Comparative Structural Characterization of Spiral Dextrin Inclusion

Sep 14, 2017 - In this study, the preparation and structural properties of spiral dextrin (SD)/vitamin E and SD/soy isoflavone inclusion complexes wer...
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Comparative Structural Characterization of Spiral Dextrin Inclusion Complexes with Vitamin E or Soy Isoflavone Ping-Ping Wang,† Xin-Sheng Qin,† Qing-Yu Yang,†,‡ Zhi-Gang Luo,*,† Zhi-Gang Xiao,‡ and Xi-Chun Peng§ †

School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China ‡ College of Grain Science and Technology, Shenyang Normal University, Shenyang, Liaoning 110034, People’s Republic of China § Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510630, People’s Republic of China ABSTRACT: In this study, the preparation and structural properties of spiral dextrin (SD)/vitamin E and SD/soy isoflavone inclusion complexes were studied. SD was obtained from debranched normal maize starch using isoamylases. After fractionation using a novel method of gradient ethanol precipitation, SD was separated into different fractions, among which SD-40 was found to be the optimal host molecule to prepare SD inclusion complexes with vitamin E or soy isoflavone. X-ray diffraction (XRD) and 13C cross-polarization magic angle spinning nuclear magnetic resonance (NMR) suggested that the crystalline structures of SD-40/vitamin E and SD-40/soy isoflavone were V6II and V6III types, respectively. Small-angle X-ray scattering revealed that the SD-40/vitamin E inclusion complex formed a tighter and more compact crystallite than the SD-40/soy isoflavone inclusion complex. Furthermore, the connection structures of inclusion complexes were investigated by two-dimensional nuclear Overhauser effect spectroscopy NMR, indicating that part of vitamin E with an alkyl chain was encapsulated in the helix cavity of SD-40, whereas the aromatic ring B of the soy isoflavone molecule was complexed by the helix cavity and screw of SD. KEYWORDS: spiral dextrin, vitamin E, soy isoflavone, inclusion complexes, structural characterization

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INTRODUCTION

estrogen-related cancer, cardiovascular disease, lipid profiles, climacteric symptoms, and osteoporosis in humans. However, the bioavailability of vitamin E and soy isoflavone is limited as a result of its heat and light instability during storage.8 Our previous study investigated the formation of debraned starch/ phosphatidylcholine inclusion complexes and found that amylose could improve the oxidantive stability of antioxidants through incorporation.9 Starch is a mixture of two major polysaccharides: linear amylose and highly branched amylopectin. Amylose is known to co-crystallize with various molecules, such as alcohols,10 aromatic compounds,11 lipids, and emulsifiers,12 forming a V-amylose crystalline structure via hydrophobic and van der Waals interactions. Currently, amylose is normally obtained through separation of the two components of starch or hydrolysis of native starch using pullulanases. However, these conventional methods have disadvantages of a complicated process and low inclusion rate. In this study, linear dextrins produced from isoamylase enzymatic hydrolysis of native starch were defined as “spiral dextrin (SD)”. SD has the feature of a repeating (1,4)-α-Dglucose unit and has a lower molecular weight than amylose directly extracted from native starch. SD was designated because dextrin would form a helical structure in iodine or other guest molecule solutions, which is similar to that of β-cyclodextrin and

Antioxidants are widely used as oxidant protectors in food, cosmetic, and pharmaceutical products.1−3 Fat-soluble vitamins have a similar structure composed of a head ring and a long carbon side chain (Figure 1A). Vitamin E, as a type of fat-soluble vitamin, is particularly well-known for its potent antioxidant activity in both biological and food systems.3−5 Genistein is the most abundant isoflavone in soy. The main structural characteristics of genistein are two aromatic rings A and B, linked by a heterocyclic pyrone ring C (Figure 1B). Numerous studies6,7 have reported the health beneficial effects of soy isoflavones on

Received: Revised: Accepted: Published:

Figure 1. Chemical structures of (A) vitamin E and (B) genistein. © 2017 American Chemical Society

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July 13, 2017 September 14, 2017 September 14, 2017 September 14, 2017 DOI: 10.1021/acs.jafc.7b03242 J. Agric. Food Chem. 2017, 65, 8744−8753

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Journal of Agricultural and Food Chemistry

that maize starch has been completely debranched. Moreover, the hydrolysis percent (16.40%) of debranched starch made under the above conditions was determined using the method of Bruner.22 Fractionation of SD. The fractionation of SD was conducted by gradient ethanol precipitation based on a previous method.23 The SD solution was precipitated by slowly adding dehydrated ethanol under continuous stirring until reaching the final ethanol concentration of 40% (v/v) and kept at 4 °C for 24 h. Then, it was centrifuged at 4500 rpm for 5 min to obtain the precipitated fraction, which was defined as SD subfraction SD-40. Dehydrated ethanol was further added slowly to the supernatant, to reach a final ethanol concentration of 50% (v/v), and kept at 4 °C for 24 h. The precipitates (SD-50) were obtained by centrifugation. A further increase in the ethanol concentration to 60, 70, and 80% led to subfractions SD-60, SD-70, and SD-80, respectively. Molecular Weight Distribution of SD Fractions. The homogeneity and molecular weight distribution of SD subfractions were determined by high-performance gel permeation chromatography (HPGPC) equipped with a refractive index detector. The columns used were a 300 × 7.8 mm inner diameter, 10 μm, TSK-GEL G-5000 PWXL, and a 300 × 7.8 mm inner diameter, 5 μm, TSK-GEL G-3000 PWXL, with a guard column of the same material (Tosoh Co., Ltd., Tokyo, Japan). The mobile phase was KH2PO4 (0.02 M) and set at a flow rate of 0.6 mL/min. The temperatures of the column and detector were 35 and 45 °C. The injection volume was 20 μL. Dextrans of various molecular weights (708 000, 344 000, 200 000, 107 000, 47 100, 21 100, 9600, and 5900 Da) were used as standards.24 The molecular weight of each sample was determined according to the calibration of the standard curve. Preparation of SD/Vitamin E and SD/Soy Isoflavone Inclusion Complexes. Maize starch and SD subfractions (SD-40, SD-50, SD-60, SD-70, and SD-80) of 5 g dry basis was dissolved in 10 mL of NaOH (1.0 M), adjusted to pH 7.0 with HCl (1.0 M), and diluted with water to 30 mL. The antioxidant solutions of vitamin E (0.5 g) and soy isoflavone (0.5 g) were prepared in 5 mL of ethanol and dimethyl sulfoxide, respectively. SD subfraction solution (50 mL, 10 mg/mL) and vitamin E solution were mixed and incubated at 45 °C for 4 h to obtain the SD/ vitamin E sample, and the SD subfraction (50 mL, 10 mg/mL) and soy isoflavone solution mixture was incubated at 65 °C for 5 h to obtain the SD/soy isoflavone sample. The vitamin E and soy isoflavone payloads and inclusion rates were measured using the method of previous studies.25,26 Structural Characterization of Complexes. X-ray Diffraction (XRD) Analysis. XRD analysis was determined by a RU200R powder diffractometer (Bruker, Karlsruhe, Germany) equipped with a graphite crystal monochromator. The operating conditions were as follows: Cu Kα radiation at 40 mA, incident wavelength of 0.1524 nm, voltage of 40 kV, scanning range from 5° to 40° (2θ), scanning length of 0.02°, and scanning speed of 0.1 s/step.27 The crystalline size data and degree of relative crystallinity were calculated according to the method of Nara and Komiya28 using Jade 6.0 software (Material Date, Inc., Livermore, CA, U.S.A.). 13 C CP/MAS NMR Analysis. In this study, analysis of the solid powders was achieved by direct excitation of the 13C nuclei and 1H−13C CP 13C MAS NMR according to conditions described previously.9 The 13 C CP/MAS NMR spectra were recorded on an Avance 400 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a 4 mm MAS probe. Samples (100 mg) were packed in a 4 mm zirconia rotor and spun at 6000 revolutions/min. The spectra were achieved at ambient temperature with a sampling time of 17 ms and a relaxation delay time of 2 s. All chemical shifts were reported in parts per million (ppm). SAXS Analysis. SAXS measurements were performed using a SAXSess small-angle X-ray scattering system (Anton Paar, Graz, Austria) with an acceleration voltage of 40 kV and current of 50 mA. The X-ray source was a Cu Kα radiation with a wavelength of 0.1542 nm. Each sample was prepared by mixing starch with distilled water and filled into a capillary (0.01 mm). The sample−detector distance was 261.2 mm, and the temperature was kept at 26 °C during the measurement. The average repeat distance (i.e., thickness of the semi-crystalline

amylose. Further research is needed to investigate the structure of SD and guest molecule inclusion complexes. Earlier studies presented the existence of amylose helices with six, seven, or eight glycosyl residues per helical turn.13,14 In addition, numerous studies have shown that guest molecules could possibly position in three locations of amylose complexes: within the helices, between the helices, or dispersed in the amorphous regions.15,16 It has been reported that amylose complex formation can be influenced by the degree of polymerization of amylose, structure of guest molecules, complexation condition, and other factors. Shiran et al.17 studied different dimensions of amylose−long-chain fatty acid complexes (molecular, nano-, and micro-level characteristics) and proposed that, in the case of long-chain fatty acids, increased fatty acid unsaturation impaired the structure of amylose inclusion complexes. Garcia et al.18 reported that long amylose chains can form more crystalline and stable complexes. Bart et al.19 found that three different crystalline amylose−glycerol monostearate complexes (types I, IIa, and IIb) could form by increasing the synthesis temperature. Many studies9,20 have reported that the guest molecular structure (chain length) and complexation temperature have great effects on the structural properties of V-amylose. However, the influence of a linear alkyl chain and aromatic ring structure on amylose complexation is still not clear, such as the exact arrangement fractal structure, crystalline thickness, connection type, and position of guests. In this study, normal maize starch was hydrolyzed by isoamylase and followed with a novel method of gradient ethanol precipitation to obtain the favorable segment of SD for complexation with vitamin E and soy isoflavone. To determine the structural differences between the two inclusion complexes, the effect of the guest structure on the crystalline form, crystalline thickness, connection type, and appearance of incusion complexes was investigated by X-ray diffraction (XRD), 13C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) NMR, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). In addition, the oxidative stability of single oxidants, SD-40/vitamin E (a linear alkyl chain) and SD-40/soy isoflavone (aromatic ring), was studied.

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MATERIALS AND METHODS

Materials. Commercial food-grade maize starch (amylose content of 24.75%) was obtained from Tiancheng Maize Development Co., Ltd. (Jilin, China). Vitamin E (>98%) and soy isoflavone (>80%) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The major isoflavone used in this experiment was genistein. Isoamylase (EC 3.2.1.68, activity of 2.0 × 105 units/g) was supplied by Hubei Hongyun Long Biological Technology Co., Ltd. (Wuhan, China). One unit of isoamylase activity is defined as the amount of enzyme that liberates one glucose reducing equivalent per minute from pullulan at pH 6.0 and 50 °C. Preparation of SD. Starch (20 g, dry basis) was mixed with 400 g of water in a 500 mL three-necked flask. The slurry was adjusted to pH 6.0 with an acetate buffer solution. After gelatinization at 99.9 °C under stirring for 1 h, the mixture was cooled to 50 °C and the debranching reaction was started by adding isoamylase (12.5 units/g). The dispersion with pH 6.0 was incubated at 50 °C for 12 h to completely debranch the starch. After the reaction, the temperature was adjusted to 100 °C for 20 min to inactivate the enzyme. It was noted that the debranched reaction needed to be confirmed by 1H NMR.21 When the maize starch was hydrolyzed by isoamylase under the above reaction conditions, the α-1,6 peak located at 4.7−5.0 ppm disappeared altogether, which indicated 8745

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Journal of Agricultural and Food Chemistry lamellae) of the amorphous and crystalline lamellar of each sample can be calculated as d = 2π /q

(1) −1

where d (nm) is the lamellar repeat distance and q (nm ) is the scattering vector. The relationship between q and θ can be calculated as

q = (4π sin θ )/n

(2)

where λ (nm) is the wavelength of the X-ray source and 2θ is the scattering angle. The normalized correlation function γ1(r) is defined as

γ1(r ) =

∫0

∞

I(q)q2 cos(qr ) dq/Q

(3)

where I(q) is the scattering intensity, q is the scattering vector defined as q = 4π sin θ/λ (2θ is the scattering angle), and γ is the direction along the lamellar stack. The scattering invariant, Q, is defined as

Q=

∫0

∞

I(q)q2 dq

(4)

The parasitic scattering and thermal fluctuation were corrected using a normalized correlation function. 2D NMR Analysis. The sample solution for 2D rotating-frame NOESY analysis was prepared by dissolving samples in deuterated dimethyl sulfoxide (DMSO-d6) solvent. The spectra were recorded at 400 MHz using a Bruker ADVANCE instrument at 298.15 K. Data were reported as the chemical shift. All chemical shifts were reported in ppm. SEM Analysis. The morphological properties of SD-40/vitamin E and SD-40/soy isoflavone were measured by a 1530VP scanning electron microscope (LEO, Oberkochen, Germany). The sample powders were directly fixed on an aluminum stub with double sticky tape and sputtered with gold under vacuum. Then, the samples were measured at a 20 kV acceleration voltage. CLSM Analysis. Nile Blue and Nile Red solutions (0.1 mg/mL) were prepared using deionized water and 1,2-propylene glycol as solvents, respectively. A total of 0.005 g of SD-40/vitamin E or SD-40/soy isoflavone was dissolved in 20 μL of Nile Blue and 20 μL of Nile Red solutions and kept in the dark for 12 h.29 The sample slide was prepared and measured at the excitation wavelengths of 488 and 633 nm by a Leica TCS-SP5 (Bruker, Solms, Germany), respectively. Antioxidant Activities. The sample solutions of the SD-40/vitamin E physical mixture, SD-40/vitamin E complex, SD-40/soy isoflavone physical mixture (10:1, w/w), and SD-40/soy isoflavone complex were stored at 50 °C. Their 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity30 and hydroxyl radical (OH−) scavenging activity24 were determined at 0, 3, 7, 14, and 30 days, separately. Statistical Analysis. All experiments were conducted in triplicate and expressed as the mean ± standard deviation (SD) using the statistical software package SPSS (SPSS, Inc., Chicago, IL, U.S.A.). Analysis of variance (ANOVA) was carried out by Tukey’s test (p < 0.05).

Figure 2. HPGPC profile of (A) debranching maize starch and (B) SD fractions.

polymerization of SD-40, SD-50, SD-60, SD-70, and SD-80 was decreased from 128 to 71, 41, 29, and 21, respectively. Furthermore, the individual peaks were quite narrow, strongly suggesting that SD subfractions obtained by gradient ethanol precipitation not only gave a low-molecular-weight polymer but also had better homogeneity. Payload and Inclusion Rate of Antioxidants. The complexation of the antioxidant with SD is influenced by many factors. The molecular weight of SD had a great influence on the antioxidant payload and inclusion rate. As shown in Table 1, the vitamin E and soy isoflavone payloads and inclusion rates displayed a gradual downtrend with the decrease of the molecular weight of SD. When the molecular weight increased from 3.35 to 20.9 kDa, the payload of vitamin E rose from 10.81 to 140.50 mg/g and the payload of soy isoflavone rose from 0.97 to 111.83 mg/g. Meanwhile, the inclusion rate of vitamin E enhanced from 4.09 to 64.67%, and the inclusion rate of soy isoflavone enhanced from 0.01 to 68.77% (Table 1). Therefore, the SD-40 inclusion complexes with the highest vitamin E and soy isoflavone payloads were chosen as the optimal molecule for further analysis. XRD Analysis. XRD was used to verify the formation of the SD-40/antioxidant inclusion complexes. As shown in Figure 3A, the diffractograms of SD-40, vitamin E, and SD-40/vitamin E physical mixture showed a big and broad peak, which indicated that they all exhibited amorphous characteristics. The diffraction peaks at 7.34°, 12.99°, 17.17°, 19.96°, 22.02°, and 24.06° in the

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RESULTS AND DISCUSSION Characterizations of SD Fractions. As shown in Figure 2, different SD fractions showed great differences in molecular distribution and homogeneity. Figure 2A showed that SD had a wide molecular distribution. However, an increase in the ethanol concentration from 40 to 80% resulted in a decrease of the weight-average molecular weight (Mw) from 20.9 to 3.35 kDa (Figure 2B). The solution of SD was fractioned by a stepwise increase in ethanol concentrations, giving five subfractions, SD40, SD-50, SD-60, SD-70, and SD-80, with yields of 60.4, 5.2, 9.1, 8.8, and 9.7% of dry matter, respectively. SD-40 obtained at an ethanol concentration of 40% was the major fraction. The data of molecular weight in Figure 2B showed that, with an increase in the ethanol concentration from 40 to 80%, the degree of 8746

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Journal of Agricultural and Food Chemistry Table 1. Effect of the Molecular Weight of SD on Vitamin E and Soy Isoflavone Payloads and Inclusion Ratesa

a

sample

vitamin E payload (mg/g)

vitamin E inclusion rate (%)

soy isoflavone payload (mg/g)

soy isoflavone inclusion rate (%)

maize starch SD-40 SD-50 SD-60 SD-70 SD-80

13.27 ± 0.81 a 140.59 ± 5.27 b 90.72 ± 3.90 c 61.98 ± 3.21 d 24.91 ± 1.12 e 10.81 ± 0.98 f

7.14 ± 0.73 a 64.67 ± 1.87 b 53.91 ± 1.02 c 47.72 ± 1.18 d 20.61 ± 1.09 e 4.09 ± 0.24 f

10.32 ± 0.92 a 111.83 ± 3.89 b 68.72 ± 2.12 c 31.08 ± 1.24 d 12.78 ± 1.02 e 0.97 ± 0.13 f

9.81 ± 0.62 a 68.77 ± 1.56 b 50.67 ± 1.41 c 31.26 ± 1.01 d 16.81 ± 0.92 e 0.01 ± 0.00 f

Values are the mean ± SD (n = 3). Different letters (a−e) indicate a significant difference (p < 0.05).

40/vitamin E inclusion complex formed a more perfect crystalline structure than the SD-40/soy isoflavone inclusion complex. The degrees of crystallinity of SD-40/vitamin E and SD-40/soy isoflavone inclusion complexes were 26.3 and 36.7%, respectively, which indicated that the SD-40/soy isoflavone complex was composed of a larger crystal area than the SD-40/ vitamin E inclusion complex. Peaks at ∼22° and ∼24° in diffractogram patterns of SD-40/vitamin E and SD-40/soy isoflavone inclusion complexes represented the characteristic peaks of V6II or V6III types, which confirmed that vitamin E and soy isoflavone were physically and not molecularly trapped between SD double helices within the matrix. 13 C CP/MAS NMR Analysis. The molecular structure of SD inclusion complexes was further investigated through the analysis of 13C CP/MAS NMR spectra (Figure 4). Previous work32 has reported that the peaks of the glucose unit were assigned as follows: 102.49 ppm, C-a; 71.37 ppm, C-b, C-c, and C-e; 81.20 ppm, C-d; and 60.07 ppm, C-f. The SD-40/vitamin E physical

Figure 3. XRD diagrams: (A) SD-40, vitamin E, SD-40/vitamin E physical mixture, and SD-40/vitamin E inclusion complex and (B) SD40, soy isoflavone, SD-40/soy isoflavone physical mixture, and SD-40/ soy isoflavone inclusion complex.

diffractogram of the SD-40/vitamin E inclusion complex confirmed the formation of inclusion complexes. As presented in Figure 3B, the diffractogram of soy isoflavone showed many irregular peaks and the diffractogram of the SD-40/soy isoflavone physical mixture exhibited a simple superimposition of SD-40 and soy isoflavone, which were composed of the main diffraction peaks of SD-40 and some characteristics of soy isoflavone. In comparison to the SD-40/soy isoflavone physical mixture, the SD-40/soy isoflavone inclusion complex confirmed the formation of inclusion complexes as a result of the existence of diffraction peaks at 7.62°, 13.12°, 16.69°, 19.45°, 21.77°, 23.76°, and 25.93°. The diffraction (panels A and B of Figure 3) of SD-40/vitamin E and SD-40/soy isoflavone inclusion complexes confirmed the formation of V-type structures as inferred from the peaks at 7°, 13°, and 20°, which were already extensively described.20,31 These peaks were taller and clearer in the diffraction curve of the SD-40/vitamin E inclusion complex than in that of the SD-40/soy isoflavone inclusion complex (panels A and B of Figure 3). These results indicated that the SD-

Figure 4. 13C CP/MAS NMR spectra: (A) SD-40, SD-40/vitamin E physical mixture, and SD-40/vitamin E inclusion complex and (B) SD40, SD-40/soy isoflavone physical mixture, and SD-40/soy isoflavone inclusion complex. 8747

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Journal of Agricultural and Food Chemistry mixture presented an equivalent spectrum to SD-40, except the appearance of a small peak located at 24.32 ppm, attributed to C7 and C-11 of vitamin E (Figure 4A). Interestingly, SD-40/ vitamin E inclusion complexes displayed typical V6II-type characteristics, of which the C-d and C-a peaks were sharp and moved downfield and the C-3 peak was partially resolved from the C-b and C-e peaks. The results were in agreement with previous 13C CP/MAS NMR studies of amylose complexes.33 Moreover, the peak (23.85 ppm) corresponded to C-7 and C-11 in vitamin E of the SD-40/vitamin E inclusion complex shifted upfield by 0.47 ppm, which was probably caused by the combined trans and gauche conformations of the alkyl chain of the vitamin E complexed with SD-40. Similar results had also been observed by Cheng et al.,9 who investigated the complexation of debranched starch and phosphatidylcholine. As shown in Figure 4B, the SD40/soy isoflavone inclusion complex showed typical V6III-type diffraction diagrams, where the C-c peak of SD-40 was almost completely resolved from the C-b and C-e peaks and new peaks of C-12, C-13, and C-14 (39.19, 30.28, and 24.32 ppm) appeared. These results were in agreement with previous 13C CP/MAS NMR studies of high-amylose corn starch−genistein complexes.34 Overall, the above results indicated that the discrepancy in the molecular structure of the guests probably affected the crystalline structures of the corresponding SD-40 inclusion complexes. When specific regions of the spectra were further analyzed, Cb, C-c, and C-e of SD-40, methylene groups of vitamin E, and benzene ring of soy isoflavone showed major structural differences between the two inclusion complexes. Because Snape et al.35 had demonstrated that all polar groups larger than carboxyl must lie outside the helix cavity of amylose, we could conclude that the SD-40/vitamin E inclusion complex was probably formed by the hydrophobic interactions between the alkyl chain of vitamin E and the helix cavity of SD-40, with the rest of the vitamin E molecule patterns presented outside the helix cavity. In addition to the aromatic ring B, the other parts of soy isoflavones could not be embedded in the helical structure of SD. SAXS Analysis. Characterization of the nanostructure of samples was performed using SAXS. The SAXS scattering intensity distributions for maize starch, SD-40, SD-40/vitamin E inclusion complex, and SD-40/soy isoflavone inclusion complex scattering patterns were presented in Figure 5A. It is clear that maize starch showed one characteristic peak at q = 0.67 nm−1, which corresponds to a Bragg distance of about 9.22 nm. The location of the peak was reciprocally related to the average total thickness of the crystalline and amorphous regions in lamellar arrangements. Moreover, the intensity of this peak depended upon the amount of the ordered semi-crystalline structures and/ or the differences in electron density between crystalline and amorphous lamellae of the amorphous background. The peak located at 0.67 nm−1 disappeared in SD-40, which indicated that the crystalline structure of the maize starch was completely destroyed after the gelatinization and enzymatic hydrolysis processes. Meanwhile, after combination with vitamin E and soy isoflavone, new larger peaks at 0.28 and 0.30 nm−1 were observed in the SAXS curve of the complexes. These results indicated that a new semi-crystalline structure with a longer Bragg distance (22.43 and 20.93 nm) formed in the complexes (Figure 5A). The peak intensity of the SD-40/vitamin E inclusion complex was larger than that of the SD-40/soy isoflavone inclusion complex, suggesting a gradual decrease of electron density contrast between amorphous and crystalline lamellae from the SD-40/

Figure 5. (A) SAXS patterns (log−log) and (B) Lorentz-corrected SAXS profiles of maize starch, SD-40, and SD-40/vitamin E inclusion complex, and SD-40/soy isoflavone inclusion complex.

vitamin E inclusion complex to the SD-40/soy isoflavone inclusion complex. This phenomenon indicated that the semicrystalline structure of the SD-40/vitamin E inclusion complex was arranged more tightly than that of the SD-40/soy isoflavone inclusion complex. These results were in agreement with XRD. The fractal structure of various starch analyzed using SAXS has been reported previously.9 The fractal structures characterized by the fractal dimension D can be obtained from the scattering power law equation

I ∝ qα

(5)

where α is an exponent ranging from −1 to −4 and can be obtained from the slope between log I and log q and I is the scattering intensity. In the case of −4 < α < −3, the scattering could be regarded as a reflection from the surface and the objects can be judged as possessing a surface fractal structure with the fractal dimension Ds = α + 6, suggesting the irregularity of the surface. Ds of 2 meant that the scattering surface was smooth. In the case of −3 < α < −1, the scattering objects could be seen as possessing a mass fractal structure with a Dm of −α, which referred to the degree of compactness of the physical arrangement of the mass. As shown in Figure 5B, the slope of the scattering patterns of maize starch, SD-40, SD-40/vitamin E, and SD-40/soy isoflavone were −1.79, −2.27, −1.64, and −1.54, respectively, indicating that maize 8748

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deeper inclusion of this moiety into the cavity and screw of SD40. Similar results about the formation mechanism of drug/ cyclodextrin inclusion complexes were reported.38 Furthermore, the results indicated that the regular arrangement of SD-40 was disrupted by vitamin E because its alkyl chain encapsulated in the helix cavity of SD-40 with the aromatic ring of the vitamin E molecule lying outside the helix cavity, whereas the soy isoflavone molecule was complexed with SD-40 by bonding between the single aromatic ring B and the helix cavity and screw of SD-40. The phenomena were in agreement with the conclusion of Le Bail et al.,39 who reported that the amylose− decanal complex showed the V6I type and the amylose− menthone complex revealed a diffraction diagram of V6III. On the basis of the results of NOESY, the possible interaction modes of SD-40/vitamin E (Figure 7A) and SD-40/soy isoflavone (panels B and C of Figure 7) inclusion complexes were presumed. SEM Analysis. SEM was applied to study the morphologies of the SD-40/vitamin E physical mixture, SD-40/vitamin E inclusion complex, SD-40/soy isoflavone physical mixture, and SD-40/soy isoflavone inclusion complex. The SD-40/vitamin E physical mixture displayed a rough surface with no distinct feature (Figure 8A). After complexation with antioxidants, the SD-40/vitamin E inclusion complex displayed a smooth and tight appearance with thin lamellar structures embedded in the matrix (Figure 8B). The SD-40/soy isoflavone physical mixture appeared to be smooth with a strip-like structure (Figure 8C), while the SD-40/soy isoflavone inclusion complex displayed a porous and granular structure (Figure 8D). The difference in physical mixtures and inclusion complexes was probably due to the formation of inclusion complexes between SD-40 and antioxidants. In addition, the appearances of SD-40/vitamin E and SD-40/soy isoflavone inclusion complexes exhibited great discrepancy. The possible reasons might be that the whole linear alkyl chain of vitamin E molecules was encapsulated in the helix cavity of SD in the SD-40/vitamin E inclusion complex, while larger guest molecules lie inside or outside the helix cavity in the SD-40/soy isoflavone inclusion complex, which was in agreement with the results from NOESY. As a bulky and aromatic molecule, soy isoflavone complexed with SD-40 might form a more loose semi-crystalline structure than the SD-40/vitamin E inclusion complex, which was consistent with previous results obtained from XRD and SAXS. CLSM Analysis. CLSM images of samples are showed in Figure 9. Vitamin E and soy isoflavone stained by Nile Red appeared as red-stained spots under 633 nm wavelength laser irradiation, and SD-40 stained by Nile Blue appeared as green under 488 nm wavelength laser irradiation. Laser scanning micrographs of two physical mixtures showed dispersed stained spots under the superimposed channel, which fluoresced green and red, respectively (panels A and C of Figure 9). The SD-40/ vitamin E complex exhibited yellow fluorescence concentrated in the central area of inclusion complexes under the superimposed channel (Figure 9B), which could be attributed to the presence of vitamin E inside the SD helices. However, Figure 9D shows that the red fluorescence of soy isoflavone almost covered the green fluorescence of SD-40 with a scattered distribution, which could be explained by the suggestion that parts of soy isoflavone laid between helices. The fact that SD-40/vitamin E and SD-40/soy isoflavone inclusion complexes displayed different microstructures suggested that the particle size of guests or connection type between guest and host might influence the inclusion process, which has also been confirmed by NOESY.

starch, SD, SD-40/vitamin E inclusion complex, and SD-40/soy isoflavone inclusion complex all possessed a mass fractal structure with Dm = 1.79, 2.27, 1.64, and 1.54, respectively. Therefore, the structure of the SD-40/vitamin E inclusion complex was tighter and more compact than that of the SD-40/ soy isoflavone inclusion complex, which had a lower Dm value. NOESY Analysis. To further determine the spatial closeness and relative position of SD-40 and complex formation, a 2D NOESY experiment was carried out. The nuclear Overhauser effect (NOE) cross-correlation in NOESY could be observed if two protons were closely located in space within 0.4 nm.36 It has been reported that the H-c and H-e protons were located inside the hydrophobic cavity, while the other protons (H-a, H-b, H-d, and H-f) were present outside the cavity for the SD molecule.37 In Figure 6A, the SD-40/vitamin E inclusion complex showed

Figure 6. NOESY spectra of (A) SD-40/vitamin E and (B) SD-40/soy isoflavone inclusion complexes.

appreciable correlations between the H-5 proton of vitamin E and the H-c and H-e protons in the cavity of SD-40. It suggested that vitamin E had no interactions with other external protons in the cavity of SD-40 and the alkyl chain including H-5 of vitamin E was only located in the cavity of SD-40. However, for the SD-40/ soy isoflavone inclusion complex, the NOE correlations between all protons of SD-40 and protons at the aromatic ring B of soy isoflavone were clearly observed (Figure 6B), which suggested a 8749

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Article

Journal of Agricultural and Food Chemistry

Figure 7. Schematic representation of the intermolecular forces in inclusion complexes of (A) vitamin E and (B) soy isoflavone as a front view drawing and (C) side elevation drawing with SD from the results of NOESY.

Antioxidant Property Analysis. ABTS Radical Scavenging Activity. The results of the ABTS radical scavenging activity of samples are presented in Figure 10A. At first, SD-40/vitamin E and SD-40/soy isoflavone physical mixtures showed higher ABTS radical scavenging activity than inclusion complexes.

However, after 7 days, the SD-40/vitamin E inclusion complex had higher ABTS radical scavenging activity than that of the SD40/vitamin E physical mixture, while the SD-40/soy isoflavone inclusion complex was the same after 1 day. In addition, with the time increase, the ABTS radical scavenging activity of vitamin E 8750

DOI: 10.1021/acs.jafc.7b03242 J. Agric. Food Chem. 2017, 65, 8744−8753

Article

Journal of Agricultural and Food Chemistry

Figure 8. SEM images: (A) SD-40/vitamin E physical mixture (2000×), (B) SD-40/vitamin E inclusion complex (2000×), (C) SD-40/soy isoflavone physical mixture (2000×), and (D) SD-40/soy isoflavone inclusion complex (2000×).

Figure 10. Antioxidant activities: (A) ABTS radical scavenging activity and (B) hydroxyl radical scavenging activity.

day, the hydroxyl radical scavenging activities of vitamin E physical mixtures and inclusion complexes were 25 and 52%, respectively, and the hydroxyl radical scavenging activities of soy isoflavone physical mixtures and inclusion complexes were 38 and 58%, respectively. Moreover, the decline of the hydroxyl radical scavenging ability from 1 to 30 days in SD-40/vitamin E was 15%, whereas it was 20% in SD-40/soy isoflavone. Both the ABTS and OH radical scavenging ability experiments suggested that physical mixtures showed almost equal radical scavenging activity as complexes at first. However, after several days, SD-40/antioxidant complexes showed better activity than physical mixtures. This could be explained by the suggestion that vitamin E and soy isoflavone encapsulated in SD-40 could protect them against damage, thus leading to the increase of stability. In addition, vitamin E in the SD-40/vitamin E inclusion complex was more stable than soy isoflavone in the SD-40/soy isoflavone inclusion complex. The reason might be that the SD40/vitamin E inclusion complex, with a V6II crystalline structure, had a tighter and more compact semi-crystalline structure than that of the SD-40/soy isoflavone complex, with a crystalline structure of V6III. The insights in this study may prospectively be used to design and exploit SD inclusion complexes as a novel delivery system.

Figure 9. CLSM images: (A) SD-40/vitamin E physical mixture, (B) SD-40/vitamin E inclusion complex, (C) SD-40/soy isoflavone physical mixture, and (D) SD-40/soy isoflavone inclusion complex under 633 and 488 nm wavelength superimposed laser channels.

and soy isoflavone in the physical mixtures decreased greater than that in the inclusion complexes. On the 30th day, the ABTS radical scavenging activities of vitamin E physical mixtures and inclusion complexes were 1 and 32%, respectively, and the ABTS radical scavenging activities of soy isoflavone physical mixtures and inclusion complexes were 33 and 61%, respectively. In addition, the decline of the ABTS radical scavenging ability from 1 to 30 days was 10% in SD-40/vitamin E, while it was 32% in SD-40/soy isoflavone. Hydroxyl Radical Scavenging Activity. In Figure 10B, it is obvious that the antioxidant capacity of physical mixtures and inclusion complexes were decreased with an increase in time. However, the decrease of antioxidant capacity was slower in the inclusion complexes than in the physical mixtures. After 1 day, both inclusion complexes showed higher hydroxyl radical scavenging activity than that of physical mixtures. On the 30th

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DOI: 10.1021/acs.jafc.7b03242 J. Agric. Food Chem. 2017, 65, 8744−8753

Article

Journal of Agricultural and Food Chemistry ORCID

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Zhi-Gang Luo: 0000-0001-7549-8083 Funding

This research was supported by the National Natural Science Foundation of China (21576098 and 21376097), the Key Project of Science and Technology of Guangdong Province (2016A050502005, 2017B090901002, and 2015A020209015), the Key Project of Science and Technology of Guangzhou City (201508020082), and the Project funded by the China Postdoctoral Science Foundation (2016M590787 and 2017T100616). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b03242 J. Agric. Food Chem. 2017, 65, 8744−8753