Preparation of Chitosan–TPP Microspheres as Resveratrol Carriers Ah Ra Cho, Yong Gi Chun, Bum Keun Kim, and Dong June Park

Resveratrol (3,4 ,5-trihydroxy-trans-stilbene)-loaded chitosan–sodium tripolyphosphate (TPP) microspheres using high (310 to 375 kDa) and medium (190 to 310 kDa) molecular weight chitosan and TPP in varying concentrations were produced to improve resveratrol bioavailability. A 450 μm nozzle encapsulator was used to produce the microspheres. The mean microsphere particle size was between 160 and 206 μm, and exhibited a narrower size distribution as the TPP solution concentration increased. The encapsulation efficiency increased from 94% to 99% with a decrease in chitosan concentration from 1% to 0.5% and a decrease in crystallinity of the microspheres. FTIR data showed a polyelectrolyte interaction between chitosan and TPP. X-ray diffraction patterns were matched up with DSC and FTIR, which shows decrease of crystallinity and enhancement of hydrogen bonding with TPP concentration. An increase in the concentration of TPP solution from 1% to 3% led to a lower initial burst of resveratrol release. These results suggest that chitosan–TPP microspheres could be used as a potential delivery system to control the release of resveratrol.

Abstract:

E: Food Engineering & Physical Properties

Keywords: chitosan, controlled release, microspheres, resveratrol, sodium tripolyphosphate

Introduction

Resveratrol (3,4 ,5-trihydroxy-trans-stilbene) is a nonflavonoid poly-phenolic compound abundant in grapes, peanuts, and other foods commonly consumed in the human diet (Amri and others 2012). The health-beneficial properties of resveratrol have been extensively investigated in recent years due to their potential application in pharmaceutics, nutraceuticals, and functional foods (Sessa and others 2011). Chemically, resveratrol has 2 geometrical isomers, trans-resveratrol and cis-resveratrol; trans-resveratrol is the more stable and active natural isomer form (Filip and others 2003). Since the 1990s, critical studies have demonstrated several health-promoting activities of trans-resveratrol, such as antioxidant activity (Baur and Sinclair 2006; Mahal and Mukherjee 2006), reduction of lipid peroxidation, and lowering of blood pressure (Bradamante and others 2004). However, the biomedical applications of resveratrol remain limited due to its short biological half-life, labile properties, and rapid metabolism and elimination (Baur and others 2006). Therefore, development of resveratrol as a functional food and nutraceutical ingredient is feasible only when encapsulated in a delivery system capable of stabilizing and protecting resveratrol from degradation while preserving its biological activities and enhancing its bioavailability (Sessa and others 2011). Furthermore, microencapsulation helps to control drug release rates and prolong the release time (Desai and others 2006). There are many ways to encapsulate drug based on the use of surfactants, lipids, biopolymers, or mixtures of these components. Currently, several delivery systems for resveratrol have been prepared by liposomal system, cyclodextrin complexes, solid lipid nanoparticles, and chitosan microspheres (Augustin and others 2013). Chitosan is a naturally occurring and abundantly available polysaccharide (Agnihotri and others 2004; Sinha and others 2004); it is a glucosamine and N-acetyl-d-glucosamine copolymer linked by β-(1,4)-glycosidic bonds obtained by N-deacetlyation MS 20131523 Submitted 10/23/2013, Accepted 1/13/2014. Authors are with Korea Food Research Instit, 1201-62 Anyangpangyo-Ro, Baekhyun-Dong, BundangKu, Songnam-Si, Kyunggi-Do 463-746, Republic of Korea. Direct inquiries to author Park (E-mail: [email protected]).

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of chitin (Bhumkar and Pokharkar 2006). Chitosan-based microspheres have been extensively studied for drug delivery systems due to the biological properties of chitosan, such as its relative nontoxicity, biocompatibility, biodegradability, cationic properties, bioadhesive characteristics, and permeability enhancing properties (Fan and others 2012). Chitosan microspheres have been prepared through various methods, such as spray drying, chemical cross-linking, emulsions, droplet coalescence, and ionic gelation (Agnihotri and others 2004). Some of these methods have the inherent disadvantage of undesirable effects due to cross-linking agents such as glutaraldehyde, glyoxal, and ethylene glycol diglycidyl ether (Berger and others 2004; Bhumkar and Pokharkar 2006). To overcome the disadvantages, the ionic gelation with sodium tripolyphosphate (TPP), which has been approved as a “generally recognized as safe” (GRAS 582.6810) ingredient by the Food and Drug Administration, has been investigated by many researchers (Ko and others 2002; Luo and others 2010; Jarudilokkul and others 2011; Fan and others 2012). Chitosan and TPP can form biocompatible, cross-linked microspheres through electrostatic interactions that can be efficiently employed in nutraceutical compound delivery. The cross-linking density, crystallinity, and hydrophilicity of cross-linked chitosan allow modulation of nutraceutical release and extend its range of potential applications in delivery systems (Bhumkar and Pokharkar 2006). Bodmeier and others (1989) have produced chitosan–TPP microspheres by dropping chitosan droplets into a TPP solution. In this study, resveratrol-loaded chitosan–TPP microspheres were prepared by an ionic cross-linking method with different molecular weights and concentrations of chitosan and TPP solution concentrations. The objective of this work was to study the effect of these parameters on the release of resveratrol from chitosan–TPP microspheres.

Materials and Methods Materials Medium-molecular weight chitosan (190 to 310 kDa, MMW) and high-molecular weight chitosan (310 to 375 kDa, HMW) R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12395 Further reproduction without permission is prohibited

Resveratrol-loaded microspheres . . .

Parameters

Processing condition

Drug loading Polymer

Resveratrol 1 mg/100 mL Chitosan Molecular weight: medium (190–310 kDa), high (310–375 kDa) Concentration : 0.5, 1% (w/v) Sodium tripolyphosphate (TPP) Concentration : 1, 2, and 3% (w/v) 1200 Hz 1000 V 450 μm 100 rpm

Cross-linking solution Encapsulator

Frequency Electrode tension Nozzle size Stirring speed

were purchased from Sigma-Aldrich Chemical Co. Ltd. (Genomics Ventures, Iceland). Resveratrol, TPP, and phosphatebuffered saline (PBS buffer, pH 7.4) were bought from SigmaAldrich Chemical Co. Ltd. (St. Louis, Miss., U.S.A.). Lactic acid (Junsei Chemical Co. Ltd., Tokyo, Japan) and ethyl alcohol anhydrous (Carlo Erba Reagents, Milano, Italy) were also purchased. Filter paper (ø 110 mm) was purchased from Whatman Ltd. (Maidstone, UK).

Encapsulation efficiency The amount of encapsulated resveratrol was determined indirectly by measuring the differences between total amount of resveratrol and free amount of resveratrol in supernatant. The encapsulation efficiency (EE) was expressed through the following equation (Kim and others 2009): EE(%) =

Total amount of resveratrol − Free amount of resveratrol in supernatant Total amount of resveratrol ×100

Preparation of resveratrol-loaded chitosan–TPP microspheres Different concentrations of chitosan solution (0.5% and 1%, w/v) were prepared by dissolving MMW and HMW chitosan in 1% (v/v) lactic acid solutions. Resveratrol (1 mg) was dissolved in ethanol (1 mL) and added to 100 mL of chitosan solution with mild stirring (100 rpm/min) for 10 min. Chitosan solution (100 mL) was dropped into 200 mL of different concentrations of TPP solution (1%, 2%, and 3%, w/v) through an encapsulator (Encapsulator, B-390 pro, B¨uchi, Essen, Germany). The encapsulator had a 450 μm nozzle and a vibration frequency and electrode tension fixed at 1200 Hz and 1000 V, respectively. The microspheres were stirred at 100 rpm for 30 min for hardening. Chitosan–TPP microspheres were prepared according to the ionotropic gelation process. Finally, the microspheres were filtrated using ø 110 mm filter paper, and washed twice with distilled water. Parameters for all the preparations are summarized in Table 1. All the formulations were made in triplicate.

In vitro release study An in vitro release study of resveratrol was carried out in PBS R buffer (pH 7.4). The entire release system (Vision G2 Elite 8 Dissolution Tester, Hanson Research Corp., Calif., U.S.A.) was maintained at 37 °C with continuous stirring at 100 rpm/min. Resveratrol-loaded chitosan–TPP microspheres (0.5 g) were placed in PBS buffer (500 mL) and left to react for 24 h after which 1 mL of the buffer was collected and filtered using a 0.45-μm PVDF syringe filter (Whatman Ltd., Maidstone, UK). The amount of loaded resveratrol was analyzed by HPLC as described above. The drug concentrations were calculated by measuring the peak area and compared them with the peak area of known standards. The mass of resveratrol released from microspheres at given time were determined through the following equation: Reveratrol release(%) = Mass of resveratrol released from microspheres

× 100

Mean particle size and particle size distribution Total loading amount of resveratrol in microspheres A laser diffraction particle size analyzer (CILAS 1064, Compagnie Industrielle Des Lasers, Orleans, France) was used to measure Fourier transform infrared (FTIR) spectroscopy particle size distribution and the mean particle size of chitosan– FTIR spectroscopy was used to measure changes in the chemical TPP microspheres. structure of chitosan, TPP, resveratrol, and resveratrol-loaded microspheres. The spectra were acquired at 600 to 4000 cm−1 wave −1 Determination of resveratrol content by high performance numbers with a 4 cm resolution utilizing an FTIR spectrophotometer (Vertex 70, Bruker, Rheinstetten, Germany) equipped liquid chromatography (HPLC) with an attenuated total reflectance cell. Resveratrol concentrations were obtained through the direct injection of samples into a HPCL system (L-2130, Hitachi Ltd., Tokyo, Japan). A reversed phase column (Nucleosil 100-5 C18 , Differential scanning calorimetry (DSC) Thermal transition properties of chitosan, resveratrol, and 250 mm × 4.0 mm, D¨uren, Germany) was used for separation, whereas an acetonitrile–water (60:40) solution was used as the chitosan–TPP microspheres were measured using DSC (Perkin mobile phase. The flow rate and ultraviolet wavelength were Elmer DSC7, Waltham, Mass., U.S.A.). Each sample (5 mg, dry 0.3 mL/min and 306 nm, respectively. The resveratrol concen- basis) was accurately weighed on an aluminum pan and then the trations were determined by measuring the peak areas and com- pan was sealed. The samples were heated from 20 to 430 °C at paring them with the peak areas of known standards (Kim and a rate of 10 °C/min, and an empty pan was used as a reference. The onset (To ), peak (Tp ), and conclusion (Tc ) temperatures along others 2003). Vol. 79, Nr. 4, 2014 r Journal of Food Science E569

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Table 1–Processing conditions used in the study.

Resveratrol-loaded microspheres . . . with the gelatinization enthalpy (࢞H) were determined from the electron microscope (SEM, S-3500N, Hitachi Ltd., Tokyo, Japan). The wet microspheres were attached onto a samthermograms. ple plate and the temperature of the cooling stage was fixed at −10 °C to accurately control dehydration. The samX-ray diffraction (XRD) pattern XRD patterns of chitosan and resveratrol-loaded chitosan mi- ples’ surface structure was observed at 20 kV of accelerating crospheres were obtained by a Philips X´PERT-PRO X-ray voltage. diffractomether (PANalytical, Almero, the Netherlands) equipped ˚ X-ray source. The volt- Statistical analysis with a monochromatic Cu Kα (1.5406 A) age used was 45 kV and angle range was scanned from 5° to 35° All data were expressed as the mean of triplicate and anawith step size of 0.0167°. lyzed by analysis of variance (ANOVA) using the statistical package of social science (SPSS) version 10.0 (SPSS Inc., Chicago, Micrography Ill., U.S.A.). The significant means were compared by DunThe microstructure of resveratrol-loaded chitosan–TPP mi- can’s multiple range tests. Significance was accepted at the 5% crospheres was observed by using a low-vacuum scanning level.

E: Food Engineering & Physical Properties Figure 1–Particle size distribution of resveratrol-loaded chitosan–TPP microspheres; cross-linked with 0.5% MMW chitosan and 1% (A), 2% (B), and 3% (C) TPP; 1% MMW chitosan and 1% (D), 2% (E), and 3% (F) TPP; 0.5% HMW chitosan and 1% (G), 2% (H), and 3% (I) TPP; 1% HMW chitosan and 1% (J), 2% (K), and 3% (L) TPP.

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Resveratrol-loaded microspheres . . .

Mean particle size (μm) TPP (%)

Chitosan

1 2 3 1 2 3

MMW HMW

0.5% 167.26 170.02 160.57 190.50 185.91 193.20

± ± ± ± ± ±

1.36b 1.41c 2.81a 0.09e 0.69d 1.42f

1% 161.24 174.97 175.75 206.81 163.83 178.23

± ± ± ± ± ±

1.75a 0.37c 0.91c 0.99e 0.52b 1.10d

Mean with different letters in same row is significantly different (P < 0.05) by Duncan’s multiple range test. a

Results and Discussion Mean particle size and particle size distribution Table 2 shows the microsphere mean particle size in relation to chitosan molecular weight (MMW and HMW) and concentration (0.5% and 1%) cross-linked with TPP solutions of varying concentrations (1%, 2%, and 3%). Although mean particle size did not change with increasing TPP solution concentration, it increased with the HMW chitosan (Table 2), which is in agreement with the study by Desai and others (2006), who found that the mean particle

the greater viscosity of the polymer solution due to the higher molecular weight. Mean particle size is one of the most important parameters determining microsphere biocompatibility and bioactivity (Luo and others 2010). In general, it is known that mean particle size and particle size distribution of chitosan–TPP nanoparticles depend largely upon chitosan concentration and molecular weight and mixing conditions, such as stirring (Gan and others 2005). Unlike mean particle size, particle size distribution was narrower as the TPP solution concentration increased (Figure 1). Fan and others (2012) suggested that when the TPP solution was below 2.5% in volume during the production of chitosan–TPP microspheres, the reaction solution remained opalescent because chitosan and TPP did not fully cross link. However, when volume was increased to 3.3%, the reaction solution was clear without visible particles and opacity due to an increase in cross-linking density between chitosan and TPP. Chitosan–TPP microspheres thus form dense structures at a specific TPP solution concentration. In this study, a 3% TPP solution was appropriate for chitosan–TPP interactions without TPP separation from the particle during the hardening stage, allowing for the formation of uniform particles.

Encapsulation efficiency Encapsulation efficiency is defined as the percentage of resveratrol loading content that can be entrapped into chitosan–TPP microspheres. As shown in Figure 2, encapsulation efficiency decreased as concentration of chitosan increased. An encapsulation efficiency of MMW chitosan microspheres prepared with 1% and 2% TPP solution were decreased from 99.01% to 94.59% and from 99.55% to 97.07%, respectively by increasing concentration of chitosan from 0.5% to 1% (Figure 2A). Moreover, similar results were observed for HMW chitosan–1% TPP microspheres, for which encapsulation efficiency decreased from 99.10% to 94.83%. These results are in agreement with previous studies in which encapsulation efficiency increases with increasing chitosan concentration (Sinha and others 2004; Gan and others 2005; Wu and others 2005). Wu and others (2005) reported that an increase in chitosan concentration from 1% to 3% led to a decrease in encapsulation efficiency of ammonium glycyrrhizinate and a decrease in encapsulation efficiency of BSA-loaded nanoparticles from 88.3% to 61.3% (Wu and others 2005). This indicates that the greater solution viscosity due to higher chitosan concentrations contributes to a decrease in encapsulation efficiency (Gan and others 2005). Therefore, a low chitosan concentration would increase the drug encapsulation capabilities. In addition, both MMW and HMW chitosan–TPP microspheres showed a high encapsulation efficiency between 94.59% and 99.84%. Similar results were reported in a study by Ko and others (2002) in which felodipine-loaded chitosan–TPP microspheres showed over 90% encapsulation efficiency (Ko and others 2002). Changes in chitosan concentration and molecular weight did not yield significant differences in encapsulation efficiency, however higher TPP solution concentrations produced a higher encapsulation efficiency. Finally, because resveratrol is insoluble in water, it remains undissolved during the crosslinking and hardening process and therefore there is minimal Figure 2–The effect of microsphere concentration on encapsulation ef- loss of resveratrol during the hardening and washing stages reficiency at 0.5% and 1% of chitosan (A) MMW chitosan and (B) HMW sulting in high encapsulation efficiencies (Ko and others 2002). chitosan.

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Table 2–Mean particle size of resveratrol-loaded chitosan–TPP size of chitosan–TPP microspheres increased when the molecular microspheresa . weight of chitosan increased. This particle size increase is due to

Resveratrol-loaded microspheres . . .

E: Food Engineering & Physical Properties

This is consistent with previous results, where a very high (Sinha and Trehan 2003; Agnihotri and others 2004; Gan and resveratrol encapsulation efficiency (>97.7%) was observed when Wang 2007). Ko and others (2002) reported that higher TPP solution concalcium-pectinate was used to form microspheres (Das and Ng centrations resulted in slower felodipine release from microspheres 2010). (Ko and others 2002). Moreover, Desai and Park found that the release rate of entrapped acetaminophen from chitosan microIn vitro release study Figure 3 presents the in vitro release profiles of resveratrol from spheres decreased when the TPP solution concentration increased chitosan–TPP microspheres with varying TPP solution concen- (Desai and Park 2005). These results are correlated with this study, trations. As the TPP solution concentration increased, slow re- in which slower release rate was observed with concentration of lease tendency was obtained for all samples (Figure 3). Although TPP solution (Figure 3). The release rate of drugs from chitosan– there were no significant changes during the first 30 min ex- TPP microspheres depends on the density of the chitosan–TPP cept for microspheres cross-linked with 1% HMW chitosan with matrix, which increases with increasing TPP solution concentra3% TPP solution (Figure 3), a very slow release was observed tion. Furthermore, a previous report suggested that the diffusion even after 30 min. Drug release is dependent on both diffusion of drugs from chitosan films decreased as the concentration of the through the polymer matrix and polymer degradation. The release TPP solution increased (Remunan-Lopez and Bodneier 1997). of resveratrol, or other encapsulated drugs, is due to the following The swelling and permeability characteristics of chitosan films are phenomena: (i) release from the microsphere surface; (ii) release dependent on the concentration of the cross-linking agent, and through the pores, dependent on microsphere structure; (iii) dif- concentration of 3% TPP solution shows higher film thickness and fusion through the intact polymer barrier, dependent on intrinsic puncture strength compared to other concentrations (Remunanpolymer properties and core solubility; (iv) diffusion through a wa- Lopez and Bodneier 1997). Figure 4 shows the effects of chitosan concentration and ter swollen barrier, dependent on polymer hydrophilicity, which in turn depends on polymer molecular weight; and (v) polymer molecular weight on resveratrol release at 3% TPP concentration, erosion and bulk degradation release affected by the rate of ero- indicating that a significantly similar release tendency was observed sion and hydrolysis of polymer chains, leading to pore formation for 0.5% MMW and 1% HMW chitosan microspheres. However,

Figure 3–The effect of TPP concentrations on in vitro release profile of resveratrol-loaded chitosan–TPP microspheres with 0.5% (A) and 1% (B) MMW chitosan, and 0.5% (C) and 1% (D) HMW chitosan.

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Resveratrol-loaded microspheres . . . (Huang and Brazel 2001). Because of the important roles of initial burst release, this study focused on the reduction of initial release, so 1% HMW chitosan microspheres were chosen for further study to analyze relation of release mechanism with physical properties of microspheres.

Fourier transform infrared spectroscopy The microsphere intermolecular interactions were characterized by FTIR (Figure 5). Seven characterization peaks were observed in HMW chitosan–TPP microspheres at 3358.46, 2873.31, 1646.15 to 1653.24, 1376.47 to 1587.93, 1058.24 to 1064.48, 1026.87 to 1028.81, and 886.58 to 894.85 cm−1 . According to Luo and others (2010) and Lawrie and others (2007), these peaks could be explained as O–H from H-bonded, C–H stretch form aldehyde,

Figure 4–The effect of molecular weight and concentrations of chitosan on in vitro release profile of resveratrol-loaded chitosan–3% TPP microspheres; (A) release profile over 7 h and (B) initial burst of microspheres over 1 h.

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the initial burst (from 0 min to 1 h) of HMW chitosan microspheres was significantly lower than that of MMW microspheres (Figure 4B). It is known that the viscosity of chitosan solutions is important in the formation of microspheres and that a higher viscosity leads to decreased drug release rates (Jarudilokkul and others 2011). Gan and others (2005) prepared BSA-loaded chitosan–TPP microspheres and found that HMW chitosan may be a factor in reduced burst effect and slower release (Gan and others 2005). Furthermore, addition of TPP may provide a greater cross-link density with HMW, causing an increased rigidity as well as increased inter-chain bonding, and thus reducing drug release. Based on the release results, 1% HMW chitosan–TPP microspheres showed effect on the lower burst release and slower release. Generally, higher initial burst may lead to reduction of the effective lifetime of microspheres and waste amount of drug

Resveratrol-loaded microspheres . . . C=N and N–H from amine I and amide II, -CH3 symmetrical deformation, C–N from amine, C–O stretching, and C–H from alkene or aromatic bonds, respectively. In comparison with chitosan, resveratrol-loaded chitosan–TPP microspheres showed the characteristic absorption bands at spe-

E: Food Engineering & Physical Properties

cific wavenumbers (Figure 5). As the concentration of the TPP solution increased, the peaks at 3358.46 cm−1 became wider, indicating an enhancement in hydrogen bonding. Furthermore, the peak at 1646.15 to 1650.20 cm−1 became larger than that for chitosan alone due to the electrostatic interaction between the phosphoric groups in TPP and the amino groups in chitosan (Wu and others 2005; Shah and others 2009). The stronger absorption band at the range of 1351.89 to 1376.47 cm−1 of HMW chitosan in the resveratrol-loaded microspheres was characterized as CH3 symmetrical deformation (Shavi and others 2011). The TPP peak at 1127.29 cm−1 disappeared after chitosan and TPP cross-linking due to chitosan and TPP intermolecular interactions. The decreased absorption at 890.83 to 892.93 cm−1 and 710.66 to 711.70 cm−1 in HMW chitosan microspheres corresponds to a C–H bend from the aromatic ring, providing confirmation of incorporation of resveratrol in the microsphere matrix (Shavi and others 2011).

Thermal properties DSC studies were performed to understand the behavior of the cross-linked chitosan on application of thermal energy. Figure 6

Figure 5–FTIR spectrum of cross linking material (A): (a) HMW chitosan powder, (b) TPP, (c) resveratrol, and HMW chitosan–TPP microspheres (B): (a) resveratrol unloaded chitosan–1% TPP microspheres, (b) resveratrol unloaded chitosan–2% TPP microspheres, (c) resveratrol unloaded chitosan–2% TPP microspheres, (d) resveratrol-loaded chitosan–1% TPP microspheres, (e) resveratrol-loaded chitosan–2% TPP microspheres, (f) resveratrol-loaded chitosan–3% TPP microspheres.

Figure 7–XRD patterns of resveratrol-loaded HMW chitosan–TPP microspheres: (A) chitosan, (B) TPP, (C) chitosan–TPP 1% microspheres, (D) chitosan–TPP 2% microspheres, and (E) chitosan–TPP 3% microspheres, and (F) resveratrol. Figure 6–DSC analysis of resveratrol-loaded HMW chitosan–TPP microspheres: (A) chitosan, (B) resveratrol, (C) chitosan–TPP 1% microspheres, (D) chitosan–TPP 2% microspheres, and (E) chitosan–TPP 3% microspheres.

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shows the thermal transition of HMW chitosan, resveratrol, and resveratrol-loaded chitosan–TPP microspheres. A lower ࢞H value was observed as the TPP solution concentration increased from 1% to 3% (473.89, 231.14, and 220.47 J/g, respectively; Figure 6). Polymer crystallinity has a great effect on the polymer’s barrier properties. Since the crystalline phase of the polymer is essentially impermeable to water, encapsulation is likely to occur in the amorphous region of the polymer, the higher the crystalline region the lower the encapsulation efficiency (Kim and others 2005; Youan and others 1999). The highest encapsulation efficiency was obtained when the TPP solution concentration increased from 1% to 3% (Figure 2), which indicates that the crystallinity of the wall materials is an important factor in encapsulation efficiency. Furthermore, resveratrol showed a single sharp endothermic peak corresponding to melting of its crystalline structure at 266 °C; this peak was absent in the resveratrol-loaded chitosan microsphere thermogram (Figure 6). Similarly, Isailovic and others (2013) reported the absence of the resveratrol melting peak in resveratrol-loaded liposomes due to resveratrol entrapment in the liposome and significant interactions between resveratrol and liposome structural components (Isailovic and others 2013).

reported that chitosan crystallized in an orthorhombic unit cell (Epure and others 2011; Matet and others 2013). After ionic cross-linking with TPP, no peak was found at 9.7o in the diffractograms of both resveratrol-loaded chitosan microspheres (Figure 7C and D), indicating that hydrogen bonding occurred by cross linking. These results are in agreement with the FTIR results (Figure 5), and may be explained an enhancement of hydrogen bonding by chitosan and TPP cross linking. The broader peak of chitosan microspheres was obtained at higher TPP concentration (Figure 7C–E). It is well known that the width of XRD peak is related to the size of crystallite, the broadened peak usually results from imperfect crystal (Jingou and others 2011). The increase of imperfect crystal is corresponding with DSC results (Figure 6), which show decrease of crystallinity by TPP concentration. As compared with a chitosan powder, XRD spectrum of resveratrol-loaded microspheres the characteristic at 2θ of 20.9o , confirming the presence of resveratrol within chitosan microspheres (Figure 7). This implied that the encapsulation of resveratrol resulted in a change in the chitosan–TPP encapsulated structure (Yoksan and others 2010; Hosseini and others 2013).

XRD Crystallographic structure of chitosan and resveratrol-loaded microspheres were determined by XRD and presented in Figure 7. Chitosan powder XRD shows the presence of crystalline regions with 2 main peaks at 9.7o and 19.4o (Figure 7A), and similar results were also reported by Matet and others (2013) and Yoksan and others (2010). The peak around at 10o represents hydrated crystal due to the integration of water molecules, and the peak located at 20o is assigned to the crystal lattice of chitosan, which has been

Micrography The shape and surface morphology of chitosan–TPP microspheres are shown in Figure 8. Microspheres prepared with no resveratrol loading had a smooth surface (Figure 8A and B), whereas the resveratrol-loaded microspheres showed a rough and wrinkled surface (Figure 8C and D). A similar result has been observed in resveratrol-loaded pectin-based microspheres where a rough and rugged surface was produced due to the high hydrophobicity of resveratrol thus remaining as insoluble particles

Figure 8–Scanning electron microscopy (SEM) of HMW 1% chitosan–3% TPP microsphere (A) 100× and (B) 500×, resveratrol-loaded HMW 1% chitosan–3% TPP microsphere (C) 100× and (D) 500×. Vol. 79, Nr. 4, 2014 r Journal of Food Science E575

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Resveratrol-loaded microspheres . . .

Resveratrol-loaded microspheres . . . throughout the matrix (Das and others 2011). In addition, Ramachandran and others (2011) suggested that this roughness is due to insoluble drug particles remaining in the surface pores after aqueous washing (Ramachandran and others 2011). This could explain the roughness of the drug-loaded microspheres.

Conclusions

E: Food Engineering & Physical Properties

This study showed that encapsulation efficiencies of over 94.59% were observed in all resveratrol-loaded chitosan–TPP microspheres prepared. The mean particle size ranged between 160.58 and 206.52 μm, and the particle size distribution narrowed as concentration of TPP solution increased. FTIR analysis of chitosan microspheres provided evidence of cross-linking between positively charged amino groups and negatively charged phosphate groups. The encapsulation efficiency of resveratrol increased with a decrease in crystallinity and concentration of the chitosan solution. XRD patterns were in accordance with the DSC and FTIR. Crystalline decreased and structure became dense by TPP concentration. A lower initial burst of in vitro release was displayed at higher TPP solution concentrations; however, changes in the concentration and molecular weight of chitosan had no effect on initial drug release. Therefore, the concentration of TPP solutions plays an important role on the characteristics of chitosan– TPP microspheres, allowing for high a control of resveratrol release.

Acknowledgment This work is a part of the Korea Food Research Instit. research project E 0121704.

Conflicts of Interest The authors report no conflicts of interest. The authors alone are responsible for the contents and writing of this article.

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Preparation of chitosan-TPP microspheres as resveratrol carriers.

Resveratrol (3,4',5-trihydroxy-trans-stilbene)-loaded chitosan-sodium tripolyphosphate (TPP) microspheres using high (310 to 375 kDa) and medium (190 ...
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