1. Introduction
Starch is a key energy source for humans and is an easily acquired and highly nutritional biopolymer. It displays excellent biodegradability and non-toxicity [
1]. It has been widely used in a variety of food products and industrial applications [
2]. It is composed of amylopectin and amylose, and variations in the amounts result in a variety of starches such as waxy corn, rice, wheat, etc., to name a few. Waxy cornstarch is predominantly composed of amylopectin (95–99%) [
3,
4]. Although waxy cornstarch had been identified as a useful biomaterial, its water insolubility restricts the widespread application. In this regard, addition of groups such as octenyl succinic anhydride (OSA) on the starch chains is being explored [
5]. OSA starches displays excellent emulsification properties and good encapsulation efficiency toward many sensitive and insoluble functional molecules [
6]. Amylose is also employed as a functional material, examples include bovine hemoglobin encapsulation in amylose matrix as an oxygen carrier, but not as flexible as short glucan chains (SGC). Starch could be modified by pullulanase to produce short glucan chains. Pullulanase is a well-known starch debranching enzyme [
7], and around 70–80% of starches are being treated with pullulanase to generate short glucan chains mainly for encapsulation applications. Reddy et al. used pullulanase to modify corn, potato, cassava and other plant starch to form inclusion complex with stearic acid, and they found that enzymatic debranching of starch effectively improved the complexation, crystallinity, dispersion and stability of starch–stearic acid complexes. [
8].
Curcumin is a plant phenol and makes up to 5% of the dietary spice turmeric possessing antioxidant [
9,
10], anti-inflammatory [
11,
12], anti-tumor [
13], anti-HIV [
14], anti-bacterial and anti-microbial [
15] properties. In addition, curcumin could cure respiratory diseases and improve the human health. However, curcumin is water insoluble and is not readily absorbed in the human digestive system, in neutral, acidic and alkaline conditions. In addition, its photosensitive nature constrains the general utility and limits the product development. In this regard, encapsulating curcumin into amphiphilic polymers could circumvent the issue [
16,
17,
18,
19]. Yu [
20] encapsulated curcumin into hydrophobically modified starch to enhance the in vitro anti-cancer activity. The OSA starch is also used to encapsulate water insoluble essential oil [
21].
Herein, we prepared OSA–SGC–curcumin nanoparticles to improve the water solubility and bioavailability of curcumin. Thus prepared nanoparticles have been characterized by FTIR, XRD, TGA, TEM and dynamic light scattering (DLS). The effect of pH on the curcumin release kinetics has been established in the simulated intestinal and blood environments.
2. Materials and Methods
2.1. Materials
Waxy cornstarch was a gift from Ingredion Co., Ltd. (Guangzhou, China). Pullulanase (1000 ASPU/g) was obtained from Novozymes Investment Co. Ltd. (Bagsvaerd, Denmark). OSA (≥95.0%) was purchased from Tokyo Chemical Industry Co., LTD. (TCI, Tokyo, Japan). Curcumin (≥94.0%) was obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and all chemical reagents were analytical grades.
2.2. Preparation of OSA Grafted on SGC and OSA–SGC Nanoparticles
The OSA–SGC was produced by the method described by Sun et al. [
22] with modifications. Briefly, waxy cornstarch (15 g/mL) slurry (100 mL phosphate buffer solution, pH = 5.0) was kept in boiling water under vigorous stirring for about 30–40 min. The pullulanase (0.015 g/mL) was added and the sample was incubated at 8 ≤ pH ≤ 9, 58 °C for 6 h, and centrifuged at 1300×
g for 2 min, and lyophilized for 48 h to produce SGC powder. Then 5 g of SGC powder was dissolved in 100 mL distilled water to form 5% (w/v) aqueous solution. SGC solutions were incubated in an oil bath at 121 °C for 30 min and 0.1 mol/L NaOH was added to keep the solution pH at around 8.5 to provide a suitable environment for OSA to make reaction with the SGCs. The OSAs (equivalent to 25%, 50% and 100% of the weight of SGC powder) were dispersed in about 2 mL anhydrous ethanol and added to the SGC mixture with continuous magnetic stirring with 8.5 ≤ pH ≤ 9.0 at 55 °C for 8 h, and then the pH was adjusted to 6.8 with 0.1 mol/L HCl solution to stop the reaction. The OSA–SGC were washed twice with 99.7% anhydrous ethanol and precipitates were cooled at 4 °C for 8 h before freeze-drying. The OSA–SGC polymers equivalent to 25%, 50% and 100% of the dry weight of SGC powder were recorded as OSA
0.25–SGC, OSA
0.5–SGC and OSA
1.0–SGC, respectively. Finally, 100 mg of the above three OSA–SGC polymers were weighed and dispersed in a phosphate buffer solution of pH = 7.4 to prepare a 10 mg/L solution. After the solution was stirred in a constant temperature water bath at 37 ° C for 6 h, it was cooled to room temperature to obtain an OSA–SGC nanoparticle solution. The OSA–SGC nanoparticles were then precipitated with absolute ethanol and washed with water for 2–3 times. Finally, the precipitate was freeze-dried to obtain a dry powder of OSA–SGC nanoparticles.
2.3. Determination of Degree of Substitution
NMR was a common method to determine the degree of octenyl succinate substitution of the short glucan chain. This experiment was carried out by hydrogen spectroscopy (Brook, Steffisburg, Switzerland) at 600 MHZ, weighing 20 mg of octenyl succinate and short glucan chain, respectively. Of octenyl succinate short–glucan chain nanoparticles 20 mg were put in a nuclear magnetic tube, with the addition of 0.7 mL deuterated-dimethyl sulfoxide (DMSO-d6) containing 0.5% (w/w) LiBr. Then the mixture was heated at 80 °C to dissolve those nanoparticles, finally 20 mg deuterated-trifluoroacetic acid (TFA-d1) was added in the solution. A small amount of TFA-d1 could separate the peak of the hydroxyl group of the short glucan chain and the hydroxyl group in the water molecule, so that the nuclear magnetic spectrum was clearer. The formula for calculating the degree of substitution was as follows:
where
I0.89 represented the integral of the CH
3 signal peak in OSA,
Iα-1, 4 represented the integral of the signal peak at 5.11 ppm of the proton on the α-(1→4) connected carbon atom,
Iα-1,6 represented the integral of the proton signal peak on the α-(1→6) connected carbon atom,
Ir-e corresponds to the reducing chain ends.
Ir-e was the
1H NMR integrals of the internal α and β reducing chain ends at approximately 4.28 and 4.91 ppm in a short glucan chain [
23].
The reaction efficiency formula is as follows:
Among them, the theoretical degree of substitution (DS) was based on the assumption that all added anhydride reacts with starch to form ester derivatives.
2.4. Determination of the Critical Micelle Concentration (CMC)
The critical micelle concentration (CMC) of the short glucan chain octenyl succinate solution was determined by the fluorescence probe method [
24]. Firstly, 1 mg/mL OSA–SGC nano-micelle blank solution was prepared, and the original solution was diluted with distilled water into 0.5, 0.1, 0.025, 1 × 10
−3 and 0.05 × 10
−3 mg/mL. Pyrene/acetone solution with the concentration of 6 × 10
−5 mg/mL was prepared. Of the pyrene/acetone solution 1 mL was taken in a beaker and stored in tin foil. The pyrene/acetone solution was heated at 40 °C for 30 min. Through adding 10 mL of the above different concentrations of nanomicelle solution into the pyrene/acetone solution, and the pyrene concentration in the solution was 6 × 10
−6 mg/mL. By stirring at room temperature for 24 h, the pyrene/acetone solution was fully mixed. The fluorescence intensity of the solution was measured in a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) with an excitation wavelength of 335 nm, a scanning wavelength of 350 to 550 nm, and a slit width of excitation and emission just about 5.0 nm. The fluorescence emission intensity of pyrene at 383 nm in nanoparticles was recorded as I
3, and the fluorescence emission intensity at 373 nm in water was recorded as I
1. With the increasing concentration of pyrene in the solution of nanoparticles, the fluorescence at 383 nm will increase, while the fluorescence at 373 nm would be relatively weakened. With continuous changes of the concentration, the I
3/I
1 value of pyrene in water tended to be a constant, and the solubility of pyrene in nanoparticles would increase, and a mutation value would appear in I
3/I
1, indicating that the nanoparticles were formed at this time, and the concentration of the mutation point was the critical micelle concentration (CMC).
2.5. Preparation of Curcumin OSA–SGC Complexes
Curcumin was dissolved in ethanol at the concentration of 5 mg/mL, and OSA–SGC solutions were prepared with 10 mg/mL in PBS with 2 h heating according to Sun’s [
25] method with modification, and were sonicated in an ultrasonic cleaner for 15 min. Curcumin solutions were slowly added to OSA–SGC with a ratio of curcumin: OSA-SGC 1:5 (v/v) and heated at 37 °C under constant stirring for 8 h. After that, rotary evaporation was used to remove ethanol at 38 °C for 30 min. The OSA–SGC–CUR (curcumin) were freeze-dried, and kept in a dryer for further analysis.
2.6. Encapsulation Efficiency (EE) and Loading Content (LC)
Column: Agilent HC-C18 (5 μm × 4.6 mm × 250 mm); temperature: 40 °C; injection volume: 10 μL; detection at 430 nm; flow rate; 1.0 mL/min. Mobile phase A: 5% glacial acetic acid water, mobile phase B: acetonitrile.
Curcumin was dissolved in 400 mL methanol at a concentration of 1000 μ/mL under constant stirring. The samples were divided into five parts with diluted concentrations of 1, 5, 10, 20 and 50 μg/mL. High-performance liquid chromatographic spectra were recorded with the set peak area as the abscissa and concentration as the ordinate: The curcumin standard curve equation was thus established. The OSA–SGC–CUR nanoparticle samples were added to methanol at a concentration of 1 mg/mL and shaken for 2 min. The peak area of the supernatant at t 430 nm was determined, as was the concentration of curcumin corresponding to the standard curve. OSA–SGC–CUR nanoparticle samples were added to methanol at a concentration of 1 mg/mL and mixed to release the curcumin under high-speed centrifugation (1500× g, 20 min). Then, the supernatants were reserved to measure the curcumin peak area at 430 nm, and the curcumin concentration calculated according to a standard curve.
2.7. FTIR of OSA, OSA–SGC and OSA–SGC–CUR
The molecular structures of SGC, OSA–SGC and OSA–SGC–CUR were confirmed using FTIR spectrophotometry (c). Samples were spread on the countertop and scanned under ambient conditions, and the FTIR spectra of SGC, OSA–SGC and OSA–SGC–CUR were accumulated, from 400–4000 cm−1, on an FTIR spectrophotometer with 32 scans at a resolution of 4 cm−1. Data were processed by the Origin Pro 2017C Software.
2.8. X-ray Diffraction
The X-ray patterns of SGC, OSA–SGC and OSA–SGC–CUR were carried out using a X-ray diffractometer (D8 Advance, Bruker, Rheinstetten, Germany) with Cu Kα (1.5418 Å) radiation at a voltage of 40 kV and a current of 30 mA. One gram of the sample was dispersed in the sample holder. The scanning region of diffraction angle (2θ) was set from 5 to 50° with a scanning speed of 4°/min, and a time step of 0.4 s.
2.9. Dynamic Light Scattering (DLS)
The average size and zeta potential of the samples were measured by DLS, using a Malvern Nano Zetasizer (Malvern Instruments Ltd., Malvern, UK). The method was performed on samples diluted in distilled water and ultrasonicated for 5 min before measurement at a diffraction angles at 90° before analysis at 25°.
2.10. Thermogravimetric Analysis (TGA)
The thermal stability of OSA, OSA–SGC, and OSA–SGC–CUR was assessed by TGA. Thermogravimetric analysis was conducted with a Q-5000 (TA Instruments Inc., New Castle, DE, USA) instrument, was first calibrated with indium: The 2 to 5 mg inclusion compound samples were placed in a crucible and the temperature increased from 20 to 400 °C, at 10 °C/min with nitrogen used as a protective gas. The resulting data were analyzed using the Universal Analysis V4.5A Instrument software.
2.11. Transmission Electron Microscopy (TEM)
The transmission electron microscopy was performed using a Tecnai G2 F30-TWIN from Bruker, Germany, with an acceleration voltage of 200 kV. The OSA–SGC nanoparticle dry powder was formulated into a solution with a mass fraction of 1%, and sonicated at room temperature for 10 min, then dropped onto a copper mesh with a carbon support film, and was freeze-dried for measurement. The samples were kept in a sterile state and remained transparent.
2.12. Intestinal and Blood Environment Slow Release Simulation
Around 1 mL of OSA–SGC–CUR nanoparticle solution was packed in a dialysis bags and placed in a 30 mL phosphate buffer solution at pH 6.8 and 7.4 with 3% (v/v) Tween 80, pancreatin (57.6 mg/mL) and bile salts (46.8 mg/mL). The absorbance of curcumin was measured by the 1 mL external solution of dialysis bag, which was obtained by constant temperature oscillated at 37 °C at 200 rpm for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 h. After measurement, pH 6.8 and 7.4 phosphate fresh buffer solutions, which contained 3% (v/v) Tween 80, pancreatin (57.6 mg/mL) and bile salts (46.8 mg/mL) should be supplemented in time to keep the total volume of 30 mL unchanged. The extracted solution was measured at 430 nm by a UV-vis spectrophotometer, and amount of dialyzed curcumin was calculated from the obtained concentration.
2.13. Statistical Analysis
All measurements were carried out in triplicate and average results are reported. Data were analyzed by an analysis of variance (ANOVA), followed by Duncan’s multiple range test using SPSS 22 Statistical Software Program (SPSS Incorporated, Chicago, IL, USA). A value of p < 0.05 was considered statistically significant.