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Free radical scavenging properties of hetero-chitooligosaccharides using an ESR spectroscopy
Food and Chemical Toxicology 42 (2004) 381–387 www.elsevier.com/locate/foodchemtox
Free radical scavenging properties of hetero-chitooligosaccharides using an ESR spectroscopy Jae-Young Je, Pyo-Jam Park, Se-Kwon Kim* Department of Chemistry, Pukyong National University, Busan 608-737, South Korea Received 9 April 2003; accepted 6 October 2003
Abstract Nine kinds of hetero-chitooligosaccharides (hetero-COSs) with relatively higher molecular weights (90, 75 and 50HMWCOS), medium molecular weights (90, 75 and 50-MMWCOS), and lower molecular weights (90, 75 and 50-LMWCOS) were prepared from partially deacetylated hetero-chitosans (90, 75 and 50% deacetylated chitosan), and their scavenging activities were investigated against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide and carbon-centered radicals using electron spin resonance (ESR) spin-trapping technique. Superoxide, hydroxyl and carbon-centered radicals were generated from hypoxanthine-xanthine oxidase reaction, hydrogen peroxide-ferrous sulfate (Fenton reaction) and azo compound 2,2-azobis-(2-amidinopropane)-hydrochloride (AAPH), respectively. The ESR results revealed that 90-MMWCOS, which is having relatively medium molecular weights prepared from 90% deacetylated chitosan, showed the highest scavenging activity on all tested radicals. In addition, the radical scavenging activity of hetero-COSs increased with a dose-dependent manner, and it was dependent on their degree of deacetylation values and molecular weights. # 2004 Elsevier Ltd. All rights reserved. Keywords: Hetero-chitooligosaccharides; Electron spin resonance; Free radical; Radical scavenging activity
1. Introduction Chitosan, which is a copolymer consisting of b(1!4)-2-acetamido-d-glucose and b-(1!4)-2-amino-dglucose units, is derived from chitin by deacetylation in the presence of alkali. Chitosan exhibits a wide variety of physiological activities such as antitumor activity (Sugano et al., 1992), immumo-stimulating effect (Jeon and Kim, 2001), antimicrobial effect (Sudharsham et al., 1992), and cholesterol-reducing effect (Maezaki et al., 1993). Although chitosan has very strong functional properties in many areas, its high molecular weights and viscosity might restrict the uses in vivo. Therefore, an attention in chitosan fields has recently been increased to the production of useful chitooligosaccharides (COS) because COS is not only water-soluble but also possesses versatile functional properties such as antitumor activity (Suzuki et al., 1986; Jeon and Kim, 2002), immumostimulating effect (Tokoro et al., 1988; Jeon and Kim, * Corresponding author. Tel.: +82-51-620-6375; fax: +82-51-6288147. E-mail address: [email protected] (S.-Kwon Kim). 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2003.10.001
2001), enhancement of protective effects against infections associated with some pathogens in mice (Tokoro et al., 1989; Yamada et al., 1993), antifungal activity (Kendra and Hagwiger, 1984; Hirano and Nagao, 1989), and antimicrobial activity (Kendra and Hagwiger, 1984; Kim et al., 2000; Jeon et al., 2001). Recently, the antioxidative properties of chitosan and its derivatives containing COS as natural antioxidants have remarked the most attention. However, little information on the free radical scavenging activity of hetero-COSs is available until now. In recent years, reactive oxygen species (ROS) and free radicals play an important role in many diseases such as cancer (Muramatsu et al., 1995; Leanderson et al., 1997), gastric ulcers (Sussman and Bulkley, 1990; Debashis et al., 1997), Alzheimer’s, arthritis and ischemic reperfusion (Vajragupta et al., 2000). Formation of free radicals such as superoxide anion . radical (O 2 ) and hydroxyl radical ( OH) is an unavoidable consequence in aerobic organisms during respiration. These radicals are very unstable and react rapidly with the other groups or substances in the body, leading to cell or tissue injury. Free radical
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scavenger is a preventive antioxidant. Antioxidants can act at different levels in an oxidative sequence. This may be illustrated considering one of the many mechanism by which oxidative stress can cause damage by stimulating the free radical chain reaction of lipid peroxidation. Many synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, t-butylhydroquinone and propyl gallate may be used to retard lipid peroxidation in a lot of fields (Wanita et al., 1996). However, the use of synthetic antioxidants is under strict regulation due to the potential health hazards caused by such compounds (Park et al., 2001). In the present study, we investigated the free radical scavenging activity of hetero-COSs with different degree of deacetylation values and molecular weights on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide . anion radical (O 2 ), hydroxyl radical ( OH) and carboncentered radical using electron spin resonance (ESR) spin-trapping techniques.
2. Materials and methods
90-HMWCOS, 75-HMWCOS and 50-HMWCOS), mediun molecular weights (Mw, 5000–1000 Da; 90MMWCOS, 75-MMWCOS and 50-LMWCOS), and lower molecular weights (Mw, below 1000 Da; 90LMWCOS, 75-LMWCOS and 50-LMWCOS). Nine kinds of COSs recovered were lyophilized on a freezingdrier for 5 days. 2.3. Scavenging effect on DPPH radical DPPH radical scavenging activity was measured using the method described by Nanjo et al. (1996). An ethanol solution of 60 ml of each sample (or ethanol itself as control) was added to 60 ml of DPPH (60 mM) in ethanol solution. After mixing vigorously for 10 s, the solution was then transferred into a 100 ml quartz capillary tube, and the scavenging activity of hetero-COSs on DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 11000; sweep time, 30 s.
2.1. Materials 2.4. Hydroxyl radical scavenging activity Chitin prepared from crab shells donated by Kitto Life Co. (Seoul, Korea). The chitosanase (35,000 U/g protein) derived from Bacillus sp. was purchased from Amicogen Co. (JinJu, Korea), and cellulase was donated from Pacific Chemical Co. Ltd. (Seoul, Korea). An ultrafiltration membrane (UF) reactor system for the preparation and the fractionation of hetero-COSs, based on molecular weights, was from Millipore Co. (Bedford, MA). Hypoxanthine (HPX), 1,1-diphenyl-2picrylhydrazyl (DPPH), xanthine oxidase (XOD), 5,5dimethyl-pyrrokine N-oxide (DMPO), 2,2-azobis-(2amidinopropane)-hydrochloride (AAPH) and a-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade.
Hydroxyl radicals were generated by iron-catalyzed Haber-Weiss reaction (Fenton driven Haber-Weiss reaction) and the generated hydroxyl radicals rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman, 1984). The resultant DMPO-OH adduct was detectable with an ESR spectrometer. Hetero-COSs (0.2 ml) with various concentrations was mixed with DMPO (0.3 M, 0.2 ml), Fe2SO4 (10 mM, 0.2 ml) and H2O2 (10 mM, 0.2 ml) in a phosphate buffer solution (pH 7.2), and then transferred into a 100 ml quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1200; sweep time, 4 min.
2.2. Preparation of hetero-chitosans and their COSs 2.5. Superoxide anion radical scavenging activity Three kinds of partially deacetylated chitosans designated 90% deacetylated chitosan, 75% deacetylated chitosan and 50% deacetylated chitosan were prepared from crab shell chitin, according to our previous method (Park et al., 2003). Hetero-COSs were prepared from partially deacetylated hetero-chitosans by two enzymatic reactions using chitosanase and cellulase in an ultrafiltration membrane reactor system according to a previously reported method (Park et al., 2003). Three different UF membranes ranged from molecular weight cut offs (MWCO) of 10, 5 and 1 KDa were used, and fractionated into nine kinds of COSs with relatively higher molecular weights (Mw, 10000–5000 Da;
Superoxide anion radicals were generated from HPX-XOD system (Rosen and Rauckman, 1984). HPX (4 mM, 50 ml) was mixed with phosphate buffered saline (PBS, pH 7.4, 30 ml), hetero-COSs (50 ml) with various concentrations, DMPO (4.5 M, 20 ml) and XOD (0.4 U/ ml, 50 ml), and then the mixture was transferred into a 100 ml quartz capillary tube. After 45 s, the ESR spectrum was recorded using an ESR spectrometer. Manganese oxide was used as an internal standard. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 4 mW; modulation frequency, 9.41 GHz; amplitude, 1500; sweep time, 1 min.
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2.6. Assay for carbon-centered radical Carbon-centered radicals were generated by AAPH. A PBS reaction mixture containing 10 mM AAPH, 10 mM 4-POBN and hetero-COSs with various concentrations was incubated for 30 min at 37 C in a water bath (Hiramoto et al., 1993), and then transferred to 100 ml quartz capillary tube. The spin adduct was recorded using an ESR spectrometer. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 10 mW; modulation frequency, 9.41 GHz; amplitude, 11000; sweep time, 1 min. 2.7. Statistics The data presented are means S.E. of three determinations.
3. Results DPPH radical is a stable radical which gives typical ESR spectra shown in Fig. 1(a). Fig. 1(b)–(d) shows the effects of hetero-COSs with different degree of
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deacetylation values and molecular weights. Among the nine hetero-COSs, 90-MMWCOS showed the highest radical scavenging activity against DPPH, and addition of 90-MMWCOS (5 mg/ml) scavenged 94.13% of DPPH radical. All of the hetero-COSs examined were found to possess DPPH radical scavenging activity in a concentration-dependent manner, and they eliminated the DPPH radical signal over 70% at 5 mg/ml. Specially, COS with relatively higher degree of deacetylation values and medium molecular weights showed higher scavenging activity against DPPH radical. Hydroxyl radicals generated in Fe2+/H2O2 system were trapped by DMPO forming spin adduct which could be detected by an ESR spectrometer, and the typical 1:2:2:1 ESR signal of the DMPO–OH adduct was observed as shown in Fig. 2(a). The height of the third peak of the spectrum represents the relative amount of DMPO–OH adduct. The hetero-COSs also suppressed hydroxyl radical in almost the same manner as DPPH radical. After the addition of hetero-COSs, the decrease of the amount of DMPO–OH adduct was shown on the ESR spectrum. The ESR results showed that 90-MMWCOS quenched about 95% of the hydroxyl radical at 0.075 mg/ml, while 75-MMWCOS
Fig. 1. DPPH scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of DPPH radical obtained in an ethanol solution of 30 mmol/l DPPH; (b) DPPH scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) DPPH scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) DPPH scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
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and 50-MMWCOS scavenged about 88.28 and 87.93% of the hydroxyl radical at the same concentration, respectively [Fig. 2(b)–(d)]. The addition of the heteroCOSs in the reaction system resulted in a dose-dependent scavenging on the ESR signal intensity of DMPO–OH adduct. The scavenging activity of hetero-COSs on superoxide anion radical was investigated using the HPX-XOD system as a superoxide anion radical source. The hetero-COSs suppressed the signal intensity of DMPO–OOH adduct of ESR spectra, and the scavenging activity was increased with concentration-increment of hetero-COSs (Fig. 3). 90-MMWCOS showed the highest scavenging activity among other tested agents, and it scavenged 64.73% on the superoxide anion radical at 1.00 mg/ml. The carbon-centered radical spin adduct generated by the decomposition of AAPH incubating with spin trap 4-POBN at 37 C for 30 min, was measured using an ESR spectrometer [Fig. 4(a)]. After the addition of hetero-COSs, a dose dependent decrease of ESR signal intensity was observed. In addition, the addition of 90MMWCOS showed the highest scavenging activity, and the scavenging activity increased from 27.02 to 77.98% with increasing concentrations from 0.625 to 2.500 mg/ml. Carbon-centered radical scavenging activity of
90-MMWCOS, 75-MMWCOS and 50-MMWCOS were 77.98, 70.67 and 69.60% at 2.50 mg/ml, respectively [Fig. 4(b)–(d)].
4. Discussion Free radicals with the major species of reactive oxygen species (ROS) are unstable, and react readily with other groups or substances in the body, resulting in cell damage and hence human disease (Halliwell and Gutterridge, 1989). Therefore, removal of free radicals and ROS is probably one of the most effective defence of a living body against various diseases. Beneficial effects of antioxidants are well known in scavenging free radical and ROS or in preventing oxidative damage by interrupting the radical chain reaction of lipid peroxidation (Halliwell and Gutterridge, 1998). It is generally considered that the inhibition of lipid peroxidation by an antioxidant may be due to the free radical scavenging activity. Lipids of biological membranes, especially those in the spinal cord and brain containing highly oxidizable polyunsaturated fatty acids, are particularly affected (Braughler and Hall, 1989). Moreover, the brain contains considerable amounts of prooxidant transition metal ions and utilizes
Fig. 2. Hydroxyl radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of hydroxyl radical obtained in Fenton reaction system; (b) DPPH scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) DPPH scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) DPPH scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
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Fig. 3. Superoxide radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of superoxide radical obtained in HPX-XOD system; (b) Superoxide radical scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) Superoxide radical scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) Superoxide radical scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
a lot of oxygen. These properties set the stage for the generation of free radicals and ROS, and the subsequent acute cellular injury. In this study, we found that hetero-COSs effectively scavenged on DPPH, hydroxyl, carbon-centered and superoxide radicals. DPPH, a stable radical, was scavenged by all hetero-COSs over 70% at 5.00 mg/ml. Hydroxyl radicals were generated in a Fenton reaction and were visualized by ESR spectrometer. The ESR signal is inhibited by the presence of .OH scavengers, who compete with DMPO for .OH. Hydroxyl radicals are highly reactive in causing enormous biological damage. Hetero-COSs scavenged hydroxyl radicals at relatively low concentrations. Superoxide radicals were generated from HPX-XOD system. XOD, which is one potential source of superoxide radical, catalyses the oxidation of hypoxanthine to xanthine and xanthine to uric acid (Yang et al., 2002). In order to check the inhibitory activity of hetero-COSs on XOD, the enzyme was assayed. However, heteroCOSs had no inhibitory activity on XOD (data not shown). AAPH can decompose to form carbon-centered radicals that can react swiftly with O2 to yield peroxyl
radicals to stimulate lipid peroxidation (Halliwell and Gutterridge, 1990). Hetero-COSs quenched carboncentered radicals over 65% at 2.500 mg/ml. In our previous study, chitosan with relatively higher degree of deacetylation among three kinds of partially deacetylated hetero-chitosans showed the higher radical scavenging activity on DPPH, hydroxyl, carboncentered and superoxide radicals (Park et al., 2004). Xie et al. (2001) reported that water-soluble chitosan derivatives prepared by graft copolymerization of maleic acid sodium onto hydroxyporpyl chitosan and carboxymethyl chitosan sodium, showed radical scavenging activity against hydroxyl radical. Matsugo et al. (1998) reported that three different water-soluble chitosan derivatives obtained by the acylation of chitosan inhibited thiobarbituric acid reactive substance formation in t-butylhydroperoxide and benzoyl peroxide induced lipid peroxidations. In addition, several marine polysaccharides such as alginate, alginate sulfate, propylene gucolalginate sodium sulfate, N,O-carboxymethyl chitosan and hydroxypropylated chitosan inhibited the oxidation of phosphatidylcholine-liposomal initiated by addition of AAPH (Xue et al., 1998).
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Fig. 4. Carbon-centered radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of carbon-centered radical obtained during incubation of AAPH with 4-POBN; (b) Carbon-centered radical scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) Carbon-centered radical scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) Carbon-centered radical scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
We also investigated ascorbic acid as a reference compound on DPPH, hydroxyl, carbon-centered and superoxide radical. The radical scavenging activity of ascorbic acid was a little higher than those of hetero-COSs (data not shown). Ascorbic acid was actually a potent hydroxyl and carbon-centered radical scavenger, but simultaneously formed its own radical, ascorbyl radical (Yamaguchi et al., 2000). Podmore et al. (1998) reported that an ascorbyl radical might cause oxidative reactions on other materials, and that ascorbic acid had a prooxidant activity. However, hetero-COSs didn’t form its own radical. Therefore, they can be considered as potent radical scavenger due to be less possibility that it may cause other oxidative stress. N-acetylglucosamine, which is monomer of chitooligosaccharide, showed no scavenging activity against all test radical species at 10 mg/ml. In the present study, these results showed that the scavenging activity of hetero-COSs on various radicals was dependent on the degree of deacetylation values and molecular weights i.e. 90-MMWCOS, which is with relatively medium molecular weights prepared from 90% deacetylated chitosan, showed the highest radical scavenging activity. These results indicate that the hetero-COSs quench various radicals by the action of
nitrogen on C-2 position of COS. It was also dependent on their molecular weights, but their exact role has not been elucidated. The reduction of oxygen to water must proceed via a series of sequential one-electron transfers, yielding superoxide anion, hydrogen peroxide, and hydroxyl radical as intermediate (Yang et al., 2002). In the present study, the hetero-COSs effectively scavenged hydroxyl and superoxide radical, and acted as a direct radical scavenger according to the DPPH and carboncentered radical assay. The scavenging mechanism of the hetero-COSs on free radicals may be related to the fact that free radicals can react with the residual free amino groups NH2 to form stable macromolecule radicals, and the NH2 groups can form ammonium groups NH+ 3 by absorbing hydrogen ion from the solution (Xie et al., 2001). Our results agree with this point exhibiting higher scavenging activity over all tested free radicals with the increment of deacetylation values of COSs in all free radicals assays because increased free amino groups NH2 on the C-2 position of COS. It means that the free amino groups in the hetero-COSs play an important role in free radical scavenging activity.
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In summary, free radical scavenging activity of the hetero-COSs was investigated on DPPH, hydroxyl, superoxide and carbon-centered radical using ESR spectroscopy. The ESR results showed that 90MMWCOS has the highest radical scavenging activity against all tested radicals, and the scavenging activity was dependent on their degree of deacetylation values and molecular weights.
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Free radical scavenging properties of hetero-chitooligosaccharides using an ESR spectroscopy Jae-Young Je, Pyo-Jam Park, Se-Kwon Kim* Department of Chemistry, Pukyong National University, Busan 608-737, South Korea Received 9 April 2003; accepted 6 October 2003
Abstract Nine kinds of hetero-chitooligosaccharides (hetero-COSs) with relatively higher molecular weights (90, 75 and 50HMWCOS), medium molecular weights (90, 75 and 50-MMWCOS), and lower molecular weights (90, 75 and 50-LMWCOS) were prepared from partially deacetylated hetero-chitosans (90, 75 and 50% deacetylated chitosan), and their scavenging activities were investigated against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide and carbon-centered radicals using electron spin resonance (ESR) spin-trapping technique. Superoxide, hydroxyl and carbon-centered radicals were generated from hypoxanthine-xanthine oxidase reaction, hydrogen peroxide-ferrous sulfate (Fenton reaction) and azo compound 2,2-azobis-(2-amidinopropane)-hydrochloride (AAPH), respectively. The ESR results revealed that 90-MMWCOS, which is having relatively medium molecular weights prepared from 90% deacetylated chitosan, showed the highest scavenging activity on all tested radicals. In addition, the radical scavenging activity of hetero-COSs increased with a dose-dependent manner, and it was dependent on their degree of deacetylation values and molecular weights. # 2004 Elsevier Ltd. All rights reserved. Keywords: Hetero-chitooligosaccharides; Electron spin resonance; Free radical; Radical scavenging activity
1. Introduction Chitosan, which is a copolymer consisting of b(1!4)-2-acetamido-d-glucose and b-(1!4)-2-amino-dglucose units, is derived from chitin by deacetylation in the presence of alkali. Chitosan exhibits a wide variety of physiological activities such as antitumor activity (Sugano et al., 1992), immumo-stimulating effect (Jeon and Kim, 2001), antimicrobial effect (Sudharsham et al., 1992), and cholesterol-reducing effect (Maezaki et al., 1993). Although chitosan has very strong functional properties in many areas, its high molecular weights and viscosity might restrict the uses in vivo. Therefore, an attention in chitosan fields has recently been increased to the production of useful chitooligosaccharides (COS) because COS is not only water-soluble but also possesses versatile functional properties such as antitumor activity (Suzuki et al., 1986; Jeon and Kim, 2002), immumostimulating effect (Tokoro et al., 1988; Jeon and Kim, * Corresponding author. Tel.: +82-51-620-6375; fax: +82-51-6288147. E-mail address: [email protected] (S.-Kwon Kim). 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2003.10.001
2001), enhancement of protective effects against infections associated with some pathogens in mice (Tokoro et al., 1989; Yamada et al., 1993), antifungal activity (Kendra and Hagwiger, 1984; Hirano and Nagao, 1989), and antimicrobial activity (Kendra and Hagwiger, 1984; Kim et al., 2000; Jeon et al., 2001). Recently, the antioxidative properties of chitosan and its derivatives containing COS as natural antioxidants have remarked the most attention. However, little information on the free radical scavenging activity of hetero-COSs is available until now. In recent years, reactive oxygen species (ROS) and free radicals play an important role in many diseases such as cancer (Muramatsu et al., 1995; Leanderson et al., 1997), gastric ulcers (Sussman and Bulkley, 1990; Debashis et al., 1997), Alzheimer’s, arthritis and ischemic reperfusion (Vajragupta et al., 2000). Formation of free radicals such as superoxide anion . radical (O 2 ) and hydroxyl radical ( OH) is an unavoidable consequence in aerobic organisms during respiration. These radicals are very unstable and react rapidly with the other groups or substances in the body, leading to cell or tissue injury. Free radical
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scavenger is a preventive antioxidant. Antioxidants can act at different levels in an oxidative sequence. This may be illustrated considering one of the many mechanism by which oxidative stress can cause damage by stimulating the free radical chain reaction of lipid peroxidation. Many synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, t-butylhydroquinone and propyl gallate may be used to retard lipid peroxidation in a lot of fields (Wanita et al., 1996). However, the use of synthetic antioxidants is under strict regulation due to the potential health hazards caused by such compounds (Park et al., 2001). In the present study, we investigated the free radical scavenging activity of hetero-COSs with different degree of deacetylation values and molecular weights on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide . anion radical (O 2 ), hydroxyl radical ( OH) and carboncentered radical using electron spin resonance (ESR) spin-trapping techniques.
2. Materials and methods
90-HMWCOS, 75-HMWCOS and 50-HMWCOS), mediun molecular weights (Mw, 5000–1000 Da; 90MMWCOS, 75-MMWCOS and 50-LMWCOS), and lower molecular weights (Mw, below 1000 Da; 90LMWCOS, 75-LMWCOS and 50-LMWCOS). Nine kinds of COSs recovered were lyophilized on a freezingdrier for 5 days. 2.3. Scavenging effect on DPPH radical DPPH radical scavenging activity was measured using the method described by Nanjo et al. (1996). An ethanol solution of 60 ml of each sample (or ethanol itself as control) was added to 60 ml of DPPH (60 mM) in ethanol solution. After mixing vigorously for 10 s, the solution was then transferred into a 100 ml quartz capillary tube, and the scavenging activity of hetero-COSs on DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 11000; sweep time, 30 s.
2.1. Materials 2.4. Hydroxyl radical scavenging activity Chitin prepared from crab shells donated by Kitto Life Co. (Seoul, Korea). The chitosanase (35,000 U/g protein) derived from Bacillus sp. was purchased from Amicogen Co. (JinJu, Korea), and cellulase was donated from Pacific Chemical Co. Ltd. (Seoul, Korea). An ultrafiltration membrane (UF) reactor system for the preparation and the fractionation of hetero-COSs, based on molecular weights, was from Millipore Co. (Bedford, MA). Hypoxanthine (HPX), 1,1-diphenyl-2picrylhydrazyl (DPPH), xanthine oxidase (XOD), 5,5dimethyl-pyrrokine N-oxide (DMPO), 2,2-azobis-(2amidinopropane)-hydrochloride (AAPH) and a-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade.
Hydroxyl radicals were generated by iron-catalyzed Haber-Weiss reaction (Fenton driven Haber-Weiss reaction) and the generated hydroxyl radicals rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman, 1984). The resultant DMPO-OH adduct was detectable with an ESR spectrometer. Hetero-COSs (0.2 ml) with various concentrations was mixed with DMPO (0.3 M, 0.2 ml), Fe2SO4 (10 mM, 0.2 ml) and H2O2 (10 mM, 0.2 ml) in a phosphate buffer solution (pH 7.2), and then transferred into a 100 ml quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1200; sweep time, 4 min.
2.2. Preparation of hetero-chitosans and their COSs 2.5. Superoxide anion radical scavenging activity Three kinds of partially deacetylated chitosans designated 90% deacetylated chitosan, 75% deacetylated chitosan and 50% deacetylated chitosan were prepared from crab shell chitin, according to our previous method (Park et al., 2003). Hetero-COSs were prepared from partially deacetylated hetero-chitosans by two enzymatic reactions using chitosanase and cellulase in an ultrafiltration membrane reactor system according to a previously reported method (Park et al., 2003). Three different UF membranes ranged from molecular weight cut offs (MWCO) of 10, 5 and 1 KDa were used, and fractionated into nine kinds of COSs with relatively higher molecular weights (Mw, 10000–5000 Da;
Superoxide anion radicals were generated from HPX-XOD system (Rosen and Rauckman, 1984). HPX (4 mM, 50 ml) was mixed with phosphate buffered saline (PBS, pH 7.4, 30 ml), hetero-COSs (50 ml) with various concentrations, DMPO (4.5 M, 20 ml) and XOD (0.4 U/ ml, 50 ml), and then the mixture was transferred into a 100 ml quartz capillary tube. After 45 s, the ESR spectrum was recorded using an ESR spectrometer. Manganese oxide was used as an internal standard. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 4 mW; modulation frequency, 9.41 GHz; amplitude, 1500; sweep time, 1 min.
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2.6. Assay for carbon-centered radical Carbon-centered radicals were generated by AAPH. A PBS reaction mixture containing 10 mM AAPH, 10 mM 4-POBN and hetero-COSs with various concentrations was incubated for 30 min at 37 C in a water bath (Hiramoto et al., 1993), and then transferred to 100 ml quartz capillary tube. The spin adduct was recorded using an ESR spectrometer. Experimental conditions as follows: magnetic field, 336.5 5 mT; power, 10 mW; modulation frequency, 9.41 GHz; amplitude, 11000; sweep time, 1 min. 2.7. Statistics The data presented are means S.E. of three determinations.
3. Results DPPH radical is a stable radical which gives typical ESR spectra shown in Fig. 1(a). Fig. 1(b)–(d) shows the effects of hetero-COSs with different degree of
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deacetylation values and molecular weights. Among the nine hetero-COSs, 90-MMWCOS showed the highest radical scavenging activity against DPPH, and addition of 90-MMWCOS (5 mg/ml) scavenged 94.13% of DPPH radical. All of the hetero-COSs examined were found to possess DPPH radical scavenging activity in a concentration-dependent manner, and they eliminated the DPPH radical signal over 70% at 5 mg/ml. Specially, COS with relatively higher degree of deacetylation values and medium molecular weights showed higher scavenging activity against DPPH radical. Hydroxyl radicals generated in Fe2+/H2O2 system were trapped by DMPO forming spin adduct which could be detected by an ESR spectrometer, and the typical 1:2:2:1 ESR signal of the DMPO–OH adduct was observed as shown in Fig. 2(a). The height of the third peak of the spectrum represents the relative amount of DMPO–OH adduct. The hetero-COSs also suppressed hydroxyl radical in almost the same manner as DPPH radical. After the addition of hetero-COSs, the decrease of the amount of DMPO–OH adduct was shown on the ESR spectrum. The ESR results showed that 90-MMWCOS quenched about 95% of the hydroxyl radical at 0.075 mg/ml, while 75-MMWCOS
Fig. 1. DPPH scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of DPPH radical obtained in an ethanol solution of 30 mmol/l DPPH; (b) DPPH scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) DPPH scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) DPPH scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
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and 50-MMWCOS scavenged about 88.28 and 87.93% of the hydroxyl radical at the same concentration, respectively [Fig. 2(b)–(d)]. The addition of the heteroCOSs in the reaction system resulted in a dose-dependent scavenging on the ESR signal intensity of DMPO–OH adduct. The scavenging activity of hetero-COSs on superoxide anion radical was investigated using the HPX-XOD system as a superoxide anion radical source. The hetero-COSs suppressed the signal intensity of DMPO–OOH adduct of ESR spectra, and the scavenging activity was increased with concentration-increment of hetero-COSs (Fig. 3). 90-MMWCOS showed the highest scavenging activity among other tested agents, and it scavenged 64.73% on the superoxide anion radical at 1.00 mg/ml. The carbon-centered radical spin adduct generated by the decomposition of AAPH incubating with spin trap 4-POBN at 37 C for 30 min, was measured using an ESR spectrometer [Fig. 4(a)]. After the addition of hetero-COSs, a dose dependent decrease of ESR signal intensity was observed. In addition, the addition of 90MMWCOS showed the highest scavenging activity, and the scavenging activity increased from 27.02 to 77.98% with increasing concentrations from 0.625 to 2.500 mg/ml. Carbon-centered radical scavenging activity of
90-MMWCOS, 75-MMWCOS and 50-MMWCOS were 77.98, 70.67 and 69.60% at 2.50 mg/ml, respectively [Fig. 4(b)–(d)].
4. Discussion Free radicals with the major species of reactive oxygen species (ROS) are unstable, and react readily with other groups or substances in the body, resulting in cell damage and hence human disease (Halliwell and Gutterridge, 1989). Therefore, removal of free radicals and ROS is probably one of the most effective defence of a living body against various diseases. Beneficial effects of antioxidants are well known in scavenging free radical and ROS or in preventing oxidative damage by interrupting the radical chain reaction of lipid peroxidation (Halliwell and Gutterridge, 1998). It is generally considered that the inhibition of lipid peroxidation by an antioxidant may be due to the free radical scavenging activity. Lipids of biological membranes, especially those in the spinal cord and brain containing highly oxidizable polyunsaturated fatty acids, are particularly affected (Braughler and Hall, 1989). Moreover, the brain contains considerable amounts of prooxidant transition metal ions and utilizes
Fig. 2. Hydroxyl radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of hydroxyl radical obtained in Fenton reaction system; (b) DPPH scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) DPPH scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) DPPH scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
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Fig. 3. Superoxide radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of superoxide radical obtained in HPX-XOD system; (b) Superoxide radical scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) Superoxide radical scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) Superoxide radical scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
a lot of oxygen. These properties set the stage for the generation of free radicals and ROS, and the subsequent acute cellular injury. In this study, we found that hetero-COSs effectively scavenged on DPPH, hydroxyl, carbon-centered and superoxide radicals. DPPH, a stable radical, was scavenged by all hetero-COSs over 70% at 5.00 mg/ml. Hydroxyl radicals were generated in a Fenton reaction and were visualized by ESR spectrometer. The ESR signal is inhibited by the presence of .OH scavengers, who compete with DMPO for .OH. Hydroxyl radicals are highly reactive in causing enormous biological damage. Hetero-COSs scavenged hydroxyl radicals at relatively low concentrations. Superoxide radicals were generated from HPX-XOD system. XOD, which is one potential source of superoxide radical, catalyses the oxidation of hypoxanthine to xanthine and xanthine to uric acid (Yang et al., 2002). In order to check the inhibitory activity of hetero-COSs on XOD, the enzyme was assayed. However, heteroCOSs had no inhibitory activity on XOD (data not shown). AAPH can decompose to form carbon-centered radicals that can react swiftly with O2 to yield peroxyl
radicals to stimulate lipid peroxidation (Halliwell and Gutterridge, 1990). Hetero-COSs quenched carboncentered radicals over 65% at 2.500 mg/ml. In our previous study, chitosan with relatively higher degree of deacetylation among three kinds of partially deacetylated hetero-chitosans showed the higher radical scavenging activity on DPPH, hydroxyl, carboncentered and superoxide radicals (Park et al., 2004). Xie et al. (2001) reported that water-soluble chitosan derivatives prepared by graft copolymerization of maleic acid sodium onto hydroxyporpyl chitosan and carboxymethyl chitosan sodium, showed radical scavenging activity against hydroxyl radical. Matsugo et al. (1998) reported that three different water-soluble chitosan derivatives obtained by the acylation of chitosan inhibited thiobarbituric acid reactive substance formation in t-butylhydroperoxide and benzoyl peroxide induced lipid peroxidations. In addition, several marine polysaccharides such as alginate, alginate sulfate, propylene gucolalginate sodium sulfate, N,O-carboxymethyl chitosan and hydroxypropylated chitosan inhibited the oxidation of phosphatidylcholine-liposomal initiated by addition of AAPH (Xue et al., 1998).
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Fig. 4. Carbon-centered radical scavenging activity of hetero-chiooligosaccharides. (a) ESR spectrum of carbon-centered radical obtained during incubation of AAPH with 4-POBN; (b) Carbon-centered radical scavenging activity of 90-HMWCOS, 90-MMWCOS and 90-LMWCOS in various concentrations; (c) Carbon-centered radical scavenging activity of 75-HMWCOS, 75-MMWCOS and 75-LMWCOS in various concentrations; (d) Carbon-centered radical scavenging activity of 50-HMWCOS, 50-MMWCOS and 50-LMWCOS in various concentrations. Values represent meansS.E. (n=3).
We also investigated ascorbic acid as a reference compound on DPPH, hydroxyl, carbon-centered and superoxide radical. The radical scavenging activity of ascorbic acid was a little higher than those of hetero-COSs (data not shown). Ascorbic acid was actually a potent hydroxyl and carbon-centered radical scavenger, but simultaneously formed its own radical, ascorbyl radical (Yamaguchi et al., 2000). Podmore et al. (1998) reported that an ascorbyl radical might cause oxidative reactions on other materials, and that ascorbic acid had a prooxidant activity. However, hetero-COSs didn’t form its own radical. Therefore, they can be considered as potent radical scavenger due to be less possibility that it may cause other oxidative stress. N-acetylglucosamine, which is monomer of chitooligosaccharide, showed no scavenging activity against all test radical species at 10 mg/ml. In the present study, these results showed that the scavenging activity of hetero-COSs on various radicals was dependent on the degree of deacetylation values and molecular weights i.e. 90-MMWCOS, which is with relatively medium molecular weights prepared from 90% deacetylated chitosan, showed the highest radical scavenging activity. These results indicate that the hetero-COSs quench various radicals by the action of
nitrogen on C-2 position of COS. It was also dependent on their molecular weights, but their exact role has not been elucidated. The reduction of oxygen to water must proceed via a series of sequential one-electron transfers, yielding superoxide anion, hydrogen peroxide, and hydroxyl radical as intermediate (Yang et al., 2002). In the present study, the hetero-COSs effectively scavenged hydroxyl and superoxide radical, and acted as a direct radical scavenger according to the DPPH and carboncentered radical assay. The scavenging mechanism of the hetero-COSs on free radicals may be related to the fact that free radicals can react with the residual free amino groups NH2 to form stable macromolecule radicals, and the NH2 groups can form ammonium groups NH+ 3 by absorbing hydrogen ion from the solution (Xie et al., 2001). Our results agree with this point exhibiting higher scavenging activity over all tested free radicals with the increment of deacetylation values of COSs in all free radicals assays because increased free amino groups NH2 on the C-2 position of COS. It means that the free amino groups in the hetero-COSs play an important role in free radical scavenging activity.
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In summary, free radical scavenging activity of the hetero-COSs was investigated on DPPH, hydroxyl, superoxide and carbon-centered radical using ESR spectroscopy. The ESR results showed that 90MMWCOS has the highest radical scavenging activity against all tested radicals, and the scavenging activity was dependent on their degree of deacetylation values and molecular weights.
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