…ir.yic.ac.cn/bitstream/133337/24770/1/Improved... · 2020. 7. 8. · 2.ExperimentalSection...

RESEARCH ARTICLEwww.starch-journal.com

Improved Antioxidant and Antifungal Activity of ChitosanDerivatives Bearing Urea Groups

Jingjing Zhang, Yingqi Mi, Xueqi Sun, Yuan Chen, Qin Miao, Wenqiang Tan, Qing Li,Fang Dong, and Zhanyong Guo *

Eight novel chitosan derivatives bearing urea groups are designed andsynthesized. Fourier transform infrared spectroscopy, 1H nuclear magneticresonance spectrometer, and elemental analysis are performed to confirm thestructural characteristics of chitosan derivatives. The antioxidant activities,including superoxide radicals’ scavenging activity, 1,1-diphenyl-2-picrylhydrazylradicals’ scavenging activity, and hydroxyl radical’ scavenging activity, of thederivatives are explored within different concentrations in the reaction system.In vitro fungicidal activity of these compounds is further tested againstFusarium oxysporum f. sp. niveum, Phomopsis asparagus, F. oxysporumf. sp. cucumebrium Owen, and Botrytis cinerea, respectively, particularlycompounds exhibit significant control effect at 1.0 mg mL−1. The experimentalresults indicate that the products bearing urea groups show enhancedantifungal property and antioxidant activity compared with pristine chitosan.Meanwhile, their bioactivities follow some regularity on the whole, that is, theyare related to the electron-withdrawing property of the different substitutedgroups of urea. Derivatives with stronger electron-withdrawing property willhave higher biological activities. L929 cells are used to carry out cytotoxicitytest of chitosan and chitosan derivatives by Cell Counting Kit-8 assay. Theresults indicate that some of the samples show low cytotoxicity. This researchwill be helpful to broaden the application of chitosan in materials.

1. Introduction

Chitosan is one of the most abundant renewable re-sources and is the only cationic polysaccharide in nature.[1,2]

Dr. J. Zhang, Dr. Y. Mi, Dr. X. Sun, Dr. Y. Chen, Dr. Q. Miao, Dr. W. Tan,Dr. Q. Li, Dr. F. Dong, Prof. Z. GuoKey Laboratory of Coastal Biology and Bioresource UtilizationYantai Institute of Coastal Zone ResearchChinese Academy of SciencesYantai 264003, ChinaE-mail: [email protected]; [email protected]. J. Zhang, Dr. Y. Mi, Dr. X. Sun, Dr. Y. Chen, Dr. Q. Miao, Dr. W. Tan,Dr. Q. Li, Dr. F. Dong, Prof. Z. GuoCenter for Ocean Mega-ScienceChinese Academy of Sciences7 Nanhai Road, Qingdao 266071, P. R. ChinaDr. J. Zhang, Dr. Y. Mi, Dr. X. Sun, Dr. Y. Chen, Prof. Z. GuoUniversity of Chinese Academy of SciencesBeijing 100049, China

The ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/star.201900205

DOI: 10.1002/star.201900205

Chitosan-based biomaterials have attractedincreasing attention due to the charac-teristics of abundant sources, being re-newable, low cost, non-toxicity, and goodbiocompatibility.[3–5] However, the commer-cial application of chitosan is greatly lim-ited due to its dense structure and poorsolubility, which is caused by the in-tramolecular and intermolecular hydrogenbonding of chitosan as well as the rigid crys-talline structure.[6,7] Chemical modificationis an ideal way to improve the water solubil-ity of chitosan. Indeed, attaching functionalgroups to chitosan molecules can improveits water solubility and biological activity.[8,9]

At present, the chemicalmodificationmeth-ods of chitosan mainly include acylation,esterification, etherification, quaternariza-tion, and so on.[10] With the deepening ofchitosan and its derivatives research, theutilization value of this kind of renewableresources would increase.Urea is a well-known organic compound

that is of considerable interest becauseof the diverse biological properties.[11,12]

Urea derivatives are widely used becauseof their good pharmacological properties.

The urea linkage is often present in many pharmacologi-cally active drugs.[13,14] For example, it has been reported asanti-tuberculosis,[15] antimalarial,[16] antibacterial,[17] anti-chronicmyeloid leukemia,[18] anti-inflammatory,[19] antiproliferative,[20]

and anticancer agents,[21] and so forth. In addition, a lot ofwork has been done on the development and application of ureaderivatives. Pan et al. had synthesized a series of novel C14-urea-tetrandrine derivatives. They reported that the C14-urea substi-tuted derivatives held up fairly well in anticancer activity and theycould be used as a potential anticancer drug candidate.[22] Sev-eral 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol-2-yl)ureaderivatives with remarkable effects on human chronic myeloidleukemia cell line K562 were also designed. Especially, some ofthem showed much better inhibitory activity than standard drugimatinib.[18] Inspired by that, a class of pyridine ureas were syn-thesized and grafted onto chitosan in order to obtain severalpolysaccharide derivatives with better biological activity.The aim of our work was synthesis, characterization, and

in vitro studies of novel chitosan derivatives bearing ureagroups which displayed promising antifungal and antioxidantproperties. We chose compounds with different substituents tobe able to investigate possible structure–activity relationship.

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2. Experimental Section

2.1. Materials

Chitosan with a molecular weight of 5000–8000 Da wassupplied by Golden-Shell Pharmaceutical Co. Ltd. (Zhejiang,China). The degree of deacetylation of chitosan was 73.5%.Chloroacetyl chloride (product code C104559), nicotinoyl chlo-ride hydrochloride (product code N107157), aniline (productcode A112122), 2-fluoroaniline (product code F107841), 2-chloroaniline (product code C103931), 2-bromoaniline (prod-uct code B108449), 2-aminothiazole (product code A104872),2-amino-1,3,4-thiadiazole (product code A151011), 2-amino-5-methyl-1,3,4-thiadiazole (product code A151010), and 3-amino-1,2,4-triazole (product code A107202) were purchased from theSigma-Aldrich Chemical Corp (Shanghai, China). The otherreagents were supplied by SinopharmChemical Reagent Co., Ltd.(Shanghai, China) and used without purification.

2.2. Analytical Methods

2.2.1. Fourier Transform Infrared Spectroscopy

The infrared spectra of the samples were measured by Jasco-4100 Fourier transform infrared (FT-IR) spectrometer (Japan,provided by JASCO Co., Ltd., Shanghai, China) with a resolutionof 4.0 cm−1 and a range of 4000–400 cm−1. The tested sampleswere lamellated with KBr for observations.

2.2.2. NMR Spectroscopy

The 1H Nuclear Magnetic Resonance (1H NMR) spectra of sam-ples were recorded by Bruker AVIII-500 Spectrometer (500MHz,Switzerland, provided by Bruker Tech. and Serv. Co., Ltd., Bei-jing, China) at 25 °C. The samples were dissolved in 0.6 mL D2Oor DMSO for analysis.

2.2.3. Elemental Analysis

The elemental analysis was performed on Vario Micro ElementalAnalyzer (Elementar, Berlin, Germany). The degree of substitu-tion (DS) in chitosan derivatives was evaluated by the carbon/nitrogen ratio and it was according to the following equations:

DS1 =n1 ×MC −MN ×WC∕N

n2 ×MC(1)

DS2 =MN ×WC∕N + n2 ×MC × DS1 − n1 ×MC

n3 ×MC(2)

DS3 =

n1 ×MC − n′

1 ×MN ×WC∕N − n2 ×MC ×DS1 + n3 ×MC ×DS2n′2 ×MN ×WC∕N − n4 ×MC

(3)

where DS1, DS2, and DS3 represent the deacetylation de-gree of chitosan, chloroacetyl in chitosan derivative, and ureagroups in chitosan derivatives; MC and MN are the molarmass of carbon and nitrogen, MC = 12, MN = 14; n1, n2, n3,and n4 are the number of carbon of chitin, acetamido group,chloroacetyl group, and urea group, n1 = 8, n2 = 2, n3 = 4, 2,6-(3-(benzylureido)-pyridyl)acetyl chitosan chloride (BUCACS):n4 = 24, 2,6-(3-(2-fluorinbenzylureido)-pyridyl)acetyl chitosanchloride (FBUCACS): n4 = 24, 2,6-(3-(2-chlorobenzylureido)-pyridyl)acetyl chitosan chloride (CBUCACS): n4 = 24, 2,6-(3-(2-brominbenzylureido)-pyridyl)acetyl chitosan chloride (BBU-CACS): n4 = 24, 2,6-(3-(2-aminothiazolureido)-pyridyl)acetylchitosan chloride (THUCACS): n4 = 18, 2,6-(3-(2-amino-1,3,4-thiadiazolureido)-pyridyl)acetyl chitosan chloride (TDUCACS):n4 = 16, 2,6-(3-(2-amino-5-methyl-1,3,4-thiadiazolureido)-pyridyl)acetyl chitosan chloride (MTDUCACS): n4 = 18, 2,6-(3-(3-amino-1,2,4-triazolureido)-pyridyl)acetyl chitosan chloride(TRUCACS): n4 = 16; n′1 and n′2 are the number of nitrogen ofCACS and urea group, n′1 = 1, BUCACS: n′2 = 6, FBUCACS:n′2 = 6, CBUCACS: n′2 = 6, BBUCACS: n′2 = 6, THUCACS:n′2 = 8, TDUCACS: n′2 = 10, MTDUCACS: n′2 = 10, TRUCACS:n′2 = 12; WC/N represents the mass ratio between carbon andnitrogen in chitosan derivatives.

2.3. Synthesis of Chitosan Derivatives

As shown in Scheme 1, the synthesis of chitosan derivatives wascarried out by the following steps.

2.3.1. Synthesis of Urea Groups

First, nicotinoyl chloride hydrochloride (20 mmol) was dispersedequably in 15 mL of acetone and stirred at 0 °C. The mixture wasthen added dropwise to the aqueous solution of sodium azide(3.9 g NaN3 dissolving in 12 mL of deionized water). The reac-tion mixture was stirred for 3 h under the condition of ice bath.After the reaction was finished, the solution had been stratifiedand the lower layer was removed. The remaining solution waspoured into 10 mL of methylbenzene solvent at 60 °C and stirredfor 2–3 h. Then, the mixture was cooled and some pink crystalswere precipitated out. The precipitate was filtered and pyridine-3-isocyanate was obtained.Finally, pyridine-3-isocyanate (30 mmol) and aniline, 2-

fluoroaniline, 2-chloroaniline, 2-bromoaniline, 2-aminothiazole,2-amino-1,3,4-thiadiazole, 2-amino-5-methyl-1,3,4-thiadiazole,or 3-amino-1,2,4-triazole were added to methylbenzene (20 mL)in a 50 mL flask. The mixture was stirred for 24 h at 60 °C, then alarge amount of solid formed and was filtered, which was furtherpurified by crystallization from the solvent that the ratio of waterand ethanol was 1:1 and several urea groups were synthesized.

2.3.2. Synthesis of Chloracetyl Chitosan

Chloracetyl chitosan (CACS) was synthesized according to thefollowingmethod: first, chitosan (1.61 g, 10mmol) was dispersed



O

OH

HO O nNH2

O

OCCH2Cl

HO O nNHCCH2Cl

CS

NMP

BUCACS: R= BBUCACS: R=

CBUCACS: R= FBUCACS: R=

THUCACS: R= TDUCACS: R=

MTDUCACS:R= TRUCACS: R=

Br

Cl F

N

COClNaN3

N

N C OHN C

OHN R

N

RNH2

HN C

O HN R

N

ClCH2COCl

O

OCCH2

HO O nNHCCH2

O HN C

OHN R

N

Cl

O

O

O

HN C

OHN R

NCl

DMSO

N

S

N NH

N

N N

S

N N

S CH3

CACS

Scheme 1. Synthesis routes for chitosan derivatives.

in 100 mL of N-methyl pyrrolidone (NMP) at room temperature(r.t.). Then, 20 mmol chloracetyl chloride was added. After stir-ring for 12 h at r.t., the solutionwas poured into 200mL of ether toobtain some precipitates. The precipitate was filtered and washedwith ethanol by turns. Finally, CACS was achieved after vacuumfreeze-drying for 24 h.

2.3.3. Synthesis of Chitosan Derivatives (BUCACS, FBUCACS,CBUCACS, BBUCACS, THUCACS, TDUCACS, MTDUCACS,TRUCACS)

1 mmol CACS and 6 mmol urea groups dissolved in 20 mL ofN,N-dimethylformamide (DMF) were stirred for 24 h at 60 °C.The solution was precipitated in excess acetone. Then the precip-itate was filtered andwashed with ethanol for three times. Finally,the chitosan derivatives were obtained after drying at 60 °C for6 h.

2.4. Antioxidant Assays

2.4.1. Superoxide-Radical Scavenging Activity Assay

The superoxide-radical scavenging ability was assessed follow-ing the previous method.[23] The reaction mixture, involvingtest samples (5 mg mL−1, 0.06, 0.12, 0.24, 0.48, and 0.96 mL),phenazine methosulfate (PMS, 30 µm), nitro blue tetrazolium(NBT, 72 µm), and nicotinamide adenine dinucleotide reduced(NADH, 338 µm) in Tris HCl buffer (16 mm, pH 8.0), was in-cubated 5 min at r.t. The absorbance was measured at 560 nm.Three replicates for each sample were tested and the superoxide-

radical scavenging effect was calculated according to the follow-ing equation:

Scavenging effect (%) =[1−

Asample 560 nm −Acontrol 560 nmAblank 560 nm

]× 100

(4)

where Asample 560 nm is the absorbance of the samples, Acontrol 560 nmis the absorbance of the control (NADHwas substituted with dis-tilled water), and Ablank 560 nm is the absorbance of the blank (sam-ples were substituted with distilled water).

2.4.2. Hydroxyl-Radical Scavenging Activity Assay

The test of hydroxyl-radical scavenging ability was carried outaccording to Li’s method with minor modification.[24] The reac-tion mixture, containing testing samples of chitosan or chitosanderivatives (10 mg mL−1, 0.045, 0.09, 0.18, 0.36, and 0.72 mL),safranine O (0.23 µm), EDTA-Fe2+ (220 µm), and H2O2 (60 µm)in potassium phosphate buffer (150 mm, pH 7.4), was incubatedfor 30min at 37 °C. The absorbance of themixture wasmeasuredat 520 nm against blank. Three replicates for each sample weretested and the hydroxyl-radical scavenging effect was calculatedaccording to the following equation:

Scavenging effect (%) =Asample 520 nm − Ablank 520 nmAcontrol 520 nm − Ablank 520 nm

× 100 (5)

where Asample 520 nm is the absorbance of the samples, Acontrol 520 nmis the absorbance of the control (H2O2 was substitutedwith potas-



sium phosphate buffer), and Ablank 520 nm is the absorbance of theblank (samples were substituted with distilled water).

2.4.3. DPPH-Radical Scavenging Activity Assay

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging proper-ties of the products were evaluated by the following method:[25]

the testing samples in different concentrations and 2 mL ofDPPH ethanol solution (180 µmol L−1) were incubated for20 min at room temperature. The absorbance of the remainedDPPH radical was measured at 517 nm against a blank. Thecontrol groups containing different concentrations of sam-ples and 2 mL of ethanol was measured at 517 nm. Threereplicates for each sample were tested and the DPPH-radicalscavenging effect was calculated according to the followingequation:

Scavenging effect (%)=[1−

Asample 517 nm −Acontrol 517 nmAblank 517 nm

]× 100

(6)

whereAsample 517 nm is the absorbance of the samples, Acontrol 517 nmis the absorbance of the control (DPPH was substituted withethanol), and Ablank 517 nm is the absorbance of the blank (sampleswere substituted with distilled water). Vitamin C was used as apositive control.

2.5. Antifungal Assay

The antifungal ability was carried out by the method of hy-phal measurement.[9] Briefly, the stock solutions of chitosan andderivatives were prepared with a concentration of 6 mg mL−1.Then, each sample solution was added to sterilized potato dex-trose agar (PDA) medium to obtain final concentrations of 0.1,0.5 and 1.0 mg mL−1. The culture media containing sampleswere poured into Petri dishes (7 cm). After solidification, 5.0 mmdiameter of fungi mycelium was transferred to the test plateand incubated at 27 °C for 2–3 days. When the mycelia of fungireached the edges of the control plate (without the presence ofsamples), the inhibition indices of all samples were calculated asfollows:

Antifungal index (%) =(1 − Da∕Db

)× 100 (7)

whereDa is the diameter of the growth zone in the test plates andDb is the diameter of the growth zone in the control plate.

2.6. Cytotoxicity Assay

The cytotoxicity of chitosan and synthesized chitosan derivativeson L929 cells at different concentrations (1.0, 10.0, 100.0, 500.0,and 1000.0 µg mL−1) was determined by Cell Counting Kit-8(CCK-8) assay in vitro.[26] L929 cells were cultured at 37 °C inRPMI medium (containing 1% mixture of penicillin and strep-tomycin, and 10% fetal calf serum). The cells were seeded on 96-

well flat-bottom culture plates at a density of 1.0 × 105 cells andincubated under 5% CO2 atmosphere. After 24 h of cell attach-ment, the samples with different final concentrations were intro-duced to cells, separately. Next, the cells were cultured for 24 h.Afterward, 10 µL of CCK-8 solution was added in each well andincubated for another 24 h at 37 °C. The absorbance at 450 nmwas measured. Cell viability was recorded according to the fol-lowing formula:

Cell viability (%) =Asample − AblankAnegative − Ablank

× 100 (8)

where Asample is the absorbance of the samples (containing cells,CCK-8 solution, and sample solution), Ablank is the absorbance ofthe blank (containing RPMI medium and CCK-8 solution), andAnegative is the absorbance of the negative (containing cells andCCK-8 solution).

2.7. Statistical Analysis

All the data related to antioxidant activity, antifungal activity, andcytotoxicity assay were illustrated as mean ± standard deviation(SD), n= 3. Significant difference analysis was determined usingScheffe’smultiple range test. The significant differences were de-fined at p < 0.05.

3. Results and Discussion

3.1. Chemical Synthesis and Characterization

The FT-IR, 1H NMR, and elemental analysis were performedto analyze changes that occurred in chitosan derivatives due tochemical reaction process.Figure 1 shows the FT-IR spectra of chitosan and chitosan

derivatives. In chitosan spectrum, the peaks observed at 3417,2919 and 2881, and 1596 cm−1 were assigned to O─H and N─Hstretching vibrations, ─CH asymmetric stretching, and vibrationmodes of amino group.[27,28] In spectrum of CACS, there weretwo peaks at 1741 cm−1 and 790 cm−1 corresponding to vibrationsof C═O and C─Cl.[29,30] It indicated the existence of chloroacetylgroup and CACS was obtained. In spectra of the final chitosanderivatives, there was a decrease at peak of 1749 cm−1 which re-ferred to vibration of C═O and the characteristic absorbance ofC─Cl at 790 cm−1 disappeared. Meanwhile, new peaks appearedat ≈1698, 1520, 820, and 750 cm−1, as would be expected afterthe process of insertion of urea groups into CACS. The peakobtained at 1698 cm−1 for BUCACS, FBUCACS, CBUCACS,BBUCACS, THUCACS, TDUCACS, MTDUCACS, and TRU-CACS was corresponded to ─NH─CO─NH─ bond presented inurea groups.[26] For BUCACS, FBUCACS, CBUCACS, and BBU-CACS, the peaks at 1530 cm−1, and 820 cm−1 were characteristicabsorption of benzene and pyridine of urea groups.[31] For THU-CACS, TDUCACS, MTDUCACS, and TRUCACS, the additionalpeaks at 1550 cm−1, 1520 cm−1, and 820 cm−1 were attributedto the typical absorption of nitrogen-containing heterocycles.[26]

These data preliminarily proved that the urea groupswere graftedonto CACS.



Figure 1. FT-IR spectra of chitosan and chitosan derivatives.

Figure 2 shows the 1H NMR spectra of chitosan and chi-tosan derivatives. In the 1H NMR spectrum of chitosan, the sig-nals of C-2 proton, C-3–C-6 protons, and C-1 proton appearedat 𝛿3.0 ppm, 𝛿3.6–3.9 ppm, and 𝛿5.5 ppm.[31,32] After modifi-cation, the peak at 𝛿4.3 ppm assigned to the proton signals of─COCH2Cl was observed from the

1H NMR spectrum of CACS,which indicated the existence of the chloroacetyl group.[29,30] Asfor as the 1H NMR spectrum of BUCACS, FBUCACS, CBU-CACS, BBUCACS, THUCACS, TDUCACS, MTDUCACS, andTRUCACS, the characteristic signal of ─COCH2Cl at 𝛿4.3 ppmwas weaker and new signals, which were attributed to protons ofthe pyridine ring, benzene ring, and nitrogen-containing hetero-cycles, appeared at 𝛿6.8–9.5 ppm.[26,30,33] Therefore, these resultswere enough to prove the successful synthesis of chitosan deriva-tives bearing urea groups.Table 1 shows the yields and DSs of chitosan derivatives. The

DSs of chitosan derivatives were estimated by elemental analy-sis. The DS of CACS was 0.43 calculated by the formulas. As tothe final products, derivatives containing phenylurea (BUCACS,FBUCACS, CBUCACS, and BBUCACS) possessed the higherDS, which were more than 0.7, and some even as high as 0.9.However, the DSs of THUCACS, TDUCACS, MTDUCACS, andTRUCACS were only around 0.25.

3.2. Antioxidant Activity

Reactive oxygen species (ROS) are by-products of toxic oxy-gen metabolism and the products are the result of ionizing

radiation or aging of some chemicals. Antioxidants are thesubstances which inhibit oxidation.[34] Nowadays, searching forenvironmentally friendly and efficient antioxidants is becom-ing increasingly urgent. In this paper, several chitosan deriva-tives with high antioxidant activity were synthesized and theirsuperoxide-radical scavenging activity, hydroxyl-radical scaveng-ing activity, and DPPH-radical scavenging activity are shown inFigures 3–5.Figure 3 shows the scavenging ability of chitosan and its

derivatives against superoxide radicals. According to the graph,several results could be concluded as follows. The antioxidantactivity of all samples was increasing with the concentration.Compared with the antioxidant activity of chitosan, the chitosanderivatives possessed better activity. In addition, the superox-ide radical scavenging rates of chitosan derivatives bearing ureagroups were much higher than that of CACS. The higher an-tioxidant activity was attributed to the excellent biological activityof urea groups. For example, the superoxide radical scavengingindices of chitosan, CACS, BUCACS, FBUCACS, CBUCACS,BBUCACS, THUCACS, TDUCACS, MTDUCACS, and TRU-CACS were 34.17%, 71.50%, 80.36%, 84.79%, 97.71%, 97.78%,73.43%, 77.08%, 85.73%, and 100% at 0.8 mg mL−1. The elec-trophilic groups could attract more single electron of free rad-icals to inhibit the free radical chain reaction.[35] Hence, chi-tosan derivatives with stronger electron-withdrawing propertywould have higher antioxidant property. As we can see in Fig-ure 3, the antioxidant property of chitosan derivatives followedthe rules of chitosan < CACS < BUCACS < BBUCACS < CBU-CACS < FBUCACS and chitosan < CACS < THUCACS < TD-



Figure 2. 1H NMR spectra of chitosan and chitosan derivatives.

Table 1. The yields and the DSs of chitosan derivatives.

Compounds Yields [%] Elemental analyses [%] Degrees ofsubstitution

Deacetylation

C N H C/N

CS 34.73 6.21 6.78 5.59 0.74

CACS 83.42 35.72 5.06 6.42 7.06 0.43

BUCACS 80.71 40.56 9.84 6.01 4.12 0.71

FBUCACS 79.17 40.31 9.85 6.54 4.09 0.75

CBUCACS 78.93 40.71 10.21 6.31 3.99 0.91

BBUCACS 79.46 46.64 13.51 6.40 4.05 0.81

THUCACS 69.90 37.81 10.85 6.85 3.48 0.29

TDUCACS 61.32 38.33 12.66 6.38 3.03 0.24

MTDUCACS 72.51 38.79 12.98 5.96 2.99 0.28

TRUCACS 59.78 39.34 13.98 6.34 2.81 0.21

UCACS < FBUCACS < TRUCACS. For BUCACS, FBUCACS,CBUCACS, and BBUCACS, the order of the antioxidant activ-ity was consistent with the electron-withdrawing capacity of thedifferent substituted atoms (─F >─Cl > ─Br > ─H) of the urea

groups. For instance, at the concentration of 1.6mgmL−1, the su-peroxide radical scavenging rates of chitosan, CACS, BUCACS,FBUCACS, CBUCACS, and BBUCACS were 34.72%, 79.80%,83.71%, 88.64%, 96.15%, and 100%, respectively. Similarly, thetrend of antioxidant activity of chitosan derivatives THUCACS,TDUCACS,MTDUCACS, and TRUCACSwasmainly affected bythe electron-withdrawing capacity and the detailed explanationshad been discussed in a previous paper.Figure 4 shows the scavenging ability of chitosan and its

derivatives against hydroxyl radicals. The positive control had arelatively weak hydroxyl radical scavenging ability and all chi-tosan derivatives showed stronger scavenging ability than it.According to the graph, the scavenging ability of all sample stillenhancedwith the increase of concentration. For example, the hy-droxyl radical scavenging indices of MTDUCACS were 52.21%,70.12%, 80.21%, 86.52%, and 94.84% when the correspondingconcentrations were 0.1, 0.2, 0.4, 0.8, and 1.6 mg mL−1. Addi-tionally, the hydroxyl radical scavenging rule of chitosan < CACS< BUCACS < BBUCACS < CBUCACS < FBUCACS was dis-tinct in Figure 4. However, the scavenging regularity of deriva-tives THUCACS, TDUCACS, MTDUCACS, and TRUCACSwas not obvious. But it was still TRUCACS that had the best



Figure 3. Superoxide-radical scavenging activity of chitosan and chitosan derivatives.

Figure 4. Hydroxyl -radical scavenging activity of chitosan and chitosan derivatives.

Figure 5. DPPH -radical scavenging activity of chitosan and chitosan derivatives.



Figure 6. The antifungal activity of chitosan and chitosan derivatives against F. oxysporum f. sp. niveum.

Figure 7. The antifungal activity of chitosan and chitosan derivatives against P. asparagus.

scavenging capacity, which was consistent with the previous con-clusion that chitosan derivatives containing triazole possessedbetter antioxidant capacity.[23]

Figure 5 shows the scavenging ability of chitosan and itsderivatives against DPPH radicals and the most rules discussedabove still could be appropriate for the antioxidant activity of allsamples against DPPH radicals. At the low concentrations, thescavenging ability of chitosan derivatives bearing urea groupswas lower than that of positive control. However, when the con-centration was 1.6 mg mL−1, the scavenging rates of FBUCACSand TRUCACS could be more than 90%, which was close to theantioxidant activity of positive control. It suggested that thesederivatives could be used as novel antioxidant biomaterials inproper concentration range.

3.3. Antifungal Activity

Plant mycosis is not only a notorious agricultural productionproblem, but also can produce fungal toxins harmful to animal

and human health. Effective control of fungal diseases is ofgreat significance for food safety and human health. At present,chemical pesticides are the main means of preventing variousagricultural diseases. However, the growing resistance of plantpathogens to fungicides forces researchers screening for eco-friendly antifungal agents.[36,37] In this paper, several biodegrad-able chitosan derivatives were synthesized and their antifungalactivity against Fusarium oxysporum f. sp. niveum, Phomopsis as-paragus, F. oxysporum f. sp. cucumebrium Owen, and Botrytiscinerea was studied. The test results are shown in Figures 6–9.As seen in Figure 6, the antifungal activity of each sample

against F. oxysporum f. sp. niveum was positively correlated withtheir concentration. Meanwhile, the antifungal ability of the fi-nal chitosan derivatives was higher than that of chitosan andCACS. For instance, the inhibitory indices of BUCACS, BBU-CACS, CBUCACS, FBUCACS, THUCACS, TDUCACS, MTD-UCACS, and TRUCACS were 47.41%, 49.73%, 57.62%, 59.68%,41.21%, 52.10%, 62.27%, and 63.49%, while that of chitosan andCACS were only 19.98% and 31.11%. The same phenomenoncould be seen in inhibiting the growth of other fungi. Hence, it



Figure 8. The antifungal activity of chitosan and chitosan derivatives against F. oxysporum f. sp. cucumebrium Owen.

Figure 9. The antifungal activity of chitosan and chitosan derivatives against B. cinerea.

could be concluded that the enhancement of antifungal activityof chitosan derivatives was mainly attributed to urea groups. Ad-ditionally, the antifungal rules, chitosan < CACS < BUCACS <BBUCACS < CBUCACS < FBUCACS and chitosan < CACS <THUCACS < TDUCACS < FBUCACS < TRUCACS, were alsoin accord with the order of the electronegativity of urea groupsin chitosan derivatives. The growth inhibition of fungi mighthave occurred due to the established interaction between theelectrophilic groups with substances on cell wall of microorgan-isms. This resulted in the inhibition of membrane permeability,which hindered the metabolism of cell and led to the death ofmicroorganisms.[38] Therefore, chitosan derivatives with strongelectron-absorbing ability had better antifungal activity.Figure 7 shows the antifungal activity of chitosan and its

derivatives against P. asparagus. All samples showed antifungalability against P. asparagus and the inhibitory indices increasedwith increasing concentration. The samples that exhibited rela-tively weak inhibitory activity were chitosan and CACS, whichfurther explained the important role of urea groups in improv-ing the antifungal activity of chitosan derivatives. Besides, theability of the final derivatives in inhibiting P. asparagus wasstronger than that of F. oxysporum f. sp. niveum. For example, at

1.0 mgmL−1, the inhibition rates of BUCACS, BBUCACS, CBU-CACS, and FBUCACS against F. oxysporum f. sp. niveum were47.41%, 49.73%, 57.62%, and 59.68%, while their inhibition ratesagainst P. asparagus were 64.44%, 69.17%, 70.86%, and 75.51%.Figure 8 shows the antifungal activity of chitosan and its

derivatives against F. oxysporum f. sp. cucumebrium Owen. Therules of the compounds against F. oxysporum f. sp. cucume-brium Owen were similar to that of them against F. oxysporumf. sp. niveum. For instance, chitosan derivatives bearing ureagroups with stronger electronegativity would present better an-tifungal activity. And the antifungal property of chitosan deriva-tives against F. oxysporum f. sp. cucumebrium Owen followed therule of chitosan < CACS < BUCACS < BBUCACS < CBUCACS< FBUCACS.However, the inhibition rates ofMTDUCACSwerebetter than that of TRUCACS, which was different from the pre-vious conclusion. Perhaps, the DSs also had an effect on the an-tifungal activity of the products.Figure 9 shows the antifungal activity of chitosan and its

derivatives against B. cinerea. As exhibited in Figure 9, the mostrules discussed above still could be appropriate for the antifun-gal activity of all samples against B. cinerea. All samples wereconcentration-dependent and the inhibitory property of the eight



CS CACS BUCACS BBUCACS CBUCACS FBUCACS THUCACS TDUCACSMTDUCACSTRUCACS

0

20

40

60

80

100

120

140

1000 µg/mL 500 µg/mL 100 µg/mL 10 µg/mL 1 µg/mL 0 µg/mL

ce

ll v

iab

ilit

y(%

)

Figure 10. The cytotoxicity of chitosan and chitosan derivatives on L929 cells.

final products was better than chitosan and CACS. The antifun-gal activity of chitosan and its derivatives against B. cinerea wasaffected by both DS as well as electron-withdrawing ability of dif-ferent substituted groups. The reasons had been discussed andagreed with earlier.

3.4. Cytotoxicity Analysis

In order to explore the cytotoxicity of chitosan and chitosanderivatives, the cell viability of L929 cells by CCK-8 assay wasstudied and the results are shown in Figure 10. As seen, the cellviabilities of L929 cells treated with chitosan were about 100% atall tested concentrations. Similarly, the cytotoxic effect of CACSwas low and the cell viabilities were more than 90%. However,the cytotoxic effects of BUCACS, BBUCACS, FBUCACS, andTHUCACS were significantly higher. When the concentration is1000 µg mL−1, the cell viabilities of BBUCACS and FBUCACSwere 45.52% and 42.74%. It means that the samples are toxic tonormal cells at these concentrations. As to TDUCACS, MTDU-CACS, and TRUCACS, their cytotoxic effects were very weak andcould promote cell growth in high concentrations. Hence, someof these chitosan derivatives could be considered as ideal bioma-terials.

4. Conclusions

In this paper, the method was studied to synthesize a new classof chitosan derivatives bearing urea groups and their chemicalstructure was characterized by FT-IR, 1HNMR spectroscopy, andelemental analysis. We also explored the antioxidant activity, in-cluding superoxide radical scavenging ability, hydroxyl radicalscavenging ability, and DPPH radical scavenging ability, as wellas the antifungal activity against F. oxysporum f. sp. niveum, P. as-paragus, F. oxysporum f. sp. cucumebrium Owen, and B. cinereaof these derivatives. The activity test results indicated that thechitosan derivatives bearing urea groups exhibited enhanced an-tioxidant and antifungal activity. The active urea groups playeda key role in enhancing bioactivity. Furthermore, their bioactiv-ity could be influenced by the electron-withdrawing capacity. Thechitosan derivatives with higher electron-withdrawing capacitypossessed better bioactivity. In addition, the cytotoxic effect of chi-tosan derivatives was assessed by CCK-8 method. The cytotoxicresults showed that some synthetic chitosan derivatives bearingurea groups had decreased cytotoxic effect. Therefore, these find-ings support a continued investigation on the application of chi-tosan derivatives modified with urea groups in biocompatible an-tifungal agents and antioxidants. Further studies will focus onthe antioxidant and antifungal action patterns of these chitosanderivatives.



AcknowledgementsThe authors thank the Natural Science Foundation of Shandong ProvinceScience and Technology Development Plan (2019GHY112010), NationalNatural Science Foundation of China (41576156), and Natural Sci-ence Foundation of Shandong Province of China (ZR2017BD015 andZR2019BD064).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsantifungal activity, antioxidant activity, chitosan derivatives, urea groups

Received: August 23, 2019Revised: November 19, 2019

Published online:

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