Optimization and Characterization of Curcumin Loaded in

Article pubs.acs.org/JAFC

Optimization and Characterization of Curcumin Loaded in Octenylsuccinate Oat β‑Glucan Micelles with an Emphasis on Degree of Substitution and Molecular Weight Jia Liu,† Fang Chen,† Weina Tian,‡ Yaqin Ma,† Jing Li,† and Guohua Zhao*,†,§ †

College of Food Science, Southwest University, Chongqing 400715, People’s Republic of China Institute for Food & Bioresource Engineering, College of Engineering, Peking University, Beijing 100871, People’s Republic of China § Food Engineering and Technology Research Centre of Chongqing, Chongqing 400715, People’s Republic of China ‡

ABSTRACT: The dependence of the curcumin loading capacity (CLC) of octenylsuccinate oat β-glucan (OSG) micelles on the structural parameters (degree of substitution, DS; molecular weight, Mw) of OSG was unknown and explored in this study. Meanwhile, the curcumin-loaded OSG micelle (COM) was first characterized. The results from response surface methodology revealed that the linear effects of Mw and stirrer input power, as well as the quadratic effect of DS, were significant (p < 0.05). The maximum CLC value of the OSG micelle was obtained as 4.21 μg/mg. Dynamic light scattering showed that the average size and ζ potential of the COM were 308 nm and −10.8 mV, respectively. Transmission electron microscopy and atomic force microscopy evidenced that the COM was elliptical in shape. Fourier transform infrared spectroscopy, differential scanning calorimeltry, and X-ray diffraction revealed that curcumin was loaded in OSG micelles in an amorphous form by interacting with OSG molecules. KEYWORDS: curcumin, octenylsuccinate oat β-glucan, micelles, degree of substitution, response surface methodology

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by complexing with chitosan,17,18 soy protein,19 or zein;20 and codissloved by covalently attached to hyaluronic acid21 or alginate.22 In loading curcumin into micelles formed by amphiphilic polymers, certainly, the structural characteristics of the host polymer are of much importance. Amphiphilic copolymers with varying proportions of the hydrophobic and hydrophilic parts show different curcumin loading capacities (CLCs).15 Oat soluble β-glucan is a functional and bioactive polysaccharide consisting of linear chains of β-D-glucopyranosyl units linked with (1→3) and (1→4) linkages.23 Previously published works from our studies were mainly focused on the noncovalent interactions between oat β-glucan and polyphenols.23−27 It was proven that the interaction between hydrophilic polyphenols and oat β-glucan was governed by hydrogen bonds. However, hydrophobic polyphenols showed a poor affinity binding to oat β-glucan. In our recent work, octenylsuccinate oat β-glucan (OSG) was synthesized, and the potential of OSG to increase the dispersion of curcumin in aqueous medium was proven.28 The degree of substitution (DS) and oat β-glucan molecular weight (Mw) significantly affected the size and ζ potential of OSG micelles. However, the effects of structural characteristics of OSG on the interaction between OSG and curcumin are still unknown. Besides, the effects of curcumin loading on the morphological properties of OSG micelles are not reported up to date. Thus, the aim of the present study was to investigate the effects of DS, Mw, and

INTRODUCTION Food can be used as a vehicle to deliver bioactive compounds as a daily nutrient supplement.1 Thus, incorporating bioactive compounds with health-promoting or disease-preventing effects into food has attracted much attention both in basic research and industrial applications.2,3 However, a number of bioactive compounds own a low solubility, which limits their incorporation in aqueous foods. Novel vehicles, including nanoparticles, micelles, and hydrogels, have been designed to counteract this problem.3,4 Generally, these methods successfully dispersed hydrophobic bioactive compounds in water through weak interactions (hydrophobic interactions, hydrogen bonds, and van der Waals forces) or by forming covalent bonds with hydrophilic or amphiphilic polymers.5,6 Polyphenols have been eliciting substantial interest because of the increasing number of supporting proof of their beneficial effects on human health. Curcumin is an important polyphenol monomer isolated from the rhizome of the plant Curcuma longa along with demethoxy curcumin and bisdemethoxy curcumin.7 Extensive studies confirmed diversely biological activities of curcumin, such as anti-inflammation, antioxidation, anticancer, and antimicrobiology.8,9 Regrettably, the poor water solubility (11 ng/mL, 25 °C) of curcumin badly limited its incorporation in aqueous foods. Numerous approaches have been addressed to improve the water solubility or dispersion of curcumin, such as encapsulation into polymeric micelles of beta casein,10 hydrophobically modified starch,11 or a copolymer of Nisopropylacrylamide with N-vinyl-2-pyrrolidone, poly(ethyleneglycol) monoacrylate,12 polyethylene glycol, and polyethylenimine,13 methoxypoly(ethylene glycol)-b-poly(εcaprolactone-co-p-dioxanone),14 and Pluronic (P123 and F68);15 included in cyclodextrins;16 amorphously dispersed © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7532

March July 8, July 8, July 8,

26, 2014 2014 2014 2014

dx.doi.org/10.1021/jf5014692 | J. Agric. Food Chem. 2014, 62, 7532−7540

Journal of Agricultural and Food Chemistry

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stirrer input power (IP) on the loading of curcumin in OSG micelles using response surface methodology (RSM). Moreover, the curcumin-loaded OSG was characterized by using dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction (XRD).

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Table 1. Degree of Substitution (DS) and Reaction Condition for Preparing Octenylsuccinate Oat β-Glucan (OSG)a no.

oat βglucanb

OSAc (mL)

time (h)

1 2 3 4 5 6 7 8 9

OG OG OG OG1 OG1 OG1 OG2 OG2 OG2

0.3 0.6 0.6 0.3 0.6 0.6 0.3 0.6 0.6

2 2 3 2 2 3 2 2 3

MATERIALS AND METHODS

Materials. Commercial soluble β-glucan (80 wt% β-glucan content) extracted from oats was purchased from Zhangjiakou Yikang Biological Technology Co., Ltd. (China). 2-Octen-1-ylsuccinic anhydride (product no. 416487) was purchased from Sigma Chemical Co. (USA). Curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, 98% purity) was supplied by Adamas Reagent Co., Ltd. (China). IR spectroscopy grade KBr was purchased from Guangfu Reagent Chemical Co., Ltd. (China). Chromatographic grade acetonitrile and formic acid were also used. All chemicals were used in this study without further purification. Oat β-Glucan Solution Preparation and Hydrolysis. Oat βglucan (2.500 g) was dispersed in 1 L of distilled water and heated at 80 °C with magnetic stirring for 2 h. The resultant dispersion was cooled to room temperature with tap water. An oat β-glucan solution (1.953 ± 0.075 g/L) was obtained as the supernatant by centrifuging the dispersion at 3000g for 10 min. Oat β-glucan was hydrolyzed according to a previously described method.29 Two hydrolysates of oat β-glucan (OG1 and OG2) were prepared in the present study. In detail, 100 μL (OG1) and 200 μL (OG2) of hydrochloric acid (6 M) were mixed with 100 mL of the above-prepared oat β-glucan solution (0.195 g oat β-glucan), respectively. Then, the mixtures were inoculated at 50 °C for 10 min to allow the intended hydrolysis. After hydrolysis was completed, hydrolysates were rapidly cooled to room temperature and neutralized (pH 6∼7) with 2 M NaOH. The hydrolysates were successively dialyzed against tap water and distilled water for 24 h in a dialysis bag (MD44-14, Union Carbide Co., Seadrift, TX, USA) with a size exclusion of 14 000 g/mol for globular molecules. Then, they were subjected to lyophilization to obtain OG1 (0.192 ± 0.007 g) and OG2 (0.188 ± 0.004 g). The Mw values of oat β-glucan, OG1, and OG2 were determined as 16.8 × 104, 11.9 × 104, and 7.3 × 104 g/mol, respectively, on a high-performance sizeexclusion chromatography (HPSEC) system.30 Different Mw values (4 × 104, 7 × 104, 10 × 104, and 200× 104 g/mol) of standard dextrans (Pharmacia Fine Chemicals, Uppsala, Sweden) were used as Mw standard reference. Synthesis of Octenylsuccinate of Oat β-Glucan and Its Hydrolysates. Oat glucan, OG1, and OG2 were octenylsuccinated according to our previously proposed method, and the DS of each was determined by elemental analysis using an Elementar CHNS analyzer (Vario EL III, Elementar Analysensysteme, Germany).28,31 Detailed information on reaction conditions and DS of the resultant products was shown in Table 1. Loading Curcumin into OSG Micelles. Freeze-dried OSG powder (40 mg) was dissolved in 20 mL of distilled water by heating at 100 °C for 2 min continuous stirring. After complete cooling at room temperature, the volume of the resultant solution was complemented to 20 mL by adding distilled water. Curcumin powder (10 mg) was introduced into the solution, and the suspension was homogenized at 12 000 rpm for 1 min in a homogenizer (IKA T18 basic, IKA-Werke GmbH & Co., Staufen, Germany). Then, the resultant homogenate was continuously stirred under different input power of the stirrer (IP) (Table 2) at 25 °C for 24 h to complete the transfer of curcumin into OSG micelles. Finally, the suspension was centrifuged at 5600g for 10 min to recover the supernatant for determining the CLC of OSG micelles. The CLC values of OSG micelles were determined as described previously and calculated by using the following equation:28

Mwd (g/mol)

DS 0.0076 0.0169 0.0266 0.0085 0.0175 0.0281 0.0080 0.0172 0.0273

± ± ± ± ± ± ± ± ±

0.0010 0.0038 0.0022 0.0007 0.0032 0.0021 0.0005 0.0025 0.0015

17.0 17.1 17.3 12.0 12.2 12.3 7.4 7.5 7.5

× × × × × × × × ×

104 104 104 104 104 104 104 104 104

All reactions were carried out at 45 °C. bThe molecular weights of oat β-glucan (OG), OG1, and OG2 were 16.8, 11.9 and 7.3 × 104 g/ mol. The molecular weights of OG, OG1, and OG2 were determined before derivatization with OSA. cOSA, 2-octen-1-ylsuccinic anhydride. d The molecular weight of octenylsuccinate oat β-glucan was calculated according to the DS and molecular weight of oat β-glucan. a

Table 2. Level of Various Independent Variables at Coded Values of Response Surface Methodology Experimental Design coded values independent variable

symbol

−1

0

1

degree of substitution molecular weight (104 g/mol) stirrer input power (W)

X1 X2 X3

0.0080 7.3 3.3

0.0172 11.9 3.8

0.0273 16.8 4.4

curcumin loading capacity (μg/mg) =

mass of loaded curcumin in 1 mL of OSG solution (μg) mass of used OSG in 1 mL of OSG solution (mg)

(1) Experimental Design. RSM based on the Box−Behnken design was adopted to observe the effects of DS and Mw of OSG as well as IP of operation on CLC of OSG micelles. A three-level, three-variable design was used in the present study. The three independent variables are DS (X1), Mw (X2), and IP (X3), and CLC was used as the dependent variable response. The factorial design consisted of 12 factorial points and 5 central points. The coded and uncoded independent variables used in the RSM design are listed in Table 3. To fulfill this experiment, totally, nine OSG should be synthesized. Theoretically, as the requirement of the experiment design, nine OSG could be evenly divided into three groups, and the OSGs in the same group own the identical DS but the differentiated Mw. Actually, it is an impossible task to obtain an identical DS for oat β-glucans with different Mw. Therefore, three OSG with different Mw and nonsignificantly differentiated DS were grouped in the present study (Table 3). All trials were performed in triplicate. A Design-Expert software version 7.0 (STAT-EASE Inc., Minneapolis, MN, USA) was used to generate the experimental designs, statistical analysis, and regression model. A second-order polynomial equation was used to express the predicted responses (CLC) as functions of the independent variables, as shown in the following equation:

Yi = a0 + a1X1 + a 2X 2 + a3X3 + a12X1X 2 + a13X1X3 + a 23X 2X3 + a11X12 + a 22X 2 2 + a33X32 where Xi represents the independent variables, a0 is a constant, and ai, aii, and aij are the linear, quadratic, and interactive coefficients, respectively. Characterization of OSG Micelles. OSG micelles (2 mg/mL OSG) and curcumin-loaded OSG (2 mg/mL OSG) were prepared 7533



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hermetically sealed, and were quickly transferred to the sample holder of the instrument. Then, the pans were heated from 30 to 200 °C with a heating rate of 10 °C min−1 in a nitrogen atmosphere. The recorded DSC curve of a sealed empty pan was used as reference. XRD. The patterns of native curcumin, OSG, their physical mixture (OSG/curcumin = 100:1), and curcumin-loaded OSG were obtained using an X-ray diffractometer (D8 Advance, Bruker, Germany). The measurements were performed with 40 kV and 25 mA. The scanned angle (2θ) was set from 5° to 40°, and the scan rate was 2° min−1. UV Stability Study. To verify the effective loading of curcumin by OSG micelles, a methanol−water (5:95, v/v) solution of curcumin and curcumin-loaded OSG micelles were subjected to UV light (253 nm) using a superclean bench (SW-CJ-1F, China). Sampling was carried out at various time points (10, 20, 30, 40, 50, and 60 min), and the amount of curcumin in the samples was determined by highperformance liquid chromatography.28 The percent of curcumin refers to the ratio of the content of curcumin retained in the sample after UV irradiation to the original one in the sample. Statistical Analysis. Results were expressed as the mean with standard deviation of at least three measurements. SPSS 19.0 was used to analyze the data. Analysis of variance (ANOVA) was utilized to determine the least significance at p < 0.05 by Tukey’s HSD test.

Table 3. Box−Behnken Design Matrix, Experimental Values for Three-Level, Three-Factor Response Surface Analysisa decoded valuesc

b

no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0.0076 0.0266 0.0080 0.0273 0.0085 0.0281 0.0085 0.0281 0.0169 0.0172 0.0169 0.0172 0.0175 0.0175 0.0175 0.0175 0.0175

X1

X2

X3

experiment values (μg/mg)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.3 7.3 16.8 16.8 11.9 11.9 11.9 11.9 7.3 16.8 7.3 16.8 11.9 11.9 11.9 11.9 11.9

3.8 3.8 3.8 3.8 3.3 3.3 4.4 4.4 3.3 3.3 4.4 4.4 3.8 3.8 3.8 3.8 3.8

1.28 0.55 1.59 1.78 0.15 0.04 0.30 3.22 0.20 0.65 2.86 4.30 1.99 2.22 1.78 1.80 1.78

0.0010a 0.0022c 0.0005a 0.0015c 0.0007a 0.0021c 0.0007a 0.0021c 0.0038b 0.0025b 0.0038b 0.0025b 0.0032b 0.0032b 0.0032b 0.0032b 0.0032b

predicted values (μg/mg)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.11 0.63 1.51 1.95 0.20 0.20 3.22 3.17 0.32 0.68 2.83 4.18 1.91 1.91 1.91 1.91 1.91

0.04 0.05 0.13 0.12 0.07 0.01 0.16 0.16 0.02 0.11 0.11 0.19 0.12 0.11 0.12 0.09 0.08

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RESULTS AND DISCUSSION Results of RSM. The data of 17 experiments represented in Table 1 relating the CLC value of OSG micelles were fitted to multiple regressions. A second-order polynomial function was built to describe the effects of independent factors, namely, DS (X1), Mw (X2), and IP (X3), on the dependent variable CLC (Y), which was given as follows:

a

Values bearing different on-line lowercase letters in the same column are significantly different (p < 0.05) by Tukey’s HSD test. b Experiments were conducted in a random order. cX1 = degree of substitution, X2 = weight-average molecular weight (104 g/mol), X3 = stirrer input power (W).

Y = 1.914 − 0.0113X1 − 0.4595X12 + 0.4288X 2

under above obtained optimal conditions (DS = 0.0199, Mw =16.8 × 104 g/mol, and IP = 4.4 W) and subjected to DLS, TEM, and AFM analyses. Moreover, the lyophilized micelles were analyzed by FT-IR spectroscopy, DSC, and XRD. DLS. The size and ζ potential of OSG micelles in aqueous solution were determined based on DLS and laser doppler electrophoresis by using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) equipped with a He−Ne laser (633 nm) and 90° collecting optics. The results of size and ζ potential were expressed as nanometers and millivolts, respectively. The particle size distribution is reported as a polydispersity index (PDI). The temperature was maintained at 25 °C during the measurement. These measurements were performed in triplicate. TEM. The appearance of the samples was determined by TEM measurements. A drop of the curcumin-loaded OSG solution was placed in a carbon-coated copper TEM grid, negatively stained with 1% uranyl acetate (w/v) for 10 min, and air-dried. Imaging was performed at 200 kV using a JEOL JEM-2100 instrument (JEOL, Japan) equipped with a Gatan 94 Ultrascan 1k charge-coupled device camera.32 AFM. The shape of curcumin-loaded OSG micelles (COMs) were further characterized by AFM (Dimension Icon, Bruker, Germany).33 A drop of the curcumin-loaded OSG solution (2 mg/mL) was placed on freshly cleaved mica. After 5 min of incubation at room temperature, the surface of the mica was gently rinsed with 5 mL of deionized water and blown with dry nitrogen. The sample was airdried at room temperature and mounted on the microscope scanner. The shape was observed and imaged in a noncontact mode. FT-IR Spectroscopy. FT-IR spectra of native curcumin, OSG, their physical mixture (OSG/curcumin =100:1), and curcumin-loaded OSG were obtained using an FT-IR spectrophotometer (Spectrum 100, Perkin−Elmer, Akron, OH, USA) in the range 500−4000 cm−1 through the KBr method. DSC. DSC measurements were carried out with a thermal analyzer (DSC 4000, Perkin−Elmer). Up to 5 mg of native curcumin, OSG, their physical mixture (OSG/curcumin = 100:1), and curcumin-loaded OSG were placed in an aluminum sample pan, which were

− 0.1545X 2 2 + 1.5X3 + 0.243X32 + 0.23X1X 2 − 0.0125X1X3 + 0.2475X 2X3

The coefficients of the response surface model as described above were evaluated. The ANOVA was shown in Table 4. In Table 4. Analysis of Variance of the Regression Coefficients of the Fitted Quadratic Equations for the Curcumin Loading Capacity of Curcumin-Loaded Octenylsuccinate Oat βGlucan Micelles sum of squares

df

mean

F value

p value

model fit

model

21.1375

9

2.3486

48.19

0.05). The contour plots of Mw versus DS and Mw versus IP (Figure 1a, c) revealed that CLC increased with the increase of Mw. Compared with molecular chains of OSG with higher Mw, the chains of OSG with lower Mw are relatively short, and possibly make the physical entrapment of curcumin difficult.41 The encapsulation of curcumin in the pluronic block copolymer 7535



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Table 5. Size, ζ Potential, and Polydispersity Index (PDI) of β-Glucan and Octenylsuccinate Oat β-Glucan (OSG) before and after Curcumin Loading.

a

sample

DS

size (nm)

ζ potential (mV)

PDI

β-glucana OSG curcumin-loaded OSG

− 0.0199 0.0199

691.0 ± 10.5a 404.2 ± 3.5b 308.0 ± 10.2c

−8.7 ± 0.2c −12.4 ± 0.5b −10.8 ± 0.2a

0.459 ± 0.020a 0.356 ± 0.012b 0.233 ± 0.014c

Size, ζ potential, and PDI of β-glucan was reported by Liu et al.28

Figure 2. Transmission electron micrograph, three-dimensional view, and size distribution of octenylsuccinate oat β-glucan (OSG) micelles before (a−c) and after (d−f) curcumin loading. 7536



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sample preparation. DLS determined the size in the hydrated state of the sample, whereas TEM and AFM observed the size in the dried state of the sample. FT-IR Spectra. The FT-IR spectra of curcumin, OSG, their physical mixture, and curcumin-loaded OSG were shown in Figure 3. The characteristic peaks at 1727 and 1574 cm−1 in

and PEO-PCL micelles also showed that polymers with higher molecular weights had a better CLC.42,43 Effects of IP on CLC. As revealed by the ANOVA (Table 4), the linear term of IP had a significant effect (p < 0.01) on the CLC of OSG micelles. However, its quadratic effect was insignificant (p > 0.05). CLC of OSG micelles increased as IP increased from low input power levels to high input power levels (Figure 1b, c). The OSG micelles underwent a process of disassembly and reassembly during the agitation, which allowed the loading of curcumin into the OSG micelle core. Thus, increasing IP can increase the contact opportunity of curcumin and OSG micelles. More importantly, an increase of IP can promote the diffusion of the hydrophobic compounds toward the dispersed phase.44 Predicted Model and ANOVA. ANOVA results in Table 4 indicated that the regression model was adequate for the prediction. The coefficient of determination R2 was found to be 0.9844, indicating that 98.44% of the variability in the response could be explained by the obtained model. The value of R2 was always in the range 0∼1.0, and the closer the R2 was to 1.0, the stronger the model and the better it predicted the response. The adjusted R2 (0.9644) is in good agreement with the coefficient of determination R2 (0.9844), suggesting an excellent correlation between the predicted values and the experimental results. The model showed that the p value for CLC (0.3091) was higher than 0.05, which implied that the lack-of-fit was not significant relative to the pure error. Verification of the model equation was conducted using the selected optimal conditions. Under optimal conditions (DS = 0.0199, Mw = 16.8 × 104 g/mol, and IP = 4.4 W), the maximum CLC of OSG micelle was 4.21 ± 0.16 μg/mg, which agreed with the predicted value of 4.20 μg/mg. No significant difference (p > 0.05) was observed between the experimental and predicted values. Therefore, the results indicated the suitability of the model employed and the success of RSM in the optimization of CLC. Characterization of Curcumin-Loaded OSG. Particle Size, ζ Potential, and Morphology. Particle characteristics of β-glucan and OSG before and after curcumin loading were shown in Table 5. After derivatization (OSG), the size, PDI, and ζ potential of micelles decreased due to the strong intraand intermolecular hydrophobic interactions among octenylsuccinic groups, causing the hydrophobic substituent groups to self-assemble and form a uniform distribution of size.28 DLS results (Table 5) revealed that the mean diameter and PDI of the OSG micelles were decreased from 404.2 to 308 nm and 0.356 to 0.234 after curcumin loading. The size and PDI of micelles formed by monomethoxyl poly(ethylene glycol)poly(1,3-bis(p-carboxy-phenoxy)propane) also showed a significant decrease after curcumin incorporation.45 Hong et al. ascribed the decreased size of micelles to the stronger hydrophobic interaction between curcumin and polymers.45 After loading of curcumin into OSG micelles, ζ potential values of COMs were increased from −12.4 to −10.8 mV. Furthermore, TEM showed that smooth spherical and irregular-shaped particles were observed for the OSG micelles before and after curcumin loading. AFM results confirmed that both OSG micelles and COMs were elliptical in shape with a horizontal size of 20−200 nm and a vertical size of 5−18 nm (Figure 2 b, c and e, f). The mean diameter of COMs determined by DLS was slightly larger than the size observed by TEM and AFM. The difference of reported diameter between DLS and TEM was probably due to the process of the

Figure 3. FT-IR spectra of curcumin (a), octenylsuccinate oat βglucan (b), their physical mixture (c), and curcumin-loaded octenylsuccinate oat β-glucan (OSG) (d).

OSG and curcumin-loaded OSG are attributed to the OSG carbonyl group (−CO−) and the asymmetric stretch of vibration of the carboxylate group (RCOO −). Several absorbance peaks are observed in the spectra of curcumin. Six of them are also observed in the physical mixture (curcumin and OSG). These peaks at 1505, 1430, 1207, 1029, 965, and 856 cm−1 are assigned to the vibration of CO stretching, CC stretching, (trans) olefinic CH bending stretching, and asymmetric COC stretching. The spectra of curcumin-loaded OSG nearly overlapped with that of OSG. This observation indicates that the curcumin is loaded into the micelle and no free curcumin is observed.46,47 Nevertheless, the peaks at 1505 and 856 cm−1 shifted toward 1516 and 843 cm−1 in the spectra of curcumin-loaded OSG. The peaks at 1430, 1206, 1029, and 965 cm−1 disappeared in the spectra of curcumin-loaded OSG. These shifted and disappeared vibrations in the spectra of curcumin-loaded OSG compared with curcumin and the mixture are an evidence for the successful loading of curcumin into OSG micelles. DSC. DSC studies were employed to investigate the crystal transformation of the curcumin-loaded OSG (Figure 4). The results showed that the endothermic peak of native curcumin was found at approximately at 182 °C. OSG showed a broad endothermic peak at around 75 °C. However, a weak endothermic peak at 182 °C indicates that the crystalline structure of curcumin is still present in the physical mixture of OSG and curcumin. More interestingly, the characteristic peak was not observed in the lyophilized powder of curcumin-loaded OSG. This observation indicates that curcumin is dispersed by loading into OSG micelles forming an amorphous state.48 XRD. To study the interactions between curcumin and OSG polymer, XRD was used. The characteristic peaks at a diffraction angle of 2θ (8.89°, 14.48°, 17.22°, 18.18°, 23.3°, 24.60°, and 25.52°) suggested the high crystalline structure of curcumin (Figure 5a). A weak peak at 8.89° and 17.22° 7537



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Figure 4. DSC curves of curcumin (a), octenylsuccinate oat β-glucan (b), their physical mixture (c), and curcumin-loaded octenylsuccinate oat β-glucan (OSG) (d).

Figure 6. UV stability study of curcumin (■) and curcumin-loaded octenylsuccinate oat β-glucan (OSG) micelles (□). Values are given as the mean of three independent experiments, and error bars represent the SD. Different lower case letters indicate a significant difference (p < 0.05).

curcumin-loaded OSG were first investigated in this work. CLC highly depended on the structural characteristics of OSG. The CLC increased with an increase in the DS of OSG from 0.0080 to 0.0172, indicating that the hydrophobic interaction was the driving force for the increased dispersibility of curcumin. However, a decrease in the CLC was observed as further increase in the DS, due to the smaller micelle cavity of OSG micelles and the hindrance of curcumin loading into the micelle by amounts of octenyl succinate groups on the highly substituted OSG. OSG with higher Mw, offering a longer chain to encapsulate curcumin, also resulted in a higher CLC. The FT-IR, DSC, and XRD analyses confirmed that curcumin was loaded in OSG micelles to form an amorphous complex through the intermolecular interactions. Besides, the UV stability study supported the successful loading of curcumin into OSG micelles. This study suggested that OSG could be used as a nanovehicle for the loading of hydrophobic bioactive compounds.

Figure 5. XRD patterns of curcumin (a), octenylsuccinate oat β-glucan (b), their physical mixture (c), and curcumin-loaded octenylsuccinate oat β-glucan (OSG) (d).

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indicated that the crystalline structure of curcumin was still present in the physical mixture of the OSG micelles. However, no characteristic curcumin peaks were observed in the spectrum of curcumin-loaded OSG (Figure 5d). The absence of the crystalline order of curcumin in the curcumin-loaded OSG indicated that the formation of an amorphous complex through the intermolecular interactions between curcumin and OSG.49 UV Stability Study. The data from the UV stability study were summarized in Figure 6. More than 90% of curcumin was unchanged in OSG micelles as compared to less than 20% for curcumin in methanol−water (5:95, v/v) solution at the end of 6 h. Obviously, the curcumin loaded in OSG micelles showed a better stability under UV irradiation than in methanol−water (5:95, v/v) solution. This is due to the OSG cavity originates a sort of “shield” effect of the curcumin against the UV irradiation. This phenomenon strongly supports the successful loading of curcumin into OSG micelles. In summary, the dependence of CLC of OSG micelles on the structural parameters of OSG and the characterization of

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 23 68 25 19 02; fax: +86 23 68 25 19 47; e-mail: [email protected]. Funding

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (no. 31371737). Notes

The authors declare no competing financial interest.

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