Preparation, Characterization, and Biochemical Activities of N-(2

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Preparation, Characterization and Biochemical Activities of N-(2-Carboxyethyl)chitosan from Squid Pens Jun Huang, Hai-Hua Xie, Sheng Hu, Jin-Yan Gong, Cheng-Jun Jiang, Tian Xie, Qing Ge, yuanfeng Wu, Yanli Cui, Shi-Wang Liu, Jianwei Mao, and Lehe Mei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505581n • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Submitted to Journal of Agricultural and Food Chemistry (revised)

2 3

Preparation, Characterization and Biochemical Activities of

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N-(2-Carboxyethyl)chitosan from Squid Pens

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Jun Huang,† Haihua Xie,† Sheng Hu,‡ Tian Xie,† Jinyan Gong,† Chengjun Jiang,† Qing Ge,†

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Yuanfeng Wu,† Shiwang Liu,† Yanli Cui, Jianwei Mao,*† and Lehe Mei *‡

§

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†

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of Zhejiang Province, School of Biological and Chemical Engineering, Zhejiang University

Key Laboratory of Agricultural Products Chemical and Biological Processing Technology

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of Science and Technology, Hangzhou, 310023, PR China.

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‡

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Zhejiang University, Ningbo, 315100, PR China.

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§

Department of Biological and Pharmaceutical Engineering, Ningbo Institute of Technology,

Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China.

14 15

*Corresponding author:

16

Jianwei Mao

17

Tel: +86-571-85070392; Fax: +86-571-85070392.

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E-mail address: [email protected].

19

Lehe Mei

20

Tel: +86-571-87953161; Fax: +86-571-7951982.

21

E-mail address: [email protected].

22

1 ACS Paragon Plus Environment

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ABSTRAT

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Chitosan was prepared by alkaline N-deacetylation of β-chitin from squid pens, and

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N-carboxyethylated derivatives (N-CECS) with different degrees of substitution (DS)

26

were synthesized. The carboxyethylation of the polysaccharide was identified by

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FT-IR, 1H NMR,

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calculated by 1H NMR and elemental analysis. All three N-CECS samples showed

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good water solubility at pH >6.5. The antioxidant properties and bile acid binding

30

capacity of the derivatives were studied in vitro. The highest bile acid-binding

31

capacity of all N-CECS reached 36.9 mg/g, which was 2.6-fold higher than that of

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chitosan. N-CECS showed a stronger scavenging effect on ABTS radical ability, and

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EC50 values were below 2 mg/mL. The scavenging ability of N-CECS against

34

superoxide radicals correlated well with the DS and concentration of N-CECS. These

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results indicated that N-carboxyethylation is a possible approach to prepare chitosan

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derivatives with desirable in vitro biochemical properties.

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KEYWORDS

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β-Chitin; Chitosan; N-(2-Carboxyethyl) chitosan; Antioxidant; Bile acid capacity;

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Characterization

13

C NMR, and XRD analysis. The DS of the derivatives was

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INTRODUCTION

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Chitin, the second most abundant natural polymer after cellulose, is a linear

43

chain consisting of partly deacetylated (1→4)-2-acetamido-2-deoxy-β-D-glucose

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units and classified into α and β types.1,2 α-Chitin has a structure of antiparallel

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chains and exists in the crab, shrimp and lobster. β-Chitin is found in squid pens and

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has parallel chains joined through intrasheet hydrogen bonding.3 The β-chitin has

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higher solubility, higher reactivity and higher affinity toward solvents and swelling

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than α-chitin because it has weaker intermolecular hydrogen bonding.4-6 Squid pens,

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the richest source of β-chitin, have feather-shaped internal structures and are as the

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main waste component of squid. Therefore, squid pens are valuable as a starting

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material for the preparation of β-chitin.

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Chitosan consists of β-(1,4)-2-amino-2-deoxy-D-glucan (glucosamine) and

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β-(1,4)-2-acetamido-2-deoxy-D-glucan (N-acetyl glucosamine) units. It is a cationic

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polysaccharide obtained by N-deacetylation of chitin under alkaline condition or by

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enzymatic hydrolysis in the presence of a chitin deacetylase.1,2,7-9 It has antioxidant

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and antimicrobial activity, biocompatibility, biodegradability, hemostatic activity and

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wound healing properties. Thus, chitosans are used in many areas including food,

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cosmetics, biomedical and pharmaceutical applications.1,8,10-14

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Kurita et al.5 reported that chitosan prepared from β-chitin exhibited higher

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reactivity than α-chitin. However, chitosan is soluble only in acidic aqueous media,

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which has severely limited its applications. To further extend the utilization of

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chitosan, functional groups have been introduced to the hydroxyl and amino groups


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of chitosan to make it more water-soluble. Various chemical modifications, such as

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oxidative

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hydroxypropylation,16 quaternization,17,18 and PEG-grafting19 have been widely

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carried out. Versus ordinary chitosan, chitosan derivatives have improved

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water-solubility over a wide pH range and offered special properties such as

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antioxidant activity, moisture absorption/retention, foaming capacity, as well as

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antitumor and antifungal activities.8,10,15-18

depolymerization,8

carboxymethylation,10

lauroyl

sulfonation,15

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In particular, carboxyalkylation is frequently used to impart better water

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solubility to polysaccharides. Carboxymethyl chitosan is the most investigated

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species and has been the focus of cosmetic applications. 20 Carboxyethylation of

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chitosan, as a regular nuclephilic substitution and elimination reaction, was

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accompanied by the reactions of hydrolysis and dehydrohalogenation of

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halopropionic acids.21,22 However, the information about (2-carboxyethyl) chitosan

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(CECS) is rather limited.

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CECS can be divided into three types based on their different substitution

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positions on the carboxyethyl group: O-CECS, N-CECS, and N,O-CECS. The

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substitution positions and the number of carboxyethyl groups in the polymer chain

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will directly affect the properties of CECS.

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alkaline hydrolysis of O-(2-cyanoethyl) chitosan, and N-CECS can be synthesized

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via Michael-type 1,4-conjugate addition24-26 or alkylation by halocarboxylic acids in

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neutral or mild alkaline conditions.21,22

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Lee et al.23 synthesized O-CECS by

Reactive oxygen species (ROS) have a wide variety of pathological effects,



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including in cancer, cardiovascular disease, diabetes and atherosclerosis.27

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Antioxidants can reduce oxidative damage by scavenging ROS or preventing the

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generation of ROS. The use of synthetic antioxidants such as butylated

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hydroxyanisole, t-butylhydroquinone, and propyl gallate is strictly regulated because

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of their potential health hazards. Recently, the antioxidant activity of chitosan and its

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derivatives have attracted the most attention in replacing synthetic species because of

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their nontoxic nature and natural abundance.8,10,28,29 Furthermore, the extent to which

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carboxyethyl groups have been added to the polymer chain will directly affect the

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properties of CECS. However, to the best of our knowledge, there have been very

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few attempts to evaluate the relationship between the biochemical activities of

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N-CECS and their different degrees of carboxyethyl substitution.

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In this paper, chitosan was prepared by alkaline N-deacetylation of squid pen

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β-chitin and three N-(2-carboxyethyl) chitosans with different degrees of substitution

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(DS) of carboxyethyls were synthesized to investigate the effect of substituting

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groups on the antioxidant activity and bile acid binding capacity. The antioxidant

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activity of N-CECS was investigated by scavenging ABTS radicals (ABTS·+),

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superoxide anion radicals (·O2¯) and reducing power. As well, in vitro bile acid

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binding capacity of the derivatives were evaluated.

103 104

MATERIALS AND METHODS

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Materials. Squid pens were provided by Hangzhou Baokai Biochemical

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Co.,Ltd. (Hangzhou, China). 3-Chloropropionic acid, sodium bicarbonate, sodium


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acetate trihydrate, ferrous sulfate, bile acid (derived from taurocholate), furfual, nitro

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blue

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2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic

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potassium ferricyanide (K3Fe(CN)6), nicotinamide adenine dinucleotide-reduced

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(NADH), and trichloroacetic acid were purchased from Aladdin-reagent Co., Ltd.

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(Shanghai, China). Sodium phosphate monobasic monohydrate, sodium phosphate

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dibasic anhydrous, and acetic acid were purchased from Sangon Biotech Co.,Ltd.

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(Shanghai, China). All other chemicals were of analytical grade and used without

115

further purification. All water used in the extraction and analysis was distilled and

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deionized.

tetrazolium

(NBT),

phenazine acid

methosulfate ammonium

(PMS), salt)(ABTS),

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Preparation of chitosan from squid pens. The preparation of chitosan from

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squid pens was carried out using the method of Huang et al.8 Chitosan with a degree

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of deacetylation of more than 90% was prepared from squid pens with a viscosity

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average-molecular weight of 6.5×105 Da.

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Preparation of N-CECS. N-(2-Carboxyethyl) chitosans (N-CECS) was

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synthesized as described by Skorik et al.21and Kogan et al.22 According to Figure 1,

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chitosan (2g) was dissolved in water (140 mL) containing 3-chloropropionic acid for

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1h. An amount of NaHCO3 equivalent to the 3-chloropropionic was added to the

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dissolved chitosan in batches, which was stirred for additional 30 min to remove the

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excess of CO2 (pH ~6.5). After stirring at 60 ℃ for 6 h, NaHCO3 (6 g) was added,

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and after 1~2 h, the pH of the mixture was adjusted to be 8.5 by adding 10% NaOH.

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When the reaction was complete, the product was filtered and precipitated in ethanol,



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and the precipitant was subsequently dissolved in water (20 mg/mL). The solution

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was acidified to pH 1-2 with concd HCl. The product was then dialyzed using a

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regenerated cellulose membrane (Mw cut-off 3500) against distilled water for 2–3

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days, and lyophilized to obtain purified N-CECS. The sample was kept in a

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refrigerator for 4 °C.

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Characterization of N-CECS derivatives. Fourier transform infrared (FT-IR)

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spectra of chitosan and N-CECS were obtained using a Bruker FT-IR spectrometer

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(Vertex 70, Bruker Optik GmbH, Ettlingen, Germany). The 1H NMR and 13C NMR

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spectra of N-CECS were recorded on a Bruker AVANCE III 500 MHz using D2O as

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solvent operating at 25 °C. Determination of the degree of substitution (DS) was

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estimated by the integrals of δ(-CH2COOH) in the amino group from the following

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equation: DS =

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y + 2z x+ y+z

(1)

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Where x corresponds the mole fraction of no-carboxyethylated glucosamine units, y

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is the mole fraction of mono-carboxyethylated glucosamine units, and z is the mole

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fraction of di-carboxyethylated glucosamine units.

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The X-ray diffraction spectra of the samples in powder form were performed by

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a X-ray scattering diffractometer (X’ Pert PRO, PANalytical, Holland) with Cu Kα

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185 radiation (λ=1.5444) in the range of 5–40° (2θ) at a voltage of 40 kV and a 40

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mA of current. To minimize the influence of the uneven surface of samples on the

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diffraction angle, each N-CECS was pressed into a 0.8 mm thick pellet with 10 MPa

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of pressure.


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The CHN elemental analysis of chitosan and N-CECS was performed on a

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Vario El cube CHNOS Elemental Analyzer (Elementar Analysensysteme GmbH.,

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Hanau, Germany). The degree of deacetylation (DDA) value of chitosan samples

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was calculated from the following formula30:

155 156 157

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DDA % =

6.857 − C/N 1.714

(2)

DS was designated as the average number of carboxyethyl groups on each D-glucosamine. It was calculated as C%/N%:

C [ (CECS) − 5.14] 14 N DS= × 36 DDA%

(3)

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Where the C/N is a percentage ratio of these elements in the derivative and in the

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original chitosan. The constant 5.14 is the C/N value of chitosan at a deacetylation

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degree was 100%. There were two hydrogen atoms on each amino group which

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could be substituted by the carboxyethyl groups. When the hydrogen atoms of the

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–NH2 on chitosan were fully substituted, the DS of N-CECS was 200%.

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Estimation of N-CECS water solubility. The water solubility of N-CECS at

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various pH values was determined by a turbidity measurement.31 An aqueous

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solution of N-CECS (2 mg/mL, pH 1.5) was prepared; NaOH solution (10 M) was

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added to adjust the pH. The transmittance of the solutions at different pH values was

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recorded on a METASH UV-5500PC Spectrophotometer at 600 nm.

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Bile acid binding capacity assay. The bile acid binding capacity of N-CECS

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was investigated in vitro using the method of Muzzarelli et al.32 and Zhao et al.10

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with minor modifications. An aliquot of the sample (1 mL, 10 mg/mL) was mixed

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with 0.4 mL of 5 mg/mL bile acid, and the mixtures were adjusted to a total volume



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of 5 mL with distilled water. The mixtures were incubated for 2 h at 37 °C, and then

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filtered. The resulting samples (1 mL) were mixed with 1 mL 1% (w/v) furfural and

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13 mL 45% (v/v) sulfuric acid. The samples were then incubated for 20 min at 70 °C,

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and the absorbance was measured at 605 nm.

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ABTS radical scavenging ability. The radical scavenging activity of the

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N-CECS towards ABTS was measured based on the method of Prouillac et al.33 and

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Re et al.34 with some modifications. Briefly, the ABTS radical cation (ABTS·+) was

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generated by mixing an ABTS diammonium salt solution (7.4 mM in water) with the

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same volume potassium persulfate aqueous solution (2.6 mM). The mixture was kept

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in the dark overnight until the reaction was complete. The working solution was

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prepared by diluting the ABTS stock solution in PBS solution (0.1 M, pH 7.4) to an

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absorbance of 0.7 at 734 nm. The free radical scavenging activity was assessed by

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mixing the ABTS·+ working solution (0.8 mL) with N-CECS solution (0.2 mL) and

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shaking for 30 seconds. After reaction for 20 min, the scavenging activity was

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calculated according to the following equation:

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Scavenging effect (%) = (1 -

Asample Acontrol

) × 100

(4)

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where Acontrol was the absorbance of the initial concentration of the ABTS·+ (distilled

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water instead of sample), and Asample was the absorbance of the remaining

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concentration of ABTS·+ in the presence of N-CECS. The EC50 value (mg/mL) is the

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effective concentration at which ABTS·+ radicals were scavenged by 50%.

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Superoxide radical scavenging ability. The superoxide scavenging ability of

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N-CECS was assessed by the method of Zhao et al.10 and Yuan et al.35 Each sample


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(2-10 mg/mL, 0.2 mL) were mixed separately with 0.2 mL of NADH (471.6 µM),

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0.2 mL NBT (342.5 µM) in 0.05 M Tris–HCl buffer (pH 8.0). The reaction was

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initiated by adding 0.2 mL of PMS solution (163 µM) to the mixture, then incubated

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at 25 °C for 5 min, and the absorbance at 560 nm was measured against blank

199

samples. In control, sample was substituted with Tris–HCl buffer (0.05 M, pH 8.0).

200

Decreased absorbance of the reaction mixture indicated increased superoxide anion

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scavenging activity.

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Reducing power measurement. The reducing power of N-CECS samples was

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measured as described by Huang et al.36 The reaction mixture contained different

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concentrations of N-CECS samples (0.2 mL), 0.2 M sodium phosphate buffer pH 6.6

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(0.2 mL) and 1% (w/v) potassium ferricyanide (0.2 mL). The mixtures were blended

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well and incubated for 20 min at 50 °C. Then, 10% (w/v) trichloroacetic acid (0.2

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mL) was added to the mixtures and centrifuged at 4000 rpm for 10 min. The

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supernatant (0.5 mL) was mixed with distilled water (0.5 mL) and 0.1% (w/v) ferric

209

chloride solution (0.1 mL). The absorbance values were determined at 700 nm.

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Increased absorbance of the reaction mixture indicated an increased reducing power.

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Statistical analysis. All of the analyses were performed at least in triplicate.

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Each experimental data point represents the mean from three independent

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experiments. The deviation from the mean at the 95% significance level was used for

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statistical analysis.

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RESULT AND DISCUSSION



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Characterization of N-CECS. The preparation process and reaction conditions

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of N-CECS with different DS from squid pens are shown in Figure 1. Infrared

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spectroscopy has been used to determine the structure of the chitosan and

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N-CECS.26,37 Figure 2 displays the FT-IR spectrum of chitosan and N-CECS. Both

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characteristic peaks for chitosan at 3433 and 1078 cm-1 could be attributed to the

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O–H and C–O stretching vibrations, respectively. After carboxyethylation, two new

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bands at 1585 cm−1 and 1407 cm−1 were observed. These were characteristic peaks

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of the stretching vibration of asymmetric and symmetric carboxyl groups,

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respectively.10,38 Therefore, the FT-IR data suggested that carboxyethyl groups were

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present in the derivative.

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To compare the crystalline properties between the different samples, the XRD

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patterns of the chitosan, N-CECS1, N-CECS2 and N-CECS3 are depicted in Figure 3.

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The peak registered near 19.90° highlights the relatively regular crystal lattice of

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chitosan. In comparison with unmodified chitosan, the X-ray patterns of N-CECS

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samples exhibit some changes to both their diffraction angles and peak intensity. The

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peak intensity registered near 19.90° decreased and shifted to a higher 2θ value

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(22.70°). The rigid crystalline structure of chitosan is mostly maintained by the

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intramolecular and intermolecular hydrogen bonds.39

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The introduction of carboxyethyl groups disturbed the crystal domain of

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chitosan because of the high steric hindrance. Thus, the peaks of the crystal

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associated with chitosan were weakened and broadened. The change in these

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characteristic XRD peaks demonstrated that chitosan transited from a crystalline


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structure to an amorphous state through the carboxyethylation process.

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The chemical structure of the N-CECS were further characterized by 1H NMR

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and 13C NMR spectroscopies. Figure 4 and 5 show typical 1H and 13C NMR spectra

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of reaction product. Mono- and di-carboxyethylated amines are distinguishable on

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the spectrum because of the two different chemical shifts of -CH2COONa (2.56 and

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2.80 ppm). The 13C NMR spectrum of N-CECS showed as expected chemical shifts

245

of carbonyl atoms of carboxyethyl (179.54 ppm), α-methylene of carboxyethyl of

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GlcNR2 (33.51 ppm), and α-methylene of carboxyethyl of GlcNHR (36.39 ppm).

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The spectrum was comparable to literature reports.21,22 The 1H NMR and 13C NMR

248

spectrum further proves the substitution of carboxyethyl group onto the amino

249

groups of the chitosan backbone. Skorik et al.22 reported that the chemoselectivity of

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chitosan carboxyalkylation arose from the mild alkaline reaction conditions. This

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makes it possible differentiate the reactivity of -OH and -NH2 groups. Mild condition

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(pH 8-9) facilitated alkylation of chitosan exclusively on the amino groups.

253

The high substitution found on the amino groups could hardly be achieved via

254

Michael addition reaction.24-26,40,41 Moreover, the structure of N-CECS was different

255

from that of N,O-(2-carboxyethyl)chitosan (N,O-CECS)—the methylene of the

256

carboxyethyl group substituted on C-6 also existed in the 1H NMR spectrum of

257

N,O-CECS in addition to the carboxyethylation of amide group.37 The DS values

258

were calculated by 1H NMR and elemental analysis (Table 1). The data showed that

259

the DS values from 1H NMR were similar to that from elemental analysis.

260

Water solubility. Carboxyalkylation of chitosan can greatly improve its water



261

solubility. Figure 6 illustrates the solubility of N-CECS with different DQ values at

262

different pH values. The solubility of all N-CECS samples decreased with increase

263

of pH, and the corresponding pH of N-CECS1, N-CECS2, and N-CECS3 with

264

minimum solubility is 5.6, 4.1 and 3.3, respectively. Further increasing the pH,

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however, led to a drastic increase in solubility and finally plateaued.

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Figure 6 also showed good water solubility of all N-CECS above pH 6.5, but

267

chitosan has poor solubility at pH >6.5. The possible explanation for this

268

phenomenon might be that the solubility of polyampholytes is the lowest at

269

isoelectric point (pI), and all N-CECS samples with amino group and the

270

carboxyethyl group are polyampholytes, and pI of all N-CECS is different. When

271

negative charge in N-CECS structure increases upon introduction of carboxyethyl

272

groups, the corresponding pI of N-CECS decreased. The N-CECS had a more

273

amorphous structure than chitosan, but the pI of N-CECS has positive correlation

274

with the pH of the minimum solubility.

275

In vitro bile acid binding capacity. The in vitro bile acid binding capacities of

276

N-CECS are shown in Table 1. The binding capacity of N-CECS1, N-CECS2, and

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N-CECS3 against bile acid were 27.2 mg/g, 36.9 mg/g, and 0 mg/g (1.9-, 2.6-, and

278

0-fold higher than chitosan), respectively.

279

Previous reports have showed that the charge–charge interactions are the major

280

force between the anionic bile acid and the bile acid sequestrants.36,42 Compared with

281

the native chitosan, the introduction of carboxyethyl groups severely disrupted the

282

inner structure of N-CECS. The ability of the N-CECS to form hydrogen bonds


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declined sharply, that is, the amino groups were activated, which increases the its

284

bile acid-binding capacity. When negative charge in N-CECS structure increased

285

above a certain level upon the introduction of carboxyethyl groups, the significant

286

decrease in the bile acid binding capacity could be caused by ionic repulsion due to

287

its anionic characteristics. Moreover, the solubility improvement of N-CECS also

288

enhances its biological activity including bile acid-binding capacity.43 Thus, N-CECS

289

with enhanced bile acid binding capacity may be used as an ingredient in the

290

pharmaceutical industry to possibly reduce cholesterol and blood pressure.

291

Scavenging effect on ABTS radicals. The ABTS free radicals have been

292

widely used for testing the preliminary radical scavenging capacity of plant extracts

293

or antioxidants.44 ABTS radical scavenging is an electron transfer reaction—the

294

addition of antioxidants to the preformed radical cation ABTS·+ reduces ABTS. This

295

extent of the reaction and the kinetics depend on the antioxidant activity, the

296

concentration of the antioxidant, and the duration of the reaction.33 The scavenging

297

activities of the N-CECS towards ABTS radicals with varying DS and concentrations

298

are shown in Figure 7. Figure 7 showed that the scavenging ability of N-CECS

299

towards ABTS radicals improved as a function of concentrations. The EC50 values of

300

N-CECS1, N-CECS2 and N-CECS3 towards ABTS radical were 1.83 mg/mL, 1.23

301

mg/mL, and 0.33 mg/mL, respectively. The data indicated that the addition of the

302

carboxyethyl groups to chitosan improved its scavenging activities against the ABTS

303

radicals.

304

The hydroxyl group of the chitosan and its derivatives plays an important role



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in scavenging the ABTS radicals.17 The formation of intermolecular and

306

intramolecular hydrogen bonds in chitosan inhibited the reaction of ABTS radicals

307

with the active hydroxyl groups. Compared with chitosan, the inner structure of

308

N-CECS was severely disrupted by the introduction of hydrophilic N-carboxyethyl

309

groups. The ability to form hydrogen bonds declined sharply, that is, more active

310

hydroxyl groups in the N-CECS polymer chains were exposed. As the DS increased,

311

the numbers of active hydroxyl groups in the N-CECS increased. Thus, there were

312

better scavenging abilities by N-CECS samples with a higher DS than N-CECS

313

samples with a lower DS.

314

Cheng et al.16 reported that the scavenging of ABTS radicals by hydroxypropyl

315

chitosan (HPCS) with different DS (0.42–2.25) were below 40% at 10 mg/mL.

316

Huang

317

N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC) with

318

different DS (0.77–1.06) was below 35% at 5 mg/mL. Compared with HTCC and

319

HPCS chitosans, N-CECS was more effective in ABTS radical scavenging activity.

et

al.17

showed

that

the

scavenging

of

ABTS

radicals

by

320

Superoxide radical scavenging activity. The superoxide anion formed in

321

almost all aerobic cells is known to be very harmful to cellular components and is a

322

precursor of more reactive oxidative species such as single oxygen and hydroxyl

323

radicals.44,45 Figure 8 depicts the scavenging activities of the N-CECS with varying

324

DS and concentrations on superoxide radicals. The scavenging effect of all forms of

325

N-CECS toward superoxide radical correlated well with their concentrations. The

326

correlation between the carboxyethyl content (DS) of the N-CECS and their


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superoxide radical scavenging activity was positive. At 10 mg/mL, the scavenging

328

effect on superoxide radicals of N-CECS1, N-CECS2, and N-CECS3 were 42.08%,

329

45.92%, and 50.44%, respectively. The corresponding EC50 values (mg/mL) were

330

13.32, 11.52, and 9.75. Compared with the previously reported EC50 value for

331

chitosan (14.58 mg/mL),10 N-CECS was effective than chitosan at eliminating

332

superoxide radicals.

333

Chitosan has two hydroxyl groups and one amino group in its basic unit.

334

Polysaccharides with scavenging effects towards superoxide anion all have one or

335

more alcohol or phenolic hydroxyl groups. The formation of hydrogen bonds in

336

chitosan inhibited the reaction of superoxide anion with the active hydroxyl groups

337

in the polymer chains.8,10 After etherification, more hydroxyl groups are activated,

338

and this is helpful for enhancing the scavenging effect towards the superoxide

339

radical. As an electron-donating group, the carboxyethyl group may enhance the

340

electron cloud density of active hydroxyl group in N-CECS polymer chain. Thus the

341

electron-donating activity of the N-CECS increased and the scavenging effect on

342

superoxide anion increased when the DS increased from 0.88 to 1.41. We conclude

343

that N-CECS may be an antioxidants based on its ability to scavenge ABTS and

344

superoxide radicals.

345

Reducing power. Figure 9 depicts the reducing power of N-CECS with

346

different DS values using the potassium ferricyanide reduction method. As shown in

347

Figure 9, the reducing power of the N-CECS correlated well with increasing

348

concentration. However, the correlation between the carboxyethyl content (DS) of



349

N-CECS and reducing power was not positive. At 6 mg/mL, the absorbance values

350

of N-CECS1, N-CECS2, and N-CECS3 were 0.049, 0.064, and 0.032, respectively.

351

Huang et al.8 reported that at 2 mg/mL, the reducing power of low molecular weight

352

(LMW) chitosans was more than 0.50 respectively. Compared with LMW chitosans,

353

it seems that N-CECS has poor reducing power. Previous studies have shown direct

354

correlation between the antioxidant activities and the reducing power of certain plant

355

extracts.37,46,47 The reducing properties are generally associated with the presence of

356

reductones that break the free radical chain by donating a hydrogen atom.44 The

357

reducing power of N-CECS suggested that it was likely to contribute insignificantly

358

towards the observed antioxidant effect.

359 360

ACKNOWLEDEGMENTS

361

We thank Professor Yury A. Skorik at St. Petersburg State Chemical Pharmaceutical

362

Academy (Russia) for providing the help in synthesizing the N-CECS. This work

363

was supported by the Science and Technology Department of Zhejiang Province of

364

the People’s Republic of China (No. 2008C14067), the Interdisciplinarity Research

365

Program of Zhejiang University of Science and Technology (No. 2013JC10Y), the

366

International Science & Technology Cooperation Program of China (No.

367

2010DFA34370), and the International S&T Cooperation Program of Zhejiang (No.

368

2013C14012).

369 370

ABBREVIATIONS ABTS, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid);


Page 18 of 37

Page 19 of 37


371

N-Carboxyethylated chitosan (N-CECS); XRD, X-ray diffraction; DS, degrees of

372

substitution; NMR, Nuclear magnetic resonance; FT-IR, Fourier transform infrared.

373 374

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519



FIGURE LEGENDS 520

Figure 1. Preparation of water-soluble N-(2-carboxyethyl)chitosan (N-CECS).

521

Figure 2. FT-IR spectra of the chitosan (CS) and N-CECS. DS value of the N-CECS

522

were 1.41 by 1H NMR.

523

Figure 3. The X-ray diffraction patterns of the chitosan (CS) and N-CECS with

524

different DS. DS values of N-CECS1, N-CECS2, and N-CECS3 calculated by 1H

525

NMR were 0.88, 1.06 and 1.41, respectively.

526

Figure 4. 500 MHz 1H NMR spectroscopy (δ, ppm) of N-CECS2 solution in D2O:

527

2.56 (-CH2COONa of GlcNHR, Ha), 2.80 (-CH2COONa of GlcNR2, Hb), 3.08 (H-2

528

of GlcNH2), 3.14 (H-2 of GlcNHR), 4.90 ( H-1 of GlcNHR), and 5.04 (H-1 of

529

GlcNR2). DS of the N-CECS2 were 1.06 by 1H NMR.

530

Figure 5. 125 MHz

531

33.51 (CH2COONa of GlcNR2), 36.39 (CH2COONa of GlcNHR), 44.69 (NCH2 of

532

GlcNHR), 49.03 (NCH2 of GlcNHR2), 60.29-62.53 (C-2, C-6), 65.51-76.82 (C-3,

533

C-4, C-5), 96.8-98.93 (C-1), 179.54 (COONa). DS value of the N-CECS3 were 1.41

534

by 1H NMR.

535

Figure 6. The pH dependence of N-CECS water solubility with different DS. DS

536

values of N-CECS1, N-CECS2, and N-CECS3 calculated by 1H NMR were 0.88,

537

1.06, and 1.41, respectively.

538

Figure 7. The scavenging effect of N-CECS with different DS towards ABTS

539

radicals. DS values of N-CECS1, N-CECS2, and N-CECS3 calculated by 1H NMR

540

were 0.88, 1.06 and 1.41, respectively.

13

C NMR (δ, ppm) spectroscopy of N-CECS3 in D2O at 25℃.


Page 26 of 37

Page 27 of 37


541

Figure 8. Scavenging effect of N-CECS with different DS towards superoxide

542

radicals. DS values of N-CECS1, N-CECS2, and N-CECS3 calculated by 1H NMR

543

were 0.88, 1.06 and 1.41, respectively.

544

Figure 9. Reducing power of N-CECS with different DS. DS values of N-CECS1,

545

N-CECS2, and N-CECS3 calculated by 1H NMR were 0.88, 1.06 and 1.41,

546

respectively.



Page 28 of 37

547

Table 1. The reaction conditions, DS, and bile acid binding capacity of

548

N-CECS. Product

549 550 551 552 553 554 555

C%

H%

C/N

DSb

x:y:zc

DSd

BAe

7.72

40.24

6.56

5.20

–

–

–

14.0

6

5.08

37.92

6.10

7.45

0.91

0.16:0.80:0.04

0.88

27.2

5

15

4.78

37.98

5.94

7.94

1.12

0.07:1.06:1.41

1.06

36.9

10

120

4.10

37.74

5.33

9.22

1.62

0.00:0.59:0.41

1.41

0

Reagent

Reaction

ratioa

time (h)

Chitosan

–

–

N-CECS1

5

N-CECS2

N-CECS3

N%

a

The molar ratio of 3-chloropropionic acid to D-glucosamine. DS was calculated per D-glucosamine residue of chitosan by elemental analysis. c See Figure 1. d DS was calculated per D-glucosamine residue of chitosan by 1H NMR spectrum, DS= (y+2z)/( x+y+z). e Bile acid (BA) binding capacity (mg/g) b


Page 29 of 37


556 557

Figure 1．．

558

559 560



561 562

Figure 2．

563

564 565 566


Page 30 of 37

Page 31 of 37


567 568

Figure 3．．

569

570 571



572 573

Figure 4．

574

575 576


Page 32 of 37

Page 33 of 37


577 578

Figure 5．

579

580 581



582 583

Figure 6．

584

585 586


Page 34 of 37

Page 35 of 37


587 588

Figure 7．

589

590 591



592 593

Figure 8．

594

595 596


Page 36 of 37

Page 37 of 37


597 598

Figure 9．

599

600 601



602 603

TOC Graphic

604 605 606


Page 38 of 37

Preparation, Characterization, and Biochemical Activities of N-(2

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