Chitosan is the second most abundant natural polysaccharide next to cellulose. It is a polymer consisting of N-glucosamine and N-acetylglucosamine units linked by β-1,4-glycoside. Due to its many unique properties, such as biodegradability, biocompatibility, nontoxicity, antibacterial, and antifungal properties, as well as its ability to stimulate disease resistance, among other special characteristics, chitosan has been widely applied in many fields.
Generally, chitosan obtained from the deacetylation of chitin has a high molecular weight and particular characteristics. However, chitosan can only be dissolved in an acidic environment; therefore, its application is limited . In recent years, chitosan and oligochitosan with low molecular weights have attracted a great deal of research attention because of their good solubility and better properties, such as increased disease-resistance activity, stimulating development of plants or animals, antifungal activity, antibacterial activity, and high antioxidant activity, compared with ordinary chitosan , , .
There exist a variety of techniques that can be used to prepare chitosan oligomers , , , , , . Among these methods, the gamma radiation process has many advantages. For example, it could be carried out at ambient temperature to yield products with high purity. This method can also be applied at an industrial production scale. In addition, studies on the degradation of chitosan by gamma irradiation combined with H2O2 have shown that this method can effectively degrade chitosan and reduce radiation dose, thereby creating convenient conditions for production deployment , .
On the basis of earlier reports , , , we studied the preparation of water-soluble oligochitosan by gamma Co60 irradiation method in this work. The aim of this study is to reduce energy consumption during irradiation and increase efficiency in the process of chitosan degradation. We combine the heterogeneous degradation of chitosan through the oxidation of H2O2 and the homogeneous degradation of chitosan through the radiation of γ/H2O2. We also use lactic acid as a solvent instead of acetic acid, which is the most common solvent for chitosan. The use of lactic acid helps avoid the unpleasant smell of acetic acid and facilitates the application of chitosan/oligochitosan with low molecular weight, which are obtained after irradiation, as biotic elicitors for plant. Moreover, the solubility and antioxidant activity of oligochitosan with different molecular weights are also investigated in this report.
2 Materials and methods
Chitosan made from crab shell chitin with 82.3% degree of deacetylation (DD%) and weight-average molecular weight (Mw) of 109.34 kDa was supplied by the VINAGAMMA Center. 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was obtained from Biobasic Canada. H2O2 30% (d: 1.11 g/ml) was purchased from Merck, Germany. All other chemicals, including acid lactic, NaOH, NH4OH 25%, and ethyl alcohol, were of reagent grade. Distilled water was used in all preparations.
2.2 Preparation of chitosan, chitosan solution, and γ-irradiation
2.2.1 Heterogeneous degradation of chitosan through the oxidation of H2O2:
Chitosan in flake form was soaked in a glass beaker containing H2O2 with different concentrations (w/v) of 0.5%, 1%, and 2% in 24 h. The mixtures were poured through a cloth filter and washed several times in distilled water to remove H2O2. Then, the samples were dissolved by 1% (w/v) lactic acid. The solutions were filtered through a stainless steel mesh and neutralized with 5% (v/v) NH4OH solution. Then, ethyl alcohol was added slowly while stirring. The chitosan precipitate was filtered, washed with alcohol several times, and then dried at 60°C in an oven . The dried chitosan sample was ground into powder for subsequent GPC, FT-IR, and XRD investigations.
2.2.2 Homogeneous degradation of chitosan through the radiation of γ/H2O2:
After soaking chitosan in H2O2 solution with concentrations of 1% and 2% in 24 h, 2% (w/v) lactic acid was added until the chitosan was completely dissolved. The solutions were filtered through a stainless steel mesh. Then, hydrogen peroxide (30% H2O2) and water were added to the solutions to prepare 4% (w/v) chitosan/0.5% (w/v) H2O2 solutions. The resulting solutions were irradiated on gamma SVST Co-60/B irradiator with the absorbed dose range of up to 24 kGy. CS1, CS2, CS3, CS4, CS5, and CS6 represented the chitosan samples irradiated at doses of 4, 8, 12, 16, 20, and 24 kGy, respectively. After irradiation, the solutions were neutralized with NH4OH 5%. Chitosan samples were precipitated by absolute alcohol, washed with alcohol several times, and then dried in an oven at 60°C . The dried chitosan samples were ground into powder for GPC, FT-IR, XRD, UV-Vis, and antioxidant activity analyses.
The Mw of chitosan samples were measured by Agilent GPC (LC-20AB Shimadzu, Japan) with detector RI-10 A and the column ultrahydrogel model 250 from waters (USA). The standards for calibration of columns were pullulan with Mw of 780–380,000 Da. The eluent was an aqueous solution 0.25 m CH3COOH/0.25 m CH3COONa with flow rate of 1 ml/min .
IR spectra were taken on an FT-IR 8400S spectrometer (Shimadzu, Japan) using KBr pellets. The DD% was calculated based on FT-IR spectra according to following equation :
where A1320 and A1420 are absorbances of chitosan at 1320 and 1420 cm−1, respectively. The X-ray diffraction patterns of the chitosan samples were recorded in the scattering range (2θ) of 5°–40° with steps of 0.01°/min. The spectra were recorded at 40 kV and 40 mA.
The estimation of water-solubility was carried out by adding water to 6.25 ml solution of 4% (w/v) chitosan/0.5% (w/v) H2O2 after radiation to prepare a 50-ml chitosan solution with a concentration of 0.5%. The pH levels of the five samples of 0.5% chitosan were adjusted to 3, 5, 7, 9, and 11, respectively, by using 0.1 N HCl or 0.1 N NaOH solutions. The pH dependence of the water solubility of chitosan was evaluated from the turbidimetry of a solution of chitosan. The solution transmittance at 600 nm was recorded on a UV-vis spectrophotometer (V-630, Jacco, Japan) using a quartz cell with an optical path length of 1 cm .
In vivo antioxidant assay was performed by dissolving 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in water with a concentration of 7.4 mm. Next, 2 ml ABTS solution was mixed with 2 ml K2S2O8 with a concentration of 2.6 mm to create the free radical cation, ABTS˙+. The ABTS˙+ solution was kept in the dark for 24 h. The ABTS˙+ solution was diluted by water to obtain an optical density (OD) of 1±0.1 at the wavelength of 734 nm, after which the solution was also diluted by water for the control. For studying antioxidant activity, the chitosan and oligochitosan samples with concentration of 0.2% were mixed with 0.1% acid acetic. About 0.6 ml of the solution sample was added into a cuvette, which contained 1 ml of ABTS˙+ solution. Otherwise, the control sample was created by adding 0.6 ml distilled water into a cuvette containing 1 ml of ABTS˙+ solution. The OD of the samples were measured over time on a UV-vis spectrophotometer at max=734 nm. The efficiency of free radical capture was calculated as follows:
3 Results and discussion
3.1 Effect of the heterogeneous degradation of chitosan by oxidation of H2O2 on Mw and DD
The results in Table 1 show that the molecular weight of chitosan after heterogeneous degradation tended to decrease as the concentration of H2O2 increased from 0.5% to 2%. Specifically, when soaking chitosan in H2O2 solution with a concentration of 0.5% (w/v) after 24 h, the Mw of chitosan decreased from 109.34 to 66.58 kDa. When the concentration of H2O2 solution increased by more than 0.5%, the chitosan degradation efficiency increased, and the Mw of chitosan decreased by over 50% compared with the Mw of initial chitosan. However, the structure of chitosan may be affected by vigorous oxidation conditions or when a higher concentration of H2O was used. Therefore, the concentration of H2O2 solution must not be too high. In a previous report, Tian et al.  studied the degradation of chitosan by H2O2 and selected H2O2 solution with concentrations ranging from 0.5% to 2%. Hien et al. , successfully degraded chitosan by 1.5% (w/v) H2O2. Qun et al.  also studied the degradation of chitosan with H2O2, using H2O2 with a concentration of 2.5% (w/v). Results indicated that the faster the degradation process of chitosan was, the more significantly the molecular weight was reduced, which also simultaneously affected the DD% of chitosan. Therefore, based on the results of previous studies, the concentrations of H2O2 solution used in this research ranged from 0.5% to 2% for the heterogeneous degradation of chitosan. As shown in Table 1, the concentrations of H2O2 that proved to be effective for heterogeneous degradation without affecting the DD of chitosan ranged from 1% to 2%. The mechanism of the depolymerization system of chitosan with H2O2 is shown below .
The hydroperoxide anion is very unstable and is easily decomposed to high reactive hydroxyl radical (HO˙).
The main chemical action of HO˙ with polysaccharide has been demonstrated to be hydrogen abstraction; here, HO˙ pulls off a hydrogen atom of chitosan and combines with it to form water.
3.2 Effect of the homogeneous degradation of chitosan on Mw and DD through the radiation of γ/H2O2
The degradation of chitosan by irradiation method combined with H2O2 solution is classified as homogeneous degradation. This is different from heterogeneous degradation process in the first step; thus, the concentration of H2O2 should not be too high. H2O2 solution with a concentration of 0.5% is suitable for oxidative reaction in mild conditions. In this study, we used lactic acid as a solvent instead of acetic acid, the most common solvent for chitosan. The use of lactic acid helped avoid the unpleasant smell of acetic acid. In addition, the chitosan and oligochitosan with low molecular weight, which were obtained after irradiation, can be directly used as biotic elicitors for plant.
The molecular weight changes of chitosan based on the doses are depicted in Figure 1 and Table 2. Results showed that Mw and number-average molecular weight (Mn) of the chitosan samples decreased rapidly when radiation dose was increased to 10 kGy and then decreased slowly when the dose further increased to more than 10 kGy. The polydispersity index (PI) of chitosan samples also decreased gradually with the increasing dose, indicating that the molecular distributions of chitosan and oligochitosan with low molecular weight, which were obtained after irradiation, were narrower and more homogeneous than CS02 samples obtained after soaking in H2O2 without irradiation. PI and the DD of chitosan also changed during the irradiation process. These results are the same as those obtained in previous studies , , .
The degradation of chitosan occurs via the combination of gamma Co60 radiation treatment and H2O2 based on the mechanism of synergistic degradation effect. This is a phenomenon reported in previous studies , , , which suggests that the combined treatment by H2O2 and γ-ray enhances the degradation of chitosan. This is due to the formation of hydroxyl radicals HO˙ through the radiolysis of water and H2O2.
Furthermore, during irradiation, and H˙ can react with H2O2.
Hydroxyl radicals serving as a powerful oxidative agent reacted with chitosan through the abstraction of carbon-bound hydrogens. The resulting carbonhydrate radicals can then cause the direct breakage of the glucosidic linkage by rearrangement .
3.3 FT-IR spectra
As shown in Table 2, when the chitosan sample soaked in 1% H2O2 solution was irradiated, its DD was less affected than that of chitosan samples soaked in 2% H2O2; however, the depolymezation of chitosan by gamma radiation proved to be ineffective. Oligochitosans with Mw ranging from 7.00 to 3.00 kDa were obtained by radiating chitosan samples soaked in 2% H2O2 at doses ranging from 8 to 24 kGy. According to the results previously published by Qin et al. , Feng et al. , and other authors, oligochitosan with Mw of about 2.00–3.00 kDa has good solubility in water. Thus, chitosan samples soaked in 2% H2O2 and radiated with different doses were selected in the current study for further characterization.
The FT-IR spectra of CS01 (the initial chitosan), CS02 (chitosan sample soaked in 2% H2O2 and without radiating), and chitosans with Mw ranging from 15.85 to 3.70 kDa, obtained after radiation at doses of 4–24 kGy, are shown in Figure 2. The FT-IR spectra of the original chitosan shows characteristic absorptions. The peaks at 3200–3500 cm−1 (O-H, N-H), 1655 cm−1 (amid I), 1595 cm−1 (amid II), 1420 cm−1 (symmetrical deformation of -CH3 and -CH2), and 1320 cm−1 are assigned to the absorbance of C-N of CH3CONH- (amid III). The peaks at 1072, 1028, and 1153-895 cm−1 are assigned to the stretching vibration of C-O, stretching vibration of the C-O-C in the glucose circle, and the special absorption peaks of β(1–4) glycoside bond in chitosan, respectively. Compared with the FT-IR spectra of the original chitosan (CS01), the FT-IR spectra of CS02 and of the radiated chitosan samples (Figure 2) showed that the principal functional groups of these materials were still present after irradiation, and that the main polysaccharide chain structure remained the same during the degradation process.
3.4 XRD analysis
Figure 3 shows the XRD diffraction patterns of the original chitosan and chitosan samples after irradiation with gamma ray in the presence of H2O2. As can be seen, the pattern of the original chitosan exhibited one peak at 2θ of 20.2° without a peak at about 9°–10°. This is different from the previous reports , , . Six polymorphs have been proposed for chitosan: “tendon,” “annealed,” “I-2,” “L-2,” “form-I,” and “form-II” . The form II crystal is also orthorhombic with a unit cell of a=4.4, b=10.0, c=10.3 Å (fiber axis). The strongest reflection appears at 2θ of 20.1°, which also corresponds to the (100) reflection , , . As shown in Figure 3, the original chitosan shows the strongest reflection at 2θ of 20.2°, which is coincided as a pattern with the form-II crystal. For chitosan soaked in 2% H2O2 solution (CS02), the pattern had two peaks: the peak at 2θ of 20.2° and a new appearing peak at 2θ of 10.2°. Such a pattern characterizes a chitosan polymorph, which is referred to as the “L-2 polymorph.”
Meanwhile, compared with CS02 sample, CS1 was not different from CS02: the peaks at 10.2° and 20.2° were retained. This could be because the dose of 4 kGy to obtain CS1 was too low to destroy the the crystallinity of chitosan. For CS3 and CS5, the main difference of oligochitosans seemed to be the disappearance of peak 10.2° and remaining peak 20.2° with less intensity. This results showed that the increasing irradiation dose resulted in the synergistic degradation of chitosan by γ-ray/H2O2, which in turn, caused the destruction of the crystallinity of chitosan. During depolymerization, the change in peaks can be caused by the degradation of the amorphous part of chitosan, whereas the crystal part can be temporarily maintained. However, for CS5, a peak appeared at 13.9°. This phenomenon is unexplained and needs further study.
3.5 Solubility of the degraded chitosan by γ-ray/H2O2
The water-solubility properties of oligochitosans with different molecular weights obtained from irradiation were confirmed by investigating the transmittance of the chitosan solution at different pH values. The results are presented in Figure 4. All tested samples showed good solubility at pH≤5. CS1 with Mw of 15.85 kDa obtained from irradiation at the dose of 4 kGy showed limited solubility and was easily transferred to precipitate at pH>5, whereas the oligochitosans CS2 (Mw=7.05 kDa) and CS3 (Mw=7.03 kDa) were soluble in a wider pH range. These two samples can be dissolved in both acidic and neutral environments. However, at a pH range of about 7.5–8, precipitation occurred and their solubility decreased when pH gradually increased. Compared with CS1, CS2, CS3, and CS4, the samples CS5, CS6 are water-soluble over a wide pH range of 3–11. The transmittance of CS6 in a pH range of 3–11 was more than 90%, indicating that water solubility obviously depended on the molecular weight of chitosan. It seemed that the high solubility of chitosan with low molecular weight can be attributed to the decrease of intermolecular interactions, such as Van der Waals forces and hydrogen bond . Additionally, during the degradation of chitosan by γ-ray/H2O2, its crystallinity was gradually destroyed, thereby creating an amorphous structure. Thus, CS5 and CS6 have high solubility.
From the test results of the solubility of oligochitosans in pure water, Table 3 shows that CS5 and CS6 are almost completely dissolved. However, tiny insoluble particles can still be observed in the solution. The oligochitosans are dissolved in aqueous acid, after which they adjusted to neutral pH; thus, their solubility appeared to be higher than that of in pure water. This result suggests that ion power may be the cause of this phenomenon .
3.6 ABTS radical scavenging activity
Figure 5 shows the results of the investigations on the antioxidant activity of chitosan samples with different molecular weights. As can be seen, the smaller the molecular weight of chitosan was, the higher the demonstrated antioxidant effectiveness. The efficiency of the antioxidant activity of chitosan was calculated according to equation (2). At 90 min, the efficiency of ABTS˙+ radical scavenging activities of CS01 (Mw=109.34 kDa), CS02 (Mw=35.29 kDa), CS3 (Mw=7.03 kDa), and CS5 (Mw=4.07 kDa) were 34.8%, 69.6%, 84.5%, and 99.2%, respectively.
The antioxidant activity of chitosans is clearly shown in Figure 6. As can be seen, the ABTS solution was colorless, and the ABTS˙+solution is green. In the same observation, when chitosans with different molecular weights were added to the ABTS˙+solution, chitosans with lower Mw showed higher reducibility. Specifically, the reducibility of CS5 was the highest, quickly reducing ABTS˙+into ABTS. Thus after the reaction, the solution was colorless and transparent. Meanwhile, ABTS˙+solution with the presence of CS1 was still green. This suggests that oligochitosan not only solves the limit of solubility of high molecular weight chitosan but also has special antioxidant activity. Its antioxidant activity was better than both chitosans with Mw>100.00 kDa and LMWC with 10.00<Mw<100.00 kDa. It seemed that the lower the Mw of oligochitosan, the higher the efficiency of its antioxidant activity. Tomida et al.  also released the same conclusion in their study on antioxidant activity of chitosan and oligochitosan with Mw ranging from 2.8 to 931 kDa.
Meanwhile, the study of Feng et al.  on the enhancement of antioxidant acctivity of chitosan by irradiation also showed that the chitosan sample that had been irradiated at 20 kGy exhibited the highest antioxidant activity. The scavenging percentages of chitosan irradiated for 0, 2, and 10 kGy against hydroxyl radical were 16.6%, 41.1%, and 47.1%, respectively. Yang et al.  suggested that the hydroxyl and amino groups of chitosan and oligochitosan play important roles in improved scavenging activity. Compared with chitosan, oligochitosan has a very short chain, while the ability to form intramolecular hydrogen bonds and Van der Waals forces decline sharply, that is, the hydroxyl and amino groups are activated. This facilitates effective reactions with free radical.
Oligochitosan with Mw<10.00 kDa was effectively prepared by combining the heterogeneous degradation of chitosan/H2O2 and the homogeneous degradation of chitosan/H2O2 with gamma radiation. The characterization of the structure of the degraded chitosan by FT-IR indicated that the main structure of chitosan was retained after irradiation. Oligochitosans with Mw of 4.07 and 3.70 kDa, which were obtained from irradiation at doses of 20 and 24 kGy, have good solubility in a wide pH range of 3–11. Furthermore, CS5 (Mw=4.07 kDa) has the highest antioxidant activity and the most effective scavenging behavior in the shortest amount time. This shows that oligochitosan has potential applications as an effective and natural antioxidant.
This research was funded by the Vietnam National University HoChiMinh City (VNU-HCM) under Grant No. C2016-18-09.
Hien NQ, Phu DV, Duy NN, Kim Lan NT. Carbohyd. Polym. 2012, 87, 953–938. Google Scholar
Jonathan ZK, Mohammad RK, Bui VT, Creber KAM. Can. J. Chem. 1998, 76, 1699–1706. Google Scholar
Hien NQ, Phu DV, Phu BP, Huy HT. J. Nucl. Sci. Technol. 2008, 4, 44–51. Google Scholar
Ulanski P, Sonntag C. J. Chem. Soc. 2000, 2, 2022–2028. Google Scholar
Samuels RJ. J. Polym Sci.: Polym. Chem. 1981, 19, 1081–1105. Google Scholar
About the article
Ngoc Thuy Nguyen
Ngoc Thuy Nguyen received her Bachelor’s degree in 2014 from the University of Science, HCMC, Vietnam. She is currently pursuing her Master’s degree at the University of Science, HCMC, Vietnam, focusing on the synthesis and investigation of the properties of oligochitosan/nanosilica hybrid material. Her research interests include polymers, composite materials, and biopolymers.
Dong Quy Hoang
Dong Quy Hoang holds a PhD in Polymer Science and Engineering from Sungkyunkwan University, South Korea (2009). Currently, she is a lecturer at the Department of Polymer and Composite Materials, Faculty of Materials Science, University of Science, Ho Chi Minh City, Vietnam. She is mainly engaged in the study of the synthesis of polymers, biopolymers, composites, biocomposites, and flame-retardant polymers.
Ngoc Duy Nguyen
Ngoc Duy Nguyen, PhD, is currently working as a scientific researcher at the Research and Development Center for Radiation Technology, Ho Chi Minh City, Vietnam. His research interests focus on radiation processing technology (γ-ray and electron beam) for the synthesis and modification of polymer materials, nano materials, and nano composites.
Quoc Hien Nguyen
Quoc Hien Nguyen, Associate Professor, works as a researcher at the Research and Development Center for Radiation Technology, Ho Chi Minh City, Vietnam; he is also a lecturer at the University of Science, Ho Chi Minh City, Vietnam. His research interests focus on radiation processing technology (γ-ray and electron beam) for the synthesis and modification of polymer materials, nano materials, and nano composites.
Dai Hai Nguyen
Dai Hai Nguyen obtained his PhD from Ajou University, Republic of Korea, in 2013. Currently, he works as a researcher at the Institute of Applied Materials Science, Vietnam Academy of Science and Technology. He is also an invited lecturer at TraVinh University and University of Science, Ho Chi Minh City, Vietnam.
Published Online: 2017-02-07
Published in Print: 2017-09-26