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Carbohydrate Polymers 174 (2017) 999–1017

Contents lists available at ScienceDirect

Carbohydrate Polymers

j ourna l ho me page: www.elsev ier .com/ locate /carbpol

eview

ynthesis, characterization, bioactivity and potential application ofhenolic acid grafted chitosan: A review

un Liu ∗, Huimin Pu, Shuang Liu, Juan Kan, Changhai Jinollege of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China

r t i c l e i n f o

rticle history:eceived 10 February 2017eceived in revised form 3 July 2017ccepted 6 July 2017vailable online 12 July 2017

eywords:pplication

a b s t r a c t

In recent years, increasing attention has been paid to the grafting of phenolic acid onto chitosan inorder to enhance the bioactivity and widen the application of chitosan. Here, we present a comprehen-sive overview on the recent advances of phenolic acid grafted chitosan (phenolic acid-g-chitosan) inmany aspects, including the synthetic method, structural characterization, biological activity, physico-chemical property and potential application. In general, four kinds of techniques including carbodiimidebased coupling, enzyme catalyzed grafting, free radical mediated grafting and electrochemical meth-ods are frequently used for the synthesis of phenolic acid-g-chitosan. The structural characterization of

ioactivityhitosanraft copolymerhenolic acidtructure

phenolic acid-g-chitosan can be determined by several instrumental methods. The physicochemical prop-erties of chitosan are greatly altered after grafting. As compared with chitosan, phenolic acid-g-chitosanexhibits enhanced antioxidant, antimicrobial, antitumor, anti-allergic, anti-inflammatory, anti-diabeticand acetylcholinesterase inhibitory activities. Notably, phenolic acid-g-chitosan shows potential appli-cations in many fields as coating agent, packing material, encapsulation agent and bioadsorbent.

© 2017 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10002. Synthetic methods and reaction mechanisms of phenolic acid-g-chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1000

2.1. Carbodiimide based chemical coupling method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10002.2. Enzyme catalyzed grafting method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10012.3. Free radical mediated grafting method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10022.4. Electrochemical method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1003

3. Structural characterization of phenolic acid-g-chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10033.1. Thin-layer chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10033.2. High performance liquid chromatography (HPLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10033.3. UV–vis spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10053.4. Fourier-transform infrared (FT-IR) spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10053.5. Nuclear magnetic resonance (NMR) spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10053.6. Scanning electron microscope (SEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006

4. Physicochemical properties of phenolic acid-g-chitosan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10064.1. Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10064.2. Thermal stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10064.3. Crystallinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10064.4. Rheological property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006

5. Biological activities of phenolic acid-g-chitosan . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1. Non-cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author.E-mail address: [email protected] (J. Liu).

ttp://dx.doi.org/10.1016/j.carbpol.2017.07.014144-8617/© 2017 Elsevier Ltd. All rights reserved.

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5.3. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10085.4. Antitumor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10095.5. Anti-allergic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10095.6. Anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10095.7. Anti-diabetic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10105.8. Acetylcholinesterase inhibitory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010

6. Applications of phenolic acid-g-chitosan in food technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10106.1. Coating solutions for food preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10106.2. Films as food packing materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10126.3. Nanoparticles for delivery of functional dietary ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10136.4. Hydrogels as encapsulation agents and novel biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10136.5. Microspheres as bioadsorbents and encapsulation agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1013

7. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014

. . . . . .

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References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Chitin is the second most abundant polysaccharide mainlyxtracted from the exoskeleton of sea creatures, such as crayfish,obster, prawns, crab and shrimp. Chitosan is the deacety-ated product of chitin obtained by alkaline treatment (Kumar,

uzzarelli, Muzzarelli, Sashiwa, & Domb, 2004). Chitosan is also unique cationic polysaccharide with many special featuresncluding viscosity, polyelectrolyte behavior, mucoadhesivity, filmorming and metal chelating ability (Pillai, Paul, & Sharma, 2009;hukla, Mishra, Arotiba, & Mamba, 2013). Besides, due to its non-oxic, non-antigenic, biocompatible and biodegradable properties,hitosan has wide applications in food, tissue engineering, phar-aceutical, textile, agriculture, water treatment and cosmetics

ndustries (Aider, 2010; Ngo et al., 2015; Rinaudo, 2006; Suh &atthew, 2000; Vakili et al., 2014). However, the use of chitosan

s greatly limited by its poor solubility in water, because chi-osan is only soluble in acidic media. Chemical modification isn efficient approach to improve the water solubility as well asndow some new characteristics for chitosan (Alves & Mano, 2008;rashanth & Tharanathan, 2007). Among various chemical modi-cation methods, graft copolymerization reaction has been mostidely used (Jayakumar, Prabaharan, Reis, & Mano, 2005; Thakur

Thakur, 2014). Graft copolymerization can introduce desiredhysicochemical and biological properties into chitosan, makingolecular design possible.In order to improve the physicochemical and biological prop-

rties of chitosan, increasing efforts have been made to grafthenolics, especially phenolic acids onto chitosan through graftopolymerization since 2008 (Bozic, Gorgieva, & Kokol, 2012;urcio et al., 2009; Pasanphan & Chirachanchai, 2008). Pheno-

ics are the most abundant secondary metabolites widespreadhroughout the plant kingdom, such as fruit, vegetables, cereals,lives, dry legumes, cocoa, tea, coffee, and wine (Oroian & Escriche,015). Phenolics are an essential part of human diet with variousaluable biological activities, including antioxidant, antimicrobial,nti-diabetic, anti-inflammatory, anticancer and metabolic regu-ation properties (Ali Asgar, 2013; Roleira et al., 2015; Shahidi &mbigaipalan, 2015). More than 8000 naturally occurring chem-

cal compounds belong to the category of “phenolics”, whichhare a common structural feature, i.e. an aromatic ring bear-ng at least one hydroxyl substituent (Costa et al., 2015). Basedn the number of phenol rings and the way they bond, phe-olics can be divided into five different categories includinghenolic acids, flavonoids, tannins, stilbenes and lignans (Stalikas,
007). As an important category of phenolics, naturally occur-ing phenolic acids usually possess one carboxylic acid groupith two distinctive carbon frameworks, i.e. the hydroxybenzoic
nd hydroxycinnamic structures (Khadem & Marles, 2010; Lochab,

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1014

Shukla, & Varma, 2014). Accordingly, phenolic acids can be fur-ther divided into two subcategories: hydroxybenzoic acids andhydroxycinnamic acids (Table 1). In the past decade, phenolic acidshave been demonstrated to possess potent antioxidant, antimi-crobial, anticancer, antiviral, anti-inflammatory, antimutagenic,antirheumatic, antipyretic, antiseptic, anthelmintic, neuroprotec-tive and hepatoprotective activities (Heleno, Martins, Queiroz, &Ferreira, 2015).

Nowadays, four kinds of grafting techniques including car-bodiimide based coupling, enzyme catalyzed grafting, free radicalmediated grafting and electrochemical methods are frequentlyused for the synthesis of phenolic acid grafted chitosan (pheno-lic acid-g-chitosan) (Bozic, Strancar, & Kokol, 2013; Kim et al.,2010; Liu, Lu, Kan, & Jin, 2013b; Pasanphan & Chirachanchai,2008). The introduction of phenolic groups onto chitosan back-bone not only greatly alters the structural and physicochemicalproperties (solubility, thermal stability, crystallinity and rheologi-cal properties) of chitosan, but also remarkably increases biologicalactivities (antioxidant, antimicrobial, antitumor, anti-allergic, anti-inflammatory, anti-diabetic and acetylcholinesterase inhibitoryactivity) of chitosan (Cirillo et al., 2016; Liu, Lu, Kan, Wen, & Jin,2014a; Oliver, Vittorio, Cirillo, & Boyer, 2016). Notably, pheno-lic acid-g-chitosan shows potential applications in many fieldsof food technology, which can be used as functional foods, foodcoating agents, food packing materials, encapsulation agents forfunctional dietary ingredients, and bioadsorbents for the treatmentof iron overload diseases (Aljawish, Chevalot, Jasniewski, Scher, &Muniglia, 2015; Hu et al., 2015; Liu et al., 2015b, 2016a). Therefore,the functional phenolic acid-g-chitosan needs to be extensivelyresearched and utilized in the future. This review systematicallyfocuses on the recent advances of phenolic acid-g-chitosan in manyaspects, including the synthetic method, structural characteriza-tion, physicochemical property, biological activity with detailedfunctional mechanism, potential applications in food technologyand future perspectives.

2. Synthetic methods and reaction mechanisms of phenolicacid-g-chitosan

2.1. Carbodiimide based chemical coupling method

Carbodiimide based chemical coupling reagents, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and dicy-clohexylcarbodiimide (DCC) have been widely used for thesynthesis of phenolic acid-g-chitosan (Eom, Senevirathne, & Kim,
2012; Pasanphan & Chirachanchai, 2008; Schreiber, Bozell, Hayes,& Zivanovic, 2013; Xie, Hu, Wang, & Zeng, 2014). Carbodiimidemediated grafting method is highly efficient and requires onlymild reaction conditions. The grafting reactions usually proceed

J. Liu et al. / Carbohydrate Polymers 174 (2017) 999–1017 1001

Table 1Classification and structures of the prominent naturally occurring phenolic acids.

Phenolic acidsa

Hydroxybenzoic acids Hydroxycinnamic acids

p-Hydroxybenzoic acid Salicylic acid o-Coumaric acid m-Coumaric acid

Vanillic acid Protocatechuic acid p-Coumaric acid Caffeic acid

�-Resorcylic acid Gentisic acid Ferulic acid Sinapic acid

orts (

iuwdbfiacKSW

n(rrngb2a((tstgtc&

rcodt(2crr

Gallic acid Syringic acid

a The classification and structures of phenolic acids are based on the previous rep

n acidic aqueous solutions which significantly enhance the sol-bility of chitosan reactant. In addition, the coupling reagents asell as urea derivatives can be easily removed after reaction byialysis (Guo, Anderson, Bozell, & Zivanovic, 2016). Till now, car-odiimide mediated coupling method has been successfully usedor the syntheses of several kinds of phenolic acid-g-chitosans,ncluding gallic acid-g-chitosan, caffeic acid-g-chitosan, feruliccid-g-chitosan, protocatechuic acid-g-chitosan, salicylic acid-g-hitosan and chlorogenic acid-g-chitosan (Aytekin, Morimura, &ida, 2011; Liu et al., 2016b; Pasanphan & Chirachanchai, 2008;chreiber et al., 2013; Woranuch & Yoksan, 2013; Wei et al., 2010;ei & Gao, 2016).The grafting reaction mechanism for the synthesis of phe-

olic acid-g-chitosan by using EDC and N-hydroxysuccinimideNHS) coupling reagents is shown in Fig. 1a. EDC can firstlyeact with carboxyl groups on phenolic acid to form amine-eactive O-acylisourea (intermediate 1). If this intermediate doesot encounter an amine, it will hydrolyze and regenerate carboxylroups. In the presence of NHS, amine-reactive O-acylisourea cane further converted to amine-reactive NHS ester (intermediate). Finally, the amine-reactive NHS ester can react with aminond hydroxyl groups on chitosan to yield phenolic acid-g-chitosanLiu et al., 2016b; Pasanphan & Chirachanchai, 2008). Xie et al.2014) also introduced EDC and 1-hydroxybenzotriazole (HOBt) ashe coupling reagents for the synthesis of gallic acid-g-chitosan,uggesting the grafting efficiency could be greatly enhanced inhe presence of HOBt. Moreover, some phenolic acids can be alsorafted onto chitosan derivatives, such as partially quaternized chi-osan and N, O-carboxymethyl chitosan by carbodiimide mediatedhemical crosslinking methods (Alexandrova, Obukhova, Domnina,

Topchiev, 1999; Yu et al., 2011).In general, the grafting efficiency (also expressed as grafting

atio) of phenolic acid-g-chitosan is greatly affected by reactiononditions. Many researchers suggested that the grafting ratiof phenolic acid-g-chitosan depended on the depolymerizationegree and molecular weight of chitosan, initial molar ratio of chi-osan/phenolic acid, reaction pH, reaction temperature and timeAytekin et al., 2011; Schreiber et al., 2013; Woranuch & Yoksan,
013). Recently, effect of solvent composition on the grafting effi-iency of gallic acid-g-chitosan via EDC mediated coupling waseported by Guo et al. (2016). Results showed that ethanol in theeaction solvent significantly inhibited the grafting reaction by
Chlorogenic acid

Khadem & Marles, 2010; Lochab et al., 2014).

acting as a reactant. As compared with other synthetic methods,carbodiimide based chemical coupling method posesses the high-est grafting ratio (Xie et al., 2014). Thus, this method is still the mostefficient approach for the synthesis of phenolic acid-g-chitosantill now. However, it should be noted that this method usuallyrequires a large amount of chemical crosslinking reagents, such asEDC, DCC, NHS and HOBt. These chemical crosslinking reagents aremuch expensive and environmentally disadvantageous, which maycause adverse impacts on human body when the grafted productsare used in food and pharmaceutical industries. Therefore, it is stillessential to explore other cheaper and safer grafting methods.

2.2. Enzyme catalyzed grafting method

In recent years, enzyme catalyzed grafting method has beenadopted for the synthesis of phenolic acid-g-chitosan (Aljawishet al., 2015). There are several advantages for the use of enzymecatalyzed grafting method. First, enzymes frequently used for thesynthesis of phenolic acid-g-chitosan are much cheaper than car-bodiimide. Enzymes could be continuously utilized if immobilizedenzyme technology is adopted (Vittorio et al., 2016). Moreover,the high selectivity and specificity of enzymes eliminate the needfor full protection and deprotection steps involved in chemicalcoupling reaction. Finally, enzyme catalyzed grafting method ismuch safer and more eco-friendly than chemical coupling method(Aljawish et al., 2014b). However, the enzyme catalyzed graftingmethod also has some limitations. Enzyme usually catalyzes theoxidation of phenol hydroxyl groups on phenolic acids into o-quinones, which will eventually decrease the biological activities,e.g. antioxidant and antimicrobial activities of the grafted products(Aljawish et al., 2012).

Till now, several kinds of phenolic acids including gallic acid,caffeic acid, ferulic acid, gentisuric acid, p-hydroxybenzoic acid,protocatechuic acid, chlorogenic acid and p-coumaric acid havebeen successfully grafted onto chitosan by enzyme mediated graft-ing method (Aljawish et al., 2012, 2014a; Bozic et al., 2012, 2013;Brzonova, Steiner, Zankel, Nyanhongo, & Guebitz, 2011; Chao, Shyu,Lin, & Mi, 2004; Itzincab-Mej&a, López-Luna, Gimeno, Shirai, &
Bárzana, 2013; Kumar, Smith, & Payne, 1999; Liu et al., 2014c;Vachoud, Chen, Payne, & Vazquez-Duhalt, 2001; Vartiainen, Rättö,Lantto, Nättinen, & Hurme, 2008; Wu, Chen, Wallace, Vazquez-Duhalt, & Payne, 2001; Yang et al., 2016b). In enzyme catalyzed


by EDF h et a

g1a(22arvfitNqwaBai

Fig. 1. Reaction mechanisms for the synthesis of phenolic acid-g-chitosanigure is adapted from previous reports (Pasanphan & Chirachanchai, 2008; Aljawis

rafting reactions, three kinds of enzymes including laccase (EC.10.3.2), peroxidase (EC 1.11.1.x), and tyrosinase (EC 1.14.18.1)re frequently used for the synthesis of phenolic acid-g-chitosanAljawish et al., 2012, 2014b; Bozic et al., 2012, 2013; Chao et al.,004; Itzincab-Mejía et al., 2013; Kumar et al., 1999; Liu et al.,014c; Vachoud et al., 2001). As shown in Fig. 1b, these enzymesre capable to catalyze phenol oxidation to generate o-quinoneeactive intermediate. The formed o-quinone intermediate areery reactive species and powerful electrophiles, which can beurther involved in a number of non-enzymatic reactions includ-ng covalent coupling to form dimers, oligomers and polymershrough C C, C O and C N bonds (Aljawish et al., 2015; Kudanga,yanhongo, Guebitz, & Burton, 2011). It is proposed that the o-uinone intermediate can undergo two different types of reactionith chitosan to form either Schiff-bases (C N) or Michael type

dducts (C NH) via covalent linkages (Hollmann & Arends, 2012).ozic et al. (2012) suggested that the oxidized pheonlic acid couldlso crosslink with chitosan by hydrogen bonds and electrostaticnteractions. In addition, the oligomer-forming reactions could lead

C/NHS chemical coupling (a) and enzyme catalyzed grafting (b) methods.l., 2015).

to a complex mixture of monomers or oligomers grafted onto chi-tosan. Therefore, the exact grafting patterns between chitosan andphenolic acid by enzyme catalysis are still unclear till now. Inaddition, the reaction conditions (such as the depolymerizationdegree and molecular weight of chitosan, initial molar ratio of chi-tosan/phenolic acid, reaction pH, temperature and time as wellas type of enzyme) on the grafting efficiency of phenolic acid-g-chitosan have not been studied yet.

2.3. Free radical mediated grafting method

Grafting of phenolic acid onto chitosan can be also achieved viafree radical mediated reaction. In recent years, a number of freeradical initiator systems including ceric ammonium nitrate (Nuneset al., 2013), potassium persulfate (Chiang, Li, Chao, & Su, 2011;
Shiu et al., 2010) and ascorbic acid (Vc)/hydrogen peroxide (H2O2)redox pair (Curcio et al., 2009) have been developed to initiate thegrafting copolymerization reaction between phenolic acid and chi-tosan. Among these free radical initiator systems, Vc/H2O2 redox

Polym

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air system has been most widely used. There are several advan-ages for Vc/H2O2 redox pair induced grafting method. Firstly, therafting reagents (Vc and H2O2) used to generate free radicals areuch cheaper than carbodiimide and enzymes. Secondly, Vc/H2O2

edox pair is less toxic than carbodiimide reagents. Finally, it isossible to perform the reaction at room temperature to avoid theegradation and oxidation of phenolics. Therefore, Vc/H2O2 redoxair induced grafting method is more economical and eco-friendlyhan carbodiimide based chemical coupling method. Moreover, freeadical induced grafting method can also avoid the oxidation ofhenolic acid which inevitably occurs during the enzyme catalyzedrocess.

Curcio et al. (2009) introduced Vc/H2O2 redox pair system intohe synthesis of gallic acid-g-chitosan for the first time. However,he grafting ratio of phenolic acid-g-chitosan prepared by Vc/H2O2nder atmospheric air was not very ideal (only 7 mg/g). In order to

ncrease the grafting ratio, Liu et al. (2013b) improved this methody optimizing the reaction conditions. It was found that the graft-

ng ratio of phenolic acid-g-chitosan was affected by many factorsncluding the concentrations of chitosan, phenolic acid, Vc and

2O2 as well as reaction time. Moreover, the grafting ratio of phe-olic acid-g-chitosan could be greatly enhanced to 128.3 mg/g byarrying out the reaction under nitrogen gas flow.

The possible reaction mechanism for the synthesis of pheno-ic acid-g-chitosan by Vc/H2O2 redox pair system was proposedCurcio et al., 2009; Liu et al., 2013b). As shown in Fig. 2, Vc existss the di-acid form (AscH2) in acetic acid solution, and can reactith H2O2 to generate hydroxyl radical (•OH) as well as reso-ance stabilized tricarbonyl ascorbate free radical (AscH•). As AscH•

as a pK = −0.86, it is not protonated and is present in the formf semi-dehydroascorbate radical (Asc•−) (Valko, Rhodes, Moncol,zakovic, & Mazur, 2006). Afterwards, the previously formed •OHan abstract hydrogen atom from chitosan with the formation ofhitosan macro radicals. Finally, phenolic acid monomers which aren the close vicinity of reaction sites become acceptors of chitosan

acro radicals, and thus phenolic acid-g-chitosan is synthesized.ill now, many kinds of phenolic acid-g-chitosans, such as galliccid-g-chitosan, caffeic acid-g-chitosan, ferulic acid-g-chitosan, p-ydroxybenzoic acid-g-chitosan, protocatechuic acid-g-chitosan,-coumaric acid-g-chitosan, vanillic acid-g-chitosan have beenynthesized by using Vc/H2O2 redox pair system (Chatterjee et al.,015, 2016; Eom et al., 2016; Lee, Woo, Ahn, & Je, 2014b; Liu et al.,013b, 2015b). Although •OH has been generally accepted as therimary free radical to induce the grafting process, no direct evi-ence has been proved by researchers till now. Thus, more in-depthxperiments should be carried out to further reveal the exact reac-ion mechanism of free radical induced grafting method. Beside,nly one study has focused on the reaction conditions of Vc/H2O2edox pair induced grafting process (Liu et al., 2013b). In order tobtain phenolic acid-g-chitosan with ideal grafting ratio, variouseaction conditions including the concentrations of Vc, H2O2, chi-osan and phenolic acid, reaction pH, temperature and time can beptimized in future.

.4. Electrochemical method

As chitosan is pH-responsive and possesses good film-formingbility, it can be cast, spun and electrodeposited (Suginta,hunkaewla, & Schulte, 2013). On the other hand, phenolics canccept, store and donate electrons under biologically relevant con-itions (Faure et al., 2013). Based on the characteristics of chitosan
nd phenolics, the synthesis of phenolic-g-chitosan can be achievedy electrochemical method. To date, most studies on the elec-rochemical synthesis of phenolic-g-chitosan have focused on therafting of catecholic reactants (especially catechol) onto chitosan
ers 174 (2017) 999–1017 1003

film (Gray et al., 2011; Kim et al., 2010, 2014; Ryu, Hong, & Lee,2015).

As presented in Fig. 3, catechol-g-chitosan is electrochemicallysynthesized in three steps. Firstly, chitosan film is electrodepositedby immersing a gold electrode into chitosan solution and retainingthe current density constantly with a cathodic voltage. Subse-quently, the chitosan-coated electrode is immersed in catecholsolution with an anodic voltage applied to oxidate catechol into o-quinone. Finally, the oxidized o-quinone is covalently linked withthe amine groups in chitosan backbone via Michael-type additionand Schiff base reactions (Gray et al., 2011; Kim et al., 2010). Itshould be noted that the reaction mechanisms of enzyme catalyzedgrafting and electrochemical methods are much similar. Therefore,electrochemical method is also a safe and eco-friendly approachfor the synthesis of phenolic-g-chitosan. However, this method cancause the oxidation the phenolic acids. As a result, the biologi-cal activities of phenolic-g-chitosan are greatly reduced. Besides,the electrochemical method usually requires the specific appara-tus. Till now, only caffeic acid has been successfully grafted ontochitosan by electrochemical method (Gray et al., 2011). Accordingto the grafting mechanism, it is speculated that many other phe-nolic acids bearing catecholic moieties, such as protocatechuic acidand chlorogenic acid can be also grafted onto chitosan via electro-chemical method.

3. Structural characterization of phenolic acid-g-chitosan

3.1. Thin-layer chromatography (TLC)

To confirm the successful grafting of phenolic acid onto chitosanbackbone, it is essential to characterize the conjugate by differentanalytic methods. Although less used, TLC has been adopted forphenolic analysis since 1960s. TLC is often used to simultaneouslyanalyze several phenolic samples (Stalikas, 2007). In addition, TLCcan be also used to distinguish phenolic acid-g-chitosan from freephenolic acid. Hu et al. (2015) separated gallic acid and gallic acid-g-chitosan on silica gel plates by using butyl alcohol–water–aceticacid (50:40:1) as the expansion agent. Similar procedure was devel-oped out by Cho, Kim, Ahn, and Je (2011b) and Chatterjee et al.(2015) using chloroform–ethyl acetate–acetic acid (50:50:1) asthe expansion agent. After being exposed to iodine vapors or 30%H2SO4, colored spots with different migration distances appearedon the plate. In general, free phenolic acid migrants a distance onthe plate, whereas phenolic acid-g-chitosan remains at the baseline. In future, two-dimensional TLC technology can be employedto separate different phenolic acid-g-chitosans in the same system.

3.2. High performance liquid chromatography (HPLC)

Apart from TLC, HPLC has also been used to distinguish pheno-lic acid-g-chitosan from free phenolic acid. HPLC analyis is oftencarried out after the dialysis of phenolic acid-g-chitosan’s reactionsolution. As compared with TLC, the wide range of commerciallyavailable columns and the possibility of combining two or morecolumns in a switching mode enable the widespread applicationof HPLC in the analysis of phenolic acid-g-chitosan. HPLC systemsequipped with reversed-phase C18 column and UV detector havebeen used to distinguish phenolic acid-g-chitosan from free phe-nolic acid (Curcio et al., 2009; Liu et al., 2013b).

Besides, HPLC can be also applied to determine the molecularweight of phenolic acid-g-chitosan. Size-exclusion chromatogra-
phy (SEC) or gel permeation chromatography (GPC) coupled withrefractive index detector (RID) are often adopted (Bozic et al., 2012,2013; Kim, Ryu, Lee, & Lee, 2013; Wu et al., 2016a). The molecularweight of phenolic acid-g-chitosan depends on the grafting ratio as


O O

HONH2

OH OH

O ONHCOCH3

O

O ONH

O OO

ROR

O

OR

OR

R

O

R

O

OH

O O

O ONH

O O

O ONHCOCH3

O

OHA

OO O

OHO

O

HO O2O2 OH O

OO

HO

AscAscHAscorbic acid (AscH2)

OH

Chitosan macro radicals

Phenolic acid

Fig. 2. Proposed mechanisms for the synthesis of phenolic acid-g-chitosan by Vc/H2O2 redox pair mediated grafting method. (a) Procedure for the generation of •OH viaVc/H2O2 redox pair. (b) Conjugation of phenolic acid onto chitosan by •OH mediated graft copolymerization reaction.

Figure is adapted from previous report (Liu et al., 2013b).

Fig. 3. The synthetic mechanisms of catechol-g-chitosan by electrochemical method.Figure is adapted from previous report (Kim et al., 2010).

Polym

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ell as grafting method used. In general, phenolic acid-g-chitosanynthesized by carbodiimide based coupling and enzymatic cat-lyzed grafting methods often has higher molecular weight thanhitosan, whereas phenolic acid-g-chitosan synthesized by freeadical induced grafting reaction shows lower molecular weighthan chitosan (Aljawish et al., 2014b; Bozic et al., 2012; Wei & Gao,016; Wu et al., 2016b).

.3. UV–vis spectroscopy

UV–vis spectrophotometric technique is often used for the iden-ification and quantification of phenolic acid-g-chitosan. Althoughhitosan has no absorption peak in the range of 240–400 nm, phe-olic acid-g-chitosan usually exhibits characteristic UV absorptionand due to grafted phenolic moieties. The characteristic absorp-ion peak of phenolic acid-g-chitosan depends on the type ofhenolic acid grafted. For example, gallic acid has an absorptionaximum at 262 nm, which is due to the �-system of benzene

ing. However, this absorption shifts to 272 nm when gallic acids grafted onto chitosan by EDC coupling or free radical mediatedrafting method (Liu et al., 2013b; Xie et al., 2014). The red shift ofallic acid-g-chitosan can be attributed to the less amount of energyequired for the n–�* and �–�* transition due to the covalent link-ge between phenolic acid and chitosan (Bozic et al., 2012). Similared shifts are also observed in caffeic acid-g-chitosan and feruliccid-g-chitosan (Bozic et al., 2012; Liu, Wen, Lu, Kan, & Jin, 2014b).

On the other hand, UV–vis spectrophotometric analysis is oftenpplied to determine the grafting ratio of phenolic acid-g-chitosan.olin–Ciocalteu reagent is frequently used to determine the totalhenolic content in phenolic acid-g-chitosan at 760 nm (Curciot al., 2009; Liu, Jia, Kan, & Jin, 2013a). Based on this method, therafting ratio of phenolic acid-g-chitosan can be calculated. In othertudies, some researchers also determined the grafting ratio byeasuring UV absorbance intensity of phenolic acid-g-chitosan in

thanol-HCl solution system at 320 nm (Aytekin et al., 2011). Inddition, ninhydrin assay has also been applied to analyze aminoroup content in phenolic acid-g-chitosan (Chatterjee et al., 2015;oranuch & Yoksan, 2013). This method is based on the reaction

f the primary amino groups of chitosan with ninhydrin reagent toorm chitosan–ninhydrin complexes, which have a deep blue colorith absorbance maximum at 570 nm (Sabnis & Block, 2000). How-

ver, ninhydrin assay ignores the formation of ester bond betweenydroxyl groups (at C-3 and C-6 positions) of chitosan and carboxylroups of phenolic acids. Thus, the grafting ratio of phenolic acid--chitosan determined by ninhydrin assay is normally lower thanolin–Ciocalteu assay. By contrast, Folin–Ciocalteu assay is still theost suitable method to determine the grafting ratio of phenolic

cid-g-chitosan.

.4. Fourier-transform infrared (FT-IR) spectroscopy

FT-IR spectroscopic technique is useful to confirm the successfulrafting of phenolic acid onto chitosan. Moreover, FT-IR spec-roscopy can provide structural information on the grafting patternf phenolic acid-g-chitosan. In FT-IR spectrum, chitosan usuallyhows the absorption bands at around 1650, 1550 and 1320 cm−1,orresponding to C O stretching (amide I), N H bending (amideI) and C N stretching (amide III) of the residual N-acetyl groups,espectively. The band at 1595 cm−1 is due to the N H bending ofhe primary amine (Lim & Hudson, 2004). The bands at 1420 and380 cm−1 can be attributed to CH2 bending and CH3 symmet-ical deformation, respectively (Zhang et al., 2009). As compared
ith chitosan, the N H bending of the primary amine (1595 cm−1)
n the phenolic acid-g-chitosan often decreases or even disappears,hich is due to the change of primary amine into secondary amine

n chitosan chains (Spizzirri et al., 2009). Moreover, some typi-

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cal C C stretching bands on aromatic ring can be observed within1450–1600 cm−1 (Chatterjee et al., 2015; Liu et al., 2013b, 2014b).For phenolic acid-g-chitosan synthesized by carbodiimide basedcoupling method and free radical induced grafting reaction, a newband usually appears at around 1730 cm−1, which can be attributedC O stretching of esters (Liu et al., 2013b, 2016b; Pasanphan &Chirachanchai, 2008). This new band indicates the formation ofester bond between hydroxyl groups of chitosan and carboxylgroups of phenolic acid. However, phenolic acid-g-chitosan syn-thesized by enzymatic catalyzed grafting method normally exhibitsa different FT-IR spectrum due to different grafting mechanisms(Aljawish et al., 2012; Bozic et al., 2012; Yang et al., 2016b). In somestudy, FT-IR spectrometer with attenuated total reflectance (ATR)model has been demonstrated as an effective approach to analyzethe structural characterization of phenolic acid-g-chitosan, whichcan be better utilized in future (Bozic et al., 2013).

3.5. Nuclear magnetic resonance (NMR) spectroscopy

The successful synthesis of phenolic acid-g-chitosan can be alsoconfirmed by NMR spectroscopy. 1H NMR spectrum of chitosan hasbeen assigned by several researchers (Aytekin et al., 2011; Cho, Kim,Ahn, & Je, 2011a; Liu et al., 2013b; Pasanphan & Chirachanchai,2008; Xie et al., 2014). Chitosan usually exhibits a single peak atround 2.9 ppm (H-2), multiple peaks at 3.3–3.7 ppm (H-3, H-4, H-5,H-6) and a small single peak at 4.4 ppm (H-1). The single peak atround 1.8 ppm represents protons of N-acetyl glucosamine units.As compared with chitosan, phenolic acid-g-chitosan often showsnew peaks in the range of 6.0–8.0 ppm, which can be assigned tothe protons of phenolic acid moieties (Eom et al., 2012). In gen-eral, the 1H NMR spectra of phenolic acid-g-chitosan synthesizedby carbodiimide based coupling method are much similar to con-jugate prepared by free radical induced grafting reaction (Liu et al.,2015b). This indicates that these two grafting methods might havesimilar reaction mechanism. However, phenolic acid-g-chitosansynthesized by the enzymatic catalyzed grafting method showsa totally different 1H NMR spectrum. Bozic et al. (2012) synthe-sized caffeic acid-g-chitosan by laccase catalyzed polymerizationand observed numerous peaks between 2.8 and 3.4 ppm, whichcould be attributed to oligomers/polymers of caffeic acid moieties.However, no peak was observed between 6.0 and 8.0 ppm. Notably,almost all the researchers have ignored the fact that free phenolicacid also exhibits peaks between 6 and 8 ppm. The observed novelpeaks on the 1H NMR spectra of final products cannot guarantee thesuccessful grafting of phenolic acid with chitosan, especially whenfree phenolic acid is presented in the final products. Therefore, 1HNMR must be coupled with other technologies such as TLC, HPLCand FT-IR spectrometer to confirm the successful grafting.

The structure of phenolic acid-g-chitosan can be further char-acterized by solid-state 13C NMR. According to the literature, thesolid-state 13C NMR spectrum of chitosan usually shows signals atabout 57.8, 61.1, 75.5, 83.2 and 105.3 ppm, which can be assignedto C-2, C-6, C-3 and C-5, C-4 and C-1, respectively. Signals at around174.2 ppm (C O) and 23.7 ppm (–CH3) are due to the carbonyl andmethyl groups of N-acetylglucosamine residues, respectively (Liuet al., 2013b, 2014b; Pasanphan & Chirachanchai, 2008). As com-pared with chitosan, the C-1, C-3 to C-5 and C-6 peaks of phenolicacid-g-chitosan often become enlarged and overlap the neighbor-ing C-4 and C-2 peaks (Liu et al., 2013b, 2014b; Pasanphan &Chirachanchai, 2008). Moreover, additional peaks can be observedin the range of 100−170 ppm, attributing to the carbons of phe-nolic acid moieties (Liu et al., 2013b, 2014b, 2016b; Pasanphan &
Chirachanchai, 2008). These peaks’ intensity often increases withthe increase of grafting ratio.
Although the above mentioned methods are useful to provethe successful grafting of phenolic acid onto chitosan, the detailed

1 Polym

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tructures of different phenolic acid-g-chitosans are still unclear.o better elucidate the fine chemical structure of phenolic acid--chitosan, two-dimensional (2D) NMR technologies includingH–1H correlated spectroscopy (COSY), 1H–13C heteronuclearingle-quantum coherence (HSQC), 1H–13C heteronuclear mul-iple quantum coherence (HMQC), and 1H–13C heteronuclear

ultiple-bond correlation (HMBC), nuclear Overhauser effect spec-roscopy (NOESY) and rotating frame Overhauser enhancementpectroscopy (ROESY) can be applied in future.

.6. Scanning electron microscope (SEM)

Recently, some researchers have applied SEM to observe theurface features of different phenolic acid-g-chitosans, such asrotocatechuic acid-g-chitosan, chlorogenic acid-g-chitosan andinnamic acid-g-chitosan (Liu et al., 2016b; Wei & Gao, 2016; Yangt al., 2016b). As reported, apparent differences can be observedetween chitosan and phenolic acid-g-chitosan. Chitosan usuallyhows a flaky nature with smooth surface due to strong hydro-en bond interaction among molecules. By contrast, the surfaces ofrotocatechuic acid-g-chitosan and cinnamic acid-g-chitosan areuch rough, which may be due to the grafted phenolic acid moi-

ties (Liu et al., 2016b; Yang et al., 2016b). However, Wei and Gao2016) observed that chitosan changed from a flaky nature with rel-tively rough surface to needle-like or stick-like morphology afterrafting with chlorogenic acid. These results indicate the surfaceeatures of phenolic acid-g-chitosan are closely related to the char-cteristic of native chitosan and the type of phenolic acid grafted.xcept for SEM, atomic force microscope (AFM) is another usefulool to investigate the conformation of phenolic acid-g-chitosanDu, Wang, Yuan, Wei, & Hu, 2009). Unfortunately, no report hasocused on the conformation of phenolic acid-g-chitosan by usingFM till now.

. Physicochemical properties of phenolic acid-g-chitosan

.1. Solubility

One of the main goals for the synthesis of phenolic acid-g-hitosan is to increase the water solubility of chitosan. Therefore,he solubility of phenolic acid-g-chitosan is often an impor-ant concern of most researchers. In general, chitosan usuallyhows improved water solubility after grafting with phenolic acidAljawish et al., 2014b; Chatterjee et al., 2015; Kumar et al., 1999;iu et al., 2013b; Pasanphan & Chirachanchai, 2008; Schreiber et al.,013; Woranuch & Yoksan, 2013). Some researchers further sug-est that the water solubility of phenolic acid-g-chitosan is relatedo the grafting ratio and reaction conditions (Kumar et al., 1999;chreiber et al., 2013). In addition, the freeze-drying step involvedn the preparation process of phenolic acid-g-chitosan may initiatehe packing structure, resulting in a decrease in water solubilityPasanphan & Chirachanchai, 2008). Recently, Guo et al. (2016)evealed that the solubility of gallic acid-g-chitosan was associatedith ethanol concentration used during the EDC/NHS grafting pro-

ess. Gallic acid-g-chitosan forms a stable colloidal dispersion wheneionized water is used as the solvent. However, when ethanoloncentration in the solvent increases to 50% or higher, redun-ant ethanol can prevent the hydration of chitosan and thus reducevailability of chitosan’s active sites, resulting in densely precipi-ated gallic acid-g-chitosan from the reaction solution.

.2. Thermal stability

Thermal stability and degradation of phenolic acid-g-chitosanre important issues from both scientific and industrial perspec-ives. Differential scanning calorimetry (DSC) is a conventional

ers 174 (2017) 999–1017

thermal analysis method widely used to characterize the phasetransition of chitosan derivatives (Kittur, Prashanth, Sankar, &Tharanathan, 2002). Different thermal behaviors between nativechitosan and phenolic acid-g-chitosan can be evaluated by DSCtechnique (Curcio et al., 2009; Lee et al., 2014b; Liu et al., 2013b;Woranuch & Yoksan, 2013). Liu et al. (2013b) observed the intro-duction of gallic acid moieties onto chitosan backbone caused thedecrease in the water holding capacity of gallic acid-g-chitosan,which was probably due to the decrease in the intermolecularhydrogen bond interactions. A similar result was obtained byWoranuch and Yoksan (2013).

Apart from DSC, thermo gravimetric analysis (TGA) has beenused as a popular technique to study the thermal stability anddecomposition of copolymers. The first derivative TGA (DTG) sup-plies information on the relative volatilization and decompositionrates of grafted copolymer. The peak in the DTG curve repre-sents the maximum rate of mass loss (Liu et al., 2013d). Till now,the thermal stabilities of many phenolic acid-g-chitosans includ-ing caffeic acid-g-chitosan, ferulic acid-g-chitosan, p-coumaricacid-g-chitosan, protocatechuic acid-g-chitosan and chlorogenicacid-g-chitosan have been evaluated by TGA (Aljawish et al.,2012; Liu et al., 2014b, 2016b; Pasanphan & Chirachanchai, 2008;Woranuch & Yoksan, 2013; Yang et al., 2016b). Dehydration anddecomposition have been generally considered as two major pro-cesses associated with the degradation of chitosan and phenolicacid-g-chitosan. Results showed that the introduction of the func-tional group could obstruct chitosan chain packing, resulting inthe decrease of thermal stability. Moreover, the thermal stabil-ity of phenolic acid-g-chitosan depends on the kind of phenolicacid grafted as well as the grafting ratio (Itzincab-Mejía et al.,2013; Liu et al., 2014b, 2016b; Pasanphan & Chirachanchai, 2008;Woranuch & Yoksan, 2013). However, Yang et al. (2016b) reportedthat the conjugation of cinnamic acid and its derivatives onto chi-tosan by enzyme catalyzed grafting method resulted in the increaseof chitosan’s thermal stability. This suggests the thermal stabilityof phenolic acid-g-chitosan is also related with the grafting methodused.

4.3. Crystallinity

Crystallinity is an important physicochemical property of chi-tosan and its derivatives. The crystallinity degree of chitosan isassociated with its deacetylation degree. Crystallinity is maximalfor both chitin and fully deacetylated chitosan. However, minimalcrystallinity is achieved at intermediate deacetylation degrees ofchitosan (S enel & McClure, 2004). The crystallinity of chitosan andphenolic acid-g-chitosan can be determined by X-ray diffraction(XRD). The diffraction pattern of chitosan normally shows a char-acteristic peak at around 20◦ (2�), corresponding to its crystal formII (Liu et al., 2013b). As compared with chitosan, phenolic acid-g-chitosan often exhibits relatively broader and weaker peaks on XRDspectra, indicating the grafting process could reduce the crystal-lization of chitosan to some extent (Liu et al., 2013b, 2014b, 2016b;Pasanphan & Chirachanchai, 2008; Wei & Gao, 2016; Woranuch& Yoksan, 2013; Yang et al., 2016b). This is probably due to thatthe inter- and intra-molecular hydrogen bonds inside chitosanmolecules have been greatly reduced after grafting.

4.4. Rheological property

The rheological property of phenolic acid-g-chitosan is animportant index reflecting its potential ability to be utilized in the
fields of food coating, film-forming and drug delivery. Till now, onlyfew researchers have focused on the rheological properties of phe-nolic acid-g-chitosan (Aljawish et al., 2014b; Liu et al., 2014c; Wei& Gao, 2016; Wu et al., 2016b; Xie et al., 2014). All these researchers

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eported that the viscosity of phenolic acid-g-chitosan solution wasuch higher than that of native chitosan solution. The higher vis-

osity of phenolic acid-g-chitosan solution should be due to theormation of physical crosslink resulting from hydrophobic inter-ctions between phenolic side chains (Aljawish et al., 2014b). Xiet al. (2014) and Wu et al. (2016b) observed that the viscosity ofhe gallic acid-g-chitosan solution decreased with the increase ofrafting ratio, although the synthetic methods used were totally dif-erent. Wu et al. (2016b) explained that gallic acid-g-chitosan withower grafting ratio had larger number of interactions and entan-lements between the macromolecules, which in turn enhancedhe dynamic module of the solution. Xie et al. (2014) and Wu et al.2016b) further suggested gallic acid-g-chitosan solution displayed

shear-thinning behavior of the typical non-Newtonian fluid. How-ver, an absolutely opposite phenomenon was observed by Wei andao (2016) in chlorogenic acid-g-chitosan solution. This indicates

he rheological property of phenolic acid-g-chitosan solution maye also affected by the kind of phenolic acid grafted.

. Biological activities of phenolic acid-g-chitosan

.1. Non-cytotoxicity

In order to confirm the safety of phenolic acid-g-chitosan, sev-ral conjugates have been tested by the cell viability assay. Resultshowed phenolic acid-g-chitosan did not exert any obvious toxicffect on normal cells, such as PC12 cells (Cho et al., 2011a), humanmbilical vein endothelial cells (HUVEC) (Aljawish et al., 2014c;im, Kim, Ryu, & Lee, 2015; Soliman, Zhang, Merle, Cerruti, &arralet, 2014), RAW264.7 macrophage cells (Ahn et al., 2016; Chot al., 2011b; Lee et al., 2014b; Ngo et al., 2011b), SW1353 cells (Ngot al., 2011a), RBL-2H3 mast cells (Vo, Ngo, & Kim, 2012a), Changiver cells (Senevirathne et al., 2012) and human mesenchymaltem cells (MSCs) (Aljawish, Muniglia, & Chevalot, 2016a). How-ver, further in vivo studies including short and long-term feedingxperiments are needed to fully understand the significance ofnteractions between phenolic acid-g-chitosan and relevant biolog-cal systems as well as evaluate the potential human health risksfter exposing to phenolic acid-g-chitosan.

.2. Antioxidant activity

Phenolic compounds are of considerable interest due to potentntioxidant properties and other potential health benifits. Thentioxidant potential of phenolic compounds mainly dependsn the number and arrangement of hydroxyl groups (Shahidi &mbigaipalan, 2015). It has been demonstrated that the antioxi-ant activity of chitosan can be greatly enhanced by grafting withhenolic compounds (Hu & Luo, 2016). Till now, the antioxidantctivity of phenolic acid-g-chitosan has been evaluated by numer-us in vitro assays, including oxygen-radical absorbance capacity,educing power, metal chelating ability, lipid peroxidation inhi-ition, �-carotene-linoleic acid assay, superoxide anion, hydroxyl,,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and,2-diphenyl-1- picrylhydrazyl (DPPH) radicals scavenging assaysCasettari, Gennari, Angelino, Ninfali, & Castagnino, 2012; Eomt al., 2012; Lee et al., 2014b; Liu et al., 2014b, 2016b; Schreibert al., 2013; Wei & Gao, 2016; Xie et al., 2014).

In general, many factors may affect the antioxidant activ-ty of phenolic acid-g-chitosan, such as the type of phenoliccid grafted, molecular weight and deacetylation degree of chi-
osan, the grafting method used and the grafting ratio. Eom et al.2012) compared the antioxidant activity of eight different kindsf phenolic acid (p-hydroxybenzoic acid, coumaric acid, proto-atechuic acid, caffeic acid, vanillic acid, ferulic acid, syringic
ers 174 (2017) 999–1017 1007

acid and sinapinic acid) conjugated chitooligosaccharides, anddemonstrated protocatechuic acid-g-chitooligosaccharide and caf-feic acid-g-chitooligosaccharide had higher antioxidant activitythan other phenolic acid-g-chitooligosaccharides. Casettari et al.(2012) further suggested the antioxidant activity of phenolic acid-g-chitosan strongly depended on the number of hydroxyl groupsin the grafted phenolic moieties. Similar phenomenon was alsoobserved by other investigators (Lee et al., 2014b; Liu et al., 2014b).Aytekin et al. (2011) found the antioxidant activity in vitro of caffeicacid-g-chitosan varied with the reaction conditions (pH, reactiontemperature, reaction time and molar ratio of chitosan/caffeic acid)and the molecular weight of chitosan. Similar results were observedby Schreiber et al. (2013) in gallic acid-g-chitosan. Recently, Wuet al. (2016c) found that the antioxidant activity of gallic acid-g-chitosan increased with the increase of grafting ratio and with thedecrease of grafted copolymer’s molecular weight. Among abovementioned factors, the type of phenolic acid grafted and graftingratio should be the most important aspects affecting the antioxi-dant activity of phenolic acid-g-chitosan. However, the antioxidantactivity of different kinds of phenolic acid-g-chitosans is rarelycompared (Eom et al., 2012). The relationship between graftingratio and antioxidant activity of phenolic acid-g-chitosan has notbeen well established till now. Moreover, researchers normallyprefer to adopt the free radical scavenging assays to evaluate theantioxidant activity in vitro of phenolic acid-g-chitosan. In future,different antioxidant methods should be used to fully evaluate thein vitro antioxidant activity of phenolic acid-g-chitosan.

The antioxidant activity of some phenolic acid-g-chitosanderivatives has also been evaluated by some researchers. Antiox-idant activity of gallic acid-g-quaternized chitosan was reportedby Alexandrova et al. (1999). The synthesized chitosan deriva-tives exhibited much higher peroxidation inhibition effect andH2O2 scavenging activity than native chitosan as well as gallicacid. Yu et al. (2011) synthesized gallic acid-g-N,O-carboxymethylchitosan and measured its DPPH and ABTS radical scavenging activ-ities. Liu, Lu, Kan, Tang, and Jin (2013c) further compared theantioxidant activity of three different kinds of phenolic acid-g-N,O-carboxymethyl chitosans, and found the antioxidant activityof grafted copolymers decreased in the order of gallic acid-g-N,O-carboxymethyl chitosan > caffeic acid-g-N,O-carboxymethylchitosan > ferulic acid-g-chitosan. Ren, Li, Dong, Feng, and Guo(2013) found gallic acid and caffeic acid grafted quaternized chi-tosan exerted improved antioxidant activity than chitosan as wellas quaternized chitosan. These results suggest the antioxidantactivity of chitosan derivatives can be enhanced by grafting withphenolic acid.

The antioxidant activity in vitro of phenolic acid-g-chitosan canbe also evaluated at a cellular level. Pasanphan, Buettner, andChirachanchai (2010) revealed the possible antioxidant mechanismof gallic acid-g-chitosan by using electron paramagnetic resonancetechnique, and attributed the antioxidant activity of gallic acid-g-chitosan to the H-atom transferring ability of galloyl groups.Ngo et al. (2011a, 2011b) evaluated the antioxidant activity of gal-lic acid-g-chitooligosaccharides in mouse macrophage RAW264.7cells and human chondrosarcoma SW1353 cells. Results showedthat gallic acid-g-chitooligosaccharides could inhibit oxidation ofcellular macromolecules, such as DNA, protein and lipid in cells,whereas enhance the expression levels of superoxide dismutase(SOD) and glutathione (GSH). Similar results were reported by Cho,Kim, and Je, (2011c) and Senevirathne et al. (2012) using RAW264.7macrophage cells and Chang liver cells, respectively. In future, morein-depth experiments should be carried out to evaluate the in vitro
antioxidant activity of phenolic acid-g-chitosan through differentcellular models.
Although many reports on the in vitro antioxidant activity ofphenolic acid-g-chitosan, little attention has been paid to the in vivo


F hand, phenolic acid-g-chitosan could directly scavenge free radicals to inhibit oxidationo also enhance the expression levels as well as activities of cellular antioxidant enzymes,s nd glutathione reductase (GR).

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Fig. 5. The proposed antimicrobial mechanisms for phenolic acid-g-chitosan. Phe-nolic acid-g-chitosan can increase the permeability of Gram-negative bacteria’souter membrane (OM) and cellular membrane (CM), whereas increase the per-

ig. 4. The proposed antioxidant mechanisms of phenolic acid-g-chitosan. On onef DNA, protein and lipid in cells. On the other hand, phenolic acid-g-chitosan coulduch as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) a

ntioxidant activity of phenolic acid-g-chitosan till now. Notably,here are some differences between in vitro and in vivo assays.n one hand, chemical composition of culture media is the pri-ary factor affecting the in vitro antioxidant activity of phenolic

cid-g-chitosan. However, numerous factors including digestibil-ty, bioavailability and metabolism of phenolic acid-g-chitosan maynfluence the in vivo antioxidant activity of conjugate. Thus, then vivo antioxidant activity system is much more complicated thanhe in vitro one. On the other hand, in vitro antioxidant assays usu-lly ignore the biological actions in vivo, such as the activities ofntioxidant enzymes, the oxidative-related metabolic pathways asell as the activation or repression of gene expression of antioxi-ant compounds and enzymes. Therefore, in vivo antioxidant assaysre more reliable to reflect the real antioxidant potential of pheno-ic acid-g-chitosan. Liu et al. (2014b) revealed that administrationf caffeic acid-g-chitosan and ferulic acid-g-chitosan could sig-ificantly increase antioxidant enzymes activities and decreasealondialdehyde levels in both serums and livers of D-galactose

nduced aging mice. The in vivo antioxidant activity of these twohenolic acid-g-chitosans was much higher than that of chitosan.otably, no significant difference in the in vivo antioxidant activityas observed between caffeic acid-g-chitosan and ferulic acid-g-

hitosan, although caffeic acid-g-chitosan exhibited higher in vitrontioxidant activity than ferulic acid-g-chitosan (Liu et al., 2014b).n another study, Liu, Lu, Wen, Kan, and Jin (2015a) further sug-ested the hepatoprotective effect of phenolic-g-polysaccharideas related with antioxidant activity. Based on above mentioned

iterature, the potential antioxidant mechanisms of phenolic acid--chitosan are presented in Fig. 4.

.3. Antimicrobial activity

Apart from antioxidant activity, the antimicrobial activity ofifferent phenolic acid-g-chitosans has been evaluated by severalesearchers (Aljawish et al., 2014b; Bozic et al., 2012; Chatterjeet al., 2015; Eom et al., 2016; Itzincab-Mej&a et al., 2013; Kim & Je,015; Lee & Jea, 2013; , 2014a,b; Yang et al., 2016b).

Among various phenolic acid-g-chitosans, the antimicrobial
ctivity of gallic acid-g-chitosan has been most extensively stud-ed. Itzincab-Mej&a et al. (2013) successfully grafted gallic acid ontohitosan by horseradish peroxidase biocatalyst, and evaluated thentibacterial activity against two food pathogens (Escherichia coli
meability of Gram-positive bacteria’s CM. The increased permeability of cellularstructure will result in the release of intracellular components, such as enzyme andDNA, etc.

and Listeria monocytogenes). It was found that gallic acid-g-chitosanexhibited higher antibacterial activity than native chitosan. Leeet al. (2014b) synthesized gallic acid-g-chitosan by a free radicalmethod, and evaluated its antimicrobial activity against ten kindsof foodborne pathogens. Results showed that gallic acid-g-chitosanpossessed higher antibacterial effect against Gram-positive bacte-ria than against Gram-negative bacteria. The detailed antimicrobialmechanisms of gallic acid-g-chitosan were elucidated by Leeand Je (2013). Results showed that gallic acid-g-chitosan treat-ment increased the release of intracellular components in bothE. coli and Staphylococcus aureus, and rapidly enhanced 1-N-phenylanphthylamine uptake and �-galactosidase release viaincreasing the permeability of outer and inner membrane of
E. coli. Accordingly, we proposed the potential antimicrobial mech-anisms of phenolic acid-g-chitosan (Fig. 5). Lee et al. (2014a)further revealed the synergic antibacterial effect of gallic acid-g-chitosan combined with �-lactams (ampicillin or penicillin) against

Polymers 174 (2017) 999–1017 1009

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ethicillin-resistant S. aureus. Kim and Je (2015) reported gal-ic acid-g-chitosan exhibited higher antibacterial activity againsttreptococcus parauberis than that of Streptococcus iniae and Vib-io anguillarum. Although many studies have been carried out onhe antimicrobial activity of gallic acid-g-chitosan, the relationshipetween the grafting ratio and the antimicrobial activity of galliccid acid-g-chitosan is still unknown till now.

The antimicrobial activity of different kinds of phenolic acid-g-hitosans has been reported by some researchers. Bozic et al. (2012)ynthesized gallic acid-g-chitosan and caffeic acid-g-chitosan bysing laccase as the catalyst, and suggested the antimicrobial activ-

ty of graft copolymers was closely related to sample pH value,he mode of functionalization used and the type of microorganismested. Aljawish et al. (2014a) prepared ferulic acid-g-chitosan andthyl ferulate grafted chitosan by laccase catalyis. However, theyound that chitosan and its derivatives presented similar antibacte-ial activity against E. coli, S. aureus, L. monocytogenes and Salmonellanterica. Lee et al. (2014b) further compared the antimicrobialctivity of three kinds of hydroxycinnamic acid-g-chitosans against5 different clinical isolates. Among three conjugates, ferulic acid--chitosan showed the highest antimicrobial activity. Interestingly,hree conjugates exhibited relatively higher antimicrobial activitygainst Gram-positive bacteria than that of Gram-negative bac-eria. Vanillic acid-g-chitosan, p-coumaric acid-g-chitosan, galliccid-g-chitosan and ferulic acid-g-chitosan were synthesized byhatterjee et al. (2015) via free radical mediated grafting method.hese four conjugates were demonstrated to possess antibacterialctivity against a wide range of foodborne pathogens and spoilageacteria. Among them, p-coumaric acid-g-chitosan inhibited theidest antibacterial spectrum. Recently, Yang et al. (2016b) eval-ated the antimicrobial activity of caffeic acid-g-chitosan, feruliccid-g-chitosan, chlorogenic acid-g-chitosan and p-coumaric acid--chitosan against Ralstonia solanacearum. They found chitosan andts derivatives showed similar antibacterial activity against most R.olanacearum strains tested, except for strains of R. solanacearumS-5 and GIM1.74. Above results suggest the antimicrobial activ-

ty of phenolic acid-g-chitosan depends on the grafting methodsed, the type of phenolic acid grafted and the microbial strainested. However, no study has been carried out to compare thentimicrobial activity of phenolic acid-g-chitosan synthesized byifferent grafting methods. In addition, to better understand thentimicrobial mechanisms of phenolic acid-g-chitosan, more in-epth experiments including the effect of phenolic acid-g-chitosann the damage to bacterial cytoplasmic membrane and DNA, denat-ration of protein and inactivation of enzyme should be carriedut.

.4. Antitumor activity

Phenolic compounds have been demonstrated to possess anti-umor activity against breast, cervical, colon, leukemia, liver, lung,rostate and skin cancer cells. The antitumor mechanism is nor-ally related to the cytotoxic effect of phenolics on cancer cells,hich is associated with cell cycle arresting followed by apoptosis

Roleira et al., 2015). Till, only few studies have been carried outn the antitumor activity of phenolic acid-g-chitosan (Aytekin &orimura, 2010; Hu et al., 2015; Lee et al., 2013). The antitumor

ctivity is closely related to the molecular weight of pheno-ic acid-g-chitosan. Aytekin and Morimura (2010) demonstratedhat caffeic acid-g-chitosan with high molecular weight possessedigher anti-proliferative activity on human leukaemic monoblast937 cells. Lee et al. (2013) further demonstrated the antitumor
ctivity of caffeic acid-g-chitosan against CT26 mouse colorectalarcinoma cells in a dose-dependent manner. However, the anti-umor activity of conjugate was not significantly affected by therafting ratio. Recently, nanoparticles assembled from gallic acid-g-
Fig. 6. The anti-allergic mechanisms of gallic acid-g-chitooligosaccharide in RBL-2H3 mast cells sensitized with dinitrophenylspecific IgE antibody.

chitosan and caseinophosphopeptides showed cytotoxicity againstCaco-2 colon cancer cells (Hu et al., 2015). The antitumor effect ofgallic acid-g-chitosan nanoparticles increased with the elevationof grafting ratio. Notably, existing studies have only concerned onin vitro antitumor activity of some phenolic acid-g-chitosans, suchas gallic acid-g-chitosan and caffeic acid-g-chitosan. Futher workcan be carried out to compare the in vitro antitumor activity ofdifferent kinds of phenolic acid-g-chitosans through various tumorcellular models. Moreover, the in vivo antitumor activity of differentphenolic acid-g-chitosans can be also evaluated.

5.5. Anti-allergic activity

Allergy is a disorder of immune system in which an exaggeratedresponse occurs when a person is exposed to normally harmlessenvironmental substances (Kay, 2001). Vo et al. (2012a) evalu-ated the anti-allergic activity of gallic acid-g-chitooligosaccharideagainst RBL-2H3 allergic mast cells sensitized with dinitrophenyl-specific immunoglobulin E (IgE) antibody. As presented in Fig. 6,gallic acid-g-chitooligosaccharide could suppress the expressionof high-affinity IgE receptor (Fc�RI) on mast cells’ surface, andcould significantly inhibit translocation of nuclear factor (NF)-�B byblocking the degradation of inhibitory �B-� (I�B-�) protein and thephosphorylation of mitogen-activated protein kinases (MAPKs).Thus, the expression of interleukin (IL)-4 and tumor necrosis factor(TNF)-� could be suppressed afterwards. Moreover, gallic acid-g-chitooligosaccharide could also suppress the histamine release andintracellular Ca2+ elevation. This study suggests the grafting of gal-lic acid onto chitooligosaccharide could significantly enhance theinhibitory effect of chitooligosaccharide against allergic reactionsin RBL-2H3 mast cells.

5.6. Anti-inflammatory activity

Inflammation is an important host response to various stimuliincluding physical damage, microbial invasion, ultraviolet irradia-tion and immune reactions. Excessive or prolonged inflammation isharmful and can contribute to the pathogenesis of many diseases,such as chronic asthma, rheumatoid arthritis, multiple sclerosis,inflammatory bowel disease, psoriasis and cancer (Vo, Ngo, & Kim,
2012b). Recently, Ahn et al. (2016) revealed the anti-inflammatorymechanisms of gallic acid-g-chitosan in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. As shown in Fig. 7, gallicacid-g-chitosan could suppress the activation of activator protein-1

1010 J. Liu et al. / Carbohydrate Polym

Fig. 7. The potential anti-inflammatory mechanisms of gallic acid-g-chitosan in LPS-s

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possesses better food preservation effect than chitosan coating. The

timulated RAW264.7 macrophages.

AP-1) through the MAPK signaling pathway composed of extracel-ular signal-regulated kinase (ERK1/2), p38 and c-Jun N-terminalinase/stress activated protein kinase (JNK). On other hand, galliccid-g-chitosan could inactivate NF-�B via inhibiting the phos-horylation and degradation of the NF-�B inhibitor (I�B�). The

nactivated AP-1 and NF-�B could further inhibit the expressionsf pro-inflammatory cytokines, including inducible nitric oxideynthase (iNOS) and cyclooxygenase-2 (COX-2). Therefore, the pro-uction of TNF-�, IL-1�, IL-6, nitric oxide (NO) and prostaglandin2 (PGE2) were suppressed.

.7. Anti-diabetic activity

Diabetes mellitus (DM) is the most common endocrine disor-er known and results in deficient insulin production (type I DM)r combined resistance to insulin action and the insulin secre-ary response (type II DM). Postprandial hyperglycemia plays anmportant role in the development of type II DM, as well as inomplications associated with micro-vascular and macro-vasculariseases (Kim, Hyun, & Kim, 2011). �-Glucosidase and �-amylasere two key enzymes involved in the metabolism of carbohydrates.s shown in Fig. 8a, inhibitors of these two enzymes can delay car-ohydrate digestion and prolong overall carbohydrate digestionime, causing a reduction in the rate of glucose absorption andonsequently blunting the postprandial plasma glucose rise (Leet al., 2010). Liu et al. (2013b) synthesized gallic acid-g-chitosanith different grafting ratios by free radical mediated method,

nd further evaluated the �-glucosidase and �-amylase inhibitoryffects of gallic acid-g-chitosans. Results showed that gallic acid--chitosan possessed much higher �-glucosidase and �-amylasenhibitory effects than native chitosan. Moreover, �-glucosidasend �-amylase inhibitory effects of gallic acid-g-chitosan increasedith the increase of grafting ratio. Results indicated the potential

pplication of gallic acid-g-chitosan for the development of anti-iabetic agent.

ers 174 (2017) 999–1017

5.8. Acetylcholinesterase inhibitory activity

Alzheimer’s disease (AD) is a progressive neurodegenerativedisorder that impairs memory and behavior in elderly peo-ple. Reduced acetylcholine in the brain is the most remarkablebiochemical change in AD patients. Acetylcholinesterase canterminate nerve impulse transmission by rapid hydrolysis of acetyl-choline. Therefore, acetylcholinesterase inhibitions can be usedfor the treatment of AD (Anand, Singh, & Singh, 2012). Cho et al.(2011a) evaluated the acetylcholinesterase inhibition activity ofgallic acid-g-chitosan with different grafting ratios. Gallic acid-g-chitosan exhibited potent acetylcholinesterase inhibitory activityin a dose-dependent manner. The acetylcholinesterase inhibitoryactivity was associated with the grafting ratio of gallic acid-g-chitosan. The acetylcholinesterase inhibitory mechanism of gallicacid-g-chitosan was shown in Fig. 8b. Various bioactivities withcorresponding functional mechanisms of phenolic acid-g-chitosansare summarized in Table 2.

6. Applications of phenolic acid-g-chitosan in foodtechnology

6.1. Coating solutions for food preservation

Chitosan based edible coatings have received increasing atten-tion in recent years. They can not only delay respiration rate,decrease weight loss, and prolong shelf life of food; but also pre-vent the decrease in contents of natural antioxidants during foodstorage. Recently, some phenolic acid-g-chitosan coating solutionshave been developed (Wu et al., 2016a; Yang et al., 2016a; Zhang,Zhang, & Yang, 2015b).

Zhang et al. (2015b) synthesized salicylic acid-g-chitosan andevaluated its effect on alleviating chilling injury of cucumber dur-ing cold storage. Results showed salicylic acid-g-chitosan coatingpossessed better chilling injury inhibition effect than salicylic acidor chitosan coatings. The effect of protocatechuic acid-g-chitosancoating on the postharvest quality of Pleurotus eryngii (P. eryngii)was investigated by determining various physicochemical param-eters and enzyme activities (Liu et al., 2016a). Although chitosancoating could improve the postharvest quality of P. eryngii, itseffect was not as good as protocatechuic acid-g-chitosan coating.Notably, protocatechuic acid-g-chitosan with high grafting ratiohas been demonstrated as suitable coating material for the posthar-vest storage of P. eryngii. Caffeic acid-g-chitosan coating solutionwas prepared by Yang et al. (2016a) and applied to the postharveststorage of mulberry fruit. Caffeic acid-g-chitosan coating could sig-nificantly improve the quality and extend the shelf life of mulberryfruit during storage at 4 ◦C by decreasing the rotting rate, weightloss and MDA content. Moreover, caffeic acid-g-chitosan coatingcould also maintain higher levels of anthocyanins, ascorbic acid,polyphenols and flavones in mulberry fruit than the other treat-ments. Recently, Wu et al. (2016a) prepared gallic acid-g-chitosancoating solution and used it for the postharvest storage of silverypomfret (Pampus argenteus). As compared with gallic acid/chitosanmixture coating, gallic acid-g-chitosan coating effectively inhib-ited microbial growth, and reduced contents of volatile bases andlipid oxidation products in silvery pomfret. Moreover, gallic acid-g-chitosan coating could maintain a high water-holding capacityand good sensory properties in silvery pomfret, which extendedthe shelf life of silvery pomfret by 3–6 days.

Existing studies indicate that phenolic acid-g-chitosan coating

superior preservation effect of phenolic acid-g-chitosan should bemainly attributed to its high antioxidant and antimicrobial activ-ity. However, almost all the researchers have ignored the fact that

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Table 2Various biological activities and corresponding functional mechanisms of different phenolic acid-g-chitosans.

Biological activities Phenolic acid-g-chitosans Synthetic methods Detailed functional mechanisms References

Antioxidant activity Caffeic acid-g-chitosan; Carbodiimide mediated coupling method; Potent reducing power, metal chelating ability, lipidperoxidation inhibition, free radicals scavenging activities;

Aljawish et al., 2012, 2014a, 2014b;Aytekin et al., 2011; Bozic et al.,2012; Casettari et al., 2012;Chatterjee et al., 2015; Cho et al.,2011b, 2011c; Curcio et al., 2009;Eom et al., 2012; Hu et al., 2015,2016; Itzincab-Mejía et al., 2013;Liu et al., 2014b; Liu et al., 2014b,2016; Ngo et al., 2011a, 2011b;Pasanphan and Chirachanchai,2008; Pasanphan et al., 2010;Schreiber et al., 2013; Senevirathneet al., 2012; Wei and Gao, 2016;Woranuch and Yoksan, 2013; Wuet al., 2016c; Xie et al., 2014

Coumaric acid-g-chitosan; Free radical induced grafting reaction; Inhibit oxidation of cellular macromolecules, such as DNA,protein and lipid in mouse macrophages;

Chlorogenic acid-g-chitosan; Enzymatic catalyzed method Enhance the expression levels of antioxidant enzymes,such as SOD and GSH;

Ferulic acid-g-chitosan; Decrease reactive oxygen species induced activation ofNF-�B;

Gallic acid-g-chitosan; Increase antioxidant enzymes activities and decreasemalondialdehyde levels in both serums and livers ofD-galactose induced aging mice

p-Hydroxybenzoicacid-g-chitosan;Protocatechuicacid-g-chitosan;Sinapic acid-g-chitosan;Syringic acid-g-chitosan;Vanillic acid-g-chitosan

Antimicrobial activity Caffeic acid-g-chitosan; Free radical induced grafting reaction; Increased the release of intracellular components; Aljawish et al., 2014b; Bozic et al.,2012; Chatterjee et al., 2015; Eomet al., 2016; Itzincab-Mejía et al.,2013; Kim and Je, 2015; Lee and Je,2013; Lee et al., 2014a, 2014b;Yang et al., 2016a,b

Chlorogenic acid-g-chitosan; Horseradish peroxidase-catalyzed method; Rapidly enhance 1-N-phenylanphthylamine uptake and�-galactosidase release via increasing the permeability ofouter and inner membrane;

Coumaric acid-g-chitosan; Laccase-catalyzed method Synergic antibacterial effect of phenolic acid-g-chitosanwith �-lactams, such as ampicillin and penicillin

Ferulic acid-g-chitosan;Gallic acid-g-chitosan;Sinapic acid-g-chitosan;Vanillic acid-g-chitosan;

Antitumor activity Caffeic acid-g-chitosan; EDC mediated couplingmethod

Anti-proliferative and anti-invasive effectsagainst tumor cells

Aytekin and Morimura, 2010; Huet al., 2015; Lee et al., 2013Gallic acid-g-chitosan

Anti-allergic activity Gallic acid-g-chitosanoligosaccharide

DCC mediated couplingmethod

Inhibit the histamine release and production as well asintracellular Ca2+ elevation;

Vo et al., 2012a

Suppress the expression and production of IL-4 and TNF-�;Block the degradation of I�B-� protein, translocation ofNF-�B and phosphorylation of MAPKs

Anti-inflammatoryactivity

Gallic acid-g-chitosan Free radical inducedgrafting reaction

Reduce the production of nitric oxide and prostaglandin E2by inhibiting iNOS and COX-2 expression;

Ahn et al., 2016

Suppress the production and mRNA expression ofpro-inflammatory cytokines;Inactivate NF-�B by inhibiting the phosphorylation anddegradation of the NF-�B inhibitor;Suppress the activation of activator protein-1 (AP-1)through the MAPK signaling pathways.

Anti-diabeticactivity

Gallic acid-g-chitosan Free radical induced grafting reaction Inhibit the activity of �-glucosidase and �-amylase todelay carbohydrate digestion and prolong overallcarbohydrate digestion time

Liu et al., 2013b

Acetylcholinesteraseinhibitory activity

Gallic acid-g-chitosan Free radical induced grafting reaction Suppress the reduce of acetylcholine level in the brain byinhibit acetylcholinesterase activity

Cho et al., 2011a


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hese coating materials will be eaten by people along with theoated food. Thus, the safety of these phenolic acid-g-chitosanoating materials should be evaluated beforehand. Considering theotential cost of these coating materials, the applications of somexpensive phenolic acid-g-chitosans, such as protocatechuic acid--chitosan and caffeic acid-g-chitosan as coating materials mayeem unrealistic. By contrast, gallic acid-g-chitosan can be consid-red as a good candidate of coating material for its low cost anduperior preservation effect.

.2. Films as food packing materials

Nowadays, there is increasing interest to develop bio-basedctive films in order to improve food preservation as well as toeduce the use of chemical preservatives (Aider, 2010). Owingo its non-toxic, biodegradable, biocompatible, antimicrobial andood film forming properties, chitosan has been widely used inhe preparation of edible films for food packaging (Kerch, 2015). Inecent years, several researchers have developed functional filmssing different phenolic acid-g-chitosans (Aljawish et al., 2016b;iu, Meng, Liu, Kan, & Jin, 2017; Nunes et al., 2013; Schreiber et al.,013; Woranuch, Yoksan, & Akashi, 2015; Wu et al., 2016b).

In general, the physicochemical and functional properties ofhenolic acid-g-chitosan film are associated with the grafting
ethod used and the kind of phenolic acid grafted. Gallic acid-
-chitosan film was firstly developed by Schreiber et al. (2013)nd applied as peanut powder packaging material. Gallic acid--chitosan film showed superior preservation effect on peanut

id-g-chitosan via inhibiting the activities of �-amylase, �-glucosidase and acetyl-

powder by reducing the levels of thiobarbituric acid reactivesubstances, peroxide and conjugated trienes. Wu et al. (2016b)further investigated the physicochemical properties of the gallicacid-g-chitosan film. As compared with chitosan film, gallic acid-g-chitosan film exhibited decreased mechanical strength and waterresistance, whereas enhanced antioxidant and antimicrobial activ-ities. Nunes et al. (2013) prepared caffeic acid-g-chitosan film byusing genipin as the crosslinker. The film showed improved antiox-idant activity and ideal low solubility in acidic media. However,the surface wettability, mechanical properties and thermal stabil-ity of chitosan film were not significantly influenced after graftingwith caffeic acid and crosslinked by genipin. Ferulic acid-g-chitosanfilm was developed by Aljawish et al. (2016b). By contrast withchitosan film, ferulic acid-g-chitosan film showed decreased mois-ture content, water sorption capacity and mechanical properties,but increased barrier and antioxidant properties. Woranuch et al.(2015) developed novel composite films by incorporating ferulicacid-g-chitosan with other biodegradable materials. The incorpo-ration of ferulic acid-g-chitosan into biodegradable film causeddecrease in the water vapor barrier property and extensibility,however, increase in the oxygen barrier property and antioxidantactivity. In addition, ferulic acid-g-chitosan film also exhibited celladhesion ability to HUVEC and MSCs cells, suggesting it had poten-tial applications in tissue engineering to create new functionaltissue (Aljawish et al., 2014c, 2016a). Recently, protocatechuic acid-
g-chitosan films with different grafting ratios have been preparedand characterized in terms of physicochemical, mechanical andantioxidant properties (Liu et al., 2017). Results showed that the

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Although many studies on the different kinds of phenolic acid--chitosan films, no study has compared the physicochemicalnd functional properties of phenolic acid-g-chitosan films withhose of traditional used food packaging films, such as low den-ity polyethylene, polypropylene and polyethylene terephthalatelms. On the other hand, whether phenolic acid-g-chitosan filmill undergo degradation during storage at harsh conditions is stillnclear. Some kinds of phenolic acid-g-chitosan films, such as galliccid-g-chitosan and protocatechuic acid-g-chitosan films usuallyresent yellowish color, which might influence consumers’ sensorynd prevent the application of these films.

.3. Nanoparticles for delivery of functional dietary ingredients

The encapsulation and delivery of certain labile drugs and func-ional dietary ingredients has potential in solving the problem ofheir poor bioavailability. Due to the fact that many labile drugsnd nutraceutical molecules are oxidation-sensitive, the antioxi-ant polymeric conjugates have received increasing attention asovel delivery systems to prevent drugs or nutraceuticals fromxidation damage (Ko et al., 2014; Li, Jiang, Xu, & Gu, 2015; Ting,iang, Ho, & Huang, 2014). In recent years, some phenolic acid-g-hitosan nanoparticles have been successfully developed as goodarriers for labile drugs and nutraceutical molecules. By comparingith chitosan nanoparticles, phenolic acid-g-chitosan nanoparti-

les exhibit much higher antioxidant activity, which makes themore suitable to deliver oxidation-sensitive drugs and functional

ietary ingredients.Wei et al. (2010) developed salicylic acid-g-

hitooligosaccharide nanoparticles, and investigated the effectsf chitooligosaccharide’s molecular weight and initial molecularatio of chitooligosaccharide/salicylic acid on physicochemicalroperties of nanoparticles. Wang, Zhang, Wei, and Wang (2013)urther applied these nanoparticles to encapsulate paclitaxel.e2+-triggered self-assembly of gallic acid-g-N, O-carboxymethylhitosan nanoparticles were prepared by Yu et al. (2011). Theomplex nanoparticles were radical/pH-responsive and could besed to deliver antioxidative protein or natural products. Solimant al. (2014) prepared hydrocaffeic acid-g-chitosan nanoparticlesy ionic gelation with sodium tripolyphosphate. The obtainedanoparticles showed over 6 folds enhancement in chitosanucoadhesion to rabbit intestine. Likewise, these nanoparticlesere able to open the tight junctions of Caco-2 monolayers in a

eversible manner, suggesting that hydrocaffeic acid-g-chitosananoparticles could be used in the design of drug delivery systems

or peptide and protein drugs with poor oral absorption efficiency.Recently, spherical and physicochemical stable nanoparticles

ssembled from gallic acid-g-chitosan, caffeic acid and feruliccid with caseinophosphopeptides were developed for the deliv-ry of epigallocatechin-3-gallate (EGCG) (Hu et al., 2015, 2016).he nanoparticles not only exhibited strong antioxidant activitynd cytotoxicity against Caco-2 cells, but also showed improvedelivery property on controlling the release of EGCG under sim-lated gastrointestinal environments. Abdel-Wahhab et al. (2016)repared gallic acid-g-chitosan nanoparticles and evaluated theirrotective roles against ochratoxin-A (OTA) toxicity in catfishClarias gariepinus). It was found that both chitosan and gallic acid--chitosan nanoparticles were successful in counteracting the toxicffect of OTA. Moreover, gallic acid-g-chitosan nanoparticles wereuperior due to its high phenolic content.

As for the future applications of phenolic acid-g-chitosananoparticles, massive quantities of nanoparticles are needed toe produced, which would greatly increase the potential risk ofuman exposure. Thus, the initial step should be to assess the

ers 174 (2017) 999–1017 1013

safety of these nanoparticles and understand their interactionswith relevant biological systems. In addition, many physicochem-ical properties of nanoparticles including size, surface chemistry,crystallinity, morphology, solubility, aggregation tendency, homo-geneity of dispersions and turbidity can influence the toxicity ofnanoparticles in biological systems (Hussain et al., 2009). There-fore, multidisciplinary studies are needed to characterize andchoose appropriate exposure protocols of phenolic acid-g-chitosannanoparticles, as well as establish reliable methods to assessnanoparticles’ internalization with living organisms (Oberdörsteret al., 2005).

6.4. Hydrogels as encapsulation agents and novel biomaterials

The design and preparation of hydrogels have attractedmany interests in biomedical engineering, pharmaceutical andbiomaterial science because of their tunable chemical and three-dimensional physical structure, good mechanical properties, highwater content and biocompatibility. Hydrogels can present in theform of macroscopic networks or confined to smaller dimensionssuch as microgels or nanogels, which are crosslinked polymeric par-ticles (Chacko, Ventura, Zhuang, & Thayumanavan, 2012; Farjami& Madadlou, 2017).

From the structural point of view, the amide linkages formedbetween chitosan with phenolic acid make it possible for pheno-lic acid-g-chitosan to form nanomicelles in water to encapsulatebioactive substances. Beyki et al. (2014) prepared cinnamic acid-g-chitosan nonogels through an EDC-mediated reaction followed bypolymeric gel formation by self-aggregation in aqueous medium.The obtained nonogels were further used to encapsulate Menthapiperita essential oils. Results showed the encapsulated essentialoils were capable to preserve the quality of tomato fruit during1-month’s storage period. Afterwards, caffeic acid and benzoicacid grafted chitosan nanogels were also developed by the sameresearch group for the encapsulation of other kinds of essential oils(Khalili et al., 2015; Zhaveh et al., 2015).

Kim et al. (2013) synthesized 3,4-dihydroxyhydrocinnamicacid-g-chitosan by EDC mediated coupling reaction. The obtainedwater soluble chitosan derivative could directly form hydrogels inneutral buffer solution. The formed hydrogels showed adhesiveproperties, which might be useful in areas of surface function-alization, drug delivery, tissue engineering and tissue adhesives.Kim et al. (2015) further demonstrated that the gastrointestinaltract retention of chitosan was greatly improved after graftingwith 3,4-dihydroxyhydrocinnamic acid, which was due to the for-mation of irreversible catechol mediated crosslinking with mucin.Recently, novel hydrogels composed by gelatin and phloretic acid-g-chitosan were developed by bienzymatic crosslinking approach(Zhang et al., 2015a). Mechanical testing revealed the excellentstrength of hydrogels. The cytotoxicity and proliferation assay con-firmed that hydrogels could effectively support cell adhesion andproliferation. The development of above intelligent drug deliveryvehicles requires a foundation in the physicochemical charac-terization of phenolic acid-g-chitosan hydrogels, as well as thetherapeutics to be delivered. With increased understanding thefundamental loading and release criteria of varying therapeutics,it will be able to adapt delivery systems for different drug for-mulations. Once these design parameters have been established,cheap, non-toxic and efficient phenolic acid-g-chitosan hydrogeldrug delivery systems can be clinical availability.

6.5. Microspheres as bioadsorbents and encapsulation agents

Chitosan with high contents of amino and hydroxyl groups canbe used as bioadsorbents to remove heavy metals from aqueoussolution (Wang & Chen, 2014). In recent years, some phenolic

1 Polym

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7

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cid-g-chitosan microspheres have been developed and applied fordsorption of ions and dyes.

Chao et al. (2004) grafted four kinds of phenolic acids ontohitosan via tyrosinase catalysis. The synthesized phenolic acid--chitosans were further prepared into microspheres for thedsorption of two cationic dyes (crystal violet and bismarck brown). Results showed that the maximum adsorption capacities foroth dyes decreased in the order of hydrocaffeic acid-g-chitosan3,4-dihydroxyphenyl-acetic acid-g-chitosan > protocatechuiccid-g-chitosan > p-hydroxybenzoic acid-g-chitosan microspheres.atechol, caffeic acid and 2,5-dihydroxybenzoic acid were suc-essfully grafted onto chitosan by laccase catalysis and wereurther prepared into functionalized microspheres for the adsorp-ion of iron ions (Brzonova et al., 2011). This study showed thereat potential of application phenolic-g-chitosan microspheress novel bioadsorbents for the treatment of iron overload dis-ases, because these microspheres possessed high iron-chelatingbility at gastrointestinal pH conditions. Five different kinds ofhenolic acid-g-chitosan microspheres were successfully preparedy Liu et al. (2015b) via a free radical mediated grafting methodnd further applied for the adsorption of Fe(II). These micro-pheres exhibited excellent adsorption characteristics for Fe(II)ons. Results of adsorption kinetics and isotherms showed thedsorption process was endothermic, monolayer and chemicaldsorption in nature. Moreover, the Fe(II) adsorption property oficrospheres was not only attributed to the iron-chelating ability

f phenolic acid grafted, but also related to the physical nature andhemical composition of microspheres, such as surface morphologynd pore size. Except for microspheres, phenolic acid-g-chitosanased bioadsorbents can be also prepared into other forms, suchs beads, membrane, film and even composites with other materi-ls. Thus, the selection of the most appropriate form is one of theey issues to achieve the ideal adsorption amount. Further workhould be extended from lab scale batch study to pilot-plant studyn order to check the feasibility of phenolic acid-g-chitosan basedioadsorbents on commercial scale.

Microencapsulation is a novel technology that can improve theetention time of the nutrient in food and allow controlled releaset specific times during food consumption or in the intestinalut. Recently, Chatterjee et al. (2016) prepared ferulic acid-g-hitosan microspheres and demonstrated these functionalizedicrospheres could serve as excellent encapsulating materials forater soluble vitamins, such as thiamine and pyridoxine. This sug-

est that phenolic acid-grafted chitosan microspheres could also beromising edible bioactive wall materials for the microencapsula-ion of nutrients and nutraceuticals.

. Conclusions and future perspectives

Till now, four kinds of grafting methods including carbodiimideased coupling, enzyme catalyzed grafting, free radical mediatedrafting and electrochemical methods have been used for the syn-hesis of phenolic acid-g-chitosan. Among these methods, freeadical mediated grafting reaction is the most promising approach.n future, more in-depth experiments are needed to reveal the

echanism of free radical mediated grafting reaction.Several instrumental methods including TLC, HPLC, UV–vis, FT-

R, 1H and 13C NMR spectroscopy as well SEM have been used toonfirm the successful grafting of phenolic acid onto chitosan. Toetter elucidate the fine chemical structures of phenolic acid-g-hitosan, 2D NMR technology can be applied in future. In addition,
FM can be combined with SEM to reveal the conformation of phe-olic acid-g-chitosan.
Conjugation of phenolic acid onto chitosan greatly altershe physicochemical properties (e.g. solubility, thermal stability,

ers 174 (2017) 999–1017

crystallinity and rheological properties) and increases biolog-ical activities (e.g. antioxidant, antimicrobial, antitumor, anti-allergic, anti-inflammatory, anti-diabetic and acetylcholinesteraseinhibitory activity) of chitosan. In future, more attention should bepaid to the physicochemical properties of phenolic acid-g-chitosan,as these properties are closely associated with the ultimate applica-tions of conjugate. Besides, more reliable evaluation methods, suchas cellular and animal models should be applied to fully assess thebiological activities of phenolic acid-g-chitosan.

Phenolic acid-g-chitosan can be used as food coating agents,food packing materials, encapsulation agents for functional dietaryingredients, and bioadsorbents for the treatment of iron overloaddiseases. Notably, the safety of phenolic acid-g-chitosan should beevaluated beforehand. Beside, the potential cost and efficacy ofdifferent kinds of phenolic acid-g-chitosans should be compared.It is certain that functional phenolic acid-g-chitosans need to beextensively researched and utilized in future.

Acknowledgements

This work was supported by Grants-in-Aid for scientific researchfrom the National Natural Science Foundation of China (No.31571788 and 31101216), Natural Science Foundation of JiangsuProvince (No. BK20151310), Qing Lan Project of Jiangsu Province,Jiangsu Provincial Government Scholarship for Overseas Studies,and High Level Talent Support Program of Yangzhou University.

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1 Polym

L

L

L

L

L

L

L

L

L

L

L

N

N

N

N

O

O

O

P

P

P

P

R

R

R

R

S

S

S


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Synthesis, characterization, bioactivity and potential...

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