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MODIFICATION OF CHITOSAN FOR SIMULTANEOUS

ANTIOXIDANT AND ANTIBACTERIAL FUNCTIONS

CHEN FEI

NATIONAL UNIVERSITY OF SINGAPORE 2009

MODIFICATION OF CHITOSAN FOR SIMULTANEOUS

ANTIOXIDANT AND ANTIBACTERIAL FUNCTIONS

CHEN FEI (B. ENG ECUST)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009

ACKNOWLEDGEMENT

Studying for a degree is a huge process that evolves over time and involves many role players, some of whom do not even realize the part they have played. I owe a huge debt to my supervisor Prof. Neoh Koon Gee, who gave me confidence and guidance to persist in my research works. I also want to express my thanks to my colleagues, Dr.

Shi Zhilong, Chua Poh Hui, Tan Lihan, Zhang Fan and Lim Siew Lay. Discussing and talking with them have always inspired me on my research work.

Finally, I would like to appreciate the financial support from the National University of Singapore.

i TABLE OF CONTENTS

ACKNOWLEDGEMENT i

TABLE OF CONTENTS ii

SUMMARY vi

NOMENCLATURE viii

LIST OF FIGURES ix

LIST OF SCHEMES x

LIST OF TABLES xi

1 Introduction 1

2 Literature review 5

2.1 Chitosan 5

2.1.1 Sources of chitosan 5

2.1.2 Chemistry of chitosan 7

2.1.3 Biological properties of chitosan 9

2.1.4 Applications of chitosan 11

2.2 Ascorbic acid 14

2.3 Essential oils 15

2.3.1 Major components of essential oils 15

2.3.2 Antibacterial activity of essential oils 16

2.3.3 Mode of antibacterial action of essential oils 17

2.4 Derivatization methods 19

2.4.1 Derivatization of chitosan 19

2.4.2 Derivatization of ascorbic acid 21

2.4.3 Formylation of essential oils 22

3 Antioxidant and antibacterial abilities of chitosan ascorbate 24

ii 3.1 Introduction 24

3.2 Experimental 26

3.2.1 Materials 26

3.2.2 Preparation of chitosan ascorbate 26

3.2.3 Characterization of chitosan ascorbate 26

3.2.4 Free radical scavenging ability of chitosan ascorbate 27

3.2.5 Antibacterial test of chitosan ascorbate 27

3.2.6 Cytotoxicity assay of chitosan ascorbate 28

3.3 Results and discussions 30

3.3.1 Characterization of chitosan ascorbate 30

3.3.2 Free radical scavenging ability of chitosan ascorbate 33

3.3.3 Antibacterial test of chitosan ascorbate 34

3.3.4 Cytotoxicity assay of chitosan ascorbate 35

3.4 Conclusion 37

4 Antioxidant and antibacterial activities of eugenol grafted chitosan nanoparticles

38

4.1 Introduction 38

4.2 Experimental 40

4.2.1 Materials 40

4.2.2 Synthesis of eugenol aldehyde 40

4.2.3 Preparation of chitosan nanoparticles 40

4.2.4 Grafting of eugenol on chitosan and chitosan nanoparticles through the

Schiff base reaction 41

4.2.5 Characterization techniques 41

4.2.6 Scavenging ability of eugenol grafted chitosan derivatives 42

iii 4.2.7 Determination of antibacterial activity of eugenol grafted chitosan

derivatives 42

4.2.8 In vitro cytotoxicity of eugenol grafted chitosan derivatives 42

4.3 Results and discussions 44

4.3.1 Synthesis of eugenol aldehyde 44

4.3.2 Characterization of eugenol grafted chitosan derivatives 46

4.3.3 Free radical scavenging ability of eugenol grafted chitosan derivatives

51

4.3.4 Antibacterial effects of eugenol grafted chitosan derivatives 52

4.3.5 Cytotoxicity assay of eugenol grafted chitosan derivatives 54

4.4 Conclusion 57

5 Antioxidant and antibacterial activities of carvacrol grafted chitosan

nanoparticles 58

5.1 Introduction 58

5.2 Experimental 59

5.2.1 Materials 59

5.2.2 Synthesis of carvacrol aldehyde 59

5.2.3 Preparation of chitosan nanoparticles 60

5.2.4 Grafting of carvacrol on chitosan nanoparticles through the Schiff base

reaction 60

5.2.5 Characterization techniques 60

5.2.6 Scavenging ability of carvacrol grafted chitosan nanoparticles 60

5.2.7 Determination of antibacterial activity of carvacrol grafted chitosan

nanoparticles 61

5.2.8 In vitro cytotoxicity of carvacrol grafted chitosan nanoparticles 61

iv 5.3 Results and discussions 62

5.3.1 Synthesis of carvacrol aldehyde 62

5.3.2 Characterization of carvacrol grafted chitosan nanoparticles 63

5.3.3 Free radical scavenging ability of carvacrol grafted chitosan

nanoparticles 67

5.3.4 Antibacterial effects of carvacrol grafted chitosan nanoparticles 68

5.3.5 Cytotoxicity assay of carvacrol grafted chitosan nanoparticles 69

5.4 Comparative study of carvacrol grafted chitosan nanoparticles and eugenol

grafted chitosan nanoparticles 72

5.4.1 Degree of grafting of essential oil components 72

5.4.2 Antibacterial activity 72

5.4.3 Antioxidant activity 73

5.4.4 Cytotoxicity 73

5.5 Conclusion 75

6 Conclusion and recommendations 76

6.1 Conclusion 76

6.2 Recommendations 79

7 References 81

v SUMMARY

Polysaccharides can be modified with various molecules bearing intriguing biological properties. The modified polysaccharides retain the bulk properties with additional biological activities. In this thesis, three small molecules from natural sources, namely, ascorbic acid, eugenol, and carvacrol were grafted via chemical reactions to chitosan, a popular polysaccharide. The main focus of this thesis is on the subsequent biological assays of the developed chitosan derivatives.

Firstly, ascorbic acid, a commonly used antioxidant, was grafted onto chitosan to yield chitosan ascorbate. A series of characterization techniques and biological assays were applied to identify the modified product and as well as its biological properties.

Next, aldehyde groups were introduced in eugenol and the modified eugenol was reacted with chitosan and chitosan nanoparticles (CH NPs) through the Schiff base reaction. The eugenol grafted chitosan (CHEU) and eugenol grafted chitosan nanoparticles (CHEU NPs) were then characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). CHEU and CHEU

NPs were then assayed to assess their free radical scavenging and antibacterial abilities. Their cytotoxicity towards 3T3 mouse fibroblasts was also investigated.

The modification strategy was then extended to another essential oil component, carvacrol. The formylated carvacrol was grafted onto the CH NPs. Similar characterization techniques and biological assays were then used to investigate the carvacrol grafted chitosan nanoparticles (CHCA NPs). Finally, a comparative study

vi was made between CHCA NPs and CHEU NPs to establish their suitability for potential biomedical applications.

In conclusion, the chitosan was successfully modified with ascorbic acid, eugenol or carvacrol either in the power form (CHAA and CHEU) or in the nanoparticle form

(CHEU NPs and CHCA NPs). these modified chitosan powders and nanoparticles have simultaneous antioxidant and antibacterial functions which may potentially be useful in biomedical and food packaging applications.

vii NOMENCLATURE

AA L-ascorbic acid

ATCC American type culture collection

CH Chitosan

CHAA Chitosan ascorbate

CHCA NPs Carvacrol grafted chitosan nanoparticles

CHEU NPs Eugenol grafted chitosan nanoparticles

DPPH Free radical diphenylpicrylhydrazyl

EC50 Equivalent concentration to give 50% effect

E. coli Escherichia coli

EO Essential oil

DMEM Dulbecco’s modified eagle’s medium

FTIR Fourier transform infrared spectroscopy

IC50 Half maximum inhibitory concentration

MBC Minimum bactericidal concentration

MIC Minimum inhibition concentration

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

NMR Nuclear magnetic resonance

ROS Reactive oxygen species

S. aureus Staphylococcus aureus

TEM Transmission electron microscopy

TSB Tryptic soy broth

XPS X-ray photoelectron spectroscopy

viii LIST OF FIGURES

Figure 2-1 Locations and mechanisms in the bacterial cell thought to be sites of action for EO components.

Figure 3-1 XPS N 1s core level spectra of (a) CH and (b) CHAA.

Figure 3-2 FTIR spectra of CH and CHAA.

Figure 3-3 Free radical scavenging abilities of CH and CHAA.

Figure 3-4 Viabilities of 3T3 fibroblasts (105 cells/ml) incubated with CH and CHAA dissolved in the culture medium containing 0.25% acetic acid for 72 hours. The viabilities are expressed as percentages relative to the result obtained in the control experiments without CH or CHAA.

Figure 4-1 UV-visible absorption spectra of eugenol before and after formylation.

Figure 4-2 XPS N 1s core-level spectra of (a) chitosan (CH); (b) chitosan nanoparticles (CH NPs); (c) eugenol grafted chitosan (CHEU); (d) eugenol modified chitosan nanoparticles (CHEU NPs).

Figure 4-3 FTIR spectra of CH, CHEU, CH NPs and CHEU NPs.

Figure 4-4 TEM investigations of CH NPs (a) and CHEU NPs (b). The scale bar is 1 μm.

Figure 4-5 Free radical scavenging abilities of (a) CHEU and CH; (b) CH NPs and CHEU NPs.

Figure 4-6 Viabilities of 3T3 mouse fibroblasts incubated with (a) CH and CHEU dissolved in culture medium containing 0.25% acidic acid; (b) CH NPs and CHEU NPs dispersed in the culture medium for 72 hours.

Figure 5-1 UV-visible absorption spectra of carvacrol before and after formylation.

Figure 5-2 XPS N 1s core-level spectrum of CHCA NPs.

Figure 5-3 FTIR spectra of CH NPs and CHCA NPs.

Figure 5-4 TEM investigation of CHCA NPs. The scale bar is 1 μm.

Figure 5-5 Free radical scavenging abilities of CH NPs and CHCA NPs.

Figure 5-6 Viabilities of 3T3 mouse fibroblasts incubated with CH NPs and CHCA NPs distributed in the culture medium for 72 hours.

ix LIST OF SCHEMES

Scheme 2-1 Structures of cellulose, chitin and chitosan.

Scheme 2-2 Preparation process of chitosan from chitin.

Scheme 2-3 Structural formulae of selected components of EOs.

Scheme 2-4 Derivatization of L-ascorbic acid at different carbon positions.

Scheme 2-5 L-ascorbic/L-dehydroascorbic acid interconversion.

Scheme 3-1 Molecular structure representations of modification process of chitosan by AA.

Scheme 4-1 Synthesis products of eugenol aldehydes: 1) eugenol; 2) 2-hydroxyl-3- methoxy-5-(2-propenyl) benzaldehyde; 3) 3-hydroxy-4-methoxy-6-(2- propenyl) benzaldehyde; 4) 3-hydroxyl-2-methoxy-6-(2-propenyl) benzaldehyde.

Scheme 4-2 Molecular representations of modified CH and CH NPs (5—7).

Scheme 5-1 Synthesis products of carvacrol aldehyde.

Scheme 5-2 Molecular representation of CHCA NPs.

x LIST OF TABLES

Table 2-1 MICs of chitosan in vitro against bacteria and fungi.

Table 2-2 MICs of selected essential oil components against food borne pathogens in vitro.

Table 2-3 Strategies for derivatization of chitosan.

Table 3-1 MICs and MBCs of CH and CHAA in TSB medium.

Table 4-1 Degree of acetylation of chitosan before and after grafting with eugenol and the degree of grafting.

Table 4-2 Characteristics of CH NPs and CHEU NPs.

Table 4-3 MICs and MBCs of chitosan and eugenol grafted chitosan derivatives in TSB medium against S. aureus and E. coli.

Table 5-1 Degree of acetylation of chitosan before and after grafted with carvacrol and the degree of grafting.

Table 5-2 Characteristics of CH NPs and CHCA NPs.

Table 5-3 MICs and MBCs of CH NPs and CHCA NPs in TSB medium against S. aureus and E. coli.

Table 6-1 Comparison of chitosan modified with essential oil components and

ascorbic acid.

xi Chapter 1

1 Introduction

Reactive oxygen species (ROS) and bacterial infections are two threats which the

human body, animals and even food are continuously exposed to. ROS may lead to

oxidative stress, which has been considered to be related to aging, many pathological

disorders, and even cancer (Hermans et al., 2007, Shetty et al., 2008, Reddy et al.,

2008). Food quality also deteriorates when exposed to ROS (Srinivasa and

Tharanathan, 2007). On the other hand, infections will result from bacterial

attachment and biofilm formation on surfaces in daily situations. Thus, materials

possessing antioxidant and antibacterial activities can be expected to have good

potential for applications. Of particular interest would be such materials developed

from natural sources. Materials developed from natural sources have played such an

important role in biomedical science because they are usually believed to be

biocompatible, eco-friendly and low cost. They are usually studied by two means: in

bulk form where they are modified to cater for special purposes; or as a functional group grafted on to the bulk materials to confer them with additional properties.

Three natural materials, chitosan (CH), essential oils (EOs) and L-ascorbic acid (AA),

have been selected for investigation in the current work because of their interesting

properties. CH is prepared from the chitin which is harvested from shrimp shells, crab

or fungal mycelia. CH can be easily made into nanoparticles, membranes and fibers.

With their versatile forms, CH and its derivatives are applied in drug delivery, wound

dressing and food packaging due to their biocompatible, antimicrobial and

pharmaceutical properties (Rabea, et al., 2003, Srinivasan and Tharanathan, 2007,

Kumar, 2000). Depending on the purposes of the various applications, different

1 Chapter 1 modification strategies were adopted. The basic principle for chitosan modification is to make use of either its hydroxyl groups at the C-3 and C-6 positions, or its amine groups (Rinaudo, 2008).

The richest natural sources of AA are fruits and vegetables. AA is very popular for its antioxidant property. It can also be modified and manipulated to form complex molecules for further applications. AA, can be alkylated and acylated under basic and acidic conditions, oxidized or reduced, and also be modified to form acetal and ketal derivatives.

EOs, extracted from natural plants, are known to possess medicinal properties, antimicrobial activity and even antioxidant property. The phenolic part of their structures is believed to confer their intriguing biological properties. On the other hand, their potential applications have been limited by their cytotoxicity to human cells and tissues. So there is an incentive to find a breakthrough to allow the utilization of their biological properties while concomitantly reducing the risk of damaging the normal cells and tissues.

In this work, the objective is to develop biomaterials based on CH as the bulk material, and modified with AA and EOs. The EOs and AA modified chitosan will possess both antioxidant and antibacterial activities which may be potentially useful for biomedical and food packaging applications. With this purpose in mind, a more detailed discussion of the properties of these three materials and the previous works related to the present study are given in Chapter 2.

2 Chapter 1

In Chapter 3, the modification technique used to prepare chitosan ascorbate (CHAA)

through the Schiff base reaction is presented. The CHAA was characterized by X-ray

photoelectron spectroscopy (XPS), and its biological assays showed that after

modification, CHAA possessed antioxidant properties while its antibacterial efficacy

was slightly lower compared to that of chitosan.

Essential oils contain a large number of aromatic components, which may be utilized

for functionalization purposes via appropriate strategies. Since the aromatic aldehyde formed by Schiff base reaction is more stable than CHAA, eugenol, one of the essential oil components, was grafted onto CH and chitosan nanoparticles (CH NPs) through Schiff base reaction as described in Chapter 4. The biological assays showed that the eugenol grafted chitosan (CHEU) and eugenol grafted chitosan nanoparticles

(CHEU NPs) were conferred with antioxidant and antibacterial abilities, and both of them showed lower cytotoxicity than free eugenol. CHEU NPs showed even higher biological activity efficacy due to their nanoparticulate form compared to CHEU.

The modification techniques were then extended to another essential oil component,

carvacrol. In Chapter 5, the process to introduce aldehyde group into carvacrol and

the subsequent grafting of carvacrol aldehyde onto CH NPs was describes. The

carvacrol grafted chitosan nanoparticles (CHCA NPs) were then characterized by

XPS and transmission electron microscopy (TEM). The biological assay showed that the CHCA NPs possessed both antioxidant and antibacterial activities. The cytotoxicity of CHCA NPs is significantly lower than that of pure carvacrol. The

CHCA NPs were finally compared with CHEU NPs with respect to their degrees of

grafting and biological activities.

3 Chapter 1

Finally, Chapter 6 gives the overall conclusion of the present work and the recommendations for the future work. Though the properties of chitosan have been successfully improved via chemical modifications, much work can still be done to further enhance its use in clinical and industrial applications.

4 Chapter 2

2 Literature review

This literature review focuses on the applications of three types of natural materials in

the domain of biomaterials: chitosan, EOs and AA. Their antioxidant and

antimicrobial mechanisms, and the modification strategies which are applicable to

these three materials are reviewed.

2.1 Chitosan

Chitin, or poly (N-acetyl-1, 4-β-D-glucopyranosamine) is the most abundant natural polysaccharide after cellulose. It may be regarded as cellulose with the hydroxyl group at the C-2 position replaced by an acetamido group. Chitin can be N-

deacetylated to obtain chitosan. The structures of cellulose, chitin and chitosan are

shown in Scheme 2-1. The following paragraph will focus on the chemistry,

properties and applications of chitosan.

2.1.1 Sources of chitosan

Chitin can be obtained from shrimp shells or crab and fungal mycelia (Kumar, 2000).

The N-deacetylation of chitin to obtain chitosan is usually realized by alkali treatment

as shown in Scheme 2-2. The alkali treatment can result in chitosan with different

degrees of deacetylation, depending on the alkali concentration, treatment duration

and temperature. According to Wu et al (1978), deacetylation of chitosan could reach

around 70% (degree of deacetylation, DD) in the first hour of alkali treatment in 50%

NaOH solution at 100 ºC, but it would take 5 hours to reach 80%. Further treatment

showed no increase of DD value. Domard et al. (1983) and Mima et al. (1983)

5 Chapter 2

proved their strategies to prepare highly and even fully deacetylated chitosan without much degradation of the chitosan molecular chains by repeating the alkali treatment

several times. Therefore, the preparation process of chitosan determines the resultant

DD value, which will influence the properties of chitosan.

OH OH

O O O O * OH OH

OH OH

Cellulose

OH OH

O O O O * OH OH

NHCOCH3 NHCOCH3

Chitin

OH OH

O O O O * OH OH

NHCOCH3 NH2

Chitosan

Scheme 2-1 Structures of cellulose, chitin and chitosan.

6 Chapter 2

OH OH OH OH

O O O O O O O O * OH OH NaOH * OH OH

Deacetylation NHCOCH3 NHCOCH3 NHCOCH3 NH2 Chitin Chitosan

Scheme 2-2 Preparation process of chitosan from chitin.

2.1.2 Chemistry of chitosan

Investigations of chitosan have been concerned with its preparation from chitin and its

resultant degree of deacetylation and molecular weight, as well as their effects on its

solution properties, since these chemical properties may significantly affect the

biological properties and applications of chitosan and its derivatives. The effects and

characterizations of such properties (DD, molecular weight and solubility) are briefly

discussed below.

(1) Degree of N-acetylation and degree of deacetylation

Takahashi et al (2008) and Chiu et al (2007) reported that a higher DD would lead to a higher antibacterial efficacy against Staphylococcus aureus (S. aureus) and

Escherichia coli (E. coli). According to Je et al. (2006), chitosan with a DD of 90%

has a higher scavenging reactive oxygen species (ROS) efficacy compared to those

with DDs of 75% and 50%.

Degree of N-acetylation (DA) is usually defined as the ratio of 2-acetamido-2-deoxy-

D-glucopyranose to 2-amino-2-deoxy-D-glucopyranose structural units. On the other

hand, another term DD, which is defined as the proportion of nitrogen which is in the

7 Chapter 2

form of amine groups, is more commonly used. DD can be determined by methods

including nuclear magnetic resonance (NMR), ultraviolet-visible spectroscopy (UV),

infrared spectroscopy (IR), circular dichroism (CD), colloid titration, etc (Miya et al.,

1980, Baxter et al., 1992, Rinaudo etc., 1992, Muzzarelli and Rocchetti, 1985). So far,

the most reliable method seems to be 1H NMR (Rinaudo, 2006).

(2) Molecular weight

The molecular weight of chitosan affects the antimicrobial ability (Guo et al., 2008,

Seyfarth et al., 2007, Tsai et al., 2006), drug delivery behavior (Zhou et al., 2008,

Gupta and Jabrail, 2008), hemostasis (Yang et al., 2008), as well as the antioxidant

ability of chitosan and its derivatives (Kim and Thomas, 2007, Je et al., 2004). Thus,

molecular weight is another important property of chitosan. The molecular weight

distribution can be determined by high performance liquid chromatography (HPLC)

(Wu, 1988). The term average molecular weight is often used and it can be simply

and rapidly determined by viscometry using the Mark-Houwink equation (Kumar,

2000). The Mark-Houwink equation is expressed as:

[η] = KM α

Where K and α have been determined in 0.1 M acetic acid and 0.2 M sodium chloride

solution: K=1.81× 10-3 and α=0.93. η is the intrinsic viscosity of chitosan solution

and M is the average molecular weights. (Kumar, 2000)

(3) Solubility

When the degree of deacetylation of chitin reaches about 50% , it becomes soluble in

aqueous acidic solution and is called chitosan (Rinaudo, 2006). Chitosan in acidic

media becomes a polyelectrolyte because of the protonation of the amine (–NH2)

8 Chapter 2

groups. The degree of protonation increases progressively, in tandem with the

progressive solubilization of chitosan. Complete solubilization is obtained when the

degree of protonation exceeds 50% and the stoichiometric ratio ([AcOH]/[Chit-NH2])

is 0.6 (Rinaudo et al., 1999). The solubility limits the applications of chitosan, thus

various modification techniques and derivatives have been developed to improve its

solubility. Copolymerization of maleic acid sodium onto hydroxypropyl chitosan and

carboxyethyl chitosan sodium yielded the water-soluble chitosan derivatives with antioxidant activity (Xie et al., 2001) and antibacterial activity (Xie et al., 2002).

2.1.3 Biological properties of chitosan

Chitosan is a non-toxic, biocompatible, and biodegradable amino polysaccharide with

interesting biological, physical, and pharmacological properties. It has notable

bioactivities including promotion of wound healing hemostatic activity, immunity

enhancement, hypolipidemic activity, mucoadhesion, eliciting biological responses,

and antimicrobial activity. Chitosan is also promising as a support polymer for drug

delivery, gene therapy, cell culture, and tissue engineering. In particular, the

antimicrobial activity of chitosan and its derivatives against different groups of

microorganisms, such as bacteria and fungi, have received considerable attention in

recent years (Kumar, 2000, Hirano, 1999). The antimicrobial and antioxidant

activities of chitosan and its derivatives are elaborated below.

(1) Antimicrobial activity of chitosan and its derivatives

Chitosan has been shown to be fungicidal against several fungi (Table 2-1) (Liu et al.,

2001). This antifungal property makes chitosan popular in food industry applications

such as food wraps (Jiang and Li, 2001) and food additives (Fang et al., 1994).

9 Chapter 2

Chitosan also inhibits the growth of various bacteria (Table 2-1) (Liu et al., 2001).

However, chitosan is insoluble in most solvents except dilute organic acids such as

acetic acid and formic acid. Thus, various chitosan derivatives were developed to

satisfy different applications without the loss of antibacterial activity.

Table 2-1 MICs of chitosan against bacteria and fungi in vitro.

Bacteria MICa (ppm) Fungi MIC (ppm) Agrobacterium tumefaciens 100 Botrytis cinerea 10 Bacillus cereus 1000 Fusarium oxysporum 100 Corynebacterium michiganense 10 Drechslera sorokina 10 Erwinia sp. 500 Micronectriella nivalis 10 Erwinia carotovora subsp. 200 Piricularia oryzae 5000 Escherichia coli (E. coli) 20 Rhizoctonia solani 1000 Klebsiella pneumoniae 700 Trichophyton equinum 2500 Micrococcus luteus 20 Pseudomonas fluorescens 500 Staphylococcus aureus (S. aureus) 20 Xanthomonas campestris 500

a MIC = minimum growth inhibitory concentration.

The exact mechanisms of the antimicrobial action of chitosan and its derivatives are still unknown (Rabea et al., 2003), but different mechanisms have been proposed.

Chitosan acts mainly on the outer surface of the bacteria. The positively charged amine groups of chitosan interact with the negatively charged microbial cell membrane, altering the cell membrane permeability (Chen et al., 1998, Fang et al.,

1994, Jung et al., 1999). Chitosan has also been known to act as a chelating agent which selectively binds metals and thus inhibits the production of toxins and microbial growth (Cuero et al., 1991). When chitosan is released from the cell wall of fungal pathogens by plant host hydrolytic enzymes, it penetrates into the nuclei of the fungi and interacts with RNA and protein synthesis (Hadwiger, 1985). 10 Chapter 2

(2) Antioxidant activity of chitosan and its derivatives

Free radicals, which come from ROS, may disturb the “redox homeostasis” of humans, causing oxidative stress on human; they may also deteriorate food quality upon penetrating food packages. Chitosan and its derivatives have also been evaluated as an antioxidant (Yen et al., 2008, Yen et al., 2007) to protect wounds and food from being attacked by ROS. The antioxidant activity of chitosan has been reported to depend on the preparation source, such as crab shells and Shiitake stipes, as well as its molecular weight (Sun et al., 2008, Sun et al., 2007, Koryagin et al., 2006). Chitosan can be conferred with antioxidant activity via different strategies, either by encapsulating chitosan with an antioxidant or derivatization of chitosan (Zhang et al., 2008,

Kosaraju et al., 2006, Guo et al., 2005, Xing et al., 2005b). The antioxidant activity mechanism of chitosan is still unknown. Xie et al. (2001) proposed that the amine groups of chitosan may contribute to the antioxidant activity of chitosan. However, there is also a report on the limited antioxidant activity of chitosan (Zhang et al.,

2008).

2.1.4 Applications of chitosan

(1) Chitosan nanoparticles

Chitosan is easily processed into different forms such as films, gels and nanoparticles.

The nanoparticulate system provides a particular useful platform, demonstrating unique properties with potential wide-range therapeutic applications such as drug delivery, gene delivery and cancer targeting. Yao et al. (1995) and Kas (1997) highlighted the preparation and properties of the chitosan nanoparticulate system. Due to the wide interests in the chitosan nanoparticulate system, the preparation methods are surveyed here.

11 Chapter 2

Ionotropic gelation. In this method, chitosan is dissolved in acetic acid and then

added dropwise into different concentrations of tripolyphosphate solution. The

particles are harvested by centrifugation and washed with Milli-Q water, followed by

freeze-drying (Kas, 1997, Kumar, 2000, Zhang et al., 2004). This method is the most

frequently used one.

Solvent evaporation techniques. In this method, chitosan in an aqueous acetic acid

solution is added to toluene and sonicated to form a water/oil (W/O) emulsion.

Glutaraldehyde in toluene is then added and the mixture is stirred at room temperature

to give cross-linked microspheres. The suspension is centrifuged, followed by

evaporation of the solvent. The microspheres are obtained after separation, washing

and drying (Gallo and Hassan, 1988).

Li et al. (1991) modified this solvent evaporation technique and renamed it “Dry-in-

Oil” by evaporating the W/O emulsion system at 50 °C under reduced pressure instead of adding of glutaraldehyde as the crosslinker. Pavenetto et al (1995)

developed a multiphase emulsion method using the solvent evaporation technique by a

three-step emulsification process: Step 1, aqueous drug solution and oil containing

stabilizers are combine to form a W/O emulsion; Step 2, the W/O emulsion is then

dispersed into the polymer solution; Step 3, the system is evaporated under reduced

pressure.

Precipitation/coacervation method. In this method, sodium sulphate solution is added

dropwise to chitosan in acetic acid solution with stirring and ultrasonication. The

12 Chapter 2

nanoparticles are harvested by centrifugation and washing process (Genta et al., 1995,

Berthold et al., 1996).

(2) Food packaging

Packaging is important in post-harvest preservation of fruits, vegetables and processed foods to achieve a relatively long shelf-life. Besides protection of food from physical change (mechanical damage during transit or storage, loss of consistency or crispness, loss of appearance, and sales appeal), the package should also have the ability to inhibit microbial infections and oxygen-based deterioration

(Srinivasa and Tharanathan, 2007). Due to their film-forming ability, antimicrobial ability and biodegradability, chitosan and its derivatives have been successfully used in food industry as an eco-friendly packaging material.

(3)Wound healing

Wound healing is the process of repairing injury to the skin and other soft tissues due to infections or in the normal aging process of cells (Meddahi et al., 1994). Wound healing involves three distinct phases: the inflammatory phase, the proliferative phase, and finally the remodeling phase (Calvin, 2000, Klenkler and Sheardown, 2004). The inflammatory response begins immediately after the wound happens, followed by the accumulation of bacteria and debris which need to be phagocytized or removed

(Degim, 2008). ROS released by the neutrophils can destroy viable tissues if they are not limited. Chitosan can be used via two means in the process of wound healing either in the form of wound dressings or in the form of a micro/nanoparticulate system for delivery of wound healing growth factors.

13 Chapter 2

Chitosan has been used as wound dressings because it is a natural, biocompatible,

biodegradable, hemostatic, and anti-infective mucoadhesive polymer in addition to its

positive charge property, film-forming and gelation characteristics. Chitosan as a

wound dressing has been shown to affect all stages of wound healing. Its hemostatic

activity can be seen in the inflammatory phase, and it also regulates the migration of

neutrophils and macrophages acting on repair processes such as fibroplasias and re-

epithelization, thus accelerating the wound healing process (Borchard and Junginger,

2001, Ueno et al., 2001). Deng et al (2007) have reported a chitosan-gelation sponge

wound dressing which possessed antibacterial ability and promoted the wound healing

process. The recent development in chitosan wound dressing include the

incorporation a procoagulant (polyphosphate) and an antimicrobial agent (silver) in

the chitosan dressing to improve hemostatic and antimicrobial properties (Ong et al.,

2008).

Chitosan has also been evaluated as a potential carrier for wound healing growth

factors in the form of micro- or nanoparticles. Controlled release of growth factor

from this chitosan microparticulate system has enhanced healing in the wound sites

(Degim, 2008).

2.2 Ascorbic acid

AA, also known as Vitamin C, is probably the most celebrated chemical since its

discovery in 1921 (Arrigoni and De Tullio, 2002). AA belongs to the six-carbon

lactone family which can be synthesized from glucose in the liver of most mammalian

species except humans (Padayatty et al., 2003), as humans lack the enzyme

gulonolactone oxidase, which is essential for the synthesis of 2-keto-l-gulonolactone,

14 Chapter 2 the immediate precursor of AA. The DNA encoding for gulonolactone oxidase has undergone substantial mutation, leading to the absence of a functional enzyme in humans. Therefore, without the ingestion of Vitamin C, a deficiency state with a broad spectrum of clinical symptoms will occur in humans. Scurvy, a clinical expression of Vitamin C deficiency, is a lethal condition unless appropriately treated

(Padayatty et al., 2003).

AA acts as an antioxidant which can efficiently scavenge toxic free radicals and other

ROS produced in cell metabolism. AA is an electron donor making it a reductant.

When it acts as a reductant, AA donates two electrons from a double bond between the C-2 and C-3. AA is an antioxidant because it prevents other compounds from being oxidized by donating its electrons. Thus, AA itself is oxidized in the process

(Buettner, 1993).

2.3 Essential oils

EOs are oily aromatic compounds extracted from natural plant materials. They were widely used in the food, dentistry and cosmetic industries because of their antibacterial, antifungal and antioxidant properties.

2.3.1 Major components of essential oils

The methods using for producing EOs include steam distillation and extraction by means of liquid carbon dioxide under low temperature and high pressure. The former is commercially used because of the lower cost. The major components of the economically interesting EOs are listed in Scheme 2-3.

15 Chapter 2

2.3.2 Antibacterial activity of essential oils

The antibacterial activity of EOs has been tested in vitro for many years. The antibacterial efficacies of EOs are commonly characterized by the minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC).

Though there are different definitions of these terms, MIC is generally defined as the lowest concentration resulting in no visible change in the turbidity of a suspension of the test organism for a certain incubation period, while MBC is defined as the concentration at which 99.9% or more of the initial inoculum is killed after a certain incubation period (Cosentino et al., 1999, Burt, 2004). Table 2-2 summarizes the

MICs of some EO components tested in vitro against food borne pathogens.

O

O O

O O

geranyl acetate eugenyl acetate trans-cinnamaldehyde

OH

HO OH

geraniol carvacrol thymol

OH

O O

eugenol limonene g-terpinene carvone

Scheme 2-3 Structural formulae of selected components of EOs

16 Chapter 2

Table 2-2 MICs of selected essential oil components against food borne pathogens in vitro.

Essential oil MIC, approximate Species of bacteria References components range (μl/ml) (Kim et al., 1995, Carvacrol E. coli 0.225-0.5 Cosentino et al., 1999) (Kim et al., 1995, S. typhimurium 0.224-0.25 Cosentino et al., 1999) S. aureus 0.175-0.45 (Cosentino et al., 1999) (Cosentino et al., 1999, L. monocytogenes 0.375-5 Kim et al., 1995) Eugenol E. coli 1.0 (Kim et al., 1995) S. typhimurium 0.5 (Kim et al., 1995) L. monocytogenes >1.0 (Kim et al., 1995) S. aureus 1.0 (Walsh et al., 2003) Geraniol E. coli 0.5 (Kim et al., 1995) S. typhimurium 0.5 (Kim et al., 1995) L. monocytogenes 1 (Kim et al., 1995) Thymol E. coli 0.225-0.45 (Cosentino et al., 1999) S. typhimurium 0.056 (Cosentino et al., 1999) S. aureus 0.140-0.225 (Cosentino et al., 1999) L. monocytogenes 0.450 (Cosentino et al., 1999) B. cereus 0.450 (Cosentino et al., 1999)

2.3.3 Mode of antibacterial action of essential oils

Although the antimicrobial abilities of EOs have been investigated in many research

groups, the mechanisms of action are not known in detail. Figure 2-1 shows the

possible action targets of the EOs against the bacterial cell (Burt, 2004).

17 Chapter 2

Figure 2-1 Locations and mechanisms in the bacterial cell thought to be sites of action for EO components (Burt, 2004).

It has been proposed that the hydrophobicity of EOs and their components enables

them to partition in the lipids of the bacterial cell membrane and mitochondria,

damaging the structures and making them more permeable (Knobloch et al., 1986,

Sikkema et al., 1994). Consequently, the cell constituents may leak out. The

continuing leakage would finally result in the death of the cell (Carson et al., 2002).

Generally, EOs with the strongest antibacterial activities contain a high percentage of

phenolic compounds, such as eugenol, carvacrol and thymol. Phenolic compounds are generally considered to disturb the cytoplasmic membrane, disrupt the proton motive force, electron flow, active transport and coagulation of cell contents in bacteria

(Sikkema et al., 1995, Denyer, 1991, Davidson, 1997). Thus, the phenolic structure of

EO components may contribute to the antibacterial properties of these compounds. A study on the chemical structures of individual EO components showed that the presence of hydroxyl group in phenolic compounds such as eugenol, carvacrol and thymol has been very important in the action against the bacteria (Dorman and Deans,

2000). EO components also appear to act on cell proteins embedded in the

18 Chapter 2 cytoplasmic membrane, interacting with the enzymes of the bacterial cell, causing bacteria death (Knobloch, 1989, Pol et al., 2001).

2.4 Derivatization methods

Functionalization of existing biomaterials has been studied for many years. The requirements for a normal material application always comprise both bulk and surface properties. Thus, the research on the modification of the existing natural materials is one of the foci in materials science. Compared to AA and EOs, chitosan is more extensively modified to suit various applications in a broad spectrum of areas.

2.4.1 Derivatization of chitosan

Among the many chitosan derivatives in the literature, one can differentiate between specific reactions involving the -NH2 group at the C-2 position or nonspecific reactions of hydroxyl (–OH) groups at the C-3 and C-6 positions (especially esterification and etherification) (Mourya and Inamdar, 2008). Some simple reactions involving C-2 position include the quaternization of the amine group or the reaction of an aldehydic functional group with –NH2 by reductive amination. One important thing to note is that more regular and reproducible derivatives would be obtained from highly deacetylated chitin (Domard and Rinaudo, 1983). Table 2-3 summarizes the general modification strategies for the chitosan (Mourya and Inamdar, 2008, Kumar,

2000).

19 Chapter 2

20 Chapter 2

2.4.2 Derivatization of ascorbic acid

There has been a great number of works on the chemistry of AA and its derivatives

(Tolbert, 1975, Andrews and Crawford, 1982). Based on this, many opportunities exist for the modification and manipulation of AA. Scheme 2-4 shows the potential reactivity of all the functional groups on AA (Andrews and Crawford, 1982).

6-sulfate, acyl, silyl, boryl 6 OH and methyl derivatives 5, 6-O-Ketal and acetal derivatives 5-acyl, methyl, boryl and silyl 5 OH derivatives O O proton exchange 4 1 C-2, C-3 reduction and oxidation 3 2

3-phosphate, silyl, methyl, HO OH boryl and acyl derivatives 2-sulfate, phosphate, silyl, boryl, 2, 3-O-Acetal methyl and acyl derivatives derivatives

Scheme 2-4 Derivatization of L-ascorbic acid at different carbon positions.

L-dehydroascorbic acid (DHA) (shown in Scheme 2-5) is the stable oxidation product of AA. Its three carbonyl groups can potentially react with a number of functional groups, such as amines.

OH OH

OH OH O O 2[H] O O

-2[H]

HO OH O O L-ascorbic acid L-dehydroascorbic acid

Scheme 2-5 L-ascorbic / L-dehydroascorbic acid interconversion.

21 Chapter 2

DHA still possesses reducing ability and can be degradatively oxidized by, for

example, molecular oxygen and hypoiodite ion to L-threonic and oxalic acids (Davies

et al., 1991).

2.4.3 Formylation of essential oils

The formyl group is potentially one of the most useful and versatile functional groups

to be introduced onto aromatic compounds. The accessibility coupled with versatile chemical properties makes aldehydes an important class of organic compounds (Olah

et al., 1987). As mentioned in Section 2.3, the components of EOs are mostly

aromatic compounds which can be formylated using proper strategies.

Eugenol, one of the major components of EO, is a phenolic compound. Reports about

derivatization of eugenol mostly involved the hydroxyl group on the phenolic ring

(Rahim et al., 2004, Sadeghian et al., 2008, Rojo et al., 2006). In order to preserve the hydroxyl group on eugenol, Bhagat et al (1982) synthesized four new derivatives of eugenol using halogen and N-bromo-succinimide as halogenating reagents. However,

the synthesis process required the use of tetrachloroform which is environmentally

unfriendly. The Reimer-Tiemann reaction may be used to formylate guaiacol to obtain

vanillin (Divakar et al., 1992). Since the structure of guaiacol is very similar to that of

eugenol, the Reimer-Tiemann reaction may be used to formylate eugenol to prepare eugenol aldehyde. Carvacrol, another important essential oil component, can also be formylated and reacted with amines (Knight et al., 2005).

Based on their respective properties, CH, EOs and AA are highly regarded in the biological field. With the understanding of their reaction mechanism, strategies can be

22 Chapter 2 developed to combine their intriguing properties. The following chapters will show how these modification strategies were utilized for preparing chitosan with antioxidant and antibacterial properties.

23 Chapter 3

3 Antioxidant and antibacterial abilities of chitosan

ascorbate

3.1 Introduction

There is no doubt of the importance of AA. AA, as a popular antioxidant, is commonly associated with the treatment of scurvy, once a fatal disease. The ability of

AA to scavenge free radicals, promote collagen biosynthesis, cause melanin reduction, provide photoprotection and enhance immunity, makes it widely applicable in the food, cosmetic and pharmaceutical industries (Bossi et al., 2000, Yamamoto et al.,

2002). However, AA is not just an antioxidant. Its remarkable function as a co- substrate of many important dioxygenases, in regenerating enzymes and its intriguing function in gene expression have been reviewed by Arrigoni (2002).

AA was reported to be encapsulated in CH NPs to prevent it from being decomposed into inactive compounds (Jang and Lee, 2008, Rojas and Gerschenson, 2001, Yuan and Chen, 1998). AA added into chitosan water solution was confirmed to improve chitosan solubility and form chitosan ascorbate (Muzzarelli, 1985). Chitosan ascorbate was considered to be useful in wound healing process (Pikiel and

Kopczewski, 1998). Although both the antibacterial and antioxidant properties can benefit the wound healing process, there have been no reports on these two properties of chitosan ascorbate.

The purpose of this study is to investigate the antibacterial and antioxidant abilities of chitosan ascorbate. For this purpose, chitosan ascorbate was first synthesized via the

24 Chapter 3

Schiff base reaction. The synthesis product was characterized by XPS and Fourier

Transform Infrared (FTIR) spectroscopy. The free radical diphenylpicrylhydrazyl

(DPPH) assay and MIC test were used to evaluate the antioxidant and antibacterial activities. Finally, the cytotoxicity of chitosan ascorbate towards 3T3 mouse fibroblasts was addressed.

25 Chapter 3

3.2 Experimental

3.2.1 Materials

Chitosan was purchased from CarboMer Inc and refined by dissolving in acetic acid and followed by filtration. The viscosity-average molecular weight was about 2.2

×105 and the degree of deacetylation was 84% as determined by CHN elemental analysis. AA and other chemicals were obtained from Sigma-Aldrich and used as received. S. aureus (American Type Culture Collection (ATCC) 25923), E. coli

(ATCC DH5α) and mouse fibroblast cells (3T3-Swiss albino) were supplied by

ATCC.

3.2.2 Preparation of chitosan ascorbate

Chitosan ascorbate (CHAA) was prepared as reported elsewhere (Muzzarelli et al.,

1984). Briefly, 1 g chitosan powder was suspended in 100 ml of water. Ascorbic acid was added (in an amount which is equimolar to the free amino groups of chitosan), resulting in the immediate dissolution of chitosan. The resulting light yellow solution was limpid and viscous, with pH between 4 and 5. After 6 hours of stirring, the solution was precipitated with acetone, and the raw product was dialyzed against water for 3 days. The CHAA was obtained after freeze-drying.

3.2.3 Characterization of chitosan ascorbate

The chemical composition of CHAA was analyzed by XPS on an AXIS HSi spectrometer (Kratos Analytical Ltd.) with an AlKα X-ray source (1486.6 eV photons). The XPS measurements were carried out as that reported earlier (Shi et al.,

26 Chapter 3

2005). All binding energies were referenced to the C 1s hydrocarbon peak at 284.6 eV.

In the peak analysis, the line width (full-width at half-maximum) of the Gaussian peak

was kept constant for all components in a particular spectrum. FTIR spectra of CH

and CHAA, dispersed in potassium bromide (KBr) and pelletized, were obtained

using a Bio-Rad FTIR model FT135 spectrometer under ambient condition.

3.2.4 Free radical scavenging ability of chitosan ascorbate

Predetermined amounts of the CHAA powder and CH powder were dispersed into 4

ml of DPPH ethanol solution (0.05 mM) with shaking for 2 hours. The solution was

then centrifuged and the absorbance of the supernatant was measured at 517 nm. The

inhibition ratio was expressed as follows:

Inhibition ratio % = (A0 –AS)/ A0 × 100;

Where A0 is the absorbance of the DPPH ethanol solution at 517 nm without test

sample and AS is the absorbance of the supernatant after reaction of DPPH with the

CH or CHAA.

3.2.5 Antibacterial test of chitosan ascorbate

The representative gram-positive and gram-negative bacteria (S. aureus and E. coli,

respectively) were cultured in tryptic soy broth (TSB, Sigma) medium. MIC was

determined by a turbidimetric method (Andrews, 2001, Qi et al., 2004). In present

work, since CH is insoluble in water, it was dissolved in culture medium containing

0.25% (v/v) acetic acid to obtain a stock solution of 8 mg/ml. This solution was

serially diluted in a 1:1 ratio with broth medium to obtain test sample solutions with

concentrations ranging from 0.08 mg/ml to 2 mg/ml. The bacteria suspension was

27 Chapter 3

then added to each sample solution to achieve a final bacterial concentration of 105 cells/ml. The bacterial concentration was estimated from the optical density of the suspension based on standard calibration with the assumption that the optical density of 1.0 at 600 nm for S. aureus suspension is equivalent to approximately 109 cells/ml

and the optical density of 1.0 at 540 nm for E. coli suspension is equivalent to

approximately 109 cells/ml. (Shi et al., 2005, Shi et al, 2006). All the test sample

solutions were incubated in an orbital shaker with a shaking speed of 200 rpm/min at

37 °C for 20 hours. The procedure was repeated with the test sample containing

CHAA. Solutions of culture medium containing acetic acid ranging from 0 to

0.0625% (v/v) were used as controls. The preliminary experiment showed the MIC

value of acetic acid is 0.25% (v/v). The MIC was determined as the minimum

concentration at which there is no visible change in the turbidity of the medium. The

MBC, defined as the lowest concentration of test sample that kills 99.9% of initial

inoculum, was determined in those test samples after the MIC test showed no growth.

The assay was carried out by counting the number of colonies after the bacteria were

cultured overnight on agar plates.

3.2.6 Cytotoxicity assay of chitosan ascorbate

The MTT assay is a standard colorimetric assay for measuring cellular proliferation

with the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

(Mosmann, 1983). In this work, the 3T3 fibroblasts cells were cultured in a

supplemented Dulbecco’s Modified Eagle’s Medium (DMEM) (DMEM 90%, fetal

bovine serum 10%, 1 mM L-glutamine and 100 U/ml penicillin-streptomycin) in a 5%

CO2 incubator at 37 °C. The CH and CHAA powder were dissolved in 0.25% acetic

acid solution to prepare the stock solution. Our preliminary experiment showed that

28 Chapter 3

0.25% acetic acid did not result in any cytotoxicity effects on the 3T3 mouse

fibroblasts. 100 μl cell suspension with a concentration of 105 cells/ml was seeded in a

96-well plate and the plate was incubated for 24 hours. The medium was then substituted with 100 μl medium containing the CH or CHAA at predetermined concentrations. After different periods of incubation, the medium was replaced with

100 μl medium containing the MTT reagent (0.5 mg/ml). After 2 hours incubation, the medium was removed and the formazan crystals were dissolved with 100 μl dimethyl sulfoxide for 15 minutes. The optical absorbance was then measured with a microplate reader (Tecan GENios) at a wavelength of 570 nm. Control experiments were carried out in the same manner using the complete stock solutions without test samples.

29 Chapter 3

3.3 Results and discussions

3.3.1 Characterization of chitosan ascorbate

When the AA was added into the chitosan-water suspension, chitosan dissolved

rapidly. The AA was oxidized to DHA in water solution and may react with the amine

groups on chitosan molecules through Schiff base reaction (Muzzarelli, 1985). The

possible reaction mechanism is shown in Scheme 3-1. The successful modification of

CH was first confirmed by XPS analysis. The N 1s core-level spectra of CH and

CHAA are shown in Figure 3-1. The N 1s core-level spectrum of CH (Figure 3-1(a))

can be fitted with two component peaks at 399.4 eV and 400 eV which are attributed

to NH and –O=C-N groups. The ratio of –O=C-N to total nitrogen indicates the

degree of acetylation of chitosan. The degree of acetylation of chitosan calculated

from Figure 3-1(a) is 15.7%, which is very close to the value of 16% from CHN

elemental analysis. After modification with AA (Scheme 3-1), two new peaks appears

at 398.5 eV and 401.6 eV in the N 1s core-level spectrum of CHAA, which can be

attributed to =N- (Figure 3-1(b)) and N+. The =N- species indicates the formation of

the Schiff base. On the other hand, the protonated nitrogen may be the result of ionic

interactions between chitosan and AA in the solution. The degree of acetylation

calculated from Figure 3-1(b) is 15.2%, which is also consistent with the value

obtained from elemental analysis of CH. The degree of grafting of AA on CH as calculated from the ratio of =N- species to the total nitrogen in Figure 3-1(b) is 49%.

30 Chapter 3

OH

OH O O

HO OH

L-ascorbic acid

OH OH OH

OH O O O O O O * OH OH +

NHCOCH3 NH2 O O

Chitosan L-dehydroascorbic acid

OH OH

O O O O * OH OH

NHCOCH3 N

O C C

HO O O

HO Chitosan ascorbate

Scheme 3-1 Molecular structure representation of modification process of chitosan by AA.

31 Chapter 3

Figure 3-1 XPS N 1s core level spectra of (a) CH and (b) CHAA.

The FTIR spectra of the CH and CHAA are shown in Figure 3-2. The absorption

bands at 3420 cm-1 and 1078 cm-1 can be attributed to υ(OH) and δ(C-O-C), respectively, which are the two characteristic bands of chitosan. After modification, an additional band appears at 1720 cm-1, due to the presence of carbonyl groups.

There is no absorption band at around 1760 cm-1, implying the absence of α-β

unsaturated cyclic ketones (Muzzarelli, 1984). While absorption bands due to υ(NH)

32 Chapter 3 and amide group were observed at about 1656 cm-1 and 1600 cm-1, respectively, for

CH, a broad absorption band between 1660 cm-1 and 1550 cm-1 was observed for

CHAA. This is due to the loss of the amine groups in the modification of CH to form

CHAA, as well as the formation of the C=N group, which has an absorption band at around 1640 cm-1.

Figure 3-2 FTIR spectra of CH and CHAA.

3.3.2 Free radical scavenging ability of chitosan ascorbate

The most promising property of AA is its antioxidant activity. Chitosan can be used as a wound dressing, as mentioned in Section 2.1.4. CHAA may have the potential to be used as wound dressing with enhanced wound healing ability. AA has been reported to possess a high antioxidant efficacy with an EC50 (equivalent concentration to give 50% effect) value of 5 μg/ml (Kim et al., 2002). The scavenging ability of

CHAA was compared to CH in Figure 3-3. It can be seen that CHAA shows scavenging ability while CH shows almost no scavenging ability. The EC50 value of

CHAA is determined to be 3.6 mg/ml from Figure 3-3.

33 Chapter 3

Figure 3-3 Free radical scavenging abilities of CH and CHAA.

3.3.3 Antibacterial test of chitosan ascorbate

The antibacterial activity of chitosan has been studied thoroughly, as described in

Chapter 2, while AA has not been reported to have antibacterial activity. The antibacterial activity of CH and CHAA were tested using two parameters: MIC and

MBC. The MIC and MBC values of CH and CHAA against S. aureus and E. coli are shown in Table 3-1. The MIC and MBC of CHAA are 0.25 mg/ml while the equivalent values for CH against the respective bacteria are generally lower. This means that the modification process compromised the antibacterial activity of chitosan to some degree. Since the amine groups of chitosan are responsible for the antibacterial activity, the reduced activity can be attributed to the consumption of some (but not all) of the amine groups in the modification process. However, this

34 Chapter 3

effect is compensated by the additional antioxidant properties and the CHAA still

showed significant antibacterial ability. Wounds are usually accompanied by

microbial infections and free radicals accumulation which would delay the wound

healing process (Houghton et al., 2005). Thus, the confirmed antibacterial ability of

CHAA, as well as its antioxidant property, would be beneficial the wound healing process. With CH or CHAA solution at 0.25 mg/ml, the contribution of acetic acid to the killing of the bacteria is minimal as determined from the control experiments.

Table 3-1 MICs and MBCs of CH and CHAA in TSB medium.

Sample MIC (mg/ml) MBC(mg/ml) S. aureus E. coli S. aureus E. coli CH 0.125 0.032 0.25 0.125 CHAA 0.25 0.25 0.25 0.25

3.3.4 Cytotoxicity assay of chitosan ascorbate

AA has been reported to have cytotoxic effects under certain conditions. It has been

shown to be cytotoxic to several types of normal and tumor cells in vitro (Bram et al.,

1980, Jampel, 1990). The cytotoxicity is believed to be dependent on the cell density

and AA concentration. With 105 human skin fibroblasts cells/well, a concentration of

0.1 mM (17.6 μg/ml) AA resulted in 66% cell viability (Murakami et al., 1992). Our

experiment showed that AA would cause death of 3T3 mouse fibroblast cells above a

concentration of 20 μg/ml. The MTT results of CH and CHAA towards 3T3

fibroblasts are shown in Figure 5-4. It can be seen that the cells incubated with CHAA

grew better than those incubated with CH. At concentrations close to the MIC values

of CHAA for S. aureus and E. coli, the fibroblast viability is about 105%. When the

concentration increases to 0.5 mg/ml, the fibroblast viability is still above 100%. Thus,

35 Chapter 3 by grafting AA to chitosan, antibacterial and antioxidant properties can be achieved with reduced mammalian cell cytotoxicity as compared to AA.

Figure 3-4 Viabilities of 3T3 fibroblasts (105 cells/ml) incubated with CH and CHAA dissolved in the culture medium containing 0.25% acetic acid for 72 hours. The viabilities are expressed as percentages relative to the result obtained in the control experiments without CH or CHAA.

36 Chapter 3

3.4 Conclusion

In this section, the synthesis of chitosan ascorbate (CHAA) was demonstrated. The in vitro DPPH assay and MIC test confirmed that CHAA possesses both free radical scavenging ability and antibacterial property while chitosan only has antibacterial property. The antibacterial efficacy of CHAA against E. coli and S. aureus is lower than that of CH because of the consumption of some amine groups on chitosan during the grafting process. Nevertheless, CHAA still possesses significant antibacterial efficacy with an MIC value of 0.25 mg/ml against both bacteria. The decrease in antibacterial activity is compensated by additional antioxidant ability. The modification also decreases the cytotoxicity of CHAA compared to AA. Since the grafting reaction takes place between the amine groups of chitosan and ascorbic acid, this technique can be used to modify chitosan in the form of nanoparticles, films and fibers.

37 Chapter 4

4 Antioxidant and antibacterial activities of eugenol

grafted chitosan nanoparticles

4.1 Introduction

Eugenol, as a major component of EOs, is an aromatic compound extracted from

clove, nutmeg and cinnamon. It has been widely used in food, dentistry and cosmetic

industries because of its antibacterial, antifungal and antioxidant properties (Kalemba

and Kunicka, 2003, Burt, 2004, Bakkali et al., 2008). Eugenol is one of ingredients of

zinc oxide eugenol cement (ZOE), which has been widely used in dentistry for

indirect pulp capping, and also as a temporary filling and root canal sealer (Markowitz

et al., 1992). Eugenol’s antimicrobial activity may be the result of its hydrophobicity and hydroxyl group. The hydrophobicity of eugenol may enable it to partition in the

lipids of bacterial cell membrane and mitochondria, damaging the structures and

making cell membrane more permeable (Knobloch et al., 1986). The hydroxyl group

is thought to bind to proteins, thus preventing enzyme action (Wendakoon and

Sakaguchi, 1993).

Eugenol has been reported to be a highly effective antioxidant (Ogata et al., 2000,

Atsumi et al., 2005, Mastelic et al., 2008) at low concentration, while it act as a

prooxidant at high concentration, causing tissue damage due to the formation of

harmful phenoxyl radicals (Decker, 1997, Suzuki et al., 1985). The antioxidant

potency of eugenol probably depends on its aromatic chemical structure, where the

electrons are delocalized on the aromatic nucleus (Kitagawa et al., 1992).

Recently, eugenol’s ability to inhibit cancer cell growth was reported as well

(Carrasco et al., 2008, Pisano et al., 2007). However, it has also been reported that

38 Chapter 4

eugenol irritates periapical tissue, and may have some degree of toxicity (Manabe et

al., 1987). For instance, eugenol caused a depression of cell membrane surface tension

at the concentration of 0.1 mM resulting in rat hepatocytes membrane lysis (Manabe

et al., 1987).

In present study, we investigated the combination of two natural compounds, eugenol

and chitosan, to obtain a product with antibacterial and antioxidant properties and yet

low cytotoxicity. Eugenol was first modified with aldehyde groups and then reacted

with chitosan and chitosan nanoparticles through the Schiff base reaction. The

antioxidant and antibacterial activities of eugenol grafted chitosan were assayed, and the cytotoxicity towards mouse 3T3 fibroblasts was also investigated.

39 Chapter 4

4.2 Experimental

4.2.1 Materials

Sodium tripolyphosphate (TPP) and eugenol (98%) was obtained from Sigma-Aldrich and used as received. The S. aureus, E. coli and fibroblasts were obtained from

ATCC as mentioned in Chapter 3.

4.2.2 Synthesis of eugenol aldehyde

A general Reimer-Tiemann reaction was used to synthesize eugenol aldehyde

(Divakar et al., 1992). 22 mmol eugenol was dissolved in 150 ml water at 80 ºC.

After bringing down the temperature of the mixture to 60 ºC, 400 mmol KOH and 88 mmol chloroform were added. The latter was added at a rate of 1 ml per hour over a period of 7 hours. The reaction mixture was kept at 60 ºC for another 8 hours. Post- treatment was initiated with the acidification of the solution with dilute sulfuric acid, and eugenol aldehyde was recovered by extraction with methylethyl ketone.

4.2.3 Preparation of chitosan nanoparticles

A general ionic gelation method was used to prepare chitosan nanoparticles (CH NPs)

(Zhang et al., 2004, Shi et al., 2006). The procedure was as follows: 0.5% (w/v) chitosan in 1% (v/v) acetic acid solution was adjusted to pH of about 4.1 with 10 N sodium hydroxide. 10 ml of 2.5 mg/ml TPP solution was then added dropwise into 30 ml of chitosan solution with stirring at 1000 rpm for 15 min. The nanoparticles were then separated by centrifugation at 15000 rpm for 30 min. The supernatant was removed and the CH NPs were rinsed with water followed by freeze-drying.

40 Chapter 4

4.2.4 Grafting of eugenol on chitosan and chitosan nanoparticles

through the Schiff base reaction

The Schiff base reaction was used for grafting the eugenol aldehyde onto the CH

powder and CH NPs. A 100 ml round bottom flask with stirrer was charged with 30

ml methanol and 100 mg CH powder or CH NPs, followed by addition of an excess

amount of eugenol aldehyde (based on the amount of amine group in chitosan). The

reaction mixture was refluxed for 48 hours. The eugenol grafted CH (CHEU) or

eugenol grafted CH NPs (CHEU NPs) were obtained by centrifugation at 15000 rpm

after washing with ethanol several times and drying in a vacuum oven at 40 ºC for 12

hours.

4.2.5 Characterization techniques

The UV-visible absorption characterization of the synthesized eugenol aldehyde was

investigated using a Shimadzu UV-3101 PC scanning spectrophotometer. 1H NMR

spectra were measured on a Bruker ARX 300 MHz spectrometer, using deuterated chloroform as the solvent. The chemical composition of CHEU and CHEU NPs were analyzed by XPS. The detailed process was described in Section 3.2.3. FTIR spectrum of CHEU and CHEU NPs was obtained as described in Section 3.2.3.

The nanoparticles were examined using a field-emission scanning electron

microscope (FE-SEM, JEOL JSM 6700F) under the TEM mode. The particle sizes

and zeta potentials of CH NPs and CHEU NPs were measured using a Zetasizer Nano

ZS (Malvern Instruments, Southborough MA) with a laser of 633 nm wavelength at a

90º scattering angle. The suspensions containing the different nanoparticles were

prepared with Mili-Q water. The mixture was vortexed for 30 s before the

41 Chapter 4

measurement was conducted. The size measurement was conducted at 25°C in

triplicate. The zeta potential measurement was performed using a capillary zeta potential cell in automatic mode in triplicate and the average values were reported.

4.2.6 Scavenging ability of eugenol grafted chitosan derivatives

The scavenging abilities of CHEU, CH, CHEU NPs and CH NPs were measured and expressed as described in Section 3.2.4.

4.2.7 Determination of antibacterial activity of eugenol grafted chitosan

derivatives

For the antibacterial assay, the stock solutions of CH and CHEU were prepared by dissolving them in broth medium containing 0.25% (v/v) acetic acid due to their insolubility in the water. These test samples were then diluted to obtain a series of concentrations, as described in Section 3.2.5. On the other hand, the CH NPs and

CHEU NPs were directly dispersed in broth medium. The rest of the procedures are as

described in Section 3.2.5.

4.2.8 In vitro cytotoxicity of eugenol grafted chitosan derivatives

The MTT assay was utilized to investigate the cytotoxicity of CHEU and CHEU NPs

towards 3T3 mouse fibroblasts. The stock solutions of CH and CHEU were prepared

by dissolving them in culture medium containing 0.25% acetic acid. The solution

without CH or CHEU was used as controls. The bulk solution was then diluted to

form a series of concentrations as described in Section 3.2.6. On the other hand, the

CH NPs and CHEU NPs were directly dispersed in the cell culture medium, and the

42 Chapter 4 medium without the nanoparticles was used as control. The rest of the procedures are as described in Section 3.2.6.

43 Chapter 4

4.3 Results and discussions

4.3.1 Synthesis of eugenol aldehyde

The UV-visible absorption spectra of phenolic aldehydes indicate a peak at around

310 nm due to n-π* transition of the keto group, and another at around 275 nm attributed to π- π* transition of the phenolic portion (Divakar et al., 1992). Therefore,

UV-visible absorption spectroscopy can provide direct evidence for the successful synthesis of eugenol aldehyde. The UV-visible absorption spectra of eugenol before

and after formylation are compared in Figure 4-1. The peak at 282 nm is characteristic

of the phenolic group of eugenol. After formylation of eugenol, the appearance of two

peaks at 311 nm and 350 nm indicates the formation of a mixture of phenolic

aldehyde groups.

Figure 4-1 UV-visible absorption spectra of eugenol before and after formylation.

44 Chapter 4

From 1H NMR analysis of the eugenol before and after formylation, three new signals are observed in the eugenol aldehyde products: 10.97 ppm for the ortho-aldehyde,

9.88 ppm and 9.84 ppm for two forms of meta-eugenol aldehyde (Divakar et al.,

1992). From the UV-visible absorption spectroscopy and NMR analysis, the possible synthesis products from eugenol are given in Scheme 4-1. The low NMR signal of one of the meta-eugenol aldehydes indicates its low concentration relative to the ortho- and the other meta-aldehyde products and hence its presence was not discernible from the UV-visible absorption spectrum in Figure 4-1(b). The product yield of eugenol aldehydes as estimated from the 1H NMR spectra, was about 20%.

Our preliminary experiments showed that eugenol did not react with the chitosan powder or chitosan nanoparticles. Hence, the grafting reactions with chitosan powder or chitosan nanoparticles were carried out using the eugenol aldehydes without the need for further separation from the eugenol.

O OH OH OH OH O O O O CHCl3 + + O KOH

O

1 243

Scheme 4-1 Synthesis products of eugenol aldehydes: 1) eugenol; 2) 2-hydroxyl-3-methoxy-5-(2- propenyl) benzaldehyde; 3) 3-hydroxy-4-methoxy-6-(2-propenyl) benzaldehyde; 4) 3-hydroxyl-2- methoxy-6-(2-propenyl) benzaldehyde.

45 Chapter 4

4.3.2 Characterization of eugenol grafted chitosan derivatives

Jung and coworkers (2006) prepared eugenol-grafted chitosan hydrogels by grafting

eugenol to chitosan via cerium (IV) ammonium nitrate mediated polymerization.

Their DPPH assay showed that the hydrogel possessed antioxidant activity. In our

work, eugenol aldehyde was grafted to chitosan and chitosan nanoparticles through

Schiff base reaction (Knight et al., 2005). The successful modifications of CH and

CH NPs were first confirmed by XPS analysis. The N 1s core-level spectra of CH,

CH NPs, CHEU and CHEU NPs are shown in Figure 4-2. The N 1s core-level

spectrum of CH (Figure 4-2(a)) is the same as that shown in Chapter 3. For CH NPs

(Fig 4-2(b)), an additional high binding energy (BE) peak (above 400 eV) is observed

and attributed to the positively charged nitrogen (N+), which may be the result of

ionic interaction with TPP during the nanoparticle preparation process. After Schiff

base reaction (Scheme 4-2), a new peak appears at 398.5 eV in the N 1s core-level

spectra of CHEU and CHEU NPs, which is attributed to =N- (Figure 4-2(c)). The

degree of grafting of eugenol on CH NPs is calculated from the ratio of =N- to the total nitrogen. The degree of acetylation of chitosan and the degree of grafting of eugenol as determined from the XPS analysis are summarized in Table 4-1. Under the same reaction conditions, the degree of grafting of eugenol on chitosan is half of that on CH NPs. The higher degree of grafting of the latter is likely to be the result of the increased surface area of the nanoparticles. As expected, the grafting of eugenol did not significantly affect the degree of acetylation of chitosan.

46 Chapter 4

HO

5)CH or CH NPs grafted with 2: a=OH, b=OCH3, c=H, d=2-propenyl,e=H; 6)CH or CH NPs grafted with 3: a=H, b=OH, c=OCH , d=H, e=2-propenyl; O * 3 OH 7)CH or CH NPs grafted with 4: a=2-propenyl, b=H, c=H, d=OH, e=OCH3; *

N

a

e b

d c

5-7

Scheme 4-2 Molecular representations of modified CH and CH NPs (5—7).

Table 4-1 Degree of acetylation of chitosan before and after grafting with eugenol and the degree of grafting.

Degree of grafting of Degree of acetylation eugenol CH 15.7% --- CH NPs 16.2% --- CHEU 15.7% 12.0% CHEU NPs 17.2% 26.7%

47 Chapter 4

Figure 4-2 XPS N 1s core-level spectra of (a) chitosan (CH); (b) chitosan nanoparticles (CH NPs); (c) eugenol grafted chitosan (CHEU); (d) eugenol modified chitosan nanoparticles (CHEU NPs).

Figure 4-3 FTIR spectra of CH, CHEU, CH NPs and CHEU NPs.

48 Chapter 4

The FTIR spectra of the CH, CHEU, CH NPs and CHEU NPs are shown in Figure 4-

3. The characteristic band at 3400 cm-1 corresponds to the combined peaks of the -

NH2 and -OH group stretching vibration in chitosan. The two absorption bands at

1650 cm-1 and 1592 cm-1 attributed to υ(C=O) and υ(NH), characteristic of chitosan,

are shifted to 1637 cm-1 and 1540 cm-1, respectively, after nanoparticles formation

(Knaul et al., 1999). The polyphosphoric groups of sodium polyphosphate may

interact with the ammonium groups of chitosan, enhancing both the inter- and intra-

molecular interactions between chitosan nanoparticles, resulting in the shift of the

υ(C=O) and υ(NH) absorption bands. After the Schiff base reaction, the intensity ratio of the υ(NH) band at 1540 cm-1 to the υ(C=O) band at 1637 cm-1 in the spectrum of

CHEU NPs decreased in comparison to the corresponding ratio of CH NPs. A similar

effect was observed when the spectra of CH and CHEU were compared. This

decrease arises as amine groups on the surfaces of CH NPs and CH powder are

consumed in the formation of –C=N- groups.

Table 4-2 shows the size and zeta potential of CH NPs and CHEU NPs. The average

hydrodynamic diameter of CH NPs and CHEU NPs were determined to be 217 nm

and 235 nm, respectively. The average size of CH NPs (Figure 4-4(a)) and CHEU

NPs (Figure 4-4(b)) from TEM pictures are both around 100 nm. The larger size

determined by the Zetasizer is probably due to the different measurement

environment. The NPs are suspended in water when measured by the Zetasizer, but

under the vacuum environment of the TEM, the NPs lose water, causing them to

shrink. As expected, there is no substantial change in the size of the nanoparticles

after grafting with eugenol. As shown in Table 4-2, the zeta potential of CHEU NPs

decreases to 28.4 mV compared to 37.6 mV of CH NPs and this decrease may have

49 Chapter 4

a

b

Figure 4-4 TEM investigations of CH NPs (a) and CHEU NPs (b). The scale bar is 1 μm.

50 Chapter 4

resulted from the consumption of amine groups in the Schiff base reaction. It will be shown in Section 4.3.4 that this decrease in zeta potential does not compromise the antibacterial action of CHEU NPs.

Table 4-2 Characteristics of CH NPs and CHEU NPs.

Hydrodynamic NPs Polydispersity Zeta potential (mV) size (nm) CH NPs 217±17 0.595 +37.6±1.3 CHEU NPs 235±12 0.318 +28.4±2.2

4.3.3 Free radical scavenging ability of eugenol grafted chitosan

derivatives

The process of wound healing includes inflammation, cell proliferation and contraction of collagen lattice formation. When a wound is incurred, it is accompanied, within a short time by pain, reddening and edema, which are the typical symptoms of inflammation caused by the release of eicosanoids, prostaglandins, leukotrienes and ROS (Sidhu et al., 1999, Houghton et al., 2005). These oxygen- derived species consist of oxygen radicals and certain non-radicals that are oxidizing agents and/or are easily converted into radicals. Antioxidants have been shown to play a role in delaying or preventing oxidative stress caused by free radicals (Hermans et al., 2007). Chitosan is widely used in wound healing, because it enhances the functions of inflammatory cells such as polymorphonuclear leukocytes, macrophages and fibroblasts; consequently, it promotes granulation and the organization phase of healing (Minagawa et al., 2007, Ueno et al., 2001, Degim, 2008). Conferring chitosan with eugenol ligands may retain its favorable biological properties for wound healing while providing promising antioxidant activity to accelerate the wound healing process.

51 Chapter 4

It has been reported that eugenol exhibits high free radical scavenging ability with

EC50 values of about 9 μg/ml (Mastelic et al., 2008). The free-radical scavenging

ability of CHEU and CHEU NPs are compared to CH and CH NPs, respectively, in

Figure 4-5. It can be seen that both CHEU and CHEU NPs show free radical

scavenging ability dependent on their concentrations in the range of from 0.25 mg/ml

to 4 mg/ml. The EC50 value for CHEU is above 4 mg/ml while the EC50 of CHEU

NPs is 2.6 mg/ml (Figure 4-5). The nanoparticle form of CH improved the reaction

condition and increased the degree of grafting of eugenol, resulting in a higher

antioxidant efficacy. As a control, the CH and CH NPs showed nearly no free radical

scavenging ability. Although some researchers have reported the limited antioxidant

properties of chitosan nanoparticles, their antioxidant activity largely depends on the

molecular weight and degree of deacetylation (Je and Kim, 2006, Xing et al., 2005a).

4.3.4 Antibacterial effects of eugenol grafted chitosan derivatives

The mechanisms of the antibacterial action of CH have not been fully understood, it is

believed that the interactions between the protonated amine units of CH and the

negatively charged cell membrane leads to disruption of the membrane functions and

leakage of cellular components (Raafat et al., 2008). During the antibacterial test,

chitosan was used as the control to compare with CHEU while the CH NPs acted as

the control for CHEU NPs. The MICs and MBCs of CH, CHEU, CH NPs and CHEU

NPs against S. aureus and E. coli are shown in Table 4-3. It can be seen that the MIC

and MBC values of CHEU are higher than those of CH, indicating that CHEU is less

effective as an antibacterial agent compared to CH. With CHEU at 0.5 mg/ml, the

contribution of acetic acid in the killing of bacteria is still very limited according to the control experiment. The CHEU NPs showed a higher antibacterial efficacy against

52 Chapter 4

Figure 4-5 Free radical scavenging abilities of (a) CHEU and CH; (b) CH NPs and CHEU NPs.

both gram-positive and gram-negative bacteria than CH NPs, indicating that the

modification of CH NP by eugenol did not result in loss of antibacterial efficacy. As

can be seen from Scheme 4-2, the modification of CH NPs with eugenol via the Schiff base reaction involves the amine group of CH. Thus, it might be expected that the antibacterial properties of the CH will be compromised. However, eugenol is also known to have antibacterial properties (Kim et al., 1995, Cosentino et al., 1999). The free hydroxyl group on benzene ring is essential for the antimicrobial ability of eugenol (Wendakoon and Sakaguchi, 1993, Ben Arfa et al., 2006), and since this group is not affected by the modification process, the phenolic group may compensate for the “lost” antibacterial properties of the amine group on CH NPs. As for CHEU, the grafting of eugenol rendered it less soluble even in acetic acid solution, which significantly decreased the probability of its interaction with bacteria, thus lowering

53 Chapter 4

the antibacterial efficacy when compared to chitosan. However, the CHEU NPs are

able to penetrate into cell membrane due to their small sizes, resulting in the death of

the bacterial cells. As a result, the CHEU NPs showed improved antibacterial efficacy

compared to CH NPs, even though CHEU showed lower antibacterial efficacy compared to CH.

At the site of a wound, ROS is produced in high amounts as a defense mechanism against invading bacteria. However, the process of wound healing may also be

hampered by the presence of free radicals which can damage the cells surrounding the

wound or by microbial infections (Houghton et al., 2005). The DPPH and

antibacterial assays have confirmed that the CHEU NPs possess both antioxidant and

antibacterial properties. Thus, such NPs with dual functionalities may have good

potential for enhancing wound healing.

Table 4-3 MICs and MBCs of chitosan and eugenol grafted chitosan derivatives in TSB medium against S. aureus and E. coli.

S. aureus E. coli Polymer MIC (mg/ml) MBC (mg/ml) MIC (mg/ml) MBC (mg/ml) CH 0.125 0.25 0.032 0.125 CHEU 0.5 0.5 0.125 0.25 CH NPs 0.5—1 2 0.5—1 2 CHEU NPs 0.5 1 0.25—0.5 2

4.3.5 Cytotoxicity assay of eugenol grafted chitosan derivatives

Since the ultimate goal of our research is the application of the antioxidant and antibacterial chitosan derivatives in mammalian systems, the issue of cytotoxicity has to be addressed. Pure eugenol has been reported to be toxic and the cytotoxicity value

(defined as the maximum concentration not resulting in death) of eugenol on human

54 Chapter 4

gingival fibroblasts is 1.9 μM (0.31 μg/ml) (Gerosa et al., 1996). Our results also

show that the cytotoxicity value of eugenol on 3T3 mouse fibroblasts is 0.31μg/ml.

Figure 4-6(a) shows the viabilities of the 3T3 mouse fibroblasts incubated with CH and CHEU. There is no significantly cytotoxicity for CHEU below 0.1 mg/ml. When

the concentration increases to 0.5 mg/ml (MIC value of CHEU against S. aureus), the

cell viability drops to 60%. There is no cytotoxic effect observed for CH. In fact, CH

even promotes cell growth in this experiment.

Figure 4-6(b) shows the viabilities of the 3T3 mouse fibroblasts when incubated with

CH NPs and CHEU NPs of various concentrations. There is no significant

cytotoxicity when the concentrations of CH NPs and CHEU NPs are below 0.2 mg/ml.

When the NPs concentrations are increased to 0.5 mg/ml which is close to the MIC

values of both CH NPs and CHEU NPs (except for CH NPs against E. coli where the

value is 0.125 mg/ml), the cell viability decreases to 92% for CHEU NPs but

remained at 100% for CH NPs. With further increase in NPs concentration to 1 mg/ml,

significant cytotoxicity (< 50% viability) is observed with CHEU NPs. However,

with CH NPs at a concentration of 2 mg/ml, the cell viability is still higher than 80%.

The advantages of the CHEU over eugenol can be seen by comparing their respective

MIC against bacteria and the cytotoxicity values for 3T3 mouse fibroblasts. The MICs

of eugenol against both S. aureus and E. coli are 1 mg/ml (Kim et al., 1995, Cosentino

et al., 1999). Since the cytotoxicity value of eugenol towards 3T3 mouse fibroblast is

0.31 μg/ml, the application of eugenol at their MIC values against bacteria would result in the killing of mammalian cells as well. On the other hand, the cell viability of

55 Chapter 4

3T3 fibroblasts is 60% for CHEU and 92% for CHEU NPs, respectively, at their MIC value (0.5 mg/ml).

Figure 4-6 Viabilities of 3T3 mouse fibroblasts incubated with (a) CH and CHEU dissolved in culture medium containing 0.25% acidic acid; (b) CH NPs and CHEU NPs dispersed in the culture medium for 72 hours.

56 Chapter 4

4.4 Conclusion

In this work, chitosan and chitosan nanoparticles were successfully grafted with the

essential oil component, eugenol. The grafting was facilitated by first converting the eugenol to its aldehydes. The in vitro DPPH assay and MIC test confirmed that the chitosan and chitosan nanoparticles grafted with eugenol possessed both antioxidant and antibacterial activities whereas the unmodified chitosan only have antibacterial properties. Most importantly, the antibacterial efficacy of the eugenol grafted chitosan nanoparticles against S. aureus (gram-positive bacterium) and E. coli (gram-negative

bacterium) is just as high as or even better than the unmodified chitosan nanoparticles.

The eugenol-grafted chitosan nanoparticles are significantly less cytotoxic towards

mammalian cells than free eugenol. At the MIC of the former (against S. aureus and E.

coli), fibroblasts still exhibit >90% viability whereas at the MIC of the pure eugenol,

the fibroblasts will no longer be viable. The successful modification of chitosan with

eugenol provides the incentive for similar modifications with other members of the

essential oil family. In the next chapter, another essential oil component, carvacrol, will be studied using similar techniques as described in this chapter.

57 Chapter 5

5 Antioxidant and antibacterial activities of carvacrol

grafted chitosan nanoparticles

5.1 Introduction

Natural antioxidants have been extensively studied for their ability to protect

organisms and cells from being damaged by ROS (Cozzi et al., 1997, Gordon and

Weng, 1992, Kim et al., 1994). Herbs are harmless sources of natural antioxidants.

Carvacrol is one of the essential oil components which can be extracted from thyme.

It has been reported to possess antimicrobial and antioxidant properties. Recently, carvacrol was also reported to have the ability to inhibit cancer cell growth (Seval and

Yusuf, 2006, Koparal and Zeytinoglu, 2002). However, carvacrol may have some degree of toxicity (Bimczok et al., 2008). For instance, carvacrol inhibited the proliferation of purified porcine lymphocytes with an IC50 (half maximum inhibitory

concentration) of 182 ± 67 μM in MTT assays (Bimczok et al., 2008).

In this section, carvacrol was grafted to chitosan to obtain nanoparticles with antibacterial and antioxidant properties and yet relative low cytotoxicity. Carvacrol was modified with aldehyde groups and then reacted with chitosan nanoparticles

through the Schiff base reaction. The antioxidant and antibacterial activities of these

nanoparticles were assayed, and the cytotoxicity towards mouse 3T3 fibroblasts was

investigated using similar procedures as described in the previous chapters.

58 Chapter 5

5.2 Experimental

5.2.1 Materials

The chitosan and TPP were the same as described in Chapter 4. Carvacrol (98%) were

obtained from Sigma-Aldrich and used as received. Acetonitrile, triethylamine, anhydrous magnesium chloride (MgCl2) and paraformaldehyde were also purchased

from Sigma-Aldrich. S. aureus, E. coli and mouse fibroblast cells were supplied by

ATCC.

5.2.2 Synthesis of carvacrol aldehyde

The synthesis of carvacrol aldehyde was performed under a moisture free

environment using the method described in an earlier work (Knight et al., 2005). In

this method carvacrol was formylated selectively at the position ortho to the hydroxyl

group. Briefly, a 250 ml round bottom flask was placed under an argon atmosphere

and filled with 40 mmol carvacrol, 150 ml acetonitrile, 150 mmol triethylamine and

40 mmol anhydrous MgCl2. The solution was stirred for 15 min at room temperature.

270 mmol paraformaldehyde was then added, and the reaction mixture was refluxed for 3.5 h. The solution was then allowed to cool to room temperature and acidified by

the addition of 5% (w/v) aqueous hydrochloric acid (320ml) followed by stirring for

30 min under argon atmosphere. The product was extracted by diethyl ether and the

ether portion was collected and washed with saturated sodium chloride aqueous

solution. The volatiles were removed under reduced pressure to yield the raw

carvacrol aldehyde product.

59 Chapter 5

5.2.3 Preparation of chitosan nanoparticles

A general ionic gelation method was used to prepare chitosan nanoparticles (CH NPs)

(Shi et al., 2006). The details were described in Section 4.2.3. CH NPs prepared as described in the previous chapter were used for grafting with carvacrol.

5.2.4 Grafting of carvacrol on chitosan nanoparticles through the Schiff

base reaction

Carvacrol grafted chitosan nanoparticles (CHCA NPs) were synthesized using the same method as described in Section 4.2.4, with the use of carvacrol aldehyde in place of eugenol aldehyde.

5.2.5 Characterization techniques

The as-synthesized carvacrol aldehyde was characterized by UV-visible spectroscopy and 1H NMR, as described in Section 4.2.5. The CHCA NPs were characterized by

XPS and FTIR, as described in Section 3.2.3 and 4.2.5. The particle size and zeta

potential of the CHCA NPs were determined as described in Section 4.2.5.

5.2.6 Scavenging ability of carvacrol grafted chitosan nanoparticles

The scavenging ability of CHCA NPs was measured and expressed using the method

as described in Section 3.2.4.

60 Chapter 5

5.2.7 Determination of antibacterial activity of carvacrol grafted

chitosan nanoparticles

The antibacterial efficacy of CHCA NPs was determined in the same manner as that

described in Section 4.2.7 for CHEU NPs.

5.2.8 In vitro cytotoxicity of carvacrol grafted chitosan nanoparticles

The MTT assay, as described in Section 4.2.8, was used to investigate the cytotoxic

effect of CHCA NPs.

61 Chapter 5

5.3 Results and discussions

5.3.1 Synthesis of carvacrol aldehyde

As mentioned in Section 4.3.1, the two peaks at 310 nm and 275 nm of the UV-visible absorption spectra are the two characteristic peaks of phenolic aldehydes. Hence, UV- visible absorption spectroscopy was used to characterize the synthesis product of carvacrol. The UV-visible absorption spectra of carvacrol before and after formylation are compared in Figure 5-1. The peak at 276 nm is characteristic of the phenolic groups of carvacrol. After formylation of carvacrol, a new absorption peak at

350 nm appears indicating the formation of phenolic aldehyde.

2.0

1.5

Carvacrol

1.0 Carvacrol aldehyde

Absorbance (arb. units) 0.5

0.0 250 300 350 400 450 Wavelength (nm)

Figure 5-1 UV-visible absorption spectra of carvacrol before and after formylation.

The 1H NMR analysis of the carvacrol before and after formylation showed an additional peak at 10.42 ppm for carvacrol aldehyde as compared to carvacrol (Knight

62 Chapter 5

et al., 2005). From the UV-visible absorption spectroscopy and NMR analysis, the

possible synthesis product from carvacrol is given in Scheme 5-1. The product yield of carvacrol aldehyde estimated from the 1H NMR spectra is 51.7%. Our preliminary

experiments showed that carvacrol does not react with the chitosan nanoparticles.

Hence, the grafting reactions with chitosan nanoparticles were carried out using the

carvacrol aldehyde without the need for further separation from carvacrol.

OH OH MgCl2

Et3N (CH2O)n O

carvacrol 2-hydroxy-6-isopropyl -3-methylbenzaldehyde

Scheme 5-1 Synthesis process of carvacrol aldehyde.

5.3.2 Characterization of carvacrol grafted chitosan nanoparticles

In the present work, the Schiff base reaction was chosen for grafting carvacrol to

chitosan nanoparticles due to the ease of synthesis and structural rigidity of Schiff base (Knight et al., 2005). The successful modification of the CH NPs was first confirmed by XPS analysis. The N 1s core-level spectrum of CHCA NPs is shown in

Figure 5-2. Comparing this spectrum to that of CH NPs in Figure 4-2(b), a new component peak appears at about 398.5 eV in the N 1s core-level spectrum of CHCA

NPs, attributed to =N- species formed after Schiff base reaction (Scheme 5-2). The

degree of acetylation of chitosan and the degree of grafting of carvacrol, as calculated

63 Chapter 5 from the XPS analysis, are summarized in Table 5-1. As expected, the degree of acetylation of chitosan is not significantly affected by the grafting of the carvacrol.

HO HO OH O * O * OH OH * * + N NH2 O

HO

chitosancarvacrol aldehyde Schiff base

Scheme 5-2 Molecular representations of CHCA NPs.

Table 5-1 Degree of acetylation of chitosan before and after grafted with carvacrol and the degree of grafting.

Degree of grafting of Degree of acetylation carvacrol CH 15.7% --- CH NPs 16.2% --- CHCA NPs 15.5% 50.7%

64 Chapter 5

Figure 5-2 XPS N 1s core-level spectrum of CHCA NPs.

The FTIR spectra of the CH NPs and CHCA NPs are shown in Figure 5-3. The

-1 absorption band at 3400 cm is attributed to -OH and -NH2 stretching vibration in the chitosan matrix (Xu and Du, 2003). The absorption bands at 1637 cm-1 and 1540 cm-1

attributed to υ(C=O) and υ(NH), respectively, are characteristic bands of chitosan

nanoparticles (Knaul et al., 1999). After the formylation reaction, the intensity ratio of

the υ(NH) band at 1540 cm-1 to the υ(C=O) band at 1637 cm-1 in the spectrum of

CHCA NPs decreased compared to the corresponding ratio in the spectrum of CH

NPs. This decrease is due to the consumption of amine groups on the CH NPs surface

as a result of the formation of –C=N- bonds upon grafting of carvacrol.

65 Chapter 5

Figure 5-3 FTIR spectra of CH NPs and CHCA NPs.

In the ionic gelation method, the nanoparticles were formed through interactions between the positively charged chitosan and negatively charged phosphate groups of

TPP. Table 5-2 shows the sizes and zeta potentials of CH NPs and CHCA NPs. The average hydrodynamic diameter of CHCA NPs was determined to be 260 nm, compared to 217 nm for CH NPs. The TEM images of CH NPs (Figure 4-4(a)) and

CHCA NPs (Figure 5-4) show that both the average sizes of CH NPs and CHCA NPs are around 100 nm. The size difference may originate from the different measurement techniques, as explained in Section 4.3.2. As expected, there is no substantial change

in the size of the nanoparticles after grafting with the carvacrol and there seems to be

no agglomeration. The zeta potential of the particle surface plays an important role in

the surface interactions as mentioned in Section 4.3.2. As shown in Table 5-2, the zeta

potential of CHCA NPs is lower than that of CH NPs. This decrease may result from

the consumption of the amine groups of the CH NPs in the Schiff base reaction. It will

66 Chapter 5

be shown in Section 5.3.4 that this decrease in zeta potential does not compromise the antibacterial action of the carvacrol-grafted CH NPs.

Table 5-2 Characteristics of CH NPs and CHCA NPs

Hydrodynamic NPs Polydispersity Zeta potential (mV) size (nm) CH NPs 217±17 0.595 +37.6±1.3 CHCA NPs 260±18 0.366 +18.8±1.0

Figure 5-4 TEM investigation of CHCA NPs. The scale bar is 1 μm.

5.3.3 Free radical scavenging ability of carvacrol grafted chitosan

nanoparticles

Conferring chitosan with carvacrol ligand may retain its favorable biological

properties for wound healing while providing promising antioxidant activity to

accelerate the wound healing process, as mentioned in Section 4.3.3. It has been 67 Chapter 5

reported that carvacrol exhibits free radical scavenging ability with EC50 values of

about 267 μg/ml (Mastelic et al., 2008). The free radical scavenging ability of CHCA

NPs is shown in Figure 5-5. It can be seen that CHCA NPs show free radical

scavenging ability dependent on the concentration in the range of 0.25 mg/ml to 4

mg/ml. The EC50 value for CHCA NPs is slightly higher than 4.0 mg/ml.

Figure 5-5 Free radical scavenging abilities of CH NPs and CHCA NPs.

5.3.4 Antibacterial effects of carvacrol grafted chitosan nanoparticles

The antibacterial effect of CHCA NPs was evaluated using two parameters: MIC and

MBC. The MICs and MBCs of CHCA NPs against S. aureus and E. coli are shown in

Table 5-3. It can be seen that the MICs and MBCs of CHCA NPs and CH NPs are similar against both gram-positive S. aureus and gram-negative E. coli, indicating that the modification of CH NPs by carvacrol did not result in any loss of antibacterial efficacy. As discussed earlier, the modification of CH NPs with carvacrol via the

68 Chapter 5

Schiff base reaction, similar to eugenol grafting process, also involved the amine

group and the zeta potential of the CH NPs decreased after grafting with the EO components. Thus, this may result in the loss of the antibacterial efficacy. However, like eugenol, carvacrol is also known to have antibacterial properties (Kim et al., 1995,

Cosentino et al., 1999). The free hydroxyl group on the benzene ring is essential for

the antimicrobial activity of carvacrol (Wendakoon and Sakaguchi, 1993, Ben Arfa et

al., 2006), and since this group is not influenced by the reaction, the phenol group

may compensate for the “lost” antibacterial properties of the reacted amine group on

CH NPs. As a result, the CHCA NPs showed almost the same antibacterial efficacy

as CH NPs.

Table 5-3 MICs and MBCs of CH NPs and CHCA NPs in TSB medium against S. aureus and E. coli.

S. aureus E. coli NPs MIC (mg/ml) MBC (mg/ml) MIC (mg/ml) MBC (mg/ml) CH NPs 0.5—1 2 0.5—1 2 CHCA NPs 0.5—1 2 0.5—1 2

As mentioned in Section 2.1.4, chitosan has been applied in wound healing process.

The DPPH and antibacterial assays have confirmed that CHCA NPs possess both antioxidant and antibacterial properties, which would be advantageous for scavenging the free radicals and killing bacteria at the wound site. This would promote the wound healing process by preventing ROS-caused cell damages and microbial infection.

5.3.5 Cytotoxicity assay of carvacrol grafted chitosan nanoparticles

If CHCA NPs are intended for clinic use, their cytotoxic effect towards mammalian cells should be addressed. Pure carvacrol has been reported to be toxic (Bimczok et al.,

69 Chapter 5

2008). Our results also show that the cytotoxicity value of carvacrol on 3T3 mouse

fibroblasts is 0.28 μg/ml. Figure 5-6 compares the viabilities of the 3T3 mouse

fibroblasts when incubated with CH NPs and CHCA NPs of various concentrations.

Similar to the results observed with CHEU NPs, there is no significant cytotoxicity

when the concentration of CHCA NPs is below 0.2 mg/ml. When the NPs

concentration is increased to 0.5 mg/ml (close to the MIC of CHCA NPs), the cell

viability decreases to 81%. With further increase in NPs concentration to 1 mg/ml, significant cytotoxicity (< 50% viability) is observed with CHCA NPs.

Figure 5-6 Viabilities of 3T3 mouse fibroblasts incubated with CH NPs and CHCA NPs distributed in the culture medium for 72 hours.

The advantages of the CHCA NPs over pure carvacrol can be seen by comparing their

respective MIC against bacteria and the cytotoxicity values for 3T3 mouse fibroblasts.

The MICs of carvacrol against S. aureus and E. coli are 0.175—0.45 mg/ml and

0.225—0.5 mg/ml, respectively (Kim et al., 1995, Cosentino et al., 1999). Since the

70 Chapter 5 cytotoxicity value of carvacrol towards 3T3 mouse fibroblast is 0.28 μg/ml, the application of carvacrol at their MIC values against bacteria would result in the killing of mammalian cells as well. This is similar to the case of eugenol, as discussed in

Chapter 4.

71 Chapter 5

5.4 Comparative study of carvacrol grafted chitosan

nanoparticles and eugenol grafted chitosan nanoparticles

5.4.1 Degree of grafting of essential oil components

The successful modification of chitosan nanoparticles with the two essential oil components, eugenol and carvacrol, was confirmed by the XPS. Under the same reaction condition (excess essential oil aldehyde compared to the amine groups on the

CH NPs), the degree of grafting of carvacrol is 50.7% (Figure 5-2) while the degree of grafting of eugenol is 26.7% (Figure 4-2). The degree of grafting of carvacrol is twice that of eugenol. A possible reason is that the -OCH3 group on eugenol aldehyde

causes steric hindrance during the Schiff base reaction.

5.4.2 Antibacterial activity

The antibacterial activities of CHEU NPs and CHCA NPs were conducted in the same

batches. The results have been shown in Table 4-3 and Table 5-3. It can be seen that the antibacterial efficacy of CHEU NPs is higher than that of CHCA NPs, while the

antibacterial efficacy of CHCA NPs is similar to that of CH NPs. The amine groups of

chitosan and the hydroxyl groups on the aromatic rings of eugenol and carvacrol are believed to be responsible for the antibacterial properties. Though the Schiff base

reaction compromised some of the amine groups on the chitosan, the grafted essential

oil component contains hydroxyl groups that were preserved. Since the exact

mechanisms of antibacterial activities of chitosan and essential oils are still unknown,

it is not possible to draw a conclusion regarding the relative antibacterial efficacy of

72 Chapter 5

the amine group versus that of the hydroxyl group on the aromatic ring of eugenol and carvacrol.

5.4.3 Antioxidant activity

The free radical scavenging efficacy of pure eugenol has been reported to be 30 times higher than that of pure carvacrol (Mastelic et al., 2008). The hydroxyl groups on their aromatic rings are probably responsible for the antioxidant activity. Since the modification process did not compromise the hydroxyl groups on their aromatic rings, a higher free radical scavenging ability of CHEU NPs as compared to that of CHCA

NPs is expected, and this is confirmed by comparing the DPPH assays in Figure 4-5 and Figure 5-5. The EC50 value for CHEU NPs is nearly twice that of CHCA NPs

even though the degree of grafting of essential oil component for CHEU NPs is only

half that for CHCA NPs. Though the modification process did not involve the

hydroxyl groups on the aromatic rings of carvacrol and eugenol, the introduction of

aldehyde groups linked to the amine groups of chitosan may influence the free radical

scavenging action.

5.4.4 Cytotoxicity

Pure eugenol and carvacrol are cytotoxic with very similar cytotoxicity values. The

grafting of eugenol and carvacrol onto chitosan nanoparticles lowers the cytotoxicity of CHEU NPs and CHCA NPs, compared to pure eugenol and carvacrol, respectively.

At the MIC of CHEU NPs (0.25 – 0.5 mg/ml), the cell viability of 3T3 mouse fibroblasts is still above 90%, while at the MIC of CHCA NPs (0.5 – 1 mg/ml), the

73 Chapter 5 cell viability decreased to 80% or even 50%. The CHEU NPs seem to have lower cytotoxicity than CHCA NPs.

74 Chapter 5

5.5 Conclusion

In this work, chitosan nanoparticles were successfully grafted with the essential oil

component, carvacrol. The grafting was facilitated by first converting carvacrol to

carvacrol aldehyde. The in vitro DPPH assay and MIC test confirmed that the CHCA

NPs possessed both antioxidant and antibacterial activities whereas the chitosan nanoparticles have only antibacterial properties. The antibacterial efficacies of CHCA

NPs against S. aureus (gram-positive bacterium) and E. coli (gram-negative bacterium) are very close to those of the unmodified CH NPs. The CHCA NPs are significantly less cytotoxic towards mammalian cells than free carvacrol. At the MIC of the former

(against S. aureus and E. coli), fibroblasts will exhibit >50% viability whereas at the

MIC of the pure carvacrol, the fibroblasts will be no longer viable.

When the antioxidant activities and antibacterial activities of CHCA NPs were compared to those of CHEU NPs, the use of CHEU NPs is more advantageous than

CHCA NPs because of their higher antioxidant and antibacterial efficacies and lower cytotoxicity. In addition, CHEU NPs may have potential applications in dentistry, wound healing and food packaging.

75 Chapter 6

6 Conclusion and recommendations

6.1 Conclusion

Three natural materials have been modified to develop dual functional properties:

antioxidant and antibacterial. Chitosan was utilized as the bulk material to be

modified with functional biomaterials, ascorbic acid (AA) and essential oils (EOs),

through the Schiff base reaction. Biological assessments of the functionalized systems

have been designed to evaluate their performances in vitro. The results are shown in

Table 6-1.

Table 6-1 Comparison of chitosan modified with essential oil component and ascorbic acid

Antioxidant Antibacterial Activity Chitosan and Degree of Activity MIC (mg/ml) MBC(mg/ml) its Grafting derivatives S. aureus E. coli S. aureus E. coli EC50 (mg/ml) CH -- Nil 0.125 0.032 0.25 0.125 CH NPs -- Nil 0.5—1 0.5—1 2 2 CHAA 15.2% 3.6 0.25 0.25 0.25 0.25 CHEU 12% >4.0 0.5 0.125 0.5 0.25 CHEU NPs 26.7% 2.6 0.5 0.25—0.5 1 2 CHCA NPs 50.7% >4.0 0.5—1 0.5—1 2 2 Nil: DPPH tests have shown nearly no free radical scavenging ability. --: Not applicable

First of all, AA was reacted with chitosan to prepare chitosan ascorbate (CHAA). The biological assessments confirmed that chitosan attained the additional free radical scavenging ability after modification with AA, although the modification caused a slight decrease in its antibacterial efficacy.

76 Chapter 6

Considering that phenolic aldehyde formed Schiff base should be more stable than

CHAA, the aldehyde group was introduced onto the aromatic ring of eugenol, a

component of essential oils, and the as-synthesized eugenol aldehyde was reacted

with chitosan and chitosan nanoparticles (CH NPs) through Schiff base reaction to

yield eugenol-grafted chitosan (CHEU) and eugenol-grafted chitosan nanoparticles

(CHEU NPs), respectively. The degrees of grafting of eugenol were calculated from

XPS analysis to be 12.0% and 26.7% for CHEU and CHEU NPs, respectively. The properties of CHEU NPs were found to be superior to those of CHEU. The immobilized eugenol was found to confer antioxidant ability on CH NPs with an EC50 value of 2.6 mg/ml. On the other hand, the existing antibacterial property of CH NPs was not compromised although the modification process consumed amine groups. At the MIC of CHEU NPs against S. aureus and E. coli, the cell viability of 3T3 mouse fibroblasts can be as high as 92%. On the other hand, pure eugenol at its MIC against the same bacteria would result in mammalian cell death.

Finally, the modification strategy was extended to another essential oil component, carvacrol. Aldehyde groups were first introduced on the aromatic ring of carvacrol.

The carvacrol-grafted chitosan nanoparticles (CHCA NPs) were then obtained by coupling the aldehyde groups on carvacrol aldehyde to the amine groups on CH NPs.

The biological assessments confirmed that the CHCA NPs simultaneously possess antioxidant and antibacterial abilities, with lower cytotoxicity than pure carvacrol. A comparison made between CHEU NPs and CHCA NPs showed that CHEU NPs, with only about half the degree of grafting of the essential oil component as CHCA NPs, have twice the antioxidant activity. The CHEU NPs exhibited higher antibacterial efficacy than CH NPs and CHCA NPs. With such excellent biological properties, the

77 Chapter 6

CHEU NPs still possess lower cytotoxicity than CHCA NPs, thus less damage towards human cells will be inflicted during its applications.

78 Chapter 6

6.2 Recommendations

A number of possible applications of modified chitosan with antibacterial and antioxidant activities can be foreseen. Two examples which need further study are given below:

Influence of degree of deacetylation on antioxidant and antibacterial properties of eugenol and carvacrol grafted chitosan

In this thesis, chitosan with 84% degree of deacetylation has been used so that the results obtained can be more easily compared with our previous work. As mentioned in Section 2.1.2, previous studies have shown that the degree of deacetylation has a strong correlation with the antioxidant and antibacterial properties. So, similar modifications of chitosan of different degree of deacetylation with the essential oils and ascorbic acid should be carried out.

Chitosan nanoparticulate system as a carrier to deliver growth factor for wound healing

Growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor

(FGF), can be encapsulated in CHEU NPs or CHCA NPs. With this encapsulation system, the release behavior of growth factor can be investigated, and the in vivo wound healing effect can be studied. This antibacterial and antioxidant nanoparticulate system may be an effective delivery vehicle for pharmaceutical purposes.

Essential oil component-grafted chitosan film for food packaging

79 Chapter 6

Effective food packaging should possess moisture prevention, ROS and bacteria inhibition properties in addition to the appropriate mechanical property. Since the essential oil component-grafted chitosan nanoparticles have been confirmed to possess antibacterial and antioxidant properties and the modification technique is applicable to chitosan film, the use of essential oil component-grafted chitosan film for food packaging would be a worthwhile investigation.

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