Polymer nanoparticles composed with gallic acid grafted chitosan and bioactive peptides combined antioxidant, anticancer activities and improved delivery property for labile polyphenols

Journal of Functional Foods 15 (2015) 593–603

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Polymer nanoparticles composed with gallic acid grafted chitosan and bioactive peptides combined antioxidant, anticancer activities and improved delivery property for labile polyphenols Bing Hu, Yan Wang, Minhao Xie, Guanlan Hu, Fengguang Ma, Xiaoxiong Zeng * College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

A R T I C L E

I N F O

Article history:

A B S T R A C T Polymer nanoparticles assembled from gallic acid (GA) grafted chitosan (CS, GA-g-CS for GA

Received 26 January 2015

grafted CS) and caseinophosphopeptides (CPP) were developed to deliver (−)-epigallocatechin-

Received in revised form 8 April

3-gallate (EGCG) as novel functional foods. The contents of GA in GA-g-CS copolymers were

2015

in the range of 26.5 ± 1.0–126.0 ± 1.1 mg/g, with the increase of molar ratio of GA to glu-

Accepted 9 April 2015

cosamine in CS. Compared with CS, GA-g-CS possessed much higher solubility under neutral

Available online

and alkaline environments. Spherical and physicochemical stable nanoparticles assembled from GA-g-CS and CPP were obtained with particle size around 300 nm and zeta

Keywords:

potential of less than +30 mV. The GA-g-CS-CPP nanoparticles showed strong antioxidant

Gallic acid grafted chitosan

activity and cytotoxicity against Caco-2 colon cancer cells. The EGCG-loaded GA-g-CS-CPP

Nanoparticles

nanoparticles (84–90% for encapsulation efficiency) showed improved delivery property, con-

EGCG

trolling release of EGCG under simulated gastrointestinal environments, preventing its

Antioxidant activity

degradation under neutral and alkaline environments, and amplifying its anticancer activ-

Cytotoxicity

ity against Caco-2 cells. © 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Over the last decade, significant attentions have been paid to polymer nanoparticles as oral delivery systems for

nutraceuticals to improve their bioavailability as novel functional foods (Acosta, 2009; Braithwaite et al., 2014; Ting, Jiang, Ho, & Huang, 2014). Polymer conjugates with biological activities synthesized through grafting antioxidant agents to the polymer molecular chains have drawn increasing attention for

* Corresponding author. College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. Tel.: +86 25 84396791; fax: +86 25 84396791. E-mail address: [email protected] (X. Zeng). Abbreviations: CPP, caseinophosphopeptide; DLS, dynamic light scattering; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EDC-HCl, 1-ethyl-3(3′-dimethylaminopropyl-carbodiimide) hydrochloride; EGCG, (−)-epigallocatechin-3-gallate; ET, electron transfer; GA-g-CS, gallic acid grafted chitosan; GI, gastrointestinal; HAT, hydrogen atom transfer; HOBt·H2O, 1-hydroxybenzotriazole monohydrate; LSD, least significant difference; PBS, phosphate buffer saline; PDI, polydispersity index; ROS, reactive oxygen species; SD, standard deviation; SGF, simulated gastric fluid; SIF, the simulated intestinal fluid; TEM, transmission electron microscopy http://dx.doi.org/10.1016/j.jff.2015.04.009 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 15 (2015) 593–603

their unique functions (Cirillo et al., 2010; Piras, Dessy, Dinucci, & Chiellini, 2011). Oxidative damage, related to over-production of reactive oxygen species (ROS), is always involved in the initiation and progression of many diseases and disorders, such as carcinogenesis and evolution of cancer (Fang, Seki, & Maeda, 2009; Kim et al., 2011; Miller, Albers, Pralle, Isacoff, & Chang, 2005). On the other hand, the bioactive polymer conjugates have also been paid a great attention as novel delivery systems for drugs or nutraceuticals (Ko et al., 2014; Williams, Lepene, Thatcher, & Long, 2009), because the carrier matrix materials are the major content compared with their payload content in most delivery systems. In addition, many labile drug or nutraceutical molecules are oxidation-sensitive, which is the most common cause for their deterioration during storage and/ or transport to the required target site in the body (Janesirisakule, Sinthusake, & Wanichwecharungruang, 2013). EGCG, the most abundant tea catechin in green tea, is known as a strong natural antioxidant. A lot of epidemiological and preclinical studies have demonstrated that EGCG can reduce the risk of cancer, which is mainly attributed to its inhibitory effects on enzyme activities and signal transduction pathways, resulting in the suppression of cell proliferation and enhancement of apoptosis (Yang, Wang, Lu, & Picinich, 2009). However, EGCG is very unstable in plasma and intestinal juice neutral and alkaline environments, leading to its very low bioavailability (Yoshino, Suzuki, Sasaki, Miyase, & Sano, 1999). One of the main functions of the carrier system is to deliver their payload to the desired target sites, reducing nontarget (systemic) exposure and increasing the exposure concentrations and/or duration per administered dose at the target site(s). Chitosan (CS) and its derivatives are widely used in fabrication of promising vehicle for oral delivery of therapeutics or nutraceuticals to increase their bioavailability due to their excellent mucoadhesive and absorption-enhancing properties (Chen et al., 2013; Chiu et al., 2010). However, CS based nanoparticles swell highly, even burst break, in stomach acid, caused by the strong repulsion among the highly protonated amino-groups (Gamboa & Leong, 2013). In addition, owing to its semicrystalline nature and multiple H-bond forming groups, CS is insoluble in water (when pH > 6.4), which limits adopting CS for nutraceuticals delivery. Distinct chemical modifications such as glycol CS, PEGylated CS, thiolated CS, quaternary CS, carboxymethylated CS have been synthesized to improve their solubility in neutral and alkaline pH (Al-Hilal, Alam, & Byun, 2013). Despite that CS based antioxidant polymer conjugates have been developed in previous studies (Curcio et al., 2009; Spizzirri et al., 2010), the influence of antioxidant agent grafting on the physicochemical properties of CS and the nutraceuticals delivery properties of the CS based nanoparticle carriers were scarcely addressed. Herein, gallic acid (GA)-grafted-CS (GA-g-CS) conjugates were synthesized using a chemical implanting method developed in our previous study (Xie, Hu, Wang, & Zeng, 2014). GA is a naturally occurring phenolic acid with high antioxidant activity. The phenolics-polysaccharide conjugates occur widely in natural food, which are formed through the covalent linkage between phenolics and cell wall structural components, such as cellulose, hemicellulose, lignin, pectin and rod-shaped structural proteins (Arranz, Silvan, & Saura-Calixto, 2010). In present study, GA was conjugated to CS forming amide bond which

could easily be hydrolyzed in the gastrointestinal tract and the human colon. The GA-g-CS conjugate could be safe and extensive toxicology studies will still be needed before it can be widely used in the food industry. The grafting reaction was confirmed and characterized by thin-layer chromatography, UV–vis spectroscopy and Fourier transform-infrared (FT-IR) spectroscopy. The solubility of the GA-g-CS under pH 7.0 and pH 8.4 was characterized and compared with that of CS. Novel polymer nanoparticles composed with GA-g-CS and caseinophosphopeptides (CPP) were prepared and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS) and electrophoretic mobility (ζ-potential) measurements. CPP is a group of anionic polypeptides, which are released from the N-terminus polar region during the tryptic digestion of milk casein proteins. The antioxidant activities of the GA-g-CS-CPP nanoparticles were evaluated using DPPH radicals assay and β-carotene-linoleic acid assay. The oral delivery properties of the GA-g-CS-CPP nanoparticles for phytochemicals were characterized through determining their encapsulation efficiency and release profile of EGCG, stabilization effects on EGCG in simulated gastrointestinal (GI) environment, and their anticancer activities against intestinal cancer cells loading with/without EGCG.

2.

Materials and methods

2.1.

Materials

CS (Average molecular weight, ~1.5 × 10 5 ; Degree of deacetylation, ≥90.0%), 1-ethyl-3-(3′-dimethylaminopropylcarbodiimide) hydrochloride (EDC-HCl) and Folin–Ciocalteu reagent were purchased from Kayon Biological Technology Co., Ltd. (Shanghai, China). GA·H2O, 1-hydroxybenzotriazole monohydrate (HOBt·H2O) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). CPP were prepared and identified with HPLC-MS-MS as described in our previous report (Hu, Wang, Li, Zeng, & Huang, 2011). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), EGCG (purity > 98%), β-carotene, linoleic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade.

2.2.

Synthesis and characterization of GA-g-CS

2.2.1.

Synthesis of GA-g-CS

The synthesis was performed based on the one-pot method as reported (Xie, Hu, Wang, & Zeng 2014) with some modifications. CS (0.303 g, 1.85 mmol) was stirred in deionized water (30 mL) with HOBt (0.282 g, 1.85 mmol) overnight until a clear solution was obtained. GA (0.311 g, 1.85 mmol) was introduced into the CS solution followed by the dropwise addition of an alcoholic solution of EDC (0.355 g, 1.85 mmol, 2 mL). GAg-CSs with different substitutions of GA were prepared by changing the ratio between glucosamine in CS and GA (1:0.1, 1:1, 1:3, and 1:5). The reaction was carried out for 24 h in ambient temperature and atmosphere in the dark. The resultant liquid was poured into dialysis bags (MWCO 8000–14,000 Da), dialyzed against deionized water for 6 days with four changes of water each day. The resulting solutions were firstly tested by

Journal of Functional Foods 15 (2015) 593–603

thin layer chromatography (Section 2.3) to check whether the synthesized GA-g-CS samples still contained free GA. Then, the synthesized GA-g-CS samples were frozen and dried by lyophilizer to obtain solid conjugate of GA-g-CS. Blank CS, acting as a control, was prepared in the same conditions but in the absence of GA.

2.2.2.

Thin-layer chromatography of GA and GA-g-CS

Silica gel plates (100 × 30 mm, 0.2–0.25 mm for thickness) were purchased from Qingdao Ocean Chemical Factory (Qingdao, China). Aliquots (10 µL) of GA solution and GA-g-CS solution with different glucosamine/GA ratios after dialysis were applied to the plates. The supernatant liquid of the solvent system, butyl alcohol–water–acetic acid (50:40:1), was used as the expansion agent. The chromatograms were developed at room temperature (25 °C) for 40 min and air-dried. Exposure of the developed plates to iodine vapors in an iodine-saturated chamber for about 10 min yielded yellow spots that were attributed to the presence of GA.

2.2.3.

Characterization of GA-g-CS

Characterizations of GA-g-CS were carried out by UV–vis spectroscopy and FT-IR spectroscopy. The UV–vis spectra were recorded with a UV-2600 spectrophotometer (Shimadzu, Japan) by scanning from 200 to 800 nm. The FT-IR spectra were collected under ambient conditions, using a Nicolet iS10 FT-IR spectrometer (Thermo Electron Corp., Madison, WI, USA). Each spectrum was averaged over 256 scans with 4 cm−1 resolution in the range of 500–4000 cm−1.

2.2.4. Evaluation of phenolic groups by Folin–Ciocalteu reagent GA contents were determined according to the previous method (Liu et al., 2009). Briefly, 0.5 mL of GA-g-CS solution was mixed with 1.0 mL of Folin–Ciocalteu reagent for 5 min in the dark, followed by 2.0 mL 20% sodium carbonate (Na2CO3) added. The mixture was shaken and kept at 30 °C for 1 h, absorbance (Abs) at 747 nm was measured using a vis-spectrophotometer (Jinghua Instrument 722, Shanghai, China). GA was used as a standard. The grafting contents of GA-g-CS were expressed as mg of GA equivalent per g of copolymer.

2.2.5. Characterization of GA-g-CS solubility in neutral and alkaline pH Equal amounts of GA-g-CS with different GA contents and CS were dissolved with the same concentration. Equal volume of the polymer solutions were adjusted to pH 7.0 and pH 8.4, respectively. After 2 h, the polymer solutions were centrifuged under 5000 rpm for 30 min. After centrifugation, the suspensions were removed and the precipitation of each sample was lyophilized and weighted. The ratio between the amount of dried precipitate and the amount of original sample was calculated and compared.

2.3. Preparation and characterization of GA-g-CS-CPP nanoparticles and EGCG loaded GA-g-CS-CPP nanoparticles 2.3.1.

General procedure for preparation of nanoparticles

GA-g-CS-CPP and CS-CPP nanoparticles were prepared according to our previously reported procedure (Hu et al., 2011) with

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modification. GA-g-CS was dissolved in deionized water, CS was dissolved in 1% (w/v) acetic acid solution with sonication until the solution was transparent, and the aqueous solution of CPP was obtained at a suitable concentration. GA-g-CS, CS and CPP solutions were adjusted to pH 6.2 with 1.0 N HCl or NaOH solution. Subsequently, CPP solution was added to the GA-g-CS and CS solution, respectively, under stirring at room temperature. For the preparation of EGCG-loaded nanoparticles, aqueous solution of EGCG was added to the GA-g-CS and CS solution before the addition of CPP solution. The formation of GA-gCS-CPP and CS-CPP nanoparticles started spontaneously via the CPP initiated ionic gelation mechanism.

2.3.2.

Characterization of nanoparticles

The measurement of particle size, polydispersity index (PDI) and zeta potential of the nanoparticles was performed on a Zetasizer Nano-ZS (Malvern Instruments) on the basis of DLS techniques. All measurements were made in triplicate at 25 ± 1 °C. The morphological characteristics of the nanoparticles were examined by a high performance digital imaging TEM machine (JEOL H-7650, Hitachi High-Technologies Corp., Japan). One drop of the suspension was placed on a copper grid and allowed to evaporate in the air. Once evaporated the samples were placed in a TEM for imaging. The accelerating voltage used was 100 kV and the images were taken on a Gatan electron energy loss spectrometry system using 6 eV energy slit.

2.3.3. Determination of antioxidant activity of GA-g-CS-CPP nanoparticles in vitro 2.3.3.1. DPPH radicals scavenging assay. DPPH radicals scavenging assay was performed according to the reported procedure (Spizzirri et al., 2010). GA, CS, GA-g-CSs, CS-CPP nanoparticles and the GA-g-CS-CPP nanoparticles were homogenously dispersed in deionized water on a series of concentrations (0.125, 0.25, 0.5, 1.0, 1.5, 2.0 mg/mL, respectively). A 50 µL sample was added to each well of 96-well microplate, mixed with 200 µL methanolic DPPH· solution (0.4 mM). The reactions were carried out in the dark at room temperature for 30 min. Abs was measured at 517 nm by a microplate reader (Bio-Tek µquant, USA). A − A2 ⎞ ⎛ Scavenging Activity = ⎜ 1 − 1 ⎟ × 100% ⎝ A0 ⎠ where A0 is the Abs of the control (water instead of sample), A1 is the Abs of the sample, and A2 is the Abs of the sample only (methanol instead of DPPH).

2.3.3.2. β-Carotene-linoleic acid assay. Inhibiting peroxidation effects of GA, CS, GA-g-CSs, CS-CPP nanoparticles, GA-g-CSCPP nanoparticles on a linoleic acid system were determined by β-carotene bleaching test (Spizzirri et al., 2010) with some modifications. Briefly, 2 mL of β-carotene solution (1.0 mg/mL in chloroform) was added to 40 mg of linoleic acid and 400 mg of Tween 20. After being evaporated at 40 °C in a rotary evaporator to remove chloroform, the mixture was immediately diluted with 200 mL of distilled water, followed by ultrasonication to form an emulsion. The emulsion (1.25 mL)

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was transferred to microcentrifuge tubes containing 50 µL of sample. The tubes were then shaken gently and incubated at 45 °C for 1 h. The Abs of 200 µL resulting liquid was measured at 470 nm using the microplate reader.

A − A60 ⎞ ⎛ Antioxidant Activity = ⎜ 1 − 00 × 100% 0 ⎟ ⎝ ⎠ A0 − A60 where A0 and A00 are the Abs measured at the initial incubation time for samples and control, respectively, whereas A60 0 and A60 are the Abs of the samples and control, respectively,

2.4. Determination of stability of EGCG loaded in GA-gCS-CPP nanoparticles under neutral and alkaline environments Equal volume (0.5 mL) of the EGCG and EGCG loaded GA-gCS-CPP nanoparticles with the same concentration of EGCG was dispersed in 9.5 mL PBS (pH 7.4). At the time intervals of 0, 2 and 6 h, 0.5 mL sample was withdrawn from the EGCG and EGCG loaded GA-g-CS-CPP nanoparticles solution, respectively, and extracted with the same volume (0.5 mL) of ethyl acetate twice (Lambert et al., 2003). Blank PBS (pH7.4, 0.5 mL) was fed back. After extraction, the ethyl acetate phase was rotary evaporated, re-dissolved in water, and analyzed by using HPLC according to our reported method (Hu et al., 2009).

after incubated for 60 min.

2.5. Cytotoxicity of GA-g-CS-CPP nanoparticles and EGCG loaded GA-g-CS-CPP nanoparticles against cancer cells 2.3.4. Encapsulation efficiency of EGCG and in vitro release profile of EGCG from the nanoparticles in simulated GI environments The encapsulation efficiency of EGCG in the GA-g-CS-CPP nanoparticles and the CS-CPP nanoparticles was determined according to our previous report method with minor modification (Hu et al., 2008). Briefly, the EGCG-loaded nanoparticles were carefully transferred into an Amicon Ultra-15 centrifugal filter device (Millipore Co., Billerica, MA, USA) made up of a centrifuge tube and a filter unit with low-binding Ultracel membrane (MWCO 1000). After centrifugation at 4500 g for 75 min, free EGCG penetrated through the Ultracel membrane into the centrifuge tube, and the EGCG-loaded nanoparticles in the filter unit were obtained for the determination of the in vitro release profile. The amount of EGCG in ultrafiltrate was determined by HPLC according to our reported method (Hu et al., 2009). The encapsulation efficiency of EGCG was calculated using the formula below:

Encapsulation efficiency (% ) Total amount of EGCG − amount of EGCG in ultrafiltrate = × 100 Total amount of EGCG The release profiles of EGCG from the CS-CPP nanoparticles and the GA-g-CS-CPP nanoparticles in simulated GI environments were analyzed by dialysis method. The EGCG loaded nanoparticles, obtained from the centrifugation in determination of the encapsulation efficiency, were dispersed in the simulated gastric fluid (SGF) and the simulated intestinal fluid (SIF) solutions respectively, which were further immediately placed in the dialysis bags (MWCO 3.5 kDa) (MYM Biological Technology Company, USA). SGF was 0.1 N HCl (pH 1.2) containing 0.1% pepsin, and SIF was 10 mM phosphate buffer saline (PBS, pH 7.4) with 1.0% trypsin. The dialysis bags containing SGF were immersed in acid release medium (0.1 N HCl, pH 1.2), and the ones containing SIF were immersed in neutral release medium (PBS, pH 7.4). Aliquots of dissolution media (0.5 mL) were withdrawn, and the concentration of EGCG was determined by HPLC after appropriate dilution with water. The same volume of buffer (0.5 mL) was fed back to release medium. The percent cumulative amount of EGCG released from the nanoparticles was calculated as a function of time.

Cytotoxicity was measured using the trypan blue dye exclusion test. Cells (100,000) were seeded in 6-well plates, in 2.5 mL of medium each well. After 24 h, supernatants were discarded and replaced by fresh EGCG, GA-g-CS-CPP nanoparticles, or the EGCG loaded GA-g-CS-CPP nanoparticle dilutions in cell culture medium in range concentration of 12.5–200 µg/mL for EGCG and of 12.5–200 µg/mL for the nanoparticles. After 72 h of exposure, supernatants were discarded and cells were rinsed twice with PBS. Cells exposed to EGCG, GA-g-CS-CPP nanoparticles, and EGCG loaded GA-g-CS-CPP nanoparticles were harvested with 1 × trypsin. Cells were mixed with an aqueous solution of trypan blue (0.4%) in 50/50 (v/v) (Eurobio) according to a method previously described (Grabowski et al., 2013). Cell counting was performed using Kova counting chambers (Kova Glasstic Slide, Hycor Biomedical) by microscopy. The cell viability was calculated as the ratio between uncolored cells and total cells. The cytotoxicity was calculated as the ratio between the subtraction of uncolored cells from the total cells and total cells. All measurements were performed in triplicate.

2.6.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) of triplicates. The least significant difference (LSD) test and oneway analysis of variance (ANOVA) were used for multiple comparisons by SPSS 16.0. Difference was considered statistically significant if p < 0.05, and very significant if p < 0.01.

3.

Results and discussion

3.1.

Synthesis and characterization of GA-g-CS

3.1.1.

Synthesis of GA-g-CS

GA-g-CSs were synthesized through conjugating the carboxyl group in GA to the amino-groups in CS, mediated via a water-soluble carbodiimides conjugating agent, EDC, according to our previous study (Xie et al., 2014). The yellow spots in Fig. 1 represent the presence of GA. The movement of free GA appeared and no yellow spots for free GA could be found on the base line after the development of the thin-layer

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Fig. 1 – Thin-layer chromatogram of gallic acid (A), gallic acid grafted chitosan (GA-g-CS) with different glucosamine to GA ratios, 1:1 (B), 1:3 (C), and 1:5 (D).

chromatography. On the contrary, after the development, the yellow spots for the GA-g-CS samples with different glucosamine to GA ratios only appeared on the base line and no migration could be observed. It indicated that GA was successfully chemically conjugated in the CS molecular chains and this was not a mixture condition (Cho, Kim, Ahn, & Je, 2011). There was no free GA in the GA-g-CS samples after dialysis for 6 days.

3.1.2.

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observed. The amide II bands around 1585 cm−1 arose from the N—H bending vibrations coupled to C—N stretching vibrations. Amide III bands arose from the C—N stretching vibration. This phenomenon was caused by the grafting of GA on the amino groups in the CS chain. Similar result was also reported in a previous study (Yang et al., 2011). The substitution of GA on CS was determined by Folin– Ciocalteu reaction, in which phenolic compounds underwent a complex redox reaction with phosphotungstic and phosphomolybdic acids (Siripatrawan & Harte, 2010). The transfer of electrons at basic pH reduced the phosphomolybdic/ phosphotungstic acid complexes to form chromogens, and then the color was developed. Calculated from the results from the Folin–Ciocalteu reaction, the substitutions of GA on the dry GAg-CS copolymers were 26.5 ± 1.0, 113.7 ± 1.1, 126.0 ± 1.1 and 122.6 ± 2.2 mg/g, with the molar ratios between glucosamine in CS to GA of 1:0.1, 1:1, 1:3 and 1:5, respectively. The solubility of the GA-g-CSs and CS in neutral and alkaline environments is shown in Fig. 2C. Grafting with GA, the precipitations of the copolymers under pH 7.0 and pH 8.4 significantly decreased compared with the plain CS, indicating that grafting with GA significantly increased the solubility of CS in neutral and alkaline pH conditions. In addition, the solubility of the GA-g-CS was improved with the increase of the GA substitution ratio from 113.7 ± 1.1 to 126.0 ± 1.1 mg/g. As the pKa value of the amine groups on CS was approximately pH 6.4, CS precipitated at neutral pH due to deprotonation of amine groups. This property of CS suggested that CS and the CS based biomaterials could be effective only in a limited area of the human body, such as in the stomach and duodenum, where the pH values were below or close to its pKa. In fact, the general pH value in human body, the digestive tract, blood vessel, et al., was neutral and alkalescent. Therefore, grafting with GA successfully extended the soluble pH scope of CS, which was the pre-requirement for intestinal mucosal delivery and vascular delivery of the nutraceuticals using CS based biomaterials.

3.2. Preparation and characterization of GA-g-CS-CPP nanoparticles and EGCG loaded GA-g-CS-CPP nanoparticles

Characterization of GA-g-CS

Fig. 2A shows the UV–vis spectra of CS and GA-g-CS with different glucosamine to GA ratios. After grafting with GA, two new absorption peaks appeared around 255 nm and 281 nm in the UV–vis spectra of the GA-g-CSs. The absorption peak around 281 nm originated from the π-system of the benzene ring in GA grafted in the conjugate polymer. Another new peak located at 255 nm represented the π π* absorption that arose from the unsaturated ketones (Kang & Kang, 2012) in the GAg-CS molecules, which was indicated in Fig. 2A. It could be confirmed that GA was grafted to CS. The GA-g-CSs were then characterized by FT-IR, compared with the CS. The result is shown in Fig. 2B. The main characteristic absorption bands of CS appeared at 1640, 1585, 1374 and 1150–1040 cm−1, which corresponded to amide I, amide II, amide III groups, and glycosidic linkage (C—O—C), respectively (Pawlak & Mucha, 2003). It could be found in Fig. 2B that the peak height ratio between the absorption bands of amide II and amide I increased with the decrease of glucosamine to GA ratios (increase of GA). In addition, significant changes in the bands of amide III group around 1374 cm−1 could be

3.2.1.

Physicochemical properties of nanoparticles

The particle sizes of the CS-CPP and GA-g-CS-CPP nanoparticles with different GA grafting ratios, as well as their companions loading with EGCG are shown in Fig. 3A. The particle size of the CS-CPP nanoparticles determined by DLS at room temperature was 246.4 ± 5.1 nm (n = 3), with the PDI around 0.367. With the increase of the GA grafting ratios in GA-g-CS, the particle size of the nanoparticles firstly increased sharply, and then decreased until it is of the similar value as the one of the CSCPP nanoparticles. The particle size of the GA-g-CS-CPP nanoparticles with the GA grafting ratio of 26.5 ± 1.0 mg/g was as large as 518.1 ± 23.3 nm, with the PDI around 0.530, which indicated that the obtained nanoparticles dispersion was highly heterogeneous. As the GA grafting ratio increased to 113.7 ± 1.1 and 126.0 ± 1.1 mg/g, the particle size decreased to 311.9 ± 4.9 nm and 278.0 ± 3.8 nm, respectively. Also, the PDI of the nanoparticle suspensions decreased to around 0.288 and 0.294, respectively, meaning the homogeneous dispersion of nanoparticles. Similar change trend in particle size and PDI could also be found in the nanoparticles loading with EGCG. Encapsulation of EGCG

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within the nanoparticles did not change the particle size and PDI significantly, except the case of the nanoparticles composed of GA-g-CS with the GA grafting ratio of 26.5 ± 1.0 mg/ g, in which the particle size increased significantly after loading with EGCG. The low grafting ratio of GA onto CS tended to cause the increased heterogeneity inside the GA-g-CS polymer, which might be related to the large particle size and decreased homogeneity of the nanoparticle suspension. It could be found in Fig. 3B that the surface charge of the nanoparticles increased slightly with the increase of GA substitution. Encapsulation of EGCG within the nanoparticles did not change the surface charge significantly, also except the case of the nanoparticles with the GA grafting ratio of 26.5 ± 1.0 mg/g. Fig. 3C and D shows the structure and morphology of the GAg-CS-CPP nanoparticle with GA grafting ratios of 113.7 ± 1.1 (C) and 126.0 ± 1.1 mg/g (D), which were characterized using TEM. It could be seen that the nanoparticles were regularly spherical in shape, dispersed homogenously, which was in consistence with the DLS results.

3.2.2.

Fig. 2 – UV–vis spectra (A) and FT-IR spectra (B) of chitosan (CS) and gallic acid grafted chitosan (GA-g-CS) with different glucosamine to GA molar ratios; (C) The solubility of the GA-g-CSs and CS in neutral and alkaline environments.

Antioxidant activity of GA-g-CS-CPP nanoparticles

There are various in vitro chemical-based assays that would be used to determine antioxidant activities, including oxygen radical absorbance capacity (ORAC), DPPH• scavenging method, ferric reducing capacity, β-carotene-linoleic acid system etc. These in vitro antioxidant assays can generally be classified into two types depending upon the reactions involved, the assays based on hydrogen atom transfer (HAT) reactions and the ones based on electron transfer (ET). In the present study, the DPPH assay, one represent of ET reactions, and the β-carotenelinoleic acid system assay that belongs to the HAT reactions was selected to test the antioxidant activity of the GA-g-CSCPP nanoparticles. GA-g-CS, CS, and GA were used as the controls. It could be found from Fig. 4A that the GA-g-CS-CPP nanoparticles showed significantly higher (p < 0.01) scavenging activity on DPPH radicals than the CS-CPP nanoparticles. Grafting CS with GA endowed the nanoparticles with significantly enhanced antioxidant activity. And the nanoparticles’ scavenging activity on DPPH radicals increased with the increase of the GA grafting ratios, which meant that the antioxidant activity of the nanoparticles would be adjusted through changing the GA grafting ratios. The scavenging activities of the GA-g-CS-CPP nanoparticles on DPPH radicals were lower compared with their corresponding GA-g-CSs. It might be caused by the decrease of the GA availability for the interaction with DPPH due to the embedding of parts of the GA groups inside the GA-g-CS-CPP nanoparticles. The scavenging activities of the free GA on DPPH radicals were significantly higher than that of either GA-g-CS or GA-g-CS-CPP nanoparticles. In β-carotene-linoleic acid system assay, the formed linoleic acid free radical attacked the highly unsaturated β-carotene molecules and rapidly bleached the orange color of β-carotene in the absence of an antioxidant. The lowest β-carotene discoloration rate exhibited the highest antioxidant activity. Fig. 4B and C shows the inhibition effects of the GA-g-CS and GA-gCS-CPP nanoparticles on linoleic acid autoxidation, respectively. It could be found from Fig. 4C that the inhibition effects of the GA-g-CS-CPP nanoparticles were significantly higher (p < 0.01)

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Fig. 3 – The particle size (A) and surface charge (B) of the chitosan (CS)-caseinophosphopeptides (CPP) and the gallic acid grafted chitosan (GA-g-CS)-CPP nanoparticles with different GA substitutions before and after loading with EGCG. (NP1, NP2, NP3 and NP4 represent the CS-CPP nanoparticles and GA-g-CS-CPP nanoparticles with the GA substitutions of 26.5 ± 1.0, 113.7 ± 1.1, 126.0 ± 1.1 mg/g respectively. NP1-E, NP2-E, NP3-E and NP4-E represent the CS-CPP nanoparticles and GA-g-CS-CPP nanoparticles loading with EGCG, respectively). The a, b, c in panel A represent the significant difference (p < 0.05) in particle size among the nanoparticles composed of the GA-g-CS with different GA grafting ratios. ** in panel A represents the significant difference (p < 0.01) in particle size before and after loading with EGCG. TEM image of the GA-gCS-CPP nanoparticles with the GA substitutions of 113.7 ± 1.1 (C) and 126.0 ± 1.1 mg/g (D).

than that of the CS-CPP nanoparticles. The inhibition effects of the GA-g-CS-CPP nanoparticles were similar to those of the corresponding GA-g-CSs (Fig. 4B). Different from the results derived from the DPPH assay, the inhibition effects of both GAg-CS and GA-g-CS-CPP nanoparticles on linoleic acid autoxidation were significantly higher than that of the free GA, which might be related to the higher affinity of both the GAg-CS and GA-g-CS-CPP nanoparticles with linoleic acid than that of free GA.

the encapsulation efficiency of EGCG inside the polymer nanoparticles, which might be related to the increased molecular weight of the polymers grafting with GA. Also, the encapsulation efficiency of EGCG in the GA-g-CS-CPP nanoparticles with GA substitution ratios of 26.5 ± 1.0 mg/g appeared to be the highest among the tested nanoparticles. It might be caused by the largest particle size of the nanoparticles composed of the GA-g-CS with substitution ratios of 26.5 ± 1.0 mg/g (Fig. 3A).

3.2.3. Encapsulation efficiency of EGCG in GA-g-CS-CPP nanoparticles

3.2.4. In vitro release profile of EGCG from nanoparticles in simulated GI environments

Encapsulation efficiencies of EGCG in CS-CPP nanoparticles and GA-g-CS-CPP nanoparticles with different GA substitution ratios are shown in Table 1. Grafting GA onto CS appeared to increase

The in vitro release of EGCG from the CS-CPP nanoparticles and GA-g-CS-CPP nanoparticles in simulated gastric and intestinal environments is shown in Fig. 5. Compared with the CS-CPP

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environments. Furthermore, the release or leakage of EGCG from the nanoparticle decreased to be more controllable manner when the GA grafting ratios in GA-g-CSs increased. It meant that the controlled release profile of EGCG from the nanoparticles could be modulated effectively through changing the GA grafting ratios in GA-g-CSs. This phenomenon could have been caused by the grafting GA on the CS molecular chain weakening the strong repulsion among the highly protonated amino groups in CS under strong acidic condition, leading to the decrease of nanoparticles swelling, thus the slow down of the release of the payloads. However, in the simulated intestinal environment, no significant difference in the release profile of EGCG from the CS-CPP nanoparticles and GA-g-CSCPP nanoparticles could be observed. In Fig. 5B, the decrease of the measued release rate after 120 min was mainly caused by the unstability and degration of the EGCG under neutral and alkaline environments (Yoshino et al., 1999), which also indicated that it was very necessary to encapsulate EGCG in polymer nanoparticles. In fact, prevention of the burst release of loading drugs or nutraceuticals, especially small molecular and hydrophilic ones, from the delivery systems in the GI tract was a big challenge for oral delivery. The results in the present study meant that the release profile of the payloads from the GAg-CS-CPP nanoparicles could be adjusted through changing the GA grafting ratio, which would prefer the controlled release of the payloads at the sites of their action.

3.3. Stability of EGCG loaded in GA-g-CS-CPP nanoparticles under neutral and alkaline environments EGCG is very unstable under physiological neutral and alkaline environments. It was found in previous studies that EGCG quickly degraded in plasma and intestinal juice neutral and alkaline environments, forming its autoxidation homodimers (Yoshino et al., 1999). In Fig. 6, EGCG degraded quickly under pH 7.4 environment, with only 28.5% of EGCG still remaining in the PBS buffer after incubation for 6 h. Interestingly, after encapsulating in the GA-g-CS-CPP nanoparticles, the degradation of EGCG decreased significantly (p < 0.01), indicating that the nanoparticles with antioxidant activity could stabilize EGCG under alkaline environments significantly. It is very important for the delivery of oxidation-sensitive drugs and bioactive molecules to maintain their stability, further to increase their bioavailability and bio-functions.

3.4. Cytotoxicity of GA-g-CS-CPP nanoparticles and EGCG loaded GA-g-CS-CPP nanoparticles against cancer cells

Fig. 4 – Antioxidant activity of the gallic acid grafted chitosan (GA-g-CSs) and GA-g-CS-caseinophosphopeptides (CPP) nanoparticles. (A) Scavenging activity of the GA-g-CSs and GA-g-CS-CPP nanoparticles on DPPH radicals; Inhibition effects of the GA-g-CSs (B) and the GA-g-CS-CPP nanoparticles (C) on linoleic acid autoxidation. nanoparticles, the GA-g-CS-CPP nanoparticles with the molar ratios between glucosamine in CS and GA of 1:1 (data not shown) and 1:3 significantly (p < 0.05) reduced the release or leakage of EGCG from the nanoparticle in simulated gastric acid

The bioactivities of nutraceuticals carried by nanoparticle delivery systems in different organs were mainly dependent on the amount of their active constituents that could reach the target tissue. In oral administration route, it was found that the EGCG concentration in the small intestine and colon were much higher than other organs and tissues (Lambert et al., 2003). In fact, colon or colorectal cancer was one of the most prevalent types of cancer in many countries, mostly caused by the critical factor of a poor diet regimen. GA and its derivatives, especially EGCG, have been reported to possess prevention effects against the colon cancers in in vitro and in vivo assays (Santos, Ponte, Boonme, Silva, & Souto, 2013).

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Table 1 – The encapsulation efficiencies of EGCG in the CS-CPP nanoparticles and the GA-g-CS-CPP nanoparticles with different GA substitution ratios. Samples

EGCG-CSCPP NPs

EGCG-GA-g-CS (1:0.1)-CPP NPsa

EGCG-GA-g-CS (1:1)-CPP NPsa

EGCG-GA-g-CS (1:3)-CPP NPsa

Encapsulation efficiency (%)

84.4 ± 3.4

90.1 ± 2.4

87.7 ± 2.9

88.6 ± 2.1

a

The substitutions of GA on the dry GA-g-CS copolymers were 26.5 ± 1.0, 113.7 ± 1.1, and 126.0 ± 1.1 mg/g, with the molar ratios between glucosamine in chitosan and GA of 1:0.1, 1:1 and 1:3, respectively.

To evaluate the anticancer potential of the GA-g-CS-CPP nanoparticles and the EGCG loaded GA-g-CS-CPP nanoparticles against colon cancer, we performed trypan blue assay to determine human intestinal Caco-2 cancer cell density. The viable cells could exclude the trypan blue dye, which could not be stained. The trypan blue assay showed that in the cell line, grafting of GA onto CS enhanced significantly (p < 0.01) the inhibition effect of the polymer nanoparticles on the Caco-2 cell proliferation (Fig. 7A), and the inhibition effect of the polymer nanoparticles increased with the elevation of the GA grafting ratio. The IC50 values for the GA-g-CSs with GA substitutions of 26.5 ± 1.0 and 126.0 ± 1.1 mg/g were 154.4 and 115.5 µg/mL, respectively. Fig. 7B shows the Caco-2 cancer cell viability after incubation with EGCG and EGCG loaded GA-g-CS nanoparticles for 72 h. Very interestingly, the cell growth inhibition effects of EGCG were enhanced after being associated with the GAg-CS nanoparticles, especially for the one with the GA grafting ratio of 126.0 ± 1.1 mg/g (p < 0.01). The IC50 values for EGCG, and the EGCG loaded GA-g-CS nanoparticles with GA substitutions of 26.5 ± 1.0 and 126.0 ± 1.1 mg/g were 86.6, 77.3 and 50.2 µg/mL, respectively, in terms of EGCG concentration. Therefore, it would be concluded that association of EGCG within the GA-g-CS-CPP nanoparticles could obtain synergistic cancer cell growth inhibition effects against human colon cancer cells. In previous studies, similar results in the cytotoxicity of the polyphenols loaded nanoparticles against cancer cells were also reported (Lvov et al., 2009; Majumdar et al., 2014).

4.

Conclusion

In summary, it was found that grafting GA to CS endowed the CS based nanoparticles with not only antioxidant and anticancer activities, but also significantly improved physicochemical properties as oral delivery systems for labile polyphenol, controlling release of EGCG under simulated GI environments, preventing its degradation under neutral and alkaline environments, and amplifying its anticancer activities against colon cancer cells, which could be modified through changing the GA grafting ratios. Compared with CS, the GAg-CS conjugates possessed much higher solubility under neutral and alkaline environments. Therefore, the GA-g-CS-CPP nanoparticles are very potential delivery matrix for bioactive polyphenols, which can be used as novel functional foods.

Acknowledgements This work was supported by The Natural Science Foundation of Jiangsu Province, China (BK2012367), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20120097120001).

Fig. 5 – The release profile of EGCG from chitosan (CS)-caseinophosphopeptides (CPP) nanoparticles and gallic acid grafted chitosan (GA-g-CS)-CPP nanoparticles with different GA substitution ratios in simulated gastric (A) and intestinal (B) environments. The substitutions of GA on the dry GA-g-CS copolymers were 26.5 ± 1.0 and 126.0 ± 1.1 mg/g with the molar ratios between glucosamine in chitosan and GA of 1:0.1 and 1:3, respectively.

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Fig. 6 – The stabilization effects of the gallic acid grafted chitosan (GA-g-CS)-caseinophosphopeptides (CPP) nanoparticles on EGCG in alkalescent environment (pH 7.4). **Very significant difference (p < 0.01).

Fig. 7 – Cell viability of the human colon Caco-2 cancer cells incubated with the blank chitosan (CS)caseinophosphopeptides (CPP) nanoparticles, the gallic acid grafting chitosan (GA-g-CS)-caseinophosphopeptides (CPP) nanoparticles (A) and EGCG, EGCG loaded GA-g-CS-CPP nanoparticles (B).

REFERENCES

Acosta, E. (2009). Bioavailability of nanoparticles in nutrient and nutraceutical delivery. Current Opinion in Colloid & Interface Science, 14, 3–15. Al-Hilal, T. A., Alam, F., & Byun, Y. (2013). Oral drug delivery systems using chemical conjugates or physical complexes. Advanced Drug Delivery Reviews, 65, 845–864. Arranz, S., Silvan, J. M., & Saura-Calixto, F. (2010). Nonextractable polyphenols, usually ignored, are the major part of dietary

polyphenols: A study on the Spanish diet. Molecular Nutrition & Food Research, 54, 1646–1658. Braithwaite, M. C., Tyagi, C., Tomar, L. K., Kumar, P., Choonara, Y. E., & Pillay, V. (2014). Nutraceutical-based therapeutics and formulation strategies augmenting their efficiency to complement modern medicine: An overview. Journal of Functional Foods, 6, 82–99. Chen, M. C., Mi, F. L., Liao, Z. X., Hsiao, C. W., Sonaje, K., Chung, M. F., Hsu, L. W., & Sung, H. W. (2013). Recent advances in chitosan-based nanoparticles for oral delivery of macromolecules. Advanced Drug Delivery Reviews, 65, 865– 879.

Journal of Functional Foods 15 (2015) 593–603

Chiu, Y. L., Ho, Y. C., Chen, Y. M., Peng, S. F., Ke, C. J., Chen, K. J., Mi, F. L., & Sung, H. W. (2010). The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan. Journal of Controlled Release, 146, 152–159. Cho, Y. S., Kim, S. K., Ahn, C. B., & Je, J. Y. (2011). Preparation, characterization, and antioxidant properties of gallic acidgrafted-chitosans. Carbohydrate Polymers, 83, 1617–1622. Cirillo, G., Kraemer, K., Fuessel, S., Puoci, F., Curcio, M., Spizzirri, U. G., Altimari, I., & Iemma, F. (2010). Biological activity of a gallic acid-gelatin conjugate. Biomacromolecules, 11, 3309–3315. Curcio, M., Puoci, F., Iemma, F., Parisi, O. I., Cirillo, G., Spizzirri, U. G., & Picci, N. (2009). Covalent insertion of antioxidant molecules on chitosan by a free radical grafting procedure. Journal of Agricultural and Food Chemistry, 57, 5933–5938. Fang, J., Seki, T., & Maeda, H. (2009). Therapeutic strategies by modulating oxygen stress in cancer and inflammation. Advanced Drug Delivery Reviews, 61, 290–302. Gamboa, J. M., & Leong, K. W. (2013). In vitro and in vivo models for the study of oral delivery of nanoparticles. Advanced Drug Delivery Reviews, 65, 800–810. Grabowski, N., Hillaireau, H., Vergnaud, J., Santiago, L. A., KerdineRomer, S., Pallardy, M., Tsapis, N., & Fattal, E. (2013). Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells. International Journal of Pharmaceutics, 454, 686– 694. Hu, B., Pan, C. L., Sun, Y., Hou, Z. Y., Ye, H., Hu, B., & Zeng, X. X. (2008). Optimization of fabrication parameters to produce chitosan-tripolyphosphate nanoparticles for delivery of tea catechins. Journal of Agricultural and Food Chemistry, 56, 7451– 7458. Hu, B., Wang, L., Zhou, B., Zhang, X., Sun, Y., Ye, H., Zhao, L. Y., Hu, Q. H., Wang, G. X., & Zeng, X. X. (2009). Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection. Journal of Chromatography. A, 1216, 3223–3231. Hu, B., Wang, S. S., Li, J., Zeng, X., & Huang, Q. R. (2011). Assembly of bioactive peptide–chitosan nanocomplexes. The Journal of Physical Chemistry B, 115, 7515–7523. Janesirisakule, S., Sinthusake, T., & Wanichwecharungruang, S. (2013). Nanocarrier with self-antioxidative property for stabilizing and delivering ascorbyl palmitate into skin. Journal of Pharmaceutical Sciences, 102, 2770–2779. Kang, Y. A., & Kang, F. A. (2012). A balanced linear equation of the extended Woodward UV rules for all types of α,β-unsaturated ketones. Tetrahedron Letter, 53, 1928–1932. Kim, S., Park, H., Song, Y., Hong, D., Kim, O., Jo, E., Khang, G., & Lee, D. (2011). Reduction of oxidative stress by p-hydroxybenzyl alcohol-containing biodegradable polyoxalate nanoparticulate antioxidant. Biomaterials, 32, 3021–3029. Ko, E., Jeong, D., Kim, J., Park, S., Khang, G., & Lee, D. (2014). Antioxidant polymeric prodrug microparticles as a therapeutic system for acute liver failure. Biomaterials, 35, 3895–3902. Lambert, J. D., Lee, M.-J., Lu, H., Meng, X. F., Hong, J. J. J., Seril, D. N., Sturgill, M. G., & Yang, C. S. (2003). Epigallocatechin-3gallate is absorbed but extensively glucuronidated following

603

oral administration to mice. Journal of Nutrition, 133, 4172– 4177. Liu, L., Sun, Y., Laura, T., Liang, X., Ye, H., & Zeng, X. (2009). Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng. Food Chemistry, 112, 35–41. Lvov, Y. M., Shutava, T. G., Balkundi, S. S., Vangala, P., Steffan, J. J., Bigelow, R. L., Cardelli, J. A., O’Neal, D. P., & Lvov, Y. M. (2009). Layer-by-layer-coated gelatin nanoparticles as a vehicle for delivery of natural polyphenols. ACS Nano, 3, 1877–1885. Majumdar, D., Jung, K.-H., Zhang, H. Z., Nannapaneni, S., Wang, X., Amin, A. R. M. R., Chen, Z. J., Chen, Z. G., & Shin, D. M. (2014). Luteolin nanoparticle in chemoprevention: In vitro and in vivo anticancer activity. Cancer Prevention Research, 7, 65–73. Miller, E. W., Albers, A. E., Pralle, A., Isacoff, E. Y., & Chang, C. J. (2005). Boronate-based fluorescent probes for imaging cellular hydrogen peroxide. Journal of the American Chemical Society, 127, 16652–16659. Pawlak, A., & Mucha, A. (2003). Thermogravimetric and FTIR studies of chitosan blends. Thermochimica Acta, 396, 153–166. Piras, A. M., Dessy, A., Dinucci, D., & Chiellini, F. (2011). 2-Methoxy aniline grafted poly(maleic anhydride-alt-butyl vinyl ether) hemiester: A new biocompatible polymeric free radical scavenger. Macromolecules, 44, 848–856. Santos, I. S., Ponte, B. M., Boonme, P., Silva, A. M., & Souto, E. B. (2013). Nanoencapsulation of polyphenols for protective effect against colon–rectal cancer. Biotechnology Advances, 31, 514– 523. Siripatrawan, U., & Harte, B. R. (2010). Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract. Food Hydrocolloids, 24, 770–775. Spizzirri, U. G., Parisi, O. I., Iemma, F., Cirillo, G., Puoci, F., Curcio, M., & Picci, N. (2010). Antioxidant–polysaccharide conjugates for food application by eco-friendly grafting procedure. Carbohydrate Polymers, 79, 333–340. Ting, Y. W., Jiang, Y. K., Ho, C. T., & Huang, Q. R. (2014). Common delivery systems for enhancing in vivo bioavailability and biological efficacy of nutraceuticals. Journal of Functional Foods, 7, 112–128. Williams, S. R., Lepene, B. S., Thatcher, C. D., & Long, T. E. (2009). Synthesis and characterization of poly(ethylene glycol)glutathione conjugate self-assembled nanoparticles for antioxidant delivery. Biomacromolecules, 10, 155–161. Xie, M. H., Hu, B., Wang, Y., & Zeng, X. (2014). Grafting of gallic acid onto chitosan enhances antioxidant activities and alters rheological properties of the copolymer. Journal of Agricultural and Food Chemistry, 62, 9128–9136. Yang, C. S., Wang, X., Lu, G., & Picinich, S. C. (2009). Cancer prevention by tea: Animal studies, molecular mechanisms and human relevance. Nature Reviews. Cancer, 9, 429–439. Yang, L. Q., Yang, B., Zeng, D., Wang, D., Wang, Y., & Zhang, L. M. (2011). Formation and properties of a novel complex composed of an amylose-grafted chitosan derivative and single-walled carbon nanotubes. Carbohydrate Polymers, 85, 845–853. Yoshino, K., Suzuki, M., Sasaki, K., Miyase, T., & Sano, M. (1999). Formation of antioxidants from (-)-epigallocatechin gallate in mild alkaline fluids, such as authentic intestinal juice and mouse plasma. Journal of Nutrition Biochemistry, 10, 223–229.