The effect of solvent composition on grafting gallic acid onto chitosan via carbodiimide

Accepted Manuscript Title: The Effect of Solvent Composition on Grafting Gallic Acid onto Chitosan via Carbodiimide Author: Ping Guo John D. Anderson Joseph J. Bozell Svetlana Zivanovic PII: DOI: Reference:

S0144-8617(15)01193-5 http://dx.doi.org/doi:10.1016/j.carbpol.2015.12.015 CARP 10613

To appear in: Received date: Revised date: Accepted date:

30-9-2015 23-11-2015 7-12-2015

Please cite this article as: Guo, P., Anderson, J. D., Bozell, J. J., and Zivanovic, S.,The Effect of Solvent Composition on Grafting Gallic Acid onto Chitosan via Carbodiimide, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.12.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The Effect of Solvent Composition on Grafting Gallic Acid onto Chitosan via

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Carbodiimide

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Ping Guoa, John D. Anderson b, Joseph J. Bozell b, Svetlana Zivanovica*

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Tennessee, Knoxville, TN 37996, United States b

Center for Renewable Carbon, 2506 Jacob Drive, University of Tennessee, Knoxville, TN 37996, United States

* Corresponding author. Tel.: 865-850-5484; E-mail addresses: [email protected].

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Abstract

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Department of Food Science and Technology, 2510 River Drive, University of

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The primary antioxidant (AOX) activity of chitosan can be introduced by grafting of

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phenolic compound - gallic acid (GA) to its amino and/or hydroxyl groups. The objective

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of this study was to investigate the effect of ethanol (EtOH) concentration (0%, 25%,

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50%, and 75% in water) on efficiency of grafting GA onto chitosan in the presence of 1-

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ethyl-3-(3-dimethylaminopropyl) -carbodiimide (EDC)/N-hydroxysuccinimide (NHS).

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The grafting was confirmed by FTIR and the efficiency was quantified as Folin’s total

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phenolics. When pure deionized water was used as a sole solvent (0% EtOH), GA was

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grafted to chitosan at the largest extent (285.9 mg GA/g chitosan). As the concentration

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of EtOH increased, the grafting efficiency proportionally decreased. NMR studies

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showed that EtOH inhibited grafting of GA by prohibiting the production of the

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intermediate - NHS ester. The results confirm that the concentration of EtOH in grafting

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solution significantly affects grafting efficiency of GA on chitosan.

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1. Introduction Food packaging with antioxidant (AOX) properties can extend food shelf life and

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improve safety. Developments in this field are continuously evolving in response to the

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growing demand for multifunctional active packaging (Bolumar, Andersen & Orlien,

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2011; de Kruijf, van Beest, Rijk, Sipilainen-Malm, Losada & De Meulenaer, 2002;

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Lopez-de-Dicastillo, Gomez-Estaca, Catala, Gavara & Hernandez-Munoz, 2012; Realini

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& Marcos, 2014). Chitosan, a co-polysaccharide of 2-acetamido-2-deoxy-β-D-glucose

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and 2-amino-2-deoxy-β-D-glucose, has been considered as an alternative to synthetic

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polymers in food packaging due to its biodegradability and film forming ability

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(Schreiber, Bozell, Hayes & Zivanovic, 2013; van den Broek, Knoop, Kappen & Boeriu,

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2015; Woranuch, Yoksan & Akashi, 2015). Chitosan is produced by the deacetylation of

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chitin, obtained from crustacean shells left as a waste in the seafood industry. The

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presence of amino groups enables chitosan to chelate metal ions (Guibal, 2004; Yoshida

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& Takemori, 1997) and to be chemically modified (Julkapli, Akil & Ahmad, 2011;

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Mourya & Inamdar, 2008). Chitosan has been commercially used in water purification

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(Correa-Murrieta, Lopez-Cervantes, Sanchez-Machado & Sanchez-Duarte, 2014; Karimi

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& Zamani, 2013; Vakili et al., 2014), and evaluated as an antimicrobial food packaging

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(Marin et al., 2015; van den Broek, Knoop, Kappen & Boeriu, 2015; Zivanovic, Chi &

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Draughon, 2005) and as a carrier in drug delivery systems (Lin & Lin, 2009;

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Mukhopadhyay, Sarkar, Bhattacharya, Bhattacharyya, Mishra & Kundu, 2014; Shim &

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Nho, 2003). Chitosan films have a low gas permeability (Jeon, Kamil & Shahidi, 2002),

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good mechanical properties (Gallstedt, Brottman & Hedenqvist, 2005), excellent metal

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binding potential (Julkapli, Akil & Ahmad, 2011) and with chitosan's intrinsic

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antimicrobial efficiency (Kumar, 2000), may serve as multifunctional active packaging.

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Furthermore, there is an increased interest in chemically modifying chitosan by grafting it

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with a phenolic acid in order to introduce primary antioxidant properties and thus extend

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the shelf life of packaged food (Curcio et al., 2009; Schreiber, Bozell, Hayes &

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Zivanovic, 2013; Woranuch, Yoksan & Akashi, 2015).

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Modification of chitosan films has been simply achieved by mixing or coating

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(Hromis et al., 2014; Yang et al., 2014), or by physiochemical and biochemical methods

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such as irradiation (Elbarbary & Mostafa, 2014; Iturriaga, Olabarrieta, Castellan, Gardrat 2

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& Coma, 2014), or enzymatic (Aljawish, Chevalot, Jasniewski, Revol-Junelles, Scher &

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Muniglia, 2014; Bozic, Gorgieva & Kokol, 2012; Zavaleta-Avejar, Bosquez-Molina,

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Gimeno, Perez-Orozco & Shirai, 2014) and free radical reactions (Curcio et al., 2009;

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Pasanphan & Chirachanchai, 2008; Schreiber, Bozell, Hayes & Zivanovic, 2013; Xie,

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Hu, Wang & Zeng, 2014). Mixing is an easy and fast but with a high possibility for

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antioxidants to be lost from the films by volatilization or leaching (Curcio et al., 2009).

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Radiation introduces covalent bonds between the AOXs and chitosan but often causes

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degradation of either the polymer or phenolic acid, or both. Radiation induces polymer

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degradation via chain scission, resulting in cracking of the surface and loss of mechanical

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properties (Curcio et al., 2009; Jahan & McKinny, 1999), while phenolic acid may be

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degraded by hydroxy radicals generated during radiolysis of water (Breitfellner, Solar &

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Sontag, 2002). Enzymatic methods using laccase (Aljawish, Chevalot, Jasniewski, Revol-

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Junelles, Scher & Muniglia, 2014), tyrosinase (Jus, Stachel, Fairhead, Meyer, Thony-

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Meyer & Guebitz, 2012), and horseradish peroxidase(Sakai, Khanmohammadi,

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Khoshfetrat & Taya, 2014) have been applied to functionalize chitosan with phenolic

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compounds, but can catalyse oxidation of phenolics (Zinnai, Venturi, Sanmartin,

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Quartacci & Andrich, 2013), and thus reduce their AOX properties. In contrast, 1-ethyl-

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3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS),

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extensively used in amidation of proteins (Krishnamoorthy, Selvakumar, Sastry, Sadulla,

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Mandal & Doble, 2014) offer an alternative chemical approach for the modification of

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chitosan (Pasanphan & Chirachanchai, 2008; Schreiber, Bozell, Hayes & Zivanovic,

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2013). This method requires only mild reaction conditions, does not exhibit the

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disadvantages of the other procedures, and uses reagents (EDS and NHS) that can be

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easily removed after the reaction to produce clean products.

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Gallic acid (GA, 3,4,5-trihydroxy benzoic acid) is a naturally occurring antioxidant.

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GA and its derivatives form a large family of plant secondary polyphenolic metabolites,

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and are normally present in fruits, vegetables, nuts, tea, etc (Arbos, de Freitas, Stertz &

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Dornas, 2010; Arcan & Yemenicioglu, 2009; Lu, Nie, Belton, Tang & Zhao, 2006). GA

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derivatives, such as propyl gallate (Hamishehkar, Khani, Kashanian, Dolatabadi &

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Eskandani, 2014) and eppigallocatechin gallate (Rashidinejad, Birch, Sun-Waterhouse &

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Everett, 2014), have been widely used as food additives to prevent oil rancidity. Their

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antioxidant activity is achieved by direct termination of free radicals via rapid donation of

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hydrogen atoms or electrons, so they are classified as primary AOXs (Shahidi & Naczk,

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2004). With three phenolic hydroxyl groups in its structure, GA exhibits strong AOX

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activity, while the carboxyl group enables its grafting to various matrices, including

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collagen

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(Krishnamoorthy, Selvakumar, Sastry, Sadulla, Mandal & Doble, 2014; Pasanphan,

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Buettner & Chirachanchai, 2010).

through

amidation

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esterification

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chitosan,

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GA can be grafted to chitosan via amide or ester linkages by first activating the

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carboxyl group of GA through conversion to amino or hydroxyl-reactive intermediate via

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EDC/NHS (Pasanphan & Chirachanchai, 2008; Schreiber, Bozell, Hayes & Zivanovic,

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2013). As indicated in Scheme 1, GA 1 reacts with EDC 2 to form the hydrolytically

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unstable O-acylisourea 3 (half-life, t½ = seconds in aqueous solution) (Grabarek &

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Gergely, 1990; Madison & Carnali, 2013). However, in the presence of NHS 4, the

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longer lived GA-NHS ester 5 (t½ = hours in aqueous solution) is formed (Hermanson,

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2008) and serves as an activated ester that reacts further with –NH2 and/or –OH on

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chitosan 7 to form GA-grafted chitosan 8 (Pasanphan & Chirachanchai, 2008).

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Scheme 1. EDC/NHS activation of gallic acid

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Although the reaction is simple, it is time consuming and results in relatively low

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grafting efficiency. In order to improve the reaction, studies have been conducted that

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alter the EDC/NHS ratio (Ahn et al., 2013; Sam et al., 2010; Wang, Yan, Liu, Zhou &

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Xiao, 2011), the concentration of all components (Schreiber, Bozell, Hayes & Zivanovic, 4

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2013), or the pH of the reaction (Madison & Carnali, 2013). Nonetheless, the reaction

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still suffers from a low grafting efficiency. In all of these studies and in many others

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investigating grafting of GA or other carboxylic acid to chitosan using ECD/NHS,

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aqueous ethanol served as a solvent throughout the reaction. However, the concentration

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of EtOH was not consistent across these reports, and it is not clear how is the EDC/NHS

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coupling reaction is affected by EtOH concentration, or how the EtOH concentration

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affects the grafting efficiency (Liu, Lu, Kan & Jin, 2013; Pasanphan & Chirachanchai,

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2008; Woranuch & Yoksan, 2013). Furthermore, Nam et al.(Nam, Kimura, Funamoto &

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Kishida, 2010) used EDC/NHS for collagen crosslinking and reported that the

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crosslinking rate increased as the EtOH concentration in the solvent increased up to 0.12

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M (6.9% v/v) but decreased as the concentration was increased further. Here we present

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the effect of EtOH concentration (0 - 75% v/v) on efficiency of grafting GA on chitosan

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utilizing EDC and NHS, and chemistry underlying the change in the reaction.

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2. Methods

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2.1. Materials

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Chitosan with an average molecular weight of 307 kDa and 80% degree of

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deacetylation (DDA), was donated by Primex. EDC (PubChem CID: 2723939) (99.8%

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purity) and NHS (PubChem CID: 80170) (98% purity) were purchased from Acros

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Organics. GA (PubChem CID: 370) was purchased from Sigma-Aldrich.

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2.2. Purification of chitosan

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Chitosan flakes were dissolved in 1w/w% acetic acid to form a 1w/w% chitosan

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solution. The solution was stirred overnight, filtered through Miracloth®, and chitosan

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was precipitated by adjusting pH to ~10. The precipitate was washed with deionized

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water until neutral and freeze-dried. Purified chitosan was kept in a desiccator at room

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temperature until needed.

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2.3. Synthesis of GA-grafted chitosan

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GA-grafted chitosan was prepard using a modified literature method (Pasanphan &

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Chirachanchai, 2008). GA (0.500 g, 3 mmol), EDC (0.580 g, 3 mmol) and NHS (0.340 g,

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3 mmol) were mixed as solids, added to 20 mL of various concentrations of aq. EtOH,

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and stirred in an ice bath for 1 h. Chitosan (0.32 g, 1.18 µmol), dispersed in 30 mL aq.

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EtOH of the same concentration, was added to the solution, and additionally stirred for

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0.5 h in ice-bath followed by 6 h stirring at room temperature (standard procedure). After

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the grafting was completed, the product was centrifuged at 3,315 g for 20 min, washed 3

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times with 50 mL aliquots of 75% EtOH, and freeze dried. To test the effect of EtOH

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concentration on grafting efficiency, four concentrations (0, 25, 50, and 75% v/v) of aq.

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EtOH were used. To test the effect of time on grafting efficiency, 25% EtOH was used as

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solvent and the last step (stirring in the presence of chitosan) was varied between 2, 6, 12

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and 24 h. The concentration of residual GA was determined by washing grafted chitosan

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(25% EtOH, 6 h) 7 times with 50 mL 75% aq. EtOH and analyzing the wash for total

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phenolics.

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2.4. Confirmation of grafting and characterization of GA-grafted chitosan

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2.4.1. Confirmation of grafting

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Chitosan, grafted chitosan, and a mixture of GA and chitosan (mix) prepared in the

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same ratio as found in grafted chitosan were ground to a fine powder with a mortar and

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pestle, mixed with KBr, and pelleted. FTIR spectra were acquired on the pellet (Nicolet

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NEXUS 670,Thermo, Madison, WI) between 500 and 4000 cm-1, with 128 scans and

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resolution of 4 cm-1.

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2.4.2. Solubility of grafted chitosan in 1% acetic acid

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Solubility of 0.1% w/w pure chitosan, grafted chitosan, and mix in 1% acetic acid was

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assessed as transmittance (T%) at 600 nm using a spectrometer (UV-2101PC Shimadzu,

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Columbia, MD) (Schreiber, Bozell, Hayes & Zivanovic, 2013), with transmittance of

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100% indicating complete solubility.

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2.4.3. Determination of Total Phenolics content

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Total phenolics content was determined by Folin-Ciocalteau method (Folin &

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Ciocalteu, 1927) with modification (Schreiber, Bozell, Hayes & Zivanovic, 2013).

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Briefly, the grafted chitosans were solubilized by sonication (Bradson 1510, Brason

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Ultrasonics Corp., Danbury, CT) in 0.25% acetic acid to give a 0.025% solution of

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dissolved chitosan. 1 mL of this solution was added to 7 mL DI water with 1 mL Folin-

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Ciocalteau reagent. After 3 min, 12.4% sodium carbonate solution was added to the

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mixture, and the solution was vortexed. The mixture was kept at 40°C for 30 min, after

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which the absorbance was measured at 725 nm using a spectrophotometer. Gallic acid

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standards of different concentration (0.000, 0.0125, 0.025, 0.050, 0.075 and 0.1 mg/mL)

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were prepared the same way.

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2.4.4. Measurement of AOX properties

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DPPH free radical scavenging capacity was measured using a previously reported

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method (Arbos, de Freitas, Stertz & Dornas, 2010) with modification (Schreiber, Bozell,

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Hayes & Zivanovic, 2013). Aliquots of 1 mL 0.001% each chitosan in 0.01% acetic acid

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were added to 1 mL 100 µM methanolic DPPH (2,2-diphenyl-1-picrylhydrazyl) solution.

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The mixture was stirred for 30 min in dark at room temperature, followed by absorbance

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measurement at 517 nm. The DPPH free radical scavenging capacity was calculated

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using the following equation:

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DPPH scavenging capacity (%) = (A0 – A1)/A0 × 100

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where A0 is the absorbance of the control (DI water instead of sample) and A1 is the

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absorbance of sample.

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Reducing powder was determined following a reported method (Yen & Chen, 1995).

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Aliquots of 1mL 0.025% each chitosan in 0.25% acetic acid were mixed with phosphate

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buffer (pH 6.6, 0.2 M) and 2.5 mL 1% potassium ferricyanide (K3Fe(CN)6). The mixture

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was incubated at 50 °C for 20 min followed by addition of 2.5 mL 10% trichloroacetic

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acid, and centrifuged 10 min at 3,315 g. Aliquots of 2.5 mL of the upper layer were added

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to 2.5 mL DI water, followed by addition of 0.5 mL 0.1% iron chloride solution.

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Absorbance of the solution was immediately measured at 700 nm.

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2.5. NMR characterization of EDC/NHS activation

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H, gradient HSQC (heteronuclear single quantum coherence) and HMBC

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(heteronuclear multiple bond correlation ) NMR (gHSQC, gHMBC) measurements were

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carried out on a Varian 400-MR spectrometer equipped with a broadband probe operating

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at 399.78 MHz for proton and 100.54 MHz for carbon. Solid gallic acid (25 mg), EDC

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(29 mg) and NHS (17 mg) (1:1:1 molar ratio) were vortexed for 10 s, followed by adding

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1 mL of a d4-methanol (CD3OD)/D2O solution of varying concentrations (0, 25, 50, 75,

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100% v/v). The resulting solution was stirred in an ice bath for 1 h. Maleic acid (1.1 M,

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100 μL) as internal standard, was added to the solution. The mixture was transferred into

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a 5 mm NMR tube. 1H spectra were acquired with a 25 s relaxation delay, 2 scans, and an

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acquisition time of 2.556 s. The FIDs (free induction decay) were transformed using

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Mnova (Mestrelab Research SL., Santiago de Compostela, Spain), version 10.0.1, and

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processed using a third order Bernstein polynomial baseline fit. All spectra were

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referenced to the residual D2O signal at 4.79 ppm.

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To isolate the unknown product, solid GA (500 mg), EDC (580 mg) and NHS (340

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mg) (1:1:1 molar ratio) were vortexed for 10 s, followed by addition of 20 mL 100%

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methanol. The resulting solution was stirred in an ice bath for 1 h, and 20 mL DI water

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was added to the reaction mixture. Rotovapor was used to evaporate the methanol to 20

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mL of the resulting solution. Ethyl acetate (EtOAc) (20 mL x 5 times) was used to extract

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GA, GA-NHS ester, and unknown product from the aqueous layer, and the EtOAc layer

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was washed by DI water (20 mL x 5 times). The final EtOAC fraction was concentrated

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to 10 mL and the unknown product, which is shown on the top of GA spot on thin layer

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chromatography (TLC) was isolated using preparative layer chromatography on Si gel (2

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mm plates, Analtech, Inc. Newark, DE) and toluene/ethyl acetate/formic acid/methanol

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(3:3:0.8:0.2 v/v/v/v) (Imran et al., 2011) as the eluent. The product was scraped from the

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plate and the Si gel was washed with acetone. Solvent removal on the rotary evaporator

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gave a 50 mg single product, which was analysed by NMR.

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gHSQC and gHMBC were acquired on the isolated sample dissolved in d6-acetone.

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The gHSQC experiment used 128 increments and 8 scans/increment in the F1 direction,

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giving a spectrum size of 962 x 128. A 90o pulse with a relaxation delay of 1 s, an

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acquisition time of 0.15 s, and a one-bond C-H coupling constant of 146 Hz were

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employed. Nonuniform sampling (NUS) (Barna & Laue, 1987; Rovnyak, Sarcone &

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Jiang, 2011) was employed to shorten the total experimental time. The gHMBC

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experiment was conducted using 200 increments and 4 scans/increment in the F1

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direction. A 90o pulse with a relaxation delay of 1 s, an acquisition time of 0.15 s, and the

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multiple-bond C-H coupling constant of 8 Hz were employed. Runs were carried out at

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25 oC without spinning and typically required about 10 min 47 s for gHSQC and 11 min 8

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11 s for gHMBC. The FIDs were transformed using Mnova, version 10.0.1, and

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processed using a third order Bernstein polynomial baseline fit. All spectra were

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referenced to the residual acetone-d6 signal at 2.05/206.26 ppm.

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2.6. Statistical Analysis

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All wet chemical analyses were done in triplicate. Tukey HSD (honestly significant

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difference test) comparison of means (p < 0.05) was performed using SAS (SAS

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Enterprise Guide 6_1, SAS Institute).

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3. Results and discussion

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3.1. Confirmation of grafting

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The effect of solvent composition on the efficiency of grafting GA onto chitosan and

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on the solubility of the grafted chitosan was determined by performing the reaction in 0,

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25, 50 and 75% v/v aq. EtOH. Prior to the analyses, grafting was confirmed by FTIR

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(Fig. 1). Peaks at 1645 cm-1 and 1550 cm-1 correspond to the C=O stretching in amide

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linkages and the asymmetric bending of the free –NH2 in chitosan, respectively (Kono,

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Onishi & Nakamura, 2013; Li, Kong, Cheng, Li, Liu & Chen, 2011). The reduced

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intensity of 1550 cm-1 peak relative to 1645 cm-1 peak in grafted chitosan compared to

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relative intensity of these peaks in non-grafted chitosan was consistent with grafting by

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amidation between chitosan amino groups and GA carboxyl groups. The grafted chitosan

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also showed a new absorption band at 1715 cm-1. This peak has been assigned to the C=O

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stretching vibration of the ester group (Kono & Nakamura, 2013; Li, Kong, Cheng, Li,

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Liu & Chen, 2011), and in grafted chitosan resulted from the esterification between the

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hydroxyl groups on chitosan and carboxyl group on GA. The 1715 cm-1 peak intensity

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was higher in chitosans grafted with less EtOH indicating the possibility that grafting in

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pure DI water or at lower concentration of EtOH favored esterification between GA and

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chitosan, whereas higher concentrations of EtOH in solution favored grafting by

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amidation.

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Sun Nov 09 21:15:44 2003 (GMT-05:00)

*GA_25_6hrs 90

1645

1550

0%

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88 86

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50% 80 78

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1715

75%

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Pure chitosan

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1900

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Mix 1800

1700

1600

1500

1400

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1300

Wavenumbers (cm-1)

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% Transmittance

25%

Fig. 1.

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Number of sample scans: 128 Number of background scans: 128 Resolution: 4.000 Sample gain: 1.0 Mirror velocity: 0.6329 Aperture: 100.00

3.2. The effect of EtOH concentration on grafting efficiency

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Collection time: Wed Apr 30 05:46:47 2003 (GMT-05:00)

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No peak table for the selected spectrum!

Detector: DTGS KBr

As shown in Fig. 2 (A) theBeamsplitter: highestKBrefficiency of 285.9 mg GA eq/g was achieved in Source: IR

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pure water results but decreased the EtOH concentration in grafting solution increased. NoDIsearch for theasselected spectrum!

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Thus, when the grafting solvent was 25% EtOH, the efficiency was 260.9 mg GA eq/g,

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and with 75% EtOH, the efficiency was down to 122.2 mg GA eq/g grafted chitosan.

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Applying the same reaction but in either pure EtOH or 'alcoholic solution', grafting

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efficiencies reported by other research groups were in the range of 65 - 209.9 mg GA

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eq/g (Schreiber, Bozell, Hayes & Zivanovic, 2013; Xie, Hu, Wang & Zeng, 2014). The

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grafting efficacy of 285.9 mg GA eq/g achieved in our study may be due to the higher

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level of EDC and NHS we used, which consequently activated more GA, but also due to

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interferences of EtOH with the grafting reaction that resulted in lower yield found in

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literature.

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The grafting efficiency was further indirectly assessed by determining the AOX

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properties as DPPH scavenging activity and as reducing power (Fig. 2 B, C). Antioxidant

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activity of grafted chitosan was directly related to the amount of GA grafted. Both DPPH

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scavenging activity and reducing power were the highest in chitosan grafted in pure DI

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water (60% for 0.001% chitosan, and 0.92 for 0.025% chitosan, respectively) and, as

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expected, decreased with increased EtOH concentration in reaction solvent (down to 33%

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and 0.40, respectively). 10

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287 288 289

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Fig. 2.

3.3. The recovery of grafted chitosan after grafting

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The recovery of grafted chitosan was also affected by composition of the solvent used

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for grafting. When DI water was used as grafting solvent, GA-grafted chitosan formed a

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stable colloidal dispersion (Figure 3), and was difficult to separate by centrifuging. To

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recover grafted chitosan, 187 mL 95% EtOH had to be stirred into the 50 mL reaction 11

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mixture for 30 min, followed by cooling in the refrigerator for 30 min to precipitate

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chitosan. Although grafting in water had the highest grafting efficiency, the precipitation

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of grafted chitosan was time-consuming and considered impractical for routine grafting.

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As concentration of EtOH in grafting solution increased, separation of grafted chitosan

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was easier. In 25% aq. EtOH, grafted chitosan was just slightly dispersed, and in 50%

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and 75% aq. EtOH was completely precipitated. No chitosan was dissolved (nor

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dispersed) in any of the solvents at the beginning of the grafting process, when all

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compounds were just mixed, but became dispersed in water as the reaction developed.

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Although chitosan dissolves in aqueous solutions only when pH is lower than 5, it

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extensively hydrates ("swells") in pure water. Addition of 50% or more of EtOH reduces

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chitosan's interaction with water and causes its precipitation (Nam, Kimura, Funamoto &

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Kishida, 2010). Thus, when grafting is conducted in pure water, more of chitosan's active

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sites are available for grafting with GA. Furthermore, reactivity of GA's carboxyl group

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and formation of GA-NHS ester is favored in pure water compared to aq. EtOH solutions

324

(Nam, Kimura & Kishida, 2008). As the EtOH concentration increases to 50% or higher,

325

it prevents hydration of the polysaccharide and thus reduces exposure and availability of

326

chitosan's active sites, resulting in reduced grafting efficiency and densely precipitated

327

grafted chitosan from the grafting solution (Fig. 3).

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0% aq. EtOH

50% aq. EtOH

328

25% aq. EtOH

75% aq. EtOH

Fig. 3.

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329

3.4. The solubility of grafted chitosans The solubility of freeze-dried grafted chitosan was also affected by the solvent used

331

for grafting and by efficiency of grafting. As shown in Fig. 4, when 0.1% freeze-dried

332

grafted chitosan was dissolved in 1% acetic acid, chitosans grafted in pure water and in

333

75% EtOH had better solubility in acidified water compared to those grafted in 25% and

334

50% EtOH (transmittance of ~72% vs. ~53%, respectively). Good solubility of highly

335

grafted chitosan (grafted in pure water) was most likely due to the presence of large

336

number of bulky phenolic groups of grafted GA (1 GA at every ~3.5 glucosamine units).

337

Additionally, water protected chitosan molecules from crosslinking by "shielding" its

338

hydroxyl and amino groups (Tonsomboon & Oyen, 2013; Zhang et al., 2009). However,

339

when a certain concentration of ethanol in the solvent was reached (approx. 25% - 50%),

340

EDC activated the hydroxyl groups of chitosan which formed crosslinking with the

341

amino groups (Chiou & Wu, 2004). On the other hand, when concentration of ethanol

342

during the reaction was as high as 75%, it prevented hydration of chitosan molecules,

343

EDC had limited accessibility to hydroxyl groups of chitosan what all prevented

344

crosslinking of chitosan.

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345

347 348 349 350 351 352

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Fig. 4.

353 354 355

3.5. NMR characterization of EDC/NHS activation of GA Although it is clear that presence of EtOH decreases the grafting efficiency of GA on 13

Page 13 of 24

chitosan, the factors contributing to this effect could include solubility of the

357

polysaccharide in ethanol and competition of ethanol with chitosan's hydroxyl or amine

358

groups for the activated carboxyl group on GA. To investigate possible causes, the effect

359

of solvent composition on EDC/NHS activation of GA was investigated using 1H qNMR.

360

We initially investigated the coupling in d4-methanol (CD3OD)/D2O solutions of a

361

GA/EDC/NHS mixture with varying concentrations of CD3OD, because methanol has

362

also been used as solvent for EDC/NHS reactions (Xing et al., 2007). Previous reports on

363

the structural elucidation of the reaction products between EDC/NHS and GA (Anderson,

364

2015), identified the singlet  at δ 7.04 ppm as the aromatic proton of starting GA (Fig.

365

5). Similarly, peak  at δ 7.16 ppm was identified as expected GA-NHS ester 5.

366

Integration of peak  as a function of methanol concentration showed that as the

367

methanol concentration increased, the amount of peak  produced during the reaction

368

decreased from 33.8% to 3.4 % of the amount of original GA (stacked 1H NMR spectra

369

in Fig. 6A). At the same time, increasing the methanol concentration resulted in the

370

formation of a new aromatic singlet ☐at δ 6.98 ppm. As the methanol concentration was

371

increased to 100%, peak ☐ increased from 0 to 33.9% of the amount of original GA used

372

(Fig. 6B). To investigate the unknown GA-related product, it was isolated from the

373

reaction mixture of GA and EDC/NHS in 100% methanol (MeOH), and characterized by

374

gHSQC and gHMBC (Fig. 7).

376 377 378 379

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380 381 382

14

Page 14 of 24

383 384 385

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cr

387

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388 389

an

390 391

M

392

d

393

te

394

396 397 398 399 400

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395

Fig. 5.

401 402 403

15

Page 15 of 24

404 405 406

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cr

408

us

409 410

an

411 412

M

413

d

414

Fig. 6.

416

We have identified this new compound as methyl gallate using 2D NMR

417

measurements. gHSQC shows an expected one-bond correlation between C2/C6 (108.96

418

ppm) and their attached protons at 7.12 ppm (Fig. 7A). Further, the one bond correlation

419

between C8 and H8 at 51.01/3.78 is consistent with the presence of a methoxy group in

420

the unknown product. When two bond correlations from gHMBC measurements are

Ac ce p

421

te

415

422

resulting from coupling between H8 and carbonyl carbon 7. The gHMBC spectrum also

423

shows the expected two and three bond correlations between the H2,6 and the other

424

carbons of the aromatic ring at 109.36/7.12 (H2/C6, H6/C2), 120.78/7.12 (H2,6/C4),

425

137.85/7.12 (H2,6/C1), 145.16/7.12 (H2,6/C3,5) and 166.35/7.12 (H2,6/C7). Each

426

correlation is very similar to the correlation between the GA carbons and protons

427

(Werner, Bacher & Eisenreich, 1997) and together provide support for our identification

428

of the side product formed during EDC/NHS coupling as methyl gallate (Ma, Wu, Ito &

16

Page 16 of 24

429

Tian, 2005). Observation of increasing amounts of methyl gallate as the concentration of

430

methanol in the reaction increased suggests a competitive reaction between methanol and

431

chitosan for the activated GA intermediate 5 (Scheme 1).

ip t

432 433

cr

434

us

435 436

an

437

M

438 439

8

2,6

(B)

d

440

Acetone-d6

O

441

8

5

8

7

1

O

2

3J

4

HO

442

3

OH

Ac ce p

2,6

443

6

HO

te

Acetone-d6

4 3,5 1 7

444 445 446

Fig. 7.

447

Further verification of this competitive reaction is provided when 100% MeOH was

448

substituted by 75% aq. EtOH, normally used for grafting gallic acid onto chitosan. Under

449

these conditions, ethyl gallate was isolated, dissolved in d6-acetone and measured by

450

similar gHSQC and gHMBC measurements (Fig. 8). One-bond correlation on gHSQC

451

spectrum (Fig. 8A) between C2/C6 (109.38 ppm) and H2/6 was observed at 7.13 ppm.

17

Page 17 of 24

452

Further, the one bond correlations between C8 and H8 at 60.08/4.25, C9 and H9 at

453

13.68/1.13 and two bond correlations on gHMBC (Fig. 8B) between C8 and H9 at

454

60.08/1.13, C9 and H8 at 13.68/4.25 are consistent with the presence of an ethyl group in

455

the unknown product. An additional correlation at 165.77/4.25 is observed and

ip t

456

group and carbonyl carbon 7. Similar to methyl gallate, the gHMBC spectrum also shows

458

the expected two and three bond correlations between the H2, 6 and the other carbons of

459

the aromatic ring at 108.86/7.12 (H2/C6, H6/C2), 121.22/7.12 (H2,6/C4), 137.71/7.12

460

(H2,6/C1), 145.11/7.12 (H2,6/C3,5) and 165.77/7.12 (H2,6/C7).

us

cr

457

461

an

462 463

M

464

d

465

te

466

468

Ac ce p

467

2,6

469 470

(B)

9

8 Acetone-d6

9

Acetone-d6

471

8

472

O HO

3,5

1

5

2,6

473

6

4 1

HO

4

7

O

8

9

2 3

OH

3J

7

474 475

Fig. 8.

18

Page 18 of 24

These results further indicate that EtOH (or MeOH) inhibits coupling of GA to

477

chitosan, which may proceed through a competitive conversion of the intermediate O-

478

acylisourea ester 3 to a very stable ethyl gallate (methyl gallate) instead of the target

479

compound, a more stable but still reactive NHS ester 5, which will further react with –

480

OH and –NH2 on chitosan. When used as the component of solvent for grafting GA to

481

chitosan, EtOH not only decreases the grafting efficacy by precipitating chitosan, it also

482

acts as another nucleophile, competing with NHS to attack O- acylisourea, forming ethyl

483

gallate, and negatively affecting the yield of GA-NHS ester and, thus decreasing the

484

grafting efficiency of GA at high EtOH co-solvent concentrations.

485

4. Conclusions

us

cr

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476

This study clearly showed that ethanol, as a solvent for grafting of GA onto chitosan

487

by EDC/NHS, reduces the grafting efficiency of the reaction by acting as a reactant and

488

decreasing the yield of GA-NHS ester. Although grafting in DI water without presence of

489

ethanol results in the highest reaction yield, it is impractical due to additional steps

490

needed to separate grafted chitosan from the reacting mixture. Concentration of 25%

491

EtOH in aqueous system seems the most practical due to the high grafting efficiency and

492

easily separable grafted chitosan. Utilizing these findings, a more efficient antioxidant

493

biodegradable packaging material can be created for controlling lipid oxidation and

494

extending shelf life of packaged food.

495

5. Acknowledgements

497 498 499 500

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an

486

This work was financially supported by UTIA Innovation Grant.

501 502 503 504 505

19

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Reference:

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619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663

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List of captions Scheme 1. EDC/NHS activation of gallic acid

671

Fig. 1. Confirmation of grafting by FTIR

672

FTIR spectra of GA grafted chitosan produced in pure deionized water (black), 25%

673

(red), 50% (green), 75% aq. EtOH (dark green), pure chitosan (pink), and chitosan mixed

674

with gallic acid (Mix) (blue).

675

Fig. 2. Effect of solvent composition on grafting effeciency

676

Effect of solvent composition on grafting efficiency: (A) Total phenolics (mg GA eq/g),

677

(B) DPPH scavenging (%) (0.001% grafted chitosan in 0.01% acetic acid), (C) Reducing

678

power (absorbance at 700 nm, 0.025% grafted chitosan in 0.25% acetic acid). Values are

679

presented as means with standard deviation. Bars with different letters are significantly

680

different (p < 0.05).

681

Fig. 3. Appearance of GA grafted chitosan dispersion after grafting reaction

682

Appearance

683

dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in 0%

684

(pure DI water), 25%, 50% and 75% aq EtOH immediately after grafting reaction.

685

Fig. 4. Solubility of grafted chitosan

686

Effect of solvent composition on solubility (% transmittance at 600 nm) of grafted

687

chitosan (0.1% chitosan dissolved in 1% acetic acid). Values are presented as means with

688

standard deviation. Bars with different letters are significantly different (p < 0.05).

689

Fig. 5. Quantitative 1H NMR spectra of GA, EDC and NHS reaction

690

(A) 1H NMR spectra of gallic acid (GA), EDC and NHS reaction in d4-Methanol

691

(CD3OD) /D2O solution with different concentrations, (B) Expansion of 5.8-8.6 ppm

692

region of the 1H NMR spectra of GA, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

693

(EDC) and N-hydroxysuccinimide (NHS) reaction in d4-Methanol (CD3OD) /D2O

694

solution with different concentrations (bottom upward: 0, 25, 50, 75, 100 %

695

CD3OD/D2O).  denotes peak of GA,  denotes peak of GA-NHS ester, ☐ denotes

M

acid

(GA)

d

gallic

grafted

chitosan

using

1-ethyl-3-(3-

Ac ce p

te

of

an

us

cr

ip t

670

23

Page 23 of 24

peak of unknown compound.

697

Fig. 6. Yield of GA-NHS ester and methyl gallate

698

Yield (%) of gallic acid - N-hydroxysuccinimide (GA-NHS ester) (A) and methyl gallate

699

(B) in 0, 25, 50, 75, 100 % CD3OD.

700

Fig. 7. gHSQC and gHMBC of methyl gallate

701

(A) gHSQC and (B) gHMBC of the isolation from reaction of gallic acid and EDC/NHS

702

in 100% methanol. gHSQC was acquired as 128 increments and 8 scans per increment,

703

giving an overall acquisition time of 10 min 47 s using nonuniformed sampling (NUS).

704

gHMBC was acquired as 200 increments and 8 scans per increment, giving an overall

705

acquisition time of 11 min 11 s using NUS.

706

Fig. 8. gHSQC and gHMBC of ethyl gallate

707

(A) gHSQC and (B) gHMBC of the isolation from reaction of gallic acid and EDC/NHS

708

in 75% ethanol aqueous solution. gHSQC was acquired as 128 increments and 8 scans

709

per increment, giving an overall acquisition time of 10 min 47 s using nonuniformed

710

sampling (NUS). gHMBC was acquired as 200 increments and 8 scans per increment,

711

giving an overall acquisition time of 11 min 11 s using NUS.

713 714 715 716

cr

us

an

M

d

te

Ac ce p

712

ip t

696

717 718

24

Page 24 of 24