Free radical scavenger activity of chitosan and its aminated derivative

1 Polymer research department, Advanced Technology and New Materials Research Institute, City of Scientific Research and Technological Applications. New Borg ElArab, Alexandria, Egypt. 2 Laboratory of Bioorganic Chemistry of Drugs, Institute of Experimental Pharmacology and Toxicology, SK-81404 Bratislava, Slovakia. 3 Chemistry Department, Faculty of Science, University of Jeddah, Osfan, P. O. Box: 80203, Jeddah 21589, Saudi Arabia.


INTRODUCTION
The harmful effect of free radicals attracts the attention of the scientist in last few years.The dangerous effect of free radicals reactive oxygen species (ROS) that generated by our body driving scientists to focus their studies on understanding its generation mechanisms, combined diseases and final the best methods to control it.ROS are generated by our body via various endogenous systems, exposure to different physiochemical conditions or pathological states.Generation of superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide, are normal by-products of metabolic processes in cells which may be increased under up normal environmental conditions, and uncontrolled ROS can easily react with a wide range of essential biomolecules, causing damage and ultimately leading to cell death (Anselmo and Cobb., 2004;Baek et al., 2008;Waris and Ahsan., 2006;Fernandes et al., 2010).A balance between free radicals and antioxidants is necessary for proper physiological function.(Lobo et al., 2010).Scavengers of free radicals are preventive antioxidants, as they can break the oxidative sequence by delaying or preventing ROS generation, and by competing for existing radicals and removing them from the reaction, thus protecting biomolecules from damage (Kim and Rajapakse., 2005;Xiong et al., 2007).Chitosan is biocompatible, biodegradable, nontoxic, renewable biopolymer produced by alkali treatment of chitin the second most abundant natural polysaccharide next to cellulose and found in the composition of crustacean shells.Chitin consists of ß (1→4)-2-acetamido-2deoxy-d-glucopyranose (GlcNAc) as a repeating unit.Deacetylation of chitin yields chitosan, which is a copolymer GlcNAc and ß-(1→4)-2-amino-2-deoxy-d-glucopyranose with deacetylation greater than 60%.Chitosan has a broad field applications ranging from cosmetics, artificial skin, photography, food and nutrition, wound dressing, ophthalmology and wastewater treatment (Kumar et al., 2007;Shahidi et al., 2004;Dodane et al., 1998;Jeon et al., 2000;Mohy Eldin et al 2008a;b;2013;2015).The unique properties of chitosan over the other polysaccharide were attributed to free amine groups that give it its basic character.
In this study, antioxidant evaluation of chitosan and extra-aminated chitosan was performed.

Preparation of aminated chitosan
Aminated chitosan derivative was prepared according to our previous work (Mohy Eldin et al., 2012).In detail, 4 g of chitin was dispersed in 50 mL of (6.9 mM) p-benzoquinone (PBQ)distilled water solution at pH 10 and 60 °C and was stirred for 6 h.The PBQ-conjugated chitin was separated and washed with distilled water to remove unreacted PBQ.
In the second step, PBQ-conjugated chitin was dispersed in 50 mL of (6.9 mM) EDA-distilled water at 60 °C and stirred for 6 h.The aminated modified chitin was separated and washed with distilled water to remove unreacted EDA.
In the last step, aminated modified chitin was deacetylated according to the method of Rigby and Wolfarn.The aminated modified chitin derivative was treated with 50% aqueous solution of NaOH at 120-150 o C for 12 h (see figure 1).The obtained aminated chitosan derivatives were separated and washed with distilled water to remove excess of NaOH.

ABTS method
For the ABTS decolorization assay, the radical cations were pre-formed by the reaction of an aqueous solution of K 2 S 2 O (3.30 mg) in H 2 O (5 ml) with ABTS (17.2 mg).
The resulting bluish green radical cation solution was stored overnight in the dark below 0 o C. Before the experiment, the solution.(1 ml) was diluted with a final volume (60 ml) acetic acid solution (0.5%).
The UV/VIS spectra were recorded at defined times, in 1cm quartz UV cuvette after mixing of antioxidant solution (50 µl) with an ABTS.‫‬ soln.(2 ml).

Rotational viscometry method
For the rotational viscometry measurements, a hyaluronan soln.(2.5 mg/ml) was prepared in the dark, standing at room temperature in an aqueous.NaCl soln.(0.15 mM) in two steps: first, the solvent (4.0 ml) was added to a hyaluronan powder (20 mg), and, within 6 h, the same solvent (3.85 ml) was added.The stock solutions (16 µM) of ascorbic acid, Polymer solution (0.5%) stock solutions were prepared in acetic acid (0.5%).CuCl (160 µM) were also prepared.
Before starting to monitor the kinetics of the hyaluronan degradation via rotational viscometry, the mixture (8.0 ml), consisting of the biopolymer solution of the concentration as stated above, ascorbic acid (100 µM), Cu II ions (1.0 µM), and certain amount of chitosan (or aminated chitosan) solution were transferred into the Teflon cup reservoir of the Brookfield LVDV-II‫‬ PRO digital rotational viscometer (Brookfield Engineering Labs., Inc., Middleboro, MA, USA).
The experimental set was carried out by adding the chitosan (or aminated chitosan) solution at the beginning of degradation and also after 1 hour of degradation start.The Data acquisition of the viscometer output parameters was performed by recording within 2 min after the onset of the experiment.
Time-dependent changes of the dynamic viscosity values of the system were measured at 25.0 ±0.1 within 3 min intervals for up to 5 h.The viscometer Teflon spindle rotation rate was rpm, i.e., at the shear rate equaling 237.6 s -1 .(Soltes et al., 2005;2007)

Antioxidant evaluation
In this study antioxidant of chitosan and aminated chitosan were done by measuring hydroxyl and alkyl free radical scavenger activity via rotational viscometry method beside measure the free radical scavenger activity via ABTS method.

Rotational viscometry method
Rotational viscosity of hyaluronan was studied under free radical degradation condition (i.e.; by the Weissberger's oxidative system) in presence and absence of chitosan or aminated chitosan.This standardized method is, in general, used to mimic the pathophysiological situation, which may occur at the early stage of acute joint inflammation.The chief advantage of rotational viscometry is the possibility to carry continuously out measurements at a given shear rate over extended periods of time.(Hrabarova et al., 2010).Applied Weissberger's system (ascorbic acid and Cu II) in aerobic conditions generate HO  See Figure (2).
By application of Weissberger's system, the hyaluronan degradation was evidenced by the gradual decline of its dynamic viscosity.Attacking of hydroxyl free radicals to hyaluronan backbone eliminate hydrogen atom from it that leaving the free radical on the chain.Under aerobic condition splitting chains will appear, Figure 3. Figure ( 4) showed the effect of the addition of chitosan and aminated chitosan solution (in acetic acid) atin the beginning of the test.It can show increase the rate of hyaluronan degradation in acidic condition that may be attributed to catalytic effect of H + in production of  OH radicals from Weisberg's oxidation system.Chitosan and aminated chitosan show relatively moderated hydroxyl radical scavenger in this conditions.the degradation kinetics when it adds after one hour of the test begging.At this time, the hydroxyl radicals will be consumed in hyaluronan degradation, and the dominated free radicals are alkoxyl, peroxyl and hyaluronan macroradicals that generated through propagation step (Valachová et al., 2011;Hrabarova et al., 2012).According to uninhibited/inhibited hyaluronan degradation method, the concept of antioxidant evaluation focused on scavenger of hydroxyl or alkoxyl radicals.The strategy of scavenging of these free radicals comes through donating hydrogen atom.The scavenging activities of chitosan derivatives against  OH may be derived from some or all of the following: i.The hydroxyl groups in the polysaccharide unit can react with  OH by the typical H-abstraction reaction.
ii.  OH can react with the residual free amino groups NH 2 to form stable macromolecule radicals.(Xie et al., 2001) In our testing material, the ideal source of hydrogen atom coming from -NH 2 and -OH groups that distributed along the backbone of the polymer, under acidic condition (pH ~ 4) amine groups in chitosan protonated, that decrease its contribution to scavenge radical hydroxyl compared to hydroxyl groups.So, decrease the scavenger activity of aminated chitosan rather than chitosan itself may be explained on the idea of consumption of hydroxyl group in amination process.

ABTS Method
In this method ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation (Wolfenden and Willson., 1982) forms the basis of one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of solutions of pure substances (Rice-Evans and Miller., 1995;Rice-Evans et al., 1995;Miller et al., 1996), aqueous mixtures and beverages (Salah et al., 1995).Decolorization of ABTS •+ bluish green color was taken as a monitor for measuring the ability of an antioxidant to donating an electron.ABTS •+ ‫‬ exhibits a bluishgreen color with maximum absorbance values at 645, 730, and 815 nm, this color rapidly decrease by an acceptant electron from antioxidant substance (Hrabarova et al., 2010;Re et al., 1999).The dose-response curve obtained by analysis of a range of concentrations of chitosan and aminated chitosan was measured and presented in figure (7,8).It was clear increase the removing activity by increasing concentration.
Several mechanisms were stated to explain the antioxidant activity of chitosan (Xue et al., 1998;Muzzarelli et al., 1997;Park et al., 2004;Sousa et al., 2004).The most suitable one is the interaction of free radical with hydroxyl or amine group to form stable macromolecule radicals (Xie et al., 2001).
Figure(5)  show the effect of chitosan and aminated chitosan on

Figure ( 6
Figure (6) show the effect of chitosan and aminated chitosan in decolorization of ABTS •+ color.It can recognise the scavenger activity of aminated chitosan more than that for chitosan itself.Figure (6) Show the time dependency of decolorization activity of polymer.The dose-response curve obtained by analysis of a range of concentrations of chitosan and aminated chitosan was measured and presented in figure(7, 8).It was clear increase the removing activity by increasing concentration.Several mechanisms were stated to explain the antioxidant activity of chitosan(Xue et al., 1998; Muzzarelli et al.,  1997;Park et al., 2004; Sousa et al., 2004).The most suitable one is the interaction of free radical with hydroxyl or amine group to form stable macromolecule radicals(Xie et al., 2001).

Fig. 4 :
Fig.4: Effect of chitosan and aminated chitosan dissolved in acetic acid (0.5%) on the hyaluronan degradation induced by WBOS (black) when added to the reaction system before initiating the degradation of HA.

Fig. 5 :Fig. 6 :Fig. 7 :Fig. 8 :
Fig.5: Effect of chitosan and aminated chitosan dissolved in acetic acid (0.5%) on the hyaluronan degradation induced by WBOS (black) when added to the reaction system after 1 hour of initiating the degradation of HA.