International Journal of Polymer Science
Volume 2018 (2018), Article ID 3970142, 7 pages
https://doi.org/10.1155/2018/3970142

Research Article

Synthesis, Characterization, and Anti-Phytopathogen Evaluation of 6-Oxychitosan Derivatives Containing N-Quaternized Moieties in Its Backbone

Kun Gao,1,2,3,4 Yukun Qin,1,2,3 Song Liu,1,2,3 Ronge Xing,1,2,3 Huahua Yu,1,2,3 Xiaolin Chen,1,2,3 Kecheng Li,1,2,3 and Pengcheng Li1,2,3

¹Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, No. 7 Nanhai Road, Qingdao 266071, China
²Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Qingdao 266237, China
³Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
⁴University of Chinese Academy of Sciences, Beijing 100049, China

Correspondence should be addressed to Yukun Qin; ykqin@qdio.ac.cn and Pengcheng Li; pcli@qdio.ac.cn

Received 12 April 2018; Accepted 20 June 2018; Published 1 August 2018

Academic Editor: Miriam H. Rafailovich

Copyright © 2018 Kun Gao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The structure modification of chitosan has great application potential. 6-Oxychitosan was prepared by specially oxidizing the C6-OH of chitosan, then 6-oxychitosan was reacted with three kinds of aldehydes to prepare N-quaternized 6-oxychitosan derivatives in this paper. The derivatives were characterized by FT-IR, NMR, and elemental analysis. The antimicrobial activity of these derivatives was tested against two common plant-threatening fungi and three plant disease bacteria. The results showed that N-quaternized 6-oxychitosan derivatives had good water-solubility and excellent antimicrobial activity. Moreover, derivative 3 which connected 8-hydroxyquinolines had the highest antimicrobial activity than the other derivatives. The inhibitory indices of derivative 3 against V. albo-atrum and P. hibernalis are 89.1% and 72.8% at 0.4 mg/ml. The MICs of 3 against X. oryzae, P. syringae, and E. rhapontici were 625, 625, and 156 mg/l, respectively. All the results indicate that derivative 3 has the potential of becoming an alternative to harmful agricultural chemicals.

1. Introduction

Plants are vulnerable to a variety of pathogenic microorganisms, including bacteria, fungi, and viruses. Among them, the majority of diseases are caused by fungi and bacteria. They could cause at least 10% of global food losses. Meanwhile, they would give rise to human hunger and malnutrition [1]. More than 800 million people worldwide do not have enough food and 130 million people live for less than 1 dollar a day [2]. In order to protect plants from fungal and bacterial persecution, all kinds of chemical fungicides and farm antibiotics are used in the process of planting. With the abuse of these agricultural chemicals, many serious problems were exposed, such as environmental pollution and ecological balance disorders, since they may kill the nontarget soil microorganism [3]. Meanwhile, the increase in resistance for universal fungicides and antibiotics has been affirmed [4, 5]. The safety and efficiency of agricultural chemicals have always been a major obstacle to the development of agriculture.

Chitosan, an important marine resource, is an ideal natural antimicrobial in that it is the only positively charged basic polysaccharide in nature, and it has biodegradability and biocompatibility [6]. According to reports, chitosan has inhibitory effect on fungi, bacteria, and viruses. Meanwhile, chitosan has been proved to be an inductor for plants and it can stimulate various plant defense reactions and improve plants’ resistance for these pathogenic microorganisms [7, 8]. In recent years, 6-oxychitosan has attracted much attention because it is similar to the structure of some metabolites in plants and animals, such as hyaluronic acid, salicylic acid, and abscisic acid. 6-Oxychitosan is water-soluble and has the potentiality to enhance the biological activities of chitosan [9]. Another excellent characteristic which enables chitosan to play an important role in agriculture is its ability to be chemically modified [10]. After modification, the restriction of water solubility which is its weakness will be overcome and it will obviously improve their biological activities [11]. According to previous studies, chitosan derivatives such as hydroxypropyl chitosan, O-hydroxyethyl chitosan, carboxymethyl chitosan, and quaternized chitosan, among others, also show more significant antimicrobial activity [12–14]. Quaternary ammonium salt derivatives of chitosan are very vital chitosan derivatives, because they have good solubility in an aqueous solution at neutral pH and shows excellent antimicrobial activity at this condition [15].

An important synthetic method of N-quaternized chitosan derivatives is synthesizing from reductive alkylation using various different aldehydes via the formation of Schiff base intermediates, followed by methylation with methyl iodide or ethyl iodide. The different N-quaternized chitosan derivatives which were synthesized by a series of aldehydes have diverse antimicrobial activities [16, 17]. 8-Hydroxyquinolines are an important member of natural heterocyclic substructures which play a key role in pharmaceuticals and agrochemicals [18]. The interest in 8-hydroxyquinolines has grown dramatically in the last two decades as they are privileged structures for the design of new drug candidates that exert a variety of biological activities, such as neuroprotection, anticancer, antibacterial, and antifungal activity [19, 20]. Therefore, the introduction of 8-hydroxyquinoline into the chitosan skeleton can strengthen its antimicrobial activity and chitosan could reduce the cytotoxicity owing to its special structure [21].

In this paper, we exhibited the synthesis and antimicrobial activity of a group of quaternized 6-oxychitosan derivatives. We specifically oxidized C6-OH of chitosan and prepared 6-oxychitosan firstly. Then, 6-oxychitosan was reacted with three kinds of aldehydes and NaBH₄ was added to prepare N-substitute 6-oxychitosan derivatives. Finally, the C2-NH₂ and NH₂ on a benzene ring were modified as a quaternary ammonium salt. By this way, we obtained the target chitosan derivatives which were supposed to have ideal water solubility and antimicrobial activity. What’s more, the structure and physicochemical characteristics of new chitosan derivatives were characterized using FT-IR, ¹H NMR, ¹³C NMR, and elemental analysis. The two common plant-threatening fungi, Verticillium albo-atrum (CGMCC 3.2869) and Phytophthora hibernalis (CGMCC 3.986), and three universal plant disease bacteria, Xanthomonas oryzae (CGMCC 1.0843), Pseudomonas syringae (CGMCC 1.1844), and Erwinia rhapontici (CGMCC 1.6978) were chosen to test the antimicrobial activity of the target chitosan derivatives.

2. Experimental

2.1. Materials

Chitosan was purchased from Qingdao Baicheng Biochemical Corp. Its viscosity-average molecular weight is 160 × 10⁴ Da and the degree of deacetylation is 85%. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and sodium borohydride were purchased from Aladin Chemical Corp. 8-Hydroxyquinoline with a minimum purity of 98% and the other reagents such as sodium hydrate, sodium bromide, sodium hypochlorite, hydrochloric acid, ethanol, 4-dimethylaminobenzaldehyde, and benzaldehyde were obtained from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Two plant-threatening fungi, V. albo-atrum and P. hibernalis and three plant-threatening bacteria, X. oryzae, P. syringae, and E. rhapontici, were supplied by China General Microbiological Culture Collection Center (CGMCC).

2.2. Analytical Methods

Fourier transform infrared (FT-IR) spectra was obtained on a Thermo Fisher Scientific Nicolet iS10 FT-IR Spectrometer with KBr disks ranging from 4000 cm⁻¹ to 400 cm⁻¹. ¹H NMR and ¹³C NMR were measured with a JEOL JNM-ECP 600 spectrometer and using the D₂O as the solvent. The elements analyses (C and N) were recorded on a Vario 114 EL-III elemental analyzer. We used the results to calculate the degree of substitution of the chitosan derivatives and computational formula, referring to the dos Santos et al.’s study [22].

2.3. Synthesis of Chitosan Derivatives

2.3.1. Synthesis of the 6-Oxychitosan

6-Oxychitosan was prepared referring to the methods reported by Yoo et al. [23]. Chitosan (2 g) was dissolved in 100 ml of acetic acid solution (1%, v/v). Then, a certain concentration of sodium hydroxide solution was slowly added to adjust the chitosan solution to make it neutral, and TEMPO (0.074 mmol), NaBr (6.72 mmol), and NaOCl (27.34 mmol) were added into the reaction system. The mixture was kept at room temperature at pH 10.8 with 0.4 M NaOH and stirred for 1 h. Finally, 5 ml of ethanol was added and the system was neutralized with 4 M HCl to pH 7 to stop the reaction. The neutralized reaction mixture was dialyzed and freeze-dried.

2.3.2. Synthesis of Quaternized 6-Oxychitosan Derivatives

The synthetic strategy for the preparation the quaternized 6-oxychitosan derivatives is shown in Scheme 1. The benzaldehyde and paradimethylaminobenzaldehyde were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. The mixture of 8-hydroxyquinoline-7-carbaldehyde and 8-hydroxyquinoline-5-carbaldehyde was synthesized according to the methods reported by Fan et al. [24]. Quaternized 6-oxychitosan derivatives were synthesized as follows: 3.5 grams of 6-oxychitosan (20 mmol) was dissolved in 100 ml of H₂O at rt and 10 ml of ethanol was added to the solution. Then, 60 mmol of various aldehydes were added with stirring at 50°C. After 6 h, 1.135 g of NaBH₄ (30 mmol) was added and the reaction was carried out for 2 h at rt. The solution was precipitated in ethanol and the precipitants were washed with acetone. The N-substituted 6-oxychitosan derivatives were gained after drying at 60°C for 24 h. A gram of N-substituted 6-oxychitosan was dispersed into 50 ml of N-methyl-2-pyrrolidone (NMP) for 12 h at room temperature. 1.5 g of NaI, 0.12 ml of NaOH (1 M), and 8 ml of CH₃I were added to the mixture in this order with stirring, and every reaction continued for 2 h at 60°C. The solution was precipitated by excessive ethanol and washed with acetone. The quaternized 6-oxychitosan derivatives were obtained after drying at 60°C for 24 h.

Scheme 1: Synthesis scheme for the preparation of quaternized 6-oxychitosan derivatives.

2.4. Antifungal Assays

Antifungal assays against V. albo-atrum and P. hibernalis were measured by following the plate growth rate method [25]. Briefly, the sterilized potato dextrose agars were prepared with chitosan, 6-oxychitosan, and derivatives 1–3 to gain the final concentrations of 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml. All of the samples were dispersed in distilled water solvent. Then, the mixture was cooled in the plate and a 5.0 mm diameter of fungi mycelia was transferred to the test plate and incubated at 28°C until the fungi mycelia of the blank control group reached the edge. Finally, the antifungal index was calculated according to (1).

where Da is the diameter of the growth zone in the text plates, and Db is the diameter of the growth zone in the blank control plate. Each experiment was replicated three times and the dates were analyzed according to Duncan’s method. When , the statistical significance of the result was acknowledged.

2.5. The MIC of Chitosan Derivatives on Bacteria

The minimum inhibitory concentration (MIC) of the 6-oxychitosan and chitosan derivatives against X. oryzae, P. syringae, and E. rhapontici were recorded using the typical microdilution method. The experiments were performed in 96-well polystyrene microtiter plates with minor modifications. Briefly, 100 μl of liquid nutrient medium was added to every well of the 96-well polystyrene microtiter plate, and 100 μl of 20 mg/ml 6-oxychitosan solution or chitosan derivative solution was added to the first row of the 96-well plate and mixed. Then, 100 μl of the mixture from the first row and so on was added to the second row. Finally, 100 μl of overnight-cultured bacterial suspension (OD₆₀₀ = 0.8–1.0) was added to every well of the 96-well plate to gain a final concentration of 0.078, 0.156, 0.313, 0.625, 1.250, 2.50, and 5.0 mg/ml. The blank control group just contains 100 μl of liquid nutrient medium and 100 μl of sterilized water. The 96-well plate was incubated without shaking for 24 h at 28°C. The MIC was defined as the lowest concentration of derivatives at which no bacteria survived. All assays were carried out at least three times in biological repeats.

3. Results

3.1. Chemical Synthesis and Characterization

Every chitosan derivative has to be measured by FT-IR, ¹H NMR, ¹³C NMR, and elemental analysis. The FT-IR, ¹H NMR, and ¹³C NMR spectra of chitosan, 6-oxychitosan, 1, 2, and 3 are exhibited in Figure 1, and their elemental analysis, yield, and DS are shown in Table 1.

(a)

(b)

(c)

Figure 1: The chemical structure of chitosan and chitosan derivatives. (a) FT-IR spectra, (b) ¹H NMR, and (c) ¹³C NMR of chitosan, 6-oxychitosan, 1, 2, and 3.

Table 1: The elemental analysis results, yield, and DS of the quaternized 6-oxychitosan derivatives.

Sample
Elemental analysis (%)
DS (%)
Groups
Yield (%)

C
N

6-Oxychitosan
32.33
4.19
35.7
Carboxyl
87.5

1
35.75
4.32
40.6
Quaternary ammonium salt
65.4

2
44.26
4.56
44.4
Quaternary ammonium salt
67.7

3
26.50
3.97
13.8
Quaternary ammonium salt
59.8

6-Oxychitosan is the intermediate of the quaternized 6-oxychitosan derivatives 1, 2, and 3, so the primary task is to confirm the structure of 6-oxychitosan. From Figures 1(b) and 1(c), the ¹H NMR and ¹³C NMR of chitosan and 6-oxychitosan only have a slight difference in that the chemical shift of the carboxyl group of 6-oxychitosan overlaps with the acetyl group of chitosan. In FT-IR of 6-oxychitosan, the shape of the peak at 3200 cm⁻¹ is wider than chitosan, and the signal intensity is stronger, meaning that with the formation of carboxyl groups, intramolecular hydrogen bonds increase. In addition, intense bands at 1623 cm⁻¹, assigned to the carboxyl group, partially overlaps the 1648 cm⁻¹ bands typical for chitosan resulting in enhanced signal strength. Meanwhile, 6-oxychitosan had good water solubility. All of these supports the accuracy of the structure of 6-oxychitosan.

Subsequently, 6-oxychitosan was, respectively, reacted with three types of aldehydes to gain 6-oxychitosan Schiff base derivatives. Then, 6-oxychitosan Schiff base derivatives were reduced and quaternized to obtain quaternized 6-oxychitosan derivatives 1, 2, and 3. Therefore, the derivatives 1, 2, and 3 have a phenyl group and quaternary ammonium salt besides the skeleton of 6-oxychitosan. The signal strength of the peak of the three derivatives at 1521 cm⁻¹ obviously weakened, meaning the C2-NH₂ of 6-oxychitosan had reacted. This is because 1521 cm⁻¹ is the absorption peak of −NH₂. For derivative 1, the signal strength of the peak at 1645 cm⁻¹ became stronger and the new peak at 1464 cm⁻¹ in FT-IR could prove the existence of a quaternary ammonium salt. The peak at 7.43 ppm in ¹H NMR and the several peaks at 125 ppm–135 ppm in ¹³C NMR could testify to the existence of the monosubstituted benzene ring. For derivative 2, it also applicable to the same analysis method. The peaks at 1633 cm⁻¹ and 1470 cm⁻¹ in FT-IR and the peaks at 7.78 ppm and 7.62 ppm in ¹H NMR could explain the existence of a quaternary ammonium salt and the parasubstituted benzene ring. The analytic procedure of derivative 3 is basically consistent with derivatives 1 and 2. The peaks at 1644 cm⁻¹ and 1457 cm⁻¹ in FT-IR are assigned to the quaternary ammonium salt. There are some new peaks at the ranges of 1400–1500 cm⁻¹ and 700–900 cm⁻¹ in FT-IR and several peaks at the range 7–10 ppm in ¹H NMR caused by 8-hydroxyquinoline.

3.2. Water Solubility

Figure 2(a) shows the solidity of chitosan and chitosan derivatives and Figure 2(b) shows the aqueous solution of the synthesized chitosan derivatives and chitosan (neutral water, 10 mg/ml). It is obvious that the three chitosan derivatives and 6-oxychitosan have considerable water solubility, but chitosan is insoluble in neutral water. The good water solubility of the three chitosan derivatives was due to two hydrophilic groups which are the carboxyl group with a negative charge and the quaternary ammonium salt with a positive charge.

(a)

(b)

Figure 2: (a) The solidity of chitosan and chitosan derivatives. (b) The aqueous solution of chitosan and chitosan derivatives (neutral water, 10 mg/ml).

3.3. Antifungal Activity

It was recognized that quaternary ammonium salt derivatives of chitosan have excellent antifungal activity and good water solubility. Its antifungal activity is related to the number of positive charges per unit concentration [26]. Therefore, the introduction of multiple quaternary ammonium N⁺ into each repeat unit of chitosan can effectively improve its antifungal activity. Subsequently, the C2-NH₂ of chitosan was reacted with paradimethylaminobenzaldehyde and the mixture of 8-hydroxyquinoline-7-carbaldehyde and 8-hydroxyquinoline-5-carbaldehyde. In this way, every repeat unit of chitosan which was modified has two N⁺. In addition, 8-hydroxyquinoline has an antifungal activity and has a synergistic reaction with N⁺. The antifungal activity of chitosan and all the derivatives against V. albo-atrum and P. hibernalis is shown in Figures 3 and 4.

Figure 3: The antifungal activity of chitosan, 6-oxychitosan, 1, 2, and 3 against V. albo-atrum.

Figure 4: The antifungal activity of chitosan, 6-oxychitosan, 1, 2, and 3 against P. hibernalis.

The antifungal activity against all samples is positively correlated with sample concentration. The inhibitory indices of chitosan, 6-oxychitosan, 1, 2, and 3 against V. albo-atrum are 10.5%, 39.5%, 61%, 76.2%, and 88.1% at 0.4 mg/ml, respectively. The inhibitory indices of chitosan, 6-oxychitosan, 1, 2, and 3 against P. hibernalis are 18.6%, 55.9%, 62.7%, 64.4%, and 72.8%, respectively. According to the inhibitory indices against V. albo-atrum and P. hibernalis, the three chitosan derivatives show much stronger antifungal activity than unmodified chitosan. Through the difference analysis and standard deviation, it shows that the antifungal activity of these five samples has an obvious difference. The difference is more significant with the increase of the concentration of the sample. There is a slight variance on antifungal activity among the three derivatives against P. hibernalis at 0.2 mg/ml, but a marked difference at 0.4 mg/ml. The antifungal activity of chitosan derivatives 2 and 3 was superior to that of chitosan derivative 1, affirming that the greater the number of positive charges per unit concentration, the stronger the antifungal activity of chitosan derivatives. At the same time, the inhibitory index of derivative 3 with a DS of 0.14 was slightly higher than that of derivative 2 with a DS of 0.44, inferring that chitosan derivative 3 will have a more advantageous antifungal activity if it has a higher DS. Therefore, chitosan derivative 3 has the enormous potential of becoming an effective antifungal agent.

3.4. The MIC of Chitosan Derivatives on Bacteria

The minimum inhibitory concentrations of chitosan, 6-oxychitosan, 1, 2, and 3 against X. oryzae, P. syringae, and E. rhapontici were measured using the typical microdilution method. The data obtained from Table 2 showed the same results as the antifungal activity of chitosan and chitosan derivatives. The modified chitosan had higher antibacterial activity. As shown in Table 2, chitosan derivative 3 was the most effective derivative against the three bacteria. The MICs of the 3 against X. oryzae, P. syringae, and E. rhapontici were 625, 625, and 156 mg/l, respectively. This is due to the joint efforts of the quaternary amination of chitosan and 8-hydroxyquinoline which enhanced the interaction between chitosan and anionic compounds of bacteria. This interaction can alter bacterial surface morphology and disrupt cell membranes, interact further with cellular DNA, and inhibit transcription [27].

Table 2: The minimum inhibitory concentrations (MICs) of chitosan, 6-oxychitosan, 1, 2, and 3 against X. oryzae, P. syringae, and E. rhapontici in the 96-well polystyrene microtiter plate.

MIC (mg/l)

X. oryzae
P. syringae
E. rhapontici

Chitosan
>5000
>5000
>5000

6-Oxychitosan
2500
5000
625

1
2500
1250
625

2
2500
2500
313

3
625
625
156

In summary, the quaternized chitosan derivative 3 has the excellent antifungal activity and antibacterial activity and good water solubility, confirming that the concentrations of N⁺ and the types of aldehydes which were reacted with chitosan play a vital role in the antimicrobial activity of quaternized chitosan derivatives [28]. The chitosan derivative containing 8-hydroxyquinoline significantly enhances its antimicrobial activity more than 6-oxychitosan. What’s more, chitosan can reduce the cytotoxic action of 8-hydroxyquinoline due to its special structure and biocompatibility.

4. Conclusion

In this paper, we first synthesized the water-soluble 6-oxychitosan. Then 6-oxychitosan was designed and, respectively, reacted with benzaldehyde, paradimethylaminobenzaldehyde, and the mixture of 8-hydroxyquinoline-7-carbaldehyde and 8-hydroxyquinoline-5-carbaldehyde to obtain three quaternized chitosan derivatives. Their antifungal activity against two common plant-threatening fungi was measured by hyphal measurement in vitro and their antibacterial activity against three universal plant disease bacteria was estimated by the typical microdilution method. These chitosan derivatives have good water solubility and stronger antimicrobial activity than chitosan. From the above data, the antimicrobial activity is positively correlated with the number of N⁺ of every repeat unit of quaternized chitosan derivatives. Moreover, the types of aldehyde which were reacted with C2-NH₂ of 6-oxychitosan also made a contribution to the antimicrobial activity. The chitosan derivatives containing 8-hydroxyquinoline has the most effective antimicrobial activity among the three chitosan derivatives. In addition, the DS of chitosan derivative 3 is slightly low. If the DS of chitosan derivative 3 is the same as derivative 2, it could show higher antimicrobial activity. It is worth studying further to ascertain the supposal and assess its control effect in the field. In conclusion, chitosan derivative 3 is environmentally friendly and active and has the potential of becoming an alternative to harmful agricultural chemicals.

Data Availability

The data related to this article is currently being patented, so it cannot be made public. If you want to see the original data, please e-mail Yukun Qin.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors thank the Key Research and Development Program of Shandong Province (2018GHY115017, 2018GHY115008), the Nantong Science and Technology Bureau, the Science and Technology Program of Qingdao (17-3-3-60-nsh), and the Scientific and Technological Innovation Project financially supported by the Qingdao National Laboratory for Marine Science and Technology (no. 2015ASKJ02) for financially supporting this work.

References

G. B. Martin, A. J. Bogdanove, and G. Sessa, “Understanding the functions of plant disease resistance proteins,” Annual Review of Plant Biology, vol. 54, no. 1, pp. 23–61, 2003.

R. N. Strange and P. R. Scott, “Plant disease: a threat to global food security,” Annual Review of Phytopathology, vol. 43, no. 1, pp. 83–116, 2005.

F. Mubeen, M. A. Shiekh, T. Iqbal, Q. M. Khan, K. A. Malik, and F. Y. Hafeez, “In vitro investigations to explore the toxicity of fungicides for plant growth promoting rhizobacteria,” Pakistan Journal of Botany, vol. 38, pp. 1261–1269, 2006.

G. A. Secor and V. V. Rivera, “Fungicide resistance assays for fungal plant pathogens,” Methods in Molecular Biology, vol. 835, pp. 385–392, 2012.

V. Hernandez, T. Crépin, A. Palencia et al., “Discovery of a novel class of boron-based antibacterials with activity against gram-negative bacteria,” Antimicrobial Agents and Chemotherapy, vol. 57, no. 3, pp. 1394–1403, 2013.

V. E. Tikhonov, E. A. Stepnova, V. G. Babak et al., “Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives,” Carbohydrate Polymers, vol. 64, no. 1, pp. 66–72, 2006.

S. Mandal, I. Kar, A. K. Mukherjee, and P. Acharya, “Elicitor-induced defense responses in Solanum lycopersicum against Ralstonia solanacearum,” The Scientific World Journal, vol. 2013, Article ID 561056, 9 pages, 2013.

S. N. Kulikov, S. N. Chirkov, A. V. Il’ina, S. A. Lopatin, and V. P. Varlamov, “Effect of the molecular weight of chitosan on its antiviral activity in plants,” Applied Biochemistry and Microbiology, vol. 42, no. 2, pp. 200–203, 2006.

J. Pei, Y. Yin, Z. Shen, X. Bu, and F. Zhang, “Oxidation of primary hydroxyl groups in chitooligomer by a laccase-TEMPO system and physico-chemical characterisation of oxidation products,” Carbohydrate Polymers, vol. 135, pp. 234–238, 2016.

D. de Britto, M. R. de Moura, F. A. Aouada, L. H. C. Mattoso, and O. B. G. Assis, “N,N,N-trimethyl chitosan nanoparticles as a vitamin carrier system,” Food Hydrocolloids, vol. 27, no. 2, pp. 487–493, 2012.

Q. Li, W. Tan, C. Zhang, G. Gu, and Z. Guo, “Synthesis of water soluble chitosan derivatives with halogeno-1,2,3-triazole and their antifungal activity,” International Journal of Biological Macromolecules, vol. 91, pp. 623–629, 2016.

A. F. Martins, P. V. A. Bueno, E. A. M. S. Almeida, F. H. A. Rodrigues, A. F. Rubira, and E. C. Muniz, “Characterization of N-trimethyl chitosan/alginate complexes and curcumin release,” International Journal of Biological Macromolecules, vol. 57, pp. 174–184, 2013.

W. Xie, P. Xu, W. Wang, and Q. Liu, “Preparation and antibacterial activity of a water-soluble chitosan derivative,” Carbohydrate Research, vol. 50, no. 1, pp. 35–40, 2002.

Y. Peng, B. Han, W. Liu, and X. Xu, “Preparation and antimicrobial activity of hydroxypropyl chitosan,” Carbohydrate Research, vol. 340, no. 11, pp. 1846–1851, 2005.

A. F. Martins, S. P. Facchi, H. D. M. Follmann, A. G. B. Pereira, A. F. Rubira, and E. C. Muniz, “Antimicrobial activity of chitosan derivatives containing N-quaternized moieties in its backbone: a review,” International Journal of Molecular Sciences, vol. 15, no. 11, pp. 20800–20832, 2014.

N. Vallapa, O. Wiarachai, N. Thongchul et al., “Enhancing antibacterial activity of chitosan surface by heterogeneous quaternization,” Carbohydrate Polymers, vol. 83, no. 2, pp. 868–875, 2011.

M. R. Avadi, A. M. M. Sadeghi, A. Tahzibi et al., “Diethylmethyl chitosan as an antimicrobial agent: synthesis, characterization and antibacterial effects,” European Polymer Journal, vol. 40, no. 7, pp. 1355–1361, 2004.

H. P. Zhao, Z. P. Cui, Y. C. Gu, Y. X. Liu, and Q. M. Wang, “The phytotoxicity of natural tetramic acid derivatives,” Pest Management Science, vol. 67, no. 9, pp. 1059–1061, 2011.

A. Albert, M. I. Gibson, and S. D. Rubbo, “The influence of chemical constitution on anti-bacterial activity. Part VI: the bactericidal action of 8-hydroxyquinoline (oxine),” The British Journal of Experimental Pathology, vol. 34, no. 2, pp. 119–130, 1953.

A. Albert, A. Hampton, F. R. Selbie, and R. D. Simon, “The influence of chemical constitution on anti-bacterial activity. Part VII: the site of action of 8-hydroxyquinoline (oxine),” The British Journal of Experimental Pathology, vol. 28, no. 2, article 13126414, pp. 75–84, 1954.

O. M. Saka and A. Bozkir, “Formulation and in vitro characterization of PEGylated chitosan and polyethylene imine polymers with thrombospondin-I gene bearing pDNA,” Journal of Biomedical Materials Research Part B, Applied Biomaterials, vol. 100B, no. 4, pp. 984–992, 2012.

Z. M. dos Santos, A. L. P. F. Caroni, M. R. Pereira, D. R. da Silva, and J. L. C. Fonseca, “Determination of deacetylation degree of chitosan: a comparison between conductometric titration and CHN elemental analysis,” Carbohydrate Research, vol. 344, no. 18, pp. 2591–2595, 2009.

S. H. Yoo, J. S. Lee, S. Y. Park, Y. S. Kim, P. S. Chang, and H. G. Lee, “Effects of selective oxidation of chitosan on physical and biological properties,” International Journal of Biological Macromolecules, vol. 35, no. 1-2, pp. 27–31, 2005.

L. Fan, X. H. Jiang, B. D. Wang, and Z. Y. Yang, “4-(8-Hydroxyquinolin-7-yl) methyleneimino-1-phenyl-2,3-dimethyl-5-pyzole as a fluorescent chemosensor for aluminum ion in acid aqueous medium,” Sensors and Actuators B: Chemical, vol. 205, pp. 249–254, 2014.

Q. Li, W. Tan, C. Zhang, G. Gu, and Z. Guo, “Novel triazolyl-functionalized chitosan derivatives with different chain lengths of aliphatic alcohol substituent: design, synthesis, and antifungal activity,” Carbohydrate Research, vol. 418, pp. 44–49, 2015.

K. V. Harish Prashanth and R. N. Tharanathan, “Chitin/chitosan: modifications and their unlimited application potential—an overview,” Trends in Food Science and Technology, vol. 18, no. 3, pp. 117–131, 2007.

A. F. Martins, J. F. Piai, I. T. A. Schuquel, A. F. Rubira, and E. C. Muniz, “Polyelectrolyte complexes of chitosan/heparin and N,N,N-trimethyl chitosan/heparin obtained at different pH: I. Preparation, characterization, and controlled release of heparin,” Colloid & Polymer Science, vol. 289, no. 10, pp. 1133–1144, 2011.

P. D. Chethan, B. Vishalakshi, L. Sathish, K. Ananda, and B. Poojary, “Preparation of substituted quaternized arylfuran chitosan derivatives and their antimicrobial activity,” International Journal of Biological Macromolecules, vol. 59, pp. 158–164, 2013.