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Reaction Mechanisms, Structural and Physicochemical Properties of Caffeic Acid Grafted Chitosan Synthesized in Ascorbic Acid and Hydroxyl Peroxide Redox System Jun Liu, Huimin Pu, Chong Chen, Yunpeng Liu, Ruyu Bai, Juan Kan, and Chang-Hai Jin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05135 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Journal of Agricultural and Food Chemistry

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Reaction Mechanisms, Structural and Physicochemical Properties of Caffeic

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Acid Grafted Chitosan Synthesized in Ascorbic Acid and Hydroxyl Peroxide

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Redox System

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Jun Liu,*,† Huimin Pu,† Chong Chen,‡ Yunpeng Liu,† Ruyu Bai,† Juan Kan,† and

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Changhai Jin†

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†

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Jiangsu, China

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‡

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*

College of Food Science and Engineering, Yangzhou University, Yangzhou 225127,

Testing Center, Yangzhou University, Yangzhou 225009, Jiangsu, China

Corresponding author: E-mail: [email protected]

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ACS Paragon Plus Environment


Ascorbic acid (AA) and hydroxyl peroxide (H2O2) redox pair

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ABSTRACT:

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induced free radical grafting reaction is a promising approach to conjugate phenolic

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groups with chitosan (CS). In order to reveal the exact mechanisms of AA/H2O2 redox

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pair induced grafting reaction, free radicals generated in AA/H2O2 redox system were

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compared with hydroxyl radical (•OH) produced in Fe2+/H2O2 redox system. Moreover,

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the structural and physicochemical properties caffeic acid grafted CS (CA-g-CS)

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synthesized in these two redox systems were compared. Results showed only

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ascorbate radical (Asc•−) was produced in AA/H2O2 system. The reaction between

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Asc•− and CS produced novel carbon-centered radicals, whereas no new free radicals

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was detected when •OH reacted with CS. Thin layer chromatography, UV–vis, Fourier

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transform infrared and nuclear magnetic resonance spectroscopic analyses all

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confirmed CA was successfully grafted onto CS through Asc•−. However, CA could be

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hardly grafted onto CS via •OH. CA-g-CS synthesized through Asc•− exhibited lower

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thermal stability and crystallinity than the reaction product obtained through •OH. For

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the first time, our results demonstrated the synthesis of CA-g-CS in AA/H2O2 redox

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system was mediated by Asc•− rather than •OH.

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KEYWORDS: Chitosan; Grafted copolymerization; Physicochemical properties;

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Reaction mechanisms; Structural characterization

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■ INTRODUCTION

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Chitosan (CS), the partially deacetylated product of chitin, is a unique cationic

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polysaccharide with many important characters including polyelectrolyte behavior,

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viscosity, mucoadhesivity, metal chelating and film forming abilities.1 Due to its

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biocompatibility, non-toxicity, biodegradability and low immunogenicity, CS has been

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widely used in fields of food, pharmaceutical, tissue and environmental engineering.2

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Owing to the presence of abundant functional groups (amino and hydroxyl groups),

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CS can be easily modified by many chemical methods, such as alkylation, acylation,

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quternization,

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phosphorylation and graft copolymerization.3 Among these techniques, graft

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copolymerization is frequently used to incorporate desired functionalities into CS.4

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Graft copolymerization can afford new types of tailored hybrid materials composed of

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natural polysaccharides and synthetic polymers.5

hydroxyalkylation,

carboxyalkylation,

thiolation,

sulfation,

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Phenolic compounds, the secondary metabolites of plants, have received

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increasing attention in recent years due to their valuable bioactivities.6–7 Based on the

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number of phenol rings and the way they bond, phenolic compounds can be divided

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into five different categories including flavonoids, phenolic acids, tannins, stilbenes

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and lignans.8 In the past decade, different types of phenolic compounds have been

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conjugated with CS through graft copolymerization.9–10 The conjugation of phenolic

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groups with CS not only alters the structure and physicochemical properties (e.g.

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solubility, rheology, crystallinity and thermal stability) of CS, but also greatly

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improves the biological activities of CS (e.g. antioxidant, antimicrobial and antitumor

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activities).11 Moreover, several studies have demonstrated that phenolic grafted CS

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has wide applications in fields of food and agriculture.12–15 For example, nanoparticles

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assembled from gallic acid grafted CS and caseinophosphopeptides were developed to

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deliver (−)-epigallocatechin-3-gallate as novel functional foods.12 Protocatechuic acid

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grafted CS solution was developed as a novel edible coating material for Pleurotus

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eryngii postharvest storage.13 Besides, protocatechuic acid grafted CS film is expected

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to serve as a novel food packaging material.14

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Till now, four types of grafting techniques including carbodiimide based coupling,

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enzyme catalyzed grafting, free radical mediated grafting and electrochemical

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methods have been developed to graft phenolic groups onto CS.16–19 Among these

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techniques, ascorbic acid (AA) and hydroxyl peroxide (H2O2) redox pair induced free

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radical grafting method is very promising due to its simpleness and cost saving.10

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However, the exact mechanisms for the synthesis of phenolic grafted CS through

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AA/H2O2 redox system are still unclear. Curcio et al. proposed that the interaction

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mechanism between AA and H2O2 involved the oxidation of AA by H2O2 with the

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formation of hydroxyl radical (•OH), which initiated the grafting reaction.17 Although

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this hypothesis lacks sufficient evidence, •OH has been accepted as the key factor in

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the synthesis of phenolic grafted CS for a long time.10–11 Recently, ascorbate radical

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(Asc•−) was detected in AA/H2O2 redox pair system by an electron paramagnetic

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resonance (EPR) study.20 However, whether the graft copolymerization between

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phenolic groups and CS is mediated by •OH is still unknown. Therefore, more

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in-depth experiments are needed to reveal the exact mechanisms of AA/H2O2 redox

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system mediated grafting reaction.

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The aim of this study is to demonstrate whether the graft copolymerization is

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mediated by •OH in AA/H2O2 redox system. Considering that Fe2+/H2O2 redox system

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is a classic Fenton reaction for •OH production, the free radicals generated and grafted

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coplymers produced in Fe2+/H2O2 redox system can be used to compare with those of

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AA/H2O2 redox system. Firstly, free radicals generated in Fe2+/H2O2 and AA/H2O2

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redox systems were characterized and quantified by EPR technique. Then, caffeic acid

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(CA), a typical phenolic acid, was grafted onto CS through Fe2+/H2O2 and AA/H2O2

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redox systems, respectively. Finally, the structures and physicochemical properties of

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two kinds of grafted products were characterized and compared by several

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instrumental methods. Our study provides novel information on the synthetic

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mechanisms of phenolic grafted CS through AA/H2O2 redox system.

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■ MATERIALS AND METHODS

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Materials and reagents. CS (deacetylated degree of 90% and molecular weight

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of 974.3 kDa) was purchased from Maclin Biochemical Co. Ltd. (Shanghai, China).

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CA, AA, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), deuterated acetic acid and

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deuterium oxide (D2O) were purchased from Sigma Chemical Co. (MO, USA).

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Folin-Ciocalteu reagent was purchased from Sangon Biotechnology Co. Ltd.

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(Shanghai, China). All other reagents were of analytical grade.

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Characterization and quantification of free radicals produced in two

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different redox systems. Free radicals generated in two different redox systems were

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characterized and quantified by EPR technique. In Fe2+/H2O2 redox system, 50 µL of

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FeSO4 solution was mixed with 50 µL of H2O2 solution followed by addition of 50 µL

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of 50 mM DMPO. Effects of FeSO4 concentration (0.125, 0.250, 0.375, 0.500, 0.625

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and 0.750 mM), H2O2 concentration (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM), solution pH

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value (2.0, 3.0, 4.0, 5.0, 6.0 and 7.0) and reaction time (5, 10, 15, 20, 25, 30, 35, 40,

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45, 50, 55 and 60 min) on free radical production were investigated by single factor

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experiments. In AA/H2O2 redox system, 50 µL of AA solution was mixed with 50 µL

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of H2O2 solution without addition of DMPO. Effects of AA concentration (0.075,

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0.100, 0.125, 0.150, 0.175 and 0.200 M), H2O2 concentration (0.050, 0.075, 0.100,

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0.125, 0.150 and 0.175 M), solution pH value (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0) and

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reaction time (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min) on free radical

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production were tested. EPR measurements were conducted on a Bruker A300

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spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) with detection

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conditions set as follows: microwave frequency, 9.44 GHz; microwave power, 20.017

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mW; center field, 3500 G; sweep width, 100 G; modulation frequency, 100 kHz; and

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modulation amplitude, 1.00 G. According to the literature, the whole height of the

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strongest EPR signal (the second peak in Fe2+/H2O2 redox system and the first peak in

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AA/H2O2 redox system) was taken as the relative intensity of free radical signal.20–22

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Each experiment was carried out in triplicate.

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Synthesis of CA grafted CS (CA-g-CS) in two different redox systems. The

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optimal reaction conditions for free radical production obtained above were further

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used in the synthesis of CA-g-CS. The synthesis of CA-g-CS was based on our

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previously established method with some modifications.23 For AA/H2O2 redox system,

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0.5 g of CS was thoroughly dissolved in 50 mL of 1% acetic acid solution (v/v) in a

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500 mL three-necked round bottom flask. Subsequently, 1.32 g of AA and 1 g of CA

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were added into the reactor. The solution pH value was adjusted to 6.0. A slow stream

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of nitrogen gas was allowed to pass through the reactor for 60 min. Thereafter, 0.375

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mL of 10 M H2O2 solution was added into the reactor to initiate the reaction. The

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reaction was carried out under continuous flow of nitrogen gas for 16 h. For

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Fe2+/H2O2 redox system, 7 mg of FeSO4 and 0.15 mL of 0.5 M H2O2 solution were

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added into the reactor with the solution pH value adjusted to 5.0. All other reaction

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conditions and procedures were the same as that of AA/H2O2 redox system. The

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obtained reaction mixture was dialyzed against distilled water by using a 14,000 Da

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molecular weight cutoff membrane for 72 h and the dialyzate was lyophilized. To

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remove the un-grafted homopolymer of CA, the lyophilized reaction product was

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Soxhlet extracted with ethanol for 12 h, dialyzed against distilled water for 72 h and

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lyophilized. The obtained grafted copolymers in AA/H2O2 and Fe2+/H2O2 redox

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systems were named as CA-g-CS I and CA-g-CS II, respectively. The grafting ratios

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of CA-g-CS I and CA-g-CS II were determined by the Folin–Ciocalteu method using

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CA as the standard.17, 23

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Structural characterization of CA-g-CS I and CA-g-CS II. TLC analysis was

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performed according to the reported method with some modifications.12 Equal volume

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(10 µL) of CS, CA, CA-g-CS I and CA-g-CS II solutions with the same concentration

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(0.1 mg/mL) were spotted on a silica gel plate (50 × 200 mm). The mixed solvent

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system of butyl alcohol-deionized water-acetic acid (50:40:1) was used as the mobile

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phase. The developed plate was finally exposed in an iodine-saturated chamber for

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about 10 min.

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The average molecular weight (Mw) of each sample was determined on Agilent

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1200 system (Agilent Technologies, CA, USA) equipped with a TSK gel G4000

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SWXL column (30 cm × 7.8 mm × 10 µm, Tosoh Corp., Tokyo, Japan) and

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refractive index detector (RID). Sample solution (20 µL) was injected and eluted with

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0.1 M Na2SO4 solution at the flow rate of 0.7 mL/min. The molecular weight of each

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sample was calculated from the calibration curve of T-series dextran standards

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(T-2000, T-1000, T-500, T-200, T-100, T-70, T-40 and T-10).

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For UV-vis spectroscopic analysis, 0.5 mg/mL of each sample dissolved in acetic

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acid aqueous solution (1%, v/v) was scanned on Lambda 35 spectrophotometer

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(Perkin-Elmer Ltd., MA, USA) ranging from 200 to 600 nm.

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For FT-IR spectroscopic analysis, 1 mg of dried sample was ground with about 50

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mg of dried potassium bromide powder and pressed into pellets. Then, Varian 670

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spectrometer (Varian Inc., CA, USA) was used to record the FT-IR spectrum of each

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sample in the frequency range of 4000–400 cm–1.

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For 1H NMR spectroscopic analysis, 5 mg of each sample was dissolved in 0.5

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mL of deuterated acetic acid/D2O (1%, v/v) and detected on AVANCE-600

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spectrometer (Bruker Inc., Karlsruhe, Germany) at 25 °C. The substitution degree of

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CA in the grafted product was calculated from 1H NMR spectra according to the

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previous study.24

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Determination of the physicochemical properties of CA-g-CS I and CA-g-CS

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II. The thermal stability and decomposition of sample were analyzed on Pyris 1 TGA

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instrument (Perkin-Elmer Ltd., MA, USA). Each dried sample (about 5 mg) was

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placed in the TGA furnace and heated from 30 to 800 °C under nitrogen atmosphere

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(flow rate of 10 °C/min).

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The crystallinity of each sample were recorded on Bruker AXS D8 Advance

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X-ray diffractometer (Bruker Inc., Karlsruhe, Germany) equipped with Ni-filtered Cu

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Kα radiation and operated at voltage of 40 kV and current of 40 mA (λ = 0.154 nm).

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The scanning scope (2θ) was 5–80° and the scanning speed was 3°/min.

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S-4800 SEM (Hitachi Ltd., Tokyo, Japan) was used to observe the surface

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morphology of each sample. Before observation, sample was sputtered with gold in a

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S150A putter coater unit (Edwards, England). The clear images of each sample were

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taken at the accelerating voltage of 15 kV with the magnification of 1000× and 6000×,

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respectively.

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Statistical analysis. The Duncan test and one-way analysis of variance

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(ANOVA) were used for multiple comparisons by SPSS 13.0 software package.

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Difference was considered to be statistically significant if p < 0.05.

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■ RESULTS AND DISCUSSION

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Effects of reaction conditions on •OH production in Fe2+/H2O2 redox system.

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In the homogenous Fenton reaction, Fe2+ can react with H2O2 to produce Fe3+ and

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•

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in aqueous solution. Therefore, the spin trap of DMPO is frequently used to produce

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DMPO−•OH adduct, which can be easily detected and quantified by EPR.26 As shown

OH (Equation 1).25 Due to its short lifetime, •OH cannot be directly detected by EPR

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in Figure 1A, EPR spectrum of DMPO−free radical adduct exhibited a quartet with

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relative intensity of 1:2:2:1. Besides, the gyromagnetic constant (g = 2.0099) and

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hyperfine coupling constant (aH = aN = 14.94 G) of formed adduct were consistent

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with those of DMPO−•OH adduct in the literature.25, 27, 28 These results confirmed •OH

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was successfully produced in Fe2+/H2O2 redox system.

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Fe2+ + H2O2 → Fe3+ + •OH + OH−

(1)

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•

OH + Fe2+ → OH− + Fe3+

(2)

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•

OH + H2O2 → H2O + •OOH

(3)

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In order to achieve high •OH production, the reaction conditions of Fe2+/H2O2

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redox system were investigated. As presented in Figure 2A and Figure 2B, •OH

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production was greatly increased when Fe2+ concentration increased from 0.125 to 0.5

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mM or H2O2 concentration increased from 0.5 to 1.5 mM. However, •OH production

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was gradually decreased when Fe2+ and H2O2 concentrations were beyond the optimal

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values. Notably, •OH production obtained at solution pH 5.0 was significantly higher

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than that obtained at other pH values (Figure 2C). In addition, •OH production was

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gradually decreased with the increase of reaction time (Figure 2D). The decreased

198

•

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Equations 2 and 3.29 Based on the results of single factor experiments, the optimal

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reaction conditions for •OH production in Fe2+/H2O2 redox system were determined as

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follows: Fe2+ concentration of 0.5 mM, H2O2 concentration of 1.5 mM, solution pH

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value of 5.0 and reaction time of 5 min.

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OH were probably consumed in the reactions with Fe2+ or H2O2 as presented in

To investigate the reaction mechanism between CS and •OH, CS was added into

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Fe2+/H2O2 redox system with the highest •OH production. As compared with

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Fe2+/H2O2/DMPO reaction system, CS/Fe2+/H2O2/DMPO reaction system exhibited a

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remarkable reduction in •OH production (Figure 1A). The reduced •OH production

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might be due to the •OH scavenging ability of CS. Park et al. supposed that •OH could

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react with amino groups in CS chains to produce stable macromolecule radicals.30

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However, no new free radicals were detected in the CS/Fe2+/H2O2/DMPO reaction

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system, indicating the reaction between •OH and CS should be due to other

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mechanisms. Pasanphan et al. suggested that CS could chelate transition metals (e.g.

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Fe2+), resulting in the decrease of •OH generation through Fenton reaction.31 This

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hypothesis was well supported by our results. The generation of •OH was inhibited

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probably due to the Fe2+-chelating ability of CS.

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Effects of reaction conditions on free radical production in AA/H2O2 redox

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system. AA is a water-soluble ketolactone with two ionizable hydroxyl groups. As

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presented in Scheme 1, the dissociation of 3–OH and 2–OH in AA produces ascorbate

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monoanion (AscH−, pK1 = 4.1) and ascorbate dianion (Asc2−, pK2 = 11.6).32 At

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physiological pH, AscH− is the dominant form. Notably, AscH− is an excellent

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reducing agent and readily undergoes two consecutive one-electron oxidation

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reactions to form first ascorbate radical (Asc•−) and then dehydroascorbic acid (DHA).

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Due to its relatively long lifetime, Asc•− can be easily detected by EPR in aqueous

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solution.26, 33, 34 Therefore, EPR was used to detect free radicals formed in AA/H2O2

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redox system. As shown in Figure 1B, EPR spectrum of AA/H2O2 redox system

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exhibited a duplet with gyromagnetic constant (g) of 2.0057 and hyperfine coupling

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constant (aH) of 1.802 G, which was consistent with the EPR spectrum of Asc•− in the

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literature.20, 26 These indicated that Asc•− was produced in AA/H2O2 redox system. In

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order to confirm whether •OH was also produced in AA/H2O2 system, the •OH spin

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trap of DMPO was added into the reaction solution. Surprisingly, no typical EPR

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signal for DMPO−•OH adduct was detected in AA/H2O2/DMPO system (Figure 1B).

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This indicated that •OH was not produced in AA/H2O2 redox system. Besides, the

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addition of DMPO could not significantly enhance the signal intensity of Asc•−. As

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compared with AA/H2O2 reaction system, the autoxidation of AA in aqueous solution

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yielded much lower signal intensity of Asc•− (approximately 1/100). This suggested

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H2O2 could accelerate the oxidation of AA to generate more Asc•−. Based on above

236

results, the following reaction mechanism between AA and H2O2 was proposed:

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AA + H2O2 → Asc•− + 2H2O

(4)

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To optimize Asc•− production, the reaction conditions of AA/H2O2 redox system

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were investigated by single factor experiments. As shown in Figure 3A and 3B, Asc•−

240

production was continuously increased when AA concentration increased from 0.075

241

to 0.15 M or H2O2 concentration increased from 0.05 to 0.075 M. The enhancement in

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Asc•− production was advantageous to the formation of active sites on CS backbones.

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However, further increasing the concentration of AA or H2O2 yielded a significant

244

decrease in Asc•− production. The optimal molar ratio of AA/H2O2 obtained in our

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study was 2:1, which was significantly higher than the molar ratio of AA/H2O2

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reported previously (0.052).20 The pH value of reaction solution had a profound

247

impact on Asc•− production. The highest Asc•− production was achieved at solution pH

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of 6.0 (Figure 3C). To further evaluate the effect of reaction time on Asc•− production,

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EPR spectra were collected every 5 min. As shown in Figure 3D, Asc•− production

250

was gradually decreased with the extension of reaction time. Arizmendi-Cotero et al.

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also observed that the Asc•− production was reduced when reaction time extended

252

from 15 min to 45 min.20 The decreased Asc•− production might have been converted

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into DHA and diketogulonic acid.37 Thus, the highest Asc•− production could be

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achieved under the following optimal reaction conditions: AA concentration of 0.15

255

M, H2O2 concentration of 0.075 M, solution pH value of 6.0 and reaction time of 5

256

min.

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In order to reveal the reaction mechanism between CS and Asc•−, CS and DPMO

258

were added into AA/H2O2 redox system with the highest Asc•− production. As

259

compared with AA/H2O2/DMPO reaction system, CS/AA/H2O2/DMPO reaction

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system showed a significant reduction in Asc•− production (Figure 1B). Moreover, the

261

EPR spectrum of CS/AA/H2O2/DMPO system presented a novel hextuple split with

262

gyromagnetic constant (g) of 2.0057. The hyperfine coupling constants aH and aN of

263

the hextuple split were 22.90 G and 16.58 G, respectively. Based on the gyromagnetic

264

constant and hyperfine coupling constants, the newly formed hextuple split could be

265

assigned to carbon-centered radicals.20, 35, 36 This result indicated Asc•− could abstract

266

hydrogen atom from CS and produce carbon-centered radicals along CS chains. The

267

formation of carbon-centered radicals was also observed in inulin/AA/H2O2/DMPO

268

reaction system.20 Thus, it could be concluded that the reaction mechanism between

269

Asc•− and CS was mainly based on the hydrogen donating ability of CS, which was

13



270

significantly different from the reaction mechanism between •OH and CS. Notably,

271

the newly formed carbon-centered radicals in CS/AA/H2O2 system could further react

272

with phenolic compound (e.g. CA used in this study) to produce phenolic grafted CS.

273

By contrast, it might be very difficult for phenolic compound to conjugate with CS in

274

Fe2+/H2O2 redox system due to lacking of free radicals sites on CS backbones.

275

Structural characterization of CA-g-CS I and CA-g-CS II. CA-g-CS I and

276

CA-g-CS II were individually synthesized in AA/H2O2 and Fe2+/H2O2 redox systems

277

with the optimal reaction conditions. Folin–Ciocalteu assay showed the grafting ratio

278

of CA-g-CS I was 68.5 mg/g, whereas the grafting ratios of CS and CA-g-CS II were

279

almost zero. This indicated CA could not be successfully grafted onto CS through •OH.

280

In order to further demonstrate CA was grafted onto CS through Asc•− rather than •OH,

281

the structural characterization of CA-g-CS I and CA-g-CS II were compared by

282

different instrumental methods.

283

To verify whether CA was successfully grafted onto CS, different samples

284

including CS, CA, CA-g-CS I and CA-g-CS II were analyzed on TLC. As shown in

285

Figure 4A, CA migrated a long distance and showed a big yellow spot on the silica gel

286

plate. However, CS and two CA-g-CS samples did not show any yellow spot at the

287

corresponding position of CA, confirming the absence of free CA molecules in these

288

samples. This suggested the unreacted free CA molecules had been completely

289

removed by dialysis and soxhlet extraction. For CA-g-CS I, a small yellow spot was

290

observed at the base line of silica gel plate, indicating CA was successfully conjugated

291

with CS through Asc•−. Similar phenomenon was observed on the TLC of other

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phenolic acid grafted CSs, such as gallic acid grafted CS, ferulic acid grafted CS,

293

vanillic acid grafted CS and coumaric acid grafted CS.12,

294

spot could be observed at the base line for CS as well as CA-g-CS II samples. This

295

suggested that CA could be hardly grafted onto CS through •OH. The average

296

molecular weights of CS, CA-g-CS I and CA-g-CS II were determined by SEC

297

(Figure 4B). According to the calibration curve of T-series dextran standards, the

298

average molecular weights of native CS, CA-g-CS I and CA-g-CS II were determined

299

as 974.3, 1049.0 and 898.9 kDa, respectively. As compared with CS, the enhanced

300

molecular weight of CA-g-CS I should be attributed to the grafted CA moieties. By

301

contrast, the relatively lower molecular weight of CA-g-CS II might be due to the

302

degradation effect of •OH on CS backbones.40

38, 39

However, no yellow

303

UV–vis spectrophotometer was subsequently used to characterize CS, CA-g-CS I

304

and CA-g-CS II. As shown in Figure 5A, no UV absorption peak was observed in the

305

range of 250–400 nm for CS solution. By contrast, CA-g-CS I solution exhibited two

306

UV absorption peaks at approximately 290 and 320 nm. These two UV absorption

307

peaks were the same as the absorption maxima of CA solution, indicating the UV

308

absorption peaks of CA-g-CS I were attributed to the grafted CA moieties. Similar

309

results were also observed in CA-g-CS samples synthesized by other researchers.38, 41

310

As compared with CA-g-CS I, CA-g-CS II exhibited much weaker UV absorption

311

band ranging from 250 to 400 nm. However, no obvious absorption peak could be

312

identified from the UV spectrum of CA-g-CS II. Kang et al. suggested that the UV

313

absorption band was resulted from the degradation products of CS by H2O2.42 Thus,

15



314

the UV spectra of CA-g-CS II further confirmed that CA could be hardly grafted onto

315

CS via •OH.

316

The FT-IR spectra of CS, CA, CA-g-CS I and CA-g-CS II are presented in Figure

317

5B. For CS, the broad band at 3390 cm–1 was due to the stretching vibration of O–H

318

and N–H, whereas the band at 2881 cm–1 was typical C–H vibration. Bands at 1655

319

and 1325 cm–1 were assigned to C=O stretching (amide I) and C–N stretching (amide

320

III) of secondary amide for N-acetyl glucosamine groups, respectively. A

321

characteristic band at 1597 cm–1 was attributed to N–H bending of primary amine for

322

glucosamine groups.43 Two bands at 1420 and 1380 cm–1 were assigned to –CH2

323

bending and –CH3 symmetrical deformation, respectively. Besides, three bands at

324

1155, 1094 and 1034 cm–1 were the characteristics of saccharide structure.23 For

325

CA-g-CS I, a new band appeared at 1537 cm–1, which could be assigned to C=C

326

stretching of CA moieties.16 This confirmed CA was successfully grafted onto CS

327

through Asc•−. However, the previously reported band at around 1730 cm–1

328

(corresponding to ester bond) was not observed in CA-g-CS I, which was probably

329

because the ester had been removed by Soxhlet extraction.19 Different from CA-g-CS

330

I, CA-g-CS II showed a much similar FT-IR spectrum to CS, suggesting CA could not

331

be grafted onto CS through •OH. A similar phenomenon was observed by Tian et al.40

332

They suggested that •OH could only break the 1, 4-β-D-glucosidic bond in CS,

333

leading to the depolymerization of CS chains.

334

The 1H NMR spectra of CS, CA, CA-g-CS I and CA-g-CS II are shown in Figure

335

6. The 1H NMR spectrum of CA-g-CS I was somewhat different from that of CS. On

16


Page 16 of 40

Page 17 of 40


336

one hand, CA-g-CS I retained the characteristic proton signals of CS including δ 3.1

337

ppm (H-2), δ 3.6–3.8 ppm (H-3 to H-6) and δ 1.9 ppm (proton of N-acetyl

338

glucosamine residues). On the other hand, CA-g-CS I exhibited several new proton

339

signals in the range of 6.3–7.6 ppm, which could be assigned to the methine protons

340

of CA according to the literature.41, 44, 45 This further confirmed that CA had been

341

successfully grafted onto CS through Asc•−. The substitution degree of CA-g-CS I

342

calculated from 1H NMR spectrum was 7.0%, which matched with the result of

343

Folin–Ciocalteu assay (68.5 mg/g). Different from CA-g-CS I, CA-g-CS II exhibited

344

a similar 1H NMR spectrum to CS, indicating the failure of grafting CA onto CS via

345

•

OH.

346

Physicochemical properties of CA-g-CS I and CA-g-CS II. The thermal

347

behavior of CS, CA, CA-g-CS I and CA-g-CS II was determined by TGA. As

348

illustrated in Figure 7A, the TGA curve of CS exhibited two weight loss stages: the

349

first stage (30–156 °C) was attributed to the loss of water, and the second stage

350

(157–800 °C) was due to the pyrolytic decomposition of CS.46 Similarly, CA-g-CS II

351

and CA both exhibited two stages of weight loss. By contrast, CA-g-CS I showed

352

three weight loss stages. The first stage (30–134 °C) was due to the loss of adsorbed

353

and bound water. The second stage (135–289 °C) was attributed to the degradation of

354

grafted CA moieties. The third stage (290–800 °C) was ascribed to the degradation

355

and combustion of CS backbones. At the end of 800 °C, the residual char yields of CS,

356

CA-g-CS I, CA-g-CS II and CA were 30.38%, 21.75%, 36.61% and 5.89%,

357

respectively. This indicated the decomposition rate of all samples decreased in the

17



358

order CA > CA-g-CS I > CS > CA-g-CS II. The peaks in the first derivative TGA

359

(DTG) thermograms supplied information on the maximum decomposition rate of all

360

samples. As shown in Figure 7B, the maximum decomposition rate appeared at 312,

361

263, 313 and 221 °C for CS, CA-g-CS I, CA-g-CS II and CA, respectively. This result

362

suggested CA-g-CS I had a lower thermal stability than CS. Pasanphan and

363

Chirachanchai suggested that the grafting of phenolic acid onto CS could obstruct the

364

packing pattern of CS chains, resulting in the decrease of thermal stability.19 However,

365

CA-g-CS II showed a higher thermal stability as compared with CS, indicating the

366

thermal stable part of CS was retained during the depolymerization process of by •OH.

367

The crystallinity of CS, CA, CA-g-CS I and CA-g-CS II was measured by XRD.

368

As presented in Figure 7C, CS showed a semi-crystalline structure with two

369

diffraction peaks at 2θ = 11.5° and 20.4°, corresponding to crystal forms I and II,

370

respectively.23 The crystallinity degree of CS was calculated as 28.44%, which was

371

mainly caused by inter- and intramolecular hydrogen bonds.47 By contrast, CA

372

exhibited a crystalline state with several sharp diffraction peaks ranging from 2θ =

373

10° to 50°. After bulky CA was grafted onto CS through Asc•−, the diffraction peaks

374

of CA-g-CS I became broader and weaker as compared with those of CS. In addition,

375

the diffraction peaks of CA-g-CS I shifted to 12.7 and 23.3°. The crystallinity degree

376

of CA-g-CS I was 12.52%, indicating the grafted product was almost in an amorphous

377

state. The reduction in the crystallinity of CA-g-CS I should be related the destruction

378

of inter- and intramolecular hydrogen bonds by Asc•− as well as bulky CA moieties.

379

Similar phenomenon was observed in other phenolic acid grafted CSs.19, 23, 46, 47 As

18


Page 18 of 40

Page 19 of 40


380

compared with CS, the position of diffraction peaks for CA-g-CS II was unchanged.

381

The crystallinity degree of CA-g-CS II decreased to 18.39%, which was much higher

382

than that of CA-g-CS I. Tian et al. suggested the amorphous part of CS was

383

preferentially degraded by •OH and the crystalline part was temporally maintained.40

384

After the amorphous part of CS was peeled off and dissolved in the reaction medium,

385

the crystalline part of CS started to be degraded and the crystallinity degree decreased

386

accordingly.

387

The surface morphology of CS, CA, CA-g-CS I and CA-g-CS II was observed by

388

SEM. As shown in Figure 8, CS were highly packed granules with rough surface,

389

owing to the strong inter- and intramolecular hydrogen bond interaction among

390

molecules. CA was in the crystallographic state with sharp edge. Different from CS

391

and CA, CA-g-CS I was planar flake or stick with relatively smooth surface. This

392

indicated the inter- and intramolecular hydrogen bonds in CS had been greatly

393

decreased during the grafting process, which were also evidenced by TGA and XRD.

394

Similar phenomenon was observed by Wei and Gao.48 Due to the degradation effect of

395

•

396

CA-g-CS II still retained the original spatial structure of CS granules.

OH, many cavities could be observed in the inner structure of CA-g-CS II. However,

397

Combining all above results, it could be concluded that Asc•− and •OH had distinct

398

mechanisms of action on CS granules. As shown in Figure 9, native CS granules were

399

composed of crystalline and amorphous regions. The crystalline region of CS granules

400

was maintained through inter- and intramolecular hydrogen bonds.47 Asc•− could

401

abstract hydrogen atom from CS and produce carbon-centered radicals along CS

19



402

chains, causing inter- and intramolecular hydrogen bonds greatly reduced. Afterwards,

403

bulky CA moieties could be grafted onto CS, which further reduced the hydrogen

404

bond interactions among CS chains. As a result, the original spatial structure of CS

405

granules were seriously destructed by Asc•− as well as CA moieties. Thus, CA-g-CS I

406

exhibited a planar morphology with decreased crystallinity degree and thermal

407

stability. Different from Asc•−, •OH could mainly degrade CS chains by rupturing of 1,

408

4-β-D-glucoside bond.40 The amorphous part of CS could be first degraded by •OH

409

and released from granules, resulting in many cavities in CA-g-CS II. During the

410

degradation process, the inter- and intramolecular hydrogen bonds were seldom

411

affected. Therefore, CA-g-CS II still maintained its original spatial nature. The

412

resultant porous CA-g-CS II granules (probably the crystalline part of CS) were more

413

thermal stable than native CS and CA-g-CS I. Notably, •OH could not create any

414

active site on CS chains. Thus, CA could be hardly grafted onto CS through •OH.

415

Overall, this study demonstrated the graft copolymerization reaction between CS

416

and CA in AA/H2O2 redox system was mediated by Asc•− rather than •OH. Asc•−

417

generated in AA/H2O2 redox system could abstract hydrogen atom from CS and

418

produce carbon-centered radicals along CS chains, causing inter- and intramolecular

419

hydrogen bonds greatly reduced. By contrast, •OH generated in Fe2+/H2O2 redox

420

system could mainly degrade CS chains by rupturing 1, 4-β-D-glucoside bond.

421

However, •OH could not create any active site on CS chains, making the conjugation

422

of CA with CS very difficult. Instrumental analyses further confirmed CA was

423

successfully grafted onto CS through Asc•−, whereas CA could be hardly grafted onto 20


Page 20 of 40

Page 21 of 40


424

CS through •OH.

425

■AUTHOR INFORMATION

426

Corresponding Author

427

*(J.

428

[email protected].

429

Funding

430

This work was supported by Grants-in-Aid for scientific research from the National

431

Natural Science Foundation of China (No. 31571788 and 31101216), Natural Science

432

Foundation of Jiangsu Province (No. BK20151310), Qing Lan Project of Jiangsu

433

Province, and High Level Talent Support Program of Yangzhou University.

434

Notes

435

The authors declare no competing financial interest.

436

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437

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27


13

C NMR.


FIGURE LEGENDS Scheme 1. Scheme for the oxidation of AA through ascorbate radical (Asc•−) to DHA. Figure 1. EPR spectra of •OH in Fe2+/H2O2/DMPO and CS/Fe2+/H2O2 systems (A), and Asc•− in AA/H2O2, AA/H2O2/DMPO, AA only and CS/AA/H2O2/DMPO systems (B). Figure 2. Effects of Fe2+ (A) and H2O2 (B) concentrations, solution pH value (C) and reaction time (D) on •OH production. Figure 3. Effects of AA (A) and H2O2 (B) concentrations, solution pH value (C) and reaction time (D) on Asc•− production. Figure 4. TLC (A) and SEC (B) chromatogram of CS, CA-g-CS I and CA-g-CS II. On TLC chromatogram, a, b, c and d represents CS, CA, CA-g-CS II and CA-g-CS I, respectively. Figure 5. UV (A) and FT-IR (B) spectra of CS, CA, CA-g-CS I and CA-g-CS II. Figure 6. 1H NMR spectra of CS, CA, CA-g-CS I and CA-g-CS II. Figure 7. TGA (A) and DTG (B) curves, and XRD spectra (C) for CS, CA, CA-g-CS I and CA-g-CS II. Figure 8. SEM micrographs of CS (A and B), CA-g-CS I (C and D), CA-g-CS II (E and F) and CA (G and H). Figure 9. The proposed reaction mechanisms of CS with CA through Asc•− and •OH.

28


Page 28 of 40

Page 29 of 40


OH

OH (pK1 = 4.1)

O O

HO

+

−H

OH O

(pK2 = 11.6)

O

HO

−H

O O

HO

+

+

+H

+H

HO

+

−O

OH

−O

−OH

AscH

AA

−e

−

−e

+

2−

Asc

O−

−

−H

OH

OH O O

HO −O

O• Asc•−

Scheme 1

29


−e

O

−

O

HO

O

O DHA


Page 30 of 40

Relative intensity (a.u.)

(A) 2+

Fe /H2O2/DMPO

2+

CS/Fe /H2O2/DMPO

3440

3460

3480

3500

3520

3540

3560

3540

3560

Magnetic field (Gauss)

(B)

Relative intensity (a.u.)

AA/H2O2

AA/H2O2/DMPO

AA only

CS/AA/H2O2/DMPO

3440

3460

3480

3500

3520

Magnetic field (Gauss) Figure 1

30


Page 31 of 40


(A)

(B)

220000

200000

•OH intensity (a.u.)

180000


250000

140000

100000

60000

150000

100000

50000

20000 0.125

0 0.25

0.375

0.5

0.625

0.75

0.5

1

2+

Fe concentration (mM)

(C)

1.5

2

2.5

3

H2O2 concentration (mM)

(D)

2500000

3000000 2500000



2000000

1500000

1000000

500000

2000000 1500000 1000000 500000 0

0 2

3

4

5

6

7

0

10

20

30

40

Reaction time (min)

pH value

Figure 2

31


50

60


(A)

(B)

32000

35000

30000

Asc intensity (a.u.)

28000 26000

25000

20000

•−

•−


30000

24000 22000 20000 0.075

0.1

0.125

0.15

0.175

15000

10000 0.05

0.2

0.075

(D)

150000

0.125

0.15

0.175

150000

120000


120000

90000

90000

60000

•−

60000

•−


0.1

H2O2 concentration (M)

AA concentration (M)

(C)

Page 32 of 40

30000

30000

0

0 3

4

5

6

7

8

0

10

20

30

40

Reaction time (min)

pH value

Figure 3

32


50

60

Page 33 of 40


(A)

a

b

c

d

(B)

Relative intensity

CS

CA-g-CS I

CA-g-CS II 0

2

4

6

Retention time (min) Figure 4

33


8

10


Page 34 of 40

(A) 1.2

Absorbance

1 CS CA-g-CS I CA-g-CS II CA

0.8 0.6 0.4 0.2 0 200

250

300

350

400

450

500

550

600

Waveleghth (nm)

(B) CS

895

1655 1420 1325 1597 1380

2881

1155 1034 1094

3390

Transmittance (%)

CA-g-CS I

895 1320 1537 1380 1648

3380

662

2893

CA-g-CS II

615

1045 1153 1072

1655 1420 1325 1597 1380 1152

2882

895

662

1047 1083

3395 CA

3433

900 974 816 1120 1218

3232

576

1620 1645 1450 1280

4000

3500

3000

2500

2000 -1

Wavenumber (cm )

Figure 5

34


1500

1000

500

Page 35 of 40


H-3 to H-6

OH 6 5

4 O OH

4 6

O

O OH

1 2

3

NH2

OH 5

1 2 NHCOCH3 0.1

3

0.9

H–2

O

H–Ac

CS OH 6 5 RR

4 O OH

R 2

3

4 6

O

O OH

1

H–g

CA-g-CS I

5 RR 3

NH2 0.9

H–b

H-3 to H-6

OH O R

1 2 NHCOCH 3

H–f H–e

H–2 H–Ac 0.1

H–h H-3 to H-6

4 O OH

OH 6 5

4 6

O

O OH

1 2

3

NH2

0.9

OH 5

O

H–2 1

2 NHCOCH3 0.1

3

H–Ac

CA-g-CS II e R =

HO d

f

a

O

g h

c OH

i OH

b

H–g

H–b H–f H–e H–h

CA 10.0

9.0

8.0

7.0

6.0

5.0 4.0 Chemical shift (ppm)

3.0

2.0

Figure 6

35


1.0

0


Page 36 of 40

(A) 100 CS CA-g-CS I CA-g-CS II CA

Weight (%)

80 60 40 20 0 0

100

200

300

400

500

600

700

800

Temperature (°C)

(B) 2

DTG (%/min)

-2 CS CA-g-CS I CA-g-CS II CA

-6 -10

-14 0

100

200

300

400

500

600

Temperature (°C)

36


700

800

Page 37 of 40


(C)

Relative intensity

CS CA-g-CS I CA-g-CS II CA

0

20

40

60

2q (deg)

Figure 7

37


80


(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

Figure 8

38


Page 38 of 40

Page 39 of 40


CA-g-CS I (flake or stick)

CS (granule)

CA-g-CS II (granule) CS chain

Hydrogen bond

Ascorbate radical

Hydroxyl radical

Figure 9

39


CA


Reaction Mechanisms, Structural and Physicochemical Properties of Caffeic Acid Grafted Chitosan Synthesized in Ascorbic Acid and Hydroxyl Peroxide Redox System

Caffeic acid-g-chitosan

Chitosan

Degraded chitosan

Chitosan chain

Hydrogen bond

Caffeic acid

Ascorbate radical produced from ascorbic acid/hydrogen peroxide system 2+

Hydroxyl radical produced from Fe /hydrogen peroxide system

TOC Graphic

40


Page 40 of 40

Reaction Mechanisms, Structural and Physicochemical Properties of

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