Quercetagetin-Loaded Composite Nanoparticles Based on Zein and

Jun 13, 2018 - (13,14) Moreover, the composite nanoparticles can provide a better protecting ... Hyaluronic acid (99%, molecular weight 1.0 × 105) wa...
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Food and Beverage Chemistry/Biochemistry

Quercetagetin-loaded composite nanoparticles based on zein and hyaluronic acid: formation, characterization and physicochemical stability Shuai Chen, Cuixia Sun, Yingqi Wang, Yahong Han, Lei Dai, Arzigül Abliz, and Yanxiang Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01046 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Quercetagetin-loaded composite nanoparticles based on zein and

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hyaluronic acid: formation, characterization and physicochemical

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stability

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Shuai Chen, Cuixia Sun, Yingqi Wang, Yahong Han, Lei Dai, Arzigül Abliz,

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Yanxiang Gao*

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Beijing Advanced Innovation Center for Food Nutrition and Human Health,

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Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of

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Functional Food from Plant Resources, College of Food Science & Nutritional

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Engineering, China Agricultural University, 100083, China

11 12 13 14 15 16

*Corresponding author.

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Tel.: + 86-10-62737034

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Fax: + 86-10-62737986

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Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,

20

China

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E-mail: [email protected]

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Abstract

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Zein and hyaluronic acid (HA) composite nanoparticles were self-assembly

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fabricated using anti-solvent co-precipitation (ASCP) method to deliver quercetagetin

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(Que). FTIR, CD and FS results revealed that electrostatic attraction, hydrogen

26

bonding, and hydrophobic effect were the dominant driving forces among zein, Que,

27

and HA. With the increasing of HA level, the morphological structure of

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zein-Que-HA complex was changed from nanoparticle (from 100: 5: 5 to 100: 5: 20)

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to microgel (from 100: 5: 25 to 100: 5: 30). The encapsulation efficiency of Que has

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significantly increased from 55.66% (zein-Que, 100:5) to 93.22% (zein-Que-HA, 100:

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5: 20), and Que in the zein-Que-HA composite nanoparticles exhibited obviously

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enhanced photochemical, thermal and physical stability. After 8 months of storage

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(4°C), the retention rate of Que also up to 77.93%. These findings interpreted that

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zein-HA composite nanoparticle would be an efficient delivery system for

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encapsulating and protecting bioactive compounds.

36 37

Keywords: Quercetagetin; Zein; Hyaluronic acid; Composite nanoparticles;

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Interaction.

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INTRODUCTION Quercetagetin (Que) is a kind of alcohol soluble bioactive substance, with many hydroxyl

groups.1

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phenolic

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anti-inflammatory capacities,2-3 and a potential in the prevention of tumor,

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cardiovascular disease and other chronic diseases. 4-5 Thus, Que can be applied to

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health food, however, the application of Que was limited owing to its weak chemical

49

stability, poor water solubility, and low oral bioavailability.

It

has

excellent

antioxidant,

antimicrobial,

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To overcome the aforementioned disadvantages, many delivery systems have

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been designed, e.g. hydrogel, liposome, micelle, and particle.6 Compared to other

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delivery systems, nanoparticles have unique advantages, e.g. small mean size

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(10-1000 nm), high encapsulation efficiency, slow degradation rate, effective

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targetability and penetration ability.6 Food-grade nanoparticles are usually prepared

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from protein and polysaccharide, which are non-toxic, biodegradable, and

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biocompatible,7 such as soy protein,8 lactoferrin,9 gelatin,10 chitosan,11 and alginate.12

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Zein is a vegetable protein with much hydrophobic amino acids (over 50%).12 It

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is easy to form nanoparticles by the anti-solvent precipitation (ASP) method for

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delivering polyphenols.7 However, zein nanoparticle is unstable when confronted with

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certain pH, high salt concentration, and thermal processing. In order to enhance the

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stability of zein nanoparticle, some food biopolymers have been used to coat its

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sufurce.13-14 Moreover, the composite nanoparticles can provide a better protecting

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function and higher encapsulation efficiency (EE) for bioactive components.14-15 Such

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as curcumin,16 resveratrol17, and epigallocatechin gallate.18 Recently, the investigation

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into delivery system based on the complexation between protein and polysaccharide

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has been becoming a research hotspot.

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Hyaluronic acid (HA) is a linear polysaccharide,19 which is naturally found in

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human bodies with important biological and physicochemical functions,20 such as

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anti-aging,

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angiogenesis, and embryonic development.19-20 Due to the rapid turnover (about 1/3

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of HA is degraded and regenerated daily) in human body,21 oral intake of HA can

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replenish the raw material of HA needed for metabolism. Based on the particular

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physicochemical properties and functional attributes, HA has been widely used as a

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raw material for the production of soft-tissue filler, anti-aging matrices, and drug

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delivery vehicles. 21 Previous researchers mainly concentrated on the application of

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HA in medicine through applying ointment or injection.22 Some drug or gene delivery

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systems were fabricated based on HA, such as HA-chitosan nanoparticles,23-24

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HA-silk hydrogels,21 HA-curcumin nanogels.25 However, the investigations into HA

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as a matrix to design food-grade delivery systems for oral intake are very limited.

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Little information focused on interaction among HA, protein, and polyphenols was

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found in previous literatures.

cell-matrix

interactions,

anti-inflammation,

immunoregulation,

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In the present work, the zein-Que-HA composite nanoparticles were fabricated

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using a new and simple (anti-solvent co-precipitation, ASCP) method, which was ease

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of operation and required a small amount of HA. The influence of the proportion of

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zein, Que, and HA on the physicochemical property and microstructure of

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zein-Que-HA was investigated. The physical, photochemical, thermal stability, as

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well as a long-term storage stability of Que in zein-HA composite nanoparticles were

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evaluated. The findings from present work would have a potential application in the

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field of nutraceutical delivery system.

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MATERIALS AND METHODS

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Materials. Zein (95%) was obtained from Gaoyou Co. Ltd. (Jiangsu, China).

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Hyaluronic acid (99%, molecular weight 1.0×105) was purchased from Xi’an

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Baichuan Co. Ltd. (Xi’an, China). Ethanol (99.9%) was purchased from Beijing

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chemical Co., Ltd (Beijing, China). Quercetagetin (95%, w/w) was prepared from

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marigold (Tagetes erectaL.) using the procedure described in our previous report.26

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All other chemicals were analytical grade unless stated otherwise.

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Preparation of zein-Que-HA composite nanoparticles. Zein-Que-HA composite

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nanoparticles were prepared using the ASCP method. Briefly, Different quantities (50,

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100, 150, 200, 250, and 300 mg) of HA were dissolved in distilled water (30 mL), and

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then the solutions were diluted with 70 mL of ethanol. Zein (1.0 g) and Que (50 mg)

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were added to 100 mL HA aqueous ethanol solution (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0

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mg/mL). After ultrasonic treatment for 20min, the final mixed solution (40 mL) was

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injected into the 120 mL distilled water in 2 min, and stirred for 20 min at 600 rpm.

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To obtain zein-Que-HA aqueous dispersions, ethanol was removed using rotary

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evaporation (45°C, -0.1 MPa). Zein, zein-Que, and Que-HA aqueous dispersions were

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prepared by the aforementioned method as controls. Part of the colloidal dispersions

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were stored at 4°C, and others were freeze-dried (-50°C, 48 h) for solid-state analysis.

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In the study, the different mass ratios of zein-Que-HA complexes at 100:5:5,

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100:5:10, 100:5:15, 100:5:20, 100:5:25 and 100:5:30 were termed as zein:Que:HA

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(100:5:5),

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(100:5:20) , zein:Que:HA (100:5:25) and zein:Que:HA (100:5:30), respectively.

zein:Que:HA

(100:5:10),

zein:Que:HA

(100:5:15),

zein:Que:HA

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Characterization of zein-Que-HA composite nanoparticles

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Particle size, zeta-potential, entrapment efficiency (EE) and loading capacity

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(LC). The particle size (nm) and zeta-potential (mV) of zein-Que-HA composite

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nanoparticles were analyzed according to our previous report.27 Three parallel samples

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were performed, and result was calculated as cumulative mean value. The EE and LC

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of zein-Que-HA composite nanoparticles were analyzed using our reported method.28

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They were centrifuged at 15000 g for 30 min (25 °C). The supernatant liquor was

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sucked out and diluted using ethanol aqueous solution (70%, v/v), and determined

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using a UV-1800 spectrophotometer at 360 nm. The EE and LC were calculated by

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the following equations:

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EE =

Total Que - Free Que × 100% ………………..………(1) Total Que

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LC =

Total Que − Free Que × 100% ………..… (2) Total mass of composite particle

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Field

emission

scanning

electron

microscopy

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(FE-SEM)

and

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Fourier-transform infrared (FTIR) spectroscopy. The microstructure of the

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freeze-dried zein-Que-HA composite nanoparticles was observed by the field

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emission scanning electron microscope (FE-SEM, SU8010, Hitachi). The surfaces of

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samples were sputter-coated with a gold layer, and the accelerating voltage was 5.0

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kV. The Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, U.K.) and

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the potassium bromide (KBr) pellet method were used in the work. 29 11 scans were

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taken in the range of 400-4000 cm-1, and the resolution was 4 cm-1. The infrared

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spectra of zein, zein-Que, HA-Que, and zein-Que-HA composite nanoparticles were

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analyzed using Omnic v8.0 (Thermo Nicolet, USA).

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Fluorescence spectroscopy (FS), circular dichroism (CD) and differential

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scanning calorimetry (DSC) analysis. The intrinsic fluorescence of zein-Que-HA

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composite nanoparticles was measured at 0.25 mg/mL of zein with Que (12.5 µg/mL)

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and HA at different levels. The spectral region was 290-450 nm, the scanning speed

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was 100 nm/min, excitation wavelength at 280 nm, both excitation and emission slit

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widths were set at 10 nm. CD measurement conditions: far-UV region (190-260 nm),

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path length was 0.1 cm. recorded speed was 100 nm/min, 2.0 nm band width, 20

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accumulations, and 0.2 nm resolution. The contents of secondary structure of

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zein-Que-HA

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http://dichroweb.cryst.bbk.ac.uk, which was an online Circular Dichroism Website.31

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The thermal characteristics of zein-Que-HA composite nanoparticles were analyzed

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using DSC. Briefly, the freeze-dried sample (3.0 mg) was placed into an aluminium

composite

nanoparticles

were

analyzed

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Dichroweb:

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pan and hermetically sealed. DSC measurement conditions: temperature range was

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30-150 oC, temperature rate was 10 oC/min. The thermal curve were collected by the

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DSC-60 workstation.

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Stability evaluation

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Physical stability. The physical stability of the zein-Que-HA colloidal dispersions

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were measured using the LUMiSizer (LUM, Germany) according to our previous

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reported.32 The measurement parameters were as follows: sample amount, 1.8 mL;

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rotational speed, 4000 rpm; time interval, 30 s; temperature, 25°C.

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Photochemical stability. The free Que, zein-Que, HA-Que, and zein-Que-HA

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colloidal dispersions were placed into a light cabinet. The light condition was 0.68

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W/m2 and 45°C. 32 The sampling was carried out at time points of 0, 2, 4, 6, 8 and 10

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h. In general, the degradation of flavonoids followed the first order kinetics.33 The

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half-life (t1/2) and degradation rate constant (k) were calculated using the following

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equations:

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Ln (C / C 0 ) = − kt ……………..(3)

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t1/ 2 =

Ln2 ……………………..(4) k

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Where C0 represents the concentration of Que at the initial and C represents the

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concentration of Que at time t (h).

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Thermal stability. According to the method of Liu et al., 34 50 mL samples of free

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Que, zein-Que, HA-Que, and zein-Que-HA colloidal dispersions were placed into test

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tubes and incubated in a water-bath at 60, 70 and 80 °C for 10 h. After that, the

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samples were cooled in ice water. The Que in the samples were quantified according

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to the aforementioned method.

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Storage stability. The storage stability of zein-Que-HA colloidal dispersions was

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performed on a refrigerated condition (4 °C) for 8 months. Particle size of composite

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colloidal nanoparticles was detected by the method as aforementioned above, and the

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retention rate of Que after storage was estimated by the following equation:

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Retention rate (%) =

the amount of Que after storge the initial amount of Que

×100%............(5)

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Statistical analysis. The parallel experiments were performed at least in three times.

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Results were expressed as mean ± SD. Data were analyzed by ANOVA, and p < 0.05

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was regarded as significant.

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RESULTS AND DISCUSSION

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Particle size, zeta-potential, entrapment efficiency (EE), and loading capacity

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(LC). The photographs of samples were shown in Fig. 1a. It's clearly observed that

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hyaluronic acid (HA) aqueous solution, Que in 70% ethanol aqueous solution and

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HA-Que aqueous solution were clear and transparent. However, zein-Que and

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zein-Que-HA were homogeneous colloidal dispersions. The mean size and

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zeta-potential were given in Fig. 1b and c. The zein-Que nanoparticles had a smaller

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particle size than that of zein nanoparticles. It could be explained by the fact that

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polyphenols tend to intimate connection with proline-rich zein, and form a small and

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compact structure.35 When HA was added, the particle size was significantly (p < 0.05)

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increased from 89.54 nm (zein-Que) to 315.93 nm (zein-Que-HA), and the

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zeta-potential was changed from 29.43 mV (zein-Que) to -16.47 mV (zein-Que-HA).

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The surface charge of native zein nanoparticle was positive (31.97 mV), while native

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HA was negative (-49.30 mV). In general, the charge attribute of composite

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nanoparticle was dominated by the charge characteristics of the outer layer

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biopolymer.36 It indicated that the negatively charged HA coated on the surface of

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zein nanoparticles. Similarly, Patel et al.13 had reported that positively charged zein

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nanoparticles was covered by the negatively charged sodium caseinate. On the one

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hand, the addition of HA resulted in the increased volume of zein-Que-HA spherical

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nanoparticle; On the other hand, the decrease of zeta-potential resulted in the reduced

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electrostatic repulsion, the aggregation of zein-Que-HA composite nanoparticles

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might be formed. Therefore, zein-Que-HA composite nanoparticles had a larger

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particle size than zein-Que nanoparticles.

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Continuously increasing the concentration of HA from 100:5:5 to 100:5:15

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(zein:Que:HA), both zeta-potential and the particle size were decreased. It could be

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explained by the fact that the increasing negative charge led to the increased

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electrostatic repulsion, which could prevent the aggregation of zein-Que-HA

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composite nanoparticles. However, it was interesting to find that with the mass ratio

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(zein: Que: HA) increasing from 100:5:20 to 100:5:25, the particle size was obvious

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increased from 233.73 nm to 398.9 nm. It might be caused by the crosslinking of

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excessive HA.37 A similar observation was reported that high level of shellac could

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increase the size of zein-shellac composite nanoparticles through intermolecular

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cross-linking.29

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The effects of different formulations on EE and LC were presented in Table 1. All

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the zein-Que-HA composite nanoparticles exhibited significantly (P < 0.05) higher

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EE values than that of zein-Que nanoparticles. With the mass ratio of zein: Que: HA

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increased from 100:5:5 to 100:5:20, there was a gradual increase in the EE and LC.

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Particularly, the highest EE and LC values of Que in zein-Que-HA (100:5:20)

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composite nanoparticles reached up to 93.22% and 3.85%, respectively. The result

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may be due to the long molecular chain and high molecular weight of HA (1.0×105

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Da), which was 4.5 times than zein (2.2×104 Da). Liang et al.15 reported that chitosan

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derivative could entrap more curcumin and lead to a high EE ascribing to its longer

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chain of molecule structure. They were consistent with our previous study focused on

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the Que-loaded zein-propylene glycol alginate complexes.28

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Overall, the negatively charged HA could be combined with positively charged

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zein, and formed a compact-structure nanoparticle (zein:Que:HA=100:5:20) with

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strong ability of encapsulation. These results revealed that HA and zein had a

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synergistic effect on improving the EE and LC of Que, and zein-HA binary composite

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nanoparticles could be an ideal carrier for functional component.

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Field emission scanning electron microscopy (FE-SEM) analysis. The

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morphological images of samples were shown in Fig. 2. Zein nanoparticles (Fig. 2a)

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were typically spherical, which was the same as reported in previous study.30 HA (Fig.

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2b) exhibited a fine dendritic network structure. When zein and HA were combined

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together to encapsulate Que, the morphology of zein-Que-HA composite

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nanoparticles were also spherical, but their surface was coarse (Fig. 2c, d, e, f). The

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result could be explained by that HA covered and convolved on the zein nanoparticles

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by electrostatic attraction, and Que was embedded in zein nanoparticles by

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hydrophobic interaction.38

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As shown in Fig. 2g and h, it was interesting to find that the crosslinking occurred

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among excessive HA. Zein-Que-HA complex presented a 3D network-like structure,

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and seemed to be clusters of grapes. The result demonstrated that the increasing

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concentration of HA led to the zein-Que-HA complex change from spherical particle

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to network-like microgel. It was similar to that propylene glycol alginate-human

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serum albumin complex presented a vesicular structure or a lacework-like structure

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depending on the polymer concentrations. 39

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Fourier-transform infrared (FTIR) spectroscopy. Zein nanoparticles showed a

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major band at 3313.40 cm-1 (Fig. 3a), which was ascribed to the stretching vibration of

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hydroxy groups. The band at 1659.81 cm-1 represented the C-O stretching (amide I)

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and 1533.51 cm-1 was related to the C-N stretching coupled with N-H bending modes

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(amide II).30 Compared with individual zein, the peaks of amide I and amide II of

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zein-Que were changed to 1658.88 cm-1 and 1534.63 cm-1, respectively, and their

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peak intensity was increased. The findings demonstrated that hydrophobic interaction

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might exist between zein and Que, which was ascribed to the high percentage of

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hydrophobic amino acids (over 50%) of zein and aromatic rings of Que.40 In addition,

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the presence of Que induced significant shift of –OH groups to 3310.12 cm-1, which

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revealed that the hydrogen bonding occurred between zein and Que. When HA was

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incorporated, the peak of hydrogen bond (–OH) was shifted to 3304.11 cm-1 (zein:

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Que: HA=100:5:5). These findings suggested that the intermolecular forces between

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zein and HA included not only electrostatic attraction due to their opposite charges,

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but also hydrogen bonding. Similarly, Luo et al.14 had reported that hydrogen bonding

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was one of the dominant driving forces in the formation of zein-carboxymethyl

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chitosan-vitamin D3 nanoparticle. Compared the low level (zein: Que: HA=100:5:5)

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with high level (zein: Que: HA=100: 5: 25) of HA, the peaks of amide II was changed

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from 1536.12 cm-1 to 1541.10 cm-1, the peaks of C–O stretching of –COO− was

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changed from 1451.17 cm-1 to 1450.76 cm-1, and the peaks of –OH groups was

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changed from 3304.11 cm-1 to 3311.49 cm-1. When HA was at a high level, the

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hydrogen bonding formed among the –COOH, –NHCOCH3 and –OH groups on the

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backbone of HA molecules.37 The hydrogen bonding resulted in the formation of a 3D

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network-like structure microgel, whose microtopography was confirmed by FE-SEM

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(Fig. 2g and h).

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Fluorescence spectra analysis. Fig. 3b showed that the fluorescence emission

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peak of zein nanoparticles was at 304 nm,27 and the presence of Que caused the

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fluorescence quenching. It might be attributed to multiple intermolecular interactions

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such as energy transfer, rearrangement, collisional quenching, and ground state

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complex formation.41 The incorporation of HA into zein-Que (zein: Que: HA, 100:5:5

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and 100: 5: 10) resulted in the decreased intensity of fluorescence. The result might be

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explained by the fact that a slight precipitation phenomenon occurred when zein was

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combined with the low concentration of HA, and total quantity of zein-Que-HA

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colloidal nanoparticles decreased, which led to the decrease in the fluorescence

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intensity. However, when the concentration of HA was increased (zein:Que:HA,

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100:5:15 and 100:5:20), the zein-Que-HA colloidal dispersions were stable, and the

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fluorescence intensity of zein-Que-HA colloidal nanoparticles were increased. It

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could be explained by that HA was combined with hydrophilic groups of zein, and

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induced the tryptophan residues exposed, which were originally inside the zein.36

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With the increase of HA concentration, excessive HA might wrap around zein-Que

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nanoparticles, therefore, the fluorescence intensity of zein-Que-HA (100:5:25 and

283

100:5:30) was slightly less than that of zein-Que-HA (100:5:15 and 100:5:20).

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Circular dichroism (CD) analysis. Fig. 3c showed that zein had a positive peak

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(195 nm), 2 negative peaks (208 and 224 nm), and a zero-crossing (203 nm). It was

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attributed to the high content of α-helix.42 When zein was combined with HA, the

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secondary structure was significant changed, which could be reflected by the changes

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in the ellipticities of spectral curve at both 208 and 224 nm. The interaction between

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the protein and polymer made the alteration in secondary structure of the protein was

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a common phenomenon.43-44

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The DICHROWEB procedure (SELCON3) was performed to calculate the

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percentages of α-helix, β-sheet, β-turn and unordered coil, 31 and the data were shown

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in supporting information Table 1. The secondary structure of zein contained 49.7%

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α-helix, 8.8% β-sheet, 16.8% β-turn and 25.6% unordered coil, respectively. However,

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when Que was incorporated, the percentage of α-helix was significantly (p < 0.05)

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reduced from 49.7% (zein) to 35.7% (zein-Que). At the same time, the percentages of

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β-sheet and unordered coil were significantly (p < 0.05) increased from 8.8% and 25.6%

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to 10.5% and 32.1%, respectively. The finding might be attributed to the hydrophobic

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interaction between Que and zein, which caused the change of zein structure.40 When

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HA was added (zein: Que: HA=100:5:5), the α-helix of zein was obviously decreased

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from 35.7% to 9.0%, but β-sheet increased from 10.5% to 30.2%. These results could

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be explained by that a low concentration of HA led to the slight aggregation of zein

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nanoparticles. Lefevre and Subirade

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in the aggregation of proteins. Continuously increasing the concentration of HA, the

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percentage of α-helix in zein was gradually increased, however, the other secondary

306

structure decreased. The result might be ascribed to the conformational relaxation of

307

zein-Que-HA composite nanoparticles,

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fluorescence analysis.

45

pointed that β-sheet was frequently increased

38

which was also confirmed by the

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Differential scanning calorimetry (DSC) analysis. Thermograms of zein,

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zein-Que, HA-Que, and zein-Que-HA composite nanoparticles were showed in Fig.

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3d. There was a broad endothermic peak at 95.9°C in the curve of zein, it was related

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to the denaturation of biopolymers.46 The endothermic peak temperature of zein-Que

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(100:5) complex was lower (92.8°C) than that of zein. The decrease might be

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attributed to hydrophobic effects and hydrogen bonding between Que and zein, which

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were confirmed by the FTIR result. It was analogous to that curcumin could decrease

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the melting temperature of zein though the intermolecular interactions.40 The

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endotherms peak of zein-Que-HA (100:5:5) complex was 78.6°C, the low melting

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temperature may be due to that it had the large particle size and loose network

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structure, resulting in the weak stability against the thermal treatment. With the

320

increasing of HA concentration, the endothermic peak of zein-Que-HA complex

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tended to the higher temperature. This result revealed that the incorporated HA

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enhanced the ability to resist degeneration of zein-Que-HA composite nanoparticles.

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In particular, zein-Que-HA composite nanoparticles (zein-Que-HA, 100:5:20) and

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microgel (zein-Que-HA, 100:5:25) exhibited the better thermal stability than that of

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zein-Que-HA (100:5:5, 100:5:10, and 100:5:15). It was futher cinformed that

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polysaccharides could improve the stability of the protein by disturbing its original

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denaturation behavior.47 Kittur et al. 48 has found that the alteration of endothermic

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peak temperature of chitosan was related to the polymer-water interaction, namely,

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the water holding capacity. The HA with lots of hydrophilic groups could bind some

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water molecules into the zein-Que-HA microgel, leading to the increased endothermic

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peak temperature.

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Physical stability. As shown in Fig. 4, it was obvious that zein-Que-HA colloidal

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dispersions at the mass ratios of 100:5:5 and 100:5:10 were less stable than those of

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100:5:15, 100:5:20, 100:5:25, and 100:5:30. It was attributed to the aggregation of

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zein-Que-HA nanoparticles, which was confirmed by particle size, zeta-potential and

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FE-SEM analyses in this work. For better understanding, the curves of the integrated

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transmission against the measuring time were plotted.32 Lower value of the slope

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represent more stable (Fig. 4g). It was apparent that the slopes of zein-Que-HA

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colloidal dispersions with a low level of HA (100:5:5 and 100:5:10) were significantly

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(p < 0.05) lower than the slopes of other samples. At high levels of HA, the colloidal

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dispersions became more stable. One of the reasons was that the negative charge of

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zein-Que-HA composite nanoparticles was increased; electrostatic repulsion could

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prevent the particles from being together. With the addition of HA (100:5:25 and

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100:5:30), the viscosity of the colloidal dispersions was increased, and zein-Que-HA

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microgel with 3D network-like structure was formed, thus induced more stable

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dispersions.38

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Photochemical and thermal stability. On the basis of particle size, zeta-potential,

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EE, LC, and micro-morphology analysis results, zein-Que-HA (100:5:20) composite

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nanoparticles were regarded as an ideal delivery carrier for Que. The photochemical

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and thermal stability of free Que, HA-Que (20:5), zein-Que (100:5), and

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zein-Que-HA (100:5:20) were examined in the work.

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Fig. 5a showed the relationship between the retained Que (Ln(C/C0)) and the

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time (h) at the level of 150 µg/mL Que in free or in composite nanoparticles. The

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degradation of Que followed the first order kinetics. The coefficient (R2 > 0.95),

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half-life (t1/2), and overall rate constant (k) for Que were given in supporting

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information Table 2. The protective capability of zein, HA, and zein-HA composite

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nanoparticles on Que according to t1/2 value was ranked as follows: Que < HA-Que