwell, Murcia, Chirico, & Aruoma, 1995). High levels of hydrogen peroxide can attack several cellular energy-producing systems; for example it inactivates the glycolytic enzyme glyceraldehydes3-phosphate dehydrogenase (Hyslop et al., 1988). Fig. 2 shows the dose–response effect of both the various chitosan derivatives and ascorbic acid on the H2 O2 -scavanging activity. It was found that the H2 O2 scavenging effect of the tested disaccharide chitosan derivatives increased as their concentration increased up to 500 lg/ml. At this dosage level, the chitosan derivatives, regardless of DS, showed a scavenging effect of 80% or more for H2 O2 DS was also found to influence the H2 O2 -scavenging effect of the chitosan derivatives, since increasing the DS resulted in the reduction of the scavenging activity for H2 O2 . Furthermore, it was also observed that with similar degree of DS, the chitosan derivatives with different kinds of disaccharide exhibited different extents of H2 O2 -scavenging activity. As shown in Table 2 the chitosan derivatives shows a IC50 ranging between 163.2 and 305.4 lg/ml, whereas the IC50 for ascorbic acid was found to be ca. 115.5 lg/ ml. This indicates that all the disaccharide chitosan derivatives tested have substantial H2 O2 -scavenging activity, although less than ascorbic acid. It was previously reported that oxidative destruction of chitosan occurred in presence of hydroxy radicals which were generated from H2 O2 (Kabal’Nova et al., 2001; Qin, Du, & Xiao, 2002). Therefore, the reduced amount of hydrogen after this reaction may account for the H2 O2 -scavenging effect of the chitosan derivatives observed in the present study.
Fig. 2. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid on hydrogen peroxide. Each value represents mean SD ðn ¼ 3Þ. Ascorbic acid, O; DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
3.3. Scavenging of superoxide anion radical Superoxides are radicals in which the unpaired electron is located on oxygen. Although they have limited chemical reactivity, superoxides are able to generate more dangerous species, such as highly reactive hydroxyl radicals and the protonated form of superoxide (Halliwell & Chirico, 1993).
Fig. 3 shows the dose–response curves of the chitosan derivatives on the scavenging effect for superoxide anion radicals. Contrary to the report of Matsugo et al. (1998), which indicated that the water-soluble chitosan derivatives prepared by acylation of chitosan did not show any scavenging activity toward superoxide, various extents of superoxide anion-scavenging effects were observed
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Table 2 IC50 of disaccharide chitosan derivatives with different degree of substitution and ascorbic acid in scavenging hydrogen peroxide Degree of substitution (%)
IC50 ( lg/ml)a of disaccharide chitosan derivative Lactose
20–30 40–50 60–70 Ascorbic acid
b
A 230.59 12.24b AB 255.90 19.04ab B 284.31 13.79ab 115.53 0.48
Maltose
Cellobiose
A 248.77 7.50b A 248.44 0.72a B 305.38 13.08b
A 163.16 21.49a B 285.36 13.74b B 270.62 3.76a
a
The efficient concentration of antioxidant that decreases initial hydrogen peroxide concentration by 50%. Each value is given as mean SD ðn ¼ 3Þ. Means within a column with the same captical letters are not significantly different ðp > 0:05Þ. Means within a row with the same lowercase letters are not significantly different ðp > 0:05Þ. b
DS 20–30% showed the highest scavenging effect followed by those with DS 40–50%, and the chitosan derivatives DS 60–70% exhibited little or no scavenging effect for superoxide anion.Furthermore, no significant difference was noted among the IC50 of the various disaccharide chitosan derivative with DS 20–30% ðp > 0:05Þ (Table 2). Xue et al. (1998) indicated that the scavenging activities of chitosan derivatives against hydroxyl radical, the strongest reactive oxygen species, was derived from: (1) the hydroxyl groups in the polysaccharide unit that can react with hydroxyl radical by the typical H-abstraction reaction, (2) the residual free amino groups that can react with hydroxyl radical or (3) the NH2 groups can form NHþ 3 then reacting with hydroxyl radical through addition reaction. Our results again demonstrated the important role of NH2 and NHþ 3 in exhibiting the antioxidative activity of disaccharide chitosan derivatives. The more free amino groups present (low DS) in chitosan derivatives, the higher scavenging effect toward DPPH radicals (Fig. 1) and superoxide anion radicals (Fig. 3) was noted. Furthermore, ability to dissociate the strong hydrogen bond of chitosan derivatives varies with the kinds of disaccharide on the chitosan molecules and thus might lead to the different antioxidative activities observed with various disaccharide chitosan derivatives tested in the present study. 3.4. Copper ion chelating ability
Fig. 3. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution on superoxide anion radicals. Negative value showed that the samples do not have any scavenging effects. Each value represents mean SD ðn ¼ 3Þ. DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
with the water-soluble disaccharide chitosan derivatives tested in the present study. As shown in Fig. 3, in addition to concentration, DS was also found to affect the scavenging effect of the chitosan derivatives for superoxide anion radicals. Regardless of the kinds of disaccharide on the molecules, the chitosan derivatives with
Transition metal ions can initiate lipid peroxidation and start a chain reaction, which leads to the deterioration of flavor and taste in food (Gordon, 1990). It has also been proposed that the catalysis of metal ions might correlate to cancer and arthritis (Halliwell et al., 1995). In the present study, the chelating ability of the disaccharide chitosan derivatives with different DS toward copper ions was investigated. As shown in Fig. 4, all the tested chitosan derivatives exhibited Cu2þ ion-chelating ability, which increased as the concentration increased. While their chelating activities were less than that observed with EDTA. EDTA at a concentration of 1.0 mg/ml showed a
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chelating ability of chitosan is strongly affected by the degree of acetylation, with the fully acetylated chitosan showing very little chelating activity. On the other hand, Kurita, Sannan, and Iwakura (1979) reported that although the metal ion absorption capability of chitin is closely related to its amino acid content, other factors such as affinity for water and crystallinity also affected the ion-chelating activity.
60 40 20 Lactose chitosan derivative
Scavenging effect (%)
0 80
4. Conclusions
60 40 20 Maltose chitosan derivative
0 80 60 40 20 Cellobiose chitosan derivative
0
0
1
2 3 Concentration (mg/mL)
4
Fig. 4. Scavenging effects of disaccharide chitosan derivatives with different degree of substitution and EDTA on cupric ion. Each value represents mean SD ðn ¼ 3Þ. EDTA, O; DS 20–30%, d; DS 40–50%, N; DS 60–70%, j.
This study demonstrated that the water-soluble N-alkylated disaccharide chitosan derivative has various extents of antioxidant properties, including scavenging activities for DPPH radicals, hydrogen peroxide, superoxide anion radicals and Cu2þ ion-chelating activity. These antioxidative effects may vary with the concentration, DS and the kinds of disaccharide on the chitosan derivative molecule. In general, within a certain concentration range, the antioxidative activity increased with the concentration. Furthermore, it was noted that the derivatives with low DS (20–30%) exhibited a higher DPPH radicals- superoxide anion radicals- and H2 O2 scavenging activity than those with higher DS (60–70%). In light of the antioxidative activity observed in the present study and the water-soluble characteristics (Yang et al., 2002), it is suggested that these disaccharide chitosan derivatives may be used as a source of antioxidants, as a possible food supplement or in the pharmaceutical industry, especially in areas where the use of chitosan and its derivatives is limited due to their insolubility.
References Cu2þ -scavenging effect of ca. 60%. while a dosage of 4.0 mg/ml or more of the chitosan derivatives tested was required to show similar degree of scavenge effect on Cu2þ . Unlike the effects observed on the scavenging effect for DPPH (Fig. 1, Table 1) and H2 O2 (Fig. 2, Table 2), no regular trend concerning the effect of DS on the Cu2þ -scavenging activity of the disaccharide chitosan derivatives can be drawn (Fig. 4). Among the lactose chitosan derivatives tested, those with DS 40–50% showed the least Cuþ -scavenging effect (Fig. 4). Whereas, among the cellobiose chitosan derivatives tested the lowest scavenging effect was noted with samples having a DS of 60–70%. Factors affecting the ion-chelating ability of chitosan and chitin are rather complex. Inoue, Baba, Yoshizuka, Noguchi, and Yoshizaki (1988) suggested that chelating of Cu2þ by chitosan involves the binding of Cu2þ with the hydroxyl group on C6 and amino group on C2 of the chitosan molecule. Qin (1993) indicated that the ion-
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