Characterization and antioxidant activity of the complexes of tertiary butylhydroquinone with β-cyclodextrin and its derivatives

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Food Chemistry

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Characterization and antioxidant activity of the complexes of tertiary butylhydroquinone with β -cyclodextrin and its derivatives

Hongyu Pu, Qiaomei Sun, Peixiao Tang

^⁎

, Ludan Zhao, Qi Li, Yuanyuan Liu, Hui Li

School of Chemical Engineering, Sichuan University, Chengdu 610065, China

A R T I C L E I N F O

Chemical compounds studied in this article:

Tertiary butylhydroquinone (PubChem CID:

16043)

Beta-cyclodextrin (PubChem CID: 444041) Dimethyl-beta-cyclodextrin (PubChem CID:

122143)

Hydroxypropyl-beta-cyclodextrin (PubChem CID: 14049689)

1,1-Diphenyl-2-picrylhydrazyl (PubChem CID:

2735032)

Safranin O (PubChem CID: 2723800) Pyrogallol (PubChem CID: 1057)

2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (PubChem CID:

9570474)

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (PubChem CID: 64965).

Keywords:

Tertiary butylhydroquinone Cyclodextrin

Inclusion complex Antioxidant activity

A B S T R A C T

Tertiary butylhydroquinone (TBHQ) is a water-insoluble antioxidant. In this study, three cyclodextrin inclusion complexes were prepared to improve the water solubility of TBHQ and expand its range of application. Analysis of phase solubility indicated that TBHQ can form 1:1 inclusion complex with hydroxypropyl-beta-cyclodextrin (HP-β-CD) and dimethyl-beta-cyclodextrin (DM-β-CD) and 1:2 inclusion complex with beta-cyclodextrin (β-CD).

The possible inclusion configuration between TBHQ and CDs was determined through FT-IR, PXRD, DSC, NMR, and SEM analyses. Results were validated by theoretical study of AutoDock molecular docking. The scavenging effects of the inclusion complexes were not effective on DPPH radical but higher on hydroxyl, superoxide and ABTS+ radicals than that of TBHQ monomer. Moreover, the water solubility of TBHQ increased after com- plexation with HP-β-CD and DM-β-CD. The stability of TBHQ is related to the type of storage materials used, and the stability can be improved by complexation with CDs.

1. Introduction

Oxidation is one of the major causes of food spoilage and human aging; as such, study of antioxidants has become particularly important.

Free radicals are an atom or an atom that contains an unpaired electron when atoms or molecules lose or acquire an electron for some reasons.

Free radicals, such as hydroxyl (OH), superoxide (O²⁻), DPPH, nitrogen oxide (NOx), carboxyl (ROO), and lipid radicals, exhibit strong oxida- tion ability (Kabir et al., 2015). However, free radicals are harmful to human health because they damage cells and life macromolecules, in- cluding DNA, RNA, proteins, sugars, and lipids (Amarowicz, Pegg, Rahmi-Moghaddam, Barl & Weil, 2004). In this regard, scavenging free radicals has significance for extending food shelf life, delaying human aging, and maintaining good health. Therefore, the ability of anti- oxidants to scavenge free radicals must be assessed.

Common methods of scavenging free radicals include assays for

oxygen radical absorbance capacity (ORAC) (Sueishi et al., 2012), total radical trapping antioxidant parameter (TRAP) (Pellegrini et al., 2003), Trolox equivalence antioxidant capacity (TEAC) (Brizzolari, Marinello, Carini, Santaniello & Biondi, 2016), ferric ion reducing antioxidant power (FRAP) (Han, Yang, Yang, Feng & Yang, 2011), hydroxyl radical scavenging capacity (Morelli, Russo-Volpe, Bruno & Lo Scalzo, 2003), superoxide radical scavenging capacity (Li, Chen, Mai, Wen & Wang, 2012), and DPPH radical scavenging capacity (Sun & Ho, 2005). These methods can be classified as follows depending on the reactions in- volved: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET) (Huang, Ou, & Prior, 2005).

Encapsulation is extensively used in the food, cosmetics, and drug in- dustries to improve the water solubility and other characteristics of food additives (Gouin, 2004; Zeller, Saleeb, & Ludescher, 1999).

Scholars have also investigated the use of complexation with cyclo- dextrins (CDs) to increase the water solubility and free radical

https://doi.org/10.1016/j.foodchem.2018.04.008

Received 8 December 2017; Received in revised form 2 April 2018; Accepted 4 April 2018

⁎Corresponding author.

E-mail address:tangpeixiao@126.com(P. Tang).

Available online 05 April 2018

0308-8146/ © 2018 Elsevier Ltd. All rights reserved.

T

scavenging ability of food additives (Bangalore, Mcglynn & Scott, 2005;

Karathanos, Mourtzinos, Yannakopoulou & Andrikopoulos, 2007;

Martínez-Alonso, Losada-Barreiro, & Bravo-Díaz, 2015; Zhang et al., 2015; Li, Pu, et al., 2017). Cyclodextrin possesses a hydrophobic cavity and a hydrophilic outer surface, and the cyclodextrin cavity can provide binding sites for small molecules of all sizes to enhance the solubility of guest molecules (Brewster & Loftsson, 2007).

Tertiary butylhydroquinone [TBHQ, Fig. S1(A)] is phenolic anti- oxidant with high efficiency and stability and can be used to increase shelf life and improve food safety in oil-rich foods. TBHQ is mainly used in edible oils, fried baked goods, and meat products to prevent food from being oxidized (Kashanian & Dolatabadi, 2009). TBHQ is also used as an antioxidant in cosmetic products, such as lipsticks, perfumes, and skin care preparations (Shahabadi, Maghsudi, Kiani, & Pourfoulad, 2011). However, the application of TBHQ is limited because it is dif- ficult to dissolve in water. Hence, the present work mainly aims to prepare CDs/TBHQ inclusion complexes to improve the water solubility and expand the application range of TBHQ.

In this study, the inclusion complexes of TBHQ with three kinds of CDs (β-CD, DM-β-CD, and HP-β-CD) [Fig. S1(B)] were prepared by freeze drying. Thermodynamic parameters of the CDs/TBHQ system were determined via phase solubility studies. The inclusion complexes were characterized by FT-IR, DSC, XRD, NMR, and SEM analyses. The aqueous solubility of the formed CDs/TBHQ inclusion complexes was also investigated. The antioxidant capacity of TBHQ and the inclusion complexes was evaluated by measuring the clearance of DPPH, hy- droxyl, peroxygen and ABTS+ radicals. Finally, the stability of TBHQ inclusion complexes in different materials and aqueous solution was studied.

2. Materials and methods

2.1. Chemicals

TBHQ (FW = 166.22, purity≥99%) was purchased from Huaxia Reagent Co. Ltd. (Chengdu, China) and used without further purifica- tion.β-CD (FW = 1134.98,≥98%), HP-β-CD (FW = 1541.54,≥98%) and DM-β-CD (FW = 1331.39, ≥98%) were obtained from Sigma–Aldrich Chemical Company (Shanghai, China) and used without further purification. The other reagents and chemicals used were of analytical reagent grade and were acquired from Ke-Long Chemical Reagent Factory (Chengdu, China). Tri-distilled water was used throughout the experiment.

2.2. Preparation of inclusion complexes

TBHQ inclusion complex withβ-CD was prepared in molar ratio of 1:2, and TBHQ inclusion complexes with HP-β-CD and DM-β-CD were prepared in molar ratio of 1:1 through freeze drying. TBHQ and CDs were precisely weighed in molar ratio of 1:1 or 1:2 and completely dissolved in 40 mL of 10% ethanol–water (v/v) solution. After magnetic stirring at a controlled temperature (60 ± 1 °C for theβ-CD system and 40 ± 1 °C for the other systems) for 6 h. The resulting solution was filtered. Thefiltrate was dried in a vacuum freeze dryer, and the re- sulting solid complexes were collected.

TBHQ and CDs were precisely weighed according to the reaction molar ratio and completely mixed for 5 min by using a vortex mixer to obtain the physical mixture.

2.3. Testing of the properties of inclusion complexes

Phase solubility was studied based on the method of Higuchi and Connors (Higuchi & Connors, 1965). Excess amount of TBHQ (about 30 mg) was added to 10 mL of aqueous solutions ofβ-CD, HP-β-CD, and DM-β-CD with various concentrations (0 to 10.00 mM forβ-CD and 0 to 20.00 mM for HP-β-CD and DM-β-CD). After ultrasonic irradiation for

15 min, the mixtures were left to stand for 7 days at 37 °C. After reaching the equilibrium, each suspension was filtered through a 0.45μm microporous membrane. Thefiltrate was diluted with an ap- propriate amount of water, and TBHQ concentration in the solution was measured by UV–Vis spectrophotometer (TU-1901, Peking General In- strument, China) at 288 nm. The standard curve of TBHQ is Y = 0.01817X−0.00082∗∗, R²= 0.9999 (Fig. S2).

The strength of guest molecule complexation with CDs can be de- scribed by the stability constant (KC).KCcan be calculated by the slope and intercept of the straight line in the phase solubility diagram ac- cording to the Higuchi–Connors equation:

= −

Kc slope

intercept·(1 slope) (1)

The direction of the inclusion process can be described by Gibbs free energy (ΔG). Gibbs free energy at 310 K is calculated by Eq.(2):

= − = − G H T S TlnK

Δ Δ Δ R C (2)

where R is the universal gas constant (8.314 J·mol⁻¹·K⁻¹), and T (310 K) is the experimental operating temperature.

In brief, 20 mg of the inclusion complexes were dissolved in 10 mL of tri-distilled water. After proper dilution, TBHQ concentration in the solution was measured at 288 nm. The loading efficiency (LE) of TBHQ in the inclusion complexes was calculated as follows:

= ×

LE(%) Amount of TBHQ in inclusion complexes Total amount of inclusion complexes 100%

(3) Solubility was studied based on the balance method (Grant &

Higuchi, 1990). At 25 ± 2 °C, a certain amount of TBHQ or inclusion complexes was added to 3 mL of water. The mixture was shaken for 30 s every 5 min, and dissolution within 30 min was observed. If no visible solute particles were detected, then the solution was considered to be completely dissolved. After proper dilution, TBHQ concentration in the solution was measured at 288 nm. The solubility of β-CD/TBHQ in- clusion complex was lower than that at TBHQ at 25 °C but sensitive to temperature, that is, the solubility increased rapidly with increasing temperature. In subsequent free radical scavenging experiment, the temperature was increased to increase the solubility of β-CD/TBHQ inclusion complex and achieve the test concentration.

2.4. Characterization of the complexes

The three inclusion complexes, namely,β-CD/TBHQ IC, HP-β-CD/

TBHQ IC, and DM-β-CD/TBHQ IC, were characterized.

FT-IR spectra were recorded on a Nicolet 6700 spectrometer (Thermo Fisher Scientific, USA) at a scanning range of 4000–400 cm⁻¹. In brief, 1 mg of the sample was placed in 100 mg of KBr to form a tablet for testing.

PXRD patterns were obtained using X’Pert PRO (PANalytical, Almelo, Netherlands) diffractometer. Testing was performed using Cu Kα1 radiation under 2θangular range of 5°–50°, step size of 0.01313°, and counting time of 30 ms/step. The tube voltage and current were set as 40 kV and 40 mA, respectively. The slit and the divergence slit were 1/4° and 1/8°, respectively.

The DSC curves were analyzed by DSC 8500(PerkinElmer, USA).

Samples (2.5 ± 0.5 mg each) were placed in sealed aluminum pans and heated at 10 °C/min from 50 °C to 200 °C under a nitrogen atmo- sphere.

1H-NMR spectra of TBHQ, CDs, and the inclusion complexes (dis- solved in D2O) were recorded on a 400 MHz spectrometer (Bruker Avance, Germany) at 25 °C. The chemical shift value of D2O as internal reference was set to 4.70 ppm. Chemical shift difference (Δδ) was cal- culated by:

= −

δ δ δ

Δ (complex) (free) (4)

2D ROESY spectra were recorded on a 400 MHz spectrometer

(Bruker Avance, Germany) at 25 °C. The inclusion complexes were dissolved in D2O.

SEM images were collected by a QUANTA 250 scanning electron microscope (FEI, USA) at 5.0 kV. The samples were placed on a con- ductive plate, which was surface coated with gold before measurement.

2.5. Molecular modeling of inclusion complexes

The structure of TBHQ was rendered using ChemBioDraw Ultra software and optimized by ChemBio3D Ultra through MM2 molecular mechanics method. The structures of β-CD and DM-β-CD molecules were obtained from the Cambridge Structural Database (Ref. code:

BCDEX03 forβ-CD; Ref. Code: BOYFOK03 for DM-β-CD). The structure of HP-β-CD was established on the basis ofβ-CD. Molecular modeling of TBHQ with CDs was performed using the Lamarckian genetic algorithm by Auto Dock Tools 1.5.6 software.

2.6. Antioxidant activity studies

2.6.1. DPPH free radical scavenging activity

The 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) scavenging activity of CDs/TBHQ inclusion complexes was measured by bleaching the purple-colored ethanol solution of the stable DPPH radical ac- cording to Blois method (Sun & Ho, 2005; Prior, Wu & Schaich, 2005).

In brief, 2 mL of DPPH ethanol solution (0.165 mM) was added with different volumes of sample solution dissolved in 50% ethanol solution.

The solution was added with 50% ethanol solution to obtain a total volume of 3 mL. After completely mixing, the system responded for 30 min at 25 °C. Absorbance (Ai) was recorded at 517 nm. DPPH was then replaced by absolute ethanol. After 30 min, absorbance (Aj) was recorded at 517 nm. The sample solution was replaced by 50% ethanol solution. After 30 min, absorbance (A0) was recorded again at 517 nm.

In each experiment, three parallel measurements were performed. In addition, DPPH solution was freshly prepared. The capability to sca- venge the DPPH radical was calculated using the following equation:

= − − ×

RSA% [1 (A_i A_j)/A₀] 100% (5) where A0is the absorbance of the stable DPPH radical without the test compound, Ai is the absorbance of the remaining concentration of DPPH in the presence of the test compound, and Ajis the absorbance of the test compound.

2.6.2. OH free radical scavenging activity

The hydroxyl radical (%OH) scavenging activity was determined based on the Fenton (Haber–Weiss) reaction according toSchweikert method (2000). Fe²⁺was used as catalyst to produce%OH from H2O2. Fe²⁺was then oxidized to Fe³⁺by %OH according to the following equation (Morelli et al., 2003):

→ + +

+ + HO−

Fe H O² 2 2 Fe3 ·OH (6)

Hydroxyl free radicals produced by the reaction can lead to fading of Safranine O. The reaction system with afinal volume of 8 mL in- cluded 5 mL of Safranine O (0.001 mM), 1 mL of FeSO4·7H2O (1.5 mM), 1 mL of 3% H2O2 (freshly prepared), and different volumes of the sample solution dissolved in water. After complete mixing, the system responded for 40 min at 37 °C. Absorbance was recorded at 520 nm. The blank group used 1 mL of distilled water instead of the sample, and the control group used 2 mL of distilled water instead of the sample and FeSO4·7H2O solution. In each experiment, three parallel measurements were conducted. The capability to scavenge the HO%radical was cal- culated using the following equation:

= − − ×

RSA% (A_S A)/(A₀ A) 100% (7) where ASis the absorbance of the reaction system without the test compound, A is the absorbance of the reaction system in the presence of the test compound, and A0is the absorbance of the system without the

test compound and FeSO4·7H2O solution.

2.6.3. O²⁻free radical scavenging activity

Superoxide anion radical (O²⁻·) scavenging activity was tested based on pyrogallol autoxidation according to the method ofMarklund (1974). Pyrogallol will oxidize spontaneously under weak basic con- ditions and produce O²⁻with colored intermediates (Li et al., 2012).

The colored intermediates exhibit the maximum absorption at 320 nm.

Upon addition of the antioxidants, they will quickly react with O²⁻, thereby preventing the accumulation of intermediate products and re- ducing the absorption of the solution at 320 nm. In brief, 5 mL of 50 mM Tris-HCl buffer solution (pH 8.2) was added with different volumes of the sample solution dissolved in water. The solution was added with Tris-HCl buffer solution to reach a total volume of 5.5 mL. The solution was heat preserved for 10 min at 25 °C and added with the pyrogallol solution (25 mM) pretreated at 25 °C. After complete mixing, the system responded for 40 min. Absorbance (Ai) was recorded at 320 nm. The 25 °C preheated pyrogallol solution was replaced by the 25 °C preheated HCl solution (10 mM). After 40 min, absorbance (Aj) was recorded at 320 nm. The sample solution was replaced by Tris-HCl buffer solution.

After 40 min, absorbance (A0) was recorded at 320 nm. In each ex- periment, three parallel measurements were conducted. The capability to scavenge the O²⁻% radical was calculated using the following equation:

= − − ×

RSA% [1 (A_i A_j)/A₀] 100% (8) where A0is the absorbance of the colored intermediates, Aiis the ab- sorbance of the remaining concentration of the colored intermediates in the presence of test compound, and Ajis the absorbance of the test compound.

2.6.4. ABTS+ free radical scavenging activity

The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammo- nium salt free radical (ABTS+) scavenging activity of CDs/TBHQ in- clusion complexes was measured by bleaching the green-colored ethanol phosphate buffer solution of the ABTS+ radical according to Miller and Rice-Evans (1996). In brief, ABTS+ free radical was pre- pared by 54.2 mg of ABTS powder dissolved in 10 mL of phosphate buffer (5 mM, pH 7.0) and 1 g of MnO2at room temperature for 30 min (Dolatabadi, Mokhtarzadeh, Ghareghoran & Dehghan, 2014). Then the prepared solution was centrifuged at 4000 r/min for 5 min andfiltrated.

Thefiltrate was diluted with phosphate buffer until the absorbance of solution equals with 0.70 ± 0.01 at 734 nm. 2 mL of ABTS+ solution was added with different volumes of the sample solution dissolved in phosphate buffer. The solution was added with phosphate buffer to obtain a total volume of 3 mL. After completely mixing, the system responded for 10 min at room temperature. Absorbance (A) was re- corded at 734 nm. The sample solution was replaced by phosphate buffer. After 10 min, absorbance (A0) was recorded again at 734 nm. In each experiment, three parallel measurements were performed. The capability to scavenge the ABTS+ radical was calculated using the following equation:

= − ×

RSA% (A₀ A A)/ ₀ 100% (9)

where A0is the absorbance of the colored intermediates, A is the ab- sorbance of the remaining concentration of the colored intermediates in the presence of test compound.

2.7. Cytotoxicity assay

The cytotoxicity of TBHQ and inclusion complexes to LO2 cell line was determined by MTT assay (Tang et al., 2017). The LO2 cells were cultured in medium containing DMEM, 10% FBS and 1% penicillin- streptomycin with an atmosphere of 95% air and 5% CO2 at 37 °C.

Exponentially growing cells were cultured for 12 h in 96-well plates, with a seeding density of 4.0–5.0 × 10³cells per well. The resulting

cells were then exposed to 100μL cell culture medium with different concentrations of TBHQ or inclusion complexes aqueous solution for 48 h. The 96-well plates were centrifuged at 1000 r/min for 4 min and removed the supernatant carefully. The cells were washed with PBS buffer (0.01 M, pH 7.4) twice, and then 200μL MTT reagents (0.5 mg/

mL in PBS) were added to each well. After further incubation for 4 h, the medium in each well was removed and replaced with 100μL DMSO.

The plate was gently shaken for 10 min to dissolve the formazan crys- tals and the absorbance at 492 nm was obtained using a microplate reader (Molecular Devices, USA).

2.8. Stability studies

Pure TBHQ was placed in a PVC plastic bottle and a glass bottle. The three inclusion complexes were treated with the same method. All the samples and their packages were placed in a glassware dryer and lay aside for 6 months. The morphology of the sample was visually

observed. FT-IR spectra, PXRD patterns, and DSC curves were obtained through the same method.

The particle sizes and zeta potential of the TBHQ and its inclusion complexes in water solution were analyzed using a dynamic light scattering (DLS)-based Zeta PALS + BI-90Plus (Brookhaven Instrument Co., USA) at 20 ± 1 °C, with afixed scattering angle of 90°. Prepared aqueous solution with the same weight of TBHQ and inclusion com- plexes, and lay aside for a certain amount of time. The experiments were performed after 0, 2 and 4 weeks of preparation and three parallel measurements were performed in each experiment.

3. Results and discussion

3.1. Studies of the properties of inclusion complexes

The phase solubility diagrams of TBHQ with CDs were obtained according to Higuchi and Connors method (Higuchi & Kristiansen, Fig. 1.FT-IR spectra (A), PXRD patterns (B) and DSC curves (C) of (a) TBHQ, (b)β-CD, (c)β-CD/TBHQ Physical Mixture (PM), (d)β-CD/TBHQ IC, (e) HP-β-CD, (f) HP-β-CD/TBHQ PM, (g) HP-β-CD/TBHQ IC, (h) DM-β-CD, (i) DM-β-CD/TBHQ PM, (j) DM-β-CD/TBHQ IC and¹H NMR spectra (D) of (a) TBHQ, (b)β-CD, (c)β-CD/

TBHQ IC, (d) HP-β-CD, (e) HP-β-CD/TBHQ IC, (f) DM-β-CD, (g) DM-β-CD/TBHQ IC in D₂O.

1970). Fig. S3shows the phase solubility diagram of CDs/TBHQ in- clusion complexes in water at 37 °C. TBHQ withβ-CD can be classified as BS type, and the inclusion complex stoichiometric ratio between TBHQ andβ-CD was 2:1 (guest molecule: host molecule). TBHQ with HP-β-CD and DM-β-CD can be classified as ALtype, and the inclusion complex stoichiometric ratio was 1:1.KCandΔGwere calculated, and the corresponding values are presented in Table S1. The KC of the TBHQ-HP-β-CD system is higher than that of the TBHQ/DM-β-CD system, suggesting the TBHQ-HP-β-CD system is more stable. The va- lues ofΔG are negative, indicating that the inclusion process is spon- taneous.

The loading efficiency (LE) values of β-CD/TBHQ IC, HP-β-CD/

TBHQ IC, and DM-β-CD/TBHQ IC are 11.79% (RSD = 1.18%), 10.11%

(RSD = 1.01%), and 8.48% (RSD = 0.29%), respectively.

The solubility values of TBHQ,β-CD/TBHQ IC, HP-β-CD/TBHQ IC, and DM-β-CD/TBHQ IC at 25 °C are 4.02 (RSD = 1.33%), 2.18 (RSD = 1.18%), 114.13 (RSD = 2.06%), and 143.06 (RSD = 1.06%) mg/mL, respectively. After complexation with HP-β-CD and DM-β-CD, the water solubility of TBHQ increased obviously, and the magnifica- tion times are 28 and 35, respectively.

The main parameters of the properties of the inclusion complexes are listed inTable S1.

3.2. Characterization of the complexes 3.2.1. FT-IR analysis

The FT-IR results confirmed the formation of CDs/TBHQ inclusion complexes. After TBHQ formed inclusion complexes with CDs, the characteristic peaks of TBHQ may decrease, shift, or disappear. The content of TBHQ in the CDs inclusion complexes is low; as such, the shape of the inclusion complexes is similar to that of CDs. The FT-IR spectra of TBHQ, β-CD, HP-β-CD, and DM-β-CD and their physical mixtures and inclusion complexes are shown inFig. 1(A). After forming theβ-CD/TBHQ IC, the absorption peaks of TBHQ at 1611, 1590, and 1489 cm⁻¹ correspond to benzene skeleton vibration that shifted to nearby 1638 and 1505 cm⁻¹. The absorption peaks of TBHQ at 2962 cm⁻¹for CeH asymmetric stretching vibration, at 2869 cm⁻¹for CeH symmetric stretching vibration, and at 1185 cm⁻¹ for CeO stretching vibration were masked by β-CD. The absorption peaks of TBHQ at 1443 cm⁻¹for CeH asymmetrical bending vibration and at 1388 and 1364 cm⁻¹ for CeH symmetric bending vibration showed reduced intensity and shifted to 1448 and 1368 cm⁻¹, respectively.

After forming the HP-β-CD/TBHQ IC, the absorption peaks of TBHQ at 1611, 1590, and 1489 cm⁻¹correspond to benzene skeleton vibration that shifted to nearby 1646 and 1508 cm⁻¹. The absorption peaks of TBHQ at 2962 cm⁻¹ for CeH asymmetric stretching vibration, at 2869 cm⁻¹for CeH symmetric stretching vibration, at 1443 cm⁻¹for CeH asymmetrical bending vibration, at 1388 and 1364 cm⁻¹for CeH symmetric bending vibration, and at 1185 cm⁻¹ for CeO stretching vibration were masked by HP-β-CD. The same results were obtained in the DM-β-CD/TBHQ IC. FTIR spectra indicated that there may be in- teractions between host and guest molecules, and other methods could be performed to further verify the formation of inclusion complexes.

3.2.2. PXRD analysis

PXRD is an effective supplemental technology used to confirm the formation of CDs inclusion complexes (Williams, Mahaguna &

Sriwongjanya, 1998). The PXRD patterns of TBHQ,β-CD, HP-β-CD, and DM-β-CD and their physical mixtures and inclusion complexes are shown in Fig. 1(B). TBHQ presented many sharp characteristic dif- fraction peaks at angles of 7.6, 13.6, 15.2, 16.8, 17.7, and 22.9°, in- dicating that TBHQ exists as crystal.β-CD also exists as crystal, and HP- β-CD and DM-β-CD exist in amorphous state, as evident by the lack of diffraction peaks. The diffractograms of the physical mixtures were a simple superposition of the characteristic peaks of CDs and TBHQ.

However, in the diffractograms of the inclusion complexes, the

characteristic peaks of TBHQ disappeared. The diffractograms of theβ- CD/TBHQ IC presented new peaks at 6.9, 13.1, and 19.7°, whereas those of the HP-β-CD/TBHQ IC and DM-β-CD/TBHQ IC showed an amorphous or disordered state with lack of diffraction peaks. These results indicate the successful preparation of the inclusion complexes.

3.2.3. DSC analysis

DSC analytical technique can be used to study the physical state of guest molecules present in inclusion complex systems (Li, Zhang, et al., 2017). The DSC curves of CDs, free TBHQ, their physical mixtures, and their inclusion complexes are shown inFig. 1(C). TBHQ exhibited one sharp endothermic peak near 130 °C, corresponding to its melting point.

The DSC curves of the physical mixture were a simple superposition of TBHQ and CDs. Thisfinding suggests that TBHQ and CDs molecules were physically mixed only but did not interact with each other. In the DSC curves of the CDs/TBHQ inclusion complexes, the melting peak of TBHQ near 130 °C disappeared. This result could be due to the forma- tion of the inclusion complexes of TBHQ with CDs and the consequent alteration in thermal properties.

3.2.4. ¹H and 2D ROSEY NMR analyses

1H NMR spectra are the most direct and effective evidence for the formation of the inclusion complexes between CDs and guest molecules.

When the TBHQ molecules enter the cavity of the CDs, the micro- environment of the proton will change, leading to proton chemical shifts (Ge et al., 2011). However, the change in the chemical shifts is not evident because of the non-covalent bond between the host and guest molecules (Onnainty, Longhi, & Granero, 2011). Furthermore, when the guest molecule enters the cavity of CDs, the protons inside the CD cavities (H-3 and H-5) become more sensitive to environmental changes than those of the outside protons (H-1, H-2, and H-4) (Polyakov, Leshina, Konovalova, Hand & Kispert, 2004). The¹H-NMR spectra of pure CDs, pure TBHQ, and the inclusion complexes in D2O are presented in Fig. 1(D). The chemical shifts and changes in the complexes are listed inTable 1. The hydrogen on the tert-butyl of TBHQ in theβ-CD/TBHQ IC, HP-β-CD/TBHQ IC, and DM-β-CD/TBHQ IC ex- hibited significant downward chemical shift by 0.078, 0.131, and 0.148 ppm, respectively. The other hydrogen on the benzene ring of TBHQ in the inclusion complexes exhibited significant downward or upward shift because it entered different locations in the CD cavity.

Meanwhile, the hydrogen of CDs exhibited chemical shift changes, wherein the protons inside the CD cavities (H-3 and H-5) shifted up- ward. These phenomena indicate that TBHQ may have entered into the CD cavities.

The 2D ROESY NMR spectra can provide the spatial relation be- tween the H cores of the host and the guest atoms (Yang et al., 2009).

Fig. S5illustrates the ROESY spectra of theβ-CD/TBHQ IC, HP-β-CD/

TBHQ IC, and DM-β-CD/TBHQ IC in D2O.

After the TBHQ molecules entered the CD cavities, the hydrogen on the benzene ring was associated with the H-3 and H-5 of CDs, forming

Table 1

1H NMR chemical shifts (δ, ppm) and chemical shift differences.

Hydrogen H-3 H-5 ArC(CH3)3 H-3′ H-5′ H-6′

TBHQ δ – – 1.221 6.748 6.656 6.531

β-CD δ 3.870 3.759 – – – –

β-CD/TBHQ IC

δ 3.862 3.754 1.299 6.737 6.710 6.607

Δδ −0.008 −0.005 0.078 −0.011 0.054 0.076

HP-β-CD δ 3.928 3.771 – – – –

HP-β-CD/

TBHQ IC

δ 3.914 3.729 1.352 6.711 6.669 6.539

Δδ −0.014 −0.042 0.131 −0.037 0.013 0.008

DM-β-CD δ 3.904 3.838 – – – –

DM-β-CD/

TBHQ IC

δ 3.862 3.778 1.369 6.647 6.632 6.523

Δδ −0.038 −0.060 0.148 −0.101 −0.024 −0.008

cross peak. Thisfinding indicates the successful preparation of the CDs/

TBHQ inclusion complexes. Hence, the benzene ring of TBHQ was embedded into the CD cavities to form the complexes.

3.2.5. SEM analysis

SEM can be used as an auxiliary method to confirm the formation of inclusion complexes and determine their morphology before and after formation (Liu et al., 2013). The SEM images of the samples are dis- played inFig. 2. Pure TBHQ exhibits multilayer crystalline structures of different sizes. Pure β-CD exhibits different sizes of blocky crystal structures, HP-β-CD appears as spherical shape with cavity structures, and DM-β-CD possesses a globular cavity or cavity fragment structures.

The physical mixtures show the combination of TBHQ crystal structures and CD structures. Theβ-CD/TBHQ IC appears as acicular structure, and the HP-β-CD/TBHQ IC and DM-β-CD/TBHQ IC appear as irregular flaky structure. All these phenomena indicate that the shape and size of the CDs/TBHQ inclusion complexes were completely different from those of TBHQ and CDs. Hence, the inclusion complexes were suc- cessfully formed.

3.3. Molecular modeling of inclusion complexes

The possible conformations of the inclusion complexes were de- termined through molecular docking. Fig. 3 shows the molecular structures of the β-CD/TBHQ IC, HP-β-CD/TBHQ IC, and DM-β-CD/

TBHQ IC with the lowest energy in the docking results (binding energy was −4.65, −6.19, and −5.67 kcal, respectively). As shown in Fig. 3(A), the phenolic hydroxyl group of the two TBHQ molecules combined withβ-CD through three hydrogen bonds, with distances of 1.976, 1.995, and 1.904 Å, respectively. A hydrogen bond was also

formed between the two TBHQ molecules, and the distance of the hy- drogen bond was 2.151 Å. From the plain view of theβ-CD/TBHQ IC [Fig. 3(A-a)], TBHQ completely entered the interior of theβ-CD cavity.

TBHQ combined with HP-β-CD and DM-β-CD through two hydrogen bonds. The distances of the hydrogen bonds between the phenolic hy- droxyl group of TBHQ and HP-β-CD were 1.948 and 1.958 Å, and those between TBHQ and DM-β-CD were 1.955 and 1.749 Å. In addition, the lowest binding energy of the HP-β-CD/TBHQ IC indicates that it was the most stable among the three inclusion complexes. Thisfinding is consistent with the results of the phase solubility studies.

3.4. Antioxidant activity studies

3.4.1. DPPH free radical scavenging activity

The scavenging effects of the CDs/TBHQ inclusion complexes on DPPH radicals are shown inFig. 4(A). The scavenging effect of TBHQ on DPPH radicals could be related to the concentration of the TBHQ solution. The free radical scavenging rate increased with increasing concentration of the CDs/TBHQ inclusion complexes solution; however, with increasing TBHQ concentration, the increase in the DPPH free radical scavenging rate decreased and tended to stabilized. The scavenging effects of the CDs/TBHQ inclusion complexes remained si- milar to those of TBHQ. These phenomena indicate that the free radical scavenging properties of TBHQ were not eliminated after forming in- clusion complexes with CDs.

3.4.2. OH free radical scavenging activity

The scavenging effects of CDs/TBHQ inclusion complexes on OH free radicals are shown inFig. 4(B). The free radical scavenging rate increased with increasing concentration of TBHQ and the three Fig. 2.SEM images of (a) TBHQ, (b)β-CD, (c) HP-β-CD, (d) DM-β-CD, (e)β-CD/TBHQ Physical Mixture (PM), (f)β-CD/TBHQ IC, (g) HP-β-CD/TBHQ PM, (h) HP-β- CD/TBHQ IC, (i) DM-β-CD/TBHQ PM, (j) DM-β-CD/TBHQ IC [(j) (200×), others (500×)].

inclusion complexes. The scavenging rate of the HP-β-CD/TBHQ IC increased linearly within the test concentration range. With increasing concentration of the TBHQ, the scavenging rate of theβ-CD/TBHQ IC initially increased faster than that of the HP-β-CD/TBHQ IC, but the growth rate slowed down later. Thereafter, the scavenging rate of theβ- CD/TBHQ IC was similar to that of the HP-β-CD/TBHQ IC. With in- creasing concentration of the DM-β-CD/TBHQ IC, the OH free radical scavenging rate increased rapidly at first, then increased slowly and tended to be stable. The scavenging rate of the DM-β-CD/TBHQ IC under stable condition was the same with that of TBHQ. The scavenging rate of the inclusion complexes was higher than that of TBHQ. Overall, the hydroxyl radical scavenging activities were as follows:β-CD/TBHQ IC > HP-β-CD/TBHQ IC > DM-β-CD/TBHQ IC > TBHQ. When the concentration of TBHQ was 0.015 mM, the scavenging rate of theβ-CD/

TBHQ IC and HP-β-CD/TBHQ IC was almost twice higher than that of TBHQ and the DM-β-CD/TBHQ IC. These phenomena may be due to the free radical scavenging activity of CDs, whose molecule cavity allows

the effective stabilization of radicals (Jullian et al., 2008). The forma- tion of hydrogen bonds between the hydroxyl groups of CDs and the phenolic hydroxyl groups of the TBHQ molecule contributed to anti- oxidant activity (Stražišar, Andrenšek &Šmidovnik, 2008).

3.4.3. O²⁻free radical scavenging activity

The scavenging effects of the CDs/TBHQ inclusion complexes on O²⁻free radicals are shown inFig. 4(C). The free radical scavenging rate of TBHQ appeared waved with increasing concentration and maintained at around 10%. The DM-β-CD/TBHQ IC showed a similar phenomenon, and the scavenging rate was maintained at around 20%, which is almost twice higher than that of TBHQ. These phenomena indicate the lack of dose effect relationship between the scavenging capacity and their concentrations. The ability to scavenge free radicals reached saturation. The scavenging rate of theβ-CD/TBHQ IC and HP- β-CD/TBHQ IC increased linearly with increasing concentration of the CDs/TBHQ inclusion complexes solution, indicating the presence of a Fig. 3.Molecular docking of the lowest docking energy conformation and energy distribution diagram of TBHQ withβ-CD (A); TBHQ with HP-β-CD (B); and TBHQ with DM-β-CD (C).

dose effect relationship between the scavenging capacity and their concentrations. Overall, the O²⁻radical scavenging activities were as follows: β-CD/TBHQ IC > HP-β-CD/TBHQ IC > DM-β-CD/TBHQ IC > TBHQ. In addition to the influence of CDs, the causes of different scavenging effects may be due to different formation mechanisms of CD inclusion complexes.

3.4.4. ABTS+ free radical scavenging activity

The scavenging effects of the CDs/TBHQ inclusion complexes on ABTS+ free radicals are shown inFig. 4(D). Both TBHQ and its in- clusion complexes can effectively scavenge ABTS+ radical. Besides, the scavenging effect of TBHQ on ABTS+ radicals could be related to the concentration of the TBHQ solution and the change trend of clearance rate is consistent with that of scavenging DPPH radical. At low Fig. 4.Scavenging effects of TBHQ and CDs/TBHQ IC on DPPH radical (A), OH free radical (B), O²⁻free radical (C) and ABTS+ free radical (D), and Proliferation inhibition of free TBHQ and CDs/TBHQ IC on LO2 (E).

concentration, the scavenging activities of inclusion complexes are higher than that of pure TBHQ. When the concentration of TBHQ was 0.006 mM, the scavenging rate of the β-CD/TBHQ IC and DM-β-CD/

TBHQ IC was almost twice higher than that of TBHQ. When the con- centration of TBHQ is greater than 0.3 mM, the efficiency of TBHQ and its inclusion complexes to scavenging ABTS+ is close to 100%. Gen- erally, the ABTS+ radical scavenging capability was arranged as fol- lows: DM-β-CD/TBHQ IC >β-CD/TBHQ IC > HP-β-CD/TBHQ IC >

TBHQ.

CDs could act as a reservoir of TBHQ, acting as a controlled release system, maintaining in solution a constant concentration of antioxidant.

In this sense, minor concentrations would be necessary in complex form to achieve the same effect that in free form. The scavenging effects of the three inclusion complexes on OH free radicals, O²⁻free radicals and ABTS+ free radicals significantly improved. The scavenging effects on DPPH free radicals were not significant. For OH free radicals and O²⁻free radicals, the scavenging effects ofβ-CD/TBHQ IC and HP-β- CD/TBHQ IC were similar, and both showed higher scavenging effects than those of DM-β-CD/TBHQ IC and TBHQ. For ABTS+ free radicals, inclusion complexes showed higher scavenging effects than TBHQ, especially DM-β-CD/TBHQ IC andβ-CD/TBHQ.

3.5. Cytotoxicity assay

In the published literature, it was pointed out that there was cyto- toxicity and DNA damage properties in TBHQ (Eskandani, Hamishehkar

& Dolatabadi, 2014). The cytotoxicity of TBHQ and the inclusion complexes at various concentrations was investigated. The influence of

free TBHQ and inclusion complexes on LO2 cell line is displayed in Fig. 4(E). DM-β-CD/TBHQ IC has lower cytotoxicity when the con- centration of TBHQ is greater than 0.1 mM. In addition, HP-β-CD/

TBHQ IC andβ-CD/TBHQ IC shows a lower inhibition compared to free TBHQ at various concentrations. Especially at the concentration 0.05 mM, the viability of LO2 cell line in the presence of HP-β-CD/

TBHQ IC is about 1.5 times higher than that of TBHQ. From the general trend, the cytotoxicity of the inclusion complexes was lower than that of TBHQ.

3.6. Stability studies

TBHQ metamorphosed in the plastic bottle under normal environ- mental conditions after 6 months; in this regard, the stability of TBHQ and its inclusion complexes was investigated. The TBHQ placed in the glass bottle retained its original morphology [Fig. 5(A)], whereas TBHQ placed in the plastic bottle formed a black substance after 6 months [Fig. 5(B)]. The substance was mainly produced along the inner wall of the plastic bottle. However, inclusion complexes remained unchanged both in plastic and glass bottles.

We characterized the black substance through FT-IR, PXRD, and DSC analyses. The FT-IR spectrum [Fig. 5(C-b)] show the absorption peak of TBHQ at 1611 cm⁻¹, which corresponds to the disappearance of benzene skeleton vibration, and a new peak at 1635 cm⁻¹. As shown in the PXRD pattern [Fig. 5(D-b)], a new sharp diffraction peak appears at angle of 8.6°, indicating the formation of a new substance or crystal.

The DSC curve [Fig. 5(E-b)] presents an endothermic peak at about 130 °C (melting peak of TBHQ) and a new endothermic peak at about Fig. 5.TBHQ placed in the glass bottle for 6 months (A) and TBHQ placed in plastic bottle for 6 months (B); FT-IR spectra (C), PXRD patterns (D), and DSC curves (E) of (a) TBHQ, (b) TBHQ (metamorphic), (c)β-CD/TBHQ IC, (d) HP-β-CD/TBHQ IC, and (e) DM-β-CD/TBHQ IC [(b), (c), (d), and (e) were the spectra of the samples placed in the plastic bottle after 6 months].

100 °C. All these phenomena indicate that TBHQ was not stable in the plastic bottle and produced a new substance after 6 months. However, the black substance must be identified and investigated.

The FT-IR spectra, PXRD patterns, and DSC curves of the inclusion complexes remained invariable after incubation in the plastic bottle for 6 months, indicating the stability of the complexes.

To investigate the stabilities of TBHQ and inclusion in aqueous so- lution, we evaluated the particular size and zeta potential after 0, 2 and 4 weeks of preparation. The DLS images are shown inFig. S6and the effective diameter and zeta potential are presented inTable S2. With the prolongation of the placement time, the particle size of TBHQ changed from 112.2 nm to 150.7 nm, and the zeta potential changed from−11.4 mV to−1.5 mV, indicating that TBHQ is not very stable in aqueous solution. The particular size of inclusion complexes is smaller than that of TBHQ, due to the superior solubility of inclusion com- plexes. The zeta potential of inclusion complexes basically remained unchanged after 4 weeks, which demonstrates that the inclusion com- plexes are more stable.

All the experimental data show that the stability of TBHQ improved after complexation with CDs.

4. Conclusions

This work reveals that complexation with CDs can be used as an effective strategy to broaden the application of TBHQ and provide a reference value for research and development of water-soluble anti- oxidants. The water solubility of TBHQ improved after complexation with HP-β-CD and DM-β-CD. The antioxidant activity in the aqueous solution of TBHQ increased in different degrees after complexation with the three kinds of CDs. The effects of the three inclusion complexes on OH, O²⁻and ABTS+ free radical scavenging activity were significantly improved. However, the scavenging effects of the inclusion complexes on DPPH free radicals were not evident. Besides, the stability of TBHQ in PVC plastic materials or aqueous solution improved and cytotoxicity reduced by forming inclusion complexes.

Acknowledgments

This work was supported by the Applied Basic Research Program of Science & Technology Department of Sichuan Province, China (Grant No. 18YYJC0705) and Large-scale Science Instrument Shareable Platform Construction of Sichuan Province (Grant No. 2015JCPT0005- 15010102) and the Large-scientific Instruments Sharing Platform Ability Construction of Sichuan Province (Grant No. 2016KJTS0037).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.foodchem.2018.04.008.

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