Introduction

Reactive oxygen species (ROS) are byproducts of normal metabolic process in cells or are produced by sunlight, ionizing radiation, ultraviolet and chemical reactions, which are generated continuously in the mitochondria in most cells. Although ROS may serve as intracellular signal messengers or as bactericidal agents, excess ROS also cause oxidative damage to proteins, DNA, and lipids [1]. Oxidative stress arises when rates of ROS production surpasses the rates of removal. Oxidative stress is associated with various degenerative diseases, including cataract, macular degeneration, cancer, and arteriosclerosis. Reactive oxygen species levels are normally controlled by intracellular antioxidant defense mechanisms that include low-molecular-weight antioxidants and antioxidant enzymes. Many synthetic chemicals such as phenolic compounds are found to be strong radical scavengers; however, the use of synthetic antioxidants is under strict regulation due to their potential health hazards [2]. Therefore, the search for natural antioxidants as alternatives to synthetic product is of great importance.

Chitosan is derived from chitin by deacetylation in the presence of alkali which is a copolymer consisting of β-(1→4)-2-acetamino-d-glucose and β-(1→ 4)-2-amino-d-glucose units with the latter usually exceeding 85%, is a natural polysaccharide that occurs mainly in fungi, invertebrates and yeasts. It is the second most abundant natural polymer after cellulose [3–5]. Recent studies on chitosan have attracted interest for conventing it to chitooligosaccharide, because chitooligosaccharide is not only water-soluble but also possesses versatile function properties such as antitumor activity, immuno-enhancing effects, antifungal activity, etc. [6]. Some research has shown that the antibacterial effect of chitooligosaccharide was greatly dependent on their degree of polymerization (DP) or molecular weight (M w) and requires chitooligosaccharide with DP 6 or greater [7]. It is noted that the antioxidant activities of chitobiose and chitotriose have been already reported [8].

In the present work, we prepared two chitooligosaccharides with different molecular weights and investigated their antioxidant activity with scavenging activity of chitooligosaccharide against superoxide radical using phenazin methosulphate (PMS)–β-nicotinamide adenine dinucleotide (NADH)–nitroblue tetrazolium (NBT) system, scavenging activity against carcinogen-induced active oxygen species and scavenging activity against hydrogen peroxide released from polymorphonuclear leukocytes stimulated by phorbol-12-myristate-13-acetate (PMA).

Materials and methods

Chemicals and reagents

Chitosan was obtained from Nantong Xincheng Biochemical Company (Jiangsu, China). Chitosan was refined twice by dissolving it in 10 g/l acetic acid solution, filtered, precipitated with 50 g/l NaOH, and finally dried in vacuum at room temperature. Its degree of deacetylation was 92.3% measured by titration and its average molecular weight (M w) was 2.8×104 by measuring the viscosity. Phenazin methosulfate (PMS), β-nicotinamide adenine dinucleotide (NADH) were purchased from Sigma-Aldrich Chemical Co., USA, NBT from Shanghai Biochemical Factory, Shanghai, China, PMA from Calbiochem, La Jolla, CA, USA and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) from Sigma-Aldrich, St. Louis, MO, USA. All the other chemicals used in this study were of research purity grade.

Preparation of chitooligosaccharide

We prepared chitooligosaccharide according to the method we have published [4]. Chitosan (10 g) was placed in a flask with 5% H2O2 (250 ml), the flask was placed in hot water, and the reaction mixture was stirred and reacted for 10 h. After this, the reaction mixture was filtered through a Buchner filter under reduced pressure. The filtrate was concentrated to about one-tenth under reduced pressure, and then ethanol was added to obtain a white precipitate. The precipitate was washed with ethanol and dried in vacuum at room temperature.

Separation of different molecular weight chitooligosaccharide

The produced chitooligosaccharide (30 mg) was dissolved in a minimum volume of distilled water about 1.5 ml, and then filtered by filter paper. The filtrate was subsequently subjected to a Sephadex-25 separated column (column size: 1.6×85 cm bed volume). The sample was placed on the top of the column and eluting with a distilled water with a flow rate of 10 ml/h. Fractions of 3 ml each were collected and 0.5 ml portions were analyzed according to the total carbohydrate content quantified by the anthrone-sulphuric acid method. Two distinct, well-separated peaks, according to molecular weight of chitooligosaccharide, were observed in each hydrolysate. The obtained two major peaks were combined to yield solution of chitooligosaccharide 1 and chitooligosaccharide 2. The chromatographic procedure was repeated to accumulated individual chitooligosaccharide in quantity. Then the solution was placed into a lyophilization flask and was lyophilized under 35–45 mTorr vacuums.

Determination of molecular weight of chitooligosaccharide

The number average molecular weight (W M) of chitooligosaccharide was determined by the method of end group analysis we reported previously [4]. Color-producing reagent: 0.5 g of potassium ferricyanide was accurately weighed and dissolved in 0.5 mol/L sodium carbonate solution (1 l). Standard solution: d-glucosamine hydrochloride (GAH) 1 g/100 ml. Several different volumes of standard solution and 2.0 ml of color-producing reagent were accurately added to tubes (10 ml), respectively. The final volume was adjusted to 5 ml by adding distilled water. Then the tubes were put into a boiling water bath for 15 min, and cooled to room temperature in cold water immediately. After that, the absorbance values of the solution in each of the comparison tubes were determined at 420 nm with distilled water as a control solution. The regressive equation (absorbance value versus the amount of GAH) was obtained. A desired weight of chitooligosaccharide was accurately weighed and dissolved in 2.0 ml of color-producing solution. The final volume of the solution was adjusted to 5 ml by adding distilled water. The following steps were as same as those mentioned earlier. The amount of GAH corresponding to this chitooligosaccharide could be consulted from the regressive equation, and the W M of chitooligosaccharide was then calculated by the following equation: W M=(W 1/W 2)×215.5, where W 1 is the weight of chitooligosaccharide (g); W 2 the amount of GAH corresponding the oligoglucosamine from the regressive equation.

By determination, the W M of resulted white two solids, chitooligosaccharide 1 and chitooligosaccharide 2, were 1,100 and 500, respectively. Hereafter, they are called Ch1100 (high-molecular-weight fraction, M w: 1,100; average DP: 7) and Ch500 (low-molecular-weight fraction, M w: 500; average DP: 3), respectively.

Scavenging activity of chitooligosaccharide against superoxide radical with PMS–NADH–NBT system

The superoxide radical scavenging activity of chitooligosaccharide was performed as described elsewhere [9]. In brief, the superoxide radical was generated in 3.0 ml of 16 mmol/l Tris–HCl buffer (pH 8.0) containing 78 μmol/L NADH, 50 μmol/L, NBT and 10 μmol/L PMS. After addition of chitooligosaccharide at varying concentrations (1,000, 100, 10 and 1 μmol/L), the color reaction of the rest superoxide radical with NBT was detected at 560 nm using Hitachi U-3000 spectrophometer. Deionized water and ascorbic acid were used as the blank and positive controls. The scavenging activity of superoxide radical (Inhibition%) was thus calculated with the equation: [(A blank−A sample)/A blank]×100. Values are means±SD of three determinations.

Scavenging activity of chitooligosaccharide against carcinogen-induced active oxygen species

Phorbol-12-myristate-13-acetate was dissolved in dimethylsulfoxide (Me2SO) at a concentration of 10 μmol/L. 2′,7′-Dichlorodihydrofluorescein diacetate was dissolved in ethanol at a concentration of 5 mmol/L and stored in the dark at −20°C. B16BL6 melanoma cells were grown in DMEM with 10% FCS at 37°C, in 95% air and 5% CO2 atmosphere in 96-well flat bottom microtiter plates (105 cells/well). The intracellular formation of reactive oxygen species was detected using the fluorescent probe DCFH-DA. The fluorescence was measured at 534 nm emission with an excitation wavelength of 488 nm in a Spectra-Max GeminiXS fluorometer from Molecular Devices (USA, using SOFTmax PRO 4.3.1, Life Sciences Edition). Cells were incubated in the dark with PMA (1 μmol/L) and fluorescent probe DCFH-DA (5 μmol/L), Ch1100, Ch500, aminoglucose (1,000, 100, 10, 1 μmol/L) or not for 30 min prior to the measurement of ROS concentrations. Vitamin C (1,000, 100, 10, 1 μmol/L) was used as a positive control, and Me2SO as a negative control. Values are means±SD of three determinations [10].

Scavenging activity of chitooligosaccharide against hydrogen peroxide (H2O2) released from polymorphonuclear leukocytes (PMNs) stimulated by PMA

Adult male Sprague-Dawley (SD) rats weighing 180–200 g were selected, the animals were housed in groups cages, purina diets and tap water were supplied to them ad libitum. Prior to the commencement and throughout the experiment the rats were housed at 20–24°C room temperature and 12 h light/dark cycles. Rat mononuclear leukocytes were isolated from the heparinized blood samples according to a previously described procedure [11]. The mononuclear leukocytes oxidative burst stimulated with PMA (1 μmol/L) and Ch1100, Ch500, aminoglucose (100 μmol/L) or not, then incubated with the DCFH-DA (5 μmol/L) for 30 min at 37°C, washed and resuspended in physiological salt solution. The level of DCF fluorescence was monitored with Spectra-Max GeminiXS fluorometer. Retinoic acid (RA, final concentration 83.3 μmol/L) was used as a positive control, and Me2SO as a negative control. Experimental results were recorded as means±SD of three parallel measurements.

Results and discussion

FT-IR spectra of the chitooligosaccharide

FTIR spectra were taken using Nexus model 870 Fourier Transform IR Spectrophotometer. Dry products powder was ground with KBr powder and compressed into discs for FTIR examination.

Figure 1 shows FT-IR spectra of Ch1100 and Ch500, respectively. We can see that the FT-IR spectrum of both chitooligosaccharide was almost the same. From Fig 1, the characteristic absorptions were displayed at 1656.5 and 1598.6 cm−1 attributable to amide bands, respectively. No band is observed between 1670 and 1900 cm−1, which indicated that oxidated groups such as the carboxylic or aldehyde groups do not exist in both chitooligosaccharides. Which indicated that the –OH and –NH2 group in chitosan are not destroyed by oxidation of H2O2. It also suggested that in the procedure of chitooligosaccharide preparing no other chemical material was brought into products. So Ch1100 and Ch500 have same the chemical structure.

Fig. 1.
figure 1

FT-IR spectra of (a) Ch1100 and (b) Ch500

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Scavenging activity of chitooligosaccharide against superoxide radical with PMS–NADH–NBT system

Figure 2 shows the superoxide radical scavenging activity of Vitamin C, Ch1100, Ch500 and aminoglucose at the concentration of 1,000, 100, 10 and 1 μM, respectively, in a PMS–NADH system and assayed by the reduction of NBT. The antioxidant activity of Ch1100 was 63.9, 54.8, 41.4 and 28.3% at the concentration of 1,000, 100, 10 and 1 μmol/L, respectively. While the antioxidant activity of Vitamin C was 75.7, 57.4, 47.7 and 35.0% at the concentration of 1,000, 100, 10 and 1 μmol/L, respectively. Vitamin C is known to be an antioxidant. The results also indicated that the antioxidant ability of all tested compounds was dose-dependant activity in a PMS–NADH system, but it was nonlinear. It also showed that the antioxidant activity of Ch1100 was much more effective than that of Ch500.

Fig. 2.
figure 2

Scavenging activity of chitooligosaccharide against superoxide radical with PMS–NADH–NBT system. Ch1100 means chitooligosaccharide with molecular weight of 1,100; Ch500 means chitooligosaccharide with molecular weight of 500

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Scavenging activity of chitooligosaccharide against carcinogen-induced active oxygen species

2′,7′-Dichlorodihydrofluorescein diacetate can be oxidized by various ROS including hydrogen peroxide, organic hydroperoxides, nitric oxide and peroxynitrite. It is a nonpolar compound that readily diffuses in to cells, where it is hydrolyzed to the nonfluorescent polor derivative DCFH and thereby is trapped within the cells. In the presence of ROS, DCFH is oxidized to the highly fluorescent (dichlorofluorescein) DCF. The level of DCF fluorescence reflected the concentration of ROS [10].

The results of inhibition by Ch1100, Ch500, aminoglucose of PMA-generated activated oxygen species (ROS) in B16BL6 melanoma cells presented in Fig. 3. We can see that aminoglucose only possesses a little antioxidant activity at very concentration; and the activity of Ch500 was high (30.8%) only at high concentration of 1,000 μmol/L, while the radical scavenging activity of Ch1100 was up to 26.1% even at the concentration of 1 μmol/L. Incubation of DCFH-loaded B16BL6 with increasing concentrations of Ch1100 led to a comparatively concentration-dependent decrease in DCF fluorescence. These results suggested that Ch1100 was a good radical scavenger than Ch500 and aminoglucose.

Fig. 3.
figure 3

Scavenging activity of chitooligosaccharide against superoxide radical with 2′,7′-dichlorofluorescindiacetate fluorescent probe. Ch1100 means chitooligosaccharide with molecular weight of 1,100; Ch500 means chitooligosaccharide with molecular weight of 500

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Scavenging activity of chitooligosaccharide against Release of hydrogen peroxide (H2O2) from polymorphonuclear leukocytes (PMNs) stimulated by PMA

The results of inhibition by Ch1100, Ch500, aminoglucose of PMA-generated activated oxygen species (ROS) in PMNs is presented in Fig. 4. The inhibition of aminoglucose, Ch500, Ch1100 was 38.9, 18.2 and 10.9%, respectively, at the concentration of 100 μmol/L. In mean time, we take RA as positive control. Its inhibition was 39.5% (100 μmol/L). This result further suggested that Ch1100 was a potent antioxidant.

Fig. 4
figure 4

Scavenging activity of chitooligosaccharide against release of hydrogen peroxide from polymorphonuclear leukocytes stimulated by PMA. The concentration of sample was 100 μmol/L. Ch1100 means chitooligosaccharide with molecular weight of 1,100; Ch500 means chitooligosaccharide with molecular weight of 500. RA means retinoic acid

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It is known that phenclic compounds from the plant, such as phenylpropaniod glycosides, may react with superoxide radical by one-electron transfer mechanism or hydrogen abstraction mechanism to form the semiquinones [9]. It is not very clear whether the mechanism of superoxide and hydroxyl radical scavenging by chitooligosaccharide was similar to that of plant phenolic compounds [12], which reported that the scavenging mechanism of chitosan is related to the fact that the free radical can react with the residual free amino group NH2 to form ammonium groups NH3 + by absorbing a hydrogen ion from solution.

Our results indicated that chitooligosaccharide showed higher scavenging activity. It means that the free amino group in chitooligosaccharide plays an important role in free radical scavenging activity. Compared with chitooligosaccharide, chitosan itself has the highest content of hydroxyl and amino groups and should have the strongest scavenging ability. But the antioxidant activity of chitosan was almost equal to zero [3]. This may be related to the formation of strong intermolecular and intramolecular hydrogen bonds that reduced the reactivity of hydroxyl and amino groups in the polymer chains. Compared with chitosan, chitooligosaccharide has very short chain and the ability to form intramolecular hydrogen bonds declines sharply, that is, the hydroxyl and amino groups are activated and this is helpful to the reaction with superoxide anions. And we also find that the radical scavenging activity of Ch1100 was higher than that of Ch500 and aminoglucose. This finding is similar with the result of Chen et al. [8]. This mechanism remains to be further investigated. These results indicate that high-molecular-weight chitooligosaccharide can be considered as potent radical scavengers to explore. Antioxidant exerts its role in vivo or in food mostly via inhibiting generation of ROS, or scavenging free radicals. The levels of endogenous antioxidants may also be up-regulated by increased expression of the genes encoding the antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase.

Conclusions

In summary, the antioxidant activity of chitooligosaccharides with different molecular weights was examined with scavenging activity of chitooligosaccharide against superoxide radical using PMS–NADH–NBT system, scavenging activity of chitooligosaccharide against carcinogen-induced active oxygen species and scavenging activity of chitooligosaccharide against hydrogen peroxide released from polymorphonuclear leukocytes stimulated by PBA. By the aforementioned three methods, the radical scavenging activity (Inhibition%) of Ch1100 was determined to be 54.8, 35.3 and 38.9%, respectively, at the concentration of 100 μmol/L, while that of Ch500 was 40.1, 13.9 and 18.2%, respectively, at the same concentration. These results clearly suggest that Ch1100 is a potent radical scavenger, which has the high radical scavenging activity.