Encapsulation of Vitamin E and Soy Isoflavone Using Spiral Dextrin

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Functional Structure/Activity Relationships

Encapsulation of vitamin E and soy isoflavone using spiral dextrin: Comparative structural characterization, release kinetics and antioxidant capacity during simulated gastrointestinal tract Ping-Ping Wang, Zhigang Luo, and Xichun Peng J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Journal of Agricultural and Food Chemistry

Encapsulation of vitamin E and soy isoflavone using spiral dextrin: Comparative structural characterization, release kinetics and antioxidant capacity during simulated gastrointestinal tract

Ping-Ping Wang,† Zhi-Gang Luo,*,†, ‡ Xi-Chun Peng§

†

School of Food Science and Engineering, South China University of Technology,

Guangzhou, 510640, China. ‡

South China Institute of Collaborative Innovation, Dongguan, 523808, China

§

Department of Food Science and Engineering, College of Science and Engineering,

Jinan University, Guangzhou, 510630, China

*Corresponding author: Tel: +86-20-87113845, Fax: +86-20-87113848, E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Spiral dextrin subfraction (SD-40) obtained through enzyme

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debranching and gradient ethanol precipitation could interact with vitamin E (VE) or

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soy isoflavone (SIO) to form V-type inclusion complexes. The formation of two

4

inclusion complexes was confirmed by Fourier transform-infrared spectroscopy,

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atomic force microscopy and differential scanning calorimetry. In this study, an in

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vitro gastrointestinal model was used to investigate the breakdown of inclusion

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complexes and release behavior of bioactive compounds. The results indicated that

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the two inclusion complexes exhibited a controlled and sustained release behavior

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during digestion. In addition, the SD-40/VE inclusion complex presented higher

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stability and stronger antioxidant capacity than the SD-40/SIO inclusion complex.

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Furthermore, the first and zero order models were applied to understand the release

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kinetics of VE and SIO from inclusion complexes in the stomach. While the first

13

order model was chosen to describe the release of VE and SIO from inclusion

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complexes in the intestine.

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KEYWORDS: Spiral dextrin; Inclusion complex; In vitro digestion; Release

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kinetics; Antioxidant activity

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INTRODUCTION

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Vitamin E (VE) refers to a group of fat soluble compounds that naturally exists

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in the form of eight different isomers including α, β, γ, and δ derivatives of

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tocopherol and tocotrienol (Figure 1A).1 VE has been confirmed to possess many

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biological activities, including strong antioxidant activity, antitumor properties,

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anti-aging effects, and improvement of cardiovascular health.2,3 Soy isoflavone

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(SIO), mainly composed of genistein and daidzein, is a compound that is

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ubiquitously distributed in foods, which affects a wide-variety of body systems

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(Figure 1B). Numerous studies have reported the beneficial health effects of SIO on

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estrogen-related cancer, cardiovascular disease, lipid profiles, climacteric symptoms,

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and osteoporosis in humans.4 Unfortunately, the incorporation of VE and SIO into

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commercial products is a challenge due to their relatively low chemical stability,

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water-solubility, and bioavailability.2,5 In addition, the low chemical stability of VE

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and SIO under acidic conditions results in their low bioavailability after oral

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administration. To enhance the stability and bioavailability of VE and SIO in the

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gastrointestinal tract, extensive strategies, including liposome, nanoparticles,

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nanoencapsulation and carbohydrate biopolymers encapsulation, have been

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developed to protect them from the acidic conditions of the stomach.6-8

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Amylose inclusion complexes are attractive starting materials in the field of

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bioactive compounds delivery due to their biocompatibility, biodegradability, low

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immunogenicity, and ability to be readily modified by various functional moieties.9

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In previous studies, some complexes based on amylose or amylose analogues 3



40

(debranched starch10, spring dextrin11 and spiral dextrin4 ) have been obtained to

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create a new delivery system to protect volatile and sensitive guest molecules, such

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as vitamins, fatty acids, and flavones. For example, Cheng et al.10 found that the

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stability of phosphatidylcholine was significantly improved and it could be gradually

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released with pancreatin treatment after being encapsulated by debranched-starch

45

with single helixes. Xu et al.11 revealed that spring dextrin and its complexes with

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α-linolenic acid or linoleic acid not only improved the stability, but also achieved the

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targeted delivery of functional lipids or other bioactive components.

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Spiral dextrin (SD), which is characterized by a repeating (1,4)-α-D-glucose

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unit that can be obtained from the hydrolysis of native starch with debranching

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enzymes (Figure 1C), has the ability to form helical structure by complexing with

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guest compounds. In our previous study, five subfractions of SD were obtained by

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gradient ethanol precipitation. Among them, the SD-40 inclusion complexes with the

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highest VE and SIO payload were chosen as the optimal molecule for further

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analysis.4 The influence of linear alkyl chain (VE) and aromatic ring structure (SIO)

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on the structure of SD inclusion complexes is significant, such as the resulting exact

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arrangement fractal structure, crystalline thickness, connection type, and position of

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guests. However, whether bioactive compounds VE and SIO could remain stable

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during gastrointestinal (GIT) digestion after encapsulation with SD-40 is still

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unknown. And no further discussion or comparison of SD-40 inclusion complexes

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with different structures as a delivery system during GIT has been reported. Thus, it

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is crucial to determine how the SD-40 inclusion complexes influence the

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bioaccessibility of bioactive components during the digestion process.

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In this study, to evaluate the protective effect of SD-40 on VE and SIO, two

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inclusion complexes were prepared and digested under simulated GIT. The structures

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of the two complexes were characterized by Fourier transform-infrared spectroscopy

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(FT-IR), atomic force microscopy (AFM) and differential scanning calorimetry

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(DSC). In addition, to determine the functional difference between the two inclusion

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complexes as affected by crystalline structure during GIT tract, the properties of

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particle size, digestibility, releasing rate and change in antioxidant activity were also

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studied. Different kinetic models were used to study the controlled release kinetics of

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VE and SIO from SD-40 inclusion complexes during gastric and intestinal digestion

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stage, respectively. These results of this study will be useful for designing effective

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amylose analogues-based delivery systems for lipophilic bioactive components.

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MATERIALS AND METHODS

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Materials. Maize starch (amylose content 24.75%) was purchased from

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Tiancheng Maize Development Co., Ltd. (Jilin, China). Isoamylase (EC 3.2.1.68

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activity of 2.0 × 105 units/g) was purchased from Hubei Hongyun Long Biological

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Co., Ltd. (Wuhan, China). Vitamin E (>98%) and soy isoflavone were obtained from

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Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other chemicals

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used were of analytical grade. SD, SD-40, SD-40/VE inclusion complex (SD-40/VE)

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and SD-40/SIO inclusion complex (SD-40/SIO) were prepared as previously

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described.4 5



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Structural characterization of complexes. Fourier transform-infrared (FT-IR)

84

spectroscopy. The infrared spectra of the samples were taken by using a FT-IR

85

spectrometer (VERTEX 33, Bruker, Germany). The samples were mixed with

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anhydrous KBr, compressed into thin disk-shaped pellets, and then scanned from

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4000 to 400 cm−1.

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Differential scanning calorimetry (DSC). Thermal properties of the samples

89

were analyzed by DSC (DSC 214, Netzsch, Germany). DSC analyses were carried

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out in modulated mode under N2 atmosphere. The lyophilized samples were

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accurately weighted into aluminum pans, and the pans were then hermetically sealed.

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Finally, samples were equilibrated at 20 °C, and then heated to 400 °C at a heating

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rate of 10 °C/min.

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Atomic Force Microscopy (AFM). According to a previous method12 with some

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modifications, AFM images of samples were determined using a Nanoscope IIIa

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Multimode scanning probe microscope (Nanoscope 3A Multimode, Veeco, USA).

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The SD-40, SD-40/VE and SD-40/SIO samples (10 µg/mL, 10 µL) were deposited

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onto a freshly cleaved mica surface, dried in a desiccator, and captured in the tapping

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mode.

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Simulated GIT Analysis. The samples were digested in a simulated GIT model

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that consisted of mouth phase, gastric phase and small intestine phase under the

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protection from light and oxygen. The simulated GIT studies were carried out

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according to a previously described method with modifications.13 The constituents

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and concentrations of mouth phase, gastric phase and small intestinal phase are

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presented in Table 1.14,15

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SD-40, SD-40/VE and SD-40/SIO were subjected to in vitro digestion based on

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a previous method with modifications.16 Each sample was digested at 37 °C in a

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flask with a magnetic stirrer. A schematic representation of the optimized in vitro

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digestion model is presented in Table 1. The digestion was started by adding 160 mL

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of saliva (pH 6.8 ± 0.2) to 8 g of samples (dry weight). The mixture was rotated

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head-over-heels for 5 min at 100 rpm. Subsequently, 160 mL of gastric juice (pH 1.2

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± 0.02) was added and rotated for 2 h. The mixture collected from the gastric phase

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was adjusted to pH 7.0 ± 0.2 using NaOH (2 M). Finally, 56 mL of bile extract

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solution, 24 mL of saline solution and 40 mL of pancreatic lipase were added, and

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the mixture was rotated for another 2 h. The microstructure, particle size distribution,

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glucose content, release behavior and antioxidant activity were characterized at each

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stage.

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Determination of glucose content during in vitro digestion. The glucose

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contents were determined during the digestion of SD-40 inclusion complexes. At

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each time intervals, sample aliquots (0.5 mL) were withdrew and immediately mixed

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with 4 mL anhydrous ethanol to inactivate the enzymes. After centrifugation (5000

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rpm, 5 min), the glucose content in the supernatant was determined using the

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Megazyme GOPOD kit.17

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In vitro release study. The content of VE and SIO were determined during the

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digestion of their complexes in the simulated GIT model. At predetermined time 7



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intervals, sample aliquots (5 mL) were withdrew and immediately mixed with 5 mL

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anhydrous ethanol to inactivate the enzymes, followed by centrifugation (5000 rmp,

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5 min). The whole VE and SIO in the sediment were extracted absolutely by ethanol

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and dimethyl sulfoxide, respectively. And then, the VE and SIO concentrations were

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measured using the method described in previous studies.4

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Release Kinetics Analysis. The release kinetics of VE and SIO were evaluated

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by fitting their release rates during simulated gastric and intestinal stage into

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different kinetic models including zero order (cumulative amount of drug released

134

versus time), first order (log cumulative percentage of drug remaining versus time),

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Higuchi (cumulative percentage of drug released versus square root of time), and

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Korsmeyer–Peppas model (log cumulative percentage of drug released versus log

137

time), using the eqs. (1) - (4) below.

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Zero order model kinetic: Qt=Q0+K0t

(1)

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First order model kinetics: ln(1-Qt)=-K1t

(2)

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Higuchi model kinetics: Qt= Kht1/2

(3)

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Korsmeyer-Peppas model kinetics: Mt/M∞= Kptn

(4)

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where Qt is the percentage of drug release at time t, Q0 is the initial amount of drug

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in the microspheres and K0, K1, Kh are the release constant in zero order, first order

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and Higuchi model, respectively. In the Korsmeyer–Peppas model, Mt is the amount

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of drug released at time t, M∞ is the amount of vitamin E released at infinite time,

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Mt/M∞ is the fraction of drug released at time t. Kp is the kinetic constant and n is the

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release exponent. The estimated n value is used to assess the release mechanism.18 8


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Antioxidant activity evaluation of VE and SIO. Antioxidant activities of VE,

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SIO and their inclusion complexes exposed to GIT conditions were evaluated

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through investigating their free radicals-scavenging effect on ABTS•, DPPH• and

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•OH. Samples were obtained from various stages of digestion: (i) immediately after

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preparation, (ii) after 5 min incubation at pH 6.8 in simulated saliva, (iii) after 2 h

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incubation at pH 1.2 in simulated gastric juice, and (iv) after 2 h incubation at pH 7

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in simulated intestinal fluid. At different time intervals (0, 5, 125 and 245 min),

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sample aliquots (10 mL) were withdrew and the sample of SD-40/VE was

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immediately mixed with 10 mL anhydrous ethanol, while the SD-40/SIO sample was

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added into dimethyl sulfoxide to absolutely dissolve SIO. The ABTS•, DPPH• and

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•OH radical scavenging activities of VE and SIO were measured using the method

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previously recorded.19,20

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Particle Size Analysis. The size distribution and average diameter of initial

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inclusion complexes and samples exposed to GIT conditions were determined by a

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laser particle size analyzer (Mastersizer 2000, Malvern, UK) equipped with Sirocco

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2000.

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Confocal Laser Scanning Microscopy (CLSM) Analysis. Confocal imaging

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of the inclusion complexes was carried out with an oil immersion objective lens

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using a Leica TCS SP5 confocal microscope (Leica TCS-SP5, Bruker, Germany) at

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ambient temperature. After freeze drying, the samples (0.002 g) were dissolved

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separately in 20 µL of Nile blue and 20 µL of Nile red solution, and the solutions

9



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were kept in the dark for 12 h. The Helium Neon 202 laser (He-Ne) with excitation

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at 488 nm and 633 nm was used for the mixed dyes.

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Statistical Analysis. All data were expressed as the means ± standard

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deviations of triplicate experiments determined using the statistical software package

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SPSS (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was carried

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out by Tukey’s test (p < 0.05).

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RESULTS AND DISCUSSION

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Formation of Inclusion Complexes. Helical SD has a hydrophobic cavity that

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can encapsulate hydrophobic guest molecules to form SD inclusion complexes. The

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SD-40/VE and SD-40/SIO were synthesized according to our previously reported

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protocols, and the highest payloads of VE and SIO were 140.50 and 111.83 mg/g,

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respectively.4 The formation of SD-40/VE and SD-40/SIO complexes were further

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confirmed by FT-IR, DSC and AFM.

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Direct evidence of the successful encapsulation of VE and SIO by SD-40 and

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molecular changes in the inclusion complexes (Figure 2) were confirmed by the

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FT-IR spectroscopy. For SD-40, the major absorption peaks were recorded at around

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1020, 1081, and 1153 cm−1 corresponding to the coupled C−C/C−O stretching

186

vibrations and the antisymmetric stretching vibration of the C−O−C glycosidic

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bridge. The characteristic FT-IR absorption bands of VE at 1371 cm−1 corresponded

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to the phenyl skeletal, the overlap of asymmetrical methyl ending and methylene

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scissoring vibration, and the band at 1462 cm−1 was attributed to symmetrical methyl

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bending.6 The FT-IR spectrum of SD-40 and VE physical mixtures (SD-40+VE) 10


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showed no shift in the characteristic bands of the individual spectrum of SD-40 and

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VE. However, for SD-40/VE, it was clear that all the characteristic absorption peaks

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of VE were obviously weakened compared to the physical mixture, suggesting that

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VE was already entrapped into the cavity of SD-40. These results were in agreement

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with previous papers that studied the complex formation between large-ring

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cyclodextrin and vitamin E acetate.21 Besides, the characteristic absorption peaks of

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VE in the SD-40/VE spectrum shifted from 1462 cm-1 and 1371 cm-1 to 1469 cm-1

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and 1381 cm-1, respectively. The increase in these shifts indicated the formation of

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hydrogen bond and the presence of van der Waals forces between VE and SD-40

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during the formation of inclusion complex. Similar results were found by Rosanna et

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al.22 who studied the complex formation of genistein and daidzein with substituted

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sulfobutyl ether-β-cyclodextrin.

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The C=C stretching vibrational frequencies of SIO were observed at 1625 cm-1,

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and the peak at 1515 cm-1 corresponded to vibrations involving the carbon and

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hydrogen atoms of phenolic ring. Similarly, the FT-IR spectrum of SD-40 and SIO

206

physical mixtures (SD-40+SIO) showed approximate superimposition and no shifts

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in the characteristic bands compared to the individual spectrum of SD-40 and SIO. It

208

can be seen clearly that the characteristic absorption peaks of SIO in SD-40/SIO

209

spectrum increased from 1625 cm-1 and 1515 cm-1 to 1651 cm-1 and 1563 cm-1,

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respectively, suggesting the successful formation of SD-40/SIO inclusion complex.

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Similar results were found by Cheng et al.10 who investigated the formation of

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inclusion complexes of debranched-starch and phosphatidylcholine. 11



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DSC is also applied to verify the formation of inclusion complexes. As shown

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in Figure 3, SD-40, SD-40+VE and SD-40/VE all had a broad endothermic peak

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between 50 and 150 °C. Deng et al.23 revealed that this peak was produced by the

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release of water molecules. The endothermic peak in SD-40 corresponding to

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dehydration temperature of water molecules was 125.46 °C, while the broad

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endothermic peak in SD-40/VE was around 90.84 °C. The decrease in endothermic

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peak indicated that the dehydration process was changed after the formation of

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inclusion complexes, which was in agreement with the results from Deng et al.23

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Emilio et al.24 have reported that the differences in thermograms between the

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polyphenols (orogenic acid, rutin and quercetin)–β-cyclodextrin physical mixture

223

and their complexes indicated the combination of the host and guests. The other two

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small peaks at ~194.94 and 230.74 °C may be related to the elimination of SD-40.

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Compared to the thermograms of SD-40 and SD-40+VE, the disappearance of

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endotherm at 193.28 °C in the thermogram of SD-40/VE could be due to a major

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interaction between SD-40 and VE, which confirmed the formation of inclusion

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complex. These results were in agreement with the reports of Zhang et al.25 who

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studied the physicochemical characteristics of complexes between amylose and

230

garlic bioactive components generated by milling activating method. Meanwhile, the

231

DSC thermogram of SD-40+SIO showed similar endothermic peaks to SD-40, while

232

the endotherm of SD-40/SIO disappeared at 184.54 °C. This result also proved the

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formation of SD-40/SIO inclusion complexes.

234

AFM is performed to confirm the successful formation of inclusion complexes. 12


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As presented in Figure 4A, the particle sizes of SD-40 distributed uniformly. The

236

presence of large particle may be caused by the aggregation of SD-40, which was in

237

agreement with previous studies.8 It was observed that the surface of SD-40 particles

238

was sharp and rough with no distinct features. Compared to SD-40, the SD-40/VE

239

displayed a larger and tighter granular structure with a flat surface. SD-40/SIO

240

showed more regular shape and flatter than SD-40. Lesmes et al.26 have reported that

241

the hydrophobic interactions between host and guests were contributed to an

242

improvement in granule size, compactness and flat surface to a certain degree.

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Therefore, the great differences in Figure 4A, 4B and 4C strongly supported the

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formation of the SD-40/VE and SD-40/SIO.

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Particle Size Analysis. The particle size distribution of the samples at different

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stages in the simulated GIT model was observed. As shown in Figure 5, the particle

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size distribution of SD-40/VE and SD-40/SIO presented almost bimodal distribution

248

before digestion. However, after exposure to artificial saliva, the SD-40/VE and

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SD-40/SIO exhibited a slightly decrease in surface-weighted average particle size

250

(D[3,2]). The decrease in particle size might be attributed to the fact that the SD-40

251

inclusion complexes were partly destroyed to small particles by physical mechanical

252

action or α-amylose in mouth. While the d(0.1) of SD-40/SIO, which means 10% of

253

the particle size is below this value, exhibited slightly increase (Table 2), which may

254

be due to the swelling and aggregation.15 After passing through the simulated gastric

255

stages, both surface- and volume-weighted average particle size of SD-40/VE and

256

SD-40/SIO decreased (Figure 5), which was related to the inclusion complexes 13



257

destroyed by vortexing and degraded by gastric acid. Interestingly, the particle size

258

distribution of SD-40/SIO changed from bimodal to monomodal, while the

259

SD-40/VE remained bimodal particle size distribution with small change. According

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to Wang et al.4, the SD-40/VE formed more compact crystalline structure than

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SD-40/SIO. The larger decrease in the particle size of SD-40/SIO suggested that

262

SD-40/VE was more stable in the acid condition of stomach than SD-40/SIO. These

263

transition from bimodal to monomial for SD-40/SIO might be explained by the fact

264

that the loose crystalline structure of SD-40/SIO large particle size were more easily

265

destroyed by gastric juice.4

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After exposure to the simulated small intestinal fluid, the particle size

267

distribution of two SD-40 inclusion complexes became broader and larger, indicating

268

that the inclusion complexes digested by small intestinal fluid had tendency to

269

aggregate or swell to form a loose structure. This aggregation might be due to the

270

presence of calcium ions and bile salts in the simulated small intestinal fluid.27 The

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specific change and discrepancy in particle size of SD-40/VE and SD-40/SIO during

272

small intestinal digestion would be further studied in this paper.

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Glucose content Analysis. The change of glucose content of SD-40, SD-40/VE

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and SD-40/SIO during different digestion stages is presented in Figure 6. After oral

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digestion, the glucose contents of SD-40, SD-40/VE and SD-40/SIO were 0.33, 0.45

276

and 0.36 mg/mL, respectively. Although low content of glucose was detected, all of

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the samples were rapidly digested by the α-amylase within the first 5 min. In

278

addition, the larger increase in glucose content after mouth digestion of SD-40/SIO 14


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with 0.234 than SD-40/VE with 0.230 indicted that the SD-40/SIO was more easily

280

digested by α-amylase in mouth. In the gastric stage, the glucose content had very

281

slight increases of 0.22, 0.38 and 0.46 mg/mL for SD-40, SD-40/VE and SD-40/SIO,

282

respectively. The reason why the SD-40 and SD-40 inclusion complexes were slowly

283

and slightly influenced by gastric juices was probably due to the absence of

284

amylase.28 However, the SD-40/SIO showed larger increase in glucose content than

285

SD-40/VE during gastric stage, which suggested that the SD-40/VE exhibited a

286

stronger resistant to acid environment than SD-40/SIO during gastric stage. For the

287

intestinal digestion, another rapid increase in the glucose content profiles was

288

observed for all samples from 125 to 225 min, which might be ascribed to the

289

degradation of SD-40 by pancreatic enzymes. However, at the end of digestion from

290

225 to 245 min, the increase in glucose content of all samples became flat due to the

291

decrease in the concentrations of SD-40, SD-40/VE and SD-40/SIO. Mirpoor et al.29

292

found similar results that the quercetin was protected by nanocarrier in the upper

293

GIT and then released in the intestinal tract. Meanwhile, the glucose content profiles

294

during intestinal digestion showed similar trend as simulated saliva and gastric stage,

295

in which the SD-40/SIO also presented faster digestion rate than SD-40/VE. The

296

reason might be that the SD-40/VE with V6ΙΙ crystalline structure had tighter and

297

more compact semicrystalline structure than SD-40/SIO with crystalline structure of

298

V6ΙΙΙ.4 The results of the whole digestion profiles indicated that SD-40 had a better

299

protection for VE than SIO against the gastric juice.

15



300

In Vitro Release Study. The release profiles of SD-40/VE and SD-40/SIO are

301

shown in Figure 7. There was an initial burst release of VE (28.31%) and SIO

302

(34.18%) in mouth. The reason was that the saliva enzyme degraded the wall

303

material of SD-40, which led to the partial release of VE and SIO. This result was

304

consistent with the change of glucose content during oral digestion that both glucose

305

content of SD-40/VE and SD-40/SIO had large increases with 0.230 and 0.234,

306

respectively. However, in gastric stage, the release curve became steady at a lower

307

rate and ended with 38.08% of VE and 46.49% of SIO release. The low release rate

308

was probably related to the resistance of SD-40 inclusion complexes to the acid

309

condition of the stomach digestion. At the beginning of the intestinal digestion, a

310

quick increase in the release profiles was observed for VE and SIO, which could be

311

explained by the fact that the wall materials SD-40 had been destroyed by digestive

312

enzymes in simulated intestinal fluid. Similar quick increase in intestinal digestion

313

had been reported for folic acid encapsulated by horse chestnut starch and

314

β-cyclodextrin.7 At the end of the intestinal digestion, the ultimate release rate of VE

315

and SIO was found to be 95.67% and 98.47%, respectively. After 80 min duration in

316

the intestinal digestion, the release rates became significantly slow, which may be

317

due to the fact that almost no VE and SIO were left in the inclusion complexes. On

318

the whole, the above results clearly indicated that encapsulation of VE and SIO

319

within SD-40 enabled them to achieve target release in the intestine. Additionally,

320

the SD-40/VE showed slower release rate than SD-40/SIO during the simulated

321

saliva and gastric juice, suggesting the higher stability of SD-40/VE in oral and 16


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gastric digestion. The previous study found that encapsulating and stabilizing

323

capacities of inclusion complexes were closely related to the structure of inserted

324

compounds. The properties of encapsulation materials were greatly affected by the

325

guests with different structures.4,30 VE with an alkyl chain was encapsulated in the

326

helix cavity of SD-40, whereas the single aromatic ring of SIO was complexed by

327

helix cavity and screw of SD. However, the similar release rate of SD-40/VE and

328

SD-40/SIO in intestine indicated that the SD-40 inclusion complexes with different

329

crystalline structures were completely destroyed during intestinal digestion, and the

330

enzymatic hydrolysis rate was little affected by the crystalline structure of inclusion

331

complexes.

332

Release kinetics Analysis. To understand the mechanism of bioactive

333

compounds release, in vitro release data was analyzed using the kinetics equations

334

that are empirical in nature. The cumulative release of VE and SIO in simulated GIT

335

tract were curved fitted to zero order model, first order model, Higuchi model and

336

Korsmeyer-Peppas model. And the results are presented in Table 3. In gastric stage,

337

the highest value of correlation coefficient (r2) was observed in the case of the Zero

338

order equation to describe the SIO release from the SD-40/SIO, which suggested that

339

cumulative release amount of SIO was proportional to time during the stomach.

340

However, the release data of SD-40/VE during the stomach were fitted to the First

341

order mode kinetic equation with the highest correlation coefficient (r2) (0.9788).

342

The Zero order model was existed in cumulative amounts of antioxidants released

343

versus time, while First order was present in the form of log cumulative percentage 17



344

of bioactive amounts remaining versus time. The two different kinetics equations of

345

SD-40/VE and SD-40/SIO during the stomach indicated that the different structure

346

of two SD-40 inclusion complexes exerted great influence on their resistant ability to

347

gastric juice. Furthermore, based on the correlation coefficient (r2) shown in Table 3,

348

the First order model was the best model to describe the release curves of VE and

349

SIO from the SD-40 inclusion complexes in intestinal stage. The same model was

350

also used by Anilkumar et al.31 to describe retarding materials released from

351

β-cyclodextrin. The first order release rate constant for SD-40/VE and SD-40/SIO

352

was 0.0245 and 0.0331, respectively. The discrepancy in release rate content also

353

revealed that SD-40 had stronger protection to VE than SIO in the small intestine.

354

CLSM Analysis. CLSM images of two inclusion complexes and samples

355

collected after different stages of the simulated GIT model are presented in Figure 8.

356

Basically, SD-40 was labeled by Nile Blue (green color), while VE and SIO were

357

labeled by Nile Red stain (red color).4 The yellowish green area in the overlay

358

images (Figure 8) indicated the formation of complexes between SD-40 and

359

antioxidants, which was similar to the result of Aceituno-Medina et al.32, who

360

investigated the potential of amaranth protein-based electrospun fibers for the

361

encapsulation and protection of quercetin and ferulic acid.

362

The images of SD-40/VE and SD-40/SIO showed that there was a

363

homogeneous particle size distribution of the inclusion complexes during oral

364

digestion, which was in accordance with the previous results of particle size analysis.

365

Additionally, after incubation in the stomach, the SD-40/VE was transformed to 18


Page 18 of 43

Page 19 of 43


366

elongated and loose structures, suggesting that the structure of SD-40/VE was

367

affected by simulated gastric fluid. Noticeably, the SD-40/VE still exhibited yellow

368

fluorescence concentrated in the central area under the superimposed channel

369

(Figure 8). And the green fluorescence of SD-40 was distinctly observed at the edge

370

of area, suggesting the little release of VE from inclusion complex. This is consistent

371

with our digestion and release study that SD-40 had protective effect on VE during

372

gastric digestion. However, the red fluorescence of SIO almost covered the entire

373

green fluorescence of SD-40 and even existed at the outer edge of SD-40/SIO after

374

gastric digestion, which indicated that SIO was about to be released from SD-40/SIO.

375

Furthermore, the images demonstrated that structure of SD-40/SIO was influenced

376

more seriously than SD-40/VE by the acid condition of the stomach. The images of

377

the samples in the small intestine stage clearly showed that the red fluorescence of

378

VE and SIO almost covered the whole scope with a scattered distribution, suggesting

379

that the encapsulated antioxidants had been entirely released from SD-40. In addition,

380

the green fluorescence of SD-40 had no regular shape, which might be due to the

381

destruction of SD-40 by digestive enzyme in the intestine. These results were in

382

agreement with the release profiles that almost all of VE and SIO were released from

383

the SD-40 inclusion complexes after intestinal digestion.

384

Antioxidant Activity Evaluation. The chemical properties of bioactive

385

compounds may change during the gastrointestinal digestion since they were greatly

386

affected by their structures.32 However, there is little report about the bioactivities

387

variation of SD-40 inclusion complexes after digestion. In this study, the antioxidant 19



388

activities of VE and SIO were investigated using a digestion model in vitro (Figure

389

9).

390

DPPH• Radical Scavenging Activities. The results of the DPPH• radical

391

scavenging activity of samples are presented in Figure 9A. Before digestion, both

392

SD-40/VE and SD-40/SIO presented slightly lower DPPH• radical scavenging

393

activity than free VE and SIO. However, after oral digestion, the DPPH• radical

394

scavenging activities of free VE, SD-40/VE, free SIO and SD-40/SIO were 38.94%,

395

38.48%, 81.02% and 82.10%, respectively. Interestingly, both SD-40/VE and

396

SD-40/SIO had higher DPPH• radical scavenging activity than that of free VE and

397

SIO in the end of gastric digestion. As the time increasing, the DPPH• radical

398

scavenging activities of free VE and SIO decreased greater than that in their

399

inclusion complexes. In Figure 9A, the DPPH• radical scavenging activities of free

400

VE, SD-40/VE, free SIO and SD-40/SIO were reduced by 23.56%, 10.63%, 44.36%

401

and 17.90% after digestion, respectively.

402

ABTS• Radical Scavenging Activity. In Figure 9B, it is obvious that ABTS•

403

radical scavenging activity of free VE, SIO and their inclusion complexes decreased

404

with an increase in digestion time. However, the decrease of ABTS• radical

405

scavenging activity was slower in inclusion complexes than that in free VE and SIO.

406

In the initial digestion, the ABTS radical scavenging activities of free VE, SD-40/VE,

407

free SIO and SD-40/SIO were 84.53%, 80.97%, 93.28% and 90.09%, respectively.

408

However, after digested by mouth, SD-40 inclusion complexes showed better ABTS

409

radical scavenging activity than free VE or SIO. Moreover, in the end of digestion, 20


Page 20 of 43

Page 21 of 43


410

the ABTS• radical scavenging activities of free VE and inclusion complexes were

411

decreased to 30.92 and 66.39%, respectively, and the ABTS• radical scavenging

412

activities of free SIO and inclusion complexes was 32.92 and 63.98%, respectively.

413

Hydroxyl Radical Scavenging Activity. As displayed in Figure 9C, hydroxyl

414

radical scavenging activity of samples showed a continual decrease with increase of

415

incubation time. The OH• scavenging activity of free VE, SD-40/VE, SIO and

416

SD-40/SIO reduced from 60.64%, 59.27%, 63.29%, and 62.03% to 18.29%, 42.89%,

417

21.38%, and 43.29% after 245 min incubating.

418

It is noteworthy that the SD-40/VE and SD-40/SIO exhibited a lower radical

419

scavenging activity than free VE and SIO before GIT incubating. There are two

420

possible explanations for this observation. First of all, it may be due to the partly

421

degradation during the preparation of inclusion complexes. During thermal

422

processing or under oxidative conditions, VE or SIO can be (reversibly) oxidized

423

and lost antioxidant capacity. The second reason could be that VE or SIO molecules,

424

which are more deeply incorporated in the spiral cavity of SD-40, are not accessible

425

for radicals, resulting in (apparent) lower radical scavenging activity.8 In the

426

digestion from saliva to intestine, the radical scavenging activities of all samples

427

decreased continually, which indicated that the GIT environment caused great

428

damage to bioactive compounds.33 However, the two inclusion complexes exhibited

429

a lower decrease rate than free bioactive compounds, which was attributed to the

430

protective effect of SD-40 on VE and SIO. This was in good agreement with other

431

studies that inclusion complexes were beneficial to enhance the antioxidant stability 21



432

of bioactive compounds.6,8 In addition, DPPH•, ABTS• and OH• radical scavenging

433

activities of the SD-40/SIO showed greater decrease than SD-40/VE, which further

434

indicated that the SD-40 had a stronger protective effect on VE than SIO. These

435

results are promising since these SD-40 inclusion complexes show the ability to

436

protect VE and SIO during the in vitro digestion.

437

In this work, a simple and eco-friendly procedure for producing a novel dextrin

438

SD-40 was designed by enzyme debranching and gradient ethanol precipitation as

439

wall material to encapsulate VE and SIO. The formation of SD-40/VE and

440

SD-40/SIO inclusion complexes was confirmed by FTIR, DSC, and AFM. It is

441

noteworthy that this study served as first trial of functional comparison between

442

SD-40/VE and SD-40/SIO during the gastrointestinal system. In addition, the

443

relationship between the chemical structure of inclusion complexes and functional

444

characteristic was revealed. By comparing with SD-40/VE, the SD-40/SIO showed

445

faster digestion rate, lower release rate and stronger antioxidant capacity. Besides,

446

the in vitro release kinetics of VE and SIO from SD-40 inclusion complexes

447

demonstrated that the release mechanism highly depended on their molecular

448

structures. Additionally, more detailed studies indicated that SD-40 exhibited

449

stronger protection to guest compounds with alkyl chain (VE) than aromatic ring

450

(SIO) during GIT. The insights in this study is prospectively to be used for designing

451

and exploiting SD-40 as a novel material for delivering bioactive compounds with

452

alkyl chain.

453 22


Page 22 of 43

Page 23 of 43


454

AUTHOR INFORMATION

455

Corresponding Authors

456

*Tel: +86-20-87113845. Fax: +86-20-87113845. E-mail: [email protected]

457

(Zhi-Gang Luo).

458

Funding

459

This research was supported by the National Natural Science Foundation of China

460

(21576098 and 21376097), Fundamental Research Funds for the Central Universities,

461

SCUT (2018ZM0124), the Key Project of Science and Technology of Guangdong

462

Province (2017B090901002, 2016A050502005, and 2015A020209015), the Key

463

Project of Science and Technology of Guangzhou City (201508020082), and the

464

Project funded by the China Postdoctoral Science Foundation (2016M590787 and

465

2017T100616).

466

Notes

467

The authors declare no competing financial interest.

468 469

23



Page 24 of 43

470

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29



FIGURE CAPTIONS Figure 1. The chemical structures of (A) vitamin E, (B) soy isoflavone, (C1) side elevation drawing of spiral dextrin and (C2) front view drawing of spiral dextrin Figure 2. FT-IR spectra of samples in full range (4000−500 cm−1) and expanded range (2000−800 cm−1) Figure 3. DSC profiles of SD-40, SD-40+VE, SD-40/VE, SD-40+SIO and SD-40/SIO Figure 4. Typical AFM height images of (A) SD-40, (B) SD-40/VE and (C) SD-40/SIO Figure 5. Particle size distribution of (A) SD-40/VE and (B) SD-40/SIO before and after the in vitro digestion Figure 6. Hydrolysis kinetics of SD-40, SD-40/VE and SD-40/SIO during an in vitro digestion study Figure 7. Release kinetics of VE and SIO from SD-40 inclusion complexes during an in vitro digestion study Figure 8. CLSM images of SD-40/VE and SD-40/SIO during in vitro digestion Figure 9. DPPH radical scavenging activity (A), ABTS radical scavenging activity (B) and hydroxyl radical scavenging activity (C) of VE, SD-40/VE, SIO and SD-40/SIO during an in vitro digestion study

30


Page 30 of 43

Page 31 of 43


Table 1. Constituents and Concentrations of the Mouth phase, Gastric Phase and Small Intestine Phase of Simulated GIT Saliva

Gastric Juice

Small intestine Juice

Chemical

NaCl

NaCl

2 g/L

2.5 mL pancreatic lipase

composition

58.5 mg/500mL

HCl

7 mL/L

(187.5 mg/2.5mL)

KCl

Pepsin

3.2 g/L

3.5 mL bile extract solution

74.5 mg/500mL

(187.5 mg/3.5mL)

NaHCO3

1.5 mL saline solution (10

1.05 g/500mL

mM CaCl2+150mM NaCl)

α-amylase Time

5 min

2h

2h

pH

6.8 ± 0.2

1.2 ± 0.02

7.0 ± 0.2

31



Page 32 of 43

Table 2. Particle Size Characterization of SD-40/VE and SD-40/SIO before and after the in vitro Digestion

SD-40/VE

SD-40/SIO

D [3, 2] (µm) a

D [4, 3] (µm) b

d (0.1) (µm) c

d (0.5) (µm) d

d (0.9) (µm) e

Initial

24.786

73.694

9.855

50.84

172.596

Mouth

21.419

97.144

7.758

73.361

224.021

Stomach

15.165

36.86

6.749

23.737

85.951

Intestine

47.544

407.469

19.507

248.391

1059.923

Initial

26.766

110.371

9.237

79.86

260.793

Mouth

26.581

126.215

10.283

103.194

277.326

Stomach

15.947

59.575

5.881

41.646

141.519

Intestine

42.773

336.517

17.472

191.015

891.765

a

D[3,2] Surface area mean diameter (Sauter Mean Diameter): is the weighted average surface diameter, assuming spherical particles of the same surface area as the actual particles. b D[4,3] Volume mean diameter (De Brouckere Mean Diameter): is the weighted average volume diameter, assuming spherical particles of the same volume as the actual particles. c-e d(0.1), d(0.5) and d(0.9): a particle size value indicating that 10%, 50% and 90% of the distribution is below this value, respectively.

32


Page 33 of 43


Table 3. Different Kinetic Models Evaluation of VE and SIO Release Formation codes

SD-40/VE

SD-40/SIO

Zero model

order

First model

order

Higuchi model

Korsmeyer–Peppas model

r2

K0 a

r2

K1 b

r2

Kh c

r2

Kp d

ne

Gastric stage

0.9745

0.0007

0.9788

0.0011

0.9697

0.0091

0.865

0.2548

0.0779

Intestinal stage

0.8281

0.0044

0.9718

0.0245

0.9427

0.0605

0.9672

0.281

0.2687

Gastric stage

0.9922

0.001

0.9899

0.0017

0.9223

0.0125

0.8412

0.2785

0.0973

Intestinal stage

0.8371

0.0041

0.9813

0.0331

0.9429

0.056

0.9719

0.3382

0.2347

a-d

K represents the kinetic constant of Zero order model, First order model, Higuchi model and Korsmeyer-Peppas release kinetics model. e n represents release exponent of Korsmeyer-Peppas release kinetics model.

33



Page 34 of 43

Figure 1

A

HO

O

HO

O

B

OH

O

OH

C1

C2

OH

H

H O

O O

HO

H

HO

H

H

H H

OO

H H

O

H OH

HO

HO

H

H

O

O H

H

OH

OH

O H

H

HO H

O

H H H

H

OH

H

OH O

O

O

OH

H

34


H

Page 35 of 43


Figure 2 5.5

SD-40/SIO

5.0 SD-40+SIO 4.5 4.0

SIO

3.5 SD-40/VE

3.0 2.5

SD-40+VE

2.0

VE

1.5 SD-40

1.0 0.5 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber(cm ) 5.5

SD-40/SIO

1625

1153 1081 1020

1515

5.0 SD-40+SIO 4.5 4.0

SIO

3.5 3.0

SD-40/VE

2.5

SD-40+VE

2.0

VE

1.5 1.0

SD-40

1462

1371

0.5 2000

1800

1600

1400

1200

1000

-1

Wavenumber(cm )

35


800


Page 36 of 43

Figure 3 97.54

7

225.76

SD-40/SIO

79.91

6 184.54

5

222.81

SD-40+SIO

90.84 227.21

4

SD-40/VE 3

98.33 193.28 227.21

SD-40+VE

2 125.46

1

194.93 230.74

SD-40 0 0

50

100

150

200

250

300

T（ °C）

36


350

400

Page 37 of 43


Figure 4

37



Page 38 of 43

Figure 5

A 5

Intensity(%)

4

Initial Mouth Stomach Intestine

3

2

1

0 1

10

100

1000

Particle Diameter(um)

B

5

Intensity(%)

4

Initial Mouth Stomach Intestine

3

2

1

0

1

10

100

Particle Diameter(um)

38


1000

Page 39 of 43


Figure 6

2.5

SD-40 SD-40/VE SD-40/SIO

Glucose Content(mg/mL)

2.0

1.5

1.0

0.5

0.0

0

50

100

150

200

Time(min)

39


250


Page 40 of 43

Figure 7

VE

100

SIO

Antioxidant release ratio(%)

80

60

Oral stage

Gastric stage

Intestinal stage

40

20

0 0

50

100

150

Time(min)

40


200

250

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Figure 8

Initial

Mouth

SD40/ VE

SD40/S IO

41


Stomach

Intestine


Figure 9

A DPPH radical scavenging activity (%)

100

VE SD-40/VE SIO SD-40/SIO

90 80 70 60 50 40 30 20 10 0

Initial

Stomach

Mouth

Intestine

Stage

B

100


ABTS radical scavenging activity (%)

90 80 70 60 50 40 30 20 10 0

Initial

Stomach

Mouth

Intestine

Stage

C

100


OH radical scavenging activity (%)

90 80 70 60 50 40 30 20 10 0

Initial

Stomach

Mouth

Stage

42


Intestine

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Table of contents graphic

43


Encapsulation of Vitamin E and Soy Isoflavone Using Spiral Dextrin

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